Summary Microglia, the resident macrophages of the central nervous system (CNS), engage in various CNS-specific functions that are critical for development and health. To better study microglia and the properties that distinguish them from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly ramified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components, CSF-1/IL-34, TGF-β2, and cholesterol. Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic and lasting change in phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia requires continuous instructive signaling from the intact CNS.

Article Nanoscale Surveillance of the Brain by Microglia via cAMP-Regulated Filopodia Graphical Abstract Highlights d Microglia use actin-dependent filopodia to efficiently sample the brain parenchyma d Intracellular cAMP drives filopodia growth but induces large process retraction d Norepinephrine and nitric oxide contribute to cAMP-driven filopodia extension d Fine filopodia and large processes establish dual-scale surveillance by microglia This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). relative to variations in the brain parenchyma environment. STAR+METHODSDetailed methods are provided in the online version of this paper and include the following:TABLE d CONTACT FOR REAGENTS AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS B Animals B Acute slice preparation and maintenance B Isolation of rat microglia d METHOD DETAILS B Open cranial window surgery and in vivo two-photon imaging B Brain perfusion, immunofluorescence and confocal imaging B Acute slice two-photon imaging B Slice fixation and immunolabelling B Rat primary microglia live imaging B Western blotting d QUANTIFICATION AND STATISTICAL ANALYSIS B Image processing and analysis B Statistical analysis SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.

Summary Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we develop a rapid magnet-based phenotypic screening strategy, and perform eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused mini-screens, orthogonal assays and primary human macrophages, we demonstrate that 1) the previously-uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, 2) very long chain fatty acids are essential for efficient phagocytosis of certain substrates, and 3) the previously-uncharacterized Alzheimer’s disease-associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

SUMMARY Nervous system function depends on proper myelination for insulation and critical trophic support for axons. Myelination is tightly regulated spatially and temporally, but how it is controlled molecularly remains largely unknown. Here, we identified key molecular mechanisms governing the regional and temporal specificity of central nervous system (CNS) myelination. We show that transcription factor EB (TFEB) is highly expressed by differentiating oligodendrocytes and that its loss causes precocious and ectopic myelination in many parts of the murine brain. TFEB functions cell-autonomously through PUMA induction and Bax/Bak activation to promote programmed cell death of a subset of premyelinating oligodendrocytes, allowing for selective elimination of oligodendrocytes in normally unmyelinated brain regions. This pathway is conserved across diverse brain areas and is critical for myelination timing. Our findings define an oligodendrocyte-intrinsic mechanism underlying the spatiotemporal specificity of CNS myelination, shedding light on how myelinating glia sculpt the nervous system during development.

It was recently found that nociceptive sensations (stinging, pricking, or burning) can be evoked by cooling or heating the skin to innocuous temperatures (e.g., 29°, 37°C). Here we show that this lowthreshold thermal nociception (LTN) can be traced to sensitive 'spots' in the skin equivalent to classically defined warm spots and cold spots. Because earlier work had shown that LTN is inhibited by simply touching a thermode to the skin, a spatial search procedure was devised that minimized tactile stimulation by sliding small thermodes (16 mm 2 and 1 mm 2 ) set to 28° or 36°C slowly across the lubricated skin of the forearm. The procedure uncovered three types of temperature-sensitive sites (thermal, bimodal and nociceptive) that contained one or more thermal, nociceptive or (rarely) bimodal spots. Repeated testing indicated that bimodal and nociceptive sites were less stable over time than thermal sites, and that mechanical contact differentially inhibited nociceptive sensations. Intensity ratings collected over a range of temperatures showed that LTN increased monotonically on heat-sensitive sites but not on cold-sensitive sites. These results provide psychophysical evidence that stimulation from primary afferent fibers with thresholds in the range of warm fibers and cold fibers is relayed to the pain pathway. However, the labile nature of LTN implies that these lowthreshold nociceptive inputs are subject to inhibitory controls. The implications of these findings for the roles of putative temperature receptors and nociceptors in innocuous thermoreception and thermal pain are discussed.