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Seizure susceptibility and anticonvulsant activity of carbamazepine, diphenylhydantoin and phenobarbital in rats with selective depletions of brain monoamines
Nwrophenmculoy?. Vol Il. fop 64-647 0 Pergamon Press Ltd1978Printed inGreatBritain
SEIZURE
SUSCEPTIBILITY AND ANTICONVULSANT ACTIVITY OF CARBAMAZEPINE, DIPHENYLHYDANTOIN AND PHENOBARBITAL IN RATS WITH SELECTIVE DEPLETIONS OF BRAIN MONOAMINES
A. QUATTRONE,* V. CRUNELLI and R. SAMANIN Istituto di Ricerche Farmacologiche “Mario Negri”, Via Eritrea 62, 20157 Milan, Italy (Accepted 12 January
1978)
Summary-The effects of selective lesioning of brain catecholamine-or serotonin--containing neurones on electroshock seizure thresholds and anticonvulsant activity of diphenylhydantoin, phenobarbital and carbamazepine were studied in rats. An intraventricular injectton of 6-hydroxydopamine, which markedly decreased brain catecholamine concentrations, lowered seizure thresholds and decreased the anticonvulsant effect of the antiepileptic compounds under study. Pretreatment with desipramine, which protected noradrenergic neurones from the neurotoxic action of 6-hydroxydopamme, did not significantly affect seizure thresholds and drug activity with respect to controls. Raphe lesions, which selectively decreased brain serotonin, also did not significantly modify seizure thresholds or drug anticonvulsant activity. The findings are compatible with the hypothesis that brain catecholamines, particularly noradrenaline, play a role in the control of seizure susceptibility as well as in the anticonvulsant activity of diphenylhydantoin, phenobarbital and carbamazepine in the rat.
There is evidence that brain monoamines may be involved in the electroconvulsive threshold as well as in the anticonvulsant activity of antiepileptic drugs in laboratory animals. Thus, increased susceptibility to electroconvulsions has been reported in mice and rats treated with drugs which deplete brain monoamines (Chen, Ensor and Bohner, 1954; Koe and Weissman, 1968; Rudzik and Johnson, 1970; Kilian and Frey, 1973; Azzaro, Wenger, Craig and Stitzel, 1972) while inhibitors of monoamine oxidase or amine precursors such as L-DOPA and 5-hydroxytryptophan (5HTP) have been reported to raise the electroconvulsive threshold (Prockop, Shore and Brodie, 1959; Chen, Ensor and Bohner, 1968; Rudzik and Johnson, 1970; Kilian and Frey, 1973). As regards anticonvulsant activity, it has been suggested that catecholamines (CA) may be preferentially involved in the anticonvulsant action of diphenylhydantoin (DPH) (Meyer and Frey, 1973), or carbamazepine (CBZ) (Quattrone and Samanin, 1977), and evidence has been presented indicating that CA and serotonin (5-HT) may be involved in the mechanism of action of phenobarbital (PB) (Frey and Magnussen, 1971; Meyer and Frey, 1973). According to other investigators, however, neither, CA nor 5-HT play any role in the anticonvulsant action of DPH or PB (Rudzik and Mennear, 1965; Rudzik and Mennear, 1966; Gray and Rauh, 1971; Przegalinski, 1976). In the present experiments the relative role of brain monoamines in the electroconvulsive threshold and in the anticonvulsant activity of CBZ, DPH and PB * Visiting scientist from Clinica Neurologica, Universita di Messina, Messina, Italy. 643
was further explored, using either intraventricular injections of 6-hydroxydopamine (6-OHDA) or raphe lesions which are known to cause selective degeneration of brain CA- and 5-HT-containing neurones respectively (Uretsky and Iversen, 1970; Samanin, Ghezzi, Valzelli and Garattini, 1972). In addition, one group of animals was injected with desipramine (DMI) before the 6-OHDA administration in order to protect the noradrenergic neurones from the neurotoxic action of this compound (Breese and Traylor, 1971). METHODS Female
Charles-River
rats (20&225 g), fed ad libitemperature (22’C + 1) (60%), were used in all experi-
turn and housed at constant and relative humidity ments. Electrolytic
and chemical
2OOpg of 6-OHDA
lesions
(calculated as free base) were administered intraventricularly (according to Noble, Wurtman and Axelrod, 1967) to animals pretreated 30min earlier with pargyline (50 mg/kg i.p.) to increase the effect of 6-OHDA on brain dopamine (DA) (Breese and Traylor, 1971). Another group of animals received an intraperitoneal injection of DMI hydrochloride (30 mg/kg), 30 min before the pargyline + 6OHDA treatment. 6-OHDA was dissolved in 20~1 of saline containing ascorbic acid (1 mg/ml). Controls received pargyline or DMI + pargyline and were injected intraventricularly with an equal volume of vehicle. Electrolytic lesions of the nucleus raphe medianus
