Purkinje cell degeneration and control mice: responses of single units in the dorsal cochlear nucleus and the acoustic startle response

Hearing Research 148 (2000) 137^152 www.elsevier.com/locate/heares

Purkinje cell degeneration and control mice: responses of single units in the dorsal cochlear nucleus and the acoustic startle response K. Parham a , G. Bonaiuto a , S. Carlson b;1 , J.G. Turner b;2 , W.R. D'Angelo a , L.S. Bross b , A. Fox a;3 , J.F. Willott b , D.O. Kim a; * a

Division of Otolaryngology and Surgical Research Center, Department of Surgery and Neuroscience Program, University of Connecticut Health Center, Farmington, CT 06030-1110, USA b Department of Psychology, Northern Illinois University, DeKalb, IL 60115, USA Received 29 September 1999; accepted 7 June 2000

Abstract The cartwheel cell is the most numerous inhibitory interneuron of the dorsal cochlear nucleus (DCN). It is expected to be an important determinant of DCN function. To assess the contribution of the cartwheel cell, we examined the discharge characteristics of DCN neurons and behavioral measures in the Purkinje cell degeneration (pcd) mice, which lack cartwheel cells, and compared them to those of the control mice. Distortion product otoacoustic emissions and auditory brainstem-evoked response thresholds were similar between the two groups. Extracellularly recorded DCN single units in ketamine/xylazine-anesthetized mice were classified according to post-stimulus time histogram (PSTH) and excitatory^inhibitory response area (EI-area) schemes. PSTHs recorded in mouse DCN included chopper, pauser/buildup, onset, inhibited and non-descript types. EI-areas recorded included Types I, II, III, I/III, IV and V. There were no significant differences in the proportions of various unit types between the pcd and control mice. The pcd units had slightly lower thresholds to characteristic frequency tones; however, they had spontaneous rates, thresholds to noise, and maximum driven rates to noise that were similar to those of the control units. Pcd mice had smaller startle amplitudes, but startle latency, prepulse inhibition/augmentation and facilitation by a background tone were comparable between the two groups. From these results, we conclude that DCN function in response to relatively simple acoustic stimuli is minimally affected by the absence of the cartwheel cells. Future studies employing more complex and/or multimodal stimuli should help assess the role of the cartwheel cells. ß 2000 Elsevier Science B.V. All rights reserved. Key words: Acoustic startle response; Dorsal cochlear nucleus; Purkinje cell degeneration

1. Introduction Among the dorsal cochlear nucleus (DCN) interneurons, cartwheel cells are the most common class of inhibitory interneurons (Mugnaini, 1985; Wenthold et al., 1987 ; Osen et al., 1990; Berrebi and Mugnaini, 1991).

* Corresponding author. Tel.: +1 (860) 679 3690; Fax: +1 (860) 679 2451; E-mail: [email protected] 1 Present address: Department of Psychology, Bethel College, Mishawaka, IN 46545, USA. 2 Present address: Department of Psychology, University of Tennessee, Martin, TN 38238, USA. 3 Present address: Department of Otolaryngology, SUNY HSCSyracuse, Syracuse, NY 13210, USA.

Cartwheel cells are located in molecular and upper fusiform cell layers, where few auditory nerve ¢bers terminate (Osen, 1970 ; Lorente de No, 1981 ; Brown and Ledwith, 1990 ; Ryugo and May, 1993). Their main excitatory input is via glutamatergic endings of granule cells in the molecular layer of the DCN (Kane, 1974; Godfrey et al., 1977 ; Oliver et al., 1983; Wouterlood and Mugnaini, 1984; Wouterlood et al., 1984; Golding and Oertel, 1996). Cartwheel cells synapse principally on the fusiform cells (DCN's main projection neurons) and other cartwheel cells (Berrebi and Mugnaini, 1991). Because they outnumber the fusiform cells, cartwheel cells are expected to play a signi¢cant role in shaping the DCN output. In vitro intracellular recording and labeling studies

0378-5955 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 0 0 ) 0 0 1 4 7 - 7

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suggest that the complex spike discharge in the DCN is exclusively associated with cartwheel cells (e.g. Zhang and Oertel, 1993a ; Manis et al., 1994). Based on this association, in vivo DCN recordings of complex spike discharges have been ascribed to cartwheels cells (Parham and Kim, 1995; Davis and Young, 1997). A preliminary in vivo intracellular recording and labeling study further established this association (Ding and Voigt, 1996). The majority of complex spiking cells are poorly driven by pure tones and broad-band noise (Parham and Kim, 1995; Davis and Young, 1997). In response to electrical stimulation of the somatosensory nuclei or parallel ¢bers, the cartwheels cells are believed to be responsible for long-latency inhibition of the fusiform cells (Young et al., 1995; Davis et al., 1996a). Details of cartwheel cell interaction with other cells in encoding stimuli remain unclear. A classic method of assessing the contribution of an element of a circuit to the overall function of the whole to which it belongs is to study the circuit in the presence and absence of that element. This task is feasible when the elements of that circuit are physically separate. When they are closely intertwined (i.e. consisting of neighboring cells) then selective removal of a single element represents a major challenge. In the present context, such a challenge exists in assessing the contribution of the cartwheel cells to DCN function. Several murine mutations, such as lurcher, Purkinje cell degeneration (pcd), staggerer, nervous, and tambaleante, have been identi¢ed as having postnatal degeneration of cerebellar Purkinje cells (Green, 1989). Among these, the pcd mouse shows rapid and nearly complete loss of Purkinje cells between 17 and 45 days of age (Mullen et al., 1976; Landis and Mullen, 1978). The pcd mutation is associated with a spontaneous mutation in the pcd gene located on chromosome 13 (Green, 1989; Campbell and Hess, 1996; Sweet et al., 1996) with a recessive mode of inheritance. The cartwheel cell of the DCN, the proposed homologue of the cerebellar Purkinje cell, also undergoes degeneration in the pcd mouse (Berrebi et al., 1990). The pcd mouse, therefore, o¡ers a unique opportunity to evaluate the contribution of cartwheel cells to DCN function. The overall goal of this study is to take an initial step in evaluating DCN function in mice with (i.e. control) and without (i.e. pcd) cartwheel cells. Toward this objective, we have adopted a multidisciplinary approach by using both electrophysiological and behavioral measures. The electrophysiological characteristics of the DCN neurons in vivo have been documented in several species in both post-stimulus time histogram (PSTH) and excitatory^inhibitory response area (EI-area) schemes (cat: e.g. Pfei¡er, 1966; Young and Brownell, 1976 ; Shofner and Young, 1985; Rhode and Smith, 1986 ; chinchilla : Kaltenbach and Saunders, 1987 ; rab-

