Isolation and Whole-Cell Patch-Clamp Recording of Hippocampal Microglia from Adult Mice

Summary

The present protocol describes how to isolate and purify primary hippocampal microglia from adult mice, followed by instructions for conducting whole-cell patch-clamp recordings on these acutely isolated cells.

Abstract

Microglia are resident immune cells in the brain that interact with neurons to maintain the homeostasis of the central nervous system (CNS). Studies show that the microglial surface expresses potassium channels that regulate microglial activation, while abnormalities in these potassium channels can lead to neural diseases. Currently, whole-cell patch-clamp recordings of microglia are mostly performed on cultured primary microglia from fetal or newborn mice due to difficulties in conducting electrophysiological evaluations on acutely isolated microglia. This study introduces an easy-to-follow protocol for isolating hippocampal microglia from adult mice and performing whole-cell patch-clamp recordings on the isolated cells. Briefly, the brain was removed from a mouse after decapitation, the hippocampus was dissected bilaterally, and microglia were isolated using an adult mouse brain dissociation kit. The microglia were then purified using a magnetic-activated cell sorting (MACS) method and seeded onto coverslips. Successful microglial isolation was confirmed by immunofluorescent staining with anti-CD11 and anti-Iba1 antibodies. A cover slip was placed in a recording chamber, and the whole-cell potassium currents of the acutely isolated microglia were recorded under voltage-clamp conditions.

Introduction

Microglia, which derive from myeloid progenitors in the primitive yolk sac, are resident in the CNS and comprise about 10% to 15% of total nerve cells^1,2. Functionally, in addition to surveilling the local environment, performing immune defense functions, and providing nutrition and support for neurons^3,4, microglia can also directly contact neurons to regulate neuronal surface receptors and corresponding ligand binding, and indirectly interact with neurons via the secretion of cytokines⁵. Studies in vitro and in vi....

Protocol

All experiments were approved by the Life Science Animal Care and Use Committee of South China Normal University, and appropriate standards of animal welfare were maintained (ethical approval: SCNU-SLS-2023-048). Male or female 3-5-month-old C57BL/6J mice were used for this study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Preparation of solutions

1. Prepare 50 mL of intracellular solution containing 160 mM.......

Discussion

Cultured microglia from fetal or newborn mice are clearly unsuitable for studying adult microglia. Additionally, given the heterogeneity of microglia across different brain areas²¹, microglia isolated from the whole brain may not accurately represent the characteristics of microglia in a specific brain structure. This protocol provides a method for isolating hippocampal microglia specifically to evaluate the electrical properties of these cells. It includes methods for recording the currents gover.......

Materials

Name	Company	Catalog Number	Comments
100 mm Petri dish	Corning	353003
15 mL Falcon tubes	BD	352096
24-well plates	BD	353047
35 mm Petri dish	Corning	353001	-
50 mL Falcon tubes	BD	352070
70 μm cell screening	Miltenyi	130-095-823	To remove cell clumps before cell sorting
Adult Mouse Brain Dissociation Kit	Miltenyi	130- 107-677
Anti-CD11b Antibody	Bio-Rad	MCA74	Goat Anti-mouse IgG also available. For blocking endogenous immunoglobulins.
Anti-Iba 1 Antibody	SYSY	234308	Goat Anti-guinea pig IgG) also available. For blocking endogenous immunoglobulins.
Axon Digidata 1440A	USA
Axon MultiClamp 700B Amplifier	USA
BSA Albumin Fraction V	BioFrox	4240GR500	Serum
C57BL/6 mice	Guangdong Medical Laboratory Animal Center
CD11 b/c MicroBeads	Miltenyi	130-105-634
Clampfit 10.6	USA
Confocal Microscope	Zeiss	LSM 800
Culture medium	Fisher Scientific	C11995500BT	-
DAPI dye	Beyotime	C1002
Electrode puller	Narishige	PC-10
Ethyl carbamate	Sigma	51-79-6
HBSS	Servicebio	G4203
Horizontal shaker	SCILOGEX	SLK-O3000-S
Image analysis software	Fiji
MACS Columns	Miltenyi	130-042-201
MACS Separators	Miltenyi	130-042-102
Paraformaldehyde	Fisher Scientific	T353-500	Use fresh 4% solution in 1X PBS, pH 7.2-7.4.
Poly-L-lysine	Beyotime	C0313	Coverslip coating
Triton X-100	Sigma	X100