Deuterium content of water increases depression susceptibility: the potential role of a serotonin-related mechanism.

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ContentslistsavailableatScienceDirect

Behavioural

Brain

Research

j o ur na l h o me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / b b r

Research

report

Deuterium

content

of

water

increases

depression

susceptibility:

The

potential

role

of

a

serotonin-related

mechanism

Tatyana

Strekalova

a,b,c,∗

_,

_Matthew

_Evans

a

_,

_Anton

_Chernopiatko

d,e

_,

_Yvonne

_Couch

a

_,

João

Costa-Nunes

b

_,

_Raymond

_Cespuglio

f

_,

_Lesley

_Chesson

g

_,

_Julie

_Vignisse

h

_,

Harry

W. Steinbusch

c

_,

_Daniel

_C.

_Anthony

a

_,

_Igor

_Pomytkin

d,e

_,

_Klaus-Peter

_Lesch

c,i,∗∗ a_Department_of_{Pharmacology,}_Oxford_University,_Oxford,_UK

b_Institute_for_Hygiene_and_Tropical_Medicine,_New_University_of_Lisbon,_Portugal

c_School_for_Mental_Health_and_{Neuroscience,}_Department_of_{Neuroscience,}_Maastricht_University,_Maastricht,_Netherlands d_Laboratory_of_Cognitive_{Dysfunctions,}_Institute_of_General_Pathology_and_{Pathophysiology,}_Moscow,_Russia

e_Timantti_AB,_Stockholm,_Sweden

f_Claude_Bernard_University,_Faculty_of_Medicine,_EA₄₁₇₀_Lyon,_France g_IsoForensics_Inc.,_Salt_Lake_City,_UT,_USA

h_GIGA_{Neuroscience,}_University_of_Liege,_Liege,_Belgium

i_Division_of_Molecular_Psychiatry,_Laboratory_of_{Translational}_{Neuroscience,}_Department_of_Psychiatry,_{Psychosomatics}_and_{Psychotherapy,}

UniversityofWuerzburg,Wuerzburg,Germany

h

i

g

h

l

i

g

h

t

s

•Geographicaldistributionofdeuteriumcorrelateswithdepressionrate.

•Deuteriumdepletedwaterreducesstress-induceddepressive-likesignsinmice.

•Hippocampalproliferationafterstressisrescuedbydeuteriumdepletedwater.

•DeuteriumdepletedwaterinducesSSRI-likechangesinEEGparametersofsleep.

•Above-indicatedeffectsmaybeduetonormalizationofhippocampal5-HTTlevel.

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received21April2014

Receivedinrevisedform22July2014 Accepted23July2014 Availableonlinexxx Keywords: Depression Chronicstress SERT

Hippocampalcellproliferation Deuterium

Sleep

a

b

s

t

r

a

c

t

Environmentalfactorscansigniﬁcantlyaffectdiseaseprevalence,includingneuropsychiatricdisorders suchasdepression.Theratioofdeuteriumtoprotiuminwatershowssubstantialgeographical varia-tion,whichcouldaffectdiseasesusceptibility.Thusthelinkbetweendeuteriumcontentofwaterand depressionwasinvestigated,bothepidemiologically,andinamousemodelofchronicmildstress.We performedacorrelationanalysisbetweendeuteriumcontentoftapwaterandratesofdepressionin regionsoftheUSA.Next,weuseda10-daychronicstressparadigmtotestwhether2-week deuterium-depletedwatertreatment(91ppm)affectsdepressive-likebehaviorandhippocampalSERT.Theeffect ofdeuterium-depletiononsleepelectrophysiologywasalsoevaluatedinnaïvemice.Therewasa geo-graphiccorrelationbetweenacontentofdeuteriumandtheprevalenceofdepressionacrosstheUSA. Inthechronicstressmodel,depressive-likefeatureswerereducedinmicefedwithdeuterium-depleted water,andSERTexpressionwasdecreasedinmicetreatedwithdeuterium-treatedwatercompared withregularwater.Fivedaysofpredatorstressalsosuppressedproliferationinthedentategyrus;this effectwasattenuatedinmicefedwithdeuterium-depletedwater.Finally,innaïvemice, deuterium-depletedwatertreatmentincreasedEEGindicesofwakefulness,anddecreaseddurationofREMsleep, phenomenathathavebeenshowntoresultfromtheadministrationofselectiveserotoninreuptake inhibitors(SSRI).Ourdatasuggestthatthedeuteriumcontentofwatermayinﬂuencetheincidence ofaffectivedisorder-relatedpathophysiologyandmajordepression,whichmightbemediatedbythe serotoninergicmechanisms.

