Topical Application of Indicators for Calcium Imaging at the Drosophila Larval Neuromuscular Junction

Abstract

Calcium imaging is a technique in which Ca2+-binding molecules are loaded into live cells and as they bind Ca2+ they “indicate” the concentration of free calcium through a change in either the intensity or the wavelength of light emitted (fluorescence or bioluminescence). There are several possible methods for loading synthetic Ca2+ indicators into subcellular compartments, including topical application of membrane-permeant Ca2+ indicators, forward-filling of dextran conjugates, and direct injection. Calcium imaging is a highly informative technique in neurobiology because Ca2+ is involved in many neuronal signaling pathways and serves as the trigger for neurotransmitter release. This article describes the topical application of Ca2+ indicators at the Drosophila larval neuromuscular junction (NMJ). This loading technique is simple to execute and yields data quickly. The drawback is that the data can be difficult to interpret, primarily because it is difficult to ascertain which cellular and subcellular compartment(s) are loaded (e.g., muscle, nerve, or glia; cytosol, mitochondrion, or endoplasmic reticulum).