While the ultimate dependence of brain function on its energy supply is evident, how basic neuronal parameters and network activity respond to energy metabolism deviations is unresolved. The resting membrane potential (E m ) and reversal potential of GABA-induced anionic currents (E GABA ) are among the most fundamental parameters controlling neuronal excitability. However, alterations of E m and E GABA under conditions of metabolic stress are not sufficiently documented, although it is well known that metabolic crisis may lead to neuronal hyper-excitability and aberrant neuronal network activities. In this work, we show that in slices, availability of energy substrates determines whether GABA signaling displays an inhibitory or excitatory mode, both in neonatal neocortex and hippocampus. We demonstrate that in the neonatal brain, E m and E GABA strongly depend on composition of the energy substrate pool. Complementing glucose with ketone bodies, pyruvate or lactate resulted in a significant hyperpolarization of both E m and E GABA , and induced a radical shift in the mode of GABAergic synaptic transmission towards network inhibition. Generation of giant depolarizing potentials, currently regarded as the hallmark of spontaneous neonatal network activity in vitro, was strongly inhibited both in neocortex and hippocampus in the energy substrate enriched solution. Based on these results we suggest the composition of the artificial cerebrospinal fluid, which bears a closer resemblance to the in vivo energy substrate pool. Our results suggest that energy deficits induce unfavorable changes in E m and E GABA , leading to neuronal hyperactivity that may initiate a cascade of pathological events. Keywords: brain slices, cortex, energy substrates, GABA, hippocampus, network oscillations. mechanism provides all the necessary energy and cofactors for normal fetal development. At birth this transplacental supply of nutrients ends and crucial changes in the energy supply occur. Following a brief pre-suckling period (postnatal starvation) there is an adaptation to a fat-rich diet (Girard et al. 1992;Medina and Tabernero 2005;Ward Platt and Deshpande 2005). Immediately after birth but before suckling, KBs are not available and lactate is the main energy substrate to the newborn (Girard et al. 1992;Medina et al. 1996;Medina and Tabernero 2005;Ward Platt and Deshpande 2005). The rate of lactate utilization by neurons in the early neonatal rat brain is significantly higher than that of glucose or beta-hydroxybutyrate [BHB, the predominant ketone body in the blood (Bough and Rho 2007)] ( Arizmendi and Medina 1983;Fernandez and Medina 1986;Vicario et al. 1991) and recent results showed the importance of lactate as a cerebral oxidative energy substrate (Schurr and Payne 2007;Bak et al. 2009;Castro et al. 2009).In the postnatal developing rat brain, blood glucose levels are close to those in adults (Pereira de Vasconcelos and Nehlig 1987;Nehlig and Pereira de Vasconcelos 1993). However, glucose utilization is limited and is o...

Network activation triggers a significant energy metabolism increase in both neurons and astrocytes. Questions of the primary neuronal energy substrate (e.g., glucose vs. lactate) as well as the relative contributions of glycolysis and oxidative phosphorylation and their cellular origin (neurons vs. astrocytes) are still a matter of debates. Using simultaneous measurements of electrophysiological and metabolic parameters during synaptic stimulation in hippocampal slices from mature mice, we show that neurons and astrocytes use both glycolysis and oxidative phosphorylation to meet their energy demands. Supplementation or replacement of glucose in artificial cerebrospinal fluid (ACSF) with pyruvate or lactate strongly modifies parameters related to network activity-triggered energy metabolism. These effects are not induced by changes in ATP content, pH i , [Ca 2 þ ] i or accumulation of reactive oxygen species. Our results suggest that during network activation, a significant fraction of NAD(P)H response (its overshoot phase) corresponds to glycolysis and the changes in cytosolic NAD(P)H and mitochondrial FAD are coupled. Our data do not support the hypothesis of a preferential utilization of astrocyte-released lactate by neurons during network activation in slices-instead, we show that during such activity glucose is an effective energy substrate for both neurons and astrocytes. Keywords: astrocytes; energy metabolism; glycolysis; lactate; network activity; neurons INTRODUCTION High cellular energy demands during network activation are met by upregulation of cytosolic glycolysis and mitochondrial oxidative phosphorylation. Mitochondrial metabolism provides most of the ATP but glycolysis is also enhanced and may contribute to the energy production. Journal of Cerebral Blood1-4 For elucidation of the cellular basis of neuroenergetics, measurements of metabolic signals including the oxygen utilization and NAD(P)H/FAD autofluorescence provide valuable information for connecting energy metabolism with neuronal activity. NADH (reduced form) is fluorescent when excited with UV light whereas NAD þ is not, leading to a decrease in observed fluorescence as a result of NADH oxidation. In contrast, FAD (oxidized form) is fluorescent, so the oxidation of FADH 2 to FAD causes an increase in fluorescence. The fluorescence of NADH cannot be separated from that of NADPH and their emission is measured in concert (NAD(P)H). NAD(P)H fluorescence represents a 'mixed' signal since this cofactor can be produced by both glycolysis and mitochondria, whereas FAD fluorescence is entirely mitochondrial. 5,6 Measurements of these parameters in combination with electrophysiological recordings have been used in many studies to monitor the energy status during neuronal activity in brain tissues.Typically, NAD(P)H transients induced by synaptic stimulation have a characteristic biphasic waveform: the initial short dip is followed by a long-lasting overshoot. While there exists a common

