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In vitro assay system for synapse formation between cultured neurons of mice
s115
OPTICAL DEMONSTRATION OF THE FUNCTIONAL EMBRYONIC CHICK SPINAL CORD SLICE.
163 YOSHIYASU Dept. Physiol.,
ARAI, YOKO MOMOSE-SATO,
KATSUSHIGE
FORMATION
OF NEURAL CIRCUITS IN THE
SATO, KOHTARO KAMINO
Tokyo. Med. & Dent. Univ. Sch. Med., Tokyo 113-8519, Japan
We ha1.e apphed multiple-site
optical recording of transmembnne
potential changes to recordmg of neuronal ctrcuit electrical
acttvtty from embryontc chick spinal cord shce preparations. The slice preparations were drssected from B-day old chtck embryos at the level of the 13th of the cervtcal spinal nerve or the 2nd or 5th of the lumbosacral spmal nerve, and they were statned wtth a voltage-senstttve
dye (NK2761).
Transmembrane
voltage-related
optical changes evoked by spinal nerve
sttmulation wtth postttve square current pulses using a suctton electrode were recorded stmultaneously from many loci m the preparation, usmg a 128- or 10%element photodtode array. In the dorsal region, the fast sptke-like stgnal was identified as the presynapttc actton potential m the sensory nerve, and the slow signal as the postsynaptic potential. In the ventral regton, the fast sptke-hke signal rcilects the antidromrc actton potential m motoneurons, and the slow stgnal represents the postsynapttc potential evoked in the motoneuron. Synaptic fatigue induced by repetitive sttmuli in the ventral synapses was more rapid than in the dorsal synapses. The ventral neural response was also more sensitrve to cooling than the dorsal response. Furthermore, we have been able to demonstrate formed tn the X-day old embryonic chtck spinal cord.
164
that functtonal monosynaptic
and polysynaptic
connections
are
CORPUS GLOMERULOSUM IN A TELEOST BRAIN: NOVEL PREPARATION FOR THE STUDY OF NEURONAL PROCESSING IN BRAIN NUCLEI
‘H.Tsutsui, *N.Yamamoto, *HIto and ‘YOka ‘Misaki Marine Biological Station, University of Tokyo, Misaki, Miura, Kanagawa 238-0225, Japan Department of Anatomy, Laboratory of Comparative Neuromorphorgy, Sendagi, Tokyo113-0022, Japan
‘Nippon Medical School,
Studies of higher neumnal function and celIular/sub-cellular mechanisms of synaptic transmissions are some of the frontiers in neuroscience. However, very few model systems provide opportunities for the study of neumnal processing in brain nuclei. On the other hand, most teleosts have a characteristic large nucleus, corpus glomemlosum (CG) which is considered to relay mainly visual information to the hypothalamus. CG is classified into 3 types by morphological criteria, and especially the type III CG may serve as an excellent experimental model for the study of neuronal processing in brain nuclei at system and ceIlular levels, because of its welldefined laminar structure, synaptic organization, and inputioutput fiber connections. As a first step to characterize this model system, we newly developed a brain slice preparation of CG for electrophysiological analysis. Evoked post-synaptic potentials from the tip of ‘giant dendrites’ of large cells were recorded by intracellular microelectrode. We find that 1) the giant dendrite receives ghttamatergic input from the nucleus cortical, 2) the giant dendrite has at least two subtypes of glutamate receptors, the non-NMDA and NMDA receptors and 3) local synaptic connections exist between large cells mediated by NMDA and GABA* receptors. The present results suggest that CG may serve as a good model to study the role of synaptic plasticity involved in neumnal processing in brain nuclei.
165 AKIHIRO
IN VITRO ASSAY SYSTEM FOR SYNAPSE OF MICE MIZUTANI
‘, JIN-ICHI INOKUCHI
2, YOICHIRO
FORMATION
KURODA
BETWEEN
CULTURED
NEURONS
d
I Seikagaku Corp.,Tokyo Research Inst.,Higashiyamato-shi,Tokyo 207-0021, 2 Dept.of Biomembrane and Biofunctional Chemistry,Graduate School of Pharmaceutical Sciences,Hokkaido Univ.,Sapporo-shi 060-0812, 3 Dept.of Molecular and Cellular Neurobiology,Tokyo Metropolitan Inst.for Neuroscience,Fuchu-shi,Tokyo 183-8.526 Productions of various transgenic mice and knockout mice enable us to investigate the crucial molecules in various brain functions. To investigate the nature of the neuronal circuit and synaptic functions in gene manipulated mice, we tried to establish the assay system for the functional synapse formation in cultured mouse cortical neurons. Cerebral cortices were dissected from 14%17-day fetal mice, then cells were dissociated with papain and cultured. After several days, we observed spontaneous synchronous oscillations of Ca” transients. The frequency of oscillation was gradually increased during 7%14 days in culture. Application of tetrodotoxin or of 2-amino-phosphonovaleric acid(DAPV)inhibited the oscillation, suggesting that this oscillation of CaZ+ transients reflects synchronous bursts of firing mediated by abundant glutamatergic synapses in the cultured neuronal network. This system will provide a new method for the elucidation of functional molecules of synapses in neuronal circuits in gene knockout mice including the one without the ganglioside synthases.
