Neural timing, inhibition and the nature of stellate cell interaction in the ventral cochlear nucleus

Hearing Research

Hearing Research 216–217 (2006) 31–42

www.elsevier.com/locate/heares

Research paper

Neural timing, inhibition and the nature of stellate cell interaction in the ventral cochlear nucleus Karina Needham a

a,b

, Antonio G. Paolini

a,b,c,*

Department of Otolaryngology, The University of Melbourne, 32 Gisborne Street, East Melbourne, Vic. 3002, Australia b The Bionic Ear Institute, 384-388 Albert Street, East Melbourne, Vic. 3002, Australia c School of Psychological Science, La Trobe University, Bundoora, Vic. 3086, Australia Received 31 October 2005; received in revised form 25 January 2006; accepted 26 January 2006 Available online 22 March 2006

Abstract The ventral cochlear nucleus (VCN) stellate cell population comprises two clusters: narrowly-tuned, excitatory T stellate neurons, and D stellate neurons, a broadly-tuned population of inhibitory cells. These neurons respond to best frequency (BF) tone bursts in a chopper or onset manner, respectively. Through extensive local and commissural projections the D stellate population provides a source of fast inhibitory input to both intrinsic and contralateral T stellate neurons. Whilst the nature of interactions between intrinsic stellate populations is difficult to examine, our previous intracellular investigations of the commissural pathway have provided a means by which to study this relationship in the in vivo preparation. It is the aim of this paper to both review and extend our understanding of the link between stellate populations and their involvement in the commissural pathway by presenting an overview of the results attained in our recently expanded study. The sample of 17 intracellular and 34 extracellular onset chopper (OC) and late/ideal (OnL/OnI) neurons revealed antidromic activity in 31.4% of neurons following contralateral stimulation, providing physiological evidence that OnL/OnI neurons also contribute projections to the commissural connection. Alternatively, 64.7% of the 34 intracellularly-recorded chopper neurons displayed fast, monosynaptic inhibitory potentials. This commissural input was found to influence the timing of neural activity in chopper neurons, providing insight into the relationship that exists between T and D stellate neurons. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Cochlear nucleus; Stellate neurons; Chopper cells; Onset cells; Intracellular; Commissural

1. Introduction The ventral cochlear nucleus (VCN) comprises two populations of stellate/multipolar neuron: T and D stellate. The T stellate neuron, named for the trajectory of its axonal projection into the trapezoid body (Oertel et al., 1990), has also been termed planar due to the orientation of dendrites in line with the plane of incoming auditory nerve fibres (Doucet and Ryugo, 1997), and type I on account of its sparse somatic synaptic coverage (Cant, 1981). The *

Corresponding author. Address: School of Psychological Science, La Trobe University, Bundoora, Vic. 3086, Australia. Tel.: + 61 3 9479 2947; fax: + 61 3 9479 1956. E-mail addresses: [email protected] (K. Needham), a.paolini@ latrobe.edu.au (A.G. Paolini). 0378-5955/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.01.016

activity recorded from these neurons in response to bestfrequency (BF) tone bursts can be described as either sustained or transient chopper (CS and CT, respectively) (Feng et al., 1994; Ostapoff et al., 1994; Palmer et al., 2003; Paolini et al., 2005; Rhode and Smith, 1986; Smith and Rhode, 1989). Alternatively, axonal projections of the D stellate neuron travel dorsally (Oertel et al., 1990). Also named radiate for their dendritic organization (Doucet and Ryugo, 1997), or type II, due to their dense somatic coverage (Cant, 1981), the D stellate neuron responds to BF tones in an onset chopper (OC) or onset-late (OnL) manner (Arnott et al., 2004; Palmer et al., 2003; Paolini and Clark, 1999; Paolini et al., 2005; Rhode and Smith, 1986; Smith and Rhode, 1989; Smith et al., 2005; Winter and Palmer, 1995). These response patterns are noted for their broad tuning and large dynamic range, qualities reflective of the

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D stellate cells’ far-reaching dendritic branches, and subsequent wide-ranging afferent input. Moreover, this feature affords the OC or OnL neuron with the ability to respond more vigorously to broadband stimuli, such as noise, than pure tones (Winter and Palmer, 1995). The presence of oval/pleomorphic vesicles within the axon terminals of OC neurons (Smith and Rhode, 1989), together with the identification of glycine immunoreactivity in a population of large multipolar neurons in the VCN (Doucet et al., 1999; Kolston et al., 1992; Peyret et al., 1987; Wenthold et al., 1987), suggests that the D stellate population is glycinergic, and its input therefore inhibitory. The projections of these neurons terminate extensively across both VCN and dorsal cochlear nucleus (DCN) regions (Adams, 1983; Arnott et al., 2004; Doucet and Ryugo, 1997; Doucet et al., 1999; Palmer et al., 2003; Smith and Rhode, 1989; Smith et al., 2005), and have subsequently been implicated as the source of wideband inhibition throughout the cochlear nucleus (CN: Davis and Young, 2000; Doucet et al., 1999; Ferragamo et al., 1998; Joris and Smith, 1998; Nelken and Young, 1994; Winter and Palmer, 1995). Amongst the potential targets of D stellate projections are neurons of the T stellate population (Ferragamo et al., 1998; Smith and Rhode, 1989). This wideband inhibition of T stellate neurons has been proposed as a means of sharpening spectral contrast through suppression of off-BF (lateral) input (Rhode and Greenberg, 1994), or more recently, improve spectral coding via its influence on neural timing (Paolini et al., 2004, 2005). Whilst the presence of inhibitory input to T stellate neurons is well documented (Adams, 1993; Altschuler et al., 1986; Babalian et al., 2002; Cant, 1992; Ferragamo et al., 1998; Josephson and Morest, 1998; Juiz et al., 1996; Paolini et al., 2004, 2005; Saint Marie et al., 1993; Wenthold et al., 1988; Wickesberg and Oertel, 1990; Wu and Oertel, 1986), the nature of D stellate input to T stellate neurons is difficult to attest in the in vivo preparation. Given the weight of evidence implicating D stellate neurons as the source of commissural input (Alibardi, 1998; Arnott et al., 2004; Cant and Gaston, 1982; Davis, 2005; Joris and Smith, 1998; Needham and Paolini, 2003; Schofield and Cant, 1996; Shore et al., 1992; Smith et al., 2005; Wenthold, 1987), and the T stellate population as a target (Alibardi, 2000a; Babalian et al., 1999, 2002; Needham and Paolini, 2003; Schofield and Cant, 1996; Shore et al., 2003), the manner in which D stellate neurons influence the activity of T stellate neurons, can also be studied through an examination of the commissural projection. The following results describe the physiological properties of VCN T and D stellate activity as attained through in vivo intracellular recordings, and document the nature of the interaction between the two groups, as observed through investigations of the commissural connection. Whilst the main findings presented have been described in our previous publications (Needham and Paolini, 2003; Paolini and Clark, 1999; Paolini et al., 2004, 2005), the larger sample of neurons on which we report here is able

