Intracellular Response Properties of Units in the Dorsal Cochlear Nucleus of Unanesthetized Decerebrate Gerbil

JIANG DING 1 AND HERBERT F. VOIGT 1,2 1Department of Biomedical Engineering, 2Department of Otolaryngology, and Hearing Research Center, Boston University, Boston Massachusetts 02215-2407

Ding, Jiang and Herbert F. Voigt. Intracellular response properties cochlea and conveyed by the auditory nerve. The CN is of units in the dorsal cochlear nucleus of unanesthetized decerebrate subdivided into dorsal (DCN), anteroventral, and postero- gerbil. J. Neurophysiol. 77: 2549±2572, 1997. Intracellular recording ventral subnuclei on the basis of anatomic and cytoarchitec- experiments on the dorsal cochlear nuclei of unanesthetized decere- tural differences (Lorente de No 1981; Osen 1969; RamoÂny brate gerbils were conducted. Acceptable recordings were those in which resting potentials were 050 mV or less and action potentials Cajal 1909). Each subnucleus receives similar tonotopically (APs) were ¢40 mV. Responses to short-duration tones and noise, arranged auditory nerve input (Brown and Ledwith 1990; and to current pulses delivered via recording electrodes, were ac- Osen 1969; Rose et al. 1960) and projects to higher auditory quired. Units were classi®ed according to the response map scheme centers via different pathways (Adams 1979; Adams and (types I±IV). Ninety-two acceptable recordings were made. Most Warr 1976; Cant and Gaston 1982; Osen 1972; Ryugo and units had simple APs (simple-spiking units); nine units had both Willard 1985). simple and complex APs, which are bursts of spikes embedded on The DCN is distinguished from other cochlear subnuclei slow, transient depolarizations (complex-spiking units). Of 83 simple- by its layered structure, variety of morphologically different spiking units, 46 were classi®ed as follows: type I/III (9 units), type neurons, intrinsic neural circuitry, and diverse physiology. II (9 units), type III (25 units), type IV (2 units), and type IV-T (1 unit). One complex-spiking unit was classi®able (a type III unit); Neurons in the DCN include large projection neurons (fusi- six were unclassi®able because of weak acoustic responses. Classify- form/pyramidal and giant cells) and a group of small inter- ing 39 other simple-spiking units and 2 complex-spiking units was neurons (cartwheel, granule, Golgi, stellate, and corn or ver- impossible, because they were either injured or lost before suf®cient tical cells) (Benson and Voigt 1995; Brawer et al. 1974; data were acquired. Many simple-spiking units showed depolarization Kane 1974; Lorente de No 1981; Mugnaini et al. 1980a,b; or hyperpolarization (Ç5±10 mV) during acoustic stimulation; some Osen 1969; Osen and Mugnaini 1981; Wickesberg and Oer- were hyperpolarized during the stimulus-off period. Type I/III units tel 1988; Wouterlood and Mugnaini 1984; Wouterlood et al. were not hyperpolarized during off-best-frequency (off-BF) stimula- 1984; Zhang and Oertel 1993a±c, 1994). Extensive electro- tion. In contrast, many type II units were hyperpolarized by off-BF frequencies, suggesting that they received strong inhibitory sideband physiological studies have revealed that DCN neurons dis- inputs. When inhibited, some type III units were hyperpolarized. Type play a variety of responses to acoustic stimulation. These IV units were hyperpolarized during inhibition even at low levels responses have been classi®ed with the use of discharge (õ60 dB SPL); sustained depolarizations occurred only at higher patterns (peristimulus time histograms, regularity analysis) levels, suggesting that they receive strong inhibitory and weak excit- (Gdowski 1995; Pfeiffer 1966; Pfeiffer and Kiang 1965; atory inputs. Several intracellular response properties were statistically Rhode and Smith 1986a,b; Young et al. 1988) and/or re- different from those of extracellularly recorded units. Intracellularly sponse maps (types I±V) (Davis et al. 1996a; Evans and recorded type II units had higher thresholds and lower maximum Nelson 1973; Young and Brownell 1976; Young and Voigt BF-driven and noise-driven rates than their extracellularly recorded counterparts. Type I/III units recorded intracellularly had lower maxi- 1982). How these response properties are related to auditory mum BF-driven rates. Type III units recorded intracellularly had information processing and perception remains largely un- higher maximum noise rates compared with those recorded extracellu- known. In the past, most in vivo electrophysiological studies larly. Weaker acoustic responses most likely result from membrane of CN physiology have been extracellular recording studies disruption, but heightened responses may be related to weakened (e.g., Davis et al. 1996a; Evans and Nelson 1973; Nelken chloride-channel-dependent inhibition due to altered driving forces and Young 1994; Rhode and Smith 1986a,b; Shofner and resulting from KCl leakage. Firing rates of simple-spiking units in- Young 1985; Young and Brownell 1976; Young and Voigt creased monotonically with increasing levels of depolarizing current 1982). The few in vivo intracellular recording studies con- pulses. In contrast, many complex-spiking units responded nonmono- ducted (e.g., Rhode et al. 1983a,b; Romand 1978; Rouiller tonically to depolarizing current injection. The monotonic rate-versus- current curves and the nonmonotonic rate-versus-sound level curves and Ryugo 1984; Smith and Rhode 1985, 1989), have all of type IV and III units suggest that the acoustic behavior is the result been performed on anesthetized animals, and units were clas- of extrinsic inhibitory inputs and not due solely to intrinsic membrane si®ed on the basis of temporal discharge patterns. Electro- properties. physiological properties were not investigated in these stud- ies. Ostapoff et al. (1994) reported that a cartwheel cell recorded in an anesthetized gerbil DCN was a chopper. INTRODUCTION Recently, in vitro slice studies in mice and guinea pigs The cochlear nucleus (CN) is the ®rst nucleus in the described responses of some CN neurons to current pulse central auditory pathway to process information about the injection, to stimulation of various axon pathways, and to auditory environment transduced and encoded within the various pharmacological manipulations (Hirsch and Oertel

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1988a,b; Manis 1990; Manis et al. 1994; Oertel and Wu membrane to measure the sound pressure level during acoustic 1989; Oertel et al. 1990; Zhang and Oertel 1993a±c, 1994). calibration procedures. Pure tone stimuli were generated by a pro- Although the electrophysiology of these neurons, such as grammable (Wavetek, model 5100) or manual (Wavetek, model action potential features and membrane properties [current- 188) oscillator with low harmonic distortion (less than 060 dB). voltage (I-V ) relationship, input resistances, etc.] can be Broadband noise stimuli were digitally generated on a personal computer (Gateway 2000, 486DX) and output through a D/A well studied with this approach, assessing the cells' acoustic converter (Tucker-Davis) at a sampling frequency of 100 kHz response properties directly is impossible. (Davis et al. 1996a). Bursting stimuli were gated on and off with This study aims at a comprehensive physiological investi- 5-ms rise/fall times by the use of an electronic cosine switch gation of DCN neurons by conducting intracellular in vivo (Wilsonics, model BSIT). The signals were attenuated by a pro- single-unit recording experiments. Mongolian gerbils (Meri- grammable attenuator (Wilsonics, model PATT) to achieve the ones unguiculatus) were chosen as subjects because they desired stimulation levels. For tones, this attenuation was relative have a well-developed auditory system capable of low-fre- to the acoustic ceiling; for noise, the attenuation was relative to quency hearing (Ryan 1976), neuronal organization and cell the maximum level of noise without attenuation (Ç95 dB SPL; types similar to those of cats (Benson and Voigt 1995; bandwidth 0±25 kHz). The acoustic system used is more com- Schwartz et al. 1987), large bullae enabling easy access to pletely described in Davis et al. (1996a), which contains a repre- sentative calibration curve (see their Fig. 1). A DEC LSI 11/73 the DCN (Frisina et al. 1982; Lay 1972; Plassmann et al. computer system was used to control stimulus presentation and 1987), and a stable preparation for in vivo intracellular stud- data acquisition. ies. Our goal was to record intracellular responses to both acoustic and electrical stimuli, classify the units according Calibration to the response map scheme, and then mark the neurons so that associations could be made between neuron type and The acoustic system was calibrated in each experiment before unit response type. In this paper, we report on the intracellu- data collection to ®nd the maximum deliverable sound pressure lar responses obtained from neurons not marked or recov- level (dB SPL) for pure tones over a range of 0±25 kHz. This was ered. The response properties of identi®ed DCN neurons done by delivering an acoustic click (5V, 20 ms) and measuring the will be reported in a subsequent paper. Some of this work resulting sound pressure (impulse response) near the tympanic membrane. The system's frequency response was computed by has been reported before in abstract form (Ding et al. 1996). performing a fast Fourier transform of the impulse response. A 10-band equalizer (BSR, model EQ-3000) was used to achieve METHODS the ¯attest frequency response by modifying the frequency content of the click before delivery to the ear. To compensate for the Surgical preparation non¯at frequency response, the digital noise was created to have An unanesthetized, decerebrate preparation was selected to elim- a spectrum whose magnitude was the inverse of the magnitude of inate the in¯uence of barbiturates on neuronal response properties the system frequency response (up to 25 kHz), and the phase was (Evans and Nelson 1973; Young and Brownell 1976). All experi- randomized between {p radians. ments were performed in a sound-attenuating chamber (IAC, model 1202A) with the use of institutionally approved protocols. Electrodes Detailed surgical procedures can be found in a separate paper (Davis et al. 1996a). Brie¯y, young (Ç3 mo old) gerbils were Glass microelectrodes were pulled with the use of a Flaming- ®rst administered atropine (0.04 mg/kg im) to reduce respiratory Brown micropipette puller (Sutter Instrument, model P80/PC), mucus buildup. The animals were then anesthetized by an intraperi- and ®lled with 0.5 M KCl, 0.05 M tris(hydroxymethyl)amino- toneal injection of brevital sodium (methohexital sodium, 65 mg/ methane buffer, and 4% horseradish peroxidase. The tips were kg), an ultrashort-acting barbiturate anesthetic. Supplemental usually õ0.5 mm and the initial resistances were ú100 MV, mea- doses of brevital sodium (32 mg/kg) were administered as needed sured at 1 kHz with the use of a microelectrode impedance meter during surgery. Body temperature was monitored and maintained (Winston Electronics, model BL-1000). The electrodes were at Ç39ЊC with the use of a Harvard Apparatus heating blanket and beveled in a saline±silicon carbide (Buehler) slurry to reduce the controller. After an incision in the throat, a small slit was made resistance to Ç40 MV before use. on the ventral surface of the trachea, and the common carotid The electrode was advanced into the DCN through the para¯oc- arteries were ligated with silk. The animal was placed in a stereo- culus by a stepper motor microdrive (Kopf Instruments, model taxic device (KOPF, model 1730) after the pinnae were removed. 660). A silver±silver chloride wire was used to connect the elec- A hole was made in the skull and a supracollicular decerebration trode to the head stage of an Axoclamp 2-A (X1) ampli®er (Axon was performed by aspirating all the brain tissue rostral to the supe- Instruments). Another such wire was placed in the neck muscula- rior colliculus. This complete aspiration precludes the return of ture as a reference electrode. The electric activity recorded by the motoric activity when the anesthetic wears off (Newlands and Axoclamp was further ampli®ed by a DC ampli®er (Tektronix, Perachio 1990). The empty portion of the skull was gently packed model AM502), digitized by an A/D converter, and stored on the with gelfoam to encourage blood clotting and to provide mechani- LSI 11/73 computer system. The signals were sometimes also cal support for the remaining brain tissue. The DCN was accessed recorded on videotape by a VCR (Sony, model SL-HF 900) after with the use of a transbullar approach (Frisina et al. 1982). The being processed by a modi®ed pulse-code module (Sony, model anesthetic was discontinued and the animal was allowed to stabilize PCM-501ES) (Bezanilla 1985). for Ç30 min. Brain stem auditory evoked responses Acoustic system The click-induced brain stem auditory evoked response (BAER) Acoustic stimuli were delivered monaurally in a closed system was measured in each animal before placement of a glass micro- to the left ear by an earphone (Beyer Dynamic DT48A, 200 V) electrode into the DCN, to gauge the integrity of the auditory brain that was coupled to a hollow earbar. A probe-tube microphone stem pathways. Platinum needle electrodes were placed subder- (0.5 in. Bruel & Kjaer, model 4134) was placed near the tympanic mally at skull midline (noninverting), posterior to the ear of stimu-

