Department of Neurophysiology, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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Golding, Nace L. and Donata Oertel. Physiological identification of the targets of cartwheel cells in the dorsal cochlear nucleus. J. Neurophysiol. 78: 248-260, 1997. The integrative contribution of cartwheel cells of the dorsal cochlear nucleus (DCN) was assessed with intracellular recordings from anatomically identified cells. Recordings were made, in slices of the cochlear nuclei of mice, from 58 cartwheel cells, 22 fusiform cells, 3 giant cells, 5 tuberculoventral cells, and 1 cell that is either a superficial stellate or Golgi cell. Cartwheel cells can be distinguished electrophysiologically from other cells of the cochlear nuclei by their complex spikes, which comprised two to four rapid action potentials superimposed on a slower depolarization. The rapid action potentials were blocked by tetrodotoxin (n = 17) and were therefore mediated by voltage-sensitive sodium currents. The slow spikes were eliminated by the removal of calcium from the extracellular saline (n = 3) and thus were mediated by voltage-sensitive calcium currents. The spontaneous and evoked firing patterns of cartwheel cells were distinctive. Cartwheel cells usually fired single and complex spikes spontaneously at irregular intervals of between 100 ms and several seconds. Shocks to the DCN elicited firing that lasted tens to hundreds of milliseconds. With the use of these distinctive firing patterns, together with a pharmacological dissection of postsynaptic potentials (PSPs), possible targets of cartwheel cells were identified and the function of the connections was examined. Not only cartwheel and fusiform cells, but also giant cells, received patterns of synaptic input consistent with their having originated from cartwheel cells. These cell types responded to shocks of the DCN with variable trains of PSPs that lasted hundreds of milliseconds. PSPs within these trains appeared both singly and in bursts of two to four, and were blocked by 0.5 or 1 µM strychnine (n = 4 cartwheel, 4 fusiform, and 2 giant cells), indicating that cartwheel cells are likely to be glycinergic. In contrast with cartwheel cells, which are weakly excited by glycinergic input, glycinergic PSPs consistently inhibited fusiform and giant cells. Tuberculoventral cells and the putative superficial stellate cell received little or no spontaneous synaptic activity. Shocks to the DCN evoked synaptic activity that lasted ~5 ms. These cells therefore probably do not receive input from cartwheel cells. In addition, the brief firing of tuberculoventral cells and of the putative superficial stellate cell in response to shocks indicates that these cells are unlikely to contribute to the late, glycinergic synaptic potentials observed in cartwheel, fusiform, and giant cells.
The cartwheel cells of the dorsal cochlear nucleus (DCN) are interneurons that are known to contact other cartwheel as well as fusiform cells, the main projecting neurons of the DCN (Berrebi and Mugnaini 1991 Slice preparation and intracellular recordings
Parasagittal slices of the cochlear nuclei were cut from the brain stems of 18- to 26-day-old mice as described previously (Golding and Oertel 1996 Morphological identification of cells
Biocytin was injected into recorded cells with 0.5- to 2-nA depolarizing current pulses 100 ms in duration delivered at a rate of 2.5 pulses per second for up to 5 min. Slices were fixed in 4% paraformaldehyde and stored refrigerated for 1-21 days. Slices were then embedded in a mixture of gelatin and albumin, sectioned at 60 µm, and processed for biocytin histochemistry with the use of the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) in conjunction with Co2+ and Ni2+ intensification (Adams 1981 The conclusions of the present study are based on recordings of 58 cartwheel cells, 22 fusiform cells, 3 giant cells, 5 tuberculoventral cells, and 1 putative superficial stellate cell, all of which were anatomically identified. The resting potentials and input resistances of cells, as well as the duration of recordings, are summarized in Table 1.
