Comparative analysis of the molecular, spatial, and functional domains of vertebrate habenula

The habenula (Hb) is a diencephalic brain region implicated in adaptive behaviors and neuropsychiatric disorders. Two main Hb subdivisions, the lateral habenula and medial habenula, have been shown to play different roles in diverse behaviors, ranging from classical and operant learning to the prediction of outcomes, fear, addiction, and social interactions. However, our understanding of cytoarchitecture and topography of the vertebrate Hb, as well as how different Hb microcircuits are organized across species, remains limited. In this study, we utilized a combination of single-cell and spatial transcriptomic approaches across multiple species to investigate how habenular subdivisions are organized and how this organization compares across species in relation to Hb function. First, we demonstrated that habenular cell types are relatively conserved across species from fish to mice, with some exceptions. Subsequently, employing spatial transcriptomics at subcellular resolution, we identified numerous topographically organized subregions within the adult zebrafish and mouse Hb. Many of these subregions were found to be shared between both species. We also observed that while zebrafish Hb is known to be highly asymmetric across hemispheres, several habenular subdomains remain symmetric, resembling those of mouse Hb. Similarly, though the mouse habenula is traditionally considered highly symmetrical across hemispheres, we observed a few mouse Hb subregions with prominent asymmetries. These results suggest that the asymmetry of the vertebrate habenula exists on a continuum across species. Moreover, vertebrates share many cytoarchitectural similarities that are topographically organized. Our ongoing investigation aims to elucidate how the molecular topography of the zebrafish Hb relates to its functional architecture.