γ-aminobutyric acid, benzodiazepine binding sites and γ-aminobutyric acid concentrations in epileptic El mouse brain

European Journal of Pharmacology, 119 (1985) 217-223

217

Elsevier

~ - A M I N O B U T Y R I C ACID, B E N Z O D I A Z E P I N E B I N D I N G S I T E S A N D - y - A M I N O B U T Y R I C A C I D C O N C E N T R A T I O N S IN E P I L E P T I C El M O U S E B R A I N HARUO HATTORI *, MASATOSHI ITO and HARUKI MIKAWA Department of Pediatrics, Kvoto University Medical School, Sakyo-ku. Kvoto 606. Japan

Received 6 June 1985, revised MS received 20 August 1985, accepted 1 October 1985

H. HATTORI, M. ITO and H. MIKAWA, y-Aminobutyric acid, benzodiazepine binding sites and y-aminobutvric acid concentrations in epileptic El mouse brain, European J. Pharmacol. 119 (1985) 217-223. All E1 mice provoked by postural stimulation since the age of 4 weeks had convulsions between 22 and 24 weeks of age, the refractory period ranging from 20 to 30 min. As compared to ddY mice, the maximal number of high-affinity [3H]muscimol binding sites was larger and the affinity was lower in the brains of the El mice, which had or had not experienced repeated seizures caused by postural stimuli. The basal and '/-aminobutyric acid (GABA)-stimulated [3H]flunitrazepam binding sites, and GABA concentration in the brains of the El mice did not differ from those of the ddY mice. In E1 mice following provoked convulsions, there were no temporary changes in [3H]muscimol binding, or [3 H]flunitrazepam binding with or without exogenous GABA stimulation. The GABA concentration in the brains of the El mice increased immediately after seizures, and returned to the control values within 60 min. Epilepsy

E1 mouse

'/-Aminobutyric acid

Benzodiazepine

1. Introduction El mice are genetically inclined to exhibit generalized convulsions in response to postural stimulation (Imaizumi et al., 1959; Imaizumi and Nakano, 1964), and become increasingly sensitive with age (Suzuki and N a k a m o t o , 1982). The evoked convulsions are followed by a refractory period (Suzuki and Nakamoto, 1978), as observed after electrically and chemically induced seizures in rats (Nutt et al., 1981), and after audiogenic seizures in audiogenic seizure-susceptible rats (Tacke et al., 1984). In contrast to electrically or chemically induced convulsions, the paroxysmal discharges on the electroencephalograms of these E1 mice recorded during the ictal and interictal periods have been characterized as epileptic convulsions (Suzuki, 1976; Suzuki and N a k a m o t o , 1977). Although various changes of neurotransmitters such as acetylcholine, ami~ao acids and catecholamines have been reported in the brains of the E1 mice * To whom all correspondence should be addressed. 0014-2999/85/$03.30 © 1985 Elsevier Science Publishers B.V.

Receptor binding

(Naruse et al., 1960; Hiramatsu and Mori, 1977), the neuronal mechanism underlying genetical seizure susceptibility to postural stimuli is not known. Furthermore, there are no reports on the properties of ~,-aminobutyric acid ( G A B A ) and benzodiazepine receptor binding sites in the brains of these mice. In an attempt to define the role of the GABAergic mechanism in seizure susceptibility and the refractory period, we examined G A B A receptor binding using [3H]muscimol, benzodiazepine receptor binding using [3 H]flunitrazepam, the effect of exogenous G A B A on benzodiazepine binding to crude brain m e m b r a n e fractions, and the G A B A concentrations in the brains of El mice.

2. Materials and methods 2.1. Animals

The E1 mice used in these experiments were of the F80 to 85 generation and were inclined to

218

develop tonic or clonic convulsions or both, and running fits or other manifestations. The mice were obtained from the Psychiatric Research Institute of Tokyo, Japan, and were maintained in our animal center as an inbred strain, housed in metal cages that were cleaned only once a week so as not to excessively stimulate the mice. Pellet food and water were supplied as usual. The El mice were stimulated once a week from the age of 4 weeks according to the method developed by lmaizumi et al. (1959). That is, the mouse was taken out of its cage, placed on the metal mesh of the cage and observed for 3 min. It was then tossed up in the air about 15 cm high 30 times. When the mouse displayed seizures during the procedure, the procedure was stopped immediately. The refractory period was determined as the time when the next stimulus caused convulsions. For the experiments to determine temporary changes, the :nice were killed at 1, 10, 20, 30, 45 and 60 rain following the provoked convulsions. El mice, which had not been stimulated and therefore had not developed convulsions, were used as controls. Mice of the ddY strain were used as other controls. These animals were adults of both sexes, between 22 and 24 weeks of age, and each weighing between 25 and 40 g.

