NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones

1 NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+](i)) were determined in individual cultured rat mesencephalic neurones by the f ura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT-PCR. 2 NMDA (1-100 mu M, plus 10 mu M glycine) induced a concentration-dependent increase in [Ca2+](i) (EC50 = 5.7 mu M). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 mu M) and nitrendipine (1 mu M), but depen dent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 mu M), a specifi c antagonist at the glycine binding site on the NMDA receptor, abolished th e NMDA response. 3 Memantine, an open-channel blocker, and ifenprodil, a preferential non-co mpetitive NR1/NR2B receptor antagonist diminished the NMDA effect with an I C50 value of 0.17 and 1 mu M, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively. 4 Agarose gel analysis of the PCR products followed by ethidium bromide flu orescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3' sp lice form without both E21 and E22 exceeded that containing E21 by approxim ately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to appr oximately 1:2:1. NR2D mRNA was also detectable. 5 In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA recepto rs which might be involved in the development of ethanol dependence and wit hdrawal. The high affinity of NMDA to this receptor, its sensitivity to ife nprodil and memantine may suggest that the mesencephalic NMDA receptor comp rises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.