Imaging Mitochondrial Dynamics in the Central Nervous System (CNS).

Notable for producing ATP via oxidative phosphorylation, mitochondria also control calcium homeostasis, lipogenesis, the regulation of reactive oxygen species, and apoptosis. Even within relatively simple cells, mitochondria are heterogeneous with regard to their shape, abundance, movement, and subcellular locations. They exist as interconnected, tubular networks and as motile organelles that are transported along the cytoskeleton for distribution throughout cells. These spatial and morphological features reflect variability in the organelle's capacity to synthesize ATP and support cells. Changes to mitochondria are believed to support cell function and fate, and mitochondrial dysfunction underlies disease in the nervous system. Here we describe an in vivo time-lapse imaging approach to monitor and measure the movement and position of the mitochondria in cells of the developing brain in albino tadpoles. The unparalleled benefit of using for these experiments is that measurements of mitochondrial morphology and distribution in cells can be measured in vivo, where the surrounding neural circuitry and other inputs that influence these critical organelles remain intact. This protocol draws together techniques to label brain cells and capture the morphology of the cells and their mitochondria with 3D time-lapse confocal microscopy. We describe open-source methods to reconstruct cells in order to quantify the features of their mitochondria.