Synapse. 2018;e22059. wileyonlinelibrary.com/journal/syn  | 1 of 23https://doi.org/10.1002/syn.22059

© 2018 Wiley Periodicals, Inc

Received:8May2018  |  Revised:15June2018  |  Accepted:6July2018DOI: 10.1002/syn.22059

R E S E A R C H A R T I C L E

Cyanobacterial extract with serotonin receptor subtype 7 (5‐HT7R) affinity modulates depression and anxiety‐like behavior in mice

Neil C. Lax1,3 | Stacy‐Ann J. Parker2,3 | Edward J. Hilton1,3 | Youstina Seliman1,3 | Kevin J. Tidgewell2,3 | Benedict J. Kolber1,3

1Department of Biological Sciences, Duquesne University, Pittsburgh, Pennsylvania2Mylan School of Pharmacy, Duquesne University, Pittsburgh, Pennsylvania3Chronic Pain Research Consortium, Duquesne University, Pittsburgh, Pennsylvania

CorrespondenceKevinJ.Tidgewell,MylanSchoolofPharmacy, Duquesne University, Pittsburgh, PA;ChronicPainResearchConsortium,DuquesneUniversity,Pittsburgh,PA.Email: tidgewellk@duq.edu

BenedictJ.Kolber,DepartmentofBiologicalSciences, Duquesne University, Pittsburgh, PA;ChronicPainResearchConsortium,DuquesneUniversity,Pittsburgh,PA.Email: kolberb@duq.edu

Funding informationFogarty International Center (FIC) International Cooperative Biodiversity Group,Grant/AwardNumber:2U01TW006634-06; NationalCenterforComplementary and Integrative Health, Grant/AwardNumber:R15AT008060;AmericanPainSocietyandtheSharonS.Keller Fund for Chronic Pain Research; Duquesne University Faculty Development Fund;Smithsonian,Grant/AwardNumber:16S,1,2,3and4;TropicalResearchInstitute;AmericanPainSociety;FogartyInternationalCenter,Grant/AwardNumber:2U01TW006634-06;DuquesneUniversity;NationalInstituteofMentalHealth;UniversityofNorthCarolina

AbstractMarine cyanobacteria represent a unique source in the field of drug discovery due to the secondary metabolites they produce and the structural similarity these com-poundshavetoendogenousmammalianreceptorligands.Aseriesofcyanobacteriawere subjected to extraction, fractionation by column chromatography and screened foraffinityagainstCNStargetswithafocusonserotoninreceptors(5-HTRs).Outof276 fractions screened, 21%had activity at 5-HTRs and/or the 5-HT transporter(SERT). One sample, a cyanobacterium identified by 16S rRNA sequencing asLeptolyngbya from Las Perlas archipelago in Panama, contained a fraction with noted affinityforthe5-HT7receptor(5-HT7R).Thisfraction(DUQ0002I)wasscreenedviaintracerebroventricular (ICV) injections in mice using depression and anxiety assays includingtheforcedswim,tailsuspension,elevatedzeromaze,andlight-darkprefer-encetests.DUQ0002Idecreaseddepressionandanxiety-likebehaviorsinmalesanddidnothaveeffectsin5-HT7Rknockoutorfemalemice.AdministrationofDUQ0002ItotheCA1ofthehippocampusinducedantidepression-like,butnotanxiolytic-likebehaviors.Testingoffurtherpurifiedmaterialsshowednobehavioraleffects,leadingus to hypothesize that the behavioral effects are likely caused by a synergistic effect betweenmultiplecompoundsinthefraction.Finally,DUQ0002Iwasusedinamodelofneuropathicpainwithcomorbiddepression(sparednerveinjury—SNI).DUQ0002IhadasimilarantidepressanteffectinanimalswithSNI,suggestingaroleforthe5-HT7Rinthedevelopmentofcomorbidpainanddepression.Theseresultsdemon-strate the potential that cyanobacterial metabolites have in the field of neuropharmacognosy.

K E Y W O R D S

animal models, anxiety, behavior, cyanobacteria, depression, drug discovery, serotonin receptor 7

1  | INTRODUC TION

Filamentous cyanobacteria are small, photosynthetic prokaryotes found in diverse environments around the globe. They are knownto produce secondary metabolites that were first studied in freshwater environments due to the poisoning of domestic cattle drinking from freshwater with cyanobacterial blooms (Stewart, Seawright, & Shaw, 2008) and in marine environments for their involvement in

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“swimmer’s itch” (Osborne&Shaw,2008).Thesemetabolitesare thought tohaveoriginallyevolvedasa typeofchemicalprotectionagainstpredation(Nagle&Paul,1999).Inmarineenvironments,theseintracellularmetabolitesarereleasedwhencellsarelysed(WorldHealth Organization, 1999), as when an herbivore grazes on the organism, or are released into the water as signaling molecules between cells. Regardless of their purpose, the fact that these compounds can have direct effects on animals has led to the investigation of cyano-bacterial metabolites on cytotoxicity, including for cancer and infection (viral, microbial, malaria) and antifeedant effects, among others (Dixit & Suseela, 2013).

Whatremainsrelativelyunexploredbeyondcytotoxiceffectsofcyanobacterialcompoundsisifthesemetabolitescanmodifysignalingcascadesinmammals,especiallyinthenervoussystem.Alargenumberofcompoundsproducedbycyanobacteriaandothermicroorganismsaresmalllinearandcyclicpeptides(Ahmed,Lax,&Tidgewell,2015;Tan,2007;Yanoetal.,2015),whichstructurallyresembleendogenousligandstoG-proteincoupledreceptors(GPCRs)foundinmammaliancells.Todate,onlyahandfulcompoundsextractedfromcyanobacteriahavebeeninvestigatedforactivityatGPCRs.TheseincludeextractsfromthegenusMoorea (formerly Lyngbya (Engene et al., 2012)) acting on the cannabinoid receptors (Gutierrez et al., 2011; Han, McPhail, Ligresti, Di Marzo, & Gerwick, 2003; Montaser, Paul, & Luesch, 2012; Sitachitta & Gerwick, 1998) and an extract from the genus Oscillatoria acting on the serotonin system identified in our previous publication (Laxetal.,2016).ThegoalofthepresentresearchwastofurtherprobemetabolitesextractedfromcyanobacteriaagainstGPCRsfoundintheCNS.Wesoughttodeterminepotentialpsychoactiveeffectsofmetabolitesinaffectivedisorderssuchasdepressionandanxiety.Bydoingsowe hope to find compounds that could be used as chemical leads and as tools that lead to a better understanding of biological systems within theCNS,withaprimaryfocusonserotonin(5-HT).

5-HTisoneofthemainmonoamineneurotransmittersinthenervoussystemandplaysaroleinmanyphysiologicalprocessesincludingbehavior,mood,pain, learning,memory, sleep, andappetite (Gellyncket al.,2013;Monti& Jantos,2014;Pytliak,Vargova,Mechirova,&Felsoci,2011).5-HTbindstoserotoninreceptors,whicharebroadlyclassedintosevenfamilies(5-HT1-7R)(Pytliaketal.,2011).Theserecep-torsaremainlyfoundperipherallyintheGItract(Tuladhar,Ge,&Naylor,2003)andsmoothmuscleofbloodvessels(Bardetal.,1993)andintheCNS(Bonaventureetal.,2004).Manycommonaffectivedisorders,includingdepressionandanxiety,areknowntobeassociatedwith5-HTsignaling.Mostofthecommonanti-depressants(e.g.,selectiveserotoninreuptakeinhibitors(SSRIs))target5-HTre-uptakecausinganincreaseinsynapticlevelsof5-HTfollowingnormalrelease(Ferguson,2001).Thisincreaseinserotonincanthenbindtoserotoninreceptors(e.g.,5-HT1A,5-HT2A,5-HT2C,5-HT4,5-HT6,5-HT7)(Pytliaketal.,2011)thatareknowntohaveeffectsindepression.Ofthese,5-HT7R is arelativelyunder-studiedreceptorwithstrongpotentialtoenhancetheantidepressanteffectsofSSRIswhenpharmacologicallyinhibited(Guseva,Wirth,&Ponimaskin,2014;Tokarski,Kusek,Sowa,&Bobula,2014)andmanyanimalstudieshaveshownthattargetingthe5-HT7R receptor can modulate affective behaviors.

In rodents, administrationof selective5-HT7Rantagonistsgenerallydecreasesdepression-likebehaviors (Hedlund,Huitron-Resendiz,Henriksen,&Sutcliffe,2005;Kimetal.,2014;Sarkisyan,Roberts,&Hedlund,2010;Wesolowska,Nikiforuk,&Stachowicz,2006).Intermsofanxiety-likebehavior,theroleof5-HT7Risnotasclear.Inmice,somestudieshaveshownthatblockadeofthe5-HT7Rreducesanxiety-likebehaviorintheopenfieldtest(Guillouxetal.,2013;Hedlund&Sutcliffe,2007;Wesolowskaetal.,2006),however,anotherstudyfoundthat5-HT7Ragonistsreduceanxiety-likebehavior(Adrianietal.,2012).Inhumanstudies,themultimodalantidepressantvortioxetine(Brintellix),whichactsasa5-HT7R antagonist, while also increasing serotonin concentrations through reuptake inhibition, has been shown to reduce majordepressivedisorder(MDD)inbothshortandlong-termclinicaltrials(Pearce&Murphy,2014).Additionally,theclinicallyestablishedeffectsofsomeantipsychoticdrugs,includingamisulpride,aripiprazoleandlurasidonemostlikelyfunctionthroughthe5-HT7R(Abbasetal.,2009;Bonaventureetal.,2007;Cates,Roberts,Huitron-Resendiz,&Hedlund,2013).Overall,thesedatademonstratethattargetingthe5-HT7R can alter both affective behavior in animals and humans.

GiventhepossiblepresenceofCNSactivecompoundsincyanobacterialmetabolitesandthewell-establishedrolethatthe5-HT7R plays indepressionandanxiety,wesoughttoinvestigatethepotentialofcyanobacteriatoproduce5-HTRligandsanddiscoverspecificligandsforthe5-HT7Rthatcouldinduceantidepressantandanxiolytic-likeeffects.Inthisstudy,wesoughttoinvestigatethepotentialofmarinecya-nobacteriatoproduce5-HTRligandsbyscreeningextractstakenfrommarinecyanobacterianearPanamaandCuraçao.Crudeextractsfromcyanobacteria were fractionated using silica gel chromatography and screened in vitro for GPCR binding affinity by the Psychoactive Drug Screening Program (PDSP) (Besnard et al., 2012).

Oneparticularfractionwithsignificantbinding(>50%inprimaryassay)andselectiveaffinityforthe5-HT7R,DUQ0002I,wasinjectedinto the lateral ventricle of mice via intracerebroventricular (ICV) injections for initial screening in a series of depression and anxiety behav-ioralassays,includingtheforcedswim,tailsuspension,elevatedzeromaze,andlightdarkpreferencetests.Wehypothesizedthatthisfrac-tionwouldalteranxiety-likeanddepression-likebehaviorsintheseanimals.AftertheinitialcharacterizationviaICV,wenextexploredthespecificityoftheinvivoresultsusing5-HT7Rknockout(5-HT7R-/-)miceandinvestigatedpossiblesexdifferencesintheobservedbehavior.WeexploredtheanatomicalspecificityofthebehavioraleffectsbyutilizingbrainregionspecifictargetingofDUQ0002ItotheCA1regionofthehippocampus.Additionally,purifiedsubfractionsoftheparentfractionDUQ0002Iweretested.Finally,weutilizedDUQ0002Iasatherapeuticleadtodetermineifthisfractioncouldalleviatethedepression-likeeffectsinducedbytheneuropathicpainsparednerveinjury(SNI)model.

