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Enkephalin-containing structures in the brain of the hagfish, eptatretus burgeri
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INPUT
FROM
ENKEPHALINERGIC
TERMINALS
TO V M H
GLUCORECEPTIVE
NEURONS
SHINOB~ I N A G A K I I, YOSHI~UKI K U B O T A I , S H O I C H I S H I M A D A I*, SHOZO K I T O I, KAZUO S A S A K I - and T A K E T O S H I O N O I: Dept. I n t e r n a l M e d . , H i r o s h i m a Univ. Sch. Med., I-2-3 K a s u m i , M i n a m i k u , H i r o s h i m a 734, 2: Dept. P h y s i o l . , Facul. M e d . , Toyama Med. P h a r m a c e u t . U n i v . , T o y a m a 930-01 In the v e n t r o m e d i a l h y p o t h a l a m i c n u c l e u s (VMH) g l u c o s e r e c e p t i v e (GR) n e u r o n s and enkephalin (ENK) imrnunoreactive f i b e r s are p r e s e n t in a large number, suggesting i n t e r r e l a t i o n s b e t w e e n GR n e u r o n s a n d e n k e p h a l i n in r e l a t i o n to the VMH function. A d u l t m a l e r a t s w e r e d e c a p i t a t e d and a b o u t 500 700 um thick coronal tissue slices were cut. To i d e n t i f y the GR neurons, glucose was experimentally v a r i e d f r o m 5.5 to 20 mM. A f t e r i d e n t i f i c a t i o n of GR n e u r o n s by extracellular recording, HRP w a s i n j e c t e d i n t r a c e l l u l a r l y and processed for immunohistochemical procedures for ENK. The e f f e c t of the g~ucose level was tested on 114 V M H n e u r o n s in 18 s l i c e s . The s p o n t a n e o u s f i r i n g r a t e w a s 2.5 ~.7 spikes/s (mean + SD, n = 1 1 4 ) . W h e n g l u c o s e i n c r e a s e d to 20 mM, 27 neurons (23.7%) responded, and the r e m a i n i n g 87 (77.3%) w e r e unchanged. Of the 27 responding neurons, 16 (59.2%) i n c r e a s e d t h e i r d i s c h a r g e rates upon glucose application and 7 (25.9%) s h o w e d an i n c r e a s e f o l l o w e d by a decrease. The discharge rates of four n e u r o n s (14.8%) d e c r e a s e d . In agreement with the description by Ono et al. GR n e u r o n s w e r e 10-20 um in d i a m e t e r and multipolar with more than 3 primary dendrites ranging widely in the VMH with some p r o j e c t i n g o u t of the VMH. w i t h an e l e c t r o n m i c r o s c o p e , it c o u l d b e e n s e e n t h a t GR n e u r o n s h a d m o d e r a t e l y d e v e l o p e d c y t o p l a s m i c o r g a n s and an i n d e n t e d nucleus. They a l w a y s r e c e i v e d s y n a p t i c i n p u t s f r o m n o n - l a b e l e d t e r m i n a l s on t h e i r soma, and dendritic s h a f t s and s p i n e s . A GR n e u r o n (#13) has a dendrite extending t o w a r d the t h i r d v e n t r i c l e . T h i s d e n d r i t e w a s o b s e r v e d to be c l o s e l y a p p o s e d by 10 ENK-IR axonal b o u t o n s u n d e r l i g h t m i c r o s c o p e . It w a s then determined by examining serial u l t r a t h i n s e c t i o n s t h a t 7 of t h e s e sites received synaptic inputs directly from ENK-IR axonal boutons. This ultrastructural evidence agrees well with the d i r e c t e x c i t a t o r y e f f e c t of ENK or morphine on VMH glucoreceptive neurons. ANALYSIS
OF T H E
ENKEPHALINERGIC
AFFERENT
SYSTEM
IN T H E
RAT
INTERPEDUNCULAR
NUCLEUS
TOSHIMI MICHIGAMI*, KOH SHINODA~ MARIKO YAMANO~ YURIKO KAWAI, MASAYATOHYAMA AND YAHE SHIOTANI Department of Neuroanatomy, Institute of Higher Nervous Medical School, 4-3-57, Kitaku, Nakanoshima, Osaka 530,
