Exacerbation of Epilepsy by Astrocyte Alkalization and Gap Junction Uncoupling

Seizures invite seizures. At the initial stage of epilepsy, seizures intensify with each episode; however, the mechanisms underlying this exacerbation remain to be solved. Astrocytes have a strong control over neuronal excitability and the mode of information processing. This control is accomplished by adjusting the levels of various ions in the extracellular space. The network of astrocytes connected via gap junctions allows a wider or more confined distribution of these ions depending on the open probability of the gap junctions. K⁺ clearance relies on the K⁺ uptake by astrocytes and the subsequent diffusion of K⁺ through the astrocyte network. When astrocytes become uncoupled, K⁺ clearance becomes hindered. Accumulation of extracellular K⁺ leads to hyperexcitability of neurons. Here, using acute hippocampal slices from mice, we uncovered that brief periods of epileptiform activity result in gap junction uncoupling. In slices that experienced short-term epileptiform activity, extracellular K⁺ transients in response to glutamate became prolonged. Na⁺ imaging with a fluorescent indicator indicated that intercellular diffusion of small cations in the astrocytic syncytium via gap junctions became rapidly restricted after epileptiform activity. Using a transgenic mouse with astrocyte-specific expression of a pH sensor (Lck-E²GFP), we confirmed that astrocytes react to epileptiform activity with intracellular alkalization. Application of Na⁺/HCO₃^– cotransporter blocker led to the suppression of intracellular alkalization of astrocytes and to the prevention of astrocyte uncoupling and hyperactivity intensification both in vitro and in vivo. Therefore, the inhibition of astrocyte alkalization could become a promising therapeutic strategy for countering epilepsy development.SIGNIFICANCE STATEMENT We aimed to understand the mechanisms underlying the plastic change of forebrain circuits associated with the intensification of epilepsy. Here, we demonstrate that first-time exposure to only brief periods of epileptiform activity results in acute disturbance of the intercellular astrocyte network formed by gap junctions in hippocampal tissue slices from mice. Moreover, rapid clearance of K⁺ from the extracellular space was impaired. Epileptiform activity activated inward Na⁺/HCO₃^– cotransport in astrocytes by cell depolarization, resulting in their alkalization. Our data suggest that alkaline pH shifts in astrocytes lead to gap junction uncoupling, hampering K⁺ clearance, and thereby to exacerbation of epilepsy. Pharmacological intervention could become a promising new strategy to dampen neuronal hyperexcitability and epileptogenesis.     

Monitoring of glutamate‐induced excitotoxicity by mitochondrial oxygen consumption

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na⁺ influx increases intracellular Ca²⁺ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na⁺ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na⁺/K⁺‐ATPase exchanges intracellular Na⁺ with extracellular K⁺ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate‐mediated excitotoxicity. Under a pyruvate‐restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 μM. The OCR monitoring also revealed the contribution of the N‐methyl‐D‐aspartate receptor and Na⁺/K⁺‐ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte‐dependent protection of neurons from glutamate‐mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.     

Astrocytic poly(ADP‐ribose) polymerase‐1 activation leads to bioenergetic depletion and inhibition of glutamate uptake capacity

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP‐1‐induced depletion of cellular NAD⁺, glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP‐dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP‐1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP‐1 activation by the DNA alkylating agent, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC₅₀ = 78.2 ± 2.7 μM). HPLC revealed MNNG‐induced time‐dependent reductions in NAD⁺ (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [³H]glutamate uptake rate (V_max) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 ± 2.8 in control cells to 2.1 ± 0.8 nmol/min/mg protein 4 h post‐MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP‐1, directly indicating PARP‐1 involvement, and by adding exogenous NAD⁺ (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co‐culture with PARP‐1^−/− astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP‐1 can raise glutamate levels sufficiently to have receptor‐mediated effects at neighboring neurons. Taken together, these experiments showed that PARP‐1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity. © 2009 Wiley‐Liss, Inc.     

