Neuropharmacology 49 (2005) 367e375 www.elsevier.com/locate/neuropharm
5-HT1A receptor agonists modify epileptic seizures in three experimental models in rats Marı´ a-Leonor Lo´pez-Meraz a,b, Marı´ a-Eva Gonza´lez-Trujano b, Leticia Neri-Baza´n a, Enrique Hong a, Luisa Lilia Rocha a,* a
Departamento de Farmacobiologı´a Centro de Investigacion y de Estudios Avanzados, Czda. Tenorios 235. Col. Granjas Coapa 14330 Mexico DF, Mexico b Subdivicio´n de Investigaciones en Neurociencias, Instituto Nacional de Pslquiatrı´a ‘‘Ramo´n de la Fuente’’ Received 12 November 2004; received in revised form 4 March 2005; accepted 31 March 2005
Abstract The effects of two serotonergic (5-HT1A) receptor agonists (8-OH-DPAT; 0.01, 0.1, 0.3, 1 mg/kg, s.c., and Indorenate; 1, 3, 10 mg/kg, i.p.) were evaluated in three type of seizures in male Wistar rats: clonicetonic convulsions induced by pentylenetetrazol (PTZ, 60 mg/kg, i.p.), status epilepticus (SE) of limbic seizures produced by kainic acid (KA, 10 mg/kg, i.p.) and toniceclonic seizures by amygdala kindling. 8-OH-DPAT decreased the incidence of tonic seizures and the mortality rate induced by PTZ. Indorenate increased the latency to the PTZ-induced seizures and decreased the percentage of rats showing tonic extension and death. Concerning KA, 8-OH-DPAT augmented the latency and reduced the frequency of wet-dog shake (WDS) and generalized seizure (GS). At high doses it diminished the occurrence and delayed the establishment of SE. Indorenate augmented the latency to WDS, GS and SE, and diminished the number of GS. 8-OH-DPAT and Indorenate did not alter the expression of kindled seizures. However, Indorenate enhanced the refractoriness to subsequent seizures during the postictal depression. Some effects induced by 8-OH-DPAT and Indorenate on seizures evaluated and postictal depression were fully or partially blocked by WAY100635. These results suggest that 5-HT1A receptor agonists modify epileptic activity depending on the type of seizure. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: 8-OH-DPAT; Indorenate; 5-HT1A receptor agonist; Pentylenetetrazole; Kainic acid; Amygdala kindling; Postictal depression
1. Introduction A large number of serotonergic (5-HT) receptors with different anatomical localizations and effects have been identified (Barnes and Sharp, 1999). Although the 5-HT1A receptor subtype has been involved in the modulation of seizure activity, the results obtained are controversial. A group of evidence supports its antiepileptic effects. The administration of WAY100635, a 5-HT1A receptor antagonist, avoids the protective effect induced by the intrahippocampal infusion of serotonin * Corresponding author. Tel.: C52 55 5061 2859; fax: C52 55 5061 2863. E-mail address:
[email protected] (L.L. Rocha). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.03.020
on the development of pilocarpine-induced seizures (Clinckers et al., 2004). The compound 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT1A receptor agonist, decreases the number and duration of the electrographic seizures induced by the intrahippocampal injection of kainic acid (KA) (Gariboldi et al., 1996). Similarly, 8-OH-DPAT increases the afterdischarge (AD) threshold, reduces the duration of partial seizures, avoids generalized seizures (GS) induced by hippocampal kindling, and delays the progression of the amygdala kindling process (Wada et al., 1992, 1993, 1997). In contrast, the proepileptic effect of 5-HT1A receptors is supported due to the marked and a dose-dependent increases in number and mean cumulative duration of
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spike-wave discharges induced by the administration of 5HT1A agonists in a genetic model of absence epilepsy (Filakovszky et al., 1999; Jakus et al., 2003), effects blocked by WAY100635 (Graf et al., 2004). On the other hand, it is well known that 5methoxytryptamine-b-methylcarboxylate (Indorenate) is a 5-HT1A receptor agonist that produces antihypertensive, anxiolytic and antidepressant effects (Hong, 1981; Ferna´ndez-Guasti et al., 1992; Martınez-Mota et al., 2002). However, at present it is unknown if Indorenate is able to modify the epileptic activity. The main goal of this study was to analyze and compare the effects of 8-OH-DPAT and Indorenate on three different types of convulsive seizures in rats: primary generalized seizures, status epilepticus (SE) of limbic seizures and complex partial seizures with secondary generalization. Three experimental models were chosen as follows: (1) systemic administration of pentylenetetrazol (PTZ), which induces clonicetonic generalized seizures and represents the first choice for anticonvulsive drug screening, (2) systemic administration of KA which produces SE of limbic seizures (Lothman and Collins, 1981), and (3) electrical amygdala kindling, a model of epileptogenesis which induces complex partial seizures with secondary generalization (Sato et al., 1990) and, in addition, allows the evaluation of the refractoriness for subsequent convulsions during the postictal period following kindled seizures (Caldecott-Hazard and Engel, 1987; Engel and Ackermann, 1980).
