Download Synaptic Inputs to Stellate Cells in the Ventral Cochlear Nucleus

Synaptic Inputs to Stellate Cells in the Ventral Cochlear Nucleus
MICHAEL J. FERRAGAMO, NACE L. GOLDING, AND DONATA OERTEL
Department of Neurophysiology, University of Wisconsin Medical School, Madison, Wisconsin 53706-1532
INTRODUCTION
Auditory information is carried from the cochlea to the
cochlear nuclear complex of the brain stem by auditory nerve
fibers. In terminating on at least six types of principal cells,
auditory nerve fibers feed information to at least six parallel
ascending pathways in mammals. One pathway is mediated
by T stellate cells of the ventral cochlear nucleus (VCN),
stellate cells named for the trajectory of their axons through
the trapezoid body (Oertel et al. 1990). T Stellate cells
project to the contralateral inferior colliculus in mice and
cats (mice: Oertel et al. 1990; Ryugo et al. 1981; cats: Adams
1979, 1983; Cant 1982; Oliver 1987; Osen 1972; Roth et
al. 1978).
Even the earliest studies revealed that stellate cells were
of multiple types. Stellate (multipolar) cells were recognized
by Osen (1969) to be of multiple classes based on somatic
size. In contrast to the T stellate cells, the axons of which
leave the VCN through the trapezoid body, the axons of D
stellate cells have a dorsalward trajectory toward the intermediate acoustic stria. Stellate cells also differ in the synaptic
density distributions (Cant 1981), dendritic architecture
(Brawer et al. 1974; Oertel et al. 1990; Tolbert and Morest
1982), axonal destinations (Oertel et al. 1990; Smith and
Rhode 1989), terminal vesicle shape (Smith and Rhode
1989), immunoreactivity (Adams and Mugnaini 1987;
Wenthold et al. 1987), responses to sounds in vivo (Smith
and Rhode 1989), and shock evoked synaptic responses in
vitro (Oertel et al. 1990).
T stellate cells have both their dendrites and terminal
arbors aligned with isofrequency laminae in the tonotopically arranged caudal anterior VCN (AVCN) and posterior
VCN (PVCN) (Oertel et al. 1990). Recordings have not
been made from single, identified units in mice, but the
arrangement of dendrites with respect to the tonotopy of the
VCN suggests that T stellate cells are tuned narrowly. In
cats, stellate cells that project through the trapezoid body
respond to tones with steady firing as ‘‘choppers’’ (Smith
and Rhode 1989). Choppers in cats, rats, and chinchillas are
tuned sharply and respond to tones near the best frequency
tonically at a steady rate (Rhode and Smith 1986; Wickesberg 1996). Inhibitory sidebands flank the excitatory response area. Although an initial spike signals the onset of
the tone with temporal precision, the timing of subsequent
spikes is independent of the phase of the sound. The ultrastructure of their terminals suggests that these stellate cells
are excitatory (Smith and Rhode 1989).
D stellate cells have long, sparsely branched dendrites that
are spread across isofrequency lamina and terminate widely
in the VCN with local collaterals (Oertel et al. 1990). The
spread of dendrites and the properties of corresponding cells
in cats indicate that D stellate cells are likely to be broadly
tuned. Similar cells in cats and guinea pigs respond to sound
as ‘‘onset-choppers’’, are inhibitory and glycinergic, and
project to the contralateral cochlear nucleus (cats: Cant and
Gaston 1982; Smith and Rhode 1989; guinea pigs: Schofield
and Cant 1996; Wenthold 1987). As D stellate cells are
inhibitory and glycinergic, they are labeled immunocytochemically with antibodies to glycine conjugates (Oertel and
0022-3077/98 $5.00 Copyright q 1998 The American Physiological Society
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Ferragamo, Michael J., Nace L Golding, and Donata Oertel.
Synaptic inputs to stellate cells in the ventral cochlear nucleus. J.
Neurophysiol. 79: 51–63, 1998. Auditory information is carried
from the cochlear nuclei to the inferior colliculi through six parallel
ascending pathways, one of which is through stellate cells of the
ventral cochlear nuclei (VCN) through the trapezoid body. To
characterize and identify the synaptic influences on T stellate cells,
intracellular recordings were made from anatomically identified
stellate cells in parasagittal slices of murine cochlear nuclei. Shocks
to the auditory nerve consistently evoked five types of synaptic
responses in T stellate cells, which reflect sources intrinsic to the
cochlear nuclear complex. 1) Monosynaptic excitatory postsynaptic potentials (EPSPs) that were blocked by 6,7-dinitroquinoxaline2,3-dione (DNQX), an antagonist of a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptors, probably reflected activation by auditory nerve fibers. Electrophysiological estimates
indicate that about five auditory nerve fibers converge on one T
stellate cell. 2) Disynaptic, glycinergic inhibitory postsynaptic potentials (IPSPs) arise through inhibitory interneurons in the VCN
or in the dorsal cochlear nucleus (DCN). 3) Slow depolarizations,
the source of which has not been identified, that lasted between
0.2 and 1 s and were blocked by DL-2-amino-5-phosphonovaleric
acid (APV), the N-methyl-D-aspartate (NMDA) receptor antagonist. 4) Rapid, late glutamatergic EPSPs are polysynaptic and may
arise from other T stellate cells. 5) Trains of late glycinergic IPSPs
after single or repetitive shocks match the responses of D stellate
cells, showing that D stellate cells are one source of glycinergic
inhibition to T stellate cells. The source of late, polysynaptic EPSPs
and IPSPs was assessed electrophysiologically and pharmacologically. Late synaptic responses in T stellate cells were enhanced by
repetitive stimulation, indicating that the interneurons from which
they arose should fire trains of action potentials in responses to
trains of shocks. Late EPSPs and late IPSPs were blocked by
APV and enhanced by the removal of Mg 2/ , indicating that the
interneurons were driven at least in part through NMDA receptors.
Bicuculline, a g-aminobutyric acid-A (GABAA ) receptor antagonist, enhanced the late PSPs, indicating that GABAergic inhibition
suppresses both the glycinergic interneurons responsible for the
trains of IPSPs in T-stellate cells and the interneuron responsible
for late EPSPs in T stellate cells. The glycinergic interneurons that
mediate the series of IPSPs are intrinsic to the ventral cochlear
nucleus because long series of IPSPs were recorded from T stellate
cells in slices in which the DCN was removed. These experiments
indicate that T stellate cells are a potential source of late EPSPs
and that D stellate cells are a potential source for trains of late
IPSPs.
52
M. J. FERRAGAMO, N. L. GOLDING, AND D. OERTEL
METHODS
Tissue preparation
The cochlear nuclei were obtained from 18- to 26-day–old CBA
or ICR mice as described previously (Golding and Oertel 1996;
Zhang and Oertel 1993). The dissection was performed in carbogen-infused saline of the following composition (in mM): 130
NaCl, 3 KCl, 1.2 K2HPO4 , 2.4 CaCl2 .H2O, 1.3 MgSO4 , 3 N-2hydroxyethylpiperazine-N *-2-ethanesulfonic acid, 20 NaHCO3 ,
and 10 glucose, pH 7.4, 317C. With a single parasagittal cut, the
cochlear nuclei were removed from the brain stem with a tissue
slicer (Frederick Haer, New Brunswick, ME) in a slice that was
between 250 and 400 mm at its thickest point. The slice was immersed in oxygenated saline in a tissue chamber with a volume of
0.3 ml and continuously perfused at a rate of 10–12 ml/min (Oertel
1985). The temperature of the bath was maintained at 347C with
a thermoregulator (UW-Madison Medical Electronic Shop) with
feedback supplied by a temperature probe in the chamber (Physitemp, Clifton, NJ). The slice was allowed to ‘‘rest’’ for 60–90
min before recording.
Pharmacological agents were dissolved in normal saline or saline
in which MgSO4 was replaced with CaCl2 (0 Mg 2/ ) and introduced
into the chamber without a break in the flow. Strychnine, picrotoxin
bicuculline methiodide, DL-2-amino-5-phosphonovaleric acid (APV),
and 6,7-dinitroquinoxaline-2,3-dione (DNQX) were obtained from
Sigma (St. Louis, MO) and added to normal saline.
Electrophysiological recording
Recording electrodes were constructed of 1-mm–diam omega
dot tubing (WPI) pulled (Sutter Instruments, San Francisco, CA)
to impedances of 120–250 MV and filled with 1% biocytin
(Sigma) in 2 M K / -acetate, pH 7.0. Intracellular potentials were
amplified, low-pass filtered at 10 kHz (ICX2-700; Dagan, Minneapolis, MN), continually monitored audiovisually, and recorded on
chart paper (Gould, Valley View, OH). Data acquisition, current
injection, and shock triggering were all performed by a Digidata
1200A computer interface under control of pClamp software
(Axon Instruments, Foster City, CA) in an IBM-compatible computer (Micron, Nampa, ID). All responses to current injection and
synaptic responses °600-ms duration were sampled digitally at 25
kHz; synaptic responses exceeding 600 ms were sampled at 10
kHz. Shocks were delivered to the severed eighth nerve through a
stimulating electrode constructed from an adjacent pair of insulated
tungsten electrodes (Bak Electronics, Rockville, MD), each with
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a 50-mm exposed tip. Stimulation voltage (0.1–100 V; 100-ms
duration) was produced by an isolated DC source (S-100; Winston
Electronics, Millbrae, CA) under control of a digitally triggered
timer (A-65; Winston Electronics).
