Øivind Skare

Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema

Aquaporin-4 (AQP4) is a primary influx route for water during brain edema formation. Here, we provide evidence that brain swelling triggers Ca2+ signaling in astrocytes and that deletion of the Aqp4 gene markedly interferes with these events. Using in vivo two-photon imaging, we show that hypoosmotic stress (20% reduction in osmolarity) initiates astrocytic Ca2+ spikes and that deletion of Aqp4 reduces these signals. The Ca2+ signals are partly dependent on activation of P2 purinergic receptors, which was judged from the effects of appropriate antagonists applied to cortical slices. Supporting the involvement of purinergic signaling, osmotic stress was found to induce ATP release from cultured astrocytes in an AQP4-dependent manner. Our results suggest that AQP4 not only serves as an influx route for water but also is critical for initiating downstream signaling events that may affect and potentially exacerbate the pathological outcome in clinical conditions associated with brain edema.

Glial-conditional deletion of aquaporin-4 (Aqp4) reduces blood–brain water uptake and confers barrier function on perivascular astrocyte endfeet

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.

Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice

The aquaporin-4 (AQP4) pool in the perivascular astrocyte membranes has been shown to be critically involved in the formation and dissolution of brain edema. Cerebral edema is a major cause of morbidity and mortality in stroke. It is therefore essential to know whether the perivascular pool of AQP4 is up- or down-regulated after an ischemic insult, because such changes would determine the time course of edema formation. Here we demonstrate by quantitative immunogold cytochemistry that the ischemic striatum and neocortex show distinct patterns of AQP4 expression in the reperfusion phase after 90 min of middle cerebral artery occlusion. The striatal core displays a loss of perivascular AQP4 at 24 hr of reperfusion with no sign of subsequent recovery. The most affected part of the cortex also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the striatal core, but it shows a partial recovery toward 72 hr of reperfusion. By freeze fracture we show that the loss of perivascular AQP4 is associated with the disappearance of the square lattices of particles that normally are distinct features of the perivascular astrocyte membrane. The cortical border zone differs from the central part of the ischemic lesion by showing no loss of perivascular AQP4 at 24 hr of reperfusion but rather a slight increase. These data indicate that the size of the AQP4 pool that controls the exchange of fluid between brain and blood during edema formation and dissolution is subject to large and region-specific changes in the reperfusion phase.

Free School Fruit--sustained effect three years later.

BackgroundNorwegian children consume less fruit and vegetables (FV) than recommended. In order to increase the intake, a School Fruit subscription programme is now offered to all Norwegian elementary and junior high schools. This programme has limited effect due to low participation by schools and pupils. However, recent evaluations of the programme offered for free have reported good effects in increasing FV intake. The purpose of the present study is to evaluate the long term effects of the Norwegian School Fruit programme, provided at no-cost to the pupils, three years after it was provided for free.MethodsA total of 1950 (85%) 6th and 7th grade pupils from 38 Norwegian elementary schools participated in the project. Nine schools were selected as intervention schools and participated for free in the Norwegian School Fruit programme for a school year (October 2001 until June 2002). A baseline questionnaire survey was conducted in September 2001, and follow-up surveys were conducted in May 2002 and May 2005. FV intake was assessed by a written 24-h recall (reporting FV intake at school and FV intake all day), and by four food frequency questions (reporting usual FV intake). Data were analysed by a linear mixed model for repeated measures.ResultsThe pupils in the free fruit group increased their FV intake compared to pupils in the control group as a result of the intervention. Some of the effect was sustained three years later. The estimated long-term effects for FV all day were 0.38 and 0.44 portion/day for boys and girls, respectively.ConclusionThe results show long-term effects of a free school fruit programme.

Pairwise likelihood inference in spatial generalized linear mixed models

Spatial generalized linear mixed models are flexible models for a variety of applications, where spatially dependent and non-Gaussian random variables are observed. The focus is inference in spatial generalized linear mixed models for large data sets. Maximum likelihood or Bayesian Markov chain Monte Carlo approaches may in such cases be computationally very slow or even prohibitive. Alternatively, one may consider a composite likelihood, which is the product of likelihoods of subsets of data. In particular, a composite likelihood based on pairs of observations is adopted. In order to maximize the pairwise likelihood, a new expectation-maximization-type algorithm which uses numerical quadrature is introduced. The method is illustrated on simulated data and on data from air pollution effects for fish populations in Norwegian lakes. A comparison with alternative methods is given. The proposed algorithm is found to give reasonable parameter estimates and to be computationally efficient.

Expression of glutamine synthetase and glutamate dehydrogenase in the latent phase and chronic phase in the kainate model of temporal lobe epilepsy.

