Developmental expression of the Sturge-Weber syndrome-associated genetic mutation in Gnaq: a formal test of Happle's paradominant inheritance hypothesis.

Sturge-Weber Syndrome (SWS) is a sporadic (non-inherited) syndrome characterized by capillary vascular malformations in the facial skin, leptomeninges, or the choroid. A hallmark feature is the mosaic nature of the phenotype. SWS is caused by a somatic mosaic mutation in the GNAQ gene (p.R183Q), leading to activation of the G protein, Gαq. Decades ago, Rudolf Happle hypothesized SWS as an example of "paradominant inheritance", that is, a "lethal gene (mutation) surviving by mosaicism". He predicted that the "presence of the mutation in the zygote will lead to death of the embryo at an early stage of development". We have created a mouse model for SWS using gene targeting to conditionally express the GNAQ p.R183Q mutation. We have employed two different Cre-drivers to examine the phenotypic effects of expression of this mutation at different levels and stages of development. As predicted by Happle, global, ubiquitous expression of this mutation in the blastocyst stage results in 100% embryonic death. The majority of these developing embryos show vascular defects consistent with the human vascular phenotype. By contrast, global but mosaic expression of the mutation enables a fraction of the embryos to survive, but those that survive to birth and beyond do not exhibit obvious vascular defects. These data validate Happle's paradominant inheritance hypothesis for SWS and suggest the requirement of a tight temporal and developmental window of mutation expression for the generation of the vascular phenotype. Furthermore, these engineered murine alleles provide the template for the development of a mouse model of SWS that acquires the somatic mutation during embryonic development, but permits the embryo to progress to live birth and beyond, so that postnatal phenotypes can also be investigated. These mice could then also be employed in pre-clinical studies of novel therapies.

CVN-AD Alzheimer's mice show premature reduction in neurovascular coupling in response to spreading depression and anoxia compared to aged controls.

We compared the efficacy of neurovascular coupling and substrate supply in cerebral cortex during severe metabolic challenges in transgenic Alzheimer's [CVN-AD] and control [C57Bl/6] mice, to evaluate the hypothesis that metabolic insufficiency is a critical component of degeneration leading to dementia. We analyzed cerebral blood flow and metabolic responses to spreading depression (induced by K+ applied to the cortex) and anoxia across aging in CVN-AD + C57Bl/6 genotypes. In the CVN-AD genotype progression to histological and cognitive hallmarks of dementia is a stereotyped function of age. We correlated physiology and imaging of the cortex with the blood flow responses measured with laser doppler probes. The results show that spreading depression resulted in a hyperemic blood flow response that was dramatically reduced (24% in amplitude, 70% in area) in both middle-aged and aged CVN-AD mice compared to C57Bl/6 age-matched controls. However, spreading depression amplitude and conduction velocity (≈6 mm/min) did not differ among groups. Anoxia (100% N2 ) showed significantly decreased (by 62%) reactive blood flow and autoregulation in aged AD-CVN mice compared to aged control animals. Significantly reduced neurovascular coupling occurred prematurely with aging in CVN-AD mice. Abbreviated physiological hyperemia and decreased resilience to anoxia may enhance early-onset metabolic deficiency through decreased substrate supply to the brain. Metabolic deficiency may contribute significantly to the degeneration associated with dementia as a function of aging and regions of the brain involved.

Rapid, Dose-Dependent Enhancement of Cerebral Blood Flow by transcranial AC Stimulation in Mouse.

