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The ultrastructure of sympathetic ganglia of the lizard Cnemidophorus neomexicanus.

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The Ultrastructure of Sympathetic
of the
Lizard Cnemidophorus neomexicanus ’
Department of Anatomy, The University of New Mexico
School of Medicine, Albuquerque, New Mexico
Sympathetic ganglia of six Cnemidophorus neomexicanus lizards were
fixed by immersion with glutaraldehyde or a combination of glutaraldehyde and paraformaldehyde. All ganglia were post-fixed in 1% osmium tetroxide and embedded
in Epon.
Neuronal somata and processes of stellate ganglia were ensheathed typically by
capsular cell cytoplasm and membranes; however, parts of some processes were invested only by basement membrane. Axo-somatic, axo-dendritic and axo-axonal synapses were observed. Pre- and post-synaptic processes contained 200 A neurotubules,
100 A neurofilaments, 225-500 A presumptive glycogen granules and occasional multivesicular bodies. Presynaptic endings contained 500-700 A “clear” vesicles and a few
dense-cored vesicles of 600-1100 A.
Aggregates composed of 225-500 A granules, presumably glycogen, were frequently
found in peripheral perikaryal positions i n intimate association with lipid droplets.
Clusters of particles within somata were also found which resembled ribosomes but
were not associated with endoplasmic reticulum or vesicular membranous elements.
These clusters, perhaps “areticular Nissl substance,” were associated in some instances
with fibrillar material or lattice-like granular structures. Abundant and frequently large
lipid droplets were observed i n proximity to the ribosomal-like particulate material.
The ultrastructure of sympathetic ganglia of several submammalian vertebrates
has been described by Smith (’58,’59),
Taxi (’64, ’67), Yamamoto (’63), Szentagothai (’64), Grillo (’66) and others. Observations on the fine structure of reptilian
sympathetic ganglia have been included in
the context of some reports (Taxi, ’67;
Szentagothai, ’64); however, few studies
have been concerned primarily with the ultrastructure of lizard ganglia. Smith (’58)
briefly described inclusions and mitochondrial characteristics in perikarya and axons of Phrynosoma cornutum and Bufo
marinus. Two types of Nissl bodies in
stellate ganglia of Phrynosoma cornutum,
the horned lizard, were described also by
Smith (’59). He remarked that the fine
structure of one of the two types of Nissl
substance corresponded with previous descriptions, whereas the ultrastructure of
the other type had not been reported in
RNA-rich cytoplasm of neurons or other
cell types. Inasmuch as the constituent
particles of the latter type of Nissl body
were unassociated with elements of the
endoplasmic reticulum, this type was designated as “areticular” Nissl substance by
ANAT. REC., 164: 185-204.
Because of the meager data available
concerning the fine structure of autonomic
ganglia of reptiIes, especialIy lizards, this
study was undertaken as the first in a series of investigations of the cytology of
sympathetic ganglia in several southwestern species of lizards.
Cnemidophorus neomexicanus, the New
Mexican Whiptail, is a relatively small
lizard ( 2 1/2 to 3 inches in snout-vent
length) native to central and southern
New Mexico (Stebbins, ’54). Because the
possible effects of diet, seasonal variations
and captivity upon the fine structure of
lizard ganglia have not been determined,
it is noted that specimens utilized in this
investigation were kept at room temperature four to six months during which time
they were maintained on a diet consisting
of mealworms. Between January 17 and
March 29 the lizards were sacrificed during exposure to ice-bath hypothermia.
Received Aug. 28, ’68. Accepted Jan. 20, ’69.
1This study was performed during the tenure of a
post-doctoral fellowship to Gene L. Colborn supported
by N.I.H. grant 1-R01-GM14435-02.
2 Present address: Department of Anatomy, University of Texas Medical School at San Antonio, San
Antonio Texas.
3Part’of this study was supported by USPHS grant
I-ROl-NB 07557-01A1.
Stellate ganglia were excised bilaterally bodies differ considerably in size and infrom six specimens; ganglia from five liz- ternal structural characteristics (figs. 2,4).
