Orexins cause epileptic activity

Peptides37(2012)161–164

ContentslistsavailableatSciVerseScienceDirect

Peptides

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / p e p t i d e s

Short

communication

Orexins

cause

epileptic

activity

Haydar

Ali

Erken

_,

_Gülten

_Erken

_,

_Osman

_Genc¸

_,

_Selim

_Kortunay

_,

_Melike

_S¸

_ahiner

_,

Günfer

Turgut

_,

_Sebahat

_Turgut

a_{BalikesirStateHospital,Balikesir,Turkey}

b_{BalikesirUniversity,FacultyofMedicine,DepartmentofPhysiology,Balikesir,Turkey} c_{DumlupinarUniversity,FacultyofMedicine,DepartmentofPhysiology,Kutahya,Turkey} d_{PamukkaleUniversity,FacultyofMedicine,DepartmentofPharmacology,Denizli,Turkey} e_{AcibademUniversity,FacultyofMedicine,DepartmentofPhysiology,Istanbul,Turkey} f_{PamukkaleUniversity,FacultyofMedicine,DepartmentofPhysiology,Denizli,Turkey}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received1May2012

Receivedinrevisedform5June2012 Accepted5June2012

Availableonline4July2012 Keywords: EEG Epilepsy Hypocretin Orexin Powerspectrum Seizure

a

b

s

t

r

a

c

t

Orexinshavebeenimplicatedintheregulationofsleep–wakecycle,energyhomeostasis,drinking behav-ior,analgesia,attention,learningandmemorybuttheireffectsonepilepticactivityarecontroversial.We investigatedwhetherintracorticalinjectionsoforexinA(100pmol)andB(100pmol)causeepileptic activityinrats.Weobservedepilepticseizurefindingsonthesetwogroupsrats.OrexinAandBalso sig-nificantlyincreasedtotalEEGpowerspectrum.Ourfindingsindicatethatorexinscauseepilepticactivity. ©2012ElsevierInc.Allrightsreserved.

1. Introduction

The orexins (OXs), orexinA and B, alsoknown as hypocre-tins (hypocretin 1 and 2), are neuropeptides derived fromthe sameprecursormolecule, prepro-orexin,synthesizedin the lat-eralhypothalamicarea[5,31].Orexinergicneuronsprojectwidely tonumerous brain regions includingcerebralcortex, thalamus, hypothalamus, nucleus accumbens, brain stem and spinal cord [4,27,29].OXreceptors(OX1andOX2)areexpressedintheseareas especiallyinthecorticalregions,hippocampus,thalamic, hypotha-lamic and brain stem nuclei [22,40]. It has beenreported that OXsmayplayaroleinvariousphysiologicalfunctionsincluding theenergyhomeostasis[12,32,44],sleep–wakecycle[32], drink-ingbehavior[19],analgesia[25],attention[10],learning[36]and memory[1,15].Althoughitwasshowninnumerousstudiesthat orexinshaveneuroexcitatoryeffect[5,42],therewerefew stud-ies,which investigateorexin–epilepsy relationship[8,18,30]. In a previous study, it was shown that after generalized convul-sions,thelevelsoforexinAdecreaseincerebrospinalfluid[30]. In another studyit has beenreported that orexin A decreased

∗ Correspondingauthor.Tel.:+902662459020;fax:+902662444109. E-mailaddress:haerken@yahoo.com(H.A.Erken).

bicuculline-inducedepilepticactivityaccordingtoinvitro exper-iments[8].Ontheotherhand,inourpreviousstudy,weshowed thatorexinsenhancethecorticalepilepticactivityinducedby intra-corticalapplicationofpenicillin-G[18].Thefindingsofprevious studiesinvestigatingorexin–epilepsyrelationshipare controver-sial [8,18,30] and orexin–epilepsyrelationship is not clearyet. Based onourpreviousfindings wethoughtthatorexins induce epilepticactivitywithoutusing anyepileptogenicagent. There-foretheaimofthisstudywastoinvestigatewhetherorexinscause epilepticactivityinrats.

