Epilepsy Research 58 (2004) 155–165

Behavioral effects of bicuculline microinjection in the dorsalversus ventral hippocampal formation of rats, and

control of seizures by nigral muscimol

Marcelo Cairrão Araujo Rodriguesa,1, Rene de Oliveira Belebonib,Joaquim Coutinho-Nettob, Wagner Ferreira dos Santosa,

Norberto Garcia-Cairascoc,∗a Laboratório de Neurobiologia e Peçonhas, Departamento de Biologia, Faculdade de Filosofia,

Ciencias e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazilb Laboratório de Neuroqu´ımica, Departamento de Bioqu´ımica, Faculdade de Medicina de Ribeirão Preto,

Universidade de São Paulo, Ribeirão Preto, Brazilc Laboratório de Neurofisiologia e Neuroetologia Experimental, Departamento de Fisiologia,

Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes,3900 Ribeirão Preto, SP 14049-900, Brazil

Received 3 November 2003; received in revised form 4 February 2004; accepted 5 February 2004

Abstract

This work aims to describe behavioral/electroencephalographic (EEG) seizures induced by bicuculline microinjection intrac-erebroventricularly (ICV) and in the dorsal hippocampal formation (DHF) or ventral hippocampal formation/amygdala area(VHF-AMY). We also test if GABAergic manipulation in the substantia nigra pars reticulata (SNPR) is capable of controllingthose seizures. ICV injection of bicuculline induced a progressive sequence of convulsive responses, jumps and escapes fromthe open-field. This effect was partially reached by bicuculline injection in the DHF or VHF-AMY injection. Also: muscimolinjection, but not GABA uptake blockers (nipecotic acid or a spider venom neurotoxin FrPbA2), into the SNPR abolished seizures

Abbreviations:AMY, amygdala; ICV, intracerebroventricular (ly); DHF, dorsal hippocampal formation; DH, dorsal hippocampus; VHF,ventral hippocampal formation; VHF-AMY, ventral hippocampal formation/amygdala area; SNPR, substantia nigra pars reticulata; FrPbA2,venom fraction derived from the spiderParawixia bistriata(GABA uptake inhibitor); TLE, temporal lobe epilepsy; GABA,�-aminobutyric acid;GABAA, GABA receptor type A; AU, arbitrary units (for quantification of the FrPbA2); EEG, electroencephalographic; AP, antero-posterior;ML, meso-lateral; DV, dorso-ventral; WDS, wet dog shakes; TC, tonic–clonic seizures; WR, wild running; PBS, phosphate-buffered saline;AHiPM, amygdalo-hippocampal area posteromedial part; DG, dentate gyrus; PMCo, posteromedial cortical amygdaloid nucleus; LM, localizedmyoclonus; WAR, Wistar Audiogenic Rats; SNPRanterior, substantia nigra pars reticulata, anterior portion; SNPRposterior, substantia nigra parsreticulata, posterior portion

∗ Corresponding author. Tel.:+55-16-6023023; fax:+55-16-6330017.E-mail address:ngcairas@fmrp.usp.br (N. Garcia-Cairasco).1 Present address: Laboratorio de Neurofisiologia e Neuroetologia Experimental, Departamento de Fisiologia, Faculdade de Medicina de

Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.

0920-1211/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.eplepsyres.2004.02.001

156 M. Cairrão Araujo Rodrigues et al. / Epilepsy Research 58 (2004) 155–165

induced by bicuculline injection in the DHF. It was concluded that different neuronal circuitry in the hippocampal formation aremodulated, at least partially by nigral GABAergic mechanisms.© 2004 Elsevier B.V. All rights reserved.

Keywords:Hippocampal formation; GABAA receptor; Epilepsy; Substantia nigra; Video-EEG;Parawixia bistriata; Spider venom

1. Introduction

Animal models of induced epilepsy are consideredvery important to study, among others, the temporallobe epilepsy (TLE) in humans. TLE is the mostcommon form of epilepsy in humans (for review,seeEngel, 1996), and the hippocampus and amyg-dala (AMY) are temporal structures closely relatedto epileptogenicity (Ben-Ari, 1985; Engel, 1996).Furthermore, literature has pointed that the ventralhippocampal formation (VHF) is more excitable andepileptogenic than the dorsal hippocampal forma-tion (DHF) (Racine et al., 1977; Gilbert et al., 1985;Becker et al., 1997; Akaike et al., 2001). A classicconvulsant employed in experimental epilepsy is bicu-culline (Curtis et al., 1970), especially in systemic orin vitro protocols due its putative GABAA blockade(Macdonald and Olsen, 1994).

