Rune Enger

• Causemann, Marius; Enger, Rune & Rognes, Marie Elisabeth (2026). Stretch and flow at the gliovascular interface: High-fidelity modeling of astrocyte endfeet. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 123(11). doi: 10.1073/pnas.2517059123. Full text in Research Archive
• Bakketun, Cecilie Bugge; Roth, Lena Catherine; Bjørnstad, Daniel Marelius; Ottersen, Ole Petter; Jensen, Vidar & Enger, Rune (2025). The Impact of Aquaporin-4 Deletion on K+-Induced Astrocytic Swelling Depends on K+ Concentration. Glia. ISSN 0894-1491. 74(1), p. 1–13. doi: 10.1002/glia.70086. Full text in Research Archive Show summary

  Astrocytes swell in response to elevations in extracellular K+ concentration. This K+-induced swelling is widely believed to be due to astrocytic K+ uptake, even if the underlying mechanisms are not fully understood. Conflicting results pertaining to the role of the brain water channel AQP4 in K+-induced swelling have been presented. This calls for revisiting the effect of AQP4 on K+-induced astrocytic swelling dynamics. In this study, we performed two-photon microscopy of acute hippocampal slices from wildtype (WT) and Aqp4 −/− mice to assess astrocytic swelling in response to medium high 10 mM and pathologically high 50 mM [K+] solutions. We demonstrate that K+-induced swelling is attenuated in Aqp4 −/− astrocytes exposed to 10 mM [K+]o compared to WT. In slices exposed to 50 mM [K+]o, peak swelling was similar between the two genotypes, whereas the cell volume recovery was more complete in Aqp4 −/− astrocytes. We demonstrate that the two [K+] concentrations elicit fundamentally different astrocytic Ca2+ signaling responses, and that the Ca2+ signaling response differs between the genotypes in the 10 mM [K+]o scenario. Our findings suggest that K+-induced astrocytic swelling has different mechanistic underpinnings, depending on the K+ concentration to which the astrocytes are exposed, and that altered astrocytic Ca2+ signaling is a putative mechanism involved.

• Kristensen, Anders M.; Bordoni, Luca; Nielsen, Marie B.; Faivre, Anna; Kidmose, Hanne & Sardella, Donato [Show all 12 contributors for this article] (2025). In vivo expression of VCAM1 precedes nephron loss following kidney tubular necrosis. Science Advances. 11(43). doi: 10.1126/sciadv.adz5358. Full text in Research Archive Show summary

  Nephron loss is a key event during onset and progression of chronic kidney disease, yet the mechanisms dictating tubule repair versus atrophy remain poorly understood. While fibrosis has been proposed to drive progressive organ damage, antifibrotic therapies have failed in clinical trials. Here, we reveal that tubular vascular cell adhesion molecule 1 (VCAM1) expression precedes nephron loss, fibrosis, and long-term kidney dysfunction. Using serial intravital microscopy in transgenic mice, we track tubulointerstitial remodeling between injured and intact tissue over 3 weeks. VCAM1 is rapidly induced in a distinct subset of injured tubules, preceding atrophy with sustained fibroblast recruitment. However, fibroblasts remain confined to injury sites and do not cause secondary damage in uninjured tubules. Last, in human kidney transplant biopsies, tubular VCAM1 expression, but not kidney injury molecule 1, correlates negatively with early and 12-month graft function, underscoring its potential as a biomarker of adverse outcomes. These findings position VCAM1 as an early indicator of tubular fate and nephron loss.

• Therkelsen, Helena Eide; Enger, Rune; Eide, Per Kristian & Ringstad, Geir (2025). Evidence for cellular and solute passage between the brain and skull bone marrow across meninges: A systematic review. Journal of Cerebral Blood Flow and Metabolism. ISSN 0271-678X. 45(4), p. 581–599. doi: 10.1177/0271678X251316392. Full text in Research Archive Show summary

  A potential two-way passage of cells and substances between the brain and skull bone marrow may open for new insights into neurological disease. The arachnoid membrane was traditionally considered to restrict cells and larger molecules in CSF from entering the dura and bone marrow directly. However, new data on exchange between brain and skull bone marrow have recently emerged. Here, we conducted a systematic literature to answer the question: What is the current evidence regarding the movement of cells and molecules between the brain and skull bone marrow, spanning CSF and meninges? We excluded studies related to head or skull trauma, cranial fractures or defects, cancer invasion, CSF leakage, spontaneous intracranial hypotension, spinal dura mater, and studies solely focusing on meningeal lymphatic vessels or the passage of substances from CSF to meningeal lymphatic vessels. The review identified 16 studies that provide evidence of communication between the brain, meninges and skull bone marrow. Cells (such as B and T cells and neutrophils), bacteria, and substances (tracers, drug compounds) have been reported to pass between the brain and skull bone. However, most studies are performed in rodents, emphasizing the need for translation to humans.

