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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2012 Apr 4;32(8):1457–1467. doi: 10.1038/jcbfm.2012.45

Deletion of astroglial connexins weakens the blood–brain barrier

Pascal Ezan 1,2,3,7, Pascal André 4,7, Salvatore Cisternino 4, Bruno Saubaméa 4,5, Anne-Cécile Boulay 1,2,3, Suzette Doutremer 6,8, Marie-Annick Thomas 6,8, Nicole Quenech'du 6,8, Christian Giaume 1,2,3, Martine Cohen-Salmon 1,2,3,*
PMCID: PMC3421093  PMID: 22472609

Abstract

Astrocytes, the most prominent glial cell type in the brain, send specialized processes named endfeet, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction protein connexins 43 and 30 (Cx43 and Cx30) allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. The contribution of astroglial connexins to the physiology of the brain vascular system has never been addressed. Here, we show that Cx43 and Cx30 expression at the level of perivascular endfeet starts from postnatal days 2 and 12 and is fully mature at postnatal days 15 and 20, respectively, indicating that astroglial perivascular connectivity occurs and develops during postnatal blood–brain barrier (BBB) maturation. We demonstrate that mice lacking Cx30 and Cx43 in GFAP (glial fibrillary acidic protein)-positive cells display astrocyte endfeet edema and a partial loss of the astroglial water channel aquaporin-4 and β-dystroglycan, a transmembrane receptor anchoring astrocyte endfeet to the perivascular basal lamina. Furthermore, the absence of astroglial connexins weakens the BBB, which opens upon increased hydrostatic vascular pressure and shear stress. These results demonstrate that astroglial connexins are necessary to maintain BBB integrity.

Keywords: aquaporin-4, astrocyte, blood–brain barrier, connexin, gap junction, β-dystroglycan

Introduction

In contrast to the large majority of organs, most of the brain vascular system constitutes a solute barrier called the blood–brain barrier (BBB) between the blood and brain parenchyma, which consists of an endothelial barrier situated at capillary level, and to a lesser extent at precapillary arteriolar and postcapillary venular systems. The BBB prevents the nonspecific paracellular route of hydrophilic solutes, supplies the brain with essential nutrients, and allows excess of released neurotransmitters and toxic molecules to be transported in the bloodstream (Abbott et al, 2010). Morphological and biochemical features of the BBB include low endocytic activity, the absence of fenestrations between endothelial cells, the presence of endothelial tight junctions (TJ), a continuous basal lamina (BL) surrounding the endothelium, and the expression by endothelial cells of several nutrient and xenobiotic membrane carriers, as well as a complex enzymatic system inactivating neuroactive and toxic compounds. In the last decades, the notion has emerged that BBB development and function were not only processes intrinsic to endothelial cells, but required surrounding astrocytes and pericytes (Liebner et al, 2011). In the mouse, pericytes were recently shown to be recruited to nascent vessels during embryogenesis inducing the formation of endothelial TJs and inhibiting the vascular permeability to transcardiacally perfused tracers, while astrocytes generated in the late embryonic stages (Rowitch and Kriegstein, 2010) were shown to extend processes around vessels during the first postnatal week (Daneman et al, 2010) inducing many BBB properties such as the polarized expression of transporters in the luminal and abluminal endothelial membranes (reviewed in Abbott et al, 2010; Liebner et al, 2011). In addition, both cell types have been shown to express several BBB-inducing factors such as angiopoietin-1, transforming growth factor-β, and for astrocytes glial-derived neurotrophic factor as well as SSeCKS, a protein kinase C substrate (Abbott et al, 2006). Finally, both astrocytes and pericytes have been shown to express BL components, thus contributing to the BL surrounding the endothelium (del Zoppo and Milner, 2006; Dore-Duffy, 2008).

Importantly, astrocytes display processes called endfeet, closely apposed to the outer surface of the endothelium and almost completely sheathing the vessel walls (Mathiisen et al, 2010). Astrocyte endfeet constitute a specific subcellular domain dedicated to gliovascular interaction. Indeed, they express a large molecular repertoire involved in the control of the vascular tone, the energy supply to neurons as well as ions and water homeostasis (Tsacopoulos and Magistretti, 1996; Simard et al, 2003; Gordon et al, 2007). One of their most striking properties is their enrichment in membrane proteins connexin 30 and 43 (Cx30 and Cx43), which assemble in closely packed hexameric rings or connexons that align head-to-head between neighboring cells to form intercellular or gap junction channels (Simard et al, 2003; Rouach et al, 2008). Such molecular specialization allows astrocytes to exchange ions and small molecules (<1.5 kDa) directly around the brain endothelium within perivascular astroglial networks (Rouach et al, 2008). Cx30 and Cx43 have been shown to contribute to several aspects of brain development and functions (reviewed in Giaume et al, 2010). In particular, Cx43 and Cx30 double knockout mice exhibit altered energy metabolite trafficking (Rouach et al, 2008), neurogenesis (Kunze et al, 2009), dysmyelination, and parenchymal vacuolation (Lutz et al, 2009), and increased hippocampal synaptic transmission caused by the inability of uncoupled astrocytes to redistribute glutamate and potassium uptaken from the extracellular space during synaptic activity (Pannasch et al, 2011).

Despite the strong enrichment of Cx30 and Cx43 at the gliovascular interface, the contribution of perivascular astroglial networks to the physiology of the brain vascular system has never been addressed. Here, we studied the developmental expression of Cx30 and Cx43 in astrocyte endfeet and demonstrate that they appear postnatally during BBB maturation. We show that the absence of astroglial Cx43 and Cx30 in double knockout mice leads to endfeet morphological alterations and abnormal expression of several astrocyte perivascular endfeet markers. Altogether, these alterations weaken the BBB, which opens upon increased hydrostatic vascular pressure and shear stress.

Materials and methods

Ethics Statement on Animal Experiments

Experiments and techniques reported here complied with the ethical rules of the French agency for animal experimentation and with the IMTCE (Institut Médicament Toxicologie Chimie Environnement) animal ethics committee (Université Paris Descartes) (agreement number 86 to 23).

Mice

DKo mice have been generated by the crossing of Cx30−/− mice ubiquitously deleted for Cx30 (Teubner et al, 2003) and Cx43fl/fl/hGFAP-Cre (Theis et al, 2001) where Cx43 deletion is targeted to the GFAP (glial fibrillary acidic protein)-expressing cells.

