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Electroencephalographic studies of chlorpromazine methiodide and somatostatin-induced barrel rotation in rats
EXPERIMENTAL
NEUROLOGY
79, 704-7 13 ( 1983)
Electroencephalographic Studies of Chlorpromazine Methiodide and Somatostatin-Induced Barrel Rotation in Rats ROBERTE.BURKEAND
STANLEY FAHN'
Department of Neurology, Columbia University, College of Physicians and Surgeons, 710 West 168th Street, New York, New York 10032 Received June 28, 1982; revision received September 13. 1982 It has been reported that intraventricular injection of chlorpromazine
methiodide
(CPZMI), a quaternary ammonium derivative of chlorpromazine, in rats induces abnormal, twisting postures which may serve as an experimental model of the human movement disorder dystonia. We have shown elsewhere that the behavior induced by intraventricular CPMZI is identical to what has been called ‘barrel rotation,” first observed to follow intraventricular injection of somatostatin (SRIF), which consists of twisting about the long axis, with repetitive lateral rolling. The suitability of barrel rotation, induced by CPZMI or SRIF, as an experimental model for dystonia depends on its physiologic basis. Human dystonia is clinically not a convuIsive phenomenon. SRIF-induced barrel rotation has been reported to be associated with epileptiform activity recorded by the electroencephalogram (EEG). The purpose of this study was to investigate EEG activity during CPZMI- and SRIF-induced rotation. We found that CPZMI barrel rotation was not associated with epileptiform activity in cortex, amygdala, or hippocampus, and contrary to prior reports, neither was SRIF rotation. Both CPZMI and SRIF injected in high doses could induce epileptiform activity, but this was associated with clonic motor phenomena and not barrel rotation. We conclude that electroencephalographic criteria do not exclude either CPZMI- or SRIF-induced rotation as models for movement disorders, but their validity as such requires further study.
INTRODUCTION Rotrosen and coworkers ( 16) have reported that intraventricular injection of chlorpromazine methiodide (CPZMI), a quaternary ammonium derivative Abbreviations: CPZMI-chlorpromazine methiodide, SRIF-somatostatin. I We are grateful to Madeline Himy and Donna Johnson for preparation of the manuscript. This work was supported by the Dystonia Medical Research Foundation. Correspondence should be addressed to Dr. R. E. Burke, address above. 704 0014-4886/83/030704-10$03.00/O Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any fom resewed
EEG IN CPZM
AND SRIF BARREL
ROTATION
705
of chlorpromazine, into rats induces sustained, twisting postures which may serve as an experimental model for the human movement disorder dystonia, which is characterized by twisting involuntary movements. They showed that CPZMI, although a chlorpromazine derivative, did not alter cerebral dopamine metabolism when given intraventricularly, affect serum prolactin levels, or displace [3H]spiperone binding. It thus appears to lack dopamine antagonist activity. We have shown (3) that the twisting postures induced by CPZMI are identical in appearance to what has been called “barrel rotation,” first observed to follow intraventricular injection of somatostatin (SRIF), which consists of twisting about the long axis, with repetitive lateral rolling (4). We also showed that CPZMI-induced rotation is due to an antimuscarinic effect of CPZMI and that barrel rotation is induced by many antimuscarinics (3). CPZMI rotation is inhibited by simultaneous intraventricular injection of carbachol, a muscarinic agonist, and enhanced by atropine. CPZMI is a potent inhibitor of [3H]quinuclidynl bet&ate binding at the muscarinic receptor (3). Since its original description (4), barrel rotation has been reported by many investigators to follow intraventricular or intracerebral injection of SRIF (6, 7, 9, 12, 17). It has also been observed to follow injection of Substance P (8) and vasopressin (11). The biochemical and physiologic basis for this behavior, and its neuroanatomic substrate are unknown. The specificity of the behavior has been questioned. Cohn and Cohn found no dose-response relationship for the effect (4), and vasopressin-induced barrel rotation has been attributed to a “toxic effect” (11). In favor of specificity, however, is that other neuropeptides, including thyrotropin-releasing hormone (TRH) (4, 17), leutinizing hormone releasing hormone (LHRH) (17), angiotensin and neurotensin (12), fi-endorphin (2), ACTH, cw-melanocyte stimulating hormones, the (Ala’$SRIF analogue ( 18), L-prolylglycine, poly-L-proline, or poly-L-glutamate (12) do not induce barrel rotation. Furthermore, in the case of CPZMI-induced rotation, there is a quantitative, linear dose-response relationship and a specific pharmacologic basis, that of muscarinic antagonism (3). The suitability of CPZMI- or SRIF-induced barrel rotation as experimental models of dystonia depends on their physiologic basis. Dystonia is clinically not considered to be a convulsive phenomenon, because it is usually not paroxysmal, and it is not associated with epileptiform activity on the electroencephalogram (EEG). Prior electrophysiological studies of SRIF-induced behavior abnormalities have attributed barrel rotation to convulsive activity (7). Vasopressin-induced barrel rotation has also been proposed to be convulsive in nature (10). Intracerebral injection of the excitotoxin, kainic acid, has caused sustained twisting about the long axis late in the course of seizures
