Electrophysiological methods will be used to study the actions of opioids, cocaine and nicotine on individual rat brain neurons in vitro. Adult rats will be anesthetized and then killed; thin slices of brain tissue will be maintained at 37 degrees C in a physiological solution to which drugs and other substances can be added. The purpose of these experiments is to determine the effects of these drugs of abuse of single neurons of the rat brain and thus to make inferences about their actions in man. Studies will be performed on cells of the ventral tegmental area (VTA), nucleus accumbens, and locus coeruleus . Six main types of experiment are proposed. First, calcium currents will be recorded using the whole-cell configuration of the patch clamp method from neurons of the rat locus coeruleus neurons; the actions of opioids selective for mu, delta and kappa receptors will be studied. Second, the role of G-proteins and kinases in coupling opioid receptor to potassium and calcium channels in locus coeruleus neurons will be studied by introducing antibodies, toxins and other molecules into the cell from the patch clamp electrode. Third, the actions of opioids will be measured on potassium and calcium currents in locus coeruleus neurons that have been continuously or intermittently exposed to morphine in vitro during 24 - 48 hr. These experiments on the rat locus coeruleus are a continuation of present work and seek to determine the molecular events that immediately follow opioid receptor activation, and the changes that occur as a result of long term agonist exposure. Fourth, using intracellular recording methods principal (dopamine-containing) and secondary cells of the VTA will be distinguished, and the actions of opioids selective for mu, delta and kappa receptors will be studied on ion conductances of both types of cell, as well as on GABA-mediated and glutamate-mediated synaptic potentials. Fifth, the effectiveness of cocaine to inhibit dopamine uptake under physiological circumstances uptake will be studied by recording dopamine-mediated synaptic potentials in the VTA and the nucleus accumbens. Sixth, the action of nicotine on neurons of the VTA and nucleus accumbens will be studied both by intracellular microelectrode recording and by patch-clamp recording, particularly with respect to the receptor type and differences between principal and secondary cells.
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