Brain Research, 170 (1979) 497-507 © Elsevier/North-Holland Biomedical Press
497
C A T E C H O L A M I N E S A N D CONVULSIONS
STEPHEN T. MASON and MICHAEL E. CORCORAN* Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. V6T 1 W5 (Canada)
(Accepted November 16th, 1978)
SUMMARY Severe depletion of brain noradrenaline and separately of brain dopamine was induced in rats by intracerebral injection of the selective neurotoxin 6-hydroxydopamine, and the susceptibility of the treated animals to various seizure-inducing manipulations was examined. A significant potentiation of the seizures induced both by Metrazol and by electroconvulsive shock was found in animals depleted of brain noradrenaline, but no alteration was seen after depletion of brain dopamine on either measure. The catecholaminergic drug cocaine also induced seizures, but these were found not to depend on either brain noradrenaline or dopamine as they continued to occur in the virtual absence of either catecholamine. It is concluded that cocaine induces seizures by a non-specific toxic mechanism and that noradrenaline, but not dopamine, is involved in reducing the susceptibility of the central nervous system to the several distinct forms of seizure induction examined.
INTRODUCTION Considerable interest has focused on the functions of the catecholamines noradrenaline (NA) and dopamine (DA) as putative neurotransmitters in the central nervous system. Although less studied than their possible roles in reinforcement and learningS,a, 18, the participation of the catecholamines in the control of seizures has been suggestedln, 27. A number of pharmacological techniques for changing the functional activity in catecholaminergic systems appear to alter the susceptibility o f animals to seizures. Most recently the specific catecholaminergic neurotoxin 6hydroxydopamine (6-OHDA), which produces a severe and permanent physical * Present address: Department of Psychology, University of Victoria, P.O. Box 1700, Victoria, B.C. V8W 2Y2. Canada.
498 destruction of catecholaminergic neurons ~9, has been used. Thus, intraventricular injections of 6-OHDA were observed to markedly potentiate convulsions induced by pentylenetetrazol (Metrazol) 6,7. Rats treated with 6-OHDA showed convulsions of longer duration and of greater severity than controls6, 7. Since intraventricular 6O H D A depleted both NA and DA, however, it was not possible to determine which catecholamine was crucial for this effect. The relative participation of NA and DA in other forms of seizure activity is also unclear. Although NA appears to be involved in the postdecapitation reflex4,87, the picture is less certain with other forms of seizure activity because the treatments used to modify them resulted in depletion of peripheral as well as central catecholamines 3,19, in loss of NA together with DA 1,2,31, or, when only central NA was affected, in depletion of cerebellar and spinal as well as forebrain NAS,1L A few studies have also implicated dopamine independently of NA. Thus, apomorphine and piribedil reduce the susceptibility to photically induced convulsions in papio papio 28. The enhanced convulsion susceptibility induced by the catecholaminedepleting agent Ro4-1284 was reversed by intracerebroventricular infusion of dopamine 12 and this reversal was blocked by the dopamine receptor blocking agent, pimozide z~. Further evidence for a role of catecholamines in convulsions is found in the fact that the indirectly acting catecholaminergic agonist cocaine has been reported to cause seizuresl°,~6,z°, z~. The amount of cocaine required to induce seizures is generally very high, however, and the convulsant effects of cocaine could therefore be mediated by a non-specific toxic mechanism. In the present study we used localized injections of 6-OHDA into the ascending fibers of the NA systems in the mesencephalon, resulting in a selective depletion of forebrain NA and sparing DA and spinal or cerebellar NA 2°. A similar intracerebral injection of 6-OHDA into the nigrostriatal bundle was used to destroy the ascending DA systems selectively. METHODS
Surgical Dorsal bundle 6-OHDA Male albino Wistar rats (Woodlyn Farms, Ontario) weighing 300 g at the time of operation were anesthetized with Nembutal (50 mg/kg intraperitoneal) and positioned in a stereotaxic apparatus. Two holes were drilled in the skull, through which a 34gauge cannula was lowered to the following coordinates: AP + 2.6 mm from interaural line, ML ± 1.1 mm from the midline suture at bregma, and DV + 3.7 mm from the interaural line, with the animal's skull in the plane of K6nig and Klippel a4. Four micrograms of 6-hydroxydopamine (6-OHDA HBr, Regis Chemicals, weight expressed as free base) dissolved in 2/zl of 0.9 ~ saline with 0.2 mg/ml ascorbic acid antioxidant were infused bilaterally at the rate of 1 #l/min over 2 min. The cannula was left in for an extra minute to permit diffusion of the drug. Control animals
499 received infusion of an equal volume of saline-ascorbic vehicle. The skin was then sutured, and two weeks were allowed for completion of anterograde degeneration of the forebrain terminals before behavioral testing commenced 32. Nigrostriatal bundle 6-OHDA Similar injections of 6-OHDA were made bilaterally into the ascending dopamine bundle in the mesencephalon using the following coordinates: AP + 5.9 mm from interaural line, M L ~ 2.9 mm from midline suture at bregma, and DV q- 1.9 mm from interaural line. To prevent the marginal loss of NA which also occurs with this treatment the animals were pretreated with the NA-uptake inhibitor desimipramine (DMI 25 mg/kg intraperitoneally) 30 min prior to intracerebral injection 11. Controls received DMI pretreatment and saline-ascorbic infusion. Such marked loss of DA as caused by this operation is known to result in aphagia and adipsia, often leading to death if the animals are not tube fed as. Although recovery of feeding and drinking can occur after several weeks, similar recovery of other DA functions may also be argued to occur. We deemed it desirable to test the DA-depleted rats before any such recovery had taken place, these animals were thus tested 72 h after the operation. In an attempt to control for any aphagia-induced weight loss or any other change due to absence of food intake in the lesioned rats that might alter seizure susceptibility, the control and lesioned rats were deprived of all food from the time of operation until testing. Water was continuously available. Biochemical Following completion of behavioral testing animals were sacrificed by decapitation. Their brains were rapidly removed and dissected on ice into the following regions; hippocampus-cortex, hypothalamus, cerebellum, spinal cord and striatum 22. These regions were then assayed for endogenous catecholamines by the method of McGeer and McGeer 18. This served to confirm the extent and pattern of the amine depletions induced by the various 6-OHDA treatments. Behavioral Metrazol convulsions Nine NA-depleted and 7 control animals were used in one group and a further 10 DA-depleted and 10 control rats in another group. Each rat received a subcutaneous injection of 70 mg/kg of Metrazol, a dosage found in previous work to induce convulsions in approximately 90 % of normal rats6, 7. Each rat was then placed in a testing cage and observed for 1 h. The latencies to the first myoclonicjerk and the first generalized seizure were recorded as were the duration and number of seizures. The type of seizure, in terms of containing only clonic or both clonic and tonic components, was noted. Cocaine convulsions Three doses of cocaine hydrochloride were examined in NA-depleted rats. A
