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Pyridazinyl-GABA derivatives as GABA and glycine antagonists in the spinal cord of the cat
Neuroseience Letters, 68 (1986) 211 215
211
Elsevier Scientific Publishers Ireland Ltd. NSL 04036 P Y R | D A Z I N Y L - G A B A DERIVATIVES AS GABA A N D GLYCINE A N T A G O N I S T S IN THE SPINAL C O R D OF THE CAT
B.D. GYNTHER and D.R. CURTIS Department ~['Pharmacology, John Curtin School o['Medical Research, G.P.O. Box 334, Canberra. ,4.('. T. 2601 (Australia)
(Received April 4th, 1986; Revised version received and accepted April 8th, 1986)
hk'v wor~Zs." cat spinal cord glycine },-aminobutyricacid (GABA) pyridazinylderivatives of GABA 2-(carboxy-3'-propyl)-3-amino-6-paramethoxy-phenylpyridaziniumbromide (SR95531 )
of tw~ arylaminopyridazinederivatives of 7-aminobutyric acid (GABA) tested as antagonists of the inhibitory actions of glycine and GABA in the spinal cord of pentobarbilone-anaesthetized cats. one SR95531 was sufficientlyselective to be of use in microelectrophoreticinvestigations of GABA-mediated synaplic transmission. Identification of inhibitory synaptic transmitters in the spinal cord of the cat as either of the inhibitory amino acids, glycine and ~,-aminobutyric acid (GABA), has depended to a considerable extent on the use of the selective antagonists, strychnine and bicuculline, respectively [2]. Many spinal neurones are influenced by both glycine- and GABA-mediated inhibitory pathways [4, 8, 11], and a similar situation may occur elsewhere in the CNS. Accordingly, selective, readily reversible and stable antagonists of either amino acid are of interest for further investigation of central glycine and G A B A receptors and of synaptic inhibitory processes associated with them. The recent availability of several arylaminopyridazine derivatives of GABA, reported as antagonists at G A B A receptors and binding sites [1, 7, 9, 12], has provided an opportunity to compare the effects of these compounds with strychnine and bicuculline methochloride as antagonists of the inhibitory actions of glycine and GABA. The investigation was carried out on dorsal horn interneurones within lumbar segments of cats anaesthetized with pentobarbitone sodium, using previously described techniques [3]. Extracellular action potentials of single neurones were recorded by means of the central (3.6 M NaCI) barrel of 7-barrel micropipettes. The outer barrels contained the following aqueous solutions from which active ions were ejected microelectrophoretically: glycine (0.2 or 0.5 M, pH 3, HC1); G A B A (0.2 M, pH 3, HC1); DL-homocysteic acid (DLH, 0.2 M, pH 7.5, NaOH); NaC1 (1.8 M); strychnine hydrochloride (2 mM in 150 mM NaCl); bicuculline methochloride (BMC, 10 mM in 150 mM NaCI); 2-(carboxy-3'-propyl)-3-amino-4-methyl-6-phenylpyridaziniumchloride (SR95103, 10 or 50 mM in 150 mM NaCI) and 2-(carboxy-3'-propyl)-3-amino-6paramethoxy-phenylpyridazinium bromide (SR95531, 5 mM in 150 mM NaCI). 0304-3940/865 03.50 © 1986Elsevier Scientific Publishers Ireland Ltd.
21 ~ R e t a i n i n g voltages o f 0.5 V were used r o u t i n e l y to reduce the leakage o f a m i n o acids a n d antagonists. Cell firing rates were m a i n t a i n e d (30-60 spikes/s) by the c o n t i n u o u s ejection o f D L H (5 20 hA), and glycine a n d G A B A were ejected sequentially using e l e c t r o p h o r e t i c c u r r e n t s for p e r i o d s (5-30 s) a n d at intervals (20-30 s) which yielded s u b m a x i m a l or n e a r - m a x i m a l i n h i b i t i o n o f firing. A n t a g o n i s m o f the i n h i b i t o r y effects o f these a m i n o acids was o b s e r v e d as a slowing o f the onset and a reduction in the degree o f inhibition. The effect o f SR95531 was also e x a m i n e d on the bicuculline-sensitive d e p o l a r i z a tion o f ventral h o r n t e r m i n a t i o n s o f g r o u p l a p r i m a r y afferent fibers o f hind limb e x t e n s o r muscles by tetanic volleys in l o w - t h r e s h o l d flexor muscle afferent fibres ( p r i m a r y afferent d e p o l a r i z a t i o n , P A D ) a n d by the G A B A - m i m e t i c piperidine-4-sulp h o n i c acid (P4S, 20 m M in 150 m M NaC1, p H 3.5) ejected m i c r o e l e c t r o p h o r e t i c a l l y , using previously d e s c r i b e d techniques [5].