A. QUATTRONE,V. CRUNELLI and R
644
(MR) were produced by applying a 2.5 mA anodal current for 17 set through a stainless-steel electrode, insulated except for the tip (tip: 0.5 mm; 0.3 mm in diameter). The following stereotaxic co-ordinates given in the Kanig and Klippel
(1963) rat brain atlas
were used: A 0.4; L 0.0; H - 2.6. Control rats were similarly operated but not lesioned. Electroshock
procedure
Electroshock equipment (Basile, Milan), supplying from 5 to 100 rectangular pulses per set, 0.1-l msec in width, for a duration ranging from 0.1 to 6sec,
was used. The amperage could be accurately selected on a scale from lO-lOOmA. The electroshock threshold, defined as the amperage necessary to cause the hindleg tonic extensor component of the seizure in 50% of the animals (C& = convulsive current fifty), was determined for each experiment in groups of 10 animals by the “up and down” method described by Kimball, Burnett and Doherty (1957). Electroshock was delivered to the animals through ear clip electrodes. The stimulation parameters were similar to those described by Bogue and Carrington (1953), and consisted of 50 rectangular pulses per set, 1 msec in width for a duration of 6 sec. On the first day of the experiment, both controls and experimental animals were injected with the vehicle and subjected to electroshock in order to determine the convulsive threshold. Twenty-four hours later, the same animal groups were treated intraperitoneally with CBZ (5 and lOmg/kg) or orally with DPH (100 and 200 mg/kg) or PB (10 and 20 mg/kg) and the electroshock threshold was again determined. Preliminary experiments had shown that the CCso of the various animal groups was constant when redetermined 24 hr later without drug administration.
SAMANIN
Testing and biochemical determination were performed l&15 days after surgery. Animals, taken randomly from the various experimental groups and not submitted to the electroshock test, were used for determination of brain monoamines. 5-Hydroxytryptamine was measured fluorimetrically according to Giacalone and Valzelli (1969), and noradrenaline (NA) and DA were assayed according to Chang (1964) and Laverty and Taylor (1968). The data were analysed by a factorial analysis (twofactor repeated measures Split-Plot Design, 2 x 2) (Winer, 1971) and by Tukey’s HSD test. Student’s t-test was used for biochemical data. RESULTS
As shown in Table 1, the electroconvulsive threshold in animals which received an intraventricular injection of 6-OHDA was significantly lower than in controls. With the exception of CBZ at the Smg/‘kg dose level, the anticonvulsant action of CBZ, DPH and PB was significantly reduced in the 6-OHDA treated rats, although different levels of significance were found depending on the dose and drug used (Table 2). In particular, DPH appears to be less affected by 6-OHDA as indicated by the fact that the high level of significance (P < 0.001) found with CBZ and PB was not reached in this group. In contrast, as shown in Tables 3 and 4, neither intraventricular injection of 6-OHDA in DMI-pretreated animals nor MR lesion significantly influenced the electroconvulsive threshold or the effect of the anticonvulsant drugs. Histology showed that lesions located in the MR usually destroyed the target nucleus without damage to the surrounding areas. A few animals in which the lesion was not properly located were not included in the experiments.
Table 1. Effect of intraventricularly
injected 6-hydroxydopamine (6-OHDA) on the anticonvulsant activity of carbamazepine (CBZ), diphenylhydantoin (DPH) and phenobarbital (PB). The data were analysed by a two-factor repeated measures Split-Plot Design 2 x 2 (see Table 2)
Treatment Vehicle Carbamazepine Vehicle Carbamazepine Vehicle Diphenylhydantoin Vehicle Diphenylhydantoin Vehicle Phenobarbital Vehicle Phenobarbital
Dosage (mg/kg) 5
10 100 200 10 20
Electroconvulsive Controls 22.83 + 31.33 + 21.80 k 58.53 + 22.33 + 38.90 f 21.83 + 48.00 z 21.70 + 37.16 f 22.30 + 53.96 k
1.12 1.77 0.72 1.77 0.44 2.22 0.33 2.25 0.06 0.49 1.11 4.31
threshold (mA) 6-OHDA 16.03 k 25.23 + 15.66 + 30.46 + 13.56 f 21.50 + 15.73 + 32.16 : 16.10 + 21.76 + 16.50 + 32.50 f
0.94* 0.43 0.23* 1.93 0.57* 0.87 0.38* 1.08 0.06* 0.15 0.29* 0.87
Each figure is the mean f S.E. of 3 CCsO (convulsive current fifty) values. Animals received 50mg/kg i.p. of pargyline 30min before intracerebral administration of 6-OHDA. CBZ was dissolved in propylene glycol and administered i.p. 2 hr before the test. DPH and PB were suspended in carboxymethylcellulose (CMC) 0.5% and administered orally 6 and 3 hr respectively before the test. * P < 0.01 compared with controls (Tukey’s HSD test).
