Inxaxheba ye-hypocretin (orexin) kwindlela yokuziphatha ngokwesini (2007)

J Neurosci. 2007 Mar 14;27(11):2837-45.

I-Muschamp yeJW1, Dominguez JM, I-Sato SM, Shen RY, Hull EM.

Abstract

Indima yehypocretin (orexin; hcrt / orx) kwimigaqo yolawulo lomtshato isekwe kakuhle.

Kutshanje, i-hcrt / orx iye yanyanzeliswa kumvuzo wokutya kunye nokuziphatha okukhangela iziyobisi. Siyaxela apha ukuba kwiirowu zesilisa, i-Fos immunoreactivity (ir) kwi-hcrt / orx neurons inyuka iphawuleka ngexesha lokukhuphela, ngelixa i-castration evelisa ukunciphisa kwe-hcrt / orx neuron yeeseli kunye namanqanaba eprotheni kwikhosi yexesha ehambelana nokuchaphazeleka kokuphambuka kokuziphatha.

Iziphumo zazibuyiselwe umva endaweni ye-estradiol. I-Immunolabeling ye-androgen (AR) kunye ne-estrogen (i-ER alpha) ii-receptors azibonisanga kucalucalulo lwe-hcrt / orx ene-AR kunye ne-hcrt / orx neurons ezimbalwa ezibonisa i-alpha ye-ER, iphakamisa ukuba umgaqo wehomoni we-hcrt / orx expression uhambelana ne-afferents evela kwii-neurons ezinee-receptors. Sikwabonisa ukuba ukulawulwa kwenkqubo ye-orexin-1 receptor antagonist SB 334867 [N- (2-methyl-6-benzoxazolyl) -N ”-1,5-naphthyridin-4-yl urea] kuphazamisa isimilo sokuziphatha. Indawo enye yeziphumo ze-hcrt / orx inokuba yindawo yecandelo le-ventral tegmental (VTA). Sibonisa apha ukuba i-hcrt-1 / orx-A ivelisa ukunyuka okuxhomekeke kwidosi kwinqanaba lokudubula kunye nomsebenzi wabemi be-VTA dopamine (DA) neurons kwi vivo. Ukwenza kusebenze i-hcrt / orx ngexesha lokuqhutywa, kunye nokukhuthazeka kwe-VTA DA neurons yi-hcrt / orx, kunokuba negalelo ekunyukeni okunamandla kwi-nucleus accumbens ye-DA ebikhe yaqwalaselwa ngaphambili ngexesha lokuziphatha ngokwesini kwendoda. Ukulandelwa kathathu kwe-immunolabeling kwi-VTA yangaphambili kubonise ukuba i-Fos-ir kwi-tyrosine hydroxylase-positive neurons efakwe kwi-hcrt / orx fibers iyanda ngexesha lokudibana.

Ngokudibeneyo, ezi datha zixhasa umbono wokuba i-hcrt / orx peptides inokuthi isebenze ngendlela enovelwano nge-steroid ukuqhuba amandla azalisekisiweyo emivuzo yendalo efana nesini ngokuvula inkqubo ye-mesolimbic ye-DA.

intshayelelo

Oko kufunyanwayo kwii-1990s (de Lecea et al., 1998; USakurai et al., 1998), inkqubo yehypocretin (orexin; hcrt / orx) ijongiwe ikakhulu kwimeko yezomntu kunye nokuziphatha okungaphakathi (I-Sutcliffe kunye ne-de Lecea, 2002; I-Siegel, 2004). Iimpuku eziguqukayo ezinokungasebenzi kakuhle kwe-hcrt / i-orx ebonisa iipateni ezingalinganiyo zomhleko ophawulwe ngotshintsho oluqhubekayo phakathi kokulala nokuphaphama (Mochizuki et al., 2004). I-hcrt / orx peptides kukholelwa ukuba yenza kube lula ukuvuseleleka ngokuqhabalaka ngokwamaqela amileyo kumachule eesistim zobuchopho kwindawo yengqondo (umzekelo, locus ceruleus, raphe)I-Peyron et al., 1998; UNambu et al., 1999; Umthengisi et al., 2005). Ukongeza kwingqikelelo yabo kwiziza ezilawula ukuqina kunye nokuvuselela, i-hcrt / orx neurons ikwachaza imvelaphi yendlela ye-mesolimbic dopamine (DA) kwindawo ye-ventral tegmental area (VTA) (UFaddy kunye noDeutch, 2002). Le pateni yoqhagamshelo ikhuthaze iqela lamaqela ophando ukuvavanya indima ye-hcrt / orx kumiselo olunxulumene nomvuzo (I-DiLeone et al., 2003; Harris et al., 2005; UHarris no-Aston-Jones, i-2006).

Izifundo kwizilwanyana ezihambayo ad adum bonisa ukuba i-hcrt / orx neurons yonyusa izinga lokudubula kwabo ngexesha lokubanika amandla, isenzo sokuhlola (UMileykovskiy et al., 2005) kunye nokuphuculwa kwemveliso yesiporho emva kokufakwa ngaphakathi kwe-hcrt / orx infracioner iye yamiswa yi-receptor blockade ye-DA (UNakamura et al., 2000). Olunye uphononongo lubonise ukwanda kwemisebenzi yeseli ye-DA in vitro ngu-hcrt / orx (I-Korotkova et al., 2003), kunye nokukhululwa kwe-nucleus eqokelelweyo (NAc) ukukhutshwa kwe-DA kwi vivo emva kokusetyenziswa kwe-hcrt / orx kwi-VTA (UNarita et al., 2006). Kutshanje, izifundo zokuziphatha zifumanise ukuba ukwenziwa kwenkqubo ye-hcrt / orx kunokuba yinto ebalulekileyo kwizizathu zokusebenzisa iziyobisi zokuxhatshazwa (I-Boutrel et al., 2005; Harris et al., 2005; I-Borgland et al., 2006), kunye nembuyekezo yokutya yendalo amandla ayo omelezelelo amiselwe ekhaya zizakhiwo ze-hypothalamic (I-Hoebel, 1969; Iiroll et al., 1980; UFulton et al., 2000; UThorpe et al., 2005).

Njengoko i-hcrt yangaphandle / i-orx yangaphandle inokuphucula ukondla nokufuna ukutya (Kotz, 2006), ukuhanjiswa kwe-intrahypothalamic ye-hcrt / orx yenza ukuba indoda ibe nesimilo ngokwesiniUGulia et al., 2003). Ukukhanya kwezifundo zakudala apho ukondliwa okanye ukusebenza kwamadoda okuphenjelelwa kukhuthazwe kukuvuselelwa kombane kweendawo kwindawo ye-hypothalamic area (LHA) ngoku eyaziwa ngokuba ne-hcrt / orx neurons (UVaughan noFisher, 1962; I-Caggiula kunye neHoebel, i-1966; I-Swanson et al., 2005), siye safuna ubungqina bokusebenza kwe-hcrt / orx neurons ngexesha lokukhuphela kunye nokonakaliswa kwendlela yokuziphatha e-orexin-1 receptor (OX1) ibhloko. Siphinde savavanya ukulawulwa kwe-neuroendocrine yenkqubo ye-hcrt / orx ngokulinganisa imiphumo yokukhutshwa kunye nokufakwa kwehomoni kumxholo we-hcrt / orx kunye nokukhuphela kabini i-hcrt / orx kunye ne-steroid receptors. Okokugqibela, sivavanye ubungakanani bokuthi inkqubo ye-mesolimbic ye-DA isebenze nge-hcrt / orx kwi vivo kwanokuba ii-neurons ze-DA kwiVTA ezifumana ii-hcrt / orx zokufaka zibonisa ulwandiso lwe-Fos immunoreactivity (ir) ngexesha lokukhuphela.

Impahla nenkqubo

I-Fos immunohistochemistry, indlela yokuziphatha, kunye nokubalwa kweseli.

