Ukutya okuPhakamileyo kwexesha elide kunciphisa i-Dopamine Reuptake ngaphandle kokuguqula ukucaciswa kweGenes yeDAT (2013)

  • UJackson J. Cone,
  • Elena H. Chartoff,
  • UDavid N. Potter,
  • UStephanie R. Ebner,
  • UMitchell F. Roitman

Abstract

Uphuhliso lokutyeba okubangelwa kukutya okubangelwa kukutya (DIO) kunokutshintsha ngokubonakalayo imiba emininzi yokubonakaliswa kwe-dopamine, kubandakanya ukubonakaliswa kwe-dopamine transporter (DAT) kunye ne-dopamine reuptake. Nangona kunjalo, ixesha lexesha lokutshintsha okubangelwa kukutya kwintetho ye-DAT kunye nomsebenzi kunye nokuba olo tshintsho luxhomekeke kuphuhliso lwe-DIO luhlala lungasonjululwanga. Apha, sondla iigundane eziphezulu (i-HFD) okanye i-low (LFD) yokutya okunamafutha kwi-2 okanye kwii-6 iiveki. Ukulandela ukuvezwa kokutya, iimpuku zaye zathotywa i-urethane kunye nomsebenzi we-DAT wokubulala wavavanywa ngokuvuselela ngombane imizimba yeeseli ze-dopamine kwindawo ye-ventral tegmental (VTA) kunye nokurekhoda utshintsho olunesiphumo kugxininiso lwe-dopamine kwi-ventral striatum kusetyenziswa i-voltammetry ye-scan ekhawulezayo. Sikwalinganisile isiphumo se-HFD kwi-membrane ehambelana ne-DAT kumaqhezu eseli e-striatal ukusuka kwiqela elahlukileyo leempuku ezilandela ukuvezwa kwenkqubo yokutya efanayo. Ngokucacileyo, akukho nalinye kumaqela ethu onyango elahlukileyo kubunzima bomzimba. Sifumene intsilelo kwireyithi ye-dopamine reuptake kwiigundane ze-HFD ngokunxulumene neempuku ze-LFD emva kwe-6 kodwa hayi iiveki ze-2 zokuvezwa kokutya. Ukongeza, ukonyuka kwe-dopamine ekhutshiweyo emva komceli mngeni we-pharmacological we-cocaine yancitshiswa kakhulu kwi-HFD xa kuthelekiswa neempuku ze-LFD. Uhlalutyo lwe-blot lwaseNtshona lubonise ukuba akukho mpembelelo yokutya kwiprotheni ye-DAT epheleleyo. Nangona kunjalo, iiveki ze-6 zokuvezwa kwe-HFD zinciphise kakhulu i-50 kDa DAT isoform kwi-synaptosomal membrane ehambelana neqhezu, kodwa kungekhona kwiqhezu elihambelana nokuphinda kusetyenziswe i-endosomes. Idatha yethu ibonelela ngobungqina obongezelelweyo botshintsho olubangelwa kukutya kwi-dopamine reuptake ngaphandle kotshintsho kwimveliso ye-DAT kwaye ibonisa ukuba olo tshintsho lunokubonakaliswa ngaphandle kophuhliso lwe-DIO. 

Citation: I-Cone JJ, i-Chartoff EH, i-Potter DN, i-Ebner SR, i-Roitman MF (i-2013) Ukutya okuPhakamileyo okuPhakamileyo kunciphisa i-Dopamine Reuptake ngaphandle kokuguqula i-DAT Gene Expression. PLoS ENYE 8(3): e58251. doi:10.1371/journal.pone.0058251

umhleli: USidney Arthur Simon, iZiko lezoNyango leYunivesithi yaseDuke, eUnited States of America

I funyenwe: Oktobha 26, 2012; Zamkelwa: NgoFebruwari 5, 2013; Yashicilelwe: Matshi 13, 2013

Copyright: © 2013 Cone et al. Eli linqaku elivulekileyo lokufikelela lisasazwe phantsi kwemiqathango yeLayisensi ye-Creative Commons Attribution, evumela ukusetyenziswa okungathintelekiyo, ukuhanjiswa, kunye nokuveliswa kwakhona kuyo nayiphi na indlela, ngaphandle kokuba umbhali wokuqala kunye nomthombo banikwe ikhredithi.

