Brain Research, 106 (1976) 1 20

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© ElsevierScientificPublishing Company, Amsterdam - Printed in The Netherlands

Research Reports

C L I M B I N G FIBER RESPONSES OF C E R E B E L L A R P U R K I N J E CELLS TO PASSIVE M O V E M E N T OF T H E CAT F O R E P A W

D. S. R U S H M E R , W. J. ROBERTS AND G. K. A U G T E R

Neurological Sciences Institute of Good Samaritan Hospital and Medical Center, 1015 N.W. 22nd A venue, Portland, Oreg., 97210 (U.S.A.) (Accepted September 8th, 1975)

SUMMARY

The activity of cerebellar Purkinje cells during controlled and passive movement of the forepaw was studied in the cat. Burst responses characteristic of activation by climbing fibers were observed in Purkinje cells in lobules Vb and Vc of the cerebellar vermis and paravermis. The climbing fiber responses followed the onset of a movement with a latency ranging from 20 to 60 msec depending upon movement type and amplitude. Responsive Purkinje cells were localized in a well defined parasagittal strip very near the paravermal vein in lobules Vb and Vc. Cells within the responsive strip responded with identical response probabilities and latencies for any particular type of movement presentation. Responses were independent of starting paw position and direction of movement. Climbing fiber responses could be evoked by extremely small movements with most cells responding to displacements of 50/~m. The latencies and probabilities for climbing fiber responses were inversely related to movement amplitude with latencies as long as 80 msec for very small displacements.

INTRODUCTION

The neural circuitry of the cat cerebellum and its input pathways have been studied in detail, both morphologically and electrophysiologically. The responses of cerebellar neurons to electrical stimulation of peripheral nerves have been documentedS-l°,13,1a,26, 27,3°,35 as have their responses to natural stimulation of selected sensory modalities3,17,lS,2°,42,45-4L Although these experiments have yielded much information about the organization and properties of the cerebellar input pathways and the responses of cerebellar neurons to their activation, two important aspects of cerebellar

organization have not yet been addressed. First, experiments dealing with Purkinje cell responses to naturally coded inputs have addressed only individual modalities of sensory information 16,34. This approach ignores the possibility that the cerebellar cortex may be designed to simultaneously process information from a variety ~f sensory receptors all monitoring the state of motion of a limb. Second, the spatial and temporal distribution of cortical responses have been studied topographically in terms of active and inactive regions ; however, variations that may occur within a responsive region have not been systematically explored. The present experiments were designed to measure the responses of cerebellar Purkinje cells to controlled passive movements of the intact forepaw in cats. This paper describes the spatio-temporal distribution of climbing fiber responses to this naturally encoded, multimodal input and presents a functional interpretation for these responses. Topographical localization and description of 'simple spike' responses of Purkinje cells due to activation of the mossy fiber-granule cell-parallel fiber pathway, and the interaction of these two inputs in cerebellar information processing will be discussed in a separate publication (in preparation). METHODS

Surgical procedures Experiments were performed upon 43 cats weighing 2.5-3.0 kg. In 21 of these animals, an initial peritoneal dose of 30 mg/kg pentobarbitol was administered followed by supplemental intravenous doses given upon return of corneal reflexes. The remaining animals were initially anesthetized with halothane and subjected to a midcollicular decerebration. The decerebration was accomplished by slow insertion of a blunt instrument between the superior and inferior colliculi. No attempt was made to spare the pyramidal tracts, a technique suggested by Eccles et al. 15 to increase spontaneous cerebellar climbing fiber responses in this preparation. Halothane anesthesia was discontinued immediately following completion of the decerebration. The decerebrate animals were paralyzed with gallamine triethiodide and artificially respired only when reflex stepping and other movements interfered with recording. After decerebration, lobutes IV, V, and VI of the cerebellar cortex were then exposed from the midline to the lateral edge of the pars intermedia cortex on the side ipsilateral to the stimulated paw. Upon removal of the dura mater, the cerebellar cortex was covered with a layer of Ringer's-agar and continuously moistened with Ringer's solution. warmed to 38 °C -- 2 ~C throughout the recording period to maintain normal cortical temperature. Arterial blood pressure was monitored and expired COz concentration was maintained at 4 - 5 ~ . Animal temperature was maintained at 37.5 °C, measured rectally. Stimulation techniques The animal was placed in a prone position in a stereotaxic frame with the right forepaw on a small platform attached to a solenoid device (Fig. 1). The plantar surface

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Fig. 1. Diagram of experimental setup. of the paw rested on the platform with no other physical restraints, although the arm was taped loosely to the table below the elbow. The silent solenoid device was especially designed for this application (Dipl. lng. Detlef Burchard, Germany) with a flat frequency response of 0-65 Hz. This device contained feedback circuits which allowed computer control of either displacement or force. The net force acting on the paw was obtained by subtraction of the force needed to move the unloaded platform from that required to move both the platform and the paw together. Actual forces applied to paw receptors could be different from those measured due to the mechanical properties of the intervening tissue.

