Protocol for simultaneous in vivo two-photon imaging and locomotion quantification during olfactory stimulation and pharmacology in walking Drosophila

To decipher the circuit and molecular mechanisms underlying neural information processing, it is essential to manipulate cellular targets while simultaneously recording neural activity and behavior during sensory stimulation. Here, we present a protocol for Drosophila that temporally synchronizes olfactory stimulation and pharmacological intervention with in vivo recording as well as locomotion measurement. We detail step-by-step workflows for head-fixed walking flies, including fly preparation, surgery, two-photon imaging, and locomotion detection, providing instructions for surgery station, odor stimulator, and perfusion system setup.