Electrophysiological Recordings of Reprogrammed Neurons in a Mouse Brain Slice

Abstract

Source: Pereira, M., et al. In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors. J. Vis. Exp. (2019)

This video demonstrates a protocol to evaluate the maturation and reprogramming of glial cells into neurons in a transfected mouse striatum slice. The experiment measures resting membrane potential and response to current injections using neuron-specific fluorescent markers and electrophysiological techniques. Voltage spikes during these measurements indicate successful neuronal maturation and reprogramming.

Protocol

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

1. Whole-cell patch-clamp recordings

1. Transfer the first tissue section to the recording chamber submerged in a continuous flow of Krebs solution. Mount the section using light weights and submerge the objective.
2. Identify the striatal region in the microscope and search for GFP-positive (reprogrammed) neurons. Select a neuron that is extensive in morphology and not covered by fiber bundles or blood vessels.
3. Prepare borosilicate glass pipettes (3–7 MΩ) for patch....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Sigma-Aldrich	S3014
KCl	Sigma-Aldrich	P9333
NaH2PO4-H2O	Sigma-Aldrich	S9638
MgCl2-6 H2O	Sigma-Aldrich	M2670
CaCl2-6 H2O	Sigma-Aldrich	C8106
MgSO4-7 H2O	Sigma-Aldrich	230391
NaHCO3	Sigma-Aldrich	S5761
Glucose	Sigma-Aldrich	G7021
KOH	Sigma-Aldrich	P5958-500G
K-D-gluconate	Sigma-Aldrich	G4500 Sigma
KCl	Sigma-Aldrich	P9541
KOH-EGTA (Etilene glycol-bis-N-tetracetic acid)	Sigma-Aldrich	E3889 Sigma
KOH- Hepes acid (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid)	Sigma-Aldrich	H7523
NaCl	Sigma-Aldrich	S3014
Mg2ATP	Sigma-Aldrich	A9187
Na3GTP	Sigma-Aldrich	G8877
Ice
Borosilicate glass pipette	Sutter Company	B150-86-10
Glass capillary puller	Sutter company	P-1000
WaterBath	Thermo Fisher Scientific	TSGP02
Clampfit software	Molecular Devices