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Fig. 1. A comparison of the effects of microelectrophoretically administered strychnine (A, STRYCH), SR95103 (B, 10 mM in 150 mM NaCl) and BMC (C) on the inhibition by glycine (GL, 0.2 M) and GABA (GA, 0.2 M) of the firing of a spinal dorsal horn interneurone, maintained by the continuous administration of oL-homocysteic acid. Microelectrophoretic ejections are indicated by horizontal bars and symbols; A: GL 25 nA, GA 40 nA; before, during and 4 min after STRYCH 40 nA for 66 s. B: GL 12 nA, GA 20 nA; before, during the ejection of SR95103 40 nA for 150 s, 80 nA for 60 s, terminating at the vertical broken line and 60 s after SR95103. C: GL 9 nA, GA 20 nA; before, during the ejection of BMC; 20 nA for 240 s, terminating at the vertical broken line, and 90 s after BMC. Ordinates: firing rate (spikes/s); abscissae: time (min).
213
The effects of SR95103 and SR95531 were examined in 11 cats. With all 11 cells SR95103 reversibly reduced the inhibition of dorsal horn interneurone firing by both glycine and GABA. In only one case was the GABA-induced inhibition diminished to a greater extent than that of glycine, while with 5 cells the glycine response was more sensitive to SR95103 than was the inhibition produced by GABA. No evidence of an entirely selective inhibition of GABA or glycine was observed with SR95103 using currents of 10-40 nA. A comparison of the effects of microelectrophoretic strychnine, SR95103 and BMC is shown in Fig. 1. SR95103 was less potent than strychnine and BMC in reducing the effects of glycine and GABA, respectively, and recovery occurred at a similar rate to that of BMC, but more rapidly than that of strychnine. S R95103 also increased cell firing rates by 5-10 spikes/sec. Fig. 2 illustrates the reversible antagonism by microelectrophoretic SR95531 of glycine- and GABA-induced inhibition. As with 9 other cells, low currents (10 20 nA) of SR95531 selectively reduced inhibition by GABA without influencing that by glycine. When ejected with currents of approximately twice this value, SR95531 generally reduced the inhibitory effects of both GABA and glycine. SR95531 alone produced a small increase in cell firing rate, but rarely produced the bursts of firing which typically occur with BMC [10]. SR95531 and BMC appeared to be equipotent as GABA antagonists, and recovery occurred at similar rates. A
B
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Fig. 2. The effect of SR95531 on the inhibition by glycine (GL, 0.5 M, 7 hA) and G A B A (GA, 0.2 M, 20 hA) of the tiring of a dorsal horn interneurone, maintained by continuous administration of DE-homocysteic acid. Microelectrophoretic ejections are indicated by horizontal bars and symbols. SR95531 was ejected for a total time of 8 min; 20 nA for 2.5 rain, 40 nA for 3 min, 60 nA for 2.5 min. A: before SR9553 I. B: 1.25 rain after beginning SR95531, 20 nA. C: 1.25 rain after beginning SR95531, 40 nA. D: 1 rain aftcr beginning SR95531, 60 nA. E: 1 min after SR95531. F: 4 min after E. Ordinates: firing rate (spikes/s); abscissae: lime (30 s).