500 TABLE I Post-mortem amine assays
Post-mortem amine assays on dorsal bundle 6-OHDA and nigrostriatal 6-OHDA injected rats. Values are means with standard error of the mean in ng of amines per g wet weight of tissue. ~ column is the percentage of control values remaining in lesioned tissues and the P column gives the two-tailed probability comparing control and lesioned values using Student's t-test. Control (n = 10)
Lesioned (n = 10)
%
P
2 26 124 120
0.001 0.001 NS NS
Dorsal bundle 6-OHDA
Noradrenaline Hippocampus-cortex Hypothalamus Cerebellum Spinalcord Dopamine Striatum
264 2240 219 255
zL 6 ± 77 ± 32 ± 30
13100 zL 570
6 590 271 307
± ± ± ~
1 87 35 22
11600 ± 1190
88
NS
Nigrostriatal bundle 6-OHDA
Noradrenaline Hippocampus-cortex Hypothalamus Dopamine Striatum
287 i 28 1950 -- 115
263 ± 9 1850 ± 85
92 95
NS NS
9820 ± 475
1390 :~ 139
14
0.001
very steep d o s e - r e s p o n s e curve was f o u n d in pilot studies, with no effect at 60 mg/kg, m a r k e d convulsions at 70 mg/kg, a n d m a x i m a l response at 80 mg/kg. Five N A depleted a n d 5 c o n t r o l rats were used at the 60 a n d 80 m g / k g dosages a n d 10 N A depleted a n d 10 c o n t r o l rats at the i n t e r m e d i a t e dosage. Nine D A - d e p l e t e d a n d 5 c o n t r o l rats were e x a m i n e d at the 70 m g / k g dosage. The p r o c e d u r e was similar to t h a t described for M e t r a z o l - i n d u c e d seizures except t h a t the cocaine was a d m i n i s t e r e d intraperitoneally. Electroconvulsive shock ( ECS)
T e n N A - d e p l e t e d a n d 10 c o n t r o l animals were e x a m i n e d for their response to ECS. Three intensities o f ECS were a d m i n i s t e r e d in a s c e n d i n g order, each s e p a r a t e d by a 2-day period. Two a l l i g a t o r clips w r a p p e d in saline-soaked c o t t o n w o o l were a t t a c h e d to the a n i m a l ' s ears, a n d the following intensities o f ECS were a p p l i e d for 1 sec: 10 m A , 15 m A , a n d 22.5 m A . The p o w e r source for E C S has been d e s c r i b e d m o r e fully elsewhere 17. The a n i m a l was placed in a plastic cage; the clips were r e m o v e d ; a n d the resulting convulsion m e a s u r e d with r e g a r d to its latency o f onset, d u r a t i o n , a n d severity. The seizure was c h a r a c t e r i z e d in terms o f c o n t a i n i n g clonic elements alone or in c o m b i n a t i o n with tonic c o m p o n e n t s . Nine c o n t r o l rats a n d 10 D A - d e p l e t e d rats were also tested at the lowest ECS intensity a n d nine c o n t r o l a n d 10 D A - d e p l e t e d rats at the highest E C S intensity.
501 TABLE lI Metrazol seizures
Seizures induced in response to 70 mg/kg Metrazol in dorsal bundle 6-OHDA injected (NA-depleted) and nigrostriatal bundle 6-OHDA injected (DA-depleted) rats. Values are means with the standard error of the mean. Control (n = 7)
Lesioned (n = 9)
P
Latency to first myoclonicjerk (min) Latency to first seizure (min) Duration of first seizure (sec) No. of rats with multiple seizures No. of rats with tonic seizures
8.7 -4- 1.2 14.9 -4- 1.6 32.9 ± 3.0 0/7 0/7
8.2 -4- 1.4 NS 14.8 -4- 2.3 NS 113.2 -4- 11.7 0.01 5/9 0.03 5/9 0.03
D A depleted
n = 10
n = 10
Latency to first myoclonic jerk (rain) Latency to first seizure (min) Duration of seizures (sec) No. of seizures
7.6 11.6 119 2.6
N A -depleted
4- 1.5 -4- 2.6 -4- 47 -4- 0.8
7.5 10.0 248 3.5
-4- 1.3 -4- 1.8 -4- 86 -4- 1.0
NS NS NS NS
RESULTS Biochemical
T h e results o f p o s t m o r t e m a m i n e assays are shown in T a b l e I for a t y p i c a l g r o u p o f N A - d e p l e t e d rats a n d a g r o u p o f D A - d e p l e t e d rats. I n t r a c e r e b r a l injection o f 6O H D A into the ascending N A fiber-bundle resulted in a d e p l e t i o n o f h i p p o c a m p a l cortical N A to less t h a n 5 ~ o f c o n t r o l values a n d a loss o f h y p o t h a l a m i c N A to some 3 0 ~ o f n o r m a l . N o significant a l t e r a t i o n in striatal D A was p r o d u c e d as a consequence o f this t r e a t m e n t , a n d n o significant change in spinal or cerebellar N A occurred. I n t r a c e r e b r a l injection o f 6 - O H D A into the a s c e n d i n g D A fiber-bundle with D M I p r e t r e a t m e n t gave rise to a r e d u c t i o n in striatal D A to 14 ~ o f c o n t r o l values with no change in h y p o t h a l a m i c o r h i p p o c a m p a l - c o r t i c a l N A . D a t a on o t h e r animals ( M a s o n a n d Fibiger, u n p u b l i s h e d ) indicates t h a t these lesions fail to affect s e r o t o n i n , choline acetyltransferase o r glutamic acid d e c a r b o x y lase in a n y b r a i n a r e a e x a m i n e d , a n d a m o r e detailed discussion o f the specificity o f 6O H D A lesions is available elsewhere 23. Histological m i c r o g r a p h s o f the area o f 6O H D A injection have been p u b l i s h e d elsewhere zl. Metrazol-induced seizures
T a b l e II shows the effect o f these a m i n e d e p l e t i o n s on the seizures i n d u c e d b y M e t r a z o l injection. A m a r k e d p o t e n t i a t i o n o f the seizure was seen in the N A - d e p l e t e d rats. The d u r a t i o n a n d n u m b e r o f seizures were e n h a n c e d , a n d N A - d e p l e t e d b u t n o t c o n t r o l rats t e n d e d to d i s p l a y t o n i c convulsions. T h e N A - d e p l e t e d rats had significantly longer seizures ( M a n n - W h i t n e y 34 U = 4.5, P < 0.02) t h a n controls, a n d
502 TABLE III
Cocaine convulsions Seizures in response to various doses of cocaine in dorsal bundle 6-OHDA injected (NA-depleted) and nigrostriatal 6-OHDA injected (DA-depleted) rats. Values are means with standard error of the mean.