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2  | E XPERIMENTAL PROCEDURES

2.1 | Field sampling of cyanobacteria

Cyanobacterial sampleswerecollectedbetween2011and2016throughsnorkelingandSCUBAnearPanama (CaribbeanSeaandPacificOcean)andCuraçao.ThespecificsampleoffocusherewascollectedbysnorkelingonJanuary28,2013whileatadepthof1moffthecoastofMogoMogo(GPScoordinates:8°34’50.2”N,79°01’10.6”W)intheLasPerlasArchipelago,Panama.Adarkgreen/blackcyanobacterialbiomasswascollected.Thesamplewasfullysuspendedinethanol:seawater(50:50)mixturetopreservethesampleuntilitcouldbestoredat-20°Cpriortoextraction.Avoucherspecimen(PLP-28Jan13-2)isdepositedintheDepartmentofMedicinalChemistry,GraduateSchoolofPharmacy,DuquesneUniversityandwasgiventhelaboratoryidentifierDUQ0002.

2.2 | Extraction and in vitro characterization of metabolites

TheDUQ0002cyanobacterialbiomass(75g,drywt)wasextractedwith2:1CH2Cl2-MeOHtoafford3.3gofcrudeextract.Thiscrudeextractwasfractionatedovernormalphasesilicagelwithastepwisegradientsolventsystemofincreasingpolaritystartingfrom100%hexanesto100%MeOH,toyieldninefractions(A–I).Thefractionelutingwith100%methanol(fractionI(DUQ0002I),292mg)waschromatographedonhypersepC18reversedphase(2000mg/15mL)columnusingmethanol:water,gradient50–100%toyieldfoursub-fractions,DUQ0002I-1-4correspondingto224.9,5.7,7.9,and13.9mg,respectively.Thefractionmostsimilartothecrudefraction,sub-fractionDUQ0002I-1(167.5mg),wasfurtherpurifiedoversilicagel(90.6g)usingmethanol:dicloromethane,gradients5,8,and10%toyieldsub-fractions,DUQ0002I-1A(3.2mg),DUQ0002I-1B(4.0mg)andDUQ0002I-1C(7.9mg).

NMRspectrawererecordedwithmethanolasinternalstandard(δC49.9,δH3.34),onaBruker500MHzspectrometeroperatingat499.7MHz for 1H and 125.7 MHz for 13Cequippedwitha5mmPATXI1HD/D-13C/15NZ-GRDProbe.ColumnchromatographywasperformedusingSorbentTechnologiessilicagel(230-400mesh).SolventswereevaporatedonaHeidolphrotaryevaporator.

2.2.1 | Psychoactive drug screening program binding assays

AllbindingdataforfractionsDUQ0002A-IwereperformedandgenerouslyprovidedbytheNationalInstituteofMentalHealth’sPsychoactiveDrugScreeningProgram(NIMHPDSP)utilizingaradioligandcompetition-bindingassay.Experimentaldetailsareavailableonlineathttps://pdsp.med.unc.edu/.Inbrief,fractionsthatpassprimarybindingcriterionofgreaterthan50%inhibitionat10μM are tagged for secondary radioligand binding assay to determine IC50atthespecifictargetofinterest.Todetermineoverallhitratesat5-HTreceptorsandtheserotonintransporter(SERT)forallfractionsscreenedbetween2011and2016,wecalculatedthenumberoffractionswith>50%bindingaffinityat5-HTtargets(n = 58) compared to the total number of our cyanobacterial fractions that have been screened by the PDSP (n=276).

2.2.2 | Microscopy

SamplesofDUQ0002usedformicroscopywerepreparedusingapieceofthesamplestoredinRNAlatersolution.Filamentswerespreadoutonto a slide with distilled H2Oandimagedat20XonaNikon1AR-HDmicroscopeusingtheFITC(487nm),TRITC(560nm)andCy5(637nm)channels.

2.2.3 | 16S rRNA gene sequencing and phylogenetic analysis

GenomicDNAwasextractedfromDUQ0002thatwaspreservedinRNAlater®solution(ThermoFisher)aftercollection.Lysisbuffer (10mMTris,0.1MEDTA,0.5%(w/v)SDS,20μg/mLRNase,pH8.0)wasaddedtothecyanobacteriamassat10Xthebiomassofthecells(i.e.,100 mg cells = 1000 μLlysisbuffer).Thiswasfollowedby100μL of lysozyme solution (10 mg/mL, Sigma) for 30 minutes with an incubation at37°C.0.01XthevolumeofProteinaseK(10mg/mL,GeneLink)wasthenaddedandincubatedfor1hrat50°C.Followingthis,themix-turewascentrifugedat13,000rpmfor3minandtheremainingpelletwasusedwiththeWizardGenomicDNAPurificationKit(Promega).ExtractedgenomicDNA thenunderwentPCR toamplify the16S rRNAgenewithprimers (CYA106FandCYA1509R)previouslyutilized(Engene,CameronCoates,&Gerwick,2010;Nubel,Garcia-Pichel,&Muyzer,1997).SuccessfulPCRamplifications(~1370bp)underwentPCRpurificationusingtheMinElutePCRPurificationKit(Qiagen)andpurifiedPCRproductswereusedforTOPOcloning.OneShotE. coli cells alongwiththepCR®4-TOPOvectorwithkanamycinresistancefromtheTOPOTACloning®Kit(Invitrogen)wereusedandcellswereplatedovernightat37°ConLBKANplates.SuccessfultransformantswereselectedandusedincolonyPCRwiththeM13FandM13Rprimerstoconfirmthepresenceofthe16SrRNAsequencenowintheTOPOvector(~1535bp).PurifiedPCRproductforcorrectsampleswassenttoBeckmanCoulter(nowGENEWIZ)toundergoforwardandreverseSangersequencingwiththeM13FandM13Rprimers.Oursequencefor

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DUQ0002,alongwith16SrRNAgenesequencesthatwereobtainedwithsequencedataavailableattheNationalCenterforBiotechnology(NCBI)webpage(https://www.ncbi.nlm.nih.gov)werethenusedforphylogeneticanalysis.GenesequenceswerealignedusingtheMusclealgorithmandphylogenyreconstructionwasdoneusingmaximumlikelihoodmethodwith500bootstrapreplicatesusingtheMEGA(version5.1)program(Hall,2013).DUQ000216SrRNAGenBankdepositnumber(MH357345).

2.3 | Animals

Allanimalprocedureswerereviewedandcarriedout inaccordancewiththeNational InstitutesofHealthGuidefortheCareandUseofAnimalsandtheInstitutionalAnimalCareandUseCommitteeatDuquesneUniversity.ExperimentswereperformedoneithermaleorfemaleC57Bl/6Jmicethatwere8–10weeksold.ForexperimentsusingtheSNI/shammodel,animalswere8–10weeksoldwhenSNI/shamsurger-ieswerecompleted,andallbehavioraltestingbegan10weeksaftersurgery.For5-HT7Rknockoutexperiments,8-10weekold5-HT7R-/-(homozygote)miceand5-HT7R+/+(wildtype)siblingcontrolswereused(JacksonLaboratories(Cat:019453)Hedlundetal.,2003).5-HT7R knockoutmicewerepreviouslybackcrossedontoaC57Bl/6Jlineandwerere-derivedinC57femalemice.Allexperimentalmiceweregrouphoused up until cannulation surgeries were performed (this is necessary since group housing can cause the animals to lose their skull caps thatarepresentaftercannulation).Miceweremaintainedona12hlight/darkcycle(lightsonbetween7:00AMand7:00PM)withad libitum accesstofoodandwater.Allproceduresweredoneduringthelightcycle.Allbehaviorsperformedwererecordedandanalyzedbyamaleexperimenter(duetotheknowneffectsofhumanpheromonesonrodentbehavior(Sorgeetal.,2014)),whowasblindedtoexperimentaltreatments.

2.4 | Cannulation procedures for in vivo fraction and drug delivery

Cyanobacterialfractions,drugs,andvehiclesweredelivereddirectlyintotheCNSviastainlesssteelcannulas.Forcannulationsurgeries,ani-malswereanesthetizedwith3%isoflurane/0.6%oxygen.Intracerebroventricular(ICV)surgeriesweredoneasdescribedpreviously(Glascocketal.,2011)andCA1bilateralcannulationswereperformedassubsequentlydescribed.Briefly,micewereplacedinastereotaxicframeandsteelcannulas(one8.00mmforICVandtwo5.60mmforCA1)wereplacedintotherightlateralventricle(ICV)orbilaterallyintotheCA1regionoftheleftandrighthippocampus.Thefollowinganatomicalcoordinateswereused:ICV—0.5mmanteriortobregma,1.0mmlateraltomidlineand2.0mmventraltotheskull;CA1—1.6mmanteriortobregma,1.0mmlateraltomidlineand1.6mmventraltotheskull.Adentalcement skullcap secured with two bone screws was used to hold cannulas in place. Mice were allowed to recover on heating pads, housed in individual cages (this is required since group housed animals often remove their cagemate’s cement skullcaps through grooming or other social behavior) and were given one week of recovery prior to the beginning of behavioral testing. Following all behavioral tests, cannula placement was verified with necropsy.

2.4.1 | Fraction and compound administration for behavioral analysis

Cyanobacterialextractsandcommercialcompoundsforbehavioralcharacterizationwerepreparedasdescribedpreviously(Laxetal.,2016).Briefly,samplesfromcollectionDUQ0002,fractionI(DUQ0002I),theknown5-HT7RantagonistSB258719(SB258719hydrochloride,Tocris)andsubfractionsisolatedfromtheparentfraction(DUQ0002I-1A-C,2-4)weredissolvedinamixtureof50%DMSOand50%artificialcer-ebrospinalfluid(aCSF)containingthefollowing(inmM):125NaCl,2.5KCl,1.25NaH2PO4,25NaHCO3, 2.0 CaCl2, 1.0 MgCl2,and25D-glu-cose,bubbledwith95%O2/5%CO2for20min.priortouse.Wehavepreviouslyshownnobehavioraleffectofthisvehiclepreparationinmicecomparedto100%aCSFvehicle(Laxetal.,2016).Microinjectionswereperformedusinga32-gaugeinjectorthatextended0.5mmbeyondthetipoftheICVorCA1cannulas.Theinjectorwasattachedtoflexibletubinganda1.0μL syringe (Hamilton) that was used to deliver a total volume of 0.5 μLovera2-minperiod(twosyringes,injectors,andsetsoftubingwereusedforbilateralCA1injections;0.5μLperCA1).Theinjectors were kept in place for one minute after the injections were complete to allow for complete compound infusion and all behaviors were conducted5minutesaftercompoundadministration.Forallinjections,fractionDUQ0002IandsubfractionsDUQ0002I-1A-C,DUQ0002-2-4wereadministeredatadoseof40μg per cannula and SB258719 was administered at a dose of 5 μgbasedonpreviouslyliterature(Zhaoetal.,2014).ThefractiondosewasbasedonPDSPscreeningat4mg/mLandadjustedforICVinjectionsandintra-amygdalainjectionsthatwehavedonepreviously(Kolberetal.,2010;Laxetal.,2016).50%/50%aCSF/DMSOwasusedasavehiclecontrolinallexperiments.