Activity, Japan
Osaka
University
The interpeduncular nucleus (IPN) of the rat brain has the following three major terminal fields of enkephalin-like immunoreactive (ENKI) fibers: the rostral subnucleus, the dorsal lateral subnucleus and the central subnucleus. In order to examine the origins of these ENKI f i b e r s , several kinds of knife cuts and electrolytic lesions were made. In this study, all the rats (Wistar Strain male rats, 50-100g) were operated on o r k i l l e d under anesthesia with sodium pentobarbital (20mg/kg or 60mg/kg, i.p.). After a few days, frozen sections of the rat brains were made and processed by the indirect immunofluorescence method with the antibody against leucine-enkepalin. When t h e f r o n t a l transection was made bilaterally at the intermediate level of the dorsal tegmental nucleus (DTg), a remarkable decrease o f t h e ENKI f i b e r s occurred only in the rostral subnucleus. Also the electrolytic lesion involving the unilateral DTg a n d t h e d o r s a l raphe nucleus (DR) c a u s e d m a r k e d d e p l e t i o n s of the ENKI f i b e r s in the contralateral rostral subnucleus and the central subnucleus. On the other hand, when the lesion involved a caudal portion of the unilateral ventral tegmental nucleus ( V T g ) , o n l y t h e ENKI f i b e r s in the contralateral dorsal lateral subnucleus were markedly reduced. These results clearly demonstrated that t h e ENKI fibers in these three subnuclei o f t h e IPN w e r e c o n t r a l a t e r a l l y supplied from the ENKI n e u r o n s in the discrete regions of the midbrain tegmentum as follows: the rostral subnucleus from the cdntralateral DTg, t h e d o r s a l lateral subnucleus from the contralateral VTg a n d t h e c e n t r a l subnucleus f r o m t h e DR.
INPUT
FROM
ENKEPHALINERGIC
TERMINALS
TO V M H
GLUCORECEPTIVE
NEURONS
SHINOB~ I N A G A K I I, YOSHI~UKI K U B O T A I , S H O I C H I S H I M A D A I*, SHOZO K I T O I, KAZUO S A S A K I - and T A K E T O S H I O N O I: Dept. I n t e r n a l M e d . , H i r o s h i m a Univ. Sch. Med., I-2-3 K a s u m i , M i n a m i k u , H i r o s h i m a 734, 2: Dept. P h y s i o l . , Facul. M e d . , Toyama Med. P h a r m a c e u t . U n i v . , T o y a m a 930-01 In the v e n t r o m e d i a l h y p o t h a l a m i c n u c l e u s (VMH) g l u c o s e r e c e p t i v e (GR) n e u r o n s and enkephalin (ENK) imrnunoreactive f i b e r s are p r e s e n t in a large number, suggesting i n t e r r e l a t i o n s b e t w e e n GR n e u r o n s a n d e n k e p h a l i n in r e l a t i o n to the VMH function. A d u l t m a l e r a t s w e r e d e c a p i t a t e d and a b o u t 500 700 um thick coronal tissue slices were cut. To i d e n t i f y the GR neurons, glucose was experimentally v a r i e d f r o m 5.5 to 20 mM. A f t e r i d e n t i f i c a t i o n of GR n e u r o n s by extracellular recording, HRP w a s i n j e c t e d i n t r a c e l l u l a r l y and processed for immunohistochemical procedures for ENK. The e f f e c t of the g~ucose level was tested on 114 V M H n e u r o n s in 18 s l i c e s . The s p o n t a n e o u s f i r i n g r a t e w a s 2.5 ~.7 spikes/s (mean + SD, n = 1 1 4 ) . W h e n g l u c o s e i n c r e a s e d to 20 mM, 27 neurons (23.7%) responded, and the r e m a i n i n g 87 (77.3%) w e r e unchanged. Of the 27 responding neurons, 16 (59.2%) i n c r e a s e d t h e i r d i s c h