Fluorescent measurementof[Ca rescue scavenger-mediatedincreasesinNMDA receptorcurrents. scavenging. DiazoxideapplicationfollowingROSscavengingdidnot NMDA receptorcurrentsandwasunaffected bysubsequentROS peroxide decreasedcurrents.AMPA receptorcurrentsand[Ca NMDA receptorwhole-cellcurrentsby100%,whilehydrogen acetylcysteine (NAC).Unlikeanoxia,ROSscavengersincreased modest riseinintracellularCa in NMDA andAMPA receptorcurrentsthataredependentupona Neurological damageisavoidedthroughanoxia-mediateddecreases western paintedturtle,whichoverwintersformonthswithoutoxygen. isoxazolepropionic acid(AMPA) receptors.Thisdoesnotoccurinthe aspartate (NMDA)andalpha-amino-3-hydroxy-5-methyl-4- that [Ca prevented duringanoxiabymitochondrialCa associated withanincreaseinNMDA receptorcurrentsthatis decreases inNMDA/AMPA receptorcurrentsbutarerather decreases inROSconcentrationarenotlinkedtoanoxia-mediated oxygen species(ROS),themostprevalentandstableofwhich is reacts rapidlywithwater, leadingtotheformation ofotherreactive (Chen etal.,2003;Liu2002).Thishighlyreactivemolecule consumed (~3%)isleftpartiallyreducedasthesuperoxide anion a resultofinconsistenciesinelectronflux,portionalloxygen electron acceptorofthemitochondrialtransportchain. As *These authors contributed*These equally tothiswork authors Biology,Evolutionary Toronto, University of Toronto, ONM5S3G5, Canada. 3346 Received 24March 2014;Accepted24June2014 ‡ 1 Aerobic organisms usediatomic oxygen(O Calcium, Fluorescence Whole-cell patch-clamp,Anoxiatolerance,Channelarrest, KEY WORDS:Reactiveoxidativespecies,Pyramidalneurons, asked whethermitochondrialCa decreases in[ROS]increasedNMDA receptorcurrents,wenext concentrations wereunaffected byROSmanipulation.Because through excessivecalciumloadingviaover-activationof Oxygen deprivationtriggersexcitotoxiccelldeathinmammalneurons David JamesDukoff cell currentsinpaintedturtlecorticalneurons Scavenging ROSdramaticallyincreaseNMDA receptorwhole- RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3346-3355doi:10.1242/jeb.105825 sensitive potassium(mK potentiation duringanoxia.NormoxicactivationofmitochondrialATP- INTRODUCTION we scavengedROSwith decreased intracellular[ROS]onNMDA andAMPA receptorcurrents, mechanism toregulateglutamatereceptors.To assesstheeffects of species (ROS)generation,whichisanotherpotentialsignaling from mitochondria.Anoxiaalsoblocksmitochondrialreactiveoxygen ABSTRACT Author ([email protected]) for correspondence Ecologyand of CellandSystemsBiologyDepartment of Department 2+ ] i increases beforeROSdecreases.We concludethat N 1, ATP -2-mercaptopropionylglycine (MPG)or *, DavidWilliam Hogg ) channelswithdiazoxidedecreased 2+ concentrations ([Ca 2+ ] i 2+ and ROSlevelsdemonstrated release preventsreceptor 2+ release. 2 ) astheterminal 2+ ] 1, i ) originating N *, PeterJohnHawrysh -methyl- 2+ N D ] - - i N covered lakesandpondsforupto4 turtle, 1992). Thissequenceofeventsdoesnotoccurinthewesternpainted eventual excitotoxiccelldeath(ECD)(Bosleyetal.,1983; Choi, intracellular ROSconcentrations([ROS] glutathione peroxidase.Thisantioxidantdefensesystemmaintains including: superoxidedismutase,catalaseandglutathione/ ROS concentrationsaremanagedbyaseriesofantioxidantproteins ROS concentrationsandratesofoxidation(Ottavianoetal.,2008). membrane permeabilitytoNa 5-methyl-4-isoxazolepropionic acid(AMPA) receptors,increasing glutamate releaseover-activates postsynapticalpha-amino-3-hydroxy- potential firingandariseinexcitatoryaminoacidrelease.Excessive lost, leadingtomembranepotentialdepolarization,increasedaction hydrogen peroxide(H receptor over-activation resultsinexcessivecalcium (Ca D’Autréaux andToledano, 2007;Rheeetal.,2003). conformation andlevelsofactivity(CrossTempleton, 2006; critical cysteineresiduesontarget proteinsthatcanalterprotein and cellularsignallingprocessesthroughreversibleoxidationof ROS levelshavebeenidentifiedtoplayrolesinfeedbacksystems ROS concentrationscanoccur(Starkov, 2008).Recently, changesin H including nitricoxidefromintracellularsynthaseand et al.,2008).ROSgeneratedfromnon-mitochondrialsources, various cellularcomponents(HenzlerandSteudle,2000;Ottaviano intracellular andextracellularenvironment,wheretheycanoxidize Generated ROSdiffuse outofthemitochondriaandinto in ROSlevels,significantvariations[ROS] 2008; Sies,1993).However, despitemechanismstocontrolchanges and reversesROS-mediatedproteinoxidation(Ottavianoetal., affected bymorethanafewminutesofO unable tosurviveunderanoxicconditionsandaredeleteriously For themostpartitisanon-issueasvertebratespeciesare known whateffects thismayhaveoncellularmetabolismorhealth. depolarization andremovalofthemagnesium(Mg Na that cannotsustainthehighenergetic demandsofneuraltissue. loss ofoxidativephosphorylationreducesATP productiontolevels most rapidlyincurredwithinthecentralnervoussystem,where GABA near itsrestingmembranepotential, atthereversalpotentialfor serves tocounteractexcitatoryinputs byeffectively ‘clamping’ thecell Lutz, 1991).Theconsequentincrease inGABA receptoractivity neurotransmitter gamma-aminobutyric acid(GABA)(Nilssonand in alarge increaseintheconcentrationofinhibitory inhibitory signaling:inthecerebrocortex, theonsetofanoxiaresults withstand extendedperiodsofanoxiaisinpartduetoanincrease in tolerant (Jackson,2000;JacksonandUltsch,1982).Itsability to -methyl- 2 In theabsenceofO O + /K 2 from extracellularxanthineoxidase,alsocontributetobaseline Chrysemys picta + A -ATPase activitydecreasesandmembraneiongradientsare receptor (approximately D -aspartate (NMDA)receptors.Subsequently, NMDA 1 and LeslieThomasBuck 2 Gray 1831.Itoverwintersatthebottomofice- (anoxia) ROSproductionceasesanditisnot 2 O 2 ) (ChandelandSchumacker, 2000). + and resultinginmembranepotential − 80

months andisnaturallyanoxia- mV). Thisresults inadecrease i ) withinnon-toxicranges 2 deprivation. Damageis 1,‡ i 2+ and extracellular ) blockfromthe 2+ ) influxand

The Journal of Experimental Biology activation, Ca pyramidal neurons,whichprotectsagainstexcessivereceptor reduction inNMDA-andAMPA-receptor-evoked currentsin nature andtheirroleinECDmammals.Anoxiainducesa50% to anoxiaisofparticularinterestbecausetheirglutamatergic ~80–90% oftheneuronalpopulation(Ulinski,2007).Theirresponse (Pamenter etal.,2011; Pérez-Pinzónetal.,1992). consumption toaratemetthroughglycolyticfermentationalone in actionpotentialfrequency(75–95%),andareductionATP RESEARCH ARTICLE NMDA andAMPA receptorcurrentsistheresult ofamitochondrial- 2008b; ShinandBuck,2003).Theanoxicdownregulationof C CM-DCF ΔF (% change) AB In theturtlecerebrocortex,pyramidalneuronsaccountfor AAgamma-aminobutyricacid H GABA ECD Ṗ O reactiveoxygenspecies Ψ [ROS] ROS R mitochondrialpermeabilitytransitionpore NMDA NAC mPTP MPG mK AT 1,2-bis(o-aminophenoxy)ethane-N,N,N ′,N-tetraacetic acid alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid [Ca BAPTA AMPA aCSF MDFchloromethyl-2 CM-H CM-DCF CM-H List of symbolsand abbreviations List of –20 –10 v iii i a O 2 m 10 20 30 O 2 2+ TP AT 0 2 ] i i 2 2 oto A oeoeH Rotenone NAC Control C chloromethyl-2 5-(and-6)-chloromethyl-2′,7-dichlorodihydrofluorescein DCFDA DCF * Anoxia Normoxia 2+ MPG influx andECD(Bickler, 1998;Pamenteretal., excitotoxic celldeath mitochondrial membranepotential intracellular ROSconcentration whole-cell accessresistance partial pressureofoxygen N-methyl- N-acetylcysteine N-2-mercaptopropionylglycine mitochondrial ATP-sensitive potassium hydrogen peroxide intracellular Ca artificial cerebrospinalfluid * diacetate, acetylester * Normoxia Rotenone * D MPG -aspartate * ′,7-dichlorofluorescein ′,7-dichlorodihydrofluorescein 2+ concentration * H H 2 2 * O O 2 2 10 min # * 2 O * # 2

