Oxotremorine induced tremor in the decorticated cat

Inf. .I. Neuropharmacol..

1965,

4, 139-148

Pergamon Press. Printedin Gt. Britain. [7 figs.,29refs.1

OXOTREMORTNE INDUCED TREMOR IN THE DECORTICATED CAT* E. E. DEcrU’j’[and[R. W. RAND University of California, Los Angeles, School of Medicine, Department of Surgery/Neurosurgery and the Brain Research Institute, Los Angeles, California 90024 Summary-A technique for the experimental study of tremor is described. Injection of oxotremorine into acutely decorticated cats consistently induced a generalized tremor. The electromyographic analysis showed that the induced tremor was regular with simultaneous contractions of flexor and extensor muscles, although extensors were more affected. The frequency spectrum was wide (8-28 c/s). Tremors started first in the head and neck muscles and were visible during one to two hours as either short or long bursts, with or without waxing and waning characteristics. Frequency spectrum, phase relations of flexor and extensor activity and mode of onset of oxotremorine tremors were similar to those observed in shivering. However, atropine sulfate blocked oxotremorine tremors as it does parkinsonian tremor but it does not affect shivering. Pentobarbital in subanesthetic and anesthetic doses was able to block oxotremorine tremor. Tolerance to oxotremorine appeared to develop with administration of successive doses. INTRODUCTION UNTIL recently, neurophysiological bases of various tremors, particularly those of a pathological type (parkinsonism), were investigated by studying tremors induced experimentally either by electrical stimulation (FOLKERTS and SPEIGEL, 1953 ; JENKER and WARD, 1953), or by lesions in the midbrain reticular formation (WARD et al., 1948; and PETERSON et al., 1949). A promising pharmacological approach was introduced when EVERETT et al. (1956a) discovered tremor-inducing properties of tremorine (1, 4-dipyrrolidino-2-butyne). Subsequently, GEORGE et al. (1962) studied the central effects of oxotremorine (1-2-0x0pyrrolidino)-4-pyrrolidino-butyne-2-, a metabolite of tremorine. They found this drug capable of producing powerful generalized tremors in a variety of species. This effect, as well as the peripheral parasympathomimetic action, was blocked by atropine sulfate. Other anti-Parkinson agents also blocked this tremor. In chronic spinal animals oxotremorine did not produce tremor below the level of the spinal section (GEORGE et al., 1962). Electromyogram (EMG) techniques have not been employed until now to analyze oxotremorine-induced tremor. Such a study is important in order to compare more closely drug-induced tremor with physiological and parkinsonian tremor, the EMG characteristics of which are well-known. Immobilization of the animal in a rigid frame was desirable to record EMG. Anesthetics could not be employed for immobilization since they antagonize tremor; therefore, a technique involving acute decortication of cats was used. We have found that oxotremorine induced a consistent tremor in the acutely decorticated cat. The analysis of oxotremorine tremor by means of this method is the basis of this paper.

*Supported by a grant from the U.S. Public Health Service (NB O-3140). tPostdoctora1

Trainee supported

by U.S. Public Health Service Training Grant No. 5-TI-MH-6415. 139

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E. E. DECIMAand R. W. RAND METHODS

A preliminary survey was carried out in twenty-four cats in which different central nervous system ablations were performed acutely. The most caudal section was made at the intercollicular plane and successively higher sections were carried out up to an almost complete decortication. Oxotremorine injection in the animals with transections between the extreme levels, i.e., intercollicular and cortical, produced inconsistent results. However, a tremor was consistently produced in the decorticated cat. Therefore the latter was the experimental preparation used. Acute surgical procedures were carried out in 15 female and male cats weighing from 2.5 to 3.5 kg. Under ether anesthesia, the trachea was cannulated and slings of silk were passed around both common carotid arteries. Edin electrodes* (15 mm long uninsulated stainless steel pins) were surgically implanted in muscles of the front and hind limbs. The muscles investigated were biceps and triceps brachii, tibialis anterior, and gastrocnemius. The animal was then attached to a modified stereotactic frame fixed by the headholder, a clamp in a lumbar spinous process and two pins in the ilia. After part of the skull was removed both cerebral hemispheres were exposed and the dura was opened. Carotid circulation was stopped by closing the silk loops and then decortication was carried out

