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AMERICAN ASSOCIATION OF ANATOMISTS
ONE HUNDRED AND THIRD ANNUAL MEETING
HAHNEMANN UNIVERSITY, THOMAS JEFFERSON UNIVERSITY,
MEDICAL COLLEGE OF PENNSYLVANIA,
PHILADELPHIA COLLEGE OF OSTEOPATHIC MEDICINE,
TEMPLE UNIVERSITY, AND UNIVERSITY OF PENNSYLVANIA
SCHOOLS OF MEDICINE
APRIL 22125,1990
OFFICERS
President
JEROME SUTIN
Emory University
Atlanta
President-Emerit us
First Vice-President
Program Secretary
ROGER R. MARKWALD
JENNIFER H. LAVAIL
CHARLES E. SLONECKER
Medical College of Wisconsin
Milwaukee
University of California
San Fraiicisco
University of B.C.
Vancouver
President-Elect
Second Vice-President
Secretary- Peasurer
KAREN R. HITCHCOCK
DONALD A. FISCHMAN
ROBERT D. YATES
University of Illinois
Chicago
Cornell Medical School
New York City
Program Secretary-Elect
%lane University
New Orleans
MARILYN L. ZIMNY
Louisiana State University
New Orleans
These are abstracts of papers presented at the 103rd annual meeting of the American Association ofAnatomists in Philadelphia, Pennsylvania under the sponsorship of the Schools of Medicine at Hahnemann
University, Jefferson Medical College, Medical College of Pennsylvania, Philadelphia College of Osteopathic
Medicine, Temple University, and the University of Pennsylvania on April 22-25,1990.
The abstracts are listed in alphabetical sequence by senior author.
Names of authors who are guests of the Association are marked with an asterisk. In addition, the letter “S”
preceding the first author’s name is used t o denote a student presentation by an individual in hidher terminal
year of study, sponsored by hisher major professor. Postdoctoral Fellows are designated by an open diamond
symbol (0) next t o their names.
A reprint from
THE ANATOMICAL RECORD
Volume 226, No. 4, April 1990
Written and Compiled by
Dr. Charles E. Slonecker
1A
Introduction: Dr. Stephen T. Kitei (University of Tennessee)
Dr. AIan Peters, Chairman and Waterhouse h f e a s o r of
Anatomy (Boston University Sehwl ofMedieine) Weuronal
Inter-relations in the Cerebral Cortex'
SUYMARY OF PROGRAM
THURSDAY APRIL 19, FRIDAY APRIL 20, SATURDAY APRIL Z l , 1990
3 DAY
SATELLITE CONFERENCE ON T h e Anatomical
Temple
Basis of the Swtional Imsging Modalities.'
University
Organizer: Dr. Carson D. Schneck (Temple University)
6:OO
PM-7:00 PM
Socislircr (No Host Bar)
SATURDAY. APRIL 21. IOW
Univ. of
SATELLITE CONFERENCE ON 'Intermediate
Voltage Electron Micmsmw and 3-D
Pennsvlvonia
Imaging.'
Organizers: Dr. Keith R. Porter, Dr. Lee D. Peachy, and Karen
L. Anderson (University of Pennsylvania)
SATELLITE CONFERENCE ON 'Current
Univ. of
Approaches to Imaging using Light
Pennsylvania
Micmscopy.'
Organizer: Dr. Joseph W. Sanger (University of Pennaylvnmn)
1O:OO AM-5:30 PM
A.A.A. EXECUTIVE COMMITTEE MEETING
Salon I0
Presiding Oflieer: Dr. Jemme Sutin
(Mezzanine Levcl)
(Emory University)
6:30 PM-8:OO PM
ASSOCIATION OF ANATOMY CHAIRMEN'S MEETING
Presidmg Officer: Dr. Cornelius Rosse Philadelphia Room
(University of Washington)
(Mezzanine Level)
k a n ' a Corner
(Lobby Level)
3:30 PM-530 PM
EDUCATIONALAFFAIRS COMMITTEE
REFRESHER COURSE
Bsllmm B
'Cellular Cytoskeleton.'
Organizer: Dr. Stanley L. Erlandaen (University of
Minnesota)
Speakers: Dr. Riehard Linck (University of Minnesota) W c w
Concepts for Microtubules and Intermediate
Filaments' Dr. Wayne Vogl (University of British
Calumbia) .Actin Filaments: Intracellular
Arrangements and Functions."
Ballroom A & B
KEYNOTE ADDRESS TO THE 103RD ANNUAL MEETING
(Sponsored by JEOL Inc. & AAC)
Nob4 Laureate lbmten Wiesel (Rockefeller University)
Weural Mechanisms of Vision"
7:OO PM-8:00 PM
830 PM-1030 PM
SUNDAY. APRIL 22.1990
800 A M 4 0 0 PM
Registration
1O:OO AM4:00 PM
Spouses' Hospitality Suite
Wyndhnm Foycr
( B a l l m m Level)
Association ofAnstomy Chairmen's Reception at the Univcrsily
of Pennsylvania
Ballroom Foyer
MONDAY. APRIL 23,1990
8 1 5 A M 4 : O O PM
1990 Cajal Club Program
Presiding Oflicer: Dr. Stephen T. Kitai
(The University of Tennessee)
Ballroom A
MORNING SESSION-SIMWSIUM
Tunctional Integration in the Basal Ganglia'
CO-Chairs: Dr. Suzanne Haber (University of Rwhester) and
Dr. John R. Sladek, Jr. (University of Rwhester)
815-8:30
Introduction
Dr. John R. Sladek, Jr. (University of Rwhester)
830-900
Dr. Stephen Kitai (University ofTennessee)
Tunctional circuits of the basal ganglia"
900-930
Dr. Charles Gerfen WMH) 'Striatal
mmpartmental organization: In situ hybridization
analysis of function'
9:30-1O:OO Dr. Derek Van der K w y (University of Toronto)
T h e development of functional mmpartments in
the basal ganglia'
1000-10:30 Dr. Suzanne Haber (University of Rochesbr)
Tunctional integration VLI. segregation thmugh
basal ganglia pathways'
10:30-11:W Dr. Timothy Collier (University of Rwhester)
Tactnrs promoting survival and p w t h of
dopamine neurons: Applications Tor neural
graRing in the basal ganglia'
11:OO-11:30 Dr. Roger Albin (University of Michigan) 'The
functional anatomv of movement disorders"
11:30-11:45 Dr. John R. Sladei. Jr. (University o r b c h e s t e r )
General Discussion
11:45AM-1:00 PM
LUNCH
AFTERNOON SESSIONS
LOO PM-230 PM
MEMORIAL SYMPOSIUM IN HONOR OF HANS K W P E R S
(CO-sponsoredbyAAA& Dupont)
"Transneurnnsl Transport of Herpes Viruses'
Chair:
Dr. Peter L. Striek
LOO-1:30
J. Patriek Card. Ph.D. (E.I. du Pont de Nemours
and Company) "Uptake end trans-synaptie
transport of alpha-herpes viruses in the rat
central nervous system"
130-200
Jennifer H. L a v a 4 Ph.D. (University of
California) "EM, immunocytochemical and
autoradiographic studies of virus transport
thmugh the trigeminal system"
200-230
Peter L. Strick. Ph.D. (SUNYHealth Science
Ccnter a t Syracuse) "Transneuronal transport of
Herpes Simplex Virus in the motor system of
primates"
2 4 0 PM- 00 PM
KRIEGAWARDS CEREMONYAND SPEAKERS
240-300
Krieg Cortical Kudos Awards
3:OO-3:30 Dr. Dcepak N. Psndya (Boston University)
Cortical Discovery Award Winner
3:304:00 Dr. Migucl Marin-Padilla (Dartmouth Medical
School) Cortical Discovery Award Winncr
7:OO A M 4 3 0 AM
CANADIAN ASSOCIATION OF ANATOMY CHAIRMEN'S
BREAKFAST
care
Presiding Offieer: Dr. Stan R. Blecher (Univ. of (Lobby Levcl)
Guelph)
800 AM4:OO PM
Registration
Ballmom Foycr
8 3 0 AM-10:30 AM
PRESIDENTIAL SYMPOSIUM
Ballroom A
T h e Pliable Nervous System: Changing Synapse Efficacy."
Organizer: Dr. Jemme Sutin (Emory University)
Speakers:
830-9:00
Dr. Jemme Sutin (Emory University) 'Does
sprouting of one class of terminals change the
behanor of other types of synapses?'
Dr. Robert Mwre (SUNY a t Stony B m k ) 'Does
9:OO-9:30
sprouting change the density of synapses an target
neurons?'
9:30-10:30 Dr. Charles Stevens (Sslk Institute) "Activity
dependent modulation of synapse ellicacy: The
Nrrent status of Hebb's birds-of-a-feather
proposal.'
830 AM-500 PM
AAA Placement Service
Snlons 1/2
9:oo AM4:00 PM
Spouses' Hospitality Suite
Lngan's Corner
(Lobby Level)
900AM4:30 PM
Commercial Exhibits
Conference Centcr
(Mezzanine Level)
9:30 Ah4:30 PM
POSTER SESSIONS (Please keep
posters u p far the entire day)
Conference
(MezzanineCrnter
Lcvcl I
Cellular Bioloav and Extracellular Matrix
(Papers 1-5)
Neuropathology
p a p e r s 2e-w
Neumcytology
(Papers 3 7 4 4 )
Neumplastieity, Transplantation and Regeneration
(Papers 4 6 7 2 )
Neural Degeneration
(Papers 7 S 7 7 )
Developmental Neumbiology
(Papers 7 a 9 9 )
Neumendocrinology
(Papers 100-112)
10:30 Ah-l1:3OAM
PROGRAM TIME RESERL-ED FOR POSTER RELIEW
All authors shauldbe present a: their posters dunnq t h i s t i m r
1030 AM-1200 NOON
Association ofAnstomy Chsirmi- -Publie
Philndelphia
Affairs Committee
Room
WORKSHOP ON LOW-LEVEL RADIOACTIVE WASTE
Moderator: Dr. Gordon &ye, Chairman AAAPublic Affairs
Committee. Chairman New York State Low-Level
Waste Group
Issues:
Backgmundofthe Pmblem
States and Compacts
4:OO PM-5:OO PM
Cajal Club Business Mccting
5 0 0 PM4:OO PM
Twenty-Scmnd Annual Pinekney J . Harman Momorial
LCctUre
2A
Federal Milestones
Consequences of Failure to Meet Milestones
Panel Participants:
Hon. Angelo Orazia, Chairman, NYS Low-Level
Radioactive Waste Siting Commission
Dr. John Randall, Pmjeet Director for Low-Level
Radioactive Waste Disposal, N Y S Energy Research
and Development Authority.
Mr. William Dornsife, Chief, Division afNuelear
Safety, Department o f Envimnmental Resources.
State of Pennsylvania
Mr. John R. Vincenti, Executive Secretary,
Appalachian Compact of Users ofRadioactive
Isotopes.
1 0 3 0 AM-lZm NOON
Salon 8
Committee on Anatomical Nomenclature
Chairman: Dr. George F. Martin (Ohio State University)
1130 AM-1230 PM
R.R. BENSLEY MEMORIAL LECTURE
Ballmom A
'Structural Concerns ofthe Human Centmmere"
Dr. William C. Earnshaw (Johns Hopkins University)
1:15 PM-3:15 PM
SPECIAL TOPIC SESSIONS I
Ballmom A
"MultiDle Roles of Gmwth Factors in
Ne;mdevelopment"
Dr.
Story
Landis
(Case
Western
Reserve
Organizer:
University)
1:30-1:35
Intmductian: Dr. Story Landis
Dr. Eugene Johnson (Washington University)
1:35-215
W h y are NGF receptors everywhere (almost)?"
215-255
Dr. J a n e Dodd (Columbia University College of
Physicians and Surgeons) 'Cell patterning and
neumoal recognition in spinal cord"
Dr. Story Landis (Case Western Reserve) "Target255-315
derived fadoris) determine neurotransmitter
phenotype'
Ballmom B
Wuclear-Cytoplasmic Interactions"
Organizer: Dr. Carl Feldherr (University ofFloridaGainesville)
Speakers:
1:15-1:45 Dr. Gerd Maul (Wistar Institute) 'Morphology of
the Nuclear Pares and Related Structures"
(Paper 112A)
Dr. Carl Feldherr (University ofFlorida) "The
1:45-215
Function of the Nuclear Pares in Macrnmoleculnr
Exchange"
Dr. Pamela Silver (Rinecton University)
2:15-245
'Biochemical and Genetic Analvsis of Nuclear
Protein Import"
Mr. Rabert Mandell (University o f Florida)
245-300
'Bidirectional Exchange ofTwo hse 70-related
oocyte proteins acmss the Nuclear Envelope"
(Paper 113)
Dr. Daman C. Herbert (University ofTexas Health
3:OO-3%
Science Center) 'Regulation o f Transferrin Gene
Expression in TranSKmiC Mice"
(Paper 113A)
Dr. Rudolph Jaenisch (Whitehead Institute, Massachusetts
Institute of Biology) '"Inductionof Developmental Mutations
in Transgenic Mice."
8 3 0 AM-530 PM
AAA Placement Service
9:00AM4:30 PM
Cammereid Exhibits
900 AM4:OO PM
Spouses' Hospitality Suite
10:45 AM-1245 PM
Philadelphia Room
SPECIAL TOPIC SESSIONS I1
Mezzanine Level
Tmnticrs in Mucin Biology"
Organizer: Dr. Robert D. Specian (Louisiana
S t a t e University-Shrev&t)
Speakers:
1045-1130 Dr. Marian Neutra (Harvard Medical Schaol) "New
Approaches: Monolayer culture systems and
transgenic mice"
11:30-12:15 Dr. Daniel K. Podolsky (Harvard Medical School)
'Complexities of Mucin Glyco-pmtein S t N c t U r e "
1215-1:00 Dr. Judith St. George (University of
California-Daws) "Biology of Goblet Cclls in
Rcspiratory Airways"
"Membrane-Cytaskeleton Interactions:
Ballmom A
Mechanism and Regulation"
Organizer: Dr. Mary Beckerle (University of Utah)
Speakers:
10:45-11:15 Dr. Paul Forscher Wale University) "The
cytomechanies of neurnnal gmwth cone motility:
11:15-11:45 Dr. Sally Zigmond (University of Pennsylvania)
'Leukocyte actin assembly regulated by
ehemoattractants."
1145-1215 Dr. Mary Beckerle (University ofUtah) "Actinmembrane interaction a t sites of cell-substratum
adhesion."
1215-1245 Dr. Clayton Buck (Wistar Institute) 'Intcgrins as
transmembrane mediators ofcytoskelctanextracellular matrix interactions."
Ballmom B
"New Perspectives on the Control of Bird Flight"
(Papers 264-265B)
Organizer: Dr. Arthur English (Emory University)
Speakers:
10:45-11:15 Dr. Colin Pennycuick (Miami University)
"Structures and structural materials in the wings
of flying vertebrates."
(Paper 264)
11:15-11:45 Dr. Robert Hikida (Ohio University) "Are bird
m u d e s just a modification of the mammalian
plan?"
(Paper 265)
1145-1215 Dr. George E. Godow, Jr. (Brnwn University) "The
neural contml offlight: what we can learn fmm
the starling (Sturn& vulgaris)."
(Paper 265A)
121.5-1245 Dr. John Steevea (University ofBritish Columbia)
Weursl control o f avian locomotion: Is it
different?'
(Paper 265B)
5 3 0 PM-430 PM
Ballmom A
CHARLES JUDSON HERRICK MEMORIAL
LECTURE
'Development and Plasticity of B Neural Crest-derived
Biopotential Progenitor Cell.' Dr. David Anderson
(California Institute o f Technology)
7 0 0 PM-1030 PM
SOCIALIZER AT FRANKLIN INSTITUTE
(Refreshments and hors d'ooeuvres will bf served)
TUESDAY, APRIL 24,1990
7:OO A M 4 3 0 AM
AMERICAN ASSOCIATION OF
Salons 5w
VETERINARY ANATOMISTS BREAKFAST
Presiding Ollicer: Dr. John T Munnell ( U n \ v r r - i t y ofGear6ia)
8:OO AM4:oo PM
Registration
Ballmom Foyer
8 3 0 AM-l0:3OAM
Ballmom A
VICE PRESIDENTIAL SYMPOSIUM
'Gene Knockout in Development."
Organizer: Dr. Donald A. Finchman (Cornell University)
Speakers:
Dr. Alexandra Joyner (Mount Sinai Hospital Researeh
Institute) "Role o f t h e Mammalian Engrsiled-like Genes in
CNS Development."
Logan's Corner
(Lobby Level)
9:30 AM4:30 PM
POSTER SESSIONS
Conference Center
(Mezzanine Level)
(Posters are to be displayed for
t h e entire day. Authors are asked to be present, particularly
fmm 1003:OO pm.)
Sensory Systems
(Papers 151-175)
Neumtransmittera
(Papers 176-183)
Motor Systems
(Papers 184-190)
Neurnglis
(Papera 191-195)
Autonomic and Enterie Nervous Systems
(Papers 196-210)
Developmental Biology
(Papers 211-251)
Gmss Anatomy
(Papers 252-2631
3 3 0 PM-530 PM
PLATFORM SESSIONS
Advisory Committee ofYoung Anatomists Philadelphia Roam
South
Special Platform Session for the
J a n Langman Award Finalists
(Featuring Outstanding Graduate Student Researeh
Presentation)
(Papers 114-120)
Salon 10
Skeletal Muscle
(Papers 121-128)
Cellular Biology and Extracellular Matrix Philadelphia Room
(Papers 129-136)
North
Salons 314
Neurncytology
(Papers 137-142)
Regulation of Development
Salons 5/6
(Papers 143-150)
5:30 P M 4 4 5 PM
GRADUATE STUDENT RECEPTION
Wyndhsm Ballmom
Sponsored by the AAA and AAC. Hasted by the ACYA.
Faycr
Salons 112
Conference Center
(Mezzanine Level1
1 2 4 5 PM-200 PM
AAA BUSINESS MEETING
Ballmom C
Presiding Ollicer: Dr. Jemme Sutin (Emory University)
1 3 0 PM-330 PM
PROGRAM TIME RESERVED FOR POSTER REVIEW
(All authors should he present a t their posters during this
time)
1:304:30 PM
Franklin Room
VIDEO INTERVIEWS OF PIONEERS IN
( M n s Level)
MODERN BIOLOGY: An Interview of
Dr. Berta Schaner by Pmf. Mary A. Bonnenlle
2 3 0 PM4:OO PM
Editorial Board
The Anatomical Rccard
Salon 10
4 3 0 PM4:OO PM
Editnrial Board
The Amcrican Journal afAnatomy
Salon 10
3 3 0 PM-530 PM
EAC SyMpOSIUM
3A
Ballmom A
"Immunamntraception: Vaccines Tor the Male and Female"
Omanizer: Dr. John Herr (Universitv
. ofviruinia)
Sp;skers:
3 3 0 4 : 3 0 Dr. Bonnie Dunbar (Baylor University)
'Develoomentallv Remlated Ovarian Antigens and
Their Rbles in F&tilzy4:30-5:30
Dr.Paul PrimakoN (University of Connecticut)
%pas
toward a Birth Contml Vaccine that
Blocks Sperm Function"
1030 AM-100 PM
PLATFORM SESSIONS
Gmas Anatomy
(Papers 38-389)
Male Reproductive System
(Papers 390-396)
Lymphoid Tiasues
(Papers 391402)
Female Repmductive System
(Papers 26.5-212)
3:30 PM-5:30 PM
PLATFORM SESSIONS
Cardiovascular Development
(Papers 213-280)
Developmental Neumbiology
(Papers 281-285)
Neumendaerinalogy
(Papers 286-294)l
Philadelphia Room
Salons 3/4
Franklin Room
Salons 5/6
11:45 Ah-1:30 PM
Salons 5/6
PROGRAM TIME RESERVED FOR POSTER REVIEW
IAll authors should be presenr a t thew posters dunng this
lime i
Salons 3/4
Philadelphia Room
North
1.30 PM-3 30 PM
SPECIAL TOPIC SESSIONS 111
Philadelphia Room
ofthe Cell Cycle'
Dr Gary C Schoenwolimniversity of Utah,
%egulatian
7:30 PM-890PM
AAAHistorian's Slide Presentation
Dr. G.E. Eriksan (Haward University)
Organizer
Speakers
1 30-2 10
Ballrooms A + B
8 0 0 PM-1000 PM
2 10-2 SO
PRESIDENTIAL AWARDS SESSION
Ballmoms A + B
Presiding Officer: Dr. Jemme Sutin (Emory University)
Henry Gray Award
ACYNAAC Graduate Student Diasertation Award
J a n Langman Award
Basmajian/Williams & Wilkina Award
2 50-3 30
'Innovations in Electmnic Anatomy"
Ballmom A
(EAC SPONSORED)
Organizer: Dr. Mark Nathanson (University of Medieine and
Dentistry ofNew Jersey)
Speakers:
1:30-1:35
Introduction: Dr. Nathanson
1:35-215
Dr. Robert Chase (Stanford University) 'The
Electronic Cadaver"
2:15-2:50
Dr. D a n d Whitloek (University of Colorado)
T h r e e Dimensional Computer Images of Human
WEDNESDAY, APRIL 25,1990
7 3 0 AM-930 AM
Council ofPresident's Breakfast
800 AM-1200 NOON
Registration
Salon 10
Ballmom Foyer
8 3 0 AM-1130 AM
MORPHOGENESIS AND DEVELOPMENTAL
Ballmom A
BIOLOGY SYMPOSIUM
'Angiogenesis: The Cellular and Developmentsl Biology of the
Vascular System"
Organizer: Dr. Richard Feinberg (University of Medieine &
Dentistry oiNew Jersey)
Speakers:
830-9:30
Dr. Judah Folkman (Harvsrd University) 'Switch
tc Angiogenesis During Tumarigenesis"
9:30-1000 Dr. Drew Noden (New York State College of
Veterinary Medieinel 'Origins and Assembly of
Embryogenic Endothelid Channels"
10:00-10:30 Dr. Thomas Poole (SUNYUpstate Medical Center)
"Endothelid Cell Origin and the Marphogenesis of
Embryonic Vascular Pattern"
1030-1130 Dr. Ken Thomas (Merck, Sharp & Dohme Research
Laboratories) T h e Structure Stability and in vivo
Activities ofAcidic FGF"
8:30 AM-5:OO PM
AAA Placement Service
Dr Kathryn Swensan (Haward Medical School)
'Cyclins and Rewlation oithe Cell Cycle m Early
Embryos *
Dr Edward Salmon (University ofNorth Cnmlina
micmtubule Dynamics and Chromosome
Movement "
Dr Thomas Schmcdcr(Fndsy Harbor
Laboraranes) T h e Contractile Ring Motor for
Cytokenesrs"
Anatom9
Dr. Cornelius Rosse (University of Washington)
T h e Presentation oiSpstial and Abstract
Knowledge in Computer-Readable Form."
(A di-ssion on Innovations in Electmnic Anatomy will
continue fmm 3 3 0 4 S O for audience interaction with the=
authors and their innovative teehniques.)
250-3:30
"Scanning Tunnelling Micmsmpy"
Ballmom C
Organizer: Dr. Robert 0. Kelley (University o i New Mexico)
Sneakers:
-r------
130-2:15
Dr. John A. Panitz (University of New Mexico and
UNM School afMedicine) 'Scanning Tunnelling
Microscopy: Status and Pmapecta for Biological
Imaging"
2:15-3:00 Dr. Stewart M. Lindsay (Arizona State University)
'STM Images of Nucleic Acids in Water"
Dr. Paul E. West (Quansean, Inc., Pasadena) STM
390-330
Techniques
(A Workshop on SEanning Tunnelling Microsmpy will be a
continuation oithis program imm 330-530 PM)
Salons 112
9:00 AM4:OO PM
Spouses' Hospitality Suite
Lagan's Corner
3:30 PM-530 PM
WORKSHOPS
"Scanning Tunnelling Microscopy"
Ballmom C
Organizer: Dr. Robert 0. Kelley (New Mexico)
Participant: Dr. Paul E. West, Vice President for Technology
(Quanscm, Inc., Pasadena) "Hands-n Work\hop
lor STM"
(Lobby Level)
900 AM4:30 PM
Commercial Exhibits
Conference Center
(Mezzanine Level)
900 A M 4 0 0 PM
POSTER SESSIONS
Conference Centor
(Please keep posters up far the entire day) (Mezzanine Level)
Cardiovascular Structure. Function and Metabolism
(Papers 295-309)
Endaerine Tissues and Function
(Papers 310-311)
Female Repmductive System
(Papers 317A-333)
Male Repmductive System
(Papers 334-345)
Skeletal Muscle
(Papers 346-356)
Lymphoid Tiaaues
(qapers 357-360)
Imagmg Techniques in Anatomical Education
(Papers 36-380)
(:oniercwce
Books ofAnatomy and Medical Sciences"
Cvntcr Por1cr
Arco
Organizers: Dr. Richard Libhin (New York University)
Dr. Duane E. Haincs (University ofMississippi)
3-
THURSDAY, APRIL 26,1990
SATELLITE CONFERENCE ON 'Computer
Thomas
Jeflersan University
Assisted Instnrction."
Organizer: Dr. Ronald P. Jensh (Thomas JeNersan University)
4A
ABSTRACTS
of the
American Association of Anatomists
One Hundred and Third Annual Meeting
April 22-25, 1990 Philadelphia
ADRIAENSEN,* Dirk, Dietrich
W. SCHEUERMA”,
Jean-Pierre TIMMERMANS and Marie H.A. DE GROODTLASSEEL, Institute of Histoloav and MiCrOSCODiC
Anatomy,
University of
AfiEwerp,
Belgih.
Calcitonin gene-related peptide, enkephalin and
serotonin in pulmonary neuroepitneiiai boaies ot
tne rea-earea turtle, pseuaemys scripta eieqans.
man, serotonin (5-HT) and several neurope tides
including bombesin (BOMI, calcitonin YCAL),
enkephalin (ENKI, somatostatin (SOMI,
tokinin
(CCKI and
calcitonin
g e n ~ %
pe tide (CGRPI were demonstrated in solitar NEE
ceyls and NEBs. In amphibians, BOM. ENK an2 CCK
were only found in single NEE cells whereas 5-HT
was also observed in NEBs. In reptiles, 5-HTcontainin
NEBs were demonstrated (Scheuermann
DW et a?. (19831 Cell Tissue Res 2 3 4 : 2 4 9 - 2 6 9 ;
Pastor LM et al. ( 1 9 8 7 ) Cell Tissue Res 2 4 8 :
713-715). Nevertheless the neurope tide content
of the reptilian pulmhnarg neuroengocrine
thelial s stem remains to e elucidated. In t is
study, txe lungs of the red-eared turtle, Pseudemys scri ta elegans, were investigated using
immunocytocEemistr for 5-HT and the following
neuro eptides: CGR6 ENK BOM SOM CAL and CCK.
The fissues were fiepar&d ro6tinei for immunoc tochemical inves igation on garafxin sections.
CXRP-imdnoreactive NEBs coul be demonstrated
in
the ciliated e ithelium of the bronchi
rimary, secondary, eertlary trabeculae and inferfaveolar septa Occasional1
single NEE cells
were immunoreaitive for C G R ~ IUsing consecutive
sections, CGRP could be shown to coexist with
5;HT i n NEB cells. In addition, NEBs immunoreactive for ENK, were observed. To our knowledge,
rovides the first demonstration of
y:;:s
NK-inmunoreactivity
in
ulmonary
neuroe ithelial bodies of a lower verte&rate. So
far, EGRP was only observed in NEBs of mammals.
Hence, NEBs in the lungs of Pseudemys, a semiaquatic obligatory air-breathing reptile, also
contain multiple neuroactive
substances. It
seems, therefore, that throughout the evolution,
the pulmonary receptor mechanisms act through or
are regulated by a complexity of hormonal.
neurotransmitter or neuromodulator substances.
“E&-
g$;-
E
s
AHMAD*, Iqbal, Alan W. STEGGLES*, Judith A.
FINKELSTEIN,
Department
of
Anatomy
and
Biochemistry, Northeastern Ohio Universities
In
College of Medicine, Rootstown, Ohio,
situ hybridization studv of GH qene expression
in lean and aeneticallv obese Zucker rats.
We have shown in an earlier study an obesityassociated depression in GH mRNA levels which
may be the cause of decreased plasma GH levels
in obese Zucker rats. In order to look into the
possibility
of
a
decreased
number
of
somatotrophs in the obese rat pituitary as the
cause of the decrease in GH mRNA levels, GH gene
5A
expression was studied by in situ hybridization
on 61.1 sections using a 35S labelled GH cDNA.
Specificity of hybridization was checked by
pretreatment with RNase A and a competition
study.
Counting of somatotrophs in a given
region of the anterior pituitary showed no
significant difference in their number in lean
and obese animals.
Somatotrophs in lean
pituitary sections were found to express more GH
transcripts than in obese pituitary sections,
extending at the single-cell level the result
obtained by Northern blot analysis. Funded by
Ohio Board of Regents Research Challenge.
~
~
~
~
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~
AIRHART*,Mark J., Mary Ann ROBERTS*, Thomas B.
XNUDSEN*, and Richard G. SKALKO, Department of
A ~ t c a n y , Jams H. Qlillen College of Ndcine, East
Tennessee S t a t e University, Johnson City, Tennessee.
?he precision of fiber quidmce for a population of
adenwine deaminase irmauroreactive primry afferent
f k s in developinq muse spinal cord.
This study examined the precision of central fiber
growth in a subpopulation of dorsal root ganglion
neurons in developing mouse spinal cord.
hnunohistoci.remic1 techniques using a mmospecific,
polyclonal antiserum to mouse adenosine deaminase
(ALIA) were utilized to selectively label a Dopllation
of primary sensory affemts that have been fourd to
exclusively innemate laminae I and I1 of the dorsal
horn in adult mice.
Initial growth of ADAimumreactive (ADA-IR) primary afferents occurred
very early in developat, E10, a time coincident with
ti7e earliest settling t h of dorsal root ganglion
neurons. Adenosine deaninass imnumrextive pritMry
afferents were ohserved thrmghout the cross-sectional
area of the primordial dorsal funiculus (IF)
as early
as gestatimal day 10. These i m ~ o s t a i n e d fibers
remained quiescent in the DF during its grmrth and
change in moqblogy.
By gestational day 15 t k DF
had separated into two pathways, the tract of Lissauzr
and dorsal column pathway, and ADA-IR fibers were
observed exclusively in the tract of Lissauer. This
segregation of fibrs remained thrm-t
developnent
and reflected the adult pattern. Inmumstained fibers
initially g r e w into the dorsal horn at 216. The
label4 fikers irmnediately q e w into laminae I and I1
mimicking their adult distribution. Exuberant fiber
grcxvth was m t detected thrmghcut their developat.
These results strongly suggest that ADA-IR fibers
exhibit precise fiker guidance to a preferred patby,
the tract of Lissauer, and a precise laminar
innervation of t h e dorsal horn.
(Supprted by NIH
grant 1 -R29-HD25143)
6A
ABSTRACTSLAAA 103RD MEETING
Al-Bagdadi, F.K., B.E. Eilts", D.J. MCCOY*
and J.D. Cummings*. Department of Veterinary
Anatomy and Fine Structure and The Department
of Veterinary Clinical sciences, Louisiana
State University, Baton Rouge, Louisiana.
Scanninq Electron Microscopy of the
Endometrium of Repeatedly Infused Mares With
Gentamicin.
Nine cycling thoroughbred mares were used
to determine the effect of repeated intrauterine infusion of Gentamicin on the endometrium. Five mares were infused on two consecutive days with 40 ml Gentamicin (50
mg/ml) mixed with 80 ml of saline. Four
mares served as controls and were infused
with 120 ml of saline. Endometrial biopsies
were obtained from a l l mares before infusion
and on days 1, 3 , and 5 after the second
intrauterine infusion. Each biopsy was processed for scanning electron microscopy. The
stage of the mare's estrous cycle at biopsy
was determined by blood progesterone concentration. No difference was seen between the
endometrial appearance in estrus or diestrus.
The endometrium of the gentamicin infused
mares had cellular perforations and less
microvilli on day 1. On day 3 , the reaction
was minimal in both groups. A large number
of the endometrial secretory cells lost their
lumina1 surface on the seventh day after
infusion in both groups. This indicates that
the Gentamicin infusion induced pathologic
changes earlier than saline, but both groups
had significant endometrial changes seven
days after infusion.
ALBRIGHT, Bruce C., Michael R. BROWN*, Harvey, R. KNULL*,
Department of Anatomy and Cell Biology; Department of
Biochemistry and Molecular Biology, University of North
Dakota School of Medicine, Grand Forks, North Dakota.
Dystrophic changes in terminals in the gracile nucleus
in streptozotocin diabetic rats.
The ultrastructure of terminals in the gracile and
cuneate nuclei of STZ diabetic rats were examined at two
weeks and one month after the onset of diabetes.
Diabetes was induced by the injection of 70 mg/kg of
streptozotocin and animals were considered diabetic when
urine glucose reached 2000 mg/dl while control animals
tested negative (less than 100 mg/dl ). A1 1 experimental
animals were diabetic at 24 h post-injection. Three
experimental and two age matched control rats were
killed and processed for electron microscopy at two weeks
and one month post-injection. Dystrophic terminals were
present in both diabetic groups and none were observed
in controls. The number of dystrophic terminals
increased and the severity of their response increased
with longer diabetic periods. Dystrophic changes
involved the decrease in number of synaptic vesicles, the
presence of large possibly swollen synaptic vesicles, an
apparent reduction in the number of synaptic contacts,
and the accumulation of tubulovesicular SER. Branched
tubulovesicular SER was the best indicator of the
dystrophic response. Compared with two weeks experimental rats, the one month diabetic animals appeared to
have a greater number of both dystrophic terminals and
myelinated axons with abnormal amounts of tubulovesicular SER. Often both terminals and myelinated axons
appeared enlarged and filled with SER. More variable
morphological changes included the presence of lamellar
bodies and peroxisomes. At two weeks, many dystrophic
terminals did not appear to have the necessary organelle
complement and organization to support synaptic transmission. Many of the dystrophic terminals resembled those
of primary afferents. These findings show that severe
diabetes as modeled by the STZ diabetic rat has an early
and progressive dystrophic affect on terminals in a
somatosensory nucleus of the central nervous system.
AL-ALI, S.Y.A., I.M. HasSan*, S . Sadek*, and
M. Clavel*, Departments of Anatomy and Nuclear
Medicine, University of Kuwait and Berard Medical
Centre, Lyon, France. Scanning and transmission
electron microscopy of isolated rat liver perfused in vitro with high dose of methotrexate.
The structural integrity of isolated rat liver
was maintained for 3 hours at 3OoC in vitro by a
perfusion system. To study the direct effects of
high dose of methotrexate on the fine structure
of hepatocytes, 2000mg methotrexate was administered for 3 hours (ll.llmg/min) into the liver
via the portal vein. Treated and control livers
were processed for light, scanning and transmission electron microscopy. Survey low power
microscopy showed large part of liver plates
appeared normal. However focal changes consisted
of vacuolation and disruption of plates were seen
but without zonal preponderance. Scanning microscopy revealed extraordinary surface activity of
the plasma membrane. The canalicular surface of
the hepatocytes appeared nodular, and the sinusoidal surface exhibited tightly packed and
conglomerular transformation of microvilli, bleb
formation and fragility of plasma membrane.
Transmission electron microscopy of altered cells
showed disorganization of the GER, formation of
monoribosomes, central accumulation of mitochondria with low matrical density, hyperplasia and
fine vesicular transformation of SER, and large
aggregation of glycogen granules. The nuclei
exhibited condensation of the nuclear chromatin.
This study showed that the ultrastructural
changes, particularly the plasma membrane of the
hepatocytes are greater than that seen with the
light microscope, and reflect the disturbance in
the ionic and volume regulatory mechanisms
induced by high dose of methotrexate.
(Supported by K.U. Grant No, MA025).
s Al-Owais, H . ,
Sherif , M.F. , Al-Zuhair , A.G.H.
and Nawar, A.M*. Anatomy Department, Faculty of
Medicine, Kuwait University. Regeneration of
Axonotmised Rat Facial Nerve as Revealed by
Enzyme Tracer Transport : Quantitative study of
Retrograde Labeling with Horseradish Peroxidase
(HRP).
Compression was applied to the extramastoid
portion of the facial nerve, just distal to its
posterior auricular branch, for 10 minutes in 15
young adult Wistar rats. The force of compression
was 20.85z.22 Newtons. After 6, 12 and 18 days,
the facial nerve was re-exposed, severed distal
to the compression site and HRP was directly
applied to the proximal stump. In another group
of animals, HRP was applied similarly to the
facial nerve, without compression, and were used
as controls. After 72 hours, animals were euthanized by intracardiac perfusion, their brain
stems were serially sectioned and processed for
IlRP reaction. Recovery was determined by vibrissal movements and the return of the deviated
external nostril to its normal position. The
results revealed that 77.7~5.5% facial moto-
ABSTRACTS-AAA 103RD MEETING
neurons c o n t r i b u t e t o t h e nerve t r u n k d i s t a l t o
i t s p o s t e r i o r a u r i c u l a r branch. Application of
HRP t o t h e f a c i a l nerve a t 6 , 1 2 and 18 days
following compression r e s u l t e d i n l a b e l i n g of
39.6+2.8%, 74.8~5.358and 76.0+5.3% of i t s motoneurons r e s p e c t i v e l y , and signs of recovery were
manifested a t 1621 days. I n comparison w i t h
previous r e s u l t s from our laboratory, t h e s e
findings i n d i c a t e t h a t i n t h e r a t , neuronal-fiber
r a t i o of f a c i a l nerve is 1 : 1 . 3 . Following axontomesis, axonal t r a n s p o r t a c t i v i t y is r e s t o r e d
over t h e s i t e of t h e l e s i o n e a r l i e r than regain i n g normal nerve function and preceeds m y e l i nation. Moreover, f a c i a l nerve can function
adequately i n s p i t e of incomplete myelination
of i t s f i b e r s .
(Supported by Kuwait University Grant N o . MA027)
SAllen.* D. T. and J. A. Kiernan,* Department of
Anatomy, The University of Western Ontario.
London, Canada. (Sponsored by R..P. Singh)
&netration of e n t e r i c nervous t i s s u e hy
acer mote-.
The existence i n t h e myenteric plexus of a
b a r r i e r t o c i r c u l a t i n g p r o t e i n s i s controv e r s i a l . Gershon & Bursztajn (J. Comp. Neurol.
180, 1978) d i d n o t d e t e c t intravenously i n j e c t e d
horseradish peroxidase i n t h e e x t r a c e l l u l a r
spaces of t h e plexus i n t h e r a t , b u t Jacobs (J.
Neurocytol. 6 . 19771 did d e t e c t t h e enzyme i n a
comparable study i n t h e r a t and guinea-pig. The
discrepancy m a y have been due t o d i f f e r e n t times
of c i r c u l a t i o n of t h e t r a c e r . I n t h e present
study, horseradish peroxidase o r rhodamine Bconjugated bovine albumin c i r c u l a t e d f o r 5 o r 30
minutes a f t e r intravenous i n j e c t i o n i n r a t s .
The medulla, hypothalamus and ileum w e r e
examined by e l e c t r o n o r fluorescence microscopy.
The tracers d i d n o t c r o s s t h e blood-brain
b a r r i e r i n most p a r t s of t h e b r a i n , b u t
exudation w a s evident i n t h e area postrema and
median eminence. These regions are well known
t o have permeable blood vessels. I n t h e e n t e r i c
nervous system. peroxidase r e a c t i o n product w a s
found by e l e c t r o n microscopy i n t h e e x t r a c e l l u l a r spaces of ganglia and tracts i n t h e
myenteric plexus 5 min, b u t not 30 min a f t e r
i n j e c t i o n . Rhodamine B-albumin w a s observed as
f l u o r e s c e n t l i n e s between neurons i n t h e
myenteric and submucous ganglia a f t e r 30 min.
b u t n o t a f t e r 5 min. W e conclude t h a t t h e
e x t r a c e l l u l a r spaces of t h e e n t e r i c nervous
system are a c c e s s i b l e t o horseradish peroxidase
and t o f l u o r e s c e n t l y l a b e l l e d albumin. The
f a i l u r e t o d e t e c t horseradish peroxidase 30 min
a f t e r i n j e c t i o n may be due uptake of t h e enzyme
by phagocytic c e l l s .
(Supported by a g r a n t
from the J. P. B i c k e l l Foundation, Toronto.)
&VAREZ,* I.S.and G.C. SCHOENWOLF, Department of
Anatomy, University of Utah School of Medicine, Salt Lake
City, Utah. &re0 ithelial cell behavl'or in heterotoDiC
gpiblast erafts.
In previous studies, our laboratory has shown that avian
MHP cells, wedge-shaped neurepithelial cells that
comprise the median hinge point of the bending neural
plate, arise from a single, midline prenodal rudiment in
the epiblast of primitive streak stage embryos, whereas L
cells, lateral, spindle-shaped neurepithelial cells, arise
7A
from paired paranodal epiblast rudiments at the same
stages. Both MHP and L cells undergo division and
extensive rearrangement (intercalation) during neural
plate shaping and bending, but these two cell populations
remain spatially segregated from one another throughout
these processes; that is, MHP cells intercalate only with
MHP cells and L cells intercalate only with L cells. The
following experiment was performed to determine whether
intermixing of prospective MHP and L cells can occur when
they are given the opportunity to do so. Quail prospective
MHP and L cells were transplanted heterotopically to chick
host blastoderms (to wit, prospective MHP cells were
transplanted to the prospective L cell site and vice versa)
and the distribution and arrangement of the grafted cells
were determined (using the quail heterochromatin marker
to identify grafted cells) in chimeras obtained 24h
postgrafting. Our results demonstrate that heterotopic
MHP and L cells alter their characteristic patterns of
rearrangement such that MHP cells intermix only with
MHP cells and L cells intermix only with L cells. Thus,
MHP and L cells must each contain unique intrinsic,
identity markers that allow them to recognize their
counterparts. "his research was supported by NIH grant
no. NS 18112 and Fulbright Fellowship no. FU89-8797464.
ANDERS, Juanita, Sara WOOLERY,* Rosemary BORKE and
Willem VANDEMERWE,* Department of Anatomy and Laser
Biophysics Center, USUHS, Bethesda, Maryland. Varvina the
wavelenath of an araon ion tunable dve laser durina low
enerav irradiation alters the Dhotobiostimulatorv effect on rat
facial nerve reaeneration. Recently, we have shown that low
energy (8.5 mW, 60 min) laser irradiation caused an increase
in the rate of rat facial nerve regeneration after crush injury
(Anders et al., 1989, Neuroscience, 15 (1): 316). A wavelength
of 632nm was used in those experiments. The purpose of this
study was to establish the wavelength with the greatest
stimulatory effect on the rate of facial nerve regeneration. The
facial nerve was crushed unilaterally in anesthetited rats for 90
secs. The wound was sutured and transcutaneously irradiated
daily with an argon ion tunable dye laser at 8.5 mW for 60 min
for 7, 8 and 9 days at various wavelengths (632nm, 514nm,
457nm and 361nm). Rats were injected subcutaneously, on the
side of the face supplied by the injured nerve, with 24ul of
HRP (20%), 24 hrs before aldehyde perfusion. The number of
HRP labeled neurons in the facial nucleus was used as an
assay of the degree of regeneration. Control, uninjured rats
injected with HRP had an average of 2950 facial motor
neurons labeled. Rats in which the facial nerve was crushed
but not irradiated had an average of 24 neurons labeled on
day 7 post-crush, 18 on day 8, 89 on day 9 and 1140 on day
10. Laser irradiation at 632nm caused a faster rate of
regeneration with a %fold increase in the number of labeled
neurons on day 7. Laser irradiation at all wavelengths
examined caused an increase in the rate of nerve regeneration
as compared to the non-irradiated controls. However, 632nm
had the greatest effect on the rate of facial n e w regeneration
and decreasing the wavelength decreased the stirnulatory
effect. The effect of low energy biostimulation at wavelengths
greater that 632nm still needs to be investigated.
ANDERSON, E . , Mascus WALKER,* Mong-Ting LEE* and G. Y. LEE,*
Department of Anatomy and Cellular Biology and Laboratory of
8A
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
Human Reproduction and R e p r o d u c t i v e B i o l o g y , Harvard Medical
School, Boston, M a s s a c h u s e t t s . O v a r i a n c y s t f o r m a t i o n after
t r e a t m e n t with d e h y d r o e p i a n d r o s t e r o n e i n t h e rat.
From a p r o j e c t d e s i g n e d t o s t u d y d e h y d r o e p i a n d r o s t e r o n e
(DHEA)-induced p o l y c y s t i c o v a r i e s we documented t h e s e r u m
hormonal, macro- and m i c r o s c o p i c changes (Lee, M-T.,
Anderson and Lee, 1989, Anat. Rec. 223:66A), w e now r e p o r t
t h e f i n e s t r u c t u r a l changes of e q u i v a l e n t c y s t i c o v a r i e s .
Inmature (27 d a y s ) Sprague Dawley r a t s were i n j e c t e d d a i l y
(s.c.)
f o r 20 days w i t h 6 mg/100 g bw DHEA d i s s o l v e d i n 0.2
ml sesame o i l and c o n t r o l s were i n j e c t e d f o r t h e same t i m e
w i t h a n e q u i v a l e n t amount of sesame o i l . Animals were
k i l l e d a f t e r 0,2,3,4,5,10,15 and 20 d a y s of i n j e c t i o n and
t h e o v a r i e s removed and p r o c e s s e d f o r e l e c t r o n microscopy.
During c y s t f o r m a t i o n , g r a n u l o s a c e l l s o f t h e a f f e c t e d
f o l l i c l e s undergo what a p p e a r s t o b e s t r u c t u r a l l u t e i n i z a t i o n , i . e . , t h e r e is an a c c u m u l a t i o n o f smooth endoplasmic
r e t i c u l u m , l i p i d d r o p l e t s and many m i t o c h o n d r i a h a v e t u b u l a r
cristae. Granulosa c e l l s undergo a s y s t e m a t i c b l e b b i n g
which b e g i n s on t h e s u r f a c e f a c i n g t h e antrum. Progress i v e l y , t h e b l e b s a r e p i n c h e d o f f and removed w i t h remnants
o f g r a n u l o s a cells by macrophages, l e a v i n g a s i n g l e l a y e r of
c e l l s on t h e basement membrane. I n t h e a p i c a l and b a s a l
a s p e c t s of t h e s e g r a n u l o s a cells is a l a y e r of 6 nm ( a c t i n )
f i l a m e n t s . T h i s b a s a l l a y e r of c e l l s , i n a d d i t i o n t o b e i n g
a s s o c i a t e d v i a gap j u n c t i o n s , a c q u i r e s t i g h t i u n c t i o n s . The
b a s a l c e l l s a l s o h a v e c o a t e d a n d non-coated b a s a l i n v a g i n a t i o n s . The noncoated i n v a g i n a t i o n s , which e v e n t u a l l y become
v e s i c l e s , are b e l i e v e d t o move f l u i d from t h e i n t e r s t i t i u m
i n t o t h e c a v i t y of t h e c y s t ( v i a t r a n s c y t o s i s ) . The
v a s c u l a r t h e c a i n t e r n a a p p e a r s t o d e g e n e r a t e . What w e
f r e q u e n t l y o b s e r v e a r e l a y e r s of c e l l s r e s e m b l i n g smooth
muscle cells. Cyst s i z e v a r i e s from 0.45 mm t o 2.2 mm i n
d i a m e t e r . Cyst f o r m a t i o n presumably r e s u l t s from h i g h e r
l e v e l s o f c i r c u l a t i n g a n d r o g e n s , e s t r a d i o l , and p r o l a c t i n
Supported by
(Lee, W T . e t a l . , Anat. Rec. 223:66A).
Grants HD14574 and HD06645 from N.I.H.
ANDERSON,* James, 11, Marisela MORALES,+ and Eva
FIFKOVA, Department of Psychology, University of
Colorado, Boulder, Colorado. Microwave fixation of brain
tissue for immunoelectron microscopv.
It is generally accepted that vascular perfusion fixation with
a standard aldehyde mixture is the only adequate method for
preparing brain tissue for electron microscopy. However,
perfusion is not always possible, e.g., in the case of human
material or brain slices. Fixation by immersion is an
alternate possibility. However, in the brain it yields
preservation which is inferior to that obtained by perfusion.
Recently it has been reported that immersion into 6%
glutaraldehyde combined with microwave irradiation yields
very good results (Jensen and Harris, SOCNeurosci Abstr, 550,
1988). We have adapted this procedure for use in
immunocytochemistry. Blocks of brain tissue were
i m m e r s e d into a m i x t u r e of 4% paraformaldehyde and 1-1.5%
g l u t a r a l d e h y d e f o r 20 and 40 sec of uniform heating to 50' C.
The tissue was postfixed with uranyl a c e t a t e instead of Os04
and embedded at low temperature in Lowicryl K4M. The
actin filaments were well preserved. In order to use this
procedure for immunoelectron microscopy, we had to
establish that a n t i g e n i c i t y of the tissue did not suffer by the
microwave irradiation. Therefore, we used monoclonal
antiactin a n t i b o d i e s with a gold probe which were previously
tested on perfused material. In comparing the pattern of
actin labeling with the two fixation procedures, we could not
detect any differences in the distribution of the gold label.
Thus, it is possible to use a combined procedure of
immersion fixation, microwave irradiation, and low
temperature embedding for immunoelectron microscopy.
Supported by NIH Grant AG04804.
S
AI l C i 1 ~ S T W N * Ilrlle,
.
Samnel R. SCIINITZCR*. Emily C1 INF*. Uiaa
SOI.II\.IAN', I&IY \V.UEIB*, Marrella STEVENS', and Richaid 1;. LAHERIT,
Departments of Psychology and Life Sciences, Group on Regulationof Iminune
Function (GRIF), Indiana State University, and Terre Haute Center for Medical
Education, School of Medicine, Indiana University, Terre Haute, Indiana. Aii
examination of the [email protected] induced by the activitv stress yaradigp
The activity-stress paradigm is known to produce rapid weight loss, increased
activity and death in rats. The cause of death has been attributed to gastric ulcers,
induced by stress. Access to the activity wheel was considered essential to the
developmentof the ulcers. Others have observed atrophy of the lyniphoid
system, diminished immune function, and pulnionary heniorrhagc. 111 this stncly,
we divided our Long-Evans rats into 6 groups. Two groups were housed in their
normal cages and given food ad libitum (NC)or for only 1 hr/day (FR). The
remaining J groups were housed in smaller cages adjacent to activity wheels. Two
groups had food ad libitum,one with access to the wheelIAC) and one did not
IS(.'). 'I'he remaining two groups were given food for only 1 hr/day. One group
(lid not have access to the wheel (SCBFR)and the other did have access t o the
whecl (AS),the complete activity-stressparadigm. Animals were followed until
all l h c AS animals died, at which lime, any animals remaining alive were
sacrificed. During necropsy, the lungs, kidneys, stomach, lymph nodes, and
Ihyroid gland were removed, fixed. and examined by light microscopy. The
thyinis, spleen, liver, adrenals, gonads, ulerus, and anterior pituitary were
removed, weighed, fixed, inid examined by light microscopy. On those animals
sacrificed, trunk blood was collected in NaCitrate. Platelet counts were
performed and both prothronihin and activated thrumhoplastin tiiiies were
determined. All groups on restricted food (FR, SCBFR, AS) showed significant
weight loss. lliese groups also had atrophied livers. spleens, and thymi. AS
animals had the shortest survival times, followed by the SCBFR group. I'he
other groups had longer survival times similar in duration amongst themselves.
AS and SC&FR animals showed gastric ulceration secondary to hemorrhage in
the gastric mucusa. The lungs of these two groups of animals were filled with
blood, indicating substantial pulmonary heniurrhage. Spleens were hypoactive as
indicated by a total lack of germinal centers. Adrenals and kidneys w.:ere
heniorrhagic. Livers exhibited a loss of normal architecture with a pooling of
blood in llie portal vessels indicating a portal hypertension. Platelet counts in the
AS group were lower than the other groups and both prothronibin and activated
thromboplastin times were elevated. We conclude that activity-stresscauses a
defect i n hemostasis that is responsible for the gastric ulceration and most
prohably the canse of death due to the pulmonary heniorrhnce. Further, our data
iiidirale that while access to the activity wheel exacerbates the syndrome, the
coinhination of siiiall caging and food restriction cause the same pathology.
S
BAGNALL, Keith. Department of Anatomy and Cell Biology, University
of Alberta, Edmonlon, Alberta. Canada T6G 2H7. Jhe lemmral and
Datial distribulion of fibronain durina the m i a r a t i o n m
differentiation of sclerotome cells,
Fibroneclin has been shown to influence cell migration and
differentialion in many siluations, particularly in conneclion with
neural crest cells. However, while the neural crest cells are
migrating laterally from the neural tube. sclerolome cells are
migrating medially in Ihe same environment to surround the
nolochord and neural tube. This experiment was carried out to
delermine whelher or not fibronectin had any clear influence on the
sclerotome cells by determining the spatial and lemporal presence of
fibronectin during the migration of sclerotome cells and lheir
differentiation into the constituents of the vertebral column. Chick
embryos between 3 and 1 1 days old were exposed to anti-fibronectin
(goat anti-human with a second antibody of rabbit anti-goat IgG
conjugaled to FITC) and the relationship between the presence of
fibroneclin and the developing structures associated with the
sclerolome cells was observed. The sclerolome cells begin to exhibit
the presence of fibronectin only after Ihe dispersal of Ihe somitic
cells. Before Ihis, the fibroneclin is reslricled lo Ihe epilhelium
ABSTRACTS-AAA 103RD MEETING
surrounding the somite where it is very prominent. As the sclerotome
cells migrate medially, there is an even distribution of fibronectin
throughout the cells and intercellular matrix which is maintained
throughout the migration. This distribution of fibronectin is
maintained even after the cells have completed their migration.
Regions which clearly show a diminution of fibronectin include the
developing dorsal root ganglion, emerging neurites, and the notochord
although there is a heavy concentration around the junction between
the sclerotome and the notochord. The neural tube is also a region of
reduced fibronectin presence. At this time the fibronectin is clearly
on the cell surface and in the intercellular matrix. This situation
remains until cartilage starts to appear in the vertebral anlage, when
there is a sudden but complete absence of fibronectin wherever
carlilage appears. While there is this reduction in these areas, the
presence of fibronectin in adjacent junctional zones with other tissues
becomes more pronounced, especially near the neural tube.
Supported by a grant from the Natural Sciences and Engineering
Research Council of Canada.
BAILEY. Cleta Sue, RalpQ L. KITCHELL, Richard D. JOHNSON',
and Michael J. GUINAN, Departments of Surgery and Anatomy,
School of Veterinary Medicine, University of California, Davis,
California and Department of Physiological Sciences, College of
Veterinarv Medicine. Universitv of Florida. Gainesville. Florida.
ComDaratbe studies of the cutankous areas and the soinal root origins
gf cutaneous nerves of the hindlimb in cats and dons. Cutaneous areas
(CA) of the hindlimb were determined in deeply anesthetized dogs and
cats by exposing the cutaneous nerves, electrophysiologically recording
the neural responses elicited by brushing the hairs and listening to the
response using an audiomonitor. The spinal root origin of each
cutaneous nerve was determined by stimulating the dorsal roots and
recarding the evoked potentials from the cutaneous nerve. The amount
of CA overlap was greater in the cat than in the dog. The CA for the
lateral cutaneous femoral nerve did not reach the dorsal midline in the
cat, but did in the dog. The CA for the lateral and caudal cutaneous
femoral nerves reached further distally in the cat than in the dog. The
CA for the superficial peroneal nerve was located on the dorsal surface
of the paw, except between digits 2 and 3, and the distal aspects of the
cranial and lateral surfaces of the crus. Its CA was relatively smaller
in the cat than in the dog. The CA for the deep peroneal nerve
supplied the skin of the dorsal hindpaw between digits 2 and 3 in both
spkcies. The CA for the saphenousnerve was on the medial aspect of
the limb as far distally as the footpad of digit 2. It was completely
overlapped distal to the hock by the CA of the superficial peroneal
nerve dorsally and the plantar nerves on the plantar aspect of the
hindpaw. Two caudal cutaneous sural nerves were present in most
dogs but only 20 % of the cats. The spinal root origins for the various
nerves were as follows: Bold, most common origin; (), other origins
observed in a few animals.
Doe
Cat
-Nerve
-. _.
Lat. cut. femoral
Caud. cut. femoral
Superficial perinea1
Dorsal nerve, Penis
Saphenous
Lateral cut. sural
Caudal cut. surals
Superficial peroneal
Deep peroneal
Plantars
L3-L4
Sl-SZ
,943
S1-SZ
L5-L6
L6-S1
L7-S1
L6-L7
L6-L7
L6-SZ
(L3-LS;L4-L5)
(Sl)
(SI-S2;S2-S3)
(S2;S2-S3;SI)
(L4-L6)
(L5-L7;L6-L7)
(L6-Sl)
(L6-SI)
(L6-S1)
(L6-L7;L5-S1)
L4-LS(L5-L6;L3-L4)
Sl-S3 (SI-S2;S2-S3)
SZ-S3 (SZ;Sl-S3)
S1-SZ (SI-S3;L7-S2)
L5-L6 (L4-L6)
L6-L7 (L6-SI)
L 7 4 1 (SI;L6-L7)
L6-L7 (L6-S1)
L6-L7 (L6-LRL7-SI)
L6-SZ (L6-SIiL7-Sl)
BAKER, J a m e s H a r l e y , D a r r y l SING+, and J a m e s
C. MACKENZIE, D e p a r t m e n t o f A n a t o m y , H o w a r d
U n i v e r s i t y , W a s h i n g t o n , D.C..
Immunocytochemical l o c a l i z a t i o n of c a l c i u m
activated neutral protease in deaeneratinq
musc 1 e
T h e p u r p o s e o f t h i s study w a s t o l o c a l i z e
calcium activated neutral protease in
d e g e n e r a t i n g m u s c l e . R a t s w e r e k i l l e d 3, 5, 7,
14, 21, and 2 8 days a f t e r t e n o t o m y o f t h e
9A
s o l e u s and t h e muscles were removed and p l a c e d
I n f i x a n d p r e p a r e d f o r immunocytochemi c a l
s t a i n i n g . CANP-I & CANP-2 s t a i n i n g w a s f a i n t
and l o c a l i r e d n e a r t h e s a r c o l e m m a i n n o r m a l
soleus muscle fibers.
I n the experimental
a n i m a l s t h e r e was a S1ight i n c r e a s e i n
s t a i n i n g f o r CANP-1 i n t h e s o l e u s m u s c l e
f i b e r s 3 and 5 d a y s a f t e r t h e o p e r a t i o n .
Unl i k e t h e normal a n i m a l s t h e s t a i n i n g was
not l i m i t e d t o t h e a r e a s j u s t b e n e a t h t h e c e l l
m e m b r a n e b u t c o u l d b e s e e n throughout t h e
m u s c l e f i b e r . T h e r e w a s no d i s c e r n a b l e
i n c r e a s e i n s t a i n i n g beyond that seen i n
normal a n i m a l s a f t e r f i v e days. T h e r e was v e r y
i n t e n s e s t a i n i n g f o r CANP-2 i n t h e s o l e u s
f i b e r s t h r e e d a y s a f t e r tenotomy. In p o r t i o n s
of t h e muscle f i b e r s n e a r t h e muscle-tendon
j u n c t i o n t h e s t a i n i n g w a s p r o f u s e and a c r o s s
t h e e n t i r e w i d t h of t h e f i b e r s . Those a r e a s of
t h e f i b e r s near the tendon were undergoing
segmental n e c r o s i s . However, i n t h o s e p o r t i o n s
of t h e f i b e r s w h i c h were not u n d e r g o i n g
n e c r o s i s , s t a i n i n g was i n t e n s e but l i m i t e d t o
a regular banding p a t t e r n which crossed the
m u s c l e f i b e r s . T h e d i s c r e t e banding p a t t e r n ,
b a s e d on p r e v i o u s s t u d i e s , p r o b a b l y s u r r o u n d s
t h e a r e a o f t h e 2 - d i s c s and i s p r o b a b l y t h e
i n i t i a l a r e a of m y o f i b r i l l a r d e g e n e r a t i o n .
T h i s s t u d y w a s s u p p o r t e d by M I H G r a n t No.
S06RR08016.
S
BAKKUM,*Barclay W., Louis A. BENEVENTO and Rochelle S .
COHEN,* Natl. Coll. of Chiropractic, Lombard, Illinois;
Dept. of Anatomy, Univ. of Maryland Baltimore Coll. of
Dental Surgery, Baltimore, Maryland; and Dept. of Anatomy
and Cell Biology, Univ. of Illinois at Chicago, Chicago,
Illinois. The effects of lieht/dark-and dark-rearine on
svnaDtic morvholoev in the suverior colliculus and visual
cortex of the voune adult rat.
Studies of animals visually deprived into adulthoodhave
shown
Vnaptic morphology in the
(Gabbott and Stewart1987, U. Brain Res. &3:103-114) Our
previous studies (Bakkum et al., 1988, Soc. Neurosci. &.
14:1111) demonstrated that dark-rearing does not affect
synaptic development in the rat superior colliculus (SC)
and visual cortex (VC) during the postnatal period (up to
1 month). In order to further assess the effect of visual
deprivation on adult synaptic morphology we examined the
ultrastructure in the SC and VC of rats either light/darkor dark-reared to the ages of 28 or 56 days. The SC, VC
and, as a control, the auditory cortex (AC) of SpragueDawley rats, raised either in 14 hrs light-10 hrs dark
(Lt/Dk) or in total darkness (DK), were fixed at 28 and 56
days postnatal and processed for EM analysis. At 28 days
there were no differences in the parameters measured
between Lt/Dk and Dk rats, but at 56 days there was a
significant decrease in the number of synapses in the SC
and VC, but not AC, of Dk versus Lt/Dk rats. There was an
increase in the percentages of positively curved synapses
in all brain areas and rearing conditions at 56 as compared
to 28 days. The percentages of perforated synapses in the
VC of Lt/Dk rats was higher at 56 versus 28 days. In the
SC of Dk rats, there was a decrease in the percentages of
spinous synapses at 56 as compared to 28 days. The SC had
longer contacts and higher percentages of symmetric and
dendritic synapses versus the VC and AC in both rearing
conditions at both ages. These data demonstrate plastic
changes in the rat SC and VC aftes their appearance becomes
mature at 28 days and indicate that it is during this
period that visual deprivation affects synaptic numbers.
Supported by NIH grant NS15889.
10A
ABSTRACTSAAA 103RD MEETING
BALDWIN*. Scott. Hiroaki SUZUKI*. Michael SOLURSH,
Departments of Pediatrics and Biology, University of Iowa,
Iowa City, Iowa. In dtu hvbridization localizes
nectin RNA to the precardkc mesoderm in the rat.
--
Immunohistochemicallocalization of fibronectin to the
mesodermal-endodermalinterface coincident with the
distribution of the presumptive heart cells in the chicken
has led to the postulation that endodermally derived fibronectin rovides a haptotactic "que" for precardiac cell
migraffon gom the primitive streak into the early heart
tube. Utilizing Ln sftu hybridization with an 353 labelled
rat fibronectin riboprobe (Xrlf-1). we investigated the distribution of fibronectin RNA production in cells from presomite (midgastrulation)through 8 somite (formation of
primitive heart tube) Wistar rat embryos. Serial cross
sections demonstrated that in the presomite embryo, the
fibronectin transcript was diffuse1 distributed throughe, incLding cells in the
out the prim
primitive s t r Z . The en oderm and ectoderm showed no
specific labelling above that attributed to background
si al and there was no detectable accentuation of
laglling in the area of the precardiac mesenchyme. However, in the 3-4 somite embryo there appeared to be a regional elevation of si al in the precardiac mesodermal
cells with no detectage increase in the subjacent endodem. Labelling in the 7-8 somite embryo was
rimarily restrlcted to the endocardium and anterior
Foregut with only background signal detected in the myocardium as has been previously described in the avian
embryo. Because the primary source of fibronectin
appears to be the precardiac cells themselves, we
conclude that fibronectin does not provide an "external
signal" for directed migration in the rat. Rather. we
r l a t e that fibronectin may play a role in establishing
e "epithelial"-likesheet that is characteristic of the
precardiac mesench e. (Supported by Grants No.
HM2197, HL43600~hD05505.DE05837 from the National Institutes of Health)
mesenchr
BANICK,* Paul D., Shu-min WEI,* Steven K. AKIYAMA.* WenTien CHEN* and Branislav VIDIC, Department of Anatomy and
Cell Biology, Georgetown University. Washington DC and (SKA)
Laboratory of Molecular Biology, NCI, NIH. Bethesda. MD.
Pulmonarv exoression of fibronectin (FN) and FN-receotor (FNR)
in fibrotic areas of human larae cell carcinoma.
Tissue blocks were obtained from excised pulmonary lobules at
surgery and prepared for electron microscopic stereology and
immunological investigations. A considerable increase in the
interstitial volume density of the parenchyma, almost double of
the normal value, was observed in tissues surrounding the
malignant foci. This development was due to the infiltration of the
interstitium
by inflammatory cells and to an augmented
deposition of extracellular substances, collagen and elastin. There
was, in addition, a three fold increase in the cross diameter of
basal laminae with the resulting thicknening of the air-blood
barrier. Presence of FN and FNR, consisting of integrin a 3 or a5
and 01 submits, was analyzed by J143 mouse mAb and rat mAbs
16 and 13 respectively on 0.5 vm thin frozen sections. Malignant
large cells showed an insignificant level of FN and FNR labeling.
Only attenuated labeling was observed in those tissue segments not
undergoing fibrosis and distant from the tumor. Closer to the
malignant focus, however, a301 and a501 FNR were present on
basal laminae underlying pneumocytes and on fibroblasts and
other inflammatory cells of the fibrotic area. Semisuantitative
immunofluorescence revealed a 3.6 and 4.5 higher FNDl labeling
of basal laminae and fibroblasts of fibrotic tissue respectively than
in the normal parenchyma. It is concluded that peripheral fibrosis
around the malignant nucleus may be initiated by an induction of
FN and FNR in parenchyma1 and infiltrating inflammatory cells
by the tumor.
B A N 3 0 , A d e s e g u n 0 . D e p a r t m e n t o f A n a t o m y , O b a f e m i Awolowo U n i v e r s i t y , I l e - I f e . N i g e r i a . Venous p a t t e r n
s of t h e dorsum of t h e hand i n Africans.
The l a r g e a n d m o d e r a t e v e i n s o f t h e body a r e
c o n s t a n t s t r u c t u r e s and are g e n e t i c . T h e Basilic a
and C e p h a l i c are s u c h v e i r s on t h e m e d i a l and l a t e r a l s i d e s r e s p e c t i v e l y o f t h e u p p e r 1imb.They ori g i n a t e from t h e d o r s a l venous p l e x u s of t h e hand.
1 2 p a t t e r n s ( P 1 a t e I A t o 1 L ) o f v e n o u s p l e x u s were
observed i n Africans i n Nigeria.Each person has
e i t h e r o n e p a t t e r n i n b o t h h a n d s or d i f f e r e n t p a t t e r n s i n e a c h hand.The i n c i d e n c e o f t h e s e p a t t e r n s
a n d t h e i r i n h e r i t a n c e were s t u d i e d . T h e h a n d s o f
1 2 6 0 p e r s o n s were e x a m i n e d ( i e 2 5 2 0 h a n d s ) 6 6 0 f e m a l e s a n d 6 0 0 m a l e s . A l l were b l a c k s . T h e n u m b e r i n c luded 20 i d e n t i c a l twins and 20 families.Each fani l y h a s a minimum o f 5 m e m b e r s . 3 4 . 9 % p e r s o n s h a v e
same p a t t e r n s i n b o t h h a n d s . P a t t e r n I A t h e commone s t i s 3 2 . 5 % o f h a n d s . F o l l o w e d by IK 1 3 . 5 % , I € 11. 9 % , I G 9 . 5 8 , I i 9 . 5 % , I 3 8.7%, I F 6 . 4 % , I B 2 . 4 2 ,
I D 2.4%, I C 1 . 6 % , I H 0.8%, I L O . B % . C e r t a i n p a t t e r n s o c c u r m o r e i n women, I A 1 9 . 8 % women, 1 2 . 1 % men.
1 K 1 0 . 3 % women, 3 . 2 % m e n . I i 5 . 5 % women, 4 . 0 % men.
O t h e r s o c c u r more i n m e n , I E 8.7% m e n , 3 . 2 % women.
I F 4.0% men, 2 . 4 % women. I G 5 . 5 % men, 4 . 0 % women.
I D a n d I H r a r e i n women.IC a n d I L r a r e i n men.None
o f t h e t w i n s had i d e n t i c a l p a t t e r n s . T h e p a t t e r n s
i n families did not indicate
i n h e r i t a n c e . I t is s u g g e s t e d
t h a t w h i l e some p e r i p h e r a l vei n s are g e n e t i c . 0 t h e r s s c h a s
t h e d o r s a l venous p l e x u s are
not.Their appearances and p a t t e r n s a r e probably due t o l o c a l
conditions eg local pressures
i n - u t e r o . It is a l s o s u g g e s t e d
t h a t t h e r e m i g h t b e r a c i a l dor.
sal venous p a t t e r n s o f the
h a n d . I t may a l s o b e d u e t o
local conditions in-utero.
b -basilic vein
Grant-3ames Henry Memorial
c -cephalic vein
h o s p i t a l , Ilesa. Nigeria.
BAPTISTA, C a r l o s A.C. and L i b e r a t o J . A . DIDIO,
D e p a r t m e n t o f A n a t o m y , Medical C o l l e g e o f O h i o ,
T o l e d o , O h i o , J o s C C a r l o s PRATES*, D e p a r t m e n t o f
M o r p h o l o g y , E s c o l a P a u l i s t a d e Medicina, SPo
P a u l o , B r a z i l and G o r d a n a TEOFILOVSKI-PARAPID*,
Anatomy,
School
of
Medicine,
D e p a r t m e n t of
U n i v e r s i t y of B e l g r a d e , Yugoslavia. .The a r t e r i a
d i a u o n a l i s of t h e human heart.
The
surgical,
clinical
and
radiological
i m p o r t a n c e of t h e diagonal a r t e r y i n t h e h u m a n
h e a r t and t h e e x t r e m e v a r i a b i l i t y i n i t s
d e s c r i p t i o n prompted u s t o u n d e r t a k e i t s s t u d y .
T h e i n v e s t i g a t i o n w a s performed i n 150 h e a r t s ,
after
injection
of
colored
substance
and
radiopaque m e d i u m i n t h e c o r o n a r y a r t e r i e s v i a
aorta.
Then
t h e h e a r t s were
radiographed,
d i s s e c t e d , drawn and photographed. T h e f o l l o w i n g
r e s u l t s were o b t a i n e d : T h e l e f t c o r o n a r y a r t e r y
division:
bifurcation
p r e s e n t e d 3 t y p e s of
( 5 4 . 7 % ) , t r i f u r c a t i o n ( 3 8 . 6 % ) and q u a d r i f u r c a t i o n
(6.7%),
the latest t w o t y p e s presenting a
diagonal a r t e r y . T h e diagonal a r t e r y was f o u n d as
t h e o n l y one branch i n 3 4 . 7 % . A s a double branch
i t was found i n 6 . 7 % o f t h e cases. I t was
a s s o c i a t e d w i t h t h e f i r s t branch of t h e a n t e r i o r
i n t e r v e n t r i c u l a r b r a n c h i n 1.2%; a s s o c i a t e d w i t h
t h e f i r s t branch of t h e c i r c u m f l e x branch i n
1.2%; a s s o c i a t e d w i t h b o t h p r e c e d i n g b r a n c h e s
simultaneously
i n 1.3%. T h e length o f the
diagonal a r t e r y i n m a l e s , C a u c a s i a n s , v a r i e d f r o m
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
20.1 t o 30.0 mm (28.9%) a n d 30.1 t o 4 0 . 0 mm
(34.2%) w h i l e i n non-Caucasians i t v a r i e d f r o m
20.1 t o 30.0 mm (23.5%) and 30.1 t o 40.0 mm
(18.1%). I n f e m a l e s , C a u c a s i a n s ,
the length
v a r i e d f r o m 20.5 t o 30.0 mm (18.2%), 30.1 t o 40.0
mm (36.3%) and 40.1 t o 50.0 mm (36.3%) w h i l e i n
n o n - C a u c a s i a n s i t v a r i e d f r o m 10.1 t o 20.0 mm
(18.15), 20.1 t o 30.0 mm (36.3%) and 30.1 t o 4 0 . 0
mm (18.1%).
BARDO,* M i c h a e l T . a n d R o n a l d P . HAMMER J R , D e p a r t m e n t o f P s y c h o l o g y , U n i v e r s i t y of K e n t u c k y ,
L e s i n g t o n , K e n t u c k y ; D e p a r t m e n t of Anatomy a n d
R e p r o d B i o l , U n i v e r s i t y o f H a w a i i S c h o o l of
Medicine, Honolulu, H a w a i i .
Autoradioaraphic
l o c a l i z a t i o n of D 1 a n d D2 d o p a m i n e rec e v t o r s i n t h e m e s o l i m b i c system of r a t s .
B r a i n s f r o m a d u l t male r a t s were s e c t i o n e d b y
a c r y o s t a t a t 2 0 um t h r o u g h t h e e n t i r e r o s t r a l c a u d a l e x t e n t of t h e n u c l e u s a c c u m b e n s .
Adjac e n t s e c t i o n s were m o u n t e d o n m i c r o s c o p e s l i d e s
a n d a u t o r a d i o g r a p h i c a n a l y s e s o f D 1 a n d D2 rec e p t o r d e n s i t i e s were p e r f o r m e d .
T i s s u e sect i o n s w e r e incubated i n T r i s buffer containing
e i t h e r 1 . 6 nM 3H-SCH23390 for t h e D 1 a s s a y o r
0 . 5 nM 3 H - s p i r o p e r i d o l for t h e D2 a s s a y .
Scatchard analyses revealed that t h e s e radioligand
c o n c e n t r a t i o n s r e p r e s e n t e d 1 . 5 X K, f o r e a c h
receptor subtype.
I n e a c h a s s a y , 1 0 0 nM k e t a n s e r i n was a d d e d t o i n h i b i t b i n d i n g t o 5-HT rec e p t o r s a n d n o n s p e c i f i c b i n d i n g w a s estimated by
d e t e r m i n i n g t h e a m o u n t of b i n d i n g t h a t w a s d i s p l a c e d b y 1 uM ( t ) b u t a c l a m o l . F o l l o w i n g i n c u b a t i o n , s e c t i o n s were w a s h e d , d r i e d , a n d e x p o s e d
to tritium-sensitive film.
Computer-assisted
quantitative regional densitometry revealed that
b o t h D 1 a n d D2 r e c e p t o r s d i s p l a y e d a r o s t r a l c a u d a l g r a d i e n t w i t h i n t h e n u c l e u s accumbens,
with higher densities present i n the r o s t r a l
p o r t i o n t h a n i n t h e c a u d a l p o r t i o n . The
r o s t r a l - c a u d a l c h a n g e w a s g r e a t e r i n D2 s i t e s
t h a n i n D 1 s i t e s . A l s o , D2 s i t e s , b u t n o t D 1
s i t e s , d i s p l a y e d a g r e a t e r d e n s i t y i n t h e core
o f t h e n u c l e u s accumbens r e l a t i v e t o the s h e l l
of t h e n u c l e u s a c c u m b e n s .
I n the same sections
used f o r t h e n u c l e u s accumbens d e t e r m i n a t i o n s ,
n e i t h e r t h e caudate-putamen n o r the o l f a c t o r y
t u b e r c l e s s h o w e d a n y c h a n g e i n D 1 o r D2 s i t e s .
T h e s e r e g i o n a l d i f f e r e n c e s may h a v e i m p o r t a n t
i m p l i c a t i o n s f o r mesolimbic r e w a r d s y s t e m s .
( S u p p o r t e d b y g r a n t s DA05312 a n d DA04081.)
BARRETT, C h a r l e s P a t r i c k , Lloyd GUTH and
Rosemary P e n e l o p e REES," De p a r tm e n t o f
Anatomy, U n i v e r s i t y o f M a r y l a n d , S c h o o l o f
Medicine, B a l t i m o r e , Maryland.
A c t i v a t i o n of
m a c r o R h a g e s s t i m u l a t e s a x o n a l r e g e n e r a t i o n in
the injured spinal
The m a c r o p h a g e s t h a t a c c u m u l a t e a t s i t e s of
t i s s u e i n j u r i e s - s e c r e t e s u p e r o x i d e s and o t h e r
cytotoxic agents.
I n s p i n a l cord trauma, they
promote a p r o g r e s s i v e n e c r o s i s r e s u l t i n g i n a n
environment h o s t i l e t o axonal ingrowth.
C o n v e r s e l y , o t h e r macrophage s e c r e t i o n s
p r o m o t e m i t o g e n e s i s , a n g i o g e n e s i s and
n e u r i t o g e n e s i s , which a r e f a v o r a b l e t o t h e
Thus
r e p a i r of i n j u r e d n e u r a l t i s s u e s .
s e l e c t i v e r e g u l a t i o n of macrophage a c t i v i t y
might enhance axonal r e g e n e r a t i o n .
After
~~
11A
c r u s h i n g t h e s p i n a l c o r d o f a d u l t r a t s , we
t r e a t e d t h e m w i t h i n t r a v e n o u s E. c o l 1
a
l i p o p o l y s a c c h a r i d e ( L P S ) , 0 . 4 mg/kg b . i . d . ,
d o s e known t o p r i m e m o n o c y t e s a n d m a c r o p h a g e s
f o r s e c r e t i o n of growth f a c t o r s .
After 2 , 4 ,
o r 8 weeks, t h e s p i n a l c o r d s were examined by
LM h i s t o l o g y a n d i m m u n o c y t o c h e m i s t r y .
In
comparison with s a l i n e - t r e a t e d c o n t r o l s , t h e
l e s i o n s i t e of t h e LPS-treated r a t s e x h i b i t e d
( a ) reduced t i s s u e n e c r o s i s ; ( b ) enhanced
i n g r o w t h o f g l i a l and e p e n d y m a l c e l l s w h i c h
formed c e l l u l a r b r i d g e s a c r o s s t h e l e s i o n ; ( c )
c o n s i d e r a b l e ingrowth of r e g e n e r a t e d axons;
and ( d ) r e d u c e d d e n s i t y o f t h e g l i a l s c a r a t
t h e severed ends of t h e s p i n a l cord.
These
o b s e r v a t i o n s c o r r o b o r a t e t h e e a r l y f i n d i n g s of
W i n d l e who u s e d P i r o m e n , a b a c t e r i a l LPS, t o
s t i m u l a t e axonal regeneration i n t h e c a t
spinal cord.
We s u g g e s t t h a t t h e f a v o r a b l e
e f f e c t o f LPS r e s u l t s f r o m a l t e r e d m a c r o p h a g e
activity.
S u p p o r t e d by N I H g r a n t 1 9 - 2 1 4 6 0 .
S
BEASON,* Lori L.. Mark B . MOSS, and Douglas L. ROSENE,
Department of Anatomy, Boston University School of
Medicine, Boston, Massachusetts. A c e tvl c h o I i n e s t e r a s e
-letion
in the a m d l a of rhesus monkevs f
m
k s h s of thc basal f o r c b r i h
The cholinergic basal forcbrain (BF) of the monkey is
known to project to widespread areas of the cerebral cortex,
amygdala, and hippocampus.
As part of our studies on the
cffects of BF damage on memory function in the monkey,
acetylcholinesterasc (AChE) lcvcls of the amygdala wcre
mcasured in a series of rhesus monkcjs that reccived lesions
of the BF using the neurotoxin, ibotenic acid. The data were
then compared with AChE levels of corrcsponding sites in
normal control monkcys.
The site and sizc of the BF lesions
werc analyzed and AChE density i n the amygdala was
measured in all animals by computcr assistcd image analysis.
Four levels of the amygdala wcrc chosen for analysis and
measurements were madc in 4 nuclei: thc Accessory Basal
(AB); Lateral Basal (LB); Medial Basal (MB); and Lateral (Lt).
The optical densities (OD) of the nuclei werc calculated as a
proportion of the OD of the AChE dense putamen. which was
used as an intcrnal control since it should not be affected by a
BF lesion.
Because there wcre no obvious differences
according to antcrior/posterior (AP) lcvels in the controls.
the values of cach nuclcus wrre collapsed over AP levels. In
the AB of thc BF monkeys, thc mcan proportional OD was 0.37
as compared with 0.53 for thc normal controls. In tllc LB and
MB, the mean proportional OD for the BF was 0.45 and 0.32
rcspcctivcly whcrcas in tlic normal control monkeys these
valucs were 0.90 and 0.78. In the Lt. BF monkcys had a mcan
proportional OD of 0.31 as coinpared with 0.72 in tlic control
group.
Thus for each anipgdala nucleus measured, the AChE
value was significantly lower in monkcys with BF lesions
than in normal controls.
Together with earlier findings from
our laboratory showing loss of AChE in the hippocampus
followiiig BF damagc in the monkey, it is plausiblc that the
anterograde mcmory impairmen1 in monkcys with BF lcsions
may be due, at lcast in part, to thc disruption of the
cholincrgic
systcm
in
aniygdrla
and
hippocampus.
(Supported by NIH Grants AGO4321 and NS 19416).
&.
S
BEASTON-WIMMER,* Patricia R and Amold J. SMOLEN, Department
Philadelphia,
of Anatomy, The Medical College of. Pennsylvania,
.
in the
Pennsylvania.
12A
ABSTRACTS-AAA 103RD MEETING
At birth, superior cervical ganglia (SCG) of male and female rata contain
equal numbers of neurons. No sex difference exists in the' content of
norepinephrine (NE) in these neurons or in the enzyme activity of tyrosine
hydroxylase (TH). A sex difference does exist in the expression of THmRNA, measured by quantitative in situ hybridization, with SCG neurons
in females expressing more TH-mRNA then males. At this time, there is
no sex difference in the mass of the submandibular gland (SMG), a target
of the SCG.
During the first two postnatal weeks, natural neuron death in the SCG
results in the loss of significantly fewer neurons in males. Neurons in
females continue to express more TH-mRNA and both TH enzyme activity
and NE content per neuron are l u w r in males. Since the adult sex
difference in body weight and sympathehr. target, SMG or pineal, mass
has not yet been established, the same target mass is innervated by more
neurons in males.
In the adult, the sex difference in neuron number is maintained. By this
time, both overall body weight and target, SMG and pineal, mass is
significantly greater in males. Expression of TH-mRNA is now greater in
SCG neurons of males. Both TH enzyme activity and NE content per
neuron are now equal in males and females.
The developing nervous system must match a population of neurons with
its targets. These results demonstrate that there are changes in both cell
number and neurotransmitter TH-mRNA in the developing SCG of both
males and females before there is any sex difference in the amount of
target to be innervated. We conclude that while the development of both
TH enzyme activity and NE content in sympathetic neurons appears to
depend on target availability, naonaLal changes in cell number and
expression of TH-mRNA appear to anticipate adult target demand.
Effects of aging on both SCG neuron number and catecholamine
metabolism are under investigation. Preliminary results indicate no
difference in TH-mRVA content per neuron between 6 month and 24
month old male Fischer rats.
BELLONI,* Anna Sandra, Francesco MUSAJO*.
Paola ANDREIS',
Giuseppina MAZZOCCHIf and Gastone G. MISSDOFIFER. Department
of Anatomy, University of Padua. Padua, I t a l y .
Morphology
and function of a d r e n o c o r t i c a l t i s s u e regenerated from gland
c a p s u l a r fragments a u t o t r a n s p l a n t e d i n the rat musculus gracilis.
Regenerated a d r e n o c o r t i c a l nodules w e r e obtained by impL
a n t i n g , i n t h e musculus gracilis o f rats, s e v e r a l (6-7) frag
ments of t h e c a p s u l a r t i s s u e of their excised a d r e n a l s . Four
months a f t e r t h e o p e r a t i o n , each b i l a t e r a l l y adrenalectomi
zed rat developed 6-7 well-encapsulated a d r e n o c o r t i c a l nod!
l e s (of about 2-3 mm i n d i a m e t e r ) , and displayed an almost
complete normalization o f its b a s a l and s t i m u l a t e d blood
l e v e l s of c o r t i c o s t e r o n e . b u t n o t of aldosterone. I n v i t r o
study showed t h a t regenerated nodules were w e l l functioning
as far as g l u c o c o r t i c o i d production w a s concerned. Accordin
gly. e l e c t r o n microscopy and s t e r e o l o g y i n d i c a t e d that the
majority of the parenchymal c e l l s (independently o f t h e i r
l o c a t i o n i n the o u t e r subcapsular, middle o r i n n e r p o r t i o n s )
c l o s e l y resembled those of the zonae f a s c i c u l a t a / r e t i c u l a i s
of the a d r e n a l glands of age-matched sham-operated rats. By
c o n t r a s t , regenerated nodules evidenced a r e l a t i v e impaifl
ment i n a l d o s t e r o n e s e c r e t i o n , and this w a s coupled w i t h
the presence of only f e v zona glomerulosa-like c e l l s .
Such
c e l l s were grouped i n s m a l l islets, l o c a t e d n e a r t h e f e w
connective t r a b e c u l a e detaching from the capsule, and a u t o
radiography showed that they w e r e t h e only parenchyma1 eleq!
e n t s of t h e nodule a b l e t o bind (125II-angiotensin 11. The
p o s s i b i l i t y i s suggested t h a t the p a u c i t y of zona g l o m e e
losa-like c e l l s i n regenerated nodules could be a s c r i b e d t o
the absence of zona m e d u l l a r i s , which is c u r r e n t l y thought
t o e x e r t a p a r a c r i n e c o n t r o l on t h e growth and s e c r e t i o n of
zona glomerulosa i n t h e r a t adrenal glands.
BERINGER, Ted and Nallu S. REDDY*, Schools of Basic L i f e
Sciences and Medicine, U n i v e r s i t v of Missouri, Kansas Citv,
Missouri. Phospholipid and f a t t ; a c i d a n a l y s i s of base lateral membranes from t h e r a t r e n a l cortex.
AS a p r e r e q u i s i t e to r e c o n s t i t u t i n g t h e cerium i o n
t r a n s p o r t channel (Beringer, J. C e l l Biology. 107:799a,
'88) from b a s o l a t e r a l mendranes (BLM)of t h e r a t r e n a l
c o r t e x i n t o proteoliposomes, it w a s necessary t o analyse
t h e BLM f o r phospholipids and f a t t y a c i d s s i n c e t h i s
information is n o t known. BLM were prepared according t o
S c a l e r a et al. (MembK. Biochem. 4:49-61, '81) and
solubilized i n chloroforwmthanol f o r analysis.
Phospholipids are expressed as percentage of total
phospholipid content: lysophosphatidylcholine, 0.42%;
sphingomyelin, 21%; phosphatidylcholine (PC) , 32.7%;
phosphatidylserine ( P S ) , 9.22%: p h o s p h a t i d y l i n o s i t o l
( P I ) , 3.71%; p h o s p h a t i d y l e t h a n o l a i n e (PE), 24.74%;
lysophosphatidylethanolmine, 3.35%; phosphatidylg l y c e r o l , 4.75%. F a t t y a c i d a n a l y s e s expressed as a
f r a c t i o n of total f a t t y a c i d s i n t h e BLM were 14:O (0.7%),
16:O (26.6%), 1 6 : l (1.24%), 18:O (21.45%), 18:l (10.73%),
18:2n6 (14.7%), 18:3n3 (0.24%)) 18:4n6 (0.1%)) 20:4n6
(18.53%), 22:6n3 (2.12%). Analysis of major f a t t y a c i d s
of i n d i v i d u a l phospholipids from t h e BLM were: PC
(16:0, 37.2%; 18:0, 16.4%, 18:l. 11.33%: 18:2n6, 16.73%:
20:4n6, 10.95%); PE (16:0, 18.2%: 18:0, 23.75%: 18:lt
11.88%; 18:2n6, 8.6%; 20:4n6, 29.66%); PS + P I (16:0,
10%; 18:0, 44.33%: 18:1, 6.75%; 18:2n6, 5.7%; 20:4n6,
28.18%). The results of t h i s a n a l y s i s w i l l be used t o
formulate l i p i d and f a t t y a c i d composition of
proteoliposomes r e c o n s t i t u t e d with i n d i v i d u a l BLM
p r o t e i n s i s o l a t e d by p r e p a r a t i v e i s o e l e c t r i c focusing.
The proteoliposomes prepared i n t h i s way w i l l be submitted
t o f u n c t i o n a l and u l t r a s t r u c t u r a l a n a l y s i s of t h e cerium
t r a n s p o r t channel.
Y. YAMAMOTO* and R.R. SMITH*, Departments
of Anatomy and Neurosurgery, University of M i s s i s s i p p i
Medical Center, Jackson, M i s s i s s i p p i . T h e role of
f i b r o b l a s t s i n t h e mechanism of chronic vasosuasm.
Although s e v e r a l spasmogenic substances have been
implicated as t h e cause of chronic c e r e b r a l vasospasm
a f t e r subarachnoid hemorrhage (SAH), there has been no
conclusive i d e n t i f i c a t i o n of t h e a g e n t ( s ) responsible.
S t u d i e s have stressed t h e f u n c t i o n of smooth m u s c l e c e l l s
a s t h e c o n t r a c t i l e elements i n vessels, w h i l e ignoring
t h e r o l e of o t h e r force-generating components i n t h e
v e s s e l w a l l . Collagen f i b e r s and f i b r o b l a s t s have been
shown t o p r o l i f e r a t e a f t e r SAH. F i b r o b l a s t s a r e a b l e t o
compact t h e surrounding collagen m a t r i x w i t h a
s i g n i f i c a n t r e d u c t i o n i n volume. In our study, we
employed t h e c o n t r a c t i n g fibroblast-populated collagen
l a t t i c e (FPCL) c u l t u r e model t o t e s t CSF f r o m 10 p a t i e n t s
w i t h r e c e n t l y r u p t u r e d i n t e r c r a n i a l aneurysms, d i v i d i n g
t h e CSP i n t o 3 groups according t o degree of vasospasm.
CSF from p a t i e n t s w i t h n e u r o l o g i c a l symptomatic spasm
a c c e l e r a t e d FPCL c o n t r a c t i o n s i g n i f i c a n t l y compared t o
t h e o t h e r groups (no vasospasm; vasospasm w i t h o u t
n e u r o l o g i c a l symptoms). T h e e f f e c t i s heat r e s i s t a n t ,
smaller t h a n approximately 6000-8000 molecular w e i g h t ,
and r e s i s t a n t t o chemical reduction and i n h i b i t o r s of
smooth muscle c o n t r a c t i o n . The d a t a suggests t h a t
v a s c u l a r f i b r o b l a s t s can c o n t r i b u t e t o t h e chronic s t a g e
of s u s t a i n e d vasospasm w i t h t h e a i d of p r o l i f e r a t e d
i n t e r c e l l u l a r c o l l a g e n , and t h a t a g e n t ( s ) i n CSF a f t e r
SAH a c c e l e r a t e ( m ) t h e e f f e c t of f i b r o b l a s t s on collagen
f i b e r s i n a mechanism s e p a r a t e f r o m t h e a c t i o n of smooth
BERNANKE, D.H.,
ABSTRACTELAAA 103RD MEETING
muscle cells. The s u g g e s t e d mechanism can e x p l a i n t h e
d e l a y of o n s e t o f c h r o n i c vasospasm a f t e r SAH, t h e
s u s t a i n e d c h r o n i c s t a g e of vasospasm observed i n humans,
and t h e release of vasospasm weeks t o months l a t e r , a l l
by t h e involvement of f i b r o b l a s t s and c o l l a g e n m a t r i x i n
t h e vessel wall.
Bernstein,' Aaron, Elizabeth Preisig,' and Hubert E.
Schroeder'. Department of Oral Structural Biology, Dental
Institute, University of Zurich, Zurich, Switzerland.
vitrQ
m
n of a c
p
o
n D r e v i v
and e
x
D
e
p
.
Mechanical treatment of diseased root cementurn is known,
clinically, to favor periodontal regeneration. The present
study was performed to test whether previously diseased and
experimentally treated root surfaces can support the
formation of a new collagenous matrix in vitro Three teeth,
extracted due to advanced periodontitis, were first agitated in
5% sodium hypochlorite for 2h to render the root surface
sterile and anorganic. Following the removal of its healthy,
apical one third, the root was scaled with moderate pressure
to remove visible calculus. Non-demineralized root disks
were cul transverse to the root axis and placed on a coculture of periodontal ligament and alveolar bone-derived
cells. After seven weeks i n culture, either one of two matrix
types was found along the root surface. The most frequent
matrix consisted of clusters of cells layered within densely
aggregated collagen fibrils. The other, less frequent matrix,
consisted of loosely arranged collagen fibrils adjacent to the
cemental surface. The findings support the hypothesis that,
in-vitro, a collagenous matrix is formed in contact with
diseased and experimentally treated root surfaces. However,
the smooth, non-demineralized and scaled cemental surface
does not appear to be a suitable substrate for the
interdigitation of newly produced collagen fibrils with the
root disk cemental collagen fibrils.
.
(Supported by the Swiss National Science Foundation, Fogarty
International Fellowship Nr. 88.423.1.86)
BERNSTEIN-GORAL,* Holli, Barbara S. BREGMAN, J. Scott
McDONALD,'
and
U Blair CLARK,*
Dept. Anatomy,
Georgetown Univ., Washington
D.C.; Dept. Anatomy, Univ. of
Maryland, Baltimore, Maryland. Schwann cells and astrocvtes
oromote different Datterns of
neurite outgrowth from locus
coeruleus neurons in culture.
Schwann cells support survival and neurite outgrowth from
locus coeruleus (LC) neurons i n culture (Baharloo a n d Clark,
Neurosci. Abstr. 347.4, 1989). This study compared patterns of
neurite outgrowth from LC neurons prepared from 6d postnatal
rat brainstem, cultured with 1) Schwann cells (SC), 2) Schwann
cells with basal lamina (SC+BL), 3) astrocytes (AC) and 4) on
collagen + laminin (C+L). After 3d in culture, LC neurons were
identified by immunostaining f o r neurofilament protein and
morphometric measurements of
individual
neurofilamentimmunoreactive processewere made using the Zeiss-IBAS image
analyzer. Soma diameter, the diameter of the neuritic field, and
the total neurite length were measured and the number of neurite
branch points per neuron counted. Data are presented as the mean
2S.E.M. After 3d i n culture, there were no significant differences
in soma diameter, although there were considerable differences i n
the patterns of neurite outgrowth. The diameter of the neuritic
field for LC neurons was greater when grown with Schwann cells
or astrocytes, compared with collagen+laminin (C+L 290.50um
k32.49; SC+BL: 742.47um 2215.25; A C 565.87 2143.48; SC: 806.89
+100.04). The total neurite length of LC neurons with either
astrocytes or Schwann cells was 3 fold greater than on
collagen+laminin ( C + L 689.94um 2179.97; A C 2106.26um 2398.66;
SC+BL 1308.26um 2330.09; SC: 2174.24um 2343.60). A highly
13A
branched pattern of secondary neurites was observed for LC
neurites cultured with astrocytes (branch points: C+L: 6.75 21.69;
AC: 12.13 k2.72; SC+BL 4.00 9 . 8 2 ; S C 10.20 k1.31). These data
indicate that although both Schwann cells and astrocytes promote
neurite outgrowth, the pattern of outgrowth differs. Neurite
length is similar in both Schwann cell and astrocyte environments,
but astrocytes promote a more branched, locally arborized pattern
of neurite outgrowth and Schwann cells promote a less branched,
more distally directed outgrowth pattern. Supported by NIH grants
NS19259, NS01356 (BSB), NS24252 (MBC) a n d PVA grant NBR645
(MBC).
BIJLANI, V., A.A. KHAN* a n d S. WADHWA*, Department
of Anatomy, All-India
I n s t i t u t e of Medical Sciences,
New Delhi.
Synaptogenesis i n p r e n a t a l human lateral
g e n i c u l a t e nucleus.
Age related changes i n the volume, s y n a p t i c d e n s i t y
a n d total number of s y n a p s e s i n the p r e n a t a l human
lateral geniculate nucleus have been d e t e r m i n e d from
p a r a f f i n s e c t i o n s a n d i n m a t e r i a l prepared f o r e l e c t r o n
microscopy.
T h e human f e t a l c a d a v e r s of d i f f e r e n t
g e s t a t i o n a l a g e s ranging from 13-14 to 27-28 w e e k s
w e r e o b t a i n e d by h y s t e r o t o m y a n d a u t o p s y w i t h prior
consent a n d e t h i c a l committee c l e a r a n c e .
The results
have b e e n c o r r e l a t e d w i t h the q u a n t i t a t i v e p r o f i l e
of a x o n s i n the o p t i c n e r v e s of d i f f e r e n t gestational
a g e periods.
A t a b o u t 16-17 w e e k s of g e s t a t i o n when
there is maximum number of f i b r e s (7.2 million) i n
the o p t i c n e r v e (Wadhwa a n d B i j l a n i , 1987). t h e r e
is also a d r a m a t i c i n c r e a s e i n the volume of lateral
geniculate nucleus.
A 175% gain i n volume is noticed
between 16-17 a n d 17-18 w e e k s of g e s t a t i o n .
Synaptic
d e n s i t y a n d a b s o l u t e s y n a p t i c counts also r e f l e c t a
s p u r t d u r i n g this period and s y n a p t i c l e n g t h stabilization o c c u r s b y m i d gestation.
T h u s the period from
15 to 20 w e e k s of g e s t a t i o n f o r m s a c r i t i c a l phase
i n human lateral geniculate development.
Wadhwa. S. a n d B i j l a n i , V.
Developing human o p t i c
n e r v e i n p r e n a t a l p e r i o d : Changes i n the number of
r e t i n a 1 axons.
Ind. J. Ophthalmol. 3 5 ( 1 ) : 11-16,
1987.
BILLY,* A l l e n , Robin LILEY*, and C h r i s SHAW*, Departments
o f Zoology and Ophthalmology, Department o f Zoology,
Departments o f Ophthalmology and Physiology, U n i v e r s i t y o f
B r i t i s h Columbia, Vancouver, B r i t i s h Columbia. (Sponsored
by Charles E. Slonecker). The r e g i o n a l d i s t r i b u t i o n o f
n e u r o t r a n s m i t t e r and neuromodulator r e c e p t o r s i n t h e b r a i n s
of salmon (Oncorhynchus k i s u t c h ) and rainbow t r o u t (Salmo
gai rdneri )
While sex s t e r o i d hormone treatments e a r l y i n
development a f f e c t b r a i n d i f f e r e n t i a t i o n (e.g. McEwen e t a1
1983) and b e h a v i o r a l d i f f e r e n t i a t i o n (e.g. B i l l y and L i l e y ,
1985) i n a v a r i e t y o f v e r t e b r a t e s , t h e r e has been l i t t l e
a t t e n t i o n t o t h e r e l a t i o n s h i p between development o f n e u r a l
r e c e p t o r p o p u l a t i o n s and molecular mechanisms u n d e r l y i n g
t h e expression o f s e x u a l l y dimorphic behaviors. Our
research examines whether m a s c u l i n i z i n g o r f e m i n i z i n g
hormone treatments a l t e r development o f r e c e p t o r
p o p u l a t i o n s i n t h e b r a i n . One aspect of t h i s study i s t o
d e s c r i b e t h e complex chemical c i r c u i t r y of t h e u n t r e a t e d
salmonid b r a i n . E n t i r e b r a i n s o f j u v e n i l e salmonids have
been s e r i a l l y sectionned on a c r y o s t a t (twenty micron
t h i c k s e c t i o n s ) and i n v i t r o a u t o r a d i o g r a p h i c techniques
were used t o determine t h e normal d i s t r i b u t i o n o f v a r i o u s
r e c e p t o r populations. Receptor b i n d i n g s i t e s i n a t l e a s t
t w e n t y - f i v e b r a i n r e g i o n s and s t r u c t u r e s were assayed
u s i n g tri t i a t e d r a d i o 1 igands f o r t h e neurotransmi t t e r s
.
14A
ABSTRACTSAAA 103RD MEETING
-
Tamino butyric a c i d (GABAA subtype
muscimol) and
glutamate (NMDA subtype MK801) and f o r the
neuromodulators acetylcholine (muscarinic subtype - QNB),
dopamine (D subtype - SCH23390) and cholecystokinin (CCK).
S t r i k i n g diiferences i n receptor binding densities were
as i l l u s t r a t e d by the
found i n several brain areas
r e s u l t s obtained f o r tectum, corpus c e r e b e l l i , and i n f e r i o r
lobes. Receptor binding i n the tectum ranged from l i g h t
(MK801, CNQX) t o homogeneously moderate (CCK) t o moderate
i n only one l a y e r (CPDX, SCH23390, QNB) t o heavy i n only
one l a y e r o f the tectum (GABA ). Receptor binding i n the
corpus c e r e b e l l i displayed a g i f f e r e n t binding pattern
ranging from l i g h t (CPDX, SCH23390, QNB) t o moderate (CCK)
t o very heavy i r only the cortex (CNQX). Receptor binding
i n the i n f e r i o r obes ranged from l i g h t (CNQX, CPDX) t o
moderate (MK801, CCK) t o heavy (GABAA, QNB).
-
BLECHER. Stan R. Joachim KAPAIANGA* and Darwin
LALONDE;* School of Human Biology, University of
Guelph, Guelph, Ontario, Canada. Euidermal Frowth
factor (EGF) corrects develoDmenta1 defects. including
absence of sweat Flands. in a murine homoloeue of
human hwohidrotic ectodermal dvsplasia.
A considerable amount of genetic homology has been
demonstrated between murine (and other mammalian) and
human chromosomes. In the case of the X chromosomes,
Ohno's Principle, now well documented, predicts that
any locus in one mammal is likely to be represented by
a homologous locus on the X of other mammals. Studies
in our laboratory have established the murine X-linked
as a probable homologue of the
mutant gene Tabby (B)
gene for human X-linked hypohidrotic ectodermal
dysplasia (HED). We have also demonstrated a
relationship between B and EGF, a short chain
polypeptide which, amongst other effects, is known to
induce precocious eyelid opening and incisor eruption
in neonatal mice. We have presented evidence that
hemizygotes appear to produce reduced amounts of
EGF, and we have suggested that the traits of the
Tabby syndrome may be due to a deficiency of EGF at
molecular target sites. We recently showed that
hemizygotes have delayed eyelid opening and incisor
eruption, and that EGF reverses these delays. We
report here that the mutant hemizygotes, in addition
to lacking sweat glands (previously shown), also lack
dermatoglyphics, and postnatal EGF injection leads to
development of both dermal ridges and functional sweat
glands. These findings support the proposal that EGF
plays a morphogenetic role in mammals. It also
suggests a possible future therapeutic application
for EGF in human HED.
Supported by grants to SRB from the NSERC and the MRC
of Canada.
Microcautery was used to ablate the neural
crest bilaterally from the mid-otic placode
region to the third somite in stage 9 chick
embryos. Animals were sacrificed in successive
stages, fixed in Karnovsky's fixative, and
embedded routinely in epoxy resin. Semithin
sections were cut at intervals and observed
after toluidine blue staining. At areas of
interest, adjacent thin sections were cut for
electron microscopic study. In addition,
neural tube containing cardiac neural csest was
transplanted, along with embryonic hindgut, to
the chorioallantoic membrane of host chicks;
vessel growth was compared with hindgut
transplanted without neural crest. Inappropriate development of the first primitive
vessels was observed as early as stage 11 in
animals subjected to neural crest ablation.
Segments of vessels failed to form structures
with complete, continuous, endothelium. As a
consequence, recognizable lumens were missing
in these segments. Furthermore, the presence
of neural crest in chorioallantoic grafts
induced the formation of blood vessels in
excess of controls. Derivatives of neural
crest therefore seem critical for angiogenesis,
and appropriate cardiovascular development.
Supported by NIH grant HL36095.
S
BOMBERGER." Cntherine Elizabeth and Jack Luther HAAR,
Department of Anatomy, Medical College of Virginia.
Richmond,
Virginia. Dexamethasone Increases the
Migration of Prethymic Stem Cells in vitro.
Previous studies show that stress reduces the
migration of bone marrow cells through a 5 urn Nuclepore
membrane in blind well chambers toward a supernatant
derived from minced neonatal thymuses. The migrated
cells are enriched for a "null" cell population, i.e.
Thy 1 and sIg negative, and evidence suggests that this
in vitro assay enriches for prethymic cells. Others have
shown that glucocorticoids are released from the adrenal
cortex in response to stress and inhibit many phases of
the immunological and inflammatory response. To study
the effects of glucocorticoids, such as those released
during stress, on prethymic cell migration, pellets
containing Dexamethasone, a synthetic glucocorticoid,
and releasing approximately .07 mg/day were implanted
subcutaneously in young adult CBA/J mice for 7, 14 and
2 1 days. After 7 and 21 days, enhanced migration of bone
marrow lymphocytes occurred with a decreased lymphoid :
myeloid cell ratio after 7 and 14 days. Thymus weights
were reduced in animals treated for 7, 14 and 21 days.
As seen in light and scanning electron micrographs, the
thymus was largely displaced by adipose cells. The
accelerated migration of cells to thymus supernatant
seen in this assay may reflect an effort of the bone
marrow to restore the thymic reserves depleted by
Dexamethasone treatment. (Supported by NIH grant AG
04384)
BOCKMAN, Dale E. and Thomas H. ROSENQUIST,*
Department of Anatomy, Medical College of
Georgia, Augusta, Georgia. Alteration of
anaioaenesis as a factor in cardiovascular
defects.
Previous studies have shown that ablation of
cardiac neural crest leads to defective
septation of the outflow tract of the heart as
well as defects in the formation of the
pharyngeal arch vessel apparatus and related
vessels. The purpose of the present study was
to focus on the earliest changes which occur in
the vessels after neural crest ablation.
*
*
BOYD. Robert B., Victor W. THOMPSON and James ATKIN ,
Department o f Anatomy, Pennsyl vani a Col 1 ege o f Podi a t r i c
Medicine, Philadelphia, Pennsylvania. Permeabi 1 i t y changes
i n the glomerular c a p i l l a r y wall i n streptozotocin-induced
d i a b e t i c rats.
Increased vascular permeability i s a common a l t e r a t i o n
associated with microangiopathy i n streptozotocin(ST2) i n duced diabetic r a t s . I n t h e kidney o f human diabetics t h i s
ABSTRACTSAAA 103RD MEETING
change in permeability has been associated with glomerular
basement membrane (GBM) thickening, mesangial expansion,
endothelial cell junction widening and focal detachment of
podocyte foot processes. To determine i f these pathologies
might be associated with changes in glomerular permeabi
l i t y , anionic f e r r i t i n ( A F ) was injected into STZ induced
diabetic rats. Animals were injected with AF a t 1 wk or
15 wks after induction of diabetes and sacrificed a t 1 min
or 3 m i n a f t e r AF injection. Renal tissue samples from
diabetic and control animals were processed for transmission electron microscopy. Control animals were processed
I n a l l control
in parallel with each diabetic animal.
rats, regardless of age or tracer circulation time, the AF
did not leave the glomerular capillary lumen nor were
there any changes in the normal morphology of the glomerular capillary wall (GCW). The 1 w k / l min diabetic animals
exhibited some movement of AF from the capillary lumen
into the GBM while in the 1 wk/3 min animals the concentration of AF in the GBM increased with a concomitant
increase in i t s movement towards the abluminal surface of
the GBM. The only apparent change in the ultrastructure
was a slight thickening of the GBM. In the 15 week/l min
animals AF was distributed on a continuous gradient across
the GCW with greatest concentration toward the lumina1
surface. In the 15 wk/3 min animals the concentration of
AF shifted in the abluminal direction.
Distinct morphological changes were noted in the 15 wk animals. These
included marked widening of endothelial intercellular
junctions, focal detachment o f podocyte foot processes and
extensive thickening of the GBM. These changes appear t o
correlate with the observed increase in permselectivity of
AF across the GCW. (Supported by PCPM Institutional
Research Grant 4308.32RBB).
-
BRAEKEVELT, Charlie Roger, Department of Anatomy, The
University of Manitoba, Winnipeg, Manitoba, Canada.
Fine structure of the retinal uiment euithelium (WE) of the redtailed hawk (Buteo iamaicensis)
With the possible exception of the chicken, the structure of the
avian retina has not been extensively examined. Consequently as
part of an ongoing comparative morphological study, the !be
structure of the retinal pigment epithelium (WE) and closely
associated structures such as the choriocapillaris and Bruch's
membrane (complexus basalis) have been studied by light and
electron microscopy in the red-tailed hawk (Butee jamaicensis). In
this species the retinal pigment epithelium consists of a single layer
of large cuboidal cells which display numerous basal (scleral)
infoldings and extensive apical (vitreal) processes which enclose the
outer segments of the photoreceptors. The epithelia1 cells are
jointed laterally by a series of basally located tight junctions.
Internally smooth endoplasmic reticulum is the most abundant cell
organelle while only small amounts of rough endoplasmic reticulum
are present. Polysomes are however abundant as are mitochondria
of various shapes. The basally located W E cell nucleus is normally
large and vesicular. Melanosomes are plentiful in both the apical
region of the cell body and within the apical processes themselves.
As only light-adapted specimens were available for this study
however it is not known how extensively these melanosomes move
during photomechanicalor retinomotor movements. Myeloid bodies
are large and numerous in the light-adapted state and very often
show numerous ribosomes on their outer borders. Bruch's
membrane (complexus basalis) shows the typical pentalaminate
stnicture noted in most vertebrates but with only a poorly
represented central elastic layer.
The endothelium of the
choriocapillaris is very thin facing the WE but is only moderately
fenestrated. The choriocapillaris in this species is unusual in that
many of the fenestrae show a double-layered diaphragm.
(Supported in part by the NSERC and the MRC of Canada).
15A
BRAINARD. George. Karen STEWART', Chau NGUYEN', John
HANIFIN', Milton STETSON', and Mark ROLLAG. Dept. Neurology,
Jefferson Medical College, Philadelphia, Pennsylvania; University of
Delaware, Newark. Delaware, USUHS. Bethesda. Maryland. Differential
.. of near and far ultraviolet
sof -
Exposure of the eyes to light during the night induces a rapid
depression of meiatonin synthesis and secretion. The aim of the
following study was to determine if equal photon densities of near
ultraviolet (UV-A) and far ultraviolet (UV-C) radiation are capable of
suppressing pineal rnelatonin content. Adult (3 to 6 months) male
Syrian hamsters were adapted to a light-dark cycle of LD10:14 for a
minimum of 3 weeks. Groups consisted of 7 hamsters each. During the
night, hamsters were individually exposed to 340 nm. 320 nm or 280
nm monochromatic ultraviolet radiation (half-peak bandwidths ranging
between 9 - 13 nm) at a photon density of 1.087 x IOi5 photons/cm2
for 300 seconds. The ultraviolet radiation was measured with a Solar
Light UV-A or UV-B meter. The photic stimuli were produced by either
a 100 W tungsten-halogen source reflected by a parabolic dichroic
filter or a xenon arc monochromator that was filtered with neutral
density screens and interference filters. After exposure, the hamsters
were held in darkness for 15 minutes before pineals were removed and
frozen at -200 C. Matched groups of hamsters were handled similarly,
but were not exposed to ultraviolet radiation. Pineal melatonin content
was quantified by RIA. Groups of animals exposed to equal photon
densities of 340 or 320 nm radiation had significantly (P<O.OI)
suppressed melatonin levels compared to their matched controls.
Exposure to the same photon density at 280 nm in two separate
experiments did not significantly alter pineal melatonin content
compared to that of matched control animals. These data confirm that
UV-A stimuli, which are traditionally considered not to be visible, are
indeed capable of regulating neuroendocrine function. In contrast, an
equal photon density of UV-C radiation range does not seem to be capable
of neuroendocrine regulation. Supported by NEMA Grant (LRI
B9:DR:NEMA:l) and NASA Grant (NAGW 1196) to GCE, USUHS Grant
(C07049) to MDR, and NSF Grants (DCB84-12587 and DCB8714638) to MHS.
BRIDGMAN, C h a r l e s F. and I r a R. TELFORD, L i s t e r
H i l l N a t i o n a l C e n t e r f o r Biomedical CommuniBethesda,
Maryland;
Department of
cations,
Anatomy, Uniformed Services U n i v e r s i t y of t h e
H e a l t h S c i e n c e s , Bethesda, Maryland.
New
_
_ _aae
_ ~
r e t r i e v a l system f o r anatomic i n f o r m a t i o n .
conventional textbooks headina towards
e x t i n c t i o n or c a n t h e t e x t media be g i v e n l i f e by
combining p r i n t media w i t h o p t i c a l video d i s c
u t i l i z i n g barcodes.
A new t e x t b o o k has been
a u t h o r e d by T e l f o r d and Bridgman i n which t h e
best of old and new t e c h n o l o g i e s have been
combined f o r a n e l e c t r o n i c t e x t o f t h e f u t u r e .
O v e r 800 i n t e r p r e t i v e drawings have been p r e p a r e d
t o accompany each c o n c e p t p r e s e n t e d i n t h e t e x t .
Nearly 3000 f u l l color l i g h t photomicrographs are
available t h r o u g h b a r c o d e s f r o m a g e n e r i c v i d e o
disc on l i g h t microscopy (by Frank D. A l l a n ) t o
h e l p document t h e c o n t e n t of drawings and o v e r
600 b l a c k and w h i t e photomicrographs i n t h e t e x t .
