Ultrastuctural Analysis of Dark Microglia

Ultrathin sections from the different brain regions and experimental conditions were examined and photographed at various magnifications ranging between 440× and 9,300× using an ORCA‐HR digital camera (10 MP; Hamamatsu). Profiles of neurons, synaptic elements, microglia, astrocytes, oligodendrocytes, and myelinated axons were identified according to well‐established criteria (Peters et al., 1991). In addition to their immunoreactivity for IBA1 or GFP (in the CX₃CR1‐GFP mice), microglial cells were distinguished from oligodendrocytes by their paler cytoplasm, prevalent association with the extracellular space, distinctive long stretches of endoplasmic reticulum, frequent vacuoles and cellular inclusions, irregular contours with obtuse angles, and small elongated nucleus delineated by a narrow nuclear cistern (Milior et al., in press; Tremblay et al., 2010a). To assess colocalization of the dark microglia with various markers, ultrastructural observations were conducted at the tissue–resin border, where the penetration of antibodies and staining intensity is maximal (Tremblay et al., 2010b). This analysis was strictly conducted in tissue areas where intense immunostaining was observed. For cases where no colocalization was detected, the presence of immunostaining in the same field of view ruled out the possibility that these cells were not stained due to a limited penetration of the antibodies.
To analyze dark microglia's and “normal” microglia's density across control conditions, chronic stress, aging, fractalkine signaling deficiency, and AD pathology, one ultrathin section containing the hippocampus CA1 strata radiatum and lacunosum‐moleculare was sampled in each of three mice per group (3‐month C57Bl/6J control, 14‐month C57Bl/6J control, 3‐month CX₃CR1 knockout, 3‐month stressed C57Bl/6J, 3‐month stressed CX₃CR1 knockout, and 14‐month APP‐PS1 model), for a total neuropil surface of ∼400,000 μm² sampled in each animal. The entire section area was sequentially imaged at lowest magnification under the transmission electron microscope (440×) to determine systematically the total number of grid squares enclosing tissue from each of stratum radiatum and lacunosum‐moleculare. These two neuropil layers were identified based on their position to the CA1 pyramidal cell layer, as well as their cellular and subcellular contents. The total surface area was calculated at high precision by multiplying the number of grid squares containing each of stratum radiatum or lacunosum‐moleculare by the area of a single grid square. A schematic representation of all the grid squares included in the analysis was drawn for each section/animal. The ultrathin sections were afterward rigorously screened for the presence of dark microglia, strictly identified based on a series of ultrastructural features that are described in detail in the Results section. Only dark microglia showing a complete or a partial profile where part of the nucleus could be seen were included in the analysis, considering that the chromatin pattern is a distinctive feature of the dark microglia. Each dark microglia was photographed at magnifications between 4,600× and 9,300×, and marked on the schematic representation, for a total of 95 cells included in the analysis. Considering the heterogeneity in dark microglia's distribution, with these cells generally appearing within clusters, and the impossibility to identify them with light microscopy (see Discussion section), and hence to select the areas to examine based on their presence, their density was expressed as maximal numbers per mm² of tissue surface across three animals/experimental conditions. The density of normal microglia was assessed in the same manner to allow for comparison. We did not attempt to distinguish normal microglia from bone marrow‐derived macrophages and other types of myeloid cells in the brain. In addition, using the same samples, we determined the percentage of dark microglia that were: (1) located in stratum radiatum versus lacunosum‐moleculare, (2) directly apposing one or more blood vessel, and (3) encircling one or more synaptic element (axon terminal, dendritic spine, and excitatory synapse between axon terminal and dendritic spine) with their processes.