Anatomical and functional organization of the interpeduncular nucleus in larval zebrafish

The habenulo-interpeduncular pathway is a highly conserved neural circuit across vertebrates, but the anatomical and functional architecture of the interpeduncular nucleus (IPN) remains poorly understood. Here, we use a combination of immunohistochemistry, volumetric electron microscopy (EM), and two-photon imaging to provide the first detailed characterization of the internal organization of the IPN in larval zebrafish. We show that the IPN receives extensive projections from the tegmentum, and reveal a strict segregation between the dorsal (dIPN) and ventral (vIPN) subcircuits, with minimal cross-communication. In the dIPN, we characterise in detail the inputs and outputs of r1{pi} neurons, which have been recently identified as representing the animals heading direction. In the vIPN, we identify six distinct glomerular structures, each exhibiting specific patterns of reciprocal connections and projection pathways. Finally, we demonstrate that the connectivity and spontaneous activity patterns of habenular axons are shaped by the local anatomical features of the IPN, suggesting a role for the local interneurons in modulating presynaptic dynamics. Together, these results enhance our understanding of the internal organization of the IPN, and provide a framework for future investigations into both its physiology and its involvement in behavior.