Forward-Filling of Dextran-Conjugated 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 forward-filling of dextran-conjugated indicators at the Drosophila larval neuromuscular junction (NMJ). This technique is particularly well suited for imaging changes in cytosolic Ca²⁺ as dextran conjugation prevents compartmentalization of the Ca²⁺ indicator. The major drawback is that the nerves must be severed at the start of the loading process, several hours before nerve terminals are ready to examine.