Name: Live Imaging and Characterization of Microglia Dynamics and Interactions with Synapses in Diseased Murine Retina (Video) | JoVE Journal
Uploaded: 2026-01-16T12:50:00.000+00:00
Duration: 7 min 19 s
Description: Discover microglia dynamics and synaptic interactions in mouse retina using spinning disk confocal microscopy and machine learning segmentation. This ex vivo protocol enables high-throughput quantification of motility and synaptic contacts in neurodegenerative disease models.

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PSD-95pTagRFP was a gift from Johannes Hell (Addgene plasmid #52671; RRID: Addgene_52671), while the capsid version, 7m8, was a gift from John Flannery and David Schaffer (Addgene plasmid # 64839; RRID: Addgene_64839). We would like to thank Aparna Lakkaraju for allowing us to use their lab&#39;s spinning disk microscope and Nilsa La Cunza for guidance in live imaging. We would also like to thank Felice Dunn, Annika Balraj, and Luca Della Santina for helpful discussions and comments on this manuscript. We thank Suling Wang for assistance in data analysis on Imaris.&#160;This work was funded by NIH-NEI EY028148 and EY034973 to Y.O., the ARVO David L. Epstein award to Y.O., and NIH-NEI EY034973-S1 to C.F.. This research was also supported, in part, by the UCSF Vision Core shared resource of the NIH-NEI P30 EY002162/EY037668, and by an unrestricted grant from Research to Prevent Blindness, New York, NY. Figure 1 was created on biorender.com.

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<li>Xu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+changes+and+the+response+of+microglia+in+a+light-induced+photoreceptor+degeneration+model%5BTitle%5D)+AND+%22Mol+Vis%22%5BJournal%5D)">Synaptic changes and the response of microglia in a light-induced photoreceptor degeneration model.</a> Mol Vis. 27, 206-220 (2021).</li>
<li>Paolicelli, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+states+and+nomenclature%3A+a+field+at+its+crossroads%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Microglia states and nomenclature: a field at its crossroads.</a> Neuron. 110 (21), 3458-3483 (2022).</li>
<li>Yu, Z., Gutu, A., Kim, N., O'Shea, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synapse+weakening-induced+caspase-3+activity+confers+specificity+to+microglia-mediated+synapse+elimination%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Synapse weakening-induced caspase-3 activity confers specificity to microglia-mediated synapse elimination.</a> bioRxiv. , (2024).</li>
<li>Miller, E. B., Zhang, P., Ching, K., Pugh, E. N., Burns, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+reveals+transient+microglia+recruitment+and+functional+recovery+of+photoreceptor+signaling+after+injury%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">In vivo imaging reveals transient microglia recruitment and functional recovery of photoreceptor signaling after injury.</a> Proc Natl Acad Sci USA. 116 (33), 16603-16612 (2019).</li>
<li>Wahl, D. J., Ng, R., Ju, M. J., Jian, Y., Sarunic, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensorless+adaptive+optics+multimodal+en-face+small+animal+retinal+imaging%5BTitle%5D)+AND+%22Biomed+Opt+Express%22%5BJournal%5D)">Sensorless adaptive optics multimodal en-face small animal retinal imaging.</a> Biomed Opt Express. 10 (1), 252(2019).</li>
<li>Ou, Y., Jo, R. E., Ullian, E. M., Wong, R. O. L., Della Santina, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+vulnerability+of+specific+retinal+ganglion+cell+types+and+synapses+after+transient+ocular+hypertension%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Selective vulnerability of specific retinal ganglion cell types and synapses after transient ocular hypertension.</a> J Neurosci. 36 (35), 9240-9252 (2016).</li>
<li>Della Santina, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disassembly+and+rewiring+of+a+mature+converging+excitatory+circuit+following+injury%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Disassembly and rewiring of a mature converging excitatory circuit following injury.</a> Cell Rep. 36 (5), 109463(2021).</li>
<li>Ramírez, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+changes+in+the+early+aging+stage+in+a+healthy+retina+and+an+experimental+glaucoma+model%5BTitle%5D)+AND+%22Prog+Brain+Res%22%5BJournal%5D)">Microglial changes in the early aging stage in a healthy retina and an experimental glaucoma model.</a> Prog Brain Res. 256, 125-149 (2020).</li>
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<li>Soliño, M., Yu, A., Della Santina, L., Ou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+survey+of+excitatory+synapses+reveals+sublamina-specific+and+asymmetric+synapse+disassembly+in+a+neurodegenerative+circuit%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">Large-scale survey of excitatory synapses reveals sublamina-specific and asymmetric synapse disassembly in a neurodegenerative circuit.</a> iScience. 26 (8), 107262(2023).</li>
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</ol>

