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Responses of interpositus neurones to nerve stimulation in chloralose anaesthetized cats
Brain Research, 55 (1973) 461-466
461
© Elsevier Scienlific Publishing Company, Amsterdam - Printed in The Netherlands
Responses of interpositus neurones to nerve stimulation in chloralose anaesthetized cats
D. M. ARMSTRONG, BARBARA COGDELL* AND R. J. HARVEY Department of Physiology, University of Bristol, The Medieal School, Bristol BS8 1TD (Great Britain)
(Accepted March 8th, 1973)
Eccles et al. 6 have recently described the responses of neurones of the nucleus interpositus to stimulation of forelimb and hindlimb cutaneous afferents in decerebrate cats. The responses included two phases of excitation associated with cell discharges with latencies of about 6 and 19 msec. These were separated and succeeded by phases of inhibition. The first excitation was attributed to the action of collaterals of cuneo-cerebellar or direct spino-cerebellar axons and the second to collaterals of reticulo-cerebellar and olivo-cerebellar axons. The inhibitions were attributed to the action on the nuclear cells of the impulses from Purkinje cells 5 discharged by the afferent volleys. The present paper describes firing patterns of interpositus neurones following nerve stimulation in cats anaesthetised with a-chloralose. A somewhat surprising finding was that the cells did not always display the high level of spontaneous activity which previous authors have reported for intracerebellar nuclear neurones in unanaesthetised 15, decerebrate 6 or barbiturate anaesthetised s preparations. In addition the responses to nerve stimulation included a hitherto undescribed component consisting of a high-frequency burst of action potentials. The experiments were performed on 17 cats initially anaesthetised with ether or halothane followed by 60 mg/kg of recrystallised a-chloralose dissolved in propane1,2-diol. Some animals were paralysed with gallamine triethiodide and artificially respired. Recordings were usually begun about 7 h after the initial dose of chloralose. The left deep radial (DR), sural (SUR), femoral (FEM), gastrocnemius-soleus (GS) nerves and both superficial radial (SR) nerves were dissected and stimulated with single shocks at a repetition rate of 1/2 sec. Stimulus strengths were usually in the range 1-4 times the threshold of the most excitable fibres in the nerve, though stronger stimuli were occasionally used. Interpositus axons were stimulated in the region of the right red nucleus using a concentric stainless steel electrode. In 7 experiments a similar electrode was placed in the rostral part of the right inferior olivary nucleus. * M.R.C. Scholar.
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Its location was checked by observing potentials evoked on the cerebellar surface. All electrodes were positioned stereotaxically and their tip locations were subsequently confirmed histologically. Individual neurones in the left nucleus interpositus were recorded using glass micropipettes (filled with 4 M NaC1; tip diameter 1-2 #m). Action potentials were recorded photographically and on magnetic tape. Interval histograms of spontaneous activity and post-stimulus histograms were constructed using a Modular One digital computer (Computer Technology Ltd.). Recordings were made from 120 units responding to stimulation of one or more of the dissected nerves. Seventy-one units gave a fixed latency impulse following stimulation in the region of the red nucleus. The latency varied in different units between 0.5 and 1.5 msec when just supra-threshold stimuli were used. In many units the impulse evoked by midbrain stimulation was shown to be antidromic because it would collide with an appropriately timed action potential occurring spontaneously or in response to nerve stimulation. Such units were classified as nuclear efferent cells. For 49 units antidromic identification was not attempted, but these cells were located in the same region as those which were identified and gave similar responses to nerve stimulation. Recordings were usually begun around 7 h after the initial dose of chloralose, when, in most animals, the nuclear cells gave brisk responses to nerve stimulation but displayed little spontaneous activity. Some cells showed no spontaneous activity at all, others gave only one or two bursts of 5-10 spikes during a period of 15-20 min. Over the succeeding few hours, the level of spontaneous activity increased, apparently in parallel with the increasing frequency and intensity of chloralose seizures. This spontaneous activity was not caused by the movements taking place during the seizures as it was unaffected by paralysing the animals, and showed no temporal relation to the seizures. The initially low level of spontaneous activity could have been due to a nonspecific cause such as post-operative shock. However, it did not appear to be associated with any generalised depression of the central nervous system, and certainly Purkinje cells overlying the nucleus interpositus showed a high level of spontaneous activity. Moreover, when a dose of 2-20 mg/kg of pentobarbitone was administered intravenously, many nuclear cells began an almost continuous and fairly regular discharge at rates between 20 and 80/sec. Even when the nuclear cells already showed high levels of spontaneous activity, such a dose of pentobarbitone made the firing much more regular and also usually increased the mean frequency. Despite large quantitative differences in the responses of different nuclear cells to nerve stimulation, it was possible to distinguish a typical response pattern consisting of three phases: a short latency excitation, followed by a pause, in turn followed by a more powerful long-lasting excitation. If stimulation of one nerve evoked a response, then for most units, stimulation of other nerves also produced a response, except that stimulation of GS rarely produced any detectable effect even with strengths up to 10 times threshold for the nerve (see Fig. 1D, E and F). In 112 cells an excitation with latency less than 40 msec could be evoked from
