Epilepsia, 37(8):7 18-722, 1996 Lippincott-Raven Publishers, Philadelphia 0 International League Against Epilepsy

Age-Specific Effects of Baclofen on Pentylenetetrazol-Induced Seizures in Developing Rats

*§Jana VeliSkova, *?§Libor VeliSek, and *?$§Solomon L. Moshe

*Departments of Neurology, fNeuroscience, and $Pediatrics, Albert Einstein College of Medicine; and $Epilepsy Management Unit, Montejiore Medical Center, Bronx, NY, U.S.A.

Summary: Purpose: To determine whether seizures have age-specific features, we studied the role of y-aminobu- tyric acid, (GABAB) transmission in rats of various ages (9, 15, 30, and 60 postnatal days).

Methods: We used a GABA, receptor agonist baclofen (2 or 5 mg/kg intraperitoneally, i.p.) and a GABA, recep- tor antagonist CGP 35348 (100 or 600 mg/kg i.p.) in the pentylenetetrazol (PTZ)-induced model of clonic and tonic-clonic seizures (100 mg/kg subcutaneously, s.c.).

Results: Whereas baclofen was anticonvulsant and CGP 35348 proconvulsant in most animals, there were distinct age-related differences in the effectiveness of these drugs and the antagonist had some anticonvulsant

activity in adults. Furthermore, the two drugs acting at GABAB receptors had a different profile of action in clonic seizures as compared with tonic-clonic seizures.

Conclusions: The differences in the age-specific action of the GABAB agonist and antagonist suggest that differ- ent GABAB receptor subsets may mediate the drug ef- fects. The results indicate that putative antiepileptic drugs (AEDs) must be tested during development be- cause it may not be possible to extrapolate age-specific anticonvulsant effects from studies in adult animals.

Key Words: Pentylenetetrazol-Seizures-Baclofen- Rats-Newborn.

In humans, seizures occur more frequently in children than in adults (1 ) . Age-specific seizures also occur only in children and not in adults (2). In experimental models of seizures, several studies have shown that immature animals are more sus- ceptible to seizures and that the seizures have age- specific features (3-7). The effect of antiepileptic drug (AED) treatment can also be age-specific (8,9), suggesting that developmental seizure studies may be important in designing new effective age-specific AED regimens.

Pentylenetetrazol (PTZ) is a convulsant drug that induces primarily generalized seizures (10,ll). The behavioral manifestations include myoclonic twitches followed by clonic seizures similar to those induced by flurothyl or bicuculline (6,7,12). Tonic-

Received September 13, 1995; revision accepted April 29, 1996.

Address correspondence and reprint requests to Dr. J . VeliSkovfi at Department of Neurology, Kennedy Center R 316, Albert Einstein College of Medicine, 1300 Moms Park Ave., Bronx, NY 10461, U.S.A.

Dr. VeliSek's present address is Department of Pathological Physiology, Third Medical School, Charles University, Prague, Czech Republic, and Institute of Physiology, Academy of Sci- ences of the Czech Republic, Prague, Czech Republic.

clonic seizures occur after a longer latency or after a high dose of PTZ. In the Wistar rat strain, the incidence of clonic seizures is low during the first 2 postnatal weeks; it increases during the third post- natal week and remains high in adults. In contrast, tonic-clonic PTZ seizures occur frequently through- out the development (5). Clonic PTZ-induced sei- zures are considered a model of human myoclonic seizures. Tonic-clonic seizures represent a model of generalized tonic-clonic seizures (GTCS) (13,14).

y-Aminobutyric acid (GABA)-mediated neuro- transmission plays an important role in the regula- tion of seizures both in humans and in experimental models. Drugs that enhance GABAA receptor- mediated transmission are currently used in the treatment of human seizure disorders and in exper- imental models (15-19). Information regarding the role of GABAB receptors in seizures is limited. An- imal studies suggest that activation of the GABA, system exacerbates experimental absence seizures, whereas GABAB receptor antagonists suppress such seizures (20). In human studies in adults, both proconvulsant and anticonvulsant actions of baclo- fen, a GABA, receptor agonist (21), have been re- ported (22,23). In our previous study (9), we showed that the effects of baclofen are age-

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BACLOFEN IN PTZ-INDUCED SEIZURES 719

dependent in the flurothyl-induced seizure model. Baclofen was proconvulsant in 9-day-old rats, anti- convulsant in 15- and 30-day-old rats, and ineffec- tive in 60-day-old rats.