It has been suggested that astrocytic glutamate release or perturbed glutamate metabolism contributes to the proneness to epileptic seizures. Here we investigated whether astrocytic contents of the major glutamate degrading enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) decreases on moving from the latent phase (prior to seizures) to the chronic phase (after onset of seizures) in the kainate (KA) model of temporal lobe epilepsy. Western blotting and immunogold analysis of hippocampal formation indicated similar levels of GDH in the latent and chronic phases of KA injected rats and in corresponding controls. In contrast, the level of GS was increased in the latent phase compared with controls, as assessed by Western blots of whole hippocampal formation and subregions. The increase in GS paralleled that of glial fibrillary acidic protein (GFAP). Compared with the latent phase, the chronic phase revealed a lower level of GS (approaching control levels) but an unchanged GFAP content. The decrease in GS from latent to chronic phase was significant in whole hippocampal formation, dentate gyrus and CA3. It is concluded that kainate treated rats show an initial increase in GS, pari passu with the increase in GFAP, and a secondary decrease in GS that is not accompanied by a similar loss of GFAP. In a situation where glutamate catabolism is in high demand the secondary reduction in GS level may be sufficient to contribute to the seizure proneness that develops between the latent and chronic phases.

Mislocalization of AQP4 precedes chronic seizures in the kainate model of temporal lobe epilepsy

It has been suggested that loss of the astrocytic water channel aquaporin-4 (AQP4) from perivascular endfeet in sclerotic hippocampi contributes to increased seizure propensity in human mesial temporal lobe epilepsy (MTLE). Whether this loss occurs prior to or as a consequence of epilepsy development remains to be resolved. In the present study, we investigated whether the expression and distribution of AQP4 was altered prior to (i.e., in the latent phase) or after the onset of chronic epileptic seizures (i.e., in the chronic phase) in the kainate (KA) model of MTLE. Immunogold electron microscopic analysis revealed that AQP4 density in adluminal endfoot membranes was reduced in KA treated rats already in the latent phase, while the AQP4 density in the abluminal endfoot membrane was stable or slightly increased. The decrease in adluminal AQP4 immunogold labeling was accompanied by a reduction in the density of AQP4s anchoring protein alpha-syntrophin. The latent and chronic phases were associated with an upregulation of the M1 isoform of AQP4, as judged by semi-quantitative Western blot analysis. Taken together, the findings in this model suggest that a mislocalization of AQP4--reflecting a loss of astrocyte polarization--is an integral part of the epileptogenic process.

Deletion of aquaporin-4 changes the perivascular glial protein scaffold without disrupting the brain endothelial barrier

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.

Protein interacting with C kinase 1 (PICK1) and GluR2 are associated with presynaptic plasma membrane and vesicles in hippocampal excitatory synapses.

AMPA receptors have been identified in different populations of presynaptic terminals and found to be involved in the modulation of neurotransmitter release. The mechanisms that govern the expression of presynaptic AMPA receptors are not known. One possibility is that pre- and postsynaptic AMPA receptors are regulated according to the same principles. To address this hypothesis we investigated whether protein interacting with C kinase 1 (PICK1), known to interact with AMPA receptors postsynaptically, also is expressed presynaptically, together with AMPA receptors. Subfractionation and high-resolution immunogold analyses of the rat hippocampus revealed that GluR2 and PICK1 are enriched postsynaptically, but also in presynaptic membrane compartments, including the active zone and vesicular membranes. PICK1 and GluR2 are associated with the same vesicles, which are immunopositive also for synaptophysin and vesicle-associated membrane protein 2. Based on what is known about the function of PICK1 postsynaptically, the present data suggest that PICK1 is involved in the regulation of presynaptic AMPA receptor trafficking and in determining the size of the AMPA receptor pool that modulates presynaptic glutamate release.

Postembedding Immunogold Cytochemistry of Membrane Molecules and Amino Acid Transmitters in the Central Nervous System

This chapter deals with procedures for postembedding labeling of brain sections embedded in epoxy or methacrylate resins and focuses on protocols that are based on freeze substitution of chemically fixed tissue. When optimized for the target epitope, such protocols offer a high labeling efficiency and allow simultaneous visualization of several antigens by use of different-sized gold particles. Postembedding labeling can be combined with anterograde tracing, permitting the identification of transmitter and postsynaptic receptors of identified axons. By use of tailor-made model systems, antibody selectivity can be monitored in a quantitative manner and under conditions that are representative of the immunocytochemical procedure. Such model systems also allow the generation of calibration curves for assessment of the cellular and subcellular concentration of soluble antigens. When used in conjunction with computer programs for automated acquisition and analysis of gold particles, the postembedding immunogold procedure provides an accurate representation of the cellular and subcellular distribution of proteins and small compounds such as transmitter amino acids. The present chapter provides a quantitative analysis and critical discussion of how changes in incubation parameters influence the labeling intensity. Postembedding immunogold cytochemistry stands out as a powerful technique for analysis of the chemical architecture of the central nervous system and has proved useful for investigating disease processes at the molecular level.