Auditory nerve fibre activity in the tokay gecko - Springer Link

Journal of Comparative Physiology, A

J Comp Physiol (1981) 142:203 218

9 Springer-Verlag 1981

Auditory Nerve Fibre Activity in the Tokay Geck~ I. Implications for Cochlear Processing* R.A. Eatock**, G.A. Manley***, and L. Pawson Department of Biology, MeGill University, 1205 MCGregorAve., Montreal, Quebec, Canada H3A 1B1 Accepted November 5, 1980

Summary. Extracellular recording from single auditory nerve fibres in the lizard, Gekko gecko, revealed interesting patterns of evoked and spontaneous activity. The fibres are sensitive and sharply tuned (Figs. 1 and 3) and have characteristic frequencies (CFs) from 0.15 to 5 kHz (Fig. 2). Responses to pure tone bursts fall into three main categories with different frequency distributions (Figs. 5, 6 and 7): 1) tonic; such fibres have C F s < 0 . 7 kHz; 2) phasic, restricted to CFs > 0.7 kHz; 3) intermediate, with an intermediate frequency range: 0.4-2 kHz. As there is also some intensity dependence to these response patterns, fibres were classified according to their responses to tones at 20 dB (above threshold). Within small samples of fibres drawn from the three categories, all were susceptible to two-tone suppression (Fig. 10) and responded to a broad-band Tokay call (Fig. 11). Possible correlations between these categories and populations of cochlear hair cells that differ with respect to orientation and tectorial connections are discussed. A few fibres (10 in 410) were seen to respond to both onset and offset of certain pure tones (Fig. 9). Interspike interval histograms revealed that fibres with CFs >0.5 kHz have spontaneous discharge patterns comparable to those that have been described in other species, despite low discharge rates (Figs. 12 and 13). However, fibres with C F s < 0 . 5 kHz tend to fire spontaneously at intervals close to the CF period or integral multiples thereof (Fig. 13). We propose * This work constitutes partial fulfillment of the requirements for the M.Sc. degree of R.A.E. ** Present address: Division of Biology216-76, California Institute of Technology,Pasadena, California 91125, USA *** Present address: Zoologisches Institut der Technischen Universitfit Miinchen, Lichtenbergstrasse 4, D-8046 Garching, Federal Republic of Germany Abbreviations: CF characteristic frequency; PSTH peristimulus time histogram

that this spontaneous discharge pattern may reflect 'ringing' of hair cells at their CFs and so may provide indirect evidence that hair cell membranes possess a tuned system of conductances.

Introduction A remarkable diversity in the morphology of lizard inner ears lends special interest to the study of auditory processing in these animals. Between families, there are marked differences in, for example, the shape of the basilar papilla (homologous to the mammalian organ of Corti) and underlying basilar membrane, the pattern of hair cell orientations 1 in the papilla, and the type and arrangement of tectorial structures (Miller 1966, 1973b, 1974). Although the cochlear ducts of different species within a family are similar in many respects, they may differ in detailed morphology and in size. This diversity provides an opportunity to assess the functional contributions of various cochlear structures by comparing responses recorded from the auditory periphery - i.e., the cochlea and auditory nerve of different lizards. Many comparative studies of cochlear microphonic potentials of lizards have been done (e.g., Wever and Peterson 1963; Wever et al. 1963; Wever et al. 1964). More recently, Weiss et al. (1976, 1978) and Manley (1977) have recorded from auditory nerve fibres in the alligator lizard and monitor lizard, respectiVely, and have attempted to relate response properties to specializations in cochlear morphology. We have taken a similar approach in this study of primary auditory fibre activity in the Tokay gecko, Gekko gecko. 1 The orientation of a hair cell is defined by the position at its apical surface of its kinocilium, or, in mammals, basal body

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204

R.A. Eatock et al. : Auditory Nerve Fibre Activity in the Tokay Gecko

We chose the T o k a y for several reasons, one being the highly differentiated appearance of its cochlea (Miller 1973 a). Certain features of the Tokay's basilar papilla reflect trends that are more fully developed in birds and mammals: it is long (approximately 2 mm) compared to the papillae of other lizards, has relatively many hair cells (approximately 2100), is slightly curved, and tapers from its dorsal (apical) to ventral (basal) aspect. The tectorial structures and pattern of hair cell orientations, on the other hand, are complex and unusual (Miller 1973a, 1974). In the dorsal, 'unidirectional', third of the papilla, all hair cells are oriented in one direction, transverse to the long axis of the papilla and with the kinocilia eccentrically situated toward the medial edges of the hair cells' surfaces. The ventral two-thirds of the papilla, which Miller calls 'doubly bidirectional', is divided by a midline gap into two mirror-symmetric and highly-ordered bidirectional areas - that is, areas populated by hair cells of opposing orientations. In addition, each of these three regions - the two bidirectional and one unidirectional - has a different form of tectorial attachment. In the dorsal unidirectional third and the more medial of the ventral bidirectional areas, the hair cells are connected by tectorial membrane to the limbus, an extension of the wall of the cochlear duct. The membrane is filamentous over the dorsal region and thickened ventrally. The hair cells of the more lateral of the ventral bidirectional areas are covered by 'sallets': masses of tectorial membrane, one per transverse row of hair cells, that connect to each other and the underlying hair cells but not to the limbus (Miller 1973 a). This elaborate organization of the Tokay cochlea is particularly interesting in view of suggestions that have been made concerning the significance of specific orientation patterns and tectorial connections (Weiss et al. 1976; Manley 1977). Another advantage to studying the Tokay is that, unlike most lizards, it vocalizes; thus Tokay Calls could be used in addition to conventional pure tone stimuli. A third reason for our choice of the Tokay was that its auditory system has already been studied in some detail. Data are available on its middle-ear transfer function (Manley 1972a, b; Werner and Wever 1972), its cochlear microphonic potential (Hepp-Reymond and Palin 1968; Wever et al. 1963) and the activity of its medullary (Manley 1972a, 1974) and midbrain (Sammaritano-Klein 1976) auditory centres. The anatomy of its inner ear (Miller 1966, 1973 a), cochlear nuclei (Miller 1975) and torus semicircularis (Sammaritano-Klein 1976) has been described. Single-unit analysis of auditory nerve fibres seemed an obvious next step toward understanding processing at the Tokay's auditory periphery.

