Reactive oxygen species initiate metabolic collapse in hippocampal slices: potential trigger of cortical spreading depression

Anton E Malkov, Anton I Ivanov, Popova, Irina; Marat Mukhtarov, Olena Gubkina, Tatsiana Waseem, Piotr Bregestovski,  Yuri Zilberter*. Journal of cerebral blood flow and metabolism (accepted)
INSERM UMR1106, Institut de Neurosciences des Systèmes, Aix-Marseille University, Marseille, France.


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 FADH2 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. 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.

Glycolysis and oxidative phosphorylation in neurons and astrocytes during network activity in hippocampal slices.
Anton I Ivanov, Anton E Malkov, Tatsiana Waseem, Marat Mukhtarov, Svetlana Buldakova, Olena Gubkina, Misha Zilberter, Yuri Zilberter*

INSERM UMR1106, Institut de Neurosciences des Systèmes, Aix-Marseille University, Marseille, France.

Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism. 12/2013; DOI:10.1038/jcbfm.2013.222Source: PubMed

ABSTRACT 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, pHi, [Ca(2+)]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.
11 December 2013; doi:10.1038/jcbfm.2013.222.

A new recipe in anti-Alzheimer's cookbook

"The paper by Zilberter et al. (2012) appearing in this issue reports that specific exogenous oxidative energy substrates (pyruvate and 3-β-hydroxybutyrate) can (i) correct neuronal energy supply deficiency and reduce the amyloid-β-induced abnormal neuronal activity in vitro; and (ii) correct neuronal and metabolic dysfunctions in a mouse AD model in vivo. It is well known that deficient energy metabolism and network hyperactivity are among the early symptoms of AD. The results of Zilberter et al. (2012) suggest that early AD-related neuronal malfunctions underlying hyperexcitability and energy metabolism deficiency can be prevented by dietary supplementation with natural energy substrates..."  Editorial Highlight: Natalia V. Gulyaeva*, Mikhail Y. Stepanichev. The anti-AD cookbook: a new recipe. J. Neurochem. 2013, 125, 4–6 --> Full text

Dietary energy substrates reverse early pathology in a model of Alzheimer's disease 

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-β1–42 (Aβ1–42) 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. Aβ1–42 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, Aβ1–42failed 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 Aβ1-42 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.

Article first published online: 10 JAN 2013 Journal of Neurochemistry
DOI: 10.1111/jnc.12127
© 2012 International Society for Neurochemistry
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Research Topic: The link between brain energy homeostasis and neuronal activity.  
Topic Editor: Yuri Zilberter, INSERM UMR 1106 Institut de neurosciences des systèmes, France 
Understanding how the brain ensures its energy supply. Yuri Zilberter. 

Excitatory GABA: How a correct observation may turn out to be an experimental artifact. Piotr Bregestovski and Christophe Bernard. 
The ketogenic diet as a treatment paradigm for diverse neurological . Jong Min Rho and Carl E. Stafstrom. 
Cellular Links Between Neuronal Activity and Energy Homeostasis. Pavan K. Shetty, Francesca Galeffi and Dennis A. Turner
The nervous system and metabolic dysregulation: emerging evidence converges on ketogenic diet therapy. David N. Ruskin and Susan A Masino

Energetics based spike generation of a single neuron: simulation results and analysis. Nagarajan Venkateswaran, Sudarshan Sekhar, Thiagarajan Thirupatchur Sanjayasarathy, Sharath Navalpakkam Krishnan, Dinesh Kannan Kabaleeswaran, Subbu Ramanathan, Narendran Narayanasamy, Sharan Srinivas Jagathrakshakan and S. R. Vignesh. 
Critical State of Energy Metabolism in Brain Slices: The Principal Role of Oxygen Delivery and Energy Substrates in Shaping Neuronal Activity.Anton Ivanov and Yuri Zilberter. 
Aerobic production and utilization of lactate satisfy increased energy demands upon neuronal activation in hippocampal slices and provide neuroprotection against oxidative stress. Avital Schurr and Evelyne Gozal. 
The Energy Demand of Fast Neuronal Network Oscillations: Insights from Brain Slice Preparations. Oliver Kann. 
Metabolic studies in brain slices – past, present, and future. Leif Hertz. 
Activation of Astroglial Calcium Signaling by Endogenous Metabolites Succinate and Gamma-Hydroxybutyrate in the Nucleus Accumbens.Tünde Molnár, László Héja, Zsuzsa Emri, Ágnes Simon, Gabriella Nyitrai, Ildikó Pál and Julianna Kardos. 
Carbohydrate-Biased Control of Energy Metabolism: The Darker Side of the Selfish Brain. Tanya Zilberter. 
Deciphering the neuronal circuitry controlling local blood flow in the cerebral cortex of rodents with optogenetics. Jean Rossier, Alan Urban, Armelle Rancillac and Lucie Martinez.

