| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1994: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1993: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
In order to understand the cerebral cortex in a bottom-up manner, we have to elucidate the detail of the intrinsic circuitry in the cerebral cortex. However, since earlier Golgi impregnation studies, there have been no significant advancement in this field of study. Electron microscopic studies revealed the microcircuitry such as synapses, but were not suitable for drawing the outline of the circuit architecture of the cerebral cortex. In this study, we aimed at investigating the structure of the intrinsic circuitry of the cerebral cortex by a combined method intracellular recording, biocytin injection and immunocytochemistry using brain slices. First, the membrane properties and synaptic responses were recorded in the cortical slices. After the injection of biocytin, the slices were fixed, cut into sections and processed for immunocytochemistry. The sections were stained for glutaminase, parvalbumin, calbindin or aspartate aminotransferase by triple immunofluorescence method. The impa
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led neurons, which were labeled with avidin conjugated with fluorescent dye, were thereby studied whether they were glutamatergic or GAB Aergic. Glutaminase is a synthesizing enzyme of transmitter glutamate, and present in pyramidal neurons, but not in GAB-containing neurons of the cerebral cortex ( T.Kaneko and N.Mizuno, Neuroscience, vol.61, pp.839-849,1994) .On the other hand, parvalbumin, calbindin and aminotransferdse are localized in GAB Aergic neurons in the cerebral cortex. After the observation under a fluorescence microscope, the biocytin-injected cells were developed blue black by the ABC method with DAB/nickel. Chemically classified cortical neurons such as those expressing substance P receptors or non-phosphorylated form of neurofilament proteins were further immunostained red in the same section by the PAP method with TAPM/p-cresol. The connection of the axons from the intracellularly labeled cells onto the chemically classified neurons were investigated by camera lucida reconstruction. In this way, we could reveal how the electrically and chemically classified neurons were connected with chemically classified neurons (Kaneko et al. , Comp. Neurol. vol.345, pp.161-171,172-184) . We have now found and been investigating the two types of pyramidal cells in the layr VI of the cerebral cortex, glutaminase-positive and glutaminase-negative ones. Less
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