Antibodies

We used mouse anti-Cx30 (Invitrogen, Camarillo, CA, USA) (batch 302-76-172 (1:500); mouse anti-occludin (Invitrogen) (1:500); mouse anti-ZO-1 (Invitrogen) (1:500); rat anti-mouse CD31 (Pecam1) monoclonal (1:100) (BD Biosciences, San Diego, CA, USA); rabbit anti-integrin-β1 (Millipore, Billerica, MA, USA) (1:500); rabbit anti-Kir4.1 (Alomone Labs, Jerusalem, Israel) (1:400); mouse anti-Cx43 (BD Biosciences) (1:500); rabbit anti-aquaporin-4 (anti-Aqp4) (Sigma, St Louis, MO, USA) (1:500); rabbit anti-fibronectin (Sigma) (1:400); rabbit anti-Claudin3, and rabbit anti-Claudin5 (Invitrogen) (1:100); mouse anti-β-dystroglycan (anti-β-DG) (Novocastra, Newcastle, UK) (1:200); mouse anti-dystrophin (NCL-DYS2) (Novocastra) (1:20); rat anti-mouse Laminin-1 (RD Systems, Minneapolis, MN, USA) (1:400); rabbit anti-Agrin (1:400) kindly provided by Dr Markus A Ruegg; rat anti-mouse Perlecan (1:2) kindly provided by Dr Alexender Ljubimov.

Secondary antibodies used were: Alexa-conjugated goat anti-mouse and anti-rabbit IgG (Molecular Probes, Eugene, OR, USA) (1:2,000); horseradish peroxidase (HRP)-conjugated goat anti-mouse, rabbit, or rat antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (dilution 1/2,500).

Protein Extraction from Mouse Tissues and Western Blotting

Three-month-old mouse brains were dissected and reduced in powder at −80°C, immediately dissolved in phosphate-buffered saline (PBS) with 2% sodium dodecyl sulfate, and 1 × EDTA-free Complete Protease Inhibitor (Roche, Mannheim, Germany). Lysates were sonicated twice at 20% 10 Hz (Vibra cell VCX130, Newtown, CT, USA) and centrifuged 20 minutes at 10,000 g at 4°C. Supernatants were resuspended and boiled in 5 × Laemmli loading buffer. Protein content was measured using the Pierce 660 nm protein assay reagent (Thermo Scientific, Rockford, IL, USA). Equal amounts of proteins were separated by denaturing electrophoresis in NuPAGE 3% to 8% Tris acetate gradient gel (Invitrogen), electrotransferred to nitrocellulose membranes, first analyzed using primary antibodies (list above) and HRP-conjugated secondary antibodies. Horseradish peroxidase activity was visualized by ECL using Western Lightning plus enhanced chemoluminescence system (Perkin-Elmer, Waltham, MA, USA). Blots were reprobed with mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase peroxidase (Sigma, 1:10,000) to check the protein load. Chemoluminescence imaging was performed on a LAS4000 (Fujifilm, Stamford, CT, USA). Semiquantitative densitometric analysis was performed with ImageJ software after scanning the bands.

Immunohistofluorescence

Mice were anesthetized with ketamine-xylazine (140 to 8 mg/kg, intraperitoneally) and killed by intracardiac perfusion of PBS. The brain was dissected. In all, 10 μm-thick frozen sections were fixed by immersion in 4% paraformaldehyde-PBS at 4°C for 10 minutes, and immersed in the blocking and permeabilization solution (0.2% gelatin 0.25 × TritonX-100 in PBS) for 1 hour at room temperature. Samples were incubated with primary antibodies in the blocking solution at 4°C for 12 hours, rinsed three times in the blocking solution, incubated 1 hour at room temperature with secondary antibodies, rinsed three times in PBS, and embedded in FluoromountG (Southern Biotech, Birmingham, AL, USA). Fluorescence images were taken with a SP5 confocal microscope (Leica, Wetzlar, Germany). Specificity of antibodies against Cx43 and Cx30 was checked by Western blot of brain extracts from newborn Cx43del/del mice (Theis et al, 2001) and adult Cx30−/− (Teubner et al, 2003), respectively. For Cx43 and Cx30 immunofluorescence study mice at E18, P2, P10, P12, P15, P20, P30, and 3 months were tested.

In Situ Brain Perfusion

Surgical Procedure

This procedure has been described in details in Dagenais et al (2000). Three-month-old mice were anesthetized with ketamine-xylazine (140 to 8 mg/kg, intraperitoneally) and a polyethylene catheter was inserted into the right carotid. The heart was cut and the perfusion was started immediately (flow rate: 2.5 mL/min) to obtain a complete substitution of the blood by the artificial perfusion fluid. The mouse was perfused with a perfusion fluid which contained [14C]sucrose (0.3 μCi/mL; Perkin-Elmer Life Sciences, Courtaboeuf, France) as a vascular and integrity marker. Perfusion was terminated after 120 seconds by decapitating the mouse. The brain was removed from the skull and dissected out on a freezer pack. Tissue and two aliquots of perfusion fluid were placed in tared vials and weighed, digested with Solvable (Perkin-Elmer) and mixed with Ultima gold XR (Perkin-Elmer) for 14C counting (Tri-Carb, Perkin-Elmer).

Perfusion

The perfusion fluid was a Krebs-bicarbonate (KB)-buffered physiological saline (mmol): 128 NaCl, 24 NaHCO3, 4.2 KCl, 2.4 NaH2PO4, 1.5 CaCl2, 0.9 MgCl2, and 9 D-glucose. The solution was gassed with 95% O2/5% CO2 for pH control (7.4) and warmed to 37°C. The perfusion fluid was perfused at a flow rate of 2.5 mL/min allowing for hydrostatic pressure (100 to 110 mm Hg) (Dagenais et al, 2000). In some experiments, human serum albumin (hSA) (40 g/L) (Vialebex, Paris, France) was added in the perfusion fluid to increase its viscosity and then the hydrostatic pressure (∼180 mm Hg) according to the Poiseuille's law. As a consequence of the BBB properties (e.g., TJs, ionic/water flux…) the Starling's forces in the cerebral capillaries are mainly osmolarity and hydrostatic vascular pressures whereas oncotic pressure is tiny (Ravussin et al, 1994).