to provide a greater overview of the commissural connection and stellate cell physiology, serving to both support and extend our understanding of (i) the intracellular response properties of stellate neurons, (ii) the influence of D stellate input on T stellate activity, and (iii) the involvement of stellate populations in the commissural connection. 2. Methods 2.1. Preparation Electrophysiological experiments were performed on 90 adult Hooded-Wistar rats (250–350 g). All procedures were carried out in accordance with the Royal Victorian Eye and Ear Hospital Animal Research Ethics Committee guidelines (projects 95-037-01 and 04-105) and adhered to the guidelines of the National Health and Medical Research Council of Australia. The main components of this protocol have been described previously (Needham and Paolini, 2003; Paolini and Clark, 1999; Paolini et al., 2004, 2005). Briefly, animals were anaesthetised with intraperitoneal aqueous urethane (20% w/v: total dose 2.6 g/kg; SigmaAldridge, Sydney, NSW, Australia). Following a craniotomy, the cerebellum was aspirated to expose the brainstem. Visualisation of the CN at the lateral extreme allowed a recording microelectrode (quartz thin-walled; 1.0 mm o.d.; Sutter Instrument Company, Novato, CA, USA) to be inserted into the left (ipsilateral) VCN in a dorso-ventral direction away from the DCN and octopus cell area (OCA) of the VCN. Placement of the concentric bipolar stimulating electrode (tungsten metal 76 lm core, 3–4 lm tip diameter; World Precision Instruments, Sarasota, FL, USA) into the right (contralateral) VCN was also made under visual control. Electrodes were stabilised in tissue with 4% agar. Core body temperature, controlled by a DC homeothermic blanket, was maintained at 37 °C. 2.2. Recording The recording microelectrode, filled with 1 M potassium acetate (KAc; 70–80 MX), or 4% Neurobiotin (Vector Laboratories, Burlingame, CA, USA) in 1 M KAc, was remotely advanced in 2 lm steps by a motorised microdrive (Sutter Instrument Company). Acoustic stimuli (pure tones and noise) were digitally synthesised by a Tucker-Davis Technologies (TDT; Tucker-Davis Technologies, Gainesville, FL, USA) signal generator (TDT system 2), and delivered by Beyer DT48 transducers positioned at the end of the hollow ear bars. Activation of the bipolar stimulating electrode was controlled by the TDT unit via the in-house electrical stimulator. The ‘Neurophysiology Laboratory System’ (NLS: program by R.E. Millard), run on a PC, was used to control stimulus presentation and collate spike time information. Neural signals were amplified by the Axoclamp 2B amplifier (Axon Instruments Inc.) and

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displayed on a storage oscilloscope (Tektronix, Beaverton, OR, USA). Electrophysiological traces were stored using a MacLab 4 S data acquisition system (AD Instruments Inc, Sydney, NSW, Australia) and Scope software (v3.6.9; AD Instruments Inc). The acoustic system was calibrated using a Bruel & Kjaer (B&K) measuring amplifier (type 2606; Bruel & Kjaer S&V, Naerum, Denmark), and a B&K 12 in. condenser microphone, coupled to a small probe tube positioned within the ear bar tube 3 mm from the tympanic membrane. This enabled acoustic input to be measured in decibel sound pressure level (dB SPL). Levels of interaural crosstalk, recorded with a 2-channel dynamic signal analyser (SR785; Stanford Research Systems, Sunnyvale, CA, USA) and B&K 12 in. condenser microphone coupled to the ear bar positioned contralateral to the transducer, were measured to exclude indirect stimulation of the ipsilateral ear by contralateral acoustic stimulation. Crosstalk measured for contralateral clicks and noise presented at 80–100 dB were attenuated by 40–90 dB in a complex frequency-dependent manner. A ‘search stimulus’ of 70–80 dB noise (50 ms duration, every 500 ms) was presented continuously to the ipsilateral side with advancement of the recording electrode. Intracellular impalements were signalled by a stable drop (>30 mV) in the DC level and the presence of synaptic or large action potentials (APs; >20 mV) with monophasic rise and fall times. Intracellular recordings were typically possible for between three and 120 min. Extracellular neural activity was indicated by an elevated spike rate during noise presentation. Following stable cell isolation, single bipolar electrical shocks (varying from 10 to 800 lA, presented at a rate of 1/s; and stimulated with a 50 ls phase-period and 10 ls inter-phase gap), and click stimuli (60–100 dB; 100 ls) were delivered to the contralateral CN (ipsilateral stimuli offset by 20–30 ms). The neuron’s BF was determined from a threshold tuning curve constructed online (as described by Liberman, 1978). Rate-level functions were constructed by presenting tones (50 ms duration, with 5 ms rise and fall time, and 10 Hz repetition frequency) in 5–10 dB steps in a sequential manner from subthreshold to saturation intensity (50 repetitions per step). Spike-latency tests were performed through manipulation of the inter-stimulus interval between contralateral input (click or electrical pulse) and ipsilateral click stimulation. Additional combinations of binaural and monaural stimuli were also presented. A collision test was performed in the event of the generation of presumed antidromic activity following contralateral electrical stimulation (Needham and Paolini, 2003). Animals in which an intracellular-injection of Neurobiotin (0.2–1 nA, 250 ms positive pulses at 2 kHz; 2–10 min) was made were perfused transcardially (warmed 0.9% saline followed by 200–350 ml chilled 10% neutral buffered formalin), sectioned coronally (100–120 lm), and processed for visualisation with intensified DAB (3,3 0 -diaminobenzidine; Vector).

