entr exinn

KATPchannel-depend entregulation of or exinnell ron s

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Thehypoth alamusperform sman yfunctionsthat arevitalto anorganism's surv ival. Theseinclu de,butarenotlimitedto,theregulationandcoordi nation ofbasic functions suchas energymetabolism,thesleep-wake cycle and motivation.Asobesity, sleepdisord ersand/oraddiction sc anresultfromthed ysfunctionofspeci fic neu ral systemswithi n thehypothalamu s,it isimportanttounderstand the end ogcnou s factors thatregulatethe ir activity.Neurons containingthe orex in neu ropeptidesa rclocated exclus ive lywithin thehypothalamus,sendexcitatoryprojectionstoarousal-andreward- related brai nregions and havebeen implicated innumerou sphysiologicaland behavio ral function s includi ngfeeding, sleep-wake reg ulation and reward andadd iction.

Neighbouring neuronscontainingmelanin-concentratinghorm one(MCH)alsoproject through outmuchoftheneuroaxisandare im plicatedinsimilarfunctions.lnt hepresen t thesis,I use electrophys io logicalrec ordingsfrom acute hypothalam icslicesasthemain techniqu etoinvestigatesomeof the endog eno usregulato rsofo rexinand MCH neuron s.

Thepresent thesis shows thatnociceptin/orph aninFQ(N/OFQ),anendoge nousopioid.as wellaslactateand temperatureallactasregulators of orexinneuronactivity.

Interesti ngly,they allsha rea similarmechanism whichinvolvesthe A'I'P-sensitivc potassium(KA TP)channe l.Thesecha nnelsaremetabolically-sen sitive,are composedof aunique combinationofsubunits inorexi nneur ons andlikelyr epresentmajor contributo rs tothe determ inationoforexin neuronactivity.lnco ntras t, MCHnc urons werehyperpolarizedbyN/OFQduetotheactivationof G-protein dependentinwa rd ly rectifyingpotassiumchannels,whilebe inginsensitivetotemp eratu rechanges.With regards tobeh avior,local injectionofN/OFQ within theorexinand MCHfieldin vivo

inhibitsreward-relatedfeedingwhereasthe temperatureregulation of orexin neurons appca rs to med iate the hypo phag ia assoc iated with fever. Th rough the identificatio no f orexin neurons as both tempcraturc and cnergyse nsors, it is suggcsted bythepresent thesisthat theseneuronscangate brain activityaccording to energy suppl ywh ilc possess ing thc ability toa dapt to thci ncrcased tem pcraturesassociatcdw ith infection.

These data shed ncw light on thefunction ofthcscneuronal systemsand introducc thc KATPchannel asa criticalregulat or of orexin activit y.

Ack nowlcdgements

Firstofall. I wouldlike tothank my supervisor. Dr. Michiru Hirasawa .Youhave provided me with anexcellent researchenvironmentin which to completeathesis and withampleopportunities topresentmywork atnat ional and international conferences.

Youhave guided. encoura ged andchallenged me for the lastnumber0Iyearsina way which hasfullyc onfinnedmy interestin pursuing a career in research.I cann otthankyou cnou ghforthis.

ThankstomyformerMSc supervisor Dr. Gilbert Kirouac.Your initialguidance wascriticaltothedecisionsthatIhavemadc.

Ialso thank themembers of mysupervisory committee.Drs.XihuaChen and JohnMcLean.Your input into my work has alwaysbeengreatlyapprecia ted.

I thank Christia nAlbertoforhistechnicalassistance.You have hclped meinso manywaysand havemade mytimein the labsignificantly more enjoyable.Iconsider youto be agoodfriend rather thana co-workerand wish you all thebest in thefuture.

Ialsothankmy fellowlab mates.in particularmygoodfriendsRobert Traskand MichelleQuinlan.

Many thanks gotothose outside thelab whose support andguidance havebeen invaluab le tomyprogress.I wouldlike to specifica lly thankmyparents.Don and Marie.

aswellasmybeautiful wife.Amy.

I thankboth NSERCand the Heart and StrokeFoundati on of Canadafor supporting mefinanciall y throughout mygraduate work.

Acknowledgements-- -- -- --- -- - · ··· · · - · -- -- -- --- - - ...••...• .iv

ListofFig ures ---- - - - . - .... . •--- -- -- -- ---•• • --- - - - ---•.. .• .-xii

ListofAbb reviat ions••••. •--- - ---....--- ---- --- - -•• . . .-- - - -- - xvi

Co-aut horshipstateme nt--- - - -- ...•••.---- -- - - ----.. .. •• --- ---31

Chapter I Introdu cti on and Overvi ew 1.1 TheHypoth alamus --- ••... --- - - --- --- ---... --- - - -- - -..I 1.2 Neurope ptides ofthelateral hypothalamus/perifornicalarea-- --- -- -·4

Theorexin s -- - - --..••.. --- -- - - -- . -...•• --- --- - --- - 5

Theorexin s andenergyhomeostasis --- - - - - ··· ···- -6

Theorexinsandthe sleep-wakecycle-- -- - - ---·· · · -··7

Theorexinsand rewardladdiction---- ....--- -- - --- - ----.8

Otherfunctions of theo rexins-- - - -- ----••.•. --- --- --9

Endogenous regulatorsoforexin neurona ctivity ----··· ··I I Overallfun ction ofthe orexins- --··· - - -- --- - - ·· · · -·13

Melanin-concentr atinghormone (MCH) -- --- - --.•• --- - - 14

MCHand energyh omeostasis--- --- - - · · · - - - -- ·14

MCH andthe sleep-wakecycle--- --- ---- -- - - ---····-- - -·15

Endogenous regulatorso fMCHneuronactivity- --- -- 16

Summaryoftheorexin and MCH systems --- -- - --- - - - -17

Nociceptin/Or phaninFQ- --- --- ---- -- -- - - - -- -- -- ---- -- - - - --- - - - -18

1.-1 Rat ionale andobjectives (relevantto cha pters2and 3)--- - --- --- -20

I.S KAT Pc ha nnels-- -- - ---- - --- - --- - -- -- -- - ---- -- --- - -- - - --- - --21

OverviewofK ATPchannelstructureand function--- -- - - --21

NeuronalGlucosensing- -- - -- - --- --- --- -- --- ---23

Glucosensingin orexinneurons--- --- -- -- --- ---- - -- - - 24

Temperaturesensingneurons-- --- -- --- --- --- - - - --- - ---- --- - - - 25

Rati onale andobjectives(releva nt to chapters-land S) --- --- - - -26

1.7 Objectives summary- - -- - --- --- -- -- -- --- -- -- -- --- - --- --- -- ---27

Cha pter 2 GIR Kcha nnel-med iated inhibition of melanin- concentr at inghormone neuron sbynocieeptin/orphanin FQ (Manuscriptpublished intheJournal ofNeurophysiology) Introducti on--- - --- -- - -- -- - - --- --- --- - - - --- - - 33

Materia lsa ndMethods --- - - --- ---- - -- --- ---- - - --- -- - ---35

Electrophysiology- - --- - -- -- ---- -- --- - - - --- -- ---3 5 Post-hocimmunoOuorescence- -- --- -- --- - --- ---- --- - --37

Dataanalysis --- - -- --- -- - --- --- --- - -- - -- ---38

Drugs -- --- - --- ---- --- - - --- -- -- -- -- --- - --- - - --38

Chapter 3 Effects ofG- pro teinco upled recept or activation by Nociceptin/Or pha ninFQi n orexi n neuro ns (Manuscr ipt in preparati on)

Surgerya nd behavioraltesting- - -- --- --- - --- --- - 55

Electrophysiology --- --- --- ---56

DataAnalysis---- --- - --- - -- --- ---- --- --·58

Drugs - --- - - - - --- - - - -- - - -- -- -- - - --- --- --- --·58

Effectofintra-LH/PFAN/OFQonpalatablefoodintake--- -- ---- 58

N/OFQeffectonrator exin neurons -- --- - --- - --- - - ---- - -- ---60

N/OFQa ndfoo d intake--- ---- -- - - --- - --- ---- - --- -64

N/OFQ-induced inhibitionof orexinneurons--- - - ---- - - -- --- - 66

PKC· dependent activation of KATP channelsby N/OFQ- ---.---67

Chap ter 4 A'I'Pcsens itivepotassium cha nnel-med iated lactate effectonorcx in ncurons:implicationsforbrain cncr gcticsduring arou sal (Ma nuscr ipt puhlishcd in thc. Journa lo fNc uros cicnce) Electrophysiology-- - - --- - - -- -- - -- - -- - - --- - - - -- -- - ---- - 80

Data analysis --- - - · ·· · ··· · ·· ·· ·- - -- · ··-·- -- - -- - --- ----83

Drugs -- - -- --- - - ---- - - - -- --- - - -- --- - - - --- -- - ---- - ---84

4.3 Result s- ...--- --- - - - -- -- -- - - ---- -- --- -- - - -.--- - --- --·85

Orexinneur ons selectivelyutili zelactatetofuel spontaneousa ctivity - - -- -- -- - - - --- - - - - --- - - - -- -- - - - -- - -- -- 85

KATPchannelsm ediatel actate sensing--- --- --- - - -- ·87

Kir6.1and SUR1subunits comprise KATP channels glucose· ..·· · -·-- - -.- - ··- -..---· ···..-···-- ---·- - ---- -- - --91

Short-tennglucoseeffect - - --- - - - - -- --- --- - --- --- ---- - - 92

Orexin neuronspreferlactateoverglucoseas an energy substrate- - - --- - - - --- - -- -- -- ---94

Technical considerations---- --- - - - --- - --- - - -- - - · · . - . --95

Considerationof discrepanciesin the literature--- --- --- - - ---- --96

KATP channelsmediate energy sensing inorexinneurons -- -- ---97

PhysiologicaI Signiticance ·- -·- - - - ---- - -- ·- -- - -·--- - - --- 98

Cha pter 5 KATP-dependent thermosensitivityof orexin neurons contributesto lipopolysaccharide-induced anorexia (Manuscriptin preparation) Electrophysiology---- - --- --- ---- - -- -- --- ---118

Tracing --- -- ---- - -- -- --- --- -- ---.-....-- .. ---119

Telemetry--- - · - ·· -· -- ·- - · ···--- - ··- - ·-- - · ·- -- - - --- - -.- ---120

