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Sex-determining mechanism inBuergeria buergeri (Anura Rhacophoridae). III. Does the ZZW triploid frog become female or male

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270
K. JACOBS
JOURNAL
ETOF
AL.
EXPERIMENTAL ZOOLOGY 283:270–285 (1999)
Tympanal Receptor Cells of Schistocerca gregaria:
Correlation of Soma Positions and Dendrite
Attachment Sites, Central Projections and
Physiologies
KIRSTEN JACOBS, BEATRIX OTTE, AND REINHARD LAKES-HARLAN*
Institut für Zoologie und Anthropologie, Georg-August-Universität
Göttingen, 37073 Göttingen, Germany
ABSTRACT
By using neurobiotin as a marker in intracellular recordings, we were able to
directly correlate soma positions and dendrite attachment sites as well as axonal morphologies
and physiologies of single auditory receptor cells of Schistocerca gregaria. We could clearly discriminate three groups of receptor cells, differing in their orientation within the Müller’s organ,
their central arborizations and their physiology:
Group I comprises 20 receptor cells with their dendrites attached to the “folded body.” Their
characteristic frequencies (CFs) lie at 400–700 Hz or at 1.5–2 kHz. Group II consists of 12–14 high
frequency receptor cells (CFs 12–25 kHz) whose dendrites are attached to the “pyriform vesicle.”
Group III receptor cells dendrites are attached to either the “elevated process” (EP) or to the
“styliform body” (SB); their CFs lie at 3–4 kHz. There were no differences in physiology and central arborizations between those receptor cells of Group III whose dendrites are attached to the
SB and those whose dendrites are attached to the EP.
Our method renders it possible to combine previous classifications based on either exclusively
morphological (a-, b-, c-, d-cells) or physiological (type 1–type 4 cells) findings. In contrast to the
hitherto hypothetical indirect correlations, we correlate c-cells and type 1 cells (= group I; see
above) and d-cells to type 4 cells (= group II). Furthermore, we demonstrate that a subdivision of
a-cells and b-cells is not reflected in a subdivision of type 2 and type 3 cells. The latter have to be
combined into one group (= group III). J. Exp. Zool. 283:270–285, 1999. © 1999 Wiley-Liss, Inc.
The grasshopper Schistocerca gregaria has one
ear on each side of the first abdominal segment.
An ear consists of a tympanal membrane, tracheal
sacs and a so-called Müller’s organ, which contains the somata of the tympanal sensory cells
(a-, b-, c-, and d-cells; Schwabe, ’06; Gray, ’60).
The Müller’s organ is attached to several sclerotized structures on the inner surface of the tympanal membrane. According to Gray (’60) these
structures are called “folded body” (FB), “elevated
process” (EP), “styliform body” (SB) and “pyriform
vesicle” (PV). Their functional role is apparently
to transmit vibrations of the tympanum to the sensory cells leading to sensory transduction (Stephen
and Bennet-Clark, ’82; Breckow and Sippel, ’85).
Horridge (’61) and Popov (’65) showed first that
locusts are able to discriminate not only the amplitude, but also the pitch of sounds. Later on,
Michelsen (’71a) described different response spectra for cells situated in different parts of the
Müller’s organ. He suggested (’71b) that the sclerites to which the sensory cells are attached might
© 1999 WILEY-LISS, INC.
act as “passive observers” of tympanal motion that
he shows to be already frequency dependent.
Therefore, the frequency-dependence is transmitted to the different groups of sensory cells.
Michelsen (’71b) also presumed that the mass of
the Müller’s organ affects the mechanical frequency response of the whole system. This idea
has been confirmed by Stephen and Bennet-Clark
(’82) and by Breckow and Sippel (’85).
Using intracellular recordings of processes of
tympanal receptor cells within the metathoracic
ganglion and subsequent marking of the cells, four
groups of receptor cells have been distinguished
according to their physiological properties and central projections (type 1 – type 4 receptor cells;
Römer, ’76; Römer, ’85; Halex et al., ’88). Due to
Grant sponsor: Deutsche Forschungsgemeinschaft; Grant number:
SFB 406, Teilprojekt A6.
*Correspondence to: Reinhard Lakes-Harlan, Institut für Zoologie
und Anthropologie, Georg-August-Universität Göttingen, Berliner Str.
28, 37073 Göttingen, Germany. E-mail: rlakes@gwdg.de
Received 20 February 1998; Accepted 16 June 1998.
TYMPANAL RECEPTOR CELLS OF SCHISTOCERCA GREGARIA
methodological difficulties it has so far been impossible to determine simultaneously physiological characteristics, central arborizations and the
morphology within the Müller’s organ of single
auditory receptor cells. Therefore, to date the correlation of these features has been made not with
measurements at the same time, but only indirectly: Breckow and Sippel (’85) have measured
resonant frequencies of the Müller’s organ and of
the sclerotized regions of the tympanal membrane
on different ages and species of locusts. Afterwards, they compared changes occurring in amplitude of oscillations with changes in sensitivity
of physiologically distinguishable groups formerly
described by other authors. Based upon this comparison, they presented a hypothetical correlation
between the anatomical and physiological classification of the receptor cell groups (a-cells = type
1 cells; b-cells = type 3 cells; c-cells = type 2 cells;
d-cells = type 4 cells).
In the present paper we labeled single auditory
receptor cells with neurobiotin during physiological recordings in order to obtain their anatomy
within the Müller’s organ and their central projections together with their threshold curves.
