SUMMARY New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments.

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.

Background and Purpose Immune responses to brain antigens occur after stroke, and experimental studies show that the likelihood of developing a detrimental autoimmune response to these antigens is increased by systemic inflammation at the time of stroke. The aim of this study was to determine if patients who developed infection in the post-stroke period would be similarly predisposed to develop autoimmune responses to central nervous system (CNS) antigens. Methods We enrolled 114 patients within 72 hours of ischemic stroke. Clinical and demographic data were obtained, and cellular immune responses to a panel of CNS antigens were assessed during the initial week and again at day 90. Outcome was assessed using the modified Rankin Scale. Results Patients who developed an infection, especially pneumonia, in the 15 days after stroke were more likely to evidence a TH1(+) response to myelin basic protein (MBP) and glial fibrillary acidic protein (P=0.019 and P=0.039, respectively) at 90 days after stroke. Further, more robust TH1 responses to MBP at 90 days were associated with a decreased likelihood of good outcome, even after adjusting for baseline stroke severity and patient age (Odds Ratio = 0.477, 95% CI = 0.244–0.935; P=0.031). Conclusion This study demonstrates that immune responses to brain antigens occur after stroke. And while these responses are likely to be an epiphenomenon of ischemic brain injury, the response to MBP appears to have clinical consequences. The potential role of post-ischemic autoimmune mediated brain injury deserves further investigation.

Background and Purpose-Animals subjected to an inflammatory insult at the time of stroke are predisposed to the development of an inflammatory autoimmune response to brain. This response is associated with worse neurological outcome. Because induction of immunologic tolerance to brain antigens before stroke onset is associated with improved outcome, we sought to determine whether this paradigm could prevent the deleterious autoimmune response to brain provoked by an inflammatory stimulus at the time of ischemia. Methods-Male Lewis rats were tolerized to myelin basic protein (MBP) or ovalbumin by intranasal administration before middle cerebral artery occlusion. At the time of reperfusion, all animals received lipopolysaccharide (1 mg/kg intraperitoneal). Behavioral tests were performed at set time intervals. Results-One month after middle cerebral artery occlusion, lymphocytes from the spleens of MBP-tolerized animals were less likely to evidence an autoimmune response and more likely to evidence a regulatory response (TREG) toward MBP than those from ovalbumin-tolerized animals. Animals that had an inflammatory response toward MBP (a TH1 response) performed worse on behavioral tests than those that did not. Fractalkine, a surrogate marker of inflammation, was elevated in animals with a TH1 response to MBP. Conclusions-These data extend our previous findings and suggest that deleterious autoimmunity to brain antigens can be prevented by prophylactically inducing regulatory T-cell responses to those antigens.