bit: Hui and Disterhoft, 1980; gerbil : Gdowski and Voigt, 1997; Davis et al., 1996b ; guinea pig : Stabler et al., 1996). To date, no systematic account of response characteristics of mouse DCN neurons exists. Therefore, the ¢rst goal of this paper is to document the response characteristics of mouse DCN neurons in the PSTH and EI-area schemes. The second goal of this paper is to assess whether response properties of DCN neurons in pcd mice di¡er from those of control mice. Because of the absence of inhibitory cartwheel neurons in the pcd mutant, we have also employed several behavioral tests that involve the acoustic startle response. The startle response is mediated by relatively short excitatory neural pathways in the lower brainstem : neurons in the ventral cochlear nucleus (VCN) project either directly and/or with one or two intermediary synapses to the caudal pontine reticular nucleus (PnC), whose e¡erent axons descend the spinal cord and trigger the re£ex (Davis et al., 1982; Davis, 1984; Pellet, 1990; Kandler and Herbert, 1991 ; Lingenho«hl and Friauf, 1994). Modulation of the acoustic startle response, on the other hand, may involve an interaction of excitatory and inhibitory circuits. The startle response can be modi¢ed in three ways using moderately intense, nonre£exogenic acoustic stimuli. The ¢rst is prepulse inhibition (PPI), in which a tone prepulse (S1 ) is presented 10^200 ms before an intense startle-evoking sound (S2 ) (Ho¡man and Ison, 1980). During the S1 ^S2 interval, the S1 activates neural circuits in the auditory brainstem and the resulting signals descend to the PnC, inhibiting the startle response evoked by S2 (Leitner and Cohen, 1985; Carlson and Willott, 1998 ; Li et al., 1998a,b). The second is prepulse augmentation (PPA), where a short S1 ^S2 interval ( 6 5 ms) results in a larger startle response (Willott and Carlson, 1995). PPA is thought to involve `priming' of the excitatory startle pathway by the S1 , which lasts for a few ms (Ho¡man and Ison, 1980; Ison et al., 1973). However, PPA is also likely to be countered by inhibitory circuits within the cochlear nucleus (cf. Oertel and Wickesberg, 1993; Osen et al., 1990 ; Wickesberg et al., 1994). There is evidence that inhibition is diminished in the cochlear nucleus of hearing-impaired C57BL/6J mice, and PPA is enhanced in these animals (Willott and Carlson, 1995). Third, the presence of a moderately intense background tone or noise can facilitate startle amplitude (Ho¡man et al., 1969 ; Davis, 1974 ; Corey and Ison, 1979 ; Ison and Russo, 1990). The mechanism(s) responsible for this e¡ect are not fully understood. To the extent that the above three phenomena may involve an interaction between excitatory and inhibitory neural circuits, given the well-established anatomical and physiological interconnections between the VCN

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and DCN (e.g. Wickesberg and Oertel, 1990; Saint Marie et al., 1991 ; Ostapo¡ et al., 1999), it is possible that pcd mice lacking the inhibitory cartwheel neurons may exhibit abnormal responses regarding PPI, PPA or background facilitation. The third goal of this paper is to determine whether PPI, PPA or background facilitation of the acoustic startle re£ex is di¡erent between pcd and control mice. Preliminary ¢ndings of these studies were presented at the Association for Research in Otolaryngology (Bonaiuto et al., 1996; Willott et al., 1997a). 2. Materials and methods 2.1. Physiological experiments Mice belonging to two groups were tested : pcd and control. The pcd group consisted of 13 mice that were homozygous (3/3) for the pcd mutation (on a C57BL/ 6J background). The control group consisted of 17 mice : four CBA/J (CBA), seven CBA/CaJ and six C57BL/6J (C57) mice heterozygous (3/+) for the pcd mutation. The CBA and C57 mice were obtained from Jackson Laboratories, Bar Harbor, ME, USA and Charles River Laboratories, Kingston, NY, USA. The homozygous and heterozygous pcd mice were obtained from the New England Regional Primate Research Center, Harvard Medical School, Southborough, MA, USA and Jackson Laboratories, Bar Harbor, ME, USA. All mice were between 60 and 120 days of age at the time of testing. C57 mice older than 120 days of age are expected to exhibit high frequency hearing loss (e.g. Willott, 1986; Li and Borg, 1991; Parham, 1997). The homozygote pcd mice were distinguished from their heterozygote littermates based on their ataxia, resting tremor and smaller size. The mice were anesthetized by intraperitoneal injection of a mixture of ketamine/xylazine (0.12 and 0.01 mg/g body weight, respectively). Supplemental injections were given at 10^20% of the initial dose, as needed. Body temperature was maintained near 37³C with a regulated heating pad and monitored using a thermoprobe (Harvard Apparatus). Prior to single unit recordings, ¢ve control and 10 mutant ears underwent testing of distortion product otoacoustic emissions (DPOAE) and auditory brainstem-evoked potentials (ABR). The details of the DPOAE and ABR recording procedures in mice are described elsewhere (Parham, 1997 ; Parham et al., 1999). Brie£y, DPOAE recordings used f2 /f1 = 1.2 and L1 3L2 = 20 dB. 2f1 3f2 DPOAE input/output growth functions were obtained at f2 = 12, 13.4 and 16 kHz. The 2f1 3f2 DPOAE level was recorded in response to primaries at f2 = 8, 9.5, 10, 11.3,

