| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
We have developed a system for the culture of chick dissociated chick cerebral neurons in which glutamatergic synaptogenesis proceeds with the passages of embryonic equivalent days (the sum of the embryonic age and days in vitro). In general, glutamatergic synapses contain two types of ionotropic glutamate receptor, N-metyl-D-aspartate receptors (NMDARs) and non-NMDA receptors. Whereas, we showed that the ratio of these receptors at synaptic sites was controlled by the developmental stage of postsynaptic neurons. Next, we confirmed that long-term potentiation (LTP) was induced in this culture system after brief exposure to Mg^<2+> -free medium. The potentiation was inhibited by a specific antagonist of NMDARs and by inhibitors of protein and RNA synthesis. In the potentiated neurons, synaptic transmission increased in terms of the frequency of miniature excitatory postsynaptic currents but not in terms of amplitude. In addition, a diffusible molecule (s) that promoted the potentiation
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appeatred to be involved in conditioned medium. Then, we have established four methods to isolate differentially expressed cDNAs. At first, the subtraction method accomplished by avidin-biotin binding and the differential display system were attempted, however, both methods were found out to have a high level of false positives. Secondarily, we tried the suppression subtractive hybridization (SSH). The SSH method overcomes the problem of differences in mPNA abundance by incorporating a hybridization step. We demonstrate the effectiveness of this method by northern blot analysis. Finally, we have introduced a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In RLCS,mRNA is simply converted to cDNA without PCR amplification, false positives were excluded and the gel spots were obtained with higher reproducibility. Using the latter two methods, we are now isolating differentially expressed genes depending on the LTP. Less
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