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J. Phyeiol. (1979), 289, pP. 403-423 With 7 text-figuree Printed in Great Britain

RESPONSES OF NEURONES IN NUCLEUS INTERPOSITUS OF THE CEREBELLUM TO CUTANEOUS NERVE VOLLEYS IN THE AWAKE CAT

BY D. M. ARMSTRONG AND J. A. RAWSON* From the Department of Physiology, Medical School, University of Bristol, Bristol BS8 lTD

(Received 26 May 1978) SUMMARY

1. A method is described which permitted stable extracellular recordings to be made from 115 neurones in nucleus interpositus of the cerebellum in unanaesthetized free-to-move cats. At least 95 % of the neurones were cerebellar efferent cells since they were antidromically invaded following electrical stimulation of the brachium conjunctivum in the region of the contralateral red nucleus. 2. In cats in a state of quiet wakefulness the majority of interpositus neurones were tonically active at rates ranging from 12 to 77 impulses/sec (over-all mean 34/sec). The remaining neurones were silent or discharged only a few impulses throughout observation periods of a few minutes. 3. Cutaneous afferent volleys elicited by single shocks to the superficial radial nerves in the forearm at intensities too weak to evoke a flexion reflex or behavioural arousal produced changes in firing frequency in 62 % of eighty-one cells tested. Response patterns varied widely but in 86 % of the responding cells the earliest change was a short latency (6-20 ms) increase in discharge probability which from post-stimulus time histograms was found usually to average around one impulse per stimulus. In only four cells (8 %) the earliest response was a depression of the tonic firing. However, in many cells the initial acceleration was followed by a reduction in firing frequency which lasted between 10 and 85 ms. 4. In 56 % of the responding cells a longer latency (25-80 ms) acceleration was present. Such accelerations varied widely in duration (from 55 to 550 ms) but most commonly lasted 100-200 ms. These responses were usually the most prominent feature in the response pattern: in the majority of neurones between two and five impulses were added per stimulus. 5. Considering the whole time course of the responses, the net effect of nerve volleys was to produce an increase in nuclear cell output. 6. These neurones which were influenced by nerve stimulation also discharged in response to taps to the forepaws. 7. The responses to nerve stimulation are compared with those encountered in previous studies using cats anaesthetized with chloralose or barbiturates and with the responses of Purkinje (P) cells and it is suggested that the longer latency excitatory * M.R.C. Scholar. Present address: Department of Physiology, Monash University, Clayton, Victoria, Australia. 0022-3751/79/2950-0495 $01.50 © 1979 The Physiological Society

404 D. M. ARMSTRONG AND J. A. RAWSON responses result in large part from a reduction in the tonic inhibitory action exerted on the interpositus neurones by Purkinje cells. 8. The possibility is discussed that interpositus responses to cutaneous input from the limbs might contribute (via the rubrospinal system) to the regulation of spinal flexor mechanisms during locomotion and/or contact placing reactions. TNTRODUCTION

Neurones of the intracerebellar nuclei provide the main output channel from the mammalian cerebellum and a knowledge of their firing patterns is clearly essential for progress in understanding the cerebellar contribution to motor control. In many species the neurones of nucleus interpositus (ip.) constitute an important fraction of the total population and their importance is emphasised by evidence that the nucleus projects to and powerfully excites the contralateral red nucleus (r.n.) and the ventrolateral subnucleus of the thalamus (for references see Allen & Tsukuhara, 1974). Micro-electrode recordings have previously been made from interpositus neurones (ip.n.s) in unaesthetized monkeys (e.g. Thach, 1968, 1970; Mortimer, 1975) and cats (McElligott, 1976; Burton & Onoda, 1977) and these studies have revealed brisk tonic activity in stationary animals plus phasic alterations in the discharge of many units during conditioned movements of the limbs. Burton & Onoda (1977) have also presented evidence that activity patterns during forelimb movement in the cat are strongly influenced by input from rate sensitive movement detectors in the moving limb. This last is in good accord with the abundant evidence (see Bloedel, 1973; Oscarsson, 1973; Armstrong, 1974) that there are powerful inputs from deep (and cutaneous) receptors in the limbs to the 'intermediate' zone of the cerebellar cortex, the P cells of which project to and inhibit the ip. neurones (Voogd, 1964; Armstrong & Schild, 1978; Ito, Yoshida, Obata, Kawai & Udo, 1970). So far, studies in free-to-move animals have not been concerned to define discharge patterns evoked by well-defined somatosensory inputs. However, discharges evoked by cutaneous inputs from the limbs have been studied in decerebrate cats by Eccles, Ros6n, Scheid & Taborikova (1974a, b) and Eccles, Rantucci, Rosen, Scheid & Taborikova (1974). Electrical stimulation of cutaneous nerves and adequate stimulation of mechanoreceptors were shown to evoke similar patterns of response in which there was typically a brief increase in discharge rate (latency about 16 ms) followed by a phase of depression lasting approximately 50-100 ms. These changes were attributed to the effects of afferent volleys in climbing fibres of the spino-olivocerebellar paths (see Oscarsson, 1973; Armstrong, 1974) and in mossy fibres of spinoreticulocerebellar paths (see Bloedel, 1973; Oscarsson, 1973). The facilitation was attributed to a direct excitatory influence exerted on the ip.n.s via collateral branches of the cerebellar afferents and the depression to inhibitory input from P cells excited by the arrival of the afferent volleys in the intermediate zone of the cerebellar cortex. In some cells this excitation-inhibition sequence was preceded by a similar though briefer sequence (latency 6 ms) which was attributed to comparable mechanisms operating through the faster spino-cerebellar mossy fibre paths (cuneo-cerebellar and rostral spino-cerebellar paths).

