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The green fluorescent protein (GFP) is a powerful reporter protein that allows labeling of specific proteins or entire cells. However, as GFP is a small soluble protein, it easily crosses membranes if cell integrity is disrupted, and GFP signal is lost or diffuse if the specimen is not fixed beforehand. While pre-fixation is often feasible for histological analyses, many molecular biology procedures and new imaging techniques, such as imaging mass spectrometry, require unfixed specimens. To be able to use GFP labeling in tissues prepared for such applications, we have tested various protocols to minimize the loss of GFP signal. Here we show that, in cryocut sections of snap-frozen brain tissue from two GFP reporter mouse lines, leaking of the GFP signal is prevented by omitting the commonly performed drying of the cryosections, and by direct post-fixation with 4% paraformaldehyde pre-warmed at 30–37 °C. Although the GFP staining does not reach the same quality as obtained with pre-fixed tissue, GFP localization within the cells that express it is preserved with this method. This protocol can thus be used to identify GFP positive cells on sections originating from unfixed, cryosectioned tissue.

Microglia, the resident immune cells of the brain, are increasingly implicated in the regulation of brain health and disease. Microglia perform multiple functions in the central nervous system, including surveillance, phagocytosis and release of a variety of soluble factors. Importantly, a majority of their functions are closely related to changes in their metabolism. This natural inter-dependency between core microglial properties and metabolism offers a unique opportunity to modulate microglial activities via nutritional or metabolic interventions. In this review, we examine the existing scientific literature to synthesize the hypothesis that microglial phagocytosis of amyloid beta (Aβ) aggregates in Alzheimer’s disease (AD) can be selectively enhanced via metabolic interventions. We first review the basics of microglial metabolism and the effects of common metabolites, such as glucose, lipids, ketone bodies, glutamine, pyruvate and lactate, on microglial inflammatory and phagocytic properties. Next, we examine the evidence for dysregulation of microglial metabolism in AD. This is followed by a review of in vivo studies on metabolic manipulation of microglial functions to ascertain their therapeutic potential in AD. Finally, we discuss the effects of metabolic factors on microglial phagocytosis of healthy synapses, a pathological process that also contributes to the progression of AD. We conclude by enlisting the current challenges that need to be addressed before strategies to harness microglial phagocytosis to clear pathological protein deposits in AD and other neurodegenerative disorders can be widely adopted.

Microglia, the innate immune cells of the central nervous system, actively participate in brain development by supporting neuronal maturation and refining synaptic connections. These cells are emerging as highly metabolically flexible, able to oxidize different energetic substrates to meet their energy demand. Lactate is particularly abundant in the brain, but whether microglia use it as a metabolic fuel has been poorly explored. Here we show that microglia can import lactate, and this is coupled with increased lysosomal acidification. In vitro, loss of the monocarboxylate transporter MCT4 in microglia prevents lactate-induced lysosomal modulation and leads to defective cargo degradation. Microglial depletion of MCT4 in vivo leads to impaired synaptic pruning, associated with increased excitation in hippocampal neurons, enhanced AMPA/GABA ratio, vulnerability to seizures and anxiety-like phenotype. Overall, these findings show that selective disruption of the MCT4 transporter in microglia is sufficient to alter synapse refinement and to induce defects in mouse brain development and adult behavior. The role of lactate in the control of microglial function remains poorly investigated. Here, the authors show that lactate promotes lysosomal acidification in microglia, and that mice lacking the lactate transporter MCT4 in these cells display defective brain development and anxiety-like behavior.

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

[...]computational and modeling approaches, alongside with integrative –omics (epigenetic, transcriptomic, proteomic, and metabolomic) will come to maturity and help us understand the impact of microglia in brain physiology. [...]a series of papers deal with microglial biology, including the study of autophagy (Plaza-Zabala et al.); and microglial function, including their role in synapse remodeling (Brioschi et al.,Morini et al.) and as therapeutic targets in disease (Chen et al.,Liu et al.,Aires et al.). [...]we hope this collection of systematic reviews and original methods may serve as a reference for the field, and may promote a culture of exchange for synergistically improve the current techniques available to assess microglial identity and function. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 1GinhouxFGreterMLeboeufMNandiSSeePGokhanS.Fate Mapping Analysis Reveals That Adult Microglia Derive From Primitive Macrophages.Science(2010)330:841–5. doi:10.1126/science.1194637 2GoldmannTWieghoferPMüllerPFWolfYVarolDYonaS.A New Type of Microglia Gene Targeting Shows TAK1 to be Pivotal in CNS Autoimmune Inflammation.Nat Neurosci(2013)16:1618–26. doi:10.1038/nn.3531 3YonaSKimK-WWolfYMildnerAVarolDBrekerM.Fate Mapping Reveals Origins and Dynamics of Monocytes and Tissue Macrophages Under Homeostasis.Immunity(2013)38:79–91. doi:10.1016/j.immuni.2012.12.001 4DavalosDGrutzendlerJYangGKimJVZuoYJungS.ATP Mediates Rapid Microglial Response to Local Brain Injury In Vivo.Nat Neurosci(2005)8(6):752–8. doi:10.1038/nn1472 5NimmerjahnAKirchhoffFHelmchenF.Resting Microglial Cells are Highly Dynamic Surveillants of Brain Parenchyma In Vivo.Science(2005)308(5726):1314–8. doi:10.1126/science.1110647 6CserépCPósfaiBLénártNFeketeRLászlóZILeleZ.Microglia Monitor and Protect Neuronal Function Through Specialized Somatic Purinergic Junctions.Science(2020)367(6477):528–37. doi:10.1126/science.aax6752 7SierraAPaolicelliRCKettenmannH.Cien Años De Microglía: Milestones in a Century of Microglial Research.Trends Neurosci(2019)42(11):778–92. doi:10.1016/j.tins.2019.09.004

