The objective of this research proposal is to study the regulatory mechanisms of brain neurons that are implicated in major mental disorders. The proposed experiments will be done mainly by using primary cultured dopaminergic (DA) neurons from the substantia nigra and the ventral tegmental area of the rat brain. The methods will be electrophysiological techniques combined with molecular biological methods. The first project is concerned with excitation of these DA neurons. Neurotensin produces a slow excitation of DA neurons by activating a non-selective cation channel. The analysis of the ionic mechanism has revealed that this non-selective channel is different from the well-known non-selective ion channels such as the CAN channels (the calcium-activated non-specific cation channels) or the cyclic nucleotide-gated channels. The proposed experiments will shed light on the signal transduction mechanisms of the neurotensin-induced activation of this non-selective channel. The role played by G proteins will be critically evaluated, and the identity of the G protein will be determine. The other project deals with a slow inhibition of the DA neurons by dopamine itself. This self-inhibition results from an activation of an inward rectifier K+ channel by the G protein betagamma- subunits. The project will investigate the mechanisms of the interaction of the betagamma-subunits and the inward rectifier molecule. It is proposed to find a convenient way to transfect primary cultured DA neurons with plasmid cDNA. Then the DA neurons (also HEK293 cells) will be transfected with CDNAs encoding the G protein beta-subunit and the gamma-subunit. The effects of site-directed mutations of the beta- subunit will be evaluated. The investigation will show which regions of the beta-subunit molecule are responsible for the association with and activation of the inward rectifier molecule.
