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Antagonism between strychnine and glycine, and bicuculline and GABA, in the ventromedial hypothalamus
SHORTCOMMUNICATIONS
599
Antagonism between strychnine and glycine, and bicuculline and GABA, in the ventromedial hypothalamus Among the great variety of amino acids tested within the mammalian central nervous system, the available neurochemical and electrophysiological evidence favours a major role for glycine and y-aminobutyric acid (GABA) as inhibitory synaptic transmitters TM. Furthermore, strychnine, when administered electrophoretically, suppresses the inhibitory action of glycine upon cat spinal cord neurones. Concentrations of the alkaloid adequate to abolish near-maximal inhibition of firing produced by glycine do not reduce the action of equally effective amounts of GABA1, 4. It has therefore been suggested that strychnine affects selectively the postsynaptic glycine receptors. Conversely, bicuculline has been shown to antagonize the action of GABA at the crayfish neuromuscular junction 10, and upon certain neurones in the mammalian spinal cord 2 and brain 3, while having little effect on the action of glycine. In view of the possibility that GABA and/or glycine may be important inhibitory synaptic transmitters in the functional control of hypothalamic regulatory mechanisms 7,9, a study has been made of the effects of bicuculline and strychnine on the action of GABA and to glycine on neurones located in the hypothalamus, which is known to receive a powerful inhibitory synaptic input from the amygdal#, s. Experiments were performed on 21 rats of a Carworth strain, 3(~)--450 g body weight. Initial anaesthesia by intraperitoneal injection of a mixture of urethane and sodium pentobarbitone (400 and 50 mg/kg b.w. respectively) was supplemented by 5 mg doses of sodium pentobarbitone as required every 2-3 h. Animals were fixed supine by a simple headholder, and the base of the skull was approached surgically by dividing the palate and retracting the hemisected mandibles laterally and the partially freed tongue caudally. Under the binocular microscope, the base of the brain was exposed by removing the sphenoid bone. Extracellular action potentials from neurones in the ventromedial region of the hypothalamus were recorded 0.5-1.5 mm from the midline, at a depth of 0.5-2.2 mm below the surface of the brain, by means of the centre barrel (4 M NaC1) of 5-barrel micropipettes of tip diameter 5-12 #m and resistance 2-10 MD~. The outer barrels of the micropipettes contained aqueous solutions of glycine (Fluka, 1.0 M, pH 3), GABA (Fluka, 1.0 M, pH 3), monosodium-L-glutamate (Fluka, 1.0 M, pH 7.5), strychnine sulphate (kindly supplied by Sandoz Ltd., Basle, 5 mM in 165 mM NaCI), and bicuculline hydrochloride (K & K Laboratories, 5 m M in 165 mM NaC1, pH 3). These compounds were ejected electrophoretically by appropriately directed currents, and their effects were assessed on neurones firing spontaneously, or in response to the continuous ejection of L-glutamate. The rate of firing of each neurone was displayed on a pen recorder, the action of the depressant amino acids being tested before, during, and after the application of antagonists. Stable recordings were obtained from 116 hypothalamic neurones. GABA depressed the firing of all but 2 out of a total of 72 cells, while having no effect in the 2 remaining ones. Glycine reduced the firing of all 34 cells recorded in 7 animals in which the action of this amino acid was tested. L-Glutamate excited 68 of a total of Brain Research, 45 (1972) 599-603
600
SHORT COMMUNICATIONS
A
B
13.]mV
Fig. 1. Effect of electrophoretically administered strychnine on the inhibitory action of glycine and GABA on a hypothalamic neurone. Photographic records from a spontaneously firing cell located in the ventromedial hypothalamus 1.0 mm below the ventral surface of the hypothalamus showing reduc tion of discharge rate during electrophoretic ejection for 10 sec periods of glycine (80 nA, upper traces in A-C) and of GABA (28 hA, lower traces in A-C). A, control; B, approximately 2 min after beginning ejection of strychnine sulphate (60 nA, 5 mMin 165 mM NaC1); C, 12 min after termination of strychnine ejection. Time mark: sec.
