Sensory representation in the cerebellum of the catfish

Sensory representation in the cerebellum of the catfish

0306-4522184 $3.00 f 0.00 Pergamon Press L&d 1984 IBRO ~euros~je~re Vol. 13, No. I. pp. 157-169, 1984 Printed in Great Britain SENSORY REPRESENTATI...
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0306-4522184 $3.00 f 0.00 Pergamon Press L&d 1984 IBRO

~euros~je~re Vol. 13, No. I. pp. 157-169, 1984 Printed in Great Britain

SENSORY

REPRESENTATION IN THE CEREBELLUM THE CATFISH L. T.

LEE

and T. II.

OF

BULLOCK

Neurobiology Unit, Scripps Institution of Oceanography and Department of Neurosciences, School of Medicine, University of California, San Diego, La Jolla, CA 92093, U.S.A. representation in the catfish cerebellum was studied with physiological stimuli, with electric shocks applied to peripheral nerves and with electric shocks to central structures of the brain. Evoked field potentials and unit responses were recorded in different places in the cerebellum. In the catfish, different sensory modalities are represented by discrete, only partly overlapping areas of the cerebellum. Most units are unimodal by present criteria; only in the valvula a fraction of the units are responsive to two or more modalities, Visual and somatosensory areas are the largest and they occupy the bulk of the corpus cerebelli and the valvula. In the corpus, most of the visual units are near the midline and in the dorsal tier while the somatosensory units are more lateral and ventral. Mechanical lateral line input is represented in the eminentia granularis and the valvula. Acoustic units are found in the valvula. Electroreceptive units are recorded from the lateral lobus caudalis, and to a lesser degree, from the eminentia granularis and valvula. Sinusoidal tilting and vibration units are in the lobus caudalis pars medialis. Receptive fields of units, regardless of modality, are generally large and diffuse. Some visual units respond best to moving objects. Topographical organization of receptive fields only exists among the somatosensory units. Resides these findings of modality segregation, the features of interest for comparative neurology are the following. Most units are identifiable as Purkinje cells with a characteristic mossy fiber-granular cell pattern, and but in contrast to most experience with mammals, in response to direct brain or nerve stimulations, the simple spikes have different dynamic responses for different modalities. Some are first excited, then inhibited, others vice versa. Some units are not responsive to any sensory input we delivered, Some units not meeting the criteria for Purkinje cells meet several criteria for eurydendroid cells; they give large spikes and are influenced by sensory stimuli after relative long latencies. Complex spikes, however, were only consistently observed in some of the visual units. Abstract-Sensory

The

cerebellum of teleosts can be divided into three major parts, the vestibulolateral lobe, the corpus cerebelli and the valvula.‘s~22While the fine structure of the cerebellar cortex is consistent throughout the vertebrates,28 the cerebellum of fish has the greatest range of structural variation to be found in any class.39 From time to time, speculations have been made about the relationship between the relative size of the cerebellum and its component structures and the general behavioral characteristics of various fishes.‘as32 While the actual role of the cerebellum, in fish or in other vertebrates is still the subject of conjecture, it appears to function as an integration center where sensory information from various inputs is processed and relayed to the motor centers.8~13~‘6~‘8+23 Whereas something is known about the cerebellar responses to electroreceptive input in fishes,2,3.4,38.44 much less is known about inputs from other sensory modalities. The sensory inputs to the different component parts of the cerebellum is also not known. Some classical and recent anatomical studies do indicate that different sensory inputs terminate in different parts of the cerebellum.22.3s This is in contrast with the mammalian cerebellum where some studies, using field potentials’8,26.4’ or unitary responses,‘~‘3,30~37have shown that inputs from different sensory modalities terminate and interact in the cerebellum.

This study is therefore aimed to investigate the sensory representation of the different component structures of the catfish cerebellum. Electrical shocks applied to the peripheral nerves and to central structures were employed to provide synchronized activation and produce a coarse-grained map of sensory representation in the cerebellum. The sensory specificities, receptive fields and dynamic characteristics of single units and multiple units were then investigated by more physiological stimuli belonging to different sensory modalities, presented simultaneously or in sequence. A similar study has been carried out in the elasmobranch in this laboratory;45 a preliminary report of the catfish study has been published.47 In this study, evoked potentials and unit spikes will be recorded in an attempt to determine whether different sensory inputs occupy different parts of the cerebellum and whether the cerebellar units in catfish verteother as in some are multimodal, brates , 1.5.13.18.26.30~37~4Iare or unimodal.

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EXPERIMENTAL

PROCEDURES

material

Catfish (Ictaluvus ~e~~~~~~} ranging in length from 19 to 22 cm were obtained from a fish farm and maintained at 22°C. Ammonia and nitrite as well as temperature were regulated in the laboratory aquaria. The fish was lightly anesthetized either with tricaine methansulfonate (MS 222) (Argent Chemical Laboratory Inc.. Redmond, VA) or by