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Fig. 1. A: responses of an interpositus nucleus neurone to a single stimulus to ipsilateral sural (SUR) nerve. The stimulus occurred at the beginningof the sweep as arrowed. The first phase of the response was a single spike with latency 30 msec. The third phase shows 62 spikes beginning 220 msec after the stimulus. B-J: post-stimulus histograms of interpositus neurones derived from 75 (B and C) or 50 (D-J) successive responses. In each case the horizontal axis is divided into 100 bins. The arrow at the left hand side of each histogram indicates the mean rate of spontaneous activity for the unit. Histograms based on many responses of the kind shown in A. B, C: response of unit illustrated in A evoked from ipsilateral SUR nerve. C is from same data as B, but on an expanded time scale. D, E, F: responses of another unit to stimulation of ipsilateral SR (ISR), deep radial (DR) and gastrocnemius-soleus (GS) nerves respectively. G, H, I, J: responses of another unit to contralateral SR (CSR) and contralateral inferior olivary (IO) stimulation. I and J are respectively from the same data as G and H, but on an expanded time scale. one or more peripheral nerves. The usual response consisted of one or two impulses (see Fig. 1A) b u t there was considerable variation between units. The unit shown in Fig. 1A, B a n d C gave 5 or more spikes in m a n y trials, b u t in other units the response occurred so infrequently that it could n o t be identified with certainty even on a poststimulus histogram (e.g. Fig. 1D a n d E). The latency of the earliest impulse following ipsilateral SR stimulation ranged from 6 to 34 msec in different units. The ranges for both the contralateral SR a n d the ipsilateral D R nerves were from 8 to 30 msec. Responses to h i n d l i m b nerves had longer latencies; the latency to S U R stimulation ranged from 15 to 35 msec. This first phase o f the responses appears to include b o t h excitations described by Eccles et al}. It was followed by a second phase in which the firing rate fe!l towards or often went below its pro-stimulus level (Fig. 1A, B, D, E a n d G).
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The third and most dramatic phase of the response of the interpositus neurones was a 'late burst' of action potentials (see Fig. 1A, B, D, E and G). This phase occurred in 105 cells. It had a mean latency of 150 msec. However, there was considerable variability in the latency and bursts occasionally began as early as 50 msec or as late as 500 msec after the stimulus. The duration and spike content of the burst varied considerably between units and even in a single unit the response to a constant stimulus was sometimes very variable. In some trials the burst failed to appear whilst at the other extreme the same cell might produce more than 70 spikes in a burst lasting up to 600 msec. The average amplitude of the burst also varied with the nerve stimulated (Fig. 1D and E). The bursts were abolished by small doses of pentobarbitone (2-10 mg/kg i.v.). The origin of the late burst was investigated by recording simultaneously from the cerebellar surface and from interpositus neurones. This showed that nerve stimulation evoked the expected potentials on the cerebellar surface including components mediated via mossy fibres (MF) of the cuneo-cerebellar, direct spino-cerebellar 5 and spino-reticulo-cerebellar2 paths and via climbing fibres (CF) of the spino-olivocerebellar 1A1 paths. Following these potentials there was a reduction in the neural noise recorded from the cerebellar surface which lasted for approximately the same duration as the interpositus late bursts. There were no mossy fibre or climbing fibre evoked potentials coinciding with or immediately preceding the late bursts in the nuclear cells. It thus seemed possible that despite their long latency the late bursts were not caused by a delayed input to the cerebellum but were a consequence of the train of events set up in the cerebellar cortex by the initial afferent input along the mossy and climbing fibres. The possible role of the input via the climbing fibres of the spino-olivo-cerebellar paths was investigated by directly stimulating this nucleus with single shocks of duration 0.3 msec or with two such shocks 2 msec apart. Stimulus intensity never exceeded 400 #A and was usually in the range 75-250 #A. 40 interpositus cells were studied, and in 27 the responses were directly compared with the responses to nerve stimulation. Olivary stimulation invariably evoked a response very similar in its timing and other characteristics to the late burst from nerve stimulation and, in spontaneously firing cells, this response was usually preceded by a reduction in firing rate (Fig. 1H). Comparison of Fig. 1G and H illustrates the marked similarity between the late phases of the responses evoked from the olive and from nerve stimulation. The main difference was that olive stimulation evoked a short-latency discharge in only a few of the units studied (compare Fig. 1I and J). The threshold for responses to olivary stimulation was low (around 100 #A) and the size of the late bursts was shown to increase in parallel with the stimulus current. Responses equal to those evoked from nerves were produced by currents between 150 and 300 #A. Our olive stimuli produced large CF evoked potentials on the cerebellar surface, but no MF responses were detected. In a sample of interpositus neurones the responses to olivary stimulation were directly compared with those which were produced when the electrode was moved to a position just beyond the dorsal or ventral limit of the olive. All these cells gave late bursts following olivary stimulation but stimulation outside the olive
