Atypical Course of the Habenulo-Interpeduncular Tract in Chick Embryos

Classical studies of the avian diencephalon hardly mention the habenulo-interpeduncular tract (a.k.a. retroflex tract), although both the habenula (HB) (its origin) and the interpeduncular nuclear complex (its target) are present. Retroflex tract fibers were described at early embryonic stages but seem absent in the adult in routine stains. However, this tract is a salient diencephalic landmark in all other vertebrate lineages. It typically emerges out of the caudal HB, courses dorsoventrally across thalamic alar and basal plates just in front of the thalamo-pretectal boundary, and then sharply bends 90° caudalwards at paramedian basal plate levels (this is the "retroflexion"), to approach longitudinally via paramedian pretectum and midbrain the rostralmost hindbrain, specifically the prepontine median interpeduncular complex across isthmus and rhombomere 1. We systematize this habenulo-interpeduncular course into four parts named subhabenular, retrothalamic, tegmental, and interpeduncular. We reexamined the chicken habenulo-interpeduncular fibers at stages HH30 and HH35 (6.5- and 9-day incubation) by mapping them specifically with immunoreaction for BEN protein, a well-known marker. We found that only a small fraction of the stained retroflex tract fibers approaches the basal plate by coursing along the standard dorsoventral pathway in front of the thalamo-pretectal boundary. Many other habenular fibers instead diverge into atypical dispersed courses across the thalamic cell mass (implying alteration of the first subhabenular part of the standard course) before reaching the basal plate; this dispersion explains their invisibility. A significant number of such transthalamic habenular fibers cross orthogonally the zona limitans (ZLI) (the rostral thalamic boundary) and invade the caudal alar prethalamus. Here, they immediately descend dorsoventrally, just rostrally to the ZLI, until reaching the prethalamic basal plate, where they bend (retroflex) caudalwards, entering the thalamic basal paramedian area. These atypical fibers gradually fasciculate with the other groups of habenular efferent fibers in their final longitudinal approach to the hindbrain interpeduncular complex. We conclude that the poor visibility of this tract in birds is due to its dispersion into a diversity of atypical alternative routes, though all components eventually reach the interpeduncular complex. This case merits further analysis of the diverse permissive versus nonpermissive guidance mechanisms called into action, which partially correlate distinctly with successive diencephalic, mesencephalic, and hindbrain neuromeric fields and their boundaries.