DESCRIPTION (Investigator's Abstract): The hippocampus has a high density of nicotinic acetylcholine receptors(NnAChRs) and rich cholinergic innervation, but the roles of the NnAChRs are not well understood. Using patch-clamp techniques, fluorescent Ca2+ measurements, and NnAChR labelling methods, we have obtained compelling preliminary results to support a working hypothesis for investigation of nicotinic mechanisms in the hippocampus. The applicants hypothesize that a Ca2+ influx initiated by presynaptic NnAChRs enhances the synaptic release of glutamate. Furthermore, intense synaptic activity decreases external Ca2+ and, consequently, diminishes the efficacy of NnAChRs. The aims are to investigate the hypothesis by assessing the roles of NnAChRs and by examining the underlying hippocampal slices are used to provide a more biologically relevant preparation that offers greater experimental versatility. They will determine whether NnAChR activity alters glutamatergic synaptic transmission and synaptic plasticity. Timed local applications of nicotinic agonists will be used to judge whether presynaptic NnAChRs help regulate intra-terminal Ca2+ and, thereby, modulate glutamate release. They will directly test whether NnAChRs can mediate a presynaptic Ca2+ influx by monitoring presynaptic NnAChR currents and intra-terminal Ca2+ in tissue culture and at an intact mossy-fiber terminal in the hippocampal slice. Because other presynaptic terminals in the hippocampus are small, they can only use the relatively large mossy-fiber terminals to monitor intracellular Ca2+ with fura-2 while simultaneously measuring NnAChR currents with patch-clamp techniques. Another influence on the action of NnAChRs occurs because the high current density at an active synapse not only elevates intracellular Ca2+ but also locally lowers extracellular Ca2+. Our preliminary results indicate that physiological decreases in extracellular Ca2+ diminish the efficacy of NnAChRs. In summary, a presynaptic Ca2+ influx initiated by NnAChRs could spur glutamate release. Intense synaptic stimulation, however, decreases external Ca2+, which may turn off the NnAChRs, producing a negative feedback onto glutamatergic activity.