• Bojarskaite, Laura; Nafari, Sahar; Ravnanger, Anne Katrine; Frey, Mina Martine; Skauli, Nadia & Åbjørsbråten, Knut Sindre [Show all 13 contributors for this article] (2024). Role of aquaporin-4 polarization in extracellular solute clearance. Fluids and Barriers of the CNS. 21(1). doi: 10.1186/s12987-024-00527-7. Full text in Research Archive Show summary

  Waste from the brain has been shown to be cleared via the perivascular spaces through the so-called glymphatic system. According to this model the cerebrospinal fluid (CSF) enters the brain in perivascular spaces of arteries, crosses the astrocyte endfoot layer, flows through the parenchyma collecting waste that is subsequently drained along veins. Glymphatic clearance is dependent on astrocytic aquaporin-4 (AQP4) water channels that are highly enriched in the endfeet. Even though the polarized expression of AQP4 in endfeet is thought to be of crucial importance for glymphatic CSF influx, its role in extracellular solute clearance has only been evaluated using non-quantitative fluorescence measurements. Here we have quantitatively evaluated clearance of intrastriatally infused small and large radioactively labeled solutes in mice lacking AQP4 (Aqp4–/–) or lacking the endfoot pool of AQP4 (Snta1–/–). We confirm that Aqp4–/– mice show reduced clearance of both small and large extracellular solutes. Moreover, we find that the Snta1–/– mice have reduced clearance only for the 500 kDa [3H]dextran, but not 0.18 kDa [3H]mannitol suggesting that polarization of AQP4 to the endfeet is primarily important for clearance of large, but not small molecules. Lastly, we observed that clearance of 500 kDa [3H]dextran increased with age in adult mice. Based on our quantitative measurements, we confirm that presence of AQP4 is important for clearance of extracellular solutes, while the perivascular AQP4 localization seems to have a greater impact on clearance of large versus small molecules.

• Bordoni, Luca; Thorén, Anna Elisabeth; Gutiérrez-Jiménez, Eugenio; Åbjørsbråten, Knut Sindre; Bjørnstad, Daniel Marelius & Tang, Wannan [Show all 12 contributors for this article] (2023). Deletion of aquaporin-4 improves capillary blood flow distribution in brain edema. Glia. ISSN 0894-1491. 71(11), p. 2559–2572. doi: 10.1002/glia.24439. Full text in Research Archive Show summary

  Brain edema is a feared complication to disorders and insults affecting the brain. It can be fatal if the increase in intracranial pressure is sufficiently large to cause brain herniation. Moreover, accruing evidence suggests that even slight elevations of intracranial pressure have adverse effects, for instance on brain perfusion. The water channel aquaporin-4 (AQP4), densely expressed in perivascular astrocytic endfeet, plays a key role in brain edema formation. Using two-photon microscopy, we have studied AQP4-mediated swelling of astrocytes affects capillary blood flow and intracranial pressure (ICP) in unanesthetized mice using a mild brain edema model. We found improved regulation of capillary blood flow in mice devoid of AQP4, independently of the severity of ICP increase. Furthermore, we found brisk AQP4-dependent astrocytic Ca2+ signals in perivascular endfeet during edema that may play a role in the perturbed capillary blood flow dynamics. The study suggests that astrocytic endfoot swelling and pathological signaling disrupts microvascular flow regulation during brain edema formation.

• Naik, Aditi; Jensen, Vidar; Bakketun, Cecilie Bugge; Enger, Rune; Hrabetova, Sabina & Hrabe, Jan (2023). BubbleDrive, a low-volume incubation chamber for acute brain slices. Scientific Reports. 13(1). doi: 10.1038/s41598-023-45949-9. Full text in Research Archive Show summary

  Abstract Acute brain slices are a common and useful preparation in experimental neuroscience. A wide range of incubation chambers for brain slices exists but only a few are designed with very low volumes of the bath solution in mind. Such chambers are necessary when high-cost chemicals are to be added to the solution or when small amounts of substances released by the slice are to be collected for analysis. The principal challenge in designing a very low-volume incubation chamber is maintaining good oxygenation and flow without mechanically disturbing or damaging the slices. We designed and validated BubbleDrive, a 3D-printed incubation chamber with a minimum volume of 1.5 mL which can hold up to three coronal mouse slices from one hemisphere. It employs the carbogen gas bubbles to drive the flow circulation in a consistent and reproducible manner, and without disturbing the brain slices. The BubbleDrive design and construction were successfully validated by comparison to a conventional large-volume incubation chamber in several experimental designs involving measurements of extracellular diffusion parameters, the electrophysiology of neuronal and astrocytic networks, and the effectiveness of slice incubation with hyaluronidase enzyme.

• Bojarskaite, Laura; Vallet, Alexandra Honorine Emilie; Bjørnstad, Daniel Marelius; Binder, Kristin Maria Gullestad; Cunen, Celine & Heuser, Kjell [Show all 9 contributors for this article] (2023). Sleep cycle-dependent vascular dynamics in male mice and the predicted effects on perivascular cerebrospinal fluid flow and solute transport. Nature Communications. 14. doi: 10.1038/s41467-023-36643-5. Full text in Research Archive Show summary

  Perivascular spaces are important highways for fluid and solute transport in the brain enabling efficient waste clearance during sleep. However, the underlying mechanisms augmenting perivascular flow in sleep are unknown. Using two-photon imaging of naturally sleeping male mice we demonstrate sleep cycle-dependent vascular dynamics of pial arteries and penetrating arterioles: slow, large-amplitude oscillations in NREM sleep, a vasodilation in REM sleep, and a vasoconstriction upon awakening at the end of a sleep cycle and microarousals in NREM and intermediate sleep. These vascular dynamics are mirrored by changes in the size of the perivascular spaces of the penetrating arterioles: slow fluctuations in NREM sleep, reduction in REM sleep and an enlargement upon awakening after REM sleep and during microarousals in NREM and intermediate sleep. By biomechanical modeling we demonstrate that these sleep cycle-dependent perivascular dynamics likely enhance fluid flow and solute transport in perivascular spaces to levels comparable to cardiac pulsation-driven oscillations.

