Intracellular responses of the rat anteroventral cochlear nucleus to intracochlear electrical stimulation

Brain Research Bulletin, Vol. 46, No. 4, pp. 317–327, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/98 $19.00 1 .00

PII S0361-9230(98)00017-3

Intracellular responses of the rat anteroventral cochlear nucleus to intracochlear electrical stimulation Antonio G. Paolini* and Graeme M. Clark Department of Otolaryngology, The University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, Australia [Received 1 September 1997; Accepted 9 February 1998] ABSTRACT: The anteroventral cochlear nucleus (AVCN) is the first central processing site for acoustic information. The influence and extent of convergent auditory nerve input to AVCN neurons was investigated using brief (<0.2 ms) intracochlear electrical activation of spiral ganglion cells. In 40 neurons recorded in vivo, the major intracellular response to stimulation was an excitatory postsynaptic potential (EPSP) with short latency (;1 ms) and fast rise time (<1 ms). Graduated EPSP amplitude increases were also seen with increasing stimulation strength resulting in spike generation. Hyperpolarization followed excitation in most neurons, its extent distinguished three response types: Type I showed no hyperpolarization; Type II and Type III displayed short (<10 ms) and long (>19 ms) duration hyperpolarization, respectively. Hyperpolarization was attributed to an inhibitory postsynaptic potential (IPSP) in addition to spike after hyperpolarization. Neurobiotin filling identified Type I and II neurons as stellate and Type III as bushy cells. These results suggests that AVCN neurons receive direct, possibly convergent, excitatory input from auditory nerves emanating from spiral ganglion cells with hyperpolarization resulting from polysynaptic inhibitory input. © 1998 Elsevier Science Inc.

convergence of auditory nerve fibers onto neurons in the AVCN has been studied primarily using anatomical [21,37] and in vitro physiological investigations [30], the extent and influence of this input is unclear. Auditory processing may require in addition to convergent input the integration of excitatory and inhibitory mechanisms within the AVCN. In in vitro experiments both stellate and bushy cells within the ventral cochlear nucleus have been shown to receive early monosynaptic excitatory followed by late polysynaptic inhibitory inputs when the auditory nerve was electrically stimulated [28,29,54,55]. Inhibitory processes have been shown in extracellular studies of unit responses in the AVCN to mask response activity at frequencies away from the characteristic frequency (CF) [11,25,36] or act to suppresses the response of units at their best or CF [8]. This inhibition may be responsible for fine tuning excitation from auditory nerve activation. In the present investigation we used intracochlear electrical stimulation and intracellular in vivo recording in the AVCN to examine the extent and influence of excitation, inhibition, and convergent auditory nerve input on both stellate and bushy cells. Past in vivo studies investigating electrical stimulation of the cochlea have centered on its effect on the auditory nerve response [15,17,18,20,41,47], and to a lesser extent the cochlear nucleus [10,13,14,23,42,52]. All these studies have employed extracellular recording techniques from which limited conclusions can be made about the possible neural mechanisms involved in the production of the unit response. By varying stimulation strength and intracellularly examining graded changes in excitatory postsynaptic potential (EPSP) amplitudes both convergence and coincident detection of auditory nerve input can also be investigated using intracochlear electrical stimulation, which allows direct stimulation of spiral ganglion cells for short periods unlike auditory click stimuli. This intracellular study demonstrates convergence in three distinct neural response types to intracochlear electrical stimulation correlated with neuron morphology.

KEY WORDS: Auditory nerve, Auditory neurophysiology, Cochlear nucleus, Electrical stimulation, Intracellular recording.

INTRODUCTION The intracellular response of ventral cochlear nucleus neurons to auditory stimulation has been extensively investigated [12,32,35, 43,44], where it has been shown that different neuron types within the cochlear nucleus respond differently to acoustic input. However, it is still unclear how sound information processed by spiral ganglion cells is encoded by central auditory processing. There is considerable physiological and psychophysical evidence that the frequency of sound is coded by place and temporal codes [9]. Although place coding relies on frequency specific tonotopic projections within the auditory pathway, there is increasing evidence to suggest that temporal processing may rely on coincident detection of converging neural input [7,43]. The anteroventral cochlear nucleus (AVCN) contains two predominant cell types, the bushy and stellate cells, both of which receive direct input from auditory nerve fibers [37]. Although