2. Methods 2.1. Subjects Male Wistar rats initially weighing 300 g, housed at 22 C and maintained on 12:12-h light/dark cycle were used in the present study. The animals had free access to food and water. Procedures involving animal care were conducted in agreement with the Mexican Official Norm (‘Norma Oficial Mexicana’ NOM-062-ZOO-1999) and the Ethical Committee of the Center for Research and Advanced Studies (Centro de Investigacio´n y de Estudios Avanzados). 2.2. Drugs The following drugs were used. Ketamine (KetalinÒ from Probiomed) and xylazine (Sigma) were applied to anesthetize the rats; PTZ (Sigma) and KA (Sigma) were used as convulsants; (G) 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin HBr, Sigma) and Indorenate (5-methoxytryptamine-b-methylcarboxilate HCl, Miles Laboratories and Cinvestav, Mexico) were used as 5HT1A agonists. When significant effects were induced
with 8-OH-DPAT or Indorenate, additional groups (nR6) were designed to apply WAY100635 (N-[2-(4-[2methoxyphenil]-1-piperazinyl)ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate, Sigma) alone or immediately after the 5-HT1A agonists with the purpose of blocking their effects. All drugs were dissolved in saline solution (NaCl 0.9%, SS). 2.3. Experimental groups 2.3.1. PTZ-induced clonicetonic seizures Rats received daily administration of SS (1 ml/kg i.p.) for 5 days to adjust to manipulation. Twenty-four hours after the last SS injection, animals received 8-OH-DPAT (0.01, 0.1 or 1 mg/kg, s.c., 20 min before PTZ; nZ7 per dose), Indorenate [1 (nZ12), 3 (nZ17) or 10 (nZ23) mg/ kg i.p. 90 min before PTZ] or WAY100635 (1 mg/kg, s.c. 20 min before PTZ, nZ11). Then, PTZ was administered at 60 mg/kg, i.p. This dose produces the following behavioral changes: myoclonus, defined as a whole-body twitch; clonic seizures, manifested by clonic spasms often followed by stupor or unusual posturing; and tonic seizures consisted of tonic hindlimb extension, which is usually the lethal component in approximately 50% of the rats under normal conditions (Yonekawa et al., 1980). Control groups were pretreated with SS (1 ml/kg, i.p., nZ21; or s.c, nZ8). The following changes were visually evaluated during 30 min after PTZ administration: the latencies to the first myoclonus, to the first clonic seizure and to the tonic extension; the total number of clonic seizures as well as the percentage of animals presenting clonic and tonic seizures and death rate. 2.3.2. KA-induced status epilepticus Rats received daily i.p. administration of SS for habituation (see above). Then, KA (10 mg/kg, i.p.) was applied in animals pretreated with 8-OH-DPAT, Indorenate, WAY100635 or SS (nR6 per dose for each drug) at the doses and routes of administration described previously (see PTZ-induced clonicetonic seizures). It is known that KA causes stereotyped behaviors including staring, wet dog shakes (WDS), automatisms, myoclonic jerks of head and upper limbs, and generalized seizures (GS) involving rearing, bilateral upper extremity clonic movements, falling and salivation. Finally, the seizure activity culminates into a partial SE, i.e., the seizures are repeated frequently enough that recovery between attacks does not occur (Lothman and Collins, 1981). The total number, latencies and the percentage of animals presenting WDS, GS and SE were evaluated continuously during 3 h after KA injection. 2.3.3. Amygdala-kindled seizures Rats were anesthetized with a combination of ketamine (100 mg/kg, i.p.) and xylazine (20 mg/kg, i.m.).