Control
Lesioned
P
60 mg/kg
(n - 5)
(n = 5)
Animals showing convulsions 70 mg/kg Animals showing convulsions Latency to first convulsion (sec) Duration of seizures (sec)
0/5 (n -- 10) 9/10 505 ± 65 35 ± 19
1/5 (n = 10) 8/10 779 ± 172 34 ± 12
NS NS NS
80 mg/kg Animals showing convulsions Latency to first convulsion (sec) Duration of seizures (sec)
(n 5) 5/5 135 -- 78 33 ± 20
(n 5) 4/5 176 ± 78 33 zk 48
NS NS NS
(n -- 5) 3/5 186 ± 104 125 ± 120
(n - 9) 8/9 257 ± 105 105 ± 20
NS NS NS
NA -depleted NS
D A -depleted 70 mg/kg Animals showing convulsions Latency to first convulsions (sec) Duration of seizures (sec)
significantly more animals in this group showed multiple seizures (Fisher's exact probability 34 = 0.03). Significantly more rats in the NA-depleted group also showed tonic extension of the forelimbs (Fisher's exact probability = 0.03) as compared to controls, who showed only clonic components. Despite a loss of striatal DA to less than 15 ~ of control, no change in Metrazol-induced seizures was seen in the DAdepleted group on any measure.
Cocaine convulsions In almost every case following a convulsion induced by cocaine the animal died within seconds of the onset of the convulsion. Duration values are thus curtailed by the onset of mortality and probably do not reflect the intensity of the seizure. No change was seen in any parameter measured after cocaine injection in either the NAdepleted or the DA-depleted groups (Table III). Electroconvulsive shock No change in the latency to onset of convulsion or the type of convulsion was seen (Table IV) but, as shown in Fig. 1, a marked increase in the duration of the convulsion occurred in the NA-depleted rats at the two lower intensities of ECS. The durations of convulsions in the NA-depleted rats differed significantly from those of their controls at the 10 mA intensity (Mann-Whitney U = 20, P < 0.05) and at the 15 mA intensity (U = 15, P < 0.01) of ECS. No alterations in any parameter of the ECSinduced convulsion was seen in the DA-depleted rats (Table IV).
503 TABLE IV Electroconvulsive shock seizures
Convulsions induced by three intensities of electroconvulsive shock (ECS) in dorsal bundle 6-OHDA injected animals (NA-depleted) and nigrostriatal bundle 6-OHDA injected (DA-depleted). Values are means with standard error of the mean. Control (n = 10)
Lesioned (n = 10)
10 mA ECS No. of animals showing tonic hindlimb extension Duration of convulsion (sec) Latency to tonic extension (sec)
4/10 23.4 ± 4.3 3.5 4- 1.1
2/10 35.4 ± 2.6 3.8 ± 0.9
NS 0.05 NS
15 mA ECS No. of animals showing tonic hindlimb extension Duration of convulsion (sec) Latency to tonic extension (sec)
(n = 10) 8/10 24.3 4- 3.4 3.7 4- 0.5
(n = 10) 7/10 35.2 4- 2.3 3.6 4- 0.6
NS NS 0.01 NS
22.5 mA ECS No. of animals showing tonic hindlimb entension Duration of convulsion (sec) Latency to tonic extension (sec)
(n = 10) 9/10 23.8 4- 3.2 3.6 4- 0.6
(n -- 7) 5/7 27.8 4- 2.2 2.6 4- 0.7
NS NS NS
10 mA ECS No. of animals showing tonic hindlimb extension Duration of convulsion (sec) Latency to tonic extension (sec)
(n = 9) 4/9 42.8 4- 5.1 6.5 4- 0.5
(n = 10) 3/10 47.7 4- 6.3 7.3 4- 1.7
NS NS NS
22.5 mA ECS No. of animals showing tonic hindlimb extension Duration of convulsion (sec) Latency to tonic extension (sec)
(n = 9) 9/9 24.1 ± 2.0 3.3 4- 0.8
(n = 10) 6/10 26.3 4- 3.3 2.5 4- 0.6
NS NS NS
NA-depleted
DA-depleted
DISCUSSION The present experiment confirms the i n h i b i t o r y role o f b r a i n catecholamines in convulsions that was suggested by previous workers18, ~7 a n d greatly extends these findings by showing that the catecholamine of i m p o r t a n c e is N A rather t h a n DA. T h u s severe depletion o f b r a i n D A to less t h a n 15 ~ o f c o n t r o l values was w i t h o u t effect o n Metrazol-induced, cocaine-induced or ECS-induced convulsions. C o n t r a s t e d to this is the m a r k e d increase in the d u r a t i o n of c o n v u l s i o n elicited by b o t h Metrazol a n d ECS in the animals with depletions o f forebrain N A . T h a t this effect occurred for b o t h d r u g - i n d u c e d a n d electrically induced seizures suggests that it is a general effect o n the susceptibility of b r a i n circuitry to seizure activity rather t h a n a change in the p e n e t r a t i o n of Metrazol into the b r a i n due to damage to the b l o o d - b r a i n barrier in the lesioned group. This is further b o r n e out by the unaltered latency to onset o f the M e t r a z o l - i n d u c e d convulsions, which should have been shortened if the lesion merely facilitated e n t r y of the d r u g into the brain. The present findings also indicate that the
504
ELECTROCONVULSIVE
SHOCK
C: C o n
40
t r o Is, n=lO
T: O B l e s i o n e d ,
n=lO
u) (J UJ
U) 3 0 Z O J ~x.x
Z 0 0
20
0 Z 0
~'~x
~XN
a
C
T
10 mA
C
T
15 mA
C
T
22.5 mA
Fig. 1. Duration of convulsion induced by ECS in control (C) and NA-depleted rats (T). Values are means, with S.E.M. indicated by vertical bars, of 10 control and 10 NA-depleted rats in seconds at three intensities of ECS. Stars indicate that the control and lesioned groups differed at the following two-tailed significance levels: * P < 0.05; ** P < 0.01.
