| Project/Area Number |
11680794
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neuroscience in general
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| Research Institution | Fujita Health University |
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Principal Investigator |
MIYACHI Ei-ichi School of Medicine, Fujita Health University Professor, 医学部, 教授 (90129685)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAUCHI Masamitsu School of Medicine, Fujita Health University Assistant Professor, 医学部, 講師 (30278303)
HIDAKA Soh School of Medicine, Fujita Health University Assistant Professor, 医学部, 講師 (00228735)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | retina / brain / gap junction / connexin family / connexin 36 / RT-PCR / Western blot / intercellular communication / 細胞間連絡 |
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| Research Abstract |
Connexins constitute the protein subunits of gap junctions. In the present study the expression and localization of neuronal connexins participating in electrotonic synapses were examined in adult mammalian retina and brain regions where gap junction networks of neurons are present. Connexin 36 (Cx36), a candidate for neuronal connexin, was cloned to amplify a cDNA by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA in Wister rat retina. To define cellular origin of Cx36 expression, antibodies were raised in rabbits directed against a synthetic peptide corresponding to the predicted cytoplasmic loop of the Cx36 with the 321 amino acid alignment, deduced from the Cx36 cDNA sequence. The affinity-purified antibodies recognized a 36-kDa protein in tissue extract from the retina, olfactory bulb, hippocampus, neocortex, cerebellum, medulla and spinal cord in Western blots. Cx36-immunoreactivity was found as numerous puncta in the inner plexiform layer of the retina an
… More
d in the brain regions by immunocytochemical labeling. We identified transient expression of Cx36 mRNA and proteins when the rat Cx36 cDNA subcloned into a vector was introduced into COS cells, and these transfected cells formed functional intercellular channels between the cells. Somata and dendrites from amacrine and ganglion cells in the retina, from nonpyramidal cells in the hippocampus and neocortex, and from granular cells in the olfactory bulb demonstrated cytoplasmic immunoreactivity. Developing and adult brain regions including the neocortex were shown to express certain level of Cx36 mRNA transcript by RT-PCR analysis. Cx36-intercellular channels constructing gap junctions may provide extensive spread of neuronal signals through coupled neurons in the brain as well as in the retina. Permeability of gap junctions is regulated by phosphorylation of connexin proteins via activation of protein kinases. We predicted potential phosphorylated sites in the Cx36 protein. Study of phosphorylation of the Cx36 protein is necessary for regulation of intercellular communication between neurons. Less
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