Network-level encoding of local neurotransmitters in cortical astrocytes.

Astrocytes, the most abundant non-neuronal cell type in the mammalian brain, are crucial circuit components that respond to and modulate neuronal activity through calcium (Ca2+) signalling1-7. Astrocyte Ca2+ activity is highly heterogeneous and occurs across multiple spatiotemporal scales-from fast, subcellular activity3,4 to slow, synchronized activity across connected astrocyte networks8-10-to influence many processes5,7,11. However, the inputs that drive astrocyte network dynamics remain unclear. Here we used ex vivo and in vivo two-photon astrocyte imaging while mimicking neuronal neurotransmitter inputs at multiple spatiotemporal scales. We find that brief, subcellular inputs of GABA and glutamate lead to widespread, long-lasting astrocyte Ca2+ responses beyond an individual stimulated cell. Further, we find that a key subset of Ca2+ activity-propagative activity-differentiates astrocyte network responses to these two main neurotransmitters, and may influence responses to future inputs. Together, our results demonstrate that local, transient neurotransmitter inputs are encoded by broad cortical astrocyte networks over a minutes-long time course, contributing to accumulating evidence that substantial astrocyte-neuron communication occurs across slow, network-level spatiotemporal scales12-14. These findings will enable future studies to investigate the link between specific astrocyte Ca2+ activity and specific functional outputs, which could build a consistent framework for astrocytic modulation of neuronal activity.

Imaging in vivo acetylcholine release in the peripheral nervous system with a fluorescent nanosensor.

The ability to monitor the release of neurotransmitters during synaptic transmission would significantly impact the diagnosis and treatment of neurological diseases. Here, we present a DNA-based enzymatic nanosensor for quantitative detection of acetylcholine (ACh) in the peripheral nervous system of living mice. ACh nanosensors consist of DNA as a scaffold, acetylcholinesterase as a recognition component, pH-sensitive fluorophores as signal generators, and α-bungarotoxin as a targeting moiety. We demonstrate the utility of the nanosensors in the submandibular ganglia of living mice to sensitively detect ACh ranging from 0.228 to 358 µM. In addition, the sensor response upon electrical stimulation of the efferent nerve is dose dependent, reversible, and we observe a reduction of ∼76% in sensor signal upon pharmacological inhibition of ACh release. Equipped with an advanced imaging processing tool, we further spatially resolve ACh signal propagation on the tissue level. Our platform enables sensitive measurement and mapping of ACh transmission in the peripheral nervous system.

Reversible silencing of endogenous receptors in intact brain tissue using 2-photon pharmacology.

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 µm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles.

Dynamism of an Astrocyte In Vivo: Perspectives on Identity and Function.

Astrocytes are an abundant and evolutionarily conserved central nervous system cell type. Despite decades of evidence that astrocytes are integral to neural circuit function, it seems as though astrocytic and neuronal biology continue to advance in parallel to each other, to the detriment of both. Recent advances in molecular biology and optical imaging are being applied to astrocytes in new and exciting ways but without fully considering their unique biology. From this perspective, we explore the reasons that astrocytes remain enigmatic, arguing that their responses to neuronal and environmental cues shape form and function in dynamic ways. Here, we provide a roadmap for future experiments to explore the nature of astrocytes in situ.

A roadmap to integrate astrocytes into Systems Neuroscience.

Systems neuroscience is still mainly a neuronal field, despite the plethora of evidence supporting the fact that astrocytes modulate local neural circuits, networks, and complex behaviors. In this article, we sought to identify which types of studies are necessary to establish whether astrocytes, beyond their well-documented homeostatic and metabolic functions, perform computations implementing mathematical algorithms that sub-serve coding and higher-brain functions. First, we reviewed Systems-like studies that include astrocytes in order to identify computational operations that these cells may perform, using Ca2+ transients as their encoding language. The analysis suggests that astrocytes may carry out canonical computations in a time scale of subseconds to seconds in sensory processing, neuromodulation, brain state, memory formation, fear, and complex homeostatic reflexes. Next, we propose a list of actions to gain insight into the outstanding question of which variables are encoded by such computations. The application of statistical analyses based on machine learning, such as dimensionality reduction and decoding in the context of complex behaviors, combined with connectomics of astrocyte-neuronal circuits, is, in our view, fundamental undertakings. We also discuss technical and analytical approaches to study neuronal and astrocytic populations simultaneously, and the inclusion of astrocytes in advanced modeling of neural circuits, as well as in theories currently under exploration such as predictive coding and energy-efficient coding. Clarifying the relationship between astrocytic Ca2+ and brain coding may represent a leap forward toward novel approaches in the study of astrocytes in health and disease.

Optical Probes for Neurobiological Sensing and Imaging.

