Because it lacks a lymphatic circulation, the brain must clear extracellular proteins by an alternative mechanism. The cerebrospinal fluid (CSF) functions as a sink for brain extracellular solutes, but it is not clear how solutes from the brain interstitium move from the parenchyma to the CSF. We demonstrate that a substantial portion of subarachnoid CSF cycles through the brain interstitial space. On the basis of in vivo two-photon imaging of small fluorescent tracers, we showed that CSF enters the parenchyma along paravascular spaces that surround penetrating arteries and that brain interstitial fluid is cleared along paravenous drainage pathways. Animals lacking the water channel aquaporin-4 (AQP4) in astrocytes exhibit slowed CSF influx through this system and a ~70% reduction in interstitial solute clearance, suggesting that the bulk fluid flow between these anatomical influx and efflux routes is supported by astrocytic water transport. Fluorescent-tagged amyloid β, a peptide thought to be pathogenic in Alzheimer’s disease, was transported along this route, and deletion of the Aqp4 gene suppressed the clearance of soluble amyloid β, suggesting that this pathway may remove amyloid β from the central nervous system. Clearance through paravenous flow may also regulate extracellular levels of proteins involved with neurodegenerative conditions, its impairment perhaps contributing to the mis-accumulation of soluble proteins.

Membrane water transport is critically involved in brain volume homeostasis and in the pathogenesis of brain edema. The cDNA encoding aquaporin-4 (AQP4) water channel protein was recently isolated from rat brain. We used immunocytochemistry and high-resolution immunogold electron microscopy to identify the cells and membrane domains that mediate water flux through AQP4. The AQP4 protein is abundant in glial cells bordering the subarachnoidal space, ventricles, and blood vessels. AQP4 is also abundant in osmosensory areas, including the supraoptic nucleus and subfornical organ. Immunogold analysis demonstrated that AQP4 is restricted to glial membranes and to subpopulations of ependymal cells. AQP4 is particularly strongly expressed in glial membranes that are in direct contact with capillaries and pia. The highly polarized AQP4 expression indicates that these cells are equipped with specific membrane domains that are specialized for water transport, thereby mediating the flow of water between glial cells and the cavities filled with CSF and the intravascular space. Key words: aquaporin-4 water channel; brain water permeability; glial cells; ependymal cells; immunogold electron microscopy; CSFWater metabolism is of major importance in a number of physiological processes in the CNS including CSF production and absorption, fluid transport across neuropil and vascular endothelium, and cell volume regulation (Fitzsimons, 1992;Robertson, 1992). In addition, water transport may serve to compensate for local changes in osmolality associated with potassium siphoning, which is essential for synaptic transmission. Alterations in water distribution in brain and CSF compartments is a common occurence in multiple neuropathological conditions including brain edema, brain tumors, stroke, hyponatremia, head injuries, and hydrocephalus. Despite its importance, little is known about the cellular and molecular mechanisms involved in transmembrane water movements in brain.Discovery of aquaporin-1 (Preston et al., 1992) answered the long-standing biophysical question of how water crosses plasma membranes (for review, see Agre et al., 1993;Knepper, 1994). Characterization of aquaporins provided molecular insight into fundamental processes of normal water balance and disorders of water balance outside brain (for review, see Nielsen et al., 1996). A cDNA for aquaporin-4 (AQP4) water channel protein was isolated recently from rat brain (Hasegawa et al., 1994;Jung et al., 1994), and abundant AQP4 was noted in brain including in cerebellum, hypothalamus, spinal cord, and ependymal cells lining the ventricles (Jung et al., 1994;Frigeri et al., 1995). Nevertheless, the cellular and subcellular distributions of AQP4 in brain remain unknown, and definition of the sites of AQP4 expression will be essential for understanding its physiological and pathophysiological roles.Immunocytochemistry and high-resolution immunogold electron microscopy were used to define the sites of AQP4 in brain. AQP4 expression is restricted to ependymal cell lining of ...

(AQP4) is one of the most abundant molecules in the brain and is particularly prevalent in astrocytic membranes at the blood-brain and brain-liquor interfaces. While AQP4 has been implicated in a number of pathophysiological processes, its role in brain physiology has remained elusive. Only recently has evidence accumulated to suggest that AQP4 is involved in such diverse functions as regulation of extracellular space volume, potassium buffering, cerebrospinal fluid circulation, interstitial fluid resorption, waste clearance, neuroinflammation, osmosensation, cell migration, and Ca 2ϩ signaling. AQP4 is also required for normal function of the retina, inner ear, and olfactory system. A review will be provided of the physiological roles of AQP4 in brain and of the growing list of data that emphasize the polarized nature of astrocytes.