BACKGROUND: Transcranial electrical stimulation at an appropriate dose may demonstrate intracranial effects, including neuronal stimulation and cerebral blood flow responses. OBJECTIVE: We performed in vivo experiments on mouse cortex using transcranial alternating current [AC] stimulation to assess whether cerebral blood flow can be reliably altered by extracranial stimulation. METHODS: We performed transcranial AC electrical stimulation transversely across the closed skull in anesthetized mice, measuring transcranial cerebral blood flow with a laser Doppler probe and intracranial electrical responses as endpoint biomarkers. We calculated a stimulation dose-response function between intracranial electric field and cerebral blood flow. RESULTS: Stimulation at electric field amplitudes of 5-20 mV/mm at 10-20 Hz rapidly increased cerebral blood flow (within 100 ms), which then quickly decreased with no residual effects. The time to peak and blood flow shape varied with stimulation intensity and duration, showing a linear correlation between stimulation dose and peak blood flow increase. Neither afterdischarges nor spreading depression occurred from this level of stimulation. CONCLUSIONS: Extracranial stimulation amplitudes sufficient to evoke reliable blood flow changes require electric field strengths higher than what is tolerable in unanesthetized humans (<1 mV/mm), but less than electroconvulsive therapy levels (>40 mV/mm). However, anesthesia effects, spontaneous blood flow fluctuations, and sampling error may accentuate the apparent field strength needed for enhanced blood flow. The translation to a human dose-response function to augment cerebral blood flow (i.e., in stroke recovery) will require significant modification, potentially to pericranial, focused, multi-electrode application or intracranial stimulation.

Small ubiquitin-like modifier 2 (SUMO2) is critical for memory processes in mice.

Small ubiquitin-like modifier (SUMO1-3) conjugation (SUMOylation), a posttranslational modification, modulates almost all major cellular processes. Mounting evidence indicates that SUMOylation plays a crucial role in maintaining and regulating neural function, and importantly its dysfunction is implicated in cognitive impairment in humans. We have previously shown that simultaneously silencing SUMO1-3 expression in neurons negatively affects cognitive function. However, the roles of the individual SUMOs in modulating cognition and the mechanisms that link SUMOylation to cognitive processes remain unknown. To address these questions, in this study, we have focused on SUMO2 and generated a new conditional Sumo2 knockout mouse line. We found that conditional deletion of Sumo2 predominantly in forebrain neurons resulted in marked impairments in various cognitive tests, including episodic and fear memory. Our data further suggest that these abnormalities are attributable neither to constitutive changes in gene expression nor to alterations in neuronal morphology, but they involve impairment in dynamic SUMOylation processes associated with synaptic plasticity. Finally, we provide evidence that dysfunction on hippocampal-based cognitive tasks was associated with a significant deficit in the maintenance of hippocampal long-term potentiation in Sumo2 knockout mice. Collectively, these data demonstrate that protein conjugation by SUMO2 is critically involved in cognitive processes.

Chemogenetics-mediated acute inhibition of excitatory neuronal activity improves stroke outcome.

BACKGROUND AND PURPOSE: Ischemic stroke significantly perturbs neuronal homeostasis leading to a cascade of pathologic events causing brain damage. In this study, we assessed acute stroke outcome after chemogenetic inhibition of forebrain excitatory neuronal activity. METHODS: We generated hM4Di-TG transgenic mice expressing the inhibitory hM4Di, a Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-based chemogenetic receptor, in forebrain excitatory neurons. Clozapine-N-oxide (CNO) was used to activate hM4Di DREADD. Ischemic stroke was induced by transient occlusion of the middle cerebral artery. Neurologic function and infarct volumes were evaluated. Excitatory neuronal suppression in the hM4Di-TG mouse forebrain was assessed electrophysiologically in vitro and in vivo, based on evoked synaptic responses, and in vivo based on occurrence of potassium-induced cortical spreading depolarizations. RESULTS: Detailed characterization of hM4Di-TG mice confirmed that evoked synaptic responses in both in vitro hippocampal slices and in vivo motor cortex were significantly reduced after CNO-mediated activation of the inhibitory hM4Di DREADD. Further, CNO treatment had no obvious effects on physiology and motor function in either control or hM4Di-TG mice. Importantly, hM4Di-TG mice treated with CNO at either 10 min before ischemia or 30 min after reperfusion exhibited significantly improved neurologic function and smaller infarct volumes compared to CNO-treated control mice. Mechanistically, we showed that potassium-induced cortical spreading depression episodes were inhibited, including frequency and duration of DC shift, in CNO-treated hM4Di-TG mice. CONCLUSIONS: Our data demonstrate that acute inhibition of a subset of excitatory neurons after ischemic stroke can prevent brain injury and improve functional outcome. This study, together with the previous work in optogenetic neuronal modulation during the chronic phase of stroke, supports the notion that targeting neuronal activity is a promising strategy in stroke therapy.

