Journal of Physiology (1992), 445, pp. 49-68 With 7 figures Printed in Great Britain

49

A FREQUENCY ANALYSIS OF NEURONAL ACTIVITY IN MONKEY THALAMUS, MOTOR CORTEX AND ELECTROMYOGRAMS IN WRIST OSCILLATIONS

BY E. G. BUTLER*, M. K. HORNE* AND P. R. CHURCHWARD From the Department of Clinical Neurophysiology, Alfred Hospital, Commercial Road, Prahran 3181, Victoria, Australia (Received 6 December 1990) SUMMARY

1. Extracellular recordings were made in three monkeys while recording from neurones in the motor cortex (eighty-four cells), ventro-posterior lateralis pars caudalis (VPLC, forty-two cells) and cerebellar thalamus (seventy-seven cells). 2. This experiment was designed to produce active and reflex movements of varying velocities in order to study the relationship between amplitude of velocity and magnitude of neuronal discharge of thalamic neurones. The active movements were voluntary rapid alternating movements (RAMs) of the wrist and the reflex movements were produced by forcibly oscillating the wrist joint between frequencies of 1 and 7 Hz (forced oscillations). 3. This study was also designed to examine cerebellar influences on a reflex path, namely the transcortical reflex loop. Forced oscillations were predicted to provide circumstances where active damping was required to prevent excessive oscillations in the reflex path. Rapid alternating movements of the wrist were predicted to provide circumstances where oscillations at the natural frequency in that reflex path would support and propagate the movements. 4. Forced oscillations from 1 to 7 Hz produced movements of different velocities. VPLC and cerebellar thalamic neurones discharged in relation to the duration of movement in a particular direction, but their discharge levels were unrelated to the magnitude of the velocity. Motor cortex neurones fired in a pattern which was related to the timing but not the magnitude of the acceleration. 5. In forced oscillations of the wrist the resonant frequency was between 3 and 7 Hz. They may be controlled in part by a transcortical reflex. The cerebellar thalamic neurones did not fire before motor cortex neurones. Therefore, it is unlikely that the cerebello-thalamo-cortical pathway is necessary to damp these potentially unstable oscillations by an effect on antagonist-related cortical neurones. 6. Rapid alternating movements (RAMs) of monkeys' wrists were performed in a stereotyped fashion over a narrow range of frequencies with the greatest displacement in joint angle and peak velocity at the natural frequency of 3-5 Hz. * Present address: Department of Neurology, Monash Medical Centre, Locked Bag 29, Clayton 3168, Victoria, Australia.

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E. G. BUTLER, M. K. HORNVE ANVD P. R. CHURCHWARD

7. During the performance of RAMs, neuronal discharge modulated sinusoidally in the VPLC, cerebellar thalamus and motor cortex. There was no relationship between velocity and neuronal discharge of the cerebellar thalamic and motor cortical neurones but there did appear to be a relationship between velocity and VPLC neuronal discharge. 8. The onset of electromyogram (EMG) discharge changed earlier than neuronal discharge in the motor cortex and thalamus during the performance of RAMs. When the frequency at which RAMs were performed increased, mean rectified integrated EMG discharge rose, while neuronal discharge in the cortex and cerebellar thalamus had constant discharges across the frequency range. Therefore the motor cortex and cerebellar thalamus are not part of an oscillating closed loop for the generation of RAMs. RAMs appear to be generated by subcortical mechanisms. 9. Cerebellar thalamic neuronal discharge is related to the duration of movement in one direction (or the duration of the velocity signal) but not to the magnitude of velocity in both voluntary and reflex movements. INTRODUCTION

We have presented evidence that ventro-posterior lateralis par caudalis (VPLC) and the cerebellar thalamus (ventro-posterior lateralis oralis, VPLo; ventro lateralis caudalis, VLc; area X; ventro lateralis pars postrema, VLPS) receive information about the velocity of a movement (Butler, Horne & Hawkins, 1992a). From this study we found that when a voluntary movement was made, cerebellar thalamic neurones discharged before the movement, whereas VPLC neurones fired just after the onset of movement. The duration of the burst of discharge was similar at both sites and closely related to the duration of the velocity signal. However, when the limb was unexpectedly perturbed, the duration of the cerebellar thalamic and VPLC discharges remained at a higher level for approximately the velocity duration, but the cerebellar thalamic discharge was later and occurred after the VPLC discharge commenced (Butler, Horne & Rawson, 1992 b). Since reflex and voluntary movements were stereotyped, there was little variation in their amplitude or duration and hence little variation in velocity. As a result a relationship could not be established between the amplitude of velocity and neuronal discharge (Butler et al. 1992a, b). To overcome this difficulty we designed tasks where it was expected that the resultant movements would be performed with varying velocities. The first task was a voluntary self-paced rapid alternating movement in which the animal made wrist excursions of different frequencies. The second task consisted of forced limb oscillations of frequencies between 1 and 7 Hz. At most of these frequencies forced oscillations were a reflex task which resulted in movements with different velocities and accelerations. These tasks were also designed to consider the cerebellar control of reflex paths. MacKay & Murphy (1979) suggested that the cerebellum participates in movement control by altering the gain and stability of various reflexes without being a direct component of these reflex paths. Much of the evidence for this theory is derived from studies of the control of the vestibulo-ocular reflex by the cerebellum (Ito, Shiida, Yagi & Yamamoto, 1974; Robinson, 1976). Vilis & Hore (1977, 1980) provided