Abantu abadala (∼325 g), amadoda abelana ngesondo ama-Long-Evans amagundane (Harlan, Indianapolis, IN) agcinwa kwaye asetyenziswa ngokungqinelana neZiko lezeMpilo leSizwe Izikhokelo zoKhathalelo kunye nokuSetyenziswa kwezilwanyana zaselebhu, kwaye zonke iinkqubo zavunywa ziiKomiti zeyunivesithi zokuNakekela iZilwanyana kunye neeKomiti zokuSebenzisa. Kwiveki engaphambi kovavanyo, zonke izilwanyana zazivunyelwe ukuba neeseshoni ezine zemihla ngemihla ze-1 h zesini kunye nomntu obhinqileyo owaziswe i-estrus yi-estradiol benzoate (10 μg, sc) kunye neprogesterone 48 h kamva (400 μg, sc; inikwe i-4 h ngaphambili ukuvavanya; iSigma, iSt. Louis, MO). Uvavanyo lokuziphatha kuwo onke amalinge lwenziwa i-2 h kwithuba lesiqhelo lasebusuku lezilwanyana kwikhaya labo phantsi kokukhanya ukusuka kwi-40 W ebomvu incandescent bulb. Kuvavanyo lokulingisa, iqela elinye (n = 6) yamadoda yavunyelwa ukuba ilandele kwikhaya labo kunye nomntu obhinqileyo ongena kwi-ejaculation enye, emva koko ibhinqa lisuswe. Zonke izilwanyana zanyuka phantse kwangoko kwaye zangeniswa ngaphakathi <4 min. Intsingiselo ye-ejaculation latency yeli qela yayingu-632.6 ± 169.64 s (kuthetha ± SEM; n = 6). Iqela lolawulo (n = 6) yayisetyenziselwa ukulawula inqanaba lomsebenzi. Ezi zilwanyana zajongwa ngokubonakalayo ukuqinisekisa ukuba zivukile kwaye ziyasebenza kangangexesha le-15 min, elihambelana nexesha lokuvavanywa kweqela lokuvavanywa. Iziciko zeekheji zavulwa zavalwa ekuqaleni nasekupheleni kweli xesha le-15, kodwa kungenjalo, izilwanyana zazishiywa zingaphazanyiswanga. Ulawulo lwezilwanyana ezazingxamisekile ngeli xesha zazingasetyenziswanga. Izilwanyana ziye zagwetywa ukuba ziyangxama xa zibonakala ngathi ziphumlile ecaleni lazo okanye kwimo yokubekwa entloko Espana et al., 2003). Imizuzu engamashumi amathandathu emva kokuqala kweeseshoni zokushicilela okanye zokulawulwa kokuqwalaselwa, izilwanyana zazifakwe ngaphakathi ngepentobarbital (100 mg / kg) zaza zafakwa nge-0.1 m PBS, pH 7.4, kunye ne-4% paraformaldehyde kwi-PBS. Iibroys zaziculwa kwi-20% sucrose-PBS, kwaye lonke elinye icandelo le-40 μm le-cryostat (i-30 ngesilwanyana ngasinye) kufutshane ne-hypothalamic hcrt / orx cell yabantu babethelelekile kwi-prero-hcrt / orx (1: 100; Millipore, Billerica, MA) kunye neFos (1: 10,000; EMD Biosciences, San Diego, CA) nge3 ′, 3'-diaminobenzidine (DAB) kunye nickel-intensified DAB, ngokwahlukeneyo, ngokweenkqubo ze-immunohistochemistry ezichazwe ngaphambili (Espana et al., 2003; Sato et al., 2005). Amanani eseli enziwa phantsi kobukhulu obukhulu besebenzisa imicroscope kunye nekhamera kwicandelo elinye kwinqanaba elilinganayo le-rostrocaudal (jonga Ikhiwane. 1B,C) (2.45 mm emva kwangoko kwi-bregma) (Swanson, 2004). Kwi-hemisphere nganye, iiseli eziwele phakathi kwamacala amabini okubalwa kwe-470 × 630 μm zazimakishwe kusetyenziswa isoftware yokuhlaziya imifanekiso (Umzobo-Pro Plus; Imidiya ye-Media cybernetics, iSilver Spring, MD), ngumvavanyi ongaboniyo kwiimeko zovavanyo. Kuzo zombini iihemispheres, amasimi abalekayo asebusweni asebenzisa ii-fornices njengomda wawo we-medial. Amabala okubala amaMedi asebenzisa ii-fornices njengomda wawo odlulileyo. Amanani ee-Fos-kuphela, hcrt / orx-kuphela, kunye neelebheli ezineeleybhile ezibini kunye ne-hcrt / orx kunye ne-Fos-ir zarekhodwa.

Umzobo 1. 

Ukonyusa ukungasebenzi kwe-Fos kwi-hcrt / orx neurons ngexesha lokukhuphela. A, I-Hcrt / orx neuron (ngasekunene) ibonisa uFos-ir. Ibha yesikali, i-25 μm. B, Ummeli-bandla omelekileyo ukusuka kumhlaba wokubala osekunene kumhlaba osekhohlo wolawulo lobudoda obungenanto. Ibarikali yesikali (ngaphakathi A), I-200 μm. C, I-Micrograph evela ekuprinteni oyindoda. Ibha yesikali, i-200 μm. Imisonto ibonisa i-hcrt / orx neurons ebonisa iFos-ir; iasteriski zibonisa i-Fos-ir nuclei kwiiseli ezingekho-hcrt / orx; fx, ifoni.

Uvavanyo lokugudluza kunye nokuntywila.

Indoda endala (∼325 g ekuqaleni kovavanyo) Iigundane ezinde-ii-Evans zazingafakwanga (75 mg / kg ketamine HCl kunye ne-10 mg / kg xylazine HCl, ip) kunye ne-castated. Izilwanyana zazivunyelwe i-7, i-14, okanye i-28 d yexesha lokusinda emva kokuba zenziwe i-anesthetised kwaye zenziwa i-cosmetics kwaye iingqondo zazo zaxhonywa ngenxa ye-prero-hcrt / orx njengoko ngasentla (onke amaqela, n = 5). Ezi zilwanyana zazithelekiswa neqela lonyango lwe-sham elifumene isigxina sesifo phantsi kweanesthesia (n = 5). Iimpawu zabulawa nakwi-28 d. Amanani eseli enziwa njengasentla kwindiza enye yeekoroli phantsi kwesikhulisi esisezantsi.

Kwilingo olwahlukileyo, i-28 d castraten = 5) kunye nolawulo oluphathwe nge-sham (n = 5) zazicinezelwe ngokunzulu nge-sodium pentobarbital (100 mg / kg), kwaye iingqondo zazo zasuswa kwaye zakhenkcezwa ngokukhawuleza kwi-2-methylbutane ebandiswe ngumkhenkce owomileyo. I-hypothalami efriziweyo emva koko yathintelwa kukusikwa kwe-coronal ezimbini, enye yommandla we-medial preoptic (i-mPOA) ye-rostral yokugqitywa kwezinto zangaphambili, kunye nenye kwi-posterior hypothalamic nucleus ye-caudal yokwahlulahlula i-ventricle yesithathu kwi-hypothalamic yayo kunye ikhefu mammillary. Iibhloksi zacuthwa ngokusikwa kabini kwe-sagittal kwisiphelo sendlela yokuba kukho i-cerebral peduncle kunye nokusikwa okuthe tyaba ekupheleni komqolo wesiphelo se-ventricle's hypothalamic recess. Iibhloko zeethishu emva koko zahlanjululwa zenziwa ngoonyana, iprotein yatsalwa yatshintshwa kwi-radioimmunoprecipitation buffer, pH 8.0, kunye neprotease inhibitors (Umxube weRoche Complete Mini protease; iRoche Diagnostics, iIndianapolis, IN), kunye nezicatshulwa zakhutshelwa emva kwecentrifugation. Emva kokumiselwa kweeprotein zoxinaniso kwizicatshulwa kwisifundo ngasinye, i-40 μg yeprotheni kwisilwanyana ngasinye yafakwa kwimithombo eyahlukeneyo ye-15% ye-SDS-PAGE yejeli. Ukwahlulwa kwe-Electrophoretic, ukudluliselwa kwiimbumba ze-polyvinylidene difluoride (i-Bio-Rad, iHercules, CA), kunye ne-immunolabeling yenziwa ngokwendlela yeLaemmli (UCleveland et al., 1977), usebenzisa i-1: I-1000 ye-anti-prepro-hcrt / orx antibody eyaziwa ngaphambili, kwaye kamva i-1: i-2000 anti-β-Actin (Sigma) kunye ne-1: I-5000 yeebhokhwe ezichasene nomvundla IGG (Santa Cruz Biotechnology, Santa Cruz, CA ) kwi-5% ubisi olungumgubo kwiTris-buffered saline. Ibhendi yeeprotheyini zabonwa ngocingo oluphuculweyo lwe-chemiluminescence kit (ECL; GE Healthcare, Piscataway, NJ) kunye neKakak (Rochester, NY) iifilimu zeBioMax. Iifilimu zaziveliswa nge-40 s kwaye zasonjululwa ngokwedijithali, kwaye uxinano lwamehlo lwalinganiswa kusetyenziswa isoftware ye-ImageJ esesidlangalaleni. Kwisilwanyana ngasinye, amaxabiso ezixineneyo ze-hcrt / orx njengepesenti yokulawulwa kokulayishwa kwe-β-actin amiswa kwaye afakwa kuhlalutyo lweenkcukacha.