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Then a bipolar electrode, consisting of two twisted strands of stainless steel wire, insulated except at the cross section of their tips, was stereotactically implanted into the right amygdala. Coordinates in millimeters were as follows: 2.5 caudal to bregma, 4.5 from the midsagittal plane and 8.5 from the skull surface (Paxinos and Watson, 1998). The electrode was attached to male connector pins, which were inserted into a connector strip. Stainless steel screws were threaded into the cranium over the cortex. The electrode assembly was then fixed to the skull with dental acrylic. One week after surgery, animals received daily kindling stimulation (1 strain of 1 ms pulses at 60 Hz and 400 mA, which were produced with a Grass S-48 stimulator) until reaching the kindled state, i.e. three consecutive stage V kindled seizures. Twenty-four hours after the last kindled seizure, they were divided into two experimental subgroups as follows. 2.3.3.1. KindledD8-OH-DPAT group (n[7). Kindled rats received one of the following treatments consecutively, and each one every 48 h: (1) SS (1 ml/kg, i.p.); (2) WAY100635 (1 mg/kg, s.c.); (3) 8-OH-DPAT (0.1, 0.3 and 1 mg/kg s.c.); (4) WAY100635 (1 mg/kg, s.c.) immediately after 8-OH-DPAT (1 mg/kg, s.c.); and (5) manipulation without any administration. A kindling stimulation was applied 20 min after each treatment. 2.3.3.2. KindledDINDO group (n[7). Animals were manipulated as described above for the KindledC8OH-DPAT group, except that they received Indorenate (1, 3 and 10 mg/kg i.p., 90 min before the electrical stimulation) instead of 8-OH-DPAT. Following the kindling stimulation, behavioral responses were rated according to Racine’s scale (Racine, 1972), whereas AD duration and spike frequency were determined from the electrical activity recorded by a Grass model 8-18D EEG machine. Immediately after the ictal event, refractoriness to subsequent seizures during the postictal period was estimated using the recycling paradigm described previously by Engel and Ackermann (1980). It comprised a series of seven kindling stimulations, including the one applied after the treatments (see above), and spaced at 2 min following the end of each preceding AD. Behavioral changes induced after each stimulation were evaluated according with the scale described by Racine (1972). Then, the seizure stages were averaged for each rat across the seven stimulation trials, the mean being referred to as the seizure index (SI). A high SI indicated a weak and short-lasting seizure inhibiting capacity during the postictal period, whereas a low SI indicated a strong and long-lasting seizure inhibiting capacity (Engel and Ackermann, 1980). At the end of the kindling experiments, animals were killed by decapitation and the brain removed. Frozen
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coronal sections of 20 mm were stained with cresyl violet and examined with a light microscope to verify electrode implantation within the amygdala. 2.4. Statistical analysis The percentage of animals presenting myoclonus, clonic seizures, tonic extension and mortality rate produced by PTZ as well as the percentage of rats showing SE induced by KA were statistically analyzed using the c2 test. Latencies to the different behavioral changes produced by PTZ and KA were analyzed by oneway analysis of variance (ANOVA) testing followed by the Bonferroni test. Data obtained from kindled rats were evaluated using one-way ANOVA for repeated measurements followed by the Bonferroni test. A two-way ANOVA followed by Bonferroni test was done looking for drug interaction between 5-HT1A agonists (8-OHDPAT and Indorenate) and antagonist (WAY100635). All statistical analyses were carried out with GraphPad Prism softwareÔ version 4.00 for Windows, and significances were assigned at the 0.05 level.
