Deletion of aquaporin-4 improves capillary blood flow distribution in brain edema.

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.

Increased occurrence of pathological mitochondria in astrocytic perivascular endfoot processes and neurons of idiopathic intracranial hypertension.

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.

Loss of perivascular aquaporin-4 in idiopathic normal pressure hydrocephalus.

Idiopathic normal pressure hydrocephalus (iNPH) is a subtype of dementia that may be successfully treated with cerebrospinal fluid (CSF) diversion. Recently, magnetic resonance imaging (MRI) using a MRI contrast agent as a CSF tracer revealed impaired clearance of the CSF tracer from various brain regions such as the entorhinal cortex of iNPH patients. Hampered clearance of waste solutes, for example, soluble amyloid-ß, may underlie neurodegeneration and dementia in iNPH. The goal of the present study was to explore whether iNPH is associated with altered subcellular distribution of aquaporin-4 (AQP4) water channels, which is reported to facilitate CSF circulation and paravascular glymphatic drainage of metabolites from the brain parenchyma. Cortical brain biopsies of 30 iNPH patients and 12 reference individuals were subjected to AQP4 immunogold cytochemistry. Electron microscopy revealed significantly reduced density of AQP4 water channels in astrocytic endfoot membranes along cortical microvessels in patients with iNPH versus reference subjects. There was a significant positive correlation between density of AQP4 toward endothelial cells (perivascular) and toward parenchyma, but the reduced density of AQP4 toward parenchyma was not significant in iNPH. We conclude that perivascular AQP4 expression is attenuated in iNPH, potentially contributing to impaired glymphatic circulation, and waste clearance, and subsequent neurodegeneration. Hence, restoring normal perivascular AQP4 distribution may emerge as a novel treatment strategy for iNPH.

Aquaporin-4-independent volume dynamics of astroglial endfeet during cortical spreading depression.

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.

Astroglial endfeet exhibit distinct Ca2+ signals during hypoosmotic conditions.

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.

Ca2+ Signals in Astrocytes Facilitate Spread of Epileptiform Activity.

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.

Deletion of Aquaporin-4 Curtails Extracellular Glutamate Elevation in Cortical Spreading Depression in Awake Mice.

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.

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 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.

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 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.

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 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.

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 Ca(2+) 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 Ca(2+) spikes and that deletion of Aqp4 reduces these signals. The Ca(2+) 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.

Astrocytes as critical players of the fine balance between inhibition and excitation in the brain: spreading depolarization as a mechanism to curb epileptic activity.

Spreading depolarizations (SD) are slow waves of complete depolarization of brain tissue followed by neuronal silencing that may play a role in seizure termination. Even though SD was first discovered in the context of epilepsy research, the link between SD and epileptic activity remains understudied. Both seizures and SD share fundamental pathophysiological features, and recent evidence highlights the frequent occurrence of SD in experimental seizure models. Human data on co-occurring seizures and SD are limited but suggestive. This mini-review addresses possible roles of SD during epileptiform activity, shedding light on SD as a potential mechanism for terminating epileptiform activity. A common denominator for many forms of epilepsy is reactive astrogliosis, a process characterized by morphological and functional changes to astrocytes. Data suggest that SD mechanisms are potentially perturbed in reactive astrogliosis and we propose that this may affect seizure pathophysiology.

Role of aquaporin-4 polarization in extracellular solute clearance.

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.

MAIN POINTS: Solute clearance is reduced in mice lacking AQP4 Polarization of AQP4 to the endfeet may have a greater impact on clearance of large versus small molecules Clearance of large but not small solutes is correlated with age within adult age.

BubbleDrive, a low-volume incubation chamber for acute brain slices.

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.

Sleep cycle-dependent vascular dynamics in male mice and the predicted effects on perivascular cerebrospinal fluid flow and solute transport.

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.

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 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.

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 ß-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.

Validating a Computational Framework for Ionic Electrodiffusion with Cortical Spreading Depression as a Case Study.

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.

Impaired astrocytic Ca2+ signaling in awake-behaving Alzheimer's disease transgenic mice.

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.

Neurodegenerative conditions such as Parkinson's or Alzheimer's disease are characterized by neurons dying and being damaged. Yet neurons are only one type of brain actors; astrocytes, for example, are star-shaped 'companion' cells that have recently emerged as being able to fine-tune neuronal communication. In particular, they can respond to norepinephrine, a signaling molecule that acts to prepare the brain and body for action. This activation results, for instance, in astrocytes releasing chemicals that can act on neurons. Certain cognitive symptoms associated with Alzheimer's disease could be due to a lack of norepinephrine. In parallel, studies in anaesthetized mice have shown perturbed astrocyte signaling in a model of the condition. Disrupted norepinephrine-triggered astrocyte signaling could therefore be implicated in the symptoms of the disease. Experiments in awake mice are needed to investigate this link, especially as anesthesia is known to disrupt the activity of astrocytes. To explore this question, Åbjørsbråten, Skaaraas et al. conducted experiments in naturally behaving mice expressing mutations found in patients with early-onset Alzheimer's disease. These mice develop hallmarks of the disorder. Compared to their healthy counterparts, these animals had reduced astrocyte signaling when running or being startled. Similarly, a fluorescent molecular marker for norepinephrine demonstrated less signaling in the modified mice compared to healthy ones. Over 55 million individuals currently live with Alzheimer's disease. The results by Åbjørsbråten, Skaaraas et al. suggest that astrocyte­norepinephrine communication may be implicated in the condition, an avenue of research that could potentially lead to developing new treatments.