|Budget Amount *help
¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1998 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Glutamate (Glu), a major excitatory neurotransmitter in the central nervous system (CNS), has been known to enhance synaptic efficacy that is most pronounced in the hippocampus, indicating that Glu has a key role in learning and memory function. Recently, on the other hand, the inhibitory Glu receptor (GluR) which hyperpolarizes membrane potential in the CNS has founded and this finding suggests a new mechanism for modulating excitability in the neuronal networks. However, molecular cloning of the inhibitory GluR has not yet succeeded because of its scattered and limited presence in the CNS.In the previous study we have demonstrated the presence of the inhibitory GluR, which is linked to potassium channel controlled by unknown but probably a Gq protein, in the identified neurons of Euhadra ganglia. The purpose of the present study is to detect the genes coding this inhibitory GluR using the combination technique of patch-clamp recording and RT-PCR analysis in single live neurons.
ing whole-cell recording of the inhibitory response induced by Glu in the identified neuron using patch-clamp method, the cytoplasma of the cell was aspirated into the patch pipette whose internal solution contains required reagents including reverse transcriptase and aligo-T7 to facilitate cDNA synthesis. After the first PCR reaction with sense and antisense primers from subunits of ionotropic type of GluRs (GluR1-4 coding sequence), the amplified DNA was analysed on agarose gel elctrophoresis. But no band corresponding to the GluR1-4 was detected. This result was expected because that the electrophysiological and molecular biological characteristics of the inhibitory GluR was quite different from those ionotropic subtypes of GluRs. On the other hand, in the analysis of amplified DNA followed the first PCR reaction with the primers from metabotropic types (common segment of mGluR1-6), some similarity was founded. These PCR products were subcloned. Following their sequencing, at least two unknown bands were detected. A screening test of cDNA library obtained using these PCR products as a probe is continued.
【平成10年度】問題を解決後、再度既知受容体との相同性を検索した。ionotropic typeのGlu受容体との比較では相同性は認められなかった。一方、metabotropic typeとの比較では、mGluR1〜6に共通するセグメントを基に作製したプライマーでPCR増幅を行ったときに近似のバンドが認められ、これをサブクローニングしシークエンシングしたところ、未知のバンドが少なくとも2つ見つかった。現在このPCR産物をプローブにライブラリーのスクリーニングを行っている。 Less