DataAnalysis-- --- - - -- -- ---- - - - ---- - -- -- - -- --- - --- -- - -121

Drugs - - --- - --- ---- ---- - --- - - ·-- - - ----· - - - - · - · · · · - -.- . - . -122

5.3 Result s--- -- - ---- - - --- - - ---- ---- - - -- -- --- -- --- - - -122

Orexin neurons aretemperature-sensitive---- - - --- --- -- -- ---122 Increased temperature activatesATP-sensitivepotassium

Temperaturesensit iveorexin neuron s include those proje ctingto

Temperature-indu cedhyperp olarizationin theLH/PFAis specific to orex in neurons -- -- - --- - --- - --- - - ---- - - --- - - --- 126 Orexinthermosen sitiv ityi simplicatedin LPS-induced

Temperature-inducedinhibition of orexin neurons

Ch a pte r6 Sum mary

Brief summ ary ofthem ainfind ings-- - - -- -- --- - -- - -- - -- - - --- --- 146 Implicati on s ontypica l patchclamp experimenta l

protocols- ---- - - -- -- - - --- - - - --- - - -- - - -- --- - -- -- --- -- -147 N/OF Q-ind ucedin hi hitio noforexinand MCHneu ron s --- -- - - ---148

Future directions:Investigatingwhetherthe sustainedeffect ofN/OFQo no rex in neurons is physiologically-relevant--- ---149 Future directions:FUltherex plorationo fN/OFQ-induced hypophagia--- --- -- --- - --- - - -- --- - - --- -- --- - - - --151

Future directions:Additionaleffectsof intra-LH/PFAN/OFQ -- --152 6.4 KATPc ha nnelsascritiealregulat or s oforexin neu ron function---- --152

Future directions:Role ofK ATP channelsinorexin

6.5 Dat adiscrepancy - --- --- --- -- - - --- -- -- ---- - --- -·155 6.6 Concludi ng remark s --- - --- - --- ---- - --- -- - -- - - ---- -- -- ----156

ListofFigu res

Figurc !. l Overvicw of somc oflh c criticalproj ect iont argets a nd rclatcd functionali mplicatio nsof theo rex insystem.

Figure 1.2 Ovcrview of som e ofthe critica lendoge nousregulatorsofthc orex insys tem.

Figure 1.3 Overview of KATPchannel regulationin thepancreati c

Figure 2.1 Identificationof MCHneuronsinvitro.

Figure2.2 N/OFQhyperpolarizesMCHneurons.

Figure2.3 N/OFQinducesa NO P receptor- mediated outwa rd

Figure2.4 N/OFQactiva tesG-protein dependent inwardly rectify ingpotassium (GIRK)channelsin MCHneurons.

Figure2.5 N/OFQeffect dcsens itizesandoccludes dynorph in effect.

Figure 3.1 Identificati on oforexin neuronsin vitro.

Figu re3.2 Intra-LH/PFAN/OFQdecreasespalat ablefoodintake.

Figure 3.3 N/OFQinhibits orexin neuron svia NOP receptor activatio n.

Figure 3.4 N/OFQeffectsusingdifferentintern al solutions.

Figure 3.5 N/OFQ-inducedorexin inhibitionisdependent on PKC

Figure 3.6 N/O FQ has noe ffecto n excitatorysy naptic transmission

Figure 4.1 Lactate is necessaryandsuffic ienttomaint ainbasallevel s of spontaneo us activityinorex in neurons.

Figure 4.2 Astrocyte s suppl ylactate to suppo rt the spontaneo us activity oforexin neuron s.

Figure4.3 KATPchannel smediatehyperpolari zationinducedby a lack of energysubstrate .

Figure 4.4 KAT P channel composi tio ninore xin neuro ns.

Figure 4.5 Orex in neurons are lactatesensors.

Figure 4.6 Orexi n neurons arelessactive inlow glucose

Figure4.7 Lactate increasestheexcitabilityoforexinneurons.

Figure 4.8 Short-termand long-term effectsof variousglucose

Figure 4.9 Lack of consistentshort-termglucoseeffectsonaction potential firing.

Figure4.10 Recordingmethodsdonot affect the baselinefiring rate

Figure4.1 I Orexinneuronsaslactate sensors.

Figure5.1 Orexin neuronsare tem perat ure-sensitive.

Figure 5.2 Orexin neuronsare intrinsicallytemperature-sensitive.

FigureS.3 PGE2doesnotdirectlyintluence orexinneurons.

Figure S.4 Temperatureactivates KATPchannelsinorexin neurons.

FigureS.S KATPch annelsm ediate orexinn euron thermosensitivity overa physiologicalra nge

FigureS .6 Orexinneur onspro jectingt oth el ocus coeruleus are temperature-sensitive

FigureS.7 Neighbouring MCHand unknown 3'dpartyneuronsdonot display orexin-like thermosensitive properties

FigureS.S Blockade oft emperatllre-induced orexinn ellron inhibitionpartiallyalleviatesLPS-inducedhypophagia.

FigureS.9 Orexin activity in differenttemperatures

4-CI N,alph a· cyano-4-hyd roxycinnamate;

5-HT,5 -hydrox ytriptamine(serotonin);

ACSF,a rtificialce rebrospinal fluid;

ACT H,adre nocorticotropic hormone Arc,arcuate nucleus;

BF,b asalforebr ain;

CA,ce ll.a ttached;

CalC,calphostinC;

CCCP;carbo nylcya nide m-chlorophenyl hydrazone CeA,central nucleusofthea mygda la;

CRF,corticotro pin-releas ingfactor;

CSF,cerebrospina l fluid;

CTL,co ntro l;

CWC,conventional whole-cell ; DA,d opam ine;

DMN,dorsom edial nucleusofthe thalamus;

DR,dorsal raph e;

DYN,dynorph in;

Dz,d iazoxide;

FAC,fluoroacetate;

GABA,gamma-aminobutyric acid;

GE, glucose-excited ; Gl.glucos e-inhibited;

GIRK,G-protein dependent inwardlyrectifyin g GLP-l , glucag on-Iikepeptidel ; Glib,glibenclamide;

GLU,g lucose GLUT,glucose transpo rter;

HE,hi ghenergy;

HI'A,hypotha lamicpituitaryadrenal;

IML,intermed iolateral cell column;

KATP,ATP-sensit ivc potassium K-Glu,potassiumgl ucona tc;

LAC,lactatc LC,locusc ocrulcus;

LDTg,latcrodorsal tegmentalnucleus;

LE,l ow encrgy;

LH,lateral hypothalamu s;

LPS,lipopolysacch aride;

LT,long term;

MCH ,melanin concentrat ingh orm onc;

MCHR I,melaninconcentratin ghorm onereceptorI;

mEPSC,m iniature excitat oryp ostsynapt ic curr ents;

met-Enk.rnet-Enk ephalin.

mIPSC,miniatureinhibitory postsynapticcurrents;

acSh,nucie usa ccumbe ns shell;

E,norepinephrine;

N/OFQ ,nociceptinio rphaninFQ;

NOP,noc iceptino pioid peptide;

NPY,Neurope ptideY; ORX,orexi n;

PFA,perifomic alarea;

PGE2,prostaglandin E2;

PKC,proteinkinase C;

POAH ,preopti canteriorhypoth alamu s;

POMC,proopiomel anocortin : PTX,picrotoxin;

PVN,p araventricul arnucleusof the thalamu s;

PVT,paraventricular nucleu sof thethalamu s

REM,rapideyemovement ST,sho rt terrn;

SUR,s ulphonylurearece ptor;

'I'M,luberomammillary nucleus;

Tol,tolbuta mide TQ,tertiapin Q TRH,thyrotro pin-releas inghorrnone;

TTX,tet rodo tox in;

VTA,ventral tegmentalarea;

WC,whole-cell.

Chapte r I

1.1 Thehypoth al amus

The hypothalamus maintains homeostasis by interactingwithendocrine, autono micand motivat ional systems.Thesignificanceof the hypothalamus cannotbe overstatedasthisrelatively smallareaofbraintissue critica lly regulatesene rgy hom eostasis,th e sleep -wake cycle,the stress-response,bodyt emp emtu re,h ean rate, bloodpressure,blood osmo lality,motiv ated behav iorsand rep redu ct ion,amo ngstothers.

Overall ,th eh ypothalamu sintegratesinfoml ationrelatedto an organism'sinternal and externalenvironment s,respondstothis convergent inform ationrapidly, andsends its output totarget systems whichinfluence thevery environmentalfactors thehypoth alamu s respond edto in the firstplace.Many ofthe processesmed iatedbythehypoth alam us are regulat ed according to a setpoint;adesired basalvalueofa controlled physio logica l variable. It isthe roleofthehypothalamu sto detect thepolarity and magn itude of any deviationsaway froma panic ularsetpoi ntandtoi nducea nappropriatehomeostatic response.Bodytem peratu re andfluidosmolalityaregood examplesof physiological processesthat areregulatedina hypothalamic-depe ndent manner according toa sci point

The anatomyofthehypothalam us is criticalto its homeosta ticfunction.ln terms of inputs,bothsynapticand humomIinformationconvergewithinthehypothalamus.

Mono- andpolysynaptic projectionsexist that connect much of theneuroaxis to the

hypolhalamu s andi l islh rough suchproj ecl ionslh allh eh ypolh alamu s rece ivesinpul fromolfactory,visualandvisceralsensorysystems aswellas frommultimodal brainstem afferentsandemotional limb ic regions.Humoralfactors can alsoreach thehypothalamus lhroughmedialedtransportac rosslheb lood-brai nba rrierorv iac ircumven lricularorgans;

selectregion s ofthe brainthatlack asignificantblood-brai nbarrier.Many of'these humoral factorsare themselve sinfluencedbyhypothalamic activity and thusprovidethe necessary feedback10thehypothalamusthat is critical10homeostasis.