This method has been successfully used in
bushcrickets to characterize the anatomy of
tympanal receptor cells (Stumpner, ’96). With
this method, it was possible to check how a classification of sensory cells within the Müller’s
organ translates into a somatotopic or tonotopic
organization within the auditory neuropil. Additionally, we investigated response characteristics of these cells to frequencies in a range
between 100 Hz and 40 kHz, thus also concerning very low frequencies that have not been tested
in all previous physiological studies on tympanal
receptor cells. To a great extent, our results are
not in accordance with the indirect correlation
made by several authors. Therefore, we present
and discuss an updated grouping of tympanal sensory cells.
271
ings of nerve 6 of the metathoracic ganglion (tympanal nerve) with neurobiotin. The nerve was cut
about 500 µm distal to the ganglion using a fine
pair of scissors. The cut ends of the nerve were
immersed in a neurobiotin solution (5% neurobiotin in 1 M potassium acetate [KAc]) for 12 hr
at 4°C. Afterwards, the thoracic ganglia and the
subesophageal ganglion as well as the ear were
removed from the animal and fixed for 1 hr in 4%
paraformaldehyde in PB (0.5 M Na2HPO4; 0.5 M
NaH2PO4 × 2H2O). To permeabilize the tissue, the
preparations were dehydrated, incubated in xylene for 5min, rehydrated and treated for 1 hr with
collagenase/hyaluronidase (Sigma; each 1 mg/ml
in PB). After this treatment, the preparations
were incubated overnight in a solution of avidin
with biotinylated horseradish peroxidase (Vector
Elite Kit) at room temperature. The preparations
were washed in PB several times and incubated
in diaminobenzidine and nickel chloride (Vector
Kit SK 4100) for 1 hr at room temperature before
transferring the preparations to a solution of H2O2
in PB. When labeled neurons became visible, the
reaction was stopped with PB. The preparations
were again dehydrated, cleared in methyl salicylate and viewed using a Leica DMR microscope.
Acoustic stimulation
Recordings were performed in an unechoic
chamber. Sound stimuli were presented either by
a high-frequency loud speaker (Dynaudio DF21;
2–40 kHz flat frequency response) or by a low frequency speaker (W111; 100 Hz – 7 kHz flat frequency response). Pure sine-wave tones (100 Hz
steps within 100 Hz to 1 kHz, 500 Hz steps within
1 to 4 kHz, 1 kHz steps within 4 to 12 kHz, and 5
kHz steps from 15 to 40 kHz) were generated by
a personal computer (Lang et al., ’93). They were
presented ipsilateral to the recording site at intensities of 35–90 dBSPL (5 or 10 dB increments).
Duration of tone pulses was either 100 ms or 20
ms with a repetition rate of 3.3 Hz.
MATERIALS AND METHODS
Animals
Recording of tympanal nerve activity
All experiments were performed with adult male
and female Schistocerca gregaria (L.), 90 animals
in total. The animals were obtained from crowded
cultures at the I. Zoological Institute, University
of Göttingen.
For extracellular recordings, animals were fixed
ventral side up on a platform. A small window in
the sternum was made by cutting the cuticle with
a razor blade, and a hook electrode made of steel
wire (diameter 30 µm) was placed underneath the
tympanal nerve. Silicone paste (Roth) was applied
to insulate the recording from the hemolymph and
protect the nerve from drying out. The reference
electrode was placed in the adjacent hemolymph.
Neurobiotin backfills
The anatomy of tympanal receptor cells was revealed by retrograde and orthograde axonal fill-
272
K. JACOBS ET AL.
Fig. 1. A: Schematic drawing of housing the somata of
auditory sensory cells in the Müller’s organ (MüO) and of
their projections in the central nervous system. On the left
side the metathoracic (TH3), mesothoracic (TH2), prothoracic
(TH1) and subesophageal ganglion (SEG) are given in whole
mount representation. Within the metathoracic ganglion, the
auditory sensory fibers form two arborization areas, one
within the first abdominal neuromere (caudal auditory neuropil; cNP) and one within the metathoracic neuromere (frontal auditory neuropil; fNP). In the other ganglia, only one
arborization area can be found. B, C: Sagittal sections through
the subesophageal (B) and the metathoracic (C) ganglion at
the levels indicated in A (arrows). Fibers arborize in all ganglia within the ventral association centre (VAC) or within the
median ventral association centre (mVAC). aVAC: anterior
ventral association centre; c: caudal; d: dorsal; DIT: dorsal
intermediate tract; DMT: dorsal median tract; f: frontal; Lb:
labial neuromere; MVT: median ventral tract; Md: mandibular neuromere; Mx: maxillar neuromere; TyN: tympanal
nerve; v: ventral; VIT: ventral intermediate tract; VMT: ventral median tract.
Fig. 2. Photographs of Neurobiotin-marked auditory sensory neurones of Schistocerca gregaria. A, B: Zentrifugal backfill of the tympanal nerve (n) reveals positions of somata (s)
and dendrites (d) of sensory neurones within the Müller’s
organ. The dendrites are either projecting in direction of the
folded body (FB), the elevated process (EP) or the styliform
body (SB); dendrites projecting in direction of the pyriform
vesicle are not shown. Cell bodies that belong to cells with
dendrites that are attached to the EP or the SB cannot clearly
be separated. Neurobiotin marks the whole dendrite up to
its outer segment with the scolopale (arrowhead) and sometimes also the scolopale cell (c). Scale bars: 100 µm. C: Central projection of tympanal fibers within the subesophageal
ganglion (arrowhead) in wholemount. The fibers arborize
within the labial neuromere. In addition to tympanal sensory fibers, the cell body of one interneuron (in) is marked.