MATERIALS AND METHODS Preparation All experiments were performed on 21 male Long Evans rats anesthetized with intraperitoneal (i.p.) urethane in water (1.3 g/Kg)

* Address for correspondence: Dr. Antonio G. Paolini, Department of Otolaryngology, University of Melbourne, Royal Victorian Eye and Ear Hospital, 32 Gisborne Street, East Melbourne, 3002 Australia. Fax: 61-3-9663 1958; E-mail: [email protected]

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318 and breathing spontaneously, and supplemental doses were administered if at any time during the experiment a strong corneal or paw reflex was observed. All efforts were made to minimize animal suffering in accordance with the Royal Victorian Eye and Ear Hospital animal ethics guidelines (Grant 95037). Surgery After a midline incision, the underlying muscle was cleared from the bone and the cranium and dura removed over the cerebellum. The cerebellum was aspirated on one side to expose the cochlear nucleus. A further incision was made behind the ear ipsilateral to aspiration and the bulla drilled away to expose the round window. A scala tympani stimulating electrode array was inserted into the cochlea through the round window. The array consisted of two platinum rings (diameter 0.3 mm, 0.45 mm apart) on a Silastic carrier connected to platinum/iridium wire. Intracellular recording electrodes for the AVCN were aimed under visual control. Body temperature was maintained at approximately 37°C with a DC homoeothermic blanket. Any brain swelling following surgery was reduced by 0.3 ml of Dexomethasone phosphate (4mg/ml Decadron, MSD) injected into the tail vein. Recording Microelectrodes, made from thin-walled (1.0 mm outside diameter) quartz glass (Sutter Instrument Company, CA, USA), were filled with 1 M potassium acetate (70 – 80 MV) or in some cases 4% Neurobiotin in 1 M potassium acetate. Electrodes were advanced into the AVCN in 2 mm increments using a microelectrode stepper. Intracellular stable impalements were signalled by a prolonged (.3 min), stable drop (.30 mV) in the DC level and the presence of synaptic or large action potentials (.20 mV) with monophasic rise and fall times. A MacLab 4S data acquisition system (AD Instruments, Sydney, Australia) was used to store electrophysiological traces at a bandwidth of 20 or 40 kHz. The cochlea was stimulated with biphasic, constant current pulses (charged-balanced) delivered at 100 ms per phase, 0 –2.5 mA intensity at 5 Hz repetition. Latency of EPSPs and inhibitory postsynaptic potential (IPSP) responses were measured from stimulus onset to the onset of membrane potential rise above (EPSP) or decrease below (IPSP) resting level. The EPSP thresholds were assessed to 0.1 mA resolution. These responses were distinguished from noise (pulsations or spontaneous fluctuation in membrane potential) by averaging and analyzing repeated presentations of stimuli. Previous research has demonstrated that electrical stimulation of the cochlea can produce a response in the auditory nerve in either of two ways: (1) by direct activation of spiral ganglion cells, or (2) by normal transduction of a mechanical event precipitated by the electrical current (electrophonic activation) [47]. Electrophonic activation was reduced in all but one experiment by intentionally damaging the basilar membrane upon insertion of the scala tympani electrode array. Acoustic stimuli were delivered to the animal to assess hearing loss associated with microelectrode insertion trauma. A Beyer DT48 transducer was positioned at the end of a hollow ear bar. A PDP-11/34 computer was used to generate acoustic stimuli (either pure tones or white noise, 50 ms burst, 5 ms rise-fall time, 5 Hz repetition). A second Bruel and Kjaer (B&K) 1/2 inch condenser microphone was coupled to a small probe tube that was positioned within the ear bar tube approximately 3 mm from the tympanic membrane. This second microphone allowed the precise measurement of the level of the acoustic stimuli being presented. The acoustic system was calibrated using

