EpilepsyRes., 8 (1991) 11-20
11
Elsevier EPIRES 00380
Anticonvulsant actions and interaction of GABA agonists and a benzodiazepine in pars reticulata of substantia nigra
H o n g Z h a n g , H o w a r d C. R o s e n b e r g a n d E l i z a b e t h I. T i e t z Department of Pharmacology, Medical Collegeof Ohio, Toledo, OH 43699 (U.S.A.) (Received 19 July 1990; revision received 17 September 1990; accepted 27 September 1990)
Key words: Benzodiazepines; Substantia nigra pars reticulata; Muscimol; Anticonvulsant; Pentylenetetrazol; GABA
In a previous study, infusion of flurazepam and midazolam, but not GABA or muscimol into the pars reticulata of substantia nigra (SNpr), blocked the tonus induced by a high dose of pentylenetetrazol (PTZ). It was hypothesized that the divergence of GABA A agonist and benzodiazepine (BZ) actions might be due to a complex action of the GABA A agonists in SNpr that is not shared by the BZs, and which is specific for certain experimental seizures. This was tested in the present study in which GABA, muscimol, midazolam, and combinations of midazolam with GABA and with muscimol were tested against seizures induced by PTZ (40 or 100 mg/kg, i.p.), bicuculline (0.5 mg/kg, i.v.) and maximal electroshock (MES). Intranigral midazolam was effective against PTZ, bicuculline and MES seizures. Intranigral GABA or muscimol, infused together with midazolam, prevented midazolam from blocking tonus induced by the high dose of PTZ. For bicuculline seizures, midazolam alone had a better overall anticonvulsant effect than did the combination of midazolam plus muscimol. Intranigral muscimol, midazolam and a combination of the two were equally effective against the low dose PTZ (40 mg/kg, i.p.) and MES seizures. Since the effect of intranigral drugs was model-specific, it was suggested that different populations of nigral output neurons were involved in regulating the generalization of these seizures.
INTRODUCTION The pars reticulata of substantia nigra (SNpr) is a critical site involved in the regulation of seizure generalization and propagation 14. In several types of experimental seizures, intranigrally applied GABA agonists are anticonvulsant. For example, intranigral muscimol inhibited the seizures induced by bicuculline 16'2°, flurothy141, ethanol withdrawa118, maximal electroshock (MES) 2°, sound 13
Correspondence to: Howard C. Rosenberg, M.D., Ph.D., Department of Pharmacology, Medical College of Ohio, P.O. Box 10008, Toledo, OH 43699, U.S.A.
and amygdala kindling27. However, there is evidence that the effect of an intranigral GABA agonist depends on the seizure model being used as well as the seizure component being evaluated 9'1°'28. One example may be seizures produced by pentylenetetrazol (PTZ). Iadarola and Gale 2° reported that intranigral y-vinyl-GABA (GVG, an inhibitor of GABA transaminase) and muscimol could block the tonic-clonic seizures induced by PTZ (40 mg/kg, i.v.). Okada et al. 32 found that intranigral muscimol decreased the total seizure score over 10 min following 30 mg/kg PTZ, i.p. However, in other studies, intranigral GVG failed to protect against tonic and clonic seizures induced by PTZ (75 mg/kg, i.p.) 29'3°. In our previous
0920-1211/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
12 study 45, neither intranigral G A B A nor muscimol blocked the tonic component of PTZ (70 and 100 mg/kg, i.p.) seizures. In addition, it has been suggested that enhancing GABAergic function in SNpr can facilitate as well as suppress seizure development under particular experimental conditions31,35.39. Benzodiazepines (BZs) exert their effects by potentiating GABAergic transmission 19'33. The BZ receptor is a modulatory site of the G A B A A receptor, which is a complex containing an integral CIionophore, a GABA binding site and modulatory sites 19'33. Like G A B A A agonists, an intranigrally infused BZ, clonazepam, attenuated amygdala kindled seizures 24. In our previous study45, intranigral infusions of two