Survey of intracellular recording in the cochlear nucleus of the cat

Brain Research, 148 (1978) 43-65 © Elsevier/North-Holland Biomedical Press

43

SURVEY OF I N T R A C E L L U L A R R E C O R D I N G IN T H E C O C H L E A R N U C L E U S OF T H E CAT

RAYMOND ROMAND* Eaton-Peabody Laboratory of Auditory Physiology, Massachusetts Eye and Ear Infirmary, Boston, Mass. 02114, and Department of Otolaryngology, Harvard Medical School, Boston, Mass. 02115 (U.S.A.), and Laboratoire de Neurophysiologie, Universitd des Sciences et Techniques du Languedoc, 34060 Montpellier Cedex, (France)

(Accepted September 29th, 1977)

SUMMARY Intracellular recordings were made in the cochlear nucleus of anesthetized cats, In anterior passes, one never obtained sustained depolarizations from 'primary-like' units. For 'chopper' units, however, it was possible to record sustained depolarizations accompanied by spikes that lasted as long as the tone burst. 'Pauser', 'buildup' and 'on' units also had spike responses that could be accompanied by sustained depolarizations. For 'pauser', 'buildup' and 'on' units, hyperpolarization was not seen during the times when no spike discharges appeared so long as the tone bursts were at the characteristic frequency of the units.

INTRODUCTION Recent studies on the cochlear nucleus2,3,14,15, 8° have yielded some information on how the signals are processed in the cochlear nucleus at the single cell level. Cochlear nucleus units give a variety of discharge patterns in response to short tone bursts even though they presumably receive nearly the same input from the auditory nerve. One reason for making intracellular recordings is to gain a better understanding of the cellular mechanisms that underlie the generation of discharge patterns in cochlear nucleus cells. Although intracellular recordings have been reported previously for cochlear nucleus units4,5,6, la, there are many unanswered questions as to the nature of intra* Current address: Laboratoire de Neurophysiologie, Universit6 des Sciences et Techniques du Languedoc, Place E. Bataillon, 34060 Montpellier Cedex, France.

44 cellular responses for units having distinctively different response patterns to short tone bursts. It was the purpose of this study to supply such data, METHODS The data for this report were obtained from 41 cats with clean external ears. All animals were anesthetized (75 mg/kg) with diallyl barbituric acid in urethane solution injected intraperitoneally. Body temperature was maintained between 37 and 39 °C. The lateral portion of the cerebellun was removed to expose the right cochlear nucleus. Pulsations of the cochlear nucleus were stabilized by putting either mineral oil or agar agar in saline solution over the cochlear nucleus. In most of the later experiments, after covering the nucleus with a layer of warm mineral oil, the micropipet was introduced and the opening in the skull was sealed with warm paraffin which solidified, resulting in a closed chamber, which permitted more stable recordings. The preparation was placed in an electrically shielded 'sound-proofed' chamber 29. The acoustic stimuli used in this study included rarefaction clicks produced by delivering electric pulses to a 1" condenser earphone (duration: 50 #sec; rate 10/sec; reference level: 100 V) and short tone bursts at the characteristic frequency (STBCF) of the unit (duration: 25 msec; rise-fall time: 2.5 msec; rate: 6 bursts/sec; reference level: 200 V p-p). Occasionally the tone burst duration was changed to 100 msec in order to check whether response duration was the same as tone burst duration. The acoustic system was calibrated for frequencies between 10 and 25,000 Hz. Gross cochlear responses were monitored with a wire electrode placed on the bone near the round window. The recording microelectrodes were either bevelled or unbevelled pipets filled with either 1.5 M potassium citrate or 2-3 M potassium chloride. In earlier experiments, most of the time, the micropipets were filled with 2 M KCI and had impedances between 40 and 200 MO, tested in a 0.9 ~o NaC1 solution. After experience indicated that better recordings were obtained with the higher electrode impedances, electrodes with impedances higher than 80 Mr2 were selected. The tip diameters of these electrodes were well below 0.5 #m, when examined with a scanning electron microscope. The frequency response of the microelectrodes was almost flat up to 1 kHz. The negative capacitance of the input stage was adjusted using a square wave signal. The rise time of the square wave at the output stage was approximately 100 #sec which is shorter than the rise times of most action potentials. In the initial experiments, microelectrodes were mounted on a hydraulic microdrive controlled from outside the 'sound-proofed' chamber 26. Later the hydraulic system was replaced by a stepping motor microdrive. The microelectrodes were positioned in the cochlear nucleus using a block model 2,19 for general orientation. Electrode tip location was checked on a few occasions by injecting dye and processing the tissue histologically. Resting potentials were measured with a DC voltmeter and monitored with an inkwriter recorder. The output of the microelectrode was fed to a capacitanceneutralized, high-input impedance, DC-coupling preamplifier connected to an AC

