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Microglia sculpt developing neural circuits by eliminating excess synapses in a process called synaptic pruning, by removing apoptotic neurons, and by promoting neuronal survival. To elucidate the role of microglia during embryonic and postnatal brain development, we used a mouse model deficient in microglia throughout life by deletion of the fms-intronic regulatory element (FIRE) in the Csf1r locus. Surprisingly, young adult Csf1r ΔFIRE/ΔFIRE mice display no changes in excitatory and inhibitory synapse number and spine density of CA1 hippocampal neurons compared with Csf1r + /+ littermates. However, CA1 neurons are less excitable, receive less CA3 excitatory input and show altered synaptic properties, but this does not affect novel object recognition. Cytokine profiling indicates an anti-inflammatory state along with increases in ApoE levels and reactive astrocytes containing synaptic markers in Csf1r ΔFIRE/ΔFIRE mice closely resemble the effects of acute microglial depletion in adult mice after normal development. Our findings suggest that microglia are not mandatory for synaptic pruning, and that in their absence pruning can be achieved by other mechanisms.ΔFIRE/ΔFIRE mice closely resemble the effects of acute microglial depletion in adult mice after normal development. Our findings suggest that microglia are not mandatory for synaptic pruning, and that in their absence pruning can be achieved by other mechanisms., (© 2024. The Author(s).)

Neuroinflammation is highly influenced by microglia, particularly through activation of the NLRP3 inflammasome and subsequent release of IL-1β. Extracellular ATP is a strong activator of NLRP3 by inducing K + efflux as a key signaling event, suggesting that K + -permeable ion channels could have high therapeutic potential. In microglia, these include ATP-gated THIK-1 K + channels and P2X7 receptors, but their interactions and potential therapeutic role in the human brain are unknown. Using a novel specific inhibitor of THIK-1 in combination with patch-clamp electrophysiology in slices of human neocortex, we found that THIK-1 generated the main tonic K + conductance in microglia that sets the resting membrane potential. Extracellular ATP stimulated K + efflux in a concentration-dependent manner only via P2X7 and metabotropic potentiation of THIK-1. We further demonstrated that activation of P2X7 was mandatory for ATP-evoked IL-1β release, which was strongly suppressed by blocking THIK-1. Surprisingly, THIK-1 contributed only marginally to the total K + conductance in the presence of ATP, which was dominated by P2X7. This suggests a previously unknown, K + -independent mechanism of THIK-1 for NLRP3 activation. Nuclear sequencing revealed almost selective expression of THIK-1 in human brain microglia, while P2X7 had a much broader expression. Thus, inhibition of THIK-1 could be an effective and, in contrast to P2X7, microglia-specific therapeutic strategy to contain neuroinflammation., (© 2024. The Author(s).)

In microglia, changes in intracellular calcium concentration ([Ca 2+ ] i ) may regulate process motility, inflammasome activation, and phagocytosis. However, while neurons and astrocytes exhibit frequent spontaneous Ca 2+ activity, microglial Ca 2+ signals are much rarer and poorly understood. Here, we studied [Ca 2+ ] i changes of microglia in acute brain slices using Fluo-4-loaded cells and mice expressing GCaMP5g in microglia. Spontaneous Ca 2+ transients occurred ~ 5 times more frequently in individual microglial processes than in their somata. We assessed whether microglial Ca 2+ responses change in Alzheimer's disease (AD) using App NL-G-F knock-in mice. Proximity to Aβ plaques strongly affected microglial Ca 2+ activity. Although spontaneous Ca 2+ transients were unaffected in microglial processes, they were fivefold more frequent in microglial somata near Aβ plaques than in wild-type microglia. Microglia away from Aβ plaques in AD mice showed intermediate properties for morphology and Ca 2+ responses, partly resembling those of wild-type microglia. By contrast, somatic Ca 2+ responses evoked by tissue damage were less intense in microglia near Aβ plaques than in wild-type microglia, suggesting different mechanisms underlying spontaneous vs. damage-evoked Ca 2+ signals. Finally, as similar processes occur in neurodegeneration and old age, we studied whether ageing affected microglial [Ca 2+ ] i . Somatic damage-evoked Ca 2+ responses were greatly reduced in microglia from old mice, as in the AD mice. In contrast to AD, however, old age did not alter the occurrence of spontaneous Ca 2+ signals in microglial somata but reduced the rate of events in processes. Thus, we demonstrate distinct compartmentalised Ca 2+ activity in microglia from healthy, aged and AD-like brains., (© 2023. The Author(s).)

Microglia sense their environment using an array of membrane receptors. While P2Y 12 receptors are known to play a key role in targeting directed motility of microglial processes to sites of damage where ATP/ADP is released, little is known about the role of P2Y 13 , which transcriptome data suggest is the second most expressed neurotransmitter receptor in microglia. We show that, in patch-clamp recordings in acute brain slices from mice lacking P2Y 13 receptors, the THIK-1 K + current density evoked by ADP activating P2Y 12 receptors was increased by ~50%. This increase suggested that the P2Y 12 -dependent chemotaxis response should be potentiated; however, the time needed for P2Y 12 -mediated convergence of microglial processes onto an ADP-filled pipette or to a laser ablation was longer in the P2Y 13 KO. Anatomical analysis showed that the density of microglia was unchanged, but that they were less ramified with a shorter process length in the P2Y 13 KO. Thus, chemotactic processes had to grow further and so arrived later at the target, and brain surveillance was reduced by ~30% in the knock-out. Blocking P2Y 12 receptors in brain slices from P2Y 13 KO mice did not affect surveillance, demonstrating that tonic activation of these high-affinity receptors is not needed for surveillance. Strikingly, baseline interleukin-1β release was increased fivefold while release evoked by LPS and ATP was not affected in the P2Y 13 KO, and microglia in intact P2Y 13 KO brains were not detectably activated. Thus, P2Y 13 receptors play a role different from that of their close relative P2Y 12 in regulating microglial morphology and function., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

