Magnetoelectricmaterialsforminiature,wirelessneuralstimulationat1therapeuticfrequencies23AmandaWickens1,2,BenjaminAvants2,NishantVerma3,EricLewis2,JoshuaC.Chen3,4ArielK.Feldman4,5,ShayokDutta2,JoshuaChu2,JohnO’Malley6,MichaelBeierlein6,5CalebKemere2,3,JacobT.Robinson1,2,3,7671AppliedPhysicsProgram,RiceUniversity,Houston,Texas,USA,2DepartmentofElectricaland8ComputerEngineering,RiceUniversity,Houston,Texas,USA,3DepartmentofBioengineering,Rice9University,Houston,Texas,USA,4DepartmentofComputerScience,RiceUniversity,Houston,Texas,10USA,5DepartmentofCognitiveScience,RiceUniversity,Houston,Texas,USA,6Departmentof11NeurobiologyandAnatomy,McGovernMedicalSchoolatUTHealth,Houston,Texas,USA,127DepartmentofNeuroscience,BaylorCollegeofMedicine,Houston,Texas,USA1314Afundamentalchallengeforbioelectronicsistodeliverpowertominiature15devicesinsidethebody.Wiresarecommonfailurepointsandlimitdevice16placement.Wirelesspowerbyelectromagneticorultrasoundwavesmust17overcomeabsorptionbythebodyandimpedancemismatchesbetweenair,18bone,andtissue.Magneticfields,ontheotherhand,sufferlittleabsorptionby19thebodyordifferencesinimpedanceatinterfacesbetweenair,bone,and20tissue.Theseadvantageshaveledtomagnetically-poweredstimulatorsbased21oninductionormagnetothermaleffects.However,fundamentallimitationsin22thesepowertransfertechnologieshavepreventedminiaturemagnetically-23poweredstimulatorsfromapplicationsinmanytherapiesanddiseasemodels24becausetheydonotoperateinclinical“high-frequency”rangesabove20Hz.25Hereweshowthatmagnetoelectricmaterials–appliedforthefirsttimein26bioelectronicsdevices–enableminiaturemagnetically-poweredneural27stimulatorsthatoperateatclinicallyrelevanthigh-frequencies.Asan28example,weshowthatMEneuralstimulatorscaneffectivelytreatthe29symptomsofaParkinson’sdiseasemodelinafreelybehavingrodent.Wealso30showthatME-powereddevicescanbeminiaturizedtosizessmallerthana31grainofricewhilemaintainingeffectivestimulationvoltages.Theseresults32suggestthatMEmaterialsareanexcellentcandidateforwirelesspower33deliverythatwillenableminiatureneuralstimulatorsinbothclinicaland34researchapplications.3536Wirelessneuralstimulatorshavethepotentialtoprovidelessinvasive,longer37lastinginterfacestobrainregionsandperipheralnervescomparedtobattery-38powereddevicesorwiredstimulators.Indeed,wiresareacommonfailurepointfor39bioelectronicdevices.Percutaneouswirespresentapathwayforinfection1and40implantedwirescanalsolimittheabilityofthestimulatorstomovewiththetissue,41leadingtoaforeignbodyresponseorlossofcontactwiththetargettissue2,3.42Additionally,chronicstressandstrainonwires,particularlyfordevicesinthe43periphery,canleadtofailureinthewireitselforitsconnectiontothestimulator4.In44smallanimalslikeratsandmice,wiresusedtopowerneuralstimulatorscan45interferewithnaturalbehavior,particularlywhenstudyingsocialinteraction46betweenmultipleanimals5.47

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

48Oneoftheprimarychallengesforwirelessneuralstimulatorsistocreateefficient49miniaturedevices(<1cminlength)thatoperatereliablybeneathboneandtissue50asananimalorhumanpatientengagesinnormalactivity.Atlengthsoflessthan151cm,devicescouldbefullyimplantedintheperipheryandbelightenoughtoallow52forunrestrictedanimalbehavior;howeverfordevicesthissmall,powerdelivery53remainsachallenge.Efficientpowertransferwithpropagatingelectromagnetic54wavesrequiresantennaswithfeaturesizescomparabletotheelectromagnetic55wavelength.Thus,forsub-millimeterdevices,suchastheproposedRFpowered56“neurograins6,”effectivepower-transferfrequencieslieintheGHzrange,where57electromagneticradiationisabsorbedbythebody7.Absorptionofthisradio-58frequencyelectromagneticenergylimitstheamountofpowerthatcanbesafely59deliveredtoimplantsdeepinsidetissue7.Asaresult,researcherstypicallyturnto60magneticinductionorbatteriestopowerimplanteddevices;however,these61techniquesalsolimitthedegreeofminiaturization.Batteriesincreasethesizeofthe62deviceandaddconsiderableweight.Additionally,batteriesrequirereplacementor63charging,whichcanlimitthepotentialuses.Inductivelycoupledcoils,ontheother64hand,canbemadesmallerandlighterthanbatteries,however;thepowera65receivingcoilcangenerateisdirectlyrelatedtotheamountoffluxcapturedbythe66areaofthecoils.Thus,whenthereceivercoilsareminiaturized,theoutputpower67reducesandbecomesmoresensitivetoperturbationsinthedistanceorangle68betweenthetransmitterandreceiver8.Forexample,Freemanetal.demonstrated69thatsmallinductivecoilslessthan1mmindiametercanpowerstimulatorsforthe70sciaticnerveinanesthetizedrats9;however,initspresentform,thisdevicewould71havedifficultyprovidingstablestimulationinfreelymovinganimalsduetothe72reducedpowercouplingefficiencythataccompanieschangesintheangleand73distancebetweenthereceiverandtransmittercoils.7475Additionally,forneuralstimulatorstotreatanumberofneurologicaldisorderslike76Parkinson’sDisease(PD),obsessive-compulsivedisorder,andepilepsy,theymust77operatesafelyandeffectivelyinthehigh-frequency“therapeuticband”between10078and200Hz10–12.Thistypeofhigh-frequencyneuralstimulationischallenging79becausechargeontheelectrodemustbedissipatedbetweensuccessivestimulation80pulsestopreventelectrolysis,tissuedamage,andchangestothelocalpH13.Charge81dissipationathigh-frequenciesisaccomplishedbyusingabiphasicstimulus82waveformthatactivelyorpassivelychargesanddischargestheelectrodewitheach83cycle.Indeedallclinicallyapprovedelectricalneuralstimulationtherapiesinthis84therapeuticbandusevariousformsof“chargebalanced”biphasicstimulation85waveforms14.8687Recently,severalpromisingalternativestomagneticinductionandbatterieshave88enabledminiatureneuralstimulators;however,theseapproacheshaveyetto89demonstrateinvivooperationinthetherapeutichigh-frequencybandinfreely90movinganimals.Montgomeryetal.andHoetal.haveshownthatonecanusethe91mousebodyasanelectromagneticresonantcavitytoeffectivelytransferenergyto92sub-wavelengthscaledevicesimplantedinsidetheanimal15,16.Thisapproachis93