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failed to produce any late bursts except when it produced CF evoked potentials on the cerebellar surface, presumably as a result of stimulating afferents to the olive. The low level of spontaneous activity in some interpositus neurones under chloralose raises the possibility that in the unanaesthetised animal some cells may be silent for long periods. In previous investigations6,S, a5 only tonically active units have been detected and it has been envisaged that the Purkinje cells modulate this tonic discharge of the nuclear cells by 'inhibitory sculpturing 's. The present results suggest that in addition there could be some cells which are only recruited when there is a marked reduction in the tonic inhibition exerted on them from the overlying cortex. Our findings confirm those of Eccles e t al. 6 with regard to short-latency discharges following nerve stimulation. In addition, D R stimulation at Gp lI strength has shown that deep as well as cutaneous receptors can evoke discharges at quite short latency. This is not surprising if reticulo-cerebellar collaterais are involved in generating such discharges (see ref. 12). However, the results of GS stimulation showed that single Gp II volleys in muscle nerves are not always effective. The second phase of the response described here corresponds in time with the later inhibition attributed by Eccles e t al. 6 to inhibitory action exerted on the nuclear cells by Purkinje cells discharged via the spino-reticulo-cerebellar path and the spino-olivo-cerebellar paths. Recordings from the cell bodies and axons of rubro-spinal neurones in chloralose anaesthetised cats have shown that nerve stinmlation evokes a brisk discharge similar to our late burstg, a3. Since interpositus neurones are known to excite rubrospinal cells powerfully 16, our results can be taken to demonstrate that the rubrospinal discharge is due to the late burst in nucleus interpositus neurones. The rubrospinal discharge has indeed been shown to depend on the integrity of nucleus interpositus and the cortex overlying it 9. The effects of olivary stimulation suggest that the third (or second and third) phases of the interpositus responses to nerve stimulation could be a consequence of activity mediated via climbing fibres from the inferior olive. In view of the long and variable latencies involved it is difficult to be certain of the mechanism by which climbing fibre volleys evoke late bursts, but the latter might represent dis-inhibition due to the interruption in Purkinje cell tonic firing which occurs after CF activation (the CF pause). However, as measured in barbiturate anaesthetised and decerebrate animals, CF pauses are too short (typically up to 100 mseca,4,7,a°). The duration in chloralose anaesthetised animals has been given as "several hundred milliseconds 'a4 which is more consistent with this hypothesis. We are, therefore, currently studying the CF pauses evoked in Purkinje cells by nerve and olivary stimulation in chloralose anaesthetised animals. The results confirm that the durations of evoked CF pauses are both long and variable. In 60 cells so far studied the average durations have ranged from 150 to 600 msec. It is possible that the late bursts are a product of chloralose anaesthesia. However, the rubro-spinal late bursts are produced by nerve stimulation in unanaesthetised as well as in chloralose anaesthetised animals 9. This suggests that the late bursts may in fact have some physiological significance in the normal operation of the nervous system. If the bursts are indeed a result of CF pauses in Purkinje cells, then some of the
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variability of the responses will be due to the variability in the C F pauses in different trials. It has been suggested that some of this variability is due to variations in the M F i n p u t reaching the Purkinje cells d u r i n g the pause 10. However, the pauses are t h o u g h t to be generated by mechanisms involving the excitation of basket 3,4 a n d p r o b a b l y also of Golgi cells3, v by C F collaterals. The d u r a t i o n of the pauses m a y therefore also reflect the excitability of these interneurones. I n a sense, therefore, the d u r a t i o n of the pause could be regarded as a "read-out' of the net balance of excitation a n d i n h i b i t i o n in several types o f cerebellar cortical cell. D u e to the convergence in the cortico-nuclear projection, the late burst in an interpositus n e u r o n e would then represent an averaged (and sign-reversed) read-out from a large assembly o f P u r k i n j e cells a n d their associated i n t e r n e u r o n e s in the intermediate cortex.