Microglia provide immune surveillance of the CNS. They display diverse behaviors, including nondirectional and directed motility of their processes, phagocytosis of targets such as dying neurons or superfluous synapses, and generation of reactive oxygen species (ROS) and cytokines. Many of these functions are mediated by ion channels and cell surface receptors, the expression of which varies with the many morphological and functional states that microglial cells can adopt. Recent progress in understanding microglial function has been facilitated by applying classical cell physiological techniques in situ, such as patch-clamping and live imaging, and cell-specific transcriptomic analyses. Here, we review the contribution of microglial ion channels and receptors to microglial and brain function., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Microglia, the brain's innate immune cells, are extremely motile cells, continuously surveying the central nervous system (CNS) to serve homeostatic functions and to respond to pathological events. In the healthy brain, microglia exhibit a small cell body with long, branched, and highly motile processes, which constantly extend and retract, effectively "patrolling" the brain parenchyma. Over the last decade, methodological advances in microscopy and the availability of genetically encoded reporter mice have allowed us to probe microglial physiology in situ. Beyond their classical immunological roles, unexpected functions of microglia have been revealed, both in the developing and the adult brain: microglia regulate the generation of newborn neurons, control the formation and elimination of synapses, and modulate neuronal activity. Many of these newly ascribed functions depend directly on microglial process movement. Thus, elucidating the mechanisms underlying microglial motility is of great importance to understand their role in brain physiology and pathophysiology. Two-photon imaging of fluorescently labeled microglia, either in vivo or ex vivo in acute brain slices, has emerged as an indispensable tool for investigating microglial movements and their functional consequences. This chapter aims to provide a detailed description of the experimental data acquisition and analysis needed to address these questions, with a special focus on key dynamic and morphological metrics such as surveillance, directed motility, and ramification.

Microglia, the brain's innate immune cells, have highly motile processes which constantly survey the brain to detect infection, remove dying cells, and prune synapses during brain development. ATP released by tissue damage is known to attract microglial processes, but it is controversial whether an ambient level of ATP is needed to promote constant microglial surveillance in the normal brain. Applying the ATPase apyrase, an enzyme which hydrolyzes ATP and ADP, reduces microglial process ramification and surveillance, suggesting that ambient ATP/ADP maintains microglial surveillance. However, attempting to raise the level of ATP/ADP by blocking the endogenous ecto-ATPase (termed NTPDase1/CD39), which also hydrolyzes ATP/ADP, does not affect the cells' ramification or surveillance, nor their membrane currents, which respond to even small rises of extracellular [ATP] or [ADP] with the activation of K + channels. This indicates a lack of detectable ambient ATP/ADP and ecto-ATPase activity, contradicting the results with apyrase. We resolve this contradiction by demonstrating that contamination of commercially available apyrase by a high K + concentration reduces ramification and surveillance by depolarizing microglia. Exposure to the same K + concentration (without apyrase added) reduced ramification and surveillance as with apyrase. Dialysis of apyrase to remove K + retained its ATP-hydrolyzing activity but abolished the microglial depolarization and decrease of ramification produced by the undialyzed enzyme. Thus, applying apyrase affects microglia by an action independent of ATP, and no ambient purinergic signaling is required to maintain microglial ramification and surveillance. These results also have implications for hundreds of prior studies that employed apyrase to hydrolyze ATP/ADP., Competing Interests: The authors declare no conflict of interest.

Microglia exhibit two modes of motility: they constantly extend and retract their processes to survey the brain, but they also send out targeted processes to envelop sites of tissue damage. We now show that these motility modes differ mechanistically. We identify the two-pore domain channel THIK-1 as the main K + channel expressed in microglia in situ. THIK-1 is tonically active, and its activity is potentiated by P2Y 12 receptors. Inhibiting THIK-1 function pharmacologically or by gene knockout depolarizes microglia, which decreases microglial ramification and thus reduces surveillance, whereas blocking P2Y 12 receptors does not affect membrane potential, ramification, or surveillance. In contrast, process outgrowth to damaged tissue requires P2Y 12 receptor activation but is unaffected by blocking THIK-1. Block of THIK-1 function also inhibits release of the pro-inflammatory cytokine interleukin-1β from activated microglia, consistent with K + loss being needed for inflammasome assembly. Thus, microglial immune surveillance and cytokine release require THIK-1 channel activity., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Microglia, the innate immune cells of the CNS, play a pivotal role in brain injury and disease. Microglia are extremely motile; their highly ramified processes constantly survey the brain parenchyma, and they respond promptly to brain damage with targeted process movement toward the injury site. Microglia play a key role in brain development and function by pruning synapses during development, phagocytosing apoptotic newborn neurons, and regulating neuronal activity by direct microglia-neuron or indirect microglia-astrocyte-neuron interactions, which all depend on their process motility. This review highlights recent discoveries about microglial dynamics, focusing on the receptors, ion channels, and signaling pathways involved., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)