Analysis
The slope was measured of the linear portion of the rise of the
excitatory postsynaptic potential (EPSP) from rest. When an EPSP
was not detectable measurements of amplitude and slope were
performed during a 0.5-ms window starting at the point when the
resting potential was restored after the shock. K-means cluster
analysis was performed by Statistica (Statistica, Rockville, MD).
Histology
Anatomic labeling was accomplished by iontophoretic injection
of biocytin with depolarizing current steps (0.5–2 nA; 150–200
ms) at a rate of 2 Hz for roughly 2 min. At the termination of the
experiment, the slice was fixed in 4% paraformaldehyde, 0.1 M
phosphate buffer, pH 7.4, and stored at 47C for 24 h to 2 wk. For
histological reconstruction, the tissue was embedded in a mixture
of gelatin and albumin cross-linked with glutaraldehyde and sectioned at 60 mm on a vibratome. Sections were reacted with avidin
conjugated to horseradish peroxidase (Vector ABC kit, Vector
Laboratories, Burlingame, CA) and processed for horseradish peroxidase with Co 2/ and Ni 2/ intensification. Sections mounted on
coated slides were conterstained with cresyl violet.
RESULTS
The present experiments are based on recordings from 54
T stellate cells and 3 D stellate cells. All were labeled with
biocytin and identified morphologically according to the criteria of Oertel et al. (1990). The resting potentials of
T stellate cells ranged from 051 to 068 mV [mean Å
056.7 { 4.9 (SD)]. Input resistance, measured from the
magnitude of the response to injection of 00.1 nA current,
ranged from 44 to 151 MV (mean Å 89.4 { 24.4). The
average resting potential and input resistance for D stellate
cells were 057 { 4.2 mV and 96.2 { 27.8 MV, respectively.
T stellate cells
The cell bodies of T stellate cells make up a large proportion of the large cells of the PVCN. Anatomical reconstructions of T stellate cells in this study resembled those reported
previously (Oertel et al. 1990). Many lay medially, well
away from the superficial granule cell domain on the lateral
surface of the VCN. The dendrites of T stellate cells commonly were arranged parallel to the projection of auditory
nerve fibers, putting the dendrites into the path of relatively
few of the tonotopically arranged auditory nerve terminals,
and ended in characteristic tufts that often came near but
did not intermingle with the superficial granule cells. The
axon of each cell was cut as it exited the VCN medially and
entered the trapezoid body. All T stellate cells had local
axonal collaterals restricted to roughly the same isofrequency lamina as its parent soma and dendrites. Because
the region is populated primarily by other T stellate cells, it
is likely that T stellate cells contact other T stellate cells.
Many T stellate cells also had a collateral that projected to
the fusiform cell layer of the dorsal cochlear nucleus (DCN).
This projection maintained tonotopy and occupied a nar-
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Wickesberg 1993; Wenthold 1987; Wickesberg et al. 1994).
Such labeling stains õ0.1 of cells in the dorsocaudal AVCN
and PVCN. In cats, too, corresponding cells represent a small
fraction of stellate cells (Cant 1981).
Although it is clear that T stellate cells transform the
‘‘primary-like’’ firing patterns of auditory nerve fibers to
chopper patterns that are different, it is not at all clear what
the brain accomplishes in making that transformation. To
gain a better understanding of the integrative processes that
contribute to this pathway, we have examined functionally
and in detail the synaptic inputs to T stellate cells. We show
that T stellate cells are subject to synaptically mediated
feedforward excitation and inhibition that is under
GABAA ergic control and the action of which continues over
a time course one order of magnitude greater than that previously reported (Oertel et al. 1990). We suggest that T
stellate cells integrate input from the auditory nerve with
input from other T and D stellate cells.
SYNAPTIC INPUTS TO VCN MULTIPOLAR CELLS
53
rowband (50–75 mm) spanning the fusiform cell layer from
its most rostral to its most caudal end.
Spontaneous EPSPs were recorded in 50 T stellate cells,
in 9 of which spontaneous inhibitory postsynaptic potentials
(IPSPs) also were detected. Spontaneous PSPs occurred infrequently and singly (not in bursts).
The synaptic responses of T stellate cells to shocks of the
auditory nerve have five components: monosynaptic EPSP,
disynaptic IPSP, long, slow depolarization, late EPSPs, and
trains of late IPSPs. The first two of these components are
highly consistent. They have been described previously and
therefore will be discussed only briefly (Oertel 1983; Oertel
et al. 1990; Wickesberg and Oertel 1990; Wu and Oertel
1984, 1986). The later three components are more variable
and have not been described before.
Shocks to the auditory nerve evoked EPSPs the amplitude
of which increased monotonically with the strength of the
shock. The delay between beginning of the shock and the
rise of the EPSPs was constant except in responses to the
weakest shocks where delays were a little longer. The minimum latencies ranged between 0.48 and 0.92 ms (mean Å
0.70 { 0.13, n Å 53). These latencies indicate that the
EPSPs were monosynaptic and therefore that they reflect
direct input from the auditory nerve.
A consistent feature of T stellate cells was that the EPSP
grows stepwise with increasing shock strength, indicating that
relatively few inputs converge on one cell. A superposition of
traces selected at every other stimulation voltage reveals groups
of similarly shaped EPSPs (Fig. 1). The accompanying plots
of peak amplitudes also reveal the stepwise growth of EPSPs,
indicating that relatively few inputs are recruited sequentially.
The total number of steps, however, is obscured by the presence
of an action potential in suprathreshold responses. The stepwise
increases in amplitude were accompanied by stepwise increases
in the slope of the rise of the EPSPs, revealing in addition
recruitment of inputs in suprathreshold responses. Because it is
possible that several auditory nerve fibers have similar thresholds, that contributions are too small to be resolved clearly and
because it is conceivable that auditory nerve inputs might have
been damaged in the preparation of slices, the number of resolved
steps reflects a minimum number of auditory nerve inputs. The
dotted lines in each plot indicate the mean of each cluster, determined by K-means cluster analysis. Analysis of variance was
performed on the resultant clusters; these were found in each
case to differ significantly (P õ 0.001). If each cluster with a
mean greater than Ç0 represents a separate input, then the clusters in the plots of slope in Fig. 1, A and B, indicate that these
cells received five and four inputs from the auditory nerve,
respectively. Similar experiments were conducted on two additional cells in which six and five steps were resolved, contributing
to an average of about five ( {0.8, n Å 4) resolvable steps.
Long, slow depolarization
A slow, long-lasting depolarization followed the early responses in 43 T stellate cells. The depolarization was generally observed only in responses to strong shocks. It followed
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FIG . 1. Number of converging auditory nerve fibers was estimated from
the number of steps in the growth of responses with increasing shock strength
for 2 cells. A and B, left: first response at every other stimulation voltage was
selected and superimposed to demonstrate that there are distinct groups of
similarly shaped excitatory postsynaptic potentials (EPSPs). Right: scatter
plots of the maximal amplitude of each EPSP and of the initial slope of both
the subthreshold (solid circle) and suprathreshold (open circle) responses.
Dashed line, mean of each cluster determined by K-means cluster analysis.
Total number of sub- and suprathreshold steps for the cells plotted in A and
B are 5 and 4, respectively. Two (A) and 3 repetitions (B) were performed
at each stimulation voltage. All were used in the statistical analysis: A slope,
6 clusters, F(5,58) Å 1,293.3, P õ 0.001; amplitude, 4 clusters, F(3,43) Å
335.9, P õ 0.001; B slope, 5 clusters, F(4,52) Å 1,378.2, P õ 0.001; amplitude, 4 clusters, F(3,38) Å 354.6, P õ 0.001. The bath contained 1 mM
strychnine to avoid distortion of EPSPs by IPSPs. The cell in B was hyperpolarized with a 00.1-nA current pulse during synaptic stimulation to enhance
resolution of subthreshold events.
suprathreshold EPSPs and early IPSPs, becoming evident as
the cell repolarized after the combination of the undershoot
of the action potential and the early IPSP and lasting between
100 and 500 ms (Fig. 2A). The depolarizing hump could
be suprathreshold, causing the T stellate to fire late action
potentials in response to the shock.
The appearance of the slow, long depolarization was variable in responses to single shocks, being more prominent in
some responses than others in a single cell and being more
consistent in some cells than others. Consistently, however,
repetitive stimulation promoted its appearance. Figure 2B
illustrates the responses of a T stellate cell the response of
which to a single 5-V shock did not have a detectable long
depolarizing hump. Repetitive stimulation revealed the presence of the slow depolarization and showed that its amplitude and duration increased with the rate of repetitive stimulation. Twenty shocks at 200/s evoked a long, suprathreshold, depolarization as well as late IPSPs.
