Opioid receptors are targets of narcotic analgesic drugs. Implicated in their category these drugs cause serious side effects of tolerance and physical and psychological dependence. Two general mechanisms are thought to underlay tolerance. One is an associative or conditioned form that involves alteration of neuronal networks. The other is a non-associative form where adaptive regulation occurs in the opioid-sensitive neurons during continued uses of opioid drugs. This proposal aims to study the basic regulation of opioids that may involve the non-associative tolerance. The project will focus on mechanisms of receptor desensitization and internalization, the two important steps that are believed to mediate the early stages of tolerance. Single neurons from locus coeruleus (LC) cultures will be used as a model system. It has been proposed that noradrenergic neurons in LC contain only mu opioid receptors (MORs). This receptor subtype has been recognized to be important for analgesia, tolerance and dependence to opioids. MOR is a member of G protein-couple receptors (GPCRs) superfamily. Upon activation, MOR becomes a good substrate for phosphorylation by G protein-coupled receptor kinase (GRK), the recruitment of beta-arrestins and subsequent association with clathrin-coated pits. These sequential events are believed to not only imtiate receptor internalization but are also involved in desensitization. An important question however remains whether there is a close relationship between receptor internalization and desensitization. By using a series of opioid agonists the hypothesis that desensitization and internalization are linked in a serial pathway can be tested directly. In the past, this hypothesis has been tested in recombinant systems but little or no work has been done in neuronal preparations. This proposal will use electrophysiology and fluorescent microcopy to study the relationships of MOR activation, desensitization and internalization in dissociated cell cultures of LC neurons. There are three distinct advantages of using primary cultures over previous work in brain slices. First, high-resolution imaging can be done because the preparation is only a single cell thick. Second, the pharmacological analysis will be improved because the rapid application and removal of opioid agonists are possible. Third, """"""""autapses"""""""" are formed in these cultures such that a single neuron innervates itself through recurrent axon collaterals. In this way presynaptic inhibition of neurotransmitter release by opioids can be studied. An array of opioid agonists will be tested for their efficacy in inducing MOR desensitization and internalization. The most critical plan in this study will be the simultaneous measurement of MOR activation, desensitization and internalization by using electrophysiological recording and a fluorescent agonist in a single neuron. The results from this study will provide better understanding in the early events that may lead to tolerance to opioids. The work could offer insights to the development of more effective and safer analgesics for clinical therapy.
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