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Gamma aminobutyric acid mediation of the anticonvulsant effect of clonidine on pentylenetetrazol-induced seizures in mice
273
GAMMA AMINOBUTYRIC ACID MEDIATION OF THE ANTICONVULSANT EFFECT OF CLONIDINE ON PENTYLENETETRAZOL-INDUCED SEIZURES IN MICE GEORGE Dcpar-tmrnt
AMABEOKU,
ORDIAS
CHTKUNI
and ETHEL
of Clinical
BWAKURA
Phut-mac~olo~g_y, Medical School. University PO Bar Al 78. A\totzdale, Harrct-e, Zimbcrbwe
of Zimbabwe,
SUMMARY The effects of muscimol, amino-oxyacetic acid (AOAA), diamino-N-butyric acid (DABA), bicuculline, picrotoxin, diazepam and phenobarbitone on the protective effect of clonidine against pentylenetetrazol-induced seizures were studied in mice. Muscimol, AOAA, DABA, phenobarbitone and diazepam significantly protected mice against pentylenetetrazol-induced seizures and also significantly potentiated the protective effect of clonidine against the seizures. Bicuculline and picrotoxin significantly potentiated seizures induced by pentylenetetrazol and significantly attenuated both the protective effects of muscimol and clonidine against the seizures. These data suggest that activation of gamma-aminobutyric acid systems may underlie the protective effect of clonidine against seizures induced by pentylenetetrazol in mice. KEY WOKDS:clonidine,
GABAergic
neurotransmission,
leptazol, seizures.
INTRODUCTION Clonidone, an antihypertensive agent, is a selective alpha-2 adrenoceptor agonist. Central alpha-2 adrenoceptors have been implicated in seizure mechanisms. Clonidine has been shown to protect against leptazol-induced seizures in rats [l] and against audiogenic seizures in mice [2]. In contrast, proconvulsant properties of selective alpha-2 adrenoceptor antagonists, yohimbine [3,4] and idazoxan [S, 61 have been described. Clonidine enhances the release of endogenous gammaaminobutyric acid (GABA) in various brain areas [7, 81. It has also been reported that clonidine protected against metaldehyde-induced seizures by raising brain concentrations of GABA and inhibiting the increase in brain monoamine oxidase enzyme activity [9]. In fact, it has been suggested that some effects of clonidine in mammals may occur through activation of the GABA system [S, lo]. Since GABA, a major inhibitory neurotransmitter in mammalian brain has been shown Correspondence 1043-661
to: G. Amabcoku
X/‘)4/030273-08/$08.00/0
0 I994
The Italian Pharmacological
Society
to exert an inhibitory influence on pentylenetetrazol (PTZ)-induced seizures [ 1 I-131, it is possible that the anticonvulsant effect of clonidine on PTZ-induced seizures may be mediated by its ability to alter the activity of GABA-containing neurons in the brain. This project was intended, therefore, to investigate the role of GABA in the protective effect of clonidine against PTZ-induced seizures by studying the effects of muscimol, amino-oxyacetic acid (AOAA), diamino-Nbutyric acid (DABA), bicuculline, picrotoxin, phenobarbitone and diazepam on the anticonvulsant effect of clonidine in mice.
MATERIALS
AND METHODS
Animals
Male albino mice bred in our Animal House, weighing 22-26 g, were used in this study. The animals were housed in groups of eight per cage and allowed free access to water and food. l!higs Pentylenetetrazol (Leptazol, Sigma Chemical Co.), clonidine hydrochloride (Sigma), muscimol (Sigma), amino-oxyacetic acid hemihydrochloride (AOAA, Sigma), DL-2, 4-diamino-N-butyric acid dihydrochloride (DABA, Sigma), picrotoxin (Sigma) and phenobarbitone (Paris Chemical) were all dissolved in physiological saline. (+) Bicuculline (Sigma) was suspended in Tween 80 and adjusted to the appropriate volume with physiological saline. A commercial preparation of diazepam (Diazam, Ceddes Zimbabwe) was dissolved in a minimum amount of polyethylene glycol 400 (Fluka AG, Buchs) and adjusted to the appropriate volume with physiological saline. All drugs were administered intraperitoneally (i.p.) in a volume of 1 ml per 100 g body weight of animal. Control animals received equal-volume injections of the vehicle which included saline and Tween 80 and polyethylene glycol dissolved in saline. Fresh drug solutions were prepared each day of the experiment. The drug pretreatment times prior to the injection of pentylenetetrazol were clonidine (45 min), muscimol (1 h), AOAA (20 min), DABA (30 min), bicuculline (10 min), picrotoxin (10 min), phenobarbitone (10 min) and diazepam (20 min). The pretreatment times used were as described by Amabeoku [14] and Amabeoku and Chikuni [15]. However, the doses used were established by preliminary studies.