500 AFU vi iv ii CM-DCF ΔF (% change) 100 25 50 75 0 10 1 log H Anoxia +Rotenone i.1A,Ci), indicating maintenanceofcellularredoxhomeostasis. Fig. brain, weproposethatthiswilltriggeranincreasein[Ca However, becauseROSlevelsnaturallydecreaseintheanoxicturtle al., 1989;Bodhinathanet2010;ChoiandLipton,2000). significantly increasedbyadecreaseinROSlevels(Aizenmanet vertebrate speciesdemonstratethatNMDA receptoractivityis pyramidal neuronshasnotbeenexplored,althoughstudiesinother receptor currents,wefirstconfirmedthat[ROS] Although themechanismthroughwhichanoxiaactivatesmK whether mitochondrialCa and AMPA receptor currentsinturtlepyramidalneurons,(2) dependent onmitochondrialROSproduction. and (3)usingacomplexIinhibitor, whethertheseresponsesare on NMDA/AMPA receptoractivityor[Ca al., 2008a;ZivkovicandBuck,2010).Theeffect ofchanging[ROS] significantly during1 pharmacological ROSmodulation.Fluorescencedidnotchange brain sheetsduringnormoxiaandanoxia,withwithout ROS-sensitive dye,wemeasuredchangesinfluorescencecortical in mitochondrialmembranepotential( channels hasyettobeestablished,itsactivationinitiatesadecrease based Ca To investigatearolefor[ROS] of 2′ our anoxicexperimentalprotocol.Usingthechloromethylderivative the mitochondrialATP-sensitive potassium(mK mitochondrial Ca (1) whetherdecreasing[ROS] (Pamenter etal.,2007).Theaimsofthisstudyweretodetermine: decrease inNMDA andAMPA receptorwhole-cellcurrents Pharmacological ROS scavengers alter[ROS]Pharmacological RESULTS Anoxia Anoxia +MPG 2 O ,7′ 2 (µmoll -dichlorodihydrofluorescein diacetate(CM-H

2+ 100 1000 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105825 signalling cascadethatisinitiatedbytheactivationof –1 H H ) 2 2 O O 2+ 2 2 release (HawryshandBuck,2013;Pamenteret

h ofnormoxicperfusion(0.46±0.5%; 2+ [95% O treated asindicated.Treatments: (i)normoxia DCF fluorescencerecordingsfromA;neurons relationship of[H fluorescence units. no newROSgeneration).AFU,arbitrary horizontal linearportionofthetraceindicates anoxic controls( normoxic controls. as means±s.e.m.*Significantdifference from fluorescence. DatainA andBareexpressed treatment. Arrowsindicatetheonsetofa5 rotenone. Blackbarsrepresentdurationof rotenone and(vi)anoxiaplus25 anoxia plus0.5 N concentrations ofROS([ROS] (ROS) scavengingdecreaseintracellular Fig. DCF) fluorescence( chloromethyl-2 (A) Summaryoftreatment-inducedchangesin N cerebrospinal fluid(aCSF)],(ii)anoxia(95% application of50 2 -2-mercaptopropionylglycine (MPG),(iv) release isinducedbyROSscavenging, /5% CO i

.Anoxiaandreactiveoxygenspecies 1. decrease whole-cellevokedNMDA 2 /5% CO i 2 in modulatingNMDA/AMPA bubbled aCSF),(iii)0.5 Ψ ′

,7′ mmol m 2 P μmol 2 -dichlorofluorescein (CM- ) andtriggerssubsequent O bubbled artificial <0.05). (C)SampleCM- # 2 Significant difference from Δ ] versusCM-DCF l 2+ F − l − 1 ). (B)Dose–response i 1 ] MPG, (v)25 μmol i were eliminatedby H in turtlecortical 2 i O 2 TP AT (note: the 2 μmol i DCFDA), a ). ) channel.

mmol 2+ ] l − i 1 and a 3347 n l −

l =8; min 1 − TP AT 1

The Journal of Experimental Biology increased evokedNMDA receptorcurrentsafter20(201.4±7.1%; presented insummarygraphs.)ROSscavengingduringnormoxia not significantlydifferent; therefore, onlythe20 washout (Fig. function, weaddedrotenone(25 mitochondrial ROSproductionandNMDA/AMPA receptor signaling cascade.To investigatetheconnectionbetweendecreased making itapotentiallyimportantcomponentofanyROS-mediated transport chainistheprimarysourceofROSgenerationincell, (82.7±12.2%; (58.6±9.0%; ( with 50 μmol hl-elcnutne(S .±. 4.4±0.5 4.3±0.6 Action potentialthreshold(mV) Whole-cell conductance(nS) P significantly decreasedCM-DCFfluorescenceby normoxic artificialcerebrospinalfluid(aCSF)plusrotenone dehydrogenase) inhibitor, totheperfusate.A 30 respectively; normoxia plusROSscavengers( decrease fluorescencebeyondtheeffects ofeitheranoxiaaloneor addition ofMPGorNACduringanoxiadidnotsignificantly Fig. and 3348 RESEARCH ARTICLE aae(al 1).A 5 damage (Table action potentialthreshold,indicatingthatitdidnotinduceoxidative neurons, suchasmembranepotential,whole-cellconductanceand did notaffect baselineelectrophysiologicalpropertiesofpyramidal Lei etal.,1998;Milton2007).Inaddition,thisconcentration Data shownrepresentmeans±s.e.m. ( Membrane potential(mV) currents after20(61.9±8.6%; 2A,Bi).AnoxicperfusiondecreasedevokedNMDA receptor Fig. significantly after90 Evoked NMDA receptorcurrent amplitudesdidnotchange H and firstdeterminedanappropriatephysiologicalconcentrationof (0.5 mercaptopropionylglycine (MPG)or normoxic controls.A 30 A 30 fluorescence inadose-dependentmanner( found thedatatonot besignificantlydifferent. agreement withnormoxicmeasurementsofH could detectasignificantchangeinfluorescence.Thisfindingis parameters ofcorticalpyramidalneuronsinthewesternpainted and 25.4±1.3%,respectively; increased fluorescenceduringbothnormoxiaandanoxia(23.6±1.1 turtle Table of mammalianneuronal[H cultured turtleneuronsandisnearthereportedphysiologicalrange activity ROS manipulation modifiesNMDAPharmacological receptor comparing normoxia withH that ROSlevelscouldbeexperimentallyincreased,weusedH no additionaleffect ( − ≤ 2 7.2±1.2%; O 0.001; Fig. mmol 2 1A,Ciii) comparedwithnormoxiccontrols.Conversely, the

− 1. Effectof50 min anoxictreatmentsignificantlydecreasedfluorescence to apply. DripapplicationofH 7.3±0.5%, respectively;

l − 1 n l n each) significantlydecreasedfluorescence( 1A,Cv), whileadditionofrotenoneduringanoxiahad =7, 2A,Bii). (Note:20and40 − =5 each;Fig. n 1 n =6) and40 [H =8, P 2 =0.008) oftreatment,andwerereversedafter20 µmol O − 2 9.1±0.6%; ] beingthelowestconcentrationatwhichwe P

min ofnormoxicperfusion( 2 ≤ O

l min applicationofH 0.001; Fig. − 2 1 2 within eachmeasured parameternormoxic

H O min normoxicperfusionwith

min (98.2±4.2%; 1Ciii,iv). Themitochondrialelectron n 2 2 O ] =5 each, i 2 (1–20 μmol n on electrophysiological n μmol =4; Fig. n =5 each,P =4 pertreatment).A t n =7, − Normoxia − − 43.5±1.4 86.3±0.6 1A,Cii) comparedwith P 9.2±0.9 and−

l min treatmentvalueswere − N ≤ 2 1 O 1A,Cvi). To demonstrate 0.001 forboth;Fig. P ), acomplexI(NADH -acetylcysteine (NAC) 2 =0.004) and40 n l 2 − =4–8 each;Fig. O 1 increased CM-DCF ) (Hoytetal.,1997; 2 n ≤ from themediaof =5) ofnormoxic 2