In the search for anticonvulsant therapies, re-searchers have addressed the possible existence ofendogenous neuronal circuits that could controlseizures, and thesubstantia nigra pars reticulata(SNPR) is involved in this phenomenon (Iadarola andGale, 1982; Gale, 1985; Maggio and Gale, 1989;Depaulis et al., 1994). The SNPR has been identi-fied as a critical site at which�-aminobutyric acid(GABA) agonist drugs act to reduce susceptibility toa number of types of experimentally induced gener-alized seizures (Gale, 1985). It has been shown that,for the maximal electroshock model of seizure induc-tion, nigral microinjection of GABA uptake blockershave an anticonvulsant effect in infant rats (Dalby,2000).

Therefore, understanding the differential roles ofDHF and ventral hippocampal formation/amygdalaarea (VHF-AMY) in the behavioral and electro-graphic expression of seizures, and the possibility tocontrol these seizures by GABAergic manipulation ofthe SNPR has scientific and clinical relevance. Thus,the aims of the present work are: (1) to investigate thebehavioral effects of bicuculline microinjection in thelateral ventricle (ICV), DHF, and VHF-AMY; (2) to

evaluate, because its response stability and reliability,which hippocampal subregion (DHF or VHF-AMY)is more suitable to help in the development of nigralanticonvulsant assays; (3) to test if two GABA uptakeblockers (nipecotic acid or the neurotoxin FrPbA2 de-rived fromParawixia bistriataspider venom,Cairrãoet al., 2002), as well as the classic GABAA agonist(muscimol), microinjected in the SNPR of adult Wis-tar rats are effective in preventing seizures initiated inthe hippocampal formation.

2. Material and methods

2.1. Animals and drugs

Male adult Wistar rats weighing 250–300 g from themain animal house of the Campus of Ribeirão Pretoat the University of São Paulo were used. All exper-iments were done in accordance with the recommen-dations of the Brazilian Society for Neuroscience andBehavior for animal experimentation and all effortswere made in order to avoid any unnecessary sufferingto the animals.

The doses of bicuculline (20 nmol/0.2�l per ratfor ICV and 4 nmol/0.2�l per rat for intrahippocam-pal experiments) were standardized in our laboratoryin order to induce seizures in all tested animals.The dose of nipecotic acid (12 nmol/0.2�l per rat;60 mM) is believed to completely block all GABAtransporters (IC50 = 328 nM,Andersen et al., 2001).The dose of muscimol (200 ng/0.2�l per rat) wasadapted from literature, since it is believed to pro-duce a strong anticonvulsant effect when injected inthe SNPR (25–200 ng,Sperber et al., 1989; 25 ng,Maggio and Gale, 1989). Bicuculline was injectedunilateral (DHF, VHF-AMY, or ICV), whereas theother drugs (muscimol, nipecotic acid and FrPbA2)were injected bilaterally in the SNPR. All drugs werepurchased from Research Biochemicals International(Natick, MA, USA) and freshly prepared by dilutionin phosphate buffered saline (PBS 0.05 M, pH 7.4).

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FrPbA2 is a GABA uptake blocker neurotoxinisolated from theP. bistriata spider venom (Cairrãoet al., 2002). The concentration of the isolated fractionFrPbA2 was determined arbitrarily (Cairrão et al.,2002). The value of one optical density at 215 nm wasdefined as 1000 arbitrary units (AU). The solution ofFrPbA2 (1000�l) absorbed 2.05, so it corresponds to2050 AU. In vivo experiments were carried out with0.410 AU/0.2�l per rat of FrPbA2.

2.2. Stereotaxic surgery, experimental groups andbehavioral evaluation

Stereotaxic surgery was used to place 26-gaugestainless steel guide cannulae in the hippocampalformations and brainstem (unilaterally in the DHFor VHF-AMY and bilaterally in the SNPR). Thestereotaxic target coordinates (Paxinos and Watson,1986) were (from bregma): DHF (AP:−3.8 mm, ML:−4.6 mm, DV: 4.0 mm); VHF-AMY (AP:−4.8 mm,ML: −1.4 mm, DV: 9.2 mm); SNPR (AP:−5.8 mm,ML: ±2 mm, DV: 8.2 mm). All microinjections(0.2�l in 1 min) were made with a 10�l microsyringe(Hamilton Company, Reno, NV, USA). The animalsused in video-EEG experiments were implanted withtwo chemitrodes, exactly the same cannula used inthe other animals, but with a 0.2 mm teflon coatedstainless steel twisted electrode coupled, one directedto the DHF and one to the right SNPR, and also oneguide cannula to the left SNPR.