Materials and Methods Gekko gecko weighing 40-200 g were obtained from Thailand. The data in this paper are from 410 units in 44 animals. 297 of these units are from 33 experiments whose main purpose was to observe temperature effects on tuning curves. These animals, therefore, contributed mostly tuning curve data; the temperature effects are described elsewhere (Eatock and Manley 1981). The animals were anaesthetized with intraperitoneal injections of 20% urethane (ethyl carbamate) at an initial dosage of 10 ml/kg. One or two supplemental injections at 1 ml/kg were necessary in experiments lasting more than 12 h. Artificial respiration was unnecessary. In the temperature-effect experiments, the gecko's oral temperature was varied between 20 and 30 ~ in other experiments, the animals were maintained at about 24 ~ A ventral surgical approach was used to expose the eight nerve: muscle, bone and dura overlying the ventral surface of the rostral medulla on its left side were removed, and a hole made in the arachnoid to reduce the cerebrospinal fluid pressure and create a space between the medulla and left otic capsule. Small pieces of tissue paper could then be inserted in this space, thus pushing the medulla slightly towards the animal's right side and exposing the proximal part of the left vestibulocochlear nerve (VIII), between its exit from the otic capsule and its entry on the dorsal side of the rostral medulla. Glass microelectrodes, filled with 4 M NaC1 or 3 M KC1 and having impedances of 5-40 M~2, were used to record from the posterior half of the nerve. The electrode was advanced through the nerve by means of a remote hydraulic microdrive located outside the sound-attenuating, semi-anechoic chamber containing the animal. Sound stimuli were delivered free-field from a speaker 1 m from the gecko's head, facing its left ear. White noise pulses (50 ms, 2/s) were presented as search stimuli, while pure tone pulses (50 ms, 2/s) with trapezoidal rise-fall characteristics (5 ms rise-fall time) were used to obtain frequency-threshold tuning curves. Sound pressure levels were calibrated using a Br/iel and Kjaer 0.5 in. condenser microphone placed next to the left eardrum, and a narrow-band wave analyser. Response thresholds were assessed from unit activity monitored with an oscilloscope and an audio monitor. The data were recorded on tape for off-line computer analysis using interspike interval histogram (time interval histogram, TIH) and peristimulus time histogram (PSTH) programs.

Results

At the beginning of each electrode pass, the electrode was positioned over the ventral surface of the posterior half of the eighth nerve. In penetrations that were normal to the nerve or nearly so, all units encountered were auditory and easily identifiable as such by their time-locked responses to the search stimuli. When the electrode was angled caudo-rostrally, non-auditory units were obtained dorsal to auditory units, indicating that the electrode had passed out of the auditory part of the nerve. The non-auditory units often showed regular spontaneous 2 discharge and were presumed to be vestibular. The transition from auditory to vestibular nerve was fairly abrupt. 2 Activity is considered spontaneous when recorded in the absence of controlled sound stimulation, and at subthreshold ambient sound levels


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Fig. 1. Some tuning curves for Tokay gecko auditory nerve fibres, obtained from several animals. Sound pressure is expressed in this and subsequent graphs as dB S P L = dB re. 20 gP. Also illustrated is the velocity response of the Tokay's columella (stapes) vs. stimulus frequency. The curve is an average from 3 Tokays

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Light-microscopic histology indicated that the area of the nerve penetrated contained almost exclusively fibres and not cell bodies of the cochlear ganglion or cochlear nuclei. In electrode passes at the distal end of the exposed nerve, a few units were encountered that differed from the others in spike waveform and could be held over much longer distances (tens of microns rather than several). These were thought to be primary cell bodies. Their responses, at least to pure tones, were not obviously different from those of the majority.

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To obtain a tuning curve for a unit, threshold intensities of pure tone stimuli at various frequencies were estimated. A threshold intensity was defined as that which evokes the smallest perceptible increase in discharge rate over the spontaneous level. The frequency to which a unit is most sensitive is its characteristic frequency (CF). Tokay tuning curves have a fairly simple V-shape (Fig. 1). The CFs of the fibres studied ranged from 0.15 to 5 kHz. A 'sensitivity curve' for the auditory nerve was obtained by plotting CF threshold vs. CF for each tuning curve recorded (n = 427) and drawing a curve through the lowest threshold values (Fig. 2). The curve is probably most reliable between 0.3 and 2.5 kHz, because of the large number of data points ( n = 372) in this range. It is possible that sensitivity has been underestimated at the extreme frequencies, represented by fewer units (n = 55). In Fig. 2, the sen-

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Fig. 2. Auditory sensitivity curves for the Tokay gecko. The cochlear microphonic sensitivity functions are taken from Hepp-Reym e n d and Palin (1968) [] . . . . m, and Werner and Wever (t972) 9 . . . . 9 Both indicate the intensities required to produce a 'standard' microphonic response amplitude of 0.1 gV R M S (recorded at the round window) at different frequencies of pure tone stimuli. The sensitivity curves derived from tuning curves of cochlear nucleus neurons ( = . . . 9 from Manley 1972) and auditory nerve fibres ( e - - e , present data) were arrived at by drawing curves through the most sensitive CF thresholds across the Tokay's frequency range