1 ARMSTRONG, D. M., HARVEY, R.J., AND SCHILD, R. F., Spino-olivocerebellar pathways to the posterior lobe of the cat cerebellum, Exp. Brain. Res., 18 (1973) in press. 2 BLOEDEL, J. R., AND BURTON, J. E., Electrophysiological evidence for a mossy fiber input to the cerebellar cortex activated indirectly by collaterals of spinocerebellar pathways, J. Neurophysiol., 33 (1970) 308-320. 3 BLOEDEL, J. R., AND ROBERTS, W. J., Action of climbing fibers in cerebellar cortex of the cat, J. Neurophysiol., 34 (1971) 17-31. 4 BURG, D., AND RUBIA, F.J., Inhibition of cerebellar Purkinje cells by climbing fibre input, Pftiigers Arch. ges. Physiol., 337 (1972) 367-372. 5 ECCLES,J. C., ITO, M., AND SZENT,~GOTHAI,J., The Cerebellum as a Neuronal Machine, Springer, Berlin, 1967, 257 pp. 6 ECCLES, J. C., ROS~N, [., SCHEID,P., AND T/,BO0,iKOVX,H., Cutaneous afferent responses in interpositus neurones of the cat, Brain Research, 42 (1972) 207-211. 7 LATPIAM,A., AND PAUL, D. H., Spontaneous activity ofcerebellar Purkinje cells and their responses to impulses in climbing fibres, J. Physiol. (Lond.), 213 (1971) 135 156. 8 LATHAM,A., PAUL, D. H., AND POTTS, A. J., Responses of fastigial neurones to stimulation of a peripheral nerve, J. Physiol. ([ond.), 206 (1970) 15-17P. 9 MASSION,J., ET ALBE-FESSARD, D., Dualit6 des voles sensorielles aff6rentes contr61ant l'activit6 du noyau rouge, Electroenceph. clin. Neurophysiol., 15 (1963) 435454. 10 MURPHY, J.Z., AND SABAH, N.H., Cerebellar Purkinje cell responses to afferent inputs. I. Climbing fiber activation, Brain Research, 25 (1971) 449467. l l OSCARSSON,O., The sagittal organisation of the cerebellar anterior lobe as revealed by the projection patterns of the climbing fiber system. In R. LLIN~,S (Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. Med. Ass./Educ. and Res. Found., Chicago, Ill., 1969, pp. 525-537. 12 OSCARSSON,O., AND ROSt~N, 1., Response characteristics of reticulo-cerebellar neurones activated from spinal afferents, Exp. Brain Res., I (1966) 320-328. 13 STENHOUSE, D., AND ECCLES, R. M., Activity in descending tract fibres in cats anaesthetised with chloralose, Brain Research, 35 (1971) 127-135. 14 TALBOTT, R. E., TOWE, A. L., AND KENNEDY, T. T., Physiological and histological classification of cerebellar neurons in chloralose-anaesthetized cats, Exp. Neurol., 19 (1967) 46-64. 15 THACH,W. T., Discharge of Purkinje and cerebellar nuclear neurons during rapidly alternating arm movements in the monkey, J. Neurophysiol., 31 (1968) 785-797. 16 TSUKUHARA,N., TOYAMA,K., AND KOSAKA,K., Electrical activity of red nucleus neurones investigated with intracellular microelectrodes, Exp. Brain Res., 4 (1967) 18-33.
