Establishing a Whole-Cell Configuration for Two-Photon Calcium Imaging of Brain Slices

Abstract

Source: Camiré, O., et al. Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices. J. Vis. Exp. (2018).

This video demonstrates a protocol for whole-cell patch-clamp recording and two-photon calcium imaging in brain hippocampal slices. This method allows to study the dynamics of local Ca²⁺ transients (CaTs) in dendrites of different neuronal types in acute brain slices.

Protocol

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

1. Preliminary Preparation (Optional: Prepare 1 Day in Advance)

1. Prepare three types of artificial cerebrospinal fluid (ACSF) solution (Normal, Sucrose, and Recovery Solutions, 1 L each; see Table 1). Adjust the osmolality of the solutions to 300 ± 10 mOsm and cool the ACSF-Sucrose down to a near-freezing point (0-4 °C).
   NOTE: The use of a low-sodium cutting solution (ACSF-Sucrose) helps preserve the viability of neurons in the superficial layers of acute slices.
....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Animal Strain: Mouse CD1	Charles River	22
Calcium chloride	Sigma-Aldrich	C4901
D-(+)-Glucose	Sigma-Aldrich	G8270
HEPES	Sigma-Aldrich	H3375
Magnesium chloride	Sigma-Aldrich	M8266
Magnesium sulfate heptahydrate	Sigma-Aldrich	230391
Paraformaldehyde powder, 95%	Sigma-Aldrich	158127
Potassium chloride	Sigma-Aldrich	P3911
Potassium gluconate	Sigma-Aldrich	P1847
Sodium azide	Sigma-Aldrich	S2002
Sodium bicarbonate	Sigma-Aldrich	S8875
Sodium chloride	Sigma-Aldrich	S5886
Sucrose	Sigma-Aldrich	S9378
Triton X-100	Sigma-Aldrich	T9284
Trizma base	Sigma-Aldrich	T1503
Trizma hydrochloride	Sigma-Aldrich	T3253
Sodium phosphate dibasic dihydrate	Sigma-Aldrich	71643
Sodium phosphate monobasic monohydrate	Sigma-Aldrich	S9638
Biocytin	Sigma-Aldrich	B4261
Alexa Fluor 594 Hydrazide	ThermoFisher Scientific	A10438
SR95531 (Gabazine)	Abcam	ab120042
Adenosine triphosphate (ATP)-Tris	Sigma-Aldrich	A9062
Guanosine (GTP)-Na+	Sigma-Aldrich	G8877
Oregon Green BAPTA-1	ThermoFisher Scientific	O6812
Phosphocreatine di(tris) salt	Sigma-Aldrich	P1937
Streptavidin-conjugated Alexa-546	ThermoFisher Scientific	S11225
Patch Borosilicate Glass Capillaries	World Precision Instruments	1B100F-4
Theta Borosilicate Glass Capillaries	Sutter Instrument	BT-150-10
P-97 Flaming/Brown Micropipette puller	Sutter Instrument
TCS SP5 Confocal Multiphoton Microscope	Leica Microsystems
Chameleon Ultra II Ti:Sapphire multiphoton laser	Coherent
LAS AF Imaging Acquisition Software	Leica Microsystems
Temperature Controller TC-324B	Warner Instruments
MultiClamp 700B Amplifier	Molecular Devices
Digidata 1440A Digitizer	Molecular Devices
Confocal Translator	Siskiyou
Micromanipulator	Siskiyou
pClamp Data Acquisition Software	Molecular Devices
A365 Constant Current Stimulus Isolator	World Precision Instruments
Vibraplane Optical Table	Kinetic Systems