2.5 | Behavioral testing

Wedeterminedthat5–8animalswouldbeneededpergroupbasedonpreviouslypublishedbehavioraldata(Laxetal.,2016;Lax,George,Ignatz,&Kolber,2014)and,inpart,onana‐priori poweranalysisusingasignificancelevel(alpha)of0.05(two-tailed)at95%power.Somebe-havioral tests (mainly from those during initial characterization with ICV injections) have higher values since those experiments were done in

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duplicatetoverifytheveracityoftheeffects.Forallbehavioraltests,micewerehabituatedfor1-2hrinthebehaviorroomwith60dBofwhitenoisetoblockambientbackgroundnoise,temperatureof22°C–25°C,humidityof40%–60%.Experimenterswaitedanadditional30minutesafterenteringtheroomtobeginbehavioraltestingduetotheknowneffectsofhumanpheromonesonrodentbehavior(Sorgeetal.,2014).Allanimalsreceivedfraction/subfractions/drug(DUQ0002I,DUQ0002I-1A-C,DUQ0002I-2-4,orSB258719)orvehicle5minutespriortothebeginningoftesting,wheretheywerereturnedtotheirhomecagesduringthe5minutes.Allcompounddeliverywasdoneblindedtofractionversusvehicledeliveredandsurgerytype(e.g.,SNIvssham,whereapplicable).Treatmentgroupswererandomlyassignedtoanimalsbyathird-partyexperimenterduringblinding.Animalswereeachtestedinthesamebehavioralapparatusoneatatime(notinparallel).Foranimalsthatwere tested in more than one assay, testing was done with one week between assays and testing proceeded from the least stressful to most stressfultest.Thisorderbeganwithelevatedzeromaze,followedbylight-darkpreference,tailsuspensiontestandendedwithforcedswimtest.AmaximumoffourtestswerecompletedongroupsofnaivemiceforICVtesting.Animalswereindividuallyhousedforuptofourweeks,includingtheoneweekofrecoverypost-surgeryplusoneweekbetweeneachofthebehavioraltests.Insubsequenttesting,onlycertaintestswereusedindifferentcontextsbuttestingorderalwaysproceededfromleaststressfultomorestressful.Noanimalsunderwenttestinginthesameassaytwice.Allbehavioraltestingandanalysisofbehaviorwasdoneblindedtotreatmentandsurgerytype(whereapplicable).

2.5.1 | Elevated zero maze (EOM)

Theelevatedzeromazewasperformedlargelyasdescribedpreviously(Kulkarni,Singh,&Bishnoi,2007).Inthisassay,micewereplacedonanelevatedcircularplatform(50cmoffground,4.5cmwideplatform,63.5cmdiameter)withtwo“arms”thatareopen,andtwo“arms”thatcontainahighwall(wallsare16cmhigh)enclosingtheplatform.Animalswererecordedwithanover-headvideocamera(LogitechPro9000)andlocomotionwastrackedwithANY-mazesoftware(StoeltingCo.,version4.98,WoodDale,IL).Timespentanddistancetraveledintheopen and closed arm were recorded for the 10 minute long test.

2.5.2 | Light‐dark preference (LDP) test

Thelight-darkpreferencewasperformedasdescribedpreviously(Bourin&Hascoet,2003).Inthisassay,abox(46cmLx27cmWx31cmH)witha“dark”and“light”sidethattakeup1/3and2/3oftheboxspace,respectively,isused.Adoor(5cmx6cm)separatesthetworegions.First,miceareplacedinthedarksidewiththelidonanddoortothelightsideclosedforoneminute.Aftertheoneminute,thedoorisopenedandanimalswererecordedwithanover-headcameraandtrackedwithANY-mazesoftware(StoeltingCo.,version4.98,WoodDale,IL).Timespent and mean visit time to the light side was recorded for the 10 minute long test.

2.5.3 | Tail suspension test

Thetailsuspensiontestwasperformedasdescribedpreviously(Can,Dao,Terrillion,etal.,2012).Briefly,miceweresuspendedbythedistalendsoftheirtailinaPlexiglasenclosure(35×25×25cm)withwhitesides.Theanimals’movementswererecordedwithwebcamforscoringofflineinoneminutelongbinsforatotalof6minutes.Immobilitywasdefinedastimewhenthemousedidnotexhibitanyoutwardmovementof its limbs or body.

2.5.4 | Forced swim test

Theforcedswimtestwasperformedasdescribedpreviously(Can,Dao,Arad,etal.,2012).Briefly,micewereplacedinabeakerofwater(~750mLina1.0Lbeaker,temperature26°C–30°C),withallbuttheirheadssubmerged.Theanimals’movementswererecordedwithaLogitechPro9000webcamforscoringofflineinoneminutelongbinsforatotalof6minutes.Immobilitywasdefinedastimewhenthemousedidnotexhibit any outward movements or when it only exhibited only movements necessary to maintain buoyancy (i.e., one paw gently moving in water).

2.5.5 | Mechanical sensitivity test

Mechanical sensitivitywasmeasuredwithvonFrey filaments (TouchTest) ranging from0.02g–2.56g todetermine the50%withdrawalthresholdsusingtheup/downmethod(Chaplan,Bach,Pogrel,Chung,&Yaksh,1994;Dixon,1980).AnimalswereplacedinPlexiglasboxesonawiremeshsurfacefortwohourspriortotesting.Pawwithdrawalwasdefinedasafullliftingofthefootoffofthewire-meshsurfacewhenafilamentwasappliedtothelateraledgeofthepaw(areainnervatedbytheintactsuralbranchofsciaticnerve).Afterbaselinetesting,miceunderwentSNI/shamsurgery(seebelow).Oneweekafterthesurgery,miceweresubjectedtovonFreytestingagaintoobservewithdrawalthresholds.Additionaltestingwasdoneattwo,three,five,sixandtenweeksaftersurgery.

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2.6 | Spared nerve injury (SNI) surgery

ForSNI,maleanimalswereanesthetizedwith10:1ketamine/xylazinemixtureinjectedintraperitoneallyintothemice(10μL/g) and surgery wasperformedasdescribedpreviously(Decosterd&Woolf,2000;Richner,Bjerrum,Nykjaer,&Vaegter,2011).Briefly,asuturethreadwastiedaroundtibialandcommonperonealbranchesofthesciaticnerve,bothofwhichwereligated2mmdistaltothesuture.Thesuralbranchofthe sciatic nerve was not manipulated. Sham surgeries followed the same procedure, without manipulation of the sciatic nerve. Mice recovered onheatingpadsandweregrouphouseduntilcannulationsurgerieswereperformed9weeksafterSNI/sham.

2.7 | Statistical analysis

GraphPadPrismversion6forMacOSXwasusedforallstatisticalanalysis.Alldataarepresentedasmean±SEM.Statisticalsignificancewasdeterminedbetweengroupsusingun-pairedt-testsor2-ANOVAsfollowedbyBonferronipost hoc tests. Statistical significance was estab-lishedata95%confidenceinterval(p < 0.05).

2.7.1 | Z‐score analysis

Z-scoreswerecalculatedforeachanimalineachbehaviortestedbasedonthepublishedmethod(Guilloux,Seney,Edgar,&Sibille,2011)whereapplicable.Thismethodofanalysisonlyworksifeachanimalreceivesthesametreatmentacrosseachbehavioralexperiment(thiswasnotdone during the initial screening with ICV injections and therefore this analysis was not done for those data). From each parameter measured (X=timeimmobileforFSTandTST,openarmtimeandpercentdistancetraveledinopenforEOMandtimeinlightsideandmeanvisittolightsideforLDP),anindividualZ-score(Z)wascalculatedtodeterminehowmanystandarddeviations(σ) each animal was above or below the mean of the control group (μ)usingthefollowingformula:Z=(X-μ) / σ.Z-scoresforindividualparameterswerethenaveragedtogenerate“depres-sion”and“anxiety”emotionalityscores.Withthistypeofanalysis,thecontrolgroupalwayshasanoverallZ-scoreofzero.Fortheexperimentalgroups,positiveZ-scoresreflectpositiveemotionalitychanges(i.e.,antidepressionorantianxiety),whereasnegativeZ-scoresreflectnegativeemotionality changes (i.e., depression, anxiety).

3  | RESULTS

3.1 | Screening marine cyanobacterial fractions against serotonin receptors (5‐HTRs)

Toevaluatethepotentialofmarinecyanobacteriaasasourcefornovelligandsto5-HTRsandtransporters,aseriesofcyanobacterialsamplesfromPanamaandCuraçaomadefrom2011to2016werescreenedfortheirabilitytobindto5-HTRsandthe5-HTtransporter(SERT)inaradioligandbindingassays.Theresultsofthisbroadscreening,consistingof276cyanobacterialfractions,wereobtainedfromtheNationalInstituteofMentalHealth’sPsychoactiveDrugScreeningProgram(NIMHPDSP).Thisinitialscreenindicatesthat21%ofthecyanobacterialfractionsscreenedcontaincompoundsthatcouldmodulatesignalingof5-HTRsorSERT(Figure1).Thesedatashowtheapparentevolution-arypressurethatexiststoproduce5-HTRmetabolites.Thispaperfocusesonaspecificcollectionwithafractionshowingspecificityforthe5-HT7R.

3.2 | 5‐HT7R cyanobacterial collection, phylogeny and screening

Adarkgreen/blackcyanobacterialmatwascollectedin~0.6mofwateroffMogo-MogointheLasPerlasArchipelago,Panama(8°34’50.2”N,79°01’10.6”W)andwasgiventheextractionIDofDUQ0002(Figure2a).Confocal(Figure2b)andtransmittedlight(Figure2c)microscopywas performed in order to observe the microscopic structure of the organism. Based on the morphology of the specimen, this sample was field identified as belonging to the genera of Oscillatoria or Moorea. In order to more accurately determine the species of cyanobacteria, sequencing ofthe16SrRNAgenewasperformed.WecomparedtheDUQ0002sequencewithaseriesofpreviouslypublished16SrRNAsequencesthathighlightedtherelatednessofmarinecyanobacterialstrainsthatproducenaturalproducts(Engene,Gunasekera,Gerwick,&Paul,2013).WefoundthatDUQ0002groupswithintheLeptolyngbya clade, showing that our collection most likely belongs to this genus (Figure 2d, GenBank NumberMH357345).

ToevaluatethepotentialofextractsfromDUQ0002forleadcompoundstotreatpainanddepression,fractions(namedDUQ0002A-I)werescreenedusingradioligandcompetition-bindingassays.TheresultsfortheninefractionsscreenedaresummarizedinTables1and2.“Hits”fortheprimaryassay,highlightedinyellowinTable1,wereobservedforallfractionsscreenedatoneormorereceptorsand/ortransporters.

Fractionsshowinggreaterthan50%inhibitionofbindingofradioligandintheprimarybindingscreenweresubjectedtosecondarybindingassay for IC50/Kidetermination(Table2).TheapparentKicurveforDUQ0002Iandacomparisontoclozapine,aserotoninreceptorantagonist,

     |  7 of 23LAX et al.

isshowninFigure2e.TheseapparentKi values allow us to determine if the inhibition of binding is caused by a potent compound with a low apparent Ki or a large amount of a weakly binding compound with a high apparent Ki.FractionDUQ0002Idisplayedtwo-foldselectivityfor5-HT7R (Ki/IC50 =749ng/mL)over5-HT1AR (Ki/IC50 =1746ng/mL),five-foldselectivityovermGluR5(IC50 =3676ng/mL),andthirteen-foldselectivityoverthenorepinephrinetransporter(NET;Ki/IC50 = >10,000 ng/mL). Generally, the lack of selective agonists and antagonists have beenattributedtothehighsequencehomologyamongst5-HTRsubtypes (Hoyeretal.,1994).Both5-HT1ARand5-HT7R target adenylyl cyclase(AC)withopposingeffects;5-HT7RactivatesACincreasingtheconcentrationofcyclicadenosinemonophosphate(cAMP)whereas5-HT7RinhibitsAC,decreasingcAMPconcentrations(Hannon&Hoyer,2008).