The authors have no conflicts of interest to declare.

This protocol enables the visualization and tracking of individual microglia interaction with postsynaptic sites along the dendrites of RGCs. In a neurodegenerative disease such as glaucoma, microglia colocalize with synapses in the inner plexiform layer (IPL) of the retina. The role of microglia in synapse disassembly is not well understood. However, microglia potentially contribute via several potential mechanisms, including aberrantly engulfing synapses, passively clearing debris from apoptosing cells, or a combinatio...

Circuit function and homeostasis rely on highly regulated synapse firing and other unique interactions among various cell types1. Serving as the resident macrophages of the central nervous system (CNS), microglia are a non-neuronal population in the retina that play a unique role in synaptic pruning. During CNS development, microglia prune non-functional or weak synapses to refine neural circuitry2. Microglia in the developing brain survey their environment and are primed towards a neuroprotective role during critical periods3. In visual system development, microglia are key regulators in the development of cortical neurons and interneurons, in addition to exhibiting morphological changes during critical periods of synaptic remodeling4. Similarly, in the developing retina, microglia clear apoptotic cells to make way for new synaptic connections, confirmed with upregulation of homeostatic marker P2Ry12 during these clearing events5. However, recent findings highlight that microglia may not play a definitive role in refining the neural circuitry in the developing visual system6, emphasizing that there is controversy regarding the role microglia play in visual circuit development.
In injury contexts, microglia change morphology to an active state and can play a neurodegenerative role. For example, microglia in Alzheimer&#39;s disease models demonstrated increased engulfment of amyloid-beta plaques7 and synaptic markers such as SPH and PSD958. In retinal neurodegeneration, many research groups have found microglia with engulfed synaptic material, but no clear evidence of active engulfment of intact synapses9,10,11,12,13. For example, He et al. found that microglia increase in number and have increased volume of engulfed synaptic material in a model of retinitis pigmentosa9, but it is not clear whether there was phagocytosis of intact synapses versus clearance of debris. Additionally, traditional microglial classifications -- such as &#34;resting vs. activated&#34; or &#34;M1 vs. M2&#34; -- are oversimplified and increasingly inaccurate given modern insights. Microglia exist in a gradient of states, not discrete categories12. Hence, Paolicelli et al. called for a shift from outdated binary classifications toward a more nuanced, rigorously defined nomenclature for microglial states to better understand and define microglia morphology and activity12. Therefore, strategies to evaluate microglia function ex vivo and in vivo will augment the field&#39;s ability to discern microglia states and activity in both development and disease contexts.
While strategies exist to study microglia function both ex vivo and in vivo, ex vivo live imaging has several benefits. First, corneal clarity or cataract development will not impede retinal imaging, creating clear visualization of minute processes and synaptic puncta. Further, in vivo imaging&#160;requires more technical skill to set up, which makes ex vivo preparations&#160;higher throughput setup, resulting in more animals imaged per experiment. Despite the theoretical benefits that in vivo imaging may allow, high laser power (100-230 &#181;W) is required to capture microglia process motility for longitudinal imaging14,15. However, ex vivo imaging of microglia with a lower laser power and adequate imaging media can provide the same imaging quality as in vivo imaging with the right preparation, although the tissue is no longer in situ.
We have demonstrated that microglia increase in number, complexity, and process movement after transient intraocular pressure (IOP) elevation10. In fixed tissue, this microgliosis results in increased colocalization with synaptic proteins10. Previous work has also demonstrated early synapse loss and dendrite degeneration of retinal ganglion cells (RGCs) in the murine laser-induced ocular hypertension model (LIOH) and other neurodegenerative models10,16,17,18,19,20. However, it is still unknown whether they play an active role in pruning synapses or a more passive role in clearing cellular debris, including disassembled synapses. As synapse disassembly is an early hallmark of neurodegeneration, understanding microglia dynamics and their role in synapse disassembly is critical8,10. Here, we use live imaging to understand microglia dynamics and their interaction with excitatory postsynaptic density protein-95 (PSD95) on the dendrites of RGCs.

