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The appearance of the monosynaptic reflex in the spinal cord of the rat foetus
$42 DEVELOPMENT OF S U S C E P T I B I L I T Y CEREBELLAR NEURONS
TO G A B A
AND
GLYCINE
JUNKO MORI-OKAMOTO AND JIRO TATSUNO Department of P h y s i o l o g y , National Defense Medical N a m i k i 3-2, T o k o r o z a w a , S a i t a m a 359, J a p a n .
IN C U L T U R E D
College,
T h e e f f e c t s of i o n t o p h o r e t i c a l l y a p p l i e d G A B A a n d g l y c i n e on chick cerebellar neurons cultured for 7-12 ( i m m a t u r e ) and 25-40 (mature) days were intracellularly investigated. All neurons t e s t e d , r e g a r d l e s s of t h e i r m a t u r i t i e s , dose-dependently responde d to b o t h G A B A a n d g l y c i n e . These amino acids produced either hyperpolarization or d e p o l a r i z a t i o n of m e m b r a n e p o t e n t i a l , associated with the m e m b r a n e conductance increase. As n e u r o n s m a tured, the r e v e r s a l p o t e n t i a l s for G A B A a n d g l y c i n e w e r e s h i f t e d more negatively, and the conductance increases became greater. The r e v e r s a l potentials for GABA and glycine in immature and mature neurons were not significantly different from each other, a n d s h i f t e d to 0 m V b y l o w e r i n g t h e e x t e r n a l Cl concentration. In the l o w C1 condition, furthermore, the c o n d u c t a n c e changes were not so p r o m i n e n t . The conductance increases induced by GABA and g l y c i n e in i m m a t u r e a n d m a t u r e n e u r o n s w e r e r a t h e r u n s e l e c t i v e l y blocked by picrotoxin, bicuculline and strychnine; though mature neurons showed some selectivity for G A B A to p i c r o t o x i n or b i c u c u lline, a n d for g l y c i n e to s t r y c h n i n e . These unselective antagonistic e f f e c t s m a y be due to the f a c t t h a t the i o n o p h o r e s l i n k e d to G A B A a n d g l y c i n e r e c e p t o r s h a v e t h e s a m e ion s e l e c t i v i t y and exert similar conductance changes. Possibly, these ionic channels could develop into selective types coupled specifically with the G A B A or g l y c i n e r e c e p t o r in the c o u r s e of n e u r o n a l m a t u r a t i o n .
THE APPEARANCE OF THE MONOSYNAPTIC REFLEX IN THE SPINAL CORD OF THE RAT FOETUS
NORIO KUDO* and TOSHIYA YAMADA*, Department of Physiology, Institute of Basic Medical Sciences, University of Tsukuba, Niihari-gun, Ibaraki 305, Japan. The development of the monosynaptic reflex pathway was studied histologically and electrophysiologically in foetal and new born rats. The lumbar spinal cord was isolated and incubated in a chamber perfused with Krebs solution saturated with 95% 02-5% CO 2. The L4 dorsal root ganglion was immersed in 50% HRP solution for 3-10 hours. After fixation, HRP was reacted using the tetramethylbenzidine (TMB) or "Hanker-Yates" procedure. It was revealed that primary afferent fibres began to enter the gray matter at embryonic day 16.5 (E16.5). At E17.5, many fibres had reached the ventral horn, and a few fibres with growth cones on their endings had entered the motor nucleus. At E18.5, many afferent fibres had reached the motor nucleus and had begun to ramify in the nucleus. Boutons en passant and boutons t e ~ i n a ~ were frequently found in the nucleus at this stage. The average number of boutons per one fibre in the nucleus increased markedly as the age increased, i.e. 0.6 at E17.5, 3.2 at E18.5, 4.9 at E19.5 and 10.7 at E20.5. The reflex response evoked by stimulation of the L4 dorsal root was recorded from the L4 ventral root through the suction electrode, in the isolated preparation of various ages. The segmental latency of the reflex response significantly changed between E17.5 (12.5 msec) and E18.5 (7.2 msec), indicating that monosynaptic transmission began to appear at E18.5. The number of motoneurones fired by the monosynaptic excitatory effects was estimated by comparing the amplitude of the reflex response in the L4 ventral root with that of the response evoked directly by stimulation of the L4 ventral root. 11% of L4 motoneurones were activated monosynaptically at E18.5, 21% at E19.5, 28% at E20.5 and 34% at 0 to 1 day old.