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chilling to CCI I C. It was then immobilized with 0.1 mg tubocurarine chloride, injected intramuscularly. Administration of MS 222 was terminated following surgery, while the level of curarization was maintained through repeated injections. The fish was then mounted in a plastic head holder and positioned in a tank of aerated water with the water line just below the exposed skull. Artificial respiration was provided by flushing the gills with a stream of aerated water through the mouth. The brain was exposed dorsally from the cerebrum to the level of the lateral line lobe. To permit direct stimulation, the spinal cord was exposed for a few segments anterior to the dorsal fin and the posterior lateral line nerve was exposed behind the gill cover. Wound margins were infiltrated with lidocaine (Xylocaine Eli Lilly, Indianapolis, IN). In some of the experiments, catfish Ringer’s was used to cover the exposed brain; this was not always necessary because of adequate cerebrospinal fluid. The vitality of the animal was monitored qualitatively by observing the blood flow in vessels on the cerebellar surface. Preparations can be maintained in good condition for as long as 24 h. Stimulalion For direct brain and cranial nerve stimulation, electrical pulses of 0.01&0.3 ms duration and from a few volts to several tens of volts in amplitude at a rate of 0.5-20 pulses/s were delivered to the spinal cord and to the medial lateral line lobe through a pair of tungsten electrodes with their tips separated by 200 pm or less. and to the optic tract through a pair of hooked wire electrodes, insulated except at the ends, Stimulation current, when monitored, was usually less than 20pA. Visual stimuli were provided physiologically by (1) a stroboscopic flash directed to one eye of the fish through a light pipe and (2) a moving white plastic disc 1cm in diameter or rectangular black plastic strips, IO cm in length with widths of 2-8 mm placed several centimeters away

from the eye of the fish. The moving objects were driven back and forth along the long axis of the fish by a servo-mechanism and a function generator. Both (I) and (2) were triggered by the stimulus generator. The best distance from the eye was determined by trial and error. Manually moving visual stimuli. difficult to quantify, were provided by a flash light or a small object displaced across the visual field of the fish. Tactile and proprioceptive inputs were provided manually by touching the skin with a soft brush and by moving the barbels, fins and tail of the fish. Mechanical receptors of the lateral line system were stimulated by water currents injected through a syringe whose piston was servo-controlled by a function generator. Another mechanical stimulus was provided by drops of water from a pipette of known height above the water. falling 6-20cms from the fish. Electroreceptors were stimulated by homogeneous longitudinal or transverse electric current delivered, through a stimulus isolation unit and high series resistance. to one of two pairs of 25 cm long carbon rods at the ends and the sides of the tank. Two kinds of vestibular stimuli were used. (1) A servocontrolled, rubber covered plastic rod tapped on the side of the experimental tdnk and introduced vibrations of cd 50-60 Hz of a much higher intensity than normal acousttc stimuli. (2) Angular acceleration was accomplished by placing the fish out of water on a board which moved up and down sinusoi~ally about the long axis of the fish at 0. i-O.7 Hz and up to 30 amplitude.‘h Acoustic stimulation was provided by clicks. A square pulse of I ms or a single sine wave of I ms duration was delivered to a speaker in the air 30cm above the fish. The sound pressure under water. monitored by hydrophone. was usually 15-30dB rc I pbbnr. All units were tested with ali the ditferent sensory modalities. simul~neously or seouentially.

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Fig. 1. Left: dorsal view of the catfish cerebellum. Right: parasagittal view of the cattish cerebellum cut at the plane indicated by the straight line in the dorsal view. showing the distribution of the different sensory areas in the cerebellum. Clear areas at sides and caudal boundary of corpus are zones of mainly molecular layer, with few large cells. Dotted area in parasagittal section is granule cell layer, with central zone of nerve fibers. Dashed lines mark boundaries of corpus cerebelli with lobus caudalis and valvula. A, acoustic; E, etectroreceptive; M, lateral line mechanoreceptive; s, somatosensory: v, visual; Ve. vestibular; CC, corpus cerebellum; EG, eminentia granularis; LC, lobus caudalis; OT. optic tectum: pf, posterolateral fissure; TL. torus longitudinalis; V. ventricle; VC, valvula cerebehi.

Catfish cerebellar sensory representation Recordings Evoked potentials and unit activities were recorded with either a tungsten electrode, tapered and insulated to the tip (3-12 MR, measured at 22 Hz) or saline-filled glass pipettes (resistance 3-15 MQ) inserted in various places and at different depths in the cerebellum. The reference electrode was placed at the wound margin or in the water bath. Identification of the part of the cerebellum from which the recordings were taken depended primarily on recognizing visual landmarks over the cerebellar surface and measuring the depth of the recording electrode. The transverse plate (lobus caudalis) is delimited by the posterolateral fissure and is distinguished by its white coloration (Fig. I). Eminentia granularis appears as a slight bulge at the posteroiateral part of the cerebellum. Exact boundaries between corpus cerebelli. valvula, and vestibufolateral lobe cannot be drawn and fine subdivisions have not been made. Recording depths refer to readings from the micrometer of the micromanipulator; they were checked noting the spontaneous activity of the cerebellum, which is different for the three laminae of the cortex (L. Lee, submitted for publication). Electrolytic lesions were made in several animals, and they were subsequently sacrificed and their brains processed. Agreement between the inferred and the actual recording sites was always good. Conventional equipment was employed for impedance matching and amplification. Evoked held potentials were band pass filtered between 1 Hz and 1 kHz, and led to a digital averager. Averaging was done for 8-64 consecutive responses. Trains of stimuli were given at different rates with ample recovery time (20 s-5 min) between trains. Spikes of single units or a small number of units were band pass filtered between 3 Hz and 3 kHz and were used either as raw waveforms on single sweep or 4-20 sweeps superposed, or as triggered pulses for the construction of poststimulus time histograms.