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live imaging and characterization of microglia dynamics and interactions with synapses in diseased murine retina

Live Imaging and Characterization of Microglia Dynamics and Interactions with Synapses in Diseased Murine Retina

Microglia are the resident macrophages of the central nervous system (CNS) that respond to tissue infection and injury. In addition to their role in inflammation, microglia play a developmental role in circuit refinement through synaptic pruning. However, the mechanisms of synaptic pruning in neuroinflammation and neurodegeneration remain unknown. In this protocol, we use a mouse retina explant model to study microglia dynamics ex vivo. To examine microglia motility and their interactions with postsynaptic proteins, we label synapses with AAV-PSD95-RFP and record timelapse videos of motile microglia colocalized with postsynaptic proteins using spinning disk confocal microscopy. We then create surface and spot reconstructions of microglia and PSD95 using image analysis software. Data such as microglia displacement length, process speed, and contact with postsynaptic puncta can then be extracted from these surfaces to understand microglia behavior both in homeostatic states and after neuronal injury. This protocol can be useful in examining the role of microglia in synaptic pruning in retinal neurodegenerative diseases.

Microglia from CX3CR1-GFP murine retinas were imaged using the described protocol. Mice ranged from P30 to P60 at the time of AAV-PSD95-RFP injection, and at least 21 days had elapsed to allow for AAV expression. Representative images of microglia and their interactions with PSD95 puncta before and after analysis are shown in Figure 2. To induce microglia activation, we used the murine laser-induced ocular hypertension (LIOH) model, a model that transiently e...

This paper demonstrates a method for visualizing and quantifying microglia motility and contact with postsynaptic puncta in the retina using spinning disk confocal microscopy.

Microglia are the resident macrophages of the central nervous system (CNS) that respond to tissue infection and injury. In addition to their role in ...