3.3 | Initial characterization of DUQ0002I in male mice via intracerebroventricular (ICV) injections

AfterdeterminingDUQ0002Iaffinityforthe5-HT7R,5-HT1AR,andmGluR5receptors,webegantocharacterizeDUQ0002I’spotentialef-fects using in vivoassaysinmalemice.SincethespecificnatureandsitesofactioninthenervoussystemoftheactivecompoundinDUQ0002Iwereunknown,webeganourcharacterizationofthisfractionusingICVinjections.ThismethodofadministrationallowsforthedeliveryofcompoundstothelateralventricleofthebrainanddispersesthecompoundthroughoutthecerebrospinalfluidoftheCNS(Glascocketal.,2011).Thisadministrationroutewasalsoutilizedduetothefactthatitwasunknownwhethertheactivecompound(s)hadtheabilitytocrosstheblood-brainbarrierandeliminatedanypossibleinterferenceoffirstpassmetabolismofanyactivecomponents.

Inthisinitialscreening,wetestedwhetherthefractionDUQ0002Icouldalterdepression-likeandanxiety-likebehaviorsinagroupofmalemice. Each animal was randomly assigned to a testing group, and all animals underwent the forced swim test, tail suspension test, elevated zero maze,andlight-darkpreferencetest.Forallbehavioraltests,adoseof40μgofDUQ0002Iwasused.Thisdosewasbaseduponthedosethatwas used for the initial in vitroPDSPscreening,whichwasdoneat4mg/mL,andthenadjustedforICVinjectionsandintra-amygdalainjectionsinmicethatwehavedonepreviously(Kolberetal.,2010;Laxetal.,2016).Startingwiththedepressionassays,theforcedswimtestwasused.Thisstandardtestofdepressionmeasurestheamountoftimeananimalspendsimmobileafteritisplacedinabeakerofwater,fromwhichitcannotescape.Longertimespentimmobileinthewateriscorrelatedwithgreaterdepression-likebehaviorwhilelesstimespentimmobileiscor-relatedwithgreaterantidepression-likebehavior(Can,Dao,Arad,etal.,2012).Followingafive-minutepretreatmentwith40μgofDUQ0002Iorvehicle,animalstreatedICVwithDUQ0002I(n = 18) spent a statistically significantly lower time immobile compared to control animals

F I G U R E 1   Primary binding results for initial screening of cyanobacteria collected2011-2016.Ahitisdefinedasgreaterthan50%inhibitionofbinding.Serotonin and serotonin transporter hits compromise21%ofhitsfromatotalof276fractionsscreened.SERT-serotonintransporter

5-HT1AR 4%

5-HT4R 0%5-HT3R

3%

5-HT2CR 3%

5-HT2BR 5%

5-HT2AR 2%

5-HT1DR 0%

5-HT1ER 0%

5-HT1BR 1%

Other 79%

SERT 2%

5-HT7R 1%

5-HT5AR 0%

5-HT6R 0%

Hit Rates at 5-HTRs and SERT

8 of 23  |     LAX et al.

(n=18)(Figure3a,unpairedt-test,**p=0.0019).Thetailsuspensiontestwasnextusedtofurthermeasurechangesindepression-likebehav-ior. Similar to the forced swim test, this assay measures the amount of time an animal spends immobile after being suspended upside down bythedistalendofitstail.Aswiththeforcedswimtest,longertimespentimmobileisindicativeofdepression-likebehaviorwhilelesstimespentimmobileisassociatedwithantidepression-likebehavior(Can,Dao,Terrillion,etal.,2012).MicepretreatedwithDUQ0002I(n = 9) did notshowanychangesintotaltimespentimmobilecomparedtovehicle-treatedanimals(n=9)(Figure3b,unpairedt-test,p = 0.5520).

Inadditiontostudyingdepression-likebehaviors,wealsolookedforchangesinlocomotorandanxiety-likebehaviorusingseveraldifferentassays. First, we used the elevated zero maze (EOM), a standard anxiety test which measures the amount of time and distance traveled in a cir-cularmazewithhigh-walledandlow-walledregions,inadimlylitroom.Increasedtimeandpercentoveralldistancetraveledintheopenarms(low-walledregions)iscorrelatedwithanti-anxiety-like(anxiolytic)effects,whiledecreasesarecorrelatedwithanxiogenicbehavior(Kulkarnietal.,2007).Sincethisassayalsomeasurestotaldistancetraveled,itcanalsobeusedtomeasurelocomotoreffects.AnimalspretreatedwithDUQ0002I(n = 18) showed no difference in the overall distance traveled in the EOM compared to control animals (n = 17) (Figure 3c, un-pairedt-test,p=0.1807),suggestingthatthefractiondoesnotcausehyperactivity.However,thesesameanimalspretreatedwithDUQ0002IshowedanoverallstatisticallysignificantlygreateramountoftimespentintheopenarmsoftheEOM(Figure3d,unpairedt-test,*p = 0.0188) and significantly greater overall percent distance (distance traveled in open/total distance traveled × 100) traveled in the open arms (Figure 3e, unpairedt-test,*p=0.0166).

Second,weusedthelight-darkpreference(LDP)test,anothertestofanxiety-likebehaviorwhereanimalsareplacedinatwo-chamberboxwith a brightly illuminated side and a completely dark side, separated by a small door. Increases in total time spent in the light side of the box arecorrelatedwithdecreasedanxiety-likeeffects,whiledecreasesarecorrelatedwithincreasesinanxiety-likeeffects(Bourin&Hascoet,2003).PretreatmentwithDUQ0002I(n = 19) caused animals to trend towards spending more total overall time spent in the light side com-pared to control animals (n=15)inthelatterhalfofthetest(Figure3f,unpairedt-test,p=0.1203).DUQ0002Ialsocausedanimalstohavea

F I G U R E 2   Pictures of cyanobacteria DUQ0002andphylogeny.(a)MacroscopicpictureofsampleDUQ0002beforecollection. (b) Confocal microscopy imageofDUQ0002at20XmagnificationwithFITC(487nm),TRITC(560nm)andCy5(637nm)channels.Scalebar= 100 μm.(c)TransmittedlightimageofDUQ0002.Scalebar=100μm. (d) Phylogenetic tree showing the closest relatedspeciestoDUQ0002basedoffof16SrRNAsequencing.(e)ApparentKi curveforfractionDUQ0002Ishowingthestandard5-HT7R ligand clozapine for comparison

(a) (b) (c)

(d)

(e)

(a) (b) (c)

(d)

     |  9 of 23LAX et al.

TAB

LE 1

 PDSPprimarybindingassayofDUQ0002fractions.PDSP“hits”arehighlightedinyellowandrepresentfractionsshowing>50%inhibitionofbinding.Datarepresentedasmean

%inhibition(N=4separatedeterminations).Negativeinhibitionrepresentsstimulationofbinding,meaningthatthefractionincreasedbindingofthestandardcompoundfrombaseline.

Abbreviations:mGluR5-metabotropicglutamatereceptor5;DOR-deltaopioidreceptor,KOR-kappaopioidreceptor;MOR-muopioidreceptor;SERT-serotonintransporter;NET-

norepinephrinetransporter;ND-notdetermined

DU

Q00

025‐

HT 1A

5‐H

T 1B5‐

HT 1D

5‐H

T 1E5‐

HT 2A

5‐H

T 2B5‐

HT 2C

5‐H

T 35‐

HT 5A

5‐H

T 65‐

HT 7

mG

luR5

DO

RKO

RM

OR

SERT

NET

A–6.4

–1.5

–3.6

–1.3

–6.9

–4.2

–12.4

7.5

–0.9

–8.7

–9.6

ND

–16

–8.6

7.7

91.7

9.2

B3.

79.

812

.1–7.2

–9.2

–7.9

–12.7

15.1

–4.8

–10.9

–13.4

ND

–4.7

–6.7

–2.7

86.4

2.5

C14.6

6.4

15.4

–1.4

–8.5

8.2

–1.2

5.9

9.6

8.2

5.5

75.5

1.9

–2.3

9.0

80.3

9.9

D53

.028.6

12.1

0.6

3.7

16.6

10.2

15.2

16.2

22.2

20.5

75.4

9.3

4.8

16.7

50.0

4.7

E7.

917

.715

.8–1.8

–12.4

5.6

–7.1

6.5

1.3

1.4

–3.2

58.1

3.5

0.6

8.8

57.7

22.9

F–3.4

13.9

13.7

11.6

–12.5

8.9

–17.9

4.3

–8.2

–4.7

–18.1

76.5

0.2

10.2

12.6

22.2

50.0

G3.

212

.522.4

24.6

–5.2

3.6

–15.9

13.5

–5.0

–2.0

–8.2

76.4

7.6

25.0

10.2

14.6

50.0

H0.

5–3.8

1.8

29.0

7.4

0.5

–10.2

14.0

3.8

0.6

–2.0

73.4

17.5

19.5

21.0

–19.0

50.0

I58

.323

.816.5

32.3

5.5

19.9

38.6

15.5

20.1

5.1

71.2

65.2

9.7

13.8

20.6

24.0

50.0

10 of 23  |     LAX et al.

TA B L E 2  PDSPsecondarybindingassayofDUQ0002fractionsshowingsignificantinhibitioninprimaryassay.RepresentativeIC50 valuesarederivedfromradiolabeled-liganddose-responsecurvesforcyanobacterialfractions(DUQ0002A-I).Allvaluesareinng/ml(N = 3 separatedeterminations).Openboxeswith(-)indicatethatfractionsfailedprimarybindingcriterionof>50%inhibitionat10μM. Reference compounds:8-OH-DPAT(5-HT1A, Ki=0.76nM);clozapine(5-HT7, Ki = 8 nM); fenobam (mGluR5, Ki=1.5nM);desipramine(NET,Ki=4.7nM)andamitriptyline(SERT,Ki=6.3nM)

DUQ0002 5‐HT1A IC50 5‐HT7 IC50 mGluR5 IC50 SERT IC50 NET IC50

A – – – >10000 –

B – – – >10000 –

C – – 4662 >10000 –

D >10000 – 37 >10000 –

E – – >10000 >10000 –

F – – >10000 – >10000

G – – >10000 – 6945

H – – >10000 – >10000

I 1746 749 3676 – >10000

F I G U R E 3   Initial characterization of theantidepression-likeandanxiolytic-like effects of cyanobacterial fraction DUQ0002IinmaleanimalsviaICVadministration. (a) Mice treated with DUQ0002I(n = 18) decrease time immobile compared to vehicle (n = 18) in the forced swim test. (b) Mice treated with DUQ0002I(n = 9) show no change in time immobile compared to vehicle (n = 9) in the tail suspension test. (c) Mice treated withDUQ0002I(n = 18) show no changes in total distance traveled compared to vehicle (n = 17) in the elevated zero maze. (d)MicetreatedwithDUQ0002I(n = 18) show increased time spent in the open arms of the elevated zero maze (EOM) assay compared to vehicle (n = 17). (e) MicetreatedwithDUQ0002I(n = 18) also travel a greater percent of total distance traveled in the open arms of the EOM compared to vehicle (n = 17). (f) Mice treatedwithDUQ0002I(n = 19) show a trend (p = 0.1203) of spending more timeinthelightsideoftheLight-DarkPreference (LDP) test during the latter half of the test compared to vehicle (n = 15).(g)MicetreatedwithDUQ0002I(n = 19) also show a higher average mean visit to the light side of the LDP test during the latter half compared to vehicle (n = 15). Un-pairedt-tests,*p<0.05,**p < 0.01. Datashownasmean±SEM

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     |  11 of 23LAX et al.

greater mean visit (how long the animal spends on average when it enters the light side) to the light side compared to animals pretreated with vehicleinthelatterhalfofthetest(Figure3g,unpairedt-test,**p=0.0025).Together,thesedatafromboththeEOMandLDPindicateananxiolytic-likeeffectofDUQ0002I.Finally,weobservednochangesinbodytemperatureinanimalsthatreceivedDUQ0002IviaICVinjectioncompared to vehicle.