A modified version of the method of Vellucci and Webster [16] was used to assess the convulsant activity of pentylenetetrazol. Eight mice per dose of- drug were used. The animals were kept individually in transparent perspex cages (25x 15x15 cm) for 30 min to acclimatise to the new environment prior to the administration of drug. Mice were observed for seizures for 30 min following the administration of pentylenetetrazol. The time taken for the onset of tonic convulsions and the proportion of mice convulsing were recorded. Animals that did not convulse within a 30 min period were recorded as not convulsing. The behaviour of animals after drug administration was also observed. The control and
Phar-muc~olo~iculResearch. Vol. 29, No. 3. 1994
275
test experiments were performed between 12.00 hrs and quiet room with an ambient temperature of 21+2”C.
17.00 hrs each
day
in a
Statistical anulysis The data on the time taken for the onset of tonic convulsions was compared by one way analysis of variance (ANOVA) followed by Duncan’s test. Analysis of the number of mice convulsing was carried out using the chi-squared test with Yate’s correction for continuity.
RESULTS
Convulsant effect of pentylenetetrazol and the effect of clonidine on pentylenetetrazol-induced seizures in mice Table I shows that clonidine (0.5 mg kg-‘) significantly delayed the onset and reduced the incidence of pentylenetetrazol (90 mg kg-‘)-induced tonic seizures. Clonidine (1 mg kg-‘) significantly delayed the onset of the seizures but did not affect the incidence significantly. However, clonidine (0.125-0.25 mg kg-‘) did not alter the onset or the incidence of the seizures significantly. Clonidine, in all the doses used, did not affect the myoclonic jerks which preceded the tonic seizures elicited by pentylenetetrazol (results not shown).
Table I Convulsant effect of pentylenetetrazol (PTZ) and the effect of clonidine on PTZ-induced seizures in mice Drug and dose (rng kg-‘, i.p.) PTZ 60 70 75 SO 90 100 90 90 90 90 90 *PiO.Ol tPiO.05
Clonidine
No. convulsed/ no. used
Onset of tonic convulsion: meanfsEM (min)
O/8
O/8 l/8 -
0.125 0.25 0.5 1.0 vs PTZ (90 mg kg-‘) control, I’S PTZ (90 mg kg-‘) control,
4/8 8/8 8/8 8/8 718 4/8 3/8t 4/8
15.0+0 7.5+1.09 5.5f0.64 4.5f0.50 4.5kO.56 4.83LkO.59 7.5f0.83 16.33*f2.23 12.25*+1.34
Duncan’s test. chi-squared test.
Ejfects ofmuscimnl, AOAA and DABA and their inter-actions with clonidine on pentylenctruzol-induced seizures in mice Table II shows that muscimol (0.5-l mg kg-‘), AOAA (lo-20 mg kg-‘) and DABA (8-16 mg kg-‘) significantly delayed the onset of seizures induced by pentylenetetrazol (90 mg kg-‘) and significantly reduced the number of animals
Table II Effects of muscimol, aminooxyacetic acid (AOAA), diamino-?;-butyric (DABA) and their interactions with clonidine on pentylenetetrazol (PTZ)-induced seizures in mice
acid
Onw of tonic con~‘ulsioJ1: n?L’UKkSEM (MiJ?) 90
-
90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90
0.125 0.5
0.125 0.5
-
-
0.25 0.5 1.0 0.25 0.25
5
0.125 0.5
-
10 20 5 5
-
-
0.125 0.5
-
318” 518 l/8**” 3/s*
2188 O/8
-
-
X/8 718
4/x 2/8** 2/8**
4 8 16 4 4
l/8?? O/8 518 3/X” O/8*+“* 2189 O/8
4.63fO.M 5.17?0.76 ls.61+0.723 5.4 f0.12 I 1 .o fOi 9.33*0.54+ 13.0~0.71$ 5.75kO.41 9.5 +0.3si1I IM.7 I -i12.0 _+o,t 5.0 +I.23
I I .33k1,66f-
12.5_+1.06$
i-P
convulsing. However. muscimol (0.25 mg kg-‘), AOAA (5 mg kg ‘) and DABA (4 mg kg-‘) neither affected the onset nor the incidence of the seizures significantly. Muscimol (0.25 mg kg-‘) potentiated the effect of subeffective dose of clonidine (0.125 mg kg ~‘) on pentylenetetrazol-induced tonic seizures. The onset of the seizures and the incidence were significantly delayed and reduced respectively. Similarly, combined injection of AOAA (5 mg kg ‘) or DABA (4 mg kg-‘) and a subeffective dose of clonidine (0.125 mg kg-‘) significantly delayed the onset of pentylenetetrazol (90 mg kg-‘)-induced seizures and significantly reduced the incidence. Furthermore, combined injection of either muscimol (0.25 mg kg’), AOAA (5 mg kg-‘) or DABA (4 mg kg ‘) and an effective dose of clonidine (0.5 mg kg-‘) completely abolished the tonic seizures induced by pentylenetetrazol. The myoclonic jerks preceding the tonic seizures were also completely abolished.
