Astrocyte ERK phosphorylation precedes K+‐induced swelling but follows hypotonicity‐induced swelling

Hypotonicity following water intoxication and/or salt loss leads to mainly astrocytic brain swelling. Astrocytic swelling also occurs following brain trauma or ischemia, together with an increase in extracellular K⁺ ([K⁺]_o), stimulating a bumetanide/furosemide/ethacrynic acid‐inhibitable cotransporter, NKCC1, that accumulates Na⁺ and K⁺ together with 2 Cl^‐ and osmotically obliged water. Either type of swelling may become fatal and is associated with phosphorylation of extracellular regulated kinases 1 and 2 (ERK_1/2). Only the swelling associated with elevated [K⁺]_o, leads to an increase in astrocytic proliferation and in expression of the astrocytic marker, glial fibrillary acidic protein. These differences prompted us to investigate key aspects of the molecular pathways between hypotonicity‐induced and high‐K⁺‐mediated swelling in primary cultures of mouse astrocytes. In the latter Ca²⁺‐mediated, AG1478‐inhibitable transactivation of the epidermal growth factor (EGF) receptor leads, via bumetanide‐inhibitable activation of the mitogen activated protein (MAP) kinase pathway to ERK phosphorylation and to NKCC1‐mediated swelling. In the former, inhibition of the MAP kinase pathway, but not of EGF receptor activation, abolishes ERK phosphorylation, but has no effect on swelling, indicating that activation of ERK is a result, not a cause, of the swelling.     

Contributions of the Na+/K+‐ATPase,NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

Network activity in the brain is associated with a transient increase in extracellular K⁺ concentration. The excess K⁺ is removed from the extracellular space by mechanisms proposed to involve Kir4.1‐mediated spatial buffering, the Na⁺/K⁺/2Cl^? cotransporter 1 (NKCC1), and/or Na⁺/K⁺‐ATPase activity. Their individual contribution to [K⁺]_o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na⁺/K⁺‐ATPase and to resolve their involvement in clearance of extracellular K⁺ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K⁺]_o increases above basal levels. Increased [K⁺]_o produced NKCC1‐mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K⁺ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K⁺ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K⁺]_o increase. In contrast, inhibition of the different isoforms of Na⁺/K⁺‐ATPase reduced post‐stimulus clearance of K⁺ transients. The astrocyte‐characteristic α2β2 subunit composition of Na⁺/K⁺‐ATPase, when expressed in Xenopus oocytes, displayed a K⁺ affinity and voltage‐sensitivity that would render this subunit composition specifically geared for controlling [K⁺]_o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na⁺/K⁺‐ATPase accounted for the stimulus‐induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity‐induced extracellular K⁺ recovery in native hippocampal tissue while Kir4.1 and Na⁺/K⁺‐ATPase serve temporally distinct roles. GLIA 2014;62:608–622     

Glial Na+‐dependent ion transporters in pathophysiological conditions

Sodium dynamics are essential for regulating functional processes in glial cells. Indeed, glial Na⁺ signaling influences and regulates important glial activities, and plays a role in neuron‐glia interaction under physiological conditions or in response to injury of the central nervous system (CNS). Emerging studies indicate that Na⁺ pumps and Na⁺‐dependent ion transporters in astrocytes, microglia, and oligodendrocytes regulate Na⁺ homeostasis and play a fundamental role in modulating glial activities in neurological diseases. In this review, we first briefly introduced the emerging roles of each glial cell type in the pathophysiology of cerebral ischemia, Alzheimer's disease, epilepsy, Parkinson's disease, Amyotrophic Lateral Sclerosis, and myelin diseases. Then, we discussed the current knowledge on the main roles played by the different glial Na⁺‐dependent ion transporters, including Na⁺/K⁺ ATPase, Na⁺/Ca²⁺ exchangers, Na⁺/H⁺ exchangers, Na⁺‐K⁺‐Cl^? cotransporters, and Na⁺‐ cotransporter in the pathophysiology of the diverse CNS diseases. We highlighted their contributions in cell survival, synaptic pathology, gliotransmission, pH homeostasis, and their role in glial activation, migration, gliosis, inflammation, and tissue repair processes. Therefore, this review summarizes the foundation work for targeting Na⁺‐dependent ion transporters in glia as a novel strategy to control important glial activities associated with Na⁺ dynamics in different neurological disorders. GLIA 2016;64:1677–1697     

