This program project merges two long-term and actively ongoing interests of this laboratory: the quantitative determination of muscarinic receptors in the brain, and the biochemistry of receptor activation via the phosphoinositide cleavage pathway. These approaches to understanding cholinergic signal transduction in the CNS, the first extracellular and the second intracellular, and here combined to explore experimental models of cholinergic dysfunction. Much of our biochemical knowledge regarding receptor function up to the present has been restricted to the receptor binding of labeled ligands in vitro to membranes, synaptoneurosomes or brain sections following acute or chronic exposure to a neuroactive agent. We here examine the effects of chronic in vivo conditions, such as deafferentation or exposure to drugs, not only upon the regulation of receptor number and affinity, but also on the regulation of the function of PPI-related second messengers, a consequence of receptor activation. These include: the diacylglycerol-activated protein kinase C and the mobilization of Ca2+ following inositol trisphosphate release. In vitro models of chronicity involve long-term exposure of neuroblastoma and primary neuronal or glial cultures to various anticholinergic agents, antidepressants and to Li+. A proposed developmental model is based on our observed enhanced coupling of muscarinic receptors to the cell response in fetal and neonatal brain. Possible interaction of muscarinic receptor regulation with other neurotransmitter-effector systems will also be explored. We will also investigate the role of phosphoinositides in presynaptic function, as well as the effects of the various drug treatments on acetylcholine release. Techniques that will be brought to bear upon this problem include 32P-labeled phosphoinositide turnover, (3H)inositol phosphate accumulation, and chemical determination of diacylglycerol and of the phosphoinositides. Inositol phosphate metabolism will be investigated, including the formation and breakdown of inositol trisphosphate and inositol tetraphosphate. Protein phosphorylation under control and receptor-regulated conditions will be examined with particular emphasis on protein kinase C- mediated reactions in synaptic vesicles and growth cones. Changes in receptor number and affinity will be localized via quantitative receptor autoradiography.
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