Brain Research, 88 (1975) 233-241 © Elsevier ScientificPublishing Company, Amsterdam - Printed in The Netherlands
233
T I M I N G OF ACTIVITY IN C E R E B E L L A R D E N T A T E N U C L E U S A N D C E R E B R A L M O T O R CORTEX D U R I N G P R O M P T V O L I T I O N A L MOVEMENT
W. T. THACH Departments of Physiology and Neurology, Yale Medical School, New Haven, Conn. 06510 (U.S.A.)
(Accepted November 26th, 1974)
SUMMARY
The discharge of neurons in cerebellar dentate nucleus and cerebral motor cortex was recorded on alternate days in each of 3 monkeys in association with prompt arm movement in response to a light signal. The time of change of the discharge of each neuron in relation to arm movement was computed. The distributions of the time of change for cerebellum and cerebrum overlapped, but the cerebellar distribution was shifted slightly earlier.
INTRODUCTION
Evarts has shown that neurons o f the precentral motor cortex 4,5 change discharge frequency in relation to and prior to promptly initiated volitional arm movement. A similar study 13 of the dentate nucleus of the cerebellum showed that its neurons also change prior to movement. A comparison of dentate and motor cortex in the two different studies (on different monkeys performing different tasks) revealed such overlap of timing that it was impossible to decide whether one was changing before the other. The present study was undertaken to look for timing differences between dentate and motor cortex by recording the discharge of single neurons in each of the two areas in the same monkey. METHODS
Three monkeys (Macaca mulatta) were trained to move the right arm promptly in response to a light signal. A monkey had to insert its hand through a hole in its chair, grasp an immovable vertical rod and exert force against it ( > 200 g) by flexing or extending the wrist. The wrist fit snugly in the hole and was stabilized by it. Force
234 was measured by strain gauges attached to the rod. After a period of 2 -5 sec (randomly varied v) of maintained flexion, a light came on, signalling the monkey to reverse the force by extending the wrist. The cycle was then repeated for alternate directions, holding in extension and quickly flexing, holding in flexion and quickly extending, etc. Though the rod was fixed and the measured parameter of response was a change in force, the task was not strictly an isometric contraction of muscle, since during performance the fingers, wrist, elbow, shoulder and trunk moved. When the monkey reversed force within 300 msec of the light onset, it was rewarded with fruit juice. The light was from a tungsten bulb behind a white shield 0.5 in. in diameter. Light intensity was 5.5 ft. Lamberts, with 1 0 ~ of emission after 8 msec and 9050 after 35 msec. The room was maintained in semidarkness (0.125 ft. Lamberts); the light was placed 1.5 m from the monkey. While waiting for the light to come on, the monkey fixed its gaze (demonstrated in several electro-oculogram recordings) upon the darkened bulb; after the light came on, the monkey occasionally and at varying latency shifted its gaze. Delivery of the juice was delayed 500 msec after a rewarded arm response in order to delay the muscular activity of the mouth, tongue, head and neck use din drinking the juice. After a monkey was fully trained, it was anesthetized with thiopental and steel bolts were attached to the skull, restraining the head in the primate chair. Holes were cut in the skull over the dentate ipsilateral and the motor cortex ('arm' area) contralateral to the trained arm, and steel cylinders were placed over the holes. Upon full recovery from the operation (2-3 days), the monkey was placed in the primate chair with its head restrained and was permitted to perform the task while a modified Evarts-Narashige microdrive advanced a glass-covered platinum-iridium microelectrode through the dura and into dentate and motor cortex on alternate days. The single unit action potentials, the light signal to move, and the first change in force on the rod were prepared for computer analysis (PDP-12) by a neurophysiological analysis program ('NAP') developed at the N I M H by William Sherrif, William Vaughan, and their associates. Action potentials were converted to standard 1-msec pulses and recorded on one channel of magnetic tape. A signal representing certain aspects of the performance (start and stop of performance, signal to move, and change of force for flexor and extensor trials) was recorded on a second channel of the magnetic tape. Recording sessions lasted 3-4 h each day for approximately a month; the monkey was allowed to work for as long as it wished each day, and was returned to its cage each night. The movements were overlearned (at least 500,000 trials) and were highly stereotyped: reaction time of about 250 msec varied from trial to trial, hour to hour, day to day, and monkey to monkey, but was not significantly different for cerebellar and cerebral recording sessions. After final completion of recording, the monkey was narcotized with pentobarbital, and the brain prepared for histological analysis. Penetration sites were then localized when visible by microscopic inspection o f the sections or interpolated from the microdrive coordinates used in making the penetration (Fig, 1). Data recorded on analog tape were digitized by the program referred to above,
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/' ~,-~......,.'/x Fig. 1. Locations of penetrations in which units were encountered in the 3 (top, middle, and bottom) monkeys. A: dorsal view of the right cerebellar nuclei (fastigial, interposed, and dentate). B: left cerebral hemisphere, showing location of cortical cylinder. C: cerebral cortex underlying the cylinder (dashed lines indicates approximate width of cortex in anterior wall of precentral sulcus). Scale at 1 mm intervals.