effect is unrelated to the development of receptor supersensitivity. Metrazol has been suggested to act on N A systems in some fashion to cause its convulsant and amnestic effects 29. Our data show that Metrazol can induce seizures in the near absence of N A and suggest that this action does not require the integrity of N A systems. However, if Metrazol were a direct N A agonist a potentiation of Metrazol-induced seizures might occur as a consequence of the development of receptor supersensitivity following denervation. That a similar potentiation is seen with ECS-induced seizures makes this less likely and suggests that N A may function to reduce the susceptibility of the brain to all seizures, no matter how induced. Whether this is a specific function for which the widely distributed N A innervation developed during the course of evolution or an accidental consequence of the largely inhibitory synaptic effect of N A 33 cannot be answered from the present data. That highly complex and specialized functions ~-a5 have been ascribed to brain N A on other grounds may suggest that the effect on seizure susceptibility is largely accidental. Nonetheless, a malfunction in the NA
505 systems may play a causative role in the neuropathology of the human epileptiform disease states. The finding that cocaine, a catecholamine uptake inhibitor, can cause convulsions10,26,30,a5 was cited in the Introduction as possible evidence for a role of catecholamines in convulsions. However, the present experiment demonstrated that cocaine can induce convulsions in the near total absence of either NA or DA, suggesting that the mechanism of seizure induction does not involve reuptake inhibition and is unrelated to the catecholamine systems. Rather, it appears more likely that a general, nonspecific toxic effect is responsible for cocaine-induced convulsions. That potentiation of cocaine-induced convulsions was not seen in the NA-depleted group, as it was with Metrazol-induced and ECS-induced seizures, is most probably due to the sudden decease of animals within a few seconds of the onset of the convulsion. The high mortality with cocaine further suggests a non-specific toxic mechanism in seizure induction. Only one animal died after displaying a Metrazolinduced seizure, and no mortalities occurred following ECS convulsion. On the other hand, if an animal showed a convulsion in response to cocaine it almost invariably died thereafter; animals that survived the lower dosage (60 mg/kg) of cocaine did not show seizures. Again, this suggests a toxic mechanism of seizure induction by cocaine unrelated to its ability to block catecholamine uptake. No evidence was obtained to support an involvement of brain DA in convulsions, in contradiction to some previous indications in the literature lz,28,36. The 6-OHDA technique used here spared spinal and cerebellar NA, unlike previous techniques for 6-OHDA administration 5-7,15. Nonetheless, a marked potentiation of seizure activity was seen, highlighting the role of the NA innervation of forebrain areas such as the amygdala and hippocampus, two areas particularly susceptible to seizure activity, in the development of and susceptibility to epileptiform disruption of brain functioning. A role of brain NA in the clinical epilepsies is an intriguing suggestion meriting additional investigation. ACKNOWLEDGEMENTS Supported by grants from the Medical Research Council of Canada and the National Institute of Neurological and Communicative Diseases and Stroke (U.S.) awarded to J. A. Wada, and by a grant from the MRC awarded to H. C. Fibiger. S. T. Mason is an M R C Fellow. We gratefully acknowledge the able technical support of Betty Richter.
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506 4 Bourne, W. M., Geiger, P. F. and Jobe, P. C., Influence ofnorepinephrine and 5-hydroxytryptamine in post-decapitation convulsions in rats, Res. Commun. Psychol. Psychiat. Behav., 2 (1977) 9-19. 5 Browning, R. A. and Maynert, E. W., Increased seizure susceptability in 6-hydroxydopamine treated rats, Fed. Proc., 29 (1970) 417. 6 Corcoran, M. E., Fibiger, H. C., McGeer, E. G. and Wada, J. A., Potentiation of Leptazol seizures by 6-hydroxydopamine, J. Pharm. Pharmacol., 25 (1973) 497-499. 7 Corcoran, M. E., Fibiger, H. C., McCaughran, J. A. and Wada, J. A., Potentiation of amygdaloid kindling and Metrazol-induced seizures by 6-hydroxydopamine in rats, Exp. Neurol., 45 (1974) 118-133. 8 Crow, T. J., Catecholamine-containing neurons and electrical self-stimulation. 2. A theoretical interpretation and some psychiatric implications, Psychol. Med., 3 (1973) 1-5. 9 Crow, T. J., Catecholamine-containing neurons and electrical self-stimulation. 1. A review of some data, Psychol. Med., 2 (1972) 414-417. 10 Eidelberg, E., Lesse, H. and Gault, F. P., An experimental model of temporal lobe epilepsy : studies of the convulsant properties of cocaine. In G. H. Glaser (Ed.), EEG and Behavior, Basic Books, New York, 1973, pp. 272-283. 11 Evetts, K. D. and Iversen, L. L., Effects of protriptyline on the depletion of catecholamines induced by 6-hydroxydopamine in the brain of the rat, J. Pharm. Pharmacol., 22 (1970) 540-543. 12 Jobe, P. C., Stull, R. E. and Geiger, P. F., The relative significance of norepinephrine, dopamine and 5-hydroxytryptaminein electroshock seizure in the rat, Neuropharmacology, 13 (1974) 961-968. 13 Kety, S. S., The biogenic amines in the central nervous system: their possible roles in arousal, emotion and learning. In F. O. Schmitt (Ed.), The Neurosciences, Rockefeller University Press, New York, 1970, pp. 324-336. 14 Ktinig, J. F. and Klippel, R. A., The Rat Brain, .4 Stereotaxic Atlas, Williams and Wilkins Co., Baltimore, 1963. 15 Lehmann, A., Mechanisms underlying modifications in the severity of audiogenic convulsions, Life Sci., 20 (1977) 2047-2060. 16 Lovell, R., Some neurochemical aspects of convulsions. In A. Lajtha (Ed.), Handbook of Neurochemistry, Vol. 6, Plenum Press, New York, 1971, pp. 63-102. 17 McCaughran, J. A., Corcoran, M. E. and Wada, J. A., Anticonvulsant activity of 2x8- and A~tetrahydrocannabinol in rats, Pharmacol. Biochem. Behav., 2 (1974) 227-233. 18 McGeer, E. G. and McGeer, P. L., Catecholamine content of the spinal cord, Canad. J. Biochem., 40 (1962) 1141-1151. 19 McKenzie, G. M. and Soroko, F. E., Inhibition of the anticonvulsant activity of L-DCPA by FLA-63, a dopamine-beta-hydroxylase inhibitor, J. Pharm. PharmacoL, 25 (1973) 73-77. 20 Mason, S. T. and Corcoran, M. E., Forebrain noradrenaline and Metrazol-induced seizures, Life Sci., 23 (1978) 167-171. 21 Mason, S. T. and Fibiger, H. C., Noradrenaline and avoidance learning in the rat, Brain Research, (1978) in press. 22 Mason, S. T. and Iversen, S. D., Effects of selective forebrain noradrenaline loss on behavioral inhibition in the rat, J. camp.physiol. Psychol., 91 (1977) 165-173. 23 Mason, S. T. and Iversen, S. D., An investigation of the role of cortical and cerebellar noradrenaline in associative motor learning, Brain Research, 134 (1977) 513-527. 24 Mas•n• S. T. and •versen• S. D.• Behavi•ura• basis •f the d•rsa• bund•e extincti•n e•ect• Pharmac••. Biochem. Behav., 7 (1977) 373-379. 25 Mason, S. T. and Iversen, S. D., Reward, attention and the dorsal noradrenergic bundle, Brain Research, 150 (1978) 135-148. 26 Matsuzaki, M. and Misra,A. L., Comparison ofthe convulsant effects ofcocaine and pseudococaine in the rhesus monkey, Brain Res. Bull., 2 (1977) 417-424. 27 Maynert, E. W., The role of biochemical and neurochemical factors in the laboratory evaluation of antiepileptic drugs, Epilepsia, 10 (1969) 145-162. 28 Meldrum, B., Anlezark, G. and Trimble, M., Drugs modifying dopaminergic activity and behaviour, the EEG and epilepsy in Papio papio, Europ. J. Pharmacol., 32 (1975) 203-213. 29 Palfai, T., Kurtz, P. and Gutman, A., Effect of Metrazol on brain norepinephrine: a possible factor in amnesia produced by the drug, Pharmacol. Biochem. Behav., 2 (1974) 261-262. 30 Post, R. M., Kopanda, R. T. and Black, K. E., Progressive effects of cocaine on behavior and central amine metabolism in rhesus monkeys: relationship to kindling and psychosis, Biol. Psychiat., 11 (1976) 403-4 19.