FIG. 1. Diagram of the decorticated preparation. Broken line represents area of brain removed. Structures in the middle part of the section are the head of caudate nuclei anteriorly and dorsal hippocampus posteriorly. Notice that only remaining cortex is piriformis and some of temporal lobe. by aspiration. An important landmark was the floor of the lateral ventricle; the aspiration progressed in the medial side until the dorsal hippocampus was seen. From this point the aspiration extended radially in the hemisphere in successive steps. The removal of nervous tissue was more extensive laterally than medially in order to excise as much cortex as possible (Fig. 1). Provisory hemostasis was done with small cotton balls above the bleeding area after the procedure was completed in one hemisphere. The same steps *Epsco Inc. Medical Division - 275 Massachusetts Avenue, Cambridge 39, Massachusetts. No.4128 Needle Electrodes (formerly Edin pins).

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induced tremor in the decorticated cat

were then performed on the opposite side. Carotid flow was established and after removal of the cotton balls the hemorrhage was controlled by the use of Gelfoam. To check any bleeding points, we waited 20 min before attempting to close the skin over the skull. Elimination of blood clots from the subarachnoidal space was important. The skin was then closed and 1% procaine solution was injected at pressure points and incision. The animal was allowed to recover in a warm environment. Saline solution (up to 10 cc) was given during and immediately after the operation. In its final position in the frame the cat’s four paws were touching the frame base. Recording was started not less than three hr after ether was withdrawn. Body temperature was maintained between 36-37.5 degrees centigrade rectally throughout the experiment. Oxotremorine was administered intravenously (i.v.) or intraperitoneally (i.p.) at doses of 250-300 pg/kg; larger doses were used 4-5 hr after the first injection. Methantheline bromide* (B-diethylaminoethyl xanthene -9-carboxilate methybromide, 5 mg/kg intramuscularly) was administered 20 min before each injection of oxotremorine in order to block the peripheral (parasympathetic) effects of the latter. Muscle electrical activity was displayed in two double beam cathode ray oscilloscopes and a continuous record was taken with a Grass kymograph camera. Film speeds were 25, 50 and 100 mmjsec. Frequency analyses of the tremor were made by visually evaluating the middle of each action potential group, after the pattern of the tremor became consistent at varying times following injection of oxotremorine. RESULTS

As is well known, the decorticated (thalamic) animal exhibits spontaneous walking movements (DUSSER DE BARENNE, 1920; BARD and RIOCH, 1937). The electromyogram (EMG) during these movements showed the usual bursts of action potentials alternately in flexor and extensor muscles. On some occasions spontaneous groupings of action potentials, 12-16 per set, without visible tremor were observed (Fig. 2, A). On the other hand, when the animal was resting its paws on the frame, the EMG showed discrete activity in either flexor or extensor muscles or in both, which represented the usual muscle tone background in these preparations (Fig. 2, B). Finally, a marked increase of the extensor

UPPER

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FIG. 2. Patterns of muscle electrical activity seen in the decorticated cat. A. Shows spontaneous rhythmical firing in gastrocnemius muscle. B. Usual muscle background activity. C. Burst of activity in gastrocnemius muscle induced by touching pinna. *Banthine, Trademark by Searle and Company.