Dysregulation of oxygen hemodynamic responses to synaptic train stimulation in a rat hippocampal model of subarachnoid hemorrhage.

We investigated microvascular reactivity to synaptic train stimulation after induction of subarachnoid hemorrhage in adult rats, analyzing tissue oxygen levels [pO2] in intact hippocampus. In control rats, hippocampal pO2averaged 11.4 mm Hg whereas hemodynamic responses averaged 13.1 mm Hg (to a 25 s train). After subarachnoid hemorrhage (at 2 days), we recorded a dramatic elevation in baseline pO2in the hippocampus (to 68.4 mm Hg) accompanied by inverted pO2responses to synaptic train stimulation (-9.46 mm Hg). These significant changes in baseline hippocampal pO2and inverted pO2responses after subarachnoid hemorrhage indicate severe alterations of neurovascular coupling and neuronal viability.

Age-related metabolic fatigue during low glucose conditions in rat hippocampus.

Previous reports have indicated that with aging, intrinsic brain tissue changes in cellular bioenergetics may hamper the brain's ability to cope with metabolic stress. Therefore, we analyzed the effects of age on neuronal sensitivity to glucose deprivation by monitoring changes in field excitatory postsynaptic potentials (fEPSPs), tissue Po2, and NADH fluorescence imaging in the CA1 region of hippocampal slices obtained from F344 rats (1-2, 3-6, 12-20, and >22 months). Forty minutes of moderate low glucose (2.5 mM) led to approximately 80% decrease of fEPSP amplitudes and NADH decline in all 4 ages that reversed after reintroduction of 10 mM glucose. However, tissue slices from 12 to 20 months and >22-month-old rats were more vulnerable to low glucose: fEPSPs decreased by 50% on average 8 minutes faster compared with younger slices. Tissue oxygen utilization increased after onset of 2.5 mM glucose in all ages of tissue slices, which persisted for 40 minutes in younger tissue slices. But, in older tissue slices the increased oxygen utilization slowly faded and tissue Po2 levels increased toward baseline values after approximately 25 minutes of glucose deprivation. In addition, with age the ability to regenerate NADH after oxidation was diminished. The NAD(+)/NADH ratio remained relatively oxidized after low glucose, even during recovery. In young slices, glycogen levels were stable throughout the exposure to low glucose. In contrast, with aging utilization of glycogen stores was increased during low glucose, particularly in hippocampal slices from >22 months old rats, indicating both inefficient metabolism and increased demand for glucose. Lactate addition (20 mM) improved oxidative metabolism by directly supplementing the mitochondrial NADH pool and maintained fEPSPs in young as well as aged tissue slices, indicating that inefficient metabolism in the aging tissue can be improved by directly enhancing NADH regeneration.

Nicotinamide pre-treatment ameliorates NAD(H) hyperoxidation and improves neuronal function after severe hypoxia.

Prolonged hypoxia leads to irreversible loss of neuronal function and metabolic impairment of nicotinamide adenine dinucleotide recycling (between NAD(+) and NADH) immediately after reoxygenation, resulting in NADH hyperoxidation. We test whether the addition of nicotinamide (to enhance NAD(+) levels) or PARP-1 inhibition (to prevent consumption of NAD(+)) can be effective in improving either loss of neuronal function or hyperoxidation following severe hypoxic injury in hippocampal slices. After severe, prolonged hypoxia (maintained for 3min after spreading depression) there was hyperoxidation of NADH following reoxygenation, an increased soluble NAD(+)/NADH ratio, loss of neuronal field excitatory post-synaptic potential (fEPSP) and decreased ATP content. Nicotinamide incubation (5mM) 2h prior to hypoxia significantly increased total NAD(H) content, improved neuronal recovery, enhanced ATP content, and prevented NADH hyperoxidation. The nicotinamide-induced increase in total soluble NAD(H) was more significant in the cytosolic compartment than within mitochondria. Prolonged incubation with PJ-34 (>1h) led to enhanced baseline NADH fluorescence prior to hypoxia, as well as improved neuronal recovery, NADH hyperoxidation and ATP content on recovery from severe hypoxia and reoxygenation. In this acute model of severe neuronal dysfunction prolonged incubation with either nicotinamide or PJ-34 prior to hypoxia improved recovery of neuronal function, enhanced NADH reduction and ATP content, but neither treatment restored function when administered during or after prolonged hypoxia and reoxygenation.