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS 51 evidence for the cerebellar control of a transcortical reflex loop. Nashner and colleagues showed that an unwanted and presumably transcortical long-latency response could not be suppressed in patients with cerebellar lesions but was suppressed in normal subjects (Nashner, 1976; Nashner & Grimm, 1978). Although the existence and proposed function of the transcortical reflex is controversial (see review by Marsden, Rothwell & Day, 1983), recent evidence from recordings of corticomotoneuronal spike-triggered averages of electromyogram (EMG) during limb perturbations strongly favours the existence of this loop (Cheney & Fetz, 1984). In addition, when the deep cerebellar nuclei were cooled, 3-4 Hz limb oscillations were seen, with neuronal activity in the motor cortex at the same frequency (Conrad, Matsunami, Meyer-Lohmann & Brooks, 1974; Meyer-Lohmann, Conrad, Matsunami & Brooks, 1975; Vilis & Hore, 1980). The sinusoidal oscillations of cerebellar tremor could be reset by perturbations applied during the tremor, thereby providing evidence strongly suggestive of reflex involvement in the generation of these oscillations (Vilis & Hore, 1977). Vilis & Hore (1977, 1980) suggested that the transcortical reflex was damped by early cerebellar-dependent Activity in the antagonist or braking muscle. When the agonist muscle is stretched a message is sent to the cerebellum from motor cortical cells activated by the muscle stretch. According to their model the signal is then returned, via the cerebellar thalamus, to motor cortical cells related to the antagonist muscle. Hence, antagonist-related cortical cells fire before the antagonist muscle is passively stretched by the perturbing oscillation and could be responsible for the early antagonist EMG activity that damps the movement. Cooling the cerebellum removes this path, so that antagonist-related neurones in the cortex are dependent solely on late, reflex information from the periphery arriving after the antagonist EMG burst. This late activity tends to perpetuate the movement by producing a burst of neuronal discharge in the cortex and subsequent late EMG activity which is timed to perpetuate oscillations. The loop is therefore unstable and cerebellar tremor results (Vilis & Hore, 1980; Hore & Vilis, 1984a, b). If the mechanism proposed by Vilis & Hore (1980) is correct, then the signal generated by the cerebellum ought to travel through the thalamus on its path to the motor cortex. Presumably the cerebellum or the thalamus would be responsible for receiving a command from the agonist-related cortical cell to fire, and so damp the movement. The thalamus is an obvious location for switching from agonist- to antagonist-related cortical neurones. Any perturbation that produces oscillations of potentially unstable long-loop reflexes would require damping, especially near the corner frequency, to overcome limb instability. If this damping is provided by the cerebellum it must travel through the cerebellar thalamus. Also, the discharge of thalamic neurones should be appropriately timed to activate the antagonist-related cortical neurones. It has been suggested that rapid alternating movements (RAMs) such as writing, tapping or hopping required oscillations of a functional stretch reflex which involved a long-loop pathway (Melvill Jones & Watt, 1971). These repetitive movements were hypothesized to be the result of the natural oscillatory frequency of the neuronal and mechanical properties of the limbs and transcortical reflex pathways (Stein & Oguztoreli, 1976). These movements were frequently around 3 Hz. Further support

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E. G. BUTLER, M. K. HOR.X.E AND P. R. CHURCHWARD

for this hypothesis was the finding that cerebellar nuclear lesions in monkeys caused 3 Hz tremors (Goldberger & Growdon, 1973; Gilman, Carr & Hollenberg, 1976; Vilis & Hore, 1977). Extrapolating from these previous studies we predicted that RAMs would be generated as a result of the undamped properties of transcortical long-loop reflexes, and so would require little cerebellar influence. It would also be expected that the amplitude of the cortical signal would increase to be maximum at the natural frequency. This study was designed firstly to provide active and reflex movements with varying velocities to study the relationship between the amplitude of velocity and the magnitude of the neuronal discharge of thalamic neurones. The second aim was to examine cerebellar influences on a reflex path, the transcortical reflex loop. It was predicted that forced oscillations would provide circumstances where active damping was required to prevent excessive oscillations in the reflex path. Rapid alternating movements of the wrist would provide circumstances where oscillations in that reflex path would be responsible for production of the movements, and under these circumstances most cerebellar influences on the reflex would be excluded. METHODS

Animal training, surgery, equipment, data acquisition, histological techniques and location of thalamic recordings by cerebellar nuclear stimulation are detailed in accompanying papers (Butler et al. 1992 a, b). We followed the terminology of these studies and refer to the VPL., VL, area X and VLP, nuclei of the thalamus collectively as the cerebellar thalamus. The protocols described in this study were performed on each cell concurrently with the protocols and clinical examination described in Butler et al. (1992a, b). Identical selection criteria described in those papers for cells in the VPLC, cerebellar thalamus and motor cortex were applied in this study.

