Loss or Mislocalization of Aquaporin-4 Affects Diffusion Properties and Intermediary Metabolism in Gray Matter of Mice.

The first aim of this study was to determine how complete or perivascular loss of aquaporin-4 (AQP4) water channels affects membrane permeability for water in the mouse brain grey matter in the steady state. Time-dependent diffusion magnetic resonance imaging was performed on global Aqp4 knock out (KO) and α-syntrophin (α-syn) KO mice, in the latter perivascular AQP4 are mislocalized, but still functioning. Control animals were corresponding wild type (WT) mice. By combining in vivo diffusion measurements with the effective medium theory and previously measured extra-cellular volume fractions, the effects of membrane permeability and extracellular volume fraction were uncoupled for Aqp4 and α-syn KO. The second aim was to assess the effect of α-syn KO on cortical intermediary metabolism combining in vivo [1-13C]glucose and [1,2-13C]acetate injection with ex vivo 13C MR spectroscopy. Aqp4 KO increased the effective diffusion coefficient at long diffusion times by 5%, and a 14% decrease in membrane water permeability was estimated for Aqp4 KO compared with WT mice. α-syn KO did not affect the measured diffusion parameters. In the metabolic analyses, significantly lower amounts of [4-13C]glutamate and [4-13C]glutamine, and percent enrichment in [4-13C]glutamate were detected in the α-syn KO mice. [1,2-13C]acetate metabolism was unaffected in α-syn KO, but the contribution of astrocyte derived metabolites to GABA synthesis was significantly increased. Taken together, α-syn KO mice appeared to have decreased neuronal glucose metabolism, partly compensated for by utilization of astrocyte derived metabolites.

Real-time analysis of microglial activation and motility in hepatic and hyperammonemic encephalopathy.

Hepatic encephalopathy (HE) is a potentially fatal complication of acute liver failure, associated with severe neurological dysfunction and coma. The brain's innate immune cells, microglia, have recently been implicated in the pathophysiology of HE. To date, however, only ex vivo studies have been used to characterize microglial involvement. Our study uses in vivo two-photon imaging of awake-behaving mice expressing enhanced green fluorescent protein (eGFP) under the Cx3cr1 promoter to examine microglial involvement in two different models of encephalopathy - a slower, fatal model of azoxymethane-induced HE and a rapid, reversible acute hyperammonemic encephalopathy (AHE) induced by an ammonia load. To investigate the potential contribution of microglia to the neurological deterioration seen in these two models, we developed a software to analyze microglial activation and motility in vivo. In HE, we found that microglia do not become activated prior to the onset of neurological dysfunction, but undergo activation with mildly impaired motility during the terminal stage IV. We demonstrate that this microglial activation coincides with blood-brain barrier (BBB) opening and brain edema. Conversely, both microglial activation and motility are unchanged during AHE, despite the mice developing pathologically increased plasma ammonia and severe neurological dysfunction. Our study indicates that microglial activation does not contribute to the early neurological deterioration observed in either HE or AHE. The late microglial activation in HE may therefore be associated with terminal BBB opening and brain edema, thus exacerbating the progression to coma and increasing mortality.

Phenotypic characteristics of temporal lobe epilepsy: the impact of hippocampal sclerosis.

OBJECTIVES: Whether mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) is a condition with a unique biological background that can be delineated from other TLE, is unresolved. Here we performed a comparative analysis of two TLE patient cohorts - one cohort with HS and one without HS - in order to identify phenotypic characteristics specifically associated with MTLE-HS. METHODS: Epidemiological data and clinical and diagnostic features were compared between patients with MTLE-HS and TLE patients without HS. When appropriate, data were compared with healthy controls. RESULTS: Fifty-six (26%) patients were diagnosed with MTLE-HS and 162 (74%) with other TLE. Age at epilepsy onset was lower in patients with MTLE-HS (P = 0.003) than in TLE patients without HS. Incidence of simple partial seizures was higher in the MTLE-HS group (P = 0.006), as were complex partial seizures (P = 0.001), ictal psychiatric symptoms (P = 0.015), and autonomic symptoms (P < 0.001). Interictal psychiatric symptoms, including depression, were less frequent in MTLE-HS (P = 0.043). MTLE-HS patients had a higher incidence of childhood febrile seizures (FS; P = 0.043) than TLE patients without HS. In contrast, the former group had the lower frequency of first-grade family members with childhood FS (P = 0.019). CONCLUSIONS: We identified phenotypic characteristics that distinguish MTLE-HS from other types of TLE. These characteristics will be important in diagnostics, treatment, and determination of prognosis, and provide a basis for future phenotype-genotype studies.