Inkxaso: Iprojekthi echazwe ixhaswa yi-National Institutes of Health (NIH) izibonelelo ze-DA025634 (MFR) kunye ne-T32-MH067631 kwiNkqubo yoQeqesho lwe-Biomedical Neuroscience (JJC). Inkxaso eyongezelelweyo yanikezelwa yiZiko leSizwe leMithombo yoPhando kunye neZiko leSizwe loPhando lweNzululwazi yokuGuqulela, i-NIH, ngesibonelelo se-UL1RR029877 (JJC) kunye ne-Chicago Biomedical Consortium ngenkxaso evela kwi-Searle Funds kwi-Chicago Community Trust (JJC). Umxholo kuphela uxanduva lwababhali kwaye akufuneki ukumela iimbono ezisemthethweni ze-NIH okanye iChicago Biomedical Consortium. Abaxhasi bemali babengenayo indima ekuyilweni kokufunda, ukuqokelela idatha kunye nohlalutyo, isigqibo sokupapasha, okanye ukulungiswa kombhalo wesandla.

Injongo yokunyanzela: Ababhali baye bavakalisa ukuba akukho mfuno ekhuphisanayo.

intshayelelo

Ukutyeba kakhulu kunye nokutyeba kubonisa ipesenti ekhulayo ye-United States kunye nabemi behlabathi jikelele [1], [2]. Ngelixa kukho iindlela ezininzi zokutyeba, mhlawumbi enye yezona zinto ziyingozi kakhulu kubunzima bomzimba obusempilweni kukuxhaphaka kunye nokusetyenziswa kokutya okunencasa kakhulu, okunecaloric eninzi. [3]. Inene, uxinaniso lwamandla (kcal/g) lokutya lunegalelo elikhulu ekutyebeni nasekutyebeni kwabantu abadala. [4], [5]. Ukutya okunencasa kuvuselela ukukhutshwa kwe-dopamine kwi-striatum yabantu kunye nezilwanyana ezingezozamntu [6], [7], [8], [9] kunye neereyithingi ezizimeleyo zokutyeba ekutyeni zinxibelelene kakuhle namandla eempendulo ze-neural kwi-ventral striatum. [10]. Ke, i-dopamine kunye ne-striatum zibonakala zinegalelo ekukhethweni kokutya okuxineneyo kwamandla. Kutshanje, kwaboniswa ukuba iyantlukwano ekutyeni inokubangela utshintsho ngaxeshanye kwisekethe ye-striatal kunye nokuziphatha okukhokelwa kukutya. [11]. Nangona kunjalo, mhlawumbi obungaxabisekanga bubungqina obukhulayo bokuba iyantlukwano ekutyeni okutyiweyo, ngakumbi ngokubhekiselele kumafutha, kunokunika ingxelo kunye nokutshintsha ukubonakaliswa kwe-striatal dopamine.

Ukubonakaliswa kweStriatal dopamine kulawulwa zizinto ezininzi ezibandakanya ukuveliswa kwe-dopamine nge-enzyme tyrosine hydroxylase, i-pre- kunye ne-postsynaptic dopamine receptors, kunye nabathuthi be-presynaptic dopamine (DATs), zonke eziye zabandakanyeka ekutyebeni. [12], [13]. Utshintsho kwinombolo ye-DAT okanye umsebenzi unokutshintsha inqanaba lempembelelo ye-dopamine ekhutshiweyo kwaye ngenxa yoko umsebenzi wokubetha. [14], [15]. I-insulin, ekhutshwe ngokuphendula ukutya okutyayo, ibonakaliswe ukuba inefuthe kumsebenzi we-DAT [16], [17]. Ngaloo ndlela, i-DAT ngomnye wabaviwa ekunokwenzeka ukuba iziphumo zokutya.

