Performing Electrophysiology and Two-Photon Calcium Imaging in the Adult Drosophila Central Brain During Walking Behavior

Continuing improvements in genetically encoded calcium indicators (GECIs) make imaging an increasingly attractive method to observe neural activity in the Drosophila brain. Two-photon imaging with GECIs allows calcium signals to be monitored in the entire adult fly central brain in vivo. It has recently become possible to perform two-photon imaging and electrophysiology in behaving flies during tethered flight (Maimon et al., Nat Neurosci 13:393–399, 2010) and walking (Seelig et al., Nat Methods 7:535–540, 2010). Here we elaborate on methods to perform two-photon calcium imaging with GCaMP, and GFP-/GCaMP-guided whole-cell patch clamp and loose-patch electrophysiological recordings in a head-fixed walking fly. We discuss how loose-patch recording can be used simultaneously with two-photon imaging to relate GECI signals to spiking activity in the central brain. Finally, we present a protocol for the loading and use of synthetic calcium dyes in combination with neurons labeled with a spectrally separated fluorescent marker to perform higher temporal resolution recordings from the central brain.