3. Results 3.1. Effects of 8-OH-DPAT and Indorenate on PTZ- induced clonicetonic seizures In rats pretreated with SS, the latencies for PTZinduced changes were as follows: myoclonic seizures at 0.93G0.05 min (s.c.) and 1.1G0.13 min (i.p.); clonic seizures at 1.9G0.23 min (s.c.) and 1.4G0.22 min (i.p.); and tonic extension at 4.5G1.4 min (s.c) and 2.1G0.14 min (i.p.). All animals (100%) pretreated with SS showed myoclonus and clonic seizure, whereas the tonic extension was produced in 48% (s.c.) and 63% (i.p.). Death rate was 50% (s.c.) and 57% (i.p.). Animals pretreated with the different doses of 8-OHDPAT failed to present significant changes in the latencies to the PTZ-induced myoclonus, clonic and tonic seizures, when compared with its control group (SS, s.c.) (Fig. 1). However, the percentage of rats with tonic extension and the mortality rate decreased in a dose-dependent way (Table 1). At 1 mg/kg, 8-OHDPAT significantly enhanced the number of PTZ-induced clonic seizures, an effect blocked by WAY100635. The administration of WAY100635 alone did not induce significant changes (Fig. 2). Indorenate at 1 and 3 mg/kg did not modify the PTZinduced seizures when compared with SS i.p. pretreatment. In contrast, at 10 mg/kg, this drug increased the latencies to the myoclonus (213%, P!0:05), to the clonic seizures (175%, not significant, n.s.) and the tonic extension (257%, P!0:05) induced by PTZ, effects that were blocked with WAY100635 (Fig. 1). In addition,
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Fig. 2. Effects of 8-OH-DPAT on the number of clonic seizures induced by PTZ. Values are represented as meanGS.E.M. from nR6 rats per group. The data were analyzed by two-way ANOVA followed by the Bonferroni test. *P!0:05.
Fig. 1. Effects of 8-OH-DPAT (above) and Indorenate (below) on the latency (min) to the convulsions induced by PTZ in rats. Each data point represents the meanGS.E.M. values for nR6 rats per dose. *Significant effect compared with the SS group (Two-way ANOVA followed by the Bonferroni test, *P!0:05).
Indorenate dose-dependently reduced the percentage of rats presenting clonic seizures, tonic extension and death (Table 1). 3.2. Effects of 8-OH-DPAT and Indorenate on KA-induced limbic seizures and SE In the group pretreated with SS, KA induced the first WDS at 26.9G1.8 min (100%), the first GS at 70.2G2.9 Table 1 Effects of 8-OH-DPAT and Indorenate on the percentage of animals presenting myoclonus, clonic seizures, tonic extension and death after the administration of PTZ (60 mg/kg, i.p.) Treatment (mg/kg)
Myoclonus
Clonic seizure
Tonic extension
Death
SS 8-OH-DPAT 0.01 8-OH-DPAT 0.1 8-OH-DPAT 1
100 100 100 100
100 100 100 100
63 50 ) 43 50
50 43 43 ) 13
SS Indorenate 1 Indorenate 3 Indorenate 10
100 100 100 100
100 91.7 82.4 82.4
48 50 47 ) 27
48 50 41 ) 27
The values indicate the percentage of rats per group. The data were analyzed by c2 test. * P!0:05 vs. SS group.
min (100%) and SE at 93G4.3 min (78%). The animals presented an average of 129G13.8 of WDS and 20.4G3 of GS during the recording time. In contrast to the SS group, rats pretreated with 8-OH-DPAT showed dose-dependent enhancement in the latency to the first WDS, to the first GS and to the SE. 8-OH-DPAT also decreased the total number of WDS and GS during the period of evaluation. At 0.01 mg/kg this drug enhanced the rate of animals presenting SE (22%; P!0:05), whereas at 0.1 and 1 mg/kg it decreased this parameter (11%, n.s. and 17%, P!0:05, respectively) (Fig. 3). WAY100635 fully blocked the effects induced by 8-OH-DPAT on the latencies to the WDS, to the GS and to the SE, but it did not modify the changes induced on the total number of WDS and GS. WAY100635 alone did not alter the KA-induced seizures (Table 2). Indorenate caused significant dose-dependent increases in the latencies to the first WDS, to the first GS and to the SE induced by KA, in comparison with the pretreatment with SS. This 5-HT1A agonist also diminished the total number of GS, but it did not modify the total number of WDS and the rate of animals showing SE (Fig. 3). WAY100635 fully blocked these effects, except those associated with the GS (Table 2). 3.3. Effects of 8-OH-DPAT and Indorenate on kindled seizures and postictal depression After the SS administration, all kindled animals presented stage V seizures with an AD duration of 89.4G9.26 min (KindledC8-OH-DPAT group) and 60G8.6 min (KindledCINDO group), and spike frequency of 9.7G0.92 cps (KindledC8-OH-DPAT group)
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Fig. 3. Effects of 8-OH-DPAT and Indorenate on the latency (left) and number of events (right) of WDS (upper panel) and GS (middle panel) induced by KA. Latency to the SE and the percentage of rats showing SE are indicated in the lower panel. Each data point represents the meanGS.E.M. values for nR6 rats per dose. The data were analyzed by one-way ANOVA followed by the Bonferroni test (latencies and number of events) or c2 test (percentage of rats presenting SE). *P!0:05 vs. SS.
and 7.8G1.2 cps (KindledCINDO group), respectively. The SI after SS administration (basal level) was 2.15G0.21 for the KindledC8-OH-DPAT group and 2.36G0.21 for the KindledCINDO group.
