Ultrastructural localization of the α4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra

The distribution of the alpha4-subunit of the neuronal nicotinic acetylcholine receptor (nAChR) in the rat brain was examined at light and electron microscopy levels using immunohistochemical staining. In the present study we demonstrate the specificity, in both tissue homogenates and brain sections, of a polyclonal antibody raised against the rat nAChR alpha4-subunit. The characterization of this antibody involved: (1) Western blot analysis of rat brain homogenates and membrane extracts from cells previously transfected with diverse combinations of neuronal nAChR subunits, and (2) immunohistochemistry using transfected cells and rat brain tissue. At the light microscope level, the alpha4-subunit-like-immunoreactivity (LI) was widely distributed in the rat brain and matched the distribution of the alpha4-subunit transcripts observed previously by in situ hybridization. Strong immunohistochemical labeling was detected in the mesencephalic dopaminergic nuclei. The nAChRs in this region are thought to be responsible for the modulation of dopaminergic transmission. The neurotransmitter identity of alpha4-immunolabeled neurons in the substantia nigra pars compacta (SNpc) and the ventral tegmental area was thus assessed by investigating the possible colocalization of the nAChR alpha4-subunit with tyrosine hydroxylase using confocal microscopy. The double labeling experiments unambiguously indicated that the alpha4-subunit-LI is present in dopaminergic neurons. At the electron microscope level, the neurons in the SNpc exhibited alpha4-subunit-LI in association with a minority of postsynaptic densities, suggesting that the alpha4-subunit may be a component of functional nAChRs mediating synaptic transmission between midbrain cholinergic neurons and mesencephalic dopaminergic neurons.This research was supported by grants from The College de France, the Association Française contre la Myopathie, the Council for Tobacco Research, Reynolds Pharmaceutics, the European Union (Biomed BMH1-CT94–1060 and Biotech 960236), the French Embassy in Spain, the Ministerio de Educacion y Cultura (PB94–0219-CO2-01), the Comunidad de Madrid (AE00268/95), and the National Alliance for Research on Schizophrenia and Depression.Peer reviewe