Table III shows that bicuculline (2.5-5.0 mg kg-‘) potentiated tonic seizures induced by pentylenetetrazol (75 mg kg’) by significantly shortening the onset of the seizures and increasing the number of mice convulsing. A dose of 2.5 mg kg’
Effects
Table III of bicuculline, picrotoxin and their interactions with clonidine pentylenetetrazol (PTZ)-induced seizures in mice
against
Onset of tonic convulsion: meanfsEM (min) IS 90 YO 90 75 15 90 90 90 75 7s 00 90 90
0.5
0.5
2.5 5.0 2.5 2.5 2.5
-
8/8llll 8/8nll 8/8 8/8***
0.5 0.5 0.5 1.0
1.0 1.0 I .o
0.5
218 X/8 2/811 II S/811
8/8$$ 6/8(
-
8/8llll 8/8 6/g**
0.5
X/833
11.50+1.06 4.88f0.82 14.50~1.06ti 11.33+0.72tt 6.75+0.70* 5.88f0.60* 2.75*0.42t 7.88f0.28$ 5.15kO.233 7.1lf0.12* 6.13f0.45* 2.3s+0.39t 9.38+0.44$ 7.38kO.688
*P
bicuculline also effectively shortened the onset of pentylenetetrazol (90 mg kg-‘)induced seizures. Bicuculline (2.5 mg kg~‘) effectively antagonised the protective effects of both muscimol (0.5 mg kg-‘) and clonidine (0.5 mg kg-‘) against pentylenetetrarol (90 mg kg-‘)-induced seizures by significantly shortening the onset and increasing the incidence. Picrotoxin (0.5-1.0 mg kg-‘) augmented pentylenetetraLol (75 mg kg~‘)-induced tonic seizures by significantly shortening the onset of the seizures and increasing the incidence. ( 1 .O mg kg ‘) significantly shortened the onset of pentylenetetrazol Picrotoxin (90 mg kg-‘)-induced seizures. 1 .O mg kg-’ picrotoxin antagonised the protective effects of both muscimol (0.5 mg kg-‘) and clonidine (0.5 mg kg-‘) against tonic seizures induced by pentylenetetrazol (90 mg kg-‘) by significantly shortening the onset and increasing the incidence of the seizures. Bicuculline and picrotoxin when given alone did not produce any seizures in all the doses used (results not shown).
Table 1V shows the phcnobarbitone onset and reduced the incidence seizures. 10 mg kg-’ phenobarbitone
(12.5-I 5 mg kg ‘) significantly delayed the of pentylenetetrazol (90 mg kg-‘)-induced significantly prolonged the onset but did not
Table IV Effects of phenobarbitone, diazepam and their interactions with clonidine on pentylenetetrazol (PTZ)-induced seizures in mice Drug and dose (mg kg-‘, ip) PTZ 90
90 90 90 90 90 90 90 90 90
Pkenohur-hitone
Dias-pam
-
-
10
-
12.5 15 10 -
0.1 0.2 0.4 0.1
Clonidine
0.125 0.125 0.125
No. convulsed/ no. used
Onset of tonic convulsion: mean+sm (min)
K/8
5.26+0.66 5.29f0.69 10.57f0.97” 14.66&l .08* 18.0+0* 14.0+0t 5.38kO.73
718 7/8 3/Q l/8$3$ 1/85§ 8/8 4/8 2/8$$ 2/8§
11.5+0.X3* 15.0+0.71* 12.5+0.35*
*P
affect the incidence of the seizures. Phenobarbitone (10 mg kg-‘) potentiated the effect of subeffective dose of clonidone (0.125 mg kg-‘) on pentylenetetrazolinduced seizures by significantly delaying the onset and reducing the incidence. Diazepam (0.4 mg kg-‘) significantly prolonged the onset of pentylenetetrazol (90 mg kg-‘)-induced seizures and reduced the number of animals convulsing. A dose of 0.2 mg kg-’ diazepam significantly prolonged the onset but weakly reduced the incidence of the seizures. However, diazepam (0.1 mg kg-‘) did not alter the onset or incidence of the seizures significantly. The effect of a subeffective dose of clonidine (0.125 mg kg-‘) on the seizures was potentiated by diazepam (0.1 mg kg-‘) which significantly prolonged the onset and reduced the incidence. The myoclonic jerks preceding the onset of tonic seizures were not significantly affected by either phenobarbitone and diazepam. However, the combined injection of phenobarbitone or diazepam and clonidine significantly reduced the myoclonic jerks.