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2.3. Classification Neural activity was classified according to, (i) intracellular profile to BF-tones and/or noise; (ii) response patterns displayed in post-stimulus time (PST), and inter-spike interval (ISI) histograms to BF-tones (30 dB re threshold); and/or (iii) coefficient of variation (CV) measurements, as determined by dividing the standard deviation by the mean ISI during successive 10 ms time windows over the duration of the tone burst (50 stimulus repetitions). Chopper neurons were identified by their regular discharge pattern in response to both BF tones and noise (see results for intracellular profile); where determined, neurons with CV values consistently <0.3 were classified as CS, whilst those with low CV at onset (<0.25), rising up to or above 0.3 for the remainder of the tone, were classified as CT (Blackburn and Sachs, 1989, 1992; Paolini et al., 2005; Young et al., 1988). Onset neurons were identified by their prominent and precisely-timed onset AP; neurons classified as OC displayed one to three regular APs after the onset spike, followed by low levels of late irregular discharge (see results for intracellular profile), whilst those defined as OnL/OnI displayed a prominent onset AP without regular firing thereafter, and, on occasion, late irregular discharge. Neurobiotin-filled neurons were identified by cellular morphology and dendritic arborizations (Doucet and Ryugo, 1997; Oertel et al., 1990; Palmer et al., 2003). As per Paolini et al. (2005), the intracellular response of CT and OC neurons in response to BF-tones (averaged over 25 stimulus repetitions) were measured for: (i) depolarisation peak at onset (calculated as difference in depolarisation amplitude at onset (MPt5-10) relative to activity 20–30 ms post-tone onset (MPt20-30)); (ii) adaptation of sustained depolarisation (difference in depolarisation amplitude at 20 ms (MPt20) versus 50 ms (MPt50) after stimulus onset); (iii) post-tone hyperpolarisation (difference in membrane potential 10 ms post-stimulus offset (60 ms after stimulus onset; MPt60) from resting membrane potential (RMP)), and (iv) membrane potential changes with stimulus duration, plotted as the voltage difference between RMP and membrane potential at 20 ms (MPt20), and 50 ms (MPt50) post-tone onset as a function of sound pressure level (SPL). Membrane potential levels at 20 ms, 50 ms and 60 ms post-tone onset were determined by averaging membrane potential over a 1 ms period (i.e. 20–21 ms, 50–51 ms and 60–61 ms, respectively). 3. Results Intracellular recordings were obtained for 17 onset and 34 chopper neurons. The mean (± standard error (SE)) RMP for all intracellularly recorded neurons was 49.4 ± 1.3 mV, with mean AP amplitude of 36.8 ± 1.5 mV, mean BF of 16.6 ± 1.3 kHz, and mean threshold at BF of 38.2 ± 3.6 dB. The activity of an additional 34 onset neurons was obtained with extracellular recordings: mean BF 19.0 ± 1.4 kHz and mean threshold 47.0 ± 2.8 dB. Of the

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85 neurons described here, eight onset neurons (three intracellular; five extracellular) and nine intracellular chopper neurons contributed to the results presented in Needham and Paolini (2003), whilst the responses of two chopper neurons were previously reported by Paolini and colleagues (2004). 3.1. Onset neurons The mean RMP for neurons described as onset was 50.4 ± 2.4 mV, with an average AP amplitude of 35.0 ± 2.7 mV. The mean BF was 22.2 ± 1.6 kHz, with a mean firing threshold of 30.3 ± 4.2 dB. The majority of onset neurons responded to BF tones and/or noise in an onset chopper manner (12 intracellular; 20 extracellular). As displayed by single (Fig. 1A and B; left) and averaged (Fig. 1A and B; right) intracellular traces in response to BF tones, the OC response was characterised by the presence of a prominent, precisely-timed onset spike, regular second AP and sustained depolarisation of the membrane throughout stimulus presentation. In some neurons, low levels of irregular late discharge were also observed in response to higher intensity BF tones (Fig. 1A). The presentation of noise bursts evoked a similarly well-timed onset AP and sustained depolarisation (Fig. 1C), but was accompanied by more vigorous chopping and late discharge. Contour plots depicting changes in membrane

potential as a function of stimulus SPL and duration (Fig. 1D) in response to BF tones (upper and middle) and noise (lower) demonstrated a narrow band of depolarisation at onset, decreasing in latency with increasing SPL. AP threshold was reduced, and chopping activity increased in noise relative to the BF response at all SPLs. The activity of an OC neuron in response to pure tones presented from 11 to 27 kHz at 60 dB (Fig. 2A), and 10– 28 kHz at 75 dB (Fig. 2B) demonstrated the broad response area typical of OC neurons. The latency of onset activity (denoted by first peak in depolarisation) was consistent across the majority of frequencies presented. A second band of depolarisation was also evident during BF and below-BF input, but rarely observed in response to above-BF stimuli. Additionally, sustained depolarisation was consistently smaller in amplitude in response to above-BF frequencies, and typically accompanied by a narrow band of depolarisation at stimulus offset (Fig. 2A–C). Morphological reconstruction of this neuron revealed a D stellate morphology, with broad dendritic branching, and axonal projections found throughout the VCN, traced into the DCN, and exiting the CN dorsally (Fig. 2D). In the remaining sample of neurons (five intracellular; 14 extracellular), a combination of onset response patterns were observed. These were typified by a prominent onset AP in response to BF tones, and occasional late discharge, but only exhibited a 2nd regular AP after onset in the pres-