/ 9k11$$my27J460-6 08-08-97 12:41:09 neupal LP-Neurophys INTRACELLULAR RESPONSE PROPERTIES OF GERBIL DCN UNITS 2551 lation (inverting), and at the midline 2-cm posterior to the base by dividing it by the maximum discharge rate (Young and Voigt of the neck (common). Ampli®cation gain was set to 10 5 by cou- 1982) and is called the normalized tone slope. The relative noise pling a Grass P15d bioampli®er to a Tektronix AM502 differential response, r, which is the ratio of the maximum response to noise ampli®er. The resulting signal was ®ltered from 0.1 to 10 kHz and minus SpAc to the maximum response to BF tones minus SpAc, sampled at 25 kHz for 10.2 ms with the LSI 11/73 computer was also measured (Young and Voigt 1982). system. Each BAER was the average of 350 click responses. Clicks Responses to 50-ms current pulses were examined for signs of were produced by routing a 4-V, 50-ms pulse through a PATT postcurrent hyperpolarizations, anode break excitation, and sags, attenuator to the DT 48A earphone. Click level was manually and were used to construct rate-versus-current level (RCL) curves. adjusted in 5- to 10-dB steps to determine click-evoked BAER For current values that did not give rise to action potentials, esti- threshold approximately. The experiment would continue if click- mates of the I-V curves were made. In all cases, the cells' steady- evoked BAER threshold was within normal range (Burkard and state voltage responses were measured near the end of the current Voigt 1989). Click-evoked BAER thresholds were checked during pulse injection. Estimates of the cell's input resistance were made the experiment if unit thresholds appeared to increase; experiments from these I-V curves. In cases in which the ampli®er bridge was were ended if BAER thresholds increased by 30 dB. not balanced (normal case), estimates of the electrode's contribu- tion to the voltage response to current injection were made by Unit recording ®tting the voltage response to the sum of three decaying exponen- tials. The coef®cient of the fastest-decaying exponential was taken As a microelectrode was advanced through the cerebellum's to represent the resistance of the electrode, and this component was para¯occulus into the DCN, broadband noise bursts were used to subtracted from the voltage response to yield the cell's response to detect background driving. Once background responses to the noise the current pulse (DV in Figure insets). stimuli were observed, tone bursts were used as search stimuli. A recording was considered intracellular and acceptable if the resting membrane potential was 050 mV or less and the maximum action Analysis of membrane voltage drops in response to step potential size was ¢40 mV. After an acceptable neuron penetration, hyperpolarizing current injection the unit's best frequency (BF) and threshold (u) were estimated audiovisually. Three trials of 50- or 100-ms tone bursts (250-ms It has been shown, under appropriate constraints, that a neuron's or 1-s interstimulus interval, stimuli began 10 ms after the start of transmembrane voltage change in response to a step hyperpolariz- a trial) were presented at three frequencies (BF and BF { 1 octave ing current can be described by the sum of an in®nite number of or BF { 0.7 octaves) and 17 sound pressure levels each (0±80 exponential decays (Rall 1969, 1977). The equalizing time con- dB SPL, 5-dB steps). This was followed by three trials of 50- or stants and the corresponding coef®cients in this series re¯ect the 100-ms broadband noise bursts (250-ms or 1-s interstimulus inter- passive properties of the neuron's membrane. Under shunt-free val, stimuli began 10 ms after the start of a trial) at each of 17 conditions, the largest time constant is the membrane time constant levels (0±80 dB SPL, 5-dB steps). Occasionally, longer-duration (Durand 1984; Rose and Dagum 1988). tone and/or noise bursts (duration 200 ms, interstimulus interval In the present study, a three-term exponential model was used 1,000 ms) were also used and the unit's spike times were recorded. to ®t the data recorded from DCN intracellular units. Higher orders To assess the unit's response to current stimulation, 50-ms electric of exponentials cannot be reliably estimated because of practical current pulse bursts (250-ms interstimulus interval, stimuli began limitations (e.g., bandwidth, sampling rate). Because the fastest 10 ms after the start of a trial) were delivered through the electrode change occurs at the beginning of voltage drop, the ®rst sample of (16 trials, from 01.0 to 1.0 nA in equal steps). Sometimes this data can have large errors if it is not sampled exactly at the stimulus DC paradigm was repeated for the same current levels and/or for onset (t Å 0). To avoid such error, a delay parameter was added current levels ranging from 02.0 to 2.0 nA in equal steps. to the model and ®tted to the data after skipping the ®rst one or two data points. The mathematical description of the model is

(t/d)/t Data processing V (t) Å DC / C1[1 0 e 1]

(t/d)/t (t/d)/t In classifying a unit physiologically, deviations from spontane- / C2[1 0 e 2] / C3[1 0 e 3](2) ous activity (SpAc) were used to determine excitation or inhibition (Davis et al. 1995). A driven rate that is larger (smaller) than where V (t) is the observed voltage response, DC is the known SpAc by ú2 SD of the SpAc was regarded as re¯ecting excitation resting membrane potential before the current injection, ti and Ci (inhibition). For intracellular data obtained with 50-ms (or (i Å 1,2,3) are equalizing time constants and coef®cients (t3 ú 100-ms) stimuli, driven rate was estimated over a 40-ms (or t2 ú t1 ), and d is the delay. 80-ms) period starting 10 ms after stimulus onset. SpAc was esti- Estimates of Ci , ti , and d were based on a modi®ed Levenberg- mated during a no-stimulus condition, or if this was not available Marquardt algorithm (More et al. 1980) and executed on a DEC over a 100-ms interval, at the 0 dB SPL condition. For extracellu- 3000 AXP workstation. To assess the quality of the results, a 95% larly recorded spike time data obtained with 200-ms stimuli, driven marginal con®dence interval was approximated for each estimate rate was computed over the last 80% of the stimulus duration (160 with the use of the following method (Bates and Watts 1988): let T ms) and SpAc was computed over the last 160 ms of the 1,000 s the parameter vector be u Å [d t1 t2 t3 C1 C2 C3 ] , then trial. Rate-versus-sound pressure level (RSL) curves were con- * T structed from these data. Unless stated otherwise, these curves were Éui 0 u i É É sqrt [J(u*) J(u*)]iit(N 0 P, 0.025)s (3) smoothed with the use of a three-point ®lter (Eq. 1) to reduce rate variations where u* is the optimal estimate of u, u i* is the ith element of u*, R (k) Å [R(k 0 1) / 2R(k) / R(k / 1)]/4 (1) J(u) is the Jacobian matrix whose elements are the values of F the derivatives of the voltage drop with respective to individual T where R(k) and RF (k) are the kth rate in the data array before and parameters at different time lags, [J(u*) J(u*)]ii is the ith diago- after ®ltering, respectively. As a measure of nonmonotonicity, a nal element of J(u*)TJ(u*), N is the number of data points used, straight-line (least-squares) ®t was applied to the portion of the P is the number of parameters (P Å 7), s is the square root of RSL curve between the maximum (the 1st sharp change in the variance estimate based on N 0 P degrees of freedom, and t(N 0 slope) or the transition to negative slope and the next minimum P, a/2) is the a/2 quartile for Student's t-distribution with N 0 or sudden change in slope. This estimate of slope was normalized P degrees of freedom.

