Opiate drugs affect nervous system development by disrupting endogenous opioid systems (endogenous opioids and opioid receptors). Opiates can profoundly inhibit growth. Despite wide-spread licit and illicit use, the developmental mechanisms of opiate action are not understood. This proposal examines the developmental neurobiology of opiate drugs with abuse liability. Interrelated cell and molecular biological experiments will primarily use primary cultures of highly purified populations of cells from the mouse cerebellum and cerebral cortex to explore how opiates directly regulate brain growth. Growth, and opioid receptor and gene expression, will be studied in identified populations of dividing neuroblasts (cerebellar external granular layer cells) and glia (i.e., astrocytes, oligodendrocytes, and microglia) and their progenitors, in vitro, and to a limited extent in vivo. Alterations in growth will be demonstrated to be opioid receptor specific by showing that they are dose-dependent, antagonist-reversible, and stereospecific.
Specific aim 1 will determine the effects of opiates on the generation (proliferation and survival) of identified populations of neural cells.
Specific aim 2 will identify which opioid receptor types mediate growth. Growth will be manipulated using prototypic opioid receptor agonists and antagonists for both traditional mu (mu), delta-, and kappa- as well as non-traditional """"""""immature', opioid receptor types and subtypes. Opioid receptors will be localized in developing neural cells using (i) a wipe-count method and radioligand autoradiography, (ii) fluorescent-labeled ligands and scanning laser confocal microscopy, and (iii) in situ hybridization for opioid receptor mRNA.
Specific aim 3 will determine whether dividing cells intrinsically express opioids by examining opioid gene expression in identified cells in culture using in situ hybridization and immunocytochemistry. The production of opioids by dividing cells is proposed as an autocrine/paracrine mechanism of growth regulation, and is likely to be an important site for opiate drug action. Determining the direct mechanisms of action is crucial towards understanding the etiology of opiate-dependent abnormalities in neural and behavioral development. Our hypothesis is that opiates inhibit nervous system maturation by affecting the production of neurons and/or glia. The results will obtain a unified picture of how opiate regulate growth, correlating developmental effects with cellular sites of opioid gene and receptor expression. This study will have far- reaching significance towards understanding the basic mechanisms by which opiates regulate nervous system growth, and perhaps towards designing therapeutic interventions to prevent/correct developmental abnormalities in children resulting from maternal drug abuse.
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