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

particularlyeffectivetodrivetinyLEDsforoptogeneticstimulation.However,94becausetheelectricalwaveformismonophasic,electricalstimulationhasbeen95limitedto<20Hz.Usingsuperparamagneticnanoparticlestoabsorbenergyfrom96high-frequency(500kHz)magneticfields17,onecanheatspecificregionsofthe97brain18,19infreelymovinganimals19.Thislocalheatcanstimulateneuralactivity98whenthetargetedbrainregionisgeneticallymodifiedtorespondtochangesin99temperature18,19.Howeverthisapproachrequirestransgenesis,whichadds100regulatorycomplexityandhasyettoshowhigh-frequencyoperationduethe101requirementforthetissuetocoolbetweenstimulationintervals.Ultrasound102providesapromisingandefficientmethodtopowerbioelectronicimplantsbecause103ultrasoundwavelengthsare105timessmallerthanelectromagneticwavesatthe104samefrequencyallowingsub-millimeter-sizeddevicestohavewavelength-scale105piezoelectricantennas20,21.However,implementationofthese“neuraldust”motes106canbechallenginginfreelymovinganimalsbecausetheimpedancemismatch107betweenair,bone,andtissuerequirescontactbetweensofttissueandthe108ultrasoundtransducerforefficientpowertransfer.Asaresult,therehasyettobea109demonstrationofultrasound-poweredneuralstimulatorsinfreelymoving110animals22.111112Hereweshowthatmagnetoelectric(ME)materialsenablethefirstmagnetically113poweredminiatureneuralstimulatorsthatoperateinthetherapeutichigh-114frequencyband.Similartoinductivecoils,thesematerialstransformamagnetic115fieldtoanelectricfield,butinsteadofusinganimplantedcoilweuseamaterialthat116generatesavoltageviamechanicalcouplingbetweenmagnetostrictiveand117piezoelectriclayersinathinfilm.Namely,themagneticfieldgeneratesstraininthe118magnetostrictivelayerasthemagneticdipolesalignwiththeappliedfield.That119strainexertsaforceonthepiezoelectriclayer,whichgeneratesavoltage(Fig.1).By120exploitingthistransductionmechanism,magnetoelectricsdonotsufferfromthe121sameminiaturizationconstraintsascoilsandcanbedrivenbyweakmagneticfields122ontheorderofafewmillitesla.Thesepropertieshaveledresearcherstopropose123magnetoelectricsasapromisingmaterialforbioelectronicimplants23–27.Herewe124demonstratethefirstproof-of-principlewirelessneuralstimulatorsbasedonME125materialsinafreelybehavingrodentmodelforParkinson’sDisease(PD),andthat126thesematerialscouldpowerminiaturedevicesdeepwithinthehumanbrain.127128FabricationandcharacterizationofMEstimulators129130Wefabricatedproof-of-principleMEstimulatorsbybondingarectangular131magnetostrictivelayer(Metglas)toaplatinumcoatedpiezoelectriclayer,132polyvinlydinefluoride(PVDF).Wethenencapsulatedthefilmsinaprotective133parylene-Clayer(8-10μmthick)(Fig.1a,seeMethods).WeusedPVDFlayers134between28and110μm,whichyieldedtotaldevicethicknessesbetween50-150135μm.Whenwemeasuredthevoltageacrossthefilm,wefoundadramaticvoltage136increasewhentheappliedmagneticfieldfrequency,matchesanacousticresonant137frequency(Fig.1b).Becausetheresonantfrequencyisproportionaltotheinverseof138thefilmlength,wecandesignmultiplefilmsandselectivelyactivatethemby139

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

changingthestimulusfrequency(Fig.S2b).Usingthisprinciple,wecanusedifferent140magneticfieldfrequenciestoactivateseparatedevicesthatmaybeindifferentareas141ofthebody,orcreatebiphasicstimulatorsbyinterleavedresonantstimulationof142twodifferentfilms,witheachfilmdrivingeitherthepositiveornegativephaseof143theneuralstimulus.144

Figure1|MEfilmsconvertalternatingmagneticfieldsintoavoltage.(a)DiagramofaMEdeviceonafreelymovingratforwirelessneuralstimulation.TheactiveMEelementconsistsofpiezoelectricPVDF(blue)andMetglas(gray)laminateencapsulatedbyParylene-C.Insetshowstheoperatingprinciplewherebythestrainproducedwhenmagnetizingthegraymagnetostrictivelayeristransferredtothebluepiezoelectriclayer,whichcreatesavoltageacrossthefilm.(b)ExampleofaresonantresponsecurveforaMEfilmshowingthatthemaximumvoltageisproducedwhenthemagneticfieldfrequencymatchesanacousticresonanceat171kHz.PhotographinsetshowsanexampleofanassembledMEstimulator.The“Stressprofile”insetshowsatopviewofthestressproducedinaMEfilmascalculatedbyafiniteelementsimulationonandoffresonance(COMSOL).(c)Devicetestingsetupwithapermanentmagnettoapplyabiasfieldandanelectromagneticcoiltoapplyanalternatingmagneticfield(scalebars:upper=1cm,lower=2mm)(d)Maximumstimulationduration(usinga400μs/phasepulserepeatedatincreasingfrequencies)foraMEdeviceinbiphasicandmonophasicoperation.Maximumstimulationtimeisdeterminedbytimeofelectrolysisonastereotrodeinsalineasevidencedbygasbubbles(errorbars+/-1standarddeviationforn=4trials).Dashedlinesindicatefrequenciesofelectricalstimulationusedinvariousclinicalapplications,showingthatbiphasicoperationisnecessaryformanyclinicallyrelevantapplications.Romannumeralsindicatestimulationfrequenciesdemonstratedbypreviouslypublishedminiaturemagneticstimulators(i:Magnetothermal,Chenet.al,2015,ii:Magnetothermal,Munshiet.al,2017,iii:Mid-FieldOptogenetics,Montgomeryet.al,2015,iv:RFInductiveCoupling,Freemanet.al,2017).

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

145WecanfurtherenhancethevoltagegeneratedbytheMEfilmsbyapplyinga146constantbiasfieldwithapermanentmagnetoranelectromagnet(Fig.1c).Because147thestraininthemagnetostrictivematerialisasigmoidalfunctionofthemagnetic148fieldstrength,thechangeinvoltageproducedbythealternatingfieldislargest149whenthefieldoscillatesaboutthemidpointofthesigmoid(Fig.S1)28,29.Thus,we150useabiasfieldtooffsetthemagneticfieldnearthecenterofthesigmoidal151magnetostrictiveresponsecurve.Thisbiasfieldallowsustogeneratetherapeutic152voltagelevelswhileapplyingasmall(fewmT)alternatingmagneticfieldusingan153electromagneticcoilandcustomcontrolcircuitrythatspecifiesthefrequencyand154timingofthealternatingmagneticfield(Fig.S3).155156ToidentifytheoperationalfrequenciesforourMEstimulatorswetestedthemin157salineandfoundthatwithabiphasicstimulationwaveformwecouldapplyconstant158stimulationuptoatleast800Hzwithoutsignificanthydrolysis.Forthistestwe159operatedeitheronefilmformonophasicstimulationortwofilmsforbiphasic160stimulationattachedtoastereotrode(Microprobes)insaline(seeMethods).We161thenmeasuredthetimeatwhichwecouldseebubblesattheelectrodetipresulting162fromhydrolysis.Thishydrolysiseventindicatesconditionsthatwouldlesionthe163surroundingtissue.Wefoundthatwithamonophasicstimulationwaveform164stimulationfrequenciesabove50Hzproducedhydrolysiswhilebiphasiccharge-165balancedstimulationshowednohydrolysisuptothemaximumtestedfrequencyof166800Hz.Comparedtopreviouslydemonstratedminiaturemagneticneural167stimulators,thebiphasicMEdevicesshownherearethefirsttoaccessthehigh-168frequencybandsusedforclinicalapplicationslikethetreatmentofParkinson’s169diseaseandobsessive-compulsivedisorder(Fig1d).170171Anadditionalchallengeforanywirelesslypoweredneuralstimulatoristomaintain172awell-regulatedstimulationvoltage.Thischallengeisespeciallyprevalentas173devicesbecomesmall,whichoftenreducesthepowertransferefficiencyresultingin174agreatersensitivitytothealignmentbetweenthedeviceandpowertransmitter.ME175materialsoffertwomainadvantagesthatcanenablestableandeffectivestimulation176evenasdevicesbecomesmallandmovewithrespecttothedrivercoils:177178First,MEdevicesgeneratevoltageswellinexcessoftheeffectivestimulation179potential,allowingthemtobeeffectiveevenifthematerialsaremisalignedwiththe180drivercoils.Atresonance,wehavemeasuredMEvoltagesinexcessof30Vatafield181strengthofonly1mT(Fig.S2c,d).Becauseeffectivestimulationvoltagesareusually182between1-5V,wecancaptheappliedvoltagetothiseffectivestimulationrange183usinganLEDorZenerdiode.AslongasthevoltagegeneratedbytheMEfilmis184greaterthanorequaltothecappingvoltage,wewillapplyapproximatelythesame185stimulusvoltageregardlessoftheangleordistancebetweenthedrivercoilandthe186MEfilm.Foratypicalfilmwefoundthatwecouldreorientthefilmby+/-80187degreesandmaintainvoltagesinexcessof3V(Fig.S1h).Thislargeangular188toleranceisaidedbythelargemagneticpermeabilityoftheMetglaslayer,which189

