A simple bioluminescence-based method for recording Ca²⁺ signaling in the cytosol or mitochondria of neural progenitor cells from the adult zebrafish brain

Neurospheres derived from the adult zebrafish brain are a convenient model for investigating the initial stages of neurogenesis. Neurospheres are composed of stem cells, which have the ability to either self-renew or differentiate into specialized cells (i.e., neurons and glial cells) if cultured in an appropriate medium. It has previously been suggested that Ca2+ signaling in the cytosol and mitochondria might play some role in neurogenesis. Ca2+ signals can be recorded using the non-toxic bioluminescent Ca2+ reporter complex, holoaequorin. We have developed a novel electroporation protocol to load neurospheres with cDNA encoding either mitochondrial-targeted EGFP-apoaequorin (MitGA) or the cytosolic-targeted EGFP-apoaequorin (CytGA). We show that within ~24 h after electroporation, each of these apoaequorin proteins were expressed in ~20% of the neurosphere cells. In addition, after reconstitution of holoaequorin by incubation of the cells with its luminophore, coelenterazine, dynamic changes in [Ca2+] in the cytosol and mitochondria were imaged using a custom-built electron multiplying charge coupled device (EMCCD)-based photon imaging microscope (PIM) system or measured using a photomultiplier tube (PMT)-based luminescence detection system, after activation of storeoperated Ca2+ entry (SOCE).