Fluorescent nanosensors and molecular probes are next-generation tools for imaging chemical signaling inside and between cells. Electrophysiology has long been considered the gold standard in elucidating neural dynamics with high temporal resolution and precision, particularly on the single-cell level. However, electrode-based techniques face challenges in illuminating the specific chemicals involved in neural cell activation with adequate spatial information. Measuring chemical dynamics is of fundamental importance to better understand synergistic interactions between neurons as well as interactions between neurons and non-neuronal cells. Over the past decade, significant technological advances in optical probes and imaging methods have enabled entirely new possibilities for studying neural cells and circuits at the chemical level. These optical imaging modalities have shown promise for combining chemical, temporal, and spatial information. This potential makes them ideal candidates to unravel the complex neural interactions at multiple scales in the brain, which could be complemented by traditional electrophysiological methods to obtain a full spatiotemporal picture of neurochemical dynamics. Despite the potential, only a handful of probe candidates have been utilized to provide detailed chemical information in the brain. To date, most live imaging and chemical mapping studies rely on fluorescent molecular indicators to report intracellular calcium (Ca2+) dynamics, which correlates with neuronal activity. Methodological advances for monitoring a full array of chemicals in the brain with improved spatial, temporal, and chemical resolution will thus enable mapping of neurochemical circuits with finer precision. On the basis of numerous studies in this exciting field, we review the current efforts to develop and apply a palette of optical probes and nanosensors for chemical sensing in the brain. There is a strong impetus to further develop technologies capable of probing entire neurobiological units with high spatiotemporal resolution. Thus, we introduce selected applications for ion and neurotransmitter detection to investigate both neurons and non-neuronal brain cells. We focus on families of optical probes because of their ability to sense a wide array of molecules and convey spatial information with minimal damage to tissue. We start with a discussion of currently available molecular probes, highlight recent advances in genetically modified fluorescent probes for ions and small molecules, and end with the latest research in nanosensors for biological imaging. Customizable, nanoscale optical sensors that accurately and dynamically monitor the local environment with high spatiotemporal resolution could lead to not only new insights into the function of all cell types but also a broader understanding of how diverse neural signaling systems act in conjunction with neighboring cells in a spatially relevant manner.

Astrocytes regulate cortical state switching in vivo.

The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation-dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations.

Two-photon neuronal and astrocytic stimulation with azobenzene-based photoswitches.

Synthetic photochromic compounds can be designed to control a variety of proteins and their biochemical functions in living cells, but the high spatiotemporal precision and tissue penetration of two-photon stimulation have never been investigated in these molecules. Here we demonstrate two-photon excitation of azobenzene-based protein switches and versatile strategies to enhance their photochemical responses. This enables new applications to control the activation of neurons and astrocytes with cellular and subcellular resolution.

Astrocytic regulation of cortical UP states.

The synchronization of neuronal assemblies during cortical UP states has been implicated in computational and homeostatic processes, but the mechanisms by which this occurs remain unknown. To investigate potential roles of astrocytes in synchronizing cortical circuits, we electrically activated astrocytes while monitoring the activity of the surrounding network with electrophysiological recordings and calcium imaging. Stimulating a single astrocyte activates other astrocytes in the local circuit and can trigger UP state synchronizations of neighboring neurons. Moreover, interfering with astrocytic activity with intracellular injections of a calcium chelator into individual astrocytes inhibits spontaneous and stimulated UP states. Finally, both astrocytic activity and neuronal UP states are regulated by purinergic signaling in the circuit. These results demonstrate that astroglia can play a causal role in regulating the synchronized activation of neuronal ensembles.

Astrocyte Calcium Signaling.

Astrocytes are predominant glial cells that tile the central nervous system and participate in well-established functional and morphological interactions with neurons, blood vessels, and other glia. These ubiquitous cells display rich intracellular Ca2+ signaling, which has now been studied for over 30 years. In this review, we provide a summary and perspective of recent progress concerning the study of astrocyte intracellular Ca2+ signaling as well as discussion of its potential functions. Progress has occurred in the areas of imaging, silencing, activating, and analyzing astrocyte Ca2+ signals. These insights have collectively permitted exploration of the relationships of astrocyte Ca2+ signals to neural circuit function and behavior in a variety of species. We summarize these aspects along with a framework for mechanistically interpreting behavioral studies to identify directly causal effects. We finish by providing a perspective on new avenues of research concerning astrocyte Ca2+ signaling.

Fast, Accurate, and Versatile Data Analysis Platform for the Quantification of Molecular Spatiotemporal Signals.