Recording techniques Extracellular single unit recording was accomplished using glass micropipettes filled with 3 M NaC1 or Tungsten microelectrodes (Frederick Haer, Inc.). The former had tip diameters of approximately 1.0 # m and an impedance of 8-12 MfL A capacity neutralized, AC-coupled high impedance preamplifier and other conventional recording equipment was used. Recording microelectrodes were mounted on a Kopf hydraulic microdrive and stereotaxic frame. Electrode position on the surface of the cerebellar cortex was observed with a surgical microscope and the stereotaxic coordinates of each sampled cell were recorded. The locations of individual cells in relation to each other could be computed using visual observation and stereotaxic coordinates. Subsequent histological verification of electrode tracts was accomplished using 15 # m parasagittal sections mounted and stained with Luxol fast blue. A PDP-12 digital computer was used for analysis of single unit data and generation of the input signals for the solenoid driver. Computer programs allowed the on-

line computation of interspike interval histograms (IH) to assay spontaneous activity and peristimulus time histograms (PSTH) to establish correlation between single unit activity and movement presentation. Subroutines for high frequency burst recognition were included which permitted the separation of Purkinje cell climbing fiber responses from the simple spike activity due to excitation via the parallel fibers. These subroutines discriminated CFRs from single spike responses on the basis of interspike interval. In general, bursts of spikes with interspike intervals of tess than 5 msec were classified as CFR bursts. Each burst response was counted only once in computing the histogram regardless of the number of spikes contained within it. Separate histograms were computed for each response type. While peristimulus histograms were being computed, the computer generated voltage waveforms for movement presentation. Three types of movement waveform could be generated: (1) a linearly rising 'ramp' voltage reaching a plateau followed by a linear fall to baseline (see Fig. 2A2); (2) an exponential rise to a plateau followed by an exponential fall (see Fig. 2B2); and (3) a pair of 10-msec rectangular pulses. The rectangular pulses were attenuated by the solenoid (see Fig. 2C2). Duration of the waveform plateau (or interpulse interval in the case of the pulse pair), waveform amplitude and baseline position could be specified under program control. Movements were generally presented to the paw at a rate of 1 every 2 sec. Raw single unit data and pulses for movement presentation were recorded on analog tape. Histograms were stored on digital tape.

Cell identification Purkinje cells were identified electrophysiologically by the unique burst response of these cells to activation by climbing fibers 21. In the decerebrate preparations in which climbing fiber responses sometimes failed to assume the burst configuration observed in anesthetized preparations, it was sometimes necessary to use the larger positivity associated with CFRs to distinguish them from simple spikes 48,49. RESULTS

Responses of Purkinje cells to climbingfiber synaptic input The responses of a total of 220 Purkinje cells were studied in lobules Vb, c, d, and lobute VI of cerebellar vermis and paravermis of 33 cats. In 132 cells, passive movement of the ipsilateral forepaw, using any of the 3 modes of movement presentation evoked climbing fiber responses from Purkinje cells (Fig. 2A~, B1, C1). The climbing fiber responses usually followed the onset of movement with a latency ranging from 20 to 60 msec depending on movement type and amplitude as shown in the PSTHs of Fig. 2 (Fig. 2A3, Bs, Ca). All histograms shown were computed from Purkinje cell responses to 100 movement presentations. The spontaneous firing level can be determined from the initial 60 msec of the PSTH before movement presentation has begun. Simple spike histograms are not shown. The 4 dashed lines are markers used to denote (1) onset of movement, (2) attainment of plateau position, (3) onset of return movement, and (4) attainment of baseline, respectively, except in Fig. 2C, where the markers denote the occurrence of the two 10-msec square wave movements.

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Fig. 2. Climbing fiber responses to passive forepaw movements. Trace 1 is in each case an extracellular recording from a single Purkinje cell showing climbing fiber burst responses in a pentobarbital anesthetized cat. Trace 2 is platform position recorded during 3 different movement presentations. Upward deflection indicates dorsiftexion. The maximum excursion is 3 mm in A and B and 2 mm in C. Trace 3 is the peristimulus time histogram of the climbing fiber responses to the 3 movements. The time base noted in A is the same in all traces. The binwidth is 1 msec. The C F R s which c o u l d be e v o k e d by small p a w m o v e m e n t s could also be e v o k e d by lightly t a p p i n g one or m o r e o f the f o o t p a d s in every case tested. M e c h a n i c a l s t i m u l a t i o n o f the f o r e p a w hair was often ineffective as was m a n u a l r o t a t i o n o f the ankle j o i n t , p r o v i d e d t h a t no pressure was a p p l i e d to the f o o t p a d s . The C F R s e v o k e d by f o o t p a d pressure were r a p i d l y a d a p t i n g in t h a t the response to an a p p l i e d a n d maint a i n e d pressure was m o s t c o m m o n l y a single C F R . The n o t a b l e exceptions were the C F R ' d o u b l e t s ' occurring at an i n t e r b u r s t interval o f a b o u t 20 msec, to be described below. A l l o f these tests were n o t a p p l i e d to each cell since the p r i m a r y interest centered on m u l t i m o d a l response characteristics.