Rapid alternating movements (RAMs) The animals were trained to perform self-paced, alternating flexion/extension movements of the wrist, and each movement was at least 50 deg of joint angle. There were no visual targets or movement cues, although the cursor which represented the animal's wrist joint angle was displayed on the oscilloscope screen directly in front of the animal. Eight trials were performed, each trial containing between three and five movements. The animals quickly learned to perform the movements rapidly to ensure a steady supply of a fluid fruit-juice reward (hence they became known as rapid alternating movements). Forced oscillations The monkeys were trained to hold the cursor within a target centred about the neutral wrist position while sinusoidal oscillations were applied by a torque motor about the wrist at frequencies varying between 1. 3, 5 and 7 Hz for examination of neuronal discharge. The range from 1 to 7 Hz was adopted because it represented the range at which the wrist joint was regularly used, and included the resonant frequency of the limb. Higher frequencies were not usually examined because there was a limited period of time to record each neurone. perform a sensory examination and apply cerebellar nuclear stimulation. Howsrev-er, both forearm EMiG and limb mechanics were examined during oscillations up to 16 Hz. The target was sufficiently narrow so that incorrect trials could occur unless the animal used a strategy such as stiffening the wrist joint to prevent the cursor straying outside the target, thus making the trial invalid. To force the animal to use this strategy, the target width was different for each frequency and varied between 20 and 30 deg of wrist joint angle. The force applied by the torque motor was the same across all frequencies. Each frequency was applied in blocks of four to eight trials, and each trial lasted between 1 and 3 s (three oscillations at 1 Hz; seven oscillations at 7 Hz).

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS

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Data analysis The cellular activity, joint position, acceleration and velocity were averaged at each frequency of forced oscillations and for RAMs (Fig. 1). This produced a series of sinusoids which were analysed to identify the phase shift or temporal delay between cellular activity and the three movement

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commercially available statistics package (Wilkinson, 1988). Only correlations which exceeded 2 S.E.M. errors were accepted and the time lag and sign of the greatest correlation was used to determine the phase shift. In cyclical movements such as RAMs it is difficult to know whether the neuronal signal is phase leading or lagging behind the movement. To resolve this, we assumed that as RAMs and the standard reaction-time task were both voluntary movements, cellular discharge

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E. G. BUTLER, M. K. HORNE AND P. R. CHURCHWARD

would be related to the same direction of movement in both movements (Butler et al. 1992 a) (Fig. 1). For this reason we only selected for analysis those neurones with reciprocal response to the reaction-time movement and one burst of neuronal activity per movement oscillation (singleresponse patterns; see definition in Results). A

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Fig. 2. A, the number of RAMs plotted against frequency obtained from monkey 2 over twenty-two recording session. The graph demonstrates that the monkey performed RAMs over a narrow range of frequencies, with most RAMs performed at the natural frequency. B, the amplitude of movement of each RAM plotted against frequency. Data from thirty consecutive RAMs. Amplitude is relatively constant over the central frequencies but is markedly reduced at the extremes of the frequency range. Monkey 1. In a similar way, the direction of movement to which neuronal discharge was best related during forced oscillations was determined from the response of the cell to a single torque-pulse perturbation (Butler et al. 1992 b). Since both perturbations and forced oscillations produced reflex responses, it was assumed that neuronal discharge would be related to the same direction of movement in both instances. Time series analyses was used to calculate phase shift and determine whether neuronal activity led or lagged behind the movement by examining the response to perturbations. For this reason we selected for analysis only neurones with a reciprocal response to torque-pulse perturbations and one burst of neuronal activity per movement oscillation (single-response patterns; see definition in Results). These criteria permitted comparisons of neuronal responses with discharge patterns of neurones from other sites and with forearm EMG. The relationship between timing of neuronal discharge and

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS

55

forearm EMG with movement kinematics was compared between recording sites using analysis of variance (ANOVA) and the Newman-Keuls multiple comparison test. Statistical level of significance was set at P = 0 05.

Normalization of data Rapid alternating movements. The number of discharges occurring during each RAM was divided by the period of the RAM, expressed as discharges per second, and plotted against the frequency of each movement. Because each cell had different firing rates, it was difficult to make direct comparisons between cells regarding discharge modulation with frequency of movement. To allow comparison between cells, the discharge rate at the common frequency of movement (see Fig. 2A) was arbitrarily expressed as 100. The discharge rates at other frequencies were then expressed as percentages of this figure (normalized discharge rate or NDR). It is important to note that the relative changes across the frequency range are more important than the absolute values. This method made it possible to draw together the data from neurones from each site and make comparisons with other recording sites (Fig. 4). Forced oscillations. Data were normalized by the following method. The total discharge over one cycle of oscillation was divided by the period to obtain a discharge rate (in Hz). The discharge rate at 5 Hz was set at 100 (because this was at or near the corner frequency; see Fig. 5) and the values obtained for the discharge rate at other frequencies were expressed as a percentage of this value (ND). RESULTS

Rapid alternating movements Studies of rapid alternating movements (RAMs) were carried out in three monkeys, recording from cerebellar thalamus and motor cortex in monkey 1 from only the motor cortex in monkey 2 and from cerebellar thalamus and the VPLC in monkey 3. When data collected over several months of recording were pooled it was found that RAMs were performed over a narrow frequency range, between 1-6 and 5-6 Hz, with a clear peak around the most common frequency of oscillations (Fig. 2A) which was referred to as the natural or resonant frequency of the wrist joint in this task. Amplitude of movements also tended to remain constant across the frequency range but with a rapid decrease in amplitude at the extremes of the frequency range

(Fig. 2B).

