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The increase in intracellular Ca2+ concentration induced by perfusion of Mg2+-free medium and the establishment of LTP
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GLUTAMATE-INDUCED SWELLING OF CULTURED ASTROCYTE IS DEPENDENT ON EXTRACELLULAR Ca 2+ AND Cl-, TOMOSADA SUGIbIOTO I, YUTAKA K O Y A ~ .2, AKEMICHI BABA .2, YOSHIO SHIGENAGA 1 AND HEITARO IWATA .2, 12nd Department of Oral Anatomy, Faculty o f Dentistry, an___dd2Department o_~fPharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565, Japan. The glutamate-induced astrocytic swelling was examined light- and electron-microscopically. Primary astrocyte cultures were prepared from the cerebral cortex of 1-2 day-old rats and grown in Eagle-minimum essential medium with 10% fetal bovine serum in poly-lysine-coated multiple well plates. At 2 weeks in vitro, 0.25mM dibutyryl cyclic AMP was added to the culture medium and the cells were grown for another 2 weeks. Thus prepared astrocytes were pre-incubated in HBKR (HEPESbuffered Krebs-Ringer solution for 2 h and incubated with 0.1mM L-glutamate in HBKR for 60 min. Control astrocytes were incubated with H B K R w i t h o u t L-glutamate. Thereafter, the astrocytes were fixed with aldehydes, osmicated, dehydrated and embedded in an epoxy resin. The astrocytes were block-stained with toluidine blue and, after light-microscopic examination, thin sectioned for electron-microscopy. The Glu treatment caused marked swelling of the cells characterized by reduction in cytoplasmic affinity for toluidine blue stain and disappearance of the cytoplasmic granular ground substance. The mitochondrion and nucleus also swelled, and the chromatin dispersed. The above changes were prevented by the removal of Na +, Ca 2+ or Cl- from the incubation medium for Glu treatment. However, the Glu treatment in a Cl--free medium caused conspicuous aggregation of I0 nm filaments, suggesting abnormality in the cytoskeletal elements of the affected astrocytes. The Ca 2+- and Cl-- mechanisms of Glu-induced astrocytic swelling are discussed.
THE INCREASE IN 1NTRACELLULAR Ca 2÷ CONCENTRATION INDUCED BY PERFUSION OF Mg2+-FREE MEDIUM AND THE ESTABLISHMENT OF LTP. TAKESHI NAKAMURA*, KYOKO AKITA*, AND YOSHIHISA .KUDO, Department of Neuroscience, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi. Tokyo 194, Japan. We have established a fluorometric image analyzing method for whole hippocampal slices loaded with fura-2 using a low magnification objective lens (x 4), and examined the relationship between the establishment of long term potentiation (LTP) and the change in the Ca 2÷ concentration ([Ca2÷]i). A high frequency stimulation (50Hz, 2see) applied to Schaffer/commissural pathways induced LTP and an increase in [Ca2÷]i at the proximal portion of the stratum radiatum in CAl. Pcrfusion of Mg2÷-free medium for 30 min also resulted in the increase in [Ca2÷]i at the proximal portion of the stratum radiatum in CA1. The amplitude of population spikes induced by test stimulations (0.1 Hz) was augmented during the exposure to the Mg2÷-free medium. The augmentation lasted more than 40 min (the establishment of LTP) even after reperfusion with normal medium. In MK-801 (101aM) treated slices, the perfusion with Mg2÷-free medium induced neither the LTP nor the increase in [Ca2÷]i. The region where Ca 2÷ concentrations increased by tetanic stimulation and by the exposure to the Mg2+-free medium coincided well with the region which was sensitive to NMDA (100~tM) in the same slice. The results confirmed that the LTP induced by the exposure to the Mg2+-free medium was dependent upon NMDA receptor-mediated increase in the [Ca2+]i through the same mechansim as that induced by tetanic stimultion.