Complex spikes in cartwheel cells
The ability to generate complex spikes distinguishes cartwheel cells from all other cell types in the cochlear nuclei thus far recorded. Complex spikes consist of a burst of fast, large action potentials superimposed on a slower, smaller depolarization (Manis et al. 1994
Synaptic inputs to cartwheel, fusiform, and giant cells mirror the firing of cartwheel cells
Spontaneous synaptic activity in the targets of cartwheel cells should reflect the temporal pattern of the spontaneous firing of cartwheel cells. Cartwheel cells receive spontaneous depolarizing PSPs that may lead to spontaneous firing of simple and complex spikes at rates generally <10 Hz. Complex spikes presumably lead to bursts of synaptic potentials in the targets of cartwheel cells. Three cell types received spontaneous bursts of PSPs: cartwheel, fusiform, and giant cells. Figure 3 provides a comparison of the timing of spontaneous complex spikes in three cartwheel cells with the timing of spontaneous bursts of PSPs in six cartwheel, six fusiform, and three giant cells. The timing of the clusters of PSPs is remarkably similar despite the fact that they were recorded from different cell types in different preparations. The interval between the peaks of PSPs within a burst varied within a given cell, as did the interspike intervals of fast action potentials within the complex spikes of cartwheel cells. In cartwheel cells, the complex spikes can themselves be triggered by bursts of PSPs (Fig. 3, top left and top right traces), consistent with the earlier conclusion that input from cartwheel cells to other cartwheel cells is weakly excitatory at membrane potentials near rest (Golding and Oertel 1996 Synaptic inputs to putative superficial stellate and tuberculoventral cells
In contrast to cartwheel, fusiform, and giant cells, the putative superficial stellate and tuberculoventral cells showed neither spontaneous synaptic activity (Fig. 8A) nor late synaptic activity in responses to shocks of the DCN (Fig. 8B). In the putative superficial stellate cell, neither 1 µM strychnine nor 100 µM picrotoxin noticeably affected the cell's synaptic responses (not shown). Although inhibition was not detected in the synaptic responses of the tuberculoventral cell in Fig. 8B, strychnine-sensitive IPSPs were observed in two other cells. However, these IPSPs occurred no later than 8 ms after the shock in both cases. These results indicate that the putative superficial stellate and tuberculoventral cells are unlikely to serve as targets of cartwheel cells.
Cartwheel cells could be the source of late, glycinergic PSPs in cartwheel, fusiform, and giant cells
Cartwheel, superficial stellate, and tuberculoventral cells are likely to be inhibitory interneurons and thus serve as a potential source of inhibition for other cells. A comparison of the timing of firing of these potential sources of PSPs (rasters) with potential targets (traces) is presented in Fig. 9. Each trace and each line in the raster were recorded from a separate cell. To be comparable, each response was chosen to be the longest of those recorded in that cell. The traces from the three cartwheel cells were recorded while the cells were hyperpolarized with current to reveal subthreshold PSPs. The raster plot below shows firing after a single shock in cartwheel cells, tuberculoventral cells, and the putative superficial stellate cell. The filled diamonds within each row represent the timing of action potentials within the longest response recorded in one cell. Although cartwheel cells, without exception, fired trains of spikes lasting tens to hundreds of milliseconds, the tuberculoventral and putative stellate cells fired a single action potential that occurred no later than 5 ms after the stimulus. Although the population of tuberculoventral cells and putative superficial stellate cells sampled is considerably smaller than that of cartwheel cells, these findings are consistent with a previous study in which the temporal responses of these cell types to shocks of the auditory nerve were analyzed (Zhang and Oertel 1994 Morphology of DCN neurons
Examples are shown in Figs. 10-12 of cartwheel, putative superficial stellate, fusiform, giant, and tuberculoventral cells that were recorded, labeled with biocytin, and reconstructed with a camera lucida. The dendrites are the potential targets and the terminal arbors the potential sources of some of the connections that were examined electrophysiologically above.
We conclude that the major integrative contribution of the superficial layers of the DCN to principal cells is inhibitory and primarily mediated by cartwheel cells. The cartwheel cells' distinctive patterns of simple and complex spikes are reflected in the patterns of glycinergic PSPs in cartwheel, fusiform, and giant cells. Glycinergic PSPs are inhibitory in fusiform and giant cells, but are depolarizing in cartwheel cells. Depolarizing, glycinergic PSPs can excite cartwheel cells to threshold while inhibiting other targets with the same neurotransmitter (Golding and Oertel 1996 Complex spikes in cartwheel cells
Cartwheel cells are the only cells in the cochlear nuclei known to fire complex spikes. Complex spikes occur spontaneously as well as in synaptic responses to shocks of the auditory nerve, VCN, and DCN, and in responses to depolarizing current injected through the recording electrode (Golding and Oertel 1996 Glycinergic synaptic interactions between cartwheel cells
Connections among cartwheel cells have been demonstrated anatomically (Berrebi and Mugnaini 1991 Feedforward inhibition of fusiform and giant cells by cartwheel cells
The present results support the conclusion that cartwheel cells inhibit fusiform cells (Berrebi and Mugnaini 1991 Superficial stellate and Golgi cells
The superficial stellate and Golgi cells reside in the molecular layer and could potentially provide feedforward inhibition onto fusiform and giant cells (Osen et al. 1990 Other inhibitory interneurons
Tuberculoventral cells are glycinergic interneurons that likely contact fusiform and giant cells (Oertel and Wickesberg 1993 Functional significance of the circuitry in the DCN
In considering these results in the context of the DCN as a whole it is useful to think of neurons not just singly but in arrays (Fig. 13). The principal cells combine information from two anatomically and functionally distinct neuronal circuits, one conveying multimodal sensory information through the molecular layer and one conveying tonotopic acoustic information through the deep layer. In the molecular layer, the network of cartwheel cells is excited by an array of glutamatergic parallel fibers (Manis 1989
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Mugnaini et al. 1987
). Fusiform cells are in addition innervated tonotopically by auditory nerve fibers. The cartwheel cells thus form a network of interconnected cells that lies poised to influence the topographic array of fusiform cells as it transmits acoustic information to the contralateral inferior colliculus (Adams 1979
; Oliver 1984
; Ryugo et al. 1981
).
