Characteristics of tremor in cats following injections of carbachol into the caudate nucleus

EXPERIMENTAL

14, 371-382

NEUROLOGY

Characteristics

of

Injections

(1966)

Tremor

of

Carbachol

Caudate J. D.

CONNOR,

in Cats

G. V.

into

Following the

Nucleus ROW,

AND

W. W.

BAKER]

Department of Neuvopharmacology, Eastern Pennsylvania Psychiatric Department of Pharmacology, Philadelphia College of Pharmacy Philadelphia, Pennsylvania Received

September

Institute; and Science,

and

24, 1963

Bilateral hind-limb tremor was produced in unanesthetized cats by injecting small amounts of carbachol into the caudate nucleus. These involuntary movements were recorded and their parameters characterized. Comparable injections into several other brain areas previously implicated in tremor mechanisms failed to evoke similar involuntary activity. Relative tremorogenic specificity of intracaudate carbachol was suggested by the inability of locally injected pentylenetetrazol (neuronal stimulant) or procaine (neuronal depressant) to initiate tremor. However, antagonism of established carbachol tremor by intracaudate procaine, or by systemically administered pentobarbital or atropine sulfate demonstrated that these involuntary movements could be reversed pharmacologically. Classically, the caudate nucleus has been assigned a role as a modulator of extrapyramidal function. Thus, it seems reasonable to propose that locally injected carbachol reduces or eliminates this regulatory influence of the caudate over other motor centers, causing an imbalance which results in tremor. Introduction

Although involuntary movements have been produced experimentally in several species by systemic administration of tremorogenic agents, such as reserpine, chlorpromazine, nicotine and Tremorine (11)) it has been difficult to use these as consistent pharmacologic models for analyzing tremor in the cat (1, 19). Involuntary movements are regulated to a large extent 1 This study was supported by USPHS Grant MH 08833 from the National Institute of Mental Health. A preview of this material was presented at the meeting of the American Society for Pharmacology and Experimental Therapeutics, August 1965. This research was submitted by Mr. Connor to the Philadelphia College of Pharmacy and Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 371

372

CONNOR,

ROSSI,

AND

BAKER

by the extrapyramidal motor system but the precise role of the caudate nucleus in this regulation is unsettled. The caudate has considerable cholinergic representation (5, 12), and in electrographic and behavioral studies has proven highly responsive to locally injected agents ( 15, 17). In the present study, carbachol, an acetylcholine analog known to be a potent stimulant of the caudate ( 18)) elicited pronounced and reproducible tremor when injected into the caudate nucleus of unanesthetized cats. This report will describe this local pharmacologic method for producing tremor, and the procedures for recording and semiquantitatively analyzing this involuntary movement. Methods

and

Materials

Implantation of Electrode-Injection Guides. Nine adult male cats (2.43.6 kg) were used in this study. While the cats were anesthetized with pentobarbital (35 mg/kg, ip), injection guides, which also functioned as monopolar recording electrodes, were implanted so that injections could be delivered to the caudate sites defined by the coordinates ,415, R5 or L5, D + 5 (7). At the same time, guides were implanted in other neurophysiologically significant sites (dorsal hippocampus, red nucleus, mesencephalic reticular formation, anterior hypothalamus, and third ventricle). The guides consisted of epoxy-insulated, 20-gauge stainless steel tubing with a connecting wire soldered to the exposed end. These were stereotaxically lowered through hollow screws tapped into the skull. When properly positioned, the guides were fixed to the surface screws with acrylic cement. A steel phonograph needle driven into the frontal bone served as the reference electrode. To insure support and insulation, the parts of the guides and reference electrode extending above the skull were partially imbedded in hard wax. The openings of the guides were accessible for injection, and the connecting wires were used to record local brain electrical activity. To prevent leakage of the cerebrospinal fluid and consequent occlusion of the lumen of the guides, 24-gauge solid wire stylets were inserted so that they extended 1 mm beyond the inner tips of the guides. Drug solutions were injected by inserting a stainless steel delivery cannula into the guide after removing the stylet. A soldered metal shim, functioning as a stop, permitted the cannula to extend 1 mm below the end of the guide. Small volumes (l-10 1~1) were delivered at the tip of the cannula by coupling to a calibrated micrometer-driven injector. Since volume, pH and tonicity are critical when drugs are injected intra-