Vascular Volume Calculation

The brain vascular volume (Vv) (μL/g) was calculated using the distribution of [14C]sucrose: Vv=Xv/Cv where Xv (d.p.m./g) is the [14C]sucrose measured in the right hemisphere and Cv (d.p.m./μL) is the concentration of [14C]sucrose in the perfusion fluid (Dagenais et al, 2000).

Electron Microscopy

Three-month-old mice were anesthetized with ketamine-xylazine (140 to 8 mg/kg, intraperitoneally) and transcardially perfused with 0.9% NaCl for 30 seconds and then with Karnovsky fixative (paraformaldehyde 2%, Gluta 2.5% in 0.1 mol/L phosphate buffer, pH 7.4) for 12 minutes. The brains were removed and 1 mm3 brain fragments were postfixed in the same fixative for 1 hour at 4°C and stored in PBS overnight at 4°C. The brain samples were then rinsed briefly in water, fixed in 2% aqueous OsO4 for 45 minutes at 4°C, and finally rinsed in water. After dehydration in graded ethanol, followed by propylene oxyde, the fragments were embedded in Epon (Electron Microscopy Sciences, Hatfield, PA, USA). Ultrathin (80 nm) sections were prepared, stained in 2% uranyl acetate and lead citrate and photographed in a Jeol S100 transmission electron microscope (Croisy-sur-Seine, France).

For ultrastructural analysis following an acute increase in vascular pressure, in situ mouse carotid perfusion was performed as described above. Multiple-way valves and -syringe pumps were connected to the carotid catheters, allowing for sequential perfusions of diverse fluids with selective duration as follows: (1) KB (30 seconds), (2) KB + hSA (40 g/L) + HRP (3.5 mg/mL) (2 minutes); (3) KB + HRP (3.5 mg/mL) (30 seconds), (4) Karnovsky fixative (15 minutes). The brains were removed, postfixed for 1 hour at 4°C, and rinsed in PBS. In all, 200-μm vibratome brain sections were prepared and the presence of HRP was revealed by incubating the sections in 3,3′-diaminobenzidine (DAB) (Sigma Fast, Sigma) containing 0.05% Nickel ammonium sulfate for 45 minutes at room temperature. After being rinsed in PBS, sections were postfixed for 3 hours at 4°C. In all, 1 mm2 fragments were excised and prepared for transmission electron microscopy as indicated above.

Statistical Analysis

All values are mean values±s.d. Student's unpaired t-test was used to identify significant differences between groups when appropriate. The tests were two-tailed and statistical significance was set at P<0.05.

Results

Developmental Expression of Cx30 and Cx43 in Mouse Cortical Astrocyte Endfeet

In the brain, Cx30 expression starts around the second postnatal week and increases until 1 month of age, being stably expressed in the adult (Kunzelmann et al, 1999; Nagy et al, 1999). In contrast, Cx43 expression starts during embryogenesis around E12 in the rat brain, continuously increases until the second postnatal week and remains stable onward (Nadarajah et al, 1997). Here, we addressed the developmental expression pattern of Cx43 and Cx30 at the level of the perivascular endfeet in the mouse brain. We studied together Cxs and astrocyte endfeet in the mouse cortex, by performing coimmunofluorescent labeling of Cxs and Aqp4, a member of the water channel family highly expressed in the astrocyte endfeet membrane outlining vascular walls (Simard et al, 2003). An endothelial counter-staining was also performed by immunodetecting the endothelial-specific protein Pecam1. Connexin 43 and Cx30 were imaged from the stage of their onset at the perivascular level to the stage corresponding to their full perivascular expression (Figures 1 and 2). Aquaporin-4 developmental expression has been shown to start during the first postnatal week, paralleling the formation of astrocyte endfeet as well as the reduction of the perivascular space, both of which are necessary for BBB differentiation (Wen et al, 1999). Accordingly, we determined that Aqp4 perivascular expression was almost absent at embryonic day 18 (E18) and started to be detectable at birth when astrocyte endfeet began to contact vessels (Daneman et al, 2010) (Supplementary Figure S1). At this stage, Aqp4 labeling outlined incompletely the brain vascular system, reflecting a discontinuity in astrocyte endfeet vascular coverage (Supplementary Figure S1). In contrast, at P5, it formed an almost uniform and continuous labeling around all vessels (Supplementary Figure S1), indicating that astrocyte endfeet vascular coverage was nearly complete from this stage. Connexin 43 perivascular immunoreactivity started at P2 (Figure 1). No Cx43 perivascular labeling was detected before (data not shown). At this stage, Cx43 immunoreactive puncta were mostly present in the parenchyma, but started to concentrate at the level of large (diameter >20 μm) and medium-sized (10 to 20 μm) vessels. In contrast, around capillaries (5 μm), Aqp4 labeling was weak and no Cx43 labeling was present. At P10, while Aqp4 labeling completely covered all vessels, Cx43 perivascular immunostaining increased and started to concentrate also around capillaries (Figure 1). At P15, Cx43 immunoreactivity formed large puncta around all types of vessels, and was organized in a honeycomb-like pattern around large and medium-sized vessels (Figure 1) as described previously (Yamamoto et al, 1992). In contrast, within the brain parenchyma outside the vessel walls, Cx43 immunoreactivity was evenly distributed in smaller dots lacking an apparent pattern of organization. From this stage onward, perivascular Cx43 immunoreactivity remained stable and unchanged (data not shown). Connexin 30 immunoreactivity was weaker compared with Cx43 and started to be detectable in the parenchyma at P10 (data not shown), but began to concentrate around all vessels (large, medium-size, and capillaries) only at P12 (Figure 2). From this stage to P20, Cx30 perivascular immunoreactivity increased progressively. It remained stable onward (data not shown). These results suggest that gap junction communication between perivascular astrocytes endfeet underlain by Cx30 and Cx43 is initiated postnatally during the BBB maturation phase (Liebner et al, 2011), and is fully mature around P20, being present around all vessels.

Figure 1.

Figure 1

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Developmental expression of Cx43 in astrocyte endfeet. Confocal microscopy projections in the mouse cortex performed at the indicated ages. Cx43 immunostaining is shown in green. Endothelial cells are labeled with Pecam1 (red), and astrocyte endfeet enwrapping blood vessels are labeled with aquaporin-4 (Aqp4) (blue). Images in (a) and (b) are enlarged views of the boxed areas, which correspond to vessels with a diameter of 10 to 20 μm and capillaries (5 μm), respectively. Scale bars: 10 μm.

