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Cellular metamorphosis is closely associated with intracellular oxidoreduction ( oxidation-reduction ) balance. Here, we describe cytoplasmic oxidation-reduction alterations in civilized hippocampal nerve cells exposed to glutamate. Nerve cells were transfected with HyPer, a genetically encoded oxidation-reduction biosensor for H peroxide which allows real-time imagination of the oxidation-reduction province. HyPer increases its signal with H peroxide in a dose-dependent mode meanwhile the decay of the fluorescence informs about the cut downing capableness of the cytol. The rate of decay was found to be augmented by glutamate ( 10 i?­M ) every bit good as by pharmacological stimulation of NMDA glutamate receptors. Acute chelation of extracellular Ca2+ abolished the glutamate-induced consequence observed on HyPer fluorescence. Further experiments indicated that mitochondrial map and therefore energetic substrate handiness commands the redox province of nerve cells and is required for the glutamate consequence observed on the biosensor signal. Finally, our work pointed out that astrocytic metamorphosis is involved in the alterations of neural oxidation-reduction province observed with glutamate.

Introduction

The encephalon is one of the most metabolically expensive tissues of the organic structure. It possess a high rate of glucose and O ingestion, although matching merely to 2 % of the whole organic structure weight [ 1,2 ] . The metabolic demand of the encephalon shows regional and spacial forms determined by neural transmittal, a characteristic that has been exploited in clinical diagnosing and basic research.

At resting conditions, encephalon cells supply their energy demand from glucose as the alone metabolite available for oxidization [ 3 ] . A different scenario is established when glutamate, the chief excitatory neurotransmitter is released to the synaptic cleft. Initially, a high energy demand occurs in encephalon cells due to the recovery of ionic gradients dissipated during the synaptic transmittal. For case, astrocytes respond to glutamate by exciting their glucose conveyance and glycolytic flux to guarantee uninterrupted ATP production, a phenomenon coupled to Na+ gradient-driven glutamate re-uptake and the plasma membrane Na+/K+ ATPase pump activity [ 4-8 ] . On the other manus, nerve cells seemingly do non to trip their glycolytic flux under neurotransmission. Recent grounds provided by Chuquet et Al. bespeak that nerve cells from the barrel cerebral mantle do non increase their glucose metamorphosis as astrocytes do upon hair’s-breadth stimulation in rats [ 8 ] , corroborating differential forms of glucose metamorphosis observed before in primary civilizations and encephalon pieces [ 7,9 ] . These findings indicate that nerve cells, despite their higher energetic demand [ 10 ] , do non obtain the energy from metabolising glucose, they instead prefer an alternate C beginning to fuel the costs of synaptic transmittal, likely lactate. Indeed, many studies in vitro have shown that lactate and glucose are every bit effectual to prolong synaptic map [ 11-13 ] . All these findings fit good with the thought proposed by Magistretti et Al. where glutamate increases astrocytic glycolysis giving a net release of lactate to the extracellular infinite, which is taken up by nerve cells to be oxidized by the chondriosome. This thought is better known as the astrocyte to neuron shuttle hypothesis, ANLSH [ 4 ] .

In the encephalon, blood-borne glucose metamorphosis produces the primary energy beginning, adenosine triphosphate ( ATP ) , and reduced nicotinamide A dinucleotide ( NADH ) . Oxidative phosphorylation of glucose-derived metabolites, either lactate or piruvate, besides render NADH and ATP in the Krebs rhythm ( referencia libro ) . Alternatively, glucose-6-phosphate ( glc-6-P ) can besides be metabolized by pentose phosphate tract to bring forth biosynthetic molecules and decreased nicotinamide dinucleotide phosphate ( NADPH ) , the cofactor necessary for the regeneration of reduced glutathione ( GSH ) by glutathione reductase [ 14-16 ] . Despite of NADH and NADPH portion similar oxidation-reduction features their maps are divergent. Whereas NADH is advocated to energetic maps, NADPH seems to be destined to cellular redox maps. NADPH is non merely indispensable for the regeneration of all antioxidant defence systems, such as GSH, thioredoxins and peroxiredoxins, but besides participates in detoxification with cytocrome p450 and in the “ oxidative explosion ” mediated by NADPH oxidase in immune cells [ 17-19 ] . These compartmentalised functions find convergence with the NAD+ kinase ( NADK ) , which phosphorylates NAD+ and has shown to exercise control over the cellular content of NADPH in mammalian cells [ 17,20 ] .