TO G A B A
AND
GLYCINE
JUNKO MORI-OKAMOTO AND JIRO TATSUNO Department of P h y s i o l o g y , National Defense Medical N a m i k i 3-2, T o k o r o z a w a , S a i t a m a 359, J a p a n .
IN C U L T U R E D
College,
T h e e f f e c t s of i o n t o p h o r e t i c a l l y a p p l i e d G A B A a n d g l y c i n e on chick cerebellar neurons cultured for 7-12 ( i m m a t u r e ) and 25-40 (mature) days were intracellularly investigated. All neurons t e s t e d , r e g a r d l e s s of t h e i r m a t u r i t i e s , dose-dependently responde d to b o t h G A B A a n d g l y c i n e . These amino acids produced either hyperpolarization or d e p o l a r i z a t i o n of m e m b r a n e p o t e n t i a l , associated with the m e m b r a n e conductance increase. As n e u r o n s m a tured, the r e v e r s a l p o t e n t i a l s for G A B A a n d g l y c i n e w e r e s h i f t e d more negatively, and the conductance increases became greater. The r e v e r s a l potentials for GABA and glycine in immature and mature neurons were not significantly different from each other, a n d s h i f t e d to 0 m V b y l o w e r i n g t h e e x t e r n a l Cl concentration. In the l o w C1 condition, furthermore, the c o n d u c t a n c e changes were not so p r o m i n e n t . The conductance increases induced by GABA and g l y c i n e in i m m a t u r e a n d m a t u r e n e u r o n s w e r e r a t h e r u n s e l e c t i v e l y blocked by picrotoxin, bicuculline and strychnine; though mature neurons showed some selectivity for G A B A to p i c r o t o x i n or b i c u c u lline, a n d for g l y c i n e to s t r y c h n i n e . These unselective antagonistic e f f e c t s m a y be due to the f a c t t h a t the i o n o p h o r e s l i n k e d to G A B A a n d g l y c i n e r e c e p t o r s h a v e t h e s a m e ion s e l e c t i v i t y and exert similar conductance changes. Possibly, these ionic channels could develop into selective types coupled specifically with the G A B A or g l y c i n e r e c e p t o r in the c o u r s e of n e u r o n a l m a t u r a t i o n .
THE APPEARANCE OF THE MONOSYNAPTIC REFLEX IN THE SPINAL CORD OF THE RAT FOETUS
NORIO KUDO* and TOSHIYA YAMADA*, Department of Physiology, Institute of Basic Medical Sciences, University of Tsukuba, Niihari-gun, Ibaraki 305, Japan. The development of the monosynaptic reflex pathway was studied histologically and electrophysiologically in foetal and new born rats. The lumbar spinal cord was isolated and incubated in a chamber perfused with Krebs solution saturated with 95% 02-5% CO 2. The L4 dorsal root ganglion was immersed in 50% HRP solution for 3-10 hours. After fixation, HRP was reacted using the tetramethylbenzidine (TMB) or "Hanker-Yates" procedure. It was revealed that primary afferent fibres began to enter the gray matter at embryonic day 16.5 (E16.5). At E17.5, many fibres had reached the ventral horn, and a few fibres with growth cones on their endings had entered the motor nucleus. At E18.5, many afferent fibres had reached the motor nucleus and had begun to ramify in the nucleus. Boutons en passant and boutons t e ~ i n a ~ were frequently found in the nucleus at this stage. The average number of boutons per one fibre in the nucleus increased markedly as the age increased, i.e. 0.6 at E17.5, 3.2 at E18.5, 4.9 at E19.5 and 10.7 at E20.5. The reflex response evoked by stimulation of the L4 dorsal root was recorded from the L4 ventral root through the suction electrode, in the isolated preparation of various ages. The segmental latency of the reflex response significantly changed between E17.5 (12.5 msec) and E18.5 (7.2 msec), indicating that monosynaptic transmission began to appear at E18.5. The number of motoneurones fired by the monosynaptic excitatory effects was estimated by comparing the amplitude of the reflex response in the L4 ventral root with that of the response evoked directly by stimulation of the L4 ventral root. 11% of L4 motoneurones were activated monosynaptically at E18.5, 21% at E19.5, 28% at E20.5 and 34% at 0 to 1 day old.