There were six experimental groups.Group 1: ICVgroup (n = 7). Animals received PBS in the right lat-eral ventricle and, 10 min later, the same volume ofbicuculline.Group 2: DHF group (n = 13). Animalswere injected bilaterally with PBS in the SNPR and,5 min later, with bicuculline in the cannula directed tothe DHF.Group 3: VHF-AMY group (n = 12). Ani-mals received PBS in the SNPR and then bicucullinein the cannula directed to the VHF-AMY area.Groups4–6: Muscimol (n = 5), nipecotic acid (n = 5), andFrPbA2 (n = 6). Animals received one of either drugin the SNPR (bilaterally) and, 5 min later, bicucullinein the cannula directed to the DHF.

Behavioral evaluation of animals was made as fol-lows: thirty minutes before the experiment, animalswere placed individually in a cylindrical acrylic trans-parent circular arena (height: 30 cm, diameter: 60 cm)for habituation. The animals that were used for video-

EEG studies (one animal from the Group 2, threefrom the Group 4, and two from the Group 5) wereplaced in a similar arena, but with different diameter(25 cm). After microinjection, the animals were ob-served for 30 min. The presence of myoclonus, wetdog shakes (WDS), tonic–clonic seizures (TC), wildrunning (WR), escape from test arena and the latencyto seizures were evaluated for 30 min after bicucullinemicroinjection. The classic limbic seizures (face, head,and forelimb myoclonus, rearing and falling: Classes1–5 of Racine’s scale, 1972) were seen in someanimals after bicuculline injection (seeSection 3),so the presence of this kind of seizure was alsoevaluated.

After the experiments, rats were deeply anesthetizedwith sodium thiopenthal and perfused transaorticallywith PBS and 4% paraformaldehyde solution in PBS.The prosencephalic and mesencephalic tissues werecut in serial histological sections to confirm the injec-tion sites. After perfusion, the animals of the Group 1received ICV injection of dye (toluidine blue 0.15%,1�l), their brains were removed and frozen in−20◦Cfor 2 h, then cut manually to see whether or not theventricles were stained.

2.3. Video-EEG recording

Video-EEG recording was performed as previ-ously described (Moraes et al., 2000). EEG data wassampled in 500 Hz. Much of the noise, due to cablemovement, had to be eliminated, in order to recorda trustworthy EEG from seizures. A source followercircuit, developed with miniaturized components, wasbuilt on the rat’s helmet connector. This pre-amplifierstage, which included a built-in energy source (two1.5 V batteries), gave the desired high impedanceinterface between the brain and the cable, withoutcompromising the next amplification stage (Moraeset al., 2000). The signal recorded from such a circuitwas relatively artifact-free even during severe mo-tor seizures such as, for example, wild running andtonic–clonic seizure episodes (Moraes et al., 2000).

2.4. Statistical analysis

Statistical analysis of the latency of seizures wasmade by one-way analysis of variance (ANOVA).The incidences of localized myoclonus, WR, TC, or

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C seizures, limbic seizures, and escape from arenaand immobility between groups were analyzed byFisher exact test. For both statistical tests, a two-tailand a 5% of significance level (P < 0.05) were consi-dered.

