Measuring Neural Activity in a Mouse Brain Slice after Prolonged Incubation

Samenvatting

Source: Cameron, M. A., et al., Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging. J. Vis. Exp. (120), (2017)

This video demonstrates a technique for measuring neuronal activity in mouse brain slices using calcium imaging and electrophysiological recordings. By perfusing the tissue with potassium ions and recording intracellular calcium changes through high-speed digital imaging, this method reveals real-time neuronal responses.

Protocol

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

1. Calcium Dye Loading

1. Select calcium dyes based on the preference of the individual researcher. Here we use Fluo-8AM or Fluo-4AM. However, this method can be applied to other dyes as well: dissolve calcium dyes in DMSO to a 1 mM solution and 1% block copolymers (e.g., pluronic acid-127) to a final volume of 50 µL and sonicate for 10 min.
2. Add solution to aCSF to a final concentration of 10 µM, 0.01% block copolymers for brain slices.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Sodium Chloride	Sigma Aldrich VETEC	V800372
Potassium chloride	Sigma Aldrich	P9333
D-glucose	Sigma Aldrich	G5767
Sodium bicarbonate	Sigma Aldrich	S6014
Sodium phosphate monobasic	Sigma Aldrich	S2554
Magnesium chloride	Sigma Aldrich	M9272
Calcium chloride	Sigma Aldrich	223506
Dimethyl sulfoxide anhydrous	Sigma Aldrich	276855
Pluronic F-127 Low UV absorbance*	Life technologies	P-6867	20% solution in DMSO
Fluo-4 AM	Life Technologies	F23917
Fluo-8 AM	Abcam	Ab142773
Fixed Stage Upright Microscope	Olympus	BX51WI
Microscope Objective	Olympus	XLUMPlanFLN 20x/1.00w	20X
Microscope Objective	Olympus	LUMPlanFLN 60x/1.00w	60X
CCD Camera	Andor	Ixon +
Patch clamp amplifier	Molecular Devices	MultiClamp 700B
Data acquisition system	Molecular Devices	Digidata 1440A	Axon Digidata® System
Borosilicate glass capillaries	Sutter Instrument Co	BF150-86-10
Microelectrode puller	Sutter Instrument Co	P-97	Flaming/Brown type micropipette puller
Recording/perfusion chamber	Warner Instruments	RC-40LP	Low Profile Open Bath Chambers
Software	Molecular Devices	pClamp 10.2
Andor iQ Live Cell Imaging Software	Andor	Andor iQ3
Temperature Controller	TE Technology	TC-36-25 RS232