706
BURKE
AND FAHN
in rats (1). Such observations suggest that CPZMI barrel rotation also may be a convulsive phenomenon and unlikely to pertain to the study of dystonia. The present study investigated the electrophysiological correlates of CPZMIand SRIF-induced barrel rotation. In particular we wished to learn if any of the behavioral manifestations of barrel rotation were associated with ictal activity in cortex or limbic structures. MATERIALS AND METHODS Male Sprague-Dawley rats (Camm Research Institute, Inc., Wayne, N.J.) weighing 250 to 300 g were individually housed, with free access to food and water before and after experimentation, and were maintained on a 12: 12 1ight:dark cycle. Each animal was prepared for intraventricular injection of CPZMI or SRIF by stereotaxic placement of an indwelling intraventricular guide cannula. At the time of cannula placement, recording electrodes were implanted either in the amygdala or hippocampus. In addition, each animal had four epidural recording electrodes placed over the frontal and occipital cortex bilaterally. The animals were anesthetized with pentobarbital(40 mg/kg, i.p.), and given atropine sulfate (0.05 mg, i.p.) preoperatively. They were then positioned in a stereotaxic apparatus (David Kopf Instruments) with the incisor bar 5.0 mm above the interaural line. Bregma was used for anterior-posterior and lateral zero coordinates and dura for vertical. Coordinates for the intraventricular cannulae were A + 0.8, L - 1.3, V - 3.3 mm; for the amygdala electrodes A - 1.0, L f 3.5, V - 9.5; and for the hippocampal electrodes A - 4.0, L f 4.5, V - 6.0 mm. Cortical epidural electrodes were placed through 2.0-mm burr holes. After implantation, the cannulae and electrodes were secured by skull screws and dental acrylic. The guide cannulae were 22-gauge stainless-steel with a plastic screw thread affixed (Plastic Products Co., Roanoke, Va). After implantation of each guide cannula, a stainless-steel stylet with a protective plastic cap was inserted, extending 1.0 mm beyond its tip. Depth electrodes were made from 0.36mm stainless-steel wire insulated except at the tip. Cortical electrodes were fashioned from nylon-insulated stainless-steel wire connected to bare stainless-steel pads 1.6 by 1.0 mm (both electrode types from Plastic Products). Electrical activity was recorded on a Grass Model 6 EEG machine. Because of space limitations imposed by the intraventricular cannula, the six recording electrodes, and the need to limit skull cap size for the sake of behavioral observations, we did not use an additional extracranial reference electrode. All recordings were bipolar. Cortical recordings were bilateral frontal to occipital pairs. Amygdala and hippocampal recordings were from depth electrodes to the contralateral occipital cortex electrode. Band pass was 1 to 75 Hz.
EEG IN CPZMI AND SRIF BARREL
L cortex
ROTATION
707
b
R Amygdala
L Amygdolo
A. Rat 6. Baseline.
R Cortex
mwT$j*
L Cortex
mm
8. Rat 6. During
TlOOpV
6
barrel
rotation
induced
by 20pg
CPZMI.
FIG. I. Cortical and amygdala recording during chlorpromazine metbiodide (CPZMI)induced barrel rotation. No epileptiform discharges were seen. Low-voltage fast activity seen in all our recordings, before and after drug injection, was not al&ted by 60qcle filter and it was not related to muscle activity. Its presence in baseline recordings makes its occurrence after drug injection unlikely to be epileptiform activity.
Intraventricular injections were made through a 28-gauge internal cannula connected to a Hamilton microliter syringe by a 3O-cm length of polyethylene tubing. This length allowed injection while the rat was free roaming, without disrupting behavioral or electrophysiologic observations. The internal cannula was lilled to the tip with injection solution, and extended 1.O mm beyond the guide cannula tip, so that there was no dead space for the injection. The injection volume was 10 ~1, administered during 15 s. Vehicle was 0.9% NaCl for CPZMI and phosphate-buffered saline (pH 7.4) for SFUF. The CPZMI was kindly provided by Smith Kline and French Laboratories and SRIF was purchased from Beckman Instruments, Inc. Our prior dose-response studies of CPZMI (3) and pilot studies of SRIF indicated an appropriate dose range to consistently elicit barrel rotation. To study CPZMI-induced rotation each rat was injected with 10,20, and 40 pg in sequence, with at least 6 h between injections, until barrel rotation 00 curred. No rat was given more than three injections, except one rat with hippocampal electrodes was given a 60-pg injection. To study SRIF, each rat was given 10 pg and then, if necessaq to elicit rotation, a 20-pg injection
708
BURKE AND FAHN
pw R Hippoc.
;
L Hippoe. A.Rot
4. Bowline.
+J200pV I s,
A COrwK L Carter
R Hippoc.
h
L Hippoc.
8. Rot 4.
During
barrel
rototion
induced
by 4OPg
CPZMI.