When compared with SS administration, the treatment with 8-OH-DPAT (0.1, 0.3 and 1 mg/kg) failed to modify the behavioral and electrographic expression of kindled seizures and the SI obtained with the recycling
Table 2 Effects of WAY100635 on changes produced by 8-OH-DPAT and Indorenate on the seizures and SE induced by KA Treatment
Latency to WDS (min)
Latency to GS (min)
Latency to SE (min)
No. of WDS
No. of GS
SS (1 ml/kg) 8-OH-DPAT 1 mg/kg Indorenate 10 mg/kg WAY100635 1 mg/kg 8-OH-DPATCWAY100635 IndorenateCWAY100635
26.9 G 1.8 ) 44.5 G3.2 ) 49.7 G 2.7 30 G 2.7 29 G 2.7& 36 G 2.7#
70.2 G 2.9 ) 98.6 G 7.3 ) 94.6 G 5.4 74.5 G 6.8 62.3 G 10.7& 79 G 3.3
93 G 4.3 ) 124.4 G 8.5 ) 128 G 8.8 96.6 G 8 62.2 G 11& 94.3 G 5.6#
129 G 13.8 ) 80.9 G 10.6 90.6 G 17.8 96 G 10.7 82.4 G 18.8 106.4 G 15.6
20.4 G 3 ) 8.6 G 1.4 ) 10.3 G 1.8 14.4 G 3.4 11.7 G 3.1 8.8 G 1.4
Data are the meanGS.E.M. from nR6 rats per group. Values were analyzed by two-way ANOVA followed by the Bonferroni test. * P!0:05 vs. SS. & P!0:05 vs. 8-OH-DPAT. # P!0:05 vs. Indorenate.
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paradigm (Fig. 4). Indorenate (1, 3 and 10 mg/kg) did not change the expression of kindled seizures, but at 10 mg/kg, it decreased the SI (35%, P!0:05), an effect that was blocked with WAY100635 (Fig. 4). The SI values obtained during the last trial (manipulation without treatment) were similar to those obtained during the first trial after SS administration, supporting that the pharmacological manipulation used in this experiment did not modify seizure susceptibility. The histological analysis revealed that the implanted electrodes were located at the central and basolateral amygdala nuclei.
4. Discussion The present study evaluated the effects of 5-HT1A receptor agonists in the convulsive seizures induced by
three different experimental models. We found that the protective effects of 8-OH-DPAT and Indorenate were different and depended on the type of convulsive activity evaluated. PTZ is a non-competitive g-aminobutyric acid receptor antagonist that may produce generalized seizures (Yonekawa et al., 1980). In this study, 8-OH-DPAT did not modify the latency of the PTZ-induced seizures, but decreased the mortality rate. In contrast, Indorenate augmented the latencies to the different convulsions produced by PTZ and decreased the percentage of rats with tonic extension as well as the mortality rate. The changes induced by 8-OH-DPAT and Indorenate were partially blocked by WAY100635, suggesting that their protective effects on the PTZ-induced convulsions were not totally mediated by 5-HT1A receptors. It has been described that the forebrain is involved in the expression of clonic seizures, whereas the activation
Fig. 4. Effects of 8-OH-DPAT (left) and Indorenate (right) on the afterdischarge duration (upper panel) and spike frequency (middle panel) during the kindled seizures, as well as on the Seizure Index (lower panel) during the postictal period. Values are represented as meanGS.E.M. of seven rats and were analyzed by two-way ANOVA for repeated measurements followed by the Bonferroni test. *P!0:05.
