| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1989: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Choline acetyltransterase (ChAT), the biosynthetic enzyme for ACh, is currently the most specific marker for the cholinergic neuron. cDNAs for ChAT were isolated from spinal cords of rat, mouse and human.
To investigate the development of the cholinergic neuron histochemically, we introduced molecular biological techniques into our anatomical studies. One method involves expression of ChAT protein from cDNA for rat ChAT introduced into Echerichia coli and the production of antisera against the protein. Using an antiserum obtained, immunohistochemistry (IHC) was established. The second method involves the development of an in situ hybridization (ISH) histochemistry, which enables the precise localization and indentification of individual cells in which a particular gene is transcribed. We have succeeded in detecting mRNA for ChAT of rat and human.
Using IHC and ISH, we clarified the postnatal development of the cholinergic neuron in the rat CNS. During the course of this study, we found a transient appearance of ChAT-immunoreactive neurons in the layer VI of the neocortex between postnatal day 2 and day 13.
This phenomenon is suggested to be due to the change of phenotype in these neurons rather than the death or migration of the neurons.
We have also succeeded in detecting mRNA for ChAT in motoneurons in the spinal cord of the human embryo, and this study will be continued.
On the other hand, molecular genetical analyses of ChAT gene revealed that structures of mRNA differ between the fetal and adult mouse spinal cords and there are several forms of mRNAs generated by a differential splicing in the adult mouse spinal cord. In addition, we have succeeded in cloning cDNAfor human ChAT and demonstrated that it is far larger than those for rat and mouse ones.
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