72 cells. These effects were usually o b t a i n e d with ejection c u r r e n t s o f the o r d e r o f 5-50 nA. A s a rule, G A B A a p p e a r e d to be s o m e w h a t m o r e p o t e n t t h a n glycine in reducing the firing o f m o s t h y p o t h a l a m i c neurones at lower ejection currents than those n e e d e d to achieve a c o m p a r a b l e r e d u c t i o n o f firing rate with glycine in the same cell. In all 14 neurones where strychnine sulphate was administered, e l e c t r o p h o r e t i c ejection o f the a l k a l o i d r e d u c e d o r c o m p l e t e l y a b o l i s h e d the effect o f glycine, but had little o r no action on the r e d u c t i o n o f cell firing o b s e r v e d d u r i n g the a p p l i c a t i o n o f G A B A . P h o t o g r a p h i c records f r o m one such n e u r o n e are shown in Fig. 1. This n e u r o n e fired s p o n t a n e o u s l y at a rate o f a p p r o x i m a t e l y 20 action potentials/sec, a n d r e s p o n d e d r e p r o d u c i b l y to glycine a n d G A B A (Fig. IA). A t a time when strychnine Brain Research, 45 (1972) 599-603
SHORT COMMUNICATIONS
601
GLY 35 GABAIO I
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STRYCHNINE 30 I
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GLY 10
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BICUCULLINE
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Fig. 2. Effects o f antagonists on the inhibitory action of G A B A and glycine on hypothalanucneurones. The depressant amino acids were administered alternately by electrophoresis for 30 sec periods indicated by the short horizontal bars. A, spontaneously active neurone (located in the hypothalamus
2.2 mm below its ventral surface), whose firing was depressed by glycine (GLY, 35 nA, white bar), and almost completely suppressed by GABA (10 nA, black bar). Strychnine sulphate (5 mM in 165 mM NaCI) was administered with a current of 30 nA for a period of 4 min, indicated by the hatched bar. B, spontaneously firing ventromedial neurone (located at a depth of 0.5 mm) from another rat, depressed by GABA (10 nA, black bar) and glycine (10 nA, white bar). Bicuculline-HC! (45 nA, 5 mM in 165 mM NaCI) was administered for approximately 5 rain, as indicated by the hatched bar. Ordinates: firing rate (spikes/sec); abscissae: time (min.)
completely blocked the inhibitory action of glycine, that of G A B A was unaffected (Fig. 1B). After termination of strychnine application the inhibitory response to glycine recovered (Fig. 1C). In Fig. 2A this antagonistic effect of strychnine upon the glycine- but not the GABA-induced depression of hypothalamic discharge is illustrated by recording the firing rate of another hypothalamic unit. Glycine administered with a current of 35 nA for 30 sec reduced the firing rate of this cell by approximately 50 ~ , whereas G A B A (10 nA) produced a near-maximal suppression. Strychnine, applied for some 4 min, completely antagonized the action of glycine, but did not affect the response of the neurone to GABA. The action of strychnine was slowly reversible. In 14 hypothalamic neurones, electrophoretically ejected bicucuUine (30--50 nA) reversibly blocked the action of GABA. In the 4 cells where it proved possible to assess the action of the alkaloid on the reduction of cell firing in response to G A B A and glycine, bicuculline had little or no effect upon glycine inhibition. The interaction between bicuculline and G A B A is illustrated in Fig. 2B. G A B A (10 nA) and glycine (GLY, 10 hA) were administered alternately for 30 sec periods, at 1 min intervals, and produced marked inhibition of firing of the neurone. Bicuculline, ejected with a current Brain Research, 45 (1972) 599-603
602
SHORT COMMUNICATIONS
._<2 .D
60
e-
40
•
•
20
0
15 30 Ejection current (hA)
45
Fig. 3. Reversible effect of bicuculline on the sensitivity of a hypothalamic ventromedial neurone to electrophoretically applied GABA. Same cell as Fig. 2B. The dose-response curvesrelate the percentage reduction of firing (response, ordinate) to the electrophoretic current used to eject GABA (dose, abscissa). Filled circles, control; open circles, during the administration of bicuculline-HC1(45 nA); open triangles, 10 min after termination of bicuculline administration.