In the current study, we tested the effect of a GABAB agonist (baclofen) and a GABAB antagonist (CGP 35348) (24) in the PTZ seizure model to de- termine whether there are age-specific features in- volving GABA, transmission in the modulation of PTZ-induced seizures.

METHODS

We used 221 male Sprague-Dawley (Taconic Farms) rats aged 9, 15, 30, and 60 days. The day of birth was counted as day 0. The 30-day-old rats and adult rats were housed in groups of 4; rat pups were housed with their respective dams on 12-h dark/l2-h light cycle in our accredited animal facility. All rats had free access to food and water. Rats were pre- treated with L( -) baclofen, (a GABAB receptor ag- onist; 2 or 5 mg/kg intraperitoneally, i.p.) or CGP 35348 (a GABA, receptor antagonist; 100 or 600 mg/kg i.p., a gift from Ciba-Geigy) 30 min before the PTZ challenge [lo0 mg/kg subcutaneously (s.c.); volume 1 ml/kg; (5 ) ] . Control rats were pretreated with 1 ml/kg i.p. saline 30 min before PTZ admin- istration. There were at least 8 rats in each sub- group.

Immediately after PTZ administration, the rats were placed in separate cages and observed for the incidence and latency to onset of clonic and tonic- clonic seizures in a 30-min interval, as previously described (5) . The results were evaluated using analysis of variance (ANOVA) with post-hoc Dun- nett’s test or chi-square test with post-hoc Fisher’s Exact test was calculated for each seizure type within each age group. The level of significance was preset to p < 0.05.

RESULTS

Controls PTZ-induced clonic seizures consisted of clonus

of facial and forelimb muscles associated with vibrissae movements. The righting reflex was pre- served. The seizure was accompanied by a Straub tail. Tonic-clonic seizures consisted of wild run- ning, loss of the righting reflex, and tonic flexion or extension of all limbs, followed by prolonged clo- nus.

PTZ-induced clonic seizures occurred in all rats aged 9-30 days and in 95% of adults. Tonic-clonic seizures occurred in all 9- to 15-day-old rats, in 80% of 30-day-old rats, and in 59% of adults.

In 30- and 60-day-old rats, there was a significant

time lag between the onset of clonic seizures and tonic-clonic seizures. In 9- and 15-day-old rat pups, the time lag was very short, only 32.5 ? 14.2 s (mean k SEM) and 7.1 * 6.7 s, respectively. In 30-day-old rats, this difference was 225.8 ? 45.6 s; in the adult rats, it was 276.6 * 69.1 s.

Effects of baclofen on PTZ seizures The larger dose of baclofen (5 mg/kg) induced

hindlimb ataxia in all age groups. The ataxia was severe in 9- and 15-day-old rats and mild in the older age groups. There were no apparent side effects of the lower dose (2 mg/kg) of baclofen.

Baclofen had age- and dose-specific effects on seizures. In 9-day-old rats, neither dose of baclofen had any effect on the incidence or latency of clonic and tonic-clonic seizures. In 15-day-old rats, baclo- fen did not change the incidence of clonic or tonic- clonic seizures. However, both doses significantly increased the latency to the onset of clonic (Fig. 1) (F(4,46) = 15.4, p < 0.05) and tonic seizures (Fig. 2) (F(4,4,, = 18.6, p < 0.05) as compared with age- matched controls. In 30-day-old rats, the dose of 5 mg/kg baclofen suppressed the occurrence of clonic seizures (by 33%; data not shown), whereas the 2 mg/kg dose suppressed the occurrence of tonic- clonic seizures (Table 1). Neither dose had any effect on seizure latencies (Figs. 1 and 2). In 60- day-old rats, baclofen pretreatment had no effect on the incidence and latency of clonic seizures; however, the higher dose of baclofen decreased the incidence and increased the latency to