TREMOR

373

cerebrally (16), these factors were controlled. To minimize local brain damage, volumes less than 10 ~1 were injected. Drugs were dissolved in sterile distilled water. Control solutions adjusted for equivalent pH (modified Sorenson’s phosphate buffer) and tonicity (sodium chloride) were also injected. A thermocouple probe was used to monitor rectal temperature before and after injections. The cats recovered rapidly from the operative procedures and showed no apparent adverse effects to repeated intracerebral injections during the 3 to 4 month experimental period. Each animal was rested for 1 week between injections. At the end of the study the animals were killed; the positions of the cannula tips were marked with electrolytic lesions and subsequently verified in the formalin-fixed brain. Recording and Analysis of Tremor. The unanesthetized cat with chronically implanted guides was suspended in a canvas sling while involuntary movements resulting from intracaudate injections were recorded (Fig. 1). Only the head and hind limbs of the animal protruded. A modified lightweight phonocartridge was mounted on the dorsum of the shaved right hind foot over the metatarsal surface. Phonocartridges have been used extensively as transducers for recording different types of motion (9). Foot movements caused stylus deflection and the resulting voltage outputs were recorded with an electroencephalograph. The tremor parameters selected for study were latency of onset, time for maximal response, frequency, amplitude, number of tremor bursts per minute, and the percentage of time actually spent tremoring during peak carbachol effect. Paper speeds were calibrated so that both tremor frequency and the number of tremor bursts could be counted directly from the tracings. The durations of the individual bursts were measured, and the actual tremor time (TT) was derived by adding together all these episodes. This fraction of a minute was multiplied by 100 and reported as percentage. Observed amplitudes (&) were measured and adjusted to differential amplitudes (A,,) by subtracting background activity (Ab), thus, A,, = A, - Ah. Results

Carbachol-induced tremor is characterized in Table 1 and illustrated in Fig. 2. Intracaudate injections of 7 pg quantities of carbachol produced consistent bilateral tremor with minimal cholinergic side effects. Tremor persisted for 1.5-3 hr (mean = 2) after a latency of 9-21 min (mean = 14). Peak effect occurred 20-25 min after injection (Fig. 2D). Therefore,

374

CONNOR,

ROSSI,

AND

BAKER

agents which might modify carbachol tremor were customarily administered 20-40 min after carbachol injection. Characteristically, involuntary movement consisted of fine head tremor and a coarser tremor of the limbs. Hind-foot movements were nonrotational and in a plane parallel to the

8. HIND-FOOT

MAI1

FIG. I. Technique for tremor measurement in the unanesthetized cat. Head detail (A) illustrates intracerebral injection and recording of local brain electrical activity using the combination injection-electrode. To protect the electrode-guides between experiments, a plexiglass shield is positioned on the large center bolt and held in place with a wingnut. Hind-foot detail (B) shows the position of the transducer for recording movement.

body. When injected into other brain sites (hippocampus, red nucleus, reticular formation, hypothalamus, and third ventricle), carbachol failed to elicit tremor. Tremor induced by intracaudate carbachol occurred as discrete episodes at a rate of 1628/min (mean = 21). In some of the experiments, these episodes were essentially uninterrupted at peak carbachol activity, and tremoring was almost continuous; however, mean TT for the experimental

375

TREMOR

series was 74%. Thus, even during maximal response, involuntary movement was absent approximately one-quarter of the time. Dominant hindfoot frequencies ranged from 16-23 cycle/set (mean = 20), while net amplitude A,, varied between S-30 mm (mean = 11). TABLE CHARACTERIZATION

Tremorogenic

OF THE TREMOR

PRODUCED

1 BY INTRACAUDATE

CARBACHOL

INJECTION

Mean valuesa (Range)

parameters:

7 V-8)

1. Dose (ug) 2. Latency

(min)

3. Duration

(hr)

14 (9-21) (1.k3)

4.

Tremor characteristicsa: i. Frequency (cycle/set) ii. Amplitude

20 (16-23)

differential

(mm)

11 (S-30)

iii. Burst

episodes

(per min) (ii-28)

iv. Percentage

tremor

time

a Mean values were derived from eighteen experiments except was calculated from six experiments. b Characteristics (i-iv) calculated during maximal carbachol-tremor.