Fig. 1. Intracellular profile of the OC response. Single (left) and averaged (right) intracellular response to 50 ms BF tone bursts (A–B) or noise (C) presented at 40–50 dB re threshold (BF = 25 kHz in A; 19 kHz in B). Horizontal dashed line represents RMP; horizontal bar indicates stimulus duration. (D) Contour plots depicting changes in membrane potential from RMP (averaged over 20–25 repetitions) in response to BF tones (upper and middle) and noise (lower) as a function of SPL over both initial 10 ms (left) and duration (right) of stimulus presentation. Key shows change in membrane potential from RMP over range of 5 to +15 mV (upper) and 6 to +20 mV (middle and lower).

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Fig. 2. Response area and morphology of an OC neuron. Averaged membrane potential recorded over 11–27 kHz at 60 dB (A) or 10–28 kHz at 75 dB (B). Horizontal dashed line indicates BF (19 kHz). Key shows membrane potential changes from RMP over range of 5 to +15 mV. (C) Single (left) and averaged (right) intracellular response to 50 ms tone bursts (75 dB) presented at below-BF (10.5 and 14 kHz), at BF (19 kHz) and above-BF (21 and 27 kHz). (D) Morphological reconstruction of neuron 04-600-004 following intracellular Neurobiotin injection.

ence of noise. Whilst this activity fits the pattern described as OnL, responses to both BF tones and noise were not recorded in all such neurons. In addition, the high BF (>24 kHz), and/or threshold (>50 dB) displayed by many neurons within this group, prevented a response to BF tones at 30 dB or above, from being recorded. As such, these neurons were broadly characterised as onset late or onset ideal (OnL/OnI). 3.2. Chopper neurons Intracellular recordings were obtained for 34 chopper neurons. The mean RMP was 48.9 ± 1.6 mV, with average AP amplitude of 37.6 ± 1.1 mV, mean BF of 14.0 ± 1.5 kHz, and mean firing threshold of 41.8 ± 4.8 dB SPL. The chopper intracellular profile was characterised by a precisely-timed onset AP, followed by synchronised chopping activity, firing at a rate unrelated to stimulus frequency (Fig. 3). In CS neurons, firing regularity was maintained for the duration of stimulus presentation, as illustrated in both single (Fig. 3A) and averaged (Fig. 3B) intracellular traces recorded in response to BF tones. Alternatively, an intracellular profile displaying a reduction in regularity over time, with sustained depolarisation throughout, was identified as CT (Fig. 3C– D). Similar response profiles were also observed in response to noise (CS – Fig. 3E; CT – Fig. 3F). Membrane potential changes, as depicted in contour plots (Fig. 3G),

illustrated clear distinctions between the response properties of CS (upper) and CT neurons (lower): the CS response displayed distinct peaks in depolarisation interspersed by regions of hyperpolarisation, whilst CT activity demonstrated a well-timed onset peak in depolarisation followed by diffuse periods of activity as well as sustained depolarisation for the remainder of the tone burst. From the sample of 34 intracellularly-recorded neurons displaying responses akin to those described above, four were identified as CS, whilst 15 were classified as CT. The remaining 15 neurons, identified by their intracellular profile in response to noise only, could not be definitely classified as CS or CT on the basis of CV calculations, and were therefore defined simply as chopper neurons. 3.3. Intracellular response profile Given that both OC and CT response patterns are noted for their loss of firing regularity with stimulus duration, late irregular activity, and sustained depolarisation, a scheme devised by Paolini et al. (2005) to quantify different aspects of the intracellular response profile, provided a means to differentiate the intracellular activity of OC and CT neurons. The OC population was primarily distinguished from the sample of CT neurons by the characteristic peak in depolarisation at onset (Fig. 4A–B). Calculated as the difference in membrane potential from onset (MPt5-10) to 20–30 ms post-tone onset (MPt20-30), the OC peak in

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Fig. 3. Intracellular response of chopper neurons. Single (A) and averaged (B) intracellular response to 50 ms BF tone bursts (7 kHz) presented at 30 dB re threshold for sustained chopper (CS). Single (C) and averaged (D) intracellular response to 50 ms BF tone bursts (15 kHz) presented at 30 dB re threshold for transient chopper (CT). Single intracellular response of CS (E) and CT (F) to 50 ms noise burst. (G) Contour plots depicting average change in membrane potential from RMP in response to BF tone bursts across SPL over initial 10 ms of stimulus presentation for CS (upper) and CT (lower) neuron. Key for CS shows range of 2 to +6 mV; Key for CT shows range of 4 to +11 mV.

Fig. 4. Intracellular response profile properties of OC and CT neurons. (A) Mean (±standard error) change in averaged membrane potential from stimulus onset (5–10 ms after stimulus onset: MPt5-10) to 20–30 ms post stimulus onset (MPt20-30) as a function of tone SPL for OC (open circles) and CT (closed circles) populations. (B) Example of intracellular profile of OC (grey) and CT (black) response to BF tone burst at 10 dB re threshold (averaged over 20 stimulus presentations). Individual examples of typical change in membrane potential from RMP at 20 ms (circle), 50 ms (triangle) and 60 ms (square) post-tone onset for OC (C) and CT (D–E) neurons. Shaded regions denote adaptation of depolarisation seen with stimulus duration. Horizontal dashed line represents RMP.

