The production of tremor by cholinergic drugs: Central sites of action

Int. I. Neuropharmacol., 1966, $, 27-34 PergamonPress. Printedin Gt. Britain.

THE

PRODUCTION OF TREMOR BY CHOLINERGIC DRUGS: CENTRAL SITES OF ACTION R.

GEORGE,W. L. HASLETTand D. J. JENDEN

Department of Pharmacology, The Center for the Health Sciences and Brain Research Institute, University of California, Los Angeles (Received 4 August 1965)

SummarywThe tremorogenic effect of systemically administered oxotremorine has been examined in rats following brain transections and after placement of electrolytic lesions. Transections passing through the caudal midbrain or anterior pens prevent oxotremorineAnduced tremor. Lesions placed in an area extending between the rostral midbraln and caudal pens, in all cases involving portions of the dorsal tegmentum, abolish or reduce this tremor. Lesions of diencephalic structures are without effect. Microinjections of oxotremorine or carbachol into four areas of the cat brain produce tremor: the basal forebraln, near the diagonal band of Broca; the medial preoptic area; the anteromedial hypothalamus; and the mesencephalo-pontine junction. Atropine sulfate administered into the same sites or systemically reverses this effect. INTRODUCTION

ALTHOUGHit is known that the tremor induced by tremorine is of central origin, the exact level at which it originates has not been established. EVERFrT and co-workers reported that it is blocked below the level of spinal cord transaction (1956a) but is not prevented by either decerebration (rats, mice and rabbits) or decerebellation (dogs) (1965b). In contrast, CHALMVa~Sand Y ~ have reported that chronic spinal rats (1962) and chronic low spinal dogs (1963) show tremor and other movements below the level of section. Experiments undertaken in cats have failed to resolve this dichotomy; whereas NASH and EMEatSON (1959) found that spinal cord transection was ineffective in preventing tremor, KAELBER and HAMEL (1960) reported blocking with all transections from T-1 up to the level of a plane extending between the diagonal band of Broca and the subcaUosal gyrus. The latter authors have reported more recently (KAELBERand HAMEL, 1961a) that electrolyric lesions involving the posterolateral and/or dorsal hypothalamus, and ventromedial and midline nuclei of the thalamus are effective against tremorine-induced tremor. During the past few years it has been demonstrated that the principal pharmacologic properties of tremorine are attributable to a metabolic transformation product (1-(2-oxypyrrolidino)--4-pyrrolidino-2-butyne) (oxotremorine) CHO et al., 1961). This is a highly stable tertiary amine with purely muscarinic properties (CHO et al., 1962). It is equivalent in potency to acetylcholine or carbachol with regard to its actions on the autonomic nervous system but, unlike these quaternary compounds, is capable of entering the central nervous system where it acts to cause a variety of effects, including tremor and rigidity (GEog~ et al., 1962). Oxotremorine is highly active also when applied in minute quantities directly to specific central structures; rage, parasympathetic stimulation, loss 27