74 (66-85)

for

duration

which

Hyperpyrexia occurred in all animals after intracaudate carbachol. Average rectal temperatures rose from a control value of 38.8”C to a maximum of 40.2 “C. Elevations in temperature paralleled tremor production and gradually returned to control levels when tremor stopped. In eleven of eighteen experiments, local electrographic changes were monitored 40-60 min after intracaudate carbachol. These responses developed initially as a seizure discharge lasting 0.5-l min, followed by discrete spikes with afterdischarges. The spikes fired at a rate of about 20/ min, and persisted for more than an hour, often outlasting the tremor. In four experiments, the seizure discharge was not followed by spikes, but by bursts of high, slow waves with a repetition rate of S/min. The effects of some nonspecific neuronal excitatory and depressant agents, administered either locally or systemically, are contrasted in Table 2, and illustrated in Fig. 3 and 4. Pentylenetetrazol, a potent but

OF STIMULANTS

n b c d

AND

300 pg in each caudate neither induced nor 300 pg in each caudate was nontremorogenic. 5 mg/kg, ip, was nontremorogenic. 0.5 mg/kg, ip, was nontremorogenic.

Antagonism of carbachol tremor: No. of different cats Tremor-abolishing dose No. tremor antagonisms No. of cats tremoring

Tremorogenic activity: No. of different cats Tremor-producing dose No. tremoring responses Total No. experiments

EFFECTS

antagonized

tremor.

O/6

O/9

.

. . .. ..

(-) 6 . . .a

9 .

(-)

17/18

O/6

O/9

(I

(-) 6

(+I 9 7M

Cholinergic stimulant, pentylene~ tetrazol carbachol (Intracaudate) -

AND

C-1 9 ..

Control P

2 ON PRODUCTION

Nonspecific stimulant,

TABLE DEPRESSANTS

6 .b

6/6

(+) 6 300 PP

O/6

(-)

Local, procaine

c

5/6

(+) 6 5 w/kg

O/6

C-1 6

6/6

(+) 6 0.5 mg/kg

O/6

(6) 6 . d

Cholinergic antagonist, Systemic, pentoatropine barbital sulfate +(Intraperitoneal) -

depressant

OF TREMOR

Nonspecific

ANTAGONISM

g % 2 m k 3

“-

”

8 1: 3

377

TREMOR

CONTROL FOOT -ACTIVITY

I 10th

I

------=zf,o1N S.?C

FIG. 2. Tremorogenic response to carbachol. Upper tracings: caudate electrograms; lower tracings: hind-limb movements. Intracaudate (ic) injection of carbachol (B) caused tremor after 10 min latency (C). Maximal response (D) occurred 20 min after injection ; tremor characteristics analyzed during this phase. Local electrographic activity was recorded monopolarly. Indicated time sequence is continuous from control.

A

f&#&*~ CONTROL FOOT ACTIVITY

(70

J

SO.” wx

I

nin)

.

FIG. 3. Comparative effects of pentylenetetrazol, carbachol, and atropine on tremorogenic activity. Intracaudate injection of 300 ug pentylene tetrazol (B) failed to elicit involuntary movement during an hour observational period. Carbachol, however, injected into the same site (C), elicited tremor (D & E). Systemic injection (ip) of atropine (E) antagonized this tremor after 5 min (F).

378

CONNOR,

ROSSI,

AND

BAKER

relatively nonspecific neuronal stimulant, failed to produce involuntary movement, even after 300 ug was injected into each caudate (Fig. 3, B & C). Yet, subsequent injection of carbachol into the same site promptly induced tremor (Fig. 3, C-E). Intraperitoneal injections of atropine sulfate (0.5 mg/kg) abolished carbachol tremor within S-10 min (Fig. 3, E Pr F), RT. CAUD. #4 ELK-

,irl

( 5 mid

CONTROL FOOT AcTlvlTv

CARBACHOL

A

*o,Y *cc

FIG. 4. Antagonism of carbachol tremor by intracaudate procaine or systemic pentobarbital. Intracaudate injection of carbachol (B) elicited tremor (C & D). When procaine was injected into the same site (D), the tremor response was inhibited (E). Within 50 min, the tremor returned along with the initiation of local spike activity (F & G). Systemic injection (ip) of pentobarbital (G) blocked the tremor and altered the caudate electrogram after 5 min (H).

whereas atropine methylbromide (same dose and route) did not alter the existing carbachol tremor. Procaine hydrochloride (300 ug) , a local neuronal depressant, was nontremorogenic when injected into the caudate nucleus. When injected into the caudate during peak carbachol tremor, however, it abolished involuntary movement (Fig. 4E). After 4.5 min, the carbachol tremor reappeared (Fig. 4, F & G). Smaller quantities of procaine failed to completely antagonize tremor. Pentobarbital, a general central

TREMOR

379

nervous system depressant, was nontremorogenic at the slightly sedative dose of 5 mg/kg i.p. When this injection was repeated at peak carbachol effect, tremor was completely abolished (Fig. 4H). Discussion

