The basic hypothesis of this application is that central cholinergic signaling constitutes an important modulatory control-mechanism for the fine-tuning of pre- and postsynaptic excitability in the CNS. The goal of proposed studies is to ascertain how nicotinic acetylcholine receptors (nAChR) influence synaptic activity at specific CNS relays involved in nociception, attention and arousal. Unraveling the effects of acetylcholine (ACh) in the brain is complicated by the diffuse nature of cholinergic projections and the wide variety of ionotropic (nicotinic) and metabotropic (muscarinic) ACh receptors. Further complexity is indicated by evidence that this diverse array of ACh receptors can be targeted to axonal and/or soma-dendritic domains. Hence, synaptically released ACh can directly and indirectly alter neuronal excitability by activation pre- and postsynaptic nAChR-linked mechanisms. Finally, the magnitude and duration of nAChR-mediated effects are also subject to fine-tuning by endogenous modulators as common as divalent cations and as exotic as snake toxin-like peptides. Despite the daunting nature of the task, ascertaining how cholinergic pathways participate in shaping CNS outputs is essential to understanding functions as basic as appetite, arousal, sleep, smell and pain, and others as elaborate as memory, motivation and graceful, planned movements. The current proposal: (1) determines the role of specific nAChR subtypes in tuning glutamatergic and GABA-ergic transmission at selected cholinoceptive relays and (2) probes the mechanisms by which the activation and inactivation of native nAChRs may be modulated at intracellular and extracellular sites. In this regard we assess the contribution of individual nAChR subunits to presynaptic nAChRs by comparing transmission at cholinoceptive synapses in normal animals with those from mutant mice lacking particular alpha or beta subunits. A detailed molecular dissection of specific nAChR subunits and sequence domains potentially involved in the modulation of nAChR channels by intracellular calcium and by the endogenous toxin, lynx1, examines both recombinant and native nAChRs. The effects of these modulators on native nAChRs will be examined at identified synapses invitro and, ultimately, by comparing normal mice with animals genetically modified to either lack or overexpress lynx1.
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