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Age-related changes of amino acid receptors in the rat brain studied by quantitative in vitro autoradiography
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AGE-RELATED CHANGES OF AMINO ACID RECEPTORS IN THE RAT BRAIN STUDIED BY QUANTITATIVE IN VITRO AUTORADIOGRAPHY. RIE MIYOSHI*, SHOZO KITO AND NAOMI DOUDOU*, Third Department of Internal Medicine, Hiroshlma University School of Medicine, i-2-3 Kasumi, Minamiku, Hiroshlma 734, Japan. Receptors for excitatory amino acid, L-glutamate, have been thought to be divided into three subclasses referred to as N-methyl-D-aspartic acid (NMDA)-, quisqualic acid (QA)-, and kainlc acid-sensltlve sites. NMDA sites have been advocated to have a functional relationship with strychnlne-insensltive glycine receptors. In the present study, to elucidate a role of both glutamate- and glycine-neuronal systems in the normal aging process, we examined age-related changes of strychnine-insensltive glycine receptors and QA sites in the rat brai~ through quantitative in vitro autoradiography. Glyclne and QA receptors were labelled with -H-glycine and H-AMPA, ~ v e l y . With use of Triton X-100 treated brain sections to remove possible endogenous ligands, these two ligands had a single binding site whose Kd values were 80.0 and 74.1 nM, respectlv~ly. These receptors were highly concentrated in the hlppocampus and cerebral cortex. However, H-g~yclne binding sites had the highest densities in the stratum radiatum and stratum oriens, while ~H-AMPA binding sites were rich in the stratum pyramidale. -H-AMPA binding sites showed a layered pattern in the cerebral cortex, whereas H-glycine did not. In general, the bralnstem3and cerebellum exhibited low levels of binding of these two llgands. In parallel with aging, H-glyclne bi~dlng sites in the telencephalic regions decreased, but there was no significant difference in H-AMPA binding sites. No change of these receptors in the brainstem and cerebellum were observed. These results indicate that dysfunction of glycine receptors in telencephaltc regions may be related to the aging process.
RELATIONSHIP BETWEEN CONFORMATION OF A GLUTAMATE AGONIST AND ACTIVATION OF RECEPTOR SUBTYPES.
MICHIKO ISHIDA AND HARUHIKO SHINOZAKI r The Tokyo Metropolitan
I n s t i t u t e o f Medical Science, 3-18-22, Honkomacjome, B u n k y o - k u ,
Tokyo 113, Japan.
Eight conformationally r e s t r i c t e d analogs of glutamate, isomers of a - ( c a r b o x y c y c l o p r o p y l ) g l y c i n e (CCG), demonstrated a large v a r i e t y of depolarizing activities in the newborn rat spinel cord and two novel potent NMDA-type agonists were discovered. causing depolarizations.
They were more potent than NMDA in
All D-isomers were NMDA-type agonists.
Furthermore, the (2S,3S,4S)
isomer ( L - C C G - I ) caused a l a r g e r depolarization than L-glutamate, but the depolarization was almost insensitive to o r d i n a r y glutamate blockers such as CPP, CNQX, or DNQX. the possibility of a new type o f glutamate agonist. type.
This suggests
The conformation of L-CCG-I is an extended
A f t e r application of the (2S,3SfllR) isomer o f L-CCG ( L - C C G - I I I ) ,
depolarizing responses to
L-glutamate were markedly enhanced for more than several hours without f u r t h e r application. CCG-III is expected to be a potent uptake i n h i b i t o r of L-glutamate.
L-
CCG isomers will be useful
tools f o r elucidating glutamate function.
EFFECTS OF EXCITATORY AMINO ACIDS ON MONOSYNAPTIC EPSPs OF SPINAL MOTONEURONS. FUSAO NAKAMURA* AND MIYUKI KUNO. Dept. of PhFsiol.~ Osaka CitF Univ., Med. Sch.,Abeno-ku, Osaka 545r Japan.