; Kane 1974
; Manis 1989
; Mugnaini et al. 1980a
,b
; Wouterlood and Mugnaini 1984
; Wouterlood et al. 1984
). The granule cells receive afferents from the cochlea, cochlear nucleus, superior olivary complex, inferior colliculus, auditory cortex, dorsal column nuclei, saccule, and vestibular nerve root(Berglund and Brown 1994
; Brown and Ledwith 1990
; Brown et al. 1988a
,b
; Burian and Gestoettner 1988
; Caicedo and Herbert 1993
; Feliciano et al. 1993
; Golding et al. 1995
; Itoh et al. 1987
; Kevetter and Perachio 1989
; Spangler et al. 1987
; Weedman and Ryugo 1996
; Weedman et al. 1996
; Weinberg and Rustioni 1987
; Wright and Ryugo 1996
; Zhao et al. 1995
). Any tonotopic arrangement of auditory inputs to granule cells ultimately is disrupted, because the trajectory of the parallel fibers is orthogonal to the isofrequency laminae of the DCN. The parallel fibers are thus in a position to spread excitation to cartwheel cells that lie over wide expanses of the tonotopic axis.
; Ding and Voigt 1996
; Parham and Kim 1995
). Cartwheel cells are also driven by electrical activation of somatosensory inputs from the dorsal column nuclei (Davis et al. 1996
), and are likely to participate in the inhibition of principal cells (fusiform and/or giant cells) mediated by these same stimuli (Young et al. 1995
).
). Because the reversal potential for glycinergic and GABAergic postsynaptic potentials (PSPs) was only slightly above threshold (about
53 mV), however, these PSPs could also suppress rapid firing during strong membrane depolarizations. By contrast, glycinergic and GABAergic inputs to fusiform cells are conventionally inhibitory, with reversal potentials of about
68 mV (Golding and Oertel 1996
; Zhang and Oertel 1994
).
). By correlating the distinctive temporal firing patterns of cartwheel cells with corresponding patterns of synaptic activity in putative targets, we show that not only cartwheel and fusiform cells but also giant cells receive glycinergic PSPs that could have arisen from cartwheel cells. The present results, together with those of prior studies, indicate that cartwheel cells, using a single neurotransmitter, have different synaptic actions on principal cells and other cartwheel cells (Berrebi and Mugnaini 1991
; Golding and Oertel 1996
; Zhang and Oertel 1994
).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Mice were decapitated and dissected under oxygenated (95% O2-5% CO2) normal saline at ~31°C. The saline contained (in mM) 130 NaCl, 3 KCl, 1.3 MgSO4, 2.4 CaCl2, 20 NaHCO3, 3 N-2-hydroxyethylpiperazine-N
-2-ethanesulfonic acid, 10 glucose, and 1.2 KH2PO4, pH 7.4. Slices were cut at ~300 µm thickness with an oscillating tissue slicer (Frederick Haer, New Brunswick, ME). The presence of the underlying restiform body on the medial face of the slice indicated that most of the circuitry remained intact in these slices. A slice was allowed to recover for
1 h in the recording chamber, where it was continuously superfused with saline at a rate of 9-12 ml/min and maintained at 34°C.
. Voltages were recorded by a high-impedance amplifier (Dagan IX2-700, Minneapolis, MN) and filtered at 10 kHz. Membrane potentials were continuously monitored on a chart recorder. Data acquisition and analysis were performed with the use of a Digidata 1200 interface in conjunction with pClamp software (Axon Instruments, Foster City, CA).
-aminobutyric acid-A (GABAA), N-methyl-D-aspartate, and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. In some experiments calcium was nominally eliminated by exchanging CaCl2 for MgSO4.
). Sections were mounted on coated slides and counterstained with cresyl violet. Reconstructions were made with a camera lucida.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
TABLE 1.