Kwilingo lesithathu, iigundane ezingamadoda njengoko kuchaziwe apha ngasentla zanikwa utyando olunomsindo (n = I-3) okanye isuswe kwaye yajongwa yonke imihla yesibini ye-28 d nge-dihydrotestosterone (500 μg, sc; n = 4), estradiol benzoate (20 μg, sc; n = 3), okanye inqwelo yeoyile (0.1 μl; n = 3). Zombini iihomoni zithengiwe kwaSigma. Iidosi zehormone zonyulwa ngenxa yesakhono sazo kwizifundo zangaphambili ukuze kugcinwe ubungqina bokukhuphela kwii-castrate (UPutnam et al., 2005). Ngomhla we-28th emva kotyando, izilwanyana zabulawa, kwaye i-hypothalami yazo yavalwa ngenxa yokulinganiselwa komxholo we-pre-hcrt / orx yi-immunoblot yaseNtshona njengoko kuchaziwe ngasentla.

I-Steroid hormone receptor kunye ne-hcrt / orx ephinda-kabini i-immunohistochemistry.

Amacandelo obuchopho avela kwi-LHA ye-intact, ngokwesondo engenasidala indoda eyindoda (∼350 g) Iigundane ezinde ze-Evans zazigcinelwe i-androgen receptor (AR) (1: 750; n = 6; I-Santa Cruz Biotechnology) okanye i-estrogen receptor (ERcy) (1: 2500; n = 9; I-Santa Cruz Biotechnology) isebenzisa i-nABEL eyandisiweyo ye-DAB. Emva koko, amacandelo afakwe ilebheli ye-AR abhalwa iilebheli ezimbini zepro-hcrt / orx (njengasentla), kwaye amacandelo anombhalo obhalwe i-ERcy abekwa kabini ngenxa yepro-hcrt / orx (n = 5). Emva kokunyuswa, amacandelo ahambelana ngokusondeleyo nolunye lwee-coronal ze-LHA kwii-atlas ze I-Swanson (2004) zathatyathwa ngesandla phantsi kwemakroskopu kusetyenziswa ikhonkco ye-khamera ye-lucida, kwaye iiseli ezabhalwa kabini kunye nezo zazineelebheli elinye kwicandelo ngalinye zazimakishwa. Imizobo yaphononongwa ngokwamanani kwaye yabekwa kumanqanaba e-atlas kusetyenziswa i-Adobe (San Jose, CA) Illustrator, kwaye inani ngalinye leeseli lakhonjiswa kwimifanekiso ye-atlas (Swanson, 2004). Kwimeko ye-ERcy-plus-hcrt / i-orx-labels, inani lamanani aphindwe kabini kunye ne-single-labels hcrt / orx.

I-Pharmacology kunye nokuziphatha kokuziphatha.

Indoda endala (∼350 g ekuqaleni kweemvavanyo) Iigundane ezinde ze-Evans (n = 9) banikwa iiseshoni zamava ezesondo ezine ze-1 kunye nowasetyhini owamkela ngokwesondo phakathi evekini ngaphambi kovavanyo lokuziphatha. Izilwanyana ezingaphumelelanga ukukhupha ubuncinci kube kanye ngexesha lokuqala kwe-30 min yovavanyo lokugqibela azifakwanga kuvavanyo. Amaxesha amava kunye novavanyo lokuziphatha lwenziwa phantsi kwe-40 W ebomvu ukukhanya kwe-incandescent 2 h kwixesha lesilwanyana ebusuku. Uvavanyo lokuziphatha ngokunyanzelwa lwenziwa kwikheji yendoda eyindoda kwaye yayiyi-30 min ngexesha. Iimpawu ezahlukileyo zokuziphatha kwamadoda (okt, ukunyuka, ukungena ngaphakathi, kunye ne-ejaculations) zafumana amanqaku ngamehlo angaboniyo kunyango kusetyenziswa isoftware yekhompyuter yesiqhelo erekhodayo idatha yedatha kunye ne-latency yesiganeko ngasinye sokuziphatha. Imizuzu engamashumi amathathu ngaphambi kovavanyo lokuziphatha, izilwanyana zazifakwe kwi-OX1 umchasi N- ((2-methyl-6-benzoxazolyl) -N ″-1,5-naphthyridin-4-yl urea (SB 334867) (20 mg / kg, ip) okanye isithuthi se-DMSO (0.5 ml / kg). Uvavanyo lulandele ulungelelwaniso olulula olulinganisiweyo ngaphakathi kwezifundo ezifana nokuba izilwanyana ezintlanu zifumane inaliti yeziyobisi kuvavanyo losuku lokuqala kunye nezine ezifumanekileyo zesithuthi. Emva kwexesha lokuhlamba iziyobisi i-48 h, izilwanyana ezanyangwa ngamachiza ngosuku lokuqala zanikwa isithuthi kunye nokunye.

I-Electrophysiology.