2. Materialsandmethods

2.1. Animalsandstudydesign

Allof the experimentswere approved bythe Committeeof AnimalCareatPamukkaleUniversityandtheexperimentswere performed according to the guidelines (NIH, UCSF) on animal use. Twenty adultmale Wistar Albino rats weighing243±26g (mean±SD)wereused.Alloftheratsweremaintainedina12-h light/darkcycleenvironment(lightson7:00–19:00h)ata temper-atureof23±2◦Cand50%humidity.Ratshadaccesstofoodand wateradlibitum.

0196-9781/$–seefrontmatter©2012ElsevierInc.Allrightsreserved. http://dx.doi.org/10.1016/j.peptides.2012.06.012

162 H.A.Erkenetal./Peptides37(2012)161–164

Table1

EEGpowerspectrumvaluesofexperimentalgroups.

Group Before 30min 60min 90min 120min

OXA 8.31×10−11_±1.88×10−11 _1.32×10−09_±2.45×10−10 _1.94×10−09_±3.51×10−10 _3.65×10−09_±6.18×10−10a _3.89×10−09_±5.07×10−10a

OXB 5.30× 10−11_{±1.18× 10}−11 _{9.05× 10}−10_±3.08×10−10 _{1.37× 10}−09_±4.13×10−10 _2.19×10−09_±4.61×10−10b _2.53×10−09_±4.08×10−10a

Saline 6.52×10−11_±1.42×10−11 _8.23×10−11_±2.37×10−11 _1.05×10−10_±3.17×10−11 _1.26×10−10_±2.63×10−11 _8.44×10−11_±1.41×10−11

Control 9.90×10−11_±2.85×10−11 _7.67×10−11_±1.97×10−11 _1.20×10−10_±3.51×10−11 _5.18×10−11_±1.87×10−11 _4.24×10−11_±1.09×10−11

Valuesarepresentedasmean±S.D.OXA,orexinA;OXB,orexinB.

a_{p<0.01,differentfrombeforeorexininjection.} b _{p<0.05,differentfrombeforeorexininjection.}

n=5foreachgroup.

Theratswererandomlyassignedtothefollowingfourgroups (n=5foreachgroup).Intracortical(i.c.)orexinA(OXA,100pmol, dissolvedin2␮lsaline),orexinB(OXB,100pmol,dissolvedin2␮l saline)andsaline(2␮l)wasadministeredintotheratsinthegroups 1,2and3,respectively.Group4receivednodrugorsaline. 2.2. Anesthesiaandexperimentalprocedure

Therats were anesthetized with ketamine/xylazine (90 and 10mg/kgrespectivelyi.p.)andtheirheadswereshaved.Then,the ratswereplacedonastereotaxicinstrument(StoeltingCo.,USA) andtheirhead’sweredisinfectedwithbatticon(Batticon,Adeka Co.,Turkey)andincisedfrommid-frontaltomid-occipital.After thebregmawasexposed,aholewasdrilledbyadentaldrilltoa pointthatwasdeterminedtobetheratbrainatlasofPaxinosand Watson[28](frombregma:0.7mmanterior,2.0mmrightlaterally, 2.0mmvertically).100pmolorexinAand100pmolorexinBwere dissolvedin2␮lsalineandwereadministeredtotheprimarymotor cortexbymicroinjector(HamiltonCo.,USA)togroup1andgroup 2,respectively.Similarly,2␮lsalineinjectionwasadministeredto thesameareaingroup3.Aliquotsoforexinswerepreparedand frozenat−20◦_{Cforeachexperimentandthawedanddissolvedin}

2␮lsalineimmediatelybeforeuse.OrexinAandBwerepurchased fromSigma–AldrichCo.,Germany.

2.3. EEGrecordandanalyses

TwoAgClflat electrodeswere placed onthescalp for bipo-larEEGrecording;oneofthemwasplaced ontherightparietal area,andtheotheronthemid-occipitalarea.Agroundelectrode wasplacedonthetailoftherat.EEGwasrecordedbyPowerLab 8/SPdataacquisitionsystemandChart5.2.2program (ADInstru-mentsCo.,Australia).Therecordingparameterswereasfollows: 0.3–100Hzlowandhighfrequencyfilter,50Hznotchfilter,anda recordingspeedof25mm/s.Wheneveradditionalanesthesiawas needed,itwasadministeredtotherats.Theratswereobservedand recordedduringtheexperimentalperiod.Thirtysecond artifact-freeepochswerechosenfromtheEEGrecordingsasthesamplesof corticalactivity,atbeforetheorexin/salineinjectionand30th,60th, 90thand 120thminoforexin/salineinjection. Thepower spec-trumanalysisoftheseEEGsampleswasperformedbyChart5.2.2 softwareprogram(ADInstrumentsCo.,Australia).Spectralpower valuesweretransferredtotheSPSS10.0programandanalyzedby repeatedmeasuresANOVAandPosthocTukeytest.Pvalueof<0.05 beingconsideredassignificant.