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Monosynaptic EPSPs reflect the convergence of few inputs
from the auditory nerve
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M. J. FERRAGAMO, N. L. GOLDING, AND D. OERTEL
Late EPSPs reveal the existence of excitatory interneurons
A single shock commonly evoked occasional unitary
EPSPs °500 ms after a shock to the auditory nerve. Figure
3 ( ● ) illustrates examples of late EPSPs recorded in six T
stellate cells. Even relatively weak, subthreshold shocks
could activate late EPSPs (Fig. 3A). Late EPSPs occurred
between tens and hundreds of milliseconds in 44 of 54 T
stellate cells; in some cells, the presence of IPSPs obscured
late EPSPs. The long latency of late EPSPs indicates that
they are polysynaptic and, therefore, that there exist excitatory interneurons that contact T stellate cells in slices of
the cochlear nuclei. Responses such as those illustrated in
Fig. 3A show that these excitatory interneurons are activated
by shocks to the auditory nerve.
The existence of excitatory interneurons was confirmed
FIG . 4. An inhibitory postsynaptic potential (IPSP) with a threshold
lower than that of the monosynaptic EPSP was observed consistently after
auditory nerve stimulation. The 1.5- to 2-ms latency indicated that the IPSP
was disynaptic.
with another series of experiments. The finding that excitatory interneurons can be activated polysynaptically with
shocks indicates that their dendrites lie in the slice and suggests that they might be activated chemically through excitatory synaptic receptors. Figure 3B shows that the application of glutamate does indeed increase the frequency of
EPSPs. This experiment was done in the presence of strychnine and bicuculline to eliminate inhibition.
Disynaptic IPSPs in responses to shocks to the auditory
nerve confirm the existence of inhibitory interneurons
IPSPs were evoked in 54 of 54 labeled T stellate cells by
weak shocks to the auditory nerve. As described previously,
the fact that their latencies were between 1.2 and 2 ms in
most cells indicates that they are disynaptic (Wu and Oertel
1986). Disynaptic IPSPs recorded from one T stellate cell
are shown in Fig. 4. The threshold of IPSPs in this cell, as
in most, was slightly lower than that of EPSPs. The result
that thresholds of EPSPs and IPSPs were not identical indicates that different populations of auditory nerve fibers mediate EPSPs and the IPSPs with the lowest thresholds.
Trains of late IPSPs
FIG . 3. T stellate cells received input from excitatory interneurons that
could be activated electrically and pharmacologically. A: examples of
EPSPs ( ● ) recorded from 6 different cells that occurred hundreds of milliseconds after the stimulus. Resting membrane potential is indicated ( left).
Stimulation voltages (top to bottom) were /7, 2, 5, 7.5, 5, and 5. B:
spontaneous EPSPs were not frequent and may have been masked by inhibition when the slice was bathed in normal saline. Blocking all inhibition
with 1 mM strychnine (STR) and 10 mM bicuculline (BIC) increased the
frequency of spontaneous EPSPs. Addition of 1 mM glutamate (GLU) to
the bath excited intact neurons, including excitatory interneurons, in the
slice and resulted in the summing of many EPSPs occasionally evoking a
discharge (bottom).
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Strong shocks to the auditory nerve also evoked trains of
late IPSPs that in some cases lasted for ú500 ms. In 49 of
54 T-stellate cells, shocks evoked trains of late IPSPs. For
an individual cell, the duration of the trains of IPSPs varied
on a trial-to-trial basis at a single shock strength. The cell
the responses of which are shown in Fig. 5 ( left) responded
to shocks of 65 V with trains of IPSPs in 4 of 5 trials; the
duration of the trains of IPSPs varied between 100 and 400
ms. The probability of evoking a train of IPSPs increased
with increasing shock. Examples of responses from the same
cell to a series of shocks increasing in 10-V increments show
that generally the longest trains of IPSPs are evoked with
the strongest shocks strength (Fig. 5, middle). As in all 49
cells, the threshold and the range of stimulus strengths over
which the trains of IPSPs grew was higher than the threshold
of mono- and disynaptic PSPs. Examples of trains of IPSPs
from six other cells are shown in Fig. 5, right. The beginning
of the trains coincided with other synaptic inputs and could
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FIG . 2. A: in 1 cell, monosynaptic EPSPs were followed by a long,
slow depolarization, which sometimes elicited discharges. Shock occurred
at 0 ms, and its strength (in V) is indicated above each trace. Spikes
are digitally truncated. B: responses from another cell illustrate that the
appearance of late synaptic events was enhanced by repetitive stimulation.
SYNAPTIC INPUTS TO VCN MULTIPOLAR CELLS
55
FIG . 5. Characteristics of trains of IPSPs
after auditory nerve stimulation. Left: appearance of trains of IPSPs was variable from trial
to trial. Five successive responses to identical
shocks show that the trains of IPSPs occurred
in an all-or-none fashion and that the duration
of trains of IPSPs was variable. Middle: in
the same cell, the threshold of the trains of
IPSPs was higher than the threshold of the
monosynaptic EPSP. Right: examples of
trains of IPSPs recorded from 6 cells show
that there is considerable variability from 1
cell to another. In some cells, there was a
mixture of both late excitation and late inhibition.
dorsal to the granule cell region at the VCN-DCN border. All
T stellate cells recorded in such slices exhibited late IPSPs,
indicating that they received inhibition from neurons in the
VCN (Fig. 7; n Å 6/6 in 4 slices). The disynaptic IPSP
(Fig. 7A) and trains of IPSPs (Fig. 7B) in VCN slices were
identical to those observed in slices of the entire cochlear
nuclear complex. In each case, removal of the DCN was
verified histologically (Fig. 7C). In addition to this VCN
source of glycinergic inhibition, a DCN contribution has
been demonstrated to arise from the tuberculoventral cells
of the DCN (Wickesberg and Oertel 1990), indicating that
disynaptic IPSPs probably have multiple components. The
long trains of IPSPs, on the other hand, are unlikely to represent summed components from multiple sources. They are
so unusual that they serve as physiological tags of those
interneurons. Their presence in the isolated VCN shows that
the interneurons which mediate trains of IPSPs lie in
the VCN.
Monosynaptic and late EPSPs are glutamatergic
In the presence of Mg 2/ , all synaptic responses in 5 of 5
cells to stimulation of the auditory nerve were blocked by
10 mM DNQX, a blocker of a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptors (Fig. 8A) (Honoré
et al. 1988). This finding confirms the conclusion that input
from the auditory nerve is glutamatergic (Raman et al. 1994;
Wenthold 1985; Wickesberg and Oertel 1988; Zhang and Trussell 1994). It also indicates that late responses are consequences
of the activation of AMPA receptors.
Long, slow depolarization in T stellate cells is mediated
through NMDA receptors
FIG . 6. Repetitive stimulation lowered the threshold of trains of IPSPs
in a T stellate cell. Weak shocks evoked trains of IPSPs when they were
presented repetitively but not when they were presented singly.
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Application of 100 mM APV, an antagonist of NMDA
receptors, abolished the long, slow depolarization in 6/8
cells tested (Fig. 8A). In the two cells in which the slow
depolarization was not abolished, it was a reduced and shortened. The slow depolarization after repetitive stimulation
also was blocked by APV (Fig. 8B). A shorter-lasting depolarization that was not studied further remained in the presence of APV.
Currents through NMDA receptors are known to be af-
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not be resolved. As other synaptic responses subsided, the
trains of regularly occurring IPSPs emerged, lasting between
50 and 600 ms.
The long trains of late IPSPs had three features that are
consistent with their arising from one or few inhibitory interneurons. First, the IPSPs were rapid and almost stereotyped
in their shape. Second, they occurred in an all-or-none fashion as if an inhibitory interneuron was either activated or not
activated. Third, the latest IPSPs occurred with remarkable
regularity, the interval between them increasing with time.
The long trains of late IPSPs behave as if they were generated by interneurons that fired long and regularly in responses to shocks.
Repetitive shocks evoked trains of late IPSPs in T stellate
cells more effectively than single shocks. The shock strength
required to evoke late IPSPs was consistently greater than
that required to evoke a monosynaptic EPSP. Shocks too
weak to evoke trains of IPSPs singly often activated them
when applied in rapid succession and the rate of occurrence
and the duration of the train of IPSPs increased with the
frequency of shocks (Fig. 6).
It is known that there are several populations of glycinergic and GABAergic neurons in the cochlear nuclei (Adams and Mugnaini 1987; Mugnaini 1985; Oertel and Wickesberg 1993; Osen et al. 1990). To determine whether the
interneurons that generate the long trains of IPSPs lie in the
VCN or in the DCN, recordings were made from T stellate
cells in slices in which the DCN was removed with a cut just
56
M. J. FERRAGAMO, N. L. GOLDING, AND D. OERTEL
FIG . 7. Recordings from T stellate cells in
slices of the isolated ventral cochlear nucleus
(VCN) show that glycinergic interneurons reside in the VCN. A: responses of 1 cell to a
series of shocks to the auditory nerve show that
the disynaptic IPSP was present. B: trains of
IPSPs recorded from 3 cells, each located in
a different VCN slice, were identical to those
observed in slices of the entire cochlear nuclear
complex. Stimulation voltages (top to bottom)
were /65, 50, and 65. C: these experiments
were repeated for 6 cells, the location of which
is indicated ( ● ) in 4 slices in which the absence
of the DCN was assessed histologically. Remaining granule cell border between the VCN
and dorsal cochlear nucleus (DCN) is indicated
in 3 slices.