of 45 nA for approximately 5 min completely and reversibly abolished the action of GABA, without changing markedly the neuronal response to glycine. As illustrated in Fig. 3, the GABA dose-response curve obtained from the same hypothalamic neurone was reversibly displaced to the right during the administration of bicuculline, with little change in the slope of the sigmoid curve. This was a spontaneously firing neurone, and the response is the percentage decrease of firing rate produced by the ejection of GABA for a 1 min period at each particular current intensity. GABA was retained by a current o f - - 1 5 nA. Reduction of the current to - - 3 nA resulted in approximately 15 ~ inhibition prior to the administration of bicuculline, and maximal inhibition was obtained with 15 nA. Three curves are plotted from observations made before (filled circles), during the administration of bicuculline (open circles), and approximately 10 min after termination of bicuculline administration (open triangles). It is readily apparent that the effect of bicuculline was to increase several fold the amount of GABA required to achieve a comparable reduction in rates of cell firing. These results provide evidence that strychnine reversibly blocks the action of glycine upon hypothalamic ventromedial neurones, whereas bicuculline antagonizes the reduction in cell firing observed during application of GABA. Thus in the hypothalamus, as elsewhere in the mammalian central nervous system, the action of these antagonists appears to permit a distinction to be made in electrophoretic experiments between GABA-receptors and glycine-receptors. These convulsant alkaloids may therefore prove to be of considerable value in assessing whether a particular pathway impinging upon hypothalamic neurones could function by releasing GABA or glycine as an inhibitory transmitter. Brain Research, 45 (1972) 599-603
SHORT COMMUNICATIONS
603
This work was supported by grants from the Swiss National Science Foundation (No. 3.556.71) and the F. Hoffmann-La Roche Foundation (No. 117). Department of Physiology, University of Geneva Medical School, 1211 Geneva 4 (Switzerland)
J. J. DREIFUSS E. K. MATTHEWS*
1 CURTIS,D. R., DUGGAN,A. W., AND JOHNSTON,G. A. R., The specificity of strychnine as a glycine antagonist in the mammalian spinal cord, Exp. Brain Res., 12 (1971) 547-565. 2 CURTLS,D. R., DUGGAN,A. W., FELIX,D., ANDJOHNSTON,G. A. R., Bicuculline, an antagonist of GABA and synaptic inhibition of the spinal cord of the cat, Brain Research, 32 (1971) 69-96. 3 CURTIS,D. R., DUGGAN,A. W., FELIX,D., JOHNSTON,G. A. R., ANDMCLENNAN,H., Antagonism between bicucuUine and GABA in the cat brain, Brain Research, 33 (1971) 57-73. 4 CURTIS,D. R., H&SLI,L., ANDJOHNSTON,G. A. R., A pharmacological study of the depression of spinal cord neurones by glycine and related amino acids, Exp. Brain Res., 6 (1968) 1-18. 5 DREIFUSS,J. J., KELLY, J. S., AND KRNJEVIt~,K., Cortical inhibition and y-aminobutyric acid, Exp. Brain Res., 13 (1969) 137-154. 6 DREIFUSS,J. J., MURPHY,J. T., AND GLOOR,P., Contrasting effects of two identified amygdaloid efferent pathways on single hypothalamic neurones, J. Neurophysiol., 31 (1968) 237-248. ', NICOLL, R. A., AND BARKER,J. L., The pharmacology of recurrent inhibition in the supraoptic neurosecretory system, Brain Research, 35 (1971) 501-511. 800MURA, Y., ONe, T., AND OOYAMA,H., Inhibitory action of the amygdala on the lateral hypothalamic area in rats, Nature (Lend.), 228 (1970) 1108-1110. 900MURA, Y., OOYAMA,H., YAMAMOTO,T., ONe, T., AND KOBAYASm,N., Behaviour of hypothalamic unit activity during electrophoretic application of drugs, Ann. IV. Y. Acad. Sci., 157 (1969) 642-665. 10 TAKEUCHI,A., AND ONODERA, K., Effect of bicuculline on the GABA receptor of the crayfish neuromuscular junction, Nature New Biol., 236 (1972) 55-56. I1 WERMAN,R., DAVIDOFF,R. A., AND APRISON, M. H., Inhibitory action of glycine on spinal neurons in the cat, J. Neurophysiol., 31 (1968) 81-95. (Accepted August 1st, 1972)
* Invited Professor, University of Geneva. Permanent address: Department of Pharmacology, University of Cambridge, England.
