ALL-OPTICAL MAPPING OF THE MESENCEPHALIC LOCOMOTOR REGION IN THE LARVAL ZEBRAFISH

In vertebrates, stimulation of the mesencephalic locomotor region (MLR) can reliably elicit forward locomotion and represents a promising avenue to recover motor deficits in Parkinson’s disease. Yet, MLR neurons are genetically diverse, spatially intermingled, and project widely throughout the brain. Consequently, the effects of MLR stimulation largely vary according to the location and cell type targeted. Linking the soma location of individual MLR neurons with their neurotransmitter phenotype and projection profile is essential at the population level to resolve their contribution and perform efficient stimulations for motor recovery. Here, we leverage the small size and transparency of the larval zebrafish whose MLR contains ~ 200 neurons to unravel their molecular and functional diversity. We first reconstruct in 3D the spatial organization of MLR subpopulations by imaging transgenic lines labeling neurons expressing distinct neurotransmitters or transcription factors. Using photoactivable GFP, we then perform optical retrograde labeling of descending MLR neurons based on their projections in the medulla and spinal cord to link neurotransmitter phenotypes with structural organization. Finally, we implement columnar optogenetic stimulations with digital holography to selectively activate genetically and spatially defined MLR subtypes. We combine this approach with high-speed recordings of tail movements to probe the contribution of MLR neurons to movement initiation, steering modulation or motor arrest. Altogether, our anatomical and functional mapping approaches will clarify the spatial and functional organization of the MLR in a small model organism and should help refining deep brain stimulation strategies.​