AGE

FIG. 1. The effects of baclofen and CGP 35348 on the la- tency to the onset of clonic pentylenetetrazol (PTZ)-induced seizures (mean t SEM): x axis, age groups (9, 15, 30, and 60-day-old rats); y axis, latency to onset of clonic seizures measured in seconds from the time of the administration of the convulsant PTZ. The longer the latency, the greater the resistance of the rats to seizures; i.e., the increase in the latency to onset of seizures as compared with controls indi- cates anticonvulsant effects, whereas the decrease in the la- tency to seizure onset represents a proconvulsant action. Latency to seizure onset in control rats pretreated with saline (open columns). One-way analysis of variance with post hoc Dunnett’s test was computed within each age group. ‘Signif- icant difference (p < 0.05) as compared with age-matched controls.

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720 J . VELfSKOVA ET AL.

AGE

FIG. 2. The effects of baclofen and CGP 35348 on the la- tency to the onset of tonic-clonic pentylenetetrazol (PTZ)- induced seizures (mean f SEM): x axis, age groups (9, 15, 30, and 60-day-old rats); y axis, latency to onset of tonic- clonic seizures measured in seconds from the time of the PTZ administration. *Significant difference (p < 0.05) as compared with age-matched controls.

onset of tonic-clonic seizures as compared with controls (F(4,34) = 6.49, p < 0.05).

In all age groups, the pretreatment with baclofen consistently increased the time lag between clonic and tonic-clonic seizures. In 9- and 30-day-old rats, the 2-mg/kg dose of baclofen increased the time dif- ference between clonic and tonic-clonic seizure on- set to 180 ? 26 s (F(4,41) = 16.1, p < 0.05) and 734 ? 213 s (F(4,35) = 2.2, p < 0.05), respectively. In 15- and 60-day-old rats, the 5-mg/kg dose of baclofen increased the time lag between clonic and tonic- clonic seizures to 93 ? 32 s (F(4,45) = 6.3, p < 0.05) and 978 ? 214 s (F(4,34) = 5.7, p < 0.05), respec- tively.

Effects of CGP 35348 on PTZ seizures The lower dose of CGP 35348 (100 mg/kg) had no

apparent behavioral side effects. However, the higher dose (600 mg/kg) induced ataxia in 9- and 15-day-old rats.

Pretreatment with CGP 35348 did not alter the

TABLE 1. Incidence of PTZ-induced tonic-clonic seizures

Treated rat groups

Baclofen CGP 35348 (mg/kg) (mdkg) Rat age

(days) Controls 2 5 100 600

9 818 loll0 12/12 818 818 15 18/18 718 819 9/12 818 30 12115 2/10" 719 718 12/12 60 13/22 518 4/16" 9/12 518

PTZ , pent y lenetetrazol . Data in the fields are in format xln, where x represents the

number of rats in which the seizure occurred from the total n rats in that particular subgroup. The lower the value of x, the higher the resistance of the rats to FTZ-induced seizures. Chi-square test with posthoc Fisher's Exact test was calculated for each seizure type within each age group.

a Significantly different from age-matched controls (p < 0.05).

incidence of seizures in any age group. However, CGP 35348 pretreatment influenced seizure laten- cies as a function of age, dose, and seizure type. In 9-day-old rats, both doses of CGP 35348 shortened the latency to onset of clonic (Fig. 1) (F(4,41) = 14.6, p < 0.05) and tonic-clonic seizures (Fig. 2 and Table 1) (F(4.41) = 14.2, p < 0.05). In 15-day-old rats, both doses of CGP 35348 shortened the latency to onset of clonic seizures (F(4,46) = 15.4, p < 0.05), but only the high dose significantly accelerated the onset of tonic-clonic seizures (F(4,45) = 18.6, p < 0.05). In 30-day-old rats, the dose of 600 mg/kg significantly decreased the latency to onset of clonic seizures as compared with controls (F(4,46) = 3.98; p < 0.05). Neither dose altered the latency to tonic-clonic sei- zures. In 60-day-old rats, CGP 35348 had no signif- icant effects on clonic seizures. Unexpectedly, the lOO-mg/kg dose of CGP 35348 was anticonvulsant against tonic-clonic seizures and delayed their onset as compared with controls (Fig. 2) (F(4,34) = 6.49, p < 0.05). CGP 35348 had inconsistent nonsignificant effects on the time lag between clonic and tonic- clonic seizures irrespective of dose and age group.

