Proc Natl Acad Sci US A. 2014 Epreli 22; 111(16): E1648–E1655.
Ipapashwe kwi-2014 ye-Mar 24. doi: 10.1073 / pnas.1315542111
PMCID: PMC4000785
Neuroscience
John W. Muschamp,a,1 Jonathan A. Hollander,b UJennifer L. Thompson,c UGeorge Voren,b ULinda C. Hassinger,a USara Onvani,a Theodore M. Kamenecka,b UStephanie L. Borgland,c UPaul J. Kenny,b kwaye UWilliam A. Carlezon, Omnci.a
Jonga "Iindima ezichasayo ze-cotransmission ye-dynorphin kunye ne-hypocretin kumvuzo kunye nenkuthazo” kumqulu 111 kwiphepha 5765.
Jonga "PNAS Plus Iingxelo ngokubaluleka” kumqulu 111 kwiphepha 5771.
Eli nqaku liye khankanywe ngu amanye amanqaku kwi-PMC.
Ukubaluleka
I-Hypocretin (i-orexin) kunye ne-dynorphin zi-neuromodulators ezidlala indima ebalulekileyo ekulawuleni impembelelo kunye nokukhuthaza. I-Orexin ibaluleke kakhulu kumvuzo kwaye ibandakanyeka ekufuneni iziyobisi, ngelixa i-dynorphin idibanisa isimo sengqondo esibi kwaye ibandakanyeka kwiimeko ezifana noxinzelelo. Ukuqwalasela ezi ziphumo zichasayo, iingxelo zokuba zombini iipeptide zibonakaliswe kwi-neurons efanayo kwaye zi-coreleased ziphikisana. Apha, sibonisa ukuba i-orexin kunye ne-dynorphin zidityanisiwe ngaphakathi kwe-synaptic vesicles kwaye oku kuhlanganiswa kunempembelelo enkulu kumvuzo, ukuthatha iziyobisi, kunye nokuziphatha okungxamisekileyo. Inyani yokuba i-orexin ivalela iziphumo ezidakumba njenge-antireward ye-dynorphin itshintsha kakhulu indlela esiyijonga ngayo indima esebenzayo ye-orexin engqondweni.
Abstract
I-Hypocretin (i-orexin) kunye ne-dynorphin zii-neuropeptides ezinezenzo ezichasayo ekuziphatheni okukhuthazwayo. I-Orexin ibandakanyeka kwimiba yokuvuswa kunye nomvuzo, ngelixa i-dynorphin ibandakanyeka kwiimeko ezifana noxinzelelo. Sibonisa ukuba, ngaphandle kwezenzo zabo ezichasayo, ezi peptides zipakishwe kwii-synaptic vesicles ngaphakathi kwe-hypothalamus. Ukuphazamiseka komsebenzi we-orexin kuphazamisa iziphumo ezinomvuzo ze-lateral hypothalamic (LH) yokuvuselela, kuphelisa i-cocaine-induced impulsivity, kunye nokunciphisa ukuzilawula kwe-cocaine. Ukuphazamiseka okuhambelanayo komsebenzi we-dynorphin kubuyisela olu tshintsho lokuziphatha. Sikwabonisa ukuba i-orexin kunye ne-dynorphin zineentshukumo ezichaseneyo malunga nokuchulumanca kwendawo ye-ventral tegmental (VTA) i-dopamine neurons, eyona nto iphambili ekujoliswe kuyo ye-orexin equlathe ii-neurons, kwaye inkcaso ye-intra-VTA orexin ibangela ukuncipha kokuzilawula kwe-cocaine kunye nokuzivuselela kwe-LH. ezithi zijikwe yi-dynorphin antagonism. Iziphumo zethu zichonga inkqubo yeselula ekhethekileyo apho i-orexin inokuvala isiphumo sokunyusa umvuzo we-coreleased dynorphin kwaye ngokwenza oko isebenze ngendlela evumelayo ukuququzelela umvuzo.
I-Orexin ikhuthaza ukuvuswa (1) kwaye iye yabandakanyeka kwimiphumo enomvuzo yokutya (2, 3), ukuziphatha ngokwesondo (4), kunye neziyobisi zokusetyenziswa kakubi (5, 6). Iveliswa ikakhulu kwi-hypothalamus (7), kwaye isebenza kwi-orexin 1 receptor (OX1R) kunye ne-OX2I-R (eyaziwa nangokuthi i-Hcrt-R1 kunye ne-Hcrt-R2), ebonakaliswa kwiindawo ezininzi zobuchopho, kubandakanywa indawo ye-ventral tegmental (VTA) ye-midbrain (8). I-Dynorphin, ngokuchaseneyo, ibonakaliswa ngokubanzi, ikhuthaza ukuziphatha okudandathekileyo, kwaye idlala indima ephambili ekulungelelaniseni imiphumo ephazamisayo yoxinzelelo (9, 10). Ukusebenza kwe-kappa-opioid receptor (KORs), i-receptors apho i-dynorphin yenza khona (11), inokuthomalalisa imiphumo enomvuzo yokusetyenziswa kakubi kweziyobisi (12, 13) ngezenzo ezilamlisiweyo, ubuncinci ngokuyinxalenye, ngaphakathi kwenkqubo ye-midbrain dopamine (DA) (14, 15). Ngaphandle kweziphumo ezibonakala zichasene nenkuthazo, kukho ubungqina bokuba ezi peptides zinokusebenza ngokuhambelanayo; umzekelo, zombini i-orexin kunye nedynorphin zikhululwa ngexesha lokuvuselela umbane kwi-hypothalamus (16). Njengee-neurons ze-DA, i-orexin kunye ne-dynorphin neurons zonyusa umsebenzi wazo ekuphenduleni ukuvuselela okufana nemivuzo kunye noxinzelelo (17). Iziphumo ezisebenzayo zale pateni ye-neuropeptide coexpression kwiinkqubo zomvuzo wobuchopho, kwaye, ekuziphatheni okukhuthazwayo, aziqondwa kakuhle kuba i-orexin kunye ne-dynorphin azifundwa kunye ngokwesiko. Ngenxa yeziphumo ezichasayo zokuziphatha kunye ne-neuronal physiology xa ifundwa yodwa, kunokucingelwa ukuba ukongamela kwiziphumo zepeptide enye ngaphezulu kwenye kunokubangela ukwahluka kokuziphatha kwephenotypes kubuntununtunu bomvuzo. Umzekelo, ukubonakaliswa kwe-orexin okubalaseleyo kunokuphucula ubuntununtunu bomvuzo kunye nokufuna umvuzo, ngelixa ukubonakaliswa kwedynorphin kunokubangela ukuncipha kobuntununtunu bomvuzo kunye ne-anergia. Ngenxa yokuba la mazwe abaluleke kakhulu kwizigulo zengqondo ezinjengomlutha kunye nokudakumba, apho ukusetyenzwa komvuzo kuphazamisekile, siye safuna ukujonga ukuba ezi peptides, zodwa kwaye zidibene, zizichaphazela njani izimilo ezikhuthazayo kunye nesekethe ye-VTA DA ebalawulayo. Ukwenza oku, sasebenzisa i-EM ukubonisa i-peptide colocalization kwinqanaba le-microstructural, kunye neendlela zokuziphatha ezivavanya uvakalelo lokujikeleza komvuzo wengqondo, ulawulo lwempembelelo, kunye nokuthatha iziyobisi, emva kokuguqulwa kwe-pharmacological okanye imfuzo ye-orexin-dynorphin inkqubo. Ukongezelela, sasebenzisa i-electrophysiology ukufumanisa indlela ubukho obuhambelanayo be-orexin kunye ne-dynorphin, yodwa okanye ngokudibanisa nabachasayo kwii-receptors zabo, kuchaphazela ukuxhamla kwe-VTA DA neurons.