45 amplifier with a bandwidth of either 0.1-30 kHz or 0.1-3 kHz. In order to check that the AC coupling did not distort waveforms enough to affect the results, we used a DC amplifier and compared its output to the signal coming from the AC amplifier. The filtering introduced by the AC coupling can produce subtle, but important, artifacts. For example, low-pass filtering a spike train can result in a baseline shift with an amplitude proportional to the spike discharge rate. With such an arrangement, a burst of spikes in response to a tone burst can produce an apparent 'slow potential' with the duration of the tone burst. Depending on the impulse response of the filter, negative afterpotentials can also be produced in the same way. These artifactual baseline shifts will be indistinguishable in form from true slow potential changes across the cell membrane. In photographing some of the traces for this paper, low-pass (1 kHz) filtering was occasionally used, but the effects on the responses were carefully checked. The amplitudes of spikes were reduced by the filtering but the slower potential changes were not greatly affected. The recordings were considered to be intracellular only if there was a sudden large drop in the resting DC level in combination with large positive polarity action potentials. Small resting and action potentials are thought to be obtainable when microelectrode have not penetrated the cell membrane (quasi-intraceUular recordings of Mcllwain and Creutzfeldt21), although such recordings might also be interpreted as intracellular recordings from a damaged cell. When we used electrodes with relatively large tip diameters (1/zm), the resting and action potentials were usually smaller and less stable than with electrodes having tip diameters less than 0.5/,m. It may also be that the resting and action potentials vary with the type of cell. The stimulus and response data were recorded with an FM tape recorder for off-line processing. During the experiments, PST histograms were computed so that the units could be classified. Results were more systematically examined off-line, from the recorded data, either by photographing the recordings or by processing the data with a general purpose digital computer.

Histological localization of recording electrodes Between 2 and 24 h after dye injection, animals were perfused intracardially with either a buffered paraformaldehyde solution or a 10 % formalin-saline solution. After perfusion, the head was removed and stored in the fixative overnight. The brain was then dissected from the skull, and a block of tissue containing the experimental cochlear nucleus was transferred to a solution of 85 parts fixative to 15 parts glycerine for 3-4 h. Serial frozen sections were cut at 40/zm and were mounted onto slides directly from the fixative. Sections were viewed with a fluorescence microscope using an excitation wavelength of 460 nm and a barrier filter. Even when the dye was not confined to single cells, the general locations of the electrodes in the cochlear nucleus could be ascertained by the position of the dye spots. RESULTS Data on 754 units from the cochlear nucleus are included in this report: 466 units

46 were from anterior passes through the anterior (AA) and antero-posterior (AP) parts of the anterior division of the anteroventral cochlear nucleus (AVCN)a; 288 units were from posterior passes through the dorsal cochlear nucleus (DCN) and the posteroventral cochlear nucleus (PVCN). To compare responses of auditory-nerve fibers recorded under the same conditions, we studied 25 units in passes through the auditory nerve. Since the micropipets used in intracellular recordings might also record from cell processes (including axons of both spiral ganglion and cochlear nucleus cells), the results will be reported in a way that does not assume the nature of the elements studied. Intracellular recordings might be expected to register a sizeable resting potential whether the electrode is in the cell body or a process. For the present data, 71 units had resting potentials between 50--80 mV. Most of the units had resting potentials between 20-40 mV; 125 units had resting potentials below 10 mV. Thus relatively few of our units had resting potentials as high as those found in squid axons 16, Betz cells 24, or spinal motoneurons a. Since it is not yet demonstrated that the low resting potentials are all due to damage, few units were discarded solely because of the small size of the resting potentials. Even if the resting potentials are small because of cell damage, some useful data might still be extractable 1°. Unless otherwise indicated, the resting potential recorded at the beginning of contact with each unit is given. The basic criteria for including units in the data pool are (l) resting potentials must be at least - - 10 mV; (2) action potentials must be at least 5 mV; and (3) suificient data must be available to compute a PST histogram of responses to short tone bursts (usually at least one minute of recordings) so that unit type can be determined.

(A ) Recordings from anterior passes (1) Primary-like responses. Of the units recorded in AVCN, 61 showed primarylike response patterns to STBCF (Fig. 1). None of these had baseline shifts that lasted as long as the tone bursts. 'Primary-like' units in the cochlear nucleus represent a special problem in that they can be either cells in the spiral ganglion or the cochlear nucleus. If the recordings could be established as coming from fibers, the fibers could still be from either spiral ganglion or cochlear nucleus cells. Even if the recordings could be established as being from cochlear nucleus cells, the interpretation would be vastly different depending upon whether the recordings are from the cell bodies or axons. In 22 of the units, we had click latency data to help determine whether a particular unit might be an auditory-nerve fiber. For 11 of these units, the latency was too short to be of cochlear nucleus cells; for 6 units the latency was too long to be of auditory-nerve fibers. In 5 cases, the latencies fell into a range (1.8-2.2 msec for units with CF above 1.8 kHz) that could have been from either group, considering the variability of latencies for responses to clicks across animals. Although the latency test can establish that at least some of the primary-like units must have been cochlear nucleus units, it cannot be used to determine whether a particular recording is from cell body or axon. Some workers in similar situations

47

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Fig. 1. Intracellular tracings and PST histograms of recordings from the auditory nerve and AVCN. Each row shows, for a single unit, 3 tracings of intracellular responses for consecutive tone bursts and a corresponding PST histogram. (The number at the top of each histogram is the instantaneous spike discharge rate for full vertical scale.) The voltage calibrations at the left represent 10 mV for this figure and also for all subsequent figures. The traces at the bottom show the electric input to the earphone generating the 25-msec tone bursts. The information to the left of each row is coded as follows: R R for the cat and unit number; RP for the resting potential; A P for the approximate size of the action potential; CF for the characteristic frequency; and L for the stimulus level in dB SPL (re 0.0002 dynes/cm2). The top two units were recorded with a micropipet inserted into the auditory nerve. The bottom three units were recorded with a micropipet inserted into the rostral pole of the anteroventral cochlear nucleus (AVCN).

