Intracellular calcium dynamics in cortical microglia responding to focal laser injury in the PC::G5-tdT reporter mouse

Abstract

Microglia, the resident immune cells of the brain parenchyma, are highly responsive to tissue injury. Following cell damage, microglial processes redirect their motility from randomly scouting the extracellular space to specifically reaching toward the compromised tissue. While the cell morphology aspects of this defense mechanism have been characterized, the intracellular events underlying these responses remain largely unknown. Specifically, the role of intracellular Ca2+ dynamics has not been systematically investigated in acutely activated microglia due to technical difficulty. Here we used live two-photon imaging of the mouse cortex ubiquitously expressing the genetically encoded Ca2+ indicator GCaMP5G and fluorescent marker tdTomato in central nervous system microglia. We found that spontaneous Ca2+ transients in microglial somas and processes were generally low (only 4% of all microglia showing transients within 20 min), but baseline activity increased about 8-fold when the animals were treated with LPS 12 h before imaging. When challenged with focal laser injury, an additional surge in Ca2+ activity was observed in the somas and protruding processes. Notably, coherent and simultaneous Ca2+ rises in multiple microglial cells were occasionally detected in LPS-treated animals. We show that Ca2+ transients were pre-dominantly mediated via purinergic receptors. This work demonstrates the usefulness of genetically encoded Ca2+ indicators for investigation of microglial physiology.

Microglia in neuropathology caused by protozoan parasites

“…Many of the molecular pathways involved require intracellular Ca 2+ signaling and therefore changes in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) might be able to provide an early and rather sensitive measure of microglial activation. Indeed, surveillant microglia in the healthy brain are known to maintain a low basal [Ca 2+ ] i accompanied by rare 'spontaneous' Ca 2+ transient increases in a minority of cells (Eichhoff, Brawek & Garaschuk, 2011;Pozner et al, 2015;. However, in vivo damage of even a single cell was shown rapidly (on a scale of milliseconds to seconds) and reliably to produce a so-called damage-associated [Ca 2+ ] transient in nearby microglia (Eichhoff et al, 2011).…”

“…This process was driven by ATP/ADP-induced P2Y 12 receptor signaling, activation of phospholipase C and Ca 2+ -dependent phosphorylation of serine/threonine kinase (Akt) as well as phosphoinositide-3-kinase (PI3K)-mediated Akt phosphorylation (Eichhoff et al, 2011;Madry & Attwell, 2015). Both acute and chronic (neuro)inflammation were shown to be accompanied by an increase in 'spontaneous' Ca 2+ signaling of microglia Pozner et al, 2015) again pointing to changes in microglial [Ca 2+ ] i as a universal hallmark of activated microglia.…”

“…For example, intra-vital imaging using minimally invasive two-photon microscopy allows longitudinal single-cell-level monitoring of microglial structure and function in the living brain. Structural changes in microglia have been assessed previously using mice expressing green fluorescent protein (GFP) in the presence of C-X3-C motif chemokine receptor 1 (CX 3 CR1) or the Iba-1 promoter, while cells expressing genetically encoded Ca 2+ -sensitive dyes were recently used for in vivo monitoring of microglial function (Pozner et al, 2015;. In vivo analysis of experimentally infected animals accompanied by post-hoc immunohistochemistry using recently discovered microglia-specific markers could enable a better understanding of the role of microglia in protozoan infections.…”

Microglial Calcium Signaling is Attuned to Neuronal Activity

“…Herein, we describe a series of observations regarding microglial calcium signaling in response to shifts in neuronal activity. In general, microglia are considered to have the lowest levels of spontaneous calcium activity in “resting” or “baseline” states from reports in anesthetized mice (Brawek et al, 2017; Eichhoff et al, 2011; Pozner et al, 2015). We observe that microglial calcium activity is similarly low in awake, chronic window mice.…”

“…Early work in situ suggested that calcium activity was critical for microglia, with calcium signaling underlying key physiological functions such as motility, cytokine release, and receptor trafficking/diffusion (Färber and Kettenmann, 2006; Korvers et al, 2016; Toulme and Khakh, 2012). However, more recent in vivo studies have demonstrated that spontaneous calcium signaling is nearly absent in microglia (Brawek et al, 2017; Eichhoff et al, 2011; Pozner et al, 2015). Such observations downplay the potential utility of calcium signaling for microglial function.…”