Figure 2.

Figure 2

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Developmental expression of Cx30 in astrocyte endfeet. Confocal microscopy projections in the mouse cortex performed at the indicated ages. Cx30 immunostaining is shown in green. Endothelial cells are labeled with Pecam (red), and astrocyte endfeet enwrapping blood vessels are labeled with aquaporin-4 (Aqp4) (blue). Images in (a) and (b) are enlarged views of the boxed areas, which correspond to vessels with a diameter of 10 to 20 μm and capillaries (5 μm), respectively. Scale bars: 10 μm.

Ultrastructural Characterization of the Gliovascular Interface in the Absence of Astroglial Connexins

Electron microscopy analyses of mice lacking both Cx30 (Teubner et al, 2003) and Cx43 in astrocytes (Theis et al, 2001) (Cx30−/−Cx43fl/fl/hGFAP-Cre, DKo mice), which exhibit a complete lack of dye coupling in astrocytes (Rouach et al, 2008), have shown that astrocytes are hypertrophic and reactive (Lutz et al, 2009; Pannasch et al, 2011). Thus, we hypothesized that astroglial endfeet morphology in DKo mice may be altered as well. Cerebral cortex and hippocampus ultrathin sections of 3-month-old mice, a stage where perivascular coupling is fully mature, were examined under transmission electron microscopy (Figure 3). Observations were similar in the cortex and the hippocampus. In wild-type (WT) mice, astroglial perivascular endfeet were in close contact with the BL and showed thin processes with a dense intracellular content. In striking contrast, in DKo, all microvessels were surrounded by swollen edematous astroglial endfeet, which at times could appear totally devoid of organelles. However, they always stayed closely apposed to the vessels. The BL, forming a single thin layer around the vessels displayed a continuous appearance in both DKo and WT. Finally, no difference could be seen in the overall structure of endothelial cells. In particular, TJs extended from the vessel lumen to the endothelial basement membrane with no apparent discontinuity of the paracellular spaces between endothelial cells in both genotypes (Figure 3). These results show that astrocytes lacking Cx43 and Cx30 feature edematous perivascular endfeet. However, the overall structure of the cerebral endothelium is unaffected.

Figure 3.

Figure 3

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Ultrastructural characterization of the gliovascular interface in wild-type (WT) and double knockout mice for astroglial Cx43 and Cx30. In the cortex of WT mice, thin perivascular endfeet with a dense intracellular content were in close contact with the basal lamina surrounding the microvessels. In DKo mice cortex, perivascular astrocytes appeared systematically swollen and edematous (asterisks). Images on the right are enlarged views of the endothelial tight junction (TJ) areas (rectangles) in WT and DKo. Note that the morphology of TJs was comparable in WT and DKo. a, denotes astrocyte endfeet; b, denotes basal lamina; e, denotes endothelial cell. Scale bars: 1 μm (left images) and 100 nm (right images).

Biochemical Features of the Gliovascular Interface

We next investigated whether the astroglial morphological changes observed at the vascular interface in the brain of 3-month-old DKo mice could be associated with changes in the expression of proteins expressed at the gliovascular interface. Immunoblotting for the TJ proteins CLN-3, occludin, and ZO-1 did not show any differences between WT and DKo (Supplementary Figure S2). Consistently, the distribution of these proteins revealed by immunofluorescence was identical in both genotypes (data not shown). Similarly, immunoblotting for Kir4.1 potassium channels and dystrophin expressed at the level of astrocyte endfeet membrane facing the vessels indicated the same level of expression in WT and DKo (Supplementary Figure S2). In contrast, the level of Aqp4, that colocalizes with Kir4.1, was decreased by half in DKo compared with WT (Figure 4). We next addressed the expression level of β-DG and β1-integrin, two molecules linking astrocyte and endothelial cytoskeleton to the perivascular BL (del Zoppo and Milner, 2006). Although β1-integrin level was equivalent in both strains (Supplementary Figure S2), β-DG was reduced by half in DKo mice (Figure 4). This result prompted us to test the expression and tissular distribution of BL molecules interacting with the DG complex: perlecan, laminin-1, and agrin (Henry and Campbell, 1999). No change was noted in DKo mice, neither in their level of expression assessed by Western blotting (Supplementary Figure S2) nor in their perivascular distribution (Supplementary Figure S3). Similarly, the expression level of fibronectin, enriched in the BL, was unchanged in DKo compared with WT (Supplementary Figure S2).

Figure 4.

Figure 4

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Comparison of aquaporin-4 (Aqp4) and β-dystroglycan levels in hippocampus of wild-type (WT) and double knockout mice for astroglial Cx43 and Cx30. Protein extracts from hippocampus were analyzed on Western blots using anti-β-dystroglycan anti-Aqp4, and anti-GAPDH (loading control) antibodies. The averaged ratio of β-dystroglycan (β-DG) or Aqp4 with GAPDH was calculated from three independent experiments. In DKo mice, β-DG and Aqp4 quantity was reduced by about half compared with WT. Data are mean values±s.d., *P<0.05 comparing WT and DKo mice. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Astroglial Connexins Are Required for Blood–Brain Barrier Integrity