TYMPANAL RECEPTOR CELLS OF SCHISTOCERCA GREGARIA
Scale bar: 100 µm. D: Sagittal section through the subesophageal ganglion shown in C. The arborization area of the
marked fibers (arrowheads) dorsal to the ventral association
centre (VAC) and the median ventral tract (MVT) might be
homologous to the median VAC of the thoracic ganglia. Scale
bar: 50 µm. E–J: Morphology within the Müller’s organ (F,
G, I) and central projection within the metathoracic ganglion
(E, H, J) of single tympanal sensory fibers. E: Arborization of
group I sensory fibers within the caudal auditory neuropil
(cn) and the frontal part of the frontal auditory neuropil (fn).
Scale bar: 100 µm. H, J: Arborizations of group II sensory
273
fibers (J) and group III sensory fibers (H) within the frontal
auditory neuropil (dotted line). Group II fiber arborizations
are found caudal within the frontal auditory neuropil; group
III fiber arborization lies between the arborization areas of
the other two groups. Scale bars: 50 µm. F, G, I: The dendrite of a group I sensory fiber (F) projects to the folded body
(FB), that of a group II sensory cell (G) to the pyriform vesicle
(PV) and the dendrite of a group III sensory cell (I) either to
the elevated process (EP) or the styliform body (SB). Scale
bars: 100 µm. DMT: dorsal intermediate tract; VMT: ventral
intermediate tract.
274
K. JACOBS ET AL.
Single cell recording and marking
A combined recording and staining technique
was used to generate information about the central projections, the soma position within the
Müller’s organ and the physiology of single receptor cells. The recordings were performed using
thick-walled glass microelectrodes (borosilicate)
filled with 5% neurobiotin in 1 M KAc (resistance:
50–200 MΩ) positioned in the tympanal nerve
about 500 µm distal of the metathoracic ganglion.
For stabilization of the recording, a spoon made
of steel was placed beneath the nerve and slightly
raised. The spoon also served as reference electrode. The responses of the single receptor fibers
to tone pulses were tracked on an oscilloscope either and stored on magnetic tape (Racal Store
4DS) for subsequent analyses. Threshold was defined as an average of one spike per stimulus
above spontaneous activity.
After physiological characterisation, the receptor cells were stained by switching to positive current of >1 nA for at least 5 min. The preparation
was then transferred to a moist chamber for 24–
48 hr to allow the neurobiotin to spread out within
the whole cell. Afterwards, the ganglia and ears
were processed as described above. For histology,
ganglia were embedded in polyester wax (for a
detailed description see Jacobs and Lakes-Harlan,
’97) and cut into parasagittal sections of 14 µm.
In addition to these findings, which are in agreement with results of Halex et al. (’88), we were
able to stain fiber arborizations within the subesophageal ganglion by using neurobiotin: In 10
out of 15 preparations one or two ascending fibers were marked rostrally to the prothoracic ganglion. In most of these preparations the staining
of fibers ended within the connective between the
prothoracic and the subesophageal ganglion. Nevertheless, in one preparation three fibers reaching the subesophageal ganglion were stained (Fig.
1; Fig. 2C). However, at least one of the stained
arborizations in this preparation might belong to
an interneuron, which has its soma located within
the subesophageal ganglion. This interneuron was
probably stained transsynaptically in the mVAC
of the prothoracic or mesothoracic ganglion. Such
a transsynaptic staining of interneurons occurrs
only in very rare cases. The remaining two fibers
showed some short arborizations within a neuropil region situated within the labial neuromere
(Fig. 1; Fig. 2D). This neuropil region is located
ventral to the VIT, lateral to the VMT and dorsal
to the MVT, and it might be homologous to the
mVAC of the thoracic ganglia. One of these two
marked fibers ascended within the VIT up to the
maxillar neuromere, where it ended without further arborizations. We found no marking within
the mandibular neuromere.
RESULTS
Threshold curves of the whole
tympanal nerve
Central projection of tympanal sensory cells
The axons of all tympanal sensory cells enter
the metathoracic ganglion via the tympanal nerve,
and once within the metathoracic ganglion the
axons join the ventral intermediate tract (VIT;
Halex et al., ’88; Pflüger et al., ’88). Here, they
form two arborization areas, the caudal and frontal auditory neuropil (Fig. 1). The caudal auditory neuropil is build up by collaterals projecting
into the ventral association centre (VAC) of the
first abdominal neuromere. The frontal auditory
neuropil is restricted to a neuropil region called
the median VAC (mVAC), which is located within
the metathoracic neuromere. The mVAC is situated between the median ventral tract (MVT), the
ventral median tract (VMT) and the VIT. Some
axons of tympanal sensory cells ascend to form
further arborization areas within the meso- and
the prothoracic ganglion. These arborizations
are situated in neuropil areas which are serially homologous to the mVAC of the metathoracic ganglion.
In order to extend threshold curves into the low
frequency range, we stimulated tympanal sensory
fibers with sound frequencies from 200 Hz to 7
kHz. Extracellular recordings from the tympanal
nerve of 12 different animals revealed the highest sensitivity of the hearing-threshold-curve between 2 and 5kHz with a threshold between 37–40
dB SPL (Fig. 3). Receptor cells reacted to frequencies as low as 200 Hz with a threshold between
60 and 70 dB SPL.
