Disinhibition during waking in motor cortex neuronal chains in cat and monkey

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Disinhibition during waking in motor cortex neuronal chains in cat and monkey Studies on mass and unit activities in the visual cortex have suggested increased thalamically induced inhibition during waking as compared to sleep 4,6. A similar inference, concerning the recurrent inhibition (believed to be a frequency-limiting mechanism) in the motor cortical area, was drawn from analysis of temporal patterns of spontaneous discharges in pyramidal tract (PT) neurons during the sleep-wakefulness continuum in chronically implanted monkeys7. Release from inhibition during slow sleep was also thought to occur, considering the recovery of the short-delayed potential from a pair of mass responses evoked in the motor cortex by specific thalamic stimulationlT,1L On the other hand, a reduced effectiveness of medial thalamic-inducedla and of antidromically elicited recurrent inhibition2,18 acting on relay cells of the thalamic ventrolateral (VL) nucleus was obtained both with mass and unit recordings during reticular-induced or spontaneous arousal, as compared to periods of EEG synchronization, in acute experiments on paralyzed cats. Since no direct evidence had been offered on changes in inhibitory sequences set in motion in motor cortex units during waking and sleep, it seemed worthwhile to perform such experiments both in immobilized and behaving preparations. Stimulation of the medullary pyramid or the pes pedunculi elicits antidromic spikes followed by IPSPs in PT neurons of the motor cortical area 12,16. Long-lasting inhibition was also obtained in sensorimotor cortical neurons following afferent stimulation of specific thalamic nucleig,11. Evidence has been presented to suggest that IPSPs following antidromic invasion or orthodromic activation are mediated through collaterals which engage local inhibitory interneurons5. The data reported in this paper show obvious depression, during reticular arousal or natural waking, both of recurrent inhibition following antidromic invasion of PT cells and of feedforward inhibitory events induced by thalamic stimulation, thus resulting in disinhibition of cortical neurons during waking as compared to synchronized sleep. In one series of experiments, cats were surgically prepared under ether and Surital anesthesia, immobilized by Flaxedil or a bulbospinal cut, and artificially ventilated so that the COg content of the expired air was 4.0 ± 0.2 ~o. All incisions and pressure points were carefully infiltrated with a long-lasting local anesthetic; the EEG and pupilar signs indicated that the animals fell into long periods of synchronized sleep. They awoke spontaneously or after short trains of high frequency pulses were applied to the mesencephalic reticular formation (RF). Stainless steel microelectrodes having a tip diameter of less than 2/~m were used to record extracellularly in the lateral precruciate gyrus. Antidromic invasion and subsequent inhibition of PT cortical neurons was carried out by stimulating with coaxial electrodes in the medullary pyramid. Afferent cortical inhibition was induced by stimulating the VL or the ventrobasal (VB) complex. In the other series of experiments, Macaca mulatta were chronically implanted with a plastic cylinder over the arm area of the precentral gyrus, and with coaxial stimulating electrodes in the pes pedunculi and VL nucleus. The animal sat in a primate chair and could move his limbs; only Brain Research, 30 (1971) 211-217

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the h e a d was restrained by the plastic cylinder. T h e cylinder was filled with paraffin oil a n d sealed by a plastic cover. Stainless steel microelectrodes (1-2 F m at the tip) passed t h r o u g h the intact dura. This system 10 p e r m i t t e d long periods o f extracellular r e c o r d i n g o f the same unit during n a t u r a l sleep a n d waking, with o r without movement. T h e p r e s e n t d a t a concern changes in i n h i b i t o r y events set in m o t i o n by afferent V L s t i m u l a t i o n d u r i n g slow wave sleep and quiet wakefulness. U n i t potentials (bandwidth between I a n d 30,000 c/sec), pulses s y n c h r o n o u s with testing stimuli, a n d E E G waves f r o m s e n s o r i m o t o r cortex were recorded on m u l t i c h a n n e l magnetic tape.