Hypolh alamicoulpUloccu rslh roughlhree well-established roules:(I ) Neural inforrnalionfromthehypothalamuscan beconverted10humoral signals whichactupon targetperipheral organsystems, In thiscase, spec ific hypothalarnictransmittersare releasedintothe portalcapillarysystemin themedian eminence wheretheyare carriedto lheanleriorpiluilarygland .lnlhepiluilary,lheselransmiller s act10stimulateor inhibit lherelease of ac orre spondin gh orrnoneinlolhebl oodslreamwhiehcanthenact upon speeifie largelo rgans;(2) Neurolransmillers from lhehypolhalamus canbereleased directl y intotheblood stream via the poste rior pituita ry.This system.thehypothalam ic- neurohypophysealsystem,consisls of oxyt ocinand vasopressinneuronsin thesuprao ptic and parave nlricularn uclei projeetingdirecllYlolheposleriorpiluilarywherelheir conle nls arereleased inlocirculalion.Thesepepl idesarec ril icallyinvolvedin osmo regulation(vasopressin) and lactati onin females(oxyt oein); (3)Oulpulfromlhc hypothalamuscanalsofunctionindependentlyof the pituitaryglandvia extensive projectionsthroughout muchof theneuroaxisincludingaulonomicand motivational systemsin thebrainandspinalcord.Thus,byintegrating ahost of inforrnation regard ing anorga nism' sinternal andexternalenvironmenlsandby communicatingwiththebrain

and periph erythrou gh synapticand humoralmeans,thehypothal am usiswell-suitedfor itsrolein theregulationof physiology and behavior.

Thehypothal amusconsistsofclustersof specificneuronalphenoty pes with distincttransm ittersystems,proje ctiontarget s andendogeno usregu lators.Asreflected by the differe ntoutp utpathwaysdescribedabo ve,the func t ionof thehypoth al amu sisnot carriedout by thehypot halamusasa whole,butratherbythe actionsofmultiplediffe rent neuronal ensemb leswhic heachsubservespecific funct ions.Theseneuronal ensemb les havethedaun tingtask of integrat ingahost of centra land peripherali nformationwh ile sendingapp ro priateresp onsesto auto no mic, endocrineand/oremotion/motivati on systems that are essentialtoanorganism'ssurv iva l. Forexam p le,calorichomeo stasis is maintai ne dbytheactions of mult iplesat iety signa lsonavariety ofana bo licand cata bolic hypoth alam ic neuro na lensembleswhose outputsultimat elycontrolsubsequent feeding beh aviour andenergy expe nd iture.Ga in ingafullunderstand ing0fthercgulatory mechanisms of hypotha lamic funct ionscould,intheory,allev iatecount less diso rd ers includ ing obesi ty,addict io n,anxietyandslee p disturbancestonam ea few.Suchale vel of understandin g requiresanappreciation ofnot onlythe effectorsystems upon whichthe hypothaiamLISact sbutalsothepreciseendogenou sregulato rsthatinfluence theact iv ity ofspec ifichypothal amicneuronalnet w orks. The latt er isthefocu softheprese ntthesis . More specifically,thisthesis invest igates theregu lati onof twomajorneuro pe ptidc systems that havev irt llallyco meto definethelateral hypothalamus(L H):the or exins and melanin conce ntrat inghormone(MC H).

1.2 Neu rope ptidesof the latera l hy pot ha la m us/per ifo rn iea l a rea The LHwasidentified asthe brain'sfeedin g centreover50 yea rsago.Dram atic hypophagiaand weight loss was observed followingLH lesionswhileelectrical stimulationof thisregioncouldinduce feedingbehaviour (Stellar,I994:Hetheringtonand Ranson,I940:Anandand Brobeck,195 1).ltis no w known that thei deaof a prec ise

"feeding-centre"is outdated(FlierandMaratos-Flier,1998)and thatfeeding is eontrolled bythe coordi natedactions ofa growing numberofneuropeptidesandhorm ones actingin anumberofdifferentbrainareas (Schwartzeta l.,2000).N onetheless,the roleo fthe LlI inthe contr olof foodintakeand energyhomeostasisisundisput ed.Furthermore , self- stimulationsites within the LH werefoundto significantly overlapwithsitesthat prom otedfeedin gbehavioruponstimulation(MargulesandOlds,1962),suggesting a roleofthisregion in morethan justthehomeostatic controloffoodintake.Based upon lheassociation ofth eLH withobe sityand,m orereeentl y, addicti on,it is ofinterestto determ ineth e relevantneuronal phenotypesthatexist withint hisareaand howthese neuron sareregulatedonacellular level.Importantly,overthe last decade or so.ithas

act ion s ofthe twoknownmajorneuropeptid e systemswhose cellbodies residemore -or- lessexclusivelywithinthis region ofthe hypothalamus:theorexinsystem, resid ing inthe lateral hypothalamu s and perifomicalarea (LHIPFA)andthe MCH system,resid ing in lhe LH/PFAandzona incerta.Rou ghly10- 15 yearsof researchonthesetwopeptid e systems haveshownpromisingresultsforthe generationofnot onlyanti-obes ity pharm aceuticalintervention sbutalso medicationsthat canalleviatestressandanxiety, drug add ictionaswellas sleep disturbances,amongst others. Given themultiple rolesof

theseneuropeptide s, aswill bediscussedbelow,itis of utmostimportancet ou nderstand howthey are regulated ata cellular level.Theref ore,thefocusof thepresent thes isison theregulati on ofthe sen euronalphenotypes,with emph asis onthe orexin system .

Theo rexin neuropeptides, orexi n-Aa ndorexin -B,w ere discovered simultaneouslybytwo inde pendentgroupsin 1998 (Sakurai eta!.,I998;de Lecea L.et al.,J998).Onthebasis oftheirstructural simil arityt o secretin andtheirhypoth alam ic localization,de Leceaeta!. namedthe peptidesvhypocretin- l"and"hypocretin-2".On theother hand.Sakurai eta!. named thepeptides"orexin-A" and"orexin-B"due totheir abilitytoinduce feedingwheninjected centrally.Forthe sake0fsimpli city,thepresent thesiswill referto theseneuropeptidesastheorexins.

Theorexins are cleavedfromasingle precursorpolyp eptid eprepro-orexin and bind totwoG-proteincoupledrecept ors,termedorex in recept orIando rex in rece ptor2.

Orex in receptor1iscoupl ed to Gqproteinswhile orexinreceptor2hasbeen observedto be able to coupleto eitherGqorG.oproteins (Zhueta !.,2003) . A hhoughprepro-orex in mRNA andorex inpeptideexpression is found localizedtothe LH/PFA.orex in varicositiesando rexi nrece ptorexpressioncanbe found throughout thebrain (Peyronet al.. 1998;Trivedieta l.,1998). Forexample, hea vyorexinproj ect ions arefoundinmost majorarousa l-re latedcellgroupsinclud ing thelocusc oerul eus,raphe nuclei and basal forebrai n.Withinthehypothalamu s, orex in proj ectionsareseen inregions such asthe ventromedia l, tuberomammill ary, arcuateand preopt icnuclei.Orexinfibersalsoappear in lowdensity,but diffusely,throughoutthe cortex.Such projectionpattem shave

implicatedtheorexinsystem in a widevariet y of physiologicalfunct ions incl udingthe regulat ion of energybalanc e and the sleep-wa kecycleaswellasreward andaddiction.

1.2,1.1Theorexi nsandene rgy hom costasis

Considering their restrictedlocalizat ion within the LHIPFA, it isnot surprising thatmanyearly studieson thephysiologicalroles ofthe orexins focu sed onfeed ing behavior .Duringthelightphase,injection s of ore xin-A orBintothelateral ventricle dose-dep endentl yinducedhyperph agiawithin I hourpost-injecti on,with the clTcctof orexin.A lasting longer than thatof B( Sakurai eta l., 1998).The hyperphag icetTcc tofthc orcx insco uld befurtherdemonstratedbyinjectionsdircctlyintotheparavcntricular nucleus,dorsomedial nucleus,LH/PFAornucleus accumbens (Dubeet aI.,I999;Sweetet al., 1999;Thorpe andKotz, 2005),s uggesting criticalsites of action.An interactionwith theorexigenic neuropeptide Y (N PY)n euronsisdemonstrated byth e findingthat orex in-

ind uced feedin gisblockedby a NPY receptor antago nist (Jaineta1., 2000 ).

Additionald ata supportarole oftheend ogenous orexin system infeedin g behavior . Fastingis associatedwithan upregulation of orexin mRNA (Sakura ictal., 1998) andpeptide (Parketal., 2004) asw ell as an increasein c.F os expre ssionin orexin neurons. Administrationof anorexinreceptorantago nist decreasesfoodintakc( lIay ncsct al.,2000) and micewitha specific ablat ion of orex in neurons arehypophag ic,althoug h theydodevelop late-onsetobes ity(Haraetal.,200 1).Thisisexplainedbythe fact that thcorex ins noto nlys timulatcfood intakebutalsoenergy expenditure,

1.2.1.2Theorex insand the sleep-wa kecycle

maintenance of wakefulnessand thestabilizationofthesleep-wake cycle.The importance oforexin neuronstosleep-wakeregulation is evidencedbythen arcolep syth atresult s fromalossoforexin neurons.Injustoneyearfollowin gthe discoveryoftheorexins, it wasrecognizedthat canine narcolepsythathadshowed upsome 30 yearspreviouslyhad resulted fromamutationofth e orexinreceptor 2 gene (Linet al.,1999).Subsequently, symptoms thats trikinglyresembled humannarcolepsy could beseenino rexinneuron ablated(Haraetal.,200I) and knockoutmice (Cheme llietal.,1999).Narcoleptic patients have 10wer CSFlevelsoforexin thancontrols(Nishinoeta l.,2000)which is caused by theselective death of orexinneurons(Thannickaleta l.,20 00;Crockerctal.,200 5).

Narcolepsyischaracterizedbythe intrusionof rapid eyemovement (REM)sleepinto wakef ulness,fragment edsleep/wake cyclesand theinab ilitytomaintain vigilance.The narcolepticphenotypeinorex in-compromisedanima ls suggeststhattheendogenous orexinsyste m playsa criticalrolein themaintenance of wakefulnessandaidsinthe stab ilizationofsleep/wakestates.lnterestingly,like orexina blated mice( Haraetal., 200 1),narcoleptichumans have ahigher bodymassindexdespitelower caloricintake (Schulde ta l.,2000;Lammersetal.,1996),furtherco nfirming theroleplayedby endogenous orexi n neuronsin boths leep/wake rcgulationa ndenergyhomeostasis.

