We propose to investigate a novel form of microglia Ca2+ signal that we have discovered in awake behaving mice. It is the first microglia Ca2+ signal identified so far that represents a direct response to active, vigilant behavior and neuronal activity within short latency (<10 seconds). Due to the complexity of experimental procedures and the paucity of available data very little is known about microglia Ca2+ dynamics in awake behaving mice. This is in striking contrast to the expected importance of Ca2+ signaling for microglia function. From work on cultured microglia it is known that basic cellular processes such as cytoskeletal rearrangements, which are a prerequisite for cell motility and phagocytosis, as well as most mechanisms underlying the secretion of signaling molecules such as cytokines depend on intracellular fluctuations of Ca2+. Microglia express numerous membrane proteins to potentially translate extracellular signals into intracellular Ca2+ elevations. Among these are purinergic receptors, immune system-related receptors as well as ion channels. Specifically, microglia express L-type Ca2+ channels (LTCCs). LTCCs have attracted special attention because antagonists of this class of ion channels, traditionally applied to control blood pressure, have demonstrated therapeutic potential for limiting neuroinflammation and cognitive impairments accompanying neurodegenerative diseases. The contribution of microglia LTCCs to this therapeutic potential is not yet well established and it is not clear when these ion channels become normally activated. For this proposal we will employ two-photon microscopy on transgenic mice with microglia-specific expression of the genetically-encoded Ca2+ indicator GCaMP6f. The mice will be head-fixed on a motorized linear treadmill for precise control over speed and duration of enforced locomotion events in addition to the monitoring of voluntary locomotion events. Locomotion represents a reliable means to induce moderate arousal, a behavioral state of heightened vigilance. To avoid a bias by the experimental conditions the studies will include imaging of microglia through chronic cranial windows several weeks following surgery as well as acute surgery experiments for topical pharmacology. Our preliminary pharmacological experiments suggest that locomotion-induced microglia Ca2+ elevations depend on LTCCs. In Aim 1 we will conduct a systematic characterization of the newly discovered locomotion-induced microglia Ca2+ elevations. We will investigate the kinetic constraints of the responses and test whether V1 microglia Ca2+ responses are affected by visual stimulation. In Aim 2 we will combine microglia-selective gene deletion and pharmacological experiments to determine which LTCC subtype predominantly contributes to the responses. Upon successful completion of the proposed experiments we will have established a rigorous foundation for rational design of future studies aimed at unraveling the consequences of vigilance-dependent microglia Ca2+ responses for circuit activity and behavior. These findings may then open new avenues for microglia-centered therapeutic interventions in neurodegenerative and neuropsychiatric diseases.

Microglia are the resident immune cells of the brain and express receptors and ion channels that can lead to intracellular Ca2+ rise. Even though investigations of cultured microglia have suggested that Ca2+ dynamics are important for crucial microglia functions such as cell motility, endocytosis and cytokine secretion, it is not known if, when and how microglia experience Ca2+ responses which are temporally directly related to ongoing behavior. The proposed work will define the molecular mechanism of vigilance-dependent microglia Ca2+ elevations and thereby reveal a potential target for therapeutic modulation of microglia activity in neurodegenerative and neuropsychiatric diseases.