An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Summary

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

Abstract

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer’s disease) to development of the healthy brain (e.g., synaptic pruning)^1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system⁷. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Introduction

Synaptic circuits remodel throughout the life of an animal. In the developing brain, synapses form in excess and must undergo synaptic pruning which involves the selective removal of a subset of synapses and the maintenance and strengthening of those synapses that remain^8-10. This process is necessary to achieve the precise connectivity characteristic of the adult nervous system. In the adult, synapses can also be plastic, particularly in the context of learning and memory. The structural correlates of this plasticity are thought to include the addition and/or elimination of dendritic spines and presynaptic boutons^11-13. In addition to these role....

Protocol

1. Anterograde Labeling of RGC Presynaptic Inputs

Note: All experiments involving the use of animals were reviewed and overseen by the institutional animal care and use committee (IACUC) in accordance with all NIH guidelines.

1. Sterilize field and instruments.
2. Anesthetize mouse with 4 vol % isoflurane in a Plexiglas induction chamber (this vol % isoflurane works for neonatal-adult mice). Observe mice closely to avoid over-anesthetizing. CAUTION: Avoid inhalation with a vacuum waste gas evacuation.
3. After 1-3 min, (older mice will require less time) ensure appropriate level of anesthesia is achieved by pinching t....

Results

Recently, we used this engulfment assay to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing retinogeniculate system (Figure 1)⁷. RGCs from CX3CR1-EGFP heterozygous mice were anterogradely traced with CTB-594 and CTB-647 into the left and right eyes, respectively. Following this tracing, EGFP-positive microglia within the dLGN were imaged. These images were subsequently surface-rendered for volume measurements.

Using this t.......

Discussion

In order to accurately measure phagocytosis, engulfed material must be labeled in such a way that the researcher can visualize it once lysosomal degradation has occurred. In addition, high resolution imaging is required, followed by the use of software that will enable the researcher to visualize the volume of the entire cell and quantify its contents. In this protocol, we describe a highly reliable and quantitative method for measuring phagocyte-mediated engulfment using CTB conjugated to Alexa dyes to label engulfed ma.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Heat pad	Vet Equip, Inc.	965500
Warm water source for heat pad	Kent Scientific	TP-700
Stereo microscope	DSC Optical	Zeiss Opmi -6 Surgical Microscope
Sliding microtome with freezing stage	Leica	SM2010 R
Microtome blade	Leica	14021607100
Fluorescent dissecting microscope	Nikon	SMZ800 with Epi-fluorescence attachment
Spinning disk confocal microscope	Perkin Elmer	UltraView Vox Spinning Disk Confocal
10 µl Hamilton gas tight syringes	Hamilton	80030	Use a different syringe for each color dye/tracer
Hamilton needles	Hamilton	7803-05, specifications: blunt, 1.5"
Alexa-conjugated cholera toxin β subunit (CTB)	Invitrogen	488: C22841	Reconstitute in sterile saline, 80 µl (488), 100 µl (594), 20 µl (647)
594: C22842
647: C34778
Phosphate Buffered Saline (PBS)	Sigma	P4417-50TAB
Neomycin and Polymyxin B Sulfates and Bacitracin Zinc Ophthalmic Ointment USP (antibiotic ointment)	Bausch & Lomb	24208-780-55
30.5 G needle	Becton Dickinson	305106
Spring scissors	Roboz	RS-5630
Cotton-tipped applicator	Fisher	23-400-125
Paraformaldeyde (PFA)	Electron Microscopy Sciences	15710	Dilute 16% to 4% in PBS. Paraformaldehye is toxic, use in a fume hood and wear personal protective equipment.
Dissection tools – scissors, forceps, spatula	Small scissors: Fine Science Tools	Small scissors:14370-22
Large scissors: Roboz	Large scissors: RS-6820
#55 forceps: Fine Science Tools	#55 forceps: 11255-20
Spatula: Ted Pella, Inc.	Spatula: 13504
Sucrose	Sigma	S8501-5KG	Make 30% sucrose in PBS (weight/vol)
OCT Compound	VWR	25608-930
Weigh boat	USA Scientific	2347-1426
24-well plates	BD Biosciences	353047
Sodium phosphate monobasic	Sigma	S6566-500G	Make 0.2 M sodium phosphate monobasic (PB-A) in ddH2O and 0.2 M sodium phosphate dibasic (PB-B) in ddH2O. To make 0.1 M PB, combine 19 ml PB-A and 81 ml PB-B, fill to 200 ml with ddH2O
Sodium phosphate dibasic	Sigma	S5136-500G
Coverslips, 22 X 50 mm, No. 1.5	VWR	48393 194
Charged microscope slide	VWR	48311-703
Vectasheild	Vector Laboratories	H-1200