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12, 13.4, 14.4 and 16 kHz, with L1 ¢xed at 50 dB SPL. ABR thresholds were determined using pure-tone bursts of 20 ms duration delivered in 5^10 dB steps between 0 and 80 dB SPL (re: 20 WPa) at 2, 4, 8, 10, 12, 14, 16, 24 and 32 kHz. For single unit recordings, the scalp was removed after subcutaneous lidocaine injection. The head of the mouse was secured by inserting its incisors into a bite bar and fastening a head bar to the skull using anchor screws and dental cement. A craniotomy was performed over the left posterior fossa and a portion of the cerebellum overlying the DCN was aspirated. Micropipettes ¢lled with 3 M NaCl with resistances of 15^50 M6 were lowered into the DCN for extracellular single unit recordings. In some penetrations the recording location was marked by iontophoretic injection of 10% biotinylated dextran amine (BDA) in 0.5 M potassium acetate in Tris bu¡er. As the electrode was advanced, a frequency-modulated tonal search stimulus was applied. The search stimulus had a frequency that was swept with a triangular modulating waveform over 0^50 kHz with a modulation repetition period of 4 s. The level of the swept tone was in the range of 40^60 dB SPL. The search stimulus aided in the isolation of individual units and provided a preliminary indication of the tuning characteristics of each unit. For each DCN neuron encountered, we recorded the following basic response characteristics similar to those studied in the cat cochlear nucleus (Parham and Kim, 1992 ; Ghoshal and Kim, 1997): (1) a 10 s sample of spontaneous discharges. (2) Response areas representing discharges induced by tone bursts (rise/fall time of 5 ms, tone duration of 200 ms and repetition period of 1 s) presented at various frequencies at a constant dB SPL typically 10^70 dB SPL. From the response areas, the characteristic frequency (CF) was de¢ned as the stimulus frequency which elicited the largest driven rate at a low stimulus level. (3) Response to pure tones (40^100 repetitions) having a frequency equal to the CF at a level of 60 dB SPL ; the tone burst for this measurement had rise/fall times of 5 ms, a duration of 50 ms and a repetition period of 500 ms. (4) Rate-level function for broadband noise covering 0^100 dB SPL (rms); the noise bursts had a rise/fall time of 5 ms, a duration of 200 ms and a repetition period of 1 s. For each unit, the spike waveform displayed on the oscilloscope was characterized as simple or complex (Parham and Kim, 1995). All spontaneous and evoked discharges were recorded on digital audio tapes. Further details about data analysis are described elsewhere (Parham and Kim, 1992, 1993; Ghoshal and Kim, 1997). At the conclusion of the single unit recording session, mice with BDA deposits were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital

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(0.15 mg/g body weight) and transcardially perfused with normal saline followed by 0.1% glutaraldehyde in 0.1 M sodium phosphate-bu¡ered saline. Frozen sections of the brainstem were cut at 50 Wm in the transverse plane. The sections were processed for visualization of the BDA-labeled injection sites using avidin^ biotin^peroxidase complex chemistry (Vector Laboratories) and alternate sections were stained with cresyl violet. 2.2. Behavioral experiments Five mice homozygous for the pcd gene (`mutants') and six heterozygotes (`controls') were obtained from the Jackson Laboratory, Bar Harbor, ME, USA. They were 54^90 days of age when tested. These mice were used only in the behavioral studies. The startle stimulus (S2 ) was a 100 dB SPL tone pip of 8 kHz (10 ms duration, 1 ms rise/fall time). Prepulse stimuli (S1 ) were tone pips (10 ms, duration, 1 ms rise/ fall time) of 4, 8, 12, 16 or 24 kHz at an intensity of 70 dB SPL. The stimuli were channeled to a Radio Shack Super tweeter speaker ¢tted at the top of the startle chamber (see Parham and Willott, 1988; Willott et al., 1994 for details). S1 ^S2 intervals, de¢ned with respect to the onset of S1 and the onset of S2 , were 1, 2, 3, 50 and 100 ms. Note that when the 1^3 ms intervals were used, S1 and S2 partially overlapped, but this had a minimal e¡ect on the intensity of S2 (less than 2 dB change in S2 SPL, which has no measurable e¡ect on startle amplitude). Mouse movements were measured using a stabilimeter described in an earlier paper (Parham and Willott, 1988), which converted mouse movements to voltages that were recorded on a storage oscilloscope. Acoustic startle re£ex (ASR) amplitude was de¢ned as the largest peak-to-peak voltage de£ection occurring within 30 ms of S2 onset. The latency was de¢ned as the time after onset of S2 at which the initial voltage de£ection began. Stimuli were presented in quiet; the ambient noise level was less than 50 dB SPL for octave bands centered at each target frequency. Procedures for acoustic calibration of the chamber are described elsewhere (Parham and Willott, 1988 ; Willott et al., 1994). A mouse was placed in the startle chamber. Testing was begun after initial exploratory behavior had diminished (typically 1^2 min) and after the animal had been presented with several introductory S2 -only stimuli to accommodate it to the testing environment. For testing, stimuli were presented when the mouse was not moving or grooming itself, with the between-trial intervals typically 20^25 s. The testing of each mouse was conducted over the course of several days with the order of presentation of S1 frequency and S1 ^S2 intervals varied across subjects.