The intracellular oxidation-reduction province is fundamentally determined by the ratio of redox-active braces NADH/NAD+ , NADPH/NADP+ , GSH/glutathione disulfide ( GSSG ) and thioredoxin/oxidized thioredoxin. Among those braces, GSH with an copiousness of 2 to 3 millimeters and a comparative ratio of GSH/GSSG around 100:1 is the major anti-oxidant in the cells encephalon [ 21 ] . Healthy cells maintain the cytoplasmic environment at cut downing potency of -250 millivolt, this balance is held despite of the endogenous coevals of reactive species of O ( ROS ) generated by chondriosomes as by merchandises from decrease of O2 and H2O during oxidative phosphorylation. Moderate displacements toward more oxidative potencies are thought to advance cell distinction. For case, Sundaresan and co-workers demonstrated that additions in H peroxide were indispensable for signaling induced by thrombocyte derived growing factor on vascular smooth cells [ 22 ] , many other studies have established a physiological function for ROS in cellular events as good [ 23 ] , for reappraisal refer to [ 24 ] . More terrible additions in ROS formation become harmful to cellular constituents. DNA harm, lipid peroxidation and broad thiol oxidization of proteins are known factors which finally affect structural and enzymatic maps taking to cell prostration and decease ( ref ) . Together, the grounds indicates that oxidation-reduction province is a dynamic variable in the cell and can be affected by metabolic penchants and signaling triggered by external factors such as growing factors.

Remarkable betterments in mensurating fluctuations in the oxidation-reduction province in existent clip manners have been possible thanks to fluorescent protein-based oxidation-reduction investigations. Basically, green and xanthous fluorescent proteins ( FPs ) have been chosen as templet to put cysteine braces able to organize disulfide bonds under oxidative environments, which in bend produce alterations in the fluorescent spectrum of the protein, giving the belongings to follow alterations in the oxidation-reduction province with alterations in fluorescent strength of FPs, revised by Meyer and Dick [ 25 ] . Furthermore, genetically encode biosensors have been destined to cell organs in order to obtain existent clip measurings of redox province of endoplasmic Reticulum lms [ 26 ] and chondriosome [ 27,28 ] . Herein, we took advantage of the fluorescent protein-based oxidation-reduction biosensor, HyPer, which was designed to supervise specifically intracellular H peroxide and allows ratiometric measurings [ 27 ] . However, one time HyPer molecule is oxidized, its disulfide bond is susceptible to be reduced back to free thiol groups by the action of cut downing mechanisms nowadays in the cell instead than a mere H peroxide clearance. Therefore, HyPer does non merely informs about intracellular H peroxide formation, but gives valuable information about the cut downing capacity of the environment every bit good. Based on those belongingss, we studied the alterations in the cut downing cytosolic capacity of nerve cells submitted to energy demand conditions induced by glutamate, this excitant neurotransmitter alters encephalon cell metamorphosis and in effect, redox province alterations are expected to happen harmonizing to metabolic substrate handiness.

Materials and methods

MReagents and plasmids. DL-Lactate, Na piruvate, gramicidin, Na monensin, H peroxide, reduced glutathione and standard chemicals were purchased from Sigma ( St. Louis, MO ) . Fetal bovine serum, Minimal Essential Medium, Trypsin, penicillin-estreptomycin, Glutamax, Neurobasal and B27 addendum and fluorescent investigations like Fluo-3 AM were purchased from Invitrogen ( Carlsbad, CA, USA ) . pHyPer-cito plasmid was purchased from Evrogen JSC ( Moscow, Russia ) .

