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Characterization of the responses of inferior colliculus neurons of the chicken to electrical stimulation… Neufeld, Peter Richard 1994

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CHARACTERIZATION OF THE RESPONSES OF INFERIOR COLLICULUS NEURONS OF THE CHICKEN TO ELECTRICAL STIMULATION OF THE COCHLEAR NERVE  by  PETER RICHARD NEUFELD B.Sc. (Hon), Queen’s University at Kingston, 1991  A THESIS SUBMiTTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES  Department of Physiology  We accept this thesis as conforming to the required standard  THE UNWERSITY OF BRITISH COLUMBIA July 1994 © Peter Richard Neufeld, 1994  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  Pkc.  The University of British Columbia Vancouver, Canada Date  DE-6 (2188)  AL  2’I  J9L/  11  ABSTRACT  Responses of inferior colliculus neurons of the anaesthetized, cochlea-ectomized chicken to electrical stimulation of the cochlear nerves were recorded extracellularly. At least three physiologically distinct cell types were found in the central nucleus of the inferior colliculus. Type 1 fired randomly and with a high spontaneous rate, exhibiting a Poisson distribution in the spike interval histogram. Stimulation of either cochlear nerve produced an inhibition lasting 3 to 46 ms (mean of 16.7, n=16). Type 2 exhibited little or no spontaneous activity, and responded to a short stimulus with a single spike or burst of spikes (n=21). Type 3 exhibited regular, spontaneous firing with preferred intrinsic frequencies in the audio range (n=26), usually resulting in multimodal spike interval histograms. Single pulse stimulation of the contralateral nerve reset the firing rhythm, resulting in periodic post-stimulus time histograms (PSTH). The intermodal interval for a PSTH of a type 3 cell was identical to the intermodal interval for a spontaneous interval histogram. A reverse correlation of a random sequence of stimulus pulses with the response spikes revealed preferred frequencies in the input which were similar to the output frequencies seen in post-stimulus time and interval histograms. Thus, these type 3 neurons exhibited both an oscillatory spontaneous and evoked firing pattern, and an intrinsic frequency selectivity which is presumed to give rise to the observed oscillation. These cells were found to be grouped together in a relatively small portion of the inferior colliculus. The location of several type 3 cells was identified using WGA-HRP injected from the recording electrode. These cells were found to be in the core of the central nucleus of the inferior colliculus. These findings suggest the existence of an intrinsic mechanism for frequency filtering and time coding in the CNS.  111  TABLE OF CONTENTS  ABSTRACT  .  TABLE OF CONTENTS  .  ii iii  LIST OF FIGURES  v  ACKNOWLEDGMENTS  vi  iNTRODUCTION  1  I. Time Resolution of Hearing  1  II. Auditory Signal Transduction and Temporal Coding  1  A. Signal transduction and the auditory filters  1  B. Limitations of spatial discrimination of frequency  2  C. Phase locking and temporal discrimination  3  D. Limitations of temporal discrimination  4  E. Summary  5  III. Signal Processing in the CNS  5  A. Auditory neuroanatomy and the time pathway  5  B. Directional hearing  7  C. The time resolution of directional hearing  9  IV. Summary and Hypothesis  9  V. Neuronal oscillations and resonance  11  VI. Experimental Rationale  13  A. The choice of an in vivo approach to the ICC  13  B. The role of the inferior colliculus  13  C. Animal protocol  14  METHODS  16  I. Animals  16  II. Surgical preparation  16  III. Recording electrodes  17  iv IV. Stimulus generation  .  17  V. Cell isolation and spike discrimination  18  VI. Data Acquisition  18  VII. Estimation of inherent frequency  20  VIII. Histological verification of recording sites  20  RESULTS  22  DISCUSSION  37  I. Comparisons with Previous Studies  37  II. Type 1 cells  38  A. Spontaneous activity  38  B. Response to stimuli  38  III. Type 2 cells  39  A. Response to stimuli  39  B. Interaural time sensitivity  40  C. Oscillatory activity  41  IV. Type 3 cells  43  A. Spontaneous oscillations  43  B. Oscillations in the impulse response  44  C. Response to a random pulse sequence  45  D. Frequency preferences  45  V. Conclusions REFERENCES  47 49  V  LIST OF FIGURES  Figure 1: Spontaneous interspike interval histogram and temporal response histogram characterizing ‘type 1’ cells 23 Figure 2: Temporal response histogram characterizing ‘type 2’ cells  25  Figure 3: Graphs of interaural time delay sensitivity for three ‘type 2’ cells  26  Figure 4: Spontaneous interspike interval histograms characterizing ‘type 3’ cells  27  Figure 5: Distribution of intrinsic frequencies of ‘type 3’ cells  30  Figure 6: Temporal response histogram characterizing ‘type 3’ cells  31  Figure 7: Spontaneous interspike interval histogram, temporal response histogram and reverse correlation histogram of a single ‘type 3’ cell 33 Figure 8: Photograph of sagittal section of optic tectum showing horseradish peroxidase staining at recording sites  35  Figure 9: Photograph of sagittal section of optic tectum showing horseradish peroxidase staining at recording sites  36  vi ACKNOWLEDGMENTS  Many thanks are due to my supervisor, Dr. Schwarz, for making this work possible with his support and encouragement, not only of my scientific efforts, but of my interests in music and sailing. All graduate students should be so lucky... Thanks also to Eleanor To for providing assistance with the histology, and Dr. Finlayson for leniency during the final months of writing when I should have been working for him. To Dr. Mathers, thank-you for chairing my advisory and examination committees with an experienced hand. Thanks also to Drs. Puil, Church and Vincent for their advice and criticisms during the evolution of this project into its fmal form. I would also like to thank the entire physiology department for making my time at UBC enjoyable and, some say, educational. From a memorable trip to the chocolate buffet in my first week to the pot lucks this summer, with all of the relays, longboat races and wall stormings in between, the graduate students and faculty have made a newcomer feel at home. Joe Tay worked miracles in the darkroom in turning my hastily prepared figures into beautiful slides.. .“No, I don’t need them yesterday. Anytime before noon today would be fine, Joe.” Thanks to Dr. Pederson for always being available with information or a calm word of advice, and for hosting the annual retreat. These pages record such a small part of my experiences in this department...  1 INTRODUCTION I. Time Resolution of Hearing: The auditory system is capable of incredibly fine time resolution. Seemingly simple tasks such as listening to music or turning towards the source of a sound require discrimination of events in the cochlea and the CNS to within 10 J.ts or less. The ear itself has not been shown to be capable of this resolution, so it must lie with some as yet undiscovered property of the auditory pathway.  II. Auditory Signal Transduction and Temporal Coding: A. Signal transduction and the auditory filters: Sounds which arrive at the ear cause displacements of the tympanic membrane (eardrum) which are translated via the middle ear and oval window into displacements of fluid (perilymph) in the cochlea. Movement of this fluid in turn causes displacement of the basilar membrane, on which the sensory hair cells are fixed. Thus, a sound wave whose displacement may be expressed as a complex of sinusoids causes a displacement of the basilar membrane with a very similar complex sinusoidal pattern. This movement of the basilar membrane moves the hair cells relative to their sensory cilia, which are fixed at the tectorial membrane. This movement causes receptor potentials in the hair cells, leading to synaptic generation of one or more action potentials in the auditory nerve afferents (cf. Moore, 1989). The basilar membrane does not simply mimic the vibrations of the tympanum. At the base of the cochlea (closest to the oval window), the membrane is stiff, and vibrates more easily at higher frequencies. At the apex of the cochlea (furthest from the oval window), it is less rigid, and vibrates more easily at lower frequencies. As a result, a sinusoidal stimulation arriving at the oval window travels up the basilar membrane from the base towards the apex, causing a maximum displacement of the membrane at the region “tuned” to the frequency of the sine wave. Different points on the membrane along the path of the traveling wave will also oscillate at the frequency of the stimulus, but with a smaller amplitude.  2 This “tuning” of the basilar membrane results in filtering of sound signals. A complex signal, composed of many sine waves of different frequencies and phases (typical of most natural sounds), is separated into its component frequencies by virtue of the tuning properties of the basilar membrane. A given region of the membrane vibrates maximally only if there exists a frequency component of reasonable amplitude near the preferred frequency of that region (termed the characteristic frequency, or CF). Just what is meant by ‘near’ depends on several factors. First of all, as mentioned previously, any sine wave has the capacity to excite the entire membrane if it is intense enough. Secondly, CF appears to be represented on the membrane as increasing logarithmically with distance towards the base, so that the high frequency regions near the basal end appear compressed relative to the low frequency regions at the apex. The term ‘critical band’ has been developed to describe the frequency resolution around a given centre frequency. The critical bandwidth increases linearly on a log-log plot with increasing CF and is roughly 160 Hz for a CF of 1000 Hz. Each point on the basilar membrane can be thought of as a filter with a centre frequency determined by its position and a bandwidth equal to the critical band. These filters have been termed the ‘auditory filters’ (cf. Moore, 1989). Note that the critical bandwidth and CF both increase logarithmically with distance along the basilar membrane, so that the critical band can be expressed as a fixed distance on the membrane. This width has been measured in humans and found to be about 0.9 mm (cf. Moore, 1989).  B. Limitations of spatial discrimination of frequency: The auditory filters give rise to a spatial resolution along the basilar membrane. Different frequency components of a complex sound excite different regions of the membrane, which in turn lead to different hair cells passing their excitation to auditory nerve fibres. The resolution is limited by the width of the auditory filters, which at high frequencies can be very large. Thus the high frequency overtones of a musical note can occupy the same critical band and be difficult or impossible to resolve, and two simultaneous tones lying within a critical band will be heard with only one pitch. This has been supported psychophysically (cf. Moore, 1989).  