DISCUSSION The data demonstrate that the GABAB receptor

agonist baclofen produced some anticonvulsant ef- fects against PTZ-induced seizures, either on sei- zure incidence or latency in all age groups except the group aged 9 days. In contrast, the GABAB re- ceptor antagonist CGP 35348 produced proconvul- sant effects, especially in the two youngest age groups, but in adult rats an anticonvulsant effect was evident.

Baclofen had the most consistent anticonvulsant effects in 15-day-old rats. This age-specific action of baclofen against seizures correlates well with re- sults of studies in which localized infusions of bac- lofen were used in other seizure models. These studies demonstrated that intranigrally infused bac- lofen is anticonvulsant in 15-day-old rat pups but not in adult rats in the flurothyl seizure model (25). Similar results were reported in the amygdala kin- dling model with systemic administration of baclo- fen (26). An explanation may be that there exists a developmental period during which the efficacy of the GABAB system in seizure control is enhanced. This period may correlate with the postnatal devel- opment of GABAB receptors, which are abundant in the CNS early postnatally (27). Specifically, the density of GABAB receptors is almost threefold higher in the 15-day-old substantia nigra than in adult substantia nigra (28). This developmentally transient feature may explain the anticonvulsant ac- tions of baclofen in 15-day-old rats.

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BACLOFEN IN PTZ-IND UCED SEIZURES 721

Baclofen had anticonvulsant effects against PTZ- induced tonic-clonic seizures throughout develop- ment except for the youngest group of rats. In a previous study, we also noted age-specific effects of baclofen on flurothyl-induced seizures (9). These results suggest that both clonic and tonic-clonic sei- zures have age-dependent features, which may re- sult from specific maturational patterns of the struc- tures underlying particular seizure types. Clonic seizures are believed to begin in forebrain struc- tures. In contrast, tonic-clonic seizures probably originate in brainstem structures (29,30). These parts of the brain differ in their postnatal matura- tion. The effects of baclofen on PTZ and flurothyl seizures are not identical in all age groups (9); e.g., in 9-day-old rats, baclofen has no effect on PTZ seizures, whereas it is proconvulsant in the fluro- thy1 model. Conversely, in adult rats, baclofen has anticonvulsant effects in the PTZ but not in the flu- rothyl model. Therefore, the effects of baclofen may be age- and seizure-specific.

In the PTZ model, CGP 35348 had proconvulsant effects until age 30 days against clonic seizures and until age 15 days against tonic-clonic seizures. These data are similar to the data obtained in the flurothyl seizure model. One notable exception was the anticonvulsant effect of the lOO-mg/kg dose of CGP 35348 in 60-day-old rats. An explanation may be that CGP 35348 at low doses in this age group is insufficient to act postsynaptically, although it may prevent GABA binding to presynaptic GABAB re- ceptors, thus enhancing the release of endogenous GABA. An alternative explanation may be that the role of postsynaptic GABAB sites in seizures de- creases with development but that a significant in- fluence of the presynaptic GABAB receptors still exists.

The developmental difference between the anti- convulsant effects of baclofen and proconvulsant effects of CGP 35348 suggests that there may be a difference in the receptor subsets influenced by these drugs. This conclusion is supported by our previous results showing that two GABAB antago- nists (CGP 35348 and CGP 36742) have different effects on flurothyl seizures in the various age groups. Further studies using combination treat- ment of agonists and antagonists may be necessary to clarify the existence of these subsets. Moreover, in the PTZ model, the effects of GABAB drugs in developing rats do not exactly match the effects of GABA, drugs in adult rats. Therefore, the devel- opmental effects of AEDs cannot be reliably pre- dicted from their effects in adults.

Acknowledgment: This work was supported by an Ep-

ilepsy Foundation of America Research Grant and by Grants No. 305/95/0588 from the Grant Agency of the Czech Republic (to L.V.), No. NS-20253 from NINDS, and a grant from Ciba-Geigy (to S.L.M.).

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