Disynaptic and late IPSPs are glycinergic
Consistent with earlier findings, all early and late IPSPs
were blocked by 0.5 or 1 mM strychnine, indicating that they
were glycinergic (Wu and Oertel 1986). The existence of
the trains of IPSPs raises the question how effective they
are in blocking excitation. This question was addressed by
comparing responses in the absence and presence of strychnine (Fig. 9). In slices, the balance of excitation and inhibition could tilt either toward excitation or inhibition. Most
commonly, shocks strong enough to evoke trains of IPSPs
evoked a suprathreshold monosynaptic EPSP even when the
shocks occur in rapid succession, indicating that the synchronous excitation was more potent than the inhibition (Fig. 6).
With weaker shocks, however, IPSPs prevented subsequent
EPSPs from reaching threshold (Fig. 9). Application of 1
mM strychnine blocked all IPSPs and tipped the balance
toward excitation, so that each shock evoked a spike.
GABAergic inhibition of T stellate cells is subtle
Applications of picrotoxin and bicuculline, blockers of
GABAA receptors, also were made to T stellate cells to
test whether GABAergic inhibition played a role. Although
generally all IPSPs were eliminated by strychnine, these
experiments revealed the existence of GABAergic inhibition. The clearest manifestations were polysynaptic and will
be discussed below. If there was a direct effect on GABAA
receptors of T stellate cells, that effect was subtle. We could
not demonstrate blocking of visible IPSPs, but we cannot
exclude the possibility that small, slow IPSPs, such as those
that might be generated in distal dendrites, were blocked.
Pharmacological manipulations to characterize
interneurons that impinge on T stellate cells
The late synaptic responses of T stellate cells, those that
are mediated by interneurons, serve as assays of the activity
FIG . 8. Excitation was mediated by a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. A: DL-2-amino-5-phosphonovaleric acid (APV) abolished the slow
depolarization, indicating that the slow depolarization was mediated by
NMDA receptors. 6,7-dinitroquinoxaline-2,3-dione (DNQX) eliminated the
rapid, monosynaptic EPSP, indicating that it was mediated by AMPA receptors. B: in a different cell, APV partially blocked the slow depolarization
and associated action potentials after repeated shocks (200 Hz; 95 ms). C:
hyperpolarization ( 00.05 and 00.1 nA; 300 ms) during synaptic stimulation
( /1 V) shortened the depolarization, showing that some NMDA currents
were intrinsic to T stellate cells.
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FIG . 9. Trains of IPSPs inhibited the monosynaptic EPSPs evoked with
weak shocks. EPSPs were reduced and prevented from reaching threshold
by IPSPs in response to the later shocks of a repetitive stimulus (100 Hz;
90 ms). Removal of glycinergic IPSPs with strychnine enabled a one-toone discharge to each shock as well as firing after the shock train.
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fected by a voltage-dependent block by Mg 2/ (Nowak et al.
1984). The enhancement of late PSPs, however, raises the
possibility that the depolarization arises from interneurons.
To test whether the action of NMDA receptors was intrinsic
to the T stellate cell from which the depolarization was
measured or on excitatory interneurons, the voltage sensitivity of the response was measured in the presence of Mg 2/
(Fig. 8C). The duration of the synaptically evoked late depolarization shortened as the cell was hyperpolarized, indicating that NMDA receptors were intrinsic to the recorded T
stellate cell and not on an excitatory interneuron.
SYNAPTIC INPUTS TO VCN MULTIPOLAR CELLS
57
repetitive shocks evoked long-lasting excitation that resulted
from the slow depolarization and from late EPSPs. Interestingly, bicuculline enhanced that excitation indicating that
GABAA ergic inhibition affected excitatory interneurons and
perhaps also the recorded T stellate cell.
D stellate cells
of the interneurons and reveal how pharmacological manipulations affect interneurons. To test the possibility that
NMDA receptors mediate long-lasting excitation in the excitatory and inhibitory interneurons, their action was unblocked by removing extracellular Mg 2/ and their action
was blocked by 100 mM APV. Experiments from two cells
illustrated in Fig. 10 show that the firing of both excitatory
and inhibitory interneurons is influenced by NMDA receptors. In the absence of Mg 2/ , even weak shocks evoked
trains of IPSPs that were blocked by APV (Fig. 10A). A
similar experiment in another cell shows that excitatory interneurons also are influenced by NMDA receptors. In a cell
that did not respond with late EPSPs or late IPSPs to single
shocks at low voltages in normal saline, the removal of Mg 2/
caused an increase in late excitation that was blocked by
APV (Fig. 10B). Late inhibition was present but not as
dramatic as in some other cells. The experiments illustrated
in Fig. 10 were chosen to illustrate that both excitation and
inhibition were affected by NMDA receptors and illustrate
extremes in the range of responses that were recorded in six
experiments.
The contributions of GABAA ergic inhibition to the late
PSPs also was examined. One experiment is illustrated in
Fig. 11. Under normal conditions, this cell revealed a slow
depolarization that lasted Ç200 ms in responses to single
shocks and Ç500 ms in responses to repetitive shocks as
well as late IPSPs and late EPSPs. Bicuculline (10 mM) had
no effect on the monosynaptic spike but reversibly enhanced
late IPSPs. In the presence of bicuculline, single and repetitive shocks evoked trains of late IPSPs that overcame the
long, slow depolarization and caused the cell to hyperpolarize. The slow depolarization and late EPSPs were evident
after the end of the train of late IPSPs. Strychnine eliminated
all visible IPSPs. In the presence of strychnine, single and
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Synaptic responses
Figure 12 displays responses of a D stellate cell to shocks
delivered to the auditory nerve. Weak shocks evoked a sub-
FIG . 11. GABAergic inhibition was subtle in T stellate cells. Bicuculline
(BIC) promoted IPSPs (f ), enhanced the long depolarization, and promoted
late EPSPs (*) in response to both single shocks and repetitive stimuli
(100 Hz; 90 ms). – – – , resting potential. Strychnine (STR) abolished
all IPSPs.
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2/
FIG . 10. Removal of Mg
from the extracellular saline promoted trains
of IPSPs, late EPSPs, and the long, slow depolarization. A: a weak shock
( /1 V) evoked few IPSPs and EPSPs when the slice was bathed in normal
saline. When Mg 2/ was removed, trains of IPSPs were evoked consistently
with each trial. They subsequently were abolished by APV, indicating that
they were mediated by NMDA receptors. B: in a different cell, the balance
favored excitation when Mg 2/ was removed, although IPSPs were promoted
as well. Late EPSPs and IPSPs were blocked completely, and the long
depolarization was blocked partially by APV. These manipulations were
reversed.
The cell bodies of each of the labeled D stellate cells
lay just beneath the superficial granule cells, resembling D
stellate cells described before (Oertel et al. 1990). The dendrites radiated from the cell body, independently of the tonotopic organization, and spanned extensively the dorsoventral
and rostrocaudal axis of the VCN. Collaterals of D stellate
cells were intermingled with the large cells of the PVCN
and also invaded the granule cell domains. Terminals were
observed in regions densely populated by T stellate cells
(Oertel et al. 1990; this study). In two cases where the axon
was not severed, it could be traced to the deep layer of the
DCN before its exit through the intermediate acoustic stria.
The recordings in T stellate cells predict the existence of
inhibitory interneurons that respond to strong shocks with
long-lasting trains of action potentials. Those long trains
depend on the activation of NMDA receptors and are countered by the activation of GABAA receptors. In the present
series of experiments, recordings were made from three anatomically identified D stellate cells in which some of these
tests were made. The results support the hypothesis that D
stellate cells are the source of the long trains of IPSPs but
does not prove it.
58
M. J. FERRAGAMO, N. L. GOLDING, AND D. OERTEL
DISCUSSION
FIG . 12. Responses to shocks in 1 D stellate cell. Strong shocks evoked
an initial burst of spikes that was followed by subthreshold synaptic input
that could last for hundreds of milliseconds. IPSPs (f ) occasionally occurred
in short bursts. Late EPSPs ( ● ) often resulted in discharges.
threshold depolarization of 30- to 40-ms duration. Stronger
shocks elicited suprathreshold initial depolarizations and a
long, slow depolarization the amplitude and duration of
which grew with shock strength. The step-like manner in
which the cell was depolarized suggests that it might be
excited polysynaptically through excitatory interneurons.
Strong shocks produced long trains of spikes that lasted
hundreds of milliseconds. Inhibition was present but relatively inconspicuous in comparison with T stellate cells.
The long-lasting depolarization in responses to strong shocks
occasionally was interrupted by a burst of IPSPs. The pattern
of activity, a long-lasting burst of spikes with increasing
spike intervals, mirrored that of the IPSPs observed in T
stellate cells.
D stellate cells received glycinergic inhibition. Figs. 13A
and 14 show that 1 mM strychnine eliminated the occasional
IPSPs but that glycinergic inhibition in D stellate cells did
not prominently affect synaptic responses.
