We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in vivo.
Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells’ migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.
As resident macrophages in the central nervous system (CNS), microglia represent a distinct non-neuronal population that accounts for up to 15% of all glial cells in the adult brain. Studying microglia biology has gained increasing attention in recent years due to their established importance in development, physiology, and disease1. Under physiological conditions, microglial cells are highly dynamic, continuously surveying the brain parenchyma2,3. This behavior allows microglia to colonize the brain and play pivotal roles in its development such as shaping neuronal circuitry4, synaptic pruning5, and vasculogenesis6. Furthermore, this inherent dynamic nature allows microglia to constantly monitor the CNS for signs of infection, injury, or any deviations from homeostasis7. To dissect these intricate cell dynamics, live imaging of microglia across space and time is indispensable. Fortunately, the optical transparency of externally developing zebrafish embryos, coupled with the availability of well-characterized transgenic reporter lines that fluorescently label microglia, positions zebrafish as an ideal vertebrate model for such investigations. Live imaging in zebrafish embryos offers a non-invasive approach that does not require surgery or extensive tissue manipulation, minimizing potential perturbations to the CNS status. This is a critical consideration when studying microglial cells, as they are highly sensitive to even subtle changes in the extracellular environment8.
Here, we provide a guideline to successfully track 3D microglial cell movements in the zebrafish embryo, allowing for an unprecedented view of microglia behavior within the intact architecture of the developing brain parenchyma (see Figure 1 for a graphical overview of the protocol). This step-by-step protocol details how to set up and image zebrafish microglia at different developmental stages and how to extract high-resolution data on microglia cell motility to provide valuable insights into their migratory patterns and responses to environmental cues. We also demonstrate this protocol can be adapted to perform live multi-color imaging, thus extending its applicability to studying microglia in combination with transgenic lines that mark neighboring cells, including neurons3, oligodendrocytes9, and endothelial cells10 (as shown in Figure 2). By adding to the toolbox that allows one to directly observe and characterize the dynamics of microglia behavior in real time and in their natural environment, this protocol will likely contribute to better elucidating microglia functionality during early development, both in physiology and disease.
The current protocol enables in vivo imaging of microglia dynamics in a vertebrate embryo and visualization of the acquired motility data. Microglia colonization of the developing brain occurs very early during embryogenesis and precedes critical events such as peaks of neurogenesis, astrogliogenesis, oligodendrogenesis, and many other cellular processes17. It is therefore not surprising that microglia play important functions in shaping specific aspects of brain development18, for example, through the regulation of neuronal differentiation, migration, and survival19,20,21, as well as synaptic pruning5 and myelination22,23,24.
The contribution of dysfunctional microglia to the pathogenesis and/or progression of neurodevelopmental disorders is also increasingly being recognized25. Indeed, the early presence of microglia in the forming brain exposes these cells to distinct physiological states26 and environmental changes. This can have a significant impact given that microglia are long-lived cells in both rodents and humans, being maintained during the lifespan through self-renewal of local progenitors27,28,29. We believe this protocol could serve as a powerful tool to better characterize the behavior of microglia in these distinct physiological states, as they develop, mature, and establish their network during the successive steps of brain morphogenesis.
Using the setup described here, we have successfully imaged and acquired data on zebrafish larvae as old as 6 dpf. Extending the analysis to later developmental stages will likely succeed but will require adjusting the imaging setup to take into consideration the increased sample size, especially along the z-axis. In attempting this, we suggest focusing on maintaining a low signal-to-noise ratio and fast scanning time, as they are key parameters for a successful analysis.
We suggest a minimum imaging time of 1 h to allow for microglia tracking; the longest imaging window that has been tested with this protocol is 8 h. Moreover, it is important for the tracking analysis to keep the time interval between frames as short as possible, ideally between 30 s and 60 s. This will allow for more accurate and detailed tracking data in downstream analyses. Therefore, especially if detecting more than one fluorophore, it is fundamental to avoid spectral overlap and ensure enough separation between the two fluorophore emission spectra to allow for a simultaneous acquisition, without signal bleedthrough.
Other protocols for high-quality timelapse recording of the zebrafish brain are available30, but this is the first one showing how to successfully track all microglia movement during embryonic development over an extended period. Although the workflow presented here focused on tracking microglia in a physiological context, it can be easily applied to the analysis of microglia in pathology. Indeed, several models of neurodevelopmental disorders, such as autism31, epilepsy32, and schizophrenia33, but also neurodegeneration34 and cancer35, have been established in zebrafish that provide unique opportunities for determining microglial response and behavior in disease conditions.
Notably, this tracking protocol is highly versatile and could also be instrumental for shedding light on the migration patterns of various cell types across diverse anatomical regions of the zebrafish embryo, thus potentially opening up avenues for additional applications, beyond the microglial investigation scope described in this article. Moreover, by harnessing the ability to combine multiple fluorescent transgenic lines, we gain the ability to discern the spatial relationship between microglia and other cell types of the brain microenvironment, with the potential to visualize cellular interactions and cross-talks throughout timelapse recordings, in a non-invasive way. This could be instrumental in unraveling the physiological significance of microglia behavior and contribute to a deeper characterization of these highly motile cells.
The authors have nothing to disclose.
The authors would like to express their sincere gratitude to Professor Nicolas Bayens for generously providing access to the confocal microscope essential for this study. This work was funded in part by the Funds for Scientific Research (FNRS) under Grant Numbers F451218F and UG03019F, the Alzheimer Research Foundation (SAO-FRA) (to V.W.), A.M. is supported by a Research Fellowship from the FNRS. Figure 1 was created on biorender.com.
1 L Breeding tanks | Tecniplast | ZB10BTE | |
1-phenyl-2-thiourea (PTU) | Sigma-Aldrich | P7629 | Diluted to 0.2 mM in E3 to prevent embryo pigmentation |
Bottom glass imaging dish | FluoroDish | FD3510-100 | |
Disposable Graduated transfer pipette | avantor | 16001-188 | |
Dry block heater | Novolab | Grant QBD4 | To keep low melting agarose at 37 °C |
Ethyl 3-aminobenzoate methanesulfonate (Tricaine) | Sigma-Aldrich | E10521-50G | |
Imaris 10.0 | Oxford Instruments | analysis software | |
Imaris File Converter | Oxford Instruments | https://imaris.oxinst.com/big-data | |
Laser-scanning confocal microscope | Nikon | Eclipse Ti2-E | |
Methylene blue | Sigma-Aldrich | M9140-25G | |
microloader tips | Eppendorf | 5242956003 | |
NuSieve GTG Agarose | Lonza | 50081 | |
Petri dishes (90 mm) | avantor | 391-0559 | |
Pronase | Sigma-Aldrich | 11459643001 | |
Stainless Steel Forceps Dumont No. 5 | FineScienceTools | 11254-20 | |
Stereo microscope | Leica | Leica M80 | To mount the embryos |
teasing needle | avantor | 76549-024 |
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