Functional and immunocytochemical characterization of D‐serine transporters in cortical neuron and astrocyte cultures

D‐serine is an endogenous coagonist of N‐methyl‐D‐aspartate (NMDA) receptors that plays an important role in synaptic function, neuronal development, and excitotoxicity. Mechanisms of D‐serine transport are important in regulation of extracellular D‐serine concentration and therefore of these critical processes. D‐serine can be transported with low affinity through the Na⁺‐dependent amino acid transporter termed ASCT2, whereas high‐affinity D‐serine uptake has been reported through the Na⁺‐independent transporter termed asc‐1. We investigated immunoreactivity for ASCT2 and asc‐1 and D‐serine transport kinetics in cultured cortical neurons and astrocytes to gain insight into how D‐serine transporters regulate CNS D‐serine levels. Both neurons and astrocytes exhibited low‐affinity Na⁺‐dependent D‐serine uptake (K_T > 1 mM) with broad substrate selectivity that was consistent with uptake through ASCT2. Both neurons and astrocytes also stained positively for ASCT2 in immunocytochemistry studies. Neurons but not astrocytes stained positively for the high‐affinity D‐serine transporter asc‐1, but no evidence of functional asc‐1 could be detected in neurons with conditions that produced such activity in cortical synaptosomes. These data support ASCT2 function in both neuron and astrocyte cultures and identify a discrepancy between observed asc‐1 immunoreactivity and lack of functional asc‐1 activity in neuron cultures. Together these findings further our knowledge of the processes that govern D‐serine regulation. © 2009 Wiley‐Liss, Inc.     

Two sides of the same coin: Sodium homeostasis and signaling in astrocytes under physiological and pathophysiological conditions

The intracellular sodium concentration of astrocytes is classically viewed as being kept under tight homeostatic control and at a relatively stable level under physiological conditions. Indeed, the steep inwardly directed electrochemical gradient for sodium, generated by the Na⁺/K⁺‐ATPase, contributes to maintain the electrochemical gradient of K⁺ and the highly K⁺‐based negative membrane potential, and is a central element in energizing membrane transport. As such it is tightly coupled to the homeostasis of extra‐ and intracellular potassium, calcium or pH and to the reuptake of transmitters such as glutamate. Recent studies, however, have demonstrated that this picture is far too simplistic. It is now firmly established that transmitters, most notably glutamate, and excitatory neuronal activity evoke long‐lasting sodium transients in astrocytes, the properties of which are distinctly different from those of activity‐related glial calcium signals. From these studies, it emerges that sodium homeostasis and signaling are two sides of the same coin: sodium‐dependent transporters, primarily known for their role in ion regulation and homeostasis, also generate relevant ion signals during neuronal activity. The functional consequences of activity‐related sodium transients are manifold and are just coming into view, enabling surprising and important new insights into astrocyte function and neuron‐glia interaction in the brain. The present review will highlight current knowledge about the mechanisms that contribute to sodium homeostasis in astrocytes, present recent data on the spatial and temporal properties of activity‐related glial sodium signals and discuss their functional consequences with a special emphasis on pathophysiological conditions. GLIA 2013;61:1191–1205     

The “protected” glucose transport through the astrocytic endoplasmic reticulum is too slow to serve as a quantitatively-important highway for nutrient delivery