∗ Correspondingauthorat:DepartmentofPharmacology,UniversityofOxford,MansﬁeldRoad,OX13QT,UK.Tel.:+441865281136;fax:+441865271853.

∗∗ Correspondingauthorat:DivisionofMolecularPsychiatry,LaboratoryofTranslationalNeuroscience,DepartmentofPsychiatry,PsychosomaticsandPsychotherapy, UniversityofWuerzburg,Fuechsleinstrasse,15,97080Wuerzburg,Germany.Tel.:+4993120177600/10;fax:+4993120177620.

E-mailaddresses:bioc0541@pharm.ox.ac.uk(T.Strekalova),kplesch@mail.uni-wuerzburg.de(K.-P.Lesch).

http://dx.doi.org/10.1016/j.bbr.2014.07.039

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BBR-9058; No.ofPages8

2 T.Strekalovaetal./BehaviouralBrainResearchxxx(2014)xxx–xxx

1. Introduction

Majordepressivedisorder,acommonandrecurrentdisorder, hasbeenprojectedtobecomethesecondleadingcauseofdisability worldwide by2020. It is associated withconsiderable morbid-ityandincreasedmortalityandischallengingtotreateffectively. Indeed,environmentalfactors,suchasinsolationandamountof daylight[1,2],annualﬂuctuationsofairtemperature[3],acontent ofcertainmineralsinsoilandwater[4,5]wereshowntobe impor-tantinteractingfactors forneuropsychiatricdisorders including depression.Isotopecontentofnaturalwatersisoneofthefactors thatgreatlyvariesacrossdifferentgeographicalareas[6,7]andcan profoundlyaffectbasicphysiologicalprocesses[8].However,itis unclearwhetherisotopecontenthasanimpactintheprevalence ofaffectivedisorders,orwhetheritmightalterthephenotypein animalmodelsofdepression.

Naturalwaterisamixtureofninewaterisotopologuesformed bystableisotopesofhydrogen[1_H,_protium_(H)_and2_H,_deuterium

(D)]andoxygen(16_O,17_O,18_O)._The_term_{‘isotopologue’}_refers_to_a

molecularentitythatdiffersonlyinisotopiccomposition[9].The abundanceofninewaterisotopologuesinenvironmentalwater, expressedasthedeviation(_ı)relativetotheinternationalstandard ‘ViennaStandardMeanOceanWater2(VSMOW2)standard,varies systemicallybylocationandclimaticconditions duetoisotopic fractionation accompanying evaporation-condensation process, whentheair mass moveinland over topographicfeatures. The contentofheavyisotopologuesdecreasesproportionallywiththe distancefromtheoceanandaltitude.Itisalsoinﬂuencedbyother factors,suchas latitude, humidity,and a seasonaltemperature [7,10–12].Thespatialisotopicdistributionintapwatersreﬂects theregionalvariationofisotopesinthelocalenvironment [13], whichshowsconsiderablevariationacrosstheUnitedStates(US). However,thelowestlevelsofheavyisotopologuesaredetectedin theplateauofAntarcticawhosewaterisusedasanother interna-tionalstandard, StandardLightAntarcticPrecipitation2(SLAP2) (InternationalAtomicEnergyAgency,2009).Thiswateris charac-terizedby43%reductionindeuteriumcontent(D/Hratio89.1vs. 155.8ppm)and by5%reduction inoxygen-18content(18_O/16_O

ratio1894.9ppmvs.2005.2ppm),ascomparedtoVSMOW2.

2. Objectives

Here,weperformedanepidemiologicalanalysisofthe relation-shipbetweendeuteriumcontentofdrinkingwaterintheUS,the mostabundantisotopeafterprotiumandisotopesofoxygen,and prevalenceofdepression.We thenassessedthe antidepressant-like properties of deuterium depleted water (D91=91.7ppm) againstthepharmacologicalreferencecitalopramusingthreewell establishedparadigmsofdepression-likestateandstressinmice: achronicstressdepressionmodel[14],stress-inducedsuppression ofhippocampalneuralproliferation[15]andEEGanalysisofsleep [16].

3. Methods

3.1. Epidemiologicalanalysis

DepressionrateswereobtainedforeachstateintheUSfrom themorbidityandmortalityweeklyreport(MMWR)on depres-sionin adultsintheUSintheperiod2006–2008[17].Multiple samplesoftapwaterwerecollectedovertheperiod2004–2013, andtheproportionofdeuteriumwasaveragedtogiveavaluefor eachstateinthecontinentalUSA.Wethenperformedacorrelation analysis(Pearson’sr)betweencontentofdeuteriumintapwater andprevalenceofdepressionacrosstheUSA.