BZs, midazolam and flurazepare, showed clear, dose-dependent anti-PTZ effects. Ro15-1788, a BZ receptor antagonist, which alone had no effect, reversed the anticonvulsant effects of midazolam when both were infused intranigrally, showing that intranigral BZs act via BZ receptors. Unexpectedly, but in keeping with the observation of Miller et al. 29, microinjection of either GABA or muscimol, over a wide dose range, into the SNpr failed to significantly alter PTZ convulsive activity. Several factors may explain why intranigral G A B A agonists did not share the anti-PTZ effect of intranigral BZs. Since it has been reported that not all BZ actions can be easily explained by interactions with GABA A receptors 37, it may be that BZs suppress PTZ-induced seizures by a non-GABA mediated effect. It has also been shown that not all the G A B A h receptors are linked to BZ receptors 43. Thus, another possibility in the divergence of G A B A A agonist and BZ actions is a G A B A A receptor-mediated effect of G A B A agonists in SNpr which is not shared by BZs. Such an effect of G A B A agonists might counteract the anticonvulsant effect mediated by the GABAA-BZ receptor complex. To examine these possibilities, the interaction between intranigral midazolam and G A B A agonists (GABA and muscimol) was evaluated. Since there is only limited information on the anticonvulsant effect of intranigral BZs, multiple seizure models were used in this study to observe this effect of intranigral BZs, and to determine if there is a model specificity of the divergence of G A B A A
agonist and BZ actions. GABA, muscimol, midazolam, and combinations of midazolam with each of the GABA agonists were infused into SNpr prior to eliciting seizures by PTZ (100 mg/kg, i.p.), bicuculline (0.5 mg/kg, i.v.) and MES. These intranigral infusions were also tested against a lower dose of PTZ (40 mg/kg) to see if the reported effectiveness of intranigral muscimo132 might have been a function of this lower PTZ dose or the seizure scoring method employed. MATERIALS AND METHODS
Chemical-induced seizures Stereotaxic surgery and drug injections. Stereotaxic surgical techniques were used as previously described 45. Briefly, Sprague-Dawley rats (male, 275-300 g) were deeply anesthetized (Na pentobarbital, 65 mg/kg, plus atropine sulfate, 0.5 mg/kg, i.p.). Stainless steel guide cannulas with close-fitting obturators were implanted 2 mm above SNpr, bilaterally. Coordinates for the guide cannulas were, from lambda, 3.1 mm anterior, 2.0 mm lateral and 6.0 mm below skull surface, head level 36. A week after surgery, 1 #1 of drug solution was infused over 2 min into each SNpr using stainless steel injectors which were 2 mm longer than the guide cannulas. The drugs used were GABA, muscimol, midazolam and a combination of midazolam with either G A B A or muscimol. The injectors were left in place for another minute, then withdrawn slowly and replaced with the obturators. After intranigral drug infusion, seizures were induced by i.p. injection of PTZ, 40 or 100 mg/kg, or by injection of 0.5 mg/kg bicuculline through the lateral tail vein. PTZ and bicuculline solutions were made fresh daily and were injected in a volume of 1 ml/kg. Bicuculline was diluted from stock solution a few minutes before use. The interval between intranigral drug infusion and injection of the convulsant drug was 10 min for all drugs. All the rats were returned to the home cage immediately after PTZ or bicuculline injection. Sodium pentobarbital, 40 mg/kg i.p., was immediately given to the rats which had maximal (tonic-clonic) seizures to prevent death so that rats could more
13 readily be used for histological confirmation of injection sites. Each rat was used for only 1 trial except those used for low dose PTZ seizures. In the low dose PTZ seizure experiments, rats were tested with intranigral drug (or vehicle) twice at an interval of 4-6 days. A different drug, randomly assigned, was infused into SNpr for the second test.