E x c i t a t o r y amino a c i d s a r e c a n d i d a t e s f o r t r a n s m i t t e r s a t synapses on s p i n a l motoneurons. E f f e c t s of e x c i t a t o r y amino a c i d s on monosynaptic EPSPs were s t u d i e d on i n t r a - a r t e r i a l l y p e r f u s e d f r o g s p i n a l cord, u s i n g i n t r a c e l l u l a r r e c o r d i n g s . The e n t i r e spinal cord was i s o l a t e d under a n e s t h e s i a with p e n t o b a r b i t a l . E l e c t r i c a l s t i m u l i were applied a t the l a t e r a l column. Glutamate (lmM) d e p o l a r i z e d c e l l s both in Mg2*-free and high d i v a l e n t c a t i o n (4.5mM Ca 2÷, S ~ Mg2÷) s o l u t i o n s . NMDA(5OsM) induced d e p o l a r i z a t i o n only in Mg~+-free medium. 1-D-glutamylglycine , a n a n t a g o n i s t for g l u t a m a t e r e c e p t o r s , g r e a t l y s u p p r e s s e d EPSP amplitudes, while the e f f e c t o f 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e , an a n t a g o n i s t for NMDAr e c e p t o r s , was i n c o n s i s t e n t . During g l u t a m a t e - i n d u c e d d e p o l a r i z a t i o n , t h e amplitude and h a l f width of EPSPs g r a d u a l l y decreased in both ~ g 2 * - f r e e and high d i v a l e n t c a t i o n s o l u t i o n s . The h a l f width decreased to 30-50 ~ of the controls, while t h e EPSP a m p l i t u d e was reduced to 50~ of the c o n t r o l s . Synaptic p o t e n t i a t i o n evoked by a p a i r e d s t i m u l u s was a l s o s u p p r e s s e d in t h e presence of glutamate in Mg~*-free and high divalent cation solutions. These r e s u l t s s u g g e s t t h a t monosynaptic EPSPs a r e mediated mainly v i a non-NMDA r e c e p t o r s and t h a t p r e s y n a p t i c glutamate a c t i o n c o n t r i b u t e s to a u t o modulation.
AGE-RELATED CHANGES OF AMINO ACID RECEPTORS IN THE RAT BRAIN STUDIED BY QUANTITATIVE IN VITRO AUTORADIOGRAPHY. RIE MIYOSHI*, SHOZO KITO AND NAOMI DOUDOU*, Third Department of Internal Medicine, Hiroshlma University School of Medicine, i-2-3 Kasumi, Minamiku, Hiroshlma 734, Japan. Receptors for excitatory amino acid, L-glutamate, have been thought to be divided into three subclasses referred to as N-methyl-D-aspartic acid (NMDA)-, quisqualic acid (QA)-, and kainlc acid-sensltlve sites. NMDA sites have been advocated to have a functional relationship with strychnlne-insensltive glycine receptors. In the present study, to elucidate a role of both glutamate- and glycine-neuronal systems in the normal aging process, we examined age-related changes of strychnine-insensltive glycine receptors and QA sites in the rat brai~ through quantitative in vitro autoradiography. Glyclne and QA receptors were labelled with -H-glycine and H-AMPA, ~ v e l y . With use of Triton X-100 treated brain sections to remove possible endogenous ligands, these two ligands had a single binding site whose Kd values were 80.0 and 74.1 nM, respectlv~ly. These receptors were highly concentrated in the hlppocampus and cerebral cortex. However, H-g~yclne binding sites had the highest densities in the stratum radiatum and stratum oriens, while ~H-AMPA binding sites were rich in the stratum pyramidale. -H-AMPA binding sites showed a layered pattern in the cerebral cortex, whereas H-glycine did not. In general, the bralnstem3and cerebellum exhibited low levels of binding of these two llgands. In parallel with aging, H-glyclne bi~dlng sites in the telencephalic regions decreased, but there was no significant difference in H-AMPA binding sites. No change of these receptors in the brainstem and cerebellum were observed. These results indicate that dysfunction of glycine receptors in telencephaltc regions may be related to the aging process.
