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The effects of depleting GABA on cuneate presynaptic inhibition
530
SHORT COMMUNICATIONS
The effects of depleting GABA on cuneate presynaptic inhibition Afferent cutaneous volleys from the forelimb produce a prolonged depolarization of the presynaptic terminals of cuneate tract fibers 2. This depolarization can be recorded at the dorsal surface of the cuneate nucleus as a prolonged positive wave 1 (P wave) and can be studied by the method of excitability testing of cuneate presynaptic fibers at their terminalsL Microiontophoretic application of ~-aminobutyric acid (GABA) in the cuneate nucleus produces potent inhibition of cuneate neurons 6, and perfusion of the cuneate nucleus with a GABA solution increases the excitability of primary afferent terminals 5,7. GABA has therefore been suggested as a transmitter candidate mediating postsynaptic inhibition as well as presynaptic depolarizationL In the following experiments, an agent known to decrease GABA levels in the central nervous system was used to study the effects of depleting GABA on cuneate P waves and on the excitability of cuneate afferent terminals. Experiments were performed on 8 cats anesthetized with Nembutal (35 mg/kg, i.p.), one cat anesthetized with a-chloralose (35 mg/kg, i.p.), and two unanesthetized cats decerebrated under ether anesthesia after ligation of both carotid arteries. The superficial radial nerve was isolated, sectioned, and immersed in a pool of mineral oil of constant temperature (37°C). Body temperature was maintained between 37 ° and 39.5°C. The dorsal column nuclei were exposed and intermittently flushed with warm mineral oil for the duration of the experiment. A bilateral pneumothorax was performed, and the cats were artificially respired (4 % CO2 in expired air). Gallamine (Flaxedil) was used to immobilize the cats. Unless otherwise indicated, drugs were administered intravenously. Two types of procedures were followed 3. First, the superficial radial nerve was stimulated supramaximally at frequencies of 12 c/min, and gross potentials were recorded through a monopolar electrode placed on the surface of the ipsilateral cuneate nucleus. The indifferent electrode was attached to exposed skin. Second, a glass microelectrode filled with 4 M NaC1 was inserted into the cuneate nucleus and used for stimulation. The monophasic antidromic response was recorded in the ipsilateral superficial radial nerve (platinum hook electrodes). Conditioning stimuli were applied to the skin or pad of the ipsilateral forearm. Semicarbazide (200 mg/kg) was administered intravenously over a 5-min period. It produced no immediate effects on the positive surface wave (P wave)recorded after peripheral stimulation. Depression of the P wave started within 60-75 min, and in 3 h, the P wave was either completely blocked or decreased to a very small size in all experiments (Fig. 1A). Frequently, negative deflections were observed in place of the previously positive potentials, and background activity became quite pronounced. No recovery occurred even 5 h after the administration of semicarbazide. Intravenously administered Nembutal (10-15 mg/kg), which increases the size and duration of P waves in the cuneate nucleus a, antagonized the effects of semicarbazide only when the depression of the P wave had not reached maximal levels. Four hours after semicarbazide administration, Nembutal failed to antagonize this depression. No consistent alterations in the amplitude of the N wave were seen after semicarbazide administration. In 3 experiments, however, a decrease in its duration was observed. Brain Research, 33 (1971) 530-532
531
SHORT COMMUNICATIONS
._._]i.1mV 50 rnsec
B~ 14o
200?glkg~il
I.°'~mY
semicarbazide
C
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I
"~1=0o>_zl I
°
\
~ \
o---0
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--
Controt 150
min after
~,.,icarl,''i~,
£, 100
6
~ 5O TIME - msec
~ -z~ 100