405 INTERPOSITUS RESPONSES Similar responses have since been observed in animals anaesthetized with achloralose (Armstrong, Cogdell & Harvey, 1975; Cody & Richardson, 1978) but in many ip.n.s they were overshadowed by a prominent burst of impulses (peak frequency up to 200/sec) which followed the second period of inhibition and lasted up to 300 ms. This striking response was tentatively identified as a disinhibitory phenomenon resulting from prolonged depression of the tonic background discharge in the P cells. Such depression is commonly observed following a cutaneous afferent volley in animals anaesthetized with c-chloralose (Freeman, 1968; Talbott, Towe & Kennedy, 1967; Armstrong, Cogdell & Harvey, 1979). In view of this difference between the response patterns of ip.n.s studied under different conditions of anaesthesia, together with other evidence that cerebellar cortical and deep nuclear activity is heavily influenced by type and depth of anaesthesia (e.g. Bloedel & Roberts, 1969; Gordon, Rubia & Strata, 1973; Eccles, Sabah & Taborikov6, 1974) it seems desirable that further studies should be made in the absence of anaesthetics. In the present study, therefore, extracellular recordings have been made from ip.n.s in free-to-move cats. Analyses have been made both of the tonic background activity of the neurones and of the responses evoked by weak electrical stimulation of a cutaneous nerve (superficial radial, s.r.) in the forelimb via an implanted cuff electrode. In previous studies ip.n.s have been identified solely on histological grounds but it is now known that the nucleus contains local interneurones as well as neurones projecting outside the cerebellum (Chan-Palay, 1977). We have therefore tested all units for the presence of an antidromic impulse following stimulation in the region of the contralateral r.n. and on this basis the majority of recordings were from cells projecting to the mid-brain. Some of the results have been published previously in brief (Armstrong & Rawson, 1976). METHODS

Operative techniques, Experiments were performed on seventeen adult cats of both sexes, weighing between 2-5 and 3-5 kg. At an initial aseptic operation each animal was deeply anaesthetized with sodium pentobarbitone (administered intraperitoneally), the head was immobilized in a stereotaxic head holder fitted with atraumatic ear bars and a craniotomy was performed to allow access to the left side of the cerebellum. The cranium was entered between the lambdoidal ridge and the junction of the bony tentorium with the parietal bone: the centre of the craniotomy was between 4 and 5 mm lateral to the mid line and lay between antero-posterior stereotaxic planes P 14 and P 15. A lightweight titanium cylinder with internal diameter 10 mm was cemented over the craniotomy and served to carry a micro-electrode manipulator during subsequent recording sessions. In view of the curvatures of the skull it proved convenient to fix the cylinder to the skull via a preformed base or adaptor moulded from dental acrylic cement. The bone surface was thoroughly roughened using a dental burr, small undercut holes were drilled and additional acrylic was then used to secure the adaptor to the skull. The choice of a post-tentorial approach to the cerebellum necessitated angling the micro-electrodes forwards by 300. This was achieved by holding the acrylic base and titanium cylinder in an appropriately angled stereotaxic holder during their implantation.

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Micromanipulator Between recording session the titanium cylinder was capped, but for recording the cap was replaced by a lightweight micro-electrode manipulator constructed throughout of autoclaveable materials. The manipulator was manually controlled. One complete turn of the control advanced the micro-electrode tip by 250 pin and eccentric mounting of the electrode ensured that by rotating the manipulator in the titanium cylinder, penetrations could be made into different loci within the exposed area of dura. Further details have been published elsewhere (Armstrong, Leonard & Rawson, 1975). The useful life of the preparation was usually limited by progressive thickening of the dura but could be extended to around 50 days by smearing the dura with corticosteroid cream containing antibiotics and oversealing with medical grade silicone elastomer (Dow Corning 382 elastomer) diluted x 3 with medical grade R.T.V. thinner to ensure that the cured material was a soft gel readily penetrated by micro-electrodes. At the end of each recording session one or two drops of a solution with corticosteroid and antibiotic activity (Betnesol N; Glaxo Ltd) were introduced into the chamber. Stimulating techniques A second small craniotomy was made to allow stereotaxic insertion of a stimulating electrode into the right r.n. The electrode consisted of two stainless-steel wires 250 pm in diameter and teflon insulated except over the final 250 pm. The wires were glued together (using silicone medical adhesive; type A, Dow Corning) with the tips staggered by 1 mm. The mid-brain was stimulated using 0-2 ms pulses supplied from an isolated constant current stimulator. Currents employed normally ranged from 20 to 600 pA and single stimuli rarely had any visible effects on the behaviour of the animals. Brief trains of shocks at high frequencies (up to 1000/sec) sometimes evoked a twitch of the contralateral eyelid or a brief flexion of the contralateral forelimb but neither of these responses aroused the animals. Bipolar cuff electrodes were also placed subcutaneously in the forelimbs to allow stimulation of the right and left s.r. nerves in continuity. These electrodes consisted of a short cylindrical sheath (10 mm long) of silicone medical elastomer which incorporated in its wall two nylon insulated multistrand stainless-steel wires (Bergen Wire Rope Co., Lodi, New Jersey). The ends of the wires were bared of insulation and teased slightly proud of the inner surface of the cuff. The wall of the cuff was split lengthways on one side so that it could be temporarily unfurled and positioned around a length of nerve dissected free a few centimetres above the paw. The split in the wall was then sealed with elastomer. The proximal ends of the nylon insulated wires were led subcutaneously to the head where they and the intracranial stimulating electrodes were crimped into subminiature plugs (Amphenol, Reliatac) and inserted into a receptacle strip cemented to the acrylic base. The peripheral nerves were stimulated using 0-1 ms pulses from isolated stimulators (Devices Ltd). Care was taken to maintain stimulus intensities at levels which caused little or no flexor withdrawal reflex and no sign of discomfort (pupillary dilatation or behavioural arousal). Some indication of the effectiveness of the stimuli in evoking nerve volleys was obtained from a terminal experiment in which the animal was anaesthetized to permit exposure of the s.r. nerve proximal to the implanted cuff. Recordings were made from the nerve to determine the threshold for the most excitable fibres when stimulated via the cuff. Stimulus intensities used in the recording sessions could then be expressed as multiples of this threshold (T). In view of the connective tissue reaction which usually occurred within and around the cuff it is quite possible that stimulus effectiveness declined during the life of the preparation and T values cited should therefore be regarded as very approximate measures. However it was noted that in general the stimulus required to elicit a minimal flexion reflex did not increase appreciably during the period over which micro-electrode recordings were made.