The natural frequency at which RAMs were performed varied between animals from 2-6 to 5-4 Hz. There was also some tendency for variation in the natural frequency of up to 1-4 Hz in each animal over the 6-18 months of recording, but there was no appreciable variation from day to day. Indeed the variation in frequency while recording from a single neurone was often as little as 0-5 Hz. Because of this a meaningful relationship between velocity and the neural discharge of a single cell was difficult to obtain. However, a loose inference about velocity and neuronal discharge can be made from pooled data. As pooled data demonstrated that amplitude was constant across the frequency range (Fig. 2B), then velocity of movement is related to the frequency. In other words, if there is a relationship with velocity of movement, neuronal activity should increase as the frequency of RAMs increase.

Electromyogram During RAMs, the forearm flexor and extensor EMG discharged sinusoidally and phase led the movement by 196 deg (see Table 1). This phase lead was calculated using time series analyses (as described in the Methods section). EMG activity

E. G. BUTLER, M. K. HORNE AND P. R. CHURCHWARD

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Fig. 3. A, the discharge pattern of a single-response cerebellar thalamic neurone during RAMs, firing in only one direction of wrist movement. During the standard reaction-time task this cell had increased firing during flexor movement and decreased firing with extensor movement (see text). Neuronal discharge peaks by the time the flexor RAM commences. The timing of neuronal discharge was linked best to the positive sign of the acceleration of the movement and was 180 deg out of phase with direction. B, a doubleresponse cerebellar thalamic neurone which fired during both flexor and extensor RAM excursions. This neurone also fired in association with both flexor and extensor ballistic movements (not shown). The dashed line marks commencement of extensor directed RAMS. Abbreviations as in Fig. 1 legend. Position scale, 10 deg; velocity scale, 600 deg s-1; acceleration scale, 8000 deg S-2; ND tick interval, 20 impulses s-1 in A, 10 impulses s-1 in B. Bin width, 20 ms in A, 30 ms in B. Monkey 3. TABLE 1. Timing of neuronal discharge or EMG relative to movement kinematics in RAMs Phase lead Phase lead Phase lead on position on velocity on acceleration (deg) (deg) (deg) EMG 196+5 112+6 27 +5 (n= 13) Cerebellar thalamus 192+22 107 + 19 27 + 20 (n = 21) VPLC neurone 146+9 62+11 -26+11 (n = 12) Motor cortex 62+12 145+10 -18+15 (n = 27)

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS 57 consistently increased as the frequency of oscillations increased (Fig. 4A). This suggests that there was a relationship between velocity and the amplitude of EMG activity. Using time series analyses, neuronal discharge was cross-correlated with limb position, velocity and acceleration. A cell was considered to be correlated with a movement parameter if the crosscorrelation of neuronal discharge with joint position exceeded background noise by 2 S.E.M. Many TABLE 2. RAM responses A

Well Single Reciprocal n correlated response with jumps RAMs 21 47 Cerebellar thalamus 73 57 21 12 VPLC neurone 40 38 33 27 Motor cortex 48 33 B Reciprocal Forced Well Single correlated response with perturbations n oscillations 32 17 Cerebellar thalamus 51 40 19 16 29 26 VPLC neurone * 15 27 17 Motor cortex * Perturbations were not performed in the motor cortex. neurones had only one burst of discharge during each complete movement cycle and will subsequently be referred to as single-response cells (Fig. 3A). Some neurones had two bursts of activity per movement cycle and will be referred to as double-response cells (Fig. 3B). The discharge pattern of single-response neurones resembled forearm EMG. As outlined in the Methods section (Data analysis), neurones were selected for further analysis only if the discharges were correlated with joint position, there was a reciprocal response to the reaction time movement and a single-response pattern during RAMs.

Cerebellar thalamus Recordings of the discharges of seventy-three cerebellar thalamic cells were made from monkeys 1 and 3 during the performance of RAMs. Twenty-one of these seventy-three neurones (29 %) met the selection criteria for analyses (see previous section and Methods) (Table 2A). It could not be predicted from the neuronal discharge pattern in the standard reaction-time task (Butler et al. 1992a) or from the cell's sensory receptive field (Butler et al. 1990b) whether a cell would have a single or double response to each RAM cycle. Using time series analyses the onset of the discharge bursts of selected neurones were found to phase lead the movement, with a slight tendency (non-significant, ANOVA and Newman-Keuls multiple comparison test) to fire soon after the agonist muscle commenced discharge, although the scatter of phase shifts of cerebellar thalamic neuronal discharge was substantial (Table 1). Discharge of the cerebellar thalamic neurones phase led the onset of movement (phase lead to 192 deg) and velocity (107 deg). These neurones fired just before the change in acceleration (Table 1). There was a non-significant trend (ANOVA and Newman-Keuls multiple comparison test) for this population of cells to fire before corresponding VPLC and cortical neurones.

K G. BUTLER, M. K. HORNE AND P. R. CHURCHWARD The discharge rate of cerebellar thalamic cells was almost constant across the frequency range (Fig. 4B), unlike forearm EMG which under similar circumstances rose with increasing frequency (Fig. 4A). In fact, some cerebellar thalamic neurones had a total discharge which was least at the most common or natural frequency.