3. Results

We observed epileptic seizure findings on thegroup 1 and group2rats,whichwereapplied100pmol(i.c.)OXAand100pmol (i.c.) OXB, respectively. After 25–32min OXs administrations, tonic–cloniccontractionsontheleftanteriorextremitiesoftherats wereobserved.Thecontractionsspreadedtotheleftposterior,right anteriorandposteriorextremities,tailandwholebodyoftherats.

Theseverityofthecontractionsincreasedandcontinuedtothe end-ingoftheexperiments(Until120minafterOXsadministrations). Inthegroup1rats(OXAapplied)thecontractionswereobserved moreseverethangroup2rats(OXBapplied).Howevertherewere noepilepticseizurefindingsinthesalineandcontrolgroups.Also, totalEEGpowerspectrumwasincreasedsignificantlyintheorexin AandorexinBgroups,at90and120minaftertheorexin injec-tionscomparedtothevaluesofbeforeorexinapplication(Table1). Whereas,totalEEGpowerspectrumdidnotsignificantlychangein thecontrolandsalinegroups(Table1).

4. Discussion

In this study, it was shown that orexin applications caused apparentincreaseontotalEEGpowerspectrum.InadditiontoEEG findings,duringexperimentsepilepticactivityfindingsincluding tonic–cloniccontractionsonthewholeextremities,tailandbody oftheratswereobservedbyphysicalobservations.Also,similarly topreviousstudies[6]theeffectofOXAwasmorepotentthan thatofOXBinthisstudy.Ontheotherhandinthesalineand con-trolgroupsneithercontractionsnorchangeinEEGfindingswere observed.

Inthepreviousstudiesusing100pmolorexinAorBepileptic contractionsorepilepticseizurefindingsinEEGwerenotreported [6,7,24,37,38,43].Theremaybenumerousreasonsforthissituation. Oneofthemcanbethelocalizationofthebrainareawhereorexins wereinjected.Inthepresentstudy,orexinswereinjectedtothe pri-marymotorarea.Ontheotherhandinthepreviousstudiesorexins wereinjectedatthesamedosetodifferentbrainareasincluding thebasalforebrain,nucleusbasalis,substantiainnominata, mag-nocellularpreopticnucleus,nucleus accumbens,lateralcerebral ventricleand rostrallateralhypothalamicarea[6,7,24,37,38,43]. Severalfactors(e.g.orexinreceptorsdensity)intheseareasmay changetheeffectsoforexins.Thesecondreasonmaybetheorigin oftheorexinswhichwereprovidedfromdifferentcompanies hav-ingpossiblydifferentefficiency.Inthisstudy,orexinswerebought fromSigma–Aldrich.Inthepreviousstudiesorexinswerebought fromdifferentcompanies(AmericanPeptides,Sunnyvale,CA,USA andPeptideInstitute, Minoh,Japan)[7,24,37,38,43].Thorpeand Kotzreportedthatorexinswhichhavebeenboughtfrom differ-entcompanieshadnosimilareffectsonappetitestimulation[38]. Thereforeorexins,whichwereprovidedfromdifferentcompanies, mayhavedifferenteffectsonepilepticactivity.Thethirdreason maybe,inthepreviousstudies,thesamedosesoforexinsmight haveinducedfocalepilepticactivitybutitcouldnotbeobserved. Inourstudy,becauseoforexins’injectiontotheprimarymotor areawecouldobservephysicallyapparentepilepticactivity.Also, inthedetermination offocalepilepticactivity,appropriateEEG recordingisimportant.Howeverinsomeofthepreviousstudies using100pmolorexin,EEGwasnotrecorded[24,37,38,43].Onthe otherhandinthetwoofthestudiesusing100pmolorexin,EEGwas recordedandchangesonarousalpatternofEEGcausedbyorexins werereported.Butinthesestudies,epilepticactivityfindingsin EEGwerenotreported[6,7].