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Unit typing Precise localization of the units included in Table 1 is problematic because these units are those for which intracel- Units were classi®ed physiologically according to the response lular marking was either not attempted or unsuccessful. Esti- map scheme established for cats (Evans and Nelson 1973) as modi- mates of unit localization can be inferred in some cases by ®ed by others (Shofner and Young 1985; Young and Brownell 1976; Young and Voigt 1982) and adapted for gerbils (Davis et referencing the locations of unlabeled cells to those of la- al. 1996a). Type I/III units have a SpAc rate °2.5 spikes/s, an beled units. Of the 53 units in Table 1, 17 units could be excitatory response to BF tones and noise, and no detectable side- located to a speci®c DCN layer because a deposit of horse- band inhibition. Type II units have little or no SpAc (°2.5 spikes/s), radish peroxidase was found at the sight of the recording an excitatory response to BF tones, and little to no noise response. (although no neuron was recovered). An additional 19 units Type III units have SpAc rates ú2.5 spikes/s, excitatory responses were recorded along an electrode track õ200 mm away from to BF tones and noise, and sideband inhibition. Type IV units have a track where a deposit of horseradish peroxidase was found an island of excitation near BF, inhibition at higher levels, and within the DCN. The difference in recording depth (roughly inhibitory sidebands, and respond to noise; type IV-T units have dorsal laterally and ventral medially) between these units BF-tone-driven rates that drop below half of the sum of the maxi- and the neighboring deposits was õ200 mm. Considering mum driven rate and the SpAc within 35 dB SPL of the peak. Two unit subclasses have been identi®ed recently (Davis et al. 1996a): that the gerbil DCN spans Ç1 mm rostrocaudally, 1.2 mm type III-i units, which have type III features, except that they are in the dorsomedial to ventrolateral direction, and 0.5 mm inhibited by noise at low levels and excited at higher levels, and in the dorsolateral to ventromedial direction (unpublished type IV-i units, which have type IV properties, except that they results), it is likely that most of these units were recorded are inhibited by noise at high levels. in DCN. Presented next are three detailed examples from each unit class (Figs. 1, 2, 5, 7, 9, and 10). These examples were RESULTS selected to represent typical units from different BF regions. During these experiments, many units were recorded ex- Because the response map scheme was not applicable to tracellularly to establish a normative data base for the DCN most complex-spiking units, these units were grouped sepa- in the decerebrate gerbil. These data were presented in a rately from simple-spiking units. Tabular summaries of sev- separate paper (Davis et al. 1996a). A major goal of our eral physiological parameters are provided for all classi®ed intracellular studies was to record units intracellularly and units (Tables 2±6). then mark them to ®nd the neurons' structure. All success- fully labeled units (i.e., cells recovered and associated with Type I/III units recorded physiology with high con®dence) will be reported in a subsequent paper. Presented in this paper are results Type I/III units (Table 2, Fig. 1) had resting membrane from 92 acceptably impaled spiking units (see criteria for potentials that ranged from 056 to 072 mV (average: 063.8 { acceptability in METHODS) from 69 gerbils for which mark- 5.9 mV, mean { SD) and action potential sizes that averaged ing was unsuccessful. Most of these units (83) had simple 57.6 { 9.4 mV. Most type I/III units (7 of 9) exhibited some action potentials (simple-spiking units); 9 units had both sustained depolarization during BF stimuli, although in only simple and complex action potentials (complex-spiking four cases did this exceed 10 mV. These same seven units also units). Complex action potentials consist of bursts of spikes showed sustained depolarization for the below-BF stimuli, but superimposed on slow and transient depolarization lasting only three of the units had this feature for the above-BF stimu- Ç10±40 ms; examples may be seen in Fig. 10. Table 1 lus. In addition, ®ve of nine units had sustained depolarizations shows the distribution of unit response types for simple- and of about the same size when noise was presented. None of the complex-spiking units. Type III units are the most commonly type I/III units showed any sign of hyperpolarization during encountered, whereas type IV and IV-T units are the least either tone or noise stimulation; however, small (°5mV) common. Only one complex-spiking unit could be classi®ed; hyperpolarizations were seen in ®ve of nine units after BF it was a type III unit. The other complex-spiking units were stimuli [as seen in the responses of one of the units shown in unclassi®able because of their high acoustic thresholds, weak Fig. 1A (unit J72293-6-1)], in four of nine units after either responses, and broad tuning. An additional 39 simple-spik- the below-BF tone or noise stimuli, and in two of nine units ing units and 2 complex-spiking units could not be classi®ed after the above-BF tones. Action potentials with undershoots because of incomplete data acquisition (units were either were seen in all but two type I/III units (e.g., unit lost or injured during the recording). Intracellularly recorded J72293-6-1). nonspiking and nonresponsive (to either acoustic or electri- All type I/III units had low rates of SpAc (õ7 spikes/s). cal stimulation) units were frequently impaled; presumably Their average BF tone threshold was 26 dB SPL. Discharge these were glial cells. rates increased monotonically in response to BF tone and

TABLE 1. Distribution of unit response types

Weak, Unit Type I/III II III IV IV-T Broad Total

Simple spiking 9 9 25 2 1 0 46 (87) Complex spiking 0 0 1 0 0 6 7 (13) Total 9 (17) 9 (17) 26 (49) 2 (4) 1 (2) 6 (11) 53 (100)

Values in parentheses are percentages.

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TABLE 2. Type I/III units

BF, RMP, APS, SpAc, uS , RS , uI , RI , RN , t, PCI, File ID Unit ID kHz mV mV spikes/s dB SPL spikes/s nA spikes/s MV ms, % jo0992 16-01 17.4 063 44 3.3 25 183 0.2 100 13 10.5, 5 j62593 13-01 2.3 061 64 3.3 10 181 0.07 80 14 11.2, 16 j72293 06-01 0.7 065 46 3.3 30 175 0.07 160 26 2.4, 8 j82593 12-01 8.2 058 69 6.7 20 373 0.07 540 47 3.2, 9 j31694 02-01 2.3 069 64 0 25 175 0.4 50 16 j30795 08-02 1.9 059 52 0 25 138 j73195 01-01 5.3 072 51 0 35 168 0.13 380 62 9.8, 5 j80295 16-01 2.4 056 66 0 20 147 0.25 100 14 2.6, 4 j82195 03-01 7.6 072 63 0 45 46 0.13 300 63 4.2, 2 Mean { SD 063.8 { 5.9 57.6 { 9.4 1.9 { 2.4 26 { 10 176 { 85 0.17 { 0.12 214 { 175 32 { 22.1 6.3 { 4.0

RMP, resting membrane potential; APS, maximum action potential size; SpAc, spontaneous activity; uS , threshold to best frequency (BF) tone bursts; RS , maximum BF tone driven rate (see text for details); uI , threshold to depolarizing current pulse injection; RI , driven rate at 1.0-nA depolarizing current pulse input; RN , cell input resistance; t, largest equalizing time constant; PCI, 95% parameter con®dence interval on the estimate of t. broadband noise bursts (Fig. 1B), and the RSL curves either ®ve of nine type II units were hyperpolarized after either saturated (e.g., unit J82593-12-1) or showed sloping satura- the BF or below-BF tones. tion (e.g., unit J72293-6-1). Maximum BF-driven rates av- Most type II units had no SpAc and a majority had high eraged 176 spikes/s. Noise responses were comparable with BF tone thresholds (¢35 dB SPL). Type II units' discharge tonal responses, but noise thresholds could be 25±35 dB rates increased monotonically to increasing levels of BF higher than BF tone thresholds. Unit J82593-12-1 is the only tones, noise (Fig. 2B), and current injection (Fig. 2C). The type I/III unit with an exceptionally high driven rate (373 maximum BF-driven rates, however, were consistently low spikes/s; highest of all the response types) and had equal (õ150 spikes/s for 8 of 9 units) compared with maximum thresholds for both BF tones and noise. All type I/III units discharge rates measured in extracellularly recorded type II responded monotonically to increasing levels of 50-ms depo- units. Only one unit (Jd0293-5-2) had a maximum discharge larizing current injection. The RCL curves usually did not rate ú200 spikes/s, resulting in an average for the group of saturate at 1.0 nA, as seen in Fig. 1C, and the initial slope 112 { 52 spikes/s. Although type II units generally gave of the RCL curve averaged 312 Hz/nA. Figure 1C, insets, poor responses to noise bursts, their membrane potential are estimates of the units' I-V curves. Estimates of the cells' showed clear signs that it was weakly depolarized at supra- input resistance, ranged from 13 to 63 MV, with an average threshold levels (Fig. 3, Ad and Bd). Similarly, all type II of 32 { 22 MV. Estimates of the largest equalizing time units were poorly excited by depolarizing current injection constants ranged from 2.4 to 11.2 ms, with an average of and required higher levels of current to reach action potential 6.3 { 4.0 ms. The 95% marginal con®dence bounds on these threshold than other unit classes (average current threshold: time constant estimates were never ú16% of the estimated 0.84 { 0.19 nA). The slopes of the RCL curves of type II value. One type I/III unit was localized in the fusiform cell units were the smallest of all unit classes (average: 113 { layer. 85 Hz/nA). Figure 2C, insets, are estimates of the units' I-V curves. Estimates of the input resistance for type II units were also lower than those for other unit types (15 { 4.7 Type II units MV). The estimates of the largest equalizing time constant Type II units (Table 3, Figs. 2±4) typically had large, were also very low, averaging 2.9 { 2.1 ms. This is consis- over- and undershooting action potentials (action potential tent with an electrode-induced shunt compromising the in- size: 71.3 { 13.1 mV), whereas their resting membrane tegrity of the cell's membrane, and is discussed further potentials were nearly identical to those of type I/III units below. (resting membrane potential: 063.7 { 6.9 mV). Sustained Figure 3 shows more intracellular responses to tones, depolarization was seen in eight of nine type II units during noise bursts, and current pulses from unit J60393-2-2. Data BF tone stimuli, and in all type II units responding to the in Fig. 3A were acquired for three frequencies [1.4, 2.8 below-BF and noise stimuli, but for only one of nine units (BF), and 5.6 kHz] and noise, each at eight levels from 0 presented with the above-BF tones. For many type II units, to 70 dB SPL. There were no action potential responses at such as J60393-2-2 and Jd0293-5-2 (Fig. 2A), the depolar- 5.6 kHz (an octave above BF) up to 70 dB SPL and few ization was ¢10 mV at 60 dB SPL. The membrane potential responses at 1.4 kHz (an octave below BF). Notice, how- of one type II unit (not shown) responded to below-BF tones ever, the depolarization during the acoustic stimuli and the of increasing level by changing from no response below slow hyperpolarization during the stimulus-off period for threshold to hyperpolarized responses and ®nally to depolar- two frequencies (1.4 and 2.8 kHz; Fig. 3, Ba and Bb) and ized responses, although no action potentials were produced. noise (Fig. 3Bd), although in the latter case there were no Hyperpolarized responses were never seen for BF stimuli action potential responses at all. The deviations in membrane and only rarely seen for either the below-BF (1 of 9) or potential began at sound levels well below those required above-BF (3 of 9) tones. Hyperpolarizations were more for action potential generation. The membrane behavior at common after acoustic stimuli. Eight of nine type II units the higher frequency, however, was quite different. Instead were hyperpolarized after noise presentations, whereas only of depolarizing, the cell remained at its resting position until

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FIG. 1. Intracellular responses (A), driven discharge rate-vs.-sound level curves (B), and discharge rate-vs.-current level curves (C) from 3 type I/III units, J72293-6-1 (left), J73195-1-1 (middle), and J82593-12-1 (right, localized to the fusiform cell layer). A: 3 intracellular responses from each unit to 50- or 100-ms, best frequency (BF) tone bursts at 70, 60, and 60 dB SPL, respectively. Responses are displaced from each other to aid visualization. Bar along time axis: stimulation interval. B: driven discharge rate-vs.-sound level curves for each unit derived from responses to 50- or 100-ms BF tones and noise bursts from 0 to 80 dB SPL in 5-dB steps. Data were smoothed with a 3-point ®lter. BF is indicated on each plot. C: non®ltered discharge rate- vs.-current level curves for each unit derived from responses to 16 or 17 trials of 50-ms current pulses from 01.0 to 1.0 nA in equal steps. Hyperpolarizing currents had negative values. Insets: estimated current-voltage (I-V ) curves. Input resistance estimates for these 3 units are 26, 62, and 63 MV (left to right). SpAc, spontaneous activity. DV, change in transmembrane potential.