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EMG amplitude and heightened muscle tone could be noted, e.g. when the pinna’s reflex was elicited (Fig. 2, C). Administration of oxotremorine to the decorticated cat protected by methantheline bromide produced tachypnea and a general increase in motor activity in addition to tremor. Tachypnea was observed immediately or after a five to ten min delay following i.v. and i.p. injections, respectively. Shortly thereafter, long-lasting periods of typical running movements and powerful extensor thrust were seen. Superimposed on the latter, some tremor could already be seen at this time. When this stage subsided, five to ten min after i.v. and 20 to 30 min after i.p. administration of oxotremorine, the tremorogenic action of the drug was readily seen. The tremor appeared in the head and neck muscles and later spread caudally. It was usually very fine with small displacement of the leg. Figure 3 shows

2*

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B4

I- BICEPS 2-TRICEPS

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Strips of film showing EMG of front and hind legs at different moments after oxotremorine injection. A. Control before administration of oxotremorine. B. Tremor in biceps and triceps but not in hind leg. C. Tremor in biceps, triceps, tibialis anterior and gastrocnemius muscle.

of film at different times of oxotremorine administration of the drug, tremors of relatively muscles of the front leg, but no activity is present injection, tremors are present in both. The total duration of this tremorogenic effect i.p. injection; if the drug was given i.v., shorter strips

100

injection. In B, taken 40 min after i.p. constant pattern can been seen in two in the hind leg. In C, taken 45 min after was one and one-half to two hr after the durations were observed. During these

Oxotremorine

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cat

periods all possible combinations of tremor activity could be seen at various times, e.g. tremor either in one leg, or in both front or both right legs, etc. Regarding the tremor activity of a given muscle, two modalities were observed : (I) long periods of tremor characterized by action potentials of relatively constant amplitude alternating with periods of EMG silence, sometimes of the same duration as the tremor period. (2) Short bursts of tremor lasting 0.5-2 set each separated by similar periods of silent EMG (Fig. 4, A, B and D). A slight variant of this modality was possible with altemations of small and large amplitudes (waxing and waning) of the tremor activity (Fig. 4, C). A

UPPER CHAN.-TIS. ANT. LOWER CHAN -GASTROC.

FIG. 4. Continuous

200mr C

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os~lio~aphic record (A, B, C, and D). Notice bursts of tremor (A, B, and D) and waxing and waning characteristics (C).

During the first 5-15 min of the tremorogenic stage, the tremor observed was rather irregular. In addition, the intervals between the large action potentials of the rhythmic activity (tremor) were seldom silent, and some minor electrical activity was often present (Fig. 3, C). After the first few minutes the tremor became increasingly regular and more often than not the rhythmical discharges were separated by periods of EMG silence. A wide frequency spectrum from a minimum of 8 c/s to a maximum of 28 c/s (Fig. 5) was then observed. Simultaneous recording in two legs showed that their tremors might be at the same or at different frequencies.

FIG. 5. A. B.

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Shows a 9 per set tremor. Notice simultaneous contractions on flexor and extensor muscles. Shows a 26 per see tremor, mainly in extensor muscle.

of equal amplitude

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Oxotremorine tremor was more pronounced in extensor muscles (triceps brachii and gastrocnemius). The contractions of the flexor muscles were generally either of small amplitude or absent (Figs. 3, B, and C; 4, 6, 7); on rare occasions the flexor muscles contracted as much, if not more, than the extensors (Fig. 5, A). Whenever the tremor occurred both in flexor and extensor muscles, it was in phase, i.e., both muscles contracted simultaneously. This in-phase relationship was present throughout the whole frequency spectrum (Fig. 5, A, and B); on the other hand, the pattern of time relationships, in-phase or otherwise, between the tremors recorded in the corresponding muscles of different legs, e.g., right and left gastrocnemius, was not fixed. When oxotremorine was administered the second time, larger doses were required to At the doses used (400-500 pg/kg), the total period of tremor activity was induce tremor. shorter than the one produced by the first injection, lasting from 40 to 50 min for the intraperitoneal route or less if the drug was given intravenously.