Understanding how the brain ensures its energy supply 
Yuri Zilberter*
Institut de Neuroscience des Systèmes, INSERM URM 1106, Aix-Marseille Université, Marseille, France

The theme of this research topic emerged in the hope of elucidating the mechanisms of energy supply dictated by costly neuronal activity. The versatility of the papers accepted to the topic is surprisingly broad. Three trends became evident, presumably reflecting the most vivid interests in the field: (1) the “in vivo versus in vitro” problem; (2) the role of particular energy substrates; and (3) the macro-level of energy homeostasis and how it applies to the dietary manipulations aimed at treatment of neurodegenerative disorders.
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Lactate fuels the neonatal brain 
A commentary on
Lactate effectively covers energy demands during neuronal network activity in neonatal hippocampal slices
by Ivanov, A., Mukhtarov, M., Bregestovski, P., and Zilberter, Y. (2011). Front. Neuroenerg. 3:2. doi: 10.3389/fnene.2011.00002

The brain is a highly oxidative organ and the prevailing dogma is that under normal conditions glucose is its principal metabolic substrate. However, it is well established that the brain is capable of oxidizing alternative substrates such as acetate, glutamate, lactate, and ketone bodies. The brain’s capability to use alternative substrates may enable adaptation to extraordinary metabolic conditions such as fasting with enhanced ketogenesis or special dietary conditions during the suckling period with enhanced availability of fatty acids and ketone bodies. During early postnatal development the plasma levels of lactate, acetoacetate, and hydroxybutyrate are elevated and they replace glucose as the primary metabolic fuel until the weaning period is reached. This period is further characterized by hyperexcitability with network driven bursts of neuronal activity and calcium oscillations (Erecinska et al., 2004). Thus, glucose alone may not be sufficient to sustain the energy demands of the postnatal brain. A related question is whether alternative oxidative substrates such as lactate or hydroxybutyrate per se can sustain synaptic function in this developmental period.

To address this question, Ivanov et al. (2011) simultaneously recorded oxygen tension, NAD(P)H fluorescence transients and local field potentials during electrical stimulation of the hippocampal Schaffer collateral pathway in neonatal brain tissue slices from mice. From the very beginning, the authors took great care to ensure both viability and functionality of their preparations. They convincingly demonstrated that surprisingly high superfusion rates with standard artificial cerebrospinal fluid (ACSF) in the slice chamber are required to ensure adequate oxygenation and complete electrical function in blood-free tissue slices. An important implication of this methodological tour de force is that under many previously reported experiments the requirements for viability may been met while the functionality may have been compromised.

After ideal experimental conditions were established, hippocampal slices were first superfused with ACSF containing 10 mM glucose and then with modified ACSF containing 5 mM glucose and 5 mM L-lactate. Their reasoning was if glucose alone is sufficient to sustain enhanced network activity, the addition of lactate should not change tissue metabolic states and synaptic function. To the contrary, the addition of lactate significantly increased oxygen consumption (+31%) and enhanced local field potentials (+41%) during the train stimulation (10 s, 10 Hz) and radically modified the biphasic NAD(P)H signaling: the early oxidation phase was strongly augmented while the overshoot was attenuated.

The authors went on to investigate whether lactate (10 mM) can sustain synaptic function in the absence of glucose as has been reported for mature brain tissue (Schurr et al., 1988). When lactate was the sole oxidative substrate, the changes were even more pronounced during a prolonged 30 s stimulus train: oxygen consumption increased by 54%, the local field potential by 39%, the oxidative phase of the NAD(P)H response was augmented threefold and the NAD(P)H overshoot essentially abolished. Furthermore, the progressive decay in the local field potential at the end of the 30-s stimulus period was less pronounced when lactate replaced glucose in the perfusate. When the experiments were repeated with the ketone body β-hydroxybutyrate (10 mM) as alternative oxidative substrate, similar effects on all measured parameters were observed.

