Ex Vivo Imaging of Mitochondrial Dynamics and Trafficking in Astrocytes

Mitochondria are essential organelles involved in energy supply and calcium homeostasis. The regulated distribution of mitochondria in polarized cells, particularly neurons, is thought to be essential to these roles. Altered mitochondrial function and impairment of mitochondrial distribution and dynamics is implicated in a number of neurologic disorders. Several recent reports have described mechanisms regulating the activity-dependent distribution of mitochondria within astrocyte processes and the functional consequences of altered mitochondrial transport. Here we provide an ex vivo method for monitoring the transport of mitochondria within the processes of astrocytes using organotypic "slice" cultures. These methods can be easily adapted to investigate a wide range of mitochondrial behaviors, including fission and fusion dynamics, mitophagy, and calcium signaling in astrocytes and other cell types of the central nervous system. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of brain slices Basic Protocol 2: Preparation of gene gun bullets Basic Protocol 3: Gene gun transfection of slices Basic Protocol 4: Visualization and tracking of mitochondrial movement Alternate Protocol: Transduction of EGFP-mito via viral injection of the neonatal mouse brain.