The p r e s e n t a t i o n w i l l i l l u s t r a t e some of t h e
p o t e n t i a l of barcoded t e x t b o o k s t o r e t r i e v e still
and l i v e - a c t i o n s e q u e n c e s , computer-generated
a n i m a t i o n of a b s t r a c t c o n c e p t s , l o o k - a l i k e image
comparisons, and e l e c t r o n i c i n d e x i n g . I n t e r a c t i v e
t e s t i n g of l e a r n i n g c a n a l s o be accomplished.
A l l p r o c e d u r e s a r e s i m p l e b e c a u s e o f encoded
b a r c o d e s t h a t a r e embedded i n t e x t b o o k d e s i g n .
The p r e s e n t a t i o n w i l l a l s o t r y t o a n a l y z e t h e
p r a c t i c a l i t y of e l e c t r o n i c i n n o v a t i o n i n t o d a y ' s
education.
Are
16A
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
BRIGHTON,* Brian W., Niru K. MOHAPATRA,* Stephen P.
SCHNEIDER* and Kim B. SEROOGY, Department of Physiology,
University of North Carolina, Chapel Hill, North Carolina.
Distribution of somatostatin messeneer RNA in hamster
brain. soinal cord and dorsal root eanvlia.
Gene expression for the neuropeptide somatostatin was
examined in the hamster central nervous system and spinal
ganglia using in situ hybridization. A 41 base synthetic
oligodeoxyribonucleotide probe complementary to
nucleotides 174-214 of rat preprosomatostatiq2messenger
RNA (mRNA) was labeled on the 5' end with 7 [ PIATP using
T4 polynucleotide kinase. Fresh, unperfused tissue
sections were hybridized with the labeled probe overnight,
washed and processed for both film and liquid emulsion
autoradiography. Probe specificity was verifiqd by
RNA.
Northern blot anaysis of hamster brain poly(A)
Neuronal perikarya expressing somatostatin mRNA were
widely distributed throughout the hamster forebrain
including the olfactory bulb, anterior olfactory nucleus,
olfactory tubercle, lateral septal nucleus, caudateputamen, nucleus accumbLns, amygdala, hippocampus, zona
incerta, certain thalamic and hypothalamic nuclei and
extensively in the cerebral cortex. In the brainstem,
somatostatin MA-containing neurons were mainly found in
the interpeduncular nucleus, reticular formation, certain
raphe nuclei, dorsal tegmental regions, spinal nucleus of
V, nucleus tractus solitarius, ventrolateral medulla and
dorsal column nuclei. Spinal cord somata expressing the
peptide mRNA were restricted primarily to the superficial
layers of the dorsal horn and around the central canal.
Small-sized cells comprising about 10-15% of the dorsal
root ganglia cell population were labeled with the probe.
These results are consistent with previous descriptions of
somatostatin mRNA distribution in the rat, with the
following additional areas of expression in the hamster:
thalamic reticular nucleus, dorsal column nuclei and
certain raphe nuclei. Thus, a similar, but not identical,
cellular expression of the somatostatin gene exists across
two different rodent species. (Supported by fellowship NS
08525 and grant NS 10321 from NINCDS.)
OBRODY.* Rachel I.and V i r g i n i a H. BLACK, Department o f
C e l l Bioloav. New York U n i v e r s i t v School o f Medicine. New
York, New 6 r k . Immunodetectable 1 eve1 s o f D r o t e i ns
i n v o l v e d i n c h o l e s t e r o l and s t e r o i d svnthesis i n auinea
p i a adrenal o u t e r zone c e l l t w e s : D i f f e r e n t i a l ACTH
response.
I n t h e guinea p i g adrenal t h e c e l l s o f t h e o u t e r zone
s e c r e t e h i g h l e v e l s o f s t e r o i d and respond t o ACTH w i t h
increased s y n t h e s i s o f c h o l e s t e r o l and s t e r o i d . The
o u t e r zone c o n s i s t s o f two c e l l types: zona f a s c i c u l a t a
To determine t h e
(ZF) and zona glomerulosa (ZG).
r e l a t i v e c o n t r i b u t i o n o f t h e two o u t e r zonal c e l l s types
t o t h e ACTH response, p u r i f i e d p o p u l a t i o n s o f each c e l l
type were prepared from ACTH-treated and c o n t r o l
animals. Levels o f p r o t e i n s p o t e n t i a l l y i n v o l v e d i n t h e
ACTH response were measured by ELISA i n c e l l homogenates
from t h e p u r i f i e d p o p u l a t i o n s . HMG CoA reductase, t h e
r a t e l i m i t i n g enzyme o f c h o l e s t e r o l synthesis, and two
cytochrome P45Os. P45017a and P45021, were studied.
P 4 5 0 1 7 ~i s r e q u i r e d f o r p r o d u c t i o n o f c o r t i s o l , b u t n o t
f o r c o r t i c o s t e r o n e or aldosterone. P45021 i s r e q u i r e d
f o r p r o d u c t i o n o f a l l o f these c o r t i c o s t e r o i d s . ZF c e l l s
had a 4-5 f o l d g r e a t e r c o n c e n t r a t i o n o f a l l t h r e e
p r o t e i n s , b u t t h e p r o t e i n s i n ZG c e l l s showed a g r e a t e r
response t o ACTH ( ~ f o3l d ) . T h i s response o f ZG c e l l s
t o ACTH and t h e i r possession o f P45017 i s c o n s i s t e n t
w i t h o b s e r v a t i o n s made i n v i t r o t h a t ZG c e l l s synthesize
c o r t i s o l and respond t o ACTH w i t h increased o u t p u t o f
c o r t i s o l as w e l l as o f c o r t i c o s t e r o n e and aldosterone.
I n ZG c e l l s t o a g r e a t e r e x t e n t than ZF c e l l s , t h e
response t o ACTH i n v o l v e d increases i n l e v e l s o f enzymes
o f b o t h c h o l e s t e r o l and s t e r o i d synthesis, suggesting
t h a t new p r o t e i n s y n t h e s i s i s an i m p o r t a n t component o f
t h e ACTH response o f ZG c e l l s . On t h e o t h e r hand, t h e
f a c t t h a t ZF c e l l s can i n c r e a s e s t e r o i d o u t p u t i n
response t o ACTH w i t h a l e s s e r increase i n these p r o t e i n s
suggests a d i f f e r e n t mechanism o f r e g u l a t i o n . I t may be
t h a t m o b i l i z a t i o n o f s t o r e d c h o l e s t e r o l i s more important
i n t h e ACTH-reponsiveness o f t h e l i p i d - f i l l e d ZF c e l l s
than i n t h e l i p i d - p o o r ZG c e l l s . (Supported by N I H DK
32802 and ACS BC-593)
Brown, H. Keith, J. Schellenberg*, L. Clarke*,
and M. Silbiger*, Departments of Anatomy and
Radiology, University of South Florida, Tampa,
Florida Diaqnostic utilitv of color comDosites
senerated from Maunetic Resonance Imaqes.
Currently, MRI data interpretation requires
sequential observation and integration of
several different images of the same scene. The
complex pattern of contrast behavior in several
different images reflects the biophysical
characteristics of various tissues and fluids
present.
Our efforts to compress this image
data to increase diagnostic efficiency and
accuracy have included multispectral image
analysis techniques such as Nearest-Neighbor,
Maximum Likelyhood (ML), and Composite methods.
Using a DEC Microvax I1 with Interactive Digital
Language, these methods have been applied to MRI
images obtained from the General Electric Signa
and Siemens Magnatome imagers. Pulse sequences
used for our image sets include: proton density,
T1 and T1 weighted, and both high and low flip
angle Gradient-Echo sequences. The advantage of
feature analysis methods such as ML is that the
conspicuity o f a given tissue type is
dramatically improved.
However, we have found
that at the overwhelming disadvantages of misclassification of tissues, significant loss of
other image data, and susceptability to poor
image standardization are quite troublesome.
The advantages of our Composite Generation
Methods are that no image information is lost,
computer misclassification is eliminated and the
specific conspicuity of tissues is maintained.
Also, composite images may produce color
renditions which approach natural or histologic
stain appearances. Availability of these color
composites improves the efficiency and accuracy
of diagnostic interpretation. (Supported by the
USF President's
Council & Moffitt Cancer
Research Institute)
.
S
BROWN," Richard E., and Robert D. KLEMM, Department of
Anatomy and Physiology,Kansas State University, Manhattan,
Kansas. The ProDataniurn: evolutionary anatomical adaptation
for flieht in Aves.
Although vertebrate flight has evolved independently in
widely divergent taxa, there is a common evolutionary
feature; an adaptation for increased surface area to provide
for lift. Specifically, an expanded dermal surface is the
common anatomical adaptation of all flying vertebrates. The
thoracic appendage of birds supports broad derrnalmembranes,
patagii, that convert it into a structure capable of
producing lift. The propatagium of birds is a f o l d of
normal integument and a system of supporting ligaments,
extending from the carpus proximally to the body wall, and
filling the cranial angle formed by the flexed elbow. A
pair of interactive ligaments, originating from the cranial
point of the shoulder, supports and shapes the propatagium.
Lig. propatagiale supports the leading edge of the
propatagial fold, serves as the cranial attachment for the
ABSTRACTSAAA 103RD MEETING
tissue layers creating the cambered shape, and inserts on
the carpometacarpus. The Elbow Extension Limiting Ligament
fixes the maximum angle of elbow extension, and inserts into
the proximal antebrachial fascia. That angle is crucial as
extension beyond a certain point decreases the surface area
of the propatagium. We have studied the anatomy of the
propatagium and its supporting structures in 21 orders of
birds; modifications and adaptations in structure can be
correlated with
flight style and with phylogenetic
patterns. The sequence of anatomical adaptations of the
propatagium across the orders of Aves allows us to project
back in evolutionary time, and to suggest an outline of the
events leading to the evolution of the first stage of avian
flight, i.e. gliding. We have demonstrated, via computer
modeling, the aerodynamic advantages of the propatagium
versus the elongation o f flight feathers. Results of flight
trials with sparrows, and the effects on their flying
abilities after removal of flight feathers,propatagium. or
both, demonstrates the positive contribution of the
propatagium to flight and the production of lift.
17A
zona fasciculata cells is about 30% more than that of
isolated zona nlomerulosa cells (Loesser and Malamed,
Anat. Rec. 218: 80A, 1987). In an extension of these
studies we have used scanning electron microscopy. Our
results confirmed the findings from transmission
electron microscopy. The surfaces of isolated
glomerulosa cells had bare patches devoid of microvilli,
as well as areas with an abundance of microvilli,
whereas the surfaces of isolated a fasciculata cells
were evenly and densely covered with microvilli.
Similar results werc obtained with cells in situ. The
differences in the inicrovilli of the two zones may be
related to different modes of hormone release for
aldosterone and for corticosterone (Sibley, et al., J .
Steroid
Biochem. 13: 1231; 1980; J . Endocr. 91: 313;
__
In both isolated and in situ cells, certain
1981).
unexpected features were also noted:
many surface
elevations appeared as ridges rather than a s typical
microvilli, and some long microvilli appeared to contact
neighboring cells. This raises the possibility that
these structures are involved in intercellular
communication, (Supported by NIH grant # 5 T32 NS0722906).
sBUSH,*
Kevin, Francis LYNCH,* Hsin-yi LEE,* and Robert
NAGELE,* Department of Pediatrics, University of Medicine
and Dentistry of New Jersey, Camden, New Jersey;
Department of Biology, Rutgers University, Camden, New
Jersey. (Sponsored by Robert Trelstad) Chanves in
microfilament (MF) ornanization and alimment in neuroeuithelial (NE) cells associated with successive uhases of
neural tube formation in chick and mouse embryos: a study
usine fluorescent (FITC-labeled)Dhalloidiq.
During neural tube formation, a flat sheet of columnar
NE cells bends along the embryo midline to form a hollow
cylinder. Constriction of the apical ends of NE cells at
specific sites within the wall of the forming neural tube
is now thought to play a major role in bending of the
neuroepithelium. In the present study, we used
FITC-labeled phalloidin, which binds specifically to
filamentous (F) actin, to study the temporal relationship
between the organization and alignment of MF bundles in NE
cells, the degree of apical constriction, and the local
extent of bending of the neuroepithelium. Chick and mouse
embryos at various stages of neurulation were fixed in 2%
paraformaldehyde and stained with FITC-labeled phalloidin.
Some embryos were treated briefly with cytochalasin D (CD)
prior to staining. Results confirmed that bending of the
neuroepithelium is accompanied by a local constriction of
the apical MF bundle array. These bundles are often
aligned in tandem from one cell to another, giving the
impression that a single, straight MF bundle passes
through several adjacent cells. The fluorescence
intensity of individual MF bundles was found to be
inversely proportional to the cell apical width,
suggesting that apical constriction may be accomplished
through a sliding of F-actin filaments past one another,
resulting in MF overlap. Brief treatment of embryos with
CD resulted in an immediate (within minutes) reversal of
neural fold elevation, broadening of the apical ends of NE
cells, and a decrease in the fluorescence intensity of the
apical MF array throughout the neuroepithelium. The
effects of CD were most pronounced in regions of the
neuroepithelium exhibiting bending. These findings
provide strong evidence that apical MF bundles of NE cells
play an active role in bending and shaping of the
neuroepithelium during neural tube formation.
Supported by Grant No. 23200 from the NIH.
CAIN, Lisa D. and Sasha MALAMED, Department of Anatomy,
UMDNJ-Robert Wood Johnson Medical School, Piscataway,
New Jersey. A scannine.electron microscouic comparison
of the microvilli of zona nlomerulosa and zona
fasciculata cells of the adrenal cortex.
Transmission electron microscopy has shown that the
mean aggregate microvillar surface length of isolated
S
CAMPBELL,* Stephen S. and Bruce J. CRAWFORD, Department
of Anatomy, University of British Columbia. Vancouver,
British Cblumbia. An -ultrastructural comoarison of the
extracellular matrix of embryos of the starfish Pisaster
gchraceus that have been fixed bv conventional methods
and a freeze substitution method.
Five day old embryos of the starfish Pisaster
ochraceus consist of three cellular layers and three
regions of extracellular matrix (ECM): an outer coating,
the hyaline layer (HL); an inner epithelia1 lining. the
basal lamina (BL); and a third component which fills the
blastocoel. When the embryos are conventionally fixed in
glutaraldehyde and osmium tetroxide, electron microscopy
of the HL reveals two layers associated with the tips of
the microvilli. If anionic dyes are included in the
fixation, three additional layers are seen; one below the
microvilli and two above consisting o f fibrous strands
encrusted with an amorphous material. Within the embryo,
strands of a similar nature are scattered throughout the
blastocoel but tend to concentrate more heavily in two
areas - the dorsoposterior region and the middle o f the
embryo, radially interconnecting the esophagus to the
ectoderm. A fixation technique that produces favourable
results in vertebrate tissue is freeze substitution.
When asteroid embryos which have been cryoprotected with
propylene glycol are rapidly frozen in liquid propane and
freeze substituted with ethanol, the cellular and ECM
preservation i s excellent. The results show that the HL
is twice as thick as that seen after conventional
fixation and the ECM in the blastocoel consists of
diffuse elements which are distributed evenly throughout
suggesting the presence of a gel-like material. Because
this fixation technique, which gives excellent
ul trastructural preservation, does not require the use of
gl utaraldehyde, paraformaldehyde or osmium tetroxide,
there would be much less destruction of antigenic sites.
It should therefore not only be useful for further
of
ECM
but
also
for
morphological
studies
immunocytochemical studies. Supported by Grant No.
MA0032 from NSERC.
S
CHEN,* D.F. and F.J. ROISEN, Department of Anatomical
Sciences & Neurobiology, Univ. of Louisville School of Medicine,
Louisville, Kentucky. (Sponsored by J.B. Longley) The effect of
neurotrowhic aaents on cvtoskeletal components in sensory
neuvrites.
18A
ABSTRACTSAAA 103RD MEETING
A variety of neurotrophic factors have been shown to play a
key role in the development of the nervous system. Chick
embryonic sensory ganglia (DRG) exposed to Nerve Growth
Factor (NGF) or an NGF-independentneurotrophic factor found
in medium conditioned by C, glioma cells (GCM) undergo
extensive neuritic development. Our previous studies have
shown that these two neurotrophic agents produced unique
neuritogenic patterns. The ganglioside GM1 potentiated the
action of these agents by altering the degree of neuritogenesis
but not the embryonic receptive period. This study aimed to
characterize the cytoskeleton of neurites formed in response to
these two agents alone or in combination with GMI.
Ultrastructural morphometric studies demonstrated that although
NGF and GCM both enhanced neuritogenesis, the neurites
formed in response to these agents were different. NGF-induced
neurites contained 2x the microtubules (MT) and 1 . 5 ~the
neurofilaments (NF) per pm' over controls. In contrast, GCMinduced neurites had 0 . 7 ~the MT per pm' with no alteration of
NF; GM1-induced neurites were equivalent to the controls. The
simultaneous exposure of DRG to NGF and GM1 increased
neurite production, however the neurites contained MT and NF
densities similar to untreated controls. Treatment with GCM and
GMl reversed the inhibitory action of GCM on MT and resulted
in neurites containing higher (1.8~)MT densities. Our data
demonstrate that each trophic agent produced neurites with a
unique cytoskeletal framework and that GM1 acted to normalize
these differences. Supported by NIH Grant NS24524.
S
CARhIICHAEL, Stephen W., Louis E. DE LANNEY., Donato
A. DI MONTE., Ian IRWIN., and J. William LANGSTON.,
Department of Anatomy, Mayo Clinic, Rochester, Minnesota;
California Parkinson's Foundation, San Jose, California Effect pf
MFTP on the U l t r ~ c treu of the Drimate adrenal m&la1 .
Fifteen squirrel monkeys were treated with 1-methyl-4-phenyl1,2,3,6-tetrahydropyn'dine
2.5 mg/kg, s.c.), rendering the
animals parkinsonian. Animals were sacrificed 1 to 41 days after
the injection. One adrenal medulla was processed for electron
microscopy, the other was assayed for MF'P'.
Only slight
morphologic changes were noted in specimens from animals
treated 24 hours earlier. By three days post-injection, morphologic
changes suggestive of toxic stress could be seen. Most of the
chromaffin cells from animals treated 5 or 10 days earlier were
necrotic or showed morphologicdamage, although some cells were
nearly normal. In one animal treated 41 days earlier, some
chromaffin cells were necrotic and others were depleted of
chromaffin vesicles, suggestingthat the toxic effects of MPT" can
be long lasting, although recovery appears to occur. Mean
concentrations of MPP in the adrenal medulla were 296
nmol/gm (N=3) by 1 day, 395 nmol/gm (N=3) by 3 days, 491
nmol/gm (N=2) by 5 days, and 248 nmol/gm (N=2) at 10 days.
Since the morphologic damage was heterogeneous, it is suggested
that some chromaffh cells accumulated extremely high
concentrations of this toxin. The data also suggest that the
adrenal medulla actively takes up MYIT or MPP' from the
circulation. These studies suggest that in vivo MPTP is removed
from the blood by chromaffin cells of the adrenal medulla,
converted to MPP' by MAO B, and sequestered within
chromaffinvesicles. This is exaggerated in some cells, leading to
toxic damage and death.
(m
S
Capehart", Anthony A. and David M. Biddulph, Department
of Neurobiology and Anatomy, Bowman Gray School of
Medicine, Winston-Salem, North Carolina. (sponsored by
David M. Biddulph).
E, and
Res onsiveness of aden late cyclase
for:kolin g ~
isolated ceyls from
micromass cultures
- of
-chick 1 imb mesenchyme durinq chondrogenesis.
Stimulation of adenylate cyclase (AC) by prostaglandin E, (PGE,) i n micromass cultures of mesenchyme
derived from the distal t i p of stage 25 chick limb buds
was examined over a six day period of culture. Responsiveness of PGE, was measured in b o t h dissociated and
intact cell layers in an effort t o determine whether an
inhibitory interaction occurred between PGE, receptors
of differentiating chondrocytes and the extracellular
matrix (ECM). PGE, activation of AC was equivalent in
dissociated and intact cell layers a t a l l time points
examined with maximal stimulation of AC occurring in
prechondrogenic mesenchyme after 24 hours in culture.
Responsiveness t o PGE, i n b o t h groups declined signific a n t l y i n differentiating chondrocytes on days 3 and 6.
Dose response experiments revealed dose-dependent
ins ease in AC activity over a concentration range of
10 '-lO-'
M a t a l l time points examined with prechondrogenic cells on day 1 significantly more responsive t o
PGE, a t concentrations tested than cells on days 0 , 3
and 6. I n contrast t o the reduced responsiveness of
differentiating chondrocytes t o PGE,, stimulation of AC
by forskolin resulted i n progressively increased levels
of activity in dissociated and intact cell layers. The
results of the present s t u d y demonstrate that the
reduction i n AC responsiveness t o PGE, by differentiating chondrocytes i s n o t due t o an inhibitory
interaction of PGE, receptors w i t h the cartilagespecific ECM, t o a decrease in the total pool of AC
activity in differentiating c e l l s , nor t o a reduction i n
the number of AC coupled PGE, receptors in differentiating chondrocytes. The decrease i n AC responsiveness during chondrogenesis may reflect developmentallyregulated changes in the affinity of the PGE, receptor.
Supported by Grant HD 19017 from the NIH.
~
S
CARUBELLI,* C e c i l i a M . , Tod M . DOH* and Anly L.
AULTHOIJSE , Anatomi c a 1 S c i e n c e s , U n i v e r s i t y o f
Oklahoma H e a l t h S c i e n c e C e n t e r , Oklahoma C i t y ,
The e f f e c t s o f r e t i n o l on c u l t u r e d
Oklahoma.
human c h o n d r o c y t e s .
___
Vitamin A ( r e t i n o l ) and i t s analogues have
been shown t o be t e r a t o g e n i c b o t h i n humans and
animals.
There i s a wide spectrum o f b i . r t h
d e f e c t s , one of which i s limb t r u n c a t i o n .
We
have t r e a t e d human c h o n d r o c y t e s grown i n a g a r o s e
c u l t u r e w i t h v i t a m i n A t o s t u d y t h e e f f e c t s of
r e t i n o i d s on human chondrogenesi s . C u l t u r e s were
t r e a - t e d d a i l y w i t h 5119, 7ng and 10 ng/ml r e t i n o l
i n Dul.becco' s Modified E a g l e s Media c o n t a i n i n g
10% f e t a l c a l f serum.
Treatment of 1 0 ng/ml
retinol was n o t l e t h a l t o c e l l s a s d e t e c t e d w i t h
trypan blue staining.
R e t i n o l was d i s s o l v e d i n
a b s o l u t e e t h a n o l ( p r i m a r y s t o c k lmg/ml).
To
d e t e r m i n e the e f f e c t o f e t h a n o l on chondrocytes
i n c u l t u r e , some c u l t u r e s were t r e a t e d d a i l y w i t h
equivaLent voliimes o f e t h a n o l .
Ethanol t r e a t e d
c u l t u r e s were a i m i . l a r t o u n t r e a t e d c u l t u r e s b o t h
i n mit.otic a c t i v i t y and t h e amount o f a l c i a n b l u e
s t a i n i n g m a t r i x . Moreover, ethanol. d i d n o t a l t e r
c o l l a g e n s y n t h e s i s a s d e t e c t e d by
imiunocytochemistry.
However. t h o s e c u l t u r e s t r e a t e d
w i t h r e t i n o l showed d e c r e a s e d m i t o s i s and a l c i a n
blue staining matrix.
The d e c r e a s e i n p r o t e o g l y c a n s y n t h e s i s was confirmed by t r a n s m i s s i o n
e l e c t r o n microscopy u s i n g Ruthenium Red.
The
e f f e c t s of
r e t i n o l w e r e more e v i d e n t w i t h
increasing concentrations.
A f t e r discontinued
t r e a t m e n t , c e l l s appeared t o r e c o v e r from t h e
e f f e c t s of r e t i n o l .
The amount of a l c i a n b l u e
s t a i n i n g niotrix was l e s s t h a n u n t r e a t e d c u l t u r e s
but greater than cultures t h a t w e r e treated
continuously with r e t i n o l .
(Supported i n p a r t by
OCAST g r a n t HN9-019 and OU C o l l e g e of Medicine
Alumni
Association
and
Presbyterian Harris
Research Foundation. )
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
19A
S
CHAMBERS*, John, Mark F. JACQUIN, and William E. RENEHAN,
Departmentof AnatomicalSciences and Neurobiology,University
of Louisville School of Medicine, Louisville, Kentucky; and
Department of Anatomy and Neurobiology,St. Louis University,
St. Louis, Missouri. Quantitative analvsis of individuallv labeled
neurons in rat triaeminal subnucleus interDolaris ISDVi).
Previous qualitative analysis of SpVi neurons has shown
that cells with a certain projection status, receptive field size and
peripheral receptor association have a stereotypic morphology.
To investigate this phenomenon further, and as a prelude to
similar investigationsin deafferentedanimals, we have employed
a computerized three dimensional reconstruction system to
quantitatively analyze a sample population of SpVi neurons.
Physiologically characterized local circuit (LC; cells with axons
retricted to the trigeminal brainstem nuclear complex) and
cerebellar-projecting (CB) neurons in SpVi were labeled with
iontophoretic injections of horseradish peroxidase. Confirming
previous observations, we found that projection neurons had
significantly (T-test, two tailed, p < .01) larger somata and dendritic
fields than LC neurons. In addition, we established that surface
area, volume, total fiber length and area of influencemeasurements
were larger for projection neurons. Our preliminaryresults indicate
that some of these parameters may be abnormal following
neonatal deafferentation. Many of these attributes cannot be
measured with conventional two dimensional morphometric
examination, suggesting that computerized three dimensional
quantitative analysis may identify structure-functionrelationships
which might be missed by using more rudimentary techniques.
Supported in part by DE07734 and a University of Louisville
Medical School Research Award.
CHAVEZ, Dan, Department of Anatomy, Southern Illinois
Universitv School of Medicine, Carbondale, Illinois. Another
look at the female reoroductive tract of the Rockv M
m
Pi
h t n rin
%&
G
;*!k:a
is a Lagomorph, Family
Ochotonidae, whose habitat is limlted to higher elevations
within the Rocky Mountains. For this reason, observationsand
collection have been mainly limited to the summer season when
access to the high country is facilitated by the lack of snow
cover and reduced avalanche hazard. The animal does not
survive well in captivity. The histology and morphology of the
female reproductivetract has been described (Kirkpatrick and
Satterfield, J. Mammalogy 54:855, 1973; Duke, Anat. Rec.
112:737, 1952) durin the follicular and luteal phases with the
majority of attention ledicated to the short reproductiveseason
which occurs between the vernal equinox and summer solstice.
No studies have been published regarding the ultrastructureof
the uterus. The present study expands the previous
observations to include electron microscopy and collections
during anestrus between summer solstice and vernal equinox,
Animals collected in late July possessed no corpra lutea but did
have antral follicles. No pregnant animals were collected in July
but the presence of antral follicles indicatedthat late season
pregnancies were still possible. Uteri appeared grossly
avascular and quiescent. The epithelium was columnar; s.e.m.
revealed that each epithelial cell had a single cilium. Uteri.from
animals collected in winter appeared morphologicallysimilar to
those collected in late summer except that there was even less
vasculari and thickness of the endometrium was reduced to
about ha?the thickness observed during the summer months.
The uteri presented only three infoldingsthroughout with only a
hint of secondary foldings which were present during the
reproductivemonths. Single cilia remained on each epithelial
cell throughout the uterus. These observa!ions suggest that
these seasonally polyestrus animals experience annual uterine
regressionwhich persists through late winter. These results
were neither unexpected nor surprising. The significant
observationwas the presence of a sin le cilium on epithelial
cells. The significanceof this unusual?eature remains to be
investigated.
CHEN,* D.F. and F.J. ROISEN, Department of Anatomical
Sciences & Neurobiology, Univ. of Louisville School of Medicine,
Louisville, Kentucky. (Sponsored by J.B. Longley) The effect of
neurotroDhic aaents on cvtoskeletal comoonents in s e w
neurites.
A variety of neurotrophic factors have been shown to play a
key role in the development of the nervous system. Chick
embryonic sensory ganglia (DRG) exposed to Nerve Growth
Factor (NGF) or an NGF-independent neurotrophic factor found
in medium conditioned by C, glioma cells (GCM) undergo
extensive neuritic development. Our previous studies have
shown that these two neurotrophic agents produced unique
neuritogenic patterns. The ganglioside GM1 potentiated the
action of these agents by altering the degree of neuritogenesis
but not the embryonic receptive period. This study aimed to
characterize the cytoskeleton of neurites formed in response to
these two agents alone or in combination with GM1.
Ultrastructuralmorphometricstudies demonstratedthat although
NGF and GCM both enhanced neuritogenesis, the neurites
formed in responseto these agents were different. NGF-induced
neurites contained 2x the microtubules (MT) and 1 . 5 ~the
neurofilaments (NF) per pmZover controls. In contrast, GCMinduced neurites had 0 . 7 ~the MT per pm2with no alteration of
NF; GM1-induced neurites were equivalent to the controls. The
simultaneous exposure of DRG to NGF and GM1 increased
neurite production, however the neurites contained MT and NF
densities similar to untreated controls. Treatment with GCM and
GM1 reversed the inhibitory action of GCM on MT and resulted
in neurites containing higher (1.8~)MT densities. Our data
demonstrate that each trophic agent produced neurites with a
unique cytoskeletalframework and that GM1 acted to normalize
these differences. Supported by NIH Grant NS24524.
CHEN, I-li and R.D. YATES, Department of Anatomy, Tulane
Medical School, New Orleans, LA. Localization of choline
acetvltransferase in the rat carotid bodv using
immunocvtochemistru.
a-bungarotoxin binding sites on plasma membranes of
glomus cells of the rat carotid body have been demonstrated
(Chen et al., Cell Tissue Res., 219:609, 1981; Chen and
Yates, J . Neurocytol. 13:281, 1984). These findings may
indicate nicotinic acetylcholine receptor sites on the
cells. In this study we have attempted to localize and
identify which structures synthesize acetylcholine in the
carotid body of the rat using immunocytochemical
localization of choline acetyltransferase. Preliminary
data were presented at the International Anatomical
Congress in Rio de Janeiro, Brazil in 1989.
Reference tissue consisted of sections of superior
cervical sympathetic ganglia (SCG) known to contain
cholinergic preganglionic nerve fibers.
Choline
acetyltransferase-like immunoreactivity
(CAT-I) was
observed in some axons and nerve terminals in the SCG.
Some ganglion cells and nerve fibers associated with
ganglion cells in the carotid body showed CAT-I. Some of
the interstitial connective tissue cells with attenuated
processes displayed staining but appreciable labeling was
not consistently detected in the parenchyma1 cells of the
carotid body at the LM level. Electron microscopic studies
revealed reaction product in small vesicles and ER in some
glomus cells as well as occasional labeled profiles packed
with small clear vesicles and dense-cored ones in contact
with glomus cells. Recently we have attempted to use
monoclonal antibody to more convincingly demonstrate CATI in the CB glomus cells at the EM level. These studies
have, so far, been unsuccessful and, as before, we
speculate that the amount of CAT in the rat glomus cells
may not be sufficient in amount to be demonstrated
consistently with the techniques employed and that
interstitial cell staining probably due to contaminated
antibodies in the polyclonal CAT-antiserummasked the faint
2QA
ABSTRACTS-AAA 103RD MEETING
staining of glomus cells at the light microscopic level.
(Supported in part by NIH Grant EY-03731,and the American
Heart Association.)
Chen, L.T.. R.N. ShenP, M.W. Bryant*. N.B. HornbacP, L. [email protected], and Broxmyerlk. Cepartmnt of hatmy, University of South Florida, Tanpa,
Florida, and Cepartmnts of Radiation fhcalcgy and Micine. Indiana
University, Indianapolis, Indiana. Effects of lw dose total M y
irradiation on the appearance of virus particles in the spleen of mice
infected with Friend Virus Carplex.
It has been s h in a previous study that untreated Friend Virus
infected mice died within 40 days post-infection, *reas infected
mice given low dose total body irradiation exhibited long-term survival.
(Shen,et al., Cancer Research 48:2399, 1988). In the present report
we describe effects of a single low dose total M y irradiation (TBI)
on the appearance of type C virus particles in the spleen of mice infected with Friend virus carplex (FVC). Female N/Z mice wre injected
with FVC and 5 days later were irradiated on an x-ray therapy machine
with a dosage of 150 or 403 cGy TBI. Nowinfected mice irradiated
with sam dosage of TBI served as controls. Spleens wre m v e d and
wighed two or six and a half w e k s after TBI. Slices of spleens wre
processed for light and electron microscopy. Tw w e k s after
irradiation the spleen weights of FVC infected mice wthout TBI, with
150 cGy TBI or with 400 cGy TEiI wre 30, 10 or 2 tims that of normal
mice (0.051%4.0094n) respectively. Six and a half w e k s after
irradiation, the spleen weights of all FVC-infeded mice treated with
TBI were 30 tiws that of norm31 mice. Two weks after irradiation, type C virus particles wre observed in the spleens of FVC
infected mice without TBI or with 150 cGy TBI, but not in the spleens
of FVC infected mice with 400 cGy TBI. Hauever, six and a half weks
after irradiation, virus particles wre observed in the spleens of
FVC infected mice with 403 cGy lBI. FVC infected mice with 150 cGy
lBI or 4M) cGy TBI survived 50 to 60 days. This study indicates that
a single dose of 150 cGy lBI is not able to suppress the appearance
of virus particles in the spleen of FVC infected mice. Although a
single dose of 403 cGy TBI is able to delay the appearance of virus
particles in the spleen for several weks. it is unable to extend the
survival tiw of FVC infected mice for mre than M days.
CHINTANAWONGES,* Chakchat, Mark J. HORACEK, Thomas K. BORG*
and Louis T E M C I O , Departments of Anatomy and Pathology,
University of South Carolina, Columbia, South Carolina.
Laser scanninc confocal analvsis of rat heart development.
During heart development mechanical forces are thought
to influence the fiber pattern in the myocardium as well as
the overall shape of the ventricles and atria. The long
range goal of our research is to model the effects of
mechanical stimulation on heart development. In order to
accomplish this, one must have accurate images of the fiber
pattern in the developing myocardium. In this study we
have used confocal microscopy and whole embryo staining to
vi:ualize the fiber patterns in the intact developing rat
he;rt. Sprague-Dawley rats at 10-18 days of gestation were
sacrificed by lethal injection of anesthetic to the mothers
and fixed in 4.0% paraformaldehyde in 0.1 M Hepes for 2
hours at 22°C. The embryos were washed in PBS (1 hr.)
treated with 0.1% Triton-X-100in PBS (1 hr.), washed with
PBS (1 hr.), digested with hyaluronidase (1500 units/ml)
far 1 hrs at 2 2 " C , washed in PBS (1 hr.), incubated in 10100 pg/ml of primary antibody (against alpha-actinin or
vinculin) or in Rhodamine labeled Phalloidin (4-8 hrs.),
washed in PBS ( 8 - 1 2 hrs.), incubated in 10-100 pg/ml of
FITC-labelled secondary antibody ( 8 - 1 2 hrs.), washed in PBS
(4-8 hrs.) and mounted in glycerin.
The embryos were
examined with a Bio-Rad MRC-600 confocal microscope.
Optical sections up to 700 pm deep into the tissue were
obtained with a 4x objective and the digital images were
stored. At the low magnification necessary to visualize
th? entire heart, all 3 stains resulted in very similar
im,ges. The fiber patterns in the atria were more delicate
an<,honeycombed in appearance than the denser ventricular
patterns. With increasing age, the atrial patterns took on
the appearance of the pectinate muscles of the mature
atrium. The patterns in the ventricle showed the extensive
trabeculations and complex interactions of the overlapping
fiber layers seen in the mature ventricular myocardium.
These results indicate that whole embryo staining and
confocal microscopy may be useful techniques for studying
the changes in pattern formation in the developing
myocardium. Supported in part by NIH Grants HI37669 and HU2249.
CHRISTENSEN, A. Kent, Department of Anatomy
and Cell Biology, University of Michigan Medical
plasmic retichum are observed in suiface view in
EM tissue sections, the ribosomes are seen to be
arranged in characteristic patterns such as
circles, spirals or other forms. The individual
ribosomes usually appear oblong, with the long
axis oriented perpendicular to the presumed path
of the mFWA. Unwin (1977, Nature 269:118) has
shown that small subunit of bound ribosomes lie
alongside the large subunits, in the plane of the
RER membrane. However, it has not been clear how
the subunits are oriented in relation to the
overall organization of the polysome.
Rough microsomes (RM) were prepared from rat
liver (Gaetani et al., 1983, Meth. Enzymol. 96:3)
using triethanolamine (TEA) buffered saline
(Walter and Blobel, 1983, Meth. Enzymol. 96:84).
RM vesicles were fixed with 4% formaldehyde (TEA
buffer, 0.12 M sucrose, 5 mM MgClZ), centrifuged
onto EM grid membranes, and then were negativelystained with 2 % phosphotungstic acid (containing
5 mM MgC12). In these preparations, viewed with
the electron microscope, many of the vesicles
become flatten, and polysomes are often clearly
discernible. The individual ribosomes in the
polysomes commonly show clearcut small and large
subunits. The small subunits are uniformly
oriented toward the inside of the polysomal
curve, or in other words toward the center of the
polysome. The boundary between these small and
large subunits occurs at the same level in the
ribosome where inter-ribosomal strands have
previously been described in bound polysomes
(Christensen et al., 1987, Amer. J. Anat. 178:l).
and the present observations therefore tend to
confirm the identity of the strands as mRNA,
perhaps thickened by adsorbed proteins.
CHUNG, Kyung W. Department of Anatomical Sciences,
College of Medicine, University of Oklahoma Health Sciences
Center, Oklahoma City, OK. Effects of ethanol on hepatic
estrogen receptors in female rats.
The binding of hepatic estrogen receptor sites was
investigated in control and ethanol-fed female rats with or
without hypophysectomy o r ovariectomy. Sixty days old
female rats were fed a liquid diet for 3 months containing
36% of total calories as ethanol or the isocaloric equivalent
of sucrose.
Animals were hypophysectomized o r
ovariectomized 2 weeks prior to being sacrificed. Some of
operated animals received daily 100 pg of hCG and FSH or
100 pg of estradiol for 2 weeks from the time of operation.
Ammonium sulfate fractioned hepatic cytosols were
incubated with various amounts ( 0 . 1 - 3 . 0 nM) of 3H-estradiol
21A
ABSTRACTSAAA 103RD MEETING
in the presence or absence of excess nonradioactive
estradiol.
Bound and f r e e ligands were separated by
charcoal adsorption a n d data were analyzed by Scatchard
method.
The number of estrogen receptor sites was
significantly increased in ethanol-fed rats (36 t 0.4 fmollmg
protein) when compared to that of icocaloric controls (27 f
0.3 fmollmg protein), with no significant change in K d
value (0.9 f 0.1 nM).
Hypophysectomy resulted in a
significant decrease in estrogen receptor contents in
controls (11 f 0.2 fmollmg protein) and ethanol-fed rats (8
f 0.1 fmollmg protein). However, ovariectomy caused a
significant increase in this steroid hormone receptor content
in controls (39 f 04 fmollmg protein) and ethanol-fed rats
(41 f 0.5 fmollmg protein). When hypophysectomized
animals were injected with gonadotmpins, hepatic estrogen
receptor contents were slightly increased, whereas
administration of estradiol to ovariectomized animals caused
a decrease in estrogen receptor contents. The findings
indicate that in female rats hepatic estrogen receptor sites
are under the control of the pituitary gland and the ovary
and that ethanol causes a n increase in hepatic estrogen
receptor sites, interfering with hepatic functions in terms
of steroid metabolism. Supported in part by NIAAA G r a n t
R01 AA066448.
CLE!4ENTB,X F. Richard, and Daniel H. MATULIONIS, School of
P h y s i c a l Therapy, Slippery Rock University, Slippery Rock,
Pennsylvania, and Department of Anatomy and Beurobiology,
University of Kentucky, Lexington, Kentucky. The e f f e c t s
of transcutaneouslv a w l i e d 2500 Hz freauencv e l e c t r i c a l
stimulation on the denree of microvascular perfusion in
s k e l e t a l muscle of autonomic nannlionectomized r a t s .
The purpose of t h i s study was t o determine i f t h e
increase in t h e degree of microvascular perfusion produced
by transcutaneously applied 2500 Hz e l e c t r i c a l stimulation
(2500-TES) is dependent on the influence of sympathetic
e f f e r e n t outflow. Twelve male r a t s were treated w i t h
chlorlsondamine which produced an e f f e c t i v e blockade of
t h e autonomic ganglia. S i x ganglionectomized a n i m l s were
treated w i t h 2500-TES a t current i n t e n s i t i e s 3 X t h a t
needed t o evoke minimum v i s i b l e contraction of the
t i b i a l i s anterior muscle (TA) (Ch-W). The other s i x
chlorisondamine treated rats received no ES treatment and
served a s shams (Ch-Sh). Perfused microvessels i n t h e TA
and t h e extensor digitorum longus (EDL) muscles were
labeled with fluorescein isothiocyanate-bovine s e r u m
albumin (FITC-BSA) infused 2 . 5 minutes prior t o removing
t h e muscles. The degree of microvessel perfusion was
determined by calculating the perfused microvessel/muscle
fiber r a t i o (PKV/MFF). The PMV/HF of t h e whole TA muscle of
Ch-Sh and Ch-MS animals showed a significant increase
compared t o controls. The PKV/wF of t h e Ch-MS r a t s a l s o
represents a s i g n i f i c a n t increase compared t o Ch-Sh. The
degree of microvessel perfusion in t h e EDL muscle i n both
the Ch-Sh and Ch-MS animals were s t a t i s t i c a l l y greater
than c o n t r o l s , however Ch-Sh and Ch-MS were s t a t i s t i c a l l y
similar. The changes i n the degree of microvascular
perfusion i n s k e l e t a l muscle of autonomic
ganglionectomized r a t s v a r i e s somewhat depending on t h e
muscle f i b e r type composition. These data indicate t h a t
the microvascular response t o interrupted sympathetic
outflow is variable, t h a t sympathetic outflow is involved
i n t h e increase in mlcrovascular perfusion a f t e r 2500-TES
and t h a t other mechanisms are involved i n t h e noted
hyperemic response of the microvasculature.
COLLINGE', Cory A. and Laura SCHWEITZER,' Department of
Anatomical Sciences and Neurobiology, University of Louisville School
of Medicine, Louisville, Kentucky. (Sponsored by Kunwar Bhatnagar)
. .
of the
inDut in the
achlear
of the
bv Dil.
T h e topographical organization of axonal projections from the
cochlea to the cochlear nucleus of the rodent has not been well
elucidated because of the smallness of the cochlea and the difficulty of
placing tracer substances in replicable positions along the cochlea. A
new fluorescent tracer -Dil- may be used in perfused animals,
making placements in small structures much less difficult, as well as
more exact and easily replicable. We have mapped the cochlear nerve
terminations in the cochlear nucleus with Dil and using threedimensional reconstructions of the nucleus we have demonstrated the
geometry of the cochlear input. From the apex of the cochlea, the
fibers enter the cochlear nerve root where they bifurcate and
terminate ventrally in the cochlear nucleus in a sheet-like
arrangement. The middle and basal regions of the cochlea send axons
that terminate progressively more dorsally, so that different strata of
the cochlear nucleus receive terminations from different regions of
the cochlea. By placing Dil in more than one region of the cochlea. we
have demonstrated that terminations from several regions of the
cochlea form stacked sheets of inputs. In addition, the Dil revealed
several aspects of topographic organization that have not been
described previously. When many ganglion cells were labeled in an
individual turn of the cochlea. a well-delineated stripe of label was
found in each section of the cochlear nucleus that crosses
cytoarchitectonic zones but avoids the granule cell rind in the ventral
cochlear nucleus. A small "hook" of label, discontinuous from the
stripe, could often be seen in the granule cell rind. This label may be
contained in the Type II axons described by Brown et al., 1988. When
only a few axons were labelled in the cochlear nerve, discontinuous
patches of label were found spanning the sheet suggesting that
individual axons terminate in restricted regions within rather than
throughout an entire sheet. In conclusion, we have found that labelling
with Dil provided a detailed view of small neuronal pathways in the
adult mammalian auditory system.
(NIH grant NS-20162)
COILINS,* J a w s Douglas, Poonan Batra,* Rathleen
Brown,* and Warla Shaver*. Departnent of Radiological
Sciences, UCLA School of nedicine, Los Angeles,
California. (Sponsored by Carnine D. Clemente) The
__anatonv of the lvnohatic drainaue as displaved by
)lain x-ray and maanetic resonance inauinu IWRI1.
Radioiogy specializes in the display of anatomy and the
correlation of anatoay uith pathology. Haqnetic
resonance iaaging (XRI) allows the teaching of anatomy
to nedical studezts and aiiatomists and other health
professionals. HRI todels demonstrate surface anatomy
and anatoiical structures [The Anatoaical Recad,
2i8:26A,;997). The selective placeaent of curso: lines
enables the radiologist to outline various planes of
anarony :or description and staging of disease. The
introduction of water bags inro the HR gantry increases
the signal-to-noise ratio rllouing sharper image
display of iiervs ar.2 vascular structures. Hodels of
rajiologica! anatomical correlation have been
constructed with a 0.3 R s l a Ponar permanent magnet to
obtain inages ienanstrrting the possible application of
XRI in the study si disease. The detailed inages
displayed by IRI have increased interest in the
application of contrast agents to enhance the
anatomical display of disease. Currently at BCLL the
follouinq HR! studies are being investigated: patients
with oossible netastati; i:i;st in soft tissses,
staging 0: lymphonas and 3f head and neck cancer, nerve
injuries, soft tissue Ebnormalities, and injection of
drugs into the lymphatic system for cancer treatment.
Since HRI demst:ates anaroaical landmarks, the
display of the lymphatic drainage in combination with
p!ain radisgraphs is possible. The purpose of this
presentation is to dispiay lymph drainage in order to
ailqment the teaching of the lynphatic system as
displayed by piain x-ray an2 KRI.
22A
ABSTRACTSAAA 103RD MEETING
*
CONWAY,* Francis J . and Bruce E. HIKSCH,
Department o f Anatomy, Pennsyl vani a Col 1ege o f Podi a t r i c
Medicine, Philadelphia, Pennsylvania. (Sponsored
Gibley) A user i n t e r f a c e f o r
by Charles W.
computer-assisted teaching i n a n a t o w .
I n an i n c r e a s i n g number o f medical schools, comp u t e r s are being u t i l i z e d t o a s s i s t students i n
l e a r n i n g t h e form and s t r u c t u r e o f t h e human body.
A program has been developed a t t h i s c o l l e g e t o
a i d i n t h e teaching o f t h e gross anatomy of t h e
lower extremity.
This abstract describes the
the visual representation
programs's i n t e r f a c e
o f t h e program on t h e CRT screen. The s c r e e n
presents t h e user w i t h a main menu t o choose from
among muscles, bones, blood vessels, nerves, o r
j o i n t s o f t h e f o o t , l e g , knee, t h i g h , and hip.
T h i s menu i s o f t h e pull-down v a r i e t y , a l s o known
as a frame menu. The choice i s made by moving a
h i g h l i g h t bar t o the t o p i c o f choice and p r e s s i n g
t h e ENTER key.
A f t e r t h e u s e r has chosen, say,
BONES, he i s presented w i t h another menu l i s t i n g
a l l t h e bones o f t h e l o w e r e x t r e m i t y . A g a i n ,
c h o i c e i s made by m o v i n g a h i g h l i g h t b a r and
p r e s s i n g ENTER.
The bone o f c h o i c e i s t h e n
d i s p l a y e d i n a c o l o r g r a p h i c and t h e u s e r can
chose t o a l t e r t h e graphic t o show muscle
attachments, tendons, anatomical r e l a t i o n s h i p t o
other structures, or a r t i c u l a t i o n with other
bones.
A d d i t i o n a l pop-up h e l p s c r e e n s can be
c a l l e d up t o g i v e i n f o r m a t i o n on t h e s t r u c t u r e s
under consideration.
The program i s w r i t t e n i n C
language and can be r u n on any I B M - c o m p a t i b l e
computer w i t h an EGA o r VGA graphics c a r d and a t
l e a s t 640K o f memory. A hard d i s k i s recommended
but not required.
With s l i g h t a l t e r a t i o n , t h e
program can be used as a t e s t i n g instrument.
-
COOK, Mark S . and William M. FALLS, Department of Anatomy,
Michigan State University, East Laming, Michigan.
Efferent projections of neurons in the ventrolateral
magnocellular region of rat trigeminal nucleus
interpolaris.
Efferent projections of neurons in the ventrolateral
magnocellular region of rat trigeminal nucleus interpolaris
(vlVimc) were studied by use of anterograde axonal transport of Phaseolus vulgaris leucoagglutinin (PHA-L). Light
microscopic analyses revealed that PHA-L injections into
vlVimc resulted in heavy labeling of efferent parent axon5
and their terminal arbors in several areas along the
neuras:s. Areas receiving the densest projections included
the doxsomedial portion of ventral posteromedial thalamic
nucleus (VPM), central portion of caudal zona incerta (ZI),
medial portion of anterior pretectal area (APT), ventrolat(era1 superior colliculus (SC) , parvocellular red nucleus
(RNpc), dorsal accessory olive (DAO), paramedian lobule
(PML) of the cerebellum and cervical dorsal horn (CDH).
Projections to VPM, ZI, APT, SC and RNpc were contralateral, those to DAO and CDH were bilateral, and those to PML
were ipsilateral. Neurons in vlVimc also projected to the
ipsilateral principal sensory nucleus (Vp), ipsilateral
nucleus oralis (Vo), contralateral nucleus interpolaris
(Vi) and ipsilateral medullary dorsal horn (MDH) of the
sensory trigeminal complex. Differences in vlVimc
efferents ending in each target region were based on axonal
diameter, branching pattern of the terminal arbors, and
bouton frequency and size. Efferents terminating in VPM,
ZI, APT, SC and CDH were of more than one type, while no
more than one type was found in W p c , DAO, Vp, Vo, Vi, MDH
and PML. Efferents terminating in the granule cell layer
of PML had an unbranched terminal strand with one or two
large bulbous boutons. Areas which received sparse projections included paracentral, parafascicular and posterior
thalamic nuclei, caudal pontine and intermediate reticular
nuclei, spinal vestibular nuclei and the central gray.
Based on these results, vlVimc must play a major role in
dispersing orofacial tactile input to diverse areas
throughout the diencephalon, brainstem and CDH. (Supported
by N.I.H. Grant DEO6725).
COTANCHE', Douglas A., Department of Anatomy, Boston
University School of Medicine, Boston, Massachusetts.
enhanced convast DIC imaaino of the structure of the u&&d
iectorial membrane in the embrvo nic and adult chick cochla.
The tectorial membrane is an acellular extracellular matrix
in the scala media of the chick cochlea which attaches to the
tips of the hair cell stereocilia. It is a crucial element in the
mechanical-to-electrical transduction of sound into neural
impulses performed by the hair cells in the ear. Previous
studies have utilized scanning and transmission electron
microscopy to discern the ultrastructural organization of the
tectorial membrane. However, a major drawback to these
techniques is that the tectorial membrane is a highly hydrated
gel and preparation techniques for electron microscopy require
dehyration and critical point drying or embedding in plastic to
evaluate its substructure. It has been thought that these
procedures radically change the supramolecular structure of
the tectorial membrane, and thus our interpretations of its role
in transduction have been tempered by our lack of hard data
on the normal structure of the unfixed tectorial membrane. We
have developed a technique for studying the structure of the
tecorial membrane in an unfixed, hydrated state using videoenhanced contrast differential-interference-contrast(DIC) light
microscopy. These studies have demonstratedthat the normal,
unfixed structure of the tectorial membrane does not differ
significantly from the fixed, dehydrated state seen with the
scanning or transmission electron microscope. Moreover, it
has been possible to determine the relationship between the
components of the tectorial membrane and the tall and short
hair cells in the chick basilar papilla during embryonic
development of the cochlea and during regeneration of hair
CellS following acoustic trauma. (Supported by NIH Grant No.
DC00412)
+
COTTER, John R. and T i a n - L o n g DU*, D e p a r t m e n t o f
Anatomical Sciences, S t a t e U n i v e r s i t y o f New York a t
B u f f a l o , B u f f a l o , New York.
Does r e t i n a l dvstroDhy
-a l t e r t h e cvtochrome o x i d a s e h i s t o c h e m i s t r v o f t h e
brain?
I n h e r i t e d r e t i n a l dystrophy i s characterized not
only by d e s t r u c t i o n o f photoreceptors but by
p r o g r e s s i v e d e t e r i o r i z a t i o n o f t h e electroretinogram.
T h i s i s accompanied b y decay o f t o t a l c y t o c h r o m e
o x i d a s e l e v e l s ( H u r l e y and C o t t e r , J. C e l l B i o . ,
109:1989). A n a l y s i s o f a l l l a y e r s o f t h e r e t i n a shows
t h a t t h e r e d u c t i o n i n enzyme l e v e l i n v o l v e s several
c e l l populations i n c l u d i n g those o f t h e ganglion c e l l
l a y e r . I n t h i s s t u d y cytochrome oxidase a c t i v i t y o f
c e n t r a l v i s u a l s t r u c t u r e s was s t u d i e d f o r e f f e c t s
secondary t o t h o s e observed i n t h e r e t i n a . Normal
t pt) and d y s t r o p h i c r a t s (RCS - pt) were
(RCS-&
used.
I n normal animals t h e n e u r o p i l e s t a i n e d
i n t e n s e l y and c e l l bodies were n o t e a s i l y i d e n t i f i e d .
Regional d i f f e r e n c e s were noted. I n p a r t i c u l a r , t h e
e x t e r n a l d i v i s i o n o f t h e v e n t r a l l a t e r a l geneculate
n u c l e u s s t a i n e d more i n t e n s e l y t h a n t h e i n t e r n a l
d i v i s i o n and t h e dorsomedial and ventromedial aspect
o f t h e d o r s o l a t e r a l g e n e c u l a t e nucleus s t a i n e d more
ABSTRACTS-AAA 103RD MEETING
intensley than other p a r t s .
I n t h e pretectum t h e
n u c l e u s o f t h e o p t i c t r a c t and p r e t e c t a l o l i v a r y
n u c l e u s s t a i n e d i n t e n s e l y and i n t h e s u p e r i o r
c o l l i c u l u s , t h e stratum opticum, stained l e s s
i n t e n s e l y t h a n t h e more c e l l u l a r l a y e r s .
In
dystrophic r a t s ranging from 3 t o 9 months o f age, t h e
p a t t e r n o f s t a i n i n g was s i m i l a r t o t h a t seen i n 7 and
11 month o l d c o n t r o l s . These r e s u l t s i n d i c a t e t h a t
r e t i n a 1 d e g e n e r a t i o n has no o b s e r v a b l e e f f e c t , a t
l e a s t a t t h e l i g h t m i c r o s c o p i c l e v e l s , on t h e
cytochrome o x i d a s e h i s t o c h e m i s t r y o f s u b c o r t i c a l
v i s u a l centers.
Coughlin, Patrick, and Mark Hysell$Department of
Anatomy, Philadelphia College of Osteopathic
Medicine, Philadelphia, Pennsylvania. fhe axial
&&&&jn thre e dimensions with basic vertebral
23A
t h e deep p i n e a l g l a n d o v e r t h e p i n e a l recess of
t h e t h i r d v e n t r i c l e . Some of t h e n e r v e f i b e r s ent e r i n g t h e deep p i n e a l g l a n d showed t h e p r e s e n c e
of boutons e n p a s s a g e . A few f i b e r s w e r e d e t e c t e d
i n t h e p i n e a l s t a l k , i n t h e d i r e c t i o n of t h e sup e r f i c i a l p i n e a l gland.The i n t e r g e n i c u l a t e leaflet
of t h e l a t e r a l g e n i c u l a t e body i s d i r e c t l y connec
t e d t & t h e s u p r a c h i a s m a t i c n u c l e i . The n e u r a l p r o
j e c t i o n s h e r e r e p o r t e d from t h e i n t e r g e n i c u l a t e l 2
a f l e t t o t h e p i n e a l complex s u g g e s t t h e p o s s i b i l i
t y of a d i r e c t involvement of t h e p i n e a l complexi n t h e e n t r a i n m e n t of c i r c a d i a n rhythms. Although
t h e p i n e a l gland i s b e l i e v e d t o be key-regulated
by o r t h o s y m p a t h e t i c f i b e r s o r i g i n a t i n g i n t h e sup e r i o r c e r v i c a l g l a n g l i a , t h e p r e s e n t s t u d y supp o r t s former e v i d e n c e s i n d i c a t i n g a n a d d i t i o n a l i n
n e r v a t i o n o f t h e p i n e a l complex by n e r v e f i b e r s
r i g i n a t i n g i n thalamic s t r u c t u r e s .
mechsnics.
We are developing an interactive computer
tutorial for students of Gross Anatomy which
emphasizes the neuromusculoskeletal system. The
current module focuses on the axial skeleton. and
the muscles which act upon it. Using the Amiga
system with 3D modeling software, static three
dimensional renderings of the vertebral column
and occiput have been completed. In addition,
animation files have been created (using
animation software) of individual vertebrae,
groups of vertebrae, and selected intersegmental
motions. We are currently in the process of
adding the axial musculature into the animation
capability, which will allow for demonstration of
intersegmental motion as a direct result of the
contraction of individual muscles, or muscle
groups. A controller program will allow for
selection of the specific area of interest via
regional field buttons. Individual menu buttons
will control access to animation files and deeper
levels of the program, as well as pop-up text
windows which will describe the active animation.
The program is intended to more fully acquaint
the student of Anatomy (at any level) with aspects
of vertebral mechanics and may provide additional
insights for the clinician dealing with patients
experiencing musculoskeletal problems in this
area.
COZZI, Bruno, MIKKELSEN,* J e n s Damsgaard and
MOLLER*, Morten, I n s t i t u t e of Anatomy of D o m e s t i c
Animals, U n i v e r s i t y of Milan, I t a l y ; I n s t i t u t e of
Medical Anatomv. DeDartment B. U n i v e r s i t v of
CRAWFORD, Bruce J. and Stephen S. CAMPBELL,* Department
o f Anatomy, U n i v e r s i t y o f B r i t i s h Columbia, Vancouver,
B r i t i s h Columbia. A sfmole freeze s u b s t i t u t i o n techniaue
f o r marine i n v e r t e b r a t e embrvos.
A simple technique has been developed f o r f l a s h
f r e e z i n g and f r e e z e - s u b s t i t u t i n g small (0.5um) marine
embryos u s i n g inexpensive equipment.
Embryos o f t h e
s t a r f i s h , P i s a s t e r ochraceus, were r a p i d l y frozen by
t r a p p i n g them i n copper freeze f r a c t u r e EM g r i d s . The
g r i d s were subsequently plunged i n t o a cup of l i q u i d
propane k e p t a t t h e f r e e z i n g p o i n t (-19200
by
immersion i n a Dewar f l a s k o f l i q u i d n i t r o g e n (LN) w i t h
constant s t i r r i n g , f o l l o w e d by storage i n LN. They were
then placed i n sealed v i a l s c o n t a i n i n g ethanol a t
-90%.
The v i a l s were placed i n a wide mouth Thermos
f l a s k c o n t a i n i n g a s l u r r y o f acetone and d r y i c e , cooled
t o -95oC by t h e a d d i t i o n o f LN and placed i n a -7OoC
r e f r i g e r a t o r . The temperature was monitored morning and
evening and LN was added as necessary t o keep t h e
temperature below -8OoC.
Following s u b s t i t u t i o n , which
was marked b y t h e embryos f a l l i n g o u t o f t h e g r i d ( c i r c a
5 days): t h e Thermos was warmed t o -2OoC over 2h. A t
t h i s p o i n t t h e embryos could be t r a n s f e r r e d t o 1% OsO4
i n ethanol,
b u t t h i s was n o t necessary f o r good
preservation.
Following t h i s , t h e embryos were brought
t o room temperature over 4h and embedded i n e i t h e r Epon,
Lowcryl o r LR w h i t e r e s i n s and sectioned f o r TEM. The
technique gave good c e l l u l a r p r e s e r v a t i o n of embryos
frozen i n sea water alone b u t ECM p r e s e r v a t i o n was poor.
E x c e l l e n t p r e s e r v a t i o n o f both c e l l s and ECM could be
achieved
b y adding
15% propylene g l y c o l
as
a
cryoprotectant.
The technique should be useful f o r
s t u d i e s o f both ECM morphology and immunocytochemistry i n
general as i t i s simple, r e q u i r e s l i t t l e equipment and
gives e x c e l l e n t p r e s e r v a t i o n o f both c e l l s and ECM
w i t h o u t t h e use o f aldehydes o r osmium t e t r o x i d e .
Supported b y g r a n t #A0032 from NSERC.
nection's between t h e i n t e r g e n i c u l a t e l e a f l e t of
t h e l a t e r a l g e n i c u l a t e body and t h e p i n e a l c o m p h
i n two p h o t o s e n s i t i v e Rodents, t h e g o l d e n hamster
( M e s o c r i c e t u s a u r a t u s ) and t h e Mongolian g e r b i l
(Meriones u n g u i c u l a t u s ) . By u s e of t h e anterogmde
tracer P h a s e o l u s v u l g a r i s (PHA-L) i t h a s been pog
s i b l e t o detect t h e e x i s t e n c e of a d i r e c t p r o j e c t i o n from t h e i n t e r g e n i c u l a t e l e a f l e t t o t h e deep
p i n e a l g l a n d . Nerve f i b e r s o r i g i n a t i n g from t h e
i n j e c t i o n s i t e were d i r e c t e d towards t h e m i d s a g i t
t a l p l a n e and t h e p r e t e c t a l area, and r e a c h e d t h e
h a b e n u l a r r e g i o n a n d t h e p o s t e r i o r commissure.
From t h i s s i t e , small b u n d l e s of f i b e r s e n t e r e d
CROCKE'IT,* David P., Jun ZHANG* and M. David EGGER,
Department of Anatomy, UMDNJ-Robert Wood Johnson Medical
School, Piscataway, New Jersey. A modified cytochrome oxidase
stainine urocedure reveals "uatches" of intense metabolic activitv in the
cuneate nucleus of the adult rat.
We examined the pattern of cytochrome oxidase (CO) activity in the
cuneate nucleus (CN) of 16 adult Long-Evans rats of both sexes, using
an enhanced staining technique. The chromogen was intensified with
heavy metals, cobalt and nickel, resulting in higher contrast and
sensitivity between areas of intense and weak CO activity. Within a
discrete region caudal to the obex (-0.7 mm), there were patches of
intense CO-activity. Caudal and rostra1 to this region of CO patches,
the CN was uniformly stained. In contrast, the gracile nucleus showed
little detectable differentiation in CO staining. Comparisons between
24A
ABSTRACTS-AAA 103RD MEETING
the CO staining and our mapping of primary afferents to the CN (Maslany et al., Brain Res., in press) suggest that the C O patches are
associated with dense concentrations of cutaneous afferent input,
particularly from the digits. Desfferentation (amputation of the digits
or of the entire forepnw of hypothermicatly anesthetized pups) on
postnatal day 0 disrupted the C O staining pattern in the ipsilateral C N
in 16 rats examined as adults. Complete removal of the forepaw led to
a complete or nearly complete suppression of the CO patches. In
summ:wy, (I) functionally related maps were observed in the CN when
stained with a metabolic marker and (2) these maps were significantly
altered following perinatal deafferentation. The observed changes in
the CO maps following deafferentation appear not to be due primarily
to cell death in CN. In 2 adult rats in which the forepaw had heen
amputated perinatally, when WGA-HRP was injected bilaterally into
the ventrotxisal complex of the thalamus, the density and distribution
of retrogrotlely stained neurons in CN appeared qualitatively equivalent
on Imth the intact and deafferented side.
CUMMINGS, S . L . , G.A. BISHOP, and J.S. K I N G , Dept.
of Anatomy and Neuroscience Program, The Ohio
State University, Columbus, Ohio. Corticotropin
releasins factor (CRF) and enkephalin ( E N K ) :
Colocalization and effects on Purkinie cell
firinq rate.
The distribution of CRF and E N K containing
climbing and mossy fibers is coextensive within
several regions of the opossum's cerebellum
(Cummings & King, Synapse, In Press). Within the
vermis, climbing fibers are either immunoreactive
for CRF alone or demonstrate colocalization of
CRF and E N K . Mossy fibers within the vermis are
immunoreactive for CRF alone, for E N K alone, or
demonstrate colocalization of CRF and E N K .
Iontophoretic application of CRF and E N X reveals
that these peptides have opposite effects on
Purkinje cell activity within the vermis. CRF
increases the firing rate of Purkinje cells. In
contrast, E N K decreases Purkinje cell activity.
When the peptides are applied simultaneously, CRF
is capable of partially overcoming the E N K
induced suppression. We have also analyzed the
interactions between the peptides and the amino
acids glutamate and aspartate which are presumed
to be excitatory neurotransmitters in mossy
fibers and climbing fibers, respectively. CRF
and ENK have opposite effects on both glutamate
and aspartate induced activity. CRF potentiates
and E N K suppresses the excitatory effects of both
amino acids. The present findings suggest that
cerebellar afferents likely contain one or more
peptides in addition to the amino acids glutamate
and aspartate.
It is not known, at present,
under what conditions the interactive substances
are released from the afferent terminals in vivo
or if they are co-released. (Supported by N S 08798)
Curtis,* M. and R.C.Borke, Dept. of Anatomy, Uniformed
Services University of the Health Sciences, Bethesda, Maryland.
lrnrnunoreactive chanqes in the hvpoalossal nucleus after nerve
iniury.
lmrnunocytochemical methods were used to examine the
interrelationship of two endogenous substances in normal and
injured motoneurons: choline acetyltransferase (ChAT). an
enzyme involved in the synthesis of acetylcholine, the
neurotransmitter of hypoglossal motoneurons and calcitonin
gene-related peptide (CGRP), a peptide coexistent with
acetylcholine. Rats were subjected to three types of
hypoglossal nerve injuries (resection, transection and crush)
and sacrificed at 1,3,7,20 and 50 days postoperative (dpo).
Reinnervation was documented by horseradish peroxidase
injections into the tongue musculature. Animals were fixed by
intracardiac perfusion and vibratome sections were processed
for single and double label immunocytochemistry. Maximal
reduction of ChAT immunostaining occurred in neuronal
somata and neuropil on the operated side at 7 dpo with each
type of nerve injury. At 20 dpo the lowest level of ChAT
staining was maintained with nerve resection, whereas ChAT
staining was moderate after nerve transection and returned to
control levels in nerve crush. CGRP immunostaining increased
in the somata and neuropil on the operated side, with the
maximal response at 3-7 dpo. At 20 dpo, CGRP staining
returned to control levels in nerve resection, yet remained
increased after nerve transection and crush. Return to control
levels of ChAT staining was related to axonal regeneration to
tongue musculature; staining levels remained low without
reinnetvation. Conversely, high CGRP staining levels were
maintained in animals where reinnervation occurred. ChAT and
CGRP changes appear to be indicators in a diametric fashion
of the neuron's regenerative ability.
DANIEIS, Eugena and Jagjit GILL,* Department of Anatany,
hf&ill university, Montreal, Canada.
'cal
characteristics of the cellular micrwMironnent in Ioou9e
fetal liver.
'
ybchmioal studies
Light and ultraswere parfonned to identify the cellular populations which
canprise the active liver hepopoietic.t-im
Cytospot preparations from BAIg/c fetal livers (Bays ii18) were incubated w i t h specific antibodies (alphafetoprotein, cytokeratin, laminin, fihronactrn8 -18
F4/80) to quantitate the specific raicroemrironment a l cell
populations during hepopoiesis. In stanQrdr'zed cytowts,
distinctive cell associations cmld be identified
throughout hepopoiesis. The large central cell of type 1
cell association was AFF+, cytokeratint, m p h a p q t , i c
and cantained all stages of -id
cells (pemxidase
positive and blast cells) ermbedded in its peripheral cytoplasm. The large central cell of type 2 cell association
Was l&K!-l+t
F4/8Dc1
and Wntained
Phagocytosed erythmid cells apart fran the peripheral rosette
of maturing eryulroid cells. The presence of these
physically-presanred cell associations was canfinaed by
ultrastrudural studies on enriched c a n c e n t r a t i ~ of
physically-separated liver a g p y i t a s .
charactanStiC
lipid inclusions, glycyen partacles, positive AFP
reactions w e r e localized rn tha type 1 cell associations
sugyesting that these were hepatocyte cell associations.
This type predani~ted in earlier developaent (days iiU). The hepatocytes caqletely -fed
primitive
hanopoietic cells, exhibited &ara&en stics of active
synthesis (atnm&nt W, Golgi, grayi~es?, and also
contained ferritin particles suggesting i t s lmrolvement in
hwopoiesis. Fibroneztin was identified in the E m of
such cell associations. Macrophage cell associations,
Which were lIDm tlater in devel-t
(dam 14181, -S
Phasocytosea eqthmid cells and
contained distinct w d a s e activity in the ER and
nuclear membrane. (xu observations raise the question
wfiether the hepatocyte and maczpphage cell associations
represent the sites of discrete functional interactions in
the fetal hepopoietic microemrirorrpent. (supported by the
Medical Rasearch Council of Canada).
twp
ABSTRACTS-AAA 103RD MEETING
S
DEBOOM.' Todd, Kristy KULTAS-ILINSKY and lgor ILINSKY,
Department of Anatomy, University of Iowa, Iowa City, Iowa.
The ultrastructure of svnaotic reoraanization in the feline motor
thalamus followina lesions in the basal aanalia.
The objective of this study was to determine what kind synaptic
remodeling occurs in the cat thalamus after its deafferentation
from inhibitory pathways originating in the basal ganglia.
Unilateral injections of kainic acid were made in the substantia
nigra pars reticularis (SNr) and entopeduncular nucleus (EPN).
Quantitative morphometric analysis of the ultrastructure and
GAD immunocytochemistry were performed in the ipsilateral
ventral anterior thalamic nucleus (VA) where these afferent
systems overlap. At long-term (1 year) postlesion survival times
significant decrease in the density of synaptic boutons on
primary and secondary projection neuron dendrites in the VA
was found. In contrast, the apposition of these dendrites by
vesicle-containing dendrites of local circuit neurons (LCN) was
increased significantly as was the number of dendrodendritic
synapses. There was also a moderate increase in glial
apposition. These changes were accompanied by remarkable
alteration of the patterns of GAD irnrnunoreactivity. The results
suggest that GABAergic LCN in the adult thalamus undergo
plasticity type changes in response to deafferentation from
extrinsic GABAergic afferents with dendrodendritic synapses
occupying deafferented sites.
Supported by NS R 0 1 19280.
Dechow, Paul C., Department of Anatomy, Baylor College of
Dentistry, Dallas, Texas. Contractile Droperties and threedimensional structure of rat temDoralis muscles.
Masticatory muscle contractile properties measured in vitro
or in situ underestimate maximum muscle forces. This study
used a biomechanical model, occlusal force measurements, and
three dimensional reconstruction techniques to study the
structure and contractile properties of rat temporalis muscles.
Evoked occlusal force readings were taken at the incisors in 30
adult female Sprague Dawley rats, at 5 gapes from molar
occlusion to full gape. Animals then had their temporalis
muscles surgically removed and the measurements were
repeated. Thirty control animals also had measurements taken,
followed by mock surgery, and a second group of
measurements. Differences between pre- and postoperative
readings were coupled with a biomechanical model, and data
taken from radiographs of 20 rats to determine estimates of
maximum temporalis muscle force. Also, 3 micron corona1 serial
sections of the heads of 5 rats were taken at 450 micron
intervals.
These sections were used to construct three
dimensional images of the internal structure of the temporalis
muscles, using Boulder HVEM 3-D ReconstIuction Software.
Radiographic data demonstrated that temporalis muscles of rats
lengthened on average from 15 to 20 mm throughout the range
of gapes.
The biomechanical analysis showed that the
mechanical advantage of the temporalis muscles for producing
incisal forces did not vary significantly throughout this range of
gapes. The orientation of the muscles did vary slightly,
suggesting that positions near occlusion were more optimal for
producing incisal forces. The occlusal force measurements
indicated that the temporalis muscles produced maxima! incisal
forces at an interincisal gape of 6 mm. On average, this force
was 15.4 newtons (SD=2.5 n). The biomechanical model
estimated actual peak unilateral temporalis muscle force as 65.3
25 A
n (SD = 10.6). Three dimensional reconstruction showed two
interconnected tendons in the rat temporalis muscles. One
provided attachment of the lateral portion of the muscle to the
zygomatic arch, while the second connected the central portion
of the muscle to the internal aspect of the coronoid process.
Further study of fiber length and direction will contribute to a
comprehensive model of temporalis muscle contraction.
Supported by NIH Grant DE07761.
DBEON,* Jean M., Daniel L. FEEBACK, Richard W.
LEECH,* and Roger A. BRUMBACK,* Departments of
Anatomical Sciences and Pathology, University of OklaL~oma
Health Sciences Center and Veterans Affairs Medical Center,
Oklahoma City, Oklahoma.
A systemic disorder of
mitochondria produced by experimental administration of an
inhibitor of creatine entry into cells.
Disorders of mitochondrial metabolism have been
increasingly recognized as causes of human disease with
mitochondrial abnormalities recognizable in muscle, kidney,
liver, and brain (Neuml Clin 7:123, 1989). F'reviously,
experimental models have been developed in which
morphologic changes are apparent in muscle but not systemic
mitachondria (Locklear I , Feeback DL, Pitha JV, Brumback
RA: Anat Rec 223(4):69, 1989). W
e have evaluated systemic
organs in rats given daily intraperitoneal 0-guanidinopmpionic acid (B-GPA, a cmatine analogue that displaces
creatine and inhibits its uptake by cells) which has been
reported to produce mitochondrial abnormalities in rat soleus
but not EDL muscle fibers (Br J Exp Path 69:639, 1988).
Male W i s t a r rats were injected each day for up to 8 weeks
with either E-GPA (600 Ilg/kg) OP saline. At weekly
intervals, randomly selected rats either were perfusion fixed
for EM studies or had tissues quick frozen for biochemical
analysis. Compared to control rats, EM sections from liver
showed glycogen accumulation and increased numbers of
enlarged, pleomorphic mitochondria with electron-dense
matrices and indistinct cristae. Biochemiail studies of liver
reveal& progressive increase over the 8 week period in
tissue levels of 0-GPA and phosphorylated 0-GPA while
levels of creatine and phoephocreatine declined. Similar
changes were noted in kidney, but no abnormalities have yet
been identified in brain.
[Supported by the PresbyterianlHamis Foundation]
DESMOND, Mary E., Melinda S. NegBernard G. Martirand William M.
Fleischman* Departments of Biology and Mathematics, Villanova
University, Villanova, Pennsylvania. A RaDid Reliable Calculation of
Brain FxDansion in I ivina Chick Embrvos to Reflect Fluid TransDa
the NeuroeDithelium .
Early brain expansion in the chick embryo appears to occur as a
result of positive pressure created by an accumulation of fluid in the
brain vesicles subsequent to closure of the anterior neuropore and
occlusion of the spinal cord. In order to examine the physiological
mechanismls by which fluid crosses the neuroepithelium, there needs
to be a reliable way to determine brain volume from photographs of
living embryos. To derive such a method, it was assumed that changes
in total brain volume driven by fluid hydraulics would be similar to
changes in volume of the mesencephalon, i.e.. the primary brain vesicle
having the simplest geometric shape.
Preliminary calculations of
midbrain volumes from photographs of 17 living chick embryos show
that volume can be most reliably represented by the mathematical
formula for an oblate spheroid, 4/3 II abc, where a=c>b. Chick eggs
were incubated at 380 C in shelless culture to stage 17 or 23, excised
from the yolk and their heads photographed in the lateral, dorsal,
ventral and frontal planes. From identically magnified photographs,
median anteroposterior (a) dorsoventral (b) and bilateral (c) axes
26A
ABSTRACTSAAA 103RD MEETING
were measured and the volumes calculated assuming the midbrain was a
sphere or oblate spheroid. Volume comparisons confirmed that a # b.
Although upper and lower hemispheres were non-congruent, volume
differences based on the midbrain having congruent hemispheres vs
non-congruent hemispheres were negligible. Differences in volumes
based on a& compared to those where a # c were also negligible. Thus,
the lateral view delineating a and b can be singularly used to determine
midbrain volume which allows calculation of changes in midbrain
volume for the same embryo over time. Using this method. the mean
volume plus s.d. of the midbrain for 24 paired embryos at to (HH 20)
was 0.167 mm3 + 0.061 and 24 h r s later (HH 24) was 0.318 mm3 +
0.74, representing a 50% increase in volume. The proportional change
in volume of the mesencephalon from stage 16 to 20 (24 hr period) for
15 embryos treated with 200 pM ouabain was significantly less than
the proportional change for 15 control embryos at the same stages over
the same time period.
DESMOND, Nan I, Michelle S. HEYDENREICH* and
William B LEVY,%e artment of Neurosurgery, University of
Virginia, Charlottesde, Virginia. Ouantitative characterization of
the hippocampal CA1 pyramidal cell dendritic field in stratum
moleculare.
As part of our ongoing studies to understand hi ocampal
function, we are quantifying the apical dendritic fiepof hippocampal
CA1 pyramidal cells in order to characterize the average CA1
pyrarmdal cell. Of particular interest here is the distal a ical
dendritic field located in stratum moleculare (s. mol.), t f e
termination zone of entorhinal cortical afferents to the CA1 region.
Linearized hippocampi dissected from adult, male S rape-Dawley
rats were impregnated usin a ra id Golgi method. !he hippocampi
were sectioned at a nominaf thiclness of 200 pm in one of two
orthogonal planes, either perpendicular to or parallel to the
longitudinal axis of the hippocampus. Well-impregnated CA1
pyramidal cell apical dendritic fields were drawn using a camera
ucida and then quantified using [email protected] mol., the distal
CA1 dendrites are either thin (ca. 0.8-1.5 pm) and sparsely spined, or
thicker and more dense1 spined. These dendrites may cross the
obliterated hippocampaffissure to intermingle with the distal
dendrites of the dentate granule cells. Often, though not always, the
main apical dendrite bifurcates in the vicinity of the s. radiatum (s.
rad.)/s. mol. interface, a region suspected to be a spike initiation
zone. Re ardless of the location of this bifurcation, however, the
dendritic tranching patterns distal to the bifurcation are quite
similar. Those dendritic processes entering s. mol. (generally the
apical dendrite or its sibling branches) then branch multiply. The
avera e CA1 pyramidal cell has 4th order dendritic branches in s.
mol. tange: 1-8) which implies the possibility of 16 terminal
dendritic segments. Total dendritic length ins. mol., uncorrected for
cut segments or foreshortening and thus underestimatin the actual
length, averages 1215 f 97 pm (mean sem; N = 14). Tfe distal
dendritic field in s. mol. ap ears cone-shaped and begins to fan out
Dendritic field spread there
near the s.rad./s. mol. inter!ace.
averages 68 p m with a range of 16-115pm in the transverse
(CA3-subicular) plane. There is a similar spread in the longitudinal
plane. In distal s. mol., dendritic field spread averages 278 pm in the
transverse plane and 125 pm in the longitudinal lane. Supported by
NIH grant NS26645 to NLD and NS15488 to .,I&
i ~ b L (U ~ P I C JJosrph
.
H . 1 ,
vlcholas A. Lanson, J r . 2 ,*
Harh k. bteinhelper’ f Loren J. Eieldd ,*and
billiam C. Claycomb* f Uepartments of Anatomv’ and
Biochemistry and Molecular B i o l o g y z , Louisiana
State bniversity Xedical Center. hew Orleans.
Louisiana, and Cold Spring Harhor Laboratori.3 ,
C o l d Spring Harbor, hew ’York. Ultrastructural
analysis o t cultured cardiac muscle Cell5 deri\ea
t rom atrial tumors o t transgenic mi ce.
Prolireratinp cardiac muscle cell+ exhibiting
ultrastricctur~l ieatures typical of adu1 t atrial
muscle cells In v i v o were established in culture
from cells enzymatically isolated from transg e n i c mouse atrial tumors (Field, L., Science,
2 3 9 : 1 0 2 9 , 1988). These derived cardiocytes
proliferate in culture and differeniate into
beating muscle cells which exhibit all of the
characterist1.c~typical of adult atrial
cardiocytes in vivo. Perinuclear regions contain
well developed golgi, smooth endoplasmio
reticulurn, and areas rich in atrial granules. The
nucleus is euchromatic, indicative of a highly
synthetic state, and contains one or two nucleoli
In peripheral regions of cardiocytes active
synthesis of myosin is evident as shown by the
presence of numerous arrays of linearly arranged
polysomes and newly synthesized thick (myosin)
filaments. Futhermore, these filaments are
apparently being inserted into nascent myofibrils
adjacent to the plasma membrane. In many cells
well developed myofbrils and transverse tubules
are observed. Cardiocytes, linked by intercalated
discs and gap junctions, are arranged into
confluent synchronously beating sheets. Under the
phase-contrast light microscope cells can be
observed undergoing cytokinesis. Cultures are
extremely pure and very few non-muscle cells are
observed.
DELLMA”, H.-Dieter,Jacqueline GABRION*, Alain PRIVAT*
and Marie-Jeanne DRIAN*, Department of Veterinary
Anatomy, Iowa State University, Ames, Iowa; Laboratoire
de Neurobiologie Endocrinologique, URA CNRS 1197, USTL.
Montpellier; Laboratoire de Neurobiologie du
Developpement, INSERH U249, CNRS UPR41, Montpellier.
Fine structural chanaes in the neural lobe of the rat
auouhvsis in oreanotwic culture.
In an attempt to obtain a purified population of
pituicytes, pieces of neural lobes (since efforts to
establish primary dissociated cell cultures of pituicytes
were unsuccessful) of young (15 to 35 days) or adult rats
were dissected free of the pars intermedia and incubated
on polylysine-coated plastic or glass surfaces in a
variety of culture media that were changed every 4 days.
They were fixed at regular intervals between 7 and 50
days of incubation. Many neurosecretory axons survived
for up to 21 days without any apparent signs of
degeneration. Most axons, however, degenerated and were
progressively phagocytized and subsequently eliminated by
pituicytes. Lysed neurosecretory axons that were not
eliminated, persisted as dense bodies or paracrystalline
inclusions. After 30 days of culture, pituicytes
predominated the explants. They formed clusters, with
fibroblasts, strands of connective tissue and occasional
endothelial cells scattered among them. Pituicytes
underwent morphologic changes, such as medium-dependent
decrease or increase of lipid inclusions, Golgi
activation, process extension and interdigitation,
formation of gap junctions and bundles of intermediate
filaments. At the explant surface in contact with the
culture medium, pituicytes differentiated into an
epithelia1 layer of ciliated and microvilli-bearing cells
linked by junctional complexes. Occasionally,
fibroblasts without special surface differentiations made
up this layer. Long-term neural lobe explants are a
relatively pure source of viable pituicytes that is
expected to be useful for further studies on the
functional significance of these cells.
DZKLICH, R.V.W., J.T. KELLER, T.A. HORES*, and M . J .
FRITTS*. Depts. of Emerg. Med., Anat./Cell Biol., and
Neurosurg., U. of Cincinnati, and J . N . Gamble Inst. Med.
Res.. Cincinnati, Ohio. Linear arravs of homoeeneoug
mast cells in the dura mater (uachvmeninx) of the rat,
27A
ABSTRACTS-AAA 103RD MEETING
Mast cells of the dura mater may contribute to the
pathogenesis of headache and inflammatory responses as
well as to normal regulatory functions governing blood
flow. Our previous study identified a large population
of mast cells in the dura of the rat. Since there is
more than one type of mast cell, the goal of this study
was to investigate the heterogeneity and distribution of
mast cslls in the dura of the rat. Neurolipomastocytes,
a purported type of mast cell in the pia and arachnoid
(leptomeninges), were not observed in the dura
(pachymeninx).
Hypothetically there are two types of
dural mast cells based on shape. In this study, mast
cell profiles were round, oval, or indented, and each
shape could be attributed to the presence or absence of
collagen and other neighboring tissue components. These
data suggest that there is no heterogeneity of dural mast
cells based on shape.
Ultrastructurally mast cells
appeared homogeneous. Few organelles and numerous large
dense granules indicate a resting cell that is ready to
degranulate. This is consistent with a guardian function
for these cells. Supportive of that hypothesis is the
distribution of these cells in the layers of the dura.
Cells in the outer layer were oriented parallel to the
meningeal neurovascular triad and those in the inner
layer were parallel to branches of the trigeminal nerve
that course tangentially to that triad. This suggests
that embryologically there are two layers of mesenchyme
that contribute to the formation of the dura. The
orientation of these layers at an angle to one another
would strengthen the dura and enhance its barrier
function. The majority of mast cells in each layer were
aligned in a plane parallel and adjacent to the interface
between the two layers.
Most of the cells were
distributed evenly and were not apposed to vessels or
nerves. This linear array of cells near the dural layer
interface and their resting appearance support a role for
the dural mast cell in combating pathologies that could
affect the underlying brain rather than in regulating
normal physiological functions. Supported in part by NIH
Grants NS25635 (RVWD) and NS22969 ( J T K ) .
important in cell-matrix interactions, and that corneal
and tendon fibroblasts retain their tissue phenotype,
namely their ability to orient themselves and the matrix
which they deposit, when grown in this gel system.
S
DOERR*, T.A., ROSOLIA*, D.L., GEE*, M.H., and ALBERTINE,
K.H. Departments of Physiology and Medicine, Jefferson Medical
College, Philadelphia, Pennsylvania. Prostaelandin E1 IPGEI) infusion
asswiated with decreased attachment of neutrophils (PMNs) to air
emboli in vivo durine complement-inducedacute lung iniury.
An hypothesized effect of PGEl is prevention of PMN attachment to
pulmonary microvascular endothelial cells during complement-induced
acute lung injury (Circ Res 61:420, 1987). Subsequently we found,
using quantitative morphological methods, that PGEl did not interfere
with the ability of PMNs to attach to a foreign surface (i.e.,air bubble)
in vivo (FASEB L 2:A724,1988). That negative result may be attributed
to an inadequate degree of complement activation by the air emboli, as
compared to direct infusion of complement anaphylatoxins. In the
present study we combined the use of zymosan-activatedplasma (ZAP;
to generate complement anaphylatoxins) and air emboli (AE; to be
intravascular targets for PMN attachment) to determine whether PGEl
would prevent complement-induced attachment of PMNs to air bubbles
in vivo. Eight anesthetized sheep were divided into two groups (n=4
each): PGElEAP + AE and Vehicle (saline)/ZAP + AE. PGEl was
continuously infused for 1.5 hours [30 ng/(kg x min), iv]. During the
last hour, ZAP + AE were co-infused (1.91 ml/min, iv). The lungs were
fixed by tracheal insufflation and PMN linear density was determined by
digitization (Biquant System IV)of 10 AE/sheep (1-2 AE/tissue section;
5-10 tissue sections/sheep). The table summarizes the data (normalized
1
1
Condition (n)
No. of bubbles
PMN Linear Density
(No./lmm of bubble uerimeter)
40
8 k 4s
PGElLZAP + AE (4)
VehicleEAP + AE (4)
40
20+ 1
SScatisticalIydifferent from the vehicle-treatedgroup by unpaired t-test, p0.01.
0
DOANE, Kathleen J. and David E. BIRK, Department of
Pathology, Robert Wood Johnson Medical School,
Piscataway, New Jersey. Cell orientation and matrix
deDosition in vitro: A comparison between corneal and
teridon fibroblasts.
The morphology of connective tissues is determined
both by the arrangement of cells and the matrix which
they deposit. In the present study, we compared cell
orientation and matrix deposition in vitro by fibroblasts
from two connective tissues with different arrangements
of cells and matrix: tendon, a uniaxial connective
tissue, and corneal stroma, in which the cells and matrix
are orthogonally arranged. Fibroblasts were grown in a
three-dimensional bovine type I collagen gel matrix,
which approximates the in vivo environment of the cells
better than conventional planar substrata. Cornea1
fibroblasts begin to orient perpendicular to one another
at Id in vitro. This orientation becomes more pronounced
and at 10d in vitro the cells have often formed dense
sheets at right angles to each other. Tendon fibroblasts
begin to align after 3d in vitro, with dense sheets of
cells oriented parallel observed after 10d. Type VI
collagen is a non-fibril-forming collagen found in both
tendon and cornea which as been postulated to function in
cell-matrix interactions. To determine whether type VI
collagen is also important in cell orientation, we
localized type VI collagen within these gels using
immunofluorescence techniques. These experiments
demonstrated that the fibroblasts also deposit a matrix
similar to that seen in vivo, although tissue specific
cell orientation is observed prior to any appreciable
amount of type VI deposition. Attachment assays
indicated that both corneal and tendon fibroblasts
attached to pepsin-solubilized type VI collagen, although
corneal fibroblasts apparently spread more than tendon
fibroblasts. We conclude that type VI collagen may be
There was a 40% reduction in the number of PMNs at the AE:blocd
interface in the PGE1-treated sheep as compared to the matched vehicletreated (control) sheep. Our results indicate that PGEl interferes with the
ability of PMNs to attach to a surface in vivo when complement
anaphylatoxins are also present in the pulmonary circulation. We
suggest that the protective effect of PGEl in models of complementinduced acute lung injury may be the consequence of diminished
attachment of activated PMNs to endothelial cells. [Supported by NIH
grants HL38075 and HL36237, and the Center for Critical Care
Research]
DONCARLOS, L.L., K. MALIK* and J . I . MORRELL. Inst
Animal Behav, Rutgers Univ, Newark, New Jersey. Steroid
hormones regulate estrogen receptor-inmunoreactivity
in a site-specific manner in the forebrain of the female
guinea pig.
We have investigated the effect of ovarian steroid
hormones on estrogen receptors (ER) in the brain to
determine where, and to what degree, cellular concentrations of steroid receptors change in response to
hormone exposure. Adult female guinea pigs were ovariectomized for one week, then treated with estradiol
benzoate (EB: 10 ug; s.c.) followed 40 h later by
progesterone (0.5 mg). or EB followed by the oil vehicle,
or vehicle at both time points (5 animalsltreatment
group, N=15).
Animals were sacrificed by perfusion
fixation 46h after the first treatment and the brains
processed for immunocytochemistry. ER was detected using
monoclonal antibody H222 (gift from Abbott Lab.) and the
Vectastain-Elite kit with nickel-intensified DAB. To
quantify the relative amounts of ER-immunoreactivity
(IR) within cells, micro-densitometric readings of
individual cells (50 cells per cell group per animal)
28A
ABSTRACTS-AAA 103RD MEETING
in three cell groups (one preoptic, one hypothalamic, and
one limbic) were made using a Bioquant system. Estrogen
decreased the optical density of ER-IR in the ventrolateral hypothalamus (VL) by 19.4%, and in the bed nuc.
stria terminalis (BNST) by 23%, but had no effect on
ER-IR in the rostra1 preoptic area (POA). Estrogen plus
progesterone further decreased ER-IR in the VL and BNST
(by 12% and 22% respectively, compared to estrogen
only), but again, had no effect in the POA. The relative
amount of ER-IR in control animals was lower in the BNST
compared with the POA and VL. These data suggest that
alterations in estrogen receptors in response to ovarian
steroids may be site-specific and depend, in part, on
non-steroidal influences. Supported by HD22983 to
J.I.M. and NS07997 to L.L.D.C.
DORNFEST, Burton Saul, David Marvin LAPIN,* Maurice
Ernest BUSH* and Brian Arthur NAUGHTON, Departments of
Anatomy and Cell Biology and Medicine, State University
of New York Health Science Center at Brooklyn, Brooklyn,
New York; Department of Biology, Touro College, and
DepartmentofMedical Laboratory Science, Hunter College
School of Health Sciences, New York, New York.
Phenvlhvdrazine induces blastoaenesis and activation of
lvmuhocvtes in the Lone Evans rat.
Long Evans rats administered a single injection of
PHZ (4mg/100gm body wgt) developed anemia and a
lymphocytosis which were maximal 2 to 4 and 4 to 6 days
later, respectively. Blood cell values returned to
normal by the 11th post-injection day. To assess
proliferative responses of peripheral blood mononuclear
cells (PBMC) to PHZ, PBMC of untreated rats isolated by
Ficoll-Hypaquewere incubated with PHZ. For comparison,
PBMC of untreated rats and rats administered PHZ were
incubated
with
pokeweed
mitogen
(PWM)
or
phytohemagglutinin (PHA). Incubation of PHZ with PBMC
of untreated rats significantly increased blastogenesis
in 5 day cultures of PBMC in RPMI-1640 media in
microtiter plates( 1x106 ce~s/0.2 ml per well) as
indicated by [3H] thymidine incorporation.PBMC obtained
from rats 4 to 5 days after PHZ-injection as compared
to PBMC of untreated rats when incubated with PWM or
PHA, showed significant increases in blastogenesis.
Analysis by flow cytometry of PBMC isolated from PHZtreated rats and PBMC of untreated rats cultured with
PHZ showed a decreased T to B cell ratio. Transmission
EM studies revealed an increase in blastogenic lymphoid
cells in the blood, lymph nodes and spleens of PHZtreated rats and in PBMC incubated with PHZ. Serum of
PHZ-treated rats contained elevated immunoglobulin
titers as measured by radialimmunodiffusion which
correlated well with the degree of anemia. Dexamethasone
(3 injections, 0.6 mg /100 gm body wgt) repressed the
humoral immune response, lymphocytosis and anemia
induced by PHZ. The findings indicate that immune
activation is associated with the generation of PHZinduced anemia.
DOUGLAS', G.C. AND B.F. KING, Department of Human
Anatomy, School of Medicine, University of
California, Davis, California. Isolation of
cytotroahoblast cells from rhesus monkev alacental
vlere cytotrophoblast based on positive cytokeratin
staining. In an effort to improve the purity of the
cells, the continuous Percoll density gradient was
fractionated, whereupon it was confirmed that the
monkey trophoblast cells sedimented predominantly
between densities of 1.040 and 1.053 g / m l . This is
in contrast to human cytotrophoblast cells which
sediment between 1.048 and 1.063 g/ml. By
generating the gradient from a 34.5% Percoll
solution instead of the original 3 8 % a cell
population containing about 95% cytokeratinpositive cells was obtained. The yield of cells
ranged from 0.2 - 1.0 X 106 cells per g tissue.
When placed in culture the cells adhered and by 2 4
h had aggregated to form colonies. Staining with
anti-desmoplakin and
anti-nuclear antibodies
indicated most cells were mononucleate at this
time. By 5 days, however, the staining pattern had
changed and colonies consisted mostly of large
multinucleated
masses
resembling
syncytiotrophoblast. Trophoblast cells have been
successfully maintained for 12 days. These studies
demonstrate trophoblast cells from monkey placental
villi can be isolated with a high degree of purity
and undergo morphological differentiation in vitro.
Supported by NIH grants HD 11658 and RR00169.
Drake,* Christopher J., Davis,* Lynn A. and Charles D. Little,
Department of Anatomy and Cell Biology, University of Virginia
Charlottesville, Virginia. lntearin mediated cell:ECM
interactions are essential for vascular morDhoaenesis.
lntegrins are cell surface receptors for extracellular
matrix components. We designed experiments to test the role
of integrins during morphogenesis, specifically the formation of
the dorsal aorta. Whole quail embryos were cultured using a
recently developed method in which embryos were secured to
paper rings via their vitelline membranes. The embryo/ringassemblies were placed ventral side up on an agar bed.
Stage 8 embryos were injected between the splanchnic
mesoderm and endoderm just caudal and lateral to the
somites with a monoclonal antibody specific for avian p,integrins (CSAT). Embryos were cultured ventral side down
on an agar/yolk bed, and examined 4-6 hours later. The
dorsal aorta forms between stages 7-10, and along with other
early vessels, its assembly can be monitored by immunofluorescence staining using QHI. an antibody marker for early
vascular endotheliurn (Pardanaud, et al, 1987, Development
100, 339). The CSAT injected embryos all displayed abnormal
vessel patterning, especially in their dorsal aortae, which were
fragmented. In addition, the aortic endothelial cells of injected
embryos had dramatically disrupted shapes and were poorly
spread. This is in contrast to control embryos in which normal
vasculogenesis was consistently observed. Control injections
included non-immune IgG, and a monoclonal antibody (IG)
specific for p,-integrins which has no biological activity in cell
adhesion studies (Jaffredo, et al, 1988, Development 103, 431).
These data demonstrate that perturbation of p,-integrin function
results in abnormal vessel formation, and suggests that these
ECM receptors function during normal vasculogenesis.
U.
A procedure has been developed which yields a
highly enriched population of cytotrophoblast from
near-term rhesus monkey placental villi. When
methods
originally
described
for
human
cytotrophoblast (Douglas and King, 1989, J.
Immunol. Methods, 119: 259) were applied to monkey
placentas, less than 80% of the resulting cells
DRAKE, Richard L., Stephen D. HSU and Robert R.
CARDELL, Jr.
Department of Anatomy and Cell Biology,
University of Cincinnati College of Medicine. Cincinnati,
Ohio. Activation of auiescent heDatic alvcoPen svnthase
in the uostnatal mouse.
ABSTRACTS-AAA 103RD MEETING
Glycogen synthase, the rate limiting enzyme for
glycogen biosynthesis, exists in several isoforms. In the
liver, a suggestion has been made that hepatic glycogen
synthase may have fetal and adult isoforms.
In fetal
mouse liver, substantial amounts of hepatic glycogen
synthase protein are synthesized during the final days of
gestation and the protein remains at these high levels
through birth.
However, this newly synthesized glycogen
synthase does not seem to be involved in fetal hepatic
glycogen synthesis, but instead remains in a nonfunctional
quiescent form since enzyme activity during the last days
of gestation is decreasing. The studies described in this
abstract were initiated to answer the question of when
this quiescent enzyme becomes active. Liver tissues were
obtained from mice on various postnatal days. The tissue
was homogenized and centrifuged. To evaluate the quantity
of the enzyme, the supernatant was subjected to SDS-PAGE
followed by electro-transfer of the separated proteins to
a nitrocellulose membrane.
The membrane was incubated
with rabbit anti-rat adult glycogen synthase IgG, and the
bands visualized. To determine enzymatic activity, liver
supernatant was incubated with and without glucose6-phosphate in an assay mix containing 14C-UDP-glucoseand
glycogen.
The results of these experiments demonstrate
that the amount of glycogen synthase protein remains at
high levels through the postnatal period.
There is no
further increase in the quantity of glycogen synthase
protein.
However, the total activity of the enzyme
increased steadily through the postnatal period reaching
adult levels prior to weaning. These results suggest that
the nonfunctional quiescent form of glycogen synthase,
synthesized during late gestation, undergoes an uncertain
maturation process and is activated in the early postnatal
period. "Support by Grant No. DK27097 from NIH"
DUONG, Taihung. Robin S. Fisher and Michael S. Levine, Mental
Retardation Research Center and Department of Anatomy, UCLA,
Los Angeles, California. The rodent substantia niera in aeinc.
The effect of aging on the cellular morphology of the substantia
nigra in young (3 months) and aged (24 months) Fischer 344 rats was
studied by immunohistocheinicallabeling of the rate-limiting enzyme
in dopamine synthesis: tyrosine hydroxylase (TH). Animals were
deeply anesthetized with sodium pentobarbital and intracardially
perfused with 4% paraformaldehyde @H 7.4). The brains were
removed, cryoprotected in 30% sucrose and cut on the cryostat (40
Fm section thickness) in the coronal, sagittal, and horizontal planes.
Sections were processed free-floating by the peroxidaseantiperoxidasemethod, using a commercial polyclonal antibody to TH
(Eugene Tech Int.) . Some. sections were further silver-enhanced for
visualization of cell processes. TH-immunoreactive neurons were
qualitatively studied in the substantia nigra pars compncta (A9), pars
reticulata, pars lateralis, and in the area tegmentalis ventralis (A10).
In coronal sections, TH-immunoreactive neurons in the aged rats
exhibited distortions andor atrophy in the overall appearance of the
somata. Filopodia and stubby projections from the cell bodies were
also noted. In silver enhanced sagittal sections, TH-immunoreactive
nerve cells in the A9 group often exhibited processes with grossly
swollen and distorted proximal portions, which were more obvious in
the rostra1 and caudal portions of the substantia nigra pars compacta
where they are oriented in the sagittal plane.These processes were
noted as aspiny in their swollen portion but were branched and
sparsely spiny in their distal segments. They may be dendrites which
were often directed towards the pars reticulnta. Dendritic anomalies
were not observed in neurons located in nigral pars reticulata, lateralis
and A10 dopaminergic group in the aged and young rats. We are
thus reporting morphological alterations in the somata of
TII-immunoreactive neurons of the substantia nigra and
proximal dendritic distortions in the pars compacta (AV)
in aged Fischer 344 rats. A9 has been established by previous
studies as having extensive connections with the dorsal striaturn. We
are presently investigating 1) the ultrastructure of the nigral dendritic
distortionsand 2) potential changes in the dopaminergicinnervationof
the striatum related to the aging process. Supported by Grants No.
AG7462 and HD 07032 from USPHS.
29A
EBBESSON, Sven 0. E. and Gary T. BAZER, Institute of
Marine Science, University of Alaska Fairbanks, Alaska.
Transient retinal proiections in coho salmon (Oncorhvnchus
kisutch) and the 'midlife neuroembrvonic period.'
Smolt transformation (ST) in coho salmon occurs midlife,
just before downstream migration and the subsequent ocean
odyssey. It is known from other studies that ST, which occurs
during a period of approximately 30 days, is associated with a
plasma thyroxin (PT4)surge, olfactory imprinting on the natal
stream and changes in behavior. We have examined the brain
during ST and found that the period shares many features with
critical periods of development in other vertebrates, includin
sequential surges of epinephrine, serotonin, 5HIAA an!
glutamine, changes in immunoreactivity of several neuroactive
peptides, and dramatic increases in cell proliferation. We
report here, on the basis of cobalt lysine studies, the selective
sprouting of retinal fibers to unusual structures during ST.
These unusual projections, contralateral to the eye, which
largely disa pear during the y ear following ST, ar e to a
ventrolateray region of pars dorsalis of the telencephalon
extending from the caudal ti to the anterior commissure, the
pars ventralis of the telenceppalon, the reoptic area, the most
medial thalamic and hypothalamic nucfei, the inferior lobe of
the hypothalamus and some portions of torus semicircularis.
Ipsilateral projections to these structures and to the optic
tectum are also largely transient. These studies suggest that
ST i n salmon is a unique and promising model for studying
what turns on nerve fiber growth and what factors play.roles in
neural plasticity.
Supported by grants from NIH and the Alaska Sea Grant
College Program.
EGAR, Mar4aret W. and M. S. JARIAL. Department of
Anatomy,
Indiana
University
School of
Medicine.
Indianapolis. Indiana: Department of Anatomy, Center for
Medical Education, Ball State University, Muncie. Indiana.
Chanees
proximal tubule of axolotld homozveous for 2
recessive lethal mutation.
A recessive lethal mutation in axolotls. called "short toes".
that involved the kidneys. the Mullerian hucts and the Limbs
was described by Humphrey (1%7). No linkage to other known
genes was found and although a few homozygous recessive
animals did live to maturity. none reproduced. The edema.
ascites, subcutaneous hemorrhage and resulting lethality from
kidney malfunction was expressed at variable ages from
several months to three years. The severity of expression was
also variable in different spawnings. We have studied the
ultrastructure of the mesonephric kidney of short-toed axolotls
with and without ascites andlor edema and hemorrhage. and of
normal siblings possibly heterozygous for the s gene from a
total of 7 different affected spawnings. In those short-toed
animals that express ayites or edema the proximal kidney
tubule showed excessive pathology.
There was no
morphological evidence of kidney abnormality in the siblings
having normal limbs. Mesonephroi from anunals with asites
from other gene pools (lacking the short-toed mutation)
exibited a sirmlar phenotype and even more extreme proximal
tubule pathology.
S
EISNER, Shirley, Inder J. SINGH, Shamshad H.
GILANI, and Ladislao A. GUERRA, D ep ar t m en ts o f
Anatomy, N e w York U n i v e r s i t y D en t al C e n t e r , N e w
York C i t y and U n i v e r s i t y o f Medicine and D e n t i s t r y
o f N e w J e r s e y , N e w a r k . T e r a t o q e n i c i t y of c o c a i n e
i n t h e c h i c k e n embryo.
E i g h t h u n d r ed s i x t y t w o f e r t i l i z e d W h i t e
Leqhorn c h i c k e n e g g s were i n j e c t e d w i t h 0 . 1 m l of
c o c a i n e HC1 i n s t e r i l e p h y s i o l o g i c a l s a l i n e t o
d e t e r m i n e i t s t e r a t o g e n i c i t y . F i f t e e n doses of
c o c a i n e r a n g i n g from 0 . 0 2 5 t o 0.80 mglegg w e r e
30A
ABSTRACTELAAA 103RD MEETING
used. One dose was injected into the air space of
each egg at 48 ox 12 hrs of incubation. Control
eggs were either uninjected or saline-injected.
Eggs were maintained at 99.5F and 60-65% relative
humidity. On day 12 of incubation, live embryos
were weighed, measured, examined for gross
malformations, and fixed in 10% NBF. Survival rate
of embryos at day 12 of incubation was largely
dose-dependent; in embryos injected with cocaine
at day 3, survival rates ranged from 90% (lowest
dose) to 26% (highest dose). The LD50 on day 3 was
0.48 mglegg. Day 3-injected embryos alive on day
12
showed
significantly
more
malformations
including: exencephaly, anencephaly,microcephaly,
hydrocephaly,
anophthalmia,
microphthalmia,
cranial hemorrhage, adactyly, micromelia, twisted
limbs and digits, abnormal beaks and tails, short
twisted necks, extruded viscera, edema, and
reduced body size. Brain and eye malformations
were seen most often; 34% of a l l malformed embryos
had at least one CNS malformation while the saline
group had none. Results of cocaine exposure at 48
hrs were more variable.
At 2 days, chicken
embryos were more sensitive to both cocaine and
saline; however, cocaine caused significantly more
malformed embryos and 41% of those had one or more
CNS malformations.
Histologic analysis is in
progress. This research was supported in part by
BRSG Grant RR05332, N I H .
S
ELLIS,* L.C. and J.H. YOUSON, Department of Anatomy
and Scarborough Campus .Univepity of Toronto, Toronto,
Canada. An ultrastructurai investigation of the anionic sites
in the Droneuhric renal coruuscle 6f the sea lamure .
~t
membrane (GBM) q d epithelial. odocytes combine to
form a charge-selective ermeabiEty bamer in the
vertebrate renal corpuscfe. This barrier is due in part to the
negative char e from heparan sulfate and sialic acid
residues foun8 withm these components. In the present
study we have used transmission electron microicopy and
the cationic probe pol eth leneimine (PEI) to study the
charge bamer of the dievegpin and regressing ronephric
kidney durin the life cycle of h e sea lamprey, Ferromyzon
marinus L. Aqthough the renal tubules eventually
degenerate, the renal corpuscle persists throu hout the life
cycle but undergoes si nificant alterations wfich mimic
renalpathologies in otker vertebrates. In larval and 'uvenile
individuals there is intense, regularly-spaced (40 - do nm)
PE1 staining of the lamina rara intema and extema of the
GBM, and no staining of the lamina densa. PE1 particles are
also observed on the surface of the endothelium, epithelium
and the foot processes of the podocytes. In the upstreammigrant eriod, the laryina rara extema exhibits regu1a;lyspaced &I tracer articles and the lamina rara intema is no
longer clearl de&ed with PE1 tracer present throughout
the ex andeJmesangia1 matrix. The typical banding pattern
of coligen fibrils in this region is accented b the presence
of PE1 tracer particles at the periphery.of .the Kbrils..
Electron-dense deposits ossibly fibnn) in the region of the
GBM do not stain with P I, although a few traces of the
cationic probe are localized at their periphe . The absence
of PE1 staining in the lamina densa of the G?lM and in the
electron-dense deposits suggests that these two structures
ma share some similar structural components. Although
PEfstaining was consistent in the lamina ?a! extema
throughout the life cycle the lack of a defininte rara mtema
in the upstream mi rant iamprey indicates that chan es
likely occur in the h r i b u t i o n of the anionic molec3es
durin regression of the pronephros. Supported by NSERC,
Cana%a.
(E
ENGLISH, Arthur W., John G. WOOD, and Gail SCHWARTZ*
Department of Anatomy and Cell Biology, Emory
University, Atlanta, Georgia Distribution of the
Microtubule-Associated Protein. Tau. at the DeveloDing
Neuromuscular Junction.
To investigate the role it might play in synaptic
stabilization, we examined the distribution of the
microtubule-associated protein, tau, in motor nerve
terminals of the soleus and sternomastoid muscles of rat
pups during the period of postnatal synapse elimination
(birth (PO) until P21), and in adults. Tau was visualized
by indirect immunofluorescence and compared to the
extent of motor nerve terminal arborizations which were
labelled with fluorescent alpha bungarotoxin. In adults,
tau is distributed throughout the terminal arborizations
of
neuromuscular
junctions.
Using
double
immunohistochemical labelling, this distribution largely
co-exists with that of tubulin (alpha, beta, and
tyrosinated), is more extensive than that of
neurofilament proteins (NF's), and slightly overlaps the
distribution
of the synaptic vesicle protein,
synaptophysin.
At all postnatal ages examined, this
extensive distribution of tau in the nerve terminal
arborization was noted, even though the arborizations
themselves undergo remarkable changes during the ages
studied. In young animals most muscle cells receive
more than one synaptic input, but only a small portion of
the those cell receive more than one tau-containing
input. Motor nerve terminals containing NFs but not tau
can be found at young ages, but not in older animals.
Thus the appearance of tau in a motor nerve terminal
might be associated with the beginning of its maturation.
Supported by NS 20545 and NS17731 from the USPHS.
.-
3
ENGSTROM,' C.M., G.E. LOEB', J.G. REID', W.J. FORREST and L.
AVRUCH', Departments of Anatomy, Physiology and Physical Education,
Queen's University, Kingston, Ontario and Magnetic Resonance Unit, Ottawa
General Hospital, Ottawa. Ontario. W
r
v of the
ICT) [email protected]
Valid data on the morphometiy of muscle-tendon units are important for
understanding the force generating properties of these structures. Noninvasive techniques are required to obtain these data in live human subjects.
Recently, investigators interested in the mechanics of the human
musculoskeletal system have used imaging such as CT and MR to obtain in vivo
motphometric data on muscle-tendon units. particularly for the cross-sectional
area (CSA) of the quadriceps femoris complex. However, there have been no
systematic, quantitative studies to determine the relative accuracy of these
measurements with respect to direct anatomical measurements. In the present
study, we compared muscle-tendon CSA values obtained from the thigh of
human cadavers using anatomic, CT and MR cross-sections. Embalmed lower
limbs from three male cadavers were mounted securely in a wooden brace and
serial transverse images were obtained at lOmm intervals with MR (Siemens,
1.5T Magnetom) and CT (Toshiba, 900s scanner) prior to anatomical sectioning
of the frozen limb into the same number of similarly oriented and located slice
planes. Tracings from photographic records of the sections were then enlarged
(130% real size) and the CSAs of individual muscle-tendon units were
calculated by computerized planimetiy. The muscle-tendon CSA data obtained
for the individual structures by the three techniques were normally within 10%
agreement over the entire length of each structure. However, there were two
situations where discrepancies between the measurements tended to be
greater. First, for smaller structures such as tendons of insertion (e.g., gracilis.
semilendinosus), there were differences up to 10-20% between the
anatomical and imaging values, due principally to the difficulty of accurately
locating the fuzzy boundaries of these structures in the imaging records.
Second, there is an uncertainty in localizing bifurcations of one entity into two
subentities (e.g., biceps femris splitting distally into separate long and short
ABSTRACTS-AAA 103RD MEETING
heads). This is because each image is averaged over a finite section thickness
(in this case equal to the section spacing) while the anatomical sections are
aligned on the center of this thickness, which may or may not contain the start
of the bifurcation. There are also small distorlions such as swelling during
freezing and decreasing resolution at the edges of the CT and MR bores which
limit the ultimate accuracy of each method. In general, MR imaging seems to
offer a satisfactory source of motphometric data for most biomechanical
modeling without exposing subjects to Ionizing radiation.
Supported by a grant from the Muscular Dystrophy Association of Canada.
ERLANDSEN, S.L., B. Schmidtt, W.J Bemrickt. J.
Sauchl, and B.L. Jarrollt, 'Department of Cell
Biology k Neuroanatomy, and Department of
Veterinary Pathobiology, University of Minnesota.
Environmental Monitoring Systems Laboratory, U . S .
B.P.A., Cincinnati. Ohio, and Department of
Biology, Cleveland State University, Cleveland,
Ohio. High resolution low voltage SE4 of th
all: Immunogold localization o
genq in the intact cyst and du
in vitro encysteent.