3.2. Animals

Studieswere performedusing 3.5-month-oldmale C57BL/6J mice.3.5-month-oldmaleCD1micewereusedasresident intrud-ersforsocialstressand2–5-month-oldWistarratswereusedfor predatorstress.Animalswerekeptinstandardlaboratory condi-tionsaspreviouslydescribed[14].Detailsontheanimals’housing canbefoundinSupplementarymethods.Experimentswerecarried outinaccordancewiththeEuropeanCommunitiesCouncil Direc-tiveforthecareanduseoflaboratoryanimals,andwereapproved byrespectivelocalgovernmentalbodies.

3.3. Waterisotopologuesanddrugtherapy

Experimentalsolutionsreplacednormaldrinkingwater. Con-trolwater(D140=140.3ppmdeuterium)anddeuterium-depleted water (D91=91.7ppm deuterium) were obtained from Almaz KotovskTambovReg/TimanttiAB,Stockholm,Sweden(fordetails, see Supplementary tables). Citalopram was dissolved in tap water; the solutions were changed every 3 days. The citalo-pram dose (15mg/kg/day), in D140 drinking water, was based onthe previouslyvalidated dosing method[18].We measured waterintakebetweenD91andD140groups;nosigniﬁcant differ-encebetweenthesegroupswasfound(p>0.05)(Supplementary Fig.1).

3.4. Chronicstressstudy

Micewereeitherassignedtocontrol(n=30)orstress(n=63) groups.Thegroupmeanswerematchedformousesocialbehavior, bodyweightandsucrosepreference,afterStrekalovaetal.[19].25 ofthestressedmiceweretreatedwithD140,19withD91,and19 weretreatedwiththeantidepressant,citalopram(15mg/kg/day, viadrinkingwater).ControlmiceweretreatedeitherwithD91, D140orcitalopram(N=10ineachgroup).D91,D140and citalo-pram were administrated starting 7 days prior the onset of stressandlastedfortheentiredurationofthestressprocedure (seebelow).

Weemployeda10-daystressprotocol[14]comprisingadark cycleratexposurestress(predatorstress)andlight cycle semi-randomapplicationoftwoofthreestressors:socialdefeatstress, restraint stress and tailsuspension stress. Brieﬂy, betweenthe hoursof09:00and18:00twostressorsperdaywereemployed inthefollowingsequence:socialdefeatfor30min,restraintstress for2handtailsuspensionfor40minwithaninter-session inter-valof atleast4h.Allmicewere weighedonthe7th and 10th dayofstress,andscoredforacoatstate15hafterthe termina-tionof thelast stressor.12hafterthelast stressor,afterbeing weighedandscoredforcoatdisintegration,allmiceweretested in8-hsucrosetest,toassesshedonictraits.Immediatelythereafter theirforcedswimmingwasassessedtoevaluatechangesin affec-tivebehavior.Chronicallystressedmicewerekilled∼30hafterthe laststressor,andtheirhippocampiwereisolated,quantitative RT-PCRofSERTexpressionwasperformedasdescribedelsewhere[14]. Fordetailsonthebehavioralandmolecularmethods,see Supple-mentarymethods.

3.5. Hippocampalneuralcellproliferationaftera5-daypredation stress

Micewererandomizedtotwogroups;one receivedD91 for 2 weeksand the otherD140. Both groupsunderwent predator (rat exposure) stress for 5 consecutive nights; treatment with D91 or D140 continued throughout the stress exposure (see Supplementary methods). Before the ﬁrst stress session, mice received four intraperitoneal injections of Bromodeoxyuridine

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Fig.1. Depressionratesanddeuteriumcontentoftapwater.(A)CorrelationbetweenreportedratesofmajordepressivedisorderinUSstatesandcontentofdeuteriumin tapwaterintheUSA.(B)Thereportedprevalenceofindividualswithdepressioninamongadultsaged≥18yearsineachUSstate[CentersforDiseaseControlandPrevention. MentalillnesssurveillanceamongadultsintheUnitedStates.MMWR2011;60(Suppl):1–30].(C)Geographicaldistributionofaveragedeuteriumcontentinthetapwater inthecontinentalUSAfromsamplescollectedfromeachstatefor2004–2013.

(Sigma–Aldrich, St. Louis,MO, USA;200mg/kg/kg)dissolved in 0.9%NaCland0.007MNaOH,spacedonefromtheotherby2h. Theywerekilled24hafterthelaststresssession,perfusedwith 4%-paraformaldehyde,andbrainswere removedand treatedas describedelsewhere[20];seeSupplementarymethodsformore detail.Immunoﬂuorescence,confocalmicroscopyand quantiﬁca-tionfordetectionofBrdUandKi67wereperformedasdescribedin Supplementarymethods.