F I G U R E 4  FractionDUQ0002Ishowsnoeffectsin5-HT7R knockout animals or female animals via ICV administration. (a) Male homozygote5-HT7Rknockout(KO)micetreatedwithDUQ0002I(n=6)donotshowanydifferencesintimeimmobilecomparedtovehicle(n=6)intheforcedswimtest.(b)Malehomozygote5-HT7RKOmicetreatedwithDUQ0002I(n=6)donotchangetheamountoftimespent in the open arms of the EOM compared to vehicle (n=6).(c)5-HT7RKOmicetreatedwithDUQ0002I(n=6)alsodonotchangepercent of overall distance traveled in the open arms of the EOM compared to vehicle (n=6).(d)5-HT7RKOmicetreatedwithDUQ0002I(n=6)donotchangetheamountoftimespentinthelightsideoftheLDPtestduringthelatterhalfofthetestcomparedtovehicle(n = 6).(e)5-HT7RKOmicetreatedwithDUQ0002I(n=6)donotchangethemeanvisittothelightsideoftheLDPtestduringthelatterhalfof the test compared to vehicle (n=6).(f)Malewildtypemicetreatedwiththe5-HT7R antagonist SB258719 (n = 15) show a decrease in time immobile compared to vehicle (n=17)intheforcedswimtest.(g)FemalewildtypemicetreatedwithDUQ0002I(n = 8) do not show any differences in time immobile compared to vehicle (n = 8). (h) Female wildtype mice treated with SB258719 (n = 8) do not show any differences in time immobile compared to vehicle (n=7).Un-pairedt-tests,*p<0.05.Datashownasmean±SEM

(a)

(c)

(e)

(g)

(b)

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Forced Swim Test (KOs)

EOM - Open Arm Distance (KOs) LDP - Light Side Time (KOs)

LDP - Mean Visit to Light Side (KOs)

Forced Swim Test (Females)

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Forced Swim Test (Females)

150

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Vehicle

)ces(eli bo

mmI

emi T

SB258719

Vehicle DUQ0002I

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mmI

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ecnatsiDl at oTt necr eP Vehicle DUQ0002I

40

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150

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2

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)ces(e

mi T

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12 of 23  |     LAX et al.

3.4 | Characterization of the specificity of DUQ0002I effects

DUQ0002Ishowedsecondarybindingaffinityfor5-HT1AR,5-HT7R,andmGluR5.Giventhe2-foldselectivityof5-HT7Rover5-HT1AR and mGluR5,wetestedtoseeifDUQ0002Ihadeffectsin5-HT7Rknockoutanimals.Iftheactivecompound(s)inDUQ0002Iwasselectiveforthe5-HT7R,thenwewouldpredictthattherewouldbenobehavioralchangesinanyoftheassayswehadpreviouslytestedwithDUQ0002Iusingwildtypemalemice.Hereagroupofbothwild-typeand5-HT7R knockout male animals were tested in the forced swim test, elevated zero maze,andlight-darkpreferenceassays.Intheforcedswimtest(Figure4a,unpairedt-test,p=0.2490),DUQ0002I(n=6)didnotcauseanychange on overall time immobile when compared to vehicle injected knockout mice (n=6).Similarly,DUQ0002I(n=6)didnotcauseanimalstospendmoretimeintheopenarms(Figure4b,unpairedt-test,p=0.7712)ortravelagreaterpercentdistanceintheopenarms(Figure4c,unpairedt-test,p=0.5599)whencomparedtovehicle-treatedknockoutanimals(n=6)intheelevatedzeromaze.Finally,inthelightdarkpreferenceassay,KOanimalspretreatedwithDUQ0002I(n=6)didnotspendagreateramountoftimeinthelightside(Figure4d,unpairedt-test,p = 0.1929) when compared to KO mice that received vehicle (n=6).ThesesameanimalsalsodidnothaveagreatermeanvisittothelightsidewhencomparedtocontrolKOanimals(Figure4e,unpairedt-test,p=0.4342).Together,thesedataintheKOanimalssuggestthattheanti-depressiveeffectsobservedfromDUQ0002Iarecausedbyselectiveantagonismofthe5-HT7R.

Asanadditionalmeasureof5-HT7RspecificityandtohelpdeterminewhetherDUQ0002Iisanagonistoranantagonist,wecomparedtheeffectsofDUQ0002Iwithaknown5-HT7Rantagonist,acompoundthathasbeenpreviouslyshowntohaveantidepression-likeeffectsintheforcedswimtest(Guscottetal.,2005).InamannersimilartoDUQ0002I,afiveminutepretreatmentof5μgofSB258719causedwild-typemice (n = 15) to exhibit statistically significant lower levels of immobility in the forced swim test when compared to animals that received con-trol vehicle injections (n=15)(Figure4f,unpairedt-test,*p=0.0275).Sinceantagonismofthe5-HT7Risknowntocauseantidepressant-likeeffects,thesimilarbehavioraleffectsofSB258719suggestedasimilarantagonisticmechanismasDUQ0002I.

Duetotheknownsexdifferencesindepressionandanxiety(Kokras&Dalla,2017),wenexttestedDUQ0002IinfemaleanimalsviaICVinjectiontoseeifitinducedthesameantidepression-likeandanxiolytic-likeeffectsaswehadpreviouslyshowninmaleanimals.Interestingly,administrationofthesame40μgdoseofDUQ0002Iinfemalemice(n = 8) did not induce any changes in immobility time in the forced swim testwhencomparedtocontrolvehicle-injectedmice(n=8)(Figure4g,unpairedt-test,p=0.7675).Similarly,nodifferenceswereseenbe-tweenfemalestreatedwithDUQ0002Iorvehicle inthetailsuspensiontest,elevatedzeromaze,and light-darkpreferencetest (datanotshown).Todetermineifthesesexdifferenceswerespecifictoourcyanobacteriafractionorwasabroadereffectof5-HT7R antagonists, we alsotestedtheeffectsofSB258719inaseparategroupofwild-typefemalemice.AswithDUQ0002I,SB258719(n = 8) failed to alter im-mobilitytimecomparedtocontrolvehicle-injectedanimals(n=7)(Figure4h,unpairedt-test,p=0.5986).Thesedatasuggesttheremaybeapreviouslyunidentifieddifferencebetweenmalesandfemalesinthewaytheyrespondto5-HT7Rantagonistsorinthedistributionof5-HT7R in their brains.

3.5 | Anatomical characterization of DUQ0002I in males via hippocampal administration

AfterICVandspecificitycharacterizationofDUQ0002I,weexploredwhichareaofthebrainwascontributingtotheobservedpheno-types.Previousstudieshaveshownthatantagonistsofthe5-HT7Rcauseantidepressionandanxiolytic-likeeffects(Guscottetal.,2005)whenspecificallyadministeredtotheCA1regionofthehippocampus(Wesolowskaetal.,2006).WedeliveredDUQ0002Iviaintrahip-pocampalinjectionstoseeifthisbrainregionwassufficienttoinducetheobservedeffectsofDUQ0002I.OnlymaleanimalswereusedsinceDUQ0002Iwasshowntonotalterbehaviorinfemales(Figure4g).Here,agroupofanimalsunderwenttestingusingtheforcedswimtest,tailsuspensiontest,elevatedzeromazeandlight-darkpreferencetest.Beginningwiththeforcedswimtest,weshowedthatCA1administrationofDUQ0002I(n=5)causedastatisticallysignificantdecreaseinoveralltimeimmobilecomparedtovehicle-treatedanimals (n=5)(Figure5a,unpairedt-test,*p=0.0450);incontrasttoICVdelivery,wefoundthathippocampal-deliveredDUQ0002I(n =5)alsocausedanantidepression-likeeffectinthetailsuspensiontestcomparedtovehicle-treatedanimals(n = 5) (Figure 5b, unpaired t-test,**p=0.0046).

ToshowoveralleffectsofDUQ0002Iindepression-likebehavior,weutilizedtheZ-scoreanalysistechnique(Guillouxetal.,2011).Thismethod allows for the comparison of results across different, but related, sets of data through standardization by comparison to the control group. Itenablesus toseetheoverallantidepression-like (or lateranti-anxiety-like)effectsofDUQ0002Ibyaveragingtheeffectsof thedepression behavioral tests (time immobile in forced swim and tail suspension tests). Here, the parameter measured for each animal (time im-mobile,X)wasgivenanindividualZ-score(Z)todeterminehowmanystandarddeviations(σ) each animal was above or below the mean of the control group (μ)usingthefollowingformula:Z=(X-μ) / σ.Withthistypeofanalysis,thecontrolgroupalwayshasanoverallZ-scoreofzero.Fortheexperimentalgroups,positiveZ-scoresreflectpositiveemotionalitychanges(i.e.,antidepressionorantianxiety),whereasnegativeZ-scorereflectnegativeemotionalitychanges(i.e.,depression,anxiety).Notsurprisingly,aZ-scoreanalysisofCA1administrationshowedthatDUQ0002I(n=5)causedastatisticallysignificantantidepression-likeeffectwhencomparedtovehicle-treatedanimals(n = 5) (Figure 5c, un-pairedt-test,**p=0.0045).ThesedatashowthatCA1administrationofDUQ0002Iissufficienttoinducestrongantidepression-likeeffects.

     |  13 of 23LAX et al.

Next,wetestedtoseeiftheanxiolytic-likeeffectsofICV-deliveredDUQ0002Iweremediated,inpart,by5-HT7RintheCA1ofthehip-pocampus.IntheEOMtest,weshowedthatDUQ0002I(n = 5) did not change the total amount of time spent in the open arms of the maze (Figure5d,unpairedt-test,p=0.7875)whencomparedtocontrolvehicle-injected(n=5)animalsviaCA1injection.Thefractionalsodidnotchangetheoverallpercentdistancethattheanimalstraveledintheopenarms(Figure5e,unpairedt-test,p=0.7806).WealsotestedCA1administrationofDUQ0002Iinthelight-darkpreferencetestandfoundsimilarnegativeresults.DUQ0002I(n = 5) did not change the overall amountoftimespentinthelightsideofthelight-darkpreferencetestwhencomparedtovehicle-treatedanimals(n = 5) (Figure 5f, unpaired t-test,p=0.8695)duringthelatterhalfofthetest,nordidDUQ0002Ichangetheoverallmeanvisittothelightsideofthebox(Figure5g,

F I G U R E 5  AdministrationoffractionDUQ0002IbilaterallytotheCA1regionofthehippocampusissufficienttoinduceantidepression-likeeffectsinmaleanimals.(a)MicetreatedwithDUQ0002I(n = 5) decrease time immobile compared to vehicle (n = 5) in the forced swim test.(b)MicetreatedwithDUQ0002I(n = 5) decrease time immobile compared to vehicle (n = 5) in the tail suspension test. (c) Mice treated withDUQ0002I(n = 5) show a positive increase in emotionality when looking at the effects of the depression assays (forced swim and tail suspension) compared to vehicle (n=5).(d)MicetreatedwithDUQ0002I(n = 5) show no difference in time spent in the open arms of the EOM compared to vehicle (n=5).(e)MicetreatedwithDUQ0002I(n = 5) show no difference in overall percent distance traveled in the open arms of the EOM compared to vehicle (n=5).(f)MicetreatedwithDUQ0002I(n = 5) show no difference in time spent in the light side of the LDP box during the latter half of the test compared to vehicle (n=5).(g)MicetreatedwithDUQ0002I(n = 5) show no difference in the mean visit to the light side of the LDP box compared to vehicle (n=5).(h)MicetreatedwithDUQ0002I(n = 5) show no changes in emotionalitywhenlookingattheeffectsofthecombinedanxietyassays(elevatedzeromazeandlight-darkpreference)comparedtovehicle(n=5).Un-pairedt-tests,*p<0.05,**p<0.01.Datashownasmean±SEM

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unpairedt-test,p =0.5067).Finally,aswasdoneforthedepressiontests,anoverallanxietytestZ-scorewascalculatedfortheCA1admin-istrationofDUQ0002I(calculatedusingthetimeandpercentdistancetraveledinopenfromtheEOMandtimeandmeanvisittothelightside). In contrast to what was shown for depression behaviors, the overall emotionality score related to anxiety behaviors showed no overall differencebetweencontrolandDUQ0002Itreatedanimals(Figure5h,unpairedt-test,p = 0.9127). Combined, these data show that while DUQ0002IadministrationtotheCA1issufficienttoinduceantidepression-likeeffects,itisnotsufficienttoinducetheanxiolytic-likeeffects.