3 The low frequency components of most musical pitches generally fall into separate bands and therefore should be easily discriminable. What has been found, though, is that for low frequencies (around 1 kHz), humans are able to distinguish differences in the frequencies of two tones of as little as 2 Hz. Assuming that frequencies are perceived as different when they excite different critical bands, such small frequency changes should be imperceptible. The difference in the basilar membrane excitation patterns between a 1000 Hz tone and a 1002 Hz tone cannot account for the observed resolution. An additional discrepancy arises when attempting to describe the perception of complex tones in terms of spatial resolution. The perceived pitch of a complex tone does not necessarily correspond to the position of maximum displacement of the basilar membrane. As an example, a complex tone consisting of all of the harmonics of a musical note but lacking the fundamental frequency will be perceived as having a pitch equal to the missing fundamental.  C. Phase locking and temporal discrimination: There exists another mechanism in the peripheral auditory system which is thought to contribute to frequency discrimination, particularly at the low frequency end. In contrast to the spatial discrimination arising from auditory filters, we will call this second mechanism temporal discrimination. As was stated in section II.A., each region of the basilar membrane has a particular CF. Similarly, nerve fibres possess CFs by virtue of their connections to individual hair cells. A given nerve fibre will exhibit an increase in average firing rate in response to any tone at a frequency within that fibre’s response area, and a maximal firing rate when stimulated at the CF of the unit. The rate of firing is not the only parameter of nerve firing which is dependent upon frequency. Temporal discrimination rests on the ability of nerve fibres to encode frequency information in the timing of spikes. In response to a pure tone, nerve firings tend to be synchronized to the phase of the stimulus waveform. This phenomenon, which is critical to temporal discrimination, is called phase locking. A phase locked fibre will not necessarily fire  4 at every cycle of the stimulus, but when it does fire, spikes occur at (roughly) the same phase of the stimulus waveform each time. Consider the effect of phase locking on the interspike intervals of a nerve fibre when stimulated by a 1 kHz tone. The minimum interspike interval would be 1 ms, corresponding to one period of the stimulus waveform. In addition, there would be interspike intervals occurring at multiples of this period (2 ms, 3 ms etc.). Thus, the period of the stimulating waveform is carried unambiguously in the firing pattern of one neuron over time, and a population of neurons taken together would produce spikes at every cycle of the stimulus. Phase locking can also explain the perception of a pitch at a frequency not present as a sinusoidal component in a complex tone (e.g. the phenomenon of the missing fundamental). For example, an amplitude modulated tone with a carrier frequency of 1000 Hz and a modulation frequency of 200 Hz consists of three components: 800, 1000 and 1200 Hz. It is found that a portion of the excited neurons phase lock to the overall repetition rate of the stimulus (200 Hz), which is equal to the ‘missing fundamental’ of these three frequency components. This phenomenon is thought to give rise to the perception of a tone at that frequency.  D. Limitations of temporal discrimination: The highest frequency to which a population of fibres can phase lock depends upon a number of factors and varies considerably between species. Phase locking in owls has been measured for tones up to 8-9 kHz (Sullivan and Konishi, 1984), whereas in some laboratory mammals the high-frequency limit is 3-4 kHz (Rose, et al., 1974). Temporal discrimination would seem to be able to provide the necessary resolution at the low frequency end of the spectrum (i.e. where spatial resolution is insufficient). One difficulty which is commonly expressed is that there is no evidence for a physiological mechanism which is able to carry out the interspike interval measurements with sufficient accuracy (cf. Moore, 1989). For the discrimination of a 1000 Hz tone from a 1002 Hz tone, the interspike interval of 1 ms must be measured with an accuracy of 2 ts.  5 E.  Summary: The auditory filters produce a spatial code of frequency and a means of separating  components of a complex tone. This spatial code is insufficient to account for the observed resolution of the auditory system, particularly at low frequencies. Phase locking produces a temporal code of frequencies with high resolution. It extends up to 3-5 kHz for mammals, and 89 kHz for owls, which use auditory cues to locate prey. No physiological mechanism has yet been found which is able to make use of the phase locked information. Such a mechanism must be located in the CNS, and the following section reviews the anatomy and physiology of the CNS with a focus towards temporal coding in avian species. The choice of birds as the focus of this study is due largely to the anatomical simplicity of the avian nucleus laminaris as compared with its mammalian analogue, the medial superior olivary nucleus, and is explained below in detail.  III.  Signal Processing in the CNS:  A. Auditory neuroanatomy and the time pathway: The auditory pathway has been mapped out in considerable detail in a number of species. One of the principal pathways from the periphery to the auditory cortex in birds is as follows: First order auditory nerve fibres synapse on the ipsilateral nucleus magnocellularis (NM); second order neurons send collaterals to both nuclei laminaris (NL); third order neurons synapse principally contralaterally (with a small ipsilateral projection) on the central nucleus of the inferior colliculus (ICC); fourth order neurons synapse on the ipsilateral nucleus ovoidalis of the thalamus; the thalamus sends projections to the auditory cortex. For reviews of these projections, see Yin and Chan (1988), Konishi, et al. (1988), and Conlee and Parks (1986). The ascending connections reported above are known in some detail. Less is known about descending connections, although some of the efferent projections have been mapped in the chicken: The IC projects to the caudal pontine reticular formation, which in turn projects down to the cochlea (Schwarz, et al., 1992). The structures described above are each presumed to subserve a different function, and investigations at the cellular level may provide clues to those functions. The nucleus  6 magnocellularis is made up predominately of ovoid cells with very short dendrites (Jhaveri and Morest, 1982), receiving large calycine endings of cochlear nerve fibres on their somata (Konishi, et al., 1988). The nucleus laminaris is a monolayer of fusiform cells (expanded into several layers in the owl), with dorsal and ventral dendritic arborizations, receiving excitatory inputs from ipsilateral and contralateral magnocellularis on the dorsal and ventral sides, respectively (Pallotta and Peres, 1989, Parks, et al., 1983, Smith and Rubel, 1979). As mentioned previously, it is the structure of the NL which makes it ideal for study, particularly when compared with the structure of its mammalian counterpart, the medial superior olivary nucleus (MSO). The MSO is a complex of several cell types, one of which, the principal cell, extends dendritic arborizations medially and laterally (Tsuchitani and Boudreau, 1964). The inferior colliculus is divided into several parts, a central (ICC), external (ICX) and shell (ICS) nucleus (Knudsen, 1983). The ICC of chickens contains, among other cell types, large GABAergic neurons (Granda and Crossland, 1989), some of which may project up to the nucleus ovoidalis (Muller, 1988). Glycinergic projections have been traced retrogradely from the ICC of chickens to three lower nuclei on the ipsilateral side (Schwarz, et al., 1994). The pathway outlined above is often called the time pathway because it preserves and processes temporal information as phase locked firing. This is to distinguish it from an alternative anatomical route termed the intensity pathway. The nuclei and interconnections making up the intensity pathway will not be detailed in this review, except to say that neurons in these structures generally show sensitivity to sound intensity, and phase locked firing is not observed. As a result, nuclei of the intensity pathway are not suitable subjects for a study of the fine temporal resolution referred to in section II. The time pathway has typically been studied for its relationship to directional hearing, a phenomenon which, like frequency discrimination, requires very fine resolution. A review of the functioning of this system must therefore start with an overview of directional hearing, but it must be remembered that these two tasks, directional hearing and frequency discrimination, are intimately related by the cues upon which they operate (timing and distribution of action potentials) and the structures which carry out the processing (the nuclei of the time pathway).  7 B.  Directional hearing: Directional hearing, or sound source localization, is a task which humans carry out  reasonably well without effort, and which is developed exquisitely in specialized animals such as the owl and bat. As a result of extensive psychacoustic studies, the directional cues which are available to the ear are well characterized. Most everyday sounds possess a very complex frequency spectrum, and a number of directional cues are available. For pure tones there are only two cues for the definition of the sound incidence angle in the horizontal plane (or ‘azimuthal angle’), but these are sufficient to allow localization (Blauert, 1983). They are the interaural intensity difference (11D) and interaural time or phase difference (lTD or IPD). An lID is the difference in the intensity (i.e. volume) of a sound between the two ears, and is caused by the acoustic shadowing of the head. A sound located to one side of the head will be less intense at the opposite ear providing its wavelength is less than ca. ten times the diameter of the head (Michelsen, 1992). Intensity differences are thought to be processed and compared by the intensity pathway referred to in section II.A. (Sullivan and Konishi, 1984, Takahashi, et al., 1984), and contribute to localization in the azimuthal plane for high frequencies (i.e. short wavelengths) and, in barn owls with asymmetrically located ears, the vertical plane (Volman and Konishi, 1989). It should be noted that, just as the auditory filters can discriminate frequencies exceeding the phase locking range, liDs allow azimuthal localization of high frequency tones. An lTD usually refers to the ongoing difference in timing of a sound between the two ears, and is caused by a difference in path length from the source to each ear. It is expressed in units of time (usually ,is). A difference in timing of a sinusoid produces a difference in phase, therefore the term IPD (interaural phase difference) may be used for steady state tones in place of lTD. which is sometimes reserved for differences in the time of arrival of a sound between the two ears. An IPD is expressed in units of radians, but can be converted to the equivalent units of time if the frequency is known. For our purposes, lTD will be used to indicate the ongoing time difference. The value of the lTD depends on the spacing between the two ears and the azimuthal angle of the sound source. For example, a sound source located at an azimuth angle of 90° to the right  8 of a human (head diameter of 23 cm) would reach the left ear about 700 ts later than the right (the time it takes sound at 340 mIs to travel around a sphere with a diameter of 23 cm). Phase locking of auditory nerve fibres will produce impulses in the auditory nerves with the same time discrepancy. For