- Email: [email protected]
Chloride-dependent stimulation of GABA and benzodiazepine receptor binding by pentobarbital
212
Brain Research, 225 (1981) 212-216 Elsevier/North-Holland Biomedical Press
Chloride-dependent stimulation of GABA and benzodiazepine receptor binding by pentobarbital
TOM1KO ASANO and NOBUAKI OGASAWARA Department of Biochemistry, Institute ]'or Developmental Research, Aichi Prefecture Colony, Kasugai, .4ichi 480-03 (Japan)
(Accepted August 6th, 1981) Key words: GABA receptor - - benzodiazepinereceptor - - pentobarbital - - picrotoxinin - - chloride ion - - Triton X-100 treatment
Pentobarbital reversibly increased Na+-independent GABA binding to bovine cerebral cortex membranes. This effect was chloride-dependent. Kinetic analysis of [3H]GABAbinding revealed an increase of the apparent number of binding sites. Pretreatment of the membranes with Triton X-100 extremely diminished the effect of pentobarbital on both [aH]GABA and [3H]flunitrazepam binding. Barbiturates are general central nervous system depressants that are used clinically for anesthetic, sedative-hypnotic, and anticonvulsant actions. Electrophysiological studies suggest that barbiturate can enhance or mimic the postsynaptic response to the inhibitory neurotransmitter y-aminobutyric acid (GABA)5,s,12,14. Attempts to demonstrate enhancement by pentobarbital of GABA binding to synaptic membranes have been thus far unsuccessful 4,13, except that Willow and Johnston 19 have shown 3 0 - 5 0 ~ enhancement of GABA binding by pentobarbital with a relatively crude and carefully prepared membrane. The benzodiazepine receptors are at least partly coupled to GABA receptor binding sites in the central nervous system, as shown by allosteric activation by GABA of benzodiazepine binding in membranes 16. It was recently shown that pentobarbital enhanced benzodiazepine receptor binding and the effect was chloride-dependentg, 15. In this paper, we show that pentobarbital markedly increases Na÷-independent GABA binding in the presence of chloride ions and the effects of pentobarbital on GABA and benzodiazepine binding are found at similar concentrations. The results are compatible with a possible existence of the GABA/benzodiazepine/barbiturate receptor complex. Bovine cerebral cortex was homogenized in 10 vols. of 0.32 M sucrose in a glass homogenizer with a Teflon pestle. The homogenate was centrifuged for 10 min at 1000 g and the supernatant was recentrifuged for 20 min at 20,000 g. The pellet from the second centrifugation (P2 pellet) was washed once with 50 mM Tris-HC1 (pH 7.4) and frozen at - - 2 0 °C. After thawing, the P2 pellet was washed 5 times by resuspension in 0006-8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
213 50 mM potassium phosphate buffer (pH 7.4) and centrifugation at 30,000 g for 20 min. The pellet was resuspended to a final protein concentration of 4.0-4.5 mg/ml in 50 mM potassium phosphate buffer (pH 7.4). Studies of Na+-independent [3H]GABA binding were performed as follows: the samples of the membrane suspension were incubated in 200 #1 of 50 mM potassium phosphate buffer (pH 7.4), containing 5 nM [SH]GABA (58 Ci/mmol, Amersham) in the absence or presence of 100 mM KC1. After incubation at 0 °C for 20 min the reaction was terminated by centrifugation at 30,000 g for 20 min. The supernatant was discarded, the pellet was superficially rinsed with cold distilled water, solubilized in Protosol (New England Nuclear), and the radioactivity measured. Benzodiazepine binding was determined by the method described previously2 using 0.25 nM [3H]flunitrazepam (84 Ci/mmol, New England Nuclear). Protein was determined by the method of Lowry et al. 1°. Sodium pentobarbital (Abbott) stimulated Na+-independent [3H]GABA binding in both chloride-free and chloride-enriched buffer (Fig. 1). However, both the maximum stimulation by pentobarbital (98 %) and ECs0 (concentration required for half-maximal stimulation) (320/~M) in the chloride-free medium were enhanced in the presence of 100 mM KCI (340% and 150 #M, respectively). ECs0 values for the stimulation of GABA binding determined in each condition are in good agreement with those for the stimulation of benzodiazepine receptor binding in previous reportsgJL The effect of pentobarbital on [ZH]GABA binding was reversible since preincubation with pentobarbital followed by centrifugation of the membranes and resuspension in drug-free buffer reduced [3H]GABA binding to control levels. As calcium pentobarbital (Tanabe Pharmaceutical) also stimulated [ZH]GABA binding, the effect of sodium pentobarbital did not involve the effect by low concentration of sodium ion. Ion requirement did not involve the cation, because MgCb was equally effective. The anions such as bromide, iodide and thiocyanate can substitute for
400 o 300 'S 200
100 lO
100 Pentoborbltol (pM)
1000
Fig. 1. Effect of pentobarbital on [aH]GABA binding to bovine cerebral cortex membranes. Pentobarbital enhances laHIGABAbinding in the presence (O) and absence (Q) of 100 mM KCI. Values represent the mean of 3 experiments.