Summary of characteristics of intracellularly recorded cells
; Zhang and Oertel 1993a
), and were common both in the spontaneous activity and in responses to shocks. Figure 1A shows the characteristic simple and complex spikes fired by a cartwheel cell in response to depolarizing current pulses. The ionic basis of complex spikes was explored in experiments in which voltage-sensitive sodium and calcium currents were successively eliminated (Fig. 1B). The fast, large action potentials of complex spikes that were evoked with current pulses were sodium dependent, because they were reversibly blocked by TTX, an antagonist of voltage-gated sodium channels (n = 17). The slower, smaller action potential was calcium dependent, because it was reversibly eliminated by the removal of calcium from the bathing medium (n = 3).
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FIG. 1.
Simple and complex spikes in cartwheel cells. A: response of a cartwheel cell to a series of depolarizing current pulses comprising both simple and complex spikes. Cartwheel cells responded to depolarization with slow action potentials that triggered bursts of fast action potentials (complex spikes) as well as with rapid, all-or-none action potentials (simple spikes). Frequency of simple and complex spikes increased with magnitude of current pulse. B: ionic basis of complex and simple spikes. Cartwheel cell responded to a 0.4-nA depolarizing current pulse with a complex spike followed by a train of simple spikes. Large, fast action potentials were eliminated by tetrodotoxin (TTX), a blocker of voltage-gated sodium channels, indicating that they were sodium dependent. Smaller, slower action potential that remained was abolished when calcium was removed from bath.
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FIG. 2.
Synaptic responses of a cartwheel cell to shocks of its inputs. Each group of traces shows responses to identical shocks whose timing is indicated by arrows and whose strength is indicated in V. Rate of rise of initial excitation and number of postsynaptic potentials (PSPs) increased with shock strength, indicating that additional inputs were recruited. Synaptic responses evoked both simple and complex spikes whose rate and timing of firing were variable even in responses to identical shocks. Resting potential of cartwheel cell: 65 mV. Stimulating electrodes were placed onto the lateral surface of the VCN to stimulate the superficial granule cells.
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FIG. 3.
Comparison of complex spikes in cartwheel cells and bursts of PSPs in cartwheel, fusiform, and giant cells. Complex spikes shown in top row of traces (C) occurred spontaneously in each cartwheel cell. Leftmost and rightmost complex spikes were triggered by bursts of spontaneous PSPs. Bottom rows of traces: spontaneous bursts of PSPs; each trace was recorded from a separate cell in a separate slice. PSPs were depolarizing in cartwheel cells (C) and hyperpolarizing in fusiform (F) and giant (G) cells. Slow rates of rise and rounded shapes of inhibitory PSPs (IPSPs) in the leftmost and rightmost traces of the giant cells are suggestive of dendritic filtering. Resting potential is indicated at beginning of each trace.
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FIG. 4.
Spontaneous IPSPs in giant cells show evidence of dendritic filtering. A: spontaneous IPSPs in a giant cell show heterogeneity in their shapes and durations. Whereas bursts of individual IPSPs are clearly resolved within some faster IPSPs (e.g., bottom trace), they are more difficult to distinguish from single synaptic events in the more slowly rising IPSPs (e.g., 3rd and 4th traces). Resting potential: 56 mV. Dotted line: beginning of each spontaneous PSP. B: spontaneous IPSPs in a different giant cell also show heterogeneity in their temporal patterns and in their rates of rise and decay. Morphology of this cell is presented in Fig. 11B. Resting potential:
53 mV.
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FIG. 5.
Spontaneous synaptic activity in cartwheel, fusiform, and giant cells is largely glycinergic. Each of these cells received spontaneous PSPs that occurred singly as well as in bursts of summing PSPs (a few are indicated by *). Much of this spontaneous activity remained in the presence of the glutamatergic antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and D,L-2-amino-5-phosphonovaleric acid (APV). The remainder of spontaneous activity was eliminated by strychnine (STR), indicating that it was glycinergic. Recorded cells were in different slices. Concentration of antagonists: DNQX, 40 µM; APV, 100 µM; strychnine, 0.5 or 1 µM.
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FIG. 6.
Late PSPs in responses to shocks of dorsal cochlear nucleus (DCN) in cartwheel, fusiform, and giant cells are at least in part glycinergic. In a cartwheel cell (C), shocks gave rise to a train of simple spikes lasting 200 ms. In the presence of strychnine, the same stimulus evoked a complex spike followed by a subthreshold depolarization. In a different cartwheel cell (C*), a DCN shock given while the cell was hyperpolarized with 0.2 nA current evoked a series of late depolarizing PSPs. These late PSPs were reversibly eliminated by strychnine. In the fusiform (F) and giant (G) cells, late IPSPs were also reversibly eliminated by strychnine. Synaptic activity was evoked with constant voltage shocks (
) to DCN in cartwheel and fusiform cells; synaptic activity was evoked from auditory nerve (
) in the giant cell. Cells were recorded from different slices. Concentration of strychnine: 0.5 or 1 µM.