Golgi formations consisting of aggregaards were fixed by immersion in cold
6.25% glutaraldehyde buffered with 0.15 tions of cisternae, saccules and vesicles are
M cacodylate-HCl to pH 7.1. The ganglia widely distributed within neuronal somata
from the sixth animal were immersed in (figs. 2, 4, 17). Vesicles of 600-1100 A diKarnovsky's ('65) cacodylate buffered mix- ameter possessing a dense central core of
ture of paraformaldehyde and glutaralde- 350-950 W are associated frequently with
hyde at pH 7.3. After 24 hours fixation, the Golgi system (fig. 4). Ribosome-studall tissues were washed in a dozen changes ded cisternae in parallel array (Nissl subof 0.075 M sodium cacodylate buffer for a stance) occupy large portions of perikarya
total period of one hour and subsequently (fig. 5). Preferential peripheral, or parapost-fixed in phosphate-buffered 1% os- nuclear localization of the granular endomium tetroxide (pH 7.4). Dehydration plasmic reticulum in the perikaryon was
and embedding of ganglia in Epon 812 not noted in the animals studied.
Lipid droplets are widely distributed in
were according to Luft ('61). Thick sections (0.5-1 u ) were stained with Azure I1 the neuronal cytoplasm and, although they
and methylene blue for light microscopy. may be found in any portion of the soma,
Silver and gray sections were cut with a occur with greatest frequency in the pediamond knife on a Porter-Blum MT-2 ul- riphery of the perikaryon (figs. 5, 6 , 8).
tra-microtome. Contrast of sections was Such inclusions may attain diameters in
enhanced by staining for ten minutes in excess of 5 LI and often are clearly visible
with the light microscope. High magnificaa saturated solution of uranyl acetate in tion micrographs of lipid droplets do not
50% ethanol followed by three minutes in reveal the presence of delimiting trilamlead citrate (Reynolds, '63). Some sections inar membranes.
were stained only with uranyl acetate or
Granules of 225-500 A, presumably glywith lead citrate. Electronmicrographs cogen, appear frequently and in considerwere made with an Hitachi HS-7S electron able number in the periphery of perikarya
(figs. 6, 7). An aggregation of such granules, 6 by 3.5 v (in the plane of the photoOBSERVATIONS
graphed section) is illustrated in figure 6.
Neuronal cell bodies of the stellate gan- Presumptive glycogen often is associated
glia of Cnemidaphorus neomexicanus are topographically with variable numbers of
approximately 20-35 v in diameter. From lipid droplets. Granules of similar dimenobservations of sequential thin sections sions and density are disposed singly and
with phase-contrast optics it appears that in small clusters at other sites in the perithe ganglia contain a mixed population of karyon. As shown in figures 17 and 19,
unipolar and multipolar neurons. A multi- presumptive glycogen is situated also in
polar neuron is illustrated in figure 1. Neu- diverse processes within the ganglia, parronal nuclei of the stellate ganglia are ticularly within terminal neurites and syntypically eccentrically situated and irreg- aptic bulbs.
Peripherally located collections of parularly spherical or somewhat elongate in
contour and possess a conspicuous nucleo- ticulate material occur which are dissimilus. The nuclear envelope exhibits numer- lar to the presumptive glycogen (figs. 8,
ous pores with an internal diameter of 9). The particulate aggregates are composed o f : ( 1 ) granules of approximately
about 650 A (fig. 3).
Mitochondria within the cell bodies the same density and dimensions (200 A)
are generally unremarkable, although as neuronal ribosomes; (2) particles which
branched or rather elongate forms are ob- are generally less dense and smaller than
served occasionally. All neurons observed ribosomes. The two granular types are ascontain inclusions resembling lysosomes sociated characteristically with lipid drop(figs. 4, 18), consisting of an electron lets. The 200 W particles are observed at
dense internal matrix and an investing times in intimate association with whorled
trilaminar membrane. The lysosome-like configurations of fibrillar material (figs.