High dose PTZ (100 mg/kg) and bicuculline induced seizures. Convulsive activity was recorded for 20 min after PTZ or bicuculline injection. The seizures were ranked as: 0, no seizure; 1, jerks only; 2, forelimb clonus; 3, 4-leg clonus without falling; 4, 4-leg clonus with falling, no tonic seizures; 5, 4-leg clonus with falling, tonic flexion of the front legs; 6, as in 5, plus tonic extension of the front legs. The greatest seizure ranks obtained from the rats in each treatment group were tabulated and analyzed with the Kruskall-Wallis test to determine any overall treatment effect. When an overall effect was found, specific comparisons between intranigral treatment groups were performed with the Kruskall-Wallis test. For each convulsive activity (jerking, forelimb clonus and tonic extension of the front legs), the effects of different treatments on the time to onset were compared by one-way ANOVA. In these, as in all the statistical comparisons in this study, P
11
Elsevier EPIRES 00380
Anticonvulsant actions and interaction of GABA agonists and a benzodiazepine in pars reticulata of substantia nigra
H o n g Z h a n g , H o w a r d C. R o s e n b e r g a n d E l i z a b e t h I. T i e t z Department of Pharmacology, Medical Collegeof Ohio, Toledo, OH 43699 (U.S.A.) (Received 19 July 1990; revision received 17 September 1990; accepted 27 September 1990)
Key words: Benzodiazepines; Substantia nigra pars reticulata; Muscimol; Anticonvulsant; Pentylenetetrazol; GABA
In a previous study, infusion of flurazepam and midazolam, but not GABA or muscimol into the pars reticulata of substantia nigra (SNpr), blocked the tonus induced by a high dose of pentylenetetrazol (PTZ). It was hypothesized that the divergence of GABA A agonist and benzodiazepine (BZ) actions might be due to a complex action of the GABA A agonists in SNpr that is not shared by the BZs, and which is specific for certain experimental seizures. This was tested in the present study in which GABA, muscimol, midazolam, and combinations of midazolam with GABA and with muscimol were tested against seizures induced by PTZ (40 or 100 mg/kg, i.p.), bicuculline (0.5 mg/kg, i.v.) and maximal electroshock (MES). Intranigral midazolam was effective against PTZ, bicuculline and MES seizures. Intranigral GABA or muscimol, infused together with midazolam, prevented midazolam from blocking tonus induced by the high dose of PTZ. For bicuculline seizures, midazolam alone had a better overall anticonvulsant effect than did the combination of midazolam plus muscimol. Intranigral muscimol, midazolam and a combination of the two were equally effective against the low dose PTZ (40 mg/kg, i.p.) and MES seizures. Since the effect of intranigral drugs was model-specific, it was suggested that different populations of nigral output neurons were involved in regulating the generalization of these seizures.
INTRODUCTION The pars reticulata of substantia nigra (SNpr) is a critical site involved in the regulation of seizure generalization and propagation 14. In several types of experimental seizures, intranigrally applied GABA agonists are anticonvulsant. For example, intranigral muscimol inhibited the seizures induced by bicuculline 16'2°, flurothy141, ethanol withdrawa118, maximal electroshock (MES) 2°, sound 13
Correspondence to: Howard C. Rosenberg, M.D., Ph.D., Department of Pharmacology, Medical College of Ohio, P.O. Box 10008, Toledo, OH 43699, U.S.A.
and amygdala kindling27. However, there is evidence that the effect of an intranigral GABA agonist depends on the seizure model being used as well as the seizure component being evaluated 9'1°'28. One example may be seizures produced by pentylenetetrazol (PTZ). Iadarola and Gale 2° reported that intranigral y-vinyl-GABA (GVG, an inhibitor of GABA transaminase) and muscimol could block the tonic-clonic seizures induced by PTZ (40 mg/kg, i.v.). Okada et al. 32 found that intranigral muscimol decreased the total seizure score over 10 min following 30 mg/kg PTZ, i.p. However, in other studies, intranigral GVG failed to protect against tonic and clonic seizures induced by PTZ (75 mg/kg, i.p.) 29'3°. In our previous
0920-1211/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