Positions of tympanal sensory cell somata
and dendrites within the Müller’s organ
Since there are four sites of attachment of dendrites to the tympanal membrane, it has been postulated that there are four different groups of
sensory cells within the Müller’s organ. However,
zentrifugal filling of the tympanal nerve with
neurobiotin suggests a division of the sensory receptor cells into three instead of four groups (Fig.
2A, B; Fig. 4):
Group I: This very homogenous group comprises
TYMPANAL RECEPTOR CELLS OF SCHISTOCERCA GREGARIA
275
about 20 sensory cells whose cell bodies lie in the
ventral and laterocaudal direction towards the
exit of the tympanal nerve (Fig. 4B). All cells from
this group have dendrites that are attached to
the “folded body” (FB; Fig. 2A).
Group II: This second very homogenous group
of tympanal sensory cells consists of 12–14 sensory cells with a laterocaudal and dorsal soma position within the Müller’s organ (Fig. 4C). The
dendrites of these cells run exclusively in a small
protrusion of the Müller’s organ named the “fusiform body” (FuB; Gray, ’60) which is connected to
the “pyriform vesicle” (PV).
Group III: The remaining cells belong to the
third and last receptor cell group (Fig. 4D). The
dendrites of these cells run perpendicular to those
of the other two groups of auditory receptor cells
and project either in the direction of the “elevated
process” (EP) or in the direction of the “styliform
body” (SB). The dendrites can be seen as a unit
with respect to their spatial arrangement, as they
are both arranged perpendicular to the dendrites
of the other two cell groups. A subdivision of the
somata of these tympanal sensory cells into two
Fig. 4. Schematic drawing of tympanal sensory cells
within the Müller’s organ as revealed by zentrifugal backfilling the tympanal nerve (tyn). The Müller’s organ is connected
to four sclerotized structures of the tympanal membrane, the
“elevated process” (EP), “folded body” (FB), “pyriform vesicle”
(PV) and “styliform body” (SB). A: Distribution of somata and
dendrites of all tympanal sensory cells within the Müller’s
organ (view from caudal). B: Tympanal sensory cells of group
I with a dendrite projection to the FB are drawn in black.
The inset shows the position of these cells in a transverse
section of the Müller’s organ (as indicated by arrow). C: Soma
position and dendrite projection of tympanal sensory cells of
group II. The scolopidia of these cells form a structure called
“fusiform body” (FuB). D: Somata of group III tympanal sensory cells are arranged in a ring, their dendrites are attached
either to the EP or the SB. More frontal cells are drawn in
black, more caudal ones in dark gray. c: caudal; d: dorsal; f:
frontal; l: lateral; m: median; v: ventral.
Fig. 3. Threshold curve for the response of the tympanal
nerve to acoustic stimuli with frequencies from 200 Hz to 7
kHz. For 12 animals, the mean (±S.D.; shaded area) is shown.
276
K. JACOBS ET AL.
groups due to the insertion sites of their dendrites
is not practicable as they are arranged in an almost ring-like structure: this arrangement starts
at the border to the cell bodies of the first group,
ventral and laterocaudal within the Müller’s organ, proceeds from there dorsally and to the most
median and frontal part of the Müller’s organ (Fig.
4D, black cells) and finally returns in lateral and
caudal direction again, but further dorsally (Fig.
4D, dark grey cells). Somata of cells with dendrites
attached to the EP as well as of those cells with
dendrites attached to the SB can both be found
at nearly every position in this arrangement.
Single cell physiology and morphology
Intracellular recordings from auditory receptor
cells and simultaneous application of the marker
neurobiotin render it possible to correlate three
features of these cells: the position of the soma
and dendrite within the Müller’s organ, the central projection and the physiological qualities. We
performed recordings from each of the three
groups of receptor cells just described in the
Müller’s organ. Examples of four cells of each
group are depicted in Figures 5–7.
Group I: The central projections (n = 15; morphology within the Müller’s organ see above; Fig.
2F; Fig. 5) of these cells show strong arborizations in the caudal auditory neuropil, whereas arborizations in the frontal auditory neuropil are
more sparse in comparison to that of tympanal
receptor cells of group II and group III (see below; Fig. 5; cell 1–4; Fig. 2E). Only 1–3 branches
grow from the primary axon into the frontal part
of the mVAC. They show few varicosities. 60% of
the investigated cells ascend to the mesothoracic
ganglion where they terminate in the VAC with
few branches. In one preparation, a cell of this
group was marked up to the prothoracic ganglion.
According to the projection of their dendrites
and their pattern of central projections within the
caudal auditory neuropil, cells of group I can be
further divided into two subgroups: those cells
with dendrites inserting in the caudal part of the
FB have caudal arborizations which are limited
to the VAC of the first abdominal neuromere (Fig.
5, cells 1 and 2; also Fig. 2E). Cells with dendrites
projecting to the more frontal part of the FB have
caudal arborizations in the first abdominal neuromere and additional single collaterals that run
into the second (Fig. 5; cell 3) or even near to the
third abdominal neuromere (Fig. 5; cell 4).