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237 and the digitized tapes were used to display 4 rasters for each unit (Fig. 2). The program also computed the average maintained discharge frequency for a control period of 500 msec prior to the light stimulus, the monkey's reaction time (light onset to change in force), and the time of change of neural discharge with respect both to the light onset and the change in force. A change of neural discharge frequency was detected in the following way: for the 500-msec prestimulus control period displayed in the rasters, the average spike content per each 20 msec was calculated. Eight values were then computed that deviated (4 above and 4 below) from the mean number of spikes/20 msec of the control period corresponding to the probabilities of 0.01, 0.001, 0.0001, and 0.00001. Using the 8 values as criteria for significant change in neural discharge frequency, a 20-msec scanning window moved across the data displayed in the response-aligned raster in increments of 2 msec and detected those bins that contained more (or less) spikes than the computed deviant values. The time o f change of neural discharge frequency was defined as the time at which a change that reached P < 0.00001 first crossed the P < 0.01 level of significance. The program and a complete description of the statistical and computational procedures can be obtained from the Program Library of the Digital Equipment Corporation. F o r this report neurons that were selected (a) were recorded for 25 trials (for flexion and extension) without evidence of injury, (b) showed a change judged by eye from the rasters to be consistently temporally related to the onset of movement trial after trial, and (c) underwent a change in frequency significant at the P < 0.00001 level o f probability. RESULTS
Using the above criteria, 291 neurons recorded in the cerebellar nuclei and m o t o r cortex of the 3 monkeys were selected for inclusion in the final tabulation o f results. The estimated locations of penetrations are given in Fig. 1. Fig. 2 shows rasters of the discharge of an early dentate neuron and the earliest m o t o r cortical neuron in the third monkey. As in previous experiments, both types o f neuron changed with temporal consistency better related to the movement than to the signal to move. The dentate neuron changed prior to the m o t o r cortical neuron - - for the dentate neuron 166 msec (flexion) and 180 msec (extension), and for the m o t o r cortical neuron 124 msec (flexion) and 144 msec (extension) prior to the change in force. Fig. 3 shows the distributions of the times o f change of discharge frequency of all cerebellar and cerebral m o t o r cortical neurons in the 3 monkeys. I f a unit changed
Fig. 2. Rasters of the discharge of an early dentate neuron (top 4) and the earliest motor cortical neuron (bottom 4) recorded in the third monkey. Each dot represents an action potential; each row of dots, the discharge during a trial. The upper two of each 4 rasters are for flexion (+); the lower two, extension (--). 'S rasters' on the left are aligned on the stimulus (center bar), and the response is indicated by a short vertical bar. 'R rasters' on the right are aligned on the response (center bar). Time scale is 1 sec for each raster. Both neurons changed in better relation to the response than the stimulus. The dentate neuron changed prior to the motor cortical neuron.
238
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Fig. 3. Time-of-change histograms summarizing the timing results. The 3 sets of histograms are from the 3 monkeys and show the distribution of time of change (relative to change of force) of unit discharge in cerebellar nuclei and arm area motor cortex, and for the third monkey, muscles in the arm, shoulder and trunk. Abscissa is time of change (in msec) before or after change of force (~), scale is 20 msec/division. Ordinate is number of neural (and EMG) changes; scale is 5 neural changes/ division. Units that changed for both flexion and extension are represented twicel For the first monkey, the histograms are comprised of 91 neural changes (48 neurons, 12 penetrations) in cerebellar nuclei and 52 changes (34 neurons, 10 penetrations) in motor cortex. Within the cerebellar nuclei, 10 penetrations (37 neurons, 70 changes) were in dentate and 2 penetrations (11 neurons, 21 changes) were in interpositus. For the second monkey, the histograms are comprised of 35 changes (22 neurons, 6 penetrations) in the cerebellar nuclei and 47 changes (32 neurons, 6 penetrations) in motor cortex. Within the cerebellar nuclei, 2 penetrations (4 neurons, 6 changes) were in interpositus; 3 penetrations (24 neurons, 35 changes) were at the junction of interpositus and dentate, and only one penetration (4 neurons, 6 changes) was well lateral in dentate. For the third monkey, the histograms are comprised of 130 changes (77 neurons, 19 penetrations) all in dentate, and 126 changes (78 neurons, 21 penetrations) in motor cortex.