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Pig. 1. Changes of recurrent and feed-forward inhibition in motor cortex neurons during reticular arousal. Two PT cells (A and B). A (left and right columns: recordings with bandwidths of 50-10,000 c/sec and 1-700 c/sec, respectively): a train of 7 shocks at 320/sec, applied to the medullary pyramid, evoked antidromic spikes without failure at constant (1.2 msec, evaluated at faster speed) latency, followed by inhibition (lasting 190 msec) and subsequent rebound; high-frequency (300/see) RF stimulation (between arrows; lack of shock artifacts in right column recordings are due to attenuation of high-frequency components) strikingly reduced the duration of the inhibitory slow negativity following antidrornic invasion (see right column) and increased the spontaneous discharges. B: antidromic spike (4 msec latency) evoked by the first shock in a train of 4 stimuli at 100/sec applied to the medullary pyramid, followed by no responses to the following shocks (1), and inhibition of spontaneous firing of the same unit induced by 2 VB shocks (dots), without prior activation of the cell (2); highfrequency (320/see) RF stimulation, preceding the testing stimuli by about 15 msec, induced full responsiveness to all 4 antidromic shocks (1) and restored spontaneous discharges following VB stimulation (2). In this and following figures, positivity downwards. Brain Research, 30 (1971) 211-217

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Later suitable episodes were examined in detail with an oscilloscope and EEG recorder. Fig. 1A depicts a PT cell in cat's precruciate gyrus which, following antidromically elicited spikes, exhibited long-lasting (about 200 msec) inhibition of background activity, simultaneous with a slow negative wave of the same duration. As judged by the initial positivity of the spike and its amplitude (4 mV), the recording must have been juxtacellular and, thus, the subsequent slow negativity associated with abolition of spontaneous discharges might be regarded as reflecting the IPSP of the same cell. Indeed, slow negative shifts of this cell were invariably observed to interrupt the spontaneous firing. During spontaneous EEG desynchronization, or more efficiently during RF stimulation, the duration of the inhibitory period subsequent to antidromic invasion was reduced (from 200 to 50 msec) and the spontaneous neuronal discharges were restored or even increased above the mean rate observed prior to stimulation. Reticular inhibition of the recurrent inhibitory mechanisms was also shown by comparing the lack of antidromic spikes to PT shocks after the first response in a train during EEG synchronization and, on the other hand, the possibility of the cell to follow without failure all testing shocks of the train when preceded by arousing reticular pulses (Fig. 1B1). Finally, the cortical inhibition induced by stimulating the VL nucleus or VB complex was erased or greatly reduced, whether this inhibition was preceded or not by orthodromic neuronal activation (Fig. 1B2). Similar results were obtained by studying the changes in inhibitory phenomena occurring in monkey's precentral neurons following afferent stimulation of the appropriate thalamic relay during natural waking and different stages of behavioral sleep with EEG synchrony (Fig. 2). The degree of inhibition was estimated from: (1) the duration and amplitude of the field positive slow wave following a single shock or a brief train of fast pulses to the VL (this wave was ascribed to hyperpolarization in a pool of cortical neurons and, thus, it has the same significance as the 'P' wave induced in recurrent VL circuitry by antidromic cortical stimulation1), whether or not preceded by short-latency discharges of the cell under observation, associated with (2) abolition of unit discharges, and followed by (3) cyclic rebound activity in the form of clustered spikes superimposed on the negative, depolarizing slow waves. As reported for recurrent inhibition in the VL units is, sleep with EEG synchronization was accompanied with long-lasting cortical inhibition following VL stimulation. Rhythmic sequences of spike clusters and preceding double or multiple positive slow waves were particularly evident during the period of falling into sleep with EEG spindles. Such events, associated with transient periods of closing and re-opening the eyes peculiar to sleepiness, sometimes preceded the clear-cut EEG signs of sleep by 1-2 min. These findings are consistent with our previous data from behaving cats showing that the synaptically relayed activity in the VL nucleus is inhibited to the highest degree during falling into sleep (drowsiness) and related EEG spindles 19. At deepened stages of slow sleep, VL-induced repetitive positive waves and subsequent rebound unit activities are less pronounced, whereas inhibition of spontaneous firing is more powerful (Fig. 2B). Conversely, spontaneous or naturally induced arousal and the subsequent steady state of waking were associated with obvious Brain Research, 30 (1971) 211-217

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Fig. 2. Changes in inhibition induced by afferent specific thalamic stimulation in precentral cortical neurons of the behaving Macaca mulatta during natural waking and synchronized sleep. Recordings of unit discharges and slow waves using a bandwidth of 1-10,000 c/sec, A, Inhibition induced by a single VL shock (dot) during slow sleep (s) and on awakening (w) from sleep. Note clustered firing superimposed on depolarizing slow negative waves during sleep and regularization with increased discharge frequency during waking; reduction during waking of rhythmic slow positive waves and of rebound sequences following VL testing stimulation. B, Changes in the spontaneous firing and inhibition evoked by a train of 3 VL shocks during steady state of waking (w), drowsiness (d) and slow sleep (s). Two simultaneously recorded neurons (positive-negative and negative-positive spikes). Ink-written traces show 10 testing stimulations (top), spontaneous firing of the positive-negative spike (middle) and EEG waves (bottom). The oscilloscopic traces depict in each of the 3 behavioral states the unit and slow activities elicited by the 10 testing VL stimulation (from top to bottom). Note increased inhibition during drowsiness and slow sleep as compared to waking; rebound (spike clusters) following the initial slow positive wave (indicated by an arrow) during drowsiness; rhythmic inhibition (double or triple slow positive waves) during drowsiness in spite of no significant changes in spontaneous firing; clear-cut decrease in the mean rate of firing during slow sleep. Time calibration: 100 msec (A), 4 sec (EEG traces in B) and 100 msec (oscilloscopic traces in B).