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© Elsevier Scienlific Publishing Company, Amsterdam - Printed in The Netherlands
Responses of interpositus neurones to nerve stimulation in chloralose anaesthetized cats
D. M. ARMSTRONG, BARBARA COGDELL* AND R. J. HARVEY Department of Physiology, University of Bristol, The Medieal School, Bristol BS8 1TD (Great Britain)
(Accepted March 8th, 1973)
Eccles et al. 6 have recently described the responses of neurones of the nucleus interpositus to stimulation of forelimb and hindlimb cutaneous afferents in decerebrate cats. The responses included two phases of excitation associated with cell discharges with latencies of about 6 and 19 msec. These were separated and succeeded by phases of inhibition. The first excitation was attributed to the action of collaterals of cuneo-cerebellar or direct spino-cerebellar axons and the second to collaterals of reticulo-cerebellar and olivo-cerebellar axons. The inhibitions were attributed to the action on the nuclear cells of the impulses from Purkinje cells 5 discharged by the afferent volleys. The present paper describes firing patterns of interpositus neurones following nerve stimulation in cats anaesthetised with a-chloralose. A somewhat surprising finding was that the cells did not always display the high level of spontaneous activity which previous authors have reported for intracerebellar nuclear neurones in unanaesthetised 15, decerebrate 6 or barbiturate anaesthetised s preparations. In addition the responses to nerve stimulation included a hitherto undescribed component consisting of a high-frequency burst of action potentials. The experiments were performed on 17 cats initially anaesthetised with ether or halothane followed by 60 mg/kg of recrystallised a-chloralose dissolved in propane1,2-diol. Some animals were paralysed with gallamine triethiodide and artificially respired. Recordings were usually begun about 7 h after the initial dose of chloralose. The left deep radial (DR), sural (SUR), femoral (FEM), gastrocnemius-soleus (GS) nerves and both superficial radial (SR) nerves were dissected and stimulated with single shocks at a repetition rate of 1/2 sec. Stimulus strengths were usually in the range 1-4 times the threshold of the most excitable fibres in the nerve, though stronger stimuli were occasionally used. Interpositus axons were stimulated in the region of the right red nucleus using a concentric stainless steel electrode. In 7 experiments a similar electrode was placed in the rostral part of the right inferior olivary nucleus. * M.R.C. Scholar.
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Its location was checked by observing potentials evoked on the cerebellar surface. All electrodes were positioned stereotaxically and their tip locations were subsequently confirmed histologically. Individual neurones in the left nucleus interpositus were recorded using glass micropipettes (filled with 4 M NaC1; tip diameter 1-2 #m). Action potentials were recorded photographically and on magnetic tape. Interval histograms of spontaneous activity and post-stimulus histograms were constructed using a Modular One digital computer (Computer Technology Ltd.). Recordings were made from 120 units responding to stimulation of one or more of the dissected nerves. Seventy-one units gave a fixed latency impulse following stimulation in the region of the red nucleus. The latency varied in different units between 0.5 and 1.5 msec when just supra-threshold stimuli were used. In many units the impulse evoked by midbrain stimulation was shown to be antidromic because it would collide with an appropriately timed action potential occurring spontaneously or in response to nerve stimulation. Such units were classified as nuclear efferent cells. For 49 units antidromic identification was not attempted, but these cells were located in the same region as those which were identified and gave similar responses to nerve stimulation. Recordings were usually begun around 7 h after the initial dose of chloralose, when, in most animals, the nuclear cells gave brisk responses to nerve stimulation but displayed little spontaneous activity. Some cells showed no spontaneous activity at all, others gave only one or two bursts of 5-10 spikes during a period of 15-20 min. Over the succeeding few hours, the level of spontaneous activity increased, apparently in parallel with the increasing frequency and intensity of chloralose seizures. This spontaneous activity was not caused by the movements taking place during the seizures as it was unaffected by paralysing the animals, and showed no temporal relation to the seizures. The initially low level of spontaneous activity could have been due to a nonspecific cause such as post-operative shock. However, it did not appear to be associated with any generalised depression of the central nervous system, and certainly Purkinje cells overlying the nucleus interpositus showed a high level of spontaneous activity. Moreover, when a dose of 2-20 mg/kg of pentobarbitone was administered intravenously, many nuclear cells began an almost continuous and fairly regular discharge at rates between 20 and 80/sec. Even when the nuclear cells already showed high levels of spontaneous activity, such a dose of pentobarbitone made the firing much more regular and also usually increased the mean frequency. Despite large quantitative differences in the responses of different nuclear cells to nerve stimulation, it was possible to distinguish a typical response pattern consisting of three phases: a short latency excitation, followed by a pause, in turn followed by a more powerful long-lasting excitation. If stimulation of one nerve evoked a response, then for most units, stimulation of other nerves also produced a response, except that stimulation of GS rarely produced any detectable effect even with strengths up to 10 times threshold for the nerve (see Fig. 1D, E and F). In 112 cells an excitation with latency less than 40 msec could be evoked from
