Nicotinic acetylcholine receptor subunit mRNA expression and channel function in medial habenula neurons

Relationships between nicotinic acetylcholine receptor (nAChR) channel func tion and nAChR subunit mRNA expression were explored in acutely isolated ra t medial habenula (MHb) neurons using a combination of whole-cell recording and single cell RT-PCR techniques. Following amplification using subunit-s pecific primers, subunits could be categorized in one of three ways: (i) pr esent in 95-100% cells: alpha3, alpha4, alpha5, beta2 and beta4; (ii) never present: alpha2; and (iii) sometimes present (approximate to 40% cells): a lpha6, alpha7 and beta3. These data imply that alpha2 subunits do not parti cipate in nAChRs on MHb cells, that alpha6, alpha7 and beta3 subunits are n ot necessary for functional channels but may contribute in some cells, and that nAChRs may require combinations of all or subsets of a3, a4, alpha5, b eta2 and beta4 subunits. Little difference in the patterns of subunit expre ssion between nicotine-sensitive and insensitive cells were revealed based on this qualitative analysis, implying that gene transcription per se may b e an insufficient determinant of nAChR channel function. Normalization of n AChR subunit levels to the amount of actin mRNA, however, revealed that cel ls with functional channels were associated with high levels (>0.78 relativ e to actin; 11/12 cells) of all of the category (i) subunits: alpha3, alpha 4, alpha5, beta2 and beta4. Conversely, one or more of these subunits was a lways low (<0.40 relative to actin) in all cells with no detectable respons e to nicotine. Thus the formation of functional nAChR channels on MHb cells may require critical levels of several subunit mRNAs. (C) 2000 Elsevier Sc ience Ltd. All rights reserved.