Direct Injection 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 direct injection of Ca²⁺ indicators at the Drosophila larval neuromuscular junction (NMJ). This technique allows rapid loading of most Ca²⁺ indicators, but there are drawbacks in that it is a difficult technique to master and requires additional electrophysiological equipment. Also, Ca²⁺ indicators that are easily injected are usually susceptible to compartmentalization.