Recording techniques During recording sessions micro-electrodes were push-fitted into the manipulator and advanced cautiously through the elastomer gel and the intact meninges. Initial experiments using tungsten electrodes insulated with varnish (B. & K. Resins Ltd, London) or epoxy resin (Araldite, PZ 985; Ciba Geigy Ltd) gave disappointing results as the insulation was frequently damaged when the dura was penetrated. Most of the results were therefore obtained with the

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use of glass insulated tungsten micro-electrodes manufactured by a variant of the process described by Merrill & Ainsworth (1972). Electrodes with tip diameter 1-3 ,am and impedance 0-5-5 M0 at 1 kHz were highly successful and usually withstood many encounters with the dura. Neural activity was recorded differentially between the micro-electrode and a platinum wire cemented so as to contact the dura near the edge of the craniotomy. A unity gain head-stage preamplifier utilizing field effect transistors was mounted on the acrylic base and connected to the main amplifier via flexible cable. The preamplifier weighed 7 g and the whole assembly on the skull (acrylic base, preamplifier, receptable strips, titanium cylinder and micromanipulator) weighed only 25 g. The animals appeared oblivious of the assembly except when their attention was caught by the stimulating and recording leads. The animals sat within a box measuring 1 x 0-5 x 0-5 m and were earthed via a stainless-steel wire cemented into the bone of the skull. Each recording session lasted 1-3 h after which the animal was returned to a large communal pen. Action potentials were led to an oscilloscope for visual observation and filming, to a loudspeaker and to a tape recorder for storage. Off-line analysis of taped spike trains was performed using a digital computer (Modular One; Computer Technology Ltd). Programmes were available to compute mean interspike intervals (plus standard deviation and coefficient of variation of the intervals), interspike interval histograms and autocorrelograms of tonic activity and also postand peri-stimulus time histograms of up to fifty successive responses to peripheral nerve stimuli delivered at a rate of 0 5/sec. Histograms were displayed (Tektronix 611) and were also plotted permanently using a computer-controlled X-Y plotter (Bryans Ltd). Additional analyses were performed using measurements by hand from filmed records of oscilloscope traces.

Histoogy During the terminal experiment the site of the mid-brain electrode tip was marked by passing direct current to deposit iron which was then stained by the Prussian-blue reaction. The approximate location of the cerebellar recording sites was also marked by introducing a micro-electrode into the cerebellum until a large antidromic field potential was recorded in response to midbrain stimulation. A current of 60 ,uA was then passed for 90 s to produce a small lesion. Brain stem and cerebellum were sectioned after celloidin embedding. All mid-brain electrodes were within or very close to the caudal half of r.n. Most cerebellar lesions were within the caudal two-thirds of interpositus anterior but a few were in the rostral half of interpositus posterior. No signs of gross cerebellar damage were observed despite the extensive tracking done, but the dura and pia were always thickened under the craniotomy and signs of small haemorrhages were sometimes observed in the underlying cortex.

RESULTS

Location and identification of neurones Nucleus interpositus was localized by the presence of a short-latency negativegoing field potential (on which single unit action potentials were superimposed) following mid-brain stimulation. Such responses were located at depths between 6-0 and 9-0 mm beneath the cerebellar surface (as estimated from the electrical transients which occurred as the electrode tip crossed the meninges). Stable recordings were obtained from 115 units in this region; action potential amplitudes ranged from 0.5 to 2-0 mV and the responses were either positive-negative or negative-going. In the hope of minimizing micro-electrode damage to the cells no particular efforts were made to maximize the size of the unit spikes. One hundred and nine of the units (94-8 %) gave a single impulse at short fixed latency following r.n. stimulation. Such responses are illustrated for two units in Fig. 1 A and B. All seventy units tested were able to respond to each stimulus in a short train delivered at high frequency (800-1000/sec; Fig. 1C). In each of thirtythree units tested, the impulse evoked by r.n. stimulation was shown to be due to

408 D. M. ARMSTRONG AND J. A. RA WSON antidromic invasion because it showed occlusive interaction with spontaneous action potentials and this interaction followed a time course indicative of impulse collision in the axon. On this evidence it is likely that the responses to r.n. stimulation were antidromically evoked in all or most of the units and therefore that the units A

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projected to the mid-brain. The latency of the action potentials ranged from 0-4 to 1-8 ms and the frequency distribution for the sixty-four units recorded in two cats is shown in Fig. 1D. The latency values were measured at just suprathreshold levels of stimulus intensity and are in good agreement with values reported from anaesthetized cats (Armstrong et al. 1975; Ito et al. 1970). Fig. 1 E shows the electrical thresholds of forty interpositus axons studied in one animal in which the mid-brain electrode was in the caudal part of r.n. Although the antidromic responses showed no A-B inflexion (which would indicate that the recordings were from cell bodies) individual units could be recorded