FIG. 6. Effects of

atropine upon tremor. Upper channel is biceps and lower channel is triceps

muscle. A. Control. B. Oxotremorine induced tremor. C. 1 min after atropine sulfate (1 mg/kg, i.p.). D 5 min after administration of atropine.

The blocking effect of atropine upon this tremor in the intact animal (EVERETT et al., 1956a; GEORGE et al., 1962) was present also in these preparations. Figure 6, C shows the EMG one minute after administration of 1 mg/kg, i.p. of atropine sulfate. Most of the rhythmical activity had disappeared, although some irregular groupings of action potentials could still be observed. Four min later (Fig. 6, D) no tremor was observed. The tremor disappeared immediately upon i.v. administration of atropine. Tremor could not be

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reproduced in atropinized animals by additional injections of oxotremorine, even if the doses used were four times the usual amount. The tremor was extremely sensitive to sodium pentobarbital, i.e., it was abolished by subanesthetic doses. The duration of the blockade was found to be dose related: 5 mg/kg, i.v. stopped tremor for 5-7 min, 15 mg/kg stopped it up to 20 min (Fig. 7). Anesthetic doses (35 mg/kg) abolished the tremor for several hours.

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FIG. 7. Effects of pentobarbital upon tremor. A. Control B. Oxotremorine induced tremor. C. 1 min after i.v. administration of pentobarbital

ms

[

500 E.rv

sodium (15 mg/kg).

DISCUSSION

Small amplitude rhythmical firing of the EMG was found occasionally in the acutely decorticated cat under control conditions. This activity was apparently similar to that described in human subjects during maintenance of normal posture and has been thought by L~PPOLDet al. (1957) to underlie physiologic tremor. The tremors produced by oxotremorine have features in common with a variety of tremors, but especially with those observed in shivering and Parkinson’s disease. Simultaneous contractions of flexor and extensor muscles occur in shivering dogs (KAWAMURA,1961) and in anxiety tremor of man (BRAZIER,1945). As in these conditions, the in-phase relationship between flexors and extensors of the oxotremorine tremor results in the fine character of the tremor and the small limb displacement observed. The larger amplitude of tremor in the extensor muscles reported herein occurs also in the shivering dog (KAWAMURA,1961). The onset of oxotremorine tremor in head and neck muscles was similar to that seen by BURTONand BRONK(1937) in the shivering cat. The wide range (8-28 c/s) frequency spectrum seen in these experiments fell within that shown by LIPPOLD et al. (1959) in the tibialis anterior muscle of anesthetized shivering cat (2-30 c/s). Although some frequency studies of tremorine tremor have been reported, and YIM comparison is difficult due to differences in the techniques employed. CHALMERS (1962) observed a ten c/s tremor in rats injected with tremorine. The method used recorded tremor of the whole limb (one extremity was placed inside a coil with a magnet attached to it). In this situation, the recorded tremor would be the composite result of tremors in each muscle; however, tremor frequency in each muscle does not need to be the same as that of the whole limb. On the other hand, species differences might play a role. Other investigators (KAELBERand CORRELL,1958) studying tremorine effects in intact