In comparison to conventional SBM, the use of
low voltage SHM (due to lower beam voltage, probe
size, and increased contrast) has revealed that
the outer cyst wall of the genus G i a r d i a
consisted a layer of interlacing filaments. This
layer, composed of individual filaments ranging
from 7-20 nm in diameter, formed a dense meshwork
with interconnections that often appeared
arranged in circular whorls. High resolution
immunogold immunocytochemistry using polyclonal
and monoclonal antibodies specific for the cyst
wall of Ciardia, revealed that the labeling was
restricted to filament surfaces. Examination of
trophozoites undergoing in vitro encystment
showed that extracellular filament initiation
sites first appeared between 10-12 hours
following induction of encystment. Fully formed
cysts were detected at 14-16 hours postinduction.
indicating that extracellular assembly of the
filamentous cyst wall required 4 hours. Filament
initiation sites on the trophozoite membrane at
first resembled small filamentous protruslons.
buL later appeared globular, rangins! up to 300 nm
in diameter, and were detected on intermediate
stages in cyst formation. These results would
Suggest that cyst wall precursors, after
secretion by the trophozoite, undergo both
attachment and extracellular assembly into
filaments at multiple initiation sites on the
cell surface. (Supported by BPA CR-184622)
FARNUM, Comelia E. and Kathy Beck*, Departmentsof Anatomy
and Clinical Sciences, College of Veteriny Medicine, Comell .
University, Ithaca. New York.
v and-r
learnine in veterinam medicine
The power of cross-sectionalradiography as an adjunct to the study
of normal anatomy is that the shapes of gross anatomical s t ~ c m and
s
their spatial relationshipsto each other can be visualized as they are in
the living animal: i.e., the lungs and airways filled with air,the hem
filled with blood The reality of the form of these structures in the
living state and their positionalrelationshipsto each other are dimcult
for the first year anatomy student to grasp because the principal learning
tool is the dissection cadaver. The objective of this project was to
develop a series of teaching units designed for small group learning in a
problem solving approach to 1)Assist veterinary students in the first
year of the curriculumin learning normal anatomical positional
relationships by studying cross-sectionalCT scan images of the canine
head; 2) Assist students in the 3rd and 4th years in making the
transition from the 2-dimensionalradiograph back to the 3-dimensional
31A
animal by using anatomic and CT cross-sectionalimages; 3) By the
design of the teaching modules, to encourage independentand small
group learning. Each unit consisted of a series of 3 panels focussing
on a particular aspect of anatomy and the positionalrelationshipsof
nofinal s t ~ c mins three dimensionalspace, by making comparisons
between anatomical cross sections, CT images, normal radiographs,
and transected skulls. Four students were expected to answer all
questions of the unit, including a clinical case, within a half hour period.
The relatively short time l i t was designed to motivate students to use
the time efficiently,and to maximize their interaction with each other
during the learning process. The head was chosen since it is a difficult
body region for students to dissect, and an even more difficult one for
them to visualize in 3 dimensionson radiographs. Additionally,it is an
area where both CT scans and oblique views are used fairly routinely in
diagnostic radiology in veterinary medicine. To date we have
completed five units, focussing on the eye, !arynx, soft tissues,
airways, and ear. Student evaluations of this learning experience are
exuemely positive. Since the radiographiccorrelatesmake the
anatomical presentationclinicallyrelevant, first-year students are highly
motivated to complete the units successfully.
Supported by a grant from the Alumni Fund, New York State College
of Veterinary Medicine.
FEINBERG, Richard N. and Drew M. NODEN. Depaltmentsof Anatomy, New
Jersey MedicalSchool. Newark, New Jersey and New York State College of
Veterinary Medicine, Comell University, lthaca, New York. Devalooino
During the devebpment of many organs. avascular zones appear in circumscribed regions that previously containedpatent bbod vessels. In the limb,
avascularity is evident beginningat stage 18 immediately beneath the sudace
ectoderm and, later, appears coincidentwith the onset of mesenchymal aggregation at the sites where skeletal tissues will develop. The objective of this
research was to determine whether the formation of avascular zones was associated with a loss of viable endothelialcells.
Quail embryos at stages 16 to 26 were injected with India ink to visualize the
appendiilar vascular network. Unlabeled (i.e. avascular) regions of wing or
leg primordiawere dissected apart from deeper, ink-filled regions. Quail tissue
fragments from these separate regionswere implanted into pockets cut in
somitomeres 3 and 4 of stage 9 chick embryos. Most hosts were fixed 3-4
days later at stages 22-25; a few were maintained to stage 35. Transplantderived cells were identified using the Feulgen stain, and quail endothelialcells
were localized using polycbnal antibodies specific for this population. A total of
22 vascular and 36 avascular grafts were anaiyzed.
Through stage 18 both types of implant gave similar results. Quail endothelial
cells were found in blood vessels both near the implant and farther away in the
maxillary process. periocular regions, and meningeal plexus of the midbrain.
As previously reported. this pattern of dispersal is typical of embryonic angioblasts. However, beginningat stage 20 implants of avascular tissues gave different results; mesenchyme from avascular regions progressively bses the
capacity to form endothelialcells. Implants of stage 22 or oMer avaScular
mesenchyme lack angioblasts.
These results indiite that the the formation of local avascular zones is
associated with a bss of viable endothelialcells. Whether these cells degenerate or emigrate is currently under investigation.
Supported by Grants 89031 from AHA-NJ (RNF) and DE06632 from NIDR
(DMN).
FENTIE, I.H., S.P. MAHADIK* and F.J. ROISEN, Department of
Anatomical Sciences and Neurobiology, School of Medicine,
University of Louisville, Louisville, Kentucky and Division of
Neuroscience, New York State Psychiatric Inst., New York, New
York. Role of the cytoskeleton in aanalioside distribution on
Neuro-2a neuroblastoma membranes: An irnmuncg&&gkd
studv.
Neuro-2a murine neuroblastoma (N2A) cells exposed to the
ganglioside GM1 undergo increased neuritogenesis in vitro. To
determine if this increase is related to the distribution of GM1 in
the neurilemma, localization experiments were performed on
GM1-treated N2A cells employing monoclonal and polyclonal
32A
ABSTRACTS-AAA 103RD MEETING
antibodies against GM1. For ultrastructural studies protein Ggold was used to label the antibody. Although a relatively diffuse
labelling was observed over the entire neuronalmembrane, some
colloid was found arranged in short linear arrays. These linear
patterns suggested that cytoskeletal components such as
microtubulesor microfilaments might influence the distributionof
GMl in the membrane. To examine this possibility, N2A cells
were exposed to agents which alter cytoskeletal structure
followed by GMl localization employing immunofluorescence
microscopy. In cells treated with Colcemid, to disrupt
microtubules,the majority of GM1-positive label was found in the
perikarya.
In contrast, Taxol treatment, which stabilizes
microtubules, diminished perikaryal fluorescence and increased
neuritic labelling. Although labelling was reduced in the soma,
some punctate fluorescence remained. Similarly, occasional
punctatefluorescence was observedin the perikarya of Colcemid
treated cells. Studies with cytochalasin 0,aimed to disrupt
microfilaments, did not appear to alter the distribution of GM1.
These studies suggest that microtubules, in addition to their
cytoskeletal function, may play a role in determining the
distribution of gangliosides in neuronal membranes. Supported
by NIH Grants NS24524 and DE07734.
S
FESSLER, Richord D. and Jerald A. MITCHELL, Departments of Anatomy &
Cell Biology and Neurosurgery, Wayne State University School of Medicine,
Detroit, Michigan. The effects of d-fenfluromineand 5.7dihydroxyhyptamine on the morpholoav of suDroependvmo\ neurons in the
third ventricle of the hamster. LMesocricetus auratusl.
Previous studies have shown 5,7-dihydroxyiryptamine (5.7-DHI) to
possess potent neurotoxic effects on ventricular supraependymol
serotonergic (5-HT) neurons in numerous species. In addition, dfenfluromine, a halogenated rnethomphetamine derivative used clinically as
an anorectic, has been shown to cause degeneration of forebrain 5-HT
oxons. This study seeks to document the effects of these compounds on
the ventricular suproependymal neurons in the Qolden hamster. Female
homsters (80-1009, Charles River) were dosed with either intraventricular
5,7-DHT (1 OOug, following administration of desmethylimipramine) or
introperitoneol d-fenfluromine (25mg/kg BID X 4 days, n=10; or 75mg/kg
BID X 4 days, n=5). Animals were sacrificed 3 days following their last
dose of 5.7-DHT and on days 1.3 or 7 following the final dose of dfenfluramine. They were anesthetized with ether ond perfused with saline
followed by Kornovsky's aldehyde fixative via cardiac intraventriculor
puncture. Brains were prepared in routine fashion for scanning electron
microscopy (SEM). Following 5,7-DHT administrotion, SEM revealed
widespread degeneration of supraependymal fibers lining the floor of the
third ventricle and the orgonum vasculosum of the lamina terminalis. SEM
of specimens which received d-fenfluramine, exhibited no gross
morphologic evidence of supraependymal axonol degeneration. Specimens
did exhibit a tendency toward increased blebbing over the lateral walls and
floor of the third ventricle. In addition, severol specimens were noted to
have markedly increased numbers of phogocytic-type cells which spread
over the ependymol floor of the third ventricle rostra1 to the median
eminence. lntroventricular 5,7-DHT resulted in degeneration of ventricular
supraependymal fibers in the hamster. D-fenflurornine, previously reported
to result in degeneration of 5-HT oxons in the forebrain of rats and
primates, does not oppear to result in acute morphologic alterations in the
supraependymal neurons of the homster up to 7 days post-dosing. This
roises the possibility that dorsal raphe cell bodies with supraependymal
fibers are more resistant to high doses of d-fenfluromine thon those
supplying forebrain axons. Correlative transmission electron microscopy is
being performed to ascertain ultrastructural changes induced by either
compound in suproependymal fibers. In addition, studies to compare
supraependymol versus forebrain effects of d-fenfluromine in the hamster
are in progress.
FISHER, Robin s. Departments of Psychiatry and Biobehavioral
Sciences/Anatomy and Cell Biology, Mental Retardation Rcsearch Ccntcr,
UCLA School of Medicine, L o s Angeles, California. The anatomv of
claustrocortical neurons in cats.
The projections from the claustrum to the neocortex constitute a
large but obscure corticopetal input. The objectives of this investigation
were to identify the neuronal origins of the claustrocortical projections
and t o characterize their somatodendritic architecture by morphological
means. Wheat germ agglutinin lectin-bound horseradish peroxidase(a
rctrograde connectivity marker) was injected unilaterally into the preand postcruciate gyri of the neocortex of 5 adult cats. The undecussated
claustrocortical neurons were demonstrated by peroxidasc histochcmical
methods. These cells were then Golgi impregnated and gold-toncd by
single-section methods to delineate thc structure of the claustral
projcction neurons.
Similar doubly labeled claustrocortical neurons were ohserved in the
dorsal and ventral divisions of the claustrum in all of the tested cases.
Two types of projection cells were revealed by corrclative light and
electron microscopy. 1)The principal claustrocortical neurons were small
They composed
and medium sized cells with spiny dendrites.
approximately 80% of the doubly labelcd cells. Thcir somatic sizes
rangcd from 10-35 pm. Their dendrites were usually confined to the
width of the claustrum but occasionally extended into adjacent white
matter tracts. They resembled the medium sized spiny neurons of the
ncostriatum. These observations are significant because they providc the
initial direct demonstration of a sizable corticopctal input from a
subcortical, as opposed to an infracortical, class of spiny neurons. 2)The
acccssory claustrocortical neurons were small and medium sized cells with
bcadcd, sparsely spiny dendrites. Contrary to previous speculation, at
least two different morphological types of neurons give rise to the
asccnding output of the claustrum. Both of thesc ccll types receivcd
numerous reciprocal corticoclaustral inputs. A third type of claustral
neuron, medium sized cells with smooth and sparsely spiny dcndritcs. was
gold-toned but not doubly labeled. These infrcqucnt cells and/or the
local collaterals of the projection neurons, may form local circuits in the
claustrum. Supported hy USPHS Grants HD05958 and NS24596.
Eileen C. Foley* and Thomas A. Marino. Department of
Anatomy, Temple University School of Medicine, Philadelphia,
Pennsylvania. Fibroblast growth factor regulationof embwonic
and neonatal cardiocvte Droliferation.
Fibroblast growth factor (FGF) stimulates skeletal muscle
cell division and concurrently there is a repression of terminal
differentiation. The focus of this study was to examine the
effects of acidic (aFGF) and basic (bFGF) FGF on dividing,
embryonic cardiocytes and non-dividing neonatal cardiocytes
that are still undergoing DNA synthesis. Embryonic and
neonatal cardiocyte cultures were supplemented with serum
for only 24 hours, after which the medium was changed only
on days 6. 9, 15 and 20. At these time points, serum, aFGF
or bFGF was added with 3H-thymidine. 24 hours later
cultures were fixed, autoradiography done and the percentage
labeled cardiocytes was determined. On day 0, 32% of the
embryonic and 12% of the neonatal cardiocytes were labeled.
By day 15, 12.8% of the embryonic cardiocytes were labeled
and serum (22.4%). aFGF (20.5%) or bFGF (20.6%)
significantly (P 5 0.05) elevated the percentage of labeled
cardiocytes. 4.9% of the neonatal cardiocytes were labeled on
day 15 and the percentage of labeling was not increased by
serum, aFGF, or bFGF. By day 20, only 7.3% of the
embryonic cardiocytes were labeled and neither serum, aFGF
or bFGF increased the percentage of labeling.
Our
conclusion is that serum, aFGF, and bFGF can stimulate
embryonic cardiocytes to proliferate, but can not initiate cell
division in neonatal cardiocytes, which may indicate that DNA
synthesis is under a different control mechanism in embryonic
Versus neonatal cardiocytes.
(Supported in part by NIH grant HL29351 and grant 88-1147
from the American Heart Association and the Pennsylvania
ABSTRACTSAAA 103RD MEETING
FORSSMANN. Wolf-Gcorg, Markus MEYER *, Christoph DAFFNER
and Frdnz HERBST,' Lower Saxony Institute for Peptide Research (IPF),
Medial School of Hannover, Fcodor-Lyncn-Str5, D-3000 Hannowr 6 1
(FW
The endocrine heart:
- na r
biochemical aooroach to the secretory a r w of m*nca nie m
v
w
cclls.
Cardiac atria exhibit a pcptide producing endocrine organ which is
constituted by myocndocrine cells. Peptides of Merent sizes of the
ANP/CDD (atrial natriuretic polypeptidc./cardiodilatin) family were thought
to be processed in these cells. We have earlier claimed that the prohormone
of ANPKDD contains 126 amino acids and only during secretion the
posttranslationally processed pcptidc of 28 C-terminal amino acid is found in
blood circulation. The present paper dcals with the study of guinea pig heart
using the in vitro LANGENDORFP perfusion. Biochemically the peptide
prohormone of the yet unkown amino acid acqucnec of this species was
studied using gel electrophoresis with immunoblottiag, amino acid
sequencing, and cDNA analyst The secretion appearing in the eluate of the
LANGENDORFF perfurion was compared with the molecule revealed in
freshly extraded atrial tissue. It shows that the molcculc is readily converted
from a 13 kD molecule to a 35 kD rnolccule in tbc in vitro preparation. Tbic
holds also true for comparison between tissue and plasma ANPKDD of
guinea pig in vivo. Immunocytochcmistry shows that cpitope specific
antibodies can be used for the colloidal gold technique to show that aII
secretion granules contain the large molecular, 13 kD related
immunorcactivity. The eluate was atso extracted from a larger batch of
perfused hearts in order to dcterminc the amino acid sequencn of the
secreted bioactivc molecule. The prosent work confirms furthermore that
ANPKDD is conwted from its prohormonal form to the circulating form
after secretion because exogenously applied prohormone is proewsed when
added to the perfusion solution. The investigation shows that cardiac
hormones may be stored only i0 the prohormonal form of 126 amino acids
and quickly processed into the circulating, 28 amino acids containing form
while the granules release their content by wcocytosk.
-
Supported by a grant of the VolkswagenStiftung
S
0
33A
follcklim treatments with hitar X-114
nu?hullan sperm intra-acroscmal p&in
S P l O is
ccnsidered a primary amtmcqtive vaccine irmrp.logen
the kim taskfom on oxtraaqtive vaccines A
(&
ISU,I
et
al., 1987. J.&pl'd.h~~r~l
10:231-57).
Fixed,
permeabfiizei sperm inmmstained with a mAb to -10
--lo)
ckmwkmted a bright acxsaue shapes cap.
m i i c w h g the ~ c l d ~ o mreacticm,
e
faint QFS ~IXI/W
equatoridL bars were sem, indicating that sane S P ~ O
m i n s associated with
reacted spenn (m,et
al., 1990. Biol.Reprcd.inpress).
In the present s t a t y
ejaculated qenn trpBated with l'rit.cn X-114 or TX-100
exhibited a large
of fa-te
cap Wor
b r i g h t quatxrial bars by inmunoflwm. M
ildicatedthattheiwreracxsamlmars3rane(IAM)ard
equatorial sepent persisted in TX-100 treated cells.
often acrcsaml matrix m a t e r i a l femained as well on the
IAM. Sperm extracted four times with TX-IW retain >709
faint caps ani bars indicatin3 that sP-10 persisted M
the IAN&
equatorial segment after this m t r n e n t .
treatuent with 150 mM NasQJ anp1etely rarrnred
all fluoresceme whereas 150 mM NaQ prpduoea cnly a
slight redudion in fluoresa3ue. 'Ibis finding arggests
that scdim thiccyariate s p e c i f i c a l l y d i s q d s an ionic
associaticnr between S F 1 0 an3 another raM*
aapscmal
constituent, ie. part of the ULM or pezinp a ~ ~ - 1 0 0
insoluble mtrix carponent. 'lb further investigate the
associatfon of -10 with the acrpsanal rmdmnes, the
h m t h y of S P l O Was shdied USTX-114 extracticol
ard @ase separaticm. western blot andlysis shwd
imannreactive S P l O in the aquerws fraction and I K J t in
thehydqhcbicfractian. mgether,theseresultsargue
against SPlO beins an ir&qIal manbrane p m t e i n
suggest that it is p e r i m l y assaciated w i t h the IAM
or a TX-114 insoluble ODlFtitUMt of the a a x s u a l
matrbc. [Sqpxt:NIH W16767 and 23789.1
S
FOSTER,' Davld John and Nobuyoshl HAGINO, Department d Cellular
and Structural Bidogy. Unhrersky d Texas Health Sclence Center at
dooamlng
San Antonlo, San Antonlo. Texas. Jhe effect of -1
dedetlon on wsmataI develotiment d d m mIne 0-2 receDtws In r&
sa&f!m
The effect of suppressing dopamlnerglcneurons by prenatal admlnlstratlon of alpha-methyl-para-lytyrodne(AMPT) on postnalal development
of 3H-splperonebinding to dopamine 0 2 receptors In fat caudates was
lnvesti ted. Pregnant SpragueDaAey albkro rats receked either
AMFT&ctkm (200 mg/kg, 1.p.) or d n e lnJedlonson day 15 and 20
of gestatlon.Their offspring were then sacrKlced on the flrst, fifth or tenth
daydllfe. AMPT treatmentd~nncantlydecreasedscrktdB~wfth
no effect on
In both flve and ten day dd offsprhg. as compared to
contrds.
ng In the strkta d one day dd Crrspting. however, was
m t i c and could not be relhbly quantbted. Because depletion d
catechdamlne synthesis delayed the maturation d D-2 receptors, we
hypothesized that dopamine may have a neurotrophic effecton D-2
receptor development and that there may be a critical perlod durlng
which thls effect occurs To better define the critical period involved.
pregnant rats were InJected with AMPT or saline on day 15 d gestatlon
only. Asgays d strlatal tksue from ten day old offspring faUed to show
any significant difference between AMPT-treated and the control
"ffspri? It was thus apparent that a &tical perlcd exlsts at the termlnal
stage fjestation In which dopmlne depletion can edsy D.2 receptor
development However, the presence of dopamlne 0-2 receptors has
been detected Inrat stfiaturn as early as embryonic day 14 (Sales et al.,
1989). The fact that doparnlnedepletion by AMPT failed to decrease D-2
receptors at gestational day 15 suggests that the lnthl appearance of D2 receptors Is not dependent on the presence d endogenam
dopamine. In conttast the sensltkky of D-2 receptors to dopamlne depletion at gestational day 20 leads us to mclude that dopamlne may
play a neurotrbphlc rde in later strlatal D 2 receptor expression In the
developing rat brain. (Supported by the Central Research Instlute for
Electrkal Power, Inc., Japan.)
Bai
FREDRICKSON*, Eric R. and Nancy E. J. BERMAN*,
Department of Anatomy and Cell Biology, University of K a n w
Medical Center, Kansas City. Neurowutide Y immunoreactivg
neurons and cvtochrome oxidase Datches in human striate
-cortex.
The cytochrome oxidase patches of striate cortex represent
clusters of neurons which respond selectively to colored stimuli
(Livingstone and Hubel, '87). Recently, Kuljis and Rakic ('89)
found that neuro eptide-Y immunoreactive (NPY-ir) neurons
are selectively locarked outside these patches in macaque striate
cortex, suggesting that patch and non-patch regions contain
specialized populations of cortical interneurons.
We
investigated the relationship between NPY-ir neurons and
cytochrome oxidase patches in human striate cortex. Speamens
of calcarine cortex were obtained from 3 autopsies performed at
the University of Kansas Medical Center. Postmortem times
ranged from 6-14 hours. Tissue blocks were immersion fixed,
frozen, sectioned, and adjacent transverse sections were reacted
histochemically for cytochrome oxidase using the method of
Horton and Hedley-Whyte ('841, immunohistochemically stained
using a polyclonal antibody directed against NPY, and stained
with thionin. Drawings of adjacent sections were aligned using
blood vessels and pial surface features as landmarks. NPY-ir
neurons displaying a variety of sizes and morphologies were
found in all cortical layers and in the white matter, and they
were similar to those desuibed by Kuljs and Rakic ('89).
Cytochrome oxidase patches averaged 0.55 mm in width, and
non-patch regions averaged 0.48 mm in width. Patch and nonpatch territories were reconstructed using radial striations seen
in adjacent thionin-stained sections, and N Y P e l l s located in
Equivalent
patch and non-patch regions were counted.
numbers of NPY-ir neurons were located in patch and nonntch regions. Our results suggest that the morphology and
Eminar location of NPY-ir neurons are similar in human and
macaque striate cortex. However, the specialized distribution of
34A
ABSTRACTSAAA 103RD MEETING
this population with respect to the cytochrome oxidase patches
seen in macaque is not present in human striate cortex. In this
respect, the human striate cortex may be less specialized than
that of the macaque. Supported by MH38399 and BNS 881997.
oFXENZ.*12*1Dorothy A., lhomas R. VAN DE WATER”’ and James D.
WLLIAMS
bboratorv d Develonmental ,,Otobioloav, Deots. of
Otolaryngolo&’, Neuropathdogy’. and Ne$oscience”. Alkrt Ektein Cbllege of
Medicine, Bronx, New York. Transforming mowth factor - beta: a srenal
plypeptide that modulates otic capsule formation.
respect to one another. Using a monoclonal antibody
produced against GBL, endogenous GBL is detected a t the
peripheries of cells in the walls of the developing heart a t
these stages of morphogenesis. These results suggest that
GBL and its endogenous galactoside-bearing receptors a r e
involved in heart development in Xenoous. Supported by the
Medical Research Council of Canada and the Alberta Heritage
Foundation for Medical Research.
..
Mophogenesis of the otic capsule is driven by epithelial-mesenchymal tissue
interactions that occur between the epithelial anlage of the membranous labyrinth
(omyst) and iu associated periotic mesenchyme (Van De Water et al., h a t . Rec.
199262,1981). Temporal-spatial diseibution of [email protected] growth factor beta
(TGF-B ) in both epithelial and mesenchymal components In the developing mousk
su ests a role for this growth factor in formation of the otic capsule (Van
inner
De Water PGalinovic-Schwa, Annals of NY Acad Sci., in press). In hlghdensity cultures of periotic mesenchyme, exogenous TGF-B can modulate the
expression of the cellular phenotype by periotic mesenchymal Jells by acting either
as an enhancer or suppressor of chondrogenesis (Frenz & Van De Water, Annals of
NY Acad Sci., in press). This modulation is dependent upon the state of
mesenchymal cell commitment and closely mimics the enhancement or suppression
of chondrogenesis that results when epithelial tissue is inlrcduced into cultures of
periotic mesenchyme. Utilizing polyclonal antibodies directed against TGF-B
(supplied by the courtesy of Drs. M. Sporn & K. Flanders, Chemopreventio6
Laboratory, National Cancer Institute), we have demonstrated by indirect
immunofluorescence that TGF-B is localized in otic epithelium in 10.5 gestation
day (gd) and 14.0 gd epitheliaVmksenchyma1 micromass cultures. When antibodies
directed against TGF-B are added to these cultures, epithelial-inducedmodulation
of chondrogenesis is Inhibited. In cultured 10.5 gestation day (gd) periotic
mesenchyme, the influence of otic epithelium is necessary to initiate chondrogenesis
(Frenz & Van De Water. unpublished results). When 10.5 gd periotic mesenchyme
and otic epithelium are cultured in the presence of anti-TGF-B (10 uglml), a
marked but not total suppression of chondrogenesis results, as indcated both by a
decrease in cell condensation formation and a reduction in cartilage-specific
proteoglycan production. At 14.0 gd, the influence of otic epithelium on cultured
14.0 gd periotic mesenchyme results in suppression of chondrogenic differentiation
(Frenz & Van De Water, unpub!ished results). However, in the presence of antiTGF-B , this suppression is significantly blocked. We hypothesize Lhat during otic
capsuld formation, TGF-B is localized within the otic epithelium and acts in a
paracrine manner to mehiate the pattern of chondrogenesis by enhancing
chondrogenesis in early periotic mesenchyme and later suppressing this process
during perilymphatic space formation and sculpturing of the presumptive otic
capsule.
-
(Work supported by MEHS Pathology Training Grant NS07089 & a Deafness
Research Foundation Grant)
FRUNCHAK‘, Yvette N., Kenneth D. McFADDEN and Nadine C.
MILOS., Department of Anatomy and Cell Biology, University
of Alberta, Edmonton, Alberta, Canada. Electron microscoor
studies on the effects of endogenous galactoside-binding
k c t i n on heart develooment in Xenoous laevis.
_X.-laevis embryos (stages 24-26) contain endogenous
galactoside- binding lectin (GBL). Development of several
neural crest derivatives (melanophores a n d neurites) is
altered by purified GBL (Milos et al., 1987 a n d 1989).
Regions of the heart with neural crest components
(conotruncus) a r e also altered by treatment with GBL or its
sugar hapten inhibitor thiodigalactoside (TDG) (Frunchak e t
al., 1989). We have investigated GBL a n d TDG effects on the
conotruncus using electron microscopy. Gill groove area
ectoderm was removed from stage 24-26 embryos exposing
posterior cranial neural crest t h a t contributes to the
heart. Embryos were incubated in 4 hemagglutinating units
of GBL or 2.5 mM TDG. With GBL or T D G treatment,
conotruncal surface morphology became irregular in 70% of
trcated animals. Conotruncal positioning shifted in 20% of
treated animals. Atria and sinuses also enlarged compared
to controls. With TEM, control conotruncal regions
consisted of concentric cell layers separated by fibrillar
extracellular matrix with cell-cell attachment by adherens
junctions. With GBL or TDG treatment, extracellular matrix
became sparse and cells were more loosely arranged with
S
FUNK*, Greg D., William K. MILSOM’ & John D. STEEVES’,
Department of Zoology, University of British Columbia,
Vancouver, British Columbia. Is the svnchronization of wing
beat and resDiration durina fliaht in aeese due to feedforward
or feedback activitv?
To describe the relationship between wingbeat and
respiratory pattern, wingbeat frequency (f,)
and breathing
frequency (f,) were recorded during free-flight in 5 (4-5 month
old) trained Canada geese. The two patterns were
predominantly coupled at 3 wingbeats per breath, but ranged
from 2:l to 4:l. To assess whether afferent activity could
account for the coupling of the two systems, we examined the
relationship between f, and ,f during passive wing flapping.
Results indicate that; (1) passive flapping will entrain
respiratory rhythm over small ranges, and (2) removal of wing
afferent activity by dorsal root section of the brachial plexus
has no effect on this entrainment. This implicates chest wall
and/or lung afferent feedback in this coupling. We were also
interested in whether the afferent activity associated with wing
flapping, although sufficient to entrain respiration and
wingbeat, was necessary for this coupling. The relationship
between ,f and 1, was then examined in 16 decerebrate birds
in which wing flapping was induced through electrical
stimulation of the dorso- and ventrolateral medullary reticular
formation and the medial mesencephalic reticular formation.
The range of entrainment under these conditions was 1:l to
4.1. Nine of these birds were then paralyzed and pectoralis
nerve (main wing depressor nerve) and respiratory nerve
(internal and external intercostal, and cranial nerve IX)
discharges were recorded during ”fictive” flight. A very tight
1:1 coupling was observed between pectoralis and respiratory
nerve discharge. These results indicate that there is a central
program synchronizing the outputs of the locomotor and
respiratory systems independent of
feedback.
The
synchronization expressed during free-flight, we believe, is the
product of an interaction between this central program
(feedforward) and the peripheral feedback. Supported by the
NSERC of Canada.
S
GAGXA, Claude Eugene, Deprtmmt of Anatmy, New York
University Dentdl.Center, New York City, New York. E
LNAof a bovine CTYstalline lens
qencnnic double-stranded DIA library.
Pilot studies, imrolving navel techniques, have been
undertaken to reveal the presence of htact doublestranded 2-m sequencss in a Wine (-)
lens
gemnic CNA Libraq.
Plaques containing Z-ui~
sequences have been identified, using a variety of
different anti-2-LNA antibodies, (whole sera, purified
IgG plyclonal and mnoclonal antibodies), and
isolated, under both high (4 M NaC1) and physiological
(150 nt-l NaC1) ionic conditions. To my kmmledge, this
study represents the first such attempt to d i r e c t l y
identify genomic lens LNA (intact double-stranded base
pairs) library sequences in the left-handed
confomtion.
?he present study has demonstrated:
(a) m e presence of z-LNA sequences or potential z - m
sequences in a gencanic bovine lens LNA Library.
Allawed for the isolation of 2 - m sequences w i u!b)
ch
ABSTRACTS-AAA 103RD MEETING
w x e dmzcterized by using CNA sequencing te&nique+
parallel. We have investigated how these areas are related in
These 2-helix base pairs w i l l be caplter analyzed 111
New World monkeys by assessing the electrophysiological effects
order to c&tain information on their possible roles
of ablations of parts of areas 3a and 3b on the responsivity of
during gene expression and possibly used as
neurons in area 1. We found that the removal of parts of the
hybridization probes in future experimentatim to
representations in areas 3a and 3b resulted immediately in the
explore the m l d a r biolcgy of the normal and
deactivation of corresponding parts of the body representation in
c a t a r a ~ l e n s . ~ s t u d y f u r m e r ~ area
~ 1. Thus, area 1 is dependent upon inputs from anterior
laboratoq's d u s i m that 2 - m is pnzsent
the
parietal areas 3a and/or 3b f o r its cutaneous activation, and area
lens am3 may play a role in its gene expression.
1 most likely functions as a "higher order" station than area 3b
S
GALINDEZ,* Orlando A . and J o e A . Mascorro,. Department of
Anatomy, Tulane Medical School, New O r l e a n s , L o u i s i a n a .
The e f f e c t s of
(Sponsored by Mary B. Anderson)
hypothermia on u a r a e a n a l i o n chromaffin c e l l s : comuarison
t o t h e a d r e n a l medulla." Endocrine c e l l s o f the a d r e n a l
medulla
respond
to
hypothermia
by
releasing
catecholamines (CAM). This response i s c l e a r l y e v i d e n t
a t t h e u l t r a s t r u c t u r a l l e v e l by t h e appearance of a l t e r e d
cytoplasmic g r a n u l e s known t o c o n t a i n CAM. S i m i l a r CAM
c e l l s g e n e r a l l y known as " p a r a g a n g l i a " a r e widely
d i s t r i b u t e d i n e x t r a a d r e n a l l o c a l e s . However, it i s n o t
known i f t h e s e chromaffin family c e l l s a l s o w i l l r e l e a s e
hormones under s t r e s s . Young r a b b i t s were s u b j e c t e d t o
mild ( 4 h r s . @ 4 O C , 34OC r e c t a l temperature) o r s e v e r e (10
mins
i n 0%
ice bath,
15°C r e c t a l temperature)
hypothermia. Animals were p e r f u s e d w i t h g l u t a r a l d e h y d e ,
t h e chromaffin t i s s u e mapped a n a t o m i c a l l y w i t h potassium
dichromate, and t h e n processed f o r f i n e s t r u c t u r a l s t u d y .
Dichromate mapping produced a g r o s s chromaffin r e a c t i o n
which r e v e a l e d t h e t r u e expanse o f e x t r a a d r e n a l
chromaffin t i s s u e . I n a d d i t i o n t o p a r a g a n g l i o n b o d i e s ,
e l o n g a t e d p a r a a o r t i c chromaffin organs o c c u r r e d a l o n g s i d e
t h e abdominal a o r t a , and l a r g e chromaffin c e l l c l u s t e r s
( > 100 c e l l s ) r e s i d e d w i t h i n t h e autonomic g a n g l i a .
Hypothermic s t r e s s (mild o r s e v e r e ) produced d r a m a t i c
changes i n a d r e n a l medullary c e l l s , i e . , CAM g r a n u l e s
appeared t o t a l l y devoid o f their c o n t e n t , o r o t h e r w i s e
p r e s e n t e d a c e n t r a l c o r e much reduced i n d e n s i t y from t h e
normal s t a t e . Paraganglion and p a r a a o r t i c c e l l g r a n u l e s
maintained t h e i r u s u a l i n t e g r i t y and dense c o r e f o l l o w i n g
cold s t r e s s .
I n t r a g a n g l i o n chromaffin c e l l s a s s o c i a t e d
with t h e c e l i a c g a n g l i o n a l s o r e t a i n e d normal g r a n u l e
morphology, b u t t h e s e p a r t i c u l a r chromaffin c e l l s showed
abundant
cytoplasmic
vacuolization
after
severe
hypothermia. Chromaffin c e l l s of t h e a d r e n a l medulla a r e
w e l l i n n e r v a t e d ; p a r a g a n g l i o n and p a r a a o r t i c c e l l s
probably a r e n o t , and t h o s e i n t h e c e l i a c g a n g l i o n o n l y
s p a r s e l y s o . These f a c t o r s may account f o r t h e d i f f e r i n g
response o f i n t r a - and e x t r a a d r e n a l chromaffin c e l l s t o
a similar stimulus.
GARRAGHTY, Preston E., Sherre L. FLORENCE* and Jon H.
KAAS',
Department of Psychology, Vanderbilt University,
Nashville, Tennessee. Serial Drocessine within anterior Darietal
cortex of monkevs: the deactivation of area 1 bv acute ablation5
of areas 3a and 3b,
Cortex traditionally referred to as S-I in monkeys is a
composite of four separate and complete representations of the
contralateral body surface, one in each of the four
cytoarchitectonic fields (areas 3a, 3b, 1, and 2) comprising 5-1."
Similarities in somatotopic organization, cytoarchitecture, location
relative
to
motor
cortex,
and
connections
with
the
ventroposterior nucleus (VP) of the thalamus suggest that area 3b
of monkeys is the homologue of S-I in most other mammals.
This hypothesis implies that the cutaneous representation in area
I reflects a further step in a processing hierarchy. However,
area I , like area 3b, receives substantial direct inputs from
neurons in the VP, and these connections could allow area 1 to
function independently of area 3b. Thus, the question remains
open as to whether the processing of somatosensory information
in anterior parietal cortex is predominantly hierarchical or
in a processing sequence across anterior parietal cortex.
Presumably, the VP inputs to area I play a modulatory role, with
the main activating influence stemming from corticocortical inputs
from the more anterior somatosensory fields. This hierarchical
view is consistent with the laminar arrangements of cortical
interconnections; the relative paucity and laminar locations of
thalamic inputs to area 1; the relatively more complex receptive
ficlds of neurons in area I; the nature of the behavioral deficits
which accompany ablations of single architectonic areas of
anterior parietal cortex; and the difference between latencies of
evoked potentials attributed to areas 3b and 1 in humans.
Supported by N.I.H. NS16446.
GEDUSPAN,* J a n e S. and SOLURSH, Michael. Department o f
&
Biology, U n i v e r s i t y o f Iowa, Iowa C i t y , Iowa. A
t i v e i n f l u e n c e on limb outerowth i n t h e d o r s a l Dresumut i v e limb r e e i o n .
W e e v a l u a t e d t h e c a p a c i t y of t h e presumptive limb
r e g i o n t o r e g u l a t e i n t h e p r e s e n c e of e x c e s s t i s s u e .
To do t h i s , d o r s a l o r v e n t r a l h a l v e s o f t h e r i g h t p r e sumptive wing r e g i o n from s t a g e 14-16 q u a i l embryos
were g r a f t e d t o e i t h e r t h e d o r s a l o r v e n t r a l r e g i o n o f
t h e p r o s p e c t i v e l e g a r e a of t h e same s t a g e c h i c k
embryos. The t r a n s p l a n t e d d o r s a l o r v e n t r a l t i s s u e i n
t h e d o r s a l r e g i o n o f t h e h o s t ' s presumptive hindlimb
formed w e l l - d e v e l o p e d wing buds b u t no l e g . H i s t o l o g i c a l examination of t h e s e wings show t h a t t h e b u l k of
t h e mesenchyme c o n s i s t s p r i m a r i l y o f q u a i l c e l l s which
e x t e n d from t h e d o r s a l proximal h a l f o f the wing t o t h e
d i s t a l r e g i o n . The c h i c k ( h o s t ) c e l l s are found mainly
i n t h e v e n t r a l proximal h a l f o f t h e limb. However, t h e
h o s t ' s l e g developed normally a f t e r d o r s a l o r v e n t r a l
q u a i l wing t i s s u e w a s g r a f t e d t o t h e v e n t r a l r e g i o n of
t h e presumptive hindlimb.
H i s t o l o g y of t h e s e limbs
show t h a t g r a f t e d q u a i l c e l l s a r e l o c a l i z e d i n t h e vent r a l proximal r e g i o n and most o f t h e c e l l s i n t h e l e g
a r e d e r i v e d from t h e h o s t .
The p o s i t i o n - d e p e n d e n t
growth o f t h e g r a f t s i m p l i e s t h a t some p o s i t i v e i n f l u ence e x i s t s i n t h e d o r s a l presumptive limb r e g i o n which
The r e s u l t s
promotes outgrowth o f t h e t r a n s p l a n t s .
a l s o s u g g e s t t h a t normally t h e c e l l s t h a t form t h e limb
a r e p r i m a r i l y from t h e d o r s a l r e g i o n of t h e presumptive
limb bud.
The d o r s a l l y - p l a c e d c e l l s appear t o d e t e r mine t h e wing o r l e g c h a r a c t e r o f t h e experimental
limb.
S t u d i e s a r e i n p r o g r e s s t o determine t h e n a t u r e
of the p o s i t i v e i n f l u e n c e on t h e d o r s a l presumptive
limb r e g i o n .
( T h i s work was s u p p o r t e d by N I H g r a n t
HD05505. )
S
GEHRIS,* Amy L., and Robert M. GREENE,* Department of
Anatomy,
Thomas
Jefferson
University, Philadelphia,
m e 1 Pcne exuression in develoDine murine
Pennsylvania.
palatal tissue.
Transforming growth factor-p has been shown to have an
important
role
in
developmentally
regulated
events
including a possible role in palate development.
Its
expression in this embryonic tissue at both biochemical and
In order to
molecular levels remains to be elucidated.
determine TGFp 1 gene expression in murine palatal TGFp 1
gene expression in murine palatal tissue during its
development, total RNA was isolated and screened by
36A
ABSTRACTSLAAA 103RD MEETING
northern blot analysis. A gel purified fragment from a TGFpl
cDNA plasmid clone was labeled with 32P-dCTP and used to
screen northern blots of total R N A isolated from primary
cultures of murine embryonic palate mesenchymal (MEPM)
cells as well as RNA isolated from embryonic palatal tissue
from days 12, 13, and 14 of gestation. RNA was prepared by
acid guanidinium thiocyanate-phenol-chloroform extraction
and quantitated by measurement of absorbance at 260nm.
R N A was denatured and fractionated on a 1.0% agarose gel
a n d transferred to nitrocellulose.
Preliminary results
demonstrate the presence of TGFpl mRNA on all three days of
gestation. TGFpl mRNA was also detected in RNA extracted
from MEPM cultures and R N A from murine thymus, a TGFp
positive control. The size of the TGFpl mRNA hybridizing
with our probe was approximately 2.5Kd.
The size of this
transcript compared favorably with t h a t reported for o t h e r
tissue and cell types for TGFpl. In addition, temporal changes
in the level of TGFpl message in embryonic palatal tissue
were evident with the highest level on day 14 of gestation.
These data, which indicate the presence and probable
tcrnporal variation of TGFp 1 gene expression during palate
development will be pursued by f u r t h e r examination of
transcriptional rates and localization of the TGFp message in
palatal tissue. Supported in part by NIH Grants DE05550 and
DE08 199.
S
GERRAS;G.G.,
S.D. HAM,' A.I. CANADY' and J.A. MITCHELL,
Departments of Neurosurgery and Anatomy & Cell Biology, Wayne State
University, School of Medicine, Detroit, Michigan. The effects of
kaolin-induced hvdr-s
on t h e
of t h e MetA
yentricles of the hamster.
The effects of kaolin-induced hydrocephalus on the ependyma of the
lateral ventricles of the hamster were determined. Hydrocephalus was
induced in 20 adult female hamsters (Mesocri c W auratus) by
intracisternal injection of 0.03 ml sterile kaolin (250mg/ml) in
saline; controls were sham operated (5)or vehicle injected (5). Brains
were harvested at 30 days post-treatment. Animals were anesthetized
and perfused with Karnovsky's aldehyde fixative. Ventricular surfaces
were prepared in routine fashion for examination by correlative
scanning and transmission electron microscopy. The ependyma was
intact and normal in controls: the lateral wall (LW) was uniformly
densely ciliated: apical cell surfaces had microvilli but few
supraependymal neuronal fibers. The medial wall (MW) was similar
except for the normal presence of less densely ciliated areas and well
organized networks of supraependymal nerve fibers. In hydrocephalic
animals extensive ventriculomegaly produced marked alterations in the
ependyma of both ventricular walls. On the LW, cilia were much reduced
in density and extensive areas of smooth apical cell surfaces occurred;
microvilli persisted but nerve fibers appeared less numerous. The MW
sustained more extensive damage. Not only was the density of cilia
reduced, but discontinuties of the ependyma occurred. Breaches in
ependyma ranged from small punctate openings to extensive areas (900
x 600 p)where the ependyma was so tenuous as to expose masses of
subependymal cells and processes. The supraependymal neuronal
plexuses were disturbed and the supraependymal neuronal network was
attenuated. lntraventricular macrophage-type cells, rarely seen in
controls, were observed in hydrocephalic animals. Studies are in
progress to determine t h e time-course of hydrocephalus-induced
changes and to analyze the ultrastructural features of alterations in the
ependyma and the supraependymal neuronal network.
(Supported by a grant from the Childrens Hospital of Michigan).
GEST. Thomas Rlchard and Gene Louis COLBORN, Center for
Clinical Anatomy, Medical College of Georgia. Augusta. Georgia.
Teaching clinicallv oriented human gross anatomv with Dersonal
computers and interactive videodisc.
The current decltne of graduate training in gross anatomy in
many American medical schools may ultimately result in a shortage
of well-trained instructors for human gross anatomy. This,
combined with the need for more self-paced instruction in medical
curricula as well a s an increased need for computer literacy among
medical students, is incentive for the creation of computer assisted
instructional (CAI) materials for medical education and specifically
for instruction of human gross anatomy. At the Center for Clinical
Anatomy. we are actively developing CAI materials through several
projects. One project in which we have become involved is the
development of Level 111 Macintosh software for The Anatomy Project.
The Anatomy Project is a series of 25 laserdiscs. produced by
Scholastech Limited. London, which covers completely all subjects of
clinical gross anatomy. We are using the Slice of Life IV videodisc
and the Medical Disc Reporter Shared Disc Ill videodisc for the
creation of interactive videodisc tutorials, using Hypercard for the
Macintosh. We are using the MacAnatomy collection of anatomical
computer illustrations (produced by MacMedic Publications1 to
create Hypercard interactive tutorials for gross anatomy. In
collaboration with colleagues in the Department of Anatomy and Cell
Biology, University of Michigan. we have created a database of 2.200
review questions for gross anatomy and embryology. for use on
either PC or Macintosh platforms. The text Practical Gross Anatomy
by Gene Colborn. used a s the textbook for medical gross anatomy at
MCG. was produced using a desktop publishing package on the
Macintosh. We are currently transporting the text and illustrations
into Hypercard. and will produce an interactive electronic textbook
for human gross anatomy. Richard Fbwe. Carrie DiLorenzo. and
Gary Berlin, also of the Center for Clinical Anatomy, have produced
an electronic atlas of neuroanatomy and are developing animated
tutorials of neuroanatomical subjects. Several other CAI projects are
in initial stages. Analysis of the enectiveness of the various CAI
materials will be performed in an attempt to quantitate the possible
benefit of such materials. both for the student and for the instructor.
OGIIEEFW,*
nierry,
LE BMC* and mique
RoDRIGuEZ-E03LAN*, kpartnent of C e l l Biolcqy and
Anatomy, Cornell University Medical College, New-York,
New-York. Induction of micrwillus inclusion i n an
e D i t h e l i a 1 cell line.
Using different drugs disrupting microfilaments o r
microtubular network such as cytwhalasine D (1CpM) or
nocodazole (33w) and colchicine ( I W ) respctively,
w e induced the appeafance of vacuolar apical
compartment (VAC) in an mtestinal cell l i n e Caco 2.
These inclusions w i t h inwardly facing brush-border
microvilli are similar t o those normally observed in
other epithelium under hormonal stimulation and in
patholcgical situations as cancer and the enteropathy
named congenital microvillus atrophy. O u r puzpose was
f i r s t t o characterize these vacuoles i n this i n v i t r o
system using electron
microsc~py and
innnunocyty+mist~~.
second, w i t h a lanthanum n i t r a t e
stamlng w e confirmed that these VAC structures were
not contiguous with the external medium when they were
induced by microtubules-disrupting agents.
Third,
using laser scanning confocal microscopy, we examined
the origin of these microvillus inclusions, i.e. were
they synthesized de nwo o r derived from the apical o r
basolateral plasma membrane domain.
When either of
these two different damins w e r e selectively labeled
before the drug-treatwnt, it appeared that these VAC
structures are synthesized de novo. After reroving the
"g
,M allowing the cells t o rewver,
these
nucrovillus inclusions rarely fused w i t h the apical
plasma membrane but appeared t o be degraded. Fourth,
some preliminary biccheinical data suggest that the
t o t a l pattern of plasm membrane proteins is perturbed
a f t e r cytochalasine D treatment. Finally the presence
of VAC structures in this in v i t r o system provides a
useful tool t o study the respnse of an epithelium t o
this mdification and shade soiw l i g h t on the origin
and the mintenance of epithelia1 polarity.
ABSTRACTSAAA 103RD MEETING
GILLOTEAUX, J., VELUCHAMY*, I., KOSEK*, E., KELLY*, T.R.,
Departments of Anatomy and Surgery, NortheasternOhio
Universities College of Medicine, Rootstown, Ohio. Gallbladder
BDithelial surface chanaes and aallstone formation in the Svrian
hamster as a result of sex steroid treatment.
Ultrastructuralgallbladder surface epithelium changes
associated with stones inducement by either estradiol (E) or by
both E and medroxyprogesterone (P) were studied by TEM and
SEM. Forty-eight 35-day-old male Syrian hamsters were
randomly assigned to treatment groups (ea of 16) and sacrificed
after respectively 1 and 2 months of treatment: all groups were
fed with rodent diet and water ad libitum (12:12 I:d cycle).
Control hamsters received saline; E-group was subcutaneously
injected every 5 days and a EP-group was injected like group 2
and also received P every third day following E injection.
Gallbladder epithelial surfaces and blood samples were analyzed.
After 30-days, EP cells showed apical bulging apices while Etreated cells accumulate large number of apical vesicles. In EP,
epithelial surfaces show amorphous and crystalline-likestones.
Following 60-day treatment, E cells appear with marked crowding
and have short-blunted and tall microvilli, and marginal blebs.
Lumen shows many minute electron dense deposits. EP cells
also reveal crowding and typical bulging apices; numerous
vacuoles containing a fuzzy component resemblingthat of the
cell coat are surrounded by an abundant cytoskeleton. Large
and numerous crystalline and amorphous stones are detected.
Ten-day E-treatment on a few hamsters induces apical vesicles.
C gallbladders always show well-defined, apical cell margins and
no lumina1 deposits. Without changing the diet and using a 4week long sex hormone treatment, we were able to induce
gallstones in hamsters. This was confirmed by serum lipid profile
changes. One wonders whether mucus is needed to facilitate the
nucleating process for the production of cholesterol as proposed
in other species and in man. Meanwhile, steroid treatment
stimulates the production of small secretory vesicles which
contribute to or are associated with gallstone formation.
Supported by Akron City Hospital Research Foundation and
Academic Challenge of Ohio to NEOUCOM.
GINN*, Sheryl R., George W. LANFORD* and Gary M.
PETERSON, Department of Anatomy and Cell Biology, East
Carolina University School of Medicine, Greenville, North
Carolina. Hiuuocamual fibers immunoreactive for nerve erowth
factor receutor have a uattern of innervation similar to AChEpositive fibers.
Numerous reports have indicated that nerve growth factor
(NGF) exerts neurotrophic effects on the cholinergic neurons of
the basal forebrain. Receptors for NGF (NGFr) have been
demonstrated on cholinergic perikarya in the medial septum,
diagonal band of Broca and basal nucleus of Meynert. These
neurons provide the major cholinergic innervation to the
cerebral cortex and hippocampus.
The present paper
demonstrates the presence of an NGFr-immunoreactive fiber
plexus in the rat hippocampus which is strikingly similar to the
pattern of cholinergic innervation.
Sections through the
hippocampus were stained immunocytochemically for NGFr
using the monoclonal antibody 192 IgG, and adjacent sections
were processed for acetylcholinesterase (AChE) histochemistry.
NGFr and AChE reactive fibers were found in the strata oriens,
pyramidale and radiatum of hippocampal subfields CA1 and
CA3. The most intense staining was found in the stratum
granulosum and both blades of the molecular layer as well as
in the hilus of the dentate gyrus. Transections of the fimbriafornix, which disrupt fibers projecting from the medial septum
to the hippocampus, virtually abolish the innervation pattern of
37A
both NGFr and AChE. These results provide additional
evidence that NGFr are associated with septohippocampal
fibers.
Supported by the Alzheimer’s Disease and Related Disorders
Association.
GLENDENNING, K. K., R. B. MASTERTON, K. A.
HUTSON’, R. J . NUDO*, Department of Psychology,
Florida State University, Tallahassee, Florida;
Department of Anatomy, University of Connecticut
Health Center, Farmington, Connecticut;
Department of Neurobiology & Anatomy, University
of Texas Health Science Center, Houston, Texas.
Ventral nucleus of the lateral lemniscus: nontonotopic organization.
Although the demonstration of tonotopic
organization in central auditory nuclei was once
a favored topic of auditory electrophysiology,
the ventral nucleus of the lateral lemniscus
(VLL) stood alone in defying demonstration of
tonotopicity. On the chance that an
electrophysiological map in VLL might have been
missed due to the presence of several small maps
instead of a single large one, we collected
several kinds of tract-tracing materials
pertinent to the question. In addition to
retrograde tract-tracing cases (with HRP
injections in the high and low frequency parts of
the inferior colliculus) and anterograde tracttracing cases (with tritiated leucine injections
in the cochlear nuclei o r superior olives), we
examined 2-DG cases using low-pass o r high-pass
noise for stimulation (i.e., trans-synaptic
orthograde tract-tracing) and retrograde tracttracing cases with 2 o r 3 distinctive tracers
injected into different loci within the
colliculus. Each of these types of experiment
verify the tonotopicity previously demonstrated
in the cochlear nuclei, the lateral superior
olive and the dorsal nucleus of the lateral
lemniscus. However, no frequency map nor
combination of several maps could be found in
VLL. Therefore, we conclude that though VLL may
be a complex of several nuclei, probably none are
tonotopically organized in the usual sense.
Supported by Grant No. DC-197 from NIH-NIDCD.
GOLDEN,* Gregory T., Patricio F. REYES,* Angelina W. CASAS,*
George G. SMITH.* James H. KLILP,* Ilona E. BAUER* and Shu
Tung LI,* Research Service, Veterans Administration Medical
Center, Coatesville & Neurology, Thomas Jefferson Medical
College, Philadelphia, Pennsylvania. (Sponsored by Joseph
T. Weber) A comparison of porous bovine type I collagen
conduits with peripheral nerve grafts in a transected rat
spinal cord model. To date, no one has been able to demonstrate growth of significant numbers of axons intoor across
a lesion of the mammalian spinal cord. The goal of this
study was to test the hypothesis that porous collagen type
I conduit is equivalent to sciatic nerve graft in promoting
and guiding axonal growth in damaged spinal cord. Nineteen
rats were anesthetized and had either complete (14) or
partial (5) spinal cord transections performed. Completeness of the spinal cord transection was verified by examining the tissue gap. Immediately after spinal cord transection, rats had a porous collagen conduit (Colla-Tec. Inc.)
placed to connect both ends of the cut spinal cord. Penetration of the collagen conduit was assured by using a
ABSTRACTS-AAA 103RD MEETING
glass rod with a 15011tip to push the conduit into the cut
faces of the spinal cord. This resulted in some local
damage at the site of insertion of the conduit, but assured
a good interface between conduit and spinal cord. Two
additional spinal cord transected rats had autologus sciatic nerve grafts inserted into the two cut ends of the
spinal cord in an identical manner. At varying times,after
transection and placement of conduits (9 - 180 days), animals were anesthetized, perfused with saline followed by
10% formalin and the spinal cords removed. The thoracolumbar cord was sectioned (5-1Ou) horizontally. Sections
stained with Kluver-Barrera's LFB-CV, H&E. Masson's trichrome, Van Gieson and Bodian's silver were examined by
light field microscopy. Results showed that the presence
of a collagen conduit retarded necrosis, at least at the
proximal end, of the transected spinal cord. In some cases
clusters of neurons, neurites, astrocytes, fibroblasts,
and chronic inflammatory cells occupied the lumen of the
conduit. Cooling the spinal cord prior to transection, to
reduce necrosis around the area of transection, retarded
any reorganization in the collagen conduit. Supported by
VA funds.
GOLDFINE, Steven M. and Donald A. FISCHMAN, Department of Cell
Biology and Anatomy, Cornell University Medical College, New
York, New York. Mvosin h e a w chain release from mvofibrils and
synthetic thick filaments
We have developed a cell-free system using rabbit reticulocyte lysates (Bouche et al., 1988 JCB 107:587) for analyzing
the protein interactions involved in myofibrillar assembly and
turnover. Previously, we used this system to study the
incorporation of newly synthesized myosin heavy (MHC) and
light (MLC) chains into myofibrils or synthetic myofilaments
prepared from purified myosin (Goldfine et al., 1989 UCLA
Symp. Mol. Cell. Biol. 93NS:271). Here we report on the
release of MHC from these substrates. Myofibrils or myofilaments were incubated for 1 h in lysates after which
samples were clarified by centrifugation (60 m at 100,000x
g), The amount of soluble MHC released into the lysates was
determined by an immuno-slot-blotassay using the monoclonal
antibody MF20 and an alkaline phosphatase conjugated second
antibody. Results were quantified by densitometry. -7 Mg/ml
of soluble MHC was released from 2 mg/ml myofibrils. The
released MHCs were not proteolyzed as judged by Western blots.
Assuming myosin accounts for 40% of all myofibrillar protein,
-1% of the added myosin was released. No MHC was detected when
myofibrils were incubated in LSB (100 mM KC1, 5 mM MgCl,, 5mM
EGTA, 1 mM DTT, 30 mM NaN,) although -0.1% of the added myosin
was released into LSB with 5 mM MgATP. Significantly more MHC
was released from synthetic thick filaments under the same
conditions. -10% of the filamentous myosin added to lysates
was released while -0.3% and -1.25% was released into LSB or
LSB with MgATP, respectively. This suggests that thick
filament structure and/or composition effects the rate of
myosin subunit release. In addition. more MHC was released
from both substrates into lysates than into physiological
saline. Preliminary results with muscle lysates demonstrate
the same phenomenon. These observations suggest that cellular
extracts contain factors which facilitate MHC release. Such
factors may be of importance during muscle growth, atrophy or
hypertrophy. (Supported by NIH grants AR32147 to DAF and
AR07923 to SMG).
Goslow,* G.E., Jr., A.A. Biewewer, K. P. Dial* and
F.A. Jenkins, Jr., Section of Population Biology,
Morphology and Genetics, Brown University,
Providence, RI; Department of Organismal Biology and
Anatomy, the University of Chicago, Chicago, IL;
Division of Biological Sciences, University of
Montana, Missoula, MT; and Museum of Comparative
2b%
w.bL!desx.nJ.E4.Mfu2m
(Sturnusvuloaria1 .
Zoology, Harvard University, Cambridge, MA.
L4eUalcontrolpfw:
iasstarllno
.
An understanding of the mechanics and neural
control of flapping flight requires knowledge of
muscle contractile patterns, force generation and
the kinematics of the shoulder and wing. European
starlings (Sturnus vulaarls
' ) flying in a wind tunnel
were filmed and simultaneous electromyograms (EMGs)
from select flight muscles recorded. In some
experiments, in Yiyp strain gage measurements from
the deltopectoral crest of the humerus were also
recorded. The primary downstroke and upstroke
muscles begin their electrical activity midway
through the upstroke (M. pectoralis) and downstroke
(supracoracoideus)which suggests that each muscle
undergoes an U stretch - shorten contraction
with each wingbeat. Although EMG activity in the M.
pectoralis ceases during the first third of the
downstroke, strain is maintained through 85% of the
downstroke. These findings are consistent with the
electromechanical delays associated with M.
pectoralis contractions and when coupled with rates
of muscle length change, can provide an estimate of
power. Supported by NSF grants: BSR-87-06820, DCB87-18727, & BNS-89-08243.
S
GRAAAM,* Charles A. and Peeyush K. IALA, Department of
Anatomy, University of Western Ontarjo, London, Canada.
Molecular mechanisms controllinq trouhoblast jnvasion.
Studies from this laboratory have shown that first
trimester human trophoblast cells share in vitro invasive
properties of malignant cells. However, trophoblast
invasion of the pregnant uterus is highly controlled. The
present study examined the role of first trimester decidua
in the control of trophoblast invasion. First trimester
human trophohlast cells from third and tenth passage
cultures were laheled with 1251-deoxyur5dine and examined
for their ability to invade an epithelium-free human
amniotic membrane in vitro under various conditions. The
degree of invasion was determined as the percentage of the
radioactjvity retained wjthin the membrane. Third passaqe
trophoblast cells exhibited a high deqree of invasion
(11.24% 5 2.97) when assayed alone in RPMJ/lO% FCS medium.
However, addition of 7d culture supernatant (15%) of first
trimester human decldual cells reduced this invasion to
0.36% 5 0.09 (p = 0 . 0 0 2 5 ) . This suppression was prevented
by addition of neutralizing anti-TGFg antibody (25pg/ml)
(15.71% 50.86, p = 0.0034) and was mimicked by addition of
porcine TGFBl (10ng/ml) (0.36% +- 0.01). Tenth passage
trophoblast cells had an invasion index of 7.15% 5 0.76
when assayed alone. Addition of decidual supernatant
reduced invasion to 2.63% 5 0.44 (p = 0.0004). However,
anti-TGF B antibody in this instance produced only a minor
relief of the anti-invasive effect of decidual supernatant
(4.11% +- 0.95 vs 2.63% +- 0.44 for trophoblast + Cecidual
supernatant. p = 0.1680). Furthermore, trophoblast cells
assayed in the presence of T G F B alone exhibited an
incomplete reduction in their invasiveness (5.24% 50.37 vs
These
7.15% +- 0.76 for trophoblast alone, p = 0 . 0 5 3 4 ) .
results indicate that: (a) first trimester decidua prduces
molecules which can control trophoblast invasion: (b) TGPB
is a key member of this family of molecules: (c)
trophoblast sensitivity to anti-invasive action of TGFB may
vary after long term culture. The anti-invasive mechanisms
of TGFB and other products of the decidua remain to be
elucidated. (Supported by the MRC and NCI Canada).
Green.' C. Todd, Jsines J. Tomasek, Robert D. Fllgate,' and
Ronald L .
Shew, Department of Anatomical S c i e n c e s ,
ABSTRACTSAAA 103RD MEETING
S
Universjty of Oklahoma Hcalth Sciences Center, Oklahoma
City, Oklaiioma . (Sponsored by Kimiko Dugan) AcJe:depeIngIt
-i-ncrease
.
of type I collaqen fluorescence in-ra_t_-c.o.rJj&zc.l
bone.
Humin skirl collagen and dura mater increase in
fluorescence with age.
It is brlieved this greater
flusrescence results from an increase in nonenzymatic
cross-links i n collagen. Nonenzymatic cross-links are
derived from tlie reaction of glucose with free NZI, arsups
in collagen to form a Schiff base adduct which subsequently
iinrlergoes a series of reactions that result in the
formation of Advanced Clpcosylation End-products (AGEs).
AGES rapidly reart with free NH, groups of other collagen
moiecules to form nonenzymatic derived covalent crosslinks. In this study we examined whether collagen of bone
undergoes an increase in nonenzymatic cross-links with age
as reflected by an increase in fluorescence. Femurs and
tibiae were obtained from 3 and 30 month old Fisher 344
rats. Demineralized cortical bone powder was obtahed from
femurs, sol.ubilized wilh col.lagenase and then measured for
fluorescence at 440 nm upon excitation at 370 nm. The
amount of collagen was determined bi hydroxyprolirie assay
and the level of fluorescence expressed per mg of rollagen.
The fluorescence of the collagen from 30 month cortical
bone was significantly greater (pc0.001) than at 3 months
(25.36 t L.D7 vs 12.63 f 0.84 mg/collaqen: n=10). In all
experiment.s tlie 30 to 3 month ratio was approximately 2:l.
We have demonstrated for the first time: 1) that cortical
bone type 1 collagen possess AGEs; and 2,) these
norienaymatic cross-links increase with age. The increased
collagen cross-linking in bone matrix may play an important
role in age-associated alterations in borie, such as
derreased mineral content and breaking strength. (0. Todd
Green was supported by a Presbyterian Hefris Fellowship.)
GULATI, A.K., Department of Anatomy, Medical
College of Georgia, Augusta, Georgia. A
comparison of skeletal muscle reaeneration in
normal and streptozotocin induced diabetic
39A
HAAS.Kent S. *, Steven J. PHILLIPS, Anthony J. COMEROTA', John V.
WHITE'. Departments of Vascular Surgery and Anatomy,Temple
University, Philadelphia, Pennsylvania.
The-e
brchltecture of t he sdventltla of canlne I nfrarenal a b m
a-!ml
The elastin and collagen architectureof the arterial wall is the mapr
deteminant of its stfudual integrity. visco-elastic properties, and
hemodynamics. Considerable investigationhas focused on the
morphology of the intimal and medial layers of vessels in normal and
diseased states. The importanceand possiblycritical contributionof the
adventitiallayer has not been well characterized. We have studied the
morphological configuration of collagen and elastin in the adventitiaof
canine infrarenalabdominal aorta. A special technique was devebped to
examine adventitia prepared with the Weigert Van Geison elastin stain
using a combination of high resolutioin light and SEM. Segments of canine
infrarenalaorta fixed at 0 mmHg or 100 mmHg were sectioned in
transverse, longitudinal,and frontal planes and stained. The histological
preparations were examined with high resolutionlight microscopy ,
photographed, and the presence and configuration of collagen and elastin
recorded. These same sections were then demounted, prepared, and
examined with SEM to permit 3-0 reconstrudion of the identical area of the
sections which had been visualized with LM. To further characterize the
elastin network, additional segments of vessel were digestedwith 88%
formic acid at 45C- for 72 and 96 hours. prepared and analyzedwith SEM.
Collagen fibers are arranged predominantly in circumferentially aligned
layers displaying a wavy crimp with a periodidty of 67T7um, consistent with
Type Icollagen. Elastin appears in fibrillar sheets with fenestrations. The
axis of the fibrillar component of elastin is primarily in the longitudinal
direction. The adventiiia consists of a regularly alternatingpattern of these
two proteinswith longitudinaland circumferentialaxes approximately
perpendicular to each other. The 72 hour fonnic acid preparations
confirms the 3-D architecture of elastin. Further digestion with formic acid
causes adventitial elastin sheets to appear as a longitudinalfibrillar matrix.
Although the absolute elastin content appears to be significantly less than
the collagen content, its constant and intimate association with the latter
supports the importance of the interdependence of these two proteins in
the adventitia. These studies demonstrate an anatomic interrelationshipof
collagen and elastin in the adventitia of canine infrarenalaorta. Previous
studies analyzing the mechanics and protein composition of the arterial wall
must be interpretedwith this critical point in mind. We hypothesize that
the adventitia may function similarly to the media as a WOphase" material.
While visco-elastic properties of the media are most important at bwer
pressures, adventitialmechanics may be predominant when higher arterial
pressures and wall tension are present.
rats.
Regeneration of diabetic skeletal muscle
was investigated in rats. Intravenous
injection of streptozotocin ( 6 5 mg/kg, 0.1M
citrate buffer) was used to induce diabetes.
Extensor digitorum longus (EDL) muscles from
six-week diabetic rats, were either
transplanted into diabetic or normal hosts to
initiate regeneration. Normal EDL muscle
transplants in normal and diabetic hosts were
also performed for comparison. One, 2, 4 and
12 weeks after transplantation the EDL muscle
regenerates were morphologically analyzed. A
significant reduction in the mass of diabetic
muscle as compared to normal muscle was
observed. Regeneration and formation of
neuromuscular junctions were observed in all
transplants, including diabetic regenerates in
diabetic host. The overall mass of the
diabetic EDL regenerate in the diabetic host
was significantly reduced in spite of complete
regeneration. Recovery of the diabetic muscle
mass and myofiber size was observed after
transplantation into normal hosts. The results
demonstrate the regenerative ability of
diabetic muscle. It is concluded that poor
recovery of diabetic muscle is related to
altered metabolism in the diabetic host, rather
than to innate capacity of the diabetic muscle
to per se undergo regeneration and
reinnervation. The observed enhancement in
recovery of diabetic muscle after
transplantation in a normal host indicates that
the host metabolic environment determines the
success of muscle regeneration.
HAINES, D.E. and R.M. LIBBIN, Department of Anatomy,
University of Mississippi Medical Center, Jackson,
Mississippi; VA Medical Center, Brooklyn. New York and
Department of Anatomy, New York University College of
Dentistry, New York. Some conceDts of neural structure
and function, 1853-1918.
While recognizing that the cerebellum may function in
motor and sensory areas Carpenter (1853) offered
evidence, both basic science and clinical, that this
part of the nervous system is "the organ of sexual
instinct". He further described the case of a military
officer who, on the eve of his marriage, received a blow
to the occiput which rendered him impotent. The young
man was so distressed that he committed suicide on the
morning of his wedding. Although some early concepts on
structure and function in the nervous system have stood
the test of time (Brodmann, 1909 on the cerebral cortex)
others, which at one point were dogma, did not hold up
under later scientific scrutiny. This rare book display
will highlight selected ideas and passages on the
structure and function of the nervous system as seen
through the works of W.B. Carpenter (1853, 1856 Principles of Human Physiology), R. Dunglison (1860 - A
Dictionary of Medical Science), C. Jakob (1901 - Atlas
of the Nervous System), K. Brodmann (1909 Vergleichende Lokalisalionslehre der Grosshirnrinde),
and E. Villiger (1918 - Brain and Spinal Cord: A Manual
for the Study...of the Central Nervous System).
Excerpts from these books (text and illustrations) will
be featured on the poster and the books themselves will
be available for first hand examination. In addition
40A
ABSTRACTS-AAA 103RD MEETING
-
t h i s d i s p l a y w i l l a l s o f e a t u r e R . L i s t o n (1842
P r a c t i c a l Surgery), G . McClellan (1892 - Regional
The Anatomy
Anatomy, Vol. XI), and H. C . Raven (1950
of The G o r i l l a ) . This l a t t e r book h a s a n unusual
f e a t u r e of f o l d - o u t pages showing t h e e x t r e m i t i e s i n
f u l l s i z e . O f i n t e r e s t is the f a c t t h a t some of t h e s e
volumes were published by Blanchard and Lea (1853, 1856,
1860), W. B . Saunders (1901), and J. B. L i p p i n c o t t
(1918); t h e s e houses continue t o s e r v e t h e medical
e d u c a t i o n community.
-
HALATA', Z., R.D. JOHNSON*I and T. STRASMANN*',
'Division of Functional Anatomy, Department of Anatomy, University of Hamburg, West Germany, and Tollege of Veterinary
Medicine, Gainesville, University of Florida. ToDomaphv and
ultrasuucture of sensorv nerve endings in the rat Denis.
Three types of sensorynerveendings supply the penis of the
white laboratoryrat: free nerve endings, lamellatedcorpusclesand
Ruffinicorpuscles.Free nerve endings are situated (1)in the epithelium of the glans penis between the epithelial spines, (2) in the
epithelium of the urethra and (3) in the papillary layer of the
dermis. They are derived from A-delta or from C fibers,respectively. Free nerve endingsof the epithelium reach the granular layer
and reveal button-like axonal thickenings. In the dermis, free
nerve endings are only partly covered by Schwann cell processes.
Some terminalsabut directly upon the basal lamina of the epidermis or of the mucosa. The lamellated corpusclesare situated (1)
in the papillary layer of the dermis between the basal parts of the
epithelial spines (i.e., not beneath the spines),(2) in the connective
tissue septa of the corpus cavernosum, and (3) in the connective
tissue beneath the mucosa of the urethra. The terminal axons are
within inner cores. 4-10inner cores twisted spirally around each
other can be found per corpuscle. A complete penneural capsule
surroundsthe corpuscle. 1or 2 myelinatedparent axons (diameter
about 3-5 pm) innervate one corpuscle. Rufini corpuscles are
present within the connective tissue between the OS penis and the
urethra. These corpuscleshave an incomplete capsule, one type is
composed of fibroblasts, the second type of perineural cells.
Ruffini corpuscles are supplied by myelinated afferent axons of
about 4-6 pm in diameter. These axons branch into terminals
anchored between collagen fiber bundles. Neither Merkel nerve
endings nor large Vater-Pacini corpuscles are present.
S
HALLIDAY,' Mancy L. and Janies J . TOMASEK, Department of
Anatomical Sciences, l l n i v e r s i t y of Oklahoma Health
Sciences
Center,
Oklahoma
City,
Olrlahoma.
Qeqelymerization of microtubules can enhance r a ~ d
c o n t r a c t i o n of c o l l a a e n l a t t i c e s by fibcoklefis.
S p e c i a l i z e d C i b r t b l a s t s have been proposed t o p l a y a
r o l e i n the c o n t r a c t i o n of t h e palmar f a s c i a i n
Dupuytren's d i s c a s e . These c e l l s c o n t a i n bundles of a c t i n
microfilaments with a s s o c i a t e d nonmuscle myosin ( s t r e s s
f i h e r s ) . To study the c e l l u l a r mechanisms important i n
t i s s u e c c n t r a c t i o n , f i b r o b l a s t s from Dupuytren's d i s e a s e d
palmar f a s c i a were c u l t u r e d w i t h i n a t t a c h e d 3-dimensional
hydrated c o l l a g e n l a t t i c e s . F i b r o b l a s t s c u l t u r e d i n t h e s e
l a t t i c e s f o r 5 days w i l l form s t r e s s f i b e r s . Upon r e l e a s e
from t h e underlying substratum, c o l l a g e n l a t t i c e s
c o n t a i n i n g f i b r o b l a s t s w i l l undergo a very r a p i d
c o n t r a c t i o n ; a t l e a s t a 40% reduction i n l a t t i c e diameter
w i t h i n 10 minutes.
There i s a w e l l spread microtubule
network a s s o c i a t e d with t h e s t r e s s f i b e r s i n t h e s e c e l l s .
Tlrf r o l e of microtubules i n c e l l c o n t r a c t i o n i s not !mown.
In t h i s study we used the drug nocodazole t o depolymerize
the microtubule rietwork. Depol.ymcrization of micmtuhiiles
with 5 ug/ml of nocndaaole p r i o r t o r e l e a s e o f the
collagen l a t t i c e ,
increases
the amount of
rapid
c o n t i a c t i o n . I t was a l s o observed t h a t the phorbol e s t e r
tumor promotor, phorbol 12-myristate 1 3 - a c e t a t e (TPA), a t
a c o n c e n t r a t i o n of 100 ng/ml, will. t o t a l l y i n h i b i t r a p i d
c o n t r a c t i o n by an unknown mechanism. Rapid c o n t r a c t i o n of
c o i l a g e n l a t t i c e s by f i b r o b l a s t s t r e a t e d with TPA i s
r e s t o r e d t o c o n t r o l l e v e l s when t r e a t e d with nocodazole.
These r e s u l t s suggest t h a t microtubules may play a r o l e i n
r e g u l a t i n g t h e amount of c o n t r a c t i o n generated by s t r e s s
fibers.
~licrotubu1.e r e g u l a t i o n could occur by two
d i f f e r e n t mechanisms: 1) microtubule elongation may
count.erhalance t h e c o n t r a c t i l e f o r c e e x e r t e d by s t r e s s
f i b e r s , o r 2 ) depolyjinerization of microtubules may r e l e a s e
associated
proteins
which
regulate
stress
riber
c o n t r a c t i o n . Experiments a r e undei-way t o examine tiiese
t o o p o s s i b l e c o n t r o l mechanisms.
(Supported by a Grant
Prom the P r e s b y t e r i a n Realth Foundatlon.)
HPMLFPT, William C.. . Demrtnmt
of Anataw,
. . Medical College of Ohio.
mledo, Ohio. Electrol MiQosmpic Investigations of the Fetal hkrdxnes
and uterus i n the Bladawe ShKk, Carchamnus acZKmJtu.5.
Ihe blackuse shark is a viviparms d o t e that develan
epitheliochorial yolk sac placenta. The surface cells of the mhilical
wrd are stratified, generally forming three layers.
A triplrtite
-ex
of [email protected], axldens and adherens jmcticns join the surface
cells a t their 1umxe.l buder, W e ram~mus
desmxnes attach cells in
a l l three layers. The surface c e l l s are udestly flattened w i t h apical
[email protected]
', UIIEW
IS
lateral h f o w s and a prachent terminal web.
ELectron dense, memtorane-limited
granules oaxlr i n the qtcplas of
cells of the top two layers. There is a large poprlaticn of these
vesicles W t e l y kneath the apical plasmlemm of the surface e l l s
and l i t t l e evidence of Mdocytcsis or exocytosis. The u n h i l i c a l card
p w consists of an d i l i c a l vein, umbilical artery, ciliated ciuctus
v i t w o i n t e s t i m l i s and e x t r a d x y n i c welon, all joined by d
ocnnective w e investxent rich i n wllaqen, fiLnohlasts, wth
M e and a rich, a w e n t l y h-ated,
extracwular matrix. me fetal
p r t i m of the uteroplacental c a p l e x oansists of a pxdml prtim
that forns saccular evaginations. The cells are stratified squanous
w i t h surface micmvjJ.li
and lateral hfoldicgs with diLated
inte~~%Uular
spaces. This segmmt aFpears to be involved in H 0 reghtion. The egg envelm inteKVmes between the distal
of the
placenta and uterus. It has delaninations m the uterine srnface and is
solid m the placental surface. Ihe disw p r t i m of the placenta is a
bilayer. !Tm &ace
layer is p r t i a l l y ercded ~ c myh be associated
w i t h placental detadment a t parturitim.
An elatorate array of
micrarilli between the two layers form an intaface m.Cells of the
lower layer are characterized by pauinent RER ad Golgi.
Dense
ncn-mdxane tand graiwJ.es oaxlr in the interspc? between the eg3
envelop and the distal placenta. Tnis m a t e r i a l , preSmb1y of uterine
origin, is e x k q t m d i n srmth-walled vesicles of the placental czlls.
Ihe en%thefium of the capillaries is fenestrated.
Ihe uterim
epithelium is a k o i c b l to wllmnar and is characterized by pmchent
RER, Golgi, plyxues and secretion vesicles w i t h electrcn dense
Ccntents.
The capillary endothelim is ccmtinmxs.
Nutrient ad
respiratory excharge is effected ktween the uterine epithaiwn, the egg
envelop and the distal -on
of the placenta.
prtik
HAMMER, Ronald P., JR., Craig P.S. KIM* and Jody E. MARGULIES,* Department of Anatomy & Reproductive Biology,
University of Hawaii, Honolulu, Hawaii. Sexuallv dimomhic nuclei
associated with cvclical u-ooiate receotors in rat oreootic are&
A population of p-opiate receptors which is cyclical and
sexually dimorphic has been identified in the medial preoptic area
(MPOA) of the rat (Hammer, Brain Res., 308: 172, 1984). The
density of these receptors in the MPOA is gonadal steroid hormone-dependent (Mateo, et al., this volume) and varies across the
estrous cycle in females. In order to define the anatomy underlying
this receptor population, males or females in proestms or diestrus
ABSTRACTSLAAA 103RD MEETING
were either perfused with 4% paraformaldehyde in 0.1M phosphate
buffer or decapitated, brains were removed and frozen with or
without prior 12.5% buffered sucrose treatment, sectioned at 40 pm
in a -14°C cryostat and thawmounted on gel-coated glass slides.
Serial autoradiographs through the entire MPOA were examined
using a high resolution, wide-field microscope equipped with
camera lucida. The regions containing dense preceptors were
traced together with regional landmarks, which were used t o align
the Nissl-stained sections. Boundaries of the medial preoptic
nuclear divisions determined o n the basis of cytoarchitechtonic
features were then superimposed onto autoradiographic tracings.
Serial neuroanatomical and autoradiographic data were digitized
using a Summagraphics Bitpad and HVEM 3-D Reconstruction
software. Several serial sections contained medial, lateral ( I ) and
central components of the medial preoptic nucleus (MPN) and in
each section, the region of densest preceptor labeling was located
in the MPNl only in diestrus females. Caudally, an oblique
receptor-dense region extended dorsolaterally toward the preoptic
continuation of the bed nucleus of the stria terminalis in all
animals. Thus, sexually dimorphic and cyclical preceptors located
in the MPNI represent the rostral extension of a receptor continuum present in both sexes. Supported by USPHS Awards DA04081,
NS01161 and RR08125.
S
HANES,* M.A., R.T. ROBERTSON and J. YU, Departments of
Anatomy and Neurobiology and Physical Medicine and Rehabilitation,
College of Medicine, University of California, Irvine, California.
visual cortex: imulications for the timecourse of veniculocortical
develoument>
Paiterns of acetylcholinesterase (AChE) histochemical staining in
primary visual cortex of developing animals appear quite different from
those seen in mature animals. AChE in visual cortex of mature rats
appears primarily in infragranular layers, and is found associated with
terminal fields of axonal projections from basal forebrain. In developing
rats, particularly during the 2nd and 3rd postnatal weeks, a transient
pattern of AChE is found in the deep part of layer I n and in layer IV.
This transient AChE activity is associated with terminal fields of
geniculocortical projections. The present studies were undertaken to
determine the time course of change from developing to mature AChE
patterns in rat visual cortex.
Adult Sprague-Dawley rats and pups aged 8-40 days (P8-40) were
used in these experiments. Animals were anesthetized with ether or
sodium pentobarbital and chloral hydrate. Using a stereotaxic approach,
electrolytic lesions were placed in the basal forebrain, including the
nucleus of the diagonal band, or in the dorsal thalamus, including the
lateral geniculate body. Following survival times of 4-5 days, animals
were perfused with aldehydes and processed for AChE histochemistry.
Lesions of the basal forebrain result in an almost complete loss of
AChE in visual cortex of adult rats, while lesions of the lateral geniculate
body had no detectable effect. In contrast, lesions involving the lateral
geniculate body of P-10 rats resulted in a pronounced loss of AChE
activity in layer 111-IV of visual cortex while basal forebrain lesions had
minimal effect. In developing animals, effects of thalamic lesions on
visual cortex AChE activity were progessively reduced, while effects of
basal forebrain lesions were progressively increased. The mature pattern
of AChE activity in visual cortex, as demonstrated by susceptibility to
basal forebrain lesions and absence of impact by thalamic lesions, was
not achieved until PND 40.
We have suggested previously that transient AChE activity i s
characteristic of developing geniculocortical projections (Robertson et
al., Dev. Brain Res., 1988). In this light, the present data suggest that
development of geniculocortical connectivity continues well into the
second month of postnatal life.
Supported in part by NSF grant 87-08515.
HARDY, Steven Gary Patrick, Department of Anatomy,
University of Mississippi Medical Center, Jackson,
41A
Mississippi. The distribution of reticulosDina1 and
reticulohwothalamic neurons within the ventrolateral
medulla.
There is considerable evidence to support the notion
that reticulospinal neurons, within the ventrolateral
medulla, alter autonomic activity by directly
influencing preganglionic neurons of the thoracic spinal
cord. I n addition to reticulospinal neurons, the
ventrolateral medulla also contains neurons that project
to the posterolateral hypothalamus, an area with known
autonomic involvement. Consequently, this latter group
of neurons might also influence autonomic functions.
The purpose of t h i s study was to compare in detail the
distributions of these two neuronal populations within
the ventrolateral medulla. To achieve this,
microinjections of fluorescent tracers, diamidinodihydrochloride yellow or fast blue, were made into
either the thoracic spinal cord or posterolateral
hypothalamus in a series of anesthetized rats.
Following a 3-5 day survival period, each rat was reanesthetized and processed according to routine
methodology, whereupon it was possible to plot the
distribution of neurons projecting to the thoracic
spinal cord and compare it to the distribution of
neurons projecting to the posterolateral hypothalamus.
Many neurons, labeled from either the spinal cord or
hypothalamus, were observed in close proximity within
the ventrolateral medulla. These neurons were
particularly numerous at, or near, the level of the
obex. At this level of the ventrolateral medulla,
although double labeled neurons were not present,
neurons projecting to the hypothalamus were clustered
ventrolaterally to neurons projecting to the spinal
cord. These findings suggest that functional
interrelationships may exist between medullary neurons
capable of influencing the hypothalamus and medullary
neurons capable of influencing the spinal cord.
Supported by BRSG Z-SO7-RR05386.
HAROIAN,
Alan J.
and Kevin
M. BAUMLIN*,
Devartment of Anatomv.
_ . Hahnemann Universitv.
Philadelphia, Pennsylvania. The rootlets of
all: the human brain stem.
----We
have developed
a
computer assisted
instructional program that reviews the anatomy
of the medulla, pons and midbrain. This is a
tutorial to be used by the beginning student in
neuroanatomy prior
to learning neurological
pathways.
The gross topography of the dorsal,
ventral and lateral surfaces of each brain stem
level is presented.
This is followed by a
series
of histological cross sections from
representative levels through the brain stem.
Successive sections from caudal to rostral are
examined and correlated with the surface topography to assist in building a 3-dimensional
image of the brain.
Emphasis is placed on
surface landmarks, cranial nerves and their
functional components and some of the major
pathways. This program is written in HyperCard
for a Macintosh SE computer.
It utilizes
computer graphics to teach the surface topography and cross sectional anatomy of the brain
stem. The program is linked to a high resolution monitor which is attached to a videodisc
player. Gross anatomical specimens and histological cross sections are coordinated to appear
in concert with the material depicted on the
Macintosh screen. The program includes a glossary of terms and concepts called "Questions of
Interest".
This glossary can be accessed by
"clicking" on boldface words that appear in the
text.
The names of structures to be learned
appear in italics.
Labels will appear on the
drawings when one "clicks" on the italicized
5
42A
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
words. A q u i z a t t h e end of t h e program w i l l
allow s t u d e n t s t o s e l f assess t h e i r p r o g r e s s .
"Supported by the H e a l t h S c i e n c e s L i b r a r i e s
Consortium
in
conjunction
with
The
Pew
C h a r i t a b l e T r u s t s . I'
S
HARRIS," C l i f f o r d H., E l i z a b e t h L . PAGAN*, Ronald L . SHEW
and Daniel L. 1,lcNEILL. Department of Anatomical Sciences,
U n i v e r s i t y of @klaho:na Health Sciences C e n t e r , Oklahoma
C i t y , Oklahoma. ? E 8 0 1 induces i n t r a s p i n a i s p r o u t i n a by
CGRP
-. iinmunoreactive
.
pimarv afferent fibers.
Previous s t u d i e s have demonstrated t h a t the NMDA
r e c e p t o r a n t a g o n i s t , MK-801 induces n e u r i t e extension 4
E%&.
I n t h i s study, t h e p o t e n t i a l f o r MK-801 t o induce
i n t r a s p i n a l s p r o u t i n q by primary a f f e t e n t f i b e r s i n v i v o
was examined. Twelve male Spraqus-Dawlep r a t s were d i v i d e d
Group 1 r a t s (n=4) were u n t r e a t e d and
i n t o 3 groups.
pervsd a s c o n t r o l s . Group 2 r a t s (n=4) r e c e i v e d a riaily
i n j e c t i o n of Wi-801 (1.0 mg/kg) f o r 30 consecutive deys.
Group 3 r a t s (n=4) received an i d e n t i c a l treatment with MK801. However, on day 23 a u n i l a t e r a l d o r s a l rhizotomy (Ll54) was performed.
On day 30, a l l r a t s were perfused and
thc i5 spj.naJ. cord segment removed. Cryostat s e c t i o n s were
o b t a i n e d , immunostaiiied for c a l c i t o n j n y e n e - r e l a t e d p e p t i d e
(CGRP) and v i s u a l i z e d by epi-fluorescence microscopjj. I n
s e c t i o n s frorti group 1 ai?jmals, the d i s t r i b u t i o n of CGRP
immunorcactivity was confined p r i m a r i l y t o laminae I , I1
and o u t e r V . A small number of f i b e r s were a l s o p r e s e n t i n
t.he d o r s a l grey coinmissure.
Following MK-801 treatment
(group 2 ) , t h e CGRP imml?nostaining p a t t e r n i n laminae I ,
I1 and 17 was i d e n t i c a l t o c o n t r o l s .
However, a marked
i n c r e a s e i n t.he d e n s i t y of CGRP i m r l n o r e a c t i v e f i b e r s was
p r e s e n t along the n e d i a l a s p e c t of the d o r s e l horn and i n
the dorsal. grey coinmissure.
Dorsal rhizotomy (group 3)
r e s u l t e d i n a l o s s of jmmunoreartivit.y i p s i l a t e r a l t o t h e
t r a n s e c t i o n s i n d i c a t i n g t h a t the i n c r e a s e i n immunostaininy
was primary a f f e r e n t i n 0rigj.n.
These d a t a suggest t h a t
MK-60: induces i n t r a s p i n a l s p r o u t i n g of primary a f f e r e n t
f i b e r s i n vivo.
S
HELGREN,*Maureen E. and Michael E. GOLDBERGER,.
Anatomy Department, Medical College of Pennsylvania,
Philadelphia, Pennsylvania. (Sponsored by Leonard Ross)
Enhanced afferent control contributes to the recovec of postural
reflelfes and comDlex motor functions in the adult cat after mina1
a r d iniurv,
One compensatorymechanism thought to mediate recovery of
motor function following spinal cord injury is sprouting of
undamaged dorsal root pathways below the lesion. This might
result in increased afferent control of movement. To test this
hypothesis, postural reflexes dependent on afferent input were
analyzed quantitatively pre-operatively and for 3 weeks
ptoperatively following low thoracic hemisection. Initially after
emisection there is reflex depression. The placing response and
monopedal hopping begin to recover on the 3rd dpo, but are
abnormal in amearance. For examde. a dramatic increase in
stimulus intensity required to elicit response is observed and the
flexion and support phases are exaggerated. There is partial
recovery as demonstrated by a decrease in threshold and response
values shift toward normal values by 14 d 0. This refinement of
reflex activity implies afferent control. d e time course of
enhanced reflex control arallels recovery of complex locomotor
behaviors. To test if unfamaged descending athways underlie this
recovery a contralateral hemisection made ager recovery from the
first hemisection was made. No reinstatement of the initial reflex
deficit occurred. These experiments provide additional evidence
that the recovery of reflex control is mediated by afferent systems.
Mapping studies using immunocytochemistry for single lesioned
animals reveal a permanent loss of descending serotonergic and
noradrenergic input to the hemisected side of the s inal cord.
However, using Mab Rat102 from Martin & Hocdeld there is a
compensatoryincrease in centrally projecting dorsal root in ut
which is accompanied by an increase in GAP43 staining. &ese
anatomical results suggest a process of reactive reinnervation by
primary afferent systems occurs in response to hemisection and
may be one mechanism mediating recovery of motor control
consistent with behavioral results.
Supported by NIH Grant NS24707.
S
HENDERSON,* Margaret J e a n and Sandra J e a n OLNEY,*
Department of Anatomy and School of R e h a b i l i t a t i o n
Therapy,
Queen's
University,
Kingston,
Ontario.
(Sponsored by W i l l i a m J . F o r r e s t ) DeveloDment of a
moment-EMG a n k l e muscle model,
The purpose of the study w a s t o develop a model t o
d e r i v e t h e d o r s i f l e x o r and p l a n t a r f l e x o r moments
o c c u r r i n g a t t h e a n k l e j o i n t during walking u s i n g
electromyography (EMG) and kinematics. EMG w a s recorded
from d o r s i f l e x o r and p l a n t a r f l e x o r muscle groups as t h e
s u b j e c t walked.
The s u b j e c t then matched these EMG
amplitudes d u r i n g a l t e r n a t i n g s t a t i c d o r s i f l e x i o n and
p l a n t a r f l e x i o n c o n t r a c t i o n s a t s p e c i f i c a n g l e s , while
f i x e d i n a c a l i b r a t i o n bench w i t h a f o r c e c e l l a t a
known d i s t a n c e from t h e ankle j o i n t .
Using t h e EMG
recordings from opposing muscle groups and t h e
concurrent f o r c e , a r e g r e s s i o n e q u a t i o n was d e r i v e d t h a t
weighted t h e c o n t r i b u t i o n s o f t h e opposing muscle groups
t o t h e f o r c e (moment) o u t p u t and j o i n t a n g l e .
The
e q u a t i o n f o r each
muscle group was of t h e form
M(t)-[K+a(e)-b(e)']EMG where M(t) is t h e instantaneous
muscle group moment, K i s a c o n s t a n t , a , b a r e
c o e f f i c i e n t s , f3 i s t h e i n s t a n t a n e o u s a n g l e , and EMG i s
t h e i n s t a n t a n e o u s amplitude of EMG. Addition of t h e
moments f o r each i n s t a n t i n time from each muscle group
w i t h t h e i r r e s p e c t i v e p o l a r i t i e s gave t h e n e t moment.
The e q u a t i o n w a s a p p l i e d t o t h e EMG and j o i n t a n g l e s
from walking t r i a l s t o d e r i v e t h e instantaneous
d o r s i f l e x o r and p l a n t a r f l e x o r n e t moments. To v a l i d a t e
t h e method, t h i s d e r i v e d n e t moment w a s compared w i t h
t h e n e t moment c a l c u l a t e d from a s t a n d a r d link-segment
( L - S ) model u s i n g c i n e and f o r c e p l a t e d a t a .
The
d e r i v e d moments were less t h a n t h e moments from t h e LS model. The mean RMS e r r o r w a s 1 1 . 7 2 (f3.14) N . m .
The
mean RMS e r r o r as a p e r c e n t of t h e maximum L-S moment
was 15.11 (f13.59) N.m.
This model h a s shown promise
as a method of d e r i v i n g i n d i v i d u a l muscle group moments
about t h e a n k l e j o i n t .
Supported by t h e A r t h r i t i s S o c i e t y and Grant No. MA-8178
from t h e Medical Research Council (Canada)
Mary, Rebecca WOOD,* E l i s a b e t ? SEFTOR,*
*
Ronald
* Sandra
*
Avraham
Tucson,
Houston,
Michigan
Cancer Foundation, D e t r o i t , Michigan.
Retinoic
acid i n h i b i t i o n of human melanoma c e l l i n v a s i o n
t h r o u a h a r e c o n s t i t u t e d basement membrane and i t s
r e l a t i o n t o decreases i n t h e e x v r e s s i o n of
p r o t e o l v t i c enzvmes and m o t i l i t v f a c t o r r e c e v t o r .
The a n t i n e o p l a s t i c e f f e c t s o f r e t i n o i d s have
been r e c o g n i z e d b o t h i n v i v o and i n v i t r o :
however, l i t t l e is known a b o u t t h e i r mechanism of
a c t i o n . Our s t u d y e v a l u a t e d t h e e f f e c t s of B-aal
HENDRIX,
LOT$N,
Motowo
NAKAJIMA,
MISIOROWSEI,
W i l l i a m STETLER-STEVENSON
BEVACQUA,
Lance LIOTTA,
Mark SOBEL,
RA2 and Reuben LOTAN, Univ. o f Arizona,
Arizona; M.D. Anderson Cancer C e n t e r ,
Texas; N C I / N I H , Bethesda, Maryland: and
Dafna
*
ABSTRACTS-AAA 103RD MEETING
trans retinoic acid (RA), ranging from 0.01 to 10
uM, on the invasiveness of four human melanoma
Cell lines in vitro and showed a dose- and timedependent inhibition of the ability of these
Cells to penetrate matrigel-coated filters. The
possible mechanisms of action responsible for the
anti-invasive effect were further investigated,
and the data showed that RA-treated cells:
a)
secreted lower levels of collagenolytic enzymes
detected
in
type
IV
collagen-containing
polyacrylamide gells compared with control cells,
which was demonstrated by a decreased ability to
degrade [3H]proline-labeled type IV collagen
substrate and by a reduction in the acitivity of
the 8 8 and 64 Kd collagenolytic enzymes; b)
showed a reduction in the mRNA expression of the
human type IV collagenase gene and a reduction in
plasminogen activator activity; c) were less
adhesive to matrigel; and d) showed an increase
in the high affinity metastasis-associated cell
surface laminin receptor.
Collectively, these
data demonstrate the multiple inhibitory effects
of RA on invasiveness.
Research supported by NIH/NCI 1R0142475 and the
Robert Bruno Family.
HERBERT. Damon C., Peter J. Sheridan, Frank J. WEAKER,
Christi A. WALTER,* Gwendolyn S. ADRIM.* and Barbara H.
BOWMAN,* Department of Cellular and Structural Biology,
The University of Texas Health Science Center, San Antonio,
Texas.
Reaulation o f t r a n s f e r r i n aene exoression i n
transaeni c mice.
Transferrin (TF) is an iron-binding protein synthesized
in the liver and reported to be associated with cellular
proliferation and differentiation.
It is present in
several tissues including the oligodendrocytes of the brain
and Sertoli cells of the testes. Transgenic mice have been
developed in our laboratory for the study of TF gene
expression during aging. They carry different sequences of
a chimeric gene containing either 670 or 1200 base pairs of
the 5' flanking region of human TF fused to the protein
coding region of the reporter gene chloramphenicol
acetyltransferase (CAT).
Tissue-specific gene expression
has been observed in a variety of organs such as the liver,
cerebral hemispheres, cerebellum and testes of nontransgenic mice as well as mice carrying either of the two
transgene constructs [TF(0.67)CAT or TF(1.2)CATI.
The
present study was undertaken to ascertain whether TF gene
expression could be modulated in the transgenic mice by
exogenous administration of testosterone propionate (TP) or
follicle stimulating hormone (FSH).
Adult male transgenic
mice were injected with either 0.5pg TP for 3 consecutive
days or lOpg ovine FSH daily for 4 days and sacrificed 6
hrs. (FSH-treated mice) or 24 hrs. (TP-treated mice)
following the last injections. The cerebellum and testes
were removed and transgene activity determined by a
radioenzymatic assay specific for CAT.
A significant
enhancement of TF gene expression was observed in the
cerebellum of the TP-treated transgenic mice carrying both
the TF(0.67)CAT and TF(1.2)CAT transgenes, while no change
was noted in the testes. By comparison, FSH treatment led
to a marked increase in expression in the testes of the
line of transgenic mice expressing the TF(1.2)CAT
construct. These results demonstrate that the endocrine
system can regulate TF gene activity. (Supported by NIH
Grants AGO6872 and HD10202 and by the March of Dimes)
HERMO. Louis, Richard OKO* and Norman B. HECHT*',
Department of Anatomy, McGill University, Montreal,
43A
Canada and 'Department of Biology, Tufts University,
Massachusetts, USA.
Differential uost-translational
modification of microtubules in the seminiferous
euithelium of the rat.
The cells of the testis are a rich source of
microtubules and contain unique microtubular structural
components such as the meiotic spindle and manchette.
Microtubule diversity can be maintained by differential
genetic expression of both a and R tubulin polypeptides
or by post-translational modification of tubulins. eg.
acetylation, tyrosination and detyrosination. In this
study we utilized antibodies that specifically recognize
acetylated (anti-acetyl), tyrosinated (anti-tyr) and
detyrosinated (anti-detyr) a tubulins, to examine the
post-translational modifications of microtubules of cells
of the seminiferous epithelium of the rat. The analysis
was performed by both iinmunoperoxidase staining of
paraffin and immunogold labeling of Lowicryl K4M embedded
tissue. A distinct pattern of immunolabeling was found
with all three antibodies.
The anti-detyr antibody
heavily labeled microtubules of the manchette and
axoneme. Substantial labeling was also seen over the
microtubules of the proximal and distal centrioles and
centriolar adjunct. In contrast to the unreactivity of
the anti-detyr antibodies to microtubules of Sertoli
cells, meiotic spindle and midbody, the anti-tyr
antibody labeled these structures heavily. The anti-tyr
antibody also labeled the microtubules of the axoneme,
centrioles and centriolar adjunct, but to a lesser extent
than the anti-detyr antibody; the manchette was only
faintly reactive. The anti-acetyl antibody, on the other
hand, showed the weakest labeling. The microtubules of
the axoneme, centrioles, and midbody labeled relatively
weakly whereas the manchette and microtubules of the
Sertoli cell were unreactive. These results thus suggest
that post-translational modifications of a tubulin play
an essential role in the functional microtubular
diversification of the seminiferous epithelium.
(Supported by MRC of Canada and NIH grant GM29224).
HERRING, Susan W., Department of Oral Anatomy,
University of Illinois at Chicago, Chicago,
Illinois. Periosteal misration during qrowth
of the mammalian mandible.
During the growth of long bones, the
periosteum stretches over newly added
epiphyseal tissue. Muscles attaching via the
periosteum migrate along the bone as the
periosteum distorts and grows interstitially
(DOrfl, 1980). This process of periosteal
stretching and muscle migration may also occur
in the skull, but a systematic study has never
been carried out. Of the various skull bones,
the mandible should most resemble long bones in
its growth pattern, because it has a growth
cartilage at one extremity, the condylar
cartilage. The technique of Grimm and Katele
(1979) was used to test whether growth at the
condyle distorts the periosteum analogously to
the distortion of long bone periosteum by
growth at the epiphysial plate. Titanium
granules in gelatin matrix were implanted into
the ascending ramus of growing pig mandibles,
and distortion due to growth was monitored
radiographically. Since the condyle grows in a
posterior and superior direction, the
stretching should be most posteriorly and
superiorly and least anteriorly and inferiorly.
As predicted, granules placed in the superior
part of the ramus migrated superiorly, whereas
those in the inferior part did not. However,
the contrast between anterior and posterior
placement did not show the expected
differences. All granules migrated posteriorly
44A
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
at about the same rate. This may be because
only a limited part of the anteroposterior
extent of the mandible was examined (the
ascending ramus). Nevertheless, it is clear
that growth at the condyle does distort the
mandibular periosteum, causing muscle
migration. Supported by PHS DE 08513.
HIGHISON, Gregory J., F.Donald TIBBITTS, Eugene G. FULLER*,’
and Karen L. McCOY*, Department o f Anatomy, U n i v e r s i t y of
Nevada Sfhool o f Medicine, Reno, Nevada and Department o f
Biology,
Boise State U n i v e r s i t y , Boise, Idaho.
S t r u c t u r a l re-establishment o f t h e normal vasculature i n
the postoartum hamster uterus.
E n d a r t e r i a l c y t o t r o p h o b l a s t g i a n t c e l l s have been
examined d u r i n g g e s t a t i o n w i t h respect t o t h e i r o r i g i n ,
p a t t e r n o f m i g r a t i o n and u l t r a s t r u c t u r a l changes as these
c e l l s migrate i n t o and invade t h e u t e r i n e vessel w a l l
( O r s i n i , Am J Anat.94:273,1954 and Carpenter, Placenta.3
:219,1982).
However, l i t t l e i s known about t h e postpartum
changes which occur i n these u t e r i n e vessels. Twenty-four
sexually mature v i r g i n female hamsters were evening mated
and maintained under a 12-hour l i g h t cycle. Counting from
the day o f pregnancy, animals ranging from 1-17 days
postpartum were anaesthetized and subsequently perfused
w i t h a m o d i f i e d Karnovsky’s s o l u t i o n .
U t e r i were then
processed and s t a i n e d by conventional methods f o r l i g h t
microscopy and TEM.
I n a d d i t i o n , p a r a f f i n sections were
stained w i t h Verhoeff’s e l a s t i c s t a i n , Gomori’s aldehyde
fuchsin, and Gomori’s aldehyde f u c h s i n p r e o x i d i z e d w i t h a
10% potassium peroxymonosulfate s o l u t i o n (Alexander and
Cytotrophoblast c e l l s
Garner, EXP Eve Res,36:305,1983).
were observed l i n i n g t h e lumen o f t h e u t e r i n e a r t e r i e s
through t h e s i x t h day postpartum.
Although n o t seen as
o f t e n , these l a r g e c e l l s were a l s o found i n the venous
vessels.
During t h i s same period, the i n t e r n a l e l a s t i c
laminae o f t h e invaded a r t e r i e s were discontinuous,
fragmented, o r absent. The amorphous m a t e r i a l w i t h i n t h e
t u n i c a media, described by O r s i n i as t h e s c l e r o t i c
connective t i s s u e sheath, was i d e n t i f i e d by TEM and special
l i g h t microscopy s t a i n s t o be p r e - e l a s t i n (elaunin).
Smooth muscle c e l l s o f t h e d i s r u p t e d t u n i c a media were seen
i n the process o f forming t h i s p r e - e l a s t i n m a t e r i a l . Nondegranulated mast c e l l s i n c l o s e v i c i n i t y o f t h e u t e r i n e
vessels were a c o n s i s t e n t postpartum f e a t u r e . Although t h e
p l a c e n t a l scar s t i l l remained a t day 17 postpartum, t h e
u t e r i n e vessels r e t u r n e d t o normal w i t h respect t o
s t r u c t u r a l f e a t u r e s by postpartum day 8.
HIKIDA,
Robert S.
Department o f
Zoological
and
Biomedical Sciences. Ohio U n i v e r s i t y , Athens, Ohio. Are
b i r d muscles j u s t a . m o d i f i c a t i o n o f -the mammalian Dian?The o r o o e r t i e s o f a v i a n s k e l e t a l muscles and t h e
responses’ 0; t h e neuromuscular system t o experimental
study i n e v i t a b l y r e s u l t i n comparison w i t h mammalian
briefly
s k e l e t a l muscles.
This p r e s e n t a t i o n w i l l
compare a v i a n and mammalian muscles i n t h e i r morphol o g i c a l and histochemical p r o p e r t i e s , t h e i r r e g e n e r a t i v e
responses t o f r e e g r a f t i n g , and the e f f e c t s o f i n a c t i v i t y and resumption o f a c t i v i t y on avian versus mammaA major d i f f e r e n c e between avian and
l i a n muscles.
mammalian muscles i s the presence o f m u l t i p l e i n n e r v a t i o n i n many a v i a n muscles. This p r o p e r t y e s t a b l i s h e s
d i s t i n c t muscle f i b e r types and i n f l u e n c e s t h e regene r a t i o n process. There a r e two avian muscle f i b e r types
t h a t a r e m u l t i p l y innervated.
One resembles
the
mammalian slow-twitch f i b e r i n i t s histochemical propert i e s , and t h e o t h e r i s a t o n i c f i b e r type.
Since t h e
s i t e o f i n n e r v a t i o n on a f i b e r i s important i n ree s t a b l i s h i n g i n n e r v a t i o n o f r e g e n e r a t i v e muscle, incomp a t i b l e i n n e r v a t i o n paradigms r e s u l t i n i n t e r e s t i n g
p a t t e r n s o f regeneration n o t seen i n mammals.
The
e f f e c t s o f i n a c t i v i t y on human muscles d i f f e r from avian
muscles. Avian muscles p r e s e n t a h i g h l y p l a s t i c system
whereby t h e y show extreme responses, b u t a l s o a more
complete and r a p i d recovery from i n a c t i v i t y .
In
c o n t r a s t , i n a c t i v i t y i n humans produces a l t e r a t i o n s t h a t
a r e v e r y slow t o r e t u r n t o normal for extended periods
a f t e r resumption o f normal a c t i v i t y . These comparisons
i n d i c a t e t h a t t h e a v i a n neuromuscular system should be
s t u d i e d as d i s t i n c t b i o l o g i c a l e n t i t i e s t h a t do n o t i n
many cases represent f l y i n g mammals.
Significant
b i o l o g i c a l questions can be answered u s i n g avian models
t h a t would n o t be p o s s i b l e u s i n g mammals.
HILLOOWAIA, Rumy and Roger TRENT*, Department
of Anatomy, West Virginia University and Dept.
of Health Services, State of California. The
Mandible
a measure of the nasoDharvnx.
The purpose of this study is to determine
the dimensions of the nasopharynx from that of
the mandible. The mandible (or parts thereof)
is most commonly available in forensic cases
and its dimensions can be easily ascertained
radiography in clinical medicine. The
%erand intra-correlations between the
height, length and width of the nasopharynx
and that of the mandible were established.
Correlations between mandibular and pharyngeal
dimensions are most pertinent to the purpose
of this study. Sixty-three adult human skulls,
of undetermined gender, with the third molars
erupted
were
measured
using
standard
anthropometric methods. Relationships between
dimensions are assessed with the correlation
coefficient (Pearson’s r) and its associated
significance test. Correlations (r values)
between all six dimensions are statistically
significant (p<.05) except that between the
width and the length of the nasopharynx. For
estimation of missing dimensions, bivariate
linear regression equations using a least
squares criterion are used. By adding and
subtracting the error term (standard error of
slope coefficient), a range is produced that
will encompass about 67% of all estimates. By
doubling the error term one achieves a 95%
confidence level. (Supported by the Department
of Anatomy, WW and Dept. of Health Services,
CA).
-
HILTON, Frederick K., Kunwar P. BHATNAGAR, Mary A. HILTON*
and Walter D. MONTGOMERY*, Departments of Anatomical
Sciences and Neurobiology and of Biochemistry*, University
of Louisville, Louisville, Kentucky.
The influence of
photoueriod on taurine concentrations in the hearts an4
plasma of the bip. brown bat. Eutesicus fuscus,
Twenty male big brown bats were collected in late
winter, housed in a laboratory hibernaculum at 2°C for 3
weeks, and randomly grouped into four photoperiod cycles:
24 light (L)-0 dark (D); 16L-8D; 1L-23D; OL-24D. Each
photoperiod cell contained a wire-mesh cage (10x10~18in),
exposed to 375 lux, with continuous flow of air maintaining the temperature at 22-30’ C. All bats were fed during
the light period of the 1L-23D group, with bacs in the
OL-24D group fed under dim red light. Daily feedings
consisted of a minimum of 10 mealworms coated with Stewart
formula liquid multivitamins. After 4 weeks of regulated
photoperiod, heart ventricles were dissected free of
atria, cleared of blood, frozen and lyophilized. Blood,
collected from the trunk in heparinized tubes, was centrifuged to separate plasma which was frozen. Taurine, in
ABSTRACTSAAA 103RD MEETING
plasma and e x t r a c t e d from v e n t r i c l e s , was converted t o the
fluorescent o-phthaldialdehyde d e r i v a t i v e and determined
by high performance l i q u i d chromatography. High m o r t a l i t y
i n the 1L-23D group and l o s s of h e a r t s from the 16L-8D
group precluded complete analysis f o r a l l groups. Values
a r e g i v e n ? standard deviation.
GrOuD
Taurine
plasma
ventricles
PM
nmol/mg f r e e z e - d r i e d w t
0 L-24 D
358.3 f 30.0
130.4 t 36.4
16 L-8 D
300.9 f 0.7
24 L-0 D
448.0 f 38.0
88.3 f 28.9
We have previously e s t a b l i s h e d t h a t the mean value f o r
t a u r i n e i n v e n t r i c l e s from wild, f r e e - f l y i n g normothermic
b i g brown b a t s is 120.3 f 12.3 nmol/mg f r e e z e - d r i e d w t .
While these r e s u l t s a r e preliminary, they suggest t h a t
photoperiod may influence the cardiac/plasma r a t i o of
taurine.
(Supported by g r a n t 628-347 from the Graduate
Research Council, University of L o u i s v i l l e . )
Jayaram K. UDUPA,* and David
HIRSCH,* Bruce E l l i o t ,
RORERTSX, Department of Anatomy, Pennsylvania College of
Podiatric Medicine; Medical
Image Processing
Group,
Department o f Radiology, University of Pennsylvania; and
School of Dental Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania. (Sponsored by Edvin M. Mast e r s ) J o i n t Kinematics Can Be Determined BY Comouterized
Three Dimensional Imaqinq of MRI Data.
We are applying the technique of 3-D reconstruction,
and the information revealed in the process, t o biomechan i c a l s t u d i e s of foot j o i n t s . The kinematics of a j o i n t
a r e those mechanical f a c t o r s determined by the geometry
of t h e j o i n t , independent of force, mass, and energy.
They a r e described by determining the position of the
bones a t various i n t e r v a l s through t h e i r range of motion,
and c a l c u l a t i n g such f a c t o r s a s axes of r o t a t i o n , transl a t i o n of components, and angles of movement. The small
j o i n t s of the f o o t a r e t o o small, t o o close together, and
t o o moveable i n r e l a t i o n t o the s k i n f o r conventional
kinematic a n a l y s i s . Hovever, the process of 3-D recons t r u c t i o n from s e c t i o n a l imaging data provides a nev vay
of gathering such information. Three-dimensional recons t r u c t i o n includes information on the p o s i t i o n o f each
voxel forming each bone, vhich a l l o v s for the c a l c u l a t i o n
of the bones’ positions i n the image space. I t is, i n
f a c t , capable o f more accurate kinematic data than other
methods because of the inherent resolution of the sect i o n a l imaging systems, and because the normally used
Alprojection radiographs lack the t h i r d dimension.
though computed tomography i s the e a s i e s t modality f o r
identifying bone, i t cannot be used on normal volunteers
because of the r a d i a t i o n r i s k . Magnetic resonance imaqing has no known dangers t o normal individuals. We developed a computer program vhich c r e a t e s 3-D reconstructions
from MRI data, allows u s t o mark i d e n t i f i a b l e landmarks
on each bone, and uses those landmarks t o r e g i s t e r images
of the bones from scans taken i n various j o i n t p o s i t i o n s .
The l i n e a r and angular adjustments necessary t o r e g i s t e r
the images a r e calculated, and these values describe the
motions of the j o i n t .
Animations can be created, vhich
permit e a s i e r v i s u a l i z a t i o n of t h e movements occurring.
HOVERSLAND,Roger C.. and K. D. Beaman’, Dept. of Anatomy, Indiana
University School of Medicine,Fort Wayne, Indiana and Deppr. of Immunology and
Microbiology,University of Health Scienceme Chicago Medical School. North
Chicago, Illinois -of
Mm’
v 14-30on Mouse Embm
Previous expedimentshave demonstratedthat the injection of monoclonalantibody
against antigen specifk T e l l suppressor factors (TsF) into pregnant mice was
detrimental to pregnancy. Counting the morning of vaginal plug as Day 1of
45A
pregnancy the effects of the monoclonal antibody were greatest when the injections
were given in a sequence hat included Day 3.4 & 5 of pregnancy. One possible
explanation for the deleterious effects of the monoclonal antibody are direct stage
specific embryo toxic effects rather than any interference with the immune
suppression. The followingexperiments were undertaken to examine the effects of the
monoclonal antibody on the development of preimplantation mouse embryos in vitro.