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publication), but in brief, simple spikes of the Purkinje cells are identified by their “on beam“ excitatory and “off beam“ inhibitory responses following parallel fiber stimulation, by their spontaneous rate which is between 10 and lOO,%, and by the recording depths which correspond to the histological sites of Purkinje cells. Discharges of the eurydendroid cells are recognized by their spontaneous rate (0.3-0.5/s), by the recording depths, by the long duration (about 20ms) of the spikes, and by their responses to peduncle and to natural stimulations which are of longer latencies and opposite to those of the Purkinje cells (Fig. 5G,H). “Complex” spike is readily recognized by its peculiar configuration (one to three spikes followed by or riding on a slow potential wave); they also have relatively low spontaneous rate (1-2/s). Of all the units recorded, 284 Purkinje cells with simple spike only, 8 eurydendroid cells and 18 units with “complex” spikes were found to be unimodal, that is, the units responded only to one sensory modality; other modalities neither excited nor inhibited them. Units specific to different modalities are located in different parts of the cerebellum with little overlap, except in the valvula where a small portion of multimodal units are located. The distribution of evoked field potentials following direct electrical stimulation applied to the spinal cord, posterior lateral line nerve, optic tract and medial nucleus of the lateral line lobe are in concordance with the distribution of the units.

RESULTS

The corpus cerebellum of rcruI~rMs ne~u~us~~ is large and elongated, it has a distinguishable upper and lower cortex (dorsal and ventral tier or dorsal and ventral corpus), continuous at the sides and anteriorly (Fig. 1). Posteriorly the corpus is continuous with the lobus caudalis. The relatively simple but large valvula, forms an anterior projection from the base of the corpus. The vestibulolateral lobe consists of the eminentia granularis and the lobus caudalis;3’ the latter is equivalent to the transverse plate of Larsell.” The eminentia granularis is a large cluster of granule cells dorsolateral to the caudal margin of the granular layer of the corpus cerebelli. Lobus caudalis borders the fourth ventricle and has two parts. Its pars medialis consists of a fibro-molecular layer, an inter-auricular band and a deep layer of large cells. The pars iateralis, or the auricle according to Larsell,Z2 borders the pars medialis laterally. Sensory representation

in the cerebellum

Spike responses were recorded from 304 Purkinje cells with simple spikes only, 18 units with “complex” spikes and 44 eurydendroid cells (large cells below the Purkinje cells believed to represent the deep cerebellar nuclei in the teleosts’2.35). Classification of units is to be reported in another paper (L. Lee, submitted for

Visual area The visual area includes most of the medial corpus cerebelli and the valvula (Fig. 1). In response to a brief shock applied to the optic tract, two positive waves with peak latencies of about 12 and 38 ms are observed in the molecular layer of the dorsal corpus. In the granular layer, they are replaced by a larger negative wave peaking at about 30ms and a small spike-like potential with a latency of about 5 ms. At still deeper levels (1.2-1.5 mm), the larger granular negativity gives way to a deep positive wave with a peak latency 3-5 ms shorter (Fig. 2A). The surface positive wave and the large granular negative wave are not the result of volume conduction from the tectum; the latencies and the configurations are different from the evoked field potentials recorded from the optic tectum following optic tract stimulation (ref. 48 and L. Lee, submitted for publication). The cerebellar waves are re!atively restricted in distribution. The deep positive wave reaches maximal amplitude in loci well beyond the border of the cerebellum; it therefore may represent extracerebellar activity.@ The best response is recorded from sites near the middle of the longitudinal extent of the corpus and about 0.3-OSmm lateral to the midline. The amplitude of the response attenuates rapidly anteriorly and laterally. Little or no response is recorded from the

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corpus ipsilateral to the optic tract. A true valvular field potential was not observed; it is probably obscured by the extracerebellar deep positive wave. One hundred and six units were sensitive to flash or moving visual stimuli. Of these visual units, 94 were from loci near the midline of the corpus. More than 80% of these were less than 0.9 mm deep. The dorsal medial part of the corpus, therefore, appears to be the major target of visual input. The remaining 12 units were found in the valvula. Ninety of the visual units were Purkinje cells with simple spikes only. These units respond to optic tract stimulation with a consistent pattern of inhibition at a latency of about 3640 ms (about 5-9 ms longer

A

.M

*O”

than the granular negativity of the slow potential response) and with a duration of about 50 ms. This may or may not be followed by postinhibitory excitation (Fig. 2A). The responses of these units to flash are more variable. The slow firing cells (n = 28) with spontaneous rates of 15/s or lower respond to a brief flash with either one, two, or three spikes. The spikes, if more than one, are always separated by at least 50ms. The initial synchronization is followed by a relatively long period of inhibition which may last hundreds of milliseconds. The first spike of these units has a latency of 35-45 ms (Fig. 2C). They can follow flashes presented at rates up to 6/s with at least