On the basis of their physiological properties,
these cells can be characterized as insensitive low
frequency receptors, since their thresholds always
lie above 60 dB SPL. The response of these cells
is generally limited to a frequency range from 300
to 400 Hz up to 4 kHz, in few cases up to 7–10
kHz (Fig. 5, cell 4). In general, all cells of group I
have two areas of high sensitivity: a characteristic frequency of 1.5 kHz and a second range of
high sensitivity at 400–700 Hz. Cells of the first
subgroup tend to be more sensitive at 1.5 kHz
than at 400–700 Hz (Fig. 5, cells 1 and 2); cells of
the second subgroup seem to react the other way
round (Fig. 5, cells 3 and 4).
Group II: Tympanal sensory cells of the second
group (n = 8; morphology within the Müller’s organ as described above; Fig. 2G) have numerous
and thin arborizations in the frontal neuropil (Fig.
6; Fig. 2H). These emerge from a primary axon at
the caudal end of the mVAC and cover the complete caudal portion of the mVAC. The arborizations
extend dorsally into the mVAC and terminate close
to the midline of the metathoracic ganglion. Additional smaller and less dense arborizations in
more frontal areas of the mVAC can also be found
(Fig. 6, cells 1, 3, and 4). All group II cells recorded
from, ascend towards the meso- or, in most cases,
even the prothoracic ganglion.
All group II cells respond to a very broad frequency range (4–7 kHz up to over 40 kHz). Their
characteristic frequencies lie in the high frequency
range between 12 and 25 kHz; in this range the
thresholds are between 40 dB SPL (Fig. 6, cell 4)
and 60 dB SPL (Fig. 6, cell 3). This variation in
sensitivity is also visible in recordings from several high frequency cells within the same animal.
Group III: Tympanal sensory cells of the third
group (n = 26; morphology within the Müller’s organ as described above; Fig. 2I) have thick and
numerous central arborizations in the frontal auditory neuropil. They extend to the midline of the
ganglion and cover the intermediate space between cells of groups I and II in caudal-frontal
direction. The arborization patterns, as well as
the density of arborizations, are variable: cells 1
and 2 (Fig. 7) have at least two first order projections that branch off from the primary axon. They
spread within the mVAC in dorso-ventral direction and send off neurites of higher order that arborize extensively. In contrast, in cells 3 and 4
(Fig. 7) a single first order branch extends from
the primary axon within the mVAC and sends off
a variable but high number of branches of higher
order in the frontal direction. Only 31% of the cells
of group III have arborizations in the caudal neuropil. Forty-six percent of the marked cells ascend
TYMPANAL RECEPTOR CELLS OF SCHISTOCERCA GREGARIA
Fig. 5. Central projection, physiology and morphology
within the Müller’s organ of four different tympanal sensory
cells belonging to group I. Central projection is shown both in
whole mount preparation of the metathoracic ganglion and in
sagittal section through the frontal auditory neuropil (section
level: arrow). On the figure’s right is shown the soma and dendrite of the cell within the Müller’s organ. Each cell is shown
277
in median (top of the two organs) as well as in caudal (bottom
of the two organs) view of the Müller’s organ. For better comparison two preparations have been put into one metathoracic
ganglion and the left and right cell of the metathoracic ganglion has been drawn as if belonging to a right Müller’s organ.
c: caudal; d: dorsal; f: frontal; l: lateral; m: median; mVAC:
median ventral association centre; v: ventral.
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K. JACOBS ET AL.
Fig. 6. Central projection, physiology and morphology
within the Müller’s organ of four different tympanal sensory
cells belonging to group II. For details see Fig. 5. Soma posi-
tion and dendrite projection of cell 3 confirms its belonging
to group II, but it had not been drawn.
towards the mesothoracic ganglion, 8% extend to
the prothoracic ganglion.
We could not find any correlation between the
attachment of dendrites to either the EP or the
SB and the caudal-frontal extension of the central arborizations within the frontal auditory neuropil. For example, the morphology of the cells 1
and 2 (Fig. 7) within the Müller’s organ are simi-
TYMPANAL RECEPTOR CELLS OF SCHISTOCERCA GREGARIA
279
Fig. 7. Central projection, physiology and morphology within the Müller’s organ of four
different tympanal sensory cells belonging to group III. For details see Fig. 5.
lar, but the central projection of cell 1 runs more
caudally in the mVAC than that of cell 2. In contrast, cells 3 and 4 (Fig. 7) have different attachment sites of their dendrites (the dendrite of cell
3 projects towards the SB, that of cell 4 towards
the EP), though they share the same area of arborization in the mVAC and have the same pattern of arborization. Only the density of their
branching differs slightly. Figure 2J shows the
central projections of two cells of this group that
280
K. JACOBS ET AL.
have been recorded from in the same animal. The
somata of these cells have clearly different positions within the Müller’s organ (not shown); the
dendrite of one of these cells projects to the EP,
the dendrite of the other to the SB. Within the
mVAC though, the central projections of these two
cells cover the same intermediate area.
Physiologically the cells of group III can be described as low-frequency receptors with characteristic frequencies between 3 and 4 kHz. Within
this group the threshold values as well as the covered frequency spectra vary considerably. Some
cells are very insensitive and respond to a narrow range of frequencies between 2–7 and 10 kHz
with a threshold of more than 60 dBSPL (Fig. 7;
cells 1 and 3). With respect to their physiological
properties these cells almost resemble group I
cells. Other cells with a threshold of 40–50 dBSPL
respond to frequencies up to 20–30 kHz (Fig. 7;
cell 4). Most cells have been found in their sensitivity and covered range of frequencies to show
values between these two extremes. There was
no correlation between the dendrite projection
sites and the sensitivities of these cells. Recordings and stainings from different cells of the same
animal confirm the observation that cells of this
group cover a wide range of frequencies and sensitivities.