The results of the study by Ivanov et al. (2011) are clear-cut: (1) lactate, whether alone or in the presence of glucose, sustains and even augments synaptic activity and oxidative metabolism in excited neonatal brain tissue. (2) Metabolic recovery pathways are fundamentally altered when lactate or hydroxybutyrate replaces glucose as the primary oxidative substrate.

The implications of this study reach beyond enhancing our understanding of how the neonatal brain functions... Read the rest of commentary

Lactate effectively covers energy demands during neuronal network activity in neonatal hippocampal slices
Anton Ivanov, Marat Mukhtarov, Piotr Bregestovski and Yuri Zilberter*

Although numerous experimental data indicate that lactate is efficiently used for energy by the mature brain, the direct measurements of energy metabolism parameters during neuronal network activity in early postnatal development have not been performed. Therefore, the role of lactate in the energy metabolism of neurons at this age remains unclear. In this study, we monitored field potentials and contents of oxygen and NAD(P)H in correlation with oxidative metabolism during intense network activity in the CA1 hippocampal region of neonatal brain slices. We show that in the presence of glucose, lactate is effectively utilized as an energy substrate, causing an augmentation of oxidative metabolism. Moreover, in the absence of glucose lactate is fully capable of maintaining synaptic function. Therefore, during network activity in neonatal slices, lactate can be an efficient energy substrate capable of sustaining and enhancing aerobic energy metabolism.

Citation: Ivanov A, Mukhtarov M, Bregestovski P and Zilberter Y (2011) Lactate effectively covers energy demands during neuronal network activity in neonatal hippocampal slices.Front. Neuroenerg. 3:2. doi: 10.3389/fnene.2011.00002

Received: 21 March 2011; Accepted: 25 April 2011;
Published online: 06 May 2011.
Edited by:
Sebastian Cerdan, Instituto de Investigaciones Biomedicas Alberto Sols, Spain
Reviewed by:
Tibor Kristian, University of Maryland School of Medicine, USA
Karl A. Kasischke, Rochester University, USA
Copyright: © 2011 Ivanov, Mukhtarov, Bregestovski and Zilberter. This is an open-access article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and other Frontiers conditions are complied with.
*Correspondence: Yuri Zilberter, Faculté de Médecine Timone, Institut National de la Santé et de la Recherche Médicale U751, Université de la Méditerranée, 27 Bd; Jean Moulin, 13385 Marseille Cedex 05, France, e-mail:

Inhibition of spontaneous network activity in neonatal hippocampal slices by energy substrates  
INSERM-U901, Université de la Méditerranée, Marseille, France.
Several energy substrates complementary to glucose, including lactate, pyruvate and ß-hydroxybutyrate, serve as a fuel for neurons. It was reported recently that these substrates can substantially modulate cortical excitability in neonatal slices. However, complementary energy substrates (CES) can also induce an intracellular acidification when added exogenously. Therefore, action of CES on the neuronal properties governing excitability in neonatal brain slices may be underlain by a change in the cell energy status or by intracellular acidification, or both. Here, we attempt to elucidate these possibilities in neonatal hippocampus by recording neuronal population activity and monitoring intracellular pH. We show that a spontaneous network activity pattern, giant depolarizing potentials (GDPs), characteristic for the neonatal hippocampal slices exposed to artificial cerebrospinal fluid, is strongly inhibited by CES and this effect is unlikely to be caused by a subtle intracellular acidification induced by these compounds. Indeed, a much stronger intracellular acidification in the HCO(3) -free solution inhibited neither the GDP frequency nor the GDP amplitude. Therefore, modulation of neuronal energy homeostasis is the most likely factor underlying the effect of lactate, pyruvate and ß-hydroxybutyrate on network excitability in neonatal brain slices.

Journal of neurochemistry.(2011) 2:116 DOI: 10.1111/j.1471-4159.2010.07111.x

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The principal role of oxygen delivery and energy substrates in shaping neuronal activity 
Anton Ivanov and Yuri Zilberter*
INSERM UMR751, Université de la Méditerranée, Marseille, France

