Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo Red-labeled crude synaptosomes using flow cytometry.
Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.
Microglia are the resident immune cells of the central nervous system (CNS)1. They constantly scan their microenvironment and provide surveillance1,2. Moreover, they frequently interact with synapses and mediate a fine-tuning of the synaptic activity3. Thus, they have emerged as a key player in the process of synaptic refinement.
The role of microglia in synaptic refinement through the engulfment of synapses has been shown by various research groups3,4,5,6,7. Disruptions in this process can contribute to the pathology of neurodevelopmental and neurodegenerative disorders such as schizophrenia and Alzheimer's disease8. Aberrant synaptic refinement by microglia has already been detected in various murine models of neurological disorders5,9,10. Therefore, identification of distinct mechanisms underlying microglial engulfment of synapses is paramount to understanding the pathophysiology of neurodevelopmental and neurodegenerative disorders8.
Targeting microglial engulfment of synapses holds great potential for both intervening in disease progression and gaining insights into the underlying mechanisms of neurodevelopmental and neurodegenerative disorders. To facilitate such investigations, there is a need for fast and high-throughput approaches. Current methodological approaches encompass in vivo, ex vivo, and in vitro assays that enable the detection of synaptic material within microglia. Generally, the detection of microglial engulfment of synapses relies heavily on immunohistochemistry (IHC) and microscopy-based approaches5,6,11, which are labor-intensive and show limitations in analyzing a large number of microglia.
Given these technical limitations, the exploration of alternative methodologies is imperative. To overcome this, we have optimized a flow cytometry-based approach, which enables an efficient, unbiased, and high-throughput analysis of microglial engulfment of synapses. We chose the hippocampus as the main region of interest due to its high degree of synaptic remodeling and plasticity12, but the protocol can be adapted to various brain regions. While flow cytometry has already been used in previous studies to detect microglial engulfment of synapses13,14,15, we herein provide a step-by-step methodology employing a currently commercially available, fluorophore-conjugated vGLUT1 antibody. We, moreover, provide a complementary in vitro approach for high-throughput screening of microglial engulfment of synaptic material by using crude synaptosomes.
A general view of the experimental procedure is graphically illustrated in Figure 1A. All experiments involving the handling of living animals used were performed in strict accordance with the German Animal Protection Law and were approved by the Regional Office for Health and Social Services in Berlin (Landesamt für Gesundheit und Soziales, Berlin, Germany). The mice were group-housed in ventilated cages under standard laboratory conditions with a 12:12 h light/dark cycle at the animal core facility of the Max Delbrück Center for Molecular Medicine (MDC). Food and water were provided ad libitum. See Table 1 for the composition of buffers and reagents and the Table of Materials for details related to all reagents, instruments, and materials used in this protocol. For the vGLUT1-specific assay, we used the term in vivo* throughout the manuscript to acknowledge that flow cytometry requires tissue homogenization and cell isolation, and microglia exhibit approximately 95% viability after the isolation procedure (Figure 1B and Supplementary Figure S1). Therefore, they retain their ability to engulf synaptic material ex vivo, for a short period, until the fixation. Thus, the quantification of vGLUT1+ microglia comprises both in vivo and short-term ex vivo engulfment until the fixation step.
1. Intracellular vGLUT1 staining for the detection of in vivo* engulfment of glutamatergic synapses by microglia
NOTE: The following cell isolation procedure is adapted from16. All steps of cell isolation should be carried out on ice.
2. Detection of in vitro engulfment of crude synaptosomes by microglia
In this project, we optimized and presented two protocols to measure in vivo* and in vitro engulfment of synapses by microglia. In the first protocol, we focused on in vivo* engulfment of vGLUT1-positive synapses. As a starting point, we used a previously published protocol14. However, the FACS antibodies used in this protocol are discontinued and we added many optimization steps as well as a novel method for microglia isolation16. That is why the protocol presented here is worth sharing with the scientific community as a comprehensive update to the protocols that are already published.
To quantify microglial engulfment of synapses, we used C57BL/6N male mice aged 11-14 weeks. The hippocampus was selected as the main region of interest due to its high degree of synaptic remodeling and plasticity12. We analyzed %vGLUT1-positive microglia as well as microglia-specific vGLUT1-PE fluorescence intensity (MFI) in the hippocampus of C57BL/6N mice. Spleen macrophages derived from the same animals were used as a biological negative control per experiment. We tested the vGLUT1 antibody by demonstrating a higher vGLUT1-PE fluorescence signal from the hippocampal microglia compared to the isotype control and spleen macrophages (Figure 1B–E)
Furthermore, we compared the microglial engulfment of synapses in the cerebellum as well as in the olfactory bulb (as another reference for high synaptic plasticity)20. We found a higher vGLUT1 fluorescence signal in the microglia from the olfactory bulb and a lower signal in the cerebellum compared to the hippocampus (Figure 1F). The lowest signal intensity was detected in the spleen macrophages, serving as the internal negative control (Figure 1E). Additionally, we used Vglut-IRES-Cre/ChR2-YFP mice to test the immunoreactivity of our vGLUT1 antibody. YFP is expressed by the glutamatergic neurons of these mice, indicating that the YFP-positive population should also include a vGLUT1-positive fraction. Using this staining protocol, we detected 98.7% of the YFP-positive population as vGLUT1-positive, validating the efficiency of our antibody (Supplementary Figure S3).