Brain cell civilization. Sprague-Dawley rats were obtained from the Universidad de Chile. Mixed civilizations of neural and glia cells were prepared from encephalons 1-3 yearss old newborns following the same indicant as in Loaiza et Al. 1 with minor alterations. In brief, hippocampi were dissected in HankA?s buffer, incubated in 1.25 % trypsin for 10 min at 37°C, and automatically dissociated in MEM-10 % foetal bovine serum: The cells were plated on poly-L-lysine coated ( 0.1 mg/ml ) coverslips and maintained at 37°C in a humidified ambiance with 5 % CO2/95 % air. After one hr, the media was replaced by Neurobasal/B27 and the cell readying was maintained by reclamation of media every 3 yearss. Experiments were performed after 7 to 14 yearss in KRH buffer ( in millimeter, 140 NaCl, 4.7 KCl, 20 HEPES, 1.25 MgSO4 and 1.25 CaCl2. ) .

PC12 Cell civilization. Rat phaeochromocytoma PC12 cells ( from American Type Culture Collection ) were cultured in DMEM supplemented with heat-inactivated 10 % Equus caballus serum, 5 % FBS, 50 units/mL penicillin and 100 mg/mL streptomycin ( Invitrogen, Carlsbad, CA ) . PC12 differentiated nerve cells were obtained by plating cells onto poly-L-Lysine coated coverslips in the presence of 50 ng/mL NGF ( Sigma-Aldrich, St Louis, MO ) for a period of 5 yearss in DMEM medium supplemented with 1 % heat-inactivated Equus caballus serum and 1 % FBS.

HyPer imagination. Cultured cells were transfected with pHyPer-cyto, a plasmid encoding a specific H peroxide detector with cytosolic look for mammalian cells. The transfection method used was either lipofectamine 2000 or Ca phosphate, both rendering similar per centum of neural cells showing HyPer. The ratiometric measuring of H peroxide was achieved in a Olympus microscope equipped with a whirling disc ( DSU Model ) . The readying was excited at 403/12 nanometers and 480/20nm ( MT20E emanation wheel filter ) and emanation was detected at 555/28 nanometer with a CCD camera.

Calcium Imaging

Consequences

Glutamate induces a rapid lessening in HyPer fluorescent signal in hippocampal nerve cells.

The cytoplasmic oxidation-reduction province was monitored by agencies of HyPer, a genetically encoded fluorescent index which has been proven to be specific to hydrogen peroxide and no other extremist O species [ 27 ] . HyPer ratio increased in a dose-dependent mode demoing impregnation at H2O2 doses over 50 i?­M. In add-on, consecutive pulsations of H peroxide ( 100 i?­M ) induced comparable increases in the ratio fluorescence although with some loss in the signal amplitude during the clip class ( Auxiliary figure 1 ) . Besides it can be noted that the biosensor turns spontaneously back to pre-pulse province. Complementarily, the add-on of exogenic glutathione ( 1 and 5 millimeter ) decreased the HyPer signal at radical degree every bit good as from a pre-oxidized province, ( Auxiliary figure 1 ) .

In order to look into the impact of glutamate on the cut downing capableness of nerve cells, a pre-pulse of H2O2 100 i?­M was applied to oxidise the biosensor, after this process the fluorescence started to decrease with a changeless rate ( VALOR ) named here as ( incline a, figure 1A ) . The add-on of glutamate ( 10 i?­M ) accelerated the decay of the signal ( incline B, figure 1A ) . This consequence was observed in ~80 % of the nerve cells studied and in norm, the neurotransmitter increased the incline in about seven times.

At this point, is of import to clear up that the decrease in the fluorescence from the oxidized HyPer molecule does non obey to a cytoplasmic clearance of H peroxide, but instead to decrease of disulfide bonds, as suggested by Meyer and Dick and besides, confirmed by the pre-treatment with the catalase inhibitor, 3-amino-triazol, which did non interfere with the glutamate consequence on the HyPer signal ( informations non shown ) .

Following, we tested if the pharmacological stimulation of the NMDA-sensitive glutamate receptors could mime the glutamate consequence observed supra. As shown in the figure 1B and C, NMDA was every bit effectual as glutamate to diminish the Hyper signal in a nerve cell antecedently treated with H peroxide. Furthermore, the pretreatment of ionotropic glutamate receptors blockers, amino-5-phosphonovaleric acid and 6,7-dinitroquinoxaline-2,3-dione, avoided the glutamate-dependent HyPer signal diminishment ( figure 1C ) . Together, the consequences suggest the glutamate consequence on the oxidation-reduction biosensor is mediated by the activation of NMDA-sensitive glutamate receptors.