sounds whose half-wavelength is equal to or less than the head diameter, the phase difference would be greater than 180°, resulting in ambiguity as to which ear was closer to the sound. In practise, ilDs or head movements could resolve the ambiguity. For higher frequency sounds, the wave length of the sound becomes less than the path difference between the two ears, so that the same phase difference could be produced by a number of different source locations. Using the human as an example, the IPD is an unambiguous cue at frequencies below about 1500 Hz. For animals with smaller heads, the maximum path difference is smaller, so the highest frequency which provides unambiguous cues increases, as does the lowest frequency which provides useful lID cues. The largest lTD which a chicken experiences has been measured to be about 300 ts, so that phase ambiguity should arise for frequencies above 3.4 kHz. In fact, lTD cues cease to become detectable above 2 kHz, as this is the highest frequency for which phase locking has been observed in the chicken (Warchol and Dallos, 1989). The manner in which the CNS makes use of lTD cues has been investigated in detail, and the following conclusions can be drawn. The lowest point in the auditory pathway which receives binaural information (i.e. convergence of fibres from both ears) is the NL, which receives second order projections from the NM (see section III.A.). Information in the form of phase locked spikes arrives on the dorsal surface of the NL from the ipsilateral NM, and on the ventral surface from the contralateral NM (see section Ill.A.). The convergence of many phase locked inputs results in a conspicuous field potential which has been termed the neurophonic potential. This potential reproduces faithfully the waveform of the sound stimulus (Schwarz, 1992). To what extent this neurophonic potential is generated by phase-locking remains unclear, as does the extent to which this field potential actually influences the output of the NL. The significance of the neurophonic for the present study is that it interferes with single unit recording in the NL, effectively excluding this nucleus from single unit extracellular studies.  9 Anatomical and physiological evidence has shown a delay line along the NL of chickens and owls, which is thought to compensate for an lTD at one ear by delaying the impulses from the other ear (Schwarz, 1992, Sullivan and Konishi, 1984, Takahashi, et al., 1984, Smith and Rubel, 1979, Smith, 1981, Carr, 1988). The magnitude of the lTD. in conjunction with the delay lines, determines which cells in the NL receive coincident inputs. The physiological studies cited above have demonstrated that the NL is capable of resolving ITDs in the physiologically relevant range. The ability of these cells to detect coincidence with high resolution is central to the operation of this mechanism.  C.  The time resolution of directional hearing: The localization of a 1000 Hz tone requires that the NL neurons be able to resolve a  difference in the arrival time of spikes of much less than 1 ms (the period of the tone). This is remarkable given that the input to the coincidence detector is in the form of action potentials that have durations of the order of hundreds of microseconds (Irvine, 1986), or EPSPs with durations of several milliseconds. Expressed another way, given that the longest lTD that a human experiences is 700 ps, and an EPSP lasts more than 1 ms, the entire array of neurons will experience simultaneous EPSPs for every possible lTD. Thus, it is unclear just how time resolution on the order of a few microseconds is possible.  IV.  Summary and Hypothesis: The separation of a complex tone into frequency components which occurs in the  perception of pitch, and the discrimination of two tones of very similar frequency, can be accounted for by a central processor capable of accurately measuring interspike intervals of phase locked neurons. The localization of a sound source in azimuth can be accounted for by a coincidence detection mechanism capable of detecting the relative arrival times of phase locked spike trains from each ear. These two tasks are not independent of one another. In fact, they appear to converge within the NL for the perception of a signal in noise. Consider a complex  10 sound such as speech, and a background noise, both containing many of the same frequencies. Providing that they do not originate from the same source, the various frequency components will have different ITDs. Those arising from the speech sounds will all have the same ITD, and those from the noise will share a different lTD. The discrimination of frequency components, and the sorting according to ITDs, enables the auditory system to perceive speech sounds which would otherwise be obscured by noise. The time resolution displayed by these two processes (on the order of jis) cannot be explained in terms of traditional neuronal processes (i.e. action potentials and chemical synapses lasting ms) given what is known about the auditory pathway. We propose that an additional mechanism, neuronal frequency selectivity or resonance, operates within this system, particularly in the NL due to the key role this nucleus plays in temporal processing (see section III). The membrane potential of neurons receiving phase locked information resonates electrically at auditory frequencies. This resonance would act as a filter, separating out a single frequency from an auditory nerve fibre phase locked to more than one frequency, and increasing the resolution of the coicidence detector (NL) to less than the period of resonance of the cell. As an example, consider a cell with an electrical membrane resonance at 1000 Hz receiving phase locked input. A depolarizing deflection caused by synaptic input is more likely to exceed spike threshold if it occurs in phase with the cell’s membrane potential deflections arising from resonant properties. A deflection occurring out of phase with the cell’s evoked or spontaneous oscillation is less likely to result in spike firing, and an optimal response would require a match between intrinsic and input frequencies. Convergence of many phase locked inputs could further enhance the frequency resolution by averaging the signals tuned to the same frequency in many fibres. One of the predictions of this hypothesis is that the intrinsic membrane properties of cells of the NL are tuned to different frequencies, such that the nucleus as a whole spans the entire relevant frequency- and lTD-sensitive range. Intrinsic frequency preferences could be evident as resonance in the neurons’ response to an input pulse, and possibly also in the spontaneous activity of the neurons as oscillations in the firing pattern.  11 Evidence for the existence of resonant properties of neurons has been provided by numerous studies, some of which are outlined below.  V.  Neuronal oscillations and resonance: The existence of intrinsic resonant properties has been established for several neuron  populations. These resonances, when studied extracellularly, are expressed as inherent oscillations in the firing pattern. Some cells are said to be  “pacemakers”  when they spread their  rhythm to large areas of the CNS (Metherate, et al., 1992, Steriade, et al., 1985, Steriade and Llinás, 1988, Pinault and Deschênes, 1992, Nuñez, et al., 1992, Nuñez, et al., 1992). In addition to these widespread oscillations, visible in the EEG as, for example, delta, spindle or 40 Hz waves, a number of other systems possess inherent oscillations due to underlying resonances or particular synaptic organizations. Neurons in the vestibular system of mammals (Serafm, et al., 1992), cochlear hair cells of reptiles (Fuchs and Evans, 1988), avian auditory system (Langner, 1983, Fuchs, et al., 1988)(and see section VI.C.), mammalian auditory system (Eggermont, 1992) and non-excitable cells (Pallotta and Peres, 1989), display frequency preferences in their firing patterns and/or input-output relationships. In recent years, much work has focused on these oscillations in the thalamus, thalamocortical connections and the cortex of mammals. Intracellular recordings from these neurons have demonstrated the involvement of low-threshold calcium spikes (LTSs) and a hyperpolarization-activated cation current, termed ih in the generation of delta oscillations ,  (McCormick and Pape, 1990, Llinás, et al., 1991, Gutnick and Yarom, 1989). Together, these currents result in an after-hyperpolarization followed by a rebound depolarization which, if strong enough to generate another LTS, leads to oscillations (Llinás, 1988). In the case of spindles, a calcium-dependent potassium conductance may interact with a persistent sodium conductance and IPSPs fed back to thalamic relay cells from the reticular nucleus (Llinás, 1988). Oscillations in the auditory system are not well characterized. The present study represents the first detailed extracellular evidence for oscillations in the auditory system in the absence of cochlear effects. Some of these data were previously published (Schwarz, et al., 1993).  12 Neuronal oscillations may be independent of any inherent resonance of the cell in question. If a cell receives oscillatory synaptic input, it may exhibit oscillatory firing. The frequency in this case could depend upon a number of factors, including the number of presynaptic cells and their respective frequencies. In addition, intrinsic oscillations may or may not exceed threshold, so that the absence of oscillations in extracellular records does not preclude their existence. Similarly, inherent resonant properties may be present in a given cell with a stable membrane potential at rest (i.e. no oscillations visible either intra- or extra-cellularly). By definition though, if the cell resonates, the membrane potential will oscillate at the cell’s ‘preferred’ frequency when perturbed in either direction by a stimulus. These oscillations may or may not exceed threshold, so that an oscillatory firing pattern is not necessarily detected. The preferred frequency of a resonant cell can change if the relative ion concentrations across the membrane or the membrane potential change, or in response to any substance (such as a neuromodulator) which alters ion channel properties. The technique employed in any study of neuronal resonance must take account of the information sought. An in vitro approach provides a convenient framework for intracellular or whole cell patch recordings, and a means of applying drugs both extra- and intra-cellularly. Such an approach is essential for an understanding of the mechanisms involved in resonance (i.e. the currents, second messengers, etc.). Unfortunately, network connections are lost, and questions of function cannot be easily addressed. Due to some unknown properties of the tissue, the NL of the chicken appears not suitable for an in vitro slice, whole cell patch approach (unpublished observations). An in vivo approach preserves network connections, and therefore permits the study of suprathreshold oscillations in resonating neurons or at the target(s) of their output. The frequency and lTD selectivity reviewed in section III.B. suggests that the most reasonable location at which to find neuronal resonance is the NL. Unfortunately, for the reasons outlined above and in section Ill.B., this nucleus is not a suitable subject of study. Assuming that the oscillatory firing patterns of NL neurons are preserved at the next level in the pathway, the core of the central nucleus of the inferior colliculus (ICCc), this nucleus would be the logical  13 choice for investigation. In addition, cells of the ICC may themselves possess resonant properties. A brief review of previous studies of the avian ICC is presented below.  