LOCALIZATION OF RECEPTOR ACITIVITY FOR DRUGS OF ABUSE

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DESCRIPTION: (Applicant's Abstract) A novel technique has been developed using [35S]GTPgS autoradiography to localize receptor-activated G-proteins. This technique is based upon previous studies using agonist-stimulated [35S]GTPgS binding, in the presence of excess GDP, to measure receptor-activated G-proteins in membranes. The advantage of [35S]GTPgS autoradiography over other techniques is the ability to localize functionally active G-proteins. The technique has been used to demonstrate G-proteins activated by several receptors: opioid, cannabinoid and GABA-B. [35S]GTPgS autoradiography has also been used to identify ORL1 peptide-stimulated [35S]GTPg binding. This demonstrates another advantage of the technique; since [35S]GTPgS is the radioligand used, any unlabeled agonist can be used in this technique. [35S]GTPgS autoradiography has also been used to examine G-protein activity following chronic drug treatment. In these studies, chronic morphine treatment decreased mu opioid-stimulated [35S]GTPgS binding in specific brainstem nuclei: the dorsal raphe nucleus, locus, coeruleus, parabrachial nucleus and commissural nucleus tractus solitarius. The proposed studies will begin with experiments to develop [35S]GTPgS autoradiography for additional receptor systems. Studies will then be performed to examine the effects of chronic cannabinoid or cocaine administration on receptor activation of G-proteins. Initially, studies will examine cannabinoid and dopamine-stimulated [35S]GTPgS binding, however, subsequent studies will examine other receptor systems as well. The third series of experiments will use a novel anatomical approach in which specific regions are lesioned, then [35S]GTPgS autoradiography is performed to 1) identify the origin of afferents to the nucleus and 2) determine whether the receptors on those fibers are pre- or post-synaptic. These studies will focus on the ORL1 and cannabinoid systems, since little anatomical information is available regarding these systems. This project will provide an excellent opportunity for Dr. Sim to utilize her neuroanatomical training, while developing training in the areas of signal transduction and pharmacology. Dr. Sim will conduct this research under the mentorship of Dr. Childers, an established investigator in the field of signal transduction mechanisms of drug of abuse. In addition collaborations have been established with members of the Neuroscience of Drug Abuse Research Center, with faculty specializing in behavior, pharmacology, physiology and molecular biology. This project will allow Dr. Sim to develop into an independent investigator trained in anatomy, pharmacology and signal transduction.