Theo rex inshavebeenshown tohave anexcitatory etTecto n thearousal-related noradrcne rgiclocuscoeruleus(Haganet al.,1 999;Horvathet al.,1 999), serotonergic dorsalraphe(Liuetal.,2002).histaminergictuberomamm illary nucleus (Yamanakaet al., 2002) andcholinergicbasalforebrain (Eggerma nn eta l., 2001).Thus,itisnot

surprising thatcentral orex inadministration increasesarousa landlocom otor acti vity (Haganetal.,1999).Studiesusingc-Fosa samarkerof neuronalactivityhave demonstratedthatorex inactivityinratsisincreasedduringthedark phase,whenarou sal level ishigh (Estabrookee ta l.,2001).Insupportofthis,in vivorecordi ngsfrom unanesthetized,unrestrained ratsshowthatorexin neuronsare mostactiveduringactive waking,less active duringquietwakingandvirtually silent duri ngsleepsavefor afew burstsin REM sleep(Mileykovskiyetal.,2 005).Moreover , optogenetic controlover orexin neurons usinglentiviral-mediated expressio nofthelight-sens itive channelrhodo psin-2 ino rex in neuronsdemonstrated thats pec ificactivationoforex in neuronsat 5hertzorgreatersignifica ntly decreasedthe latencyfrom sleep toa waking state (Adamantidiset aI., 2007).Thus,ample evidenceexistsdemonstratin g an undisputableroleof the orex ins in the maintenance of wakefulness and the contr ol ofthe sleep/wake cycle.

The mesolimb icdopam ine system,consisting ofdopam inergicprojectionsfrom the ventra l tegmental area(VTA)tothenucleus accumbensandprefronta lcortex,is consideredtobethebrain' sreward system.Drugs of abuseallactto increase dopamin e release and addic tion isassociatedwithplastic changes within this system(Kauerand Malenka,2007).A roleoftheorex insin rewardand addic tionissuggestedbytheirdi rect excitatoryeffecton dopamin eneurons ofthe VTA(NakamuraetaI., 2000; Korotko vaet al.,2003 )and was firstconfirmed bythedemonstration of anorex in involvementin morphine add iction (Georgescuetal.,2003).Further studies show thatorex inactivi ty is

associated with cuespredictingeitherfoodor drugreward and thatintra-VTAorexin administration reinstates extinguisheddrug-seekingbehavior(Harrisetal.,2005). Orexin- Aalsoappearstobe criticaltothe cocaine-induced plastic changesin theVTAthat contributetobehavioral sensitization(Borglandetal.,2006).Recently,itwas shownthat theorexinsareinvolvedinstress-inducedreinstatementofnicotine-se eking(Plaza-Zabala eta l.,2010).Supportingarole ofthehumanorcx insysteminrewardandaddiction isthe observationthat narcolepticpatients,inwhichorex inneurons are compromi sed, show resistance todrug abuse (Guillem inau ltetaI.,1974).Together,these datas uggestt hat the orexinsystem,asidefroma role ine nergyhomeostasis andsleep/wakeregulation,is criticallyimportant tothebehavioralresponsestoboth naturalrewards anddrugs of

Orexinactivity hasalsobeenrecentlylinkedto stressandanxiety.Thebody's stress respon se,character ized bytheacti vationof thehypothalamic-pituitary-a drena l (HPA) axis,isinitiatedbythereleaseofcortic otro pin releasing factor(CRF) fromthe paraventr icularnucieus ofthehypothalamus(PVN),which acts inth eanter iorlobe ofthe pituitarytoreleaseadrenocorticotropichormone (ACTH)intothecirculationwhich subsequentlyactsonthe adrenalcorticestoincreasecorticosterone(cortisolinhumans) release.That theorexins areinvolvedin the stress responseisevidencedbythetindi ng

plasmalevels ofbothACTIIandcorticosterone(KuruetaI.,2000).Theorexin neurons themselvesare depolar izedby CRF,whichisthought to contributetothemaintenance of

arou salduringstress (Winsky-So mmerere tal.,2004).In termsofanx iety,an increased orex in-A cerebrospinal fluidlevel is seen in humansubj ectswith panic anxietywhen compared to contro lsubjectsandorexinsignalling isrequiredtoinduceapanic-prone stateinaratpan icmodel (Johnsonetal.,20 10).Moreover,orexin infusionintothe paraventri cularnucleus ofthethalamu s(PVT)increases,whereas intra-PVTorexin antagonistinfusion decreases,anxiety measures(Li eta l.,2 0 l 0), suggestingthat endogenousorex in actsin thePVTtoregulateanxietylevels.

Theorex insalso havewell-docum entedrolesin autonomic regulation.For example,centraladministration increasesheartrateand bloodpressure (Shirasakaet aI., 200 1) andbasal bloodpressureislowerinorexin-de ficient mice(Kayaba etaI., 2003).

Theorexinshavebeenimplicatedin thestressres ponseand havealsobeenshown to potentiat esexual behaviorin malerats (Guliaetal., 2003).Orexinneuronsappear tobe criticalfor stress-induced thermogenesis (Zhanget aI.,20 10)althoughtheirpreciserolein bodytemp eratur e regulationisunclearasbothanincrease(Yoshimichi etal., 200 1;Mo ndaetal.,200 1) and decrease (Balaskoet aI.,I999;Jaszberenyietal.,2002 ) in bodytemp eratu rehasbeenobservedfollowing orexinadministration .P ainr egulationalso appea rstobemed iatedbyorexinsignaling asintrathecalorexinadm inistration produced analgesia(Yamamotoetal.,2002)andstress-induced analgesiaisabsentinanimalswith a compromised orexinsystem (Xie etal.,2008).Anoverviewofthekeyproject ionsand related functionalimp licationsof theorex insyste mcanbesecnin Figure1.1.

1.2.1.4End ogenous regulators of orexlnneuronactivity

Asorexinactivity islinked toimportantphysiologicalfunctionssuch as feed ing andsleep-wakeregulation,rewardand addiction aswellasstressandanxiety. itis important tounderstandhowthis system isregulatedendogen ously.Asmentioned. orexin neuron activity shows acircadianrhythm,with higherlevels ofactivityduringtheact ive phase (Mileykovskiy et al., 2005).Importantl y,maximal activationwas observed during active waking,suggesting that theorex ins are regulatedbymorethanju stcircadian influence fromthe suprachiasmaticnucleus (Zhange t al., 2004). Inthe pastdecade,much allentionhasbeen giventoidentifyingthe factorsthatregulate orexin activityand the mechanismsby whichtheydo so.Contributingtothisliteraturewasofgreat interestto myselfand thusbecamethe overly ingthemeof thepresentthesis.

GlutamateandGABAhave excitatoryand inhibitory effectsonorexi nneurons, respectivel y.Glutamateantagonistswere foundtoinhibitorexinneurons, suggesting that, atleast invitro,orexinneurons areunder tonic stimulation byg Ilitamate(Lielal.,2002).

An importantGABAergic projecti onhasbeennoted fromthesleep-promo ting ventrolateralpreoptic area(Sakurai et al.,2 005).Orexin neuronsalsoappeartobe innervatedbymany of the monoaminerg icandcholinergicsystems that theythem selves innervate.Noradrenaline,serotonin and dopamin eneuron s send negative feedba ck projecti on s asthesetransm itterswere each found todirectlyinhibit orexinneu rons (Alberto et al.,2 006;Li etal.,2002;Liand van denPol,2005).On the0ther hand, histamineappearstoh avenoeffect (Yamanak aeta l.,2003)whileinhibitory, excitat ory and null effectshave beenobserved with the cholinergicagonistc arbachol(Sa kuraietal.,

2005).Such reciprocalprojections are likelyto play acriticalroleinstabilizingthesleep- wakecycle(Saperetal.,2005).

Variousendogenousneuropeptide regulators of the orexinsystemhave also

beenobserved.Vasopressina ndoxytocin,peptidescritica lforwa ter homeostasis,aswell as social,sexualand matem albehavior,bothhave a directexcitatoryeffectonorexin neurons(TsunematsuetaI.,2008).Thesameauthorsdemonstratedthatwater deprivation, which nonnallyinduceslocomotor activ ity,hasno effecton locomotionino rex in knockoutmice,suggestinga role forthe orexin sinth irst-induced arousal.Thyrotropin releasing honno ne(TRH)wasalsofoundtohave adirectexcitatoryeffectonorexin neuronsand mayplayaroleinthe increasedarousalobservedfollowing centrali nject ion s ofTRH(Hara etaI.,2009) (GonzalezetaI.,2009a).Theendogenous opioids dynorph in and met-enkepha linboth havedirectinhibitoryeffects thatinvolve theac tivationo f a potassiumcurrent(Li andvandenPol, 2008;Liandvanden Pol,2006),thefunction al consequences of which have yetto be fullyelucidated.Furthermore, CRF hasan excitatoryeffectonorexinneurons,suggestinga positivefeedbackrelationshipwhich likelyfunct ion sto aidinthemaintenanceofcognitivearousal du ringstressfulevents (Winsky-Sommereretal., 2004).

orex insystem,specifichumoralfactorsappearto becritical regulatorso forex inactivity.

For example,leptin,whichc irculatesinproporti on withadiposemass,hasa direct inhibitory effectonorexi nneurons(YamanakaetaI.,2003).The orexins havealso been reported tobeinhibitedby glucose (Burdakov et al., 2006)andexcitedby ghrel in,an appetite-stimulating honnone(Yamanakaet al., 2003).Thus,eireulatingfactors

signallingenergystatuscanintluenceorexi n neur ons.Asummaryoftheendoge no us regulators of orex in neuronsareshownin figu re1.2.

Theaforementionedrolesof orexin neuronsin energy balance,sleep/wake regulat ionandreward-based behavioral respo ndingarelikelynotent irely independent functions.Inotherwords, the orexins likely donothave aprimary feeding-stimulatory (orex igenic)or wake-p romotin g role.Rather,it has been proposedthat thissystemis critica l totheorchestrationof appropriatesyste ms requir ed for the executionof relevant behaviorswhenfaced withaparticularenvironmentalchallenge (Boutrel et al.,2010).