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The water permeability of cell membranes differs by orders of magnitude, and most of this variability reflects the differential expression of aquaporin water channels. We have recently found that the CNS contains a member of the aquaporin family, aquaporin-4 (AQP4). As a prerequisite for understanding the cellular handling of water during neuronal activity, we have investigated the cellular and subcellular expression of AQP4 in the retina and optic nerve where activity-dependent ion fluxes have been studied in detail. In situ hybridization with digoxigenin-labeled riboprobes and immunogold labeling by a sensitive postembedding procedure demonstrated that AQP4 and AQP4 mRNA were restricted to glial cells, including Mü ller cells in the retina and fibrous astrocytes in the optic nerve. A quantitative immunogold analysis of the Mü ller cells showed that these cells exhibited three distinct membrane compartments with regard to AQP4 expression. End feet membranes (facing the vitreous body or blood vessels) were 10-15 times more intensely labeled than non-end feet membranes, whereas microvilli were devoid of AQP4. These data suggest that Mü ller cells play a prominent role in the water handling in the retina and that they direct osmotically driven water flux to the vitreous body and vessels rather than to the subretinal space. Fibrous astrocytes in the optic nerve similarly displayed a differential compartmentation of AQP4. The highest expression of AQP4 occurred in end feet membranes, whereas the membrane domain facing the nodal axolemma was associated with a lower level of immunoreactivity than the rest of the membrane. This arrangement may allow transcellular water redistribution to occur without inducing inappropriate volume changes in the perinodal extracellular space.

Recovery from neuronal activation requires rapid clearance of potassium ions (K ؉ ) and restoration of osmotic equilibrium. The predominant water channel protein in brain, aquaporin-4 (AQP4), is concentrated in the astrocyte end-feet membranes adjacent to blood vessels in neocortex and cerebellum by association with ␣-syntrophin protein. Although AQP4 has been implicated in the pathogenesis of brain edema, its functions in normal brain physiology are uncertain. In this study, we used immunogold electron microscopy to compare hippocampus of WT and ␣-syntrophin-null mice (␣-Syn ؊/؊ ). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the ␣-Syn ؊/؊ mice, whereas labeling of the inwardly rectifying K ؉ channel, Kir4.1, is largely unchanged. Activity-dependent changes in K ؉ clearance were studied in hippocampal slices to test whether AQP4 and K ؉ channels work in concert to achieve isosmotic clearance of K ؉ after neuronal activation. Microelectrode recordings of extracellular K ؉ ([K ؉ ]o) from the target zones of Schaffer collaterals and perforant path were obtained after 5-, 10-, and 20-Hz orthodromic stimulations. K ؉ clearance was prolonged up to 2-fold in ␣-Syn ؊/؊ mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the ␣-Syn ؊/؊ mice. These studies lead us to propose that water flux through perivascular AQP4 is needed to sustain efficient removal of K ؉ after neuronal activation.

Calcium signaling represents the principle pathway by which astrocytes respond to neuronal activity. General anesthetics are routinely used in clinical practice to induce a sleep-like state, allowing otherwise painful procedures to be performed. Anesthetic drugs are thought to mainly target neurons in the brain and act by suppressing synaptic activity. However, the direct effect of general anesthesia on astrocyte signaling in awake animals has not previously been addressed. This is a critical issue, because calcium signaling may represent an essential mechanism through which astrocytes can modulate synaptic activity. In our study, we performed calcium imaging in awake head-restrained mice and found that three commonly used anesthetic combinations (ketamine/xylazine, isoflurane, and urethane) markedly suppressed calcium transients in neocortical astrocytes. Additionally, all three anesthetics masked potentially important features of the astrocyte calcium signals, such as synchronized widespread transients that appeared to be associated with arousal in awake animals. Notably, anesthesia affected calcium transients in both processes and soma and depressed spontaneous signals, as well as calcium responses, evoked by whisker stimulation or agonist application. We show that these calcium transients are inositol 1,4,5-triphosphate type 2 receptor (IP 3 R2)-dependent but resistant to a local blockade of glutamatergic or purinergic signaling. Finally, we found that doses of anesthesia insufficient to affect neuronal responses to whisker stimulation selectively suppressed astrocyte calcium signals. Taken together, these data suggest that general anesthesia may suppress astrocyte calcium signals independently of neuronal activity. We propose that these glial effects may constitute a nonneuronal mechanism for sedative action of anesthetic drugs.