0.001 forboth; min perfusionof − O 8.6±1.5% (n 2

min dataare -test significantly n 8.7±0.5%, =8 each; − − H − 39.3±1.2 80.3±3.1 9.1±1.5 2 O 2

N 1A). 1B), min 2 =4, -2- O 2 and 40 n=6, Fig. 20 ROS. after 20and40 maintained, normoxicwashout failedtoreversetheeffects of MPG in ~50%ofrecordings.In situations wherethepatchwas hyperactivity, depolarizationinallpatchesandthelossof patch (Fig. application (Fig. normoxic control( expressed asmeans±s.e.m.*Significantdifference fromthepaired normoxic controls;dashedlinerepresentsanoxiccontrols.Dataare indicated treatment(i–vi).The20and40 recordings ofthenormoxicbaselinecurrentandfollowing20 did notdiffer statistically;therefore, onlythe20 each, min oftreatmentand20

2. P=0.001) and40 min(195.1±16.3%; (A) Whole-cellNMDA receptorpeakcurrent amplitudesfollowing 2A,Biv). Theeffects oftheseincreasesresulted in P N A B <0.001 forboth) butatrendtowardsrecovery was observed -methyl- i v iii

min (208.9±37.3%; NMDA receptor current (% normalized) 100 150 200 250

150 pA 200 pA Normoxia 50 0 100 pA The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105825 20 s 20 s 20 s

D min (182.8±16.3and186.7±5.3%, respectively; P -aspartate (NMDA)receptorcurrentsaremodulatedby

<0.05). (B)SamplepairedNMDA receptor current 2A,Biii) orafter20(192.5±19.9%; Recovery Treatment Anoxia

MPG min ofwashout.Continuouslinerepresents * H Normoxia Control 2 O Control 2 n Control

=4, MPG * *

P iv vi ii min treatmentandrecoveryvalues =0.035) ofNACapplication 500 pA 200 pA 200 pA 20 s 20 s

20 s NAC *

min datahavebeenincluded. * n=4,

H 2 *

P≤0.001) ofMPG O Rotenone 2

Anoxia Rotenone NAC Control

n min ofthe =6, Control Control * P =0.046) n =3

The Journal of Experimental Biology (93.2±7.1 and99.3±7.8%,respectively; which wasreversedafter20and40 resulted inaslow increaseinfluorescence(11.4±0.9% over10 139.6±24.7%; (103.5±2.8%; (Fig. n effect onevokedAMPA receptorcurrentsafter20(105.4±4.6%; in summarygraphs.)ROSscavengingduringnormoxiahadno significantly different; therefore,onlythe20 97.04±3.86%, respectively; and 3,respectively)NACapplications(98.62±2.81 following MPG(95.26±3.29and97.81±12.60%,respectively; remained unchangedthrough20and40 baseline following20 ROS modulation.Fluorescencedidnotchangesignificantly from fluorescence incorticalbrainsheetswithandwithoutpharmacological (1.0±1.3%; scavenging withMPG(0.6±0.6%; n fluorescence (16.9±3.3%; 4 A,Bi). Anoxicperfusionresulted inasignificantincrease Fig. Using theCa n decreased evokedNMDA receptorcurrentsafter20(80.2±3.1%; Fig. 104.6±3.4%, respectively; reversed after20and40 To investigatewhether[ROS] currents after20(100.0±3.4%; addition duringnormoxiahadnoeffect onevokedAMPA receptor currents at20(69.95±5.69%; 3A,Bi).AnoxicperfusiondecreasedevokedAMPA receptor Fig. significantly after90 Evoked AMPA receptorcurrentamplitudesdidnotchange 40 receptor currentsafter20(216.6±30.1%; mitochondrial ROSformation.RotenoneincreasedevokedNMDA rotenone wasadministeredundernormoxicconditionstoprevent regulation ofNMDA receptoractivity, thecomplexIinhibitor In ordertoevaluatetheroleofmitochondrialproducedROSin after 20and40 n normoxic washout(106.33±3.91and100.00±2.95%,respectively; of treatment,andremainedunchangedthrough2040 Fig. 40 (55.16±6.04%; RESEARCH ARTICLE assessed theeffect ofpharmacological ROSscavengingon[Ca Pharmacological ROS manipulation on[Ca hasnoeffect Pharmacological activity receptor ROS manipulation doesnotmodifyAMPAPharmacological currents receptor mitochondrial NMDA ROS increases production Inhibition of treatment (datanotshown). after 20(106.9±3.9%; 0.05% chloroformandthisdidnotaffect NMDA receptorcurrents rotenone, neuronswereexposedtoasalinesolutioncontaining that thiswasnotaresultofthechloroformusedtosolubilise =9, =5 and3,respectively;Fig. =6) and40 Exposure toMPGorNACbeyond the40 min ofnormoxicwashout(98.8±4.9and101.3±5.2%; min (232.3±28.5%; 4A,Bv) didnotsignificantlychange fluorescence. 3A,Bii). (Note:20and40 P 3A,Biii) orafter20(100.5±3.2%; =0.023) and40 n 2+ =6; Fig. -sensitive dyeOregonGreen,wemeasuredchangesin n n min (94.7±7.3%; =3 each;Fig. =6) ofNACapplication(Fig. n =5,

min ofNACwashout(168.4±26.1and 4A,Biv) oradditionofH

P min (78.4±4.4%; min ofnormoxicperfusion(0.6±0.8%;

min ofnormoxicperfusions( =0.001), andwerereversedafter20

n min ofnormoxicwashout(100.1±2.9and n =3) or40 =4, n n n =6,

=4 and3,respectively;Fig. 3A,Bv). =3 each;Fig. 2A). H i P n modulates Ca =11) and 40 P =0.01) oftreatment,whichwas

min treatmentvalueswerenot =0.001; Fig. n n 2 min (109.5±0.18%; =5, O =4) ofMPGapplication

=6; Fig. min ofnormoxicwashout n 2 n min ofnormoxicwashout =5; addition duringnormoxia =4 each;Fig. P

2A,Bvi). To determine

=0.006) and40 P min dataarepresented 2 min (99.2±2.4%; 2+ O min treatmentperiod =0.036) oftreatment, n 4A,Bii) whileROS 2 n =5,

signalling, wenext 3A,Biv). Currents 4A,Biii) orNAC (0.84±1.39%; =7) and40 P =0.017) and

2A,Bv).

n 3A). H n =6 each; =4 each; n

min of =3) of 2+

n n min; n min min n 2+ =7) 2 =6; =6; ] =4 O i ] i 2 . not shown),indicatingthatCa over-activation ofNMDA receptorsand (2)thatROS-scavenger- long-term ROSscavengerperfusion istoxictoneuronsbecauseof likely thesourceofthisincrease. Thissupportsourfindingsthat(1) n 20 manipulation. (AMPA) receptorcurrentsareunaffectedbypharmacologicalROS Fig. reduced thefluorescentincrease (5.1±0.5%over10 phosphonovaleric acid,aselective NMDA receptorinhibitor] normoxic control( expressed asmeans±s.e.m.*Significantdifference fromthepaired normoxic controls,dashedlinerepresentsanoxiccontrols.Dataare recordings ofthenormoxicbaselineandfollowing20 differ statistically;therefore, onlythe20 treatment (i–v).The20and40 =4; datanotshown).Dripperfusion ofAPV [(2R min oftreatmentand20

B A .Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid 3. iii i AMPA receptor current (% normalized) 100 125 25 50 75

200 pA 0 100 s 200 pA 100 s Normoxia The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105825 (A) Whole-cellAMPA receptorpeakcurrentamplitudesafter P <0.05). (B)PairedsampleAMPA receptorcurrent 100 pA Recovery Treatment v 50 s