Iigundane zabantu abadala iSprague Dawley (∼350 g) zazingafakwanga i-chloral hydrate (400 mg / kg, ip ukuze ifundiswe; i-100 mg · kg-1 · H-1 emva koko kulondolozo) kwaye ifakwe kwisixhobo esine stereotaxic. Isgebenga kunye nedura ngaphezulu kwengingqi yobuchopho ene-VTA yasuswa. Isilwanyana ngasinye saqala ukufumana ukumiliselwa kwe-CSF yokufakelwa (aCSF) [0.5 μl / 5 min; ukusuka kwi-lambda, i-anteroposterior, + 2.6 okanye + 3.4 mm; i-Mediolateral, ± 0.8 mm; dorsoventral, −7.0 mm; isikhumba esithe tyaba ngokwe-stereotaxic atlas (I-Paxinos ne-Watson, i-1998)], ngesirinji ye-32 ga Hamilton (0.5 μl / 5 min) kwicala elinye le-VTA. Emva koko, i-10 min kamva, inkqubo yesampulu ye-cell-track-track eyenziweyo yenziwa kwi-ipsilateral kwicala le-VTA. Izilwanyana emva koko zifumene ukuthotyelwa kwe-hcrt-1 / orx-A (0.014 nmol, n = 4; I-1.4 nmol, n = 5; okanye i-140 nmol, n = I-6 ichithwe kwi-aCSF; I-American Peptide, iSunnyvale, CA) kwi-VTA yesivumelwano ngaphambi kokuba inkqubo-yokulandela umkhondo weseli iphindaphindwe kwelinye icala. Izitofu zichasene ne-hemisphere kunye nendawo yokubulala intsholongwane okanye indawo yangasemva. Ushicilelo lwe-extracellular single-yunithi lwenziwa nge-micropipettes eneglasi enye (i-1.5 mm ububanzi bangaphandle ngaphambi kokutsala; Izixhobo zeHlabathi zokuLungisa, iSarasota, FL) ezaliswe yi-2 m NaCl kunye naphukileyo. Ukunyanzeliswa kwe-Electrode ukusuka ku-2 ukuya kwi-4 MΩ kwi-135 Hz. I-neurons ze-DA zichongiwe ngeendlela ezinokubakho ezingezizo ezongeziweyo, ezihlala zinesahlulo sokuqala / somatodendritic (IS / SD) ikhefu, ixesha elinokwenzeka lokuthatha ixesha, inqanaba lokudubula kancinci, kunye ne-spike engadibaniyo okanye indlela yokuqhushula (UGrace kunye noBunney, 1983). Ukwenza uvavanyo lweeseli-ngomkhondo-ngamnye, i-electrode yokurekhoda idluliselwe kwibhlokhi echazayo ye-VTA (2.8-3.4 mm ingaphambli ukuya kwi-lambda; 0.6-1.0 mm emva kwexesha ukuya kumbindi; 6.5-8.5 mm ngezantsi kobuchopho) Amaxesha amathandathu. I-neuron nganye echongiweyo ye-DA yarekhodwa kwi-2-5 min kwi-line kusetyenziswa inkqubo yokufumana idatha yeChart (Izixhobo ze-AD, Ukujonga intaba, CA). Inani eliphakathi le-neurons ezisebenzayo ze-DA ezihlangane ngakwicala ngalinye le-electrode kwisilwanyana ngasinye (iiseli ezilandelelweyo) yayisisalathiso somsebenzi we-VTA DA we-neuron. Inani lokudubula kwi-neurons ye-DA limiselwe kuzo zonke i-neurons ze-DA ezisampuli kuzo zonke izilwanyana kwiqela ngalinye.

Ukuvavanya ukungasebenzi kakuhle kwi-inactivation kwi-140 nmol hcrt / orx group, apomorphine HCl (20 μg / kg, ip; n = 4; I-Sigma) yalawulwa ngokukhawuleza emva kokugqitywa kwe-post-hcrt-1 / orx-A isampulu. Imizuzu elishumi emva kwenal injani yeapomorphine, iitrodi ezongeziweyo zedrode eneesampulu ecaleni kweVTA eyayifumene ngaphambili i-hcrt-1 / orx-A.

Ukuziphatha, ukubethelwa kwelebheli kathathu immunohistochemistry, kunye nokubalwa kweseli.

Abantu abadala (∼300 g) iigundane ezinde zabantu abangamadoda i-Long-Evans yanikwa iiseshoni ezine ze1 h zesondo ngamava omfazi owamkelekileyo njengalapha ngasentla. Ngaphambi (i-1 h) i-anesthesia, i-perfusion, kunye nokulungiswa kwezicubu ze-immunolabeling, izilwanyana (n = 6) bavunyelwe ukuba bakhuphele kumjikelo omnye. Njengoko ngasentla, izilwanyana ziphakanyisiwe kwaye zangenwa kamsinya kwangoko, kwaye zithetha ukuba i-ejaculation latency yeli qela yayiyi-588.50 ± 85.03 s (kuthetha ± SEM). Iqela lovavanyo lafaniswa nolawulo olunamava olwabelana ngesondo olungazange lunikwe ukufikelela kumabhinqa akrelekrele ukuba akopelwe (n = 6). Njengasentla, ukuvavanywa kwenzeka i-2 h kwixesha lesilwanyana lesiqhelo lesilwanyana phantsi kokukhanya okubomvu kwe-incandescent. Emva kokuba izilwanyana zibulewe, zilungisiwe, kwaye kubanda imicrotome (njengentla), amacandelo amane kwisilwanyana ngasinye abonisa amanqanaba amane e-rostrocaudal e-VTA akhethelwa i-immunohistochemistry. Inani lezifundo (n) esetyenziselwa ukubalwa kweseli kwinqanaba ngalinye, yabona 4 Table. Amacandelo abhalwe iFos (1: 7500; iSanta Cruz Biotechnology) ene-DAB njengasentla. Amacandelo abhalwa i-Prepro-hcrt / orx (1: 500) kunye ne-tyrosine hydroxylase (TH; 1: 2000; Millipore) kunye ne-cyanine-conjugated fluorescent yesibini antibodies (1: 200; Cy3 kunye neCy2, ngokulandelanayo; iJackson ImmunoResearch, eWest Grove, PA). Ubalo lweeseli lwenziwa phantsi kobukhulu obuphezulu (40 ×) kwi Leica (Nussloch, Jamani) I-DM 4000B imakroskowupu isebenzisa Umphandi we-Stereo isoftware (MBF Bioscience, Williston, VT) ngumvavanyi ongaboniyo kwiimeko zonyango. Umphandi we-Stereo isoftware yayisetyenziselwa ukuchaza indawo yokubala ebandakanya zonke iiseli ze-DA ngaphakathi kwinqanaba ngalinye le-VTA. Kwindawo enye ejolise kuyo nganye kumanqanaba amane e-Vostrocaudal ye-VTA, iindidi ezintlanu zamanqaku zafakwa: I-neurons ye-TH, i-neurons ye-TH ebonisa i-Fos-ir, ii-neurons ze-TH ezibonisa ngokuthe ngqo (kwi-somatic plasmalemma) ii-appositions ezi-hcrt / I-orx fibers, ii-neurons ze-TH ezibonakalayo ezibonisa zombini i-Fos-ir kunye ne-hcrt / orx appositions, kwaye ekugqibeleni, i-nolo Fos-positive nuclei engekho kwii-neurons ze-TH.

Uhlalutyo lwedatha.

Iqela lithetha ukubalwa kweeseli kulingo lwe-Fos kunye neendlela zoxinano lwee-blots zaseNtshona ukusuka kulingo lokuqala olo xa kuthelekiswa neesampulu ezizimeleyo t vavanyo. Iisampulu ezitshatileyo t Uvavanyo lwenziwa ngeendlela ezivela kwi-OX1 uvavanyo lokuziphatha Amanani eseli ekuhanjisweni, kuvavanyo lweelebheli lokuphinda-phinda kathathu, kwaye kuthetha ukuba kwinqanaba lokudubula kunye neeseli zomkhondo ngokulandelelana kwazo ziye zafakwa kwi-ANOVA.

iziphumo

Ukuprinta kukonyusa ukungasebenzi kwe-Fos kwi-hcrt / orx neurons

Ipesenti yeeseli ze-hcrt / orx-ir ezibonisa ukuba i-Fos-ir yahluke kakhulu phakathi kwamaqela, kunye nezilwanyana ezilandulayo zibonisa ukunyuka kwe-Fos-ir kwiiseli ze-hcrt / orx (t(10) = 7.71; p <0.01) (1 Table, Ikhiwane. 1). Iinombolo zamagama ezinelebhile ephindwe kabini hcrt / orx nazo zahluke kakhulu phakathi kwamaqela (t(10) = 6.03; p <0.001). Okumangalisayo kukuba, ekulingiseni izilwanyana, akukho mahluko kwinani le-Fos-positive hcrt / orx neurons yafunyanwa phakathi kwamacandelo ama-medial kunye necala lokubala (idatha engaboniswanga) (UHarris no-Aston-Jones, i-2006). Inani elipheleleyo le-Fos-ir nuclei (i-Fos-kuphela kunye ne-hcrt ene-orx neurons ephindwe kabini) yayilikhulu kakhulu ekuphelekeni kwezilwanyana kunakwizinto ezingalawulwa.t(10) = 16.97; p <0.001) (1 Table).

Ithebula 1. 