214
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Fig. 3. Comparison of the effects of microelectrophoretic SR95531 (40 hA) and BMC (20 nA) on the electrical threshold of a group Ia gastrocnemius afferent fibre termination excited in the ventral horn and on changes in threshold (ordinates: AT%) produced by P4S (40 nA for 40 s) and by tetanic stimulation (PAD, 4 volleys, 3-ms intervals, 2T) of low-threshold posterior biceps-semitendinosus afferent fibres, 30 ms before the testing pulse, for 8 s. A: threshold pulse 1.10 laA. B: threshold pulse 1.19 I~A. Abscissae: time (min).
SR95531 and BMC reversibly increased the threshold of group Ia primary afferent terminations and reduced the decrease in threshold produced by P4S and PAD, as illustrated in Fig. 3. Indeed, during microelectrophoretic SR95531 (and BMC), P4S often increased termination thresholds. Such an increase is the only effect of amino acids, such as glycine, for which Ia terminations apparently have no specific receptors, and is a consequence of the active uptake of these amino acids by neurones and glia (see ref. 6). The reversible increase in termination threshold by SR95531 and BMC presumably indicates a reduction in tonic PAD. It is not surprising that PAD generated by tetanic stimulation of flexor muscle afferent fibres was diminished but not abolished by SR95531 or BMC. When administered microelectrophoretically neither agent would be expected to influence all of the terminations of a single Ia fibre, yet PAD generated at axo-axonic synapses on each termination would contribute electrotonically to the reduction in threshold assessed by electric stimulation of one termination. Since only amino acids structurally related to GABA depolarized Ia terminations [6], the selectivity of SR95531 as a GABA antagonist could not be determined in these tests. In the rat cuneate nucleus SR95531 reduced the inhibition by GABA of the spontaneous or glutamate-evoked firing of neurones but infrequently reduced the inhibition produced by glycine, while SR95103 diminished the effects of both GABA and glycine [12]. The present findings in the cat spinal cord differ in that SR95531 ejected with currents only approximately twice those which produced concentrations selectively reducing the inhibitory action of G A B A also reduced that of glycine. Species or regional differences in GABA receptors may account for this disparity. The appar-
215
ent interaction of SR95103 with glycine and G A B A receptors may be related more to the presence in this c o m p o u n d of a methyl group at the 4-position of the pyridazinc ring than to substituents, such as the methoxy group in SR95531, at the paraposition of the phenyl ring. Substituents at this latter site, however, are clearly important for the interaction of these c o m p o u n d s with binding sites for G A B A on rat brain membranes [9]. SR95531 appears to be a soluble, potent, reversible and reasonably selective G A B A antagonist in the cat spinal cord. Reduction of the inhibitory effect of G A B A in the absence of bursts of cell firing, such as occur with BMC and presumably reflect a direct excitatory action, could be a useful attribute in microelectrophoretic investigations of GABA-mediated synaptic transmission. Further studies are required, however, in other regions of the cat central nervous system of the effect of SR95531 on the inhibitory actions of GABA-like and glycine-like amino acids. The authors are grateful to Professor C.G. Wermuth and Dr. K. Biziere for gifts of SR95103 and SR95531, to Mrs. P. Searle and Mr. T. Van Arkel for skilled technical assistance and to Mrs. J. Harries for word-processing the manuscript. 1 Chambon, J.P., Feltz, P., Heaulme, M., Restle, S., Schlichter, R., Biziere, K. and Wermuth, C.G., An arylaminopyridazine derivative of 7-aminobutyric acid (GABA) is a selective and competitive antagonist at the GABAA receptor site, Proc. Natl. Acad. Sci. USA, 82 (1985) 1832 1836. 2 Curtis, D.R. and Johnston, G.A.R., Amino acid transmitters in the m a m m a l i a n central nerwms system, Ergebn. Physiol., 69 (1974) 97 188. 3 Curtis, D.R.. Duggan, A.W., Felix, D. and Johnston, G.A.R., Bicuculline, an antagonist of G A B A and synaptic inhibition in the spinal cord, Brain Res., 32 (1971) 69 96. 4 Curtis, D.R., Game, C.J.A., Lodge, D. and McCulloch, R.M., A pharmacological study of Renshaw cell inhibition. J. Physiol. (London), 258 (1976) 227-242. 