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Fig. 2. Responses to visual stimulation recorded in medial corpus. (A) Slow field potential and unit responses to optic tract stimulation. The depths of the field potential responses are indicated at the right in pm. Each trace is an average of 8 sweeps. Unit response is represented by the poststimulus time histogram. Number of repetitions, 32; bin width, 5 ms. (B) Complex spikes in a Purkinje cell, elicited by flash (arrow). The first four traces represent four consecutive trials. The lower two traces are poststimulus time histograms of the same unit. Note that the response has a latency of about 400 ms after the stimulus. Number of repetitions, 64; bin width, 1 ms. (C) Mossy fiber activation of Purkinje cell in medial corpus showing differential responses to flash delivered to one eye of the fish through a light pipe from different directions. Each trace on the left represents a single response after the flash is delivered from the direction indicated. Traces on the right are the corresponding poststimulus time histograms. Number of repetitions, 32; bin width, 2 ms (D) Simple spikes inhibited by flash; single sweep. (E) Poststimulus time histogram of another Purkinje cell with simple spikes inhibited by flash showing two periods of inhibition. Number of repetitions, 16; bin width, I ms. (F) Lower two traces, poststimulus time histograms showing the response of a direction selective unit to a moving visual stimulus (pattern of 8 mm black and white stripes, translated parallel to the long axis of the fish, 3 cm lateral to it). T, movement caudalward; H, movement rostralward. Number of repetitions, 2; bin width, 6ms.

Catfish cerebellar sensory representation one spike. At higher rates, the spikes are less synchronized and have longer latencies. The rest of the Purkinje cells with simple spikes (n = 62) responded to a brief flash with a complex pattern of inhibition and excitation. Initial inhibition of unit activity takes place with an average latency of 80 ms after the flash and lasts for 60-100 ms (Fig. 2D). The initial depression is followed by postinhibitory excitation. When flashes are presented repetitively at the rate of 4/s, the postinhibitory excitation phase of the response remains large while the initial depression disappears. In some of the units, the first postinhibitory excitation is followed by a second period of inhibition, which lasts for about 60 ms and has an average latency of 180 ms (Fig. 2E). “Complex” spikes were observed in 16 cells and their discharges were time locked to flash with an average latency of 134 ms (range 80-400 ms) without any increase in the average discharge rate (Fig. 2B). Stimulus following becomes irregular at rates higher than 2/s. The receptive fields of the visual units are in general large. The majority of them have a visual field of about 20-50” in diameter; some have no restrictive receptive fields. Those with receptive field centers anterior and lateral to the fish usually have smaller receptive fields. In general, cells of neighboring areas have a large part of their receptive fields overlapping. In many of the units, the best responses are elicited when the light source is from the front or from the side (Fig. 2C). The data from this study do not reveal a retinotopic organization of the visual input into the corpus cerebelli. The flash sensitive units also respond, and more vigorously in many units, to a small light source or object moving at velocity of about l-6O”/s. Best velocities, as evaluated by the mean discharge rate, in Purkinje cells are between 10 and 3O”/s. Directional sensitivity was found in 12 (4 with “complex” spike and 8 with simple spike) of the 32 investigated units. A small object moving in one direction may excite these units while the same object moving in the opposite direction may cause a smaller excitatory response (Fig. 2F), no increase above the spontaneous rate or inhibition.

Tactile-proprioceptive area Field potential responses can be recorded from the corpus and from the valvula, but not from the lobus caudalis, following a brief shock applied to the spinal cord. The response begins as a fast spike-like wave peaking at about 4ms. The polarity remains unchanged and the amplitude remains more or less the same at all depths of the corpus. This fast potential is followed by a larger negative wave with a peak latency of about 9ms. This slower negative wave reaches maximal amplitude in the granular layer. In the molecular layer, there is a positive wave peaking at about 15 ms (Fig. 3A).

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A total of 125 Purkinje cells with simple spike only in the corpus and the valvula, were found sensitive to light touch applied to the skin of the fish and to proprioceptive stimuli (indentation of body surface; movements of the barbels, fins, or tail). “Complex” spikes were only found in two units; they followed spinal cord stimulation with a latency of about 15 ms. Seventeen of the 73 proprioceptive units and 10 of the 42 tactile units were recorded from the valvula. The somatosensory units are distributed laterally in the corpus cerebelli. Except in the anterior corpus, most of them are located in the ventral fold of the cortex deeper than 0.8 mm. The response of these units following spinal cord stimulation consists of an initial excitatory phase, followed by a period of inhibition and by still another phase of postinhibitory excitation (Fig. 3B,C). The initial excitation has a minimal latency of about 5 ms and lasts for about 25 ms. The inhibition occupies the following 3&50 ms. The final phase can be as long as 300 ms. The initial excitatory and the subsequent inhibitory phases are much attenuated at repetition rates higher than 4/s. With physiological stimuli, 70% of these units responded with the activation or acceleration of spike discharges (Fig. 3D,E,F). In those cells with lower rates of spontaneous discharge, light touch applied to the body surface or the barbels elicited only a brief burst of one to three widely separated spikes (Fig. 3D). The other units responded with more closely spaced spike discharges (Fig. 3E,F). Fourteen per cent of the somatosensory units were inhibited by the stimulation. The remaining 16%, all of them proprioceptive, responded bidirectionally. Their spike discharges were accelerated when the fin or barbel was moved in one direction and depressed when its was moved in the opposite direction (Fig. 3G). Most of the proprioceptive units appear to have tonic responses. Inhibition or excitation of spike discharges in these units can be as long as 5 s while the position of the fins or barbels is maintained. Cells of different response character do not distribute themselves in a recognizable pattern on the cortex. Often, cells with similar, or even the same receptive field but different forms of response are observed along the same electrode tract perpendicular to the cerebellar surface. The receptive fields of the somatosensory units are different. Some units are bilateral, others unilateral. The receptive fields of the proprioceptive units are more well defined in size, of the tactile units more diffuse. Among the tactile units, the receptive fields of those cells sensitive to light touch applied to the barbels have the smallest receptive fields and those sensitive to light touch or brushing applied to the body or tail have the largest receptive fields, some as large as the whole caudal half of the body. A rough somatotopic organization, however, can be recognized. At successive recording sites from the anterior part of the dorsal fold of the corpus around the rostra1 pole to the anterior part of the ventral corpus