214 TABLE I Effect of pretreatment of the membranes with Triton 3(-100 on the stimulation of [aHfGABA and [aHJflunitrazepam binding by pentobarbital
Non-treated membrane was prepared as described in the text. Triton X-100 treated membrane was prepared as follows: the P~ pellet was incubated in 20 vols. of 50 mM Tris-HCl (pH 7.4) containing 0.05 700Triton X-100 for 30 min at 37 °C, centrifuged at 30,000 g for 20 min, and washed 4 times in 50 mM potassium phosphate buffer without Triton X-100. Specific [aH]GABA and [aH]flunitrazepam binding in the presence of 100 mM KCI and the absence of pentobarbital was taken as 100 ~. Concentration of pentobarbital
0.1 mM 1 mM
% Control [ZH]GABA binding
/ 3H]Flunitrazepam binding
Non-treated membrane
Triton X - I O 0 Non-treated treatedmembrane m e m b r a n e
Triton X-IO0 treatedmembrane
204 418
115 146
109 118
127 163
chloride, but acetate, fluoride and sulfate ions cannot. The selectivity of the effective anions on G A B A binding is in agreement with that on the stimulation of benzodiazepine binding by pentobarbital 9, and also with the selectivity for penetrating the activated inhibitory postsynaptic membranes of cat motoneurons measured electrophysiologically I. The increases in [3H]GABA binding elicited by pentobarbital (200 /~M) were antagonized by picrotoxinin. IC50 value (concentration that inhibits binding by 50 ~ ) of 1.4 # M was obtained in the presence of 100 m M chloride. Pretreatment of the membranes with the detergent Triton X-100 extremely diminished the effect of pentobarbital on both [aH]GABA and [aH]flunitrazepam binding (Table I). Ticku et al. 18 reported that Triton X-100 treatment inhibited picrotoxinin binding to rat brain membranes. These results indicate that the stimulation of both G A B A and benzodiazepine binding by pentobarbital is mediated by picrotoxinin (barbiturate) binding sites. Kinetic analysis of the effect of pentobarbital (200 ~M) on Na+-independent [3H]GABA binding in the presence of chloride revealed an increase of binding sites (Bmax = 2.61 :k 0.14 pmol/mg protein) over control samples (Bmax ---- 0.91 ~ 0.08 pmol/mg protein). In contrast, the apparent affinity constant was slightly increased with pentobarbital (Ka = 109 :k 6 nM with pentobarbital, Kd = 98 + 6 nM without pentobarbital), but this increase was not statistically significant (Fig. 2). The concentrations of pentobarbital which enhance G A B A binding in the present study are comparable with those which enhance GABA-mediated postsynaptic inhibition in tissue cultured mouse spinal neurons 14, and GABA-induced depolarization of frog sympathetic ganglion cells 11 and dorsal root fibers in the immature rat spinal cord in vitroL Pentobarbital is known to inhibit the binding of dihydropicrotoxinin to fresh rat brain membranes 17. Dihydropicrotoxinin binding is considered to reflect binding to chloride ionophores normally linked to G A B A receptors. Fifty per cent inhibition of dihydropicrotoxinin binding occurred at 50/~M pentobarbital and 0.4 # M picrotox-
215
.x =~ 2
200
qO0
600
800
Bound (fmole/assay)
Fig. 2. Scatchard plot of [aH]GABA binding to bovine cerebral cortex membranes. [3H]GABA binding was measured in the presence of 100 mM KCI with ((3) or without (O) 200/~M pentobarbital. The data shown are the means of 3 experiments.