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

helpstodirectthemagneticfieldlinesalongthelongaxisofthefilm,wheretheyare190mosteffectiveatcreatingamagnetostrictiveresponse.191192Second,thevoltagegeneratedbyapiezoelectricmaterialdependsonthethickness193ofthepiezoelectriclayerandnottheareaofthefilm30,allowingustofabricatesmall194magnetoelectricfilmsthatgenerateroughlythesamestimulationvoltageaslarger195devices.FigureS2showsthepeakvoltagegeneratedandqualityfactorforMEfilms196ofdifferentareas.Wefoundthat,fora52μmthickPVDFlayer,thevoltageremains197around10Vevenasthefilmlengthdecreases.Variationsof+/-40%inpeak198voltageandqualityfactorsarelikelyduetodefectsproducedduringfilm199fabrication,whichmaybereducedwithimprovedmanufacturing.Wealsoverified200thattheoutputvoltagedependsonlyonthepiezoelectricfilmthicknessby201measuringthepeakvoltagesfromMEdeviceswiththreedifferentthicknessesof202PVDF:28μm,52μm,and110μm.Asexpected,weseethatthepeakvoltage203increaseslinearlywiththePVDFthicknessandisindependentofthefilmlength.We204calculated(andexperimentallyconfirmed)thatthepowergeneratedbyaMEdevice205isproportionaltothefilmwidthforagiventhicknessandalength-to-widthratio>3206(seeFig.S2f).Despitethedecreaseinpowerasfilmsbecomesmaller,wecalculate207thatfilmslessthan1cmlongcangenerateupto4mW,whichismorethan208sufficientformanywirelessapplicationsincludingneuralstimulation31.209210MonophasicstimulationbyMEfilmsevokeactionpotentialsinvitro211212Usingfluorescencemicroscopytoimagevoltageinculturedcells,wefoundthat213monophasicstimulationfor50msat100HzbyMEfilmsreliablystimulatedaction214potentials(APs).Fortheseexperimentsweused“spiking”humanembryonickidney215(HEK)celllinesthatweremodifiedtoexpresssodiumandpotassiumchannels.216Thesecellshavespike-likeelectricalwaveformsthatarerectangularinshapeand217canlastforafewsecondsdependingontheconfluencyoftheculture32.To218determinetherelativetimingbetweenmagneticstimulationandactionpotential219generation,wetransfectedthesecellswithArcLight33-ageneticallyencodedvoltage220indicatorthatallowsustomeasureactionpotentialsusingfluorescencemicroscopy.221222

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

Toimagefluorescencewhileweappliedmagneticfields,wedevelopedan223experimentalsetupthatallowsustoplacecellsandMEfilmsbeneathanobjective224lensatthecenterofa10cmlongsolenoidwitha3cmgapinthecenter.This225configurationallowedustoplaceMEfilms,cells,andtheobjectivelensatthecenter226oftheappliedmagneticfield(Fig.2a).Twoslightlylargercoilsplacedoneitherside227ofthegapprovidetheconstantbiasfield.228229WethenapproximatedanimplantedMEstimulatorusingtwoexperimental230configurations:1)growingcellsdirectlyontheMEfilm(Fig.S4)and2)layinga231coverslipwithadherentcellsontopoftheMEfilm(Fig.2).Toculturecellsdirectly232ontheMEfilm,wecoatedthetopparylenelayerwithpoly-l-lysine.Thehealthy233proliferationofHEKsontheMEdeviceindicatesthatthisencapsulationapproach234preventstheMEmaterialsfromlimitingcellgrowth(Fig.S4b).However,inatypical235usecase,thetargetcellsmaynotadheretotheMEstimulator,sowealsotestedthe236responseofcellslaidontopoftheMEmaterials.Inthisconfigurationwefirstgrew237thecellsoncoverslipsfor3-5daysbeforeinvertingthecoverslipsandlayingthem238ontheMEfortesting(Fig.2,seeMethods).239

Figure2|MonophasicMEstimulatorsactivatecellsinvitro(a)Schematicoftheexperimentalsetup(b)MicroscopeimageofholesstampedintotheMEfilmandfiniteelementsimulationoftheelectricfieldshowsthattheholesproducefringingelectricfieldsthatoverlaptheculturecells(c)VoltageacrosstheMEfilmwhenthemagneticfieldisonresonanceand(d)offresonance.Insetsshowazoominofthehighfrequencycarrierwaveform.(e-g)FluorescencefromspikingHEKstransfectedwithArcLightshowactionpotentialsaretriggeredbytheMEfilmdrivenatresonance(e),butnotwhenthefilmisdrivenoffresonance(f).FluorescencefromHEKcellstransfectedwithGFPshownoresponsewhentheMEfilmisdrivenonresonanceconfirmingthatthemeasuredArcLightresponseistheresultofachangeintransmembranepotentialandnotanartifactofthemagneticfieldoracousticresonanceoftheMEfilm.

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

240Tocreatefringingelectricfieldsthatinteractwiththeculturedcells,westamped241holesintheMEfilm(Fig.2b).Thefilmswereotherwisefabricatedasdescribed242above(Fig.1,Methods).InexperimentsusingMEfilmsandPtelectrodeswefound243thathigh-frequencybiphasicstimulationattheMEresonancefrequency(typically24420-150kHz)wasnoteffectivetostimulateAPsinculturedHEKs,aspredictedbythe245low-passfilteringpropertiesofthecellmembrane9.Tocreateaneffective246monophasicstimuluswaveform,weusedaSchottkydiodetorectifythevoltageto247createentirelypositiveornegativevoltagewaveformsdependingonthediode248direction.Thisrectifiedwaveformhasaslowlyvaryingmonophasicenvelopeinthe249

Thetransistorsblockcurrentsgeneratedbyonefilmfrompropagatingthroughthe286circuitryattachedtotheotherfilm,ensuringthatonlyonehalfofthecircuitisactive287atatime.ByswitchingthemagneticfieldfrequencybetweenthetwoMEresonance288frequencies,wecanalternatepositiveandnegativephasestimulationtocreatea289biphasicneuralstimulator(Fig.3b-d).Inthiscasetheresidualchargeof-2.3nC,290whichdischargesin500Hzwithoutaccumulatingcharge.292293WefoundthatourbiphasicMEstimulatoriscapableofrepeatableneural294stimulationusingneocorticalbrainslicesderivedfrommicethatexpressthe295geneticallyencodedcalciumindicatorGCaMP3inGABAergicneurons.Toimage296neuralactivityfollowingMEstimulationweinsertedastereotrodeattached297biphasicMEstimulatordescribedabovewhileweimagedGCaMPactivityusing298fluorescencemicroscopy(Fig.3e-g,Methods).Wechoseneuralstimulation299parameterssimilartothosecommonlyusedfordeepbrainstimulation34:100300biphasicpulsesat200Hzwitheachphaselasting175μs.Whenthemagneticfield301wasonweobservedacorrespondingincreaseinfluorescenceinn=23recordingsin302neocorticallayer5consistentwithactivity-mediatedcalciumincreases303(SupplementaryVideo2).Followingbathapplicationoftetrodotoxin(TTX,500304nM)fluorescenceincreaseswerecompletelyblockedinn=9recordingsconfirming305thatMEstimulationreliablyevokedsodium-channeldependentactionpotentialsin306nearbyneurons.307 308