• Ellingsrud, Ada; Dukefoss, Didrik; Enger, Rune; Halnes, Geir; Pettersen, Klas & Rognes, Marie (2022). Validating a computational framework for ionic electrodiffusion with cortical spreading depression as a case study. eNeuro. 9. Full text in Research Archive
• Åbjørsbråten, Knut Sindre; Skaaraas, Gry Helen Enger Syverstad; Cunen, Celine Marie Løken; Bjørnstad, Daniel Marelius; Binder, Kristin Maria Gullestad & Bojarskaite, Laura [Show all 15 contributors for this article] (2022). Impaired astrocytic Ca2+ signaling in awake-behaving Alzheimer’s disease transgenic mice. eLIFE. doi: 10.7554/eLife.75055. Full text in Research Archive Show summary

  Increased astrocytic Ca2+ signaling has been shown in Alzheimer’s disease mouse models, but to date no reports have characterized behaviorally induced astrocytic Ca2+ signaling in such mice. Here, we employ an event-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake-behaving tg-ArcSwe mice and non-transgenic wildtype littermates while monitoring pupil responses and behavior. We demonstrate an attenuated astrocytic Ca2+ response to locomotion and an uncoupling of pupil responses and astrocytic Ca2+ signaling in 15-month-old plaque-bearing mice. Using the genetically encoded fluorescent norepinephrine sensor GRABNE, we demonstrate a reduced norepinephrine signaling during spontaneous running and startle responses in the transgenic mice, providing a possible mechanistic underpinning of the observed reduced astrocytic Ca2+ responses. Our data points to a dysfunction in the norepinephrine–astrocyte Ca2+ activity axis, which may account for some of the cognitive deficits observed in Alzheimer’s disease.

• Ellingsrud, Ada Johanne; Dukefoss, Didrik Bakke; Enger, Rune; Halnes, Geir; Pettersen, Klas Henning & Rognes, Marie Elisabeth (2022). Validating a Computational Framework for Ionic Electrodiffusion with Cortical Spreading Depression as a Case Study. eNeuro. 9(2). doi: 10.1523/ENEURO.0408-21.2022. Full text in Research Archive Show summary

  Cortical spreading depression (CSD) is a wave of pronounced depolarization of brain tissue accompanied by substantial shifts in ionic concentrations and cellular swelling. Here, we validate a computational framework for modeling electrical potentials, ionic movement, and cellular swelling in brain tissue during CSD. We consider different model variations representing wild-type (WT) or knock-out/knock-down mice and systematically compare the numerical results with reports from a selection of experimental studies. We find that the data for several CSD hallmarks obtained computationally, including wave propagation speed, direct current shift duration, peak in extracellular K+ concentration as well as a pronounced shrinkage of extracellular space (ECS) are well in line with what has previously been observed experimentally. Further, we assess how key model parameters including cellular diffusivity, structural ratios, membrane water and/or K+ permeabilities affect the set of CSD characteristics.

• Bjørnstad, Daniel Marelius; Åbjørsbråten, Knut Sindre; Hennestad, Eivind; Cunen, Celine; Hermansen, Gudmund Horn & Bojarskaite, Laura [Show all 9 contributors for this article] (2021). Begonia - a two-photon imaging analysis pipeline for astrocytic Ca2+ signals. Frontiers in Cellular Neuroscience. 15. doi: 10.3389/fncel.2021.681066. Full text in Research Archive Show summary

  Imaging the intact brain of awake behaving mice without the dampening effects of anesthesia, has revealed an exceedingly rich repertoire of astrocytic Ca 2+ signals. Analyzing and interpreting such complex signals pose many challenges. Traditional analyses of fluorescent changes typically rely on manually outlined static region-of-interests, but such analyses fail to capture the intricate spatiotemporal patterns of astrocytic Ca 2+ dynamics. Moreover, all astrocytic Ca 2+ imaging data obtained from awake behaving mice need to be interpreted in light of the complex behavioral patterns of the animal. Hence processing multimodal data, including animal behavior metrics, stimulation timings, and electrophysiological signals is needed to interpret astrocytic Ca 2+ signals. Managing and incorporating these data types into a coherent analysis pipeline is challenging and time-consuming, especially if research protocols change or new data types are added. Here, we introduce Begonia, a MATLAB-based data management and analysis toolbox tailored for the analyses of astrocytic Ca 2+ signals in conjunction with behavioral data. The analysis suite includes an automatic, event-based algorithm with few input parameters that can capture a high level of spatiotemporal complexity of astrocytic Ca 2+ signals. The toolbox enables the experimentalist to quantify astrocytic Ca 2+ signals in a precise and unbiased way and combine them with other types of time series data.