3.6 | Bioassay‐guided fractionation of DUQ0002I

Allofthealterationsindepressionandanxiety-likebehaviorseenthusfarwereproducedusingafractionofcyanobacterialextract(DUQ0002I),which contained a mixture of various compounds. In an effort, to move beyond simply testing extracts for behavioral effects and identify specific active compoundswithin this fraction,DUQ0002Iwaspurified into four subfractions (namedDUQ0002I-1, -2, -3 and -4) usingreverse-phasecolumnchromatography.Sub-fractionsDUQ0002I-2,DUQ0002I-3andDUQ0002I-4wereallobtainedasgreenpastes.Incontrast,sub-fractionDUQ0002I-1wasobtainedasayellowpasteand,unlikefractionsDUQ0002I-2,-3and-4,wasshownbyNMRtobemostchemicallysimilartoDUQ0002I.Duetoitschemicalsimilaritytotheparentfraction,andthefactthatthesesub-fractionsstillcontainedmixturesofcompounds,sub-fractionDUQ0002I-1wasfurtherpurifiedusingsilicagelchromatographytoyieldthreemorepurifiedsub-frac-tions,(namedDUQ0002I-1A,-Band-C).AflowchartillustratingthefractionationofDUQ0002IcanbeseeninFigure6.Allsub-fractionsandpurifiedsub-fractionsdescribehereinweretestedin vivo (see Figure 7) after the initial characterization of the parent fraction described below in an attempt to determine if these purified fractions contained the active compound(s).

Thethreesubfractions(DUQ0002I-2,-3and-4)andthethreepurersubfractionsofsubfractionDUQ0002I-1(DUQ0002I-1A,-1Band-1C)weretestedviaCA1injectionsinseparategroupsofmaleanimalsthatunderwenteitherthetailsuspensionorforcedswimtests.Wepre-dictedthatsincesignificantantidepressant-likeeffectswereseenwiththeparentfraction(DUQ0002I)viaCA1injections(seeFigure5),thenanysubfraction(s)withtheactivecompound(s)shouldhavethesame,orperhapsstrongerbehavioraleffects.SincesubfractionDUQ0002I-1hadchemicallyuniquecharacteristicssimilartotheparentfraction,wepredictedthatoneifitspurifiedsubfractions(eitherDUQ0002I-1A,-1Bor-1C)wouldcontainanactivecompoundandinducethesameantidepression-likeeffectsastheparentfraction.

Interestingly,noneofthesubfractionsorpurifiedsubfractionsexhibitedanystatisticallysignificantbehavioraleffects(DUQ0002I-2(n = 11, vehicle group, n=12)(Figure7a,unpairedt-test,p=0.1810),DUQ0002I-3(n=6,vehiclegroupn=5)(Figure7b,unpairedt-test,p =0.6954)orDUQ0002I-4(n=6,vehiclegroupn=6)(Figure7c,unpairedt-test,p=0.2545;DUQ0002I-1A(n=6,vehiclegroup,n=6)(Figure7d,un-pairedt-test,p=0.5780),DUQ0002I-1B(n=6,vehiclegroup,n=5)(Figure7e,unpairedt-test,p=0.8271),orDUQ0002I-1C(n = 7, vehicle group, n=7)(Figures7f,unpairedt-test,p=0.2145)).Followingthesenegativefindings,wereconfirmedtheactivityoftheparentDUQ0002IfractionbyscreeningitonceagainusingNMR.Nochangeswereobserved,suchthatboththeoriginalNMRandthissecondNMRthatrecon-firmedactivityhadthesameactivepeaks.Thissuggeststhattheparentnorsubfractionshaddegraded.Thismaysuggestthattheactivityseen in the parent fraction is caused by a synergistic effect between two or more unidentified compounds in different subfractions that do not havesub-therapeuticactivityalone.ForoursubsequentSNIstudies,wechosetotesttheparentfraction,whilestructureelucidationeffortscontinue to identify the individual synergistic components that could become lead compounds in drug design.

F I G U R E 6  FlowchartofthefractionationofDUQ0002I

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3.7 | Utilizing DUQ0002I as a therapeutic lead in the spared nerve injury (SNI) model of comorbid pain and depression

Thusfarinourstudies,weshowedthatDUQ0002Icausesanxiolyticandantidepression-likeeffectsthatarespecifictomaleanimalsandlikelyactthrough5-HT7R.Wealsoshowedthattheantidepression-likeeffectswerelikelycaused,inpart,throughtargetingtheCA1ofthehippocampus. In all of these experiments, the animals that were tested were naïve mice; they did not have any underlying manipulation that causedthemtobeinaheightenedstateofdepression.Inordertounderstandmoreabouthowtargetingthe5-HT7R may be involved in alter-ingdepression-likebehavior,wedecidedtotestDUQ0002Iinanimalswithanunderlyingdepressionphenotype.Here,onlymaleanimalsweretestedsinceDUQ0002Iwasfoundtonothaveanyeffectsinfemales(seeFigure4g).Sincethereissignificantevidencethatchronicpainoftenoccurs with depression symptoms and that each condition can exacerbate the effects of the other (Bair, Robinson, Katon, & Kroenke, 2003; VonKorff,Ormel,Katon,&Lin,1992),wedecidedtousethewellcharacterizedsparednerveinjury(SNI)model(Decosterd&Woolf,2000)ofneuropathic pain with comorbid depression.

First,wewantedtodetermine ifandwhenanimals that receivednocompoundswithSNIexhibitedchanges inmechanical sensitivity(i.e.,pain-likebehavior)andaffectivebehaviors(i.e.,depression-likebehaviors)whencomparedtocontrolshamanimals.Consistentwiththerobustpain-likephenotypeinSNI,wefoundthatatone,three,five,sixandtenweekspost-surgery,maleSNIanimals(n=6)hadstatisticallysignificant lower withdrawal thresholds when compared to sham animals (n=6)intheipsilateralpaw(Figure8a,2-wayANOVA,overalleffectoftime***p<0.0001,overalleffectofsurgery***p<0.0001,Bonferroniposthoctests,**p<0.01,***p<0.001).WenextdeterminedwhenSNIanimalsdevelopeddepression-likeeffectsintheforcedswimandtailsuspensiontestswhencomparedtoshamanimals.Usingthesamegroupofanimalsthatunderwentmechanicalsensitivitytesting,wefoundthatbythefive-weekpost-surgerytimepoint,SNIanimalsshowed

F I G U R E 7  AdministrationofpurifiedsubfractionsofDUQ0002IbilaterallytotheCA1regionofthehippocampushasnoeffectsinmales.(a)MicetreatedwithDUQ0002I-2(n = 11) show no difference in time immobile compared to vehicle (n = 12) in the forced swim test. (b)MicetreatedwithDUQ0002I-3(n=6)shownodifferenceintimeimmobilecomparedtovehicle(n = 5) in the forced swim test. (c) Mice treatedwithDUQ0002I-4(n=6)shownodifferenceintimeimmobilecomparedtovehicle(n=6)intheforcedswimtest.(d)MicetreatedwithDUQ0002I-1(n=6)shownodifferenceintimeimmobilecomparedtovehicle(n=6)inthetailsuspensiontest.(e)MicetreatedwithDUQ0002I-1B(n=6)shownodifferenceintimeimmobilecomparedtovehicle(n = 5) in the tail suspension test. (f) Mice treated with DUQ0002I-1C(n = 7) show no difference in time immobile compared to vehicle (n=7)intheforcedswimtest.Un-pairedt-tests.Datashownasmean±SEM

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F I G U R E 8  Sparednerveinjury(SNI)inducesmechanicalhypersensitivityandincreasesdepression-likeeffects10weekspostsurgery;DUQ0002Iadministrationreversesthesedepression-likeeffectswhenadministeredtotheCA1inmaleanimals.(a)MicethatreceiveSNIsurgery(n=6)havelowerwithdrawalthresholdsforupto10weekspostsurgerycomparedtosham(n=6).(b)MicethatreceiveSNIsurgery (n=6)showatrendofincreasingtimeimmobilecomparedtosham(n=6)intheforcedswimtest.(c)MicethatreceiveSNIsurgery(n=6)increasetimeimmobilecomparedtosham(n=6)inthetailsuspensiontest.(d)MicethatreceiveSNIsurgery(n=6)haveanegativedepression emotionality score when looking at the effects of the combined depression assays (tail suspension and forced swim tests) compared to sham (n=6).(e)SNImicethatreceiveDUQ0002I(n=12)andshammicethatreceiveDUQ0002I(n = 10) both decrease time immobilecomparedtoSNIanimalsthatreceivevehicle(n = 10) and sham animals that receive vehicle (n=12)intheforcedswimtest.(f)SNImicethatreceiveDUQ0002I(n=12)andshammicethatreceiveDUQ0002I(n=10)bothdecreasetimeimmobilecomparedtoSNIanimalsthat receive vehicle (n = 10) and sham animals that receive vehicle (n=12)inthetailsuspensiontest.(g)SNImicethatreceiveDUQ0002I(n=12)andshammicethatreceiveDUQ0002I(n = 10) both show a positive increase in emotionality score when looking at the combined effectsofthedepressionassays(forcedswimandtailsuspension)comparedtoSNIanimalsthatreceivevehicle(n = 10) and sham animals that receive vehicle (n=12).A-2-wayANOVA,maineffectoftimeandsurgery,Bonferronipost-hoctest*p<0.05,***p<0.001;B-un-pairedt-test,p=0.0834;C-un-pairedt-test,**p<0.01;D-un-pairedt-test,*p<0.05;E-2-wayANOVA,overallmaineffectofcompound;Bonferroniposthoctestn.s.andsham-unpairedt-test,+p=0.0658andSNI-unpairedt-test,*p=0.0458;F-2-wayANOVA,overallmaineffectofcompound;Bonferronipost-hoctest**p<0.01andsham-unpairedt-test,*p=0.0271andSNI-unpairedt-test,**p=0.0032;G-2-wayANOVA,overallmaineffectofcompound,Bonferronipost-hoctest*p<0.05,***p<0.001.Datashownasmean±SEM

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nodifferencesinoveralltimeimmobileintheforcedswimandintailsuspensiontests(datanotshown).However,bythe10weekpost-surgerytimepoint,SNIanimals(n=6)trendedtowardsshowinghigherlevelsofimmobilitywhencomparedtoshamanimals(n=6)intheforcedswimtest(Figure8b,unpairedt-test,p=0.0834).SNIanimals(n=6)alsoshowedsignificantlyhigherlevelsofimmobilityinthetailsuspensiontest when compared to control sham animals (n=6)(Figure8c,unpairedt-test,**p=0.0037).CombiningtheresultswithaZ-scoreanalysis(calculatedusingthetimeimmobileintheforcedswimandtailsuspensiontests)showedthatoverall,SNIanimalsshowednegativedepressionemotionality(i.e.,greaterdepression-likebehavior)comparedtoshamanimals(Figure8d,unpairedt-test,*p = 0.0110).

