Microglial Interactions with Synapses Are Modulated by Visual Experience methods

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    "name": "Microglial Interactions with Synapses Are Modulated by Visual Experience methods",
    "description": "Evidence-backed execution summary for Microglial Interactions with Synapses Are Modulated by Visual Experience methods from Microglial Interactions with Synapses Are Modulated by Visual Experience.",
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        "@type": "HowToStep",
        "position": 1,
        "name": "Dynamics of the Structural Relationships between Microglia and Synapses",
        "text": "To characterize the structural dynamics of microglia-synapse interactions, we used two-photon in vivo imaging of layers I/II of V1 in juvenile CX 3 CR1-GFP/Thy1-YFP mice,, in which both microglia and layer V neurons are fluorescently labeled. We utilized a thinned-skull preparation, which minimizes brain injury and allows long-term tracking of microglial dynamics without causing microglial activation ( ). Even though the resolution of two-photon microscopy (see for details on measurement of the experimental point spread function) prevents visualization of direct contacts between fluorescently labeled elements, it enables the study of their structural dynamics and determination of close proximity (the apparent colocalization of fluorescence for microglial and neuronal elements was considered putative contact). A recent study demonstrated a relatively constant duration (4.60\u0011..."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Dynamics of the Structural Relationships between Microglia and Synapses",
        "text": "(A) Time-lapse images showing three dendritic spines (green; red arrowheads) contacted by microglia (yellow; white arrowheads) over 45 min. Scale bar = 5 µm. (B) Dendritic spine size changes during microglial contact. (C) Dendritic spine size before, during, and after contact. (D) Correlation between the initial dendritic spine size and the change in spine size during contact. (E) Average size changes in the presence versus in the absence of microglial contact for large and small dendritic spines (mean ± SEM). (F) Change in small dendritic spine size during microglial contact. (G) Images from chronic experiments showing the elimination of a dendritic spine that had been contacted by a microglial process over 2 d (white arrowhead during the contact; orange arrowhead after the contact), while the other contacted (white arrowheads during the contact; red arrowheads..."
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        "@type": "HowToStep",
        "position": 3,
        "name": "Dynamics of the Structural Relationships between Microglia and Synapses",
        "text": "To verify whether microglial processes target specific subsets of synapses, we measured the size of dendritic spines and axon terminals in the presence and absence of a putative microglial contact. In this analysis, we noticed that dendritic spines that were in close proximity to microglial processes at any point during the imaging session were generally smaller than the rest of the spine population. We quantitatively recorded spine size at the beginning of imaging and found that dendritic spines receiving putative contact during imaging were significantly smaller than spines remaining non-contacted ( p <0.001; n = 31 contacted spines in five animals and 45 non-contacted spines in three animals, with infrequent stubby spines excluded; see ). When microglia came into close proximity with these synaptic structures, individual pre- and postsynaptic elements both expanded and..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Dynamic Relationships between Microglia and Synapses during Alterations in Sensory Experience",
        "text": "To assess the dynamic changes in microglia-synapse interactions during visual deprivation, we used two-photon imaging of layers I/II of V1 in juvenile mice that were subjected to DA for 8-10 d, from the beginning to the peak of the critical period. Microglial processes appeared thickened and sparse, and more often terminated into crown-like structures resembling phagocytic cups than in control animals (;, as well as and and for comparison of microglial morphology in control and DA animals). We also found that the average motility of microglial processes was significantly reduced ( ) when assayed in two ways: comparing morphology over a 5-min interval (motility index; control: 8%±0.8%; DA: 6%±0.5%; p <0.05; n = 10 microglia in four control animals and 8 microglia in four DA animals) and over a 25-min interval, where the difference between control a..."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Dynamic Relationships between Microglia and Synapses during Alterations in Sensory Experience",
        "text": "(A) Time-lapse images taken in a DA animal showing a microglial process that terminates into a structurally stable phagocytic cup (white arrow), while another process dynamically contacts two dendritic spines (red arrowheads before the contact; white arrowheads during the contact). Scale bar = 5 µm. (B) Time-lapse images showing another example of a structurally stable phagocytic cup (white arrow) in a DA animal. Scale bar = 10 µm. (C) Time-lapse images showing the motility of microglia during normal visual experience over three time points, 0, 5, and 25 min. Images from each time point were colored in red, green, and blue, respectively, and then merged to reveal microglia-associated pixels that were unchanged throughout the three time points (stable; white), changed in one of the three time points (dynamic; yellow and fuchsia), or changed in two of t..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "Microglial Apposition and Phagocytosis of Synaptic Elements",