DISCUSSION The present study shows that clonidine (0.5 mg kg-‘) has a protective effect against pentylenetetrazol-induced seizures which is consistent with the observations of Papanicoloau it ul. [ 1J who reported the anticonvulsant effect of clonidine. Muscimol, a selective GABA,, receptor agonist, mimics the inhibitory effects of GABA by interacting with GABA,% receptors in the brain. Moreover, GABA is a major inhibitory neurotransmitter in the brain and the impairment of its neurotransmission has been associated with epilepsy [ 171. In this study, muscimol potentiated the protective effect of clonidine against pentylenetetrazol-induced
seizures. These data implicate GABA neurotransmission in the effect of clonidine against seizures induced by pentylenetetrazol. Bicuculline, a potent GABAA receptor antagonist, in the present study, inhibited the protective effects of muscimol and clonidine against seizures induced by pentylenetetrazol in a similar manner. This further implicates GABA receptor activation in clonidine effect against pentylenetetrazol-induced seizures. This hypothesis is further supported by the observation that picrotoxin, a GABA antagonist, which acts by blocking chloride channels linked to GABA, receptors, antagonised the effects of muscimol and clonidine against seizures induced by pentylenetetrazol in a similar way. AOAA is a potent inhibitor of GABA transaminase (GABA-T), an enzyme which metabolises GABA thereby leading to an increase in brain GABA levels. The present data show that AOAA potentiated the protective effect of clonidine against pentylenetetrazol-induced seizures. The increased GABA level as a result of the activity of AOAA may be responsible for the potentiation of clonidine effect against pentylenetetrazol seizures. Similarly, DABA was shown in this study to augment the protective effect of clonidine against seizures induced by pentelenetetrazol. DABA is a potent and selective inhibitor of GABA uptake, which results in the accumulation of GABA in the brain. It is, therefore, probable that the potentiation by DABA of clonidine effect may be due to an increase in the brain levels of free GABA available at the postsynaptic receptor sites. This still supports the hypothesis that GABA system activation may be involved in the protective effect of clonidine against pentylenetetrazol-induced seizures. Post-synaptic GABA, receptors are functionally linked to benzodiazepine receptors, barbiturate receptors and chloride channets to form a GABA-chloride ionophore complex which is involved in the modulation of GABAergic inhibitory transmission [ 181. Barbiturates and benzodiazepines have been reported to increase chloride flux through chloride channels at GABA, receptor sites to enhance GABAergic inhibition [18]. It is important that in this study, phenobarbitone, a barbiturate, and diazepam, a benzodiazepine, protected mice against pentylenetetrazol-induced seizures and also potentiated the protective effect of clonidine against the seizures. In conclusion, the data obtained in this study suggest that enhancement of GABA neurotransmission potentiated the protective effect of clonidine against pentylenetetrazol-induced seizures while inhibition attenuated the protective effect of clonidine.
ACKNOWLEDGEMENTS This project was supported by the Research Board, authors wish to thank Miss C. Zambezi for excellent
University secretarial
of Zimbabwe. assistance.
The
REFERENCES 1. Papanicolaou J, Summers KJ. Vajda FJ, Louis WJ. Anticonvulsant effects of clonidine mediated through central alpha-2 adrenoceptors. EM/. .I Pl~rrwcrc~ol l%2: 77: I K-6.