Cellular Links between Neuronal Activity and Energy Homeostasis.

Neuronal activity, astrocytic responses to this activity, and energy homeostasis are linked together during baseline, conscious conditions, and short-term rapid activation (as occurs with sensory or motor function). Nervous system energy homeostasis also varies during long-term physiological conditions (i.e., development and aging) and with adaptation to pathological conditions, such as ischemia or low glucose. Neuronal activation requires increased metabolism (i.e., ATP generation) which leads initially to substrate depletion, induction of a variety of signals for enhanced astrocytic function, and increased local blood flow and substrate delivery. Energy generation (particularly in mitochondria) and use during ATP hydrolysis also lead to considerable heat generation. The local increases in blood flow noted following neuronal activation can both enhance local substrate delivery but also provides a heat sink to help cool the brain and removal of waste by-products. In this review we highlight the interactions between short-term neuronal activity and energy metabolism with an emphasis on signals and factors regulating astrocyte function and substrate supply.

Exploiting metabolic differences in glioma therapy.

Brain function depends upon complex metabolic interactions amongst only a few different cell types, with astrocytes providing critical support for neurons. Astrocyte functions include buffering the extracellular space, providing substrates to neurons, interchanging glutamate and glutamine for synaptic transmission with neurons, and facilitating access to blood vessels. Whereas neurons possess highly oxidative metabolism and easily succumb to ischemia, astrocytes rely more on glycolysis and metabolism associated with synthesis of critical intermediates, hence are less susceptible to lack of oxygen. Astrocytoma and higher grade glioma cells demonstrate both basic metabolic mechanisms of astrocytes as well as tumors in general, e.g. they show a high glycolytic rate, lactate extrusion, ability to proliferate even under hypoxia, and opportunistic use of mechanisms to enhance metabolism and blood vessel generation, and suppression of cell death pathways. There may be differences in metabolism between neurons, normal astrocytes and astrocytoma cells, providing therapeutic opportunities against astrocytomas, including a wide range of enzyme and transporter differences, regulation of hypoxia-inducible factor (HIF), glutamate uptake transporters and glutamine utilization, differential sensitivities of monocarboxylate transporters, presence of glycogen, high interlinking with gap junctions, use of NADPH for lipid synthesis, utilizing differential regulation of synthetic enzymes (e.g. isocitrate dehydrogenase, pyruvate carboxylase, pyruvate dehydrogenase, lactate dehydrogenase, malate-aspartate NADH shuttle) and different glucose uptake mechanisms. These unique metabolic susceptibilities may augment conventional therapeutic attacks based on cell division differences and surface receptors alone, and are starting to be implemented in clinical trials.

Pyruvate incubation enhances glycogen stores and sustains neuronal function during subsequent glucose deprivation.

The use of energy substrates, such as lactate and pyruvate, has been shown to improve synaptic function when administered during glucose deprivation. In the present study, we investigated whether prolonged incubation with monocarboxylate (pyruvate or lactate) prior rather than during glucose deprivation can also sustain synaptic and metabolic function. Pyruvate pre-incubation(3-4h) significantly prolonged (>25 min) the tolerance of rat hippocampal slices to delayed glucose deprivation compared to control and lactate pre-incubated slices, as revealed by field excitatory post synaptic potentials (fEPSPs); pre-incubation with pyruvate also reduced the marked decrease in NAD(P)H fluorescence resulting from glucose deprivation. Moreover, pyruvate exposure led to the enhancement of glycogen stores with time, compared to glucose alone (12 µmol/g tissue at 4h vs. 3.5 µmol/g tissue). Prolonged resistance to glucose deprivation following exogenous pyruvate incubation was prevented by glycogenolysis inhibitors, suggesting that enhanced glycogen mediates the delay in synaptic activity failure. The application of an adenosine A1 receptor antagonist enhanced glycogen utilization and prolonged the time to synaptic failure, further confirming this hypothesis of the importance of glycogen. Moreover, tissue levels of ATP were also significantly maintained during glucose deprivation in pyruvate pretreated slices compared to control and lactate. In summary, these experiments indicate that pyruvate exposure prior to glucose deprivation significantly increased the energy buffering capacity of hippocampal slices, particularly by enhancing internal glycogen stores, delaying synaptic failure during glucose deprivation by maintaining ATP levels, and minimizing the decrease in the levels of NAD(P)H.