60 dB SPL; at higher levels the neuron was hyperpolarized 50-ms current pulse injections ranging from 02to2nAin during the stimuli (Fig. 3Bc). equal steps. The responses are overplotted to aid compari- Figure 3C shows this unit's responses to 16 trials of sons. The sudden shift of the recorded potential in each trial

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TABLE 3. Type II units

BF, SpAc, uS , RS , uI , RI , R2 RN , t, PCI, File ID Unit ID kHz RMP, mV APS, mV spikes/s dB SPL spikes/s nA spikes/s MV ms, % j60393 02-02 2.8 065 75 0 45 92 0.93 16, 175 11 3.3, 4 j62993 12-01 7.2 062 78 0 15 102 0.93 10, 100 16 jd0293 05-02 12.9 077 95 0 25 233 0.93 21, 150 14 jd2193 13-01 4.3 060 60 0 45 92 0.67 37, 100 9 1.7, 14 j30294 01-02 2.6 059 52 2.9 10 142 0.9, 25 j31694 07-01 1.5 066 82 2.1 20 125 0.93 8, 175 10 2.5, 29 j41394 07-01 2.9 065 68 0 55 85 0.47 0, 125 24 j22795 02-01 3.4 069 60 0 35 83 1.00 7, 150 17 j92595 01-01 4.7 052 71 0 35 55 17 6.3, 22 Mean { SD 063.7 { 6.9 71.3 { 13.1 0.6 { 1.1 32 { 15 112 { 52 0.84 { 0.19 14, 139 15 2.9 12, 32 4.7 2.1

Data in the RI , R2 column are for both I Å 1.0 and I Å 2.0 nA. For remaining abbreviations, see Table 2. was due to the voltage drop across the electrode (the bridge els, saturating at high levels with high maximum rates was not balanced before delivery of the current pulses (220 { 62 spike/s; e.g., units J52793-5-1 and JN2393-11- through the electrode). The unit responded with action po- 1 in Fig. 5B). Unit J80295-11-1 is an example of a type III tentials for depolarizing currents ¢1.2 nA. Some action po- unit with a nonmonotonic rate-versus-level curve (Fig. 5). tentials had strong overshoots, and the neuron became more Its RSL curve shows a negative slope at middle levels. Like depolarized during high-level current injection. type I/III units, type III units showed excitatory responses r (Young and Voigt 1982) is the ratio of a unit's maxi- to noise at all levels with thresholds Ç15±25 dB higher than mum response to noise less SpAc to its maximum response their BF tone thresholds. to BF tones less SpAc, and can be used to distinguish type Type III units discharge monotonically in response to in- I/III units from type II units in decerebrate cats and gerbils creasing levels of current pulse injection. Note in the three (Davis et al. 1996a). Figure 4A shows r plotted against examples in Fig. 5C that the units were inhibited by hyperpo- normalized tone slope for type I/III and type II units. The larizing current injection and excited by depolarizing current r values ranged from 0.3 to 1.39 for type I/III units and injection (SpAc is shown in the plot as the rate at 0-current from 0 to 0.29 for type II units. The normalized slope values level). The threshold to current was õ0.3 nA for all type for the type I/III units are within the range of values seen III units. The slopes of the type III RCL curves averaged with extracellularly recorded type I/III units, but this is not 235 { 85 Hz/nA. Figure 5C, insets, are the units' I-V curves. so for the type II units. These differences between extracellu- The input resistances for this unit class ranged from 7 to 50 larly recorded units and intracellularly recorded units are MV with an average of 24 { 10.3 MV. Estimates of the discussed below. units' largest equalizing time constants ranged from 1.9 to Three type II units were localized. One was found in the 6.6 ms with an average of 3.9 { 1.6 ms. fusiform cell layer and the other two were found deep in Shown in Fig. 6 are additional intracellular responses of the DCN bordering the intermediate acoustic stria. unit J80295-11-1 to tones and noise bursts (A and B) and current pulse injections (C). Data are presented in the same Type III units format used in Fig. 3. This is a typical type III unit, with a Type III units (Table 4, Figs. 5 and 6) make up more than high driven rate and a very regular ®ring pattern. Note that half the classi®able unit population. The resting membrane the responses at BF (12.22 kHz) decreased as sound levels potentials for the 25 type III units ranged from 051 to 074 rose from 20 to 50 dB SPL, thus resulting in a nonmonotonic mV (average: 060.6 { 5.3 mV) and their action potential RSL curve (also see Fig. 5B, right). The cell was depolar- sizes ranged from 42 to 79 mV (average: 61.0 { 9.2 mV). ized during suprathreshold stimuli and strongly hyperpolar- Most type III units (16 of 25) had undershooting action ized when the stimulus was turned off, especially at high potentials. Sustained depolarizations or hyperpolarizations levels. Inhibitory responses are seen at both below-BF (7.52 were observed in 20 of 25 type III units for some stimulus kHz) and above-BF (19.85 kHz) tones, with sustained hy- condition. Sustained depolarizations during BF tone stimuli perpolarizations evident during the stimulus at 40 dB SPL typically increased as a function of level from a few milli- for the lower tone (Fig. 6Bb) and at ú50 dB SPL for the volts at threshold to a maximum of 5±10 mV in 14 of 25 higher tone (Fig. 6Bc). The responses to 7.52-kHz tones units. Although hyperpolarizations were never seen during became excitatory as sound levels exceeded 50 dB SPL. BF stimuli, poststimulus hyperpolarizations were seen in 9 These excitatory responses were followed by strong down- of 25 units. Two of these type III units had poststimulus ward shifts in membrane potential that exceeded 10 mV at hyperpolarization but no obvious depolarization during the 70 dB SPL (Fig. 6Ba). BF tone stimuli. The examples in Fig. 5A show both sus- In response to the above-BF stimuli, none of the type III tained depolarizations and poststimulus hyperpolarizations. units showed sustained depolarizations. About half of the Type III units had a wide range of SpAc (50 { 31 spikes/s) units (13 of 25) had sustained hyperpolarizations between and sound thresholds (15 { 15 dB SPL). In response to BF 5 and 15 mV. For the below-BF stimuli, however, only three tone bursts, most of the type III units (20 of 25) increased of the type III units showed sustained hyperpolarizations. their discharge rate monotonically with increasing sound lev- After the below-BF stimuli, hyperpolarizations were seen

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FIG. 2. Intracellular responses (A), rate-vs.-sound level curves (B), and rate-vs.-current level curves (C) of 3 type II units, J60393-2-2 (left, localized to the intermediate acoustic stria), JD2193-13-1 (middle), and JD0293-5-2 (right). A:3 intracellular responses of each unit to 50-ms, 60-dB SPL BF tone bursts displaced from each other to aid data visualization. Bar along time axis: stimulation interval. B: discharge rate-vs.-sound level curves for each unit derived from responses to 50-ms BF tones and noise bursts from 0 to 80 dB SPL in 5-dB steps. Data were smoothed with a 3-point ®lter. BF is indicated on each plot. C: non®ltered discharge rate-vs.-current level curves for each unit derived from responses to 16 trials of 50-ms current pulse from 02.0 to 2.0 nA in equal step size. Hyperpolarizing currents had negative values. Insets: estimated I-V curves. Input resistance estimates for these 3 units are 11, 9, and 14 MV (left to right). more frequently (9 of 25). These would often occur without Type III units never had poststimulus hyperpolarizations fol- preceding sustained depolarizations (5 of 9), but when they lowing the above-BF stimuli. did follow a stimulus-evoked depolarization, they were typi- The responses to noise were similar to the responses to cally larger than the depolarizations by Ç5mV(8of9). BF tones. These included sustained depolarizations of 5±10

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FIG.3. A: intracellular responses of a type II unit (J60393-2-2) as a function of sound pressure level (from 0 to 70 dB SPL in 10-dB steps) for 3 frequencies [1.40, 2.80 (BF), and 5.60 kHz] and noise (50 ms in duration). At each frequency and level, 3 responses were collected and are displaced from each other to aid data visualization. B: 4 intracellular responses from A, enlarged for clarity: Ba, 1.40 kHz, 70 dB SPL; Bb, 2.80 kHz, 30 dB SPL; Bc, 5.60 kHz, 70 dB SPL; Bd, noise, 70 dB SPL. C: unit's responses to 16 trials of 50-ms current pulses from 01.0 to 1.0 nA in equal steps. Traces are overplotted such that response to larger current is placed higher. Sudden shift in voltage was due mainly to voltage drop across the recording electrode. Bar along time axis: stimulation interval. This unit was localized to the intermediate acoustic stria. mV (8 of 25) and larger (5±15 mV) afterhyperpolarizations tion potentials (Fig. 7A). Although depolarizations were (Fig. 6, A and Bd). Hyperpolarizations were never observed not observed during tonal stimulation for either type IV during the noise. unit, hyperpolarizations were seen during tonal stimuli in Figure 6C shows a type III unit's responses to 16 trials all three units and during the stimulus-off period in one of current pulse injections ranging from 01 to 1 nA in equal unit (Jd2193-4-1). In contrast, the type IV-T unit (Fig. steps. The unit ®red regularly in response to depolarizing 7, J31494-19-1) had sustained depolarization during tonal currents (similar to its responses to acoustic stimuli) and it stimulation (Fig. 8Bd). Unlike most other units, the depo- was inhibited by hyperpolarizing current pulses. larization for the type IV-T unit took an abrupt course: Seven of the nine localized type III units were found in Ç10 ms after stimulus onset, the cell was quickly depolar- the fusiform cell layer; the other two were in the deep DCN. ized (within 5 ms) and remained so until the stimulus was turned off. The cell then gradually returned to the resting Type IV and type IV-T units potential without hyperpolarization. The type IV units had high SpAc (57 and 60 spikes/s). At Type IV and type IV-T units (Table 5, Figs. 7±9) make BF, both units were excited at low sound levels (beginning at up only Ç6% of the classi®able simple-spiking unit popu- 10 dB SPL) and then were inhibited at higher levels (Fig. lation. The two type IV units and the type IV-T unit re- 7B). One unit (J71593-10-1) could be further excited at corded in this study had undershooting action potentials; levels ú60 dB SPL. The type IV-T unit had similar discharge one type IV unit (Jd2193-4-1) also had overshooting ac- rate characteristics; however, its rate did not fall below

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IV-T unit. The discharge rates obtained with 1-nA current pulses exceeded the maximum tone responses by a factor of 2 or 3, and the slopes of the RCL curves were 191 and 166 Hz/nA for the type IV units and 520 Hz/nA for the type IV-T unit. Figure 7C, insets, are I-V curves for the three units. The input resistances for these units are 14, 30, and 30 MV (left to right) and the largest equalizing time con- stants are 2.6, 10.6, and 12.9 ms, respectively. Additional data from the type IV-T unit are shown in Fig. 8. Figure 8A shows intracellular responses to tones of three frequencies and noise at eight sound pressure levels. At BF (3.0 kHz) there were fewer spikes at 60 dB SPL than at 40 and 50 dB SPL. Responses below BF (1.85 kHz) were much stronger than those at BF, although the threshold was 20 dB higher. Sustained depolarization is clearly seen during the stimuli at and below BF, at 70 dB SPL for the above-BF tones, and for noise (Fig. 8B). Figure 8C shows this unit's responses to 16 trials of 50-ms current pulses ranging from 01 to 1 nA in equal steps. In this case, the unit did not produce action potentials until the current level reached 0.5 nA. Postsynaptic potentials (PSPs) are clearly seen in this ®gure (arrow). Figure 9 shows a set of intracellular responses from one of the type IV units (J71593-10-1) to tones and noise bursts and current pulses. At BF (4.7 kHz), the unit was excited at 10 and 20 dB SPL and then was strongly inhibited from 30 to 50 dB SPL. At higher levels it was excited again. Excitatory responses are observed at the below-BF (2.89 kHz) tone condition at levels of ¢40 dB SPL. Signs of depolarization during tonal stimuli at levels õ60 dB SPL were not obvious (Fig. 9, Ba and Bb). Hyperpolarizations, however, are seen at BF where the unit was inhibited (Fig. 9, Bc and Bd). Note that there was more signi®cant depolar- ization during high-level noise bursts, but the unit's action potentials decreased in size and got wider, taking on an injured appearance. Figure 9C shows this unit's responses to 16 trials of 50-ms current pulses ranging in amplitude from 01 to 1 nA in equal steps. Notice the regular ®ring pattern. Current pulses ú0.07 nA would cause the unit's discharge rate to exceed the SpAc rate. This type IV unit and the type IV-T unit were found in the deep DCN.