• Heuser, Kjell & Enger, Rune (2021). Astrocytic Ca2+ Signaling in Epilepsy. Frontiers in Cellular Neuroscience. 15. doi: 10.3389/fncel.2021.695380. Full text in Research Archive Show summary

  Epilepsy is one of the most common neurological disorders – estimated to affect at least 65 million worldwide. Most of the epilepsy research has so far focused on how to dampen neuronal discharges and to explain how changes in intrinsic neuronal activity or network function cause seizures. As a result, pharmacological therapy has largely been limited to symptomatic treatment targeted at neurons. Given the expanding spectrum of functions ascribed to the non-neuronal constituents of the brain, in both physiological brain function and in brain disorders, it is natural to closely consider the roles of astrocytes in epilepsy. It is now widely accepted that astrocytes are key controllers of the composition of the extracellular fluids, and may directly interact with neurons by releasing gliotransmitters. A central tenet is that astrocytic intracellular Ca 2+ signals promote release of such signaling substances, either through synaptic or non-synaptic mechanisms. Accruing evidence suggests that astrocytic Ca 2+ signals play important roles in both seizures and epilepsy, and this review aims to highlight the current knowledge of the roles of this central astrocytic signaling mechanism in ictogenesis and epileptogenesis.

• Ellingsrud, Ada Johanne; Enger, Rune; Dukefoss, Didrik Bakke; Halnes, Geir; Pettersen, Klas Henning & Rognes, Marie Elisabeth (2021). Validating a computational framework for ionic electrodiffusion with cortical spreading depression as a case study. BioRxiv. ISSN 0362-4331. doi: 10.1101/2021.11.29.470301. Full text in Research Archive
• Hagen, Espen; Chambers, Anna; Einevoll, Gaute; Pettersen, Klas Henning; Enger, Rune & Stasik, Alexander Johannes (2021). RippleNet: a Recurrent Neural Network for Sharp Wave Ripple (SPW-R) Detection. Neuroinformatics. ISSN 1539-2791. 19, p. 493–514. doi: 10.1007/s12021-020-09496-2. Full text in Research Archive Show summary

  Hippocampal sharp wave ripples (SPW-R) have been identified as key bio-markers of important brain functions such as memory consolidation and decision making. Understanding their underlying mechanisms in healthy and pathological brain function and behaviour rely on accurate SPW-R detection. In this multidisciplinary study, we propose a novel, self-improving artificial intelligence (AI) detection method in the form of deep Recurrent Neural Networks (RNN) with Long Short-Term memory (LSTM) layers that can learn features of SPW-R events from raw, labeled input data. The approach contrasts conventional routines that typically relies on hand-crafted, heuristic feature extraction and often laborious manual curation. The algorithm is trained using supervised learning on hand-curated data sets with SPW-R events obtained under controlled conditions. The input to the algorithm is the local field potential (LFP), the low-frequency part of extracellularly recorded electric potentials from the CA1 region of the hippocampus. Its output predictions can be interpreted as time-varying probabilities of SPW-R events for the duration of the inputs. A simple thresholding applied to the output probabilities is found to identify times of SPW-R events with high precision. The non-causal, or bidirectional variant of the proposed algorithm demonstrates consistently better accuracy compared to the causal, or unidirectional counterpart. Reference implementations of the algorithm, named ‘RippleNet’, are open source, freely available, and implemented using a common open-source framework for neural networks (tensorflow.keras) and can be easily incorporated into existing data analysis workflows for processing experimental data.

• Bojarskaite, Laura; Bjørnstad, Daniel Marelius; Pettersen, Klas Henning; Cunen, Celine; Hermansen, Gudmund Horn & Åbjørsbråten, Knut Sindre [Show all 12 contributors for this article] (2020). Astrocytic Ca2+ signaling is reduced during sleep and is involved in the regulation of slow wave sleep. Nature Communications. 11. doi: 10.1038/s41467-020-17062-2. Full text in Research Archive Show summary

  Astrocytic Ca2+ signaling has been intensively studied in health and disease but has not been quantified during natural sleep. Here, we employ an activity-based algorithm to assess astrocytic Ca2+ signals in the neocortex of awake and naturally sleeping mice while monitoring neuronal Ca2+ activity, brain rhythms and behavior. We show that astrocytic Ca2+ signals exhibit distinct features across the sleep-wake cycle and are reduced during sleep compared to wakefulness. Moreover, an increase in astrocytic Ca2+ signaling precedes transitions from slow wave sleep to wakefulness, with a peak upon awakening exceeding the levels during whisking and locomotion. Finally, genetic ablation of an important astrocytic Ca2+ signaling pathway impairs slow wave sleep and results in an increased number of microarousals, abnormal brain rhythms, and an increased frequency of slow wave sleep state transitions and sleep spindles. Our findings demonstrate an essential role for astrocytic Ca2+ signaling in regulating slow wave sleep.

