Long-term imaging of the ventral nerve cord in behaving adult Drosophila

The dynamics and connectivity of neural circuits continuously change during an animal’s lifetime on timescales ranging from milliseconds to days. Therefore, to investigate how biological networks accomplish remarkable cognitive and behavioral tasks, minimally invasive methods are needed to perform repeated measurements, or perturbations of neural circuits in behaving animals across time. Such tools have been developed to investigate the brain but similar approaches are lacking for comprehensively and repeatedly recording motor circuits in behaving animals. Here we describe a suite of microfabricated technologies that enable long-term, minimally invasive optical recordings of the adult Drosophila melanogaster ventral nerve cord (VNC)—neural tissues that are functionally equivalent to the vertebrate spinal cord. These tools consist of (i) a manipulator arm that permits the insertion of (ii) a compliant implant into the thorax to expose the imaging region of interest; (iii) a numbered, transparent polymer window that encloses and provides optical access to the inside of the thorax, and (iv) a hinged remounting stage that allows gentle and repeated tethering of an implanted animal for two-photon imaging. We validate and illustrate the utility of our toolkit in several ways. First, we show that the thoracic implant and window have minimal impact on animal behavior and survival while also enabling neural recordings from individual animals across at least one month. Second, we follow the degradation of chordotonal organ mechanosensory nerve terminals in the VNC over weeks after leg amputation. Third, because our tools allow recordings of the VNC with the gut intact, we discover waves of neural population activity following ingestion of a high-concentration caffeine solution. In summary, our microfabricated toolkit makes it possible to longitudinally monitor anatomical and functional changes in premotor and motor neural circuits, and more generally opens up the long-term investigation of thoracic tissues. ### Competing Interest Statement The authors have declared no competing interest.