507 31 Quattrone, A. and Samanin, R., Decreased anitconvulsantactivity of carbamazepine in 6-hydroxydopamine-treated rats, Europ. J. Pharmacol., 41 (1977) 333-336. 32 Ross, R. A. and Reis, D. J., Effects of lesions of locus coeruleus on regional distribution of dopamine-beta-hydroxylase activity in rat brain, Brain Research, 73 (1974) 161-166. 33 Segal, M. and Bloom, F. E., The action of norepinephrinein the rat hippocampus. I. Iontophoretic studies, Brain Research, 72 (1974) 79-97. 34 Siegel, S., Nonparametric Statistics, McGraw-Hill, 1956. 35 Stripling, J. S. and Ellinwood, E. H., Potentiation of the behavioral and convulsant effects of cocaine by chronic administration in the rat, Pharmacol. Biochem. Behav., 6 (1977) 571-579. 36 Stull, R. E., Jobe, P. C. and Geiger, P. F., Brain areas involved in the catecholamine mediated regulation of electroshock intensity, J. Pharm. Pharmacol., 29 (1977) 8-11. 37 Suenaga, N., Yamada, K. and Fukuda, T., Correlation between central catecholamine level and post-decapitation convulsion in rats treated with 6-hydroxydopamine, Brain Research, 122 (1977) 165-169. 38 Ungerstedt, J., Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigrostriatal dopamine system, Actaphysiol. scand., Suppl. 367 (1971) 95-122. 39 Uretsky, N. J. and Iversen, L. L., Effects of 6-hydroxydopamine on catecholamine containing neurons in the rat brain, J. Neurochem., 17 (1970) 269-278.
497
C A T E C H O L A M I N E S A N D CONVULSIONS
STEPHEN T. MASON and MICHAEL E. CORCORAN* Division of Neurological Sciences, Department of Psychiatry, University of British Columbia, Vancouver, B.C. V6T 1 W5 (Canada)
(Accepted November 16th, 1978)
SUMMARY Severe depletion of brain noradrenaline and separately of brain dopamine was induced in rats by intracerebral injection of the selective neurotoxin 6-hydroxydopamine, and the susceptibility of the treated animals to various seizure-inducing manipulations was examined. A significant potentiation of the seizures induced both by Metrazol and by electroconvulsive shock was found in animals depleted of brain noradrenaline, but no alteration was seen after depletion of brain dopamine on either measure. The catecholaminergic drug cocaine also induced seizures, but these were found not to depend on either brain noradrenaline or dopamine as they continued to occur in the virtual absence of either catecholamine. It is concluded that cocaine induces seizures by a non-specific toxic mechanism and that noradrenaline, but not dopamine, is involved in reducing the susceptibility of the central nervous system to the several distinct forms of seizure induction examined.
INTRODUCTION Considerable interest has focused on the functions of the catecholamines noradrenaline (NA) and dopamine (DA) as putative neurotransmitters in the central nervous system. Although less studied than their possible roles in reinforcement and learningS,a, 18, the participation of the catecholamines in the control of seizures has been suggestedln, 27. A number of pharmacological techniques for changing the functional activity in catecholaminergic systems appear to alter the susceptibility o f animals to seizures. Most recently the specific catecholaminergic neurotoxin 6hydroxydopamine (6-OHDA), which produces a severe and permanent physical * Present address: Department of Psychology, University of Victoria, P.O. Box 1700, Victoria, B.C. V8W 2Y2. Canada.