SNAT3‐mediated glutamine transport in perisynaptic astrocytes in situ is regulated by intracellular sodium

The release of glutamine from astrocytes adjacent to synapses in the central nervous system is thought to play a vital role in the mechanism of glutamate recycling and is therefore important for maintaining excitatory neurotransmission. Here we investigate the nature of astrocytic membrane transport of glutamine in rat brainstem slices, using electrophysiological recording and fluorescent imaging of pH_i and . Glutamine application to perisynaptic astrocytes induced a membrane current, caused by activation of system A (SA) family transporters. A significant electroneutral component was also observed, which was mediated by the system N (SN) family transporters. This response was stimulated by glutamine (K_M of 1.57 mM), histidine, and asparagine, but not by leucine or serine, indicating activation of the SNAT3 isoform of SN. We hypothesized that increasing the [Na⁺]_i would alter the SNAT3 transporter equilibrium, thereby stimulating glutamine release. In support of this hypothesis, we show that SNAT3 transport can be driven by changing cation concentration and that manipulations to raise [Na⁺]_i (activation of excitatory amino acid transporters (EAATs), SA transporters or AMPA receptors) all directly influence SNAT3 transport rate. A kinetic model of glutamine fluxes is presented, which shows that EAAT activation causes the release of glutamine, driven mainly by the increased [Na⁺]_i. These data demonstrate that SNAT3 is functionally active in perisynaptic astrocytes in situ. As a result, astrocytic signaling, as would be stimulated by neighboring synaptic activity, has the capacity to stimulate astrocytic glutamine release to support glutamate recycling.     

Importance of astrocytes for potassium ion (K+) homeostasis in brain and glial effects of K+ and its transporters on learning

Initial clearance of extracellular K⁺ ([K⁺]_o) following neuronal excitation occurs by astrocytic uptake, because elevated [K⁺]_o activates astrocytic but not neuronal Na⁺,K⁺-ATPases. Subsequently, astrocytic K⁺ is re-released via Kir4.1 channels after distribution in the astrocytic functional syncytium via gap junctions. The dispersal ensures widespread release, preventing renewed [K⁺]_o increase and allowing neuronal Na⁺,K⁺-ATPase-mediated re-uptake. Na⁺,K⁺-ATPase operation creates extracellular hypertonicity and cell shrinkage which is reversed by the astrocytic cotransporter NKCC1. Inhibition of Kir channels by activation of specific PKC isotypes may decrease syncytial distribution and enable physiologically occurring [K⁺]_o increases to open L-channels for Ca²⁺, activating [K⁺]_o-stimulated gliotransmitter release and regulating gap junctions. Learning is impaired when [K⁺]_o is decreased to levels mainly affecting astrocytic membrane potential or Na⁺,K⁺-ATPase or by abnormalities in its α2 subunit. It is enhanced by NKCC1-mediated ion and water uptake during the undershoot, reversing neuronal inactivity, but impaired in migraine with aura in which [K⁺]_o is highly increased. Vasopressin augments NKCC1 effects and facilitates learning. Enhanced myelination, facilitated by astrocytic-oligodendrocytic gap junctions also promotes learning.     