Ukunyusa i-Fos-ir ngexesha lokuprinta kwii-hcrt / orx neurons zegundane lesilisa

Ukuhanjiswa kunciphisa i-hcrt / orx immunoreactivity kunye neprotein kwi-hypothalamus

Xa kuthelekiswa nolawulo oluphathwe nge-sham, ukubalwa kwe-hcrt / orx-ir neurons kubonise ukwehla okukhulu kwenombolo yeeseli 28 d emva kokukhutshwa (Ikhiwane. 2A) (F(3,16) = 7.60; p <0.005). Oku kubonisa ukwehla ngama-31.8% kwinani leeseli kubemi abaqwalaselweyo. Post post (Tukey) kuvavanyo akubonakalanga mahluko omkhulu phakathi kwe-rats-rats-rats okanye 7 okanye 14 d castrate. Uhlalutyo olulandelayo lwaseNtshona lwe-immunoblot ye-hypothalamic prepro-hcrt / orx ifumene ukwehla okubonakalayo kokuqina kobubanzi be-hcrt / orx band ukusuka kwi-28 d castrate xa kuthelekiswa nolawulo oluphathwe nge-sham (Ikhiwane. 2B) (t(8) = 2.99; p <0.05.).

Umzobo 2. 

Ukufuduswa kunciphisa i-hcrt / orx-ir kwi-hypothalamus yamadoda. AI-micrographs emelekileyo ye-hcrt / orx-ebhalwe i-neurons kwimozulu enye ibonisa ukwehla okukhulu kwenombolo yeseli yi-28 d emva kokuhanjiswa. Amaxabiso asezantsi athetha ukubalwa kweseli kuzo zombini iihemispheres ± SEM; **p <0.005. Ibha yesikali, i-200 μm; fx, ifom. B, I-immunoblots yaseNtshona ibonisa ukwehla okukhulu kwi-hypothalamic prepro-hcrt / orx ye28 d castrate. Ibhendi nganye imela uphawu lwesilwanyana esinye kwelinye iqela. Amaxabiso athetha ntoni units I-SEM ye-dense optical density (od) ye-hcrt / orx isihlobo se-β-actin; *p <0.05. C, I-Immunoblots ye-prepro-hcrt / orx kwi-28 d castrate ibonisa u-E2 ukugcina umxholo we-hypothalamic hcrt / orx elingana neyo shams xa kuthelekiswa nolawulo oluphathwe yioli. Amaqela anonobumba omncinci omnye akahluke kakhulu (p <0.05).

I-Estradiol ibuyisela amanqanaba e-hypothalamic hcrt / orx

Kwi-28 d emva kokusasazwa okanye ukwenziwa utyando ku-sham, amanqanaba e-prero-hcrt / orx kwizilwanyana eziphathwa ngeoyile zazingaphantsi kakhulu kunaleyo ye-sham- kunye ne-estradiol (E2-amaqela akhulayo (F(3,9) = 8.47; p <0.005) (Ikhiwane. 2C). Inye indlela ANOVA nge iposi (Tukey) iimvavanyo zifumanise ukuba amanqanaba e-prepro-hcrt / orx alinganiswa ku-sham- kunye no-E2Izilwanyana ezikhulisiweyo azifani kwaye enye ye-E2 Iqela alizange lahluke kwizilwanyana eziphathwa nge-DHT.

I-ERcy iyadibana kunye ne-hcrt / orx kwaye idityaniswa kwindawo engafaniyo yabemi be-hcrt / orx neurons

I-Nuclear AR yasuswa ngaphakathi kwindawo ephambili ye-hcrt / orx ye-neuron, isasazeka ngokuphakathi ukusuka kwi-arcuate nucleus ukuya kwi-optic tract. Akukho matyala ii-ARS zenyukliya kunye ne-hcrt / orx zifunyenwe kwi-neuron efanayo (idatha ayiboniswanga). Nangona i-ERα ibonise iphethini efanayo yokuhanjiswa kwinqanaba le-hypothalamus, iilebula ze-ERα zazinkulu kakhulu kwi-nucleus ye-ventromedial. Ngapha nangapha, iilebheli ze-ERα zafunyanwa kwiibhendi ezinamathambo zeeseli ezandisiweyo phakathi kwiphilisi yangaphakathi phantsi kwe-zona incerta ukuya kwi-dorsomedial hypothalamus (DMH). Ilebheli ye-ERα yayingafane ifumaneke kwi-hcrt / orx neurons (<1% yabemi abavavanyiweyo). Xa i-ERcy yabonwa icolococize nge-hcrt / orx, yayiqhelekile kwii-neurons ngaphakathi okanye kwi-rostral nje kwi-DMH (Ikhiwane. 3). Ke, nangona imbalwa kakhulu i-hcrt / orx neurons kwi-hypothalamus ichaza i-ERcy, umlinganiselo omkhulu (∼65%) walawo abekwe ngaphakathi okanye kufutshane ne-DMH enza (Ikhiwane. 3).

Umzobo 3. 

I-ERcy iyadibana kunye ne-hcrt / orx. I-nercy nuclei imelwe zizangqa ezivaliweyo, ii-hcrt / orx neurons zimelwe zizangqa ezivulekileyo, kunye neeseli ezibhalwe kabini i-ERcy plus hcrt / orx iiseli zimelwe zinkwenkwezi. Amanani akwiimilimitha ezi-caudal ukuya kwi-bregma. I-AHN, i-Anterior hypothalamic nucleus; fx, fornix; ot, umthambo I-PVN, i-nucleus ye-patriyo I-VMH, i-hemothedial hypothalamus; I-3v, i-ventricle yesithathu.

I-Hcrt / orx receptor blockade imaketha ikopi

Xa kuthelekiswa nesithuthi, isenzo sokuxela kunye ne-OX1 I-antagonist SB 334867 inyuse intsingiselo yentshukumo ye-intromit kunye nokwehla kwesantya sokuhamba kwe-ejaculation (2 Table). Iisampulu ezibini t Iimvavanyo ziveze ifuthe elibalulekileyo le-SB 334867 kwii-latency zokungena ngaphakathi (t(8) = 3.31; p <0.05) kunye ne-ejaculation frequency (t(8) = 2.40; p <0.05). Unyango lweziyobisi lukwabonakala ngathi luvelisa ukunyuka okungabalulekanga kwiintsingiselo zentsingiselo yokunyuka kunye ne-ejaculate kunye nokuhla kwenani lokungeniswa (2 Table).

Ithebula 2. 

OX1 I-SB 334867 ye-antagonist ichaphazela indlela yokuziphatha kwamadoda

I-Hcrt-1 / orx-A inyusa izinga lokudubula le-VTA DA ye-Von kunye nomsebenzi wabemi kwaye yenza i-depolarization inactivation

Idosi ephantsi ye-hcrt-1 / orx-A (0.014 nmol) inyukayo ye-VTA DA ye-neuron yereyithi yokudubula ngokuthelekiswa nolawulo oluphethwe zizithuthi (Ikhiwane. 4B,C) (ukuthelekisa kabini; F(1,18) = 13.83, p <0.01; emva kokudityaniswa kwe-2 × 3 ANOVA, F(1,10) = 13.67, p <0.005), kodwa ayikho enye idosi ye-hcrt-1 / orx-A enale mpembelelo. Le dosi ibonakala inoxanduva lwesiphumo esiphambili se-hcrt / orx kwinqanaba lokudubula elifunyenwe kwi-2 × 3 ANOVA. A iposi (Tukey) uvavanyo aluvezi umahluko obonakalayo kumanqanaba aphakathi kweqela lolawulo oluphathwe yi-CSF kwidosi yonke. Idosi ye-1.4 nmol yedosi ye-hcrt-1 / orx-Into eyonyuswe kakhulu ye-VTA DA ye-neuron yenani labasebenzi (inani le-neurons elisebenza ngokuzimeleyo elifunyenwe kwingoma nganye yombaneIkhiwane. 4D) (ukuthelekisa ngambini, F(1,24) = 6.42, p <0.05; emva konxibelelwano olubalulekileyo kwi-2 × 3 exutywe ne-ANOVA, F(2,10) = 16.71, p <.001). Elona thamo liphezulu le-hcrt-1 / orx-A kuvavanyiwe (i-140 nmol) inciphise kakhulu umsebenzi wabemi bee-neurons ze-DA (iiseli ngengoma nganye) (Ikhiwane. 4D) (Amanyathelo aphindaphindeneyo yendlela i-ANOVA kwi-140 nmol idosi, F(2,6) = 12.20; p <0.01). Oku kuncipha kuguqulwe yinkqubo yenaliti ye-apomorphine (Tukey's iposi vavanyo).