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

309 310

Figure3|BiphasicMEstimulatorsactivateneuronsinexvivobrainslices(a)SchematicofexperimentalsetupwithtwoMEfilmsforbiphasicstimulation(b)Measuredvoltagewaveformproducedbyourmagneticfieldgenerator.Whencoupledtothemagneticcoils,thiswaveformproducesmagneticfieldsthatalternatebetweentheresonantfrequenciesofthetwoMEfilms(c)Measuredvoltageacrossthestereotrodeshowstheexpectedbiphasicpulseshape(d)Calculatedcurrentbasedonmeasuringthevoltageacrossourloadresistor(VR)showsnearlyperfectchargebalancingwithonly2.3nCaccumulatingontheelectrodeperpulsetrain.(e)Brightfieldimageofstereotrodeinmousecortex(scalebar=1mm)withinsetofGCaMPsignalaveragedovera600μmx600μmregionaroundstereotrodetip.Arrowindicatingafluorescingcellbodynearthestereotrode(f)AverageGCaMPsignalwhenresonantmagneticfieldisappliedbefore(f)andafter(g)addingTTXshowsneuralactivityisinducedbytheMEstimulator.Thingreentracesrepresentseparateexperimentsfromtwodifferentbrainslices,andthickblacktracesrepresentthemeanofallexperiments.

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

MENeuralStimulationinFreelyMovingRatsShowsBehavioralEfficacy311312AmajoradvantageofourMEstimulatorsisthefactthatremoteactivationenables313experimentswithfreelybehavinganimals.Asaproof-of-principleweadaptedour314biphasicstimulatorfordeepbrainstimulation(DBS)infreelymovingrats(Fig.4).315TotestMEstimulatorefficacy,weusedapreviouslyreportedprotocoltotestDBSin316hemi-parkinsonianrats36.Intheseexperimentsratsareinjectedwith6-OHDAinthe317leftmedialforebrainbundle(MFB)tocreateaunilaterallesionofthesubstantia318nigraparscompacta(SNc).Theanimalsarethenplacedina30cmdiametercircular319enclosure.Followingadoseofmethamphetamine,thehemi-parkinsonianratshave320beenshowntorotateipsilateraltotheinjection(e.g.leftforinjectionintotheleft321MFB).Duringtheserotations,theratprimarilymovesusingitscontralateral(right)322forepaw,rarelyplacingtheipsilateral(left)forepawontotheground.Whena323biphasicstimulusisappliedat200Hzinthesub-thalamicnucleus(STN)usinga324tetheredelectrodearraystimulator,ratstypicallystopturningtotheleftandexhibit325morenormalbehaviorsuchasmovingwithbothforepaws,maintainingasteady326orientation,orturningtothecontralateralside34.327328Tocreateawireless,biphasicMEstimulatorforfreelymovinganimalsweaddeda329smallpermanentmagnettotheMEstimulatortogenerateabiasfield,andwrapped330thebehavioralchamberwith18AWGcopperwiretocreateasolenoid(Fig.4a,S5).331Byintegratingthesmallpermanentmagnet(<0.25g)intotheMEstimulator,we332couldensurethatthebiasfieldwasconstantlyalignedwithMEfilmsastheanimal333movedwithintheenclosure.Wecouldalsoensurethatthepositiveandnegative334stimulihadequalamplitudesbyindependentlyadjustingthedistancebetweeneach335filmandthepermanentmagnet.ThisMEstimulatorwasthenconnectedtoa336commercialelectrodearray(Microprobes)implantedintheSTN(Fig4b,see337Methods).Weensuredthatthestimulationvoltageandcurrentwerewithinthesafe338andtherapeuticrangebymeasuringtheoutputoftheMEstimulatorconnectedto339anequivalentcircuitmodelofthebrain(Fig.4c,seemethods).Specifically,we340observedpeakvoltagesofapproximately+/-1.5Vandpeakcurrentsof341approximately+/-100μAfor400usatapproximatelya50%dutycycle(200μsof342overallcurrentperphase),whichiswithintheeffectivestimulationrangereported343forconventionalwiredstimulators36.Whenwetunethemagneticfieldfrequencyoff344resonanceweobservealmostnogeneratedvoltageorcurrent(Fig.4c).345

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

346WethentestedthewirelessversionofourbiphasicMEstimulatormountedtothe347headofafreelybehavingratandfoundthatMEstimulationshowedefficacy348comparabletopreviouslyreportedwiredDBSstimulators(Fig.4).Withamagnetic349fieldappliedatresonance,wefoundthatone-minuteperiodsof200Hzbiphasic350pulsesresultedinasignificantdecreaseintheanimal’srotationrate(Fig.4dgreen351intervals).Thisdecreasedrotationwasnotobservedwhenthemagneticstimulus352frequencywastunedoffresonance(Fig.4dblueintervals).Plotsofthehead353

Figure4|EffectiveDBSinafreelymovingratusingawirelessMEstimulator(a)Experimentalsetupshowingratinacircularenclosurewrappedwithmagnetwire.InsetshowsabiphasicMEstimulatoronaonecentcoin(b)SchematicofthebiphasicMEstimulatorattachedtotheelectrodearraythatisimplantedintotheSTN(c)MeasuredvoltagegeneratedbytheMEdeviceandthecurrentappliedtothebrainonresonance(green)andoffresonance(blue).Approximately100μAbiphasicstimulationisappliedonlywhenthenthemagneticfieldfrequencymatchestheresonancecondition.(d)Angularvelocityofthehemi-Parkinsonianratovera40minuteDBStrialwithintervalsofresonantandnon-resonantstimulationshowsthatrotationsarereducedonlywhenthestimulatorisactivatedbyaresonantmagneticfield(e)Typicaltrajectoriesshowthelocationoftheanimal’sheadovertwo30-secondintervalsdenotedinc(scalebar=5cm)(f)Averageangularvelocityoftheratduringthe30secondsbeforestimulationandthefirst30secondsofstimulationforeachintervalduringthe40-minexperimentshowsaclearreductioninangularvelocityonlywhentheMEfilmisactivatedonresonance(***P=4x10-7,n.s.=notsignificantP=0.70,pairedt-test)