Glycogen levels in resting brain and its utilization rates during brain activation are high, but the functions fulfilled by glycogenolysis in living brain are poorly understood. Studies in cultured astrocytes have identified glycogen as the preferred fuel to provide ATP for Na⁺,K⁺-ATPase for the uptake of extracellular K⁺ and for Ca²⁺-ATPase to pump Ca²⁺ into the endoplasmic reticulum. Studies in astrocyte–neuron co-cultures led to the suggestion that glycogen-derived lactate is shuttled to neurons as oxidative fuel to support glutamatergic neurotransmission. Furthermore, both knockout of brain glycogen synthase and inhibition of glycogenolysis prior to a memory-evoking event impair memory consolidation, and shuttling of glycogen-derived lactate as neuronal fuel was postulated to be required for memory. However, lactate shuttling has not been measured in any of these studies, and procedures to inhibit glycogenolysis and neuronal lactate uptake are not specific. Testable alternative mechanisms to explain the observed findings are proposed: (i) disruption of K⁺ and Ca²⁺ homeostasis, (ii) release of gliotransmitters, (iii) imposition of an energy crisis on astrocytes and neurons by inhibition of mitochondrial pyruvate transport by compounds used to block neuronal monocarboxylic acid transporters, and (iv) inhibition of astrocytic filopodial movements that secondarily interfere with glutamate and K⁺ uptake from the synaptic cleft. Evidence that most pyruvate/lactate derived from glycogen is not oxidized and does not accumulate suggests predominant glycolytic metabolism of glycogen to support astrocytic energy demands. Sparing of blood-borne glucose for use by neurons is a reasonable explanation for the requirement for glycogenolysis in neurotransmission and memory processing.     

High potassium enhances secretion of neurotrophic factors from cultured astrocytes

Elevation of extracellular potassium concentration ([K⁺]_o) in the central nervous system (CNS), which is observed such after physiological stimuli and during ischemia, is known to be regulated by astrocytes. We suspected that in response to increased [K⁺]_o, astrocytes might secrete some neurotrophic factor(s) to promote the survival of active and/or ischemically damaged neurons. In the present study, we examined neurotrophic activity contained in HK-ACM, i.e., astrocyte-conditioned medium (ACM) obtained after culturing astrocytes in 40 mM potassium-containing medium (HK medium). Addition of HK-ACM to basal forebrain cultures from postnatal 2-week-old (P2w) rats increased both the choline acetyltransferase (ChAT) activity (4.40-fold) and the number of ChAT-positive neurons (2.01-fold) as compared with non-conditioned HK medium. On the other hand, the neurotrophic effects of LK-ACM, i.e., ACM collected after culturing astrocytes in 4 mM potassium-containing medium (LK medium), were much weaker (2.85- and 1.41-fold for ChAT activity and number of ChAT-positive neurons, respectively) than those of HK-ACM. The neurotrophic effects of ACMs increased in a manner dependent on potassium concentration and on astrocyte culture time. Addition of an antibody against nerve growth factor (NGF) neutralized the neurotrophic effects of HK- and LK-ACMs. Direct quantification of NGF protein in ACMs by the two-site ELISA method demonstrated that a high concentration of potassium enhanced NGF secretion from cultured astrocytes. These results suggested that astrocytes secrete NGF in response to [K⁺]_o elevation in the CNS.     

Depression of neuronal excitability and epileptic activities by group II metabotropic glutamate receptors in the medial entorhinal cortex

Whereas the ionotropic glutamate receptors are the major mediator in glutamatergic transmission, the metabotropic glutamate receptors (mGluRs) usually play a modulatory role. Whereas the entorhinal cortex (EC) is an essential structure involved in the generation and propagation of epilepsy, the roles and mechanisms of mGluRs in epilepsy in the EC have not been determined. Here, we studied the effects of activation of group II metabotropic glutamate receptors (mGluRs II) on epileptiform activity induced by picrotoxin or deprivation of extracellular Mg²⁺ and neuronal excitability in the medial EC. We found that activation of mGluRs II by application of the selective agonist, LY354740, exerted robust inhibition on epileptiform activity. LY354740 hyperpolarized entorhinal neurons via activation of a K⁺ conductance and inhibition of a Na⁺‐permeable channel. LY354740‐induced hyperpolarization was G protein‐dependent, but independent of adenylyl cyclase and protein kinase A. However, the function of Gβγ was involved in mGluRs II‐mediated depression of both neuronal excitability and epileptiform activity. Our results provide a novel cellular mechanism to explain the antiepileptic effects of mGluRs II in the treatment of epilepsy. © 2015 Wiley Periodicals, Inc.     