MPG Anoxia Control

min ofwashout.Continuouslinerepresents * Normoxia

min treatmentandrecoveryvaluesdidnot Control 2+

influx throughNMDA receptorsis MPG

min datahavebeenincluded. ii iv 500 pA

100 s 200 pA H 50 s 2 Control O 2 NAC

min oftheindicated * NAC Anoxia

Control min; H 2 O

)-amino-5- 2 Control n =4; data 3349

The Journal of Experimental Biology mK mitochondrial Ca tested theeffect ofpharmacologically increasingmitochondrialCa assessed theeffect onwhole-cell NMDA receptorcurrents.First, we receptor currents,wepharmacologicallymodulatedbothsignals and 3350 RESEARCH ARTICLE [Ca release priortoROSscavenging. To achieveasmallincrease in Anoxia-mediated activationofmK Subsequent anoxicH P evoked NMDA receptorcurrents after20(69.6±3.5%; receptor currentsweremeasured.Anoxicperfusionalonedecreased applied followingthetransitiontoanoxiaandchangesinNMDA current amplitudeswereaffected byincreasesinROS,H To determinewhetheranoxicdownregulationofNMDA receptor (5 ethane-N,N,N′ clamp experimentswhereinclusionof1,2-bis(o-aminophenoxy) likely thatthiswasafactor. Thisis supportedbywhole-cellpatch scavenger-mediated increaseinCa treated neuronsaredifficult torecover. BecausethereisaROS- mitochondrial Ca currents. To betterunderstand theinteractionofanoxic decreasing [ROS] and Buck,2010).However, thisoccurs atthesametimeas currents (HawryshandBuck,2013;Pamenteretal.,2008a;Zivkovic prior totheROS-scavenger-mediated increasein[Ca signaling proteins.However, becauseexperimentswerecompleted n decreased evokedNMDA receptorcurrentsafter20(66.9±3.9%; al., 2008a;ZivkovicandBuck, 2010).Diazoxideadministration receptor currentsafter20(205.37±23.58%;n in NMDA receptorcurrentamplitudes after20(58.6±6.73%; at the40 Mitochondrial Ca byH unaffected Anoxia-mediated changes inNMDA activityare receptor increases inNMDAincreases currents receptor (Fig. (222.65±33.24%; Normoxic washoutdidnotreversetheanoxiceffects after20 changes werenotsignificantlydifferent fromanoxiaalone( P (59.00±11.48%; (75.36±10.10%; A =5, =0.031) and40 =0.001) and40 Oregon green ΔF (% change)

mmol Normoxia 2+ 10 15 20 25 TP AT 0 5 P 5A,Bii). MPGapplication followingdiazoxide treatmentdid ] i =0.049) and40 channel activatordiazoxidewas administered(Pamenteret and generatereductionsinNMDA receptorcurrents,the

l

Anoxia − min timepointcouldbemodulatedbyCa 1 ) didnotpreventMPG-mediatedincreasesinNMDA * ,N′ MPG n n 2+ 2 min (54.36±5.57%; -tetraacetic acid(BAPTA) inthepipette n 3 i.5). =3; Fig. i 2+ =3, min (66.6±6.0%; O 2+ , whichwefoundtoincreaseNMDA receptor =4, release andpreventsexcessiveNMDA receptor 2 2

release preventsrelease ROS-scavenger-induced NAC release anddecreasing[ROS] application O min (62.6±5.7%; P P 2 =0.005) butdidreversethemafter40 =0.012; datanotshown). administration didnotreversethedecrease H 2 O

2 2+ , itispossiblethatdatacollected n iii B v ii n =5, =5, n TP AT =5, P P =4, =0.013) oftreatment. P <0.001) oftreatment; channels leadsto =0.022) oftreatment P MPG Normoxia =0.020) or40 H 2 O i 2 2+ 2+ on NMDA ] -activated i , itisnot P 2 O >0.05). 2 n n

was min min min =5, =5, 2+ (101.6±11.4 and104.2±8.0%,respectively; effects werereversedafter20and40 anoxic perfusionandrecovery. Bathchamber alone (P P effects ofROSscavengingarereversedbyanincreasein[Ca respectively; 40 P reverse MPGeffects significantlyafter20(180.4±11.8%; P evoked NMDA receptorcurrentsafter 20 to NMDA receptorover-activation. MPGadministrationincreased encountered inpreviousROSscavengingexperimentsandattributed was limitedto20 diazoxide wasappliedaftertheadditionofMPG.MPGapplication and steady-statechangesin increase inrhodaminefluorescence( the mitochondriaandisresultofdepolarization NMDA andAMPA receptorcurrentsduringanoxiaoriginatesfrom assessed thechangesinbathchamber 20 (57.3±5.5%;n not produceincreasesinNMDA receptorcurrentamplitudesafter Ṗ the switchtoanoxicperfusion,onsetofdecreaseinchamber levels tothepre-anoxicvalues(146.4±2.7 probe placedinthecenterofbathchamberduringa30 in partialpressureofoxygen( AMPA receptorinhibition,wenextassessedthetimelineofchanges To understandthesequenceofeventsleadingtoanoxicNMDA and formation 597±62.8 anoxic switchandreacheda plateauindicatingnonewROS rate ofROSgenerationstarted todecrease120.2±7.6 196±19.9 s aftertheswitchtoanoxia( 70.4±5.6 123 andchangesin[Ca 607±63.7 investigate thetimelineofthisCa i.6A).ReperfusionwithnormoxicaCSFreturnedchamber Fig. 0.52±0.3 mmHg duringa30minanoxictreatment( decreased fromanormoxicvalueof147.32±2.8 Oregon Greenfluorescence increase (elevated[Ca 355±25.1 70.6±5.1 Increases in[Ca Increases [ROS] iv =0.003) or40 =0.036) orproducechangessignificantlydifferent fromdiazoxide =0.001; Fig. i O 2 min ofnormoxicwashout(83.7±21.2and89.8±22.6%, occurred in6.0±0.7 i decreases during anoxiadecreases >0.05; Fig. Anoxia s aftertheswitchtoanoxia and reachedsteadystate s aftertheswitchtoanoxiaandreachedasteadystate s (n NAC s aftertheswitch( The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105825 =5 each).TheCa n 5A,D). Subsequentdiazoxideapplicationdidnot 10 min min (175.4±19.8%; =4 each;Fig. 2+ min inordertotryandpreventthelossofpatch =5, ]

5A,Bii). Theeffects werereversedafter20and s aftertheanoxic switch( i

and decreases in and decreases 200 AFU P 2+ =0.008) and40

s andreachedasteady-statelevelby ] Ψ i with OregonGreen.Theonsetofthe m Ṗ

5A,Bii). To determinewhetherthe n using thefluorescentdyerhodamine- O 2+ Fig. ( from thepairednormoxiccontrol as means±s.e.m.*Significantdifference Green fluorescence.Dataareexpressed treatment-induced changesinOregon illustrated inBii. two steady-stateparalleltangents,as fluorescence werecalculatedbetween duration oftreatment.Changesin indicated (i–v).Blackbarsrepresent traces fromA;neuronstreatedas effect on[Ca P =11 each;Fig. 2 ), <0.001). (B)Drift-correctedsample signal responsibleformodulating

2+ Ψ n .Manipulationof[ROS] 4. =4, m signal, weassessedtheonset , [Ca Ψ n=5 foreach;Fig.

