Optogenetic interrogation of the zebrafish lateral line reveals brain-wide neural circuits involved in pattern separation.

An animal's ability to interact with its environment relies on the brain's capacity to distinguish between patterns of sensory information. To investigate this, we used the posterior lateral line system of larval zebrafish, composed of mechanosensory neuromasts innervated by neurons from the posterior lateral line ganglion. Using single-neuromast optogenetic stimulation and whole-brain calcium imaging, we developed a precise and flexible approach to examine sensory processing. Stimulating individual neuromasts revealed that second-order circuits show diverse selectivity despite lacking topographic organization. We further show that complex combinations of neuromast stimulation are encoded by sparse neuronal ensembles within the medial octavolateralis nucleus (MON) and that neuromast input integrates nonlinearly. This approach provides a powerful method for spatiotemporal interrogation of the zebrafish lateral line, sheds light on how neuromast input is integrated in the brain, and positions this system as a valuable model for studying whole-brain sensory encoding.