48

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Fig. 2. Waveforms of spikes recorded from the auditory nerve and the cochlear nucleus. Units 45-2 and 42-7 were recorded from the auditory nerve. Units 43-17, 43-18, and 42-13 were recorded from AVCN, and 49-10 was recorded from the DCN. The top 4 units have primary-like response patterns to tone bursts. The bottom two units have chopper response patterns. Although the two middle units (43-17 and 43-18) were 'primary-like', they were not auditory-nerve fibers because their latencies of responses to clicks were too long.

49 have tried to use spike waveforms to make such a distinction 25. For instance, it has been suggested that short duration spikes are more likely to be from axons than from cell bodies 12. Presumably, at least some of the cochlear nucleus units and none of the auditory-nerve units would be cell bodies, so that one might expect some differences in measures such as slope, rise-time, or width of the action potential between populations of auditory-nerve and cochlear nucleus units if these measures differentiated between cell bodies and fibers. Fig. 2 shows examples of primary and second order primary-like units that exhibit action potentials without sustained depolarization as well as chopper units that have sustained depolarization. There was no strict relation between spike rise time and presence of sustained depolarization. Moreover, the resting potentials, depolarizations, and afterpotentials do not offer any obvious way to distinguish cell bodies and axons in our data. In 25 units from the auditory nerve, the resting potentials ranged between --5 mV and 30 mV. Most of the units showed a negative afterpotential (Unit 32-6, Fig. 1) that ranged from less than 1 mV to more than 5 mV. Some units did not show afterpotentials (Unit 32-9, Fig. 1), and no unit showed sustained depolarization. It may be that almost all of the primary-like recordings from AVCN are from axons. If so, then there has to be some methodological bias against recording from the cell bodies, because this region is tightly packed with cells. Either the cell bodies in this region give intracellular responses that resemble those of auditory-nerve fibers or our sampling of primary-like units in the AVCN selects against recordings from cell bodies. (2) Primary-like responses with a notch. As was described earlier 2°, some units in the cochlear nucleus have a notch in the PST histogram that appears immediately after the initial peak in an otherwise primary-appearing histogram. Although there may be a few instances of auditory-nerve fibers that show such response patterns, they would be extremely rare. Five 'primary-like-with-notch' units were found in the present sampling of AVCN. Two of these showed baseline depolarizations occurring a few msec after the first spike (e.g. Unit 30-10, Fig. 3). Both of these units had large resting potentials and two sizes of spikes. The large spikes were approximately 25 inV. Fig. 3 shows that for different triggering levels, the shape of the PST histograms remains primary-likewith-notch. Three other units that had primary-like-with-notch response patterns showed no baseline depolarization lasting as long as the tone bursts. These units did not show the systematic presence of both large and small spikes (Unit 24-10, Fig. 3). Another 3 units showed depolarization and two sizes of spikes, but because they had CF below 1 kHz, synchrony of responses to individual cycles of the stimulus prevented unequivocal identification of unit type. Current was injected through the micropipet for the two 'primary-like-withnotch' units showing depolarization and for the 3 low-CF units showing depolarization. In each case the result was like that shown for Unit 30-10 in Fig. 3. When no current was injected, the depolarization was rather small, but when --5 nA was injected through the micropipet, depolarization was increased. When + 5 nA was injected, both depolarization and spike amplitudes decreased.

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Fig. 3. Intracellular recordings and PST histograms of responses to tone bursts from the AVCN. Detecting primary-like-with-notch response patterns in the PST histograms requires a greater time resolution than a 50-msec time scale can give. For this reason, histograms are shown with 20-msec time scales also. Unit 30-10: recording A shows intracellular responses recorded in the usual way without injecting current. The upper histograms were made by triggering on the large spikes and the lower ones by triggering on both the large and small spikes. The arrows to the right of the A records show the approximate triggering levels. Recording B shows responses during injection of a --5 nA current through the micropipet. C shows responses during injection of a +5 nA current. Unit 24-10. A simpler set of results is shown for a second unit. Only one spike size was involved, and no current injections were made. F r e q u e n t l y recordings were characterized by a rapid decrease in the values of both the resting a n d action potentials after an initial appearance of a large resting potential. This p h e n o m e n o n is p r e s u m a b l y due to cell injury lz. The sudden rapid increase in spike rate that is also frequently attributed to injury l°,z~ was n o t seen for 'primary-like' units, with or without notches. I n a few cases there would be a sudden appearance of spike responses to tone bursts without a resting potential, followed by a n a b r u p t disappearance of spikes with a simultaneous appearance of a resting potential of more t h a n 50 mV. (3) Chopper-type S. Thirty-five units with chopper-type S response patterns 2