2.2. /*H/Muscimol binding Membranes were prepared by the method of Enna and Snyder (1977) after modification. The mice were decapitated and their brains were rapidly removed and homogenized in 15 volumes of icecold 0.32 M sucrose in a glass homogenizer fitted with a Teflon pestle. The homogenate was centrifuged at 1 000 × g for 10 rain, and the supernatant was centrifuged at 2 0 0 0 0 × g for 20 min and resuspended in 20 volumes of 50 mM Tris-citrate, pH 7.1, before freezing at - 2 0 ° C for at least 18 h. The frozen membrane was thawed, centrifuged at 27000 × g for 20 min, and suspended in 20 volumes of 50 mM Tris-citrate, pH 7.1, containing 0.05% Triton X-100 at 37°C for 30 min. After centrifugation at 27000 × g for 20 min, the pellet was resuspended in 50 volumes of 50 mM Tris-citrate, pH 7.1. The high-speed centrifugation

and resuspension were repeated 3 times and the pellet was finally resuspended in this buffer. Binding assays for the high-affinity sodium-independent [3H]muscimol receptor were performed using the filtration assay method of Williams and Risley (1979) after modification. Portions of membranes (0.09-0.11 mg of protein, in duplicate) were incubated with [~H]muscimol (20.6 Ci/mmol, New England Nuclear) at 4°C for 20 min. The assay was terminated by the addition of 4 ml of ice-cold 50 mM Tris-citrate, pH 7.1, and rapid filtration through Whatman G F / B filters under suction. After washing twice with 4 ml of the buffer, the filters were dried and the radioactivity was counted in 8 ml of a toluene scintillation cocktail in a liquid scintillation counter and corrected for counting efficiency. Specific binding, defined as that displaceable by 10 btM GABA and always representing over 90% of the total binding, was determined at 8 [~ H]muscimol concentrations (0.2-6.5 nM). Scatchard analysis of the data was done using the least square linear regression method.

[~H]Flunitra=epam binding and exo
2.3.

Membranes were prepared according to the method of Horton et al. (1982) after modification. The mice were decapitated and their brains were rapidly removed and homogenized in 50 volumes of 50 mM Tris-citrate, pH 7.1. The homogenate was centrifuged at 27000 × g for 20 min. The pellet was rehomogenized and recentrifuged twice as above, and resuspended in 20 volumes of the buffer before freezing - 2 0 ° C for at least 18 h. The frozen membrane was thawed, centrifuged at 2 7 0 0 0 × g for 20 min, washed twice with 50 volumes of 50 mM Tris-citrate, pH 7.1 and finally resuspended in this buffer. Portions of membranes (0.08-0.11 mg of protein, in duplicate) were incubated with [~H]flunitrazepam (77.4 Ci/mmol, New England Nuclear) at 4°C for 20 min. Bound radioactivity was separated and determined by the method mentioned before. Specific binding, defined as that displaceable by 10 /,M diazepam and always representing over 90% of the total binding, was determined at 7

219 concentrations (0.6-10.2 nM) for Scatchard analysis, or at a fixed concentration of [3H]flunitrazepare (1.1 nM) in the presence and absence of exogenous G A B A (0.1-500 ~M).