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Frequency (Hz) Fig. 4. A, mean discharge rate for a typical forearm EMG recording plotted against the frequency at which RAMs were performed, normalized to 3-8 Hz. Data obtained from one recording session when RAMs were performed between frequencies of 2-3-8 Hz. Panels B-D show the normalized discharge rate (see text) plotted against frequency of each movement. This manoeuvre made it possible to draw neurones from each site together for analysis, in addition to enabling comparison of sites of recording during RAMs. Error bars indicate 1 S.E.M. on each side of the mean. Data are normalized about 3-8 Hz. allowing data from different sites to be compared in different animals who performed the task at varying frequencies. B, data from twenty cerebellar thalamic neurones normalized at 3-8 Hz. The trend was for constant firing across the frequency range. C, data from twelve VPLC neurones normalized at 3-8 Hz. Normalized neuronal discharge rate increased with rising frequency. D, data from twenty motor cortex neurones normalized at 3-8 Hz. Normalized neuronal discharge rate was constant across the frequency range.

Because amplitude was constant across the frequency range, increasing frequency resulted in increasing velocity of movement. This indicates that even using pooled data there is no relationship between amplitude of velocity and neuronal activity.

Ventro-posterior lateralis pars caudalis Recordings during the performance of the RAM task were made from forty wristrelated VPLC neurones in monkey 3. Twelve of the forty neurones (30 %) (Table 2A) met the selection criteria for further analyses (see Methods) because neuronal discharge was well correlated, there was a single response to RAMs and reciprocal

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS

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firing pattern during the reaction-time task. These neurones all had deep receptive fields and the remaining single-response neurones which did not fully meet the criteria had similar characteristics and behaviour to these neurones. Double-response cells usually had purely cutaneous sensory receptive fields on the hand (14/17, 82 %). As the animal's hand fitted snugly into a manipulandum, there was pressure on the sensory field whenever the manipulandum moved. The discharge of the selected neurones phase led the movement (Table 1), but lagged behind EMG, although this trend was not significant. Although the discharge also phase led the velocity and lagged behind the acceleration components of the movement, it did not appear closely coupled to any particular movement parameter. The normalized discharge rate generally rose with increasing frequency (Fig. 4C). This pattern was dissimilar to that observed in the cerebellar thalamus and motor cortex, but resembled forearm EMG (Fig. 4A). While we could not confirm a relationship between velocity and amplitude of neuronal response of individual neurones because of the lack of variation in movement on a day-to-day basis, pooled data do suggest that a relationship may exist. As pooled data demonstrated that amplitude of movement was constant across the frequency range (Fig. 2B), then neuronal activity should increase as the frequency of RAMs increases if there is a relationship with velocity of movement. This evidence, albeit from pooled data, indicates that, similar to EMG, the amplitude of the response of VPLC neurones to movement is related to the amplitude of velocity. Motor cortex Recordings were made from the motor cortex during the performance of RAMs from forty-eight cells in two monkeys. Twenty-seven cells (56%) met the criteria for further analyses (Table 2A). We were unable to predict from the standard reaction-time task or the sensory characteristics of each cell whether a response to RAMs would be well correlated. The activity of the selected neurones phase led joint angle but lagged behind forearm EMG and cerebellar thalamic neurones (Table 1). This trend was non-significant (ANOVA and Newman-Keuls multiple comparison test). Cortical neurones had timing characteristics similar to those of VPL, neurones, phase leading the movement velocity and just lagging behind the acceleration. A normalized discharge rate was obtained for each frequency (Fig. 4D). Motor cortex neurones had a reasonably constant firing rate across the frequency range and were similar to cerebellar thalamic neurones. Forced oscillations When the monkey's wrist was oscillated sinusoidally between 1 and 16 Hz the monkey adopted a strategy of forearm muscle co-contraction to keep the wrist stiff and prevent the cursor moving outside the target window. Total limb compliance (joint angle divided by applied torque) and the phase difference between applied torque and joint angle was calculated for each frequency (Fig. 5A and B). The corner frequency for each of the three animals used in this experiment varied from 3 to 7 Hz but was constant for each animal. Limb stiffness, which is inversely proportional to the compliance, was least at the natural frequency. Joint angle was greatest at the

G. BUTLER, M. K. HORNE AND P. R. CHURCHWARD corner frequency (Fig. 5C). The peak velocity and acceleration of forced oscillations varied with the frequency, tending to mirror the joint angle, with maximum peak velocity and acceleration at 5-7 Hz, and minimum values always 1 Hz.

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Electromyogram Forearm EMG activity changed sinusoidally and was better formed and correlated to joint angle above 1 Hz. The onset of forearm EMG discharge always phase led the A

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Fig. 5. Frequency analysis of limb mechanics during forced sinusoidal oscillations of the wrist, represented as Bode plots. The resonant or corner frequency was 5 Hz (semilogarithmic scale). A, limb compliance (joint angle/torque) is plotted against frequency of forced oscillations. Data obtained from three separate recording sessions. B, phase lead of applied torque with respect to joint angle plotted against frequency, showing a large change in phase around 5 Hz. C, joint angle plotted against frequency of oscillations. Amplitude of torque was the same for each frequency. Joint angle peaked at 5 Hz. Each symbol represents a different monkey.

movement onset by approximately 100 deg, which meant that the time lead (in ms) progressively diminished as the frequency increased (Table 3). EMG discharge also phase led the movement velocity but lagged behind acceleration. Total meanrectified, integrated EMG discharge was calculated for each frequency and this is illustrated as normalized data in Fig. 6A. Data were normalized as described in the

