Imaging Calcium Activity Patterns in the Drosophila Pupal Ecdysis Neural Circuit

Abstract

Insects execute a behavioral program, called an ecdysis sequence, to shed their cuticle at each developmental molt. The neural circuit controlling the ecdysis sequence consists of approximately 300 peptidergic neurons that express the Ecdysis Triggering Hormone receptor (ETHR) and are activated by peripheral release of Ecdysis Triggering Hormone (ETH). While several groups of neurons within the network play known roles in the ecdysis sequence, such as those expressing the hormones CCAP and bursicon, the timing of their activity is poorly understood and likely involves regulation by as yet unidentified inhibitory neurons. In general, the patterns of activity within the ecdysis network and how they give rise to motor output remains enigmatic. Using the genetically tractable Drosophila model system, and focusing on the ecdysis sequence executed at the pupal molt, we have investigated the evolution of network activity using Gal4 driver lines to express UAS-RCaMP or UAS-GCaMP6 Ca2+ biosensors in subsets of neurons in the ETHR network or its downstream motor neurons. We are able to elicit the ecdysis sequence in an excised pupal brain by application of exogenous ETH and visualize the neural activity underlying the ecdysis-specific motor patterns in targeted neurons. The quantitative temporal information provided by Ca2+ biosensors, when combined with anatomical data derived from structured illumination and diffraction-limited fluorescence microscopy techniques, provides insights into how the activity patterns of the ETHR circuit are generated.