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sitivity curve for auditory nerve fibre data is compared to that for cochlear nucleus neurons (from Manley 1972a) and to cochlear microphonic sensitivity functions (from H e p p - R e y m o n d and Palin 1968; and Werner and Wever 1972). The data f r o m primary and secondary single units are in good agreement, and suggest that sensitivity is somewhat greater from 0.5 to 0.7 kHz, and from 2 to 2.5 kHz, than at other frequencies. Campbell (1969) found similar sensitivity maxima in evoked potential recordings from the Tokay's cochlear nuclei. The curves in Fig. 2 indicate that threshold depends to some extent on the frequencies to which a unit is responsive. Sensitivity at frequencies above 0.8 k H z relative to lower frequencies is much poorer in the cochlear microphonic functions in Fig. 2 than in the single unit curves; the possible significance of this discrepancy will be discussed later. One measure of the sharpness of a unit's tuning is the Q~0ab value, obtained by dividing CF by the tuning curve bandwidth at 10 dB above CE threshold. In the Tokay, larger Q values occurred at higher frequencies, so that the range of values was greater than at lower frequencies (Fig. 3). Almost all units with Q~odb'S greater than 8 had CFs above 2 kHz, while Q10dB's of less than 4 occurred almost exclusively at CFs below 2 kHz. There are insufficient data per animal to determine the extent to which the variation in QlOOB at a given frequency is the result of pooling the data. Q~ooB does vary, however, for fibres of similar CF in a given animal. In mammals, broad tuning has been shown to be associated with elevated thresholds in the event of cochlear trauma, hypoxia, etc. (Evans 1974; Robertson and Manley 1974). In T o k a y

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fibres, there was a weak tendency for higher Q values to be associated with lower thresholds. However, in many instances low Q values were obtained from relatively sensitive units, and so presumably did not reflect damage to the cochlea. Sharpness of tuning can also be expressed in terms of the slopes of the two sides - low-frequency and high-frequency - of the tuning curve. Low-frequency slopes ranged from 20 to 240 dB/octave, and highfrequency slopes from 20 to 300 dB/octave ; all slopes were measured from 3 to 23 dB above threshold. As with Q10dB,higher values and a greater range of both low- and high-frequency slope values occurred at higher frequencies. Figure 4A illustrates that there was a pronounced overall tendency toward sharper tuning


on the high-frequency side. However, from the graph of the ratio of high-frequency to low-frequency slope ( H F : L F ) vs. CF (Fig. 4B), it can be seen that below 0.7 kHz, low-frequency slopes were, on average, as steep as high-frequency slopes. The ratios tend to be highest between 1 and 2 kHz, as a result of decreased low-frequency slopes in this range. Though not studied systematically, some tonotopic organization of the nerve was evident. This was not unexpected, since the Tokay's cochlear nuclei have at least a rough tonotopic organization (Manley 1972a). Fibres along the posterior edge of the auditory nerve had CFs from 0.15 to 0.7 kHz. Electrode passes running through the middle of the auditory nerve and normal to it contacted fibres with CFs from 0.2 to 2.5 kHz; there was some tendency for CF to increase with depth. Higher CFs, from 3 to 5 kHz, were found deep in the anterior part of the auditory nerve - i.e., around the midline of the eighth nerve. Tonotopic organization of the nerve and cochlear nuclei suggests a systematic distribution of frequencies along the basilar papilla (Konishi 1970).

2. P S T H s

Peristimulus time histograms (PSTHs) were computed off-line from the tape-recorded pure tone responses of 81 fibres from 18 animals. Each PSTH represents the summed activity of a fibre in response to 30 presentations of a tone burst, delivered at a constant intensity. The tone burst had a 5 ms rise-fall time unless otherwise stated. The beginning of fall-time

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was 50 ms after the beginning of rise-time, so that the tone burst was at full amplitude for 45 ms. The PSTHs obtained from 73 of the 81 fibres could be divided according to shape il~to three major categories: tonic, phasic, and intermediate. The tonic PSTH (Fig. 5A) indicates a response that adapted slowly, if at all; that is, the probability of the unit's firing remained fairly constant throughout the tone burst. Phasic PSTHs had one (Fig. 5C) or more (Fig. 5D) pronounced peaks at response onset followed by a considerably reduced sustained discharge to the remainder of the stimulus; the impulses forming the initial peak were highly synchronized. PSTHs with less pronounced initial peaks were classed as intermediate (Fig. 5B). The type of PSTH produced by a unit depended to some extent on stimulus intensity. The progression shown in Fig. 6 - from intermediate to a single peak to multiple peaks with increasing intensity was common. As a result, fibres were classified according to the PSTHs they produced in response to a tone at or near CF and approximately 20 dB above threshold. PSTH type was also related to CF (Fig. 7) : tonic PSTHs were only obtained from fibres with CFs of 0.7 kHz or less, while phasic PSTHs only occurred at CFs of 0.7 kHz or more. Intermediate PSTHs occurred over the range of 0.4 to 2 kHz, which overlaps the distribution of both tonic and phasic PSTHs. To determine whether this classification scheme was to any extent an artifact of the arbitrarily chosen rise-time of 5 ms, the PSTHs of 7 fibres were also studied at rise-times of 2.5 and 1.0 ms, keeping stimulus frequency and intensity constant. Three units clas-

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Fig. 5 A - D . Examples of the major categories of peristimulus time histogram (PSTH): A tonic, B intermediate, C phasic with a single peak, D phasic with multiple peaks. Each PSTH represents the s u m m e d response of a unit to 30 stimulus presentations: in these cases, the stimulus was a CF tone at 30 dB above threshold. The tone burst is depicted as beginning 16 ms later than the origin of the PSTH. This is the time delay (for a 5 ms rise-time) between the trigger for the computer and the measured time of arrival of the tone burst at the lizard's ear, including electronic delays and travel time of the sound in air

