Three-dimensional Ca2+ imaging advances understanding of astrocyte biology

Glial calcium dynamics in space and time Astrocytes use calcium signals to process information received from neighboring brain cells and thus generate modulatory responses at the local or network level. Previous studies have relied on calcium imaging in line scans or in a single focal plane mostly focusing on the cell bodies of astrocytes. Bindocci et al. used more powerful scanners that can rapidly scan many focal planes. They combined this technique with advanced genetic tools for monitoring calcium gradients with high sensitivity, which allowed three-dimensional calcium imaging of a whole astrocyte. Most of the basal calcium activity occurred in the astrocyte processes, some in the endfeet, and only a small fraction actually in the cell bodies of astrocytes. Science, this issue p. eaai8185 A two-photon imaging technique allows the measurement of calcium dynamics in neuronal glial cells in three dimensions. INTRODUCTION Astrocytes translate incoming information and generate functional outputs via Ca2+ signaling. Thereby, they respond to neuronal activity, producing downstream modulation of synaptic functions, and may participate in hemodynamics regulation. Deciphering the “Ca2+ language” of astrocytes is therefore essential for defining their roles in brain physiology and pathology. However, the specifics of astrocytic Ca2+ signaling are still poorly understood, and recent studies producing inconsistent or contradictory results have fostered debate on the actual role of astrocytes in synaptic and vascular functions. RATIONALE A neglected potential source of inconsistencies lies in the way astrocytic Ca2+ signaling has been studied to date, mostly by conventional two-dimensional (2D) imaging, which assumes that sampling a single (~1 μm) focal plane is representative of the entire astrocytic cell. This is, however, dubious given that astrocytes are highly 3D cells, entertain heterogeneous 3D relations with neighboring structures, and display Ca2+ signals on a local scale. Therefore, we developed a new method to three-dimensionally scan entire astrocytes and observe full-cell Ca2+ dynamics. RESULTS With our 3D approach, we sampled astrocytes at a sufficient rate to detect events with durations of >1.5 s throughout the cell, and faster ones in selected substructures. We found that Ca2+ activity in an individual astrocyte is heterogeneously scattered throughout the cell, largely compartmented within each region, and preponderantly local. The majority resides in the “gliapil,” the peripheral region composed of fine (optically subresolved) structures occupying ∼75% of the astrocyte volume. Within the central (resolvable) “core,” the soma is mostly inactive, whereas processes are frequently active yet show widely different activity between them. Even in individual processes, activity distributes heterogeneously, with alternating “hot” and “cold” spots. We performed 3D imaging in awake mice and in adult brain slices. Activity in vivo was faster and more frequent, particularly in endfeet, yet similar in properties and cellular distribution to slices, except for the presence of cell-wide “global” Ca2+ events mainly associated with mouse movement. Contrary to current beliefs, global events were not sweeping waves, but rather consisted of multifocal Ca2+ elevations that started at multiple gliapil loci and then spread to the core. At the vascular interface, astrocytic Ca2+ activity was mostly restricted to individual endfeet, even to their fractions, and only occasionally coordinated with the endfoot process or the rest of the astrocyte. Two or more endfeet were mainly asynchronous, even when enwrapping the same vessel. Astrocytic structures and axons intersected three-dimensionally, and minimal axonal activity (individual action potentials) produced time-correlated astrocytic Ca2+ elevations in small spots (<1% of the volume), which demonstrates that astrocytes can sense even the lowest levels of neuronal activity. CONCLUSION We provide the first comprehensive 3D map of Ca2+ activity in an individual astrocyte. Its widespread, heterogeneous, local, and mostly 3D nature confirms the appropriateness of our whole-cell imaging approach. Past 2D studies, often focusing on somatic Ca2+ dynamics, inadequately described the emerging richness and complexity of the astrocyte activity, notably at astrocyte-synapse and astrocyte-vascular interfaces, where activity is small, fast, and frequent. In this context, we can foresee future challenges in extending studies to the gliapil, whose structures fall below current optical resolution, and in reporting the complete gamut of astrocyte Ca2+ signals at the whole-cell scale, both requiring technical advances. Nonetheless, the technique demonstrated here promises to make 3D Ca2+ imaging the state-of-the-art approach for Ca2+ studies addressing the role of astrocytes in brain function. 3D Ca2+ imaging reveals the complex activity of astrocytes. (1) Representation of the 3D structural interactions among astrocyte, axons, and blood vessels. The astrocyte contains optically resolved (c, core) and unresolved (g, gliapil) regions. (2) 3D Ca2+ imaging reveals different activity (intensity color scale) in different astrocytic regions (P, processes; S, soma; EF, endfoot); (3) in different processes; and (4) in different loci of individual processes. (5) It also reveals asynchronous activity in endfeet, (6) often restricted to endfoot subdomains, and (7) local astrocyte responses to minimal axonal firing, or (8) global responses to in vivo locomotion (time color scale). Astrocyte communication is typically studied by two-dimensional calcium ion (Ca2+) imaging, but this method has not yielded conclusive data on the role of astrocytes in synaptic and vascular function. We developed a three-dimensional two-photon imaging approach and studied Ca2+ dynamics in entire astrocyte volumes, including during axon-astrocyte interactions. In both awake mice and brain slices, we found that Ca2+ activity in an individual astrocyte is scattered throughout the cell, largely compartmented between regions, preponderantly local within regions, and heterogeneously distributed regionally and locally. Processes and endfeet displayed frequent fast activity, whereas the soma was infrequently active. In awake mice, activity was higher than in brain slices, particularly in endfeet and processes, and displayed occasional multifocal cellwide events. Astrocytes responded locally to minimal axonal firing with time-correlated Ca2+ spots.