5 Curtis, D.R. and Lodge, D., The depolarization of feline ventral horn group Ia spinal afferent terminations b y G A B A , Exp. Brain Res., 46 (1982} 215 233. 6 Curtis, D.R., Lodge, D., Bornstein, J.C., Peel, M.J. and Leah, J.D., The dual effects of G A B A and related amino acids on the electrical threshold of ventral horn group la afferent terminations in the cat, Exp. Brain Res., 48 (1982) 387 400. 7 Dcsarmenien, M., Desaulles, E., Feltz, P., H a m a n n , M., Michaud, J.C. and Mienville, J.M., Electrophysiological study of pyridazine-GABA derivatives with G A B A - A antagonist properties, Br. J. Pharmacol., in press. 8 Game, C.J.A. and Lodge, D., The pharmacology of the inhibition of dorsal horn neurones by impulses in myelinated cutaneous afferents in the cat, Exp. Brain Res., 23 (1975) 75-84. 9 Heaulme, M., C h a m b o n , J.P., Leyris. R., Molimard, J.C., Wermuth, C.G. and Biziere, K., Biochemical characlcrization of the interaction of three pyridazinyl-GABA derivatives with the (}ABA-A receptor site, Brain Res., in press. 10 Johnston, G.A.R., Beart, P.M., Curtis, D.P,., Game, C.J.A., McCulloch, R.M. and Maclachlan, R.M., Bicuculline methochloride as a G A B A antagonist, Nature (London), New Biol., 240 (1972) 219 220. I I Lodge, D., Curtis, D.R. and Brand, S.J., A pharmacological study of the inhibition of ventral group la-excilcd spinal interneurones, Exp. Brain Res., 29 (1977) 97 105. 12 Michaud, .1.(,7., Mienville, J.M., C h a m b o n , J.P. and Biziere, K., Interactions of three pyridazinylG A B A derivatives with rat central G A B A and glycine receptors, an in vivo microiontophoretic study, Neuropharmacology, in press.
211
Elsevier Scientific Publishers Ireland Ltd. NSL 04036 P Y R | D A Z I N Y L - G A B A DERIVATIVES AS GABA A N D GLYCINE A N T A G O N I S T S IN THE SPINAL C O R D OF THE CAT
B.D. GYNTHER and D.R. CURTIS Department ~['Pharmacology, John Curtin School o['Medical Research, G.P.O. Box 334, Canberra. ,4.('. T. 2601 (Australia)
(Received April 4th, 1986; Revised version received and accepted April 8th, 1986)
hk'v wor~Zs." cat spinal cord glycine },-aminobutyricacid (GABA) pyridazinylderivatives of GABA 2-(carboxy-3'-propyl)-3-amino-6-paramethoxy-phenylpyridaziniumbromide (SR95531 )
of tw~ arylaminopyridazinederivatives of 7-aminobutyric acid (GABA) tested as antagonists of the inhibitory actions of glycine and GABA in the spinal cord of pentobarbilone-anaesthetized cats. one SR95531 was sufficientlyselective to be of use in microelectrophoreticinvestigations of GABA-mediated synaplic transmission. Identification of inhibitory synaptic transmitters in the spinal cord of the cat as either of the inhibitory amino acids, glycine and ~,-aminobutyric acid (GABA), has depended to a considerable extent on the use of the selective antagonists, strychnine and bicuculline, respectively [2]. Many spinal neurones are influenced by both glycine- and GABA-mediated inhibitory pathways [4, 8, 11], and a similar situation may occur elsewhere in the CNS. Accordingly, selective, readily reversible and stable antagonists of either amino acid are of interest for further investigation of central glycine and G A B A receptors and of synaptic inhibitory processes associated with them. The recent availability of several arylaminopyridazine derivatives of GABA, reported as antagonists at G A B A receptors and binding sites [1, 7, 9, 12], has provided an opportunity to compare the effects of these compounds with strychnine and bicuculline methochloride as antagonists of the inhibitory actions of glycine and GABA. The investigation was carried out on dorsal horn interneurones within lumbar segments of cats anaesthetized with pentobarbitone sodium, using previously described techniques [3]. Extracellular action potentials of single neurones were recorded by means of the central (3.6 M NaCI) barrel of 7-barrel micropipettes. The outer barrels contained the following aqueous solutions from which active ions were ejected microelectrophoretically: glycine (0.2 or 0.5 M, pH 3, HC1); G A B A (0.2 M, pH 3, HC1); DL-homocysteic acid (DLH, 0.2 M, pH 7.5, NaOH); NaC1 (1.8 M); strychnine hydrochloride (2 mM in 150 mM NaCl); bicuculline methochloride (BMC, 10 mM in 150 mM NaCI); 2-(carboxy-3'-propyl)-3-amino-4-methyl-6-phenylpyridaziniumchloride (SR95103, 10 or 50 mM in 150 mM NaCI) and 2-(carboxy-3'-propyl)-3-amino-6paramethoxy-phenylpyridazinium bromide (SR95531, 5 mM in 150 mM NaCI). 0304-3940/865 03.50 © 1986Elsevier Scientific Publishers Ireland Ltd.