2.4. GABA concentration The brains were removed rapidly (less than 30 s) and placed into a beaker of methanol precooled to approximately - 8 0 ° C on dry ice and acetone (Alderman and Shellenberger, 1974). Brain extracts for G A B A assays were prepared essentially according to the method of Enna (1985). The frozen brain tissue was dispersed with a Polytron in 50 volumes of ice-cold distilled water and the homogenate was centrifuged at 27000 x g at 4°C for 20 min. A fraction of the supernatant was removed and diluted to the equivalent of 500 volumes with ice-cold distilled water. The G A B A content was determined by using the [3H]muscimol radioreceptor assay of Bernasconi et al. (1980) after modification (Ito et al., 1984). A 50 /~1 portion of the m e m b r a n e suspension (0.09-0.11 mg of protein, in quadruplicate), which was prepared from the brain of the ddY mouse, was placed into a culture tube containing 300 ~1 of 50 m M Tris-citrate, p H 7.1, 100 t~l of the sample and 50 t~l of 12 nM of [3H]muscimol (20.6 C i / m m o l , N e w England Nuclear). Binding assays for the high-affinity [3H]muscimol receptor were performed as previously mentioned. Determinations of the inhibition of binding at different concentrations of G A B A were done routinely for a standard curve.

3. Results 3.1. El mice N o seizure was observed in the E1 mice when they were not stimulated. N o n e of the stimulated El mice died of a seizure. They began to develop convulsions after stimulation at the age of 6 weeks, and became more sensitive to stimulation as they became older, all of them developing convulsions from about the 15th week up to the 35th week (fig. 1). At about 20 weeks old, most mice began to exhibit seizures during the observation on the metal mesh. The duration of overall convulsive movements was about 15 s. The E1 mice, between 22 and 24 weeks old, used for the subsequent experiments, had 13.8 _+ 0.2 (mean _+ S.E., range 10-18) repeated seizures. Of ten 22 to 24-week-old El mice, each tested after an initial convulsion, none had a recurrent convulsion at 1, 10 and 20 min of the second stimulus; 8 had a convulsion at 30 min, and all did at 45 and 60 min. The duration of the refractory period raaged from 20 to 30 min.

3.2. [ W ] M u s c i m o l binding Table 1 shows the dissociation constant (KD) and the maximal number of binding sites (B ...... ). %

Convulsed

lOO

2.5. Protein concentration 50 Protein determinations were carried out by the method of Lowry et al. (1951).

2.6. Statistics

O-

1'0 All results are given as the means + S.E. and statistical significance was assessed by the twotailed Student's t-test. P values smaller than 0.05 were considered significant.

1'5

:)0

2'5

50

~5

Age (weeks)

Fig. 1. The percentage of E1 mice in each age group that bad convulsions. The mice had been kept as an inbred strain in our animal center and stimulated posturally once a week from the age of 4 weeks. The number of mice examined each week was 244 at 4 weeks and 68 at 35 weeks.

TA BLf,2 1 [ H]Muscnnol and [~H]flunitrazepam b i n d i n g to crude m e m b r a n e fractions fronl the brains, and G A B A c o n c e n t r a t i o n s in the brains of s t i m u l a t e d and u n s t i m u l a t e d El mice and d d Y mice. [ H l M u s c m l o l binding

Mouse strain

El (stimulated) El ( u n s t i m u l a t e d )

ddY n=number

[ ~H]Flunitrazepam binding

GABA concentration

n

K I) (n M)

Bma~ (pmol/mg protein)

n

KD (nM)

B,,,,,~ (p m o l / m g protein)

n

5 5 5

1,96+0.14 " 1,87+0.08 b 1,46 + 0 . 0 7

1.73+0.09 h 1.70+[).08 ~' 1.32+0.07

4 4 4

3.53_+0.17 3.26_+0.10 3.53-+0.25

1.51 + 0 . 0 4 1.53+0.02 1.42±0.08

10 7 10

p. m o l / g tissue

1.gl + 0 . 0 6 1.77-+0.09 1.78_+0.05

of animals. Data are m e a n s ± S . E . Significant difference between El (stimulated or u n s t i m u l a t e d ) and ddY mice is

indicated as: " P < 0.01, h p < 0.001.

The affinity of high-affinity [ ~H]muscimol binding sites was lower, but the number was larger in stimulated and unstimulated El mice than in ddY mice. Neither Kt) nor B...... values differed significantly between stimulated and unstimulated El mice. 3.3. [3H]Flunitrazepam binding and GABA concentrations The K D and B...... values of [3H]flunitrazepam binding did not differ significantly between stimulated and unstimulated El mice or ddY mice (table 1). Exogenous G A B A increased the [3H]flunitrazepam binding in E1 and ddY mice.