498 destruction of catecholaminergic neurons ~9, has been used. Thus, intraventricular injections of 6-OHDA were observed to markedly potentiate convulsions induced by pentylenetetrazol (Metrazol) 6,7. Rats treated with 6-OHDA showed convulsions of longer duration and of greater severity than controls6, 7. Since intraventricular 6O H D A depleted both NA and DA, however, it was not possible to determine which catecholamine was crucial for this effect. The relative participation of NA and DA in other forms of seizure activity is also unclear. Although NA appears to be involved in the postdecapitation reflex4,87, the picture is less certain with other forms of seizure activity because the treatments used to modify them resulted in depletion of peripheral as well as central catecholamines 3,19, in loss of NA together with DA 1,2,31, or, when only central NA was affected, in depletion of cerebellar and spinal as well as forebrain NAS,1L A few studies have also implicated dopamine independently of NA. Thus, apomorphine and piribedil reduce the susceptibility to photically induced convulsions in papio papio 28. The enhanced convulsion susceptibility induced by the catecholaminedepleting agent Ro4-1284 was reversed by intracerebroventricular infusion of dopamine 12 and this reversal was blocked by the dopamine receptor blocking agent, pimozide z~. Further evidence for a role of catecholamines in convulsions is found in the fact that the indirectly acting catecholaminergic agonist cocaine has been reported to cause seizuresl°,~6,z°, z~. The amount of cocaine required to induce seizures is generally very high, however, and the convulsant effects of cocaine could therefore be mediated by a non-specific toxic mechanism. In the present study we used localized injections of 6-OHDA into the ascending fibers of the NA systems in the mesencephalon, resulting in a selective depletion of forebrain NA and sparing DA and spinal or cerebellar NA 2°. A similar intracerebral injection of 6-OHDA into the nigrostriatal bundle was used to destroy the ascending DA systems selectively. METHODS
Surgical Dorsal bundle 6-OHDA Male albino Wistar rats (Woodlyn Farms, Ontario) weighing 300 g at the time of operation were anesthetized with Nembutal (50 mg/kg intraperitoneal) and positioned in a stereotaxic apparatus. Two holes were drilled in the skull, through which a 34gauge cannula was lowered to the following coordinates: AP + 2.6 mm from interaural line, ML ± 1.1 mm from the midline suture at bregma, and DV + 3.7 mm from the interaural line, with the animal's skull in the plane of K6nig and Klippel a4. Four micrograms of 6-hydroxydopamine (6-OHDA HBr, Regis Chemicals, weight expressed as free base) dissolved in 2/zl of 0.9 ~ saline with 0.2 mg/ml ascorbic acid antioxidant were infused bilaterally at the rate of 1 #l/min over 2 min. The cannula was left in for an extra minute to permit diffusion of the drug. Control animals
499 received infusion of an equal volume of saline-ascorbic vehicle. The skin was then sutured, and two weeks were allowed for completion of anterograde degeneration of the forebrain terminals before behavioral testing commenced 32. Nigrostriatal bundle 6-OHDA Similar injections of 6-OHDA were made bilaterally into the ascending dopamine bundle in the mesencephalon using the following coordinates: AP + 5.9 mm from interaural line, M L ~ 2.9 mm from midline suture at bregma, and DV q- 1.9 mm from interaural line. To prevent the marginal loss of NA which also occurs with this treatment the animals were pretreated with the NA-uptake inhibitor desimipramine (DMI 25 mg/kg intraperitoneally) 30 min prior to intracerebral injection 11. Controls received DMI pretreatment and saline-ascorbic infusion. Such marked loss of DA as caused by this operation is known to result in aphagia and adipsia, often leading to death if the animals are not tube fed as. Although recovery of feeding and drinking can occur after several weeks, similar recovery of other DA functions may also be argued to occur. We deemed it desirable to test the DA-depleted rats before any such recovery had taken place, these animals were thus tested 72 h after the operation. In an attempt to control for any aphagia-induced weight loss or any other change due to absence of food intake in the lesioned rats that might alter seizure susceptibility, the control and lesioned rats were deprived of all food from the time of operation until testing. Water was continuously available. Biochemical Following completion of behavioral testing animals were sacrificed by decapitation. Their brains were rapidly removed and dissected on ice into the following regions; hippocampus-cortex, hypothalamus, cerebellum, spinal cord and striatum 22. These regions were then assayed for endogenous catecholamines by the method of McGeer and McGeer 18. This served to confirm the extent and pattern of the amine depletions induced by the various 6-OHDA treatments. Behavioral Metrazol convulsions Nine NA-depleted and 7 control animals were used in one group and a further 10 DA-depleted and 10 control rats in another group. Each rat received a subcutaneous injection of 70 mg/kg of Metrazol, a dosage found in previous work to induce convulsions in approximately 90 % of normal rats6, 7. Each rat was then placed in a testing cage and observed for 1 h. The latencies to the first myoclonicjerk and the first generalized seizure were recorded as were the duration and number of seizures. The type of seizure, in terms of containing only clonic or both clonic and tonic components, was noted. Cocaine convulsions Three doses of cocaine hydrochloride were examined in NA-depleted rats. A
500 TABLE I Post-mortem amine assays
Post-mortem amine assays on dorsal bundle 6-OHDA and nigrostriatal 6-OHDA injected rats. Values are means with standard error of the mean in ng of amines per g wet weight of tissue. ~ column is the percentage of control values remaining in lesioned tissues and the P column gives the two-tailed probability comparing control and lesioned values using Student's t-test. Control (n = 10)
Lesioned (n = 10)
%
P
2 26 124 120
0.001 0.001 NS NS
Dorsal bundle 6-OHDA
Noradrenaline Hippocampus-cortex Hypothalamus Cerebellum Spinalcord Dopamine Striatum
264 2240 219 255
zL 6 ± 77 ± 32 ± 30
13100 zL 570
6 590 271 307
± ± ± ~
1 87 35 22
11600 ± 1190
88
NS
Nigrostriatal bundle 6-OHDA
Noradrenaline Hippocampus-cortex Hypothalamus Dopamine Striatum
287 i 28 1950 -- 115
263 ± 9 1850 ± 85
92 95
NS NS
9820 ± 475
1390 :~ 139
14
0.001
very steep d o s e - r e s p o n s e curve was f o u n d in pilot studies, with no effect at 60 mg/kg, m a r k e d convulsions at 70 mg/kg, a n d m a x i m a l response at 80 mg/kg. Five N A depleted a n d 5 c o n t r o l rats were used at the 60 a n d 80 m g / k g dosages a n d 10 N A depleted a n d 10 c o n t r o l rats at the i n t e r m e d i a t e dosage. Nine D A - d e p l e t e d a n d 5 c o n t r o l rats were e x a m i n e d at the 70 m g / k g dosage. The p r o c e d u r e was similar to t h a t described for M e t r a z o l - i n d u c e d seizures except t h a t the cocaine was a d m i n i s t e r e d intraperitoneally. Electroconvulsive shock ( ECS)
T e n N A - d e p l e t e d a n d 10 c o n t r o l animals were e x a m i n e d for their response to ECS. Three intensities o f ECS were a d m i n i s t e r e d in a s c e n d i n g order, each s e p a r a t e d by a 2-day period. Two a l l i g a t o r clips w r a p p e d in saline-soaked c o t t o n w o o l were a t t a c h e d to the a n i m a l ' s ears, a n d the following intensities o f ECS were a p p l i e d for 1 sec: 10 m A , 15 m A , a n d 22.5 m A . The p o w e r source for E C S has been d e s c r i b e d m o r e fully elsewhere 17. The a n i m a l was placed in a plastic cage; the clips were r e m o v e d ; a n d the resulting convulsion m e a s u r e d with r e g a r d to its latency o f onset, d u r a t i o n , a n d severity. The seizure was c h a r a c t e r i z e d in terms o f c o n t a i n i n g clonic elements alone or in c o m b i n a t i o n with tonic c o m p o n e n t s . Nine c o n t r o l rats a n d 10 D A - d e p l e t e d rats were also tested at the lowest ECS intensity a n d nine c o n t r o l a n d 10 D A - d e p l e t e d rats at the highest E C S intensity.
