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

To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because the portion of the nervous system that contains limb premotor and motor circuits is largely inaccessible to large-scale recording techniques 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 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 novel class of locomotion-associated descending neurons. This new approach enables the direct investigation of circuits associated with complex limb movements.