Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca²⁺) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca²⁺ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca²⁺ dynamics in vivo, where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca²⁺ imaging in vivo with a subcellular resolution that permits the analysis of Ca²⁺ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca²⁺ affinities are used to demonstrate the use of this protocol for measuring relative Ca²⁺ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca²⁺ handling regulates neuronal function and plasticity.