depression of the cortical inhibitory phenomena elicited by afferent stimulation, practically erasing the positive slow waves and the subsequent unit rebounds, restoring and regularizing the spontaneous discharges and, usually, increasing the mean rate of discharge (Fig. 2). The depression of recurrent and feed-forward inhibitory events in motor cortex units during reticular arousal and natural waking both in immobilized and behaving Brain Research, 30 (1971) 211-217

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animals is in line with inhibition, during arousal, of inhibitory phenomena occurring during recruiting waves 18 or evoked by antidromic stimulation in VL relay cells2,18. The present data and conclusion on disinhibition during waking drawn from unit analysis conflict with some of our inferences resulting from recovery during slow sleep of short-delayed VL-motor cortex evoked potentials 17,19. The increase during slow sleep of the second motor cortex potential evoked by paired VL shocks (see Fig. 7 in ref. 19) might now be explained by hyperpolarization following the first response. Increased extracellularly recorded slow positive field potentials reflect IPSPs in a pool of neurons (see again Fig. 2). Consequently, the EPSPs elicited by a second VL testing shock in this period of membrane hyperpolarization will be accentuated, thus enhancing the primary surface positivity of the evoked potential, consideringT M that it is composed of EPSPs in deep layers. Depression during waking of inhibitory mechanisms acting on principal cells in both thalamic2,18 and cortical (present investigation) motor relays does not necessarily imply inhibition of inhibitory interneuronal apparatus. At least in the VL, Purpura et al. 13 have shown that reticular fibers exert direct and powerful excitatory influences on the post-synaptic membrane of the relay cells. This is why conditioning reticular stimuli did not simply restore spontaneous discharges which were inhibited following antidromic invasion, but even increased the discharge frequency. Since this was observed in both the VL relay cells and the PT neurons (Fig. l A), we could postulate that reticular inhibition of inhibition results simply from strong, direct excitatory influences on principal cells in thalamic and cortical motor relays which overwhelm the effect of inhibitory interneurons. However, reduction or disappearance of recurrent and feed-forward inhibition occurred in some units during wakefulness without simultaneous increase in the firing rate, as compared with drowsiness when inhibition was obviously increased (Fig. 2B). Other observations in cat also suggest that the excitability of internuncial neurons might be inversely related to the excitability of principal neurons. Motor cortex relay ceils (which can be synaptically driven at short latencies by VL stimulation and antidromically invaded by PT shocks) were characterized by a regular discharge during EEG patterns of waking. On this background, the firing rate was clearly increased by high-frequency reticular pulses (Fig. 3A). Cortical units, which could not be invaded from the PT or unidentified by any testing stimuli, generally exhibited short or long bursts of high-frequency (200-500/see) spikes interspersed with long periods of silence even during EEG patterns of waking. This pattern of spontaneous activity was not encountered in PT cells. It was also described in the motor cortex neurons by Stefanis15 who suggested that it represents the background activity of cortical interneurons. In contrast to the effects on PT cells, described above, spontaneous discharges of some of these presumed interneurons were completely arrested during an increased vigilance induced by high-frequency reticular pulses (Fig. 3B). These observations are in agreement with those of Evarts 7 from the unrestrained monkey, where he showed that, in contrast to PT neurons, many non-PT cells tend to discharge in bursts even during wakefulness and they have a higher discharge frequency during slow wave sleep in comparison with waking. The above data suggest that motor cortex inhibitory mechanisms are depressed Brain Research,

30 0971) 211-217

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during waking. The question remains open as to the mechanism underlying this removal of inhibition by ascending fibers of diffuse systems involved in waking. This might result from inhibition of the local inhibitory interneurons or (and) by strong excitation of PT cells overwhelming inhibition. In any case, the preceding observations fit well with studies of Jasper e t al. 8 on gamma-aminobutyric acid (GABA), the chemical agent believed to be responsible for cortical inhibition: the rate of release of GABA from the cerebral cortex is 3 times higher during synchronized sleep than during wakefulness. This work was supported by the Medical Research Council of Canada (MA3689). Laboratoire de Neurophysiologie, Ddpartement de Physiologie, Faeultd de M~decine, Universitd Laval, Quebec (Canada)