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Fig. 1. A: responses of an interpositus nucleus neurone to a single stimulus to ipsilateral sural (SUR) nerve. The stimulus occurred at the beginningof the sweep as arrowed. The first phase of the response was a single spike with latency 30 msec. The third phase shows 62 spikes beginning 220 msec after the stimulus. B-J: post-stimulus histograms of interpositus neurones derived from 75 (B and C) or 50 (D-J) successive responses. In each case the horizontal axis is divided into 100 bins. The arrow at the left hand side of each histogram indicates the mean rate of spontaneous activity for the unit. Histograms based on many responses of the kind shown in A. B, C: response of unit illustrated in A evoked from ipsilateral SUR nerve. C is from same data as B, but on an expanded time scale. D, E, F: responses of another unit to stimulation of ipsilateral SR (ISR), deep radial (DR) and gastrocnemius-soleus (GS) nerves respectively. G, H, I, J: responses of another unit to contralateral SR (CSR) and contralateral inferior olivary (IO) stimulation. I and J are respectively from the same data as G and H, but on an expanded time scale. one or more peripheral nerves. The usual response consisted of one or two impulses (see Fig. 1A) b u t there was considerable variation between units. The unit shown in Fig. 1A, B a n d C gave 5 or more spikes in m a n y trials, b u t in other units the response occurred so infrequently that it could n o t be identified with certainty even on a poststimulus histogram (e.g. Fig. 1D a n d E). The latency of the earliest impulse following ipsilateral SR stimulation ranged from 6 to 34 msec in different units. The ranges for both the contralateral SR a n d the ipsilateral D R nerves were from 8 to 30 msec. Responses to h i n d l i m b nerves had longer latencies; the latency to S U R stimulation ranged from 15 to 35 msec. This first phase o f the responses appears to include b o t h excitations described by Eccles et al}. It was followed by a second phase in which the firing rate fe!l towards or often went below its pro-stimulus level (Fig. 1A, B, D, E a n d G).
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The third and most dramatic phase of the response of the interpositus neurones was a 'late burst' of action potentials (see Fig. 1A, B, D, E and G). This phase occurred in 105 cells. It had a mean latency of 150 msec. However, there was considerable variability in the latency and bursts occasionally began as early as 50 msec or as late as 500 msec after the stimulus. The duration and spike content of the burst varied considerably between units and even in a single unit the response to a constant stimulus was sometimes very variable. In some trials the burst failed to appear whilst at the other extreme the same cell might produce more than 70 spikes in a burst lasting up to 600 msec. The average amplitude of the burst also varied with the nerve stimulated (Fig. 1D and E). The bursts were abolished by small doses of pentobarbitone (2-10 mg/kg i.v.). The origin of the late burst was investigated by recording simultaneously from the cerebellar surface and from interpositus neurones. This showed that nerve stimulation evoked the expected potentials on the cerebellar surface including components mediated via mossy fibres (MF) of the cuneo-cerebellar, direct spino-cerebellar 5 and spino-reticulo-cerebellar2 paths and via climbing fibres (CF) of the spino-olivocerebellar 1A1 paths. Following these potentials there was a reduction in the neural noise recorded from the cerebellar surface which lasted for approximately the same duration as the interpositus late bursts. There were no mossy fibre or climbing fibre evoked potentials coinciding with or immediately preceding the late bursts in the nuclear cells. It thus seemed possible that despite their long latency the late bursts were not caused by a delayed input to the cerebellum but were a consequence of the train of events set up in the cerebellar cortex by the initial afferent input along the mossy and climbing fibres. The possible role of the input via the climbing fibres of the spino-olivo-cerebellar paths was investigated by directly stimulating this nucleus with single shocks of duration 0.3 msec or with two such shocks 2 msec apart. Stimulus intensity never exceeded 400 #A and was usually in the range 75-250 #A. 40 interpositus cells were studied, and in 27 the responses were directly compared with the responses to nerve stimulation. Olivary stimulation invariably evoked a response very similar in its timing and other characteristics to the late burst from nerve stimulation and, in spontaneously firing cells, this response was usually preceded by a reduction in firing rate (Fig. 1H). Comparison of Fig. 1G and H illustrates the marked similarity between the late phases of the responses evoked from the olive and from nerve stimulation. The main difference was that olive stimulation evoked a short-latency discharge in only a few of the units studied (compare Fig. 1I and J). The threshold for responses to olivary stimulation was low (around 100 #A) and the size of the late bursts was shown to increase in parallel with the stimulus current. Responses equal to those evoked from nerves were produced by currents between 150 and 300 #A. Our olive stimuli produced large CF evoked potentials on the cerebellar surface, but no MF responses were detected. In a sample of interpositus neurones the responses to olivary stimulation were directly compared with those which were produced when the electrode was moved to a position just beyond the dorsal or ventral limit of the olive. All these cells gave late bursts following olivary stimulation but stimulation outside the olive