To test whether NMDA receptors mediate the long-lasting
firing of D stellate cells in responses to shocks, 100 mM
APV was applied to the bath. Figure 13A shows that APV
reversibly eliminated the long-lasting depolarization. The
remaining early excitation was blocked reversibly by DNQX
(n Å 2/2). To determine whether NMDA receptors were
intrinsic to the recorded cell or on excitatory interneurons,
the voltage dependence of the late response was examined
(Fig. 13B). The long, late depolarization that caused the D
stellate cell to fire for Ç200 ms was shortened to Ç100 ms
when the cell was hyperpolarized, this is consistent with
synaptic excitation mediated by NMDA receptors that were
intrinsic to the D stellate cell.
GABAergic inhibition plays a prominent role in the synaptic responses of D stellate cells. Even in the absence of
visible IPSPs, picrotoxin, a blocker of GABAA receptors,
enhanced the firing of the cell (Fig. 14; n Å 1/1). Both
the frequency and duration of firing were augmented in the
presence of picrotoxin.
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FIG . 13. A: late excitation in D stellate cells was abolished by APV.
Strychnine (STR) reversibly blocked all IPSPs but had little effect on
the firing of this cell. DNQX reversibly blocked the monosynaptic EPSP
mediating the first few spikes. B: hyperpolarization during stimulation ( /10
V) shortened the depolarization and associated activity, showing that some
NMDA currents were intrinsic to the D stellate cell. Strychnine was included
to isolate excitatory inputs.
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T stellate cells form one of the parallel ascending auditory
pathways from the ventral cochlear nucleus to the inferior
colliculi. In considering the role of these neurons in the
auditory pathway, the significance of the pattern of convergence of auditory nerve fiber inputs and the interaction of
those synaptic inputs with the intrinsic electrical properties
to generate the chopper responses to tones has been appreciated (Banks and Sachs 1991; Molnar and Pfeiffer 1968;
Oertel 1983; Wang and Sachs 1995; White et al. 1994; Wu
and Oertel 1984). The present study indicates that neuronal
circuits that provide long-lasting excitatory and inhibitory
feedforward interactions also contribute significantly to the
responses of T stellate cells to activation of auditory nerve
fibers.
The new results raise the question what is the source of
the additional inputs. T stellate cells are known to receive
glycinergic inhibition from tuberculoventral cells (Wickes-
SYNAPTIC INPUTS TO VCN MULTIPOLAR CELLS
FIG . 14. Blocking the GABAA receptor with picrotoxin (PIC) prolonged
the firing in one D stellate cell, whereas strychnine (STR) had little effect.
Innervation of T stellate cells by auditory nerve fibers
Auditory nerve fibers are of two types. Type I fibers are
large and myelinated and comprise 95% of the total while
type II fibers are small, unmyelinated and comprise only
Ç5% of the total (cats: Kiang et al. 1982; mice: Ehret 1979).
On the basis of extracellular injections of auditory nerve
fibers in mice, type I auditory nerve fibers have been observed to terminate on both D and T stellate cells (M. W.
Garb and D. Oertel, unpublished observations). Most neurons in the multipolar cell area of the PVCN (probably T
stellate cells) are contacted heavily at the cell body unlike
cells that project through the trapezoid body in cats. In cats
type I fibers innervate all of the large cells, including those
that correspond to T and D stellate cells (Liberman 1991,
1993). The anatomic findings are consistent with what is
known about responses to activation of auditory nerve fibers
in vivo and in vitro. The short-latency, sharply timed responses to the onset of tones indicate that chopper and onsetchopper units receive input from the large, myelinated auditory nerve fibers (Blackburn and Sachs 1989; Rhode and
Smith 1986; Smith and Rhode 1989). In slices from mice,
both D and T stellate cells respond to shocks of the auditory
nerve with EPSPs (Oertel et al. 1990; Wu and Oertel 1986).
As thresholds for EPSPs are low and latencies are õ1 ms,
the input is probably from myelinated auditory nerve fibers.
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Anatomic and electrophysiological evidence indicates that
few auditory nerve fibers innervate a T stellate cell. The
orientation of the dendrites of T stellate cells parallel to the
path of auditory nerve fibers and spanning a small proportion
of the tonotopic axis indicates that T stellate cell dendrites
are positioned to receive input from a limited group of fibers.
The result that the amplitude of responses to shocks of the
auditory nerve grow in three or four discrete jumps with
shock strength indicates that the number of fibers innervating
one T stellate cell in a mouse is small, perhaps as small as
three or four (Fig. 1). As any of the jumps in amplitude
could have resulted from the recruitment of more than one
fiber and as it is possible that inputs might have been cut or
damaged, this estimate represents a minimum. This conclusion is in contrast with the results of similar experiments in
octopus cells, in which such subthreshold jumps cannot be
detected (Golding et al. 1995). This result also indicates
that models of choppers, based on what is known in cats,
that require the integration of many inputs might be oversimplified (Banks and Sachs 1991; Molnar and Pfeiffer 1968;
Wang and Sachs 1995).
It is intriguing that the NMDA-receptor–mediated slow
depolarizations were generated with shock strengths greater
than those required to produce apparently maximal monosynaptic EPSPs. This finding suggests that different sources
of glutamatergic input may activate different populations of
receptors. It raises the possibility that type I auditory nerve
fibers act primarily through AMPA receptors, as they are
known to do in other vertebrate cochlear nuclei (Raman et
al. 1994; Zhang and Trussell 1994) whereas other sources of
excitation, alone or in combination, are required to activate
NMDA receptors. It is conceivable that type II auditory
nerve fibers contribute to the long, slow depolarization.
Small, unmyelinated fibers would be expected to have higher
thresholds for shocks than larger, myelinated fibers and their
responses would be expected to be later.
Sources of polysynaptic excitation
The late EPSPs observed in T stellate cells indicate that
T stellate cells receive excitatory input from excitatory interneurons in the slices. In being separated from their natural
synaptic inputs, isolated axons cannot contribute to polysynaptic responses. Monosynaptic responses have latencies between 0.5 (synaptic delay) and Ç3 ms (2.5-ms conduction
delay for an unmyelinated fiber of 0.5-mm plus 0.5-ms synaptic delay). Therefore EPSPs the latencies of which are
ú3 ms are polysynaptic and must be generated by excitatory
interneurons. Two other experimental observations confirm
this conclusion. As cut axons have not been observed to fire
spontaneously, the presence of spontaneous EPSPs is an
indication of the existence of excitatory interneurons. Furthermore, the activation of EPSPs with the application of
glutamate indicates that the dendrites of excitatory interneurons are accessible from the bath.
T stellate cells are excitatory neurons known to terminate
in the vicinity of T stellate cells. T stellate cells terminate
locally in the multipolar cell area of the PVCN (Oertel et
al. 1990; this study). This area is occupied by T stellate
cells and occasional D stellate and bushy cells, some or all
of which are therefore presumably their targets. The ultra-
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berg and Oertel 1990). The present experiments show that,
in addition, T stellate cells are a possible source of feedforward excitation and D stellate cells are a possible source
of feed-forward inhibition. The finding that T stellate cells
are influenced by GABAergic neurons is particularly intriguing. Golgi cells in the superficial granule cell domain are
the only known source of GABA intrinsic to the VCN. They
do not receive input from the large, myelinated type I auditory nerve fibers but may be innervated by the small, unmyelinated, type II auditory nerve fibers (Ferragamo et al.
1997). These experiments thus raise the possibility that T
stellate cells are influenced by neurons in the superficial
granule cell layer and that they are influenced directly by
acoustic input from the large, myelinated type I auditory
nerve fibers and also indirectly by the small, unmyelinated,
type II auditory nerve fibers through Golgi cells.
59
60
M. J. FERRAGAMO, N. L. GOLDING, AND D. OERTEL
Sources of glycinergic cochlear nuclear inhibition
Glycinergic inhibition is recorded consistently in T stellate
cells spontaneously and in responses to shocks of the auditory nerve as prominent, rapid IPSPs. The latencies of IPSPs
indicate that they are polysynaptic and arise through interneurons that are intrinsic to the slice. All distinct IPSPs in
T stellate cells, as in other cells of the VCN, are blocked
by strychnine, indicating that they are glycinergic (Wu and
Oertel 1986).
An ability to label glycinergic interneurons with antibodies to glycine conjugates allows the population of glycinergic
neurons to be identified (Oertel and Wickesberg 1993).
Three groups of cells account for immunopositive labeling:
in the DCN, tuberculoventral cells (Osen et al. 1990; Saint
Marie et al. 1991; Wenthold et al. 1987; Wickesberg et al.
1994) and cartwheel cells (Osen et al. 1990; Saint Marie et
al. 1991; Wenthold et al. 1987), and in the VCN, multipolar
cells (Schofield and Cant 1996; Wenthold 1987), which
correspond to D stellate cells (Oertel et al. 1990).
Tuberculoventral cells have been shown to provide disynaptic, glycinergic inhibition to T stellate cells in responses
to shocks of the auditory nerve (Wickesberg and Oertel
1990). Although there is no doubt that tuberculoventral cells
contribute to the disynaptic IPSPs, several experimental
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findings show that they do not mediate the long trains of
IPSPs. First, tuberculoventral cells do not fire for prolonged
periods when activated through eighth nerve inputs (Golding
and Oertel 1997; Zhang and Oertel 1993). Second, long
trains of IPSPs are preserved in slices in which the DCN
was removed from the slice (Fig. 7).
