Nigral muscimol infusions facilitate the development of seizures in immature rats

Developmental Brain Research, 13 (1984) 305-308

305

Elsevier BRD 60(O9

Short Communications

Nigral muscimol infusions facilitate the development of seizures in immature rats SOLOMON L. MOSHI53,2 and BRUCE J. ALBALA1 tSaul R. Korey Department of Neurology, and 2Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park A venue, Bronx, NY 10461 ( U.S. A.)

(Accepted December 20th, 1983) Key words: flurothyl - - seizures - - epilepsy - - animal suckling - - substantia nigra - - GABA agonists - - rotational behavior-- rats

Since recent data utilizing GABAergic stimulation of the substantia nigra (SN) suggest that the SN is a crucial site in a circuitry involved in the modification of seizures in adult rats, the role of the SN was investigated in seizures of rat pups. Bilateral nigral infusions of the GABA agonist muscimol partially protected adult rats against flurothyl-induced seizures, while similar infusions actually facilitated the development of flurothyl seizures in 15-day-old rat pups, These results suggest that age-related differences in the nigral GABA sensitive system may account for the increased susceptibility to generalized seizures of the developing brain. There is evidence that in adult animals epileptiform electrical activity propagates through the basal ganglia and most notably through the substantia nigra (SN) 24,26. In addition, deoxyglucose ( D G ) autoradiographic studies of convulsing adult animals have disclosed that the SN is metabolically active during generalized seizures induced by kindling of the amygdala or by systemic injections of kainic acid, bicuculline or pentylenetetrazolS,7,17. This increase in the metabolic activity of the SN does not appear to be related to the motoric p h e n o m e n a of the seizure per se, since similar increases in the metabolic activity of the SN occur in anesthetized rats with penicillin-induced amygdaloid electrographic seizures 27. Recent data in adult animals suggest that the SN may be a crucial site in a circuitry involved in the modification of seizures in the mature CNS. Thus bilateral ablation of the SN has been reported to modulate the severity of experimentally induced seizures although the direction of this modification (i.e. protection against seizures9, tl vs facilitation of seizures 4,9,15)varied among different investigators. A m ato et al. reported that unilateral electrical stimula-

tion of the SN in cats inhibited focal paroxysmal activity of the ipsilateral ventrobasal amygdala 4. Iadarola and Gale reported that bilateral infusions of muscimol (a G A B A agonist) and gamma-vinyl G A B A (an irreversible inhibitor of G A B A transaminase and therefore an indirect G A B A agonist) in the vicinity of the SN protected rats against seizures produced by maximal electroshock, bicuculline or pentylenetetrazo112,13. These authors postulated that the G A B A e r g i c synapses in the vicinity of the SN may be critically involved in the control of seizure propagation. This hypothesis is supported by the data of Nitsch and Okada who reported differential d e creases of G A B A in the SN of rabbits during the preictal period of methoxypiridoxine induced seizures23. More recently the severity and/or the duration of generalized kindled seizures in adult rats were suppressed following bilateral microinfusions of G A B A agonists in the vicinity of the SY 16,19. We have begun investigating the role of the SN in order to clarify why immature animals have an increased susceptibility to seizures 3,2°-22. Our data with D G autoradiography demonstrated a striking age-re-

Correspondence: S. L. Moshe, Saul R. Korey Department of Neurology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A.

0165-3806/84/$03.00 © 1984 Elsevier Science Publishers B.V.