Astroglial signaling to the vascular endothelium is necessary to the BBB integrity (Abbott et al, 2006). We hypothesized that in DKo mice alteration of endfeet architecture together with the modified expression of astroglial perivascular markers might alter barrier properties of the cerebral endothelium. Blood–brain barrier integrity of 3-month-old DKo was assessed by measuring the brain Vv by in situ brain perfusion of [14C]sucrose (Dagenais et al, 2000) (see Materials and methods) and was compared with WT mice (Figure 5). [14C]Sucrose was used as a marker of the vascular space and integrity because it does not cross the BBB significantly during short exposure. The distribution of [14C]sucrose Vv was measured after 120 seconds of in situ brain perfusion in regular fluid at 2.5 mL/min. In WT, Vv (14.26±0.7 μL/g; n=5) was in agreement with the previously described [14C]sucrose Vv distribution in the mouse brain (Figure 5, white column) (Dagenais et al, 2000). In DKo mice, [14C]sucrose Vv was slightly elevated compared with WT mice (14.7±1.0 μL/g; n=5), although the difference between both genotypes was not statistically significant (Figure 5, white column). Thus, BBB integrity appeared unchanged in the absence of Cx30 and Cx43 in astrocytes. We next examined the mechanical resistance of the BBB in WT and DKo mice when submitted to higher hydrostatic vascular pressure (from 110 to 180 mm Hg) and sheer stress, performing in situ perfusions as described above, but adding hSA to the saline perfusion (Figure 5, black column) (see Materials and methods). In these conditions, [14C]sucrose Vv measured in WT mice was comparable to the WT Vv measured in the preceding experimental conditions (without albumin) (15.23±0.61 μL/g; n=5), indicating that the albumin-mediated vascular pressure increase during 120 seconds had no effect on BBB integrity. In contrast, brain [14C]sucrose Vv in DKo mice was significantly enhanced (18.6±2.1 μL/g; n=5), being 22% higher than in WT mice (P<0.05). Thus, an acute increase of the vascular hydrostatic pressure caused a statistically greater loss of vascular integrity in DKo mice. These results suggest that the absence of astroglial Cxs weakens the BBB, which becomes leaky when stressed.

Figure 5.

Figure 5

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Comparison of brain vascular volume in wild-type (WT) and double knockout mice for astroglial Cx43 and Cx30 by in situ brain perfusion assays. [14C]sucrose distribution volume in the brain of WT and DKo mice were measured with albumin-free-bicarbonate buffer (open bars) and with albumin addition (40 g/L; filled bars) delivered by in situ brain perfusion at 2.5 mL/min for 120 seconds. Data are mean values±s.d. of five animals. *P<0.05 comparing WT and DKo mice; †P<0.05 comparing DKo with and without albumin perfusion fluid.

Ultrastructural Characterization of the Gliovascular Interface in High Vascular Pressure Conditions

To better characterize the alteration of BBB integrity generated by the absence of astroglial Cxs under high vascular pressure, in situ brain perfusion was performed adding in the perfusate hSA and HRP (44 kDa), a vascular tracer that does not cross the healthy BBB. Then, HRP activity was revealed by incubating brain sections in DAB (see Materials and methods). As shown in Figure 6A, a massive HRP labeling was detected in almost every brain areas in DKo mice, as opposed to WT. Ultrathin sections of HRP and hSA in situ perfused brains were then examined under transmission electron microscopy (Figure 6B). No morphological difference could be detected compared with brain tissues not submitted to in situ perfusion (Figure 3), indicating that this experiment per se does not affect the brain architecture. Observations were similar in the cerebral cortex and the hippocampus. In WT mice, most vessels appeared devoid of HRP staining. In DKo mice, DAB reaction product was outlining the BL around microvessels and extended in the extracellular spaces of the parenchyma. No accumulation of HRP in the cytoplasm or in pinocytic vesicles could be detected in endothelial cells. Similarly, we found no obvious enlargement at the level of TJ between endothelial cells in the cerebral endothelium of DKo mice (data not shown). Altogether, these results indicate that under increased vascular pressure, BBB becomes permeable to high molecular weight molecules in the absence of astroglial Cxs.

Figure 6.

Figure 6

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Ultrastructural characterization of the gliovascular interface interface in wild-type (WT) and double knockout mice for astroglial Cx43 and Cx30 under high vascular pressure conditions. (A) The brain sections of WT and DKo mice subjected to in situ brain perfusion with albumin and horseradish peroxidase (HRP) revealed with DAB. A massive extravasation of HRP was observed in many areas of DKo brain compared with WT brain. (B) Transmission electron microscopy images of cortex ultrathin sections of WT and DKo mouse brains subjected to in situ brain perfusion with albumin and HRP. In WT mice, almost no sign of HRP extravasation was detected. In DKo mice, perivascular astrocytes appeared systematically swollen and edematous (asterisks), and a strong HRP staining was present at the level of the basal lamina around vessels (arrows) and in intercellular spaces in the parenchyma. a, astrocyte endfeet; e, endothelial cell. Scale bars: 5 μm. DAB, 3,3′-diaminobenzidine.

Discussion

The barrier properties of the cerebral vascular system greatly depend on interactions and cooperative functions of endothelial cells, astrocytes, and pericytes (Liebner et al, 2011). Astrocytes, in particular through the secretion of humoral factors, have been shown to induce barrier properties (Abbott et al, 2006). In this context, the influence of Cx43 and Cx30, the astroglial Cxs responsible for intense perivascular communication between endfeet (Simard et al, 2003; Rouach et al, 2008) on BBB physiology has never been studied. Here, we show that Cx43 and Cx30 start to be enriched at the level of perivascular endfeet from P2 and P12, respectively, suggesting the development of a high level of astroglial connectivity at the vascular interface during postnatal BBB maturation. Furthermore, we demonstrate that the deletion of Cx30 and astroglial Cx43 weakens the BBB that becomes permeable when vascular pressure increases. These results show for the first time that astroglial Cxs are necessary to BBB integrity in stress conditions. Whether the loss of BBB integrity is only related to the loss of Cx-based gap junction channel functions remains to be determined. Indeed, at the membrane, Cxs not only form intercellular channels but also hemichannels (Giaume et al, 2010), and they are the site for anchoring multiprotein complexes linking them in particular to the cytoskeleton (Olk et al, 2009). These interactions, which potentially regulate cell polarity, may be critical to maintain the gliovascular architecture. Interestingly, Cx30 has been shown to control the integrity of the stria vascularis endothelial barrier in the inner ear (Cohen-Salmon et al, 2007). However, the underlying mechanism, the transcriptional downregulation of Bhmt (betaine-homocysteine S-methyltransferase), does not occur at the gliovascular interface (Cohen-Salmon et al, 2007).

In conditions of higher sheer stress and hydrostatic vascular pressure, we observed a massive extravasation of HRP in DKo mice. Interestingly, HRP could only be detected in the extracellular space, and did not accumulate within endothelial cells. Thus, the presence of HRP in the DKo parenchyma may likely result from intercellular leaks due to the opening of endothelial TJs, rather than from an increased transendothelial transport. In DKo, neither the level of expression nor the tissular distribution of TJ-associated proteins and TJ morphology were changed compared with WT. In addition, DKo TJs were functional under normal vascular pressure. Thus, HRP leaks in DKo mice under high vascular pressure might rather result from a loss of TJ mechanical resistance, which might be general or affect a restricted number of TJs.

