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.

During DNA recombination and repair, RecA family proteins must promote rapid joining of homologous DNA. Repeated sequences with >100 base pair lengths occupy more than 1% of bacterial genomes; however, commitment to strand exchange was believed to occur after testing ∼20–30 bp. If that were true, pairings between different copies of long repeated sequences would usually become irreversible. Our experiments reveal that in the presence of ATP hydrolysis even 75 bp sequence-matched strand exchange products remain quite reversible. Experiments also indicate that when ATP hydrolysis is present, flanking heterologous dsDNA regions increase the reversibility of sequence matched strand exchange products with lengths up to ∼75 bp. Results of molecular dynamics simulations provide insight into how ATP hydrolysis destabilizes strand exchange products. These results inspired a model that shows how pairings between long repeated sequences could be efficiently rejected even though most homologous pairings form irreversible products.

The dynamics and connectivity of neural circuits continuously change on timescales ranging from milliseconds to an animal’s lifetime. Therefore, to understand biological networks, minimally invasive methods are required to repeatedly record them in behaving animals. Here we describe a suite of devices that enable long-term optical recordings of the adult Drosophila melanogaster ventral nerve cord (VNC). These consist of transparent, numbered windows to replace thoracic exoskeleton, compliant implants to displace internal organs, a precision arm to assist implantation, and a hinged stage to repeatedly tether flies. To validate and illustrate our toolkit we (i) show minimal impact on animal behavior and survival, (ii) follow the degradation of chordotonal organ mechanosensory nerve terminals over weeks after leg amputation, and (iii) uncover waves of neural activity caffeine ingestion. Thus, our long-term imaging toolkit opens up the investigation of premotor and motor circuit adaptations in response to injury, drug ingestion, aging, learning, and disease.