Optical recording of intricate molecular dynamics is becoming an indispensable technique for biological studies, accelerated by the development of new or improved biosensors and microscopy technology. This creates major computational challenges to extract and quantify biologically meaningful spatiotemporal patterns embedded within complex and rich data sources, many of which cannot be captured with existing methods. Here, we introduce Activity Quantification and Analysis (AQuA2), a fast, accurate, and versatile data analysis platform built upon advanced machine learning techniques. It decomposes complex live imaging-based datasets into elementary signaling events, allowing accurate and unbiased quantification of molecular activities and identification of consensus functional units. We demonstrate applications across a wide range of biosensors, cell types, organs, animal models, and imaging modalities. As exemplar findings, we show how AQuA2 identified drug-dependent interactions between neurons and astroglia, and distinct sensorimotor signal propagation patterns in the mouse spinal cord.

A Photoactivatable Norepinephrine for Probing Adrenergic Neural Circuits.

Norepinephrine (NE) is a critical neuromodulator that mediates a wide range of behavior and neurophysiology, including attention, arousal, plasticity, and memory consolidation. A major source of NE is the brainstem nucleus the locus coeruleus (LC), which sends widespread projections throughout the central nervous system (CNS). Efforts to dissect this complex noradrenergic circuitry have driven the development of many tools that detect endogenous NE or modulate widespread NE release via LC activation and inhibition. While these tools have enabled research that elucidates physiological roles of NE, additional tools to probe these circuits with a higher degree of spatial precision could enable a finer delineation of function. Here, we describe the synthesis and chemical properties of a photo-activatable NE, [Ru(bpy) 2 (PMe 3 )(NE)]PF 6 (RuBi-NE). We validate the one-photon (1P) release of NE using whole-cell patch clamp electrophysiology in acute mouse brain slices containing the LC. We show that a 10 ms pulse of blue light, in the presence of RuBi-NE, briefly modulates the firing rate of LC neurons via α-2 adrenergic receptors. The development of a photo-activatable NE that can be released with light in the visible spectrum provides a new tool for fine-grained mapping of complex noradrenergic circuits, as well as the ability to probe how NE acts on non-neuronal cells in the CNS.

Network-level encoding of local neurotransmitters in cortical astrocytes.

Astrocytes-the most abundant non-neuronal cell type in the mammalian brain-are crucial circuit components that respond to and modulate neuronal activity via calcium (Ca 2+ ) signaling 1-8 . Astrocyte Ca 2+ activity is highly heterogeneous and occurs across multiple spatiotemporal scales: from fast, subcellular activity 3,4 to slow, synchronized activity that travels across connected astrocyte networks 9-11 . Furthermore, astrocyte network activity has been shown to influence a wide range of processes 5,8,12 . While astrocyte network activity has important implications for neuronal circuit function, the inputs that drive astrocyte network dynamics remain unclear. Here we used ex vivo and in vivo two-photon Ca 2+ imaging of astrocytes while mimicking neuronal neurotransmitter inputs at multiple spatiotemporal scales. We find that brief, subcellular inputs of GABA and glutamate lead to widespread, long-lasting astrocyte Ca 2+ responses beyond an individual stimulated cell. Further, we find that a key subset of Ca 2+ activity-propagative events-differentiates astrocyte network responses to these two major neurotransmitters, and gates responses to future inputs. Together, our results demonstrate that local, transient neurotransmitter inputs are encoded by broad cortical astrocyte networks over the course of minutes, contributing to accumulating evidence across multiple model organisms that significant astrocyte-neuron communication occurs across slow, network-level spatiotemporal scales 13-15 . We anticipate that this study will be a starting point for future studies investigating the link between specific astrocyte Ca 2+ activity and specific astrocyte functional outputs, which could build a consistent framework for astrocytic modulation of neuronal activity.

Norepinephrine links astrocytic activity to regulation of cortical state.

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.

Dopamine activates astrocytes in prefrontal cortex via α1-adrenergic receptors.

The 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 unclear. Here, we characterize divergent signaling signatures in mouse astrocytes of the PFC and primary sensory cortex, which show differential responsiveness 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 the PFC, contributing to PFC function though neuromodulator receptor crosstalk.

Deformable mirror-based axial scanning for two-photon mammalian brain imaging.

Significance: To expand our understanding of the roles of astrocytes in neural circuits, there is a need to develop optical tools tailored specifically to capture their complex spatiotemporal Ca 2 + dynamics. This interest is not limited to 2D, but to multiple depths. Aim: The focus of our work was to design and evaluate the optical performance of an enhanced version of a two-photon (2P) microscope with the addition of a deformable mirror (DM)-based axial scanning system for live mammalian brain imaging. Approach: We used a DM to manipulate the beam wavefront by applying different defocus terms to cause a controlled axial shift of the image plane. The optical design and performance were evaluated by an analysis of the optical model, followed by an experimental characterization of the implemented instrument. Results: Key questions related to this instrument were addressed, including impact of the DM curvature change on vignetting, field of view size, image plane flatness, wavefront error, and point spread function. The instrument was used for imaging several neurobiological samples at different depths, including fixed brain slices and in vivo mouse cerebral cortex. Conclusions: Our implemented instrument was capable of recording z -stacks of 53 µ m in depth with a fine step size, parameters that make it useful for astrocyte biology research. Future work includes adaptive optics and intensity normalization.

