The functions of nicotinic acetylcholine receptors (AChRs) in the nervous system are almost completely unknown. One of the most abundant AChRs in both the CNS and PNS is a species that binds alpha-bungarotoxin (alphaBgt), contains alpha7 subunits, and has a high relative permeability to calcium. This proposal examines the function of such receptors (alphaBgt-AChRs) in a simple vertebrate neural circuit. The experiments use whole-cell patch clamp on neurons in situ to test the hypothesis that alphaBgt-AChRs generate most of the synaptic current despite having a perisynaptic location. Patch clamp recording from presynaptic calyces will be used to test the hypothesis that presynaptic alphaBgt-AChRs also are present and modulate transmitter release. Subunit-specific monoclonal antibodies (mAbs) will be used to identify presynaptic AChR species, determine their subunit composition, and visualize their locations via confocal immunofluorescence. Sharp electrode intracellular recording-will be used to determine whether alphaBgt-AChRs increase synaptic efficacy and support high frequency transmission through the ganglion. Application of alphaBgt in vivo, together with surgical manipulations, will be used to determine whether the receptors alter neuronal survival, synaptic development, or receptor distribution during embryogenesis. A major goal will be the development and use of strategies for manipulating the levels of neuronal AChR gene products in vivo and in cell culture. The approaches will include transient transfection of postmitotic neurons in culture and retroviral infection of embryos in vivo as two vehicles for introducing epitope- tagged mutant constructs, dominant negatives, and targeted ribozymes. The strategies will be employed to alter patterns of AChR expression and discover receptor roles in the development and function of identified neural circuits. The biomedical relevance of neuronal AChRs is clear: they must participate in the powerful addictive quality of nicotine responsible for vast numbers of chronic smokers world-wide. Neuronal AChRs have also been implicated in a range of brain functions including state of arousal, level of attention, and short-term memory formation. AChR changes have been reported in patients suffering from Alzheimer's disease, Parkinson's disease, and other cognitive and behavioral disorders. The studies outlined here represent basic research intended to illuminate the role of nicotinic receptors in the nervous system. More broadly, they may provide new insight into fundamental aspects of synaptic function and lay groundwork for considering therapeutic strategies involving nicotinic receptors.
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