Differential effect of alpha-syntrophin knockout on aquaporin-4 and Kir4.1 expression in retinal macroglial cells in mice.

Aquaporin-4 water channels and the inwardly rectifying potassium channels Kir4.1 are coexpressed in a highly polarized manner at the perivascular and subvitreal endfeet of retinal Müller cells and astrocytes. The present study was aimed at resolving the anchoring mechanisms responsible for the coexpression of these molecules. Both aquaporin-4 and Kir4.1 contain PDZ-domain binding motifs at their C-termini and it was recently shown that mice with targeted disruption of the dystrophin gene display altered distribution of aquaporin-4 and Kir4.1 in the retina. To test our hypothesis that alpha-syntrophin (a PDZ-domain containing protein of the dystrophin associated protein complex) is involved in aquaporin-4 and Kir4.1 anchoring in retinal cells, we studied the expression pattern of these molecules in alpha-syntrophin null mice. Judged by quantitative immunogold cytochemistry, deletion of the alpha-syntrophin gene causes a partial loss (by 70%) of aquaporin-4 labeling at astrocyte and Müller cell endfeet but no decrease in Kir4.1 labeling at these sites. These findings suggest that alpha-syntrophin is not involved in the anchoring of Kir4.1 and only partly responsible for the anchoring of aquaporin-4 in retinal endfeet membranes. Furthermore we show that wild type and alpha-syntrophin null mice exhibit strong beta1 syntrophin labeling at perivascular and subvitreal Müller cell endfeet, raising the possibility that beta1 syntrophin might be involved in the anchoring of Kir4.1 and the alpha-syntrophin independent pool of aquaporin-4.

Aquaporin-4 in the central nervous system: cellular and subcellular distribution and coexpression with KIR4.1.

Aquaporin-4 (AQP4) is the predominant water channel in the neuropil of the central nervous system. It is expressed primarily in astrocytes, but also occurs in ependymocytes and endothelial cells. A striking feature of AQP4 expression is its polarized distribution in brain astrocytes and retinal Muller cells. Thus, immunogold analyses have revealed an enrichment of AQP4 in endfeet membranes in contact with brain microvessels or subarachnoidal space and a low but significant concentration in non-endfeet membranes, including those astrocyte membranes that ensheath glutamate synapses. The subcellular compartmentation of AQP4 mimics that of the potassium channel Kir4.1, which is implicated in spatial buffering of K(+). We propose that AQP4 works in concert with Kir4.1 and the electrogenic bicarbonate transporter NBC and that water flux through AQP4 contributes to the activity dependent volume changes of the extracellular space. Such volume changes are important as they affect the extracellular solute concentrations and electrical fields, and hence neuronal excitability. We conclude that AQP4-mediated water flux represents an integral element of brain volume and ion homeostasis.

High-resolution immunogold cytochemistry indicates that AQP4 is concentrated along the basal membrane of parietal cell in rat stomach.

Gastric parietal cells secrete hydrochloric acid in stomach. Because the secreted HCl solution is isotonic with the plasma fluid, it should accompany the water transport across the membranes of parietal cells. Aquaporins (AQPs) are water channel proteins that play the central role in the cellular handling of water in various mammalian tissues. Using immunocytochemistry, we found that AQP4 was expressed only in parietal cells of rat gastric mucosa. Immunogold electron microscopy study further demonstrated that AQP4 was mostly localized at the basal membrane of parietal cells. In the basal membrane, AQP4 was prominently enriched on the portion contacting with the basement membrane surrounding gastric glands. These results suggest that the contact between basement membrane and basal membrane may generate the signal involved in the targeting of AQP4 in gastric parietal cells.

Ontogeny of water transport in rat brain: postnatal expression of the aquaporin-4 water channel.