Ṗ m min ofnormoxicwashout P O Ψ =0.006; Fig. depolarization) occurred

min (198.9±21.2%;n mmHg; 2 2+ m 2+ min (62.8±8.8%; n using afluorescentO ] ] occur priorto =4 each;Fig. i i . (A)Summaryof n and [ROS]

=8 each;Fig. 6B). Theonsetof Ṗ n O =5). Following 2 n=5, 5A,Biii). The significantly 2+

mmHg to s afterthe i . First,we ] 6C). The i P≤0.001; i

) began 5A). has no Ψ

6D). m n n

. To min 2+ Ṗ =4, =4, =4, O ] i 2 2 ,

The Journal of Experimental Biology receptor peakcurrentamplitudesafter20 changes areunaffectedbyROSmanipulation. on [Ca scavengers, wedemonstratethatdecreasesin[ROS] glutamate receptoractivityinreptiles.Usingpharmacological ROS investigation intotheeffects ofoxidizing/reducingagentson western paintedturtle.To ourknowledgethisisthefirst glutamatergic signallinginpyramidal neuronsoftheanoxia-tolerant In thisstudyweexploredtheeffects ofscavengingROSon RESEARCH ARTICLE exogenous application ofH 100% increaseinNMDA receptorwhole-cellcurrentswhile application ofROSscavenging agentsresultedinanapproximately Fig. scavenging agents(MPGorNAC) orH 2010). Undernormoxicconditions, theapplicationofROS currents duringanoxia(Pamenter etal.,2008b;ZivkovicandBuck, an increasein[Ca The 20and40 initial treatmentandafter20 current recordingsofthenormoxicbaseline,following20 normoxic control( expressed asmeans±s.e.m.*Significantdifference fromthepaired normoxic controls;dashedlinerepresentsanoxiccontrols.Dataare combination treatment2,and20 DISCUSSION therefore, onlythe20

.Anoxia-ormitochondrial-Ca 5. A B iii i

2+ 200 pA 100 pA 20 s 50 s ]

i NMDA receptor current (% normalized) 100 150 200 250 or AMPA receptorwhole-cellcurrents(Figs Anoxia + H 50 0

min treatmentandrecoveryvaluesdidnotdiffer statistically; Anoxia P <0.05). (B)Pairedsamplewhole-cellNMDA receptor * 2+

MPG

min datahavebeenincluded. Recovery Anoxia +H ]

Control 2 i

Diazoxide +MPG O * Anoxia or reductionsinNMDA andAMPA receptor Diazoxide + MPG2

Control min oftheindicatedcombinationtreatment(i–iii). Diazoxide *

min ofwashout.Continuouslinerepresents 2 O 2 O 2 2 * 2+ decreased whole-cell currentsby ii Recovery -release-mediated NMDA receptor 100 pA

* 25 s min oftreatment1,20 MPG + Diazoxide 2 O 2 (A) Whole-cellNMDA MPG did nothaveaneffect Diazoxide +MPG * Recovery Diazoxide

min oftheindicated * Control i do notinitiate

min of

3, 4).The protocol. (B)Rhodaminefluorescencetracedemonstratingthetimingof [ROS] ROS viaH approximately 20%(Fig. depolarization withanoxictreatment( oxygen (Ṗ Fig. outlining changesin[ROS] zero tosimplifyinterpretationofthefigure. corrected anddatainB–Dwereartificiallysettoabaselinefluorescence of reduce noiseandhighlightthetimelineofevents.DatainBCwere drift portions ofthetraceswerefittedwithanon-linearfour-parametercurve to average of10regionsinterestperreplicate.Theonsetandrecovery state levelsareshownineachpanel.EachtraceB–Drepresentsan response followingtheswitchtoanoxicperfusionandtimesteady- neither treatment affects AMPA receptorcurrents(Aizenman etal., agents (e.g.dithiothreitol)increase NMDA receptorcurrentsand nitrobenzoic acidorglutathione disulfide)decreaseandreducing which applicationofoxidizing agents(e.g.5,5 consistent withresultsfromstudies ofothervertebratespeciesin effect onAMPA receptorcurrentsinresponsetoROSscavengingis anoxia-mediated decreasesinNMDA receptorcurrents. trace showingchangesin[Ca Our findingsofachangeinNMDA receptorcurrentsandno

.Timeline ofanoxia-inducedchangesinpartialpressure 6. ·

i ΔF ΔF ΔF P . (A)Sampletraceofbathchamber CM-DCF (AFU) Oregon Green (AFU) Rhodamine (AFU) Chamber O2 (mm Hg) 200 400 600 200 400 600 100 200 300 100 125 150 25 50 75 0 O 0 0 0 2 ), mitochondrialmembranepotential(Ψ 2 01 02 03 04 05 60 55 50 45 40 35 30 25 20 15 10 5 0 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105825 O D C B A 2 application underanoxicconditionsdidnotreverse Anoxia i with anoxia( Steady state:597s Onset: 120s Steady state:196s Onset: 70s Steady state:355s Onset: 70s Steady state:607s Onset: 6s 2+

2). Furthermore,there-introductionof ] i with anoxia( Time (min) n =11). (C)OregonGreenfluorescence n =8). Time totheonsetof Ṗ O 2 n during anoxictreatment =5). (D)CM-H Recovery m ), [Ca 2 DCF trace ′ 2+ -dithiobis-2- ] i and 3351 Ψ m

The Journal of Experimental Biology between mitochondria,aprimarysiteforintracellular Ca ROS changesmaybeinvolvedinanegativefeedbacksystem mitochondria hasyettobeestablished;however, weproposethat function ofNMDA receptorredoxcontrolortheconnectionto modulatory sites(Mollajewetal.,2010;Ottaviano2008). The channels, inordertooxidizeextracellularNMDA receptorredox freely diffuse acrossthecellularmembrane,ormovethroughion conditions when ATP levelsarehighand are activatedduringanoxia 3352 RESEARCH ARTICLE storage/regulation, andNMDA receptor-mediated Ca scavenging orH oxidizing agents,aratiocomparabletothe5:1wefoundforROS agents isgenerallysixto11 timesgreaterthandecreasesinitiatedby NMDA receptorcurrentsresultingfromapplicationofreducing 2000; Gozlanetal.,1995;Janáky1993).Theincreasein 1989; Bodhinathanetal.,2010;Choi2001;andLipton, 2007; Duganetal.,1995).A feedbackloopbetweenCa death. storage couldbeutilizedtopreventexcessiveuptakeleading cell scavenging maypermitexcessiveCa 2000). Thelarge increaseinreceptorcurrentstriggeredbyROS (Aizenman etal.,1989;Bodhinathan2010;ChoiandLipton, mitochondrial uncouplersorCa and Verstreken, 2006).H necessary ATP forsynapticactivities(DanyszandParsons,1998;Ly excitatory synapses,asaremitochondria,whichprovidethe receptors arehighlyexpressedinthepost-synapticdensitiesof in oxidation/degradationofthescavengingagentsused.NMDA lack ofrecoveryseenduringROSscavengingistheresultdelays achieved withinthereperfusionperiod,supportingideathat NMDA receptoractivity. Fullrecoveryfromrotenonetreatment was changes inmitochondrialROSproductioncandirectlymodulate to adegreesimilarthatofROSscavenging,demonstrating inhibitor rotenoneresultedinincreasesNMDA receptoractivity degraded (Ottavianoetal.,2008). baseline concentrationsarere-establishedandscavengingagents if extracellularlevelsareeliminated,itmaytakesometimebefore 2010; Köhretal.,1994).BecausecellularROSproductionisslow, reducing agentswerereversedbywashout(Bodhinathanetal., similar tootherstudiesinwhichtheeffects ofoxidizingbutnot reversed byreperfusionbuttheeffects ofMPGandNACwerenot, result ofmK triggered hasyettobeestablished. We haveproposed thatitisthe activation unlessblockedbytheremovalofextracellular Ca Mitochondrial ROSgenerationisincreasedbyNMDA receptor Buck, 2013).mK (Pamenter etal.,2008a;Zivkovic andBuck,2010;Hawrysh conductance mitochondrialpermeability transitionpores(mPTPs) al., 2008).Thismayalsoexplainwhytheeffects ofH the cellandareslowtodiffuse out(Jonesetal.,2000;Ottaviano oxidized statebecauseextracellularantioxidantsareproducedwithin extracellular redoxsitesbeingmaintainedinapredominantly agents, comparedwithoxidizers,isthoughttobetheresultof et al.,1994).ThehighsensitivityofNMDA receptorstoreducing et al.,2001;GozlanandBen-Ari,1995;Kim1999;Sullivan collectively termedNMDA receptorredoxmodulatorysites(Choi NMDA receptorsubunits(GluN1,GluN2A andGluN2B), to theexistenceofextracellularcysteineresidueslocatedonspecific death. TheredoxsensitivityofNMDA receptorshasbeenattributed NAC/MPG applicationoftenresultedincelldepolarizationand pyramidal cellsduringNMDA application,explainingwhy The mechanismbywhichCa Blocking mitochondrialROSproductionusingthecomplexI TP AT activation andsubsequentformation oflow- 2 TP AT O 2 channels remainclosedunder normoxic application inturtlepyramidalneurons 2 O 2 produced fromthesemitochondriacan 2+ 2+ release fromthemitochondria is uniporter inhibitors(Duanetal., 2+ influx intopatched 2+ influx and 2 2+ O 2 entry. were 2+ 2+ , seen whentheanoxia-mediatedincreasein[Ca receptor activitywillriseandmayleadtoECD.Thiseffect wasalso if increasesin[Ca receptor currentsbroughtaboutbyROSscavenging,suggestingthat was notsuccessfulinreversingthelarge increasesinNMDA is importanttonotethatapplicationofdiazoxideafterMPGaddition a Ca decrease andreachessteadystate~400 result ofROSdecreases(Shinetal.,2005).FormitochondrialCa significant increasesinNMDA receptor currents,potentiallyasa downregulation oftheNMDA receptorandalsoproduced range (CanepariandMammano,1999),itislikelythat (Plášek andSigler, 1996)andOregonGreen inthemillisecond rhodamine-123 hasaresponsetimeintheseconds–minutesrange dyes occurredat~70 increase influorescenceofbothrhodamine-123andOregonGreen receptor activity. that block/inhibitNMDA receptorredoxsitesanddecreaseNMDA subunits duringanoxiamaycausechangesinproteinconformation Calmodulin bindinganddephosphorylationofNMDA receptor dependent phosphatasePP2B(calcineurin)(Shinetal.,2005). (Ca supports thehypothesisthatanoxia-mediateddepolarizationof chamber Interestingly, changesin NMDA receptorsbeforeredoxmodulationcouldoccur. steady state.We propose thatthisprovidessufficient timetoinhibit application, indicatingthatCa currents comparabletoanoxiaandwasunaffected byMPG channel agonistdiazoxideproducedadecreaseinNMDA receptor increase in[Ca (Hawrysh andBuck,2013).Themechanismthroughwhichan chelator BAPTA blocksthedecreaseinNMDA receptorcurrents permits aninfluxofK as mitochondrialATP production decreases.Openingofthechannel painted turtleare notresponsiblefortriggering downregulationof during anoxiawithinthecortical pyramidalneuronsofthewestern dependency ofCa aforementioned informationcollectively, weconcludethatthe time occurs beforetissue is thesignaltoinducemitochondrialCa depolarizion occurredpriortoCa activation ofmK release topreventredox-inducedNMDA receptorpotentiation comparable tothoseinducedbyanoxia,andtheadditionofCa decreases NMDA receptorcurrentsandincreases[Ca western paintedturtle,additionofthemPTP activatoratractyloside mediated increaseinmitochondrialCa control andinitiatesreceptorinhibition.Replicatingtheanoxia- indicates thatasecondarymechanismoverridesmodulatoryredox activity; however, receptorcurrentsdecreaseduringanoxia,which formation andCa turtle brain. ROS, andthisorderofevents mayhavebeenselectedforinthe Ca compared changesin assess thetimingofintracellularanoxia-mediatedsignals,we activity oftheCa receptor currentshaspreviouslybeenattributedtoincreasesinthe anoxia-mediated Ca We haveshownthatdecreasesin[ROS] In summary, wehavedemonstratedthatdecreasesin[ROS] 2+ 2+ /calmodulin) andsubsequentactivationoftheCa release mustalloccurbeforeanoxicdecreasesinROS.To 2+ Ṗ O chelator, whichpreventedtheanoxia-mediated The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105825 2 reached ~0 2+ TP AT ] 2+ i 2+ 2+ brings aboutadecreaseinNMDA andAMPA ] release (MurchisonandGriffith, 2000).Inthe 2+ channels, mPTP formationandmitochondrial i Ṗ release iscrucialwithrespect tochangesin 2+ do notoccurbeforeROSdecreases,NMDA O + signal occurs~40 Ψ -binding messengerproteincalmodulin

