Studying Axon-Astrocyte Functional Interactions by 3D Two-Photon Ca²⁺ Imaging: A Practical Guide to Experiments and "Big Data" Analysis

Recent advances in fast volumetric imaging have enabled rapid generation of large amounts of multi-dimensional functional data. While many computer frameworks exist for data storage and analysis of the multi-gigabyte Ca²⁺ imaging experiments in neurons, they are less useful for analyzing Ca²⁺ dynamics in astrocytes, where transients do not follow a predictable spatio-temporal distribution pattern. In this manuscript, we provide a detailed protocol and commentary for recording and analyzing three-dimensional (3D) Ca²⁺ transients through time in GCaMP6f-expressing astrocytes of adult brain slices in response to axonal stimulation, using our recently developed tools to perform interactive exploration, filtering, and time-correlation analysis of the transients. In addition to the protocol, we release our in-house software tools and discuss parameters pertinent to conducting axonal stimulation/response experiments across various brain regions and conditions. Our software tools are available from the Volterra Lab webpage at https://wwwfbm.unil.ch/dnf/group/glia-an-active-synaptic-partner/member/volterra-andrea-volterra in the form of software plugins for Image J (NIH)-a de facto standard in scientific image analysis. Three programs are available: MultiROI_TZ_profiler for interactive graphing of several movable ROIs simultaneously, Gaussian_Filter5D for Gaussian filtering in several dimensions, and Correlation_Calculator for computing various cross-correlation parameters on voxel collections through time.