Umzobo 4. 

I-Hcrt / orx ilawula umsebenzi we-VTA DA we-neuronal kwi vivo. AI-Waveform ye-VTA ye-neuron eqhelekileyo ebonisa iimpawu ezinokubakho kunye nokuqhekeka kwe-IS / SD. B, Phezulu, inqanaba lokudubula kunye nephethini ye-neuron ephathwe kwisithuthi esifanayo ibonisa isenzo esiqhelekileyo sokuqhuma; ngezantsi, i-neuron yokudubula ekhawulezayo eyabonwa emva kokungeniswa kwe-0.014 nmol ye-hcrt 1 / orx A (n = 4). C, D, Ubudlelwane bokuphulukana ne-hcrt-1 / orx-A yendawo ekuhliswe kuyo kwinqanaba lokudubula kunye nenqanaba labasebenzi be-VTA DA neurons. I-Hcrt-1 / orx-A (1.4 nmol; n = 5) yonyusa inani labemi. Umsebenzi oncitshisiweyo woluntu emva kwe140 nmol ye-hcrt 1 / orx A (n = 4) yatshintshwa yenkqubo ye-apomorphine (20 μg / kg). Inani elingaphakathi kwebar nganye libonisa inani lee-neurons ezirekhodiweyo. Ibonisiwe ziindlela ± SEM. *p <0.05; **p <0.01.

I-neurons ye-VTA eyi-VTA ene-hcrt / orx appositions ibonisa ukwanda kwe-Fos-ir emva kokukhuphela

Iindlela ezimbini ze-ANOVA kwinani elichanekileyo le-Fos-ir nuclei kwi-non-TH ye-neurons ebonakalayo yabonisa iimpembelelo zonyango (F(1,38) = 38.88; p <0.001) kunye nenqanaba leatomical (F(7,38) = 12.59; p <0.001), kunye nonxibelelwano olubalulekileyo phakathi kwezi zinto zimbini (F(7,38) = 7.45; p <0.001), ucebisa ukuba i-Fos inyanzeliswe ngexesha lokukhutshelwa kubonakala ngokukhethekileyo kumanqanaba okubala angaphandle. Oku kungqinwe yindlela elandelayo ye-ANOVA kunye iposi (Tukey) iimvavanyo, ezibonisa amanqanaba amabini e-anteriormost ("rostral" kunye "1 middle") ukuba babe namanani amakhulu e-Fos-positive nuclei (F(3,24) = 9.48; p <0.001). Kwakungeyonyani nolawulo lokungafotokiyo, apho i-basos Fos-ir yayingafaniyo ngenqanaba (3 Table). Ipesenti yee-neurons ezifakwe iilebhile nge-hcrt / orx appositions ayifani ngonyango lokuvavanywa; Nangona kunjalo, eli nyathelo libonise uphawu lwe-rostrocaudal ephawulweyo, elona nqanaba liphezulu kakhulu elinepesenti ephezulu kakhulu yezi neurons kuwo omabini amaqela (4 Table) (F(3,38) = 133.57; p <0.001). Oku kufumanisa kuyahambelana nokuxinana okuphezulu kwe-hcrt / orx fibers esiyiqapheleyo kwi-VTA yangaphambili. Ipesenti ye-TH enelebheli ye-neurons ebonisa i-Fos-ir (kodwa ingenazo i-hcrt / i-orx appositions) eyahlukileyo ngonyango okanye ngenqanaba le-rostrocaudal. Ipesenti yee-neurons ze-TH ezibonisa zombini iiFos kunye ne-hcrt / orx appositions zibonise umphumo obalulekileyo kunyango (F(1,38) = 8.62; p <0.01), inqanaba (F(3,38) = 4.53; p <0.01), kunye nonxibelelwano lwabo (F(3,38) = 4.53; p <0.01). Indlela enye ye-ANOVA kwiindlela ezivela kwiqela lokulinga icebisa ukuba i-Fos-ir ekhutshelweyo kwi-TH neurons ene-hcrt / i-orx appositions yenzeka kakhulu ngaphakathi kweeseli ezikwi-rostral VTA (F(3,24) = 4.85; p <0.05).

Ithebula 3. 

Inani le-Fos-ir nuclei kwiiseli ezingekho-ze-TH ze-VTA kumanqanaba amane e-rostrocaudal

Ithebula 4. 

Ipesenti ye-neurons ze-TH ezibonisa i-hcrt / orx appositions, Fos, okanye zombini

ingxoxo

I-Fos-ir ekhulayo kwi-hcrt / orx neurons kwi-LHA emva kokukhuphela iphakamisa ukuba ukuvulwa kwezi seli kuhamba neendlela zokuzala zamadoda (UMorgan noCurran, 1991). Ezi datha zihambelana nezifundo zangaphambilini ze-2-deoxyglucose ezixela ukwanda komsebenzi we-metabolic kwi-LHA emva kokubhengeza ivumba labesifazana (I-Orsini et al., 1985). Esi siphumo sinokubonakalisa ukuhanjiswa okuhambileyo kwe-hcrt / orx kwiindawo ezinesiphelo sendlela ye-hcrt / orx njenge-mPOA, apho kuye kwaboniswa i-hcrt / orx ukwenza ukuqhuba kokuziphatha kwendoda (UGulia et al., 2003). Ukungeniswa okunxulumene ne-Fos ngokwesondo kwi-hcrt / i-orx neurons kuyahambelana nombono wokuba i-hcrt / i-orx neurons inovakalelo kubaxhasi bendalo. Uphononongo lwamva nje lubonakalisile ukuba i-Fos-ir inyuke kwi-hcrt / orx neurons yeempuku ezimiselwe ukulindela umvuzo wokutya kwaye oku kunyuka kwe-Fos-ir kudityaniswe nenqaku lokukhetha kwezilwanyana kwindawo ekhethiweyo yeparadigm (Harris et al., 2005). Ukwenza kusebenze i-hcrt / orx neurons kunokuba yinto enxulumene nomvuzo, ngenxa yokuba olu phando lungentla lubonisa ukungabikho konyuka kwamanani kwiiseli zeFos-ir hcrt / orx kwizilwanyana ezivezwe kukhuthazo lwento yenoveli. Aba babhali baphinda baxele ipesenti ye-hcrt / orx neurons echaza i-basal (15%), i-novelty-indened (18%), kunye nemeko yokutya (50%) I-Fos-ir ehambelana nezo sizibika apha (12% basal vs 40 % uchukunyisiwe Oku kuqaphelekayo kuphakamisa ukuba i-hcrt / orx neurons isebenze ngemivuzo yendalo njengokutya kunye nesondo.