51

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Fig. 4. Intracellular recordings and PST histograms from 4 'chopper-type S' units in the cochlear nucleus. Units 14-9 and 42-13 were recorded from the ACVN. Units 37-35 and 37-33 were recorded from the DCN. Units 14-9, 42-13, and 37-35 are examples of units with sustained depolarization, whereas unit 37-33 is an example of a unit that did not show sustained depolarization.

were found in AVCN, usually when the micropipet was in a posterior location. Since no auditory-nerve fibers have such response patterns, these units had to be from the cochlear nucleus (or conceivably efferent fibers). In response to each tone burst, most of these units showed depolarization that lasted as long as the stimulus with a concomitant burst of spikes that are time-locked to the onset of the bursts (Fig. 4). The size of the depolarization was variable across units but was stable for each unit. The response to tone bursts was usually followed by a hyperpolarization that varied in amplitude and in duration for different units. The resting and action potentials of these units are usually low in comparison with those of 'primary-like-with-notch' units. It was possible to record positive spike activity without a resting potential for 'chopper-type S' units, but in some cases a resting potential would suddenly appear upon further advancement of the electrode. In a few cases, rapid spike discharges (characteristic injury discharges) and resting potentials would both appear together.

52

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Fig. 5. Intracellular recordings of a 'chopper' unit in response to clicks. Each row of intracellular recordings contai,s three groups of five superimposed tracings of responses to clicks at the level indicated by the number on the left. The corresponding PST histograms are on the right.

Sometimes injury spike discharges appeared a few minutes after the appearance of a resting potential and were rapidly followed by a marked decrease or disappearance of the resting potential. In many cases, it was possible to record the sustained depolarizations for some time after spike discharges had ceased. Depolarization without spikes can also be obtained when a stimulus is below spike threshold. Under these circumstances when stimulus level is increased, the amplitude of the depolarization increases, and the probability of spikes appearing increases. This situation is illustrated for a 'chopper' unit in response to clicks (Fig. 5). No measurable hyperpolarizations during tone bursts were noted for 'choppertype S' units in the AVCN but this may be because we made no attempt to study responses to tone bursts not at CF in a systematic way. A few 'chopper-type S' units in the AVCN showed no depolarization during tone bursts. These might have been

53 fibers, especially since their spikes had faster slopes and shorter rise-times than those of 'chopper-type S' units with depolarization. (4) Chopper-type T responses. Only 8 units with chopper-type T patterns 2 were located in AVCN. Three of these showed depolarization lasting as long as the tone bursts. Since most of the 'chopper-type T' units were from the DCN-PVCN, this type of response will be discussed in a later section. (5) On responses. Six units having an on response pattern were found in the AVCN. Four showed depolarization with either an on-type L 14 or type G spike pattern 2. The others were 'on-type A' units, an example of which can be found in Fig. 8 (Unit 53-3). This response is characterized by a spike at the beginning of the response, followed by a depolarization that decreases with time.

(B) Recordings from posterior passes For most of the posterior passes (which were in later experiments), the microelectrodes had higher impedances (presumably with smaller tips) than those used in most of the earlier AVCN experiments. Thirty-nine 'primary-like' units were encountered in posterior passes. The short latency of their responses suggests that these units were auditory-nerve fibers. Some primary-like-with-notch responses were recorded from the PVCN. In these cases, the value of the resting and action potentials for responses showing depolarization appear to be smaller than for those recorded from AVCN (Table II), but the data are too sparse to permit a definitive conclusion. (1) Chopper-type S responses. In posterior passes, 36 'chopper-type S' units with depolarization resembled 'chopper-type S' units in the AVCN (Fig. 4). The sustained depolarization is followed by a hyperpolarization when the tone burst is turned off. A significant number of 'chopper-type S' units that did not show sustained depolarization were found in both the DCN and the PVCN, especially when the microelectrodes were placed more medially, where the dorsal acoustic stria emerges. Presumably these units were fibers from cochlear nucleus cells. Some chopper-type S responses showed sustained hyperpolarization when the stimuli were high level tone bursts at frequencies close to the high-frequency edges of the units' tuning curves. A few 'chopper-type S' units even showed small sustained hyperpolarizations when the tone bursts were at low levels. (2) Chopper-type Tresponses. Twenty-one 'chopper-type T' units were found in DCN and PVCN. These units have a more transient chopper pattern than 'choppertype S' units. They have a well-defined sustained depolarization, exemplified by the recordings from Unit 51-10 in Fig. 6. It is sometimes difficult to distinguish these units from 'chopper-type S' units on the basis of PST histograms alone (Unit 51-25, Fig. 6) but the mean interval between spikes is longer for 'chopper-type T' units. (3) Pauser and buildup responses. Twenty-two units with pauser and buildup response patterns were found in the DCN both with and without depolarization. In many respects, the intracellular recordings from 'pauser' and 'buildup' units resemble those of 'chopper-type S' units. All 3 types of units have small resting and action potentials, frequent signs of injury discharges, and occasional signs of hyperpolarization. Only one unit with depolarization had a resting potential as large as --20 mV