10, 11) or with “lattices” composed of 240- (125-225 A ) which surrounds a central
300 A subunits (fig. 12). Although fre- core (350-950A). Synapses are seen frequently juxtaposed to granular endoplas- quently at points of origin of neuronal
mic reticulum (fig. 8), the aggregations of processes (figs. 15, 19). Collaterals arising
particulate material contain few intrinsic from the processes also may enter into
membranous elements.
synaptic relationships with clusters of vesSynapses of extra-somatic and somatic icle-filled nerve terminals.
types are encountered in stellate ganglia
Great numbers of axo-somatic synapses
of C. neomexicanus. Attempts were made are observed within the stellate ganglia,
to classify the extra-somatic synapses into Surfaces of somata vary considerably with
axo-dendritic and axo-axonal categories; respect to numbers and sizes of end bulbs
thus, processes containing Nissl substance in synaptic juxtaposition and in degrees of
and/or appreciable quantities of free ribo- complexity of association of pre- and postsomal particles were identified as den- synaptic membranes. Necklace-like rows
drites, and processes containing few or no of vesicle-filled nerve endings appear to
ribosomes were tentatively identified as occupy large areas of the neuronal surface
postganglionic axons when observed in (fig. 17). Profiles of synaptic bulbs (fig.
synaptic relationship with preganglionic 16) suggest that preganglionic neurites
may possess sequential, perhaps numerSome characteristics of axo-dendritic ous, bead-like enlargements of synaptic
and axo-axonal types of synapses are illus- knobs over a given perikaryon.
Many contacts between synaptic bulbs
trated by figures 13 and 14, respectively.
Both figures exhibit areas of increased and neuronal somata consist of an indendensity in the regions of apposition of pre- tation of the neuronal plasma membrane
and post-synaptic membranes . . . the “ac- into which the bulb or knob is seated (figs.
tive zones” (Taxi, ’67). Synaptic clefts in 5, 15). Adjacent to some of these contacts
the “active zones” measure approximately (figs. 17, 18,20) elaborate finger-like in175 A. Sub-synaptic formations of cister- terdigitations of plasma membranes occur.
nae, sacs, or electron-dense apparatus were It is often difficult to identify the cellular
origins of these finger-like processes.
not observed in the ganglia studied.
Pre- and post-synaptic processes contain
variable numbers of 175-225 A neurotuIn the present study, light microscopic
bules, 100 A neurofilaments and presumptive glycogen granules (figs. 14, 19). A observations of thick sections of lizard
few multivesicular bodies, lipoidal droplets sympathetic ganglia agree in general with
and dense inclusions of diverse forms are the early descriptions by Huber (1899) in
present in some processes. Presumptive studies of chelonians. Electron microscopic
post-ganglionic axons appear to contain observations of the current investigation
greater numbers of tubules and filaments indicate the presence both of unipolar and
than processes identified as dendrites. Ax- multipolar neurons in Cnemidophorus.
onal, dendritic and perikaryonal cover- The latter feature distinguishes this repings consist generally of several layers of tile from amphibia, in which autonomic
satellite membranes and cytoplasm. Infre- ganglion cells appear to be solely unipolar
quently, some portions of processes are (Huber, 1899; Pick et al., ’64; Taxi, ’67;
Grillo, ’66). Huber (1899) noted a few bicovered solely by basement membrane.
Synaptic vesicles are predominantly of polar neurons near the poles of tortoise
the “clear” type (with the techniques em- ganglia; the number of such cells, howployed), are approximately 500-700 A in ever, was not great. Bipolar neurons have
diameter and are bounded by a trilaminar not been observed in C n e m i d q h o r u s in
membrane (figs. 18,20). In figure 20, tu- the present study.