FIG. 2. Cortical and hippocampal recording during CPZMI-induced barrel rotation. There was bilateral rhythmic slow activity in cortex and hippocampus but with no epileptiform dis charge.
after a 6-h interval. A total of 12 rats was studied, 6 each for CPZMI and SRIF, and, within these groups, 3 each with amygdala and hippocampal electrodes. During experimentation the animals were placed in a glass observation cage measuring 40 X 2 1 X 25 cm. Recording electrodes and intraventricular cannula were put in place and after 30 min to habituate to the experimental setting, we recorded at least 5 min of baseline EEG. Then the injection was made while EEG recording continued during the behavioral events and until the animal’s behavior returned to normal. Onset and duration of barrel rotation behavior was marked by an event signal on the polygraph tracing. “Barrel rotation” was defined as behavior which began as unilateral forepaw and hind paw extension, followed by a twist about the long axis of the trunk in a direction opposite the side of limb extension, and culminating in repetitive lateral rolling in the direction of the trunk twist. After completion of the experiment, the rats were killed, the brains I&d in 10% buffered Formalin, and embedded in paraffin. Placement of cannula and electrodes was confirmed by examination of cresyl violet-stained lo-pm sections.
EEG IN CPZMI AND SRIF BARREL
ROTATION
709
R Wt.&& LccduW t;J'W
R wippoc.
L nippoe. A. Rot 3. During
bard
rotation
inducd
by CPZMI.
T=w Is
R Cator
L cwtrr R Hippoc. L Hippos.
6. Rat 3. Aftrr
borrrl
rotation
with clonic
motor
activity.
FIG. 3. After 60 pg CPZMI, barrel rotation occurred during which the recording in A was made. After barrel rotation ceased, the animal developed bilateral clonic jerking of the limbs associated with bursts of bilaterally synchronous ~-HZ multiple spike complexes. These appeared to be of cortical origin. No epileptiform activity was evident during barrel rotation.
RESULTS Three rats with amygdala electrodes developed barrel rotation after intraventricular injection of CPZMI. They first developed extension of the fore and hind paw on one side, either right or left. Then the trunk twisted and the rats rolled laterally in the direction opposite the side of limb extension. During this sustained abnormal posturing the animals remained alert and struggled to right themselves. They withdrew appropriately from pressure applied to the limbs, and thus appeared to have normal strength and sensation. There were no clonic movements. Figure 1 shows a representative cortical and amygdala recording during barrel rotation induced by 20 pg CPZMI. The recording was free of sustained paroxysmal activity during the rotation. All three animals showed this encephalographic pattern. Figure 2 shows cortical and hippocampal recordings during barrel rotation induced by 40 pg CPZMI. Again, no epileptiform activity was seen during
710
BURKE
AND FAHN
R Cortex L Cortex R Amypdolo
L Amypdala A. Rot 8. Boseline.
R Cortex L Cortex R Amypdala
L Amypdala 6. Rat 8. Ourin
barrel
rotation
induced
by 20~9
SRIF.
FIG. 4. Cortical and amygdala recording during somatostatin (SRlF)-induced barrel rotation. There was no epileptiform activity.
the recording. Slower frequencies appeared bilaterally. Two other animals with hippocampal recordings likewise had no paroxysmal activity. CPZMI in doses above 60 pg can induce, in addition to rotation, sustained abnormal postures, seizures, and death (3). After an intraventricular injection of CPZMI one animal (No. 3) developed barrel rotation without electrographic seizure activity (Fig. 3A). Barrel rotation then ceased, sustained abnormal posturing developed, and clonic motor activity supervened. During this behavioral seizure, epileptiform activity was observed (Fig. 3B). Behavioral observations made for SRIF-induced barrel rotation were similar to those for CPZMI rotation. The animals developed sustained extension of the fore and hind paw on the right or left, and then twisted about the long axis, rolling away from the side with limb extension. They retained full strength in all limbs, and thus the term “hemiplegia in extension” which has been applied to the fore and hind paw extension observed in SRIF rotation (15) is not accurate. Figure 4 shows cortical and amygdala recordings during barrel rotation induced by 20 /lg SRIF. As with CPZMI, no epileptiform activity was seen. Two other rats showed similar results. Figure 5 shows cortical and hippocampal recordings during barrel rotation
EEG IN CPZMI AND SRIF BARREL
ROTATION
711
A. Rat IO. Baseline.
L Cortea
#
R Hippoc.
L Hippoc.
VWB. Rat IO. During
barrel
rotation
induced
by IOtq
SRIF.
FIG. 5. Cortical and hippocampal recording during SRIF-induced barrel rotation. There was no epileptiform activity.