H.A.Erkenetal./Peptides37(2012)161–164 163 Accordingtosomeotherpreviousstudies,orexinshave

neu-roexcitatoryeffectandmaycausebehavioralconvulsionactivity [5,13,42].Ourresultsconfirmthesereports.

Itisknownthatduringepilepticactivity,balancebetween gluta-mateandGABAreleaseisabolished[2,34].Inthepreviousstudies itwasreportedthatorexinsincreasedglutamate[16,17,41]and GABA[23,41]release.On theotherhandthereareseveral stud-iesreportedthatorexinsdecreasedglutamate[11]andGABA[39] release.Inanotherstudy,ithasbeenshownthatNMDAreceptors arealsorelatedwithepilepticactivity[21].Inrats,theresultsof previousstudiesfor explanationofthemechanismsofepileptic activitycausedbyorexinsarestraightforward.

Anotherpossiblecauseoforexin-inducedepilepticactivitymay bethedirecteffectoforexinsonneuronaldepolarization. Accord-ingtoinvitroexperimentalstudies,orexinscausedepolarization inneurons[33]andincreaseinfiringfrequenciesofneurons[3,9]. Orexinsmayformdirectexcitatoryeffectsonneuronsvia increas-ingtheinfluxofsodium[20],activatesodium–calciumexchanger pump[9],increaseinfluxofcalcium[26,42]ordecreaseeffluxof potassium[14].Theimportanceofintracellularcalciumincreasein termsofneuronalexcitabilityandepilepticactivityisknown[35]. Forthisreason,thismechanismalsomediatestheeffectsof orex-insonthecentralnervoussystem.Hencetheseeffectsoforexins, whichfacilitateneuronaldepolarization,supportepilepticactivity andmayexplaintheincreaseintotalEEGpowerspectruminour study.

Inconclusion,inthisstudy,weshowedthatintracortical appli-cationsoforexinscausedepilepticactivity,butthemechanismsof thiseffectarenotclear.

Funding

ThisstudywassupportedbyPamukkaleUniversityResearch Fund.

Conflictofinterest

Theauthorsdeclarethatthereisnoconflictofinterest.

References

[1]AkbariE,NaghdiN,MotamediF.Theselectiveorexin1receptorantagonist SB-334867-Aimpairsacquisitionandconsolidationbutnotretrievalofspatial memoryinMorriswatermaze.Peptides2007;28(3):650–6.

[2]André V, Marescaux C, Nehlig A, Fritschy JM. Alterationsof hippocam-palGABAergicsystemcontributetodevelopmentofspontaneousrecurrent seizuresintheratlithium–pilocarpinemodeloftemporallobeepilepsy. Hip-pocampus2001;11(4):452–68.

[3]BurletS,TylerCJ,LeonardCS.Directandindirectexcitationoflaterodorsal tegmentalneuronsbyhypocretin/orexinpeptides:implicationsfor wakeful-nessandnarcolepsy.JNeurosci2002;22(7):2862–72.

[4]CutlerDJ,MorrisR,SheridharV,WattamTA,HolmesS,PatelS,etal.Differential distributionoforexin-Aandorexin-Bimmunoreactivityintheratbrainand spinalcord.Peptides1999;20(12):1455–70.

[5]deLeceaL,KilduffTS,PeyronC,GaoX,FoyePE,DanielsonPE,etal.The hypocre-tins:hypothalamus-specificpeptideswithneuroexcitatoryactivity.ProcNatl AcadSciUSA1998;95(1):322–7.

[6]DongHL,FukudaS,MurataE,ZhuZ,HiguchiT.Orexinsincreasecortical acetylcholinereleaseandelectroencephalographicactivationthroughorexin-1 receptorintheratbasalforebrainduringisofluraneanesthesia.Anesthesiology 2006;104(5):1023–32.

[7]DongH,NiuJ,SuB,ZhuZ,LvY,LiY,etal.Activationoforexinsignalin basalforebrainfacilitatestheemergencefromsevofluraneanesthesiainrat. Neuropeptides2009;43(3):179–85.