Swiss-Websterfemale mice were placed with males and allowed to mate. Mating was
confirmed by a vaginal plug and designated Day 1of pregnancy. Animals were killed
on Day 3 of pregnancy and morulae collected by flushing the oviducu with a stream
of incubation medii.Embryos were examined, p l e d and then incubated either in
normal media (Kreb’s Ringer bicarbonate media,1 mum1glucose, lmg/mlbovine
serum albumin, pH 7.4,37O C, 5% [email protected]) or in normal media with the addition of
monoclonal antibody 14-30in ascites fluid. control ascites fluid,or purified rat IgM at
a concentration of 100 [email protected] ul. Six of the 26 morulae incubated in the control
media had become blastocysts by the equivalent of Day 4 (fust day of incubation), all
were blastocysts by the equivalent of Day 5, and 6 of those blastocysu had hatched by
the equivalent of Day 6. Of the 25 moruhe incubated in the media containing the
monoclonal antibody 14-30.12had become blastocysts by Day 4.25 were
blastocysts on day 5, and 18 of those blastocysts had hatched by Day 6. Of the 26
morulae incubated in the media containing a similar amount of ascites fluid, 9 had
become blastocysts on day 4.24 had become blastocysts on Day 5, and 18 of those
blastocysts had hatched by day 6 with 1 dead embryo. Of the 28 morulae incubated in
the media containing purified rat IgM. 3 had become blastocysu on Day 4, 24 had
become blastocysts by Day 5 , with none of the blastocysts hatched on Day 6 with 7
dead embryos. These data suggest that the presence of the monoclonal antibody in the
ascites fluid or the control ascites fluid were not detrimental to embryo development
from the morulae stage to the blastocyst stage or in the hatching process but were in
fact somewhat beneficial to these stages of development. The presence of the purifii
rat IgM did not appear to interfere in the development of blastocysts from morulae but
was associated with a lack of hatching and an increase in embryo loss. These data
indicate that recent work demonstrating an antipregnancy effect of the monoclonal
antibody 14-30when injected into mice during early pregnancy is not due to a direct
anti-embryoeffect and thus, suppon the concept that such negative effects are. due to
interference with an essential T-cell mediated immune suppression in early pregnancy
HOWLAND*,Dena R., Barbara S. BREGMAN, Alan
TESSLER’, and Michael E. GOLDBERGER*, Department of
Anatomy, The Medical College of Pennsylvania, Philadelphia,
Pennsylvania, Department of Anatomy and Cell Biology,
Georeetown Universitv. Washington. D.C. (sponsored by Walter
Rub&) Transplants aher the &tor develofment of kiitens
after n e o n a t a i w i n a l cord transection.
T h e aim of current studies is to evaluate the survival of
homotypic embryonic transplants into the neonatally
transected kitten spinal cord and to evaluate the effect of these
transplants on motor behavior. Spinal cord from the fourth
week of gestation in the cat survives in t h e host lesion cavity.
The motor development of three kittens lesioned and given E26
transplants on the day after birth has been studied. Like
neonately transected kittens without transplants (spinal
animals), these kittens show an accelerated development of
hindlimb reflex behaviors. Unlike spinal animals, these kittens
with transplants develop overground locomotion on a runway.
Patterns requiring coordination between hind and forelimbs,
such as a diagonal pattern typical of overground locomotion in
normal adult cats, do occur. Although present, this overground
locomotion has some abnormal characteristics. The step cycle
duration is lengthened. There is not a consistent 11 relationship
(pairing) between hind and forelimb step cycles. The postural
stability of the hindquarters during locomotion is impaired
with frequent lateral deviations noted. The number of falls
and positive weight support capacity these animals exhibit
during overground locomotion changes with age. Falls are
usually followed by successful recoveries and show at least a
30% decrease in number between 6 and 23 weeks of age. Full
weight support ste cycles often exhibit an exaggerated second
“yield” (almost to tge point of collapse) in E3 when weight
support is first attempted. Hindlimb stepcycles exhibiting full
weight support increase with age. As great as 86% of all
stepcycles were full weight supporting in one animal at 23
weeks which was 56% more than at 6 weeks. These results
suggest that embryonic spinal cord transplants alter the motor
development of newborn kittens with spinal cord transections.
Supported by grant NS24707 and the Daniel Heumann
Foundat ion.
46A
ABSTRACTS-AAA 103RD MEETING
HOYT', Richard F., JR., Nancy A. McNELLY', and Sergei P. SOROKIN,
Laboratory of Pulmonary Cell Biologv, Department ofAnafoiny,Boston Universily
Sc/iool OfMedicine, Bosfon,MaFsnc/wseffs. Neuroeoithelialbodies (NEB5)
influence cell division in the uulmonarv endoderm of fetal hamster lune
Autoradiographswere made from lungs of a newborn Syrian golden hamster
continuously exposed to 3H-thymidine throughout the final 4.5 days of gestation.
The specimen was normal in terms of branching pattern, overaU histologid
appearance, and distribution and differentiationof NEBs. Silver grains were
counted over nuclei of 1,298 small-granule endocrine cells in 165NEBs and 1,005
adjacent nonendocrine epithelial cells. All nonendocrine cells were labeled
whereas many cndocrine cells were not, and mean grain counts were always lower
for NEBs than for surroundingundifferentiated endoderm. In NEBs,
background-corrected grain density (grains/um2 nucleus) averaged 36% and total
label (grains/nuclear profile) 17% of that in nearby epithelium. Along the left
axial bronchus, mean label rose tenfold between hilurn and periphery. It was
consistently 0.48 grains/itm2 higher in nonendocrine epithelium than in NEBs
lying between airway branch points, and these were more heavily labeled than the
older NEBs at correspondingbifurcations. Rarely, NEBs contained solitary,
intensely labeled endocrine cells at their edges. In a further 1,141 epithelialcells,
grain density was shown to decline with increasing distance from mature NEBs.
Rank-order correlationswere significant (p<O.M)1-0.05) for cells around 26 of 28
individual NEBs. Correlations for pooled values were significant in all regions
studied: right upper lobe (8 NEBs, p<O.Wl), right lower lobe (8 NEBs, p<O.001),
and left lung (12 NEBs, p<O.OOl). We conclude that NEBs originate from
endoderm, not neural crest. In fetal lung they develop from cells that stop dividing
well before their neighbors. They appear to play an integral role in local
expansion of the airway lining by stimulatingproliferation of adjoining
nonendocrine epithelial cells, few of which find their way into the NEBs
themselves. NEBs present between branching points of the bronchial tree
evidently serve to promote lengthening of individual airway branches, and their
central nervous connections may help to assure coordination of the process
throughout the entire developing lung.
Supported by USPHgraitt HL-19379.
S
HUNTER?* W i l l i a m L. and A. L a r r y ARSENAULT", Department o f
Anatomy,
University
of
B r i t i s h Columbia,
Vancouver,
Dvalle)
British
Columbia.
(Soonsored bv W i l l i a m K.
Metaphyseal c a p i l l a r y u l t r a s t r u c t u r e and qrowth dynamics
o f t h e epiphyseal qrowth p l a t e .
Metaphyseal blood vessels which invade t h e c a l c i f y i n g
epiphyseal growth p l a t e were examined by a v a r i e t y o f
techniques t o determine t h e i r morphology, c e l l d i v i s i o n ,
and
growth
p a t t e r n s as they r e l a t e t o endochondral
Four
regions
of
these vessels were
ossification.
t h e t e r m i n a l ends o f t h e
characterized:
Sprout T i p s
c a p i l l a r y s p r o u t s which impinge upon t h e h y p e r t r o p h i c
chondrocytes;
Region o f Extended C a l c i f i e d C a r t i l a q e
those deeper vessels w i t h i n t h e metaphysis which a r e
surrounded by an e x t r a c e l l u l a r m a t r i x composed o f extended
Region o f Bone D e p o s i t i o n
septa o f c a l c i f i e d c a r t i l a g e ;
f u r t h e r s t i l l from t h e e p i p h y s i s these microvessels a r e
contained w i t h i n a network o f a c t i v e bone d e p o s i t i o n ;
a t a d i s t a n c e o f 350-500pm
Region o f Primary Vessels
from t h e e p i p h y s i s a r e d i l a t e d vessels w i t h one or two
l a y e r s o f smooth muscle i n t h e i r walls, t h a t supply and
d r a i n t h e metaphyseal c a p i l l a r y plexus. The s p r o u t t i p s
w i t h an
are
continuous
blind-ended
vessels
lined
with
no
u n d e r l y i n g basement
attenuated
endothelium
membrane.
D i v i d i n g e n d o t h e l i a l c e l l s a r e most f r e q u e n t l y
found i n t h e r e g i o n o f bone d e p o s i t i o n 175-200pm behind
the
apices
of
the
growing s p r o u t t i p s .
Dividing
e n d o t h e l i a l c e l l s possess j u n c t i o n a l attachments between
daughter c e l l s and adjacent e n d o t h e l i a l c e l l s even b e f o r e
c e l l s e p a r a t i o n i s complete.
Numerous m i c r o v i l l i from
b o t h t h e daughter c e l l s and a d j a c e n t endothelium o f t e n
make c o n t a c t and form j u n c t i o n s w i t h t h e plasma membrane
of t h e d i v i d i n g c e l l s which m a i n t a i n s t h e i n t e g r i t y o f t h e
A
time
coursed,
autoradiographic
vascular
wall.
examination
of
cytokinesis
revealed
radio-labelled
e n d o t h e l i a l c e l l s t o appear a t t h e e p i p h y s i s a f t e r a 24
hour period.
The metaphyseal c a p i l l a r y s p r o u t s r e p r e s e n t
a continuous,
u n i d i r e c t i o n a l , angiogenic v a s c u l a r network
which
grows
by e l o n g a t i o n from t h e r e g i o n o f bone
deposition;
t h i s r e g i o n remains a f i x e d d i s t a n c e behind
the sprout tips.
Supported by t h e MRC of Canada.
-
-
-
-
INPANBUTR. Nongnuch, and Alan N. TAYLOR. Department o f
V e t e r i n a r y Anatomy and C e l l u l a r Biology, Ohio
State
U n i v e r s i t y , Columbus. Ohio and Department o f Anatomy,
Bavlor
Colleae
of
Dentistrv.
Dallas.
Texas.
in
I&unohistochem;cal
l o c a l i z a t i o n o f calbindin-D28K
avian lymphoid t i s s u e germinal centers.
Calbindin-D28K (CaBP), a 28 k i l o d a l t o n calcium-binding
p r o t e i n . i s regarded as an end-organ marker f o r t h e
endocrine
system.
Although
originally
vitamin D
discovered i n calcium t r a n s p o r t i n g organs, e.g.,
intestine
and kidney,
t h e p r o t e i n subsequently was r e p o r t e d i n
s e v e r a l o t h e r organ systems n o t recognized f o r t h e i r
calcium t r a n s l o c a t i n g capacity.
I n t h i s study we r e p o r t
f o r t h e f i r s t time t h e presence o f CaBP i n avian germinal
c e n t e r s l o c a t e d i n p e r i p h e r a l lymphoid tissue.
Selected
t i s s u e s were removed from growing w h i t e leghorn chickens
(6-12 weeks old), f i x e d by f r e e z e s u b s t i t u t i o n ,
paraffin
embedded, sectioned and processed immunohistochemically
PAP technique.
Imrnunocontrols
u s i n g Sternberger's
c o n s i s t e d o f s e c t i o n s r e a c t e d w i t h non-immune serum i n
place o f a n t i - c h i c k
i n t e s t i n a l CaBP.
Specific reaction
p r o d u c t f o r CaBP was observed e x c l u s i v e l y i n germinal
centers l o c a t e d i n aggregates o f lymphoid t i s s u e i n
testes, ovary, lung, and lamina p r o p r i a o f small and l a r g e
i n t e s t i n e . CaBP was a l s o present i n germinal centers i n
mural
lymphoid
nodules
(chickens
do
not
have
mammal ian-type lymph nodes).
Lymphocytes i n t h e germinal
c e n t e r s contained immunostain throughout t h e cytoplasm,
b u t t h e i n t e n s i t y o f s t a i n was v a r i a b l e from c e l l t o c e l l
as was t h e number o f immunopositive c e l l s .
Adjacent
lymphocytes which were n o t i n t h e germinal center proper
d i d n o t c o n t a i n CaBP. The f u n c t i o n a l s i g n i f i c a n c e o f t h e
expression o f CaBP i n lymphocytes d u r i n g c l o n a l expansion
i s n o t apparent from these i n i t i a l observations:
however,
t h e study does add another s p e c i f i c c e l l t y p e t o t h e
select l i s t o f c e l l s expressing t h i s v i t a m i n D dependent
p r o t e i n . Supported by N I H g r a n t DE07916 (ANT).
ISMAIL, Nermine a n d C a r l o s R . MORALES, Department o f
Anatomy, McGill U n i v e r s i t y , M o n t r e a l , Quebec, Canada.
E f f e c t s o f v i t a m i n A d e f i c i e n c v on t h e i n t e r S e r t o l i c e l l
t i g h t i u n c t i o n and o n t h e Perm c e l l D o o u l a t i o n .
When 20-day o l d r a t s a r e p l a c e d o n a v i t a m i n A
d e f i c i e n t d i e t (VAD) f o r a p e r i o d of 10 weeks, t h e
s e m i n i f e r o u s t u b u l e s a r e found t o c o n t a i n o n l y S e r t o l i
c e l l s , a few r e s i d u a l A o . A 1 s p e r m a t o g o n i a and p r e l e p t o t e n e s p e r m a t o c y t e s ( P L ) . The t y p e A 1 spermatogonia and
PL s p e r m a t o c y t e s are a r r e s t e d i n t h e i r G2 p h a s e .
I n VAD
r a t s t y p e Az-A4, i n t e r m e d i a t e ( I n ) and B spermatogonia
and a l l t y p e s of s p e r m a t o c y t e s ( e x c e p t PL s p e r m a t o c y t e s )
and s p e r m a t i d s are e l i m i n a t e d from t h e s e m i n i f e r o u s
tubules.
Two q u e s t i o n s were r a i s e d i n t h i s i n v e s t i g a t i o n : 1) Is t h e r e , i n VAD r a t s , any c o r r e l a t i o n between a
breakdown of t h e b l o o d t e s t i s b a r r i e r ( e . g . S e r t o l i c e l l
t i g h t j u n c t i o n s ) and germ c e l l l o s s as s u g g e s t e d by some
i n v e s t i g a t o r s , 2) Is the d i s a p p e a r a n c e o f most germ c e l l s
due t o t h e i r d e g e n e r a t i o n o r t h e r e s u l t o f t h e i r normal
d i f f e r e n t i a t i o n and e v e n t u a l l o s s from t h e s e m i n i f e r o u s
epithelium.
To i n v e s t i g a t e t h e s e q u e s t i o n s f o u r groups
o f male Sprague-Dawley r a t s (20-day o l d ) were f e d a VAD
d i e t f o r 7 t o 12 weeks.
The t e s t e s were f i x e d b y
p e r f u s i o n w i t h 2 . 5 % g l u t a r a l d e h y d e in 0 . 2 M sodium
c a c o d y l a t e c o n t a i n i n g o r n o t 2 % lanthanum n i t r a t e , a n
e l e c t r o n opaque t r a c e r u s e f u l f o r t e s t i n g t h e p a t e n c y o f
Sertoli c e l l tight junctions.
The lanthanum permeated
t h e i n t e r c e l l u l a r s p a c e o f t h e b a s a l compartment and w a s
a r r e s t e d b y normal i n t e r S e r t o l i c e l l t i g h t j u n c t i o n s ,
The s e m i n i f e r o u s e p i t h e l i u m showed numerous d e g e n e r a t i n g
germs c e l l s some b e i n g i n t e r n a l i z e d by S e r t o l i c e l l s as
membrane-bound phagosomes.
Thus, t h e s e r e s u l t s i n d i c a t e
t h a t i n t e s t e s from VAD r a t s : 1) I n t e r S e r t o l i c e l l
tight junctions
remain
intact
during vitamin A
d e f i c i e n c y ; 2 ) Non v i a b l e germs c e l l s d e g e n e r a t e i n s i t u
in
the
seminiferous
e p i t h e l i u m and
are a c t i v e l y
ABSTRACTSAAA 103RD MEETING
phagocytosed and eliminated by Sertoli cells. (Supported
by Medical Research Council of Canada and Fonds de la
recherche en sante du Qugbec).
Jaeger, C.B., L.A. Greene*, S. Winn*, P. Tresco* and P. Aebischer*,
Department of Anatomy and CPR, Purdue University, W. Lafayette,
Indiana; Department of Pathology, Columbia University, New York,
New York; and Artificial Organ Laboratory, Brown University,
Providence, Rhode Island. Ultrastructural characteristics of PC12 cell
p
p
Neural implants of packaged cell lines may provide a novel approach to neurotransmitter replacement strategies. PC12 cells (a cell line
isolated from a rat pheochromocytoma) synthesize, store, and release
high levels of dopamine -the neurotransmitter that is significantly reduced in patients with Parkinson's syndrome. This study investigated
the morphology of PC12 cells growing within a resmcted three-dimensional space of a porous polymer capsule. Permeable, polyvinylacrylic, copolymer tubing with selectivity of 50KDa was used for capsule preparation. Cell suspensions were loaded by ultrafiltration into the
polymer tubes and the ends of 3 to 5 mm segments were closed with
liquid polymer. Cell-filled capsules were maintained either in vitro or
implanted within the brain of rats or guinea pigs. Our present observations revealed morphological changes of enclosed PC12 cells cultured
from one to twenty three weeks. These changes related to size and distribution of storage vesicles and were most evident in comparisons of
long-term versus short-term encapsulated cell populations. PC12 cells
in short-term culture of three to four weeks typically contained abundant
quantities of large electron-dense storage granules that were randomly
distributed within the juxtonuclear cytoplasm. Their appearance resembled that of PC12 cells maintained in monolayer cultures. At periods of
ten weeks or more following enclosure, the electron-dense storage
vesicles had a more patchy distribution and they were often seen in
small cell processes. Increased frequency of empty coated-membrane
containers partially fused to the cell membrane suggested enhanced
vesicular release. Many of the long-term encapsulated cells also had
large numbers of mitochondria, some of which were electron-dense,
indicating changes in metabolic activity. Cell morphologies were similar
in both implanted capsules and capsules maintained in vitro. Possible
reasons for these changes in PC12 cell fine structure include alterations
of cell/cell contacts, since cells divide within the capsules. Another
cause may be that of molecular exclusion from the fluid environment
due to pore size of the capsule wall. These data are relevant in the evaluation of the usefulness of PC12 cell-filled capules for transmitter replacement. Supported by grant No. NS27694 from the NINDS-NIH.
JEW, Jean Y. and Yanfeng WANG*, Department of Anatomy,
University of Iowa, Iowa City, IA. Age-related changes
in myenteric plexus of colon of three mammalian species.
Disorders related to gastrointestinal motility, including chronic constipation, diverticulosis and achalasia, are commonly observed in elderly human patients.
The myenteric plexus plays an important role in gastrointestinal motility, controlling the patterns of movement.
Nevertheless, very Eew studies have addressed age-related
changes in the myenteric plexus or how these changes may
affect gastrointestinal function. We investigated agerelated changes in numbers and sizes of myenteric neurons
in the colons of guinea pigs, two strains of rats, and
mice. Segments of middle colon were removed from young
adult and aged animals sacrificed by Nembutal overdoses.
Whole thickness preparations were stained using Gabella's
b-nicotinamide adenine dinucleotide (NADH) method. Neuronal perikarya were counted and areas and shapes analyzed using computer-assisted image analysis (IBM-VIAS).
Gross lengths and circumferences of young adult versus
aged colons were factored into the neuronal counts. In
all species and strains, numbers of neurons per equivalent unit area were significantly reduced in the aged
colon as compared with the young. F o r example, in the
guinea pig the mean number was 9290 in the young adults
and 3762 in the aged. For guinea pigs and rats, the mean
47A
areas of perikarya were smaller in the myenteric plexus
of aged animals than of young adults. However, in the
mouse no significant difference was observed in neuronal
sizes of young adult versus aged colon. Comparisons of
the shape factors of myenteric neurons in young adult
versus aged animals did not yield conclusive results.
Nevertheless, neurons from aged colons showed a trend
suggesting that these neurons more closely approach the
shape of a circle whereas neurons from young animals were
more irregular in shape. In summary, myenteric neurons
in the aged colons were fewer in number, smaller in size,
and to some extent rounder in shape (due perhaps to loss
of processes). The significance of these changes and
whether they are the causes or the effects of age-related
alterations in metabolic and physiological functions remains to be clarified. Supported by DK 38123.
S
JOHNSON,* Julie and Juanita J. Anders, Department of
Anatomy, USUHS, Bethesda, Maryland. (Sponsored by Juanita
Anders) A alial scar from rat optic nerve transplanted to the
site of transection of a rat olfactory nerve blocks olfactory
nerve reinnervation of the olfactory bulb. The olfactory system
is the only region of the mammalian CNS in which
degeneration of the primary sensory neurons results in the
development of new neurons and reinnervation of the
secondary sensory neurons. Axotomy of the olfactory nerve at
the cribriform plate does not cause the formation of a glial
scar which blocks nerve regeneration. The purpose of this
study was to determine if a barrier scar formed in the optic
nerve would suppress axonal regeneration when transplanted
to the site of olfactory nerve axotomy. Primary olfactory
neurons were axotomized in anesthetized adult rats. A dental
drill was used to open the skull and a 0.5mm saw blade was
used to transect the nerve along the cribriform plate. A
compact glial scar, which was formed by transection of the
optic nerve 50 to 60 days prior to removal, was placed into
the olfactory nerve axotomy site. The rats were allowed to
survive for 1, 2, 3, 4 and 5 weeks. Retrograde transport of
20% horseradish peroxidase (HRP) from the nasal cavity by
the olfactory neurons was used to examine the temporal and
spatial pattern of regeneration of the olfactory nerve after
axotomy and the altered pattern after axotomy and glial scar
transplan- tation. In the axotomized rats, HRP was found in
the glomerular layer (GL), where the incoming olfactory nerve
axons synapse with the apical dendrites of the secondary
olfactory neurons, 21 days after lesioning. In the presence of
the transplanted glial scar, HRP labeling was not found in the
GL even at 5 weeks post-axotomy. Also labeled HRP axons
were not seen within the transplanted glial scar. These results
suggest that a barrier gliat scar can impede axonal elongation
from neurons having maximum regeneration potential.
JOHNSON, Roger B. and Susan P. PYLYPAS*, Department of Anatomy,
University of Manitoba, Winnipeg, MB, Canada. A re-examination of' the
oxytalan fiber system in the periodontal liaament of the mouse.
The morphological relationship between oxytalan and collagen fibersof the
periodontal ligament has been the subject of much speculation; however,
evidence from light (LM) and transmission electron (TEM) microscopy has
yielded little definitive morphological information, as the large volume of
collagen fibers makes study of the smaller oxytalan fibers difficult.The objective
of the present study was to study the oxytalan fiber system following complete
and partial removal of adjacent collagen fibers to allow a more precise study of
their distribution. Mandibles were removed from 6 week female Swiss-Webster
mice and ultrasonicated in 0.5N NaOH for either 20 (to partially remove
48A
ABSTRACTS-AAA 1 0 3 MEETING
~~
adjacent collagen fibers) or for 30 minutes (to completely remove collagen
fibers) and then fixed for 3 hours in Kamovsky's fixative. Tissues were either
prepared for scanning electron microscopy (SEM) using standard methods, or for
LM and TEM using standard morphological and histochemical techniques. The
latter methods were essential to precisely determine the structures present in
SEM. For LM, tissues were embedded in Epon-Araldite, sectioned at 1 pm and
stained with either toluidine blue (for morphology) or aldehyde fuchsin
following preoxidation (for oxytalan). For TEM tissues were osmicated, and
ultrathin sections were stained by either the high-iron diamine or TCH-SP
methods for oxytalan, or lead citrate and uranyl acetate for morphology. SEM
preparations demonstrated a network of sheet-like lamellae passing from tooth
to alveolar bone, encircling blood vessels, and containing many small
fenestrations. LM and TEM suggested that lamellae enclosed a network of
oxytalan which insened into root cementum, alveolar bone and walls of
periodontal ligament blood vessels. In partially digested specimens, lamellae
joined adjacent periodontal ligament collagen fibers. Thus, the present data
suggests a new interpretation of the structure and distribution of periodontal
oxytalan fibers; that is, they are not "tubular" (as classically described) but are
subcomponents of larger elastic lamellae attaching to both bone and cementum
and encircling collagen fibers and blood vessels. These lamellae are similar to
those described in the aorta and likely support and dissipate forces placed on
principal collagen fibers by the adjacent teeth.
Supported by the Manitoba Health Research Council and the Medical Research
Council of Canada, Grant MA-8035.
S
JOHNSON; R., S.D. HAM,' A.I. CANADY' and J.A. MITCHELL.
Departments of Neurosurgery and Anatomy and Cell Biology, Wayne State
University, School of Medicine, Detroit, Michigan. T h e occur rence
a n d oraanization sof
--rpus
on t h e
of t h e
medial a n d l at er al walls of t h e la te ra l v-icles
hamster,
The occurrence and organization of supraependymal neuronal elements
on the medial (MW) and lateral (LW) walls of the lateral cerebral
ventricles of the hamster were compared. Ten adult female hamsters
(Mesocricetus auratus) were anesthetized a n d perfused with
Karnovsky's aldehyde fixative; brains were removed and the ventricular
walls prepared in routine fashion for examination by correlative
scanning and transmission electron microscopy. The ependymal surface
of the LW was uniformly covered with cilia; apical surfaces of ependymal
cells frequently possessed microvilli but few supraependymal neuronal
fibers were evident, and complexes of such fibers were not observed.
The MW was also richly ciliated and microvilli were evident. However,
regions of less densely ciliated ependyma consistently occurred on the
LW; nerve fibers were abundant and frequently associated with plexuses.
Such plexuses were especially evident in less densely ciliated areas. The
most frequent type of plexus consisted of a radial array of individual
fibers and fascicles. Processes frequently contained varicosities, and
some appeared tethered to the ependymal surface by small lateral
branches. The second type of neuronal plexus was characterized by a
central placode-like structure (x diameter: = 10pm) from which
radiated sheet-like processes which divided into numerous individual
small calibre fibers. Such fibers often extended over 3 0 ~ mfrom the
plexus and criss-crossed other fibers from similar plexuses. Such
networks were particularly abundant on the ependyma in the region of
the caudate nucleus. Studies are in progress to determine the
ultrastructural organization of s uc h neuronal plexuses and their
relationships to subjacent neuropil.
(Supported by a grant from the Childrens Hospital of Michigan).
KAGEYAMA, G.H., K.A. GALLARDO*, M.E. GALLWAN*, K. UPPAL*,
and R.T. ROBERTSON, Dept. Anat. & Ncurobiology, Univ. of Calif., Irvine,
California. E.M. localization of transncuronal WGA-HRP in rreniculocortical
terminals in neonatal and adult rat visual and auditorv cortices: Some
correlations with cvtochrome oxidase (CO) and acetvlcholinesterase (AChEL
The distribution of transneuronal WGA-HRP in geniculocortical terminals in
visual and auditory cortices were compared with laminar patterns of CO and
A C E in developing and adult rats to study the development 0 1 geniculocorcical
projections and cortical oxidative metabolism. 2% WGA-HRP was unilaterally
injected into the eye (3 ul) or inferior colliculus (IC, 250 nl) of neonatal and
adult rats. After 3 4 days, animals were perfused with aldehydes and sections
processed for HRP (TMB, pH4.8, Naus et al. J Comp Neurol,'85). AChE or
CO histochemistry. Some tissue was further processed for E.M. using 1%0s04
followed by 1.5% potassium femcyanide. In visual area 17 and auditory area
41 of both developing and adult rats, WGA-HRP transncuronally labeled
geniculocortical terminals in layers IV, dcep 111, I and VI. At the E.M. level
transneuronal WGA-HRP was localized within oresumed ceniculocortica!
terminals. PND 0-4 fearly neonates): in areas 17 and 41 strong CO was found
in superficial dendrites of cortical plate neurons and in subplate (SPu) neurons.
Thcsc arc known sites of early cortical synapses and possible geniculocortical
synapses. AChE was localized in SPu ncurons and some fibers in the cortical
plate. PND 6-14 (older nconates): elevated CO was found in dendrites and
ncurons in gcniculoreceptive layers 111-IV and in SPu neurons. Used as a
developmental marker for presumed geniculocortical axons in cortical areas 17
and 41 (Robertson. Neurosci Letr,'87), A C E activity was found in fibers in
the geniculoreceptive layers, matching the transneuronal WGA-HRP pattern.
Strong AChE was also present in SPu neurons. After cortical HRP injections
retrogradely labeled geniculate neurons showed strong AChE activity in the
rough endoplasmic reticulum, indicating AChE synthesis. -elevated
CO
was present in gcniculoreceptive laminae, but reactive SPu neurons were not
sccn. Strong A C E was present in adult cortex, but in layers that did not match
with the geniculocortical projcction. Instead. A C E was diminished in both
gcniculate neurons and geniculocortical tcrminal fields implying that AChE is
associated with developing, but not adult gcniculate neurons and terminals.
Elcvated CO, f i s t in superficial dendrites and SPu neurons, and later appearing
in dendrites and neurons of layer 111-IV parallels laminar changes in the
developing geniculocortical projection, and the histochemical appearance of
AChE in layers 111-IV. The results suggcst that (1) transncuronal WGA-HRP is
suitable for labeling gcniculoco~ticalterminals at the E.M. level, (2) AChE is a
useful marker for developing gcniculocorlical projections and (3) the
developmental clevation of CO activity in dcndritcs and neuronal perikarya in
areas 17 and 41 may be closcly associatcd with the formation of thalamocortical
synapses. Supported by NSF Grant 87-08515 and NIH grant NS 25674.
KAISER, Hans E l q a r , Departwent o f Pa t h o l o g y , School of Medic i n e , U n i v e r s i t y o f Maryland a t B a l t i m o r e . Comparative asp e c t s of growth i n r e l a t l o n t o n e o p l a s t i c development.
Neoplasms, g e n e r a l l y known a s c a n c e r , h a v e been found i n
many s p e c i e s of a n i m a l s and p l a n t s a l i k e ; b u t t h e growth
p o t e n t i a l of t h e s p e c i e s under i n v e s t i g a t i o n f o r comparison
h a s n o t been p r o p e r l y e v a l u a t e d , Two t y p e s o f f a c t o r s p l a y
a d e c i s i v e r o l e , namely t h o s e of taxonomic-endogenous asp e c t s a s w e l l as t h e r e a c t i o n t o t h e exogenous s t i m u l i . Taxonomic-endogenous f a c t o r s a r e t h e v a r i a t i o n of t h e embryon a l growth p o t e n t i a l . t h e d i f f e r e n t f u l f i l l m e n t o f s i m i l a r
f u n c t i o n s by v a r i o u s s t r u c t u r e s , t h e t y p e of growth, t h e
s i z e of s p e c i e s , t h e l i f e s p a n of s p e c i e s w i t h s i m i l a r tiss u e s , t h e l i f e s p a n of t i s s u e s and c e l l s , t h e t y p e of dev e l o p m e n t a l growth, a n d t h e taxonomic p h y l o g e n e t i c g r a d e of
o r g a n s . To t h e s e endogenous f a c t o r s i n f l u e n c i n g n e o p l a s t i c
growth t h e r e a c t i o n to exogenous, e n v i r o n m e n t a l s t i m u l i
s h o u l d be a d d e d . More e x a c t l y m e r i s t e m a t i c tissues o f p l a n t s
e x h i b i t a t least t h e o r e t i c a l l y a n u n l i m i t e d embryonal p o t e n r
t i a l i n s h a r p c o n t r a s t t o t h e c e l l i n v a r i a b i l i t y of c e r t a i n
i n v e r t e b r a t e s and m a n 's own n e r v o u s system; f u n c t i o n s a r e
performed by d i f f e r e n t mechanisms such a s t h a t o f t h e mobili t y of a n i m a l s and p l a n t s . The t y p e of growth v a r i e s i n
s p e c i e s r e s u l t i n g i n r e m a r k a b l e v a r i a t i o n s of s i z e a s s een
i n cephalopod m o l l u s c s where t h e s m a l l e s t I d i o s e p u s s p . exh i b i t s a body s i z e of 1.5-2cm whereas t h e l a r g e s t s p e c i e s
A r c h i t h e u t i s p r i n c e p s shows a l e n g t h of 18m ( t h e body a l o n e
6.6m).
The l i f e span of t h e same t y p e s v a r i e s a l s o a s i n
t h e mouse, NUS musculus up to 6 y e a r s and i n t h e b l u e whale
B a l e n o p t e r a musculus up t o 110 y e a r s . S i z e and l i f e span
of similar t i s s u e s in v a r i o u s s p e c i e s d i f f e r a c c o r d i n g l y .
Developmental growth of a n i m a l s p e c i e s w i t h c o n t i n u e d and
c a t a s t r o p h i c development c a n b e d i s t i n g u i s h e d . These normal
a n a t o m i c a l and p h y s i o l o g i c a l v a r i a t i o n s a r e t h e background
f o r t h e n e o p l a s t i c development a s a r e t h e major abnormalit i e s of growth, A taxonomic-phylogenetic g r a d e c a n be s een
i n form of t h e development of t h e l y m p h a t i c system so imp o r t a n t f o r m e t a s t a s i s i f e.g. mammals, b i r d s , lower t e t r a p o d s , f i s h , a n d i n v e r t e b r a t e s a r e compared. These normal
a n a t o m i c a l f a c t o r s i n t e r a c t w i t h t h e environment d u r i n g
carcinogenesis.
ABSTRACTS-AAA 103RD MEETING
49A
KW,' Frederick W.K., Jean Langlais,* Normand Vigneault* and
p e r month; o f t h e s e , 2 c e l l s m i g r a t e d t o t h e p i t an d 7
c e l l s , t o the n e c k and b a s e . O l d p a r i e t a l c e l l s i n e i t h e r
Kenneth D. Roberts; Departments of Anatomy and Biochemistry,
p i t o r base underwent d e g e n e r a t i o n a n d were e l i m i n a t e d b y
University of Montreal and Makonneuve-Rosemont Hospital
e x t r u s i o n i n t o t h e u n i t lumen, p h a g o c y t o s i s b y n e i g h b o r i n g
Research Center, Montreal, Quebec, Canada. (Sponsored by
c e l l s o r d e s t r u c t i o n b y c o n n e c t i v e t i s s u e macrophages
Bernard Messier) 1
The
e n t e r i n g t h r o u g h a b r e a k i n the basement membrane.
of ohospholiuase A, in human sDermatozoa.
t u r n o v e r t i m e of p a r i e t a l c e l l s a v e r a g e d 7 1 d a y s .
Phospholipase A, (PLA,), which catalyses the hydrolysis of fatty
A s f o r mucous n e c k c e l l s , t h e e v i d e n c e i n d i c a t e d t h a t
acids linked to membrane phospholipids has been postulated to play
t h e y t r a n s f o r m e d i n t o c e l l s c a l l e d muco-zymogenic, which
a role in the acrosome reaction of mammalian sperm cells. The
t h e m s e l v e s t r a n s f o r m e d i n t o zymogenic c e l l s .
The l i f e
present study was undertaken to localize this enzyme in human
d u r a t i o n o f the l a t t e r exceeded 8 months.
spermatozoa. Polyclonal antibodies were raised in rabbits
immunized with solubilized membrane proteins extracted from
sperm heads with Triton X-100. The antibodies were recovered
from antisera using protein A Sepharose and further purified by
KARIM, Algernon, Dirk-Jan BERVOETS*, Antonius BRONCKERS', Don
chromatofocusing. Eluted fractions yielding positive responses
LYARUU' and Jo WaLTGENS*, Department of Anatomy, University of
against purified PLA, from human spermatozoa were pooled and
Manitoba, Winnipeg, Manitoba, Canada; and Laboratory of Tooth
applied to an affinity column of Affigel 10-CM Sepharose linked to
Development, Department of Oral Cell Biology, Free University of Amsterdam,
purified PLA, from human spermatozoa.
The anti-PLA,
The Netherlands. The effects of adriamvcin on tooth development in vitro.
immunoglobulins were then concentrated, dialysed and used for
In vivo, adriamycin destroys immature pulp cells and cause the production
immunofluorescentand immunoelectronmicroscopic localization of
of osteodentin in rat incisor pulp. The present study was initiated to
PLA, in human spermatozoa.
Anti-human sperm PLA,
determine whether the in vivo changes are a direct effect of adriamycin, and
immunofluorescence was brightest in the equatorial region of the
to investigate this drug's effects on tooth development. Second maxillary
sperm plasma membrane. Although less intense, anti-PLA, staining
molars of 4-5 days old golden hamsters were exposed for 2 h. in vitro to 1
was also detected along the flagellum. Label-fracture replicas
mg/L adriamycin, rinsed, and subsequently cultured up to 7 days without the
confirmed the immunofluorescent results and provided the high
drug. At 3, 5 and 7 days of culture the synthesis of extracellular tooth
resolution surface distribution of the antigen on freeze-fracture
matrices and their mineralization were examined by measuring the
images of human sperm cells. Together with results from fractureincorporation of 3H-proline (5 pCi/ml) and the uptake of 45Ca and 32P04 (5
label preparations, label-fracture established the transmembrane
pCi/ml) by the explants, after a 2 4 h. pulse labeling period. Compared with
nature of the PLA, molecule and incidentally, confirmed previous
unexposed control explants, adriamycin inhibited the overall growth of the
findings which suggested that the covalent backbone of integral
molars. This was reflected by the lower dryweight (Pz 0.0001) and total
proteins is not broken during freeze-fracture. Thin-section
protein content (p :0.001) than the control after 7 days in culture. In spite
iminiinocytochemistry revealed intracellular locations of anti-human
of this, adriamycin enhanced the biosynthesis and secretion of the watersoluble enamel matrix proteins (amelogenins). At 3 days (p s 0.0001) and
sperm PLA, over plasma membrane, acrosomal membranes and
5 days (p s 0.0001) dentin formation was inhibited, while at 5 and 7 days
matrix, nucleus as well as various tail components. Thus our results
-'Ta and 32PO, content were reduced. Also, osteodentin formation was
suggest that the role of PLA, is likely not to be restricted to
observed after 3 days in culture. Therefore, it appears that the stimulation
membrane events during acrosome reaction or sperm-eggfusion and
of extracellular enamel matrix production, which may be hypomineralized as
that P L k may also be involved in other important sperm processes.
a result of the reduced calcium uptake during its formation; the inhibition
Supported by grants from the MRC of Canada and FRSQ.
S
of dentin formation; and the stimulation of osteodentin formation may be
representative of a direct differential effect of adriamycin on various
compartments within the tooth. Moreover, since the decrease in 32P04
activity coincided with the formation of osteodentin, it is speculated that the
osteodentin matrix may not contain the highly specific phosphorylated
dentinophosphophoryns.
(Supported by grants from the MMSF and the MHRC.)
S h e r i f Mohamed a nd Charles P h i l i p p e LEBLOND,
Department o f Anatomy, M c C i l l U n i v e r s i t y , M o n t r e a l , Quebec,
.
Canada. 1
(Also Supported by The Academic C e n t e r for Dentistry, Fr e e
In the body r e g i o n o f t h e mouse stomach, t h e mucosa
University, Amsterdam)
c o n t a i n s numerous, f a i r l y s t r a i g h t u n i t s composed o f a p i t
which i s connected by an is thm us t o a g l a n d u l a r p o r t i o n
i n c l u d i n g two p a r t s : ne c k a nd base. E l e c t r o n microscopy
and SH-thymidine r a d i o a u t o g r a p h y were u s e d t o f i n d o u t how
the c e l l s o f the u n i t undergo renewal.
The i s t h m u s
c o n t a i n s , i n a d d i t i o n t o some m a ture p a r i e t a l c e l l s , four
Kawaja,* Michacl D., Fagan,* Anne M . , Fircstcin.* Bonnic L.
t y p e s o f immature c e l l s c h a r a c t e r i z e d by few o r g a n e l l e s ,
and Gage,* Fred H., (Sponsored by Glcnn R. Northcutt)
many free r i bos ome s a nd d i f f u s e n u c l e a r c hro m a t i n :
Dcpartment of Neuroscicncc, University of California, San
1) u n d i f f e r e n t i a t e d c e l l s t h a t a r e h i g h l y p r o l i f e r a t i v e
U It r a s t r u c t u ra I o r San i L a t i on of p ri m a ry
Diego, Cal i iorni a.
(25% l a b e l i n g 30 min after 3H-thymidine i n j e c t i o n ) a n d a r e
fibroblast autografts i n the rat striatum,
t a k e n t o be s t em c e l l s g i v i n g r i s e t o t h e o t h e r immature
A prcvious study from this laboratory explored parameters
c e l l s ; 2 ) immature s u r f a c e mucous c e l l s , w i t h few s m a l l
for intraccrebral g r a f t i n g of gcnctically modified skin
dense mucous g r a n u l e s ;
3) immature p a r i e t a l c e l l s ,
primary fibroblasts in the rat (Fircstein et al., Soc. Neurosci.
c h a r a c t e r i z e d by long i r r e g u l a r m i c r o v i l l i a t t h e apex and
15, 1989). There rcmains, howevcr, a paucity of information
4) immature mucous ne c k c e l l s , w i t h f e w l a r g e mucous
concerning the morphological features of autologous skin
The l a s t t h r e e immature c e l l s e v o l v e i n t o t h e
granules.
fibroblast grafts, and thc cellular interactions between graft
c o r r e s p o n d i n g m at ur e c e l l s , namely, s u r f a c e mucous, mucous
and host CNS. The purposc of the present investigation was to
n e ck, and p a r i e t a l c e l l s .
providc a dctailed examination of thc ultrastructural charactTo t r a c e the m i g r a t i o n and f a t e o f m a ture c e l l s ,
eristics of fibroblast autografts in thc rat striatum. Primary
c o n t i n u o u s i n f u s i o n of SH-thymidine w a s a d m i n i s t e r e d f o r
fibroblasts isolated from skin biopsics were grown in culturc
t i m e s v a r y i n g from 2 t o 52 da ys . S u r f a c e mucous c e l l s were
under standard conditions and takcn through 4 passagcs beforc
found t o m i g r a t e o u t o f the is thm us toward the g a s t r i c
~
implantation. Stcrcotaxic placcmcnts o l cells ( 3 ~ 1 0 cclls
lumen i n a p i p e l i n e p a t t e r n ; i n 4 d a y s , t h e y r e a c h e d the
in 3 P I ) were madc in the striatun1 of donor female Spragucs u r f a c e where t h e y were l o s t .
Mature p a r i e t a l c e l l s m i g r a t e d from t h e isthmus i n t h e
Dawlcy rats. After 8 wccks, the animals were ancsthctized and
perfused with a solution of 4% paraforrnaldchyde and 0.1%
d i r e c t i o n o f e i t h e r p i t o r ne c k. I t w a s e s t i m a t e d t h a t , on
glutaraldchydc in phospholc buffcr (pH 7.4).
Corona1 sections
the a v e r a g e , 9 p a r i e t a l c e l l s were produced i n one isthmus
KARAM,*
50A
ABSTRACTSLAAA 103RD MEETING
(50 p m ) wcre post-fixed in buffered osmium tetroxide,
dchydratcd and ernbcdded in plastic. The grafts were composed
primarily of fibroblasts and collagen. The fibroblasts possessed
cxtcnsivc rough cndoplasmic rcticulum and secrctory vesicles.
Phagocytic cclls with debris-filled cytoplasm wcre found in the
grafts as well. Astrocylic processes were also evident throughout thc grafts, but astrocytc cell bodics were found in the
ncuropil only.
Endothclial cells of small blood vessels and
capillaries within the grafts possessed junctional complexcs at
These rcsults reveal that primary fibroblast
sites of apposition.
autografts appear active and hcalthy at 8 weeks following
implantation in the striaturn, and thcrc are no signs of tumorigcncsis. The presence of host astrocytic processes and blood
vcsscls i n the grafts may scrvc as structural and nutritive
supports rcspcctivcly , and both fcaturcs indicate dynamic
From thcsc
ccllular intcraclions bctwccn graft and host CNS.
rcsulls, i t is concludcd that skin primary fibroblasts arc
[~otcntial candidatcs f o r grafting aftcr gcnetic modificalion,
2 n d the eniployrncnt of thcsc autologous cclls rcduccs lhc
likelihood of turnor formation and, possibly, inimunc rcjcction.
S
ANP is a diuretic and vasodilatory peptide normally
synthesized and secreted by the atria of the mammalian heart.
More recently it has been demonstrated that the ventricles also
produce ANP. Ventricular ANP has been demonstrated in the
fetus and neonate and under pathological conditions such as
hypertension. Right ventricular hypertrophy (RVH) and
pulmonary hypertension can be induced in rats by exposure to
hypobaric hypoxia. In order to localize ANP in the ventricles of
the heart during the onset, maintenance, and recovery from
hypobaric exposure, we subjected male Sprague-Dawley rats to
hypobaric hypoxia (0.5 atm.) for 1 to 21 days or 21 days + 21
days of normobaric recovery. RVH was detected as early as 1
day after hypobaric exposure and increased steadily between 7
and 21 days.
Using an antibody against synthetic atrial
natriuretic factor IV, we performed immunohistochemical staining
on sections containing both ventricles. ANP-immunoreactivity
(ANP-ir) was detected in the right ventricle and septum as early
as 3 days, and staining increased up to 21 days. Staining was
also detected in the left ventricle at 21 days. Staining first
appeared as specific foci at the intersection of the right ventricle
and septum and near arteries in the right ventricular and septal
walls. As hypobaric exposure continued, ANF staining increased
in intensity and appeared to move outward in all directions from
the initial foci of ANP-ir. These initial foci may be sites of
maximum wall tension due to the hypertrophy. In the recovery
group, ANP-ir was notably reduced in all ventricular areas and
the staining resembled that of hearts from rats exposed to
hypoxia less than 3 days. We conclude that there is a direct
correlation between the degree of ventricular hypertrophy and
immunocytochemical labeling for ANP in the ventricles. This
work was supported by AHAl Kansas AftXate.
KELLY*, Cheri W., Andrzej Janecki*, Anna Steinberger* and
Lonnie D. Russell, Department of Physiology, Southern Illinois
Univ., Carbondale Illinois and Department of Oh/Gyn and
Reproductive Sciences, Univ. of Texas Health Sciences
. Center.
.
Houston, Texas. Matu ration of Sertoli cells i n vivo and i n v m o a s
jndicated bv chanees in cell and nuclear voluncii.
Much of our current information about Sertoli cell function
Unfortunately. this
has been obtained from in vitro studies.
information is from cultured rat Sertoli cells of immature animals
Cells from young animals are most
(about 14-20 days of age).
readily cultured.
It was the objectives of this study to determine
if Sertoli cells grown in culture are similar to Sertoli cells grown
in vivo. and to determine how closely Sertoli cells from immature S
animals resemble those of the adult.
Nuclear and cell volumes,
determined by standard morphometric techniques for LM and EM, KEMPER, Abbie C. and Robert D. SPECIAN, De artment of
Cellular Biology and Anatomy, LSU School o f Medicine,
were used to judge cell maturation in four groups:
# l . Perfused testes from five 18 d. rats (mean testis wt. 0.09 Shreveport? Louisiana. A comparative studv of intestinal stored
mucin in iuvenile. YOU ne adult and adult ratS. As rats age,
gm.)
#2. Fixed pellets from ten 19 d. rats (mean testis wt. 0.09 gm.) developmental changes oc&r in a variety of organ systems. While
changes in the structure of the intestine may be anticipated, they
isolated according to Janecki and Steinberger (1987)
have not been well defined. The purpose of this study was to
#3. Perfused testes from five 26 d. rats
#4. Sertoli cells from ten 18-19 d. rats (mean testis wt. 0.09 gm.) quantitate and compare the volume of intestinal stored much in
juvenile (50 gm), young adult (175 gm) and adult (500 gm rats.
cultured for seven days (two chamber system) w F S H and T
Group
#I
Nuclear Volume ( p m 3 )
Cell Volume ( p m 3 )
226.5 f 7.5
884.1 i 62.0
#2
272.0 11.2
945.8 i 44.7
#3
323.6f 6.3
1,920.1 i 190.3
#4
340.9 f 17.7
2,156.1 f 118.7
adult
523 and 592
6.012 (from cited values)
Sertoli cells from immature testes either in vivo o r i n virro
display nuclear and cell volumes that are a fraction of the adult
size, indicating cell and nuclear volumes are measures of Sertoli
cell maturation.
Volumes of Sertoli cells and their nuclei
prepared for culture are not different (P<0.05)from those of the
intact animal at the same age. From day 19 to day 26 days, Sertoli
In the
cells grow in vivo to over twice their initial size.
equivalent time period, Sertoli cells in culture grow to attain the
same size as their in vivo counterpart (P>0.05). This indicates that
biochemical experiments measure not only 'time-in-culture'
responses but also maturation responses. Suppported by NIH
HD17802 (AS).
*
S
KELLEY,* Kirk B., MCKENZIE, James C., and KLEIN, Robert
M., Department of Anatomy and Cell Biology, University of
Kansas Medical Center, Kansas City, Kansas; Department of
Anatomy, Howard University, College of Medicine, Washington,
D.C.
Immunocytochemical localization of atrial natriuretic
peptide (ANP) in the ventricles of the rat heart during right
ventricular hypertrophy and recovery.
Five male, Wistar rats in each age group were fasted ovemigl! and
fixed by vascular perfusion. The entire small intestine was removed,
photographed and cut into ten equal segments, and further sliced,
processed and embedded in JJ3-4. The length of the small bowel
was determined with a digital analyzer from photographic
enlargements of the gross specimen. For morphometry, 3 pm
sections from each segment were stained with PAS to demonstrate
neutral mucins. The volume of epithelium er surface area of
epithelia1 basal lamina was calculated with a &em grid to correct
for anisotrophy and a square lattice rid was used to determine the
volume of stored mucin per epithefium. Stored mucin quantities
were then expressed in terms of basal lamina surface area. The
total length of the small intestine increased from 77.1 cm for 50
gram animals to 108.3 cm. and 102.4 cm for 175 and 500 gm animals
respectively. Although the length of the intestine increased, no
statistically significant differences were observed in the volume
density of villus stored mucin per villus basal lamina among the
three age groups. In sharp contrast however, dramatic differences
in the quantit of crypt stored mucin per crypt basal lamina were
revealed. In 80 gm rats, crypt mucin values ranged from 0.96
pms/pm2 in the duodenum to 1.44 pms/pm2 in the ileum, and in
175 gm rats, the volume density for these same segments was 0.93
pms/pmz and 1.40 pms/pm2. In 500 gm animals, values of 2.30
pms/pmz and 4.17 pms/pm2 were recorded, indicating an increase
in the volume density of > 100%. From these data it a pears that
the amount of mucin stored in villus goblet cells per s u d c e area of
villus basal lamina remains constant throughout the lifetime of
these animals, although there is an increase in the total stored
much in the small intestine as a result in an increa e in total length
of the gut from 50-175 gms. Supported by Grant JDK 33720 from
the National Institutes of Health.
ABSTRACTSAAA 1 0 3 MEETING
~ ~
51A
S
Kennedy,* Jasiri and James C. McKenzie, Dept. of
Anatomy, ColLpf Me&, Howard University,Washington,
L
. . . .
L
Originally discovered in the cardiac atria, atrial
natriuretic peptide (A")
has also been localized in the
brain where it may serve as a neurotransmitter. In our
study of the distribution of ANP in the canine brain, we
have noted populations of intenselyANP-immunopositive
perikarya in the cap regions and cellular bridges of the
Islands of Calleja. These structures may be related to
extrapyramidalor endocrin~rticostriatopallidalsystems
involved in regulation of oroingestive behavior or
reproductivefunctions. The purpose of this study was to
determinethe neurotxansmittertypesof &erents to these
neurons prior to tracing studies. Formalin-fixedsections
of dog brain were incubated in ANP antibody (1:1O,OOO) and
staining was completed using the ABC technique with
DAB as the chromagea After washing, sectiom were then
incubated with antibodies against either tyrosine
hydroxylase (TH)(l:lO,OOO)or substance P (SP)(1:2,000)
using the glucose oxidase (GO) technique with the
tetrazolium salt NBT as the chromagea These double
labelling studies revealed the absence of dopaminergic (TH)
inputs and the presence of a dense network of SP inputs to
the region of the ANP-positiveperikarya. TH-positive
afferents appeared to be most dense in the region of the
granule cell complex and the core zones. Thus, ANPpositive perikarya of the cap and bridge regions are
probably not innervatedby dopaminergicneurons of the
substantia nigra-ventral tegmental area. Supporting
studies at the ultrastructural level are currently
underway. Supportedby grantsh m the Americian Heart
Association, Nation's Capital Affiliate and from Howard
U n i ~ ~ ~ .
KIELY, Michael L. and D;...lly- R. SAWYER*, Department of Anatomy and Oral Biology and Department
of Oral Diagnosis, Pathology and Radiology,
Loyola University Chicago, School of Dentistry,
Maywood, Illinois. Interparietal bones in the
Asian Indian skull.
During development of the occipital bone,
the interparietal part of the squamosal portion
may not fuse completely thus resulting in a single, separate bone or in two or more bones. Examination of the occipital region in 234 adult
Asian Indian skulls (125 male and 109 female)
revealed the presence of separate interparietal
bones in eight skulls (3.42%). In five of these
cases, this bone was seen as a single midline
structure; in two instances, the bone was found
on the left side and in one skull, the interparietal region was divided into two separate portions. Also, in 18 skulls, single or multiple
preinterparietal bones were observed at the lambda; two of these were associated with interparieta1 bones. In numerous skulls, small sutural
bones were found in the lambdoid suture on both
sides some of which were associated with the
interparietal and preinterparietal bones. Observation of separate bones in the interparietal
and preinterparietal regions provides basic information regarding the ossification patterns of
these areas. Interpretation of the data from
this study indicates that the interparietal develops from five ossification centers i.e. one
central, two lateral and a pair in the preinterparietal region. The possibility exists, however
that the central piece may develop from two centers. The significance of multiple sutural bones
observed in the lambdoidal region and their association with incomplete fusion of the interparietal is unknown. The results of this study reveal a greater frequency of occurrence of interparietal separation than has been previously reported in other populations.
S
KLIETSCH*, Rhea P., Mary Ellen ROSSOUW and Peter A .
MERRIFIELD, Department of Anatomy, The University of
Western Ontario, London, Ontario, N6A 5C1. Phenotvuic
expression and fate of urimarv mvotubes in uresumutive
fast ind slow muscles of the JaDanese auail.
The histogenesis of muscle is characterized by the
sequ; itial appearance of two distinct populations of
muscle cells, usually referred to as primary and
secondary generation myotubes. In the Japanese quail,
___Coturnix coturnix jauonica, primary myotubes differentiate in specific muscles between embryonic day 5 (ES)
and E8, and act as a scaffolding for the subsequent
We have used
development of secondary myotubes.
monoclonal antibodies specific for slow ( S M - 1 , SM-2) and
perii,atal (fast) isoforms of myosin heavy chain (MHC) to
follow the differentiation and fate of primary myotubes
in embryonic muscles which develop into predominantly
fast (pectoralis, sartorius) or slow (medial adductor)
muscles in the adult. In all three muscles, primary
myotubes co-express both slow (SM-1) and perinatal
(fast) MHC at 8E - 10E, while secondary myotubes express
In the pectoralis
predominantly perinatal (fast) MHC.
muscl-, primary myotubes atrophy and die around hatching
whilt the secondary myotubes mature to populate the
musci? with exclusively fast (Type 11) fibers. In the
sartrrius muscle, primary myotubes down-regulate
perinatal (fast) MHC around 12E and begin to co-express
SM-2 and SM-1, suggesting maturation into slow fibers
which persist as Type I11 fibers in the adult. Primary
inyotubes in the medial adductor undergo a similar
tranformation, however in this muscle secondary myotubes
make only a minor contribution to subsequent muscle
growth.
These results suggest that primary myotubes
develop into slow fibers of adult muscles and are
dependent upon environmental cues for their survival.
Since primary myotube death and changes in MHC
expression occur concurrently with the loss of
polyneuronal innervation, we are presently examining the
role of innervation in the maintenance and maturation of
primary myotubes. Supported by a grant from MRC to PAM.
M., Norkrt J. E X J R Z m * , and Loan
iunctions in
the NOD mouse. Department of ha&&
Sciences and
Neurobiology, university of Lcprisville, Lcuisville,
XIUEBER, Kathleen
mm*. Momholcaic analysis of re-ar
Kentucky.
R a n d e l l i n g of r w m m w c d a r junctions (NMS) ccaus
to -ise,
in a nunher of disease states, as a
and b i n g aging. The process is characterized by degenerwell as an
ation of old NMJ's, formation of new ones
inmease3 fresuency of intranakl and +3?muEdspmuting
of i n t r a - F a r nerves. The plrpose of this ""r was
to deternune if NMS remoaellirq ocmrs in diabetic
neuropathy. m e extensor digitorum lciqus (EDL) f?
5 NDD
mice/ age (4, 6, 8, 10, 18 and.20 Weeks) Were exarmned
electron micrcsczpically and hmtochemically. Nm length
(N=50/ muscle) was detennind fmm whole nuunt silver/
acetylcholine stained muscles (Slack +t pnckett! F'f1qe.r~
Ach. 391: 306-308, 1981). NMS length lllcreased signifie
antly (p<.05) with age. Myofibers are thCUght to irduce an
increase in NMS size as a response to h
m
y or
patholqic conditions within the fiber. U l t r a s t r u m
52A
ABSTRACTS-AAA 103RD MEETING
examination of the EDL muscles M a t e d mkstimh'a1 degaeration of NMS'S, myofibers and capillaries w h i c h
progressed with the pathcgemsis of the disease. Erdplates
without nerve terrmMIs indicate possible retraction of
tenninal sprouts or incconplete reinnervation of the
myofiber. Myofiber -is
was indicated by distended ttubules, mitmhondrial inclusions, disruption of the
sarcolermna, and empty basal lamina1 tubes. (Funaea by a
grant from NIMlK #R29DK41553).
KNIGHT, David Stanley, Howard W i l l i a m RUSSELL* and John
Anthony BEAL, Department of C e l l u l a r Biology and Anatomy,
L o u i s i a n a S t a t e U n i v e r s i t y School of Medicine, S h r e v e p o r t ,
Louisiana.
T r a n s i t o r y inner medullary nerve terminals
i n t h e c a t kidney.
I n t r a r e n a l nerves immunoreactive f o r tyrosine hydroxylase
( T H I ) , dopamine b e t a hydroxylase ( D B H I ) , substance P ( S P I )
and c a l c i t o n i n
gene-related
peptide
(CGRPI)
were
v i s u a l i z e d by t h e ABC method i n cats 2 , 4 , 6 , 8, 10 and
1 2 weeks of age.
Animals were fixed by p e r f u s i o n w i t h
2% p a r a f o r m a l d e h y d e + 0.15% p i c r i c a c i d , and k i d n e y s were
sectioned w i t h a V i b r a t o m e a f t e r immersion i n t h e f i x a t i v e
overnight.
THI and D B H I nerve terminals innervate t h e
r e n a l p e l v i s , i n t e r l o b a r v e i n s and a r t e r i a l t r e e including
m e d u l l a r y vascular bundles of c a t s of each age studied.
In k i d n e y s of 2 , 4 and 6 week o l d c a t s , THI and D B H I axons
emerge from t h e vascular bundle and pass through t h e inner
s t r i p e as a coherent b u t loosely-organized group i n t o t h e
inner m e d u l l a .
C o l l a t e r a l s of these axons diverge from
t h e c e n t r a l group of axons and form e l a b o r a t e , varicose
plexuses among t h e surrounding t u b u l e s of t h e inner
medulla.
Such p l e x u s e s i n 8 and 10 week o l d c a t s are l e s s
e x t e n s i v e , and many terminals appear degenerate. No THI
o r D B H I nerve plexuses were found among inner m e d u l l a r y
t u b u l e s of t h r e e month o l d c a t s . Most i n t r a r e n a l S P I and
CGRPI nerve terminals i n c a t s of each age innervate t h e
r e n a l p e l v i s and i n t e r l o b a r a r t e r i e s .
Nerve terminals
immunoreactive f o r t h e s e p e p t i d e s are l e s s numerous t h a n
T H I and D B H I terminals b u t a l s o innervate t h e i n n e r m e d u l l a
of 2 , 4 and 6 week o l d c a t s .
S P I terminals are sparse
and confined t o t h e c e n t r a l g r o u p of axons t h a t emerges
from t h e v a s c u l a r b u n d l e , whereas CGRPI terminals are more
numerous and form p l e x u s e s among t h e surrounding t u b u l e s
of the i n n e r medulla.
There were no S P I o r CGRPI nerve
t e r m i n a l s i n t h e inner m e d u l l a of t h r e e month o l d c a t s .
I n n e r m e d u l l a r y innervation a p p e a r s t o b e t r a n s i t o r y .
S u p p o r t e d by N I H g r a n t HLB 34431.
KOCH,* Barbara A . and William E. KOCH, Department
of Cell Biology and Anatomy, University of North
Carolina at Chapel Hill, North Carolina. Growth
and differentiation of maxillary vrocesses during
treatment with colchicine.
Embryonic growth may include cell enlargement
and differentiation, an increase in cell number,
and/or an increase in mass or volume of extracellular materials. Earlier quantitative studies
revealed that fractions of the maxillary process
of the embryonic chicken differ in growth
potential
and
in
capability
for
forming
differentiated tissues.
The current study
assesses the importance of cell reproduction in
the early growth phase of these fractions.
Isolated maxillary processes were segregated into
equal distal and proximal halves.
Control and
experimental cultures, derived from the same
embryo, were grown in contact with complex culture
medium containing serum.
Inhibition of cell
division was achieved by addition of colchicine
(O.Olpg/ml) to the
medium during the culture
period. After two days, the mean volume of distal
control cultures was approximately 5 . 24x107wm3
whereas treated cultures measured 2. lxiO7pm3. A
similar reduction was noted in the growth of
colchicine treated proximal fractions, Volumes of
fractions exposed to colchicine at three days, and
fixed on the fifth day, showed little difference
when
compared
with
controls.
Types
of
differentiated tissues also differed in control
and
experimental
cultures.
Quantitative
measurements of growth under these conditions have
revealed that cell reproduction is a significant
aspect of the overall enlargement during early
facial morphogenesis in the chick.
Supported by NIH Research Grant DE 08035
KORDOWER, Jeffrey H., Don M. GASH, and Massimo S .
FIANDACA? Department of Anatomy and Cell Biology, University of
Illinois School of Medicine, Chicago Illinois; Department of
Neurobiology and Anatomy, University of Rochester School of
Medicine, Rochester New York; and Division of Neurosurgery,
University of Massachusetts Medical Center, Worcester Massachusetts.
T r o ~ h i cand neurite Dromotine effects of peripheral nerve erafts uDon
cholinereic septal/diavonal band neurons in monkevs.
Cholinergic basal forebrain [CBF] neurons contain receptors for nerve
growth factor (NGF) and are dependent upon NGF for their viability.
This information, coupled with the consistent observations that CBF
neurons degenerate in Alzheimer's disease has led to the speculation that
NGF may prevent the degeneration observed in this region. Schwann
cells which ensheath peripheral nerves produce NGF and other growth
factors following nerve transection. We tested the hypothesis that grafts
of autologous surd nerve [SN] could provide trophic support to
axotomized CBF neurons in Cebus monkeys. In studies conducted to
date, ten monkeys received a unilateral knife cut of the fornix using an
open microsurgical procedure. Nine monkeys received transplants at
the time of the lesion. They received either the lesion alone [n=l],
control intracerebroventricular (icv) implants of muscle [n=2], icv
implants of SN [n=4], or implants of SN placed into the lesion site
[n=2]. The final monkey received an icv implant of SN 7 days
following the fornix transection. All monkeys were sacrificed 30 days
following implantation. Sections were processed for the visualization of
the NGF receptor and acetylcholinesterase [AChE]. Lesions were
verified in all monkeys by the dramatic reduction in AChE-containing
fibers within the ipsilateral hippocampal formation and dentate gyms.
Monkeys receiving the lesion alone, control implants, SN implants into
the lesion site, or nondelayed icv SN implants all displayed a similar
reduction [mean=50%] of cholinergic medial septal neurons on the
lesioned side. In contrast, a 14% reduction in cholinergic medial septal
neurons was observed in the single animal receiving the delayed SN
transplant. The five monkeys receiving the icv SN implants also
demonstrated a vigorous AChE-sprouting response throughout the
septal region, an effect not observed in monkeys within the other
treatment groups. These fibers appeared to emanate from intact neurons
within the vertical limb of the diagonal band and abutted the icv SN
implant. These data suggest that implants of peripheral newe can induce
neurite outgrowth from intact primate CBF neurons and possibly
provide trophic influence upon axotomized CBF neurons as well.
KORTE, Gary E. Departments o f Ophthalmology and Anatomy,
A l b s r t t i n s t e i n C o l l e g e o f Medicine, Bronx, New York. High
voltage e l e c t r o n microscopy o f r e a c t i v e M u l l e r c e l l s i n
rabbit retina.
H i g h v o l t a g e e l e c t r o n m i c r o s c o p y (HVEM) was used t o
examine t h e changes i n M u l l e r c e l l (MC) shape and t h e
d i s t r i b u t i o n o f e x t e r n a l l i m i t i n g membrane (ELM) j u n c t i o n a l
components d u r i n g f o r m a t i o n o f s u b r e t i n a l s c a r s i n r a b b i t s
dosed w i t h sodium i o d a t e . T h i s chemical d e s t r o y s t h e
p h o t o r e c e p t o r s and r e t i n a 1 pigment e D i t h e l i u m (RPE), leadi n g t o s c a r f o r m a t i o n i n t h e space f o r m e r l y o c c u p i e d by
t h e damaged cells. HVEM was used t o s t u d v t h e p r o c e s s bv
which
a s c e n d i n g p r o c e s s e s o f MC t r a n s f o r m frorn s l e n d e r
ones f o r m i n g t h e ELM i n t o h y p e r t r o p h i c ones t h a t c o n t a c t
ABSTRACTS-AAA 103RD MEETING
t h e remnant RPE basement membrane. Observations were made
usinq STERECCN, a computer program t h a t r e c o n s t r u c t s
t r a c e d images o f s e r i a l t h i c k s e c t i o n s (0.5 o r lum s e c t i o n s
were used) viewed i n s t e r e o (Marko e t a l . , J. E l e c t r .
Microsc. Tech. 9:393,'88).
HVEM revealed t h a t MC ascending
processes undergo s t r i k i n g changes d u r i n g scar formation:
i ) t h e ELM j u n c t i o n a l complexes and m i c r o v i l l i a s s o c i a t e d
w i t h them become s c a t t e r e d o v e r t h e ascending process
surface. A s i m i l a r s c a t t e r i n g occurs on m i f o t i c MC t h a t
were reconstructed. i i ) The ascending processes hypertrophy
and become more i r r e g u l a r i n shape. i i i ) When t h e processes
c o n t a c t t h e remnant RPE basement membrane, however, they
become more r e g u l a r i n shaoe and lose t h e f i l i f o r m and
lamell i f o r m appendages t h e y n o r m a l l y bear. They a l s o form
attachments t o t h e basement membrane. The HVEM o b s e r v a t i o n s
c l a r i f y r o u t i n e t r a n s m i s s i o n e l e c t r o n microscopic and
l i g h t microscopic ones. They a l s o provided views o f r e a c t i v e ascending processes t h a t suggest c o n t a c t w i t h t h e
RPE basement membrane e l i c i t s s t r u c t u r a l changes i n them,
and t h a t t h i s c o u l d c o n t r i b u t e t o t h e c o n t r o l o f subr e t i n a l scar formation d u r i n g r e t i n a l disease.
Supported by Research t o Prevent Blindness, I n c . and
a g r a n t from NIH (#RR01219) supporting t h e Biotechnology
Resource i n HVEM a t t h e NY S t a t e Dept. o f H e a l t h i n
Albany, NY.
K R B B S , Wolf and Ingeborg K R E B S , *
D e p a r t m e n t of O p h t h a l m o l o g y , C o l u m b i a
U n i v e r s i t y , N e w Y o r k . A d i s t i n c t a r o u D of
n e u r o n s in the r a t r e t i n a w i t h a x o s o m a t i c
SYEL&EESE
I n the v e r t e b r a t e r e t i n a , the s o m a t a of
n e u r o n s a r e s p a t i a l l y s e p a r a t e d f r o m the
n e t w o r k of t h e i r a x o n a l a n d d e n d r i t i c
fibers. It i s g e n e r a l l y a s s u m e d that
a x o s o m a t i c s y n a p s e s d o n o t o c c u r i n the
retina. H o w e v e r , in r e t i n a s of a l b i n o
l a b o r a t o r y r a t s , a d i s t i n c t p o p u l a t i o n of
small cells with axosomatic synapses was
discovered. T h e s o m a t a of t h e s e c e l l s w e r e
l o c a t e d at the i n n e r b o r d e r of t h e
i n t e r n a l n u c l e a r l a y e r , partly p r o t r u d i n g
i n t o the i n n e r p l e x i f o r m layer. D a r k l y
stained cytoplasm and irregularly
indented, dense nuclei characterized these
cells. T h e y n u m b e r e d a p p r o x i m a t e l y 3,000
per m m a of r e t i n a . T h e s o m a t i c c r o s s
s e c t i o n s w e r e b e t w e e n 3 and 6 pm i n
diameter. S y n a p s e s w e r e s e e n r e g u l a r l y at
t h e p e r i k a r y a l m e m b r a n e of the cells. T h e
presynaptic terminals contained either
d a r k c o r e or c l e a r v e s i c l e s i n d i c a t i n g
t h a t a t l e a s t s o m e of the t e r m i n a l s
b e l o n g e d to a m a c r i n e cells. S m a l l s y n a p t i c
r i b b o n s , d i a g n o s t i c for b i p o l a r c e l l s ,
were s e e n in s o m e of the p r e s y n a p t i c
processes. T h e m o r p h o l o g y of the n u c l e u s
and the f a c t t h a t a m a c r i n e and b i p o l a r
cells made synaptic contacts to the small
c e l l s s u g g e s t t h a t they a r e a m a c r i n e s .
Richard M. KRIEBEL,* Kenneth E. MILLEQ* and James P.
McALLISTEP II,* Depts. Anatomy, PCOM and Temple U. Sch.
Med., Phila., Pennsylvania and Searle Co., St. Louis,
Niewenhuis)
Missouri. (Soonsored bv R.J.
Immunohistochemical study of the basal forebrain region
in experimental infantile hydrocephalus.
The primary goal of our studies on experimental
infantile hydrocephalus has been to provide a cellular
basis for the residual neurological deficits observed
53A
even though surgical intervention may have relieved the
ventriculomegaly. Cytoarchitectural alterations may
only provide a partial insight into the effects of the
increased intracranial pressure on the central nervous
system. It has been established that alterations in
the neuronal microenvironment may result in abnormal
phenotypic expression of neurotransmitters. Using
immunohistochemical methods we have begun to examine
alterations in choline acetyltrans-ferase(ChAT) in the
basal forebrain region of kittens which have received a
cisternal injection of kaolin to produce experimental
hydrocephalus. Rydrocephalic animals were killed ten
days after kaolin injection, fixed brains were sectioned, reacted with antisera to ChAT, and localization
of ChAT visualized with peroxidase anti-peroxidase
techniques. The basal forebrain region in kittens contains numerous neurons labelled with the ChAT antisera.
In the hydrocephalic brains, enlargement of the
anterior aspect of the lateral ventricles severely
compresses the septal forebrain region ventrally
against the cranial floor as well as pushing basal
ganglia and substantia innominata laterally. Although
grossly displaced, the substantia innominata contained
ChAT immunolabelled neurons. In contrast, there was a
decrease in ChAT containing neurons in the septal
region. However, a large population of unlabelled
neurons was still found in this region. Future studies
on hydrocephalic kittens which have received ventriculoperitoneal shunts will determine if ChAT labelled
septal neurons can be spared. Supported by NTY HD21527
to JPM.
KRAUSE,William J. and Debra M. SHERMAN., Department of Anatomy, School
of Medicine, University of Missouri, Columbia; and Department of Botany and
Piant Pathology, Purdue University, West Lafayette, Indiana. Loeslization of
relaxin in the reDroductive tract of the female owssum.
Ten adult female opossums (DidelDhis vireiniana) were examined in this study
and consisted of non-pregnant, pregnant, and lactating animals. Each p u p
consisted of at least three animals. Approximate ages of pregnancies were
determined from timed pregnancies. Female opossums were stripped of their
litters and placed in breeding pens with continuous access to males. A spermpositive date was determined by examining smears taken from the umgenital
sinus each morning. Females 10%and 11%days into pregnancy (opossums have
a 12% day gestation period) were killed by an intracardiai injeaion of Ketamine
followed by exsanguination. Ovaries, oviducts, uteri, lateral vaginal canals as
well as placentas from pregnant animals were removed as quickly as possible and
fixed by immersion in Bouln's solution for a period of 24 hrs. Tissues were
processed routineiy for paraffin embedding, sectioned at about 6 microns and
mounted on acidsleaned slides. seaions were deparaffinzed in ylene and
rehydrated to water prior to incubation in 1.0% H20Z to remove endogenous
peroxide. After blocking with normal goat serum, the sections were incubated
with primary antiserum (anti-porcine relaxin: courtesy of P.A. and MJ. Fields)
for 24 hrs. at 4'C. Immunohistochemical visualization was accomplished by the
avidin-biotin-peroxidasecomplex (ABC) procedure using a Vecta-lab "elite" kit.
Peroxidase activity was demonstrated by incubation with 3,3'daminobenzidine
tetrahydrochloride containing 0.01% H202 in 0.1M Tris-HCL bufler. Controls
consisted of pre-absorption of the primary autiserum with the carresponding
antigen as well as omission of the primary antiserum. Sections of Bouln's Iixed
porcine corpra iutea served as a positive control. Treated sections were then
dehydrated, cleared in ylene and wverslipped for examination. Relaxinimmunoreactivity was observed only in the corpra lutein cells of ovaries from
non-pregnant, pregnant and lactating opossums. Immunoreactivity for porcine
relaxin was not observed In other tissues examined. Unlike many other
methatherian species, the corpra of DidelDhis persist well into lactation and
component lutein cells remain immunoreactivefor relaxin. Loesliitiou of reiaxin
in the ovaries of a marsupial is of interest because at parturition the young pass
through a newly formed mid-line passage (pseudovaginal canal) rather than
through the vaginal canal as in eutherian mammals.
S
'KRESS, Douglas W., Patricia A. GRAHAM'. Mark H. SCHUTTA', John
P. HANIFIN', Milton H. STETSON', Fred D. LUBLIN', Robert L.
KNOBLER, George C. BRAINARD. Dept. Neurology, Jefferson Medical
54A
ABSTRACTS-AAA 103RD MEETING
College, Philadelphia, Pennsylvania. T
h
e
f
f
e
oft
sphptpDeriod
.e
.c
m o s t e r o n e on thvmic weiaht in dewIn juvenile Siberian hamsters, increased testicular weight and
concomitant thymic involution is seen with exposure to long
photoperiods while decreased testicular weight and larger thymuses
result from short photoperiod exposure. This study tested the
hypothesis that high levels of testosterone secreted during exposure to a
long photoperiod mediate thymic involution. Groups of 9 juvenile male
Siberian hamsters were kept in either a long photoperiod (L:D 14:10),
a short photoperiod (L:D 8:16), or a short photoperiod plus a daily
subcutaneous injection of testosterone cypionate (1 mg/kg).
Organ
weights were compared as percent body weights (mean L SEM).
Hamsters exposed to a long photoperiod had significantly increased
testicular weights (2.35 j- 0.1 1) and significantly decreased thymic
weights (0.14 ;t .0065) compared to the testicular(0.37 ;t 0.22) and
thymic (0.2 A .0012 ) weights seen in the short photoperiod group
(pc0.001). Those hamsters injected with testosterone had testicular
weights (0.17 5 0.04) similar to the other short photoperiod group
and significantly lighter than those from the long photoperiod group
(p<O.OOl). However, thymic weights (0.15 rt ,0078) in animals
receiving testosterone were similar to those of the long photoperiod
group and significantly lighter than those from the short photoperiod
group (p<0.005). RIA showed that the long photoperiod animals had
significantly lower plasma cortisol levels than either of the two short
photoperiod groups (pe0.025) and that the short photoperiod group had
significantly lower plasma testosterone than
either the long
photoperiod group or the group receiving testosterone (pe0.005).
Given no significant differences in cortisol levels between the two short
photoperiod groups, we conclude that the testosterone treatment in the
short photoperiod group was a primary mediator of thymic involution.
This result suggests that high levels of testosterone in hamsters exposed
to a long photoperiod may mediate thymic involution by a similar
mechanism. Supported by NEMA Grant (LRI 89:DR:NEMA:I); NASA
Grant (NAGW1196); and NSF Grants (DCB84-12587 and DCB87
-14638).
.
S
KRUK,* P a t r i c i a Ann, Ne l l y AUERSPERG, and Sarah Louise
MAINES-BANDIERA,*
Department o f Anatomy, U n i v e r s i t y o f
Human
B.C.,
Vancouver,
B r i t i s h Columbia,
Canada.
o v a r i a n surface e o i t h e l i u m i n t i s s u e c u l t u r e .
Although t h e human o v a r i a n s u r f a c e e p i t h e l i u m (OSE)
comprises o n l y a minute p o r t i o n o f t h e ovary, i t p l a y s
an i m p o r t a n t r o l e i n o v u l a t i o n and gives r i s e t o over
85% o f human o v a r i a n carcinomas.
I n spite o f i t s clini c a l importance, no animal models f o r i n v i v o s t u d i e s o f
t h i s t i s s u e e x i s t because o v a r i a n tumors i n animals
u s u a l l y a r i s e i n f o l l i c u l a r , stromal o r germ c e l l s .
Methods t h a t a r e successful f o r t h e c u l t u r e o f OSE from
o t h e r species have proved inadequate f o r human OSE.
Growth from e x p l a n t s i n medium 199/MCDB202/15%FBS/
h y d r o c o r t i s o n e (HC)/EGF (Siemens & Auersperg, J. C e l l .
P h y s i o l . 134:347, 1988) improved t h e y i e l d o f human OSE,
b u t contamination by o t h e r c e l l types and EGF-induced
changes t o a f i b r o b l a s t i c , k e r a t i n - n e g a t i v e phenotype
w e r e major problems.
I n t h i s study, c e l l s from 55
normal o v a r i a n b i o p s y specimens w e r e used t o improve and
s i m p l i f y t h e methodology f o r OSE c u l t u r e and t o d e f i n e
t h e i n f l u e n c e o f c l i n i c a l parameters on t h e c u l t u r e s .
Advantage was taken o f t h e tenuous attachment o f OSE t o
OSE was scraped o f f t h e o v a r i a n
underlying tissues:
surface, g e n e r a t i n g e p i t h e l i a 1 fragments which produced
monolayers i n c u l t u r e , w i t h l i t t l e contamination by
o t h e r c e l l types. I n a d d i t i o n t o speed, s i m p l i c i t y , and
h i g h e r p u r i t y o f t h e c u l t u r e s , t h e new scrape method
produced more c e l l s compared t o t h e e x p l a n t method. An
improved n u t r i e n t medium w i t h o u t exogenous HC & EGF
(199/MCDB105/15%FBS) r e s u l t e d i n OSE l i n e s t h a t maint a i n e d t h e o r i g i n a l k e r a t i n - p o s i t i v e e p i t h e l i a l phenoOSE remained
type f o r up t o 12 p o p u l a t i o n doublings.
viable i f frozen i n l i q u i d nitrogen p r i o r t o c u l t u r e
p r o v i d i n g OSE independently o f t h e a v a i l a b i l i t y o f
s u r g i c a l specimens. Growth was n o t i n f l u e n c e d by diagn o s i s (nonmalignant gynecological d i s o r d e r s ) , p a t i e n t
age (mean, range: 40.5, 20-62 y r s ) , o r t h e presence o f
c y s t s o r f o l l i c l e s i n t h e biopsy specimen. This c u l t u r e
system p r o v i d e s c o n d i t i o n s f o r i n depth s t u d i e s o f human
OSE p h y s i o l o g y and pathology.
Supported by MRC o f
Canada #MA-10185 & BC Foundation for Non-Animal Research.
S
KURATANI,* Shigeru and Dale E. BOCKMAN,
Department of Anatomy, Medical College of
Georgia, Augusta, Georgia. (Sponsored by G. S .
Sohal) Reauirement and sDecificitv of neural
crest contributions to thvmic develoDment.
Previous studies have shown that neural
crest ablation leads to defective thymic
development. In the present study, e/c8
monoclonal antibody (gift of G. Ciment) was
used to label neural crest-derived mesenchyme.
The quantity of these derivatives is correlated
with the size of the thymic primordium after
neural crest ablation; less elc8 localization
is observed around smaller thymic primordia.
In order to determine if all regions of neural
crest have the capacity to support thymic
development, quail neural crest from different
axial levels was transplanted into chicks,
substituting it for the neural crest that
provides the precursor cells for the thymus.
This heterotopic transplantation utilized
segments approximately the length of two
somites; neural folds were transplanted from
selected levels stretching from the forebrain
cephalically to caudal presomitic regions. The
localization of quail cells around the thymic
primordia was greatest when the donor tissue
came from the levels normally contributing to
pharyngeal arches three through six. The
capacity for neural crest derivatives to
cluster around the thymus tapered off both
cephalically and caudally from this level.
Thus, neural crest displays commitment before
migration, with the capacity to support thymic
development centered in the posterior region of
cranial neural crest. Supported by Grant No.
2332 from The Council for Tobacco Research.
O"H,*
Earbra, lknneth KLo?z,* Jahn HEPR and Charles
FUcXzINQR, Department of Anatmy and Cell Biology,
university of Virginia school of Medicine,
Charlattesville, Virginia. Localization of s ~ e antiaen
~ m
SP-10 d u r h the seminifemus cvcle in man.
SP-10 is a hunnn spenn antigen located within the
acmsae of spermatids as wdl as mature ejaculated sperm
(Herr et. al., Biol Repmd;1990; in press). It has been
classified as a antracqtive vaccine candidate by the
World Health Organization lbskfonze on owtraoeptive
Vaccines (Amkrscm et. al., J Reproa Immol, 10:231-257;
the regulation d
1987). To begin to
function of SP-10, the six stages of the saniniferrxls
cycle were studied to determine the distribution of the
antigen during genesis of the acmsae in hunnn testis.
An irrmunogold technique, usa monoclcanl antjhcdy
(MHS-10) raised against SP-10, was enployed to lccalize
the antiin sections of plastic-e&eMd testes at
both the light and electron microsapic levels. study of
10-30 sectors of seminifenxls tubules at each of the six
d g e s of the spenmtopnic cycle shcw& that s ~ i was
o
first detedable in the acrosQnal region of spermatids at
the earliest step in Spermiogenesis and mtinued to be
present during each successive step. AS the acrosome
umkwent characteristic changes in shape, the staining
ABSTRACTS-AAA 103RD MEETING
pattern represerrtative of the presenrz of SPIO cbanpfi
likewise. It was plnctate a t the earliest steps and
subsequentlyfrom--tov-shaped
as rarnd qxmatids wdement elorgatim. 'Ihe
localization of S P l O to spexmtids w i m each stage of
the seminifemus cycle, its w i n g pattern during
spermiogenesis, and its absenz fmn diploid cells
suggest that S P l O is a testicular differentiatim
antigen. sllpportea by a grant fmn the f&.llm Fanrhtion
and grants ID16767 and HD23789 fmn NM.
S
KUTSCHKE*, William J., Paul M. HEIDGER, Jr. and Ziad
BATAINEH, Department of Anatomy, Univeristy of Iowa, Iowa
City, Iowa and Jordan University of Science and
Technology,
Irbid, Jordan.
Fine structural
and
immunocvtochemical studies of Drostatic tumor metastases
in lung.
A tumor subline of the Dunning rat prostatic
adenocarcinoma has recently been established which
exhibits preferential metastasis to lung tissue (Dr.
David M. Lubaroff, U. IA). Metastatic lesions become
well-established within lung several weeks following tail
vein injection of tumor cells. The present studies were
undertaken to investigate the cellular relationships of
the tumor to lung tissue, and to determine whether sites
of metastasis may be related preferentially to lung
topography, particularly those regions which exhibit high
concentrations of extracellular matrix components. Both
normal and turnor-bearing lung were processed by routine
means for light microscopy, and for transmission and
scanning electron microscopy. Immunocytochemical studies
of fibronectin localization were performed utilizing the
avidin-biotin
technique
and
goat
anti-fibronectin
antibodies. Metastatic foci were frequently encountered
under the pleural surface, in close proximity to the
basal lamina of the visceral pleura. Deeper within lung,
metastases
were
frequently
localized
immediately
surrounding vasculature and the bronchial tree. EM
studies confirmed that the invading metastases develop
beneath the basal laminae of these structures; however,
tumors were not observed to invade the basal lamina or
epithelium. The basal laminae of the pleura, vasculature
and bronchiclar epithelium exhibited intense reactivity
for fibronectin within lung. This association suggests
the desirability of extending the present studies to
include
evaluation
of
other
components
of
the
extracellular matrix in order to probe further the
hypothesis that extracellualr matrix components may
provide a favorable interface for the seeding and growth
of metastatic lesions within host tissues. Supported by a
grant from the Milheim Foundation for Cancer Research.
LAHR,' Stephen P, and Michael E. GOLDBERGER:
Department of Anatomy, Medical Colle e of Pennsylvania,
Philadelphia, Pennsylvania. (Sponsored i y Peter D. Chanter)
Recoverv of hindlimb motor function in sDinallv transected
55A
air stepping and monopedal hopping on both sides, and
bipedal locomotion is seen during the first week p.0. following
the spinal transection, and begins on the chronic side as early
as the second day p.0. This degree of recovery is much earlier
than we (Robinson and Goldberger, 1986) and others reported
reviously for spinal transection alone. Although both
gindlimbs exhibit recocious recovery, there is also a
temporary markecfasymmetry in motor function between the
two hindlimbs The hindlimb ipsilateral (chronic) to the
original hemisection shows less initial impairment and faster
motor recovery during the first two weeks following
transection compared to the contralateral (acute) hindlimb
and the difference persists for up to a month after the
transection. We conclude that spinal shock interferes with
recovery of motor function since reducing spinal shock
facilitates hindlimb motor recovery following spinal
transection.
Supported by the American Paralysis Association and NIH
grant NS24707.
LALA, Peeyush K. and Ranjit S.
PARHAR", Department of
Anatomy, University of Western Ontario, London, Canada.
Eradication of multi-organ human melanoma metastases in
nude mice with chronic indomethacin therapy combined with
IL-2: characteristics of in situ activated killer
____We have shown that tumor-bearing leads to PGE mediated inactivation of host killer lineage cells whi?h
can be abrogated by chronic indomethacin therapy (CIT),
so that CIT combined with multiple rounds of IL-2 can
cure experimental and spontaneous metastases of a variety
of murine tumors.
We tested this therapy in nude mice
bearing multi-organ metastases (simulating human disease)
of a human melanoma line 70-W.
NIH Swiss nude mice were
injected i.v. with 5 x 106 70-W cells.
After 4-5 wks
when metastases were well established in the lungs, skin
and brain, mice were either sham-treated with vehicles
alone O K placed on CIT (14 glml in drinking water)
combined with multiple rounds of IL-2 (25000 Cetus units
8 hrly i.p. on d 5-9, 15-19, 25-29, 35-39, 47-51 and 8 1 ,
following the onset of CIT (d 0 ) .
Mice were left for
survival and autopsied on death, o r killed on d 82 of
therapy to measure the number of metastatic foci in
various organs and killer activity in splenic and
pulmonary lymphocytes and macrophages against K562 (NK
sensitive), Daudi and 70-W (NK resistant) targets. 70-W
killer cells were phenotyped for Thy-1, Lyt-2 and AGM-1
markers on the basis of ab+C' mediated deletion of killer
function.
The therapy caused a reduction in the median
number of lung colonies from 150 to 2, a regression of
skin melanotic foci, and a freedom from brain metastases.
Survival time improved from 39-108 d (81 d median) in
controls to 39-460+ d (150 median) in treated mice (37%
surviving 460+ d).
High killer activity against all
targets was generated in lymphoyctes (of AGM-1+, Thy-l+
and Lyt-2phenotype) and macrophages (of AGM-12
phenotype) in the spleen as well as the lungs. Sera from
treated mice enhanced and sham-treated mice suppresed
70-W target killing ability of these cells. Thus, CIT in
combination with IL-2 can activate LAK cells and
macrophages in situ and cure multi-organ human melanoma
metastases in nude mice.
(Supported by the N C I Canada)
e.
x
.
Previous studies showed that the reflex depression (spinal
shock) which follows spinal cord transection in adult cats can
be reduced by a rior spinal cord injury (Chambers et al., 1966).
The present s t u J e s were designed to determine if recovery
from a previous s inal cord hemisection enhances hindlimb
motor recovery a h e r spinal transection. Locomotion,
monopedal hopping and placing were tested and filmed on
video tape, pre- and postoperatively in three adult cats that
were preoperatively trained to perform bi edal and
quadripedal treadmill locomotion at 0.2-0.C?mls. Following a 12 month recovery after a low thoracic spinal hemisection, the
cord was transected one segment rostra1 to the hemisection
and the hindlimb motor recovery compared with that seen
after transection alone as described previously. Recovery of
56A
ABSTRACTS-AAA 103RD MEETING
distinct surface buains: apical, lateral and basal. We
have attenpted to isolate these membram daaains frao
bwina corneal enpthelim for future duuacbn'zation
using a lactopemoade-bead (LPD-latex) technique to bind
to call surfaces. LPD-latex bends are aispersea (1:400
dilution) and bound to the esposed apical menbraneg of a
cultured bwina e t h e l i d nwnolayer at 4 0 C to prevent
phagocytosis of the beads. Another moaolayer is trypsinized and the -latex
beads added to the cell
suspentfion to bind total cell membaanes. A third mmlayer i s first cultured onto gelatin mtil confluent and
then the gelatin is liquefied. The moaolayer is then
inverted and exposed to -latex
bends to bina basal
nsanbrane demains. Membraae orientatim is identified by
incubating the mollolayers (before inversion w o r bead
-sure)
to lectin-aRp at 370 C for 10 dnutes. This
allows endocytic uptake into the apical cell cytoplasm
before processin3 the sallples described above for lectinHRP labeland m. In the apical sidevp sample,
cells are bormd to the beads on the membrane side where
there is lectin Staining in the apical cytoplarpn. In the
inverted sallple, basal cell membaanes are bound to beads
where there is lectin stainiag in the cytoplasm oppxite
fran the bound beads. The total cell simple m i s t s of
beads bound randemty to cell meahraae surfaces. The
three membrane preparations can then be raaioiodinated
and lysed in p v t i o n for gel elecrqhoretAc and
protein &macten zatirm. This metbDd allows for islation of specific menbrane daaains for discrete
biochemical c h a n c h n'zation.
suppoaea by Eaptist Memorial Hospital, Meuphis, Tenn.
S
LAPASHA,* D. I . , D. V. PROVENZA, L. A. BENEVENTO, R. M.
MESZLER, Department o f Anatomy, D e n t a l School, U n i v e r s i t y
o f Maryland a t B a l t i m o r e , B a l t i m o r e , M a r y l a n d . (Sponsor:
L o u i s A. Benevento) Eye development i n t h e Ts 16 mouse.
Developmental d e f e c t s o f t h e v i s u a l a p p a r a t u s i n Ts 16
f e t a l mice have been d e s c r i b e d as g e n e r a i i y a f f e c t i n g t h e
r e t i n a , c h o r o i d a l l a y e r , pigment e p i t h e l i u m and l e n s .
S p e c i f i c a l l y h y p o c e l l u l a r i t y and developmental d e l a y were
observed ( O s t e r - G r a n i t e , e t al., 1983, 1986). I n t h e p r e s e n t s t u d y , male mice d o u b l y heterozygous f o r R o b e r t s o n i a n
t r a n s l o c a t i o n m e t a c e n t r i c chromosomes 16 and 17 and c h r o mosomes 6 and 16 Rb (16.17) 7 B n r / Rb (6.16) 24 Lub were
bred w i t h female mice o f a normal a c r o c e n t r i c genome t o
y i e l d o f f s p r i n g t r i s o m i c f o r chromosome 16. M u r i n e f e t a l
t r i s o m y 16 (Ts 16) and t h e i r e u p l o i d c o n t r o l l i t t e r m a t e s
g e s t a t i o n a l ages day 17-19 were e x c i s e d by m i d l i n e l a p a rotomy, k a r y o t y p e d f o r g e n e t i c a n a l y s i s , and whole heads
r o u t i n e l y processed. T i s s u e s were s e c t i o n e d f r o n t a l l y ,
h o r i z o n t a l l y and p a r a s a g i t a l l y , and s t a i n e d w i t h H a r r i s '
h e m a t o x y l i n and e o s i n , p e r i o d i c a c i d - S c h i f f r e a c t i o n and
trichrome f o r l i g h t microscopic h i s t o l o g i c a l study o f the
o c u l a r r e g i o n s . P r e v i o u s l y r e p o r t e d d e f e c t s were observed
o n l y i n t h e m i l d e s t o f cases. L e n t i c u l a r anomalies i n clude a reduction i n the s i z e o f t h e lens.
I n more s e v e r l y a f f e c t e d Ts 16 f e t u s e s , a p h a k i a (absence o f c r y s t a l l i n e
l e n s ) i s accompanied by an i n v a g i n a t i o n o f p e r i p h e r a l r e g i o n s o f n e u r a l r e t i n a and pigment e p i t h e l i u m , which t o g e t h e r f o r m a v e s i c l e - l i k e pseudolens. M a l a c c u m u l a t i o n o f
ectomesenchyme f o r m i n g t h e v i t r e o u s i s n o t uncommon. The
n e u r a l r e t i n a i s c h a r a c t e r i z e d by t h e absence o f a t r a n s i e n t n e r v e f i b e r l a y e r , i n c r e a s e d i n t e r c e l l u l a r spaces
and p y k n o t i c n u c l e i among c e l l s o f t h e i n n e r n e u r o b l a s t i c
l a y e r . The c e n t r a l r e g i o n o f t h e cornea i s comprised o f
dense a g g r e g a t i o n s o f i r r e g u l a r l y a r r a n g e d mesenchymal
c e l l s w h i l e peripheral regions e x h i b i t a p a u c i t y o f c e l l s
and i n c r e a s e d i n t e r c e l l u l a r spaces. The r e s u l t s o f t h e
p r e s e n t s t u d y demonstrate t h a t o c u l a r t i s s u e s d e r i v e d f r o m
s u r f a c e ectoderm, neuroectoderm, and t h e n e u r a l c r e s t a r e
a l l a f f e c t e d ddVerSely by t h e presence o f an e x t r a c h r o mosome 16 i n t h e m u r i n e genome b u t even more s i g n i f i c a n t l y
t h a t these defects represent d e f i n i t e malformations.
LARRIVA-SAHD, Jorge., Hector OROZCO* Alejandro
TREJO* and Roger A. GORSKI, Laboratory of
Experimental Pathology, Dept. of Pathology,
Instituto Nacional de la Nutricion S Z , Mexico,
DF, MEXICO and Laboratory of Neuroendocrinology
and Dept. of Anatomy, UCIA School of Medicine.
Morphometric studv of the vomeronasal orsan of
the rat.
Several structures of the brain implicated in
reproductive behavior have been shown to be sexually dimorphic. In a previous report (Segovia,
el al. Dev Brain Res 5:209,('82)) sex differences
in the vomeronasal organ (VO) were reported. In
the present study no sex differences were found
in normal adult rats and in rats treated with
gonadal sex steroids perinatally. Wistar albino
rats were divided into: 1 Males, 2 Females, 3
Males castrated as newborns, 4 Males castrated
as newborns and treated for ten days with 150 Wg
testosterone proprionate (TP), and 5 Females
treated for ten days after delivery with TP. Ten
rats per group were sacrificed at ten weeks of
age by perfusion with Karnovsky's fixative. Since
the VO did not differ in length, it was blocked
into anterior, middle, and posterior thirds and
embedded in epon. One pm thin sections were used.
With a camera lucida the neuroepithelium and
nuclei of sensory and supporting cells were outlined with a cursor on a bitpad. Area and perimeter of each structure were submitted to a one
way ANOVA. A difference of p< 0.05 or less was
considered as significant. No significant differences were found between sexes in any parameter. Castration of the male1 treatment of the
newborn female with TP did not modify the size of
the VO or nuclei of the sensory or supporting
cells. The data presented herein coupled with the
fact the VO neuroepithelium is renewed every 12
weeks cast doubt upon the conclusion that there
is a sex difference in the rat's VO and that this
difference is determined by organizatonal effects
of sex steroids.
Supported by CONACyT.
IARSEN,* S.A.(l) and B. BIZOT,*(Z) (1) Department of
Anatomical Sciences and Neurobiology, University of Louisville
School of Medicine, Louisville, Kentucky, (2) Department of
Internal Medicine, Yale University, New Haven, Connecticut
(Sponsored by Richard Rink) Correlationof the cytoarchitecture
of the inferior colliculus with the fifth wave of the auditory
brainstem resDonse in kittens.
The auditory brainstemresponse (ABR) is an objective method
of assessing deafness in neonates. Although it is assumed that
each successive wave is generated from a higher nucleus or
fiber bundle in the central auditory pathway, there is little direct
evidence that specific structures of the central auditory pathway
generate the individual peaks of the ABR. METHODS: We have
studied the maturation of latency and amplitude of the fifth wave
in the ABR in kittens from birth to sixty days of age in correlation
with the development of neuronal cytoarchitecturein the inferior
colliculus. RESULTS: During the first week of life, the ABR is
flat, has no repeating features and does not lend itself to latency
or amplitude measurements. At this time, neurons in the inferior
colliculus are small, densely packed and have had a rapid
increase in cytoplasmic and nuclear size. Following the rapid
neuronal growth, the ABR waveform begins to emerge. The fifth
wave of the ABR reaches adult-likelatencies and amplitudes near
the end of the first month of life when neurons in the inferior
colliculus are nearing the size found in the adult cat.
CONCLUSIONS: We interpret the results of this study as a
positive correlation of the emergence and maturation of the fifth
wave of the ABR with the growth curve of neurons in the inferior
ABSTRACTS-AAA 103RD MEETING
colliculus. We also contend that our results supply additional
information suggesting that neurons in the inferior colliculus
represent the generation site of the fifth wave of the ABR.
S
LEEPARD,* J e n n i f e r , Frank E . BARONE* and Dennis E. MORSE,
Department o f Anatomy, Medical College of O h i o , Toledo, Ohio.
Microscooic a n a l y s i s of b r e a s t imDlant/stromal c a ps ule
interface in rabbit s.
Two common problems a r e associ at ed with s u r g i c a l
1) a t h i c k f i b r o u s
implantation of b r e a s t prost heses:
capsule t e n d s t o form which e v e n t u a l l y becomes c o n t r a c t e d and
d i s f i g u r i n g ; and 2 ) a chroni c low-grade inflammation.
Manufacturers have experimented w i t h imp1 ants const ruc te d of
a v a r i e t y of m a t e r i a l s and t e x t u r e s . S i g n i f i c a n t reduc tion
in c o n t r a c t u r e s and inflammation has been report ed w i t h the
use of s y n t h e t i c implants which have a s u r f a c e texture. The
purpose of t h i s st udy was t o examine the implant/capsule
interface
in
smoothand t ext ured-surface-prosthe s e s
harvested a t i n t e r v a l s through 16 weeks of i mpl ant a tion i n
female r a b b i t b r e a s t s . The e n t i r e implant and surrounding
t i s s u e s were e x c i s ed and f i x e d f o r l i g h t and e l e c t r o n
microscopy. Wedges of t i s s u e were c u t from t h e s e specimens
with c a r e taken t o preserve t h e implant/capsule i n t e r f a c e .
Samples f o r scanning e l e c t r o n microscopic a n a l y s i s were
frozen in l i q u i d n it rogen and f r a c t u r e d perpendi cul ar t o t h e
i n t e r f a c e . Other p r eparat ory procedures were r o u t i n e . Major
d i f f e r e n c e s between t h e two t ypes of implants a r e obvious
The smooth-surface-implant has a t h i c k
a f t e r 8 weeks.
f i b r o u s c a p su l e w i t h l a m e l l a r col l agen a s the primary
component.
The f i b r o u s capsul e does n o t adhere t o the
implant. The t e x tu r e d-surface-i mpl ant develops a caps ule of
non-lamellar c o l la gen which i s much t h i n n e r t h a n t h a t
a s s o c i a t e d with t h e smooth-surface-implant. The caps ule i s
t i g h t l y adherent t o t h e implant. Other s t u d i e s have shown
t h a t t h e c r i t i c a l period f o r successful b r e a s t p r o s t h e s i s
implantation i s the f i r s t 8 weeks. This st udy sugges ts t h a t
t h e g r e a t e r su c c e ss r a t e of t h e t ext ured-surface-i mpla nt i s
due in p a r t t o i t s a b i l i t y t o r e s i s t e a r l y formation of a
t h i c k , l a m e l l a r c a psul e.
Research su p p o r t was provided f o r JL from the Dean's O f f i c e ,
Medical College of Ohio.
LEVINE, Rhea J. C. and John L. WOODHEAD', Department of
Anatomy,
Medical
College of
Pennsylvania,
Philadelphia,
Pennsylvania.
Exolorine thick filament anatomv bv electron
microscooic techniques.
Paramyosin (PM) forms the cores of invertebrate, but not
vcrtebrate, thick filaments, which have backbones formed from
myosin (M) rods. Helically arranged M heads project from the
surfaces of both filament types and, under activating conditions,
interact with actin on thin filaments. Detailed knowledge of the
organization of thick filament proteins is essential to understand
molecular behavior during contraction. All relaxed thick filaments
studied have axial repeats of 14.3 - 14.5 nm for successive crowns of M
heads, but differ in their screw and rotational symmetries. Although
two M headslsurface subunit are either apparent or suggested, the
oricntation of individual heads is not resolved, in 3D reconstructions
from images of negatively-stained filaments. To determine whether
the two heads in each subunit belong to the same, or to axially
sequential Ms, we crosslinked the active sites of nearest M heads with
bisZ2ATP (a short [48 nm] bifunctional agent) on isolated Limulus,
Aeauioecten, goldfish and frog thick filaments and stabilized the
linkage with vanadate (Vi). Controls were untreated or incubated in
DTT after crosslinking, to sever the SS bond in bis 2ATP. All
filaments were incubated for 15 min on 0 . 6 ~KCI. Incu%ations were
done on EM grids and samples taken at each step were negativelystained and examined in the TEM at 80kV. Myosin disassociated
from control filaments; those from invertebrate muscles left intact PM
57A
cores. All filaments with stabilized, crosslinked heads remained intact,
indicating that bis22ATP.Vi formed intermolecular bonds, and
supporting the suggestion that the two M headslsubunit belong to
different, axially sequential, molecules. To determine the sites of
interaction between PM and M in the cores of invertebrate filaments
in the relaxed and activated states, we are using monoclonal IgGs vs.
Limulus PM in combination with avidin-labeling of biotinylated
cysteines to map epitopes on rotary-shadowed PM molecules, and on
both negatively-stained PM paracrystals and thick filament cores in
the EM. These studies will be extended to negatively-stained, relaxed
and activated intact filaments. Together, both types of analysis should
provide necessary insights into thick filament anatomy.
Supported by HHS grants AR33302 and HL15835 to the Pennsylvania
Muscle Institute.
W.B. QUAY and P.S. TIMIRAS*, Department of
Anatomy, Michigan S t a t e U nive rs ity, E a s t Lansing, Michigan;
and Department of Molecular and C e l l Biology, U nive rsity of
C a l i f o r n i a , Berkeley, C a l i f o r n i a . Molecular a c t i o n of
5-methoxytryptamine on microtubules: changes i n t a u
p r o t e i n i n human neuroblastoma c e l l s .
LEW, G l o r i a M . ,
I n r e c e n t y e a r s 5-methoxytryptamine (5-MT) has a t t r a c t e d
a g r e a t d e a l of i n t e r e s t as it i s a p o t e n t i a l p i n e a l
hormone and i s a p o t e n t i a l pre c urs or i n t h e b i o s y n t h e s i s of
t h e psychomimetic a ge nt - 5-methoxy-N,N-dimethyltryptamine.
5-MT has a profound i n f l u e n c e on t h e s e xua l a c t i v i t y of
some mammals. I t s phys iologic a l a c t i v i t y i s independent of
b u t similar i n many ways t o t h a t of melatonin. Recent d a t a
i n d i c a t e t h a t melatonin and s e r o t o n i n a f f e c t cytochemical
and biochemical parameters i n c u l t u r e d human neuroblastoma.
I n t h i s i n v e s t i g a t i o n w e used a human neuroblastoma c e l l
l i n e , LAN-5, as a t i s s u e c u l t u r e model, t o s tudy t h e d i r e c t
e f f e c t s of 5-MT on mic rotubule s , p a r t i c u l a r l y t a u p r o t e i n ,
w h i c h a c t s i n vivo c h i e f l y t o induce t h e assembly of
t u b u l i n and i n v i t r o t o promote mic rotubule polymerization.
U ndiffe re ntia te d LAN-5 c e l l s w e r e t r e a t e d f o r 48 h r w i t h
1 x 1 0 - 5 M and 1 x 1 0 - 7 M 5-MT.
LAN-5 were d i f f e r e n t i a t e d by
c u l t u r i n g t h e c e l l s i n medium supplemented w i t h r e t i n o i c
a c i d (0.01 mM) f o r seven days p r i o r t o 48 h r tre a tme nt w i t h
5-MT.
C e l l s w e r e ha rve s te d, p r o t e i n s were separated using
SDS-PAGE, and Western b l o t s w e r e performed using a mouse
monoclonal antibody f o r t a u . 5-MT (1 x 1 0 - 5 M and ~ x ~ O - ~ M )
i n u n d i f f e r e n t i a t e d c e l l s inc re a s e d t a u p r o t e i n (50 k D ) i n
t h e c y t o lasmic f r a c t i o n : i n t h e membrane f r a c t i o n 5-MT
( 1 x 1 0 - g M ) decreased t h i s 50 kD band.
However i n
d i f f e r e n t i a t e d c e l l s 5-MT i n b o t h c onc e ntra tions decreased
t a u p r o t e i n (50 kD) i n t h e cytoplasmic f r a c t i o n . T h e s e t a u
changes w e r e accompanied by changes i n t o t a l p r o t e i n and
c e l l number i n d i f f e r e n t i a t e d and u n d i f f e r e n t i a t e d cells.
Our d a t a show t h a t 5-MT has d i r e c t e f f e c t s on t h e microtubule s of u n d i f f e r e n t i a t e d and d i f f e r e n t i a t e d c u l t u r e d
human neuroblastoma. Furthe r experimentation t o s tudy t h e
mechanism of a c t i o n i s warranted. Supported by t h e S t a t e
of C a l i f o r n i a , Department of Health Se rvic e s .
S
L I , * Kang and Marcia G . Welsh, Department of Anatomy, C e l l
Biology and Neurosciences, U n i v e r s i t y o f South Ca rolina,
School
of
Medicine,
Columbia,
South
Ca rolina.
Immunohistochemical demonstration o f t v r o s i n e hvdroxvlase
and neuropeptide-Y nerve f i b e r s i n p i n e a l e la nds i n s i t u
and i n p i n e a l p r a f t s .
The i n t a c t p i n e a l complex as w e l l as p i n e a l g r a f t s were
examined for t h e pre s e nc e of t y r o s i n e hydroxylase (TH) and
neuropeptide-Y (NPY) immunoreactivity u s i n g t h e a v i d i n b i o t i n - p e r o x i d a s e immunohistochemical technique. For t h e
p i n e a l g r a f t s , s u p e r f i c i a l p i n e a l s from 3-4-week-old
Mongolian g e r b i l s and ne ona ta l Syria n hamsters were
t r a n s p l a n t e d i n t o t h e p i n e a l r e c e s s of 8-10-week-old
g e r b i l s , and i n t o t h e i n f u n d i b u l a r recess ( I R ) and under
58A
ABSTRACTS-AAA 103RD MEETING
University, Philadelphia, Pennsylvania. (Sponsored by Mary-Clare
. .R . A
Holst).
G
A
e
r
p
ex in r&
The existence of non-thalamic neocortical afferent systems arising from monoaminergic and cholinergic nuclei in the brain stem and
basal forebrain respectively has been extensively documented. In
this report, we describe a new direct GABAergic projection from the
zona incertato the neocortex. This projectionwas demonstratedwith
fluorescent retrograde tracers, including rhodamine or fluorescein
coated microspheresand Fluoro-gold. Small tracer injections were
made in a variety of neocortical areas in Long-Evans (Hooded) rats.
In addition to adult rats (n =40),2-3 week developing rats were also
used (n = 20). Retrograde labeling was found bilaterally in the zona
incerta after injections in most neocortical areas, including
somatosensory, visual, auditory, temporal, and frontal cortices.
Though a rough topography was visible, the degree of overlap indicated a somewhat diffuse projection system. Also, the overall
strength of this connection was found to be much greater in the 2-3
week old animals, even through it was substantial in adults. Combined retrograde tracer and immunohistochemical procedures
demonstratedthat most identifiedincerto-corticalneurons react positively to antisera for glutamic acid decarboxylase (GAD), which is the
synthetic enzyme and marker for y-aminobutyric acid (GABA). The
function of this system is presently unknown. The fact that it is fairly
diffuse, however, and is greatly amplified during early development
suggests similarities with the modulatory systems arising from the
monoamine and cholinergic nuclei. Supported by FAPESP 88140449 to M.A.L.N., PHS grants NS26722, AA06965, KO2-AAOOO89,and an
award from the AFOSR.
the renal capsule of 8-10-week old hamsters, respectively.
Light microscopically, abundant TH fibers formed a dense
meshwork in the parenchyma of the superficial and deep
pineals. Ultrastructurally, TH imunoreactive fibers were
present mainly in perivascular spaces, but a number of TH
fibers were located in the parenchyma between the
pinealocytes. TH-positive cells were demonstrated in
superficial and deep pineal glands of hamsters. NPY fibers
were present in both superficial and deep pineal glands of
the gerbil and hamster. NPY fibers were distributed evenly
throughout the pineal complex of the gerbil, but they were
more often located in the central part of the superficial
pineal of the hamster. Eight weeks following transplatation
in the gerbil, pineal grafts had numerous, evenly
distributed TH imunoreactive fibers although the number of
TH fibers was less than those in the pineals in situ. Some
NPY fibers were also found in the gerbil grafts. In the
hamster, a few TH fibers were demonstrated in the renal
grafts 4 weeks after transplantation. Many TH and NPY
fibers were found surrounding the IR grafts, but were only
rarely seen entering the graft. These studies indicate that
TH and NYP immunoreactive fibers are present in the in situ
pineal complex of both the hamster and the gerbil.
Reinnervation of pineal grafts by either NPY or TH
immunopositive fibers appears to be dependent upon the
location of the grafted tissue and on the reintegration of
the graft in its new site. Supported by NIH Grant #HD24717.
LIBBIN, R.M., S.R. FRENKEL and O.G. MITCHELL, Veterans
A d m i n i s t r a t i o n M e d i c a l Center, B r o o k l y n , New York (RML)
and Department o f Anatomy, New York U n i v e r s i t y C o l l e g e
o f D e n t i s t r y , New York, New York,(RML, SRF, OGM).
O b s e r v a t i o n s on t h e r e g e n e r a t i o n o f t h e c a r p a l s k e l e t o n
o f N. v i r i d e s c e n s a d u l t s .
E x t a n t d e s c r i p t i o n s o f f o r e l i m b r e g e n e r a t i o n i n t h e newt
p r o v i d e an i n c o m p l e t e a c c o u n t o f t h e r e s t o r a t i o n o f t h e
w r i s t s k e l e t o n . S e v e r a l r e m a i n i n g gaps impede assessment
o f t h e f r e q u e n c y and s i g n i f i c a n c e o f v a r i a n t morphology
n o t e d i n t h e c a r p a l group, p a r t i c u l a r l y t h e i n t e r e l e m e n t
f u s i o n s e n c o u n t e r e d i n l a t e - t e r m r e g e n e r a t e s ( L i b b i n e t a1
Anat. Rec., 223: 68A, 1989). F i f t y a d u l t newts s u s t a i n e d
u n i l a t e r a l f o r e l i m b a m p u t a t i o n s 2.5 mm above t h e elbow
j o i n t and were k i l l e d a t 3-day i n t e r v a l s e x t e n d i n g f r o m day
2 1 t h r o u g h day 48 ( 5 newts p e r i n t e r v a l ) . R e g e n e r a t i n g
l i m b s k e l e t o n s were s i m u l t a n e o u s l y s t a i n e d en b l o c w i t h
m e t h y l green. B a s a l e 1,2 o f t h e d i s t a l row was t h e f i r s t
c a r p a l t o r e a p p e a r ( d a y 21-24), d e m o n s t r a t i n g t h a t t h e
w r i s t s k e l e t o n does n o t r e g r o w i n a p r o x i m a l - t o - d i s t a l
d i r e c t i o n as had been c l a i m e d . B e g i n n i n g on day 24
t r a n s v e r s e and l o n g i t u d i n a l c l e f t s began t o s e g r e g a t e
t h e c a r p a l p r i m o r d i a i n t o t h r e e columns and t h r e e rows.
T h i s i n v e s t i g a t i o n i n d i c a t e s t h a t t h e f r e q u e n t l y observed
variant fusions, radiale-prepollicis, centrale-intermedium and b a s a l e 3-4, r e s u l t f r o m i n c o m p l e t e , and n o t
m i s d i r e c t e d , s e p a r a t i o n b y t r a n s v e r s e c l e f t s , and t h a t
the p a r t i a l f u s i o n o f intermedium w i t h ulnare, representing
the n a t i v e pattern, a r i s e s v i a incomplete d i v i s i o n o f a
s i n g l e c a r t i l a g e by t h e p o s t a x i a l l o n g i t u d i n a l c l e f t .
The o c c a s i o n a l l y observed s e p a r a t i o n o f t h e l a t t e r elements
i s i n t e r p r e t e d t o i n d i c a t e i n a p p r o p r i a t e extension o f t h a t
c l e f t p r o x i m a l l y . T h i s s t u d y a l s o demonstrates t h a t
b a s a l e 1,2 does n o t m i g r a t e f r o m t h e p o s t a x i a l edge o f
t h e w r i s t t o t h e o p p o s i t e s i d e as e n v i s i o n e d b y Benzo
e t a l . (Anat. Rec., 183: 421, 1975). S u p p o r t e d by t h e
Veterans A d m i n i s t r a t i o n and t h e Anatomy Research Fund
o f New York U n i v e r s i t y C o l l e g e o f D e n t i s t r y .