Brain Research, 45 (1972) 599-603
599
Antagonism between strychnine and glycine, and bicuculline and GABA, in the ventromedial hypothalamus Among the great variety of amino acids tested within the mammalian central nervous system, the available neurochemical and electrophysiological evidence favours a major role for glycine and y-aminobutyric acid (GABA) as inhibitory synaptic transmitters TM. Furthermore, strychnine, when administered electrophoretically, suppresses the inhibitory action of glycine upon cat spinal cord neurones. Concentrations of the alkaloid adequate to abolish near-maximal inhibition of firing produced by glycine do not reduce the action of equally effective amounts of GABA1, 4. It has therefore been suggested that strychnine affects selectively the postsynaptic glycine receptors. Conversely, bicuculline has been shown to antagonize the action of GABA at the crayfish neuromuscular junction 10, and upon certain neurones in the mammalian spinal cord 2 and brain 3, while having little effect on the action of glycine. In view of the possibility that GABA and/or glycine may be important inhibitory synaptic transmitters in the functional control of hypothalamic regulatory mechanisms 7,9, a study has been made of the effects of bicuculline and strychnine on the action of GABA and to glycine on neurones located in the hypothalamus, which is known to receive a powerful inhibitory synaptic input from the amygdal#, s. Experiments were performed on 21 rats of a Carworth strain, 3(~)--450 g body weight. Initial anaesthesia by intraperitoneal injection of a mixture of urethane and sodium pentobarbitone (400 and 50 mg/kg b.w. respectively) was supplemented by 5 mg doses of sodium pentobarbitone as required every 2-3 h. Animals were fixed supine by a simple headholder, and the base of the skull was approached surgically by dividing the palate and retracting the hemisected mandibles laterally and the partially freed tongue caudally. Under the binocular microscope, the base of the brain was exposed by removing the sphenoid bone. Extracellular action potentials from neurones in the ventromedial region of the hypothalamus were recorded 0.5-1.5 mm from the midline, at a depth of 0.5-2.2 mm below the surface of the brain, by means of the centre barrel (4 M NaC1) of 5-barrel micropipettes of tip diameter 5-12 #m and resistance 2-10 MD~. The outer barrels of the micropipettes contained aqueous solutions of glycine (Fluka, 1.0 M, pH 3), GABA (Fluka, 1.0 M, pH 3), monosodium-L-glutamate (Fluka, 1.0 M, pH 7.5), strychnine sulphate (kindly supplied by Sandoz Ltd., Basle, 5 mM in 165 mM NaCI), and bicuculline hydrochloride (K & K Laboratories, 5 m M in 165 mM NaC1, pH 3). These compounds were ejected electrophoretically by appropriately directed currents, and their effects were assessed on neurones firing spontaneously, or in response to the continuous ejection of L-glutamate. The rate of firing of each neurone was displayed on a pen recorder, the action of the depressant amino acids being tested before, during, and after the application of antagonists. Stable recordings were obtained from 116 hypothalamic neurones. GABA depressed the firing of all but 2 out of a total of 72 cells, while having no effect in the 2 remaining ones. Glycine reduced the firing of all 34 cells recorded in 7 animals in which the action of this amino acid was tested. L-Glutamate excited 68 of a total of Brain Research, 45 (1972) 599-603
600
SHORT COMMUNICATIONS
A
B
13.]mV
Fig. 1. Effect of electrophoretically administered strychnine on the inhibitory action of glycine and GABA on a hypothalamic neurone. Photographic records from a spontaneously firing cell located in the ventromedial hypothalamus 1.0 mm below the ventral surface of the hypothalamus showing reduc tion of discharge rate during electrophoretic ejection for 10 sec periods of glycine (80 nA, upper traces in A-C) and of GABA (28 hA, lower traces in A-C). A, control; B, approximately 2 min after beginning ejection of strychnine sulphate (60 nA, 5 mMin 165 mM NaC1); C, 12 min after termination of strychnine ejection. Time mark: sec.