3.6. EEGsleepstudy

12 naïve C57BL6J mice were implanted with electrodes for polygraphic recordings as described elsewhere [16]. The citalopram-treated group was not included in the EEG experi-mentwithsleepanalysis,sincetheeffectsofSSRIsonsleepwere intenselyinvestigatedandarewelldescribedinaliteratureofthe lastdecades(forareview,seeUrsin[21]).2weeksaftersurgeryand recoveryperiod,animalswereconnectedtorecordingcablesand allowedtohabituatetotherecordingchambersfor1week. Light-ingscheduleinthechamberswaskeptreversed(12h:12h,light-off at9:00),foodandwaterwereavailableatlibitum.Thereafter,D91 orD140deliverywasstartedsimultaneouslywiththepolygraphic recordingsthatlasted14days.Datawererecordedandanalyzedas describedelsewhere[22].

3.7. Statistics

DatawereanalyzedwithGraphPadPrismversion5.0for Win-dows (San Diego, CA). One- and two-way ANOVA was used followed by a Tukey’s post-hoc comparisons (or more strictly, Tukey–Kramer,owingtounequalgroupsizes).t-testswereapplied fortwo-group,two-tailedcomparisons.AtypeIerrorrateofp<0.05 wasadopted,anddataareshownasmean±SEM.

4. Results

4.1. Depressionprevalenceanddeuteriumcontentintapwater We performedcorrelationanalysisusing Pearson’sr,forthe relationship between incidence of depression and deuterium

contentoftapwater(Fig.1).Therewasasigniﬁcantcorrelation (r=0.468; p=0.0016; F=11.49) between deuterium content of tapwaterandratesof depression.Fromthelinearequationwe estimatethatprevalenceofdepressionisincreasedby1.8%(95% conﬁdenceintervals0.7–2.9%;R2₌_0.219;_p₌_0.0016;_F₌_11.49)_for

a 10ppm increaseindeuterium levelin tapwater (see Supple-mentaryTable3state-by-statetapwaterdeuteriumcontent.)In ordertoallowmorestringentcontrolofexperimentalvariables, wepursuedfurtherworkinanimalmodels.

4.2. Assessmentofanhedoniainduction

Two-way ANOVA revealed a signiﬁcant effect of stress on sucrose preference (F1,120=37.83, p<0.001), and an interaction

between stress and treatment (F2,120=4.84, p=0.009). Post-hoc

Tukey-correctedt-testsshowedasigniﬁcantreductioninsucrose preference from baseline for the non-treated stressed group (p<0.05),butwhen treatedwithcitalopramandD91 therewas nodifferencefrombaselineinterms ofsucrosepreference.This suggeststhatD91,likecitalopram,preventsareductioninsucrose preferenceforthestressedcohort(Fig.2A).

4.3. Effectsoftreatmentonﬂoatingbehavior

‘Behavioraldespair’measuredinthemodified Porsoltforced swimtest (FST)byoccasionoffloatingbehaviorisanothersign of depressive-likestatein rodents[23].Therewasa significant maineffectofstressforlatencytofloatingintheFSTasrevealed by two-way ANOVA (F1,87=14.19, p<0.001), but there was no

signiﬁcantmaineffectoftreatment(F2,87=0.82,p=0.44)andno

signiﬁcantinteraction(F2,87=0.92,p=0.40).Post-hoctestsshowed

a significantreduction in latency to floatingfor stressed com-paredwithcontrolmicetreated withD140 (p<0.01), butthere wasnosignificantdifferencebetweenstressedandcontrolmice thatreceivedcitalopramorD91(p>0.05;Fig.2B).Therewasalsoa significantmaineffectofstressfortimespentfloating(F1,81=9.11,

p=0.0033),withstressedmiceﬂoatingforlongerthancontrols,and asigniﬁcantmaineffectoftreatment(F2,87=3.14p<0.05),butno