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS 61 Methods section (see Normalization of data). EMG discharge generally peaked at 3-5 Hz in monkey 3, coinciding with the corner frequency. There was no relationship between forearm EMG discharge and velocity magnitude (Fig. 6E). In the case of forced oscillations, time series analyses were used as previously described to determine whether neuronal discharge was cross-correlated with limb position, velocity and acceleration. A cell was correlated with a movement parameter if the cross-correlation of neuronal TABLE 3. Timing of neuronal discharge or EMG relative to movement kinematics in response to forced oscillations Oscillations 1 Hz Phase lead 3 Hz 5Hz 7 Hz of neurone (deg) (deg) (deg) (deg) P EMG 67+6 94+8 104+5 96+5 (n= 13) V 20+7 12+9 18+4 21+7 A -88+15 -68+11 -62+5 -61+8 Cerebellar thalamus P 131+28 77+13 85+15 74+18 (n= 17) V 29+23 -10+19 2+17 -27+17 A -56+37 -70+20 -87+18 -103+18 P 73+8 98+7 113+5 94+8 VPL, neurone V (n= 16) -4+5 -16+5 16+6 13+7 A -84+18 -68+6 -64+6 -72+7 p Motor cortex 111+14 98+17 110+14 126+11 V (n= 15) 42+12 -8+2 29+19 46+14 A -30+29 -8+24 -33+16 -41+14 P, position; V, velocity; A, acceleration. In the case of EMG, n = number of recordings made from forearm extensor muscles. There was no difference between forearm extensor and flexor muscle.

discharge with joint position exceeded background noise by 2 S.E.M. Many neurones had only one burst of discharge during each complete oscillation cycle and will subsequently be referred to as single-response cells (Fig. 7), but some neurones had two bursts of activity per movement cycle and will be referred to as double-response cells. In view of the explanation in the Methods section (Data analysi8), neurones were selected for further analyses only if the discharges were correlated with joint position, had a single-response pattern to forced-wrist oscillations and a reciprocal response to torque-pulse perturbations. As described in the Methods section, the response of a cell to a single torque-pulse perturbation (Butler et al. 1992 b) was used to determine the direction of movement to which neuronal discharge was related, and therefore determined whether neuronal activity led or lagged behind the movement.

Cerebellar thalamus Fifty-one cerebellar thalamic cells were examined for responses to forced-wrist oscillations (1-7 Hz), seventeen (33 %) of which met the selection criteria (Table 2 B). The activity of the remaining single-response neurones without reciprocal responses to the torque-pulse perturbations did not differ from the seventeen selected neurones. The discharge of fifteen of the seventeen cerebellar thalamic cells (88%) were approximately in phase with movement velocity at all frequencies (Table 3). Neuronal discharge lagged behind the acceleration by a substantial phase. The discharges of the two remaining neurones were in phase with acceleration. The phase lead on movement of cerebellar thalamic cells was not significantly different (ANOVA and Newman-Keuls multiple comparison test) to cells in the VPLc nucleus

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Fig. 6. Mean EMG discharge (A) and neuronal discharge (B-D) resulting from forced oscillations was normalized to 5 Hz (see text) and plotted against frequency of oscillation. Means (± 1 S.E.M.) are represented by error bars. A, forearm extensor EMG discharge was greatest at 3 Hz and least at 7 Hz. Pooled data from fifteen recording sessions. B, cerebellar thalamic neuronal discharge was constant across the frequency range. Pooled data from seventeen neurones. C, VPLC neurones showed constant discharge across the

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS

63

and forearm EMG, but with movements above 1 Hz there was a non-significant trend to fire after the motor cortex neurones (Table 3). When compared with VPLC neurones and EMG there was a large range in phase shift of cerebellar thalamic neurones with respect to the movement parameters, as evidenced by the larger standard errors. Movements produced by forced oscillations were performed at different amplitudes and frequencies, thereby leading to considerable variation in velocity. However, there was no relationship between the amplitude of acceleration, velocity or displacement and the magnitude of neuronal discharge (Fig. 6). Displacement, velocity and acceleration peaked at 5-7 Hz (Fig. 6F) whereas the discharge of cerebellar thalamic neurones remained constant over the range of frequencies (Fig. 6B). Data were normalized as described in the Methods section. The discharge rate was usually constant across the frequency range and resembled the discharge of VPLC and motor cortex neurones. However, individually four cells discharged least at the corner frequency, and six cells peaked at 3 or 5 Hz. Ventro-posterior lateralis pars caudalis Twenty-nine cells were examined for their responses to forced oscillations. The responses of VPLC neurones to forced oscillations were clearer and better defined than those of cells in the cerebellar thalamus or motor cortex. Sixteen of these twenty-nine (55%) met the selection criteria and were studied (Table 2 B and Fig. 7). The receptive fields in fifteen of nineteen single-response neurones (79 %) had some deep components, a trend also observed with single-response neurones during RAMs. Six of seven double-response neurones had purely cutaneous fields. Using time series analyses the phase of the discharge of VPLC neurones was closest to movement velocity (Table 3). The normalized discharge rate (to 5 Hz) for these neurones was calculated at each frequency. The discharge level of most neurones was constant across the frequency range (Fig. 6C), and did not change appreciably with movement acceleration, velocity or displacement (Fig. 6B), indicating that there was no relationship between the magnitude of peak velocity or acceleration and the level of neuronal discharge.