2. Horton R, Anlezark G, Meldrum B. Noradrenergic influence on sound-induced seizures. .I Phwmacol Exp Ther 1980; 214: 437-42. 3. Lloyd KG, Worms P. Differential effect of yohimbine in several chemical models of convulsions in mice. Br .I Pharmucol 1982; 77: 340P. 4. Stark K, Borowski E, Endo T. Preferential blockade of presynaptic alpha adrenoceptors by yohimbine. Eur- J Phurmacol 1975; 34: 385-8. 5. Doxey JC, Roach AG, Smith CFC. Studies on RX781094: a selective, potent and specific antagonist of alpha-2 adrenoceptors. BrJ Pharmacol 1983; 78: 489-505. A, Forster EA. Proconvulsant actions of selective alpha-2 adrenoceptor 6. Fletcher antagonists. BrJ Pharmacol 1984; 81: 39P. 7. Pittaluga A, Raiteri M. Clonidine enhances the release of endogenous gamma aminobutyric acid through alpha-2 and alpha-l presynaptic adrenoceptors differentially located in rat cerebral cortex subregions. J Pharmacol Exp Ther 1988; 245: 682-6. 8. Pittaluga A, Torelli R, Raiteri M. Clonidine differentially modulates the release of endogenous GAB A in various rat brain areas. Pharmucol Res 199 1; 24: 189-96. 9. Homeida AM, Cooke RG. Anticonvulsant activity of diazepam and clonidine on metaldehyde-induced seizures in mice: effects on brain gamma-aminobutyric acid concentrations and monoamine oxidase activity. J Vet Pharmacol Ther 1982; 5: 187-90. IO. Czyzewska-Szafran H, Jastrz:ebski Z, Remiszewska M, Wutkiewicz M. Effect of clonidine on blood pressure and GABAergic mechanisms in spontaneous hypertensive rats. Eur J Pharmacol I991 ; 24: 189-96. I 1. Kent AP, Webster RA. The role of GABA and excitatory amino acids in the development of the leptazol-induced epileptogenic EEG. Neuropharmacolo,gy 1986; 25: 1023-30. GABAergic pathway in 12. Okada R, Negishi N, Nagaya H. The role of the nigrotegmental the propagation of pentylenetetrazol-induced seizures. Bruin Res 1989; 480: 383-7. 13. De Deyn PP, Macdonald RL. Effects of antiepileptic drugs on GABA responses and on reduction of GABA responses and by pentylenetetrazol and methyl-6,7-dimethoxy-4ethyl-beta-carboline-3-carboxylate (DMCM) on mouse neurons in cell culture. Epilepsiu 1989; 30: 17-25. 14. Amabeoku G. Involvement of gabaergic mechanisms in chloroquine-induced seizures in mice. Gen Pharmacol 1992; 23: 225-9. IS. Amabeoku GJ, Chikuni 0. Chloroquine-induced seizures in mice: the role of monoaminergic mechanisms. Eur Neur-opsvc’hoi.,har.wrrcr,ol 1993; 3: 37-44. 16. Vellucci SV, Webster RA. Antagonism of caffeine-induced seizures in mice by RO 151788. Eur. J Pharmacol 1984; 97: 289-93. and gamma-aminobutyric acid-mediated inhibition. Int Rel, 17. Meldrum BS. Epilepsy Newohio
1975;
17:
l-36.
18. Olsen RW. GABA-benzodiazepine-barbiturate 37: l-3.
receptor
interactions.
.I Nelo-oc,hcn~ 19X1:
GAMMA AMINOBUTYRIC ACID MEDIATION OF THE ANTICONVULSANT EFFECT OF CLONIDINE ON PENTYLENETETRAZOL-INDUCED SEIZURES IN MICE GEORGE Dcpar-tmrnt
AMABEOKU,
ORDIAS
CHTKUNI
and ETHEL
of Clinical
BWAKURA
Phut-mac~olo~g_y, Medical School. University PO Bar Al 78. A\totzdale, Harrct-e, Zimbcrbwe
of Zimbabwe,
SUMMARY The effects of muscimol, amino-oxyacetic acid (AOAA), diamino-N-butyric acid (DABA), bicuculline, picrotoxin, diazepam and phenobarbitone on the protective effect of clonidine against pentylenetetrazol-induced seizures were studied in mice. Muscimol, AOAA, DABA, phenobarbitone and diazepam significantly protected mice against pentylenetetrazol-induced seizures and also significantly potentiated the protective effect of clonidine against the seizures. Bicuculline and picrotoxin significantly potentiated seizures induced by pentylenetetrazol and significantly attenuated both the protective effects of muscimol and clonidine against the seizures. These data suggest that activation of gamma-aminobutyric acid systems may underlie the protective effect of clonidine against seizures induced by pentylenetetrazol in mice. KEY WOKDS:clonidine,
GABAergic
neurotransmission,
leptazol, seizures.
INTRODUCTION Clonidone, an antihypertensive agent, is a selective alpha-2 adrenoceptor agonist. Central alpha-2 adrenoceptors have been implicated in seizure mechanisms. Clonidine has been shown to protect against leptazol-induced seizures in rats [l] and against audiogenic seizures in mice [2]. In contrast, proconvulsant properties of selective alpha-2 adrenoceptor antagonists, yohimbine [3,4] and idazoxan [S, 61 have been described. Clonidine enhances the release of endogenous gammaaminobutyric acid (GABA) in various brain areas [7, 81. It has also been reported that clonidine protected against metaldehyde-induced seizures by raising brain concentrations of GABA and inhibiting the increase in brain monoamine oxidase enzyme activity [9]. In fact, it has been suggested that some effects of clonidine in mammals may occur through activation of the GABA system [S, lo]. Since GABA, a major inhibitory neurotransmitter in mammalian brain has been shown Correspondence 1043-661
to: G. Amabcoku
X/‘)4/030273-08/$08.00/0
0 I994
The Italian Pharmacological
Society
to exert an inhibitory influence on pentylenetetrazol (PTZ)-induced seizures [ 1 I-131, it is possible that the anticonvulsant effect of clonidine on PTZ-induced seizures may be mediated by its ability to alter the activity of GABA-containing neurons in the brain. This project was intended, therefore, to investigate the role of GABA in the protective effect of clonidine against PTZ-induced seizures by studying the effects of muscimol, amino-oxyacetic acid (AOAA), diamino-Nbutyric acid (DABA), bicuculline, picrotoxin, phenobarbitone and diazepam on the anticonvulsant effect of clonidine in mice.