signiﬁcantinteractionbetweentreatmentandstress(F2,87=1.28,

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Fig.2. CitalopramorD91amelioratesstress-induceddepressive-likechangesinmice.(A)D140treatedstressmiceshowedasignificantreductioninsucrosepreferenceat day10(p<0.01,Tukey’sposthoctest).However,citalopram-treatedandD91-treatedstressedmicedisplayednoalterationinsucrosepreference(p>0.05,Tukey’sposthoc test).(B)Intheforcedswimtest,thelatencytofloatwassignificantlyincreasedbystressintheD140-treatedmice(p<0.01),butthiswasnotthecaseinthecitalopram orD91-treatedgroups(p>0.05;Tukey’sposthoctest).(C)ThedurationoffloatingwassignificantlyelevatedinstressedmicethatreceivedD140(p<0.001),butitwas unalteredinD91andcitalopramtreatedanimals(p>0.05;Tukey’sposthoctest).(D)Allstressedgroupsshowedasignificantdecreaseinbodyweightcomparedwith controls(p<0.001forD140andcitalopramtreatedanimals,p<0.01forD91treatedmice).Post-hoctestingrevealedthatbodyweightwassignificantlyhigherinstressed groupstreatedwithcitalopramorD91comparedtoD140(p<0.01andp<0.0001respectively).(E)Allstressedgroupshadsignificantlylowercoatscoresthancontrolswhile stressedcitalopramorD91-treatedanimalshadsignificantlyhigherscoresthantheD140-treatedgroup(p<0.001,Tukey’stest).(F)Therelative-foldmRNAexpressionlevel ofthe5-HTtransporter(SERT)wassignificantlylowerinD91-treatedstressedmice.Dataarepresentedasrelative-foldcomparedtocontrolanimalswithallexpression beingnormalizedtothehousekeepinggeneGAPDH.Dataaremean±SEM(*<0.05,Tukey’sposthoctest).Dataaremean±SEM.

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betweencontrolandstressedD140-treatedgroups(p<0.01)but notbetweenothergroups(p>0.05).

4.4. Changesinbodyweight

In terms of body weight, at the end of the chronic stress paradigmtherewasasigniﬁcantmaineffectofstress(F1,87=64.09,

p<0.0001),anear-signiﬁcantmaineffectoftreatment(F2,87=2.94,

p=0.058),butnosigniﬁcantinteractionbetweenstressand treat-ment(F2,87=1.71,p=0.0.187).However,stressedmicetreatedwith

citalopram or D91 were heavierthan those treated with D140 (p<0.01andp<0.0001,respectively,Fig.2D).

4.5. Evaluationofcoatscores

Beforetheonsetofthechronicstressprocedure,allmicehad good coat quality, with no signiﬁcant difference between the groups(data notshown). Aftercompletion ofthechronicstress procedure, a two-way ANOVA comparison showed signiﬁcant maineffectsofbothstress(F1,87=3.59,p<0.0001);andtreatment

(p=0.0317;Fig.2E),andtherewasalsoa signiﬁcantinteraction betweenstressandtreatment(F2,87=3.59, p<0.05).Allstressed

miceshowedsigniﬁcantlylowerscoresofcoatstatethancontrol treatedanimals(p<0.001).However,citalopramandD91 admin-istrationgroupsshowedhigherscoresofcoatstatecomparedwith theD140-treatedgroup(p<0.001).

4.6. Hippocampalgeneexpressionofserotonintransporter(SERT) Giventhemarkedbehavioraldifferencesbetweenthegroups, andpreviousfindingsshowingupregulationoftheserotonin trans-porter (SERT) during anhedonia [14], we sought to elucidate whethertreatmentwithcitalopramorD91ledtochangesinSERT mRNAexpression.A2-wayANOVArevealedasignificant differ-encebetweentreatmentgroupsinhippocampalexpressionofSERT mRNA(F2,47=4.11,p=0.022Fig.2F).Therewasnosignificantmain

effectofstress(p=0.542)andnosignificantinteraction(p=0.705). There was a trend to increased SERT expression for stressed compared withnon-stressed micein theD140and citalopram-treatedgroup,butthesewerenotsignificantonpost-hocanalysis (p>0.05).However,bothcitalopramandD91decreasedthe expres-sionofSERT,whichwassignificantlylowerinstressedD91-treated compared with stressed D140-treated mice (p<0.05), but the comparisonofcitalopram-treatedversusD140didnotsurvive cor-rectionformultiplecomparisons(p=0.26).Therefore,inaddition toD91treatmenthavingasignificanteffectonbehavior,italsohad asignificanteffectontheexpressionlevelofSERTmRNAinthe hippocampus.