When drugs in solutions are injected into specific areas of the brain, consideration must be given to the possibiilty that the agents act on other brain structures. Racik, Buchwald and Wyers (15) injected 100 ul volumes of eserine solution into the caudate without apparent involvement of adjoining areas. However, Stevens, Kim and MacLean (18) decided that crystal implantation was necessary to limit the spread of drugs from this site. Altough other structures bordering the ventricles have been previously implicated in tremor mechanisms (4), certain experimental factors tend to support the present interpretation that carbachol produced tremor by a primary action on the caudate nucleus. Stereotaxic coordinates were chosen so that caudate injections could be made into the center of this relatively large structure. These injection sites were subsequently verified. The small quantities (7 ug) and volumes (8 ~1) of carbachol which were injected would tend to minimize diffusion. Active cholinergic sites in the vicinity of the injection provide abundant loci for binding a quaternary ammonium compound such as carbachol. Furthermore, the consistent, predictable time course for onset and development of tremor after intracaudate carbachol suggests such a specific localized action. According to Delgado and Rubinstein (2), seepage along the guide tracts is minimal in chronic preparations because the guides become encapsulated in a glial sheath which tends to seal the tract. Perhaps most significantly, carbachol injections into the hippocampus, hypothalamus, red nucleus, reticular formation, and even directly into the third ventricle, failed to elicit appreciable involuntary movement. It is often difficult to differentiate tremor from shivering solely on the basis of gross observation of limb movements (3). However, Stuart and associates (20) noted that atropine blocked both experimental and Parkinson tremor but did not diminish the intensity of cold-induced shivering in cats. Since both shivering and tremor are apparently mediated by the reticulospinal tract, these authors proposed a site of action for atropine rostra1 to the midbrain extrapyramidal pathways. In the present study, systemically injected atropine abolished involuntary movement induced by intracaudate carbachol. Furthermore, rectal temperatures after local carbachol injection- increased about 1.4”C above that of the controls. When

380

CONNOR,

ROSSI,

AND

BAIiER

movement spontaneously ceased or was antagonized by other agents, temperatures returned to normal but never dropped below the control levels. Since shivering is apparently of hypothalamic origin, it is significant that injections of small quantities of carbachol into the hypothalamus, or even into the third ventricle, did not produce involuntary motor responses. These factors appear to indicate that the involuntary movement induced by intracaudate carbachol is tremor and not shivering. The present finding that intracaudate procaine abolishes carbachol tremor reinforces the contention that carbachol is acting locally; procaine has been used to dissect-out chemically specific brain areas whose subsequent destruction might reduce the tremor of Parkinsonism (13). In all probability, the observed reversibility of procaine suppression is due to enzymatic destruction, since this compound is readily hydrolyzed (6). Since tremor returned after procaine was presumably destroyed, carbachol apparently has a longer duration of activity at the dose levels employed. If the primary site of action for carbachol is the caudate, its role in the production of tremor must be explained. Significantly, two major efferent outflows from the caudate serve to modulate substantia nigra and reticular formation activities (8). Both of these structures have been implicated in tremor mechanisms (10, 22). Even though carbachol is relatively fast acting, there is a latency of lo-20 min before tremor onset. This might be construed to mean involuntary movement occurs due to progressive reduction of the inhibitory influences of the caudate, permitting an imbalanced condition with consequent exaggerated activity of related extrapyramidal centers. As another possibility, the carbachol-stimulated caudate may exercise a proportionately greater inhibitory influence over the rest of the motor system. Presumably, tremor could occur by tipping the extrapyramidal scale in either direction (14). It appears that neither excitation nor depression of the caudate nucleus by a nonspecific neuronal excitatory agent (pentylenetetrazol) or depressant (procaine) are in themselves sufficient to produce tremor. Yet, abolition of carbachol tremor by local procaine or by systemically administered (ip) pentobarbital or atropine demonstrates that carbachol tremor can be antagonized by pharmacologic agents. Atropine methylbromide, a quaternary derivative of atropine, does not readily pass the blood-brain barrier (2 1). When administered intraperitoneally, this agent, unlike atropine, is ineffective against carbachol tremor. This is taken as evidence that intraperitoneal atropine blocks carbachol-induced involuntary movement through a central, rather than peripheral mechanism,

