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.
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 |
.