2 mmHg, indicatingthatdepolarizationof s aftertheswitchtoanoxia.Because into themitochondriathattriggersmPTP m reaches 0 Ψ , [Ca m and [Ca 2+ 2+ release overridesredoxcontrol.It ] i 2+

mmHg. Whenconsideringthe and [ROS] release. Thisfindingfurther 2+ ] 2+ 2+ i

i s before[ROS] occurred beforethebath s before[ROS] increase NMDA receptor release. Theonsetofthe release withthemK 2+ i . Theonsetofthe ] i was blockedwith 2+ 2+ /calmodulin- ] i i i to levels begin to reaches Ψ Ψ Ψ TP AT 2+ 2+ m m m i

The Journal of Experimental Biology Pipette solutionscontained(inmmol borosilicate glasspipettes(HarvardApparatusLTD, Holliston,MA,USA). results andofparticularnote,switchingbetween95%O HEPES, 110 Kgluconate,1MgCl gassed with95%N chamber, andthe spacebetweenthefluidsurfaceandcoverwasgently with aholefortherecordingelectrodewasplacedoverperfusion 1 MgCl Normoxic aCSFwasgassedwithair/5%CO not different fromreservoir experiments wereperformedataroomtemperatureof22°C. jacketed inthesamemannerasabovetomaintainanoxicconditions. All (see detailsbelow).Fast-stepperfusionsyringeswerealsobubbled and used todeliverpharmacologicalmodifiersdirectlyabovethecortical sheet controller andSF-77Bfast-stepperfusionsystem;Warner Instruments)was Denmark). A fast-stepdrugperfusionsystem(VC-6modelvalve analyzer andWitrox-1 v1.6.0software(Witrox 1,LoligoSystems, was usedtoposition themwithinthetissue.Cell-attached 5–10 micromanipulator (Burleigh,PCS-6000 series,Thorlabs,Newton,NJ,USA) CA, USA)wasinsertedintopipettes andamotorizedpatch-clamp headstage andMultiClamp700Bamplifier (MolecularDevices,Sunnyvale, osmolarity 295–300 cerebrospinal fluid(aCSF;inmmol cortical sheetswereisolatedfromwholebrainsandbathedinartificial area ofthedorsalcortexwereobtainedusingfire-polished4–6 Whole-cell recordingsfrompyramidalneuronslocatedinthedorsomedial changes). Bathchamber and air/5%CO was rapidlyexcisedfromthecraniumwithin30 (Shin andBuck,2003).Briefly, turtlesweredecapitatedandthewholebrain recordings undernormoxicandanoxicconditionsaredescribedelsewhere Basic protocolsforcorticalsheetdissectionandwhole-cellpatchclamp Adult femaleturtles(carapacelength~15 Council onAnimalCareregardingthecareanduseofexperimentalanimals. Committee andconformstotherelevantguidelinesissuedbyCanadian This studywasapprovedbytheUniversityofToronto AnimalCare anoxia, mitochondrialCa vertebrate species.Ourfindingsindicatethatduringthetransitionto oxidative/reductive challengesinamannersimilartoother that NMDA receptorsinturtlepyramidalneuronsrespondto NMDA andAMPA receptors.Instead,wehaveprovidedevidence Turtles weregivencontinuousaccesstofoodandkeptona12 temperature wasmaintainedat~18°Candtheair20°C. heating lampandaflow-throughdechlorinatedfreshwatersystem.Thewater housed inlarge indoorponds(2×4×1.5 from NilesBiologicalInc.(Sacramento,CA,USA).Theanimalswere light:dark photoperiod. RESEARCH ARTICLE downregulation NMDA andAMPA receptoractivity. is essentialforoverridingmechanismsofredoxcontrolandthe experiment tomaintainanoxicconditions. vigorously for30 N ~0 The chamberwasgravityperfusedwithaCSFatarateof2–3 26 chamberwithaP1platform(Warner Instruments,Hamden,CT, USA). (pH and theouterjacketwasgassedwith95%N chamber, perfusion tubesfromtheintravenousbottleweredoublejacketed effect onROSproduction.To maintainanoxicconditionsinthebathing were obtainedusing blind-patchtechniquesdescribed elsewhere(Blantonet Cortical brain sheet preparation andexperimental sheetpreparation setup brain Cortical Animals MATERIALS ANDMETHODS Whole-cell patchWhole-cell clamp electrophysiology 2 /5% CO mmHg undertheseexperimentalconditionsin~10 7.4; osmolarity285–290 2 , 2NaH 2 . Preliminaryexperimentscomparingtheuseof95%O 2 demonstrated thattherewerenodifferences inanyofthe 2 PO min priortoanexperimentandgentlythroughoutthe

mOsM). AnAg-AgClelectrodeconnected toaCV-7B 2 4 /5% CO ·2H Ṗ 2 O Ṗ O, 26.5NaHCO 2+

mOsm). CorticalsheetswereplacedinanRC- 2 O 2 2 was measuredusingafluorescentoxygen release priortodepletionofROSlevels . TheanoxicaCSFreservoirwasbubbled ; seeResultsandFig. 2 , 0.3NaGTP and2NaATP (pH

l m) equippedwithabaskingplatform, −

l 1 − ): 107NaCl,2.6KCl,1.2CaCl 1 ): 8NaCl,0.0001CaCl

cm, 200–300 Ṗ 3 2 O , 10glucoseand5imidazole 2 /5% CO 2 and anoxicaCSFwith95% in bathaCSFdecreasedto

s ofdecapitation.Six

6A for timelineof min (i.e.bathṖ 2 and aplasticcover g) werepurchased 2 and airhadno