12 study 45, neither intranigral G A B A nor muscimol blocked the tonic component of PTZ (70 and 100 mg/kg, i.p.) seizures. In addition, it has been suggested that enhancing GABAergic function in SNpr can facilitate as well as suppress seizure development under particular experimental conditions31,35.39. Benzodiazepines (BZs) exert their effects by potentiating GABAergic transmission 19'33. The BZ receptor is a modulatory site of the G A B A A receptor, which is a complex containing an integral CIionophore, a GABA binding site and modulatory sites 19'33. Like G A B A A agonists, an intranigrally infused BZ, clonazepam, attenuated amygdala kindled seizures 24. In our previous study45, intranigral infusions of two BZs, midazolam and flurazepare, showed clear, dose-dependent anti-PTZ effects. Ro15-1788, a BZ receptor antagonist, which alone had no effect, reversed the anticonvulsant effects of midazolam when both were infused intranigrally, showing that intranigral BZs act via BZ receptors. Unexpectedly, but in keeping with the observation of Miller et al. 29, microinjection of either GABA or muscimol, over a wide dose range, into the SNpr failed to significantly alter PTZ convulsive activity. Several factors may explain why intranigral G A B A agonists did not share the anti-PTZ effect of intranigral BZs. Since it has been reported that not all BZ actions can be easily explained by interactions with GABA A receptors 37, it may be that BZs suppress PTZ-induced seizures by a non-GABA mediated effect. It has also been shown that not all the G A B A h receptors are linked to BZ receptors 43. Thus, another possibility in the divergence of G A B A A agonist and BZ actions is a G A B A A receptor-mediated effect of G A B A agonists in SNpr which is not shared by BZs. Such an effect of G A B A agonists might counteract the anticonvulsant effect mediated by the GABAA-BZ receptor complex. To examine these possibilities, the interaction between intranigral midazolam and G A B A agonists (GABA and muscimol) was evaluated. Since there is only limited information on the anticonvulsant effect of intranigral BZs, multiple seizure models were used in this study to observe this effect of intranigral BZs, and to determine if there is a model specificity of the divergence of G A B A A
agonist and BZ actions. GABA, muscimol, midazolam, and combinations of midazolam with each of the GABA agonists were infused into SNpr prior to eliciting seizures by PTZ (100 mg/kg, i.p.), bicuculline (0.5 mg/kg, i.v.) and MES. These intranigral infusions were also tested against a lower dose of PTZ (40 mg/kg) to see if the reported effectiveness of intranigral muscimo132 might have been a function of this lower PTZ dose or the seizure scoring method employed. MATERIALS AND METHODS
Chemical-induced seizures Stereotaxic surgery and drug injections. Stereotaxic surgical techniques were used as previously described 45. Briefly, Sprague-Dawley rats (male, 275-300 g) were deeply anesthetized (Na pentobarbital, 65 mg/kg, plus atropine sulfate, 0.5 mg/kg, i.p.). Stainless steel guide cannulas with close-fitting obturators were implanted 2 mm above SNpr, bilaterally. Coordinates for the guide cannulas were, from lambda, 3.1 mm anterior, 2.0 mm lateral and 6.0 mm below skull surface, head level 36. A week after surgery, 1 #1 of drug solution was infused over 2 min into each SNpr using stainless steel injectors which were 2 mm longer than the guide cannulas. The drugs used were GABA, muscimol, midazolam and a combination of midazolam with either G A B A or muscimol. The injectors were left in place for another minute, then withdrawn slowly and replaced with the obturators. After intranigral drug infusion, seizures were induced by i.p. injection of PTZ, 40 or 100 mg/kg, or by injection of 0.5 mg/kg bicuculline through the lateral tail vein. PTZ and bicuculline solutions were made fresh daily and were injected in a volume of 1 ml/kg. Bicuculline was diluted from stock solution a few minutes before use. The interval between intranigral drug infusion and injection of the convulsant drug was 10 min for all drugs. All the rats were returned to the home cage immediately after PTZ or bicuculline injection. Sodium pentobarbital, 40 mg/kg i.p., was immediately given to the rats which had maximal (tonic-clonic) seizures to prevent death so that rats could more
13 readily be used for histological confirmation of injection sites. Each rat was used for only 1 trial except those used for low dose PTZ seizures. In the low dose PTZ seizure experiments, rats were tested with intranigral drug (or vehicle) twice at an interval of 4-6 days. A different drug, randomly assigned, was infused into SNpr for the second test.
High dose PTZ (100 mg/kg) and bicuculline induced seizures. Convulsive activity was recorded for 20 min after PTZ or bicuculline injection. The seizures were ranked as: 0, no seizure; 1, jerks only; 2, forelimb clonus; 3, 4-leg clonus without falling; 4, 4-leg clonus with falling, no tonic seizures; 5, 4-leg clonus with falling, tonic flexion of the front legs; 6, as in 5, plus tonic extension of the front legs. The greatest seizure ranks obtained from the rats in each treatment group were tabulated and analyzed with the Kruskall-Wallis test to determine any overall treatment effect. When an overall effect was found, specific comparisons between intranigral treatment groups were performed with the Kruskall-Wallis test. For each convulsive activity (jerking, forelimb clonus and tonic extension of the front legs), the effects of different treatments on the time to onset were compared by one-way ANOVA. In these, as in all the statistical comparisons in this study, P