iziphumo
I-Orexin kunye neDynorphin ziiCotransmitters.
Siye saqinisekisa i-coexpression ye-orexin kunye ne-dynorphin ngaphakathi kwe-neurons efanayo ye-mouse lateral, i-perifornical, kunye ne-dorsomedial hypothalamus usebenzisa i-fluorescence microscopy (18) (Ikhiwane. 1A). Ubukho bee-neuron ezibonisa ii-transmitters ezininzi zichazwe kwezinye iisekethe zobuchopho kwaye zinokumela isiseko se-neural sokucoca iindlela apho ukukhutshwa kwee-neurotransmitters ezidibeneyo kwenzeka ngokwahlukileyo kumazinga okudubula (19). Ukusebenzisa i-EM, nangona kunjalo, sifumene ukuba i-orexin kunye ne-dynorphin zihlanganiswe ngaphakathi kwee-synaptic vesicles ezifanayo. Uninzi lweziganeko ze-copackaging zabonwa kwi-unmyelinated, iinkqubo ze-axonal ze-varicose, apho i-immunolabeling ifunyenwe kwi-vesicles okanye kufuphi. Ngaphakathi kweeseli ze-neuronal, ukulebhile okubalulekileyo kwakunxulunyaniswa ne-Golgi complex, ngelixa kungekho nanye ifunyenwe kwi-nuclei ekufutshane.Ikhiwane. 1 B kwaye C). I-Dendrites ikwaqulathe ilebhile enxulunyaniswa ne-vesicle yazo zombini iipeptides, icebisa ukukhutshwa kwe-dendritic okunokubakho kwaba bathumeli. Inani elincinane leeprofayili ze-microstructural ezibanjiweyo iitheminali ze-axon ezinophawu kuzo zombini iipeptides ezibekwe kwindawo enkulu (∼100 nm) iivesicle ezibekwe ngaphandle kwendawo yokukhupha yee-symmetrical synapses (Ikhiwane. 1 B kwaye C), ukubonelela ngenkxaso kwisigqibo sokuba i-orexin kunye ne-dynorphin zisebenza njenge-cotransmitters kwaye, phantsi kweemeko eziqhelekileyo, zikhutshwe kunye kunokuba zihluke njengomsebenzi wokudubula kweeseli.
Imbuyekezo yoMvuzo-Iziphumo eziPhakamisayo ze-Orexin Blockade ziguqulwa yi-Dynorphin Blockade.
Ukuphonononga ukubaluleka kokusebenza kwale pateni ikhethekileyo yentetho yokuhambisa, siye savavanya ukuba ngaba ukuphazamiseka kwe-orexin kunye nedynorphin ukusayina kunokuchaphazela isimilo esintsonkothileyo esibonisa inkuthazo eqhelekileyo kunye ne-aberrant. Kwi-C57BL/6 iimpuku eziqeqeshelwe ukwenza i-intracranial self-stimulation (ICSS) iqiniswe nge-lateral hypothalamic (LH) stimulation (LH)20), ukuvalwa kwe-OX1I-Rs ngu-N-(2-methyl-6-benzoxazolyl)-N″-1,5-naphthyridin-4-yl urea (SB334867) ngexesha lesigaba sokukhanya kubangele ukunyuka okuxhomekeke kwidosi kwimida yomvuzo (Ikhiwane. 2A; Indlela enye ephindaphindwayo imilinganiselo ye-ANOVA yeDosi: F3,12 = 4.44, P < 0.02). Ukonyuka kwimida ye-ICSS kubonisa ukucutha okubangelwa kunyango kwimpembelelo enomvuzo yovuselelo, uphawu oludakumba olubonisa ukuhla kobuntununtunu bokuvuza (20). Esi siphumo asibanga ngenxa yokuthomalalisa okanye okunye ukuphazamiseka kokuziphatha okungachanekanga, kuba iireyithi zokuphendula ze-ICSS azizange zichaphazeleke (Ikhiwane. 2B; Indlela enye ephindaphindwayo imilinganiselo ye-ANOVA yeDosi: F3,24 = 0.33, P > 0.80).
Ukuphakama kwimida yomvuzo okubangelwa yi-SB334867 kuthintelwe ngokunyangwa kwangaphambili nge-nor-binaltorphimine (norBNI) [indlela ezimbini eziphindaphindiweyo imilinganiselo ye-ANOVA yeDrug (phakathi kwezifundo) × I-Dose SB (ngaphakathi kwezifundo) ukusebenzisana: F3,24 = 3.98, P <0.01], evelisa i-blockade ehlala ixesha elide yezenzo ze-dynorphin kwi-KORs (10). Ezi datha zibonisa ukuba ilahleko yomqondiso we-orexin ityhila iziphumo ezifihlakeleyo ze-antireward ze-coreleased dynorphin. Ulawulo lwe-norBNI lulodwa aluzange lunciphise imida yokuvuza. Nangona esi siphumo sinokunxulunyaniswa ne-pharmacodynamics ekhethekileyo ye-norBNI kunye nabanye abachasi be-KOR beprototypical (10), kunokuphinda kubonise ukuba kukho ukuphindaphinda kwiinkqubo ezimodareyitha umsebenzi weesekethe zomvuzo wengqondo okanye ukunyuka kwe-phasic kwithoni ye-orexin yodwa (engachaswanga yi-coreleased dynorphin) akwanelanga ukuhambisa umqondiso womvuzo ovela kwindawo yokuvuselela kwi-hypothalamus esecaleni. Ezi ziphumo ekuqaleni zinokubonakala zingahambelani nomsebenzi wabanye abaye bavavanya i-SB334867 kumda we-ICSS ngexesha lesigaba sobumnyama (21). Nangona kunjalo, kukho ubungqina obuninzi bokuba ukwehla komsebenzi we-orexin kunokuba neziphumo ezixhomekeke ekubeni izilwanyana ziyavavanywa ngexesha lokukhanya okanye isigaba esimnyama. Umzekelo, ukutya kunye namanzi zigcina iziphumo zazo ezinomvuzo kwi-orexin KO iimpuku xa uvavanyo lwenziwa ngexesha lobumnyama kodwa hayi ngexesha lokukhanya (22), ixesha apho senza lonke uvavanyo lwethu lokuziphatha.