Motor cortex Twenty-seven neurones were recorded while the wrist was oscillated sinusoidally between 1 and 7 Hz. Fifteen neurones (56 %) met the selection criteria for further analyses (Table 2B). Cortical neuronal discharge was not well correlated with forced oscillations when compared with the cells in the VPLC or cerebellar thalamus. Perturbations were not performed during motor cortex recordings. frequency range. Pooled data from twelve neurones. D, motor cortex neurones showed a diminution in discharge at 3-5 Hz. Pooled data from fifteen neurones. Mean EMG discharge (E) and neuronal discharge (F-H) resulting from forced oscillations were normalized to 5 Hz and plotted against velocity of the movement at each frequency. Means (+ 1 S.E.M.) are represented by error bars. Forearm EMG discharge (E) plotted against peak velocity of forced oscillations at each frequency is not related to velocity (n = 15 recording sessions). Cerebellar thalamic discharge (F), VPLC discharge (G) and motor cortical discharge (H) were also unrelated to peak velocity. The same neurones listed in B-D are represented. 0, 1 Hz; *, 3 Hz; A, 5 Hz; A, 7 Hz.

E. G. BUTLER, M. K. HORNE AND P. R. CHURCHWARD

64

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200 ms 500 ms Time (ms) Time (ms) Fig. 7. A single-response VPLC neurone with entrained neuronal discharges during each movement cycle. Neuronal discharge increased following perturbation of the forearm flexors (i.e. extensor movement) and decreased following stretch of the extensors. During forced oscillations neuronal discharge phase led the extensor movement, with discharge approximately in time with the negative sign of the velocity. Extension is depicted as downward displacement. Dashed line indicates peak negative velocity. Abbreviations as

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS 65 Cortical discharge phase led movement. There was a non-significant trend (ANOVA and Newman-Keuls multiple comparison test) for cortical cells to lead the discharge in the cerebellar thalamus, VPLC and EMG (Table 3). The onset of neuronal discharge in the motor cortex was better related to the movement acceleration than were the neurones in the VPLC and cerebellar thalamus. Fifteen neurones were analysed in this way, and ten neurones were closely related to the timing of the acceleration in one direction while five neurones were closely related to the timing of the velocity of the muscle stretch. (Note that acceleration is 180 deg out of phase with movement.) The normalized discharge rate was variable from one neurone to the next, but the general pattern was of constant discharge across the frequency range of 1-7 Hz (Fig. 6D). There was no relationship between the peak magnitude of the movement kinematics and the level of cell discharge (Fig. 6H). DISCUSSION

Does the thalamus signal velocity ? Cerebellar thalamic neurones fired strongly in relation to the RAM task. Unfortunately, during the recording from a single neurone RAMs were performed over a very narrow range of frequencies and the relationship between the discharge of a single cell and neuronal discharge could not be studied. However, some evidence was gained from pooled data which suggested that in the case of the cerebellar thalamus and the motor cortex, a relationship between neuronal discharge and the magnitude of velocity is extremely unlikely. In the case of the VPLC, some relationship may exist. However, forced oscillations provided a wide range of velocities and there did not appear to be any relationship between the magnitude of neuronal response and the magnitude of displacement, velocity or acceleration of movement at any of the recording sites. It appears, at least in the cerebellar thalamus, that neuronal activity does not signal amplitude of velocity. The situation in the VPLC requires further investigation. The forced-oscillations task produced a variety of movement velocities, confirming that in contrast to amplitude of velocity, timing and duration of thalamic neuronal discharge was closely related to timing of the velocity signal. The duration of each burst of discharge of VPLC and cerebellar thalamic neurones was closest in time of onset and duration to the sign of the velocity, occurring while the velocity had one sign and ceasing as soon as velocity crossed zero (Table 3). The peak velocity usually coincided with the peak neuronal discharge (Fig. 7). The sign of the velocity changed when the movement changed direction. Another way of expressing these findings is that the phasic burst of activity in the thalamus is a measure of the duration of movement in a certain direction. An acceleration contribution to this discharge was considered unlikely because the neurone fired considerably later than the timing of the change in sign of the acceleration (Table 3). The timing of neuronal discharge in in Fig. 1 legend. Bin width, 34 ms; ND tick interval, 20 impulses s-1. Frequency (Hz): A,

1; B, 3; C, 5; D, 7. Position scale (deg): A-D, 10. Velocity scale (deg s-1): A, 60; B, 200; C, 500; D, 250. Acceleration scale (degs 2): A, 600; B, 2600; C, 11000; D, 10000. Monkey 3. 3