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Fig. 3. Responses to somatosensory stimulation recorded in mid-corpus. (A) Slow field potential responses to single shocks to the spinal cord. The depths of the field potential responses are indicated at the right in pm. Each trace is an average of 8 sweeps. (B)-(C) Purkinje cell response to spinal cord stimulation (arrow). (C) represents the corresponding poststimulus time histogram. Number of repetitions, 32; bin width, O.Sms. (D) Response of a slow discharging Purkinje cell to a light touch applied to the largest barbel. (E) Effect of bending the largest barbel on a Purkinje cell with higher rate of spontaneous discharge. (F) Acceleration of spike discharge to brushing the body surface gently. The duration of the stimulus is indicated by the black bar underneath the trace. (G) Spontaneous discharge of a unit (first and third traces) accelerated or depressed by extending the pectoral fin outward (second trace) or toward the body (fourth trace). The effect lasts as long as the position of the fin is maintained.

and then to the more posterior part of the ventral corpus, Purkinje cells sensitive to stimuli applied to the barbels, head region, body and tail of the fish, respectively, are encountered with increased probability. Units in the valvula are primarily sensitive to tactile and proprioceptive stimulation applied to the barbels, pectoral fins and the head region of the fish. Vibration and tilting A band of large cells, presumably Purkinje cells, caudal to the posterolateral fissure and about 0.5-1.50 mm below the surface, respond to tilting of the fish or to vibration. Although the rates of spontaneous discharge (ca 60/s) of these cells are not different from units located in the other parts of the cerebellum, their spontaneous activity is characterized by a higher degree of regularity (Fig. 4A,B). Several cells with only simple spikes were excited by tilting of the fish, either head up or head down, that is without directional selectivity (Fig. 4C). Tilting produces no responses in the nearby eminentia granularis or in other parts of the cerebellum. Most of the vibratory units in the lobus caudalis pars medialis have low spontaneous discharge rates.

In response to a tap applied to one side of the tank, these units give a burst of three to six regularly spaced spikes (Fig. 4D). With repeated taps, the latencies of these spikes show only a small dispersion (Fig. 4E). The number of spikes after each stimulus, however, is not constant (Fig. 4E) and to a certain extent, is graded with stimulus intensity. Another part of the cerebellum with units responding to vibration is the valvula; these units, in addition, respond to sound. Acoustic

input

Although evoked potentials to clicks are readily recorded from the deeper part of the lobus caudalis pars medialis, from the eminentia granularis, from the valvula and from the lateral part of the corpus cerebelli, comparisons with the evoked potentials recorded from the brain stem reveal that they have the same configurations and latencies. The potentials recorded in the cerebellum are smaller in amplitude and thus far we have seen no unitary discharges associated with them. They are, therefore, most likely be volume conducted from the deeply located anterior nucleus of the octavolateral lobe of the medulla (about 0.5 mm beyond the boundary of the lobus

Catfish cerebellar sensory representation

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Fig. 4. (A), (B). Spontaneous activity of units recorded from lobus caudalis pars medialis showing regularly discharging type of unit. (C) Single unit, presumably vestibular, from lobus caudalis pars medialis showing acceleration with tilting regardless of direction. Upper trace is the poststimulus time histogram. Number of repetitions, 16; bin width, 18 ms. The lower trace indicates the position of the fish. HD, head down; HU, head up. (D) Unit discharges (upper trace) following a light tap applied to the side of the experimental tank. The vibration of the tank (lower trace) was monitored by a contact microphone. (E) Poststimulus time histogram of another unit following the same stimulation. Number of repetitions, 16; bin width, 0.5ms.

caudalis pars medialis) and from the acoustic portions of the midbrain.” No acoustic units were found in the eminentia granularis. Units sensitive to acoustic stimuli were only found in the valvula. These respond to a click with a brief initial excitation beginning at about 30 ms, followed by a period of inhibition lasting for about 100 ms. As expected these units also respond to vibration, induced in the water by a light tap applied to the tank with a similar response; such a tap includes a relatively intense acoustic component.

