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, 521 (15), 3389-405

Tanycyte-like Cells Form a Blood-Cerebrospinal Fluid Barrier in the Circumventricular Organs of the Mouse Brain

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Tanycyte-like Cells Form a Blood-Cerebrospinal Fluid Barrier in the Circumventricular Organs of the Mouse Brain

Fanny Langlet et al. J Comp Neurol.

Abstract

Tanycytes are highly specialized ependymal cells that form a blood-cerebrospinal fluid (CSF) barrier at the level of the median eminence (ME), a circumventricular organ (CVO) located in the tuberal region of the hypothalamus. This ependymal layer harbors well-organized tight junctions, a hallmark of central nervous system barriers that is lacking in the fenestrated portal vessels of the ME. The displacement of barrier properties from the vascular to the ventricular side allows the diffusion of blood-borne molecules into the parenchyma of the ME while tanycyte tight junctions control their diffusion into the CSF, thus maintaining brain homeostasis. In the present work, we combined immunohistochemical and permeability studies to investigate the presence of tanycyte barriers along the ventricular walls of other brain CVOs. Our data indicate that, unlike cuboidal ependymal cells, ependymal cells bordering the CVOs possess long processes that project into the parenchyma of the CVOs to reach the fenestrated capillary network. Remarkably, these tanycyte-like cells display well-organized tight junctions around their cell bodies. Consistent with these observations, permeability studies show that this ependymal layer acts as a diffusion barrier. Together, our results suggest that tanycytes are a characteristic feature of all CVOs and yield potential new insights into their involvement in regulating the exchange between the blood, the brain, and the CSF within these "brain windows."

Keywords: area postrema; ependymocytes; organum vasculosum laminae terminalis; subcommissural organ; subfornical organ; tight junction protein.