induced by 10 Fg SRIF, and no epileptiform activity was present. This rat (No. 10) showed rhythmic slow activity; two other rats did not. We found that injection of higher doses of SRIF could induce clonic motor activity (wet dog shakes, twitching of forepaws), which was associated with epileptiform activity, as reported by others (7). However, such behavior and epileptiform activity was dissociable from barrel rotation, as with CPZMIinduced rotation. DISCUSSION These results show that CPZMI-induced barrel rotation is not a convulsive phenomenon. Atropine applied to cortex can induce epileptiform activity (5) and CPZMI injected intraventricularly can induce seizures (3). However, barrel rotation induced by CPZMI can be dissociated from paroxysmal epileptiform activity induced by antimuscarinics. Similarly, our studies show that SRIF-induced barrel rotation is not a convulsive phenomenon. Our results differ from those of Havlicek and Freisen (7) who concluded that SRIF barrel rotation is epileptic in nature. In their report, although they clearly show paroxysmal activity on an electroencephalographic recording, they do not state that barrel rotation behavior
712
BURKE
AND
FAHN
occurred during such paroxysms. In an earlier study ( 15) those investigators observed unilateral fore and hind paw extension and lateral rolling to follow intrastriatal injection of 10 Kg SRIF in the absence of electroencephalographic paroxysmal activity in cortex and striatum. Unilateral fore and hind paw extension [which those authors variously termed “hemiplegia in extension” ( 15) or “paraplegia in extension” (13)] is always seen early in the barrel rotation response. Those authors described unilateral fore and hind paw extension following injection of 10 pg SRIF into hippocampus ( 13) and amygdala (14) in the absence of sustained paroxysmal activity at cortical or injection site recordings. Thus, our recordings and, we believe, those of the above cited authors ( 13- 15) do not support their conclusion that barrel rotation is a convulsive phenomenon (7). Our study shows that CPZMI-induced barrel rotation, which Rotrosen and coworkers proposed may be an animal model of dystonia (16) cannot be excluded as such by electrophysiological criteria. Like human dystonia, it is not associated with epileptiform discharges. However, to establish either CPZMI or SRIF barrel rotation as possible valid models of human dystonia will require fulfillment of additional criteria of comparison between the animal behaviors and the clinical disorder. REFERENCES 1. BEN-Am, Y., E. TREMBLAY, AND 0. P. OTTERSEN. 1980. Injections of kainic acid into the amygdaloid complex of the rat: an electrographic, clinical and histological study in relation to the pathology of epilepsy. Neuroscience 5: 5 15-528. 2. BLOOM, F., D. SEGAL, N. LING, AND R. GUILLEMIN. 1976. Endorphins: profound behavioral effects in rats suggest new etiologic factors in mental illness. Science 194: 630-632. 3. BURKE, R. E., S. FAHN, H. R. WAGNER, AND M. SMEAL. 1982. Chlorpromazine methiodide induced barrel rotation: an antimuscarinic effect. Bruin Rex 250: 133-142. 4. COHN, M. L., AND M. COHN. 1975. Barrel rotation induced by somatostatin in the nonlesioned rat. Bruin Rex 96: 138- 141. 5. DANIELS, J. C., AND R. SPEHLMANN. 1973. The convulsant effect of topically applied atropine. Electroencephal. Clin. Neurophysiol. 34: 85-87. 6. GARCIA-SEVILLA, J., T. MAGNUSSON, AND A. CARLSSON. 1978. Effect of intracerebroventricularly administered somatostatin on brain monoamine turnover. Bruin Res. 155: 159-164. 7. HAVLICEK, V., AND H. G. FRIESEN. 1979. Comparison of behavioral effectsof somatostatin and B-endorphin in animals. Pages 381-402 in R. COLLU et al., Eds., Central Nervous System Effects of Hypothalamic Hormones and Other Peptides. Raven Press, New York. 8. JAMES, T. A., AND M. S. STARR. 1979. Effects of Substance P injected into the substantia n&a. Br. J. Pharmacol. 65: 423-429. 9. KASTIN, A. J., 0. H. COY, Y. JACQUET, A. V. SCHALLY, AND N. P. PLOTNIKOFF. 1976. CNS effects of somatostatin. Metabolism 27: 1247-1252. 10. KASTING, W., W. L. VEALE, AND K. E. COOPER. 1980. Convulsive and hypothermic effects of vasopressin in the brain of the rat. Can. J. Physiol. Pharmacol. 58: 316-319. 11. KRUSE, H., T. B. VAN WIMERSIMA GREIDANUS, AND D. DEWIED. 1977. Barrel rotation induced by vasopressin and related peptides in rats. Pharm. Biochem. Behav. 7: 3 1 l-3 13.
EEG IN CPZMI AND SRIP BARREL
ROTATION
713
12. MALTHE-S~REN~~EN, D., P. L. WOOD, D. L. CHENEY, AND E. COSTA. 1978. Modulation of the turnover rate of acetylcholine in rat brain by intraventricular injections of thyrotropin-releasing hormone, somatostatin, neurotensin, and angiotensin II. J. Neumhem.
31: 685-691. 13. REEK, M., V. HAVLICEK,
K. R. HUGHES, AND H. FRIESEN. 1976. Central site of action of somatostatin: role of hippocampus. Neurophamacology 15: 499-504. 14. REZEK, M., V. HA~LICEK, K. R. HUGHES, AND H. FRIESEN. 1977. Behavioral and motor excitation and inhibition induced by the administration of small and large doses of so matostatin into the amygdak Nemopharmacology 16: 157-162. 15. REZEK, M., V. HAVLICEK, L. LE~BIN, L. F?NSKY, E. A. KRUGER, K. R. HUGHES, AND H. FRIESEN. 1977. Neostriatal administration of somatostatin: differential effect of small and large doses on behavioral and motor control. Can. J. Physiol. Pharmacol. 55: 234-242. 16. ROTROSEN, J., M. STANLEY, C. KUHN, D. WAZER, AND S. GERSHON. 1980. Experimental dystonia induced by quaternary chlorpromazine. Neurology 30: 878-881. 17. VUAYAN, E., AND S. M. MCCANN. 1977. Suppression of feeding and drinking activity in rats following intmventricular injection of thyrotropin releasing hormone. Endocrinology
loo: 1727-1730. 18. WOOD, P. L., D. L. CHENEY, AND E. COTTA. 1979. Modulation of the turnover rate of hippocampal acetylcholine by neuropeptid~ possible site of action of a-melanocyte stimulating hormone, adrenocorticotrophic hormone and somatostatin. J. Pharmacol. Exp. Ther. 209: 97-303.