The scope of our lab's research is to study the underlying mechanisms of glaucoma and microglia in synapse remodeling during development. The current experimental challenges revolve around whether or not microglia are actively disassembling retinal ganglion cell synapses or whether they're just passively clearing debris. Begin with mouse retina positioned on a filter paper and dry it with a laboratory wipe.Invert the retina onto a MatTek Petri dish. Weigh down the retina using either metal beads or a ring. Then, add three to five drops of artificial cerebrospinal fluid.Place the sample under a spinning disc confocal microscope and adjust the settings to preview the retina. Collect images along the Z axis at 10 to 20 micrometers total depth using a step size of 0.3 micrometers. Maintain the time interval between frames at 20 to 30 seconds to allow for reliable cell tracking.To perform surface rendering of the microglia, convert the time lapse file into IMS format using the file converter software. Input the voxel sizes based on the previously acquired image properties. In the analysis software arena, click on the observe folder to open the converted file and ensure the image is in 3D view.To detect individual microglia, click the add new surface icon in the object toolbar. Select segment only a region of interest, track surfaces over time, classify surfaces, and object-object statistics under algorithm settings. Then, click the blue play button to proceed.Choose the channel for surface rendering. Optionally, enable smooth to smooth the surface creation and enter the surface detail. Under thresholding, choose machine learning segmentation for detecting cells.Under training data, use the background and foreground paintbrush tools. Draw paintbrush strokes by selecting the desired paintbrush and using the shift plus left click. Ensure interpolate display is enabled, then select train and predict.Use the yellow pointer in the middle to navigate across the Z axis. Draw multiple strokes across the X, Y, and Z planes, and across a few timeframes. Use three to five short strokes per entry, iteratively adjusting until an accurate surface is made.Use the yellow rectangle to toggle between the training display and the actual surface created as a final check. Also, check across the time lapse that the surface matches the cell morphology. Once done, deselect split objects.Adjust the voxel histogram threshold to remove small unconnected objects not part of the primary surface. Then, edit the surface manually to remove extraneous objects or cut edges using the scissor tool. If desired, set a threshold of gaps allowed for object and select the tracking algorithm for the moving cell.Filter track objects not associated with the main surface. Once finished, set the moving cells display to a transparent profile to visualize the puncta for the next steps. To detect individual PSD95 puncta, click on the blue surface icon in the object toolbar.Then, select track spots over time, classify spots, and object-object statistics and press play. Select the source channel for the synaptic marker. Turn off slicer and other channels to visualize only PSD95.Use the control button and the pointer selection tool to estimate the XY diameter of the puncta. Pick a few bright puncta that persist across frames and turn off background subtraction. Next, add a filter based on spot quality.Adjust the histogram to include accurate PSD95 puncta across all time frames. Remove the false positive spots via edit. Toggle between the cube or circle cursor to delete spots lasting only one or two frames.If desired, set a threshold of gaps allowed and choose the tracking algorithm for synaptic spots. Filter spot tracks not associated with real puncta using a histogram threshold to exclude small object tracks. Define classifications using the shortest distance to surface classification.Set engulfed as any puncta less than zero micrometers, contacted as approximately between zero to 0.5 micrometers from the surface, and uncontacted as 0.5 plus micrometers. Assign labels to events using the classifications to find above. Once the surfaces and spots appropriately represent microglia and puncta, export all statistics under the statistics tab.Microglia exhibited increased process displacement length after laser-induced ocular hypertension compared to the control. Microglial speed was significantly higher in laser-treated eyes than in controls. The number of contact events between microglia and PSD95 puncta increased following laser treatment.Time-lapse recordings showed that laser-treated retinas exhibited greater microglial motility and increased interactions with PSD95 puncta compared to control retinas. Our significant findings have shown that there's increased microglia number, complexity, and motility, and synaptic colocalization following transient intraocular pressure elevation in mice. Our protocol offers ex vivo imaging, which helps avoid cataract and corneal opacity issues, enabling easier setup for faster experiments and overall higher throughput.Our findings advance research by enabling visualization of microglia-neuron interactions, cellular communication and dynamics using transgenic fluorescent lines, as well as high-resolution time-lapse imaging.

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JoVE Journal

Neuroscience

Source: Maruoka, H., et. al. Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice. J. Vis. Exp. (2022)     This video shows a two-photon microscopy procedure for imaging neuronal activity and microglial dynamics in an awake mouse during visual stimulation, highlighting calcium transients and cellular interactions.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

Source: Bosco, A., et al. In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma. J. Vis. Exp. (2015) This video demonstrates in vivo imaging of retinal microglia in a mouse model of glaucoma. A transgenic mouse with fluorescent protein-expressing retinal microglia is positioned under an ophthalmoscope. Infrared illumination is used to locate the optic nerve head (ONH), and the focal plane is...

In Vivo Imaging of Retinal Microglia in a Mouse Model of Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

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.

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

We describe a method for repeatedly visualizing murine microglia and circulating monocytes in vivo over hours, days or weeks using transcranial two-photon microscopy. We demonstrate how to prepare a thinned-skull window that allows intermittent observation of quiescent microglia that can be activated by adjacent stereotactic injection of the HIV-1 regulatory protein Tat.

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Discover microglia dynamics and synaptic interactions in mouse retina using spinning disk confocal microscopy and machine learning segmentation. This ex vivo protocol enables high-throughput quantification of motility and synaptic contacts in neurodegenerative disease models.

Live Imaging and Characterization of Microglia Dynamics and Interactions with Synapses in Diseased Murine Retina

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