Tissue-and cell-specific deletion of the Aqp4 gene is required to differentiate between the numerous pools of aquaporin-4 (AQP4) water channels. A glial-conditional Aqp4 knockout mouse line was generated to resolve whether astroglial AQP4 controls water exchange across the blood-brain interface. The conditional knockout was driven by the glial fibrillary acidic protein promoter. Brains from conditional Aqp4 knockouts were devoid of AQP4 as assessed by Western blots, ruling out the presence of a significant endothelial pool of AQP4. In agreement, immunofluorescence analysis of cryostate sections and quantitative immunogold analysis of ultrathin sections revealed no AQP4 signals in capillary endothelia. Compared with litter controls, glial-conditional Aqp4 knockout mice showed a 31% reduction in brain water uptake after systemic hypoosmotic stress and a delayed postnatal resorption of brain water. Deletion of astroglial Aqp4 did not affect the barrier function to macromolecules. Our data suggest that the blood-brain barrier (BBB) is more complex than anticipated. Notably, under certain conditions, the astrocyte covering of brain microvessels is rate limiting to water movement. edema | electron microscopy | homeostasis | membrane | swelling M ore than 100 y have elapsed since it was first shown that some solutes present in blood are retained by brain capillaries, pointing to the existence of a barrier function at the bloodbrain interface. This early finding naturally inspired a discussion as to what could constitute the morphological substrate of this barrier (1). With the advent of the electron microscope it became clear that the blood-brain interface is composed of endothelia and pericytes, surrounded by a basal lamina and perivascular endfeet of astrocytes. After decades of intense debate, a concept emerged that the functional barrier resides at the level of the endothelial cells (2). This was consistent with morphological data, which clearly showed that endothelia were continuous and coupled by tight junctions (3). The perivascular endfeet, on the other hand, were not coupled by tight junctions and were portrayed as a discontinuous layer with spacious clefts separating the individual processes.Discussions on the morphological substrate of barrier function have been focused on solutes (4), and the field has not yet matured to provide a consistent view regarding what cellular structures, if any, restrict water movement between blood and brain. Following the discovery of water channels, it became clear that the major brain water channel AQP4 is implicated in water transport at the blood-brain interface. Thus, global Aqp4 knockout significantly limited the development of brain edema, attesting to the importance of AQP4 water channels (4). As AQP4 is strongly expressed in the perivascular endfeet (5), the interest in these processes was rekindled also in the context of their possible barrier function.Specifically, it was proposed that the endfeet could restrict water flow, most significantly in pathophysiological se...

Key roles of macroglia are inextricably coupled to specialized membrane domains. The perivascular endfoot membrane has drawn particular attention, as this domain contains a unique complement of aquaporin-4 (AQP4) and other channel proteins that distinguishes it from perisynaptic membranes. Recent studies indicate that the polarization of macroglia is lost in a number of diseases, including temporal lobe epilepsy and Alzheimer’s disease. A better understanding is required of the molecular underpinning of astroglial polarization, particularly when it comes to the significance of the dystrophin associated protein complex (DAPC). Here we employ immunofluorescence and immunogold cytochemistry to analyze the molecular scaffolding in perivascular endfeet in macroglia of retina and three regions of brain (cortex, dentate gyrus and cerebellum), using AQP4 as a marker. Compared with brain astrocytes, Müller cells (a class of retinal macroglia) exhibit lower densities of the scaffold proteins dystrophin and α-syntrophin (a DAPC protein), but higher levels of AQP4. In agreement, depletion of dystrophin or α-syntrophin – while causing a dramatic loss of AQP4 from endfoot membranes of brain astrocytes – had only modest or insignificant effect, respectively, on the AQP4 pool in endfoot membranes of Müller cells. Also, while polarization of brain macroglia was less affected by dystrophin depletion than by targeted deletion of α-syntrophin, the reverse was true for retinal macroglia. These data indicate that the molecular scaffolding in perivascular endfeet is more complex than previously assumed and that macroglia are heterogeneous with respect to the mechanisms that dictate their polarization.

Expression of the water channel aquaporin-4 (AQP4) at the blood-brain interface is dependent upon the dystrophin associated protein complex. Here we investigated whether deletion of the Aqp4 gene affects the molecular composition of this protein scaffold and the integrity of the blood-brain barrier. High-resolution immunogold cytochemistry revealed that perivascular expression of α-syntrophin was reduced by 60% in Aqp4(-/-) mice. Additionally, perivascular AQP4 expression was reduced by 88% in α-syn(-/-) mice, in accordance with earlier reports. Immunofluorescence showed that Aqp4 deletion also caused a modest reduction in perivascular dystrophin, whereas β-dystroglycan labeling was unaltered. Perivascular microglia were devoid of AQP4 immunoreactivity. Deletion of Aqp4 did not alter the ultrastructure of capillary endothelial cells, the expression of tight junction proteins (claudin-5, occludin, and zonula occludens 1), or the vascular permeability to horseradish peroxidase and Evans blue albumin dye. We conclude that Aqp4 deletion reduces the expression of perivascular glial scaffolding proteins without affecting the endothelial barrier. Our data also indicate that AQP4 and α-syntrophin are mutually dependent upon each other for proper perivascular expression.

Astrocytic endfeet cover the brain surface and form a sheath around the cerebral vasculature. An emerging concept is that endfeet control blood–brain water transport and drainage of interstitial fluid and waste along paravascular pathways. Little is known about the signaling mechanisms that regulate endfoot volume and hence the width of these drainage pathways. Here, we used the genetically encoded fluorescent Ca2+ indicator GCaMP6f to study Ca2+ signaling within astrocytic somata, processes, and endfeet in response to an osmotic challenge known to induce cell swelling. Acute cortical slices were subjected to artificial cerebrospinal fluid with 20% reduction in osmolarity while GCaMP6f fluorescence was imaged with two‐photon microscopy. Ca2+ signals induced by hypoosmotic conditions were observed in all astrocytic compartments except the soma. The Ca2+ response was most prominent in subpial and perivascular endfeet and included spikes with single peaks, plateau‐type elevations, and rapid oscillations, the latter restricted to subpial endfeet. Genetic removal of the type 2 inositol 1,4,5‐triphosphate receptor (IP3R2) severely suppressed the Ca2+ responses in endfeet but failed to affect brain water accumulation in vivo after water intoxication. Furthermore, the increase in endfoot Ca2+ spike rate during hypoosmotic conditions was attenuated in mutant mice lacking the aquaporin‐4 anchoring molecule dystrophin and after blockage of transient receptor potential vanilloid 4 channels. We conclude that the characteristics and underpinning of Ca2+ responses to hypoosmotic stress differ within the astrocytic territory and that IP3R2 is essential for the Ca2+ signals only in subpial and perivascular endfeet.