Seizure susceptibility and anticonvulsant
645
activity
Table 2. Statistical analysis of the data in Table 1 by a two-factor measures Split-Plot Design 2 x 2 Treatment
Dosage (mg/kg)
d.f.
5 10 100 200 10 20
1;4 1;4 1;4 1;4 1;4 1;4
Carbamazepine Carbamazepine Diphenylhydantoin Diphenylhydantoin Phenobarbital Phenobarbital
Biochemical data are summarized in Table 5. Forebrain levels of CA were markedly reduced in 6-OHDA-treated animals, while S-HT concentrations were not significantly different from controls. Intraventricular injection of 6-OHDA in DMI-treated rats
produced a marked depletion of brain DA levels with little or no effect on forebrain NA or 5-HT concentrations. 5-HT but not CA levels were significantly reduced in the forebrain of MR lesioned rats. DISCUSSION
Seizure susceptibility
Monoamine depletors such as reserpine, ce-methylparathyrosine, parachlorophenylalanine (PCPA) or 6-OHDA have been reported to lower the electroconvulsive threshold in laboratory animals (Chen et al., 1954; Koe and Weissman, 1968; Rudzik and Johnson, 1970; Kilian and Frey, 1973; Quattrone and Samanin, 1977). These findings, together with reports on the anticonvulsant activity of L-DOPA, d-amphetamine and 5-HTP in mice and rats (Chen et al., 1968; Rudzik and Johnson, 1970; Kilian and Frey, 1973) have led to the suggestion that brain monoamines may play a role in seizure susceptibility. Preferential involvment of NA was suggested on the basis of experiments in which a lowered electroconvulsive threshold was found in animals treated with dopamine+hydroxylase inhibitors (Rudzik and Johnson, 1970; Kilian and Frey, 1973; McKenzie and Soroko, 1973) or adrenergic blockers such as phentolamine and pro-
F interaction
repeated
P N.S.
pranolol (Kilian and Frey, 1973). On the other hand, drugs believed to specifically affect central dopaminergic mechanisms have been found to modify seizure susceptibility in rabbits and rats (De Schaepdryver, Piette and Delaunois, 1962; McKenzie and Soroko, 1972; Stull, Jobe, Geiger and Fergusson, 1973). Therefore, from these studies it is not completely clear whether NA or DA or both CA are involved in control of the electroconvulsive threshold. In the present experiments an intraventricular injection of 6-OHDA in rats pretreated with pargyline caused a marked decrease of brain CA and a significant lowering of seizure susceptibility. Together with data from previous studies with 6-OHDA (Browning and Maynert, 1970; Quattrone and Samanin, 1977) these findings strongly suggest that CA may be involved in the control of electroshock seizure susceptibility in rats. Furthermore, no changes in the electroconvulsive thresholds were found in animals in which noradrenergic but not dopaminergic neurones were protected by DMI pretreatment from the action of 6-OHDA. These data appear to support the hypothesis that NA is the particular amine involved in control of seizure susceptibility in rats. Changes of seizure threshold have also been found in mice and rats treated with compounds such as 5-HTP or PCPA, known to modify brain 5-HT concentrations (Koe and Weissman, 1968; Kilian and Frey, 1973; Przegalinski, 1976). Furthermore, cyproheptadine, a 5-HT antagonist, has been reported to lower the seizure threshold in mice (Kilian and Frey,
Table 3. Effect of intraventricular injection of 6-hydroxydopamine (6-OHDA) in desipramine (DMI) pretreated rats on the anticonvulsant activity of carbamazepine (CBZ), diphenylhydantoin (DPH) and phenobarbital (PB) Treatment Vehicle Carbamazepine Diphenylhydantoin Phenobarbital
Dosage (mg/kg) 10 200 20
Electroconvulsive threshold (mA) Controls DMI + 6-OHDA 21.5 (19.0-24.5) 51.3 (42.7-61.7) 43.7 (41.7-45.7) 50.1(42.7-58.9)
21.6(19.0-25.5) 47.9 (43.7-52.5) 49.1 (42.5-56.5) 49.0 (45.7-52.5)
Animals received 50 mg/kg i.p. of pargyline 30 min before intracerebral administration of 6-OHDA. Each figure is a CC,,, (95% confidence limits) calculated for a group of 10 animals according to the “up and down” method of Kimball et al. (1957). CBZ was dissolved in propylene glycol and administered i.p. 2hr before the test. DPH and PB were suspended in CMC 0.5% and administered orally 6 and 3 hr respectively before the test.