Huber’s (1899) observation of nuclebular profiles appear to be associated with
some of the clear vesicles. A few dense- ated capsular or satellite cells has been
cored vesicles 600-1100 A are observed in confirmed in Cnemidophorus ganglia. The
pre-synaptic endings. Figure 18 shows that thickness and numbers of the investing
such vesicles possess an outer, lucent zone layers of satellite cell cytoplasm and
membranes are subject to considerable
variation from cell to cell. The frequent
juxtaposition of satellite nuclei between
neuronal somata and the bases of emerging processes in C. neomexicanus also
duplicates Huber's earlier findings in tortoises. Neuronal processes of lizard ganglion cells are covered with variably thin
lamellae of satellite cell elements; at some
loci the investment of processes appears
to be reduced solely to a layer of basement
Axo-dendritic synapses have been the
most frequently observed synaptic type in
sympathetic ganglia of mammalian forms
(Szentagothai, '64; Grillo, '66). However,
in amphibian sympathetic ganglia, axosomatic synapses are more characteristic
(Yamamoto, '63; Szentagothai, '64; Grillo,
'66; Taxi, '67) and are particularly numerous upon root portions of nerve processes (Yamamoto, '63). Szentagothai ('64)
found very few axesomatic synapses in
cervical sympathetic ganglia of the turtle
Emys orbicularis. The majority of synaptic contacts were observed between dendrites and terminal branches of preganglionic fibers at some distance from the
cell body.
Stellate ganglia of C. neomexicanus contain axo-dendritic, axo-axonal and abundant axo-somatic synapses. Synaptic bulbs
are prevalent particularly at bases of
emerging neuronal processes. A sub-synaptic apparatus such as that described in
amphibians (Grillo, '66; Taxi, '67) has
not been observed in the present study.
There appear to be rich pericellular plexuses of non-medullated preganglionic fibers. It is presumed that neurites arising
from these plexuses end as intra-capsular
synaptic buttons which, in some planes of
section, seem to be arranged in bead-like
fashion along the neurite and resemble
en passant synaptic forms.
The presence of glycogen in autonomic
ganglia of mammalian forms has been
previously demonstrated at the light microscopic level with histochemical techniques (Sulkin and Kuntz, '50). Particles
resembling glycogen (Revel, Napolitano
and Fawcett, '60) are highly labile in neurons of the central nervous system of reptiles (Kruger and Maxwell, '67). The distribution of presumptive glycogen in c!.
neomexicanus in superficial regions of somata and within neuronal processes of
autonomic ganglia is comparable with previous observations in frog and lizard
(Yamamoto, '63; Taxi, '67). Yamamoto
('63) demonstrated that aggregates of
200 A to 400 A granules in Rana catesbi-.
m a stain intensely with lead hydroxide
and correspond in distribution to periodic
acid-Schiff (PAS) positive material which
is digestible with saliva. Yamamoto suggested that the granular aggregates are
composed either of glycogen or a related
substance such as glycoprotein or glycolipid. Taxi ('67) observed that autonomic
ganglia of poikilotherms, in contrast to
homeothermic species, contain abundant
glycogen, especially within synaptic processes.
In the lizard material used for the present study the clusters of presumptive glycogen in somata and processes are quite
large and are composed of 225 A to 500 A
granules. The granules stain intensely
with lead citrate. The morphologic and
staining characteristics of the granules are
similar to those described for glycogen in
other tissue (Revel, Napolitano and Fawcett, '60).
In an investigation of "neurosecretory"
inclusions of sympathetic ganglia of
Phrynosoma cornutum and B u f o marinus,
Smith ('58) described dense, subspherical
whorls of 60-100 A thick, moderately
dense filaments. In both species the filaments were replaced in varying degree
with granules of similar size and density.
The granules were usually more numerous
in the smallest masses and appeared to be
precursors of the filaments. The filaments
appeared to Smith to be identical with
neurofilaments. The whorled masses were
believed to accumulate as the result of
cytoplasmic streaming.
Masses composed of closely packed,
dense particles 200 A to 400 A in diameter
were found by Smith ('59) in stellate ganglia of the horned lizard, Phrynosoma cmnutum. Such masses stained with thionine
before, but not subsequent to ribonuclease
treatment; they were non-reactive to the
periodic acid-Schiff procedure and were
thus tentatively identified not as glycogen
but as a specialized RNA-containing cytoplasmic moiety. Such aggregates of par-
ticles were termed “areticular Nissl sub- progress may elicit some aspects of these
stance” by Smith, in contradistinction to as yet only suspected inter-relationships.
the classical, endoplasmic reticular riboACKNOWLEDGMENT
somal system (Nissl substance). The conThe
suggestions of Dr. L. M.
stituent particles of the areticular Nissl
material were four to eight times the di- Napolitano in the preparation of the manuameter of single ribonucleoprotein gran- script are gratefully acknowledged.
ules of the reticular Nissl substance. Near
zones of junction with the reticular Nissl,
1966 Electron microscopy of
the areticular material appeared to arise
sympathetic tissues. Pharmacol. Rev., 1 8 : 387by clustering of ribosomal granules. There
was subsequent partial dispersion of the Huber, G. C. 1899 A contribution on the
minute anatomy of the sympathetic ganglia of
substance of the areticular particles into
the different classes of vertebrates. J. Morph.,
an additional, less dense material.