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failed to produce any late bursts except when it produced CF evoked potentials on the cerebellar surface, presumably as a result of stimulating afferents to the olive. The low level of spontaneous activity in some interpositus neurones under chloralose raises the possibility that in the unanaesthetised animal some cells may be silent for long periods. In previous investigations6,S, a5 only tonically active units have been detected and it has been envisaged that the Purkinje cells modulate this tonic discharge of the nuclear cells by 'inhibitory sculpturing 's. The present results suggest that in addition there could be some cells which are only recruited when there is a marked reduction in the tonic inhibition exerted on them from the overlying cortex. Our findings confirm those of Eccles e t al. 6 with regard to short-latency discharges following nerve stimulation. In addition, D R stimulation at Gp lI strength has shown that deep as well as cutaneous receptors can evoke discharges at quite short latency. This is not surprising if reticulo-cerebellar collaterais are involved in generating such discharges (see ref. 12). However, the results of GS stimulation showed that single Gp II volleys in muscle nerves are not always effective. The second phase of the response described here corresponds in time with the later inhibition attributed by Eccles e t al. 6 to inhibitory action exerted on the nuclear cells by Purkinje cells discharged via the spino-reticulo-cerebellar path and the spino-olivo-cerebellar paths. Recordings from the cell bodies and axons of rubro-spinal neurones in chloralose anaesthetised cats have shown that nerve stinmlation evokes a brisk discharge similar to our late burstg, a3. Since interpositus neurones are known to excite rubrospinal cells powerfully 16, our results can be taken to demonstrate that the rubrospinal discharge is due to the late burst in nucleus interpositus neurones. The rubrospinal discharge has indeed been shown to depend on the integrity of nucleus interpositus and the cortex overlying it 9. The effects of olivary stimulation suggest that the third (or second and third) phases of the interpositus responses to nerve stimulation could be a consequence of activity mediated via climbing fibres from the inferior olive. In view of the long and variable latencies involved it is difficult to be certain of the mechanism by which climbing fibre volleys evoke late bursts, but the latter might represent dis-inhibition due to the interruption in Purkinje cell tonic firing which occurs after CF activation (the CF pause). However, as measured in barbiturate anaesthetised and decerebrate animals, CF pauses are too short (typically up to 100 mseca,4,7,a°). The duration in chloralose anaesthetised animals has been given as "several hundred milliseconds 'a4 which is more consistent with this hypothesis. We are, therefore, currently studying the CF pauses evoked in Purkinje cells by nerve and olivary stimulation in chloralose anaesthetised animals. The results confirm that the durations of evoked CF pauses are both long and variable. In 60 cells so far studied the average durations have ranged from 150 to 600 msec. It is possible that the late bursts are a product of chloralose anaesthesia. However, the rubro-spinal late bursts are produced by nerve stimulation in unanaesthetised as well as in chloralose anaesthetised animals 9. This suggests that the late bursts may in fact have some physiological significance in the normal operation of the nervous system. If the bursts are indeed a result of CF pauses in Purkinje cells, then some of the
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variability of the responses will be due to the variability in the C F pauses in different trials. It has been suggested that some of this variability is due to variations in the M F i n p u t reaching the Purkinje cells d u r i n g the pause 10. However, the pauses are t h o u g h t to be generated by mechanisms involving the excitation of basket 3,4 a n d p r o b a b l y also of Golgi cells3, v by C F collaterals. The d u r a t i o n of the pauses m a y therefore also reflect the excitability of these interneurones. I n a sense, therefore, the d u r a t i o n of the pause could be regarded as a "read-out' of the net balance of excitation a n d i n h i b i t i o n in several types o f cerebellar cortical cell. D u e to the convergence in the cortico-nuclear projection, the late burst in an interpositus n e u r o n e would then represent an averaged (and sign-reversed) read-out from a large assembly o f P u r k i n j e cells a n d their associated i n t e r n e u r o n e s in the intermediate cortex.