The stimulation produced by GABA was maximal at a GABA concentration of 5 /.tM and remained high up to 500/~M. The mean percentage increase ( + S . E . ) in specifically bound [3H]flunitrazepam (at the 5 highest GABA concentrations used, 5, 10, 50, 100 and 500 >M) compared with the concurrent controls did not differ significantly between E1 and ddY mice (40.4 _+ 2.1 in stimulated El mice, 39.1 -4- 2.8 in unstimulated El mice and 42.1 + 2.5 in ddY mice; [3H]flunitrazepam 1.1 nM, for 4 separate experiments performed in duplicate). Also there were no significant differences in endogenous GABA concentrations among stimulated and unstimulated E1 and ddY mice (table 1).

TABLE 2

The effect of a p r o v o k e d convulsion on [~H]muscimo]. and basal and G A B A - s t i m u t a t e d [3H]fIunitrazepam b i n d i n g to crude m e m b r a n e fractions from the brains of El mice. The c o n c e n t r a t i o n s of [3H]muscimol and [ 3 H ] f l u n i t r a z e p a m were 1.0 nM and 1.1 nM respectively. The effect of G A B A s t i m u l a t i o n on [ ~ H ] f l u n i t r a z e p a m b i n d i n g is shown as percent increase c o m p a r e d with respective controls. Time after a convulsion (min)

Specifically bound [ ~ H]muscimol

Specifically bound [3H]flunitrazepam

G A B A (50 # M ) stimulation on specifically b o u n d [ ~ H]flunitrazepam

n

( f m o l / m g protein)

n

( f m o l / m g protein)

n

(% increase)

('ontrol

10 4 4 4 4 4 4

603__+ 16 575 ± 16 615 ± 20 621 + 36 582_+22 5 8 4 ± 18 6 2 2 ± 17

7 5 5 5 5 5 5

357+ 366 ± 363_+ 353+ 338+ 365± 361 +

4 5 5 5 5 5 5

40.9+3.3 35.9 + 3.0 35.5±2.0 41.8_+ 3.3 41.0_+2.3 35.2+2.0 36.1 + 3 . 4

1 10 20 30 45 6(}

n = n u m b e r of animals. Data are m e a n s ± S.E.

6 17 10 17 9 10 1l

221

Brain GABA (~rnol,/g tissue)

2.4-

2.0-

co~',rol

~

1'o Time

:~o

~o

4g

do

(rain) after a convulsion

Fig. 2. Brain GABA concentrations in stimulated El mice at various times after a convulsion. Hatched area shows the m e a n s ± S . E , of the GABA concentrations in 10 control E1 mice. Values at various times are the means_+ S.E. of 7 animals that had convulsions for quadruplicate measurements. The brain GABA increased significantly (* P < 0.01, ** P < 0.001) from 1 min after a convulsion until 45 rain and returned to the control value at 60 rain.

3.4. Temporary changes in [¢H]muscimol binding, and [ 3H]flunitrazepam binding with or without exogenous GABA stimulation, and brain GABA concentrations in stimulated El mice after provoked convulsions The number of [3H]muscimol and [3H]flunitrazepam binding sites, and the effect of GABA stimulation on specifically bound [3H]flunitrazepam were not affected by the occurrence of a provoked convulsion (table 2). The GABA concentrations increased significantly 1 min after the provoked convulsion occurred, remained high up to 45 min and returned to the control value by 60 min (fig. 2).

4. Discussion Both audiogenic seizure-susceptible mice and E1 mice are genetically established models for epilepsy. The brain of audiogenic mice has been reported to have a smaller number and a higher affinity of the high-affinity [3H]GABA binding

