Imaging Nicotine-Induced Calcium Signaling along Ventral Hippocampal Axons in Synaptic Co-Cultures

Abstract

Source: Zhong, C., et al., Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons. J. Vis. Exp. (2015)

This video demonstrates how nicotine-mediated activation of nicotinic acetylcholine receptors in the mouse ventral hippocampus results in sustained calcium signaling along the axons.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Calcium Imaging

1. Rinse cultures (5 - 7 days in vitro) quickly with normal HBS, then load cultures with 5 µM Fluo-4 Ca²⁺ indicator (AM ester) and 0.02% Pluronic F-127 in HBS (2 ml) for 30 min at 37 °C and 5% CO₂.
2. Wash out the Fluo-4 solution with HBS (3 times/5 min). Return the cultures to the incubator (37 °C / 5% CO₂) for at least 30 min.
3. Maintain cultures (5 - 7 days in vitro) in an imaging chamber and mount the chamber on a spinning disk confocal microscope. Continuously perfuse (1 ml/min) with HBS cocktail at RT.
4. Collect Fluo-4 fluorescence images of axonal projections from the vHipp micro-slices with a Plan-Apochromat objective (60X water with 1.4 NA, excitation 488 nm, emission 530 nm) and a CCD camera.
5. Collect baseline Fluo-4 fluorescence images (every 10 sec for 2 min) as a pre-nicotine control. Apply nicotine (1 µM) by rapid perfusion (2 ml/min) for 1 min, then wash the nicotine with an HBS cocktail. Keep capturing time-lapse images before, during, and after nicotine application every 10 sec for 30 min.
6. Save all frames of the raw fluo-4 fluorescence images and export them as a series of TIFF images for further analysis.
7. Collect the integrated intensity of fluo-4 fluorescence along vHipp axons before and after nicotine application.
8. Normalize integrated fluorescence intensity with the following equation: ΔF/F₀ =  (F-F₀)/F₀, where F₀ is the background-corrected pre-nicotine integrated fluorescence intensity, and F is the integrated fluorescence intensity along vHipp axons at each time point after nicotine application. Analyze and plot normalized integrated fluorescence intensity.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HBS cocktail for live imaging pH=7.3
NaCl	Sigma	S9888	135 mM
KCl	Sigma	P9333	5 mM
Magnesium chloride	Sigma	M8266	1 mM
Calcium chloride	Sigma	C1016	2 mM
HEPES	Sigma	H3375	10 mM
Glucose	Sigma	G0350500	10 mM
Tetrodotoxin	Tocris	1078	2 µM
Bicuculline	Tocris	131	10 µM
D-AP-5	Tocris	105	50 µM
CNQX	Tocris	1045	20 µM
LY341495	Tocris	1209	10 µM