Complex-spiking units Complex-spiking units (Table 6, Fig. 10) made up 14% of the spiking units encountered in this study; however, only one of these units could be classi®ed by its responses to sound. Figure 10 shows data from three complex-spiking units; unit J61793-8-1 (middle) was classi®ed as a type III FIG. 4. Relative noise response vs. normalized tone slopes for intracellu- unit. All complex-spiking units produced both complex ac- larly recorded type I/III ( ᭺ ) and II ( ● ) units (A) and intracellularly ( ᭡ ) tion potentials and simple action potentials. Complex action and extracellularly ( ᭝ ) recorded type III units (B). See text for de®nitions. potentials are composed of bursts of simple spikes superim- posed on a slow, transient depolarization that lasts Ç10±40 SpAc, which was very low. Although the type IV-T unit ms (see the examples in Fig. 10A). The resting membrane responded to noise monotonically, the two type IV units potential (067.2 { 4.1 mV) of complex-spiking units did responded nonmonotonically. Noise thresholds were Ç5±10 not vary as much as their action potential sizes measured dB higher than BF tone thresholds. Unlike the nonmonotonic from simple spikes (58 { 14 mV). Simple action potentials RSL curves obtained with BF tones, all type IV and IV-T from most units (6 of 7) had a slow rising and falling phase units responded to current pulse injection of increasing levels without undershoots. The complex-spiking type III unit, with increased discharge rates (Fig. 7C). The minimum cur- however, had undershooting action potentials. rent level required to reach threshold was very low (Ç0.07 Complex-spiking units had some SpAc (23 { 14 spikes/s). nA) for both type IV units but high (Ç0.5 nA) for the type Unclassi®able units were poorly tuned to tonal stimuli and

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TABLE 4. Type III units

BF, RMP, APS, SpAc, uS , RS , uI , RI , RN , t, PCI, File ID Unit ID kHz mV mV spikes/s dB SPL spikes/s nA spikes/s MV ms, % j51293 10-01 1.7 065 42 90 10 285 0.07 400 28 j52793 05-01 2.2 059 51 26 20 229 0.07 240 22 3.3, 11 j61393 04-01 2.2 052 58 133 30 227 0.07 280 7 j61793 06-01 1.7 061 62 30 15 102 0.07 27 2.3, 26 j62993 08-03 6.6 062 77 17 50 308 0.2 160 j81293 04-01 1.5 060 69 83 5 319 j81993 05-01 7.3 056 58 70 10 221 0.07 280 28 4.8, 6 j81993 10-01 4.7 058 59 77 0 229 jn2393 11-01 4 061 64 83 0 323 0.07 320 15 4.6, 14 jn3093 08-01 5.9 067 79 37 0 275 0.33 160 25 1.9, 8 jd0793 19-01 8.5 064 63 40 25 240 jd0993 04-01 2.1 067 79 27 5 167 jd2193 12-01 3.6 058 53 80 0 229 0.07 240 26 j10694 02-01 3.3 060 65 30 20 135 0.07 80 9 j11794 07-03 9.9 074 65 17 35 200 0.07 260 31 2.2, 7 j22394 12-01 1.5 069 55 57 5 221 j30994 10-01 9.1 063 63 20 40 158 0.07 220 23 j31494 17-01 6 058 58 30 10 160 0.07 120 23 j42094 01-03 1.5 051 52 93 15 242 0.07 240 20 j32195 17-01 6.9 058 63 26 10 136 j32695 08-01 2.2 052 53 51 50 133 0.07 160 25 3.2, 21 j80295 11-01 12.2 061 65 56 0 215 0.07 300 28 4.9, 3 j80295 15-01 4.7 058 62 23 5 237 j82195 01-01 6.8 062 45 25 5 310 0.07 420 50 6.6, 4 jo1895 03-01 3.8 062 65 35 5 206 0.07 260 38 4.8, 5 Mean { SD 060.6 { 5.3 61 { 9.2 50 { 31 15 { 15 220 { 62 0.09 { 0.07 244 { 91 24 { 10.3 3.9 { 1.6

For abbreviations, see Table 2. had high BF thresholds (ú30 dB SPL) and low maximum throughout the stimulus. After the current pulse stops, the driven rates (õ100 spikes/s) (e.g., J41194-14-1 and cell's membrane potential drops Ç10 mV below resting po- J41394-4-1 in Fig. 10B). Noise responses were even weaker tential and reaches resting potential again after nearly 40 than tonal responses. These units usually responded to acous- ms. Afterhyperpolarization following current injection oc- tic stimuli with simple action potentials. Complex action curred in 13 of 39 units tested, with the majority appearing potentials occurred less frequently and did not appear to be in the responses of type III units. This feature is seen in acoustically driven. Type III unit J61793-8-1 was the only about half the type III population. Table 7 summarizes these one with a relatively low threshold to BF tones (15 dB SPL) and other features seen in the current pulse responses. and a maximum driven rate ú120 spikes/s. It was also the When the initial voltage response to a current pulse is only unit that responded to tone and noise bursts with com- greater than the unit's steady-state voltage response, there plex action potentials at all suprathreshold levels. is a sag in the voltage response waveform. Examples of such Unlike the monotonic responses to current pulse injections sags from a type II unit and a type III unit may be seen in for all simple-spiking units, many complex-spiking units re- Fig. 11, C and D. Sags are seen in about one third of the sponded nonmonotonically to depolarizing current pulses type III population and in three of seven type II units; they (see examples in Fig. 10C). Some of this nonmonotonicity were not seen in type I/III units (Table 7). was because the simple action potentials would decrease Anode break excitation was observed in only 3 of 39 in size with the larger depolarizing currents, making them units. An example from a type III unit is seen in Fig. 11B. dif®cult to detect. Most units, however, tended to ®re com- plex action potentials at the onset of high-current-level stim- DISCUSSION uli. Figure 10C, insets, are the estimates of the I-V curves for these units. The input resistance for the complex spiking Comparison with other intracellular studies: in vivo units averaged 40 { 18.2 MV, and the largest equalizing preparations time constants averaged 5.0 { 1.5 ms. Two complex-spiking All previous in vivo intracellular recording studies of the units were localized to the molecular layer. DCN have been done on anesthetized animals (Britt and Starr 1976; Gerstein et al. 1968; Rhode et al. 1983a,b; Ro- Additional features of current pulse responses mand 1978; Smith and Rhode 1985, 1989). Comparing the results of this study with those of previous studies is thus Occasionally, a unit's response to current pulse injection dif®cult. Another confounding problem is that units in the would contain features, such as afterhyperpolarization, earlier studies relied primarily on the temporal-response- anode break excitation, or ``sag,'' that warrant mention. Ex- based (peristimulus time histogram) classi®cation scheme amples of these three features may be seen in Fig. 11. Figure introduced by Pfeiffer (1966). Units in the current study are 11A shows unit J82593-12-01, a type I/III unit, responding classi®ed according to the response map scheme introduced to a 1-nA current pulse with a steady rate of action potentials by Evans and Nelson (1973). The response map scheme

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FIG. 5. Intracellular responses (A), rate-vs.-sound level curves (B), and rate-vs.-current level curves (C)of3type III units, J52793-5-1 (left, localized to the fusiform cell layer, JN2393-11-1 (middle), and J80295-11-1 (right). A: 3 intracellular responses for each unit to 50- or 100-ms, 60-dB SPL BF tone bursts displaced from each other to aid visualization. Bar along time axis: stimulation interval. B: discharge rate-vs.-sound level curves for each unit derived from responses to 50- or 100-ms BF tones and noise bursts from 0 to 80 dB SPL in 5-dB steps. Data were smoothed with a 3-point ®lter. BF is indicated on each plot. C: non®ltered discharge rate-vs.-current level curves for each unit derived from responses to 16 or 17 trials of 50-ms current pulse from 01.0 to 1.0 nA in equal step size. Hyperpolarizing currents had negative values. Insets: estimated I-V curves. Input resistance estimates for these 3 units are 22, 15, and 28 MV (left to right). was recently shown not to be very useful in the barbiturate- 1997a,b). So, in fact, the earlier investigators may have had anesthetized gerbil, where up to 80% of DCN neurons have no choice but to use the peristimulus time histogram scheme. almost no SpAc (Gdowski 1995; Gdowski and Voigt Gerstein et al. (1968) were the ®rst to study the intracellular

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FIG.6. A: intracellular responses of the type III unit (J80295-11-1) as a function of sound pressure level (from 0 to 70 dB SPL in 10-dB steps) for 3 frequencies [7.52, 12.22 (BF), and 19.85 kHz] and noise (100 ms in duration). At each frequency and level, 3 responses were collected and are displaced from each other to aid data visualization. B: 4 intracellular responses from A, enlarged for clarity: Ba, 7.52 kHz, 70 dB SPL; Bb, 7.52 kHz, 40 dB SPL; Bc, 19.85 kHz, 70 dB SPL; Bd, noise, 70 dB SPL. C: same unit's response to 16 trials of 50-ms current pulses from 01.0 to 1.0 nA in equal steps. Traces are overplotted such that response to larger current is placed higher. Sudden shift in voltage was due mainly to voltage drop across the recording electrode. Bar along time axis: stimulation interval. membrane potentials of DCN neurons. They described sound- tained depolarizations were from cell bodies and those without evoked depolarizations that were usually accompanied by in- were from cell processes. With better resting potentials, Rhode creased discharge rate. More often, however, increases in et al. (1983b) reported that fusiform cells with resting potentials sound-evoked ®ring rates occurred without any accompanying between 050 and 065 mV, responded to tone pips with depo- depolarizations. Romand (1978) also reported that DCN units larizations of Ç10 mV that lasted the duration of the stimulus respond to BF tones either with or without sustained depolariza- and long-lasting hyperpolarizations of similar size following tions. Romand suggested that perhaps the recordings with sus- the stimuli. Many of our units show sustained depolarizations

TABLE 5. Type IV (IV-T) units

BF, RMP, APS, SpAc, uS , RS , RI , RN , t, PCI, File ID Unit ID Type kHz mV mV spikes/s dB SPL spikes/s uI , nA spikes/s MV ms, % j71593 10-01 IV 4.7 064 61 57 10 94 0.07 220 30 12.9, 8 jd2193 04-01 IV 1.5 062 75 60 5 113 0.07 260 14 2.6, 37 j31494 19-01 IV-T 3 077 57 3 25 77 0.07 240 30 10.6, 66 Mean { SD 067.5 { 8 64.5 { 9.5 40 { 32 13 { 10 94 { 18 0.07 { 0 240 { 20 24 { 9.1 8.7 { 5.4

For abbreviations, see Table 2.