Ten trials for each S1 ^S2 interval and the control (S2 only) condition were conducted in random order each day. Each mouse was ¢rst tested using 70 dB S1 of 4, 8, 12, 16 and 24 kHz with an S1 ^S2 interval of 100 ms. This combination produces signi¢cant PPI in mice (Willott et al., 1994). Next, shorter S1 ^S2 intervals (1, 2, 3 and 50 ms) were presented using the most e¡ective S1 , 12 kHz. Finally, the e¡ects of a continuous 70 dB 4 kHz background tone were determined for S2 -only and S1 ^S2 (S1 = 12 kHz) with a 1 ms S1 ^S2 interval. As was the case in our earlier study (Willott et al., 1994), the data in each session were trimmed by eliminating the highest and lowest ASR amplitudes for S1 ^ S2 trials of each S1 ^S2 interval condition and for the control condition (two out of 10 responses for each). This was done because of the occasional occurrence of very large or small ASRs that would distort the mean. A mean startle amplitude was then computed for the eight S1 ^S2 trials of each S1 ^S2 interval and the eight control trials for each session (i.e. each S1 frequency). Startle modi¢cation for each frequency was expressed as a percentage of the mean control startle amplitude for that session (S1 ^S2 trials divided by the mean of S2 only trials). Skewed distributions of frequency type data with multiple categories were compared between the control and mutant mice using the M2 test. Comparisons between control and mutant mice that involved only one variable were analyzed with t-tests. Startle modi¢cation (relative amplitude) was evaluated using two-way analysis of variance (ANOVA), with S1 frequency, S1 ^S2 interval or background tone (presence or absence) as repeated measures, and group (mutants versus controls) as an independent measure. In all cases, K = 0.05 (twotailed). The care and use of animals reported in this study was approved by the Animal Care Committee of the University of Connecticut Health Center (Auditory Neurobiology and Biophysics, Protocol No. 89-512), and the Institutional Animal Care and Use Committee of Northern Illinois University (Neurobehavioral Testing of Mutant and Genetically Altered Mice, Protocol No. 149). 3. Results 3.1. Physiological experiments The ABR thresholds and DPOAE characteristics of control and pcd mice were comparable. Fig. 1a shows the mean ABR thresholds of control and pcd mice between 2 and 32 kHz. The di¡erences between the two groups were not statistically signi¢cant (P s 0.05). Fig.

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Fig. 1. Mean ABR thresholds (a), mean DPOAE level (b) and mean DPOAE input/output functions (c) for ¢ve control and 10 pcd ears.

1b shows the mean DPOAE level of control and pcd mice. The detection thresholds of the two groups were comparable, although at f2 6 10 kHz, pcd mice had slightly lower DPOAE levels. This di¡erence was found to be statistically signi¢cant in a two-way analysis variance (groupUfrequency ; P 6 0.05). Mean DPOAE levels as a function of L2 at f2 = 13.4 kHz are shown in

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Fig. 1c for control and pcd mice. There was no signi¢cant di¡erence between the two groups. Other mean DPOAE input/output functions of control and pcd mice at f2 = 9.5 and 13.4 kHz with L1 3L2 = 10 and 20 dB also failed to exhibit signi¢cant statistical di¡erences. These results suggest that the pcd and control groups have comparable auditory sensitivity and cochlear function. Fig. 2a shows an example of a single unit recording site labeled with BDA at a recording depth of 645 Wm. This is a representative section of the DCN of a pcd mouse which shows that the recording site was located in the deep layer of the DCN close to the granular cell layer which separates the DCN and posteroventral cochlear nucleus (PVCN) (Fig. 2b). A reconstructed electrode track, along with the position of the recorded single units and their CFs, is shown in Fig. 2c. As histological marking of all units was not feasible in the present study, we applied an additional criterion for separating DCN units from those in PVCN. The border between the DCN and PVCN was estimated by an abrupt change in a tonotopic (or CF versus depth) pattern (cat, Rose et al., 1960 ; mouse, Willott, 1983). The spontaneous rate (SR) distributions of single units in DCN of control (Fig. 3a) and pcd (Fig. 3b) mice were similar. The SRs of the majority of the units in both groups were less than 20 spikes/s, with 67^68% of the units having SR 6 5 spikes/s. A M2 test showed no signi¢cant di¡erence between the two distributions (P s 0.05). The CF distributions of control and pcd mice were qualitatively di¡erent. The CF distributions of DCN

Fig. 2. A micrograph of a single unit recording site labeled with biotinylated-dextran amine in a pcd mouse DCN (a), the outline of the subdivisions of the cochlear nucleus (b) and a reconstruction of the electrode track with location and CF of the single units recorded in this penetration (c). Section thickness is 50 Wm. Scale bar 200 Wm. DCN, dorsal cochlear nucleus; PVCN, posteroventral cochlear nucleus; RB, restiform body; FCL, fusiform cell layer; GCL, granule cell layer.