inin 17. These results are in g o o d a g r e e m e n t with the effective c o n c e n t r a t i o n s o f these substances for the m o d u l a t i o n o f G A B A binding, suggesting t h a t p i c r o t o x i n i n ( b a r b i t u r a t e ) b i n d i n g sites are c o u p l e d to G A B A receptors. P e n t o b a r b i t a l stimulates b o t h G A B A a n d b e n z o d i a z e p i n e b i n d i n g at the similar c o n c e n t r a t i o n s . B o t h stimulations were d e p e n d e n t on chloride ions a n d sensitive to T r i t o n X-100 t r e a t m e n t . These results further s u p p o r t the G A B A / b e n z o d i a z e p i n e r e c e p t o r c o m p l e x as shown b y allosteric activation b y G A B A o f b e n z o d i a z e p i n e b i n d i n g 16, b y p r o t e c t i n g b o t h b i n d i n g sites b y G A B A a n a l o g u e s a n d b e n z o d i a z e p i n e s f r o m i n a c t i v a t i o n b y h e a t a n d i o d o a c e t a m i d e 6, a n d b y c o p u r i f i c a t i o n o f b o t h b i n d i n g sites2,3,7.
1 Araki, T., Ito, M. and Oscarsson, O., Anion permeability of the synaptic and non-synaptic motoneurone membrane, J. Physiol. (Lond.), 159 (1961) 410-435. 2 Asano, T. and Ogasawara, N., Solubilization ofy-aminobutyric acid receptor from rat brain, Life Sci., 26 (1980) 1131-1137. 3 Asano, T. and Ogasawara, N., Soluble gamma-aminobutyric acid and benzodiazepine receptors from rat cerebral cortex, Life Sci., 29 (1981) 193-200. 4 Enna, S. J. and Snyder, S. H., A simple, sensitive and specific radioreceptor assay for endogenous GABA in brain tissue, J. Neurochem., 26 (1976) 221-224. 5 Evans, R. H., Potentiation of the effects of GABA by pentobarbitone, Brain Research, 171 (1979) 113-120. 6 Gavish, M. and Snyder, S. H., Benzodiazepine recognition sites on GABA receptors, Nature (Lond.), 287 (1980) 651-652. 7 Gavish, M. and Snyder, S. H., 7-Aminobutyric acid and benzodiazepine receptors: copurification and characterization, Proc. nat. Acad. Sci. (U.S.A.), 78 (1981) 1939-1942. 8 Huang, L.-Y. M. and Barker, J. L., Pentobarbital: stereospecific actions of ( + ) and (--) isomers revealed on cultured mammalian neurons, Science, 207 (1980) 195-197. 9 Leeb-Lundberg, F., Snowman, A. and Olsen, R. W., Barbiturate receptor sites are coupled to benzodiazepine receptors, Proc. nat. ,4cad. Sci. (U.S.A.), 77 (1980) 7468-7472. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275.
216 11 Nicoll, R. A., Pentobarbital: differential postsynaptic actions on sympathetic ganglion cells, Science, 199 (1978) 451-452. 12 Nicoll, R. A. and Wojtowicz, J. M., The effects of pentobarbital and related compounds on frog motoneurons, Brain Research, 191 (1980) 225-237. 13 Olsen, R. W., Ticku, M. K., Greenlee, D. and Van Ness, P., GABA receptor and ionophore binding sites: interaction with various drugs, In P. Krogsgaard-Larsen, J. Scheel-Krfiger and H. Kofod (Eds.), GABA-Neurotransmitters, Munksgaard, Copenhagen, 1979, pp. 165-178. 14 Ransom, B. R. and Barker, J. L., Pentobarbital selectively enhances GABA-mediated post-synaptic inhibition in tissue cultured mouse spinal neurons, Brain Research, 114 (1976) 530-535. 15 Skolnick, P., Moncada, V., Barker, J. L. and Paul, S. M., Pentobarbital: dual actions to increase brain benzodiazepine receptor affinity, Science, 211 (1981) 1448-1450. 16 Tallman, J. F., Thomas, J. W. and Gallager, D. W., GABAergic modulation of benzodiazepine binding site sensitivity, Nathre (Lond.), 274 (1978) 383-385. 17 Ticku, M. K., Ban, M. and Olsen, R. W., Binding of [3H]a-dihydropicrotoxinin, a 7-aminobutyric acid synaptic antagonist, to rat brain membranes, Molec. Pharmacol., 14 (1978) 391-402. 18 Ticku, M. K., Van Ness, P. C., Haycock, J. W., Levy, W. B. and Olsen, R. W., Dihydropicrotoxinin binding sites in rat brain: comparison to GABA receptors, Brain Research, 150 (1978) 642-647. 19 Willow, M. and Johnston, G. A. R., Enhancement of GABA binding by pentobarbitone, Neurosci. Lett., 18 (1980) 323-327.