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depolarisation was most prominent at depolarisation threshold (Fig. 4A). An example of the response profile exhibited by an OC neuron (Fig. 4C) showed little-to-no adaptation of depolarisation between 20 ms (MPt20) and 50 ms (MPt50) post-stimulus onset (measured re RMP), and no post-tone hyperpolarisation, measured as the change in membrane potential from RMP at 60 ms posttone onset (MPt60). Alternatively, CT neurons exhibited one of two patterns: (i) low adaptation of depolarisation over time, and no post-tone hyperpolarisation (Fig. 4D); or (ii) large depolarisation adaptation and significant post-tone hyperpolarisation (Fig. 4E). 3.4. The commissural connection between stellate populations The nature of interactions between the two stellate populations was investigated through an examination of the commissural pathway. As demonstrated previously (Needham and Paolini, 2003), antidromic APs were activated in OC neurons following electrical stimulation of the contralateral CN, indicating the presence of commissural projections. In the extended sample of 51 onset neurons presented here, antidromic activity was observed in 16 cells, including neurons from both OC and OnL/OnI groups (Table 1). As displayed in Fig. 5, antidromic APs were induced with consistently short onset latency (mean latency 1.32 ± 0.07 ms), steady with both increasing stimulus strength (Fig. 5B), and multiple presentations at a single intensity (Fig. 5C). Consistent with the all-or-none nature of antidromic activity, no excitatory synaptic potentials were observed in response to sub-threshold stimuli (Fig. 5B – 160 lA; Fig. 5C – 170 lA). Collision of the electrically-evoked antidromic AP with an ipsilaterally-induced orthodromic AP via manipulation of the inter-spike interval (Fig. 5D), eliminated the presumed antidromic AP when inter-stimulus interval was less than the sum of the orthodromic APs absolute refractory period and the onset latency of the antidromic AP (Fig. 5D iii). Importantly, antidromic activity was absent from recordings taken from the sample of chopper neurons (Table 1), thereby providing no physiological evidence of contralateral input from VCN chopper neurons. Table 1 Presence of commissurally-induced activity within onset and chopper populations with contralateral electrical stimulation Neuronal group

Total number

% Antidromic

All onset cells Intracellular onset cells Extracellular onset cells OC cells OnL/OnI cells All chopper cells Identified CS cells Identified CT cells

51 17 34 32 19 34 4 15

31.4 17.6 38.2 28.1 36.8 0

% Inhibited 0

64.7 50 80

Number of neurons within each group demonstrating antidromic activity or inhibition expressed as a percentage.

Fig. 5. Antidromic activity displayed by onset neurons in the VCN. (A) Antidromic AP seen in response to 200 lA electrical pulse presented to the contralateral CN (artefact denoted s). (B) Averaged intracellular responses (over 20 presentations) recorded in response to varying electrical stimulation strength (160–200 lA) display the all-or-none nature of the antidromic AP. Membrane potential normalised to zero. Horizontal dashed line indicates normalised RMP. (C) Single intracellular traces in response to electrical stimulation at threshold (170 lA). Horizontal dashed line indicates RMP. Asterisks denote truncated AP. (D) Collision test: as the interval between ipsilaterally-evoked activity (70 dB; vertical dashed line denoted (i), and antidromic activity decreased (i–iii), the ipsilaterally-AP collided with the antidromic AP, thereby preventing it from reaching the soma (iii). When ipsilateral input was presented following contralateral stimuli, the electrically-evoked antidromic AP returned (iv).

Instead, stimulation of the contralateral CN via electrical input induced monosynaptic (mean latency 1.52 ± 0.11 ms), fast inhibitory postsynaptic potentials (IPSPs) in 22 (64.7%) of the 34 intracellularly-recorded chopper neurons (mean amplitude 3.42 ± 0.57 mV; mean duration 9.86 ± 1.10 ms; Table 1). As displayed in Fig. 6A and B, electrical stimuli rapidly induced large IPSPs in chopper neurons, steady in onset latency, but increasing in amplitude with increasing stimulus strength. Consistent across the sample of neurons, hyperpolarisation amplitude was largest at onset, decaying rapidly thereafter. Activation of the commissural pathway with contralaterally-presented acoustic (click) input also evoked inhibition

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of a similar time course (Fig. 6C), suggesting that both stimuli activate the same pathway. Furthermore, the onset latency displayed by acoustically-induced hyperpolarisation (mean 3.77 ± 0.15 ms) was consistent with a monosynaptic connection between cochlear nuclei following transmission through the cochlea and auditory nerve. Given the chopping activity exhibited in OC and some OnL neurons in response to noise, a high frequency train of electrical pulses (Fig. 6D) and acoustic clicks (Fig. 6E) was presented to the contralateral CN in an effort to simulate this discharge pattern. High frequency stimulation resulted in the summation of inhibitory potentials, with hyperpolarisation duration and amplitude increasing linearly with stimulus frequency (Fig. 6F: intracellular response of neuron 02-716-001 previously published in (Needham and Paolini (2003)). A similar pattern of activity

Fig. 6. Inhibitory influence of commissural pathway on chopper neurons. Averaged (and normalised) intracellular response of chopper neurons 03729-003 (A) and 03-754-013 (B) to contralateral electrical stimulation (200 lA; artefact denoted s) presented over a range of strength. (C) Averaged intracellular profile recorded in response to contralateral acoustic click (90 dB; vertical dashed line denoted c). (D) Intracellular response to multiple electrical pulses (100–300 Hz). (E) Intracellular response to single (black) or multiple (grey) contralateral clicks. (F) Change in amplitude (closed circles) and duration (open circles) of hyperpolarisation with increasing stimulus frequency (100–500 Hz; electrical pulse 100 lA); intracellular recordings of neuron 02-716-001 in response to high frequency electrical stimulation previously published in Needham and Paolini (2003).