.,
LIN*, Chia-Sheng, Miguel Angelo L. NICOLELIS*, and John King
CHAPIN*, Department of Physiology and Biophysics, Hahnemann
W , E.A., S.H. TAW and W.C. KX, n2partm3lt of Anatany, k t i d
University of Singapre, Singapre. Spptic jmctions on p w y t e s
in the msnkey, k a c a f&cMs.
It is generally accepted that the p i d gland of nrnmals is
pxtganglionic nerve fibers origimting
fran the superior cervical w o n as h e l l as by fibers fran the cmtral
nervow system. Ihe present study v a s carried out to detemiw if any
of these fibers in the gland d d fonn synaptic contacts with the
prepmdmmt pinealmyte. For t h i s purpose, a total of four mnml
& mukeys, wighing 2.54.2 kg here used. In addition, four other
nonkeys were subjected to bilateral superior cervical ganglicawtmy
and Ere sxrificed 3,5,7 and 30 days after the operation. All the
anirrals here prfused with a mixed aldehyde solution d e r segatal
anagthesia.
Following perfusion the pineal gland wds remved,
p3stoslricated and bla%d in the routine olanner for electron micrcsmpy.
Ultrathin sxtions of the entire gland cut sagitally here prepared.
SyMptic contacts u s e observel b3xzn a variety of a x ~ ntem6m.l~
and the cell bodies of pinmlmytes in the mnml mukeys. Sum? of
the tarrrinals Contairpd pleclrorphic agranular vesicles with a few large
dense cored vesicles, others m e exrlusively agranular. S t i l l other
tmndrds foming synaptic junctions &zed smll granular vesicles.
?he frequency of axo-pinealoqtic mtzcts was estimated to be 2.4
per 103 pineelocytes. Folkwing gpnslionecany m y axon terminals
mdenent degewative changes : accmdation of glycqm wsses and
dense irregular bodies. fist of the degenerating terrrdnals shmed
p1-Y
agranular vesicles with soll~ smll and a few large dense
mred vmcles. Synaptic mtacts were observed benem the d-ating
axon tenrdrals and p i n d c c y t e s in 5 and 7 days pxtoperative animls.
A few c c c a s i d axordl profiles tearing s m l l h cored vesicles
persisted in the M y surviving d y . Ihe wjority of the axon
tenrdnals observed at this stage, k v e r , &wzd round agranular
vesicles and were presymptic to the intrapheal neurons.
It is
cod&
fran this study that at least sarre of the pinealocytes are
under the direct i n f l w of cells in the superior cervical ganglion
imemaid by c
i
t
0
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
through direct SyMPtic
contacts, W tk intrapineal neurons are
~ p t i c p r i w r i l y t o I ? m m n . s' w i n tk central nervous
whether l a m i n i n is n o r m a l l y a s s o c i a t e d w i t h
t h o s e g l i a l s t r u c t u r e s or is e x p r e s s e d by
SY-.
a s t r o c y t e s only after dorsal r o o t injury.
T r a n s v e r s e s e c t i o n s of normal s p i n a l c o r d s
and s p i n a l c o r d s of a n i m a l s w i t h u n i l a t e r a l
9th dorsal root crushes w e r e stained, using
t h e PAP t e c h n i q u e , w i t h a n a n t i s e r u m t o
l a m i n i n (E-Y L a b o r a t o r i e s , San Mateo, C A ) .
Laminin-positive
staining
was
widely
d i s t r i b u t e d t h r o u g h o u t t h e s p i n a l w h i t e and
g r a y matters i n both normal s p i n a l c o r d s and
i n c o r d s f o l l o w i n g d o r s a l root c r u s h . I n t h e
w h i t e matter,
l a m i n i n is a s s o c i a t e d w i t h
a s t r o c y t i c radial p r o c e s s e s while i n t h e gray
m a t t e r it s u r r o u n d s n e u r o n a l somata.
"Supported by G r a n t No. NS24309 f r o m t h e
LIU, Keh-Min, Department of Anatomy, Kaohsiung
Medical C o l l e g e , Kaohsiung, Taiwan, Republic of
China.
U l t r a s t r u c t u r a l s t u d i e s on d i f f e r e n t i a t i o n
of nerve c e l l s i n t h e cardiac g a n g l i o n of t h e p r e natal rat.
I n t h e d e v e l o p i n g c a r d i a c g a n g l i a , t h e morphol o g i c a l s t r u c t u r e of t h e g a n g i i o n i c c e l l of the
c h i c k embryo, p r e n a t a l r a t , and human f e t u s w e r e
r e p o r t e d . However, none of these s t u d i e s r e v e a l ed t h e l i n e a g e of t h e d i f f e r e n t c e l l s found i n
t h e g a n g l i a nor w e r e t h e y c o n c e r n e d w i t h morphol o g i c a l changes t h a t o c c u r r e d d u r i n g c y t o g e n e s i s
i n t h e g a n g l i a . The a i m of t h e p r e s e n t s t u d y i s
t o s t u d y t h e d i f f e r e n t i a t i o n of d e v e l o p i n g c a r d i a c
g a n g l i o n i c c e l l s and i t s r e l a t i o n t o t h e o r g a n e l l e s . T h e f e t a l rets(Sprague-Dawley s t r a i n ) f r o m
1 4 - 1 6 , 1 8 , 2 0 - 2 1 days of g e s t a t i o n w e r e p e r f u s e d
w i t h 2 % paraformaldehyde, 2 % g l u t a r a l d e h y d e and 2 %
PVP i n 0 . 1 M
cacodylate b u f f e r s o l u t i o n ( p H 7 . 4 . 4 "
C ) . A f t e r t h e p e r f u s i o n , t h e h e a r t w a s dissected
a n d processed w i t h r o u t i n e e l e c t r o n microscopy.
T h e u l t r a t h i n s e c t i o n s w e r e viewed w i t h a H i t a c h i
H-500 e l e c t r o n microscope. I n t h e d e v e l o p i n g c a r d i a c g a n g l i o n , t h e u n d i f f e r e n t i a t e d c e l l , t h e neur o b l a s t , t h e immature neuron, and s m a l l g r a n u l a r
c e l l s w e r e observed. T h e u n d i f f e r e n t i a t e d c e l l w a s
i r r e g u l a r l y shaped, c o n s i s t e d of a l a r g e , d a r k
s t a i n e d round n u c l e u s and had v e r y f e w o r g a n e l l e s .
T h e n e u r o b l a s t can be s u b d i v i d e d i n t o a p o l a r , earl y b i p o l a r , i n t e r m e d i a t e b i p o l a r , and l a t e b i p o l a r
n e u r o b l a s t s . A s t h e development of t h e n e u r o b l a s t
proceeded, t h e number of m i t o c h o n d r i a , polysomes
and rough endoplasmic r e t i c u l u m , and t h e g r o u p of
Golgi complexes i n c r e a s e d p r e d o m i n a t e l y . However,
o n l y v e r y f e w dense-core v e s i c l e s c a n be seen. T h e
l o n g process extended f r o m t h e l a t e b i p o l a r neurob l a s t . T h e immature neuron c o n s i s t e d of a c e n t r a l l y l o c a t e d n u c l e u s , numerous m i t o c h o n d r i a , w e l l
developed G o l g i complexes and v e s i c l e s , and r a n domly d i s t r i b u t e d r o u g h endoplasmic r e t i c u l u m , and
w a s surrounded by s a t e l l i t e c e l l s and nerve fibers.
F r a n c i s J o s e p h and Bruce TEDESCHI*,
Department o f Anatomy and Neurobiology,
E a s t e r n V i r g i n i a Medical School, Norfolk,
V i r g i n i a . Laminin d i s t r i b u t i o n i n t h e a d u l t
f r o a (Rana c a t e s b e i a n a ) s p i n a l c o r d .
LIUZZI,
T h e non-collagenous
glycoprotein laminin
is a n e x t r a c e l l u l a r m a t r i x molecule t h a t has
been shown t o promote n e u r i t i c e l o n g a t i o n
from both P N S and CNS neurons & v u .
a d d i t i o n , by v i r t u e of i t s p r e s e n c e
d e v e l o p i n g mammalian CNS and i n t h e a d u l t CNS
of some l o w e r v e r t e b r a t e s t h a t d i s p l a y
a
c a p a c i t y f o r CNS r e g e n e r a t i o n , l a m i n i n has
been proposed a s a n e s s e n t i a l s u b s t r a t e for
CNS a x o n a l growth and r e g e n e r a t i o n . A number
of y e a r s ago L i u z z i and Lasek (1985) showed
t h a t a f t e r c r u s h , a d u l t frog d o r s a l r o o t
axons r e g e n e r a t e i n t o t h e s p i n a l c o r d where
t h e y grow, a r b o r i z e and synapse.
Electron
microscopy of h o r s e r a d i s h p e r o x i d a s e - l a b e l l e d
r e g e n e r a t i n g axons showed t h a t t h e y g r e w
a l o n g t h e e n d f e e t and p r o c e s s e s of r a d i a l
a s t r o c y t e s . T h i s s t u d y w a s done t o d e t e r m i n e
i:
NIH.
59A
'I
LI VOLSI, Guido,* Richard J WEINBERG,* and Aldo
RUSTIONI, Dept. of Cell Biology and Anatomy, University of
North Carolina, Chapel Hill, North Carolina. Perioheral nerve
stimulation and neurotransmitters in sainal dorsal horn.
As part of a n ongoing study of putative neurotransmitters
in dorsal root ganglion neurons, we have investigated the
effects of peripheral nerve stimulation on immunohistochemical staining for substance P (SP)and glutamate (Glu)
in the dorsal horn of the r a t spinal cord. Stimulating cuff
electrodes were implanted unilaterally on the sciatic nerve of
pentobarbital-anesthetizedSprague-Dawley rats.
Immediately after 5'-30' of repetitive electrical stimulation,
they were perfused with 4% Cyanamide and 4% paraformaldehyde. Cord segments L4 and L5 were removed,
postfiied overnight, sectioned, and processed for immunocytochemistry. Staining was analyzed densitometrically with
a video microscopic system. In non-stimulated controls, antiSP stained fibers, almost exclusively in superficial laminae of
the dorsal horn. Anti-Glu stained both fibers and cells, most
prominently in superficial laminae. Following stimulation,
substance P staining in these laminae at L4-L5 was reduced
ipsilateral to the stimulated side. This effect was strongest
after stimulation at C fiber strength, presumably reflecting
depletion of neurotransmitter pool, as has been previously
suggested. Glutamate, which colocalizes with SP, is also
thought to be released by C fibers. Glu staining was more
variable, but generally, staining in superficial laminae was
increased ipsilateral to the stimulated side with C fiber
stimulation. This increased staining was observed in both
neuropil and cells, although density of cell staining was more
variable. Further experiments a r e underway to establish
whether this effect is metabolic, or perhaps related to
increases in glial content of glutamate following its release
from nerve terminals. Supported by NM award # NS-12440.
S
LU,* Chengliang, G. YORKE* and F.J. ROISEN, Department of
Anatomical Sciences and Neurobiology, School of Medicine,
University of Louisville, Louisville, Kentucky. (Sponsored by G.
Stephen Nettleton)The effect of taurine on neuronal development
invitro.
Substantial evidence implicates taurine in the development of
the nervous system although the exact nature of its action and
extent of participation remain unknown. In this study the action
of taurine on a Nerve Growth Factor (NGF)-independent murine
neuroblastoma cell line (Neuro-2a) and an NGF-responsive rat
pheochromocytoma cell line (PC12) has been evaluated
60A
ABSTRACTS-AAA 103RD MEETING
morphologically and biochemically. The neuritogeniccapacity of
taurine on these two cell lines was probed further by comparing
its action in the presence and absence of the exogenous
ganglioside GM1. Exposure of Neuro-2a cells to media
containing 1 mM taurine increased neuritogenesis and elevated
the activity of ornithine decarboxylase (ODC), the rate limiting
enzyme in polyamine biosynthesis, 3 fold. In contrast, PC12
cells exposed to media supplemented solely with taurine
remained mitotically active and did not exhibit neurite formation.
However, ODC activity was enhanced 35% after 5 h exposure.
Taurine potentiatedthe action of NGF on PC12 cells, producing
many long neurites which had greater diameters than those
formed in response to NGF alone. The simultaneous application
of NGF and taurine to PC12 cells for 5 h did not increase ODC
activity over the NGF controls. Previously, we have shown that
the ganglioside GM1 stimulates Neuro-2a neuritogenesis and
potentiates NGF's action on PC12 cells. Taurine and GM1 (100
pg/ml) were administered simultaneously to both cell lines. GMl
potentiated taurine's neuritogenic action on Neuro-2a cells but
had no effect on PC12 cells in the absence of NGF. In the
presence of NGF, taurine, and GM1, PC12 cells underwent
substantial neuritic development. These studies demonstrate
that taurine has a neurotrophic potential which produces neuron
specific responses. Supported by NIH Grant NS24524.
LUCKETT, W . P., Department of Anatomy, University of Puerto
Rico, San Juan, Puerto Rico. Developmental evidence for
dental homologies in the mammalian order Hyracoidea.
Although most fossil hyracoids possess the complete eutherian dentition, Recent species are characterized by loss
of posterior incisor loci in both jaws, and by loss of one
post-incisor tooth in each jaw quadrant, generally believed
to be the canine. The first premolar (dP1) is thought to
be replaced in hyraxes, although others believe that the
anterior "premolar" is actually a premolariform canine. The
present study analyzes prenatal dental development in an
extensive histological series of 12-125mm Procavia fetuses,
in order to identify additional criteria for assessing dental homologies in hyracoids and other mammals. Three upper
deciduous incisors are variably developed during fetal and
juvenile life, although only the large first one constantly
occurs and is replaced. A tiny deciduous canine is developmentally retarded in both jaws, probably correlated with
the excessive growth distally of the large incisors. The
first molar initiates development in 29-33mm fetuses, when
dP4 has already reached the middle-late bell stage. Such a
developmental delay for M 1 occurs in all eutherians studied
that retain dP4. Only three premolars develop anterior to
M 1 in each jaw quadrant of the fetal stages examined. This
is consistent with the hypothesis that dP1, which is the
slowest developing of the deciduous teeth when present, is
the missing postcanine tooth in living hyracoids. The
successional lamina is only moderately thickened lingual to
the deciduous teeth in the latest fetuses studied, but observations on tooth eruption and replacement in juvenile
skulls (examined at the American Museum o f Natural History)
are consistent with these suggested homologies. They also
indicate that the tiny dC is replaced by a small premolariform C in both jaws; ttis latter tooth is the one identified previously as " P 1 . Molarization of the canine is an
unusual condition in mammals, but it is already seen in
some genera of Miocene hyracoids. This study suggests that
the pattern of initiation and early differentiation of
tooth germs can be a useful criterion for assessing dental
homologies in hyracoids and other mammals. Supported by
RCMI Grant RR-03051 from the University of Puerto Rico.
LUDOLPH.* David Charles, JoAnn Cameron, and
David L. Stocum, Department of Cell and Structural
Biology, University of Illinois, Urbana, Illinois.
ventralizes ositional memory in the
dorsoventral a - n b o l o t l
li$sT
v i = d i h e
effect ofg?--(RA)
on
pattern completion in the dorsoventral (DV) axis of
regenerating axolotl limbs. Half and double half dorsal
and ventral zeugopodia (lower arms or legs) were amputated
through their distal ends, and four days later the animals
were injected intraperitoneally with 50 (large animals) or
100 (small animals) pg RA/gm body wt. Half and double
half dorsal and ventral zeugopodia of uninjected axolotls.
and sham-operated zeugopodia of RA-treated 1 imbs served as
controls. Skeletal patterns and the DV muscle patterns of
control and experimental regenerates were then analyred.
Sham-operated zeugopodia of uninjected animals regenerated
normally. Sham-operated, RA-treated zeugopodia
regenerated normally with proximodistal duplications.
Sixty percent of uninjected control dorsal half
zeugopodia, 80% of control ventral half zeugopodia. and
100% of control double dorsal and double ventral
zeugopodia regenerated distal ly, but the regenerates
failed to form the complementary half muscle pattern.
Thirty-eight percent of the RA-treated ventral half
zeugopodia and 78% of RA-treated double ventral zeugopodia
failed to regenerate distally. Of those cases that did
regenerate distal ly, none formed the complementary dorsal
half muscle pattern. By contrast, 100% of RA-treated
dorsal half zeugopodia regenerated distally and all had a
normal DV muscle pattern. Forty-one percent of RA-treated
double dorsal zeugopodia failed to regenerate, but of the
remainder that did regenerate, 50% formed the ventral half
muscle pattern. These represented six cases, four of
which regenerated single limbs, and two of which
regenerated twin limbs, each with a normal DV muscle
pattern. We interpret these data to mean that RA
ventralizes the positional memory of blastema cells in the
DV axis.
Supported by NIH Grant HD 12659 to DLS.
Retinoic acid
--
LUGO-GARCIA, Nidza, Rosa Esther BLANCO", Thomas E.
HUGHES" and Harvey KARTEN, Dept. of Anatorqy and
Institute of Neurobiology, University of Puerto Rico,
San Juan, Puerto Rico; Dept. of Neurosciences, UCSD,
La Jolla, California. Localization of G o l i k e
GABA-like inuaunoreactivity
the ground squirrel
retina.
of
retinal
The
identification
neurotransmitters/modulators remains central to a
complete understanding of the role of the retina in
visual function. Imnohistochemical techniques have
provided a precise means of identifying populations
of
retinal
neurons
which
utilize
specific
neurotransmitters/modulators.
We
have
used
immunohistochemical methods to identify and characterize
GABAergic neurons in the ground squirrel retina.
Retinas were incubated with antibodies against glutamic
acid decarboxylase (GAD) and gamma aminobutyric acid
(GABA) and processed for fluorescence and/or avidin
biotin labeling. Immunoreactivity was expressed in
the inner nuclear layer (INL), inner plexiform layer
(IPL), and ganglion cell layer (GCL) Imunoreactive
neurons in the INL were identified as small amacrines
with cell bodies near the inner nuclear/inner plexiform
border. Labeled cells in the GCL may be either ganglion
cells and/or displaced amacrines.
The number of
imnoreactive neurons was greater in retinal sections
incubated with the GABA antiserum.
Inmunoreactive
neurons in the INL and GCL gave rise to processes
that entered the IPL. Inmunoreactive processes ran
through all IPL sublayers, but the staining intensity
was highest in the innermost and outermost sub-nae.
Thus, GAD and GABA imoreactivity may be present
in amacrine, displaced amacrine, and perhaps ganglion
cells. (Supported by NIH Grant NS-07464, Navy Grant
N00014-89-5-3070 and RCMI Grant RR-03051).
.
61A
ABSTRACTgAAA 103RD MEETING
S
LUTHIN*, Gary R. and Chia-Sheng LIN*, Department of Physiology
LYSIAK,* Jeffrey J. and Peeyush K. LALA, Dept. of Anatomy,
University of Western Ontario, London, Canada. (Sponsored
and Biophysics and Institute for Neuroscience, Hahnemann Univerby Evelyn L. Shaver) In situ localization a 2 characterisity, Philadelphia, Pennsylvania 19102 (Sponsored by Peter R.
zation of bone marrow derived cells in the murine decidua.
..
Meyer). Locallzatlon of m l p
Studies usino pseudopregnant radiation bone marrow (BM)
Five subtypes of muscarinicacetylcholine receptor (MR) have been
identified, and mRNA for four of the subtypes is expressedin rat brain.
An antibody specific for the m l MR was used to localize this subtype
of MR in brain using an immunohistochemicalapproach. Areas previously shown to be rich in pirenzepine binding activity and known to
containmRNAfor the m l MR were intensely stained usingthe specific
anti-ml antibody. In hippocampus,the stratum oriens and molecular
layer containedthe highest immunoreactivity. The caudate-putamen
of the striatum was also intensely stained. The layers IV and VI of
cerebral cortex exhibited moderate staining, while only weak staining
was seen in the other cortical layers. Intense staining was seen in
the granular layer, but not other layers, of the cerebellum. Labelled
cells were detected in the pyramidal cells of hippocampus, granular
cells of the dentate gyrus ond olfactory bulb, and both superficial and
deep layers of cerebral cortex. Labelled cells were also noted scattered rather evenly in the striatum and the thalamus. These results
suggest it will be possible to study the regulation of MR expression
by identifyingboth mRNA and protein species of the MR subtypes.
LYNCH, Kathryn, and FRASER, Scoti Earl,' Department of Anatomy,
Uniformed Services University of the Health Sciences, Bethesda, Maryland;
Department of Physiology and Biophysics, University of California, Irvine,
California. Does formation of the DroneDhric duct in Xenopus involve cell
miqration?
We have used vital staining with fluorescent dyes to ask whether the
pronephric duct in Xenopus laevis forms by an active process of directed
cell movement and thus constitutes a model in which to study the
phenomenon of cell migration. Morphometric examination of fixed embryos
showed a ridge or cord of cells caudal to the developing pronephros,
beginning at St 23. From Stage 23 to 29/30, the cord of cells increased in
length at approximately the same rate as the embryonic trunk. Between St
29/30 and St 33/34, however, the cord elongated much more rapidly than
the trunk, and hollowed to form the pronephric duct. The cord thus
appeared to constitute a pronephric duct rudiment (PDR). Simple
examination did not reveal whether the rapid elongation of the PDR to form
the pronephric duct was due to active caudal outgrowth (i.e., a combination
of cell rearrangement,proliferation and migration), or to local recruitment of
cells from the lateral plate. To distinguish between these two alternatives,
we labeled the PDR or the lateral plate with the fluorescent tracer, Dil. Dil
is a lipophilic dye which is incorporated into cell membranes. Cells labeled
by pressure injection of Dil were monitored with a SIT camera on a Zeiss
epifluorescent microscope and videotaped at intervals during the
development of the embryos. PDR cells labeled as early as St 23-24 did
not move from the injection site before St 29/30, showing that the PDR
forms in situ, and not as an outgrowth of the pronephros. PDR cells
labeled with Dil prior to St 29/30 moved caudally from the injection site
during stages 29 to 33/34, indicating that the rapid elongation which occurs
at those stages is an active process involving migration and rearrangement
of PDR cells. Duct cells labeled during or after St 33/34 moved only
slightly or not at all. Cells labeled by injections into the lateral plate caudal
to the PDR did not move at any time, indicating that additional cells are
not recruited into the elongating cellular cord from the mesoderm over
which it passes. We conclude from these results that the pronephric duct
in Xenopus develops by active caudal elongation of a rudiment which itself
forms by local delamination from the lateral plate.
chimeras (Kearns & Lala 1982) and prenatally reconstituted
preanant R M chimeras (Johnson 6 Lala 1989) have demonstrated hemopoietic origin of certain murine decidual
cells isolated by collagenase dispersion. Present study
examined the in situ distributjon and properties of the
hemopoietically derived subset of decidual cells during
normal prpgnancy in mice reconstituted with BM cells
carrying a transgenic marker. RM cells from a transgenic
B-globin
CD-1 strain (CD-16: carryinq 1,000 copies of
qenes in tandem) were injected into the yolk sac of d13 to
d17 conventional CD-1 embryos accorAing to Johnson and
Lala (1989). The pregnant females were then allowed to
deliver normally, the female offsprings were mated with
CD-1 males and then sacrificed at d12 of gestation. Their
spleens, uteri and other organs were sectioned on a cryostat. The extent of chimerism was evaluated by in situ
hybridization of the sections wjth a biotinylated DNA
prohe specific for the 6 -glohin genes followed by ABCperoxidase or ABC-alkaline phosphatase staining. Tissue
controls were provided by CD-1 6 and CD-1 mice, respectively. Tissues were also processed without the application of the probe. Reconstituted mice exhibited variahle
deqrees of hemopoietic chimerism as indjcated by lahellina
of their splenocytes as well as hemopoietic cells in other
organs.
Variable cellular lahelling was also noted in
their decidua basalis and metrial glands.
These cells
were identified as typical decidual cells, macrophaqes,
and granulated metrjal gland (GMG) cells. Labellinq was
also noted in similar cells migrating out of metrial gland
explants after 24 hr. culture. The nonpregnant uterus of a
chimeric mouse revealed labelling of suhepitheljal stromal
cells indicatjve of their hemopoietic oriqin.
These
results reveal: (1) hemopoeitic orjqin of certain typical
decidual cells and G M G cells identified in situ; ( 2 )
hemopoietjc origin of certain stromal cells of the uterus.
The latter may represent precursors of drcidual cells.
(Supported by the MRC Canada).
McALLISTER,* James P.11, KATZ,* Steven D. and WAY, John
S., Dept. of Anatomy, Temple U. Sch. Med., Philadelphia, Pennsylvania. Effects of experimental infantile
hydrocephalus & ventriculoperitoneal shunts on connections of the motor cortex.
___---Our previous findings suggest that afferents of the
cerebral cortex may be irreversibly altered by hydrocephalus. To examine connectivity directly, axonal
tracer studies were performed on kittens in which
hydrocephalus was induced at 10-11 days of age by
intracisternal injection of kaolin. At 11-12 days
post-kaolin, some hydrocephalic animals received low
pressure VP shunts. Injections of horseradish peroxidase (HRP) were made unilaterally in cortical areas 4
and 6 in the following sequence: hydrocephalic animals
at 9-15 days post-kaolin; shunted animals at 1, 2 and 4
weeks post-shunt; control animals at times corresponding to the ages of hydrocephalic and shunted animals.
Within 3 days post-shunt, ventriculomegaly was reduced
with noticeable neurologic improvements. However,
during the second week several animals developed secondary ventriculomegaly as a result of shunt malfunctions
and underwent successful shunt revision. In control
animals, consistent retrograde ARP labelling was found
in contralateral areas 4 , 6 ; ipsilateral claustrum; nucleus basalis, thalamic nuclei (reticular, intralaminar,
ventral anterior, ventral lateral, some ventral posterior); dorsal raphe; midbrain reticular formation;
ventral tegmental area; locus coeruleus. Axonal label
was observed in all the same thalamic regions; internal
-_
62A
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
c a p s u l e ; c r u s c e r e b r i ; pons; d o r s a l column n u c l e i ;
pyramids; s p i n a l c o r d . In c o n t r a s t , h y d r o c e p h a l i c
b r a i n s e x h i b i t e d r e d u c t i o n s i n both c e l l u l a r and a x o n a l
l a b e l l i n g i n most of t h e s e r e g i o n s , e s p e c i a l l y t h e
c o n t r a l a t e r a l c o r t e x . I n s p i t e o f damage t o t h e i n t e r n a l c a p s u l e , t h e r e was l a b e l l i n g i n t h e thalamus. In
g e n e r a l , shunted a n i m a l s e x h i b i t e d l a b e l l i n g s i m i l a r t o
c o n t r o l s . These r e s u l t s i n d i c a t e t h a t , p r i o r t o
s h u n t i n g , some damage t o c o r t i c a l pathways o c c u r s b u t
t h a t s u r g i c a l decompression may a l l o w r e s t o r a t i o n o f
t h e s e c o n n e c t i o n s . Supported by NIH uD21527 t o JPM.
MCATEE*, Marietta M. and Barbara S. BREGMAN, Dept.
Anatomy/Cell Biology Georgetown University, Washington DC.
Time course of axonal withdrawal from inaoorooriate CNS
transolants.
Both appropriate (target-specific) and inappropriate (nontarget) CNS transplants support the survival of immature
axotomized neurons temporarily. T h e requirements for
permanent survival are, however, target-specific. These same
CNS transplants permit the growth of axotomized serotonergic
(5HT) axons into the site of neonatal spinal cord lesions, but
only the target-specific transplants maintain these projections.
The current experiments were designed to determine the time
course of axonal withdrawal of 5HT raphespinal axons from
inappropriate target tissues. Spinal cord lesions ("overhemisections," T6-T8) were made in rat pups 2-3 days postnatal, and appropriate target (spinal cord, SC; embryonic day
14; E14) or inappropriate target (hippocampus, HC or cortex,
CX; E18; or cerebellum, CB; €15) tissues were placed into the
lesion sites. Animals survived for 7, 14, 17, 21-26, 28, 30 days
and t 2 months. Immunocytochemical techniques and cresyl
violet counterstain were used to visualize 5HT axons.
Transplants survive and grow within the lesioned neonatal
spinal cord, and develop morphological features characteristic
of their development in situ. Serotonergic axons are present in
all transplants at 7-14 days post-operative (dpo). Projections are
withdrawn earliest from the CB tissue. Axonal withdrawal
from HC transplant tissue begins between 14 and 17 dpo, and
is complete by 21 dpo. CX transplants, however, support 5HT
axons as late as 26 dpo. Spinal cord transplants maintain robust
5HT growth permanently. These results indicate that axonal
projections grow into and are maintained in the inappropriate
target tissue for relatively long periods of time postnatally
(beyond 14 days), but a r e then withdrawn.
T h e various
inappropriate targets that we examined receive 5HT input in
situ, although the nuclei giving rise to the projection differ.
Clearly, transmitter specificity per se is not sufficient to
maintain 5HT projections. T h e conditions which trigger and/or
regulate subsequent axonal withdrawal are not currently
known. Appropriate target transplant tissue is required to
maintain robust 5HT axonal growth within CNS fetal
transplant tissue. Supported by NIH NS 19259 and NS 01356.
OMcCarthy,* Kevin John, Abrahamson, Dale Raymond,
and Couchman, John Robert,* Department of Cell
Biology and Anatomy, University of Alabama at
Birmingham,
Birmingham,
Alabama.
Basement
membrane-SDecific
chondroitin
sulfate
proteoslvcan
is
temDorallv
and
sDatiallv
eXDreSSed durins rat kidnev develoDment.
We recently reported the production of core
protein specific monoclonal antibodies (mAbs)
which recognize a newly described basement
membrane-specific
chondroitin
sulfate
proteoglycan (CSPG) (McCarthy et al.; J. Cell
Biol. 109: IN PRESS). In adult kidneys, antisera
against heparan
sulfate proteoglycan
(HSPG)
immunostained peripheral loop glomerular basement
membrane (GBM) and mesangial matrix: however,
anti-CSPG mAbs failed to stain GBM but did stain
mesangium.
In the present study we used
antibodies against HSPG and CPSG to examine when
the compartmentalization of these proteoglycans
occurred during glomerular morphogenesis.
Our
results showed that
temporal and spatial
heterogeneity
existed
in
the
developmental
distribution of CSPG not seen with HSPG. Unlike
HSPG, little CSPG was found in the basement
membranes
of
structures
associated
with
"vesicler1 and
stages of glomerular
morphogenesis.
CSPG
first appeared
in
the
subsequent "s-shaped" stage.
In the capillary
loop
stage
of
glomerular development,
the
distribution of CSPG and HSPG resembled that seen
in
the
adult.
Surprisingly,
unlike
the
distribution in adult glomeruli where CSPG was
absent from GBM, we found in developing kidneys
CSPG within the GBM of maturing stage glomeruli.
The duration of this transient association with
the developing GBM is uncertain. Our results
suggest that CSPG might play a role in the
assembly
of
basement
membranes
during
development, serving to temporarily stabilize the
GBM during the later maturation stages.
MACKENZIE*, L e s l i e Wayne, Department o f Anatomy and
O b s t e t r i c s and Gynaecology, Q u e e n ' s U n i v e r s i t y , Kingston,
O n t a r i o , Canada.
(Sponsored b
Madan G . J o n e j a )
C h a r a c t e r i z a t i o n of mvosin h e a w c z a i n i n human u t e r i n e
t i s s u e and c u l t u r e d mvometrial smooth muscle c e l l s .
Human u t e r i n e smooth muscle c e l l s m a i n t a i n e d i n
c u l t u r e r e t a i n biochemical processes t h a t p e r t a i n t o t h e
c o n t r a c t i l e system o f i n t a c t smooth muscle t i s s u e
(Richardson e t al. 1 9 8 6 ; I n V i t r o C e l l . Devel. B i o l .
23:521, MacKenzie e t a l , 1989; Am. J . P h y s i o l . In P r e s s ) .
I t i s n o t known, however, whether t h e s e smooth muscle
c e l l s undergo p h e n o t y p i c modulation i n c u l t u r e .
The
p r e s e n t i n v e s t i g a t i o n w a s conducted t o c h a r a c t e r i z e
myosin heavy c h a i n e x p r e s s i o n and c o n t e n t i n human
u t e r i n e smooth muscle
t i s s u e and c u l t u r e d human
myometrial smooth muscle c e l l s .
Monolayer c u l t u r e s o f
myometrial smooth muscle c e l l s were e s t a b l i s h e d a f t e r
l i m i t e d enzymatic d i g e s t i o n of human myometrial t i s s u e
o b t a i n e d a t t h e t i m e o f hysterectomy. Myosin heavy c h a i n
was e v a l u a t e d and q u a n t i f i e d by d e n s i t o m e t r y a f t e r
s e p a r a t i o n o f p r o t e i n s by sodium dodecyl s u l f a t e polyacrylamide g e l e l e c t r o p h o r e s i s .
In homogenates o f
myometrial t i s s u e , t h r e e p r o t e i n bands comigrated w i t h
t h e 200 kDa myosin heavy c h a i n s t a n d a r d . The c o n t e n t o f
t h e a p p a r e n t myosin heavy c h a i n isoforms i n myometrial
t i s s u e was 75+15 pg/mg p r o t e i n . Human myometrial smooth
muscle c e l l s i n p r i m a r y c o n f l u e n t c u l t u r e s a l s o c o n t a i n e d
t h r e e p u t a t i v e isoforms o f myosin heavy c h a i n a c c o u n t i n g
f o r 65f19 pg o f myosin/mg p r o t e i n o r 87X o f t h e myosin
heavy c h a i n c o n t e n t o f myometrial t i s s u e . The number o f
i s o f o r m s and c e l l u l a r myosin c o n t e n t were similar i n
p r e c o n f l u e n t and c o n f l u e n t c u l t u r e s .
With i n c r e a s e d
number o f c e l l p a s s a g e s , however, t h e number o f p r o t e i n
bands t h a t c o m i g r a t e d w i t h myosin heavy c h a i n s t a n d a r d
d e c r e a s e d a s d i d myosin c o n t e n t . I n c o n f l u e n t c u l t u r e s
a f t e r f o u r p a s s a g e s , o n l y one i s o f o r m o f myosin heavy
c h a i n was p r e s e n t a c c o u n t i n g f o r 41f12 pg of myosin/mg
p r o t e i n o r 542 o f t h e myosin heavy c h a i n c o n t e n t
estimated f o r i n t a c t t i s s u e .
I n comparison, c u l t u r e d
human s k i n f i b r o b l a s t s c o n t a i n e d 25+7 pg o f myosin/mg
p r o t e i n e x p r e s s e d a s a s i n g l e 200 kDa p r o t e i n band.
Thus,
this
i n v e s t i g a t i o n d e m o n s t r a t e s t h a t human
myometrial t i s s u e and c u l t u r e d myometrial smooth muscle
c e l l s c o n t a i n i s o f o r m s o f myosin heavy c h a i n and t h a t
e x p r e s s i o n and c o n t e n t o f t h e myosin heavy c h a i n isoforms
a r e a l t e r e d by r e p e a t e d c e l l p a s s a g e s .
Immunological
a n a l y s i s o f t h e myosin heavy c h a i n isoforms is c u r r e n t l y
underway.
McKenzie, James C., Tadashi Inagami* and Jane N.
Scott, Dept. of Anatomy, Howard University,
Washington,Dc;Dept. of Biochemistry, Vanderbilt Univ.,
ABSTRACTS-AAA 103RD MEETING
Nashville, Tennesee; Dept. of Anatomy, Wright State
. .
h
w
y discovered
in the cardiac atria, has potent natriuretiddiuretic and
vasorelaxant properties. In addition, ANP-LIR has been
detected in the brain, anterior pituitary, adrenal medulla,
hypertmphiedventri~kidneyandothertissues.
Inthe
present study, we investigatedthe distribution of ANPLIR in the kidneys of several adult mammals and in the
developing rat kidney. Kidneys from mouse, rat, cat, dog,
pig and monkey were fixed in Perfix or Bouin's solution,
either by perfusion or immersion, and were routinely
embedded in paraf6n. Human renal tissue was obtained
from autopsy specimens. After depamffimz
* ation, 5 p m
sections were incubated with antibdy against rat ANFIV (1:lOOO) and processed for immunohistochemical
staining using the ABC technique with kits purchased
from Vector Laboratories (Burlingame,CA). DAB was
used as the chromagea In all species, intracellular
A"-LIR was observed in intercalatedcells of cortical
and outer medullary collecting ducts, Positive cells were
only inhquently seen in the inner medulla (papilla)of
the rat kidney but extended deeply into the papilla of the
mouse kidney. Stainingwas most intense in the apical
region of the intercalated cells. The other cells of the
collecting ducts had no ANP-LIR. Staining was not
observed in the developing kidney of rats at 16 days
gestation and only a few stained cells were observed at 18
days. At 20 days gestation,ANP-LIR was observed in
cells lining the exterior of the papilla as well as in
forming collecting ducts. While the origin of ANP in
these cells is still speculative, endogenous synthesis
seems likely.
63A
MCNEILL, Thomas H., Heng-Wei CHENG', Caleb E. FINCH'
and Guilo PASINE'ITI', Andrus Gerontology Center, University
of Southern California, Los Angels, California. Morpholoeical and
mJ
Followine Neuronal Deafferentation.
The present study was undertaken to identify potential
molecular markers that may characterize the cellular mechanisms
involved in neuronal outgrowth and synaptic remodeling in the
striatum (ST) following neuronal deafferentation. In particular,
we used
hybridization and blot hybridization techniques to
examine changes in mRNA prevalence for growth associated
protein (GAP-43) and astrocytic specific glial fibrillary acidic
protein (GFAP) in the ipsilateral ST and contralateral cortex at
3, 10 and 27 days following a unilateral lesion of the corticostriate
pathway. In addition, changes in mRNA prevalence were
correlated with variations in the immunocytochemical staining
patterns for GFAP and tyrosine hydroxylase (TH) in the ST and
substantia nigra. We found that the prevalence of GFAP mRNA
in the deafferentaled striatum was increased by 3 days postlesion,
the time when phagocytic astrocytes have been shown to be
actively involved in removing degenerating presynaptic nerve
terminals and postsynaptic dendritic spines from the lesion site. In
addition, the increase in the prevalence of GFAP mRNA was
correlated with an increase in the density of immunoreactive fibers
for both GFAP and T H in the dorsolateral part of the
deafferentated ST. In contrast, the changes in the prevalence of
GAP-43 mRNA in the contralateral cortex was variable and was
significantly elevated at 3, 10 and 27 days postlesion in some but
not all of the rats examined. These data suggest that reactive
astrocytes play and important role in remodeling the synaptic
connectivity of the striatum following neuronal deafferentation by
removing degenerating axon and dendritic profiles from the lesion
site. Whether they also serve a neurotrophic role to induce axonal
sprouting and new synapse formation in the ST is currently
unknown. Supported by PHS grants AG05445, AGO0300 and a
grant from the Natiwal Parkinson's Disease Foundation.
MCMILLAN, PaulJ and John ARCHAMBEAU,' (Technical assistant,
M-H. Archambeau), Departments of Anatomy and Radiation
Sciences, Loma Linda University, Loma Linda, California.
1
.
The microvessels that supply the epidermis and the superficial dermis are intimately linked to the health and
integrity of the skin. Descriptions of these vessels based
on sections and corrosion casts have demonstrated changes
from controls in aging, in a variety of diseases and in
radiation damage.
We describe here a method of
quantitatively characterizing these microvessels so that
those processes which alter their structure can be more
precisely defined. Vertical sections (perpendicular to the
skin surface) are used in this study. The PAS stain was
used to facilitate identification of the vessels' basement
membranes. Datavoice (Support Technologies Inc., Grand
Terrace, CA.) was chosen for this study to facilitate data
collection. Rules for data entry have been drawn up for
1
reticle so that
this project using a 3 3 5 x 3 3 5 ~ ~ 1sampling
the entire epidermis and dermis is sampled to a depth of
670 pm.
Vertical line probes 670
long are
systematically applied to the skin from which the volume
fractions (V,) of epidermis proper, epidermal rete pegs,
dermis and dermal vessels are estimated. The position of
each transected blood vessel axis and the tips of epidermal
rete pegs are recorded. The length per unit volume (L,)of
microvessels in the sampled region is estimated from the
number of vessel transections. The distribution of vessels
along the plane of the section is characterized by
computing the L, of vessels in a test frame 250x670 )un which
is moved along the section in 75 p n steps. This gives a
running mean of the vessel L, so that the mean free distance
between regions of high vessel density is objectively
determined. Serial reconstructions are used to correlate
these data with the vascular configuration. This method
will be used to evaluate the changes that occur following
irradiation of swine skin.
McSPARREN', Jennifer, Theresa ECKENRODE: Forrest
HAUN, Marion MURRAY* and Leonard ROSS. Department
of Anatomy, Medical College of Pennsylvania, Philadelphia,
Pennsylvania. Habenular transplants remlate the response to
stress in adult rats with lesions of the habenulo-interueduncular
system.
The major habenular input to the interpeduncular nucleus
(IPN) of the midbrain is removed by lesions of the fasciculus
retroflexus (FR), a lesion which also disrupts normal sleep
cycles in rats. Transplants of embryonic habenula cells both reestablish normal patterns of habenula innervation of the IPN
and restore normal sleep patterns in FR lesioned animals. In
this study we measured plasma levels of norepinephrine (NE)
and epinephrine (EPI) at 2-3 months post-FR lesion in adult
rats with or without habenula cell transplants placed into the
lesion site 06 days post-lesion. Circulating catecholamine levels
were also measured immediately after the animals were
subjected to a repeated acute stress by being placed on a small
platform in a water tank for up to 3 hrs/day for 4 consecutive
days. Levels were assayed by HPLC following an intra-orbital
bleed a t the same time of day (1400-1500 hr). FR-lesioned
animals have significantly higher plasma NE (1.90 ng/ml) and
EPI (4.91 nglml) levels compared to normals (.71 ng/ml and .98
ng/ml respectively); N E and EPI levels are also significantly
elevated following the water tank stress, especially In animals
with FR lesions (1.31 nglml NE and 2.14 nglml EPI in normals,
7.42 ng/ml N E and 8.07 ng/ml EPI in F R lesioned animals). By
contrast, animals with FR lesions and habenular transplants
have near-normal lasma N E and EPI levels even after the
water tank stress 8.10 nglml N E and 1.21 nglml EPI). These
results suggest that: 1) the habenula-IPN system is involved in
central modulation of peripheral catecholamine levels,
especially under stressful stimulation; and 2) this modulatory
64A
ABSTRACTS-AAA 103RD MEETING
i n f l u e n c e is re-established in FK-lesioned animals by
transnlants
fetal
r~~~~~~ of .
. . habenular
~ . ~ . cells.
~.~~~
. ~ . ~ ~ ~ . ~ ~
Supported b y A l l e g h e n y H e a l t h Services G r a n t #31 (FH)
and NIH g r a n t NS16556 (MM).
~~
~
~~~
~~
MA*, T.G., M. ZHOU*, N.D. RADTKE* and M.T. TSENG,
Departments of Anatomical Sciences and Neurobiology,
Ophthalmology and Vision Science, University of Louisville,
Louisville, Kentucky. Effects of taurine on N-methvl-D-asDartate
1NMDAI receDtor aaonists and antaaonists induced calcium
transDort in bovine retinal membranes.
Evidence suggests that taurine may have a regulatory role in
excitable tissues as a modulator of calcium fluxes. At low Cat'
concentrations, it stimulates calcium movement (T.G. Ma et al.,
J Cell Biol 109 [4, pt2]:334a, 1989), but the mechanism
governing this stimulation is not known. In this study, it was
found that calcium movement in bovine retinal membranes was
also stimulated by the NMDA receptor agonists (L-aspartate and
NMDA) and the modulator glycine. Enhancements of calcium
uptake were 253%, 355% and 89% for L-aspartate, NMDA and
glycine, respectively. In contrast, the antagonists phencyclidine
and ketamine decreased calcium uptake by 88% and 84%,
respectively. When taurine was introduced into the preparations
containing these agonists, calcium uptake was additionally
stimulated. However, taurine did not block the action of
ketamine and phencyclidine. It is known that the NMDA
receptor complex comprises an ion channel with a modulatory
glycine binding site in addition to the agonist recognition site.
Occupation of the agonist binding site facilitates opening of the
channel, whereas agonist occupation of the glycine site
increases the probability of channel opening. The noncompetitive antagonist acts on a third site by binding in the ion
channel. Present results indicate that taurine may not exert its
modulatoryeffect on calcium movement at these sites on NMDA
receptor complex. These data are consistent with our previous
finding in which taurine has no effect on the inhibition of calcium
influx caused by calcium channel blocker verapamil.
MA, Terence P., Ann M. GRAYBIEL and Robert H. WURTZ,' Lab of
Sensorhotor Research, National Eye Institute. N.I.H., Bethesda, Maryland;
and Department of Brain and Cognitive Science, Massachusetts Institute of
Technology. Cambridge, Massachusetts. Distribution of saccade-related
neurons in the macaque superior colliculus.
We studied the localization of saccade-related neurons in the
superior coiiicuius of two adult rhesus monkeys (Macaca mulatta). Actlvlty
for visually-guided saccades was recorded In trained. awake, behaving
monkeys. Eye position was monitored using the scleral search coil
technique. Data were collected with a PDP 11/73 computer that also
controlled the presentation of stimuli. At each location where we recorded
saccade-related responses, we made electrolytic lesions (10 p m p negative
current, 15-30 s). Subsequently, the animals were sacrificed and the
superior coiliculi were serially sectioned. Alternate sections were
processed for cytochrome oxidase or acetyicholnesterase (AChE) activity
and the electrode penetrations were reconstructed with the aid of an
overhead projector. We were able to identify histologically 39 of 42 leslon
sites. Nine (23.1%) of the recording sites were located In the ventral optic
layer; 13 sites (33.3%) were In the intermediate gray layer; 12 sites (30.8%)
were In the intermediate white layer; 1 site (2.6%) was at the border
between the Intermediate white and deep gray layers; and 4 sltes (10.3%)
were in the dorsal deep gray layer. We conclude, therefore, that saccade
related neurons occur at depth-levels extending from the ventral half of the
optic layer through the dorsal half of the deep gray layer. We examined
the relationship between the motor response characteristics and their
locations within the superlor coiiicuius. We did not flnd that neurons having
different discharge characteristics were clearly related to spedfic tectai
layers. However, in single penetrations normal to the surface of the
coiiicuius, neurons having discharges that end by the end of the saccade
(clipped) tended to be encountered first and neurons having discharges
which extended beyond the end of the saccade (partially clipped and
unciipped) were encountered last. Therefore, we condude that neurons
having different discharge characteristics were organized along a
dorsoventral axls. Last, we examined the distribution of saccade-related
neurons with different motor characteristics in relation to the distribution of
AChE-positive puffs. We did not find any clear relationship, whlch lends
support to the idea that the function of tectofugal neurons Is not solely
dependent on the afferent terminal regions in which their somata lie.
(Supported in part by NRSA F32-EY05950 to TPM and 2-R0i-EY02866IOAI to AMG.)
MAGNEY,* Jean E. and Charles KNOX,* Department o f C e l l
B i o l o g y and Neuroanatomy, U n i v e r s i t y o f Minnesota,
Minneapolis, Minnesota; Department o f Physiology,
U n i v e r s i t y o f Minnesota, Minneapolis, Minnesota.
(Sponsored by Robert L. Sorenson) "The Anatomy o f the
Kidney", a l e v e l 111 i n t e r a c t i v e videodisc program f o r
medical students.
To h e l p medical students t o acquire s k i l l s i n a c t i v e ,
independent, s e l f - d i r e c t e d learning, so important t o
p h y s i c i a n s i n an expanding technological environment (as
emphasized i n the AAMC-GPEP Report), medical schools
must p r o v i d e a p p r o p r i a t e resource m a t e r i a l s . This
prompted us t o design a computer-assisted program which
i n t e g r a t e s gross, microscopic, and developmental
anatomical concepts w i t h neuroendocrine c o n t r o l systems
and r e q u i r e s the student t o c o r r e l a t e basic science w i t h
m o r p h o l o g i c a l l y o r i e n t e d c l i n i c a l m a t e r i a l . Touch
screen and keyboard enable the l e a r n e r to i n t e r a c t w i t h
questions w i t h i n the program, and t o c o n t r o l the pace
and d i r e c t i o n o f the lesson. Visual m a t e r i a l s on
v i d e o d i s c i n c l u d e medical i l l u s t r a t i o n s , l i g h t and
e l e c t r o n micrographs, videotaped cadaver prosections,
non-invasive imaging, and 30 computer graphics. 2D
g r a p h i c s a r e supplied by the software. A few copies o f
the program w i l l be made a v a i l a b l e f o r evaluation;
p r e s e n t a t i o n r e q u i r e s an I B M Infowindow display, model
4055, l i n k e d t o e i t h e r an IBM/AT w i t h hard d i s k and a
5.25 (1.2 megabyte) d i s k d r i v e o r a PS2 model 50 ( o r
g r e a t e r ) and a videodisc p l a y e r .
Supported by the Department o f C e l l B i o l o g y and
Neuroanatomy, U n i v e r s i t y o f Minnesota Medical School ;
t h e I B M Advanced Education Project; the Minnesota
Medical Foundation Herz Award;
the U n i v e r s i t y o f
Minnesota Health Science Learning Resources
I n s t r u c t i o n a l Computing Grant; and a U n i v e r s i t y o f
Minnesota Educational Development Grant.
MALAMED, Sasha, Jean A . GIBNEY," and Sergio R. OJEDA,"
UMDNJ-Robert Wood Johnson Medical School, Piscataway,
New Jersey and Division of Neuroscience, Oregon Regional
Primate Research Center, Beaverton, Oregon.
Ovarian
of
innervation
develops
before
initiation
folliculopenesis.
Although
evidence
is growing that prepubertal
development is influenced by direct neural inputs of the
ovary, morphological evidence for the appearance of
nerve fibers in the ovary during early development has
yet to be reported. The present irnmunohistochemical and
electron
microscopic
studies were
undertaken
to
demonstrate chemically defined nerve fibers in the
perinatal rat ovary and to determine if they innervate
the gland before the formation of primordial follicles
whose differentiation they may influence ( E . B . A . S .
84:
5803, 1987).
With electron microscopy we have
found nerve fibers containing clear and dense core
vesicles in the ovary of the 22 day rat fetus. Within
20 hours after birth the ovary was found to be sparsely
innervated by fibers containing immunoreactive tyrosine
hydroxylase. Close examination of all sections of an
ovary typically revealed up to 3 labelled nerve fibers
per ovary.
Some fibers were associated with blood
ABSTRACTS-AAA 103RD MEETING
vessels and some were not. This labelling was confirmed
with electron microscopy. Serial sections of 24 hour
ovaries revealed a negligible frequency of follicles at
this time (about 1 questionable primordial follicle per
ovarv). Onlv 24 hours later (i.e. 48 hours after birth)
there were at least 3000 follicles per ovary, indicating
a n abrupt period of differentiation between the ends of
postnatal days 1 and 2 . These results demonstrate that
the catecholaminergic innervation of the ovary develops
before follicular formation and raises the possibility
that the extrinsic innervation of the ovary contributes
to the initiation of folliculogenesis. (Supported by
NIH erant HD 2 4 8 7 0 ) .
I
reach an apparent equilibrium between the nucleus and cytoplasm 3-6 hours
post-injection. The N/C ratios at equilibrium were approximately 40X greater
than that found for BSA (.08 vs. ,0019). These results are consistent with those
found for endogenous B3/B4, and suggest that B3, B4 and hsc73 are transported
into the cell nucleus. When the iodinated proteins were microinjected directly
into oocyte nuclei, they entered the cytoplasm at significantly greater rates than
BSA controls. At two hours post-injection, approximately 45% of the injected
B3/B4 and 60% of the hsc73 were located in the cytoplasm, compared to only
6.2% of the BSA. These results suggest that these proteins are transported out
of the cell nucleus. Experiments have also been performed to determine if
separate nuclear and cytoplasmic populations of B3/B4 exist in the cell.
Oocytes were metabolically laheled with 'H-leucine or microinjected
cytoplasmically with iodinated B3/B4 and allowed to equilibrate for 16 hours.
These cells were then bisected. under oil. alone their animal oole-veeetal Dole
boundaries. Labeled vegetal poles were 'then Tused with unlibeled, iucleated
animal poles. After 4 hours, the nuclei were isolated and analyzed by 2D-gel
fluorography (for endogenously labeled proteins) or by measuring 251 (for
injected B3/B4). The results demonstrated that cytoplasmic B3/B4 can enter
the cell nucleus long after equilibration has been reached, arguing against the
existence of two separate populations of B3/B4. Collectively, these results show
that B3, B4 and rat hsc73 are capable of bidirectional exchange across the
nuclear envelope, and suggest that they are continuously recycling by a transportmediated mechanism. This work supported by NIH Grant GM43065-04.
.
Y
MALLONGA, * Rosa L. and Marcia ONTELL, Department of
Neurobiology, Anatomy and Cell Science, University of
Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
Taraet dependence of murine fetal motor neurons.
Little is known about the target dependence of
developing mammalian motor neurons, because of the
difficulty in manipulating mammalian fetuses. We have
developed a technique to transect the sciatic nerve in
fetal mice without interfering with fetal viability. The
uterine wall and fetal membranes are incised, and the
limb is externalized. The sciatic nerve is exposed and
cut, and the limb is returned to the amniotic sac. The
embryo, attached to the placenta, is allowed to develop
in the abdominal cavity. This procedure permits
evaluation of the effects of nerve transection on the
survival of sciatic motor neurons. Moreover, we can
examine the ability of the axotomized motor neurons to
regenerate their axons and to reinnervate their target
muscles. The sciatic nerve on one side was cut at
midthigh level in 5 Swiss Webster fetal mice at 17 days
in utero; the contralateral side served as a control. At
6 - 8 weeks of age both extensor digitorum longus (EDL)
muscles of each of these mice were injected with
horseradish peroxidase (HRP). Lumbosacral spinal cord
sections (50 @m thick) were processed for HRP-reaction
product with tetramethylbenzidine (TMB). The mean number
of HRP-labeled motor neurons on the operated side was 47%
of the value seen on the intact side (operated side=
7.2t1.6; intact side= 1 5 . 4 k 1 . 7 ) . The normal EDL motor
neuron pool was located at lumbar segments 3 - 5 . However,
we consistently found that the reinnervated EDL motor
neuron pool was shifted cranially to lumbar segments 2 - 4 .
The position of the p o o l within the ventral horn did not
seem to change. Thus fetal motor neurons whose axons had
been cut in utero were able to survive and re-establish
neuromuscular connections. However, the specificity of
spinal motor neurons for individual muscles does not
appear to he as rigid as has been demonstrated for normal
developing motor neurons. We are investigating the
effects of transient denervation on the developing
muscle. Supported by grants from MDA and March of Dimes.
S
MANDELL,' Robert B. and Car1 M. FELDHERR; Department of Anatomy
and Cell Biology, University of Florida, GainesviUe, Florida. (Sponsored ,by
Michael H. Ross) Bidirectional exchanee of two hsc70-related oocvte oroteins
across the nuclear envelove.
proteins, B3 and B4
We have verified that two 70,000 M W Xenouus
(PI 5.58 and 5.75, respectively), located in both the nucleus and cytoplasm,
rapidly exchange between these two intracellular compartments. We isolated
these proteins using DEAE column- and ATP-Agarose affdty-chromatography,
a general method employed to isolate Hsp70-related proteins.
These
polypeptides have identical CNBr peptide maps indicating that they are closely
related. Their CNBr peptide maps are also identical to that of hsc73, a heatshock cognate protein isolated from rat brain. These results indicate that B3
and B4 are members of the Hsp7O family. B3, B4 and rat hsc73 were iodinated
and microinjected into Xenoous oocyte cytoplasms. The cells were manually
enucleated at 1-15 hours after injection, and radioactivity was measured in the
nuclei and their respective cytoplasms. Results indicated that B3, B4 and hsc73
65A
S
MARCH*, Demck, Chris SHAW*, Department of Ophthalmology,
University of British Columbia, Vancouver, British Columbia.
(Sponsored by Charles E. Slonecker). Is the eeniculo-share transmitter a
tachvkinin ?
Work within our group involves the study of neurotransmitter
receptors and the cortical chemical circuitry in the mammalian visual cortex
(Shaw et a1.,1986). An unsolved problem in this field is the identity of
the geniculo-striate transmitter. Recently, evidence from other groups has
shown that members of a family of small peptides, the tachykinins have
receptors in the intermediate layers of the rat cerebral cortex (Cascieri e t
a1.,1984,1986 and Mantyh e t al., 1984). Considering that visual input
from the lateral geniculate nucleus synapses on cells in layer 4 of the visual
cortex, these neuropeptides are possible candidates for the geniculo-striate
transmitter. The present study explores this probability using an iodinated
Bolton-Hunter labelled eledoisin ([1251]-BH-ED) applied to corona1
sections of cat and rat visual cortex. Eledoisin is a substance P
homologue obtained from species of octopcds. Cryostat sections of 20p
were incubated with ['25r]-BH-ED using a modified protocol from
Cascieri et al., 1986. Preliminary results in cat visual cortex show high
binding in layer 4 and moderate binding in layer 1 in young cats aged 30
days, while adults show mainly layer 4 binding. Binding of [1251]-BH-ED
in rat cerebral cortex is similiar to those found by the previous groups
mentioned, ie. very high binding centered in layer 4.
In a second experiment, quinolinic acid (QA) was used to destroy
cell bodies within cat visual cortex. [lZI]-BH-ED binding was abolished
in the lesion zone. Since QA does not affect axon terminals, glia or fibers
of passage, the loss of binding suggests that these receptors are located on
intrinsic cortical neurons. These results lend support to the possibility that
eledoisin, or a related tachykinin, is a geniculesmate neurotransmitter
candidate or modulator. Further lesion and characterization studies in
progress will determine if tachykinins are located in terminals originating
from the lateral geniculate nucleus. Together with the present localization
of these receptors to cells of layer 4, these data may strengthen the
contention that a tachykinin serves as the geniculo-striate neurotransmitter.
S
MARION,* M. Susan and Edward C. CARLSON, Department o f
Anatomy and Cell B i o l o g y , U n i v e r s i t y of North Dakota,
Grand Forks, North Dakota. Nonenzymatic g l y c a t i o n i n
c a l f and a d u l t bovine ocular basement membranes.
T h i s s t u d y examined e a r l y and advanced (AGE)
g l y c a t i o n e n d p r o d u c t s i n l e n s c a p s u l e ( L C ) and
Descemet's membrane (DM) i n calves and a d u l t c a t t l e .
Calf and a d u l t LC and DM were i n c u b a t e d w i t h 200M
66A
ABSTRACTSAAA 103RD MEETING
glucose f o r 10 days o r 35 days a t 37OC with the 10 day
samples reduced w i t h NaCNBH3 throughout the incubation.
An antibody directed a g a i n s t g l u c i t o l l y s i n e ( C u r t i s s
and Witztum, 1983) and immunogold were employed t o
l o c a l i z e e a r l y glycation products. Electron microscopy
demonstrated increased binding i n a l l experimental
basement membranes (BMs) compared t o c o n t r o l s incubated
under the same conditions with t h e exception of sugar.
Differences in AGE content can be estimated by
fluorescence and absorbance c h a r a c t e r i s t i c s of t h e
protein (Monnier, e t a1
1984). Accordingly, a d u l t
LCs and DMs incubated 35 days with no reducing agent
were digested with trypsin and examined by fluorometry
(excitation/emission max 370/440nm). Data obtained
from supernatants indicated increased fluorophore
content in sugar incubated BMs fver nonincubated
c o n t r o l s . Analysis of "control t r y p s i n d i g e s t s showed
higher r e l a t i v e fluorescence values in a d u l t vs. c a l f
BMs and i n DM vs. LC. Absorbance data (UV max 350nm)
on the same supernatants gave r a t i o s comparable t o
fluorescence values. Amino acid a n a l y s i s of each BM
indicated a reduction in concentration of a t l e a s t one
of the basic amino a c i d s in a d u l t BM when compared t o
c a l f . These data suggested d i f f e r e n t i a l concentrations
of e a r l y nonenzymatic glycation products a s well as AGE
in ocular BMs. These products may be involved in
protein c r o s s l i n k formation leading t o BM s t r u c t u r a l
changes, including thickening, which a r e known t o occur
during aging. Supported in p a r t by the North Dakota
Lions Foundation.
.,
MARTIN, Vicki Joan, Department of Biological Sciences,
University of Notre Dame, Indiana. Examination of stem
cell differentiation and migration during embryogenesis.
The differentiation and migration behaviors of a population of migratory stem cells, interstitial cells, were
examined in marine hydrozoan embryos (planulae) using a
BrdU/anti-BrdU technique. Embryonic interstitial cells
divide to maintain the population or differentiate into two
classes of progeny: nerve cells or nematocytes. I-cells
and their progeny were marked with the thymidine analogue
bromodeoxyuridine (BrdU). Early cleaving embryos were immersed in 200 p M BrdU in seawater and continuously cultured
to the desired planula stage. 95-100% of the I-cells and
100% of their derivatives were labelled using this procedure. BrdU-marked cells were visualized immunocytochemically using an anti-BrdU monoclonal antibody. BrdU marked
I-cells and their derivatives were identifiable based upon
nuclear morphology. I-cell nuclei were larger than those
of the differentiated progeny and I-cells occurred singly.
Nematoblast/nematocyte nuclei were small, found in groups,
and crescent shaped. Ganglionic nerve cell nuclei were
extremely small and spindle shaped. The I-cell system of
embryos was eliminated by treating 16-24 hour planulae with
0.2% colchicine in seawater for 2 hours. After a recovery
period treated planulae were found to consist entirely of
epithelial cells. Pieces of different aged BrdU labelled
planulae ( 1 6 hour and 36 hour) were grafted to pieces of
epithelial planulae; one day later epithelial pieces were
examined for labelled cells. Epithelia1 pieces contained
labelled I-cells, nematoblasts/nematocytes and ganglionic
nerve cells. The numbers of labelled I-cells and their
progeny found in epithelial pieces was dependent upon the
age of the labelled I-cell donor. Young 16 hour donor
I-cells showed a greater tendency to migrate into epithelial tissue than did older 36 hour I-cells. Also more
labelled derivatives were found in epithelial pieces repopulated with young 16 hour I-cells than in epithelial
pieces repopulated with 36 hour I-cells. Results from
the repopulation studies suggest that embryonic I-cells
exhibit temporal restrictions in their developmental potentials and their migration patterns. Supported by NSF
grants DCB-8702212, DCB-8711245, and DCB8942149.
S
MASLANY,* Steven, David P. CROCKET* and M. David
EGGER, Department of Anatomy, UMDNJ-Robert Wood Johnson
Metlicd School, Piscataway, New Jersey. The proiection of cutaneous
primary afferents to the dorsal column nuclei and dorsal horn in the
rat: WGA-HRP vs. B-HRP.
The projections from cutaneous afferents in the forelimb to the
dorsal column nuclei (DCN) and dorsal horn (DH) were examined
with 25% free HRP, 2.5% WGA-HRP, a mixture of 25% free HRP
and 2.5% WGA-HRP (WGA-HRPIHRP), or 0.1% B-HRP. The
tracer was injected intracutaneously into the forelimb digits. Three
days later, the rats were perfused transcardially, horizontal sections
(60-pm thick) were cut and the HRP was reacted using the TMBmethod. The location of the label was reconstructed by camera
lucida drawings. Data from 30 rats were analyzed. In rats which
received a n injection of only 25% free HRP, no label was detected in
the DCN or DH. WGA-HRP/HRP injected animals produced
projection patterns similar to WGA-HRP injected animals. The
WGA-HRP and t h e B-HRP labelled afferents in the DCN projected
more or less to the same locations for each digit, but B-HRP labelling
was more intense. The DCN labelling was precisely restricted
metliolaterally, with little or no overlap into adjacent areas. Afferents
from digit 1 projected ventrolaterally in the cuneate nucleus; those
from digit 5 dorsomedially to those from digit 1. In the DH, in
contrast, patterns of labelling with WGA-HRP differed markedly
from those with B-HRP. WGA-HRP labelled cutaneous atlerents
projected to Rexed's laminae 1-111, with the densest label in lamina
11. In contrast, B-HRP labelled cutaneous afferents projected to
laminae 11-V, with the densest label in laminae 111-IV. Little or no
labelling was found in lamina I. Comparisons of the projection maps
from each of the five digits, using either WGA-HRP or B-HRP,
indicated that, in the cervical spinal cord, there was extensive
overlapping among the labelled cutaneous afferent fihers from these
digits. In addition, rostrocaudal regions of heaviest B-HRP label
coincided closely with those of the WGA-HRP labelled afferents.
Projections from digit 1 extended rostrocaudnlly from c;lud;rl C4 to
caud;il C6; projections from digit 5 extended from c;~udalC6 to
rostra1 C8. I n summary, at least for afferents projecting to the spinal
cord, WGA-HRP and B-HRP labelled different subpopulations, with
the B-HRP subpopulation apparently biased toward afferents o f
1:irger diameter.
S
MATEO,* Annabelle R., Jody E. Margulies' and Ronald P.
Hammer, Jr., Department of Anatomy & Reproductive Biology,
University of Hawaii, Honolulu, Hawaii. Dvnamic oatterns of
medial oreootic rr-ooiate receotor rermlation bv gonadal hormones.
The medial preoptic area of rat hypothalamus (MPOA)
contains a population of p-opiate receptors whose density varies
across the estrous cycle, and is increased by hormone priming in
ovariectomized females. The relationship of gonadal steroid
hormone environment to MPOA preceptors was studied over time
in ovariectomized females to determine the dynamic effect of
changing hormone levels. Females were ovariectomized and
implanted two weeks later with either blank or filled silastic
capsules containing 2 mm p-estrddiol (E2). Capsules were removed
or sham surgery was performed and 2.5 mg progesterone (P) or
propylene glycol vehicle was administered 48 hr later, and animals
were subsequently decapitated and trunk blood collected 3, 27, or
51 hr after surgery. Brains were frozen and sectioned, and sections
were incubated in ['HIDAGO to selectively label preceptors, and
exposed to 'H-sensitive X-ray film. Computer-assisted quantitative
densitometry was used to measure radioligand binding. Plasma
levels of E,, P and prolactin were assessed by RIA. ANOVA
showed a significant main effect of treatment on MPOA preceptor
density, which peaked 27 hr following E,,P treatment and subsequently declined. This decline occurred in the presence or absence
of E,. These changes were not, however, observed in the lateral
preoptic area. Moreover, a significant correlation existed between
MPOA preceptor density and plasma P level, but not with other
hormones. W e conclude that changing hormone levels, particularly
ABSTRACTS-AAA 103RD MEETING
P administered following E, priming, affect MPOA preceptor
density over time. T h e presence of E, promotes P effects, but
apparently does not alter preceptor level. MPOA preceptor
density could decline following E,,P exposure due either to
dynamic effects of other hormones or receptor turnover. Supported
by USPHS Awards RR08128, NS01161 and RR03061.
MASTEHION, R. B., R . J . NUDO*, and D. P.
SUTHERLAND',
Department of Psychology, Florida
State University, Tallahassee, Florida;
Department of Neurobiology & Anatomy, University
o f Texas Health Science Center. Houston. Texas.
Morphometric relationships among four forebrain
and midbrain tracts descending to the spinal
cord.
In each of 24 mammals the cells originating
the corticospinal, hypothalamospinal,
tectospinal, and rubrospinal tracts (i.e., C S T ,
HYT, TST, RST) were retrogradely labeled witn HRP
applied to the spinal cord after hemisection a t
the CI-C2 level. The resulting labeled cell
profiles were tnen counted in systematic sets of
sections and the total number of labeled cells
originating each tract was estimated. These
numbers proved to range from 16 cells in the
tectospinal tract of rabbit to over 91,000 cells
in the corticospinal tract o f raccoon. Over the
24 species, the CST has by far the largest number
of cells of the four tracts, averaging over
19,600 cells or about 7.4X more than the average
of 2650 cells in the HST. The average number of
RST cells is more than 4 X larger than the average
of 600 HST cells which in turn is more than twice
as many as the average of 268 cells in the TST.
Strict statistical comparisons of the four tracts
among the species show that the CST and RST are
related to their host's body size and brain size
and also reliably and directly related to each
other. In contrast, the number of HST cells is
reliably related to the number of TST cells but
neither is related to the CST or RST nor to brain
size or body size.
Supported by Grant No. NS7726 from NIH-NINDS.
MATTHIES, Donald L. and Kap J . LEE*, Oepartment
of Anatomy, University o f North Dakota School of
Medicine, Grand Forks, North Dakota.
Microtubule involvement as a common factor in
testicular seminiferous tubule damage.
A variety of disturbances produce damage to
seminiferous epithelium but the formation of
multinucleate cells (MN cells) is a common
denominator. These large cells have few to
dozens of ring-like nuclei, are rarely found
free in tubular lumen or epididymis, and are
apparently confined in Sertoli cells. In
searching for the mechanism and the cells
involved in producing this response and with the
knowledge that colcemide prevents polymerization of intracellular microtubules we injected
it into marked locations of the subtunical space
of rat testes. We used 50 ug and 2 0 pg doses in
50 p 1 saline with saline only for controls a t the
opposite pole o f the testis from the colcemide
injection. Injection site tissue was fixed for
serial sectioning 2 4 , 48, and 72 hours after
injection. M-N cells were found in the colcemide treated tissue only and the numbers encountered reflected both a dose and time dependency.
67A
Germinal cells retain a unique relationship to
each other by intercellular bridges and to
Sertoli cells by being nearly but not completely
surrounded by them. It is also known that
Sertoli cells survive germinal cells in testicular damage. We postulate therefore that in
damaged testes microtubules release their
conformational force o n germ cells whose intercellular confluences reopen to restore haploid
nuclei into a common nuclear bag or M - N cell for
destruction by lysosomes. This, plus other
mechanisms e.g. blood testis barrier, which
confine germ cells to the tubule and duct system
could act to prevent autoimmune responses to
antigens from damaged sperm.
MAUL, Gerd G . , Ph.D. The Wistar Institute of Anatomy and
Biology, 3601 Spruce Street, Philadelphia, PA 19104.
Nuclear Core Comolex Structure.
The two membranes of the nuclear envelope separate the
two major compartments of the cell - the nucleus and the
cytoplasm. This barrier is perforated where the two
membranes are fused to facilitate exchange between the
compartments. A channel of communication is maintained
through the pores by a complex arrangement of proteins
called the nuclear pore complex.
Its structural
characteristics are an intracisternal ring which may
prevent the membranous pore from enlarging and to which
is attached the non-membranous part of the pore complex,
by proteins that pass through the membrane.
The
nonmembranous pore complex has a symmetric arrangement of
traverse fibrils that pass through the waist of the pore
and two rings on either side of the waist. Other fibers
act as a sieve to prevent free diffusion of larger
molecules.
Several models of the nuclear pore complex have been
proposed from structural data, but none has successfully
predicted or even suggested the functional properties
that can account for active and selective transport
demonstrated with molecular techniques.
Different
fixation techniques produce varying images that are
probably derived from artifactual precipitation of the
same structure. Averaging techniques have a1 so been
detrimental when coaxing information out of isolated and
therefore modified material. Aggregating effects of
bivalent ions change the filamentous structures to
globular granules.
The
present
image of the
three-dimensional arrangement of pore complex components
is therefore determined by the techniques used, and is
not yet useful to define properties of an assumed
bidirectional active selective transport mechanism.
MAY, Paul J., John D. PORTER and Paul D. R. GAMLIN*. Depts. of
Anatomy
and Ophthalmology, U. of Mississippi Med. Ctr.,
Jackson, Mississippi; Depts. of Ophthalmology, and Anatomy and
Neurobiology, U. of Kentucky Med. Ctr., Lexington, Kentucky;
Dept. of Physiological Optics, U. of Alabama at Birmingham,
Alabama. Connections between
the Macaque cerebellum and
oculomotor repion: Pathwavs subservins the near resuonse.
?he components of the near response are controlled by
motoneurons in the oculomotor and Edinger-Westphal nucleus, and
vergence related cells in the supraoculomotor area, but the
cerebellum also plays a role by modulating this response. The
pathways subserving this cerebellar modulation were investigated using WGA-HRP in cynomolgous monkeys. Following injections
of the physiologically identified Edinger-Westphal nucleus and
surrounding supraoculomotor area, retrogradely labelled neurons
were concentrated bilaterally, with a contralateral predminance, at two cerebellar sites: in the rostra1 fastigial nucleus and at the border between the anterior and posterior interposed nuclei.
Investigation of the terminal distributions of
68A
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
these two populations produced corroborating r e s u l t s . Specific a l l y , a f a s t i g i a l nucleus injection labelled terminals b i l a t e r a l l y i n the Edinger-Westphal nucleus, the supraoculomotor
area and immediately adjacent portions of the oculomotor nucleus.
I n addition, retrogradely labelled neurons were found i n
the i p s i l a t e r a l supraoculomotor area and b i l a t e r a l l y i n the
Edinger-Westphal nucleus. A n injection centered i n the posteri o r interposed nucleus labeled terminals i n the l a t e r a l supraoculomotor
area contralaterally, but retrogradely labelled
c e l l s throughout the supraoculomotor area and Fdinger-Westphal
An anterior interposed nucleus injection produced
nucleus.
terminal labelling b i l a t e r a l l y i n and between the oculomotor
No labelling was
nuclei and i n the anteromedian nucleus.
observed a f t e r an injection of the dentate nucleus. As a
whole, these r e s u l t s indicate the presence of reciprocal connections between portions of the f a s t i g i a l and interposed
nuclei and the midbrain regions subserving the near response.
The pathways from and t o these two cerebellar nuclei may underl i e the adaptive and dynamic modulation of lens, pupil and
vergence a c t i v i t y by the cerebellum. Supported by NE1 Grants
EY07166 (PJM), EX05464 (JDP) and EX07558 (PDRG).
S
MECHALCHUK,* Cheri
Lynn and Bernard H.
BRESSLER,
Department o f Anatomy, U n i v e r s i t y o f B r i t i s h Columbia,
Vancouver, B.C.
Contractile orooerties o f fast-twitch
s k e l e t a l muscle o f t h e mdx mouse f o l l o w i n q denervation
and devascul a r i z a t i o n .
Changes i n c o n t r a c t i l e p r o p e r t i e s o f t h e f a s t - t w i t c h
extensor d i g i t o r u m longus (EDL) muscle o f normal mouse
and o f t h e mutant mdx mouse were studied f o l l o w i n g
The nerve and
denervation and d e v a s c u l a r i z a t i o n (DD).
vessels o f EDL were severed a t 4 weeks o f age and then
operated and c o n t r o l muscles o f each s t r a i n were s t u d i e d
a t 7 weeks and 16 weeks o f age. A t 7 weeks, f o l l o w i n g
DD, EDL o f both s t r a i n s showed s i g n i f i c a n t l y decreased
i s o m e t r i c t w i t c h and tetanus tensions compared t o t h e i r
7 week non-DD c o n t r o l s although t e n s i o n values d i d n o t
By 16 weeks,
d i f f e r between DD EDL o f t h e two s t r a i n s .
however, normal operated muscle e x h i b i t e d a recovery o f
57% and 58% o f absolute tetanus and t w i t c h tensions
r e s p e c t i v e l y w h i l e the mdx EDL recovered remarkably t o
96% and 99% o f i n i t i a l 7 week values. W i t h i n t h e non-DD
EDL t h e r e was no change i n time t o peak (TTP) o r one-half
r e l a x a t i o n time (112 RT) between s t r a i n s o r between
ages. A t 7 weeks the DD mdx EDL e x h i b i t e d s i g n i f i c a n t l y
slower TTP and 112 RT w h i l e t h e normal EDL showed slower
112 RT. By 16 weeks a l l t h e values f o r TTP and 1/2 RT o f
the DD EDL o f both s t r a i n s no longer d i f f e r e d from non-DD
controls.
No d i f f e r e n c e was found i n t h e maximum
of
shortening
(Vo)
or
in
post-tetanic
velocity
p o t e n t i a t i o n (PTP).
Following DD t h e r e was an increase
i n r e s i s t a n c e t o f a t i g u e i n both s t r a i n s a t 7 weeks.
This r e s i s t a n c e p e r s i s t e d a t 16 weeks i n t h e normal mouse
b u t a t 16 weeks the operated mdx EDL had r e t u r n e d t o a
I t would appear t h a t
normal l e v e l o f f a t i g u a b i l i t y .
f o l l o w i n g a denervationldevascularization i n s u l t t h e mdx
EDL i s a b l e t o recover c o n t r a c t i l e c h a r a c t e r i s t i c s such
as t e n s i o n production and f a t i g u a b i l i t y t o a remarkably
l a r g e r e x t e n t than normal EDL, l i k e l y i n d i c a t i n g an
to
regenerate
skel eta1
muscle.
enhanced
a b i 1 it y
Supported by the Muscular Dystrophy Association o f Canada.
MEEK,* W i l l i a m , S a b r i n a LAWSON,* B r i a n RABER,*
a n d Oza NCCLAIN,* D e p a r t m e n t o f Anatomy,
C o l l e g e o f O s t e o p a t h i c Medicine-Oklahoma S t a t e
U n i v e r s i t y , T u l s a , Oklahoma. ( S p o n s o r e d by
Gerald R.-Kirk). Spheroidal d e n s i t i e s of k e r a t i n o b s e r v e d d u r i n g m i t o s i s of t h e human
amnion c e l l line--WISH.
Brightly f l u o r e s c e n t s p o t s are p r e s e n t i n
WISH m i t o t i c c e l l s , a s r e v e a l e d w i t h a k e r a t i n
p o l y c l o n a l a n t i b o d y i n b o t h i n d i r e c t immunof l u o r e s c e n c e a n d avidin-biotin-peroxidase
techniques.
I n comparing o t h e r e p i t h e l i o i d
c e l l l i n e s , t h e s p o t s a r e seen i n HeLa but n o t
WISH a l s o c o n t a i n s v i m e n t i n ;
i n CHO-K1.
however, t h e s p o t s a r e n o t d i s p l a y e d w i t h a
monoclonal a n t i b o d y .
In addition, the keratin
s p o t s a r e p r e s e n t a r o u n d t h e p e r i p h e r y of some
i n t e r p h a s e c e l l s . When viewed w i t h
t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y (TEN),
electron dense spheroids are present near the
c e l l membrane o f m i t o t i c c e l l s . T h e s e m e a s u r e
130-180nm i n d i a m e t e r w i t h some h a v i n g
elongated p r o f i l e s .
They a r e s e e n i m m e d i a t e l y
beneath t h e c o r t i c a l r i n g of microfilaments;
and i n some a r e a s , s p e c i f i c a l l y n e a r t h e c e l l
substrate, intermediate filaments (IFS)
interlink the densities.
I n t a c t IFS appear t o
b e more p r e v a l e n t i n t h e s e a r e a s o f c e l l - t o s u b s t r a t e i n t e r a c t i o n . The f l u o r e s c e n t s p o t s
a n d t h e d e n s i t i e s a p p e a r t o b e t h e same s t r u c tures.
T h e s e f o c i may r e p r e s e n t o r g a n i z a t i o n
s i t e s o r d e p o t s f o r t h e k e r a t i n IFS d u r i n g
mitosis.
There presence during i n t e r p h a s e a l s o
s u g g e s t s a s i m i l a r f u n c t i o n i n a r e a s of c e l l s
undergoing a n i n t e r n a l r e s t r u c t u r i n g of t h e
cytoskeleton, i.e., lamellipodia, cell borders,
s u b s t r a t e a t t a c h m e n t s i t e s . The r e l a t i o n s h i p
t o the vimentin I F S i n such coexpressing cells
and o t h e r c y t o s k e l e t a l p r o t e i n s i s of i n t e r e s t .
S u p p o r t b y G r a n t n o . 89-09-245 from t h e
American O s t e o p a t h i c A s s o c i a t i o n .
0 MENDEZ,* I., N. RAJMUMAR*, K. ELISEVICH*and B. FLUMERFELT,
Departments of Anatomy and Clinical Neurological Sciences, University
of Western Ontario, London, Canada.
GAD-uositive nimo-rectal
neurons receive a direct GABAermc inuut: A combined ultrastructural
immunocvtochemical and WGA-HRP tracinn study.
A projection from the substantia nigra pars reticulata to the superior
colliculus has been well established by means of anatomical tracing
methods. The existence of GABAergic neurons in the pars reticulata is
also well known, and recent evidence has suggested that the nigral
projection to the superior colliculus is GABAergic in nature. The aim
of this study was to determine if the GABAergic nigral neurons receive
an input from GAD-positive axons, and if the same neurons give rise
to the nigral projection to the superior colliculus. To label the nigrotectal neurons, wheat germ agglutinin-conjugated HRP (WGA-HRP) was
injected stereotaxically into the superior colliculus of adult Wistar rats
by means of the diffusion placement procedure. After a survival period
of 48 hrs. the animals were perfused transcardially with a solution of
4% paraformaldehyde and 0.08% glutaraldehyde in 0.1M cacodylate
buffer (pH 7.2). The primary fixative was followed with a solution of
4% paraformaldehyde and 0.1% zinc salicylate in 0.1M cacodylate buffer
(pH 6.8). WGA-HRP activity was demonstrated using 3,Y-diaminobenzidine tetrahydrochloride (DAB), and GAD immunoreactivity was
localized with the peroxidase-antiperoxidase technique following the
protocol of Mugnaini and Oertel. All tissues wLie prepared for
examination at both the light and electron microscope level. Cell bodies
labeled retrogradely from the superior collicuIus were found within the
ventolateral region of the pars reticulata of the substantia nigra. The
WGA-HRP reaction product took the form of discrete punctate granules
which appeared as labeled lysosomal profiles with the EM. Axons and
perikarya within the same area were clearly labeled with the diffuse
reaction product characteristic of DAB hunohistochemistry and
synaptic interactions between the two were frequently seen. In some
cases, neurons were found to possess diffuse immunoprecipitate for GAD
and punctate reaction product for WGA-HRP. These results suggest the
presence of a GAD-positive input onto GAD-stained nigral neurons
which in turn provide a GABAergic projection to the superior colliculus.
(Supported by the Medical Research Council of Canada)
69A
ABSTRACTS-AAA 103RD MEETING
MERCHENTHALER, Istvan and Jerome L. MADERDRUT, Functional
Morphology Section, LMIN, NIEHS, Research Triangle Park,
North Caroliona; Bolinwood St. Chapel Hill, North Carolina
Immunocvtochemical localization of neurokinin B in the rat
central nervous svstem.
Two mammalian genes (preprotachykinin A and B) code for
precursors containing the sequences of one or more
tachykinins. The sequences of two tachykinins, substance P
(SP) and neurokinin A (NA) are contained in the protachykinin
A prohormone, while the sequence of only one tachykinin,
neurokinin B (NB) is contained in the protachykinin B
prohoromone. The organization of perikarya and fibers that
contain protachykinin A-derived peptides has been mapped with
inmunocytochemistry using antisera directed toward the
carboxyl terminus of SP. However, the distribution of
protachykinin B-derived peptides has not previously been
mapped because antisera directedtowardthe carboxyl terminus
of NB have significant cross-reactivity withNA. One feasible
strategy that can circumvent this obstacle is to use antisera
directed toward the amino terminus of NB (Kager and Conlin:
Peptides 10: 71 713-716, 1989). The distribution of NB-like
immunoreactivity was clearly different from the distribution
of
SP-like
immunoreactivity
when
studied
with
immunocytochemistry in adjacent vibratome sections from
previously colchicine-treated rats. The distribution of NBimmunoreactive perikarya was similar to the distribution of
preprotachykinin B-containing perikarya visualized with
situ hybridization (Warden and Young: J. Comp. Neurol.
272:90-113, 1988). For example, numerous NB-immunopositive
perikarya were seen in the cyngulate and piriform cortex, the
bed nucleus of the stria terminalis, striatum, medial
habenula, superior colliculus, central gray and reticular
formation of the lower brainstem, and the area postrema. In
addition, inmunoreactive fibers and terminals were detected
in the lateral septum, different regions of the hypothalamus,
the external cuneate nucleus, the spinal trigeminal nucleus
and the superficial layers of the posterior horn of the
spinal cord. These experiments indicate that NB is cleaved
from the prohormone in most perikarya containing tachykinin
mRNA. These experiments also reveal for the first time the
distribution of NB-containing fibers and nerve terminals.
MIKAIJA," Takashi, Andrei BORISOV,* Anthony BROWN* and
Donald A. FISCHMAN, Department of Cell Biology and
Anatomy, Cornell University Medical College, New York,
New York. Cell lineage analysis of avian cardiac
development with recombinant retrovirus.
The heart is probably the first organ system to
differentiate and function during embryogenesis of
higher vertebrates. To analyze cell lineages of all
cardiac tissues, we have modified the avian SNV (spleen
necrosis retrovirus)-based vector by inserting the
reporter gene, bacterial R-galactosidase gene, lacZ.
This new retroviral vector, designated CXL, was
introduced into chicken and quail embryos on 1st and
2nd days o f incubation. Most of endodermal, mesodermal
and ectodermal cells of these early embryos could be
infected with CXL. Infection of specific cardiac
progenitors has been achieved by microinjection of
viruses at low concentration into the precardiac
mesodermal area. We stained embryos with X-gal and
chose those with R-galactosidase positive clones only
in the heart wall, Using serial sections of stained
heart we have been analyzing clonal shape and size to
develop a three dimensional map of progenies derived
from a single infected progenitor. (Supported by the
Aaron Diamond Foundation and NIH/RFA HL37675.)
and Claude E. G?GNA, Department of
AMtcsny, New York university Dental center, New York
MFTCHEU, Onmtxl G.
City, New York.
2-DNA innnmoreactivitv in fixed
mammalian cataractous crvstalline lens tissue
sections.
Using the peroxiaase-antiperoxidase (PAP) method,
along with anti-Z-m whole antisera, anti-2-DNA
purified I ~ Gpolyclonal antibodies and anti-2-DNA
monoclonal antibcdies, a,
p
v of 2-DNA was
established for the first tune m bath human and calf
cataract lens tissues. Nuclear fixatives (e.g.,
clarke's and carnuy's solution) allawed for the
developwnt of Z-DNA innnmoreactivity. Nonnuclear
fixatives (e.g., Buffered neutral folmalin solution)
resulted in poor or m 2-DNA inmnmreactivity. 2-DNA
antibozty binding patterns were visualized by the PAP
technique in the lens cortex region, but not in the
lens nuclear region.
No Z-CNA iImum-ctivity
occurred with unfixed lens tissue sections.
pretreatnent of fixed lens tissue sections with mase
I, and actinanych D, before the (semndazy)
application of anti-2-m antibodies, resdtd in 110
z-DNA hmnoreactivity. FNase pretreatment had no
effect on 2-DNA immunol-Bactivity.
Ihe data
accumulated in tkis pilot study indicates that Z-DNA
sequences directly exist or have the potential to form
in human and calf catiuactcus DNA, and may play a
significant role in the development of
cataractcgenesis
.
-
MOORE, N. Anthony and Carroll R. BALL, Department of
Anatomy, University of Mississippi Medical Center, Jackson, Mississippi. Observations 0"
Structure of
Myoepithelial Junctions in Cat Middle Cerebral Arteries.
Since myoepithelial
junctions were first described by
- Rhodin, close membrane appositions of endothelial cells
and vascular smooth muscle cells have been reported in
numerous tissues and organs. They are said to be more
numerous in the smaller resistance vessels of the arterial system, i.e., the arterioles and their immediate
branches, however, we have observed a significant population of these junctions in the middle cerebral arteries
of control cats. The endothelial cells possess foot-like
processes which extend either into the underlying internal elastic lamina to contact intimal smooth muscle cells
or through the elastic lamina to contact medial smooth
muscle cells. The base of the endothelial cell process
is frequently filled by a thick microfilament bundle
(stress fiber) capped in a unique relationship by a flattened profile of endoplasmic reticulum. A comparison of
cross-sections and longitudinal sections of these
endothelial projections reveals that they are ridges
flattened in the longitudinal axis rather than spherical
fingers. The projections are paralleled by extracellular
condensations of anchoring filaments. This arrangement
should provide adaptability to changes in the vessel
diameter. At the point of cell-to-cell contact, the
basal lamina of the endothelial cell and the external
lamina of the smooth muscle cell are absent. In many
instances the junction appears to be a typical gap-type
junction, but the length of the contact varies greatly.
This junction has been proposed to be a site for electrical or ionic communication between the endothelial and
smooth muscle cell. We have also observed a more typical
"synaptic" communication which contains a variety of
intracellular vesicles including Weibel-Palade bodies.
In view of recent proposals that endothelial cells themselves regulate the character of the vascular wall by
producing chemical agents to modulate smooth muscle function and growth, the myoendothelial junction is an obvious candidate to facilitate this interaction.
MORGAN,C.W., L.A.FELKINS,* S.J.ZHANG,* and
W.C.DeGROAT,* Departments of Anatomy and
70A
ABSTRACTSLAAA 103RD MEETING
Neurobiology and Urology, Eastern Virginia
Medical School, Norfolk, Virginia and
Department of Pharmacology, University of
Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania. Axon collaterals of sacral
precranalionic neurons identified by
intracellular injection of HRP.
Previous electrophysiological studies in
the cat sacral spinal cord demonstrated
recurrent inhibition of bladder preganglionic
neurons (PGN) during electrical activation of
preganglionic axons, a mechanism attributed
in part to PGN axon collaterals. Yet
numerous retrograde HRP studies of these
neurons demonstrated axons but no
collaterals.
In the current experiments the more
sensitive technique of intracellular
injection of HRP into single neurons revealed
axon collaterals in 7 of 17 PGN in the 52
spinal cords of 8 cats. These PGN were
identified by characteristic conduction
velocities of 4-6 M/Sec and thresholds 10
times greater than motor neurons during
antidromic activation of the S2 ventral
roots. Three different patterns were seen:
(1) numerous boutons spread throughout the
autonomic nucleus; (2) moderately complex
collaterals with fewer boutons in the lateral
and/or medial ventral horn; and ( 3 ) very
simple collaterals with few branches and
boutons in the lateral ventral horn.
While it remains to be determined which
type of PGN and its collaterals subserve
recurrent inhibition of bladder neurons, the
emergence of more than one pattern suggests
that a variety of other functions may be
served by these axon collaterals.
Supported by NINCDS Grant R01 NS26585.
Moses", R.L., and William C. Claycomb", Departments
of Anatomy, and Biochemistry and Molecular Biology,
Louisiana State University Medical Center, New
Orleans, Louisiana. Retention of M-lines in adult
rat cardiac myocytes cultured on laminin.
-Laminin, an extracellular matrix component, has
been shown to enhance the attachment of cultured
cardiac muscle cells to substrata and to affect the
morphology of the myocytes. The rapid attachment
and subsequent failure of many of the myocytes to
round up leaves open the question of whether or not
sarcomeres remain intact in myocytes spreading on
laminin-coated substrata. In the absence of
laminin, preexisting sacomeres are clearly not
utilized intact, becoming disorganized and then
reorganizing as the cells spread out. One characteristic of that reorganization is the absence of Mlines in the resulting sarcomeres. In order to
determine if the same sort of reassembly occurs in
myocytes cultured on laminin, we examined by in situ
en face TEM, cells cultured for 4 and 6 days on
either laminin or plastic substrata. M-lines were
evident in many of the cells cultured on laminin for
both time periods, but not in the cells cultured on
plastic. In optimally sectioned cells the M-line
was resolvable into five separate sublines. Cells
which initially assumed a bipolar shape by becoming
attached at their ends appeared to retain M-line
structure most consistently. These studies indicate
that some myocytes cultured on laminin retain intact
sarcomeric structure for at least 6 days in vitro
and may indicate differences in myofilament dynamics
in myocytes cultured on different substrata.
MuEHLEMAN,* Carol and Fred RAHIMI,* Department of
Anatcmical Sciences. Dr. Willian M. Scholl Colleqe of
Pcdiatric Medicine,.Chicago, Illinois. The effectiveness
of "epineurial capping" in limiting axonal regeneration
in severed peripheral nerves in the rat.
The severinq of peripheral nerves is a c m n
procedure for fhe &val
of stump n e m , MOrton's
n e m , or in anputation procedures. This severing
process typically results in axonal regeneration and/or
overproliferation of Schwann cells at the proximal stump.
A true n e u m is described as a neoplasm of nerve fibers
and this neoplasm is often the cause of pain,
particularly in weight bearing sites. In order to test
the effectiveness of the epineurim as a barrier to axon
regeneration, 31 rats w e r e anesthetized and their right
sciatic nerves severe3 just above the level of
bifurcation. In 17 of the rats no further surgical
treabnt was performed and the incision was sutured.
In the experimental group of 14 rats the epineurim was
At the
sutured tightly over the proximal nerve s-.
end of 3, 6 or 8 mnths the animals were anesthetized
and the severed nerve was reexposed and evaluated. A
section o f each nerve was resected to include p m x k l
middle and distal portions, fixed in forrnalin and
processed for light microscopic study. All control
animals shawed extensive axonal regeneration with Schwann
Cell proliferation and tough, fibrous adhesions to
surrounding mscle tissue. Experin-ental animals showed
no axonal regeneration beyond the "epineurial cap" and
little or no adhesion to surrounding connective tissue
or mscles. Microscopic examination of the stump,
however, shcrwed that m s t of the experhntal animals
displayed overproliferation of Schwann Cells and at least
sane axonal g m h within the confines of the "epineurial
cap". The results suggest that although "epineurial
capping" is not ccnpletely effective in eliminating
n e u m M fornution, it may be useful in limiting the
regenerating tissue and confining it away fran a site
in which weight bearing may cause pain. Supported by
Dr. Willim M. Scholl College of Pcdiatric Medicine.
MUNGER, Bryce L. and Gary J. BENNETT*, Department of
Anatomy, The Milton S . Hershey Medical Center, The
Pennsylvania State University, Hershey, PA and National
Institute o f Dental Research, N I H , Bethesda, MD. The
periuheral axonal uatholonv in the constrictive model of
peripheral neurouathv.
A model of peripheral neuropathy characterized by
hyperalgesia, allodynia, and spontaneous pain has been
created in adult rats by placing loosely constrictive
ligatures around the sciatic nerve. The abnormal
responses to innocuous stimuli are apparent by 2 days.
The present study analyzed the sciatic nerve and cutaneous and muscular nerves distal to the ligature. A l l
large diameter Group I and I1 fibers degenerate distal to
the ligature in all rats studied 10 days after the ligature. Remnants of degenerating myelin and axonal
fragments were identified in silver-stainedparaffin
sections and by electron microscopy. Small diameter
myelinated fibers ( A delta) and unmyelinated C fibers
were demonstrated distal to the ligature in light and
electron microscopic preparations in all cases studied.
The extent of preservation of small diameter myelinated
fibers varied somewhat from case to case. In the cutaneous and muscular nerves, only small diameter myelinated
fibers and C fibers were present. Some intact axons
evidenced some disruption of microtubules and nerve filaments. The sensory terminals of these axons were free
nerve endings (FNE's) on hair follicles and in dermal
papillae in the skin and in the perimysium of muscle.
These findings document a selective partial deafferentation of hairs as well as Meissner and Merkel terminals
in skin, and the afferent as well as efferent innervation
of muscle except for FNE's of A delta and C fibers.
(Supported in part by USPHS Grant NS 19462.)
ABSTRACTS-AAA 103RD MEETING
71A
S
NAPlERALSKI,* Julie A. and Leonard M. EISENMAN, Department
of Anatomy, Jefferson Medical College, Thomas Jefferson
University, Philadelphia, Pennsylvania.
T h e develpxoression of Zebrin 1 and CaBP in the cerebel lum of n o r m a l
and lurcher mutant m i c e
Purkinje cell (PC) heterogeneity has been previously
demonstrated by his toc he m ic a l and immunohistochemical
techniques.
The survivability of the different PC populations in
neurologically mutant mice can also be observed using these
techniques.
The cerebellar defect of the mutant mouse lurcher
involves the steady loss of P C beginning at approximately
poslnatal day 10 (P10) and progressing to a total loss of PC in the
adult mouse. The expression of Zebrin 1 which is recognized by
thc antibody Q113 in P C was analyzed in the developing
ccrcbcllum of normal and lurcher mice. A PC specific antibody,
vitamin D-dependent calcium binding protein (CaBP), was used
to label surviving PC.
In the adult cerebellum the monoclonal
antibody 4113 recognizes Zebrin 1 that is confined to a subset of
PC clustered in parasagittal bands interposed by non-reactive
bands of PC.
At P11 in the normal mouse, a pattern of
allernating 4 1 1 3 positive and Q113 negative PC i s apparent in
the vermis of lobules VIII and IX while all of the PC in the
vcrmis of lobules VII and X are immunopositive. Subsequent to
this time, the staining pattern progresses rostrally and laterally
to the hemispheres until the adult pattern is formed.
In the
lurchcr animal, most of the P C never express the Q113 antigen.
Occasionally a few isolated cells will be immunopositive. but no
clcar cvidence of banding is apparent. The CaBP antibody labels
PC in the cerebellum of the normal and lurcher animals.
At
P11, the PC of the lurcher appear in a continuous monolayer
with their dendrites extended.
By P16, small gaps in this
monolayer can be seen in the vermis.
The number of P C
prcscnt continues to decrease steadily with increasing age
which coincides with previous data concerning PC loss in this
neurologically mutant mouse.
However, the CaBP positive cells
are distributed in a way as to suggest random loss of Pc‘s. These
preliminary data suggest that the survivahility of PC in lurcher
(Our thanks
is not correlated with their Zebrin I heterogeneity.
to Dr. Richard Hawkes for the 4 1 1 3 monoclonal antibody and
support from NIH grant 22093.)
NAUGHTON, B r i a n A . a n d C a i l K . NAUGHTON*, M e d i c a l
Laboratory Sciences Department, Hunter College
S c h o o l o f H e a l t h S c i e n c e s , New Y o r k , New Y o r k ;
a n d Marrow-Tech I n c . , E l m s f o r d , New York.
E v a l u a t i o n of s u s p e n d e d n y l o n s c r e e n long-term
r a t bone marrow c u l t u r e s by flow c y t o m e t r y .
C u l t u r e s of Long-Evans r a t bone marrow c e l l s
w e r e e s t a b l i s h e d o n s u s p e n d e d n y l o n s c r e e n temp l a t e s and s u b j e c t e d t o phenotypic a n a l y s e s u s i n g
mouse(Ms) m o n o c l o n a l a n t i b o d i e s ( A b ) t o r a t b l o o d
Monolayer
c e l l s p r o d u c e d b y S e r o t e c I n c . , UK.
c u l t u r e s o f r a t bone marrow s t r o m a w e r e l i f t e d
and seeded onto nylon f i l t r a t i o n screens w i t h
g r i d o p e n i n g s o f 2 1 0 um. When 6 0 - 7 5 % o f t h e m e s h
openings were spanned b u t not completely covered
by s t r o m a l c e l l p r o c e s s e s t h e c u l t u r e s w e r e i n o c u l a t e d w i t h marrow e n r i c h e d f o r h e m a t o p o i e t i c
c e l l s a n d s t u d i e d f o r up t o 28 w e e k s . The t y p i n g
Abs ( w i t h a p p r o p r i a t e s p e c i f i c i t i e s ) w e r e OX-33
(B), W 3 / 2 5 ( T q ) , O X - 8 ( T g ) , M O M / 3 F 1 2 / F Z ( g r a n u l o c y t e ) , and ED-l(mon0cyte).
Single color analysis
o f enzyme s e p a r a t e d a d h e r e n t c e l l s w a s p e r f o r m e d
using an EPICS C flow cytometer a f t e r a p p l i c a t i o n
of s h e e p a n t i - M s IgG1-F IT C a n d c o m p a r e d t o i d i o t y p i c c o n t r o l c e l l s . T h e n a t u r a l k i l l e r (NK) c e l l
p o p u l a t i o n was d e f i n e d u s i n g a two c o l o r
analysis.
The r e s u l t s i n d i c a t e t h a t s u s p e n d e d
n y l o n s c r e e n bone marrow c u l t u r e s c o n t i n u e t o
s u p p o r t t h e p r o d u c t i o n of a l l c e l l l i n e a g e s t e s t e d , e v e n a f t e r 28 weeks.
T he r a n g e s o f p o s i t i v i t y f o r e a c h Ab w e r e : OX-33 ( 3 - 8 % ) , W3/25 ( 2 - 5 % ) ,
OX-8 ( 3 - 9 % ) , MOM/3F12/F2 ( 9 - 1 5 % ) , E D - 1 ( 6 - 1 2 % ) ,
(2-5%). Bone marrow c u l t u r e s
a n d NK 3 . 2 . 3 .
e s t a b l i s h e d w i t h marrow which w a s lymphocyted e p l e t e d v i a t h e AET-rosette p r o c e d u r e showed a
r e b o u n d i n p h e n o t y p i c a l l y m a t u r e T a n d B c e l l s by
Suspended nylon screen
2-3 w e e k s o f c u l t u r e .
c u l t u r e s of r a t bone marrow c e l l s m a n i f e s t
p e r s i s t a n t multilineage expression over long
terms and permit a t l e a s t t h e terminal s t a g e s of
T lymphocyte d i f f e r e n t i a t i o n .
and D a n i e l J.
Belliveau,*
NAUS,
C h r i s t i a n C.G.
Department of Anatomy, U n i v e r s i t y of Western O n t a r i o ,
London, Canada.
C e r e b e l l a r somatostatin: changing
e x p r e s s i o n d u r i n g development.
The p o s t n a t a l a p p e a r a n c e of t h e mRNA f o r
p r e p r o s o m a t o s t a t i n , as w e l l a s t h e p r e s e n c e of t h e
p r o s o m a t o s t a t i n - d e r i v e d p e p t i d e s , s o m a t o s t a t i n 2 8 and
w a s examined i n t h e r a t
s o m a t o s t a t i n 28 (1-12),
RNA b l o t t i n g a n a l y s i s i n d i c a t e s t h a t
cerebellum.
t h e r e i s a d r a m a t i c d e c r e a s e i n t h e l e v e l of t h i s
mRNA
during c e r e b e l l a r maturation.
In s i t u
h y b r i d i z a t i o n r e v e a l s t h a t many c e l l s c o n t a i n t h i s
mRNA a t e a r l y p o s t n a t a l s t a g e s ,
with progressively
fewer c e l l s b e i n g l a b e l e d a f t e r p o s t n a t a l day 20.
Immunohistochemical a n a l y s i s r e v e a l s t h a t t h e r e i s a
c h a n g i n g p a t t e r n of d i s t r i b u t i o n of t h i s p e p t i d e
t h r o u g h c e r e b e l l a r development.
I n i t i a l l y , cells i n
and f i b e r
t h e granule cell layer a r e s t a i n e d ,
i m m u n o r e a c t i v i t y i s l o c a l i z e d p r i m a r i l y i n t h i s same
layer.
During t h e second p o s t n a t a l week,
this
granule c e l l layer s t a i n i n g decreases,
and some
Purkinje c e l l s d i s p l a y immunoreactivity, p a r t i c u l a r l y
i n t h e f l o c c u l u s and p a r a f l o c c u l u s .
This Purkinje
c e l l s t a i n i n g i s h e t e r o g e n e o u s and d e c r e a s e s by
p o s t n a t a l day 30, b u t immunoreactive c e l l s a r e s t i l l
present i n t h e granule cell layer.
In t h e a d u l t
cerebellum,
s o m a t o s t a t i n mRNA i s s t i l l d e t e c t a b l e ,
and some of t h e s e c e l l s remain immunoreactive f o r
these peptides.
Further s t u d i e s a r e i n progress t o
c h a r a c t e r i z e t h e phenotype of t h e s e c e l l s . Supported
by Grant No. OCP0041612 from t h e N a t u r a l S c i e n c e s and
En g i n e e r i n g Research Council of Canada.
NEWTON, Bruce W. Department of Anatomy, University of Arkansas
Sexuallv dimorDhiC
for Medical Sciences, Little Rock, Arkansas.
immunohistochemical distribution nf catecholamine-svnthesizing
enzvmet in lamina IX gf the rat lumbar soinal cord.
Rat lumbar spinal cord contains three known sexually dimorphic
motonuclei which innervate abdominal and perinea1 mm.: cremaster
nucleus (CN), spinal nucleus of the bulbocavernosus (SNB), and the
dorsolateral nucleus (DLN). Previous studies indicate that the male
CN, SNB, and DLN receive a greater number of 5HT- and peptidergic-immunoreactive (IR) fibers than females (Brain Res. Bull, 15:
609,’85; J,, como. Neurol, 248:235,’86; & Neurosci. && 14286,’88;
94:10,’88; Anat. R& 22383A,’89; Dev. Brain Res, 47:
Neurosci.
226,’89). This study examines the distribution of tyrosine hydroxylase
(TH-IR), dopamine-beta-hydroxylase (DBH-IR), and phenylethanolamine-N-methyltransferase (PNMT-IR): enzymes of the catecholamine
(CA) synthetic pathway. To examine TH-, DBH-, and PNMT-IR,
adult male and female rats were fixed with 4% p-formaldehyde and
Vibratome sections were processed using the PAP technique. Results
indicate that in either sex the number of TH-IR fibers > DBH-IR >
PNMT-IR. This agrees with previous studies which indicate that
adrenaline, synthesized by PNMT, is sparse in lamina 1X; whereas,
noradrenaline, synthesized by DBH, is the predominant CA in lamina
IX (Histochem, 8 1:237,’84; L corno. Neurol. 242:358,’85). Overall, the
male CN, SNB, and DLN contain more TH- and DBH-IR fibers than
females and vice versa for PNMT-IR. The distribution of male THIR, the rate limiting enzyme for CA synthesis, is DLN > SNB = CN;
for females: DLN > CN > SNB. The distribution of male DBH-IR is
CN > DLN > SNB; for females: CN > DLN = SNB. The distribution
of male PNMT-IR is C N > SNB > DLN; for females: CN > SNB =
72A
ABSTRACTS-AAA 103RD MEETING
nocturnal melatonin peak (03:OO-06:00), hamsters were
individually exposed to the 620 nm light for 5 minutes, at
irradiances of 348, 116. 58, 29, 11.6, 5.8, 1.16, or 0.116
pW/cmz. The animals were sacrificed and pinealectomized after a
15 minute rest in darkness following the 5 minute exposure.
Control animals were handled similarly but received no
experimental light exposure. Pineal glands were frozen and stored at
-7OOC and later assayed for melatonin content using RIA. All data
were subjected to one-way ANOVA and differences between means
were determined by Newman-Keuls multiple range tests. Unexposed
control groups exhibited typically high nocturnal levels of pineal
melatonin. In animals exposed to irradiances of 348, 116, or 58
pW/cmz (photon densities of 326.10, 108.70, and 54.35 x 1015
photons/cmz, respectively), pineal melatonin contents were
significantly suppressed as compared to their matching unexposed
controls (p~0.001, p<O.OOl, and p<O.OI, respectively).
NICOLEl$*, Miguel Angelo L., Chia-Sheng LIN*, and John King lrradiances of 29 pWlcm2 or weaker produced no significant
suppression.
This experimenl demonstrates the ability of
CHAPIN , Department of Physiology and Biophysics, Hahnemann monochromatic red light at 620 nm to suppress nocturnal pineal
University, Philadelphia, Pennsylvania. (Sponsored by Antony melatonin in Syrian hamsters and that 58 pW/cmz may be close to
threshold level for its suppressing capacity. Supported by NASA
Grigonis). )
Grant NAGW 1196, USUHS Grant C07049, NEMA Grant LRI
89:DR:NEMA:l, and NSF Grants DCB84-12587 and DCB87-14638.
DLN. In either sex, there are greater numbers of TH- and DBH-IR
fibers within CN, SNB, and DLN than in the remainder of lumbosacral
lamina IX. For PNMT-IR, the CN, SNB, and DLN do not preferentially contain more IR fibers than other lamina IX motonuclei. These
data indicate that the sexually dimorphic lumbar nuclei are surrounded
by fibers which have the potential to produce all of the CA, but that
synthesis of noradrenaline and/or dopamine would predominate over
adrenaline production. Furthermore, since females appear to have
more PNMT-IR than males, females would have a greater potential for
producing adrenaline within lamina IX than males. Supported by an
Arkansas Caduceus Club Junior Faculty Research Grant and BRSG
RR05350.
B!s.
Little is known about the effects of early peripheraldeafferentation
on the developmental specification of thalamocortical connections
in the somatosensory system. One theory is that adequate peripheral
innervation is necessary for thalamocorticalafferents to successfully
compete for cortical targets. In the current study we have investigated this problem by injecting retrograde tracers (rhodamine
coated microspheres (RCM)) in the somatosensory (Sl) cortices of
developing and adult normal rats, and also rats peripherally deafferented by neonatal destructionof the mystacial vibrissae. Small inof RCMs in the SI barrelfieldregions of normal
jections (0.5 - 1.O
2-3 week old rats (n = 20) revealed a strong projectionfrom the magnocellular subdivision of the medial geniculate nucleus (MGN). This
projection from the MGN to the SI cortex is apparently transitory,
since it was not seen after equivalent injections in the SI cortex of normal adults. It m observed, however, after equivalent SI cortex injections in adult rats (n= 12) whose mystacial vibrissae had been
removed on post-natal day 1. Interestingly, these peripherally
deprived SI cortices still maintained an apparently normal projection
from the ventral posteromedial (VPM) thalamus. In conclusion, the
preservationof connections from the MGN suggests that the region
normally destined to be SI cortex may, after the peripheral denervation, hwe a more polysensory or associative function. Supportedby
FAPiSP 8814044-9to M.A.L.N., PHS grants NS26722, AA06965, K02AAOOO89, and an award from the AFOSR.
S
Nierenhuis, Robert. F a l t e r Prozialeck* and Greg
C h r i s t i a n s e n * . D e p a r t m e n t of Anatomv a n d D e p a r t m e n t o f
Phvsio1oqy;Pharmacolo~~. P h i l a d e l p h i a C o l l e g e o f Ost e o p a t h i c Y e d i c i n e , P h i l a d e l p h i a , Pennsvlvania. EffecLs
o f Cadaium (Cd'*) on LLC-PKi C e l l s i n C u l t u r e .
Although t h e t o x i c e f f e c t s of cadmium ( C d Z + )on o r g a n s
s u c h as t h e testes, l u n g s and kidney are well known, t h e
s p e c i f i c c e l l u l a r mechanisms u n d e r l y i n g t h e s e t o x i c e f f e c t s have j e t t o be e l u c i d a t e d . I n t h e s t u d i e s d e s c r i b e d
h e r e , w e have examined t h e e f f e c t s of Cd'+ on t h e g e n e r a l
m o r p h o l o e y , c e l l v i a b i l i t y and t h e s t r u c t u r e o f j u n c t i o n a l complexes and m i c r o f i l a m e n t s i n c e l l s of t h e est a b l i s h e d p o r c i n e r e n a l e p i t h e l i a 1 c e l l l i n e , LLC-PBI.
T h e s e c e l l s were grown t o c o n f l u e n c y i n alpha-MEM s u p p l e mented w i t h 10% c a l f serum, exposed t o v a r y i n g c o n c e n t r a t . i o n s of C d C l z a n d t h e n e x a m i n e d by p h a s e c o n t r a s t a n d
e l e c t r o n microscopy. I n a d d i t i o n m i c r o f i l a m e n t s were
v i s u a l i z e d by u s i n g r h o d a m i n e - c o u p l e d p h a l l o t o x i n s a n d
v i n c i i l i n was v i s u a l i z e d by immunofliiorescent t e c h n i q u e s .
The i n t e g r i t y of i n t e r c e l l u l a r j u n c t i o n s was a s s e s s e d by
n o n i t o r i n g t h e C d L + - i n d u c e d c o l l a p s e o f domes a n d by
measuring changes i n t h e t r a n s e p i t h e l i a l e l e c t r i c a l
r e s i s t a n c e . Exposure t o Cd2& caused a r a p i d d e c r e a s e i n
t r a n s e p i t h e l i a : r e s i s t a n c e and t h e concomitant c o l l a p s e
o f domes. The e f f e c t s o c c u r r e d a t Cd"
concentrations
(10-60 microY) and d u r a t i o n s o f e x p o s u r e ( a s l i t t l e as 1
h r . ; t h a t d i d riot a l t e r c e l l u l w ATP l e v e l s or k i l l t h e
c e l l s . E x p o s u r e t o E0 m i c r o # CdL' from 1-8 h r s . c a u s e d
many c e l l s t o d e t a c h from e a c h o t h e r v i t h time and assume
a s p h e r i c a l s h a p e . Q u a n t i t a t i v e and morphologic changes
i n m i c r o f i l a m e n t s were s e e n b u t no o b v i o u s d i f f e r e n c e s
were d e t e c t e d i n v i n c u l i n . E l e c t r o n m i c r o s c o p y showed
q u a l i t a t i v e time dependent changes i n j u n c t i o n a l int e g r i t y . T h e s e r e s u l t s i n d i c a t e t h a t CdL+ h a s a r e l a t i v e l y s p e c i f i c d a m a g i n g e f f e c t on t h e o c c l u d i n g j u n c t i o n s between LLC-PBi c e l l s and t h a t i t may i n v o l v e t h e
d i s r u p t i o n of j u n c t i o n a l complexes and/or t h e m i c r o f i l a ment component of t h e c y t o s k e l e t o n .