21 ~ R e t a i n i n g voltages o f 0.5 V were used r o u t i n e l y to reduce the leakage o f a m i n o acids a n d antagonists. Cell firing rates were m a i n t a i n e d (30-60 spikes/s) by the c o n t i n u o u s ejection o f D L H (5 20 hA), and glycine a n d G A B A were ejected sequentially using e l e c t r o p h o r e t i c c u r r e n t s for p e r i o d s (5-30 s) a n d at intervals (20-30 s) which yielded s u b m a x i m a l or n e a r - m a x i m a l i n h i b i t i o n o f firing. A n t a g o n i s m o f the i n h i b i t o r y effects o f these a m i n o acids was o b s e r v e d as a slowing o f the onset and a reduction in the degree o f inhibition. The effect o f SR95531 was also e x a m i n e d on the bicuculline-sensitive d e p o l a r i z a tion o f ventral h o r n t e r m i n a t i o n s o f g r o u p l a p r i m a r y afferent fibers o f hind limb e x t e n s o r muscles by tetanic volleys in l o w - t h r e s h o l d flexor muscle afferent fibres ( p r i m a r y afferent d e p o l a r i z a t i o n , P A D ) a n d by the G A B A - m i m e t i c piperidine-4-sulp h o n i c acid (P4S, 20 m M in 150 m M NaC1, p H 3.5) ejected m i c r o e l e c t r o p h o r e t i c a l l y , using previously d e s c r i b e d techniques [5].
A
STRYCH 40
GL o
I
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Fig. 1. A comparison of the effects of microelectrophoretically administered strychnine (A, STRYCH), SR95103 (B, 10 mM in 150 mM NaCl) and BMC (C) on the inhibition by glycine (GL, 0.2 M) and GABA (GA, 0.2 M) of the firing of a spinal dorsal horn interneurone, maintained by the continuous administration of oL-homocysteic acid. Microelectrophoretic ejections are indicated by horizontal bars and symbols; A: GL 25 nA, GA 40 nA; before, during and 4 min after STRYCH 40 nA for 66 s. B: GL 12 nA, GA 20 nA; before, during the ejection of SR95103 40 nA for 150 s, 80 nA for 60 s, terminating at the vertical broken line and 60 s after SR95103. C: GL 9 nA, GA 20 nA; before, during the ejection of BMC; 20 nA for 240 s, terminating at the vertical broken line, and 90 s after BMC. Ordinates: firing rate (spikes/s); abscissae: time (min).