Regulatory volume increase in astrocytes exposed to hypertonic medium requires β1‐adrenergic Na+/K+‐ATPase stimulation and glycogenolysis

The cotransporter of Na⁺, K⁺, 2Cl^–, and water, NKKC1, is activated under two conditions in the brain, exposure to highly elevated extracellular K⁺ concentrations, causing astrocytic swelling, and regulatory volume increase in cells shrunk in response to exposure to hypertonic medium. NKCC1‐mediated transport occurs as secondary active transport driven by Na⁺/K⁺‐ATPase activity, which establishes a favorable ratio for NKCC1 operation between extracellular and intracellular products of the concentrations of Na⁺, K⁺, and Cl^– × Cl^–. In the adult brain, astrocytes are the main target for NKCC1 stimulation, and their Na⁺/K⁺‐ATPase activity is stimulated by elevated K⁺ or the β‐adrenergic agonist isoproterenol. Extracellular K⁺ concentration is normal during regulatory volume increase, so this study investigated whether the volume increase occurred faster in the presence of isoproterenol. Measurement of cell volume via live cell microscopic imaging fluorescence to record fluorescence intensity of calcein showed that this was the case at isoproterenol concentrations of ≥1 µM in well‐differentiated mouse astrocyte cultures incubated in isotonic medium with 100 mM sucrose added. This stimulation was abolished by the β₁‐adrenergic antagonist betaxolol, but not by ICI118551, a β₂‐adrenergic antagonist. A large part of the β₁‐adrenergic signaling pathway in astrocytes is known. Inhibitors of this pathway as well as the glycogenolysis inhibitor 1,4‐dideoxy‐1,4‐imino‐D‐arabinitol hydrochloride and the NKCC1 inhibitors bumetanide and furosemide abolished stimulation by isoproterenol, and it was weakened by the Na⁺/K⁺‐ATPase inhibitor ouabain. These observations are of physiological relevance because extracellular hypertonicity occurs during intense neuronal activity. This might trigger a regulatory volume increase, associated with the post‐excitatory undershoot. © 2014 Wiley Periodicals, Inc.     

Molecular mechanisms of K+ clearance and extracellular space shrinkage—Glia cells as the stars

Neuronal signaling in the central nervous system (CNS) associates with release of K⁺ into the extracellular space resulting in transient increases in [K⁺]_o. This elevated K⁺ is swiftly removed, in part, via uptake by neighboring glia cells. This process occurs in parallel to the [K⁺]_o elevation and glia cells thus act as K⁺ sinks during the neuronal activity, while releasing it at the termination of the pulse. The molecular transport mechanisms governing this glial K⁺ absorption remain a point of debate. Passive distribution of K⁺ via Kir4.1-mediated spatial buffering of K⁺ has become a favorite within the glial field, although evidence for a quantitatively significant contribution from this ion channel to K⁺ clearance from the extracellular space is sparse. The Na⁺/K⁺-ATPase, but not the Na⁺/K⁺/Cl⁻ cotransporter, NKCC1, shapes the activity-evoked K⁺ transient. The different isoform combinations of the Na⁺/K⁺-ATPase expressed in glia cells and neurons display different kinetic characteristics and are thereby distinctly geared toward their temporal and quantitative contribution to K⁺ clearance. The glia cell swelling occurring with the K⁺ transient was long assumed to be directly associated with K⁺ uptake and/or AQP4, although accumulating evidence suggests that they are not. Rather, activation of bicarbonate- and lactate transporters appear to lead to glial cell swelling via the activity-evoked alkaline transient, K⁺-mediated glial depolarization, and metabolic demand. This review covers evidence, or lack thereof, accumulated over the last half century on the molecular mechanisms supporting activity-evoked K⁺ and extracellular space dynamics.     

Clearance of activity‐evoked K+ transients and associated glia cell swelling occur independently of AQP4: A study with an isoform‐selective AQP4 inhibitor