28

R. GEOROE, W. L. HASLETT and D. J. JENDEN

of muscular tone and alterations of ¢lectroencephalographic (EEG) patterns all have been observed (HASLETTet al., 1963; GEORGn etal., 1964). The present work was undertaken in order to identify the central structures responsible for the tremorogenic action of oxotremorine. Initially, brain transections were performed in rats as a means of delimiting grossly the level at which oxotremorine was acting. To complement these studies brainstem lesions were made in a series of rats. Finally, small amounts of oxotremorine were injected directly into various widespread areas of the brainstem in conscious cats in an effort to induce tremor. METHODS Experiments involving transection of the brain stem or the placement of electrolytiy lesions were carried out on adult male and female rats (190-230 g) of the Sprague-Dawlec strain. Anesthesia was maintained with methohexital (Brevital): females 35 mg/kg; males 45 mg/kg, intraperitoneally (i.p.) or ether in the former case, while pentobarbital (Nembutal) (35 mg/kg, i.p.) was used in the latter. Transections were accomplished with the animals mounted in a Krieg-Johnson stercotaxic instrument. The dorsal surface of the skull was removed with the aid of a dental burr, and a blunt blade was passed through the brain at the desired level; all nervous tissue rostral to the section was then removed by aspiration. Bleeding was arrested by applying gel foam to the cut surface; in laterexperiments the wound was also washed with a thrombin solution. The vacant portion of the cranial cavity was filledwith cotton, the scalp being closed with small wound clips when it was certain that bleeding had ceased. Immediately following the operation, each animal was given 5-10 ml of glucose (i.p.)in order to compensate for fluid loss incurred at the time of transection. Rats maintained for longer than 24 lax were given 10 ml of 5 % glucose daily (i.p.). Body temperatures were held as near normal as possible during the survival period by placing the animals under a weak heat source. Electrolytic lesions were made by delivering an anodal current (2 m A for 10-15 scc) via an unipolar, stainlesssteelclectrodewhich was insulated to within 1 m m of the tip; the indifferentelectrodewas placed inside the rectum. Electrode placement was accomplished by using the Krieg-Johnson stercotaxic instrument in conjunction with the coordinate system described for the rat by K R m O (1946). In order to be certain that a typical oxotremorine response could be obtained in each experimental animal control observations were taken 4-7 days before operation (transection or lesioning). Banthine (5 mg/kg, i.p.)was given 20 rain prior to the administration of oxotremorinc (200/~g/kg, i.v.)in order to prevent peripheral parasympathetic Stimulation. This same procedure was followed postoperatively. Oxotremorine was administercd to transcctcd animals 1-48 hr after the operation. Animals with electrolytic lesionswere allowed to recover for 3-7 days before they were challenged with oxotremorine. Drugs were injected into the brainstems of conscious cats via stainlesssteelcannulae, equivalent in diameter to 25 gauge hypodermic tubing. Guides for these cannulae, consisting of 20 gauge stainlesssteel tubing, were implanted permanently by the stereotaxic technique such that their tips lay within 3-10 m m of the finalinjectionsites. In order to prevent foreignmaterialsfrom occluding the guides when not in use stilettos,which extended I m m below the tips of the guides, were inserted (see DECIMA and GEORGE (1964) for a detailed description of this system). Drugs were made up in 0"9~/o NaCI solution (pH 7.0-7.4) and wore delivered in a total volume of I-3 ~l.

Cholinergicdrugs and tremor

29

Electromyographic (EMG) activity of opposing muscle groups in cats (flexors and extensors of a forelimb) was recorded on magnetic tape. Recording electrodes consisted of platinum iridium wires coated with Insl-X to within 0.5 mm of their tips. These were stitched permanently into the muscles and connected to a Winchester plug on the skull by fine insulated wires which were led beneath the skin. Tremor in rats was recorded on a Grass electroencephalograph (model IIIG) after amplifying the current induced by a magnet moving within a coil of wire. The coil consisted of a cardboard drum 10 inches in diameter, around which had been wound 1000 turns of lacquered copper wire (resistance 1.5 ~/ft). The output of this coil was led through a 60 cycle filter and then into the final amplification stage of the electroencephalograph. The rat was held around the chest by a padded wooden collar, such that its hind limbs hung freely within the coil. A small bar magnet was taped to the right hind foot. Brains were perfused via the carotid arteries with normal saline solution, then perfused and fixed with formalin (10%). The extent of functional material presen t in transected brains was determined by taking frozen sections (80/*) from the cut face of the brain stem until the first intact section was encountered. These received a Nissl stain. All other rat brains were cut (40-50/z sections) on a freezing microtome and stained with hematoxylin and eosin. Cat injection sites were reconstructed by cutting either frozen or paraffin sections (80 and 30/, in thickness, respectively) and staining according to the Weil or Nissl techniques. RESULTS Transections of the rat brainstem Brainstem transections were performed on twelve rats. These experiments Showed that transections passing through the caudal midbrain or anterior pons prevent the tremor which is normally caused by oxotremorine. All effective sections lay caudal to the red nucleus and rostral to the nucleus of the abducens (VI) nerve. Tremor appeared in every animal sectioned above this level. Figure 1, showing the various levels of transection, is based upon gross inspection of the fixed brainstems in conjunction with low power microscopic examination of frontal sections.