Brain Research, 225 (1981) 212-216 Elsevier/North-Holland Biomedical Press
Chloride-dependent stimulation of GABA and benzodiazepine receptor binding by pentobarbital
TOM1KO ASANO and NOBUAKI OGASAWARA Department of Biochemistry, Institute ]'or Developmental Research, Aichi Prefecture Colony, Kasugai, .4ichi 480-03 (Japan)
(Accepted August 6th, 1981) Key words: GABA receptor - - benzodiazepinereceptor - - pentobarbital - - picrotoxinin - - chloride ion - - Triton X-100 treatment
Pentobarbital reversibly increased Na+-independent GABA binding to bovine cerebral cortex membranes. This effect was chloride-dependent. Kinetic analysis of [3H]GABAbinding revealed an increase of the apparent number of binding sites. Pretreatment of the membranes with Triton X-100 extremely diminished the effect of pentobarbital on both [aH]GABA and [3H]flunitrazepam binding. Barbiturates are general central nervous system depressants that are used clinically for anesthetic, sedative-hypnotic, and anticonvulsant actions. Electrophysiological studies suggest that barbiturate can enhance or mimic the postsynaptic response to the inhibitory neurotransmitter y-aminobutyric acid (GABA)5,s,12,14. Attempts to demonstrate enhancement by pentobarbital of GABA binding to synaptic membranes have been thus far unsuccessful 4,13, except that Willow and Johnston 19 have shown 3 0 - 5 0 ~ enhancement of GABA binding by pentobarbital with a relatively crude and carefully prepared membrane. The benzodiazepine receptors are at least partly coupled to GABA receptor binding sites in the central nervous system, as shown by allosteric activation by GABA of benzodiazepine binding in membranes 16. It was recently shown that pentobarbital enhanced benzodiazepine receptor binding and the effect was chloride-dependentg, 15. In this paper, we show that pentobarbital markedly increases Na÷-independent GABA binding in the presence of chloride ions and the effects of pentobarbital on GABA and benzodiazepine binding are found at similar concentrations. The results are compatible with a possible existence of the GABA/benzodiazepine/barbiturate receptor complex. Bovine cerebral cortex was homogenized in 10 vols. of 0.32 M sucrose in a glass homogenizer with a Teflon pestle. The homogenate was centrifuged for 10 min at 1000 g and the supernatant was recentrifuged for 20 min at 20,000 g. The pellet from the second centrifugation (P2 pellet) was washed once with 50 mM Tris-HC1 (pH 7.4) and frozen at - - 2 0 °C. After thawing, the P2 pellet was washed 5 times by resuspension in 0006-8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
213 50 mM potassium phosphate buffer (pH 7.4) and centrifugation at 30,000 g for 20 min. The pellet was resuspended to a final protein concentration of 4.0-4.5 mg/ml in 50 mM potassium phosphate buffer (pH 7.4). Studies of Na+-independent [3H]GABA binding were performed as follows: the samples of the membrane suspension were incubated in 200 #1 of 50 mM potassium phosphate buffer (pH 7.4), containing 5 nM [SH]GABA (58 Ci/mmol, Amersham) in the absence or presence of 100 mM KC1. After incubation at 0 °C for 20 min the reaction was terminated by centrifugation at 30,000 g for 20 min. The supernatant was discarded, the pellet was superficially rinsed with cold distilled water, solubilized in Protosol (New England Nuclear), and the radioactivity measured. Benzodiazepine binding was determined by the method described previously2 using 0.25 nM [3H]flunitrazepam (84 Ci/mmol, New England Nuclear). Protein was determined by the method of Lowry et al. 1°. Sodium pentobarbital (Abbott) stimulated Na+-independent [3H]GABA binding in both chloride-free and chloride-enriched buffer (Fig. 1). However, both the maximum stimulation by pentobarbital (98 %) and ECs0 (concentration required for half-maximal stimulation) (320/~M) in the chloride-free medium were enhanced in the presence of 100 mM KCI (340% and 150 #M, respectively). ECs0 values for the stimulation of GABA binding determined in each condition are in good agreement with those for the stimulation of benzodiazepine receptor binding in previous reportsgJL The effect of pentobarbital on [ZH]GABA binding was reversible since preincubation with pentobarbital followed by centrifugation of the membranes and resuspension in drug-free buffer reduced [3H]GABA binding to control levels. As calcium pentobarbital (Tanabe Pharmaceutical) also stimulated [ZH]GABA binding, the effect of sodium pentobarbital did not involve the effect by low concentration of sodium ion. Ion requirement did not involve the cation, because MgCb was equally effective. The anions such as bromide, iodide and thiocyanate can substitute for
400 o 300 'S 200
100 lO
100 Pentoborbltol (pM)
1000
Fig. 1. Effect of pentobarbital on [aH]GABA binding to bovine cerebral cortex membranes. Pentobarbital enhances laHIGABAbinding in the presence (O) and absence (Q) of 100 mM KCI. Values represent the mean of 3 experiments.