Ratherthanbeinginvolved spec ifically in drug-seek ingbehavior,theo rex insarev iewed asasystem that can inducearou sal/vigilanceand increasem otivation atappropriate times (e.g.duringhunger or thirst)tohelpdirect goal-oriented behaviorsand,ultimately,aid in the organism'ssurviva l. Thus,theorexins coordinate multiple externa l andinternal signals and respondbyincreasingordecreas ingarousaland mot ivationaccord ingly.This iswell-exemplifiedby astudy show ing that theanticipatoryincreasesin locomotor activityobserved inanimalson a food-restricteddiet islost afterorex inne uronablation (Akiyamaetal.,2004).

1.2.2 Mela nin-concentratinghormone

MCH was originally isolated in the salmon pituitary (Kawa uchietal.,1983) and itsexpressioninthe hypothalamu swasdemonstrated almost twodecadesago (Bittencourt eta I., 1992).MCH-c ontainin g cellbodi es,Iikethe orexins, are restrictedto thehypoth alamus.They are foundto co-ex ist (but donot co-Iocalize) withorexin neurons in theLH/PFAbutalsoextendslightly moredorsallyintothezona incerta.Anexcellent figure showingthedistribut ion oforex in versusMCHneuronswasrecentlypublished (Hahn,20 10).Liketheorexi ns,MCHfibersandvaricositiesare foundthroughoutmuch oftheneuroax is (Bittencourtet aI.,1992) asisitsG-proteincoupled recept or MCHR I (Saito etaI.,2001 ).Although themainthemeofthepresentthesisregards orexinneuron regulation,MCHneuronsareviableinourexperimentalsetupand were also ofinterest duetotheir own physiological and behavioralimplication s.Nonetheless,asthe majority of the presentthesisfocuses on the orex insystem,the followingintroduct ionofthe MCH systemisbrief incompar ison.

1.2.2.1MCH andenergy homeostasis

Aswiththe orex ins,the ratherrestrictedlocal izat ion ofMC H neuronswithinthe lateralhypoth alamu spromptedinvestigat ion oftheroleof thispeptid ein the contro lof food intake.It was found thatMCHactivityhas a stimulating effectonappetite(Qu etal., 1996) and MCH overexpression causesobesity(Ludwig etaI.,200 1).MCHknockout mice are hypophagic and lean andalsodisplayincreased locomotor activity,ahighbasal metabolicrate andare resistant todiet-indu ced obesity(ShimadaetaI., 1998;Kokk otouet aI.,2005) .Food depr ivat ionincreasesboth orexinand MCHmRNA expression(QuetaI.,

1996) via anucleartranslocation ofthetranscript ionfactor foxa2 (Silvact al.,2009). The preciseprojection targets ofM CHn euronsth atm ediateth efeed ing effect arenot entirely understood, although there appea rsto beat leastapartial roleofi nhibitory MCH project ions tomedium spinyneuronsin thenucleus accumbe nsshell(Georgescu et al., 2005;Searset al.,2010).Nonetheless,itis well-acce pted thatMCHneur on activity promotes energy intake whiledecreasingenergyexpenditure.Thisis in contrastwith orex in neurons,which increasebothe nergyi ntakeande nergyexpenditure.

1.2.2.2.MCHneuronsandth e sleep -wak ecyci e

MCHneuronsarequietduringact ive or quiet wakefulnessbut areactiveduring REMsleep(HassanietaI.,2009). Thispatternofactivityacross thesleep-wakecycle is different fromthatof the orexin neuronsaswellasthatof the wake-prom otin g noradr energic, serotonergieandhistaminergicneuronsinthe10euscoeruleus, dorsal raphe and tuberomamm illary nucle us,respectively (Gervasonietal.,I998;Gerva soniet al.,2000;Taka hashietal.,2006).Central MCH administration increases bothslow-wave and REM sleepwhereas MCHknockoutmicesleeplessduringboththe activeand inactiveperiods(Willieetal.,2008),suggest ingarole ofendogenous MC Hi ns leep- wakeregulation.Althoughspeculative, MCH islikelyto exertitseffectonthesleep- wake cycle throughtheinhibitionof wake-promot ingareas suchasthelocuscoeruleus, dorsal rapheand tuberom am millarynucl eus (PeyronetaI., 2009).

Liketheorexins,M CHneuronsha ve alsobeenimpl icatedin anxiely anddrug add iction.MCHreleasehas becnshowntoactivate the HPA axis (Kennedyet al.,2003) whileMCH antago nistsareconsistentlyfoundtohave potentanxiolytic effects (Borowsky eta l.,2002;Chakie ta l.,2005 ;Sm ithe ta l.,2006;Georgesc ue ta l.,2005).Mice lackin gth eMCHreceptor subtypeIdisplay anxiolyticbe haviors in an um berof tests rout inelyusedtoquantifyfear andanxietyin rode nts(RoyetaI.,2006).M Cll hasalso beenimplicatedin the con sumpti on of rewardings uhstances includingpalata ble food (Mo rcnsetaI.,2005 ),sucrose(Sakamakietal.,2005) and ethanol (Duncane taI.,2006) andcanincrease the rewardvalueof cocaineviaprojectionstothenucleu s accumbens (ChungetaI.,2009). Anadd itio nal roleof MCHin increasinglearningand memoryhas beensuggested (Adamantidis andde,2009).This was recentlys upportedbythe observationofhippocampal synaptic plasticitydeficits in MCHRI knockoutmice (Pacho udetaI.,2010).Itissuggested thatMCHprojecti onstothe hippocampusmay prom otememory consolidation during sleep(Pacho udetaI.,20(0).

1.2,2,~Endoge no us regul al or s of l\l CIIneuron activit)

Much lessisknown regarding theregu lators ofMCHneuronsincomparison10 orcxinneurons.One report (vandcn Pol etal.,2004)demonstratedthatMCH neuronsare excitedby glutamate,ATPandorexin-AwhereasGABA,norepi nephrine,acetylcholine, serotoninandNPYall inhibitMCH neurons.MCH neuron s arealsoinhibitedbythe opioid dy norphin( LiandvandenPol, 2006).Cannabinoids, which increase fecd ingand decrease arousal. depolarize MCH neuronsthrough areduct ion in presynaptic GABA

release(Huanget aI.,2007). Ouroverallcomprehensio nofMCHfunctionwoul d benefit greatly from studiesaddress ingsomeadditionalendogenous regulators ofthi s syste m.

1.2.3Sum maryof the orexinand MCH syste ms

The orexin and MCHsystemsreside side-by-side within theLH/PFAwith MCH neuron s extending dorsallyintothe zona incerta.In term s of energyhom eostasis, orex in neurons stimulatefood intake aswell asenergy expenditure whereasMCHneu rons act liketypic al anabo lic pcptide sbyincreasin gfood intakeand deereasinge nergy expend iture.Theorexinsare linked to anetnegat ive energy balance whereasMCHis linkedto anet positiveenergybalanc e.Asthese peptidesalsostimul ate elassicreward pathw aysin the brain,their rolein hedonicfeedinghasnotbeenove rlooked(Zhenge tal..

2007; Mo rens eta l..2005).Theyhavealsobeen shown to regulatestress andanxiety levels. Given theobes ity,add iction andanxiety thatcan result fromthemanipulati on of eitheroneof thesesystems.it is of interest tounderstandhow orexinand MCHneu rons are norm allyregulated at the cellular level.WhenIbeganmyPhD,thenewly-de fined rolesof the orex inand MCH systems in reward were becom ing ofgreat interest tothe field . At thetime,verylittlewasknownregardinghow orexinand MCHneuron s are regul atedby endogenous opio ids, an eur opept ide famil yintimatelytied torew ard . One particularopioid, termed nocieeptin/orphanin FQ(N/O FQ). was of particularinterest due toitsfunctionaloverlap witha numberof aspects of boththeorex in and MCH syste ms includ ing foodintake, reward andadd ictionas wellas stressandanxiety. Theregulation of orexi nandMCH neuronsby N/OF Qbecamethe initialfocus of mythesis andis presented as separatem anuseripts in chapters 2 and 3 .

I.3Nocicept in/Orphani n FQ

N/OFQis an endogenou s opioid (Meunieretal.,1995;Reinscheidetal.,1995)that bindstoitsown receptor,termed the noeiceptin opio id peptide (NOP)receptor.N/O FQ- containingcell bodies aswellasN/O FQfibers and NOPreceptors are foundthroughout the brain andspinal cord,which isin contra st10theorex in and MCH neuronswhose somaare found exci usively in the hypothalamus.E lectrophysio logicalstud iesof thc cellularactionsof N/OFQ generally includepotassiumcurrentactivationand/orcalcium currentinhibition(Meis,2003).N/OFQhasbeenshownto activate aninwardly-rectifying pota ssiumcurre nt,therebyexerting a directinhibitoryeffect,inc ellsfrom a numberof brainregion sincludin gthelocus coeruleus(Connor etal.,1996b),p araventricular nucleus (Shirasakaetal., 200I) andVTA (Zhonget al.,2002)amongstothers.

Nociceptin is so-named duetoinitialobservatio nsofthe hyperalgesia produ ced whenthepeptid e wasinj ected centra lly(Meunieretal.,1995).However,further investigationdemonstrated apotentanalgesiceffect wheninjected intothe spinal cord and the centra leffect haslargelybeen reinterpretedas a result0fa decreaseinstress- inducedanalgesia (Mogilet al.,1996).

Interestingly, IO FQhasalsobeendescribedasthe brain' santi-opi oid (Mog il et al.,1996),whichise speei allytruein terms ofreward and addiction. Centralinfusion o f N/O FQi tself does not res ult in a conditioned place preferenceoravoidancc(C iccoeio ppo etal., 2000).ltdoes,howe ver, aboli shtheplacepreferenc einducedbycocain e (Sakoori and Murphy,2004),amphetamine (Kotlinskaetal., 2003),morphine (Ciccocioppo etal., 2000) andalcohol(Ciccocioppoct al.,1999).An interactionbetweenN/OF Qand the mesolimb ic dopamine systemhasbeenshown,suggesting a mechanismforN/O FQ's

inhibitoryeffect on thebrain'srewardcircuitr y.Forexample, VTAdopamin eneurons contai nNOPreceptor mRNA (Ma idmentetaI.,2002),N/OFQapplication directly inhibits VTAdopam ineneurons(ZhengetaI.,2002)and intra-VTA infusionofN/OFQ decreasesdialysatelevels ofdopamin ein thenucleus accumbens(Murphyand Maidment, 1999) .

