The nucleus locus coeruleus is a rather uniform collection of noradrenaline containing nerve cells. In the rat, there are about 1500 on each side, approximately half of the noradrenaline containing cells in the brain. These cells have been implicated in several of the acute actions of opioids, and their increased activity during withdrawal from opioids may contribute to several of the physical signs of withdrawal. Our previous work has determined that rat locus coeruleus neurones express Mu receptors; there is no evidence for delta or kappa receptors. Activation of the Mu receptors on these cells results in an increase in membrane potassium conductance. The objective of the proposed experiments is to determine the properties of this potassium conductance at the molecular level, to determine how this conductance is linked to the opioid receptor through a guanine nucleotide binding protein (G-protein), and to investigate the changes in this coupling which accompany, or cause, tolerance to the action of opioids. Electrophysiological methods will be used (whole cell recording with intracellular perfusion, and single channel recording) on cells dissociated from the locus coeruleus of rats and maintained in culture for periods of up to several days. The effects of full and partial agonists at the Mu receptor, with and without experimental manipulation of the coupling G protein will be studied. These experiments will be carried out on neurons removed from normal rats, neurons removed from morphine- treated rats, and on neurons removed from normal rats and maintained in primary culture for several days with and without morphine. The experiments form a test of the hypothesis that the Mu receptors couple directly through a G protein to a potassium channel, and impairment of this coupling is the primary defect in opioid tolerance.
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