VI.  Experimental Rationale:  A. The choice of an in vivo approach to the ICC: We have established that the auditory system exhibits a time resolution which is not easily explained by known neuronal mechanisms. Studies of the anatomy and physiology of the auditory pathway point to the NL and its target, the ICC, as being most likely instrumental in the fine temporal discrimination, but the NL itself is not an ideal structure for single unit recording. The hypothesis under investigation is that neurons in the central auditory pathway, particularly in those regions involved in temporal processing, possess inherent resonances in the auditory range. This property would allow such cells to act as filters for frequency analysis of sound signals, thereby assisting in the combination of the filtered output from both cochleae. This might equip these cells with a better time resolution for coincidence detection than they would possess in the absence of resonance. Due to the need to study properties of the ICC which potentially arise from the NL, an in vivo approach, which preserves the interconnections, is necessary.  B.  The role of the inferior colliculus: Functionally and anatomically distinct regions of the avian IC (also called the nucleus  mesencephalicus lateralis pars dorsalis or MLD) have been demonstrated (Knudsen and Konishi, 1978, Dezsö, et al., 1993). Since then, these regions have been better characterized, and some of their connections mapped. The cells of the central nucleus (ICC) are arranged tonotopically in the owl and are sensitive to interaural time differences in sound stimuli (Wagner, et al., 1987). The ICC receives inputs primarily from the contralateral NL and from neurons in the intensity pathway (Conlee and Parks, 1986, for the chicken), and has been shown in owls to be sensitive to lTD (Wagner, et al., 1987, Takahashi and Konishi, 1988, Takahashi and Konishi, 1983). Commissural projections from the core of the ICC (ICCc) to the opposite lateral shell of the ICC (ICCs) are thought to assist in the representation of contralateral space by the lateral ICCs in owls  14 (Takahashi, et al., 1989). The remainder of the ICCs is not sensitive to interaural time and does not contain a representation of azimuth (Takahashi and Konishi, 1983). Chickens also possess conmuissural projections between the ICC on each side (Conlee and Parks, 1986), but studies complementary to those in the owl have not been carried out. The external nucleus (ICX) is a region overlying the ICC laterally and anteriorly, and individual cells in the owl’s ICX receive inputs from an array of cells in the ICC representing the entire frequency spectrum but only a single lTD (Konisbi, et al., 1988). The result is a topographic arrangement of space-specific neurons in the ICX forming a map of auditory space (Knudsen and Konishi, 1978, Knudsen and Konisbi, 1978). It is important from the perspective of the present study to note that these spacespecific neurons derive their lTD sensitivity from ongoing time disparities (IPDs), and are insensitive to transient time disparities (ITDs) (Moiseff and Konishi, 1981). None of the studies outlined in the preceding paragraph preclude the existence of oscillations in the firing patterns of IC neurons. In addition, these studies support the position that the ICC is the principle target of the NL and that it may preserve the fine time sensitivity seen in the NL. The present study represents the first characterization of the time patterns of chicken ICC neuronal responses to electrical pulse stimuli of the cochlear nerves.  C.  Animal protocol: The reason for the choice of an avian subject for this study relates to the difference  between the NL and the MSO of mammals (see sections II.E. and III.A.), and the chicken is an economical choice. The in vivo approach to the ICC is rationalized in section VI.A.. It was decided as well that an anaesthetized preparation was more humane than an alert restrained preparation, particularly considering that the vestibular system was damaged during the preparation, and a decerebrate preparation is not possible for recordings from the IC (located in the midbrain). Studies carried out on anaesthetized and unanaesthetized animals are compared in the discussion.  15 Electrical stimuli of the cochlear nerves (see methods) were used instead of acoustic stimuli for several reasons. Firstly, removal of the cochleae completely eliminates unwanted acoustic background noise, including physiological noise (see Moore, 1989 for a discussion of physiological noise). Secondly, stimulating the cochlear nerves with brief voltage pulses allows for precise control of the timing of stimuli. Thirdly, spontaneously active, rhythmic firing has been documented in avian cochlear nerve fibres and ascribed to rhythmical electrical potentials (oscillations) of the hair cells which they innervate (cf. Manley and Gleich, 1992). In order to eliminate oscillations of cochlear origin and limit the input to single, precisely timed spikes, experiments addressing intrinsic neuronal frequency selectivity in the auditory pathway must be carried out on animals without cochleae.  16 METHODS I.  Animals: Female domestic chickens (Gallus domesticus) were used in all experiments. They were  obtained as adults from a local supplier and maintained in animal care facilities for up to 2 weeks until needed. In general, the animals were not fasted before experiments. Animals ranged in size from 1.1 to 2.0 kg. A total of 23 animals were used for this study.  II.  Surgical preparation: Animals were anaesthetized with a single intra-muscular injection of a xylazinelketamine  mixture; 7.3 mg/kg xylazine (Rompun) and 57.2 mg/kg ketamine (Ketalean). Anaesthesia was maintained throughout all experiments with intra-muscular injections of 3.5 mg xylazine/25 mg ketamine approximately every two hours as required. Cloacal temperature was monitored periodically and maintained between 40 and 41.5°C using a heating pad. In a few cases, when the airway was obstructed by vomitus or saliva, the trachea was cannulated but the animals were not artificially ventilated. Once anaesthetized, the chicken was placed in a stereotaxic head holder. The head was held between two ear bars and a bit was placed at the joint of the beak. The head was then tilted forward such that the upper mandible was inclined downwards at a 45° angle (Kuenzel and Masson, 1988). The skin was removed from the top of the skull and the back of the neck over an area bordered by the comb, the ears and the foramen magnum. All muscle was removed from the bone above the ears and on the neck. All of the trabecular bone posterior to the forebrain and lateral to the sagittal sinus was removed without exposing the dura. The bone overlying the ampulla of the lateral semicircular canal was lifted off and the membranous labyrinth was removed, including the cochlea and the entire contents of the lateral and anterior semicircular canals and vestibule. The resulting hole into the cochlea was enlarged sufficiently to provide a clear view of the auditory nerve. This procedure was carried out for both cochleae. A pair of stimulus electrodes was placed against the cochlear nerve near the internal acoustic canal. The stimulus electrodes were prepared in the following manner: silver wire  17 (99.99%, Medwire Corp.) of 76 jim diameter, coated in Teflon (total diameter 114 jim) was stripped of insulation for the terminal 35 to 70 jim and coated in chloride by dipping in 2M NaCl while applying 10 V anodic DC current (Hewlett-Packard Harrison 6200B DC power supply). The chloride coating was inspected under 40x magnification to insure an even and complete covering. Once in place on the nerve, the wires were cemented against the skull using dental acrylic cement at a point approximately 0.5 to 1 cm from the nerve. The condensation was removed from the wires using a gauze wick, and the region overlying the cochlea filled in with petroleum jelly from a syringe to stabilize the electrodes and prevent moisture build-up around the tips. Once both ears had been prepared in this way, the bone overlying the tectum of one side was removed, and the dura incised to expose a roughly rectangular region of tissue approximately 2 mm square. The recording microelectrode was then inserted into the tectum using a threedimensional mechanical micro-manipulator supporting a Trent Wells Mark ifi hydraulic microdrive.  III.  Recording electrodes: Recording electrodes were glass micropipettes pulled from 3 mm diameter glass capillary  tubes (1.5 mm inside diameter) using a Narishige vertical one-stage electrode puller. The length of the shaft from shoulder to tip was about 10 mm. The electrode was then broken to an outside diameter of between 3 and 6 jim, with an impedance of 0.8 to 1.2 M2 to a 1 kHz square wave. Electrodes were filled with the recording solution consisting of 2M NaC1. For horseradish peroxidase (HRP) labeling of recording sites (see below), the solution consisted of 0.5 mg of protein dissolved in 50-60 jiL of either 1.0 M NaC1 or normal saline.  IV.  Stimulus generation: A NeuroLog system was used for stimulus generation. TTL pulses were used to trigger  the pulse buffers, digital oscilloscope (Nicolet) and the recording scope (EGAA computerscope, RC electronics). Two pulse buffers allowed for independent control of stimulus delay and  18 amplitude for the right and left ear electrodes. These pulses were then fed into NeuroLog stimulus isolation units, which generated short (0.1 ms) electrical pulses of 1-200 pA. Pulses were presented at a rate of 2/s or as a random sequence. The random sequence was generated by a noise generator (Coulbourn Instruments S81-02), the output of which was low-pass filtered at 1 kHz (Krohn-Hite model 3343 filter) and fed into a NeuroLog spike trigger unit before passing into the pulse buffers. The output pulses to the auditory nerves were presented with a Poisson distribution possessing a modal interval of 2 to 3 ms. For interaural time series experiments (see below), the stimulus to one or the other ear was delayed at the pulse buffer with a resolution of 10 ps between 20 ps and 3 ms. Delay times were monitored with the aid of the oscilloscope.  V.  Cell isolation and spike discrimination: The electrode was oriented on the tectal surface according to anatomical cues. In general,  the target was as far medial and rostral as possible while avoiding sinuses and blood vessels. Once on the surface of the tectum, the electrode was advanced approximately 1 mm before beginning recording. The ICC was identified physiologically as a region with a visible field potential evoked by electrical stimulation of the auditory nerve. Individual cells were isolated by forward and backward movement using the microdrive. The spike discrimination was carried out by the EGAA software using an adjustable voltage threshold. A recording was considered to be due to a single unit when all of the spikes crossing the threshold were of similar amplitude (±10%) and duration and the interval histogram showed no interspike intervals less than 2 ms.  VI.  