Striatal astrocytes transdifferentiate into functional mature neurons following ischemic brain injury

To determine whether reactive astrocytes stimulated by brain injury can transdifferentiate into functional new neurons, we labeled these cells by injecting a glial fibrillary acidic protein (GFAP) targeted enhanced green fluorescence protein plasmid (pGfa2‐eGFP plasmid) into the striatum of adult rats immediately following a transient middle cerebral artery occlusion (MCAO) and performed immunolabeling with specific neuronal markers to trace the neural fates of eGFP‐expressing (GFP⁺) reactive astrocytes. The results showed that a portion of striatal GFP⁺ astrocytes could transdifferentiate into immature neurons at 1 week after MCAO and mature neurons at 2 weeks as determined by double staining GFP‐expressing cells with βIII‐tubulin (GFP⁺‐Tuj‐1⁺) and microtubule associated protein‐2 (GFP⁺‐MAP‐2⁺), respectively. GFP⁺ neurons further expressed choline acetyltransferase, glutamic acid decarboxylase, dopamine receptor D2‐like family proteins, and the N‐methyl‐d ‐aspartate receptor subunit R2, indicating that astrocyte‐derived neurons could develop into cholinergic or GABAergic neurons and express dopamine and glutamate receptors on their membranes. Electron microscopy analysis indicated that GFP⁺ neurons could form synapses with other neurons at 13 weeks after MCAO. Electrophysiological recordings revealed that action potentials and active postsynaptic currents could be recorded in the neuron‐like GFP⁺ cells but not in the astrocyte‐like GFP⁺ cells, demonstrating that new GFP⁺ neurons possessed the capacity to fire action potentials and receive synaptic inputs. These results demonstrated that striatal astrocyte‐derived new neurons participate in the rebuilding of functional neural networks, a fundamental basis for brain repair after injury. These results may lead to new therapeutic strategies for enhancing brain repair after ischemic stroke. GLIA 2015;63:1660–1670     

Potassium channel activity and glutamate uptake are impaired in astrocytes of seizure‐susceptible DBA/2 mice

Purpose: KCNJ10 encodes subunits of inward rectifying potassium (Kir) channel Kir4.1 found predominantly in glial cells within the brain. Genetic inactivation of these channels in glia impairs extracellular K⁺ and glutamate clearance and produces a seizure phenotype. In both mice and humans, polymorphisms and mutations in the KCNJ10 gene have been associated with seizure susceptibility. The purpose of the present study was to determine whether there are differences in Kir channel activity and potassium‐ and glutamate‐buffering capabilities between astrocytes from seizure resistant C57BL/6 (B6) and seizure susceptible DBA/2 (D2) mice that are consistent with an altered K⁺ channel activity as a result of genetic polymorphism of KCNJ10. Methods: Using cultured astrocytes and hippocampal brain slices together with whole‐cell patch‐clamp, we determined the electrophysiologic properties, particularly K⁺ conductances, of B6 and D2 mouse astrocytes. Using a colorimetric assay, we determined glutamate clearance capacity by B6 and D2 astrocytes. Results: Barium‐sensitive Kir currents elicited from B6 astrocytes are substantially larger than those elicited from D2 astrocytes. In addition, potassium and glutamate buffering by D2 cortical astrocytes is impaired, relative to buffering by B6 astrocytes. Discussion: In summary, the activity of Kir4.1 channels differs between seizure‐susceptible D2 and seizure‐resistant B6 mice. Reduced activity of Kir4.1 channels in astrocytes of D2 mice is associated with deficits in potassium and glutamate buffering. These deficits may, in part, explain the relatively low seizure threshold of D2 mice.     