NEUROLOGY
79, 704-7 13 ( 1983)
Electroencephalographic Studies of Chlorpromazine Methiodide and Somatostatin-Induced Barrel Rotation in Rats ROBERTE.BURKEAND
STANLEY FAHN'
Department of Neurology, Columbia University, College of Physicians and Surgeons, 710 West 168th Street, New York, New York 10032 Received June 28, 1982; revision received September 13. 1982 It has been reported that intraventricular injection of chlorpromazine
methiodide
(CPZMI), a quaternary ammonium derivative of chlorpromazine, in rats induces abnormal, twisting postures which may serve as an experimental model of the human movement disorder dystonia. We have shown elsewhere that the behavior induced by intraventricular CPMZI is identical to what has been called ‘barrel rotation,” first observed to follow intraventricular injection of somatostatin (SRIF), which consists of twisting about the long axis, with repetitive lateral rolling. The suitability of barrel rotation, induced by CPZMI or SRIF, as an experimental model for dystonia depends on its physiologic basis. Human dystonia is clinically not a convuIsive phenomenon. SRIF-induced barrel rotation has been reported to be associated with epileptiform activity recorded by the electroencephalogram (EEG). The purpose of this study was to investigate EEG activity during CPZMI- and SRIF-induced rotation. We found that CPZMI barrel rotation was not associated with epileptiform activity in cortex, amygdala, or hippocampus, and contrary to prior reports, neither was SRIF rotation. Both CPZMI and SRIF injected in high doses could induce epileptiform activity, but this was associated with clonic motor phenomena and not barrel rotation. We conclude that electroencephalographic criteria do not exclude either CPZMI- or SRIF-induced rotation as models for movement disorders, but their validity as such requires further study.
INTRODUCTION Rotrosen and coworkers ( 16) have reported that intraventricular injection of chlorpromazine methiodide (CPZMI), a quaternary ammonium derivative Abbreviations: CPZMI-chlorpromazine methiodide, SRIF-somatostatin. I We are grateful to Madeline Himy and Donna Johnson for preparation of the manuscript. This work was supported by the Dystonia Medical Research Foundation. Correspondence should be addressed to Dr. R. E. Burke, address above. 704 0014-4886/83/030704-10$03.00/O Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any fom resewed
EEG IN CPZM
AND SRIF BARREL
ROTATION
705
of chlorpromazine, into rats induces sustained, twisting postures which may serve as an experimental model for the human movement disorder dystonia, which is characterized by twisting involuntary movements. They showed that CPZMI, although a chlorpromazine derivative, did not alter cerebral dopamine metabolism when given intraventricularly, affect serum prolactin levels, or displace [3H]spiperone binding. It thus appears to lack dopamine antagonist activity. We have shown (3) that the twisting postures induced by CPZMI are identical in appearance to what has been called “barrel rotation,” first observed to follow intraventricular injection of somatostatin (SRIF), which consists of twisting about the long axis, with repetitive lateral rolling (4). We also showed that CPZMI-induced rotation is due to an antimuscarinic effect of CPZMI and that barrel rotation is induced by many antimuscarinics (3). CPZMI rotation is inhibited by simultaneous intraventricular injection of carbachol, a muscarinic agonist, and enhanced by atropine. CPZMI is a potent inhibitor of [3H]quinuclidynl bet&ate binding at the muscarinic receptor (3). Since its original description (4), barrel rotation has been reported by many investigators to follow intraventricular or intracerebral injection of SRIF (6, 7, 9, 12, 17). It has also been observed to follow injection of Substance P (8) and vasopressin (11). The biochemical and physiologic basis for this behavior, and its neuroanatomic substrate are unknown. The specificity of the behavior has been questioned. Cohn and Cohn found no dose-response relationship for the effect (4), and vasopressin-induced barrel rotation has been attributed to a “toxic effect” (11). In favor of specificity, however, is that other neuropeptides, including thyrotropin-releasing hormone (TRH) (4, 17), leutinizing hormone releasing hormone (LHRH) (17), angiotensin and neurotensin (12), fi-endorphin (2), ACTH, cw-melanocyte stimulating hormones, the (Ala’$SRIF analogue ( 18), L-prolylglycine, poly-L-proline, or poly-L-glutamate (12) do not induce barrel rotation. Furthermore, in the case of CPZMI-induced rotation, there is a quantitative, linear dose-response relationship and a specific pharmacologic basis, that of muscarinic antagonism (3). The suitability of CPZMI- or SRIF-induced barrel rotation as experimental models of dystonia depends on their physiologic basis. Dystonia is clinically not considered to be a convulsive phenomenon, because it is usually not paroxysmal, and it is not associated with epileptiform activity on the electroencephalogram (EEG). Prior electrophysiological studies of SRIF-induced behavior abnormalities have attributed barrel rotation to convulsive activity (7). Vasopressin-induced barrel rotation has also been proposed to be convulsive in nature (10). Intracerebral injection of the excitotoxin, kainic acid, has caused sustained twisting about the long axis late in the course of seizures
