Mapping of presynaptic nicotinic acetylcholine receptors using fluorescence imaging of neuritic calcium.
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, Mn(2+), into the postsynaptic neuron. After quenching, nicotine treatment elicits calcium transients that can be observed in spatially distinct regions of neurite bundles contacting the Mn(2+)-infused neuron. Thus, the approach described allows one to readily map the distribution of activated nAChRs on presynaptic inputs in vitro.
Girod
Romain
R
Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, 1051 Riverside Drive PI, Annex 807, New York, NY 10032, USA.
Jareb
Mark
M
Moss
Jason
J
Role
Lorna
L
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D016257:Fura-2; D005728:Ganglia, Sympathetic; D008161:Lumbosacral Region; D008856:Microscopy, Fluorescence / Q000379:methods*; D009046:Motor Neurons / Q000166:cytology* / Q000378:metabolism*; D009538:Nicotine / Q000008:administration & dosage; D011978:Receptors, Nicotinic / Q000032:analysis / Q000378:metabolism*; D017661:Receptors, Presynaptic / Q000032:analysis / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013569:Synapses / Q000502:physiology; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology
D011978:Receptors, Nicotinic; D017661:Receptors, Presynaptic; D009538:Nicotine; D002118:Calcium; D016257:Fura-2