in r e l a t i o n b o t h to flexion a n d extension it was represented twice in these h i s t o g r a m s , t h o u g h the o u t c o m e is the same if the m o v e m e n t s are a n a l y z e d separately. The first set o f h i s t o g r a m s is for the first m o n k e y : the d i s t r i b u t i o n s o v e r l a p greatly as expected, b u t the cerebellar d i s t r i b u t i o n a p p e a r s earlier. The second set o f h i s t o g r a m s is for the second m o n k e y : the overlap is m o r e c o m p l e t e ; the p e a k o f the cerebellar d i s t r i b u t i o n a p p e a r s slightly earlier. The t h i r d set o f h i s t o g r a m s is for the t h i r d m o n k e y : the cerebellar d i s t r i b u t i o n again overlaps the cerebral, b u t a p p e a r s slightly earlier. T a b l e I gives some statistical m e a s u r e s o f the d i s t r i b u t i o n s . T h e d i s t r i b u t i o n s
239 TABLE I STATISTICAL MEASURES OF THE TIMING RESULTS
N = total number of neural changes for both flexion and extension. Medians and interquartile ranges are in msec before or after (--) change of force. Z is the standard normal deviate and P the corresponding probability as computed by the Mann-Whitney rank tests of the null hypothesis that the two compared distributions were sampled from identical populations. N
Monkey No. 1 Cerebellum Cerebrum Monkey No. 2 Cerebellum Cerebrum Monkey No. 3 Cerebellum Cerebrum Arm Shoulder and trunk
Median
lnterquartile Range
91 52
76 43
96 68
46 20
35 47
56 44
68 66
36 18
130 126 41 44
69 54 40 11
98 88 76 36
38 --6 16 --8
Mann-Whitney rank test
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P
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< 0.0001
1.14
0.16
3.34
< 0.001
4.13
< 0.0001
by eye and by measure 1~ deviate from normal (skewness, P < 0.01 ; positive kurtosis, P < 0.05), so median and interquartile range are given and the Mann-Whitney non-parametric rank method 8 used to test the null hypothesis that the two compared distributions were sampled from identical populations. Comparison of the means and a parametric t test of the difference of the means gave similar conclusions. In all 3 monkeys, the median (and the mean) and the interquartile range were earlier for the cerebellar distributions. The difference between the cerebellar and motor cortex distributions was highly significant by the Mann-Whitney test (and the t-test) for the first and third monkeys, but not for the second. The lack of significance in the second monkey could have resulted from the smaller sample size and its contamination by the inclusion of interpositus neurons which have been reported to change later during this type of behavior than dentate la. To investigate the possibility that dentate neurons were preferentially connected to an earlier contracting set of muscles and the motor cortex to a later contracting set and that this was the basis of the observed timing differences, in the third monkey the electromyographic activity of muscles in the arm, shoulder, and trunk was recorded before and after the brain recording. Electrodes consisted of two tefloncovered stainless steel wires bared 1 mm back from the tips, that were fish hooked into a muscle. The muscles studied were finger and wrist flexors and extensors of the forearm, biceps and triceps in the upper arm, and deltoid (3 divisions), pectoralis, latissimus, supra- and infraspinatus, trapezius, rhomboid, neck extensors, sternocleidomastoid and erector spinae of the shoulder and trunk. Through multiple unit recordings, E M G records were processed in the same way as for brain single units. The time of change distributions are shown in the third set of histograms in Fig. 3.