646
A. QUATTRONE,V. CRUNELLIand R.
SAMANIN
Table 4. Elect of median raphe (MR) lesions on the ant~convulsant activity of carbamazepine (CBZ), diphenylhydantoin (DPH) and phenobarbital (PB)
Dosage Treatment Vehicle Carbamazepine Diphenylhydantoin Phenobarbital
Electroconvulsive Controls
(m&kg) 10 200 20
24.0 (20.5-28.6) 67.1(63.572.5) 49.0 (43.552.5) 51.3 (44.7-58.9)
threshold (mA) MR 24.5 (22.c27.5) 66.2 (60.3-72.6) 49.0 (43.0-56.5) 53.7 (48.G60.5)
Each figure is a CC,, (95% confidence limits) calculated for a group of 10 animals according to the “up and down” method of Kimball et al. (1957). CBZ was dissolved in propylene glycol and administered i.p. 2 hr before the test. DPH and PB were suspended m CMC 0.5% and administered orally 6 and 3 hr respectively before the test.
1973). However, some difficulties exist in interpreting the resufts obtained with these drugs. 5-Hydroxytryptophan, in fact, can interfere with the metabolism of CA in the brain (Johnson, Kim and Boukma, 1968) and PCPA has been reported to display a proconvulsive effect by causing phenylketonemia in rats (Alexander and Kopeloff, 1970). With regard to cyproheptadine, besides its relative specificity, it is not clear to what extent peripheral 5-HT antagonists can efficiently block central 5-HT receptors (Haigler and Aghajanian, 1974). In the present experiments MR lesion did not modify the electroconvulsive threshold, suggesting that serotoninergic neurones in the brain are not particularly important in the control of seizure sus~ptibility in rats. These data agree with the conclusions of other authors (Rudzik and Johnson, 1970; Jobe, Stull and Geiger, 1974) who did not find evidence of 5-HT involvement in control of the electroconvulsive threshold in rats. Anticonvulsant activity It has been suggested by some authors that brain monoamine may also play a role in the anticonvulsant effect of antiepileptic drugs. Noradrenaline has been involved in the effect of DPH, while both NA and 5-HT appear to be important for the anticonvulsant action of PB (Frey and Magnussen, 1971; Meyer and Frey, 1973). A preliminary report on a decreased anticonvulsant effect of CBZ in 6-OHDA treated rats has recently been published (Quattrone and Samanin,
1977). Other authors, however, did not find any evidence for a role of monoamine in the action of these drugs (Rudzik and Mennear, 1965; Rudzik and Mennear, 1966; Gray and Rauh, 1971; Przegalinski, 1976). In most of these studies the effects on monoamine manipulations on electroshock threshold and on anticonvulsant activity were studied in separate experiments. Therefore it was difficult to establish to what extent changes in seizure susceptibility could affect the activity of anticonvulsant drugs. One of the objectives of the present experiments was to overcome this difficulty by studying seizure susceptibility and effect of CBZ, DPH and PB in the same animals, evaluating the electraconvulsive threshold before and alter drug treatment. The data obtained in the present study, analysed as a 2 x 2 factorial design using Fischer’s F ratio for significant interaction testing, showed that 6-OHDA treatment significantly affected the action of CBZ, DPH and PB. This was particul~ly significant for CBZ and PB (P < 0.001) while lower levels of significance were reached in DPH group. These findings suggest that CA can play a role in the anticonvulsant activity of these drugs. Brain levels of CBZ were not significantly affected by 6-OHDA injection (Pantarotto, Crunelli, Quattrone, Negrini, Frigerio and Samanin, 1977) indicating that the treatment had not interfered with avaiIability of this drug for the brain. As in seizure susceptibility, the particular CA involved appears to be NA, since protecting norad-
Table 5. Effect of the various treatments on levels of noradrenaline Experimental group Controls Pargyline + 6-OHDA DMI + pargyhne + 6-OHDA Sham-operated MR-lesioned
NA
“/;,change
0.46 f 0.01
-
(NA), dopamine (DA) and serotonin (S-NT)
Forebrain levels @g/g + SE.) 7; change DA
5-HT
1.38 f 0.03
-
0.41 +_0.01
0.07 + 0.01t
- 84.8
0.25 + 0.02t
-81.9
0.40 f 0.02
0.37 + 0.01* 0.49 f 0.01 0.47 * 0.01
- 19.6 -
0.42 f 0.02t 1.40 + 0.03 1.42 f 0.02
- 70.0 -
0.42 & 0.02 0.39 * 0.02 0.12 f 0.01t
Each figure is the mean & SE. of 10 animals. * P c 0.01 compared with controls (Student’s f-test). t P i 0.001 compared with controls (Student’s r-test).
% change
-69.3
Seizure susceptibility and anticonvulsant renergic neurones with DMI led to no significant differences between 6-OHDA treated animals and their controls. Serotonin does not appear to be involved in the activity of CBZ, DPH and PB, judging from the failure of MR lesion to modify drug effects on electroshock seizures. However, it cannot be excluded that 5-HT neurones originating in other areas may play a role in both seizure susceptibility and the action of CBZ, DPH
and PB.