MATERIALS
AND METHODS
Animals
Male albino mice bred in our Animal House, weighing 22-26 g, were used in this study. The animals were housed in groups of eight per cage and allowed free access to water and food. l!higs Pentylenetetrazol (Leptazol, Sigma Chemical Co.), clonidine hydrochloride (Sigma), muscimol (Sigma), amino-oxyacetic acid hemihydrochloride (AOAA, Sigma), DL-2, 4-diamino-N-butyric acid dihydrochloride (DABA, Sigma), picrotoxin (Sigma) and phenobarbitone (Paris Chemical) were all dissolved in physiological saline. (+) Bicuculline (Sigma) was suspended in Tween 80 and adjusted to the appropriate volume with physiological saline. A commercial preparation of diazepam (Diazam, Ceddes Zimbabwe) was dissolved in a minimum amount of polyethylene glycol 400 (Fluka AG, Buchs) and adjusted to the appropriate volume with physiological saline. All drugs were administered intraperitoneally (i.p.) in a volume of 1 ml per 100 g body weight of animal. Control animals received equal-volume injections of the vehicle which included saline and Tween 80 and polyethylene glycol dissolved in saline. Fresh drug solutions were prepared each day of the experiment. The drug pretreatment times prior to the injection of pentylenetetrazol were clonidine (45 min), muscimol (1 h), AOAA (20 min), DABA (30 min), bicuculline (10 min), picrotoxin (10 min), phenobarbitone (10 min) and diazepam (20 min). The pretreatment times used were as described by Amabeoku [14] and Amabeoku and Chikuni [15]. However, the doses used were established by preliminary studies.
A modified version of the method of Vellucci and Webster [16] was used to assess the convulsant activity of pentylenetetrazol. Eight mice per dose of- drug were used. The animals were kept individually in transparent perspex cages (25x 15x15 cm) for 30 min to acclimatise to the new environment prior to the administration of drug. Mice were observed for seizures for 30 min following the administration of pentylenetetrazol. The time taken for the onset of tonic convulsions and the proportion of mice convulsing were recorded. Animals that did not convulse within a 30 min period were recorded as not convulsing. The behaviour of animals after drug administration was also observed. The control and
Phar-muc~olo~iculResearch. Vol. 29, No. 3. 1994
275
test experiments were performed between 12.00 hrs and quiet room with an ambient temperature of 21+2”C.
17.00 hrs each
day
in a
Statistical anulysis The data on the time taken for the onset of tonic convulsions was compared by one way analysis of variance (ANOVA) followed by Duncan’s test. Analysis of the number of mice convulsing was carried out using the chi-squared test with Yate’s correction for continuity.
RESULTS
Convulsant effect of pentylenetetrazol and the effect of clonidine on pentylenetetrazol-induced seizures in mice Table I shows that clonidine (0.5 mg kg-‘) significantly delayed the onset and reduced the incidence of pentylenetetrazol (90 mg kg-‘)-induced tonic seizures. Clonidine (1 mg kg-‘) significantly delayed the onset of the seizures but did not affect the incidence significantly. However, clonidine (0.125-0.25 mg kg-‘) did not alter the onset or the incidence of the seizures significantly. Clonidine, in all the doses used, did not affect the myoclonic jerks which preceded the tonic seizures elicited by pentylenetetrazol (results not shown).
Table I Convulsant effect of pentylenetetrazol (PTZ) and the effect of clonidine on PTZ-induced seizures in mice Drug and dose (rng kg-‘, i.p.) PTZ 60 70 75 SO 90 100 90 90 90 90 90 *PiO.Ol tPiO.05
Clonidine
No. convulsed/ no. used
Onset of tonic convulsion: meanfsEM (min)
O/8
O/8 l/8 -
0.125 0.25 0.5 1.0 vs PTZ (90 mg kg-‘) control, I’S PTZ (90 mg kg-‘) control,
4/8 8/8 8/8 8/8 718 4/8 3/8t 4/8
15.0+0 7.5+1.09 5.5f0.64 4.5f0.50 4.5kO.56 4.83LkO.59 7.5f0.83 16.33*f2.23 12.25*+1.34
Duncan’s test. chi-squared test.