Localization of climbing fiber responses Purkinje cells which exhibited these climbing fiber responses were localized in a sagittally oriented strip o f cerebellar cortex a p p r o x i m a t e l y 4 m m f r o m the midline ipsilateral to the m o v e m e n t . Fig. 3A is a schematic r e p r e s e n t a t i o n o f a d o r s a l view o f the cerebellar cortex showing t h a t the responsive strip is very near the p a r a v e r m a l vein in lobules Vb a n d Vc. The strip is a p p r o x i m a t e l y 0.6-1 m m in width a n d is a p p r o x i m a t e l y 9 m m in length in the p a r a s a g i t t a l direction (Fig. 3B). Purkinje cells giving low t h r e s h o l d C F R s to p a w m o v e m e n t were n o t f o u n d in areas either m e d i a l or lateral to this p a r a s a g i t t a l strip in lobules V b a n d Vc or in l o b u l e VI. O c c a s i o n a l cells were

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Fig. 3. Location of responsive forepaw strip. Diagram A is a schematic representation of a dorsal view of cat cerebellar cortex, right side, lobules V and VI. The shaded cortical strip having climbing fiber responses is centered in the pars intermedia under the paravermal vein. The strip illustrated is a composite of the responsive regions in all cats studied and is wider than that found in any one animal. Diagram B is traced from a parasagittal section made through the responsive strip in one cat. The crosshatched region was responsive to forepaw movements. Recording was not attempted in Va or rostral Vb.

f o u n d in these other areas, however, t h a t r e s p o n d e d with C F R s to high intensity taps or pressure on the forelimb. R e c o r d i n g was not a t t e m p t e d in lobule Va. L o c a t i o n s o f responsive Purkinje cells in two animals are s h o w n in the schematic d r a w i n g o f the u n f o l d e d cortex o f lobules Vb a n d Vc in Fig. 4A. I n these p a r t i c u l a r instances the strip was less t h a n 1 m m wide a n d m o r e t h a n 9 m m long. F o u r representative cells f r o m the strip in one cat, all r e c o r d e d f r o m one e l e c t r o d e track, are localized in the p a r a s a g i t t a l section in Fig. 4B d r a w n f r o m histological sections. The m o s t c a u d a l cell here was unresponsive b u t the rostral extent o f the strip was n o t determined. D u r i n g searches for Purkinje cells responsive to p a w m o v e m e n t s , it was f o u n d in two a d e q u a t e l y tested animals t h a t some cells l o c a t e d 200-400/~m lateral to the ' p a w ' strip w o u l d r e s p o n d to t a p s delivered to the foreleg a b o v e the paw. I n the 3 animals tested, searches medial to the ' p a w ' strip revealed C F R s in response to t a p s o f the ipsilateral hindleg a b o u t 500/~m f r o m the ' p a w ' strip. I n the one cat studied using

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Fig. 4. Location and response characteristics of Purkinje cells within the forepaw strip. Map A sho0vs the relative locations of responsive and unresponsive Purkinje cells from 2 cats. The map represents the unfolded Purkinje layer in pars intermedia. The cells denoted X were from the second cat. The medio-lateral location of this population was shifted arbitrarily in order to align with the responsive cells in the first cat. B shows the location of 4 Purkinje cells from one cat as viewed in parasagittal histological sections. The CF responses of the 3 cells denoted C, D, and E obtained with a 3-ram ramp forepaw movement are shown in the histograms C, D, and E respectively. The numbers in the histograms give the number of CFRs for 100 movements. Note the similarity in response probability and latency in the 3 widely spaced cells. Pentobarbital preparation.

a l p h a - c h l o r a l o s e anesthesia, individual Purkinje cells in the lateral vermis or p a r a vermis in l o b u l e V could be activated by weak m e c h a n i c a l s t i m u l a t i o n o f most o f the ipsilateral b o d y surface, an effect a t t r i b u t e d to the anesthetic.