Due to ionotropic glutamate receptors activation, and in particular NMDA-sensitive receptors, would take to a Na and Ca inflow, we investigated if bring oning a glutamate-independent Na entry could trip a decrease in the biosensor fluorescence. First, nerve cells were exposed to monensin, a Na+/H+ antiporter, such intervention provoked a rapid diminishment in the HyPer signal ( figure 2A ) . In excitable cells, like nerve cells, membrane possible can be perturbed by distinct cation entry, which in bend will open electromotive force dependent Ca channels, among others voltage dependent channels. Sing this point, cytosolic Ca was imaged in nerve cells exposed to monensin to corroborate cytosolic Ca disturbances by monensin. The figure 2C shows that a monolithic addition in the cytoplasmic Ca occurred upon monensin exposure, go forthing the Ca signaling as a valid option to explicate the consequence of this Na ionophore. Of the note, the efficiency of monensin to pervade Na+ and so, to trip a cytoplasmic Ca addition in nerve cells, should be accompanied by a cytoplasmic proton bulge. The predicted alkalinization of cytol was confirmed by lading nerve cells with the cell permeating pH-sensitive dye BCECF, monensin induced a rapid and transeunt alkalinization as it can be observed in the auxiliary figure 2. Alternatively, nerve cells were exposed to veratridine, an alkaloid that interacts with voltage-dependent Na+ channels in their unfastened province, which induces a gradual Na inflow with the attendant cytosolic Ca addition [ 9 ] , but without the pH perturbations generated by monensin. This compound was every bit effectual as monensin to speed up the decrease in the HyPer signal ( see the figure 2B and C ) .

To prove straight the function of Ca inflow in the glutamate consequence, we performed experiments with an extracellular buffer without Ca and supplemented with the Ca sequestering agent, ethene ethanediol tetraacetic acid ( EGTA, 5 millimeter ) . Under nominal nothing Ca status, glutamate was non able to arouse a diminishment in the HyPer fluorescency. Furthermore, the neurotransmitter recovered its capableness to impact HyPer signal when Ca was placed back to the extracellular media ( Figure 3 ) . Together, these consequences indicate that a Ca inflow is a cardinal cellular courier in the glutamate-evoked decrease of the biosensor HyPer at the cytoplasmic neural environment.

Astrocytic metamorphosis is connected with the neural oxidation-reduction position

Intriguingly, the lessening in the biosensor fluorescency either induced by a Ca inflow with ionophores or by exposure with the neurotransmitter glutamate, could be observed in the absence of any metabolic fuel ( e.g. glucose 5 millimeter ) . Sing that nerve cells are non good equipped with energetic reservoirs, it is sensible to presume that after 10 proceedingss without glucose the glycolitic flux has diminished to negligible degree. However, chondriosomes still can be bring forthing cut downing power by feeding Krebs rhythm with mediators. To buttockss that the mitochondrial machinery was involved in the biosensor signal diminishment, the readying was pre-incubated with the mitochondrial toxicant, Antimycin A. The figure 4 shows that under this status, glutamate was non able to impact the fluorescence of the biosensor. This determination unveils that mitocondrial map is necessary to detect the glutamate consequence on the HyPer, but which metabolite or from where it comes, are non informed.

The co-culture mode used here allowed us to look into if astrocytic metamorphosis is connected in some manner with neural oxidation-reduction position. In order to impair merely the glial metamorphosis, cells were treated with fluoroacetate ( 5 millimeter ) for at least 2 hours before the imaging process. This compound is merely metabolized by astrocytes, where it is converted in fluorocitrate, which eventually exerts the toxic action specifically on the aconitase enzyme holding the Krebs rhythm [ 29,30 ] . In order to guarantee the animal starch ingestion by astrocytes, 30 proceedingss before the experiment started, glucose was washed out and the experiment was carried out. The figure 5A shows a clip class of HyPer signal recorded from cells which were energetically deprived with fluoracetate, such status was effectual in avoid the glutamate consequence on HyPer described before, no important alterations in the basal rate of HyPer recovery before and after glutamate exposure were detected ( figure 5C ) , proposing that the astrocytic metabolic machinery is polar in the HyPer ratio lessening. These consequences suggest an intercellular transportation from astrocytes to nerve cells, where the Hyper molecule is located.