306 lated difference in the metabolic pattern of the SN during generalized seizures induced either by kindling~ or by systemic administration of kainic acid:t With either type of induced seizures, there were no changes in the D G uptake in the SN of rat pups. In contrast, there were m a r k e d increases in the D G uptake within the SN of the adult rats 1,3-s-7 ~'r Since in adult rats the nigral effects on seizures a p p e a r to be m e d i a t e d by a G A B A - s e n s i t i v e system 12,l~.1'~-I~ the present study reports the effects of nigral G A B A e r gic stimulation on behavior and seizures in immature rats. Initially the effects of unilateral nigral muscimol infusions on behavior were d e t e r m i n e d in 3 adult and 3 15-day-old rats. Acute unilateral nigral muscimol infusions were m a d e under halothane anesthesia. A 1 !d Hamilton syringe was stereotaxically lowered through a trephine hole using the following coordinates (from B r e g m a reference): adult A P = ~ / . 5 cm, lateral = - - 0 . 1 7 cm, depth = 0.87 cm; pup A P 0.5 cm, lateral = - - 0 . 1 7 cm, depth = 0.71 cm. One hundred ng of muscimol (total volume 11.5 el) were infused over a 5 rain period to minimize drug spread. Two minutes after the completion of the infusion, the syringe was withdrawn, the r a f s incision sutured and the anesthesia was removed. Each rat was observed for 5 h. Minimal behavioral differences were observed between the rat pups and the adult rats. All the animals begun standing and ambulating a p p r o x i m a t e l y 15 min after removal of the halothane anesthesia. A t about 30 min, turning, contralateral to the injected site, was observed in all rats. This turning was the p r e d o m i n a n t behavioral response although the rats displayed other stereotypic responses such as sniffing, purposeless gnawing or chewing, head tremors and tilting of the head towards the uninjected side. Due to anesthetic aftereffects, the turning behavior was at first sporadic, but by 1 h postinjection, the rats were uniformly turning contraversively. The turning consisted of the rat making very tight circles with most of the m o v e m e n t occurring in the forelimbs and head. Often, there was virtually no m o v e m e n t in the hindlimbs. This turning persisted for periods of 10-15 rain at 1-2 h postinjection; it occurred in sporadic 2-5 rain bursts thereafter and was rarely seen after 4 h. The only difference between the rat pups and adult rats was the intensity of the turning. A d u l t rats often -

had 2(~-30 r p m , w h c r c a ~, rat pups rar~,i,~ :xc~'cdc,,i 10-15 r p m .

Twenty-four hours after the musdm~t ~ni~i~)~. the rats were observed again to assess whel her ~heru were any long-lasting or p e r m a n e n t changes which might have been the result of an artificial lesion rhc rats exhibited normal behavior lor thci~ rcspcctwc ages with no compulsive turning or stcreotypics S~t;sequently, the rats were killed, the brains removed and sectioned. The tip of the injecting needle w:l~ found to be in the rostromedial SN m all the r;~ts These results suggest that the G A B A - s e n s i t i x c mgral output system which can induce c:,mtr:d~tc~al turning and stereotypies is mature or at least drivable in rat pups. Since it hats been p r o p o s e d lha! ;tculc muscimol-elicited contraversivc turning i.~ ~e[ated to dopaminergic nigrostriatal stimulation in adult r,als j:~, the present findings suggest that the nigrostriataf pathway is functional in the rat pup. Fhis hypothesis is consistent with the l i t e r a t u r d '.~. The difference m the intensity of turning m the tx~o age group~ was probably due to the incomplete myclination of the motor systems -',~:. In the second experiment the effects of acute hilateral nigral infusions of muscimol on seizures were studied in adult rats and rat pups. The infusion procedures were similar to those described above except that the infusions were made bilaterally, the ,ccond immediately after the first. Since the initial muscimol behavioral study d e m o n s t r a t e d that the effects of halothane had dissipated 1 h after the intusion and at the same time there is a peak of muscimol-elicited turning and stereotypies, the seizure susceptibility ot each rat to flurothyl convulsions was assessed t h postinfusion. Tbe latency to the onset of a ~.cncralized clonic-tonic convulsion with a concurrent ](~ss of posture was taken as the convulsant end-point -':'. Some of the rats were re-exposed to flurothy] 24 h tater at which time any of the previous muscimot effects should have dissipated. The rats were then killed and histological verification was obtained, with the tips of the needle tracts found to be at the rostromediat SN. All the rats in both age groups rejected with nmscimol exhibited stcreotypies. M o r e o v e r bilateral infusions of muscimol in or adjacent to the SN afforded significant protection against flurothvl seizures in the adult rats. The mean latency (~ ± S.E.) to the onset of seizures in the muscimol group (n = 4) was 14.~) _+

307 TABLE I Effects o f bilateral nigral muscimol infusions (100 ng in 0.5 ld) on the latency to flurothyl induced seizures in rat pups

The latency values are means _+S.E. 1 h, 1 h after the infusions; 24 h, 24 h after the infusions, * P < 0.01 from the 3 control groups tested at 1 h; ** P < 0.01 from rats infused with muscitool and retested at 24 h (t-test, see text for details). 1h n