• Eide, Per Kristian; Hasan-Olive, Md Mahdi; Hansson, Hans-Arne & Enger, Rune (2020). Increased occurrence of pathological mitochondria in astrocytic perivascular endfoot processes and neurons of idiopathic intracranial hypertension. Journal of Neuroscience Research. ISSN 0360-4012. 99(2), p. 467–480. doi: 10.1002/jnr.24743. Full text in Research Archive Show summary

  Idiopathic intracranial hypertension (IIH) primarily affects fertile, overweight women, and presents with the symptoms of raised intracranial pressure. The etiology is unknown but has been thought to relate to cerebrospinal fluid disturbance or cerebral venous stenosis. We have previously found evidence that IIH is also a disease of the brain parenchyma, evidenced by alterations at the neurogliovascular interface, including astrogliosis, pathological changes in the basement membrane and pericytes, and alterations of perivascular aquaporin-4. The aim of this present electron microscopic study was to examine whether mitochondria phenotype was changed in IIH, particularly focusing on perivascular astrocytic endfeet and neurons (soma and pre- and postsynaptic terminals). Cortical brain biopsies of nine reference individuals and eight IIH patients were analyzed for subcellular distribution and phenotypical features of mitochondria using transmission electron microscopy. We found significantly increased prevalence of pathological mitochondria and reduced number of normal mitochondria in astrocytic endfeet of IIH patients. The degree of astrogliosis correlated negatively with the number of normal mitochondria in astrocytic endfoot processes. Moreover, we found significantly increased number of pathological mitochondria in pre- and postsynaptic neuronal terminals, as well as significantly shortened distance between mitochondria and endoplasmic reticulum contacts. Finally, the length of postsynaptic density, a marker of synaptic strength, was on average reduced in IIH. The present data provide evidence of pathological mitochondria in perivascular astrocytes endfeet and neurons of IIH patients, highlighting that impaired metabolism at the neurogliovascular interface may be a facet of IIH.

• Hagen, Espen; Chambers, Anna; Einevoll, Gaute; Pettersen, Klas Henning; Enger, Rune & Stasik, Alexander Johannes (2020). RippleNet: A Recurrent Neural Network for Sharp Wave Ripple (SPW-R) Detection. BioRxiv. ISSN 0362-4331. doi: 10.1101/2020.05.11.087874. Full text in Research Archive Show summary

  Hippocampal sharp wave ripples (SPW-R) have been identified as key bio-markers of important brain functions such as memory consolidation and decision making. SPW-R detection typically relies on hand-crafted feature extraction, and laborious manual curation is often required. In this multidisciplinary study, we propose a novel, self-improving artificial intelligence (AI) method in the form of deep Recurrent Neural Networks (RNN) with Long Short-Term memory (LSTM) layers that can learn features of SPW-R events from raw, labeled input data. The algorithm is trained using supervised learning on hand-curated data sets with SPW-R events. The input to the algorithm is the local field potential (LFP), the low-frequency part of extracellularly recorded electric potentials from the CA1 region of the hippocampus. The output prediction can be interpreted as the time-varying probability of SPW-R events for the duration of the input. A simple thresholding applied to the output probabilities is found to identify times of events with high precision. The reference implementation of the algorithm, named ‘RippleNet’, is open source, freely available, and implemented using a common open-source framework for neural networks (tensorflow.keras) and can be easily incorporated into existing data analysis workflows for processing experimental data.

• Hasan-Olive, Md Mahdi; Enger, Rune; Hansson, Hans-Arne; Nagelhus, Erlend Arnulf & Eide, Per Kristian (2019). Pathological mitochondria in neurons and perivascular astrocytic endfeet of idiopathic normal pressure hydrocephalus patients. Fluids and Barriers of the CNS. 16:39, p. 1–16. doi: 10.1186/s12987-019-0160-7. Full text in Research Archive Show summary

  Background A growing body of evidence suggests that the accumulation of amyloid-β and tau (HPτ) in the brain of patients with the dementia subtype idiopathic normal pressure hydrocephalus (iNPH) is associated with delayed extravascular clearance of metabolic waste. Whether also clearance of intracellular debris is affected in these patients needs to be examined. Hypothetically, defective extra- and intra-cellular clearance of metabolites may be instrumental in the neurodegeneration and dementia characterizing iNPH. This study explores whether iNPH is associated with altered mitochondria phenotype in neurons and astrocytes. Methods Cortical brain biopsies of 9 reference (REF) individuals and 30 iNPH patients were analyzed for subcellular distribution and morphology of mitochondria using transmission electron microscopy. In neuronal soma of REF and iNPH patients, we identified normal, pathological and clustered mitochondria, mitochondria-endoplasmic reticulum contact sites and autophagic vacuoles. We also differentiated normal and pathological mitochondria in pre- and post-synaptic nerve terminals, as well as in astrocytic endfoot processes towards vessels. Results We found a high prevalence of pathological mitochondria in neuronal soma and pre- and post-synaptic terminals, as well as increased mitochondrial clustering, and altered number of mitochondria-endoplasmic reticulum contact sites in iNPH. Non-fused autophagic vacuoles were more abundant in neuronal soma of iNPH patients, suggestive of cellular clearance failure. Moreover, the length of postsynaptic densities was reduced in iNPH, potentially related to reduced synaptic activity. In astrocytic endfoot processes, we also found increased number, area and area fraction of pathological mitochondria in iNPH patients. The proportion of pathological mitochondria correlated significantly with increasing degree of astrogliosis and reduced perivascular expression of aquaporin-4 (AQP4), assessed by light microscopy immunohistochemistry. Conclusion Our results provide evidence of mitochondrial pathology and signs of impaired cellular clearance in iNPH patients. The results indicate that iNPH is a neurodegenerative disease with close similarity to Alzheimer’s disease.