240 The earliest muscles to change were in the arm, followed by changes in the shoulder and trunk. Since it is known that arm area motor cortex is preferentially connected to muscles of the arm, and since these muscles changed actively before any other. it is unlikely that the dentate was connected to an earlier set of muscles than motor cortex. An attempt was also made in the third monkey to show that the region in which the earliest dentate changes were recorded was in fact concerned with arm movements. A 2-mm lesion was made in dentate (Fig. 1, bottom, A) by passing current (800 #A, 10 min) through the recording electrode. As in Holmes' studies 9, this resulted in persistently (one week of observation) prolonged reaction time. Muscular activation was irregular, with motor units frequently dropping entirely out of the performance of some trials. Nevertheless the pattern of muscle activation, with arm preceding more proximal muscles, was preserved. Observation of the monkey's behavior in its cage revealed scant deficits: the monkeys had slight ataxia of its right arm in palping and picking at its head gear, but not in grooming and scratching its body. The most obvious deficit was in reaching for its food: whereas previously the monkey had used either arm interchangeably, after the lesion the monkey invariably reached with the left arm and supported its body with the right. With regard to still other muscles of the body, the sporadic eye movements usually followed the change in force exerted by the arm, and drinking movements were even more delayed. Some units (about 5 ~ of all recorded) fired short bursts in apparent relation to eye (or attempted head) movements or the drinking movements. These units were automatically rejected by the selection criteria because of their lack of consistent temporal relation to the movement of the arm. DISCUSSION
Two hypotheses on the function of the dentate nucleus hinge on its reciprocal connections with motor cortex (see ref. 6) and on the question of timing. One hypothesis z,1°,11 proposes the following sequence of events: a command for movement occurs in the motor cortex prior to any change in dentate; the command then goes down not only to inter- and motorneurons but also (via pons) to dentate; the dentate feeds back a signal to motor cortex to cause or modulate later components of the motor command. The loop would provide a system of rapid internal feedback that could correct output of motor cortex perhaps before any feedback from the periphery. The second hypothesis 1,2,6,9 proposes that a command for movement (or some aspect of it) occurs in cerebral association cortex and then sequentially feeds through pons, dentate, VA-VL thalamus, and motor cortex, gaining specifications at each stage. The dentate, prior to any involvement of motor cortex, would compute major features of the motor program. In the first hypothesis, dentate would change activity in relation to movement after motor cortex; in the second, before. If there had been a clear separation of cerebellar and cerebral timing distributions without any overlap, this study might have served to reject one o f the two hypotheses. As it stands, the large overlap and small difference are consistent with
241 both hypotheses. It remains for other experiments employing different methods to corroborate the timing difference observed here and to aid in its interpretation. ACKNOWLEDGEMENTS
Thanks are due to Harry Fein for instrument design, Mahlon DeLong for thoughts on task training and monitoring, and E. V. Evarts for help with every aspect of this work. Supported by NINDS Research Grant No. 5-R01-NS-09984.
REFERENCES 1 CARMAN,J. B., Anatomic basis of surgical treatment of Parkinson's disease, New Engl. J. Med., 279 (1968) 919-930. 2 DEECKE,L., BECKER,W., GROZINGER,B., SCHEID,P., AND KORNHUBER,H., Human brain potentials preceding voluntary limb movements, Electroenceph. clin. Neurophysiol., Suppl. 33 (1973) 97-104. 3 ECCLES,J. C., Circuits in the cerebellar control of movement, Proc. nat. Acad. Sci. (Wash.), 58 (1967) 336-343. 4 EVARTS, E. V., Pyramidal tract activity associated with a conditioned hand movement in the monkey, J. Neurophysiol., 29 (1966) 1011-1027. 5 EVARTS, E.V., Contrasts between activity of precentral and postcentral neurons of cerebral cortex during movement in the monkey, Brain Research, 40 (1972) 25-31. 6 EVARTS, E.V., AND THACH, W.T., Motor mechanisms of the CNS: cerebrocerebellar interrelations, Ann. Rev. Physiol., 21 (1969) 451-498. 7 FEIN, H., A method for generating random time intervals, Electroenceph. clin. Neurophysiol., 33 (1972) 433-434. 8 FREUND,J. E., Mathematical Statistics, Prentice Hall, Englewood Cliffs, N.J., 1971, pp. 347-349. 9 HOLMES,G., The cerebellum of man, Brain, 62 (1939) 1-30. 10 ITO, M., Neurophysiological aspects of the cerebellar motor control system, Int. J. Neurol., 7 (1970) 162-176. 11 RUCH, T. C., 'Motor systems'. In S.S. STEVENS(Ed.), Handbook of Experimental Psychology, Wiley, New York, 1951, pp. 154-208. 12 SNEDECOR, G.W., AND COCHRAN, W.G., Statistical Methods, 6th ed., Iowa State University Press, Ames, Iowa, 1972, pp. 86-88. 13 THACH, W.T., Discharge of eerebellar neurons related to two maintained postures and two prompt movements. I. Nuclear cell output, J. Neurophysiol., 33 (1970) 527-536.