Data Acquisition: IC neurons were recorded with glass pipette microelectrodes as described above. The  electrical activity was amplified and filtered in one of 2 ways: preamplified lOOx (BAK electronics preamplifier), band-pass filtered between 100 Hz and 10 kHz (NeuroLog filter) and amplified 50x (NeuroLog amplifier) or; preamplified lOOx and band-pass filtered between 300 Hz and 10 kHz (A-M Systems microelectrode AC amplifier model 1800) and amplified 50x (NeuroLog amplifier). The spikes in the resultant signal were transformed into TTL pulses and sampled with  19 a 20 jis resolution (EGAA computerscope software on a 386 clone). The data were collected and stored as one of the following types of histograms: 1. Interval histogram (111): except where noted, this histogram is constructed without any stimulus to the cochlear nerves. The time interval between consecutive spikes is plotted on the X axis, and the number of such intervals recorded is plotted on the Y-axis. This histogram was used to characterize the intrinsic periodicity (if present) in the spontaneous activity of the neuron. The sampling window was 160 ms long, allowing detection of interspike intervals of 160 ms (corresponding to a frequency of 6.25 Hz) 2. Post-stimulus time histogram (PSTH): this histogram is constructed in the presence of regular stimuli at one or both nerves. The X-axis is time following the trigger. The stimulus falls at approximately 2 ms on the X-axis, and every spike occurring over the following 350 ms is plotted on the time axis. The number of spikes occurring at a particular post-stimulus time is plotted on the Y-axis. In general, PSTHs were generated from up to 100 stimuli. This histogram was used to characterize ability of a stimulus pulse to entrain the activity of the neuron. 3. Reverse correlation histogram (RCH): this histogram is constructed in the presence of a random train of pulses delivered to one cochlear nerve. The Y-axis is the number of stimuli occurring at a particular time. The X-axis is the time before each spike. Every spike is assigned t =  0, and the timing of every stimulus in the random train preceding the spike is plotted on the X  axis. Generation of a RCH required that the amplitude of the spike potentials be greater than the stimulus artifact. This was necessary due to manner in which this histogram was constructed. The computer scope was triggered by a voltage deflection exceeding the preset threshold (presumed to be a spike), and plotted the pre-trigger time of every TTL pulse. Stimulus artifacts exceeding the voltage threshold of the computer scope would trigger the scope and be treated as spikes. Thus, the computer scope voltage threshold had to be set above the maximum displacement produced by stimulus artifacts and below the minimum displacement produced by spikes. This histogram was used to measure the intrinsic frequency preference (if any) in the input to the neuron.  20 For some cells, an interaural time series was performed. This consisted of a series of binaural PSTHs, constructed from 30 stimuli. The stimulus amplitudes were adjusted such that a monaural stimulus was just below threshold for the cell under study. Each PSTH was constructed with a different interaural delay, ranging from up to 3000 jis ipsilateral ear leading to 3000 jis contralateral ear leading. The time delays used were based on several considerations: 1. As the maximal interaural time discrepancy that a chicken experiences is approximately 300 Is (based upon head diameter), relatively more points were chosen in this range. 2. The oscillating cells that were found had on average a period of approximately 10 ms (see results), suggesting that the oscillatory period of the type 2 cells described in the results might have a similar value (see discussion), so the range of points used should cover at least half of that period (5 ms). 3. The entire experiment should be done as quickly as possible because of the constant risk of losing the cell. The resulting compromise consisted of steps of 40 jis around 0 ITD, with larger steps (100-1000 is) at larger ITDs.  VII.  Estimation of inherent frequency: For cells exhibiting regularly spaced peaks in either their IH, PSTH or RCH, their  inherent oscillatory period was taken to be the average distance (on the time axis) between successive peaks. This distance was measured with the aid of an analysis routine in the computerscope software. A vertical line was placed through the centre of each peak (as estimated by eye) and the value on the time axis of each line was displayed with a resolution of 0.04 ms. Due to the width of the peaks and the difficulty in discerning the precise centre of each peak, this procedure generated a value for the oscillatory period with a precision of ± 0.5 ms, depending upon the sharpness and number of peaks.  VIII.  Histological verification of recording sites: For selected experiments in which a region of oscillating neurons were found, wheat germ  agglutinin conjugated to horseradish peroxidase (WGA-HRP type VI, Sigma) was dissolved in  21 the recording solution as described above. The protein was ejected from the electrode tip using 2 hA current pulses (tip positive) delivered by a NeuroLog system NL800 stimulus isolator. The pattern of current pulses was generated by a 0.25 Hz square wave (Simpson model 420 function generator) fed through a NeuroLog pulse buffer into the stimulus isolator, resulting in current pulses of 2 seconds in duration separated by 2 second pauses. The ejection was maintained for 10-15 minutes at the desired site, then the electrode was withdrawn at about 4 jim/s (while maintaining the current pulses) to mark the track. Following WGA-HRP ejection, the animal was perfused under deep anaesthesia with 0.5% sodium nitrite in .9 N saline at ca 35°C followed by paraformaldehyde/gluteraldehyde. The brain was removed and sectioned sagittally on a freezing microtome into 50 jim thick sections. These were then incubated for horseradish peroxidase (HRP) and counterstained with the Nissl stain thionine. Black and white photographs were taken under white light using a photomacroscope (Wild M400).  22 RESULTS  A total of 81 neurons were isolated. Of these, 69 were classified into 1 of 3 types on the basis of their spontaneous and evoked activity. The remaining 12 could not be classified. Type 1 cells exhibited spontaneous activity without an intrinsic frequency preference (see below) and were briefly suppressed by stimulation of one or both cochlear nerves. Type 2 cells exhibited little or no spontaneous activity, and responded to stimulation of either cochlear nerve with a single spike or short burst of spikes. Type 3 cells exhibited a high degree of spontaneous activity and displayed a frequency preference in their firing pattern (see below). This same frequency preference was seen in response to single stimuli or random trains of stimuli of the cochlear nerve. Type 1: These cells fired spontaneously and randomly, producing a roughly Poisson distribution in the interval histogram (IH). Figure la shows the typical appearance of an IH of a type 1 cell. There is only one, broad peak, extending over a 20 ms period. The firing rate (not shown) was normally quite high, often approaching 100 spikes/s, but without a dominant frequency in the firing pattern. Type 1 cells responded to inputs from both nerves with a period of suppression lasting 3 to 46 ms (mean 16.7, S.D. 11.3, n=16) after a latency of 3 to 14 ms (mean 7.5, S.D. 3.3). During this period, these cells were completely silent. Figure lb shows a typical example of a PSTH from a type 1 cell. The thin peak occurring at 2 ms represents the stimulus artifact. A very slight increase in firing probability is visible at approximately 5 ms (3 ms post-stimulus), suggestive of a type 2 response (see below). A profound suppression was often the only indication that the cell was responsive to stimulation of the cochlear nerve. In this example, the silent period extends from approximately 5 to 15 ms on the time axis. Following this period, the cell resumes firing with random probability, visible here as an approximately flat line along the time axis. Type 1 cells were found distributed throughout the region yielding field potentials following cochlear nerve stimulation (the presumed ICC) and were often the only cell type in the  23  70-  A  60-  I  5040-  30-  z  20-  L..LL. I.  01 0  1.1  I  I  I  I  I  20  40  60  80  100  Time (ms)  20-  B 15-  C  10-  Iz 5 [IIii IIIIII liii liii IIIIIIII I III  I  0 0  20  40  60  80 100 Time (ms)  II ill IUhIIII IIIIIIII NI III 1 120  140  160  Figure 1 A: An interval histogram of a type 1 cell. The time between every 2 consecutive spontaneous action potentials (spikes) is plotted on the X-axis. Histogram constructed from 150 sweeps. The bin width is 320 jis. Cell 9.3.H., file EL91531H. B: Post stimulus time histogram of a type 1 cell. The stimulus artifact is visible at time = 2 ms. A region of suppression is visible between time = 5 and 15 ms. Histogram constructed from 100 stimuli. The bin width is 20 is. Cell 2.18.C., file ELi l/69PS.  24 area. A total of 16 such cells were recorded, although many more were isolated, suggesting that this is a common cell type in the ICC. Type 2: The second type exhibited very little or no spontaneous activity. Out of 21 type 2 cells isolated, 14 exhibited no spontaneous activity and the remaining 7 fired less than 5 spikes per second. Their response to a single pulse stimulus was phasic, that is, a single AP or a short burst of APs after a latency of approximately 6 ms. The response latency was similar for contralateral and ipsilateral stimuli (contralateral mean 6.06 ms, S.D. 1.8, ipsilateral mean 6.71 ms, S.D. 2.1, n  =  13). Figure 2 shows a typical PSTH of a type 2 cell. A single very sharp peak  occurs at a relatively fixed interval (latency) after the stimulus. Following this onset response, this cell remained silent until the next stimulus. For some type 2 cells, an interaural time series was performed as described in the methods. When recording extracellular activity, oscillations are only visible when there is steady state spike activity. A type 2 cell, without any spontaneous activity, might still oscillate at subthreshold levels. Such oscillations could be revealed by repeated stimulation, such as occurs during an lTD series. Figure 3 shows the results from three such experiments. The series shown in figures 3a and 3c were extended to ±3000 p.s, but showed no change in response between ±1000 and 3000 Jis, and so the plots were truncated to allow a reasonable resolution around zero. Although type 2 cells did respond differently at different 1TDs, no cyclic pattern was visible in the lTD series. Type 2 cells were found spaced throughout the presumed ICC and tended to have large amplitude field potentials, suggesting very large cells. They were the most common cell type isolated, suggesting that this may be the dominant cell type in the ICC under anaesthesia. Type 3: These cells exhibited a high spontaneous rate with a periodicity in their firing pattern, as shown in the IH. Figure 4 shows three examples of IHs from type 3 cells. The first one (fig. 4a) shows only a single peak with a very narrow base (compare fig. la). This is representative of a cell whose firing rate is equal to the inherent frequency. The interspike interval for this cell is given by the time of the single peak, which is centred around 15 ms. This corresponds to an inherent frequency of 67 Hz. The second one (fig. 4b) exhibits several modes,  25  8-  6-  —  0  I  I  I  I  I  20  40  60  80  100  Time (ms)  Figure 2 Post stimulus time histogram of a type 2 cell. The stimulus occurs at time = 2 ms (not marked). All of the cell’s responses occur at approximately 8 ms (6 ms after the stimulus). Histogram constructed from 50 stimuli. The bin width is 20 jis. Cell 12. 