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Fig. 3. SR distribution for DCN units recorded in 17 control (a) and 13 pcd (b) mice. CF distribution for DCN units recorded from 17 control (c) and 13 pcd (d) mice. Number of neurons (n) are shown in each panel.

single units recorded in the control and pcd mice are shown in Fig. 3c and d, respectively. CF distribution of the control mice was unimodal. The mode of this distribution was located between 10 and 15 kHz. In contrast, the CF distribution of the pcd mice was bimodal, with peaks at 10^15 and 20^25 kHz. These differences were statistically signi¢cant (M2 = 14.8, df = 6, P 6 0.05).

Distributions of threshold at CF, noise threshold and maximum driven rate to noise are shown in Fig. 4 for control (top) and pcd (bottom) mice. Thresholds at CF of the majority (92^95%) of the units ranged from 0 to 70 dB SPL. The CF thresholds of the pcd DCN units (Fig. 4b) were lower than those of the controls (Fig. 4a), such that 62 and 77% of control and pcd units, respectively, had thresholds less than 30 dB SPL. A

Fig. 4. Distributions of threshold to pure tone at CF (left column, panels a and b), threshold to noise (middle column, panels c and d) and maximum driven rate to noise (right column, panels e and f) for DCN units of 16 control (top row, panels a, c and e) and 13 pcd (bottom row, panels b, d and f) mice. n, number of neurons.

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Fig. 5. Examples of PSTHs recorded from DCN units of the control (left column) and pcd (right column) mice.

M2 analysis showed a signi¢cant di¡erence between the two distributions (M2 = 13.1, df = 6, P 6 0.05). The noise threshold distributions of the two groups were similar (Fig. 4c,d). The mode of both distributions was between 30 and 40 dB SPL. A majority of the control (62%) and pcd (68%) units had noise thresholds less than 40 dB SPL. A M2 analysis showed no statistically signi¢cant di¡erence between the two distributions (P s 0.05). The distributions of maximum driven rate to noise for control and pcd mice are shown in Fig. 4e,f. In both distributions, the majority of the units had maximum driven rates below 200 spikes/s (81% for control and 89% for pcd mice). The mode of the control distribution was 50^150 spikes/s, whereas that of the pcd

mice was between 0 and 50 spikes/s. A M2 analysis showed that this di¡erence between the two distributions was not statistically signi¢cant (P s 0.05). Examples of DCN PSTH types recorded from single units in the DCN of control and pcd mice are shown in Fig. 5. Discharges of all units were examined for regularity based on the coe¤cient of variation (CV) of interspike intervals as a function of time (e.g. Young et al., 1988 ; Parham and Kim, 1992). Chopper units were subdivided into three subtypes : chop-S (CV 6 0.35), chop-T (CV increased from 6 0.35 to s 0.35 within the ¢rst 15 ms of the response) and chop-O (CV s 0.35). Examples of chop-S and chop-T units are shown in the top two rows of Fig. 5 for control mice (left column) and pcd mice (right column).

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Fig. 6. Examples of EI-areas recorded from DCN units of control (left column) and pcd (right column) mice. The stimulus for rate-level functions shown was broadband noise.

Examples of buildup, onset and inhibited units are shown in the lower part of Fig. 5. The pauser PSTH was also observed, although not shown. Onset variants observed included onset chopping and onset responses followed by inhibition or low level of sustained activity. Examples of EI-area Type I/III, II, III and V units

recorded from DCN of control mice (left column) and pcd mice (right column) are shown in Fig. 6. Both Type I/III and II units had SR 6 5 spikes/s. Type I/III and II units in control (panels a and e, respectively) and pcd (panels c and g, respectively) had narrow regions of excitation near threshold that broadened slightly as

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the stimulus level was increased. The two unit types were di¡erentiated based on their responses to broadband noise. Type I/III units had maximum driven rate to broadband noise s 20 spikes/s, whereas Type II units were de¢ned as units with maximum driven rate to broadband noise 6 20 spikes/s (panels f and h for control and pcd mice, respectively). Rate-level functions in response to broadband noise are shown for the two Type I/III units in Fig. 6b (control) and d (pcd). Both groups of mice had monotonic (e.g. Fig. 6b) and nonmonotonic (e.g. Fig. 6d) rate-level functions. Examples of rate-level functions in response to broadband noise are shown for Type II units in Fig. 6f and h for control and pcd mice, respectively. Type III EI-areas were distinguished by a central region of excitation surrounded by inhibitory sidebands (Fig. 6i and k for control and pcd mice, respectively). Type III units typically had strong responses to broadband noise (Fig. 6j and l for control and pcd mice, respectively), but the noise rate-level functions of both groups included monotonic (Fig. 6j) and non-monotonic (Fig. 6l) types. Type IV and V EI-areas were dominated by broad regions of inhibition. Type IV units were distinguished from Type V units based on narrow regions of excitation at CF near threshold. Type V examples are illustrated for control (Fig. 6m) and pcd (Fig. 6o) mice. The region of inhibition typically became clearer and broader as the stimulus level increased. Type IV and V units could be excited or inhibited by broadband noise in both control and pcd mice. Noise rate-level functions for the control (panel n) and pcd (panel p) Type V units are shown in Fig. 6 where inhibitory responses are seen for both. Distributions of PSTH and EI-area types of control and pcd mice are shown in Fig. 7. Because of the small number of samples, units with pauser and buildup PSTH types and those with Type IV and V units were combined (PB and IV/V, respectively). In both control

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Fig. 7. Distributions of DCN PSTHs (a) and EI-areas (b) recorded in the control (black bars) and pcd (gray bars) mice. Inset in panel (a) shows the distribution of chopper subtypes in the control and pcd mice. Nondes., non-descript, PB, pause-build, PL, primary-like, ND, not driven. Because of the small number of units in the Type IV and V EI-areas, these two classes were combined into one group, IV/V.