233
T I M I N G OF ACTIVITY IN C E R E B E L L A R D E N T A T E N U C L E U S A N D C E R E B R A L M O T O R CORTEX D U R I N G P R O M P T V O L I T I O N A L MOVEMENT
W. T. THACH Departments of Physiology and Neurology, Yale Medical School, New Haven, Conn. 06510 (U.S.A.)
(Accepted November 26th, 1974)
SUMMARY
The discharge of neurons in cerebellar dentate nucleus and cerebral motor cortex was recorded on alternate days in each of 3 monkeys in association with prompt arm movement in response to a light signal. The time of change of the discharge of each neuron in relation to arm movement was computed. The distributions of the time of change for cerebellum and cerebrum overlapped, but the cerebellar distribution was shifted slightly earlier.
INTRODUCTION
Evarts has shown that neurons o f the precentral motor cortex 4,5 change discharge frequency in relation to and prior to promptly initiated volitional arm movement. A similar study 13 of the dentate nucleus of the cerebellum showed that its neurons also change prior to movement. A comparison of dentate and motor cortex in the two different studies (on different monkeys performing different tasks) revealed such overlap of timing that it was impossible to decide whether one was changing before the other. The present study was undertaken to look for timing differences between dentate and motor cortex by recording the discharge of single neurons in each of the two areas in the same monkey. METHODS
Three monkeys (Macaca mulatta) were trained to move the right arm promptly in response to a light signal. A monkey had to insert its hand through a hole in its chair, grasp an immovable vertical rod and exert force against it ( > 200 g) by flexing or extending the wrist. The wrist fit snugly in the hole and was stabilized by it. Force
234 was measured by strain gauges attached to the rod. After a period of 2 -5 sec (randomly varied v) of maintained flexion, a light came on, signalling the monkey to reverse the force by extending the wrist. The cycle was then repeated for alternate directions, holding in extension and quickly flexing, holding in flexion and quickly extending, etc. Though the rod was fixed and the measured parameter of response was a change in force, the task was not strictly an isometric contraction of muscle, since during performance the fingers, wrist, elbow, shoulder and trunk moved. When the monkey reversed force within 300 msec of the light onset, it was rewarded with fruit juice. The light was from a tungsten bulb behind a white shield 0.5 in. in diameter. Light intensity was 5.5 ft. Lamberts, with 1 0 ~ of emission after 8 msec and 9050 after 35 msec. The room was maintained in semidarkness (0.125 ft. Lamberts); the light was placed 1.5 m from the monkey. While waiting for the light to come on, the monkey fixed its gaze (demonstrated in several electro-oculogram recordings) upon the darkened bulb; after the light came on, the monkey occasionally and at varying latency shifted its gaze. Delivery of the juice was delayed 500 msec after a rewarded arm response in order to delay the muscular activity of the mouth, tongue, head and neck use din drinking the juice. After a monkey was fully trained, it was anesthetized with thiopental and steel bolts were attached to the skull, restraining the head in the primate chair. Holes were cut in the skull over the dentate ipsilateral and the motor cortex ('arm' area) contralateral to the trained arm, and steel cylinders were placed over the holes. Upon full recovery from the operation (2-3 days), the monkey was placed in the primate chair with its head restrained and was permitted to perform the task while a modified Evarts-Narashige microdrive advanced a glass-covered platinum-iridium microelectrode through the dura and into dentate and motor cortex on alternate days. The single unit action potentials, the light signal to move, and the first change in force on the rod were prepared for computer analysis (PDP-12) by a neurophysiological analysis program ('NAP') developed at the N I M H by William Sherrif, William Vaughan, and their associates. Action potentials were converted to standard 1-msec pulses and recorded on one channel of magnetic tape. A signal representing certain aspects of the performance (start and stop of performance, signal to move, and change of force for flexor and extensor trials) was recorded on a second channel of the magnetic tape. Recording sessions lasted 3-4 h each day for approximately a month; the monkey was allowed to work for as long as it wished each day, and was returned to its cage each night. The movements were overlearned (at least 500,000 trials) and were highly stereotyped: reaction time of about 250 msec varied from trial to trial, hour to hour, day to day, and monkey to monkey, but was not significantly different for cerebellar and cerebral recording sessions. After final completion of recording, the monkey was narcotized with pentobarbital, and the brain prepared for histological analysis. Penetration sites were then localized when visible by microscopic inspection o f the sections or interpolated from the microdrive coordinates used in making the penetration (Fig, 1). Data recorded on analog tape were digitized by the program referred to above,
i
/' ~,-~......,.'/x Fig. 1. Locations of penetrations in which units were encountered in the 3 (top, middle, and bottom) monkeys. A: dorsal view of the right cerebellar nuclei (fastigial, interposed, and dentate). B: left cerebral hemisphere, showing location of cortical cylinder. C: cerebral cortex underlying the cylinder (dashed lines indicates approximate width of cortex in anterior wall of precentral sulcus). Scale at 1 mm intervals.