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

trajectoriesshowthatthepathologicalrotationsobservedduringoff-resonant354magneticfieldstimulationarenotpresentwhentheMEstimulatorisactiveduring355resonantmagneticfieldstimulation(Fig.4e,Methods).Whenaveragedoverall356trials,averagerotationrateduringthefirsthalfofstimulationfelltoastatistically357significant1.6rotationsperminute(rpm),comparedto9.3rpmintheabsenceof358stimulation,or9.4rpmduringoff-resonantstimulation(pairedt-test,Fig.4f).We359furtherdemonstratedtherepeatabilityofthisstimulatorbyrepeatingthis360stimulationprotocolonasecondratandfoundsimilarresults(FigS5b).361362Withaweightof0.67g,theMEstimulatorsdescribedherearethefirstreported363miniature,magnetic,highfrequencystimulator.Furthermore,bychangingthe364frequencyandtimingoftheexternaldrivecoils,wecangenerateavarietyof365stimulationpatternsthroughoutthetherapeuticwindowof100-200Hzwith366applicationstootherdiseasemodels.Additionally,calculationsofthemagneticfield367strengthssuggestthatwecanreconfigurethedrivecoilsforanumberofbehavioral368experimentsbyplacingcoilsbeneaththefloorofananimalenclosure.Finite369elementsimulationsandmeasurementsshowthatevenatdistance4-5cmabovea370drivecoil,MEfilmsgeneratesufficientvoltageforstimulation(Fig.S6).This371distancecouldbefurtherimprovedbyoptimizingthegeometryofthecoilsor372increasingthepowerofthemagneticfield.373374Demonstrationofmultichanneldeepbrainstimulationinskullphantomusing375rice-sizedMEfilms376377Inadditiontosupportingexperimentsinfreelymovingrodents,MEmaterialscould378enableminiaturizedwirelessstimulatorsthatoperatedeepinthebrainoflarge379animalsorhumanpatientsandareindividuallyactivatedwithanexternal380electromagnet.Toourknowledge,thisisthefirsttechnologythatenables381independentexternalwirelesscontrolofmultipleminiaturestimulatorsdeep382beneathahumanskullphantom.Figure5ashowsthepredicteddepththatvarious383miniatureantennascouldbesafelyimplantedundertheskullandgenerate1mWof384power,whichisintheapproximatemaximumpowerrequiredforhigh-frequency385continuousneuralstimulation31.Asmentionedabove,radio-frequency(RF)386poweredantennasthatoperateatfrequenciesabove~1MHzhavelimitationsinthe387amountofpowerthatcansafelybedeliveredtoanimplanteddevicewithout388causingpotentiallyharmfultissueheating.Simulationsshowthatwhenoperating389withthesafepowerlimits,RF-antennasmustbeplacedonthesurfaceofthebrain390orinveryshallowregionstoharvest1mWofpower.“Mid-field”techniques37,391improvetheRFcouplingefficiencyenablingdeepoperation,butbecausethis392approachoperatesatafixedfrequencytherehaveyettobedemonstrationsof393individuallyaddressablemotesorbiphasicstimulation.Othertechniquesfor394wirelesspowerdeliverydiscussedpreviously,suchmagneticinduction,alsocannot395achievedeepmultichannelstimulation.Forexample,evenusingahigheroperating396frequencyof1MHzaninductivecoilwiththesameorientationandcross-sectional397areaastheMEfilmsshownherewouldrequireaminimumof500turnsofwireto398generate2Vusingthesame0.5mTfieldusedhere(assumingatypicalQ-factorof399

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

10).Thus,devicesbasedonmagneticinductorscannotbeminiaturizedwithout400sacrificingavailablepowerasdescribedpreviously9.401402Asaproof-of-conceptdemonstrationweshowthattworice-sizedMEfilmscanbe403individuallyaddressedatthecenterofahumanskullphantomusinganexternal404electromagnet.Thesetwo-filmswithlengthsof8mmand10mmhaveacoustic405resonantfrequenciesof180and200kHz,whicharedeterminedbythefilmlength.406WhenthesefilmsareattachedtoanorangeLED,theiroutputvoltageiscappedat407approximately1.8V,whichhelpstoregulatethestimulationvoltageandallowsus408tovisualizefilmactivation.MEfilmsofthissizearesmallerthancurrentDBSleads409andcouldpotentiallybeimplantedintodeepbrainareasasshowninFig5c.410Additionally,themagneticstimulationcoilissmallenoughtobeincorporatedintoa411stylishhatorvisorthatcouldbeworncomfortablybyapatient.Whenweplaced412

Figure5|MiniaturizedMultichannelStimulationinHumanSkullPhantom(a)ComparisonofeffectivedepthbeneathahumanskullphantomforMEdevicescomparedtootherminiaturewirelessstimulators.Depthlimitisbasedonsafetylimitstogenerate1mW.(i:Parket.al,ProcNatAcSci,2016,ii:Yazdandoostet.al,AsiaPacMicrowConf,2009,iii:Yazdandoostet.al,Proc37EuropMicrowConf,2007,iv:Agrawalet.al,NatBiomedEng,2017)(b)PhotosofMEfilmsnexttoagrainofriceandthecorrespondingvoltageasafunctionofmagneticfieldfrequency(fieldstrength1mT,scalebars2mm)(c)SchematicofshowingpotentialapplicationoffullyimplantedMEfilmswiththemagneticfieldgeneratedbyanexternalcoilthatcanbeincorporatedintoahatorvisor(d)Frontviewand(e)topviewofskullphantomwiththetopremovedtoviewLEDs(filmlocationsindicatedbyarrows,scalebar1cm)(f)PhotoofLEDsattachedtoMEfilmswiththemagneticfieldsatappliedat180kHzand(g)200kHz.SelectiveilluminationoftheLEDscorrespondingtheresonantfrequenciesofthefilmsdemonstratessuccessfulmultichannelactivationofindividualfilms(scalebars1cm).Magneticfieldstrengthwasmeasuredtobe0.5mTatthelocationoftheMEfilms.

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

thetwoMEfilmsatthecenterofaskullphantomwefoundthatwecould413individuallyilluminatetheLEDsoneachfilmwhenweappliedamagneticfieldat414theresonantfrequencyoftheselectedfilm(Fig5d-g).Forthisexperimentweuseda415400Wpowersupply,whichproducedafieldofapproximately0.5mTatthecenter416oftheskullphantom.Thetopoftheskullphantomwasremovedforvisualization,417buthadnoaffectonourabilitytodrivetheLEDindicators.Thenumberof418stimulationchannelscouldbeincreasedwiththeadditionofMEfilmswithdifferent419resonantfrequencies.420421Outlook422423Toourknowledge,thisisthefirstdemonstrationofaminiature,magneticneural424stimulatorthat1)operatesinthetherapeuticband(100-200Hz)infreelymoving425animalsand2)enablesindividuallyaddressableminiaturestimulatorsdeepwithin426ahumanskullphantom;however,theadvantagesofMEmaterialsextendbeyond427theseproof-of-principledemonstrations.428429MEstimulatorssuchastheonedescribedintheinvivoratexperimentcouldhave430animmediateimpactonthestudyofDBStherapiesusingrodentdiseasemodels.431BecausetheMEstimulatoriscompatiblewithcommercialimplantedelectrodes,and432themagneticstimulatorscanbeadaptedtoanumberofstandardbehavioral433experimentsoranimalenclosures,ourMEstimulatorscouldreadilyreplacethe434wiredDBSstimulatorscurrentlyinuse.Asaresult,newexperimentscanbe435developedtoprobetheeffectsofchronicandcontinuousDBSorDBSinsocial436contextswherewiredDBSstimulatorswouldbeimpracticable.437438Additionally,MEmaterialshavethepotentialtoenableminiatureneuralstimulators439thatcanbeimplanteddeepinthebrainoflargeanimalsorhumansandaddressed440externallywithasmallelectromagnet.Asshownhere,rice-sizedfilmscanbe441selectivelyactivatedbasedonuniqueresonantfrequencies.Additional442miniaturizationisnotexpectedtoreducethevoltageproducedbythesefilmssince443thevoltagedependsonthethicknessofthepiezoelectricfieldandnotthefilm444length(Fig.S2c),suggestingthatevensmallerfilmscouldserveaseffective445stimulators.446447WealsoforeseeapplicationsforMEmaterialsasawirelesspowertechnologyfor448morecompleximplantablebioelectronicdevices.Forexample,thedemonstrated449abilityofMEfilmstopowerLEDsimpliesthatMEmaterialscouldpower450implantableoptogeneticstimulators,orsmallintegratedcircuitsforphysiological451monitoring.452453Torealizethesefullyimplantablebioelectronicdevices,workisneededtoimprove454MEmaterialsandfabricationprocessestoreliablyproducehigh-qualityminiature455MEfilms,andencapsulatethemforchronicuse.Forwearabletechnologies,itisalso456necessarytofurtherminiaturizemagneticfieldgeneratorssothattheycanbe457

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

batterypoweredandcomfortablyworn.Theseadvancesmustalsobeaccompanied458byinvivotestingtoshowsafetyandefficiencyforchronicuse.459460Overall,MEmaterialshavethepotentialtofillakeyneedforwirelesspower461deliverytominiaturebiphasicneuralstimulatorsandotherbioelectronicdevices462wherethemajorchallengeistransferringenergyoverdistancesofseveral463centimeterswithoutheatingthetissueorsufferinglossatinterfacesbetweentissue,464bone,andair.465466References467

1. Hargreaves,D.G.,Drew,S.J.&Eckersley,R.KirschnerWirePinTractInfection468Rates:Arandomizedcontrolledtrialbetweenpercutaneousandburiedwires.469J.HandSurg.Br.Eur.Vol.29,374–376(2004).470

