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.
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).
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 remain8-10. 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 boutons11-13. In addition to these roles in the healthy nervous system, synaptic remodeling is also involved in nervous system disease/injury12,14,15. For example, following spinal cord injury, severed axons must subsequently remodel and form new synapses to achieve functional recovery16-19.
Emerging as an important aspect of synaptic plasticity is the process of phagocytosis or engulfment of synapses destined for removal3,5,20. We recently showed this phenomenon in the context of synaptic pruning in the healthy, postnatal mouse brain7. Specifically, microglia, the resident CNS immune cells and phagocytes, were shown to engulf presynaptic inputs during a peak period and in a region of developmental synaptic pruning, the postnatal dorsal lateral geniculate nucleus (dLGN) of the thalamus. Genetic or pharmacological blockade of this engulfment resulted in sustained deficits in synaptic connectivity.
In this protocol, we describe a reliable and highly quantitative assay to measure phagocyte-mediated engulfment of presynaptic inputs. For the purposes of this article, this assay will be presented in the context of the developing retinogeniculate system, which includes retinal ganglion cells (RGCs) residing in the retina that project presynaptic inputs to the dLGN (Figure 1A). To begin, a lysosomal degradation-resistant anterograde labeling strategy will be described, which is used to visualize RGC-specific presynaptic inputs in the dLGN (Figure 1)7,21. Following this description, a detailed methodology for imaging and quantitatively measuring engulfment using confocal microscopy combined with 3 dimensional (3D) surface volume rendering will be given. This methodology is based on fixed tissue preparation but may also be adapted for use in live imaging studies. Importantly, while the assay has been validated in the context of the healthy, postnatal retinogeniculate system, one could apply the same techniques to assess other phagocyte-neuron interactions throughout the brain and during disease, as well as phagocyte function in other organ systems.
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.
2. Prepare Tissue for Imaging
This tissue preparation protocol is used for reporter mice in which the phagocytes are labeled with fluorescent markers (e.g., CX3CR1-EGFP for microglia). If a reporter line is not available, the investigator can immunostain tissue sections (see Discussion).
3. Imaging Tissue
All images are acquired on a spinning disk confocal microscope (UltraView Vox spinning disk confocal microscope equipped with diode lasers (405 nm, 445 nm, 488 nm, 514 nm, 561 nm, and 640 nm)). Images can also be acquired on any microscope with the ability to acquire high resolution z-stacks (e.g., laser scanning confocal microscope, epifluorescent microscope followed by deconvolution, etc.). Frame size is typically 1,000 x 1,000 pixels.
4. Prepare Images for Quantification (ImageJ)
5. Prepare Image for Quantification in Imaris
6. 3 Dimensional (3D) Surface Rendering of the Phagocyte
7. 3D Surface Rendering of Engulfed Material
8. Calculate the Total Volume of the Field of View
9. Calculate the Amount of Phagocytosed Material
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 technique, we found that during a peak period of developmental synaptic remodeling within the dLGN (P5), presynaptic inputs are engulfed by microglia (Figure 6). As few as 4 days later when a large amount of remodeling is nearly complete (P9), there is a dramatic reduction in the amount of engulfed inputs. In addition, engulfment and pruning are disrupted in mice deficient in proteins belonging to the classical complement cascade (Figure 6) as well as following manipulation of neuronal firing (data not shown)7.