2 ml G /5% CO 2 , 10Na Ω

O min h:12 seals 2

M 7.4; was Ṗ − O Ω 2 1 h 2 2 , . (22°C). Duringloading,theacetategroupsonCM-H 50 μmol generation, thefluorescence attreatmentsteadystate wascomparedwitha change inCM-DCF fluorescence.To assess treatmenteffects onROS solution inDMSO)for30 30 wavelength of520 wavelength of495 a subsequentincreaseinfluorescence. CM-DCFwasexcitedwitha second controlvalue(t subsequent recordingswerenormalizedtothatfirstcontrolvalue.The perfusion. Theinitialcurrentrecordingwassettoavalueof100%andall at thestartofexperiment( recordings. ControlevokedNMDA/AMPA receptorcurrentswererecorded period wasallowedforpatchstabilizationpriortocommencementof Following membraneruptureandformationofthewhole-cellpatch,a5 R was determinedbeforeeachmeasurementandrecordingswerediscardedif compensation, typicalwhole-cellaccessresistance( the whole-cellpatch-clampconfiguration.Followingcapacitance al., 1989).Uponsealformation,negativepressurewasappliedtoachieve Changes in[ROS] comparison andproduceaveragetraces(Fig. regression linefittothe10 Oregon Greenandrhodaminetracesweredriftcorrectedtoa linear Easy RatioProimagingsoftwaretoreducenoiseandsimplifyinterpretation. Brightly fluorescingcellswereavoided.Sampletracessmoothedusing interest fromthecenterofcellbodywasusedasasinglereplicate. cortical sheetwerechosenatrandomandtheaveragechangeinregionsof subtracted fromfluorescentdata.Forstatisticalanalysis,10neuronsper constant witheachtreatment;therefore,backgroundfluorescencewasnot fluorophores. Thebackgroundfluorescencewasminimalandremained cortical sheetswereexposedtoeachtreatmentintheabsenceof fluorescence ofcorticalsheetsaffects fluorescencemeasurements,control experiments ofuptoanhourinlength.To assesswhetherendogenous camera (Rolera-MG International). FluorescenceemissionsweredetectedwithanEMCCD controlled byEasyRatioProimagingsoftware(PhotonTechnology monochromonator (PhotonTechnology International,London,ON,Canada), using aFITCfilterset(Semrock,Rochester, NY, USA)anda an Olympus0.8NA,40×waterimmersionobjective.Dyeswereimaged bath chamberofanuprightmicroscope(OlympusBX51WI)equippedwith In allfluorescenceexperiments,corticalsheetswereplacedinaflow-though generation ofROSresultsinoxidation oftheCM-H by intracellularesterases,preventingdyeleakage.Steady-statenormoxic sensitive fluorescentindicator5-(and-6)-chloromethyl-2 aCSF containing5 Invitrogen, Burlington,ON,Canada).Corticalsheetswereincubated in and analyzedusingClampex10software(MolecularDevices). an Axopatch-1Damplifier, aCV-4 headstageandaDigidata1200interface, pyramidal cellswerediscarded.Alldatacollectedat5–10 described elsewhere(ShinandBuck,2003),patchesfromnon- of experiments,astepprotocoltoidentifycelltypewasperformedas than 30 excited for0.5 for 40 following theexperimentaltreatmentperiodwithcontrolnormoxicaCSF recordings weretakenat20 Experimental conditionsweremaintainedfor40–80 perfused withexperimentalbulkaCSFtreatmentsand/ordripperfusions. normoxic recordingsandforfuturestatisticalanalysis.Cellswerenext dichlorodihydrofluorescein diacetate,acetylester(CM-H Evoked NMDA andAMPA recordings current receptor CM-DCF fluorescence measurements for measurements [ROS]CM-DCF fluorescence measurements Fluorescence a changed bymorethan20%orwhole-cellleakcurrents min thenreperfusedwithcontrol aCSFfor20 min andcurrentrecordingsweretakenat20 pA duringthecourseofexperiment.Priortocommencement l − 1 H The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105825 2 O

s every10 2 for 5 nm. Corticalsheetswereexposedto treatmentaCSFfor i μmol were assessedusingthemembrane-permeableROS- i

, QImaging,Burnaby, BC,Canada).Neuronswere nm andfluorescenceemission was detectedat =10 min. CessationofROSgeneration resultsinno

l min) wasusedtoconfirmconsistencywithinthe min (4°C)followedbya30 − s topreventbleachingofthedyeandpermit 1

min normoxicportionofthetracetoenable

CM-H min intervals.Thetissuewasreperfused t =0 min) andfollowing10 2 DCFDA (froma1

6).

min intervals. min andevokedcurrent R 2 2 DCF toCM-DCFand

DCFDA areremoved a min ortreatedwith i ) was20–25

min washinaCSF

min ofnormoxic mmol

kHz using 2

DCFDA; l − M 1 3353 Ω stock

′ min . ,7′ R - a

The Journal of Experimental Biology dissolved inDMSOtoastockconcentrationof25 20–40 wavelength of520 wavelength of488 indicating sufficient dyeuptake.OregonGreenwasexcitedwitha The doubleloadingperiodresultedinelevatedbaselinefluorescencelevels, mK dye rhodamine-123(Invitrogen)for50 Buck, 2013).CorticalsheetswereincubatedinaCSFcontaining5 included intherecording electrodesolution.Tetrodotoxin waspurchased of mitochondrial[Ca activator ofmK used werebasedonpreviousinvestigations(HawryshandBuck,2013). complex Iinhibitorrotenone(25 2008). Mitochondrial-specificROSproductionwashaltedusingthe primary mitochondrialROSproduct(Chenetal.,2003;Ottaviano used inothervertebratetissuestodecrease[ROS] through dripapplicationonly. Inaddition,H 3354 RESEARCH ARTICLE Decreases in[ROS] Cortical neuronswereloadedwiththemembrane-permeable fluorescence attributedtochangesin[Ca value wassubtractedfromallrecordingsduringanalysistoisolatethe fluorescence signalandobtainavalueforbackgroundfluorescence.This Oregon Green(froma1 Changes in[Ca regression line(Croweetal.,1995). are presentedaspercentchangeexpressedrelativetothatfittednormoxic linear regressionlinefittothe10 5 aCSF flowwashaltedandtissueswereincubatedinionomycin(2 fluorescence toreturnbaseline.Onceeachexperimentwascompleted, consecutive 1 cell-permeable ROSscavengers:MPG(0.5 (0.5 ( were dripandbulkperfused.Diazoxide andH aCSF. Duringwhole-cellrecordings,bothROSscavengersandrotenone to becomecloudy. Allotherpharmacologicalcompoundsweredissolvedin not utilizedtodissolverotenoneas the combinationoftencausedsolution before beingdilutedfurtherinaCSF(0.05%finalsolution).DMSO was DMSO (1%infinalsolution)androtenonewassolubilizedchloroform cortical tissue(Pamenteretal.,2007).Diazoxidewasinitiallysolubilized in rotenone anddiazoxidewerebasedonpreviousexperiments turtle Pamenter etal.,2008a;ZivkovicandBuck,2010).Concentrations of to levelscomparablethoseseenduringanoxia(HawryshandBuck, 2013; 520 495 in aCSF(22°C)for20 to 50 μmol (Invitrogen). OregonGreenwasselectedbecauseofitshighCa sensitive fluorescentindicatorOregonGreen488BAPTA-1 AM application ofcell-permeableH et al.,2002;Liu1993).[Ca For experiments involvingCa 5 Oregon Green fluorescence measurements for measurements [Ca fluorescence Green Oregon application toturtlecorticalneuronsresultsinadepolarizationof than thesarcolemma(Garlidetal.,1997).Furthermore,diazoxide mitochondria, whichpossessa1000-foldgreatersensitivitytodiazoxide administration anddrug Pharmacology potential ( membrane for measurements mitochondrialRhodamine-123 fluorescence investigations (HawryshandBuck,2013). baseline. Theprotocolandconcentrationsusedwerebasedonprevious reperfused withcontrolaCSFfor30 K