The mammalian brain consists of 80% water, which is continuously shifted between different compartments and cellular structures by mechanisms that are, to a large extent, unresolved. Aquaporin 4 (AQP4) is abundantly expressed in glia and ependymal cells of the mammalian brain and has been proposed to act as a gatekeeper for brain water dynamics, predominantly based on studies utilizing AQP4‐deficient mice. However, these mice have a range of secondary effects due to the gene deletion. An efficient and selective AQP4 inhibitor has thus been sorely needed to validate the results obtained in the AQP4^?/? mice to quantify the contribution of AQP4 to brain fluid dynamics. In AQP4‐expressing Xenopus laevis oocytes monitored by a high‐resolution volume recording system, we here demonstrate that the compound TGN‐020 is such a selective AQP4 inhibitor. TGN‐020 targets the tested species of AQP4 with an IC₅₀ of ~3.5 μM, but displays no inhibitory effect on the other AQPs (AQP1‐AQP9). With this tool, we employed rat hippocampal slices and ion‐sensitive microelectrodes to determine the role of AQP4 in glia cell swelling following neuronal activity. TGN‐020‐mediated inhibition of AQP4 did not prevent stimulus‐induced extracellular space shrinkage, nor did it slow clearance of the activity‐evoked K⁺ transient. These data, obtained with a verified isoform‐selective AQP4 inhibitor, indicate that AQP4 is not required for the astrocytic contribution to the K⁺ clearance or the associated extracellular space shrinkage.     

Astrocytic and neuronal oxidative metabolism are coupled to the rate of glutamate–glutamine cycle in the tree shrew visual cortex

Astrocytes play an important role in glutamatergic neurotransmission, namely by clearing synaptic glutamate and converting it into glutamine that is transferred back to neurons. The rate of this glutamate–glutamine cycle (V_NT) has been proposed to couple to that of glucose utilization and of neuronal tricarboxylic acid (TCA) cycle. In this study, we tested the hypothesis that glutamatergic neurotransmission is also coupled to the TCA cycle rate in astrocytes. For that we investigated energy metabolism by means of magnetic resonance spectroscopy (MRS) in the primary visual cortex of tree shrews (Tupaia belangeri) under light isoflurane anesthesia at rest and during continuous visual stimulation. After identifying the activated cortical volume by blood oxygenation level‐dependent functional magnetic resonance imaging, ¹H MRS was performed to measure stimulation‐induced variations in metabolite concentrations. Relative to baseline, stimulation of cortical activity for 20 min caused a reduction of glucose concentration by ?0.34 ± 0.09 µmol/g (p < 0.001), as well as a ?9% ± 1% decrease of the ratio of phosphocreatine‐to‐creatine (p < 0.05). Then ¹³C MRS during [1,6‐¹³C]glucose infusion was employed to measure fluxes of energy metabolism. Stimulation of glutamatergic activity, as indicated by a 20% increase of V_NT, resulted in increased TCA cycle rates in neurons by 12% ( , p < 0.001) and in astrocytes by 24% ( , p = 0.007). We further observed linear relationships between V_NT and both and . Altogether, these results suggest that in the tree shrew primary visual cortex glutamatergic neurotransmission is linked to overall glucose oxidation and to mitochondrial metabolism in both neurons and astrocytes.     

Principles of sodium homeostasis and sodium signalling in astroglia

Sodium homeostasis is at the center stage of astrocyte (and brain) physiology because the large inwardly directed Na⁺ gradient provides the energy for transport of ions, neurotransmitters, amino acids and many other molecules across the plasmalemma and endomembranes. Cell imaging with commercially available chemical indicators allows analysis of dynamic changes in intracellular Na⁺ concentration (Na⁺]_i), albeit further technological developments, such as genetically‐controlled or membrane targeted indicators or dyes usable for advanced microscopy (such as fluorescence‐lifetime imaging microscopy) are urgently needed. Thus, important questions related to the existence of Na⁺ gradients between different cellular compartments or occurrence of localised Na⁺ micro/nanodomains at the plasma membrane remain debatable. Extrusion of Na⁺ (and hence Na⁺ homeostasis) in astrocytes is mediated by the ubiquitously expressed Na⁺/K⁺‐ATPase (NKA), the major energy consumer of the brain. The activity of the NKA is counteracted by constant constitutive influx of Na⁺ through transporters such as the NKCC1 (Na⁺‐K⁺?2Cl^‐‐co‐transport) or the NBC (Na⁺?2 ‐co‐transport). In addition, Na⁺‐permeable ion channels at the plasma membrane as well as Na⁺‐dependent solute carrier transporters provide for Na⁺ influx into astrocytes. Activation of these pathways in response to neuronal activity results in an increase of [Na⁺]_i in astrocytes and there is manifold evidence for diverse signalling functions of these [Na⁺]_i transients. Thus, in addition to its established homeostatic functions, activity dependent fluctuations of astrocyte [Na⁺]_i regulate signalling cascades by feeding back on Na⁺‐dependent transporters. The Na⁺ signalling system may be ideally placed for fast coordinating signalling between neuronal activity and glial “homeostatic” Na⁺‐dependent transporters. GLIA 2016;64:1611–1627     