Ukubeka iziphumo zenkqubo ye-SB334867 kunye nolawulo lwe-norBNI kwi-ICSS, iqela elahlukileyo leempuku zafakwa kunye ne-LH evuselela i-electrode kunye ne-VTA yesikhokelo se-cannulae. I-Microinfusion ye-SB334867 kwi-VTA ibangele ukonyuka okuphawulweyo kwimida yomvuzo, ebonisa ukuhla kobuntununtunu bomvuzo. Nangona i-intra-VTA norBNI iyodwa ingenayo impembelelo kwi-ICSS thresholds, ithintele imiphumo yokunyuswa kwe-SB334867 elandelayo (Ikhiwane. 2C; Indlela enye ephindaphindwayo imilinganiselo ye-ANOVA yeChiza: F3,9 = 10.98, P <0.01). Nangona ukufakwa kweziyobisi kwi-intracranial kuthande ukuvelisa ukuncitshiswa okuthobekileyo kumazinga aphezulu okuphendula xa kuthelekiswa neenaliti zenkqubo yeziyobisi, ezi ziphumo azizange zifikelele kukubaluleka kwezibalo (Ikhiwane. 2D; Indlela enye ephindaphindwayo imilinganiselo ye-ANOVA yeChiza: F3,9 = 1.03, P = 0.112).
Impulsivity ilawulwa yi-Orexin kunye neDynorphin Transmission.
Impulsivity iphawulwa ngokusilela ekucinezeleni iindlela zokuziphatha ezifuna umvuzo, kunye namanqanaba aphezulu okungxama ibe luphawu oluqhelekileyo kwizigulo zengqondo ezininzi (23). Iziyobisi zokusetyenziswa gadalala, kubandakanya icocaine, zinokubangela ukonyuka kokungabinamdla, okucingelwa ukuba kuqhube ukukhula komlutha (24). Ukuthathela ingqalelo indima ephambili ye-coreleased orexin kunye ne-dynorphin ekulawuleni ubuntununtunu kwisiphumo esivuzayo sovuselelo lwe-LH kuvavanyo lwe-ICSS, sicinge ukuba intsebenziswano phakathi kwezi zimbini ze-neuropeptides inokuphembelela isiseko sokungabi namdla kunye nokusilela okubangelwa yi-cocaine kolu kuziphatha. Impulsivity inokulinganiswa kwiigundane ngokulinganisa iimpendulo zangaphambi kwexesha kwi-5-choice serial reaction time task (5-CSRTT) (25), imodeli yezilwanyana efana novavanyo oluqhubekayo lokusebenza olusetyenziselwa ukufunda ingqalelo ebantwini. Ukuphendula kwangaphambi kwexesha kolu vavanyo luhlala luphantsi phantsi kweemeko eziqhelekileyo kwaye lugqithiswa ngamachiza aphakamisa ukuhanjiswa kwe-DA (26). Sisebenzise i-5-CSRTT ukujonga igalelo lenkqubo ye-orexin-dynorphin kukuziphatha okuzenzekelayo kunye ne-cocaine-induced impulsive. Xa ilawulwa yodwa, i-SB334867 iphinde yanciphisa inani esele liphantsi leempendulo zangaphambi kwexesha (Ikhiwane. 3A; F3,21 = 4.89, P <0.01). Oku kuncitshiswa kwenzeke ngokungabikho kweziphumo ekuchanekeni kwempendulo (F3,21 = 1.45, P = 0.25), ukubambezeleka kokubuyiswa kwepellet (F3,21 = 0.91, P = 0.44), okanye inani leemvavanyo zokuvuselela ezigqityiweyo (F3,21 = 1.46, P = 0.25), ebonisa ukuba babengekho ngenxa yokuthotywa komlindo okanye amandla eemoto. Ulawulo lwe-norBNI, nangona kunjalo, luguqule iziphumo ze-SB334867 ekuphenduleni kwangaphambi kwexesha (Ikhiwane. 3B; F3,18 = 0.45, P = 0.71), ebonisa ukuba ukuhanjiswa kwe-dynorphin okungachaswanga kubaluleke kakhulu ekulungelelaniseni le miphumo echaseneyo. Inikwe yodwa okanye idityaniswe ne-SB334867, i-norBNI ayivelisanga ziphumo kwimilinganiselo yokuchaneka kwempendulo (F3,18 = 0.66, P > 0.58), ukubambezeleka (F3,18 = 3.09, P > 0.06), okanye inani leemvavanyo zovuselelo ezigqityiweyo (F3,18 = 2.38, P > 0.10). Unyango lwangaphambili nge-SB334867 luthintele ukonyuka okuphindwe kabini ekuphenduleni kwangaphambi kwexesha okubangelwa yi-cocaine (Ikhiwane. 3C; F6,24 = 5.84, P <0.01). Ezi datha zibonelela ngobungqina bokuba i-orexin neurotransmission inokulawula indlela yokuziphatha engxamisekileyo phantsi kwesiseko kunye neemeko ezivuselelwe yi-cocaine ngendlela ye-dynorphin-sensitive.
I-Dynorphin iMedia iyanciphisa i-Cocaine Self-Administration kwi-OX1R-Null Iimpuku.