706
BURKE
AND FAHN
in rats (1). Such observations suggest that CPZMI barrel rotation also may be a convulsive phenomenon and unlikely to pertain to the study of dystonia. The present study investigated the electrophysiological correlates of CPZMIand SRIF-induced barrel rotation. In particular we wished to learn if any of the behavioral manifestations of barrel rotation were associated with ictal activity in cortex or limbic structures. MATERIALS AND METHODS Male Sprague-Dawley rats (Camm Research Institute, Inc., Wayne, N.J.) weighing 250 to 300 g were individually housed, with free access to food and water before and after experimentation, and were maintained on a 12: 12 1ight:dark cycle. Each animal was prepared for intraventricular injection of CPZMI or SRIF by stereotaxic placement of an indwelling intraventricular guide cannula. At the time of cannula placement, recording electrodes were implanted either in the amygdala or hippocampus. In addition, each animal had four epidural recording electrodes placed over the frontal and occipital cortex bilaterally. The animals were anesthetized with pentobarbital(40 mg/kg, i.p.), and given atropine sulfate (0.05 mg, i.p.) preoperatively. They were then positioned in a stereotaxic apparatus (David Kopf Instruments) with the incisor bar 5.0 mm above the interaural line. Bregma was used for anterior-posterior and lateral zero coordinates and dura for vertical. Coordinates for the intraventricular cannulae were A + 0.8, L - 1.3, V - 3.3 mm; for the amygdala electrodes A - 1.0, L f 3.5, V - 9.5; and for the hippocampal electrodes A - 4.0, L f 4.5, V - 6.0 mm. Cortical epidural electrodes were placed through 2.0-mm burr holes. After implantation, the cannulae and electrodes were secured by skull screws and dental acrylic. The guide cannulae were 22-gauge stainless-steel with a plastic screw thread affixed (Plastic Products Co., Roanoke, Va). After implantation of each guide cannula, a stainless-steel stylet with a protective plastic cap was inserted, extending 1.0 mm beyond its tip. Depth electrodes were made from 0.36mm stainless-steel wire insulated except at the tip. Cortical electrodes were fashioned from nylon-insulated stainless-steel wire connected to bare stainless-steel pads 1.6 by 1.0 mm (both electrode types from Plastic Products). Electrical activity was recorded on a Grass Model 6 EEG machine. Because of space limitations imposed by the intraventricular cannula, the six recording electrodes, and the need to limit skull cap size for the sake of behavioral observations, we did not use an additional extracranial reference electrode. All recordings were bipolar. Cortical recordings were bilateral frontal to occipital pairs. Amygdala and hippocampal recordings were from depth electrodes to the contralateral occipital cortex electrode. Band pass was 1 to 75 Hz.
EEG IN CPZMI AND SRIF BARREL
L cortex
ROTATION
707
b
R Amygdala
L Amygdolo
A. Rat 6. Baseline.
R Cortex
mwT$j*
L Cortex
mm
8. Rat 6. During
TlOOpV
6
barrel
rotation
induced
by 20pg
CPZMI.
FIG. I. Cortical and amygdala recording during chlorpromazine metbiodide (CPZMI)induced barrel rotation. No epileptiform discharges were seen. Low-voltage fast activity seen in all our recordings, before and after drug injection, was not al&ted by 60qcle filter and it was not related to muscle activity. Its presence in baseline recordings makes its occurrence after drug injection unlikely to be epileptiform activity.
Intraventricular injections were made through a 28-gauge internal cannula connected to a Hamilton microliter syringe by a 3O-cm length of polyethylene tubing. This length allowed injection while the rat was free roaming, without disrupting behavioral or electrophysiologic observations. The internal cannula was lilled to the tip with injection solution, and extended 1.O mm beyond the guide cannula tip, so that there was no dead space for the injection. The injection volume was 10 ~1, administered during 15 s. Vehicle was 0.9% NaCl for CPZMI and phosphate-buffered saline (pH 7.4) for SFUF. The CPZMI was kindly provided by Smith Kline and French Laboratories and SRIF was purchased from Beckman Instruments, Inc. Our prior dose-response studies of CPZMI (3) and pilot studies of SRIF indicated an appropriate dose range to consistently elicit barrel rotation. To study CPZMI-induced rotation each rat was injected with 10,20, and 40 pg in sequence, with at least 6 h between injections, until barrel rotation 00 curred. No rat was given more than three injections, except one rat with hippocampal electrodes was given a 60-pg injection. To study SRIF, each rat was given 10 pg and then, if necessaq to elicit rotation, a 20-pg injection
708
BURKE AND FAHN
pw R Hippoc.
;
L Hippoe. A.Rot
4. Bowline.
+J200pV I s,
A COrwK L Carter
R Hippoc.
h
L Hippoc.
8. Rot 4.
During
barrel
rototion
induced
by 4OPg
CPZMI.
