1996 Fiscal Year Final Research Report Summary
Analysis of differentially expressed genes depending on the synaptic long-term potentiation
| Project/Area Number |
07808075
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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 |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
UYEDA Atsuko Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Research assistant, 基礎工学部, 教務職員 (90252634)
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| Co-Investigator(Kenkyū-buntansha) |
KASAI Michiki Department Biophysical Engineering, Faculty of Engineering Science, Osaka Univer, 基礎工学部, 教授 (40022595)
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| Project Period (FY) |
1995 – 1996
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| Keywords | long-term potentiation / cerebral neuron / Dissociated culture / Differential Display system / Subtraction method / Suppression subtractive hybridization / Restriction landmark cDNA scanning |
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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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