• Rosic, Brana; Dukefoss, Didrik Bakke; Åbjørsbråten, Knut Sindre; Tang, Wannan; Jensen, Vidar & Ottersen, Ole Petter [Show all 8 contributors for this article] (2019). Aquaporin-4-independent volume dynamics of astroglial endfeet during cortical spreading depression. Glia. ISSN 0894-1491. 67(6), p. 1113–1121. doi: 10.1002/glia.23604. Full text in Research Archive Show summary

  Cortical spreading depression (CSD) is a slowly propagating wave of depolarization of gray matter. This phenomenon is believed to underlie the migraine aura and similar waves of depolarization may exacerbate injury in a number of neurological disease states. CSD is characterized by massive ion dyshomeostasis, cell swelling, and multiphasic blood flow changes. Recently, it was shown that CSD is associated with a closure of the paravascular space (PVS), a proposed exit route for brain interstitial fluid and solutes, including excitatory and inflammatory substances that increase in the wake of CSD. The PVS closure was hypothesized to rely on swelling of astrocytic endfeet due to their high expression of aquaporin‐4 (AQP4) water channels. We investigated whether CSD is associated with swelling of endfeet around penetrating arterioles in the cortex of living mice. Endfoot cross‐sectional area was assessed by two‐photon microscopy of mice expressing enhanced green fluorescent protein in astrocytes and related to the degree of arteriolar constriction. In anesthetized mice CSD triggered pronounced endfoot swelling that was short‐lasting and coincided with the initial arteriolar constriction. Mice lacking AQP4 displayed volume changes of similar magnitude. CSD‐induced endfoot swelling and arteriolar constriction also occurred in awake mice, albeit with faster kinetics than in anesthetized mice. We conclude that swelling of astrocytic endfeet is a robust event in CSD. The early onset and magnitude of the endfoot swelling is such that it may significantly delay perivascular drainage of interstitial solutes in neurological conditions where CSD plays a pathophysiological role.

• Hasan-Olive, Md Mahdi; Hansson, Hans-Arne; Enger, Rune; Nagelhus, Erlend Arnulf & Eide, Per Kristian (2019). Blood-Brain Barrier Dysfunction in Idiopathic Intracranial Hypertension. Journal of Neuropathology and Experimental Neurology. ISSN 0022-3069. 78(9), p. 808–818. doi: 10.1093/jnen/nlz063. Full text in Research Archive
• Eilert-Olsen, Martine; Hjukse, Jarand Berg; Thoren, Anna; Tang, Wannan; Enger, Rune & Jensen, Vidar [Show all 8 contributors for this article] (2019). Astroglial endfeet exhibit distinct Ca2+ signals during hypoosmotic conditions. Glia. ISSN 0894-1491. 67(12), p. 2399–2409. doi: 10.1002/glia.23692. Full text in Research Archive Show summary

  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.

• Hasan-Olive, Md Mahdi; Enger, Rune; Hansson, Hans-Arne; Nagelhus, Erlend Arnulf & Eide, Per Kristian (2018). Loss of perivascular aquaporin-4 in idiopathic normal pressure hydrocephalus. Glia. ISSN 0894-1491. 67(1), p. 91–100. doi: 10.1002/glia.23528. Full text in Research Archive
• Heuser, Kjell; Nome, Cecilie; Pettersen, Klas; Åbjørsbråten, Knut Sindre; Jensen, Vidar & Tang, Wannan [Show all 10 contributors for this article] (2018). Ca2+ signals in astrocytes facilitate spread of epileptiform activity. Cerebral Cortex. ISSN 1047-3211. 28(11), p. 4036–4048. doi: 10.1093/cercor/bhy196. Full text in Research Archive Show summary

  Epileptic seizures are associated with increased astrocytic Ca2+ signaling, but the fine spatiotemporal kinetics of the ictal astrocyte–neuron interplay remains elusive. By using 2-photon imaging of awake head-fixed mice with chronic hippocampal windows we demonstrate that astrocytic Ca2+ signals precede neuronal Ca2+ elevations during the initial bout of kainate-induced seizures. On average, astrocytic Ca2+ elevations preceded neuronal activity in CA1 by about 8 s. In subsequent bouts of epileptic seizures, astrocytes and neurons were activated simultaneously. The initial astrocytic Ca2+ elevation was abolished in mice lacking the type 2 inositol-1,4,5-trisphosphate-receptor (Itpr2−/−). Furthermore, we found that Itpr2−/− mice exhibited 60% less epileptiform activity compared with wild-type mice when assessed by telemetric EEG monitoring. In both genotypes we also demonstrate that spreading depression waves may play a part in seizure termination. Our findings imply a role for astrocytic Ca2+ signals in ictogenesis.

• Enger, Rune (2017). Automated gold particle quantification of immunogold labeled micrographs. Journal of Neuroscience Methods. ISSN 0165-0270. 286, p. 31–37. doi: 10.1016/j.jneumeth.2017.05.018. Full text in Research Archive Show summary

  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.

• Eidsvaag, Vigdis Andersen; Enger, Rune; Hansson, Hans-Arne; Eide, Per Kristian & Nagelhus, Erlend Arnulf (2017). Human and mouse cortical astrocytes differ in aquaporin-4 polarization toward microvessels. Glia. ISSN 0894-1491. 65(6), p. 964–973. doi: 10.1002/glia.23138. Full text in Research Archive Show summary

  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 AQP4's 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.