Fig. 1. The effects of semicarbazide on cuneate surface potentials and on excitability of cuneate afferent terminals. A, Surface potentials shown before and 3 h after semicarbazide (decerebrate unanesthetized cat). B, Antidromic response in superficial radial nerve after direct cuneate stimulation, showingcontrol response at left, then response 2 h after semicarbazide (nembutalized cat). C, Plot of the time course of the increase in excitability of superficial radial nerve terminals in the cuneate nucleus after conditioning stimulation (2 pulses, 250/see) of the ipsilateral forepaw at various intervals in a nembutalized cat. This depression of excitability increase was observed at all depths within the nucleus. In 3 cats, anesthetized with Nembutal, surface potentials were recorded over a period of 4-5 h after administration of an equivalent volume of physiological saline. These cats, which were also paralyzed with Flaxedil, served as controls for the anesthetized, semicarbazide-treated cats. N o significant changes in the depth o f the P wave of these controls could be observed. In two cats anesthetized with Nembutal, the excitability of cuneate afferent terminals was tested by passing brief submaximal pulses through a glass microelectrode in the cuneate nucleus (12 c/min) and recording the antidromic potentials in the superficial radial nerve. These potentials consist of two spike complexes, the first resulting from stimulation of the terminals and the second representing the dorsal column reflex 2 (DCR). Since it is difficult to maintain the exact location of the microelectrode during the prolonged time course o f development of semicarbazide effects, the excitability of cuneate terminals was tested at various depths falling within Brain Research, 33 (1971) 530-532
532
SHORT COMMUNICATIONS
0.8-1.5 m m f r o m the surface. T h e D C R was depressed within 75 rain o f s e m i c a r b a z i d e a d m i n i s t r a t i o n a n d c o m p l e t e l y b l o c k e d in 2 h (Fig. 1B). N o D C R could be evoked at a n y d e p t h within the nucleus. The increase in excitability o f cuneate terminals by c o n d i t i o n i n g s t i m u l a t i o n a p p l i e d at various intervals to the ipsilateral f o r e p a w was greatly r e d u c e d within 150 rain after d r u g a d m i n i s t r a t i o n (Fig. 1C). In one experiment, pyridoxine, a d m i n i s t e r e d 150 rain after semicarbazide, a n t a g o n i z e d its effects within lh. Thus it can be seen t h a t the slowly d e v e l o p i n g effects o f s e m i c a r b a z i d e in the cuneate nucleus resemble those observed after p i c r o t o x i n 3. S e m i c a r b a z i d e is k n o w n to deplete G A B A f r o m the c e n t r a l nervous system by blocking its synthesis 8. The slow time course o f d e v e l o p m e n t o f the effects o f s e m i c a r b a z i d e m a y result f r o m the slow rate o f d e p l e t i o n o f G A B A 4 in the absence o f synthesis. These d a t a s u p p o r t the h y p o t h e s i s t h a t G A B A m a y function as a t r a n s m i t t e r in the cuneate nucleus, effecting d e p o l a r i z a t i o n o f p r e s y n a p t i c t e r m i n a l s at a x o - a x o n i c synapses. Department of Pharmacodynamics and Toxicology, School of Pharmacy, and Department of Physiology, School of Medicine, American University of Beirut, Beirut (Lebanon)
N. R. BANNA S. J. JABBUR
1 ANDERSEN, P., ECCLES, J. C., SCHMIDT, R. F., AND YOKOTA, T., Slow potential waves produced in the cuneate nucleus by cutaneous volleys and by cortical stimulation, J. Neurophysiol., 27 (1964) 78-91. 2 ANDERSEN,P., ECCLES, J. C., SCHMIDT, R. F., AND YOKOTA, T., Depolarization of presynaptic fibers in the cuneate nucleus, J. Neurophysiol., 27 (1964) 92-106. 3 BANNA, N. R., AND JABnUR, S. J., Pharmacological studies on inhibition in the cuneate nucleus of the cat, Int. J. Neuropharmacol., 8 (1969) 299-307. 4 BELL, J. A., AND ANDERSON, E. G., A comparison of the actions of semicarbazide and picrotoxin on spinal synaptic activity, Pharmacologist, 12 (1970) 252. 5 DAVIDSON, N., AND SOUTHWICK, C. A. P., The effect of topically applied amino acids on primary afferent terminal excitability in the rat cuneate nucleus, J. Physiol. (Lond.), 210 (t970) 172P-173P. 6 GALINDO,A., KRNJEVI~, K., AND SCHWARTZ,S., Micro-iontophoretic studies on neurones in the cuneate nucleus, J. Physiol. (Lond.), 192 (1967) 359-377. 7 GALINDO, A., GABA-picrotoxin interaction in the mammalian central nervous system, Brain Research, 14 (1969) 763-767. 8 KJLLAM,K. F., ANDBAIN, J. A., Convulsant hydrazides. I. In vitro and in vivo inhibition of vitamin B6 enzymes by convulsant hydrazides, J. Pharmacol. exp. Ther., 119 (1957) 255-262. (Accepted August 16th, 1971)