NGUYEN,' Dong-Chau, John P. HANIFIN,' Mark D. ROLLAG. Milton H.
STETSON,' and George C. BRAINARD, Department of Neurology,
Jefferson Medical College, Philadelphia, Pennsylvania.
..
. . D
pf different D
i
Svrian hamsNocturnal pineal melatonin synthesis and secretion in hamsters can
be suppressed by unexpected light exposure during the dark phase of
the 1ight:dark cycle. Both the irradiance and the wavelength are
important parameters in light-induced suppression of melatonin and
wavelengths above 610 nm have been thought to have no significant
influence on rodent neuroendocrine system. The purpose of this
study was to examine the effect of monochromatic red light at 620
NODEN. Drew M.. Department of Anatomy, NYS College of Veterinary Medinrn (10 nm half-peak bandwidth) on the suppression of nocturnal
cine, Cornell University,Ithaca, New York.
melatonin production. Each control and experimental group consisted
of 7 adult male Syrian hamsters (Mesocricefus aurafus, 3 to 5
Beginning at stage 18 endocardial cells at specific sites in the cardiac outmonths old). All animals were adapted to a LD10:14 1ight:dark cycle
flow tract delaminate to form mesenchymalcells of the endocardial cushions.
(lights on 08:OO-18:OO) for at least 4 weeks.
During their
The objectives of this research have been to identify the embryonic origins of
73A
ABSTRACTS-AAA 103RD MEETING
these and adjacent endocardial cells, and to determine when endocardial cells
become spatially programmedto undergo this transformation.
Two sets of transplants were performed: orthotopic grafts of somitomeres 5,
6 or 7 and adjacent lateral mesoderm (n=15), and heterotopic grafts in which
cervical segmental plate mesoderm was placed in the same sites as in set 1
(n=31). Tissues were grafted from stage 8.5to 10 quail donors into stage 9
chick embryos, which were then fixed in Carnoy's between stages 16 and 26.
All endocardial and mesenchymalcells derived from the transplant were
identifiedon deparaffinbed sections using chicken polyclonal antibodies that
recognize quail cell surface antigens.
The results of both sets are similar. In 39 of 46 survivors quail mesodermal
cells invaded the developing oulflow tract and contributed to endocardium, but
not myocardium. This contribution ranged from a few isolated cells to the
erdire bulbo-truncalendocardium on the side ipsilateralto the implant. Neither
ventricular nor atrial endocardiumwas labeled, but the ipsilateralcommon
cardinal vein and sinus venosus contained quail endothelial cells in 3 cases.
Implants of mesoderm immediately rostra1or caudal to somitomeres 5-7 did
not result in outflow tract labeling. These results unexpectedly indicate that
precursors for outflow tract and aortic sac endocardium arise within periotic
paraxial and lateral mesoderm, not within the cardiogenic plate.
In 6 cases (4 = heterotopic) immunopositive mesenchymal cells were found
in developing endocardial cushions beneath patches of quail outflow tract
endocardium. Thus, endothelial precursorsfrom non-cardiogenic regions of
the embryo can, if grafted beneath or beside the otic region, form outflow tract
endocardium and subsequently participate in endocardial cushion formation.
Therefore, the spatial programmingthat determines which endocardial cells
will form cushion mesenchyme must occur during or after the formation of the
definitive outflow tract.
Supported by Grant No. DE06632 from the NIH (NIDR).
NODEN, Drew M. and LI Xia', Department of Anatomy, NYS College of Veterinary Medicine, Cornell University, Ithaca, New York.
(sponsoredby W. 0. Sack)
Grafts onto the chorio-allantoicmembrane (CAM) have often been used to
study the ability of tissues to develop independent of their normal environment.
Blood vessels found within grafts are presumed to be CAM-derived. Following
the discovery that many embryonic primordia contain endogenous angioblasts,
however, this presumption is open to challenge.
The objective of this research has been to examine the relative contributions
of CAM- and graftderived endothelial cells to the definitive vascular network
found in grafted limb buds. Quail limb buds at stage 16 to 19 were explanted
onto the 8.5day chick CAM and allowed to develop for 4 to 11 days. Explants
were fixed in Carnoy's and paraffin sections were treated with polyclonal antibodies that recognize quail endothelial cells. Only grafts containingwelldefined connective tissues and feather primordia were processed.
Blood vessels lined with quail, i.e. donor-derived, endothelium were found in
48 of 53 cases. As summarized below, these limbs ranged from having their
vessels entirely of donor origin to having a mixture of quail and chick vessels.
Donor-derived vessels were preferentiallylocated in the deeper and also more
distal parts of the limbs, with chick endothelialtissues more abundant closer to
the limb-CAM interface.
NORVELL,
John
E.,
and
Robert
G.
MACBRIDE,
Department
of
Anatomy,
Cell
Biology
8
Neuroscience,
Oral
Roberts, University,
Tulsa,
Oklahoma.
P o s t m o r t e m s t a b i l i t v o f N e u r o D e D t i d e \L
fl u o r e s n
Current techniques t o study neuropeptides
involve t h e perfusion of l i v i n g animals w i t h
various
fixatives
at
4-C
to
stabilize
the
neuroactive substances.
Unaffected kidneys from
dogs and r a t s s a c r i f i c e d f o r other s t u d i e s w e r e
u s e d t o d e t e r m i n e t h e p o s t m o r t e m stabi 1i t y o f
N e u r o p e p t i d e Y (NPY) a t room t e m p e r a t u r e i n s i t u .
N o n p e r f u s e d renal t i s s u e s w e r e f i x e d a t t h e t i m e
o f r e m o v a l , f r o z e n and c r y o s t a t s e c t i o n s made.
was
The
NPY-Like
immunoreactivity
(NPY-LI)
determined
by
the
indirect
immunofluorescent
technique.
K i d n e y s f r o m t w o animals o f b o t h
s p e c i e s w e r e r e m o v e d a t t h e t i m e of d e a t h , as
w e l l as 2 , 4, 6 , 8, 1 8 , a n d 2 4 hours p o s t m o r t e m .
R e n a l n e r v e s were l o c a t e d i n nerve bundles i n t h e
adventitia
of
arcuate
arteries
and
their
branches.
T h e r e w a s no e s s e n t i a l d i f f e r e n c e
between t h o s e tissues removed i m m e d i a t e l y upon
s a c r i f i c e o f t h e animal a n d t h o s e r e m o v e d u p t o
2 4 hours p o s t m o r t e m .
A
slight decrease i n
i n t e n s i t y o f f l u o r e s c e n c e f o r t h e l a t t e r t i m e was
o b s e r v e d i n some c a s e s , b u t n o t e n o u g h t o a f f e c t
evaluation for
t h e presence of
NPY-LI
nerve
fibers.
T h e r e s u l t s o f t h i s s t u d y show t h a t c o l d
p e r f u s i o n o f l i v i n g a n i m a l s i s not n e c e s s a r y for
d e t e c t i o n of N P Y - L I f i b e r s .
It a l s o showed t h a t
t i s s u e s may b e s u s t a i n e d a t 2 3 ' C f o r up t o 24
hours a f t e r d e a t h and s t i l l
y i e l d acceptable
results.
It f u r t h e r raises t h e p o s s i b i l i t y o f
u s i n g human t i s s u e s o b t a i n e d a t a u t o p s y f o r
further study.
v.
all donor (quail)
19
majority donor
54
majority host
18
9
all host (chick)
The relative distribution of donor and host endothelial cells did not change
with duration on the CAM nor with age of the initial explant. However, the
penetration by CAM vessels was clearly greatest in those cases in which the
graft-CAM interface was large and irregular. Most of the chick endothelial
tissues in the graft were continuouswith CAM vessels; suggesting that the
CAM does not contain isolated invasive angioblasts similar to those found
within the body of the embryo. Invasion of the CAM by donor-derivedangiogenic precursors was an infrequent finding, and was similarly correlatedwith
the extent of the limb-CAM interface.
These resuns indicate that the blood vessels which develop in CAM-grafted
limb buds are mostly of donor origin. These endothelial tissues may depend
upon the formation of anastornoses with CAM vessels for their assembly and
branching, but penetration by CAM endothelial cells is not necessary for graft
survival and development.
Supported by Grant No. DE06632 from NIH (NIDR).
ODonnell, Elsa H.J. Biosciences, SC/€NCf CONSORTIUM,
248 Tom Hill Drive Boulevard, Macon, Georgia.
Rina Ordination of Mammalian ChromosomeS.
Human and mouse chromosomes arranged in quasi-planar ring
confi urations in which concentric rows of chromatids ran in
paral el from midinterphase to early prophase.The ri orous ordination
by side, established periodidties in which lengtfts of condensing
chromatids showed as electron dense ribbing, raising above the
surface of rings and alternatin with an interchromatid constituent
of lower electron density an interpreted to be less condensed
chromatin. The quasi-planar rings showed a slight curvature
suggestive of tori. A single nucleolus occupied the center of a ring.
The concentric parallel arrangement of chromatids spanned a
hierarchy of distances, the widest observed at early intephase. The
interchromatid constituent was consistently present presumably
serving a putative matrical, supportive role. Chromosomes were
found to be polarized with condensation starting at the telomeric end
of the long arm of chromosomes and proceeding sequential1 towards
the telomere at the short arm of chromosomes. Condknsation
pro ressed not by supercoiling of chromatids but by their wrapping
an I or layerin at ri ht angles to the inner and outer rims, in
changing handedlness. !oral sectors in which chromatids condensed
along the outer rim, showed high electron transparenc . The nonrandomized distribution of these eukaryotic comp ements is
interpreted to manifest early interphase ordination. It is also
suggestive of being the support, or alternatively, the result of the
propagation of chemical waves during interphase syntheses
proceeding in circular or elliptical patterns, as previously proposed by
ODonne11,'1983, 1989. Non-randomized chromosomal ordination
introduces topological constrains to the randomness of chromosomal
9
L9
8
Y
74A
ABSTRACTS-AAA 103RD MEETING
exchanges by facilitating translocations between neighboring
chromosomes but restricting, if not impeding, translocations between
non-neighboring chromosomes. 'Arn.Ass.Anat., 205(3)146A and
International Conference on the Human Genome I, 9.
ODOR, D.L. and James R. AUGUSTINE, Department of
Anatomy, University of South Carolina, Columbia, South
Carolina. Cyclic chanpes in baboon oviductal epithelium.
We studied the olive baboon, Papio cvnocephalus to
determine the extent of cvclic deciliation and reciliation of
oviductal epithelium among primates. The animals became
available from other studies. Oviducts were surgically
removed a t different menstrual stages, based on the onset
of bleeding. Light, transmission, and scanning electron
microscopy (EM) were performed. In 20 oviducts, cells of
each segment were counted to determine percent of
nonciliated, ciliated and ciliogenic cells of fimbrial,
ampullar and isthmic epithelial cells. The means of the
percents of nonciliated epithelial cells for the fimbria ( F ) ,
ampulla (A) and isthmus ( I ) were: Group 1-early
proliferative stage, N=3; F=89.1, A189.3, H 9 . 2 .
Group
2-mid-proliferative stage, N=3; F161.2, A=66.0, I=69.7.
Group 3-late proliferative & early secretory stages, N=7;
F=53.1, A=60.8, I=66.9. Group 4-late secretory stage
N=7 ; F=82.6, A.80.5, I=70.3. Statistical comparisons were
made with a one-way analysis of variance and the General
Linear Model Procedure from SAS to account for unequal
sample sizes. A significant difference existed in percent
of fimbrial and ampullar nonciliated cells between Groups 1
& 3 and between Groups 3 & 4.
N o significant difference
occurred between isthmic groups. SEM of the fimbrial
and ampullar epithelium of early proliferative and late
secretory groups revealed many nonciliated but few
ciliated cells. Large, flat apices indicated some deciliated
cells and reciliation occurred in similar cells. Many
nonciliated cells had raised borders outlining their
polygonal apices with moderate numbers of microvilli. In
mid-proliferative phase there were more reciliating and
ciliated cells than earlier. A maximum percent of ciliated
and a minimum of nonciliated cells occurred during the
late proliferative and early secretory stages ; deciliated
and reciliating cells were rare. Light and TEM correlated
with the SEM images. Cyclic deciliation and reciliation
occur in the fimbriae and ampullae of the baboon oviduct
but no significant changes in ciliation in the isthmus.
OGILVIE, Robert W. and FEEBACK, Daniel L.. Department
of Anatomical Sciences, University of Oklahorfm Health
Sciences Center, Oklahoma City, Oklahonm. Molybdate
sta-tion
of s t a i u i n ~in the metachmmatic method for
skeletal muscle fiber typing.
A modification of the histochemical method for skeletal
muscle fiber typing substitutes metachmnmtic dyes for
sulfide (Doriguzzi
Histochem. 79:289-294, 1983).
Further madification by incubating at a lower temperature
preserved differential staining sufficient to identify skeletal
muscle fiber types I, IIA, IIB, and IIC in a single section
(Ogilvie and Feeback, submitted November, 1989). However,
this method requires rapid, carefully timed dehydmtion in
ethanol to preserve the differential staining because the
metachromatic dye used, Toluidine Blue 0, is soluble in
alcohol and acetone. Standardization of the method would be
possible if the dye-tissue complex could be made insoluble in
alcohol6 or acetone. Miiller (Stain Technol. 64:93-96, 1989)
found that treatment of methylene blue stained nerve tissue
with 10%aqueous ammonium molybdate preserved the staining
with alcohol dehydration. The present study investigated
whether metachmmatic dye staining of human, rat, and
mouse skeletal muscle could be made alcohol stable with
ammonium molybdate. Frozen sections were cut at 10 pm and
air dried prior to the histochemical procedures. The trials
consisted of testing 1%,5%,10%,and 20%ammoDium molybdate
in water. Sections were stained by the metachmmatic dye
method for mJrofibrillar ATPase histochemistryas modified bY
Ogilvie and Feeback, rinsed briefly in wnter, and i m m e d
in molybdate for one minute. Treatment with ammonium
molybdate resulted in retention of dye within the muscle
fibers when exposed to ethanol for two hours.
Without
molybdate treatment all dye was extnrcted w i t h i n 2 hours
from sections in 95% ethanol. Sections immersed in a 20%
solution of ammonium molybdate for one minute were superior
to those in 1%.5%or 10%solutions. The density in the four
fiber types was sufficient to allow identification even in black
and white photographs. (Supported in part by NIH grant
#HL 37387-03 and BRSG grant # SO7RR05411-27).
OKO,* Richard, Mireille LIMOGES* and Yves CLERMONT,
Department of Anatomy, McGill University, Montreal,
Canada.
Bioeenesis of uerforatorial uroteins in the
seminiferous epithelium of the rat: a 1iPht and electron
microscooe immunocvtochemical investieation.
The perforatorium is a rigid cytoskeletal element
which underlies the acrosomic system of mammalian
spermatozoa and binds the inner acrosomal membrane to the
nuclear membrane. The rat perforatoriwn is composed of
several polypeptides which can be divided into two groups
on the basis of their distribution. A lower mol. wt.
group (ie. Mr ,< 16,000) is restricted to the thicker
apical part of the perforatorium, while a higher mol. wt.
group (ie. Mr >, 34,000) is distributed throughout the
In this study polyclonal antibodies,
perforatorium.
raised against the entire perforatoriwn and affinity
purified from polypeptides of Mr 16,000 and 34,000. were
used to find the distribution of perforatorial proteins
in germ cells at the various stages of differentiation.
level and
Immunoperoxidase staining at the W
quantitative immunogold labeling at the EM level were
used.
There was a similar distribution pattern of
The
labeling with a l l three antibody preparations.
immunolabeling appeared in early spermatocytes and
increased progressively in both the nucleus and cytoplasm
until step 10 spermatids. Up to this step the labeling
was more intense over the nucleus than over the
cytoplasm. During nuclear condensation in step 11 and 12
spermatids there was a progressive loss of all the
labeling over the nucleus and an increase of labeling
over the cytoplasm.
Thereafter cytoplasmic labeling
progressively increased in the subacrosomal space. In
summary proteins destined to form the perforatorium
appear early during spermatogenesis and are found in
both the nucleus and cytoplasm of spermatocytes and early
In elongating spermatids these proteins
spermatids.
seemingly leave the nucleus and concentrate in the
subacrosomal cytoplasmic space where at the very end of
spermiogenesis they condense to form the perforatorium.
(Supported by the Medical Research Council of Canada).
a-.
S
OLIVER, Mary G. and Robert D. SPECIAN, Department of
Cellular Biology and Anatomy, Louisiana State University Medical
line secretorv Dathwav
mucous granules along the periphery of the apical granule mass.
The factors that affect this phenomenon are unknown. The purpose
of this study was to determine the role that classical second
messengers and the cytoskeleton play in baseline secretion. To
determine the rate of mucous granule translocation under control
conditions, rabbit colonic mucosal explants were pulse-labeled with
H-glucosamine,then maintained on normal culture medium for 1,
75A
ABSTRACTS-AAA 103RD MEETING
2,4, or 6 hours. Maximal granule movement was quantitated by LM
autoradiographic analysis. To determine possible regulation by the
{hosphoinositide siBnaling system, comparisons were made
etween granule mi ration in controls and mucosal explants
exposed t o diacylgtcerols and {horbol esters at var ing
concentrations in the presence and a sence of calcium ionopiore
A23187. No significant acceleration or inhibition of granule
translocation resulted from exposure to any of the agents. To
determine the role of microfilaments in baseline secretion, granule
migration was analyzed in mucosal explants exposed to
cytochalasins and phalloidin. Exposure to halloidin results in no
significant alteration in granule transtcation; exposure to
cytochalasin D and dihydrocytochalasin B results in significant
acceleration in granule migration to the cell surface. To determine
the role of microtubules, granule migration was analyzed in
mucosal explants exposed to nocodazole or taxol prior to or
subsequent to pulse-labeling. Exposure to nocodazole after
pulse-label results in significant inhibition of granule translocation;
exposure to taxol has no significant effect. Pretreatment of explants
with nocodazole results in virtual1 total cessation of granule
movement and retention of radiolabeikd mucins in the Golgi region
of the cell; pretreatment with Tax01 impedes the movement of
radiolabeled mucins from the Golgi region to the apical granule
mass, but upon reaching the apical granule mass the rate of granule
translocation is the same as controls. These data suggest
that the baseline secretory pathway in mature goblet cells from
rabbit olon is constitutive rather than regulated. Supported by
Grant DK 33720 from the National Institutes of Health.
6
S
OSBORNE,. John G. and Kurl F. HAUSER, Department of Anatomy and
Neurobiology, University of Kentucky School of Medicine, Lexington, Kentucky.
(Sponsored by Harold H. Traurig) Evidence for onioid expression b
proliferatine neural cells in vim: 13HI-Thvmidine incorooration bv enkeohali:
immunoreactive cells In oreanotwie cultures of rat cerebellum.
The neuronal progenitors of the rat cerebellar external granule layer (EGL)
are reported to transiently express enkephalin immunoreactivity during
development in vivo (Zagon et al., Science, 227 1049, 1985). To better understand
the role of endogenous opioids and the mechanisms governing transient opioid
expression in developing neural cells, enkephalin immunoreactivity was examined
in organotypic cultures of neonatal rat cerebellum. Cerebella from male, SpragueDawley rats were isolated from the brainstem, dissected into 12 explants per
cerebellum, and maintained in serum-containing nutrient medium for 10 days on
ACLAR plastic coverslips in Maximow double-coverslip assemblies or in 12-well
(22 mm diameter) tissue culture chambers. Explants were treated with 0.25
pCi/ml of [3H]-thymidine for 16 h prior to harvesting, fixed in 4%
paraformaldehyde, cryoprotected, and frozen-sectioned
10 pm thick).
Immunocytochemistry was performed with antisera against [met$snkephalin (gift
from Dr. Bruce E. Maley), which has limited cross reactivity to [leu5]-enkephalin,
using diaminobenzedine as a chromogen. For subsequent autoradiography,
immunostained sections were coated with NTB-2 emulsion and exposed for 4
weeks at 4%
Based on perikaryal size, morphology, and distribution, a
subpopulation of enkephalin immunoreactive cells could be identified as EGL
cells (or their progeny). These cells were undifferentiated (lacking neuritic
processes) and most often did not incorporate [3H] thymidine; however, some
displayed both enkephalin IR and [3H]-thymidme labeling in the same cell. The
relative immaturity of the enkephalin immunoreactive cells indicates that
enkephalin is expressed during the earliest stages of cellular differentiation. In
addition, the close temporal proximity (< 16 h) between [3H]-thymidine treatment
and time of fuation suggests that enkephalin immunoreactivity can be expressed
during cell division. Thus, the presence of enkephalii immunoreactivity appears
to be an intrinsic property of some populations of immature neural cells even
when they are isolated in culture. Although endogenous opioids are involved in
numerous neuromodulatory and neuroendocrine functions, recent reports that
opioids directly influence the growth of mammalian neural cells in primary culture
(Stiene-Martin and Hauscr, Life Sci., 46: 91, 1990) indicate that the increased
expression of opioid peptides by developing neural cells may be important in the
regulation of nervous system maturation. (Supported by NIH RR-05374, and PSP
and UKMC Research Funds from the University of Kentucky).
*-
OSMXD, Dennis 0.. J d e Tww,*
Raffi
mren JXXXSEN,* Dqprhent of AMtpny, m i l l
and
University,
Montreal, canada. biiiQpemrirormental studies of the
proliferation and deletion of B ~ Z O + B lwphocvte
prosenitor cells in mouse bone mat-rw.
Tha B220 glycaprotain &Ara&en' Z e S the B 1ynpaoCYte
lineage in mice.
It is expressecl both on precursor B
cells, includixq pro-B cells before the expression of p
hchains and p-B
cells having c y t 4 p l d C p thaw,
as well as on matura B lymphocytas. We have localized
B220'
cells in the bone m a r z ~ wOf 3-5 wlr old mice by the
-b
of 1251-labaled IIIO&&
antibcdy (mAb) 14.8
administered iv and detected by light and electron microscope radioautography of fenoral diaphyseal sections. m
define precursor B cell stages in situ we have examind
B220+ cells in 1) mutant mice with severe Cunbined
irmamodeficiency (Scm) in which all differentiating B
precursors abort at the late pro-B cell stage 2) mti-IFpr
treated mice in which all newly-fonned B lymphocytas are
deleted 3) mice undergoing regeneration after 8 irradiation (150r) 4) mice treated w i t h vincristine to
cause dividing cells to ammulate in metaphase.
Marphanetric analysis of entire sections revealed that the
incidences of labeld B220+ precursor cells, including
mitotic folms, were higheat in patchy areas in peripheral
rqions of the marrow. The earliest B precursor cells vmre
often located w i t h i n the bonelining cell layers
suplolllded by a distinctive electron-dense matrix. mny
~ 2 2 0 + cells were associated w i t h strcmal retic~larcells
and macrophages. The latter assxdations were especially
frequent in Scm mice and anti-IFpr treated mice, while in
nomice lateled N Z O +material appeared w i i t a i n macmphages at time intervals (10-20m) after 1 2 5 1 - 1 ~14.8
administration. The results suggest that proliferatiq
early precursor B cells in mou58 bone plEvlcRT 7
microemn
'nmJnents near the
*
-, possibly
influenced by the local reticular cells and extramllular
matrix, and that defective precursor cells a r i s i q in both
noxmal and aysresulated B cell genesis are deleted by
macmphages (supported by the Madical Reseatch council of
Canada).
OKERS, Noel O., and Robert J. DeLORENZO*, Department
of Anatomy, Department of Neurology, Medical College
of Virqinia, Virginia Cornonwealth University,
Richmond; Virginia. Dimensions and variations of the
internal carotid arter
In the maioritv of 'cases of thromboembolic stroke.
the middle ceredral artery is involved. The site
where the thrombus is lodped, in many cases, is in the
carotid siphon or the proximal end of the middle
cerebral artery before it gives off its numerous
branches to the brain. Removal of the thrombus could
be accomplished by the placement of a thrombolytic
agent at the site of the c l o t via a specially
developed catheter, the size and length of which would
be limited by the diameters and lengths of the vessels
it has to traverse. Measurements were made on the
cervical, petrous, cavernous and cerebral parts of the
internal carotid artery (I.C.A.) using about 100 half
heads. The cervical part was 7 cm long (range 5.3 to
8 cm) and the internal diameter (I.D.) at its proximal
end was 4 mm (range 3 . 2 to 6 rn). The petrous and
cavernous parts together were 7 cm long (range 6 to 8
cm). The cerebral part (middle cerebral artery) up to
the posterior end of the lateral fissure measured 10
cm (range 9 to 11 cm) and its proximal I.D. was 3 m
(range 2 to 4 mn). The length and diameter of the
catheter to be used for depositina a thrombolytic
agent at the site of the clot will therefore be
limited by the measurements of the I.C.A. as given
above. Variations of the I.C.A. are found mainly in
the cavernous part, where one to five half circle
I n several
loops are found (carotid siphon).
specimens, the walls of the cerebral part were
.
76A
ABSTRACTS-AAA 103RD MEETING
unusually t,hin, b u t no rupture was observed.
instance, the cervical part had atrophied.
In one
Supported by University Grant- i n-Aid.
PADYKULA. Helen A. and Ita R. Kaiserman-Abramof,
Department o f Cell Biology, University of Massachusetts
Medical School, and Case Western University School of
Medicine, Cleveland, Ohio. Cellular analvsis of the
primate menstrual cvcle: Menses. The close o f the
menstrual cycle is marked by a complex combination of
catabolic and anabolic events in the endometrium. The
stimulus for regression is the waning of the
progesterone surge. During menses, estrogenic action is
on the endometrium at the basal E2 level (70-100pg/ml).
Endometrial biopsies were obtained by hysterotomy for
the 4 days of menses. Serum levels o f E2 and P4 were
measured. An intravenous injection (3H)TdR was made at
1 hour prior to hysterotomy to label stem-progenitor
cells. The nomenclature of primate endometrial zonation
is used as described by Padykula et al., 1984. On DAY
1,the endometrium is composed of a disintegrating
functionalis with dissolution o f the stromal compartment
whereas the basalis is anabolic. Glycogen occurs as
rich deposits of stored energy i n basalis gland cells.
The continued presence of leucocytic infiltration
indicates that the clean-up operation in the
functionalis continues. The glandular cells do not
incorporate (3H)TdR. but subjacent fibroblasts do.
W . The luminal epithelium and underlying stroma is
removed. Functionalis I1 glands undergo changes in
conformation. Basalis I11 epithelium stores glycogen
whereas Basalis IV lacks glycogen. U. Glandular
zones 11, 111, and IV are present with zonal structural
differences. W. The primordia of functionalis I
and I1 have reappeared. The luminal epithelium consists
of irregularly shaped cells low in glycogen content. In
the basalis, glycogen storage is greater in Zone I11
than IV. A s the cycle closes, the serum E2 level
remains at the basal level. The postmenstrual
endometrium is stripped of transient epithelia1 cells
whereas the germinal cells of basalis I11 and IV possess
the potential for reconstruction o f the next
functionalis. The onset o f a new ovarian dominant
follicle secretes estradiol that will reconstruct the
endometrium o f the next cycle, from day 4 onward
(Supported by NICHD Program Project HD20290-05).
PAPKA, R.E.,
Department of Anatomical Sciences,
University of Oklahoma, Oklahoma City, Oklahoma.
A
The pelvic paracervical autonomic ganglia (PG) of
female rats were studied for the presence of
subpopulations of synaptic terminals that could be
derived from sensory nerve fibers. Immunohistochemical
staining for the synaptic-terminal protein, synapsin I
was used to identify synaptic endings. Antiserum against
the neuropeptide calcitonin gene-related peptide (CGRP)
was used as a marker for a subpopulation of sensory
nerves. Immunohistochemical double labeling was used to
examine the existence of CGRP in synapsin Iimmunoreactive synaptic terminals. The uterine cervix
was also examined for the coexistence of CGRPimmunoreactivity in synapsin I-immunoreactive nerve
fiber varicosities. Synapsin I-immunoreactivity was
present as abundant punctate, granules around principal
neurons in the PG.
These terminal-like structures
appeared predominantly associated with, and encircling,
neuron somata; thus it is presumed that they represent
primarily axosomatic synapses, or axodendritic terminals
at the base of dendrites. Synapsin I-immunoreactivity
was also present in nerve fibers of the myometrium,
vasculature and epithelium of the cervix.
CGRPIimmunoreactivity
coexisted
with
synapsin
immunoreactivity in subpopulations of the terminals in
the PG and nerves in the cervix. It is suggested that
synapsin I is a useful immunohistochemical marker of the
synaptic terminal population in this ganglion and that
immunostaining f o r CGRP used in conjunction with
immunostaining for synapsin I labels a subpopulation of
synaptic terminals. On the basis of pervious studies in
our laboratory, these CGRP-immunoreactive terminals in
the PG, and uterine cervix, appear to be sensory fibers
and originate from dorsal root ganglia. (Supported by
NIH 1 R01 NS22526, Presbyterian Health Pdn. and OU
Medical Alumni Grant).
PAPPAS, G.D., Jacqueline SAGEN, and John D. ORTEGA', Dept.
of Anatomy and Cell Biology, Univ. of Illinois at Chicago,
Chicago, Illinois. Xenoaraft-hostrelationshiDs in rat CNS
pain modulatory reaions.
The transplantation of chromaffin cells from the
adrenal medulla into pain modulatory regions of the CNS
(periaqueductal gray and spinal cord) has been shown
previously to reduce pain sensitivity, most likely via
local release of neuroactive substances from the
transplanted cells.
The ready availability of bovine
adrenal glands, as well as the high levels of opioid
peptides produced by their chromaffin cells, make these
glands a potentially valuable donor source for
antinociception studies. However, the success of these
xenografts depends on their ability to survive and
integrate within the host CNS. The aim of the present
study was to assess host-graft relationships of bovine
chromaffin cells transplanted to the rat CNS. We have
found that isolated bovine chromaffin cells survive for at
least three months in the rat periaqueductal gray, with no
evidence of immunologic response following a short-term
course of immunosuppressant treatment. In the early stages
following transplantation, only minor pathology is found at
the injection site, which apparently recovers completely at
later stages.
The host-graft borders are not well
demarcated, in contrast to solid tissue grafts. Neuronal
processes of host origin that form numerous synapses
contacts with the transplanted bovine chromaffin cells are
apparent by three weeks following transplantation.
Migration also occurs from the graft into the host
parenchyma, as evidenced by individual chromaffin cells
found in the neuropil and near host parenchyma1 blood
vessels. The clusters of chromaffin cells found in the
graft itself are generally not very vascular, in contrast
to solid tissue grafts. The chromaffin cell clusters are
surrounded by blood vessels of the non-fenestrated CNS type
at the host graft border. It is likely that the small size
of the graft does not require extensive angiogenesis. The
lack of fenestrated peripheral type endothelial capillaries
normally seen in the adrenal medullary tissue graft may
contribute to the survival of these xenografts in the rat
brain.(Supported by NIH Grants GM37326 and NS25054)
PARKINSON. Dwight. Department of Anatomy, Faculty of
Medicine, University o f Manitoba, Winnipeg, Manitoba.
Sub-Arachnoi d Seota and Trabeculae.
This study was undertaken because o f the confusion
arising from the diversity of names, descriptions, and
drawings of the spinal subarachnoid septae and trabeculae in the standard texts and dictionaries, even
from page to page in the same texts. Forty-two
autopsy cords from 30 weeks gestation to age 60 with
ABSTRACTSAAA 103RD MEETING
equal sex d i s t r i b u t i o n were examined.
We found t h e
a n t e r i o r aspect i s v i r t u a l l y f r e e o f any attachments
between t h e c o r d s u r f a c e and t h e surrounding arachnoid
sheath w h i l e p o s t e r i o r l y i n t h e c e r v i c a l r e g i o n t h e r e
i s a s c a n t y p a r a - s a g i t t a l s e r i e s o f connecting f i b e r s
and f e n e s t r a t e d membranes which become p r o g r e s s i v e l y
more e x t e n s i v e i n t h e l o w e r c e r v i c a l cord, remain so
throughout t o t h e
lumbar enlargement where t h e y
dwindle then end a b r u p t l y a t t h e f i l u m t o c o n t i n u e
w i t h a few s l e n d e r s t r a n d s i n t h e cauda t o i t s t e r m i n ation.
V a r i a t i o n s i n t h e s i z e and arrangement o f
c o n s t i t u e n t f i b e r s o f v a r i o u s s t r u c t u r e s i s noted.
There was no sex d i f f e r e n c e noted.
I n infancy there
i s no s e p a r a t i o n between t h e meninges u n t i l about 20
weeks.
From then u n t i l s h o r t l y a f t e r b i r t h t h e r e i s
poor s e p a r a t i o n o f t h e arachnoid from t h e o v e r l y i n g
dura.
k l i t h i n c r e a s i n g age t h e t i s s u e s i n c r e a s e i n
t h i c k n e s s b u t n o t a p p a r e n t l y i n number o f connections.
There were
numerous
unexplained
"Rogue"
fibers.
The f i n d i n g s a r e s u b s t a n t i a t e d w i t h numerous
d i s s e c t i n g scope photographs.
S
PARLOW.* Mary H., David L. BOLENDER and John LOUGH,
Department o f Anatomy and Cellular Biology, Medical College
o f Wisconsin, Milwaukee, Wisconsin. Localization of bFGF in
the mvocardium o f the earlv embrvo.
The heart is unique among developing organs, i n that i t
must begin functioning soon after the intitation o f
cytodifferentiation.
Recent strdies on the effects o f growth
factors in development have led us to hypothesize that the
differentiation and maturation o f the myocardium may be
enhanced by endogenous growth factors. such as basic
fibroblast growth factor (bFGF). For example, bFGF has been
indentified in cardiac muscle cells of H&H stage 12-29
chicken embryos (Joseph-Silverstein, et al.. J. Cell Biol.
108:2459,'89).
However, its presence in the early stages o f
cardiac morphogenesis has not been investigated.
In this
study, we have mapped the appearance o f bFGF between
stages 4-16 o f chicken embryogenesis.
Tissue sections from
cryopreserved embryos were reacted with polyclonal
antibody prepared against recombinant human bFGF,
followed by indirect localization using fluoroscein-labeled
secondary antibody. As early as stage 9 , bFGF was observcd to
be restricted to cells of the developing myocardium, as
evidenced by i t s co-localization with myosin heavy-chain.
Within the myocardial cells, the bFGF staining pattern was
punctate to globular suggestive o f packaging in secretory
vesicles. In addition to its intracellular localization. bFGF was
also detected at stage 15 in the adjacent myocardial basement
membrane as a dispersed particulate. Staining within the
myocardial basement membrane declined in intensity near
the cndothelium.
These results suggest paracrine and/or
autocrine roles for bFGF during cardiac morphogenesis.
Supported by NIH grant HL 39829.
PARSONS, Jonathan A, and Stephen FALCONER*, Department of Cell
Biology and Neuroanatomy, University of Minnesota, Minneapolis,
Minnesota. "Mac-cross sect ional- anatomv"- A comDuter a s s i w
instruction wroaram.
To facilitate learning of anatomical relationships evident in cross
sections (CX) of the body and to encourage use of computer-aided
medical self-instruction programs at an early stage in their program,
an interactive computer assisted instruction (CAI) program has been
written to accompany our gross anatomy course. The program uses both
CT scans of body regions and atlas drawings of extremities CX. The CA1
was developed for Macintosh computers using Supercard. 32 cross
sections of body regions (extremities, 11; head and neck, 8; torso, 13)
were digitized (Hewlett Packard ScanJet Plus) in CT scan orientation,
pixel painted, and prominent features designated by numbered leaders.
A set of menus was developed which allows users to reveal or hide
77A
structure identifications, obtain specific CX information or select
"Testing" or "Help" modes. In the primary learning mode, a user can
obtain an anatomical, embryological or functional "hint" for each
enumerated structure by "clicking" its button. With appropriate key
strokes, the identity of each structure is revealed.and highlighted. In
the evaluation mode, the user may select either an "Identification" or a
"Questions and Answers" test. which appears in a field adjacent to the
CX. Identification tests require typing of responses ; spelling is not a
consideration as an "Answers" field appears after completion of the test.
Questions and answers tests consist of 3 to 5 functionally or embryologically oriented questions per CX. In this mode, the user may have a
hint shown, try several responses or have the correct answer
displayed: both numerically and by structure highlighting. To date,
only the extremities unit of this CA1 has been tested by student use.
Judged by checkout records and questionaires, reception by 1st year
medical and dental students has been good. Both increased appreciation
of anatomical relationships and new interest in the use of CIA programs
have been reported outcomes. Although still in its formative stages,
this CX-anatomy CA1 has gained acceptance by our students as a useful
adjunct in learning anatomical relationships - knowledge which is
becoming ever more important with the advent and increasing use of
new imaging modalities. (Extremities CX reproduced with permission
from "Anatomy: Atlas of Topographical and Applied Human Anatomy" by
Eduardo Pernkopf, 3rd English Edition. 1989, Urban & Schwarzenberg,
Baltimore-Munich. Project supported in part by a Health Science
Learning Resources Instructional Grant, University of Minnesota).
S
PATESTAS,' Maria, James L. H I A l l , Leslie P. GARTNER, and D.
Vincent PROVENZA. Department of Anatomy, Dental School, University
of Maryland at Baltimore, Maryland. (Sponsored by Leslie P. Gartner).
Palatwenesis in the trisomv 16 mouse.
Based on the genetic homology between murine chromosome 16 and
human chromosome 21, experimentally induced murine trisomy 16 (Ts
16) has been considered to serve as a suitable experimental model of
Down's syndrome. Ts 16 fetuses were obtained following two consecutive
breedings utilizing Rb(16.17j78nr and Rb(6.16)24.Lub mice (procured
from Jackson Laboratories) to produce progeny doubly heterozygous for
chromosome 16. F, males thus derived were mated with karyotypically
normal C57BU6.J females to generate trisomy 16 offsprings. Ts 16
fetuses and their normal littermates were extirpated via laparotomy on
days 14 to 18 of gestation. All fetuses were karyotyped by obtaining a
biopsy of liver tissue to determine their chromosomal complement. Fetal
heads were prepared for routine histology to obtain 6um thick corona1
sections. Alternate slides were stained with H&E, PAS, or trichrome.
Palatogenesis in Ts 16 mice was compared with palate development of
normal littermates from the 14th to the 18th days of gestation to
determine if an extra chromosome 16 contributes to the incidence of
morphologicalvariations in fetal palatal tissues. Secondary palatal closure
in normal fetuses is completed between the 14th and 15th gestational
days. No clefting was noted in any of the normal fetuses. However, 80%
of the trisomic fetuses (15 to 18 days) displayed variable degrees of
palatal clefling. Although palate development in trisomic fetuses without
clefting closely paralleled that of controls, a developmental delay of at
least one day was noted. Futthermore, an abnormally shaped tongue
might have contributed to palatal clefting by interfering with normal shelf
elevation. The intermaxillary segment and nasal septum of clefted
trisomics was hypoplastic and grossly misshapen. Preliminary results
appear to indicate that genetic imbalance resulting from an extra
chromosome 16 may indirectly interfere with palatogenesis by stunting
shelf growth andlor delaying reorientation and midline fusion by at least
one day.
PATRICKSON*, John W., Thornas E. SMITH* and Shan-Shan
ZHOU*, Department of Anatomy, Lama Linda University,
Lorna Linda, California.
The central afferent and
efferent comDonents of the swerior and recurrent
larvnseal nerve.
78A
ABSTRACTS-AAA 103RD MEETING
The larynx reflexively protects the pulmonary airway
against the aspiration of foreign materials during
deglutition, phonation and respiration. The reflexes
are believed to be mediated via the superior (SLN) and
recurrent laryngeal nerves (RLN). Sprague-Dawley rats
(250-310g; n-34) were anesthetized with chloral hydrate
(0.5 g/kg). The superior and recurrent laryngeal nerves
were cut proximal to the larynx and placed in sylastic
tubing containing 10% WGA-HRP in saline and sealed with
non-toxic dental impression material (Coltene). Fortyeighthours post-application the animal was perfused and
30 um frozen sections in the coronal or horizontal plane
were made and processed for peroxidase. SLN: Labelled
terminals were found bilaterally in the interstitial and
the dorsomedial pole of the medial subnuclei of nucleus
tractus solitarius (NTS), the ipsilateral being more
dense.
Labelled perikarya were distributed ipsilaterally in the rostral ventrolateral subdivision of
nucleus ambiguus (NA) and in the dorsal motonucleus of
vagus (DMX).
RLN: Sparsely labelled terminals were
located ipsilaterally in the interstitial and the
dorsomedial subnuclei of NTS. Two distinct populations
of labelled motoneurons were found to exist within NA;
a rostral as those in SLN, and a caudal at the level of
obex.
S
PATTEN,* R.M., and W.K. OVALLE, Department of Anatomy,
University of British Columbia, Vancouver, Canada.
Three dimensional moroholoav of intact muscle soindles
isolated from the hamster tenuissimus.
The tenuissimus is a long, thin hindlimb skeletal
muscle which in hamsters contains about 200 extrafusal
muscle fibers. Embedded in this richly innervated muscle
is a continuous array of 16-20 closely packed muscle
spindles making it ideal for the isolation and harvesting
of these sensory receptors. In this correlative light
and electron microscopic study, freshly frozen or epon
embedded specimens were first prepared for serial microscopic analysis. Camera lucida reconstruction of spindle
distribution showed a close proximity to the main arterial and nervous supply in the cental core o f the muscle.
Oxidative enzyme and myosin ATPase staining varied along
the lengths of the three intrafusal fiber types. For
scanning electron microscopy, isolated spindles were
first fixed in 2.5% buffered glutaraldehyde, followed by
1% osmication, and mechanical disruption of the outer
capsule under the dissecting microscope. Some spindles
were treated with EN HCl at 60oC to more clearly expose
the outer surfaces of the intrafusal fibers and associated nerve endings. In these preparations, a highly
registered sarcomere banding pattern was visualized in
subsarcolemmal areas with coiled sensory-nerve fibers
intimately attached to their outer surfaces. Punctate
neural expansions adhered to selected regions along the
equatorial length o f the intrafusal fibers. By transmission electron microscopy, these expansions appeared
crescent-shaped, and were enveloped by external lamina.
Each profile contained a plethora of mitochondria and
cytoskeletal organelles. Elements of the capsular sleeve
showed a multilayered pattern, enveloping groups o f
intrafusal fibers for varying distances. Myosatellite
cells were also encountered on the surfaces of nuclear
bag and nuclear chain fibers and each was, in turn,
invested by a delicate connective tissue coat. The
methodology used in this study provides a novel view o f
the exquisite three dimensional architecture of this
neuromuscular receptor. (Supported by grants from NSERC
and the Muscular Dystrophy Association of Canada)
PAULSEN, Douglas F. and Lei PANG*, Department of Anatomy,
Morehouse School of Medicine, Atlanta, Georgia.
Position-relatedeffects of retinoic acid ( R A ) on chick
limb-bud chondrogenesis in serum-free microculture.
An anteroposterior (AP) RA gradient appears to control
AP limb-skeletal polarity via concentration- (and thus
position-) related effects on mesenchymal cell growth and
differentiation. Yet regional differences in how cells
respond to RA may also contribute to skeletal patterning.
The present study extends to the AP axis earlier in vitro
analyses of proximodistal (PD) differences in chick limb
mesenchyme responses to RA. Stage 21-22 distal (250um tip)
and proximal wing-bud regions, and stage 23-24 distal,
proximal (with precartilage condensations) and intervening
(subdistal) wing-bud regions, were divided into anterior
and posterior halves. High-density, 4-day cultures of
cells from each region were grown in defined medium containing 0, 5 , or 50 ng of RA/ml. These were analyzed for
accumulated Alcian-blue stainable matrix or DNA to measure
chondrogenesis or proliferation respectively. At 5 ng/ml,
RA enhanced chondrogenesis in anterior and posterior distal
cultures from both stages and in stage 23-24 subdistal cultures. Subdistal cultures showed greater chondrogenic enhancement by RA than did distal cultures. Stage 23-24
posterior distal and subdistal cultures showed greater
chondrogenic enhancement than did the corresponding anterior cultures. No AP differences in RA effects were seen
in stage 21-22 distal cultures. RA exposure enhanced DNA
accumulation in stage 23-24 distal and subdistal, but had
little effect in proximal cultures; no significant AP differences in total DNA were seen. DNA analyses of stage 2122 cultures are underway. The results confirm previously
demonstrated proximodistal differences in RA effects on
limb chondrogenesis, which may be related to the proximalto- distal progression of differentiation. Since AP differences in RA effects on chondrogenesis do not appear in
distal and subdistal mesenchyme until stage 23-24, these
differences may be induced by a period of in vivo exposure
to the anteroposterior RA gradient, which is known to be
present by stage 21. Supported by NIH/MBRS RR08248
PAWLINA, Wojciech, Lynn H. IARKIN, Susan OGILVIE' and
Susan C. FROST', Department of Anatomy and Cell Biology
and Department of Biochemistry and Molecular Biology,
University of Florida, Gainesville, Florida. Effect of svnthetic
human relaxin on proliferation and differentiation of 3T3-Ll
preadiDocvte5.
Relaxin is a hormone primarily associated with the events
of parturition and has been shown to be a member of the
insulin-like growth-peptide family. Using the differentiating
3T3-Ll mouse cell line, we have reported that porcine relaxin
interferes with the proliferative but not the adipocyte
conversion response to inducing agents. This results in cells
which are nearly twice the size of normal 3T3-LI adipocytes
at maturation, an observation noted in mammary adipocytes
when treated with relaxin, in vivo. Recently, synthetic human
relaxin has been provided to us by GenentechJnc., San
Francisco, CA. We undertook the present study to compare
the biological effects of human relaxin with that of porcine
relaxin. Our results show that human relaxin was an order of
magnitude more effective than porcine relaxin in blocking the
proliferative response of 3T3-Ll preadipocytes but had no
effect on differentiationbased on accumulation of lipid and the
ability of insulin to stimulate glucose transport in mature cells.
As with porcine relaxin, treatment with human relaxin during
differentiation resulted in larger cells. The effects of relaxin
were negated by treatment with specific antibodies against
both relaxins. These studies indicate that synthetic human
relaxin is more potent than porcine relaxin and has effects that
are similar to porcine relaxin. Also, some of the effects of
relaxin noted with 3T3-Ll cells are similar to those reported in
vivo. This work was supported in part by a grant provided by
the Division of Sponsored Research, University of Florida and
NIH Grant DK-39135 (to S.C.F.).
ABSTRACTS-AAA 103RD MEETING
79A
S
PENDINO', Kimberly J., Richard R. SCHMID'P, Pierette SHIPMANAPPASAMY', and Kenneth P. CHEPENIK, Department of Anatomy,
lefferson Medical College and Department of Pathology and Laboratory
Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
Localization of cvclooxveenase within the me- and uostnatal murine
m.
Previous work in our laboratory has implicated arachidonic acid
metabolites, particularly the prostaglandins, as modulators of thymocyte
proliferation/differentiation within the organ-cultured fetal thymus.
Further, eicosanoid profiles (thin layer radiochromatography) for murine
thymic lobes obtained on several days of p r e and postnatal development
show distinct temporal patterns which may have developmental
significance. Current studies focus on determining the cell type(s)
responsible for eicosanoid synthesis within the fetal murine thymus.
Immunohistochemistry has been utilized to localize cyclooxygenase. the
enzyme which converts arachidonic acid to the prostaglandin
endoperoxide derivatives, within the thymus. Briefly, embryonic day 14
and day I8 embryos, and postnatal day 8 and 7 week thymic lobes were
embedded in paraffin, sectioned, and deparaffinized prior to staining.
Sections were incubated overnight in humidified chambers with rabbit
anti-cyclooxygenase (1:loOO). Following three 10-minute washes in PBS,
sections were incubated with goat anti-rabbit IgG for 60 minutes (1:60),
washed and incubated for an additional 60 minutes with rabbit peroxidase
anti-peroxidase solution (1:50). Finally, slides were washed and
developed with a 0.05% solution of 3,3'-diaminobenzidine (DAB)
containing 0.01% hydrogen peroxide. Controls received a solution of
authentic cyclooxygenase adsorbed with the primary antibody or two of
the above reagents plus DAB, in different combinations. Cells of ED18,
PND8, and 7 week thymic lobes treated with rabbit anti-cyclooxygenase
exhibited differential positivity compared to control lobes. Cells of ED14
liver and notochord, ED18 submandibular gland and choroid plexus, and
adult lung also stained positively at a dilution of 1:loOO. Furthermore,
staining in thymic tissue appeared to be localized to the medullary region,
more specifically, to the larger, nonlynrphoid thymic cells. Our findings
demonstrate that thymic tissue obtained at various developmental stages
expresses cyclooxygenase. Further studies will involve double-labeling of
cells within the thymus using anti-cyclooxygenase and antibodies to cell
surface antigens to determine the exact cell type(s) capable of
metabolizing arachidonic acid.
PENG, Isaac and Donald A. FISCHMAN, Department of
Anatomy, UMDNJ-RWJMS Piscataway, New Jersey and
Department of Cell Biology and Anatomy, Cornell
The
Univ. Med. Coll., New York, New York
insertion of newly synthesized actin into
mvofibrils
The constant replacement of proteins is a
feature of all intracellular structure. For
myofibrils the process of protein removal and
insertion must occur in the context of pngoing
contractile function. This process is not
understood. We have examined entry and release of
newly synthesized proteins from myofibrils using
an in vitro system (Bouche et al., JCB 107:587).
Proteins are translated in a reticulocyte lysate
in the presence of 35Smet. The newly synthesized,
proteins are incubated with myofibrils. The
myofibrils are then harvested by centrifugation,
and analyzed by SDS PAGE and fluorography. Using
this system, we have shown that radiolabeled
actin which has incorporated into myofibrils can
be released into solution when incubated with a
reticulocyte lysate but not when incubated in a
physiological
salt
solution
(LSB).
When
myofibrils are incubated in reticulocyte lysate
and assayed for released actin, approximately 3%
of myofibrillar actin is released within 1 h at
4 ' ~ . In contrast, little if any release 1s
detectable when myofibrils are incubated in LSB.
When, fluorescein-labeled actin (FITC-actin) is
incubated with myofibrils in a reticulocyte
lysate, strong fluorescence is observed in the Zand I-bands. No incorporation into 2 - and Ibands is seen in LSB. When ATP, but not ADP, AMP,
GTP or AMP-PNP, is added to LSB, FITC-actin
incorporation into 2- and I-bands is identical to
that seen in the reticulocyte lysate. All of
these observations are consistent with an ATPdependant process for the incorporation and
release of actin from myofibrils (Supported by
NIH AR32147 and MDA) .
PENNYCUICK,* Colin James, Department. of Biology,
University of Miami, Coral Gables, Florida.
es ands-urts
in t.he w i n e
inn v e r t e w .
The wings of flying vertebrates are slender
structures that have to carry large bending and
torsional loads. In bats and pterosaurs the
structure is hanglider-like, with a flexible
membrane (basically collagen) stretched between
stiff bony supports. Bird wings are pure
cantilever structures, made of bone proximally
and keratin distally. The flight performance of
the animals is ultimately limited by the wing
geometry that is possible with these two
fundamentally different types of structure. The
geometry is itself limited by the properties of
the structural materials.
PBRSAUD, T.V.N., J.E. BRUNI and G. FROESE,*p'Department of Anatomy
and Manitoba Cancer Foundation'. University of Manitoba. Winnipeg,
Manitoba. Canada. The effects of low doses of ionizing radiation on the
develoument of the rat CNS.
Exposure of pregnant animals to ionizing radiation causes resorption,
retardation and developmental defects in offspring. There is little
information, however, on the effects of low levels of x-irradiation on early
CNS morphogenesis. Pregnant S/D rats were exposed to 50 rads on
embryonic day 9.5 and along with controls were killed at post-irradiation
intervals of 4, 48 hrs, and 10 days. No statistically significant differences
were observed in the number of embryos per litter, and in crown-rump or
head length of embryos born to controls and irradiated dams killed 4 and
48 hrs post-irradiation. Although gross morphological abnormalities were
observed in both controls and irradiated embryos killed 48 hrs
post-irradiation, the malformations appeared not to be treatment-induced.
The mean developmental stage attained by controls and irradiated embryos
was not different for any of the parameters selected to assess embryonic
development. Controls, however, showed a greater range in the extent of
development of the forebrain, olfactory system, midbrain, hindbrain, and
caudal neural tube. In all cases, development of these regions was slower
in controls than their irradiated counterparts. On day 9.5 of development
the embryonic nervous system consisted of a single pseudostratified layer
of mitotic and intermitotic columnar neuroepithelial cells. In irradiated
embryos, mitoses were reduced within the neuroepithelium and evidence
of pyknosis and some necrosis was seen a t 4hrs post-irradiation. By 48 hrs
post-irradiation, however, the nervous system of irradiated embryos did not
differ significantly from controls. Neuropathological changes such as
abnormal structure, ectopic growth, retarded development, arrest of
proliferation and/or migration of cells were not seen. In animals killed at
term, 151 live fetuses were born to 12 controls and 206 to 15 irradiated
animals, resulting in a mean of 12.6 and 13.7 per litter respectively. A
higher incidence of developmental defects were observed in irradiated than
control fetuses a t term. The most common malformations were defects of
the eye and of spinal curvature.
(Supported by Unsolicited Proposals Fund, Supply and Services, DSS;
Atomic Energy Control Board; Health and Welfare - Canada)
PETRAS,, J.M., and S.M. ANDERSON*, Department of Medical
Neurosciences, Division of Neuropsychiatry, Walter Reed Army
80A
ABSTRACTS-AAA 103RD MEETING
..
Institute of Research. Washineton
D.C. (Soonsored bv J.
v
Astruc) Hereditary cerebellar ataxia: neuroanatomical and
clinical findings in the rat.
Cerebellar pathology and malformations characterize a
neurological mutation occurring in a subset (NHS/Nts) of the
N/Nih heterogenous rat stock. Breeding data are consistent
with the mode of transmission for a recessive trait expressed by
a single autosomal gene. Trunkal ataxia, intention tremors,
falling and lateropulsion are severe in affected rats. After
falling, recovery is difficulty. Abduction of all limbs results in a
broad based stance during standing and locomotion.
With
significant abduction of the limbs a t the shoulder, elbow, knee
and hip joints, the pelvis and abdomen may rest on the ground
and a hopping locomotion ensues as the impairments progress
with age.
Videographic analysis supplemented direct
observations. Clinically affected rats have lower body and brain
weights. Brain weights are 86%, and body weights are 62% of
their matched clinically unaffected littermates. Affected rats
live shorter lives (mean of I I mo). Cerebellar hypoplasia is not
present. Microscopically, the cerebellar cortex is thinner. Axon
degeneration (Nauta) when present is sparse in the cerebellar
cortex and the white matter of the folia. There is a marked loss
or degeneration of Purkinje neurons, and loss of granule cells.
Among surviving Purkinje cells, there appears t o be evidence
(Golgi-Cox) of forshortened dendrites, and ectopically located
neurons in the granule cell layer in affected rats. In genetically
heterozygous matched littermates without clinical impairments,
Purkinje neuron pathology is present although sparse relative to
homozygous affected littermates. Axon degeneration in the
forebrain is present dorsolaterally in the convexity of the
frontal cortex, the fimbria and columns of the fornix, the
hippocampal formation, and the septum. These mutants may
provide a model for studying hereditary cerebellar degenerative
disease.
I n t e r s t i t i a l c e l l s of Leydig c h a r a c t e r i s t i c a l l y occur
in c l u s t e r s . Often these clusters remain i n t a c t when
i n t e r s t i t i a l tissues a r e mechanically separated from
other components of t h e t e s t i s . T h e presence of strong
i n t e r c e l l u l a r attachments i s most l i k e l y one of the
factors responsible f o r maintaining the i n t e g r i t y of
Leydig cell c l u s t e r s . In t h i s study we present evidence
t h a t a c t i n associated adhesion junctions are present a t
s i t e s of i n t e r c e l l u l a r attachment between ground squirrel
Leydig
cells.
Fixed,
detergent
extracted
and
mechanically
separated
tissue
was
probed,
by
the
f-actin
specific
stain
fluorescence,
with
NBD-phallacidin and with an a f f i n i t y purified antibody
raised against human p l a t e l e t vinculin.
In regions of
intercellular
contact, NBD-phallacidin staining was
intense and appeared as a solid linear band. A similar,
although l e s s intense, pattern was present in t i s s u e
stained with the vinculin probe. Control s l i d e s f o r b o t h
NBD-phallacidin and vinculin were negative.
Zones of
i n t e r c e l l u l a r contact, similar t o those seen by LM, were
At
certain
sites
of
viewed
ultrastructurally.
intercellular
contact,
subsurface
filaments
were
present. These filaments, interpreted by us as a c t i n ,
formed a dense carpet adjacent t o the plasma membrane on
each side of the junction and appeared t o be organized
i n t o networks rather than b u n d l e s . Our r e s u l t s indicate
t h a t Leydig c e l l s may possess actin associated adhesion
junctions similar to other cell types.
T h a t these
junctions are l i k e l y "adhesive" in nature i s indicated by
the observations t h a t : 1 ) vinculin, a marker f o r actin
associated i n t e r c e l l u l a r adhesion s i t e s , i s present a t
t h e junctions, 2)
following mild mechanical t i s s u e
separation.
i n t e r c e l l u l a r adhesion i s maintained in
regions
where
the
junctions
occur,
and
3)
ul t r a s t r u c t u r a l l y ,
the
junctions
resemble
actin
associated adhesion junctions. This work i s supported by
grant #MT-B020 from the MRC.
S
P f e i f f e r , * David C. and A. Wayne Vogl, Department of
Anatomy, The University of British Columbia, Vancouver,
British Columbia, Canada.
Ectoolasmic ("iunctional")
soecializations in Sertoli c e l l s of the rooster and
turtle.
In t h i s study we present evidence t h a t apically
situated ectoplasmic specializations in Sertoli c e l l s of
two non-mammalian vertebrates are contractile.
Ectoplasmic specializations in t h e turtle and rooster
occur adjacent t o s i t e s of attachment t o elongate spermat i d s and are characterized by a layer of "loosely" crosslinked actin filaments t h a t l i e s next t o the plasma
membrane.
In t h e t u r t l e , a cistern of endoplasmic
reticulum i s associated with t h e cytoplasmic face of the
filament layer. In fixed frozen sections of t u r t l e and
rooster t e s t e s , immunologi cal probes f o r non-muscl e
myosin ( g i f t from Or. K. Fujiwara) react with epithelia1
regions t h a t also s t a i n with NBD-phallacidin and t h a t are
known t o contain ectoplasmic specializations. Moreover,
when clusters of glycerinated models of t u r t l e ectoplasmic specializations are exposed t o standard contraction buffers, t h e overall s i z e of t h e clusters i s reduced
and individual ectoplasmic specializations become smaller
in diameter. We i n t e r p r e t both of these changes as being
produced by t h e contraction of actin bundles in ectoplasmic specializations.
Our r e s u l t s a r e consistent with the general hypothesis
that mammalian ectoplasmic specializations m?y have
e v o l v e d from a c t i n associated adhesion junctions i n which
the actin bundles were i n i t i a l l y contractile. This work
i s s u p p o r t e d by grant #MT-8020 from t h e MRC.
S
Pfeiffer*, David C. and A.W.
Vogl, Department of
Aridtomy. T h e University of British Columbia, Vancouver,
British Columbia, Canada. Evidence t h a t around sauirrel
W i a c e l l s oossess actin associated adhesion iunctions.
S
P f e i f f e r , * David C. and A.W. Vogl, Department of Anatomy,
The University of British Columbia, Vancouver. British
containing structures found a t s i t e s of attachment t o
spermatids and t o neighbouring Sertoli c e l l s . We suspect
t h a t these cytoskeletal structures are a form of actin
associated adhesion junction. If t h i s i s true, molecular
components, such as vinculin, t h a t characterize actin
associated adhesion junctions in general
should be
present in ectoplasmic specializations. In the ground
s q u i r r e l , ectoplasmic specializations associated with
spermatids are large and t h e actin filament bundles form
elaborate patterns t h a t change during spermatogenesis.
To t e s t the prediction that vinculin i s present in ectoplasmic specializations, we probed t h e ground squirrel
system with an a f f i n i t y purified antibody raised against
human p l a t e l e t vinculin. Testes were perfusion fixed
with buffered 3% paraformaldehyde then minced i n t o small
pieces.
The pieces were extracted with 0.1% Triton
X-100, washed with PBS, t h e n fragmented by gently asperating the material through a large bore syringe needle.
The fragments, containing spermatids together with
attached ectoplasmic specializations, were applied t o
polylysine coated s l i d e s then processed f o r vinculin
localization using indirect immunofluorescence or for
actin localization using NBD-phallacidin.
Adjacent t o
spermatids, l i n e a r patterns obtained with t h e vinculin
probe were similar t o those obtained with t h e probe for
filamentous a c t i n . T h e fluorescence patterns were eliminated by pre-adsorption of the antibody with antigen and
were e i t h e r d i f f e r e n t or absent when the primary antibody
was replaced with non-adsorbed serum from the a f f i n i t y
column or when the primary antibody was omitted from the
protocol respectively. Our r e s u l t s s u p p o r t t h e conclu-
81A
ABSTRACTS-AAA 103RD MEETING
sion t h a t v i n c u l i n i s present i n ectoplasmic specializations.
F u r t h e r , t h e y i n d i c a t e t h a t v i n c u l i n i s co-dist r i b u t e d w i t h a c t i n b u n d l e s w i t h i n each e c t o p l a s m i c
s p e c i a1 iz a t i o n . T h i s w o r k i s s u p p o r t e d b y g r a n t #MT-8020
f r o m t h e MRC.
PHAM, Tuan D., Michael D. GERSHON and Taube P. ROTHMAN, Department 01
Anatomy and Cell Biology, Columbia University, College of Physicians
. and. Surgeons,
New York, New York. Birthdavs of e
p
amoarison with Kotideroic n
m
Phenotypic diversity in enteric nervous system (ENS) transcends that of
other regions of the PNS. All of the classes of putative and established
neurotransmitters that are found in the CNS are also found in the bowel. In mice, the
neural crest-derived enteric neuronal precursors colonize the gut as early as day E9.
in contrast, the first neuronal properties that can be detected, which include specific
uptake of 3H-5-hydroxytryptamine (3H-5-HT), uptake of 3H-choline, conversion of
3H-choline to 3H-acetylcholine, and the expression of neumpeptae Y (NPY) cannot be
detected until day E12. Other neuropeptides, including substance P, vasoactive
intestinal polypeptide (VIP), or calcitonin-gene related peptide (CGRP) all appear at
later times. The current experiments were done to test the hypothesis that some
enteric neurons withdraw earlier from the mitotic cycle than others so as to produce
a pool of terminally differentiated neurons that coexists with dividing neural precursor
cells in the developing ENS. In order to determine the time when neurons undergo their
iinal mitotic division, gravid mice were injected with 3H-thymidine (5 pCVg at 4-8 hr
intervals for 24 hrs) on days E8-EI8. Pups were injected similarly on days P1-5, P9,
P i 5, and P2i. Colchicine (5 mgkg [i.p]) was administered on day P29, to enhance the
detection of neuropeptides, and the animals were killed 24 hrs later. Neuropeptides,
choline acetyltransferase (ChAT), and 5-HT were demonstrated by
immunocytochemistry simultaneously with the radioautographicvisualization of cells
iabeled by 3H-thymidine in dissected laminar preparations of the bowel wall.
Birthdays of serotonergic neurons were observed as early as day E8, occurred in
greatest numbers on day Ei0, and declined in frequency on day E l l . Birthdays of
cholinergic neurons also occurred as early as day E8, but in contrast to serotonergic
neurons, birthdays of cholinergic neurons continued to occur in increasing numbers at
day Ell. No peptidergic neurons were found to be born earlier than day E10.
Oiilerent peptidergic neurons were seen to withdraw from the mitotic cycle at
different times, some during postnatal life. These observations demonstrate that
some of the crest-derived precursors of enteric neurons are already postmitotic at
the time they colonize the bowel. Other such cells continue to proliferate after birth,
when the ENS is organized into ganglia and is functional. Serotonergic and some
cholinergic neurons become postmitotic before any peptidergic neurons are born and
before any peptides are expressed in enteric neurons. The precocious timing of the
final mitosis of serotonergic and cholinergic neurons is consistent with the possibility
that the birthdays of peptidergic neurons may be influenced by the early-developing
neurons of the ENS. Supported by NIH grants HD21032, HD20470, NS15547.
OPHELAN, Kevin D., Michael J. TWERY* and Joel P.
GALLAGHER,* Department of Pharmacology and
Toxicology, University of Texas Medical Branch,
Galveston, Texas. MorDholouical evidence for
electrotonic couDlinq of rat dorsolateral sevtal
_
nucleus
___
neurons.
__
It is widely believed that the dorsolateral
septal nucleus (DLSN) plays an essential role in
the septal driving and modulation of hippocampal
theta rhythm. We have previously demonstrated
that DLSN projection neurons possess extensive
recurrent axon collaterals which may contribute
to the rhythmical output of this nucleus (Anat.
Rec. 223:90A).
The
present
study
uses
intracellular recording and
labeling with
Lucifer yellow in an in vitro slice preparation
of rat septum to evaluate the incidence of dyecoupling among DLSN neurons. Twenty percent (9
out of 4 5 ) of the injections resulted in the
labeling of pairs of dye-coupled neurons. These
neurons were morphologically heterogeneous and
could not be distinguished from non-coupled
neurons, The cell coupling occurred at dendro-
dendritic rather than somato-somatic junctions.
Several dye-coupled neurons gave rise to
recurrent axon collaterals indicating that they
function both as projection neurons and local
circuit interneurons. The dye-coupled neurons
exhibited passive membrane properties which did
not differ significantly from
non-coupled
neurons. Many cells were spontaneously active at
rest and exhibited a burst firing pattern. The
present results provide indirect morphological
evidence suggesting that some DLSN projection
neurons are electrotonically coupled. This
coupling would provide an effective means of
synchronizing the occurrence of the high
frequency bursts that have been proposed to
underlie the septal maintenance and modulation
of hippocampal theta rhythm. Supported by NIMH
Grant MH-39163 to J.P.G.
PICKARD, Gary Edward, and Eckhard FRIAUF,*
Department of Anatomy, West Virginia University,
Morgantown,
West
Virginia;
Department
of
Neurobiology, Stanford University, Stanford,
California;- Morvholosical features of qanalion
cells afferent &Q the SUDraChiaSmatiC nucleus
a livinq retina preDaration.
revealed
Retina1
innervation of the hypothalamic
suprachiasmatic nucleus (SCN), a biological clock,
synchronizes the endogenous circadian system to
the environmental 1ight:dark cycle.
Although
processing of photoperiodic information begins in
the retina, very little is known of the retinal
circuitry underlying this process. We have begun
to describe this circuitry in the golden hamster
by analyzing the morphology of retinal ganglion
cells (RGCs) afferent to the SCN.
RGCs were
identified following the retrograde transport of
rhodamine-labeled microspheres injected into the
SCN.
The morphology of identified RGCs was
revealed by intracellular injection of Lucifer
yellow (LY) in living retina maintained in vitro.
LY filled RGCs were subsequently photoconverted
and logged into a computer. RGCs afferent to the
SCN are few in number and have a large soma
(13.7+0.4 pm: N=7) with 3-4 primary dendrites
which branch sparingly.
The dendrites are
unistratified in the inner plexiform layer (IPL)
although the depth of stratification in the IPL
varies among RGCs; the majority stratifying in the
proximal half of the IPL (the ON sublamina). The
dendritic field is relatively large (average 280
w )
This
first
complete
morphological
description of RGCs afferent to the SCN is
consistent with their presumed role of detecting
changes in ambient illumination.
The long
unbranched dendrites imply a high degree of
convergence with a subsequent loss of spatial
resolution. This work was supported by NIH grant
NS 21165 to GEP.
.
S
POMERANZ. Howard D., Taube P. ROTHMAN, R. Jawt JACOBS-COHEN, and Michael D.
rettndars.
Although the enteric neurons and glia of the hindgut have been shown in avian embryos to be
formed by emigrbs from the neural crest, the axial levels from which these precursors originate
is Lontroversial. We have previously demonstrated that the postumbilical bowel contains
neurogenic cells before it is colonized by cells from the vagal crest, suggesting that the Sacral
as well as the vagal crest contributes precursors to the hindgut: however, this evidence is
indirect. The nondeleterious fluorescent carbocyanine dye, Dil, and a replicationdeficientavian
retrovirus, U-10(from Dr. Joshua Sanes. of Washington Univ., St. Louis), were therefore
82A
ABSTRACTS-AAA 1WRD MEETING
employed lo trace the migration of cresl cells diredy. Dil is advantageous in hat H can be
used to label rehtively hrge wmben of crestderbed calls; however, It may beaxne 6luled by
prqressive cell divislon. The retrovirus o v e m e s Uis pot9nUal limitation because 1 iS -My
incorporated into the DNA of the cells II infects and is thus transmilled lo all W
U progeny.
The retrovirus, however,infects only anal nunbrs of d s . Dil Q( U-10was inpcted with
pressure lhroogh a mimpipetle Inlo the neural crest 01 chick embryos. The e m t W s were
then incubated for 24-120 hrs post-injection. Infected cells were delected
immunocytochernidlywn
i antibodiesto pgaladou’dase([email protected]) and Dil-hbeiedcells by the
fiwrescenced tkir pbma membranes Cells were also simultaneouslybnmmiateMwiR iiw
monoclonal antibody NC-1, which recognizes migrating uestderived cells as well as mose
commitled to neuronalMglial phenotypes. hjecrionof Dil lnto the pal (mites3.7)or sacral
(somiles28-36) crest led to Vle appearance of Dillbeled d l s within Ihe bawel wall. Many
cells had a mesenchymal appearance. but some had a multipolar neuronal mrpholopy and
exhik’led fluorescenl Mricose axons. lngclion 01 Dil into Um saaal uest Id to the appearance
of Dil-fluorescenlcells in Me bowel caudal,M no1 rosval,to the umbiEcal flexure. Fdlowing
injection of U-10into the sacral crest.Bgaliabeled cells were observedin dorsal mot Banglia
(DRG). the ganglion of Remak and the enlericmesenchyme d the post-umbilicalbowel. h the
DRG and the hindgut p-gal-labeledcells were found to co-express NC-1. confirming their
neural crest origin. The Bgaliabeled cells were olen fourd In coherent &ins or dusters.
suggesting lhat the labeled cells were subcbnef orlplnating from a common prewnor. In
summary,we have found that when the sacral crest was Qected with Dil or a replicationdeficient retrovirus (emding Bgal),Dil- or pgalhbeled&Ih migaled to Vle poslumbilical
bowel. Many of the cells infectedwith Me retrovirus m+xps!essedNC1. These studies
confirm that Iha avian postumbilical gut is mbnized by cells derived lrom the sacral fegion of
the neural crest. The pcoporlion ofenteric neumns in Ihe hindput derived from vagal or sacral
crest emgr6s remains to be determined,as does Ihe possik4ity lhat dls Ran Ihe sacral cresl
contributeto non-neuronalcomponentsofIt18bowel wall. Supporled by NIH grants GM 07367,
HD 17736.and NS 15547.
POPOFF. Steven N.. Mark A. GRISE* and Sandy C. MARKS, JR..
Dept. of Anatomy, Temple U. Sch. Med.. Philadelphia,
Pennsylvania; Dept of Cell Biology. U. Mass. Med. Sch.,
Worcester, Massachusetts. The effects of 1.25-dihydroxyvitamin 2 0” osteoclast number and cytochemistry in normal
and osteopetrotic rabbits.
Osteopetrosis is a metabolic bone disease characterized
by defective osteoclast-mediated bone resorption which coexists with elevated serum lr25-dihydroxyvitamin D
(calcitriol or 1.25) levels in some osteopetrotic children
and animals. Based on the hypothesis that vitamin D resistance may be a component of the osteopetrotic syndrome, we
examined the effects of high-dose calcitriol therapy on
osteoclast number and cytochemistry in normal and osteopetrotic (0.) rabbits. 1.25 was continuously infused at
doses of 0.5. 2.5 or 25 pgfkgfday via subcutaneously implanted osmotic minipumps for a period of 7 days. Following
treatment, the proximal tibia1 metaphyses were processed
for histomorphometric and cytochemical analyses. Sections
were stained for tartrate-resistant acid phosphatase (TrAP)
or acid ATPase (TraATPase). Osteoclasts were significantly
rabbits compared to age-matched
reduced in untreated
normal littermates between birth and 3 weeks of age (41-462:
of normal). While most normal osteoclasts (85%) stained
heavily for TrAP or TraATPase, less than half of 0s osteoclasts were heavily stained for these acid hydrolases.
1.25 infusions resulted in elevations of osteoclast number
in both normal and 0s rabbits, but the number of osteoclasts remained significantly lower in mutant compared to
normal rabbits at any given dose. 1.25 infusions also
resulted in a significant increase in the percentage of 0s
osteoclasts staining heavily for TrAP and TraATPase. These
results suggest that inspire of high endogenous levels of
1.25,
rabbits can respond to exogenous 1.25 as evidenced
by increased osteoclast number and cytochemical staining,
even though these osteoclasts fail to resorb the excess
skeletal matrix (Popoff et al, 1989).
Thus, the defect
appears to be in the initial cytodifferentiation of osteoclasts in the mutant skeleton. not in their responsiveness
to endogenous regulators of bone resorption.
Supported by NIH Grants DE07444 (SCM) and DE 05461 (SNP).
S
POZZO MILLER,* Lucas and Agustin AOKI, Centre
of Electron Microscopy, School of Medicine,
Cordoba National Universitv. Cordoba. Araentina.
The synaptic organization of the
ventrolateral subdivision of the hypothalamic
ventromedial nucleus (VL-VMN) was examined
morphometrically in rats of both sexes at
different postnatal ages. The axodendritic
synapses were classified and quantified as
synapses on dendritic spines and synapses on
dendritic shafts. A sexual dimorphism was found
throughout the postnatal life in spine synapses,
the nunher of which is lower in females than in
males. By contrast, the density of shaft
synapses was also lower in females but only
after 100 days. Neonatal treatment (day 5 ) with
testosterone stimulated the synaptogenesis in
the VL-VMN of female rats. The number of spine
synapses increased significantly in comparison
to untreated controls, even exceeding that of
males. T h i s difference observed from 10-day
continued until adulthood. The injection of 100
ug of tamoxifen to newborn male rats interfered
with synaptogenesis in the VL-VMN. The number of
spine synapses in these rats was significantly
lower than in the controls and the pattern
resembles that of the females. The djminution in
density of spine synapses was found throughout
the prepubertal life. These results suggest that
spine synapses are the structures of the VL-VKN
sensitive to the organizational effect of
gonadal steroids. A reversion of the. synaptic
organization in both sexes can be induced by
experimental injections of androgens or estrogen
antagonists during the critical period of sexual
differentiatlon of the brain.
PTASHEKAS.Julius Ruvinas.bID.SR..
Lab. Electron
.USSK.
nt.
ital vi-microscopical
study revealed uitrastructural evidence of the
gastric diffuse endocrine system reaction after
herbicide bentioks exposure.WISTAR rats 300-330
gr b.w. were tube fed with bentioks twice a week
for 40 days at the doses 7.70 and 700 mg/kg b.w.
Samples of gastric mucosa (pyloric) were prepared
for transmission electron microscopy.Ultrastructurally A(enterog1iucagon) and G(gastrin,enkephalin) endocrine cell types at the rates of 1/1,
1/2 were fixed according to Lausanne Enteroendocrine Cell Classification.Their ultrastructure
and function were estimated when electronmicroSCOpiCQl image was videoprocessed at the monitor
and distinct density as well as size (nm) and
number of hormones containing secretory granules
within 1 mkm2 of endocrine cell cytoplasme were
fixed.Al1 data obtained had been statistically
processed by software package. Thus, after exposure of 7 mg/kg dose, amount of secretiques
within A and G cells decreased while originating
exocytosis of hormones.Secretory granules of low
density were found interacting to Golgi complex
structures.After 70 mg/kg bentioks treatment A
and G cells were accumulating secretory granules
both decreased their functional activity. 700 mg
per kg exposure enhanced hormones accumulation
in enterogliucagon containing A cells up to com~
ABSTRACTS-AAA 103RD MEETING
plete extrusion stoppage. G cells on the other
hand were extruding hormones out of cell again.
Effect of intense vacuolization followed break
down of mature secretory granules number within
cytoplasma.Given data illustrate structural basis of enteroendocrine cell adaptation fenomenon
after environmental chemical influence.
S
Douglas J. and Paul 3. GARDNER. Department of
RABURN.*
Anatomy, U n i v e r s i t y of Nebraska Medical Center. Omaha,
The
role
of
testicular
macrophages
in
Nebraska.
steroidogenesis.
Recent evidence s u g g e s t s t h s t s t e r o i d b i o s y n t h e s i s i n
Leydig c e l l s can b e - i n f l u e n c e d by f a c t o r s - o t h e r t h a n
l u t e i n i z i n g hormone (LH) , s p e c i f i c a l l y , t h o s e produced by
macrophages of t h e t e s t i c u l a r interstitium. Because normal
t e s t o s t e r o n e b i o s y n t h e s i s i s e s s e n t i a l t o male development
and f u n c t i o n , it i s important t h a t p o s s i b l e Leydig c e l l mscrophage i n t e r a c t i o n i n t h i s p r o c e s s be examined.
The
p r e s e n t i n v e s t i g a t i o n employed as an i n v i t r o model coc u l t u r e s of Leydig c e l l s and macrophages o b t a i n e d from
testes of 64-69 day o l d , male Fisher-344 r a t s .
Leydig
c e l l s c o l l e c t e d from c o n t i n u o u s P e r c o l l g r a d i e n t s were c u l t u r e d a l o n e o r i n combination w i t h p e r i t o n e a l o r t e s t i c u l a r
macrophages i s o l a t e d by d i f f e r e n t i a l attachment.
The
v a r i o u s c u l t u r e s were incubated f o r 2 hours i n t h e
presence of LH (10 ng/ml).
f o l l i c l e - s t i m u l a t i n g hormone
(FSH. 10 ng/ml) or i n t e r l e u k i n - 1 b e t a (IL-16. 20 u n i t s /
ml). A 1 m l sample of t h e medium from each of t h e c u l t u r e s
was t h e n removed and analyzed f o r t e s t o a t e r o n e (T) and
dihydro t es t o s t e rone (DHT) c o n t e n t by radioimmunoas say. The
production of T and DHT by e a c h of t h e c o - c u l t u r e t y p e s w a s
compared t o t h a t of Leydig c e l l s a l o n e s t i m u l a t e d by t h e
same
factors.
Comparisons were
a l s o made between
co-cultures containing e i t h e r t e s t i c u l a r o r p e r i t o n e a l
macrophages.
Values were c o n s i d e r e d s i g n i f i c a n t w i t h P <
0.05.
Preliminary d a t a s u g g e s t t h a t t h e p r e s e n c e of e i t h e r
t e s t i c u l a r o r p e r i t o n e a l macrophages i n f l u e n c e s t h e proI n t h e p r e s e n c e of
d u c t i o n of DHT but n o t n e c e s s a r i l y T.
LH. FSH. o r IL-1B. DHT p r o d u c t i o n w a s decreased i n c u l t u r e s
c o n t a i n i n g t e s t i c u l a r macrophages when compared t o t h o s e of
p e r i t o n e a l macrophages.
S i g n i f i c a n t d i f f e r e n c e s occurred
only i n t h o s e c u l t u r e s t r e a t e d w i t h FSH. However, i n t h e
absence of exogenous f a c t o r s . DHT production was s i g n i f I n c o n t r a s t , T production w s s i n c r e a s e d
i c a n t l y increased.
i n t h e s e same c u l t u r e s i n t h e presence of LH. FSH and IL-1B
and s i g n i f i c a n t l y d e c r e a s e d i n t h e i r absence.
These
r e s u l t s suggest t h a t t h e presence of b o t h macrophages and
FSH may modulate e i t h e r t h e s e c r e t i o n o r conversion of T
and DHT.
83A
were determined by counting the number of nuclei of each group of
cells per five crypts. The volume fractions occupied by vacuoles and
mucin granules in the epithelium were determined using the point
counting method. Maintenance on HF induced dramatic changes in
the crypt epithelium for both descending and sigmoid colon, but not
in the transverse colon. In the decending colon, there was a
decrease in the number of vacuolated cells in the crypt (12% in HF
rabbits vs. 42% in control rabbits; p<O.O5), with a concomitant
increase in the number of oblet cells (53.7% in HF rabbits vs.
31.5% in control rabbits; pilf.05). The volume fraction occupied by
both vacuoles and granules in the crypt showed comparable trends.
Vacuoles occupied only 13.5% of the epithelium in HF rabbits vs.
41.5%. in the control animals (ps0.05), with the volume fraction
occupied by goblet cell granules increasin from 21.2% in control
rabbits to 43.3% in HF rabbits (pSO.05). h e s e changes occurred
significantly in the distal colon (descending and sigmoid), while
there were no significant changes in the transverse colon. These
data suggest that altering the colonic lumina1 environment by
increasin the concentration of dietary fiber has a dramatic effect
on the eflular distribution of the colonic epithelium. Supported by
Grant DK 33720 from the National Institutes of Health.
a
RANA, M.W.,
A. AYALA," R.E.
DEAN* and I . H . CHAUDRY".
Department of
Anatomy
and Neurobiology,
St.
Louis
U n i v e r s i t y , S t . Louis, Missouri and Department of Surgery,
Michigan S t a t e U n i v e r s i t y , E a s t Lansing, Michigan.
The e f f e c t
hemorrhage on t h e f u n c t i o n of Kupffer
and s p l e n i c a d h e r e n t c e l l s a s demonstrated b~ scanning
inununo e l e c t r o n microscopy.
I t i s w e l l known t h a t t h e r e is i n c r e a s e d s u s c e p t i b i l i t y
t o s e p s i s following hemorrhage.
The mechanism of t h i s i s
n o t w e l l understood. Recent s t u d i e s u s i n g inununo-chemical
a s s a y s have demonstrated t h a t macrophage (M0) a n t i g e n
p r e s e n t a t i o n f u n c t i o n is depressed f o l l o w i n g hemorrhage and
p e r s i s t s d e s p i t e adequate r e s u s c i t a t i o n .
The aim of t h i s
s t u d y w a s t o determine, by u s i n g scanning immuno e l e c t r o n
microscopic t e c h n i q u e s , t h e number of a c t i v e [email protected] a f t e r
hemorrhage. S i x t e e n male C3H/Hen mice, a n o n l e t h a l model
of hemorrhage e s t a b l i s h e d i n our l a b o r a t o r i e s , w e r e bled t o
t h e blood p r e s s u r e of 35mm Hg and were maintained a t t h a t
p r e s s u r e f o r 1 hour, t h e n r e s u s c i t a t e d , w i t h t h e i r own
blood and adequate f l u i d s .
Mice s e r v i n g as c o n t r o l were
sham o p e r a t e d . Twenty f o u r hours a f t e r , Kupffer c e l l s (Kc)
from l i v e r s and s p l e n i c adherent cells (Sac) w e r e incubated
and t h e n were exposed t o p o l y s t e r e n beads conjugated w i t h
anti-mouse IgG which s p e c i f i c a l l y b i n d s t o FC r e c e p t o r s .
A f t e r washing t h e c e l l s were prepared for o b s e r v a t i o n s . A t
l e a s t one hundred c e l l s from each animal were examined.
Although both l a b e l e d and u n l a b e l e d c e l l s were found i n a l l
c u l t u r e s , t h e number of Kc from post-hemorrhage mice which
e x h i b i t e d s p e c i f i c r e c e p t o r l a b e l i n g was s i g n i f i c a n t l y
decreased (41.0 2 2.6 mean SE) compared t o c o n t r o l (64.2 2
7.5 P>0.05 by unpaired t - t e s t ) .
The number of Sac from
post-hemorrhage mice was a l s o s i g n i f i c a n t l y decreased (35.7
- 2.5) compared t o c o n t r o l (61.2 2 3-9P>0.05). The
i n t e r n a l i z a t i o n of marker was seen i n some c e l l s .
The
cause of t h i s may be t h e l o s s , i n a c t i v a t i o n a n d l o r
W
e suggest t h a t
the
internalization
of
receptors.
decreased number of a c t i v e M0 may c o n t r i b u t e t o t h e
d e p r e s s i o n of a n t i g e n p r e s e n t a t i o n and consequently may
enhance s u s c e p t i b i l i t y t o s e p s i s .
---
+
S
RADWAN, Khaled and Robert D. SPECIAN, De artment of
Cellular Bioloey and Anatomy, LSU School o f Medicine,
Shreveport, Louisiana. The effect of a hieh-fiber diet on colonic
i h 11 1 11
1
The colonic epithelium is composed of
responsible for fluid secretion and fluid
absorption, respectively. In the rabbit, the cry t portion is
populated by both goblet and vacuolated cells, wiereas on the
surface, the vacuolated cells mature, altering their structure and
function to become columnar absorptive cells. The purpose of this
study was to examine the effects of increased dietary fiber on
colonic epithelial cell populations. New Zealand white rabbits 2-3
months old weighing an average of 2.4 k were maintained on
either rabbit chow containin high-fiber (25k fiber, HF) or normal
rabbit chow (18% fiber) for 80 days. At sacrifice,segments from the
transverse, descending and sigmoid colons were excised and
processed for histolo Methacrylate sections were cut and stained
with toluidine blue.%e
ercentage and distribution of oblet,
vacuolated and surface coyumnar cells in the colomc epitfelium
RASWEILER. John J . , I V , Department of O b s t e t r i c s and
Gynecology. C o r n e l l U n i v e r s i t y Medical College, New York,
New York. Evidence f o r a r o l e of endometrial e n d o t h e l i a l
c e l l s i n t h e c o n t r o l of t r o p h o b l a s t i c growth.
I n an e f f o r t t o f u r t h e r d e f i n e t h e f a c t o r s t h a t c o n t r o l
t r o p h o b l a s t i c growth, p l a c e n t a l development w a s examined
h i s t o l o g i c a l l y throughout pregnancy i n captive-bred b l a c k
m a s t i f f b a t s , Molossus a t e r . The morphogenesis of t h e
ABSTRACTSAAA 103RD MEETING
definitive discoidal hemochorial chorioallantoic placenta
was of particular interest, because it always forms at the
cranial end of the right uterine horn. This positioning
was related to the presence of an unusual vascular tuft
which developed in the endometrium immediately around the
uterine end of each oviduct following ovulation. This tuft
consisted of a cluster of capillaries and possibly endothelial tubules connected to specialized arterioles and
venules which ran parallel to the intramural oviduct. As
decidual reactions occurred, the endothelial cells of the
tuft vessels hypertrophied, and their basal laminae appeared much more prominent in sections stained for glycoproteins. Similar changes were observed in the vascular tufts
of non-pregnant uteri which had decidualized spontaneously.
When a conceptus was present, cytotrophoblast proliferated
preferentially around the vascular tuft in the right horn,
and its vessels (many of which did not contain blood)
became surrounded by trophoblastic cuffs. A functional
placenta was subsequently formed when trophoblastic tubules
grew out from these cuffs, developed lumina, and began to
carry maternal blood. These observations suggest that the
endothelial cells of the tuft vessels may secrete factors
which influence trophoblastic growth and are at least
partially incorporated into the vessels' basal laminae.
Endometrial endothelial cells may be playing a similar
morphogenetic role during placental development in many
other mammals with invasive trophoblast. In some species
extracellular material apparently secreted at least in part
by these cells is abundant in the interhemal barrier of the
chorioallantoic placenta, or is retained there following
loss of the maternal endothelial cells, for reasons which
heretofore have not been completely understood. Supported
by NIH grants HD-17739 and RR-05396.
REIDENBERG, Joy S . and Jeffrey T. LAITMAN,
Departments of Cell Biology/Anatomy and
Otolarvngolom, Mount Sinai School of Medicine, New
York,-N&w f & k . Histological changes in the skull base
of the rat (Rattus [email protected]) after surgical removal of
the spheno-occipital synchondrosis
The human skull possesses many characteristic
features, such as marked basicranial flexion coupled
with a pronounced sella turcica, a prominent dorsum
sellae, and a steeply sloped clivus. The biomechanical
forces which create these features are, however, not
well understood. This study examines the artificial
formation of these traits in rats as a model for
understanding their development in humans.
The spheno-occipital synchondrosis was surgically
ablated in 89 infant rats at 1 3 days. Ten sham
operated and 6 normal rats were used as controls.
Anesthetized rats were laterally radiographed a t 40, 60,
80, 100, and 120 days. Skull base shape was recorded
via angular measurements. Rats were killed at 130 days
and perfused with buffered formalin. The heads were
removed whole, decalcified, embedded in celloidin and
paraffin, sagittally sectioned at 6 microns, and stained
with hematoxylin and eosin for histological analysis.
Results indicate that ablation of the spheno-occipital
synchondrosis induces the premature formation of a
spheno-occipital synostosis. This premature synostosis
reduces basisphenoidal and basioccipital growth,
resulting in basicranial shortening. This shortening is
coupled with basicranial flexion and cranial vaulting, as
revealed by radiographic analysis. These changes
produce traits reminiscent of the human condition: 1)
The shortened basicranium of experimentals reveals a
sphenoid sinus located under the pituitary, as in
humans, rather than anterior to it, as in controls. 2 )
The synostosis is often accompanied by an endocranial
elevation resembling the human dorsum sellae and
clivus. This elevation may be a form of "buttressing"
formed for structural support. This formation may
result from mechanical stresses inherent in a flexed
basicranium and/or a vaulted calvaria.
.
RIEKE, G a r l Kalman and Benedicta KURUNWNE,*
Department of Anatomical S c i e n c e s , Meharry
Medical C o l l e g e , N a s h v i l l e , Tennessee.,Ls t h e
e x c i t a t o r v amino a c i d r e c e v t o r a n t a a o n i s t
Kvnurenic a c i d a n e u r o t o x i n ?
E x c i t a t o r y amino a c i d s can a c t a s neurot o x i n s and may p l a y a role i n neurodegenera t i v e d i s o r d e r s s u c h a s Huntington's d i s e a s e
( H D ) . The e x p r e s s i o n of n e u r o t o x i c i t y can be
blocked o r s u p p r e s s e d by t r e a t m e n t w i t h recept o r a n t a g o n i s t s . Kynurenic a c i d ( K Y N ) is a
non-specific glutamate receptor antagonist,
y e t it is n o t c l e a r whether neurons c h a l l e n g e d
w i t h KYN w i l l b e d e s t r o y e d i n r e s p o n s e t o t h i s
p o t e n t i a l n e u r o t o x i n . c o n t i n u o u s i n j e c t i o n of
KYN (3.32 mg/100 m l , pH 7 . 5 , 5x t h e endogenous
KYN level/g r a t f o r e b r a i n ) by means of an A l z e t
o s m o t i c pump c o u p l e d t o a c a n n u l a implanted i n
t h e s t r i a t u m produced a l e s i o n . The l e s i o n showed t h r e e c o n c e n t r i c r e g i o n s : a c e n t r a l n e c r o t i c
a r e a f i l l e d w i t h macrophages, a narrow p y k n o t i c
r e g i o n and a p e r i p h e r a l s p o n g i o s e zone. I n t h e
s p o n g i o s e zone t h e r e w a s s e l e c t i v e neuron
s p a r i n g s i m i l a r t o t h e neuropathology s e e n i n
HD. The s p a r e d n e u r o n s were t h e NADPH d i a p h o r ase p o s i t i v e , somatostatin-like-immunoreactive,
medium s i z e d a s p i n y 11, n o n - p r o j e c t i o n s t r i a t a l
neurons. Our o b s e r v a t i o n s show t h a t KYN is a
n e u r o t o x i n , and raise a q u e s t i o n a s t o t h e
e f f i c a c y of f u t u r e t h e r a p e u t i c u t i l i z a t i o n of
e x c i t a t o r y amino a c i d r e c e p t o r a n t a g o n i s t s .
The s e l e c t i v e n e u r o t o x i c i t y of KYN i s f u r t h e r
s i g n i f i c a n t i n t h a t KYN l e v e l s are e l e v a t e d i n
t h e b r a i n s of p a t i e n t s w i t h HD. T h i s s u g g e s t s a
p o s s i b l e r o l e of KYN i n t h e e t i o l o g y of HD.
Supported by MBRS g r a n t S06RR08037.
RINAMAN,* Linda and Pat LEVITT, Depanment of Anatomy,
Medical College of Pennsylvania. Philadelphia, Pennsylvania.
R a u i d depeneration d a x o t o m ized mmotoneuronsin
neonatal
Nerve injury in neonatal animals often leads to motoneuronal
at
early
degeneration,
reflecting target
dependence
developmental stages.
Mature motoneurons generally survive
axotomy, but injured preganglionic parasympathetic neurons
in the adult dorsal motor nucleus of the vagus (DMV) are
unique: for unknown reasons, most die within a few months
after vagal injury. To delerminc whcthcr DMV motoneurons
arc also unique in their neonatal injury response. we compared
the survival of vagal motoneurons in the DMV and nucleus
ambiguus (NA) following vagal nerve crush in I-day-old rats.
Rat pups were anesthetizcd by hypothermia. With the aid of a
dissecting microscope, the left ccrvical vagus nerve was
thoroughly crushed at the level of the omohyoid muscle and
injected with cholera toxin-horseradish peroxidase (CT-HRP) or
Fluorogold (FG). One to 20 days following ncrve injury and
tracer injection. animals were anesthetizcd and perfusionfixed. Corona1 sections through the medulla were examined for
Both the
thc presence of retrogradely labeled motoncurons.
DMV and NA were filled with labeled motoneurons in rats
sacrificed 1 day after nerve crush. Labeled neurons in the DMV
markedly decreased in number arter 3 and 4 days, and after 6
days the DMV was complctely devoid of lnbcled neurons.
Examination of Nissl-stained material confirmed this neuronal
loss. In contrast, labclcd neurons wcrc prcscnt in the NA even
at thc longest survival times. Degeneration of injured neonatal
DMV motoneurons was far more rapid and complete than has
been reported for any other brainstcm or spinal motoneuronal
population. further demonstrating their unique vulncrability
to ncrve injury. DMV motoneurons target postsynaptic neurons
in Ihc gastroinlestinal entcric nervous systcm, and at least some
of them receive dircct synaptic fcedback from gastric vagal
sensory neurons, whose axons arc also injurcd by ncrvc crush.
We have begun experiments to test whether injured D M V
moloneurons dcgeneratc bccausc they are deprived of uniquc
m.
ABSTRACTSAAA 103RD MEETING
ncurally-transported factors normally supplied by
nervous system and/or by vagal sensory neurons.
the
enteric
Supporred
by NIMH Grant MH45507 and NIH Training Grant NS07287.
S
Reimer. C.L. and Crawford, B.J. Department of Anatomy,
University of British Columbia, Vancouver British
Columbia, Canada. Monoclonal antibodv to a olasma
membrane-associated molecule may identifv a new kind of
cell adhesion molecule in the embrvonic asteroid,
(Pisaster ochraceus).
Intercellular interactions are believed to play
fundamental roles during embryogenesi s and in the adult
organism. At the molecular level, cell-cell adhesions
iave been shown to depend on the presence of specialized
contact receptors" known as cell adhesion molecules or
CAMS. This study has produced a monoclonal antibody to
a plasma membrane-associated molecule present in embryos
of the asteroid, P. ochraceus. To prepare the antibody,
whole embryo homogenates from the late gastrula stage
were injected into Balb/C mice, and hybridomas were
produced by fusion of mouse lymphocytes and myeloma
Immunohistochemi stry was performed on
cells (FOX NY).
thin sections o f resin-embedded material. The embryos
were fixed by the freeze substitiution method as
described by Campbell and Crawford (1989). involving
liquid propane as a freezing medium, and ethanollalcian
blue as a substituting medium. Os04 was omitted from
the standard protocol to preserve tissue antigenicity.
This fixation/embedding protocol allowed for both good
ultrastructure and optimal
antigen preservation.
Preliminary results with the antisera showed a
concentration of labelling at the intercellular
boundaries of epithelium, a labelling pattern similar to
that seen with some anti-CAM antibodies (e.g.
anti-E-Cadherin in embryonic mouse hepatocytes). Cell
surfaces facing the blastocoele which are not in contact
with other cells generally did not stain as intensely.
Immunolabelling for N-CAM, A-CAM and E-Cadherin showed
no cross reactivity with this tissue, indicating that
this antibody may be against a new kind of CAM.
Preliminary results also showed that this molecule is
1
day
present
in
embryos
as
early
as
Further experiments involving
post-ferti 1 ization.
anti body perturbation studies and cell reagregation
studies will help to elucidate the function o f this
molecule. Supported by grant #MA0032 from NSERC.
Rennels, H a r s h a l l L. and O.R. Blaumanis*,Departments of
Anatomy and Neurology, U n i v e r s i t y of Maryland School of
Medicine, Baltimore, Maryland. Rauid, lame s c a l e
paravasoular
e x t r a c e l l u l a r spread of t r a c e r p r o t e i n
throughout
f o r e b r a i n from t h e subarachnoid space.
Horseradish peroxldase (HRP), is widely d i s t r i b u t e d
throughout t h e cat f o r e b r a i n w i t h i n minutes a f t e r
i n f u s i o n of t r a c e r s o l u t i o n i n t o t h e subarachoid space
(SAS). Adult c a t s were a n e s t h e t i z e d and an 18 gauge
needle was introduced i n t o t h e c i s t e r n a magna. CSF e f f l u x
was measured and an e q u a l volume of 4% HRP (Sigma Type
11) i n balanced s a l t s o l u t i o n was infused through t h e
same needle by g r a v i t y flow. The t r a c e r s o l u t i o n was
permitted t o c i r c u l a t e i n t h e SAS f o r p e r i o d s of 4, 10,
15, 20, 30, 60 o r 120 minutes. Animals were t h e n perfused
through t h e ascending a o r t a with aldehyde s o l u t i o n s and
f r o n t a l o r h o r i z o n t a l b r a i n s e c t i o n s were c u t with an
Oxford Vibratome. HRP w a s l o c a l i z e d u s i n g t e t r a m e t h y l benzidine o r 3'-5' diaminobenzidine as chromogens. Rapid
tracer I n f l u x occurred along t h e deep p e r i v a s c u l a r s p a c e s
(PVS) t h a t accompany t h e major p e n e t r a t i n g arteries from
t h e c i r c l e of Willis, with spread through t h e b a s a l
laminae (BL) around t h e i r c a p i l l a r y branches. From t h e s e
"paravascular" f l u i d pathways (PVS and BL), t h e t r a c e r
85A
e n t e r e d t h e e x t r a c e l l u l a r spaces (ECS) o f t h e i n t e r n a l
capsule and i n t r a c e r e b r a l white matter. Within t h e s e
l i n e a r channels, t h e t r a c e r was d i s t r i b u t e d throughout
t h e hemispheres 5 minutes a f t e r i n f u s i o n i n t o t h e SAS.
The d e n s i t y of HRP r e a c t i o n product i n c r e a s e d with
p r o g r e s s i v e l y longer p e r i o d s of t r a c e r c i r c u l a t i o n . From
t h e s u b c o r t i c a l white m a t t e r , t r a c e r extended through t h e
c o r t e x t o t h e p i a 1 s u r f a c e in many areas, p o s s i b l y
reflecting transcortical f l u i d efflux i n t o t h e
hemispheric SAS. F l u i d / t r a c e r movements w i t h i n t h e
f o r e b r a i n t h u s appear t o be i n f l u e n c e d by t h e d i s t r i b u t i o n of t h e p a r a v a s c u l a r pathways, t o g e t h e r with t h e
r a d i a l l y o r i e n t e d ECS of t h e hemispheric white m a t t e r .
The h y p o t h e s i s is proposed t h a t t h e s e t r a c e r movements
reflect an on-going c i r c u l a t i o n of e x t r a c e l l u l a r
fluid/CSF through t h e f o r e b r a i n . (NIH Grant No. 16332.)
R I G A M O N T T , D.D.,
J.B. L O N G , C.P. WINGFIELD A N D R.F.
GENOVESE*, Department of Medical Neurosciences, Walter Reed
Army Institute of Research, Washington, D . C . (Sponsored by
Thomas Crisp). ~ o p r o t e c t i v ee f f e c e r M K801 in a rodent
model of spinal cord ischemia: Anatomical and behavioral
correlation.
Dynorphin A 1-17 ( D Y N ) causes dose dependent spinal cord
loss,
and
ischemia,
hindlimb
paralysis,
nociceptive
neuropathological changes when injected into t h e lumbar cistern.
Since N M D A receptors
have
been implicated in t h e
pathophysiology of ischemic CNS injury, w e examined t h e effects
of M K801 on D Y N-induced neuropathological changes and
behavioral performance. Adult Sprague Dawley r a t s were divided
int3 five groups and iqjected as: I, saline; II, M K 801 alone (1 50
nmoles); E
i
, DYN+MK801 (300 nmoles); I V , DYN+MK801 (150
nmoles) and V , D Y N alone. The dose of D Y N used throughout w a s
20 nmoles, an amount known t o cause paralysis. Animals were
given a battery of behavioral testa prior t o , and subsequently a t 1,
24, 40 and 72 hours after injection. Following t h e final test
session, anesthetized animals were perfused and paraffin
embedded sections of the lumbosacral cord examined and scored
by an observer blinded t o t h e t r e a t m e n t condition. T i s s u e w a s
typically stained
with H & E , N i s s l ,
Kluver-Barrera
and
Bielschowsky techniques. A modified Tarlov motor score from
zero t o five, t h e latter being normal produced t h e following mean
scores a t 72 hours: Group I and II, 5; Group m, 3.36; Group I V ,
1.75 and Group V, 0.83. The inclined plane results (mean score in
degrees) were as follows: Group I, 55; U, 52; m, 50; I V , 41, and V,
33. A n i m aLs w i t h t h e greater dose of t h e M K 801 were typically
weight bearing and performed substantially better on these tests.
There
were
strong
positive
correlations
between
neuropathological changes in t h e spinal cord segments examined,
w i t h either preservation or destruction of neural tissue, and
behavioral performance. Maximum neural tissue damage w a s
observed in the conus medullaris and lumbar enlargement i n
animals treated with D Y N alone and maximum neural protection
was observed in Group Ill animals. Thus, t h e N M D A receptor
antagonist MK801 offers neural protection in t h e spinal cord
following ischemia produced by the peptide, dynorphin, a s
evidenced by both anatomical and behavioral m easures.
S
RIZZO,
Victor
J.,
Richard N.
FEINBERG a n d
New
D a v i d 0 . DEFOUW,
D e p a r t m e n t o f Anatomy,
J e r s e y M e d i c a l S c h o o l , Newark,
New J e r s e y .
Structure-function
analysis of microvascular
the c h i c k c h o r i o a l l a n t o i c
endothelium
membrane.
The
extraembryonic
chorioallantoic
(CAM) o f t h e c h i c k p r o v i d e s a model
membrane
to
evaluate
development
of
the
were
microcirculation.
Chick
embryos
incubated
using
established
shell-less
c u l t u r e techniques f o r observation a t day 6
86A
ABSTRACTSAAA 103RD MEETING
of
n o r m a l 21 d a y i n c u b a t i o n s . A g r a d e d
s e r i e s o f FITC t a g g e d d e x t r a n s a n d a l b u m i n
was l o c a l l y m i c r o i n j e c t e d a t 0 . 3 u l l s e c for 30
sec. Macromolecular f l u x a c r o s s p r e c a p i l l a r y
1
and
2),
ca i l l a r y ,
and
(orders
p o s t c a p i l l a r y ( o r d e r s 1 a n d 27 s e g m e n t s o f
the
microvascular
unit
was
observed
qualitatively
with
videofluorescence
microscopy. A t day 6, e f f l u x a c r o s s t h e
s e g m e n t a l e n d o t h e l i a was n o t o b s e r v e d .
Since
previous
data
were
consistent
with
continuous c a p i l l a r y angiogenesis u n t i l day
1 0 (DeFouw e t a l . , M i c r o v a s c . R e s .
38:136,
1989),
these
results
suggest
that
morphogenesis of e n d o t h e l i a l p e r m s e l e c t i v i t y
i s a n o t a b l e f e a t u r e of normal angiogenesis.
U l t r a s t r u c t u r a l immunochemical l o c a l i z a t i o n
of
t h e macromolecular probes served
to
support t h i s concept.
S u p p o r t e d by AHA
Grant-in-Aid.
ROBERTSON, R.T., G.H. KAGEYAMA, F. MOSTAMAND*, K.A.
GALLARDO* and J. YU, Departments of Anatomy and Neurobiology
and Physical Medicine and Rehabilitation, College of Medicine,
University of California, Iryine, California. Transient patterns of
acetvlcholinesterase activitv in geniculocortical proiections to nrimary
puditorv cortex in develouinz rats.
Transient patterns of acetylcholinesterase (AChE) activity are
characteristic of some developing thalamocortical systems. We present
here the results of studies of AChE activity in medial geniculate and
primary auditory cortex of developing rats.
Subjects were Sprague-Dawley rats, both adults and laboratory born
pups aged 0-40 postnatal days (PO-40). Some PO-P5 pups were
anesthetized by hypothermia and the inferior colliculus or medial
geniculate body was removed unilaterally by aspiration. Other pups
(PND 5-12)or adults were anesthetized with ether or sodium barbiturate
and chloral hydrate and received injections of horseradish peroxidase
(HRP) or HRP conjugated to wheat germ agglutinin (WGA-HRP) in the
inferior colliculus or medial geniculate body. Following survival times
of 2-5 days, animals were perfused with aldehydes and sections
processed for AChE or HRP histochemistry.
AChE activity in primary auditory cortex of developing rats occurs as
a dense fiber-like plexus in the deep part of layer III and layer IV. This
pattern is transient, appearing about PND 5, showing peak intensity at
PND 9-12, and declining to the adult pattern by PND 20. In mature rats,
AChE activity is found throughout cortical laminae but especially in the
infragranular layers. Injections of WGA-HRP in the inferior colliculus
result in anterograde transneuronal labeling of geniculocortical terminals
in auditory cortex: the pattern of HRP labeling is coextensive with the
transient AChE activity indicating that transient AChE activity occurs in
primary auditory cortex. Further, placement of lesions in the medial
geniuclate body in P8-10 rat pups is followed by marked reduction of
transient AChE staining in ipsilateral auditory cortex.
We have suggested previously that transient AChE activity is
characteristic of developing geniculocortical projections of the visual
system (Robertson et al., Dev. Brain Res., 1988). The present data
suggest that developing geniculocortical neurons of the primary auditory
system also display transient AChE activity. AChE activity appears
characteristic of these neurons during the time when geniculocortical
axons are growing into auditory cortex and forming synaptic connections
with cortical neurons.
Supported in part by NIH grant NS 25674.
ROGERS, Carla Susan and Lydia DiDio SCHAFER*,
Departments of Anatomy and Pathology, Medical College
of Ohio, Toledo, Ohio. TEM studv of t h e e f f e c t s of DON
on t h e develooinq a t r i a l seDtum.
An investigation of t h e t e r a t o g e n i c e f f e c t s of 6diazo-5-0x0-L-norleucine (DON) on t h e developing atrium
of t h e Sprague-Dawley embryonic r a t heart was performed.
Pregnant female r a t s were injected on day 11 with .15,
. 2 , o r . 2 5 mg/kg of DON. Female r a t s were s a c r i f i c e d
on day 13 t o f i r s t determine t h e number of implantations
vs. t h e number of surviving embryos.
The following
r e s u l t s were obtained: 5% resorption/95% survival with
.15 mg/kg, 15%resorption/85% survival with .2mg/kg, and
28%/72% with .25 mg/kg of DON injected on day 11.
Embryonic h e a r t s were prepared f o r scanning and
transmission e l e c t r o n microscopy by standard methods and
observed with a Cambridge Stereoscan 180 and P h i l l i p s
CM10, r e s p e c t i v e l y . No abnormalities were noted i n t h e
However, o f t h e 50
groups i n j e c t e d with .15 mg/kg.
specimens injected with .2 o r .25 mg/kg and examined by
SEM, 29 demonstrated an abnormality of septum primum as
seen on g e s t a t i o n a l day 13 in which t h e length was
i n s u f f i c i e n t and did not meet and fuse with t h e endocardi a1 cushions. The second opening, foramen secundum,
developed in t h e same ventrocranial area, however t h e
s i z e of t h e opening was diminished.
Transmission
e l e c t r o n microscopic s t u d i e s of t h e endocardial c e l l s
of septum primum which a r e instrumental in establishing
foramen secundum e x h i b i t differences from those o f t h e
control specimens. Cell shape and endoplasmic r e t i c u l a
of t h e drug-treated specimens e x h i b i t profound d i f f e r ences. Large areas of extremely d i l a t e d endoplasmic
r e t i c u l a may be r e l a t e d t o changes in glycoprotein
production.
DON has been shown t o a l t e r sulfated
glycosaminoglycan and fibronectin production, which may
be a f f e c t i n g t h e a l t e r e d septum.
(Supported by a g r a n t from
Association, Ohio A f f i l i a t e ) .
the
American
Heart
ROLLAG, Mark D., Michael N. SHERIDAN, Cynthia P.
FREEMAN*, Stephen J. ROSEN*, Frank J. TOTH*, and
Sherry L. BRAHMAN*, Departments of Anatomy,
Radiology, and the Center for Interactive Media
in Medicine, Uniformed Services University,
Bethesda, Maryland. MediOuiz Gross Anatomv: an
interactive videodisc txoaram for Dresentation of
anatomical auestions in a aame format.
Computer programs, which use interactive
video-disc technology to present medical
information and clinical scenarios, promise to be
a valuable aid in the medical school teaching
environment. We have utilized the interactive
attributes of this technology to present
anatomical questions in a quiz game format.
Students currently use this program to quiz
themselves as they prepare for exams administered
by the gross anatomy faculty. The MediQuiz Gross
Anatomy program (to be commercially published)
runs on the IBM's InfoWindow system: any IBM
system with ONline Corporations's GL-512 Genlock
card: or the Electronic Information Delivery
System. Typically, a game takes about 30 min. An
initial menu allows the player to select 5 of 25
categories (20 correspond to gross anatomical
regions and 5 to embryonic development). The
player competes with 2 computer-generated
opponents to select questions of varying levels
of difficulty presented in either a textual or
radiographic image format. Once chosen there is a
timed opportunity to answer the question. If the
answer is correct, points are credited to the
player account: if incorrect, points are deducted
from the player account. There is an intermediate
spelling round and a final bonus question. At the
end of the session, players have the opportunity
to review the questions they miss.
-
ABSTRACTS-AAA 103RD MEETING
S
ROSOLIA*, D.L., GEE*, M.H., and ALBERTINE, K.H.,
Departments of Physiology and Medicipe, Jefferson Medical,College,
Philadelphia, Pennsylvania.. Cvtochemical and structural evldenceof
activation of neutroDhils m vivn in the DUlmonaxv. In virro activation of neutrophils (PMNs) using a variety of stimuli
(C5a, WLP, PMA) is associated with biochemical, cytochemical, and
structural changes in PMNs, including release of granule enzymes and
increased cell area. However, there is a dearth of information regarding
the activation responses of PMNs in vivo. Since the lung is a primary
organ site of PMN sequestration during systemic inflammatory
responses, we hypothesized that the activated PMNs in the pulmonary
microcirculation would degranulate and increase in size (area). PMN
degranulation in vivo was assessed in three anesthetized sheep, two of
which had zyniosan-activatedplasma (ZAP; to activate C5a) infused for
30 minutes, by reacting lung tissue sections with Fast Blue BB
diazonium salt (Beckstead,
57:1088, 1981) to demonstrate
neutrophil alkaline phosphatase (NAP) activity. A lung biopsy was
taken from each sheep before the infusion of either ZAP or untreated
plasma followed by lung fixation in sifu during the infusion. One
hundred PMNs in alveolar capillaries were rated on a scale of 0 (no
reaction *oroduct)
to 4+ (most intense and uniform reaction oroduct). The
:
the averaee NAP activitv results.
Dble -S
Condition (n)
Period
NAP Activitv Scale I% of PMNs)
(min.)
0
1+
2+
3+
4+
Baseline (3
0
2s
49
14
in
__
-2
..
ZAP (2j '-'
30
84
16
0
0
0
Plasma I1)
16
8
3
Degranulation of intravascular PMNs in the lung was evident at 30
minutes (100% of PMNs had a NAP score ~ l + as
, compared to the
baseline and plasma control samples). PMN area in vivo was determined
in 4 anesthetized sheep (two infused with ZAP for 30 minutes and two
infused with plasma). Bioquant System IV was used to digitize the area
of 10 PMNs in alveolar capillaries in each of 8 random tissue
blockslsheep. PMN area increased from 49 pm2 in the control sheep to
61 pm2 in ZAP-mated sheep. Ourresults indicate that PMN activation in
vivo is associated with both acute degranulation and increased cell area.
We suggest that sequestration of PMNs in the lung during acute lung
injury is caused by entrapment of enlarged, activated PMNs. [Supported
by NIH grants HL36237, HL34014, HL38075, and the Center for
Critical Care Research]
- 1
ROWLAND; Jeffrey R., Pamela E. PETERSON,' Andrew G.
HENDRICKX. and Doris B. WILSON, California Primate Research
Center, University of California, Davis. California; Division of
Anatomy, Department of Surgery, School of Medicine, University of
of thQ
California, San Diego, La Jolla, California.n 8 - 10 in the fy&agu.
The extracellular matrix (ECM) is now considered to play a key
role in normal morphogenesis by providing the framework for cell
migration, adhesion and proliferation. The regional and temporal
distribution of the ECM was studied in embryos of the long-tailed
monkey (Macaca fascicularis) during the period of primitive streak,
notochord and early neural tube development (stages 8-10).