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sified as tonic were found to be tonic at the shorter rise-times as well. With phasic PSTHs, individual peaks became sharper and in two cases the number of peaks increased from one to two. Thus, though no intermediate units were tested, at least the tonic and phasic categories remained stable over different rise-times. Several observations lead to the conclusion that single-peak and multiple-peak PSTHs have similar origins: 1) in one unit that produced two peaks at 5 ms rise-time, the second peak was greatly reduced at 2.5 ms but more pronounced at 1 ms; 2) the frequency distributions of the two PSTH types were nearly identical (Fig. 7C, D); 3) although changes with intensity were not always examined, our impression was that phasic units produced single peaks at low intensities and multiple peaks at moderate-tohigh intensities. Incipient second and third peaks were often visible at lower intensities, and the rate at which such peaks developed with intensity determined whether a unit was a single-peak or multiple-peak unit at 20 dB above stimulus threshold. In PSTHs with more than one peak, the interval between peaks varied from 2.5 to 4 ms for different units, remaining constant over different stimulus intensities for the same unit. Responses to CF tones also were analysed in the form of interspike interval histograms (time interval histograms, TIHs). This analysis revealed that the responses of tonic units were phase-locked to the stimulus; i.e., they tended to fire at intervals correspond-


ing to the stimulus period or integral multiples thereof. This behaviour was not observed in the phasic units sampled, including a relatively low-frequency fibre ( C F = 0 . 6 kHz). Phase-locking was evident in some intermediate PSTHs, but was less precise than that exhibited by tonic units. Although the sample examined was too small (n=40) to allow us to be conclusive, the data suggest that the CF range of 0.6-0.8 kHz represents a transition zone below which phase-locking consistently occurs and above which it does not occur. This is also the range of overlap between the frequency distributions of tonic and phasic units (Fig. 7). Although the difference in frequency distribution of the two types is an important factor, since it is presumably easier for fibres to follow low frequencies, there may be a difference in the sensitivity to phase of the two PSTH categories over and above considerations of CF. It is worth noting that mammalian cochlear nerve fibres phase-lock up to 4 or 5 kHz (Kiang et al. 1965). Rate-intensity functions graphs of average discharge rate vs. stimulus intensity - were obtained for nine fibres with CFs ranging from 0.425 to 2.9 kHz. In each case a series of ten or more PSTHs was taken, using the same tone (at or near CF) at intensities made progressively higher in increments of 3 4 dB. Examples of the different types of function obtained are shown in Fig. 8. The discharge rates of the two low-frequency tonic units increased linearly with slopes of 12 and 14 spikes/s/dB, reaching maxima of over 200 spikes/s. Discharge rate increased uniformly throughout the tone burst, so that the PSTH continued to be tonic9 Rates increased more slowly for phasic units, with an average slope of approximately 6 spikes/s/dB, and usually plateaued at lower values. In these units the initial peak became more prominent with increased intensity, while discharge rate over the rest of the histogram did not increase significantly. In two cases, however, spike rate began to rise again at intensities of 30-40 dB above threshold, after having plateaued. This second rise reflected the growth of second and third peaks within the histogram. The unit with CF of 2.9 kHz (Figs. 6B, 8), although phasic, never plateaued because the later peaks grew with the same time course as the initial peak. A fourth category of unit, the on-off type, was encountered rarely in our sample (Fig. 9). Of the 410 fibres in this study, ten were observed to respond both at onset and offset of stimulus. PSTH analysis was done for five of these units (Fig~ 9): two were on-off at all frequencies within their tuning curves (Fig. 9A) and three were on-off only at certain frequencies removed from CF. In one case, a unit with CF of 0.3 kHz had a tonic response to CF stimulation

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but was on-off at 0.25 kHz. Figure 9C depicts an on-off response to 1.6 kHz tones, 0.5 kHz below the moderately high CF (2.1 kHz). The PSTH in Fig. 9 D was obtained at 0.4 kHz above a low CF (0.3 kHz). Since, in all five cases, threshold was above 60 dB SPL at the frequencies that evoked the on-off response, the units that were on-off at CF were relatively insensitive. The use of a tone burst with trapezoidal rise-fall characteristics rather than a Gaussian envelope raises the possibility that the off discharge was in response to new frequencies introduced during the off-ramp of the stimulus. However, if bandspread were the source of the off responses reported here, they would be expected to occur much more commonly. In addition, discharge following the onset peak was reduced in on-off PSTHs relative to phasic PSTHs, a feature which cannot be related to bandspread in the stimulus. Eight fibres (CFs ranging from 0.45 to 2 kHz) were tested for two-tone suppression: the reduction in a unit's response to an excitatory tone (tone 1) caused by simultaneous delivery of a second tone

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Fig. 9 A - D . On-off PSTHs. A Response of a unit with C F = 0.15 k H z to stimulation with 0.15 k H z tones at 62 dB SPL. B As in A but with stimulus intensity raised to 72 dB SPL. C A unit with C F = 2 , 1 kHz, CF t h r e s h o l d = 3 6 dB SPL, responding to 1.6 kHz tones at 70 dB SPL (1 dB above threshold at that frequency). This unit had a phasic P S T H at higher stimulus frequencies. D Response of a unit with C F = 0 . 3 kHz, CF t h r e s h o l d = 33 dB SPL, to 0.7 k H z tone bursts at 89 dB SPL (5 dB above threshold at that frequency)

(tone 2). In this study, tone 1 was a 60-ms CF tone at 10 dB above threshold. Tone 2 was varied in frequency, intensity, duration (30, 50 or 70 ms), and time of onset relative to tone 1. F o r each combination, a P S T H of the fibre's responses to 60 two-tone stimulus presentations (2/s) was computed and tone 2 was judged effective at reducing the response to tone 1 if the histogram revealed a clear decrease in response rate during tone 2. The range of effective second tones could then be expressed graphically as 'suppressive' areas m a p p e d relative to the unit's tuning curve (Fig. 10). Checks on the acoustic system indicated that distortion could not be responsible for these ef-

fects. F o r four fibres, the suppressive area was larger on the high-frequency side of the tuning curve. One unit was slightly more sensitive to low-frequency second tones than to higher-frequency tones. In three cases, contact with the fibre was lost before the relative size of its low- and high-frequency suppressive areas could be assessed. The responses of 20 fibres (CFs from 0.3-3.6 kHz) to a tape-recorded Tokay call were also examined. The entire call was 750 ms long, and consisted of a 150 ms component followed by a 190 ms pause and then a 400 ms component. A wide-band sonagram of the call showed that it was broad-band and that each component comprised a number of brief pulses. Its energy was concentrated below about 1.7 k H z (Fig. l lA), although higher frequencies (up to 3.5 kHz) occurred in the early part of each of the two main components. As expected with a broadband call, responses were obtained from all 20 fibres tested. The PSTHs consisted of successive peaks (Fig. 11 B), reflecting the pulsed nature of the call. PSTHs from different units with similar CF had superimposable peaks, indicating that each peak was a response to a specific pulse within the call. A unit responded maximally, of course, to those parts of the call with most energy around its CF. For instance, while 0.5 k H z units (Fig. 11 B) responded throughout both components of the call, the 219 k H z unit responded only to the first part of each component,