Figures

Figure 1
Figure 1
Photomicrographs showing the association of vimentin-positive cells bearing processes with MECA 32-immunoreactive fenestrated capillaries in sagittal sections of mouse CVOs. A: Low-magnification photomontage of Hoechst counterstaining (blue) showing the location of CVOs in the mouse brain. B,C,E,F,H: High-magnification images showing the distribution of vimentin (red) and MECA 32 (white) immunoreactivity in each CVO (B, ME; C, OVLT; E, SFO; F, AP; H, SCO). Insets in B,E: High-magnification images of the areas indicated. D,G: High-magnification images corresponding to the areas indicated in C,F. All sections were counterstained using Hoechst (blue) to visualize cell nuclei and determine the morphological limits of each brain structure. Vimentin immunoreactivity (red) is distributed throughout the cells lining the third (3V in B–E,H) and fourth ventricles (4V in F,G). Notably, vimentin-positive ependymal cells send processes into the parenchyma of the CVOs (asterisks in B–H). MECA 32-positive vessels (white) are observed in the choroid plexus (CP in E) and CVOs (B–G), but not in the SCO (H). Remarkably, high-magnification images show the association between vimentin-positive cells and MECA 32-immunoreactive fenestrated capillaries via vimentin-positive processes (arrowheads in B,D,E,G). 3V, third ventricle; 4V, fourth ventricle; ME, median eminence; SCO, subcommissural organ; SFO, subfornical organ; OVLT, organum vasculosum laminae terminalis; AP, area postrema; CP, choroid plexus; PC, posterior commissure. Scale bars = 1,000 μm in A; 100 μm in B,C,E,F,H; 10 μm in D,G and inserts in B,E.
Figure 2
Figure 2
Photomicrographs showing the distribution of vimentin and detyrosinated-tubulin immunoreactivity in coronal sections of mouse CVOs. A,D,G,J: Low-magnification photomontage of detyrosinated tubulin (green) and vimentin (red) immunofluorescence in each CVO, with Hoechst counterstaining (blue) (A, OVLT; D, SFO; G, AP; J, SCO). B,C,E,F,H,I,K,L: High-magnification images of areas indicated in A,D,G,J, respectively. Vimentin immunoreactivity is distributed throughout the cells lining the third (3V in A–F,J–L) and fourth ventricles (4V in G–I). Note that detyrosinated-tubulin-immunoreactive cilia (green) observed along the ventricular wall (open arrowhead in B,E,H,K) are absent in ependymal cells bordering the OVLT, SFO, and AP (C,F,I). Interestingly, ependymal cells lining the SCO display numerous detyrosinated-tubulin-positive structures (green) that coat the ventricular wall and project into the ventricular lumen (L). Coordinates relative to bregma for coronal sections of CVOs: OVLT (+0.35), SFO (−0.60), AP (−7,20), SCO (−2.55). 3V, third ventricle; 4V, fourth ventricle; SCO, subcommissural organ; SFO, subfornical organ; OVLT, organum vasculosum laminae terminalis; AP, area postrema. Scale bars = 100 μm in A,D,G,J; 20 μm in B,C,E,F,H,I,K,L.
Figure 3
Figure 3
Expression of the tight junction protein ZO-1 in vimentin-positive ependymal cells bearing processes in sagittal sections of mouse CVOs (section adjacent to that shown in Fig. 1). A: Low-magnification photomontage of Hoechst counterstaining (blue) showing the location of CVOs in the mouse brain. B,C,E,F,H: High-magnification images showing the distribution of vimentin (red) and ZO-1 (green) immunoreactivity in each CVO (B, ME; C, OVLT; E, SFO; F, SCO; H, AP). Insets in B,E,H: High-magnification images corresponding to the areas indicated. D,G: High-magnification images corresponding to the areas indicated in C,F, respectively. All sections were counterstained with Hoechst (blue). ZO-1 is expressed in brain capillaries (green, open arrows in B,C,E,F,H) and in the choroid plexus (CP in E), which are known to display well-differentiated tight junction complexes. Notably, in the ME (inset in B), the OVLT (D), the SFO (inset in E), the AP (inset in H), and SCO (G), vimentin-positive ependymal cells (red) express ZO-1 (green) in a distinct honeycomb pattern around their cell bodies (arrow). 3V, third ventricle; 4V, fourth ventricle; ME, median eminence; SCO, subcommissural organ; SFO, subfornical organ; OVLT, organum vasculosum laminae terminalis; AP, area postrema; CP, choroid plexus; PC, posterior commissure. Scale bars = 1,000 μm in A; 100 μm in B,C,E,F,H; 10 μm in D,G and inset in B (applies also to E,H).
Figure 4
Figure 4