Age-Induced Alterations in Hippocampal Function and Metabolism.

As the nervous system ages, a variety of changes occur in metabolism supporting glial and neuronal function, resulting in greater susceptibility to disease conditions. Changes with aging in the metabolic unit (i.e., neurons, glial cells and blood vessels) have been reported to include alterations of vascular reactivity, impaired transport of critical substrates underlying metabolism, enhanced reactive oxygen species production and alterations in calcium signaling. Some diseases are focused on the elderly, particularly cerebral ischemia, cognitive limitations, iatrogenic hypoglycemia, malignant brain tumors (i.e., glioblastoma), and Alzheimer's disease, partly due to metabolic alterations with aging. These metabolic changes with aging are discussed in light of primary theories of aging of the brain, which include mitochondrial, calcium dysfunction and enhanced oxidative damage. Here we focus on metabolic changes with aging which can influence the susceptibility of the brain to ischemia and cognitive function. Lastly, we describe treatment possibilities for these abnormal responses to aging, particularly the topic of caloric/dietary restriction, and possible mechanisms underlying this treatment direction.

Simultaneous monitoring of tissue PO2 and NADH fluorescence during synaptic stimulation and spreading depression reveals a transient dissociation between oxygen utilization and mitochondrial redox state in rat hippocampal slices.

Nicotinamide adenine dinucleotide (NADH) imaging can be used to monitor neuronal activation and ascertain mitochondrial dysfunction, for example during hypoxia. During neuronal stimulation in vitro, NADH normally becomes more oxidized, indicating enhanced oxygen utilization. A subsequent NADH overshoot during activation or on recovery remains controversial and reflects either increased metabolic activity or limited oxygen availability. Tissue P(2) measurements, obtained simultaneously with NADH imaging in area CA1 in hippocampal slices, reveal that during prolonged train stimulation (ST) in 95% O(2), a persistent NADH oxidation is coupled with increased metabolic demand and oxygen utilization, for the duration of the stimulation. However, under conditions of either decreased oxygen supply (ST-50% O(2)) or enhanced metabolic demand (K(+)-induced spreading depression (K(+)-SD) 95% O(2)) the NADH oxidation is brief and the redox balance shifts early toward reduction, leading to a prolonged NADH overshoot. Yet, oxygen utilization remains elevated and is correlated with metabolic demand. Under these conditions, it appears that the rate of NAD(+) reduction may transiently exceed oxidation, to maintain an adequate oxygen flux and ATP production. In contrast, during SD in 50% O(2), the oxygen levels dropped to a point at which oxidative metabolism in the electron transport chain is limited and the rate of utilization declined.

Lactate uptake contributes to the NAD(P)H biphasic response and tissue oxygen response during synaptic stimulation in area CA1 of rat hippocampal slices.

Synaptic train stimulation (10 Hz x 25 s) in hippocampal slices results in a biphasic response of NAD(P)H fluorescence indicating a transient oxidation followed by a prolonged reduction. The response is accompanied by a transient tissue PO(2) decrease indicating enhanced oxygen utilization. The activation of mitochondrial metabolism and/or glycolysis may contribute to the secondary NAD(P)H peak. We investigated whether extracellular lactate uptake via monocarboxylate transporters (MCTs) contributes to the generation of the NAD(P)H response during neuronal activation. We measured the effect of lactate uptake inhibition [using the MCT inhibitor alpha-cyano-4-hydroxycinnamate (4-CIN)] on the NAD(P)H biphasic response, tissue PO(2) response, and field excitatory post-synaptic potential in hippocampal slices during synaptic stimulation in area CA1 (stratum radiatum). The application of 4-CIN (150-250 micromol/L) significantly decreased the reduction phase of the NAD(P)H response. When slices were supplemented with 20 mmol/L lactate in 150-250 micromol/L 4-CIN, the secondary NAD(P)H peak was restored; whereas 20 mmol/L pyruvate supplementation did not produce a recovery. Similarly, the tissue PO(2) response was decreased by MCT inhibition; 20 mmol/L lactate restored this response to control levels at all 4-CIN concentrations. These results indicate that lactate uptake via MCTs contributes significantly to energy metabolism in brain tissue and to the generation of the delayed NAD(P)H peak after synaptic stimulation.