Considerable experimental evidence indicates that D stellate cells are the source of the trains of IPSPs. First, it is the
only class of glycine-immunopositive neurons in the VCN.
Furthermore, pharmacological manipulations produce parallel changes in the firing of D stellate cells and the appearance
of IPSPs in T stellate cells. 1) Late IPSPs in T stellate cells
were evoked by strong shocks that lasted for hundreds of
milliseconds. D stellate cells fire for long periods in responses to strong shocks. 2) Both the trains of IPSPs of T
stellate cells and the late firing of D stellate cells were
blocked by APV. 3) Both the trains of IPSPs in T stellate
cells and late firing of D stellate cells were promoted by
application of GABAA antagonists. The results that D stellate
cells contact T stellate cells and that they respond to weak
shocks with single spikes monosynaptically indicate that
they contribute to the disynaptic IPSP.
GABAA ergic influence
Markers of GABAergic neurotransmission in the cochlear
nucleus reveal the presence of both cell bodies and terminals
that could be GABAergic. Antibodies to GABA conjugates
and to glutamate decarboxylase (GAD) generally label neurons that are functionally GABAergic. Occasionally GAD
and GABA are associated with neurons that are functionally
glycinergic; cartwheel cells of the DCN, for example, are
labeled for GABA and GAD yet seem to be glycinergic
(Golding and Oertel 1997; Golding et al. 1996). Functionally GABAergic neurons and their terminals are labeled consistently for GABA and GAD, however, indicating that the
source of GABAergic input in T stellate cells would be
expected to be labeled. GABAergic input could arise from
neurons intrinsic to the cochlear nuclei or from sites external
to the nucleus, such as the superior olivary nucleus (Saint
Marie et al. 1989). Only GABAergic neurons in the cochlear
nuclei can function in polysynaptic circuits in slices as was
observed in the present study, however, isolated terminals
of extrinsic sources cannot be activated synaptically.
Labeling for GAD and GABA is associated strongly with
regions that contain granule cells, the molecular and fusiform
cell layers of the DCN and the superficial granule cell domain of the VCN. In cats and guinea pigs, antibodies to
GABA conjugates and to GAD, a biosynthetic enzyme, have
been shown to label specific groups of cells and terminals
(GABA: Kolston et al. 1992; Osen et al. 1990; Wenthold et
al. 1986; GAD: Adams and Mugnaini 1987; Moore and
Moore 1987; Mugnaini 1985; Saint Marie et al. 1989). In
the DCN, the majority of cell bodies and puncta that were
labeled with antibodies against GABA and GAD lie in the
superficial and fusiform cell layers (Adams and Mugnaini
1987; Kolston et al. 1992; Moore and Moore 1987; Mugnaini
1985; Osen et al. 1990; Saint Marie et al. 1989; Wenthold
et al. 1986). Labeled neurons are cartwheel, stellate, and
Golgi cells. As none of these neurons make direct or indirect
connections with the VCN, it is unlikely that cartwheel,
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structure of T stellate cell terminals and functional studies
of the inputs to the inferior colliculi is consistent with their
being excitatory (Oliver 1984, 1987; Smith and Rhode
1989).
The present experiments provide functional evidence in
support of the conclusion that T stellate cells mediate late
EPSPs. If T stellate cells are excited by other T stellate cells,
then disynaptic EPSPs that reflect the firing of other stellate
cells should be observed under similar conditions as stellate
cell firing. The present experiments reflect the parallel nature
of T stellate cell firing and late EPSPs under five experimental conditions. 1) Stellate cells consistently are brought to
threshold Ç1 ms after shocks to the auditory nerve. Disynaptic EPSPs with latencies of Ç1.6 ms are observed but in the
presence of monosynaptic EPSPs and disynaptic IPSPs the
early disynaptic EPSPs are sometimes difficult to resolve.
2) Strong shocks evoke a long, slow depolarization in T
stellate cells that causes T stellate cells to fire hundreds of
milliseconds after a strong shock to the auditory nerve.
Strong shocks also evoke very late EPSPs in T stellate cells.
3) APV reduces late firing and late EPSPs in T stellate cells.
4) The removal of extracellular Mg 2/ enhances firing as
well as late EPSPs. 5) Strychnine and bicuculline enhance
firing as well as late EPSPs in T stellate cells. In summary,
although the results of the present experiments are consistent
with the conclusion that T stellate cells excite one another,
it does not rule out the possibility that other, hitherto unknown, cells contribute to the excitation.
The only other known excitatory neurons that terminate
in the vicinity of T stellate cells are granule cells. The dendrites of T stellate cells end in bushy branches, some of
which often come near, but never penetrate, the layer of
superficial granule cells that overlies them. It is conceivable,
therefore, that granule cells could provide polysynaptic excitation.
SYNAPTIC INPUTS TO VCN MULTIPOLAR CELLS
61
the same group of auditory nerve fibers innervates tuberculoventral cells which, in turn, provide delayed, glycinergic
inhibition (Wickesberg and Oertel 1988, 1990). D stellate
cells contribute to the disynaptic IPSP and at high shock
strengths can provide trains of late IPSPs to T stellate cells.
D Stellate cells are driven by type I auditory nerve fibers
(Oertel et al. 1990; this study), and they receive GABAergic
inhibition, of which Golgi cells are a likely source (Mugnaini
1985). Golgi cells lie in the granule cell domain, away from
the terminals of type I auditory nerve fibers. The finding
that they are activated by shocks to the auditory nerve more
slowly than that to T or D stellate cells in the vicinity suggests that they are activated by type II auditory nerve fibers
(Benson et al. 1996; Ferragamo et al. 1997).
superficial stellate or Golgi cells of the DCN contribute to
GABAergic inhibition in T stellate cells of the VCN.
GABAergic input to T stellate cells of the VCN could
arise from Golgi cells in the superficial granule cell domain
either mono- or disynaptically. Labeled cell bodies identified
as Golgi cells were observed to be associated with the superficial granule cell layer (Mugnaini 1985). These neurons
terminate locally in the superficial granule cell layer with
very dense terminal arbors that abut the underlying large
cell area (Ferragamo et al. 1997). The dendrites of D stellate
cells lie just beneath the superficial granule cell domain,
poised to be contacted by Golgi cells proximally and distally,
indicating that D stellate cells could mediate GABAergic
responses. Furthermore, some of the branches of the distal
dendrites of T stellate cells approach the superficial granule
cell domain. If Golgi cells contact T stellate cells directly,
those contacts can only be on distal dendrites. In contrast
with glycinergic IPSPs, GABAergic IPSPs were not prominent in T or D stellate cells; IPSPs that remained in the
presence of strychnine were small and inconspicuous, if
present. There are four possible reasons for this observation:
the synaptic currents associated with GABAergic inputs
were relatively slower and weaker, they were generated relatively far from the somatic recording site, they were mediated through an excitatory interneuron, or there were presynaptic GABAergic receptors present.
Proposed neuronal connections
The present considerations have provided evidence for the
connections that are summarized in Fig. 15. We propose that
T stellate cells receive excitatory, glutamatergic input from
a small number of type I auditory nerve fibers (monosynaptic
EPSPs) as well as through collaterals of other T stellate
cells (late EPSPs) (Oertel et al. 1990). The topographic
arrangement of tuberculoventral cells indicates that roughly
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Implications for acoustic processing
T stellate and D stellate cells, identified in vitro in mice
correspond to cells in vivo in cats as choppers and onsetchoppers, respectively (Oertel et al. 1990; Smith and Rhode
1989). Although response patterns to tones have not been
measured in mice, it is likely that all mammals have units
with common characteristics. Chopper and onset-chopper
units with similar characteristics have been made in cats
(Blackburn and Sachs 1989; Rhode and Smith 1986; Smith
and Rhode 1989) and rats (W. S. Rhode, personal communication). Choppers have been recorded in chinchillas
(Wickesberg 1996) and gerbils (Frisina et al. 1990). Choppers fire regularly in response to short tone bursts, are tuned
narrowly with prominent inhibitory sidebands, and have dynamic ranges that average 30 dB but rarely exceed 40 dB
(Evans and Nelson 1973; Rhode and Greenberg 1994; Rhode
and Smith 1986; Shofner and Young 1985). Onset choppers
fire only at the beginning of sound pulses with precisely
timed action potentials and have dynamic ranges that average
60 dB but can span 90 dB (Rhode and Smith 1986).
The present results suggest that choppers excite other
choppers tuned to similar frequencies. In responses to tones,
auditory nerve fibers excite choppers most strongly at the
beginning of the pulse when the firing rates of auditory nerve
fibers are highest (Rhode and Smith 1986). Presumably
other similarly tuned choppers boost excitation after the initial transient and account for the ability of choppers to respond with steady firing rates when their primary afferent
inputs have a strong transient. This circuit raises the questions whether the mutual excitation in choppers could be
self-sustaining and how chopper responses are terminated.
Probably, in vivo as in vitro, the excitation is too weak
to be self-sustaining; inhibition from tuberculoventral cells
could terminate responses (Wickesberg and Oertel 1990).
Our results also suggest that onset-choppers inhibit choppers. The possibility that onset-choppers inhibit choppers
has been proposed before (Smith and Rhode 1989) and is
consistent with what is known about their responses in vivo.
