DEVELOPMENTAL BRAIN

RES~RCH

ELSEVIER Developmental Brain Research 79 (1994) 297-300 . . . . . . . . . . . .

Short Communication

Further evidence of involvement of substantia nigra GABA B receptors in seizure suppression in developing rats

Jana Veli~kovfi a,d,*, Douglas S. Garant a,d, Shu G. Xu a,d, Solomon L. Mosh6 a.b.c.d a Departments of Neurology, b Neuroscience and c Pediatrics, and d The Montefiore / Einstein Epilepsy Management Center ,l

Albert Einstein College of Medicine. 1410 Pelham Parkway South, Bronx. NY 104 6L USA

Accepted 8 February 1994

Abstract

To determine whether the substantia nigra GABAergic anticonvulsant effects depend on GABA a receptor activation, we tested the effects of intranigral CGP 35 348 (a GABA a receptor antagonist) alone or in combination with gamma-vinyl-GABA (GVG; a GABA-transaminase inhibitor) on flurothyl seizures in rat pups and adult rats. In pups, nigral infusions of CGP 35 348 decreased the thresholds for clonic and tonic seizures and attenuated the anticonvulsant effects of GVG. In adults, nigral infusions of CGP 35 348 did not alter seizure thresholds. The data suggest that, in rat pups, nigrai GABAB receptors regulate, in part, flurothyl-induced seizures.

Key words: y-Aminobutyric acid receptor; Substantia nigra; Seizures; y-Aminobutyric acid; Rat; Development

Epileptic seizures occur more frequently in children than in adults [5]. Experimental studies have also shown that immature animals are more susceptible to seizures than mature animals and the expression of seizures is age-dependent [1,8,10,19,20]. There is evidence that seizure activity can be controlled by GABA receptors. The substantia nigra (SN) has been identified as one of the critical sites for GABA-mediated anticonvulsant activity [2,6]. In adult rats, bilateral infusions of gamma-vinyl-GABA (GVG), a GABA-transaminase inhibitor, into the substantia nigra pars retieutaris (SNR) protected the rats against both clonic and tonic seizures induced by maximal electroshock, kindling or chemical-induced seizures including flurothyMnduced seizures [6,9,18,21]. In rat pups, intranigral GVG infu- sions also had anticonvulsant effects against flurothyl- induced seizures [23]. G V G increases the levels of endogenous GABA, thereby affecting both GABA A and GABA a receptors. Muscimol (both a high- and low-affinity GABA A receptor agonist) and baclofen (a GABA B receptor agonis0 were used to demonstrate the role of the respective binding sites in the SNR in

* Corresponding author. Fax: (1) (718) 824 3058; Internet: velisek@aecom.yu.edu.

0165-3806/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD! 0t 65-3806(94)00026-V

the flurothyl model of seizures. In adult rats, intrani- gral infusions of muscimol were antieonvulsant [11,17], however, similar doses were proconvulsant in rat pups [15,16]. In contrast, baclofen exerted n o effects in adult rats [17], but was anticonvulsant in rat. pups [16]. Previ- ous binding experiments from our laboratory have demonstrated the anatomically selective overproduc- tion of GABA a receptors in the immature SN [4]. We tested the hypothesis that the nigral G#~BA B receptors may be involved in seizure expression in rat pups but not in adults by using a new selective GABA a receptor antagonist CGP 35 348 [12].

We used male Sprague-Dawley rats from Taconic Farm at age 15 days (pups; weight: 28-32 g) and adults 60 days (adults; weight: 200-225 g). The day of birth was counted as day 0 (PN). The pups were operated under ketamine and xylazine anesthesia (70 m g / k g and 10 mg/kg, i.p.) at PN 13; the adults at PN 58. Stain- less-steel cannulae (C315G, Plastics One) were stereo- taxicalty implanted into both SNR. For the pups we used the verified coordinates from our previous study: posterior = 5.1 mm, laterally = 3.5 mm, with reference to bregma; depth from the skull = 5.0 mm. For the adults, the coordinates were: posterior --- 5.3 ram, later- ally = 4.0 mm, with reference to bregrna; depth from the skull = 7.8 ram. The cannulae in both age groups

298 J. Veli'~koc~ et al./Decelopmental Brain Research 79 (1994) 297 300

were angled at 15 ° laterally and the incisor bar was set at - 3 . 5 mm [21,23]. The cannulae were fixed to the skull with dental acrylic cement and stainless-steel screws. The pups were then returned to their respec- tive dam to recuperate. The adults were housed 2 -3 per cage with free access to food and water.