TREMOR

381

On the basis of the known autonomic pharmacology of carbachol, it would be reasonable to speculate that the tremorogenic actions of the drug involve cholinergic or cholinoceptive mechanisms in the caudate. Further investigation with other cholinergic agents and antagonists are needed to characterize these mechanisms more specifically. References W. W., M. J. HOSKO, W. J. RUTT, and J. R. MCGRATH. 1960. Tremorine-induced rage and its antagonism by atropine. Proc. Sot. Exptl. Biol. Med. 104: 214-217. DELGADO, J. M. R., and L. RUBINSTEIN. 1964. Intracerebral release of neurohumors in unanesthetized monkeys. Arch. Intern. Pharmacodyn. 150: 530-546. DOMER, F. R., and W. FELDBERG. 1960. Tremor in cats: The effect of administration of drugs into the cerebral ventricles. Brit. J. Pharmacol. 15: 578-587. “Pharmacological Approach to Brain,” pp. 64-65. Williams FELDBERG, W. 1963. and Wilkins, Baltimore, Maryland. FELDBERG, W., and M. VOGT. 1948. Acetylcholine synthesis in different regions of the central nervous system. J. Physiol. London 107: 372-381. GOODMAN, L. S., and A. GILMAN. 1955. “The Pharmacological Basis of Therapeutics,” 2nd ed., p. 365. Macmillan, New York. JASPER, H. H., and C. AJMONE-MARSAN. 1953. “A Stereotaxic Atlas of the Diencephalon of the Cat.” National Research Council of Canada, Ottawa. The extrapyramidal motor system, p. 869. JUNG, R., and R. HASSLER. 1960. In “Handbook of Physiology, Sect. 1, Neurophysiology,” Vol. 2, J. Field Led.]. American Physiological Society, Washington, D.C. JURKO, M. F., and D. P. FOSHEE. 1962. Tremor: Consideration of recording techniques. Am. J. Electroencephalog. Technol. 2: 82-89. KAFLBER, W. W. 1963. Tremor at rest from tegmental lesions in the cat. J. Neuropathol. Exptl. Neurol. 22: 695-701. MALONE, M. H., F. C. ARZT, R. A. BRAGAN, and L. D~c.4~0. 1965. Reserpinenicotine induced Parkinsonism in mice. Arch. Intern. Pharmocodyn. 154: 69-81. MCLENNAN, H. 1964. The release of acetylcholine and of 3-hydroxytyramine from the caudate nucleus. J. Physiol. London 174: 152-161. OBRAWR, S. A. 1957. A simplified neurosurgical technique for approaching and damaging the region of the globus pallidus in Parkinson’s disease. J. Neurol. Neuvosurg. Psychiat. 20: 47-49. PAPEZ, J. W. 1959. Caudate nucleus and putamen; Their connections, inhibitory functions, and mobilization of cholinesterase. Recent Advan. Biol. Psychiat. 1: 17-19. (Proc. Ann. Conv. Sot. Biol. Psychiat., 14th, Atlantic City, 1958.) RAKIC, L., N. A. BUCHWALD, and E. J. WYERS. 1962. Induction of seizures by stimulation of the caudate nucleus. Electroencephalog. Clin. Neurophysiol. 14: 809-823. RECH, R. H., and E. F. DOMINO. 1959. Observations on injections of drugs into the brain substance. Arch. Intern. Pharmacodyn. 121: 429-442. BAKER,

2. 3. 4. d 3. 6. 7. 8.

9. 10. 11. 12. 13.

14.

15.

16.

382 17. 18. 19. 20.

21.

22.

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AND

BAKER

SPIEGEL, E. A., and E. G. SZEKELY. 1961. Prolonged stimulation of the head of the caudate nucleus. Arch. Neural. 4: 55-65. STEVENS, J. R., C. KIM, and P. D. MACLEAN. 1961. Stimulation of caudate nucleus. Arch. Neural. 4: 47-54. STRASSBURGER, R. H., and L. A. FRENCH. 1961. Experimental production of tremor. J. Lancet 81: 86-89. STUART, D. G., R. GEORGE, W. J. FREEMAN, A. HEMINGWAY, and W. M. PRICE. 1961. Effects of anti- and pseudoparkinson drugs on shivering. Exptl. Neurol. 4: 106-114. VERNIER, V. G., and K. R. UNNA. 1963. The central nervous system effects of drugs in monkeys with surgically-induced tremor: Atropine and other antitremor agents. Arch. Intern. Pharmacodyn. 141: 30-53. WARD, A. A., W. S. MCCULLOCH, and H. W. MAGOUN. 1948. Production of alternating tremor at rest in monkeys. J. Neurophysiol. 11: 317-330.