The widely tuned onset-choppers could provide choppers
with inhibitory sidebands. Near the characteristic frequency
excitation from auditory nerve fibers is strong and can overcome inhibition by onset-choppers. At the edges of the response area such a model predicts that excitation by the
auditory nerve would provide an onset transient that is cut
short by inhibition from onset-choppers. As predicted, chop-
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FIG . 15. Summary of the proposed connections to T stellate cells (T
St). A small number of auditory nerve fibers excite T stellate cells monosynaptically. Those auditory nerve fibers also excite tuberculoventral cells
(TV), which inhibit T stellate cells, contributing to the disynaptic IPSP. A
different population of type I auditory nerve fibers excites D stellate cells (D
St), which contribute to the disynaptic glycinergic IPSPs and occasionally
provide long trains of late IPSPs. Golgi cells (Go) are driven through type
II auditory nerve fibers and inhibit D stellate cells. T stellate cells excite one
another. Glutamatergic inputs are indicated with clear symbols, glycinergic
inputs are indicated with stippled symbols and GABAergic inputs are shown
with black symbols.
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M. J. FERRAGAMO, N. L. GOLDING, AND D. OERTEL
pers do respond with onset transients away from the characteristic frequency (W. S. Rhode, personal communication).
The suggestion that Golgi cells inhibit D stellate cells,
onset-choppers, is also consistent with what is known about
their responses to sound. GABAergic inhibition could contribute to the cessation of firing after the two or three chopping responses at the onset. Consistent with this role, application of bicuculline converted a phasic pattern to a tonic
one in response to tone bursts in the PVCN (Palombi and
Caspary 1992). It also has been suggested that GABAergic
inhibition modifies the gain of activity in the ventral cochlear
nucleus (Caspary et al. 1994; Evans and Zhao 1993, 1997).
These findings together with the present results suggest that
a role of type II auditory nerve inputs might be in regulating
the gain of the circuits of the cochlear nuclei through Golgi
cells.
Received 11 April 1997; accepted in final form 5 August 1997.
REFERENCES
ADAMS, J. C. Ascending projections to the inferior colliculus. J. Comp.
Neurol. 183: 519–538, 1979.
ADAMS, J. C. Multipolar cells in the ventral cochlear nucleus project to the
dorsal cochlear nucleus and the inferior colliculus. Neurosci. Lett. 37:
205–208, 1983.
ADAMS, J. C. AND MUGNAINI, E. Patterns of glutamate decarboxylase immunostaining in the feline cochlear nucleus complex studied with silver
enhancement and electron microscopy. J. Comp. Neurol. 262: 375–401,
1987.
ALTSCHULER, R. A., BETZ, H., PARAKK AL, M. H., REEKS, K. A., AND WENTHOLD, R. J. Identification of glycinergic synapses in the cochlear nucleus
through immunocytochemical localization of the postsynaptic receptor.
Brain Res. 369: 316–320, 1986.
BANKS, M. I. AND SACHS, M. B. Regularity analysis in a compartmental
model of chopper units in the anteroventral cochlear nucleus. J. Neurophysiol. 65: 606–629, 1991.
BENSON, T. E., BERGLUND, A. M., AND BROWN, M. C. Synaptic input to
cochlear nucleus dendrites that receive medial olivocochlear synapses.
J. Comp. Neurol. 365: 27–41, 1996.
BERGLAND, A. M. AND BROWN, M. C. Central trajectories of type II spiral
ganglion cells from various cochlear regions in mice. Hear. Res. 75:
121–130, 1994.
BLACKBURN, C. C. AND SACHS, M. B. Classification of unit types in the
anteroventral cochlear nucleus: PST histograms and regularity analysis.
J. Neurophysiol. 62: 1303–1329, 1989.
BRAWER, J. R., MOREST, D. K., AND KANE, E. C. The neuronal architecture
of the cochlear nucleus of the cat. J. Comp. Neurol. 155: 251–300, 1974.
BROWN, M. C., BERGLUND, A. M., KIANG, N.Y.S., AND RYUGO, D. K. Central trajectories of type II spiral ganglion neurons. J. Comp. Neurol. 278:
581–590, 1988.
CANT, N. B. The fine structure of two types of stellate cells in the anterior
division of the anteroventral cochlear nucleus of the cat. Neuroscience
6: 2643–2655, 1981.
CANT, N. B. Identification of cell types in the anteroventral cochlear nucleus
that project to the inferior colliculus. Neurosci. Lett. 332: 241–246, 1982.
CANT, N. B. AND GASTON, K. C. Pathways connecting the right and left
cochlear nuclei. J. Comp. Neurol. 212: 313–326, 1982.
CASPARY, D. M., BACKOFF, P. M., FINLAYSON, P. G., AND PALOMBI, P. S.
Inhibitory inputs modulate discharge rate within frequency receptive
9k23
/ 9k22$$de05 J294-7
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We are grateful to I. Siggelkow, J.A. Ekleberry, and J. Meister for flawless histological processing and to P. Heinritz for administrative support. We
also are indebted to J. M. Wotton for advice on statistics and to colleagues of
the Friday morning ‘‘Hearing and Donuts’’ for comments on this work.
This study was supported by National Institute of Deafness and Other
Communications Disorders Grant DC-00176.
Address for reprint requests: D. Oertel, Dept. of Neurophysiology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI
53706-1532.
fields of anteroventral cochlear nucleus neurons. J. Neurophysiol. 72:
2124–2133, 1994.
EHRET, G. Quantitative analysis of nerve fibre densities in the cochlea of
the house mouse (Mus musculus). J. Comp. Neurol. 183: 73–88, 1979.
EVANS, E. F. AND NELSON, P. G. The responses of single neurones in the
cochlear nucleus of the cat as a function of their location and the anaesthetic state. Exp. Brain Res. 17: 402–427, 1973.
EVANS, E. F. AND ZHAO W. Varieties of inhibition in the processing and
control of processing in the mammalian cochlear nucleus. Prog. Brain
Res. 97: 117–126, 1993.
EVANS, E. F. AND ZHAO W. Onset units in guinea pig ventral cochlear
nucleus: neuropharmacological studies (Abstract). Assoc. Res. Otolaryngol. 20: 116, 1997.
FERRAGAMO, M. J., GOLDING, N. L., GARDNER, S. M., AND OERTEL, D.
Golgi cells in the superficial granule cell domain over the VCN (Abstract). Assoc. Res. Otolaryngol. 20: 44, 1997.
FERRAGAMO, M. J., GOLDING, N. L., AND OERTEL, D. A possible ventral
cochlear nucleus source of inhibition upon T-stellate cells. Soc. Neurosci.
Abstr. 22: 647, 1996.
FRISINA, R. D., SMITH, R. L., AND CHAMBERLAIN, S. C. Encoding of amplitude modulation in the gerbil cochlear nucleus. I. A hierarchy of enhancement. Hear. Res. 44: 99–122, 1990.
GOLDING, N. L. AND OERTEL, D. Context-dependent action of glycinergic
and GABAergic inputs in the dorsal cochlear nucleus. J. Neurosci. 16:
2208–2219, 1996.
GOLDING, N. L. AND OERTEL, D. Physiological identification of the targets
of cartwheel cells in the dorsal cochlear nucleus J. Neurophysiol. 78:
248–260, 1997.
GOLDING, N. L., ROBERTSON, D., AND OERTEL, D. Recordings from slices
indicate that octopus cells of the cochlear nucleus detect coincident firing
of auditory nerve fibers with temporal precision. J. Neurosci. 15: 3138–
3153, 1995.
GREENBERG, S. AND RHODE, W. S. Periodicity coding in cochlear nerve and
ventral cochlear nucleus. In: Auditory Processing of Complex Sounds,
edited by W. A. Yost and C. S. Watson. Hillsdale, NJ: Erlbaum, 1987,
p. 225–236.
HONORÉ, T., DAVIES, S. N., DREJER, J., FLETCHER, E. J., JACOBSEN, P.,
LODGE, D., AND NIELSEN, F. E. Quinoxalinediones: potent competitive
non-NMDA glutamate receptor antagonists. Science 241: 701–703, 1988.
KIANG, N.Y.S., RHO, J. M., NORTHROP, C. C., LIBERMAN, M. C., AND RYUGO, D. K. Hair-cell innervation by spiral ganglion cells in adult cats.
Science 217: 175–177, 1982.
KOLSTON, J., OSEN, K. K., HACKNEY, C. M., OTTERSEN, O. P., AND STORMMATHISEN, J. An atlas of glycine- and GABA-like immunoreactivity and
colocalization in the cochlear nuclear complex of the guinea pig. Anat.
Embryol. (Berl.) 186: 443–465, 1992.
LIBERMAN, M. C. Central projections of auditory-nerve fibers of differing
spontaneous rate. I. Anteroventral cochlear nucleus. J. Comp. Neurol.
313: 240–258, 1991.
LIBERMAN, M. C. Central projections of auditory nerve fibers of differing
spontaneous rate. II. Posteroventral and dorsal cochlear nuclei. J. Comp.
Neurol. 327: 17–36, 1993.
MOLNAR, C. E. AND PFEIFFER, R. R. Interpretation of spontaneous spike
discharge patterns of neurons in the cochlear nucleus. Proc. IEEE 56:
993–1004, 1968.