sites than the controls with no difference in the K D or Bmax values of [3H]flunitrazepam binding sites or GABA contents (Ticku, 1979; Horton et al., 1982; Sykes and Horton, 1982). In this study, we found no increase in brain GABA contents in E1 mice aged at least 12 weeks as compared to the controls (Naruse et al., 1960). The reason for this discrepancy is unknown, but could be the use of animals of a different age or a different stimulation method. Furthermore, our results do not preclude differences in uptake and release of this neurotransmitter, or in the distribution in localized areas of the brain. We observed a significantly higher number and lower affinity of [3H]muscimol binding sites in E1 mice than in ddY mice, but there was no difference in the characteristics of [3H]flunitrazepam binding. The differerfce in the [3H]muscimol binding sites in E1 mice does not seem to be related to the repeated convulsions because unstimulated El mice, which did not develop convulsions, also showed the same difference in [~H]muscimol binding. Therefore, the difference in [)H]muscimol binding may be due to the genetic difference between these two strains of mice. Since the functional significance of the high- and low-affinity GABA binding sites is still unclear, the results from the present study are difficult to evaluate. However, since an increased number of binding sites is usually associated with an elevated physiological response (Burt et al., 1977), the increased number of GABA binding sites could indicate an enhanced GABAergic mechanism in the brains of El mice. As GABAergic neurotransmission is a major inhibitory mechanism in the mammalian central nervous system, it is tempting to speculate that the observed increase in the receptor density reflects an inhibitory compensation for increased overall excitability in the brain of this strain. Exogenous GABA enhanced the specific [3H]flunitrazepam binding in E1 and ddY mice to well-washed, frozen and thawed membranes as previously reported (Chiu and Rosenberg, 1979). The extent of maximal stimulation of [~H]flunitrazepam binding by exogenous GABA in E1 and ddY mice was quite similar to that reported by Horton et al. (1982). However, the lack of difference between El and ddY mice is not con-

22,2 sistent with the greater maximal stimulation of [3H]flunitrazepan binding by GABA in audiogenic-susceptible mice than seizure-resistant mice (Horton et al., 1982). There was no difference m GABA-stimulated [3H]flunitrazepam binding between El and ddY mice, although a significant difference in [~H]muscimol binding was observed. These high-affinity [3H]muscimol binding sites in El and ddY mice might not be coupled to the benzodiazepine binding sites. This may be explained by the finding that the low-affinity, but not high-affinity, GABA receptor is coupled to the benzodiazepine receptor (Olsen, 1982; Butch et al., 1983). GABA or benzodiazepine receptors have been reported to show rapid temporary changes following seizures in experimental animals. Ross and Craig (1982) reported that the maximal number of high-affinity sodium-independent [3H]GABA binding sites increased temporarily following a seizure induced by electroconvulsive shock in the rat cerebral cortex. Whereas, Atterwill et al. (1981) did not find a transient increase in [~H]muscimol binding in th rat cerebral cortex after an electrically induced convulsion; this was the case in the El mouse after a provoked convulsion. There is some evidence that [3H]GABA and [3H]muscimol may not label the same population of binding sites and that displacement of [aH]muscimol by GABA may lead to an overestimation of GABA receptor density (DeFeudis et al., 1979). Concerning the benzodiazepine receptor, the maximal number of [3H]diazepam binding sites in the rat cerebral cortex is reported to have increased temporarily after a seizure induced by an electroconvulsive shock (Paul and Skolnick, 1978), Asano and Mizutani (1980) reported a transient increase in the affinity of the [3H]diazepam receptor in the brain of the Mongolian gerbil following the occurfence of a seizure. There were no temporary changes of basal or GABA-stimulated [3H]flunitrazepam receptor binding in the brains of El mice after a provoked convulsion This was in accordance with the findings of Bowdler and Green (1982) that electrically induced convulsions had no effect on [3H]diazepam receptor binding to a well-washed membrane preparation obtained from rat brain. We also used the membranes after ex-

tensive washing. Thus, modulatory compounds on benzodiazepine binding sites may have been washed out (Napias et al., 1980). Furthermore, Nutt and Minchin (1983) found no transient change in [3H]diazepam or [3H]ethyl-,8-carboline carboxylate binding in the rat brain in vivo after a seizure induced by an electroconvulsive chock, after which any endogenous compounds would still be present. A change constantly observed following provoked convulsions in El mice was a rise in the brain GABA concentration. It seems unlikely that the increase was due to postictal hypoxia, since no change in GABA concentration was observed in ddY mice 20 min after they had been placed for 15 s in a desiccator in which the air was replaced by 100% nitrogen (data not shown). These results are in accordance with the report of a transient increase in the GABA concentrations in the brain of rats following electroconvulsive shock (Bowdler and Green, 1982). There was no clear relationship between increased seizure threshold and brain GABA in this case since a flurothyl-induced seizure and repeated electroconvulsive shocks did not raise brain GABA, while both increased the seizure threshold. On the other hand, an increase in whole brain GABA concentrations caused by inhibition of GABA-transaminase (4-aminobutyrate-2-oxoglutarate aminotransferase, EC 2.6.1.19) could inhibit the seizures in audiogenic mice (Schechter et al., 1977). A rapid temporary change in brain GABA concentrations after a provoked seizure in E1 mice may cause the refractory period by elevating the seizure threshold. Although the elevated seizure threshold returned to normal after 45 min, the GABA concentrations in the brains were still elevated. This indicates that the increase in the seizure threshold is not related simply to an increase in brain GABA concentration. The role of GABA-mediated neurotransmission in the elevated seizure threshold during the refractory period remains to be identified.