[8]Doreulee N, Alania M, Vashalomidze G, Skhirtladze E, Kapanadze TS. Orexinergic systemandpathophysiologyofepilepsy.GeorgianMed News 2010;188:74–9.

[9]Eriksson KS, Sergeeva O, Brown RE, Haas HL. Orexin/hypocretin excites the histaminergic neurons ofthe tuberomammillary nucleus. J Neurosci 2001;21(23):9273–9.

[10] FadelJ,BurkJA.Orexin/hypocretinmodulationofthebasalforebrain choliner-gicsystem:roleinattention.BrainRes2010;1314:112–23.

[11]Haj-DahmaneS,ShenRY.Thewake-promotingpeptide orexin-Binhibits glutamatergic transmission to dorsal raphe nucleus serotonin neurons through retrograde endocannabinoid signaling. J Neurosci 2005;25(4): 896–905.

[12]HaynesAC,JacksonB,OverendP,BuckinghamRE,WilsonS,TadayyonM,etal. Effectsofsingleandchronicintracerebroventricularadministrationofthe orex-insonfeedingintherat.Peptides1999;20(9):1099–105.

[13] IdaT,NakaharaK,KatayamaT,MurakamiN,NakazatoM.Effectoflateral cerebroventricularinjectionoftheappetite-stimulatingneuropeptide,orexin andneuropeptideY,onthevariousbehavioralactivitiesofrats.BrainRes 1999;821(2):526–9.

[14] Ivanov A, Aston-Jones G. Hypocretin/orexin depolarizes and decreases potassium conductance in locus coeruleus neurons. Neuroreport 2000;11(8):1755–8.

[15]JaegerLB,FarrSA,BanksWA,MorleyJE.Effectsoforexin-Aonmemory pro-cessing.Peptides2002;23(9):1683–8.

[16]JohnJ,WuMF,KodamaT,SiegelJM.Intravenouslyadministeredhypocretin-1 altersbrainaminoacidrelease:aninvivomicrodialysisstudyinrats.JPhysiol 2003;548(Pt2):557–62.

[17] KodamaT,KimuraM.Arousaleffectsoforexin-AcorrelatewithGLUrelease fromthelocuscoeruleusinrats.Peptides2002;23(9):1673–81.

[18]KortunayS,ErkenHA,ErkenG,Genc¸O,S¸ahinerM,TurgutS,etal.Orexins increasepenicillin-inducedepilepticactivity.Peptides2012;34(2):419–22. [19]Kunii K, Yamanaka A,Nambu T, Matsuzaki I, Goto K,Sakurai T.

Orex-ins/hypocretinsregulatedrinkingbehaviour.BrainRes1999;842(1):256–61. [20]LiuRJ,vandenPolAN,AghajanianGK.Hypocretins(orexins)regulateserotonin

neuronsinthedorsalraphenucleusbyexcitatorydirectandinhibitoryindirect actions.JNeurosci2002;22(21):9453–64.

[21]LoscherW.Newvisionsinthepharmacologyofanticonvulsion.EurJPharmacol 1998;342(1):1–13.

[22]MarcusJN,AschkenasiCJ,LeeCE,ChemelliRM,SaperCB,YanagisawaM,etal. Differentialexpressionoforexinreceptors1and2intheratbrain.JComp Neurol2001;435(1):6–25.

[23]Martin G, Fabre V, Siggins GR, de Lecea L. Interaction of the hypocre-tins with neurotransmitters in the nucleus accumbens. Regul Pept 2002;104(1–3):111–7.

[24]Matsumura K, Tsuchihashi T, Abe I. Central orexin-A augments sym-pathoadrenal outflow in conscious rabbits. Hypertension 2001;37(6): 1382–7.

[25] Mobarakeh JI,TakahashiK,SakuradaS,Nishino S,WatanabeH,KatoM, etal.Enhancedantinociceptionbyintracerebroventricularlyand intrathe-callyadministeredorexinAandB(hypocretin-1and-2)inmice.Peptides 2005;26(5):767–77.

[26] NakamuraY,MiuraS,YoshidaT,KimJ,SasakiK.Cytosoliccalciumelevation inducedbyorexin/hypocretiningranulecelldomaincellsoftheratcochlear nucleusinvitro.Peptides2010;31(8):1579–88.