Mechanoreceptive

lateral line area

The lateral line nerves, anterior and posterior, convey both electroreceptive and mechanoreceptive inputs. Only the posterior lateral line nerve was stimulated in this study because of the surgical difficulties of reaching the anterior lateral line nerve. The cerebellar representation of the mechanoreceptors from the anterior half of the body was investigated by electrical stimulation applied to the medial nucleus of the lateral line lobe which is the primary relay center of the mechanoreceptive input3’ Electrical stimulation applied to the posterior lateral line nerve elicits an early triphasic spike-like activity with a peak latency of about 4-6ms in the

molecular layer overlying the eminentia granularis. It is followed by a slow positive wave peaking at about 25 ms. The lateral eminentia granularis is dominated by a large negative wave with an onset latency of about 16ms and peak latency of about 25ms followed by a positive wave peaking at about 75 ms (Fig. 5B). The fast triphasic wave and the surface positive wave can be recorded from almost the whole cerebellum but are largest in the eminentia granularis and the lobus caudalis pars lateralis. The iate negative wave, however, is seen only in the lateral part of the ipsilateral eminentia granularis. Direct electrical stimulation applied to the medial nucleus of the lateral line lobe gives rise to field potential responses in the ipsilateral and contralateral eminentia granularis (lateral part) and in addition, in the valvula (Fig. SC,D,E). The field potential response in each recording region consists mainly of a fast positive-negative spike-like wave followed by a larger negative wave with maximal amplitude in the granular layer. Recordings from the ipsilateral eminentia granularis in addition showed a preceding negative wave peaking at about 1 ms. This negative wave, however, is probably not cerebellum generated since it reaches maximal amplitude at a depth beyond the eminentia granularis. A spike-like positivenegative wave with the positive wave peaking at

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about 8 ms is also observed in the fibro-molecular layer of the transverse plate. Thus it appears that the mechanoreceptive lateral line input distributes to the lateral eminentia granularis and the valvula. The spike-like activity in the fibro-molecular layer of the transverse plate is indicative of fibers of passage. Furthermore, the valvula does not receive direct afferent fibers arising from the tail and body, conveyed by the posterior lateral line nerve. A total of 52 units, 26 in the lateral eminentia granularis and 26 in the valvula, were found to be sensitive to mechanoreceptive input of the lateral line. Of these, 44 were identified as Purkinje cells with simple spikes. These units also responded to direct stimulation applied either to the posterior lateral line

nerve or the medial nucleus of the lateral line lobe with acceleration followed by depression, sometimes with another phase of excitation. The responses of the mechanoreceptive units to physiological stimuli can be divided into two categories. Those with higher rates of spontaneous discharge are depressed (Fig. 5G,H; upper traces) but this is not obvious when they are stimulated repetitively at rates higher than 2/s. Units of lower spontaneous rates are excited; a single stimulus may elicit one or several bursts of activity depending on the intensity (Fig. 5D). This group of units follows repetitive stimulation at rates as high as S/s. Eight eurydendroid cells were recorded from the lateral eminentia granularis. These units respond to either natural stimuli or direct brain stimulation, with

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Fig. 5. Response to lateral line input. (A) Cross-section of the cerebellum showing the eminentia granularis. The stars indicate the recording sites of the field potentials shown in (B). (B) Slow field potential responses to posterior lateral line nerve stimulation. The depths of the field potential responses are indicated at the right in pm. Each trace is an average of 8 sweeps. (C)-(E) Field potential responses following stimulation applied to the medial nucleus of the lateral line lobe. The potentials are recorded from ipsilateral eminentia granularis (C), contralateral eminentia granularis (D) and from valvula (E). (F) Unit response (upper trace.) to water movement recorded from eminentia granularis. Poststimulus time histogram of the same unit is shown in the lower trace. Number of repetitions, 16; bin width, I ms. (G) Effect of water movement (local jet) on two units (second and third traces) recorded from the same tract. Each trace represents the superposition of 10 sweeps. The first trace indicates the onset of the stimulus. (H) Effect of medial nucleus stimulation on two units recorded from the same tract. Each trace represents the superposition of 10 sweeps. The straight line on each trace is the stimulus artifact. (I) Response (represented by the poststimulus time histogram) of a valvular unit to a transverse electric field in the water. Duration of the stimulus is indicated by the black bar underneath. Number of repetitions, 16; bin width, 1ms. Cg, granular layer of the corpus; Cm, molecular layer of the corpus; EC, eminentia granularis; SGN, secondary gustatory nucleus; V, ventricle.

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Catfish cerebellar sensory representation

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E 0

Fig. 6. Multimodal units. (A) Spike discharges of a valvular unit following a brief flash (third trace), medial nucleus stimulation (second trace), or no stimulus (first trace). (B) The corresponding poststimulus time histogram of the same unit. First trace: spontaneous activity. Second trace: after medial nucleus stimulation (small arrow). Third trace: after a brief flash (large arrow). Fourth trace: after medial nucleus stimulation followed by brief flash, at a certain interval. Number of repetitions, 16; bin width, 2.5 ms. (C) Poststimulus time histogram of another unit. First trace: spontaneous activity. Second trace: after spinal cord stimulation (small arrow). Third trace: after a brief flash (large arrow), and simultaneous presentation of spinal cord stimulation and a brief flash (last trace). Number of repetitions, 16; bin width, 0.5 ms.

minimum latencies slightly longer than the onset of the inhibitory phase of the Purkinje cells (usually found along the same tract) that responded to the same stimuli (Fig. 5G,H; lower traces). In the eminentia granularis, the units primarily respond to stimuli applied to the ipsilateral side. In general, the receptive fields of the mechanoreceptive units are large and difficult to delimit. They overlap with one another and do not appear to fall into any recognizable pattern. Units in the valvula, however, have their receptive fields confined to the head region. Electroreceptive input