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of brainstem structures participates in the expression of the tonic component, and that the serotonergic system is present in both brain areas (Browning, 1985; Lazarova et al., 1983; Pazos and Palacios, 1985; Yonekawa et al., 1980). The changes induced by 8-OH-DPAT and Indorenate on PTZ-induced myoclonus and clonic seizures could result from the activation of 5-HT1A receptors in forebrain, while the reduced tonic extension component and mortality rate could be due to an activation of serotonergic mechanisms in the brainstem that contains high density of 5-HT1A receptors (Pazos and Palacios, 1985). Repeated generalized seizures are associated with high intensity of the postictal depression (Mucha and Pinel, 1977). The elevated incidence of clonic seizures produced by 8-OH-DPAT could be associated with a strong postictal depression that may reduce the expression of the PTZ-induced tonic component. However, we cannot discard that 8-OH-DPAT, at 1 mg/kg, produces toxic effects associated with increased excitability and with augmented clonic seizures. The systemic injection of KA initially induces an epileptic focus in hippocampus and thereafter, seizure activity propagates to limbic and extralimbic brain areas (Lothman and Collins, 1981). In the present study, 8-OH-DPAT delayed the appearance and decreased the incidence of WDS, GS and SE induced by KA, while Indorenate retarded the appearance of WDS, GS and SE and decreased the incidence of GS. Since some of these changes were blocked by WAY100635, it is possible to support the idea that activation of 5-HT1A receptors induces protective effects on KA-induced seizures (Gariboldi et al., 1996). The ventral hippocampus and especially the dentate granule cells are essential for WDS expression and seizure spreading (Grimes et al., 1990). The KA-induced WDS represent neuronal hyperactivity in limbic structures that spread to midbrain areas and motor system (Lothman and Collins, 1981; Velı´ seˇk et al., 1988; Veliseˇk and Mares, 2004). The decreased incidence of WDS and GS induced by 8-OH-DPAT and Indorenate could result from neuronal hyperpolarization produced by activation of postsynaptic 5-HT1A receptors in the hippocampus (Lu and Gean, 1998). In fact, in normal conditions, the dentate gyrus and CA1-CA3 fields contain high density of 5-HT1A receptors (Pazos and Palacios, 1985). Interestingly, the effect of 8-OH-DPAT on KAinduced seizures depended on the dose applied, i.e., at low doses (0.01 mg/kg), it increased the percentage of animals showing SE, whereas at higher doses it produced protective effects. The exact explanation for this 8-OH-DPAT effect is unknown, and it is complicated by the fact that the precise role of 5-HT1A receptors in the etiology of SE of limbic seizures has been scarcely evaluated. However, the present results agree with those
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reported by Watanabe et al. (2000), who described that WAY100635 at 0.1 mg/kg increased the AD duration produced in hippocampus of rats, while at higher doses (0.3 and 1 mg/kg), it delayed the appearance for subsequent AD and decreased its duration. It is possible to suggest that the anti- or proconvulsant effects of 8-OH-DPAT depend on its pre- or postsynaptic actions. Further experiments applying 8-OH-DPAT in specific brain areas, such as raphe nucleus and hippocampus, will help to determine the exact participation of pre- and postsynaptic 5-HT1A receptors on the regulation of convulsive activity in SE and other experimental models. SE in humans and animals is associated with elevated systemic arterial pressure (Simon et al., 1997). The maintenance of systemic blood pressure to provide adeuate cerebral perfusion could minimize or prevent neuropathologic sequels to SE of extended duration (Walton, 1993). The cardiovascular effects of 8-OHDPAT and Indorenate were not evaluated in the present study. However, it is known that these drugs decrease the blood pressure in hypertensive animals in different experimental models (Antonaccio and Kerwin, 1981; Gradin et al., 1985), an effect that is not observed in normotensive animals after Indorenate administration (personal communication by E. Hong). In the present study, 8-OH-DPAT and Indorenate induced significant effects in KA-induced SE. It is possible that antihypertensive effects mediated by these drugs could impact directly or indirectly on seizure duration, and/or neuronal injury. Future experiments should be focused to test this hypothesis. Concerning amygdala kindling, a model of epileptogenesis that produces complex partial seizures with secondary generalization (Sato et al., 1990), it was found that 8-OH-DPAT and Indorenate did not modify the expression of generalized kindled seizures. These findings are in agreement with previous reports in which the systemic administration of 8-OH-DPAT does not change the amygdala