PAOLINI AND CLARK a B&K measuring amplifier (type 2606) to enable acoustic input to be measured in dB sound pressure level (SPL). Cells were filled with Neurobiotin by passing approximately 1.1 nA of depolarizing current pulses (500 ms duration, 1 Hz repetition) through the micropipette, for at least 3 min and optimally up to 15 min. In order to easily distinguish cells on the basis of their position and depth, up to five cells were injected per experiment in no more than three electrode penetrations. Histology After anesthetic overdose the rats were perfused transcardially with 10% formalin containing 30% sucrose. They were decapitated and the head dissected free and preserved in sucrose-formalin solution. After 4 –5 days the heads were returned to the stereotaxic apparatus, the brains blocked in either the coronal or parasaggital plane, and the brain removed from the skull and then sectioned on a freezing microtome. The cochlear nucleus was examined for Neurobiotin-filled cells in 120 mm sections. Cells filled with Neurobiotin in brain sections were processed using avidin-horseradish peroxidase (HRP). Sections were washed in 0.1 M phosphate buffer (six by 10 min) and incubated in a 1:5000 dilution of avidin-HRP in phosphate buffer and 0.03% Triton-x overnight. The sections were then washed in five 10-min washes of 0.1 M phosphate buffer and incubated in intensified cobalt-nickel 3,3 Diaminobenzidine (DAB) [1] for 20 min. The cochleae were removed and sectioned in two animals to confirm basilar membrane damage. They were decalcified in 4% ethylenediaminetetraacetic acid (EDTA) in phosphate buffered 2.5% glutaraldehyde, dehydrated, and finally embedded in Spur’s resin. The blocked cochleae were sectioned at 2 mm in the horizontal plane, and sections every 126 mm were collected and stained with hematoxylin. Imaging and Cell Location Cells were drawn from 120 mm sections using camera-lucida. Cells that could be identified and traced in continuity were reconstructed from serial sections using a drawing tube at a total magnification of 3 625 with an oil-immersion lens (planachromat, NA 1.0). During experimental recording, distance from the cochlear nucleus surface to the filled cells was noted. A surface map for each experiment acted as a guide to determine the relative positions of electrode penetrations. Neurobiotin filled cell locations could, therefore, be provisionally determined during the experiment. These predicted locations corresponded well with their actual locations after histological verification. Cells were typed into various neuronal classes based on soma size and shape, dendritic arborization, and position within the nucleus [2,6,22]. RESULTS Intracellular Recordings Stable intracellular recordings were obtained from 40 neurons, of which six were spontaneously active. Stable neurons were held for at least 3 min and had a mean (6 SEM) resting membrane potential of 257.8 6 2.4 mV with a mean (6 SEM) action potential amplitude (with corresponding ranges) of 37.1 6 2.4 mV (20 –70 mV). In all but one animal, neural responses to sound were seen only at intensities greater than 90 dB SPL. In one animal in which hearing was not affected, seven neurons were impaled. These neurons responded to intracochlear electrical stimulation in a similar fashion to those neurons recorded in deafened animals and have been included in the sample.

AVCN RESPONSES TO INTRACOCHLEAR STIMULATION Responses to Stimulation In response to electrical stimulation, 30 neurons displayed only one distinct EPSP, nine neurons showed an additional EPSP of longer latency, and one neuron responded only with hyperpolarization. Neurons responding with one EPSP only also showed, in the majority (29 out of 30), a hyperpolarizing response to stimulation that followed the EPSP response. Three response types could be distinguished based on the presence, duration, or absence of this hyperpolarization following electrical stimulation (Fig. 1A and B). Ten neurons responded with no hyperpolarization and have been classed as Type I responses (Fig. 1Ai and B). In nine of these Type I neurons, two EPSP components were seen (Fig. 1Ai). The remaining neuron classed as Type I showed only one EPSP component. In all cells showing hyperpolarization to stimulation, its duration showed a bimodal distribution allowing neurons to be classified into two additional types. Fourteen neurons, classed as Type II, showed a short duration hyperpolarization response following the initial EPSP component and action potential generation (Fig. 1Aii and B) with a mean duration (6 SEM) of 6.5 6 0.5 ms (range: 2.7–9 ms). In 12 neurons, classed as Type III and morphologically identified as bushy cells in three recordings (see later), a long lasting hyperpolarization response was seen following the initial EPSP component and action potential generation (Fig. 1Aiii, iv and B) with a mean duration (6 SEM) of 26.1 6 2.5 ms (range: 19.5–39 ms). Three neurons were not assigned to any type as the duration of the hyperpolarizing response was difficult to assess. The results are summarized in Table 1. These initial or single EPSP responses had fast rise times with peak amplitude occurring within 1 ms from EPSP onset (Fig. 1A). The latency of the EPSPs ranged from 0.4 – 4 ms, with the first and second EPSPs having mean latencies (6 SEM) of 0.8 6 0.3 and 2.3 6 0.3 ms, respectively. The EPSPs were elicited at a mean threshold stimulus intensity (6 SEM) of 0.9 6 0.1 mA (range: 0.2–2.1 mA). The duration of the first EPSP component was relatively short with a mean (6 SEM) of 3.6 6 2.3 ms (range 1.2–10 ms). Of the 39 neurons that responded with EPSP, 36 elicited an action potential to higher intensity stimulation with action potential peak latencies (6 SEM) of 1.43 6 0.1 ms. Action potentials were not seen on the second EPSP (Fig. 1Bi). In 14 neurons variations in stimulus strength produced large graduated changes in EPSP amplitude in all cell types (Fig. 2A–D) before spike activation. Type I (n 5 4), Type II (n 5 7), and Type III (n 5 3) cells had a mean 6 SEM number of graduated steps to spike generation of 7.3 6 0.3, 4.3 6 0.3, and 6.7 6 0.3. Type II neurons showed a significantly lower number of graduated amplitude rises to spike generation than Type I neurons (t(9) 5 26.94, p , 0.05). In five of six Type II neurons that were spontaneously active, a small afterpotential was seen following spontaneous action potentials (Fig. 3A), but this was smaller in amplitude and duration than the stimulus evoked response (Fig. 3B). In Type II and Type III neurons, the amplitude of the hyperpolarizing response increased upon spike generation compared to subspike threshold levels (e.g., Fig. 3C). The latency of the hyperpolarizing response also changed with changing stimulus strength (e.g., Fig. 3D) indicating a possible polysynaptic response. In 10 cells tested, the hyperpolarization seen in Type II and Type III neurons decreased in amplitude with more negative membrane potential having a reversal potential between 270 – 280 mV. In two Type III neurons examined extensively the reversal potential of the hyperpolarization became more negative upon spike generation. This is illustrated in Fig. 4A–D, which displays a Type III response to intracochlear electrical stimulation.

