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J Vis Exp. 2019 Jan 25;(143). doi: 10.3791/58873.

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine.

Author information

1
Department of Pharmacology, Northwestern University Feinberg School of Medicine.
2
Department of Physiology, Northwestern University Feinberg School of Medicine.
3
Janelia Research Campus, Howard Hughes Medical Institute.
4
Department of Pharmacology, Northwestern University Feinberg School of Medicine; drenan@northwestern.edu.

Abstract

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

PMID:
30735191
DOI:
10.3791/58873

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