Ukuba sesichengeni sokukhotyokiswa konyuka ngokubonakalayo kubantu abangangxamiyo, kwaye ukonyuka okubangelwa yi-cocaine kucingelwa ukuba negalelo ekuveleni komlutha (23, 27). Ngapha koko, ukuhanjiswa kwe-orexin kunye nokuhanjiswa kwe-dynorphin kuye kwabandakanyeka ngokuzimeleyo ekulawuleni iziphumo ezinomvuzo we-cocaine kunye nezinye iziyobisi zokusetyenziswa kakubi.28-32). Sicinge ukuba intsebenziswano phakathi kwe-orexin kunye nosasazo lwedynorphin inokulawula ngokuthe ngqo ukuthatha ichiza. Ukuphonononga oku kunokwenzeka, siye savavanya i-cocaine ye-cocaine yokuzilawula ngokwakho kwiimpuku eziguqulwe ngokwemfuza eziswele i-OX.1IiRs (OX1R−/−). Iimpuku zolu hlobo lwe-genotype zibonisa ukulawulwa kwe-cocaine ephantsi kakhulu kuluhlu olubanzi lweedosi (i-0.1-1 mg / kg nge-infusion) kodwa zibonisa ukuphendula okungaguqukiyo kwimbuyekezo yokutya phantsi kweshedyuli efanayo yokomeleza (33), ecebisa ukuba ukuncitshiswa kokuthatha i-cocaine ayisiyonto yesibini kwintsilelo kwindlela yokuziphatha. Ngaphezu koko, i-OX1R−/− iimpuku zibonisa amaxabiso aqhelekileyo okuzilawula kwe-cocaine ngexesha elimalunga neeseshini ezintathu zokuqala zofikelelo lwe-cocaine kodwa emva koko ngokukhawuleza zibonise ukuncipha kokuthatha i-cocaine (33). Siye saqinisekisa le phenotype ngedosi ye-0.3 mg / kg ngokufakwa, ebonisa ukuba umqondiso nge-OX1I-Rs idlala indima ebalulekileyo ekusekeni nasekugcineni i-cocaine yokuzilawula ngendlela yokuziphatha [Ikhiwane. 4; iindlela ezimbini eziphindaphindiweyo i-ANOVA, i-Genotype (phakathi kwezifundo) × Unyango lweziyobisi (ngaphakathi kwezifundo): F1,12 = 12.91, P < 0.01]. Kanye njengokuba unyango lwangaphambili kunye ne-norBNI lubuyisele i-ICSS eqhelekileyo kunye nokuziphatha okungxamisekileyo kwiimpuku ezinikwe i-SB334867, iphinde yabuyisela ngokuyinxenye ukuzilawula kwe-cocaine kwi-OX.1R−/− iimpuku, ukubonelela ngomzekelo okhethekileyo apho ukusilela kokuziphatha okuveliswe ngokuchithwa kofuzo kumsebenzi wenkqubo ye-neurotransmitter enye ihlangulwe ngokuvalelwa kwenye. Ezi ziphumo zibonisa ukuba, kwi-OX1R−/− iimpuku, izenzo ezingachaswanga ze-dynorphin zinciphisa iipropathi ezinomvuzo ze-cocaine, kwaye ngaloo ndlela zinciphisa ukuzilawula kwechiza. Okubangela umdla, kwi-OX1R+ / + (ulawulo) iimpuku, i-norBNI inciphise ngokungalindelekanga ukuzilawula kwe-cocaine. Enye ingcaciso enokwenzeka yesi siphumo kukuba i-dynorphin ekhutshwe yi-nonorexin neurons, efana nebizwa ngokuba yi-"ngqo" ye-striatonigral medium spiny neurons, ineziphumo ezichaseneyo ekuthatheni kwe-cocaine kwaye inokwenza lula iziphumo ezinomvuzo ze-cocaine. Ubukho babantu ababini be-KORs abaneendima ezichaseneyo kumvuzo we-cocaine buya kuchaza ukuba kutheni i-norBNI iguqule ngokuyinxenye ukusilela ekuthatheni i-cocaine okufunyenwe kwi-OX.1R KO iimpuku. Kungenjalo, ukuchasana kwe-KOR kunciphisa i-aversive okanye uxinzelelo lweziphumo zokurhoxa kwe-cocaine (34), ezinegalelo kwindlela yokusela iziyobisi (17). Nokuba, ezi datha zibonisa ukuba iziphumo ezinxunguphalisayo ezifana nedynorphin zikho ngokungabikho kokubonisa i-orexin, ivelisa ukuncipha kweziphumo ezinomvuzo ze-cocaine, ngelixa iziphumo ze-orexin ziququzelela iziphumo ezinomvuzo ze-cocaine, zandisa ixesha labo ngokungabikho kwe-cocaine. umqondiso wedynorphin.
I-Orexin kunye neDynorphin zinokuSebenzisa iZiphumo eziNchasayo eziLimeleyo kwi-Excitability ye-VTA DA Neurons.
Izakhiwo zobuchopho ezifumana igalelo kwi-hypothalamic orexin kunye ne-dynorphin neurons zinokuvezwa kuzo zombini iipeptides, kwaye ke zixhomekeke kwiziphumo ezichasayo kwi-neuronal excitability (35, 36). Ubungakanani beziphumo zepeptide enye zikoyisa ngaphezu kwezo zezinye zixhomekeke kwizinto ezininzi, kubandakanywa ubuninzi obunxulumene nepeptide nganye, ixesha elide kwindawo engaphandle kwe-extracellular, kunye nokubonakaliswa kwe-receptor kwiindawo ezahlukeneyo ze-neurons ekujoliswe kuzo, kunye nokusebenzisana phakathi ii-receptors kunye neendlela zabo zokubonisa i-intracellular kwiiseli ze-postsynaptic. Impulsivity kunye nomvuzo we-cocaine kulawulwa, ubuncinci ngokuyinxalenye, yi-DA neurons kwi-VTA (26), eyona njongo ibalaseleyo yeeseli ezine-orexin ze-hypothalamic (37). Ngaphezu koko, ukufakwa kwe-orexin kwi-VTA kuphucula ukufuna iziyobisi (6). Ukuvavanya igalelo elihambelanayo lepeptide nganye kumsebenzi we-VTA neurons, senze ukurekhoda kwe-electrophysiological ukusuka kwiiseli ze-DA kwi-C57BL / i-6 ye-mouse izilayi zobuchopho ezivezwe kwi-orexin kunye ne-dynorphin esetyenziswe ngokubodwa okanye kunye. Njengoko bekulindelekile, xa isetyenziswe ngokwahlukileyo, i-orexin yayinomdla ngokufanayo, ngelixa i-dynorphin ivelisa iziphumo zokuthintela kuphela (Ikhiwane. 5 A kwaye B; F2,50 = 18.95, P ≤ 0.01). Kubemi bee-neuron ze-DA ezirekhodiweyo, uninzi luphendule ekugxininiseni kokugcwala kwazo zombini iipeptides, nangona igcuntswana elincinci laliphendula ngokukhethayo kuphela kwi-orexin okanye idynorphin.Ikhiwane. 5B). Okumangalisayo kukuba, xa zombini iipeptides zisetyenziswa kwii-neuron eziphendula kabini (n = 10), akuzange kubekho siphumo somnatha kwizinga lokudubula (Ikhiwane. 5A), icebisa ukuba iziphumo ezichasayo zepeptide nganye ekugxininiseni okugcweleyo zirhoxisa enye kwenye ngokukhululeka. Ezine kwii-neurons ze-10 zibonise ukuvinjelwa okukhethiweyo yi-dynorphin nangona kukho i-orexin, ngelixa iseli enye yayinomdla kakhulu (> i-1.5-fold change) yi-orexin nangona kukho i-dynorphin.Ikhiwane. 5 A kwaye C). Ngokubanzi, nangona iiseli ezininzi ziphendula i-orexin kune-dynorphin, ezo seli zaziphendula kuzo zombini ii-peptides zazingenalo utshintsho olunetha kwizinga lokudubula xa i-orexin kunye ne-dynorphin ziye zadityaniswa, nto leyo ebonisa ukuba iimpembelelo ezichasayo zepeptide nganye zilungelelanisiwe ngaphakathi kweseti ye-VTA DA. imithambo-luvo ifundiwe.