Fibronectin (FN) was localized by indirect immunofluorescence,
glycosaminoglycans (GAG) were identified with alcian blue staining,
and the periodic acid-Schiff (PAS) reaction was used to stain
polysaccharide-associated constituents. In the mesoderm, a fibrillar
FN reaction was evident throughout the matrix, appearing more
prominent in lateral regions at all stages. A positive PAS reaction
originated in paraxial regions in stage 8, was distributed throughout
the mesoderm in stage 9, then became restricted to lateral regions
during stage 10. Using the alcian blue method with enrymatic
digestion and variations in pH, a predominance of neutral GAG was
observed in lateral regions of the mesoderm at stage 8. then was
distributed throughout the mesoderm in stages 9 and 10. The
concentration of sulphated GAG increased gradually with advancing
stage, and appeared to be more prominent in cranial regions. FN was
localized in the basement membrane of neuroectoderm in all stages,
and gradually increased in the basement membrane of the gut
endoderm with advancing stage. The interruption of the basement
membrane in the primitive streak region, where migratory cells are
separating from the neural ectoderm, was also well demonstrated
using the FN technique. The notochordal plate and primitive node,
sites of active cell organization and/or migration, were associated
87A
with a strong PAS reaction. This study demonstrates a temporal and
spatial pattern of ECM deposition in the primate embryo and supports
the view that these constituents play a role in cell migration and
critical epitheliallmesenchymal interactions during t h i s dynamic
period of early development. [Supported by NIH Grant #RR00169].
ROY, William A., Jayne BERNANKE*, and Norman CAPRA,
Departments of Anatomy and Physical Therapy, University of
Mississippi, Jackson, Mississippi. Prenatal development of the
masticatory muscles in Macaca nemestrina.
Macaca nemestrina fetuses timed at 62, 121, and 135 days
were delivered by Caesarian section, fixed by perfusion or
immersion in phosphate-buffered 2.0% paraformaldehyde 2.5 %
glutaraldehyde (pH 7.3). The masseter, temporal, anterior
digastric, and posterior digastric muscles were dissected,
photographed and processed into plastic for light microscopic and
ultrastructural analysis. Muscle tissue from the 62-day fetus
consisted primarily of small cylindrical myocytes and associated
satellite cells. A number of profiles suggestive of recently fused
myoblasts were present, but no mitotic figures were observed.
The smallest myocytes contained centrally located nuclei
surrounded by myofibrils. Larger fibers contained eccentric
nuclei and clusters of myofibrils which were dispersed throughout
the cytoplasm. Early stages of fascicle development were
observed with small groups of myoctes surrounded by developing
capillaries, fibroblasts, and connective tissue. Only a few
scattered bundles of unmyelinated nerve fibers were observed in
the muscles of the 62-day fetus; there was no evidence for a
functional motor innervation. By 121 days, the muscles were
organized into discrete fascicles. Individual, oval-shaped fibers
contained subsarcolemmal nuclei and, on the basis of
mitochondrial content, seemed to consist of two fiber types.
Capillaries were present within a well-developed endomysium.
Muscle spindles, containing bag and chain fibers, were observed
in the temporal and masseter muscles. Nerve fascicles contained
a number of myelinated profiles. Ultrastructural study of the
121-day muscles revealed a number of relatively simple
neuromuscular junctions which lacked the junctional folding
typical of mature muscle fibers. Muscle tissue from the 121 and
135-day animals were similar in appearance. Criteria such as
mitochondrial content of myocytes, fascicular organization, nerve
and vascular development were more reliable indices of muscle
differentiation than fiber diameter in prenatal animals.
RUNYAq, Raymond B., Jay D. PO"TS*, Ram V. SHARMA*, Dan L.
WEEKS , y d Rynesh C. BHALLA, Departments of Anatomy and
Biochemistry , University of Iowa, Iowa. City, IA. Calcium. C-kinas
prosine kinase and a o e m s toxin-sensitive G protein each plav a rolc
in signal transduction of a tissue interaction in the embrvonic h e a c
In normal heart development, endothelial cells of the atrioventicular
(AV) canal undergo cell transformation to form a mesenchymal cell
population. This was shown to be due to a complex stimulus produced
by the myocardium. One component of this stimulus appears to be a
member of the TGFP family of growth factors (Potts and Runyan, Dev.
Biol. 134, 392). By Northern blot analysis there appear to be several
different members of the TGFP family present in the heart at this time.
Cloning experiments are underway to characterize these molecules. In
order to identify additional components of the transformation stimulus,
we have begun to examine the role of signal transduction mechanisms in
this process. Since the quantity of tissue precludes biochemical
measurement, our approach has focused upon the use of activators and
inhibitors of specific mechanisms. Activators of C-kinase, PMA and
mezerein, both produce an incomplete transformation in an in vitro
bioassay. Inhibitors of both C-kinase (H-7, staurosporine) and tyrosine
kinase (genistein) block cellular transformation induced by either the
myocardium or myocardially-conditioned medium. Pertussis toxin, but
not cholera toxin, also blocks cellular transformation. In order to
88A
ABSTRACTS-AAA 103RD MEETING
directly test the apparent pathways, we examined the levels of
intracellular calcium ([Ca++].) in endothelial cells after the addition of
the transforming stimulus. A t canal endothelial cells were loaded with
the dye, fura 2, and examined by digital image analysis. These cells
demonstrated an elevation of intracellular calcium in response to the
stimulus. This response was specific when compared to non-responsive
ventricular endothelia or older AV canal endothelia. These data show
that signal transduction of the developmental stimulus is mediated by a
G protein-coupled receptor, kinase activities (C-kinase and tyrosine
kinase), and [Ca++Ii Further studies will be aimed at identifying the
components of the stimulus which initiate these processes. Supported by
N M HL 38649 (RBR), GM 40308 (DLW) and HL 35682 (RCB).
S
R U S A , * K e n a t a , I t a R. KAISERMAN-ABRAMOF, d n d L o i s
K . L A E M L E , D e p a r t m e n t s of Anatomy, UblDNJ-New
J e r s e y Medical S c h o o l , Newark, IIJ and Case Western
R e s e r v e School of M e d i c i n e , C l e v e l a n d , OH.
V a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e ( V I P ) and n e u r o p e p t i d e Y (NPY)-like immunoreactivity in t h e
s u p r a c h i a s m a t i c n u c l e u s (SCN) o f t h e m u t a n t
a n o p h t h a l m i c mouse.
C i r c a d i a n rhythms g e n e r a t e d by t h e SCN a r e e n t r a i n e d t o e n v i r o n m e n t a l l i g h t - d a r k c y c l e s by a
d i r e c t p r o j e c t i o n from t h e r e t i n a . T h i s r e t i n a l
p r o j e c t i o n o v e r l a p s t h e d i s t r i b u t i o n of VIP
n e u r o n s , and NPY t e r m i n a l s which o r i g i n a t e from
t h e l a t e r a l g e n i c u l a t e n u c l e u s . The SCN o f t h e
a n o p h t h a l m i c mouse, however, r e c e i v e s no r e t i n a l
i n p u t . The o r g a n i z a t i o n o f t h e SCN, i n p a r t i c u l a r
VIP and N P Y i m m u n o r e a c t i v i t y , was s t u d i e d i n 3
a n o p h t h a l m i c a n d 2 C57BL/6 c o n t r o l mice.
Animals
were p e r f u s e d w i t h Bouins s o l u t i o n , t h e b r a i n s
were removed, embedded i n o a r a f f i n , and s e c t i o n e d
a t 1 2 u m i n t h e c o r o n a 1 p l a n e . T h e - p e p t i d e s were
l o c a l i z e d u s i n g t h e a v i d i n b i o t i n method ( V e c t o r
L a b s ) and a n t i s e r a p u r c h a s e d from Immunonuclear
C o r p . ( V I P ) and P e n i n s u l a L a b s ( N P Y ) . S e l e c t e d
s e c t i o n s were s t a i n e d w i t h c r e s y l v i o l e t t o det e r m i n e t h e l o c a t i o n and c y t o a r c h i t e c t u r a l o r g a n
z a t i o n of t h e SCN. The SCN i n t h e a n o p h t h a l m i c
mouse e x h i b i t s a h i g h d e g r e e o f v a r i a b i l i t y .
In
t w o a n i m a l s t h e r i g h t a n d l e f t SCN were asymmetr C
and were P a r t i a l l v d i s o l a c e d i n t o t h e m i d l i n e :
t h e S C N o f t h e t h i r d a n i m a l c o u l d n o t be l o c a t e d .
Numerous VIP c e l l s were p r e s e n t i n t h e SCN of t h e
f i r s t two a n i m a l s . T h e i r p e r i k a r y a were l a r g e
and r i c h l y s t a i n e d ; t h e i r d i s t r i b u t i o n , however,
was abnormal compared t o c o n t r o l m i c e . A r i c h
p l e x u s o f VIP f i b e r s w a s p r e s e n t t h r o u g h o u t t h e
n u c l e u s . N e i t h e r VIP c e l l s n o r f i b e r s c o u l d be
demonstrated i n comparable r e g i o n s of t h e t h i r d
brain.
HPY f i b e r s were a l s o p r e s e n t i n t h e
a n o p h t h a l m i c S C N , where t h e y a p p e a r e d t o be more
numerous t h a n i n c o n t r o l m i c e . ( S u p p o r t e d by
UMDNJ F o u n d a t i o n ) .
SAAD, Anuradha D., Abraham P. MATHEWS,' lgnatius P. TAN' and
Vincenza M. SORRENTINO,*Cornell University Medical College, N e w
York, New York. Mvosin thick filament stabilitv in the absence and
presence of C-orotein.
Previous studies by us have shown that myosin thick filaments exist
in dynamic equilibrium with a small but constant pool (critical
concentration) of soluble myosin. Using a fluorescence energy transfer
(FET) assay and electron microscopy we have previously demonstrated
that myosin molecules undergo rapid and extensive exchange between
filaments. This exchange is mediated by a soluble pool of myosin. In
this study we have examined the effect of C-protein, a major
component of thick myofilaments, on the ability of myosin to exchange
between filaments. Adult chicken pectoralis myosin and C-protein were
isolated and labeled with either donor (5-(2-((iodoacetyl)aminoethyl)
aminonapthalene-I-sulfonic acid) or acceptor (5-iodoacetamido
fluorescein)fluorochromes. To monitor exchange using FET, myosin
and C-protein were combined in a 1:l molar ratio and formed into
filaments containing either donor-labeled or acceptor-labeled filaments,
and the exchange of myosin monitored by the decrease in donor
fluorescence over time. Myosin exchange in filaments containing Cprotein was significantly lower than that in myosin-only filaments. To
further characterize the effect of C-protein on myosin exchange, we
measured the critical concentration of myosin in the absence or presence
of C-protein. Myosin was mixed with varying amounts of C-protein i n
a high ionic strength buffer (0.5M KCI, lOmM Imidazole, pH 6.8).
Filaments were then prepared by rapidly diluting into physiologic ionic
strength (0.125M KCI, lOmM Imidazole, pH 6.8). Filaments prepared
with and without C-protein were identical in morphology and length.
To examine the amount of soluble myosin present at equilibrium with
fully assembled filaments, myosin filaments with or without C-protein
were sedimented for 30 and 60 min at 100,000 x g in a n airfuge and the
amount of myosin remaining in the supernatant examined by a
quantitative ELISA assay using the anti-myosin monoclonal antibody
MF20. The amount of soluble myosin present at equilibrium with
myosin only filaments was -12ug/ml. When the filaments contained Cprotein in addition to myosin (Myosin:C-protein 2 5 1 or I : I ) a
significant decrease in the soluble pool (-6ug/ml) was observed. The Cprotein dependent decline in the soluble pool of myosin and reduction
in myosin exchange between filaments suggest a possible role for Cprotein as a stabilizer of myosin thick filaments. (Supported by The
Muscular Dystrophy Association and NIH-AM37653).
S
SANNER*, Cynthia, and Michael E. GOLDBERGER:
Department of Anatomy, Medical College of Pennsylvania,
Philadelphia, Pennsylvania, (Sponsored by Pat Levitt)
Gffect of ave of axotomv on survival of cat Clarke's neurons.
It has been shown in the adult cat Clarke's Nucleus (CN)
that the closer the axotomy is to the cell body the greater the
cell death and decrease in cell size (Loewy, 77). Unilateral
dorsal lateral column lesions a t T9 and C2 were performed on
neonatal (NOS) and adult (AOs) cats to determine if this
decrease in cell size and increase in cell death was influenced
by age as well. First the CN cells were counted and their areas
measured from ?O+ serial sectioned L3 cresyl violet stained
spinal cord using an image analysis system (Bioquant). They
were then divided into groups consisting of small (A), medium
(B), and large (C) cells using the criteria of Loewy (79). MAP2
was used to differentiate between smaller A cells and large glia.
Cells smaller than [email protected], presumably glial cells, were not counted.
Neonatal C N showed a greater decrease in cell size and cell
number than adult CN after either C2 or T9 lesion; with the
greatest decrease occuring in T9 lesioned NOS (59% cell loss). In
both NOS and AOs no C-cells remained after either T9 or C2
lesions. Changes in both B-cell number and size were seen in
AOs, but the greatest change was in NOS with an almost
com lete B-cell loss (98%) and a dramatic decrease in B-cell size
(63%!.
Though A-cells were altered the least of the three
groups the greatest loss was in T9 lesioned NOS (325%) and the
least in C2 lesioned AOs (10.5%). A-cell size was decreased in T9
lesioned NOS by 14% and in C2 lesioned AOs by 11%. The agerelated decrease in cell size and increase in cell death in CN
after axotomy seems to parallel changes seen with distance.
That there is death of cells is shown by the overall decrease in
cell number; however, some of the apparant decrease in cell
number could be due to shrinkage rather than death. The
greatest change appears to be in the C-cells as they are affected
by lesions of any distance or age. The influence of age on CN
may be explained by the postnatal development of collaterals
which might protect adult but not neonatal CN neurons from
the eCfect of axotomy. T h e realtionship of distance might be
due to the ossibility that collaterals arise scvcral segments
rostra1 to tge cell body.
Supported by grants NS24707 and NS15529.
S
SAFFERSTEIN*, H.T. and F.J. ROISEN, Department of
Anatomical Sciences and Neurobiology, University of Louisville
School of Medicine, Louisville, Kentucky. Dual modulatory role
of GM1 in NCAM exmession in the neural cell line. Neuro-2a.
89A
ABSTRACTS-AAA 103RD MEETING
The ability of exogenous ganglioside GM1 to induce the
differentiation of the Neuro-Pa neuroblastoma cell line has been
well documented. Previously, we have reportedthat exposure of
Neuro-2a to GM1 produces a linear pattern formation of the
neural cell adhesion molecule NCAM as revealed by
immunofluorescence microscopy. This pattern differedfrom the
distribution revealed when cells were differentiated by serum
deprivation or dibutyryl CAMPexposure. This study focused on
elucidating the molecular mechanisms involved in GM1-mediated
modulation of NCAM. With the addition of cytoskeletal altering
agents (cytochalasin D, Colcemid, or Taxol) in the growth media,
we were able to disrupt the organization of NCAM produced by
GM1 alone. However, the simultaneous addition of cytochalasin
D
Taxol in the presence of GM1 augmented the NCAM
distributionseen with GM1 alone. The effects of these agents on
the cytoskeleton (confirmed by TEM analysis) revealed an
underlying ultrastructuralbasis for the NCAM distribution seen in
ganglioside-induceddifferentiation of Neuro-Pa. The patterns
obtained appear to be influenced by a microtubule-directed
mechanism. Furthermore, SDS-PAGE separation and western
blot analysis studies indicated that the modulation of NCAM by
GM1 may be due to changes in the molecular species of NCAM
expressed under these conditions. Exposure of Neuro-Pa to
GM1 resulted in an increase in the 180 KDa form of NCAM (36%
to 65% of reactive product) and a decrease in the 140 KDa form
(29% to 9%, control vs. GMl treatment). Exogenous GM1
exposure of Neuro-Pa cells independently influenced two
interrelated events, i.e., cytoskeletal reorganization and direct
effects on NCAM distribution and changes in the synthesis of the
140KDa and 180 KDa species of NCAM.
This study
demonstratesthat gangliosideGM 1 modulates NCAM expression
through several independent but related mechanisms.
Supported by NIH Grant NS24524 and the Herbert Wald
Memorial Fund.
SCALIA, Frank, Department of Anatomy and Cell Biology,
SUM-Health Science Center at Brooklyn, Brooklyn N.Y.
Further study of the suecificity & stability of
aberrant optic nerve proiections to the olfactory
-.
We previously reported (Scalia, J C N , '87) that
regenerating optic nerve fibers can form synaptic
connections aberrantly in the olfactory cortex of the
adult frog (& piuiens) when the growing fibers are
surgically deflected into the cerebral hemisphere. Their
failure
to
synapse
in
other
cerebral
areas distributed along their path of outgrowth suggested
the operation of highly specific mechanisms.
In the
frog, the main and accessory olfactory bulbs project
differentially to the olfactory cortex and amygdala.
Therefore, we have further tested the suecificity of the
abnormal connection by initiating the cerebropetal
outgrowth in specimens surviving ablation of the
accessory olfactory bulb (N-10).
These frogs were
allowed to survive for 12-33 weeks. Additional cases
(N-12) were studied without ablation of the accessory
bulb. To further examine the stabilitv of the aberrant
projection, frogs in the latter group were allowed to
survive for shorter (12-33 wks, N-9) or longer ( 5 8 - 7 8
wks, N=3) periods. As reported previously, the aberrant
optic projection was promoted by severing the optic nerve
at the optic chiasma, and implanting the ocular stump
into the ventral surface of the ipsilateral cerebral
hemisphere. The status of the aberrant projection was
assessed with the utoradiographic tracing method, after
leucine and proline, and by the
eye-injection of 4I'
anterograde migration of HRP applied to the affected
LM and EM
optic nerve (after intraorbital transection).
observations were made on the latter material.
The
aberrant optic projection to the olfactory cortex formed
in each specimen, and persisted through all of the longer
survival periods. The optic fibers crossed the amygdala
in all cases, but did not form a terminal neuropil in
this structure in any case. (Supported by PHS grant no.
EY-05284).
SATYAPAL, Kapi 1 and Ganas MATHURA*,
Department o f
Anatomy, U n i v e r s i t y o f Durban-Westvi 1 l e , Durban, South
A f r i c a . The r e n a l v e i n o f t h e human kidney.
Since sparse l i t e r a t u r e e x i s t s on t h e p a t t e r n s o f
venous drainage o f t h e Human Kidney, a study was undertaken t o observe i f indeed any p a t t e r n d i d e x i s t as has
been we1 1 d e f i n e d i n t h e segmental a r t e r i a l d i s t r i b u t i o n
o f the Renal A r t e r y . F o r t y p a i r s o f u n f i x e d p o s t mortem
renal b l o c k d i s s e c t i o n s were used t o prepare r e s i n casts
o f the r e n a l vasculature. I t was observed t h a t :
- The l e n g t h o f t h e Renal Vein on t h e r i g h t was 2.1 cms
and l e f t 5.2 cms.
The I n f r a r e n a l Angle on t h e r i g h t was 54.50 and t h e
l e f t 49.20.
- The L e f t Renal Vein entered t h e I n f e r i o r Vena Cava
(1.V.C.)
a t a h i g h e r l e v e l i n 62.5% w h i l e t h e r i g h t was
h i g h e r i n 32.5%.
-
80th entered a t t h e same l e v e l i n 5%. These observations
d i f f e r e d from t h e standard t e x t s . A concept o f p r i m a r y
These were i d e n t i f i e d as
t r i b u t a r i e s i s introduced.
upper, lower and p o s t e r i o r .
The Renal Vein was d e f i n e d
from t h e union o f a t l e a s t two primary t r i b u t a r i e s t o t h e
I.V.C.
A c l a s s i f i c a t i o n o f these drainage p a t t e r n s i n t o
t h r e e types i s proposed. Type I (78.75%) where t h e upper
and lower j o i n e d t o form t h e Renal Vein i n 43.75% and had
an a d d i t i o n a l p o s t e r i o r t r i b u t a r y i n 35%.
Type I 1
displayed an average o f f o u r primary t r i b u t a r i e s j o i n i n g
a t one p o i n t t o form t h e Renal Vein i n 7.5%.
Type I11
showed t h a t as w e l l as t h e Renal Vein, 13.75% had an
a d d i t i o n a l independent p r i m a r y t r i b u t a r y which drained
I t was observed t h a t 42.5% o f
d i r e c t l y i n t o t h e I.V.C.
p a i r e d r e n a l casts d i s p l a y e d t h e same p a t t e r n on both
sides. The study f a c i l i t a t e d an update o f t h e anatomy o f
the Renal Vein and confirmed t h e existence o f a p a t t e r n
o f venous drainage.
SCHECHTER, Joel, Jolene WINDLE* and Pamela MELLON*,
Department of Anatomy and Cell Biology, University of
Southern California School of Medicine, Los Angeles,
California; and The Salk Institute, La Jolla, California.
Neural
_ _tissue
present within the anterior pituitarv of
transaenic mice.
Pituitary tumorigenesis has been followed in a
transgenic line of mice, c(-T7, containing the promoter
region of the full length human glycoprotein hormone a subunit gene linked to the SV40 T-antigen gene. Tumor
foci were identified within the anterior pituitary of
both male and female transgenic mice, and were
characterized by greatly hypertrophied gonadotropes and
A
thyrotropes, and hyperplasia with mitotic figures.
loss of parenchymal cell integrity was also observed, as
well as disruptions of tissue integrity at the interface
of the parenchyma with the pericapillary spaces and
vascular disruption including a variety of hemorrhagic
lakes. Tumor characteristics were not uniform throughout
the anterior pituitary and regions of the parenchyma
which appeared normal also were apparent. Neural tissue
was evident associated with the parenchyma1 cells within
the tumor foci. Non-tumor areas were devoid of neural
tissue as were all of the anterior pituitaries of
littermate controls. In no instance was there evidence
that neural tissue had invaded the anterior pituitary
from the neural lobe.
Giant neuronal-like cells were
intermixed within the anterior pituitary parenchyma and a
diverse population of axons were apparent.
Synaptoid
contacts were evident between the neuronal processes but
not between neurons and the granulated pituitary cells.
Neuronal-like cells had close associations with
folliculo-stellate cells which occasionally appeared to
enclose bundles of axons.
Some neuronal-like cells
90A
ABSTRACTS-AAA 103RD MEETING
contained a follicular lumen, raising the possibility
that the neuronal-like cells have arisen from the
folliculo-stellate cells.
These findings demonstrate
that profound deviations from the normal pituitary
cytoarchitecture can accompany pituitary tumorigenesis in
transgenic mice. (Supported by NIH 35904, HD 20377, and
MOD 1-1182.)
Usin
a methylene-blue stainin
techni ue
Do iel 7 1 9 8 9 ) introduced his morphoyogical cyasl
sigication of enteric neurons. This classif ication is still used in a revised and extended
version based on the silver-impregnation studies
Stach ( 1 9 8 9 ) . Using immunocytochemical techzfques, an increasing number of neuropeptides
have been localized in these enteric neurons
over the past 1 0 years. Only in rare cases,
however, does immunoreactivity of the neuronal
perikar a, either with or without colchicine
t;retreaxment. permit to recognize the whole
etailed shape and size of the cell bodies. In
this respect, the combination of neuro eptide
immunocytochemistry with the immunocytocgemical
visualization of a microtubule-associated
rotein, i.e. M A P Z , offers a gossibility to extend
our knowledge concerning t e neurochemical content of some of the morphologically distinct
neuronal cell t
the polyclonal antiserum
raised against MAfTe;sed
in this stud
allows a
clear visualization of the perlkar alLdendritic
domain. Double-labellin experimenxs with Substance P !SP) indicate tzat part of the type Ineurons in the myenteric plexus, whose projections run matnly in an oral direction, are
SP-immunoreactive. Besides GABA, 5-HT, enkephalin and VIP which have been demonstrated in
morphological t pe I-neurons in the astrointestina? tract oT smaller animals, ?be present
findings allow to add SP to the chemical codin
of these neurons. Together with other neurona?
markers
such as
neurofilament protein and
neuron-s ecific enolase. MAP2 may contribute to
bridge the gaE between the conventional morphological tec niques and peptide immunocytochemistry.
S
SCHMITZ, Robert J., Rebecca A. OGLE*, and Roy C. OGLE*,
Dept. of Anatomy and Cell Biology, Medical University of South
Carolina, Charleston, South Carolina. Characterization it iid
m .u t. i o n of the non-inteerin laminin recLaminin, a imjor
component of basement membranes, is involved in many basic cellular
functions including adhesion, migration, and neurite outgrowth -processes mediated via cell surface laminin receptors. Included within
this group of proteins are members of the integrin family and the high
affinity, non-integrin laminin receptor (Mr=67 kd ) which has been
isolated from a variety of cell types. cDNA clones for the latter
protein have been isolated and found, surprisingly, to code for an
amino acid sequence with a molecular weight of 32 kd (Segui-Real,
B. et al.). Variations in molecular size and unusual structural features
such as a lack of N-linked sugars and typical signal and
transmembrane sequences have led to doubts as to whether this
protein functions as a cell surface laminin receptor. To address this
problem, monoclonal and polyclonal antibodies were raised against
the native 67,000 form from Engelbreth-Holm-Swam (EHS) tumor
and to recombinant beta-galatosidase fusion proteins expressed in H
d.
hunofluorescent and biochemical studies verify that the 67/32
kd laminin receptor complex is present on the surface of a variety of
cell types such as murine melanoma cells, chicken fibroblasts, and
endothelial cells; and shows codisaibution with laminin on the cell
surface and polymerized actin filaments inside the cells. Furthermore,
western blot analysis with these antibodies of membrane extracts of
EHS tumor, whole two-day chick embros, developing chick brain,
and 10 day rat pup brain suggested that more than one size of nonintegrin laminin binding protein exists (Mr= 32,45, 55,67, and 110
kd). To explore further the possible existence of a family of laminin
binding proteins, microsomal preparations from EHS tumor and 14
day embryonic chick brain were subjected to laminin affinity
chromatography. After washing with 1 M NaCl, the column was
then eluted with 0.2 M GlyMCl pH 2.5. All of the above size
varients have been eluted from both membrane extractions, however
the 110 kd form appears to have the highest affinity for laminin. The
exact relationship of these immunologically related forms to the 67/32
kd laminin receptor complex remains to be determined, and will be the
focus of future experiments.
SCHULZ,* Mona Lisa, Deepak N. PANDYA, and M. Marsel
MESULAM. Department of Behavioral Neuroscience and Anatomy, Boston University School of Medicine, Boston: Harvard Neurological Unit, Beth Israel Hospital, Boston.
Fronto-parietal Connections in the Rhesus Monkey.
Previous studies have shown sequential projections
from inferior parietal lobule(1PL) to the frontal lobe.
The aim of the present investigation was to delineate the
precise nature of the fronto-parietal projection. Eight
cerebral hemispheres with WGA-HRP injections in different
subdivisions of IPL including the lower banks of the
intra-parietal sulcus(1PS) were used in this study. The
results demonstrate that the ventral postcentral gyms
(areas 3 and 1) receives limited projections from areas
4, 6,the frontal operculum (ProM) and the gustatory area.
The rostral IPL(areaPF) receives its main input from
areas 6 and 46 as well as minor projections from area 4,
the gustatory area and area ProM. The midportion of IPL,
area PFG receives predominant projections from area 46
and minor input from areas 4,6,8, and the gustatory area.
Caudal IPL, area PG, receives input from caudal 46 as well
as areas 8 and 6. Area cpt receives projections from
dorsal areas 46 and 8. Like area Opt, the projection to
the lower bank of IPS are derived chiefly from dorsal 46
and 8.
The three architectonic areas in IPL have distinct
and differential connections from the frontal lobe. MI,
area 6, the gustatory area, and ProM project to rostral
PF. The caudal region of IPL, area PG differs by receiving input from 46, 8, and 6. In contrast, the IPS (lower
bank) receives projections from areas 8 and 46 but not
area 6.
Differential patterns of frontal lobe projections to
subregions of IPL are in concordance with physiolcgic
properties of these areas. Areas 3,l and PF participate
in somatosensory processing, area PG in complex visuospatial computations and IPS cortex in oculomotor programning. Supported by ENRM Veterans Hospital, Bedford,
MA, NIH grants 16481 and 20285.
SCHWEISTHAL, M . R . Department of Anatomy, Cell
Biology
and
Neuroscience.
Oral
Roberts
University, School of Medicine, Tulsa, Oklahoma.
The effects of control diet. isocaloric etdiet and m t r o l diet olus ethano1 on a d v w
Sprague-Dawley rats were used to study the
effects o f ethanol on maternal rats and their
offspring following various diets and exposure
time on those diets. Female and male rats were
randomly divided into control and experimental
groups and matched according to weight. Control
and
experimental
animals
received
both
isocaloric and nonisocaloric commercial diets.
91A
ABSTRACTS-AAA 103RD MEETING
Time o f e x p o s u r e t o a l c o h o l r a n g e d f r o m 4 3 d a y s
P r i o r t o s a c r i f i c e and c o l l e c t i o n
t o 35 weeks.
o f b l o o d samples, m a t e r n a l and n e o n a t e r a t s w e r e
given a voluntary glucose drink.
Plasma i n s u l i n
l e v e l s f o r m a t e r n a l animals and t h e i r o f f s p r i n g
were c o n s i s t e n t l y
l o w e r than t h o s e f o r t h e
control
animals.
T h e r e was
indication o f
improvement i n one e t h a n o l g r o u p ( P F E ) f o l l o w i n g
p l a c e m e n t o n a new e t h a n o l d i e t ( E C ) .
Ethanol
t r e a t e d a n i m a l s showed r e t a r d e d g r o w t h a s w e l l
as a lower s u r v i v a l r a t e t h r o u g h o u t a l l t h r e e
m a t i n g s when compared t o controls.
Increased
e t h a n o l i n t h e d i e t h a d a more d r a m a t i c e f f e c t
On t h e
d u r i n g p r e g n a n c y than d u r i n g l a c t a t i o n .
o t h e r hand, t h e l o n g e r t h e e x p o s u r e t o e t h a n o l ,
t h e more d r a m a t i c e f f e c t s were s e e n i n t h e
l a c t a t i o n p e r i o d ; m a l e s appeared t o b e a f f e c t e d
more than f e m a l e s .
These r e s u l t s i n d i c a t e a
strong
relationship
between
alcohol
and
s i g n i f i c a n t l y r e d u c e d i n s u l i n r e s p o n s e and t h e
resulting
poor
development
and
growth
of
offspring.
d e m o n s t r a t e d t h a t i m p l a n t s of e n d o c h o n d r a l bone m a t r i x
i n d u c e bone f o r m a t i o n v i a e n d o c h o n d r a l o s s i f i c a t i o n i n
c o n t r a s t t o i m p l a n t s o f intramembranous bone m a t r i x
which i n d u c e o n l y intramembranous o s s i f i c a t i o n .
This
work s u g g e s t s t h a t the m a t r i x o f e n d o c h o n d r a l bone
d i f f e r s q u a l i t a t i v e l y from t h e matrix o f intramembranous
bone due t o t h e i r r e s p e c t i v e a b i l i t i e s t o i n d u c e
c a r t i l a g e a n d / o r bone f o r m a t i o n .
Biochemical a n a l y s i s
of t h e m a t r i c e s is necessary i n o r d e r t o f u r t h e r
c h a r a c t e r i z e their i n h e r e n t inductive p r o p e r t i e s .
In
c u r r e n t c l i n i c a l p r a c t i c e , e n d o c h o n d r a l bone m a t r i x i s
u s e d t o i n d u c e new bone f o r m a t i o n i n p a t i e n t s w i t h l a r g e
bone d e f e c t s a n d f r a c t u r e s . However, t h e r e a r e s e t t i n g s
where i t may b e a d v a n t a g e o u s t o r e p l a c e bone o r i g i n a l l y
formed
via
intramembranous
ossification with
intramembranous bone m a t r i x ( f o r example, f r a c t u r e s o f
t h e cranium).
SEITZ *, Jurgen, Meena KUMARI
and Gerhard AUMULLER,
Department of Anatomy and Cell Biology, Philipps-University, Marburg, Federal Republic of Germany. Sulfhvdrvl oxidase an immuno~
S
SCHWERDTFEGER; Susan Rae, Alvin M. EARLE and Lyal G.
LEIBROCK,' Departments of Anatomy and Surgery, University
of Nebraska Medical Center, Omaha, Nebraska. Correlation
between hvoothalamic discharee with cardiac arrhvthmias
following iniection of substances into the subarachnoid sDace.
Cardiac arrhythmias following subarachnoid hemorrhages are
well documented.
Evidence indicates that changes in
posterolateral hypothalamic discharge is a causative factor in
these cardiac arrhythmias. The purpose of this study was to
assess changes in various physiological parameters after
injection of neurotransmitters and platelets into the
subarachnoid space over the circle of Willis. Using the SpragueDawley rat model, five groups of ten animals underwent
injection into the subarachnoid space of whole blood, platelets
(buffy coat), norepinephrine, serotonin, or gamma-amino-nA control group was injected with
buteric acid (GABA).
balanced salt solution. Posterolateral hypothalamic multiple
unit activity, arterial blood pressure, intracranial pressure and
electrocardiogram recordings were monitored prior to and
following injection of substances into the subarachnoid space.
Serum magnesium levels were determined both before and a f t e r
injection. Cardiac arrhythmias were graded on a 0 to 4+
severity scale following injection. Differences between groups
were determined using a paired t test. Values were considered
significant when p ~0.05. Bradycardia was observed in all
groups. The most severe arrhythmias occurred in animals
injected with whole blood, platelets, serotonin and GABA.
Significa.:t decreases in multiple unit activity were observed in
experimental groups. Each experimental group exhibited a
statistically significant post-injection increase in serum
magnesium levels, while the control group did not.
histochemical marker for lsostnatal differentiation of rat testis.
Sulfhydryl oxidase (SOx) was described as a secretory protein in rat
seminal vesicles. Using antibodies against this protein we have
shown its presence in mitochondria of spermatogenic cells in rat,
hamster and even human. In the rat, SOx immunoreactivity was specifically associated with pachytene spermatocytes from stage I onwards. During spermiation, immunoreactive SOx was retained in the
residual bodies. SOx immunoreactivity was traced in paraffin sections
of immature rat testis starting from Day 5 after birth onwards till Day
30. SOx immunoreactivity was detected at Day 5 in secondary gonocytes and prespermatogonia. In A-spermatogonia, SOx immunoreactive material was aggregated on one side of the cell.
No SOx
imrnunoreactivity was present in intermediate and B-spermatogonia.
With the appearance of pachytene spermatocytes in the tubules, SOx
immunoreactivity was seen again. Thereafter, the distribution pattern
of SOx was same as in adult animals. There was a positive, but diffuse staining in Sertoli cells at Day 5. From Day 10 onwards a slight
aggregation of immunoreactivity was observed. In 20 days old animals, only the basal portion of the Sertoli cells displayed a positive
immunoreaction. lmmunoreactive SOx disappeared from Sertoli cells
by Day 26. Our results indicate that SOx can be used as a marker
enzyme for postnatal differentiation of spermatogenesis. (Supported
S
SCOTT,* Caroline K e l l y a n d James Anderson HIGHTOWER,
Department o f Anatomy, C e l l Biology and N e u r o s c i e n c e s ,
U n i v e r s i t y o f S o u t h C a r o l i n a , Columbia, S o u t h C a r o l i n a .
The m a t r i x of e n d o c h o n d r a l bone d i f f e r s a u a l i t a t i v e l v
from t h e m a t r i x o f intramembranous bone,
Osseous t i s s u e d e v e l o p s v i a two d i s t i n c t l y d i f f e r e n t
processes:
endochondral
ossification
and
intramembranous o s s i f i c a t i o n .
The p r e s e n t s t u d y w a s
designed t o t e s t t h e hypothesis t h a t b o t h types o f
o s s e o u s t i s s u e c o n t a i n u n i q u e i n d u c i n g f a c t o r s f o r the
promotion o f bone development.
P r e v i o u s w o r k e r s have
s u g g e s t e d that i m p l a n t s of d e m i n e r a l i z e d e n d o c h o n d r a l
a n d intramembranous bone m a t r i c e s i n d u c e e n d o c h o n d r a l
ossification.
They c o n c l u d e t h a t t h e bone growth
promotion p r o p e r t i e s o f t h e r e s p e c t i v e matrices are very
s i m i l a r . However, r e c e n t work from o u r l a b o r a t o r y u s i n g
morphological
and
radiolabeling
techniques
has
by the Deutsche ForschungsgemeinschaftSe 370/2-6).
Severin*, C h a r l e s M . , Frank C . K a l l e n , J u d i t h H.
Tamburlin* and George J. Alker, Jr.**, Departments o f
Anatomical Sciences and **Radioloav, State U n i v e r s i t y
o f New York a t Buffalo. A course 7-n sectional anatomy
f o r r a d i o l o a i c imaainq.
With the advent o f comouter tomoaraohv. nuclear
magnetic resonance, and p o s i t r o n emis;ion
iransaxial
tomography s c a n n i n g , as w e l l as u l t r a s o u n d , an
understanding o f sectional anatomy has become basic t o
proper i n t e r p r e t a t i o n o f these imaging modalities. The
t i m e a v a i l a b l e i n c u r r e n t gross anatomy courses does
not a l l o w f o r adequate i n s t r u c t i o n on s e c t i o n e d
specimens, l e t alone how these r e l a t e t o s t a t e - o f - t h e -
92A
ABSTRACTS-AAA 103RD MEETING
a r t scanning techniques.
Furthermore, student
i n e x p e r i e n c e makes i t premature t o o f f e r such a course
d u r i n g t h e medical o r dental school years. Therefore a
course has been d e s i g n e d f o r r e s i d e n t s and
p r a c t i t i o n e r s w h i c h r e v i e w s g r o s s , s e c t i o n a l and
developmental anatomy, and r e 1 a t e d r a d i o l o g y , i n each
r e g i o n o f t h e body, w i t h a focus on relevance t o common
c l i n i c a l problems amenable t o r a d i o l o g i c d i a g n o s i s .
P a r t i c i p a n t s have met f o r t h r e e hours, once a week, f o r
16 weeks. Each session began w i t h a l e c t u r e conducted
j o i n t l y by an anatomist and a c l i n i c i a n . The c l i n i c i a n
was u s u a l i y a r a d i o l o g i s t b u t c e r t a i n subjects d i c t a t e d
o t h e r e x p e r t i s e , e.g. a c a r d i o l o g i s t p a r t i c i p a t e d t o
consider echocardiography. A f t e r l e c t u r e , t h e class
was d i v i d e d i n t o s m a l l g r o u p s and r o t a t e d t h r o u g h
l a b o r a t o r y demonstrations o f i n s i t u morphology,
s e c t i o n e d specimens f r o m o u r r e f e r e n c e c o l l e c t i o n s ,
re1 a t e d r a d i o g r a p h i c images and d i s c u s s i o n s o f
developmental processes which determine morphology and
common v a r i a t i o n s . Problem s o l v i n g sessions f o l l o w e d ,
i n v o l v i n g both c l i n i c a l anatomy and imaging, t o p r o v i d e
an o p p o r t u n i t y t o a p p l y t h e d e s c r i b e d and demonstrated
p r i n c i p l e s . I n i t i a l r e a c t i o n o f course p a r t i c i p a n t s
has been p o s i t i v e . Furthermore, comments by h o s p i t a l
s t a f f assigned t o t r a i n our p a r t i c i p a t i n g r e s i d e n t s
s u g g e s t t h a t t h i s c o u r s e has i n f a c t i n c r e a s e d t h e
knowledge and c o n f i d e n c e o f t h e s e r e s i d e n t s i n t h e
i n t e r p r e t a t i o n o f such scans.
SEVERSON, Arlen R., Elizabeth Kohn' and Thomas E Huntley,'
Departments of Anatomy and Cell Biology, and Biochemistry and
Molecular Biology, University of Minnesota, Duluth, School of
Medicine, Duluth. Minnesota. Fffect of mixtures of min-d
.
. bone matrix on alkaline DhosDhatase bone
and
matrix and -d
..
brtrate-resistant acid m a t a s e activity.
The type of cell recruited to subcutaneous sites of implanted
devitalized bone matrix is dependent on the implant mineral content.
Mineralized bone matrix (MBM) recruits multinucleated giant cells
(MGCs) and osteoclasts, while demineralized bone matrix (DBM)
recruits mesenchymal cells which differentiate into cartilage and
osteogenic cells, initiating both endochondral and intramembranous
bone formation. Osteoclasts can be differentiated from the MGCs by the
presence of tartrate-resistant acid phosphatase (TRAP). The present
study was undertaken to examine the role of osteogenesis on the
recruitment of osteoclasts and the presence of TRAP in the implants.
Animals received on day 0 one of 5 types of subcutaneous implants
(DBM, MBM. 1 part DBM to 3 parts MBM, 1 part DBM to 1 part MBM,
or 3 parts DBM to 1 part MBM). On day 12 the implants were
removed and processed for light microscopic study, and for
histochemical and biochemical analysis of alkaline phosphatase (AP)
and TRAP activity. Histochemical staining localized AP activity in the
cell membranes of cartilage and osteogenic cells, and in the adjacent
matrix. TRAP was found primarily in the cytoplasm of osteoclasts.
MGCs lacked TRAP activity. Alkaline phosphatase activity was highest
in the DBM implants and decreased as the proportion of DBM particles
in the implant decreased, with the lowest values in the MBM implants,
indicating a relationship between alkaline phosphatase activity and the
amount of DBM particles implanted. TRAP activity was greater in the
MBM than in the DBM. However, TRAP levels were even higher in the
1 DBM : 3 MBM and highest in the 1 DBM : IMBM mixtures. This
suggests that some factor in the DBM particles or some secretion of
differentiating mesenchymal cells stimulates the formation of TRAP.
Although the MBM particles were few in the 3 DBM : 1 MBM implants,
the implants exhibited TRAP activity only slightly less than the MBM
particle implants. This observation further supports the hypothesis
that some factor associated with the implantation of DBM stimulates
osteoclast formation and the formation of TRAP. (Supported by the
Duluth Clinic Education and Research Foundation).
SHAH, Anu and V. Sahgal*, Neuromuscular Studies Unit,
Rehabilitation Institute of Chicago, Illinois. Morphometric
studies of normal muscle mitochondria in a variety of
mammalian species.
In earlier studies, we showed that the size and shape of
subsarcolemmal and intermyofibrillary mitochondrial populations were significantly different in Ischemic and Inflammatory myopathies while they were similarly affected in
Dystrophy and metabolic muscle disorders (Anat.Record 223:
1048,1989). In this study we describe the morphometric features of mitochondria of normal adult muscles(Human,monkey,
cat and rat) and compare them with the mitochondria of immature (65 days) puppy diaphragm to study the effects of
age on mitochondrial structure. The mitochondrial mesurements (size, shape and area) were carried out as described
earlier in two regions as well as two fiber types of (1)
Human and monkey quadriceps, (2) Cat soleus (pure type 1)
and caudofemoralis (pure type Z ) , (3) Rat soleus (predominant type 1) and extensor digitorum longus-EDL(predominant
type 21, and ( 4 ) immature puppy diaphragm. The statistical
t tests and correlation studies were used for comparisons
of mitochondrial populations in two fiber types and two
regions of adult muscles as well as comparison of various
adult muscles with the immature muscle. The mitochondrial
size and area were significantly smaller (p=O.OOl) in human
quadriceps as compared to monkey quadriceps and also immature puppy diaphragm in both fiber types and regions of
fibers. The mitochondrial shape was also different(p=0.001)
(oval to round) in human quadriceps but only in both regions. The mitochondrial size, shape and area of monkey
quadriceps were not significantly different (p=O.OOl) from
immature puppy diaphragm in both fiber types and regions.
These differences could be related to species variation.
The studies on slow and fast muscles of cat and rat mitochondria are in progress. The observations so far suggest
that the normal mitochondrial size in human skeletal muscle
is smaller than the monkey. We believe that further 3-D
reconstruction studies on mitochondrial structure would
give a better insight into the organization of mitochondria
in a cell system and its relation to cell functioning.
SHAW*,
CHRIS, LINDA CAMERON*, and ANITA HENDRICKSON,
Dept. o f Ophthalmology, Univ. o f B r i t i s h Columbia and
Depts. o f B i o l o g i c a l S t r u c t u r e and Ophthalmology, Univ.
o f Washington,
Seattle.
(Sponsored by Charles E .
Slonecker).
Prenatal and oostnatal develooment o f GABA
and b e n z o d i a z w i n e r e c e o t o r s i n monkev v i s u a l c o r t e x .
Gamma-aminobutyric a c i d (GABA) has been i m p l i c a t e d as
p l a y i n g a c r u c i a l r o l e i n both c o r t i c a l i n f o r m a t i o n
processing and i n normal and abnormal development.
In
order t o examine the ontogenesis o f GABA and r e l a t e d
receptors,
we
have
used
conventional
in
vitro
autoradiographic
methods
to
study
the
laminar
GABAB.
distributions
and c h a r a c t e r i s t i c s o f GABAA,
and benzodiazepine ( B Z ) receptors a t d i f f e r e n t ages o f
pre- and p o s t n a t a l monkey s t r i a t e cortex (area V l ) .
Twenty micron t h i c k c r y o s t a t sections o f s t r i a t e c o r t e x
were incubated u s i n g [3Hl-muscimol,
C3HI-baclofen.
or
[3HI-flunitrazepam
as described p r e v i o u s l y (Shaw e t
GABAA and BZ receptors were present from
a l . , 1986).
t h e e a r l i e s t age examined, F 7 2 days, w h i l e GABAs
r e c e p t o r s were present from a t l e a s t F 126 days. GABAA
receptors were concentrated i n the c o r t i c a l p l a t e as were
BZ receptors which were a l s o found s u b c o r t i c a l l y . Both
r e c e p t o r p o p u l a t i o n s were d i s t r i b u t e d i n the f i n a l
b i n d i n g p a t t e r n s h o r t l y a f t e r b i r t h , i n which the densest
b i n d i n g was found i n lamina 4CO.
GABAs receptors were
most densely concentrated above and below lamina 4 a t a l l
ages.
Q u a n t i t a t i v e measurements o f GABAA receptor
number and a f f i n i t y showed an apparent peak i n number and
the lowest a f f i n i t y a t 18 weeks p o s t n a t a l . The present
data demonstrate t h e e a r l y p r e n a t a l occurrence o f some o f
t h e receptors associated w i t h neuronal i n h i b i t i o n i n the
ABSTRACTS-AAA 103RD MEETING
s t r i a t e c o r t e x o f t h e monkey. The observation t h a t these
r e c e p t o r p o p u l a t i o n s a r e present p r i o r t o t h e a r r i v a l o f
g e n i c u l a t e o r c a l l o s a l a f f e r e n t s t o t h e c o r t e x and b e f o r e
most synaptogenesis has occurred (Rakic e t a l . 1986).
suggests t h a t t h e i n i t i a l expression o f t h e receptors
does n o t r e q u i r e such synaptic contacts. Changes i n
GABAA c h a r a c t e r i s t i c s w i t h age may suggest, however,
t h a t some aspects o f r e c e p t o r ontogenesi s a r e dependent
on a p p r o p r i a t e s y n a p t i c a c t i v i t y .
SHEA, J.R., T.J. ALLARDYCE. and K.L. WAPNER,. Departments
of Orthopaedic Surgery a nd Anatomy, Thomas Jefferson
University, Philadelphia, Pennsylvania. Utilization of Iona flexor
tendons
f or
tibialis
Dosterior
tendon
reconstruction:
intertendinous CO nnections within the second muscular laver of
the sole.
Disruption of tibialis posterior (TP) tendon may lead to
disabling, progressive talipes equinovalgus.
Reconstructive
procedures to reinforce or replace the disrupted tendon utilize t h e
long flexor tendons, flexor hallucis longus (FHL) or flexor
digitorum longus (FDL). I n order to isolate a nd completely
mobilize these long flexor tendons in the sole, intertendinous
connections between them must be severed.
Although the
presence of such connections is generally known, their
configuration is variable a nd requires elaboration.
An
appreciation of their variational patterns is essential for a simple
and satisfactory reconstructive procedure.
T o delineate all
intertendinous connections, observations were made upon en bloc
dissections of the second muscular layer of the sole in 85 a d u l t
feet f r om embalmed cadavers.
These reveal th a t a n
intertendinous connection between F HL a nd F DL is quite
Reciprocal
constant; seldom is there no connection (2/85).
interconnections a r e sometimes observed between the long flexor
tendons (26/85). Musculotendinous faseieuli from the quadratus
plantae may contribute t o the common digital tendon (6/85), o r
digital tendons 11, 111, IV, a nd V a nd may yield accessory
attachments for lumbricales 2, 3, a nd 4. Tracts of annectent
fasciculi f r om t he hallucial tendon may contribute to digital
tendons 11, 111, and I V a n d may yield accessory attachments f o r
lumbricales 1, 2, 3, a n d 4. These observations suggest that the
FDL tendon may be more suitable than the F HL tendon f o r
repair of the ruptured T P tendon.
S h e r i f , M.F., Al-Khawad, A * . and Thomas, L*.
Anatomy Department, F a c u l t y of Medicine, Kuwait
U n i v e r s i t y . On t h e c e n t r a l Connections of t h e
Trigeminal Mesencephalic Root Neurons and
S i g n i f i c a n c e of Synapses on them : H i s t o c h e m i c a l
and R e t r o g r a d e L a b e l i n g Study w i t h H o r s e r a d i s h
P e r o x i d a s e (HRP).
I n t h r e e g r o u p s of 6 young a d u l t Wistar r a t s ,
t h e c a u d a l p o r t i o n of t h e t r i g e m i n a l mesencephal i c n u c l e u s (MesV), t r i g e m i n a l motor n u c l e u s and
cerebellar f o l i a 11, I11 and I V ( a t t h e vermal
r e g i o n ) were s t e r e o t a x i c a l l y i n j e c t e d w i t h 2 u l
of 50-60% HRP i n Ringer s o l u t i o n . S e r i a l s e c t i o n s
from t h e f o r e b r a i n , b r a i n s t e m and t r i g e m i n a l
g a n g l i o n were p r o c e s s e d f o r HRP r e a c t i o n .
D i f f e r e n t r e g i o n s of M e s V from a n o t h e r 4 a n i m a l s
were s u b j e c t e d t o a c e t y l c h o l i n e s t r a s e (AchE)
r e a c t i o n and p r o c e s s e d f o r t r a n s m i s s i o n e l e c t r o n
microscopy (TEM). The r e s u l t s r e v e a l e d t h a t some
MesV neurons were l a b e l e d f o l l o w i n g HRP i n j e c t i o n i n t o t h e cerebellum and t r i e e m i n a l motor
n u c l e u s . I n case of HRP i n j e c t i o ; i n t o t h e M e s V ,
l a b e l i n g w a s d e t e c t e d among some neurons i n t h e
g a n g l i o n i c l a y e r of t h e c o r t e x of e p s i l a t e r a l
p a r i e t a l l o b e (mainly a r e a s 39, 4 0 and 2 ) ,
~~~
~~~
93A
medullary v e n t r a l r e t i c u l a r n u c l e u s , and t r i g e m i n a l g a n g l i o n . A t TEM l e v e l , about 700 p r o f i l e s
from d i f f e r e n t M e s V n e u r o n s , s u b j e c t e d t o AchE
r e a c t i o n were examined. Dense enzymatic r e a c t i o n
p r o d u c t w a s o b s e r v e d a t t h e s y n a p t i c c l e f t s of
56% of t h e t o t a l number of s y n a p s e s s p o t t e d i n
200 p r o f i l e s . These f i n d i n g s confirmed t h a t t h e
MesV p r o j e c t s t o t h e c e r e b e l l u m and t r i g e m i n a l
motor n u c l e u s and s u g g e s t t h a t i t r e c e i v e s
a f f e r e n t i n p u t from t h e c e r e b r a l c o r t e x and r e t i c u l a r f o r m a t i o n , p r o b a b l y a l o n g t h e c o r t i c o and
r e t i c u l o - b u l b a r s y s t e m s , which e x e r c i s e modulat o r y f u n c t i o n . I n a d d i t i o n , some M e s V neurons
may a c t a s i n t e r n u n c i a l ones r e c e i v i n g a f f e r e n t
f i b e r s from t h e t r i g e m i n a l g a n g l i o n .
(Supported by Kuwait U n i v e r s i t y Grant No.MA 018)
SHEW, R.L.,
R.E.
PAPKA and D . L .
McNEILL,
Department.
of
Anatomical
Sciences,
OUHSC,
Oklahoma C i t y , Oklahoma. C a l c i t o n i n g e n e - r e l a t e d
peptide i n t h e r a t u t e r u s :
P r e s e n c e .in n e r v e s
---______________
and e f f e c t s on u t e r i n e c o n t r a c t i o n .
The i n f l u e n c e of c a l c i t o n i n g e n e - r e l a t e d
p e p t i d e (CGRP) on r a t u t e r i n e a c t i v i t y was
examined
in
concert
with
the
anatomical
d i f l t . r i b u t i o n o f CGRP-immunoreactive f i b e r s i n t h e
uterus.
CGRP was l o c a l i z e d i n n e r v e f i b e r s ;
t h e s e p e p t i d e - c o n t a i n i n g n e r v e s were abundant
t h r o u g h o u t t h e mesoinetrium o f the u t e r i n e h o r n
and appeared t o i n n e r v a t e mesometrial smooth
muscle.
I n t h e u t e r i n e w a l l , immunoreactive
f i h e r s were p r e v a l e n t i n t h e myometrium and
endometrium a s w e l l a s i n t h e e n d o c e r v i x . F i b e r s
i n t h e endometrium and e n d o c e r v i x formed a p l e x u s
s u h j a c e n t t o the e p i t h e l i u m and some p e n e t r a t e d
t h e e p i t h e l i u m a s i n t r a e p i t h e l i a l f i b e r s . CGRPimmunoreactive
nerves
in
the
myometrium
p a r a l l e l e d t h e smooth muscle f a s c i c l e s and were
i n t i m a t e l y a s s o c i a t e d w i t h t h e muscle c e l l s . The
a c t i o n o f CGF.P (10.' t o 10-6 M ) on a c e t y l . c h o l i n e
([email protected]" or lO-* M ) - s t i m u l a t e d u t e r i n e a c t i v i t y was
examined
vitro.
Exogenously a p p l i e d CGRF
induced
a
dose-dependent
relaxation
of
acetylchcline-stimulated u t e r i n e c o n t r a c t i o n s .
CGRP hacl no e f f e c t on b a s e l i n e u t e r i n e t e n s i o n .
The l o c a l i z a t i o n of CGRP-immunoreactivity i n
n e r v e s and t h e r e l a x i n g e f f e c t of t h i s p e p t i d e
s u g g e s t a r o l e f o r CGRP-containing n e r v e f i b e r s
in
t.he
regulation
of
uterine
activity.
[ S u p p o r t e d by OU Medical Alumni G r a n t s and NIH 1
P.01 NS225261
~~
0 SHINAR, Yael', Wendy BATTISTI, Michal SCHWARTZ', Pat
LEVITT and Marion MURRAY*, Department of Anatomy,
Medical College of Pennsylvania, Philadelphia, Pennsylvania.
Temuoral and sDatial transitions in non-neuronal cells,
chondroitin sulfaie Droteonlvcan and embrvonic NCAM
immunoreactivity during reeeneration of the -goldfish oDtic
W.
Axons of the goldfish optic nerve re enerate readily
following crush whereas in mammals CN! axons fail to
regenerate following a similar injury unless provided with a
supportive environment. Since the environment ap ears to be
crucial to the success of regeneration, we wished to ctaracterize
the neuronal and non-neuronal contributions to the molecular
environment of the normal and regenerating optic nerve. We
examined the distribution of non-neuronal cells and
characterized the expression of two molecules involved in
94A
ABSTRACTSAAA 103RD MEETING
neuron-glia interaction, chondroitin sulfate proteoglycan
(CSPG) and embryonic NCAM (eNCAM), following optic
nerve crush in the goldfish. I n the intact nerve, CSPG
immunoreactivity is present but eNCAM reactivit is not
detectable. In retinas explanted 7 days post crush, CSPG
antibody stained neurons and their neurites, indicating a
neuroilal source for this molecule. Within one week following
crush when the axons are degenerating distal to the crush and
regeneration initiates from the proximal stump CSPG
reactivity disappears completely from the crush site but there is
a n accumulation of glial cells, and eNCAM reactivity increases
at the crush site. Two to three weeks following crush, glial cells
are densely packed adjacent to regenerating axons. Intense
CSPG immunoreactivity a peared to be associated with
regenerating axons and low revels of eNCAM expression were
seen associated with those glial cells affiliated with
regenerating axons. The differential expression of CSPG and of
eNCAM suggests that the molecules play different roles in
regeneration. eNCAM appears t o be associated with nonneuronal cells and is expressed during regeneration. CSPG
appears to be associated with neurons and Its levels increase
during regeneration.
Supported by NIH grant NS16556.
SIMARD, ThCrBse and CERQUEIRA,* Esem, Laboratoire
d'anatomie, Facult6 de mCdecine, UniversitC de MontrCal,
Montreal, Canada. Natural indeuendency of the neuromusmlar svstem of human's thumb applied to surface anatomy.
The morphologic substratum of diverse functions attributed
to each muscle is a necessary foundation knowledge which is
practically unknown. This study endeavors to define the
neuromuscular functional system of hability of the thumb and
to relate the surface anatomy. Embalmed human specimens
without neurologic disease, were used to demonstrate the
specific groups of muscle, having their own insertions, and to
establish nerve projections and sites of hila. Among muscles
of the forearm, the Abductor Pollicis Longus has a tail shape
made of juxtaposed bipennate, unipennate and flat types
muscle groups; this suggest that they act independently in
different angles of abduction, extension and supination of the
thumb. They are innervated by single twigs from lateral and
intermediate rami of the lateral trunk of the posterior branch
of radial nerve. The Flexor Pollicis longus is made of superficial and deep short muscle fibers. In addition, there are
particularly short muscle fibers which terminate on the tendon
itself. These groups are organized to have their own role on
specific joints of the thumb. Each group is well innervated
by a proper branch of the anterior interosseus nerve. In the
hand, the deep ulnar nerve shows an oblique projection and
gives twigs to each group of the first dorsal interosseus muscle.
In the thenar region, the motor branch for the median nerve
gives rami, two of which in opposite direction are specific for
the multi-shaped Abductor Pollicis Brevis and Opponens
Pollicis groups. Surface anatomy under these theoretical bases
needs to be completed with individual parameters applied to
lively subjects. The finding of the muscle groups of the thumb
should serve to describe their independent role in each fine
movement. Granted from the FRSQ an CNPq from Brazil.
SINHA, A.A., O.F. DELEON*, M.J. WILSON* and B.F. SLOANE*,
Res. Ser., VA Medical Center and Depts. of Genetics & Cell Biol., Lab.
Med. & Pathology, Univ. of Minnesota, Minneapolis, Minnesota, and
Dept. of Pharmacology, Wayne State Univ., Detroit, Michigan.
hvbridization of a biotinvlated olieonucleotide catheosin B txobe in human
human prostate. We modified the techniques of Brigatti et al. (Virol.
126:32-50, 1983), Singer et al. (Bio.Tech. 4:230-243, 1986) and Angerer
et al. (In: In Situ Hybridization: Applications to Neurobiology, Valentino
et al., editors, Oxford Univ. Press, New York, 1987, pp 42-69) to
localize CB mRNA in prostate sections by in situ hybridization. We
synthesized the CB probe using a Biosearch Model 8750 Oligonucleotide
Synthesizer (Multi GenjBiosearch,Nevada, CA) and end-labeled it with
biotinylated dUTP (Bio-11-dUTP,Enzo Biochem., New York, NY) and
terminal transferase (Boehringer Mannheim, Indianapolis, IN).
Unincorporated bases were removed by spun column chromatography
through sephadex G-10. Prostate samples frozen in liquid nitrogen were
embedded in OCT (Lab Tek Products, Miles Lab., Naperville, IL),
sectioned with a cryostat, mounted on p l y L-lysine coated slides,
alkylated, wetted for 5' at 37OC with 25 to 501.11of hybridization mixture
(HM) containing 4XSSC (lXSSC=lSOmM Nacl, 15 mM Na Citrate, pH
7.2), 50% deionized fomamide, 10%dextran sulphate, 1X Denhardt's
solution, 0.025% yeast tRNA, 0.05% denatured salmon testis DNA, 1mM
RNA-vanadyl complexes and 3 nghl oligodeoxy thymidine-12mers
(Sigma, St. Louis, MO). Each section was hybridized with HM plus
probe covered with parafilm for 4 hours in humidified trays at 37OC. All
sections were washed with 2XSSC (several changes) and phosphate
buffer saline, pH 7.4. CB probe was localized using rabbit anti-biotin
(EnzoBiochem), goat anti-rabbitsecondary antisera and avidin biotin
complex procedures (Vectastain kit, Vector Labs., Burlingame, CA). The
preliminary result showed localization of CB mRNA in many basal and
some columnar cells of normal and hypertrophic prostates, but none in the
control sections. This research was supported by VA Research Funds.
S
SMITH, Jodi L. and Gary C. SCHOENWOLF. Departmcnt of Anatomy,
University of Utah School of Mcdicine, Salt Lake City, Utah. Eurther
$vidence of extrinsic forces in neural fold elevation .
It is likely that neurulation is driven both by intrinsic and extrinsic forces,
although it is generally espoused that only intrinsic forces are.required.
Intrinsic forces could be generated by wedging of neurepithelial cells; extrinsic
forces by growth and expansion of non-neurepithelialtissues adjacent to the
neural plate. Our previous studies have shown that during elevation of the
neural folds, significant neurepithelial cell wedging occurs only within the
median hinge point (MHP), the midline region of neural plate anchored to the
notochord. In addition. it has been shown that elevation of the neural folds
still occurs when MHF' cells are.prevented from becoming wedge-shaped by
removing the cells that give rise to the notochord. the structure that induces
wedging of MHP cells. In contrast, neural fold elevation fails to occur when
the neural plate is isolated from adjacent non-neurepithelialtissues, although
MHP cells become wedge-shaped and the midline neural plate fumws.
Collectively. these results suggest that elevation of the neural folds is not
driven by neurepithelial cell wedging but rather is driven by extrinsic forces
likely generated by adjacent non-neurepithelial tissues. However, it could be
argucd in the first expcriment that in the absence of localized neurepithclial
cell wedging, compensatory and atypical cell wedging occurred uniformly
throughout the neural plate, providing forces adequate for neural fold elevation.
Likewise, it could be argued in the second experiment that the microsurgical
manipulations carried out to achieve separation of the neural plate and adjacent
non-neurepithelialtissues damaged the neural plate to the extent that the neural
folds were unable to elevate. If extrinsic forces generated by adjacent nonneurepithelial tissues drive neural fold elevation, then the neural folds should
still elevate after the removal of MHP and flanking neurepithelial cells,
provided that adjacent non-neurepithelial tissues =main intact. The following
experiment was performed to ascertain whether extrinsic forces drive neural
fold elevation. MHP cells. the underlying notochord, Hensen's node (the
rudiment of the notochord and some MHP cells),and lateral neurepithelial
cells flanking the MHP were excised from chick embryos prior to neural fold
elevation, splitting the neural plate into two separate lateral pieces. In such
embryos, the neural folds still formed and underwent full elevation. Thus,
microsurgery alone does not cause damage sufficient to prevent neural fold
elevation. Moreover. the two lateral pieces of neural plate changed their
orientation from horizontal to vertical rather than merely curling uniformly
across thcir width. Hence, compensatory and atypical cell wedging cannot
explain neural fold elevation in such embryos. We conclude that wedging of
MHP cells does not drivc neural fold elevation and that non-neurepithelial
tissues adjacent to the neural plate provide extrinsic forces sufficient to
generate this process. This research was supported by NIH grant no. NS
18112.
m.
Our objective was to localize a 25-base biotinylated oligonucleotide
cathepsin B (CB) "sense" DNA probe in normal and benign hypertrophic
SMITH, L i z a b e t h and William M . FALLS, Department o f
Anatomy, Michigan S t a t e U n i v e r s i t y , E a s t L a m i n g , Michigan.
95A
ABSTRACTSAAA 1 0 3 MEETING
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S
Thalamic projections from neurons in rat trigeminal nucleus
oralis.
Rat trigeminal nucleus oralis (Vo) contains ventrolatera1 (VL), dorsomedial (DM), and border zone (BZ) subdivisions. Earlier studies (Kruger et al., 1977; Fukushima
and Kerr, 1979) have shown sparse projections from Vo to
the contralateral ventral posteromedial thalamic nucleus
(VPM). This light microscopic study employed the anterograde transport of Phaseolus vulgaris leucoagglutinin
(PHA-L) to determine the overall pattern, organization and
morphology of efferent axons from neurons in each Vo subdivision to VPM and other diencephalic nuclei. Injections
into VL and BZ resulted in discrete patches of labeled
parent axons and terminal arbors in contralateral VPM.
Labeling was also noted in the posterior thalamic nuclei
(PO) and zona incerta (ZI) bilaterally, with a contralateral predominance, and the parafascicular nucleus (PF)
contralaterally. Injections confined to DM also resulted
in patchy labeling of parent axons and terminal arbors in
VPM. This labeling was bilateral with the greatest density
contralaterally. Bilateral labeling with a contralateral
predominance was noted in PO, ZI, PF and the centrolateral
thalamic nucleus (CL). Injections into all three Vo subdivisions resulted in labeling in the centromedian thalamic
nucleus (CM). Morphological differences in DM, VL and BZ
efferent axons within each target nucleus were based on
parent axonal diameter, terminal arbor branching pattern,
and bouton density and size. Efferents terminating in VPM,
PO and ZI were of more than one type, while no more than
one type was found in PF, CL and CM. These results suggest
that the rat Vo trigeminothalamic projection is more extensive than previously indicated. In addition, neurons in
each Vo subdivision are involved in relaying orofacial
input to VPM as well as other thalamic and diencephalic
nuclei. Based on axonal morphology, inputs from neurons in
each Vo subdivision to VPM, PO and ZI may be of different
typeswhilethose to PF, CL and CM may be of a single type.
(Supported by N.I.H. Grant DE06725).
SMOLEN, Arnold J., Joel SWARTz*, Patricia BEASTON-WIMMER,*
Leo COSIO,* M. Nathan PINKNEY,* Laureen SENA,* Darsit SHAH'
and George L. POPKY,* Departments of Anatomy and Radiologic
Sciences, The Medical College of Pennsylvania, Philadelphia,
Pennsylvania. &
The region of the temporomandibular joint is of clinical interest, due to
its involvement in dislocations and in a syndrome producing severe pain.
It is crucial that any clinician who works in this area understands the
anatomical relationships of the temporomandibular joint with other
surrounding structures, including ligaments, muscles, and nerves.
Because of this importance, a large number of continuing medical
education programs include courses on the TMJ, many of which require
the dissection of human cadaver material. The computer based
interactive video program described here was designed to provide the
anatomical knowledge of the TMJ, without the need for repeated human
cadaver dissections.
This program combines full-motion and still-frame video, computer
graphics with animation, and computer assisted instruction into an
interactive learning experience. The program runs on an IBM AT
compatible computer with a videodisk player and an InfoWindows
monitor that is capable of superimposing full-motion or still-frame video
images with computer generated text and graphics, and is touch
sensitive.
The program consists of three modules. The first module introduces the
student to the anatomy of the TMJ. Using computer graphics, the
student is introduced to the bones, muscles, ligaments, and nerves of the
region, and to the TMJ itself. The student then performs a "dissection"
of the region of the TMJ by controlling the video presentation with the
touch screen.
The second module demonstrates the physical
examination of the TMJ using full-motion video, and introduces
diagnostic imaging modalities for the study of the TMJ. The third
module uses clinical case studies to reinforce the concepts introduced in
the first two modules, and to serve a5 a means of evaluating the
student's knowledge of the TMJ.
S
SOHN, Deborah J. and William P . FALLS, Department of
Anatomy, Michigan State University, East Lansing, Michigan.
Primary trigeminal axons terminating in the dorsomedial
subdivision of rat trigeminal nucleus oralis.
Anterograde transport of horseradish peroxidase was used
to examine the overall morphology and distribution of
primary trigeminal axons terminating in the dorsomedial (DM)
subdivision of rat trieeminal nucleus oralis (Vol. Injections into tne sensory root of the trigeminal nerve labeled
axons throughout DM in each of its three morphologically
distinct regions (caudal, middle and rostral). Light
microscopic analyses revealed that each region receives the
terminal arborizations of two different populations of
primary axons. The first population was a direct continuation of parent fibers traveling in the dorsomedial portion
of the spinal trigeminal tract (SVT).
It entered Vo by
traversing the border zone (BZ) subdivision without
branching and terminated in DM as a sparsely branched, long
(up to 650 pm in length) medially directed strand containing several axonal endings. Parent fibers and terminal
strands measured 1-2 pm in diameter and each strand contained up to 18 boutons measuring 1-4 pm along their maximum diameter. The second population arose as collaterals
from parent branches (2-5 pm in diameter) descending in the
dorsomedial portion of SVT. The collaterals traversed BZ
and entered DM where they divided into two to four
medially directed terminal strands. The resulting coneshaped dendritic arbors spread out mediolaterally, rostrocaudally and dorsoventrally for 17-93 vm. One to nineteen
boutons (1-6 vm along their maximum diameter) were located
along the collateral and their terminal strands. These
morphological findings suggest that at least two types of
orofacial sensory inputs are being conveyed to DM.
Previous data (Falls et al., 1985) has shown that the
majority of neurons in each region of DM directly innervate
one or more of the orofacial portions of four major tactile
areas of the cerebellar cortex. Based on these findings,
some trigeminocerebellar projection neurons in each region
of DM may be receiving one or more different types of orofacial tactile inputs.
(Supported by N.I.H. Grant DE06725).
SOKOLOFF, Alan J., and Terrence W. DEACON,* Biological
Anthropology, Harvard University, Cambridge, Massachusetts; and
USC Dental School, Los Angeles, California. m e r v a w
he c v n o w monkey
fascicularis).
Despite the importance of the mammal tongue in mastication,
deglutition and respiration, nothing is known about the innervation of
individual muscles of the tongue body. An understanding of the
organization of the hypoglossal nucleus (HGN) motoneurons which
innervate tongue body musculature is critical for evaluating theories
which relate the role of individual muscles to the production of specific
tongue body movements. In the present investigation, 0.3-3.0 p1 of a
5%solution of the retrograde axonal tracer WGA-HRP were injected
into the tongue in 21 macaques
fascicularis) to study the
topographic organization of HGN motoneurons. Brainstem and
tongue tissue was cut at 75 microns on a freezing microtome, reacted
with TMB, counterstained in neufraI red and observed under
brightfield, darkfield and polarized microscopy. WGA-HRP injections
into the tongue body demonstrate a musculotopic segregation of HGN
motoneurons. Motoneurons innervating the transversus and verticalis
muscles are located in the medial HGN,motoneurons innervating the
a
96A
ABSTRACTSAAA 1 0 3 MEETING
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genioglossus muscle are located in the intermediate HGN,
motoneurons innervating the hyoglossus and inferior longitudinalis
muscles are located in the ventrolateral HGN, and motoneurons
innervating the styloglossus, palatoglossus and superior longitudinalis
muscles are located in the dorsolateral HGN. Motoneurons innervating
the geniohyoid muscle are located in a cell column separated ventrally
from the HGN. Injection into the extra-lingual portion of the
palatoglossus muscle labels neurons in nucleus ambiguus, indicating a
dual motor nucleus innervation for this muscle. These results
demonstnte that the motoneurons which innervate tongue body
muscles of similar orientation are localized to a discrete subregion of
the HGN. These results also demonstrate topographic overlap of
motoneurons which innervate classically defined intrinsic and extrinsic
tongue muscles. Supported by a NSF Graduate Fellowship and NIDR
Grant R29 DE07380.
S
SOLIS-SOTO*, Juan Manuel , and Jul i o Sepirlveda-Saavedra,
Departamento d e H i s t o l o g i a , F a c u l t a d d e Medicina, UniversL
dad AutBnoma d e Nuevo Le&. Monterrey, N.L., Mlxico.
E f f e c t o f Dexamethasone on t h e l u n g ' i - c o n e c t i v e tissue
fibers d u r i n g f e t a l development i n a l b i n o mouse (Mus Muscu-
lus).
Although t h e s t i m u l a t i n g e f f e c t o f dexamethasone o v e r -s u r f a c t a n t production, and t h e i n c r e a s e i n d i f f e r e n t i a t i o n
r a t e o f e p i t h e l i a 1 components of the developing lung are
well documented, e f f e c t e x e r t e d on t h e mesenchymal compon e n t s have been poorly c h a r a c t e r i z e d . In the p r e s e n t s t u d y ,
t h e e f f e c t induced by dexamethasone given t r a n s p l a c e n t a l l y
t o developing mice was a n a l y z a d ; f o c u s s i n g on t h e amounts
o f c o l l a g e n and e l a s t i c fibers. Morphometric and biochemic a l t e c h n i q u e s were u t i l i z e d . Dexamethasone was given -s t a r t i n g on t h e 14th day o f g e s t a t i o n and up t o the 1 8 t h
day. F e t a l l u n g s were c o l l e c t e d s t a r t i n g on day 1 5 t h o f
g e s t a t i o n and u p t o t h r e e days post-partum. For morphomet r y , tissue s e c t i o n s were s t a i n e d w i t h S i r i u s red and Van
Gieson t e c h n i q u e s f o r c o l l a g e n . Resorsin-Fucsin was used
for e l a s t i c f i b e r s . Collagen was d e t e c t e d s i n c e t h e b e g i n n
ing o f t h e s t u d y , while e l a s t i n appeared u n t i l day 16. Col l a g e n was found i n c r e a s e d a t day 1 7 i n t r e a t e d animals.
E l a s t i c f i b e r s appeared i n c r e a s e d on days 17, 18 and 1 day
post-partum. In t h e biochemical s t u d y t h e c o l l a g e n c o n t e n t
was determined by the spectrophornetric q u a n t i t a t i o n o f
p r o l i n e . There was a good c o r r e l a t i o n w i t h t h e mrphomet r i c a l d a t a , showing b e s i d e s an i n c r e a s e i n c o l l a g e n cont e n t on day 16 a s compared t o c o n t r o l s . Dexamethasone i n creases t h e collagen content i n a t r a n s i t o r y fashion s i n c e
t h e amounts found l a t e r a r e t h e same a s i n non t r e a t e d
c o n t r o l s . A t t h e end o f the s t u d y t h e appearance o f t h e
lung was the same i n b o t h t r e a t e d and c o n t r o l s .
-
S
Soonpaa,* Mar< H. a n d J o h n 0. Oberpriller, Department
of Anatomy a n d Ce!l Biology, Unfversity of North Dakota,
G r a n d ForKs, North Darota.
Aqents affectinq t h e
p r o l i f e r a t i v e b e h a v i o r of t n e a d u l t n e w t c a r d i a c
mvocvte.
T o b e t t e r u n d e r s t a n d t h e mechanisms g o v e r n i n g t h e
proliferation of c a r o i a c myocytes i t i s i m p o r t a n t to
idert-fy t h e f a c t o r s c o n t r o l l i n g t h i s phenomenon, a n d to
c h a r a c t e r i z e t h e i r s p e c i f i c actions. T h e model c h o s e n to
s t u d y was tine a d u l t n e w t v e n t r i c u i a r m y o c y t e i n celi
culture.
Ventricular m y o c y t e s were
enzymatica;;y
s e p a r a t e d , s u s p e n d e d i n a solution of modified L e i b o v k z
i-:5 med:u!m a n d e i t h e r 10% f e t a l b o v i n e s e r u m (FBS) or
10% h o r s e s e r u m (HS), t h e n preplaced o n t o plastic ( d a y
one).
By d a y 3 most of t h e n o n m y o c y t e s h a d plated
o n t o t h e plast-c, leaving a relatively p u r e solution of
m y o c y t e s . T h e solution of myocytes was t h e n inoculated
i n t o laminin-coated a i s h e s , o n t o which the m y o c y t e s
plated. On d a y 5, a n d e v e r y o t n e r d a y t n e r e a f t e r , t h e
cells were f e d with medium containing e i t h e r 12-0tetradecanoyiphorDo1-13-acetate ( T P A ) or p l a t e l e t d e r i v e d g r o w t h f ctor (PDGF). On d a y 1 1 t h e cells were
g i v e n 1 vCi/ml '+I thym-dine, a n d 24 hours lacer t h e
cells were fixed a n d s t a i n e d . Dishes were t h e n coated
with p h o t o g r a p h i c emulsion, exposed, a n d developed.
T h e p e r c e n t of cells with labeled nuclei was t h e n
determined.
Electron microscopic s t u d i e s of
u l t r a s u - u c t u r e are c u r r e n t ; y b e i n g done, as are s t u d i e s
of oossible c h a n g e s of t h e mitotic k d e x d u e to t h e s e
f a c t o r s . TPA in c o n c e n t r a t i o n s of 25 ng/ml i n c r e a s e d
t h e p e r c e n t of iabe!ed c e l l s from a c o n t r o l of 25.5% to
48% with FBS, a n d from 28% to 40% with HS. PDGF a t 2
units/m! i n c r e a s e d l a b e l i r g from 28.5% to 35% with FBS.
T h e s e r e s u l t s i n d i c a t e t h a t the 3 N A s y n t h e t i c a c t i v i t y of
t h i s c u l t u r e s y s t e m c a n b e i n c r e a s e d by t h e s e f a c t o r s .
F u t u r e r e s e a r c h w i l l i n c l u d e more c o m p l e t e
c h a r a c t e r i z a t i o n of t h e s e f a c t o r s , of o t h e r f a c t o r s , a n d
of t h e i r i n t e r a c t i o n s . T h i s work was s u p p o r t e d i n p a r t
b y NSF g r a n t RI18610675 to t h e S t a t e oq North Dakota.
S
SOPPER Maggie M.*I, Peter A. MERRIFIELD' and Earl G.
NOBLE"',
Faculty of Physical Education' and Department
of Anatomy',
The University of Western Ontario, London,
Ontario, N6A 3K7. Fiber sDecific exmession of IIB
myosin h e a w chain in thvrotoxic rat soleus muscle.
Thyroid hormone is known to induce IIB myosin heavy
chain (MHC) mRNA accumulation in the soleus muscle of
adult rats (Izuno et al. Science 231: 597, 1986) yet
little is known about the expression of the accompanying
protein. We have characterized a monoclonal antibody
(Mab 212F) specific to the IIB MHC (Sopper et al. J .
Cell Biol. 197: 36a, 1989) and have used it to examine
the spatial expression of this protein in hyperthyroid
rats. Male Sprague-Dawley rats (200-25Og) were injected
with 300ug.kg-1 body weight of triiodothyronine (T3) on
alternate days for a 28 day treatment period. Western
blot and ELISA analysis of purified myosins detect the
initial appearance of 119 MHC after 5 days of T3
treatment followed by a progressive accumulation of this
isoform. Immunolocalization experiments on frozen tissue
sections demonstrate
that IIB MHC expression is
initially restricted to the histochemically defined IIA
fiber population, with additional fibers being recruited
as T3 treatment progresses. After 21 to 28 days
approximately 50% of all muscle fibers express IIB MHC.
A subset of the remaining fibers are histochemically
identified as type I1 but do not express 119 MHC. The
mechanism underlying this differential response is
currently unknown, but may be related to functional
differences that are believed to exist between primary
and secondary fibers in developing muscle. Supported by
granrs from MRC of Canada to PAM and NSERC of Canada to
EGN .
S
SOSNOWSKI,* Jeffery, Kenneth H. REID* and Jonathan
NEWMARK*
Department of Anatomical Sciences and
Neurobiologyand Department of Neurology,School of Medicine,
University of Louisville, Louisville, Kentucky. (Sponsored by
Robert V. Gregg) Anatomical and Dhvsioloaicalcharacterization
of a LPC-induced focal demvelinatina lesion in the rat fimbria.
Focal demyelination was induced in the rat fimbria by
stereotaxic injection of 0.3 uI of a concentrated micellar
suspensionof the detergent lysophosphatidylcholine (LPC). One
week after the injection, the rat was sacrificed, the brain
hemispheres divided, and the injected hippocampus-fimbria
ABSTRACTS-AAA 103RD MEETING
dissected en bloc. This preparation was submerged in
oxygenated artificialcerebrospinalfluid and maintained in vitro for
5-7 hours. Electrical responses were studied at three
temperatures: physiologic, hypothermic, and hyperthermic.
Stimulating and recording sites were then marked and the tissue
was fixed for histological analysis. LPC-induced lesions showed
an irregular region of demyelination, considerable edema, some
reactive astrocytes, and little axonal destruction. The center of
the lesion showed a near-total conduction block. This was
surrounded by regions showing a primary wave followed by a
slowly propagating secondary wave, suggesting a mixture of
normal and demyelinated fibers. At 33"C, conduction velocities
were 4-6 m/sec for the primary wave and 0.5-0.6 m/sec for the
secondary wave. Conduction velocities varied as expected with
temperature. However, the hyperthermic conduction block
predicted on clinical grounds was not seen in this lesion. Focal
demyelinated regions occur in several demyelinating diseases;
this preparation may prove useful in the evaluation of proposed
treatments for multiple sclerosis and related diseases.
SPECIAN, Robert D., Shi Jun ZHANG*,
Don A. SIBLEY*, Abbie
C. KEMPER and Mary G. OLIVER, De artment of Cellular
Biology and Anatomy, LSU School of d d i c i n e , Shreveport,
Louisiana. Recoverv of goblet cells from an accelerated secreto
aCrypt goblet cells throughout the small and large intestin?
secrete their stored intracellular mucin in response to [email protected]
stimulation. The secretory event is rapid and involves the majonty
of stored granules. The purpose of the present study is to determine
the time course of recovery of goblet cells from an accelerated
secretory event, ie. how quickly are their intracellular stores of
mucin replaced. Four experimental groups were used with five
male, Wistar rats in each group. The rats were fasted overnight
prior to sacrifice. The first group served as controls. The second
group received pilocarpine (160 mgkg body weight) for 30 minutes
to empty the stored mucous granules from crypt goblet cells. The
third group received pilocarpine for 30 minutes followed by
atropine (160 mgkg body weight) for 60 minutes to allow for a
recovery period. The final group was allowed to recover with
atropine for 120 minutes. Upon sacrifice, the intestine was removed
and a segment from duodenum, jejunum, ileum and descending
colon were fixed by immersion, processed and embedded in glycol
methacrylate. Three micrometer JB-4 sections from each segment
were stained with periodic acid-Schiff to demonstrate neutral
mucins. The volume of epithelium per surface area of epithelia1
basal lamina was calculated with a Merz grid to correct for
anisotrophy and a square lattice grid was used to determine the
volume of stored much per epithelium. Stored mucin quantities
were then expressed in terms of basal lamina surface area. Values
were compared by an unpaired T-test to determine significance.
Pilocarpine stimulation resulted in a significant decrease in the
stored mucin in the crypt of duodenum, jejunum, ileum and
descending colon, with duodenum also showing a significant
decrease in villus stored m u c h Following a 60 minute recovery
period, all tissues were statistically indistinguishable from controls,
indicating that the intracellular granules had been replenished.
Following 120 minutes of recove
villus stored mucin in the
jejunum and ileum had significanri increased over the control
tissues. These data suggest that goblet cells can recovery from an
accelerated secretory event quickly, refilling th mucous ranules
in 60 minutes or less. Sup orted by Grant %IS 33720 t o m the
National Institutes of H e a d
SPURGEON.' Thomas. Stephen KOCH.' Thomas MCCRACKEN.. and
SteDhen ROPER DeDartment of Anatomv and Neurobiolom, Colorado
Sta'te University, FOG Collins, Colorado.- Automated &&a acauisition
for the ce r
natomic image
- - e The
r ?
has identified a
major limitation in the generation of computer &ages of anatomic
specimens. namely, the initial acquisition of digitized data
97A
(digilizafion). Surface rendering by computer presently requires the
manual digitization of serial cross-sections of specimens: the contours
of corresponding structures in different sectional planes then must be
linked by the computer program to produce a three-dimensional
image. To promote the development of computer images of anatomy
we are implementing a three-dimensional obJect digitizing system (the
DlGIBOTl which will automate the inltial acquisition of surface data
from actual or modeled specimens. The DIGIBOT is a laser-based
computer peripheral which mathematically "locates" polnts on a
surlace via triangulation. The DIGIBOT achieves resolution of better
than 1/10.000 in a working volume of up to one cubic meter,
Incorporates a range of user-selected scanning modes designed to
optimbe surface sampling density and scan speed for a given specimen.
and includes an adaptive data filter which can typically reduce the
total number of data points by 80-90% with no perceptible loss of
image quality. Such data reduction greatly increases the speed at
which the computer generates or modifies an image. This system also
eliminates the separate mathematical treatment required to llnk the
geometry between adjacent sectional planes when data are gathered by
manual digitization of sectioned specimens. The DIGIBOT
automatically acquires not only the geometry of a surface but also the
topology required to reconstruct the final three-dimensional image.
The DIGIBOT has been used to produce very sophisticated images of a
number of osseus and plastinated visceral specimens: we are
developing an interface which will allow images produced in this
fashion to be combined with one another and to be enhanced through
the addition of anatomic structures (eg. blood vessels. newes. etc.) not
present on the originally-digitized specimen. This work is part of the
Vesalius Project and is supported by the Fund for the Improvement of
Posl-Secondary Education (FIPSE) of the U.S. Department of
Education.
STAGAARD, M.*, R.S, NOWAKOWSKI, and K. MLSLLGARD'.
Institute of Medical Anatomy A, Panum Institute, University of
Copenhagen, DK-2200 Copenhagen, Denmark, and Department of
Anatomy, UMDNJ-RobertWood Johnson Medical School, Piscataway,
New Jersey. Early appearance of S-100-motein in the fimhria of the
hipnocamDus in human fetuses.
The distribution of three glial cell associated markers (S-100,
vimentin and GFA) was studied by immunocytochemical techniques
in the developing brain of human fetuses ranging from 22 to 200 mm
crown-rump-length (CRL) or 7 to 23 weeks. At and prior to 52 mm
CRL, radially aligned cells extending from the ventricular zone (VZ)
into the marginal zone (MZ) of the fimbria are solely vimentinpositive. At 69 mm CRL, some radially aligned cells in the fimbria
are also GFA-positive. At 81 mm CRL, the intensity of the vimentinreactivity is markedly diminished, GFA-reactivity is unchanged, but a
high proportion of the VZ cells of the fimbria have become S-100
positive. At this stage, cells stained with both GFA and with S-100
exhibit the morphology of radial dial cells. At 109 mm CRL, in
addition t o the radially aligned VZ cells, astrocytes in the marginal
zone of the fimhria are S-100 and GFA-positive. The distribution of
the S-100-positive astrocytes and the GFA-positive astrocytes is
partially overlapping. Both are found in the fimbria and in a narrow
band extending through the intermediate zone towards CA3, whereas
S-100-positive cells are also found subjacent to the pia1 surface
extending from the finibria into the anlage of the dentate gyrus. In
the area of the overlap, double-stained preparations showed that some
but not all of the cells contain both antigens. At 156 mm and 200 mm
CRL, the distribution of S-100 positive staining in the fimbria is
unchanged from the pattern seen at 109 mm CRL, but now both S100 and GFA positive astrocytes are distributed throughout the
adjacent hippocampus.
Thus, in the developmental period examined, the distribution of S100 reactivity corresponds to the border between a cortical structure
a n d its major efferent/afferent pathway. This sugests a possible role
for S-100-positiveglial cells of the fimbria in the guidance of axons.
Supported by NlMH grant #MH44188.
I
STEECE-COLLIER, Kathy*, David M. YUREK*. Timothy J. COLLIER*.
Celia D. SLADEK. and John R. SLADEK. JR. Detmlment of Neurobiology
and Anatomy, University of Rochester School of Medicine, Rochester. NeG
York.
98A
ABSTRACTS-AAA 103RD MEETING
It has recently been shown that near-ultraviolet radiation (UV-A),
though defined as being outside the range of visual perception, can
affect neuroendocrine, circadian. and reproductive responses in some
rodent species. The purpose of this study was to investigate the
response of the pineal melatonin rhythm in Siberian hamsters
(Phodopus sungorus) to long or short day UV-A light-dark cycles.
Broadband UV-A (340-405nm) light was provided by a 20W BLB
fluorescent lamp mounted behind a UV-A glass filter (Corning BG).
Adult female hamsters (n=131) were exposed to UV-A either from
0900-1700 (LD 8:16) or from 0100 to 1700 (LD 1623) for 19
days. Groups of animals from each lighting condition were +ki,
sacrificed in darkness at 0100, 0300, 0600, 0900, i iU0, 1400.
1700, 1900, 2100. and 2300. Pineal glands were removed, frozen,
and stored at -700 C and later assayed for melatonin content using
RIA. Data were analyzed using 2-way ANOVA (photoperiod x time of
day). Comparisons were made of treatment cell means at selected
times of day, using 1-tests with Bonferroni corrections of significance
levels. There were significant main effects of both photoperiod
(F=15.84, df=l, pc.0001) and time of day (F=11.55, df=9,
pc.OOOI), and a significant interaction (F=2.635, df=9, pc.001).
Melatonin levels in both groups began to rise at 1900 and reached
peak levels at 2300. Melatonin production had ceased by 0300 in the
long photoperiod group, but was still elevated at 0300 in the short
photoperiod group (t=5.87, pc.OOOl), and at 0600 by some
individual short photoperiod subjects. Therefore, the duration of
elevated melatonin production was at least three hours shorter in
subjects exposed to long days of UV-A radiation. Melatonin was low
during the hours of UV-A radiation exposure in both groups. Thus,
the rhythm of pineal melatonin production in Siberian hamsters
appears to respond to photoperiods of UV-A light in a manner similar
to photoperiods of visible light.
(Supported by NEMA Grant
LRI89:DR:NEMA:l; NASA Grant NAGWl196; NSF Grants DCB8412587 and DCB8714638; and USUHS Grant C07049).
veoIDmer-d
embeontc
in
-Evidence suggests that chron=vated
levels of dopamine @A) may have
toxic effects on DA neurons. Several mechanisms may be involved in these
negative effects, including the metabolic generation of oxygen radicals, quinones
and even the neurotoxin 6-hydroxydopamine, and/or a receptor mediated growthinhibiting i duence of DA, as has been demonstrated by Lankford and colleagues
(PNAS 85:2839, 1988). This potential D A autotoxicity may have particular
impact on therapies for Parkinson's disease, which in general are directed at
augmenting DA system function. In conjunction with our ongoing studies of DA
neuron grafting in animal models of Parkinson's disease, we became interested in
whether the standard D A augmentation therapy employed in the disease,
levodopa administration,had any effects on the viability of grafted embryonic DA
neurons. In addition, we examined the effects of levodopa on the development of
DA neurons in culture. Adult male F344 rats received unilateral 6hydroxydopamine lesions of the nigrostriatal DA system, and 4 weeks later were
implanted with a single block graft of embryonic day 15 ventral midbrain tissue
containingthe developingA8-Al0 DA neurons placed into the denervated caudate
nucleus. Beginning the day after graftimg, animals were divided into two groups:
lhose receiving twice daily inmperitoneal injection of Sinemet (levodopa plus a
peripheral decarboxylase inhibitor), and a control group receiving injections of
saline. Treatments continued for a six week period, after which subjects were
sacrificed and the morphological development of grafted D A neurons was
assessed utilizing tyrosine-hydroxylase (TH) immunocytochemistry. Grafts in
rats receiving Sinemet treatment exhibited a marked decrease in neurite
outgrowth, decreased perikaryal size and staining for TH, and increased
infiltration by macrophages. Behavioral responses to DA agonists and
amphetamine also suggest diminished function of grafted neurons in rats exposed
to levodopa. Similar results were obtained in culture studies. Levodopa added to
embryonic midbrain cultures produced a dose-dependent decrease in the number
of TH neurons, as well as the length and complexity of their neurites (108M-10'
4M lcvodopa). These results are consistent with the view that increased
concentrations of DA, and/or the metabolic by-products of DA, in the local
environment of DA neurons can be detrimental to their morphological
development. In particular, administration of levodopa in combination with
neural grafts of embryonic DA neurons may lead to decreased survival and
integration of transplanted neurons, decreasing their potential therapeutic efficacy.
This work was supported by the United Parkinson Foundation, the PEW
Foundation, and AG00847.
S
STIENE-MARTIN,* Anne, John G. OSBORNE and Kurt F. HAUSER,
Department of Anatomy and Neurobiology, University of Kentucky School of
STEEVES, John D., Departments o f Anatomy and Zoology,
Medicine, Lexington, Kentucky. Cellular localization of ouioid eene emression
u n i v e r s i t y o f B r i t i s h Columbia, Vancouver, B.C. V6T 2A9.
in cultured astroevtes: Colocalization of oroenkeohalin mRNA in sifu
;
hvbridizationand dial fibrillaw acidic orotein imrnunoreactive oroducts.
The neural control o f vertebrate locomotion i s based
on a hierarchical organization with i n t r i n s i c spinal cord
Recent Northern analysis studies indicate that mixed-glial cultures express
networks generating the basic rhythms o f locomotor
proenkephalin gene mRNA product. To determine whether cultured astrocytes
activity.
There are a number o f descending supraspinal
express the proenkephalin gene, a method was developed to simultaneously
pathways t h a t could p a r t i c i p a t e i n the d i r e c t i n i t i a t i o n
localize the astrocytic marker, glial fibrillary acidic protein (GFAP), and mRNA
We have
and control o f spinal locomotor networks.
for proenkephalin within the same cell by immunocytochemistry and in sifu
examined the neural control o f avian locomotion (both
hybridization, respectively. Primary dissociated cultures of mixed-dial cells were
walking and f l y i n g ) and compared our r e s u l t s w i t h
obtained from cerebral cortex of 1-day old mice and grown on poly-L-lysinefindings from p a r a l l e l studies on f i s h , amphibians,
reptiles,
and
mammals.
Like
all
sub-mammalian
coated glass coverdips in a nutrient media (DMEM) containing 10% fetal calf
vertebrates, birds do not possess a telencephalo-spinal
serum for 5 or 6 days. Cells were fixed on their coverslips with 4%
pathway analogous t o the mammalian corticospinal t r a c t .
paraformaldehyde in 0.1 M phosphate buffer. GFAP immunocytochemistry was
We have i d e n t i f i e d t h a t the essential common descending
performed using diethyl pyrocarbonate treated reagents and visualized using
pathway f o r the i n i t i a t i o n o f avian locomotion originates
diaminobenzedine as a chromogen. For itt sifu hybridization, immunostained
w i t h i n the brainstem r e t i c u l a r formation.
Similar
cultureswere washed extensively (7x) with water, pretreated with acetic anhydride
r e s u l t s have been reported f o r a l l other vertebrates.
(0.25% in 0.1 M triethanolamine), proteinase K (1 pg/ml); and hybridized for 18
Our other studies have shown t h a t the coupling between
locomotor and respiratory a c t i v i t y appears t o be t i g h t l y
hours at 37O C using a 35S cRNA probe (50,ooO cpm/coverslip) provided by Dr.
controlled during f l i g h t ,
as opposed t o walking.
Michael Melner (Oregon Regional Primate Research Center). Coverslips were
Supported by the Natural Sciences and Engineering
then washed ( 0 2 SSC highest wash stringency), dried, and exposed to NTB-2
Research Council (NSERC) o f Canada.
emulsion at 4OC for 4 weeks. Proenkephalin mRNA was c o l d i e d in
approximately 70% of GFAP-immunoreactive type I astrocytes, while
approximately 30% of type I astrocytes did not contain hybridization signal. This
is the first demonstration localizing proenkephalin gene products at the cellular
level in growing cultured astrocytes. The reasons why this opioid gene is
expressed by developing astrocytes are uncertain. However, recent evidence that
0STEWART, * Karen T., Mark D. ROLLAG, Milton H. STETSON, George
the proenkephalin gene product, met-enkephalin, inhibits DNA synthesis in
C. BRAINARD, Department of Neurology, Jefferson Medical College,
cultured type I astrocytes implies that proenkephalin gene expression may be
Philadelphia, Pennsylvania; Department of Anatomy, Uniformed
involved in the regulation of astrocyte growth; and suggests that astrocytes are
Services University of Health Sciences, Bethesda, Maryland; and
critical intermediaries in opioid-dependent regulation of neural growth.
Department of Life and Health Sciences, University of Delaware,
(Supported by NIH RR-05374, and PSP and UKMC Research Funds from the
Newark, Delaware. .€ff,fsts of1-1
dav
University of Kentucky).
99A
ABSTRACTS-AAA 103RD MEETING
STODDARD, Susan L., Ronald A. KARWOSKI', Richard A.
ROBB', and Stephen W. CARMICHAEL, Department of
Anatomy, Indiana University School of Medicine, Fort Wayne,
Indiana; Departments of Physiology and Biophysics and Anatomy,
Mayo Clinic, Rochester, Minnesota. Computer-generated 3dimensional image of the brain that can be manipulated and
dissected.
A data set generated from 1.2 mm-thick magnetic resonance
images (MRI) was used to construct a 3-dimensional image of the
human head and brain using h d y z e software (" Biodynamics
Research Unit, Mayo Foundation, Rochester, MN).
The
ventricular system, internal capsule, putamen, and caudate and
lenticular nuclei were identified in coronal, transverse, or sagittal
sections generated from the MRI data set. A single structure was
traced in each MRI slice, either automatically using grey scale
boundaries, or by hand using a mouse. The information for each
object was combined to produce a three-dimensional volume or a
shaded surface display of the neuroanatomical structure. The
surface of the head and face can be visualized, rotated on any
combination of axes, dissolved away, or split open to reveal the
brain within. The surface of the brain can be rotated, rendered
transparent, and removed to reveal internal structures. Each
structure can be assigned any color and manipulated individually
or in combination with any or all of the other structures.
Structures or portions of structures can be labeled. The
relationships of internal structures to each other and the surfaces
of the brain and head are easily visualized. A videotape of all of
these features will be presented. A interactive version of this data
set for student use on a personal computer is being developed.
STRASMANN, T.*, T. FEILSCHER * and Z. HALATA, Division
of Functional Anatomy, Department of Anatomy, University of Hamburg, West Germany. Topograuhv and structure of mechafioreceptors
of intervertebral discs. ioints and muscles in the umer vertebral
column of the grey short-tailed ouossum.
The gross anatomy of the vertebral column of the grey shorttailed opossum (Monodelphis domesrim) is comparable to that of the
rat. Between the arches are synovial joints. The vertebral bodies are
united by intervertebral discs (symphyses) and covered by longitudinal ligaments that fuse with the vertebral bodies or the intervertebral
discs, respectively. In the light microscope it is discernable that the
anulus fibrosus of the intervertebral disc is connected with both the
ligaments by irregularly arranged collagen fiber bundles. In this ringlike connective tissue, close to the lateral edges of both ligaments,
about 40-60 small lamellated corpuscles per intervertebral disc are
present. Furthermore, small lamellated corpusclesoccur where muscles are inserted onto the vertebral arches and the spinous processes.
The muscles contain different amounts of muscle spindles and Golgitendon organs.The synovialjoints show all three kinds ofjoint capsule
nerve endings: free nerve endings, small lamellated corpuscles and
Ruffini corpuscles. By means of electron microscopy, the small
lamellated corpuscle innervating the intervertebral disc exhibits an
afferent myelinated axon of 3-3.5 pm in diameter, 3-5 layers of the
perineural capsule and an asymmetrical inner core. Between the
cytoplasmic lamellae of the terminal Schwanncell of the inner core, a
surprisinglylarge amount of collagen fibers are. present. By the use of
electron microscopy, free nerve endings can also be observed. They
are only in part covered by Schwann cells and show terminal axonal
swellings.The axolemmaof these swellingsdirectly contacts the basal
lamina and the axoplasm reveals electron-clear round vesicles. Supported by 'Deutsche Forschungsgemeinschaft (HA 1194/3-2).
S U G R U E , * ~P., J AVOLIO*~,K. B KALOW*~,
M.E.J. HOLWILL*I and P. SATIR1,'Dept.
of
Thysics, Kings College, London, England and
Dept.of Anatomy and Structural Biology,
AECOM, Bronx, New York. ComDuter modellinq
of the ciliarv axoneme at macromolecular
resolution.
A
computer generated model
of the
structural arrangement of the complete 9+2
axoneme of Tetrahvmena thermoDhila somatic
cilia is presented.
The model reconciles
detailed information about subcomponents
from
negative-stained,
thin
section,
intermediate voltage, and freeze-fracture
electron
micrographs,
integrating
the
images into a consistent three-dimensional
picture at 4 nm resolution.
Optical
diffraction
is used
to verify
major
periodicities and direct comparison to
micrographs
in
various
orientations
provides
visualization
of
the
correspondence of the model to the threedimensional axoneme. The model illuminates
problems such as the requirement
for
compaction of dynein to form an arm,
difficulties
in
visualization
of
the
circumferential links, construction of the
central sheath and comparison of inner vs.
outer arm periodicities. The model can be
manipulated
in various ways to make clear
specific
relationships
of
axonemal
structures. It is pragmatic, flexible and
useful in developing dynamic concepts such
as a spatial description of the dynein
cross-bridge cycle or relationships between
adjacent
doublets
during
sliding
and
bending.
S
SUN', Weidong, Marahatham IYER*, Mark MILLER' and Peter
CHANTLER, Department of Anatomy, Medical College of
Pennsylvania, Philadelphia, Pennsylvania, 19129. Molecular Cloning
and Partial Sequencine of Neuronal Mvosin cDNA.
In order to further our understanding of the role of myosin
within vertebrate neurons we are generating and sequencing cDNA
clones of neuronal myosin. A rat brain cDNA expression library
(Stratagene) was screened using our affinity-purified polyclonal
anti-myosin antibody, originally raised against myosin isolated from
mouse neuroblastoma N2A cells (Chantler et al., 1988. Abstracts
4th Intl. Conf. Cell Biol., Montreal, Canada. p265). 4 positive
plaques. Inserts, ranging in size
clones were isolated from 2.5 x
from 0.5 - 2.0 kB, were rescued into pBluescript (Stratagene)
plasmids prior to double stranded sequencing by the Sanger dideoxy
method using Sequenase 2.0 (US.Biochem.). To date, complete
sequence has been obtained from three clones. One of these (C4)
shows >90% sequence homology with part of the tail region of nonneuronal myosins, as compared using the Genepro (Riverside Sci.
Ent.) sequence analysis program. This insert, radiolabelled by
random priming, hybridizes with a band >7.0 kB in size on a
Northern blot using total RNA isolated from rat brain, this size
being consistent with it being a probe for myosin mRNA. No
hybridization was observed with RNA from kidney or liver run in
adjacent lanes, consistent with the anticipated specificity of this
probe for neuronal myosin. We intend to sequence the entire
neuronal myosin heavy-chain cDNA and to use the above, and other
1
6
100A
ABSTRACTS-AAA 103RD MEETING
probes to assess myosin function, both in cultured neuronal cells
and in the C.N.S. during development.
Supported by NIAMSD Grant # AR 32858 awarded to P.D.C.
SUWAf Fumihiko, Isumi TODA, Yoshikuni OHTA and Hitoshi
OKUDA, Department of Anatomy, Osaka Dental University,
Osaka Japan. SEM studies on t h e microvasculature of t h e
periosteum of t h e jaw and other bones in t h e dog.
The present studies have demonstrated microvascular
architectures of t h e periosteum of t h e jaw and other bones
on microcasts. These patterns are variant in histological
aspects and connecting conditions with surrounding structures
according to the form and location of the bones. On the
microvascular casts prepared by the plastic injection method
(Ohta et al., 1989), the microvascular architecture of the
buccal periosteum of the maxilla and mandible has been
surveyed from the bone side under S E M . The alveolar
periosteum consits of successively ranging from areas of t h e
attached gingivae, of the alveolar mucosa and of the
remaining onto the bone body. Generally, t h e vascular
network in the outer layer of t h e periosteum is composed of
arterioles and venules which form coarse meshes and that in
the inner layer is composed of capillary meshes in the shape
of sinusoidal postcapillary veins which form dense meshes. In
the periosteum of the attached gingivae, the network does
not form dense meshes but apart from t h e bone surface. In
that of the alveolar mucosa, the network composed of venous
capillaries expands closely on t h e bone surface with peculiar
larger meshes encircling perforating (Sharpey's) fiber
bundles. I n t h e remaining area, since the above venous
capillary network becomes coarser meshes, it may be a
transitional form to alter the network in the periosteum of
the jaw bone body. In t h e periosteum of the bone body, the
network is primarily composed of bundled arterioles and
venules, between which capillary networks are secondarily
found. This pattern resembles that in the periosteum of the
diaphysis of a long bone.
I n conclusion, the capillary network expanding closely on
the bone surface beneath the attached gingivae is difficult to
identify because of the presence of the perforating fiber
bundles, by which the bone is connected with gingivae as a
typical mucoperiosteum.
SVOBODA, Kathy, K.H., Department of Anatomy, .Boston University
Schqol of Me.dicine, Boston, Massachussetts.
Spatial l o c w o n of types I. 11. .and .IX c o l l w mRNA and
c v t o s k e w t e i n s in 6 dav embrvonic-c
Previous studies have shown that 6 day cornea1 epithelia1 cells
produce types I, 11, and IX collagen. In the current study, in situ
hybridization combined with confocal image analysis (Biorad MRC
500) was used to investigatethe distribution of mRNA and cytoskeletal
proteins in whole mount preparations of freshly isolated and cultured
cornea1 epithelia. The distribution of the fluorescentlytagged structures
were localized utilizing optical sectioning and image enhancement.
Epithelia were isolated from corneas with or without the basal lamina
(BL) intact, placed on black polycarbonate filters (Poretics), then
incubated with the appropriate marker. cDNA probes specific for types
I, 11, and IX collagen were labeled with 32P-dCTP or biotin-1GdUTP.
The biotin labeled probes were visualized with either avidin-FITC or
anti-biotin anitbody then goat anti-rabbit colloidal gold. The 32P labeled
probes were used to quantitate the amount of specific mRNA in
epithelia. Actin was labeled with rhodamine phalloidin. Cytokeratin
was visualized with primary antibody then fluorescently tagged
secondary antibody. Hybridization of cDNA probes were probe
concentrationand size dependent. Types I and II collagen were twenty
fold higher than type IX collagen and had different distribution patterns.
The actin was prominent as an organized network (actin cortical mat) in
the basal compartment of the basal cells when isolated with the BL
intact, and at the interface between the basal and periderm cells. Actin
also defined the cell borders and microvilli of the periderm cells.
Epithelia isolated without the BL did not havc an organized actin cortical
mat, rather the actin was localized in the blebs on the basal cell surface.
Epithelia isolated without the BL intact, and c u l t u d in the presence of
5pgJml laminin reorganized the actin cortical mat within 2 hours.
Whereas, epithelia isolated without BL and cultured without BL
proteins continued to have basal cell blebs, sometimes into the pores of
the filter. The cytokeratin filaments formed a basket-like network
around the nuclei of the periderm cells, and appeared to be aligned from
cell to cell. Treatment of epithelia with 2pM cytochalasin D disrupted
both the actin and cytokeratin networks in both cell layers of the
epithelia and decreased the amount of types I and I1 mRNA. In
conclusion, the use of confocal analysis increases our knowledge of the
spatial relationships between cytoskeletal proteins and diferent types of
collagen &NA. (AR38960 and Whitaker Health Science Fund)
TAGOE*, Clifford. and Robert Doyle YATES, Department of
Anatomy, Tulane University School of Medicine, New Orleans,
Louisiana. The structure of rat embryonic blood vessels.
The ultrastructure of the major blood vessels in the
11 1/2 day rat foetuses has been studied by scanning and
transmission electron microscopy.
The paired dorsal
aortae, the single dorsal aorta, and the anterior and
posterior cardinal veins consist of well-formed endothelial
tubes surrounded by sheaths of mesenchymal cells. The
endothelial cells have flat lumina1 surfaces which bear
short processes. TEM reveals that the endothelial cells
contain much rough endoplasmic reticulum, free and
polyribosomes and Golgi complexes but few pinocytotic
vesicles are present.
The endothelial tubes are associatedwith discontinuous
basement membranes which are more prominent in the
arteries. The surrounding sheath of mesenchyme consists
of cells arranged in concentric layers and elongated along
the circumference of the endothelial tube. In between the
cell layers cytoplasmic processes mingle with extracellular
matrix which consists of both fibrillar and amorphous
units. There are no clear bundles of elastic fibres. The
deepest cells are highly attenuated and are closely apposed
to the endothelial cells where the basement membrane is
deficient.
The outer cells are progressively less
flattened and after 6 or 7 (aortae) and 3 or 4 layers
(cardinal veins) become more mesenchymal in appearance.
The cells contain numerous arrays of rough endoplasmic
reticulum, prominent Golgi bodies, large numbers of
mitochondria, and most of them have patchy basement
membrane around them.
The deepest cells in addition
contain bundles of filaments in the peripheral cytoplasm.
Some of the mesenchymal cells in the interval between the
fusing dorsal aortae show signs of degeneration. Among
these are phagocytic cells. Our findings are in agreement
with those of Cayette et. al. on rabbit embryos (Basic Res
Cardiol, 8 4 : 2 5 9 - 2 6 7 , 1989). It also appears that during
fusion of the dorsal aortae "unwanted" mesenchymal cells
between them are removed in order to approximate the two
vessels.
T a r n , C . C . and % o n e , T . C . Department o f A n a t o m y ,
UniL-ersity of H o n g tiong, H o n g t i o n g . Ultrast.ruc.=
t u r a l morphometric analvsis of the lateral . x o . c
t a t q ~p f the intact .at??. castrated guine.+ap_i.g,
In the present stud>-, the lateral
pr-ostatelLPI of the guinea pig is described within
the framework o f a morphometric model. T h e morphological changes o f androgen deprivation in the
~ u i r ~ epig
a
LP w e r e analysed b y ultrastructural
morphometry. After perfusion fixation b y glutaraldehl.de, t h e LP o f both the intact and castrated
a n i m a l s were dissected out, weighed and processed
for electron microscopy. Stereological analyses
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
were performed hp a comhination of direct measuremcnt with a gr+iphics tablet and point and inters e c t i o n coiiiiting m e t h o d o n t h e e l a n d u l a r
epithel i i i m and the secretory c e l l s . Quantitative
d a t a here expressed i n the forms of relative densities and in absolute terms on a per average cell
basis. Castration u a s accompanied hy a quantitatiye decrease i n the weight of the organ 159.19%),
height of the epit.helium (30.03%) and the mean
\ o l u m r of the secretory cells ( 5 3 . 3 1 % 1 when compared with the intact, controls. The reduction in
t h e mean \ - o l u m e o f t h e secretor>- c e l l s was
r r s u l ted from a simultaneous decrease in the
average ~ o l i t m e (,f the cytoplasm ( 5 6 . 5 3 % ) and the
r i u ( . l t ~ u s ( 4 0 . 8 7 % ) . T h e ?:tent
of rrgression w a s
g r e a t i ' r in the Golqi complex than that of the
granular endoplasmic r e t i c u l i u n ( G E R I . The amount
of h i g h l y electron-dense ~ r a n u l e s decreased s i g nificantly w h i l c the low electron-dense and clear
graniiles remairied unaffected after castrat ion. The
qii;iiit~tati\ed a t a s h o w e d that the size rather than
I,hv riunitwr of t h r . mitoclionrlria w a s affected after
costrat ion. The stereoloqical data characterizing
tile intact y r i i n e n pig will serve a s R base line
f r s r fiirther stiidies. The decrease in function of
the s e c r e t - o r ? c ~ l l safter castration %as reflected
ti) A signjfjcant decrease iri the amount of ( ; E R ,
Gca19-i tomplrx an:l its a s s o c i a t e . d elrments ai~d
S P c r e t < o r \ ; $i.niliile:; o n a per cell b a s i s .
TANDLER, Bernard, and C a r l e t o n J. PHILLIPS*, Department o f
Oral Biology, School o f D e n t i s t r y , Case Western Reserve
U n i v e r s i t y , Cleveland, Ohio, and Department o f B i o l o g y ,
H o f s t r a U n i v e r s i t y , Hempstead, New York. Corn a r a t i v e
ultrastructure
s e c r e t o r y granules i n t h k u l a r
glands o f A f r i c a n m u r i d rodents.
of
Members o f several m u r i d r o d e n t species were captured i n
v a r i o u s l o c a t i o n s i n Kenya and t h e i r submandibular glands
prepared f o r comparative e l e c t r o n microscopic examination.
Acinar s e c r e t o r y granules i n Praomys (Mastom s) n a t a l e n s i s
c o n s i s t o f a b u l l ' s eye i n c l u s i o n c o m p d p t o s i x t e e n
a l t e r n a t i n g dark and l i g h t r i n g s suspended i n a dark m a t r i x .