DKo astrocyte enfeet showed a pronounced swelling, and often appeared watery and devoid of organelles. Edema of disconnected hippocampal astrocytes in DKo mice was recently attributed to their inability to redistribute potassium and glutamate released by neurons during synaptic activity (Lutz et al, 2009; Pannasch et al, 2011). Here, we show that this phenomenon extends to the enfeet. Astrocyte endfeet edema has been observed in association with BBB leakage in the dystrophic MDX mouse model (Nico et al, 2004) during hypoxia (Kaur et al, 2006) or stroke (Ding et al, 2006). Thus, in DKo mice, it may directly affect the endothelial barrier properties as well. In particular, the regulations taking place in astroglial endfeet inducing BBB phenotype might be severely compromised.

Expression of β-DG decreased in DKo compared with WT. β-Dystroglycan in association with α-DG (encoded by the same gene) constitutes the major astrocyte transmembrane receptor of the vascular BL (Durbeej et al, 1998; Milner et al, 2008). The glycosylated extracellular subunit α-DG has been shown to bind both laminin and the laminin-like globular domains of agrin, perlecan, and neurexin (Henry and Campbell, 1999; Sugita et al, 2001), while the transmembrane β-DG binds the cytoskeletal proteins dystrophin and utrophin (Ervasti and Campbell, 1993; James et al, 1996). Several studies have converged to the idea that this DG complex was crucial to BL organization as well as BBB integrity. First, BL appears discontinuous in the absence of β-DG (Moore et al, 2002; Satz et al, 2008). Second, partial loss of β-DG also affects deposition of laminin and disorganizes the fibronectin network (Sirour et al, 2011). Loss of β-DG was also observed during focal cerebral ischemia exposure, together with BBB breakdown (Milner et al, 2008). Finally, leukocyte infiltration in the brain parenchyma is associated with the proteolytic cleavage of β-DG (Agrawal et al, 2006). Thus, although the partial loss of β-DG in DKo did not appear to affect perlecan, agrin, and laminin deposition, it could likely affect the BL organization and influence endothelial mechanical resistance. It would also result in a decrease of α-DG. As α-DG represents the binding portion of the receptor complex, this would probably modify the links between astrocytes and the BL, a phenomenon known to significantly affect vascular integrity (Abbott et al, 2006).

The level of Aqp4, the water channel expressed at the perivascular astrocyte endfeet membrane and responsible for the astrocyte transmembrane water flux (Simard et al, 2003), dropped severely in DKo mice. Nevertheless, it was comparable to WT at P21 (Pannasch et al, 2011). Thus, Aqp4 decrease observed here in 3-month-old DKo mice is likely secondary to astrocyte edema. Interestingly, a coregulation of Aqp4 and Cx43 expression has already been shown in vivo and in vitro, indicating the possibility for a functional relationship between water homeostasis and gap junctional communication (Nicchia et al, 2005; Strohschein et al, 2011). Along the same line, the loss of β-DG has been shown to compromise the expression of Aqp4 in the perivascular endfeet (Noell et al, 2011). Loss of Aqp4 during astroglial (cytotoxic) edema, which reduces water permeability of the cells (Solenov et al, 2004), mitigates water accumulation in astrocytes and thus protects them against edema (Papadopoulos and Verkman, 2007). Therefore, the progressive loss of Aqp4 in DKo might reflect a protective mechanism aiming at attenuating the astroglial edema. Would the loss of Aqp4 affect BBB integrity? Aqp4 knockdown in primary cultured astrocytes has been reported to reorganize actin cytoskeleton (Nicchia et al, 2005). Since astroglial polarity and morphology are essential to the BBB development and maintenance, the loss of Aqp4 may perturb gliovascular complex functions. However, in vivo Aqp4 inactivation has no effect in physiological conditions (Ma et al, 1997) even at the level of the BBB (Saadoun et al, 2009). Thus, per se, the decreased level of Aqp4 in DKo mice may not directly affect BBB integrity. Nevertheless, the resulting uncoupling of water and potassium transport, together with the inability of uncoupled astrocytes to redistribute neuroactive substances away from synapses (Pannasch et al, 2011) might be deleterious for the brain and lead in particular to an increased susceptibility to seizures.

In conclusion, we here demonstrate for the first time that the absence of astroglial Cxs affects perivacular endfeet and results in a ‘loosening' of the BBB, which becomes permeable when vascular pressure is increased. Hypertension is a dysfunction that is associated to many pathologies of the central nervous system. Our results suggest that the decreased expression of astroglial Cxs, which occurs in several neuropathological conditions (reviewed in Giaume et al, 2010) might predispose to neurovascular defects and BBB dysfunctions.

Acknowledgments

The authors thank A Koulakoff and P Meda for helpful discussions and critical reading of the manuscript; K Willecke for providing Cx30−/−Cx43fl/fl/hGFAP-Cre DKo mice; and J Teillon for his excellent technical support in confocal microscopy.

The authors declare no conflict of interest.

Footnotes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website (http://www.nature.com/jcbfm)

This work was supported by la Fondation NRJ-Institut de France and by Agence Nationale pour la Recherche ANR-07-NEURO-004-01.

Supplementary Material

Supplementary Figures
Click here for additional data file. (4.6MB, pdf)