R.A. Eatock et al. : Auditory Nerve Fibre Activity in the Tokay Gecko 4

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Fig. 11, A Wide-band souagram of 2-part Tokay call used as a stimulus. B PSTHs of responses of 4 different units to the call in A. The CFs of the units are indicated. The m a x i m u m intensity within each component was similar. For the PSTHs shown, the m a x i m u m intensity of the call was 35-40 dB above the units' CF thresholds. The intensities of the frequency components relevant to each unit were not determined

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where higher frequencies were represented. It is interesting to note that the pulse-like substructure of each component of the vocalization allows phasic units to respond more vigorously to the call as a whole than to a continuous tone.

3. Spontaneous Activity The spontaneous activity of 64 units from 19 Tokays was recorded on tape and analysed in the form of interspike interval histograms (Fig. 13). Data were

6 s dead time, ms

usually collected for 1-6 min, and were only considered acceptable when computer analysis indicated a relatively constant discharge rate throughout the recording period. The spontaneous rates of the 64 units varied from 1.6 to 40 spikes/s. Occasionally, units were encountered that did not discharge spontaneously during the recording period. Apart from the omission of such units, the data probably represent a fairly random sample of spontaneous activity. The distribution of rates for the 64 fibres is shown in Fig. 12 together with the distributions of spontaneous

212


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0 30 60 30 Fig. 13. Representative interspike interval histograms of spontaneous activity, from 6 fibres whose CFs are indicated. Abscissa: interspike interval, divided into 0.1-ms bins. Ordinate: n u m b e r of spikes that occur at a given interval after the previous one. Note the different ordinate scales

modes and dead times. The mode is the most common, or 'preferred', interval between spontaneous impulses, and is the time period corresponding to the highest point in an interval histogram. Unlike mammalian modes, which are all within several ms of one another (e.g., Manley and Robertson 1976), modes in the Tokay varied from 2.5 to 7 0 m s (Fig. 12B). The dead time of a unit (Fig. 12C) is the smallest interspike interval in its histogram, and may indicate the interval below which the unit is refractory

to spontaneous discharge. Although mode and dead time were not consistently related to each other, both tended toward larger values at low spontaneous rates. Dead times of 4.5 ms or more were not observed at rates higher than 5/s. A bias is introduced, however, by the tendency to record fewer impulses at low discharge rates. Six representative interval histograms are illustrated in Fig. 13. (A) and (B) were from units with spontaneous rates of 29 and 11 spikes/s, and CFs of 1 and

R.A. Eatocket al. : AuditoryNerve Fibre Activityin the TokayGecko 1.05 kHz, respectively. Interval histograms for units with CFs above 0.5 kHz were generally comparable to those of mammals (Kiang et al. 1965; Manley and Robertson 1976) and birds (Manley and Leppelsack 1977) despite the lower spontaneous rates. Units with CFs below 0.45 kHz, on the other hand, produced interval histograms with unusual peaks early in the histogram (Fig. 13 C, D, E, F). These peaks occurred at regular intervals that corresponded at least roughly, and sometimes closely, to the reciprocal of the unit's CF. At first, we assumed that the units were being stimulated inadvertently. Since the trigger normally used for tones and white noise pulses was recorded continuously on tape during spontaneous data collection, it was possible to check for unwanted stimulation through the sound system by computing PSTHs for the spontaneous activity. PSTHs for all spontaneous data from low-frequency units were flat, indicating that the units had not been responding to sounds coming from the speaker. In subsequent experiments, the amplifier input to the speaker was removed, and care taken to keep noise outside the semi-anechoic sound-attenuating chamber to a minimum. In several instances, spontaneous activity was recorded both with and without background noise in the room outside the recording chamber. No differences were discernible in the overall discharge rates or when the interval histograms were compared. In summary, if these units were responding to low-level sounds, then they were doing so with a sensitivity far greater than would be expected from their tuning curves. One 0.25 kHz fibre whose interval histogram contained several prominent peaks separated by 4-4.5 ms intervals had a CF threshold of 65 dB SPL. A number of other fibres with CF-related preferred intervals in their TIHs had CF thresholds of 40-50 dB SPL. The ambient sound level, as measured by a 0.5-inch Brfiel and Kjaer condenser microphone, fell within the noise level of the instrument (30 dB SPL). Peaks at intervals corresponding to the reciprocal of the CF were obtained from all units tested with CFs of 0.4 kHz or less (n= 19), from two of six units tested with CFs of 0.45-0.5 kHz, and never from traits with higher CFs (n=39). Inter-peak intervals were consistent within a histogram, and were within + 1 ms of the estimated CF period for all but one unit (Fig. 13C). There is no obvious explanation for the discrepancy between inter-peak interval (5 ms) and CF period (3.3 ms) in this unit; it was not exceptionally broadly tuned (Q10aB=l.1) or insensitive (best threshold---43 dB SPL). Figure 13E shows that the initial peak was not always the most prominent. Peaks were apparent even in low-rate units with low counts in their histograms (Fig. 13F). Modes for these unusual histograms were estimated by reducing the reso-

213 lution along the time axis until a smoother histogram with a single maximum was obtained. Discussion