d

¢

b

a

FIo. I. Diagrammatic sagittal composite showing planes of transection of rat brains. Transections through d blocked tremor; transections through a, b and c were ineffective. Abbreviations: Cb, cerebellum; Ccr, cerebral cortex; P, pons; RN, red nucleus; VI, nucleus of abducens nerve.

30

K. G~I),o~ W. L. HASt,i~ and D. J. Jr~Di~N

Lesions of the rat brainstem Lesions were placed throughout the midbrain and pons so as to involve the plane of the most caudal transection which had abolished oxotremorine-induced tremor. In addition, the major structural entities of the diencephalon were selectively destroyed, particular attention being given t ° the basal ganglia. Out of a total of seventy-six rats a modification of oxotremorine-induced tremor or rigidity appeared in seven. All effective lesions lay within an area extending between the rostral midbrain and caudal pons, and in each case involved portions of the dorsal tegmentum. The most rostral of these impinged upon the periaqueductal gray and the medial geniculate body of one side whereas those located more caudally all lay in the floor of the fourth ventricle and involved the uncrossed superior cerebellar peduncles. Lesions of the ventral tegmentum within this same rostrocaudal zone did not prevent tremor.

Microinjection of oxotremorine and carbachol into the cat brainstem A total of twenty-eight cats was used in examining the effects of focally appfied oxotremorine or carbachol. Microinjections were made into the ponto-mesencephalic tegmenturn and into other sites which, according to histopathologic studies, most frequently appear to be involved in Parkinson's disease (i.e. eaudate nucleus, ansa lenticularis, globus pailidus, red nucleus, thalamic nuclei, subthalamus, fields of Florel, substantia nigra and rostral midbrain tegmentum). In addition, guides were implanted so that injections could be made into the posterolateral hypothalamus and basal forebrain; these two areas have been implicated as tremorogenic foci (in cats) by KAELnER and HAMEL, who have shown that lesions in the former reduce or abolish drug (tremorine)-induced tremor (1961b)while lesions in the latter may cause tremor (1961a) (a) Responses elicited by mesencephalic and rhombencephalic injections. Areas of the lower brainstem tegmentum were approached at a rostro-caudal angle (54-60 °) in order to avoid the tentorium cerebelli. In all cases guides were implanted bilaterally, 2-3 mm to either side of the midline, and extended well into the brainstem so that their tips fell within 3-5 mm of the final injection sites. The area thus explored was defined rostrally and caudally by the Horsley-Clarke coordinates O and P5, respectively. Of seventeen animals receiving injections of either oxotremorine or carbachol within this zone, striking changes in behavior and electrographic patterns were observed in fifteen. Typically, injection was followed within 1-5 rain by a complete and long lasting (30-60 rain) atonia. The animals appeared to be in a state of deep (paradoxical) sleep according to the most widely accepted definition of the term (JOUVET, 1962) (i.e. tonic muscular activity was absent, oscillatory movements of the eyeballs occurred, the EEG was desynchronized and an intense theta rhythm appeared in the hippocampus). Tremor coincided in onset with the reappearance of muscular tone. It occurred in eleven animals and ranged in severity from a scarcely detectable to a violent trembling. The injection site of one of these animals lay in the rostral pons (P1.5; L2.5, H3-5) (Fig. 2). EMG records indicated that this tremor originated primarily from the extensor muscles and ranged in frequency from 13 to 30 c/s (Fig. 3). (b) Responses elicited by diencephalic injection. Tremor was observed in two animals following injection into the basal forebrain. One site lay at the level of the diagonal band of Broca (A I6; L2.5; H3.5) (Fig. 4). Trembling of the head and forepaws, growling and moderate serous salivation appeared within 8 rain following unilateral injection of oxotremorine (15 ~g). When left to itself, the animal invariably curled up in a characteristic

FIG. 2. Section through pontine injection sites of one animal; a points to upper limit of cannula on one side and b to lower limit of cannula guide on the other side. Neur. f.p. 30

-

F~ti. 4. Section

through

----

an injection site at the level of the diagonal points to site of cannula tip.

band

of Broca.