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237 and the digitized tapes were used to display 4 rasters for each unit (Fig. 2). The program also computed the average maintained discharge frequency for a control period of 500 msec prior to the light stimulus, the monkey's reaction time (light onset to change in force), and the time of change of neural discharge with respect both to the light onset and the change in force. A change of neural discharge frequency was detected in the following way: for the 500-msec prestimulus control period displayed in the rasters, the average spike content per each 20 msec was calculated. Eight values were then computed that deviated (4 above and 4 below) from the mean number of spikes/20 msec of the control period corresponding to the probabilities of 0.01, 0.001, 0.0001, and 0.00001. Using the 8 values as criteria for significant change in neural discharge frequency, a 20-msec scanning window moved across the data displayed in the response-aligned raster in increments of 2 msec and detected those bins that contained more (or less) spikes than the computed deviant values. The time o f change of neural discharge frequency was defined as the time at which a change that reached P < 0.00001 first crossed the P < 0.01 level of significance. The program and a complete description of the statistical and computational procedures can be obtained from the Program Library of the Digital Equipment Corporation. F o r this report neurons that were selected (a) were recorded for 25 trials (for flexion and extension) without evidence of injury, (b) showed a change judged by eye from the rasters to be consistently temporally related to the onset of movement trial after trial, and (c) underwent a change in frequency significant at the P < 0.00001 level o f probability. RESULTS
Using the above criteria, 291 neurons recorded in the cerebellar nuclei and m o t o r cortex of the 3 monkeys were selected for inclusion in the final tabulation o f results. The estimated locations of penetrations are given in Fig. 1. Fig. 2 shows rasters of the discharge of an early dentate neuron and the earliest m o t o r cortical neuron in the third monkey. As in previous experiments, both types o f neuron changed with temporal consistency better related to the movement than to the signal to move. The dentate neuron changed prior to the m o t o r cortical neuron - - for the dentate neuron 166 msec (flexion) and 180 msec (extension), and for the m o t o r cortical neuron 124 msec (flexion) and 144 msec (extension) prior to the change in force. Fig. 3 shows the distributions of the times o f change of discharge frequency of all cerebellar and cerebral m o t o r cortical neurons in the 3 monkeys. I f a unit changed
Fig. 2. Rasters of the discharge of an early dentate neuron (top 4) and the earliest motor cortical neuron (bottom 4) recorded in the third monkey. Each dot represents an action potential; each row of dots, the discharge during a trial. The upper two of each 4 rasters are for flexion (+); the lower two, extension (--). 'S rasters' on the left are aligned on the stimulus (center bar), and the response is indicated by a short vertical bar. 'R rasters' on the right are aligned on the response (center bar). Time scale is 1 sec for each raster. Both neurons changed in better relation to the response than the stimulus. The dentate neuron changed prior to the motor cortical neuron.
238
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Fig. 3. Time-of-change histograms summarizing the timing results. The 3 sets of histograms are from the 3 monkeys and show the distribution of time of change (relative to change of force) of unit discharge in cerebellar nuclei and arm area motor cortex, and for the third monkey, muscles in the arm, shoulder and trunk. Abscissa is time of change (in msec) before or after change of force (~), scale is 20 msec/division. Ordinate is number of neural (and EMG) changes; scale is 5 neural changes/ division. Units that changed for both flexion and extension are represented twicel For the first monkey, the histograms are comprised of 91 neural changes (48 neurons, 12 penetrations) in cerebellar nuclei and 52 changes (34 neurons, 10 penetrations) in motor cortex. Within the cerebellar nuclei, 10 penetrations (37 neurons, 70 changes) were in dentate and 2 penetrations (11 neurons, 21 changes) were in interpositus. For the second monkey, the histograms are comprised of 35 changes (22 neurons, 6 penetrations) in the cerebellar nuclei and 47 changes (32 neurons, 6 penetrations) in motor cortex. Within the cerebellar nuclei, 2 penetrations (4 neurons, 6 changes) were in interpositus; 3 penetrations (24 neurons, 35 changes) were at the junction of interpositus and dentate, and only one penetration (4 neurons, 6 changes) was well lateral in dentate. For the third monkey, the histograms are comprised of 130 changes (77 neurons, 19 penetrations) all in dentate, and 126 changes (78 neurons, 21 penetrations) in motor cortex.
