IN VIVO IMAGING OF STATE-DEPENDENT ASTROCYTE VOLTAGE DYNAMICS AND NEURAL ACTIVITY IN MOUSE VISUAL CORTEX

SUMMARY This proposal aims to fundamentally advance our understanding of astrocyte physiology by using novel genetically encoded voltage indicators (GEVIs) to measure the coordinate of astrocyte membrane potential (Vm) and local neural activity in vivo. Historically considered “electrically uninteresting,” because of the minimal variations in somatic voltage observed in patch recordings, our preliminary data with JEDI3, a sensitive, stable, two-photon compatible GEVI, reveals robust changes in membrane voltage in vivo that track closely with variations in neural activity and behavioral state. In Aim 1, we will characterize the spatiotemporal coordination of astrocyte Vm fluctuations and neural activity across multiple contexts—cortical up/down states under anesthesia, transitions between quiet wakefulness and active locomotion, visual stimulation, and seizures—and test the extent to which these voltage changes occur independently of astrocyte calcium signaling. In Aim 2, we will determine how neuromodulatory inputs (noradrenergic and cholinergic) shape astrocyte Vm and whether manipulating astrocyte voltage can influence state-dependent neuronal dynamics. We will also validate optogenetic tools for reliably controlling astrocyte voltage, a critical advance that will enable causal tests of the effects of manipulating astrocyte membrane voltage. In Aim 3, we will resolve subcellular hotspots of astrocyte depolarization at the scale of individual dendrites and axons, and test whether presynaptic activity alone is sufficient to drive these focal changes. Finally, as part of Aim 3, we will develop and validate improved astrocyte-optimized GEVIs, ensuring that these optical methods are made accessible to the broader neuroscience community. By illuminating the “hidden” electrical dimension of astrocyte function, this work promises to transform our understanding of glia-neuron interactions, reveal novel mechanisms of brain state regulation, and open new therapeutic avenues for neurological diseases where astrocyte dysfunction is implicated.