Distinct and asymmetric responses to pitch-tilt axis and roll-tilt axis vestibular stimulation in larval zebrafish

Posture is crucial to executing motor functions and its perturbation leads to various strategies to regain preferred postures. The vestibular system plays a vital role in responding to movement-related cues to maintain preferred directions. Previous work has introduced a miniaturised, rotating, light-sheet microscope for brain-wide neuronal recordings during controlled vestibular stimulation in head-restrained zebrafish larvae to study whole-brain circuits. In this study, we present significant enhancements to such a setup to enable precise rotational movements along arbitrary axes between roll-tilt and pitch-tilt (nose moving up and down) directions. The improved design is 3D printable and incorporates a double galvanometer mirror configuration, facilitating replication and enhancing scanning capabilities. Using this design, we conducted the first mapping of larval zebrafish brain-wide responses to dynamic vestibular stimulation along the pitch-tilt axis, a dimension of vestibular processing that is important for movement initiation but hasn't been explored with whole-brain imaging. Our findings revealed an asymmetry in the number of responsive neurons between nose-up and nose-down tilt responses, displaying distinct spatial patterns. Furthermore, we conducted recordings during roll-tilt stimuli in the same fish, revealing a greater number of responsive neurons across the brain, especially in the cerebellum, during roll stimuli, compared to pitch. Also, we identified a transgenic line that exhibited significant overlap with these functional maps, offering exciting opportunities for further investigation using fluorescence-mediated tracing, optogenetics, or targeted ablation studies. These results reveal how whole-brain circuits respond to vestibular stimuli by recruiting distinct and asymmetric neuronal populations based on the axis of the stimulus.