Asidefromnocicept ion and addiction, add itionalstud ies havedemon strated arole ofN/OF Qin food intake, locomotor activity,stress andanxiety (Civelli,2008).For example,N/O FQhas ahyperphagic effect wheninj ectedeither intothe ventricles (Pomo nis etaI.,1996),the arcuate(Polidorieta l.,2000)orventrom edialhypothalamic nuclei or thenucleus accumbensshell(Stratfordet aI.,1997). CentralN/OFQalso dose- dependentl ydecrea seslocomotor activity(Reinscheidet al.,199 5),althou ghtherole of endogenousN/OFQin locomot ion isless clearas NOP receptorknockoutmicehaveno apparentchange in basalactivitylevels (Nishietal.,1997).Aclear roleofN/OFQin stressandanxiety hasemergedbased onstud iesshow ing that centralN/OFQinducesa potentanx iolyticeffect (Jenck et al.,1997) whileN/O FQknockoutanimals havehigher plasma corticoste rone levels andexhibit increased anxiety- likebehavior s (Reinscheid and Cive lli,2002;Kosteretal.,1999). Thus,N/OFQ,and itsinteractionswith orexinand MCHneurons,mayplayimportantfunction al rolesinenergy homeostasis,reward and addictio naswell asstressandanxiety.

Rationaleand objectives (relevant to chapters2 and 3)

Objec tive I: Todet erminethe effectofN/OFQonorexinandMeH neu ron s DuetoN/OFQ'sclose associationwiththephysiologicalfunctions of theorexin and MCHsystems,it was of initial interestto determine whetherN/OFQexertedaneffect on eitherof theseneuronalphenotypes ata cellularlevel.Chapter2 ofthepresentthesis describ esthecellular effectofN/OFQonMCHneurons anddiscussestheresultsin the formofa potent ial cellularmediatorof anxiety and/oradd iction.Chapter3of the present thesisdescrib esthecellulareffectofN/OFQonorexin neuron s.AsIwascompletingthis work,Xie et al(2008) reportedthatN/OFQinhibitedmouse orexin neuronsbythe activationofapotassiumcurrentand inhibitionofcalciumc urrentsandimplicatedth e inhibitionasameansby whichN/OFQcan decreasestress-inducedanalgesia. Mywork demonstratesthemolecularmechanisminvolvedinN/OFQ-inducedorexininhib ition.

We alsotookaninvivoapproachtohelpunderstandthe effectsof N/OFQactionswithin theLH/PFA.N/OFQistypicall ythoughtof as anorexigenicpeptidebasedoncentral injeetion studies(Polidorietal.,2000;Pomonisetal.,1996;Stratfordetal.,1997),which doesnotfit withinhibitory effectson the feeding-stimulatoryorexinand MCHneuronsin theLH/PFA.Thus, it wasoffurther interesttodeterminehowthelocalactionsof N/OFQ within the LH/PFAaffect foodintake.This isalsopresentedinChapter 3.

Theresult s of chapter3 generatedan interesting piece of informationt hats hifted the focusofmyPhD work.As youwillsee, theeffect ofN/OFQinorexin neurons was dependenton the activationof ATP·se nsiti ve potassium (KATP)channe ls;ionchannels gatedbyintracellul ar ATP which were yet tobe shownonorexin neurons.The properties ofthesechannels(see below)raisedsome interesting thoughtsona dd itio nalpotential regul ators of orexin neurons,incl udingenergysubstrate availability (Chapter 4) and ambient temperature(Chapter5). Thus,I decidedto focussolelyon the roleof KATP channels inorexin neuronregulation.

KATP channelswerediscoveredincardiac myocytesand have since been describedin manyexcitablecells includingneurons. These channeIsarehetero-octam ers composedof fouridenticalpore-formin g subunits(Kir6. 1or Kir6.2) alongwithfour identica lregulatorysulphonylureasubunits(SUR I, SUR2AorSUR2B). KATP channe ls in pancreaticbeta cells arecompose dofKir6.2 andSUR Isubunits whereas Kir6.2/S UR2Achannels are presentincard iacandskeletal muscle andKir6.I/SUR2B channe lsa re prese nt invascu lars moo th muscle.lta ppears that mos t neuro nsco ntai n KATPchan nelsofthe beta-ce ll type;Kir6.2/S URI(Karsc hinetaI.,I997;Thomz igetal., 2005).Although Kir6.1subunitsaremainly foundinastrocytes(Tho mzigeta l.,200 1).a Kir6.I/SURI combinatio n has beenobserved in certain hypoth alamicneuron s (Lee etal., 1999). Theactiv ityofKATP channe ls isprincipallydeterm ined by a complexinteractio n with intracellul ar ATPand Mg2+-boundnucleo tides(Nichols.2006) .Generallyspeak ing.

adecreasein theATP/ADPratiowill increasetheKATP channe l'sactivitywhereas an increasein the ratioinhibitschannelactivity.Duetotheirsensitivityt ointraceli ular ATP.

KATPchannelsact as molecular sensorsof cellularmetabolism(Nichols. 2006).Thisis bestexemplifi edbyth e canon ical beta.c ellmodelwherean increasein extracellul ar glucoseresultsinan increasein the intrace llularATP/ADPratio whichtriggersKAT P channeli nhibition.cellular depolarizationa nds ubsequentca lcium influxa nd the triggerin gof insulinrelease (Fig.1.3) (MikiandSeino,2005).

The inhibitory effectofATP isdeterm inedby an interactionwiththe cytoplasmic sideof theKir subunit whereasMgl+.boundnucleotid escanactivatethesechannelsvia interaction with nucleotid ebindingfoldsfound on the cytoplas mic sideof the SUR subunits (Nichols, 2006).Different channelcomp osition shave d ifferentsensitivitiesto inactivati onand activati onbyATPandM g-boundnucleotide s.respectiv ely (Takano ct al.,I998;Gribble etal.,1997;Tucker etal..1 997;Lisset al..1999) andsulphonylurca drugs. suchastolbut amide andglibenclamide, inhibitKATP channelsviainterac tionwith

Asthemodel inFig.1.3demonstrates a roleofKATP channelsin therelease of insulinin responsetoglucose availability. itis not surprising thatknockoutmodelshave demon stratedaclear role forKATP channelsinthecontrolof glucosehomeostasis.As predicte d bythemodel.insulinsecretion is notproperlyregu latedbyglucoseinKir6.2 knockoutmice.a dysfunction duetothelossof functional KATPchannels inpancreatic beta-cells (Miki etal.,1998).Thesemice alsohave impaired recoveryfrominsul in- induced hypoglycaemi a,suggesting aroleofKATPchannelsincountor-regulatory responsestoabnormalg lucosefluctuations.lnterestingly.central neuronsexpressing

KATP channelscan detectthelevelof extrace llularglucose and respondwithchanges in cellularexcitability.This abilityof specificneurons,termed neuronal glucosensing,is critical to glucosehomeostasis (Miki etaI.,2001). AstheworkinChapter3 ofthepresent thesisresultedintheidentificat ion offunct ion alKATPcha nnels onorexinneu rons, it became of interest toinvestigate the glucose nsing properties0forex inneurons.

1.5.2 Neuro na lg lucose nsi ng

Glucosensingisatermusedtodescribeanexcitablece ll thatcanmon itor thelevel of extracellularglucose anda lteritso utputaccordingly. Glucosensing neuron sare classifiedaseitherglucoseexc ited(G E) orglucose inhibited(GI) based on whether they areexcitedor inhibitedbyariseinglucose,respectively.Itisthoughtthat central glucosensingisresponsiblefordetectin gshifts inextracellularglucoseand to ,in turn, inducecounter-regulatory mech an ismstohelprestoreglucoseto aparticular set point.

Hypothalamic glucose nsingisKATPchannel-dependent,at leastincerta inGEcelltypes, asthe glucosensingabilitiesof neuronsin theVM Hislost in Kir6.2knockoutanima ls (MikietaI.,200I).Disrupt ion of KATPfunctionspecifica llyin POMCneuronsresulted inaloss ofPOMCglucosensingabilitiesandalsodisruptedtheanima l'scounter- regulatoryresponse to asystemic glucoseload(Parton et aI.,2007).KATPinvolv ement inglucos ensingisdependenton themetaboli sm of glucos e andita ppearsthatacritical enzy me involvedinmetabo lism-dependentglucosensing is glucokinase(Dunn-Mey nellet aI.,2002).GlucosensingintheVMHwas absentfollowingglucokinase RAinterference (Kangetal.,200 6). Thesedata suggestthatcentra l metabolism-depe ndentglucosensingi s critical towhole-body glucosehomeostasis.

1.5.2.1 Glucose nsi ng inorex in neu rons

Orexin neuro ns are known glucos enso rs. This was first shown in2003,where miceorexin neu ronswere demonstratedtobe inhibitedbyglucose elevati onsfrom10to 15or30 mM andexcited byglucos e decreasesfrom 10to5or 0mM (Yamanaka etal., 2003),definingorexin neu rons asGI.Asthese glucose co ncentrationsarenot physiologicallyrelevant,subse quentstud ieshave further char acterizedthe gluc osen sin g abilitiesoforex in neurons.Forexample,it was sho wn thatorexinneuronsareindccd GI neurons,whichrespondtoglucosechangesfrom I to 2.5mM (Burdakovetal.,2006).

Thesa me studydcmons tratcd thattheglucosen singperformed by orcxinneu ron s wasnot based on themeta bol ismofglucose butinsteadwasdue tothemetaboli sm -ind epcndent scnsingo fglucosefroman unknownsurfacereceptor.Considc ringouride nt ifica tionof KATPchannelson theseneurons and thepreviousreportde mo nstrating alackof gluco kinaseexpression inorexinneur o ns (Dunn-Mcynelleta l.,2002),wehypo thesized that orcxinneuron smay becap1olc oft hedircct mc12.bolism o rJ actate,an alt em ate energysubstrate(Pellerinand Magistretti ,1994). Altho ugh glucosehaslongbccn considcred themainprovider of rllelforactivencurons,manylines ofevidenccsupport the idea that activeneuronsreceiveasignificantamount offuel in thc form oflactatc whichis derived from the anaerobic glyco lytic processing ofglucose by as troc ytcs.Thc lactate isthen releasedfromastrocytes and takcnup byneuronstobeusedascnergy.