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FIG. 7. Intracellular responses (A), rate-vs.-sound level curves (B), and rate-vs.-current level curves (C) of 2 type IV units, JD2193-4-1 (left) and J71593-10-1 [right, localized to the dorsal cochlear nucleus (DCN) deep layer], and 1 type IV-T unit, J31494-19-1 (middle, localized to the DCN deep layer). A: 3 intracellular responses for each unit to 50-ms, 60- dB SPL BF tone bursts displaced from each other to aid visualization. Bar along time axis: stimulation interval. B: discharge rate-vs.-sound level curves for each unit derived from responses to 50-ms BF tones and noise bursts from 0 to 80 dB SPL in 5-dB steps. Data were smoothed by a 3-point ®lter. BF is indicated on each plot. C: non®ltered rate-vs.-current level curves of each unit were derived from responses to 16 trials of 50-ms current pulse from 01.0 to 1.0 nA in equal step size. Hyperpolarizing currents had negative values. Insets: estimated I-V curves. Input resistance estimates for these 3 units are 14, 30, and 30 MV (left to right). during the acoustic stimuli and long-lasting hyperpolarizations than ®ring action potentials. It is likely that the use of 3 M afterward (20 of 46). In fact, only seven of our classi®ed units KCl in the recording electrodes in some early studies made it did not exhibit a stimulus-evoked membrane response other dif®cult to observe hyperpolarizations. Although the electrodes

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FIG.8. A: intracellular responses of the type IV-T unit (J31494-19-1) as a function of sound pressure level (from 0 to 70 dB SPL in 10-dB steps) for 3 frequencies [1.85, 3.00 (BF), and 4.87 kHz] and noise (50 ms in duration). At each frequency and level, 3 responses were collected and are displaced from each other to aid data visualization. B: 4 intracellular responses from A, enlarged for clarity: Ba, 1.85 kHz, 70 dB SPL; Bb, 3.00 kHz, 70 dB SPL; Bc, 4.87 kHz, 70 dB SPL; Bd, noise, 70 dB SPL. C: same unit's response to 16 trials of 50-ms current pulses from 01.0 to 1.0 nA in equal steps. Traces are overplotted such that response to larger current is placed higher. Sudden shift in voltage was due mainly to voltage drop across the recording electrode. Bar along time axis: stimulation interval. Arrow: PSP. used in the current study were ®lled with 0.5 M KCl, it is likely et al. 1996a) had a similar unit distribution of type III and that even this concentration affected intracellular responses. III-i units (62%), type IV and IV-T units (16%), type I/III Gerstein et al. (1968) also described a situation in which units (14%), and type II units (8%), on the basis of a total a neuron, when presented a tone stimulus, depolarized for the of 133 units (see Table 8). Missing from this group, how- duration of the stimulus, but action potentials were generated ever, are complex-spiking units, which are dif®cult to iden- only after a delay. Rhode et al. (1983) reported a similar tify reliably in extracellular records. The low numbers of ®nding. Examples of this are also seen in our recordings of type II units in the present study suggest that neurons with type II units (Figs. 2, left and middle, and 3A). type II unit properties are particularly susceptible to injury by electrode impalement. Another difference between these Unit incidence results and those of Davis et al. (1996a) concerns type III- i units. Although this is a sizable population in the gerbil, Intracellularly recorded type III units make up nearly half few type III-i units were identi®ed in this study. This was (49%) of the recorded population. Type IV and IV-T units, probably due to an inadequate protocol for distinguishing on the other hand, are only 6% of the population, whereas type III-i units from type III units. These two unit types type I/III and type II units provided Ç17% each (see Table are similar to each other except for their broadband noise 1). About 11% of the units recorded had weak acoustic responses. Type III units are excited by noise at all levels responses, were broadly tuned, and had complex spikes. Ex- above threshold, whereas type III-i units are excited by high- tracellular data from the same lab and preparation (Davis level noise stimuli, and are inhibited by low-level noise

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FIG.9. A: intracellular responses of the type IV unit (J71593-10-1, localized to the DCN deep layer) as a function of sound pressure level (from 0 to 70 dB SPL in 10-dB steps) for 3 frequencies [2.89, 4.70 (BF), and 7.64 kHz] and noise (50 ms in duration). At each frequency and level, 3 responses were collected and are displaced from each other to aid data visualization. B: 4 intracellular responses from A, enlarged for clarity: Ba, 2.89 kHz, 70 dB SPL; Bb, 4.70 kHz, 70 dB SPL; Bc, 4.70 kHz, 30 dB SPL; Bd, 4.70 kHz, 40 dB SPL. C: same unit's response to 16 trials of 50-ms current pulses from 01.0 to 1.0 nA in equal steps. Traces are overplotted such that response to larger current is placed higher. Sudden shift in voltage was due mainly to voltage drop across the recording electrode. Bar along time axis: stimulation interval.

(Davis et al. 1996a). Type III-i units are more reliably iden- because usually the inhibition occurs within a narrow range ti®ed when their rate-versus-level curves are constructed of noise levels (Davis et al. 1996a). Because intracellular from responses to long-duration (200-ms) stimuli that are contact times were expected to be short, shorter-duration presented with the use of a small sound level step size, stimuli and larger sound level step sizes were used in this

TABLE 6. Complex-spiking units

BF, RMP, APS, SpAc, uS , RS , uI , RI , RN , t, PCI, File ID Unit ID kHz mV mV spikes/s dB SPL spikes/s nA spikes/s MV ms, % j61793 08-01 5.9 067 71 33 15 135 0.07 160 43 4.0, 3 jd1493 05-01 4.0 066 52 53 30 77 j32394 20-01 3.8 067 70 10 40 60 0.07 140 14 j41194 14-01 2.8 068 52 13 50 51 0.07 80 48 4.3, 3 j41394 04-01 7.0 070 77 17 30 85 0.07 40 55 6.7, 6 j30795 02-01 1.3 060 41 30 55 50 j91895 03-01 4.3 073 45 1 40 4 Mean { SD 067.2 { 4.1 58.3 { 14.1 23 { 18 37 { 13 66 { 40 0.07 { 0 105 { 55 40 { 18.2 5.0 { 1.5

For abbreviations, see Table 2.

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FIG. 10. Intracellular responses (A), rate-vs.-sound level curves (B), and rate-vs.-current level curves (C) of 3 complex- spiking units, J41194-14-1 (left, localized to the DCN molecular layer), J61793-8-1 (middle, localized to the DCN molecular layer), and J41394-4-1 (right). A: 3 intracellular responses for each unit to 50-ms BF tone, at 60 dB SPL for unit J41194- 14-1, 30 dB SPL for unit J61793-8-1, and 40 dB SPL for unit J41394-4-1, displaced from each other to aid visualization. Bar along time axis: stimulation interval. B: discharge rate-vs.-sound level curves for each unit derived from responses to 50-ms BF tones and noise bursts from 0 to 80 dB SPL in 5-dB steps. Data were smoothed with a 3-point ®lter. BF is indicated on each plot. C: discharge rate-vs.-current level curves for each unit derived from responses to 16 trials of 50-ms current pulse from 01.0 to 1.0 nA in equal step size. Hyperpolarizing currents had negative values. Insets: estimated I-V curves. Input resistance estimates for these 3 units are 48, 43, and 55 MV (left to right). study. Therefore it is very likely that some type III units in so in the extracellular recordings from the DCN in decere- this study are really type III-i units. brate cats. For example, Shofner and Young (1985) reported Type IV units are more plentiful and type III units less 31% type IV units, 23% type III units, and 19% type II

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but all these units were classi®ed as type III or type IV units. Because we only included classi®able units in our extracellular data base, some bursting units with poor acous- tic response might have been overlooked.

Sound-evoked and spontaneous intracellular membrane potentials Individual excitatory PSPs (EPSPs) and inhibitory PSPs (IPSPs) were rarely seen in these intracellular records. This is similar to the in vivo recordings of Rhode et al. (1983) but in contrast to the in vitro recordings of Oertel and coworkers. Integrated EPSPs and IPSPs evoked by sound stimuli, how- ever, were frequently encountered.

Type I/III units In general, type I/III units respond to tones and noise with sustained depolarizations that may or may not be followed by afterhyperpolarizations. Rhode et al. (1983) recorded from fusiform cells in cat and presented examples of hyperpolar- izations following sustained depolarizations, some of which extended for hundreds of milliseconds after the stimulus ended. Rhode et al. also showed examples in which the hyperpolarization begins before the stimulus ended. In these cases the tones were far from BF, and the hyperpolarizations were clearly the result of inhibitory processes. It is not clear, however, whether the afterhyperpolarizations are the result of active inhibitory processes or are due to some internal regulatory activity of the neurons themselves that compen- sates for the preceding sustained depolarization. It seems FIG. 11. Examples of poststimulus hyperpolarization (A), anodal break spikes (B), and sags (C and D) for 4 units in response to current pulse more likely that it is due to internal regulatory activity of injection. Arrows in each plot: corresponding feature. Current levels are the neurons, because they always appear with sustained de- indicated in the plots. Bar along time axis: stimulation interval. polarization. In addition, two of nine type I/III units showed hyperpolarizations after the offset of depolarizing current units. On the basis of inhibitory interactions between type pulse injection (Fig. 11A). II units and type IV units in cat (Voigt and Young 1980, Because type I/III units have little, if any, SpAc, inhibi- 1990), the paucity of type IV units in the gerbil has been tion cannot be detected simply by counting spikes. Inhibitory hypothesized as being due to a paucity of type II units or responses during acoustic stimulation can be detected, how- weak inhibitory properties of existing type II units (Davis ever, by observing hyperpolarizations. Only one type I/III et al. 1996). Simulations showing the effects of a reduced unit responded to sound with hyperpolarization, and this was population of inhibitory interneurons on the response proper- to a tone whose frequency was an octave above BF. It ap- ties of DCN projection units are consistent with this hypothe- pears then that the typical type I/III units receive few, if sis (Davis and Voigt 1996a). any, inhibitory inputs that cause the cell to hyperpolarize. Complex-spiking units were generally unclassi®able with That is, if there are inhibitory inputs to type I/III units, their the use of the standard response map scheme. Only one unit in¯uence rarely overcomes the more prominent excitatory (a type III unit) was classi®ed. If recorded extracellularly, input and they would probably be mediated by clamping the complex-spiking units would only be re¯ected by their burst- membrane at its resting potential. ing ®ring patterns. Recording from bursting units, however, does not necessarily imply recording from complex-spiking Type II units units. In fact, a few bursting units (Ç7%) were noted in the Most of the type II units exhibited strong sustained depo- experimental log and placed in the extracellular data base, larizations in response to tones. Many of these also had

TABLE 7. Summary of current pulse response features

Unit Type I/III II III IV Complex Total

Number of units 8 7 17 3 4 39 Anode break excitation 1 0 2 0 0 3 Sag 0 3 6 0 1 10 Afterhyperpolarization 2 1 9 0 1 13 Slope, Hz/nA 312 { 212 113 { 85 235 { 85 292 { 198 166 { 110

Slope values are means { SD.

