Reversible suppression of amygdaloid kindled convulsion following unilateral gabaculine injection into the substantia innominata

Brain Research, 305 (1984) 389-392 Elsevier

389

BRE 20278

Reversible suppression of amygdaloid kindled convulsion following unilateral gabaculine injection into the substantia innominata M. OKAMOTO and JUHN A. WADA Department of Neurological Sciences and Neurology, University of British Columbia, Vancouver, B. C. V6 T 2A1 (Canada)

(Accepted March 12th, 1984) Key words: kindled convulsion - - amygdala - - substantia innominata - - GABA - - gabaculine

Amygdaloid kindled generalized convulsion was reversibly blocked despite continued afterdischarge generation by amygdaloid stimulation for about 60 h following intracerebral administration of gabaculine, a GABA-transaminase inhibitor, into the substantia innominata (SI) ipsilateral to the side of amygdaloid kindling. It is suggested that the SI plays an important role in convulsive seizure generalization of amygdaloid origin. Although many studies have focused on the neuronal mechanisms underlying amygdaloid (AM) kindled seizures, anatomical substrates responsible for generalization of motor convulsion have not been defined. Kimura et al. 6 reported that among the cerebral structures semi-acutely lesioned electrolytically, the substantia innominata (SI) showed the most profound effect in diminishing the intensity of A M kindled convulsion, Based on this observation and the results of their choline acetyltransferase (CAT) histochemical study 7, Kimura et al. speculated that the cholinergic neurons in the SI which have a widespread cortical projection, play a critical role in accessing non-motor structures such as the amygdala to the motor mechanisms during A M kindling. This hypothesis had not been examined adequately due to the fact that chemical or electrolytic lesioning of the SI was associated with a high mortality rateS, 6. We now report on our observation that unilateral injection of gabaculine, a catalytic inhibitor of G A B A transaminase 12.13into the SI reversibly abolishes A M kindled convulsion in rats. Five male hooded rats of the Royal Victoria Hospital strain, weighing 280-320 g, were used. The rats were housed individually in a temperature- and lightcontrolled colony with free access to food and water,

except during experimentation. Bipolar electrodes made of twisted nichrome wire of 127/~m in diameter were implanted into the A M bilaterally and the right SI under pentobarbital (50 mg/kg, i.p.) anesthesia. A combined cannula-electrode using a No. 20 stainless steel tube insulated except at the tip, was implanted in the left SI. Two weeks following surgery, daily A M stimulation was delivered bipolarly in a 1-s train of constant current 60 Hz sine wave at an intensity of 60 ~A. The pattern of clinical seizure development was classified into 5 stages 12. For SI kindling, the threshold intensity to elicit afterdischarge ( A D ) was used for daily stimulation. The overall clinical ictal manifestations produced by SI stimulation were quite similar to those of A M kindling and details will be described elsewhere ( O k a m o t o and Wada, in prep.). All the animals were kindled initially at the left A M until a stable Stage 5 seizure was elicited for 5 successive days. Subsequently, the stimulus intensity was gradually reduced and the last intensity to induce Stage 5 seizure was designated as the generalized seizure triggering threshold (GST). Following establishment of the GST, the animals underwent further kindling at the left SI, right SI and right AM, sequentially. The number of stimulations required for the development of Stage 5 seizure was 10.5 +_ 5.1 (mean

Correspondence: J. A. Wada, Divisions of Neurological Sciences and Neurology, University of British Columbia, Vancouver, B.C. V6T 2A1, Canada.

0006-8993/84/$03.00 © 1984 Elsevier Science Publishers B.V.

390 + standard deviation) at the left AM, 1.2 + 0.4 at the left SI, 2.2 _+ 1.0 at the right S1 and 3.8 + 1.6 at the right AM, respectively. Upon completion of this sequence of kindling at different sites, the left AM was again stimulated at the GST until a Stage 5 seizure was elicited. Further effort was made to ensure that a Stage 5 seizure would be induced repeatedly with less than a 2-h interstimulus interval. Following these test stimulations, 19.2 Mg/3 #1 of gabaculine was injected through the cannula into the left S1 and the left AM was stimulated at varying intervals at the previously established GST. Gabaculine-HCl (G) was dissolved in distilled water and buffered to pH 7.2 with sodium bicarbonate. Injection of the vehicle alone produced no electroclinical change. Upon completion of the experiment, the animals were deeply anesthetized, their brains were perfused, serially sectioned and then stained by cresyl violet. Histological examination showed that all electrode tips were in the intended structures. Localization of the cannula electrodes is shown in Fig. 1. No histological changes were detected under light microscopic examination except for a mild gliosis along the electrode tracks. Behaviorally, G injection made the animals progressively less mobile without change of muscle tone. Two animals showed no further behavioral or EEG change. However, the remaining 3 animals became completely non-responsive, eyes open and immobile with loss of the rightening reflex, necessitating forced feeding beginning 2-3 days after the injection. Paralleling these behavioral changes, background EEG