Differences in O2 availability resolve the apparent discrepancies in metabolic intrinsic optical signals in vivo and in vitro.

Monitoring changes in the fluorescence of metabolic chromophores, reduced nicotinamide adenine dinucleotide and flavin adenine dinucleotide, and the absorption of cytochromes, is useful to study neuronal activation and mitochondrial metabolism in the brain. However, these optical signals evoked by stimulation, seizures and spreading depression in intact brain differ from those observed in vitro. The responses in vivo consist of a persistent oxidized state during neuronal activity followed by mild reduction during recovery. In vitro, however, brief oxidation is followed by prolonged and heightened reduction, even during persistent neuronal activation. In normally perfused, oxygenated and activated brain tissue in vivo, partial pressure of oxygen (P(O2)) levels often undergo a brief 'dip' that is always followed by an overshoot above baseline, due to increased blood flow (neuronal-vascular coupling). By contrast, in the absence of blood circulation, tissue P(O2)in vitro decreases more markedly and recovers slowly to baseline without overshooting. Although oxygen is abundant in vivo, it is diffusion-limited in vitro. The disparities in mitochondrial and tissue oxygen availability account for the different redox responses.

Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration.

Mitochondria are critical for cellular adenosine triphosphate (ATP) production; however, recent studies suggest that these organelles fulfill a much broader range of tasks. For example, they are involved in the regulation of cytosolic Ca(2+) levels, intracellular pH and apoptosis, and are the major source of reactive oxygen species (ROS). Various reactive molecules that originate from mitochondria, such as ROS, are critical in pathological events, such as ischemia, as well as in physiological events such as long-term potentiation, neuronal-vascular coupling and neuronal-glial interactions. Due to their key roles in the regulation of several cellular functions, the dysfunction of mitochondria may be critical in various brain disorders. There has been increasing interest in the development of tools that modulate mitochondrial function, and the refinement of techniques that allow for real time monitoring of mitochondria, particularly within their intact cellular environment. Innovative imaging techniques are especially powerful since they allow for mitochondrial visualization at high resolution, tracking of mitochondrial structures and optical real time monitoring of parameters of mitochondrial function. The techniques discussed include classic imaging techniques, such as rhodamine-123, the highly advanced semi-conductor nanoparticles (quantum dots), and wide field microscopy as well as high-resolution multiphoton imaging. We have highlighted the use of these techniques to study mitochondrial function in brain tissue and have included studies from our laboratories in which these techniques have been successfully applied.

Chloride transport inhibitors influence recovery from oxygen-glucose deprivation-induced cellular injury in adult hippocampus.

Cerebral ischemia in vivo or oxygen-glucose deprivation (OGD) in vitro are characterized by major disturbances in neuronal ionic homeostasis, including significant rises in intracellular Na(+), Ca(2+), and Cl(-) and extracellular K(+). Recently, considerable attention has been focused on the cation-chloride cotransporters Na-K-Cl cotransporter isoform I (NKCC-1) and K-Cl cotransporter isoform II (KCC2), as they may play an important role in the disruption of ion gradients and subsequent ischemic damage. In this study, we examined the ability of cation-chloride transport inhibitors to influence the biochemical (i.e. ATP) and histological recovery of neurons in adult hippocampal slices exposed to OGD. In the hippocampus, 7 min of OGD caused a loss of ATP that recovered partially (approximately 50%) during 3 h of reoxygenation. Furosemide, which inhibits the NKCC-1 and KCC2 cotransporters, and bumetanide, a more specific NKCC-1 inhibitor, enhanced ATP recovery when measured 3 h after OGD. Furosemide and bumetanide also attenuated area CA1 neuronal injury after OGD. However, higher concentrations of these compounds appear to have additional non-specific toxic effects, limiting ATP recovery following OGD and promoting neuronal injury. The KCC2 cotransporter inhibitor DIOA and the Cl(-) ATPase inhibitor ethacrynic acid caused neuronal death even in the absence of OGD and promoted cytochrome c release from isolated mitochondria, indicating non-specific toxicities of these compounds.