Expression pattern of tight junction proteins in vimentin-positive ependymal cells and their association with MECA 32-immunoreactive fenestrated capillaries in coronal sections of the mouse OVLT. A,B: Photomicrographs showing the distribution of vimentin (red) and occludin (green) immunoreactivity. C,D: Photomicrographs showing the distribution of ZO-1 (green) and MECA 32 (white) immunoreactivity. E,F: Photomicrographs showing the distribution of vimentin (red), claudin 1 (green), and MECA 32 (white) immunoreactivity. B,D,F: High-magnification images corresponding to areas indicated in A,C,E, respectively. A–D: Sections are counterstained with Hoechst (blue). Vimentin is expressed in both brain capillaries (red, open arrow in A,E) and ependymal cells. In the OVLT, vimentin-positive ependymal cells extend processes into the brain parenchyma (asterisks, A,B,E,F) and contact (arrowheads in F) MECA 32-positive fenestrated vessels (white) localized in the parenchyma of the CVO. Importantly, these ependymal cells display immunoreactivity for the tight junction proteins occludin (A,B), ZO-1 (C,D), and claudin 1 (E,F) (green). Tight junction proteins are expressed in a honeycomb pattern around ependymal cell bodies (arrows). Notably, brain vessels in neighboring structures are MECA 32-negative, ZO-1- and occludin-positive, and claudin 1-negative (open arrows in A,C,E). 3V, third ventricle; VmPO, ventromedial preoptic nucleus. Coordinates relative to bregma for coronal sections: A (+0.50), C (+0,35), E (+0.40). Scale bars = 100 μm in A,C,E; 10 μm in F (applies also to B,D).
Figure 5
Figure 5
Expression pattern of tight junction proteins in vimentin-positive ependymal cells and their association with MECA 32-immunoreactive fenestrated capillaries in coronal sections of the mouse SFO. A,B: Photomicrographs showing the distribution of vimentin (red) and occludin (green) immunoreactivity. C,D: Photomicrographs showing the distribution of ZO-1 (green) and MECA 32 (white) immunoreactivity. E,F: Photomicrographs showing the distribution of vimentin (red), claudin 1 (green), and MECA 32 (white) immunoreactivity. B,D,F: High-magnification images corresponding to areas indicated in A,C,E, respectively. A–D: Sections were counterstained with Hoechst (blue). Vimentin is expressed in both brain capillaries (red, open arrow in A,E) and ependymal cells. In the SFO, vimentin-positive ependymal cells extend processes into the brain parenchyma (asterisks, A,B,E,F) and contact (arrowheads in F) MECA 32-positive fenestrated vessels (white) localized in the parenchyma of the CVO. Importantly, these ependymal cells display immunoreactivity for the tight junction proteins occludin (A,B), ZO-1 (C,D), and claudin 1 (E,F) (green). Tight junction proteins are expressed in a honeycomb pattern around ependymal cell bodies (arrows). Notably, brain vessels in neighboring structures are MECA 32-negative, ZO-1- and occludin-positive, and claudin 1-negative (open arrows in A,C,E). 3V, third ventricle; CP, choroid plexus; SFO, subfornical organ; vhc, ventral hippocampal commissure. Coordinates relative to bregma for coronal sections: A (−0.60), C (−0,50), E (−0.50). Scale bars = 100 μm in A,C,E; 10 μm in F (applies also to B,D).
Figure 6
Figure 6
Expression pattern of tight junction proteins in vimentin-positive ependymal cells and their association with MECA 32-immunore-active fenestrated capillaries in coronal sections of the mouse AP. A,D: Photomicrographs showing the distribution of vimentin (red) and occludin (green) immunoreactivity. B,E: Photomicrographs showing the distribution of ZO-1 (green) and MECA 32 (white) immunoreactivity. C,F: Photomicrographs showing the distribution of vimentin (red), claudin 1 (green) and MECA 32 (white) immunoreactivity. D–F: High-magnification images corresponding to areas indicated in A–C, respectively. A,B,D,E: Sections were counterstained with Hoechst (blue). Vimentin is expressed in both brain capillaries (red, open arrow in A,C) and ependymal cells. In the AP, vimentin-positive ependymal cells extend processes into the brain parenchyma (asterisks, A,C,D,F) and contact (arrowheads in F) MECA 32-positive fenestrated vessels (white) localized in the parenchyma of the CVO. Importantly, these ependymal cells display immunoreactivity for the tight junction proteins occludin (A,D), ZO-1 (B,E), and claudin 1 (C,F) (green). Tight junction proteins are expressed in a honeycomb pattern around ependymal cell bodies (arrows). Notably, brain vessels in neighboring structures are MECA 32-negative, ZO-1- and occludin-positive, and claudin 1-negative (open arrows in A–C). AP, area postrema; NTS, nucleus of the solitary tract. Coordinates relative to bregma for coronal sections: A (−7,20), B (−7,35), C (−7,30). Scale bars = 100 μm in A–C; 20 μm in F (applies also to B,D).
Figure 7
Figure 7