Glutamate transporter‐associated anion channels adjust intracellular chloride concentrations during glial maturation

Astrocytic volume regulation and neurotransmitter uptake are critically dependent on the intracellular anion concentration, but little is known about the mechanisms controlling internal anion homeostasis in these cells. Here we used fluorescence lifetime imaging microscopy (FLIM) with the chloride‐sensitive dye MQAE to measure intracellular chloride concentrations in murine Bergmann glial cells in acute cerebellar slices. We found Bergmann glial [Cl^?]_int to be controlled by two opposing transport processes: chloride is actively accumulated by the Na⁺‐K⁺‐2Cl^? cotransporter NKCC1, and chloride efflux through anion channels associated with excitatory amino acid transporters (EAATs) reduces [Cl^?]_int to values that vary upon changes in expression levels or activity of these channels. EAATs transiently form anion‐selective channels during glutamate transport, and thus represent a class of ligand‐gated anion channels. Age‐dependent upregulation of EAATs results in a developmental chloride switch from high internal chloride concentrations (51.6 ± 2.2 mM, mean ± 95% confidence interval) during early development to adult levels (35.3 ± 0.3 mM). Simultaneous blockade of EAAT1/GLAST and EAAT2/GLT‐1 increased [Cl^?]_int in adult glia to neonatal values. Moreover, EAAT activation by synaptic stimulations rapidly decreased [Cl^?]_int. Other tested chloride channels or chloride transporters do not contribute to [Cl^?]_int under our experimental conditions. Neither genetic removal of ClC‐2 nor pharmacological block of K⁺‐Cl^? cotransporter change resting Bergmann glial [Cl^?]_int in acute cerebellar slices. We conclude that EAAT anion channels play an important and unexpected role in adjusting glial intracellular anion concentration during maturation and in response to cerebellar activity. GLIA 2017;65:388–400     

K+-induced ion and water movements in the frog spinal cord and filum terminale

The K⁺-induced changes in extracellular space were measured in the frog filum terminale, a glial preparation not contaminated by neurons. Comparison of the data with frog spinal cord, consisting of both neurons and glia, suggests that glia are the cellular entities responsible for swelling in response to K⁺. The glial response to K⁺ has four distinct phases, the first of which is due to the metabolic production of CO₂. In the spinal cord the neurons indirectly contribute to the swelling and water movement via generation of CO₂. Swelling in amphibian glia differs from that seen in mammalian glia with respect to a reduced dependence on carbonic anhydrase. Furthermore, in frog glia Cl^? movements are not sufficient to balance the net cation movements (Na⁺ + K⁺), making it necessary to postulate the accumulation of either lactate or bicarbonate.     

Both NKCC1 and anion exchangers contribute to Cl⁻ accumulation in postnatal forebrain neuronal progenitors

Neuronal progenitors are continuously generated in the postnatal rodent subventricular zone and migrate along the rostral migratory stream to supply interneurons in the olfactory bulb. Nonsynaptic GABAergic signaling affects the postnatal neurogenesis by depolarizing neuronal progenitors, which depends on an elevated intracellular Cl^? concentration. However, the molecular mechanism responsible for Cl^? accumulation in these cells still remains elusive. Using confocal Ca²⁺ imaging, we found that GABA depolarization‐induced Ca²⁺ increase was either abolished by bumetanide, a specific inhibitor of the Na⁺–K⁺–2Cl^? cotransporter, or reduced by partial replacement of extracellular Na⁺ with Li⁺, in the HEPES buffer but not in the CO₂/ buffer. GABA depolarization‐induced Ca²⁺ increase in CO₂/ buffer was abolished by a combination of bumetanide with the anion exchanger inhibitor DIDS or with the carbonic anhydrase inhibitor acetozalimide. Using gramicidin‐perforated patch‐clamp recording, we further confirmed that bumetanide, together with DIDS or acetozalimide, reduced the intracellular chloride concentration in the neuronal progenitors. In addition, with BrdU labeling, we demonstrated that blocking of the Na⁺–K⁺–2Cl^? cotransporter, but not anion exchangers, reduced the proliferation of neuronal progenitors. Our results indicate that both the Na⁺–K⁺–2Cl^? cotransporter and anion exchangers contribute to the elevated intracellular chloride responsible for the depolarizing action of GABA in the postnatal forebrain neuronal progenitors. However, the Na⁺–K⁺–2Cl^? cotransporter displays an additional effect on neuronal progenitor proliferation.     