213
The effects of SR95103 and SR95531 were examined in 11 cats. With all 11 cells SR95103 reversibly reduced the inhibition of dorsal horn interneurone firing by both glycine and GABA. In only one case was the GABA-induced inhibition diminished to a greater extent than that of glycine, while with 5 cells the glycine response was more sensitive to SR95103 than was the inhibition produced by GABA. No evidence of an entirely selective inhibition of GABA or glycine was observed with SR95103 using currents of 10-40 nA. A comparison of the effects of microelectrophoretic strychnine, SR95103 and BMC is shown in Fig. 1. SR95103 was less potent than strychnine and BMC in reducing the effects of glycine and GABA, respectively, and recovery occurred at a similar rate to that of BMC, but more rapidly than that of strychnine. S R95103 also increased cell firing rates by 5-10 spikes/sec. Fig. 2 illustrates the reversible antagonism by microelectrophoretic SR95531 of glycine- and GABA-induced inhibition. As with 9 other cells, low currents (10 20 nA) of SR95531 selectively reduced inhibition by GABA without influencing that by glycine. When ejected with currents of approximately twice this value, SR95531 generally reduced the inhibitory effects of both GABA and glycine. SR95531 alone produced a small increase in cell firing rate, but rarely produced the bursts of firing which typically occur with BMC [10]. SR95531 and BMC appeared to be equipotent as GABA antagonists, and recovery occurred at similar rates. A
B
•
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4o "~ 20
C SR95531 2O O
~/,'~,~
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0
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Fig. 2. The effect of SR95531 on the inhibition by glycine (GL, 0.5 M, 7 hA) and G A B A (GA, 0.2 M, 20 hA) of the tiring of a dorsal horn interneurone, maintained by continuous administration of DE-homocysteic acid. Microelectrophoretic ejections are indicated by horizontal bars and symbols. SR95531 was ejected for a total time of 8 min; 20 nA for 2.5 rain, 40 nA for 3 min, 60 nA for 2.5 min. A: before SR9553 I. B: 1.25 rain after beginning SR95531, 20 nA. C: 1.25 rain after beginning SR95531, 40 nA. D: 1 rain aftcr beginning SR95531, 60 nA. E: 1 min after SR95531. F: 4 min after E. Ordinates: firing rate (spikes/s); abscissae: lime (30 s).
214
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Fig. 3. Comparison of the effects of microelectrophoretic SR95531 (40 hA) and BMC (20 nA) on the electrical threshold of a group Ia gastrocnemius afferent fibre termination excited in the ventral horn and on changes in threshold (ordinates: AT%) produced by P4S (40 nA for 40 s) and by tetanic stimulation (PAD, 4 volleys, 3-ms intervals, 2T) of low-threshold posterior biceps-semitendinosus afferent fibres, 30 ms before the testing pulse, for 8 s. A: threshold pulse 1.10 laA. B: threshold pulse 1.19 I~A. Abscissae: time (min).
SR95531 and BMC reversibly increased the threshold of group Ia primary afferent terminations and reduced the decrease in threshold produced by P4S and PAD, as illustrated in Fig. 3. Indeed, during microelectrophoretic SR95531 (and BMC), P4S often increased termination thresholds. Such an increase is the only effect of amino acids, such as glycine, for which Ia terminations apparently have no specific receptors, and is a consequence of the active uptake of these amino acids by neurones and glia (see ref. 6). The reversible increase in termination threshold by SR95531 and BMC presumably indicates a reduction in tonic PAD. It is not surprising that PAD generated by tetanic stimulation of flexor muscle afferent fibres was diminished but not abolished by SR95531 or BMC. When administered microelectrophoretically neither agent would be expected to influence all of the terminations of a single Ia fibre, yet PAD generated at axo-axonic synapses on each termination would contribute electrotonically to the reduction in threshold assessed by electric stimulation of one termination. Since only amino acids structurally related to GABA depolarized Ia terminations [6], the selectivity of SR95531 as a GABA antagonist could not be determined in these tests. In the rat cuneate nucleus SR95531 reduced the inhibition by GABA of the spontaneous or glutamate-evoked firing of neurones but infrequently reduced the inhibition produced by glycine, while SR95103 diminished the effects of both GABA and glycine [12]. The present findings in the cat spinal cord differ in that SR95531 ejected with currents only approximately twice those which produced concentrations selectively reducing the inhibitory action of G A B A also reduced that of glycine. Species or regional differences in GABA receptors may account for this disparity. The appar-