Arrow

FIG. 5. Section

through

the medial

preoptic

area.

Arrow

points

to effective

inJectIon

site

FIG. 6. Section through

the anterior

hypothalamic

area. Arrows point to injection sites.

Cholincrgie drugs and tt~xaor

31

INTRACEREBRAL OXOTREMORINE ( 2 / J g )

BICEPS

'~........... "' .......... : :: :........: :::::: :"'':~

A. TRICEPS . . . . . . . . . . . . . . TIME EMG-BEFORE INJECTION

40 MINUTES AFTER INJECTION , f .~. L'!' ~" '-I' !':'~'.,..~'-:~.',, C.

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60 MINUTES AFTER INJECTION

FXo. 3. EMG records showing onset of tremor and change in tremor frequency following

Lintrapontmeinjection of oxotremorine. Time marker: 60 c/s. sleeping position. These signs persisted for 30--45 min. Carbachol (3 Fg; unilaterally) was also effective in this area, inducing an intense tremor which involved the entire body 40 min after injection. During this period the cat seemed mildly fearful, although mydriasis was not observed. Atropine sulfate (5 Fg), administered into the same site, reversed this effect within 6 rain. The other forebrain site lay in the medial preoptic area (A 14.5; L1-0; H-4) (Fig. 5). In this case growling, hissing and lip licking were the first indications of an effect. These occurred within 7-9 min after injection of oxotremorine (10/~g; unilaterally) and lasted for 25 min. The onset of tremor coincided with the disappearance of these signs. Trembling involved the head, neck and tail, but was never seen to include the limbs. This effect persisted for 20 min, at the end of which time the animal was completely normal in appearance. The final diencephalic site from which tremor was elicited lay in the anteromedial aspects of the hypothalamus (A13.5; L2.0; H-3) (Fig. 6). The pattern of response in this case was similar to that which occurred after injection into the medial preoptic area. Injection of oxotremorine (10 tzg; bilaterally) was followed within 4 min by yowling and growling. Piloerection, mydriasis and arch back were present also. These signs of 'rage' apparently occurred in the complete absence of fear or apprehension as evidenced by the cat's insistence upon being caressed and fondled. Fine tremor of the head and limbs commenced 15 min after injection, and increased in severity during the ensuing 15 min period. At the end of this time all signs abated, recovery being complete 50 min after injection. At no time was ataxia or salivation observed. Atropine sulfate (2 tzg), injected into these same sites prior to the administration of oxotremorine, prevented the appearance of all effects.

DISCUSSION The resultsof the present experiments appear to localizethe trcmorogcnic sitesof action of oxotrcmorine to four principal areas: the basal forebrain, near the diagonal band of Broca; the medial preoptic area; the anteromedial hypothalamus and the mcsencephalopontine junction. Of these the last site appears the most important. In evidence of this are the facts that oxotrcmorine-induced tremor is blocked by transections which pass through the mesencephMo-pontine junction, and by electrolyticlesionsinvolving the dorsal I:l

