Store-Operated Ca2+ Entry in Drosophila Primary Neuronal Cultures

Methods Mol Biol. 2018:1843:125-136. doi: 10.1007/978-1-4939-8704-7_11.

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 (IP3), which in turn activates endoplasmic reticulum (ER)-localized IP3 receptors. The IP3 receptor (IP3R) is a ligand-gated Ca2+ channel, which releases Ca2+ from ER stores. In Drosophila neurons it has been shown that depletion of ER Ca2+ store is followed by store-operated Ca2+ entry (SOCE) through STIM and Orai, the ER Ca2+ sensor and the plasma membrane Ca2+ channel respectively. The elucidation of this Ca2+ 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 Ca2+ measurements by imaging with standard Ca2+ indicator dyes. Protocols for measurement of IP3-mediated Ca2+ release, passive depletion of ER Ca2+ store, and SOCE in primary cultures of Drosophila neurons are described here.

Keywords: Carbachol; Fluo-4; IP3R; Indo-1; Orai; STIM; Thapsigargin; Time-lapse imaging.

MeSH terms

  • Animals
  • Calcium / metabolism*
  • Calcium Channels / genetics*
  • Calcium Channels / metabolism*
  • Calcium Signaling*
  • Cell Culture Techniques
  • Cells, Cultured
  • Drosophila / physiology*
  • Female
  • Image Processing, Computer-Assisted
  • Ion Channel Gating
  • Male
  • Molecular Imaging / methods
  • Neurons / metabolism*
  • Time-Lapse Imaging

Substances

  • Calcium Channels
  • Calcium