• Enger, Rune; Dukefoss, Didrik Bakke; Tang, Wannan; Pettersen, Klas; Bjørnstad, Daniel Marelius & Helm, Paul Johannes [Show all 11 contributors for this article] (2016). Deletion of aquaporin-4 curtails extracellular glutamate elevation in cortical spreading depression in awake mice. Cerebral Cortex. ISSN 1047-3211. doi: 10.1093/cercor/bhw359. Full text in Research Archive
• Szokol, Karolina; Heuser, Kjell; Tang, Wannan; Jensen, Vidar; Enger, Rune & Bedner, Peter [Show all 10 contributors for this article] (2015). Augmentation of Ca<sup>2+</sup> signaling in astrocytic endfeet in the latent phase of temporal lobe epilepsy. Frontiers in Cellular Neuroscience. 9. doi: 10.3389/fncel.2015.00049. Full text in Research Archive Show summary

  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 Ca(2+) signal that outlast the Ca(2+) signal in the cell bodies. While the amplitude of this Ca(2+) 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 Ca(2+) dependent proteases. Using a newly developed genetically encoded Ca(2+) sensor, the present study bolsters this hypothesis by demonstrating long-lasting, enhanced stimulation-evoked Ca(2+) signals in astrocytic endfeet.

• Enger, Rune; Tang, Wannan; Vindedal, Gry Fluge; Jensen, Vidar; Helm, Paul Johannes & Sprengel, Rolf [Show all 8 contributors for this article] (2015). Dynamics of ionic shifts in cortical spreading depression. Cerebral Cortex. ISSN 1047-3211. 25(11), p. 4469–4476. doi: 10.1093/cercor/bhv054. Full text in Research Archive Show summary

  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 Ca(2+) 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 Ca(2+) 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.

• Tang, Wannan; Szokol, Karolina; Jensen, Vidar; Enger, Rune; Trivedi, Chintan A. & Hvalby, Øyvind Christian dødsbo [Show all 10 contributors for this article] (2015). Stimulation-evoked Ca2+ signals in astrocytic processes at hippocampal CA3-CA1 synapses of adult mice are modulated by glutamate and ATP. Journal of Neuroscience. ISSN 0270-6474. 35(7), p. 3016–3021. doi: 10.1523/JNEUROSCI.3319-14.2015. Full text in Research Archive Show summary

  To date, it has been difficult to reveal physiological Ca(2+) events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca(2+) 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 Ca(2+) indicator GCaMP5E driven by the glial fibrillary acidic protein promoter. Two-photon imaging revealed global stimulation-evoked astrocytic Ca(2+) signals with distinct latencies, rise rates, and amplitudes in fine processes and somata. Specifically, the Ca(2+) 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 Ca(2+) transients by 30-40% in both astrocytic compartments. Blockage of the mGluRs alone only modestly reduced the magnitude of the stimulation-evoked Ca(2+) signals in processes and failed to affect the somatic Ca(2+) response. Local application of group I or I/II mGluR agonists or adenosine triphosphate (ATP) elicited global astrocytic Ca(2+) signals that mimicked the stimulation-evoked astrocytic Ca(2+) responses. We conclude that stimulation-evoked Ca(2+) 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.

• Enger, Rune; Gundersen, Georg Andreas; Haj-Yasein, Nadia Nabil; Eilert-Olsen, Martine; Thoren, Anna & Vindedal, Gry Fluge [Show all 11 contributors for this article] (2012). Molecular scaffolds underpinning macroglial polarization: An analysis of retinal Muller cells and brain astrocytes in mouse. Glia. ISSN 0894-1491. 60(12), p. 2018–2026. doi: 10.1002/glia.22416. Full text in Research Archive Show summary

  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. 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.

• Eilert-Olsen, Martine; Yasein, Nadia Nabil Haj; Vindedal, Gry Fluge; Enger, Rune; Gundersen, Georg Andreas & Hoddevik, Eystein Hellstrøm [Show all 14 contributors for this article] (2012). Deletion of aquaporin-4 changes the perivascular glial protein scaffold without disrupting the brain endothelial barrier. Glia. ISSN 0894-1491. 60(3), p. 432–440. doi: 10.1002/glia.22277. Full text in Research Archive
• Thrane, Alexander Stanley; Rappold, Philip M; Fujita, Takumi; Torres, Arnulfo; Bekar, Lane K & Takano, Takahiro [Show all 17 contributors for this article] (2011). Critical role of aquaporin-4 (AQP4) in astrocytic Ca2+ signaling events elicited by cerebral edema. Proceedings of the National Academy of Sciences of the United States of America (PNAS). ISSN 0027-8424. 108(2), p. 846–851. doi: 10.1073/pnas.1015217108. Full text in Research Archive
• Nase, Gabriele; Helm, Paul Johannes; Enger, Rune & Ottersen, Ole Petter (2008). Water entry into astrocytes during brain edema formation. Glia. ISSN 0894-1491. 56, p. 895–902. doi: 10.1002/glia.20664. Full text in Research Archive