Deficient energy metabolism and network hyperactivity are the early symptoms of Alzheimer's disease (AD). In this study, we show that administration of exogenous oxidative energy substrates (OES) corrects neuronal energy supply deficiency that reduces the amyloid-beta-induced abnormal neuronal activity in vitro and the epileptic phenotype in AD model in vivo. In vitro, acute application of protofibrillar amyloid-b 1-42 (Ab 1-42 ) induced aberrant network activity in wild-type hippocampal slices that was underlain by depolarization of both the neuronal resting membrane potential and GABA-mediated current reversal potential. Ab 1-42 also impaired synaptic function and long-term potentiation. These changes were paralleled by clear indications of impaired energy metabolism, as indicated by abnormal NAD(P)H signaling induced by network activity. However, when glucose was supplemented with OES pyruvate and 3-beta-hydroxybutyrate, Ab 1-42 failed to induce detrimental changes in any of the above parameters.We administered the same OES as chronic supplementation to a standard diet to APPswe/PS1dE9 transgenic mice displaying AD-related epilepsy phenotype. In the ex-vivo slices, we found neuronal subpopulations with significantly depolarized resting and GABA-mediated current reversal potentials, mirroring abnormalities we observed under acute Ab 1-42 application. Ex-vivo cortex of transgenic mice fed with standard diet displayed signs of impaired energy metabolism, such as abnormal NAD(P)H signaling and strongly reduced tolerance to hypoglycemia. Transgenic mice also possessed brain glycogen levels twofold lower than those of wild-type mice. However, none of the above neuronal and metabolic dysfunctions were observed in transgenic mice fed with the OES-enriched diet. In vivo, dietary OES supplementation abated neuronal hyperexcitability, as the frequency of both epileptiform discharges and spikes was strongly decreased in the APPswe/PS1dE9 mice placed on the diet. Altogether, our results suggest that early AD-related neuronal malfunctions underlying hyperexcitability and energy metabolism deficiency can be prevented by dietary supplementation with native energy substrates.

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Metabolic abnormalities found in epileptogenic tissue provide considerable evidence of brain hypometabolism, while major risk factors for acquired epilepsy all share brain hypometabolism as one common outcome, suggesting that a breakdown of brain energy homeostasis may actually precede epileptogenesis. However, a causal link between deficient brain energy metabolism and epilepsy initiation has not been yet established. To address this issue we developed an in vivo model of chronic energy hypometabolism by daily intracerebroventricular (i.c.v.) injection of the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) and also investigated acute effects of 2-DG on the cellular level. In hippocampal slices, acute glycolysis inhibition by 2-DG (by about 35%) led to contrasting effects on the network: a downregulation of excitatory synaptic transmission together with a depolarization of neuronal resting potential and a decreased drive of inhibitory transmission. Therefore, the potential acute effect of 2-DG on network excitability depends on the balance between these opposing preand postsynaptic changes. In vivo, we found that chronic 2-DG i.c.v. application (estimated transient inhibition of brain glycolysis under 14%) for a period of 4 weeks induced epileptiform activity in initially healthy male rats. Our results suggest that chronic inhibition of brain energy metabolism, characteristics of the well-established risk factors of acquired epilepsy, and specifically a reduction in glucose utilization (typically observed in epileptic patients) can initiate epileptogenesis.V C 2017 Wiley Periodicals, Inc.

Objective: Despite decades of epilepsy research, 30% of focal epilepsies remain resistant to antiseizure drugs, with effective drug development impeded by lack of understanding on how seizures are initiated. Here, we report the mechanism of seizure onset relevant to most seizures that are characteristic of focal epilepsies. Methods: Electric and metabolic network parameters were measured using several seizure models in mouse hippocampal slices and acutely induced seizures in rats in vivo to determine metabolic events occurring at seizure onset. Results: We show that seizure onset is associated with a rapid release of H 2 O 2 resulting from N-methyl-D-aspartate (NMDA) receptor-mediated activation of nicotinamide adenine dinucleotide phosphate oxidase (NOX). NOX blockade prevented the fast H 2 O 2 release as well as the direct current shift and seizurelike event induction in slices. Similarly, intracerebroventricular injection of NOX antagonists prevented acutely induced seizures in rats. Interpretation: Our results show that seizures are initiated by NMDA receptor-mediated NOX-induced oxidative stress and can be arrested by NOX inhibition. We introduce a novel use for blood-brain barrier-permeable NOX inhibitor with a significant potential to become the first seizure-specific medication. Thus, targeting NOX may provide a breakthrough treatment for focal epilepsies. ANN NEUROL 2019;85:907-920 A major goal of contemporary epilepsy research is the View this article online at wileyonlinelibrary.com.

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Excessive accumulation of reactive oxygen species (ROS) underlies oxidative damage. We find that in hippocampal slices, decreased activity of glucose-based antioxidant system induces a massive, abrupt, and detrimental change in cellular functions. We call this phenomenon metabolic collapse (MC). This collapse manifested in long-lasting silencing of synaptic transmission, abnormal oxidation of NAD(P)H and FADH 2 associated with immense oxygen consumption, and massive neuronal depolarization. MC occurred without any preceding deficiency in neuronal energy supply or disturbances of ionic homeostasis and spread throughout the hippocampus. It was associated with a preceding accumulation of ROS and was largely prevented by application of an efficient antioxidant Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The consequences of MC resemble cortical spreading depression (CSD), a wave of neuronal depolarization that occurs in migraine, brain trauma, and stroke, the cellular initiation mechanisms of which are poorly understood. We suggest that ROS accumulation might also be the primary trigger of CSD. Indeed, we found that Tempol strongly reduced occurrence of CSD in vivo, suggesting that ROS accumulation may be a key mechanism of CSD initiation.