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

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 Ca$^{2+}$ transients as their encoding language. The analysis suggests that astrocytes may carry out canonical computations in time scales of sub-seconds 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, are, 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 Ca$^{2+}$ and brain coding may represent a leap forward towards novel approaches in the study of astrocytes in health and disease.Junior Leader Fellowhip Program by 'la Caixa' Banking Foundation, LCF/BQ/LI18/11630006 BFU2017-85936-P BFU2016-75107-P BFU2016-79735-P FLAGERA-PCIN-2015-162-C02-02 HHMI 55008742 FPU13/05377 NIH R01NS099254 NSF 1604544 Agència de Gestio d’Ajuts Universitaris i de Recerca, 2017 SGR54

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

This is an open access article published under an ACS AuthorChoice License. See Standard ACS AuthorChoice/Editors' Choice Usage Agreement - https://pubs.acs.org/page/policy/authorchoice_termsofuse.htmlSynthetic 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

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

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

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

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 Ca2+ 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  μm in depth with a fine step size, parameters that make it useful for astrocyte biology research. Future work includes adaptive optics and intensity normalization

Live-imaging of astrocyte morphogenesis and function in zebrafish neural circuits.

How astrocytes grow and integrate into neural circuits remains poorly defined. Zebrafish are well suited for such investigations, but bona fide astrocytes have not been described in this system. Here we characterize a zebrafish cell type that is remarkably similar to mammalian astrocytes that derive from radial glial cells and elaborate processes to establish their territories at early larval stages. Zebrafish astrocytes associate closely with synapses, tile with one another and express markers, including Glast and glutamine synthetase. Once integrated into circuits, they exhibit whole-cell and microdomain Ca2+ transients, which are sensitive to norepinephrine. Finally, using a cell-specific CRISPR-Cas9 approach, we demonstrate that fgfr3 and fgfr4 are required for vertebrate astrocyte morphogenesis. This work provides the first visualization of astrocyte morphogenesis from stem cell to post-mitotic astrocyte in vivo, identifies a role for Fgf receptors in vertebrate astrocytes and establishes zebrafish as a valuable new model system to study astrocyte biology in vivo

A method for estimating intracellular ion concentration using optical nanosensors and ratiometric imaging.

Optical nanoparticle (NP)-based sensors have been widely implemented as tools for detection of targeted ions and biomolecules. The NP sensing platform offer a modular design that can incorporate different sensing components for greater target specificity and the ability to tune the dynamic range, as well as encapsulation of multiple dyes to generate a ratiometric signal with varying spectra. Despite these advantages, demonstrating quantitative ion imaging for intracellular measurement still possess a major challenge. Here, we describe fundamentals that enable intracellular validation of this approach using ion-selective nanosensors for investigating calcium (Ca2+) as a model ion. While conventional indicators can improve individual aspects of indicator performance such as Kd, wavelength, and ratiometric measurements, the use of NP sensors can achieve combined benefits of addressing these issues simultaneously. The nanosensor incorporates highly calcium-selective ionophores and two fluorescence indicators that act as signal transducers to facilitate quantitative ratiometric imaging. For intracellular Ca2+ application, the sensors are fine-tuned to physiological sensing range, and live-cell imaging and quantification are demonstrated in HeLa cells loaded with nanosensors and their responsiveness to carbachol-evoked store release (~400 nM). The current nanosensor design thus provides a promising sensing platform for real-time detection and optical determination of intracellular ions