Ejfects ofmuscimnl, AOAA and DABA and their inter-actions with clonidine on pentylenctruzol-induced seizures in mice Table II shows that muscimol (0.5-l mg kg-‘), AOAA (lo-20 mg kg-‘) and DABA (8-16 mg kg-‘) significantly delayed the onset of seizures induced by pentylenetetrazol (90 mg kg-‘) and significantly reduced the number of animals
Table II Effects of muscimol, aminooxyacetic acid (AOAA), diamino-?;-butyric (DABA) and their interactions with clonidine on pentylenetetrazol (PTZ)-induced seizures in mice
acid
Onw of tonic con~‘ulsioJ1: n?L’UKkSEM (MiJ?) 90
-
90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90 90
0.125 0.5
0.125 0.5
-
-
0.25 0.5 1.0 0.25 0.25
5
0.125 0.5
-
10 20 5 5
-
-
0.125 0.5
-
318” 518 l/8**” 3/s*
2188 O/8
-
-
X/8 718
4/x 2/8** 2/8**
4 8 16 4 4
l/8?? O/8 518 3/X” O/8*+“* 2189 O/8
4.63fO.M 5.17?0.76 ls.61+0.723 5.4 f0.12 I 1 .o fOi 9.33*0.54+ 13.0~0.71$ 5.75kO.41 9.5 +0.3si1I IM.7 I -i12.0 _+o,t 5.0 +I.23
I I .33k1,66f-
12.5_+1.06$
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convulsing. However. muscimol (0.25 mg kg-‘), AOAA (5 mg kg ‘) and DABA (4 mg kg-‘) neither affected the onset nor the incidence of the seizures significantly. Muscimol (0.25 mg kg-‘) potentiated the effect of subeffective dose of clonidine (0.125 mg kg ~‘) on pentylenetetrazol-induced tonic seizures. The onset of the seizures and the incidence were significantly delayed and reduced respectively. Similarly, combined injection of AOAA (5 mg kg ‘) or DABA (4 mg kg-‘) and a subeffective dose of clonidine (0.125 mg kg-‘) significantly delayed the onset of pentylenetetrazol (90 mg kg-‘)-induced seizures and significantly reduced the incidence. Furthermore, combined injection of either muscimol (0.25 mg kg’), AOAA (5 mg kg-‘) or DABA (4 mg kg ‘) and an effective dose of clonidine (0.5 mg kg-‘) completely abolished the tonic seizures induced by pentylenetetrazol. The myoclonic jerks preceding the tonic seizures were also completely abolished.
Table III shows that bicuculline (2.5-5.0 mg kg-‘) potentiated tonic seizures induced by pentylenetetrazol (75 mg kg’) by significantly shortening the onset of the seizures and increasing the number of mice convulsing. A dose of 2.5 mg kg’
Effects
Table III of bicuculline, picrotoxin and their interactions with clonidine pentylenetetrazol (PTZ)-induced seizures in mice
against
Onset of tonic convulsion: meanfsEM (min) IS 90 YO 90 75 15 90 90 90 75 7s 00 90 90
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bicuculline also effectively shortened the onset of pentylenetetrazol (90 mg kg-‘)induced seizures. Bicuculline (2.5 mg kg~‘) effectively antagonised the protective effects of both muscimol (0.5 mg kg-‘) and clonidine (0.5 mg kg-‘) against pentylenetetrarol (90 mg kg-‘)-induced seizures by significantly shortening the onset and increasing the incidence. Picrotoxin (0.5-1.0 mg kg-‘) augmented pentylenetetraLol (75 mg kg~‘)-induced tonic seizures by significantly shortening the onset of the seizures and increasing the incidence. ( 1 .O mg kg ‘) significantly shortened the onset of pentylenetetrazol Picrotoxin (90 mg kg-‘)-induced seizures. 1 .O mg kg-’ picrotoxin antagonised the protective effects of both muscimol (0.5 mg kg-‘) and clonidine (0.5 mg kg-‘) against tonic seizures induced by pentylenetetrazol (90 mg kg-‘) by significantly shortening the onset and increasing the incidence of the seizures. Bicuculline and picrotoxin when given alone did not produce any seizures in all the doses used (results not shown).