I n Colom s g o s l i n g i , t h e granules c o n s i s t of a dense,
i r r d a c e n t r i c a l l y placed i n c l u s i o n t o which a r e
attached stacked dense arches. I n Zenomys h i l d e ardae
t h e granules c o n s i s t o f a b u l l ' s eye over w b i a t tened l i g h t and dark r i n g s a r e draped, a l l p l a s t e r e d
a g a i n s t a dense i n c l u s i o n . I n Oenomys hypoxanthus, each
granule c o n s i s t s o f amoderately dense m a t r i x t h a t leaves a
l i g h t crescent a g a i n s t one pole; t h i s l i g h t area i s
occupied by c o n c e n t r i c a l l y disposed s t r i n g s o f small part i c l e s ; a f l a t c r y s t a l l o i d composed o f t h r e e o r f o u r e l o n gated, p a r a l l e l d e n s i t i e s i s present a t t h e j u n c t i o n o f t h e
dark and l i g h t zones. I n Mus sp., t h e granules c o n s i s t o f
many h i g h l y contorted, low-&nsity
rods i n a dense m a t r i x ;
numerous c o n f i g u r a t i o n s a r e generated by t h e manner i n
which t h e plane o f s e c t i o n passes through p a r t i c u l a r
granules. Granules i n Lo hurom s f l a v o u n c t a t u s resemble
those i n t h e species o f Mis tha: wc-ept
that
t h e low d e n s i t y rods a r e x r e r e g u l a r l y packed, so t h a t i n
c e r t a i n planes o f s e c t i o n t h e granules have a honeycomb-like
appearance. I n Aethom s k a i s a r i , t h e granules c o n t a i n a
v e r y dense, i r r e b h m h a t i s surrounded b y a l e s s
dense, ragged halo; these i n c l u s i o n s a r e suspended i n a
l i g h t , f i n e l y f i b r i l l o g r a n u l a r m a t r i x . These observations
i n d i c a t e t h a t , i n terms o f s a l i v a r y gland u l t r a s t r u c t u r e ,
A f r i c a n m u r i d rodents n o t o n l y a r e very d i f f e r e n t from
comnon l a b o r a t o r y rodents, b u t t h a t t h e y show as much
granule s t r u c t u r a l v a r i a t i o n as we have shown t o e x i s t i n
chiropterans. Supported i n p a r t by N I H g r a n t DE 07648.
101A
TANDLER Bernard G i u l i a n a SERRA* F e l i c e LOFFREDO* and
Alessandro RIVA, 'School o f D e n t i s t r y , Case Western Reserve
U n i v e r s i t y , Cleveland, Ohio, and Departments o f Zoology and
Cytomorphology, U n i v e r s i t y o f C a g l i a r i , C a g l i a r i , I t a l y .
microscopy o f t h e t o n g u e - o f t h e S a r d j n i a n
The tongue o f t h e S a r d i n i a n cave salamander, H dromantes
(Tarn-Schl.) c o n s i s t s o f a l o n g c a r t i l a g i n &
w i t h a fleshy, g l o b u l a r or, more u s u a l l y , a r o u g h l y t r i a n g u .
l a r protuberance a t i t s a n t e r i o r end t h a t i s o r i e n t e d w i t h
i t s v e r t e x forward, so t h a t t h e tongue as a whole resembles
a c r u d e l y fashioned arrow. The f l e s h y p o r t i o n i s covered
by an abundance o f s h o r t c l a v a t e o r l e a f l i k e p a p i l l a e ,
which probably a r e t h e cave salamander c o u n t e r p a r t s o f mamm a l i a n f i l i f o r m p a p i l l a e . I n t e r s p e r s e d among these p a p i l l a e
a r e a number o f dome-shaped t a s t e organs. T h e i r surface
c e l l s a r e o u t l i n e d by u n u s u a l l y l o n g m i c r o v i l l i , so t h a t
t h e t o p o f each t a s t e organ has a r e t i c u l a t e d appearance.
The remainder o f each c e l l a t t h e s u r f a c e o f t h e t a s t e
organs i s covered b y stubby m i c r o v i l l i . The deep holes
between these s u r f a c e c e l l s presumably a r e t a s t e pores.
The c a r t i l a g i n o u s p o r t i o n o f t h e tongue i s covered by a
t h i n e p i t h e l i u m c o n s i s t i n g o f l a r g e polygonal c e l l s , which
have an i r r e g u l a r s u r f a c e a r r a y o f m i c r o p l i c a e t h a t show
s l i g h t changes i n p a t t e r n proceeding p r o x i m a l l y down t h e
rod. I n t h e r e t r a c t e d c o n d i t i o n , t h e tongue s i t s i n an
appropriately-shaped pocket i n t h e f l o o r of t h e mouth.
Pressure e x e r t e d on t h e r o d by c o n t r a c t i o n o f muscles
w i t h i n t h e mandible causes t h e tongue t o p r o t r u d e f o r an
a s t o n i s h i n g l y l o n g distance. S t i c k y s e c r e t i o n s , produced
b y t h e numerous l i n g u a l s a l i v a r y glands, undoubtedly add t o
t h e e f f e c t i v e n e s s o f t h i s orqan i n caDture o f Drev.
Supported i n p a r t b y a g r a n t from t h e C o n s i g i i d N a z i o n a l e
d e l l e Ricerche.
genei
TAY, S.S.W. and W.C. WONG, Department of Anatomy, National
University of Singapore, Kent Ridge, Singapore. Short
term effects of insulin therapy on the gracile nu=
of streptozotocin-induced diabetic rats.
Recently, we reported the ultrastructural changes
in the gracile nucleus of streptozotocin-induced diabetic
rats (Tay & Wong, Anat. Rec 220 : 95A).
It is now
confirmed that these structural changes can be prevented
by means of insulin therapy in short term diabetic rats.
Under ether anaesthesia, male Wistar rats (180-2508)
were injected intravenously with streptozotocin (60 mg/kg
b.w. in 0.01M citrate buffer, pH 4 . 5 ) .
One group of
diabetic rats were further treated with protamine zinc
insulin (2-5 I.U.) daily, thereby maintaining a normal
blood glucose level. Both non-insulin treated and insulin
treated diabetic rats were killed by perfusion with a
mixed aldehyde solution at 3 and 7 days post-induction.
Saline injected rats served as controls.
Tissues
containing the gracile nucleus were processed for electron
microscopy. In non-insulin treated diabetic rats, numerous
electron dense axon terminals and dendrites were present
in the gracile nucleus at 3 and 7 days post-induction.
Degenerating axon terminals were characterized by swollen
mitochondria, small vacuoles and clustering of small
spherical agranular vesicles in an electron dense
cytoplasm. Most of these degenerating axon terminals
were in synaptic contact with normal and degenerating
dendrites.
Degenerating dendrites showed an electron
dense cytoplasm, swollen mitochondria, dilated rER, small
vacuoles and randomized ribosomes. In insulin treated
diabetic rats, degenerating electron dense axon terminals
and dendrites were absent in the gracile nucleus studied.
The neuronal cell bodies and their neuropils showed
features similar to those observed in control animals.
Insulin therapy appears to prevent the acute onset of
electron dense degeneration in the gracile nucleus of
102A
ABSTRACTS-AAA 103RD MEETING
short-term
d i a b e t i c rats.
It i s c o n c l u d e d t h a t t h e
degenerative
changes
in
the
gracile
nucleus
of
s t r e p t o z o t o c i n - i n d u c e d d i a b e t i c r a t s were t h e r e s u l t s
of d i a b e t e s m e l l i t u s and n o t d i r e c t chemical t o x i c i t y .
TENNYSON, Virginia M., Diane L. SHEWAN*, Richard R. BEHRINGERI, Ralph
L. BRINSTER', Richard D. PALMITER', Jason TASCH', David cRO?TY*, Debra
J.WOLGEMUTH* and Michael D. GERSHON, Departments of Anatomy & Cell
Biology, Pathology, and Genetics & Development, Columbia Univ. P & S, New
York, New York; Laboratory of Reproductive Physiology, Univ. Penn. School of Vet.
Med., Philadelphia; Pennsylvania; and Dept. Biochemistry, Univ. Washington, Seattle,
Washington.
'
e
p
Transgenic mice carrying multiple copies of the endogenousH Q X Ugene develop
congenital megacolon and express elevated levels of HmJ.4 mRNAs in the gut
(Wolgemuth et al., 1989).The current study was done to compare the abnormalities in
the colon of transgenic mice with those of lethal spotted (lslls) mice, in which
megacolon also arises. Studies demonstrating AChE activity showed that t h ~erminal
bowel of aansgenic mice is hypoganglionic not, like that of lslls mice, agar glionic.
Myenteric ganglia of the transgenic mouse at day 7'F were located in abnorma 1y wide
gaps in the external longitudinal muscle where they made direct contact with the
adventitia. These ganglia appeared in sporadic locations from the dilated bowel to the
recto-anal junction. Some of these abnormally located ganglia persisted into young
adulthood. Within the unusual ganglia the ultrastructure was that of exuaenteflc
peripheral nerve, rather than that of the ENS; thus, the abnormal ganglia had
perineurial sheaths, internal collagen and Schwann cells, which wrapped individual
axons. Myelinated axons were present in large nerve bundles in the intermuscular and
submucosal regions of both the transgenic and klls mice, but were never found in these
regions in normal mice. Neuronal cell bodies were first recognizable in the fetal gut of
normal and aansgenic mice at days E14-15. In the aansgenic animals, some of these
neurons had dense core granules resembling r8ose seen in sympatheticganglia. Similar
cells were not seen in the control. At this stage immunoreactive staining for laminin
and Type IV collagen was similar in conJol and aansgenic mice, but markedly
increased in lslls mice. We have proposed thdt the overabundanceof these components
of basal laminae may prevent the migration of neural crest emigres into the
presumptive aganglionic bowel of lslls miL:e. Smooth muscle cells develop at the
same fetal period as neurons; however, smooth muscle cells of both the transf :nic and
the lslls mice always appeared less mature than those of controls. Abnonnalibss of the
external longitudinal muscle persisted in the caudal colon of day P7 transger.c mice.
The cells were loosely packed and irregularly shaped and had few adherent junctions,
unlike controls, which had a closely packed regular pattern with many adherent
junctions. In a transgenic mouse that survived for 1 1/2 years, the caudal colon had
become aganglionic with hypertrophic smooth muscle. Aganglionosis in old
transgenic mice is probably due to cell death. These data suggest that congenital
megacolon develops in transgenic mice because an abnormal pattern of ganglionosis
occurs in the terminal bowel. The local reflexes upon which intestinal propulsion
depends therefore are lacking. The condition is not identical to that which appears in
lslls mice, although abnormalities of smooth muscle may interfere with neural
development in both animals. "Supported by N M grants HD 17736, NS 15547, HD
18122, HD 21032 and the Parkinson's Disease Foundation."
Gordana, C a r l O S A.C.
BAPTISTA, L i b e r a t o J . A . D I D I 0 ,
Department o f
Anatomy and C l i f t o n VAUGHAN*, Image A n a l y s i s
R e s e a r c h C e n t e r , Medical C o l l e g e o f Ohio,
membranacea a the
Toledo, Ohio. The
interventricular seDtum and its relationship
with the aortic valve in human hearts.
T h e areas and d i s t a n c e s were traced
m a n u a l l y , u s i n g t h e N I H Image program (Wayne
Rasband, NIH, R e s e a r c h Services Branch, N I M H ) .
The f o l l o w i n g forms o f PMSI were found: o v a l
(31.2%), triangular(25%),quadrangular(21.9%),
c i r c u l a r ( l 5 . 6 % ) a n d s e m i l u n a r ( 6 . 2 5 % ) : its s u r f a c e area v a r i e d from 5.65mmz t o 1 4 2 . 6 3 m m '
(37.221113'
a v e r a g e i n C a u c a s i a n s and 50. 99mm2
i n n o n - C a u c a s i a n s ) . The s u p e r i o r b o r d e r o f t h e
PMSI w a s most f r e q u e n t l y ( 4 0 . 6 % ) c l o s e l y r e l a t e d t o t h e i n s e r t i o n s o f b o t h t h e r i g h t (RC)
and p o s t e r i o r ( P C ) c u s p s o f t h e a o r t i c v a l v e ,
o r o n l y w i t h t h e PC ( 3 4 . 4 % ) . I n 18.7% o f t h e
c a s e s it w a s s i t u a t e d 2 . 7 - 3 . 6 m m below RC and
PC i n s e r t i o n s .
I n 6 . 2 5 % o f t h e cases t h e
s u p e r i o r border of t h e PMSI c o r r e s p o n d e d t o
t h e RC i n s e r t i o n a n d w a s n o t more t h a n 1 . 8 4 m m
below t h e PC i n s e r t i o n . D i f f e r e n c e s between
sex and race g r o u p s were n o t e d .
ber.
THLIVERIS, J.A., M. LUKOWSKI*, K.R. COPELAND*, and R.W.
YATSCOFF*,
Departments of Anatomy and Biochemistry*,
University of Manitoba and Health Sciences Clinical Research
Centre, Winnipeg, Manitoba, Canada.
Chronic Cvclosporine NeDhrotoxicitv: A rabbit model.
A suitable animal model which exhibits the chronic cyclosponne
(CsA) nephrotoxicity seen in humans has not been reported. We
have investigated the rabbit as a more promising model. Twentyfive age-matched male New Zealand white rabbits (2.5 - 3.5 kg)
were administered CsA I.V.at the following doses (5 animals per
group): Group A, 2.5 mg/kg/d; Group B, 5.0 mg/kg/d; Group C,
10 mg/kg/d; Group D, vehicle (cremophor) control; Group E, saline
control.
Creatinine clearance and CsA concentrations were
measured weekly. At the end of the study (30 days), blood
pressures were measured with the animals subsequently being
sacrificed and the kidneys obtained for light and electron
microscopic studies.
The results showed reduced creatinine
clearance in Group C over all other groups with no differences
among various groups in serum creatinine or blood pressure.
Morphological analysis revealed the following in a dose-related
1) leucocyte infiltration, 2) interstitial fibrosis, 3)
manner:
numerous lysosomes and vacuoles and loss of cellular integrity in
both proximal and distal tubule cells. All these results resemble
chronic CsA toxicity seen in man.
(Supported by Grant MA-10606 from the Medical Research Council
of Canada.)
TEOFILOVSKI-PARAPID*,
Improvement of t e c h n i q u e s f o r c a r d i a c v a l v e
s u r g e r y , c o r r e c t i o n s of c o n g e n i t a l h e a r t
m a l f o r m a t i o n s , and t h e s u r g i c a l t r e a t m e n t of
malignant
ventricular
arrythmia
require
c o n t i n u o u s s t u d y o f t h e s e p t a l area o f t h e
human h e a r t . Toward t h i s p u r p o s e , i n v e s t i g a t i o n s w e r e p e r f o r m e d i n 32 human h e a r t s o f
a d u l t i n d i v i d u a l s ( 1 9 C a u c a s i a n s and 13 nonC a u c a s i a n s ) . With t r a n s i l u m i n a t i o n of t h e p a r s
membranacea of t h e septum i n t e r v e n t r i c u l a r e
(PMSI) t h e s p e c i m e n s w e r e p h o t o g r a p h e d and t h e
3 5 m m s l i d e s o b t a i n e d w e r e d i g i t i z e d i n t o Apple
Macintosh I1 u s i n g a Dage Model 68 v i d e o c a m e ra and a Data T r a n s l a t i o n s DT 2 2 5 5 frame g r a b -
0 THORNE-TJOMSLAND,
G r o , Kirsten SANDVIG a n d S j u r OLSNES,
D e p a r t m e n t o f Anatomy, M c G i l l U n i v e r s i t y , M o n t r e a l ,
Canada;
Department
of
Biochemistry,
The Norwegian
Radiumhospital,
O s l o , Norway.
( S p o n s o r e d b y Yves
C l e r m o n t ) B i n d i n a . e n d o c v t o s i s a n d t o x i c i t y of v o l k e n s i n
i n Vero c e l l s .
The p r e s e n t s t u d y i n v e s t i g a t e d t h e b i n d i n g , e n d o c y t o s i s a n d t o x i c i t y o f v o l k e n s i n i n A f r i c a n g r e e n monkey
Volkensin is a r e c e n t l y p u r i f i e d
kidney cells (Vero).
p l a n t t o x i n ( S t i r p e e t a l , 1985), which i s i n t e r n a l i z e d
i n t o c e l l s a n d c a u s e s i n h i b i t i o n o f p r o t e i n synthesis.
Binding s t u d i e s ,
i n which c o n f l u e n t c u l t u r e s were
incubated a t 0°C w i t h iodinated volkensin, r i n s e d w i t h
c o l d medium, d i s s o l v e d a n d t h e r a d i o a c t i v i t y c o u n t e d ,
showed t h a t 1) t h e r e w e r e more than o n e m i l l i o n r e c e p t o r s
f o r v o l k e n s i n p e r V e r o c e l l , 2 ) t h e r e c e p t o r s were
glycoproteins,
since l a c t o s e c o m p e t i t i v e l y i n h i b i t e d
103A
ABSTRACTS-AAA 103RD MEETING
binding and 3 ) binding occurred with high affinity, since
neither lactose nor a cross-reactive antibody could
release bound volkensin.
In TEM studies of cultured
cells incubated with volkensin-HRP at 37°C for 15, 30 or
60 minutes and fixed in glutaraldehyde/ FeCN-reduced
O s O 4 , endocytosed volkensin conjugate appeared
consecutively in coated buds and vesicles, vesicular and
tubular endosomes and 1-2 elements closely associated
with the trans-aspect of the Golgi apparatus.
Temperature blocks showed that transport to the Golgi
apparatus probably was necessary for toxin activation.
The toxic effect of volkensin, measured as a decreased
ability of the cells to incorporate 3H-leucine,was most
pronounced after longer incubations (more than 12 hours)
and did not appear, from ionophore experiments, to depend
on an acid pH in the endosomal apparatus. In conclusion,
volkensin is a highly toxic compound for which there are
a large number of binding sites; it exerts its toxicity
after being
internalized by receptor-mediated
endocytosis. (Supported by the Norwegian Cancer Society
and the Medical Research Council of Canada).
Thomas A. Marino, Michael Cassidy*, Nancy L. Carson*,
Elizabeth Dinda*, Deborah R. Marino*, Steven R. Hower*.
Departments of Anatomy and Physiology, Temple University
School of Medicine, Philadelphia, Pennsylvania. Attenuation of
Cardiocvte arowth in size in a model of severe oressure
overload hvDertroDhv.
Growth in size of cardiocytes normally parallels the degree
of cardiac hypertrophy, especially when the hypertrophy is a
result of physiological or moderate overloads placed upon the
heart. In this study, we examined the size of adult cat
cardiocytes isolated from controls (C) and cats subjected to
moderate pressure overload of the right ventricle (RV)resulting
in compensated hypertrophy (H), or a severe overload
resulting in non-compensated hypertrophy (SH). After 45 - 60
days of banding, RV systolic pressure (mmHg) was elevated
in both experimental groups (C = 21.2k2.1; H = 47.7+4.7*;
SH = 62.3+0.6*; *:P 50.05). End-diastolic pressures (mmHg)
were significantly elevated in the SH group only (C = 2.021.4;
H = 2.1k0.7; S H = 7.3+1.3*). The cardiac index and heart
rate remained unchanged in both groups compared to
controls. The maximum width of H or SH cells did not change
(C = 19.0k0.4 #rn; H = 2l.lk0.8 *rn; SH = 20.3k0.6 rm) nor
did the length C = 115.6~2.9#m; H = 125.925.4 @rn;S H =
118.7k0.3 ,,rn). Thus, in compensated hypertrophy the
cardiocyte increases its size with increases in the minimum
diameter of the cell resulting in a cardiocyte with a round cross
sectional area. However,further increases in size by increases
in the maximum cell width do not occur as a consequence of
severe cardiac hypertrophy, and this may be a significant
consequence leading to congestive heart failure.
(Supported in part by NIH grant HL29351 and grant 88-1 147
from the American Heart Association and it Pennsylvania
Affiliate.)
THOMPSON, Ed W., and Ann M. SCHMEICHEL*; Hormel Institute,
university of Minnesota, Austin Minnesota. Structural
analysis of the mvocardium in the aeneticallv obese rat.
Cardiac hypertrophy and contractile dysfunction leading
to heart failure frequently develop in obesity. Reports of
myocardial structure, however, are limited to postmortem
examinations in which duration and severity of obesity are
widely diverse. In this study we have used the genetically
obese Zucker rat, which closely mimics
the
hyperplastic/hypertrophic pattern characteristic of the
childhood-onset type of human obesity, to examine and
quantify myocardial structure at 3 month intervals from 4
weeks to 18 months of age. Lean (heterozygous, FA/?) male
rats reached maximum weight of 4 5 1 2 2 9 g (x+SD) at
approximately 9 months of age and maintained this weight,
while obese (fa/fa) rats gained weight through 12 months,
772236 g, but then lost weight again. Heart weights were
higher in obese rats even after they lost weight, but heart
wt:body wt ratios were lower than in lean rats after 3
months of age. Ratios of left ventricular and right
ventricular weights to total heart weight revealed no
selectivity of the hypertrophy in the obese animals. An
increase in the extracellular matrix was noted in obese
rats at 12 months of age and increased with age, while no
such deposition was seen in lean rats through 18 months.
Morphometry revealed a trend toward increased cardiocyte
cross-sectional area in obese rats at 3 months of age
compared to lean animals of the same age, which became
statistically significant at 6 months. Stereologic
analysis of the cardiocytes revealed no changes in relative
volume densities of mitochondria, myofibrils, o r free
cytoplasm in either obese or lean rats. Paradoxically,
cardiocytes of obese animals tended to have less fat stored
as cytoplasmic droplets than did their lean counterparts.
No changes in cardiocyte ultrastructure were seen a s either
obese o r lean rats aged. These results are consistent with
early development of an eccentric pattern of hypertrophy
typical of volume overloading of the heart, followed by
moderate development of concentric hypertrophy typical of
pressure overloading. (Supported by the MN Affiliate of
the American Heart Association and the Hormel Foundation.)
TIMMERMANS,
Jean-Pierre,
Dietrich W;
SCHEUERand Marie
Histology
and Microscopic Anatomy, University of Antwerp,
Belgium; and Institute of Anatomy, WilhelmPieck-University, , Rostock, GDR. Distributional
pattern of qalanin-immunoreactive ( C i A L - l K 1 neurons ana tneir proIections in tne small intestine of the piq.
MA", Werner STACH,* Dirk ADRIAENSEN,
H.A. DE GROODT-LASSEEL, Institute of
Immunocytochemical
techniques
were used
to
the distribution of GAL in the enteric ner?%
system of the porcine small intestine.
Double-immunolabelling studies with either 5-HT,
other peptidergic substances or neuronal markers
were carried out in order to characterize these
intramural enteric neurons in relation to their
mor holo ical features After colchicine treatmeng, G%rL-IR neurons were found in the three
gangljonic nerve lexuses in the small intestine
of six-week-old gomestic ,pigs ( 6000 neurons/cm2
in the plexus submucosus internus (PSI), 160/cm2
in the externus (PSEI, 400/cmZ in the myenteric
morplexus ( M P ) ) . In a11 ganglionic networks
phologically defined minineurons are G d I R . a n d
most of,them are probabl also VIP-IR. Besides
the minineurons, a consizerable number of other
non-identified neurons are,GAL-IR too. In contrast to the other anglionic networks, where no
colocalization couyd be,demonstrated, GAL coexists with substance P in a few neurons of the
PSI. Thus far no coexistence has been observed
between GAL And 5-HT in the enteric neurons.
Double-labelling with neurofilament 200kD made
clear that the malority of the Dogiel type IIneurons are on1 weakly or, in most cases, not
GAL-IR. Type IIf-neurons were also found to be
GAL-immunone ative
Varicose GAL-IR fibres were
observed,in zhe thiee an lionic nerve ,meshworks
of the intestinal wal?. zocal disru tion of the
enteric nerve fibre athways b myecgomy or double m otomy in the 8P revealex an accumulation
G d - I R material at the oral side of the len
o
:
to 2 nun aborall
the number of IRfibr6s %as
si nificanly YAwer. The submucosal
networks did nog seem to be directly affected by
these lesions. From these findings, it can be
concluded that in the pig GAL-IR is resent in
functionall
distinct neu;onal
populagions. In
keeping wkgh recent electro h siolo ical stuno? only exdios, it is thus assumed that,&
erts several mechantsms of action but,also plays
more than one role in intestinal physiology.
104A
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
TIMMS, Barry G. and Liberato J . A . DiDIO, Department of
Anatomy, University of South Dakota, Vermillion, South
Dakota and Department of Anatomy, Medical College. .of
Ohio, Toledo, Ohio. T h e t of V
e of
Prostatic bud formation from the rat urogenital
sinus appears to be induced by a critical surge of
testosterone production between 16.5 and 19 days of
gestation. Using organ culture methods it was previously
shown that prostatic buds can be induced de nuvo by
androgens in urogenital sinuses derived from either male
or female rats.
In certain rodents, such as the
multimammate mouse, all females possess a well developed
prostate, homologous to the ventral lobe of the male. In
other species the gland is rarely seen in the adult
female. During the latter stages of pregnancy female rat
fetuses are exposed to varying levels of testosterone and
estradiol, based on their intrauterine position adjacent
to fetuses of the same or opposite sex. The present
study was undertaken to examine the development of the
prostatic anlageri between 18.5 and 21 days of gestation,
in female Sprague-Dawley rats taken from various in utero
positions. Fetuses were removed by Caesarian section and
their sex determined by measuring ano-genital distance.
The incidence and nature of prostatic bud development was
assessed
using
computer-assisted
3-dimensional
reconstruction of serially sectioned urogenital sinuses
from OM, 1M and 2n females (females that develop either
between two females, one male and one female, or two
males, respectively).
Two distinct features were
observed. Only exceptionally do OM females show evidence
of prostatic bud development. In comparison, lM and 211
females
frequently
exhibited
prostate
gland
morphgenesis, homologous to the ventral/lateral region
of the male. This development coincided temporally with
that of the male, but to a lesser degree. The results
suggest that differential exposure to androgen levels, as
a consequence of intrauterine position, contributes to
the occurrence of female prostatic bud development.
Supported by a USDSM Alumni Faculty Development Award and
the Parson-sEndowment Fund.
TODD, Mary E. and R.D. BEVAN, Department of Anatomy,
University of British Columbia, Vancouver, Canada: and
Department of Pharmacology, University of Vermont,
Burlington, Vermont.
Chanqes in SaDhenous artery durina arowth in WKY and
SHR: Effects of symDathetic nerve denervation.
Thigh vessels of spontaneously hypertensive (SHR) and
Wistar-Kyoto (WKY) rats were unilaterally surgically
denervated at 10 days of age by femoral nerve section.
The nerve stump was sealed within polyethylene tubing
within the abdominal cavity. Denervated and contralateral control segments of saphenous arteries from 3 and
6 week old rats were mounted in a small vessel myograph
for study. Both strains showed growth changes in blood
pressure, but there was no significant difference between
WKY and SHR. Both strains also had significant growth
changes in vessel dimensions and SHR vessels had a
thicker wall from in vitro measurements. Denervation did
not affect vessel size. Transmural nerve stimulation
indicated loss of innervation due to the surgical
procedure.
In the denervated vessels, both norepinephrine (NE) and 5-hydroxytryptamine (5-HT) dose
response curves were shifted to the left indicating a
postjunctional increase in sensitivity. Maximum tension
developed was in the order K+>5-HT>NE. In comparing
the two strains, vessels from 6 week old SHR were less
sensitive to 5-HT.
Relaxation to acetylcholine was
significantly decreased in denervated arteries from WKY
at both 3 and 6 weeks of age, whereas in SHR, only at 3
weeks. Denervated vessels from both rat strains at 3
weeks showed greater relaxation to 0 receptor activation,
but not at 6 weeks of age. Tension developed by passive
stretch indicated that changes in connective tissue had
occurred in segments from denervated vessels. Therefore,
the absence of functional innervation resulted in altered
structure and function of the saphenous artery wall.
Although there were no differences in blood pressure,
significant differences between the two strains were
present. (Supported by the British Columbia and Yukon
Heart Foundation and Grant No. PHS R37 32985-06 from NIH).
UDDIN, M. Departemnt of Anatomy, University of Saskatchewan, Saskatmn, Saskatchewan, Canada.
Cell surface and cytoskeletal autoantibodies directed
against glial cells in mrine lupus.
Systemic lupus erythematosus (SLE) is an autoimune
disease of grave consequences. It is a disorder in which
the balance between immunoregulatory mechanisms is disturbed.Murine lupus is very similar to human SLE and
shares many of its immunological, serological and genetic
background. Central nervous system ( C t i S ) manifestations
of human SLE are well recognized, however, underlying
mechanism of CNS irrununopathogenesis is not clear at
all.Recent studies on human SLE have suggested an
association between anti-cell membrane and anticytoskeletal auto-antibodies and CNS disorders.However,
similar investigations on mrine lupus are 1acking.In the
present report anti-cell membrane and anti-cytoskeletal
lupus autoantibodies are immunocytochemically demonstrated.
14 to 21 day old primary astrocyte cultures were
prepared from one day old mouse neopalia.To demonstrate
cell surface lupus antibodies cultures were fixed in 3.7%
paraformaldehyde for 50 seconds and treated with diluted
sera from MRL/lpr-lpr and NZBpJZW F1 mice, followed by
FITC-conjugated anti-mouse 1gG.Fixed cultures were then
treated with 0.2% Triton-X for 10 minutes and subjected to
anti-mouse GFAP antibody which is a specific marker for
astrocytes, followed by rhodaminated anti-rabbit IgG.
Reaction sites were visualized by a fluorescein microscope.To demonstrate cytoskeletal lupus autoantibodies,
cultures were incubated in lupus mice sera, followed by
anti-rabbit 1gG.Cells were then incubated either with
rabbit anti-actin or anti-vimentin antibodies, followed by
rhodaminated anti-rabbit 1gG.Fluorescence of both were
compared with lupus autoantibody pattern.
Results indicated that like human SLE, murine lupus
also produced antibodies against cell surface and
cytoskeletal elements of astrocytes.The possibility was
CNS
raised
about
their
involvement
in
the
pathogenesis.(Supported by University of Saskatchewan
Research Fund #7-78048).
UDDIN, M.
Department of Anatomy, University of
Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N WO.
(Sponsored by DK. G.D. Burkholder). DEMONSTRATION OF W S E
SUBMANDIBULAR KALLIKREIN GENE (ffiK-5) FXPRESSICN BY IN SITU
HYBRIDIZATION WITH OLIGCNUCLEOTIDE PROBE.
Mouse submandibular gland kallikreins are a group of
serine proteases which are encoded by a sub-family of
twenty five closely related kallikrein genes. These genes
are located on chromosome number seven. M~mbeKSof this
family which have been characterized include the genes
encoding a-(mGK-4) and y-(mGK-3) nerve growth factor,
epidermal growth factor (&K-13), renal kallikrein (nGK-6)
and glandular kallikrein (&K-5).
Although kallikreins are closely related at the amino
acid sequence level, they exhibit a high degree of
substrate specificity. Thus, the analysis of the sites and
regulation of gene expression of these serine proteases is
of considerable functional importance.
In the present report the expression and site of muse
submandibular gland kallikrein gene(ffiK-5) is visualized by
ABSTRACTSAAA 103RD MEETING
in situ hybridization histochemistry.
A
27-mer
oligonucleotide probe was chemically synthesized against
nucleotide sequence of the exon-5 of the mouse
submandibular gland mGK-5 gene (from nucleotide number 1564
to 1591). For controls a non complementary sequence of the
same region was also synthesized and,additionally, a 24-mer
oligonucleotide was also made against the intron-1 (from
nucleotide number 696 to 720).These probes were tailed with
the digoxygenin-11-dUTP. Hybridization was visualized with
alkaline phosphatase tagged antibody against digoxygenin.
Results indicated that expression of the mGK-5 gene was
confined to the striated duct cells in the adult female and
convoluted duct cells in the adult male submandibular
gland. The gene specific mRNA in the male was substantially
more than in female. No reaction was observed in l'l-day
embryonic and 6-day post natal submandibular gland duct
cells.
It was concluded that with this method mGK-5 specific
mRNA can be precisely localized in individual cells with no
background whatsoever. Only adult mice expressed mGK-5 in
their submandibular gland and that expression was confined
to striated and convoluted duct cells.
(Supported by
University of Saskatchwan Research Fund #4-15098).
UGAILY-THULESIUS, Layla, P. Sivanandasingham and
Hanan Khalaf*, Department of Anatomy, Faculty of
Medicine, Kuwait University, Kuwait. Mast cells
A comparative study.
and La cells in the ureter
-
Recently, Ugaily-Thulesius 5 3, 1988,
described the unusual occurence of mast cells in
the human ureter. These cells were found either
as part of the subepithelial connective tissue
o r in close association with smooth muscle cells.
The majority were found in close contact with a
cell resembling fibroblasts, the La cell. The
presence of mast cells o r their association with
La cells in the ureters of other species has not
been reported. This investigation is a comparative study on the occurence of mast cells and
the La cells in the ureters of different species.
Ureters from rats, rabbits, guinea pigs and pigs
were obtained and processed for electron microscopy. On examination, mast cells were not found
in the rabbit, while the guinea pig ureter
contained a very small number and these were
mainly in the lamina propria and not associated
with smooth muscle cells. In both rat and pig
ureters, a large number of mast cells was seen,
These cells were located in close proximity to
smooth muscle cells and, in the case of the rat
were also in association with La cells. The
electron microscopic features of the mast cells,
however, were different in the two species.
In conclusion: it is apparent that there is a
wide variation in mast cell population in the
ureter of different species and that their
association with smooth muscle cells and La
cells is confined to certain species.
"Supported by Grant No. MA015 from the
University of Kuwait.
URBAN, * Michele A., Kevin S. LEE and Leonard M. EISENMAN,
Department of Anatomy, Jefferson Medical College, Thomas
Jefferson University, Philadelphia, Pennsylvania. purkinie cel!
&generation in the lurcher mutant mouse: A studv us inc s i l v e r
105A
heterogeneities, one of which is the antigen Zebrin 1. The adult
lurcher mutant mouse, however, is characterized by the total
loss of these Purkinje cells. This loss has been described (Caddy
and Biscoe, '79) as beginning at about postnatal day 10 and
continuing such that almost all of these cells are gone by the
cnd of the third postnatal month. In an attempt to determine
whether there is some recognizable pattern to this loss, as
relates to Zebrin 1 distribution, we examined the degeneration
and death of Purkinje cells using a silver stain (Nadler and
Evcnson, '83). Thus far, normal and lurcher littermate mice of
agcs ranging from P11 to P24 have been studied. Degenerating
axons are recognizable in the vicinity of the cerebellar nuclci
at P11. Only axonal staining is apparent up until P I 5 at which
point degenerating Purkinje cell somas and dendrites become
visible. At this developmental stage t h e numbers of such
degenerating cells is few. In subsequent days the numbers of
degenerating
Purkinje cells
increases
throughout
the
cercbcllar cortex in an apparently random manner without any
obvious anterior-posterior o r medio-lateral differences. In
addition there does not appear to be any clear clustering of
dcgcnerating cells which could be interpreted a s representing
parasagittal bands. We conclude that there does not appear to be
any recognizable s p a t i a l pattern to the degeneration of
Pu r k i n j e c e l l s in t h e lurcher mutant. ( W e gratefully
acknowledge support from NIH grant NS-22093.)
S
VAN BOCKSTAELE, ' Elisabeth and Gary ASTON-JONES *,
Department of Mental Health Sciences, Hahnemann University,
Philadelphia, Pennsylvania.
(Sponsored by Michael C.
Kennedy) w n s from the
Suoraowlomotor nucleus of the central arav to the rostral ventrd
Retrograde transport using either wheat germ
agglutinin- horseradish peroxidase or Fluoro-Gold from the
rostral ventral medulla yields retrogradely labeled neurons in
three different subdivisions of the periaqueductal gray (PAG).
These three subdivisions include the ventrolateral and dorsal
aspects of the PAG as well as a discrete area in the rostroventral
PAG, the supraoculomotornucleus of the central gray (SOM). In
order to further establish the pathway and specificity in the
circuitry between the PAG and the rostral ventral medulla, the
anterograde tracer fhaseolus vulgaris- leucoagglutinin (PHA-L)
was iontophoretically deposited into the three subdivisions of the
PAG mentioned above and both the fiber projections and
innervation patterns were mapped. PHA-L injections into the
different subregions of the PAG yielded differential fiber
termination in the rostral ventral medulla. PHA-L injections into
the ventrolateral aspect of the PAG yielded anterograde fiber
labeling in
the medial aspect of the
nucleus
paragigantocellularis with few fibers in the rostral ventrolateral
reticular nucleus (RVL). PHA-L injections into dorsal PAG yielded
anterograde labeling in the rostral portion of the nucleus
paragigantocellularis and in the caudal portion of the ventral
medulla with few fibers in RVL. Injections into the SOM yielded
numerous, highly varicose fibers in the RVL in the region
implicated in both cardiovascular and respiratory regulation. The
projection from the SOM was examined in greater detail. To
determine whether neurons in this region projected to the area in
the nucleus paragigantocellularis containing adrenergic neurons
of the C1 cell group, tissue sections containing PHA-L fiber
terminations were immunohistochemically labeled for
phenylethanolamine-N-methyltransferase,an adrenergic marker.
Studies are now underway to determine whether stimulation of
the SOM influences cardiovascular or respiratory events.
Supported by PHS grant NS24698.
a
In the normal adult mouse cerebellum Purkinje cells form a
rnonolayer between the molecular and granular layers. These
c e lls ar e characterized by a number o f biochemical
VAN KAINEN.' Barbara R. and Jerald A. MITCHELL, Department of
Anatomy and Cell Biology,Wayne State University,School of Medicine,
106A
ABSTRACTS-AAA 103RD MEETING
Detroit, Michigan. Piverse effects 0 f alcohol o n the decldual
. .
cell reaction in the raL
The effects of ethanol (EtOH) on uterine sensitivity to deciduogenic
stimulation and deciduoma growth were determined. Mature virgin rats
(Sprague-Dawley), maintained under controlled environmental
conditions, were ovariectomized and given an estrogenlprogesterone
regimen to optimize induction and growth of deciduoma (Yochim and
DeFoe, Endocr. 72:317, 1963). Rats were randomly assigned to one of
four groups: EtOH-treated: 1) days 1-4 (pre-induction/period o f
sensitivity), 2) days 5-9 (post-induction/period of growth), 3) days 1.
9 and 4) non-EtOH treated controls (pair-fed to treated groups). EtOH,
diluted in water (15% v/v), was administered via stomach tube on the
days prescribed.
The dose was 12 ml solution/kg body wt.
Decidualization was induced in one uterine horn via injection of 0.2 ml
0.1 M PO4 buffer on day 4. Uterine sensitivity and decidual growth were
accessed as cornu weight on day 9 post-induction. Blood alcohol levels
were measured by gas chromatography. Stimulated versus nonstimulated cornu weights (mg) were: controls (12) 1280 f 82 vs. 170
+ 10; EtOH treated: days 1-4 (12) were 914 L 105 (p< 0.005) vs.
178 L 23; days 5-9 (10) were 1850 L 188 (p< 0.01) vs. 192 ;t 15;
days 1-9 (5) were 1591
111 (p NS) vs. 203
27. Blood alcohol
levels rose to 132.5 10.7 mg% at 30 min., remained elevated through
90 min. (109.6 2 8.0) and declined to 82.3 f. 8.1 at 120 min. Thus
blood alcohol levels sufficient to induce mild intoxication in humans: 1)
suppressed uterine sensitivity to decidualization, and 2) enhanced
deciduoma growth in rats. EtOH had no effect on the weight of nonstimulated cornua. Since all ovarian steroid hormone support was
exogenous, the effects of EtOH on deciduoma induction and growth were
not due to alterations in hypothalamic-ovarian interaction. Preliminary
studies indicate that EtOH markedly increases uterine blood flow. Thus,
the alcohol-induced increase in deciduoma growth may result, in part,
from increased uterine perfusion. Studies are in progress to determine
the means whereby alcohol alters uterine sensitivity to deciduogenic
stimuli and deciduoma development.
(Supported by NIAA 07606-03).
S
VAZQUEZ. 1 Marcelo Eduardo and Nestor Gabriel
CAHHI, iMBICE. La Plata, Argentina. Effect of
x-irradiation on neuritoaenesis in retina1 explants. In vitro studE.
The embryonic central nervous svstem(CNS) is
very sensitive to ionirinq radiations at different stages of development. What is not clear
yet, is wether neuritoqenesis in thr CNS is
radiosensitive. In order to understand the
effect of radiation upon the neurite outgrowth.
a bioassav of developing neural retina was used.
Retina1 exolants taken of from chick donor embrvo(E6 white Leghorn) were cultured on a hydrated collagen lattice, covered by nutrient
mmdium(Eagle’8 Basal Medium with addition of
tissue extracts). The cultures were incubated
for 5 davs (37.5 C. 5% CO2 and 80% of relative
humidity). To provide full trophic support the
medium was supplemented with E18 optic tectum
extracts. The explants were irradiated at 24
and 48 hs. of culture with 50. 1 0 0 and 200 cGy
of X-radiation. The neurites length of the leading fascicules(N1). the total number of the
fascicules and neurites (NN) and the neurite
growth i n d w (NGI), were measured daily trouqhont the 5 davs in an inverted microscope with
phase contrast. Irradiated explants showed a
significant decrease in the NGI and NL in relation with the control group with 100 and 200
CGY. The 50 cGv dose d i d not affect any parameters of measure in any of the groups (24 and
48 hs).
The statistical analisvs of these results showed a significant decrease in the values of NGI and NL (p=.CO.Ol). We could suggest
that the E6 retina have a considerable radiosensivitv. The radiation effect could alter the
steps of ganglion cell neuritogenesis(initiation and elongation). Supported by TWAS grants
RGPC89-41 and CONICET PIA 89.
~~~
~
S
VAUGHAN,* Kevin T., Steven EINHEBERx, and Donald A.
FISCHMAN. Department of Cell Biology and Anatomy,
Cornell University Medical College, New York, New York.
(Sponsored by Dana C. Brooks). Analysis of a cDNA clone
encodinn the thick-filament associated 86 KD urotein
reveals similarity to muscle C-urotein and members of
the immunovlobulin suuerfamilv,
In addition to myosin, thick myofilaments in chicken
striated muscle contain at least 6 other proteins: Cprotein, myomesin, M-protein, M-CK, titin and 86 KD
protein. Except for M-CK, none of these proteins has a
clearly defined function. 86 KD protein is a myosinbinding protein restricted to fast twitch muscles. It
is located in the C-region of the sarcomere along nine
of the 43 nm stripes, and CO-localizes with C-protein
in seven of these stripes (Bahler et al., J.Mol.Bio1.
186:381,1985). A partial clone for 86 KD protein was
isolated from a neonatal chicken muscle cDNA library.
This clone of 1 kb hybridizes with a muscle-specific
mRNA of 2.3 kb, a message size sufficient to encode the
86 KD protein. A full length cDNA was isolated from
the same library using the partial clone as a probe.
The nucleotide sequence of this full length clone
reveals significant similarity to a C-protein cDNA
(Einheber and Fischman, in press), with over 50%
identity at the 3’ end. Some regions within this
sequence show complete identity. The deduced amino acid
sequence is homologous to that of the C-protein cDNA:
49% identity over 350 amino acids, with an additional
15% conservative changes. Notable features of this
amino acid sequence include homology to the C-2 repeats
found in the immunoglobulin superfamily and to
fibronectin type 111 repeats. The similarity between 86
KD protein and C-protein at their carboxyl termini
suggests a common functional domain which may be
related to their co-localization in the A-band.
(Supported by NIH AR32147 and MDA).
VINCENT, Stephen L., Jennifer McSPARREN,’ John Paul SANGIOVANNI,’
Rex Y. WANG’ and Francine M. BENES,’ Program in Neuroscience and
Department of Psychiatry, Hatvard Medical School and Mailman Research
Center, McLean Hospital, Belmont, Massachusetts; Department of
Psychiatry and Behavioral Sciences, SUNY, Stony Brook, New York.
Alterations of a x o w dritic svnaDses in laver VI medial Drefrontal
!ollowina chronic neuroleotic administram
Evidence suggests that neuroleptic agents, used in the treatment of
psychosis, may act on the mesocortical dopamine system by competitively
inhibiting the binding of dopamine to its receptors. However, this does not
adequately explain the total range of effects elicited by these drugs, including
delayed onset for full development of antipsychotic effect and the
development and persistence of tardive dyskinesia. These findings have led
to the hypothesis that neuroleptics may induce structural alterations of
synapses in particular regions of the cerebral cortex. Previous studies in this
laboratory have shown that high dose treatment with haloperidol (HAL) for 4
months induces synaptic rearrangements in layer VI of rat medial prefrontal
cortex (MPF-VI), where the cortical dopamine projection terminates. In the
present study a quantitative electron microscopic analysis of MPF-VI was
carried out to determine the effects of low dose, long-term administration (1
yr) of HAL and clozapine (CLOZ) on the ultrastructure and arrangement of
synapses. No change was found in the size of axon terminals and dendritic
spine profiles or in synaptic vesicle density. The size of dendritic shafts was
decreased by 18% in the HAL-treated group, but decreased by only 12% in
the CLOZ-treated group. In both drug-treated groups, there was a significant
decrease in the relative percentage of axodendritic synapses displaying
asymmetric postsynaptic specializations and there was an increase in both
synapses with symmetric membrane specializations and those with no
postsynaptic membrane specializations. These data suggest that low dose,
long-term neuroleptic treatment may induce shifts among excitatory and
inhibitory synaptic elements, and such shifts may provide additional
information about the interaction between neuroleptics and intrinsic
ABSTRACTSAAA 1 0 3 MEETING
~ ~
components of MPF-VI. High resolution receptor-binding autoradiography is
being used to determine if receptors of excitatory and inhibitory
neurotransmitters are affected by neuroleptic treatment. Supported by
MH00423, MH31154, MH41440 and MH00378.
WALLACE, James A., Audrey R. ROMERO,* Agnes A. V A L L E J O S
and Jerinda K. LOBNER,* Department of Anatomy, University of
New Mexico School of Medicine, Albuquerque, New Mexico.
Characterization of tyrosine hvdroxvlase-immunoreactive neurons in
the chick dienceDhalon: Do they contain catecholamines?
While several distinct hypothalamic dopaminergic (DA) cell groups
occur in mammalian species, the existence of such diencephalic
DA-containing neurons is variable amongst lower vertebrate species.
We have previously examined the embryonic development of cells in
the chick CNS that are immunoreactive for tyrosine hydroxylase (TH),
the first enzyme in the synthesis of catecholamines (Wallace, et al.,
Soc. Neurosci. Abs. ll:lOl5,'89). By the time of hatching, at least
six groups of TH-immunostained cells can be distinguished in the
diencephalon, between the anterior commissure rostrally and the exit
of the oculomotor nerves caudally. In the current study we investigated whether these ictensely stained TH-positive neurons contain
detectable levels of DA during the first week of post-hatching
development. The presence of DA in hypothalamic cells was determined by anti-DA imniunocytochemistry and by the glyoxylic acid (GA)
histofluorescence method in non-pharmacologically pretreated
animals. The immunocytochemical detection of DA involved the use of
the ABC-peroxidase technique, including a nickel intensification of
the DAB reaction product. Although each hypothalamic group of
TH-positive neurons is comprised of numerous cells, when the
diencephalic region was examined for the presence of DA, only
occasional weakly-stained DA-irnmunopositive cells were encountered
in any group. These results can be interpreted to suggest that the
TH-inimunoreactive neurons in the chick hypothalamus may lack or
contain low levels of DA because I ) their T H enzyme is inactive or
only minimally active, 2) the cells only store small amounts of DA
within their perikarya, or 3) these neurons do not possess the
enzyme, dopa decarboxylase, to convert L-DOPA to DA. Experiments
are in progress to distinguish between these possiblities. Interestingly,
in contrast to our findings of the relative absence of DA-immunostaining
within neurons where TH-positive cells occur, intense G A histofluorescence and DA immunostaining was observed in the paraventricular organ
(PVO), where
TH-immunostained cells are found. Since numerous
serotonergic neurons exist in this organ, it is possible that the
observation of catecholamine-containing cells in the PVO may result from
the uptake of these aniines into the serotonergic neurons. Supported by
NIH grants RR-08139 and BSRG 607-05583-24.
WALRO, J., and R. HIKIDA, Department of Anatomy, Northeastern
Ohio University College of Medicine, Rootstown, Ohio; Department
of Zoological and Biomedical Sciences, Ohio University, Athens,
Ohio. Mvosin heaw chain exoression and Datterns of innervation
in avian muscle sdndles.
Myosin heavy chain (MHC) expressionand patternsof innervation
were studied in intrafusalfibers of the anterior latissimus dorsi and
extensor digitorum communis muscle of chickens (GaNus gallus),
and were compared to the corresponding features of intrafusal
fibers in mammals. (Kucera and Walro 1989, Cui and Walro 1989).
Serial transverse frozen sections of muscles were stained for
enzymes which delineated sensory endings, motor endings and
muscle fiber type, or were reacted against monoclonal antibodies
specific for avian slow, embryonic, neonatal, and adult fast MHCs.
Features of MHC expression by avian intrafusal fibers parallelled
that observedfor mammalian intrafusal fibers in that some intrafusal
fibers expressed MHCs that were not expressed by extrafusal
fibers; some intrafusalfibers expressed more than one MHC in the
same region of the fiber; and expression of some MHCs were
nonuniform along the length of fibers. However, embryonic,
neonatal, and slow-tonic MHCs were expressed in the extracapsular
107A
regions of intrafusal fibers and all fiber poles were innervated by
motor neurons at multiple sites along their length, unlike spindles in
the rat and cat in which neonatal and slow-tonic MHC are
expressed only in intracapsular regions of intrafusal fibers, and
motor neurons innervate poles of nuclear chain fibers at only one
site. Moreover, complements of fibers and the number of fibers
which expressed a particular MHC were unpredictable in avian
spindles, unlike mammalian spindles in which the complement of
intrafusal fibers and their patterns of MHC expression are
predictable. These data suggest that the sequence in which
intrafusal fibers developand the evolutionof MHC expression differs
greatly between avian and mammalian spindles. Antibodies were
furnished by the Developmental Studies Hybridoma Bank or were
graciously provided by Dr. E. Bandman. Supported in part by NIH
grant NS24684 to J.W.
Raymond J., and Ronald C. BOHN, Department
of Anatomy, The George Washington University
Medical Center, Washington, D.C. ComDuter-assisted
instruction in human clross anatomv.
The Department of Anatomy at the George
Washington
University
has
developed
eight
computer-assisted instructions (CAIs) concerning
selected topics in human gross anatomy. The
computer lessons were used by a volunteer group of
48 first-year medical students from a class of 152
students. The program lessons are highly dependent
on color graphics to explain the anatomy.
Extensive voluntary testing with immediate and
cumulative feedback is incorporated into the
lessons so that the students can evaluate their
own progress in mastering the subject matter. At
the completion of the gross anatomy course, the
student
users
were
requested
to
respond
anonymously to an questionnaire regarding their
impressions of the CAIs. In addition, test
performance on a multiple-choice examination taken
by the entire class was compared between the users
of the computer-assisted instructions and their
non-user
classmates.
The
responses
in the
questionnaire revealed a very positive attitude
regarding the value and usefulness of the
computer-assisted instructions in learning human
gross anatomy. The overall rating of the programs
on a scale of 1.0 to 10.0 was 1.8 k 1.0 with 1.0
representing "extremely helpful" and 10.0 being
"of no value". A comparison of test scores showed
no significant difference in test performance
between the users of the computer-assisted
instructions and the non-users. The results of the
study suggest that while the programs did not
appreciably augment the student's performance as
evaluated by a multiple-choice examination, the
students
perceived
the
computer-assisted
instructions as valuable educational tools in
mastering the subject of human gross anatomy.
WALSH,
WALSH, Raymond J , Luz MANGURIAN and Barry I POSNER",
Department of Anatomy, The George Washington University
Medical Center, Washington, D.C. and Department of Medicine,
Royal Victoria Hospital, Montreal, Quebec. The distribution
of lactoeen receutors in the hvuothalamus of the rabbit and
rat.
The hypothalamus contains lactogen receptors as detected
with in vitro radioreceptor assay techniques. In vitro
autoradiography employing the principles of competitive
binding was applied to frozen sections of rat and rabbit
brains in order to define the location of the lactogen
-
108A
ABSTRACTS-AAA 103RD MEETING
receptors relative to specific hypothalamic nuclei. The
lactogens human growth hormone (hGH) and ovine prolactin
(oPRL) were radiolabelled with iodine-125 and the
competition for observed binding sites was assessed with
unlabelled hGH, oPrl, and bovine growth hormone (bGH). The
autoradiographs were analyred with a microcomputer-based
densitometry system. The binding characteristics of the
lactogens indicated that the rabbit hypothalamus contains
specific
binding
sites
within
the
supraoptic,
paraventricular, suprachiasmatic.ventromedia1, arcuate, and
dorsomedial nuclei and the medial preoptic area. Unlabelled
bGH was effective in competing for binding sites in all
areas when hGH but not oPRL was used as the radiolabelled
ligand, suggesting the presence of growth hormone receptors
in the rabbit hypothalamus with a distribution similar to
that of the lactogen binding sites. In contrast to the
rabbit, no lactogen binding sites were detected in the rat
hypothalamus regardless of the ligand used in the assay. All
of the ligands were successful in detecting lactogen
receptors within the rat choroid plexus and liver. The broad
distribution of lactogen receptors observed in the rabbit
hypothalamus suggests an extensive influence of lactogens
onhypothalamic regulatory systems. The results fromthe rat
raise questions as to the nature of rat brain lactogen
receptors in comparison to lactogen receptors that exist at
peripheral target tissues in the rat.
The work was supported by NSF grant BNS-8604760 (RJW) and
a grant from the Medical Research Council of Canada (BIP).
WANG,' Jun, James L. H I A l l , Leslie P. GARTNER, and
D.Vincent PROVENZA. Department of Anatomy, Dental School,
University of Maryland at Baltimore, Maryland. (Sponsored by
James L. Hiatt) Histoloqic studies of kidney develoDment in
trisomv 16 mice.
Little research has been done on kidney development in
the trisomy 16 (Ts 16) mouse, an animal model for studying
Down's Syndrome (DS) in humans. Its prenatal development
and mechanisms of development are far from being revealed in
detail. In the pesent study, kidney development in trisomic and
control fetuses on the 18th day of gestation was examined
histologically. Trisomy 16 mice were poduced from WO
breedings of mice possessing a Robertsonian translocation
genotype for chromosome 16 (Jackson Laboratories, Bar Harbor,
ME). Mice monozygous for Rb(16.17)7Bnr were bred with others
monozygous for Rb(6.16)24Lub. F, males were mated to normal
female C57 BUGJ mice. All resulting fetuses were karyotyped to
verify trisomy genotype. Fetal bodies of trisomic and normal
littermates were processed for histologic examination, serial
sectioned at 6um in the horizontal or longitudinal Nanes, and
stained with bematoxylin and eosin. When compared to normal
littermates, kidney development in the trisomic fetuses
demonstrated: a reduction in the number and size of nephrons
and collecting tubules; decreased cortex thickness; delayed
development of renal corpuscles and tubules; reduction in tubular
branching and lumina1 diameters; less developed renal papilla;
over abundance of edernatous connective tissue throughout entire
medulla; enlarged urinary space whose covering was reduced to
a simple flattened squarnous epithelium; and kidney length which
was equal to that of 1 to 2 days earlier in controls. These
findings suggest that kidney development in the trisomy 16
mouse is less organized and differentiated. Further studies are
planned regarding mechanisms of development,
S
WANG,* Xin, William Kenneth METCALF a n d N o r a h F r a n c e s
METCALF, Department of Anatomy, University of N e b r a s k a Medical
Center, O m a h a , Nebraska. T h e role of 2.3-DPG a n d A T P in t h e
w u l a t i o n of hemoelobin o x i d a t i o n in ? h e neonate.
2,3-diphosphoglycerate (2,3-DPG) a n d A T P a r e t h e t w o major
i n t r a c e l l u l a r organic p h o s p h a t e compounds in erythrocytes. 2,3-DPG
decreases a n d A T P increases t h e oxidation sensitivity of a d u l t
hemoglobin. T h e present study was u n d e r t a k e n t o determine w h e t h e r
these e f f e c t s a r e also present i n n e o n a t a l blood, which contains
about 75% f e t a l hemoglobin. Nine fetal cord blood samples were
obtained f r o m t h e University of N e b r a s k a Hospital d u r i n g t h e t h i r d
stage of delivery, a n d s t o r e d in EDTA vacutainers f o r n o longer
t h a n 24 hours a t 4OC p r i o r t o testing. A f t e r washing t h e r e d cells
3 times (with p h o s p h a t e b u f f e r e d saline, 0.5 mM, p H 7.0). t h e
washed cell suspension was hemolyred a n d adjusted t o a s t a n d a r d
absorbance, 1.8, 1 cm, 420 nm, a t 9OC a n d p H 7.0, i n a t e m p e r a t u r e
controlled spectrophotometer cuvette. Oxidation of t h e hemoglobin
t o methemoglobin was e f f e c t e d by adding 2.0 ml (Hb: 3.4x10-' M) of
hernolysate t o 3.125 ug sodium nitrite in 50 ul water. T h e 2,3-DPG
a n d .4TP were added immediately prior t o the addltian at t h e
nitrite; 0.0 of 2,3-DPG a n d A T P in t h e control group, 3.4x10-' M of
3,3-DPG in group one, 1 3 . 6 ~ 1 0 -M
~ of 2,3-DPG in group two, a n d
3.1~10-~M
of A T P i n group three.
T h e a d d i t i o n o f 2,3-DPG
increased t h e time to 50% conversion, a n d t o t a l conversion time
f r o m 6.1i1.1 a n d 1 0 . 4 ~ 1 . 7min to 8.0+1.6 a n d 13223.3 min (PCO.05)
respectively. T h e opposite e f f e c t was observed by a d d i t i o n of ATP,
decreasing t h e time t o 50% conversion a n d t o t a l conversion time,
f r c m 6.1k1.1 a n d 10.4i1.7 min t o 4.6k0.9 a n d 7.1i1.5 min (PCO.01).
T h e e f f e c t o f DPG, approximately x1.3. on cord blood resistance t o
oxidation is less t h a n t h a t on adult blood, approximately x1.45;
while A T P h a d a s i m i l a r e f f e c t on cord a n d a d u l t samples,
approximately x0.7 in e a c h case. T h e d i f f e r i n g effectiveness of DPG
in t h e two cases may well be d u e to t h e known decreased DPG
binding cLpacity of f e t a l hemoglobin.
WARREN, Susan, Department of Anatomy, University of
Mississippi Medical Center, Jackson, Mississippi.
Or aniza i n ef halamocortical connectivi in ventral
w c l e u s f of an Old Wor&&copithecus
aethiops.
poarchitectural analysis of the postcenal gyrus of 6
aethiops (African green) has defined four diverse areas,
which comprise the primary somatosensory (SI) cortex.
Micromapping techniques used to study the topography of
primary somatosensory cortex in this species have delineated
a separate hand representation within each area, suggesting
differential functions for each. In contrast, the resence
of functionally specific, multiple hand areas in tge ventral
posterior lateral nucleus (VPL), the thalamic source of input
to primary somatosensory cortex, has not been investigated in
this species. The connectivity between VPL and SI cortex was
studied by making injections of different retrograde
neuroanatomical tracers into separate, functionally defined,
SI hand area sites. Tissue was processed for combined
fluorescence and WGA-HRP histochemistry. Following
individual digit re resentation injections in SI cortex,
labelled neurons grmed 'C-shaped lamellae with medially
directed concave surfaces that spanned the dorsal-ventral
extent of VPL. Digit 5 was medial and the thumb lateral.
Despite their presence in SI cortex, separate submodality
specific hand re resentations were not found in VPL. Single
VPL neurons o?differing modalities (ef, rardly,adapting,
slowly adapting and joint) or receptive ield ocations
(e.g., glabrous, hairy, proximal or distal) intermingled
within the digit lamella, forming a single topographic
pattern. There was, however, no convincing evidence for
doubly labelled neurons, suggesting collateral projections to
distinct SI areas arisinF from individual VPL neurons are not
typical of thalamocortical input in this species.
Furthermore, there was no evidence in the African green
monkey of a 'core' and 'shell' organization, as has been
reported in macaques. Thus, the thalamocortical organization
ABSTRACTSAAA 103RD MEETING
of this species appears to differ from that of other Old
World Monkeys, implying that many of the fine details of
thalamocortical connectivity are s ecies specific.
Supported by BRSG Grant #2 S8RR05386.
WEINBERG Richard J," David J TRACEY," and Aldo RUSTIONI,
Department of Cell Biology and Anatomy, University of North
Carolina, Chapel Hill, North Carolina. Ultrastructural studv of
anteromade labeline with WGA-HRP.
Horseradish peroxidase conjugated to wheat germ agglutinin
(WGA-HRP)is a major tool for neuroanatomical pathway tracing.
This tracer is subject to transneuronal transport, and exhibits a
relative selectivity for unmyelinated fibers, at least after injections
in dorsal root ganglia (DRGs). The present study was performed to
gain insight into mechanisms underlying these phenomena.
Laminectomies were performed on pentobarbital-anesthetized
Sprague-Dawleyrats; 0.25-0.5 pl of 2% WGA-HRP were injected
mto L5 DRGs. After 2-24 h survival, animals were sacrificed by
aortic perfusion of saline, followed by 2.5% glutaraldehyde-0.54
paraformaldehyde in phosphate buffer. Vibratome sections of L5
spinal segments and dorsal roots were reacted for HRP, using
TMB histochemistry with molybdate or tungstate stabilization.
Selected sections for electron microscopic study were further
stabilized with DAB, osmicated, dehydrated in ascending alcohols
to propylene oxide, and embedded in Epon-Spurr. Thin sections
were mounted on copper grids and poststained with lead citrate
and uranyl acetate. Label in the spinal cord was concentrated in
the substantia gelatinosa, where unmyelinated primary afferents
terminate. Label was barely detectable after 4h, but intense after
8h; the 25-30 mm transport distance implies a transport rate of
approximately 100 mm/day, suggesting fast axoplasmic transport.
Ultrastructural examination of dorsal roots revealed many labeled
myelinated and unmyelinated axons. Examination of the substantia gelatinosa revealed many labeled dendrites and glia, but
few labeled axons or terminal boutons. However, label was
prominent just outside the plasma membrane of unmyelinated
fibers and boutons. The pronounced extracellular label in the
present material remains to be explained. Evidence to date
suggests that transported WGA-HRP is efficiently released at
terminals in the spinal cord. WGA-HRP not taken up by glia or
postsynaptic elements may diffuse extracellularly and bind to
axolemmal sialoglycoproteins of unmyelinated fibers, thus
producing more detectable labeling of unmyelinated than
myelinated fibers at the light microscopic level. This work was
supported by NIH award # NS-12440.
S
WEIR,*Faith I . and L o i s K . L A E M L E , Department of
Anatomy, UMDNJ-Graduate School of Biomedical
S c i e n c e s a n d Hew J e r s e y Medical S c h o o l , Newark,
New J e r s e y . Development of n e u r o p e p t i d e Y - l i k e
immunoreactivity in t h e r a t telencephalon.
N e u r o u e o t i d e Y ( N P Y ) - l i k e i m m u n o r e a c t i v i t v was
l o c a l i z e d \ n t h e c e r e b r a l c o r t e x of r a t s from
embryonic day 14 ( E 1 4 ) t o p o s t n a t a l day 2 1 ( P 2 1 )
u s i n g l i g h t m i c r o s c o p i c immunocytochemistry and
t h e a v i d i n b i o t i n method. Timed p r e g n a n t r a t s
were a n e s t h e t i z e d w i t h sodium p e n t a b a r b i t a l .
Embryos w e r e removed by c a e s a r i a n s e c t i o n and f i x ed i n Bouins s o l u t i o n o r i n 4 % p a r a f o r m a l d e h y d e /
0 . 5 % g l u t a r a l d e h y d e ; p o s t n a t a l a n i m a l s were a n e s t h e t i z e d and p e r f u s e d t r a n s c a r d i a l l y w i t h p h y s i o l o g i c a l s a l i n e f o l l o w e d by f i x a t i v e . Whole
embryos, or p o s t n a t a l b r a i n s , were t h e n embedded
in p a r a f f i n , s e c t i o n e d a t 14um in t h e corona1 o r
s a g i t t a l p l a n e , mounted o n g l a s s s l i d e s , and
processed f o r immunocytochemistry.
NPY-immunoreactive n e u r o n s a p p e a r e d f i r s t i n
t h e p r i m a r y o l f a c t o r y c o r t e x . Afew n e u r o n s were
s e e n i n t h i s r e g i o n a t E16. A t E 1 7 t h e number of
n e u r o n s i n t h e o l f a c t o r y c o r t e x had i n c r e a s e d a n d
109A
i m m u n o r e a c t i v e c e l l s and f i b e r s were s e e n i n t h e
c a u d a t e - p u t a m e n and a m y g d a l o i d n u c l e u s . A t E19
i m m u n o r e a c t i v e c e l l s i n t h e s e a r e a s were more
a b u n d a n t , and a few i m m u n o r e a c t i v e c e l l s were
p r e s e n t i n t h e d e e p e s t l a y e r of t h e c o r t e x j u s t
d o r s a l t o t h e r h i n a l f i s s u r e . O n t h e day of b i r t h
c o n s i d e r a b l e numbers of N P Y c e l l s were p r e s e n t i n
t h e hippocampal f o r m a t i o n , and an a b u n d a n c e of
l i g h t l y s t a i n e d , b i p o l a r c e l l s were p r e s e n t
t h r o u g h o u t t h e r o s t r o - c a u d a l e x t e n t of t h e
cortical plate.
Deeply-stained, non-bipolar c e l l s
appeared in t h e deeper c o r t i c a l l a y e r s on P 4 . On
s u b s e q u e n t d a y s i m m u n o r e a c t i v e c e l l s were found
i n i n c r e a s i n g numbers and i n more s u p e r f i c i a l
c o r t i c a l l a y e r s . O n P15, t h e number a n d d i s t r i b u t i o n o f NPY n e u r o n s a p p r o x i m a t e d t h a t s e e n i n t h e
adult.
WELSH,* Alerick 0. and Allen C. Enders, Department of Human
Anatomy, School of Medicine, University of California,
e of s
-tu
in e v u
Davis, California. m
.
.
in
- t
af t.he rat
to fcumatim of the
In studies of animals with hemochorial placentation the
invasive qualities of trophoblast are often cited without
giving adequate attention to the possible active role played
by maternal tissues. This study, in conjunction with
previous studies, attempts to elucidate the cooperative
nature of interactions between trophoblast and uterine
tissues during placentation in the rat. On days 8-12 of
pregnancy rats were perfused via the abdominal aorta with
buffer, then fixative and gestation sites examined by light
microscopy, and transmission and scanning electron
microscopy. On day 8, a large portion of the uterine
luminal epithelium mesometrial to the conceptus degenerates
and is sloughed into the lumen. Although morphologically
this degeneration resembles necrosis, it is controlled
necrosis since it does not involve all the epithelial cells
in the chamber. This degeneration differs from the
apoptotic degeneration of epithelial cells juxtaposed to the
conceptus. Uecidual cell processes penetrate the uterine
luminal basal laminas beneath degenerating and healthy
epithelial cells similar to the way they penetrate the basal
lamina in the region juxtaposed to the conceptus. After
epithelial cells are sloughed, maternal sinusoids open
directly into the former uterine lumen and, by day 9, the
lumen becomes lined with hypertrophied endothelial cells.
Vascular perfusion indicates that maternal blood flows
through this luminal space. When first formed, mesometrial
uterine tissues are not in direct contact with the conceptus
which occupies an antimesometrial position. As the
conceptus grows within the confines of the gestation
chamber, the Trager moves into and, by day 10, occupies most
of the uterine luminal blood space. As it does so,
trophoblast giant cells of the outer layer of the Trager tap
maternal sinusoids along the lateral wall of the chamber by
displacing endothelial cells and phagocytizing apoptotic
decidual cells. The manner in which uterine tissues in the
mesometrial region of the gestation chamber differentiate
and undergo cell death suggests internal controls (possibly
triggered by trophoblast) rather than direct external
factors. Rat trophoblast may be migratory and phagocytic but
probably only indirectly destructive to uterine tissues.
Supported by NICHD Grant No. HD10342.
WEINBERG, Joanne, Department of Anatomy, The University
of British Columbia,
Vancouver,
British Columbia,
Canada. Effects of chronic ethanol consumotion on thg
adrenocortical resaonse t o r e s t r a i n t s t r e s s .
Chrmic ethanol consumption has been shown t o elevate
basal levels o f corticosterone and t o attenuate the
adrenocortical response t o a challenge dose of ethanol.
However, the e f f e c t s of chronic ethanol consumption on
adrenocortical responsiveness t o s t r e s s o r s has not been
llOA
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
w e l l documented.
I n t h e present study we examined t h e
adrenocortical response t o r e s t r a i n t s t r e s s i n animals
c h r o n i c a l l y consuming ethanol.
Sprague-Dawley
female
r a t s were randomly assigned to:
Alcohol ( A b l i q u i d
ethanol d i e t , ad l i b ; Pair-fed ( P F ) - l i q u i d c o n t r o l d i e t
i n t h e amount consumed by an alcohol p a r t n e r ( g l k g body
w t ) each day; o r Control (Cl-lab chow and water, ad l i b .
A f t e r 3 wk and 6 wk o f alcohol intake, animals were
subjected t o 2h continuous r e s t r a i n t s t r e s s and a
c o r t i c o s t e r o n e time course was
determined.
Basal
c o r t i c o s t e r o n e l e v e l s were elevated i n A females compared
t o PF and C females, both a t 3 wk and 6 wk. A t 3 wk, A
and C females had somewhat g r e a t e r c o r t i c o i d l e v e l s than
PF females a t 30 min. b u t c o n t r o l s had s i g n i f i c a n t l y
h i g h e r c o r t i c o i d s than A and PF females a t 60 and 120
min. A t 6 wk, c o n t r o l s had h i g h e r c o r t i c o s t e r o n e l e v e l s
than PF animals throughout t h e 120 min s t r e s s p e r i o d .
However, both PF and C females showed a s i g n i f i c a n t
c o r t i c o i d decrease from 30 t o 60 min w h i l e A females
showed no change from 30 t o 60 min. These data i n d i c a t e
t h a t chronic ethanol consumption d i d n o t a l t e r t h e
i n i t i a l c o r t i c o s t e r o n e response t o stress, b u t d i d a l t e r
t h e p a t t e r n o f c o r t i c o s t e r o n e s e c r e t i o n over time.
Supported by g r a n t AA07789 from NIAAA.
the ankle (40'
of dorsiflexion) with 5-10' range of m t i o n (NRI).
The
contralateral hind lirrb served as a noninmobilized control.
A l l rabbits uere
then weighed and placed i n separate cages and allowed free activity.
Aninals
uere checked daily t o ensure p r q x r placement and f i t o f the cast.
Rabbits uere
12 (n.4); 16 ( ~ 4 or
) 21 days ( w 4 )
sacrificed after 1 ( ~ 3 ) ; 4 (n.5); 8 (n.4);
of NRI. An additional 3 rabbits were immbilized as described, but with no freed m of j o i n t motion (RI = r i g i d inmobilization). These animals, which served as
Rawe of m t i o n was measured
positive controls, were sacrificed after 21 days.
before application of the cast and at sacrifice.
A t sacrifice both the l e f t and
right knees and ankles were viewed grossly and prepared for histologic staining
u i t h Safranin-0 and fast green or Toluidine Blue to reveal matrix proteoglycens.
A I 1 animals lost weight during the period o f i m b i l i t y .
No gross changes
were noted i n animals sacrificed after 1, 4 and 8 days of NRI. Two and 3 rabbits
sacrificed after 12 and 16 days, respectively, of N R l had a mild decrease i n
range of motion i n the knees and ankles.
A l l animals sacrificed 21 days after
N R I or R I showed moderate decreases i n range o f rmtion i n the l e f t ankles and to
a lesser extent i n the knees. An intact articular surface, normal matrix staining and normal c e l l morphology were noted i n a l l tissues (patella. fernoral
condyles, t i b i a l plateaus and ankle) frm the noninmobilized control and NRI
knees and ankles fran a l l t i m e periods.
In sharp contrast, matrix proteoglycan
staining was severely depleted i n the articular cartilage fran a l l RI knees and
ankles after 3 weeks.
The articular surface was intact and c e l l mrphology norm1 i n these animals. The results fran the present study suggest that allowing a
small amount of j o i n t rmvement during i m b i l i z a t i o n of a joint i n flexion does
not result i n the loss of m a t r i x proteoglycans seen i n joints which are r i g i d l y
inmobilized f o r the Same length of time.
Supported i n parts tv grants AG-04736 and l-P50-AR39239 and the I l l i n o i s Chapter
of the A r t h r i t i s Foundation.
References.
1. J. Anat. 108:497 - 507, 1971.
2. J . Bone Joint surg. 42:31 - 49, 1960.
3. Clin. Orthop. 34:184 -195, 1964.
4. Anat. Rec. 149: 113 - 134, 1964.
5. Ann. Rheun. Dis. 29 : 634 -642, 1970.
6 . Clin. Orthop. 107:249
257, 1975.
.
S
W I L L I W , * ' James D.,Dorothy A. FRENZ*133,and Thomas R. V A N Dp
WATER ' , $abratory o f Developyental Otobiology, Depts. o f Otolaryngology ,
Newscience , and Neuropathology , Albert Einstein College o f Medicine, Bronx,
New York. Fibroblast growth factor: A n early initialor o f otic capsule
chondrogenesis.
Fibroblast growth factor (FGF) slimulates proliferation anUor induces
differentiation in a variety o f neurwtoderm and mesoderm-deriveQ cells
(Gospodarowicz et al, Endca. Review 8:95-114, 1987). Int-2 proto-oncogene
encodes a growth factor closely related to FGF which has similarly been suggested
to function as an inducer o f mesodermal differentiation in mammalian
embryogenesis and Int-2 is known to be expressed i n the rhombencephalon adjacent
to the developing otocyst (Wilkinson et al. EMBO J. 7691-95, 1988). A t 10.5 days
of embryonic development otocysts are dependant on the inductive influences of
rhombencephalic tissue for advances in morphogenesis (Van De Water and Conley,
Anat Rec 202:195A. 1982). Previous micromass culture studies have indicated that:
1) periotic mesenchyme isolated from 10.5 GD mouse embryos is no1 capable o f
differentiating into cartilage in these high density cultures; 2) when otOcyst
epithelium is added to 10.5 GD mesenchyme cultures chondrogenesis does occur
(Frenz and Van De Water, unpublished results). The present study seeks to
determine a role for FGF in the sequence o f events leading to otic capsule formation.
Periolic mesenchy9e removed from 10.5-11.0 G D mouse embryos was plated at a
density o f 2.5 x 10 cells in a 10 ul drop. These high density cultures were exposed
to FGF (bovine) at a concentration of 1.0 n d m l for days C-1 (48 H) i n viuo. The
cultures were maintained for a total of 7 days at which lime the presence of
cartilage-specific proteoglycans was determined b y the ability o f the cellular matrix
10 bind Alcian blue stain @H 1.0). SpecuophotomeUic quantification o f the bound
Alcian blue stain extracted from the micromass cultures with 8M guanidinium H C I
provided the means to evaluate the extent o f chondrogenesis. As previously
observed, periotic mesenchyme cultured without the addition of exogenous FGF
does not undergo differentiation as evidenced b y the absence of chondrogenic
nodules and by the failure of the matrix to bind Alcian blue stain after 7 days in
viuo. Periotic mesenchyme cells cultured i n the presence (48 H) of exogenous FGF
form chondrogenic nodules by day 5-6 in v i u o which bind Alcian blue stain after I
days i n culture. These results. i n Combination with the observed expression o f the
Inr-2 protwoncogene in rhombomeres 5 and 6. suggest a role for FGF in omcystmesenchyme interaction as a possible iniliator o f chondrogenesis in the early stages
of otic capsule development. These i n viuo results demonstrate that the addition o f
exogenous FGF is able to overcome periotic mesenchyme's dependance on omcyst
epithelium to induce chondrogenesis i n culture.
Work supported b y NIEHS Pathology Training Grant NS07089 & a Deafness
Research Foundation Grant)
YILLIAMS, Jams M., Dennis R. ONGCHl' and Eugene J-M. A. THONAR',
Departnents of
Anatomy, Biochemistry and Internal Medicine. Rush Presbyterian S t . Luke's Medical
Center, Chicago, I l l i n o i s . Non-riqid i n m b i l i r a t i m Preserves the intearitv of
rabbit knee and ankle articular cartilaqe.
Degenerative changes i n the articular cartilage have been noted i n a variety
of animals using different modes of j o i n t inmDbilization (1-6).
These changes
have inpcrtant inplications with respect t o i n m b i l i z a t i m o f hunan joints (e.g.
fracture reduction, inhibitive splinting, serial casting). The present s t w investigated the effects of non-rigid inmobilization (NRI) of the ankles and knees
of rabbits i n their anatmic position for a period of 21 days.
Adolescent New Zealand white male rabbits ( 3 kg) were used i n t h i s study. Using a plaster cast, the l e f t knee was inmobilized (135' of f l e x i m ) a l q with
WILLIAMS, Terence H., Jean C. FOLAN, YanFeng WANG and
Jean Y. JEW. Department of Anatomy, University of Iowa,
Iowa City, IA. Mitral valve innervation: a model f o r
neural aaina in the autonomic nervous svstem?
Species variations in atrioventricular valve innervation was the subject of a recent report (Williams et al.,
Amer. J. Anat., in press). The present study compared the
innervation patterns of mitral valves in four species of
young and old mammals: guinea pig, rat, mouse, and opossum. Human mitral valve disease is a prevalent disorder
which has a number of causative factors, one of which may
be merely an acceleration of the aging process. The heart
valves of experimental animals are uniquely suitable f o r
studying the aging of autonomic nerve nets which can be
stained and their distributions examined as whole mounts.
The hearts were removed from deeply anesthetized animals
and, using whole mount preparations, nerves of the mitral
valves were stained to localize acetylcholinesterase
(AChE). The valve nerve networks were drawn using a Nikon
Optiphot system with camera lucida attachment. Nerve networks in the valves of young and old animals were compared. In the young specimens, the valves were richly innervated. The basic pattern of innervation was a dense
basal zone plexus with nerve fiber bundles which extended
towards and became thinner in the intermediate zone of
the valve. In the distal zone, the nerves again appeared
as a concentrated but finer plexus. Fine nerve bundles
extended from this distal plexus towards and, in the case
of the guinea pig, reached the free edges of the valve
cusps and into the chordae tendineae. In the mitral
valves of old guinea pig, rat, mouse, and opossum,
innervation was relatively sparse. Strikingly substantial
declines were found in the densities of nerve fibers
across the entire valve but were most evident near the
free edges of all the valve cusps. Whatever the
functional effects, the loss of valvar nerves appears to
typify the normal aging process, whose time course in the
human has yet to be elucidated. To what extent there
exists a link between the age-related changes in valvar
innervation and the increased incidence of mitral valve
dysfunction and pathology in aged human subjects warrants
investigation. Supported by DK38123.
ABSTRACTS-AAA 1 0 3 MEETING
~ ~
WILSON, Frank J.. DavidJ. RILEY,* RenaF. BANNEn,* BonnieW. PENG,*
Sandra A. HAYES* and Carol A. TOZZI,* Departments of Anatomy and
Medicine,University of Medicine and Dentistry of New Jersey-Robert Wood
Johnson Medical School, Piscataway, New Jersey and Lyons VA Medical
Center, Lyons, New Jersey. lmmunocvtochernical localizationof collaaenase in aranular cells in rat hvoertensive oulmonaw arteries.
The amount of connective tissue in the main pulmonary arteries (PAS)of
rats increaseswithin 10 days of exposure to hypoxic conditionsand returns
to normal levelswithin one week after recoveryin air. Concomitantwith the
decrease in connective tissue during recovery is the rapid increase in
collagenolyticand elastolytic activities in the main PAS which indicatesthat
proteolysisaccountsfor the breakdownof connective tissue. To identifythe
source of collagenase activity in blood vesselsduringrecoveryfrom hypoxic
hypertension,we have studied the imrnuno-localizationof collagenase in
small PAS (<300pm diameter). Rats (200gm) were exposed to 10% 0, for
10 days or recovered in air for 1, 3, 7 or 14 days. Pulmonary tissue was
fixed, inflated and prepared for light microscopy by cryosectioning and for
electron microscopy by Lowicryl embedment. Two different primary
antibodieswere used: sheep anti-rabbit procollagenaseand rabbit anti-rat
procollagenase. An appropriate secondary antibody was either conjugated
with rhodamine or with 15nm gold particles. In normotensive animals,
immunoreactivitywas distributed diffusely throughout the tunica media of
the PAS. In hypertensivevesselsand in those examined 1, 3 and 7 days
after recovery, the antibody was localized in granular cells within the tunica
mediaand perivascularconnective tissue. The immunostainingpattern was
similar for both primary antibodies. Absorption of the primary antibody with
the antigen abolished the specific irnrnunofluorescence. lmmunoelectron
rnicroscopy demonstrated the localization of the enzymes in inclusion
granules of large, single-nucleatedcells. In addition, antibody was also
depositedamong collagen fibrils in the extracellularmatrix. Other cell types
in the vicinity, e.g., alveolar Type I1 pneumocytes and goblet cells in the
walls of bronchioles.were unreactive. The immunoreactive granular cells
were associatedwith smooth musclefibers and fibroblastsin the walls of the
vessels. They appeared in greater numbers in hypertensive and early
recovery rats. Enzyme histochemical studies characterized these cells as
originating from monocytes/macrophages. These results suggest that
interstitialtissue macrophagescontaining collagenase are present in small
PAS of rats with hypoxic pulmonary hypertension. Furthermore, these cells
may be the source of increased collagenolyticactivity in PAS during early
connectivetissue regression of structural changes following recovery from
hypoxia. Supported by grants HL24264, HL07426, American Heart
Association/New Jersey Affiliate, Medical Research Service of the VA.
WIYESKI, Lawrence E., Sidney A. PITTS,* and Ophelia I.
WEEKS,
Department
of
Anatomy,
Morehouse
School of
Medicine,
Atlanta,
Georgia;
Department
of Biological
Sciences.
Florida
International
Universitv.
Miami.
F l o r i d a . ' H i s t o c h e m i c a l o r g a n i z a t i o n of t h e k b r i s s a e l
o p e r a t i n g f a c i a l muscles i n t h e golden hamster and r a t .
The golden hamster (Mesocricetus auratus) and t h e Norway
r a t (Rattus norve i c u s 3 e x h i b i t two types o f e x p l o r a t o r y
b e h a v i z a s d e f i 2 e d b y t h e use o f t h e m y s t a c i a l (snout)
vibrissae.
The hamster
demonstrates -rapid,
complex
whisking o f t h e v i b r i s s a e ; t h e r a t demonstrates slow,
r e l a t i v e l y simple whisking.
The d i f f e r e n t
behaviors
p r e d i c t i n t e r s p e c i f i c v a r i a t i o n s i n t h e morphology o f t h e
v i b r i s s a l motor system, thus, d i f f e r e n t ( p o s s i b l y overl a p p i n g ) a n a t o m i c a l designs u n d e r l y i n g t h e o r g a n i z a t i o n o f
t h e face.
The gross morphology o f the s u p e r f i c i a l f a c i a l
muscles that operate t h e v i b r i s s a e is v i r t u a l l y i d e n t i c a l
i n the two species; however, t h e muscles may d i f f e r i n
their
physiological
characteristics
(e.g.,
twitchc o n t r a c t i o n times,
oxidative capacities).
Preliminary
histochemical
tests
indicate
two major p a t t e r n s o f
organization.
Myosin ATPase r e a c t i o n s show t h a t t h e
muscles t h a t a c t t o p r o t r a c t t h e v i b r i s s a e ( n a s o l a b i a l i s
profundus; v i b r i s s a l capsular muscles) a r e composed almost
The
e n t i r e l y (95+%) of t y p e I I a f i b e r s i n b o t h species.
in
retraction
of
the
vibrissae
muscles
involved
( n a s o l a b i a l i s ; m a x i l l o l a b i a l i s ) a r e more heterogeneous i n
t h e i r composition (10-30% t y p e I;70-90% t y p e I I a ) .
Succinic
dehydrogenase
(SDH)
and
NADH
tetrazolium
reductase r e a c t i o n s show t h a t a l l v i b r i s s a l muscles i n t h e
hamster a r e composed e n t i r e l y of highly o x i d a t i v e f i b e r s .
The muscles i n the r a t , e s p e c i a l l y the r e t r a c t o r muscles,
lllA
contain
large
populations
of
nonoxidative
andfor
I n general, the v i b r i s s a l
intermediate-staining fibers.
muscles i n t h e hamster a r e composed almost e n t i r e l y of
fast-switch,
fatigue-resistant
(FR) f i b e r s w i t h m i n o r
populations
of
slow-twitch,
fatigue-resistant
(SRI
fibers.
The homologous muscles i n t h e r a t a r e composed
l a r g e l y o f FR f i b e r s , w i t h s i g n i f i c a n t p o p u l a t i o n s o f
fast-twitch,
f a t i g a b l e (FF) f i b e r s .
Supported by NIH
(NIDR) DE09038.
WINTERSTEIN." James F. and William E. BACHOP. Departments of
Anatomy and Diagnostic Radiology. The National College of Chiropractic,
Lombard. Illinois.
The coroorotransverse liqament at
L
5
J
g
intervertebral foramen: a qross anatomical-radioqraphic comparison,
During the past quarter century, more and more articles, abstracts,
and books have been reporting ligament-like bands in and about the
intervertebral foramina of the lumbosacral region (e.g., Macnab, 1969;
Golub and Silverman, 1969; Nathan et al., 1982; Lombardi et al., 1984;
Janse et al., 1985; Amonoo-Kuofi et al., 1988). One such ligament, often
found on one or both sides of the cadaver to be spanning the
inteNertebral foramen between the L5 vertebra and the sacrurn,
frequently takes the form of a rigid strut that in appearance and
mechanical properties resembles the bone of both the vertebral body and
the transverse process to which the strut connects. But in radiographic
properties, such rigid struts do not resemble bone. They appear
radiolucent when the spine bearing them is x-rayed using standard
equipment and procedures. Despite such a strut's own radiolucency, its
position can be rendered visible in a plain radiograph of the L5/S1
intervertebralforamen by placing a lead strip over the rigid strut prior to
using the same equipment and protocols. The photographs and
radiographs herewith displayed depict the gross anatomical and
radiographic appearance of the rigid struts found traversing the L S E 1
intervertebralforamina of nine midsagittally sectioned lumbar spines from
dissected cadavers. The radiolucency of the rigid strut would presumably
allow it to go undetected in plain radiographs of patients presenting with
low back pain and/or sciatica. Macnab, an orthopaedic surgeon, in 1977
hypothesized that such a rigid strut, i.e., a 'corporotransverse ligament.'
could exert a 'guillotine effect' on the ventral ramus of the L5 spinal
nerve.
S
WOLF,* C u r t i s V. and K y u l i g W. CIIUNG, D e p a r t m e n t o f
Anatomical Sciences, College of Medicine, U n i v e r s i t y o f
Oklahoma H e a l t h Sciences Center. Oklahoma C i t v . OK.
E f f e c t s o f e t h a n o l o n b i o s y n t h e s i s and a c t i o n o f < s t e r o i d
hormones in female r a t s .
O v a r i a n s t r u c t u r e and f u n c t i o n in t e i m s o f s t e r o i d o geriesis and u t e r i n e e s t r a d i o l r e c e p t o r c o n c e n t r a t i o n s w e r e
i n v e s t i g a t e d in ethanol-fed and c o n t r o l r a t s . Y o u n g adult
female King-Holtzman r a t s were f e d a liquid d i e t f o r 3
months c o n t a i n i n g e i t h e r 6% (36% o f t o t a l calories) e t h a n o l
or t h e isocaloric e q u i v a l e n t o f sucrose. U l t r a s t r u c t u r e o f
o v a r i a n s t e r o i d p r o d u c i n g cells o f ethanol-fed r a t s was
c h a r a c t e r i z e d by t h e presence of numerous pleomorphic
mitochondria, d i l a t e d c i s t e r n a e o f smooth endoplasmic
r e t i c u l u m , and i n c r e a s e d n u m b e r s o f lipid d r o p l e t s . To
determine o v a r i a n p r o g e s t e r o n e and e s t r o g e n b i o s y n t h e s i s ,
o v a r i a n t i s s u e homogenates o f ethanol-fed a n d c o n t r o l r a t s
were i n c u b a t e d with 2 pM o f 3H-pregnenolone f o r 1 h o u r at
3 7 k . Progesterone a n d e s t r a d i o l w e r e t h e n isolated by thin
l a y e r c h r o m a t o g r a p h y and q u a n t i t a t e d by r e v e r s e isotope
dilution.
F o r m a t i o n o f p r o g e s t e r o n e and e s t r a d i o l was
s i g n i f i c a n t l y decreased in ethanol-fed r a t s w h e n compared
t o controls. Radioimmunoassay r e v e a l e d that c o n c e n t r a t i o n
of serum p r o g e s t e r o n e was s i g n i f i c a n t l y l o w e r in ethanolf e d r a t s ( 4 . 4 n g l m l ) than in c o n t r o l animals (17.9 n g l m l )
Similarly,
s e r u m l e v e l (29.5 pglml) o f e s t r a d i o l was
decreased in e t h a n o l - f e d r a t s w h e n compared t o t h e c o n t r o l
Rat u t e r i n e e s t r a d i o l r e c e p t o r s i t e s
yalue (77.7 p g l m l ) .
w e r e t h e n measured by charcoal a d s o r p t i o n technique.
.
112A
ABSTRACTS-AAA 103RD MEETING
b ~ t m s t r w m eof the i n ~ s t i t i a lcells of Cajal (102) in
the nm!ey goresenbled that of other nenmalian species, but
differed in their pEnvlity and alrrost lack of slooth endoplaslric
reticulun, caveolae and filarpnts. ?he plasmlam of the cpplasnic
prccEsss of the Icc ws in close contact (20-30 m
l gaps) w i t h that
of mth MsdLe cells. lhis m y OCCaSiOnaUy take the f m of a
w,but gap juxtim k v e mt been o k d . Vesiculated axm
profiles, cmtaining large granular or agranuk vesicles w r e in c l e
cmtact (Z-3 nu gap) with the plasrelema o f ICE. In a f w
vesiculated profiles a presynaptic density could be [email protected] Ihe
intercalation of the Icc tEtu%nthe vesiculated axm profiles and the
m
t
h rmscle cells suggests a role in esophageal m o t i l i t y . Ektw3z-n
3 and 21 days following bilateral vagtmy scme 10: s h e d regressive
changessuchasincreesed electrcn density a d shrinkage o f the
o f the organelles and dissolutiQl of the nuclear
cytoplasn, U-&amtin mterials. Axcn profiles in the vicinity of the affected
Wong, Y.C. and Chan,* L., Department of Anatomy, Faculty of Icc contained glycogen gr?mules sugemng
'
injury. In late stages,
Medicine, University of Hong Kong, Hong Kong. 8-D-xrloside the nunber of ICC and m
t
h rmscle contacts xas reduced. Ihe results
mrtiallv inhibited the androgen induced growth of _& suggest that the ~ g u snerves exert a trowc inflwxrx on the I C a n d
1atera1
-Ermstate of the prembcr.t.cll y .castrated_guinga pi&
that the intercellular relaticmtlips betksen Icc and snooth mrle cells
Stromal-epithelia1 interactions are important for the m y be plastic. It is tentatively svggested that these Mgal effects
morphogenesis and cytodifferentiation of the prostate gland. my be m d i a t d via the go&geal myenteric ganglia.
The aim o f this study was to examine the effects of
B-D-syloside ( X Y L ) , a compound which interferes with the
supported by Grant W46/85fran the N.U.S.
st romal proteoglycans ( PGs) synthesis, on androqen induced
growth of thr lateral prostate (LP). Young male guinea pigs
werc castrated at 3 wks of age and divided into three groups
s i h wlts after castration. In g r o u p one, the animals were
in.jected (s.c.) daily with 80 mg/kg of XYL followed b y , 3
da>--slater, a daily dose of 10 mg/kg of DHT for two more
W R E E * , A n d r e a s , K a r l Z I L L E S * a n d A x e 1 SCHLEICHER*
wks. The second group served as control and received DHT
( S p o n s o r e d b y T.H. SCHIEBLER) D e p a r t m e n t s o f
only. I n the t.hird group, animals were treated first with
Anatomy o f t h e U n i v e r s i t i e s o f W u r z b u r g and K o l n ,
XYL like those in group one, and then folloxed by DHT alone
F e d e r a l R e p u b l i c o f Germany) L o c a l c e r e b r a l g l u for 2 wks to check the reversibility of XYL effect. At the
cose u t i l i z a t i o n o f t h e c o r t i c a l areas i n t h e
end of the experiment the LP k-as removed and processed for
morphological and cytochemical examination. The results r a t b r a i n : a r e a l a n d l a m i n a r p a t t e r n
showed that XTL inhibited the growth of the LP to DHT
On t h e b a s i s o f q u a n t i t a t i v e B y t o r a d i o g r a p h y
stimulation. The fibroblasts showed dilated GER filled with
f o l l o w i n g a d m i n i s t r a t i o n o f [ C]-2-deoxy-D-glugranular substances. I n the interstitial spaces, there was a
cose (J.Neurochem. 28:897-917), t h e i s o c o r t e x o f
drastic increase in Cuprolinic Blue (CB) positive filaments t h e r a t c a n b e s u b d i v i d e d i n t o r e g i o n s o f d i f f e aild polygonal granules believed to be PGs o r GAGslsL. Their
r e n t l o c a l c e r e b r a l g l u c o s e u t i l i z a t i o n (LCGU).
number was much greater than the control. The distribution B o r d e r s d e f i n e d o n t h e b a s i s o f r e g i o n a l LCGU
and density of the collagen fibres appeared similar to the
d i s t r i b u t i o n match t h e r e s p e c t i v e a r c h i t e c t o n i coiitrol. The secretory alveoli were lined by epithelium with
c a l borders. Nearly a l l o f t h e architectonifrw secretory granules of low electron density and a larqer
c a l l y d e f i n e d a r e a l borders ( p a r c e l l a t i o n a c c o r iiulnber of clear vesicles. There was 8 slight reduction in
d i n g t o Z i l l e s , K., The c o r t e x o f t h e r a t .
ylycoconjusate react ivities in the epithelia1 cells. The
S p r i n g e r 1 9 8 5 ) a r e v i s i b l e i n t h e LCGU p i c t u r e s .
lect i n binding patterns and the structural features were
F u r t h e r m o r e , t h e r e g i o n a l LCGU d i s t r i b u t i o n r e comparable between the control and recovery groups
v e a l s m o r e c l e a r l y some o f t h e a r e a l b o r d e r s ,
indicating that S T L effects were reversible. The results
which a r e d i f f i c u l t t o d e f i n e i n t h e adjacent
suggezt that stromal PG biosynthesis may play a role in
N i s s l s t a i n e d s e c t i o n s ; i . e . , t h e temporal areas
epi thelial function and an altered stromal matrix would
can be c l e a r l y d e l i n e a t e d f r o m t h e s u r r o u n d i n g
haniper t h e effects of DHT on the tarqet organ. (Supported by
p a r i e t a l f i e l d s and t h e l a t e r a l secondary v i s u a l
HKU, CRCG No. 335/031/0015).
a r e a . As t h e a r e a l p a t t e r n o f t h e c o r t e x i s
Re ferences:
g e n e r a l l y c o r r o b o r a t e d b y t h e LCGU p a t t e r n , i t
1. Chan, L. and Wong, Y. C. The Prostate 14:145-162, 1989.
can be concluded t h a t t h e c o r t i c a l p a r c e l l a t i o n
2. Chan. L. and Wong, 1.. C. The Prostate 14:133-145, 1989.
r e f l e c t s m e t a b o l i c and f u n c t i o n a l a s p e c t s .
Uterine cytosol was incubated with increasing amounts of
3H-estradiol (0.1-3 nM) in the presence or absence of nonradioactive steroid.
Scatchard analysis indicated that
uterine cytosol receptor content was 46.2 fmol/mg protein
in ethanol-fed rats which was significantly lower than that
(80.6 fmollmg protein) of controls. The present findings
suggest that ethanol alters ovarian structure and function
as well as steroid hormone action in the uterus, leading to
a female reproductive dysfunction. Supported in part by
NIAAA Grant R01 AA066448
W, W.C., S.H. TAN", T.Y.
and E.A. LUG, Ikprhmnt of h t a n y ,
k t i d M v e r s i t y of S i n g p r e .
vagotmised interstitial cells o f
Ultmstmzture of mmd and
in the mdcey esxhagw.
Eleven mxlkeys
fascicularis) of either sex and wAghjng
1.2-4.2 kg here used. In 6 anids, mder intrapsitmeal segatal
( d i m pentobarbital 33 nlg/kg) anesthesa
'
and aseptic conditions, a
o r e s t a g mid-cal
bilateral vagotcray ws done with survival tims
of 1,3,5,7,10and 21 days. of the 5 remining mmkeys, 3 here shan
operated with slwival tines of 5.7 and 21 days. Ihe other 2 rronkeys
here used as unoperated controls.
At sacrifice, each a n i m l xas
rmnestktised and while under arti€icial respiratim receivecl routinely
intracadiac i n j x t i o n s o f heparin and 1% d u n nitrite. Follaring
a Ringer wa&, the aniwl ms perf&
via the left ventride with a
odxed aldehyde m l u t i m in 0.m cacodylate buffer (pH 7.4) and p-ocessed
for conventional transmssl
. 'on electrm ndcrascopy.
The l a m i n a r LCGU d i s t r i b u t i o n w i t h i n a c o r t i c a l
a r e a m e a s u r e d f r o m t h e l a y e r 1/11 b o r d e r t o t h e
c o r t e x / w h i t e m a t t e r boundary a l s o r e v e a l s a r e a l
s p e c i f i c l a m i n a r p a t t e r n s . A l l t h e p r i m a r y sensory areas e x h i b i t t h e highest values i n t h e
range of l a y e r I V , w h i l e t h e primary motor cort e x shows h i g h v a l u e s p r e d o m i n a n t l y i n l a y e r V .
Supported by g r a n t s o f t h e Deutsche Forschungsgemeinschaft.
S
WU*, Wutian, David E . SCOTT, Department of
Anatomy and Neurobiology, Eastern Virginia
The
Medical School, Norfolk, V i r g i n i a .
transplanted p i n e a l gland a s a neuroendocrin;
model of reinnervation, p l a s t i c i t y and funct i o n a l recovery.
ABSTRACTS-AAA 103RD MEETING
Twenty p i n e a l e c t o m i z e d r a t s were d i v i d e d
i n t o 3 g r o u p s and underwent v a r i o u s manipulations.
F i v e remained u n t r e a t e d , 6 r e c e i v e d
sham t r a n s p l a n t a t i o n s w i t h f r a g m e n t s o f occ i p i t a l c o r t e x i n t o t h e t h i r d c e r e b r a l vent r i c l e , 9 received p i n e a l t r a n s p l a n t s . Seven
normal r a t s w e r e used a s c o n t r o l s . S i x weeks
following transplantation,
serum m e l a t o n i n
was measured by R I A , and h o s t b r a i n t i s s u e s
were p r e p a r e d f o r e i t h e r I C C w i t h a n t i - s e r u m
a g a i n s t t y r o s i n e h y d r o x y l a s e (TH) and/or
t r a n s m i s s i o n e l e c t r o n microscopy (TEM). Four
out of n i n e pinealectomized, p i n e a l - t r a n s p l a n t e d h o s t s demonstrated a s i g n i f i c a n t increase i n t h e serum m e l a t o n i n c o n c e n t r a t i o n
comparable t o c o n t r o l s and i n c o n t r a s t t o
t h a t o f u n t r e a t e d r a t s and p i n e a l e c t o m i z e d
s h a m - t r a n s p l a n t e d h o s t s . No s i g n i f i c a n t d i f f e r e n c e w a s o b s e r v e d between sham-transp l a n t e d h o s t s and u n t r e a t e d p i n e a l e c t o m i z e d
r a t s . P i n e a l o c y t e s i n v i a b l e g r a f t s demonstrated morphological criteria c o n s i s t e n t
w i t h an a c t i v e s e c r e t o r y process.
TH p o s i t i v e n e u r i t e s were o b s e r v e d t o i n v a d e p i n e a l
g r a f t s from t h e h o s t median eminence.
With
TEM, a d r e n e r g i c n e r v e e n d i n g s w i t h d e n s e core
v e s i c l e s w e r e o b s e r v e d t o t e r m i n a t e between
p i n e a l o c y t e s and i n p e r i v a s c u l a r s p a c e s of
fenestrated capillaries i n viable grafts.
The f u n c t i o n a l
recovery of
transplanted
p i n e a l o c y t e s may b e l i n k e d d i r e c t l y t o r e i n n e r v a t i o n of t h e g l a n d by n e r v e f i b e r s from
t h e h o s t e n d o c r i n e hypothalamus.
(Supported
by NSF G r a n t BNS 8709687).
113A
S u r g e r y , Mercer U n i v e r s i t y School o f Medicine,
Macon, Georgia. Endoqenous s r o w t h f a c t o r a f f e c t s
mvosenic d i f f e r e n t i a t i o n
uncommitted v l u r i p o t e n t and linease-committed s t e m cells.
I n s u l i n - l i k e growth f a c t o r - I ( I G F - 1 ) h a s been
p o s t u l a t e d t o a f f e c t t h e i n v i v o r e p a i r of
damaged s k e l e t a l muscle. I n c u b a t i o n o f c u l t u r e d
s k e l e t a l muscle s a t e l l i t e c e l l s and t r a n s f o r m e d
cell l i n e s w i t h IGF-1 demonstrated a n i n c r e a s e
i n myogenesis. The r e s u l t s s u g g e s t e d t h a t IGF-1
may d i r e c t l y s t i m u l a t e s t e m c e l l s t o
d i f f e r e n t i a t e i n t o m u l t i n u c l e a t e d myotubes. A
c o m p a r a t i v e i n v i t r o model system c o n s i s t i n g of
myogenic lineage-committed and uncommitted
p l u r i p o t e n t s t e m c e l l s w a s used t o t e s t t h i s
hypothesis. S t e m cells w e r e plated a t d e n s i t i e s
of 5 , 0 0 0 c e l l s p e r w e l l i n 2 4 - w e l l 1%g e l a t i n c o a t e d p l a t e s and switched t o d e f i n e d medium 2 4
h r s p r i o r t o t e s t i n g . Myosin c o n t e n t p e r w e l l
was measured u s i n g a m y o s i n - s p e c i f i c monoclonal
antibody-based E L I S A p r o c e d u r e . DNA c o n t e n t p e r
w e l l was measured u s i n g f l u o r o m e t r i c a n a l y s i s .
I n c u b a t i o n o f t h i s d u a l i n v i t r o system w i t h a
0 . 1 ng t o 500 ng d o s e r a n g e of I G F - 1 i n d e f i n e d
medium e l i c i t e d a n i n c r e a s e i n DNA c o n t e n t i n
b o t h s t e m c e l l s y s t e m s , an i n c r e a s e i n myosin
c o n t e n t i n t h e lineage-committed s t e m cells, b u t
no i n c r e a s e i n myosin c o n t e n t (or myogenic d i f f e r e n t i a t i o n response) i n t h e pluripotent s t e m
c e l l s . S i n c e myosin c o n t e n t was i n c r e a s e d o n l y
i n stem c e l l s a l r e a d y committed t o t h e myogenic
phenotype, t h e s e r e s u l t s s u g g e s t t h a t t h e
p r e v i o u s l y proposed myogenic-inducing a c t i v i t y
of I G F - 1 may reside i n d i r e c t l y w i t h s t i m u l a t i n g
stem c e l l s t o p r o l i f e r a t e p r i o r t o f u s i o n r a t h e r
than d i r e c t l y stimulating t h e i r d i f f e r e n t i a t i o n
i n t o m u l t i - n u c l e a t e d myotubes. Supported by t h e
Rubye Ryle S m i t h C h a r i t a b l e T r u s t .
XU,' Xiangyang, Pam PETERSON'. Doris B. WILSON and Andrew G.
HENDRICKX, Department of Histology and Embryology. Shandong Medical
University,Jinan, Shandong,P.R. China; Division of Anatomy, Department
of Surgery, School of Medicine, University of California. San Diego. La Jolla,
California; and California Primate Research Center, University of California,
Davis,
of the
. . . California.
itive W
The period of trilaminardisc formation is a cmcial time involving rapid cellular
proliferation when the embryonic axis is established and organ anlagen are
formed. The current study was undertaken to obtain data on the pattern of
[3H]thymidineincorporation in the primitive streak and notochord in early
rhesus monkey embryos. Three stage 11 embryos were labeled
intrachorionicallyfor 1, 4 and 8.5 hr, respectively, with 100 pd [3H)hymidine
and processed for autoradiography. Incorporation of label was examined in
live regions of the notochord which were divided along the rostrocaudal axis
as lollows: cranial foregut, caudal foregut, somites (3-5 and 10-13) and
region caudal to the last intersomitic groove. The longitudinal axis of the
primitive streak was divided into three equal regions; transverse sections in
each region were further divided into dorsal, medial and ventral areas.
Labeling indices (LI) were calculated from individual values in the 5
notochordal and 9 primitive streak regions. Higher LI occurred in the foregut
(42.8-48.5%)and 10-13 somites (47.3%) regions compared to the 3-5
somites (29.0%) and caudal (34.1%) regions. These elevated proliferation
rates correspond with the extensive growth and elongation in the cranial
region and formation of new somites caudally. In the primitive streak, the
increased LI in the cranial regions (53.7-65.8%)as opposed to the caudal
regions (48.4-55.2%)indicates a rostrocaudal gradient of proliferation. In
addition, more active proliferation was evident in the dorsal portion of the
primitive streak (Ll=55.2-65.8%)
compared to the medial and ventral portions
(Ll=48.4-55.9%).These observations are consistent with findings in other
species which indicate that presumptive mesoderm originates from rapidly
dividing cells which ingress through the primitive streak. [Supported by NIH
Grant #RR00169 and a scholarship from Boehringer Ingelheim, Federal
Republic of Germany]
Henry E . and Donna C. MORRISON*, D i v i s i o n
of B a s i c Medical S c i e n c e s and Department of
YOUNG,
S
ZHANG, Bin", Michael E. GOLDBERGER, Liang-fang WU
and Marion MURRAY*, Department of Anatomy, Medical
College of Pennsylvania, Philadelphia, Pennsylvania;
Histological Institute, West China University of Medical
Sciences (Sponsored by Leonard L. Ross) Plasticitv of complex
terminals in substantia eelatinosa of adult cat spinal cord
Plasticity of dorsal root projections has previously been
described after partial unilateral deafferentation of cat spinal
cord (spared root preparation: all lumbosacral roots cut
extradurally except L6), using light microscopy. Quantitative
electron microscopy was used to examine changes in a class of
dorsal root terminals, the complex or glomerular terminals, in
the spared root preparation of adult cats. We compared the
mean cross-sectional area of the complex terminals and the
len th of postsynaptic densities associated with them in 2 acute
a n f 4 chronic cats in lamina I1 at the L5 and L6 levels on the
control and deafferented sides. In th acute group the mean
number of complex terminals /100pm9 decreased, while the
mean length of postsynaptic densities increased in partially
deafferented lamina I1 compared to the control side. I the
chronic group the number of complex terminals/[email protected]
9 was
restored to normal levels, and both the mean number of
ostsyna tic densities per complex terminal and the mean
fength orpostsynaptic densities increased. Our results suggest
that spared root deafferentation elicits plasticity of the spared
root and its terminals and also in neurons post synaptic to the
spared root. The plasticity includes a compensatory increase in
the number of spared root complex terminals and an increase
in the number of s napses made by each complex terminal.
Supported by e r a n t No. NS24707 from National Institutes
of Health.
114A
ABSTRACTS-AAA 103RD MEETING
S
ZHANG,*Ming Q . , Jean Y. JEW and Terence H. WILLIAMS.
Department of Anatomy, University of Iowa, IOWa City,
Iowa. Resvonses of mventeric neurons to intestinal
obstruction.
Intestinal hypertrophy is a feature of a number Of
gastrointestinal diseases that are associated with disorders of motility. Obstructive lesions and subsequent
hypertrophy of the adjacent gastrointestinal segment affect not only the smooth muscle layers of the enteric
wall but also the myenteric (MP) and submucous nerve
plexuses. Our earlier studies of hypertrophied rat ileum
after experimentally-induced stenosis showed that M P
perikarya increased in size and became more irregular in
shape compared to control preparations. These changes
were consistent with the hypothesis that, in the obstructed gut, M P neurons act in synergism with cells in
the muscle coat. We have recently identified other
neural changes which represent adaptive interactions of
MP neurons to environmental perturbations, but which may
not necessarily be termed synergistic. In experimental
preparations we observed at the EM level: 1) injury to
neuronal perikarya, evidenced by degenerative and reactive changes including membranous dense bodies and irregularly-shaped mitochondria; 2 ) reactive, degenerating
and dystrophic axons in nerve bundles, as well as pyriform enlargements (growth cones) which indicated
regenerating axons (which by light microscopy were not
distinguishable from other axonal enlargements); 3 )
using computer-assisted analysis (VIAS) of EM profiles,
that the mean area of axonal varicosity profiles located
at neuromuscular interfaces was increased by 50%; 4 )
that the number of varicosities per unit length of axon
was increased by 50% in the nerve bundles and the number
of mitochondria was increased per unit length of axon by
100%. The mean thickness of intervaricose segments of
axons was similar to controls. The increase in numbers
and areas of varicosity profiles, specifically the
enlarged varicosities at neuromuscular interfaces, is
strong evidence for enhanced potential of the myenteric
nerves to deliver synaptic andlor trophic stimuli to the
muscle. Supported by DK38123.
Zhso*,f.Y., Wong, Y.C. and Tam*,C.C., Department of Anatomy,
University of Hong Kone;, Hong Kong. D2v.eloament and ductal
moruhogeriesj-s .&theprostatic
complex of the guinea Dig.
The changes of Slandular architecture of the prostate
qland of the guinea pig from 40 days(D) of gestation to 90D
postnatally ( F " ) were studied. The prostate qland o f the
gninea pig can be divided into lateral (LP) and dorsal (DP)
lobes and coagulatinq gland ( C G I . The morphometric measurements o f the ductal elements, after the removal of
meseiichymal/stromal tissue hy collagenase. were carried out
on a graphic tablet. The numbers of primary ducts and ductal
tips per lobe were quantified. The results showed that the
growth and ductal morphogenesis fell into two distinctive
periods. At 40D of gestation the dorsolateral prostate consisted of 5 to 8 main ducts with well developed secondary
and tertiary hranchings while the CG had 1 to 6 still unhraiiched mail! ducts. Ductal branching continued and extended
into the surrounding mesenchyme over the next 15 days. At
55D of gestation, approximately 60%, 79% and '71% of the number of adult, ductal tips of the LP. DP and CG respectively
were formed. The ductal morphogenesis was nearly complete at
birth and there was little increase in number of ductal tips
thereafter. The growth of the gland Ph; was accompanied by
the increase in volume of the epithelial ducts. By contrast,
only 80% of the ductal tips and 76% of the adult branching
points of the mouse prostate gland were attained by l 5 D PW'.
Histological study revealed that the ducts first appeared as
epithelial cords which became canalized and by D1 and D10 PN
the ducts were lined by yseudostratified 10% columnar
epithelium. The secretory alveoli with varying sizes were
developed hp D1 and by D10 PR, the epithelial folds began to
appear in both LP and DP. #ore extensix-e epithelial foldings
were found in LP and DP 20D after birth. The alveolar lumen
increased in size with evidence of secretion in the lumen.
I n CG the epithelial foldings and secretory product, though
present, but did not match the level o f development of LP
and DP until about 30D postnatally. The results indicate
that the ductal morphogenesis of the guinea pig prostate
gland may he different from other rodents and the development of the CG appears to be later than the LP and DP.
References: 'Sugimura, Y., C,unha, G. R . and Donjacour, A. A.
Biol. Reprod. 31:961-971, 1986.
S
ZHOU,* M., T.G. MA,* N.D. RADTKE,* and M.T. TSENG,
Departments of Anatomical Sciences and Neurobiology,
Ophthalmology and Vision Science, University of Louisville,
Louisville, Kentucky. (Sponsoredby Delores Schroeder)
of taurine on modulation of I'HjMK-801 bindino the N-methvCDasoartate INMDA) receator COmDlex bv L-alutamate. NMDA. Lamartate and alvcine.
Evidence indicates that taurine is an inhibitory
neurotransmitter and able to act as a putative hypoxic protectant.
However, the mechanism underlying its ability to serve as a
hypoxic protectant is not known. In bovine retina1 membranes,
using [3H]MK-801, a potent non-competitive antagonist for
NMDA receptors, we examined the effects of taurine on
cooperative modulation for ['H]MK-801 binding to the NMDA
receptor-ionchannelcomplex induced by L-glutamate,NMDA, Laspartate, and glycine. It was found that enhancement of
['H]MK-801 binding was 40%, 6%, ~ W O
and, 15% for 10 WMLglutamate, NMDA, L-aspartate, and 30 pM glycine, respectively.
No obvious effect was found for taurine (1 mM). The binding of
['HI MK-801 was additionally stimulated when glycine was
combined with these agents, and enhancement of 160%, 113%,
and 150% was found with L-glutamate, NMDA, and L-aspartate,
respectively. The addition of taurine did not change the potency
of the compounds either presentedalone or in combinationwith
glycine. The present results ~Q&Q show that taurine may not
exert its modulatory effects under ischemic conditions either at
the agonist-recognitionsite or at the glycine modulatory site on
the NMDA-receptor complex directly.
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