3. Results

3.1. Injection sites

All animals considered in the Group 1 had theirventricles stained by the dye (Fig. 1A, schematic).Histological analysis revealed that the animals of theGroup 2 (totaln = 13) were injected with bicucullinein medial CA1 (n = 10, Fig. 1B-I), dentate gyrus(n = 1, Fig. 1B-I) or subiculum (n = 2, Fig. 1B-II).The Group 3 animals (totaln = 14) were injectedwith bicuculline in the amygdalo-hippocampal areaposteromedial part (AHiPM,n = 9, Fig. 1C-I), den-tate gyrus (DG) (n = 2, Fig. 1C-I) or in the postero-medial cortical amygdaloid nucleus (PMCo,n = 3,Fig. 1C-I). Although the behavioral response to thebicuculline injection in the AHiPM, DG, or PMCo wasidentical, only the animals injected in the AHiPM wereconsidered in Group 3 for statistical analysis (latenciesand behavior,Fig. 2). All animals from Groups 2 and3 were injected with PBS in the SNPR (Fig. 1B-IIIand C-II, respectively), except for two animals inGroup 2 and seven animals from Group 3, that re-ceived PBS outside of the SNPR (mainly in thecerebral peduncle,Fig. 1C-III and C-IV). But, as ourresults did not show any difference between theseanimals and assuming that PBS has no effect whetherinjected inside the SNPR or the cerebral peduncle,and that our interest in these groups was the DHFor VHF-AMY bicuculline injection, all animals wereincluded in the statistical analysis.

Fig. 1. Injection sites. Insets are histological micrographs of representative animals from each group. (A) Group 1 (ICV). The hachuredventricles represent the presence of toluidine blue dye. (B-I–B-III) Group 2 (SNPR PBS+ DHF bicuculline). (B-I, inset) Medial CA1.(B-II, inset) Subiculum, note the clear difference to panel B-I (inset). At this saggital plane, the injection site does not reach the CA1field. (C-I–C-IV) Group 3 (SNPR PBS+ VHF-AMY bicuculline). (C-I, insets) AHiPM injection site (above) and PMCo (bottom). For thestatistical analysis, only the animals injected in the AHiPM were considered. (D-I–D-II) Group 4 (SNPR muscimol+ DHF bicuculline).(E-I–E-II) Group 5 (SNPR nipecotic acid+ DHF bicuculline). (F-I–F-II) Group 6 (SNPR FrPbA2+ DHF bicuculline). Saggital planes(mm from bregma): (A)−0.92. (B-I, D-I, and F-I)−3.80. (B-II) −5.30. (B-III, C-II, C-III, D-II, E-II, and F-II) −5.80. (C-I) −4.80.(C-IV) −6.72. (�) Injection sites. All insets were originally colored by the methylene blue or Nissl technique. Drawings were adaptedfrom the atlas ofPaxinos and Watson (1986).

Groups 4 and 5 were injected with bicuculline onlyin medial CA1 (Fig. 1D-I and E-I, respectively), andthe animals of the Group 6 (totaln = 6) were injectedin medial CA1 (n = 4, Fig. 1F-I) or dentate gyrus(n = 2, Fig. 1F-I). Only animals correctly injected inthe SNPR (for muscimol, nipecotic acid or FrPbA2)were admitted in Groups 4–6 (Fig. 1D-II, E-II, andF-II, respectively).

3.2. Behavioral effect of central bicucullineinjection

ICV bicuculline injection (Group 1) induced aprogressive sequence of responses: exophthalmia, pi-loerection, WDS, localized myoclonus (LM, mainlyin the left ear, contralateral to the injection site), co-ordinated running (with jumps and escapes from theopen-field) evolving to an “explosive” wild running(WR) with vocalization, followed by TC, C, anddeath. Animals also turned anti-clockwise (gyrus)(Fig. 2A). The incidences of: escapes, gyri, WR, TC,and C were significantly higher if compared to Group2; and the LM, crawling, TC, and C were also higherif compared to Group 3. This ICV ceiling effect waspartially reached by DHF bicuculline injection (Group2) or AHiPM animals (Group 3). The Group 2 animalshad almost only the LM (left ear, but also in the backor tail of the animal) and crawling, both with a sig-nificantly higher incidence when compared to Group3 (Fig. 2B). The majority of animals in Group 3 ex-hibited WDS (n = 6), and in some of them (n = 4) itwas not seen any other convulsive behavior. But theother animals (n = 3) evolved to escapes from the testarena, WR, TC, and C seizures (Fig. 2C). The laten-cies of the experimental groups were not statisticallysignificant (P = 0.08), but nevertheless there was aclear trend: lower latency to Group 1 (ICV bicuculline)(Fig. 2, inset). A single comparison between the ICV

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Fig. 2. Behavioral effects of bicuculline microinjection. (A) ICV; (B) DHF; (C) AHiPM.∗P < 0.05; ∗∗P < 0.01, Fisher exact test. Lettersinside brackets indicate statistically significant difference against the same behavior of a different group. (Inset) Latencies to seizures(mean± S.E.M.) induced by ICV, DHF, or VHF-AMY bicuculline microinjection.

group and DHF or VHF-AMY groups separatelyresulted in a significant difference (Student’st-test,P < 0.05—data not shown), but ANOVA is still thebest method to make comparisons between multiplegroups.