The interactive vasculo-neuro-glial system controlling energy supply in the brain is absent in vitro where energy provision is determined by experimental conditions. Despite the fact that neuronal activity is extremely energy demanding, little has been reported on the state of energy metabolism in submerged brain slices. Without this information, the arbitrarily chosen oxygenation and metabolic provisions make questionable the efficient oxidative metabolism in slices. We show that in mouse hippocampal slices (postnatal day 19–44), evoked neuronal discharges, spontaneous network activity (initiated by 4-aminopyridine), and synaptic stimulation-induced NAD(P)H autofluorescence depend strongly on the oxygen availability. Only the rate of perfusion as high as ~15 ml/min (95% O2) provided appropriate oxygenation of a slice. Lower oxygenation resulted in the decrease of both local field potentials and spontaneous network activity as well as in significant modulation of short-term synaptic plasticity. The reduced oxygen supply considerably inhibited the oxidation phase of NAD(P)H signaling indicating that the changes in neuronal activity were paralleled by the decrease in aerobic energy metabolism. Interestingly, the dependence of neuronal activity on oxygen tension was clearly shifted toward considerably larger pO2 values in slices when compared to in vivo conditions. With sufficient pO2 provided by a high perfusion rate, partial substitution of glucose in ACSF for β-hydroxybutyrate, pyruvate, or lactate enhanced both oxidative metabolism and synaptic function. This suggests that the high pO2 in brain slices is compulsory for maintaining oxidative metabolism, and glucose alone is not sufficient in fulfilling energy requirements during neuronal activity. Altogether, our results demonstrate that energy metabolism determines the functional state of neuronal network, highlighting the need for the adequate metabolic support to be insured in the in vitro experiments.

Citation: Ivanov A and Zilberter Y (2011) Critical state of energy metabolism in brain slices: the principal role of oxygen delivery and energy substrates in shaping neuronal activity. Front. Neuroenerg. 3:9. doi: 10.3389/fnene.2011.00009

Received: 21 September 2011; Accepted: 10 December 2011;
Published online: 29 December 2011.
Edited by:
Sebastian Cerdan, Instituto de Investigaciones Biomedicas Alberto Sols, Spain
Reviewed by:
Sebastian Cerdan, Instituto de Investigaciones Biomedicas Alberto Sols, Spain
Elizabeth Hillman, Columbia University, USA
Juan C. Saez, Universidad Catolica de Chile, Chile
Copyright: © 2011 Ivanov and Zilberter. This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.

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Neuronal activity in vitro and the in vivo reality: the role of energy homeostasis. 
The energy demands of the brain are exceptionally high compared with any other organ of the body. A complex control system maintains brain energy homeostasis, mobilizing appropriate energy substrates to satisfy the energy requirements. It is a common belief that many fundamental neuronal properties, including those governing excitability, are dependent on the energy supply. However, surprisingly little is known about how the specific factors underlying neuronal activity are affected by energy status. Most of these parameters have been studied in acute brain slices, in which the homeostatic system is absent and neurons in the artificial extracellular milieu are arbitrarily supplied with energy substrates. In this paper, we discuss the relationships between availability of energy substrates and neuronal excitability, and suggest that for in vitro studies, it is crucial to optimize the composition of the energy pool in the extracellular milieu.

Trends in Pharmacological Sciences (2010)
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Not only ketone bodies: on neuroprotective effects of energy substrates
In the previous post On the mechanisms of brain protection by ketones, it was described how a shortage of ketones caused pathological changes in brain cells resulting in abnormal behavior of GABA, the principal brain chemical helping to resist hyperactivity. In the second article, the authors showed that not only ketone bodies, but also other energy carriers such as lactate and pyruvate (or their shortages) can make all the difference between smoothly functioning brain networks and pathologically overly excited ones.

"We suggest that metabolic deficits induce changes in intrinsic neuronal properties resulting in hyperactivity in single neurons and aberrant neuronal network behavior. This hyperactivity in turn increases neuronal energy demands, which cannot be met due to metabolic pathologies and a vicious cycle occurs. Our hypothesis predicts that the adequate delivery of energy substrates may interrupt this pathological spiral of events and provide therapeutic options targeting the cause of pathologies rather than their symptoms" concluded the authors.

Holmgren CD, Mukhtarov M, Malkov AE, Popova IY, Bregestovski P, Zilberter Y.. Energy substrate availability as a determinant of neuronal resting potential, GABA signaling and spontaneous network activity in the neonatal cortex in vitro. J Neurochem. 2009 Nov 24.

Institut de Neurosciences des Systèmes - Inserm UMR1106 - Aix-Marseille Université - Faculté de Médecine, 27, Boulevard Jean Moulin - 13005 Marseille, France. Sub-group 5; PI: Y. Zilberter; Metabolism and Neuroprotection