Evans blue dye permeability studies associated with HuC/D and ZO-1 immunolabeling in mouse CVOs. A,E,I,M: Low-magnification photomontages showing HuC/D immunoreactivity in coronal sections of each CVO. All CVOs (OVLT, A; SFO, E; AP, I) except the SCO (M) display HuC/D-positive neuronal cell bodies (gray). B,F,J,N: Low-magnification photomontages showing fluorescence after an intravenous injection of Evans blue dye (red). When injected into the blood, Evans blue reaches the parenchyma of the OVLT, SFO, and AP, but is confined to brain vessels in neighboring structures. Evans blue diffusion is limited to the parenchyma of the CVOs (dotted line), delineating the CVOs and matching the distribution of HuC/D immunoreactivity. Note that the parenchyma of the SCO is free of the blood-borne dye. C,G,K,O: Low-magnification photomontages showing fluorescence after an i.c.v. injection of Evans blue dye (red) in association with ZO-1 immunoreactivity (green). D,H,L,P: High-magnification images corresponding to the areas indicated in C,G,K,O, respectively. Evans blue reaches and crosses the walls of the third and fourth ventricles (open arrowheads), except at the level of the CVOs, where both the ventricular wall and parenchyma (asterisk) remain dye-free. Importantly, the honeycomb pattern of ZO-1 expression (green, arrow in D,H,L,P) corresponds to the nondiffusion of the dye across the ventricular wall. OVLT, organum vasculosum laminae terminalis; SFO, subfornical organ; AP, area postrema; SCO, subcommissural organ; 3V, third ventricle; 4V, fourth ventricle; CP, choroid plexus; iv, intravenous injection; icv, intracerebroventricular injection. Coordinates relative to bregma for coronal sections: OVLT (+0.35), SFO (−0.60), AP (−7,30), SCO (−2.55). Scale bars = 100 μm in A−C,E−G,I−K,M−O; 20 μm in P (applies also to D,H,L).
Figure 8
Figure 8
Photomicrographs showing the distribution GFAP and ZO-1 immunoreactivity in coronal sections of each CVO. A: ME, B: OVLT. C: SFO. D: AP. E: SCO. Insets in A−E: High-magnification images of the areas indicated. The tight junction protein ZO-1 is expressed in blood-brain barrier capillaries (green, open arrow in A−E) and in the choroid plexus (CP in C,D). Notably, ZO-1 expression displays a typical honeycomb pattern at the ventricular wall (green, arrow in A−E). GFAP immunoreactivity is observed throughout the nervous tissue, with an increase in the signal around ZO-1-positive brain capillaries (white, open arrow in A−E), which are known to display well-differentiated tight junction complexes. Importantly, at the level of the CVOs, although GFAP-positive cells were found throughout the parenchyma of the CVOs, predominant GFAP immunoreactivity was observed close to the ventricular surface forming a dense ribbon associated with the honeycomb pattern of tight junction proteins displayed by ependymal cell bodies (arrows, insets in A−E). In the SCO, GFAP is also expressed in some cell bodies (inset in E). ME, median eminence; OVLT, organum vasculosum laminae terminalis; SFO, subfornical organ; AP, area postrema; SCO, subcommissural organ; 3V, third ventricle; 4V, fourth ventricle; CP, choroid plexus. Coordinates relative to bregma for coronal sections: ME (−1,70), OVLT (+0.35), SFO (−0.50), AP (−7,20), SCO (−2.55). Scale bars = 100 μm in A–E; 20 μm in insets.
Figure 9
Figure 9
Expression pattern of tight junction proteins in vimentin-positive cells in coronal sections of the mouse SCO. A,D: Photomicrographs showing the distribution of vimentin (red) and occludin (green) immunoreactivity. B,E: Photomicrographs showing the distribution of ZO-1 (green) and MECA 32 (white) immunoreactivity. C,F: Images showing the distribution of vimentin (red), claudin 1 (green), and MECA 32 (white) immunoreactivity. D–F: High-magnification images corresponding to areas indicated in A–C respectively. A,B,D,E: Sections were counterstained with Hoechst (blue). No MECA 32 immunoreactivity was observed in SCO and neighboring structures. Vimentin immunoreactivity is distributed throughout the cells lining the third ventricle (3V in A,C,D,F). Remarkably, vimentin-positive elongated cell bodies (red in A,C,D,F) delimit the SCO in the brain parenchyma and line the ventricular wall. High-magnification images of the ventricular wall of the SCO showing occludin (A,D), ZO-1 (B,E), and claudin 1 (C,F) immunoreactivity organized in a continuous belt around vimentin-positive cell bodies, giving rise to the characteristic honeycomb pattern (arrows in D–F). These cells display also vimentin-positive basal processes extending into brain parenchyma (asterisks in A,C) and for a few of them projecting to occludin-positive capillaries (open arrow in A). Notably, brain vessels in neighboring structures are ZO-1- and occludin-positive and claudin 1-negative (open arrows in A–C). 3V, third ventricle; PC, posterior commissure; PAG, periaqueductal gray. Coordinates relative to bregma for coronal sections: A–C (−2.55). Scale bars = 100 μm in A–C; 20 μm in D–F.
Figure 10
Figure 10
Representative figure summarizing the type of ependymal cells and the distribution of tight junction proteins in each CVO (A, OVLT; B, SCO; C, SFO; D, AP) of the mouse brain. OVLT, organum vasculosum laminae terminalis; SCO, subcommissural organ; SFO, subfornical organ; AP, area postrema; 3V, third ventricle; PO, preoptic nucleus; OC, optic chiasma; MPA, medial preoptic area; PC, posterior commissure; PrC, precommissural nucleus; PAG, periaqueductal gray; vhc, ventral hippocampal commissure; CP, choroid plexus; sm, stria medullaris; 4V, fourth ventricle; NTS, nucleus of the solitary tract; TJ: tight junction.

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