Overall, these results validate the efficiency of the vGLUT1 antibody and the presented staining protocol. We demonstrate that this protocol and the antibody can be confidently used to quantify in vivo* engulfment of synapses in a high-throughput and fast manner compared to other experimental approaches.
Moving on to the in vitro method, we isolated adult microglia and incubated them with freshly isolated pHrodo Red-labeled synaptosomes isolated from the same animals to quantify their in vitro engulfment (Figure 2A). We labeled synaptosomes with pHrodo Red, which naturally increases the fluorescence signal in acidic surrounding pH21. We freshly isolated synaptosomes and exposed them to different pH values (pH = 4 and pH = 11). After confirming the increase in fluorescence signal in low pH as a proof-of-principle experiment (Figure 2B), we incubated these synaptosomes with freshly isolated microglia for 1.5-2 h. As a control, we incubated microglia with unlabeled synaptosomes. Next, we analyzed the pHrodo Red-PE fluorescence signal from CD11b++/CD45+ microglia and observed a positive PE fluorescence, which was comparable to that obtained from synaptosomes at pH = 4 (Figure 2C). Thus, this method provides a fast and high-throughput analysis of the in vitro engulfment of synaptosomes and can be extended to amyloid plaques or the engulfment of other potential targets following necessary optimization steps. Indeed, Rangaraju et al. quantified engulfment of amyloid beta by microglia using a similar flow cytometry-based approach22. In conclusion, these two methods provide robust, efficient, and high-throughput quantification of microglial engulfment of synapses both in vivo* and in vitro.
Figure 1: Analysis of microglial engulfment of vGLUT1+ synapses in vivo*. (A) Graphical illustration of the experimental workflow depicting steps of intracellular vGLUT1 staining. (B) Gating strategy to define single/CD11b++/CD45+/ viable cell population from the hippocampus. This population was used to analyze vGLUT1-MFI as well as to quantify the percentage of vGLUT1+ microglia in the hippocampus. The gate shown with the red rectangle indicates the vGLUT1+ cell fraction in the total sample. (C) The histogram indicates vGLUT1-PE fluorescence intensity. (D) The gate shown with the red rectangle indicates no positive cell fraction showing Isotype-PE immunoreactivity. The histogram indicates Isotype-PE fluorescence intensity. (E) The gate shown with the red rectangle indicates no positive cell fraction showing vGLUT1-PE immunoreactivity in the spleen macrophages. The histogram indicates vGLUT1-PE fluorescence intensity. The gate indicated on the histogram starts at the level, where the vGLUT1-MFI from the spleen terminates (~104) and is used to analyze the vGLUT1 positive fraction in the brain samples. (F) The overlayed histogram shows the comparison of PE fluorescence intensity of spleen macrophages (grey) and microglia from the hippocampus (red), cerebellum (purple), and olfactory bulb (light blue). Please click here to view a larger version of this figure.
Figure 2: Analysis of microglial engulfment of synaptosomes synapses in vitro. (A) Graphical illustration of the experimental workflow depicting steps of the in vitro synaptosome engulfment assay. (B) Synaptosomes incubated at two different pH values show a low pHrodo Red-PE fluorescence signal at pH = 11 and a high pHrodoRed-PE fluorescence at pH = 4. (C) Single/CD11b++/CD45+ cell population was used to analyze pHrodo Red-PE fluorescence intensity. Microglia incubated with unstained synaptosomes were used as a negative control. Please click here to view a larger version of this figure.
Table 1: List of buffers and reagents used in this protocol. Please click here to download this Table.
Supplementary Figure S1: Representative image of freshly isolated adult microglia. Image acquired using a light microscope with 20x objective following the papain-based tissue dissociation protocol and MACS-based isolation of CD11b+ microglia. Scale bar = 50 µm. Please click here to download this File.
Supplementary Figure S2: Representative FACS plots demonstrating the gating strategy to define spleen macrophages. Spleen was used as a negative control in the experiments per experimental run while testing microglial engulfment of synapses in the hippocampus. FACS plots given above define the spleen macrophages as CD11b++/CD45++/viable population. This population was used to set a threshold to quantify vGLUT1+ microglia in the brain samples that reside above this threshold gate. Please click here to download this File.
