Using Volumetric 3D Microscopy Techniques to Characterize Astrocyte Morphologies over Large Spatial Scales

Abstract

Astrocytes are a type of glia found in the central nervous system with many roles, and have recently been reported to be directly involved in modulating and scaling synaptic transmission. Neurotransmitters released in synaptic activity induce astrocyte calcium signaling, which then induces the release gliotransmitters that can act on neighbouring pre- and postsynaptic neuronal elements. Astrocytes are highly 3-dimensional and form large syncytium of diffusion-coupled cells via their many long protrusions. It has been proposed that astrocytes may use these large networks to communicate and correlate to distant synapses. When studying astrocyte network interactions, it is important to establish astrocyte morphology at the the level of single cells and of populations, as this structure is directly related to the astrocytes’ ability for intercellular communication, and speed and frequency of calcium signal propagation. On-going advances in volumetric 3D imaging techniques, such as light-sheet microscopy, now allow for imaging complete murine brain sections, and obtaining a better understanding of how astrocytes are organized and interact in situ on a larger spatial scale. Here, a pipeline for characterizing morphology for both single astrocytes and their networks using volumetric 3D microscopy techniques was optimized using 500 µm and 1 mm thick mouse cortex slices. Various structural features were measured and quantified including territorial volume, cell orientations, number of connections, process arborisations and lengths. This characterization pipeline can be extended to cortical organoids, larger tissue sections, or other brain regions, and is a helpful tool for studying astrocyte network structure and functionality.