Climbing .fiber response characteristics Purkinje cells within a responsive strip all r e s p o n d e d with the same response p r o b a b i l i t i e s and latencies for any p a r t i c u l a r m o v e m e n t precentation. The peristimulus h i s t o g r a m s o f Fig. 4C, D, a n d E d e m o n s t r a t e t h a t the latencies and p r o b a b i l i t i e s (in r e s p o n s e to the same p a w m o v e m e n t s ) are essentially identical for those 3 widely spaced cells. In response to m o v e m e n t s greater t h a n 1 mm, the p r o b a b i l i t y and l a t e n c y o f the C F R s were quite similar in all a n i m a l s p r e p a r e d in the same way (e.g., for the 21 cells studied with a 3 - m m r a m p m o v e m e n t in decerebrate cats, response p r o b a b i l i t y was 0.8-1.0, m e a n latency was 25.9 msec, and S.D. was 3.2 msec). F o r r a m p m o v e m e n t s o f less t h a n 1 mm, the latencies a n d p r o b a b i l i t i e s showed greater v a r i a t i o n between animals. The responses to a r e t u r n m o v e m e n t i m p o s e d less t h a n 200 msec after an initial m o v e m e n t also s h o w e d greater variability (cf Figs. 2, 4, 5, 7, 8 a n d 10). In m o s t P u r k i n j e cells, the climbing fiber responses to the initial d y n a m i c pha~e

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Fig. 5. Climbing fiber responses to plantar and dorsal movements from different starting positions in one Purkinje cell. The 4 histograms were obtained with two dorsal-plantar movements (A and 13) and two plantar-dorsal movements ((2and D) illustrated by the interrupted lines. Note tbedifferent starting positions. The numbers of CFRs in each of the prominent peaks are shown, each for t00 movement presentations. Note also the relatively high rate of spontaneous and stimulus-related CF activity in this decerebrate preparation and the similarity of responses to the different movements. of any 3-mm ramp movement were essentially independent of both the starting paw position and the direction of movement as illustrated in Fig. 5. Response probabilities and latencies of the Purkinje cell shown here were essentially identical for the 4 different stimuli illustrated: two upward movements which had starting positions 3 m m apart (A and B); and two downward movements from different starting positions (C and D). The forces imposed on the paw foot pads during the passive movements used in this experiment were considerably less than those found in normal locomotion. The m a x i m u m transient force developed was just under 100 g for a 3-mm ramp displacement and only 2 g during the 50-#m movement (Fig. 6). In contrast, the force developed during walking in a 3-kg cat reaches a maximum of about 2 kg in 80 msec a2. Note that the forces imposed on the footpads are related to both platform position and velocity for these ramp inputs (position waveform given by interrupted line below force records). The times of occurrence of the C F R for each of the 4 inputs are indicated by the gaps in these force records. Latencies and probabilities of climbing fiber responses were inversely related to ramp movement amplitude as shown in Fig. 7. Movement amplitudes of 3 m m resulted in climbing fiber response latencies ranging from 25 to 30 msec as shown in Fig. 7A. As the displacement amplitude is decreased (Fig. 7B-F), the response latency becomes longer and the probability for response occurrence decreases. Thus, in Fig. 7F, the latency for response to a 50-ffm movement is approximately 80 msec, although this very small movement was adequate to evoke climbing fiber responses with a probabili-

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Fig. 6. Forces on the plantar forepaw surface during ramp movements of different amplitudes. The force exerted by the foot on the supporting platform is plotted as a function of time for movements whose time course is shown by the interrupted line below the force records. The movement amplitude relative to each force record is given. The gap in each force record shows the time of occurrence of the CFR in one Purkinje cell for the respective movements in this decerebrate preparation. The force trace labeled 'walking' is an approximation of the force exerted by the forepaw of a walking 3-kg cat on the same time scale (see text). ty o f 0.39 in this animal. The g r a p h in Fig. 7 G d e m o n s t r a t e s (a) t h a t the C F R latencies in d e c e r e b r a t e p r e p a r a t i o n s are less t h a n those observed in p e n t o b a r b i t a l p r e p a r a t i o n s , a n d (b) t h a t as the a m p l i t u d e o f the m e c h a n i c a l stimulus is increased the C F R latency a s y m p t o t i c a l l y a p p r o a c h e s a m i n i m u m . This m i n i m u m is on the o r d e r o f 18-20 msec a c c o r d i n g to the impulse response d a t a f r o m o u r studies in d e c e r e b r a t e p r e p a r a t i o n s (see Fig. 10). Results o f a n e x p e r i m e n t designed to d e m o n s t r a t e excitability changes in the olive following a c o n d i t i o n i n g climbing fiber response are shown in Fig. 8. I n a p e n t o b a r b i t a l p r e p a r a t i o n , a 10-msec ' c o n d i t i o n i n g ' m o v e m e n t o f 1 m m a m p l i t u d e was p r e s e n t e d to the p a w every o t h e r second. A n identical ' t e s t ' m o v e m e n t followed each ' c o n d i t i o n i n g ' m o v e m e n t at v a r i o u s latencies. The p r o b a b i l i t y for climbing fiber responses following the ' c o n d i t i o n i n g ' m o v e m e n t a p p r o a c h e d 1.0 in all h i s t o g r a m s o f the figure a n d the C F R l a t e n c y was 20-22 msec following the ' t e s t ' m o v e m e n t at intervals between ' c o n d i t i o n i n g ' a n d ' t e s t ' p r e s e n t a t i o n s o f longer t h a n a p p r o x i m a t e l y 100

