Rune Enger

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.

Water entry into astrocytes during brain edema formation

The process of brain edema formation has been studied extensively at the macroscopic level. In contrast, little is known about water fluxes and volume changes at the cellular level in the initial phase of brain edema. Insight in these “microscopic” events could pave the way for more efficient prevention and therapy. Here, we report measurements of brain cell volume responses recorded in vivo in a model of systemic hyponatremia. Transgenic mice expressing fluorescent proteins in astrocytes were subjected to hypo‐osmotic stress and two photon laser scanning microscopy. Volume measurements of glial cells in the cerebellum and the visual cortex indicate that individual astrocytes undergo a position‐dependent increase in cell volume by a factor of two or more during edema formation. Our data are the first to show that volume changes can be monitored at the cellular level in vivo and demonstrate that astrocytes are sites of water entry in the initial phase of brain edema formation. The uptake of water in astrocytes is likely to reflect the strong expression of aquaporin‐4 in these cells.

Dynamics of Ionic Shifts in Cortical Spreading Depression

Cortical spreading depression is a slowly propagating wave of near-complete depolarization of brain cells followed by temporary suppression of neuronal activity. Accumulating evidence indicates that cortical spreading depression underlies the migraine aura and that similar waves promote tissue damage in stroke, trauma, and hemorrhage. Cortical spreading depression is characterized by neuronal swelling, profound elevation of extracellular potassium and glutamate, multiphasic blood flow changes, and drop in tissue oxygen tension. The slow speed of the cortical spreading depression wave implies that it is mediated by diffusion of a chemical substance, yet the identity of this substance and the pathway it follows are unknown. Intercellular spread between gap junction-coupled neurons or glial cells and interstitial diffusion of K+ or glutamate have been proposed. Here we use extracellular direct current potential recordings, K+-sensitive microelectrodes, and 2-photon imaging with ultrasensitive Ca2+ and glutamate fluorescent probes to elucidate the spatiotemporal dynamics of ionic shifts associated with the propagation of cortical spreading depression in the visual cortex of adult living mice. Our data argue against intercellular spread of Ca2+ carrying the cortical spreading depression wavefront and are in favor of interstitial K+ diffusion, rather than glutamate diffusion, as the leading event in cortical spreading depression.

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.

Stimulation-Evoked Ca2+ Signals in Astrocytic Processes at Hippocampal CA3–CA1 Synapses of Adult Mice Are Modulated by Glutamate and ATP

To date, it has been difficult to reveal physiological Ca2+ events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca2+ signals at glutamatergic synapses of adult mice. We stimulated the Schaffer collateral/commissural fibers in acute hippocampal slices from adult mice transduced with the genetically encoded Ca2+ indicator GCaMP5E driven by the glial fibrillary acidic protein promoter. Two-photon imaging revealed global stimulation-evoked astrocytic Ca2+ signals with distinct latencies, rise rates, and amplitudes in fine processes and somata. Specifically, the Ca2+ signals in the processes were faster and of higher amplitude than those in the somata. A combination of P2 purinergic and group I/II metabotropic glutamate receptor (mGluR) antagonists reduced the amplitude of the Ca2+ transients by 30–40% in both astrocytic compartments. Blockage of the mGluRs alone only modestly reduced the magnitude of the stimulation-evoked Ca2+ signals in processes and failed to affect the somatic Ca2+ response. Local application of group I or I/II mGluR agonists or adenosine triphosphate (ATP) elicited global astrocytic Ca2+ signals that mimicked the stimulation-evoked astrocytic Ca2+ responses. We conclude that stimulation-evoked Ca2+ signals in astrocytic processes at CA3–CA1 synapses of adult mice (1) differ from those in astrocytic somata and (2) are modulated by glutamate and ATP.

Molecular scaffolds underpinning macroglial polarization: an analysis of retinal Müller cells and brain astrocytes in mouse.

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 Alzheimers 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. In addition, 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.