214 TABLE I Effect of pretreatment of the membranes with Triton 3(-100 on the stimulation of [aHfGABA and [aHJflunitrazepam binding by pentobarbital
Non-treated membrane was prepared as described in the text. Triton X-100 treated membrane was prepared as follows: the P~ pellet was incubated in 20 vols. of 50 mM Tris-HCl (pH 7.4) containing 0.05 700Triton X-100 for 30 min at 37 °C, centrifuged at 30,000 g for 20 min, and washed 4 times in 50 mM potassium phosphate buffer without Triton X-100. Specific [aH]GABA and [aH]flunitrazepam binding in the presence of 100 mM KCI and the absence of pentobarbital was taken as 100 ~. Concentration of pentobarbital
0.1 mM 1 mM
% Control [ZH]GABA binding
/ 3H]Flunitrazepam binding
Non-treated membrane
Triton X - I O 0 Non-treated treatedmembrane m e m b r a n e
Triton X-IO0 treatedmembrane
204 418
115 146
109 118
127 163
chloride, but acetate, fluoride and sulfate ions cannot. The selectivity of the effective anions on G A B A binding is in agreement with that on the stimulation of benzodiazepine binding by pentobarbital 9, and also with the selectivity for penetrating the activated inhibitory postsynaptic membranes of cat motoneurons measured electrophysiologically I. The increases in [3H]GABA binding elicited by pentobarbital (200 /~M) were antagonized by picrotoxinin. IC50 value (concentration that inhibits binding by 50 ~ ) of 1.4 # M was obtained in the presence of 100 m M chloride. Pretreatment of the membranes with the detergent Triton X-100 extremely diminished the effect of pentobarbital on both [aH]GABA and [aH]flunitrazepam binding (Table I). Ticku et al. 18 reported that Triton X-100 treatment inhibited picrotoxinin binding to rat brain membranes. These results indicate that the stimulation of both G A B A and benzodiazepine binding by pentobarbital is mediated by picrotoxinin (barbiturate) binding sites. Kinetic analysis of the effect of pentobarbital (200 ~M) on Na+-independent [3H]GABA binding in the presence of chloride revealed an increase of binding sites (Bmax = 2.61 :k 0.14 pmol/mg protein) over control samples (Bmax ---- 0.91 ~ 0.08 pmol/mg protein). In contrast, the apparent affinity constant was slightly increased with pentobarbital (Ka = 109 :k 6 nM with pentobarbital, Kd = 98 + 6 nM without pentobarbital), but this increase was not statistically significant (Fig. 2). The concentrations of pentobarbital which enhance G A B A binding in the present study are comparable with those which enhance GABA-mediated postsynaptic inhibition in tissue cultured mouse spinal neurons 14, and GABA-induced depolarization of frog sympathetic ganglion cells 11 and dorsal root fibers in the immature rat spinal cord in vitroL Pentobarbital is known to inhibit the binding of dihydropicrotoxinin to fresh rat brain membranes 17. Dihydropicrotoxinin binding is considered to reflect binding to chloride ionophores normally linked to G A B A receptors. Fifty per cent inhibition of dihydropicrotoxinin binding occurred at 50/~M pentobarbital and 0.4 # M picrotox-
215
.x =~ 2
200
qO0
600
800
Bound (fmole/assay)
Fig. 2. Scatchard plot of [aH]GABA binding to bovine cerebral cortex membranes. [3H]GABA binding was measured in the presence of 100 mM KCI with ((3) or without (O) 200/~M pentobarbital. The data shown are the means of 3 experiments.