72 cells. These effects were usually o b t a i n e d with ejection c u r r e n t s o f the o r d e r o f 5-50 nA. A s a rule, G A B A a p p e a r e d to be s o m e w h a t m o r e p o t e n t t h a n glycine in reducing the firing o f m o s t h y p o t h a l a m i c neurones at lower ejection currents than those n e e d e d to achieve a c o m p a r a b l e r e d u c t i o n o f firing rate with glycine in the same cell. In all 14 neurones where strychnine sulphate was administered, e l e c t r o p h o r e t i c ejection o f the a l k a l o i d r e d u c e d o r c o m p l e t e l y a b o l i s h e d the effect o f glycine, but had little o r no action on the r e d u c t i o n o f cell firing o b s e r v e d d u r i n g the a p p l i c a t i o n o f G A B A . P h o t o g r a p h i c records f r o m one such n e u r o n e are shown in Fig. 1. This n e u r o n e fired s p o n t a n e o u s l y at a rate o f a p p r o x i m a t e l y 20 action potentials/sec, a n d r e s p o n d e d r e p r o d u c i b l y to glycine a n d G A B A (Fig. IA). A t a time when strychnine Brain Research, 45 (1972) 599-603
SHORT COMMUNICATIONS
601
GLY 35 GABAIO I
I
I
I
I
Ii
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I
STRYCHNINE 30 I
40
I ,
30 s p i k e s 20 per second 10 0 i
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GABA
10
GLY 10
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BICUCULLINE
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Fig. 2. Effects o f antagonists on the inhibitory action of G A B A and glycine on hypothalanucneurones. The depressant amino acids were administered alternately by electrophoresis for 30 sec periods indicated by the short horizontal bars. A, spontaneously active neurone (located in the hypothalamus
2.2 mm below its ventral surface), whose firing was depressed by glycine (GLY, 35 nA, white bar), and almost completely suppressed by GABA (10 nA, black bar). Strychnine sulphate (5 mM in 165 mM NaCI) was administered with a current of 30 nA for a period of 4 min, indicated by the hatched bar. B, spontaneously firing ventromedial neurone (located at a depth of 0.5 mm) from another rat, depressed by GABA (10 nA, black bar) and glycine (10 nA, white bar). Bicuculline-HC! (45 nA, 5 mM in 165 mM NaCI) was administered for approximately 5 rain, as indicated by the hatched bar. Ordinates: firing rate (spikes/sec); abscissae: time (min.)
completely blocked the inhibitory action of glycine, that of G A B A was unaffected (Fig. 1B). After termination of strychnine application the inhibitory response to glycine recovered (Fig. 1C). In Fig. 2A this antagonistic effect of strychnine upon the glycine- but not the GABA-induced depression of hypothalamic discharge is illustrated by recording the firing rate of another hypothalamic unit. Glycine administered with a current of 35 nA for 30 sec reduced the firing rate of this cell by approximately 50 ~ , whereas G A B A (10 nA) produced a near-maximal suppression. Strychnine, applied for some 4 min, completely antagonized the action of glycine, but did not affect the response of the neurone to GABA. The action of strychnine was slowly reversible. In 14 hypothalamic neurones, electrophoretically ejected bicucuUine (30--50 nA) reversibly blocked the action of GABA. In the 4 cells where it proved possible to assess the action of the alkaloid on the reduction of cell firing in response to G A B A and glycine, bicuculline had little or no effect upon glycine inhibition. The interaction between bicuculline and G A B A is illustrated in Fig. 2B. G A B A (10 nA) and glycine (GLY, 10 hA) were administered alternately for 30 sec periods, at 1 min intervals, and produced marked inhibition of firing of the neurone. Bicuculline, ejected with a current Brain Research, 45 (1972) 599-603
602
SHORT COMMUNICATIONS
._<2 .D
60
e-
40
•
•
20
0
15 30 Ejection current (hA)
45
Fig. 3. Reversible effect of bicuculline on the sensitivity of a hypothalamic ventromedial neurone to electrophoretically applied GABA. Same cell as Fig. 2B. The dose-response curvesrelate the percentage reduction of firing (response, ordinate) to the electrophoretic current used to eject GABA (dose, abscissa). Filled circles, control; open circles, during the administration of bicuculline-HC1(45 nA); open triangles, 10 min after termination of bicuculline administration.