I. Tuning Curves Comparison with data from other animals indicates that auditory nerve fibres in the Tokay are sensitive and relatively sharply tuned. Tuning curves are Vshaped, as has been demonstrated for primary auditory fibres in cats (e.g., Evans 1972; Kiang et al. 1965), birds (Manley and Leppelsack 1977; Sachs et al. 1974), other reptiles (Klinke and Pause 1977; Manley 1977; Weiss et al. 1976), and amphibians (e.g., Capranica 1976). Q~odB values are larger, on average, than have been reported for auditory nerve fibres in the alligator lizard (Weiss et al. 1976), and monitor lizard (Manley 1977), but are comparable in both magnitude and frequency distribution to birds' (Manley and Leppelsack 1977; Sachs et al. 1974). The frequency distributions of Q~o,~Bvalues and tuning curve slopes in the Tokay also differ from those in the monitor and alligator lizards in that the latter species both have distinct fibre populations: a low-frequency, sharply-tuned population and one that is higher-frequency and more broadly tuned. In the Tokay, Q~odB and tuning curve slopes change gradually with CF, so that separate fibre populations are not apparent. The graph of high-frequency vs. low-frequency slopes (Fig. 4B) indicates an overall tendency toward steeper high-frequency slopes, as is the case for cochlear nerve fibres in mammals (e.g., Kiang et al. 1965) and caiman (Klinke and Pause 1977). Tuning curves of primary fibres in the monitor lizard (Manley 1977), starling (Manley and Leppelsack 1977) and pigeon (Sachs et al. 1974), on the other hand, have no consistent asymmetry. Comparison of the present results with data from guinea pig cochlear nerve fibres over the same range of CFs (0.1-6.0 kHz) reveals that tuning in the guinea pig is less sharp at these low frequencies (Evans 1972; R. Harrison, personal communication). The frequency response of the Tokay's middle ear contributes to the frequency dependence of QlodB and low- and high:frequency slopes. The velocity response of the columella (stapes) to constant sound pressure (Fig. 1) has been examined with the M6ssbauer technique (here taken from the velocity data used to calculate the displacements shown in Manley 1972 b). The response falls off at rates of approximate 10 dB/octave between 0.4 and 0.1 kHz, and 16 dB/octave above 2 kHz. For tuning curves above 2 kHz, the mechanical coupling through the columella tends to increase the high-frequency slope and decrease the lowfrequency slope, thus contributing to the elevated

214 HF: LF ratio in this range. Below 0.5 kHz, the reverse is true, causing H F : L F ratios to be smaller in this range than they would be if the middle ear response were completely fiat. The variety of PSTH types reported here contrasts with the rather uniform shape o f histograms recorded from mammalian cochlear nerve fibres (Kiang et al. 1965), and indicates a greater emphasis in the Tokay on peripheral filtering. At least some aspects of this filtering presumably can be related to the morphological specializations of its inner ear. All lizard basilar papillae have discrete areas of unidirectional and bidirectional hair cell orientation (Miller 1974). In the monitor lizard, a correlation between orientation and PSTH type was revealed by recording from afferent fibres adjacent to the papilla and, therefore, immediately opposite the hair cells they innervate (Manley 1977). As in the Tokay, tonic, intermediate and phasic PSTHs were obtained from fibres with low (< 0.6 kHz), intermediate and " h i g h " (>0.9 kHz) CFs, respectively. The low-frequency tonic fibres arose from a central unidirectional region of the papilla, while the higher-frequency intermediate and phasic fibres innervated bidirectional areas to either side of the central region. This led to the suggestion that in the monitor lizard, hair cell orientation pattern is the factor determining the observed histogram shapes (Manley 1977). If this were true for the Tokay, with its similar types of PSTH but different cochlear morphology, then tonic low-frequency fibres would originate in the basal unidirectional third of the papilla, and higher frequencies would be represented along the apical, bidirectional region. With this scheme, the smaller range of frequencies (CFs less than 0.75 kHz) is assigned to the smaller region of the papilla. Such a distribution would contrast with that along the mammalian cochlea, which is tuned to low frequencies apically and high frequencies basally. This arrangement is dictated by the mechanical response of the mammalian basilar membrane; whether the Tokay's basilar membrane is mechanically tuned is not known. Using the M6ssbauer technique to measure the velocity response of the small (0.4 mm) basilar membrane of the alligator lizard, Weiss et al. (1978) reported that the tuning of the membrane did not vary substantially with position along its length. The alligator lizard's basilar papilla, on the other hand, does have some degree of tonotopic organization: recordings from hair cells (Weiss et al. 1974) and from primary fibres as they emanate from the papilla (Weiss et al. 1976) have shown that, as in the monitor lizard, low frequencies are represented on the unidirectional portion of the papilla, with higher frequencies on the bidirectional area. Since the two areas also differ with

R.A. Eatocket al. : AuditoryNerve Fibre Activityin the Tokay Gecko respect to tectorial attachments in the alligator lizard, it cannot be said whether the correlation between frequency range and hair cell orientation pattern is meaningful or coincidental in this species. In the monitor, however, the tectorial membrane forms uniform attachments along the entire papilla (Miller 1978). The discrepancy between cochlear microphonic and single-unit sensitivity curves (Fig. 2) lends indirect support to the suggestion that tow and intermediate-to-high frequencies are located on unidirectional and bidirectional regions of the Tokay's papilla, respectively. Such an arrangement, together with the fact that cochlear microphonic functions are based on the fundamental component of the response, could account for the discrepancy. In round-window recordings from the Tokay, Hepp-Reymond and Palin (1968) found that the magnitude of the second harmonic often equalled or exceeded the fundamental component. Populations of hair cells of opposing orientations produce microphonic potentials that are 180~ out of phase (Flock and Wers/ill 1962) and saturate asymmetrically (Hudspeth and Corey 1977). Thus the opposing responses cancel at low amplitudes and produce a second harmonic at saturating stimulus intensities: Sensitivity estimates based on the fundamental component of the microphonic are therefore of dubious value when the auditory papilla possesses bidirectional areas, as in lizards. (The second harmonic is not a valid measure of sensitivity either, since its amplitude will vary with the relative phase and magnitude of the potentials produced by the opposing hair cell populations.) This reasoning provides a fairly simple explanation for the differences between the cochlear microphonic and single-unit sensitivity curves in Fig. 2: the fall-off of the fundamental above 0.6-0.9 kHz would be expected if higher frequencies are represented on a bidirectional area, and low frequencies on a unidirectional area, of the papilla. PSTHs with one or two initial peaks have been recorded previously in the Tokay from cochlear nucleus neurons and a small number of primary fibres (Manley 1974). In the torus semicircularis (a midbrain auditory centre) of the Tokay, only PSTHs with single initial peaks have been recorded (Sammaritano-Klein 1976). Discharge following the initial peak is more reduced in the secondary neurons than in the primary fibres, and even more reduced in the higher-order cells of the midbrain. Above the level of the primary fibres, this suppression might be mediated by contralateral inhibitory inputs. The increasing emphasis on the onset peak at progressively higher levels of the auditory pathway suggests that it is important to the animal's perception, but we can only speculate as to its function. A role in sound localization has been suggested for a similar response pattern in the monitor


