Ultrastructural localization of the alpha 4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra

The distribution of the alpha 4-subunit of the neuronal nicotinic acetylcho line receptor (nAChR) in the rat brain was examined at light and electron m icroscopy levels using immunohistochemical staining. In the present study w e demonstrate the specificity, in both tissue homogenates and brain section s, of a polyclonal antibody raised against the rat nAChR alpha 4-subunit. T he characterization of this antibody involved: (1) Western blot analysis of rat brain homogenates and membrane extracts from cells previously transfec ted with diverse combinations of neuronal nAChR subunits, and (2) immunohis tochemistry using transfected cells and rat brain tissue. At the light microscope level, the alpha 4-subunit-like-immunoreactivity (L I) was widely distributed in the rat brain and matched the distribution of the alpha 4-subunit transcripts observed previously by in situ hybridizatio n. Strong immunohistochemical labeling was detected in the mesencephalic do paminergic nuclei. The nAChRs in this region are thought to be responsible for the modulation of dopaminergic transmission. The neurotransmitter ident ity of alpha 4-immunolabeled neurons in the substantia nigra pars compacta (SNpc) and the ventral tegmental area was thus assessed by investigating th e possible colocalization of the nAChR alpha 4-subunit with tyrosine hydrox ylase using confocal microscopy. The double labeling experiments unambiguou sly indicated that the alpha 4-subunit-LI is present in dopaminergic neuron s. At the electron microscope level, the neurons in the SNpc exhibited alpha 4 -subunit-LI in association with a minority of postsynaptic densities, sugge sting that the alpha 4-subunit may be a component of functional nAChRs medi ating synaptic transmission between midbrain cholinergic neurons and mesenc ephalic dopaminergic neurons.