SincetheSNImodelcausedbothpain-likeanddepression-likeeffects10weeksaftersurgery,wetestedtheeffectsofDUQ0002Iatthistimepoint.ThesedataallowedustodetermineifDUQ0002IwouldhaveanoverallgreatereffectonSNIanimalswhencomparedtoshammiceduetotheunderlyingmodelofcomorbidity.Here,agroupofanimalsunderwenteitherSNIorshamsurgery10weekspriortothebeginningofbehavioraltesting,whichincludedtheforcedswimandtailsuspensiontests.DUQ0002ItargetedtotheCA1hadnobehavioraleffectonSNI-inducedmechanicalhypersensitivity(datanotshown).However,DUQ0002Ihadanoveralleffectinreducingimmobilityintheforcedswimtest in both sham (n=10)andSNImaleanimals(n=12)comparedtovehicle-treatedSNI(n = 10) and sham (n=12)animals(Figure8e,2-wayANOVA,overalleffectofcompound**p=0.0063,overalleffectofsurgeryp=0.2143,Bonferroniposthoctestsn.s.).Whenlookingateachindividualgroupofanimals,bothsham(Figure8e,unpairedt-test,+p=0.0658)andSNIanimals(Figure8e,unpairedt-test,*p=0.0458)shownearorsignificantlylowerlevelsofimmobilitywhenDUQ0002IisadministeredtotheCA1comparedtovehicle-treatedanimals.Similarly,DUQ0002I delivered to theCA1 reducedoverall immobility in the tail suspension test for both shamandSNI animals (Figure8f, 2-wayANOVA,overalleffectofcompound***p = 0.0002, overall effect of surgery p=0.4385,Bonferroniposthoctest,**p<0.01).Whenlookingateachindividualgroupofanimals,bothsham(Figure8f,unpairedt-test,*p =0.0271)andSNIanimals(Figure8f,unpairedt-test,**p = 0.0032) showsimilarlevelsofimmobilitywhenDUQ0002IisadministeredtotheCA1whencomparedtovehicle-treatedanimals.

TolookattheoveralleffectthatDUQ0002IhadonbothSNIandshamanimals,aZ-scoreanalysisfordepression-likebehaviors(usingtimeimmobileintheforcedswimandtailsuspensiontests)wascalculated(Figure8g,2-wayANOVA,overalleffectofcompound***p < 0.0001, overall effect of surgery p=0.1728,Bonferroniposthoctests,*p<0.05-sham,***p<0.001-SNI).ThisZ-scoreanalysisshowsthatoverall,DUQ0002Ireduceddepression-likebehaviorsinbothSNIandshamanimalsandthattheseantidepression-likeeffectsmaybegreaterinSNIanimals when compared to sham animals.

4  | DISCUSSION

Overall,ourdatashowthatcyanobacteriamayrepresentanimportantunexploredresourcefor5-HTspecificligands.Wefoundanextractfrom a marine cyanobacterium of the genus Leptolyngbyainducesantidepressantandanxiolytic-likeeffectsthatarespecifictothe5-HT7R and male animals using in vivomodelsofbehavior.Additionally,wewereabletoshowthatadministrationofourextracttotheCA1ofthehippocampusissufficienttoinducetheseantidepressant-likeeffectsandthattheeffectsarelikelycausedbyamixtureofcompoundsinthefraction.Finally,usingthisfraction,wedemonstratedthattheactivecompoundsinvolvedhadsimilareffectsinanimalswithcomorbidpain-likeanddepression-likebehavior.

Thegenusfromwhichourextractoriginated,Leptolyngbya, has global distribution in terrestrial and aquatic ecosystems (Shimura et al., 2015)andisoneofthemajornaturalproduct-producinggeneraofmarinecyanobacteria(N.Engeneetal.,2013).Whileitisunknownifthesample is still growing at the same coordinates, Leptolyngbya growing under similar environmental conditions would be predicted to produce the same types of compounds since Lyngbya, a closely related genus of cyanobacteria, has been found to grow in the same location (and pro-ducethesamecompoundsuponrecollection)twoyearsapart(Rossi,Roberts,Yoo,&Gerwick,1997).ThefactthatourphylogeneticanalysisshowedthatDUQ0002belongedtoLeptolyngbya, a different genus from the field identification (Oscillatoria or Moorea), highlights the impor-tanceofusing16SrRNAgenesequencestoobtainmoreaccurateidentificationsoffieldcollectedcyanobacteria.Recentstudiesusingwhole-genomeand16SrRNAsequencingapproacheshavechangedthetraditionalphylogeneticclassificationofcyanobacteria(Wilmotte,1994).Known compounds extracted from LeptolyngbyaincludepalmyrolideA,whichhaspotentsodiumchannelblockingactivitywithoutcytotoxic-ity(Pereiraetal.,2010),crossbyanolsA-Dwithantibioticactivity(Choi,Engene,Smith,Preskitt,&Gerwick,2010)andhonaucinsA-Cwiththeabilitytoinhibitproinflammtorycytokines(Choietal.,2012).Anothercompound,coibamideA,apotentantiproliferativecyclicdepsipeptide,was also extracted from a LeptolyngbyaspeciesfromthePacificsideofPanamabutfromtheothersideoftheAzueropeninsulanearCoibaIsland(Medinaetal.,2008).DUQ0002,however,representsthefirstLeptolyngbyaextractwithGPCR-specificpsychoactiveeffects.Overall,these data suggest that Leptolyngbya species have significant potential in the field of neuropharmacognosy.

Theobservedaffinitiesforcyanobacterialsecondarymetabolites(Figure1;Table2), includingthosefromDUQ0002Ifor5-HT1AR and 5-HT7R,areveryencouragingfromapharmacologicalviewpoint(Naumenko,Popova,Lacivita,Leopoldo,&Ponimaskin,2014)andmayrep-resentachemicalecologyrolefortheseserotoninreceptorligands.Apossibleexplanationfortheirpresencecouldbelinkedtothepreda-tor-preyrelationshipbetweenAplysia sea hares and marine cyanobacteria. In their natural environment, colonial marine cyanobacteria are preyed upon by Aplysiaseahares.Thisecologicalrelationshipcouldlinkthebiosynthesisof5-HTRligandsbymarinecyanobacteriatothese

18 of 23  |     LAX et al.

marine mollusks due to the simple structure of their nervous system. In Aplysia,theentireCNSconsistsofonlyasmallnumberofneurons,whichcontain5-HTRandhavebeenshowntobeinvolvedinlearningandmemory(Barbas,DesGroseillers,Castellucci,Carew,&Marinesco,2003).Thecompoundsfromthecyanobacteriacouldbeaffectingthefeedingbehavioroftheseaharesbytargetingtheirnervoussystem,andprovidesapossibleexplanationfortheirpresence.Theresultsofthescreeningofall276fractionsfromPanamaandCuraçao,showing21%ofallfractionshave5-HTRor5-HTtransporteractivity,suggestastrongevolutionarypressuretoproduce5-HTmetabolitesintheseorganismsand show the potential for cyanobacteria to be used in the drug discovery space.

TheinitialscreeningoftheDUQ0002fractionusingtheICVadministrationrouteallowedustohavethebroadestpossibleapplicationof the fractions across the brain, brainstem and spinal cord, while overall reducing the total amount needed to see effects. It also allowed us to avoid potentially harmful systemic effects of targeting serotonin receptors (Hutcheson, Setola, Roth, & Merryman, 2011) and allowed us to test compounds that might not cross the BBB. Other broad administration routes, including intravenous, intraperitoneal or oral gavage, whilemoreclinicallyrelevant,wouldhavelimitedourtesting.Additionally,wearenotoverlyconcernedaboutBBBpermeabilityoralternateadministrationroutesofourfractions.Alongwiththis,onlyonedose(40μg)oftheparentfraction(DUQ0002I)wastested.Sincethisinitialdoseworkedtoinducebehavioralchangesfromthefirstscreeningtests,itwasthedosethatwasusedfortherestofthestudies.Testingdifferent doses of the parent fraction would not have provided any additional insight into the specific dose of active compound(s) needed since DUQ0002Iitselfisamixtureofcompounds.Rather,ouroverallfocusisondiscoveringandidentifyingcompoundswithefficacyinanimalmodels at an early stage before dealing with lead optimization, modifications that could circumvent administration issues and dosing.

OurresultsfromtheinitialscreeningoffractionDUQ0002IwithICVadministrationcorrespondwellwithotherstudiesofthe5-HT7R usingselectiveligandsinanimalmodelsofdepressionandanxiety.Whilethemodelsusedarenotmodelsoftheanhedonicprofileorchronictreatment,theassaysthatwereusedarewell-characterizedandshowastrongpredictiveefficacyintermsofhowcompoundswouldworkinhumans(Wang,Timberlake,Prall,&Dwivedi,2017).Intermsofdepression,inrodents,administrationoftheselective5-HT7R antagonist SB269970decreasesdepression-likebehaviorsintheforcedswim,tailsuspension,andconflictdrinkingtests(Hedlundetal.,2005;Sarkisyanetal.,2010;Wesolowskaetal.,2006).Othernovelantagonistshaveshownsimilarantidepression-likeeffects(Kimetal.,2014).Moreover,male5-HT7Rknockoutanimalsalsoexhibitasimilarphenotype(Guscottetal.,2005).Thissuggeststhatthesechangesinbehavioraretrulydependentonthe5-HT7R.Lookingatanxiety-likebehavior,inmice,pharmacologicalblockadeofthe5-HT7Rreducesanxiety-likebehaviorintheopenfieldtest(Guillouxetal.,2013).OtherstudieshaveshownsimilarresultswithSB269970causinganxiolytic-likeeffectsintheVogeldrinkingtestandelevatedplusmazeinratsandthefour-platetestinmice(Wesolowskaetal.,2006).SB269970hasalsobeenshowntocauseareductioninmarbleburyinginmice,abehaviorshowingananxiolytic-likeeffect(Hedlund&Sutcliffe,2007).Combined,thesedatashowthat,ingeneral,antagonistsofthe5-HT7Rdecreasebothdepressionandanxiety-likebehaviors.Therefore,sincethebehavioralphenotypeofDUQ0002Imimicstheeffectsoftheseknownantagonists,itsuggeststheactivecompound(s)inDUQ0002Iismostlikelyactingasanantag-onistofthe5-HT7R.Ourdataalsosupportanoverallanti-depressantandanti-anxietyroleofinhibiting5-HT7R since we were able to induce thesamebehavioraleffectwithICVinjectionsofaknown5-HT7RantagonistSB258719.Moreover,thefactthatDUQ0002Ifailedtoinduceantidepressantandanxiolytic-likeeffects in5-HT7Rknockoutanimalssuggeststheselectivityoftheactivecompounds inDUQ0002Iforthe5-HT7Randindicatesthattheactivecompoundsmaynotbeactingonanyotherreceptors,includingthe5-HT1AR,NETormGluR5.OneexplanationforthisisthatthebehavioraleffectsofDUQ0002Iwereoccludedbygeneticablationofthe5-HT7R,suggestingthatDUQ0002Iwasactingasanantagonistofthe5-HT7R.However,itshouldbenotedthatintheseknockoutanimals,alreadyshowinganantidepression-likephenotype,itmaybedifficulttoseefurtherantidepression-likeeffects,evenifDUQ0002Iwasactingonadistinctreceptor.