Brain Research, 33 (1971) 530-532
SHORT COMMUNICATIONS
The effects of depleting GABA on cuneate presynaptic inhibition Afferent cutaneous volleys from the forelimb produce a prolonged depolarization of the presynaptic terminals of cuneate tract fibers 2. This depolarization can be recorded at the dorsal surface of the cuneate nucleus as a prolonged positive wave 1 (P wave) and can be studied by the method of excitability testing of cuneate presynaptic fibers at their terminalsL Microiontophoretic application of ~-aminobutyric acid (GABA) in the cuneate nucleus produces potent inhibition of cuneate neurons 6, and perfusion of the cuneate nucleus with a GABA solution increases the excitability of primary afferent terminals 5,7. GABA has therefore been suggested as a transmitter candidate mediating postsynaptic inhibition as well as presynaptic depolarizationL In the following experiments, an agent known to decrease GABA levels in the central nervous system was used to study the effects of depleting GABA on cuneate P waves and on the excitability of cuneate afferent terminals. Experiments were performed on 8 cats anesthetized with Nembutal (35 mg/kg, i.p.), one cat anesthetized with a-chloralose (35 mg/kg, i.p.), and two unanesthetized cats decerebrated under ether anesthesia after ligation of both carotid arteries. The superficial radial nerve was isolated, sectioned, and immersed in a pool of mineral oil of constant temperature (37°C). Body temperature was maintained between 37 ° and 39.5°C. The dorsal column nuclei were exposed and intermittently flushed with warm mineral oil for the duration of the experiment. A bilateral pneumothorax was performed, and the cats were artificially respired (4 % CO2 in expired air). Gallamine (Flaxedil) was used to immobilize the cats. Unless otherwise indicated, drugs were administered intravenously. Two types of procedures were followed 3. First, the superficial radial nerve was stimulated supramaximally at frequencies of 12 c/min, and gross potentials were recorded through a monopolar electrode placed on the surface of the ipsilateral cuneate nucleus. The indifferent electrode was attached to exposed skin. Second, a glass microelectrode filled with 4 M NaC1 was inserted into the cuneate nucleus and used for stimulation. The monophasic antidromic response was recorded in the ipsilateral superficial radial nerve (platinum hook electrodes). Conditioning stimuli were applied to the skin or pad of the ipsilateral forearm. Semicarbazide (200 mg/kg) was administered intravenously over a 5-min period. It produced no immediate effects on the positive surface wave (P wave)recorded after peripheral stimulation. Depression of the P wave started within 60-75 min, and in 3 h, the P wave was either completely blocked or decreased to a very small size in all experiments (Fig. 1A). Frequently, negative deflections were observed in place of the previously positive potentials, and background activity became quite pronounced. No recovery occurred even 5 h after the administration of semicarbazide. Intravenously administered Nembutal (10-15 mg/kg), which increases the size and duration of P waves in the cuneate nucleus a, antagonized the effects of semicarbazide only when the depression of the P wave had not reached maximal levels. Four hours after semicarbazide administration, Nembutal failed to antagonize this depression. No consistent alterations in the amplitude of the N wave were seen after semicarbazide administration. In 3 experiments, however, a decrease in its duration was observed. Brain Research, 33 (1971) 530-532
531
SHORT COMMUNICATIONS
._._]i.1mV 50 rnsec
B~ 14o
200?glkg~il
I.°'~mY
semicarbazide
C
/
i~
I
"~1=0o>_zl I
°
\
~ \
o---0
~
--
Controt 150
min after
~,.,icarl,''i~,
£, 100
6
~ 5O TIME - msec
~ -z~ 100