G V G and CGP 35 348 were dissolved in saline. In the pups, G V G (10 /zg /0 .25 /z l per site) or saline (0.25 /~1 per site) were simultaneously infused into both SNR on the first postoperation day (PN 14). All infusions were performed over a 2-rain period and the internal cannulae were left in the guide cannulae for an addi- tional 1 min. On PN 15, the pretreated pups were infused bilaterally with CGP 35 348 (2 /~g/0.25 p.1 per site) or saline over a 2-min period. The pups were challenged with flurothyl on PN 15, 30 rain after the saline or CGP 35 348 infusions. Flurothyl is a volatile convulsant liquid [13,14]. When flurothyl is delivered to an air-tight chamber, it evaporates, enters the body by the respiratory system and induces seizures. The ad- vantage of flurothyl is that seizures will eventually occur, if flurothyl is infused for a sufficient time into the chamber. We measured the latency to the onset of clonic and tonic seizures. Changes in latencies repre- sent changes in the amount of flurothyl necessary to evoke a seizure. Thus, the flurothyl threshold for clonic and tonic seizures can be calculated by multiplying the latency (from the beginning of infusions to seizure onset) with the infusion rate and expressed as the amount of flurothyl (pA) used to induce clonic and tonic seizures, respectively (latency x rate of infusion).

Adult rats were infused with CGP 35 348 (2/~g/0 .25 /~1 per site over a 2-rain period) or saline and tested 30 rain later. Upon completion of the experiments, the cannulae placements were histologically verified. Only data obtained at sites located in the SNR bilaterally were included in the statistical evaluation (Fig. 1).

In the pups we used one-way A N O V A with post-hoc comparisons (Fisher PLSD) to compare the thresholds for clonic and tonic seizures. In adult rats, the un- paired Student 's t-test was used. All the experimental values shown are mean ± S.E.M. Significance was ac- cepted at P < 0.05.

In the control pups (infused with saline; n = 15), the threshold for ctonic seizures was 161 +4 .4 p.l of flurothyl. Tonic seizures followed almost instantly with an average threshold 167 ± 5.1 p,1 of flurothyl. There were significant effects of the intranigral infusions of the drugs on flurothyl thresholds for both clonic (Fx34 = 20.18; P = 0.0001) and tonic (F3.34 = 26.63; P = 0.0001) seizures. Post-hoc tests showed that bilateral infusions of GVG alone increased significantly the thresholds for both clonic (214 ± 15.2 p.l) and tonic (250 ± 14.4 p.I) seizures (n = 6). Thus, clonic and tonic seizures were separated in G V G infused pups. CGP 35 348 was proconvulsant in both clonic (103 +- 8.8 /zl)

ADULT PUP

, . . . . . ... ~ . . k ~ L ' . . )

Fig. t. Locations of the cannula tips in the substantia nigra of adult rats (left) and of the 15-day-old pups (right). AP coordinate 1950/zm according to K6nig and Klippel [7] which corresponds to AP coordi- nate 5.3 mm from bregma in adult rats and 5.1 mm from bregma in 15-day-old pups. Although some cannula tips were slightly more frontal or more posterior to the plane of the figure, we are present- ing the plane with an average AP coordinate.

and tonic (118 +_ 9.0/~1) seizures (n = 9). In pups with combined intranigral infusions of G V G and CGP 35 348 (n = 8), CGP 35 348 abolished completely the anticonvulsant effect of G V G on clonic seizures (175 ± 13.9 p.1). In this group of pups, CGP 35 348 signifi- cantly decreased the tonic seizure threshold, however this newly established tonic seizure threshold (195 ± 14.1 /~1) was still significantly higher than that in con- trols (Fig. 2).

In adult rats, we tested the action of CGP 35 348. We did not find any significant effects on either type of flurothyl-induced seizures (control clonic seizure threshold 178 +- 21.9/xl, control tonic seizure threshold 311+_21.8 /~1, n = 11; CGP 35348 clonic seizure threshold 136_+8.1 /zl, CGP 35348 tonic seizure threshold 269 +_ 24.1 /~1, n = 8). Thus in adult rats, the nigral GABA~ system is probably not involved in flurothyl-induced seizures. Therefore, we did not per- form the experiments with G V G and G V G / C G P 35 348 treatments.

Our data provide additional evidence of the role of the nigral GABA system and especially GABA B recep- tors in the control of seizure in developing rats. In a recent paper, it was reported that nigral G V G infu- sions protected developing rats only against tonic seizures [23]. In the present study, however, we found that G V G increased the threshold for both clonic and tonic seizures, although the increase was larger for tonic seizures. This is also evident from the separation of the latencies of onset of clonic and tonic seizures. The difference between the two studies might be due to the smaller number of rats used in the previous study.