Supplementary Figure S3: Representative FACS plots demonstrating the gating strategy to test the efficiency of the vGLUT1 antibody. (A) Graphical illustration of the experimental workflow depicting steps of the vGLUT1 staining. YFP+ glutamatergic neurons were used to test the immunoreactivity of the vGLUT1 antibody. (B) Gating strategy to define the YFP+ population from the hippocampus of Vglut-IRES-Cre//ChR2-YFP mice that were used as a positive control for testing the efficiency of the vGLUT1 FACS antibody. YFP+ fraction was gated to specify glutamatergic synapses. In this population, the immunoreactivity of the vGLUT1 antibody was analyzed to test the immunoreactivity of the antibody. Compared to the (C) Isotype control; 97.9% of YFP-positive cell fraction is detected as (D) vGLUT1-positive. (E) The overlayed histogram indicates the comparison of the PE fluorescence between the isotype and vGLUT1 antibody. Please click here to download this File.
Synaptic refinement through microglia-synapse interaction is an intriguing area of study within the field of neuroimmunology, offering promising insights into the role of microglia in neurodegenerative and neurodevelopmental disorders. In 2011; Paolicelli et al. provided evidence of the presence of synaptic material within microglia, shedding light on their involvement in the process of synaptic engulfment4. Another intriguing study employed time-lapse imaging and an ex vivo organotypic brain slice culture model and reported that microglia engage in a phagocytic process known as trogocytosis, where they engulf presynaptic structures rather than the entire synaptic structure23. A very recent publication using a new transgenic mouse model that enables measurement of phagocytosis in intact tissue showed pruning by Bergmann-glia in vivo upon motor learning24. Thus, there is sufficient evidence indicating the involvement of glial cells in synaptic engulfment, including microglia. However, the extent to which this microglial function impacts the dynamic, and selective process of synaptic pruning requires further evidence.
Nevertheless, the quantification of microglial engulfment of synapses serves as a valuable indicator and provides partial insight into the complex dynamics of microglia-synapse interactions, especially synaptic refinement. A comprehensive review has summarized current protocols used to investigate microglia engulfment of synapses25. We would like to emphasize that our protocols are optimized based on existing protocols that are already in use. The methods presented in this study provide fast and high-throughput quantification microglial engulfment of synapses in various dissected brain regions. Depending on the brain region, an analysis of at least 10,000 microglial cells in a maximum of two days is possible for both methodologies, making them valuable for testing multiple mouse models in parallel.
We acknowledge that the quantification of vGLUT1+ microglia comprises both in vivo and short-term ex vivo engulfment until the fixation step. Therefore, we suggest that our assay presents a fast and reliable way to quantify synaptic material inside microglia as an initial step prior to in vivo validation using approaches such as IHC.
Another disadvantage of the flow cytometry analysis is the limited availability of antibodies for synaptic markers, particularly for inhibitory synapses. It is challenging to find commercially available, directly conjugated antibodies that show a bright signal for these markers. Given the extensive optimization time required to test different antibodies targeting synaptic markers, it is important to share the well-optimized procedures with the scientific community for intracellular staining with different antibodies as we do here with this study.
Regarding data analysis in this study, we used Isotype controls as technical negative controls to account for non-specific bindings of the vGLUT1 antibody, since they provide an estimate for nonspecific binding of an antibody in a sample while optimizing flow cytometry-based assays26. However, isotype controls have been mostly optimized to detect the nonspecific background signal from the surface staining procedures and are not optimal for intracellular staining controls27,28. Therefore, they should not be relied upon to distinguish between the negative and positive populations when performing intracellular staining, which involves fixation and permeabilization steps that can impact antigen detection, autofluorescence, and fluorophore brightness29. Such intracellular staining procedures require the use of appropriate biological internal controls to define the positive cell population stained for an intracellular marker29. Thus, considering that we use an intracellular staining protocol, we employed an internal biological negative control (spleen macrophages) and defined the boundary between the positive and negative populations according to the spleen macrophages isolated from the same mice. We distinguished the positive population above the gate, at which there are no vGLUT1 positive events from the spleen macrophages that serve as the biological negative control (Figure 1).
Both methods presented in this study offer great potential for initial analysis of microglial engulfment of synapses in a fast and high-throughput manner, analyzing over 10,000 cells from small brain regions and this is not achievable with standard microscopy techniques. Therefore, these methods offer a significant advantage over labor and time-intensive methods and further, provide a more comprehensive analysis of synaptic engulfment by allowing an analysis of a greater number of microglia. Additionally, the in vitro method presented in this study is particularly useful for testing the impact of different treatments on the microglial engulfment of synapses. It enables direct quantification of the effect of treatment on microglia without the confounding factors associated with other cell types. In addition, it serves as an indirect approach to proving a potential effect of microenvironment or other cell types on the process of synaptic engulfment. Therefore, we conclude that these methods, especially when used in parallel, offer intuitive and advantageous alternatives for the analysis of microglial engulfment of synaptic materials.