501 TABLE lI Metrazol seizures
Seizures induced in response to 70 mg/kg Metrazol in dorsal bundle 6-OHDA injected (NA-depleted) and nigrostriatal bundle 6-OHDA injected (DA-depleted) rats. Values are means with the standard error of the mean. Control (n = 7)
Lesioned (n = 9)
P
Latency to first myoclonicjerk (min) Latency to first seizure (min) Duration of first seizure (sec) No. of rats with multiple seizures No. of rats with tonic seizures
8.7 -4- 1.2 14.9 -4- 1.6 32.9 ± 3.0 0/7 0/7
8.2 -4- 1.4 NS 14.8 -4- 2.3 NS 113.2 -4- 11.7 0.01 5/9 0.03 5/9 0.03
D A depleted
n = 10
n = 10
Latency to first myoclonic jerk (rain) Latency to first seizure (min) Duration of seizures (sec) No. of seizures
7.6 11.6 119 2.6
N A -depleted
4- 1.5 -4- 2.6 -4- 47 -4- 0.8
7.5 10.0 248 3.5
-4- 1.3 -4- 1.8 -4- 86 -4- 1.0
NS NS NS NS
RESULTS Biochemical
T h e results o f p o s t m o r t e m a m i n e assays are shown in T a b l e I for a t y p i c a l g r o u p o f N A - d e p l e t e d rats a n d a g r o u p o f D A - d e p l e t e d rats. I n t r a c e r e b r a l injection o f 6O H D A into the ascending N A fiber-bundle resulted in a d e p l e t i o n o f h i p p o c a m p a l cortical N A to less t h a n 5 ~ o f c o n t r o l values a n d a loss o f h y p o t h a l a m i c N A to some 3 0 ~ o f n o r m a l . N o significant a l t e r a t i o n in striatal D A was p r o d u c e d as a consequence o f this t r e a t m e n t , a n d n o significant change in spinal or cerebellar N A occurred. I n t r a c e r e b r a l injection o f 6 - O H D A into the a s c e n d i n g D A fiber-bundle with D M I p r e t r e a t m e n t gave rise to a r e d u c t i o n in striatal D A to 14 ~ o f c o n t r o l values with no change in h y p o t h a l a m i c o r h i p p o c a m p a l - c o r t i c a l N A . D a t a on o t h e r animals ( M a s o n a n d Fibiger, u n p u b l i s h e d ) indicates t h a t these lesions fail to affect s e r o t o n i n , choline acetyltransferase o r glutamic acid d e c a r b o x y lase in a n y b r a i n a r e a e x a m i n e d , a n d a m o r e detailed discussion o f the specificity o f 6O H D A lesions is available elsewhere 23. Histological m i c r o g r a p h s o f the area o f 6O H D A injection have been p u b l i s h e d elsewhere zl. Metrazol-induced seizures
T a b l e II shows the effect o f these a m i n e d e p l e t i o n s on the seizures i n d u c e d b y M e t r a z o l injection. A m a r k e d p o t e n t i a t i o n o f the seizure was seen in the N A - d e p l e t e d rats. The d u r a t i o n a n d n u m b e r o f seizures were e n h a n c e d , a n d N A - d e p l e t e d b u t n o t c o n t r o l rats t e n d e d to d i s p l a y t o n i c convulsions. T h e N A - d e p l e t e d rats had significantly longer seizures ( M a n n - W h i t n e y 34 U = 4.5, P < 0.02) t h a n controls, a n d
502 TABLE III
Cocaine convulsions Seizures in response to various doses of cocaine in dorsal bundle 6-OHDA injected (NA-depleted) and nigrostriatal 6-OHDA injected (DA-depleted) rats. Values are means with standard error of the mean.
Control
Lesioned
P
60 mg/kg
(n - 5)
(n = 5)
Animals showing convulsions 70 mg/kg Animals showing convulsions Latency to first convulsion (sec) Duration of seizures (sec)
0/5 (n -- 10) 9/10 505 ± 65 35 ± 19
1/5 (n = 10) 8/10 779 ± 172 34 ± 12
NS NS NS
80 mg/kg Animals showing convulsions Latency to first convulsion (sec) Duration of seizures (sec)
(n 5) 5/5 135 -- 78 33 ± 20
(n 5) 4/5 176 ± 78 33 zk 48
NS NS NS
(n -- 5) 3/5 186 ± 104 125 ± 120
(n - 9) 8/9 257 ± 105 105 ± 20
NS NS NS
NA -depleted NS
D A -depleted 70 mg/kg Animals showing convulsions Latency to first convulsions (sec) Duration of seizures (sec)
significantly more animals in this group showed multiple seizures (Fisher's exact probability 34 = 0.03). Significantly more rats in the NA-depleted group also showed tonic extension of the forelimbs (Fisher's exact probability = 0.03) as compared to controls, who showed only clonic components. Despite a loss of striatal DA to less than 15 ~ of control, no change in Metrazol-induced seizures was seen in the DAdepleted group on any measure.
Cocaine convulsions In almost every case following a convulsion induced by cocaine the animal died within seconds of the onset of the convulsion. Duration values are thus curtailed by the onset of mortality and probably do not reflect the intensity of the seizure. No change was seen in any parameter measured after cocaine injection in either the NAdepleted or the DA-depleted groups (Table III). Electroconvulsive shock No change in the latency to onset of convulsion or the type of convulsion was seen (Table IV) but, as shown in Fig. 1, a marked increase in the duration of the convulsion occurred in the NA-depleted rats at the two lower intensities of ECS. The durations of convulsions in the NA-depleted rats differed significantly from those of their controls at the 10 mA intensity (Mann-Whitney U = 20, P < 0.05) and at the 15 mA intensity (U = 15, P < 0.01) of ECS. No alterations in any parameter of the ECSinduced convulsion was seen in the DA-depleted rats (Table IV).
