Identification of Ca2+ signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres

The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells(NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3 r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor(IP3 R) type-1 and ryanodine receptor(RyR) type-2 are differentially expressed in cells with neuron-or radial glial-like properties. Furthermore, ATP but not caffeine(IP3 R and RyR agonists, respectively), induced the generation of Ca^(2+) transients in cells exhibiting neuron-or glial-like morphology. These results indicate the differential expression of components of the Ca^(2+) -signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca^(2+) signaling as a read-out.