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Development of GABAA and benzodiazepine receptors in primary cultured neurons from mouse cerebral cortex
S6 THE EFFECT OF OF EXCITATORY
w-PHOSPHONO-&-AMINO AMINO ACIDS, IN E L
CARBOXYLYC ACID, MOUSE CONVULSIONS.
ANTAGONISTS
* AKITANE MORI, ISAO YOKOI, HIDEAKI KABUTO Department of Neurochemistry, Institute for Neurobiology, University Medical School, 2-5-] Shikata-cho Okayama 700,
Okayama Japan
Convulsions in E1 m i c e w e r e c o m p l e t e l y suppressed by 1.8 ~mol of 2-amino-5phosphonovaleric a c i d (APS) i n j e c t e d intracerebroventricularly 60 m i n u t e s before the mice were examined. The ED50 was 0.072 ~mol. The brain glutamate level was significantly l o w e r in t h e A P 5 a d m i n i s t e r e d mice compared to the controls. 2-Amino-3-phosphonopropionic acid ( AP3 ; 1.04 ~mol ) and 2-amino-7-phosphonoheptanoic acid ( AP7 ; 1.27 ~mol ) used in the same experimental conditions indicated a sedative rather than an anticonvulsive effect. The brain glutamine level was significantly higher in t h e b r a i n s o f A P 3 a d m i n i s t e r e d mice, and the glutamate level was significantly lower in t h e b r a i n s o f A P 7 a d m i n i s t e r e d miced t h a n in t h e c o n t r o l s . E1 m i c e i n j e c t e d w i t h 0.I ~ m o l o f A P 3 w e r e i n d u c e d r u n n i n g fits. E1 m i c e i n j e c t e d w i t h 0 . 0 2 ~jmol of A P 3 w e r e i n d u c e d p a r t i a l seizures in four limbs and the tail. 2-Amino-4-phosphonobutyric acids (AP4;1.02 Nmol) and 2-amino-6-phosphonohexanoic acid (AP6;0.8 ~mol) used under the same experimental conditions induced seizures in E 1 m i c e . The brain aspartate level was significantly lower i n t h e b r a i n of A P 4 administered mice t h a n in t h e c o n t r o l s , and glutamate, glutamine, GABA, glycine, and alanine levels were significantly lower in t h e b r a i n o f A P 6 a d m i n i s t e r e d m i c e t h a n in t h e c o n t r o l s . Our findings suggest that AP3, AP5, and AP7 occupying N-methyl-D-aspartate receptor sites may act as antagonists of t h e excitatory receptor sites, and that AP4 and AP6 may act rather as agonists of the same receptor sites.
DEVELOPMENT OF GABA A AND BENZODIAZEPINE CORTEX.
RECEPTORS
IN PRIMARY CULTURED NEURONS FROH MOUSE CEREBRAL
SEITARO OHKUHA*, SHOICHI TOMONO*, MASATAKA KISHI* and TSUNBICHI HASHIMOTO* (Sponsored by KINYA KURIYAMA). Department of Pharmacology, Kyoto Prefectura] University of Medicine, Kamikyo-Ku, Kyoto 602, Japan Developmental changes of GABA A and benzodiazepine (BZP) receptors in primary cultured cerebral cortical neurons, which possess morphological features typical of neurons, inciuding synaptic forma tJons and GABA metabolizing system were studied. Cerebral cortical neurons used for primary culture were dissociated from the neopa]]ium of a 15 day-old mouse fetus of STD:ddy strain by trypsin treatment. Developmental changes in GABA A and BZP receptors were examined using [~H]muscimol, a specific agonist for GABA A receptor, and [~H]flunitrazepam ([~H]FLN), a specific ]igand for the central type of BZP receptor, respectively. These primary cultured neurons had two different types of binding sites for [3H]muscimo] with high and low affinities. During neuronal growth in vitro, the binding of [~H]muscimo] to the particulate fraction obtained from primary cultured neurons showed a progressive increase. This increase of [~H]muscimol binding was found to be due to the increase in the number of binding sites (Bmax) but not its affinity (Kd). In addition, it was found that the [3H]muscimol binding was sensitive to bicuculline, a GABA A receptor antagonist, and these deve]opmenta] patterns of the binding were similar with those found in age-matched mouse fetal and neonatal cerebral cortices. On the other hand, it was found that [~H]FLN binding in primary cultured neurons was displaced dose dependently by other benzodiazepine, such as clonazepam and diazepam, and consisted of one binding site with a high affinity. This [3H]FLN binding also showed a tendency to increase its Bmax value during neuronal development in vitro, and these developmenta] patterns of [3H]FLN binding were also found to be similar with those found in age-matched mouse fetal and neonatal cerebral cortices. The [3H]FLN binding was enhanced by 10 -4 10 -5 M unlabe]ed GABA in both 7 and 14 days-old neurons in primary culture. These results indicate that primary cultured neurons used in the present study possess functional]y coup]ed GABA and BZP receptors. The above results a]sn suggest that these neurons may be useful as an experimental model for investigating the effect of various centrally acting drugs on the cerebral GABA and BZP receptor complex.
w-PHOSPHONO-&-AMINO AMINO ACIDS, IN E L
CARBOXYLYC ACID, MOUSE CONVULSIONS.