The incidence of limbic seizures (Racine, 1972),was present in all animal groups, if we consider thatthe myoclonus of the left ear is a Class 1 seizure. Butthe more severe 2–3 and 4–5 seizures were seen onlyin Group 3, mostly in those animals that presented

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the escape-WR-TC seizures. Interestingly, Group 1animals presented the escape-WR-TC, without limbicseizures.

The incidence of WDS was significantly higher inGroup 3, if compared to Groups 1 and 2 (Fig. 2C). Theanimals of Group 1 did not present WDS (Fig. 2C).

3.3. Nigral GABAergic manipulation

Since the microinjection of bicuculline in theDHF (Group 2) caused a more homogeneous re-sponse (LM and crawling, the only convulsive be-haviors seen in 84% of the animals), this site waschosen for nigral anticonvulsant experiments. Ni-gral injection of muscimol blocked the totality ofmyoclonus induced by microinjection of bicucullineinto the DHF (Table 1), although intense stereo-typic behaviors (sniffing, biting, and head nodding)appeared. No other treatment (nipecotic acid andFrPbA2) resulted in such protection against seizuresinduced by microinjection of bicuculline in the DHF(Table 1).

3.4. Video-EEG, qualitative analysis

The injection of PBS in the SNPR or in the me-dial CA1 (DHF, Group 2) did not alter the EEGbasal activity or the behavior of the animal (Fig. 3Aand B, respectively). The injection of PBS in the SNPRand bicuculline in the DHF caused the appearance ofelectric spikes in the SNPR (Fig. 3C) and in the hip-pocampus (Fig. 3D). Sometimes the electric spikes of

Table 1Anticonvulsant test of muscimol, nipecotic acid, or FrPbA2 bilat-eral nigral microinjections after DHF bicuculline

SNPR treatment Seizure Protect Total

PBS (controls) 13 0 13Muscimol 1 4∗∗ 5Nipecotic acid 5 0 5FrPbA2 6 0 6

SNPR treatment: drugs injected bilaterally in the SNPR 5 min priorthe microinjection of bicuculline in the DHF; Seizure: number ofrats presenting seizures after bicuculline injection; Protect: numberof rats without seizures up to 30 min after bicuculline injection;Total: total number of animals in the group. The number of animalsin each treatment was compared to control with Fisher’s exacttest. **p< 0.01 (two-tailed).

Fig. 3. EEG data. (1) Representative control animal injected withPBS in the SNPR (A) and DHF (B), 4 min after injections. (2) Thesame animal of (1), but now injected with PBS in the SNPR (C)and bicuculline in the DHF (D), 60 s after bicuculline injection.(3) The same animal, 4 min after bicuculline injection. (4) Animalinjected with muscimol in the SNPR (G) and bicuculline in theDHF (H), 7 min after bicuculline injection. (5) Animal injectedwith nipecotic acid in the SNPR (I) and bicuculline in the DHF(J), 9 min after bicuculline injection. Both recordings (SNPR andDHF) of each rat were simultaneous. BIC: bicuculline; NIPEC:nipecotic acid; MUSC: muscimol.

the hippocampus apparently disappeared (Fig. 3F) butthey were increased in the SNPR (Fig. 3E). The injec-tion of muscimol in the SNPR, a treatment that blockedbehavioral seizures induced by bicuculline, did notblock nigral or hippocampal EEG spikes. (Fig. 3Gand H, respectively). Nipecotic acid, that could notblock bicuculline seizures, also did not change theEEG spikes from SNPR or medial CA1 (Fig. 3I and J,respectively).

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4. Discussion

The main finding of the current work is that ICVinjection of bicuculline in rats induced a progressivesequence of responses: exophthalmia, piloerection,WDS, left ear localized myoclonus, coordinated run-ning (with jumps and escapes from the open-field),WR, vocalization, TC seizures, C seizures, and death.This ICV ceiling effect could not be totally mimickedby focal bicuculline. Instead, DHF bicuculline in-duced almost only the ear myoclonus, or VHF-AMYbicuculline injection induced only WDS in one sub-group, or escapes plus WR, TC, and C in the other.

