Ancestral organisation and asymmetric circuit architecture of the habenula revealed in a basal ray-finned fish.

The habenula is a conserved dorsal diencephalic hub that integrates information related to motivational state, behavioural flexibility, and cognitive control. In many vertebrates, it displays pronounced left-right asymmetry, yet how structural lateralisation is reflected in circuit architecture remains poorly understood, partly because phylogenetically informative lineages remain undersampled. Here, we characterise habenular connectivity in the Senegal bichir (Polypterus senegalus), a member of an early-diverging ray-finned fish lineage with marked habenular asymmetry. Using neuronal tracing, histology, and immunohistochemistry, we show that Polypterus retains a conserved vertebrate connectivity scaffold, with afferents arising from subpallial, hypothalamic, and posterior tubercular territories and efferents projecting primarily to the interpeduncular and raphe nuclei. Layered onto this scaffold are distinctive organisational features, including traits shared with teleosts that may represent an ancient actinopterygian condition, as well as apparent lineage-specific specialisations of bichirs. Notably, the enlarged right habenula receives both ipsilateral and contralateral inputs, whereas the left habenula is largely restricted to ipsilateral afferents, revealing a right-dominant mode of hemispheric integration. At the output level, left and right habenular efferents terminate in segregated dorsal and ventral territories of the interpeduncular nucleus, respectively. This organisation indicates that asymmetric integration within the habenula is associated with segregated engagement of downstream pathways. Together, these findings establish bichirs as a key comparative model for reconstructing early osteichthyan habenular circuitry and provide insight into the evolutionary origins of mechanisms linking habenular asymmetry to lateralised circuit organisation across vertebrates.