4.7. Neuralcellproliferationafteraﬁve-daystress

The number of BrdU-positive cells per mm3 _of _in _the

den-tate gyruswassigniﬁcantly reduced in thestressed D140-and D91-treatedgroups (mean:613.6±42.59 and838.8±99.18), as comparedtothenon-stressedcontrolgroup(mean:1578±147.1; p=0.0003,F=18.06,R2₌_0.766,_q₌_5.58_and_q₌_4.30,_{respectively,}

one-way ANOVA). There was a signiﬁcant difference in BrdU-positive cellsnormalized tocontrolbetweenstressedD91- and D140-treatedmice (p=0.041, t=2.086,Fig. 3A).The number of Ki67-positivecellspermm3_in_the_dentate_gyrus_was_lower_in_the

stressedD140-treatedgroup,comparedtothenon-stressedcontrol group(mean:1405_±104.05,p=0.0008,t=5.201),butthe differ-encebetweenstressedD140-treatedgroup(mean:648.3±80.1) and D91-treated mice (mean: 1371.0±558.5) did not reach

Fig.3.EffectsofD91onhippocampalSERTandneurogenesis.Instressedanimals, normalizedtocontrol,(A)thenumberofBrdU-positivecellspermm3_in_the

den-tategyruswassigniﬁcantlyhigherintheD91-treatedthaninD140-treatedmice (*p<0.05,unpairedtwo-tailedt-test).(B)ThenumberofKi67-positivecellsper mm3_in_the_dentate_gyrus_was_not_{signiﬁcantly}_different_between_D91-_treated_and

D140-treatedmice.

statistical signiﬁcance in this measure normalized to control (p=0.102,t=1.28,Fig.3B).

4.8. EEGparametersofsleep

EEG analysis was performed two weeks after surgery and recovery/adaptation period (Fig. 4A). On day 3 following D91 administration,we notedsigniﬁcantchanges inthe durationof wakefulness(W)(5–7%,p=0.044,t=2.30,unpairedtwo-tailed t-test,Fig.4B)andslowwavesleep(SWS)(5-7%,p=0.040,t=2.35, unpairedtwo-tailedt-test,Fig.4C)comparedtoD140-treated ani-mals, and this phenomenon remained constant throughout the recordingperiodtillday14(p=0.031,t=2.50,unpairedtwo-tailed t-test,Fig.4AandB).D91administrationsigniﬁcantlysuppressed REMsleepafterday4(p=0.046,t=2.27,unpairedtwo-tailed t-test,Fig.4D);thiswasmoremarkedthantheeffectontheother parameters,andwasmaximalonday11oftherecordingperiod (40%,p=0.0086,t=3.26,unpairedtwo-tailedt-test).Nodifferences betweenthegroupswereobservedinthepowerspectraatanytime periodof thelight cycle,or onanyday(p>0.05,unpaired two-tailedt-test),includingtheday11,whenmaximalchangesinsleep parameters were detected(Supplementary Fig. 2).The spectral

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Fig.4.EffectsofD140andD91onEEGparametersofsleepofnaïvemice.(A) SchematictimelineoftheEEGexperiment.Afterelectrodeimplantation, habitu-ationtotherecordingchambersandbaselineEEGregistrationontheday1,animals werecontinuouslytreatedwithD140orD91during14-dayEEGrecordings.(B) EffectsofD140andD91onwakingstate.Therewasasigniﬁcantincreaseofthe durationofthewakingstateinD91-treatedmiceincomparisontoD140-treated animalsthroughoutthetreatmentperiod.(C)EffectsofD140andD91ontheslow wavesleep.IncomparisontoD140-treatedanimals,D91-treatedmicehad signif-icantlyshorterdurationoftheslowwavesleepondays3–13.(D)EffectsofD140 andD91ontheREMsleep.AsigniﬁcantdecreaseofthedurationoftheREMsleepin D91-treatedmiceincomparisontoD140-treatedanimalswasdetectedthroughout thetreatmentperiod.*p<0.05;unpairedtwo-tailedt-test.Dataaremean±SEM, expressedaspercentagefromthemeansobtainedontheday1.

bandsoftheEEGconsideredwere:delta,0.5–4Hz;theta,4–8Hz, alpha,8–11.5Hz, sigma,11.5–14.5Hz;beta-1,14.5–18.6Hz, and beta-2,18.6–30Hz.

5. Discussion

We show a positive correlation between rates of affective disorder-related behavior and pathophysiology deﬁned by the MMWR [17] and deuterium content of water, on a state-by-state basis in the USA. Given that the average daily water intake in adults ranges between 2.7 and 3.7L per individual per day [24], the absolute consumption of the isotopologue hydrogen–oxygen–deuterium (HOD) in deuterium-containing water is around 0.7–1.2mL for water close to the VSMOW2 standard.Assuch,evenrelativelysmallchangesinthedeuterium contentofconsumedwatercouldresultinsubstantialvariations ofintake.However,itshouldbepointedoutthatsocial,economic andotherdemographicfactorsshowstate-to-statevariation,which couldbeaconfoundingfactor,anditisnotpossibletohighlight a causalrelationshipfrom correlationalstatistics. However,the strengthoftheassociationpromptedfurtherinvestigationsinmice, whichrevealedthatreduceddeuterium intakehasthepotential toreversethenegativeaffect,andaltersleeppatternsandgene expressionassociatedwithserotonergicneurotransmission.

