Live Imaging and Characterization of Microglia Dynamics and Interactions with Synapses in Diseased Murine Retina

Summary

This paper demonstrates a method for visualizing and quantifying microglia motility and contact with postsynaptic puncta in the retina using spinning disk confocal microscopy.

Abstract

Microglia are the resident macrophages of the central nervous system (CNS) that respond to tissue infection and injury. In addition to their role in inflammation, microglia play a developmental role in circuit refinement through synaptic pruning. However, the mechanisms of synaptic pruning in neuroinflammation and neurodegeneration remain unknown. In this protocol, we use a mouse retina explant model to study microglia dynamics ex vivo. To examine microglia motility and their interactions with postsynaptic proteins, we label synapses with AAV-PSD95-RFP and record timelapse videos of motile microglia colocalized with postsynaptic proteins using spinning disk confocal microscopy. We then create surface and spot reconstructions of microglia and PSD95 using image analysis software. Data such as microglia displacement length, process speed, and contact with postsynaptic puncta can then be extracted from these surfaces to understand microglia behavior both in homeostatic states and after neuronal injury. This protocol can be useful in examining the role of microglia in synaptic pruning in retinal neurodegenerative diseases.

Introduction

Circuit function and homeostasis rely on highly regulated synapse firing and other unique interactions among various cell types¹. Serving as the resident macrophages of the central nervous system (CNS), microglia are a non-neuronal population in the retina that play a unique role in synaptic pruning. During CNS development, microglia prune non-functional or weak synapses to refine neural circuitry². Microglia in the developing brain survey their environment and are primed towards a neuroprotective role during critical periods³. In visual system development, microglia are key regulators in the deve....

Protocol

Animals were housed at the University of California, San Francisco, under light cycles of 12 h light and 12 h dark and given water and standard diet ad libitum, unless specified. All procedures were approved by the Institutional Animal Care and Use Committees at the University of California, San Francisco. See the Table of Materials for materials and instruments used in this protocol. An overview of the experimental procedure, from intravitreal injection to imaging and analysis, is shown in Figure 1.

1. AAV-PSD95-TagRFP virus production and efficiency

1. Construct AAV-PSD95-ta....

Results

Microglia from CX3CR1-GFP murine retinas were imaged using the described protocol. Mice ranged from P30 to P60 at the time of AAV-PSD95-RFP injection, and at least 21 days had elapsed to allow for AAV expression. Representative images of microglia and their interactions with PSD95 puncta before and after analysis are shown in Figure 2. To induce microglia activation, we used the murine laser-induced ocular hypertension (LIOH) model, a model that transiently e.......

Discussion

This protocol enables the visualization and tracking of individual microglia interaction with postsynaptic sites along the dendrites of RGCs. In a neurodegenerative disease such as glaucoma, microglia colocalize with synapses in the inner plexiform layer (IPL) of the retina. The role of microglia in synapse disassembly is not well understood. However, microglia potentially contribute via several potential mechanisms, including aberrantly engulfing synapses, passively clearing debris from apoptosing cells, or a combinatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
25 Gauge Needle	Alcon	8065420920
26s Gauge 10 µL Syringe	Hamilton	701RNWG
7m8	Addgene	64839	http://n2t.net/addgene:64839
Bend-and-Stay Multipurpose 304 Stainless Steel Wire	Stanford Advanced Materials	SS3368	Steel wire to make rings
Calcium Chloride Hexahydrate	Sigma-Aldrich	C5080-500G
Dissection Microscope	Amscope		To dissect and mount retinas
Dissection Scissors	FST	15024-10
D-(+)-Glucose	Sigma-Aldrich	G7528-250G
Forceps	Dumont	11252-21
HEPES	Sigma-Aldrich	H3375-500G
Imaris 10.0.2	Oxford Instruments		Analysis software
Imaris File Converter	Oxford Instruments
Loctite Super Glue Ultra Gel Control	Loctite	1363589	Glue for making rings
Magnesium Chloride Hexahydrate	Sigma-Aldrich	M9272-100G
MCE Membrane filter paper (13 mm)	Millipore	HABG01300	0.45 μm Pore Size
Microenvironmental Chamber	Tokai Hit	STXG-TIZBX-SET
Nikon Eclipse Ti2-E inverted microscope	Nikon
NIS Elements	Nikon
Neomycin and Polymyxin B Sulfaes and Bacitracin Zinc Opthalamic Solution Ointment, USP	Bausch + Lomb	24208-780-55
Paintbrush Size 00	Amazon
Prism 8.0	GraphPad
Proparacaine Hydrochloride Opthalmic Solution USP, 0.5%	Sandoz	61314-0016-01
PDL-coated petri dish (35 mm)	Matek	P35GC-1.5-14-C	No 1.5 Coverslip
PSD95_pTagRFP	Addgene	52671	http://n2t.net/addgene:52671
Potassium Chloride	Sigma-Aldrich	P9333-500G
Sodium Chloride	Sigma-Aldrich	S7653-1KG
Sodium Phosphate Monobasic Monohydrate	Sigma-Aldrich	S9638-25G