These results suggest that group III is very heterogeneous with respect to various parameters.
No correlations between a given cell’s morphology in the Müller’s organ, its central projection
pattern, and its characteristic frequency, threshold and frequency range were observed. Due to
these results we can not corroborate with the often described subdivision of this group on the
grounds of different insertion sites of the dendrites
and different physiological properties of the cells.
Fig. 8. Schematic summary of morphological and physiological characteristics of the three groups (I, II, III) of tympanal sensory cells. Group I comprises both the formerly
described type-1 cells and c-cells, group II the formerly described type 4 cells and d-cells and group III the formerly
described type-2 and -3 cells and a- and b-cells. A: Soma positions and dendrite attachment sites (EP: elevated process;
FB: folded body; PV: pyriform vesicle; SB: styliform body)
within the Müller’s organ. Their axons run within the tympanal nerve (TyN). B: Within the metathoracic ganglion, tympanal sensory cells form a caudal auditory neuropile (cNP)
and a frontal auditory neuropile (fNP). They arborize tonotopic
and somatotopic within the fNP. C: Schematic drawing of
threshold curves.
DISCUSSION
Using neurobiotin as an intracellular marker we
have determined central projections, morphologies
within the Müller’s organ and physiologies of individual tympanal sensory cells. This correlation resulted in a classification of three groups of receptor
cells primarily according to the positions of their
somata and the attachment sites of their dendrites.
This division is clearly reflected in the central projections and the physiological properties of the cells
(Fig. 8). Since our results are not in accordance with
TYMPANAL RECEPTOR CELLS OF SCHISTOCERCA GREGARIA
the common ideas about the morphology of the
Müller’s organ, the arguments in favour of a division of the auditory receptor cells into three groups
will be discussed later on.
Classification of tympanal sensory cells
The division of the tympanal receptor cells of
the Müller’s organ into three groups differs from
the former division into four groups that has been
proposed by several authors. As early as 1960,
Gray grouped the tympanal sensory cells of the
Müller’s organ according to the insertion sites of
their dendrites on the tympanal membrane (“styliform body,” “elevated process,” “folded body,” and
“pyriform vesicle”). Gray named the four morphological groups a-, b-, c-, and d-cells (Table 1). Other
authors divided the auditory sensory cells into four
groups as well, by considering differing physiological characteristics and the arborization pattern
within the frontal neuropil of the metathoracic ganglion (Table 2; type 1–type 4 cells; Römer, ’76;
Petersen et al., ’82; Halex et al., ’88) as characteristics. Through a comparison of the physiological
properties of cells with the resonance frequencies
of the tympanal membrane at the attachment site
of the different cell groups, (Michelsen, ’71b;
Stephen and Bennet-Clark, ’82; Breckow and
Sippel, ’85) to date Gray’s a-cells and the type 1
cells, his b-cells and the type 3 cells, his c-cells
and the type 2 cells, and his d-cells and the type
4 cells have been correlated (Breckow and Sippel,
’85; Halex et al., ’88). These correlations were not
completely confirmed by our experimental strategy of recording from the auditory sensory cells
and marking them individually and completely
with neurobiotin. Based on simultaneous examination of their soma postions, their central pro-
281
jections and their physiologies, we propose a subdivision into only three groups (Fig. 8).
1. One group of tympanal sensory cells has
their somata positioned laterocaudally and far
ventrally within the Müller’s organ and their dendrites project onto the FB. Within the frontal auditory neuropil of the metathoracic ganglion they
form but few arborizations more frontally. They
respond preferentially to stimuli of very low frequencies and high intensity, their characteristic
frequency lying between 400–700 Hz and at 1,500
Hz respectively. The positions of their somata and
the fact that their dendrites project onto the FB
indicate that they correspond to the tympanal
sensory cells that have been described by Gray
as c-cells (’60). Michelsen (’71a) recorded extracellularly from these cells in the isolated ear close
to the soma. According to his results their frequency of highest sensitivity lies at 1.5 or 3.5 kHz;
he describes these cells as the least sensitive of
all tympanal receptors. On account of their central projections and physiological characteristics
group I cells match the cells that have been described as type 1 cells by Römer (’85) in Locusta
migratoria and by Halex et al. (’88) in Locusta
migratoria and Schistocerca gregaria. Type 1 cells
form sparse arborizations frontal within the
mVAC and stronger caudal arborizations. They
belong to the least sensitive cells with the lowest
characteristic frequency of all tympanal sensory
cells (Römer ’76). The characteristic frequencies
and thresholds that have been described for this
cell-type differ slightly between the different species that were investigated, but also from those
that were found in our experiments (Table 2).
