Many recent studies have focused on the ontogeny of central opioid systems and their functional roles in development. Although data from in vivo and in vitro studies suggest that opioid peptides influence growth and differentiation, analysis of underlying mechanism in heterogeneous systems is complex. We therefore propose to use a neurochemically identifiable population of neurons, those of the noradrenergic brainstem nucleus, locus coeruleus (LC), as a model with which to examine the possible developmental role of opioid peptides. The maturation of LC and the effects of opioid receptor manipulation, will be examined in vitro in dissociated cells in culture. Morphological development will be examined in cells labelled with [3H] norepinephrine (NE) and with antibodies directed towards the principal catecholamine synthetic enzyme, tyrosine hydroxylase (TH). Indices of functional maturation, including TH mRNA expression and stimulus-evoked [3H]NE release, will also be assessed. In preliminary studies, we have found that chronic treatment of embryonic rhombencephalic cells with Beta-endorphin produces a significant enhancement of LC cell growth, as determined by [3H]NE uptake. The mechanism and pharmacology of this opioid effect will be studied in detail. The potencies of selective opioid receptor antagonists to antagonize this Beta-endorphin effect will be examined, as will the effects of other endogenous opioid peptides. Since the growth-promoting action of Beta-endorphin is diminished in defined medium, we propose to test the hypothesis that opioid effects on LC development are mediated indirectly via glia. We will also test the hypothesis, derived from in vivo data, that opioids potentiate the degenerative effect of catecholamine neurotoxins. In order to determine the cellular specificity of opioid actions on the processes of LC differentiation, the effects of opioid peptides on cultured mesencephalic dopamine neurons and rhombencephalic serotonin cells will also be examined. The use of these dissociated culture models will permit greater control of experimental variables, and analysis of cellular mechanisms of development. These studies are of both physiological and clinical interest, in that they address mechanisms underlying normal brain development and the possible influence of environmental factors on these processes.
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