INTERPOSI TUS RESPONSES 409 over distances of 60-100 #sm and electrical contact was readily maintained for many minutes and sometimes for 2 h. These features suggest that the recordings were made from substantial generators, presumably cell bodies. This conclusion is supported by the finding that spikes recorded in the white matter above the nucleus were small, were monophasic positive or triphasic and could be recorded only for a short time. 20 15 C

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Fig. 2. Frequency histogram for the mean rates of discharge of ninety-four neurones which discharged tonically. Mean rate calculated for each unit from one or more samples of 1000 successive impulses. Four units with spike shapes and discharge patterns similar to those for antidromically identified units could not be invaded even with stimulus strengths of 2 mA. The identity of these units was deemed uncertain and as they were unresponsive to nerve stimulation they were not studied further. Two additional units were activated by r.n. stimulation at latencies of 2-2 and 2-3 ms. They could not be studied long enough to permit antidromic occlusion tests and it is possible the responses were trans-synaptically generated. Two possible paths are the sparse (Courville & Brodal, 1966) and probably excitatory (Yu, Tarnecki, Chambers, Liu & Konorski, 1973) projection from r.n. to ip. and the recurrent collaterals of the interposito-rubral axons (Matsushita & Iwahori, 1971; Chan-Palay, 1977). Evidence for synaptic action exerted on projection ip.n.s via either of these paths was sought in a random sample of fifteen neurones by compiling post-stimulus time histograms (p.s.t.h.s) to assess changes in probability of firing following single shocks to r.n. No excitatory or inhibitory effects were noted until the stimulus strength was sufficient for antidromic activation of the test cell when a brief silent period of 5-20 ms followed the antidromic spike. This period was not graded in duration with stimulus strength and it may therefore be ascribable to refractoriness at the site of generation of orthodromic impulses rather than to post-synaptic inhibition.

Background discharge of interpositus neurones All recordings were made from cats which appeared to be in a state of quiet wakefulness. Under these conditions the large majority of the neurones projecting to the mid-brain (98 out of 109; 90 %) displayed a tonic discharge. Mean firing rates were computed from samples of 1000 successive interspike intervals and a frequency histogram of the mean rates for ninety-four units is shown in Fig. 2. The over-all mean rate was 33-5 impulses/sec and although the range was from 11-6 to 77-0 impulses/sec only 10 % of the units discharged at rates above 50/sec. In general the discharge was rather irregular: the standard deviation of the interspike intervals was

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D. M. ARMSTRONG AND J. A. RAWSON often larger than the mean and interval autocorrelograms displayed no prominent recurring peaks and troughs. Firing patterns for three typical units are illustrated by the interval distributions and autocorrelograms of Fig. 3. 50

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Although the main impression was of brisk tonic activity, eleven cells (10%) showed little background activity. Nine of these units remained silent throughout a recording period of several minutes and the remaining two discharged only 1 or 2 spikes/min during the few minutes they were observed. It should be noted that the proportion of poorly active cells may have been underestimated because a mid-brain 'searching' stimulus was not used in all penetrations. It is possible that the inactive units constitute a functionally discrete population within the nucleus but when a small number of tonically active units were studied

411 INTERPOSITUS RESPONSES over long periods (30-80 min) some were observed to display a mixed nature: occasionally the steady discharge would decline or stop for periods ranging from 30 sec to several minutes.

Interpositns responses to volley in cutaneous afferents of the ipsilateral forelimb The effects of stimulating the ipsilateral s.r. nerve with single shocks at intensities up to 4 T (see Methods) were tested in eighty-one ip.n.s, fifty of which were influenced to an extent detectable from p.s.t.h.s of fifty successive responses. Fig. 4 presents sample p.s.t.h.s from six cells and Fig. 5A summarizes results from all 30

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412 D. M. ARMSTRONG AND J. A. RAWSON fifty units. In twenty-five units (50 %) the responses consisted of only one or more phases of increased discharge but in the remaining units the responses included mixtures of acceleration and depression. It is also clear from Fig. 5A that response durations varied widely. In twenty-four units (48 %) the discharge returned to background level in 100 ms or less after the stimulus, whilst in the remainder the (A)

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evoked activity persisted longer, lasting over 400 ms in four units. Although no 'typical' pattern of response could be defined, certain features were often present though in different combinations. It is important to note that excitatory responses were more frequent, and longer in duration than inhibitory components. They were also usually larger in amplitude.

Short-latency excitatory responses In forty-six of the fifty responding units (92 %) the first response to the stimulus was an increase in the probability of discharge relative to the background level. In all but three units the latency, as measured from p.s.t.h.s such as those of Fig. 4A,

INTERPOSITUS RESPONSES 413 C, E and G, was between 6 and 28 ms and the frequency distribution for the latencies is plotted above the base line in Fig. 6. In most cases (thirty-three; 66%) these increases in discharge probability were of brief duration (6-20 ms as estimated from the p.s.t.h.) and dividing the number of additional impulses (above background) by the number of stimulus presentations used to construct the p.s.t.h. showed that 15 10

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most of the responses involved the addition of no more than one impulse per stimulus; indeed, in many units the probability of evoking an impulse was well below unity. Although the short latency excitation was usually brief, in ten units (20 %) it lasted 50 ms or more (see Fig. 5A). However, although the discharge probability never fell to pre-stimulus levels during this period, there was usually a trough in the p.s.t.h. to suggest an underlying division of the response into a brief short-latency and a more delayed component. Thus, in toto, brief excitations with latency 20 ms or less were certainly or probably present in forty-three units (86 % of those responding). In the large majority of cases such responses comprised only a single peak in the p.s.t.h. but in a minority of five units two successive peaks were recognizable indicating the existence, despite the relatively low probability of obtaining an impulse, of two preferred latencies for the discharge. Such behaviour, as shown by two of the units, is visible in the histograms of Fig. 4C and G. In Fig. 4 C the two peaks have latencies of 7 and II ms and are just separate whilst in Fig. 4G the separation is much more marked. In the remaining three cases it was reinforced by an intervening period of a few milliseconds during which the firing rate was depressed well below the pre-stimulus level. Such biphasic excitation was somewhat more widespread amongst ip.n.s studies in anaesthetized cats by Eccles, Ros6n, Scheid & Taborikova (1974a) and was taken by these workers to indicate that two groups of afferent paths were involved in producing short-latency discharges, namely the direct spino-cerebellar paths and rather slower pathways relaying via the inferior olive and the medullary reticular formation (see Introduction).