32

R. GEOR6e,W. L. HASt~Trand D. J. JENDEN

tegmentum. Moreover, it has been shown that whole body tremor may be induced by injecting oxotremorine or carbachol directly into the pontine reticular formation. A growing volume of evidence has accumulated during the past 10 years supporting the concept of a tremorogenig~focus in the lower brainstem. It was originally demonstrated by WARD et al. (1948), and later by others (PETERSON et al., 1949; CARREAand METTLER, 1955; CARPENTER, 1958; POIRIER, 1960), that a sustained Parkinson-like syndrome could be caused in monkeys by placing electrolytic lesions at the level of the pons and mesencephalon. The tremor was thought to be mediated through the still intact cortico-spinal or reticulo-spinal systems, damage to the ventral tegmentum being considered the most significant contributing factor to the symptomatology. The seemingly contradictory observation that rhythmic limb movements can be generated in both monkeys (JENKNER and WARD, 1953) and cats (FOLKERTS and SPIEGEL, 1953; WYCIS et al., 1957; PINEDA and PETMECKY, 1962) by electrical stimulation of this tegmental area has been resolved hypothetically by JENKNER and WARD (1953). These authors speculate that the tremor resulting from tegmental lesions is caused by a denervation sensitivity of bulbar reticular cells to endogenous acetylcholine; thus it is possible to cause tremor either by interrupting reticular pathways to the bulb or by direct electrical stimulation of the bulbar reticular formation itself. It has been suggested (JENKNER and WARD, 1953) that such a mechanism is operative in Parkinson's syndrome, an idea consistent with the efficacy of atropinic compounds in this disorder, and in accord with the present observation that chohnergic substances cause tremor when applied focally to the reticular formation. An interesting observation in the course of these experiments was the finding that after the intracerebral injection of oxotremorine, tremor was nearly always preceded by a reduction of muscular tone associated with sleep or drowsiness. This effect was particularly noted following pontine injections, when oxotremorine induced paradoxical sleep (GEORGE et al., 1964). The animals would appear drowsy also after injections into the basal forebrain and medial preoptic areas. These data suggest that there is some anatomic rek.tLonship between drug-induced sleep and tremor. More evidence relating these neuroanatomic sites to sleep and tremor has been notzd both clinically and experimentally. Clinical observations on extrapyramidal hyperkinesis (tremor, chorea, athetosis, and ballism) in man have long been known to be dependent upon the waking state; the hyperkinesis disappears during sleep and reappears with arousal. Pallidotomy in human extrapyramidal hyperkinesis has resulted in drowsiness often progressing to deep sleep (HASSLER and .lUNG, 1960). Electrical stimulation of the caudate in cats (HESs, 1954),monkeys and man (HEATH and HODES, 1952) has produced sleep or sleeplike behavior, and electrical stimulation of the basal forebrain-preoptic areas has been shown to produce cortical synchronization and even paradoxical sleep (STERMAN and CLEMENTE, 1962 a,b). HESS (1954) noted that electrical stimulation of the supraoptic and preoptic areas in the lateral anterior hypothalamus resulted in diminution of motor behavioral pattern. Stimulation of the pontile reticular formation has been shown to trigger paradoxical sleep in the cat (JOUVET, 1962). Similarly, sleep has been induced in cats following the insertion of cholinergie agents into eannulae implanted along the Nauta limbic forebrain-limbic midbrain circuit from the preoptic region through the hypothalamus and into the midbrain and pons (HERNANDEZ-PEON et al., 1963). Finally, the complementary nature of the evidence provided by lesion and microinjection experiments requires comment. The influence of transections and discrete lesions upon the effects of a drug may provide evidence of the involvement of a neural structure in its

Cholinergic drugs and tremor

33

m e c h a n i s m s o f action, b u t c a n n o t a l o n e decide whether the structure represents the site o f action, lies o n a p a t h w a y a l o n g which the d r u g effects are t r a n s m i t t e d , or exerts a tonic influence u p o n which the a c t i o n o f the d r u g depends. While the microinjection technique also has limitations, the c o o r d i n a t e d use o f b o t h approaches m a y allow the definitive localisation o f target structures.

A c k n o w l e d g e m e n t s - - T h J s w o r k was s u p p o r t e d by U S P H S g r a n t B--03007 a n d was c o n d u c t e d d u r i n g the t e n u r e o f U S P H S P o s t d o c t o r a l Fellowship by Dr. W. L. HASLETr.