In order to extinguish such transferring, the entire obliteration of glial population from our civilizations had been ideal to fling any astrocytic part to the glutamate consequence on the biosensor, but utilizing cytosine arabinofuranoside, an agent widely used to enriched neural civilizations is non the best option because it does non vouch the entire astrocytic riddance, but besides provokes neural toxicity as an unsought secondary consequence [ 31 ] . Alternatively, we took advantage of PC12 cells, a cell line which can be differentiated to a neural phenotype with NGF [ 32 ] , which deficiency of any other cellular type that could interfere through intercellular metabolic trafficking. In the figure 5B, double monitoring of cytosolic Ca and HyPer fluorescency was achieved by lading the readying with the Ca dye FuraRed, which respond in return to Ca additions. As a general observation, the typical dosage of glutamate ( 10 i?­M ) used during this survey was uneffective to lift the Ca in differentiated PC12, therefore a higher dosage ( 100 i?­M ) was applied to arouse a Ca addition, see figure 5. Even when a Ca addition was evoked, probably by NMDA activation [ 32 ] , no warp in the HyPer signal was recorded, bespeaking that non merely the glutamate-sensing machinery on the PC12 cells is required to modify the oxidation-reduction environment at the cytol, but an external factor is necessary to arouse a decrease in the biosensor province.

Discussion

Many cellular alterations take topographic point in nerve cells upon glutamate reaching. From those, alterations in the metabolic penchant of nerve cells have been a controversial point since the ANLS hypothesis proposed in 1994. Since so, several studies have given support to the thought that encephalon cells adapt their metamorphosis under glutamatergic neurotransmission ( monton de referencias ) . Here, we have shown that neural oxidation-reduction province is another cellular facet which is modulated by glutamate and besides that, alterations in the cut downing capableness of neural cytol depend on the astrocytic metamorphosis.

To our cognition, this is the first clip that the YPF-based oxidation-reduction detector, HyPer, has been used to supervise the oxidation-reduction province in nerve cells. The experimental scheme consisted in pre-oxidize the biosensor with a H peroxide pulsation in order to supervise the rate of fluorescent ratio decay as a physiological reaction of the cut downing capableness of the cytol, where the detector was located. The intracellular decrease of HyPer molecule, and specifically its disulfide bond, is non likely related to hydrogen peroxide clearance nor catalase activity, this procedure is instead governed by glutathione-mediated decrease mechanisms [ 25 ] . A recent work with roGFP1 in nerve cells, a oxidation-reduction biosensor that like HyPer, besides has a cysteine brace to derive sensitiveness to oxidation/reduction reactions [ 33,34 ] showed no dramatic alterations in its baseline fluorescence nor in its response to hydrogen peroxide pulsation ( merely 20 % addition in oxidation grade ) by chronic intervention with 3-amino-1,2,4-triazole, reenforcing the thought that the intracellular clearance of the oxidant agent has small impact in the decrease of biosensors based in cysteine braces ( funke 2011 ) .