MOORE, J. K. AND MOORE, R. Y. Glutamic acid decarboxylase-like immunoreactivity in brainstem auditory nuclei of the rat. J. Comp. Neurol.
260: 157–174, 1987.
MUGNAINI, E. GABA neurons in the superficial layers of the rat dorsal
cochlear nucleus: light and electron microscopic immunocytochemistry.
J. Comp. Neurol. 235: 61–81, 1985.
NOWAK, L., BREGETOVSKI, P., ASCHER, P., HERBET, P., AND PROCHIANTZ,
A. Magnesium gates glutamate-activated channels in mouse central neurones. Nature 307: 462–465, 1984.
OERTEL, D. Synaptic responses and electrical properties of cells in brain
slices of the mouse anteroventral cochlear nucleus. J. Neurosci. 3: 2043–
2053, 1983.
OERTEL, D. Use of brain slices in the study of the auditory system: spatial
and temporal summation of synaptic inputs in cells in the anteroventral
cochlear nucleus of the mouse. J. Acoust. Soc. Am. 78: 328–333, 1985.
OERTEL, D. AND WICKESBERG, R. E. Glycinergic inhibition in the cochlear
nuclei: evidence for tuberculoventral neurons being glycinergic. In: The
Mammalian Cochlear Nuclei: Organization and Function, edited by
SYNAPTIC INPUTS TO VCN MULTIPOLAR CELLS
9k23
/ 9k22$$de05 J294-7
SMITH, P. H. AND RHODE, W. S. Structural and functional properties distinguish two types of multipolar cells in the ventral cochlear nucleus. J.
Comp. Neurol. 282: 595–616, 1989.
TOLBERT, L. P. AND MOREST, D. K. The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve
root: electron microscopy. Neuroscience 7: 3053–3067, 1982.
WANG, X. AND SACHS, M. B. Neural encoding of single-formant stimuli in
the cat. II. Responses of anteroventral cochlear nucleus units. J. Neurophysiol. 71: 59–78, 1994.
WANG, X. AND SACHS, M. B. Transformation of temporal discharge patterns
in a ventral cochlear nucleus stellate cell model: implications for physiological mechanisms. J. Neurophysiol. 73: 1600–1616, 1995.
WENTHOLD, R. J. Glutamate and aspartate as neurotransmitters of the auditory nerve. In: Auditory Biochemistry, edited by D. G. Drescher. Springfield, IL: Charles C. Thomas, 1985, pp. 125–140.
WENTHOLD, R. J. Evidence for a glycinergic pathway connecting the two
cochlear nuclei: an immunocytochemical and retrograde transport study.
Brain Res. 415: 183–187, 1987.
WENTHOLD, R. J., HUIE, D., ALTSCHULER, R. A., AND REEKS, K. A. Glycine
immunoreactivity localized in the cochlear nucleus and superior olivary
complex. Neuroscience 22: 897–912, 1987.
WENTHOLD, R. J. AND HUNTER, C. Immunocytochemistry of glycine and
glycine receptors in the central auditory system. In: Glycine Neurotransmission, edited by O. P. Ottersen and J. Storm-Mathisen. New York:
Wiley, 1990, p. 392–416.
WENTHOLD, R. J., ZEMPEL, J. M., PARAKK AL, M. H., REEKS, K. A., AND
ALTSCHULER, R. A. Immunocytochemical localization of GABA in the
cochlear nucleus of the guinea pig. Brain Res. 380: 7–18, 1986.
WHITE, J. A., YOUNG, E. D., AND MANIS, P. B. The electrotonic structure
of regular-spiking neurons in the ventral cochlear nucleus may determine
their response properties. J. Neurophysiol. 71: 1774–1786, 1994.
WICKESBERG, R. E. Rapid inhibition in the cochlear nuclear complex of the
chinchilla. J. Acoust. Soc. Am. 100: 1691–1702, 1996.
WICKESBERG, R. E. AND OERTEL, D. Tonotopic projection from the dorsal
to the anteroventral cochlear nucleus of mice. J. Comp. Neurol. 268:
389–399, 1988.
WICKESBERG, R. E. AND OERTEL, D. Delayed, frequency-specific inhibition
in the cochlear nuclei of mice: a mechanism for monaural echo suppression. J. Neurosci. 10: 1762–1768, 1990.
WICKESBERG, R. E., WHITLON, D., AND OERTEL, D. In vitro modulation of
somatic glycine-like immunoreactivity in presumed glycinergic neurons.
J. Comp. Neurol. 339: 311–327, 1994.
WU, S. H. AND OERTEL, D. Intracellular injection with horseradish peroxidase of physiologically characterized stellate and bushy cells in slices of
mouse anteroventral cochlear nucleus. J. Neurosci. 4: 1577–1588, 1984.
WU, S. H. AND OERTEL, D. Inhibitory circuitry in the ventral cochlear
nucleus is probably mediated by glycine. J. Neurosci. 6: 2691–2706,
1986.
ZHANG, S. AND OERTEL, D. Cartwheel and superficial stellate cells of the
dorsal cochlear nucleus of mice: intracellular recordings in slices. J.
Neurophysiol. 69: 1384–1397, 1993.
ZHANG, S. AND TRUSSELL, T. O. A characterization of excitatory postsynaptic potentials in the avian nucleus magnocellularis. J. Neurophysiol. 72:
705–718, 1994.
12-08-97 08:35:37
neupa
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.33.4 on April 29, 2017
M. A. Merchan, J. J. Juiz, D. A. Godfrey, and E. Mugnaini. New York:
Plenum, 1993, p. 225–238.
OERTEL, D. AND WU, S. H. Morphology and physiology of cells in slice
preparations of the dorsal cochlear nucleus of mice. J. Comp. Neurol.
283: 228–247, 1989.
OERTEL, D., WU, S. H., GARB, M. W., AND DIZACK, C. Morphology and
physiology of cells in slice preparations of the posteroventral cochlear
nucleus of mice. J. Comp. Neurol. 295: 136–154, 1990.
OLIVER, D. L. Dorsal cochlear nucleus projections to the inferior colliculus
in the cat: a light and electron microscopic study. J. Comp. Neurol. 224:
155–172, 1984.
OLIVER, D. L. Projections to the inferior colliculus from the anteroventral
cochlear nucleus in the cat: possible substrates for binaural interaction.
J. Comp. Neurol. 264: 24–46, 1987.
OSEN, K. K. Cytoarchitecture of the cochlear nuclei in the cat. J. Comp.
Neurol. 136: 453–484, 1969.
OSEN, K. K. Projection of the cochlear nuclei on the inferior colliculus in
the cat. J. Comp. Neurol. 144: 355–372, 1972.
OSEN, K. K., OTTERSEN, O. P., AND STORM-MATHISEN, J. Colocalization of
glycine-like and GABA-like immunoreactivities: a semiquantitative study
of individual neurons in the dorsal cochlear nucleus of cat. In: Glycine
Neurotransmission, edited by O. P. Ottersen and J. Storm-Mathisen. New
York: Wiley, 1990, p. 417–451.
PALOMBI, P. S. AND CASPARY, D. M. GABAa receptor antagonist bicuculline
alters response properties of posteroventral cochlear nucleus neurons. J.
Neurophysiol. 67: 738–746, 1992.
RAMAN, I. M., ZHANG, S., AND TRUSSELL, L. O. Pathway-specific variants
of AMPA receptors and their contribution to neuronal signaling. J. Neurosci. 14: 4998–5010, 1994.
RHODE, W. S. AND GREENBERG, S. Encoding of amplitude modulation in
the cochlear nucleus of the cat. J. Neurophysiol. 71: 1797–1825, 1994.
RHODE, W. S. AND SMITH, P. H. Encoding timing and intensity in the ventral
cochlear nucleus of the cat. J. Neurophysiol. 56: 261–286, 1986.
ROTH, G. L., AITKIN, L. M., ANDERSEN, R. A., AND MERZENICH, M. M.
Some features of the spatial organization of the central nucleus of the
inferior colliculus of the cat. J. Comp. Neurol. 182: 661–680, 1978.
RYUGO, D. K., WILLARD, F. H., AND FEKETE, D. M. Differential afferent
projections to the inferior colliculus from the cochlear nucleus in the
albino mouse. Brain Res. 210: 342–349, 1981.
SAINT MARIE, R. L., BENSON, C. G., OSTAPOFF, E.-M., AND MOREST, D. K.
Glycine immunoreactive projections from the dorsal to the anteroventral
cochlear nucleus. Hear. Res. 51: 11–28, 1991.
SAINT MARIE, R. L., OSTAPOFF, E. M., MOREST, D. K., AND WENTHOLD,
R. J. Glycine-immunoreactive projection of the cat lateral superior olive:
possible role in midbrain ear dominance. J. Comp. Neurol. 279: 382–
396, 1989.
SCHOFIELD, B. R. AND CANT, N. B. Origins and targets of commissural
connections between the cochlear nuclei in guinea pigs. J. Comp. Neurol.
375: 128–146, 1996.
SHOFNER, W. P. AND YOUNG, E. D. Excitatory/Inhibitory response types in
the cochlear nucleus: relationships to discharge patterns and responses
to electrical stimulation of the auditory nerve. J. Neurophysiol. 54: 918–
939, 1985.
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