319 When the membrane potential was varied by constant current injection the hyperpolarizing response following the EPSP, evoked at just below spike threshold, reversed at 275 mV (Fig. 4B,C). This was closer to resting than the reversal potential of the hyperpolarization following spike generation that reversed between 285–290 mV (Fig. 4D). Morphological Correlation Of 18 attempted cellular fills, 11 neurons could be identified morphologically and correlated to neuronal response (Table 2). Three neurons were identified as bushy (Fig. 5A and B) and eight neurons as stellate cells (Fig. 6A and B). In response to stimulation bushy cells showed a Type III response while stellate cells exhibited Type I and II responses. Stellate cells showed large dendritic fields ranging from 190 – 400 mm from the cell body measured along the coronal or parasaggital plane (Table 2). Bushy cells showed smaller dendritic fields of approximately 120 mm. No morphological differences were seen between Type I and Type II responders although Type II responders tended to have larger dendritic fields (Fig. 6A). DISCUSSION This in vivo investigation extends previous in vitro findings demonstrating inhibition following an initial EPSP evoked by electrical stimulation of the auditory nerve trunk [28,54,55]. However, the duration and extent of this inhibition was not uniform across cells. Not all neurons in our sample demonstrated inhibition and some showed inhibitory influences of shorter duration than others. This response inconsistency may be related to the extent to which auditory nerve fibers were stimulated or to the different cell morphologies and their complex intrinsic interactions that are left intact in this in vivo experiment. Response Characteristics In response to electrical stimulation three response types could be distinguished based on the presence or absence of hyperpolarization following the stimulus evoked response. Type I neurons receive no inhibitory influences as a result of electrical stimulation, responding only with EPSPs. The majority of these Type I neurons responded with two EPSP components. The first component most likely originates from direct monosynaptic auditory nerve input while the second EPSP component may be due to recurrent excitatory circuits within the cochlear nucleus or from auditory nerve fibers with slow conduction velocities. In Type II and Type III neurons, spike activation usually resulted in an increase in the amplitude of the hyperpolarization. It was not possible to determine whether this increase in amplitude was due to a mixed potential resulting from hyperpolarization following spike and inhibitory influences or due to recurrent feedback inhibition. In two type III neurons, however, where this was examined extensively. The hyperpolarization following spike generation had a more negative reversal potential than the hyperpolarization occurring in the absence of the action potential (see Fig. 4A–D), adding support for the presence of a mixed potential upon spike generation. Type II neurons exhibited a short duration hyperpolarizing response following the short latency EPSP component. The hyperpolarization occurring in the absence of a preceding spike was most probably a polysynaptic IPSP response. The latency of this inhibitory response suggests that these neurons receive short duration inhibitory input not directly from the auditory nerve but from local circuit neurons. This is consistent with the findings of Wu and Oertel [55] who showed early monosynaptic excitatory

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FIG. 1. Response just below (A) and just above (B) spike threshold in four neurons showing the three response types distinguished on the basis of hyperpolarization extent following electrical stimulation. Type III response was typical to both spherical and globular bushy cells depicted in Fig. 5. S, stimulus artefact; asterisk, hyperpolarization; arrow, beginning of discernible rise of EPSP.