of 45 nA for approximately 5 min completely and reversibly abolished the action of GABA, without changing markedly the neuronal response to glycine. As illustrated in Fig. 3, the GABA dose-response curve obtained from the same hypothalamic neurone was reversibly displaced to the right during the administration of bicuculline, with little change in the slope of the sigmoid curve. This was a spontaneously firing neurone, and the response is the percentage decrease of firing rate produced by the ejection of GABA for a 1 min period at each particular current intensity. GABA was retained by a current o f - - 1 5 nA. Reduction of the current to - - 3 nA resulted in approximately 15 ~ inhibition prior to the administration of bicuculline, and maximal inhibition was obtained with 15 nA. Three curves are plotted from observations made before (filled circles), during the administration of bicuculline (open circles), and approximately 10 min after termination of bicuculline administration (open triangles). It is readily apparent that the effect of bicuculline was to increase several fold the amount of GABA required to achieve a comparable reduction in rates of cell firing. These results provide evidence that strychnine reversibly blocks the action of glycine upon hypothalamic ventromedial neurones, whereas bicuculline antagonizes the reduction in cell firing observed during application of GABA. Thus in the hypothalamus, as elsewhere in the mammalian central nervous system, the action of these antagonists appears to permit a distinction to be made in electrophoretic experiments between GABA-receptors and glycine-receptors. These convulsant alkaloids may therefore prove to be of considerable value in assessing whether a particular pathway impinging upon hypothalamic neurones could function by releasing GABA or glycine as an inhibitory transmitter. Brain Research, 45 (1972) 599-603
SHORT COMMUNICATIONS
603
This work was supported by grants from the Swiss National Science Foundation (No. 3.556.71) and the F. Hoffmann-La Roche Foundation (No. 117). Department of Physiology, University of Geneva Medical School, 1211 Geneva 4 (Switzerland)
J. J. DREIFUSS E. K. MATTHEWS*
1 CURTIS,D. R., DUGGAN,A. W., AND JOHNSTON,G. A. R., The specificity of strychnine as a glycine antagonist in the mammalian spinal cord, Exp. Brain Res., 12 (1971) 547-565. 2 CURTLS,D. R., DUGGAN,A. W., FELIX,D., ANDJOHNSTON,G. A. R., Bicuculline, an antagonist of GABA and synaptic inhibition of the spinal cord of the cat, Brain Research, 32 (1971) 69-96. 3 CURTIS,D. R., DUGGAN,A. W., FELIX,D., JOHNSTON,G. A. R., ANDMCLENNAN,H., Antagonism between bicucuUine and GABA in the cat brain, Brain Research, 33 (1971) 57-73. 4 CURTIS,D. R., H&SLI,L., ANDJOHNSTON,G. A. R., A pharmacological study of the depression of spinal cord neurones by glycine and related amino acids, Exp. Brain Res., 6 (1968) 1-18. 5 DREIFUSS,J. J., KELLY, J. S., AND KRNJEVIt~,K., Cortical inhibition and y-aminobutyric acid, Exp. Brain Res., 13 (1969) 137-154. 6 DREIFUSS,J. J., MURPHY,J. T., AND GLOOR,P., Contrasting effects of two identified amygdaloid efferent pathways on single hypothalamic neurones, J. Neurophysiol., 31 (1968) 237-248. ', NICOLL, R. A., AND BARKER,J. L., The pharmacology of recurrent inhibition in the supraoptic neurosecretory system, Brain Research, 35 (1971) 501-511. 800MURA, Y., ONe, T., AND OOYAMA,H., Inhibitory action of the amygdala on the lateral hypothalamic area in rats, Nature (Lend.), 228 (1970) 1108-1110. 900MURA, Y., OOYAMA,H., YAMAMOTO,T., ONe, T., AND KOBAYASm,N., Behaviour of hypothalamic unit activity during electrophoretic application of drugs, Ann. IV. Y. Acad. Sci., 157 (1969) 642-665. 10 TAKEUCHI,A., AND ONODERA, K., Effect of bicuculline on the GABA receptor of the crayfish neuromuscular junction, Nature New Biol., 236 (1972) 55-56. I1 WERMAN,R., DAVIDOFF,R. A., AND APRISON, M. H., Inhibitory action of glycine on spinal neurons in the cat, J. Neurophysiol., 31 (1968) 81-95. (Accepted August 1st, 1972)
* Invited Professor, University of Geneva. Permanent address: Department of Pharmacology, University of Cambridge, England.
Brain Research, 45 (1972) 599-603