a r g e rates upon glucose application and 7 (25.9%) s h o w e d an i n c r e a s e f o l l o w e d by a decrease. The discharge rates of four n e u r o n s (14.8%) d e c r e a s e d . In agreement with the description by Ono et al. GR n e u r o n s w e r e 10-20 um in d i a m e t e r and multipolar with more than 3 primary dendrites ranging widely in the VMH with some p r o j e c t i n g o u t of the VMH. w i t h an e l e c t r o n m i c r o s c o p e , it c o u l d b e e n s e e n t h a t GR n e u r o n s h a d m o d e r a t e l y d e v e l o p e d c y t o p l a s m i c o r g a n s and an i n d e n t e d nucleus. They a l w a y s r e c e i v e d s y n a p t i c i n p u t s f r o m n o n - l a b e l e d t e r m i n a l s on t h e i r soma, and dendritic s h a f t s and s p i n e s . A GR n e u r o n (#13) has a dendrite extending t o w a r d the t h i r d v e n t r i c l e . T h i s d e n d r i t e w a s o b s e r v e d to be c l o s e l y a p p o s e d by 10 ENK-IR axonal b o u t o n s u n d e r l i g h t m i c r o s c o p e . It w a s then determined by examining serial u l t r a t h i n s e c t i o n s t h a t 7 of t h e s e sites received synaptic inputs directly from ENK-IR axonal boutons. This ultrastructural evidence agrees well with the d i r e c t e x c i t a t o r y e f f e c t of ENK or morphine on VMH glucoreceptive neurons. ANALYSIS
OF T H E
ENKEPHALINERGIC
AFFERENT
SYSTEM
IN T H E
RAT
INTERPEDUNCULAR
NUCLEUS
TOSHIMI MICHIGAMI*, KOH SHINODA~ MARIKO YAMANO~ YURIKO KAWAI, MASAYATOHYAMA AND YAHE SHIOTANI Department of Neuroanatomy, Institute of Higher Nervous Medical School, 4-3-57, Kitaku, Nakanoshima, Osaka 530,
Activity, Japan
Osaka
University
The interpeduncular nucleus (IPN) of the rat brain has the following three major terminal fields of enkephalin-like immunoreactive (ENKI) fibers: the rostral subnucleus, the dorsal lateral subnucleus and the central subnucleus. In order to examine the origins of these ENKI f i b e r s , several kinds of knife cuts and electrolytic lesions were made. In this study, all the rats (Wistar Strain male rats, 50-100g) were operated on o r k i l l e d under anesthesia with sodium pentobarbital (20mg/kg or 60mg/kg, i.p.). After a few days, frozen sections of the rat brains were made and processed by the indirect immunofluorescence method with the antibody against leucine-enkepalin. When t h e f r o n t a l transection was made bilaterally at the intermediate level of the dorsal tegmental nucleus (DTg), a remarkable decrease o f t h e ENKI f i b e r s occurred only in the rostral subnucleus. Also the electrolytic lesion involving the unilateral DTg a n d t h e d o r s a l raphe nucleus (DR) c a u s e d m a r k e d d e p l e t i o n s of the ENKI f i b e r s in the contralateral rostral subnucleus and the central subnucleus. On the other hand, when the lesion involved a caudal portion of the unilateral ventral tegmental nucleus ( V T g ) , o n l y t h e ENKI f i b e r s in the contralateral dorsal lateral subnucleus were markedly reduced. These results clearly demonstrated that t h e ENKI fibers in these three subnuclei o f t h e IPN w e r e c o n t r a l a t e r a l l y supplied from the ENKI n e u r o n s in the discrete regions of the midbrain tegmentum as follows: the rostral subnucleus from the cdntralateral DTg, t h e d o r s a l lateral subnucleus from the contralateral VTg a n d t h e c e n t r a l subnucleus f r o m t h e DR.