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Inhibitory response In only four out of fifty units, the first response to the stimulus was a deceleration of the tonic discharge. One response of this kind is illustrated by the p.s.t.h. of Fig. 4 B and the four latencies are plotted below the base line in Fig. 6. In twenty-one additional units (42%) the initial excitatory response was followed by a period during which the discharge rate fell below the pre-stimulus level. This feature is illustrated for one unit by Fig. 4C and D. The times of onset of such periods of inhibition or disfacilitation varied between 10 and 30 ms and in all but one unit the depression terminated less than 100 ms after the stimulus (Fig. 5A). Complete suppression of the background discharge rarely persisted for longer than 10-20 ms (see, e.g. Fig. 4C). Delayed accelerations In four units in which the brief initial excitation was followed by depression, this second component terminated the response. However, in seventeen other cases (34 %) the inhibition gave way to a second increase in the probability of discharge 11_

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50 60 70 80 90 100 110 120 Latency (ins) Fig. 7. Relationship between latency and amplitude of excitatory responses to stimulation of the superficial radial nerves. Data for both nerves are plotted. Response amplitudes were derived from post-stimulus histograms by measuring the area of each excitatory peak and dividing the total number of impulses (added above pre-stimulus level) by the number of presentations of the stimulus (fifty in each case). In the few cases in which two short latency peaks were present only the larger has been plotted. One response with latency > 120 ms omitted, some short latency (< 20 ms) excitations omitted at random to reduce overcrowding, and where a prolonged short latency excitation was present, only the first peak plotted. See text for further explanation.

415 INTERPOSITUS RESPONSES (latency 25-120 ms; mean 60 ms). When such facilitatory peaks were present in the p.s.t.h. they were usually much more prolonged than the short-latency excitation (see Fig. 4 D), though, as Fig. 5A shows, the duration varied very widely in different cells (from 50 to 410 ms; mean 160 ms). Responses similar in latency and duration were also present in six cells in which the initial brief acceleration was followed by a temporary return to background firing rate rather than by a depression (e.g. Fig. 4E and F). Similar responses were also present in the four cells showing an initial deceleration (e.g. Fig. 4B) and in three further cells a 'delayed' excitation (latency 30, 40 and 80 ms) was the first detectable response (e.g. Fig. 4H). Late facilitations were therefore observed rather frequently, being present in no less than 62 % of the responding units. In addition, it should be noted that in the ten other cells in which an excitatory response occurred with brief (20 ms or less) latency but long duration (see above and Fig. 5A) there was a trough after the initial response which suggested that the subsequent part of the response could be ascribed tentatively to a delayed phase of excitation. In general, 'delayed' responses were more substantial than the short-latency excitations. This was quantified by determining from the p.s.t.h.s the magnitude of the responses in terms of the average number of impulses added (above background) per stimulus presentation and by plotting this value against the latency of each response. The results are shown in Fig. 7 where it is clear that, except in one case, excitations with a latency of less than 25 ms never exceeded 1 5 'additional' impulses per stimulus and in the majority of cases an impulse occurred in only a proportion of the trials. By contrast twenty-five out of thirty-six responses with longer latency (69%) averaged two or more impulses per stimulus and in seven units (19%) more than five impulses were elicited.

Separate origins of the short-latency and delayed accelerations The presence of delayed accelerations in the four cells which showed an initial inhibition and in three cells in which there was no response with latency less than 30 ms indicates that late responses were not necessarily dependent on the prior occurrence of short latency accelerations or decelerations in the same unit. This provides some justification for regarding the late responses as comprising a separate (though not necessarily homogeneous) category and further evidence is provided by the differential responsiveness of short-latency and delayed excitations to changes in stimulus strength. Thus, in four units in which both types of response were present it was possible to investigate the effect of small changes in the intensity of the shock delivered to the superficial radial nerve. In two units an increase in stimulus intensity from 2 to 3T produced a substantial increase in the delayed excitation but no significant increase in the early responses, whilst in a third an increase from 1-5 to 2T increased the late response but produced little change in the early component. Conversely, in the fourth unit an increase in intensity from 1-5 to 2T produced a doubling of the early excitation but virtually no increase in the delayed response.

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Responses to stimulation of the contralateral superficial radial nerve In thirty-eight of the units which responded to stimulation of the ipsilateral nerve, the effect of stimulating the contralateral s.r. nerve was also investigated. Responses were detected in nineteen cases (50 %) and as may be seen by comparing Fig. 5A and B the types of response observed were essentially similar. Furthermore, for individual cells which responded to stimulation of each nerve both the amplitude and time course of the two responses were usually similar. The only significant difference observed was that stimulation of the contralateral nerve evoked no responses with latency less than 10 ms whereas the ipsilateral nerve elicited (excitatory) responses with latency less than 10 ms in 28 % of the units.