Table 1 Distribution of response types in control mouse DCN PSTH type Chop S Chop T Chop O PB Nondescript On Inhib PL Not driven Not done Total Percent

EI-area type I

II

I/III

III

IV/V

unclear

not done

total

percent

0 0 0 0 0 0 0 0 0 0 0 0

2 0 1 2 4 0 0 0 0 1 10 18.5

7 1 5 4 5 1 0 0 0 3 26 48.1

2 2 0 1 3 1 0 1 0 1 11 20.4

1 0 1 0 1 0 2 0 0 0 5 9.2

0 0 0 0 2 0 0 0 0 0 2 3.7

1 1 2 2 1 0 0 1 0 0 3

13 4 9 9 16 2 2 2 0 0 57

22.8 7 15.8 15.8 28 3.5 3.5 3.5 0

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and pcd PSTH distributions (Fig. 7a) the chopper units formed the largest group. There were minor di¡erences in the proportion of units belonging to di¡erent PSTH types between the control and pcd mice. For example, control mice appeared to have more chopper units than pcd mice, whereas pcd mice had more onset units than control mice. A M2 test showed that the di¡erence in the two distributions was not statistically signi¢cant (P s 0.05). The distribution of chopper subtypes for control and pcd mice is shown in the inset of Fig. 7a. The two distributions were nearly identical. A M2 test showed no signi¢cant di¡erence (P s 0.05). The EI-area type distributions of the control and pcd mice were very similar (Fig. 7b). In both control and pcd mice, nearly one half of all units were classi¢ed as Type I/III ; about one ¢fth of the units belonged to Type II or III EI-areas and less than one tenth of the units had Type IV/V EI-areas. A M2 test showed no signi¢cant di¡erence (P s 0.05). Tables 1 and 2 show the distribution of various PSTH types across di¡erent EI-area types for control and pcd mice, respectively. There were di¡erences in the number of various PSTH types in each EI-area type between the two groups. These di¡erences partly re£ect the larger sample size of the pcd group. Because of the small number of units in each subtype, statistical comparisons were not possible. In general, pauser/buildup responses were associated with EI-area Types II, I/III and III. While most chopper units were associated with EI-area Types II, I/III and III, examples with EI-area Type IV were recorded. Onset units had EI-area Type IV/V, as well as, Types I/III and III. A total of nine complex spiking units were recorded in 17 control mice compared to one unit in 13 pcd mice. The SRs of the control complex spiking units ranged from 0^16 spks/s (mean = 6.5 spikes/s, S.E.M. = 2.1), whereas that of the pcd complex spiking unit was 80 spikes/s. Four out of the 10 complex spiking units, including the one encountered in a pcd mouse, did not

Fig. 8. PSTH of a complex spiking unit recorded from a control mouse DCN. The complex spike waveform of this unit is shown in the inset.

have su¤cient data to be classi¢ed in the PSTH or EIarea schemes. The PSTH of a sample complex spiking unit recorded from the DCN of a control mouse in response to CF tone is shown in Fig. 8. The complex spike waveform of this unit is shown in the inset. Complex spikes consisted of bursts of two to ¢ve action potentials whose size gradually decreased during the burst. The discharges of this unit to CF tones were characterized by a long latency (20 ms) and a low rate, giving rise to a PSTH displaying responses mostly at time = 20^40 ms followed by a lower rate of discharges at longer times. This unit was weakly driven by pure tones and was placed in the unusual PSTH category. Three of the six complex spiking units (including the one of Fig. 8) had similar PSTHs. Two other complex spiking units exhibited a long-latency, sustained, strong response to pure tones at their CFs with long latencies (10^15 ms) which were classi¢ed as non-descript. The sixth complex spiking unit exhibited an on-inhibition-o¡ response at CF. One weakly driven unit was classi¢ed as Type II EI-area, and two others had unclear EI-area types. The two strongly driven complex spiking units had Type III EI-areas. The com-

Table 2 Distribution of response types in pcd mouse DCN PSTH type Chop S Chop T Chop O PB Nondescript On Inhib PL Not driven Not done Total Percent

EI-area type I

II

I/III

III

IV/V

unclear

not done

total

percent

0 0 0 0 1 0 0 0 0 0 1 1.2

0 1 1 2 9 3 0 0 0 1 17 21.2

10 3 7 7 4 0 0 5 2 1 39 48.7

5 2 1 2 2 1 0 0 0 3 16 20

0 0 0 0 0 1 5 0 0 0 6 7.5

0 0 0 0 0 0 0 1 0 0 1 1.2

0 0 1 1 1 2 0 0 0 0 0

15 6 10 12 17 7 5 6 2 0 80

18.7 7.5 12.5 15 21.2 8.7 6.2 7.5 2.5

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responses were observed compared to S2 -only. S1 did not in£uence startle in either group for intervals of two and three ms. The 50 and 100 ms S1 ^S2 intervals produced excellent PPI, but it was almost identical for mutants and controls. When startle responses were evoked in the presence of a continuous 4 kHz, 70 dB tone, amplitudes were slightly enhanced. The degree of enhancement, however, did not di¡er signi¢cantly between mutants and controls. For the three control mice tested, the mean amplitude increased by 27% (S.E.M. = 20) compared to 13% (S.E.M. = 6) in the three mutants tested. The background tone had no in£uence on PPA with the 1 ms S1 ^ S2 interval. Fig. 9. PPI as a function of S1 frequency in ¢ve pcd and six control mice. The S1 ^S2 interval was 100 ms.