Ukucacisa ngakumbi ukusebenzisana kwe-orexin-dynorphin ngaphakathi kwe-VTA DA neurons ezazinovelwano kuzo zombini i-orexin kunye nedynorphin, siye sazama, ngenye indlela, ukwandisa iziphumo zokuthintela i-dynorphin esetyenziswa ekuhlambeni ngonyango nge-SB334867.Umzobo weS2A; F5,25 = 2.13, P <0.01) okanye ukunyusa iziphumo zokuvuselela i-orexin efakwe kwi-bath-applied orexin ngonyango kunye ne-norBNI (Umzobo weS2B; F3,27 = 5.48, P <0.01). Kuzo zombini iimvavanyo, i-OX1I-R kunye ne-KOR blockade ayiphumelelanga ukuvelisa ezi ziphumo, iphakamisa ukuba i-SB334867 kunye ne-norBNI ayenzi ziphumo ngezenzo ezingezizo. Okubaluleke ngakumbi, ezi datha zibonisa ukuba ithoni yepeptide nganye kwi-vitro ayanelanga ukuba iphenjelelwe kukusetyenziswa kwe-antagonists encinci ye-molecule efana ne-SB334867 kunye ne-norBNI. Oku kufunyanisiweyo kuyahambelana nomsebenzi wangaphambili obonisa ukuba i-exocytosis ye-vesicles enkulu, ene-peptide yenzeka kuphela kwi-frequencies ephezulu yokudubula engazange ibekho kumalungiselelo esilayi (38).
Ukuqinisekisa ukuba i-norBNI ayizange ibe nampembelelo kwindlela yokuziphatha ngokusebenzisa "izinto ezijoliswe kuzo" ngokuthe ngqo kwi-OX1Rs, ngokulandelayo sivavanye iziphumo zalo mchasi kwi-OX1R umqondiso. Ngokukodwa, sisebenzise i-fluorometric imaging plate reader (FLIPR) ukuvavanya amandla e-orexin A, i-SB334867, okanye i-norBNI, okanye i-norBNI ukuze ifake i-intracellular calcium transients kwiiseli ze-CHO zenkcubeko ezibonisa i-OX yomntu.1I-Rs. Nangona i-orexin A ivelise ukonyuka okulindelekileyo kwi-intracellular calcium (EC50 = 0.01 μM) kunye ne-SB334867 idosi-ixhomekeke esi siphumo (EC50 = 0.035 μM), i-norBNI ayiphumelelanga ukuvelisa naziphi na iziphumo kwisiseko okanye i-orexin A-evoked ukunyuka kwi-intracellular calcium. Oku kuphakamisa ukuba iimpembelelo ze-norBNI kwi-VTA DA neuronal physiology zihamba kuphela ngeendlela ezicetywayo ze-KOR kwaye ichiza alinampembelelo ngqo kwi-OX.1R (39) (Umzobo weS3 A-C).
Ukusebenzisana kwe-Orexin-Dynorphin kwi-VTA Ukulawula iCocaine Self-Administration.
Izifundo zethu ze-electrophysiology zibonisa ukuba ukusebenzisana okuguquguqukayo phakathi kwe-orexin kunye nedynorphin kulawula umsebenzi we-VTA we-DA, kunye nokuba ii-neuron ze-VTA zinokusebenza njengenxalenye ephambili yeziphumo zenkqubo ye-orexin-dynorphin ekuziphatheni okukhuthazwayo. Ukuvavanya le ngqikelelo ngokuthe ngqo, sivavanye iziphumo zokungeniswa kwe-intra-VTA ye-SB334867 kwi-iv cocaine yokuzilawula ngokwakho kwiimpuku. Xa kuthelekiswa nokungeniswa kwemoto ye-intra-VTA, i-intra-VTA SB334867 ibangele ukuncitshiswa okubonakalayo kokutya kwe-cocaine eyayivalwe yi-norBNI (Ikhiwane. 6; Indlela enye ANOVA: F3,24 = 11.56, P <0.01), icebisa ukuba izenzo zedynorphin ezingachaswanga kule ndawo yobuchopho zijonge umvuzo wecocaine. Ezi ziphumo zibonakala ngathi ziyangqubana nezo zibonise ukungabikho kwe-intra-VTA SB334867 ekuzilawuleni kwe-cocaine kumzamo ophantsi omiselweyo we-1 (FR1) iishedyuli zokomeleza (40). Nangona kunjalo, iingxelo ezininzi zibonise ukuba njengoko iimfuno zomsebenzi zisanda, i-SB334867 isebenza ngakumbi ekunciphiseni ukuthatha iziyobisi.2, 33). Ngenxa yokuba iimpuku kolu vavanyo zenza umzamo ophezulu we-FR5 ishedyuli, iziphumo zangoku ziyahambelana nolu ncwadi. Ezi datha zibonelela ngobungqina obuthe ngqo bokuba indalo echasayo ye-orexin kunye ne-dynorphin kwi-VTA DA neuronal physiology inokusebenzisa iziphumo ezibalulekileyo kukuziphatha okuqhutywa ngumvuzo.