min priortotetrodotoxinadministration inordertolimitpotentialtoxicity. min, followedbyapplicationofMnCl d ≈ TP AT nm. CorticalsheetswereexposedtotreatmentaCSFfor30 nm andfluorescenceemissionsweredetectedatawavelengthof mmol 170 min thenreperfusedwithcontrolaCSFforatleast10 channel agonistdiazoxide(100 nmol

l l − − 1 1 ). DirectincreasesinROSwereinducedthroughdrip

h periodsat4°Cfollowedbya30 in aCSF. Followingdyeloading,corticalsheetswerewashed l − 2+ 1 TP AT ) andprevioususeinturtlecorticaltissue(Hawrysh ]

i nm. CorticalsheetswereexposedtotreatmentaCSFfor

i channels asdemonstratedbyK were assessedusingthemembrane-permeableCa nm andfluorescenceemissionsweredetectedata 2+ was achievedthroughtheseparateapplicationoftwo min. Rhodamine-123wasexcitedatawavelengthof ], andadecreaseinNMDA/AMPA receptorcurrents Ψ m mmol ) μmol 2+

2 l min normoxicportionofthetrace.Data O − 2+ 1 chelation, BAPTA (5

2 ] min toallowfluorescencereturn i stock solutioninDMSO)fortwo (50 μmol l increases werereplicatedusingthe − 2+ 1 2 μmol

). Rotenonehasbeensuccessfully min (4°C).Rhodamine-123was ]. Theprotocolandconcentrations (2 2 mmol O l 2 −

l min washinaCSF(22°C). 1 was administeredforonly −

i ). Diazoxideisapotent mmol 1 (Li andTrush, 1998;Liu

) asitrepresentedthe 2 l mmol O −1 + ), toquenchtheCa 2 flux inbovineheart

were administered l −

1 l − 2+ and thendiluted

1 mmol ) andNAC ] i

min toallow min andthen Ψ Ψ m μmol) for 2+ -sensitive

m l μmol − , release affinity 1 ) was 2+ l 2+ − 1 - un . rs,R .adSe,S.-S. Sheu, and R.A. Gross, Y., Duan, manuscript forpublication. the manuscript.L.T.B. andD.W.H. revisedandpreparedthefinalversionof performed theexperimentsandanalyseddata.D.J.D.wrotefirstdraftof L.T.B. andD.J.D.conceiveddesignedthestudy. D.J.D.,D.W.H. andP.J.H. The authorsdeclarenocompetingfinancialinterests. measurements inthetissuerecordingchamberandsalinereservoirs. The authorsthankAaronChowdhuryforassistancewithfiber-opticoxygen Inc., SanJose,CA,USA).Fluorescenceand Data wereanalyzedusingSigmaPlotsoftwareversion11.0 (SystatSoftware, obtained fromSigma-Aldrich(Oakville,ON,Canada). from Tocris Bioscience(Ellisville,MO,USA)andallotherchemicalswere Research CouncilofCanadatoL.T.B. Research fundingwasprovidedbytheNaturalSciencesandEngineering Results areexpressedasmeans±s.e.m. treatment groups.Significanceforalldatawasdeterminedat used tocomparethemeansofnormoxiccontrolsandtreatmentswithin normally distributethedatapriortostatisticalanalysis.AnANOVA was measures ANOVA. Dataweredividedby1000andarcsinetransformedto cell peakcurrentamplitudedatawereanalyzedusingaone-wayrepeated- between treatmentandcontrolgroups.NMDA andAMPA receptorwhole- way ANOVA followedbyaTukey’s posthoc ald .D,Pue,P,YrvYrvy . ury .N,Drezo R.B., Darbenzio, H.N., Murray, V., Yarov-Yarovoy, P., Paucek, K.D., Garlid, aeai .adMmao F. Mammano, and M. Canepari, R.H. Loring, and S.A. Lipton, E., Aizenman, hne,N .adShmce,P. T. Schumacker, and N.S. Chandel, ua,L . es,S . azneo .M,Hnrn .D,Rtmn .M,Lin, S.M., Rothman, S.D., Handran, L.M., Canzoniero, S.L., Sensi, L.L., Dugan, Author contributions Competing interests Acknowledgements Statistical analysis References Funding hn . aqe,E . ohda,S,Hpe,C .adLsesy E.J. Lesnefsky, and C.L. Hoppel, S., Moghaddas, E.J., Vazquez, Q., Chen, oly .M,Wohm,P . odn .D n aás R. Balázs, and R.D. Gordon, P. L., Woodhams, T. M., Bosley, T. C. Foster, and A. Kumar, K., Bodhinathan, A.R. Kriegstein, and J. LoTurco, M.G., Blanton, P. E. Bickler, hi .B n itn S.A. Lipton, and Y. B. Choi, D.W. Choi, rs,J .adTmltn D.J. Templeton, and J.V. Cross, S.A. Lipton, and H.V. Chen, Y., Choi, ’urax .adTldn,M.B. Toledano, and F. J. B. Alvarez-Leefmans, and D’Autréaux, L. Huerto, J., Altamirano, W. E., Crowe, ayz .adPros C.G. Parsons, and W. Danysz, 597-664. Neurosci. high spatio-temporalresolution. polymerase-1 activationduringglutamate excitotoxicity. mitochondrial reactiveoxygenspecies servesasasignalforpoly(ADP-ribose) physiological significanceandpossibletherapeuticapplications. ’lno .J,Lde .J,Sih .A n rvr G.J. Grover, and M.A. Smith, N.J., Lodge, A.J., D’Alonzo, mitochondria: oldquestions,newinsight. in vitro anoxia onthestimulatedreleaseofaminoacidneurotransmittersincerebellum kinase II.J.Neurosci. NMDA receptorresponseduringagingthroughCa2+/calmodulin-dependentprotein Methods from neuronsinslicesofreptilianandmammaliancerebralcortex. ( responses byreductionandoxidation. .S,Glbr,M .adCo,D.W. oxygen speciesincorticalneuronsfollowing exposureto Choi, and M.P. Goldberg, T. S., complex III.J.Biol.Chem. (2003). Productionofreactiveoxygenspeciesbymitochondria:centralrole Mol. LifeSci. protein cysteineoxidation. 400. mediate bothredoxandzn2+modulationofthenmdareceptor. 8 mechanisms thatgeneratespecificityinROShomeostasis. probe. Volume changesinsingleN1E-115 neuroblastomacellsmeasuredwithafluorescent Chrysemys picta , 813-824. . J.Neurochem. Neuroscience 30, 203-210. (1992). Excitotoxiccelldeath. 15, 6377-6388. (1998). ReductionofNMDA receptoractivityincerebrocortexofturtles The JournalofExperimentalBiology(2014)doi:10.1242/jeb.105825 57, 1535-1541. ) during6wkofanoxia. 69, 283-296. 30, 1914-1924. 40, 189-201. Antioxid. RedoxSignal. 278 (2000). RedoxmodulationoftheNMDA receptor. , 36027-36031. (1998). Glycineand J. Neurosci.Methods (1999). Imagingneuronalcalciumfluorescenceat (2006). Regulationofsignaltransductionthrough Neuron J. Neurobiol. (1995). Mitochondrialproductionofreactive J. Appl.Physiol. Am. J.Physiol. (2001). Threepairsofcysteineresidues (2007). ROSassignallingmolecules: (2007). Ca (2000). Cellularoxygensensingby (1989). SelectivemodulationofNMDA (2010). Intracellularredoxstatealters 2 , 1257-1263. Ṗ O 8 2 , 1819-1827. N test toidentifydifferences 23, 1261-1276. data wereanalyzedbyone- 87, 1-11. -methyl- (1989). Wholecellrecording J. Physiol. 2+ 275 -dependent generationof 88, 1880-1889. N Nat. Rev. Mol.CellBiol. , R86-R91. -methyl- D -aspartate receptors: J. Neurosci. Pharmacol. Rev. 585 (1983). Effects of D , 741-758. -aspartate. J. Neurosci. P 21, 392- <0.05. (1997). (1995). Cell. 50, J.

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