Optical Recording of Brain Neuron Activities from a Male Drosophila Behaving on a Treadmill

A wide array of genetic tools is available for monitoring and manipulating the activities of identified neurons in the fruit fly Drosophila melanogaster to investigate the mechanisms of neural circuit operation underlying behavior. Although contemporary genetically encoded calcium indicators have attained the high sensitivity that allows detection of a single-action potential, applying them to a behaving adult fly is still challenging because conditions ideal for optical recordings from the brain may constrain movements of the fly. Here we describe a protocol for recording brain neuron activities from a tethered male Drosophila in courtship pursuit on a treadmill, with emphasis on reproducible preparation making that takes advantage of a simple custom rig for fly tethering. The method can be applied to other experimental approaches, such as electrophysiology or optogenetic neural activity manipulation with minor modifications.