M. STERIADE P. WYZINSKI* M. DESCHENES* * M. G U E R I N

1 ANDERSEN,P., BROOKS, C. M., ECCLES, J. C., AND SEARS,T. A., The ventro-basal nucleus of the thalamus: potential field, synaptic transmission and excitability of both presynaptic and postsynaptic components, J. Physiol. (Lond.), 174 (1964) 348-369. 2 BREMER,F., Inhibitions intrathalamiques r6currentielles et physiologic du sommeil, Electroenceph. clin. Neurophysiol., 28 (1970) 1-16.

* Holder of a Centennial Scholarship (NRC). Holder of a post-graduate NRC Scholarship.

**

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3 CREUTZFELDT,O. D., WATANABE,S., AND LUX, H. D., Relation between LEG phenomena and potentials of single cortical cells. I. Evoked responses after thalamic and epicortical stimulation, Electroenceph. clin. Neurophysiol., 20 (1966) 1-18. 4 DEMETRESCU,M., DEMETRESCU,M., AND IOSIF, G., Diffuse regulation of visual thalamo-cortical responsiveness during sleep and wakefulness, Electroenceph. clin. Neurophysiol., 20 (1966) 450469. 5 ECCLES,J., The Inhibitory Pathways of the Central Nervous System (The Sherringtonian Lectures IX), Thomas, Springfield, Ill., 1969, 135 pp. 6 EVARTS,E. V., Effects of sleep and waking on spontaneous and evoked discharge of single units in visual cortex, Fed. Proc., 19, Suppl. 4 (1960) 828-837. 7 EVARTS, E. V., Temporal patterns of discharge of pyramidal tract neurons during sleep and waking in the monkey, J. Neurophysiol., 27 (1964) 152-171. 8 JASPER, H. H., KHAN, R. T., AND ELLIOTT, K. A. C., Amino-acids released from the cerebral cortex in relation to its state of activation, Science, 147 (1965) 1448-1449. 9 KRNJEVI(, K., RANDIC, M., AND STRAUGHAN, D. W., An inhibitory process in the cerebral cortex, J. Physiol. (Lond.), 184 (1966) 16-48. 10 LAMARRE,Y., JOFEROY,A. J., FILION, M., AND BOUCHOUX,R., A stereotaxic method for repeated sessions of central unit recording in the paralyzed or moving animal, Rev. canad. BioL, 29 (1970) 371-376. I 1 LI, C. L., Cortical intracellular synaptic potentials in response to thalamic stimulation, J. cell. comp. Physiol., 61 (1963) 165-179. 12 PHILLIPS, C. G., Intracellular records from Betz cells in the cat, Quart. J. exp. Physiol., 44 (1959) 1-25. 13 PURPURA,D. P., MCMURTRY,J. C., AND MAEKAWA,K., Synaptic events in ventrolateral thalamic neurons during suppression of recruiting responses by brainstem reticular stimulation, Brain Research, 1 (1966) 63-76. 14 PURPURA, D. P., SHOVER, R. J., AND MUSGRAVE,F. S., Cortical intracellular potentials during augmenting and recruiting responses. II. Patterns of synaptic activities in pyramidal and nonpyramidal tract neurons, J. Neurophysiol., 27 (1964) 133-151. 15 STEFANiS,C., Interneuronal mechanisms in the cortex. In M. A. B. BRAZIER (Ed.), The Interneuron, Univ. of California Press, Berkeley and Los Angeles, 1969, pp. 497-526. 16 STEFANIS,C., AND JASPER, H., Intracellular microelectrode studies of antidromic responses in cortical pyramidal tract neurons, J. Neurophysiol., 27 (1964) 828-854. 17 STERIADE,M., Ascending control of thalamic and cortical responsiveness, Int. Rev. NeurobioL, 12 (1970) 87-144. 18 STERIADE,M., APOSTOL, V., AND OAKSON, G., Control of unitary activities in the cerebellothalamic pathway during wakefulness and synchronized sleep, J. NeurophysioL, 34 (1971) 389-413. 19 STERIADE,M., IOSIF, G., AND APOSrOL, V., Responsiveness of thalamic and cortical motor relays during arousal and various stages of sleep, J. Neurophysiol., 32 (1969) 251-265. (Accepted April 6th, 1971)

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