lizard (Manley 1977). Lizards are poorly equipped to handle sound localization cues available to other animals; they lack pinnae and have small heads that create little sound shadow over the relatively low frequencies to which they are sensitive. Our phase-locking data suggest that they may be able to utilize binaural phase differences to localize very low frequencies (less than 0.7 kHz). Above 0.7 kHz, where no phase-locking occurs and all PSTHs can be classed as intermediate or phasic, highly synchronized onset peaks might serve to emphasize the very small differences in time of arrival of sounds at the two ears (on the order of 150 gs for a 5 cm head), thereby assisting localization by means of temporal cues. The extent to which a frequency-dependent interaction between sounds arriving at the two eardrums occurs in the buccal cavity, as recently demonstrated in birds (Hill et all 1979), is not known. It is possible that such interactions could also strongly influence the discharge properties of primary auditory nerve fibres. The multiple peaks in many PSTHs from units with CFs above 0.8 kHz have not been reported for primary fibres in other species. Since (1) the interval between peaks was a constant value between 1.5 and 4 ms for a given unit and (2) spontaneous dead times for fibres with CFs greater than 0.8 kHz were generally 2 ms or more, it is conceivable that multiple peaks reflect a tendency of these units to fire as rapidly as possible in response to the early part of the stimulus. Alternatively, multiple peaks may be related to the presence over the bidirectional two-thirds of the Tokay papilla of large tectorial masses, or 'sallets' (Miller 1973a), which only connect to one another and to the eilia of the underlying hair cells, and may thus be relatively free to oscillate (Wever 1967). The multiple peaks might thus reflect oscillations of sallets at frequencies ~determined by their inertial properties. The enhancement of multiple peaks with increased stimulus intensity (Fig. 8) would then be expected since such oscillations would damp out more slowly at higher intensities. It should be noted that the bidirectional region of the papilla is bisected longitudinally into two areas, one with sallets and one with more conventional tectorial attachments to the limbus. As the fibres innervating this region do not show a simple dichotomy of response properties, at least for pure tone stimuli, it may be that each fibre innervates hair cells from both regions, as lbund in a single transverse row.

On-off responses have been reported in auditory nerve fibres of a C F - F M bat (Suga et al. 1975) and the green frog (Sachs 1964), being apparently as rare in the latter as they are in the Tokay. The on-off responses in the bat are obtained from units with

215

best frequencies around the constant frequency component of the orientation pulse. Specialization of the area of basilar membrane on which these frequencies are represented provides a simple mechanical explanation for the off response, which cannot be applied in the present case. Although two-tone rate suppression, or two-tone 'inhibition', has been investigated thoroughly in mammals (e.g., Sachs and Kiang 1968), the mechanisms underlying it are not well understood. Its apparent relationship to tuning, and its occurrence in the Tokay and the alligator lizard (Holton and Weiss 1977), suggest that it may be a basic feature of vertebrate tuning systems. As has been observed for mammalian primary auditory neurons, suppressor tones were most effective at frequencies above CF for at least 4 of the 8 Tokay units studied. Another feature of two-tone suppression in mammals is that it disappears under certain conditions that produce broad, insensitive tuning - e.g., removal of perilymph from, or physical trauma to, the cochlea (Robertson 1976 a). We may have been observing a similar effect in the case of a rather insensitive, broadly-tuned fibre ( Q I 0 d B = 1.6): suppression could not be obtained with second tones at frequencies below CF, where the tuning was especially broad. It is worth noting that in the alligator lizard, the low-frequency, more sharplytuned fibres demonstrate two-tone rate suppression, whereas the population with higher CFs and broader tuning does not (Holton and Weiss 1977).

2. Spontaneous Activity The distribution of spontaneous rates in Fig. 12A indicates a much lower average rate and smaller range than are found in cochlear nerve fibres in mammals (Kiang et al. 1965; Manley and Robertson 1976) and birds (Manley and Leppelsack 1977; Sachs etal. 1974). The range of spontaneous activity recorded in auditory nerve fibres in the monitor lizard (Manley 1977) did not differ greatly from the range in the Tokay. In caiman (Klinke and Pause 1977) and alligator lizard (Weiss et al. 1976), the upper limits of the range were higher (70 and 80 spikes/s, respectively). These comparisons may not be very meaningful, however, since spontaneous rates could be sensitive to such factors as the type and level of anaesthesia used; for instance, urethane, used in both the Tokay and the monitor lizard experiments, might have a depressive effect on spontaneous activity. The low probability that a unit will fire at intervals shorter than its preferred interval, or mode, has been supposed to indicate that the unit is to some extent refractory to spontaneous discharge at such intervals (Kiang et al. 1965). It seems unlikely that this expla-