Brain water transport is poorly understood at the molecular level, and marked changes occur during brain development. As the aquaporin-4 (AQP4) water channel protein is abundant in brain, the expression levels and subcellular distribution of this protein were examined during postnatal development. This study focused on the cerebellum, which showed the same pattern of AQP4 development as the rest of the brain. Semiquantitative immunoblotting revealed very low levels of AQP4 in the first postnatal week. A pronounced increase was noted in the second week, from 2% of adult level at postnatal day 7 (PN7) to 25% at PN14. At PN1 and PN3 immunofluorescence microscopy revealed weak labelling, mainly in radial processes (Bergmann fibres) and at the pial surface. Between PN7 and PN14 the labelling underneath the pia showed a strong increase, and immunoreactivity also appeared around blood vessels throughout the cerebellum. High-resolution immunogold electron microscopy revealed that the subpial and perivascular labelling was restricted to glial end feet, notably to those plasma membrane domains that were apposed to the basal laminae. At no stage was there any evidence of neuronal AQP4 labelling, and AQP1, -2, -3 and -5 proteins were not detected in the neuropil. Riboprobes to AQP4 mRNA produced a particularly strong in situ hybridization signal in glial cells between PN7 and PN14, corresponding to the stage of the most rapid increase of AQP4 protein. The time course and pattern of AQP4 expression suggests that this aquaporin plays an important role in brain water and K+ homeostasis from the second week of development.

Cellular and subcellular expression of monocarboxylate transporters in the pigment epithelium and retina of the rat.

The cellular and subcellular expression of the monocarboxylate transporters MCT1, MCT2 and MCT4 [corresponding to MCT3 of Price N. T. et al. (1998) Biochem. J. 329, 321-328] were investigated in the pigment epithelium and outer retina of rats. Immunofluorescence and postembedding immunogold analyses revealed strong MCT1 labelling in the apical membrane of the pigment epithelial and no detectable signal in the basolateral membrane. In contrast, antibodies to the glucose transporter GLUT1 produced intense labelling in both membranes. Neither MCT1 nor GLUT1 was enriched in intracellular compartments. The monocarboxylate transporter MCT4 was very weakly expressed in the retinal pigment epithelium of adult animals, but occurred at higher concentrations at this site in 14-day-old rats. However, even at the latter stage, the immunolabelling of MCT4 was weak compared to that of MCT1. In the neural retina, the data were consistent with a predominant glial localization of MCT1. Specifically, immunogold particles signalling MCT1 occurred in Müller cell microvilli and in the velate processes between the photoreceptors. No labelling was obtained with antibodies to MCT2. Taken together with previous biochemical analyses, the present findings indicate that MCT1 is involved in the outward transport of lactate through the retinal pigment epithelial cells, and in the transfer of lactate between Müller cells and photoreceptors.

Immunogold evidence suggests that coupling of K+ siphoning and water transport in rat retinal Müller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains.

Postembedding immunogold labeling was used to examine the subcellular distribution of the inwardly rectifying K+ channel Kir4.1 in rat retinal Müller cells and to compare this with the distribution of the water channel aquaporin-4 (AQP4). The quantitative analysis suggested that both molecules are enriched in those plasma membrane domains that face the vitreous body and blood vessels. In addition, Kir4. 1, but not AQP4, was concentrated in the basal approximately 300-400 nm of the Müller cell microvilli. These data indicate that AQP4 may mediate the water flux known to be associated with K+ siphoning in the retina. By its highly differentiated distribution of AQP4, the Müller cell may be able to direct the water flux to select extracellular compartments while protecting others (the subretinal space) from inappropriate volume changes. The identification of specialized membrane domains with high Kir4.1 expression provides a morphological correlate for the heterogeneous K+ conductance along the Müller cell surface.

Subcellular compartmentation of glutathione and glutathione precursors. A high resolution immunogold analysis of the outer retina of guinea pig.

Selective antibodies were used to assess the cellular and subcellular localization of glutathione, and the glutathione precursors gamma-glutamylcysteine, glutamate, and cysteine, in neuronal (photoreceptors) and non-neuronal (pigment epithelial cells and Müller cells) cell types in the outer retina of the guinea pig. In each cell type the highest level of glutathione immunoreactivity occurred in the mitochondria. The labeling density in the cytoplasmic matrix was higher (and the mitochondrial-cytoplasmic gold particle ratio lower) in pigment epithelial cells than in Müller cells and photoreceptors. The latter two cell types showed a mitochondrial-cytoplasmic gold particle ratio of 15.5 and 21.7, respectively. In contrast to glutathione, gamma-glutamylcysteine seemed to be enriched in the cytoplasmic matrix relative to the mitochondria. The immunogold labeling for this dipeptide was stronger in the pigment epithelial cells than in Müller cells and photoreceptors. Glutamate immunoreactivity was high in photoreceptors, intermediate in pigment epithelial cells, and low in Müller cells, while the cysteine immunogold signal was low in each cell type and cell compartment. The present results suggest that glutathione is concentrated in mitochondria but to different degrees in different cells. The low mitochondrial content of gamma-glutamylcysteine (the direct precursor of glutathione) is consistent with biochemical data indicating that glutathione is synthesized extramitochondrially and transported into the mitochondrial matrix. Judged from the immunocytochemical data, cysteine may be a rate-limiting factor in glutathione synthesis in each cell type while glutamate can be rate limiting only in Müller cells.

Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye.

Multiple physiological fluid movements are involved in vision. Here we define the cellular and subcellular sites of aquaporin (AQP) water transport proteins in human and rat eyes by immunoblotting, high-resolution immunocytochemistry, and immunoelectron microscopy. AQP3 is abundant in bulbar conjunctival epithelium and glands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering the trabecular meshwork and Schlemm's canal. Although AQP1 is plentiful in ciliary nonpigmented epithelium, it is not present in ciliary pigmented epithelium. Posterior and anterior epithelium of the iris and anterior lens epithelium also contain significant amounts of AQP1, but AQP0 (major intrinsic protein of the lens) is expressed in lens fiber cells. Retinal Müller cells and astrocytes exhibit notable concentrations of AQP4, whereas neurons and retinal pigment epithelium do not display aquaporin immunolabeling. These studies demonstrate selective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocular epithelia, predicting specific roles for each in the complex network through which water movements occur in the eye.

Aquaporin-4 water channel protein in the rat retina and optic nerve: polarized expression in Müller cells and fibrous astrocytes.

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 MHller cells in the retina and fibrous astrocytes in the optic nerve. A quantitative immunogold analysis of the MHller 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 MHller 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.

Select types of supporting cell in the inner ear express aquaporin-4 water channel protein.

Aquaporins (AQPs) confer a high water permeability on cell membranes and play important parts in secretory and absorptive epithelia in kidney and other organs. Here we investigate whether AQPs are expressed in the sensory epithelia of the inner ear, where a precise volume regulation is crucial. By use of specific antibodies it was found that the inner ear contains AQP1 and 4 while being devoid of detectable levels of AQP2, 3 or 5. Immunofluorescence and postembedding immunogold labelling revealed a strictly non-epithelial distribution of AQP1, confirming previous data. In contrast, AQP4 protein and mRNA (visualized by in situ hybridization) were concentrated in select types of supporting cell, including Hensen's cells and inner sulcus cells. Immunogold particles signalling AQP4 were confined to the basolateral plasma membrane of Hensen's cells and to the basal plasma membrane of Claudius cells and inner sulcus cells. AQP4 was also found in supporting cells of the vestibular end organs, but was absent from transitional epithelial cells and dark cells. Strong labelling for AQP4 and AQP4-mRNA was associated with the central part of the cochlear and vestibular nerves. Hair cells were consistently unlabelled. Our findings indicate that AQP4 may facilitate osmotically driven water fluxes in the sensory epithelia of the inner ear and thus contribute to the volume and ion homeostasis at these sites.

Cellular and subcellular expression of the monocarboxylate transporter MCT1 in rat heart. A high-resolution immunogold analysis.

An antibody to the C-terminus of the monocarboxylate transporter MCT1 was used to study the precise cellular and subcellular distribution of this transporter in rat heart. Postembedding immunogold procedures revealed that the labeling in the heart was restricted to cardiomyocytes and concentrated along the plasma membrane, including the transverse tubules. Gold particles occurred with highest densities in intercalated disks, where they avoided desmosomes and gap junctions. Labeling was also associated with plasmalemmal invaginations having ultrastructural features typical of caveolae. Internal membrane compartments were unlabeled. Quantitative analyses following postembedding labeling showed that the distribution of gold particles across the plasma membrane was nearly symmetrical, indicating that the C-terminus of the transporter is situated very close to the cell membrane. In preembedding immunogold experiments, the gold particles were localized at the external aspect of the plasma membrane, suggesting that the C-terminus is extracellular. From the present data, it can be concluded that even under basal conditions the majority of the MCT1 molecules in heart is present in the myocyte plasma membrane, implying that there is a constitutive functional expression of this transporter. It follows that the increased transmembrane flux of lactate during exercise or in pathological conditions such as ischemia must be a result of altered substrate gradients rather than of translocation of MCT1 molecules to the plasma membrane.

Molecular organization of cerebellar glutamate synapses.