Thishasbecnaptlynamcdthe "astrocytcneurcr. la~ tate shulliehypothcsi s"(Pe llerinand Magistreni,1994).Chapter4 0flhepresentthcsis invest igateswhether orexinneuronsare sensitive tothe level of celluiarructabolismandwhethertheyrelyonlactate everglucos e astheir preferredenergysubstrate.

1.5.3Temperat ure -senslng neu rons

After investigating the effect of intrace llular metabolism andextrace llulare nergy substrateonorexin neurons,wethen askedwhetherthereareanyadditional potenti al regulatorsof KATPchannels inorexin neurons.It wassugges ted thatlocaltemp eratur e changesmaybe able to influenceKATPchannelactivation(QuetaI.,2007b),raisingthe possibilitythat orexin neurons maybetemperature-sensitive. Certainneuronsinthebrain, inparticu larinthe preopticanteriorhypothalam us(POAH), aredirectly responsive to 10caltemper atur e changes.Temp eraturesensitive ncllronsinciude warm-sensitive and cold-sensitive neuronswhichi ncreaseanddec reasetheirtiringrates,respect ively,withan increaseintemperature (Boulant,2000).In the caseof'the POAH,thcse neuronsdetect changesinexternalandinternaltcmpera tureand projcctto thermoeffectorsystems such asbrownadi posetisslleandskinvascll latllrein~netforttom 3 intain arelativelystable inte rna ltemperaturedespite dramaticchangesinexternaltemperaturefkomanovskyet al.,2005). Forexample, warm-sens itivenellrons are depolarizedby anincreasein tempe raturea ndw henactivc ,thesenellronspromote heatlossthroughs kinvasclllatllre vasodilationwhilethermogenesisinbrownadiposetissueis inhibited.Conversely,when cold-sens itive neuronsareactivatedbylow temperatures,anincreasein bod ytempe rature ispromotedbyvasoconstrictionandsh iveringwhile thermogenesis is induccdinbrown adipose tissue (Rornanovsky,2007).

Such body temperatureregulationis offsetbypyrogens.Byinhibitingwarm- sensitiveneuronsof thePOAH,pyrogenscaninereasebody temperature,resultingin fever.Interestingly,orcxin neuroninhibitionduring fever is suggested by studiesusingc- Fosas amarker ofneuronalactivation (Gaykemaand Goehler,2009 .Becskeietal..

2008;Parket aI.,2008)andsuch inhibitionisfittingconsideringthe physiological associati ons of theorex insystemand the sickness behaviorsthataccompany fever.Thus.

we asked whetherorexinneuronsareinhibited bytemperature and,if so,couldthis contribute to sicknessbehaviors associatedwith fever.Thisisdiscussedin moredetailin Cha pte r5 of thepresentthesis.

I.6 Rationaleandobj eetivesIre levunt to chapters4 and5)

ObjectiveZeTodet erm inethe effect of cllcrgysllbstrat e on or cxin neurons Asmentioned,theresultsof Chapter2determin edthat function alKATPchannels cxisto no rex in neurons,channelswhichcanlillke llergy metaboJism to firing frequellcy inother cell types.Thus,wesoughttodetermine whether KATPchannels inorexin neuronsrespondtometabol ic manipu lationsandwhetherorexin ncuronscan act as metaboJism-depe ndent£enson: ofextracell lllarencrgy.Thesed ata arepresented in Chapter4.

Obj ecti ve 3: To determi nethe effectoftem pera tu re on orexin neuron s

Orexinneuron activitypromote s arousal,food intakeand motiv atcd behavioral respon ses.Feverandhypertherm ia areassoc iated with behavioral depression,anore xia andalackof motivation.Based on

a

Thesedataare presented inChapter 5.

1.7 Objectives summary

The initial focusof mythesis invo lvedthe regulationof orexin and MCHneur ons byN/OFQ.This ispresented inchapters2 and3.respectively.Theresults ofchapter3 demonstrated aregulatoryrole of KATPchannels inorex in neuron activity. Thisresulted in ani nvestigationo ft heeffectsofee llular metabolismande nergysubstrate (Chapter-l) andambient temperatu re (Chapter5)onorex in neurons.

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(ArOUScii/) Goai=-. ted~ Sympathetic

l':'!-~~ behaviors outflow

Figuret.I O verv iew ofsomeof lhe er iticalp rojcctionta rgets aml re latffifun cti onal imp licatio nsofthe orexlnsystem.PVN,paraventricular nucleus of the thalamus:DMN.

dorsomedialnucicus ofth e thalamus;Arc, arcualc nucleus;NacSh,n ucleusaccumbens shell; DR,dorsal raphe;OF,basal forebrain;LC,locus coeruleus;TMN, tubcromamm illarynucleus;VTA ,ventra l tegmental area;CeA, centra l nucleus ofthe amygda la;PVT,para ventricularnucleusof the thalamu s;IML,intermed iolate ralcell

Aminoacidandsmall molecule transrnJtters Glutama te

Humoral Factors

Vasopress in.Oxytocin

- - - (Excitatory - - IInhibitory

Figurc l.20vcrvicwof som c ofihc,critic al" ndo gcn olls rcgulalorsoft hcorcxi n syste m.GABA, garnma-aminobutyricacid;NE,no;'Cpinc~hrine;5-HT. 5- hydroxytr iptarnine(serotonin);DA. dopamine;GLP- I.glucago n-like peptide1;eRF, corticotropin-rel easing factor; TRH,thyrot ropin-releasinghormone; NPY.neuropept ide Ytmet-Enk. rnet-Enkcphalin.

tGlucose

- 1'1

InSUlin

l -

(l

Ilt)+·

l \ : ..

~

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vOltage-gat;dIII - -- - -

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Figu re 1.3Overv iew ofl< ATI'channe l regula tioninthe pa nc realicbeta-cc ll, Increasedglucoseissensedby thepancreatic beta-celland insulin isreleasedin response.

Theincreaseinextracellularglucose is transpo rted insidcthecell viaglucose transporters (GLUT)whereitisultimatelymetabolizedtogenerate ATP.Thisil~creasestheA1'1'/ADP ratiowhichactsto closeKA1'1'clianne ls. Thisleadsto cellulardepo'l~rization.activat ion of voitage-gatedcalciumc hannels, calciuminflllx andthecalc ium-depe nde ntexocytosis ofinsulin .Ada ptedfrom(Mikiand Seino,2005).

Co-a uthors hipstatement

I, Matthew Parson s,hold principle authorstatus forallthe manuscript chaptersin thisthesis (chapters2-5).Iamalsotheco-fi rstauthoronamanuscriptin preparat ion, secondauthoron a publishedrcview articleand secondauthor onareviewarticle in preparationthatdonot appea r inthisthesis. All manuscript s were writtenbyme in their initial form sandwererevised overtimeby myselfandDr.Hirasawa,Below ,I have acknowle dgedadditio na lconlribut orstoeachchapter.

Chap ter2, entitlcdOoGIRKchanne l-mediated inhibition ofmelanin-concent rating hormone neurons"ispublishedin the Journal ofNeurophysiology.lappear astirst a uthor and Dr. MichiruHirasawaappearsastheother (and corres ponding)author.lcondu cted alloftheexperimentsin this chapter,withtechnicalassistancefromChristia nAlberto.

Chapter3,entitled"HypophagiaandKATP-dependentinhibitionoforexin neuronsinducedbynociceptin/orphaninI'Q" is amanu script in preparation in wh ichI appea rastirst author,followedbyJul ia Burt,Katrin ZipperienandMichiru Ilirasawa.In this chapter, Iconductedallof theexperi ments withthe exceptionof thefoodintake study (Figure3.1).ThiswasdonehyJuliaBurtand KatrinZipperlen undermy supervision.Techni cal assistance(includingsterotaxicsurgery)wasprov idedby

Chapter 4,entitled"KATPchannclmediated lactateeffectonorexin neurons:

Implicationsforbrainenergeticsduring arousal"ispuhlishcdin the Joumalof euroscience.la ppearasfi rstauthoran d Dr. Michiru Hirasawaa ppearsa stheother(and corresponding)author.Iconductedallofthcexperimentsin thischapter witht he exceptionofthe immunofluorescencedata,whichwascompletedwithhelpfromDr.

Hirasawa.ChristianAlbertoprovidedtechnicalassistancewhile Drs.Jackie Vanderluit and Quentin Pittmanprovidedsuggestions thathelpedimprovethemanuscript.

Chapter5,e ntitled"KATP-dependent thennosensitivity of orexinneurons:

Implicationsin lipopolysaccharidc-inducedanorexia"isam anuscriptinpreparationin whichIappear asfirstauthor.ChristianAlbertoappearsas sccond authorand Michiru Hirasawaaslast and correspondingauthor.Christian providedhistechnicalskillsforthe stereotaxicsurgeriesandcontributedsignificantlyto the design ofthcLPS experiments.

Dr.Hirasawadidthe tracerinjectionsintothelocuscoeruleus.Iconducted therestofthc cxperimentsin thisChaptcr.

(l' u hlishcd in thc, Jour nalofNcu ro phys iology)

Melanin-concentratinghormone (MC I1)neuro ns are locatedexclusivelywithin thelateralhypothalamus/perifom icalarea(LH/PFA) andzonaincerta,Despitethis restr ictedlocalization,MCIl··containingfibers andMCI-Ireceptors(MCHI R)can beseen inwidespreadregionsthroughoutthecentralnervoussystem (Bitt encourtetal., 1992;llervie ueta l.,2000).Likelyowi ngtoitsbroaacentraldistriblltion,MC Hhasbeen implicated in many physiol ogicalfunctions.For example.MCIl-deficientmice arc hypophagic,lean (Shimadae tal.,1998 ),hypermetabolic:andre sistanttodiet-induced obes ity(Kokkotou etal.,2005 ).On theotberhand.ovcrexprcssionofth ispeptide genera tessusce ptibility toobesity(Ludwigetal..1001).-MCH has alsobeen linked to reward andadd ictionas wellasstress andanxiety.MCIIwasrecently shown topotentiate cocai nc reward while bothcocai ne-induced pl~ceprcferenceand locomotorsensit ization werediminishedinmicelacking MCHIR (Chun getal.,2009). Further mo re,MCH hasa stimulatorycffect on thehypothalamic-pituitary-adrcnalaxis(Kennedy ctal.,2003) and MCHantagoni sts actas potentanxiolytics'when injected centrally(Borowsky etaI., 2002).AsincreasedMCH activityis associated withobesity. drug addictionandanxiety disorders,itisimportanttorecognize the endogenousfactorsthat regulatethese neurons.