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unanesthetized cats reported a tremor of “either about 8 or 16 per sec.” Unfortunately, they did not specify the technique used for such analysis. The isolated spikes observed between the action potentials groups at the beginning of the tremorogenic stage may represent the incomplete synchronization of the motorneuron pool at that moment (Fig. 3, C). The same has been reported in physiologic tremor by LIPPOLD et al. (1957). Decrease of effectiveness of oxotremorine upon second administration could be due to methantheline bromide gaining entry into the central nervous system when the latter was given for the second time, since under our experimental conditions the blood brain barrier was not intact. However, GEORGEet al. (1964), working with local microinjections of oxotremorine in the mesencephalic reticular formation in chronically implanted cats, observed the development of a local refractoriness to successive injections of this drug lasting from four to five days. Therefore, the relative ineffectiveness of the second injection of oxotremorine in the current studies was probably due to a tolerance phenomenon. The main difference between oxotremorine tremor and shivering was in the response to atropine sulfate. STUARTet al. (1961) showed that atropine did not have any effect on the cold-induced tremor of cats; on the other hand, atropine blocked the tremor induced by oxotremorine in the decorticated cat. It is well known that parkinsonian tremor is also blocked by atropine (SOLMAN,1957). However, the tremor observed in Parkinson’s disease has quite different characteristics from that induced by oxotremorine. It is a tremor of rather slow frequency (4-8 c/s) with extensor and flexor muscles contracting alternatively (HOEFERand PUTNAM,1940), and removal of precentral motor cortex will abolish it (BUCY and CASE, 1939). As stated by EVERETT(1961) tremorine, of which oxotremorine is the active metabolite, is being widely used at the present time for screening potential anti-Parkinson drugs. The results reported herein would suggest a word of caution in the interpretation of such pharmacologic tests. Barbiturates block parkinsonian tremor in man as well as in the case of pentobarbital, oxotremorine tremor in the decorticated cat. However, paradoxically they are also able to induce a generalized tremor resembling shivering at certain stages of general anesthesia (VON EULER and SOLDERBERG, 1957; LIN, 1960; HEMINGWAY,1963). Regarding the site of action of the tremorogenic effects of oxotremorine most of the published data is rather conflicting. EVERETTet al. (1956b) stated that tremorine-induced tremor was absent below the level of section in acute spinal rats. However, NASH and EMERSON(1959) reported opposite results working with cats with a chronic spinal section and suggested that the negative findings of EVERETTet al. were due to spinal shock. Tremor below the section was observed also in rats with a chronic spinal section by CHALMERS and YIM (1962). Nevertheless, KAELBERand HAMEL (1960), working with tremorine and GEORGEet al. (1962) using oxotremorine, failed to see tremor below the spinal section in chronic spinal cats. In regard to supraspinal centers related to the tremorogenic effects of tremorine, EVERETTet al. (1956a) reported that tremor was observed in decerebrate rats, mice and rabbits. No further technical details were given although it can be supposed that the authors used the sherringtonian decerebration (intercollicular section). KAELBERand HAMEL(1960), working with transections of the neuroaxis in the cat, reported that all but one prevented tremorine tremor. The only area that could be removed (without abolishing tremor) was the “frontal lobe”, i.e., that part of the cerebral hemispheres cephalad to a plane tangential to the genu of the corpus callosum.

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The results reported herein are in part a confirmation and extension of the results of and HAMEL,i.e., not only the “frontal lobe” but practically all neocortex can be dispensed with and yet tremor will still be induced by oxotremorine. It should be mentioned in this regard that the neocortex is also not essential for the production of shivering. (DUSSERDEBARENNE,1920; PINKSTONet al., 1934; ARING, 1935 and BARDand RIOCH, 1937). On the basis of frequency and phase relations of oxotremorine tremor, it may be then suggested that oxotremorine activates neural mechanisms normally used in shivering. On the other hand, shivering and oxotremorine tremor could be well differentiated pharmacologically because, like a parkinsonian tremor, oxotremorine action could be readily blocked by atropine.