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Sinika ingxelo yokuba i-orexin kunye ne-dynorphin, i-neuropeptides enokuvelisa iziphumo ezichaseneyo nokukhuthaza, zifumaneka kwii-synaptic vesicles ezifanayo. Ukufumanisa ukuba ezi neuropeptides zihlanganiswe kwaye mhlawumbi zikhutshwe phantsi kweemeko ezifanayo zomzimba (16) ineempembelelo ezinzulu kuba iphakamisa ukuba kunokwenzeka ukuba le nkqubo yenzeke kwiinkqubo ngokwesithethe ezicingelwa ngokuxhomekeke ikakhulu kwizithuthi zomntu ngamnye. Sikwabonisa ukuba i-orexin, isayina nge-OX1I-Rs, inciphisa iziphumo eziphambili zokusebenza kunye nokuziphatha kwe-cotransmitter dynorphin yayo. I-Orexin-dynorphin neurons ivakalisa amanqanaba awonyukayo emfuza kwangoko ye-c-Fos ekuphenduleni imivuzo kunye neengqikelelo zokuqikelela umvuzo (4, 6, 22), ebonisa amanqanaba aphezulu e-neuronal activation ethanda ukukhutshwa kwe-neuropeptide. Emva koko sinika ubungqina bokuba i-corelease ye-orexin inokuvala iziphumo ze-dynorphin ekuziphatheni okukhuthazwayo ngezenzo zayo kwii-neurons ze-DA kwi-VTA. Ukuvalwa kwe-orexin kunokuvelisa i-dynorphin- okanye i-KOR agonist-efana neziphumo kwi-ICSS kunye nokuziphatha okunxulumene ne-cocaine eguqulwayo ne-KOR antagonism (13, 41, 42). Uphononongo lwangaphambili lwepeptide nganye kwezi zibekwe zodwa zixhasa ezi zigqibo: Ukufakwa ngokuthe ngqo kwe-orexin kwi-VTA kubuyisela ukufuna ichiza (6), ngelixa i-intra-VTA infusion ye-KOR agonists ivelisa iziphumo ezidakumba, ezifana ne-dysphoria (43). Sicinga ukuba i-orexin iqhele ukwenza kunye negalelo lokuphendula lomvuzo kwi-VTA [umzekelo, i-glutamate evela kwi-prefrontal cortex kunye nezinye izakhiwo.44, 45)] ukoyisa impembelelo yokuthintela i-dynorphin kunye nokuhanjiswa kwe-GABA yendawo kwi-DA neurons, ukuphucula ukukhululwa kwe-DA ye-forebrain ehambelana nomvuzo kunye nokuziphatha okukhuthazwayo.
Kubalulekile ukugxininisa ukuba nangona ukonyusa ukuhanjiswa kwe-orexin kubonakala kukwazi ukuphelisa iziphumo ezidakumbisayo zokwenziwa kusebenze kwe-KOR, inkcaso ye-KOR ayivelisi siphumo sokubuyelana (umsebenzi onyusiweyo womvuzo). Sicinga ukuba oku kubangelwa, ngokuyinxenye, kwiiprofayili ezahlukeneyo ze-pharmacodynamic kunye ne-pharmacokinetic ye-SB334867 kunye ne-norBNI. Ichiza langaphambili libonisa umsebenzi wakudala kunye a t1/2 ye-∼24 imiz (46), ngelixa inaliti enye yokugqibela ivelisa ukuchasana okusebenzayo kwee-KORs eziqhubekayo iiveki (10). Ukongeza, abachasi be-KOR abafanekiselayo njenge-norBNI "ngababe-agonists becala" abanokuthi ngaxeshanye basebenze ezinye iindlela zokubonisa, ezifana ne-c-Jun kinase (39), ngaloo ndlela ivelisa iziphumo ezibukhali okanye ukulungelelaniswa kwembuyekezo okwaneleyo ukuphelisa amanqanaba aphezulu ethoni ye-orexin. Izigqibo ezicacileyo malunga nokuba ezi ziphumo zihambelanayo zilindele uphuhliso lwabachasi be-KOR abafutshane abangenzi kwezinye iindlela zokubonisa i-intracellular; iikhompawundi ezinjalo azikho okwangoku (10). Ngaphaya koko, iimvavanyo zangoku zijolise kwi-VTA kwaye ayinakukhupha ukuba kunokwenzeka ukuba iziphumo ze-orexin kunye ne-dynorphin zingabikho kwi-dichotomous kwezinye izakhiwo okanye ukuba i-VTA kuphela kwesakhiwo apho ukusebenzisana kwe-orexin-dynorphin kuphembelela ukuziphatha. Ngokomzekelo, kukho ubungqina bokuba i-orexin ibandakanyeka kwimpendulo yoxinzelelo kwaye inokuthatha inxaxheba kunye ne-dynorphin ukuvelisa amazwe angalunganga ahamba kunye nokurhoxiswa kweziyobisi (40, 47). Ngokucacileyo, umsebenzi owongezelelweyo uyimfuneko ukumisela iimeko, indawo ye-anatomical, kunye neendlela ezibonakala zivumela izenzo ezidibeneyo kunye ne-orexin kunye ne-dynorphin kwii-paradigms zokuziphatha ezahlukeneyo.
Idatha yethu ikwaxhasa imbono yokuba isenzo sazo zombini iipeptide simodulatory, ngenxa yokuphazamiseka kwe-OX1Ii-Rs okanye ii-KORs zincitshisiwe kodwa azizange ziphelise iindlela zokuziphatha ezivavanyiweyo. Umzekelo, ukuziphatha kwe-ICSS kuqhubekile nakwidosi ephezulu ye-SB334867, ebonisa ukuba i-orexin iyodwa ayanelanga ukunika ingxelo ngeziphumo ezinomvuzo zovuselelo lwe-LH. Enye into enokwenzeka kukuba nangona i-orexin isenokungayigcini impatho ye-ICSS, iyanciphisa ukuphazamiseka kwayo ngokuphelisa izenzo ze-dynorphin. Ukubonakaliswa okungafaniyo kwe-orexin kunye ne-dynorphin ngabantu abafanayo be-hypothalamic neurons ingaba yindlela apho i-excitability ye-DA neurons kwi-VTA inokulawulwa yi-stimuli yangaphandle, kunye namava okanye izifo. Njengomzekelo omnye, amanqanaba e-orexin mRNA ancipha ngokulandela uhlobo loxinzelelo lwentlalo olungapheliyo olukhokelela kuxinzelelo olufana nephenotype kwiimpuku (48) kunye neegundane (49). Le phenotype efana noxinzelelo inokubangelwa, ubuncinci ngokuyinxenye, ukunciphisa inkcazo ye-orexin eyenza izenzo ze-dynorphin zingaphikiswa. Ezi ziphumo zineempembelelo ezibalulekileyo zokutolika idatha ebandakanya i-orexin kunye ne-dynorphin ngokuzimeleyo, kuba ukulungelelaniswa kwenkqubo enye kunokuphikisana ngokulungelelanisa kwenye. Basenokongeza ukuguquguquka kuyilo lwezicwangciso zonyango ukunyanga ukuphazamiseka ukusuka kwi-narcolepsy ukuya kwisimo sengqondo kunye nokuphazamiseka kokulawula impembelelo. Umzekelo, indlela eyodwa yokunyanga iimeko ezibangelwa kukungasebenzi kakuhle kwe-orexin inokuba kukuxhaphaza ukusebenza kwe-KOR, kwaye, ngokuchaseneyo, ukuphazamiseka okuphawulwa ngumsebenzi otshintshileyo we-dynorphin kunokupheliswa kukukhohlisa kweenkqubo ze-orexin.