Nocicept inlorphaninFQ(N/OFQ)isthemostrecentl ydiscovered endogen ous opioidand bindstothenociceptinopioid peptide (NOP) receptorwhichisexpr essed throughoutthe brain(Meunieret aI.,1995;Reinscheidct aI.,1995).N/OFQis aproductof preproN/OFQ ,whichis structurall yr elat edtothe otheropioidpreeursors,inparticular preprodynorphin.Further more,theNOP receptor sharesroughly60%homologywiththe classicu.fiandxopioidreceptors.DespitesuchsimiJarities, theNOP receptordoesnot bindotherendogenous opioids andN/OFQhasnosignifieantaffinityfortheu. fiandx receptors (Meunier,1997).However,unlikeotheropioidfamilymembe rs, IOFQhas beenreferredto asthebrain's"anti-opioid"asitcandiminishthe reward ingvaJueof variousdrugs ofabuseincludingcocaine,amphetamine,morphine andethanol (Sa koori and Murphy,2004;KotlinskaetaI., 2003;Cic cociopp oet aI.,2004).N/OFQpeptide also playsarolein feedin g (Stratfordet al.,199 7) andbchavioralresponsesto stress(Kostcr ct al.,199 9).Thus,manyphysiologicalfunctionsofN/OFQoverlap with thoseoftheMCH system. OP receptor expressionhas beendemonstrated toexistwithin the LH/PF Aand zonaincerta(Neal,Jr.etal.,1999a) andN/O FQisco-expre ssedinorexin(hypoc retin ) neurons(Maoloodand Meister,2010)whichareknowntoformsynapticappositions onto MCH neurons(van denPolet al.,2004), sugges ting thatN/OFQisreleasedontoMCH neurons.However,whetheror howN/OFQregulatesMCH neuronsisunknown . There fore,the presentstudy used conventionalwholecell patchclamp recordin gsfrom acute hypoth alamic slicestoinvestigate thecellulareffectof N/OFQon MCHneurons.

Allexperimentsfollowed theguidelinessetbytheCanadianCounc ilonAnimal Care and were approved bytheMemorialUniversityInstitutional AnimalCare Committee. MaleSpragueDawleyrats(60-70g) wereobtained from the breedin g colony at MemorialUniversity.

2.2.1Electrophysiolog y

Anima lswere deeplyanesthetized with halothane,decapitatedand brainswere quickly removed.Coronalhypothalamic slices(250urn) were sectioned using a vibratome(Le ica).Sectioningtookplace in ice-coldartificialcerebrospina l tluid(ACSF) composed of(inmM):126NaCl, 2.5KCI,1.2NaH2P04,1.2MgCh,25 NaHCOJ,2 CaCI2,\0glucose,pH7.3-7.35. Followingdissection, slices wereincu bated inACSF at 32-35°Cfor30-45min,then at roomtemperatureuntilrecording. ACS F was continuouslybubbled withO2(95%)/C02(5%).

Conventionalwhole-cell patch-clamprecordingswere performed0nbrainslices perfusedwithACSFat1.5-2ml/min,26°C,usinga Multiclamp700B amplifie rand pClamp9.2software (Molecular Devices,Sunnyvale, CAl.Theinte rnalsolution conta ined (in mM):123Kgluconate,2MgClz,8 KCI,0.2 EGTA,10 HEPES,4Naj- ATP,0.3 Na-GTP,pH7.3-7.35.Biocytin(1-1.5mg/ml)wasincludedintheintern al solutio n tolabela subsetof cellsfor post-h ocimmu nohi stochemicalphenotyping and these sect ions wereprocessedtovisualizebiocytin,MCHandorexin-A(Fig.2.1A,B, orexin-Astainingnotshown).Targeted neurons,visualizedusinganinfrared differential- interference contrastm icroscope(Leica),werelocated inth e LH,PFAorzon aincerta.

Uponatta iningwhole cellaccess,eachneuron'selectrophysiologicalcharacteri sticswere obse rved byaseriesof3 00-mshyperpolarizing(-200a nd - 100 pA)and depolarizing(100 and200 pA) currentinjectionsincurrent clamp mode.Cellsthatdid notdisplayvoltage respon sestypicalof MC H neuronswerenotusedin thepresent study.Theseinclude sp ike adaptation uponpositive current inject ionand alackofspontaneous action potent ials,Ihandrebound currents(Fig.2.IC)(Eggermann etal.,2003;Albertoetal., 2006).Duringthe courseof thepresent experiments,27cellsdisplayingcharacteristic MCHelectroph ysiologicalpropert iesweresuccessfullyfilledwith biocyt in andidentified immunohistochemicall y.Of these 27 cells,26wereMCH -immun opositive suggesting thatthed etect ionofM CHneuronsbasedonelectrophysiological criter ia isaccurate (96%)inour hands.All cellsdisplayingtheaforementionedelectrophysiologicalcritcria werethus included in thepresentstudy.The300-mscurrentinjectionsde scr ibed above were also performed every30secondson cells recorded incurrentclamptomonit orinput resistanceaswellasaction potent ial responsestopositivecurrent inject ion s.AsMCII neuronsrarelyfirespontaneouslyinvitro(EggermannetaI.,2003),aIIana lyses of action potentialsrepre sentrespon sestothe100 pA currentinjec tion.Insome cases,during N/O FQapplication,the300-mscurrent inj ection swereinsufficienttofire action potentialsin MCHneuron s.For these cells,valuesof 0 Hzand300 mswere givenfor action potential frequencyand latency,respectively.All voltageclampexperimentswere performed ataholdin gpotent ial of-70mY,with theexceptionofvoltage ramp s.To determinetheeffectofN/OFQoncurrent-voltagerelationships,the membr anepotenti al wasramped from-140to-20mY (600 ms)in the absenceand presenceofN/OFQ.To measurecalcium currents,MCHneuronswere firstidentifiedbytheir

electro phys iolog ical charact eri sticsusin gth e afo rementi onedintern al and ACS F solutions . Whenan MCHneuronwas obta ined, the pipett ewas care fully rem oved from the celland theACSF was sw itched to oneconta ining(inmM):100 NaCI, 40TEA-CI, 2.5KCI,2MgCI2,5BaCI2,10 HEP ES,10Glucose, 0.001tetrod otoxin (TTX). Thesame cellwasthenre-p atchedusing an intern al solutio nconta ining(in mM):120 CsCI, I MgC h,10 HEP ES, 5 EGTA,4 Mg- ATP, 0.5 Na2-GT P. Involtage clam pmod e, voltage steps(100ms) from -80 to0 mV were appliedevery5 seconds to activatevoltage - depend ent calci umchanne ls.Cellsin which a sign ifica ntcalciumcurrent rundownwas obse rvedwere not included for analysis.

Immunohistochemica l phenotypin gwasperformed aspreviouslydescribed (Alberto etaI.,2006). Immediately follow ing recordin g,the 250 lUTIsections were fixed ineither4%

parafo rma ldehydeor I0%formalinfo r>18 ho ursat4°Cbefore being was hed(3x 10-15 minutes)in 0.1MPBS. Sectionswereincubated witha cocktailofgoatanti-orexi nA (I:2,000;SantaCruz Biotechnology, SantaCruz,CA, USA) and rabbit anti- MCH(I:2000;

Phoeni x Pharmace uticals,Belmont,CA,USA)primary antibodiesfor3days at4°C.Sections were then washed in PBS and incubat ed ina cocktailofCy3 -conj ugated donkeya nti-goa t, Cy2-conjugateddonkeyanti-rabbitandstreptavidin-conj ugatedAMCA(I:500;Jackson ImmunoResearch,West Grove,PA,USA) secondaryantibodies for 3hours atroom temp erature.Allantibodies weredilutedin 0.1MPBS with 0.05% tritonX-I00.

Sectionswerewas hed,mounted,covers lippedandvisualized using afluorescence microscopetodetect MCH(Cy2),Orexin-A(Cy3) and biocytin (AMCA).

2.2.3Data analysi s

Actionpotentia lfreq uency,membranepotent ial andholdin g currentwere

measuredusing Clampfit9.2(MolecularDevices;Sunnyvale,CAl. Data areexpressedas mean±S.E.M.The statistical testsusedinc ludedone-way ANOYA with Dunnett'spost test for multiplegroupcompari sonsand paired orunpairedStudent t-testsfortwo-group compari sons.Avalueofp<0.05wasconsideredsignifica nt.Thereport edn-valu esfor all theelectrophysiologydatathrou ghou tthisentirethesisrepresentthenum ber of cells rec ordedwhichisnotnecessarilythesame asthe numberofanima lsused .Although multipleexperimentsweresometimescarrie doutfrom differentslices fromthesame anima l,in nocasedida single animalcontributetoall n-valuesforaparti cular expe rimentset.

2.2.4Drugs

1000xfrozen aliq uots ofdrugswere thaw ed anddiluted withACS Ftotheirfinal concentrationimmed iately priortoexperimentat ion.TTX was obtained fromAlom one Labs (Jerusalem,Israel)andpicrotoxi n (PTX) was obta inedfromSigm a-Aldrich(St.

Louis,MO).N/OFQ(1- 13)NH"UFP-I0I,tertiapinQ andglibenc lamidewere obtained from TocrisBioscience(Ellisville,MO).N/OFQ(1-13)NH,is abioactivemetab olit e of N/OFQandwasusedinthepresentstudyasa pot entNOPreceptor agonist.N/OFQ(1- 13)NH,isreferr edto asN/OFQthroughout the Results section.