Ourdatashowapotentiallyinterestinganduncharacterizedsexdifferenceintermsofresponsetoinhibitionofthe5-HT7R in depression andanxiety-likebehaviors.Allofourinitialantidepressantandanxiolytic-likeDUQ0002Ieffectswerefoundinmaleanimalsandwerenotreplicatedinfemalemice.Likewise,femalemicedidnotshowanybehavioralchangeswithadministrationofthe5-HT7R antagonist SB258719. Despite the known sex differences in depression and anxiety (Kokras & Dalla, 2017), all of the depression studies (Hedlund et al., 2005; Kim et al.,2014;Mnie-Filalietal.,2011;Sarkisyanetal.,2010;Wesolowskaetal.,2006)andanxietystudies(Guillouxetal.,2013;Hedlund&Sutcliffe,2007;Wesolowskaetal.,2006)thathavelookedintothebehavioraleffectsof5-HT7Rantagonistsweredoneusingonlymaleanimals.Thisisalsotruefor5-HT7KOstudieswhereonlymalemicehavebeentestedincognitiveassays(Guscottetal.,2005;Robertsetal.,2004).Theonlyexceptionisastudythatfoundtherewasnohypothermicresponseto5-HTinmaleorfemalemice(Hedlundetal.,2003).Itisknownthatestrogenscaninterferewiththeeffectsofantidepressants(Keers&Aitchison,2010)andthatfemalescanexhibithigherlevelsofserotoninandsynthesizeitmoreslowly(Nishizawaetal.,1997).Additionalworkisneededtodeterminethemechanismbehindtheobservedsexdiffer-ences.Inthepainliterature,severalstudieshavefoundthat5-HT7Rantagonistsincreasepain-likebehaviorsinmales(Brenchatetal.,2009;Liu,Reid,&Sawynok,2013),whereasantagonistsdecreasepain-likebehaviorsinfemales(Amaya-Castellanosetal.,2011;Godinez-Chaparro,Barragan-Iglesias,Castaneda-Corral,Rocha-Gonzalez,&Granados-Soto,2011).Our failure toseeaneffectofDUQ0002I inSNI-inducedhypersensitivitymayhavebeencausedbyaflooreffectintheassaywheremaleSNI-treatedmicecouldnotshowadditionalpainbehavior.FuturestudieswithDUQ0002Iwillexplorepotentialeffectsinthecontextofothermodelsofacuteandchronicpain.

TheresultsfromouranatomicalstudieshaveprovidedinsightintowherethecompoundspresentinDUQ0002Imaybeactingtoinducetheeffectsobserved.The initial characterizationexperimentswith ICV injectionsallowedus tobroadly target the5-HT7R in the central

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nervoussystem.ThelackofanatomicalspecificitywiththeICVinjectionsismostlikelythereasonbehindthewiderangeofeffectswesawinthebehavioralassays.5-HT7Rexpressioniswell-characterizedinrodentsandhumansandisknowntobeexpressedinavarietyofregionsincludingthecortex,hippocampus,amygdala,hypothalamusandthalamus(Bonaventureetal.,2004;Gellyncketal.,2013;Martin-Cora&Pazos,2004;Varnas,Thomas,Tupala,Tiihonen,&Hall,2004).Thehippocampusispartofthelimbiccircuitryofthebrainandserotonininthisregionisinvolvedinemotionalstates(Hensler,2006)andmayplayaroleintheactionofantidepressants(Nestleretal.,2002;Vythilingametal.,2004).Moreover,the5-HT7Rhasbeenshowntoplayaroleinhippocampalneuronalexcitability(Tokarski,Zahorodna,Bobula,&Hess,2003),glutamatergicsynaptictransmission(Andreettaetal.,2016;Costa,Trovato,Musumeci,Catania,&Ciranna,2012;Vasefietal.,2013),synapticplasticity (Costa,Sardone,Lacivita,Leopoldo,&Ciranna,2015)andhippocampal-dependentmemory (Eriksson,Golkar,Ekstrom,Svenningsson,&Ogren,2008;Sarkisyan&Hedlund,2009).Previousstudieshaveshownthatblocking5-HT7RintheCA1regionofthehippo-campusdecreasesdepression-likebehaviorsinrodents(Wesolowskaetal.,2006),andourresultsobtainedintheforcedswimandtailsuspen-siontestsshowthesameeffectwithadministrationofDUQ0002I.Ofnote,wesawantidepressant-likeeffectsinbothFSTandTSTwithCA1administrationofDUQ0002I;withICVinjection,effectswereonlyseeninFST.ThisTSTdifferencecouldbearesultofaninsufficientdoseoftheactivecompoundsinDUQ0002IreachingtheareaofactivationwiththeICVinjections.WealsocompletedaZ-scoreanalysis(Guillouxetal.,2011)of“depression”behaviors.TheZ-scoreanalysisisapowerfulthatallowsforthecomparisonofresultsacrosssimilarassaysandhasbeenusedinstudiesthatusemultiplemeasuresofbehavior(deSa-Calcadaetal.,2015;Maluachetal.,2017;Mendez-Davidetal.,2017;Sargin,Oliver,&Lambe,2016).TheZ-scoreanalysishighlightstherobustanti-depressanteffectthatDUQ0002IhadwhenadministeredtotheCA1.

Thestronganti-depressanteffectwasincontrasttoourfailuretoobserveanxiety-likebehaviorswithCA1administrationofDUQ0002I.Weshowedthattherewerenoeffects ineithertheelevatedzeromazeor lightdarkpreferencetests.ThecontrastbetweentheZ-scoreanalysesofthedepressionbehaviorsandanxietybehaviorshighlightsthisdifferenceveryclearly.Thefactthatanxiolytic-likeeffectswerenotseenviaCA1administrationbutwereseenwithICVadministrationalsotellsusthatDUQ0002IhasthepotentialtoactatmultipleCNSsites.Notably,theamygdalaisoneofthecentralbrainregionsknowntobeinvolvedwithanxietyandfear(Adhikarietal.,2015).Thefactthat5-HT7Rareexpressedintheamygdala(Bonaventureetal.,2004)andthatitisclosetothecerebrospinalfluidsystemtargetedwithICVinjectionsmeansthatitcouldpossiblybethesiteofactionfortheanxiety-likeeffectsofDUQ0002I.

Interestingly,inourattempttofindtheactivecompoundinDUQ0002Iresponsibleforinducingthebehavioraleffectsshown,wefoundthatnoneofthesubfractionsDUQ0002I-1A,BandCand2,3and4inducedanyantidepressant-likeeffectswithCA1administration.Thissuggeststhatamixtureofoneormorecompoundsfoundintheoriginalparentfractionitself(DUQ0002I)isresponsibleforthechangesinbehavior.Whilenodefinitiveconclusionscanbedrawnaboutthenumberofcompoundsordoseofeachactivecompoundrequiredtocauseeffects with our cyanobacterial fraction, this type of synergistic effect from natural products is not uncommon. Isoflavone and curcumin, two natural products, inhibit the growth of pancreatic cancer cells and alter gene expression in vitrobutonlyincombination(Wangetal.,2008).Other examples include the wide variety of plant extracts, such as terpenoids, glycosteroids, flavonoids and polyphenols, that all have weak lit-tle to no antibiotic activity on their own, but have strong synergistic antibiotic effects on a wide range of microbes (Hemaiswarya, Kruthiventi, &Doble,2008).AsimilartypeofsynergisticeffectispossiblyoccurringwiththemixtureofcompoundsfoundinDUQ0002I.

Finally,ourworkindicateshownaturalproductscanbeusedformorethansimplydrugdiscovery.Naturalproductresearchcanbeusedtodeveloptoolstounderstandbiologicalsystems.Inthiscase,wechosetouseDUQ0002Iinthecontextofthesparednerveinjury(SNI),awell-establishedmodelofneuropathicpainwithcomorbiddepression(Normanetal.,2010;Suzukietal.,2007),tolearnmoreabouttheroleofthe5-HT7Rinthedevelopmentofthiscondition.Ratesofmajordepressivedisorder(MDD)anddepressionsymptomsareveryhigh(13%–82%)inchronicpainpatientsdependingonthetypeofpainstudied(Bairetal.,2003).Incontrast,MDDonlyexistsatarateof6.7%inisolationinU.S.adults(Kessler,Chiu,Demler,&Walters,2005)andratesofchronicpainconditions,suchasfacialpain(4%)andfibromyalgia(2.5%)arealsofairlylowinisolation(NCHS,2006).Similartopreviousfindings(Suzukietal.,2007),SNImicedidnotdevelopsignificantdepression-likesymptomsuntilten-weekspostsurgery,suggestingthatthedevelopmentof“depression”isslowtodevelopandissecondarytotheprimaryhypersensitivity.TheZ-scoreofdepression-likebehaviorsinSNIwhencomparedtoshamanimalshighlightthesignificantbehavioralchangesthatoccurinSNIanimals.Withthismodelestablished,weshowedthatnotonlydoesDUQ0002Idecreasedepression-likebehaviorsintheforcedswimandtailsuspensiontestsinshamanimals,butitalsohasasignificanteffectinSNIanimals.Thisissimilartoresultsthatwerefoundinanothermodelofdepression,theolfactorybulbectomyparadigminrats,whereblockadeofthe5-HT7R with the selective antagonist SB269970wasfoundtocounteractthemodel’sdepression-inducingeffects(Mnie-Filalietal.,2011).Futurestudieswillhavetoinvestigatewhatotherchangesoccurwith5-HT7R in the context of comorbidity.

Overall,ourstudydemonstratesthepowerofutilizingnaturalproductsderivedfrommarinecyanobacteriatoprobetheCNS.Here,wewere able to show that a synergistic mixture of compounds from a species of Leptolyngbya couldinducerobustantidepressantandanxiolytic-effectsinanimalmodelsofbehavior.Whilethefactthatourextractinducedbehavioraleffectsthroughamixtureofunidentifiedcompoundsdoesleadtosomelimitations,itneverthelessprovidedinsightintosomeofthebiologyoftheserotoninsystem.Thisextractshedlightontotheanatomical specificityof thedepression-likeeffectsofblocking the5-HT7R, helped us to discover a possible fundamental difference betweenmalesandfemalesinthewaythe5-HT7Rsystemworkstoinducetheseeffects,andprovidedevidencethatthe5-HT7R may play a

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roleincomorbidpainanddepression.ThesediscoveriesillustratethepotentialthatthesemicrobialsecondarymetaboliteshaveforCNSdrugdiscoveryand,inthefuture,couldpossiblybeusedfortreatmentofCNSdisordersrelatedtoGPCRmalfunction.

CONFLIC T OF INTERE S T S TATEMENT

Theauthorsreportnoconflictofinterest.

ACKNOWLEDG EMENTS

WethanktheAutoridadNacionaldelAmbiente (ANAM),PanamaforpermissiontomakeandexportcollectionsofmarinecyanobacteriaandtheSmithsonianTropicalResearchInstitute(STRI)foruseofboatandfacilitiesinPanama.WewouldalsoliketothankKhTanvirAhmedforsamplepreparationsusedinbehavioraltesting,AnaliseZapadkaforherworkwith16SrRNAsequencing,andAnnaPolaskiandHeatherAllenfortheirmanuscriptediting.Projectwasfundedthrough(1)agrantprovidedbytheAmericanPainSocietyandtheSharonS.KellerFundforChronicPainResearch(BJKandKJT), (2)agrantfromtheNIH(NCCIHR15AT008060;BJKandKJT), (3)aFogartyInternationalCenter(FIC)InternationalCooperativeBiodiversityGroup(ICBG)grantbasedinPanama(2U01TW006634-06;KJT),and(4)agrantfromtheDuquesneUniversityFacultyDevelopmentFund(KJTandBJK).KideterminationsandreceptorbindingprofilesweregenerouslyprovidedbytheNationalInstituteofMentalHealth’sPsychoactiveDrugScreeningProgram,Contract#HHSN-271-2008-025C(NIMHPDSP).TheNIMHPDSPisDirectedbyBryanL.RothMD,PhDattheUniversityofNorthCarolinaatChapelHillandProjectOfficerJamieDriscolatNIMH,BethesdaMD,USA.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.

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How to cite this article:LaxNC,ParkerS-AJ,HiltonEJ,SelimanY,TidgewellKJ,KolberBJ.Cyanobacterialextractwithserotoninreceptorsubtype7(5-HT7R)affinitymodulatesdepressionandanxiety-likebehaviorinmice.Synapse. 2018;00:e22059. https://doi.org/10.1002/syn.22059