Electroreceptive input has been studied in detail by Tong and Bullock.44 In agreement with their study, we find electroreceptive input largely terminates in the lobus caudalis pars lateralis and in the lateral eminentia granularis in catfish cerebellum. In addition, four electroreceptive units, Purkinje cells with simple spikes, are found in the valvuia. They had a latency of about 25 ms and were sensitive to the longitudinal electric field (Fig. 5I), either head positive or head negative. MultimodaI units

Thirteen units in five different fish, all of them Purkinje cells with simple spikes only, were found to be multimodal; all of them located in the valvula. Four responded to visual and tactile or proprioceptive inputs (or spinal cord stimulation); four re-

sponded to visual and lateral line mechanosensory inputs (or equivalent electrical stimulation); three responded to tactile or proprioceptive and lateral line mechanoreceptive inputs (or equivalent electrical stimulation); and two responded to vibratory and visual inputs. All of these units can be identified as Purkinje cells and have a relatively high rate of spontaneous activity. The responses of these units, as in the unimodal units, consist of inhibition which may or may not be preceded by excitation (Fig. 6A). To prevent contamination of stimuli, interactions between two sensory inputs were studied with a combination of direct electrical and physiological stimulation. Presentation of two kinds of inputs apparently does not result in facilitation of any kind or show occlusion or alter the way the information is processed by the Purkinje cells. The two responses simply summate (Fig. 6B,C). The receptive field properties of each modality in these units are similar to those of the unimodal units. All of them have their receptive fields confined to the head region of the animal. Units not driven by sensory input

Fourteen units in lobus caudalis pars medialis and 38 units in the corpus and valvula did not respond to any of the stimuli presented. Some of them, especially those recorded from lobus caudalis pars medialis, can be identified as Purkinje cells. The rest of them are eurydendroid cells.

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The results of this study indicate that the cerebellum of catfish receives visual, somatosensory, electroreceptive, lateral line mechanoreceptive, vestibular and acoustic input. Except for the valvula where multimodal units are found and inputs from all sensory modalities converge, the other parts of the cerebellum appear to receive inputs from a single modality with little overlapping. The corpus cerebelli receives visual and somatosensory inputs, partially segregated so that visual units are more medial and dorsal. Lateral eminentia granularis is the target of lateral line mechanoreceptive input and lobus caudalis pars lateralis is the major target of electroreceptive input. Lobus caudalis pars medialis receives vestibular (tilt) input. We have not examined the response of the cerebellum to yaw and to roll; the possibility that they are represented separately in the cerebellum remains a significant question. We also failed to find any acoustic response in the medial eminentia granularis. However, in agreement with Tong and Finger,4h this area does not respond to lateral line nerve input. Cerebellar responses to sensory stimulation Under the experimental conditions employed in this study, only relatively few cells showed spontaneous “complex” spikes: 16 out of the 106 cells in the visual area and 2 out of the 127 cells in the somatosensory area, and none in the other parts of the cerebellum. If the “complex” spikes originate from climbing fibers as in mammals, their scarcity may be due to a restricted localization of the climbing fiber system, or to an absence of spontaneous activity, or a high threshold to sensory input in most climbing fibers.35~42,43 It is unlikely that the scarcity is due to the preparation being in poor condition since ongoing activity and responses appear stable and similar to those in fresh preparation. The predominance of the mossy fiber activity is also reflected in the configuration of the evoked field potential responses. Despite the differences in amplitudes and latencies, the evoked field potential responses to stimulation of the spinal cord, optic tract, and medial nucleus of the lateral lobe are characterized by an early spike-like response followed by a larger negative wave in the granular mass. As interpreted by Lee from peduncle stimulation (L. Lee, submitted for publication) and in studies by other authors,y,50 the initial spike-like activity probably represents the summed action potential of the mossy fibers, and the larger granular negative wave probably arises from the synaptic activation of the granule cells. The molecular layer positive wave after stimulation of optic tract, posterior lateral line nerve or spinal cord may be attributed to the activation of the granule cell axons. The spike-like potential in the fibro-molecular layer can likewise be interpreted as activity in fibers of passage. A concomitant positive wave was not found in the valvula and eminentia

granularis following lateral line stimulation. This may be due to the orientation of the neural elements there” or the effectiveness of the stimulation in activating the granule cell axons. We have not observed the signs expected if climbing fiber activity were more common. Although most of the Purkinje cell responses in the catfish cerebellum are mediated by the mossy fiber-granule celllPurkinje cell pathway, Purkinje cells of different modalities are found to respond with different dynamic patterns. In units sensitive to either a brief flash or optic nerve stimulation, an initial excitation was not observed. Initial excitation, however, was readily observed following spinal cord, posterior lateral line or medial nucleus stimulation. The difference between visual and other inputs persists even when they converge on the same cell (Fig. 6). Furthermore, units sensitive to visual input, but not units of other sensory modalities, frequently exhibited a second period of inhibition following the first postinhibitory rebound. The responses do not change with stimulus strength. Comparison among species also reveals some differences in their responses to sensory stimulation. In contrast to the spinal cord response in catfish, spinal cord and even direct cerebellar surface stimulation in rainbow trout depress the simple spikes of the Purkinje cells without an initial excitatory phase”’ and are capable of generating a second period of inhibition. In cats, detailed analysis of the simple spike responses to different stimuli: limb nerve shocks, brief flash and click, revealed that they all have an initial excitation followed by inhibition. Furthermore, there is a high degree of similarity between the different responses in terms of the duration of the early burst either under local anesthesia or under general chloralose anesthesia.13 These differences serve to emphasize that effects produced by different sensory inputs may be another important but hiterto unstudied aspect of cerebellar evolution. Sensory representation in the cerrbellum In agreement with previous findings in goldfish,” the receptive fields of the Purkinje cells in catfish are usually large, diffuse, and with considerable overlap between neighbors. Topographical organization is only evident among somatosensory units. If the corpus cerebelli were cut horizontally along the middle of the granular layer such that the dorsal and the ventral tier were separated, and the whole corpus, together with underlying valvula were stretched out longitudinally, a rough topographical map of the body surface with the fins and the barbels overrepresented would be recognized along this sheet of tissue. The anterior end of this sheet, the valvula, represents the head, including the pectoral fins. The tail and the body are represented in progressively more posterior sites. The head is represented a second time in still more posterior sites where the eyes are also represented.