When the convulsant is injected by ICV route, ithas the possibility to reach many and distant brain ar-eas but, when it is focally administered (0.2�l), onlylocal nuclei are disinhibited. This leads to the conclu-sion that circuitries originating in the vicinity of theDHF, at least those areas close to the ones injectedin the present work, may be linked principally torestricted motor-controlling areas, what would origi-nate such localized behavioral expression, myoclonusonly in the contralateral ear, back or tail muscles. Itis possible that in the VHF-AMY group there wasa sequential recruitment of forebrain to midbrainstructures, which could be responsible for the limbicseizures and the WR and TC seizures, respectively.One example of forebrain/midbrain ictal spread isseen after exposure of mice to the convulsant gasflurothyl (Ferland and Applegate, 1998). We have alsoexamples of opposite direction recruitment (brain-stem to forebrain), such as in the case of audiogenickindling (Garcia-Cairasco et al., 1996; Moraes et al.,2000; Romcy-Pereira and Garcia-Cairasco, 2003).Although the flurothyl gas acts systemically andthe audiogenic seizures involves initially only brainstem substrates, the comparison between these proto-cols is valid, as the behavioral expressions of limbicand brain stem circuitry—myoclous (limbic)× wildrunning/clonic/tonic–clonic (brain stem)—is the same.

In the VHF-AMY bicuculline injected group(Group 3) and ICV group (Group 1), nevertheless, wehave seen an interesting flight-like picture precedingthe tonic–clonic seizures: exophthalmia, piloerection,coordinated run with escapes from the arena. It issuitable to observe here that the animals of the Group1 have already been in the test arena for 30 min be-fore they were injected with PBS, and only 10 min

later they were reinjected with bicuculline. This timeis sufficiently long for the animals to habituate to thepresumably quiet and “safe” test place, full of theanimals’ own smells. No other animals ever tried tojump out of the test arena. Both situations (flight-likeresponse and seizures) might share or have over-lapped neural substrates. We might be reminded thatthe amygdala central nucleus seem to amplify theaversive responses integrated in the inferior collicu-lus (Brandão et al., 1988; Maisonnette et al., 1996;Coimbra et al., 2000; Brandão et al., 2001), a regiontypically involved in acute audiogenic seizures, forexample in Wistar Audiogenic Rats (WAR,Garcia-Cairasco, 2002) or in non-audiogenic animals mi-croinjected with bicuculline (Tsutsui et al., 1992).Furthermore, after audiogenic kindling, amygdalaand hippocampal recruitment are effective both inbehavioral and EEG patterns, strongly supporting theoverlapping and integration of acoustic-limbic net-works (Moraes et al., 2000; Garcia-Cairasco, 2002;Romcy-Pereira and Garcia-Cairasco, 2003).

In our hands, the two GABA uptake blockers mi-croinjected bilaterally in the SNPR did not preventbehavioral seizures induced by bicuculline microin-jection in the DHF. Present results are in contrast tothe seizure protection described byDalby (2000), whoused juvenile Sprague–Dawley rats microinjected inthe SNPR with two GABA uptake blockers and alsowith muscimol (250 pmol); all these treatments pro-tected animals against maximal electroshock (Dalby,2000). The discrepancy betweenDalby (2000)dataand ours might be related to the model-dependent(Maggio et al., 1991) or age-dependent (Wurpel et al.,1992; Velısková et al., 1996) variations in the abilityof the SNPR to control seizures. Generally, infant ratsare less susceptible to the nigral control of seizures(Wurpel et al., 1992; Velısková et al., 1996).

The protection of animals by muscimol in theSNPR described here are similar to many other works(Iadarola and Gale, 1982; Gale, 1985; Maggio andGale, 1989), confirming that nigral inhibition hasanticonvulsant effects. Nevertheless, there is a subre-gional difference between the anterior and posteriorSNPR, where -in adult rats- the injection of GABAer-gic agonists (muscimol and tetrahydroisoxazolo[5,4-c]pyridin-3-ol, the THIP) or benzodiazepine agonists(zolpidem) in the SNPRanterior have an anticonvulsanteffect in the flurothyl model (these manipulations raise