AVCN RESPONSES TO INTRACOCHLEAR STIMULATION TABLE 1 RESULTS SUMMARY Response Type

1 EPSP

I II III No spike response Total no.

1 14 (5) 12 3 30 (5)

2 EPSPs

9 (1) 0 0 0 9 (1)

Total no.

10 (1) 14 (5) 12 3 39 (6)

Number of spontaneously active cells indicated in brackets.

and late polysynaptic inhibitory influences on both stellate and bushy cells recorded from cochlear nucleus slices in vitro. Just over one-third of these Type II neurons in the present study also showed spontaneous activity suggesting that they receive some tonic excitatory input, perhaps from spontaneously firing auditory nerve fibers. Type III neurons showed a long lasting hyperpolarizing response to stimulation that followed the EPSP response. The hyperpolarization occurring in the absence of a preceding spike had a reversal potential between 270 –280 mV indicating that a

321 chloride conductance may have been responsible for this hyperpolarization, suggesting an IPSP component. In vitro evidence suggests that this IPSP may be mediated predominantly by glycine. Wu and Oertel [55] have shown that an IPSP electrically evoked by stimulation of the auditory nerve in vitro could be blocked by strychnine, a blocker of glycine mediated inhibition. g-aminobutyric acid (GABA) may also be involved as antagonists specific to GABA also blocked the IPSP response although its action was not consistent [55]. Morphological Characteristics The stellate cell has a cell body with three but sometimes two radially extending dendrites. These dendrites usually are slender and branch once or twice ending with relatively small terminal elaborations [12]. Stellate cells have been divided into two classes based on morphological distinctions of noncochlear inputs; with one class receiving terminations predominantly on the proximal dendrites and the second receiving synapses on their soma [3,44]. In this investigation stellate neurons, exhibited both Type I and Type II responses to stimulation. Whether these two response types are related to the two morphological stellate classes described in the ventral cochlear nucleus is not clear. No morphological differences at the light microscope level were seen between Type I and Type II responders in our investigation, although there

FIG. 2. (A–D) Response types to electrical stimulation from just above EPSP threshold to spike threshold. EPSP amplitude rises occurred in a graduated fashion.

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FIG. 3. Stellate cell Type II responses to intracochlear electrical stimulation. (A) This neuron fired spontaneous action potentials; (B) spike response at just above threshold (1.3 mA) showing short duration hyperpolarization following spike. An EPSP response was seen at stimulus levels below spike threshold (C). An IPSP was present in the absence of a spike response (Ci) suggesting that it may contribute to hyperpolarizing response following a spike. The amplitude of the hyperpolarization increased in magnitude upon spike generation (Cii). (D) The IPSP response (average of two traces) may be polysynaptic in origin with a change in latency (indicated by arrow in ii) observed with increasing stimulus strength (i, 1.2 mA; ii, 1.1 mA). At higher stimulus intensities (Di) the latency of the IPSP was difficult to determine as it occurred directly after the EPSP response. At lower stimulus intensity the latency was longer (Dii). This neuron was morphologically identified as a stellate neuron (Fig. 6A). “S” Stimulus artefact. Calibration: A as in B.

was a tendency for Type II neurons to have larger dendritic fields and exhibit a lower number of graded steps to spike generation than Type I neurons. If Type II stellate cells receive the bulk of their convergent input through axodendritic synapses then dendritic filtering of EPSPs may account for the small number of distinguishable graduated steps to spike generation. In addition, the Type I EPSP response and the tendency for a smaller dendritic field, suggests that these stellate neurons may receive axosomatic termination of convergent auditory nerve input. Smith and Rhode [44] demonstrated that multipolar (stellate) neurons with terminals on the soma may have axons that contain pleomorphic vesicles terminating on stellate cells with sparse somatic input. These Type I neurons possibly receiving axosomatic auditory nerve input may, therefore, be responsible for the hyperpolarization seen in Type II and Type III neurons.

