Home › Neuroscience › Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine
Neuroscience JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/58873-v
What you'll learn
  • ✓ Prepare photoactivatable nicotine for uncaging experiments
  • ✓ Image neurons using 2-photon laser scanning microscopy
  • ✓ Calibrate uncaging laser galvanometers for precise stimulation
  • ✓ Combine nicotine uncaging with patch clamp recordings
Protocol

Biopharma Insights This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

Difficulty
advanced
Total time
~1 day per sample (including slice preparation and calibration)
Model organism
Mouse (C57BL/6J)
Biosafety
BSL-1

Steps

1
Prepare photoactivatable nicotine (PA-Nic)

Synthesize or reconstitute PA-Nic and prepare it for use in uncaging experiments on brain slices.

▶ 00:39
2
Image neurons with 2-photon microscopy

Visualize neurons in mouse brain slices using 2-photon laser scanning microscopy to identify cells for recording.

▶ 03:23
3
Calibrate uncaging laser and pair with recordings

Align galvanometers for precise laser flash photolysis of PA-Nic and synchronize with electrophysiological patch clamp recording.

▶ 05:28
4
Perform nicotine uncaging and record responses

Apply laser flash photolysis to uncage nicotine while recording nAChR currents and imaging cellular morphology.

▶ 07:47
5
Analyze results and conclude

Interpret representative imaging and electrophysiological data to assess nAChR function in brain slices.

▶ 09:47
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