16.C., file ELi 1/32PS.  26  12  A  10•  B I)  C  11:  I  •  I  I  •  I  C  ‘I  C  4t:  -800  -400  0  400  800  Contralateral Delay (jis)  Figure 3 Interaural time series for 3 cells. Each point represents the sum of responses to 30 stimuli at a given interaural time discrepancy shown on the X-axis. A positive contralateral delay indicates that the stimulus to the cochlear nerve contralateral to the recording site was delayed relative to the stimulus to the ipsilateral nerve. A negative contralateral delay indicates that the contralateral stimulus led the ipsilateral one by the specified amount. A: Cell 11 .27.C. B: Cell 10.2.D. C: Cell 12. l0.E. The rationale for the interaural delays chosen is given in the methods.  27 Figure 4 Interval histograms from three type 3 cells. The time between every 2 consecutive spontaneous action potentials (spikes) is piotted on the X-axis. A: This cell exhibits a single mode at approximately 15 ms, corresponding to its most common interspike interval. 50 sweeps, bin width 20 pUs. Cell 4.20.D., file EL12/811H. B: This cell exhibits 4 modes with an intermodal interval of 8.5 ms and a most common interspike interval of 8.5 ms. 100 sweeps, bin width 320 ps. Cell 2.24.B., file ELi l/721H. C: This cell exhibits 10 modes with an intermodal interval of 8.5 ms and a most common interspike interval of 25.5 ms. 100 sweeps, bin width 320 its. Cell 4.20.C., file EL12/801H.  28  25-  A  20 -4  C  10-  z  5II  0-  •  0  I 20  I 60  I 40  •  100  80  Time (ms) 120-  B  100• -4  80C  60-  z  40200  -  ••  0  20  -  •  60  40  i 80  ‘  ‘  - I 100  Time (ms) 25 20 C,)  15  E  z  10 5  IUiii Iii IJ M  0 0  20  40  60  Time (ms)  E11 80  100  29 with the first one larger than the others, and all of them equally spaced along the time axis. This indicates that this cell does skip beats, but that the intrinsic frequency (given by the intermodal interval or the time to the first peak) is still the most common firing frequency. Thus this type 3 cell differs from a type 1 cell in that all spikes fall into one of the equally spaced modes of the JR. The third one (fig. 4c) exhibits several modes, with the third one being the highest. The most common interspike interval for this cell is approximately 25 ms (the interval of the third peak), but the intrinsic period, which is represented by the intermodal interval, is 8.5 ms, corresponding to a frequency of 118 Hz. This pattern would occur if an oscillating membrane potential reaches threshold more frequently after missing one or two periods. A total of 26 type 3 cells were isolated. Their intrinsic frequencies ranged from 67 Hz to 250 Hz. The distribution of frequencies among the cells is shown in figure 5. The majority of cells possessed an intrinsic frequency near 100 Hz, with none exhibiting frequencies below 60 or above 260 Hz. These oscillatory neurons typically responded to stimulation of the contralateral cochlear nerve (and occasionally the ipsilateral as well) and were entrained to the stimulus while retaining their inherent frequency. As a result, the PSTH of a type 3 cell was multimodal, with the same intermodal interval as the 1}I. Figure 6 illustrates this. This PSTH is taken from the same cell whose JH is shown in fig. 4a, and displays the same intrinsic period (15 ms). Every stimulus pulse appeared to reset the rhythm of the cell, after which it resumed its regular firing until the next stimulus. An onset response is visible after a short latency. This may be a response of the oscillating cell, or the response of a nearby type 2 cell, or a combination of both. The possible contamination of the recording by a type 2 cell is suggested by the strong onset response without any subsequent activity between stimuli. Increasing the intensity of the stimulus had no effect on the intrinsic firing frequency of the cell (results not shown). In order to determine whether these cells can serve as filters by extracting a specific frequency from a random input, a RCH was constructed from individual type 3 cells. Thus, a random train of stimuli was applied to the cochlear nerve, and the timing of these stimuli was plotted with reference to every spike. As expected, the same intrinsic oscillatory frequency found  30  7 6  00 I  C  C C I  C 00  C CN I  C C (“1  Frequency (Hz)  Figure 5 Distribution of intrinsic oscillatory frequencies of the 26 type 3 cells isolated. The frequencies were measured as described in the methods.  31  50 40  I  20 Z  I  0  ‘  20  40  .4. . 1A, ‘.4. .’ 60  100 80 Time (ms)  4  120  140  160  Figure 6 Post-stimulus time histogram of the same cell pictured in fig. 4a. The stimulus artifact is visible as a vertical line at time 2 ms. An onset response is visible at 4-5 ms. The intennodal interval  is 15 ms, corresponding to a frequency of 67 Hz. Histogram constructed from 200 stimuli. Bin width 160 Ls, cell 4.20.D., file EL12/82PS.  32 in the IH (fig. 7a) and PSTH (fig. 7b) was seen in the response to a random train of stimuli (RCH), demonstrating that these cells respond preferentially to stimuli which occur at their preferred interval (fig. 7c). Type 3 cells were found exclusively in a small region of the ICC and relatively few were found in any given animal. They tended to have small amplitude spike potentials, making them difficult to isolate from background activity, and were more difficult to maintain close to the electrode tip than the other cell types. In order to determine histologically the location of this region, WGA-HRP injections were made as described in the methods. Figures 8 and 9 show sections through WGA-HRP labeled sites from two animals. The section shown in figure 8 was taken from an animal which received 2 separate injections approximately 100 jim apart rostro caudally. Both injections were made at the deepest (i.e. most ventral) point of oscillatory activity. The two spots shown by arrows are located in the core of the ICC (ICCc). They are roughly 100 jim apart on the rostro-caudal axis and could therefore represent the separate tracts. The section shown in figure 9 was taken from an animal which received 2 injections from 2 separate electrodes. Due to differences between electrodes, and the manipulations required to change electrodes during an experiment, the distance between tracts produced by two different electrodes could not be reliably measured. The spots marked “A” and “B” are both located in the ICCc, and may represent 2 separate tracts or uneven ejection and/or diffusion from a single tract. Every HRP labeled tract was located either within the ICCc or along a line directed towards the ICCc.  33 Figure 7 Inherent frequency of a type 3 neuron expressed in the interval histogram, post-stimulus time histogram and reverse correlation histogram. A: IH constructed from 100 sweeps, in the presence of a random train of stimuli, showing a large peak at 4.5 ms and a smaller one at 9 ms. The intervening noise could be caused by a poor isolation of the oscillating neuron or doublet firing by this cell, file EL9/68111, bin width 80 ps. B: PSTH from the same cell exhibiting the same intrinsic frequency in response to 100 individual pulse stimuli applied to the contralateral cochlear nerve. File EL9/64PS, bin width 320 us. C: RCH constructed simultaneously with the N shown in A. It exhibits the same periodicity in its responses. File EL9/67RC, bin width 4Ojis. Cell 9.3.M.  34  80—  A CID  60—  I  40—  z  20—  0 -r1’  I  I  I  I  I  20  10  0  II  30  I  I  II  I  I  40  II  60  50  Time (ms) 50  B  40 cdD  1) C,)  0  I.  0  10  A  •  I  40  I  II  Time (ms) 30  C  20—  I  5  -20  -15  -10 Time (ms)  -5  I  60  50  25  I  35  Figure 8 Sagittal section through the right optic tectum of a chicken which was injected with HRP from two separate electrode penetrations as outlined in the methods. The electrode tracts were made approximately 100 .Lm apart rostro-caudally. The 2 spots labeled by arrows indicate regions of HRP activity located in the core of the central nucleus of the inferior colliculus (ICCc). Scale: each axis arrow is 280 j.tm long. ICCs: shell of the central nucleus of the inferior colliculus; D: dorsal; R: rostral. Experiment 6.11.  36  I  %_ -  •.‘i•._  -.:  a  ‘-  —  icce ‘i.r;  tt:e  :  -  •  :  -  :e  ••  /  -  o .  *  1  -  k ‘1  .%%..  t.%  --a  t  -  Figure 9 Sagittal section through the right optic tectum of a chicken which was injected with HRP from two separate electrodes as outlined in the methods. The two spots labeled A and B indicate regions of HRP reactivity located in the core of the central nucleus of the inferior colliculus (ICCc). Scale: each axis arrow is 245 jam long. ICCs: shell of the central nucleus of the inferior colliculus; D: dorsal; R: rostral. Experiment 5.19.  %.  37 DISCUSSION  I.  Comparisons with Previous Studies: Most studies of auditory neurophysiology used sound stimuli, delivered either in the free-  field or from closed systems within the ear canal to permit dichotic investigations. Stimuli have taken the form of noise, clicks, or pure tones and had durations much longer than 1 ms, allowing the coding for ongoing sound properties (as opposed to purely the presence of the signal) to be studied. This included phase locking where possible. The interaural time sensitivity demonstrated in the owl IC (see introduction) was elucidated under such conditions. The approach of the present study differs significantly from most of the literature in this field because of the experimental protocol. The removal of the cochlea and use of electrical pulse stimuli is “unphysiological” in that the signals traveling to the CNS are not likely to resemble anything the animal could experience naturally. The impulsive stimulus is shorter than the duration of an action potential, and not repetitive, excluding phase-locking in the CNS. The excitation of all auditory nerve fibres is assumed to be simultaneous. It is presumed that the signal in the cochlear nerve is a single spike traveling simultaneously in most or all intact fibres. This paradigm was designed to reveal inherent frequency selectivity, including oscillations, of central auditory neurons, and exclude contamination due to basilar membrane ringing, hair cell resonance, and periodic responses due to phase locking to a periodic stimulus. One important aspect of this paradigm, which does lend itself to comparison with a number of previous studies, is the use of anaesthetic during recording. While it is reasonable to assume that anaesthetics will alter certain neuronal properties, it was felt that their use was justified on several grounds. Firstly, limited studies comparing anaesthetized preparations with others (decerebrate or awake) have found most results to be similar. In particular, the main response patterns characterizing auditory brainstem neurons have been found in all of these three different paradigms without qualitative difference (Rhode and Kettner, 1986, Ritz and Brownell, 1982, Webster, 1977). Secondly, as stated in the introduction, a decerebrate preparation is not compatible with recordings in the midbrain, and an unanaesthetized preparation following  38 extensive surgery was not possible (see introduction). Finally, given the extensive use of anaesthetics in the literature, comparisons with previous studies are easier to make using data from an anaesthetized animal.  II.  Type 1 cells:  A.  