Cortical astrocytes independently regulate sleep depth and duration via separate GPCR pathways.

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.

Sleep has many roles, from strengthening new memories to regulating mood and appetite. While we might instinctively think of sleep as a uniform state of reduced brain activity, the reality is more complex. First, over the course of the night, we cycle between a number of different sleep stages, which reflect different levels of sleep depth. Second, the amount of sleep depth is not necessarily even across the brain but can vary between regions. These sleep stages consist of either rapid eye movement (REM) sleep or non-REM (NREM) sleep. REM sleep is when most dreaming occurs, whereas NREM sleep is particularly important for learning and memory and can vary in duration and depth. During NREM sleep, large groups of neurons synchronize their firing to create rhythmic waves of activity known as slow waves. The more synchronous the activity, the deeper the sleep. Vaidyanathan et al. now show that brain cells called astrocytes help regulate NREM sleep. Astrocytes are not neurons but belong to a group of specialized cells called glia. They are the largest glia cell type in the brain and display an array of proteins on their surfaces called G-protein-coupled receptors (GPCRs). These enable them to sense sleep-wake signals from other parts of the brain and to generate their own signals. In fact, each astrocyte can communicate with thousands of neurons at once. They are therefore well-poised to coordinate brain activity during NREM sleep. Using innovative tools, Vaidyanathan et al. visualized astrocyte activity in mice as the animals woke up or fell asleep. The results showed that astrocytes change their activity just before each sleep­wake transition. They also revealed that astrocytes control both the depth and duration of NREM sleep via two different types of GPCR signals. Increasing one of these signals (Gi-GPCR) made the mice sleep more deeply but did not change sleep duration. Decreasing the other (Gq-GPCR) made the mice sleep for longer but did not affect sleep depth. Sleep problems affect many people at some point in their lives, and often co-exist with other conditions such as mental health disorders. Understanding how the brain regulates different features of sleep could help us develop better ­ and perhaps more specific ­ treatments for sleep disorders. The current study suggests that manipulating GPCRs on astrocytes might increase sleep depth, for example. But before work to test this idea can begin, we must first determine whether findings from sleeping mice also apply to people.

Discrete residues in the c(2)b domain of synaptotagmin I independently specify endocytic rate and synaptic vesicle size.

It has been demonstrated that synapses lacking functional synaptotagmin I (Syt I) have a decreased rate of synaptic vesicle endocytosis. Beyond this, the function of Syt I during endocytosis remains undefined. Here, we demonstrate that a decreased rate of endocytosis in syt(null) mutants correlates with a stimulus-dependent perturbation of membrane internalization, assayed ultrastructurally. We then separate the mechanisms that control endocytic rate and vesicle size by mapping these processes to discrete residues in the Syt I C(2)B domain. Mutation of a poly-lysine motif alters vesicle size but not endocytic rate, whereas the mutation of calcium-coordinating aspartate residues (syt-D3,4N) alters endocytic rate but not vesicle size. Finally, slowed endocytic rate in the syt-D3,4N animals, but not syt(null) animals, can be rescued by elevating extracellular calcium concentration, supporting the conclusion that calcium coordination within the C(2)B domain contributes to the control of endocytic rate.

Synaptotagmin I is necessary for compensatory synaptic vesicle endocytosis in vivo.

Neurotransmission requires a balance of synaptic vesicle exocytosis and endocytosis. Synaptotagmin I (Syt I) is widely regarded as the primary calcium sensor for synaptic vesicle exocytosis. Previous biochemical data suggest that Syt I may also function during synaptic vesicle endocytosis; however, ultrastructural analyses at synapses with impaired Syt I function have provided an indirect and conflicting view of the role of Syt I during synaptic vesicle endocytosis. Until now it has not been possible experimentally to separate the exocytic and endocytic functions of Syt I in vivo. Here, we test directly the role of Syt I during endocytosis in vivo. We use quantitative live imaging of a pH-sensitive green fluorescent protein fused to a synaptic vesicle protein (synapto-pHluorin) to measure the kinetics of endocytosis in sytI-null Drosophila. We then combine live imaging of the synapto-pHluorins with photoinactivation of Syt I, through fluorescein-assisted light inactivation, after normal Syt I-mediated vesicle exocytosis. By inactivating Syt I only during endocytosis, we demonstrate that Syt I is necessary for the endocytosis of synaptic vesicles that have undergone exocytosis using a functional Syt I protein.