L-CCG-I, A NEW METABOTROPIC GLUTAMATE RECEPTOR AGONIST. MICHIKO ISHIDA, YUKIKO ,
SHIMADA AND HARUHIKO SHINOZAKI, The Tokyo Metropolitan I n s t i t u t e of Medical Science, B u n k y o - k u , Tokyo 113, J a p a n . 2 - ( C a r b o x y c y c l o p r o p y l ) g l y e i n e ( C C G ) is a conformationally r e s t r i c t e d analogue of glutamate. The ( 2 S , 3 S , 4 S ) isomer of CCG ( L - C C G - I ) is more p o t e n t t h a n t h e metabotropie a g o n i s t , trans-(_*)ACPD, in e a u s l n g depolarization in t h e r a t isolated spinal c o r d , a c t i v a t i n g non-NMDA, n o n - k a i n a t e or non-AMPA t y p e r e c e p t o r s . synaptoneurosomes.
L-CCG-! stimulated ionsitol p h o s p h a t e formation in r a t hippoeampal
Peak amplitudes of r e s p o n s e s to trans-(_+)-ACPD a n d L-CCG-I were
s i g n i f i c a n t l y d e c r e a s e d when t h e t e m p e r a t u r e was d e c r e a s e d , while t h o s e of o t h e r e x c i t a t o r y amino acids were m a r k e d l y i n c r e a s e d .
Application of L-CCG-I i n d u c e d t h e oscillatory r e s p o n s e s in
Xenopus ooeytes injected with mRNA from the r a t b r a i n , s u g g e s t i n g t h a t L-CCG-I is a metabotropic r e c e p t o r a g o n i s t .
L-CCG-I d e p r e s s e d monosynaptic r e f l e x e s of t h e r a t isolated
spinal cord at c o n s i d e r a b l y low c o n c e n t r a t i o n s , h o w e v e r , it is not clear w h e t h e r t h i s d e p r e s s i o n of m o n o s y n a p t i e r e f l e x e s is t h r o u g h activation of metabotropie r e c e p t o r s .
GLUTAMATE-INDUCED SWELLING OF CULTURED ASTROCYTE IS DEPENDENT ON EXTRACELLULAR Ca 2+ AND Cl-, TOMOSADA SUGIbIOTO I, YUTAKA K O Y A ~ .2, AKEMICHI BABA .2, YOSHIO SHIGENAGA 1 AND HEITARO IWATA .2, 12nd Department of Oral Anatomy, Faculty o f Dentistry, an___dd2Department o_~fPharmacology, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565, Japan. The glutamate-induced astrocytic swelling was examined light- and electron-microscopically. Primary astrocyte cultures were prepared from the cerebral cortex of 1-2 day-old rats and grown in Eagle-minimum essential medium with 10% fetal bovine serum in poly-lysine-coated multiple well plates. At 2 weeks in vitro, 0.25mM dibutyryl cyclic AMP was added to the culture medium and the cells were grown for another 2 weeks. Thus prepared astrocytes were pre-incubated in HBKR (HEPESbuffered Krebs-Ringer solution for 2 h and incubated with 0.1mM L-glutamate in HBKR for 60 min. Control astrocytes were incubated with H B K R w i t h o u t L-glutamate. Thereafter, the astrocytes were fixed with aldehydes, osmicated, dehydrated and embedded in an epoxy resin. The astrocytes were block-stained with toluidine blue and, after light-microscopic examination, thin sectioned for electron-microscopy. The Glu treatment caused marked swelling of the cells characterized by reduction in cytoplasmic affinity for toluidine blue stain and disappearance of the cytoplasmic granular ground substance. The mitochondrion and nucleus also swelled, and the chromatin dispersed. The above changes were prevented by the removal of Na +, Ca 2+ or Cl- from the incubation medium for Glu treatment. However, the Glu treatment in a Cl--free medium caused conspicuous aggregation of I0 nm filaments, suggesting abnormality in the cytoskeletal elements of the affected astrocytes. The Ca 2+- and Cl-- mechanisms of Glu-induced astrocytic swelling are discussed.