Glial swelling and astrogliosis produce diffusion barriers in the rat spinal cord

Cell swelling and astrogliosis (manifested as an increase in GFAP) were evoked in isolated rat spinal cords of 4–21-day-old rats by incubation in either 50 mM K⁺ or hypotonic solution (235 mosmol kg^-1). Application of K⁺ and hypotonic solution resulted at first in a decrease of extracellular space (ECS) volume fraction α (ECS volume/total tissue volume) and an increase in tortuosity λ (λ² = free/apparent diffusion coefficient) in spinal gray (GM) and white matter (WM). These changes resulted from cell swelling, since the total water content (TW) in spinal cord was unchanged and the changes were blocked in Cl⁻-free solution and slowed down by furosemide and bumetanide. Diffusion in WM was anisotropic, i.e., more facilitated along fibers (x-axis) than across them (y- or z-axis). The increase of λ_y,z was greater than that of λ_x, reaching unusually high values above 2.4. In GM only, during continuous 45 min application, α and λ started to return towards control values, apparently due to cell shrinkage of previously swollen cells since TW remained unchanged. This return was blocked by fluoroacetate, suggesting that most of the changes were due to the swelling of glia. A 45 min application of 50 mM K⁺ and, to a lesser degree, of hypotonic solution evoked astrogliosis, which persisted after washing out these solutions with physiological saline. During astrogliosis λ increased again to values as high as 2.0, while α either returned to or increased above control values. This persistent increase in λ after washout was also found in WM, and, in addition, the typical diffusion anisotropy was diminished. Our data show that glial swelling and astrogliosis are associated with a persistent increase in ECS diffusion barriers. This could lead to the impairment of the diffusion of neuroactive substances, extrasynaptic transmission, “crosstalk” between synapses and neuron-glia communication. GLIA 25:56–70, 1999. © 1999 Wiley-Liss, Inc.     

Neuron type‐ and input pathway‐dependent expression of Slc4a10 in adult mouse brains

Slc4a10 was originally identified as a Na⁺‐driven Cl^?/HCO₃^? exchanger NCBE that transports extracellular Na⁺ and HCO₃^? in exchange for intracellular Cl^?, whereas other studies argue against a Cl^?‐dependence for Na⁺–HCO₃^? transport, and thus named it the electroneutral Na⁺/HCO₃^? cotransporter NBCn2. Here we investigated Slc4a10 expression in adult mouse brains by in situ hybridization and immunohistochemistry. Slc4a10 mRNA was widely expressed, with higher levels in pyramidal cells in the hippocampus and cerebral cortex, parvalbumin‐positive interneurons in the hippocampus, and Purkinje cells (PCs) in the cerebellum. Immunohistochemistry revealed an uneven distribution of Slc4a10 within the somatodendritic compartment of cerebellar neurons. In the cerebellar molecular layer, stellate cells and their innervation targets (i.e. PC dendrites in the superficial molecular layer) showed significantly higher labeling than basket cells and their targets (PC dendrites in the basal molecular layer and PC somata). Moreover, the distal dendritic trees of PCs (i.e. parallel fiber‐targeted dendrites) had significantly greater labeling than the proximal dendrites (climbing fiber‐targeted dendrites). These observations suggest that Slc4a10 expression is regulated in neuron type‐ and input pathway‐dependent manners. Because such an elaborate regulation is also found for K⁺–Cl^? cotransporter KCC2, a major neuronal Cl^? extruder, we compared their expression. Slc4a10 and KCC2 overlapped in most somatodendritic elements. However, relative abundance was largely complementary in the cerebellar cortex, with particular enrichments of Slc4a10 in PC dendrites and KCC2 in molecular layer interneurons, granule cells and PC somata. These properties might reflect functional redundancy and distinction of these transporters, and their differential requirements by individual neurons and respective input domains.     

