Imaging neural activity in the ventral nerve cord of behaving adult Drosophila

Abstract

To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because limb premotor and motor circuits have been largely inaccessible for large-scale recordings in intact, moving animals—a constraint that is true for both vertebrate and invertebrate models. Here, we introduce a method for 2-photon functional imaging from the ventral nerve cord (VNC) of behaving adult Drosophila melanogaster. We use this method to reveal patterns of activity across nerve cord populations during grooming and walking and to uncover the functional encoding of moonwalker ascending neurons (MANs), moonwalker descending neurons (MDNs), and a previously uncharacterized class of locomotion-associated A1 descending neurons. Finally, we develop a genetic reagent to destroy the indirect flight muscles and to facilitate experimental access to the VNC. Taken together, these approaches enable the direct investigation of circuits associated with complex limb movements.

Reconstruction of motor control circuits in adultDrosophilausing automated transmission electron microscopy

“…Here, we present a connectomic dataset that will enable complete mapping of connectivity of the neuronal circuits that control the legs and wings of an adult Drosophila melanogaster. Combined with recent advances in the ability to record activity from genetically identified VNC neurons during behavior (Azevedo et al, 2019;Chen et al, 2018;Mamiya et al, 2018), we expect that a greater understanding of the circuit basis for complex motor control is within reach.…”

“…Drosophila melanogaster is a particularly appealing system for dissecting mechanisms of motor control because it is a genetically accessible model system and has many complex and well-characterized behaviors including walking, flight, escape responses, grooming, and courtship. Recent advances allow in vivo electrophysiological recordings and calcium imaging of genetically identified VNC neurons in behaving adult Drosophila (Chen et al, 2018;Mamiya et al, 2018;Tuthill and Wilson, 2016b), providing an increasingly better grasp of motor and premotor neuron activity during locomotor behavior. Furthermore, the small size of the Drosophila nervous system makes it suitable for comprehensive connectome mapping using electron microscopy (EM).…”

A size principle for leg motor control inDrosophila

“…This principle provides a useful framework for understanding the organization and function of motor circuits that flexibly control a wide range of fly behaviors, from walking to aggression to courtship. Studies of motor control in the fly VNC are now possible thanks to an increasing catalog of genetically-identified cell types (Lacin et al, 2019;Shepherd et al, 2019, Namiki et al, 2018, connectomic reconstruction with serial-section EM and the ability to image from VNC neurons in walking flies (Chen et al, 2018). We anticipate that Drosophila will provide a useful complement to other model organisms in understanding the neural basis of flexible motor control.…”

A single microRNA-Hox gene module controls complex movement in morphologically-distinct developmental forms ofDrosophila

“…To prepare flies for in vivo imaging in the ventral nerve cord (VNC) ( Figure 5F) we adapted existing methods (Chen et al, 2018;Seeholzer et al, 2018). In brief, a single fly (3-5 days old) was cold-anaesthetised after eclosion and tethered using UV-curable glue to a piece of aluminium foil that covered a hole in the bottom of a modified polystyrene weighing dish.…”