16: 27-90.
In addition to the densely staining gran- Karnovsky, M. J. 1965 A formaldehyde-gluules of 2 2 5 A to 5 0 0 A , presumed to be
taraldehyde fixative of high osmolality for
use in electron microscopy. J. Cell Biol., 27:
glycogen, the present material contains a
second population of masses comprised of Kruger,
L., and D. S. Maxwell 1967 Comparaparticulate material which upon first intiie fine structure of vertebrate neuroglia:
spection bears some resemblance to the
Teleosts and reptiles. J. Comp. Neur., 129: 115142.
“areticular Nissl substance” described by
Smith (’59). Two types of particles are Luft, J. H: 1961 Improvements in epoxy resin
embedding methods. J. Biophys. Biochem. Cyobserved in C . n e m e x i c a n u s . The first
tol., 9: 409-414.
stains densely, measures approximately Pick, J., C. De Lemos and C. Gergin 1964 The
fine structure of sympathetic neurons in man.
200 A and closely resembles ribosomes of
J. Comp. Neur., 122: 19-67.
the Nissl system. The second component
J. P., L. Napolitano and D. W. Fawcett
measures 150 A or less, is not as electron Revel,
1960 Identification of glycogen i n electron
dense as ribosomes and in some specimens,
micrographs of thin tissue sections. J. Biophys.
Biochem. Cytol., 8: 575-589.
is associated with fibrillar material of similar density and transverse diameters. In Reynolds, E. S. 1963 The use of lead citrate at
high pH as a n electron-opaque stain in electron
one specimen fibrillar material was obmicroscopy. J. Cell Biol., 17: 208-212.
served in whorled configurations (figs. 10, Smith, S. W. 1958 Fine structure of “neurosecretory” inclusions in sympathetic neurons
1 1 ) reminiscent of the description by
of a lizard and a toad. Abstract. Anat. Rec.,
Smith (’58) of subspherical whorls of fila130: 464.
ments in P h r y n o s m a cornutum and B u f o
1959 “Reticular” and “areticular” Nissl
bodies i n sympathetic neurons of a lizard. J.
Taxi (’67) commented briefly upon the
Biophys. Biochem. Cytol., 6 : 77-84.
presence of large lipid droplets within neu- Stebbins, R. C. 1954 Amphibians and Reptiles
of Western North America. McGraw-Hill, New
ronal somata of frogs. He observed that
the quantities of lipid appeared greater at Sulkin, N. M., and A. Kuntz 1950 A histocertain times of the year, particularly in
chemical study of the autonomic ganglia of
the cat following prolonged preganglionic stimsummer, and suggested that the presence
ulation. Anat. Rec., 108: 255-277.
of such large amounts of lipid might be Szentagothai,
J. 1964 The structure of the aurelated to degenerative processes. Large
tonomic interneuronal synapse. Acta Neurolipid droplets are observed in sympathetic
veg., 26: 338-359.
ganglia of C . neomexicanus and, in almost Taxi, J. 1964 Etude de certaines synapses interneuronales du systeme nerveux autonome.
all instances, are closely associated with
Acta Neuroveg., 26: 360-372.
presumptive glycogen granules and/or the
1967 Observations on the ultrastrucsmaller 100 A to 200 A particulate mature of the ganglionic neurons and synapses of
the frog Rana escukntu L. In: The Neuron
terial. It is suggested that there may be
(Holger Hyden, ed.). Elsevier Publishing Comfunctional as well as topographical repany, Amsterdam, London, New York, 221-254.
lationships between the lipid droplets, pre- Yamamoto, T. 1963 Some observations on the
sumptive glycogen and particulate matefine structure of the sympathetic ganglion of
the bullfrog. J. Cell Biol., 16: 159-170.
rials. Histochemical experiments now in
1 Multipolar neuron of stellate ganglion of Cnemidophorus neomexi.
canus. N, neuronal nucleus; P, neurcnal process; SN, satellite cell
nucleus. x 8,750.
