Opiate drugs, as well as endogenous opioids (i.e., the body's own morphine- like substances), inhibit brain development in children. During development, components of endogenous opioid systems (i.e., endogenous opioids and opioid receptors) are present in high levels in many growing neural cells and tissues, and are likely to be the target of opiate drug action. By experimentally manipulating opioid-opioid receptor interactions it is possible to dramatically retard or accelerate neural and behavioral maturation. Despite the recognized importance of opiates in modifying growth, the mechanisms of their action are poorly understood, e.g., the specific developmental events that are influenced, the opioid-receptor containing targets, the critical periods of opioid sensitively, and the identity of the specific exogenous and endogenous opioids that influence maturation are not known. Our strategy is to explore the cellular and molecular mechanisms by which opiate drugs with abuse liability regulate the growth of the central nervous system. Our hypothesis is that opiates will selectively inhibit certain event(s) during neuro-ontogeny (e.g., cell proliferation or a selective aspect of cell differentiation) by acting through a specific opioid receptor (sub)type. To address these problems, organotypic (primary explant) cultures of the mouse cerebellum and spinal cord will be used as a model to directly assess the local role of opiates per se on the development of identified neurons in culture. Neuroblast proliferation will be examined in cerebellar explants using [3H]-thymidine impregnated Purkinje cells in cerebellar explants and motoneurons in explants of spinal cord.
Specific aim 2 will directly effect neural growth in culture.
Specific aim 3 will explore whether opiate drugs influence endogenous opioid gene and peptide expression by using Northern analysis and/or in situ hybridization, and immunocytochemistry. The opiate- dependent changes that are observed will be demonstrated to be specific at the level of the opioid receptor, i.e., dose-dependent, reversible by opioid antagonist, and stereospecific. The proposed studies will be the first to systematically explore each of these fundamental questions by directly assessing the cellular and molecular mechanisms of opiate action in vitro. Considering the ubiquity of opioid involvement in growth regulation, our results will have far- reaching significance towards understanding the basic mechanisms governing neuro-development. Moreover, these findings may provide the critical insight needed to design therapies to prevent correct developmental abnormalities in offspring resulting from maternal drug abuse.
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