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• Beckheim, Henrik & Enger, Rune (2024). Søvnforskning og hjernevask. [Internet]. Henrik Beckheim Podcast. Full text in Research Archive
• Enger, Rune (2024). Dynamics of the gliovascular interface across the sleep-wake cycle. Full text in Research Archive
• Enger, Rune (2024). Gliovascular dynamic in sleep. Full text in Research Archive
• Enger, Rune (2024). The role of astrocytes and the gliovascular interface in sleep. Full text in Research Archive
• Naik, Aditi; Jensen, Vidar; Bakketun, Cecilie Bugge; Enger, Rune; Hrabetova, Sabina & Hrabe, Jan (2024). Author Correction: BubbleDrive, a low-volume incubation chamber for acute brain slices (Scientific Reports, (2023), 13, 1, (20005), 10.1038/s41598-023-45949-9). Scientific Reports. 14(1). doi: 10.1038/s41598-024-52441-5. Full text in Research Archive
• Causemann, Marius; Enger, Rune & Rognes, Marie (2023). Computational Modeling of Astrocyte Endfeet: Insights into their Mechanical Buffering Function. Full text in Research Archive Show summary

  Astrocyte endfeet encompass cerebral blood vessels, forming a nearly complete sheet, yet their functional significance remains incompletely understood. Current hypotheses include the facilitation of solute clearance [1] and regulation of arteriole vasodilation and vasoconstriction [2]. Furthermore, the abundance of Aquaporin-4 (AQP4) water channels on the endfoot side facing blood vessels raises questions about their physiological function. Recent imaging studies have demonstrated significant endfoot deformations during pronounced vasomotion during sleep [3], suggesting a potential mechanical buffering function that mitigates the mechanical forces exerted on surrounding brain tissue. In this study, we employ a biomechanical modeling approach, utilizing a geometrically- explicit representation of astrocyte endfeet, interstitial and perivascular spaces, to compute fluid flow and cell deformation in response to vasodilation and constriction. Our model characterizes the intracellular and extracellular domains as porous media separated by a permeable membrane, enabling the prediction of time-evolving extracellular and intracellular fluid flow, hydrodynamic pressure, mechanical stress, and displacement. By applying this model, we examine the impact of vasomotion and perivascular flow on the mechanics of the surrounding tissue and quantitatively compare the hydrodynamical and mechanical effects of varying AQP4 density on astrocyte endfeet. Through our comprehensive model, we aim to gain insights into the physiological role of endfeet and the involvement of AQP4 water channels, with particular emphasis on their potential mechanical buffering function during vasomotion. **References** 1. Abbott, N. Joan, et al. "The role of brain barriers in fluid movement in the CNS: is there a ‘glymphatic’system?." Acta neuropathologica 135 (2018): 387-407., 10.1007/s00401-018-1812-4 2. Haidey, Jordan N., et al. "Astrocytes regulate ultra-slow arteriole oscillations via stretch-mediated TRPV4-COX-1 feedback." Cell reports 36.5 (2021)., 10.1016/j.celrep.2021.109405 3. Bojarskaite, Laura, et al. "Sleep cycle-dependent vascular dynamics in male mice and the predicted effects on perivascular cerebrospinal fluid flow and solute transport." Nature communications 14.1 (2023): 953., 10.1038/s41467-023-36643-5

• Enger, Rune (2023). Frokostmøte om hjernevask. Full text in Research Archive
• Kaalstad, Jørn E. & Enger, Rune (2023). Forskning om hjernen og søvn: Kan hindre utvikling av Alzheimers. [Journal]. Verdens Gang. Full text in Research Archive
• Enger, Rune (2023). Astrocytic calcium signaling in natural sleep and brain state changes. Full text in Research Archive
• Isachsen, Henriette Bertheussen & Enger, Rune (2023). Norsk studie kan bidra til å forebygge Alzheimers: Fant at søvn gir mer effektiv «hjernevask». [Journal]. Dagens Medisin. Full text in Research Archive
• Enger, Rune (2023). Hjernen i sentrum: Hjernens kapasitet. Full text in Research Archive
• Engelsvold, Sven Ole & Enger, Rune (2022). Spørretime om søvn. [Radio]. NRK. Full text in Research Archive
• Engelsvold, Sven Ole & Enger, Rune (2022). Musehjerner viser vei Alzheimer-forskning. [Radio]. NRK. Full text in Research Archive
• Enger, Rune (2022). Astrocytes and neurons: cortex and sleep. Full text in Research Archive
• Brækhus, Leni Aurora & Enger, Rune (2022). Studerer hjernecellene til våkne Alzheimers-mus for å forstå sykdommen bedre. [Journal]. Dagens Medisin. Full text in Research Archive
• Enger, Rune (2022). Imaging the nightlife of astrocytes - roles in Alzheimer’s disease and sleep physiology. Full text in Research Archive
• Enger, Rune (2022). Studying Astrocytes Physiology in Behaviour and Natural Sleep. Full text in Research Archive
• Enger, Rune (2022). Hva vet vi egentlig om søvn? Full text in Research Archive
• Ellingsrud, Ada; Farrell, Patrick; Dukefoss, Didrik; Enger, Rune; Pettersen, Klas & Halnes, Geir [Show all 7 contributors for this article] (2021). Accurate numerical simulation of electrodiffusion and water movement in brain tissue with cortical spreading depression as a case study. Full text in Research Archive
• Enger, Rune & Bojarskaite, Laura (2021). Hjernens vaktmestere - nattlig hjernevask og søvnregulering. SØVN. ISSN 1891-5493. Full text in Research Archive
• Bojarskaite, Laura; Enger, Rune; Hu, Hua & Boccara, Charlotte (2020). Hvorfor sover vi? Full text in Research Archive
• Boccara, Charlotte N; Hu, Hua; Enger, Rune & Bojarskaite, Laura (2020). Hvorfor sover vi? Full text in Research Archive

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