Treatment of neuropathic pain by transfection of mutant NMDA receptor gene to the spinal cord of the rat

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As a preliminary study, we planed to study the effects of mutant NMDA receptor expression on opioid tolerance of cultured cells.N616R CDNA, 616th codon from N-terminal of NR1 cDNA, which is one of a NMDA subunit cDNA, has been mutated from AAC to CGC, was ligated into pcDNA3.1 vector. NR2A cDNA, which is a cDNA of another NMDA receptor subunit, was also ligated into pcDNA3.1 vector.Neuroblastoma x glioma(NG108-15) hybrid cells were groum as monolayers in Dulbecco's modified Eagle's medium, containing 10 % fetal bovine serum. The cells were treated with control medium or medium containing delta-selective agonist (D-Pen2, D-Pen5)-enkephalin (DPDPE) for 4 to 12 hours. Thereafter, the cells were challenged with medium containing 10 μM forskolin and 500 μM 1-methyl-3-isobutylxanthine for 10 minutes. Production of cAMP was measured by enzymeimmunoassay. So far, DPDPE treatment did not cause forskolin stimulaeted hyper production of CAMP, which we planed to use as a measure for the opioid tolerance.