Ammonium‐evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity

The clearance of extracellular glutamate is mainly mediated by pH‐ and sodium‐dependent transport into astrocytes. During hepatic encephalopathy (HE), however, elevated extracellular glutamate concentrations are observed. The primary candidate responsible for the toxic effects observed during HE is ammonium (NH₄⁺/NH₃). Here, we examined the effects of NH₄⁺/NH₃ on steady‐state intracellular pH (pH_i) and sodium concentration ([Na⁺]_i) in cultured astrocytes in two different age groups. Moreover, we assessed the influence of NH₄⁺/NH₃ on glutamate transporter activity by measuring D ‐aspartate‐induced pH_i and [Na⁺]_i transients. In 20–34 days in vitro (DIV) astrocytes, NH₄⁺/NH₃ decreased steady‐state pH_i by 0.19 pH units and increased [Na⁺]_i by 21 mM. D ‐Aspartate‐induced pH_i and [Na⁺]_i transients were reduced by 80–90% in the presence of NH₄⁺/NH₃, indicating a dramatic reduction of glutamate uptake activity. In 9–16 DIV astrocytes, in contrast, pH_i and [Na⁺]_i were minimally affected by NH₄⁺/NH₃, and D ‐aspartate‐induced pH_i and [Na⁺]_i transients were reduced by only 30–40%. Next we determined the contribution of Na⁺, K⁺, Cl^?‐cotransport (NKCC). Immunocytochemical stainings indicated an increased expression of NKCC1 in 20–34 DIV astrocytes. Moreover, inhibition of NKCC with bumetanide prevented NH₄⁺/NH₃‐evoked changes in steady‐state pH_i and [Na⁺]_i and attenuated the reduction of D ‐aspartate‐induced pH_i and [Na⁺]_i transients by NH₄⁺/NH₃ to 30% in 20–34 DIV astrocytes. Our results suggest that NH₄⁺/NH₃ decreases steady‐state pH_i and increases steady‐state [Na⁺]_i in astrocytes by an age‐dependent activation of NKCC. These NH₄⁺/NH₃‐evoked changes in the transmembrane pH and sodium gradients directly reduce glutamate transport activity, and may, thus, contribute to elevated extracellular glutamate levels observed during HE. © 2008 Wiley‐Liss, Inc.     

Endogenous rhythm generation in the pre-Bötzinger complex and ionic currents: modelling and in vitro studies

The pre‐Bötzinger complex is a small region in the mammalian brainstem involved in generation of the respiratory rhythm. As shown in vitro, this region, under certain conditions, can generate endogenous rhythmic bursting activity. Our investigation focused on the conditions that may induce this bursting behaviour. A computational model of a population of pacemaker neurons in the pre‐Bötzinger complex was developed and analysed. Each neuron was modelled in the Hodgkin–Huxley style and included persistent sodium and delayed‐rectifier potassium currents. We found that the firing behaviour of the model strongly depended on the expression of these currents. Specifically, bursting in the model could be induced by a suppression of delayed‐rectifier potassium current (either directly or via an increase in extracellular potassium concentration, [K⁺]_o) or by an augmentation of persistent sodium current. To test our modelling predictions, we recorded endogenous population activity of the pre‐Bötzinger complex and activity of the hypoglossal (XII) nerve from in vitro transverse brainstem slices (700 µm) of neonatal rats (P0–P4). Rhythmic activity was absent at 3 mm [K⁺]_o but could be triggered by either the elevation of [K⁺]_o to 5–7 mm or application of potassium current blockers (4‐AP, 50–200 µm , or TEA, 2 or 4 mm ), or by blocking aerobic metabolism with NaCN (2 mm ). This rhythmic activity could be abolished by the persistent sodium current blocker riluzole (25 or 50 µm ). These findings are discussed in the context of the role of endogenous bursting activity in the respiratory rhythm generation in vivo vs. in vitro and during normal breathing in vivo vs. gasping.     