plex spiking unit with on-inhibition-o¡ was classi¢ed as Type III. 3.2. Behavioral experiments Startle responses were small in pcd mutants. The mean amplitude of the startle response to S2 -only in control mice was 9.3 voltage units (S.E.M. = 1.6), and that for mutants was 2.8 voltage units (S.E.M. = 1.0). This di¡erence was statistically signi¢cant (t(9) = 3.25, P = 0.01). Startle latency tended to be longer in the pcd mutants, 13.3 ms (S.E.M. = 1.8), than in control mice, 10.1 ms (S.E.M. = 0.2), but the di¡erence was not signi¢cant (t(9) = 1.98, P = 0.08). Fig. 9 presents the mean values for PPI for various frequencies. The degree of inhibition was quite similar for both groups and did not di¡er signi¢cantly. Fig. 10 shows that for both mutants and controls, a 1 ms S1 ^S2 interval resulted in PPA, as larger startle

Fig. 10. PPI and PPA as a function of S1 ^S2 interval frequency in ¢ve pcd and six control mice. S1 was a 12 kHz, 70 dB tone.

4. Discussion The main ¢ndings of the present study were : (1) single units recorded extracellularly from the DCN of the ketamine/xylazine-anesthetized mouse could be classi¢ed into chopper, pauser/buildup, onset, inhibited and non-descript PSTH types and Types I, II, III, I/III and IV/V EI-areas. (2) There was no signi¢cant di¡erence in the proportions of various unit types between the control and pcd mice. (3) The pcd DCN units had lower thresholds to CF tones. They did, however, have SRs, thresholds to noise, and maximum driven rates to noise that were similar to those of the control DCN units. (4) The pcd mice had smaller startle responses than the control mice. The two groups, however, did not di¡er on any type of startle modi¢cation, such as PPI, PPA or facilitation by a background tone. Prior to this study, few reports of response properties of mouse CN units in vivo were available. Willott (1983) noted the presence of various PSTH types in mouse CN and a study by Willott et al. (1988) comparing PSTH types in the CN of young and old mice was reported in an abstract. Ehret and Mo¡at (1984) examined noise masking of tone responses and critical ratios in mouse CN units but did not utilize the PSTH or EIarea classi¢cation schemes. Whereas in vivo studies of mouse CN have been limited, numerous in vitro studies (Oertel, 1983, 1985; Wu and Oertel, 1984; Hirsch and Oertel, 1988a,b; Oertel and Wu, 1989; Wickesberg and Oertel, 1988 ; Oertel et al., 1990 ; Zhang and Oertel, 1993a,b,c, 1994 ; Golding et al., 1995, 1999 ; Golding and Oertel, 1996, 1997; Agar et al., 1996, 1997; Ferragamo et al., 1998a,b) have extensively characterized the mouse CN. In comparing the present ¢ndings with those of previous DCN studies, with respect to distribution of PSTH and EI-areas in the control mouse DCN (Table 1), two factors should be taken into account: species and anesthesia. The in£uence of species is illustrated by

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comparing existing EI-area results obtained in DCN of decerebrate cat and gerbil. In the decerebrate cat, Type II, I/III, III and IV EI-areas comprised 19, 22, 23 and 34% of the units, respectively (Shofner and Young, 1985). In the decerebrate gerbil, Type II, I/III, III and IV EI-areas comprised 8, 14, 62 and 16% of the units, respectively (Davis et al., 1996b). Thus, some of the di¡erences between the distribution of various units between the present study and previous studies is likely attributable to the di¡erence in species. The proportion of units exhibiting various EI-areas is also highly variable, depending on the choice of anesthetic. For example, in the pentobarbital-anesthetized cat, Type II, III and IV/V EI-areas comprised 38, 52 and 10% of units exhibited, respectively (Joris, 1998). In contrast, under chloralose anesthesia Type II, III and IV/V EI-areas comprised 16, 43 and 41% of the units exhibited, respectively (Joris, 1998). The data of Joris (1998) did not designate Type I/III as a separate group. Data are not available for EI-area types in the ketamine/xylazine-anesthetized cat. Thus, making direct comparison between the cat and mouse DCN under the same anesthetic (i.e. ketamine/xylanzine) is not possible. It is of note, however, that in the ketamine/xylazine-anesthetized chinchilla, 67% of DCN units exhibited Type I/III-like features as compared to 37% under chloralose (Kaltenbach and Saunders, 1987). In the DCN of the ketamine/xylazine-anesthetized control mouse, 48% of the units exhibited Type I/III EI-area (Table 1). Based on the above comparisons, we anticipate that the proportion of units exhibiting di¡erent EIareas in the ketamine/xylazine-anesthetized mouse DCN would be di¡erent from those of the unanesthetized mouse. In the current study of the mouse DCN we did not ¢nd a simple relationship between PSTH and EI-area types. This ¢nding is consistent with that of a previous study in the decerebrate cat (Shofner and Young, 1985). A limitation of the physiological portion of this study is that the control group consisted of three di¡erent strains of mice (CBA/J, CBA/CaJ and heterozygous pcd on C57 background). The auditory measures of these mice, within the age range examined, are comparable (e.g. neural thresholds in the inferior colliculus, Willott, 1986 ; ABR thresholds, Li and Borg, 1991 ; DPOAE thresholds, Parham, 1997; Parham et al., 1999). Nonetheless, a potential impact on the variability of the results of the control group cannot be ruled out. The expected degeneration of cartwheel cells in the pcd DCN was consistent with the absence of normal complex spiking units. The one complex spiking unit recorded in pcd DCN had an unusually high SR. This was uncharacteristic of the ketamine/xylazine-anesthetized mouse DCN in general and of cartwheel cells in particular (see Parham and Kim, 1995; Davis and