2. Roy,B.,C.,M.D.&A.,T.P.Thebraintissueresponsetoimplantedsilicon471microelectrodearraysisincreasedwhenthedeviceistetheredtotheskull.J.472Biomed.Mater.Res.PartA82A,169–178(2007).473

3. Markwardt,N.T.,Stokol,J.&Rennaker,R.L.Sub-meningesimplantation474reducesimmuneresponsetoneuralimplants.J.Neurosci.Methods214,119–475125(2013).476

4. Sahin,M.&Pikov,V.WirelessMicrostimulatorsforNeuralProsthetics.Crit.477Rev.Biomed.Eng.39,63–77(2011).478

5. Pinnell,R.C.,Vasconcelos,A.P.De&Cassel,J.C.AMiniaturized,479ProgrammableDeep-BrainStimulatorforGroup-HousingandWaterMaze480Use.Front.Neurosci.12,1–11(2018).481

6. Nurmikko,A.V.Approachestolargescaleneuralrecordingbychronic482implantsformobileBCIs.in20186thInternationalConferenceonBrain-483ComputerInterface(BCI)1–2(2018).doi:10.1109/IWW-BCI.2018.8311503484

7. International,I.&Safety,E.IEEEStandardforSafetyLevelswithRespectto485HumanExposuretoRadioFrequencyElectromagneticFields,3kHzto300GHz.4862005,(2006).487

8. Fotopoulou,K.&Flynn,B.W.WirelessPowerTransferinLooselyCoupled488Links :CoilMisalignmentModel.IEEETrans.Magn.47,416–430(2011).489

9. Freeman,D.K.etal.Asub-millimeter,inductivelypoweredneuralstimulator.490Front.Neurosci.11,1–12(2017).491

10. DeHemptinne,C.etal.Therapeuticdeepbrainstimulationreducescortical492

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

phase-amplitudecouplinginParkinson’sdisease.Nat.Neurosci.18,779–786493(2015).494

11. Alonso,P.etal.Deepbrainstimulationforobsessive-compulsivedisorder:A495meta-analysisoftreatmentoutcomeandpredictorsofresponse.PLoSOne10,4961–16(2015).497

12. Theodore,W.H.&Fisher,R.S.ReviewBrainstimulationforepilepsy.Lancet4983,111–118(2004).499

13. Merrill,D.R.,Bikson,M.&Jefferys,J.G.R.Electricalstimulationofexcitable500tissue:Designofefficaciousandsafeprotocols.J.Neurosci.Methods141,171–501198(2005).502

14. Parastarfeizabadi,M.&Kouzani,A.Z.Advancesinclosed-loopdeepbrain503stimulationdevices.J.Neuroeng.Rehabil.14,79(2017).504

15. Montgomery,K.L.etal.Wirelesslypowered,fullyinternaloptogeneticsfor505brain,spinalandperipheralcircuitsinmice.Nat.Methods12,969–974506(2015).507

16. Ho,J.S.etal.Self-TrackingEnergyTransferforNeuralStimulationin508UntetheredMice.Phys.Rev.Appl.24001,1–6(2015).509

17. Carrey,J.etal.Simplemodelsfordynamichysteresisloopcalculationsof510magneticsingle-domainSimplemodelsfordynamichysteresisloop511calculationsofmagneticsingle-domainnanoparticles :Applicationto512magnetichyperthermiaoptimization.J.Appl.Phys.83921,(2015).513

18. Chen,R.,Romero,G.,Christiansen,M.G.,Mohr,A.&Anikeeva,P.Wireless514magnetothermaldeepbrainstimulation.Science347,1477–80(2015).515

19. Munshi,R.etal.Magnetothermalgeneticdeepbrainstimulationofmotor516behaviorsinawake,freelymovingmice.Elife6,1–26(2017).517

20. Johnson,B.C.etal.StimDust :A6.5mm3,WirelessUltrasonicPeripheral518NerveStimulatorwith82%PeakChipEfficiency.2018IEEECust.Integr.519CircuitsConf.(2018).520

21. Seo,D.etal.WirelessRecordinginthePeripheralNervousSystemwith521UltrasonicNeuralDust.Neuron91,529–539(2016).522

22. Piech,D.K.etal.StimDust:A2.2mm3,precisionwirelessneuralstimulator523withultrasonicpowerandcommunication.1–35524

23. Nan,T.etal.Acousticallyactuatedultra-compactNEMSmagnetoelectric525

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

antennas.Nat.Commun.8,1–7(2017).526

24. O’Handley,R.C.,Huang,J.K.,Bono,D.C.&Simon,J.ImprovedWireless,527TranscutaneousPowerTransmissionforInVivoApplications.IEEESens.J.8,52857–62(2008).529

25. Yue,K.etal.Magneto-ElectricNano-ParticlesforNon-InvasiveBrain530Stimulation.PLoSOne7,1–5(2012).531

26. Guduru,R.etal.Magnetoelectric‘spin’onstimulatingthebrain.Nanomedicine53210,2051–2061(2015).533

27. Ribeiro,C.,Correia,V.,Martins,P.,Gama,F.M.&Lanceros-Mendez,S.Proving534thesuitabilityofmagnetoelectricstimulifortissueengineeringapplications.535ColloidsSurfacesBBiointerfaces140,430–436(2016).536

28. Zhai,J.,Dong,S.,Xing,Z.,Li,J.&Viehland,D.Giantmagnetoelectriceffectin537Metglas/polyvinylidene-fluoridelaminates.Appl.Phys.Lett.89,8–11(2006).538

29. Kulkarni,a.etal.Giantmagnetoelectriceffectatlowfrequenciesinpolymer-539basedthinfilmcomposites.Appl.Phys.Lett.104,0–5(2014).540

30. Wan,C.&Bowen,C.R.Multiscale-structuringofpolyvinylidenefluoridefor541energyharvesting:theimpactofmolecular-,micro-andmacro-structure.J.542Mater.Chem.A5,3091–3128(2017).543

31. Amar,A.Ben,Kouki,A.B.&Cao,H.Powerapproachesforimplantablemedical544devices.Sensors(Switzerland)15,28889–28914(2015).545

32. Park,J.etal.Screeningfluorescentvoltageindicatorswithspontaneously546spikingHEKcells.PLoSOne8,1–10(2013).547

33. Jin,L.etal.SingleActionPotentialsandSubthresholdElectricalEvents548ImagedinNeuronswithaFluorescentProteinVoltageProbe.Neuron75,549779–785(2012).550

34. So,R.Q.,Mcconnell,G.C.&Grill,W.M.Frequency-dependent,transient551effectsofsubthalamicnucleusdeepbrainstimulationonmethamphetamine-552inducedcirclingandneuronalactivityinthehemiparkinsonianrat.Behav.553BrainRes.320,119–127(2017).554

35. Sun,Y.etal.WirelesslyPoweredImplantablePacemakerwithOn-Chip555Antenna.IEEE1242–1244(2017).556

36. Summerson,S.R.,Aazhang,B.&Kemere,C.T.CharacterizingMotorand557CognitiveEffectsAssociatedWithDeepBrainStimulationintheGPiofHemi-558

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

ParkinsonianRats.IEEETrans.NeuralSyst.Rehabil.Eng.22,1218–1227559(2014).560

37. Agrawal,D.R.etal.Conformalphasedsurfacesforwirelesspoweringof561bioelectronicmicrodevices.Nat.Biomed.Eng.1,1–16(2017).562

38. Schabrun,S.M.,Jones,E.,ElguetaCancino,E.L.&Hodges,P.W.Targeting563chronicrecurrentlowbackpainfromthetop-downandthebottom-up:a564combinedtranscranialdirectcurrentstimulationandperipheralelectrical565stimulationintervention.BrainStimul7,451–459(2014).566