Acknowledgements-We are grateful to M. Recchia for his assistance with statistical analysis and to J. Baggott for revision of the manuscript. REFERENCES
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SEIZURE
SUSCEPTIBILITY AND ANTICONVULSANT ACTIVITY OF CARBAMAZEPINE, DIPHENYLHYDANTOIN AND PHENOBARBITAL IN RATS WITH SELECTIVE DEPLETIONS OF BRAIN MONOAMINES
A. QUATTRONE,* V. CRUNELLI and R. SAMANIN Istituto di Ricerche Farmacologiche “Mario Negri”, Via Eritrea 62, 20157 Milan, Italy (Accepted 12 January
1978)
Summary-The effects of selective lesioning of brain catecholamine-or serotonin--containing neurones on electroshock seizure thresholds and anticonvulsant activity of diphenylhydantoin, phenobarbital and carbamazepine were studied in rats. An intraventricular injectton of 6-hydroxydopamine, which markedly decreased brain catecholamine concentrations, lowered seizure thresholds and decreased the anticonvulsant effect of the antiepileptic compounds under study. Pretreatment with desipramine, which protected noradrenergic neurones from the neurotoxic action of 6-hydroxydopamme, did not significantly affect seizure thresholds and drug activity with respect to controls. Raphe lesions, which selectively decreased brain serotonin, also did not significantly modify seizure thresholds or drug anticonvulsant activity. The findings are compatible with the hypothesis that brain catecholamines, particularly noradrenaline, play a role in the control of seizure susceptibility as well as in the anticonvulsant activity of diphenylhydantoin, phenobarbital and carbamazepine in the rat.
There is evidence that brain monoamines may be involved in the electroconvulsive threshold as well as in the anticonvulsant activity of antiepileptic drugs in laboratory animals. Thus, increased susceptibility to electroconvulsions has been reported in mice and rats treated with drugs which deplete brain monoamines (Chen, Ensor and Bohner, 1954; Koe and Weissman, 1968; Rudzik and Johnson, 1970; Kilian and Frey, 1973; Azzaro, Wenger, Craig and Stitzel, 1972) while inhibitors of monoamine oxidase or amine precursors such as L-DOPA and 5-hydroxytryptophan (5HTP) have been reported to raise the electroconvulsive threshold (Prockop, Shore and Brodie, 1959; Chen, Ensor and Bohner, 1968; Rudzik and Johnson, 1970; Kilian and Frey, 1973). As regards anticonvulsant activity, it has been suggested that catecholamines (CA) may be preferentially involved in the anticonvulsant action of diphenylhydantoin (DPH) (Meyer and Frey, 1973), or carbamazepine (CBZ) (Quattrone and Samanin, 1977), and evidence has been presented indicating that CA and serotonin (5-HT) may be involved in the mechanism of action of phenobarbital (PB) (Frey and Magnussen, 1971; Meyer and Frey, 1973). According to other investigators, however, neither, CA nor 5-HT play any role in the anticonvulsant action of DPH or PB (Rudzik and Mennear, 1965; Rudzik and Mennear, 1966; Gray and Rauh, 1971; Przegalinski, 1976). In the present experiments the relative role of brain monoamines in the electroconvulsive threshold and in the anticonvulsant activity of CBZ, DPH and PB * Visiting scientist from Clinica Neurologica, Universita di Messina, Messina, Italy. 643
was further explored, using either intraventricular injections of 6-hydroxydopamine (6-OHDA) or raphe lesions which are known to cause selective degeneration of brain CA- and 5-HT-containing neurones respectively (Uretsky and Iversen, 1970; Samanin, Ghezzi, Valzelli and Garattini, 1972). In addition, one group of animals was injected with desipramine (DMI) before the 6-OHDA administration in order to protect the noradrenergic neurones from the neurotoxic action of this compound (Breese and Traylor, 1971). METHODS Female
Charles-River
rats (20&225 g), fed ad libitemperature (22’C + 1) (60%), were used in all experi-
turn and housed at constant and relative humidity ments. Electrolytic
and chemical
2OOpg of 6-OHDA
lesions
(calculated as free base) were administered intraventricularly (according to Noble, Wurtman and Axelrod, 1967) to animals pretreated 30min earlier with pargyline (50 mg/kg i.p.) to increase the effect of 6-OHDA on brain dopamine (DA) (Breese and Traylor, 1971). Another group of animals received an intraperitoneal injection of DMI hydrochloride (30 mg/kg), 30 min before the pargyline + 6OHDA treatment. 6-OHDA was dissolved in 20~1 of saline containing ascorbic acid (1 mg/ml). Controls received pargyline or DMI + pargyline and were injected intraventricularly with an equal volume of vehicle. Electrolytic lesions of the nucleus raphe medianus
A. QUATTRONE,V. CRUNELLI and R
644