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FIG. 7.
Monosynaptic glycinergic inputs to cartwheel, fusiform, and giant cells were activated by shocks to DCN. Shock to DCN evoked a train of action potentials in a cartwheel cell (C) and trains of IPSPs in a fusiform (F) and giant (G) cell that lasted 200 ms. When glutamatergic excitation was blocked with DNQX and APV, monosynaptic PSPs remained, which were depolarizing in the cartwheel cell and hyperpolarizing in the fusiform and giant cell. These residual PSPs were glycinergic, because they were eliminated by strychnine. Monosynaptic response in cartwheel cell had a GABAergic component that is not shown. In this cell a monosynaptic glycinergic PSP was isolated and identified by including the
-aminobutyric acid-A (GABAA) receptor antagonist picrotoxin at 50 µM in the DNQX/APV and DNQX/APV/strychnine solutions. Other drug concentrations: DNQX, 40 µM; APV, 100 µM; strychnine, 1 µM. Arrows: timing and strength of shocks.
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FIG. 8.
Synaptic activity in superficial stellate and tuberculoventral cells. A: spontaneous PSPs were not observed in putative superficial stellate cell (S) or tuberculoventral cell (T). B: shocks to DCN evoked action potentials within 5 ms in both putative superficial stellate and tuberculoventral cell.
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FIG. 9.
Comparison of synaptic responses evoked by shocks to DCN in potential targets of cartwheel cells with timing of firing of potential sources of glycinergic input. Traces: responses of 3 cartwheel (C), 3 fusiform (F), and 2 giant (G) cells show that late PSPs occur nearly 300 ms after shocks to DCN. Cartwheel cells were hyperpolarized with current (between 0.2 and
0.4 nA) to reveal late depolarizing PSPs in the absence of firing. Longest duration of synaptic responses recorded in each cell is represented. Raster plots: firing patterns of 18 cartwheel cells, 3 tuberculoventral cells, and 1 putative superficial stellate cell. Each row of filled diamonds represents timing of action potentials in longest of the responses recorded in 1 cell.
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FIG. 10.
Morphology of interneurons in superficial layers of DCN. A: cartwheel cell. Cell body was located close to border of molecular and fusiform cell layers. Dendrites of cell (thicker processes) were densely covered with spines. Axon (thinner processes) arborized locally and had numerous swellings in molecular and fusiform cell layers. B: putative superficial stellate cell. Cell body was located entirely in molecular layer. Dendrites were smooth and extended laterally in molecular and fusiform cell layers. Border between layers is indicated for section that contained cell body (as a consequence of the curvature of the DCN, that border moves from section to section). Axon arborized profusely in molecular and fusiform cell layers. Cell was stained lightly, making the finest branches of the axon difficult to resolve. ML, molecular layer; FCL, fusiform cell layer; GCL, granule cell lamina.
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FIG. 11.
Morphology of DCN principal cells, fusiform and giant cells. A: fusiform cell. Cell's soma was located in fusiform cell layer. Its spiny apical dendrites spanned the molecular layer, whereas its smooth basal dendrites lay in a narrow band across the deep layer, roughly parallel to isofrequency axis of DCN. Axon of cell was cut in its medial course from cell body. DL, deep layer. B: giant cell. Cell body was located in deep layer of DCN. Its dendrites extended from deep layer to molecular layer, and were not oriented along isofrequency axis of DCN. A few spines were observed on the portion of 1 dendrite that protruded into the molecular layer. Axon made 4 hairpin turns in the deep DCN, giving off collaterals in deep layer, fusiform cell layer, and granule cell lamina before being cut in its dorsomedial trajectory out of the nucleus.
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FIG. 12.
Tuberculoventral cell. Cell's soma was located in deep layer of DCN. Its dendrites were oriented rostrocaudally, parallel to isofrequency axis of DCN, and were restricted to deep and fusiform cell layers. Axon of cell gave off local collaterals in a band near cell's dendrites as well as in a secondary cluster at a more ventral location in DCN. Main axon projected to ventral cochlear nucleus (VCN), where it ended in a narrow band of terminals in both posterior and anterior subdivisions. VIII N, 8th nerve root.
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FIG. 13.