Human and mouse cortical astrocytes differ in aquaporin-4 polarization toward microvessels

Aquaporin‐4 (AQP4), the predominant water channel in the brain, is expressed in astrocytes and ependymal cells. In rodents AQP4 is highly polarized to perivascular astrocytic endfeet and loss of AQP4 polarization is associated with disease. The present study was undertaken to compare the expression pattern of AQP4 in human and mouse cortical astrocytes. Cortical tissue specimens were sampled from 11 individuals undergoing neurosurgery wherein brain tissue was removed as part of the procedure, and compared with cortical tissue from 5 adult wild‐type mice processed similarly. The tissue samples were immersion‐fixed and prepared for AQP4 immunogold electron microscopy, allowing quantitative assessment of AQP4s subcellular distribution. In mouse we found that AQP4 water channels were prominently clustered around vessels, being 5 to 10‐fold more abundant in astrocytic endfoot membranes facing the capillary endothelium than in parenchymal astrocytic membranes. In contrast, AQP4 was markedly less polarized in human astrocytes, being only two to three‐fold enriched in astrocytic endfoot membranes adjacent to capillaries. The lower degree of AQP4 polarization in human subjects (1/3 of that in mice) was mainly due to higher AQP4 expression in parenchymal astrocytic membranes. We conclude that there are hitherto unrecognized species differences in AQP4 polarization toward microvessels in the cerebral cortex.

Augmentation of Ca2+ signaling in astrocytic endfeet in the latent phase of temporal lobe epilepsy

Astrocytic endfeet are specialized cell compartments whose important homeostatic roles depend on their enrichment of water and ion channels anchored by the dystrophin associated protein complex (DAPC). This protein complex is known to disassemble in patients with mesial temporal lobe epilepsy and in the latent phase of experimental epilepsies. The mechanistic underpinning of this disassembly is an obvious target of future therapies, but remains unresolved. Here we show in a kainate model of temporal lobe epilepsy that astrocytic endfeet display an enhanced stimulation-evoked Ca2+ signal that outlast the Ca2+ signal in the cell bodies. While the amplitude of this Ca2+ signal is reduced following group I/II metabotropic receptor (mGluR) blockade, the duration is sustained. Based on previous studies it has been hypothesized that the molecular disassembly in astrocytic endfeet is caused by dystrophin cleavage mediated by Ca2+ dependent proteases. Using a newly developed genetically encoded Ca2+ sensor, the present study bolsters this hypothesis by demonstrating long-lasting, enhanced stimulation-evoked Ca2+ signals in astrocytic endfeet.

Deletion of Aquaporin-4 curtails extracellular glutamate elevation in cortical spreading depression in awake mice

Abstract Cortical spreading depression (CSD) is a phenomenon that challenges the homeostatic mechanisms on which normal brain function so critically depends. Analyzing the sequence of events in CSD holds the potential of providing new insight in the physiological processes underlying normal brain function as well as the pathophysiology of neurological conditions characterized by ionic dyshomeostasis. Here, we have studied the sequential progression of CSD in awake wild‐type mice and in mice lacking aquaporin‐4 (AQP4) or inositol 1,4,5‐triphosphate type 2 receptor (IP3R2). By the use of a novel combination of genetically encoded sensors that a novel combination ‐ an unprecedented temporal and spatial resolution, we show that CSD leads to brisk Ca2+ signals in astrocytes and that the duration of these Ca2+ signals is shortened in the absence of AQP4 but not in the absence of IP3R2. The decrease of the astrocytic, AQP4‐dependent Ca2+ signals, coincides in time and space with a decrease in the duration of extracellular glutamate overflow but not with the initial peak of the glutamate release suggesting that in CSD, extracellular glutamate accumulation is extended through AQP4‐dependent glutamate release from astrocytes. The present data point to a salient glial contribution to CSD and identify AQP4 as a new target for therapy.

Automated gold particle quantification of immunogold labeled micrographs

BACKGROUND Immunogold cytochemistry is the method of choice for precise localization of antigens on a subcellular scale. The process of immunogold quantification in electron micrographs is laborious, especially for proteins with a dense distribution pattern. NEW METHODS Here I present a MATLAB based toolbox that is optimized for a typical immunogold analysis workflow. It combines automatic detection of gold particles through a multi-threshold algorithm with manual segmentation of cell membranes and regions of interests. RESULTS The automated particle detection algorithm was applied to a typical immunogold dataset of neural tissue, and was able to detect particles with a high degree of precision. Without manual correction, the algorithm detected 97% of all gold particles, with merely a 0.1% false-positive rate. COMPARISONS WITH EXISTING METHOD(S) To my knowledge, this is the first free and publicly available software custom made for immunogold analyses. The proposed particle detection method compares favorably to previously published algorithms. CONCLUSIONS The software presented here will be valuable tool for researchers in neuroscience working with immunogold cytochemistry.