1 ARMSTRONG, D. M., HARVEY, R.J., AND SCHILD, R. F., Spino-olivocerebellar pathways to the posterior lobe of the cat cerebellum, Exp. Brain. Res., 18 (1973) in press. 2 BLOEDEL, J. R., AND BURTON, J. E., Electrophysiological evidence for a mossy fiber input to the cerebellar cortex activated indirectly by collaterals of spinocerebellar pathways, J. Neurophysiol., 33 (1970) 308-320. 3 BLOEDEL, J. R., AND ROBERTS, W. J., Action of climbing fibers in cerebellar cortex of the cat, J. Neurophysiol., 34 (1971) 17-31. 4 BURG, D., AND RUBIA, F.J., Inhibition of cerebellar Purkinje cells by climbing fibre input, Pftiigers Arch. ges. Physiol., 337 (1972) 367-372. 5 ECCLES,J. C., ITO, M., AND SZENT,~GOTHAI,J., The Cerebellum as a Neuronal Machine, Springer, Berlin, 1967, 257 pp. 6 ECCLES, J. C., ROS~N, [., SCHEID,P., AND T/,BO0,iKOVX,H., Cutaneous afferent responses in interpositus neurones of the cat, Brain Research, 42 (1972) 207-211. 7 LATPIAM,A., AND PAUL, D. H., Spontaneous activity ofcerebellar Purkinje cells and their responses to impulses in climbing fibres, J. Physiol. (Lond.), 213 (1971) 135 156. 8 LATHAM,A., PAUL, D. H., AND POTTS, A. J., Responses of fastigial neurones to stimulation of a peripheral nerve, J. Physiol. ([ond.), 206 (1970) 15-17P. 9 MASSION,J., ET ALBE-FESSARD, D., Dualit6 des voles sensorielles aff6rentes contr61ant l'activit6 du noyau rouge, Electroenceph. clin. Neurophysiol., 15 (1963) 435454. 10 MURPHY, J.Z., AND SABAH, N.H., Cerebellar Purkinje cell responses to afferent inputs. I. Climbing fiber activation, Brain Research, 25 (1971) 449467. l l OSCARSSON,O., The sagittal organisation of the cerebellar anterior lobe as revealed by the projection patterns of the climbing fiber system. In R. LLIN~,S (Ed.), Neurobiology of Cerebellar Evolution and Development, Amer. Med. Ass./Educ. and Res. Found., Chicago, Ill., 1969, pp. 525-537. 12 OSCARSSON,O., AND ROSt~N, 1., Response characteristics of reticulo-cerebellar neurones activated from spinal afferents, Exp. Brain Res., I (1966) 320-328. 13 STENHOUSE, D., AND ECCLES, R. M., Activity in descending tract fibres in cats anaesthetised with chloralose, Brain Research, 35 (1971) 127-135. 14 TALBOTT, R. E., TOWE, A. L., AND KENNEDY, T. T., Physiological and histological classification of cerebellar neurons in chloralose-anaesthetized cats, Exp. Neurol., 19 (1967) 46-64. 15 THACH,W. T., Discharge of Purkinje and cerebellar nuclear neurons during rapidly alternating arm movements in the monkey, J. Neurophysiol., 31 (1968) 785-797. 16 TSUKUHARA,N., TOYAMA,K., AND KOSAKA,K., Electrical activity of red nucleus neurones investigated with intracellular microelectrodes, Exp. Brain Res., 4 (1967) 18-33.