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Fig. 7. Climbing fiber responses in a Purkinje cell to movements of different amplitudes. The histograms A-F were obtained with 6 different movements illustrated by the respective interrupted lines and amplitude notations. The numbers near the CFR peaks give the number of evoked CFRs from the onset of the dorsiflexion movement (vertical dashed line) to the beginning of the plantar flexion movements (100 presentations). Note the increasing latency and decreasing probability of response associated with decreasing movement amplitude. The graph G indicates the mean CFR latency as a function of the linear movement amplitude (dorsiflexion) as obtained from Purkinje cells in 10 decerebrate and 5 pentobarbital anesthetized cats. Note that the response latency is nearly independent of amplitude for the larger movements. msec (Fig. 8A). The response probability for the test m o v e m e n t is much less for shorter intervals (Fig. 8B, C and D). The results o f this experiment for 3 different animals are s h o w n graphically in Fig. 8E. Variation between animals was high, but the c o m p o s i t e result is consistent with the observed m a x i m u m following frequency o f 10-15 responses/sec in the climbing fiber system as seen by other experimenters 4.1~ N o t e the 20-msec latency difference in the C F R following the test m o v e m e n t s in A and B. This bimodal latency distribution is consistent with that observed in decerebrate preparations as described below. In decerebrate preparations a ' C F R doublet' was frequently observed to follow

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Fig. 8. C l i m b i n g fiber response p r o b a b i l i t y f o l l o w i n g previous C F activity. T h e histograms A D were o b t a i n e d f r o m a single cell in a p e n t o b a r b i t a l cat in response to b r i e f c o n d i t i o n i n g and test 2-ram movements illustrated by the interrupted fine in A. T h e vertical dashed lines denote the times o f presentation o f the 2 movements. T h e interval between the onsets o f each m o v e m e n t pair is listed. N o t e the decreasing p r o b a b i l i t y o f response to the later test m o v e m e n t as the Jnterstimu]us interval is decreased. G r a p h E gives the n u m b e r o f C F R s in response to 100 presentations o f this test m o v e m e n t in 3 P u r k i n j e cells f r o m 3 cats, each denoted by a different symbol. The dashed line in E connects the data points f r o m the cell illustrated in A - D .

a single brief paw movement. The extracellular unit record in Fig. 9A exemplifies one such doublet in which the first CFR began 18 msec after the onset of a single pulsatile paw movement of 10-msec duration (not shown). The two identical burst responses occurred at an interburst interval of 23 msec. The activity in this Purkinje cell to two pulsatile movements is shown at a slower sweep speed in Fig. 9B. The doublet response to the first movement (x ÷ y) was followed by a single C F R after the second movement (z) occurring at longer (38 msec) latency similar to that observed in Fig. 8B. The

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TIME (10 msec/div) Fig. 9. Climbing fiber doublet response to single movement presentation. Trace A is an extracellular recording from a Purkinje cell. The first of the two complex climbing fiber responses followed by 18 msec the onset of a brief 10-msec, 2-ram movement identical to that illustrated in Fig. 8 (not shown here). The first and second CFRs in A correspond to the CFRs denoted X and Y respectively in the slow sweep speed record in B. The times of presentation of the two movements made during the recording in B are shown by the short dashed lines below the record. Note that the two CFRs at an interburst interval of 23 msec follow the first movement. The CF histogram obtained from the same cell is shown in C. The numbers of CFRs in each of the 3 peaks are given. These peaks correspond in time to the responses in B labeled X, Y and Z. The vertical dashed lines in C denote the two movement presentations. Decerebrate preparation. P S T H in Fig. 9C describes the C F activity to 100 consecutive pairs of movement. These C F R doublets could also be evoked by the other types of m o v e m e n t presentations utilized (see Fig. 2) a n d were n o t c o n d i t i o n a l u p o n the p r e s e n t a t i o n of two consecutive force changes at the 20-25 msec i n t e r b u r s t interval. Olivary excitability was tested in the decerebrate p r e p a r a t i o n using a procedure identical to that described above. I n this p r e p a r a t i o n as i n the p e n t o b a r b i t a l preparation, the latency for the initial climbing fiber response was 20-22 msec a n d the probability for response approached 1.0 following the c o n d i t i o n i n g m o v e m e n t a n d long latency test m o v e m e n t s (Fig. 10A). The results of these experiments were complicated, however, by a second peak of C F R probability with a latency of 40-45 msec