ANTAGONISTS
* AKITANE MORI, ISAO YOKOI, HIDEAKI KABUTO Department of Neurochemistry, Institute for Neurobiology, University Medical School, 2-5-] Shikata-cho Okayama 700,
Okayama Japan
Convulsions in E1 m i c e w e r e c o m p l e t e l y suppressed by 1.8 ~mol of 2-amino-5phosphonovaleric a c i d (APS) i n j e c t e d intracerebroventricularly 60 m i n u t e s before the mice were examined. The ED50 was 0.072 ~mol. The brain glutamate level was significantly l o w e r in t h e A P 5 a d m i n i s t e r e d mice compared to the controls. 2-Amino-3-phosphonopropionic acid ( AP3 ; 1.04 ~mol ) and 2-amino-7-phosphonoheptanoic acid ( AP7 ; 1.27 ~mol ) used in the same experimental conditions indicated a sedative rather than an anticonvulsive effect. The brain glutamine level was significantly higher in t h e b r a i n s o f A P 3 a d m i n i s t e r e d mice, and the glutamate level was significantly lower in t h e b r a i n s o f A P 7 a d m i n i s t e r e d miced t h a n in t h e c o n t r o l s . E1 m i c e i n j e c t e d w i t h 0.I ~ m o l o f A P 3 w e r e i n d u c e d r u n n i n g fits. E1 m i c e i n j e c t e d w i t h 0 . 0 2 ~jmol of A P 3 w e r e i n d u c e d p a r t i a l seizures in four limbs and the tail. 2-Amino-4-phosphonobutyric acids (AP4;1.02 Nmol) and 2-amino-6-phosphonohexanoic acid (AP6;0.8 ~mol) used under the same experimental conditions induced seizures in E 1 m i c e . The brain aspartate level was significantly lower i n t h e b r a i n of A P 4 administered mice t h a n in t h e c o n t r o l s , and glutamate, glutamine, GABA, glycine, and alanine levels were significantly lower in t h e b r a i n o f A P 6 a d m i n i s t e r e d m i c e t h a n in t h e c o n t r o l s . Our findings suggest that AP3, AP5, and AP7 occupying N-methyl-D-aspartate receptor sites may act as antagonists of t h e excitatory receptor sites, and that AP4 and AP6 may act rather as agonists of the same receptor sites.
DEVELOPMENT OF GABA A AND BENZODIAZEPINE CORTEX.
RECEPTORS
IN PRIMARY CULTURED NEURONS FROH MOUSE CEREBRAL
SEITARO OHKUHA*, SHOICHI TOMONO*, MASATAKA KISHI* and TSUNBICHI HASHIMOTO* (Sponsored by KINYA KURIYAMA). Department of Pharmacology, Kyoto Prefectura] University of Medicine, Kamikyo-Ku, Kyoto 602, Japan Developmental changes of GABA A and benzodiazepine (BZP) receptors in primary cultured cerebral cortical neurons, which possess morphological features typical of neurons, inciuding synaptic forma tJons and GABA metabolizing system were studied. Cerebral cortical neurons used for primary culture were dissociated from the neopa]]ium of a 15 day-old mouse fetus of STD:ddy strain by trypsin treatment. Developmental changes in GABA A and BZP receptors were examined using [~H]muscimol, a specific agonist for GABA A receptor, and [~H]flunitrazepam ([~H]FLN), a specific ]igand for the central type of BZP receptor, respectively. These primary cultured neurons had two different types of binding sites for [3H]muscimo] with high and low affinities. During neuronal growth in vitro, the binding of [~H]muscimo] to the particulate fraction obtained from primary cultured neurons showed a progressive increase. This increase of [~H]muscimol binding was found to be due to the increase in the number of binding sites (Bmax) but not its affinity (Kd). In addition, it was found that the [3H]muscimol binding was sensitive to bicuculline, a GABA A receptor antagonist, and these deve]opmenta] patterns of the binding were similar with those found in age-matched mouse fetal and neonatal cerebral cortices. On the other hand, it was found that [~H]FLN binding in primary cultured neurons was displaced dose dependently by other benzodiazepine, such as clonazepam and diazepam, and consisted of one binding site with a high affinity. This [3H]FLN binding also showed a tendency to increase its Bmax value during neuronal development in vitro, and these developmenta] patterns of [3H]FLN binding were also found to be similar with those found in age-matched mouse fetal and neonatal cerebral cortices. The [3H]FLN binding was enhanced by 10 -4 10 -5 M unlabe]ed GABA in both 7 and 14 days-old neurons in primary culture. These results indicate that primary cultured neurons used in the present study possess functional]y coup]ed GABA and BZP receptors. The above results a]sn suggest that these neurons may be useful as an experimental model for investigating the effect of various centrally acting drugs on the cerebral GABA and BZP receptor complex.