OPTICAL DEMONSTRATION OF THE FUNCTIONAL EMBRYONIC CHICK SPINAL CORD SLICE.
163 YOSHIYASU Dept. Physiol.,
ARAI, YOKO MOMOSE-SATO,
KATSUSHIGE
FORMATION
OF NEURAL CIRCUITS IN THE
SATO, KOHTARO KAMINO
Tokyo. Med. & Dent. Univ. Sch. Med., Tokyo 113-8519, Japan
We ha1.e apphed multiple-site
optical recording of transmembnne
potential changes to recordmg of neuronal ctrcuit electrical
acttvtty from embryontc chick spinal cord shce preparations. The slice preparations were drssected from B-day old chtck embryos at the level of the 13th of the cervtcal spinal nerve or the 2nd or 5th of the lumbosacral spmal nerve, and they were statned wtth a voltage-senstttve
dye (NK2761).
Transmembrane
voltage-related
optical changes evoked by spinal nerve
sttmulation wtth postttve square current pulses using a suctton electrode were recorded stmultaneously from many loci m the preparation, usmg a 128- or 10%element photodtode array. In the dorsal region, the fast sptke-like stgnal was identified as the presynapttc actton potential m the sensory nerve, and the slow signal as the postsynaptic potential. In the ventral regton, the fast sptke-hke signal rcilects the antidromrc actton potential m motoneurons, and the slow stgnal represents the postsynapttc potential evoked in the motoneuron. Synaptic fatigue induced by repetitive sttmuli in the ventral synapses was more rapid than in the dorsal synapses. The ventral neural response was also more sensitrve to cooling than the dorsal response. Furthermore, we have been able to demonstrate formed tn the X-day old embryonic chtck spinal cord.
164
that functtonal monosynaptic
and polysynaptic
connections
are
CORPUS GLOMERULOSUM IN A TELEOST BRAIN: NOVEL PREPARATION FOR THE STUDY OF NEURONAL PROCESSING IN BRAIN NUCLEI
‘H.Tsutsui, *N.Yamamoto, *HIto and ‘YOka ‘Misaki Marine Biological Station, University of Tokyo, Misaki, Miura, Kanagawa 238-0225, Japan Department of Anatomy, Laboratory of Comparative Neuromorphorgy, Sendagi, Tokyo113-0022, Japan
‘Nippon Medical School,
Studies of higher neumnal function and celIular/sub-cellular mechanisms of synaptic transmissions are some of the frontiers in neuroscience. However, very few model systems provide opportunities for the study of neumnal processing in brain nuclei. On the other hand, most teleosts have a characteristic large nucleus, corpus glomemlosum (CG) which is considered to relay mainly visual information to the hypothalamus. CG is classified into 3 types by morphological criteria, and especially the type III CG may serve as an excellent experimental model for the study of neuronal processing in brain nuclei at system and ceIlular levels, because of its welldefined laminar structure, synaptic organization, and inputioutput fiber connections. As a first step to characterize this model system, we newly developed a brain slice preparation of CG for electrophysiological analysis. Evoked post-synaptic potentials from the tip of ‘giant dendrites’ of large cells were recorded by intracellular microelectrode. We find that 1) the giant dendrite receives ghttamatergic input from the nucleus cortical, 2) the giant dendrite has at least two subtypes of glutamate receptors, the non-NMDA and NMDA receptors and 3) local synaptic connections exist between large cells mediated by NMDA and GABA* receptors. The present results suggest that CG may serve as a good model to study the role of synaptic plasticity involved in neumnal processing in brain nuclei.
165 AKIHIRO
IN VITRO ASSAY SYSTEM FOR SYNAPSE OF MICE MIZUTANI
‘, JIN-ICHI INOKUCHI
2, YOICHIRO
FORMATION
KURODA
BETWEEN
CULTURED
NEURONS
d
I Seikagaku Corp.,Tokyo Research Inst.,Higashiyamato-shi,Tokyo 207-0021, 2 Dept.of Biomembrane and Biofunctional Chemistry,Graduate School of Pharmaceutical Sciences,Hokkaido Univ.,Sapporo-shi 060-0812, 3 Dept.of Molecular and Cellular Neurobiology,Tokyo Metropolitan Inst.for Neuroscience,Fuchu-shi,Tokyo 183-8.526 Productions of various transgenic mice and knockout mice enable us to investigate the crucial molecules in various brain functions. To investigate the nature of the neuronal circuit and synaptic functions in gene manipulated mice, we tried to establish the assay system for the functional synapse formation in cultured mouse cortical neurons. Cerebral cortices were dissected from 14%17-day fetal mice, then cells were dissociated with papain and cultured. After several days, we observed spontaneous synchronous oscillations of Ca” transients. The frequency of oscillation was gradually increased during 7%14 days in culture. Application of tetrodotoxin or of 2-amino-phosphonovaleric acid(DAPV)inhibited the oscillation, suggesting that this oscillation of CaZ+ transients reflects synchronous bursts of firing mediated by abundant glutamatergic synapses in the cultured neuronal network. This system will provide a new method for the elucidation of functional molecules of synapses in neuronal circuits in gene knockout mice including the one without the ganglioside synthases.