Spontaneous activity: Cells described in this study as exhibiting a high rate of irregular spontaneous activity  were classified as type 1. The pattern of activity of a typical type 1 cell is illustrated in the interspike interval histogram shown in figure 1 a. The distribution of interspike intervals can be approximated by a Poisson distribution, thus this cell type fires in a random pattern. The average spontaneous firing rate of these cells was usually between 20 and 120 spikes/s. While this activity could arise from spontaneous fluctuations of the membrane potential of these cells independently of synaptic inputs, the interval histograms are reminiscent of many central neurons receiving a continuous synaptic barrage on their dendritic trees (eg. motoneurons). A high rate of stochastic spontaneous activity at lower levels is prevalent in birds with intact cochleae (Sachs, et al., 1974, Sachs and Sinnott, 1978), suggesting that this activity may be inherited from below. In order to determine the functional significance of this pattern of activity, recordings must be made from these cells in the presence of acoustic stimuli. The origin of the observed activity is uncertain. In the context of the processing of the auditory codes it is of interest that, in contrast to the type 2 cells described below, this cell type is not tonically inhibited.  B.  Response to stimuli: The only physiological evidence that type 1 cells receive auditory input is the suppression  of firing seen in the response to pulse stimuli. As shown in the PSTH (fig. ib), there was a period of post-stimulus suppression of firing. This suppression may be due to inhibition from GABAergic cells within the IC reported by Granda and Crossland (1989), or projections from lower centres. The avian nucleus intermedius of the lateral lemniscus contains primarily GABA  39 immuno-reactive cells, and projects to the contralateral ICC (MUller, 1987, Muller, 1988), and therefore is a likely candidate for inhibitory input. A glycine-transporting projection system to the ICC from several lower centres was recently described for the chicken (Schwarz, et al., 1994), suggesting that suppression may also be glycinergic. There was considerable variability in the latency to the onset of suppression recorded for these cells; latencies varied between 3 and 14 ms, with a mean of 7.5 ms. The significance of this variability cannot be determined from the results presented here, but several possible sources of variability can be suggested from the literature. IPSPs at various regions of a cell’s dendritic tree can certainly give rise to spike train suppressions at different latencies. The suppression may furthermore be caused by synaptic inhibition arising from one or both of the transmitter specific pathways listed above (or other transmitter systems as yet undocumented in the ICC may be involved), and may originate at one of several possible sites in the auditory pathway. These sites in turn vary in their interconnections, introducing another source of variability in transmission delay. Alternatively, if the spontaneous activity of these cells arises at lower centres, there may be synaptic inhibition occurring at those centres. A characterization of the underlying mechanisms requires intracellular recording from these cells with application of GABA and glycine agonists and antagonists. Data were obtained from 16 type 1 cells, which were found throughout the region of the ICC (judged to be that region of the IC yielding field potentials following cochlear nerve stimulation). This small number is not indicative of the actual proportion of type 1 cells in the ICC. Many more were isolated but not recorded since they were not a focus of this study of neuronal frequency selectivity. It is estimated that this cell type is more numerous than type 3 cells described below.  III. A.  Type 2 cells: Response to stimuli: Cells in the present study which exhibited little or no spontaneous activity and which  responded to an impulse stimulus with a single spike or short burst of spikes were classified as  40 type 2. This simple response pattern is illustrated by the PSTH shown in figure 2. The response latency was roughly 6 ms and relatively constant, and the response was invariably followed by a period devoid of spike firing. Previous studies in the owl IC have documented the existence of ‘silent’ neurons at this level: Wagner (1990, 1992) found that IC neurons generally displayed no spontaneous activity. No comparable studies have been carried out on the chicken. Although this study did not address whether the response (fig. 2) was followed by inhibition, or was merely brief due to the duration of the stimulus, preliminary studies have investigated this (Schwarz, et al., 1993). It was found that the response is followed by a period of suppression lasting between 40 and 200 ms during which a stimulus of similar intensity will not evoke a response. As with the suppression of type 1 cells discussed above, this may be due to GABA or glycine release from local cells or from lower centres, or to inhibition of cells in lower nuclei providing inputs to the ICC. The PSTH of a type 2 cell is very similar to the PSTH of a class of cells termed “on” cells which were originally recorded in the mammalian cochlear nucleus under the influence of sound stimuli (Pfeiffer, 1966). The term ‘on’ type response has since been applied to any response which exhibits a firing pattern similar to the PSTH in figure 2 when stimulated acoustically. Typically, a longer stimulus (—200 ms) is used, and an “on” cell responds only to the initial portion. The type of stimulus used in the present study, and the relative locations of the two classes of cells (mammalian cochlear nucleus and avian midbrain), precludes any direct comparison of the two cell types. To prevent confusion of these two cell types, the term ‘on’ response has been avoided when referring to type 2 cells.  B.  Interaural time sensitivity: Cells in the inferior colliculus of the owl create a map of auditory space by firing  maximally to a particular frequency (defined as the cell’s characteristic frequency, CF) at a particular IPD. As the IPD for a tonal stimulus is varied, the response cycles from maximal to minimal to maximal again with the period of the stimulus tone. This is a simple consequence of the repetitive nature of a cyclic stimulus. An IPD of it is often indistinguishable from an 1PD of  41 3it,  therefore one would expect a similar response in the IC to both IPDs. The spike probability  is usually highest at the onset of the response, particularly for owl IC cells, with the result that a favourable IPD produces an “on” type response, whereas an unfavourable IPD does not evoke any response (Wagner, 1990). Cells of the owl’s ICC produce a more sustained response to favourable IPDs, similar to the “on-type L” units described in the PVCN (Godfrey, et aL, 1975). These units maintain a low level of activity for the duration of the stimulus. In order to pursue the possibility that the type 2 cells described here possess an intrinsic interaural time sensitivity independently of the periodic nature of acoustic stimuli, interaural time series were performed on several type 2 cells. We predicted a cyclic response to varying ITDs as was reported above for IPDs, with the period of the cycle dependent upon the intrinsic filter function of the unit (the CF could not, of course, be determined in this study). At least one response maximum should occur within the physiological range experienced by chickens (i.e. 300 jis). Twice that range (between -600 ts and +600 ps) was studied using small lTD increments (see methods). The negative results shown in figure 3 must be considered preliminary since we abandoned a search for intrinsic oscillations in these cells. No clear response maximum or periodicity was found at less than ±600 jis of lTD. There is, therefore, no evidence for subthreshold oscillatory responses which could have served to produce an intrinsic interaural time selectivity, or for network mechanisms accounting for it. The negative results do not rule out a role for these cells in interaural time delay processing. lTD selectivity could well depend upon oscillatory responses available only when using sound stimuli. It is therefore possible that these cells require an ongoing binaural stimulus from which to extract the IPD. This latter conclusion is supported by Wagner (1990), who found that the IPD sensitivity of owl ICC “on” neurons improved with time during a 120 ms long stimulus.  C.  Oscillatory activity: The principal purpose of the lTD series trials was to test for the existence of subthreshold  oscillations of type 2 cells. The possibility exists that the membrane potential of this cell type oscillates below threshold, such that stimuli which are timed in phase with these intrinsic  42 oscillations will be more likely to elicit spikes. This increased firing probability should be visible in the summed responses to 30 stimulus presentations making up a PSTH. To investigate this hypothesis, it is not necessary that the stimuli come from separate ears, or that they be presented in pairs (rather than triplets or quartets etc.), or that the amplitude of each one be subthreshold to the generation of spikes when presented alone. Nevertheless, it was decided that the same paradigm used for the lTD sensitivity could be used for a preliminary study (see methods for a description of the lTD series paradigm). The time between stimuli (ITD) was varied over a larger range in order to test for oscillations as low as 100 Hz. This value of 100 Hz (corresponding to a period of 10 ms) was chosen because it was the most common frequency expressed by type 3 cells (described below, and see figure 5). Thus a range of ±5 ms lTD was needed. The results shown in figure 3 suggest that no periodicity was present in the probability of spike firing over the time intervals studied. These results do not rule out the possibility that these cells possess oscillating potentials. The parameters listed above in particular the stimulus ,  amplitude, may have been inappropriate for demonstrating the desired response. In order to properly characterize the response properties, these experiments should be repeated with different parameters. In particular, the stimulus amplitude should be varied, and include pairs of stimuli which are suprathreshold when presented alone, to test the hypothesis that the stimuli used in the present study were not sufficiently intense to evoke a response. Monaural stimuli should also be applied, because it cannot be assumed that the signals originating at the two cochlear nerves exert the same effect on this cell type. The number of stimulus presentations at each lTD should be increased above 30, and smaller lTD steps used to increase the resolution. Unfortunately, it was generally not possible to maintain the cell isolation long enough to carry out all of these measurements. The large number of these cells suggests that they play a prominent role in the auditory pathway. A total of 21 were examined in this study, but many more were isolated. It is estimated that this may be the most common cell type in the ICC. The role that these cells play in auditory signal processing could not be determined in the present study.  43 IV.  Type 3 cells:  A.  