Muscimol Saline Sham Naive

24h Latency

7 7.0 + 0.4min*.** 4 9.5 _+0.8 min 3 10.2 _+0.1 min 7 8.8 + 0.4 min F(3,17) = 7, P < 0.01

n

Latency

6 3

10.0 + 0.7min 10.8 _+0.3 min

3 10.2 + 1.4 rain F(2,9) = 0.26, P > 0.5

1.1 min as c o m p a r e d to a mean latency of 8.1 _+ 0.5 min in the naive rats (n = 3, t-test = 5.13, P < 0.01). That this protection was the result of the specific action of muscimol as o p p o s e d to some sort of irreversible lesion is evidenced by the 24 h retest data for this group of rats. A t 24 h, the previously t r e a t e d animals had shorter latencies to seizures (9.3 ± 0.6 min) as c o m p a r e d to the day before (t-test = 4.4, P < 0.01). F u r t h e r m o r e , at 24 h, the muscimol-injected rats did not differ from the controls (muscimol = 9.3 + 0.6 min, naive = 9.3 + 0.3 min). These results suggest that nigral muscimol infusions can partially protect adult rats against flurothyl seizures and are consistent with the recent literature 12,13,16,19. In contrast, bilateral muscimol infusions in or adjacent to the SN did not protect the rat pups against flurothyl seizures (Table I). The muscimol-treated rats (n = 7) had significantly shorter latencies (7.0 _ 0.4 rain) than the saline-injected (n -- 4, 9.5 + 0.8 rain), sham (n = 3, 10.2 _+ 0.1 min) and naive rats (n = 7, 8.8 + 0.4 min; F (3,17) = 7.0, P < 0.01). The 3 control groups did not statistically differ from each other. F u r t h e r m o r e 24 h later, the 6 m u s c i m o M n j e c t e d rats that were re-exposed to flurothyl exhibited longer latencies (10 _+ 0.7 min) than the day before (6.8 + 0.4 min, t-test = 3.97, P < 0.01; one rat was not retested) and did not differ from the saline and naive controls ( F (2,9) = 0.26, P > 0.5). T h e r e f o r e bilateral nigral muscimol infusions actually facilitated the d e v e l o p m e n t of flurothyl-induced seizures in immature rats. O u r previous studies utilizing electrical kindling of the amygdala20-22 or kainic acid-induced seizures 3 indicate that rat pups are more susceptible to the devel-

o p m e n t of generalized seizures and m o r e prone to the d e v e l o p m e n t of status epilepticus than adult rats. In addition, deoxyglucose autoradiographic studies of convulsing 15-16-day-old rat pups revealed that there were no changes in the deoxyglucose uptake in the SN 1,3 in contrast to the m a r k e d increases observed under similar conditions in adult ratsl,3,5,7,17. If the lack of increased SN metabolism during seizures in rat pups were due to a functional immaturity of the SN afferents it would have been anticipated that muscimol infusions into the SN would have either averted or attenuated the ensuing seizures as in the adults as long as the SN efferent systems were functional. H o w e v e r our results indicate that bilateral infusions of muscimol into the vicinity of the SN facilitate flurothyMnduced seizures in rat pups while similar infusions partially p r o t e c t e d adult rats against the same seizures. The data then suggest that the G A B A - s e n s i t i v e nigral efferent system which results in suppression or at least attenuation of seizures in adult rats is altered in rat pups. Such d e v e l o p m e n t a l changes in the functional activity of the SN are plausible and indeed Fisher et al. have r e p o r t e d that 'the initial responses of [SN] neurons to caudate and cortical stimulation changed with age with excitatory and inhibitory responses in kittens vs almost entirely inhibiting responses in adults 'lo. We therefore propose that the increased susceptibility of the developing brain to generalized seizures may be due in part to age-related differences in the functional activity of the SN and its G A B A - s e n s i t i v e efferent system. F u r t h e r m o r e , our results indicate that this G A B A - s e n s i t i v e nigral system is different from the G A B A - s e n s i t i v e nigral system that mediates stereotypy and turning (nigrostriataliS) since the latter is mature6,8 or at least drivable in rat pups. Delineation of the neural mechanisms responsible for seizure suppression is important for the development of novel therapeutic approaches which must also take into account the maturational stage of the CNS, thereby achieving better control of age specific seizure disorders. This investigation was s u p p o r t e d by a Teacher-Investigator D e v e l o p m e n t A w a r d NS 00482 from the N I N C D S (S.L.M.). W e wish to thank Ms. Lola L e o n for her expert technical assistance and Ms. T. Castracan for her help in the p r e p a r a t i o n of this manuscript.

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