Long-term imaging of the ventral nerve cord in behaving adultDrosophila

Abstract

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.

Ascending neurons convey behavioral state to integrative sensory and action selection centers in the brain

“…Thus, future work should examine the encoding of ANs in a variety of contexts including tethered flight. Finally, it would also be interesting to test the degree to which AN encoding is genetically hardwired or capable of adapting following motor learning or after injury [53, 54]. In summary, here we have shown that ANs encode high-level behaviors that they convey to distinct integrative sensory and action selection centers in the brain.…”

Descending neuron population dynamics during odor-evoked and spontaneous limb-dependent behaviors

“…Additionally, because DN cell bodies and neurites are distributed across the brain ( Namiki et al, 2018 ), relatively invasive ( Mann et al, 2017 ) volumetric imaging approaches would be required to simultaneously record the activity of many DNs at once. As an alternative, we previously developed a thoracic dissection approach that enables the optical recording of descending and ascending axons within the cervical connective in tethered, behaving animals ( Chen et al, 2018 ; Hermans et al, 2021 ; Figure 1a ). Here, we combined this imaging approach with genetic tools ( Asahina et al, 2014 ) that restrict the expression of neural activity reporters to the brain.…”

A toolbox of engineered mosquito lines to study salivary gland biology and malaria transmission

“…The localization of most sporozoites, separated from the objective by several cell layers, was a limiting factor in our settings, which could be improved by using two-photon microscopy instead of confocal microscopy, thus providing a better resolution when imaging in deep tissues. Recently, this method has been used for long-term monitoring of the ventral nerve cord in live adult Drosophila , although the cuticle was previously partially removed for this approach [ 35 ]. Sample selection and preparation, like partial embedding of mosquitoes as well as the choice of the immersion medium, could also lead to significant improvements.…”