Spontaneous oscillations: These cells exhibited a robust oscillatory activity at higher frequencies than most  oscillating neurons reported in the literature. The spontaneous activity of type 3 cells exhibited preferred interspike intervals, as illustrated in the interval histogram (fig. 4). For some type 3 cells, only a single peak was visible in the IH (fig. 4a). In contrast to the IH of a type 1 cell (fig. 1), the peak shown in figure 4a is much taller relative to its width at the base, and does not approximate a Poisson distribution. The firing pattern of this cell is similar to that of a metronome, which never skips a beat. Expressed another way, the ‘rate’ of this cell, given by the total number of spikes averaged over the total time (67 spikes/s), was equal to its preferred ‘frequency’, given by the inverse of the interspike interval (67 Hz). The firing rate of type 3 cells was not always equal to the preferred frequency. More commonly, these cells tended to ‘skip beats’, implying that they did not fire with every cycle of oscillation. This resulted in multimodal interval histograms in which the peaks were equally spaced along the time axis (fig. 4b and c). The cell illustrated in figure 4b expressed an intrinsic period of 8.5 ms, which corresponds to a frequency of 118 Hz. The tallest peak of an interval histogram indicates the most common interspike interval, which for this cell was also 8.5 ms. The existence of a peak at 17 ms indicates that this cell sometimes skipped one cycle, and the small peak at 25.5 ms indicates that it rarely skipped two cycles. The firing pattern of the cell illustrated in figure 4c was much more complex. The large number of peaks indicates a wide variety of interspike intervals. In this case, the tallest peak was located at 25.5 ms, corresponding to the most common interspike interval. The inherent frequency is given by the time to the first peak or the intermodal interval, which for this cell was 8.5 ms, corresponding to 118 Hz. Although each type 3 cell exhibited a single robust frequency in its spontaneous activity, the population of type 3 cells isolated (26 in total) covered a wide range of frequencies. Figure 5 is an histogram illustrating the distribution of frequencies. The equipment was set up to detect frequencies down to 6.25 Hz, but this was not thought to represent a serious limitation, as the lowest frequency observed was 67 Hz.  44 We predicted the existence of inherently oscillating cells to assist the fine time resolution observed in psychophysical studies of directional hearing, and to assist in spectral processing by providing a multiple-band frequency filter. Membrane potential oscillations at auditory frequencies in cells of the NL, in concert with convergence of many phase-locked inputs, could improve the resolution provided by traditional neural mechanisms such as spike trains and EPSPs. A requirement arising from this hypothesis is that the entire frequency spectrum for which timing information is desired be represented by inherent oscillations. For the chicken, phase-locking in auditory nerve fibres is found up to at least 2 kHz (Warchol and Dallos, 1989). With a wavelength of 17 cm, sounds of this frequency are unlikely to produce much of an I]D, and therefore require fine time information to permit accurate localization. Unfortunately, out of 26 oscillating neurons isolated, none exhibited inherent frequencies higher than 250 Hz. Preliminary studies found higher inherent frequencies of up to 840 Hz (Schwarz, et al., 1993).  B.  Oscillations in the impulse response: Oscillations of type 3 cells were entrained by impulse stimuli. As a result, the post  stimulus time histograms of type 3 cells were multimodal, with the intermodal interval equal to the inherent frequency demonstrated in the cell’s IH. Figure 6 shows a PSTH from a type 3 cell. Spikes occurred at 15 ms intervals following each stimulus, corresponding to a frequency of 67Hz. Entrainment of this cell to the stimulus is indicated by the relatively constant latency to the first response following 200 stimuli. The interval histogram for the same cell is shown in figure 4a (see section IV.A.). The intermodal interval is identical in the two histograms, demonstrating that this cell expressed the same inherent frequency both spontaneously and in response to impulse stimuli. Increasing the amplitude of the stimulus had no effect on the intermodal interval (results not shown). These findings demonstrate that the oscillatory activity of type 3 cells is robust. The same inherent frequency found in the Ill (see section IV.A.) is also seen in the PSTH and is independent of stimulus amplitude. Support for the hypothesis that these cells are able to extract a  45 specific frequency from a pulse train requires the demonstration of a preferred frequency in a random pulse sequence input.  C.  Response to a random pulse sequence: Two type 3 cells were isolated which had spike amplitudes greater than the stimulus  artifact and were maintained at the electrode tip long enough to perform a reverse correlation histogram. The RCH for these cells was multimodal, and displayed the same intermodal interval as was shown in the cells’ N and PSTH (fig. 7). In contrast to the PSTH and N, in which the intermodal interval represents a commonly occurring interspike interval, the intermodal interval of a RCH represents a preferred inter-stimulus interval, and the absolute value of the position of each mode on the x-axis represents a commonly occurring response latency. Type 3 cells preferred a frequency in the stimulus input that was equal to the spontaneous and entrained inherent frequency of the cell. This property could enable these cells to signal the existence of specific frequencies in a complex input. With a population of such neurons having different inherent frequencies, a complex wave input could be filtered into its component frequencies by a running “Fourier transform” similar to that resulting from the auditory filters (see introduction). A use for such a filter has previously been proposed in a theoretical model put forth by Srulovicz and Goldstein (1983), although the range of the results presented here is not sufficient to support the model proposed.  D.  Frequency preferences: mechanisms and implications The mechanism underlying the inherent frequency preference cannot be determined from  the results of this extracellular recording study, but there are only a limited number of likely possibilities. These cells may possess inherently resonant membrane potentials. Given the proper combination of ion channels and external conditions (i.e. presence or absence of transmitters or modulators), the membrane potential of these cells could resonate within a very narrow frequency range, and the constant barrage of inputs generated spontaneously from lower centres could disturb the potential enough to maintain oscillations at the resonant frequency. The  46 extent of spontaneous activity in the auditory pathway would certainly be sufficient. Alternatively, the oscillations may persist in the absence of synaptic input, once again due to the particular properties of these neurons. Intracellular recordings from these cells could solve many of these mysteries. Another possibility altogether is that these cells are merely reproducing spike train activity received from lower centres, notably the nucleus laminaris. This possibility is supported by the finding that the NL generates an oscillating neurophonic potential in response to tonal stimuli (Schwarz, 1992), and that this nucleus sends projections directly to the ICCc (Takahashi and Konishi, 1988, Wagner, et aL, 1987, Conlee and Parks, 1986), the location of the cells reported here (figs. 8 and 9, and see below). While it seems clear that these cells could serve as a frequency filter within the CNS, we have not demonstrated that they filter coded sound frequencies. Their PSTHs (fig. 6) resemble those of chopper cells reported in the manmialian cochlear nucleus (CN, Pfeiffer, 1966, Banks and Sachs, 1991), with two important differences. Firstly, the chopping frequency of cells in the CN is independent of stimulus frequency, and may change with stimulus intensity (Oertel, et al., 1988, Young, et al., 1988). In contrast to choppers, the ICC cells reported here oscillate at relatively fixed frequencies which are stable with changing stimulus intensity. Secondly, the chopper response persists only as long as the stimulus, whereas ICC cells exhibit a steady-state oscillating background activity which is reset by an impulse (figs. 4 and 6). In addition to the differences in the response to stimuli, there is another important difference between type 3 cells of the ICC and chopper cells of the cochlear nucleus: chopper cells show no or little irregular spontaneous activity, in contrast to the regular spontaneous firing of type 3 cells. For these reasons, we have avoided applying the term “chopper” when describing the responses of these cells. It would be useful to establish whether this cell type possesses any sensitivity to ITDs. A combination of a simple spike count over the entire histogram period and a measure of sharpness of the 111 or PSTH peaks (vector strength) could provide a parameter for comparison at different ITDs. It should be noted that almost all of the type 3 cells isolated were responsive only to  47 stimulation of the contralateral electrode, discouraging any lTD studies. The significance of this contralateral selectivity, if any, is not known. The histological determination of the location of type 3 cells shows clearly that their distribution is restricted to the ICCc. Figures 8 and 9, taken from two different animals, and additional sections from two other animals (not shown), all point to a relatively small region rostro-ventrally in the medial ICC. An interesting follow-up to this would be to combine HRP labeling of oscillating neurons with anterograde tracing from the NL, particularly from the contralateral side. The NL receives excitatory inputs from both ears, and has been shown to project primarily to the medial aspect of the ICC (Conlee and Parks, 1986). Given that the type 3 cells characterized in the present study were responsive solely to contralateral stimulation, it is likely that type 3 cells do not mediate the output of the NL.  V.  Conclusions: The ICC contains at least 3 types of neurons based upon their responses to electrical pulse  stimuli of the auditory nerve. One of these exhibits spontaneous oscillations at auditory frequencies that are robust. These oscillations persist at the same frequency in response to stimuli, and as a result are able to extract frequency-specific information from a random input (noise). This supports our hypothesis that neurons in the central auditory pathway possess inherent resonances at auditory frequencies. Type 3 cells differ from NL neurons in their connectivity with the ears, however. Their frequency selectivity can, therefore, not explain the interaural time selectivity and its fine resolution in directional hearing. A large degree of inhibition is evident in the firing patterns of the other cell types. This phenomenon deserves further study to establish the cause and behaviourailfunctional significance. The findings presented here (and published previously by Schwarz, et al., 1993) demonstrate the first extracellular evidence for inherent oscillations in the central auditory pathway. The oscillatory frequencies reported for the type 3 cells are one to two orders of magnitude higher than those reported in the mammalian thalamus, suggesting that the cellular  48 mechanisms underlying the oscillations in these two systems are different. 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