There is an overall consensus about that excitant neurotransmission is extremely expensive in footings of energy. Most of the ATP is consumed by nerve cells to power ionic pumps in order to reconstruct the debauched ionic gradients [ 10,35 ] . Glucose and lactate could supply the sufficient energy to let release and re-uptake of neurotransmitter in stray nervousnesss [ 36 ] and besides, let synaptic malleability in hippocampal encephalon pieces [ 12,37 ] . However, recent in vivo grounds obtained from rat sensory cerebral mantle indicates that nerve cells do non modify their glucose consumption during hair’s-breadth stimulation as astrocytes do [ 8 ] . This determination, together with old work done in civilized encephalon cells and encephalon pieces [ 5,7 ] , supports the necessity of an alternate fuel for nerve cells during activity, most likely, breastfeed ( foremost proposed by Magistretti and Al. [ 4 ] ) . In effect, nerve cells should metabolise lactate to piruvate, a procedure catalyzed by the enzyme lactate dehydrogenase, which renders a NADH molecule. Then, piruvate would be farther oxidized by chondriosomes in the Krebs rhythm, where negatrons will eventually cut down O2 to H2O bring forthing more NADH and ATP in the procedure, doing nerve cells more aerophilic than astrocytes during neurotransmission. Consequently with this vision, our experiments indicate that the ague alterations in redox province of neural cytol toward more cut downing possible have a metabolic beginning, they were dependant of the mitochondrial map and besides, were sensitive to the specific damage of the astrocytic metamorphosis. First, we postulate that mitochondrial map, stimulated by exogenic piruvate or lactate, addition cut downing possible non merely into chondriosome, but besides in the cytoplasmic compartment. The redox communicating between these two compartments has been found in HeLa cells, where mitochondrial depolarisation was accompanied temporally with the cytosolic GSH oxidization, both cellular parametric quantities measured by agencies of TMRM and glutaredoxin-roGFP biosensor fluorescency, severally [ 28 ] . Another experimental grounds of the mitocondrial map impact over cytol oxidation-reduction province was found in civilized cortical nerve cells. Here, the writers demonstrated that extracellular glucose is necessary for cytoplasmic oxidization of dihydroethidium by NMDA. Furthermore, the suppression of mitochondrial map increases the dye oxidization and oppositely, piruvate 1mM avoided the consequence of NMDA when was applied in the absence of extracellular glucose [ 38 ] . The mechanisms implicated in the oxidation-reduction transportation between mitochondrial to cytosolic compartments are varied [ 39 ] . Furthermore, the presence of enzymatic mechanism that interchanges NADH for NADPH in eucaryotic cells [ 20 ] plus other beginnings of NADPH coevals besides the classical pentose phosphate pathway do the oxidation-reduction cellular scene notably complex [ 17 ] .

Glutamate and NMDA excitotoxic vision

low dosage of glutamate

short times of exposure,

rapid consequence

Our experimental theoretical account to work

Coexistence of astrocytes and nerve cells, advantages of this theoretical account: astrocytes are necessary for neuron ripening ( cholesterin, synapsis ripening ) .

Use f fluroacetate.Discussion: The energetic depletion protocol has been used before to turn out the astrocytic lactate reassigning a piece theoretical account, where the diffusion is a factor that could impair the effectivity of this manoeuvre. Matthew Parsons 2010, J neurosci vol 30.

pH issue

comparatively minimum acidification with glutamate 10 uM, merely 0.1 pH units

even when is impossible to except the indirect protons reassigning in the “ lactate bringing ” , cut downing displacement of HyPer were recorded in pH clinch experiments with NMDA.

It is deserving to advert that the glucose remotion induces a rapid bead in the intracellular pH, the magnitude making approximately 0.4 point of pH, likely mediated by Na/K pump, due to the suppression of that entity generates an acidification in buffer HEPES on cortical nerve cells, experimental conditions comparables to ours. Toxicon

Volume 50, Issue 4, 15 September 2007, Pages 541-552. However, this experimental manoeuvre did non impact notoriously the HyPer signal in.

another statement is respect to the deficiency of consequence in pc12 cells at glutamate 100 uM, even when these cells count with the glutamate conveyance capableness and in effect the opportunity to sour their cytol with the neurotransmitter outside ( j neurosci 2005-25 ( 26 ) 6221 and Science 2000 289 ( 957-960 )

Sodium piruvate is transported by the facilitative monocarboxylate bearers in its protonated signifier with the attendant acidification of cytol, this scenery that could interfere with HyPer feeling capableness, an issue that was besides exposed before [ 25 ] . To govern out the intracellular acidification as the cause of an impaired H2O2 detection of HyPer molecule, intracellular pH was clamped to 7.1, such value is near to the acidification observed by adding extracellular lactate 10 millimeter at two proceedingss ( non shown ) . Under controlled pH, H peroxide pulsation of 100 i?­M increased the Hyper ratio in approximately 3 units, an increase comparable to the measurings obtained in standard conditions, see auxiliary figure 1. This observation allows us to set up that the feeling belongingss of the HyPer are non affected by such moderate acidification.

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