13 following movement onset as in Fig. 9C. This late response is seen as a peak with lower response probability than that of the early response. As the interval between the conditioning and the test movements is decreased, the response probability of the early climbing fiber response to the test movement decreases to nearly zero (Fig. 10D) like that in the pentobarbital preparation (Fig. 8B). As that probability decreases, the response probability for the late response increases (Fig. 10B-D) until such time as the conditioning-test movement interval is so short that both test responses fail to appear (Fig. 10E). It was observed that climbing fiber responses in the decerebrate preparation often did not take their classic form (i.e., an initial full-size spike followed by a burst of smaller spikes), but were sometimes observed as a single full-size bi- or triphasic spike followed by a small amplitude oscillation or as a short burst of full-size spikes. Fluctuations in the form of the CFR potential for a single recording were sometimes observed to correlate with spontaneous changes in the background simple spike rate. The classic CF burst occurred during periods of low simple spike rate when the Purkinje cell is presumably more hyperpolarized as found by Martinez et a l Y . Either non-classic configuration could be converted to the classic form by infusion of pentobarbital. The single spike configuration was similar to those noted by Thach 4s and Van Gilder and O'Leary 49. DISCUSSION

The data obtained in this study describe the stimulus-response characteristics of climbing fiber mediated inputs to Purkinje cells following mechanical movement of the forepaw in cats. Before describing the spatial localization of the evoked activity cr its functional implications, it is instructive to review the stimulus-response characteristics. Stimulus-response characteristics

Climbing fiber responses (CFRs) in Purkinje cells located within the ipsilateral forepaw strip (see below) could be evoked in both unanesthetized decerebrate or pentobarbitol anesthetized cats by very small passive movements of the paw. The C F R threshold was generally less than 100 #m, in agreement with the findings of Eccles et al. 1~ and Leicht et al. zs. The maximum transient forces exerted on the footpads by such 100-/~m movements in our study with only the plantar foot surface restrained are < 1 0 g wt., probably somewhat less than that which occurred with similar stimuli in the experiments of Eccles et al. x5 and Leicht et al. zs in which the foot dorsum was restrained. We conclude, in agreement with the above authors that the extreme sensitivity of this pathway to pressures on the footpads ensures that these CFRs will commonly occur during locomotion and exploring movements unless the pathway is gated off (see below). This view contrasts with the previously stated view that all spino-olivocerebellar pathways are remarkably difficult to activate by natural stimulation25, 3v although responses to foot tapping were reported in those publications.

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Fig. 10. Climbing fiber doublet responses to brief test movement following conditioning movement. A - E are CF histograms from one Purkin)e cell in a decerebrate preparation. The times of occurrence of the 10-msec, 2-mm movements are shown by the vertical dashed lines. The numbers of CFRs in each of the peaks are given. Note that a C F R doublet commonly occurred in response to the earlier conditioning movement. As the interval between the conditioning and test movements was decreased, the probabilities of response in the early and late peaks following the test movement changed inversely.

