A visualization pipeline for in vivo two-photon volumetric astrocytic calcium imaging

Astrocytes,the multi-functional glial cells with the most abundant population in the brain,integrate information across their territories to regulate neuronal synaptic and cerebrovascular activities.Astrocytic calcium(Ca^(2+))signaling is the major readout of cellular functional state of astrocytes.The conventional two-photon in vivo imaging usually focuses on a single horizontal focal plane to capture the astrocytic Ca^(2+)signals,which leaves>80%spatial information undetected.To fully probe the Ca^(2+)activity across the whole astrocytic territory,we developed a pipeline for imaging and visualizing volumetric astrocytic Ca^(2+)time-lapse images.With the pipeline,we discovered a new signal distribution pattern from three-dimensional(3D)astrocytic Ca^(2+)imaging data of mice under isoflurane anesthetic states.The tools developed in this study enable a better understanding of the spatiotemporal patterns of astrocytic activity in 3D space.