inin 17. These results are in g o o d a g r e e m e n t with the effective c o n c e n t r a t i o n s o f these substances for the m o d u l a t i o n o f G A B A binding, suggesting t h a t p i c r o t o x i n i n ( b a r b i t u r a t e ) b i n d i n g sites are c o u p l e d to G A B A receptors. P e n t o b a r b i t a l stimulates b o t h G A B A a n d b e n z o d i a z e p i n e b i n d i n g at the similar c o n c e n t r a t i o n s . B o t h stimulations were d e p e n d e n t on chloride ions a n d sensitive to T r i t o n X-100 t r e a t m e n t . These results further s u p p o r t the G A B A / b e n z o d i a z e p i n e r e c e p t o r c o m p l e x as shown b y allosteric activation b y G A B A o f b e n z o d i a z e p i n e b i n d i n g 16, b y p r o t e c t i n g b o t h b i n d i n g sites b y G A B A a n a l o g u e s a n d b e n z o d i a z e p i n e s f r o m i n a c t i v a t i o n b y h e a t a n d i o d o a c e t a m i d e 6, a n d b y c o p u r i f i c a t i o n o f b o t h b i n d i n g sites2,3,7.
1 Araki, T., Ito, M. and Oscarsson, O., Anion permeability of the synaptic and non-synaptic motoneurone membrane, J. Physiol. (Lond.), 159 (1961) 410-435. 2 Asano, T. and Ogasawara, N., Solubilization ofy-aminobutyric acid receptor from rat brain, Life Sci., 26 (1980) 1131-1137. 3 Asano, T. and Ogasawara, N., Soluble gamma-aminobutyric acid and benzodiazepine receptors from rat cerebral cortex, Life Sci., 29 (1981) 193-200. 4 Enna, S. J. and Snyder, S. H., A simple, sensitive and specific radioreceptor assay for endogenous GABA in brain tissue, J. Neurochem., 26 (1976) 221-224. 5 Evans, R. H., Potentiation of the effects of GABA by pentobarbitone, Brain Research, 171 (1979) 113-120. 6 Gavish, M. and Snyder, S. H., Benzodiazepine recognition sites on GABA receptors, Nature (Lond.), 287 (1980) 651-652. 7 Gavish, M. and Snyder, S. H., 7-Aminobutyric acid and benzodiazepine receptors: copurification and characterization, Proc. nat. Acad. Sci. (U.S.A.), 78 (1981) 1939-1942. 8 Huang, L.-Y. M. and Barker, J. L., Pentobarbital: stereospecific actions of ( + ) and (--) isomers revealed on cultured mammalian neurons, Science, 207 (1980) 195-197. 9 Leeb-Lundberg, F., Snowman, A. and Olsen, R. W., Barbiturate receptor sites are coupled to benzodiazepine receptors, Proc. nat. ,4cad. Sci. (U.S.A.), 77 (1980) 7468-7472. 10 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275.
216 11 Nicoll, R. A., Pentobarbital: differential postsynaptic actions on sympathetic ganglion cells, Science, 199 (1978) 451-452. 12 Nicoll, R. A. and Wojtowicz, J. M., The effects of pentobarbital and related compounds on frog motoneurons, Brain Research, 191 (1980) 225-237. 13 Olsen, R. W., Ticku, M. K., Greenlee, D. and Van Ness, P., GABA receptor and ionophore binding sites: interaction with various drugs, In P. Krogsgaard-Larsen, J. Scheel-Krfiger and H. Kofod (Eds.), GABA-Neurotransmitters, Munksgaard, Copenhagen, 1979, pp. 165-178. 14 Ransom, B. R. and Barker, J. L., Pentobarbital selectively enhances GABA-mediated post-synaptic inhibition in tissue cultured mouse spinal neurons, Brain Research, 114 (1976) 530-535. 15 Skolnick, P., Moncada, V., Barker, J. L. and Paul, S. M., Pentobarbital: dual actions to increase brain benzodiazepine receptor affinity, Science, 211 (1981) 1448-1450. 16 Tallman, J. F., Thomas, J. W. and Gallager, D. W., GABAergic modulation of benzodiazepine binding site sensitivity, Nathre (Lond.), 274 (1978) 383-385. 17 Ticku, M. K., Ban, M. and Olsen, R. W., Binding of [3H]a-dihydropicrotoxinin, a 7-aminobutyric acid synaptic antagonist, to rat brain membranes, Molec. Pharmacol., 14 (1978) 391-402. 18 Ticku, M. K., Van Ness, P. C., Haycock, J. W., Levy, W. B. and Olsen, R. W., Dihydropicrotoxinin binding sites in rat brain: comparison to GABA receptors, Brain Research, 150 (1978) 642-647. 19 Willow, M. and Johnston, G. A. R., Enhancement of GABA binding by pentobarbitone, Neurosci. Lett., 18 (1980) 323-327.