Umgaqo we-estrogenic we-hcrt / orx echazwe apha unika ubungqina obengezelelweyo bolawulo lwe-hypocretinergic yokuziphatha kwezesondo zamadoda. Kuyaphawuleka ukuba ikhosi yexesha lokulahleka kwe-hcrt / orx ichazwe apha iyahambelana nedatha yokuziphatha yokuziphatha ebonisa ukuba ukuziphatha ngokwesondo kwamadoda kuthatha iiveki ukwehla emva kokuphoswa, nangaphezulu, kukuba ngu-E2 endaweni ye-DHT eyimfuneko ekubuyiseleni isimilo kwakhona (Hull et al., 2006). Ngaphandle kokulinga ngakumbi, sinokuqikelela kuphela kwiindlela ezisisiseko sokubakho kwe-hcrt / orx xa kungekho E.2. Eyona nkcazo ilula inokuba ixesha eli-lag lokunciphisa amanqanaba e-hcrt / orx libonisa ulwazelelelo lokuya kuthi ga ngokukuko ngokudodobala kozinzo kwiivenkile ezi-vesicular zomdlulisi. Njengoko kuxoxwe ngezantsi, ukungabikho kwee-ERs kwii-hcrt / orx neurons kuphakamisa ukumiswa kwe-hcrt / orx expression ngamagalelo avela kwii-neurons eziqukethe ezo receptors. Kungenxa yokuba i-neuropeptide synthesis (Enyeart et al., 1987), motility (UShakiryanova et al., 2005), kunye nokukhululwa kweziganeko ezixhomekeke kwimisebenzi (UFulop et al., 2005), naluphi na usasazo lwasemva kokuhla kwe-hcrt / orx neuronal (Smith et al., 2002) inokuchaphazela kakubi ezi nkqubo, ukucutha i-kinetics yokukhutshwa kwe-peptide okunokuthi kuncitshiswe imigangatho yokuqulunqwa kungabonakali kangangexesha elithile. Ngayiphi na indlela, kubonakala ngathi isenzo se-steroid yinto yokuqala yekhasino eyinkimbinkimbi enoxanduva lokugcina amanqanaba asiseko e-peptide.

Njengoko i-hcrt / orx neurons ibonakala ilawula ukutya ukutya ekuphenduleni kwizinto ezihlazisayo ezinxulumene nokulingana kwamandla (I-Olszewski et al., 2003; Burdakov et al., 2006), ii-hcrt / orx neurons nazo zibonakalisa ukuba zinobuthathaka kwimitha ye-hormonal kwaye zinokuqhubela phambili ukuzala ngendlela efanayo. Idatha eboniswe apha ibonisa ukuba i-basal hcrt / orx expression igcinwe ngu-E2. Kwizilwanyana ezime kakuhle ezibonisa ukugcwaliseka ngokupheleleyo kwe-hcrt / orx, lo mhambisi unokuhambisa inkqubo kulwakhiwo olubalulekileyo ekuziphatheni ngokwesini kwamadoda kunye nomvuzo. I-hcrt / orx neurons iyakonwabela ukunxibelelana okukhulu okubuyayo kunye nendawo ezinjenge-mPOA, i-nucleus yebhedi ye-stria terminalis (BNST), kunye neVTA (I-Peyron et al., 1998; USakurai et al., 2005), ezaziwa ngokuba zibalulekile ekubonakalisweni kokuziphatha kwamadoda ngokwesondo (ukuphononongwa kwakhona, bona Hull et al., 2006). Ukuncipha kwe-hcrt / orx emva kokukhutshwa kuya kulindeleka ukuba kunciphise umthombo obalulekileyo wongenelelo kwezi zinto, ngokuziphatha konakalisayo.

Indlela yokusebenza kwe-ER egcina i-basal hcrt / i-orx expression ilindele ukufundelwa okongezelelweyo; Nangona kunjalo, kunokwenzeka ukuba iqhutywa zii-arhente ezivela kwiindawo zobuchopho ze-ERcy-ezichaza ubuchopho kwiprojekthi kwi-LHA (Simerly et al., 1990; Yoshida et al., 2006), ngakumbi ezo zixhobo zifunyenwe zinotyekelo oluthile [umzekelo, BNST kunye neMPOA (I-Georges kunye ne-Aston-Jones, 2002; UHenny noJones, 2006)]. Asikuxeli ukuba i-colocalization ye-AR ene-hcrt / orx, kwaye, nangona ii-neurons ezimbalwa ze-hcrt / orx zazine-ERcy-immunopositive, ezi seli azikho zininzi ngokwaneleyo ukuba zichaze iziphumo eziphawuliweyo zokuhanjiswa, okanye zingabhalisi kunye nezo zibonwe zinciphisa umxholo we-hcrt / orx emva kokukhutshwa (Amakhiwane. 2A, 3). Sikwaxela ukuba i-ERcy-ir nuclei kunye ne-hcrt / orx neurons zihlala zisenziwa, kuphakamisa ubukho bolawulo lwasekhaya lwe-hcrt / orx neuronal kunye nomsebenzi wentetho yobunjineli kweeseli ezikufutshane ne-ERcy. Ukubaluleka komsebenzi wesekethe wasekupheleni wolu hlobo sele uchaziwe kwinkqubo ye-hcrt / orx (Li et al., 2002). Nangona kunjalo, kude kufuneke ukuba uvavanyo lwe-anatomical lweemvavanyo lubonise ii-synapses ezivuselelayo ezenziwe ziiseli eziqukethe i-ERcy kwi-hcrt / orx neurons, ukuxhomekeka kwehormoni, intetho ehambelana nayo ye-hcrt / orx ayinakuthathwa. IHcrt ukuguquguqulwa kwamagama ngokuguquguqukayoI-Taheri et al., 2000), ngexesha lokukhulelwa (Kanenishi et al., 2004), kunye nokuphendula kwiindlela ezahlukeneyo zokutya (UCai et al., 1999; I-Griffond et al., 1999). Iindlela zeemolekyuli ezilawula amandla ezixhobo hcrt intetho ayonakaliswanga, ngaloo ndlela ukulandelela umendo osuka kwi-nucleus ukuya kulungelelwano, kunye nokuthitha amagama atyhila iimolekyuli ezinokuchaphazela i-hcrt / orx expression ngeli xesha linzima.

Uluvo lokuba ukuhanjiswa kwe-hcrt / orx lubandakanyeka ekomelezeni indlela yokuziphatha njengezesondo kuyaxhaswa yidatha ebonisa ukungalungiseki kwindlela yokuziphatha ngokwesondo emva kokunyangwa kunye ne-OX1 I-SB 334867 ye-antagonist2 Table). Kwidosi efana naleyo isetyenziselwa ukuvimba ukubuyiselwa koxinzelelo lwe-cocaineI-Boutrel et al., 2005), I-SB 334867 inyuse kakhulu i-latency yokungena kunye nokunciphisa inani le-ejuko, ukucebisa ukuba ukuvinjwa kwe-hcrt / i-orx yokuhanjiswa kunokuchaphazela iipropati zokukhuthaza abafazi ababhinqileyo.

I-DA ibalulekile kwi-neurotransmitter yomvuzo, inkuthazo yenkuthazo, kunye nokuziphatha okuguqukayo (EBerridge noRobinson, i-1998; I-Ikemoto nePanksepp, i-1999; Ubulumko, 2004). Siqwalasele isiphumo sethamo lokuxhomekeka ku-hcrt / orx kwimisebenzi ye-VTA DA ye-neuron. Oku kufumanisa kuxhasa indima ye-hcrt / orx ekuqiniseni kwaye iphakamisa ukuba inkqubo ye-mesolimbic ye-DA yindawo engaphandle kwe-mPOA apho uqikelelo lwe-hcrt / orx lunokuthi lisebenze ukuphucula isimilo sabesilisa. Kwidosi ephantsi ehlolwe (i-0.014 nmol), i-hcrt / okanye i-xx inyusa izinga lokudubula ngaphandle kokuchaphazela umsebenzi wabantu (iiseli / umkhondo) (Ikhiwane. 4C). Kwidosi ephakathi (i-1.4 nmol), ukwanda kwenqubo ye-neurons ye-DA yonyuswe, kubonisa ukuba ngaphambili i-quiescent, i-hyperpolarised neurons yavuselelwa (Ikhiwane. 4D). Kwidosi ephezulu (i-140 nmol), umsebenzi woluntu we-VTA DA neurons wehlisiwe. Into enomdla kukuba, ukuncitshiswa kwe-hTA ye-honc / i-orx ebangele umsebenzi we-Von DA yabuyiselwa umva kulawulo olungxamisekileyo lwe-apomistphine ye-DA (Ikhiwane. 4D). Kwizilwanyana eziqhelekileyo, i-apomorphine hyperpolarizing i-neurons ye-DA ngokuvula ii-autoreceptors kunye nokunciphisa izinga labo lokudubula kunye nenani labasebenzi. Nangona kunjalo, emva kokunyanga ngokungapheliyo kwe-antipsychotic okanye unyango oluphindaphindiweyo ngamachiza okuphatha gadalala, i-apomorphine inokubuyisela umva ukuncipha okwenziwe ziyobisi.UGrace et al., 1997; I-Shen kunye neChoong, i-2006). I-Apomorphine icingelwa ukuba ingaguqula ukungabinamsebenzi kokuphinda ispacarization inactivation ngokufaka kwakhona iiseli ezi-overexcited ngokwaneleyo ukuze ziphinde ziqale ngokudubula. Ngokudibeneyo, le datha iphakamisa ukuba i-hcrt / i-orx ikhupha isiphumo sokuxhomekeka kwidosi kwi-VTA DA neurons, ngokuhambelana nexesha elidlulileyo in vitro sebenza (I-Korotkova et al., 2003).