Two types of bushy cells have been described as spherical in the anterior AVCN or globular in the posterior division of the AVCN [4 – 6,46]. Both neuron types in this investigation exhibited a Type III response to stimulation suggesting that these neurons receive long duration polysynaptic inhibitory input. It has been shown that a large proportion of the primary dendritic and cell body surface of bushy cells is covered with synaptic terminals, with the majority originating from the auditory nerve [31,38,43, 45], although a substantial number of terminals contain flat or pleomorphic vesicles [43] suggesting inhibitory synaptic contacts on these neurons. Inhibitory input to AVCN neurons may originate extrinsically from cells in the dorsal cochlear nucleus (DCN), a pathway referred to as the “lateral ventrotubercular tract” [22]. Wickesberg and Oertel [49] revealed projections from DCN to

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FIG. 4. Spherical bushy cell response (Fig. 5A; Type III) to intracochlear electrical stimulation. (A) Response to 1.2 mA stimulation at resting membrane potential (dashed line, insert). Spike was followed by a long lasting hyperpolarizing potential (arrow, insert); (B) in response to electrical stimulation just below spike threshold a depolarization was seen preceding the hyperpolarization. Upon injection of hyperpolarizing current [0.2 nA, stimulation protocol shown below trace (i, ii)] the depolarizing and hyperpolarizing responses increased and decreased (a1 , a2) in amplitude respectively suggesting an EPSP followed by an IPSP response to stimulation (dashed line shows passive membrane potential during current pulse). When membrane potential was varied by constant current injection at just below spike threshold the hyperpolarization response reversed at 275 mV (C, membrane potential of neuron induced by constant current injection indicated by dashed line). (D) At just above spike threshold (spike indicated by asterisk) the hyperpolarizing potential increased in magnitude and upon injection of hyperpolarizing current (protocol shown at the bottom of trace i, ii, iii) it reversed in the opposite direction. This neuron was morphologically identified as a spherical bushy neuron (Fig. 5A). “S” Stimulus artefact.

AVCN in the mouse that are frequency specific and tonotopic. Past research has shown that globular bushy cells are prominent targets of tuberculoventral neurons [51]. However, evidence also suggests DCN innervation to both spherical bushy [53] and stellate neurons [51]. The projection from DCN to AVCN is

likely to be inhibitory and primarily mediated by glycine [39, 50]. An in vitro study by Wickesberg and Oertel [50] demonstrated that glutamatergic excitation of DCN neurons results in trains of IPSPs in the AVCN. This inhibition from DCN to AVCN may underlie the probable polysynaptic inhibition in

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PAOLINI AND CLARK TABLE 2

STIMULATION RESPONSE TYPE, SOMA DIAMETER (LONG AXIS), AND APPROXIMATE MINIMUM DENDRITIC FIELD VISIBLE OF MORPHOLOGICALLY IDENTIFIED NEURONS IN THE AVCN

Neuron

Response Type

Morphological Type

Soma Diameter (mm)

Dendritic Field (mm)

223-003 223-002 224-003 242-012 242-014 242-017 242-018 242-019 243-002 243-005 259-008

III I II II I II II III EPSP only I III

Spherical bushy Stellate Stellate Stellate Stellate Stellate Stellate Globular bushy Stellate Stellate Spherical bushy

23 22 18 22 22 20 30 27 22 16 21

110 250 400 270 250 320 225 130 190 225 120

antagonist [16]. However, NMDA receptors on the postsynaptic terminal of bushy cells decrease in number as the rat matures to adult [16]. It is possible then, that the fast EPSP component is mediated by glutamate acting on non-NMDA receptors. This fast EPSP may act to boost any subsequent inputs to threshold producing an action potential at low stimulus levels. Fast EPSPs may also play an important part in temporal coding. Recent extracellular evidence has shown that neurons in the AVCN are able to phase-lock in a more precise manner and show higher entrainment capabilities than auditory nerve fibers [19], which may be related to both convergence and the fast nature of synaptic responses seen in this present investigation. Type III neurons identified as globular bushy cells differed from the responses seen in other cell types, showing the fastest rise time to a clearly definable EPSP peak in addition to having the highest amplitude EPSP before spike generation (see Fig. 2D). Globular bushy neurons have also been shown to respond with EPSPs on successive cycles of acoustic input up to frequencies of 2500 Hz [34]. In addition, intracellular recordings by Smith and Rhode [43] have shown globular bushy cells respond with fast synaptic po-