Layers of DCN allow superposition of arrays of sensory information. Array of parallel fibers excites network of cartwheel cells through glutamatergic (Glu) synapses. Spatiotemporal pattern of excitation is modified in cartwheel cells by glycinergic (Gly), context-dependent excitation or suppression. Network of cartwheel cells imposes that modified pattern on tonotopic array of fusiform cells and on giant cells in the form of inhibition.
). Some spontaneous bursts are not preceded by bursts of PSPs (Fig. 3, top middle trace).
). It seemed surprising, therefore, that despite the fact that obvious bursts of PSPs were observed in every giant cell, they were observed frequently in only one of the three examples. All three cells, however, received slow, irregular hyperpolarizations, many of which are probably bursts of PSPs smoothed by dendritic filtering. Figure 4 shows both single and bursts of spontaneous PSPs in two giant cells exhibiting a range of different rates of rise and decay. In both cells, it is difficult to distinguish whether some of the slower PSPs represent single events or bursts. The slow PSPs shown in Fig. 4A, fourth trace, and Fig. 4B, third trace, may, for example, reflect three individual PSPs, the summation of which comprises the slowly rising edge. Slower PSPs were common in the cell shown in Fig. 4A, whereas more rapid PSPs predominated in the cell shown in Fig. 4B, suggesting that the proximity of spontaneous synaptic input to the presumed somatic recording site varies across giant cells.
). When present, these PSPs occurred infrequently and singly. No spontaneous GABAergic PSPs were observed in the giant cells whose spontaneous activity was examined pharmacologically (n = 2).
; Zhang and Oertel 1993a
) or to the DCN or surface of the VCN (Golding and Oertel 1996
) (Fig. 2), cartwheel cells generate trains of action potentials that are longer than those of most cells in the cochlear nuclei. Their targets should thus receive long trains of PSPs and those PSPs should all be eliminated by a common receptor antagonist. Figure 6 shows that this is indeed the case. Late PSPs in cartwheel, fusiform, and giant cells were eliminated by strychnine, indicating that they were mediated by glycine receptors. In the cartwheel cell shown in Fig. 6C, a shock to the DCN evoked a train of action potentials that lasted 200 ms. The addition of strychnine to the bath increased the magnitude of the initial depolarization but, significantly, also abbreviated the duration of firing. It is not surprising that the removal of inhibition causes an increase of excitation, as that observed early, but the loss of late excitation is inconsistent with a direct inhibitory action of glycinergic input on cartwheel cells. It is, however, consistent with the conclusion that glycinergic inputs are excitatory in cartwheel cells (Golding and Oertel 1996
). The effects of strychnine were not reversed in every cell tested because >1 h was required to wash strychnine out of the slices. To resolve subthreshold synaptic responses in cartwheel cells, it was necessary to hyperpolarize them with current to reduce firing, as shown in Fig. 6C*. This record shows that strychnine eliminated late, depolarizing PSPs reversibly. In fusiform (Fig. 6F) and giant (Fig. 6G) cells, strychnine eliminated both early and late inhibition. In the fusiform cell, an additional GABAergic inhibitory influence was revealed by subsequent addition of 10 µM picrotoxin to a 1 µM strychnine solution (not shown).
). However, late, presumably polysynaptic, GABAergic PSPs were never observed in response to shocks.
). Cartwheel cells, but not tuberculoventral or superficial stellate cells, fire late enough after a shock to the DCN to produce the late PSPs in cartwheel, fusiform, or giant cells.
, their Fig. 9). On the other hand, the dendrites shown in Fig. 10B are less straight and the axon terminates not only in the molecular but also in the fusiform cell layer. The differences between these two cells raise the possibility that the cell shown in Fig. 10B is a Golgi cell (Mugnaini and Floris 1994
; Mugnaini et al. 1994
). The fact that the identification of Golgi and superficial stellate cells is based mainly on electron microscopic evidence and that the sample of singly labeled superficial stellate and Golgi cells is small makes the positive identification of this cell impossible.
; Zhang and Oertel 1994
), does not arborize in the DCN and was cut medially as it traversed the deep layer, presumably in its path to the dorsal acoustic stria. The cell bodies of giant cells lie in the deep layer (Fig. 11B). The dendrites of giant cells are not restricted in an isofrequency lamina in the deep layer but extend widely. Some dendrites extend through the fusiform cell layer and into the molecular layer. Because the axons of cartwheel cells are largely restricted to the molecular and fusiform cell layers in mice, their input must be located largely on distal dendrites of giant cells. The axon of this giant cell is unusual in that it is the only one (of 8 total) (Zhang and Oertel 1993b
; present study) that had local collaterals. These collaterals terminated in the fusiform cell and deep layers of the DCN as well as in the lamina of granule cells that separates the DCN from the VCN. The main axon was cut as it entered the dorsal acoustic stria. Although some principal cells have axon collaterals in the DCN, the small number of such cases indicates that fusiform and giant cells are not a major source of input to neurons in the DCN.
