RESEARCH ◥ the peripheral region composed of fine (optically RESEARCH ARTICLE SUMMARY subresolved) structures occupying ∼75% of the astrocyte volume. Within the central (resolvable) “core,” the soma is mostly inactive, whereas pro- NEUROSCIENCE cesses are frequently active yet show widely dif- ferent activity between them. Even in individual 2+ processes, activity distributes heterogeneously, Three-dimensional Ca imaging with alternating “hot” and “cold” spots. We performed 3D imaging in awake mice and advances understanding of in adult brain slices. Activity in vivo was faster ◥ and more frequent, particu- astrocyte biology ON OUR WEBSITE larly in endfeet, yet similar Read the full article in properties and cellular Erika Bindocci,* Iaroslav Savtchouk,* Nicolas Liaudet,* Denise Becker, at http://dx.doi. distribution to slices, except Giovanni Carriero, Andrea Volterra† org/10.1126/ for the presence of cell- science.aai8185 wide “global” Ca2+ events .................................................. mainly associated with INTRODUCTION: Astrocytes translate incom- which assumes that sampling a single (~1 mm) mouse movement. Contrary to current beliefs, ing information and generate functional out- focal plane is representative of the entire astro- global events were not sweeping waves, but puts via Ca2+ signaling. Thereby, they respond cytic cell. This is, however, dubious given that rather consisted of multifocal Ca2+ elevations to neuronal activity, producing downstream mod- astrocytes are highly 3D cells, entertain heter- that started at multiple gliapil loci and then ulation of synaptic functions, and may participate ogeneous 3D relations with neighboring struc- spread to the core. in hemodynamics regulation. Deciphering the tures, and display Ca2+ signals on a local scale. At the vascular interface, astrocytic Ca2+ activ- “Ca2+ language” of astrocytes is therefore essen- Therefore, we developed a new method to three- ity was mostly restricted to individual endfeet, tial for defining their roles in brain physiology and dimensionally scan entire astrocytes and observe even to their fractions, and only occasionally on May 24, 2017 pathology. However, the specifics of astrocytic Ca2+ full-cell Ca2+ dynamics. coordinated with the endfoot process or the rest signaling are still poorly understood, and recent of the astrocyte. Two or more endfeet were mainly studies producing inconsistent or contradictory RESULTS: With our 3D approach, we sampled asynchronous, even when enwrapping the same results have fostered debate on the actual role of astrocytes at a sufficient rate to detect events with vessel. Astrocytic structures and axons intersected astrocytes in synaptic and vascular functions. durations of >1.5 s throughout the cell, and faster three-dimensionally, and minimal axonal activity ones in selected substructures. We found that (individual action potentials) produced time- RATIONALE: A neglected potential source of Ca2+ activity in an individual astrocyte is heter- correlated astrocytic Ca2+ elevations in small inconsistencies lies in the way astrocytic Ca2+ ogeneously scattered throughout the cell, largely spots (<1% of the volume), which demonstrates signaling has been studied to date, mostly by compartmented within each region, and prepon- that astrocytes can sense even the lowest levels conventional two-dimensional (2D) imaging, derantly local. The majority resides in the “gliapil,” of neuronal activity. CONCLUSION: We provide the first com- http://science.sciencemag.org/ prehensive 3D map of Ca2+ activity in an individual astrocyte. Its widespread, het- erogeneous, 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 rich- Downloaded from ness and complexity of the astrocyte ac- tivity, notably at astrocyte-synapse and astrocyte-vascular interfaces, where activ- ity is small, fast, and frequent. In this con- text,wecanforeseefuturechallengesin extending studies to the gliapil, whose struc- tures fall below current optical resolution, and in reporting the complete gamut of astro- cyte 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 3D Ca2+ imaging reveals the complex activity of astrocytes. (1) Representation of the 3D structural role of astrocytes in brain function.