Open-Field Locomotion Assay with Optogenetic Stimulation v1

This protocol describes the open-field assay used to quantify locomotor responses during optogenetic manipulation of the VTA–PnO pathway. Experiments were conducted under controlled illumination conditions during the light phase and synchronized with optical stimulation delivered either to VTA somata (DAT-Cre experiments) or to presynaptic terminals (GAD2 experiments). The VTAPnO projection-defined subpopulation was selectively labeled using a retrograde Flp-dependent targeting strategy, enabling specific manipulation of VTA neurons projecting to the PnO. Locomotor behavior was recorded using overhead video acquisition and analyzed offline for directional movement parameters.