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TABLE 8. Summary of physiological features for extracellularly recorded units (average values)

Unit Type I/III II III III-i IV IV-T

Number of units 19 10 58 25 6 7 Percent of units 15 8 46 20 5 6 BF threshold, dB SPL 22.7 19 11.6 15.1 11.3 12.2 BF maximum rate, spikes/s 260.4 260.5 226.2 217.8 130.0 132.6 Noise threshold, dB SPL 37.6 39.6 28.2 23.2 15.7 24.9 Noise maximum rate, spikes/s 147.4 63.6 139.1 122.2 133.4 87.9 Spontaneous activity, spikes/s 0.4 0.8 36.7 42.5 48.1 13.9

BF, best frequency. afterhyperpolarizations with the depolarizations. Type II this and to appear as positive de¯ections. Leakage of KCl units, like type I/III units, lack enough SpAc to detect side- from the recording electrode may also cause disruption of band inhibition. The fact that they respond weakly to broad- inhibitory mechanisms, making identi®cation of these small band noise implies that they receive inhibition strong enough potentials problematic. to overcome the excitatory input from near BF energy. In fact, type II units depolarize slightly when broadband noise Complex-spiking units is presented, just not enough to elicit action potentials. Shofner and Young (1985) showed that type II units were Zhang and Oertel (1993a) identi®ed cartwheel neurons inhibited by shocks to the VIIIth nerve. Spirou and Young as a source of complex-spiking units in the mouse DCN (1991) have shown that this inhibitory input can be observed slice preparation. Manis et al. (1994) reported the same in if one ®rst activates the type II unit with a low-level BF the guinea pig DCN slice preparation; however, simple tone before presenting a second tone whose frequency and spikes were only found in 3 of 29 complex-spiking units. level falls within the inhibitory sideband. The intracellular The complex-spiking units of this study all generated simple data of the present study con®rm hyperpolarization of many spikes as well. Parham and Kim (1995) reported that 80% type II units during off-BF stimuli, providing strong evi- of the extracellularly recorded burstinglike units in the cat dence of the existence of inhibitory sideband inputs. DCN, which they presumed were from cartwheel cells, re- It is widely thought that type II units, which in cat may be sponded weakly to sound stimuli. This is true of all but one antidromically activated from the anteroventral CN (Young of our complex-spiking units. In addition, preliminary data 1980), are tuberculoventral neurons of Oertel (also called from this laboratory support the notion that cartwheel cells corn cells or vertical cells). are a source of complex-spiking neurons (Ding et al. 1994b). The weak acoustic responses of most complex-spiking units imply nonauditory functions for these units. In fact, it has Type III units been shown that somatosensory stimulation can activate Type III units have enough SpAc so that sideband inhibi- burstinglike units (Davis et al. 1996b) and inhibit DCN tion can be seen for both intracellular and extracellular re- principal cells (Young et al. 1995). These results suggest cording situations. When inhibited, many type III units ex- that complex-spiking units, presumably cartwheel cells, play hibited noticeable hyperpolarizations. Some units, however, a special role in processing multisensory information. lacked such hyperpolarizations. Preliminary results from our intracellular recording and Measurements obtained from intracellular current marking study have shown that many type III units are fusi- injections form cells. Unit input resistances in this study are generally small, suggesting the presence of a microelectrode-induced shunt. Type IV and type IV-T units These resistances, however, are comparable with those mea- sured by Manis in vitro from fusiform cells and cartwheel Gerbil type IV and IV-T units are the rarest units in both cells (Manis 1990; Manis et al. 1994), but lower than those the intracellular and extracellular unit groups. Unlike most reported for DCN neurons in vitro by Zhang and Oertel other units (including type IV-T units), the type IV units (1993a±c, 1994). Oertel has been coating electrode tips did not exhibit sustained depolarization during tonal stimula- with dichlorodimethyl silane, which Oertel claims facilitates tion at levels õ60 dB SPL. Hyperpolarizations, on the other sealing of the membrane around the penetrating electrode. hand, were seen in the type IV units at lower levels at which This may be the reason for the disparity in reported mem- the units were inhibited. Such membrane behavior suggests brane resistances in DCN units. either a weak excitatory input or a relatively strong inhibitory The majority of I-V curves (65%) from units in this study input onto type IV units. In contrast, strong onset depolariza- appears linear within 10±15 mV below the resting membrane tion during tonal stimulation in one type IV-T unit suggested potential. This is similar to results obtained by Zhang and that it could receive a very strong excitatory input. This cell Oertel (1993a±c, 1994). also showed spontaneous PSPs throughout its record that usually did not result in action potentials. Such PSPs may Intracellular versus extracellular data be EPSPs, but the relatively low resting potential (077 mV) argues that even IPSPs may cause the membrane potential Neurons impaled with microelectrodes are sometimes in- to drive to an equilibrium potential that is less polarized than jured, and therefore responses from such neurons may be

/ 9k11$$my27J460-6 08-08-97 12:41:09 neupal LP-Neurophys 2568 J. DING AND H. F. VOIGT slightly or even dramatically different from their natural re- TABLE 9. Intracellular vs. extracellular response property sponses. Although such damage may occur with extracellular differences methods as well, it is worth checking for obvious differences in response properties of neurons recorded intracellularly Unit Type Type II Type I/III Type III with those recorded extracellularly. Figure 12 shows the BF tone threshold F* N.D. N.D. intracellularly recorded unit's BF tone threshold plotted Noise threshold N.D. N.D. N.D. against its BF. BFs typically ranged from 2 to 10 kHz and Maximum rate to BF tones f² f* N.D. the thresholds ranged from 0 to 55 dB SPL. Except for type Maximum rate to noise f* N.D. F² II units and the complex-spiking units, most of the other Relative noise index N.D. F* F² units are within the threshold-BF boundary for auditory Normalized slope F³ N.D. N.D. Spontaneous activity N.D. N.D. N.D. nerve ®bers (dotted lines, from Schmiedt 1989) and also overlap with the thresholds of extracellularly recorded units Arrows: direction of change in property of intracellular units compared obtained from the same preparation (dash-dot lines, from with units of the same class recorded extracellularly. BF, best frequency; Davis et al. 1996a). N.D., no difference. * P õ 0.05. ² P õ 0.01. ³ P õ 0.001. To assess the possible ill effects of intracellular penetra- tion on the response properties of these physiologically iden- corded extracellularly. This difference is signi®cant at the ti®ed units, comparisons were made of BF tone threshold, P õ 0.05 level (Table 9) and suggests that the microelec- noise threshold, maximum discharge rates to BF tone and trode penetration of type I/III units affected at least some noise stimuli, r, normalized slope, and SpAc for units re- units' acoustic properties. Because of the decreased response corded intracellularly against those recorded extracellularly. to BF tones, the relative noise index for type I/III units also Differences in these various parameters were tested for sig- changed signi®cantly (P õ 0.05). ni®cance with the use of a two-tailed t-test; P values õ0.05 were taken to be signi®cant. The results of these tests are Type II units summarized for type I/III, type II, and type III units in Table 9 and are discussed below. The effects of intracellular impalement are even greater on the acoustic response properties of type II units. Intracel- Type I/III units lularly recorded type II units had on average twice the thresh- old to BF tones of extracellularly recorded type II units (32 BF tone thresholds and SpAc rates of type I/III units vs. 19 dB SPL). The average maximum BF-driven rate was recorded intracellularly are similar to those of type I/III less than half as much as that of extracellular units (112 vs. units recorded extracellularly (see Table 2, 7, and 9). The 261 spikes/s, see Table 3, 7, and 9). Although r values average maximum driven rates to noise stimuli are also com- were within normal range for type II units, the normalized parable. The average maximum BF-driven rate, however, tone slopes were much smaller and, often, positive. The was only 176 spikes/s when the type I/III units were im- differences in type II units' BF tone thresholds, maximum paled compared with 263 spikes/s when the units were re- rates to both BF tones and noise, and the normalized tone slopes are all statistically signi®cant (Table 9). Because most type II units in this study had large, overshooting action potentials (largest average APs of all units types), the pene- tration of the microelectrode probably did not damage the units' spike initiation mechanism. In addition, the type II units appeared healthy with respect to resting potential and action potential shape. We consider how the electrode may be affecting some acoustic response properties of these units below.