in r e l a t i o n b o t h to flexion a n d extension it was represented twice in these h i s t o g r a m s , t h o u g h the o u t c o m e is the same if the m o v e m e n t s are a n a l y z e d separately. The first set o f h i s t o g r a m s is for the first m o n k e y : the d i s t r i b u t i o n s o v e r l a p greatly as expected, b u t the cerebellar d i s t r i b u t i o n a p p e a r s earlier. The second set o f h i s t o g r a m s is for the second m o n k e y : the overlap is m o r e c o m p l e t e ; the p e a k o f the cerebellar d i s t r i b u t i o n a p p e a r s slightly earlier. The t h i r d set o f h i s t o g r a m s is for the t h i r d m o n k e y : the cerebellar d i s t r i b u t i o n again overlaps the cerebral, b u t a p p e a r s slightly earlier. T a b l e I gives some statistical m e a s u r e s o f the d i s t r i b u t i o n s . T h e d i s t r i b u t i o n s
239 TABLE I STATISTICAL MEASURES OF THE TIMING RESULTS
N = total number of neural changes for both flexion and extension. Medians and interquartile ranges are in msec before or after (--) change of force. Z is the standard normal deviate and P the corresponding probability as computed by the Mann-Whitney rank tests of the null hypothesis that the two compared distributions were sampled from identical populations. N
Monkey No. 1 Cerebellum Cerebrum Monkey No. 2 Cerebellum Cerebrum Monkey No. 3 Cerebellum Cerebrum Arm Shoulder and trunk
Median
lnterquartile Range
91 52
76 43
96 68
46 20
35 47
56 44
68 66
36 18
130 126 41 44
69 54 40 11
98 88 76 36
38 --6 16 --8
Mann-Whitney rank test
Z
P
4.01
< 0.0001
1.14
0.16
3.34
< 0.001
4.13
< 0.0001
by eye and by measure 1~ deviate from normal (skewness, P < 0.01 ; positive kurtosis, P < 0.05), so median and interquartile range are given and the Mann-Whitney non-parametric rank method 8 used to test the null hypothesis that the two compared distributions were sampled from identical populations. Comparison of the means and a parametric t test of the difference of the means gave similar conclusions. In all 3 monkeys, the median (and the mean) and the interquartile range were earlier for the cerebellar distributions. The difference between the cerebellar and motor cortex distributions was highly significant by the Mann-Whitney test (and the t-test) for the first and third monkeys, but not for the second. The lack of significance in the second monkey could have resulted from the smaller sample size and its contamination by the inclusion of interpositus neurons which have been reported to change later during this type of behavior than dentate la. To investigate the possibility that dentate neurons were preferentially connected to an earlier contracting set of muscles and the motor cortex to a later contracting set and that this was the basis of the observed timing differences, in the third monkey the electromyographic activity of muscles in the arm, shoulder, and trunk was recorded before and after the brain recording. Electrodes consisted of two tefloncovered stainless steel wires bared 1 mm back from the tips, that were fish hooked into a muscle. The muscles studied were finger and wrist flexors and extensors of the forearm, biceps and triceps in the upper arm, and deltoid (3 divisions), pectoralis, latissimus, supra- and infraspinatus, trapezius, rhomboid, neck extensors, sternocleidomastoid and erector spinae of the shoulder and trunk. Through multiple unit recordings, E M G records were processed in the same way as for brain single units. The time of change distributions are shown in the third set of histograms in Fig. 3.