was also evident following the presentation of noise to the contralateral ear (Fig. 7), with three distinct periods of hyperpolarisation evident in both the averaged intracellular response (Fig. 7A), and contour plot (Fig. 7B). In order to ascertain the influence of commissurallyderived inhibition on the activity of chopper neurons, the onset latency of ipsilaterally-evoked APs were examined through the manipulation of the inter-stimulus interval between contralateral stimulation (electrical, clicks or noise), and ipsilateral input (clicks or BF-tone bursts). When ipsilateral APs were induced during periods of commissurally-evoked hyperpolarisation (shaded regions), onset latency was significantly delayed relative to APs induced prior to, or without, contralateral stimulation (Fig. 8A–D; Kruskal–Wallis one-way ANOVA; p = < 0.001; results for neuron 02-712-002 and 03-729-005 previously published in Paolini et al. (2004)). However, this effect was neither uniform nor linear across the shaded region, with AP latency delay differing between neurons (Fig. 8A–D), as well as different stimulus conditions (Fig. 8B). In all examples, latency was delayed to the largest degree when hyperpolarisation of maximum amplitude occurred simultaneous to AP generation. In two neurons,

Fig. 7. Response of chopper neuron to presentation of contralateral noise. (A) Averaged intracellular response to 50 ms contralateral noise burst (80 dB). Asterisk denotes period of hyperpolarisation at stimulus onset. Horizontal dashed line indicates RMP. (B) Contour plot illustrating changes in membrane potential at onset of contralateral noise presentation. Asterisks denote three distinct periods of hyperpolarisation. Key shows change in membrane potential from RMP over range of 1 to +3.5 mV.

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Fig. 8. Effects of commissural input on spike timing in intracellular chopper neurons. Mean onset spike latency (normalised) is plotted as a function of stimulus-interval for neurons 02-712-002 (A), 03-729-003 (B), 03-729-005 (C) and 04-761-002 (D). Significant delays in AP onset were seen during coincident stimulation of ipsilateral activity and contralaterally-evoked hyperpolarisation (Kruskal–Wallis one-way ANOVA; p = < 0.001): the time period over which the ipsilaterally-driven AP and contralaterally-induced hyperpolarisation overlap is denoted by the shaded region. Positive values indicate an increase in mean onset latency, whilst negative values denote a decrease. Open circles indicate click-driven ipsilateral response during contralateral electrical stimulation; closed circles denote BF-tone driven ipsilateral response during contralateral electrical stimulation; and closed triangles indicate click-driven ipsilateral response during contralateral click stimulation. (E) Mean onset latency in response to ipsilateral BF tone presented alone (left) and during simultaneous presentation of contralateral noise (90 dB; right). Onset latency was significantly delayed during binaural stimulation (Mann–Whitney rank sum test; **p = < 0.001). (F) Onset latency in response to ipsilateral BF tone of increasing sound pressure level presented alone (grey circles) and during simultaneous presentation of white noise (90 dB; black circles). Onset latency was significantly delayed during all binaural conditions (Mann–Whitney rank sum test; **p = < 0.001). Spike timing results and intracellular recordings of neurons 02-712-002 and 03-729-003 previously published in Paolini et al. (2004).

contralaterally-presented noise was also able to significantly delay first spike latency when presented simultaneous to an ipsilateral BF tone burst (Fig. 8E and F; **Mann–Whitney rank sum test; p = < 0.001). 4. Discussion This study examines the intracellular activity of T and D stellate neurons in the VCN, describing the nature of the interaction between these populations through activation of the commissural pathway. The more extensive overview provided here by our increased sample of stellate neurons is able to both confirm and expand upon the results of our previous studies by illustrating that (i) D stellate neurons of both OC and OnL/OnI response types contribute projections to the commissural connection; (ii) around two-thirds of T stellate (chopper) neurons receive fast monosynaptic inhibitory input from the contralateral CN; and (iii) inhibitory commissural input is able to influence the timing of activity in chopper neurons. 4.1. Intracellular activity of onset neurons The intracellular profile exhibited by onset neurons, consistent with previous findings (Feng et al., 1994; Paolini

and Clark, 1999; Paolini et al., 2004; Paolini et al., 2005; Rhode and Smith, 1986; Rhode et al., 1983; Smith and Rhode, 1989), is typified by a prominent precisely-timed onset AP followed by sustained depolarisation for the remainder of the stimulus presentation. In the OC response (the predominant onset type recorded), onset activity is accompanied by a brief period of firing regularity, and on occasion, low levels of irregular late discharge at higher stimulus SPLs. As established by Paolini et al. (2005), this intracellular profile can be identified by a peak in depolarisation at onset, a near-absence of depolarisation adaptation between 20 and 50 ms post-stimulus onset, and negligible post-tone hyperpolarisation. These neurons respond to a broad range of frequencies, and are vigorously activated by broadband stimuli due to the extensive and wide range of afferent auditory input received by the D stellate neuron (Doucet and Ryugo, 1997; Palmer et al., 1996; Palmer et al., 2003; Paolini and Clark, 1999; Smith and Rhode, 1989; Smith et al., 2005; Winter and Palmer, 1995). As shown both here and previously (Paolini and Clark, 1999), the behaviour of OC activity changes somewhat over its broad response area, with depolarisation decreasing in amplitude in response to above-BF tones, and increasing slightly below-BF. The reduction in depolarisation aboveBF is also accompanied by a precisely-timed AP at stimulus