R6sum6--L'effet tr6morog6niqu¢ de l'oxotr&norine, administr6~ par voie syst6mique, a 6t6 6tudi6 chez le rat aI~s transsections et 16sions 61ectrolytiques c6r6brales. Les transsections int6tessant la pattie caudale du m6seac6phale et ie pont ant6rieur pr6vielment le tremblement induit par l'oxotr6morine. Les 16sionseffectu6es dans l'aire s'6tendant entre le m6senc6phale rostral et le pont caudal, int6ressant dans tous les cas des poltions du tegmentum dorsal, suppdment ou r6duiscnt c¢ trembleme,nt. Les 16sionsdes structures dienc6phaliques sent ineffectives. Des micro-injections d'oxotr6anorine ou de carbachol darts quatre aires c6r6bralcs du chat entrainent le trOnor: le cerveau ant6deur basal pros de la bande diagonale de Brocca; raire pr~-optique mbiiane; rhypothalamus antero-m&iian et la jonction m6senc6phalicopontique. Le sulfate d'atropin¢, administr6 aux m~mes endroits, produit l'effet syst6matiquemerit inverse.

Zusammenfassung--Foigend Gehirntransektion und elektrolytischen l_Asionen hat m a n die Zitter hervorbringende Wirkung von parenteral injiziertem Oxotremorin beobachtet. Transektionen, die durch das untere Mittelhim oder durch die anterior Pens gehen, verhindern oxotremorin induzierte Zitter. I.Asionen in dem Gegiet, das zwischen dem oberen Mittelhirn und dcm unteren Pens liegt, und in allen FAllen, die Portionen des dorsal Tegmentum enthalten vernichten oder verringern das Zittem. L~sionen yon dienzephalischen Strukturen ~haderen diese Wirkung nicht. Mikroinjektionen yon Oxotremorin oder Carbachol in vier Zonen des Katzenhirns erursachen einen Zitter: das basal Vorderhirn in der Ntihe des Gyrus diagonalis Broca; in diemedial prtioptische zone; die vorder-medial Hyvothalamus; und die mesenzephalischepontine Verbindung. Atropin Sulfat wirkt antagonistisch, wenn man es in die Nervenzentren oder parenteral injiziert. REFERENCES CARPENTER M. B. (1958). The neuroanatomical basis o f dyskinesia, In: Pathogenesis and Treatment of Parkinsonism, FmLoS, S. F., ed.. Soringfield, Illinois, C. C. Thomas, pp. 50-58. CARREA R. M. E. and ME'rTLER F. A. (1955). Function of the primate brachium conjunctivum and related structures. J. Comp. Neurol. 102: 151-522. CHALMEIL$R. K. and YIM G. K. W, (1962). Tremorine tremor in chronic spinal rats. Prec. Soc. exp. Biol. Med. 109: 202-205. CHALMER$R. K. and YIM G. K. W. (1963). Spinal action of tremorine in the dog. Archs. int. Pharmacodyn. 145: 322-333. CHO A. K., HASLE'FrW. L. and JENDEND. J. (1961). The identification of an active metabolite of tremorine. Biochem. Biophys. Res. Commun. 5: 276-279. CHO A. K., HASLETrW. L. and JENHEND. J. (1962). The peripheral actions of oxotremorine, a metabolite of tremofine. J. Pharmacol. 138: 249-257. DEClMA E. and GEORGE R. (1964). A simple cannula for intracerebral injections in chronic animals. Electroenceph. clin. Neurophysiol. 17: 438-439. EVERETTG. M., BLOCKUSL. E. and SHEPHERDI. M. and TOMANJ. E. P. (1956a). Production of tremor and a Parkinson-like syndrome by I, 4-dipyrrolidino-2-butyne, 'Tremorine'. Fed. Prec. 15: 420. EVV.RETr G. M., BLOCKUSL. E. and SmSl,I,~RD I. M. (1956b). Tremor induced by tremorine and its antagonism by anti-parkinson drugs. Science 124: 79. FOLKERTSJ. F. and SPmOl~LE. A. (1953). Tremor on stimulation of the midbrain tegmentum. Confinia Neurol. 13: 193-202. (~EORGE R., HASLETTW. L. and JE~'DEND. J. (1962). The central action of a metabolite of tremorine. Life Sci. 1: 361-363.