; Zhang and Oertel 1993c
), terminates in the deep layer of the DCN as well as in the VCN. The terminal arbors of tuberculoventral cells lie mingled among dendrites of fusiform and giant cells but are separated from the dendrites of cartwheel and superficial stellate cells.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
). The present results explore further the consequences of the connections of cartwheel cells on the output of the DCN.
; Manis et al. 1994
; Zhang and Oertel 1993a
). The present results show that complex spikes in cartwheel cells comprise fast, sodium-dependent action potentials superimposed on slower, calcium-dependent depolarizations. Complex spikes in cartwheel cells resemble those of the cerebellar Purkinje cells (Llinás and Sugimori 1980
), consistent with other studies showing extensive ontogenetic, morphological, and immunocytochemical similarities shared by these two cell types (Altman and Bayer 1985
; Berrebi and Mugnaini 1991
; Berrebi et al. 1990
; Mugnaini and Morgan 1987
; Pierce 1967
; Ryugo et al. 1995
; Wouterlood and Mugnaini 1984
). In Purkinje cells, calcium spikes are generated in the dendrites, and undergo passive electrotonic decay to the soma, where the recorded waveform appears small and slowly rising (Lev-Ram et al. 1992
; Llinás and Sugimori 1980
; Tank et al. 1988
). The similar shape of calcium spikes in cartwheel cells suggests that the underlying voltage-gated channels may also be dendritically located.
; Mugnaini et al. 1987
) and physiologically (Golding and Oertel 1996
; present results). The synaptic activity in cartwheel cells that mirrors the firing patterns of cartwheel cells is blocked by strychnine. This result, together with immunocytochemical evidence demonstrating that cartwheel cells show strong glycinelike immunoreactivity, support the conclusion that cartwheel cells are glycinergic (Gates et al. 1996
; Oertel and Wickesberg 1993
; Osen et al. 1990
; Wenthold et al. 1987
; Wickesberg et al. 1994
). Golding and Oertel (1996)
demonstrated that the reversal potential for glycinergic PSPs (estimated to be
53 mV) was near the firing threshold of cartwheel cells. In cartwheel cells, glycinergic synaptic input from other cartwheel cells can either promote or suppress firing, depending on how the synaptic inputs interact with the intrinsic conductances. These findings indicate that cartwheel cells form an interconnected network with both positive and negative feedback. The network is driven by excitatory input from parallel fibers (Kane 1974
; Wouterlood and Mugnaini 1984
) that is probably glutamatergic (Golding and Oertel 1996
; Hunter et al. 1993
).
; Golding and Oertel 1996
; Mugnaini et al. 1987
; Zhang and Oertel 1994
). The electrophysiological observation that the temporal patterns of glycinergic PSPs mirror the firing patterns of cartwheel cells is consistent with earlier results (Zhang and Oertel 1994
) and supports the finding from anatomic studies that cartwheel cells contact fusiform cells (Berrebi and Mugnaini 1991
). The molecular layer has opposing influences on fusiform cells. Parallel fibers excite fusiform cells, whereas cartwheel cells inhibit them. The present study confirms that the inhibitory influence through cartwheel cells is strong, long lasting, and predominant under the experimental conditions of the slice.
). Indeed, clear examples of bursts of IPSPs were not common in two of the three giant cells in the present sample. More common were slow hyperpolarizations that probably represent dendritically filtered versions of such bursts. Because cartwheel cell terminals lie in the molecular and fusiform cell layers, their inputs to giant cells must commonly be to distal dendrites. It is therefore not surprising that PSPs arising from those inputs appear filtered. The conclusion that giant cells are targets of cartwheel cells is consistent with anatomic results that show that cartwheel cells contact not only fusiform cells but also large neurons in the deep layer (Berrebi and Mugnaini 1991
). A connection of cartwheel cells with giant cells is also supported by results obtained in vivo. In cats, the responses of principal cells (type IV units) to activation of parallel fibers are dominated by inhibition lasting from 10 to 30 ms (Young et al. 1995
). Such units were recorded in both the fusiform and deep layers of the DCN, and thus may have included giant cells as well as fusiform cells. The timing of this inhibition is correlated temporally with the firing of units presumed to be cartwheel cells (Davis et al. 1996
).