54

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Fig. 6. Intracellular recordings and PST histograms for 3 'chopper' units recorded from posterior passes through the cochlear nucleus. Unit 51-25 has a response pattern that could be classified either as belonging to 'chopper-type S' or 'type T' units. One would need more information than that given by the PST histogram in order to be able to classify this response definitively. Both this unit and Unit 51-10, which is unequivocally a 'chopper-type T' unit, show sustained depolarizations. Unit 51-6, which is also a 'chopper-type T' unit shows no sustained depolarization. (Unit 49-12, Fig. 7). When sustained depolarization is present for pauser units, it lasts for the duration of the stimulation, even during the pause period (Units 38-1 and 47-15, Fig. 7). Of the 4 'buildup' units in our sample, 3 showed sustained depolarization. Once again the units without sustained depolarization are presumably fibers from cochlear nucleus cells. (4) On responses. One of the most interesting findings of Godfrey et al. 14 is the finding that the octopus cell region of the P V C N contains certain types of 'on' units. The 'on-type I' units show only a single spike in response to a tone burst stimulus, and 36 units having such responses were found in the present experiments. These units were recorded mainly from P V C N in posterior passes. Responses like that of Unit 37-43 (Fig. 8) were seen only in the PVCN. This response differs from on responses recorded from the A V C N in having fast fluctuations riding on top of the sustained depolarization. This fast activity never reaches the amplitude of the initial spike-like event at the onset of the response. I f one assumes this initial peak to be a spike and the following fast activity to be not spikes, then the spike discharge pattern of these units would be of the on-type I pattern. For low-frequency tone bursts, these units behave like 'on-type I' units in that each cycle of the tone produces a spike discharge.

55

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Fig. 7. Intracellular recordings and PST histograms for 'buildup' and 'pauser' units in the DCN. The first 3 units show pauser responses. Units 38-1 and 47-15 show ~ sustained depolarization during the tone burst, including the time of the pause in the response pattern. Unit 51-9, which had no sustained depolarization, was located in the medial part of DCN, perhaps in the dorsal acoustic stria. The PST histogram of Unit 49-12 shows a buildup pattern. The intracellular recording has a sustained depolarization that starts well before the appearance of the first spike. As the tone frequency is increased, the spike discharges no longer follow each cycle of the stimulus; they occur only at the onsets of each tone burst. As the tone burst level is increased, both the initial spike-like event and the fast fluctuations riding on the sustained depolarization remain constant in average amplitude. The fast fluctuations do not seem to consist of spike discharges, since they can occur with intervening intervals shorter than 0.5 msec. The latency of the spike in response to clicks is approximately 2 msec, which is short for most cochlear nucleus units but appropriate for 'on-type I' units. A small sustained depolarization ( ~ 1 mV) lasting as long as the tone burst can sometimes be seen for a brief time in the posterior passes. These recordings resemble recordings from the 'on' units, but the small peak at the beginning is a graded potential and not an all-or-none spike, its amplitude increasing with increased click levels.

56

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Fig. 8. Intracellular recordings and PST histograms for 4 'on' units in the cochlear nucleus. Unit 53-3 is an 'on-type A' unit recorded from the rostral AVCN with a sustained depolarization and a small negative afterpotential. Units 37-43, 51-21, and 31-5 were recorded from PVCN. Unit 37-43 is an example of a unit that would be classified as an 'on-type I' unit if one considers the first positive deflection to be the one and only spike. There is a sustained depolarization with many fast excursions which do not have an obviously all-or-none character. There are no afterpotentials for this unit. Unit 51-21 is an example of an 'on' unit that exhibits hyperpolarization with a brief burst of spikes at the onset. Unit 31-5 shows an on-type I response without either sustained depolarizations or afterpotentials. A few ' o n ' units exhibit h y p e r p o l a r i z a t i o n ( U n i t 51-21, Fig. 8) that lasts as long as 25-30 msec, the d u r a t i o n being u n r e l a t e d to the d u r a t i o n o f the tone burst. This h y p e r p o l a r i z a t i o n resembles negative afterpotentials sometimes r e c o r d a b l e f r o m a u d i t o r y - n e r v e fibers. ' O n ' units w i t h o u t either sustained d e p o l a r i z a t i o n or nonsustained h y p e r p o l a r i z a t i o n can be f o u n d in the P V C N ( U n i t 31-5, Fig. 8). ' O n - t y p e L ' a n d ' o n - t y p e S' units z4 were sometimes f o u n d in the P V C N . A t o t a l o f 24 units o f b o t h types were found, o f which s o m e exhibited sustained d e p o l a rizations. U n i t s 51-7 a n d 46-14 (Fig. 9) are e x a m p l e s o f ' o n - t y p e L' units. U n i t 51-7 shows a b u r s t o f spikes at the onset o f the t o n e b u r s t with a sustained d e p o l a r i z a t i o n t h a t lasts as l o n g as the tone burst. T h e on n a t u r e o f the spike discharge p a t t e r n is

57

RR51"7 RP: - 2 0 m V CF: 13.2kHz L: 52dB

I

RR 46-14 RP: - 3 0 m V

I L _ J_L_ L _ RR 47-3 RP: -15mV CF: 7.0kHz L: 70dB

I

L_LL_ :....:....:....:....:

RR4g'I3 RP: -50mY CF: 5.9WHz L: 60dB

.