(MR) were produced by applying a 2.5 mA anodal current for 17 set through a stainless-steel electrode, insulated except for the tip (tip: 0.5 mm; 0.3 mm in diameter). The following stereotaxic co-ordinates given in the Kanig and Klippel
(1963) rat brain atlas
were used: A 0.4; L 0.0; H - 2.6. Control rats were similarly operated but not lesioned. Electroshock
procedure
Electroshock equipment (Basile, Milan), supplying from 5 to 100 rectangular pulses per set, 0.1-l msec in width, for a duration ranging from 0.1 to 6sec,
was used. The amperage could be accurately selected on a scale from lO-lOOmA. The electroshock threshold, defined as the amperage necessary to cause the hindleg tonic extensor component of the seizure in 50% of the animals (C& = convulsive current fifty), was determined for each experiment in groups of 10 animals by the “up and down” method described by Kimball, Burnett and Doherty (1957). Electroshock was delivered to the animals through ear clip electrodes. The stimulation parameters were similar to those described by Bogue and Carrington (1953), and consisted of 50 rectangular pulses per set, 1 msec in width for a duration of 6 sec. On the first day of the experiment, both controls and experimental animals were injected with the vehicle and subjected to electroshock in order to determine the convulsive threshold. Twenty-four hours later, the same animal groups were treated intraperitoneally with CBZ (5 and lOmg/kg) or orally with DPH (100 and 200 mg/kg) or PB (10 and 20 mg/kg) and the electroshock threshold was again determined. Preliminary experiments had shown that the CCso of the various animal groups was constant when redetermined 24 hr later without drug administration.
SAMANIN
Testing and biochemical determination were performed l&15 days after surgery. Animals, taken randomly from the various experimental groups and not submitted to the electroshock test, were used for determination of brain monoamines. 5-Hydroxytryptamine was measured fluorimetrically according to Giacalone and Valzelli (1969), and noradrenaline (NA) and DA were assayed according to Chang (1964) and Laverty and Taylor (1968). The data were analysed by a factorial analysis (twofactor repeated measures Split-Plot Design, 2 x 2) (Winer, 1971) and by Tukey’s HSD test. Student’s t-test was used for biochemical data. RESULTS
As shown in Table 1, the electroconvulsive threshold in animals which received an intraventricular injection of 6-OHDA was significantly lower than in controls. With the exception of CBZ at the Smg/‘kg dose level, the anticonvulsant action of CBZ, DPH and PB was significantly reduced in the 6-OHDA treated rats, although different levels of significance were found depending on the dose and drug used (Table 2). In particular, DPH appears to be less affected by 6-OHDA as indicated by the fact that the high level of significance (P < 0.001) found with CBZ and PB was not reached in this group. In contrast, as shown in Tables 3 and 4, neither intraventricular injection of 6-OHDA in DMI-pretreated animals nor MR lesion significantly influenced the electroconvulsive threshold or the effect of the anticonvulsant drugs. Histology showed that lesions located in the MR usually destroyed the target nucleus without damage to the surrounding areas. A few animals in which the lesion was not properly located were not included in the experiments.
Table 1. Effect of intraventricularly
injected 6-hydroxydopamine (6-OHDA) on the anticonvulsant activity of carbamazepine (CBZ), diphenylhydantoin (DPH) and phenobarbital (PB). The data were analysed by a two-factor repeated measures Split-Plot Design 2 x 2 (see Table 2)
Treatment Vehicle Carbamazepine Vehicle Carbamazepine Vehicle Diphenylhydantoin Vehicle Diphenylhydantoin Vehicle Phenobarbital Vehicle Phenobarbital
Dosage (mg/kg) 5
10 100 200 10 20
Electroconvulsive Controls 22.83 + 31.33 + 21.80 k 58.53 + 22.33 + 38.90 f 21.83 + 48.00 z 21.70 + 37.16 f 22.30 + 53.96 k
1.12 1.77 0.72 1.77 0.44 2.22 0.33 2.25 0.06 0.49 1.11 4.31
threshold (mA) 6-OHDA 16.03 k 25.23 + 15.66 + 30.46 + 13.56 f 21.50 + 15.73 + 32.16 : 16.10 + 21.76 + 16.50 + 32.50 f
0.94* 0.43 0.23* 1.93 0.57* 0.87 0.38* 1.08 0.06* 0.15 0.29* 0.87
Each figure is the mean f S.E. of 3 CCsO (convulsive current fifty) values. Animals received 50mg/kg i.p. of pargyline 30min before intracerebral administration of 6-OHDA. CBZ was dissolved in propylene glycol and administered i.p. 2 hr before the test. DPH and PB were suspended in carboxymethylcellulose (CMC) 0.5% and administered orally 6 and 3 hr respectively before the test. * P < 0.01 compared with controls (Tukey’s HSD test).