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66

E. G. BUTTLER, M. K. HORNE AND P. R. CHURCHWARD

the motor cortex was best related to the sign of the acceleration (Table 3), firing soon after the acceleration changed direction, a finding previously noted (Flament & Hore, 1988). The VPLC nucleus is a sensory thalamic nucleus, and probably passively signals input from the periphery. As motor cortex neurones fibre after both VPLC neurones and EMG discharge, we propose that motor cortex neurones are not driving muscle during the RAM task, but may merely be responding passively to afferent signals. These results differ from cortical activity during another voluntary movement, the standard reaction-time task, where cortical neurones fired significantly before VPLC neurones (Butler et al. 1992a). We conclude therefore that RAMs are not a typical voluntary task driven by the motor cortex and that it was not a useful task for examining a pre-programmed movement. In conclusion, there is ample evidence from a number of voluntary and reflex wrist movement protocols that the cerebellar thalamus and VPLC signal the sign of the velocity (or duration of movement in a particular direction). There does not appear to be a relationship between the magnitude of these variables and the amplitude of cerebellar thalamic neuronal discharge. The relationship between VPLC neurones and magnitude of velocity warrants further study. Does the cerebellum control long-loop reflex stability? Vilis & Hore (1980) have suggested that the cerebellum might provide a signal which predicts and precedes the afferent signal from the periphery to the neurones in the motor cortex connected to antagonist-related muscles, and that this cerebellar signal helps damp tremors of the limb by preventing oscillations of the long-loop reflex. We designed the forced oscillations experiment to test whether the cerebellothalamo-cortical pathway would influence the efferent limb of the transcortical long-loop reflex. The animals had most difficulty in preventing oscillations at the resonant frequency of the limb. An indirect measure of this difficulty was shown by a greater movement amplitude at the resonant frequency for the same applied level of torque across the frequency range (Fig. 5C). Neilson (1972 b) suggested that EMG activity elicited by forced oscillations of the elbow up to 10 Hz was produced by both segmental and long-loop reflexes. Perturbations result in rapidly damped 5-6 Hz oscillations which were thought to be due partly to transcortical paths because they were associated with appropriately timed cortical activity (Meyer-Lohmann et al. 1975; Vilis & Hore, 1980). Hence we predicted that reflex activity in forced oscillations might involve, at least in part, a transcortical loop. If this were the case then the cerebellum would fire most at the resonant frequency to damp potentially unstable oscillations, and neuronal activity in both the cerebellum and cerebellar thalamus would phase lead cortical activity (Vilis & Hore, 1980; Hore & Vilis, 1984a). However, under conditions identical to those used for EMG recordings during forced oscillations, the magnitude of neuronal activity in the cerebellar thalamus, unlike EMG, was flat across the frequency range, without any rise at the resonant frequency. Furthermore, the cerebellar thalamus did not fire before motor cortex neurones and in fact there was a tendency (although non-significant) for cortical cells to discharge first. Although the motor cortical neurones do fire in forced oscillations and phase lead the movement, we found little evidence that the cortex is part of a long-loop reflex in either forced oscillations or RAMs. Nevertheless we

THALAMUS, CORTEX, EMG AND LIMB OSCILLATIONS 67 cannot exclude the possibility that this response to forced oscillations might at least in part be due to a transcortical reflex, but that no rise in cortical response was seen at the natural frequency because of effective cerebellar damping. Removal of cerebellar influence would result in a greater cortical response at the resonant frequency and greater motor oscillations. Even so, there is no evidence to suggest that the cerebello-thalamo-cortical pathway fires predictively before motor cortex neurones to damp forced oscillations. We must therefore conclude that Vilis and Hore's theory cannot be supported and that some other mechanism must act to damp oscillations following a limb perturbation. The evidence regarding the timing of thalamic discharge does not support the notion of a contribution from the cerebellar thalamus to antagonist-related cortical neurones. Are rapid alternating movements a resonating long-loop reflex? If movements such as writing, tapping or hopping were generated by oscillations of a functional stretch reflex which involved a long-loop pathway, we would expect that components of the loop would have greatest discharge at the loop's natural frequency. Forearm EMG discharge rises with increasing frequency (Fig. 4A), an expected finding since muscle is an integral part of an oscillating closed-loop system with a natural frequency above that examined in this experiment (Stein & Lee, 1981). The discharge in VPLC neurones mirrored the EMG results, but because of the limited frequencies that resulted in this self-paced task it is not known at which frequency VPLC activity would have peaked (Fig. 4C). However, neurones in the motor cortex and cerebellar thalamus demonstrated reasonably constant discharge levels across the frequency range in which RAMs were performed (Fig. 4B and D), with discharge sometimes being least at the resonant frequency. This was despite the movement amplitude reaching a maximum at the resonant frequency. On this evidence the motor cortex and cerebellar thalamus were not part of an oscillating closed loop for the generation of RAMs. It is possible that motor cortical neurones passively signal what has happened in the RAMs, receiving information about the movement after EMG has commenced firing. This evidence supports the notion that 2-6 Hz repetitive movements are open loop and pre-programmed (Neilson, 1972a). Unlike the voluntary reaction-time task where the motor cortex fired well before EMG and was responsible for EMG generation (Fetz & Cheney, 1980), RAMs do not appear to require the motor cortex for their generation. Because their resonant frequency is about 3-5 Hz -it is unlikely that spinal reflexes primarily generate RAMs (Stein & Oguztoreli, 1976) and if this were so one might expect cortical discharge to rise in frequency as in the EMG and VPLC. The results of this study indicate that RAMs are not an example of the long-loop transcortical reflex, and argue against the proposal that alternating movements such as hopping, writing and dancing are due to transcortical oscillations. We wish to acknowledge the assistance of Dr J. Rawson, H. Clark, J. Buttress, P. Burmeister, L. Duncan, Dr R. N. Kulkarni and D. Finkelstein. Dr S. Cheema provided invaluable assistance with histology. Funding was received from the Van Cleef Foundation and the National Health & Medical Research Council of Australia. 3-2

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E. G. BUTLER, M. K. HORNE AND P. R. CHURCHWARD REFERENCES

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