39. Mulpuru,S.,Madhavan,M.,McLeod,C.,Cha,Y.-M.&Friedman,P.Cardiac567Pacemakers :Function,Troubleshooting,andManagement.J.Am.Coll.568Cardiol.69,(2017).569

40. Kesar,T.M.etal.NovelPatternsofFunctionalElectricalStimulationHavean570ImmediateEffectonDorsiflexorMuscleFunctionDuringGaitforPeople571Poststroke.Phys.Ther.90,55–66(2010).572

41. Bewernick,B.H.etal.NucleusAccumbensDeepBrainStimulationDecreases573RatingsofDepressionandAnxietyinTreatment-ResistantDepression.Biol.574Psychiatry67,110–116(2010).575

42. Park,S.Iletal.Stretchablemultichannelantennasinsoftwireless576optoelectronicimplantsforoptogenetics.Proc.Natl.Acad.Sci.113,E8169–577E8177(2016).578

43. Yazdandoost,K.Y.A2.4GHzantennaformedicalimplantedcommunications.579APMC2009-AsiaPacificMicrow.Conf.20091775–1778(2009).580doi:10.1109/APMC.2009.5384240581

44. Yazdandoost,K.Y.&Kohno,R.Anantennaformedicalimplant582communicationssystem.Proc.37thEur.Microw.Conf.EUMC968–971(2007).583doi:10.1109/EUMC.2007.4405356584

585

586

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

Methods587588GeneralStatisticalMethods589ErrorbarsinFigureS2fgdenote+/-onestandarddeviationforn=~50datapoints.590WefurthermoreperformedaTukey’sHonestSignificantDifferencetestonthedata591inFigureS2g,whichindicatedthatthevoltageproducedateachdifferentPVDF592thicknessissignificantlydifferent.Pairedt-testswereusedfortherotationtestsin593figure4e.594595FilmFabrication596TofabricateMEfilms,weusedMetglasSA1alloy(MetglasInc)forthe597magnetostrictivelayerandpolyvinylidenefluoride“PVDF”(precisionacoustics)for598thepiezoelectriclayer.ThePVDFfilmsusedfortheseexperimentswerepre-599stretchedandpoledbythemanufacturer.Thetwolayerswerebondedtogether600usinganepoxycapableoftransferringthemechanicalstressbetweenthetwolayers601(Hardmandoublebubbleredepoxy).Priortobondingthetwolayerstogether,we602sputteredathinlayerofplatinum(

633Tomaintainsimplicity,efficiency,andlowcostthecoilsweredrivenwithfullH-634Bridgestyleswitchingcircuits.Thedriversaredesignedtodeliverhighcurrentsto635thedrivecoilsintheformofbi-phasicpulsetrains.Thisreducesthecostand636complexityofthedriveritself,aswellasthepowersupplyandcontrolcircuitry637whencomparedtoarbitraryfunctiongenerators.Thedesignalsohaspotentialfor638improvedoperationalefficiencythroughimpedancematchingwiththedrivecoils.639Furthermore,itisalsopossibletoregulatepowerdeliveredtothedrivecoilsonthe640flybyadjustingthedutycycleofthecurrentpulses,allowingpowerbeingdelivered641totheMEfilmtobeeasilycontrolleddigitallywhilemaintainingtheresonant642carrierfrequency.Theoutputcarrierandpulsefrequenciesofthemagneticfieldare643generatedusingaTeensyLCboardandcustomArduinocodetogeneratethespecific644pulsetimingstodelivercontrolledMEstimulation(Fig.S3c,d).645646Thesecoilsanddriverscanbecombinedindifferentwaystogeneratethe647appropriatefieldforagivenexperiment.Forexample,thesetupusedtogenerate648thealternatingfieldintheinvivorotationexperimentsconsistedoffoursetsofcoils649eachwithfiveturnspoweredbyonedriverwithallfourdriverssyncedtothesame650outputsignal.InthiswaywecangeneratesufficientpowertogenerateamT-scale651magneticfieldsoverthewholebehavioralarea(Fig.S5a).652653CellCulture654Forexperimentsperformedoncoverslips,HEKcellsexpressingsodiumchannel655Na1.3andpotassiumchannelK2.1weregrownon12mmpoly-l-lysinecoated656coverslipstoapproximately30%confluency.Thecellswerethentransfectedwith657thegeneticallyencodedvoltageindicatorArcLightusingLipofectamine(Invitrogen)658followingmanufacturer’srecommendations.Twotothreedaysaftertransfection659thecoverslipswereinvertedontoMEfilmsfortesting.PreparationofGFPcontrols660followedthesameprocedurewiththeexceptionofreplacingtheArcLightvector661(AddGene)withaGFPexpressionvector(AddGene).Forexperimentsperformed662withcellsgrownonthefilms,HEKcellstransfectedwithArcLightwereplacedonto663parylenecoatedpoly-l-lysinetreatedfilms.Thefilmswereplacedincellularmedia664overnightandtestedthefollowingday.665666ArcLightandGFPwereexcitedat460nmwithanLEDlightsource.Fluorescence667imageswerecollectedat33fpsusingaCCDcamera.Imageswereanalyzedusing668Matlabtoquantifyfluorescencechangesinindividualcells.Invitrotestingwas669performedinextracellularbuffer(ECB,inmM:NaCl119,KCl5,Hepes10,CaCl22,670MgCl21;pH7.2;320mOsm)671672FigureS4bwasobtainedbygrowingunmodifiedHEKcellsonafilmsubmergedin673cellularmediaforfivedays.ThecellswerethenstainedwithHoechstandCalcein-674AMtolabelthenucleusandmembranerespectivelyinlivingcells.Thecellswere675thenfixedandimagedusingaconfocalmicroscope.676677678

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

MouseBrainSliceProcedures679Weused40dayoldGAD2-GCaMP3mice,generatedbycrossingGAD2-Cre(JAX#68010802)withflox-GCaMP3(JAX#14538)animals.Preparationofbrainslices681followedproceduresdescribedbyTingetal.45andwascarriedoutinaccordance682withNationalInstitutesofHealthguidelinesandapprovedbytheUTHealthanimal683welfarecommittee.MiceweredeeplyanesthetizedwithIsofluraneandperfused684withicecoldNMDG-basedsolutionconsistingof(inmM):92NMDG,2.5KCl,1.25685NaH2PO4,10MgSO4,0.5CaCl2,30NaHCO3,20glucose,20HEPES,2thiouera,5Na-686Ascorbate,3Na-pyruvate,saturatedwith95%O2and5%CO2.,atarateof~6687ml/min.Coronalbrainslices(300µm)werecutusingavibratome(LeicaVT1200S),688incubatedfor15minat35°CinNMDGsolution,andthentransferredtoachamber689heldatroomtemperaturecontaining(inmM):92NaCl,2.5KCl,1.25NaH2PO4,2690MgSO4,2CaCl2,30NaHCO3,25glucose,20HEPES,2thiouera,5Na-Ascorbate,3Na-691pyruvate,saturatedwith95%O2and5%CO2.Forexperiments,sliceswereplaced692intoarecordingchamberperfusedwithACSFcontaining(inmM):126NaCl,2.5KCl,6931.25NaH2PO4,2MgCl2,2CaCl2,26NaHCO3,10glucose),heldat32-34°Cusingan694inlineheater.NBQX(10µM)wasincludedinthebathsolutiontoblockAMPA695receptor-mediatedsynaptictransmission.Thestereotrodewasplacedinlayer5of696somatosensory(barrel)cortex.697698GCaMP3wasexcitedat460nmwithanLEDlightsource.Fluorescenceimageswere699collectedat9.8fpsusingaCCDcameraattachedtoanOlympusBX51WImicroscope.700ImageswereanalyzedusingMatlabtoquantifyfluorescencechangesin600x600701μmregionsaroundthestereotrodetips.702703704ImplantDesignandRatSurgicalProcedures705706TwomaleLong-Evansrats(n≈1,400g)wereanesthetizedwithisofluranegas.Five707percentisofluranewasusedtoinduceanesthesiaandtwopercentwasusedto708maintainanestheticdepth.Buprenorphine(0.04mg/kg)wasadministered30709minutespriortoearbarsforanalgesia.5-7skullscrewswereplacedtoanchorthe710electrodearray.SkullscrewswereboundtoskullwithMetabonddentalacrylic.A711craniotomywasmadetoaccommodatethemicroelectrodearrayandexposean712injectionsiteforneurotoxin.A30gaugeneedlebentatthetipcutandpulledaway713theduramatercoveringofthebrain.Desipramine(DMI)reconstitutedinsalineata714concentrationof15mg/mLwasinjectedIPtoprotectnoradrenergnicneurons.The715doseofDMIwasapproximately15mg/kgandinjectedapproximately30minutes716priortoadministrationofneurotoxin.Toinduceahemiparkinsonianlesion,8ugof7176-hydroxydopamine(OHDA)at2ug/uLinsalinewasinjectedat0.2uL/minintothe718midforebrainbundle(MFB-1.2ML,-4AP,and-8.1DV).STNstimulationwas719deliveredviaa2x2platinumiridiummicroelectrodearray(Microprobes)with600x720600μmspacingof75μmelectrodes.Eachelectrodehadanominal10kOhm721impedance.Theelectrodearraywasloweredto-2.6ML,-3.6AP,andapproximately722-8.2DVfrombregma.Thearraywasfixedtotheskullwithdentalacrylic.All723