Changes in intracellular chloride after oxygen-glucose deprivation of the adult hippocampal slice: effect of diazepam.

Ischemic injury to the CNS results in loss of ionic homeostasis and the development of neuronal death. An increase in intracellular Ca2+ is well established, but there are few studies of changes in intracellular Cl- ([Cl-]i) after ischemia. We used an in vitro model of cerebral ischemia (oxygen-glucose deprivation) to examine changes in [Cl-]i and GABA(A) receptor-mediated responses in hippocampal slices from adult rats. Changes in [Cl-]i were measured in area CA1 pyramidal neurons using optical imaging of 6-methoxy-N-ethylquinolinium chloride, a Cl--sensitive fluorescent indicator. Oxygen-glucose deprivation induced an immediate rise in [Cl-]i, which recovered within 20 min. A second and more prolonged rise in [Cl-]i occurred within the next hour, during which postsynaptic field potentials failed to recover. The sustained increase in [Cl-]i was not blocked by GABA(A) receptor antagonists. However, oxygen-glucose deprivation caused a progressive downregulation of the K+-Cl- cotransporter (KCC2), which may have contributed to the Cl- accumulation. The rise in [Cl-]i was accompanied by an inability of the GABA(A) agonist muscimol to cause Cl- influx. In vivo, diazepam is neuroprotective when given early after ischemia, although the mechanism by which this occurs is not well understood. Here, we added diazepam early after oxygen-glucose deprivation and prevented the downregulation of KCC2 and the accumulation of [Cl-]i. Consequently, both GABA(A) responses and synaptic transmission within the hippocampus were restored. Thus, after oxygen-glucose deprivation, diazepam may decrease neuronal excitability, thereby reducing the energy demands of the neuron. This may prevent the activation of downstream cell death mechanisms and restore Cl- homeostasis and neuronal function

Modulation of the GABA(A)-gated chloride channel by reactive oxygen species.

The accumulation of reactive oxygen species during cellular injury leads to oxidative stress. This can have profound effects on ionic homeostasis and neuronal transmission. Gamma-aminobutyric acid (GABA) neurotransmission is sensitive to reactive oxygen species, but most studies have indicated that this is due to alterations in GABA release. Here, we determined whether reactive oxygen species can alter GABA(A) receptor-gated Cl- channels in the adult hippocampus. First, we measured the effects of hydrogen peroxide on intracellular Cl- using UV laser scanning confocal microscopy and the Cl(-)-sensitive probe, 6-methoxy-N-ethylquinolium iodide (MEQ). Superfusion of adult rat hippocampal slices with hydrogen peroxide for 10 min decreased MEQ fluorescence (elevation in [Cl-]i) significantly in area CA1 pyramidal cell soma. Alterations in [Cl-]i were prevented by the vitamin E analog Trolox, an antioxidant that scavenges free radicals. After exposure of slices to hydrogen peroxide, the ability of the GABA agonist muscimol to increase [Cl-]i was attenuated. To determine if GABA(A) receptors were sensitive to oxidative insults, the effect of hydrogen peroxide on the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to GABA-gated Cl- channels was measured using receptor autoradiography and homogenate binding assays. Hydrogen peroxide inhibited [35S]TBPS binding in a regionally selective manner, with the greatest inhibition in cerebral cortex, hippocampus and striatum, areas vulnerable to oxidative stress. Similarly, xanthine and xanthine oxidase, which generate superoxide radicals, reduced [35S]TBPS binding in these regions. The effect of hydrogen peroxide on [35S]TBPS binding was non-competitive and was prevented by Trolox and the iron chelator, deferoxamine. We conclude that reactive oxygen species may compromise GABA(A)-mediated neuronal inhibition via interaction with pre and postsynaptic sites. A reduction in GABA(A)-gated Cl- channel function during periods of oxidative stress may contribute to the development of neuronal damage.