A cknowledgements We thank Dr. Jiro Suzuki (Division of Neurophysiology, Psychiatric Research Institute of Tokyo, Japan) for the gener-

223 ous supply of E1 mice and Dr. Junko Ochi (Department of Pediatrics, Kyoto University Medical School) for her co-operation in maintaining the strain. We also thank Dr. Ted H. Chiu (Department of Pharmacology, Medical College of Ohio, U.S.A.) for critical reading and discussion of the manuscript.

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lto, M., H. Mikawa and T. Taniguchi, 1984, Cerebrospinal fluid GABA levels in children with infantile spasms, Neurology 34, 235. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Napias, C., M.O. Bergman, P.C. Van Ness, D.V. Greenlee and R.W. Olsen, 1980, GABA binding in mammalian brain: inhibition by endogenous GABA, Life Sci. 27, 1001. Naruse, H., M. Kato, M. Kurokawa, R. Haba and T. Yabe, 1960, Metabolic defects in a convulsive strain of mouse, J. Neurochem. 5, 359. Nutt, D.J., P.J. Cowen and A.R. Green, 1981, Studies on the post-ictal rise in seizure threshold, European J. Pharmacol. 71,287. Nutt, D.J. and M.C.W. Minchin, 1983, Studies on [3H]diazepam and [3H]ethyl-/~-carboline carboxylate binding to rat brain in vivo. I1. Effects of electroconvulsive shock, J. Neurochem. 41, 1513. Olsen, R.W., 1982, Drug interactions at the GABA receptorionophore complex, Ann. Rev. Pharmacol. Toxicol. 22, 245. Paul, S.M. and P. Skolnick, 1978, Rapid changes in brain benzodiazepine receptors after experimental seizures, Science 202, 892. Ross, S.M. and C.R. Craig, 1982, Changes in high affinity sodium independent gamma-aminobutyric acid binding in cerebral cortex and hippocampus of the rat following electroshock, Life Sci. 31, 2499. Schechter, P.J., Y. Tranier, M.J. Jung and P. B6hlen, 1977, Audiogenic seizure protection by elevated brain GABA concentration in mice: effects of y-acetylenic GABA and -{-vinyl GABA, two irreversible GABA-T inhibitors, European J. Pharmacol. 45, 319. Suzuki, J., 1976, Paroxysmal discharges in the electroencephalogram of the El mouse, Experientia 32, 336. Suzuki, J. and Y. Nakamoto, 1977, Seizure patterns and electroencephalograms of E1 mouse, Electroencephalogr. Clin. Neurophysiol. 43, 299. Suzuki, J. and Y. Nakamoto, 1978, Sensory precipitating epilepsy focus in E1 mice and Mongolian gerbils, Folia Psychiat. Neurol. Jap. 32, 349. Suzuki, J. and Y. Nakamoto, 1982, Abnormal plastic phenomena of sensory-precipitated epilepsy in the mutant El mouse, Exp. Neurol. 75, 440. Sykes, C.C. and R.W. Horton, 1982, Cerebral glutamic acid decarboxylase activity and ¥-aminobutyric acid concentrations in mice susceptible or resistant to audiogenic seizures, J. Neurochem. 39, 1489. Tacke, U., A. Paananen and J. Tuomisto, 1984, Seizure thresholds and their postictal changes in audiogenic seizure (AGS)-susceptible rats, European J. Pharmacol. 104, 85. Ticku, M.K., 1979, Differences in ~,-aminobutyric acid receptor sensitivity in inbred strains of mice, J. Neurochem. 33, 1135. Williams, M. and E.A. Risley, 1979, Characterization of the binding of [3H]muscimol, a potent 7-aminobutyric acid agonist, to rat brain synaptosomal membranes using a filtration assay, J. Neurochem. 32, 713.