Gene L. Colborn and Norma Jean Adamo
Neuronal perikaryon and nucleus of stellate ganglion of Cnemidophorus neomexicanus. DB, dense body; G, Golgi elements; M, mitochondrion; N, nucleus. X 10,000.
Tangential section of nuclear envelope. NP, nuclear pores. X 46,250.
Neuronal cytoplasm, illustrating Golgi elements, G; DB, dense bodies;
DV, dense-cored vesicles; M, mitochondrion. X 24,600.
Peripherally situated Nissl substance ( N S ) i n a neuron of stellate
ganglion of C. nemexicanus. G , Golgi; L, lipid; S, satellite coverings
of neuron; SB, synaptic bulb. X 14,600.
Gene L. Colborn and Norma Jean Adamo
Aggregation of presumptive glycogen granules, Gly, associated with
lipid droplets, L. X 20,700.
Higher magnification of presumptive glycogen shown in preceding
figure. x 45,800.
Nissl substance, NS, a n d peripherally situated particulate material,
AN (“areticular Nissl substance”), consisting of dense and less dense
granular types. L, lipid; N, nucleus. x 10,000.
Higher magnification of the inscribed area of figure 8. AN, “areticular Nissl substance”; L, lipid; R, ribosomes. X 41,400.
Gene L. Colborn and Norma Jean Adamo
Whorled configuration, W, consisting of fibrillar material, at periphery
of neuronal soma. L, lipid. x 12,500.
Higher magnification of the inscribed area of figure 10. L, lipid; W,
whorled configuration. X 51,750.
Lattice-like structure associated with 200 A particles of “areticular
Nissl substance.” Notice the blending of the particles at the periphery
of the lattice (arrows). L, lipid. X 41,400.
Gene L. Colborn and Norma Jean Adamo
Axo-dendritic synapse. Note “active zones” of increased density, AZ;
B, basement membrane; CV, clear vesicles; D, dendritic process; DV.
dense-cored vesicles; M, mitochondrion. x 14,600.
Axo-axonal synapse. Post-synaptic process, P; c l e x vesicles, CV, in
presynaptic process. X 14,600.
15 Axo-somatic synapses at base of emerging neuronal process. NS, Nissl
substance; SB, synaptic bulbs. X 15,200.
16 Axo-somatic synapse. Note continuity of nerve terminal, SB, with
axonal process, AX. AZ, “active zone”; NS, Nissl substance. X 24,600.
Gene L. Colborn and Norma Jean Adamo
17 Bead-like arrangement of synaptic bulbs, SB, at periphery of soma.
AZ, “active zone”; G, Golgi; Gly, glycogen; N, nucleus; S, satellite
x 15,720.
Higher magnification of area inscribed in preceding figure. The unit
membranes of the clear vesicles, CV, may be clearly seen. Note the
comglex interdigitation of satellite and neuronal plasma membranes,
S. AZ, “active zone”; DB, dense body; DV, dense-cored vesicles.
x 51,750.
Gene L. Colborn and Norma Jean Adamo
19 Axo-axonal synapse. Note prevalence of glycogen, Gly, both in preand post-synaptic processes. AZ, active zone; NF, neurofilaments;
NT, neurotubules. X 30,750.
Electron micrograph illustrating (1) the indentation of neuronal
plasma membrane by synaptic bulb; ( 2 ) interdigitation of neuronal
and satellite membranes, at arrows; ( 3 ) tubular profiles in synaptic
bulb, T; CV, clear vesicles; NS, Nissl substance i n neuronal soma:
SB, synaptic bulb. x 45,000.
Gene L. Colborn and Norma Jean Adamo
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ultrastructure, neomexicanus, lizard, cnemidophorus, ganglia, sympathetic
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