Recent work examining astrocytic physiology centers on fluorescence imaging, due to development of sensitive fluorescent indicators and observation of spatiotemporally complex calcium activity. However, the field remains hindered in characterizing these dynamics, both within single cells and at the population level, because of the insufficiency of current region-ofinterest (ROI)-based approaches to describe activity that is often spatially unfixed, size-varying, and propagative. Here, we present an analytical framework that releases astrocyte biologists from ROI-based tools. The Astrocyte Quantitative Analysis (AQuA) software takes an event-based perspective to model and accurately quantify complex calcium and neurotransmitter activity in fluorescence imaging datasets. We apply AQuA to a range of ex vivo and in vivo imaging data, and uncover novel physiological phenomena. Since AQuA is data-driven and based on machine learning principles, it can be applied across model organisms, fluorescent indicators, experimental modes, and imaging resolutions and speeds, enabling researchers to elucidate fundamental neural physiology. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Non-rapid eye movement (NREM) sleep, characterized by slow-wave electrophysiological activity, underlies several critical functions, including learning and memory. However, NREM sleep is heterogeneous, varying in duration, depth, and spatially across the cortex. While these NREM sleep features are thought to be largely independently regulated, there is also evidence that they are mechanistically coupled. To investigate how cortical NREM sleep features are controlled, we examined the astrocytic network, comprising a cortex-wide syncytium that influences population-level neuronal activity. We quantified endogenous astrocyte activity in mice over natural sleep and wake, then manipulated specific astrocytic G-protein-coupled receptor (GPCR) signaling pathways in vivo. We find that astrocytic Gi- and Gq-coupled GPCR signaling separately control NREM sleep depth and duration, respectively, and that astrocytic signaling causes differential changes in local and remote cortex. These data support a model in which the cortical astrocyte network serves as a hub for regulating distinct NREM sleep features.

Cortical state, defined by population-level neuronal activity patterns, determines sensory perception. While arousal-associated neuromodulators—including norepinephrine (NE)—reduce cortical synchrony, how the cortex resynchronizes remains unknown. Furthermore, general mechanisms regulating cortical synchrony in the wake state are poorly understood. Using in vivo imaging and electrophysiology in mouse visual cortex, we describe a critical role for cortical astrocytes in circuit resynchronization. We characterize astrocytes’ calcium responses to changes in behavioral arousal and NE, and show that astrocytes signal when arousal-driven neuronal activity is reduced and bi-hemispheric cortical synchrony is increased. Using in vivo pharmacology, we uncover a paradoxical, synchronizing response to Adra1a receptor stimulation. We reconcile these results by demonstrating that astrocyte-specific deletion of Adra1a enhances arousal-driven neuronal activity, while impairing arousal-related cortical synchrony. Our findings demonstrate that astrocytic NE signaling acts as a distinct neuromodulatory pathway, regulating cortical state and linking arousal-associated desynchrony to cortical circuit resynchronization.

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SummaryThe prefrontal cortex (PFC) is a hub for cognitive control, and dopamine profoundly influences its functions. In other brain regions, astrocytes sense diverse neurotransmitters and neuromodulators and, in turn, orchestrate regulation of neuroactive substances. However, basic physiology of PFC astrocytes, including which neuromodulatory signals they respond to and how they contribute to PFC function, is lacking. Here, we characterize divergent signaling signatures in astrocytes of PFC and primary sensory cortex in mice, which are linked to differential responsivity to locomotion. We find that PFC astrocytes express receptors for dopamine, but are unresponsive through the Gs/Gi-cAMP pathway. Instead, fast calcium signals in PFC astrocytes are time-locked to dopamine release, and are mediated by α1-adrenergic receptors both ex vivo and in vivo. Further, we describe dopamine-triggered regulation of extracellular ATP at PFC astrocyte territories. Thus, we identify astrocytes as active players in dopaminergic signaling in PFC, contributing to PFC function though neuromodulator receptor crosstalk.Graphical Abstract