The specific aim is to culture dissociated neurons from the locus coeruleus (LC), substantia nigra (SN) and dorsal raphe (DR) and study of the action of cocaine on single isolated neurons. With isolated cells the direct action of cocaine on membrane properties and exogenously applied transmitters can be studied quantitatively since diffusion barriers and endogenous transmitters will be limited. Agonists can be applied in known concentration close to the cell. The effect that cocaine has on the kinetics of ligand activated conductances will be studied to determine if cocaine changes the affinity of the receptor for the transmitter. In addition, the use of cultured cells permits whole cell recording with patch electrodes such that more control of the intracellular ion content and second messengers is gained. The long-term goal is to use the strengths of each; the brain slice and isolated cell preparations to study action of drugs of abuse at the cellular level. In the work proposed here, the acute and chronic actions of cocaine will be determined in cells from relatively homogenous nuclei (LC, SN and DR). The knowledge gained in these simple nuclei will be applied to the action(s) of cocaine in more complex areas such as the ventral tegmental area, nucleus accumbens and prefrontal cortex. Studies from cells taken from animals which have self- administered cocaine may lead to an understanding of some of the changes which cause or result from chronic cocaine use. Whole cell recordings will also eventually be used to study the changes which occur with other drugs of abuse such as opioids. Such recordings from LC neurons with patch electrodes are vital to determine the nature of the reduction in the opioid 'spare-receptor' population following chronic opioid treatment. This award will afford the PI the time to learn and become competent with the tissue culture and whole cell recording methods. The proposed experiments are a natural progression of those which are in progress in the slice preparations. The knowledge and experience gained by the work carried out on LC and DR neurons in the slice over the past 8 years will be directly applicable to work on isolated cells. This coupled with the expertise of members of the Vollum Institute in recording from cultured neurons provide an optimistic setting from which the PI can learn and apply this knowledge to the understanding of cellular mechanisms of drug abuse.