Seizure susceptibility and anticonvulsant
645
activity
Table 2. Statistical analysis of the data in Table 1 by a two-factor measures Split-Plot Design 2 x 2 Treatment
Dosage (mg/kg)
d.f.
5 10 100 200 10 20
1;4 1;4 1;4 1;4 1;4 1;4
Carbamazepine Carbamazepine Diphenylhydantoin Diphenylhydantoin Phenobarbital Phenobarbital
Biochemical data are summarized in Table 5. Forebrain levels of CA were markedly reduced in 6-OHDA-treated animals, while S-HT concentrations were not significantly different from controls. Intraventricular injection of 6-OHDA in DMI-treated rats
produced a marked depletion of brain DA levels with little or no effect on forebrain NA or 5-HT concentrations. 5-HT but not CA levels were significantly reduced in the forebrain of MR lesioned rats. DISCUSSION
Seizure susceptibility
Monoamine depletors such as reserpine, ce-methylparathyrosine, parachlorophenylalanine (PCPA) or 6-OHDA have been reported to lower the electroconvulsive threshold in laboratory animals (Chen et al., 1954; Koe and Weissman, 1968; Rudzik and Johnson, 1970; Kilian and Frey, 1973; Quattrone and Samanin, 1977). These findings, together with reports on the anticonvulsant activity of L-DOPA, d-amphetamine and 5-HTP in mice and rats (Chen et al., 1968; Rudzik and Johnson, 1970; Kilian and Frey, 1973) have led to the suggestion that brain monoamines may play a role in seizure susceptibility. Preferential involvment of NA was suggested on the basis of experiments in which a lowered electroconvulsive threshold was found in animals treated with dopamine+hydroxylase inhibitors (Rudzik and Johnson, 1970; Kilian and Frey, 1973; McKenzie and Soroko, 1973) or adrenergic blockers such as phentolamine and pro-
F interaction
repeated
P N.S.
pranolol (Kilian and Frey, 1973). On the other hand, drugs believed to specifically affect central dopaminergic mechanisms have been found to modify seizure susceptibility in rabbits and rats (De Schaepdryver, Piette and Delaunois, 1962; McKenzie and Soroko, 1972; Stull, Jobe, Geiger and Fergusson, 1973). Therefore, from these studies it is not completely clear whether NA or DA or both CA are involved in control of the electroconvulsive threshold. In the present experiments an intraventricular injection of 6-OHDA in rats pretreated with pargyline caused a marked decrease of brain CA and a significant lowering of seizure susceptibility. Together with data from previous studies with 6-OHDA (Browning and Maynert, 1970; Quattrone and Samanin, 1977) these findings strongly suggest that CA may be involved in the control of electroshock seizure susceptibility in rats. Furthermore, no changes in the electroconvulsive thresholds were found in animals in which noradrenergic but not dopaminergic neurones were protected by DMI pretreatment from the action of 6-OHDA. These data appear to support the hypothesis that NA is the particular amine involved in control of seizure susceptibility in rats. Changes of seizure threshold have also been found in mice and rats treated with compounds such as 5-HTP or PCPA, known to modify brain 5-HT concentrations (Koe and Weissman, 1968; Kilian and Frey, 1973; Przegalinski, 1976). Furthermore, cyproheptadine, a 5-HT antagonist, has been reported to lower the seizure threshold in mice (Kilian and Frey,
Table 3. Effect of intraventricular injection of 6-hydroxydopamine (6-OHDA) in desipramine (DMI) pretreated rats on the anticonvulsant activity of carbamazepine (CBZ), diphenylhydantoin (DPH) and phenobarbital (PB) Treatment Vehicle Carbamazepine Diphenylhydantoin Phenobarbital
Dosage (mg/kg) 10 200 20
Electroconvulsive threshold (mA) Controls DMI + 6-OHDA 21.5 (19.0-24.5) 51.3 (42.7-61.7) 43.7 (41.7-45.7) 50.1(42.7-58.9)
21.6(19.0-25.5) 47.9 (43.7-52.5) 49.1 (42.5-56.5) 49.0 (45.7-52.5)
Animals received 50 mg/kg i.p. of pargyline 30 min before intracerebral administration of 6-OHDA. Each figure is a CC,,, (95% confidence limits) calculated for a group of 10 animals according to the “up and down” method of Kimball et al. (1957). CBZ was dissolved in propylene glycol and administered i.p. 2hr before the test. DPH and PB were suspended in CMC 0.5% and administered orally 6 and 3 hr respectively before the test.
