Accurate quantification of the mRNA of NMDAR1 splice variants measured by competitive RT-PCR.

N-methyl-D-aspartate receptors (NMDAR) belong to the subclass of ionotropic glutamate receptors and are widely distributed in the vertebrate brain. Molecular cloning has revealed the existence of six NMDAR subunits: one NMDAR1 (NR1), four different NMDAR2 (NR2A-D) and one NMDAR3A (NR3A). Alternative splicing of the single NR1 gene generates eight isoforms with distinct functional properties [M. Hollmann, J. Boulter, C. Maron, L. Beasley, J. Sullivan, G. Pecht, S. Heinemann, Zinc potentiates agonist-induced currents at certain splice variants of the NMDA receptor, Neuron 10 (1993) 943-954 [8]; R.S. Zukin, M.V.L. Bennett, Alternatively spliced isoforms of the NMDAR1 receptor subunit, TiNS 18 (1995) 306-313 [20]]. Despite the progress made in the functional analysis of NMDARs the molecular architecture of this receptor remains to be elucidated. In situ hybridization studies have already indicated that splicing of the NR1 gene is regionally regulated in the rodent brain, which may contribute to functional diversity of NMDARs in distinct brain areas [D.J. Laurie, P.H. Seeburg, Regional and developmental heterogeneity in splicing of the rat brain NMDAR1 mRNA, J. Neurosci. 14 (1994) 3180-3194 [10]; D.G. Standaert, C.M. Testa, A.B. Young, J.B. Penney Jr., Organization of N-methyl-D-aspartate glutamate receptor gene expression in the basal ganglia of the rat, J. Comp. Neurology 343 (1994) 1-16 [18]; M. Hollmann, S. Heinemann, Cloned glutamate receptors, Ann. Rev. Neurosci. 17 (1994) 31-108 [9]]. Since in situ hybridization techniques do not allow accurate quantification of distinct NR1 splice variants and are also very time-consuming, an accurate and sensitive competitive RT-PCR assay was developed. This method was then used to study the distribution of three NR1 splice variants in the rat brain, and the results are compared with former in situ hybridization studies.