[27]NambuT,SakuraiT,MizukamiK,HosoyaY,YanagisawaM,GotoK. Distri-butionoforexinneuronsintheadultratbrain.BrainRes1999;827(1–2): 243–60.

[28]PaxinosG,WatsonC.Theratbraininstereotaxiccoordinates.4thed.SanDiego: AcademicPress;1998.

[29] PeyronC,TigheDK,vandenPolAN,deLeceaL,HellerHC,SutcliffeJG,etal. Neuronscontaininghypocretin(orexin)projecttomultipleneuronalsystems. JNeurosci1998;18(23):9996–10015.

[30]Rejdak K,Papu ´c E, Grieb P, StelmasiakZ. Decreased cerebrospinal fluid hypocretin-1(orexinA)inpatientsafterrepetitivegeneralizedtonic–clonic seizures.Epilepsia2009;50(6):1641–4.

[31]SakuraiT,AmemiyaA,IshiiM,MatsuzakiI,ChemelliRM,TanakaH,etal.Orexins andorexinreceptors:afamilyofhypothalamicneuropeptidesandG protein-coupledreceptorsthatregulatefeedingbehavior.Cell1998;92(4):573–85. [32]SakuraiT.Rolesoforexin/hypocretininregulationofsleep/wakefulnessand

energyhomeostasis.SleepMedRev2005;9(4):231–41.

[33]ShirasakaT,MiyaharaS,KunitakeT,JinQH,KatoK,TakasakiM,etal.Orexin depolarizesrathypothalamicparaventricularnucleusneurons.AmJPhysiol RegulIntegrCompPhysiol2001;281(4):R1114–8.

[34]Sitges M, Sanchez-Tafolla BM, Chiu LM, Aldana BI, Guarneros A. Vin-pocetine inhibits glutamate release induced by the convulsive agent 4-aminopyridinemorepotentlythanseveralantiepilepticdrugs.EpilepsyRes 2011;96(3):257–66.

[35]SpeckmannEJ, StraubH,Köhling R. Contributionof calciumions tothe generationofepilepticactivityandantiepilepticcalciumantagonism. Neu-ropsychobiology1993;27(3):122–6.

[36]TelegdyG,AdamikA.TheactionoforexinAonpassiveavoidancelearning: involvementoftransmitters.RegulPept2002;104(1–3):105–10.

[37]ThorpeAJ,ClearyJP,LevineAS,KotzCM.CentrallyadministeredorexinA increasesmotivationforsweetpelletsinrats.Psychopharmacology (Berl) 2005;182(1):75–83.

[38]ThorpeAJ,KotzCM.OrexinAinthenucleusaccumbensstimulatesfeedingand locomotoractivity.BrainRes2005;1050(1–2):156–62.

[39]ThorpeAJ,DoaneDF,SweetDC,BeverlyJL,KotzCM.OrexinAintherostrolateral hypothalamicareainducesfeedingbymodulatingGABAergictransmission. BrainRes2006;1125(1):60–6.

[40]TrivediP,YuH,MacNeilDJ,VanderPloegLH,GuanXM.Distributionoforexin receptormRNAintheratbrain.FEBSLett1998;438(1–2):71–5.

[41] vanden PolAN, GaoXB,ObrietanK,KilduffTS, BelousovAB. Presynap-tic and postsynaptic actionsand modulation ofneuroendocrine neurons

164 H.A.Erkenetal./Peptides37(2012)161–164 byanewhypothalamicpeptide,hypocretin/orexin.JNeurosci1998;18(19):

7962–71.

[42]van den Pol AN, Ghosh PK, Liu RJ, Li Y, Aghajanian GK, Gao XB. Hypocretin(orexin)enhancesneuronactivityandcellsynchronyin devel-oping mouseGFP-expressing locus coeruleus. J Physiol 2002;541(Pt 1): 169–85.

[43]Wang J, Osaka T, Inoue S. Orexin-A-sensitive site for energy expendi-ture localized in the arcuate nucleus of the hypothalamus. Brain Res 2003;971(1):128–34.

[44]YokoboriE,KojimaK,AzumaM,KangKS,MaejimaS,UchiyamaM,etal. Stim-ulatoryeffectofintracerebroventricularadministrationoforexinAonfood intakeinthezebrafish,Daniorerio.Peptides2011;32(7):1357–62.