240 The earliest muscles to change were in the arm, followed by changes in the shoulder and trunk. Since it is known that arm area motor cortex is preferentially connected to muscles of the arm, and since these muscles changed actively before any other. it is unlikely that the dentate was connected to an earlier set of muscles than motor cortex. An attempt was also made in the third monkey to show that the region in which the earliest dentate changes were recorded was in fact concerned with arm movements. A 2-mm lesion was made in dentate (Fig. 1, bottom, A) by passing current (800 #A, 10 min) through the recording electrode. As in Holmes' studies 9, this resulted in persistently (one week of observation) prolonged reaction time. Muscular activation was irregular, with motor units frequently dropping entirely out of the performance of some trials. Nevertheless the pattern of muscle activation, with arm preceding more proximal muscles, was preserved. Observation of the monkey's behavior in its cage revealed scant deficits: the monkeys had slight ataxia of its right arm in palping and picking at its head gear, but not in grooming and scratching its body. The most obvious deficit was in reaching for its food: whereas previously the monkey had used either arm interchangeably, after the lesion the monkey invariably reached with the left arm and supported its body with the right. With regard to still other muscles of the body, the sporadic eye movements usually followed the change in force exerted by the arm, and drinking movements were even more delayed. Some units (about 5 ~ of all recorded) fired short bursts in apparent relation to eye (or attempted head) movements or the drinking movements. These units were automatically rejected by the selection criteria because of their lack of consistent temporal relation to the movement of the arm. DISCUSSION
Two hypotheses on the function of the dentate nucleus hinge on its reciprocal connections with motor cortex (see ref. 6) and on the question of timing. One hypothesis z,1°,11 proposes the following sequence of events: a command for movement occurs in the motor cortex prior to any change in dentate; the command then goes down not only to inter- and motorneurons but also (via pons) to dentate; the dentate feeds back a signal to motor cortex to cause or modulate later components of the motor command. The loop would provide a system of rapid internal feedback that could correct output of motor cortex perhaps before any feedback from the periphery. The second hypothesis 1,2,6,9 proposes that a command for movement (or some aspect of it) occurs in cerebral association cortex and then sequentially feeds through pons, dentate, VA-VL thalamus, and motor cortex, gaining specifications at each stage. The dentate, prior to any involvement of motor cortex, would compute major features of the motor program. In the first hypothesis, dentate would change activity in relation to movement after motor cortex; in the second, before. If there had been a clear separation of cerebellar and cerebral timing distributions without any overlap, this study might have served to reject one o f the two hypotheses. As it stands, the large overlap and small difference are consistent with
241 both hypotheses. It remains for other experiments employing different methods to corroborate the timing difference observed here and to aid in its interpretation. ACKNOWLEDGEMENTS
Thanks are due to Harry Fein for instrument design, Mahlon DeLong for thoughts on task training and monitoring, and E. V. Evarts for help with every aspect of this work. Supported by NINDS Research Grant No. 5-R01-NS-09984.
REFERENCES 1 CARMAN,J. B., Anatomic basis of surgical treatment of Parkinson's disease, New Engl. J. Med., 279 (1968) 919-930. 2 DEECKE,L., BECKER,W., GROZINGER,B., SCHEID,P., AND KORNHUBER,H., Human brain potentials preceding voluntary limb movements, Electroenceph. clin. Neurophysiol., Suppl. 33 (1973) 97-104. 3 ECCLES,J. C., Circuits in the cerebellar control of movement, Proc. nat. Acad. Sci. (Wash.), 58 (1967) 336-343. 4 EVARTS, E. V., Pyramidal tract activity associated with a conditioned hand movement in the monkey, J. Neurophysiol., 29 (1966) 1011-1027. 5 EVARTS, E.V., Contrasts between activity of precentral and postcentral neurons of cerebral cortex during movement in the monkey, Brain Research, 40 (1972) 25-31. 6 EVARTS, E.V., AND THACH, W.T., Motor mechanisms of the CNS: cerebrocerebellar interrelations, Ann. Rev. Physiol., 21 (1969) 451-498. 7 FEIN, H., A method for generating random time intervals, Electroenceph. clin. Neurophysiol., 33 (1972) 433-434. 8 FREUND,J. E., Mathematical Statistics, Prentice Hall, Englewood Cliffs, N.J., 1971, pp. 347-349. 9 HOLMES,G., The cerebellum of man, Brain, 62 (1939) 1-30. 10 ITO, M., Neurophysiological aspects of the cerebellar motor control system, Int. J. Neurol., 7 (1970) 162-176. 11 RUCH, T. C., 'Motor systems'. In S.S. STEVENS(Ed.), Handbook of Experimental Psychology, Wiley, New York, 1951, pp. 154-208. 12 SNEDECOR, G.W., AND COCHRAN, W.G., Statistical Methods, 6th ed., Iowa State University Press, Ames, Iowa, 1972, pp. 86-88. 13 THACH, W.T., Discharge of eerebellar neurons related to two maintained postures and two prompt movements. I. Nuclear cell output, J. Neurophysiol., 33 (1970) 527-536.
