Two-dimensional virtual reality with optogenetic reinforcement to study landmark-guided navigation in head-fixed Drosophila
In insects the central complex (CX) is involved in various navigational behaviors. Recent calcium imaging and electrophysiology experiments in the Drosophila CX revealed a network of identified neurons that constitute an internal compass, which is updated using self-motion cues and visual landmarks. This finding suggests an involvement of the CX in guiding goal-directed navigation. Indeed, perturbing neural activity of defined cell types in the CX interferes can interfere with directed navigational behaviors such as fixation of distant visual landmarks and visual learning. An open question, however, remains how the fly’s compass is used during such directed navigational behaviors. Answering this question requires observing neural activity in the CX during a goal-directed navigational task. Current two-photon calcium imaging and electrophysiological techniques to study neural circuits in behaving flies require the animal to be head-fixed. For head-fixed Drosophila melanogaster, VR environments have been onedimensional (1D), typically providing an animal with visual feedback in response to rotation, but not forward movement. Unambiguously linking the neural representation of heading in the CX to specific visual features in a scene, however, requires 2D environments. We developed a VR system for head-fixed walking flies to study visually guided navigation in a two-dimensional (2D) world. We show that flies navigating in this VR display many of the behavioral characteristics seen in freely walking flies. We further demonstrate that the setup can be used to study visual and spatial memory by training flies to avoid virtual landmarks associated with an aversive “virtual heat” stimulus generated by optogenetically activating sensory neurons. Both the VR system and the behavioral assay are compatible with neurophysiological methods and will enable the interrogation of fly brain circuitry underlying goal-directed navigation in complex visual environments.