Figure 1. Strategy to assess microglia-mediated engulfment of RGC presynaptic inputs7. A) Schematic of anterograde tracing strategy. Left and right eye RGC inputs are traced with CTB-647 (blue) and CTB-594 (red), respectively. Microglia-mediated (green) engulfment of inputs is subsequently assessed. B) A representative low magnification image of postnatal day 5 (P5) mouse dLGN following anterograde tracing of left (blue) and right (red) eye inputs. Scale bar = 100 µm. Ci) A microglia (EGFP, green) sampled from the border region of left (blue) and right (red) eye inputs (inset in B). Cii) All CTB fluorescence outside the microglial volume has been subtracted revealing RGC inputs (red and blue) that have been engulfed (arrows, enlarged in inset). Ciii) Surface rendering of microglia and engulfed RGC inputs. Grid line increments = 5 µm. Di) A representative microglia (green, EGFP) from P5 dLGN. RGC inputs are labeled with CTB-594 (red) and lysosomes are labeled with anti-CD68 (blue). Dii) All CTB fluorescence outside the microglia volume has been removed revealing engulfed RGC inputs (red) and lysosomes (blue) within the microglia (green). Diii) Most RGC inputs (red) are completely localized within CD68-positive lysosomes (blue; white arrows). Div-v) The CD68 (Div) and CTB (Dv) channels alone. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 2. Imaris software icons.
Figure 3. General navigation windows in software. A) The Display Adjustment Window. B) The Pointer Window. Select will allow selection of specific surfaces. Navigate will allow the rotation of an image in the field of view. C) It is necessary to be in Surpass mode for surface rendering the volume.
Figure 4. Surface rendering in software. A) The Create window. B) Smoothing window. Select the channel to surface from the pull down menu (highlighted in yellow). C) Thresholding the surface to the fluorescent image. Highlighted by the red box is the threshold value that should be recorded for total engulfed and non-engulfed material. This number will be applied later to threshold and surface the engulfed material. Please click here to view a larger version of this figure.
Figure 5. Obtaining volume measurements in software. A) Tabs that appear following surface rendering. Note the Wand, Pencil, Funnel, and Graph tabs that are identified in the text. B) The Mask all feature to visualize engulfed material within the phagocyte. Note the channel selected (yellow box). C) The feature under the Funnel/Filter tab allows the selection of all surfaces in the field by sliding the histogram all the way to the left so it appears yellow. Any filter can be chosen for this. D) Under the Graph tab, volume measurements can be obtained and recorded. Please click here to view a larger version of this figure.
Figure 6. Representative data7. A) Representative surface-rendered microglia from P5 (fluorescent image is shown in Figure 1), P9, and P30 mouse dLGN. Enlarged insets denoted with a black dotted line. Grid line increments = 5 µm. B) Engulfment of RGC inputs is significantly increased during peak pruning in the dLGN (P5) versus older ages (P9 and P30). *P < 0.001 by one-way ANOVA, n = 3 mice/age. C) Microglia from mice deficient in complement receptor 3 (KO, black bar) engulf significantly fewer RGC inputs as compared to WT littermates (white bar). All data are normalized to WT control values. *P < 0.04 by Student’s t-test, n = 3 mice/genotype. All error bars represent s.e.m. Click here to view larger image.
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 material combined with high resolution confocal microscopy and 3D reconstruction. This methodology has been used in our lab to successfully analyze microglia mediated-engulfment of presynaptic inputs undergoing synaptic remodeling in the developing mouse brain (retinogeniculate system)7. In addition, it has proven to be a highly sensitive technique that can distinguish subtle changes in engulfment across different developmental ages under non-pathological conditions and between wildtype and knockout mice. With minor adjustments, this protocol can be adapted to study engulfment in live tissues by time lapse imaging. In addition, this protocol may be applied to study phagocyte-neuron interactions in disease.
Caveats
One of the most useful aspects of this assay is the ability to label neuronal projections in such a way that they remain visible after entering a lysosomal degradation pathway. However, because the neuron is labeled, it may be difficult to distinguish what part of the cell has been phagocytosed. This requires a very careful regional analysis (e.g., synaptic region versus a non-synaptic region of the dLGN) combined with electron microscopy7. In addition, this may prove more difficult in other brain regions where neuronal cell bodies and projections reside in the same area. However, alternative labeling strategies may be employed (see below). Furthermore, due to the resolution limits of light microscopy, there is a possibility that one may overestimate the amount of engulfed material. As a result, care must be taken to validate that engulfed material is within lysosomes. For this reason, we use anti-CD68 to label lysosomes within microglia, 3D rendering, and orthogonal views to validate the parameters of the assay.