We used a paradigm of stress-induced anhedonia in mice [14,19], using the established sucrose-preference test [25,26] which is a core feature of depression [27]. Citalopram is well documentedtocounteractthestress-induceddecreaseinsucrose preferenceinsimilarbehavioralparadigms[28,29].Chronicstress reduced sucrosepreference in D140-treatedmicein agreement withotherreports[30,31];however,aftertreatmentwith citalo-pramandD91therewasnolongerasignificantdifferencebetween stress groups. Importantly, neither D91 nor citalopram had an effectonsucrosetestparametersincontrolanimalsrulingouta moregeneraleffectonsucrosepreference.Ourdatausingthe Por-soltforcedswimtestshowedthatwhilstchronicstressdecreased thelatencytofloating,andincreasedthetotaltime spent float-inginmicereceivingD140, bothcitalopramandD91prevented pathologicalchangesintheseparameters,therewasnosignificant differencebetweenstressedandcontrolmicethatreceived citalo-pramorD91(p>0.05;Fig.2BandC).Previouspublicationsfromour groupshowedasimilareffectfollowingchronicadministrationof imipramineandcitalopram[18,32,33].

Allstressedanimalsshowedadecreaseinbodyweight(Fig.2D), butthiseffectwasdiminishedinD91-andcitalopram-treated ani-mals.Alltreatedmiceshowedasignificantincreaseinbodyweight comparedwithcontrolsandstressedmicetreatedwithcitalopram orD91wereheavierthanthosetreatedwithD140,whileno sig-nificantinteractionbetweenstressandtreatmentwasrevealed.A near-significantmaineffectoftreatmentwasfound.Thiswas con-sistentwithpreviousreportsofrestorationofbodyweightafter antidepressanttreatment[31,34,35].Stressedmicealsodisplayed asignificantdeteriorationofcoatstate,whichwasamelioratedby bothD91andcitalopramtreatment(Fig.2E).Coatdisintegrationis animportantfeatureofadepressive-like(anhedonic)statein pre-clinicalmodels[36,37],andimprovementsincoatstatehavebeen associatedwithantidepressanttreatment[37,38].

WenextwishedtoascertainwhetherD91treatmentresulted inacorrespondingchangeinSERTexpressioninthehippocampus. WhilsttherewasatrendtoincreasedSERTexpressioninstressed comparedwithnon-stressedD140-treatedmice,inlinewithour previousfindings[14],thiscomparisonwasnotstatistically sig-nificanthere(Fig.2F).ComparedwithstressedD140-treatedmice, expressionofSERTwassignificantlydecreasedin stressedmice treatedwithD91.Interestinglythisseemedtohaveagreatereffect sizethantheantidepressantcitalopram,whichwhilstlower,did

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notshowasigniﬁcantdecreaseinSERTexpression.Bothstressed and non-stressed animals showed a trend to decreased SERT expressionwithcitalopram/D91treatmentcomparedwithD140, soitisnotclearthatthiseffectislimitedtostressedanimalsinthis case.ChangeinSERTexpressionwithintheCNSinchronicstress anddepressionissupportedbyanumberofstudies[39–41],and havebeenassociatedwithalterationsinneuroinﬂammatory-inked pathways[14].Thus,molecularchangesrelatedtothereductionof SERTexpressionintheCNSmayunderlieantidepressant-likeaction ofbothcitalopramandD91presentedhere.

We also found that the number of BrdU-positive cells in the dentate gyrus was reduced by stress, and this was ame-lioratedbytreatment withD91compared withD140 (Fig.3A), indicating a stimulatory effectof deuterium-depletedwater on hippocampalcellproliferation.The effectsof stressand antide-pressant/anxiolytictherapyonhippocampalneurogenesisarewell establishedintheliterature[42,43],andsuggeststhatdeuterium depletioncouldfulﬁlasimilarrole.Recentﬁndingshaveshown thatantidepressants,includingSSRIs,rescueongoing neurogene-sisduringstress[44]andsuggestedalinkbetweensuppressionof SERTandtheactivationofthehippocampalneurogenesis[45]with akeyroleofBDNF-mediatedprocessesinthiseffect[46].