Neural circuits are constantly monitored and supported by the surrounding microglial cells, using finely tuned mechanisms which include both direct contact and release of soluble factors. These bidirectional interactions are not only triggered by pathological conditions as a S.O.S. response to noxious stimuli, but they rather represent an established repertoire of dynamic communication for ensuring continuous immune surveillance and homeostasis in the healthy brain. In addition, recent studies are revealing key tasks for microglial interactions with neurons during normal physiological conditions, especially in regulating the maturation of neural circuits and shaping their connectivity in an activity- and experience-dependent manner. Chemokines, a family of soluble and membrane-bound cytokines, play an essential role in mediating neuron-microglia crosstalk in the developing and mature brain. As part of this special issue on Cytokines as players of neuronal plasticity and sensitivity to environment in healthy and pathological brain, our review focuses on the fractalkine signaling pathway, involving the ligand CX3CL1 which is mainly expressed by neurons, and its receptor CX3CR1 that is exclusively found on microglia within the healthy brain. An extensive literature largely based on transgenic mouse models has revealed that fractalkine signaling plays a critical role in regulating a broad spectrum of microglial properties during normal physiological conditions, especially their migration and dynamic surveillance of the brain parenchyma, in addition to influencing the survival of developing neurons, the maturation, activity and plasticity of developing and mature synapses, the brain functional connectivity, adult hippocampal neurogenesis, as well as learning and memory, and the behavioral outcome.

Editorial on the Research Topic Cell-Cell Interactions Controlling Neuronal Functionality in Health and Disease In the central nervous system (CNS), several cell types interact with each other to promote and protect the homeostatic functions of neuronal cells. Carrier et al. discuss how the cross-talk between microglia and neurons is altered as a consequence of chronic psychological stress, which can accelerate cellular aging, cause neuronal dysfunction, and promote the development of depressive disorder and cognitive decline. [...]Kopeikina and Ponomarev highlight how platelets can infiltrate the CNS during inflammatory responses, and how they directly modulate neuronal activity and induce neurodegeneration. Funding This work was supported by: the Austrian Multiple Sclerosis Research Society and Medical University of Graz (SA), the Italian Ministry of Research and University (PRIN project 2020Z73J5A to EV and grant GR 2016-02363254 to GD'A), the Alzheimer's Association Research Fellowship (AARF, grant 2018-AARF8 588984) (IP), the Synapsis Foundation - Alzheimer Research Switzerland, the Swiss National Science Foundation (SNSF 310030_197940), and the European Research Council (ERC StGrant REMIND 804949) (RP).

C1q is released by microglia, localizes on weak synapses and acts as a tag for microglial synaptic pruning. However, how C1q tags synapses during the pruning period remains to be fully elucidated. Here, we report that C1q is delivered via extracellular vesicles by microglia to pre‐synaptic sites that externalize phosphatidylserine. Using approaches to increase or reduce vesicles production in microglia, by C9orf72 knock out or pharmacological inhibition, respectively, we provided mechanistic evidence linking extracellular vesicle release to pre‐synaptic remodelling in neuron‐microglia cultures. In C9orf72 knockout mice, we confirmed larger production of microglial extracellular vesicles and showed augmented C1q presynaptic deposition associated with enhanced engulfment by microglia in the early postnatal hippocampus. Finally, we provide evidence that microglia physiologically release more vesicles during the period of postnatal circuit refinement. These findings implicate abnormal release of microglial extracellular vesicles in both neurodevelopmental and age‐related disorders characterized by dysregulated microglia‐mediated synaptic pruning. Microglial EVs deliver C1q to pre‐synapses and promote synaptic pruning in vitro. C9orf72 KO microglia release more C1q‐storing EVs compared to WT and engulf more synapses in the hippocampus during the pruning period. Production of C1q‐storing EVs from WT microglia temporally correlates with the pruning period during postnatal development.

Chloride is the most abundant physiological anion and participates in a variety of cellular processes including trans-epithelial transport, cell volume regulation, and regulation of electrical excitability. The development of tools to monitor intracellular chloride concentration ([Cli]) is therefore important for the evaluation of cellular function in normal and pathological conditions. Recently, several Cl-sensitive genetically encoded probes have been described which allow for non-invasive monitoring of [Cli]. Here we describe two mouse lines expressing a CFP-YFP-based Cl probe called Cl-Sensor. First, we generated transgenic mice expressing Cl-Sensor under the control of the mouse Thy1 mini promoter. Cl-Sensor exhibited good expression from postnatal day two (P2) in neurons of the hippocampus and cortex, and its level increased strongly during development. Using simultaneous whole-cell monitoring of ionic currents and Cl-dependent fluorescence, we determined that the apparent EC 50 for Cli was 46 mM, indicating that this line is appropriate for measuring neuronal [Cli] in postnatal mice. We also describe a transgenic mouse reporter line for Cre-dependent conditional expression of Cl-Sensor, which was targeted to the Rosa26 locus and by incorporating a strong exogenous promoter induced robust expression upon Cre-mediated recombination. We demonstrate high levels of tissue-specific expression in two different Cre-driver lines targeting cells of the myeloid lineage and peripheral sensory neurons. Using these mice the apparent EC 50 for Cli was estimated to be 61 and 54 mM in macrophages and DRG, respectively. Our data suggest that these mouse lines will be useful models for ratiometric monitoring of Cli in specific cell types in vivo.