In the pups, the action of CGP 35 348 was procon- vulsant. A previous study showed, that in rat pups,

I. Veli'~kot'6 et al. / Det'elopmental Brain Research 79 (1994) 297-300 2tI ()

300-

250 -

200-

150-

[ ] Controls [ ] CGP353482~tg

[ ] G V G I 0 ~ g [ ] GVG+CGP35348

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elonic seizures tonic seizures

Fig. 2. Thresholds of flurothyl-induced clonic (left) and tonic (right) seizures in 15-day-old rat pups expressed in/zl of flurothyl (Mean _+ S.E.M.) Groups: controls infused bilaterally with saline/saline, 24 h apart (n = 15); GVG/saline 10/~g, a group with bilateral infusions of 10 /~g of GVG per site followed by saline the second (n = 6); saline/CGP 35 348 2/xg, a group initally infused with saline and the second day with 2 /xg of GABA B antagonist CGP 35 348 per site (n = 9); GVG and CGP 35 348, a group pretreated with GVG 10/zg followed by 2 /zg of CGP 35 348 24 h later (n = 8). * denotes a significantly different threshold compared to the control group (P < 0.05: one-way ANOVA with post-hoc Fisher PLSD test).

in t ran igra l bac to fen was an t i convu l san t aga ins t f lurothyl- induced seizures suggesting that G A B A B re- ceptors may have a crucial role in seizure control at this age [16]. Therefore , we expected that the nigral infusions of the G A B A B antagonist C G P 35 348 to be proconvulsant . Indeed, our results concurred with our predict ion; fu r the rmore C G P 35 348 completely abol- ished the ant iconvulsant act ion o f G V G against clonic seizures. This suggests that intranigral G A B A B recep- tors play an impor tant role in clonic flurothyl seizures in rat pups. In contrast , C G P 35 348 only partially abolished the ant iconvulsant effect of G V G in tonic seizures. This suggests that in tonic seizures the anti- convulsant act ion of G V G partially involves G A B A B

receptors. It may also involve the nigral G A B A A sys- tem [23]. Thus, nigral infusions of bicuculline at a dose that can facilitate both clonic and tonic flurothyl seizures [15], can only block the ant iconvulsant action of G V G in tonic seizures [23].

C G P 35 348 can ei ther block postsynaptic G A B A B sites coupled by G-prote ins to K* channels or block presynapt ic G A B A B receptors that may result in an increased release of endogenous G A B A from synaptic terminals [3], and thus increased activation of postsy- napt ic G A B A A receptors (in addit ion to GABAB) . In rat pups, nigral C G P 35 348 is proconvulsant p e r se,

and (partially) antagonizes the act ion o f G V G . This effect of C G P 35 348 is probably domina ted by postsy- naptic G A B A B blockade. This is consistent with the ant iconvulsant action o f nigral baclofen in rat pups [16]. The proconvulsant effects o f C G P 35 348 cannot

be explained by its presynapt ic action which should be additive to the anticonvulsant action of GVG. In the pups, a similar proconvulsant effect was observed after intranigral infusion of the G A B A A agonist muscimol [15,16]. Both C G P 35 348 and muscimot are proconvul- sant and antagonize G V G but nei ther converts G V G to a proconvulsant t reatment . This suggests that in the pups, nigral G V G in the presence of C G P 35 348 (a si tuation during which there should be st imulation o f only G A B A A receptors by endogenous G A B A ) is not proconvulsant like muscimol or another G A B A A ago- nist T H I P [15,16,22]. This supports our hypothesis (Garan t et al., in prep.) that in the pups, muscimol and T H I P have different actions on the G A B A A receptors than does G A B A .

In the SN in adult rats, C G P 35 348 has no signifi- cant effects on f lurothyl- induced seizures. Several fac- tors may explain this observation. First, with matura- t ion the density o f nigral G A B A B receptors decreases [4]. Second, activation of nei ther pre- nor postsynaptic G A B A B receptors in the adult SN by endogenous G A B A , has any significant influence on flurothyl-in- duced seizures. This is consistent with the ineffective- ness of baclofen in adult SN [17]. Finally, there may be ba lanced compet i t ion between post- and presynaptic G A B A B mechanisms. Al though unlikely, it is possible that postsynaptic actions of baclofen and C G P 35 348 were exactly counterac ted by their presynaptic actions affecting G A B A release.

Suppor ted by N I H Gran t NS-20253 and N R S A train- ing grant NS-07183 f rom the NINDS. G V G was a generous gift f rom Mar ion-Merre l l -Dow Research (Kansas City, MO, U S A ) and C G P 35 348 a generous gift f rom Ciba-Geigy (Basel. Switzerland).

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