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Fig. 2. Chronological change of electroclinical seizure following G injection into SI (mean clinical stage and AD duration, bars in the figure represent standard error). Note persistence of fairly long AD during complete suppression of motor seizure between 48 and 108 h.

activity was also suppressed. Stage 5 AM kindled convulsion began to be suppressed 6-20 h following G injection, and motor convulsion disappeared completely between 48 and 108 h in all the animals (Fig. 2). Despite a fairly long AD, no animal showed forelimb clonus during this period. In two animals, Stage 5 seizure reappeared between days 6 and 8, but the remaining 3 failed to show complete recovery. Throughout the course of this experiment, the ADT remained unchanged and a fairly long AD was readily induced by AM stimulation. On the other hand, the latency for the onset of Stage 3 forelimb clonus showed significant change (Fig. 3); it became

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Fig. 1. Localization of cannula electrodes in the left substantia innominata (SI). Abbreviations: CP, caudatc-putamen: GP, globus pallidus: CA, commissura anterior: AAA, anterior amygdaloid area: POA, preoptic area.

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Fig. 3. Chronological change of kindled seizure manifestations following G injection (mean latency and duration of bilateral forelimb clonus, bars in the figure represent standard errors). Note progressive prolongation and shortening of the latency to forelimb clonus prior to and following, complete seizure suppression, respectively. *Significantly prolonged compared with pre-injection value (P < 0.002).

391 extremely prolonged prior to the complete disappearance of motor convulsion and was associated with an increased AD duration. In addition, a transient and statistically non-significant increase of forelimb clonus duration was noted. When the kindled convulsion reappeared, the latency for the onset of forelimb clonus was also prolonged while duration of the forelimb clonus was short. However, the preinjection values gradually returned with seizure repetition. These changes appear significant but no statistical analysis was done due to the small number of animals involved; only one animal showed recovery to Stage 3 while two animals recovered to Stage 5 seizure state. Kindled generalized convulsion induced by AM stimulation was completely but reversibly suppressed without the blocking of AD following G injection into the ipsilateral SI. This effect could not be due to a non-specific inhibitory action on the general neuronal activity since suppression of the kindled convulsion was observed regardless of the presence or absence of an overtly apparent neurological deficit. Furthermore, the A D T remained unchanged throughout the course of this experiment in all the animals. This is in striking contrast to a marked elevation of the A D T (or GST) when G was injected into the AM (Kawahara, Seki and Wada, in prep.). Therefore, it is most unlikely that the observed effect in this study is due to diffusion of G into the AM. The chronology of the suppressive effect is considerably different from that of the GABA-elevating action of G in vitrol3. However, Gale and Iadarola 3 have shown that after inhibiting GABA-T, the onset and peak of anticonvulsant activity against maximal electroshock seizures directly parallel the time course for the increase of G A B A in nerve terminals, and that the G A B A pool associated with nerve terminals began to increase at 36 h reaching its maximum at 60 h. This chronological change roughly corresponds to the suppressive effect on kindled convulsion observed in this experiment. It is possible, therefore, the suppressive effect on AM kindled convulsion is due to the pharmacological enhancement of GABAergic transmission in the S1. The period of complete suppression of clonic convulsion was preceded and then followed by a marked prolongation of the latency for the onset of forelimb clonus which sequentially lead to kindled generalized

convulsion. This finding suggests that the G injection into the SI did not directly suppress the motor mechanism responsible for the kindled convulsion. Rather, it appears to have interfered with the mechanism of ictal linkage between the AM and the motor systems supporting the kindled convulsion. Therefore, it seems reasonable to assume that the SI by itself does not directly participate in the expression of AM kindled convulsion, but that it is critically involved in stengthening the linkage between the AM and the motor mechanism. It is not possible to explain the changes in the duration of clonic convulsion from our present data alone. However, it is possible that these changes resulted from an interaction between the motor activation mechanism responsible for Stage 5 seizure and the GABAergic inhibitory action against it. Recently, McNamara et al. l0 have demonstrated that bilateral injection of either muscimol or gammavinyl-GABA (GVG) into the midbrain tegmentum or into the substantia nigra (SN) markedly suppresses the motor manifestations of AM kindled seizures. They concluded that (a) these midbrain areas are a key site in propagation of kindled seizure activity triggered by AM stimulation, and (b) the pharmacological enhancement of GABAergic transmission in these areas is an effective method of suppressing kindled seizures. However, the finding remains controversial since Le Gai La Salle et al.8 reported that bilateral injection of G V G into the SN shortened but did not otherwise modify the motor manifestations of kindled AM seizures. Moreover, unilateral injection of the drug was not effective in suppressing kindled convulsion in either of these studies. In contrast, unilateral G injection into the SI in our study was sufficient to block the kindled convulsion completely. Furthermore, the AD duration remained largely intact despite complete suppression of the kindled convulsion. Comparable dissociation of AD and clinical seizure in AM kindled convulsion following systemic administration of gamma-acetyi-GABA has been reported ll. These findings suggest that the SI, which is known to have very high glutamic acid decarboxylase ( G A D ) activity 19, might be a key site for GABA-mediated anticonvulsive action on kindled AM seizures. In our previous studies 9.14-1s, we have shown that callosal bisection or electrolytic lesioning of the midbrain reticular formation significantly modifies the