Type III units Type III units are the largest population in both intracellu- lar and extracellular unit groups. The average BF tone thresh- olds (15 and 12 dB SPL), maximum BF-driven rates (220 and 226 spikes/s), normalized tone slopes, and spontaneous rates of activity are comparable (see Tables 2 and 3). The maximum rate to noise, however, was signi®cantly greater from type III units recorded intracellularly (Table 9). r is consequently greater for the intracellular population (Fig. 4B). These are unexpected and interesting results that are contrary to the type II unit results, where the driven rates simply decreased. To check whether the signi®cance of this result was simply spurious, the extracellular data set was FIG. 12. BF threshold vs. BF for all intracellular units. Dotted lines and dash-dot lines: boundaries of the thresholds of auditory nerves (Schmiedt divided into two equal parts and each set was tested against 1989) and of the units recorded extracellularly (Davis et al. 1996a), respec- the intracellular data set again with the use of both the tively. t-test and the Wilcoxon-Mann-Whitney rank-sum test. The

/ 9k11$$my27J460-6 08-08-97 12:41:09 neupal LP-Neurophys INTRACELLULAR RESPONSE PROPERTIES OF GERBIL DCN UNITS 2569 latter test is to determine whether these data sets could have near the cell's resting potential (Staley et al. 1992). It is come from two populations with the same mean (Diem intriguing to speculate what effects these additional conduc- 1966; Kanji 1993). In each case, the null hypothesis that tances have on DCN unit response properties. Of course, if the means are the same for the two populations was rejected the damage done by the electrode is too great, the resting at ¢95% con®dence level. potential will dissipate and the cell will die. We are not considering this case, but one in which the resting potential Type IV and IV-T units is maintained despite the electrode's presence. If we assume that the behavior of extracellularly recorded neurons is more There are too few examples of type IV units in this study similar to that of neurons recorded with patch electrodes to say what effect, if any, electrode penetration has on type than with intracellular sharp electrodes, then a smaller value IV acoustic responses. The average thresholds and maximum of electrotonic length may be assumed for extracellularly BF-driven rates of the type IV units were 11 dB SPL and recorded units. This implies that the steady-state voltage 125 spikes/s for the intracellular data and 11 dB SPL and decay over the length of the dendrites is smaller for units 130 spikes/s for the extracellular data. Type IV-T units dif- recorded extracellularly than for those recorded intracellu- fered little from type IV units in both threshold and maxi- larly. If this is true, then synapses on distal dendrites would mum rate (see Tables 2 and 3). have a more profound impact on the somas of neurons that were not impaled. Perhaps this is the reason type II and type Impact of the intracellular recording electrode I/III units give greater responses to BF tones when they are recorded extracellularly rather than intracellularly. The It appears that the effects of the intracellular recording situation involving type III units, however, is more complex. electrode on the acoustic response properties of DCN neu- A larger noise response might occur if the noise threshold rons depend to some extent on the unit type impaled. This were speci®cally reduced, but this is not so (Table 9). is interesting, especially because some differences were not Applying the logic above (i.e., distal dendrites will have less expected (e.g., type III units' maximum responses to noise in¯uence on activity near the soma of intracellularly re- increases when recorded from intracellularly). To check that corded units) to the intracellularly recorded type III units the results of Table 9 are not simply due to the inclusion of would account for the increased response to noise only if the cells with poor resting potentials, the t-tests were rerun with preponderance of distal synapses were inhibitory. Inhibitory the use of only those cells with resting potentials of 060 mV synapses on fusiform cells are thought to arise from at least or less instead of 050 mV. Except for the type II threshold three sources. Cartwheel cells of the molecular and fusiform difference now being insigni®cant, all other results remained cell layer are thought to provide inhibitory synapses to fusi- statistically signi®cant. In addition, the resting potentials at form cells (Berrebi and Mugnaini 1991). Cartwheel cells the start of data collection were compared with those at the are not often strongly activated by auditory stimuli (Parham end of data collection and were found not to be different and Kim 1995; unpublished results) and are thus unlikely (paired t-test), suggesting that the results are not due to candidates. Vertical cells are thought to have type II unit some general cell deterioration. response properties (Voigt and Young 1990; Young and The stimulus durations for the intracellular recordings were shorter than those for the extracellular recordings (50 Voigt 1982), to be glycinergic (Saint Marie et al. 1991; vs. 200 ms), and therefore the rate estimates for the two Wickesberg and Oertel 1990), and to provide synapses to situations were calculated over different intervals. To ensure the soma and proximal dendrites of fusiform cells, and are that the maximum discharge rates to tone and noise were not activated strongly by noise stimuli. Thus they are un- not different simply because of these two different rate esti- likely to play a role in the increased noise responses of mates, the discharge rates for the extracellular data were intracellularly recorded type III units. One likely possibility recalculated with the use of only the ®rst 50 ms of data. The is the wideband inhibitor (Nelken and Young 1994; Winter use of this rate estimate did not alter any of the statistically and Palmer 1995), which may correspond to the D-stellate signi®cance differences reported in Table 9 for type I/III, cells of the posteroventral CN, whose axons project into the type II, or type III units. deep DCN (Oertel et al. 1990). These projections could Staley et al. (1992) studied the membrane properties of target both the basal dendrites of the fusiform cells and the dentate gyrus granule cells with the use of both sharp elec- dendrites and somata of the vertical cells and giant cells. trodes and whole cell recordings in hippocampal slice prepa- The properties required of the wideband inhibitor include a rations. They estimated the electrotonic length of the equiva- weak response to tones and a strong response to noise, which lent cylinder representing the cell processes to be only 0.49 are precisely the response properties of the onset-C units from the whole cell data instead of 0.79 given by the sharp (Winter and Palmer 1995). Preliminary results from this lab electrode data. They also calculated from the whole cell data have shown that type III unit properties can arise from gerbil that at rest there is only a 10% decrement in the DC mem- fusiform cells (Ding et al. 1994a). If these fusiform cells brane voltage along the length of the dendrite and thus syn- are impaled with a microelectrode and the inhibitory in¯u- aptic events at the distal dendrites have a greater impact on ences of the wideband inhibitors are reduced preferentially the cell than previously thought. Staley et al. (1992) suggest to the excitatory, auditory nerve input, then a greater noise that the differences resulting from the use of sharp versus response in this cell would result. A problem with this sce- patch electrodes are due to a combined local electrode-asso- nario is that the auditory nerve input to fusiform cells is also ciated nonspeci®c leak conductance and a potassium conduc- on the basal dendrites and therefore subject to the same tance that is distributed over the cell soma and dendritic effects caused by the electrode. Thus consideration of the processes. Both conductances would have reversal potentials effects of the impaling electrode on the cable properties of

/ 9k11$$my27J460-6 08-08-97 12:41:09 neupal LP-Neurophys 2570 J. DING AND H. F. VOIGT neurons may account for the type I/III and type II unit were substantially altered by the impaling microelectrode, it responses, but not those from type III units. is likely that these slopes are only lower bound estimates. Another possible explanation for the enhanced noise re- For complex-spiking units, the action potentials were re- sponses from intracellularly recorded type III units is that lated to the current strength. High current levels were likely the 0.5 M KCl within the microelectrode had a more direct to trigger complex action potentials and could induce onset impact on the inhibition mediated by chloride conductances. responses, resulting in nonmonotonic current responses for Fromm and Schultz (1981) concluded that leakage out of many units. On the basis of this relationship between the microelectrodes ®lled with 3 M KCl was unnecessarily high spike appearance and current strength, the coexistence of and that it could be reduced ®vefold by the use of 0.5 M simple and complex action potentials during acoustic stimu- KCl. The reduced salt concentration would also yield elec- lation might suggest a difference in the strength of excitatory trodes with tip resistances or tip potentials that were not too inputs. Complex action potentials were probably triggered large. Although this concentration of KCl, which was used when strong excitatory inputs were integrated and the mem- in previous DCN studies (Manis et al. 1994; Rhode et al. brane potential reached well above the threshold, whereas 1983), is better than the 3 M KCl solutions used in the simple action potentials were probably generated when weak initial DCN studies (Gerstein et al. 1968; Romand 1978), it excitatory inputs were integrated and slightly exceeded the may still have an important impact on Cl 0-channel-mediated threshold. The strength of the excitatory input could be re- inhibitory mechanisms functioning in DCN neurons. The lated to different synaptic contacts and proportional to the impact of Cl 0 may have been compounded further in these number of the excitatory synaptic inputs as well. earlier studies by the presence of barbiturates known to affect g-aminobutyric acid receptors (Barker and Ransom 1978), Possible mechanism for controlling a unit's bandwidth which have been associated with Cl 0 channels. Although the presence of barbiturates is not a concern in this study, Depolarizations at levels below spike generation in type a reduction in the inhibitory in¯uences on the soma of a II units (example in Fig. 3A) and type IV-T units (example fusiform cell might result from KCl leakage from an impal- in Fig. 8A) indicate that these units are more sensitive than ing microelectrode. If the inhibitory action affected is nor- spike threshold reveals. This is intriguing because if there mally activated when noise is presented, as with the case of were mechanisms to modify a unit's excitability, controlling the wideband inhibitor, then an increased response to the the frequency selectivity expressed by the unit would be noise would be expected. possible. The granule cell population, with their parallel ®- bers, makes an interesting candidate for this bandwidth con- trol. Activation of parallel ®bers is thought to cause increased Responses to electric current excitability in their targets (Manis 1989; Osen and Mugnaini Despite the diversity in their acoustic responses, all sim- 1981). Thus sound levels that evoke spikeless depolariza- ple-spiking units responded monotonically to current injec- tions in these target cells (principally fusiform cells) might tion. Therefore the nonmonotonic behavior of these cells in now, with parallel ®ber activation, provoke cell discharge. response to acoustic stimulation is most likely the result At BF, this would decrease sound-evoked spike threshold. of inhibitory inputs and not due solely to the membrane Off BF, it is also likely that stimulus conditions that failed properties. For units with SpAc (type III and IV units), to generate spikes would now become active. Inactivation inhibition could also be seen when hyperpolarizing current would lead to increased spike thresholds. This is a possible was injected. Thresholds to current injection were compara- neural mechanism by which the auditory system can either ble for all units except the type II units. The high thresholds increase or decrease the bandwidth of DCN neurons. Al- of type II units are consistent with their high sound thresh- though the role of such a mechanism in audition is strictly olds (Davis et al. 1996a; Young and Voigt 1982). Data from speculative at this time, this might be an important mecha- tuberculoventral cells in the mouse slice preparation show nism for the auditory system to use in modifying signal to relatively high (Ç1 nA) current thresholds for action poten- noise ratios or for selecting various portions of the input tial generation (Zhang and Oertel 1993c). stream to analyze. Time- and voltage-dependent sags were seen in the re- sponses to current injection in 9 of 35 simple-spiking units Thanks to P. Patterson and K. Hancock for help in producing the ®gures. Thanks also to K. Hancock for comments and discussions of earlier drafts in this study. Manis (1990) reported such sags in 23 of 53 of this paper. simple spiking cells in DCN slices from guinea pig. This work was seeded by National Science Foundation Grant BNS The slopes of the frequency-current curves varied from a 8420495 and further supported by National Institute of Deafness and Other low average of 113 Hz/nA for type II units to a high average Communications Disorders Grant DC-01099 and by the Department of Biomedical Engineering at Boston University. of 312 Hz/nA for type I/III units. The simple-spiking units Address for reprint requests: H. F. Voigt, Dept. of Biomedical Engi- of Manis (1990) (mostly fusiform cells) had a mean slope neering, Boston University, 44 Cummington St., Boston, MA 02215-2407. of 116 Hz/nA, which is in the same range as type II units; however, it was much smaller than the 235 Hz/nA from Received 10 June 1996; accepted in ®nal form 21 January 1997. type III units, primarily thought to be from fusiform cells, reported here. Zhang and Oertel (1994) report slopes be- REFERENCES tween 100 and 300 Hz/nA for their population of fusiform ADAMS, J. C. 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