These differences can partly be explained from
TABLE 1. Correlation between morphology within the ear and physiology of single tympanal sensory cells as revealed
by recording from the Müller’s organ in different species of locusts*
Source
Gray (’60), (Schistocerca gregaria)
Michelsen (’71a), (Schistocerca
gregaria)
Inglis and Oldfield (’88), (Valanga
irregularis)
Nomenclature
CF
(kHz)
Threshold
(dBSPL)
Somaposition
Dendrite
attachment site
a-cells
b-cells
c-cells
d-cells
a-cells
b-cells
c-cells
d-cells
a-cells
c-cells
d-cells
—
—
—
—
3.74
3.4
1.5–3.5
10–14
4–6
2–3.5
14–25
—
—
—
—
46
51
>55
>40
>45
>70
>40
Median, dorsal
Median, ventral
Laterocaudal, ventral
Laterocaudal, dorsal
—
—
—
—
Median
Laterocaudal, ventral
Laterocaudal, dorsal
EP
SB
FB
PV
—
—
—
—
EP
FB
PV
*CF, characteristic frequency; FB, ‘‘folded body’’; EP, ‘‘elevated process’’; PV, ‘‘pyriform vesicle’’; SB, ‘‘styliform body.’’
282
K. JACOBS ET AL.
TABLE 2. Correlation between physiology and central arborization of single tympanal sensory cells
as revealed by recording and marking*
Source
Römer (’76, ’85), (Locusta migratoria)
Halex et al. (’88), (Schistocerca gregaria)
Nomenclature
CF
(kHz)
Threshold
(dBSPL)
Arborization pattern
within the mVAC
Type 1
Type 2
Type 3
Type 4
Type 1
Type 2
Type 3
Type 4
3.5–4
4
5.5–6
12–20
2–3.5
2–3.5
6
>10
50–60
>20
>40
>40
50–70
20–40
30–60
35–60
Frontal
Caudal-intermediate
Frontal-intermediate
Caudal
Frontal
Frontal-intermediate
Caudal-intermediate
Caudal
*CF: characteristics frequency; mVAC: median ventral association centre.
the fact that in the other studies the frequencyrange below 1 kHz was not tested.
Thus, the results of this study make it most
likely, that group I cells correspond to type 1 cells
and to c-cells. This directly contradicts the hypothesis of Breckow and Sippel (’85), who predicted that the cells of type 1 correspond to the
a-cells that have been described by Gray (’60) and
that the type 2 cells correspond to the c-cells.
Their results were exclusively based on a determination of differences in resonance-frequency of
the tympanal membrane and of different parts of
the Müller’s organ in different ages and species
of locusts.
2. Cells of group II are characterised in the
Müller’s organ by having laterocaudally and dorsally positioned somata, and by dendrites projecting to the PV. Therefore they correspond to
the d-cells as described by Gray (’60). Their central projection is very homogenous, forming extensive, but fine arborizations in the caudal part
of the frontal auditory neuropil. Physiologically
the cells of this group can be described as highfrequency receptors with a varying characteristic frequency between 12 and 25 kHz. The
thresholds of the single cells belonging to group
II show great variation in sensitivity. These results are in accordance with those of other authors. Group II cells correspond to the formerly
characterized type 4 high-frequency receptor
cells on the basis of physiological data and on
the grounds of their central arborization pattern
(Table 2; Römer, ’76; Petersen et al., ’82; Römer,
’85; Halex et al., ’88). Measurements of the tympanal membrane movement (Michelsen, ’71b;
Stephen and Bennet-Clark, ’82; Breckow and
Sippel, ’85) also led to the conclusion that the
insertion point of the d-cells must be the insertion point of high-frequency receptors.
3. The somata of the group III cells are arranged almost in a ring within the Müller’s or-
gan. Their dendrites project in the direction of
either the EP or the SB. Their central arborizations vary in form, but cover more or less the
part of the frontal auditory neuropil between the
other two groups. They can be classified as low
frequency receptors with a characteristic frequency of 3 to 4 kHz and very divergent thresholds. Tympanal sensory cells of this group so far
have been morphologically divided into a- and
b-cells according to the description of Gray (’60).
Other authors who have performed intracellular recordings in the tympanal nerve and the
metathoracic ganglion found two cell groups with
distinct physiological properties (type 2, type 3;
Römer, ’76, ’85; Halex et al., ’88). For this reason it will be discussed in detail why in our opinion neither the morphology of the cells in the
ear nor their physiology or central projection allow a further division and furthermore, why the
cells of type 2 and 3 and the a- and b-cells can
not be assigned.
Morphology within the ear
A morphological distinction between these cells
has been introduced by Gray (’60), who described
four separate groups of cell bodies of tympanal
sensory cells. Their dendrites are attached to one
of the four specialized structures on the tympanal membrane. However, our marking of all tympanal receptor cells within the Müller’s organ
revealed that the cell bodies of those cells that
project onto the SB and of those which connect
in the area of the EP can not clearly be separated into two groups. Therefore only the different insertion points of the dendrites would justify
segregation into two groups. When weighing this
criterion it should be kept in mind that the EP
and the SB and therefore all dendrites of this cellgroup have the same direction in space. Since this
direction differs from that of the cells of group I
and II, which are positioned perpendicular to each
TYMPANAL RECEPTOR CELLS OF SCHISTOCERCA GREGARIA
other and to group III cells, it seems justified to
view group III cells as a morphological unity.
Physiology
A clear distinction of the cells of group III,
which comprises morphologically related a- and
b-cells, on the grounds of their characteristic frequencies and their sensitivities is not possible.
The characteristic frequencies of these cells all
lie between 3 and 4 kHz with their thresholds
spreading over a wide intensity-range. Neither in
their characteristic frequency nor in their threshold a correlation with the insertion sites of the
dendrites was found. This contradicts findings of
Michelsen (’71a,c). He also found only slight differences in characteristic frequency of the a- and
b-cells he recorded from (3.4 and 3.74 kHz), but
he divided them into two physiologically distinct
groups according to their different sensitivities.
