| Co-Investigator(Kenkyū-buntansha) |
BAUGHMAN Rob ハーバード医科大学, 医学部, 副教授
SUZUKI Hidenori Tokyo Medical and Dental University Faculty of Medicine, Research Associate, 医学部, 助手 (30221328)
MURAKOSHI Takayuki Tokyo Medical and Dental University Faculty of Medicine, Research Associate, 医学部, 助手 (60190906)
YOSHIOKA Koichi Tokyo Medical and Dental University Faculty of Medicine, Assistant Professor, 医学部, 助教授 (00143579)
YANAGISAWA Mitsuhiko Tokyo Medical and Dental University Faculty of Medicine, Lecturer, 医学部, 講師 (90159252)
BAUGHMAN robert W. Harvard Medical School Faculty of Medicine, Associate Professor
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| Research Abstract |
The present research project aimed at studying the neural plasticity by means of examining the interaction between neural activities and synapse formation during development with chronically maintained central nervous system(CNS) preparations. In particular, a special emphasis was made upon investigating the modulatory effects of neurotransmitters on the synaptic plasticity. For this purpose, the following 4 in vitro CNS preparations were employed to perform electrophysiological and cell biological/biochemical analyses : 1) an isolated spinal cord preparation from newborn rats ; 2) a slice (400 mum thick) preparation from rat cerebral cortex ; 3) a dissociated neuronal cell culture, in which neurons were enzymatically isolated from the cerebral cortex, spinal cord, and spinal dorsal root ganglia of newborn rats, and maintained for 3 to 5 weeks ; 4) an organotypic culture of thin slices (100- 200 mum thick) maintained on a membrane for several days. We have been using the semi-acute pre
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parations of 1) and 2) for many years. The culture systems of 3) and 4), suitable for more chronic experiments, have been established by Dr Robert Baughman, a collaboration partner in the present project.
First, the effects of acetylcholine which is considered to promote the plasticity in developing CNS neurons were studied. A whole-cell patch-clamp recording from single neurons in the dissociated cell culture, an extracellular recording from spinal motoneurons of the isolated spinal cord preparation, and an intracellular recording from acute slice preparations all showed that acetylcholine caused a direct excitatory action on the cortical pyramidal neurons and the spinal motoneurons via activation of the muscarinic m_3 type receptor. Furthermore muscarinic agonist suppressed excitatory and inhibitory postsynaptic potentials (EPSPs & IPSPs) in the cortex, and also in the spinal cord suppressed EPSPs by a presynaptic mechanism. Next, the action of the metabotropic glutamate receptor was examined in the cortex, where glutamate is a major excitatory transmitter. Trans ACPD, an agonist for this receptor subtype, caused a membrane depolarization at the concentration of 10-100 muM.
The induction of gene expression is expected to occur during the chronic synaptic changes in response to external stimuli in order to initiate morphological and metabolic changes of the neurons. Expression of c-fos, a member of the immediate early genes, is reported in the spinal neurons after peripheral nociception, and after seizure activity in the cortical and hippocampal neurons. We examined weather this gene expression is induced by activation of the transmitter receptors using dissociated and slice culture preparations from the cortex, hippocampus, and the spinal cord. The neurons were stimulated by 1 hour application of the following agents to the medium, 1) high K+(50 mM), 2) substance P(10 muM), 3) phorbol ester (phorbol 2,3 di-acetate, 1 muM). When examined by immunocytochemistry using anti-c-fos protein, phorbol ester induced a significant increase in the c-fos expression in the dissociated cortical neurons while other stimuli did not. Substance P induced the c-fos expression in the dissociated spinal neurons. In the organotypic slice preparations, phorbol ester again triggered the c-fos expression in the cortex, hippocampus and the spinal cord neurons, however, substance P did not induce significant expression in all the neurons. Considering that substance P is one of the transmitters of the primary afferent neurons which conduct pain sensation to the spinal cord, the positive result in the dissociated spinal neurons may suggest that the present experimental paradigm is suitable for studying participation of external stimuli in the plastic changes in the CNS. Less
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