Store-Operated Ca 2+ Entry in Drosophila Primary Neuronal Cultures

Abstract

Intracellular calcium signals in neurons frequently derive from the stimulation of G protein-coupled receptors (GPCR) by neurotransmitters, neuropeptides, and neurohormones. GPCR stimulation in neurons leads to generation of inositol 1,4,5-trisphosphate (IP 3 ), which in turn activates endoplasmic reticulum (ER)-localized IP 3 receptors. The IP 3 receptor (IP 3 R) is a ligand-gated Ca 2+ channel, which releases Ca 2+ from ER stores. In Drosophila neurons it has been shown that depletion of ER Ca 2+ store is followed by store-operated Ca 2+ entry (SOCE) through STIM and Orai, the ER Ca 2+ sensor and the plasma membrane Ca 2+ channel respectively. The elucidation of this Ca 2+ signaling pathway in neurons has in part been possible due to the ease of genetic manipulation in Drosophila, which has allowed neuron-specific knockdown of various proteins of interest. This has been followed by standardization of conditions for culturing neurons from dissected brains of the relevant genotypes, such that they could be used for robust Ca 2+ measurements by imaging with standard Ca 2+ indicator dyes. Protocols for measurement of IP 3 -mediated Ca 2+ release, passive depletion of ER Ca 2+ store, and SOCE in primary cultures of Drosophila neurons are described here.