Therapidonsetintheincreaseofwakefulness,accompanied byarapiddecreaseinslowwavesleep(SWS)remainedconstant overthetreatmentperiodinD91-treatedanimals(Fig.4). Remark-ably,thechangesinREMsleepappearedprogressivelythroughout thetreatmentperiod andreachedamaximumonthe11thday whenthisstateofsleepwasdecreasedbyabout40%incomparison toabaselinevalue.AnalogouseffectsonSWSandREMsleepare reportedforantidepressantsinhibitingserotoninand noradrena-linereuptake[16,47].Bothacute[48,49]and1–3weeklong[49,50] dosingwithcitalopramorotherSSRIsreducedthedurationofREM andSWSandincreasedwakefulness.Intheclinic,theREMsleep reductionobservedwithSSRItreatmentisusedasabiomarkerof theirtherapeuticefﬁcacy[51].

InclinicaldepressionthereisdecreasedlatencytothefirstREM episode,togetherwithanenhancementofREMduringthefirstpart ofthenight.SuchaneffectiscounterbalancedbyadeclineinREM sleepduringthesecondpartofthenight,withadecreaseintotal REM-sleepinanight.Conversely,SWSisdecreasedinthefirstpart ofthenightandintermittentawakeningsincreased.Itispossible thatthechangesinSERTexpressionmayplayaroleinthechanges tosleepEEGparameters.Incontrasttothemostofstandard antide-pressants[52,53]thepowerspectraofD91-treatedanimalswasnot alteredthroughoutthetreatmentperiodregardlessthelightcycle. ThiswassurprisinggiventhereductioninREMsleepthatmight havebeenexpectedtoaltertheproportionofalphawaves.

Previousresearchhasshownakineticisotopeeffectofwateras asolvent,with100%D2Oslowingtherateofubiquinolderivative

oxidationaround400-fold[54].However,whilstdeuteriumwithin naturalwaterrange(89–155ppm)isunlikelytohaveasigniﬁcant effectonmostchemicalreactions,respirationinmitochondriamay beaspecialcase,asitinvolvesaconnectedsequenceof proton-coupledelectrontransfers.Verylowlevelsofdeuteriumhavebeen showntohaveanimpactonthisprocess[55]and mayaccount forsomeofthebiologicaleffectswehaveobservedinourmouse paradigms.

Amechanism,bywhichwaterwithreduceddeuteriumcontent exhibitsantidepressant-likeproperties,remainstobeelucidated. Basedoncurrentlyavailableliteratureandownpreliminaryresults, various speculations concerning this matter can be proposed. First,replacementofnormalwaterwithwateroflowerviscosity couldexertphysicochemicaleffects,leadingtoincreasedﬂuidity of thecell membranes and less rigid organizationof phospho-lipidbilayers[56–58]which can,inturnalterthedispersionof neurotransmitterreceptorsandincreasereceptorafﬁnity[59,60],

affectpassivebloodbrainbarrierpermeability[61]andmetabolism ofarachidonicacidandcalcium-dependentreceptorbinding[62]. Currentlyunpublishedworkfromourgroupsuggeststhat epige-neticandpost-translationalregulationmechanismsmayunderlie the effect of D91 treatment, based on limited gene expression changes,andtheremayalsobeaninvolvementoffactorsof synap-ticplasticityandBDNF/TrkBsignaling.

6. Conclusions

Takentogether,ourstudydemonstratesthatratesof depres-sioncorrelatewithgeographicaldistributionofdeuteriuminthe naturalwaterintheUSpopulation.Substitutionofnormal drink-ingwaterwithdeuterium-depletedwaterinmicecounteractsthe behavioral,transcriptionalandproliferativechangestypicalofthe depressive-likestate,whichwascomparabletotheeffectsofthe SSRIcitalopram.Innaïvemice,consumptionofdeuteriumdepleted waterresultsinchangesofEEGparametersofsleepthatare rem-iniscent of theeffects ofnoradrenaline and serotoninreuptake inhibitors.Thus,deuterium-depleteddrinkingwatercouldpresent anovelprophylacticstrategyfordepression.

Acknowledgements

Wewouldliketoacknowledgetheimportantcontributionof Drs.DoloresBonapartes,DinoraLopes,HenriqueSilveira,aswell asthetechnical helpofHelenDugua,ColetteRousset and Olga Karnauch.WethankthePortugueseFoundationforScience(FCT), NewLisbon University,theEuropeanCommunity(EC: AGGRES-SOTYPEFP7/No.602805),NARSADfortheirﬁnancialsupport,Drs. OlegDolgov,AlexanderLysko, AndreyProshin,ElenaZakharova, VladimirKovalson,AlexanderRevischin,AlmazKotovskTambov RegforsupplyingtheD91andD140waterandMr.PaulCourtel.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.bbr.2014.07.039.

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