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     

Cellular and field potential properties of epileptogenic hippocampal slices

Epileptogenic activity was induced in hippocampal slices by addition of penicillin (2.0 mM) to the binding medium. Field potential epileptiform events were recorded and single cell bursts studied with intracellular electrodes. Epileptogenic activity was seen in areas CA1 and CA3 of the slice, with bursts in CA3 always leading CA1 bursts; a cut between CA1 and CA3 abolished spontaneous bursting in CA1 but not in CA3. Increased [Mg²⁺] and decreased [Ca²⁺] abolished epileptiform discharge, thus demonstrating its dependence on synaptic activity; burst occurrence was also sensitive to [K⁺]. Measurements of single cell resting potentials, resistance, and time constant in CA1 cells revealed no difference between cells in normal medium and cells made epileptogenic by penicillin. Depolarization shifts in CA1 neurons during epileptogenesis did not behave like ‘giant EPSPs’ but rather were complexes to which depolarizing spike after-potentials, fast prepotentials, and underlying slow depolarizing events all contributed.     

Mechanistic hypotheses for nonsynaptic epileptiform activity induction and its transition from the interictal to ictal state--computational simulation

Purpose: The aim of this work is to study, by means of computational simulations, the induction and sustaining of nonsynaptic epileptiform activity. Methods: The computational model consists of a network of cellular bodies of neurons and glial cells connected to a three‐dimensional (3D) network of juxtaposed extracellular compartments. The extracellular electrodiffusion calculation was used to simulate the extracellular potential. Each cellular body was represented in terms of the transmembrane ionic transports (Na⁺/K⁺ pumps, ionic channels, and cotransport mechanisms), the intercellular electrodiffusion through gap‐junctions, and the neuronal interaction by electric field and the variation of cellular volume. Results: The computational model allows simulating the nonsynaptic epileptiform activity and the extracellular potential captured the main feature of the experimental measurements. The simulations of the concomitant ionic fluxes and concentrations can be used to propose the basic mechanisms involved in the induction and sustaining of the activities. Discussion: The simulations suggest: The bursting induction is mediated by the Cl^? Nernst potential overcoming the transmembrane potential in response to the extracellular [K⁺] increase. The burst onset is characterized by a critical point defined by the instant when the Na⁺ influx through its permeable ionic channels overcomes the Na⁺/K⁺ pump electrogenic current. The burst finalization is defined by another critical point, when the electrogenic current of the Na⁺/K⁺ pump overcomes its influx through the channels.     

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.     

Glutamine transport in cerebellar granule cells in culture

In the present study, uptake of glutamine by rat cerebellar granule cells, a predominantly glutamatergic nerve cell population, has been investigated. Glutamine is taken up by granule cells via at least three transport systems, A, ASC and L. The L-type low affinity system (K_m=2.6 mM) is the major transport system in the absence of Na⁺. The systems A and ASC represent the Na⁺-dependent transport routes, both with almost identical high affinity for glutamine (K_m=0.26 mM). Similar transport systems for glutamine are also found in cerebral cortical neurons, a predominantly GABAergic nerve cell population, and cerebral cortical astrocytes. The glutamine transport properties in granule cells, however, show a series of differences from that of cortical neurons and astrocytes: (1) uptake of glutamine by granule cells is primarily mediated by system A (54%), while contributions by system A in cortical neurons and astrocytes are less than 30%; (2) granule cells exhibit strikingly higher transport efficiency for glutamine (V_max/K_m=20 min⁻¹ for system A as compared to the V_max/K_m ratio of 5 min⁻¹ in cortical neurons and astrocytes), and (3) the initial uptake rates and the steady-state accumulation levels of glutamine are two- to threefold higher in granule cells than that of cortical neurons and astrocytes. These results taken together suggest that in accordance with the important need to replenish the neurotransmitter pool of glutamate, glutamatergic neurons exhibit highly efficient transport systems to accumulate glutamine, one of the major precursors of glutamate.     