Impahla nenkqubo
Izilwanyana.
Indoda endala ye-C57BL / 6J iigundane (i-8 wk yobudala; i-Jackson Laboratory) esetyenziswe kwiimvavanyo ze-EM zakhiwe ngamaqela (ezintathu ukuya kwezintlanu ngekheji); ezo zisetyenziselwa i-ICSS zahlaliswa zodwa emva kotyando. Amadoda amakhulu (350 g) iimpuku ze-Sprague-Dawley (iCharles River Laboratories) zasetyenziswa kwi-5-CSRTT kunye novavanyo lokuzilawula lwe-cocaine kwaye zahlaliswa ngokwamaqela amane. Indoda endala ye-C57BL/6J iimpuku (iintsuku ezisemva kokubeleka i-19-21) ezisetyenzisiweyo kuvavanyo lwe-electrophysiology zazigcinwe ngokwamaqela (ezintathu ukuya kwezintlanu ngekheji nganye). I-OX1−/− iimpuku kunye ne-OX1 yazo+ / + i-littermates (i-6 wk yobudala) esetyenzisiweyo kwizifundo zokuzilawula ifunyenwe kwi-Jackson Laboratory kwaye yawela umva ngaphezu kwezizukulwana ezisixhenxe ukuya kwi-C57BL/6 yeempuku. Ezi mpuku zagcinwa ngokwamaqela (ezimbini kwikheji nganye). Zonke izilwanyana zigcinwe phantsi kweemeko ezilawulwa ngukushisa kwi-12-h ukukhanya / umjikelezo omnyama, kunye nokuhlolwa kokuziphatha kwenzeka 4-5 h kumjikelezo wokukhanya; ukutya kunye namanzi bekukho i-ad libitum ngaphandle kokuba kuboniswe ngenye indlela. Iinkqubo zenziwe ngokuhambelana namaZiko ezeMpilo eSizwe Isikhokelo soKhathalelo kunye nokusetyenziswa kwezilwanyana zaseLebhu (50) kwaye zivunyiwe yiZiko lokuNakekelwa kwezilwanyana kunye neeKomiti zokuSebenza kwiSibhedlele saseMcLean, iYunivesithi yaseBritish Columbia, kunye neScripps Florida.
Immunohistochemistry kunye neMicroscopy.
I-Fluorescence kunye nesilivere eyongeziweyo ye-immunolabeling ye-orexin A okanye i-prodynorphin yenziwa kwamanye amacandelo engqondo yempuku ngokweenkqubo ezixelwe ngaphambili.51), kwaye kusetyenzwe ngokulandela imigaqo yeEM eqhelekileyo. Imimandla engabonakaliyo yezicubu ezigonyelweyo ziye zakhethwa ngokungenamkhethe kwaye zafotwa ukuze kulinganiswe ubungakanani beencinci zisebenzisa isoftware ye-ImageJ (amaZiko eSizwe ezeMpilo) (Izinto ze-SI kunye neendlela, i-Immunohistochemistry kunye ne-Microscopy).
I-Electrophysiology.
Iipatch pipettes (3–5 MΩ) zazaliswa nge-143 mM potassium gluconate, 10 mM Hepes, 0.2 mM EGTA, 2 mM MgATP, 0.3 mM NaGTP (ene-pH ye-7.2), kunye ne-270-280 mM mOsmol. Idatha yafunyanwa kwi-20 kHz kwaye yahluzwa kwi-2 kHz usebenzisa i-software ye-pClamp 10.0 (Izixhobo zeMolecular). Emva kokufumana ukucwangciswa kweeseli ezipheleleyo, iiseli zazigxininiswe kwi-voltage kwi--70 mV kunye nochungechunge lwamanyathelo ombane (250 ms, ukusuka -60 ukuya -130 mV kumanyathelo e-10-mV) zasetyenziswa ukufumana i-hyperpolarizing (Ih) imisinga. Ih kwamiselwa njengotshintsho lwangoku phakathi kwe ~ 30 ms kunye ne-248 ms emva kokuba kusetyenziswe inyathelo lombane. Umsebenzi wangaphakathi we-VTA DA neurons walinganiswa kwimodi ye-clamp yangoku. Iimvavanyo zaqala xa izinga lokudubula elizinzileyo laphunyezwa; I-substrates iye yasetyenziswa kwi-5 min kwaye emva koko yahlanjwa nge-cerebrospinal fluid eyenziweyo. Imizuzu emi-3 yokugqibela yecandelo ngalinye le-5 isetyenziselwe uhlalutyo lwedatha. I-Dynorphin A (1-17; 200 nM) kunye ne-orexin A (i-100 nM) zafunyanwa kwi-Peptide yaseMelika kwaye yachithwa emanzini adibeneyo. Oku kugxininiswa ngaphambili kwafunyaniswa ukuba kusebenzisa imiphumo egcweleyo kumsebenzi weseli yeVTA (5, 52). I-Thiorphan (1 μM) kunye ne-bestatin (10 μM) zafunyanwa kwi-Sigma-Aldrich, zinyibilikiswe emanzini adibeneyo, kwaye zisetyenziswe kunye nedynorphin A (Izinto ze-SI kunye neendlela, i-Electrophysiology).
Uvavanyo lweFLIPR.
OX1Umsebenzi we-R uhlolwe ngokulinganisa amanqanaba e-calcium ye-intracellular ngovavanyo lwe-FLIPR njengoko kuchaziwe ngaphambili.53) (Izinto ze-SI kunye neendlela, iFluorometric Imaging Plate Reader Assay).
ICSS.
