In Vivo Imaging of Neural Activity in Unanesthetized Drosophila Adult Flies.

In vivo imaging is crucial for studying neurobiology in Drosophila as it allows real-time visualization of neuronal activity, development, and plasticity within the intact, behaving fly. This technique provides invaluable insights into dynamic processes, such as synaptic function and circuit connectivity, which cannot be accurately captured in fixed or ex vivo preparations. Most techniques for fly preparation for in vivo imaging involve anesthetizing the flies shortly before functional imaging. Nevertheless, extensive evidence demonstrates that anesthesia impacts several cognitive and physiological processes. For this reason, in vivo imaging of adult Drosophila melanogaster without the use of anesthetics during animal preparation is a challenging yet highly desirable approach. The protocol presented here involves immobilizing the head of awake flies by suctioning the proboscis into a small metal tube connected to a vacuum system. While the head is fixed, the fly is prepared for functional imaging without the need for anesthesia. This protocol is rapid and reproducible, ensuring no harm to the fly. The main advantage of this method lies in its avoidance of anesthetic use, thereby accounting for the potential complex effects of anesthesia on neural activity. This method is both affordable and highly adaptable, utilizing inexpensive, customizable tools. It allows for the successful imaging of live flies, capturing fast changes in neural activity associated with dynamic cognitive processes.