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 th....

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