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

experimentswereapprovedbytheInstitutionalAnimalCareandUseCommitteeof724RiceUniversity.725726Priortostimulatingeachratwiththemagnetoelectricstimulator,thestimulator727powerwasestimatedviaabenchtopapproximationoftherodentelectrode728impedance.ConstantcurrentstimulationoftherodentbrainwithanA-MSystems7294100stimulatorproducedcharacteristicvoltagewaveformsthatapproximateda730simplifiedparallelRCcircuit.A56kOhmresistor,and440pFcapacitorinparallel731closelyapproximatedtheimpedancecharacteristicsoftheratbrainacrossthe732stimulatingelectrodes.Usingthiscircuitmodel,weestimatedthefieldstrengthsand733pulsedurationsnecessarytoproducethedesiredstimulationeffectsandconfirm734thatthestimulationwaschargebalancedpriortorodentexperimentation.735736RotationTestExperiments737Priortoperformingtherotationteststheratwasbrieflyanesthetizedwith5%738isofluranegasandinjectedintraperitoneally(IP)withmethamphetamine(0.31ml7391.25mg/kg)andthewirelessbiphasicstimulatorwaspluggedintotheimplanted740electrodearray.Aftertheanesthesiahadwornoff(about5-10min)theratwas741placedinthecylindricalbehavioralchamber.Themagneticfieldwasappliedover742thewholebehavioralareatothefilmsonthedevice(FigS5a).743744Themagneticfieldwasappliedonresonanceandoffresonanceforoneminuteat745varioustimesduringthe40-minutetrial.Theresonantfrequencieswere73kHzand74677kHzandtheoffresonantfrequencieswere63kHzand87kHz.747748RodentTracking749Headpositionontherotationtaskwasgeneratedusingaslightlymodifiedversion750ofDeepLabCut46totrackears,snout,andimplant.Adatasettotaling286frames751fromboththeonandoffresonancerotationtaskswashandlabeledandtrainedfor752approximately140,000iterations.753754SkullPhantomDemonstration755Atthemagneticfieldfrequenciesusedforthisexperimentboneandtissueare756effectivelytransparent47,soweselectedalifesizedskullwiththesizeofanaverage757humanadultheadasaphantom(OrientInfinityLimited).Itwaswrappedwith18758AWGmagnetwireasshowninFig5.Thecoilconsistedoffourcoilsinparalleleach759wiredtoanindividualmagneticfielddriver.Alldriverswerewiredtothesame760inputfrequencysignalandpoweredfromthesamepowersupply.Thefilmswere761suspendedatthecenteroftheskullphantom.OrangeLEDs(Chanzon)withadiode762antiparallelwereattachedtothefilmsforwirelessverificationofthevoltage763generatedbythefilms.Forvisualizationpurposestheskulltopwasremovedto764betterphotographtheLED.765766767768769

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

References77077145. Ting,J.T.,Daigle,T.L.,Chen,Q.&Feng,G.Acutebrainslicemethodsforadult772

andaginganimals:applicationoftargetedpatchclampanalysisand773optogenetics.MethodsMol.Biol.1183,221–242(2014).774

46. Mathis,A.etal.Markerlesstrackingofuser-definedfeatureswithdeep775learning.arXiv[cs.CV]1–14(2018).776

47. Bottomley,P.A.&Andrew,E.R.RFmagneticfieldpenetration,phaseshiftand777powerdissipationinbiologicaltissue:ImplicationsforNMRimaging.Phys.778Med.Biol.23,630–643(1978). 779

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

SupplementalFigure1|FilmoutputvoltageasafunctionofbiasfieldThepeakresonancevoltageissignificantlyincreasedbyamodestbiasfieldthatcanbeproducedbyapermanentmagnet.

SupplementalFigure2|MEpropertiesasafunctionoffilmsize(a)Schematicofexperimentalsetupusedtogatherdata.TestingwasperformedforMEfilmswiththreedifferentPVDFthicknesses:28(blue),52(red),and110(yellow)μm(b)Resonantfrequencyasafunctionoffilmlength(c)Outputvoltageasafunctionoffilmlength(d)Outputvoltageasafunctionoffilmsurfacearea(e)Q-factorasafunctionoffilmlength(f)Maximumpoweroutputasafunctionoffilmwidthfor52umPVDFthickness(g)Peakresonantvoltageplottedvs.PVDFthicknessshowsthatthepeakMEvoltageincreaseswiththePVDFthickness.Errorbarsindicate+/-1standarddeviationforn≈50filmsforeachthickness.(h)MEvoltageasafunctionofanglebetweenthefilmandthecoil.Blueregionshowstherangeofoperatinganglesforwhichthevoltageisgreaterthantheexpectedstimulationvoltage

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

SupplementalFigure3|MagneticFieldDriver(a)Schematicofthemajorcomponentsofthemagneticfielddriver.Dashedlinedenotescomponentsrenderedin(b).(c)Outputwaveformformonophasicstimulationandtheparametersthatcanbecontrolledbythedrivesoftware(d)Outputwaveformforbiphasicstimulation,andtheparametersthatcanbecontrolledbythedriversoftware.

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

SupplementalFigure4|MEstimulationofcellsgrowndirectlyonMEfilm(a)Schematicofexperimentalsetup(b)Microscopeimageoffixedcellsadherenttotheregionaroundastampedhole(Hoechst/Calcein-AM,cellslabeledpriortofixing)(c)ArcLightfluorescenceofspikingHEKcellswhenmagneticfieldisonresonanceand(d)offresonance.

SupplementalFigure5|MagneticFieldforDBSRotationExperiment(a)SchematicshowsthelocationandspacingofwiresandthenumberofdriversusedtogeneratethealternatingmagneticfieldoverlayingaCOMSOLsimulationofmagneticfieldstrengthinthechamber(b)ResultsfromrotationtestinRat2:Angularvelocityinthe30secondsbeforestimulationandthefirst30secondsofstimulationshowsaclearreductioninangularvelocityonlywhentheMEfilmisactivatedonresonance(***P=1.5x10-8,n.s.=notsignificantP=0.27,pairedt-test)

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855

SupplementalFigure6|MEdevicesoperateseveralcentimetersaboveasinglemagneticcoil(a)COMSOLsimulationofmagneticfieldaboveacircularcoiland(b)Measureddeviceoutputvoltageasafunctionofdistanceabovethecoil

not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted November 5, 2018. ; https://doi.org/10.1101/461855doi: bioRxiv preprint

https://doi.org/10.1101/461855