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offset. This response pattern is consistent with the nature of OC activity shown by Paolini and Clark (1999), and indicates that whilst the onset activity is maximal at BF, the propensity for neural discharge during the remainder of the tone is greater below-BF. In addition to the OC pattern of activity, responses akin to the OnL and/or OnI profile were also encountered within the VCN. OnL activity was identified by a strong onset response followed by low levels of late irregular discharge during BF tones, and the onset of a regular 2nd spike in response to noise, whilst OnI activity is noted for its solitary AP at onset (Arnott et al., 2004; Jiang et al., 1996; Rhode and Smith, 1986; Rhode et al., 1983; Smith et al., 2005; Winter and Palmer, 1995). Whilst both OnL and OnI response patterns have been associated with the octopus cell (Godfrey et al., 1975), a population of neurons restricted to the OCA at the posterior pole of the VCN (Kane, 1973; Osen, 1969), the deliberate avoidance of this region within the current study suggests that the OnL/OnI responses described here are unlikely to have originated from octopus cells. Consistent with this proposal, a number of studies have recorded OnL activity from non-OCA regions of the VCN (Arnott et al., 2004; Jiang et al., 1996; Palmer et al., 2003; Smith et al., 2005; Winter and Palmer, 1995), with some studies identifying their source as D stellate-like neurons (Arnott et al., 2004; Palmer et al., 2003; Smith et al., 2005). Furthermore, together with the findings of Paolini et al. (2005), the results provided here indicate that the OnI response pattern can be recorded from OC or OnL neurons in response to BF tones presented <30–40 dB re threshold. It is therefore surmised that responses described as OnL/OnI also represent the activity of the D stellate neuron. 4.2. Intracellular activity of chopper neurons Activity consistent with the discharge pattern described as chopper was observed in 34 intracellularly-recorded neurons in the VCN. The intracellular profile of the chopper neurons were described by sustained or transient discharge (CS and CT, respectively) on the basis of their firing regularity over the course of stimulus presentation. Neurons identified as CS displayed a regular pattern of AP’s throughout the BF-tone and/or noise presentation, with no sustained depolarisation of the membrane potential present. Alternatively, the CT profile was noted for its loss of firing regularity, and sustained depolarisation throughout. Whilst sharing some similarities with the OC intracellular profile, CT activity was differentiated by the absence of a peak in depolarisation at stimulus onset and the presence of depolarisation adaptation over time. The degree of depolarisation adaptation varied between CT neurons: responses displaying low adaptation were rarely accompanied by post-tone hyperpolarisation, whilst those neurons exhibiting a large change in depolarisation amplitude also tended to demonstrate significant post-tone hyperpolarisation. These features are consistent with those described by

Paolini et al. (2005) in a separate population of neurons, and indicate that neural discharge in the CT population is determined by the presence of BF-centred inhibitory input. 4.3. Involvement of stellate neurons in the commissural connection The commissural connection provides a monosynaptic path between cochlear nuclei, and thereby, a direct route by which contralateral input is able to influence auditory processing through the cochlear nucleus (Babalian et al., 1999, 2002; Cant and Gaston, 1982; Davis, 2005; Joris and Smith, 1998; Needham and Paolini, 2003; Schofield and Cant, 1996; Shore et al., 2003; Shore et al., 1992). The presence of this direct link has been demonstrated anatomically (Alibardi, 1998; Alibardi, 2000a; Alibardi, 2000b; Arnott et al., 2004; Cant and Gaston, 1982; Schofield and Cant, 1996; Shore et al., 1992; Smith et al., 2005; Wenthold, 1987), with the majority of studies nominating large multipolar neurons in the VCN, akin to neurons of the D stellate population, as the predominant source of commissural projections. The induction of antidromic activity in OC neurons following electrical stimulation of the contralateral CN (present results: Needham and Paolini, 2003), confirms such reports. Yet in this extended sample, antidromic activity was also observed in OnL/OnI neurons, providing physiological evidence that these neurons also contribute projections to commissural pathway. This finding is consistent with the report by Smith and colleagues (2005), of an OnL multipolar neuron providing input to the contralateral CN. Whilst there is also evidence to suggest that OnL projections do not exit the CN (Arnott et al., 2004; Palmer et al., 2003), this assumption was based on the morphology of only two OnL neurons. It is therefore likely that this sample may not be representative of the OnL population as a whole. Indeed, as demonstrated here, the presence of antidromic activity in only a portion of the onset neurons examined (31.4%) suggests that not all onset/D stellate neurons provide input to the contralateral CN. Although this result might reflect an inability of our electrical stimuli to evoke all commissural projections, this would appear unlikely given the similar percentage of chopper/T stellate neurons inhibited by electrical stimulation of the contralateral CN (64.7%) versus contralateral auditory nerve (63–67%: Babalian et al., 1999, 2002). In accord with the glycinergic nature of D stellate neurons (Doucet et al., 1999; Kolston et al., 1992; Smith and Rhode, 1989; Wenthold, 1987; Wenthold et al., 1987), activation of the commissural pathway has been shown to induce fast inhibitory potentials (Babalian et al., 1999, 2002; Needham and Paolini, 2003), and suppress both spontaneous and ipsilaterally-driven activity (Davis, 2005; Joris and Smith, 1998; Mast, 1970; Shore et al., 2003; Young and Brownell, 1976) in numerous cell types throughout the CN. The results presented here extend the findings of our previous study (Needham and Paolini, 2003), by demonstrating the activation of fast, monosynap-