Table 1V shows the phcnobarbitone onset and reduced the incidence seizures. 10 mg kg-’ phenobarbitone
(12.5-I 5 mg kg ‘) significantly delayed the of pentylenetetrazol (90 mg kg-‘)-induced significantly prolonged the onset but did not
Table IV Effects of phenobarbitone, diazepam and their interactions with clonidine on pentylenetetrazol (PTZ)-induced seizures in mice Drug and dose (mg kg-‘, ip) PTZ 90
90 90 90 90 90 90 90 90 90
Pkenohur-hitone
Dias-pam
-
-
10
-
12.5 15 10 -
0.1 0.2 0.4 0.1
Clonidine
0.125 0.125 0.125
No. convulsed/ no. used
Onset of tonic convulsion: mean+sm (min)
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affect the incidence of the seizures. Phenobarbitone (10 mg kg-‘) potentiated the effect of subeffective dose of clonidone (0.125 mg kg-‘) on pentylenetetrazolinduced seizures by significantly delaying the onset and reducing the incidence. Diazepam (0.4 mg kg-‘) significantly prolonged the onset of pentylenetetrazol (90 mg kg-‘)-induced seizures and reduced the number of animals convulsing. A dose of 0.2 mg kg-’ diazepam significantly prolonged the onset but weakly reduced the incidence of the seizures. However, diazepam (0.1 mg kg-‘) did not alter the onset or incidence of the seizures significantly. The effect of a subeffective dose of clonidine (0.125 mg kg-‘) on the seizures was potentiated by diazepam (0.1 mg kg-‘) which significantly prolonged the onset and reduced the incidence. The myoclonic jerks preceding the onset of tonic seizures were not significantly affected by either phenobarbitone and diazepam. However, the combined injection of phenobarbitone or diazepam and clonidine significantly reduced the myoclonic jerks.
DISCUSSION The present study shows that clonidine (0.5 mg kg-‘) has a protective effect against pentylenetetrazol-induced seizures which is consistent with the observations of Papanicoloau it ul. [ 1J who reported the anticonvulsant effect of clonidine. Muscimol, a selective GABA,, receptor agonist, mimics the inhibitory effects of GABA by interacting with GABA,% receptors in the brain. Moreover, GABA is a major inhibitory neurotransmitter in the brain and the impairment of its neurotransmission has been associated with epilepsy [ 171. In this study, muscimol potentiated the protective effect of clonidine against pentylenetetrazol-induced
seizures. These data implicate GABA neurotransmission in the effect of clonidine against seizures induced by pentylenetetrazol. Bicuculline, a potent GABAA receptor antagonist, in the present study, inhibited the protective effects of muscimol and clonidine against seizures induced by pentylenetetrazol in a similar manner. This further implicates GABA receptor activation in clonidine effect against pentylenetetrazol-induced seizures. This hypothesis is further supported by the observation that picrotoxin, a GABA antagonist, which acts by blocking chloride channels linked to GABA, receptors, antagonised the effects of muscimol and clonidine against seizures induced by pentylenetetrazol in a similar way. AOAA is a potent inhibitor of GABA transaminase (GABA-T), an enzyme which metabolises GABA thereby leading to an increase in brain GABA levels. The present data show that AOAA potentiated the protective effect of clonidine against pentylenetetrazol-induced seizures. The increased GABA level as a result of the activity of AOAA may be responsible for the potentiation of clonidine effect against pentylenetetrazol seizures. Similarly, DABA was shown in this study to augment the protective effect of clonidine against seizures induced by pentelenetetrazol. DABA is a potent and selective inhibitor of GABA uptake, which results in the accumulation of GABA in the brain. It is, therefore, probable that the potentiation by DABA of clonidine effect may be due to an increase in the brain levels of free GABA available at the postsynaptic receptor sites. This still supports the hypothesis that GABA system activation may be involved in the protective effect of clonidine against pentylenetetrazol-induced seizures. Post-synaptic GABA, receptors are functionally linked to benzodiazepine receptors, barbiturate receptors and chloride channets to form a GABA-chloride ionophore complex which is involved in the modulation of GABAergic inhibitory transmission [ 181. Barbiturates and benzodiazepines have been reported to increase chloride flux through chloride channels at GABA, receptor sites to enhance GABAergic inhibition [18]. It is important that in this study, phenobarbitone, a barbiturate, and diazepam, a benzodiazepine, protected mice against pentylenetetrazol-induced seizures and also potentiated the protective effect of clonidine against the seizures. In conclusion, the data obtained in this study suggest that enhancement of GABA neurotransmission potentiated the protective effect of clonidine against pentylenetetrazol-induced seizures while inhibition attenuated the protective effect of clonidine.
ACKNOWLEDGEMENTS This project was supported by the Research Board, authors wish to thank Miss C. Zambezi for excellent
University secretarial
of Zimbabwe. assistance.
The
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