The organization of key molecules at glutamatergic synapses in the rat cerebellar cortex as analyzed by high resolution immunocytochemical techniques using gold particles as markers. The distinct compartmentation of glutamate and glutamine was consistent with biochemical data indicating an active role of glia in the removal of released glutamate and in the supply of glutamine for de novo synthesis of transmitter glutamate. The presence in glial cells of two different glutamate transporters, GLT1 and GLAST, provided further support of this concept. Both transporters were selectively expressed in glial membranes and occurred at higher densities in glial processes surrounding parallel fiber synapses with spines than in glial processes associated with parallel fiber synapses with dendritic shafts. At the former type of synapse, gold particles signalling GLT1 and GLAST could be found within a few nanometers of the postsynaptic density. The rat cerebellum also contains a homologue (rEAAC1) of the glutamate transporter EAAC1, originally cloned from rabbit, mRNA encoding this transporter was restricted to neurons. The exact localization of the rEAAC1 transporter molecules at cerebellar synapses remains to be determined but immunocytochemical and physiological data from other laboratories suggest that they may be preferentially expressed in postsynaptic membranes. Gold particles representing immunoreactivity for the AMPA receptor subunits GluR2/3 were found along the entire mediolateral extent of the postsynaptic specialization of parallel fiber synapses and were rarely encountered at non-synaptic membranes. The present data show that molecules engaged in signalling at cerebellar glutamatergic synapses are precisely organized, consistent with the requirements for rapid signal transmission and efficient removal and recycling of transmitter.

Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain.

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.

Neuronal and glial handling of glutamate and glutamine during hypoosmotic stress: a biochemical and quantitative immunocytochemical analysis using the rat cerebellum as a model.

Biochemical and immunocytochemical analyses were performed to resolve how glutamate and glutamine are handled in rat cerebellar cortex in acute hypoosmotic stress. Rats were subjected to a 15-20% reduction in plasma osmolality by intraperitoneal injection of distilled water and then perfusion fixed after 4 or 8 h survival. Some rats in the latter group had their plasma isoosmolality restored by injections of hypertonic saline 4 h prior to perfusion. Water loading caused a pronounced increase in the tissue level of glutamine and an equimolar decrease in the level of glutamate after 4 h survival. The increase in glutamine was transient, as judged by analyses at 8 h survival. Light microscopic immunocytochemistry revealed a pronounced enhancement of the glutamine immunolabelling of glial cells (Golgi epithelial cells and astrocytes), including their perivascular end feet, and quantitative immunogold analyses at the electron microscopic level showed that this enhancement reflected a 50% increase in the intracellular concentration of fixed glutamine. Since water loading was associated with glial swelling this change corresponded to a several-fold increase in the glial content of glutamine. There was a modest reduction in the overall staining intensity for glutamate. The biochemical and immunocytochemical changes were reversed upon restoration of plasma osmolality by hypertonic saline. These findings suggest that hypoosmotic stress causes an increased conversion of glutamate to glutamine in glial cells and that the latter amino acid is subsequently lost from the tissue. The flux of glutamate carbon skeletons through the glutamine synthetase pathway in glia, prior to an efflux to the systemic circulation, may explain how glutamate, and excitatory transmitter and potential toxin, can be used as an organic osmolyte in brain tissue.

Light- and electronmicroscopic distribution of taurine, an organic osmolyte, in rat renal tubule cells.

Several lines of evidence suggest that taurine acts as an organic osmolyte in the kidney. We investigated the cellular and subcellular distribution of this amino acid in rat renal tubule cells. Semi- and ultrathin sections of plastic-embedded rat kidney were incubated with an antiserum against conjugated taurine, using peroxidase-antiperoxidase and immunogold procedures, respectively. Extensive control tests confirmed the selectivity of the antiserum. Our immunocytochemical preparations revealed a highly differentiated labeling pattern. Strong labeling (judged visually or by computer-aided calculation of gold particle densities) was found in collecting duct cells throughout cortex and medulla, in proximal straight tubule cells, and in cells of the descending thin limbs of Henle's loop. Intermediate gold particle densities occurred in proximal convoluted tubule cells and intercalated cells of the collecting ducts (the gold particle in the latter being 30% of that in the collecting duct cells). The distal convoluted tubules, and thick and thin ascending limbs were almost immunonegative. It cannot be excluded that the proportion of free taurine that is retained by the fixative varies somewhat among the different cell types. Yet the highly differentiated labeling pattern that was obtained suggests that taurine is heterogeneously distributed among different populations of tubule cells, and that its level varies substantially even among cells that are exposed to the same osmotic stress.