Morita et al. Cell Tissue Res., (2015) Heterogeneous vascular permeability and barrier in mouse CVOs. ____________________________________________________________________________________________ Heterogeneous Vascular Permeability and Alternative Diffusion Barrier in Sensory Circumventricular Organs of Adult Mouse Brain Shoko Morita, 1,2 Eriko Furube, 1 Tetsuya Mannari, 1 Hiroaki Okuda, 2 Kouko Tatsumi, 2 Akio Wanaka, 2 & Seiji Miyata 1,* *Corresponding to; Seiji Miyata, smiyata@kit.ac.jp 1 Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan 2 Department of Anatomy and Neuroscience, Faculty of Medicine, Nara Medical University, Shijo-cho, Kashihara, Nara 634- 8521, Japan ABSTRACT Fenestrated capillaries of the sensory circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis, the subfornical organ and the area postrema, lack completeness of the blood–brain barrier (BBB) to sense a variety of blood-derived molecules and to convey the information into other brain regions. We examine the vascular permeability of blood-derived molecules and the expression of tight-junction proteins in sensory CVOs. The present tracer assays revealed that blood-derived dextran 10 k (Dex10k) having a molecular weight (MW) of 10,000 remained in the perivascular space between the inner and outer basement membranes, but fluorescein isothiocyanate (FITC; MW: 389) and Dex3k (MW: 3000) diffused into the parenchyma. The vascular permeability of FITC was higher at central subdivisions than at distal subdivisions. Neither FITC nor Dex3k diffused beyond the dense network of glial fibrillar acidic protein (GFAP)- positive astrocytes/tanycytes. The expression of tight-junction proteins such as occludin, claudin-5 and zonula occludens-1 (ZO- 1) was undetectable at the central subdivisions of the sensory CVOs but some was expressed at the distal subdivisions. Electron microscopic observation showed that capillaries were surrounded with numerous layers of astrocyte processes and dendrites. The expression of occludin and ZO-1 was also observed as puncta on GFAP-positive astrocytes/tanycytes of the sensory CVOs. Our study thus demonstrates the heterogeneity of vascular permeability and expression of tight- junction proteins and indicates that the outer basement membrane and dense astrocyte/tanycyte connection are possible alternative mechanisms for a diffusion barrier of blood-derived molecules, instead of the BBB. Keywords; Blood–brain barrier . Tight junction . Vascular permeability . Astrocyte . Perivascular space . Mouse INTRODUCTION The blood–brain barrier (BBB) maintains homeostasis of the brain parenchyma, which is composed entirely of glial and nerve cells, by preventing the entry of certain blood-derived molecules (Engelhardt 2003; Zlokovic 2011). However, the circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO), and the area postrema (AP), lack the typical BBB (Ciofi et al. 2009). Unlike capillaries of most brain regions, the CVOs are fenestrated. This feature of their capillaries permits the two-way exchange of information between the blood and brain: the sensing of blood-derived molecules by parenchyma cells in the sensory CVOs (Johnson and Gross 1993; Fry et al. 2007) and the secretion of neurohormones into the blood circulation by the secretory CVOs consisting of the median eminence (ME) and neurohypophysis (NH; Ganong 2000). Various molecules were previously believed to be able to move freely into the parenchyma in the sensory CVOs. However, the vascular permeability of high molecular weight (MW) tracer molecules such as bovine serum albumin (BSA, MW>66,000), dextran 70,000 (Dex70k, MW=70,000) and dextran 150,000 (MW=150,000) is much lower in the AP (Faraci et al. 1989; Willis et al. 2007; Morita and Miyata 2012), OVLT, SFO, ME and NH (Morita and Miyata 2012, 2013) than in the peripheral tissues. Fenestrated capillaries of the CVOs are surrounded by a large perivascular