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R. GEORGE,W. L. HAS~'rr and D. J. JENDEN

GEORGE R., HASL~TTW. L. and JENDEND. J. (1964). A cholinergic mechanism in the brainstem reticular formation: induction of paradoxical sleep, lnt..l. NeuropharmacoL 3: 541-552. HASI~TT W. L., GEOROE R. and JE~rDL~ D. J. (1963). Localization of some central sites of action of oxotremorine. Proc. West. Pharmac. Sac. 6: 9-10. HASSLmtR.andJuNoR.(1960). HandbookofPhysloiogy;SectionI:Nevrophysiology. Editors : Field, J , Magoan, H. W. and Hall, V. Waverley Press, Inc., Baltimore 2: 910-913. HEATH R. G. and HODF.SR. (1952). Induction of sleep by stimulation of the caudate nucleus in macaqus rhesus and man. Trans. Am. Neural Assoc. 77: 204-210. I-'IERNANDEz-PEONR., CHAVEZ-IBARRA G., MORGANE P. J. and TIMO-IARIA (1963). Limbic cholinergic pathways involved in sleep and emotional behavior. ExptL NeuraL 8:93-111. HESS W. R. (1954~. Dieneephalon--Autonomie and Extrapyramidal Functions. Grune and Stratton, New York. JENKNERF. L. and WARD A., Jr. (1953). Bulbar reticular formation and tremor. Archs. Neural Psychiat. 70: 489-502. JOtrWT M. (1962). Recherehes sur les structures nerveuses et les m~anismes responsables des differentes phases du sommeil physioiogique. Archs. itaL Biol. 100: 125-296. KAet.BER W. W. and H A ~ L E. G. (1960). Drug (tremorine)-induced tremor in the cat. Archs. N~urol. 2: 338-340. KAm.~ER W. W. and HAMnBt.E. G. (1961a). Post-lesion tremor inhibition. Archs. Neural 5;: 221-226. KAm.aER W. W. and HA~mL E. G. (1961b). Observations on rest-type tremor in the cat. J. Neuropath. Exp. Neural. 20: 263-274. KRmG W. J. S. (1946). Accurate placement of minute lesions in the brain of the albino rat. Quart. Bull. Northwestern Univ. Med. School 20:199-208. NASH J. B. and EMERSONG. m. (1959). Studies on ttae mode of action of tremorine (1, 4-dipyrrolidino-2batyne.) Fed. Proc. 18: 426. PE3"ERSONE. W., MAGOUNH. W., McCuLLOCH W. S. and LINDSL~Y D. B. (1949). Production of pastural t r e m o r . . / . NeurophysioL 12: 370-384. t~),mDA A. and PEa'S~CK~eF. F. (1962). Experimental tremor following electrical stimulation of the brainstem tegrnentum of the cat. Tex. Rep. Biol. Med. 20: 79-86. Porsa~R L. J. (1960). Experimental and histological study of midbrain dyskinesias. J. Neurophysiol. 23: 534-551. STERMAN M. B. and CLEMENTEC. D. (1962a). Forebrain inhibitory mechanisms: cortical synchronization induced by basal forebrain stimulation. ExptL Neural. 6: 91-102. STERMAN M. B. and CLEMENTEC. D. (1962b). Forebrain inhibitory mechanisms: sleep patterns induced by basal forebrain stimulation in the behaving cat. ExptL Neural. 6: 103-117. WARD A. A., Jr., McCuLI.OCH W. S. and M^GOUN H. W. (1948). Production of an alternating tremor at rest in monkeys..L Neurophysiol. 11 : 317-330. WYcls H. T., SZE~ELYE. G. and SPIEOEL E. A. (1957). Tremor on stimulation of the midbrain tegrnenturn after degeneration of the brachium conjtmctivum. J. Neuropath. Exp. Neural. 16: 79-84.