Ultrastructural localisation of neuronal nicotinic acetylcholine receptor alpha4 subunit in rat nigro-striatal neurons

Somato-dendritic and presynaptic nicotinic acetylcholine receptors (nAChR) modulate dopamine release from nigro-striatal neurons. There is pharmacological evidence for α4* and α3* nAChR subtypes at striatal dopaminergic terminals, but the distribution and localisation of these subunits remains unclear. Questions such as the co-localisation or segregation of α3* and α4* subunits in the same or different neuronal populations, respectively, can be answered by analysing the distribution of subunits at the ultrastructural level.
As a first step, the α4 nAChR subunit has been localised by immunocytochemistry using antibodies targeted to both intracellular and extracellular epitopes. Tyrosine hydroxylase (TH) immunoreactivity was examined in parallel to identify dopaminergic neurons. At the light and confocal microscopy level, α4 and TH immunoreactivities co-localised in cell bodies of nigro-striatal dopaminergic neurons and in striatal synaptosomes. The subcellular distribution of the α4 subunit in relation to the local neurochemical environment has been investigated, using immuno-electron microscopy techniques. Double pre-embedding immuno-labelling, using diaminobenzidine to visualise TH and gold particles for the α4, confirmed the presynaptic location of the α4 nAChR subunit in TH positive terminals, supporting the pharmacological data. TH positive nigral cell bodies and proximal dendrites were also densely labelled for the α4 subunit. These techniques facilitate analysis of the comparative distribution of α4 and α3 nAChR subunits within nigrostriatal neurons.