KABLBER

Acknowledgements-The authors wish to express their appreciation to Dr. R. GEORGE and Dr. D. J. JENDEN,Department of Pharmacology, University of California, Los Angeles, for their helpful advice and kindness in providing the oxotremorine; and to Mrs. Grace Spears for her valuable technical assistance. Author’s address-Ermuo E. DECIMA, Department of Surgery Division of Neurosurgery, UCLA Center for the Health Sciences, Los Angeles, California 90024, U.S.A. RCsum&-Une methode d’ttude pour la production du tremhlement experimental est d&rite. Chez le chat, en preparation decortiquee aigue, l’injection d’oxotrernorine induit un tremblement gtneralisee. L’analyse EMG montre que le tremblement provoque se rep&e regulierement avec des contractions simultanees des flechisseurs et des extenseurs; cependant, les extenseurs ne sont pas touches. Le spectre de frequence est etendu (8 a 28 cps). Les tremblements debutent dans les muscles de la t&te et du cou et se manifestent pendant une ou deux heures, en bouffees soit courtes, soit longues. Les contractions prbentent des variations d’amplitude rtgulieres. Le tremor induit par I’oxotremorine montre des similitudes avec celui produit par le froid: m&ne spectre de frequence, relation de phase entre flechisseurs et extenseurs, mode d’apparition. Cependant le sulfate d’atropine qui bloque le trembletnent par l’oxotremorine ainsi que le Parkinsonisme, est sans effet sur le tremor provoque par le froid. Le Pentobarbital, en doses anesthesiques et subanesthesiques, possMe la propriete de bloquer le tretnblement a I’oxotremorine. Une tolerance a cette substance semble se developper par administration de doses successives. Zusammenfassung-Eine Technik zum experimentellen Studium des Tremors wird beschrieben. Die Injektion von Oxotremorine wurde in Katzen untemommen, deren Gehim kurz vorher von der Rinde befreit wurde, und ergab durchweg einen allgemeinen Tremor. Die elektromyographische Analyse des erzeugten Tremors zeigte einen regelmlssigen Verlauf, mit gleichzeitiger Kontraktion der Beuge- und Streckmuskehr, obgleich die letzteren starker betroffen waren. Das Frequenzspektrum war breit (8 bis 28 cps). Der Tremor begann zuerst in den Kopf und Nackenmuskeln und dauerte 1 bis 2 Stunden nach der Injektion an. Kurze oder lange Aktivitltsperioden, mit oder ohne langsamem Auf- und Abbau, wurden beobachtet. Das Frequenzspektrum, die Phasenbeziehungen der Beuge- und Streckmuskelaktivatat, und die Art des Einsetzen des Tremors, waren den Kalteschauem tilich, obwohl ein pharmakologischer Unterschied vorhanden war. Der Oxotremorine Tremor, wie der Parkinsonsche, wurde duch Atropinsulfat blockiert, wlhrend der Kalteschauer nicht beeinflusst wird. Mit Pentobarbital in anasthesierender und subanlsthesierender Dosis war es miiglich den Oxotremorine Tremor zu blockieren. Nach wiederholten Injektionen von Oxotremorine wurde ein Toleranzphlnomen beobachtet.

REFERENCES C. C. (1935). Shivering and the cerebral cortex. Amer. J. Physiol. 113: 3. BARD, P. and RIOCH, D. McK. (1937). A study of four cats deprived of neocortex and additional portions of the forebrain. Johns Hopk. Hosp. Bull. 60: 73-147. BRAZIER,M. A. B. (1945). Tremors of combat neurosis. Arch. Neural. Psychiat., Chicago 54: 175-180.