J-

.

.

.

.

5ornsec

Fig. 9. Intracellular recordings and PST histograms for 'on-type L' and 'on-type S' units with sustained depolarization. These units were from posterior passes through the cochlear nucleus. Units 51-7 and 46--14 both show on-type L response patterns with depolarizations of markedly different amplitudes. Units 47-3 and 49-13 both show on-type S response patterns, with the amplitude of the depolarization being smaller for unit 47-3. seen in PST histograms (Fig. 9). This response can be compared with that of Unit 46-14 in the same figure, where the sustained depolarization is much smaller. This difference in depolarization does not seem to be attributable to the effects of cell injury, since the resting potential is greater for the unit with the smaller depolarization. Unit 47-3, recorded from the PVCN, and Unit 49-13, from the border between the P V C N and D C N (Fig. 9) are examples of 'on-type S' responses. The intracellular recordings show the same form of sustained depolarization exhibited by 'on-type L' and 'chopper-type S' units. On-type L and -type S responses without depolarization were also recorded and were presumably from fibers.

(C) Summary of results from all units in the cochlear nucleus Table I gives ranges of resting and action potentials for the various types of units. One can seen that only 7 units had both resting and action potentials greater

58 TABLE I Abbreviations: .4, all units; D, units with sustained depolarization. Level 0]'.4 P and RP

AP RP AP RP

÷ 5 mV i --5mV i ÷ 10 mV --10mV~

AP RP AP RP

÷20mV! - - 20 mV ! -F 3 0 m V ! - - 30 mV (

Primary-like Pri-N

Chop-S

Chop-T

Pauser and buildup

On

A

D

A

D

A

D

A

D

A

D

.4

D

100

0

11

5

56

33

27

13

17

11

50

16

61

0

8

4

23

11

19

9

8

4

36

10

16 0

2

1

6

2

4

1

1

0

10

5

0

0

1

0

1

0

0

0

2

1

2

0

than 30 mV. Most units had action potentials in the 10-20 mV range. Of these units, there were many that had resting potentials of at least --40 mV. The mean values of resting and action potentials for different types of units can also be compared. Table II is organized according to whether units show sustained depolarization or not. There are no striking differences in the resting or action potentials for different types of units except that the resting potential might be somewhat larger for 'primary-like-with-notch' units. Whether a unit has sustained depolarization or not is correlated with the nature of its spike waveform. Fig. 10 shows that action potentials of units with sustained depola-

25-

'- - -; i

--

N =38

---N:83

t ....

_~ 20Z

rr"

15I0-

OI--" i I00 180

i l 260 340 VOLTS/ SEC

~'"'i 420 500

Fig. 10. Histogram of the slopes of the action potentials for units with and without sustained depolarization. Distributions of slopes for the rising phase of the spikes for all unit types. The histogram with solid lines represents the units having sustained depolarizations. The histogram with dashed lines represents units that do not have sustained depolarizations. The data for the dashed histograms come from: 16 'primary-like' units, 2 'primary-like-with-notch' units, 13 'chopper-type-S' units, 5 'choppertype T' units, 12 'on-type I' units, and 32 low-frequency and unclassified units. The comparison of slopes was done by normalizing all action potentials to an amplitude of 50 inV. For the data in this figure, the capacitance neutralization of the headstage amplifier was adjusted for each unit so that the spike waveform would be as accurate as possible.

RP-D k RP-N k AP-D k AP-N k

Type o f Response

224,11 39

234-10 39

284.19 61

144-9 61

654,15 2 484-7 3 194.6 2 124-8 3

A VCN

A VCN

DCN PVCN

Pri-N

Primary-like

234-2 3 214-7 4 114.3 3 164.6 4

DCN PVCN 17±8 14 184,6 5 8___3 14 144,4 5

,4 V C N

Chop-S

14-t-8 23 164,8 19 134,6 23 204.13 13

DCN PVCN 25-4-14 3 30±16 5 6+0 3 104-2 5

AVCN

Chop-T

194-5 11 194-11 10 264-16 ll 22+11 10

DCN PVCN

AVCN

134-11 15 17±8 7 13-1-8 15 124-5 7

DCN PVCN

AVCN

DCN PVCN 184-15 8 19-]-14 28 12~8 8 174-8 28

Pauser and buildup On-type I

Abbreviations: N, responses without sustained depolarization; D, responses with sustained depolarization; k, number in sample.

TABLE II

AVCN

25±17 13 20±10 11 174-8 13 234-14 11

DCN PVCN

On-type S and On-type L

60 rizations tend to have a smaller slope and a longer duration than those of units without. Although these data are mainly from posterior passes when the data on spike waveform were carefully obtained, there is no reason to suspect that anterior passes would give different results. Indeed, a few such passes were made towards the end of this study and the data were consistent with the above descriptions. Presumably this means that the units with sustained depolarizations tend to be cell bodies. Units without sustained depolarizations can either be fibers or injured cell bodies. With the exception of 'primary-like' units, the presence of sustained depolarizations seems to cut across all unit types.