646
A. QUATTRONE,V. CRUNELLIand R.
SAMANIN
Table 4. Elect of median raphe (MR) lesions on the ant~convulsant activity of carbamazepine (CBZ), diphenylhydantoin (DPH) and phenobarbital (PB)
Dosage Treatment Vehicle Carbamazepine Diphenylhydantoin Phenobarbital
Electroconvulsive Controls
(m&kg) 10 200 20
24.0 (20.5-28.6) 67.1(63.572.5) 49.0 (43.552.5) 51.3 (44.7-58.9)
threshold (mA) MR 24.5 (22.c27.5) 66.2 (60.3-72.6) 49.0 (43.0-56.5) 53.7 (48.G60.5)
Each figure is a CC,, (95% confidence limits) calculated for a group of 10 animals according to the “up and down” method of Kimball et al. (1957). CBZ was dissolved in propylene glycol and administered i.p. 2 hr before the test. DPH and PB were suspended m CMC 0.5% and administered orally 6 and 3 hr respectively before the test.
1973). However, some difficulties exist in interpreting the resufts obtained with these drugs. 5-Hydroxytryptophan, in fact, can interfere with the metabolism of CA in the brain (Johnson, Kim and Boukma, 1968) and PCPA has been reported to display a proconvulsive effect by causing phenylketonemia in rats (Alexander and Kopeloff, 1970). With regard to cyproheptadine, besides its relative specificity, it is not clear to what extent peripheral 5-HT antagonists can efficiently block central 5-HT receptors (Haigler and Aghajanian, 1974). In the present experiments MR lesion did not modify the electroconvulsive threshold, suggesting that serotoninergic neurones in the brain are not particularly important in the control of seizure sus~ptibility in rats. These data agree with the conclusions of other authors (Rudzik and Johnson, 1970; Jobe, Stull and Geiger, 1974) who did not find evidence of 5-HT involvement in control of the electroconvulsive threshold in rats. Anticonvulsant activity It has been suggested by some authors that brain monoamine may also play a role in the anticonvulsant effect of antiepileptic drugs. Noradrenaline has been involved in the effect of DPH, while both NA and 5-HT appear to be important for the anticonvulsant action of PB (Frey and Magnussen, 1971; Meyer and Frey, 1973). A preliminary report on a decreased anticonvulsant effect of CBZ in 6-OHDA treated rats has recently been published (Quattrone and Samanin,
1977). Other authors, however, did not find any evidence for a role of monoamine in the action of these drugs (Rudzik and Mennear, 1965; Rudzik and Mennear, 1966; Gray and Rauh, 1971; Przegalinski, 1976). In most of these studies the effects on monoamine manipulations on electroshock threshold and on anticonvulsant activity were studied in separate experiments. Therefore it was difficult to establish to what extent changes in seizure susceptibility could affect the activity of anticonvulsant drugs. One of the objectives of the present experiments was to overcome this difficulty by studying seizure susceptibility and effect of CBZ, DPH and PB in the same animals, evaluating the electraconvulsive threshold before and alter drug treatment. The data obtained in the present study, analysed as a 2 x 2 factorial design using Fischer’s F ratio for significant interaction testing, showed that 6-OHDA treatment significantly affected the action of CBZ, DPH and PB. This was particul~ly significant for CBZ and PB (P < 0.001) while lower levels of significance were reached in DPH group. These findings suggest that CA can play a role in the anticonvulsant activity of these drugs. Brain levels of CBZ were not significantly affected by 6-OHDA injection (Pantarotto, Crunelli, Quattrone, Negrini, Frigerio and Samanin, 1977) indicating that the treatment had not interfered with avaiIability of this drug for the brain. As in seizure susceptibility, the particular CA involved appears to be NA, since protecting norad-
Table 5. Effect of the various treatments on levels of noradrenaline Experimental group Controls Pargyline + 6-OHDA DMI + pargyhne + 6-OHDA Sham-operated MR-lesioned
NA
“/;,change
0.46 f 0.01
-
(NA), dopamine (DA) and serotonin (S-NT)
Forebrain levels @g/g + SE.) 7; change DA
5-HT
1.38 f 0.03
-
0.41 +_0.01
0.07 + 0.01t
- 84.8
0.25 + 0.02t
-81.9
0.40 f 0.02
0.37 + 0.01* 0.49 f 0.01 0.47 * 0.01
- 19.6 -
0.42 f 0.02t 1.40 + 0.03 1.42 f 0.02
- 70.0 -
0.42 & 0.02 0.39 * 0.02 0.12 f 0.01t
Each figure is the mean & SE. of 10 animals. * P c 0.01 compared with controls (Student’s f-test). t P i 0.001 compared with controls (Student’s r-test).
% change
-69.3
Seizure susceptibility and anticonvulsant renergic neurones with DMI led to no significant differences between 6-OHDA treated animals and their controls. Serotonin does not appear to be involved in the activity of CBZ, DPH and PB, judging from the failure of MR lesion to modify drug effects on electroshock seizures. However, it cannot be excluded that 5-HT neurones originating in other areas may play a role in both seizure susceptibility and the action of CBZ, DPH
and PB.
Acknowledgements-We are grateful to M. Recchia for his assistance with statistical analysis and to J. Baggott for revision of the manuscript. REFERENCES
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