Cell Types
In addition to microglia, this methodology could also be applied to analyze the role of other phagocytes in the brain (e.g., astrocytes) and in the peripheral nervous and immune systems (e.g., perisynaptic Schwann cells, macrophages, etc.). In addition, because CTB conjugated to Alexa dyes is so readily taken up by most cells, a similar labeling strategy may be used to label engulfed material throughout the brain and other organ systems.
Labeling of Engulfed Material
As an alternative to CTB conjugated to Alexa dyes, we have used the following strategies to label engulfed material7: 1) Fluorescent reporter mice in which RGCs were labeled with a fluorescent protein (e.g., tdTomato, EGFP, etc.). 2) Anterograde labeling with a pHrodo dye conjugated to dextran, a dye that only fluoresces once it is in a lysosome. Using these different strategies, visualization and quantification of engulfed presynaptic inputs within microglia can be performedwith the same imaging and quantification techniques. However, the Alexa dye conjugates are the most robust due to the high resistance of Alexa dye to lysosomal hydrolases7,21. In addition, labeling of engulfed material with antibodies (e.g., anti-VGlut2, a presynaptic vesicular protein) has been attempted. However, this is a relatively unreliable method of detection given that most proteins are rapidly broken down once in lysosomes. It is difficult to distinguish low fluorescence due to protein degradation over background.
Animals Used
In the current protocol, microglia are fluorescently labeled genetically using fluorescent reporter mice (CX3CR1-EGFP)22. However, there are instances in which antibody labeling versus genetic expression of fluorescent reporter constructs is necessary. For example, when fluorescent reporter mice are not available for the phagocyte of interest, the use of rats is necessary, or the researcher wishes to look at knockout mice without crossing them into a fluorescent reporter mouse line. For instances such as these, phagocytes can be labeled using immunohistochemistry and data are comparable to results from experiments using fluorescent reporter mice7. Following tissue sectioning, we typically use a standard immunostaining protocol for floating sections7. It is important to choose an antibody raised against a protein that will label the entire cell and its processes. For example, to label microglia, anti-Iba1 can be used.
Broader Applications: Live Imaging and Disease
While this protocol is based on analysis of fixed tissue, slight modifications would enable the same analysis in images acquired by live imaging. Following a time lapse imaging session, the subsequent z-stacks can be processed identical to steps 4-9 in this protocol. Furthermore, while we have applied this protocol to visualize engulfment of synapses during developmental synaptic remodeling in the healthy CNS, this protocol can also be applied in disease models. In particular, this protocol can be used to study diseases in which synapses undergo substantial remodeling, such as in the case of synapse loss and regeneration following spinal cord injury, early synapse loss associated with Alzheimer’s disease, etc12,14-19,23-26. In addition, one may also adapt this technique to study diseases in which phagocytosis of material other than synapses has been described, such as microglia/macrophage-mediated engulfment of myelin in demyelinating disease (e.g., multiple sclerosis) and engulfment of beta-amyloid in Alzheimer’s disease5,6,27-29.
In conclusion, the engulfment assay described here offers a convenient, reproducible, and sensitive technique to study phagocyte interactions with their extracellular environment. Importantly, this assay has a broad range of uses and capabilities that will serve the neuroscience community as well as those working in other organ systems.
The authors have nothing to disclose.
Work was supported by grants from the Smith Family Foundation (B.S.), Dana Foundation (B.S.), John Merck Scholars Program (B.S.), NINDS (RO1-NS-07100801; B.S.), NRSA (F32-NS-066698; D.P.S.), Nancy Lurie Marks Foundation (D.P.S.), NIH (P30-HD-18655; MRDDRC Imaging Core).
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 gauge 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 ddH20 and 0.2 M sodium phosphate dibasic (PB-B) in ddH20. To make 0.1 M PB, combine 19 ml PB-A and 81 ml PB-B, fill to 200 ml with ddH20 |
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 |