Three-dimensional Ca²⁺ imaging advances understanding of astrocyte biology

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.