Responses to tactile stimulation of the forepaws Whilst electrical stimuli applied to the s.r. nerve are useful in producing cutaneous inputs which can be precisely timed in onset and graded in size, such stimuli obviously elicit highly synchronized volleys which may be unnaturally potent in their effect. For most neurones an attempt was therefore made to observe their discharge following more natural activation of forepaw mechanoreceptors by tactile stimuli. It was found most practicable to apply these stimuli manually, by tapping the paws with a finger. Observations were therefore of a qualitative nature in which subtle changes in impulse activity would have been overlooked. In cells shown to be responsive to nerve stimulation a brisk tap to a paw often caused a change in firing frequency. The most readily detected response consisted of a long facilitation in discharge which was normally seen only in those cells that had demonstrated a delayed acceleration following nerve stimulation. In general, taps delivered either to the dorsum of the paw or to the foot pads were effective in provoking discharges, but detailed analysis of receptive fields was precluded by the rather gross nature of the stimuli. In many cells, assessment of the afferent input underlying the responses was made difficult because delivery of a tap often provoked a withdrawal of the limb and changes in posture. However, a few neurones could be studied in the absence of visible motor reactions. The responses of these cells could reasonably be ascribed primarily to inputs from cutaneous mechanoreceptors and they did not appear to differ from responses in other cells. In some cases tactile stimuli were applied for several seconds by gently squeezing the paw with a hand or by maintaining pressure on the paw with a finger. Whilst an increase in discharge was associated with the presentation of these stimuli it was not maintained for the duration of stimulus application. DISCUSSION

Location and identity of units In the present study, the absence of anaesthesia or restraint dictated recording arrangements which provide less precise control over the position of the microelectrode tip than is usual in anaesthetized preparations. Nevertheless, it is likely from the distribution of the marker lesions (see Methods) that all or most of the units were within the confines of ip. Although it was impossible to determine the position of individual units within the nucleus, a tendency was nevertheless evident

INTERPOSITUS RESPONSES 417 for neurones with similar responses to nerve stimulation to be located in clusters. Thus two or three units with similar responses were frequently isolated when tracking through a distance of less than 500 gim and as judged from multiunit activity the nearby neurones also behaved similarly. Clustering of ip.n.s with related response properties has also been detected by others, working with anaesthetized preparations (Allen, Gilbert, Martini, Schultz & Yiu, 1977). In most other studies of the intracerebellar nuclei no attempt has been made to distinguish neurones projecting outside the cerebellum from interneurones with locally ramifying axons. Since there is now good anatomical evidence for interneurones within the nuclei, we routinely stimulated the brachium conjunctivum at the mid-brain level in attempt to identify projection cells by antidromic invasion. In the event, 95 % of units were shown to project to the mid-brain, indicating that we failed to sample any significant number of interneurones, presumably because they are small (reportedly around 10 ,um in diameter; Chan-Palay, 1977). However, within the population of projection cells the axonal conduction times ranged from 1*8 to 0*4 ms, so that assuming an approximate conduction distance of 14 mm the conduction velocities ranged from 7 to 35 m/sec. If axon diameter varies with soma size as seems common elsewhere in the nervous system, then these values suggest that our recordings were not restricted to the largest nuclear neurones. Nuclear cells range in diameter from about 10 to 35 jsm in the cat and cells throughout the range provide axons to the brachium conjunctivum. Since the smallest projection cells are similar in size to the interneurones it seems safest to assume that they also were not sampled.

Tonic or 'background' discharges In most previous studies in awake animals units in the cerebellar nuclei have been detected on the basis of their brisk tonic discharge and it is generally assumed that all nuclear cells show maintained activity, presumably resulting from tonic excitatory input along collaterals of cerebellar afferent fibres. Such tonic activity is undoubtedly of great importance for signal processing in the cerebellum because it provides a base-line level which can be modulated up or down by variations in the level of input from the P cells - an operation termed 'inhibitory sculpturing' by Eccles (1973). Our results confirm that the majority of ip.n.s indeed display continuous background activity and the overall mean rate of discharge (34 impulses/sec) agrees well with the value (37 impulses/sec) reported for a mixed population of dentate and interpositus neurones recorded from awake monkeys by Thach (1968). However, we have found in addition that a significant proportion (at least 1 0 %) of nuclear cells may remain silent or essentially silent for periods of minutes. Such inactive units would be overlooked without use of an antidromic 'searching' stimulus. Their existence implies that at any one time a 'reserve' of neurones will exist which can be recruited into action by a reduction of the inhibitory influence from P cells and/or by an increase in the excitatory drive from cerebellar afferent collaterals. Our finding has recently been confirmed by a study in awake monkeys, in which a number of antidromically identified dentate and interpositus neurones were quiescent for periods of a minute or more (R. Porter & J. A. Rawson, unpublished observations). I4

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418

Activities& evoked by 8.r. 8timulation Our results demonstrate that stimulation of a single forelimb cutaneous nerve can evoke changes in the firing rate of substantial numbers of ip.n.s in awake animals. At the relatively low intensities employed it is likely that the afferent volley was confined to large myelinated fibres; in s.r. nerve these are presumably connected with cutaneous mechanosensitive receptors. The shocks used were usually well below the intensity levels required to evoke any visible or palpable reflex movements of the limbs and furthermore when they were increased to evoke a detectable reflex there was no sudden change in the responses. It therefore seems unlikely that delayed proprioceptive input resulting from reflex movements made any contribution to the responses, which nevertheless exceeded 100 ms in duration in over half the units.