DISCUSSION Since the principal objective of this study was to correlate intracellular responses with unit types as defined by spike discharge patterns, it is of first importance to establish whether unit types can be established and intracellular responses measured validly with present techniques. While the former may be cautiously regarded as practical, the latter is not so convincing. A major problem is to establish what cellular elements are being recorded from intracellularly.

( A ) Identity of cellular elements from which recordings are made The size of resting and action potentials cannot be used in this study to identify the cellular elements because the effects of injury are unclear. There does, however, seem to be a division of units into those with and without sustained depolarizations. Since it is known that micropipets of the type used in these experiments can record from both cell bodies and processes 12,2s, one possible interpretation of this dichotomy is that the units with sustained depolarization are cell bodies and the ones without are cell processes. The fact that we never recorded sustained depolarizations from the auditory nerve, where there are few if any cell bodies of neurons, is consistent with the interpretation that recordings without depolarization can be from cell processes. Primary-like recordings with short latencies and no sustained depolarization might well be from auditory-nerve fibers. Recordings with long latencies and no sustained depolarization could be from axons of cochlear nucleus cells. Since recordings with sustained depolarizations are not found in the auditory nerve, recordings with sustained depolarizations in the cochlear nucleus are likely to be from the cell bodies of cochlear nucleus cells. Recordings with extremely long latencies and with no sustained depolarization could be from axons of neurons with their cell bodies in other parts of the central nervous system. In the present study, action potentials with slower slopes and longer durations tended to be from units with sustained depolarization. If recordings with sustained depolarizations are all from cell bodies, then recording spikes with small slopes and long durations would be a convenient physiological criterion for being in a cell body. This correlation of spike shape with presence of sustained depolarization agrees with interpretations of recordings from the spinal cord lz. Spikes recorded from ventral

61 root fibers are generally shorter in duration (presumably with faster slopes) than those recorded from motoneuron cell bodies. It would be useful to make intracellular recordings from the fiber output pathways of the cochlear nucleus 1 to see if recordings with depolarization are ever found.

(B) Correlation of intracellular recording with cell types Two previous attempts to correlate cell types with response patternsS, 6 have methodological problems that could seriously affect the interpretation of their results. In the more recent study, the authors tried to correlate the shape of sustained depolarizations with response patterns. However, by using insufficiently small bin widths in calculating PST histograms, they have difficulty in distinguishing 'primarylike' units, 'primary-like-with-notch' units and 'chopper' units by the usual defining criteria. In addition, their technique of filtering their recordings before averaging can result in artifactual baseline shifts that resemble sustained depolarizations. Thus their recordings of sustained depolarizations would usually contain a spurious component. A more direct method of correlating cell types with response patterns would seem to be intracellular recordings combined with marking of cells by dye injected through the recording micropipet. Unfortunately, in the one published attempt to do so 6, the currents used for injecting dye were so high (0.1-10 #A for 15 min) that the results are of questionable validity. In our experience, currents in this range can result in more than one dye-marked cell. In at least one instance, passing only 800 nA for 5 sec resuited in the marking of more than one cell. Our present technique uses 20 nA for 5 min, but the results are too sparse to report at this time. It is interesting that sustained depolarizations of similar waveform can be seen for most types of units. Thus, 'chopper-type S', 'chopper-type T', 'pauser', 'buildup', and 'on' units all show sustained depolarizations in response to tone bursts. The glaring exception is the 'primary-like' unit. All unit types are represented in the group of units that do not show sustained depolarization. Since all the neurons involved would have axons, this latter finding is not surprising if axons do not show sustained depolarizations, as is true for auditory-nerve fibers. First, restrict attention to those 'primary-like' units that have latencies of responses too long to be auditory-nerve fibers. That sustained depolarization was not seen in any of the recordings from 'primary-like' units in the AVCN can be interpreted in several ways. One interpretation is that these units correspond to spherical or bushy cells that receive the largest endings from the auditory nerve2, 23. These large endings could generate a spike discharge in the postsynaptic cell each time a spike is transmitted from the relevant auditory-nerve fiber. If each of these spikes results in a discharge from the postsynaptic cell, there might well be no buildup of a sustained depolarization. Thus the lack of a sustained depolarization would be related to the one-for-one, input-output character of this ending-postsynaptic cell combination. An alternative explanation is that there is some bias against recording from these cell bodies. The rostral pole of the AVCN is so densely packed with these large cells that it seems unlikely that the micropipets would never penetrate any of them. One could argue, however, that the cells are particularly prone to injury or that the