RELATIONSHIP BETWEEN CONFORMATION OF A GLUTAMATE AGONIST AND ACTIVATION OF RECEPTOR SUBTYPES.
MICHIKO ISHIDA AND HARUHIKO SHINOZAKI r The Tokyo Metropolitan
I n s t i t u t e o f Medical Science, 3-18-22, Honkomacjome, B u n k y o - k u ,
Tokyo 113, Japan.
Eight conformationally r e s t r i c t e d analogs of glutamate, isomers of a - ( c a r b o x y c y c l o p r o p y l ) g l y c i n e (CCG), demonstrated a large v a r i e t y of depolarizing activities in the newborn rat spinel cord and two novel potent NMDA-type agonists were discovered. causing depolarizations.
They were more potent than NMDA in
All D-isomers were NMDA-type agonists.
Furthermore, the (2S,3S,4S)
isomer ( L - C C G - I ) caused a l a r g e r depolarization than L-glutamate, but the depolarization was almost insensitive to o r d i n a r y glutamate blockers such as CPP, CNQX, or DNQX. the possibility of a new type o f glutamate agonist. type.
This suggests
The conformation of L-CCG-I is an extended
A f t e r application of the (2S,3SfllR) isomer o f L-CCG ( L - C C G - I I I ) ,
depolarizing responses to
L-glutamate were markedly enhanced for more than several hours without f u r t h e r application. CCG-III is expected to be a potent uptake i n h i b i t o r of L-glutamate.
L-
CCG isomers will be useful
tools f o r elucidating glutamate function.
EFFECTS OF EXCITATORY AMINO ACIDS ON MONOSYNAPTIC EPSPs OF SPINAL MOTONEURONS. FUSAO NAKAMURA* AND MIYUKI KUNO. Dept. of PhFsiol.~ Osaka CitF Univ., Med. Sch.,Abeno-ku, Osaka 545r Japan.
E x c i t a t o r y amino a c i d s a r e c a n d i d a t e s f o r t r a n s m i t t e r s a t synapses on s p i n a l motoneurons. E f f e c t s of e x c i t a t o r y amino a c i d s on monosynaptic EPSPs were s t u d i e d on i n t r a - a r t e r i a l l y p e r f u s e d f r o g s p i n a l cord, u s i n g i n t r a c e l l u l a r r e c o r d i n g s . The e n t i r e spinal cord was i s o l a t e d under a n e s t h e s i a with p e n t o b a r b i t a l . E l e c t r i c a l s t i m u l i were applied a t the l a t e r a l column. Glutamate (lmM) d e p o l a r i z e d c e l l s both in Mg2*-free and high d i v a l e n t c a t i o n (4.5mM Ca 2÷, S ~ Mg2÷) s o l u t i o n s . NMDA(5OsM) induced d e p o l a r i z a t i o n only in Mg~+-free medium. 1-D-glutamylglycine , a n a n t a g o n i s t for g l u t a m a t e r e c e p t o r s , g r e a t l y s u p p r e s s e d EPSP amplitudes, while the e f f e c t o f 2 - a m i n o - 5 - p h o s p h o n o v a l e r a t e , an a n t a g o n i s t for NMDAr e c e p t o r s , was i n c o n s i s t e n t . During g l u t a m a t e - i n d u c e d d e p o l a r i z a t i o n , t h e amplitude and h a l f width of EPSPs g r a d u a l l y decreased in both ~ g 2 * - f r e e and high d i v a l e n t c a t i o n s o l u t i o n s . The h a l f width decreased to 30-50 ~ of the controls, while t h e EPSP a m p l i t u d e was reduced to 50~ of the c o n t r o l s . Synaptic p o t e n t i a t i o n evoked by a p a i r e d s t i m u l u s was a l s o s u p p r e s s e d in t h e presence of glutamate in Mg~*-free and high divalent cation solutions. These r e s u l t s s u g g e s t t h a t monosynaptic EPSPs a r e mediated mainly v i a non-NMDA r e c e p t o r s and t h a t p r e s y n a p t i c glutamate a c t i o n c o n t r i b u t e s to a u t o modulation.