In most units the earliest responses were excitatory and seemed to correspond with the initial excitations observed in decerebrate or anaesthetized animals (Eccles et al. 1974a, b; Armstrong, Cogdell & Harvey, 1975; Cody & Richardson, 1978). Excitatory responses with latency 5-10 ms can only be attributed to input via

collaterals of cuneo-cerebellar and rostral spino-cerebellar fibres: as in acute preparations, such responses were weak and present only ipsilaterally. Responses with latency 10-25 ms were bilateral and more widely distributed, findings which again parallel the results from acute preparations. Responses with this latency were attributed by Eccles et al. (1974a, b) to input via collaterals of the more slowly conducting spino-cerebellar paths including spino-reticulo-cerebellar (mossy fibre) paths and spino-olivo-cerebellar (climbing fibre) paths. In four units the earliest response to an ipsilateral input volley was a period of reduced firing and in many other responding neurones the initial excitation provoked by an ipsilateral or a contralateral volley was followed by a period of depression. The absence of detectable inhibitions following antidromic activation of the ip.n. axons would seem to indicate that recurrent inhibitory influences resulting from the initial ip.n. discharges cannot contribute significantly to these periods of depression though in view of their brevity it is possible that refractoriness played some part in their genesis. The latencies of such responses were in fact consistent with the proposal by Eccles et al. that they are due to inhibitory input from Purkinje cells discharged following arrival of the mossy and climbing fibre afferent volleys in the cerebellar cortex - the zone which provides the major cortical projections to ip. strate that appropriately timed discharges indeed occur in P cells of the intermediate cerebellar cortex - the zone which provides the major cortical projections to i.p. These discharges include both 'simple' spikes attributable to mossy fibre input and 'complex' spikes due to climbing fibre input from the inferior olive (see, e.g. Eccles, Ito & Szentagothai, 1967). In around half of the responding neurones the reactions to an input from the s.r. nerves included a period of delayed facilitation. The latencies and durations of these facilitations varied widely in different units but they were often much the most substantial component of the whole response. Similar (but often more vigorous) delayed discharges are a prominent feature of interpositus responses to nerve volleys in chloralose-anaesthetized

Richardson, 1978)

animals

but

they

(Armstrong,

are

Cogdell

much reduced

anaesthetized preparations (Eccles

et

al.

or

Harvey, 1975,

absent in

decerebrate-

1974a, b; Cody &

1979;

or

Cody

&

barbiturate-

Richardson, 1978).

INTERPOSITUS RESPONSES

419

Several explanations may be entertained to account for these accelerations but in part at least they are likely to be disinhibitory responses. Thus in chloralosed animals, recordings from P cells in the paramedian lobule portion of the intermediate cortex have demonstrated that the discharges attributed to spino-reticulocerebellar and spino-cerebellar input are followed by reductions or cessations in the tonic discharge which coincide in time with the delayed facilitations (Armstrong et al. 1978). Furthermore, in a subsequent paper we report a similar finding for P cells widely distributed in the intermediate portion of lobules V and VI of awake cats (Armstrong & Rawson, 1979). Such changes would be expected to reduce the tonic inhibitory action exerted by the cortex on the nuclear neurones. Suppression of the P cell tonic discharge could be achieved via post-synaptic inhibition of the P cells by the basket cells or via disfacilitation as a result of Golgi cell inhibition of mossy fibre-granule cell transmission (see Eccles et al. 1967 for references). It could therefore result from either mossy or climbing fibre input since both types of afferent fibre provide excitatory input to both categories of cortical inhibitory interneurone. However, Armstrong et al. (1979) have reported that similar pauses in P cell discharge (and delayed discharges of interpositus neurones) are readily produced by an electrical stimulus to the inferior olive which apparently evokes a pure climbing fibre input. This finding emphasizes the importance of climbing fibre input as a pause producing mechanism and in view of the fact that climbing fibres establish much more extensive contact with Golgi than with basket cells (Palay & Chan-Palay, 1974) it also suggests that Golgi action is a major element in pause production. If the delayed responses indeed arise via the disinhibitory mechanism proposed, then the wide range of latencies, amplitudes and durations observed is readily accounted for on the basis of the variations which exist in P cell response pattern together with the complex and specific pattern of spatial localization which exists in the highly convergent projection from the P cells to the nuclear neurones (Voogd, 1964; Chan-Palay, 1977; Armstrong & Schild, 1978). Whether the observed reductions in P cell discharge provide a complete explanation for the

delayed facilitations in ip. remains uncertain but there are other possibilities which require brief

discussion. For example, it is possible that the responses are due in part to delayed excitation via some path or paths terminating within the nuclei. One such route might be provided by the rubro-interpositus projection of Courville & Brodal (1966) but this projection is said to be sparse and in our study the only sequel detected in ip. after single shock stimulation of r.n. was antidromic invasion. Current evidence in fact suggests that the majority of excitatory nuclear afferents arise as collaterals of cerebellar afferents which terminate in the cerebellar cortex. If delayed responses were due to input via these lines it is clear they should be accompanied by excitatory responses in the cerebellar cortex. However, as recounted above, the P cells in the intermediate cortex were markedly depressed during the late facilitations. In order to invoke such a mechanism it is therefore necessary to assume the existence of cerebellar afferents which supply collaterals to ip. whilst terminating in cortical regions other than those which project to ip. This is not impossible since there is evidence that the collateral projections are less spatially localized than the inputs to the cortex (Eccles, 1973). Indeed it may be recalled that in the present study a few neurones showed short latency excitations as their sole response to s.r. stimulation. One cerebellar afferent projection which might satisfy the necessary conditions is the mossy fibre input from nucleus reticularis tegmenti pontis (n.r.t.p.). Tsukuhara, Korn & Stone (1968), have demonstrated that stimulation of this nucleus gives rise to monosynaptic activation of interpositus neurones, whilst on the other hand the terminal portions of the n.r.t.p. axons may be distributed mainly to hemispheral and vermal cortex (Jansen & Brodal, 1954; Tsukuhara, 1972). In addition, ip.n.s monosynaptically excite the n.r.t.p. neurones (Tfukuhara & Bando, 14-2

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1970) so that the possibility therefore exists for a two-neurone reverberatory loop between ip. and n.r.t.p., the operation of which would be held in check by the inhibitory action on ip. of P cells in the intermediate cortex. Tsukuhara (1972) has demonstrated the potential power of this loop by administering picrotoxin to block the inhibitory action of the P cells. Under these conditions injection of a stimulus into the loop can produce repetitive discharges in ip.n.s which may last up to 500 ms. Because of the depression of many intermediate zone P cells in our animals the circumstances are favourable for activation of this reverberatory circuit which might be set in operation by the initial discharge of the ip.n.s or possibly by peripheral input reaching n.r.t.p.