392

pattern of kindled g e n e r a l i z e d c o n v u l s i o n , suggest-

could possibly be d i f f e r e n t f r o m that of systemic

ing the i m p o r t a n c e of these structures in c o n v u l s i v e

blocking of cholinergic transmission. In addition, it is

seizure m a n i f e s t a t i o n . In this study, unilateral G in-

possible that the inhibitory effect on k i n d l e d convul-

jection into the SI a b o l i s h e d g e n e r a l i z e d c o n v u l s i o n

sion as studied h e r e is d i f f e r e n t f r o m the inhibitory

without affecting A M excitability as j u d g e d f r o m an

effect on kindling seizure d e v e l o p m e n t which was the

intact A D T during seizure suppression. Since the SI

subject of o u r p r e v i o u s study I.

has w i d e s p r e a d c o n n e c t i o n s with the c e r e b r a l cortex,

O b v i o u s l y , f u r t h e r w o r k is n e e d e d but at this p o i n t

midbrain and o t h e r limbic areas 2.4. we suspect that

we are suggesting that (a) the S1 plays an i m p o r t a n t

the SI is primarily i n v o l v e d in the process of A M - m o -

role in A M kindling by linking the A M with the m o t o r

tor linkage which m a y be necessary for convulsive

m e c h a n i s m s r e s p o n s i b l e for k i n d l e d c o n v u l s i o n , and

seizure

generalization

while

the

cerebral

cortex

and/or m i d b r a i n is s e c o n d a r i l y i n v o l v e d in the ex-

(b) G A B A - t e r m i n a l s in the SI h a v e an inhibitory action on this linkage.

pression of c o n v u l s i v e seizure. This a s s u m p t i o n is not inconsistent with o u r p r e v i o u s study~ in which atropine failed to retard A M kindling b e c a u s e it is con-

This r e s e a r c h was s u p p o r t e d by a g r a n t f r o m the Medical R e s e a r c h C o u n c i l of C a n a d a .

ceivable that the effect of local cholinergic inhibition

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Presented at annual meeting of American Epilepsy Society, Phoenix, Arizona, November, 1982. 11 Myslobodsky, M. S. and Valenstein, E. S., Amygdaloid kindling and the GABA system, Epilepsia, 21 (1980) 163-175. 12 Racine, R. J., Modification of seizure activity by electrical stimulation: II. Motor seizure, Electroenceph. clin. Neurophysiol., 32 (1972) 281-294. 13 Rando, R. R., Specific inhibitors of GABA metabolism. In P. Krogsgaad, J. Scheel-Kruger, and H. Kofod (Eds.), GABA Neurotransmitters, Munksgaard, Copenhagen, 1979, pp. 229-235. 14 Wada, J. A. and Komai, S., Effect of anterior 2/3 callosal bisection upon amygdaloid kindled primarily generalized convulsions in epileptic baboons, Papio papio. In A. G. Reeves (Ed.), Epilepsy and the Corpus Callosum, Plenum Press, New York, in press. 15 Wada, J. A. and Mizoguchi, T., Limbic kindling in the forebrain bisected photosensitive baboon, Papio papio, Epilepsia, in press. 16 Wada, J. A., Nakashima, I". and Kaneko, Y., Forebrain bisection and feline amygdaloid kindling, Epilepsia, 23 (1982) 521-530. 17 Wada, J. A. and Sato, M., Generalized convulsive seizure state induced by daily electrical stimulation of the amygdala in split brain cats, Epilepsia, 16 (1975) 417-430. 18 Wada, J. A. and Sato, M., Effects of unilateral lesion in the midbrain reticular formation on kindled amygdaloid convulsion in cats. Epilepsia. 16 (1975) 693-697. 19 Walaas, I. and Fonnum, F., The distribution of putative monoamine, GABA, acetylcholine and glutamate fibers in the mesolimbic system. In P. Krogsgaad, J. Scheel-Kruger and H. Kofod (Eds.), GA BA-Neurotransminers, Munksgaard, Copenhagen, 1979, pp. 61-73.