Aquaporin‐4 regulates the velocity and frequency of cortical spreading depression in mice

The astrocyte water channel aquaporin‐4 (AQP4) regulates extracellular space (ECS) K⁺ concentration ([K⁺]_e) and volume dynamics following neuronal activation. Here, we investigated how AQP4‐mediated changes in [K⁺]_e and ECS volume affect the velocity, frequency, and amplitude of cortical spreading depression (CSD) depolarizations produced by surface KCl application in wild‐type (AQP4^+/+) and AQP4‐deficient (AQP4^?/?) mice. In contrast to initial expectations, both the velocity and the frequency of CSD were significantly reduced in AQP4^?/? mice when compared with AQP4^+/+ mice, by 22% and 32%, respectively. Measurement of [K⁺]_e with K⁺‐selective microelectrodes demonstrated an increase to ～35 mM during spreading depolarizations in both AQP4^+/+ and AQP4^?/? mice, but the rates of [K⁺]_e increase (3.5 vs. 1.5 mM/s) and reuptake (t_1/2 33 vs. 61 s) were significantly reduced in AQP4^?/? mice. ECS volume fraction measured by tetramethylammonium iontophoresis was greatly reduced during depolarizations from 0.18 to 0.053 in AQP4^+/+ mice, and 0.23 to 0.063 in AQP4^?/? mice. Analysis of the experimental data using a mathematical model of CSD propagation suggested that the reduced velocity of CSD depolarizations in AQP4^?/? mice was primarily a consequence of the slowed increase in [K⁺]_e during neuronal depolarization. These results demonstrate that AQP4 effects on [K⁺]_e and ECS volume dynamics accelerate CSD propagation. GLIA 2015;63:1860–1869     

Astrocyte‐mediated infantile‐onset leukoencephalopathy mouse model

Astrocytes have recently been shown to provide physiological support for various brain functions, although little is known about their involvement in white matter integrity. Several inherited infantile‐onset leukoencephalopathies, such as Alexander disease and megalencephalic leukoencephalopathy with subcortical cysts (MLC), implicate astrocytic involvement in the formation of white matter. Several mouse models of MLC had been generated by knocking out the Mlc1 gene; however, none of those models was reported to show myelin abnormalities prior to formation of the myelin sheath. Here we generated a new Mlc1 knockout mouse and a Mlc1 overexpressing mouse, and demonstrate that astrocyte‐specific Mlc1 overexpression causes infantile‐onset abnormalities of the white matter in which astrocytic swelling followed by myelin membrane splitting are present, whereas knocking out Mlc1 does not, and only shows myelin abnormalities after 12 months of age. Biochemical analyses demonstrated that MLC1 interacts with the Na⁺/K⁺ ATPase and that overexpression of Mlc1 results in decreased activity of the astrocytic Na⁺/K⁺ pump. In contrast, no changes in Na⁺/K⁺ pump activity were observed in Mlc1 KO mice, suggesting that the reduction in Na⁺/K⁺ pump activity resulting from Mlc1 overexpression causes astrocytic swelling. Our infantile‐onset leukoencephalopathy model based on Mlc1 overexpression may provide an opportunity to further explore the roles of astrocytes in white matter development and structural integrity. We established a novel mouse model for infantile‐onset leukoencephalopathy by the overexpression of Mlc1. Mlc1 overexpression reduced activity of the astrocytic sodium pump, which may underlie white matter edema followed by myelin membrane splitting. GLIA 2016 GLIA 2017;65:150–168