References

  1. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ. Structure and function of the blood-brain barrier. Neurobiol Dis. 2010;37:13–25. doi: 10.1016/j.nbd.2009.07.030. [DOI] [PubMed] [Google Scholar]
  2. Abbott NJ, Ronnback L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006;7:41–53. doi: 10.1038/nrn1824. [DOI] [PubMed] [Google Scholar]
  3. Agrawal S, Anderson P, Durbeej M, van Rooijen N, Ivars F, Opdenakker G, Sorokin LM. Dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis. J Exp Med. 2006;203:1007–1019. doi: 10.1084/jem.20051342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cohen-Salmon M, Regnault B, Cayet N, Caille D, Demuth K, Hardelin JP, Janel N, Meda P, Petit C. Connexin30 deficiency causes instrastrial fluid-blood barrier disruption within the cochlear stria vascularis. Proc Natl Acad Sci USA. 2007;104:6229–6234. doi: 10.1073/pnas.0605108104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dagenais C, Rousselle C, Pollack GM, Scherrmann JM. Development of an in situ mouse brain perfusion model and its application to mdr1a P-glycoprotein-deficient mice. J Cereb Blood Flow Metab. 2000;20:381–386. doi: 10.1097/00004647-200002000-00020. [DOI] [PubMed] [Google Scholar]
  6. Daneman R, Zhou L, Kebede AA, Barres BA. Pericytes are required for blood-brain barrier integrity during embryogenesis. Nature. 2010;468:562–566. doi: 10.1038/nature09513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. del Zoppo GJ, Milner R. Integrin-matrix interactions in the cerebral microvasculature. Arterioscler Thromb Vasc Biol. 2006;26:1966–1975. doi: 10.1161/01.ATV.0000232525.65682.a2. [DOI] [PubMed] [Google Scholar]
  8. Ding YH, Ding Y, Li J, Bessert DA, Rafols JA. Exercise pre-conditioning strengthens brain microvascular integrity in a rat stroke model. Neurol Res. 2006;28:184–189. doi: 10.1179/016164106X98053. [DOI] [PubMed] [Google Scholar]
  9. Dore-Duffy P. Pericytes: pluripotent cells of the blood brain barrier. Curr Pharm Des. 2008;14:1581–1593. doi: 10.2174/138161208784705469. [DOI] [PubMed] [Google Scholar]
  10. Durbeej M, Henry MD, Campbell KP. Dystroglycan in development and disease. Curr Opin Cell Biol. 1998;10:594–601. doi: 10.1016/s0955-0674(98)80034-3. [DOI] [PubMed] [Google Scholar]
  11. Ervasti JM, Campbell KP. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol. 1993;122:809–823. doi: 10.1083/jcb.122.4.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Giaume C, Koulakoff A, Roux L, Holcman D, Rouach N. Astroglial networks: a step further in neuroglial and gliovascular interactions. Nat Rev Neurosci. 2010;11:87–99. doi: 10.1038/nrn2757. [DOI] [PubMed] [Google Scholar]
  13. Gordon GR, Mulligan SJ, MacVicar BA. Astrocyte control of the cerebrovasculature. Glia. 2007;55:1214–1221. doi: 10.1002/glia.20543. [DOI] [PubMed] [Google Scholar]
  14. Henry MD, Campbell KP. Dystroglycan inside and out. Curr Opin Cell Biol. 1999;11:602–607. doi: 10.1016/s0955-0674(99)00024-1. [DOI] [PubMed] [Google Scholar]
  15. James M, Nguyen TM, Wise CJ, Jones GE, Morris GE. Utrophin-dystroglycan complex in membranes of adherent cultured cells. Cell Motil Cytoskeleton. 1996;33:163–174. doi: 10.1002/(SICI)1097-0169(1996)33:3<163::AID-CM1>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  16. Kaur C, Sivakumar V, Zhang Y, Ling EA. Hypoxia-induced astrocytic reaction and increased vascular permeability in the rat cerebellum. Glia. 2006;54:826–839. doi: 10.1002/glia.20420. [DOI] [PubMed] [Google Scholar]
  17. Kunze A, Congreso MR, Hartmann C, Wallraff-Beck A, Huttmann K, Bedner P, Requardt R, Seifert G, Redecker C, Willecke K, Hofmann A, Pfeifer A, Theis M, Steinhauser C. Connexin expression by radial glia-like cells is required for neurogenesis in the adult dentate gyrus. Proc Natl Acad Sci USA. 2009;106:11336–11341. doi: 10.1073/pnas.0813160106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kunzelmann P, Schroder W, Traub O, Steinhauser C, Dermietzel R, Willecke K. Late onset and increasing expression of the gap junction protein connexin30 in adult murine brain and long-term cultured astrocytes. Glia. 1999;25:111–119. doi: 10.1002/(sici)1098-1136(19990115)25:2<111::aid-glia2>3.0.co;2-i. [DOI] [PubMed] [Google Scholar]
  19. Liebner S, Czupalla CJ, Wolburg H. Current concepts of blood-brain barrier development. Int J Dev Biol. 2011;55:467–476. doi: 10.1387/ijdb.103224sl. [DOI] [PubMed] [Google Scholar]
  20. Lutz SE, Zhao Y, Gulinello M, Lee SC, Raine CS, Brosnan CF. Deletion of astrocyte connexins 43 and 30 leads to a dysmyelinating phenotype and hippocampal CA1 vacuolation. J Neurosci. 2009;29:7743–7752. doi: 10.1523/JNEUROSCI.0341-09.2009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ma T, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Generation and phenotype of a transgenic knockout mouse lacking the mercurial-insensitive water channel aquaporin-4. J Clin Invest. 1997;100:957–962. doi: 10.1172/JCI231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mathiisen TM, Lehre KP, Danbolt NC, Ottersen OP. The perivascular astroglial sheath provides a complete covering of the brain microvessels: an electron microscopic 3D reconstruction. Glia. 2010;58:1094–1103. doi: 10.1002/glia.20990. [DOI] [PubMed] [Google Scholar]
  23. Milner R, Hung S, Wang X, Spatz M, del Zoppo GJ. The rapid decrease in astrocyte-associated dystroglycan expression by focal cerebral ischemia is protease-dependent. J Cereb Blood Flow Metab. 2008;28:812–823. doi: 10.1038/sj.jcbfm.9600585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moore SA, Saito F, Chen J, Michele DE, Henry MD, Messing A, Cohn RD, Ross-Barta SE, Westra S, Williamson RA, Hoshi T, Campbell KP. Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy. Nature. 2002;418:422–425. doi: 10.1038/nature00838. [DOI] [PubMed] [Google Scholar]
