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.
Highlights
Microglia are the principal immune cells in the brain
When we stained the brains of the progeny with anti-GFP antibody and anti-ionized Ca2+-binding adapter molecule 1 (Iba1) antibody, virtually all cortical microglia were co-labeled (Figures 1D,G)
Recording of intracellular signaling in microglia has been complicated by technical difficulties with dye loading and subsequent amplification of these distortions due to high immune reactivity of these cells
Summary
Microglia are the principal immune cells in the brain In their ramified, non-activated state, microglia exhibit small somas and elaborate, highly motile processes (Nimmerjahn et al, 2005). Non-activated state, microglia exhibit small somas and elaborate, highly motile processes (Nimmerjahn et al, 2005) This high degree of motility facilitates the interaction of microglial bulbous endings with numerous physiological processes in the central nervous system, ranging from response to cellular damage (Davalos et al, 2005; Wake et al, 2009) to a role in synaptic pruning and response to neuronal activity (Paolicelli et al, 2011; Schafer et al, 2012; Dissing-Olesen et al, 2014; Eyo et al, 2014). To enable more detailed studies, we have recently developed a mouse reporter of cellular activity (PC::G5tdT), which combines the use of a constitutively fluorescent protein (tdTomato) with the GCaMP5G intracellular Ca2+ indicator and displays high activity in microglia (Gee et al, 2014)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