FIG. 2. Cortical and hippocampal recording during CPZMI-induced barrel rotation. There was bilateral rhythmic slow activity in cortex and hippocampus but with no epileptiform dis charge.
after a 6-h interval. A total of 12 rats was studied, 6 each for CPZMI and SRIF, and, within these groups, 3 each with amygdala and hippocampal electrodes. During experimentation the animals were placed in a glass observation cage measuring 40 X 2 1 X 25 cm. Recording electrodes and intraventricular cannula were put in place and after 30 min to habituate to the experimental setting, we recorded at least 5 min of baseline EEG. Then the injection was made while EEG recording continued during the behavioral events and until the animal’s behavior returned to normal. Onset and duration of barrel rotation behavior was marked by an event signal on the polygraph tracing. “Barrel rotation” was defined as behavior which began as unilateral forepaw and hind paw extension, followed by a twist about the long axis of the trunk in a direction opposite the side of limb extension, and culminating in repetitive lateral rolling in the direction of the trunk twist. After completion of the experiment, the rats were killed, the brains I&d in 10% buffered Formalin, and embedded in paraffin. Placement of cannula and electrodes was confirmed by examination of cresyl violet-stained lo-pm sections.
EEG IN CPZMI AND SRIF BARREL
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6. Rat 3. Aftrr
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FIG. 3. After 60 pg CPZMI, barrel rotation occurred during which the recording in A was made. After barrel rotation ceased, the animal developed bilateral clonic jerking of the limbs associated with bursts of bilaterally synchronous ~-HZ multiple spike complexes. These appeared to be of cortical origin. No epileptiform activity was evident during barrel rotation.
RESULTS Three rats with amygdala electrodes developed barrel rotation after intraventricular injection of CPZMI. They first developed extension of the fore and hind paw on one side, either right or left. Then the trunk twisted and the rats rolled laterally in the direction opposite the side of limb extension. During this sustained abnormal posturing the animals remained alert and struggled to right themselves. They withdrew appropriately from pressure applied to the limbs, and thus appeared to have normal strength and sensation. There were no clonic movements. Figure 1 shows a representative cortical and amygdala recording during barrel rotation induced by 20 pg CPZMI. The recording was free of sustained paroxysmal activity during the rotation. All three animals showed this encephalographic pattern. Figure 2 shows cortical and hippocampal recordings during barrel rotation induced by 40 pg CPZMI. Again, no epileptiform activity was seen during
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AND FAHN
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L Amypdala A. Rot 8. Boseline.
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L Amypdala 6. Rat 8. Ourin
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rotation
induced
by 20~9
SRIF.
FIG. 4. Cortical and amygdala recording during somatostatin (SRlF)-induced barrel rotation. There was no epileptiform activity.
the recording. Slower frequencies appeared bilaterally. Two other animals with hippocampal recordings likewise had no paroxysmal activity. CPZMI in doses above 60 pg can induce, in addition to rotation, sustained abnormal postures, seizures, and death (3). After an intraventricular injection of CPZMI one animal (No. 3) developed barrel rotation without electrographic seizure activity (Fig. 3A). Barrel rotation then ceased, sustained abnormal posturing developed, and clonic motor activity supervened. During this behavioral seizure, epileptiform activity was observed (Fig. 3B). Behavioral observations made for SRIF-induced barrel rotation were similar to those for CPZMI rotation. The animals developed sustained extension of the fore and hind paw on the right or left, and then twisted about the long axis, rolling away from the side with limb extension. They retained full strength in all limbs, and thus the term “hemiplegia in extension” which has been applied to the fore and hind paw extension observed in SRIF rotation (15) is not accurate. Figure 4 shows cortical and amygdala recordings during barrel rotation induced by 20 /lg SRIF. As with CPZMI, no epileptiform activity was seen. Two other rats showed similar results. Figure 5 shows cortical and hippocampal recordings during barrel rotation
EEG IN CPZMI AND SRIF BARREL
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A. Rat IO. Baseline.
L Cortea
#
R Hippoc.
L Hippoc.
VWB. Rat IO. During
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rotation
induced
by IOtq
SRIF.
FIG. 5. Cortical and hippocampal recording during SRIF-induced barrel rotation. There was no epileptiform activity.