Epileptiform bursts elicited in CA3 hippocampal neurons by a variety of convulsants are not blocked by N-methyl-d-aspartate antagonists

Intracellular and extracellular recordings from CA₃ hippocampal neurons in vitro were used to study the ability of several NMDA (N-methyl-d-aspartate) receptor antagonists to suppress epileptiform bursts induced by NMDA and convulsants not thought to act at NMDA receptors. The antagonists, APV (d-2-amino-5-phosphonovalerate), AP-7 (d,l-2-amino-7-phosphonoheptanoate) and CPP (d,l-3-[(±)-2-car☐ypiperazin-4-yl-]-propyl-1-phosphonic acid), blocked the spontaneous and evoked bursts induced by NMDA. CPP, but not APV or AP-7, prevented the development of bursts induced by Mg-free medium. The NMDA antagonists failed to block bursting induced by kainate, 7 mM K⁺, mast cell degranulating peptide, anoxia or spontaneous bursting. In some cases the NMDA antagonists induced spontaneous bursts or enhanced burst frequency, a proconvulsant effect. It is concluded that activation of NMDA receptors is sufficient but not necessary for burst generation in the CA₃ region.     

Gray and white matter astrocytes differ in basal metabolism but respond similarly to neuronal activity

Astrocytes are a heterogeneous population of glial cells in the brain, which adapt their properties to the requirements of the local environment. Two major groups of astrocytes are protoplasmic astrocytes residing in gray matter as well as fibrous astrocytes of white matter. Here, we compared the energy metabolism of astrocytes in the cortex and corpus callosum as representative gray matter and white matter regions, in acute brain slices taking advantage of genetically encoded fluorescent nanosensors for the NADH/NAD⁺ redox ratio and for ATP. Astrocytes of the corpus callosum presented a more reduced basal NADH/NAD⁺ redox ratio, and a lower cytosolic concentration of ATP compared to cortical astrocytes. In cortical astrocytes, the neurotransmitter glutamate and increased extracellular concentrations of K⁺, typical correlates of neuronal activity, induced a more reduced NADH/NAD⁺ redox ratio. While application of glutamate decreased [ATP], K⁺ as well as the combination of glutamate and K⁺ resulted in an increase of ATP levels. Strikingly, a very similar regulation of metabolism by K⁺ and glutamate was observed in astrocytes in the corpus callosum. Finally, strong intrinsic neuronal activity provoked by application of bicuculline and withdrawal of Mg²⁺ caused a shift of the NADH/NAD⁺ redox ratio to a more reduced state as well as a slight reduction of [ATP] in gray and white matter astrocytes. In summary, the metabolism of astrocytes in cortex and corpus callosum shows distinct basal properties, but qualitatively similar responses to neuronal activity, probably reflecting the different environment and requirements of these brain regions.     

Involvement of TREK-1 Activity in Astrocyte Function and Neuroprotection Under Simulated Ischemia Conditions

Astrocytes play a fundamental role in the pathogenesis of ischemic neuronal death. The optimal operation of electrogenic astrocytic transporters and exchangers for some well-defined astrocyte brain homeostatic functions depends on the presence of K⁺ channels in the cell membranes and the hyperpolarized membrane potential. Our previous study showed that astrocytes functionally express two-pore domain K⁺ channel TREK-1, which helps to set the negative resting membrane potential. However, the roles of TREK-1 on astrocytic function under normal and ischemic conditions remain unclear. In this study, we investigated the expression of TREK-1 protein on cultured astrocytes and the effect of TREK-1 activity on astrocytic glutamate clearance capacity and release of s100β after simulated ischemic insult. TREK-1 immunoreactivity was up-regulated after hypoxia. Suppression of TREK-1 activity inhibited the glutamate clearance capability, enhanced the inflammatory secretion of astrocytes derived s100β and led to increased neuronal apoptosis after ischemic insult. Our results suggest that TREK-1 activity is involved in astrocytic function and neuronal survival. This would provide evidence showing astrocytic TREK-1 involvement in ischemia pathology which may serve as a potential therapeutic target in stroke.