External Excitation of One-Dimensional Patterned Neuronal Cultures

Abstract

Source:Stern, S., et al. External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures. J. Vis. Exp. (2017)

This video demonstrates a technique for stimulating line-patterned neuronal cultures with a uniform, unidirectional electric field.

Protocol

1. Imaging of spontaneous or evoked activity in neuronal cultures with fluorescent dyes.

1. Prepare a solution of 50 µg calcium sensitive fluorescent dye (see Table of Materials/Reagents) in 50 µL DMSO (dimethyl sulfoxide).
2. Prepare extracellular recording solution (EM) containing (in mM) 10 HEPES, 4 KCl, 2 CaCl₂, 1 MgCl₂, 139 NaCl, 10 D-glucose, 45 sucrose (pH 7.4).
3. Incubate neuronal culture in 2 mL EM with 8 µL of the calcium-sensitive fluorescent dye solution for 1 h. Protect from light and gently rotate to ensure a homogenous spread of the dye to the cells.

Results

Figure 1: Example Traces of Calcium Transients Imaged During Synchronous Network Bursts. A. An image of neurons that were dyed previous to the experiment with a calcium dye. B. Traces of intensity vs. time of the ROIs in A with the color of the tra.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Calcium chloride , 1M	Fluka	21098	Extracellular recording solution.
D-(+)-Glucose, 1M	Sigma-Aldrich	65146	Plating medium, Extracellular recording solution.
Hepes, 1M	Sigma-Aldrich	H0887	Extracellular recording solution.
KCl, 3M	Merck	1049361000	Extracellular recording solution.
Magnesium chloride , 1M	Sigma-Aldrich	M1028	Extracellular recording solution.
NaCl, 4M	Bio-Lab	19030591	Extracellular recording solution .
Sucrose, 1M	Sigma-Aldrich	S1888	Extracellular recording solution.
Electrodes wires	A-M Systems, Carlsborg WA	767000	Electric stimulation of neuronal cultures.
Signal generator	BKPrecision	4079	Shaping of the electric signal.
Amplifier	Homemade		Voltage amplification of the signal from the signal generator to the electrodes.
Power supply	Matrix	MPS-3005 LK-3	Power supply to the sputtering machine.
Platinum wires 0.005'' thick; A-M Systems,	Carlsborg WA	767000	Electric stimulation of neuronal cultures.