215
ent interaction of SR95103 with glycine and G A B A receptors may be related more to the presence in this c o m p o u n d of a methyl group at the 4-position of the pyridazinc ring than to substituents, such as the methoxy group in SR95531, at the paraposition of the phenyl ring. Substituents at this latter site, however, are clearly important for the interaction of these c o m p o u n d s with binding sites for G A B A on rat brain membranes [9]. SR95531 appears to be a soluble, potent, reversible and reasonably selective G A B A antagonist in the cat spinal cord. Reduction of the inhibitory effect of G A B A in the absence of bursts of cell firing, such as occur with BMC and presumably reflect a direct excitatory action, could be a useful attribute in microelectrophoretic investigations of GABA-mediated synaptic transmission. Further studies are required, however, in other regions of the cat central nervous system of the effect of SR95531 on the inhibitory actions of GABA-like and glycine-like amino acids. The authors are grateful to Professor C.G. Wermuth and Dr. K. Biziere for gifts of SR95103 and SR95531, to Mrs. P. Searle and Mr. T. Van Arkel for skilled technical assistance and to Mrs. J. Harries for word-processing the manuscript. 1 Chambon, J.P., Feltz, P., Heaulme, M., Restle, S., Schlichter, R., Biziere, K. and Wermuth, C.G., An arylaminopyridazine derivative of 7-aminobutyric acid (GABA) is a selective and competitive antagonist at the GABAA receptor site, Proc. Natl. Acad. Sci. USA, 82 (1985) 1832 1836. 2 Curtis, D.R. and Johnston, G.A.R., Amino acid transmitters in the m a m m a l i a n central nerwms system, Ergebn. Physiol., 69 (1974) 97 188. 3 Curtis, D.R.. Duggan, A.W., Felix, D. and Johnston, G.A.R., Bicuculline, an antagonist of G A B A and synaptic inhibition in the spinal cord, Brain Res., 32 (1971) 69 96. 4 Curtis, D.R., Game, C.J.A., Lodge, D. and McCulloch, R.M., A pharmacological study of Renshaw cell inhibition. J. Physiol. (London), 258 (1976) 227-242. 5 Curtis, D.R. and Lodge, D., The depolarization of feline ventral horn group Ia spinal afferent terminations b y G A B A , Exp. Brain Res., 46 (1982} 215 233. 6 Curtis, D.R., Lodge, D., Bornstein, J.C., Peel, M.J. and Leah, J.D., The dual effects of G A B A and related amino acids on the electrical threshold of ventral horn group la afferent terminations in the cat, Exp. Brain Res., 48 (1982) 387 400. 7 Dcsarmenien, M., Desaulles, E., Feltz, P., H a m a n n , M., Michaud, J.C. and Mienville, J.M., Electrophysiological study of pyridazine-GABA derivatives with G A B A - A antagonist properties, Br. J. Pharmacol., in press. 8 Game, C.J.A. and Lodge, D., The pharmacology of the inhibition of dorsal horn neurones by impulses in myelinated cutaneous afferents in the cat, Exp. Brain Res., 23 (1975) 75-84. 9 Heaulme, M., C h a m b o n , J.P., Leyris. R., Molimard, J.C., Wermuth, C.G. and Biziere, K., Biochemical characlcrization of the interaction of three pyridazinyl-GABA derivatives with the (}ABA-A receptor site, Brain Res., in press. 10 Johnston, G.A.R., Beart, P.M., Curtis, D.P,., Game, C.J.A., McCulloch, R.M. and Maclachlan, R.M., Bicuculline methochloride as a G A B A antagonist, Nature (London), New Biol., 240 (1972) 219 220. I I Lodge, D., Curtis, D.R. and Brand, S.J., A pharmacological study of the inhibition of ventral group la-excilcd spinal interneurones, Exp. Brain Res., 29 (1977) 97 105. 12 Michaud, .1.(,7., Mienville, J.M., C h a m b o n , J.P. and Biziere, K., Interactions of three pyridazinylG A B A derivatives with rat central G A B A and glycine receptors, an in vivo microiontophoretic study, Neuropharmacology, in press.