Iigundane zifakwe kwi-electrodes evuselelayo ye-monopolar okanye i-cannulae (i-PlasticsOne) phantsi kwe-ketamine / xylazine (i-80 kunye ne-10 mg / kg, ngokulandelanayo, ip; Sigma) iqondiswe stereotactically kwi-LH (18) kunye/okanye i-VTA echaseneyo [ukusuka kwi-bregma: anteroposterior (AP), -3.2 mm; i-mediolateral (ML), −0.5 mm; i-dorsoventral (DV), −4.7 mm ukusuka kwi-dura]. Emva kwexesha le-7-d lokubuyisela, iigundane zaqeqeshwa ukuba ziphendule ukuvuselela ingqondo njengoko kuchazwe ngaphambili (18). I-frequency ephantsi kakhulu exhasa ukuphendula (i-threshold) yahlanganiswa kusetyenziswa uhlalutyo oluncinci lwe-squares umgca we-best-fit-fit analysis. Xa iimpuku zizalisekisa inqobo yozinzo kwimiqobo ye-ICSS (± 10% ngaphezulu kweentsuku ezi-5 ezilandelelanayo), iziphumo zonyango lwamachiza zalinganiswa. I-SB334867 (i-Scripps Florida) okanye isithuthi se-DMSO sanikezelwa ngezinye iintsuku usebenzisa i-syringe ye-Hamilton (i-0.1 mL / kg ip), kwaye i-thresholds yachithwa ngokukhawuleza kwiiseshoni zovavanyo lwe-15-min. I-norBNI (10 mg / kg ip; Sigma) inikwe i-saline (10 mL / kg) i-48 h ngaphambi kokuqala kovavanyo lwe-ICSS.
I-5-CSRTT.
Iigundane zazinqatshelwe ukutya (ukuya kwi-85% yesisindo sokutya mahhala) kwaye ziqeqeshwe kumagumbi asebenzayo alawulwa yikhompyutheni ahlala ngaphakathi kweekhabhinethi ezikhupha umoya, i-sound-attenuating (i-Med Associates), kunye ne-5-CSRTT iinkqubo eziqhutywe njengoko kuchaziwe.25). I-SB334867 (kwi-DMSO, i-0.1 mL/kg) kunye/okanye i-cocaine (kwi-saline, i-1 mL/kg; i-Sigma) yanikwa ngenaliti ye-ip 10 min ngaphambi kovavanyo, njengakwezinye iimvavanyo, kwaye i-norBNI yanikwa ubuncinane i-48 h ngaphambi kokuba ukuqala kovavanyo (Izinto ze-SI kunye neendlela, 5-Ukukhetha Uthotho Reaction Ixesha Umsebenzi).
IV Cocaine Self-Ulawulo.
Iimpuku kunye neempuku zaye zafakwa i-anesthetized nge-isoflurane (1-3% vol / vol) umxube womphunga we-oxygen kwaye walungiswa ngotyando nge-Silastic (VWR Scientific) catheters kwi-jugular vein ngokwemigaqo emiselweyo (54). Ngokukhawuleza emva kokufakelwa kwecatheter kwiimpuku, i-cannulae yesikhokelo se-stainless steel stainless (23 gauge, 17 mm ubude) yafakelwa kwi-VTA (ukusuka kwi-bregma: AP, 5.3 mm; ML, ± 0.7 mm; DV, -7.5 mm ukusuka kwixesha elide ). Uvavanyo nge-SB334867 okanye i-norBNI ngexesha leeseshoni zemihla ngemihla ze-60 zenziwa emva kokuba kufunyenwe i-cocaine ezinzileyo (<20% ukuhluka ekuphenduleni iintsuku ze-3 ezilandelelanayo; Izinto ze-SI kunye neendlela, i-IV Cocaine Self-Administration).
Izibalo.
Idatha ichazwa njengentsingiselo ± SEM. Kwimifuniselo ye-ICSS, iindlela ezimbini zokuphinda-phinda imilinganiselo i-ANOVA isetyenziselwe ukuthelekisa iindlela phakathi kwe-SB334867-enyangwayo kunye neemeko zonyango ze-norBNI + SB334867. Indlela enye yokuphinda-phinda imilinganiselo ye-ANOVA ene-Newman–Keuls yovavanyo lwesithuba se-hoc yasetyenziswa ukuthelekisa iindlela ezingaphakathi kwe-SB334867 kunye neemeko ze-norBNI + SB334867. Indlela enye ephindaphindwayo yovavanyo lwe-ANOVA kunye ne-Newman-Keuls nayo yasetyenziswa ukuthelekisa iindlela kuyo yonke imifuniselo ye-5CSRTT. Amanyathelo amabini aphindaphindiweyo i-ANOVA yasetyenziselwa ukuthelekisa iindlela phakathi kwamaqela onyango kuvavanyo lokuzilawula kwe-cocaine kunye ne-OX.1R KO iimpuku. Indlela enye ephindaphindiweyo yovavanyo lwe-ANOVA kunye ne-Newman-Keuls yasetyenziselwa ukuthelekisa iimpendulo ezichanekileyo kwi-orexin kunye ne-dynorphin yi-VTA DA neurons. Indlela enye ephindaphindwayo yovavanyo lwe-ANOVA kunye ne-Newman-Keuls nayo yasetyenziswa ukuthelekisa iindlela zokusela i-cocaine kwiimpuku eziphathwe nge-SB334867 kunye ne-norBNI. Umahluko wawuthathwa njengento ebalulekileyo ukuba P <0.05.
Imibulelo
Siyabulela uDkt Garrett Fitzmaurice ngeenkcazo eziluncedo kwi-manuscript kunye no-Miranda S. Gallo kunye noMelissa Chen ngoncedo ekuqokeleleni idatha. Lo msebenzi uxhaswe ngamaZiko kaZwelonke eZibonelelo zezeMpilo ze-F32-DA026250 kunye ne-K99-DA031767 (kwi-JWM), i-F32-DA024932 kunye ne-K99-DA031222 (kwi-JAH), i-R01-DA023915 (ukuya. ) nangesibonelelo sokufunyanwa kweBhunga leNzululwazi yezeNdalo nobuNjineli boPhando (kwi-SLB).
Imihlathi
Ukungqubuzana kwenkcazo yomdla: I-WAC ibambe i-patent (i-US Patent 6,528,518; Umnikezeli: Isibhedlele saseMcLean) enxulumene nokusetyenziswa kwe-kappa-opioid antagonists kunyango lweengxaki zokudakumba. Bonke abanye ababhali babhengeza ukuba akukho mdla wemali okhuphisanayo.
Eli nqaku lingeniso ngqo lwe-PNAS.
Jonga inkcazo kwiphepha 5765.
Eli nqaku liqulethe iinkcukacha ezixhasayo kwi-intanethi www.pnas.org/lookup/suppl/doi:10.1073/pnas.1315542111/-/DCSupplemental.
Ucaphulo