ARING,

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E. E. DECIMAand R. W. RAND

BUCY, P. C. and CASE,T. J. (1939). Tremor: Physiologic mechanism and abolition by surgical means. Arch. Neural. Psychiat., Chicago 41: 721-746. BURTON,A. C. and I. W. BRONK(1937). The motor mechanism of shivering and of thermal muscular tone. Amer. J. Physiol. 119: 284. CHALMERS,R. K. and YIM, G. K. W. (1962). Tremorine tremor in chronic spinal rats. Proc. Sot. exp. Biol., N. Y. 109: 202-205. DUSSERDE BARENNE,J. G. (1920). Recherches experimentales sur les fonctions du systtme nerveux central faites en particulier sur deux chats dont le pallium et& enleve. Arch. Neerl. Physiol. 4: 31-123. EVE~IZT~,G. M., BLAXKUS,L. E. and SHEPPERD,I. M. (1956a). A tremor induced by tremorine and its antagoism by anti-Parkinson drugs. Science 124: 79. EVEREIT, G. M., BLOCKUS,L. E., SHEPPERD,I. M. and TOMAN,J. E. P. (1956b). Production of tremor and a Parkinson-like syndrome by 1-4 dipyrrolidino-2-butyne, tremorine. Fed. Proc. 15: 420-421. EVERETI,G. M. (1961). Tremorine, in Extrapyramidal System and Neuroleptics-Ed. Jean March Bordeleau. Ed. Psychiatriques, Montreal. 574 pp. FOLKERTS,J. F. and SPIEGEL,E. A. (1953). Tremor on stimulation of the midbrain tegmentum. Confinia neural., Base1 13: 193-202. GEORGE,R., HASLE~, W. L. and JENDEN,D. J. (1964). Personal communication. GEORGE,R., HASLET~,W. L. and JENDEN,D. J. (1962). The central action of a metabolite of tremorine. Lt$ Sci. 8: 361-363. HEMINGWAY,A. (1963). Shivering. Physiol. Rev. 43: 397-422. HOEFER,P. F. A. and PUTNAM,T. J. (1940). Action potentials of muscles in rigidity and tremor. Arch. Neurol. Psychiat., Chicago 43: 704-725. JENKNER,F. L. and WARD, A. A. Jr. (1953). Bulbar reticular formation and tremor. Arch. Neural. Psychiat., Chicago 70: 489-502. KAELBBR,W. W. and CORRELL,R. E. (1958). Cortical and subcortical electrical effects of psychopharmacologic and tremor producing compounds. Arch. Neurol. Psychiat., Chicago 80: 544-553. KAELBER,W. W. and HAMEL, E. G. (1960). Drug (tremorine)-induced tremor in the cat. Arch. Nemo/. 2:338-340. KAWAMURA, Y. (1961). Neuromuscular organization in shivering. In Neural Factors in Temperature Regulation. Ed. J. P. Hannon and E. Viereck. Arctic Aeromedical Laboratory. .Fairbanks, 399 pp. LIN, T. P. K. (1960). Central and peripheral control mechanisms of shivering and its effects on respiration. J. App. Physiol. 15: 567-574. LIPPOLD, 0. C. J., REDFEARN,J. W. T. and Vuco, J. (1957). The rhythmical activity of groups of motor units in the voluntary contraction of muscle. J. Physiol. 137: 473487. LIPPOLD,0. C. J., REDFEARN,J. W. T. and Vuco, J. (1959). The influence of afferent and descending pathways on the rhythmical and arrhythmical components of muscular activity in man and the anesthetized cat. J. Physiol. 146: l-9. NASH, J. B. and EMERSON,G. A. (1959). Studies of the mode of action of tremorine (l-4-dipyrrolidino-2butyne). Fed. Proc. 18: 426. PETERSON,R. W., MAGOUN,H. W., MCCULLOCH,W. S. and LINDSLEY,D. B. (1949). Production of postural tremor. J. Neurophysiol. 12: 371-384. PINKSTON,J. O., BARD,P. and RIOCH, I.McK. (1934). The response to changes in environmental temperature after removal of portions of the forebrain. Amer. J. Physiol. 109: 515-531. SOLMAN,T. (1957). A Manual of Pharmacology. Philadelphia, W. B. Saunders Co. 1535 pp. STUART,D. G., GEORGE,R., FREEMAN,W. J., HEMINGWAY,A. and PRICE, W. M. (1961). Effects of antiand pseudo-Parkinson drugs on shivering. Exp. Neural. 4: 106114. VON EULER, C. and SOLDERBERG, V. (1957). The influence of hypothalamic structures on the electroencephalogram and gamma motor activity. Electroenceph. clin. Neurophysiol. 9: 391-408. WARD, A. A., JR., MCCULLOCH,W. S. and MAOOUN,H. W. (1948). Production of an alternating tremor at rest in monkeys. J. Neurophysiol. 11: 317-330.