and hippocampal kindled seizures in rats (Cagnotto et al., 1998; Lo¨scher and Czuczwar, 1985). In contrast, experiments carried out in cats indicate that the systemic administration of 8-OHDPAT avoids the expression of hippocampal kindled seizures, whereas the intrahippocampal injection of this drug enhances the AD threshold to kindled seizures and reduces the AD duration of partial convulsions (Wada et al., 1992, 1993). It is difficult to compare the aforementioned studies because the discrepant results could be due to the different experimental designs such as the nucleus of electric stimulation, species used and route of drug administration. It is worthy of note that Indorenate augmented the refractoriness to subsequent seizures during the postictal depression, an effect that was blocked by WAY100635, whereas 8-OH-DPAT did not induce significant changes in this parameter. The hippocampus is a key structure
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for the expression of postictal depression (CaldecottHazard and Engel, 1987) and 5-HT1A receptor levels are enhanced in the rat dentate gyrus 24e48 h after the last kindled seizure (Cagnotto et al., 1998; Clark et al., 1993). The enhanced postictal depression induced by Indorenate could result from the activation of hippocampal postsynaptic 5-HT1A receptors. The fact that 8-OH-DPAT and Indorenate were more effective in reducing the incidence of generalized seizures induced by PTZ when compared with KA and kindled seizures, suggests that these drugs are more effective when the epileptic activity is initially generalized. The protective effects of 8-OH-DPAT and Indorenate could depend on the mechanisms of regulation of seizure spread in each experimental model. Thus, in PTZinduced generalized convulsions, the 5-HT1A receptor agonists may be reducing or avoiding the propagation of epileptic activity from forebrain to brainstem, whereas in KA-induced seizures these drugs only delay the spread of epileptic activity from the epileptic focus (hippocampus) to other limbic or extralimbic areas. Another interesting point is that the 5-HT1A agonists used in the present study did not modify the expression of the generalized seizures in the epileptic brain of kindled animals, although Indorenate enhanced the postictal depression. Further experiments evaluating the actions of 8-OH-DPAT and Indorenate in different experimental models of epilepsy are necessary to corroborate the role of 5-HT1A receptors in epilepsy and postictal depression. The present study demonstrates that 8-OH-DPAT and Indorenate produce different effects, that could result from the stimulation of pre- or postsynaptic 5-HT1A receptors, a situation that may depend on the nature of the convulsive activity as well as the neurotransmitters and brain areas involved in the regulation of seizure activity. Supporting this complex situation, it is currently controversial whether the effects of 5-HT1A receptor agonists in anxiety are due to somatodendritic or post-synaptic receptor activation, since results obtained depend on the behavioral paradigm, the species used and the route of drug administration (Cervo et al., 2000; De Almeida et al., 1998; Ferna´ndez-Guasti et al., 1992; Lo´pez-Rubalcava, 1996). Additionally, 8-OH-DPAT and Indorenate may produce non-specific effects. Indorenate acts with less affinity on 5-HT1B and 5-HT2A/2C receptors (Sa´nchez and Vela´zquez-Martı´ nez, 2001), whereas 8-OH-DPAT has agonist properties for 5-HT7 (Ruat et al., 1993) and a1 -adrenergic receptors (Castillo et al., 1993). It is possible that some of the effects induced by 8-OH-DPAT and Indorenate on seizure activity could be mediated by their non-specific actions. Additional experiments must be conducted to explore the possible participation of 5-HT7, 5-HT1B and 5-HT2A/2C receptors as well as the a1 -adrenergic receptor in the protective effects of 8-OHDPAT and Indorenate on the epileptic activity.
According with the data presented, it is concluded that 8-OH-DPAT and Indorenate: (a) exert differential effects on convulsive activity; (b) produce protective effects more efficiently in the PTZ- and KA-induced seizures; (c) do not modify the seizures in an epileptic brain (kindled seizures), but are able to enhance the postictal depression of kindled animals; (d) induce protective effects partially or fully mediated by 5-HT1A receptors. Further experiments should be carried out to determine whether 5-HT1A receptor agonists could be used alone or in combination with classical antiepileptic drugs to avoid or decrease seizure activity and/or improve protective mechanisms during the postictal depression.
Acknowledgements We are indebted to Mrs. Magdalena Briones and Mr. He´ctor Va´zquez for their technical assistance. This study was partially supported by Consejo Nacional de Ciencia y Tecnologıa (CONACYT) scholarship 153240.
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