503 TABLE IV Electroconvulsive shock seizures
Convulsions induced by three intensities of electroconvulsive shock (ECS) in dorsal bundle 6-OHDA injected animals (NA-depleted) and nigrostriatal bundle 6-OHDA injected (DA-depleted). Values are means with standard error of the mean. Control (n = 10)
Lesioned (n = 10)
10 mA ECS No. of animals showing tonic hindlimb extension Duration of convulsion (sec) Latency to tonic extension (sec)
4/10 23.4 ± 4.3 3.5 4- 1.1
2/10 35.4 ± 2.6 3.8 ± 0.9
NS 0.05 NS
15 mA ECS No. of animals showing tonic hindlimb extension Duration of convulsion (sec) Latency to tonic extension (sec)
(n = 10) 8/10 24.3 4- 3.4 3.7 4- 0.5
(n = 10) 7/10 35.2 4- 2.3 3.6 4- 0.6
NS NS 0.01 NS
22.5 mA ECS No. of animals showing tonic hindlimb entension Duration of convulsion (sec) Latency to tonic extension (sec)
(n = 10) 9/10 23.8 4- 3.2 3.6 4- 0.6
(n -- 7) 5/7 27.8 4- 2.2 2.6 4- 0.7
NS NS NS
10 mA ECS No. of animals showing tonic hindlimb extension Duration of convulsion (sec) Latency to tonic extension (sec)
(n = 9) 4/9 42.8 4- 5.1 6.5 4- 0.5
(n = 10) 3/10 47.7 4- 6.3 7.3 4- 1.7
NS NS NS
22.5 mA ECS No. of animals showing tonic hindlimb extension Duration of convulsion (sec) Latency to tonic extension (sec)
(n = 9) 9/9 24.1 ± 2.0 3.3 4- 0.8
(n = 10) 6/10 26.3 4- 3.3 2.5 4- 0.6
NS NS NS
NA-depleted
DA-depleted
DISCUSSION The present experiment confirms the i n h i b i t o r y role o f b r a i n catecholamines in convulsions that was suggested by previous workers18, ~7 a n d greatly extends these findings by showing that the catecholamine of i m p o r t a n c e is N A rather t h a n DA. T h u s severe depletion o f b r a i n D A to less t h a n 15 ~ o f c o n t r o l values was w i t h o u t effect o n Metrazol-induced, cocaine-induced or ECS-induced convulsions. C o n t r a s t e d to this is the m a r k e d increase in the d u r a t i o n of c o n v u l s i o n elicited by b o t h Metrazol a n d ECS in the animals with depletions o f forebrain N A . T h a t this effect occurred for b o t h d r u g - i n d u c e d a n d electrically induced seizures suggests that it is a general effect o n the susceptibility of b r a i n circuitry to seizure activity rather t h a n a change in the p e n e t r a t i o n of Metrazol into the b r a i n due to damage to the b l o o d - b r a i n barrier in the lesioned group. This is further b o r n e out by the unaltered latency to onset o f the M e t r a z o l - i n d u c e d convulsions, which should have been shortened if the lesion merely facilitated e n t r y of the d r u g into the brain. The present findings also indicate that the
504
ELECTROCONVULSIVE
SHOCK
C: C o n
40
t r o Is, n=lO
T: O B l e s i o n e d ,
n=lO
u) (J UJ
U) 3 0 Z O J ~x.x
Z 0 0
20
0 Z 0
~'~x
~XN
a
C
T
10 mA
C
T
15 mA
C
T
22.5 mA
Fig. 1. Duration of convulsion induced by ECS in control (C) and NA-depleted rats (T). Values are means, with S.E.M. indicated by vertical bars, of 10 control and 10 NA-depleted rats in seconds at three intensities of ECS. Stars indicate that the control and lesioned groups differed at the following two-tailed significance levels: * P < 0.05; ** P < 0.01.
effect is unrelated to the development of receptor supersensitivity. Metrazol has been suggested to act on N A systems in some fashion to cause its convulsant and amnestic effects 29. Our data show that Metrazol can induce seizures in the near absence of N A and suggest that this action does not require the integrity of N A systems. However, if Metrazol were a direct N A agonist a potentiation of Metrazol-induced seizures might occur as a consequence of the development of receptor supersensitivity following denervation. That a similar potentiation is seen with ECS-induced seizures makes this less likely and suggests that N A may function to reduce the susceptibility of the brain to all seizures, no matter how induced. Whether this is a specific function for which the widely distributed N A innervation developed during the course of evolution or an accidental consequence of the largely inhibitory synaptic effect of N A 33 cannot be answered from the present data. That highly complex and specialized functions ~-a5 have been ascribed to brain N A on other grounds may suggest that the effect on seizure susceptibility is largely accidental. Nonetheless, a malfunction in the NA
505 systems may play a causative role in the neuropathology of the human epileptiform disease states. The finding that cocaine, a catecholamine uptake inhibitor, can cause convulsions10,26,30,a5 was cited in the Introduction as possible evidence for a role of catecholamines in convulsions. However, the present experiment demonstrated that cocaine can induce convulsions in the near total absence of either NA or DA, suggesting that the mechanism of seizure induction does not involve reuptake inhibition and is unrelated to the catecholamine systems. Rather, it appears more likely that a general, nonspecific toxic effect is responsible for cocaine-induced convulsions. That potentiation of cocaine-induced convulsions was not seen in the NA-depleted group, as it was with Metrazol-induced and ECS-induced seizures, is most probably due to the sudden decease of animals within a few seconds of the onset of the convulsion. The high mortality with cocaine further suggests a non-specific toxic mechanism in seizure induction. Only one animal died after displaying a Metrazolinduced seizure, and no mortalities occurred following ECS convulsion. On the other hand, if an animal showed a convulsion in response to cocaine it almost invariably died thereafter; animals that survived the lower dosage (60 mg/kg) of cocaine did not show seizures. Again, this suggests a toxic mechanism of seizure induction by cocaine unrelated to its ability to block catecholamine uptake. No evidence was obtained to support an involvement of brain DA in convulsions, in contradiction to some previous indications in the literature lz,28,36. The 6-OHDA technique used here spared spinal and cerebellar NA, unlike previous techniques for 6-OHDA administration 5-7,15. Nonetheless, a marked potentiation of seizure activity was seen, highlighting the role of the NA innervation of forebrain areas such as the amygdala and hippocampus, two areas particularly susceptible to seizure activity, in the development of and susceptibility to epileptiform disruption of brain functioning. A role of brain NA in the clinical epilepsies is an intriguing suggestion meriting additional investigation. ACKNOWLEDGEMENTS Supported by grants from the Medical Research Council of Canada and the National Institute of Neurological and Communicative Diseases and Stroke (U.S.) awarded to J. A. Wada, and by a grant from the MRC awarded to H. C. Fibiger. S. T. Mason is an M R C Fellow. We gratefully acknowledge the able technical support of Betty Richter.
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