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tic inhibition in 22 of 34 (64.7%) chopper neurons following contralateral stimulation. This physiological evidence of commissural input to chopper neurons verifies anatomical observations that T stellate neurons, with which the chopper response is associated (Palmer et al., 2003; Paolini et al., 2004; Paolini et al., 2005; Smith and Rhode, 1989), are a principal recipient of direct contralateral projections (Schofield and Cant, 1996). 4.4. Nature of the interaction between stellate neurons In light of evidence identifying the D stellate population as the principal source of commissural projections, and T stellate neurons as one of their main targets (Alibardi, 1998; Arnott et al., 2004; Babalian et al., 1999, 2002; Cant and Gaston, 1982; Davis, 2005; Joris and Smith, 1998; Needham and Paolini, 2003; Schofield and Cant, 1996; Smith et al., 2005; Wenthold, 1987), the behaviour of contralaterally-induced hyperpolarisation and its influence on T stellate neurons provides an insight into the nature of interactions between the two stellate populations. Given the broad tuning characteristic of the D stellate response to acoustic input, it is of little surprise that commissurally-evoked hyperpolarisation in chopper neurons (Needham and Paolini, 2003), as well as contralaterally-induced suppression in the DCN (Davis, 2005; Joris and Smith, 1998; Mast, 1970; Young and Brownell, 1976) are more evident in response to noise, than single frequency tones. The fast, transient nature of inhibitory potentials observed following commissural stimulation is consistent with both the prominent onset response displayed by D stellate neurons, and the glycinergic nature of their projections (Babalian et al., 2002; Davis, 2005; Kolston et al., 1992; Wenthold, 1987). We have shown here, and in previous studies (Paolini et al., 2004), that activation of fast inhibitory potentials following contralateral stimulation significantly delays the onset latency of ipsilaterally-driven activity in chopper neurons. This finding demonstrates that inhibition derived from D stellate neurons is able to alter the timing of neural activity in T stellate neurons. The degree to which this is delayed is reliant on the strength (amplitude) of hyperpolarisation relative to that of the ipsilaterally-driven AP, together with the timing (or overlap) between the two events. Indeed, the summation of inhibitory potentials following high frequency contralateral stimulation, together with the distinct regions of hyperpolarisation observed during contralateral noise presentation, demonstrate that the amplitude and duration of commissural effects can be mediated by the firing activity evoked within the commissural D stellate neuron. Evidence of this connection between local T and D stellate neurons has been provided by our previous study (Paolini et al., 2004), where fast inhibition induced by off-BF tones was found to occur prior to activation of APs in chopper neurons, thereby delaying their induction. These findings have led to a time-based frequency coding theory

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being proposed in which neural delay is the principal outcome of lateral inhibition from D stellate neurons (Paolini et al., 2005), such that within the chopper population, neurons with BF’s corresponding to spectral peaks will respond faster than those in adjacent isofrequency laminae, which will be actively delayed. Likewise, commissural inhibition may provide a mechanism by which contralateral broadband stimuli is able to delay T stellate responses to weak or lateral signals relative to frequency-specific activity, thereby enhancing signal detection in noise. Acknowledgements The authors thank R.E. Millard for engineering support. Funding provided by a Melbourne Research Scholarship and the Department of Otolaryngology, The University of Melbourne. References Adams, J.C., 1983. Multipolar cells in the ventral cochlear nucleus project to the dorsal cochlear nucleus and the inferior colliculus. Neurosci. Lett. 37, 205–208. Adams, J.C., 1993. Non-primary inputs to the cochlear nucleus visualized using immunocytochemistry. In: Merchan, M.A., Juiz, J.M., Godfrey, D.A., Mugnaini, E. (Eds.), The Mammalian Cochlear Nuclei: Organization and Function. Plenum Press, New York, pp. 133–141. Alibardi, L., 1998. Ultrastructural and immunocytochemical characterization of commissural neurons in the ventral cochlear nucleus of the rat. Ann. Anatomy 180, 427–438. Alibardi, L., 2000a. Putative commissural and collicular axo-somatic terminals on neurons of the rat ventral cochlear nucleus. J. Submicrosc. Cytol. Pathol. 32, 555–566. Alibardi, L., 2000b. Cytology, synaptology and immunocytochemistry of commissural neurons and their putative axonal terminals in the dorsal cochlear nucleus of the rat. Ann. Anatomy 182, 207–220. Altschuler, R.A., Betz, H., Parakkal, M.H., Reeks, K.A., Wenthold, R.J., 1986. Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res. 369, 316–320. Arnott, R.H., Wallace, M.N., Shackleton, T.M., Palmer, A.R., 2004. Onset neurones in the anteroventral cochlear nucleus project to the dorsal cochlear nucleus. J. Assoc. Res. Otolaryngol. 5, 153–170. Babalian, A.L., Ryugo, D.K., Vischer, M.W., Rouiller, E.M., 1999. Inhibitory synaptic interactions between cochlear nuclei: evidence from an in vitro whole brain study. Neuroreport 10, 1913–1917. Babalian, A.L., Jacomme, A.V., Doucet, J.R., Ryugo, D.K., Rouiller, E.M., 2002. Commissural glycinergic inhibition of bushy and stellate cells in the anteroventral cochlear nucleus. Neuroreport 13, 555–558. Blackburn, C.C., Sachs, M.B., 1989. Classification of unit types in the anteroventral cochlear nucleus: PST histograms and regularity analysis. J. Neurophysiol. 62, 1303–1329. Blackburn, C.C., Sachs, M.B., 1992. Effects of OFF-BF tones on responses of chopper units in ventral cochlear nucleus. I. Regularity and temporal adaptation patterns. J. Neurophysiol. 68, 124–143. Cant, N.B., 1981. The fine structure of two types of stellate cells in the anterior division of the anteroventral cochlear nucleus of the cat. Neuroscience 6, 2643–2655. Cant, N.B., 1992. The cochlear nucleus: neuronal types and their synaptic organization. In: Webster, D.B., Popper, A.N., Fay, R.R. (Eds.), The Mammalian Auditory Pathway: Neuroanatomy. Springer Verlag, New York, pp. 66–116. Cant, N.B., Gaston, K.C., 1982. Pathways connecting the right and left cochlear nuclei. J. Comp. Neurol. 212, 313–326.

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