        "text": "Immunocytochemical EM and SSEM with 3-D reconstructions enabled us to analyze the morphology of microglial processes and their ultrastructural relationships with the other subcellular compartments of neuropil-astrocytic processes, axon terminals, and dendritic spines-in situ at high spatial resolution. Building on previous EM observations that microglia contact axon terminals and dendritic spines,,, our quantitative analysis revealed that most microglial processes directly appose not only axon terminals and dendritic spines, but also perisynaptic astrocytic processes and synaptic clefts. Our SSEM with 3-D reconstructions also uncovered the 3-D relationships between microglia and synapses, revealing that microglial processes contact multiple synapse-associated elements at multiple synapses simultaneously. Additionally, we found clathrin-coated pits at interfaces between..."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Motility of Microglial Processes",
        "text": "Several lines of evidence, including those presented here, suggest that microglial motility may be regulated by neuronal activity. A pioneer study reported an increase in the volume sampled by quiescent microglia in vivo over a period of 1 h after application of the ionotropic GABA receptor blocker bicuculline, whereas the sodium channel blocker tetrodotoxin had no significant effects. More recently, microglia were shown to retract their processes and reduce their frequency of contact with axon terminals in vivo, over a period of 4-6 h after binocular enucleation or intraocular injection of tetrodotoxin. In our study, two-photon analysis revealed a global decrease in microglial motility during light deprivation without correlated changes in the duration or frequency of microglial contact with individual dendritic spines. This may highlight differences between short-term (1R..."
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        "position": 8,
        "name": "Materials and Methods",
        "text": "Animals were treated in strict accordance with the University of Rochester Committee on Animal Resources and the United States National Institutes of Health standards. Light-reared animals were housed under a fixed 12-h light/dark cycle; DA animals were placed in complete darkness for 6-10 d, from P20-P22 until P28-P32 (the onset of experimentation); DA+light animals were housed under a fixed 12-h light/dark cycle for 2 d following DA until P29-P32 (the onset of experimentation). For immunocytochemical EM and SSEM, eight C57Bl/6 mice (P28) were anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and perfused through the aortic arch with 3.5% acrolein followed by 4% paraformaldehyde as previously described, or with 2.75% glutaraldehyde in 2% paraformaldehyde to compare extracellular space areas surrounding microglia between both types of fixatives. For two-..."
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    "tool": [
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        "@type": "HowToTool",
        "name": "Dynamics of the Structural Relationships between Microglia and Synapses"
      },
      {
        "@type": "HowToTool",
        "name": "Two-Photon In Vivo Imaging and Data Analysis"
      },
      {
        "@type": "HowToTool",
        "name": "Dynamics of the Structural Relationships between Microglia and Synapses"
      },
      {
        "@type": "HowToTool",
        "name": "Dynamic Relationships between Microglia and Synapses during Alterations in Sensory Experience"
      },
      {
        "@type": "HowToTool",
        "name": "Dynamic Relationships between Microglia and Synapses during Alterations in Sensory Experience"
      },
      {
        "@type": "HowToTool",
        "name": "Microglial Apposition and Phagocytosis of Synaptic Elements"
      },
      {
        "@type": "HowToTool",
        "name": "Dynamic Microglial Interaction with Subsets of Dendritic Spines"
      },
      {
        "@type": "HowToTool",
        "name": "Motility of Microglial Processes"
      }
    ],
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      {
        "@type": "HowToSupply",
        "name": "Immunoperoxidase Staining for Light Microscopy and EM"
      },
      {
        "@type": "HowToSupply",
        "name": "Immunoperoxidase Staining for Light Microscopy and EM"
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    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Microglial Interactions with Synapses Are Modulated by Visual Experience",
      "datePublished": "2010",
      "author": [
        {
          "@type": "Person",
          "name": "Marie-Ève Tremblay"
        },
        {
          "@type": "Person",
          "name": "Rebecca L. Lowery"
        },
        {
          "@type": "Person",
          "name": "Ania K. Majewska"
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      ],
      "identifier": "10.1371/journal.pbio.1000527"
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