5 An antibody specific for the neuronal nicotinic acetylcholine receptor alpha 4 subunit: High levels of expression in monoaminergic neurons

Abstract

In the brain, neuronal acetylcholine receptors (nAChRs) comprise two major types of subunits, a and p subunits. Current understanding of nAChR expression and distribution in the brain relies on localization of individual subunit transcripts or in the localization of protein based on immunohistochemistry. Different lines of evidence indicate that, in the central nervous system, nAChRs composed of a4 and e2 subunits are a majority among native nicotinic receptors. We have attempted the immunohistochemical detection of the a4 subunit using a subunit-specific antibody. We have used a polyclonal antibody raised against a peptide sequence corresponding to the cytoplasmic loop region of the rat a4 neuronal nicotinic acetylcholine receptor, comprising residues 573-592 of the cDNA deduced sequence. First, we assessed the specificity of this antibody using dil’ferent approaches. Western blot analysis of rat brain homogenates and membrane extracts of cells transfected with a4 and p4 cDNAs of nAChR subunits revealed a band of labelled protein with a molecular mass of 70 kDa. d&es, with varying intensity. The distribution of a4 like-immunoreactive (cx4-IR) cell bodies correlated well with previous in situ hybridisation results. Particularly strong labelling was detected in the monoaminergic cell groups in the brainstem. Thebe included the dopaminergic cell areas such as the substantia nigra pars compacta and ventral tegmental area, the serotoninergic neurons of the rauhe nuclei and the noradrenereic neurons of the locus coeruleus. Transmitter identity of the r&positive neurons wab tested by double-lahelling sections of the rat midbrain both for a4 and for tyrosine hydroxylase, the rate-limiting enzyme responsible for the synthesis of dopaminc, or for a4 and serotonin.