Fig. 3

AQP1 and NKCC1 are expressed by the choroidal epithelial cells and in the leptomeningeal vasculature. a–f Confocal micrograph show a leptomeningeal WGA-FITC⁺ (green) labeled vessel immunoreactive for AQP1 (magenta) and NKCC1 (orange) in the adult mouse brain (P90). In b an optical section reveals that AQP1⁺/NKCC1⁺ cells are restricted to the smooth muscle cell layer (arrowheads) and absent in the endothelial cells (curved arrowheads), which are labeled by WGA-FITC. g, h NKCC1 is detected in the choroid plexus epithelia, in ependymal cells and in the molecular layer of the cerebellum, as shown in the micrographs of the fourth ventricle. i Double labeling confirms AQP1 and NKCC1 presence in choroid plexus epithelial cells (higher magnification of the area delimited by the blue square in h). j, k Brain sections obtained from NKCC1 KO adult mice show no immunoreactivity in the brain parenchyma neither in the choroid plexus. l, m Histological sections immunolabeled with antibodies against AQP1ext (magenta), NKCC1 (yellow) and α-SMA (cyan), reveal AQP1ext⁺/NKCC1⁺/α-SMA⁺ leptomeningeal vessels around the hippocampus and third ventricle. Low magnification micrograph shows DAPI (blue) counterstaining and indicates a leptomeningeal blood vessel (asterisk) closely located to the hippocampal fissure. n–p Higher magnification of an AQP1ext⁺/NKCC1⁺ vessel (delimited by the dashed square in j. Arrowheads indicate α-SMA⁺ cells. (q) Optical sectioning reveals that both AQP1 and NKCC1 are distributed in the smooth muscle cell layer (arrowheads). r 3D rendering of the leptomeningeal vessel confirms AQP1 and NKCC1 restriction to the smooth muscle cell layer (arrowheads). ChP, choroid plexus; DG, dentate gyrus; DS, dorsal subiculum; GL, granular layer; hif, hippocampal fissure; Mol, molecular layer; SAS, subarachnoid space; 3V, third ventricle, 4V, fourth ventricle. Scale bars: a, i 20 µm; b–f, q, r 10 µm; g, h, j–p 50 µm