Studying <i>Drosophila</i> Larval Behavior in Agarose Channels

Larvae of the fruit fly <i>Drosophila melanogaster</i> are a popular and tractable model system for studying the development and function of sensorimotor circuits, thanks to the relative numerical simplicity of their nervous system and the wealth of available genetic tools to manipulate the anatomy, activity, and function of specific cell types. Researchers studying the role of a particular gene or cell type in sensorimotor circuit activity or function may wish to observe the effects of an experimental manipulation during behavior in the intact animal. Observing these effects, which may include changes in the intracellular calcium concentration or movement of small numbers of neurons, muscles, etc., typically requires high-spatial-resolution imaging, which poses several difficulties in the freely crawling larva. Freely crawling larvae can move quickly and with changeable heading, making manual or automatic tracking challenging; additionally, they may make three-dimensional movements, such as rearing, that can degrade imaging focus. These challenges are potentially solvable using advanced imaging and algorithmic tracking setups, but cost, space, or development time may be prohibitive. This protocol describes a simple and cost-effective method for placing larvae inside agarose channels, thereby restricting larval crawling to a single dimension and enabling higher-magnification time-series imaging of fluorescently labeled structures during many cycles of locomotion. By using larvae that express fluorescent calcium indicators in cells of interest, researchers can apply this method to study the effects of experimental manipulations on neural or muscular activity during behavior in the intact animal.