Young, 1997; present study). This suggests that the abnormal SR of the pcd complex spiking unit may be associated with this strain's genetically-determined degenerative process. In the present study, responses of DCN neurons to auditory stimulation were minimally a¡ected by the absence of the cartwheel cells. The proportions of various PSTH and EI-area types were comparable between the pcd and control mice. The CF distribution of the pcd units was bimodal, unlike the unimodal CF distribution of the control units. The reason for this di¡erence is unclear at present. Another di¡erence noted was that the pcd units had lower thresholds to CF tones than those of the control units. This ¢nding is consistent with the loss of an inhibitory in£uence on DCN neurons. Cartwheel cells are immunoreactive for glycine (Wenthold et al., 1987 ; Osen et al., 1990; Oertel and Wickesberg, 1993; Wickesberg et al., 1994). The glycinergic synapses of cartwheel cells have been demonstrated to inhibit DCN fusiform cells and giant cells (Golding and Oertel, 1996, 1997). Thus, the degeneration of cartwheel cells in the pcd mouse may account for the slightly lower thresholds of pcd DCN units. The ¢ndings, however, that the SRs, thresholds to noise and maximum driven rates to noise of pcd mice were una¡ected by the degeneration of cartwheel cells pose a problem for this interpretation. An alternative interpretation of the present results is suggested by studies which investigated cerebellar^vestibular interactions in the pcd mouse. In electrophysiological studies of the pcd mice, postsynaptic targets of cerebellar homologues of cartwheel cells, the Purkinje cells, in the vestibular nuclei have shown a decrease rather than the expected increase in response to vestibular stimulation (Baurle et al., 1997). This ¢nding indicates that there are compensatory reactions in the vestibular nuclei that substitute quantitatively for the Purkinje cell inhibition (Baurle et al., 1997). This conclusion is supported by the ¢nding of increased expression of the calcium-binding protein parvalbumin, which localizes predominantly in inhibitory neurons in vestibular and deep cerebellar nuclei (Baurle et al., 1997) and increased glycine-immunopositive somata and terminals in deep cerebellar nuclei (Baurle and Grusser-Cornehls, 1997). A similar compensatory mechanism may operate in the pcd DCN, thus leading to the minimal e¡ects of degeneration of cartwheel cells on DCN neuron response properties. Candidate inhibitory interneurons that may play such a compensatory role include the small stellate cells of the molecular cell layer which also receive parallel ¢ber input and project to the fusiform cells (e.g. Wouterlood et al., 1984). A third possible explanation of the minor e¡ects observed in this study is that more complex experimental paradigms may be required to uncover the role of the

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cartwheel cells. In our behavioral studies, the modi¢cation of startle associated with PPI and PPA was normal in pcd homozygotes. This ¢nding is particularly interesting with respect to PPA because this phenomenon may be mediated within the cochlear nucleus. Although there is some evidence to suggest that impairment of glycinergic tuberculoventral neurons of the DCN results in increased PPA (Willott and Carlson, 1995; Willott et al., 1997b), the same does not appear to be true of cartwheel neurons. Indeed, PPA, PPI and enhancement of startle by a background tone are possible without cartwheel neurons. This ¢nding is consistent with a recent report by Meloni and Davis (1998) that bilateral lesions of the DCN do not alter background noise facilitation, short-term habituation, PPI, PPA and fear conditioning of the startle response. The one signi¢cant behavioral de¢cit in the pcd mutants was a smaller startle response amplitude. In light of the apparent normalcy of the pcd mutant auditory system reported here, it is possible that the smaller startle response is due to motor rather than auditory de¢cits. DCN may participate in more complex behaviors such as re£exive orientation of the head to elevated sound sources (Sutherland et al., 1998). Young et al. (1995, 1997) have proposed that this may indicate a possible role for the DCN in somatosensory integration (Davis et al., 1996a). Somatosensory dorsal column and spinal trigeminal nuclei project to the DCN (Itoh et al., 1987 ; Weinberg and Rustioni, 1987). These inputs terminate as mossy ¢bers forming glomerular endings with granule cells in the CN (Wright and Ryugo, 1996). Young et al. (1995) showed that a multicomponent response to electrical stimulation of these nuclei results in a short-latency inhibitory component of unknown origin, a transient excitatory component associated with granule cell excitation of DCN type IV cells and a long-latency inhibitory component. Cartwheel cells are believed to be responsible for the long-latency inhibitory component (Davis et al., 1996a). In this regard, further investigation of the control and pcd mice subjected to tasks such as re£exive head orientation to a sound source in a freely-moving condition, where integration of somatosensory, vestibular and auditory signals is necessary, may help elucidate the role played by the DCN cartwheel cells.

Acknowledgements The physiological studies were supported in part by NIDCD, NIH grant DC 00360 to D.O.K. and the behavioral studies by NIH grant R37 AG07554 to J.F.W. G.B. was supported by the Division of Otolaryngology,

149

UCHC. W.R.D. was supported by NIDCD Training Grant DC00025. We thank Dr. Richard L. Sidman, the New England Regional Primate Research Center, Harvard Medical School, for providing the pcd mice in the initial phase of the physiological studies. We are grateful to Stephanie Bowers for her patient editing of the text of this manuscript.

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