62 afferent input from the large endings must be compromised whenever a pipet penetrates the postsynaptic cell. Whatever the correct explanation may be, it is at the moment possible to maintain that there may be a type of cell in the cochlear nucleus that would not exhibit sustained depolarization by virtue of its unusual relationship to its afferent input endings. There are indirect indications that 'chopper-type S' units in the AVCN are stellate cells 2, but definitive conclusions will have to await data such as those obtainable from dye-marking individual cells. In any case, most of the cells in the posterior parts of the AVCN, where many 'chopper-type S' units are found, receive many small endings from the auditory nerve, rather than a single or a few large endings. It is an attractive hypothesis that sustained depolarizations are produced by the summated activity of many independent endings. Such activity would presumably be less well synchronized than the activity at an equivalent number of active sites of a single large ending. It is, of course, conceivable that the time course of action of the transmitter may be different for different kinds of endings. Our recordings of 'pauser' and 'buildup' units are more compatible with the data of Britt and Starr ~ if one realizes that their 'pauser' units appear to be' buildup' units in our terminology and their 'buildup' units to be 'pauser' units 4. Since both types of units are located predominantly in the D C N and may in fact be minor variants of the same unit type, this discrepancy in terminology, however untidy, may not be important. What is important is that during both the buildup and the pause periods, there is a sustained depolarization with no evidence of hyperpolarization during these intervals. A similar situation exists for 'on-type I' and 'on-type L' units in that sustained depolarizations are seen even when there are no spike discharges following the initial spike, or burst of spikes. Britt and Starr 5 report that their onset cells generally have sustained depolarizations. In one case, however, they reported obtaining a hyperpolarization at the onset of the unit response. This response may be related to the response of Unit 51-21 (Fig. 8) which exhibited a negative afterpotential. Such an afterpotential is similar to the negative afterpotentials recorded in the auditory nerve (Unit 32-6, Fig. 1). These potentials are seen even with pipets filled with potassium citrate, so they are not due to chloride ions. Both 'on-type I' and 'on-type L' units are found in the octopus cell region14, z0 whose morphological characteristics have been studied by Osen 22 and by Kane 17. Kane described 3 types of endings on the octopus cell, two of which appear to come from auditory-nerve fibers. One hypothesis is that the on pattern of responses may be due to a combination of excitatory inputs from one type of ending and inhibitory influences from another. Such a hypothesis, though not contradicted, is certainly not supported by the present intraceUular recordings from 'on' units, which show a sustained depolarization, even during the 'silent' periods after the initial spike activity. Of course, it is possible that the effect of inhibitory synapses is to keep the depolarization from reaching the critical level at which a spike discharge is initiated. Another possible explanation for the on patterns is based on work with the sympathetic ganglion of the rabbitL The inability of the

63 ganglion cells to dischargz spikes when stimuli are delivered at high rates is thought to be due to the properties of the cell membrane. The membranes of the 'on' units may have similar properties, such that a constant excitatory input would fail to elicit continual discharges. At the moment, all that can be said is that for units showing sustained depolarization, the 'silent' period correlates with cell depolarization rather than a hyperpolarization.

(C) Suggestionsfor further work The logical next step is the identification of individual cells from which recordings have been made. This can be done if dye-marking techniques can be successfully applied to this problem. This survey of the cochlear nucleus by intracellular recording shows that most of the responses recorded in the cochlear nucleus present an excitatory pattern, at least when stimulated at CF. An inhibitory pattern is rarely seen at CF. In agreement with Gerstein et al. la, occasionally hyperpolarization was seen in 'chopper' and 'pauser' units of the DCN. Starr and Britt 27 apparently found hyperpolarization to be more common than we did. However, they averaged their recordings in order to demonstrate baseline shifts, and averages can be affected by extracellular potentials such as gross responses. In many parts of the cochlear nucleus, Starr and Britt's averaging techniques will give a small negative waveform resembling hyperpolarization, even when there is no resting potential. The whole issue of hyperpolarization needs clarification with respect to variables such as stimuli and cell type. The use of antidromic electric stimulation in combination with orthodromic stimulation seems particularly inviting, especially since several of the cell types in the cochlear nucleus have specific projection pathways. Another issue that needs to be examined more systematically is the effect of anesthesia, which can apparently markedly alter the activity of some cochlear nucleus units 11. More recently, Young and Brownell a0 have reported that barbiturate anesthesia can change an inhibitory response from the DCN to an excitatory response. The roles of various synaptic mechanisms in generating specific response patterns can be studied by passing current across the membrane or injecting chemicals near or into cells during recording7. Future work on the cochlear nucleus needs to incorporate these newer ideas and techniques of neurobiology. ACKNOWLEDGEMENTS The author wishes to thank Drs. N. Y. S. Kiang, T. R. Bourk and J. J. Guinan, Jr. for their most generous suggestions and comments received during the experiments and preparation of this manuscript. Special thanks are due to the staff of the EatonPeabody Laboratory of Auditory Physiology and in particular to S. A. Liberman, D. W. Altmann and G. S. Roberts for their assistance. This work was supported by U.S. Public Health Service Grants 1 R01 NS01344, 1 R01 NS11000, and 1 R01 NS09243, and by rlnstitut de la Sant~ et de la Recherche M6dicale, Paris, France.

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65 28 Tasaki, I., Properties of myelinated fibers in frog sciatic nerve and in spinal cord as examined with micro-electrodes, Jap. J. Physiol., 3 (1952) 73-94. 29 V6r, I.L., Brown, R. M. and Kiang, N. Y. S., Low-noise chambers for auditory research, J. acoust. Soc. Amer., 58 (1975) 392-398. 30 Young, E. D. and Brownell, W. E., Responses to tones and noise of single cells in dorsal cochlear nucleus of unanesthetized cats, J. Neurophysiol., 39 (1976) 282-300.