Supplementary Material for: Efferent Axonal Projections of the Habenular Complex in the Fire-Bellied Toad Bombina orientalis

The habenular complex and its associated axonal pathways are often
thought of as phylogenetically conserved features of the brain among
vertebrates despite the fact that detailed studies of this brain region
are limited to a few species. Here, the gross morphology and axonal
projection pattern of the habenular complex of an anuran amphibian, the
fire-bellied toad Bombina orientalis, was studied to allow
comparison with the situation in other vertebrates. Axonal pathways were
traced using biocytin applications in dissected brain preparations. The
results show that the rostral part of the left dorsal nucleus is
enlarged in this species, while the rostral ventral nucleus and caudal
parts do not show left-right size differences. Biocytin applications
revealed widespread axonal projections of the habenular complex to the
posterior tuberculum/dorsal hypothalamic region, ventral tegmentum,
interpeduncular nucleus (IPN), and raphe median. Additionally, axons
targeting the lateral hypothalamus originated from the ventral habenular
nuclei. The results also suggest an asymmetrical pattern of projection
to the IPN in the rostral part of the habenular complex, where the left
habenula preferentially targeted the dorsal IPN while the right habenula
preferentially targeted the ventral IPN. The caudal habenular nuclei
showed no asymmetry of projections as both sides targeted the ventral
IPN. Comparison of the habenular complex axonal connectivity across
vertebrates argues against strong phylogenetic conservation of the
axonal projection patterns of different habenular nuclei.