However, the analysis of freshly isolated microglia by FACS-based phagocytic assays ex vivo may pose a few disadvantages. First, it is critical to employ well-optimized protocols that generate freshly isolated microglia from the adult brain while avoiding ex vivo activation and stress response of microglia. Dissing-Olesen et al. incorporated the use of transcriptional and translational inhibitors to overcome this issue by employing a tissue dissociation procedure at 37 oC30. Mattei et al., on the other hand, presented a cold, mechanical tissue dissociation protocol to avoid inducing ex vivo expression of stress associated genes16 and we adapted this protocol in the first section to avoid ex vivo activation of stress-associated microglia response prior to intracellular vGLUT1 staining. We employed an enzymatic tissue dissociation protocol in the second section prior to the in vitro synaptosome engulfment assay considering the higher yield of microglia following papain-based tissue dissociation (data not shown). Microglia inevitably remain at 37 oC under culture conditions when incubated with synaptosomes, and incubation at 37 oC can indeed induce changes in microglia as common drawbacks of all in vitro assays and cell culture procedures. Therefore, we suggest the use of both presented protocols in parallel to reach a broader conclusion in terms of microglial engulfment of synapses.
Furthermore, it is important to carefully define the gating strategy to select CD11b++/CD45+ microglia by taking into account the presence of other immune cells in the brain parenchyma that also express these markers31. More importantly, when choosing markers to specifically target microglia (e.g., TMEM119, P2RY12), it is important to consider that they can undergo changes in their expression levels during pathological and inflammatory conditions32, and such changes should be considered prior to establishing the FACS panel to quantify microglial engulfment of synapses. Finally, it is essential to emphasize that neither of the methods discussed earlier, including the IHC- and microscopy-based in vivo approaches, can alone capture the active and selective pruning of synapses by microglia. These methods are not able to discriminate the active pruning by microglia from the passive scavenging of synaptic debris within the brain parenchyma. Therefore, when evaluating and discussing the data, it is imperative to clearly distinguish between these distinct concepts.
The authors have nothing to disclose.
We thank Regina Piske for technical assistance with microglia isolation and Dr. Caio Andreta Figueiredo for his help with microscopy image acquisition in Supplementary Figure S1. We thank the FACS facility of the MDC for their technical support. This manuscript partially presents the representative figures submitted to the Brain, Behavior and Immunity Journal in 2024. Figure 1A, Figure 2A, and Supplementary Figure S3A were created by using BioRender.com.
1 mL Dounce Homogenizer | Active Motif | Cat# 40401 | |
5 mL Tubes | Eppendorf | Cat# 0030119452 | |
Anti-CD11b | ThermoFisher Scientific | Cat# 25-0112-82 | |
Anti-CD45 | BD | Cat# 559864 | |
Anti-Ly6C | BD | Cat# 553104 | |
Anti-Ly6G | BD | Cat# 551460 | |
BCA Protein Assay Kit | Pierce | Cat# 23227 | |
C-Tubes | Miltenyi Biotech | Cat# 130-096-334 | |
CD11b MicroBeads | Miltenyi Biotech | Cat# 130-093-634 | |
CD16/CD32 Antibody | Thermo Fisher Scientific | Cat#14-0161-82 | |
Cytofix/Cytoperm Kit | BD | Cat# 554714 | |
Dulbecco's Modified Eagle Medium (DMEM) | Gibco | Cat# 41966029 | |
Dulbecco´s Phosphate Buffered Saline (DPBS) | Gibco | Cat# 14190144 | |
Falcon Round-Bottom Polystyrene Test Tubes | Thermo Fisher Scientific | Cat# 08-771-23 | |
fixable viability dye | Thermo Fisher Scientific | Cat# L34969 | |
Hibernate A medium | ThermoFisher | Cat# A1247501 | |
LS-columns | Miltenyi Biotech | Cat# 130-042-401 | |
Papain Dissociation System | Worthington | Cat# LK003150 | |
Percoll | Th.Geyer | Cat# 17-0891-02 | |
Petri dishes | Thermo Fisher Scientific | Cat# 11339283 | |
pHrodoRed | Thermo Fisher Scientific | Cat# P36600 | |
Protease inhibitor | Roche | Cat# 5892970001 | |
Red Blood Cell Lysis Buffer | Sigma | Cat# 11814389001 | |
Steritop E-GP Sterile Filtration System | Merck | Cat# SEGPT0038 | |
SynPer Solution | ThermoFisher | Cat# 87793 | |
vGLUT1 Antibody | Miltenyi Biotech | Cat# 130-120-764 |