216 nation applies to the long modes recorded in this study and from auditory nerve fibres in the monitor lizard (Manley 1977). As well, mode values obtained for units in the Tokay bore no consistent relationship to the units' dead times, which are presumably a better indicator of spontaneous refractory period. The range of dead times in the Tokay (1.1-7.7 ms) was very different from the range for guinea pig spiral ganglion cells: 0.4-3.6 ms (Manley and Robertson 1976). Comparison of spike waveforms recorded with similar microelectrodes from Tokay primary fibres and guinea pig spiral ganglion cells (Robertson 1976b) reveals a significantly longer time-course for action potentials in the Tokay. This presumably reflects the temperature dependence of the action potential mechanism, since body temperature was 10-15 ~ cooler in the Tokays than in the guinea pigs. That the peaks in interval histograms of low-frequency fibres occur at intervals approximating integral multiples of the CF period raises the interesting possibility that this is related to the tuning mechanism. The possibility remains of course that the units were responding to low-intensity sounds such as might be produced by the animal. However, this interpretation does not readily explain how units with high CF thresholds (65 dB SPL in one case) could be sensitive to such low-level stimulation, or why periodic peaks were never observed for units with CFs above 0.5 kHz. That several of the units were relatively insensitive when tested with pure tones argues against there being a low-frequency resonance in some component of the middle or inner ear: although this would amplify low ambient sound levels, one would expect its effect on pure low-frequency tones to be even greater. At the same time, some of these units were quite sensitive, with CF thresholds of 20-30 dB SPL. For these units at least, it seems unlikely that the observed oscillations were abnormal and the behaviour triggered by injury to the fibres or within the cochlea. Another possibility is that the hair cells innervated by low-frequency fibres undergo spontaneous oscillations in membrane potential at a frequency close to the optimum stimulus frequency for the cells. This suggestion receives indirect support from a variety of sources: the turtle cochlea, the bullfrog sacculus and the electroreceptive organs of gymnotids (weakly electric fish). Electroreceptor cells are homologous to the hair cells of the acousticolateralis system. Large oscillatory potentials can be recorded extracellularly from certain phasic electroreceptors under unusual conditions - e.g., when the overlying seawater is replaced by distilled water - that reduce the electrical loading of the cells (Bennett 1967). In gymnotids, these oscillations occur at the frequency of the indi-

R.A. Eatock et al. : Auditory Nerve Fibre Activityin the Tokay Gecko vidual's own electric organ discharge. Since the primary afferents from these cells are tuned to the frequency of the electric organ discharge, Hopkins (1976) suggested that regenerative membrane processes may be responsible for both the oscillatory potentials and tuning in this system. It has been established that receptor potentials of hair cells in both guinea pig and turtle cochlea are sharply tuned (Russell and Sellick 1977, 1978; Fettiplace and Crawford 1978). Fettiplace and Crawford also have shown that the turtle's hair cells respond to the injection of small current pulses of either polarity with potentials that oscillate at or near the cells' CFs. Thus, certain electroreceptor cells and hair cells, when perturbed, will 'ring' at the frequencies to which they are most responsive. In both systems, the ceils are tuned to frequencies of 1 kHz or less; the upper limit of the frequency range over which such oscillations could occur would depend upon the membrane properties of the sensory cells : membrane time constant and the kinetics of channels involved in the oscillatory potentials (see Eatock and Manley 1980). Since the turtle's hair cells do not ring when injected with hyperpolarizing pulses that are very large, Fettiplace and Crawford have suggested that a voltage-sensitive potassium conductance may be involved. Evidence for voltage-sensitive channels exists for hair cells of the bullfrog sacculus. In these cells, the occurrence of action potentials under some conditions is presumably a manifestation of voltage-sensitive Ca ++ channels (Hudspeth and Corey 1977), and an outward rectification is thought to arise from voltage-sensitive potassium channels (Corey and Hudspeth 1979). Thus, various lines of evidence raise the intriguing possibility that low-frequency tuning in acousticolateralis systems is at least partly achieved through regenerative membrane processes in the sensory cells. The accompanying paper (Eatock and Manley 1981) discusses how the similar temperature dependence of tuning in submammalian auditory organs and electroreceptors is also consistent with this possibility. An alternative possibility is that resonance of mechanically-tuned component(s) of the system - e.g., the basilar or tectorial membrane - results in low-level stimulation of the hair cells, and, again, oscillatory receptor potentials. Although papillar innervation has not been examined in the Tokay, auditory afferents are known to contact several hair cells each in all non-mammalian systems that have been studied: anuran (Frishkopf and Flock 1974; Lewis and Li 1975), bird (Takasaka and Smith 1971), and caiman (yon Diiring et al. 1974). A fibre would reproduce oscillatory receptor potentials in the presynaptic cells only if these oscillations are in phase. In this respect, resonance of the mechanical input to the hair cells pro-

R.A. Eatock et ah : Auditory Nerve Fibre Activity in the Tokay Gecko vides the easier e x p l a n a t i o n for the peaks in the interval histograms, since a c o n g r u e n c e of phase for oscillations p r o d u c e d by hair cells w o u l d require electrical i n t e r a c t i o n a m o n g the cells i n n e r v a t e d by a given afferent. However, Weiss et al. (1974) have p r o p o s e d that such i n t e r a c t i o n m a y occur via s u p p o r t i n g cells in the papilla of the alligator lizard; receptor potentials recorded in s u p p o r t i n g cells as well as hair cells imply electrical c o u p l i n g between the two types of cell. N a d o l et aI. (1976) s u b s e q u e n t l y d e m o n s t r a t e d a possible p a t h w a y for this i n t e r a c t i o n in the f o r m of gap j u n c t i o n s between s u p p o r t i n g cells a n d between hair cells a n d s u p p o r t i n g cells, again in the alligator lizard. I n starling, cochlear g a n g l i o n cells with low C F s also tend to fire s p o n t a n e o u s l y at intervals closely a p p r o x i m a t i n g the period of the C F ( M a n l e y 1979). Since in b o t h T o k a y a n d starling such preferred intervals are evident only at very low frequencies, it is possible that they have been overlooked in other animals. As in other lizards, the specialized m o r p h o l o g y of the T o k a y ' s i n n e r ear has a f u n c t i o n a l correlate in the variety o f response patterns displayed by primary afferents. A t the same time, these fibres share m a n y features of s p o n t a n e o u s a n d evoked activity with auditory n e u r o n s of higher vertebrates, reflecting the f u n d a m e n t a l l y similar design o f the cochlea in reptiles, birds a n d m a m m a l s a n d suggesting that observations f r o m this study have i m p l i c a t i o n s for peripheral a u d i t o r y processing in general. We thank D.P. Corey and D.C. Van Essen for critical reading of the manuscript. This work was supported by grants from the National Research Council of Canada and the Qu6bec Dept. of Education.

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