Possible physiological roles for the interpositus responses to cutaneous input Ip. provides a very powerful excitatory input to r.n. (Toyama, Tsukuhara, Kosaka & Matsunami, 1970), so that the ip. responses should profoundly influence the discharge patterns of rubrospinal cells. Corroborative evidence comes from the work of Massion & Albe-Fessard (1964) who observed responses similar to ours in rubrospinal cells of unanaesthetized, paralysed cats. These responses were abolished by destruction of ip., which strongly suggests they were generated via the 'intermediate' cerebellum. While it is appreciated that our responses were obtained in quietly sitting animals and may therefore be highly modified in other circumstances by additional influences acting upon the cerebellum and related structures, it seems reasonable to consider that similar responses (or similar changes in excitability) could occur when phasic mechanoreceptive inputs from the paws are relayed to the cerebellum during natural active behaviour. In every day life, phasic activation of large numbers of mechanoreceptors will occur when the paw comes sharply into contact with a substrate as during manipulatory behaviour or during footfall in locomotion. They are also likely to occur at footlift because paw mechanoreceptors display 'off' as well as 'on' discharges (see, e.g. Eccles, Sabah & Taborikovai, 1974). Although our responses were generated by stimulation of s.r. which innervates receptors distributed mainly on the dorsum of the foot, a recent report (Loeb, Bak & Duysens, 1977) has shown that cutaneous mechanoreceptor afferents with receptive fields on the dorsum of the cat hind paw do discharge during locomotion and may in some cases provide precise information regarding time of footfall and lift. Furthermore in anaesthetized animals qualitatively similar responses are evoked in ip.n.s by volleys in a range of limb nerves (e.g. Armstrong, Cogdell & Harvey, 1975). Since the ip. responses are largely excitatory (see Fig. 5) any similar responses resulting from natural behaviour would lead to a prolonged facilitation of rubrospinal activity and thus, because of the flexor facilitatory action of this tract (Massion, 1967), to facilitation of the flexor apparatus in the spinal cord. Such facilitation as a result of footfall during locomotion might well contribute to the control of the flexor motoneurone activity which sustains the next swing (flexor) phase of the locomotor cycle. The delayed facilitations we describe ranged in latency from 25 to 80 ms and in duration from 50 to 500 ms, whilst according to Engberg & Lundberg (1969) the swing phase of the step in the cat hind limb begins about 200 ms after footfall during a normal walk and about 60 ms after footfall

during a gallop. Certainly, rhythmic increases in rubro-spinal activity occur immediately preceding and during swing phases in the mesencephalic cat provoked to walk on a treadmill (Orlovsky, 1972a). Furthermore, these modulations are abolished by

INTERPOSITUS RESPONSES 421 cerebellectomy and are also abolished or severely depressed by reducing the afferent inflow associated with movement. In addition Orlovsky (1972b, c) has detected corresponding bursts of activity in ip.n.s which are mirrored by depressions of the overlying P cells. The responses described in this paper might contribute to these modulations. Another possible role of the ip. responses is in contact-placing reactions. The reaction in which contact of the dorsum of the paw with an object is followed by a withdrawal-lifting action that brings the limb into position where the paw can be placed on top of the object, is either abolished (Chambers & Sprague, 1955) or severely impaired by destruction of nucleus interpositus (Amassian, Ross, Wertenbaker & Weiner, 1972). According to Amassian et al. flexor activity in this reaction begins about 20-150 ms after paw contact, and the activity appears to last on average for about 200-300 ms. Many of the present ip. responses parallel the time course of this activity. It is also worth noting that cooling of ip. through an implanted probe, leads to a dramatic increase in the latency of flexor activity associated with the reaction (Amassian et al. 1972). Since some ip.n.s are bilaterally activated it is possible that the nucleus may contribute to the formulation of contralateral as well as ipsilateral placing reactions. Supported by the Medical Research Council. The Authors wish to thank Mrs B. Colfer for photography. REFERENCES

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THACH, W. T. (1970). Discharge of cerebellar neurons related to two maintained postures and two prompt movements. I. Nuclear cell output. J. Neurophypiol. 33, 527-536. TOYAMA, K., TSUKUHARA, N., KoSAIA, K. & MATSUNAMI, S. (1970). Synaptic excitation of red nucleus neurones by fibres from interpositus nucleus. Expl Brain Res. 11, 187-198. TsuxEuuARA, N. (1972). The properties of the cerebello-pontine reverberatory circuit. Brain Res. 40, 67-71. TsuKuHARA, N. & BANno, T. (1970). Red nuclear and interposate nuclear excitation of pontine nuclear cells. Brain Res. 19, 295-298. TsuxuaAmA, N., KORN, H. & STONE, J. (1968). Pontine relay from cerebral cortex to cerebellar cortex and nucleus interpositus. Brain Res. 10, 448-453. VOOGD, J. (1964). The CerebeUum of the Cat. Structure and Fibre Connexins. Assen: Van Gorcum. Yu, J., TARNEcxi, R., CHAMBEmS, W. W., Liu, C. N. & KoNousI, J. (1973). Mechanisms mediating ipsilateral limb hyperflexion after cerebellar paravermal ablation or cooling. Expl Neurol. 38, 144-156.