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the threshold for flurothyl-induced seizures) (Moshéet al., 1994). In the SNPRposterior, nevertheless, theinjection of muscimol or zolpidem does not protectthe rats and even has a proconvulsant effect in thesame model (it lowers the threshold). In our experi-ments, the injections were done around –5.8 AP, quitenear the middle of the AP axis of the SNPR (Groups4, 5, and 6: panels C-II, E-II, and F-II, respectivelyin Fig. 1). Although the injection-cannula position isclear in the histological section, it is impossible to seethe spreading of a non-radioactive drug (revealed byautoradiography). So, there are two alternative expla-nations for our data. First: muscimol may have spreadpreferentially into the SNPRanterior and protected theanimals. Second: in the adult Wistar rat, the protectivesubregion of the SNPR may be more extended than inthe Sprague–Dawley used byVelısková et al. (1998).The actual stage of experiments does not allow us tomake a definitive conclusion, but this fact does notinvalidate our data, since all the drugs were injectedin the same site of the SNPR. In the current work,the stereotypic behaviors induced by muscimol (headnodding, orofacial movements and sniffing) may bedue to the increase in dopamine release in forebrainstructures, which is a consequence of disinhibitionof GABAergic interneurons with cell bodies locatedin SNPR and axonal terminations in thesubstantianigra pars compacta(Martin and Haubrich, 1978).Although it has been suggested that the dopaminergiceffect of muscimol injection in SNPR is not relatedto the anticonvulsant effect of the drug (McNamaraet al., 1984), however, the possible activation of an-tagonistic neuronal networks by nigral muscimol mi-croinjection may also account for the anticonvulsanteffect of this treatment, as it has been described forhuman epilepsy (Spencer, 2002). Another curiosity isthat the peak anticonvulsant effect of nigric musci-mol is reached when orofacial stereotypy are clearlyexpressed (Gale and Browning, 1988). This is inter-esting because one of the more recent antiepilepticdrugs in use in clinical practice, lamotrigine, a potentglutamate antagonist, induces also tics in children(Menezes et al., 2000), as part of the so-called touret-tism (Lombroso, 1999), a strong indicative of its basalganglia modulatory effect.

The time used in our experiments after nigral injec-tions (5 min) was based on the literature data (Maggioand Gale, 1989), and is the minimal sufficient time

to produce an anticonvulsant effect. But many pilotworks (unpublished observations) were done (varyingthe time before the bicuculline challenge) and we con-cluded that except for muscimol, all other drugs wereineffective to produce an anticonvulsant effect.

The GABAergic antagonist bicuculline have longbeen shown to disrupt normal neurotransmission inmammal central nervous system (Curtis et al., 1970;Johnston, 1996). AlthoughVezzani et al. (1999, 2002)injected this antagonist in the dorsal hippocampus ofrats and mice to study the effect of seizures in theexpression of proinflammatory cytokines, the presentresults are the first behavioral description—andcomparison—of seizures induced by ICV and sub-regional hippocampal formation (dorsal× ventral)bicuculline microinjection in rats. Also, this is the firstattempt to block the seizures originated by focal bicu-culline in the dorsal hippocampus with GABAergicdrugs microinjected in the SNPR of rats.

5. Conclusion

The present work proposes that: (1) different neu-ronal circuitry in the DHF or VHF-AMY areas maybe responsible for the different and progressive behav-ioral seizure expression seen after ICV bicuculline in-jection, DHF been related almost only to the localizedmyoclonus and the VHF-AMY to the flight-like re-sponse, WR and TC seizures; (2) bicuculline microin-jection in different hippocampal subregions (dorsal×ventral) is a valuable model to study the spreading ofbrain ictal discharge and nigral anticonvulsant proto-cols; (3) behavioral seizures induced in the DHF areabolished by a GABAergic receptor agonist musci-mol, but not GABA uptake blockers microinjected inthe SNPR.

Acknowledgements

Marcelo C.A. Rodrigues was supported by FAPESPFellowship (#98/16010-3). N. Garcia-Cairasco holdsa CNPq Research Fellowship and is supported byFAPESP, CNPq, and PRONEX grants. Authors areindebted to Dr. Eliane C.A. Braga and Dr. Au-gusto César C. Spadaro (Faculdade de CienciasFarmaceuticas, USP), Dr. Norberto C. Coimbra

164 M. Cairrão Araujo Rodrigues et al. / Epilepsy Research 58 (2004) 155–165

(Faculdade de Medicina, USP) for critical readingof the manuscript and also to Amauri Ramos Pinhal,Antonio José Colusso, Flávio Del Vecchio and JoséAntonio Cortes de Oliveira, for technical assistance.

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