!5 The CFRs to small passive forepaw movements were readily evoked by both dorsal and plantar movements as illustrated in Fig. 5. Thus, unless gating intervenes, such responses are likely to occur after footfall as well as lift-off. This bidirectional response was also noted by Eccles et al. 1~. Failures may occur, however, for rapidly occurring events as noted in most previous studies of climbing fiber responses since the probability of occurrence of CFRs for weak inputs decreases at interstimulus intervals less than 120 msec (see Fig. 8). It was observed in our studies and by others 1~,28 that some CFRs could be evoked by footpad pressure changes on one side of the ipsilateral foot but not the other. In our paradigm the entire plantar surface was mechanically stimulated so that any spatial organization within the forepaw strip which might encode stimulus location on the foot was obscured. With this technique it appeared that essentially all Purkinje cells within the forepaw strip showed identical climbing fiber responses. The latency and probability of occurrence of CFRs within the forepaw strip were inversely related to the imposed movement amplitude or force for very small movements (eft Fig. 7). For more intense stimuli, but still weak relative to those which would occur during locomotion, the probability of occurrence of CFRs was near unity and the latency was independent of stimulus magnitude ( c f Fig. 7G). Therefore, the climbing fiber system in these preparations appears unsuitable for discriminating the magnitude of forces or movements applied to the foot during locomotion. The noted characteristics of the CFRs evoked by paw movements are most consistent with the responses expected from the dorsolateral funiculus spino-olivocerebellar pathway (DLF-SOCP) described by Larson et al. 2~. They noted that responses through that pathway were sometimes evoked by tapping footpads, and the location of their responsive strips agree with the present study (see Fig. 3). The latency of the CFR from the onset of the pulsatile movements (18-20 msec) is identical to the latency of field potentials following foreleg nerve stimulation with only the DLF-SOCP intact 2~. One would expect, however, the response latency of Purkinje cell responses to be somewhat longer for mechanical stimulation of the footpads than for electrical stimulation of the nerve more proximally if both are mediated by the DLF-SOCP. The response latency in our study is perhaps more consistent with mediation by the faster dorsal funiculus spino-olivocerebellar pathway (DF-SOCP) 36 which projects to the same cortical area; however, Oscarsson reported responses in that pathway being evoked most commonly by deep pressure and only sometimes by tapping against the paw. Therefore, the responses observed in the present study are not completely consistent in either of these pathways as previously described. We suggest instead that several pathways may be simultaneously involved and that interactions between them may occur. One characteristic of the CFR not previously reported is the CFR doublet which often occurred in our decerebrate cats (Figs. 9 and 10). The interval between the first and second CFRs to a single 10-msec pulsatile movement was 20-25 msec. Similar CFR doublets appear in Figs. 6 and 7 of Eccles et al. 1~ but those authors do not comment upon them. These CFR doublets were never observed in pentobarbital anesthetized cats; however, the latency shift which was noted in Fig. 8A and B for

16 the test movements was also 20-25 msec. This observation suggests that the mechanism responsible for the CFR doublets is also functional in the anesthetized cats but that the excitability of the olivary cells or of cells in the spino-olivary pathways is depressed by the anesthetic and prevents the doublets from occurring. The second burst in the CFR doublet does not appear to result from a recurrent excitatory phenomenon since the probability of occurrence of the second response is inversely related to the first (see Fig. 10). It is also unlikely that the double response mirrors conduction through the faster DF-SOCP followed by the DLF-SOCP, since the mean latency difference reported with electrical stimulation through those pathways is 7 msec 37, not 20-25 msec. Instead, this doublet appears to result from the influence of two sequential excitatory influences occurring 20-25 msec apart. In unanesthetized cats both may produce climbing fiber spikes; however, the inhibition which follows the first spike tends to suppress responses to the later excitation. We have found no evidence in the literature to indicate that two periods of excitation at that interval do occur following a single stimulus to mechanoreceptors 23,2s,34, peripheral nerves 5, or the cerebral cortex1,6,33 except for the extracellular CFR doublets seen in this study and in the records of Eccles et al. J5 noted above. Localization o f climbing fiber responses

Climbing fiber responses to passive ipsilateral forepaw movements are consistently found in a narrow parasagittal strip in lobutes Vb and Vc generally under or very near the paravermal vein (Fig. 3). In our most thorough explorations, this forepaw strip was found to be about 0.6 mm wide, bounded medially and laterally by unresponsive cells (Fig. 4A). A minimum estimate of the unfolded length of the strip is 9 mm, based on the data from two cats, in lobules Vb and Vc although actual strip length could be considerably longer as we did not study lobule Va in these experiments. We did not find the responsive cells to be organized in 'colonies' as reported by Eccles et al. TM. Within this forepaw strip the CFRs to forepaw movements were remarkably uniform in their probability of occurrence and latency. No response gradations within the strip were detected. For the 3-mm ramp movements the probability of occurrence was essentially one and the standard deviation of the latency in a single animal was less than 2 msec. The location of the forepaw strip in the pars intermedia is consistent with the data of Oscarsson 36 and Larson et al. 25 obtained with electrical stimulation mediated through the dorsal funiculus and dorsolateral funiculus spino-olivocerebellar pathways (DF-DLF-SOCP). The width of the forepaw strip is somewhat less than indicated in those studies which included nerves of the entire forelimb and less than the responsive area found by Murphy et al. ~4 using forelimb muscle stretch. As noted earlier, we found in some animals that the more proximal parts of the ipsilateral forelimb were mapped lateral to the forepaw strip, and the hindlimb medial to it 37. Functional conclusions

