NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones.

NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) were determined in individual cultured rat mesencephalic neurones by the fura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT–PCR. NMDA (1–100 μM, plus 10 μM glycine) induced a concentration-dependent increase in [Ca2+]i (EC50=5.7 μM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 μM) and nitrendipine (1 μM), but dependent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 μM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response. Memantine, an open-channel blocker, and ifenprodil, a preferential non-competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 μM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively. Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3′ splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable. In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively. Keywords: Cultured mesencephalic neurones, NMDA receptors, intracellular Ca2+, ethanol, ifenprodil, memantine, fura-2 method, RT–PCR, NR1 splice variants, NR2 subunits Introduction The N-methyl-D-aspartate (NMDA) receptor is essentially involved in a number of important central processes such as neuronal development, long-term-potentiation, and neuronal death (excitotoxicity) (Nakanishi, 1992). Moreover, this receptor mediates some of the acute and chronic CNS effects of ethanol (Harris et al., 1992; Hoffman, 1995; Grant & Lovinger, 1995). At concentrations associated with moderate to severe intoxications [10–100 mM, corresponding to 0.046–0.46% (w/v) in blood] ethanol acutely inhibits NMDA receptor-mediated responses in cultured neurones (Lovinger et al., 1989), brain slices (Gothert & Fink, 1989; Woodward & Gonzales, 1990; Poelchen et al., 1997; Darstein et al., 1998; Nieber et al., 1998), and transfected expression systems (Koltchine et al., 1993; Mirshahi & Woodward, 1995). The NMDA receptor is a ligand-gated ion channel highly permeable to Ca2+. In addition to glutamate, glycine is required as a co-agonist (Kleckner & Dingledine, 1988; Kemp & Leeson, 1993). Several NMDA receptor subunits (NR1, NR2A-D) have been cloned (Monyer et al., 1992; Laurie & Seeburg, 1994; Zukin & Bennett, 1995). The NR1 subunit with eight known splice variants is expressed throughout the brain, while the NR2A-D subunits display regional differences. Xenopus oocyte expression studies have shown that only the NR1 subunit is required to generate functional NMDA receptors and that co-expression with various NR2 subunits results in the assembly of ion channels with conductances that resemble native NMDA receptors. Natively expressed receptors presumably comprise two NR1 subunits (Behe et al., 1995) and at least one member of the NR2 family (Schoepfer et al., 1992). Heteromers containing different NR2 subunits display distinct pharmacological and physiological characteristics (Monyer et al., 1992). For example, ifenprodil blocks the NR1/NR2B heteromer with considerably higher potency than the NR1/NR2A heteromer (Williams, 1993; Lovinger, 1995), while ethanol preferentially affects both NR1/NR2A and NR1/NR2B receptors (Masood et al., 1994; Mirshahi & Woodward, 1995). There is considerable evidence that the ventral tegmental area (VTA)-nucleus accumbens system is implicated in the stimulant and reinforcing properties of ethanol and other drugs of abuse (Harris et al., 1992). Like morphine and nicotine, acutely administered ethanol has been shown to increase the firing rate of dopaminergic principal neurones of the VTA by a mechanism which is not completely understood. Ethanol might directly act on the dopamine neurones or inhibit GABAergic interneurones. In the present study, NMDA receptor function and mRNA subunit expression were studied on cultured rat ventral mesencephalic neurones by means of single-cell microfluorimetry (fura-2 method) and reverse transcriptase-polymerase chain reaction (RT–PCR). According to electrophysiological data, burst firing in dopamine neurones is induced by activation of somatodendritic NMDA receptors (Johnson et al., 1992). Preliminary accounts of the present results were given previously (Scheibler et al., 1998).