Spatial heterogeneity of the ventricular interface between the median eminence and arcuate nucleus revealed by an unfolded whole-mount view

BACKGROUND: The median eminence (ME) and adjacent arcuate nucleus (ARC) sit at a unique interface where blood, cerebrospinal fluid (CSF), and hypothalamic circuits meet. However, its continuous organization, postnatal maturation, and aging-related remodeling remain incompletely defined because conventional sections capture only limited spatial fragments. METHODS: A topological unfolding method was established to open and flatten the third ventricle floor for whole-mount view imaging. Whole-mount immunostaining, reporter-based anatomical mapping, coronal section validation, and tracer assays were combined to examine apical specializations, junctional architecture, vascular organization, and regional permeability along the rostrocaudal axis. Evans Blue was delivered intraventricularly or intracardially at 1, 3, and 8 weeks and at 14 months of age. WGA was delivered intraventricularly and analyzed at 4 h and 24 h after injection. RESULTS: The unfolded whole-mount view preserved the continuous surface anatomy of the third ventricle floor and enabled direct registration of ventricular and hypothalamic landmarks. A clear rostrocaudal heterogeneity was identified around the ME, including regional differences in tanycyte organization, Ki67-positive cells, vascular patterning, neuroendocrine axonal networks, ciliation, and ZO-1 junctional domains. Enlarged and more heterogeneous ZO-1 polygons were enriched toward the caudal ME and infundibular region. Tracer assays showed stronger Evans Blue access toward the caudal ME and infundibular region. WGA analysis further revealed region-dependent differences in macromolecular uptake and redistribution along the third ventricle wall. Among the ages examined, intraventricular Evans Blue permeability was greater at 1 week than at 3 and 8 weeks, became restricted by 3 weeks, and increased again in aged mice, accompanied by junctional disorganization. CONCLUSIONS: The third ventricle floor is organized as a continuous spatial gradient rather than a uniform barrier. The caudal ME and infundibular region form a more permissive CSF-facing exchange territory. Aging disrupts this regional junctional organization and is associated with increased intraventricular permeability in the ME and ARC.