▪ interactions among astrocyte, axons, and blood vessels. The astrocyte contains optically resolved 2+ (c, core) and unresolved (g, gliapil) regions. (2) 3D Ca imaging reveals different activity (intensity Department of Fundamental Neuroscience, University color scale) in different astrocytic regions (P, processes; S, soma; EF, endfoot); (3) in different of Lausanne, 1005 Lausanne, Switzerland. processes; and (4) in different loci of individual processes. (5) It also reveals asynchronous activity *These authors contributed equally to this work. †Corresponding author. Email: [email protected] in endfeet, (6) often restricted to endfoot subdomains, and (7) local astrocyte responses to minimal Cite this article as E. Bindocci et al., Science 356, axonal firing, or (8) global responses to in vivo locomotion (time color scale). eaai8185 (2017). DOI: 10.1126/science.aai8185 Bindocci et al., Science 356, 715 (2017) 19 May 2017 1of1 RESEARCH ◥ Therefore, we extracted volumes directly from RESEARCH ARTICLE fluorescence measures in the structural “core,” whereas we corrected measures done in the gliapil for volume fraction (30). The estimated average NEUROSCIENCE volume of an astrocyte was 13,019 ± 1750 mm3 (range, 3000 to 35,000 mm3), 25 ± 2.5% of which 2+ was occupied by the structural core and 75 ± 2.5% Three-dimensional Ca imaging by the gliapil. Within the core, the soma occupied 22.5 ± 2.2% of the volume, the stem processes with appendages 74 ± 2.5%, and the endfeet 3.5 ± 0.7%. advances understanding of On average, each hippocampal astrocyte had 3.5 endfeet (range, 1 to 7) and 5.8 stem processes astrocyte biology (range, 3 to 9), and each process was 20.9 mmlong (range, 5 to 64 mm) (Fig. 1). Astrocytes of the CA1, Erika Bindocci,* Iaroslav Savtchouk,* Nicolas Liaudet,* Denise Becker, CA3, and DG regions were not significantly dif- Giovanni Carriero, Andrea Volterra† ferent in their volume measures (P =0.76,0.3,and 0.3 for total, core, and gliapil volume, respectively; Astrocyte communication is typically studied by two-dimensional calcium ion (Ca2+) Kruskal-Wallis test) and were therefore grouped imaging, but this method has not yielded conclusive data on the role of astrocytes in in subsequent analyses. synaptic and vascular function. We developed a three-dimensional two-photon imaging Next, for a given astrocyte structure, we com- approach and studied Ca2+ dynamics in entire astrocyte volumes, including during pared the structure volume visible in the best axon-astrocyte interactions. In both awake mice and brain slices, we found that Ca2+ single two-photon focal plane (axial resolution, activity in an individual astrocyte is scattered throughout the cell, largely compartmented ∼1 mm) (30) to the total structure volume. A single between regions, preponderantly local within regions, and heterogeneously distributed two-photon focal plane reported at maximum 4.0 ± regionally and locally. Processes and endfeet displayed frequent fast activity, whereas the 0.30% of the volume of a hippocampal astrocyte soma was infrequently active. In awake mice, activity was higher than in brain slices, (Fig. 1 and movie S1). The best plane for endfeet on May 24, 2017 particularly in endfeet and processes, and displayed occasional multifocal cellwide events. reported 21.9 ± 2.79% of the calculated endfoot Astrocytes responded locally to minimal axonal firing with time-correlated Ca2+ spots. volume and contained at maximum 2 of 3.5 end- feet. For the soma, the best plane reported 10.44 ± 0.60% of its volume; for processes, the best plane strocytes translate information received nal elements, as well as the emerging complexity reported 6.12 ± 0.54% of their volume and con- from neighboring central nervous system of astrocytic Ca2+ activity (2). tained 2.6 of 5.8 processes with an average length cells and generate modulatory responses The reported potential feasibility of 3D two- of 9.55 mm (range, 1 to 41.9 mm). at the local or network level via Ca2+ signal- photon Ca2+ imaging in astrocytes (27)andthe A A3DCa2+ imaging and analysis ing (1–6). In response to neuronal activity, recent availability of fast scanners and sensitive they show intracellular Ca2+ elevations (7–10)that detectors led us to explore 3D scanning of entire approach for astrocyte studies result in downstream effects on synaptic trans- astrocytes. By selectively expressing the geneti- The above data prompted us to develop a 3D ap- mission and plasticity (11–15). Astrocytic Ca2+ cally encoded Ca2+ indicator (GECI) GCaMP6f (28) proach to studying astrocyte biology. We combined elevations also trigger vascular responses that in astrocytes, we succeeded in capturing whole- state-of-the-art technologies and new analytical http://science.sciencemag.org/ may be involved in the control of cerebral blood astrocyte Ca2+ activity, including in structurally methods (Fig. 2). For studying astrocyte Ca2+ flow (16–19). Therefore, deciphering the “Ca2+ unresolved regions (the so-called gliapil). We also dynamics, we generated