Studying the Regeneration of Functional Connections between Spinal Cord Slices Using a Multi-Electrode Array

Abstract

Source: Heidemann, M., et al. Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays. J. Vis. Exp. (2015).

The video demonstrates the functional regeneration of propriospinal connections using a multi-electrode array (MEA)-based assay. The spinal cord slices are placed on the MEA and are allowed to fuse. The fused slices are mechanically separated by creating a lesion, inducing propriospinal neurons to grow over the lesion and connect the slices. The electrical activity of the slices is assessed to identify the regeneration of functional connections.

Protocol

1. Preparation of MEAs

Note: MEAs are composed of a glass substrate, micro-fabricated metal electrodes and a SU-8 polymer insulation layer. For the purpose of this study, commercially available MEAs were ordered with a customized electrode array layout (Figure 1 A&B). The 68 electrodes are arranged in a rectangular grid that is split into two zones by a 300 µm wide groove free of electrodes, electrical leads and insulation. Each electrode is 40 x 40 µm in size and they are spaced apart by 200 µm from center to center. Four large ground electrodes are positioned around the r....

Results

Figure 1. Display and Analysis of Spontaneous Activity. (A) Diagram of a MEA. The platinum covered electrodes are depicted in black, the transparent wires in red and the groove in the middle of the MEA in yellow. (B) .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Planar multielectrode array	Qwane Biosciences		custom-made according to the design of our lab
Nutrient medium			For 100 ml
Dulbeccos modified Eagle's medium	Gibco	31966-021	80 ml
Horse serum	Gibco	26050-070	10 ml
distilled, sterile water			10 ml
Nerve growth factor-7S [5ng/mL]	Sigma-Aldrich	N0513	200 µl
			reconstituted in wash solution with 1% BSA
Coating solution
Extracellular matrix gel	BD Biosciences	356230	Must stay cold at all times; dilute 1:50 with medium optimized for prenatal and embryonic neurons
medium optimized for prenatal and embryonic neurons	Gibco	21103-049
Wash solution			[mg/L]
MgCl2-6H2O			100
MgSO4-7H2O			100
KCl			400
KH2PO4			60
NaCl			8000
Na2HPO4 anhydrous			48
D-Glucose (Dextrose)			1000
			According to the protocol on http://www.lifetechnologies.com/ch/en/home/technical-resources/media-formulation.152.html
Extracellular solution (pH 7.4)			[mM]
NaCl			145
KCl			4
MgCl2			1
CaCl2			2
HEPES			5
Na-pyruvate			2
Glucose			5
Chicken Plasma	Sigma-Aldrich	P2366	Lyophilized, reconstitute with wash solution
Micropipette	World Precision Instruments, Inc.	MF28G-5
Thrombin	Merck Millipore	1123740001	irritant, sensitizing, reconstitute with wash solution
Tissue culture flat tube 10 (= roller tube)	Techno Plastic Products AG	91243