The response characteristics of the climbing fiber system to passive forepaw

17 movements in the cat suggest the following functional role for this system relative to locomotion 39,4°. The climbing fiber system projecting to the ipsilateral forepaw strip in pars intermedia serves as an 'event detector' to signal that the foot has touched down or lifted off from the weight-bearing surface. The information conveyed appears to be relatively independent of the quality, magnitude or direction of the stimulus received by the plantar surface for situations comparable to locomotion. Thus, this part of the climbing fiber system seems best suited to indicate only the time of occurrence of external events taking place at the plantar surface. Our data indicate that this response would likely occur at least twice per locomotor cycle (3 or 4 times if doublets occur) rather than once as suggested by Armstrong 4. Our data are consistent with other data cited earliera3,48, 49 which indicated that a single spike is sometimes propagated down the Purkinje cell axon for each event in unanesthetized cats. This event marker concept is otherwise similar to the functional hypothesis offered by Armstrong 4 who dealt also with the post-CFR activity. As noted earlier and elsewhere 4,13, this system is incapable of repeated activation at intervals much less than 100 msec. It is, however, capable of signalling both footfall and lift-off once per locomotor cycle for the fastest gait or gallop, since the minimum half-cycle has been found to be about 100 mseO 9. If one assumes that these climbing fiber responses evoked from exteroceptors in the foot are to be used in the regulation of movement (omitting for now consideration of the simple spike pause which may follow) then one limiting factor is the minimum response time. As demonstrated, the earliest Purkinje cell response to a mechanical stimulus to the foot is about 20 msec. The minimum time required for this response to be transmitted through the interpositus and red nuclei and to reach the foreleg motoneurons is about 4 msec 44. This is sufficiently fast to influence the ongoing phase of movement immediately following the forepaw 'event' ; however, this early response contains mainly information about the time of occurrence of the event, not about other aspects of performance. This view follows from the demonstration that the CFR in the forepaw strip is rather insensitive to the stimulus characteristics and from previous studies which indicated that the Purkinje cell response to a climbing fiber input is commonly a single axonal spike in thiopentol or unanesthetized catsaa,4s, 49 which, by its unvarying nature, is independent of previous inputs (of. however, refs. 11, 12 and 24). The value of such a response which may be modulated at any of the extracortical relays, is undetermined at this time. One other function consistent with the nearly synchronous activation of an extensive strip of Purkinje cells is the suggested 'phasic-control' system al. In this hypothesis, the abrupt change in Purkinje cell activity is utilized for step-function type corrections during particular movements. Our data suggest that if such step-function corrections do obtain, they tend to be identical for each half of each step cycle since all CFs would fire with each footfall and lift-off. The value of making motor corrections based upon an unvarying response (again neglecting variations in any pauses which may ensue) is yet to be demonstrated. Oscarsson a7 suggested that the CF system might in some way signal intended movement and the MF system the actual state of the limb. This concept was based on

18 his failure to demonstrate tow threshold CF responses to peripheral nerve stimulation. The present study suggests that the CF responses evoked in this system function to signal 'status' events rather than the 'intended' events, although the experiments of Allen et al. 1,2 which demonstrate projections of limb representation areas of motor cortex upon the cells of the inferior olive receiving peripheral inputs suggest that tile latter may occur as well. Evidence has been cited above indicating that the climbing fibers in the ipsilateral forepaw strip respond in an all-or-nothing fashion to plantar events and that the initial Purkinje cell response to this input may be a single axonal spike in unanesthetized animals. This stimulus-response relationship seems in our judgment to be too stereotyped to be of major value in correcting or modifying gait in response to external demands. Thus, it appears more useful to examine the 'resetting' action of this potent excitatory input on the Purkinje cell membraneT, 22,3'~,41,43 which will erase the effects of previous inputs and in this way prepare the Purkinje cells tk)r processing inputs occurring subsequent to the 'event marker'. The length of the pause in simple spike activity which generally occurs after a CFR may be the significant parameter which is used to modify succeeding movements'l,7,~5, 41. Because the end of the post-CFR pause generally occurs some tens of milliseconds after the CFR, it is less likely that pause information is used to influence the ongoing phase of movemc~it, but it would be appropriate for modulating subsequent phases. The functional role of pauses in simple spike activity will be discussed in more detail in a subsequent paper (in preparation). The data presented earlier indicate that the CFR will occur at least twice per step cycle in the forepaw strip. There is no data available to indicate whether, in fact, this occurs in a walking cat. Gating effects of descending tracts and peripheral inputs have been demonstrated which could block these responses 1,29. Further study of this possibility is presently being investigated in records of Purkinje cell activity during locomotion to determine whether the climbing fiber responses are as predicted by the present experiments or whether the responses are significantly different in the intact animal. The experiments of Orlovskii 3a were not a sufficient test of the 'event marker' concept since the recording was not done in the forepaw strip. ACKNOWLEDGEMENTS

The authors are indebted to Ronald Newton and Marianne Hegstrom for their assistance during the early experiments in this series. This work was supported by a grant from the National Institutes of Health (NS-02289-14) and a grant to W. J. Roberts from the Millicent Foundation.

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