Catfish

cerebellar

Despite some differences, the sensory representation in the catfish cerebellum revealed by this study is generally in good agreement with the classical and recent anatomical studies,22 (see ref. 35 for a review). It supports the claim that no vestibulolateral fibers terminate in the corpus cerebelli. It also confirms the presence of spinal and trigeminal input in the corpus. But in addition, it suggests considerable spinal and trigeminal input into the valvula. In agreement with the result of Hayle,14 but at variance with that of Libouban and Szabo,27 it suggests no spinal input into the eminentia granularis. Visual input is also limited to the corpus. In contrast to the vestibular input, acoustic input is not well represented, the few units found being confined to the valvula. The catfish agrees in this respect with the thornback ray45,47but apparently contrasts with the goldfish in which a large population of units said to be acoustic is reported in the vestibulolateral lobe and the lateral corpus.”

Integration of sensory information According to LarselL2’ while the octavolateral area is derived from the special somatic sensory region of the rhombencephalon, the corpus cerebelli has a separate origin and represents a derivative of the trigeminal or general somatic sensory area. The discreteness of the sensory areas and the large percentage of unimodal units in the catfish cerebellum, therefore, may be considered to reflect a more primitive state of development and are not unexpected. However, the cerebellum is commonly regarded as coordinating motor output and the Purkinje cells are presumed to be the output of the cerebellar cortex. In view of the anatomical studies on the afferent and efferent projections and the histology of the cerebellum which point to almost unlimited facilities for the exchange of information and for close interneuronal cooperation,* the results of this study do raise the question: how and where does integration between different sensory afferents take place in the cerebellum of vertebrates like teleosts and elasmobranchs? An answer to the above question cannot at present be formulated. The following points, however, may be worth considering. (1) How the different sensory data are used by the animal may well dictate whether they will be integrated at a certain level of the central nervous system. For example, both acoustic and visual inputs are found to project to the posterior vermal region of the cat cerebellum;‘3~‘8~26 this close topographical association may be related to the possibility that when a cat responds to a sound it is likely to use the same muscle group as when the effective

sensory

167

representation

stimulus is visual. For instance, both the visual and auditory inputs may be involved in the coordination of eye movements or head turning. In line with this interpretation, all the multimodal units reported here have congruent receptive fields around the head; Bastian2 also reported units in Apteronotus with electroreceptive fields around the head and visual responses to objects moving in the same receptive field. Therefore the different inputs are conceivably to be used by the animal to achieve the same task, either sensory or motor. (2) Telencephalic input into the cerebellum may play a role in integrating information from sensory modalities. The mammalian cerebellum receives massive input from the cerebral cortex.6.7.8.“.‘3Furthermore, peripheral and cortical input of the same sensory modality converge on the cerebellum with facilitation.13 Although classical interpretations of cerebellar phylogeny” regard telencephalic input as a special development of mammals, with neocortex and neocerebellum, powerful telencephalic input into the cerebellum has recently been described in catfish.23 This input, however, arises not from the part of the pallium considered to be homologous to the mammalian isocortex but from a part homologous to the mammalian striatum.33,34The function of this input is still unknown and interaction between it and the peripheral inputs is still to be characterized. Finally, except for the large proportion of unimodal units, the organization of the catfish cerebellum does bear a certain degree of similarity to that of the mammlian cerebellum, at a coarse level. Again, if the catfish cerebellum is considered as an unfolded sheet of neural tissue, the anterior part of this sheet is invested with somatosensory input. Caudal to it and slightly intermixed with it is the medially located visual area. The vestibulolateral input occupies the caudalmost part of the cerebellum. Telencephalic input, which does not project to the valvula in this species, is not represented in the anteriormost part of this sheet. In the similarly stretched mammalian cerebellum, the anterior part also receives somatosensory input; the posterior part is the prime target of teleceptive input” while the caudalmost parts, the flocculus and nodulus are primarily vestibular. Cerebral input is represented in all parts of the cerebellum but is more concentrated in the posterior lobe. The difference between teleost or elasmobranch and mammals may be more quantitative than qualitative.

Acknowledgements-Aided by grants to T.H.B.

from the National Science Foundation and the National Institute of Neurological and Communicative Disorders and Stroke.

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