Mapping of Presynaptic Nicotinic Acetylcholine Receptors Using Fluorescence Imaging of Neuritic Calcium

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Neuronal nicotinic receptors (nAChRs) appear to function at both pre- and postsynaptic sites, to modulate the release of neurotransmitter, and to mediate synaptic transmission, respectively. Localization of functional nAChRs at presynaptic structures has only been possible under the best of circumstances where the presynaptic structure is very large allowing direct nAChR channel recording. We report here a novel and simple method that allows the visualization of stimulus-evoked changes in Fura-2 fluorescence in the presynaptic structures of essentially any neuron type in vitro. Following ‘loading’ of all neurons by incubation with the calcium-sensitive dye, Fura-2-AM, we selectively reduced the fluorescent signal in the postsynaptic neuron by injecting the Fura-2 quenching agent, Mn2+, into the postsynaptic neuron. After quenching, nicotine treatment elicits calcium transients that can be observed in spatially distinct regions of neurite bundles contacting the Mn2+-infused neuron. Thus, the approach described allows one to readily map the distribution of activated nAChRs on presynaptic inputs in vitro.