ANALYSIS BY POLYMERASE CHAIN-REACTION OF ALPHA-1 AND ALPHA-6 GABA(A) RECEPTOR SUBUNIT MESSENGER-RNAS IN INDIVIDUAL CEREBELLAR NEURONS AFTERWHOLE-CELL RECORDINGS

With the use of the single-cell polymerase chain reaction (PCR), the G ABA(A) receptor subunit mRNA content was analyzed in granule and Purki nje neurons from rat cerebellar slices. We used an experimental protoc ol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-ce ll patch-clamp technique. Based on a computer alignment of the nucleot ide sequence corresponding to alpha 1 and alpha 6 GABA(A) receptor sub units, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of sele ctive restriction sites within the targeted templates allowed us to id entify which receptor subunit mRNAs were coamplified by performing res triction enzyme-mediated cleavage of the amplification products. In al l Purkinje neurons assayed, alpha 1 subunit mRNA but not alpha 6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the alpha 1 and alpha 6 GA BA(A) receptor subunits. A comparison of the results of the PCR amplif ication and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic cur rents that clearly correlate with the presence or the absence of alpha 6 subunit mRNA.