Since in his experiments the cells were not
marked during recording, it seems possible that
a clear distinction between the two types could
not always be made, especially since they are
arranged in a continuous ring and not in two
separate groups. In addition, he performed his recordings of somata or dendrites of receptor cells
directly in the ear, which might influence sensitivity of cells.
Other publications report clear differences in
characteristic frequencies and sensitivities of type
2 and type 3 cells (Römer, ’76; Petersen et al.,
’82; Halex et al., ’88). We have never recorded
from a cell with a characteristic frequency of 6
kHz (=type 3 in Schistocerca gregaria; Halex et
al., ’88). It was possible though to show that cells
with dendrites projecting onto the SB and onto
the EP both have a characteristic frequency of 3
or 4 kHz, which corresponds to measurements of
the resonant frequency at their attachment sites
(Breckow and Sippel, ’85). Interestingly, Krahe
(’91) in Locusta migratoria and Inglis and Oldfield
(’88) in Valanga irregularis both identified only
one further group of tympanal receptor cells in
addition to the insensitive low-frequency receptor cells and the high frequency receptor cells
(cells of group I and II).
Central projection
Römer (’85) and Halex et al. (’88) made a clear
distinction between the cells of type 2 and 3, according to their central projections. The projections of both cell types were described to arborize
in the metathoracic ganglion in the intermediate area of the frontal neuropil, one more fron-
283
tal, the other more caudal. The correlation of the
frontal-intermediate and the caudal-intermediate arborizations with the corresponding cells
characterized as type 2 or type 3 in the two publications is contradictory though (Table 2): Römer
(’85) found type 2 cells arborizing more caudal
than type 3 cells; Halex (’88) described type 2 cells
to be located more frontal than type 3 cells. The
cells that have been described as group III cells
in this study arborize independently from the position of their somata and dendrites in overlapping areas in the intermediate part of the frontal
arborization area.
It can be concluded that the group I tympanal
sensory cells correspond to the physiological characterized type 1 cells and the group II tympanal
cells match the type 4 cells. Our results represent a first direct and unambiguous correlation
of the c-cells to type 1 and that of the d-cells to
type 4 cells. The a- and b-cells that have been
described by Gray (’60) on the grounds of their
morphology both belong to group III. With respect
to their central arborization pattern, group III
cells comprise both type 2 and type 3 cells. However, a-cells or b-cells do not correspond to type 2
or type 3 cells, or vice versa. Therefore, they can
not be further subdivided neither according to the
position of their somata nor to their physiology
or their arborizations.
Somatotopic order within the frontal
auditory neuropil is tonotopic
The somatotopic organization of tympanal receptor cells in the frontal auditory neuropil, which
is described in this study, leads to a clear tonotopic organisation of the mVAC. Auditory sensory
cells with a CF of 12–25 kHz arborize caudally,
sensory cells with a CF of 3–4 kHz have an intermediate representation and insensitive receptor cells with a characteristic frequency of
400–1,500 Hz project frontally. In the most anterior part of the mVAC there are no arborizations
of auditory receptor cells, but the axons of the
vibration sensitive metathoracic myochordotonal
organ and the axons of some abdominal chordotonal organs are shown to arborize in this part
of the mVAC (Bräunig et al., ’81; Bickmeyer et
al., ’92). Thus, the primary sensory information
divides the mVAC into four parts, in which information of different frequency is processed. In
1985, Römer suggested a separation of high and
low frequency information within the mVAC,
which was confirmed by Halex et al. (’88). However, Römer (’85) concluded that the frontal au-
284
K. JACOBS ET AL.
ditory neuropil is more importantly partitioned
according to the threshold intensity of the receptor cells. Considering the new information presented in our study on the response of tympanal
cells to frequencies below 2 kHz, we suggest that
the auditory neuropil is indeed subdivided according to frequency rather than to intensity.
Such a somatotopic order within a neuropil is
realized in numerous sensory systems of insects,
e.g. the cercal system of crickets (Bacon and
Murphey, ’84; Jacobs and Theunissen, ’96) and
the auditory system of bushcrickets (Römer, ’83;
Oldfield, ’88; Stumpner, ’96). In bushcrickets, a
tonotopic division of the auditory neuropil is also
achieved by somatotopic arborizations of sensory
cells which are aligned with this order (Römer,
’83; Oldfield, ’88). Auditory interneurons in the
locust also show a frequency-specific tuning
(Marquart, ’85; Römer et al., ’88). These interneurons partly achieve this tuning by having
their dendrites restricted to certain parts of the
mVAC. The best example for an overlapping of
sensory cell projection and auditory interneurons
with the same frequency tuning seems to be the
SN5, whose threshold curve resembles that of
the high-frequency receptors and whose postsynaptic structures are restricted to the caudal region of the mVAC (Römer et al., ’88). However,
many interneurons achieve their specific tuning
not by limiting their postsynaptic structures to
a distinct neuropil areas, but also by a frequency
dependent inhibition by other interneurons
(Römer et al., ’81).
ACKNOWLEDGMENTS
The authors thank Drs. F. Lang, R. Heinrich
and A. Stumpner for their critical comments on
the manuscript, Dr. G. Ganter for improving the
English language and Dr. N. Elsner for supporting the study.
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