THE INCREASE IN 1NTRACELLULAR Ca 2÷ CONCENTRATION INDUCED BY PERFUSION OF Mg2+-FREE MEDIUM AND THE ESTABLISHMENT OF LTP. TAKESHI NAKAMURA*, KYOKO AKITA*, AND YOSHIHISA .KUDO, Department of Neuroscience, Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida-shi. Tokyo 194, Japan. We have established a fluorometric image analyzing method for whole hippocampal slices loaded with fura-2 using a low magnification objective lens (x 4), and examined the relationship between the establishment of long term potentiation (LTP) and the change in the Ca 2÷ concentration ([Ca2÷]i). A high frequency stimulation (50Hz, 2see) applied to Schaffer/commissural pathways induced LTP and an increase in [Ca2÷]i at the proximal portion of the stratum radiatum in CAl. Pcrfusion of Mg2÷-free medium for 30 min also resulted in the increase in [Ca2÷]i at the proximal portion of the stratum radiatum in CA1. The amplitude of population spikes induced by test stimulations (0.1 Hz) was augmented during the exposure to the Mg2÷-free medium. The augmentation lasted more than 40 min (the establishment of LTP) even after reperfusion with normal medium. In MK-801 (101aM) treated slices, the perfusion with Mg2÷-free medium induced neither the LTP nor the increase in [Ca2÷]i. The region where Ca 2÷ concentrations increased by tetanic stimulation and by the exposure to the Mg2+-free medium coincided well with the region which was sensitive to NMDA (100~tM) in the same slice. The results confirmed that the LTP induced by the exposure to the Mg2+-free medium was dependent upon NMDA receptor-mediated increase in the [Ca2+]i through the same mechansim as that induced by tetanic stimultion.
L-CCG-I, A NEW METABOTROPIC GLUTAMATE RECEPTOR AGONIST. MICHIKO ISHIDA, YUKIKO ,
SHIMADA AND HARUHIKO SHINOZAKI, The Tokyo Metropolitan I n s t i t u t e of Medical Science, B u n k y o - k u , Tokyo 113, J a p a n . 2 - ( C a r b o x y c y c l o p r o p y l ) g l y e i n e ( C C G ) is a conformationally r e s t r i c t e d analogue of glutamate. The ( 2 S , 3 S , 4 S ) isomer of CCG ( L - C C G - I ) is more p o t e n t t h a n t h e metabotropie a g o n i s t , trans-(_*)ACPD, in e a u s l n g depolarization in t h e r a t isolated spinal c o r d , a c t i v a t i n g non-NMDA, n o n - k a i n a t e or non-AMPA t y p e r e c e p t o r s . synaptoneurosomes.
L-CCG-! stimulated ionsitol p h o s p h a t e formation in r a t hippoeampal
Peak amplitudes of r e s p o n s e s to trans-(_+)-ACPD a n d L-CCG-I were
s i g n i f i c a n t l y d e c r e a s e d when t h e t e m p e r a t u r e was d e c r e a s e d , while t h o s e of o t h e r e x c i t a t o r y amino acids were m a r k e d l y i n c r e a s e d .
Application of L-CCG-I i n d u c e d t h e oscillatory r e s p o n s e s in
Xenopus ooeytes injected with mRNA from the r a t b r a i n , s u g g e s t i n g t h a t L-CCG-I is a metabotropic r e c e p t o r a g o n i s t .
L-CCG-I d e p r e s s e d monosynaptic r e f l e x e s of t h e r a t isolated
spinal cord at c o n s i d e r a b l y low c o n c e n t r a t i o n s , h o w e v e r , it is not clear w h e t h e r t h i s d e p r e s s i o n of m o n o s y n a p t i e r e f l e x e s is t h r o u g h activation of metabotropie r e c e p t o r s .