Ukubaluleka kokuhla kwi-LHA ukuya kwi-VTA kwavavanywa kwinani lovavanyo (I-Bielajew kunye ne-Shizgal, i-1986; I-Shizgal, i-1989; Uya et al., 2001). Ezi datha zibonisa ukuba inyuka kwi-NAc DA ngexesha lokuziphatha okuhle okuchukumisayo okunjengokukhuphela (Pfaus et al., 1990; Wenkstern et al., 1993) okanye ukondla (UHernandez kunye noHoebel, 1988; URada et al., 2005) inokuxhomekeka kwezi zinto ziqikelelweyo. Oko kuqikelelwa kuqukethe i-hcrt / orx kuxhaswe kwiimvavanyo apho i-NAc DA efflux ibonwa inyuka emva kwenaliti ye-VTA ye-hcrt / orx (UNarita et al., 2006). Ngaphakathi kwe-hypothalamus, i-serotonin [5-hydroxytryptamine (5-HT)] enokubakho ngokuchasene ne-hyperpolarize hcrt / orx neurons kwi-LHA (Li et al., 2002). Ukukhetha i-serotonin reuptake inhibitors okanye i-5-HT ngokwayo, iphinde yarhoxiswa idalekile kwi-LHA kufutshane neqela eliphambili labahlali be-hcrt / orx-expression expression, yanciphisa ukukhutshwa kwe-basal kunye ne-femc ye-NAc ye-DA kunye nokulinganisa okungalunganga (Lorrain et al., 1997, 1999). Ukukhanya kwedatha eboniswe apha ngasentla ebonisa ukusebenza kwe-hcrt / orx neurons ngexesha lokukhuphela kunye ne-VTA DA neurons yi-hcrt / orx, sithi ukuhla uqikelelo lwe-hcrt / orx kwi-VTA inokulamla ukukhutshwa okunxulumene nesondo kwe-NAc DA. Ngaphaya koko, ukuthintelwa koluqikelelo lwe-intra-LHA 5-HT kunokuchaza isithintelo se-5-HT malunga nokukhululwa kwe-NAc DA kunye nokuziphatha ngokwesondo.

I-anatomical substrate yokunxibelelana kwe-hcrt / orx-DA kubonakala ekufumaneni kwethu ukuba ii-neurons ze-VTA ze-VPS zifakwa ngaphakathi kwinkqubo ye-hcrt / orx kwaye zibonise ukwanda kwe-Fos-ir ngokubhengeza kukhuthazo olunomvuzo njengokopopayi (Ikhiwane. 5, 4 Table). Esi siphumo sibonisa ukubaluleka kokuziphatha kunye nokucaciswa kwendawo, njengoko ukungena kwe-Fos ku-hcrt / orx-apposed TH neurons kwafunyanwa kuphela kwi-VTA engaphandle yezilwanyana ezikopelayo. Mhlawumbi yinto engenakukhanyelwa ukuba le patheni yokuvula isebenze kule nxalenye yeVTA, njengoko iiseli ze-DA zilala nje zibazala abona bantu bokuqala be-hcrt / orx neurons, eyehla imicu yokuhamba kule ndawo ngaphambi kokusasaza kwiithagethi zecalal ezingaphezulu kwinkqubo ye-brainstem (I-Peyron et al., 1998).

Umzobo 5. 

Ukushicilela kukwenza i-Fos-ir in hcrt / orx-ekhethiweyo ye-VTA DA neurons. AI-Micrographs ibonisa i-TH, hcrt / orx, kunye neFos ilebhile kwiVTA. Imouse lithetha i-Fos-positive TH neurons ene-hcrt / orx appositions; ii-asteris ezimakisha ii-neurons ezilebheli kabini ngaphandle kosetyenziso. Ibha yesikali, i-45 μm. B, Iinkcukacha ze-Fos-positive TH neurons eneentolo ezibonisa iindawo ze-hcrt / orx boutons kulokusetyenzwa. C, Amacandelo e-Coronal abonisa amanqanaba e-Rostrocaudal ye-VTA esetyenzisiweyo ekubaleni (kumdaka mnyama). Amanani ngasentla ngasekhohlo kwecandelo ngalinye kukwimilimitha ukusuka kwibregma. Amanani ngasekunene aphezulu athetha ± Uqikelelo lwe-SEM lokuxinana kweseli kwinqanaba. Ibhokisi ibonisa indawo yee-micrographs ngaphakathi A.

Ezi datha ziphakamisa indlela yokubonisa inoveli apho i-gonadal steroids inokuchaphazela igalelo le-hypothalamic kwiinkqubo ze-DA ezigxininiswe kukuziphatha okuvuselelayo.Ikhiwane. 6). Bakongeza ezokuziphatha ngokwesondo kuluhlu olukhulayo lweendlela zokuziphatha ezilawulwa yi-hcrt / orx, kubandakanya ukuvuka, ukungaziphathi, kunye nokufuna iziyobisi.

Umzobo 6. 

Imodeli yokulawulwa kwe-hcrt / orx yi-gonadal steroids kunye ne-VTA DA yi-hcrt / orx. I-Estradiol, eyakhelweyo evela kwi-testosterone ye-gonadal yi-aromatase, isebenza kwi-neuronal equlathe ii-neurons kwi-BNST, mPOA, kunye ne-LHA. Ezi zakhiwo zeprojekthi kwii-hypothalamic hcrt / orx neurons. Ukuqikelela okubonakalayo kwezi zinto zinokuchaphazela umsebenzi we-hcrt / orx we-neuronal kunye nenkqubo yesenzi ngendlela enobuthathaka. Ukuqikelelwa kwe-Hcrt / orx kwi-VTA ukuphucula imisebenzi ye-midbrain DA ye-neuronal ngexesha lokuziphatha ngokwesondo kwamadoda. Esi siphumo sinokuthi sithintelwe yi-infra-LHA infusions ye-5-HT enqanda umsebenzi we-hcrt / orx, imeko yokuziphatha ngokwesondo kunye nokukhululwa kwe-NAc DA.

Imihlathi

  • Ifunyenwe ngo-Epreli 21, 2006.
  • Uhlaziyo lufunyenwe ngoJanuwari 23, 2007.
  • Wamkelwe ngoFebruwari 8, 2007.

  • Lo msebenzi uxhaswe ziNkxaso-mali yeNkonzo yoLuntu yezeMpilo yaseMelika i-MH073314 (JWM), MH40826 kunye neMH01714 (EMH), kunye ne-AA12435 (R.-YS). Sibulela uGqirha Samir Haj-Dahmane ngengcebiso zakhe eziluncedo kunye noGqr Zuoxin Wang ngokubonelela ngesixhobo se microscopy.

  • Imbalelwano mayibhekiswe kuJohn Muschamp, iSebe leengqondo, iYunivesithi yaseFlorida State, Tallahassee, FL 32306-1270. [imeyile ikhuselwe]

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