induced by 10 Fg SRIF, and no epileptiform activity was present. This rat (No. 10) showed rhythmic slow activity; two other rats did not. We found that injection of higher doses of SRIF could induce clonic motor activity (wet dog shakes, twitching of forepaws), which was associated with epileptiform activity, as reported by others (7). However, such behavior and epileptiform activity was dissociable from barrel rotation, as with CPZMIinduced rotation. DISCUSSION These results show that CPZMI-induced barrel rotation is not a convulsive phenomenon. Atropine applied to cortex can induce epileptiform activity (5) and CPZMI injected intraventricularly can induce seizures (3). However, barrel rotation induced by CPZMI can be dissociated from paroxysmal epileptiform activity induced by antimuscarinics. Similarly, our studies show that SRIF-induced barrel rotation is not a convulsive phenomenon. Our results differ from those of Havlicek and Freisen (7) who concluded that SRIF barrel rotation is epileptic in nature. In their report, although they clearly show paroxysmal activity on an electroencephalographic recording, they do not state that barrel rotation behavior
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occurred during such paroxysms. In an earlier study ( 15) those investigators observed unilateral fore and hind paw extension and lateral rolling to follow intrastriatal injection of 10 Kg SRIF in the absence of electroencephalographic paroxysmal activity in cortex and striatum. Unilateral fore and hind paw extension [which those authors variously termed “hemiplegia in extension” ( 15) or “paraplegia in extension” (13)] is always seen early in the barrel rotation response. Those authors described unilateral fore and hind paw extension following injection of 10 pg SRIF into hippocampus ( 13) and amygdala (14) in the absence of sustained paroxysmal activity at cortical or injection site recordings. Thus, our recordings and, we believe, those of the above cited authors ( 13- 15) do not support their conclusion that barrel rotation is a convulsive phenomenon (7). Our study shows that CPZMI-induced barrel rotation, which Rotrosen and coworkers proposed may be an animal model of dystonia (16) cannot be excluded as such by electrophysiological criteria. Like human dystonia, it is not associated with epileptiform discharges. However, to establish either CPZMI or SRIF barrel rotation as possible valid models of human dystonia will require fulfillment of additional criteria of comparison between the animal behaviors and the clinical disorder. REFERENCES 1. BEN-Am, Y., E. TREMBLAY, AND 0. P. OTTERSEN. 1980. Injections of kainic acid into the amygdaloid complex of the rat: an electrographic, clinical and histological study in relation to the pathology of epilepsy. Neuroscience 5: 5 15-528. 2. BLOOM, F., D. SEGAL, N. LING, AND R. GUILLEMIN. 1976. Endorphins: profound behavioral effects in rats suggest new etiologic factors in mental illness. Science 194: 630-632. 3. BURKE, R. E., S. FAHN, H. R. WAGNER, AND M. SMEAL. 1982. Chlorpromazine methiodide induced barrel rotation: an antimuscarinic effect. Bruin Rex 250: 133-142. 4. COHN, M. L., AND M. COHN. 1975. Barrel rotation induced by somatostatin in the nonlesioned rat. Bruin Rex 96: 138- 141. 5. DANIELS, J. C., AND R. SPEHLMANN. 1973. The convulsant effect of topically applied atropine. Electroencephal. Clin. Neurophysiol. 34: 85-87. 6. GARCIA-SEVILLA, J., T. MAGNUSSON, AND A. CARLSSON. 1978. Effect of intracerebroventricularly administered somatostatin on brain monoamine turnover. Bruin Res. 155: 159-164. 7. HAVLICEK, V., AND H. G. FRIESEN. 1979. Comparison of behavioral effectsof somatostatin and B-endorphin in animals. Pages 381-402 in R. COLLU et al., Eds., Central Nervous System Effects of Hypothalamic Hormones and Other Peptides. Raven Press, New York. 8. JAMES, T. A., AND M. S. STARR. 1979. Effects of Substance P injected into the substantia n&a. Br. J. Pharmacol. 65: 423-429. 9. KASTIN, A. J., 0. H. COY, Y. JACQUET, A. V. SCHALLY, AND N. P. PLOTNIKOFF. 1976. CNS effects of somatostatin. Metabolism 27: 1247-1252. 10. KASTING, W., W. L. VEALE, AND K. E. COOPER. 1980. Convulsive and hypothermic effects of vasopressin in the brain of the rat. Can. J. Physiol. Pharmacol. 58: 316-319. 11. KRUSE, H., T. B. VAN WIMERSIMA GREIDANUS, AND D. DEWIED. 1977. Barrel rotation induced by vasopressin and related peptides in rats. Pharm. Biochem. Behav. 7: 3 1 l-3 13.
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12. MALTHE-S~REN~~EN, D., P. L. WOOD, D. L. CHENEY, AND E. COSTA. 1978. Modulation of the turnover rate of acetylcholine in rat brain by intraventricular injections of thyrotropin-releasing hormone, somatostatin, neurotensin, and angiotensin II. J. Neumhem.
31: 685-691. 13. REEK, M., V. HAVLICEK,
K. R. HUGHES, AND H. FRIESEN. 1976. Central site of action of somatostatin: role of hippocampus. Neurophamacology 15: 499-504. 14. REZEK, M., V. HA~LICEK, K. R. HUGHES, AND H. FRIESEN. 1977. Behavioral and motor excitation and inhibition induced by the administration of small and large doses of so matostatin into the amygdak Nemopharmacology 16: 157-162. 15. REZEK, M., V. HAVLICEK, L. LE~BIN, L. F?NSKY, E. A. KRUGER, K. R. HUGHES, AND H. FRIESEN. 1977. Neostriatal administration of somatostatin: differential effect of small and large doses on behavioral and motor control. Can. J. Physiol. Pharmacol. 55: 234-242. 16. ROTROSEN, J., M. STANLEY, C. KUHN, D. WAZER, AND S. GERSHON. 1980. Experimental dystonia induced by quaternary chlorpromazine. Neurology 30: 878-881. 17. VUAYAN, E., AND S. M. MCCANN. 1977. Suppression of feeding and drinking activity in rats following intmventricular injection of thyrotropin releasing hormone. Endocrinology
loo: 1727-1730. 18. WOOD, P. L., D. L. CHENEY, AND E. COTTA. 1979. Modulation of the turnover rate of hippocampal acetylcholine by neuropeptid~ possible site of action of a-melanocyte stimulating hormone, adrenocorticotrophic hormone and somatostatin. J. Pharmacol. Exp. Ther. 209: 97-303.