Type II and Type III neurons evoked by intracochlear electrical stimulation. But this may not be the only source of extrinsic inhibitory input. The superior olivary complex has been shown to be an important source of inhibitory input to the cochlear nucleus [33,40], which may contribute to late hyperpolarization in excess of 4 ms seen in this present investigation. Inhibitory input to AVCN neurons may also originate intrinsically (local circuit inhibition). The possible existence of local circuit neurons has been suggested by Rhode and Greenberg [36] who examined the extent of local suppression and inhibition in the cochlear nucleus. Earlier experiments by Wu and Oertel [55], and Wickesberg and Oertel [49] have also suggested the existence of local circuits within the ventral cochlear nucleus. Wickesberg and Oertel [49] injected horseradish peroxidase into the AVCN, which labeled a cluster of neurons in the AVCN dorsal to the injection site. They proposed that these cells may be interneurons relaying information between regions of the AVCN. Both excitatory and inhibitory synaptic responses have been recorded in vitro in identified stellate and bushy cells from electrical stimulation of the auditory nerve [54]. Shofner and Young [42] also described a class of neurons that showed long lasting inhibition of spontaneous activity after evoking a spike to electrical stimulation of the auditory nerve, which is comparable to the long lasting hyperpolarizing response seen in our Type III neurons. Local circuit inhibition may contribute to the bushy cell response, perhaps from neighboring stellate neurons that are known to contain GABA [27] and have axons containing pleomorphic vesicles [44] synapsing within the ventral cochlear nucleus. Fast EPSP Response Neurons recorded in this investigation also exhibited a fast EPSP response. The auditory nerve utilizes glutamate as a transmitter among other possible transmitters [48]. The existence of N-methyl-D-aspartate (NMDA), kainate, and quisqualate-nonNMDA receptors on the postsynaptic membrane of cells within the cochlear nucleus [24] make it possible for this transmitter to act slowly through NMDA receptors, and quickly through nonNMDA receptors [26]. In whole cell patch-clamp recordings from immature rats, excitatory postsynaptic currents in stellate and bushy cells were shown to contain a dual-component time course with the slow component blocked by an NMDA receptor antagonist and the fast component abolished by a non-NMDA receptor

FIG. 5. (A and B) Two identified bushy cells in the AVCN. (A) Spherical bushy cell located in the anterior AVCN (insert coronal section through the cochlear nucleus) showing spherical cell body with branching dendrites (intracellular traces shown in Fig. 1 iii; 2C; 4); (B) globular bushy cell located in the posterior division of the AVCN (insert shows a coronal section through the most lateral extent of the cochlear nucleus) characterized by thick primary dendrite ending in a tuft arrangement (intracellular traces shown in Fig. 1 iv; Fig. 2D). Calibration: B as in A, 25 mm. a, axon; V, ventral; M, medial.

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FIG. 6. (A and B) Identified stellate neurons in the AVCN filled with Neurobiotin. These neurons had axons with terminal endings (arrows) within the AVCN subdivision. Inserts show neuron position as located in coronal sections through the cochlear nucleus. (A) An example of an identified Type II responding stellate neuron with large dendritic field; (B) Type I neurons tended to have smaller dendritic fields. Calibration: 20 mm. a, axon; V, ventral; M, medial.

tentials to acoustic input. The large number of graduated steps to action potential generation also suggests that these neurons receive convergent input. This fast response coupled with convergence suggests that these neurons are particularly adept at processing temporal information. CONCLUSION In conclusion, this investigation has provided some insight into AVCN neural response to intracochlear electrical stimulation. The results from this investigation suggest that all neurons in the AVCN are able to receive direct convergent input from the auditory nerve. This input appears to be boosted by the presence of a fast EPSP in most neurons. Intracellular data obtained in this investigation also suggests polysynaptic inhibition in Type II (stellate cells) and Type III (bushy cells) responders following excita-

tion. The ability to receive convergent input and the extent of the inhibition may be important contributors to the coding of acoustic input. ACKNOWLEDGEMENTS

This work was funded by the Human Communication Research Centre, The Hearing Research Fund, and the Department of Otolaryngology.

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