Fig. 1. The effects of semicarbazide on cuneate surface potentials and on excitability of cuneate afferent terminals. A, Surface potentials shown before and 3 h after semicarbazide (decerebrate unanesthetized cat). B, Antidromic response in superficial radial nerve after direct cuneate stimulation, showingcontrol response at left, then response 2 h after semicarbazide (nembutalized cat). C, Plot of the time course of the increase in excitability of superficial radial nerve terminals in the cuneate nucleus after conditioning stimulation (2 pulses, 250/see) of the ipsilateral forepaw at various intervals in a nembutalized cat. This depression of excitability increase was observed at all depths within the nucleus. In 3 cats, anesthetized with Nembutal, surface potentials were recorded over a period of 4-5 h after administration of an equivalent volume of physiological saline. These cats, which were also paralyzed with Flaxedil, served as controls for the anesthetized, semicarbazide-treated cats. N o significant changes in the depth o f the P wave of these controls could be observed. In two cats anesthetized with Nembutal, the excitability of cuneate afferent terminals was tested by passing brief submaximal pulses through a glass microelectrode in the cuneate nucleus (12 c/min) and recording the antidromic potentials in the superficial radial nerve. These potentials consist of two spike complexes, the first resulting from stimulation of the terminals and the second representing the dorsal column reflex 2 (DCR). Since it is difficult to maintain the exact location of the microelectrode during the prolonged time course o f development of semicarbazide effects, the excitability of cuneate terminals was tested at various depths falling within Brain Research, 33 (1971) 530-532
532
SHORT COMMUNICATIONS
0.8-1.5 m m f r o m the surface. T h e D C R was depressed within 75 rain o f s e m i c a r b a z i d e a d m i n i s t r a t i o n a n d c o m p l e t e l y b l o c k e d in 2 h (Fig. 1B). N o D C R could be evoked at a n y d e p t h within the nucleus. The increase in excitability o f cuneate terminals by c o n d i t i o n i n g s t i m u l a t i o n a p p l i e d at various intervals to the ipsilateral f o r e p a w was greatly r e d u c e d within 150 rain after d r u g a d m i n i s t r a t i o n (Fig. 1C). In one experiment, pyridoxine, a d m i n i s t e r e d 150 rain after semicarbazide, a n t a g o n i z e d its effects within lh. Thus it can be seen t h a t the slowly d e v e l o p i n g effects o f s e m i c a r b a z i d e in the cuneate nucleus resemble those observed after p i c r o t o x i n 3. S e m i c a r b a z i d e is k n o w n to deplete G A B A f r o m the c e n t r a l nervous system by blocking its synthesis 8. The slow time course o f d e v e l o p m e n t o f the effects o f s e m i c a r b a z i d e m a y result f r o m the slow rate o f d e p l e t i o n o f G A B A 4 in the absence o f synthesis. These d a t a s u p p o r t the h y p o t h e s i s t h a t G A B A m a y function as a t r a n s m i t t e r in the cuneate nucleus, effecting d e p o l a r i z a t i o n o f p r e s y n a p t i c t e r m i n a l s at a x o - a x o n i c synapses. Department of Pharmacodynamics and Toxicology, School of Pharmacy, and Department of Physiology, School of Medicine, American University of Beirut, Beirut (Lebanon)
N. R. BANNA S. J. JABBUR
1 ANDERSEN, P., ECCLES, J. C., SCHMIDT, R. F., AND YOKOTA, T., Slow potential waves produced in the cuneate nucleus by cutaneous volleys and by cortical stimulation, J. Neurophysiol., 27 (1964) 78-91. 2 ANDERSEN,P., ECCLES, J. C., SCHMIDT, R. F., AND YOKOTA, T., Depolarization of presynaptic fibers in the cuneate nucleus, J. Neurophysiol., 27 (1964) 92-106. 3 BANNA, N. R., AND JABnUR, S. J., Pharmacological studies on inhibition in the cuneate nucleus of the cat, Int. J. Neuropharmacol., 8 (1969) 299-307. 4 BELL, J. A., AND ANDERSON, E. G., A comparison of the actions of semicarbazide and picrotoxin on spinal synaptic activity, Pharmacologist, 12 (1970) 252. 5 DAVIDSON, N., AND SOUTHWICK, C. A. P., The effect of topically applied amino acids on primary afferent terminal excitability in the rat cuneate nucleus, J. Physiol. (Lond.), 210 (t970) 172P-173P. 6 GALINDO,A., KRNJEVI~, K., AND SCHWARTZ,S., Micro-iontophoretic studies on neurones in the cuneate nucleus, J. Physiol. (Lond.), 192 (1967) 359-377. 7 GALINDO, A., GABA-picrotoxin interaction in the mammalian central nervous system, Brain Research, 14 (1969) 763-767. 8 KJLLAM,K. F., ANDBAIN, J. A., Convulsant hydrazides. I. In vitro and in vivo inhibition of vitamin B6 enzymes by convulsant hydrazides, J. Pharmacol. exp. Ther., 119 (1957) 255-262. (Accepted August 16th, 1971)
Brain Research, 33 (1971) 530-532