  25. Nadarajah B, Jones AM, Evans WH, Parnavelas JG. Differential expression of connexins during neocortical development and neuronal circuit formation. J Neurosci. 1997;17:3096–3111. doi: 10.1523/JNEUROSCI.17-09-03096.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nagy JI, Patel D, Ochalski PA, Stelmack GL. Connexin30 in rodent, cat and human brain: selective expression in gray matter astrocytes, co-localization with connexin43 at gap junctions and late developmental appearance. Neuroscience. 1999;88:447–468. doi: 10.1016/s0306-4522(98)00191-2. [DOI] [PubMed] [Google Scholar]
  27. Nicchia GP, Srinivas M, Li W, Brosnan CF, Frigeri A, Spray DC. New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43. FASEB J. 2005;19:1674–1676. doi: 10.1096/fj.04-3281fje. [DOI] [PubMed] [Google Scholar]
  28. Nico B, Paola Nicchia G, Frigeri A, Corsi P, Mangieri D, Ribatti D, Svelto M, Roncali L. Altered blood-brain barrier development in dystrophic MDX mice. Neuroscience. 2004;125:921–935. doi: 10.1016/j.neuroscience.2004.02.008. [DOI] [PubMed] [Google Scholar]
  29. Noell S, Wolburg-Buchholz K, Mack AF, Beedle AM, Satz JS, Campbell KP, Wolburg H, Fallier-Becker P. Evidence for a role of dystroglycan regulating the membrane architecture of astroglial endfeet. Eur J Neurosci. 2011;33:2179–2186. doi: 10.1111/j.1460-9568.2011.07688.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Olk S, Zoidl G, Dermietzel R. Connexins, cell motility, and the cytoskeleton. Cell Motil Cytoskeleton. 2009;66:1000–1016. doi: 10.1002/cm.20404. [DOI] [PubMed] [Google Scholar]
  31. Pannasch U, Vargova L, Reingruber J, Ezan P, Holcman D, Giaume C, Sykova E, Rouach N. Astroglial networks scale synaptic activity and plasticity. Proc Natl Acad Sci USA. 2011;108:8467–8472. doi: 10.1073/pnas.1016650108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Papadopoulos MC, Verkman AS. Aquaporin-4 and brain edema. Pediatr Nephrol. 2007;22:778–784. doi: 10.1007/s00467-006-0411-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ravussin PA, Favre JB, Archer DP, Tommasino C, Boulard G. Treatment of hypovolemia in brain injured patients. Ann Fr Anesth Reanim. 1994;13:88–97. doi: 10.1016/s0750-7658(94)80191-6. [DOI] [PubMed] [Google Scholar]
  34. Rouach N, Koulakoff A, Abudara V, Willecke K, Giaume C. Astroglial metabolic networks sustain hippocampal synaptic transmission. Science. 2008;322:1551–1555. doi: 10.1126/science.1164022. [DOI] [PubMed] [Google Scholar]
  35. Rowitch DH, Kriegstein AR. Developmental genetics of vertebrate glial-cell specification. Nature. 2010;468:214–222. doi: 10.1038/nature09611. [DOI] [PubMed] [Google Scholar]
  36. Saadoun S, Tait MJ, Reza A, Davies DC, Bell BA, Verkman AS, Papadopoulos MC. AQP4 gene deletion in mice does not alter blood-brain barrier integrity or brain morphology. Neuroscience. 2009;161:764–772. doi: 10.1016/j.neuroscience.2009.03.069. [DOI] [PubMed] [Google Scholar]
  37. Satz JS, Barresi R, Durbeej M, Willer T, Turner A, Moore SA, Campbell KP. Brain and eye malformations resembling Walker-Warburg syndrome are recapitulated in mice by dystroglycan deletion in the epiblast. J Neurosci. 2008;28:10567–10575. doi: 10.1523/JNEUROSCI.2457-08.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simard M, Arcuino G, Takano T, Liu QS, Nedergaard M. Signaling at the gliovascular interface. J Neurosci. 2003;23:9254–9262. doi: 10.1523/JNEUROSCI.23-27-09254.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sirour C, Hidalgo M, Bello V, Buisson N, Darribere T, Moreau N. Dystroglycan is involved in skin morphogenesis downstream of the Notch signaling pathway. Mol Biol Cell. 2011;22:2957–2969. doi: 10.1091/mbc.E11-01-0074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Solenov E, Watanabe H, Manley GT, Verkman AS. Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method. Am J Physiol Cell Physiol. 2004;286:C426–C432. doi: 10.1152/ajpcell.00298.2003. [DOI] [PubMed] [Google Scholar]
  41. Strohschein S, Huttmann K, Gabriel S, Binder DK, Heinemann U, Steinhauser C. Impact of aquaporin-4 channels on K+ buffering and gap junction coupling in the hippocampus. Glia. 2011;59:973–980. doi: 10.1002/glia.21169. [DOI] [PubMed] [Google Scholar]
  42. Sugita S, Saito F, Tang J, Satz J, Campbell K, Sudhof TC. A stoichiometric complex of neurexins and dystroglycan in brain. J Cell Biol. 2001;154:435–445. doi: 10.1083/jcb.200105003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Teubner B, Michel V, Pesch J, Lautermann J, Cohen-Salmon M, Sohl G, Jahnke K, Winterhager E, Herberhold C, Hardelin JP, Petit C, Willecke K. Connexin30 (Gjb6)-deficiency causes severe hearing impairment and lack of endocochlear potential. Hum Mol Genet. 2003;12:13–21. doi: 10.1093/hmg/ddg001. [DOI] [PubMed] [Google Scholar]
  44. Theis M, Mas C, Doring B, Kruger O, Herrera P, Meda P, Willecke K. General and conditional replacement of connexin43-coding DNA by a lacZ reporter gene for cell-autonomous analysis of expression. Cell Commun Adhes. 2001;8:383–386. doi: 10.3109/15419060109080758. [DOI] [PubMed] [Google Scholar]
  45. Tsacopoulos M, Magistretti PJ. Metabolic coupling between glia and neurons. J Neurosci. 1996;16:877–885. doi: 10.1523/JNEUROSCI.16-03-00877.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wen H, Nagelhus EA, Amiry-Moghaddam M, Agre P, Ottersen OP, Nielsen S. Ontogeny of water transport in rat brain: postnatal expression of the aquaporin-4 water channel. Eur J Neurosci. 1999;11:935–945. doi: 10.1046/j.1460-9568.1999.00502.x. [DOI] [PubMed] [Google Scholar]
  47. Yamamoto T, Vukelic J, Hertzberg EL, Nagy JI. Differential anatomical and cellular patterns of connexin43 expression during postnatal development of rat brain. Brain Res Dev Brain Res. 1992;66:165–180. doi: 10.1016/0165-3806(92)90077-a. [DOI] [PubMed] [Google Scholar]

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