). Superficial stellate and Golgi cells are small to medium-sized multipolar cells with smooth or slightly beaded dendrites that have largely been described on the basis of electron microscopy (Mugnaini et al. 1980a
; Osen et al. 1990
; Wouterlood et al. 1984
). At the light microscopic level, distinctions between these interneurons are not entirely clear, making the identification of the cell shown in Fig. 10B uncertain. In slices of the cochlear nuclei one cell has been identified as a superficial stellate cell (Zhang and Oertel 1993a
). The cell shown in Fig. 10B resembles the stellate cell in its smooth dendrites but, in contrast with the earlier example, terminates both in the molecular layer and in the fusiform cell layer; the physiological responses of the two cells were similar. With a sample of only two cells, it is not clear whether the cells are similar or different. Whether it is a superficial stellate or a Golgi cell, is it likely to be inhibitory. Both cell types were labeled with antibodies against GABA conjugates and against glutamic acid decarboxylase; Golgi cells were in addition labeled by antibodies against glycine conjugates (Adams and Mugnaini 1987
; Mugnaini 1985
; Osen et al. 1990
). The putative superficial stellate cell is unlikely to be the source of late, glycinergic PSPs because the cell fired a single, early spike in response to shocks of the auditory nerve or DCN within 5 ms of the shock. This cell could, however, be the source of GABAergic inhibition in cartwheel, fusiform, and giant cells.
; Oertel and Wu 1989
; Zhang and Oertel 1993c
, 1994
). Tuberculoventral cells are confined to the deep layer of the DCN. Their dendrites are aligned parallel to, and receive excitatory input from, a restricted group of primary auditory nerve fibers (Brown and Ledwith 1990
; Ryugo and May 1993
; Wickesberg and Oertel 1988
; Zhang and Oertel 1993c
). Their axonal arbors have one group of terminals within roughly the same isofrequency lamina as the dendrites, and another group of terminals in an isofrequency band of the VCN. Because neither their auditory nerve inputs nor any processes lie dorsal to the cell body, it is not surprising that tuberculoventral cells were activated only weakly even at the highest shock strengths. The brief firing of tuberculoventral cells is inconsistent with their giving rise to late, glycinergic activity in cartwheel, fusiform, and giant cells.
; Smith and Rhode 1989
). The firing of D-stellate cells does not account for the spontaneous and evoked bursts of PSPs recorded in cartwheel, fusiform, and giant cells, because they neither fire action potentials in bursts nor fire spontaneously in vitro (Oertel et al. 1990
; M. J. Ferragamo, N. L. Golding, and D. Oertel, unpublished results).
). The interconnections among cartwheel cells shape the spatial as well as the temporal pattern of activation of the network of cartwheel cells by parallel fibers. Activity in cartwheel cells ultimately imposes inhibition on the arrays of principal cells in the deeper layers. The array of fusiform cells preserves the tonotopic arrangement of the auditory nerve fiber inputs but not of the parallel fibers that cross the tonotopic array orthogonally. Less is known about giant cells, which also combine the same two classes of information but in different proportions and spatial patterns.
; Nelken and Young 1994
; Spirou and Young 1991
; Young and Brownell 1976
). These spectral features are produced by the passive filtering characteristics of the external ear and serve as potential cues for sound localization (Musicant et al. 1990
; Rice et al. 1992
). Young and colleagues (Kanold and Young 1996
; Young et al. 1995
) have proposed that somatosensory input to the molecular layer of the DCN could contribute information concerning the position of the pinnae and head that would be needed for the central auditory system to interpret the spectral cues encoded by DCN principal cells. Findings in vivo (Davis et al. 1996
) indicate that the cartwheel cells convert the excitation from somatosensory inputs (and their granule cell intermediaries) into robust inhibition of principal cells. The diversity of other sensory and nonsensory inputs to granule cells would suggest, however, that such inhibition could subserve a more ubiquitous role in the DCN.
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ACKNOWLEDGEMENTS |
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We thank our colleagues in the Department of Neurophysiology, especially J. A. Ekleberry, J. Meister, and I. Siggelkow in the histology lab and P. Heinritz, L. Lokken, A. Rassbach, and J. Hineline in the office. We also thank M. Ferragamo, D. Geisler, B. Rhode, P. Smith, and L. Trussell for comments on the manuscript.
This work was supported by National Institute of Deafness and Other Communications Disorders Grant RO1 DC-00176. N. Golding was supported by a predoctoral fellowship from the National Science Foundation.
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FOOTNOTES |
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Present address of N. L. Golding: Dept. of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208.
Address for reprint requests: D. Oertel, Dept. of Neurophysiology, University of Wisconsin, 1300 University Ave., Madison, WI 53706.
Received 2 July 1996; accepted in final form 17 March 1997.
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REFERENCES |
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