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The Journal of Neurophysiology Vol. 79 No. 1 January 1998, pp. 51-63
Copyright ©1998 by the American Physiological Society
Department of Neurophysiology, University of Wisconsin Medical School, Madison, Wisconsin 53706-1532
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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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-dinitroquinoxaline-2,3-dione (DNQX), an antagonist of 
-amino-3-hydroxy-5-methyl-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 Mg2+, indicating that the interneurons were driven at least in part through NMDA receptors. Bicuculline, a 
-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.
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 Tissue preparation
The cochlear nuclei were obtained from 18- to 26-day-old CBA or ICR mice as described previously (Golding and Oertel 1996 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 M 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 4°C 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 µm 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 Co2+ and Ni2+ intensification. Sections mounted on coated slides were conterstained with cresyl violet.
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) 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 Monosynaptic EPSPs reflect the convergence of few inputs from the auditory nerve
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 = 0.70 ± 0.13, n = 53). These latencies indicate that the EPSPs were monosynaptic and therefore that they reflect direct input from the auditory nerve.
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 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.
Late EPSPs reveal the existence of excitatory interneurons
A single shock commonly evoked occasional unitary EPSPs
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
Trains of late IPSPs
Strong shocks to the auditory nerve also evoked trains of late IPSPs that in some cases lasted for >500 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 not be resolved. As other synaptic responses subsided, the trains of regularly occurring IPSPs emerged, lasting between 50 and 600 ms.
Monosynaptic and late EPSPs are glutamatergic
In the presence of Mg2+, all synaptic responses in 5 of 5 cells to stimulation of the auditory nerve were blocked by 10 µM DNQX, a blocker of
Long, slow depolarization in T stellate cells is mediated through NMDA receptors
Application of 100 µM 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.
Disynaptic and late IPSPs are glycinergic
Consistent with earlier findings, all early and late IPSPs were blocked by 0.5 or 1 µM strychnine, indicating that they were glycinergic (Wu and Oertel 1986
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 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 Mg2+ and their action was blocked by 100 µM 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 Mg2+, 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 Mg2+ 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.
D stellate cells
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 Synaptic responses
Figure 12 displays responses of a D stellate cell to shocks delivered to the auditory nerve. Weak shocks evoked a subthreshold 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.
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 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 ~5% of the total (cats: Kiang et al. 1982 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 ~3 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 >3 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.
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 GABAAergic 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 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
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 onset-choppers, respectively (Oertel et al. 1990
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
rapezoid 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).
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 
orsalward 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).
). 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).
). 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 Wickesberg 1993; Wenthold 1987; Wickesberg et al. 1994). Such labeling stains <0.1 of cells in the dorsocaudal AVCN and PVCN. In cats, too, corresponding cells represent a small fraction of stellate cells (Cant 1981).
). We suggest that T stellate cells integrate input from the auditory nerve with input from other T and D stellate cells.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; 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-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid, 20 NaHCO3, and 10 glucose, pH 7.4, 31°C. 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 µm 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 34°C 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.
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 a 50-µm exposed tip. Stimulation voltage (0.1-100 V; 100-µs 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).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
. The resting potentials ofT stellate cells ranged from 
51 to 68 mV [mean = 56.7 ± 4.9 (SD)]. Input resistance, measured from the magnitude of the response to injection of 0.1 nA current, ranged from 44 to 151 M (mean = 89.4 ± 24.4). The average resting potential and input resistance for D stellate cells were 57 ± 4.2 mV and 96.2 ± 27.8 M, respectively.
). 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 narrowband (50-75 µm) spanning the fusiform cell layer from its most rostral to its most caudal end.
; 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.
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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) = 1,293.3, P < 0.001; amplitude, 4 clusters, F(3,43) = 335.9, P < 0.001; B slope, 5 clusters, F(4,52) = 1,378.2, P < 0.001; amplitude, 4 clusters, F(3,38) = 354.6, P < 0.001. The bath contained 1 µM strychnine to avoid distortion of EPSPs by IPSPs. The cell in B was hyperpolarized with a 0.1-nA current pulse during synaptic stimulation to enhance resolution of subthreshold events.
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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.
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.
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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 µM strychnine (STR) and 10 µM 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).
). 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.
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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.
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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.
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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.
; 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 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 = 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 inthe VCN.
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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.
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic 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.
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FIG. 8.
Excitation was mediated by -amino-3-hydroxy-5-methyl-4-isoxazolepropionic 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 (0.05 and 0.1 nA; 300 ms) during synaptic stimulation (+1 V) shortened the depolarization, showing that some NMDA currents were intrinsic to T stellate cells.
). 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 Mg2+ (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.
). 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 µM strychnine blocked all IPSPs and tipped the balance toward excitation, so that each shock evoked a spike.
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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-to-one discharge to each shock as well as firing after the shock train.
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FIG. 10.
Removal of Mg2+ 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 Mg2+ 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 Mg2+ 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.
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FIG. 11.
GABAergic inhibition was subtle in T stellate cells. Bicuculline (BIC) promoted IPSPs ( 
), 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.
). 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.
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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 ( ) occasionally occurred in short bursts. Late EPSPs () often resulted in discharges.
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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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FIG. 14.
Blocking the GABAA receptor with picrotoxin (PIC) prolonged the firing in one D stellate cell, whereas strychnine (STR) had little effect.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; 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 present experiments show that, in addition, T stellate cells are a possible source of feed-forward 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.
; 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 onset-chopper 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 <1 ms, the input is probably from myelinated auditory nerve fibers.
). 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).
; 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.
; 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 ultrastructure 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).
).
). 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).
). Although there is no doubt that tuberculoventral cells contribute to the disynaptic IPSPs, several experimental 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).
; 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.
; 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, superficial stellate or Golgi cells of the DCN contribute to GABAergic inhibition in T stellate cells of the VCN.
). 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.
). The topographic arrangement of tuberculoventral cells indicates that roughly 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).
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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.
; 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).
). 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).
) 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, choppers do respond with onset transients away from the characteristic frequency (W. S. Rhode, personal communication).
). 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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Address for reprint requests: D. Oertel, Dept. of Neurophysiology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706-1532.
Received 11 April 1997; accepted in final form 5 August 1997.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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