In Vivo Optical Recording of Brain Interneuron Activities from a Drosophila Male on a Treadmill

Recent development of genetically encoded calcium indicators (GECIs) allows us to directly monitor neural activities in in vivo preparations from various organisms, enabling the exploration of neural substrates for complex behaviors. As a showcase for such renovated neuroethology with GECIs, we describe a novel method to record optically interneuron activities from the brain of a tethered male fruit fly Drosophila melanogaster when contact chemosensory stimulation is given to his foreleg. In this protocol, the recording is made on the preparation in which a fly is put on an air-supported styrofoam ball, keeping the “moving” fly in position to allow monitoring of the activity of central neurons from the fly whose movement is minimally restricted. This protocol is applicable to the analysis of other behaviors induced by stimuli of different modalities with small modifications.