Kutshanje, unxulumano phakathi kokutyeba kakhulu kunye nokufumaneka kwe-DAT kunye nokuguqulwa kokutya okubangelwa kukutya komsebenzi we-DAT kuye kwajongwa. Isalathisi sobunzima bomzimba (BMI) sinxulunyaniswa kakubi nokufumaneka kwe-DAT kwi-striatum yabantu [18]. Ukubophelela kwe-DAT, kungoko ukufumaneka, kuncitshiswe kukutya okunamafutha aphezulu (HFD) ezondliwe iimpuku [19]. Ukukhuluphala okubangelwa yi-HFD (DIO) inxulunyaniswa nesantya esincitshisiweyo sokuphinda kuthathwe kwakhona i-dopamine yi-DAT kwiimpuku. [20]. Zithathiwe kunye, ezi zifundo zicebisa ukuba ukutyeba okusekwe kukusetyenziswa kwe-HFD kunokuba nefuthe elinamandla kubalawuli be-presynaptic ababalulekileyo bokusayinwa kwe-dopamine - ngakumbi i-DAT. Nangona kunjalo, ixesha lotshintsho olubangelwa kukutya ekubonakalisweni kwe-dopamine kunye nokuba uphuhliso lwe-DIO luyimfuneko ukuze utshintsho lubonakaliswe luhlala lungaziwa. Sivavanye umsebenzi we-DAT ngokukhupha ukukhutshwa kwe-dopamine kwi-ventral striatum kunye nokulinganisa izinga layo lokuphinda kuthathwe iimpuku usebenzisa i-voltammetry ekhawulezayo yokuskena. Ukufumanisa ukuba ukuhla kwe-dopamine ukuphinda kuthathwe kwakhona kubangelwe kukuncitshiswa kokubonakaliswa kofuzo lwe-DAT, silinganise i-DAT mRNA kwindawo ye-ventral tegmental kunye ne-substantia nigra sisebenzisa ixesha langempela le-qRT-PCR. Ukongeza, sisebenzise inkqubo yokwahlulwa kwe-biochemical kunye nohlalutyo lwe-blot yaseNtshona ukuvavanya amanqanaba e-DAT e-striatal kwi-synaptosomal ekrwada kunye ne-endosomal membranes. Iigundane zineeveki ezi-2 okanye ze-6 zokutya okunamafutha aphezulu okanye aphantsi, kodwa yonke imilinganiselo yenziwa ngokungabikho kwe-DIO. Iziphumo zethu zibonisa ukuba ukusetyenziswa ixesha elide kwe-HFD, ezimeleyo kwi-DIO, yehlisa isantya sokuphinda kuthathwe i-dopamine kwi-ventral striatum ngaphandle kokunciphisa ukubonakaliswa kwe-DAT.

Impahla nenkqubo

Statement Ethics

Olu phononongo lwenziwe ngokuhambelana ngqongqo kunye neengcebiso kwiSikhokelo sokuKhathalela kunye nokuSetyenziswa kweZilwanyana zeLabhoratri zamaZiko ezeMpilo eSizwe. Iprothokholi yamkelwa yiKomiti yoLondolozo lweZilwanyana kwiYunivesithi yase-Illinois, eChicago. Lonke utyando lwenziwa phantsi kwe-urethane anesthesia, kwaye zonke iinzame zenziwe ukunciphisa ukubandezeleka.

I zifundo

Iigundane eziqhelekileyo ze-Sprague-Dawley (n = 67), malunga neenyanga ze-2 ubudala kunye nobunzima be-225-275 g ekufikeni kwazo zasetyenziswa. Izilwanyana zahlaliswa ngabanye kwiikheji zeplastiki (26.5×50×20 cm) kwiqondo lobushushu- (22°C) kunye nokufuma- (30%) kubume obulawulwayo ekukhanyeni kwe-12∶12 h: umjikelo omnyama (izibane zikhanyisa kwi-07∶00 h). Iimpuku ziqhelene nesibonelelo iveki enye nge ad adum ukufikelela kwi-lab chow eqhelekileyo kunye namanzi.

Ukutya kokutya kunye neMilinganiselo yobunzima bomzimba

Emva kwe-acclimation, iigundane zilinganiswe kwaye zabelwa ngokungenamkhethe kwi-1 yamaqela e-4 aye aphikisana nobunzima bomzimba bokuqala. Amaqela amabini agcinwe kwi-fat fat diet (LFD; Izidlo zoPhando, i-New Brunswick, i-NJ; i-D12450B; i-10% ye-kilocalories kumafutha (3.85 kcal / g)). Amanye amaqela e-2 agcinwe kwi-HFD (Izidlo zoPhando; i-D12492; i-60% ye-kilocalories ukusuka kumafutha (5.24 kcal / g)). Kukutya ngalunye, iimpuku zagcinwa nokuba ziiveki ezi-2 okanye ezi-6 (iiveki). Ngaloo ndlela, amaqela e-4 aye: LFD-2 wk (n = 18), i-HFD-2 wk (n = 16), i-LFD-6 wk (n = 16) kunye ne-HFD-6 wk (n = 17). Onke amaqela ayenayo ad adum ukufikelela emanzini. Ukutya kokutya kunye nokulinganisa ubunzima bomzimba kwenziwa kathathu / i-wk kwaye idatha ichazwe ngokwahlukileyo kwiigundane eziphantsi kokurekhoda kwe-voltammetric okanye iprotheni ye-DAT / uhlalutyo lomyalezo.

Iinkqubo zoTyando kunye nemilinganiselo yeDopamine

Ukulandela ukuvezwa kokutya, i-subset yeegundane ezingazange zihluke kubunzima bomzimba zilungiselelwe ukurekhodwa kwe-voltammetric (LFD-2 wk (n = 8), HFD-2 wk (n = 6), LFD-6 wk (n = 6) , kunye ne-HFD-6 wk (n = 7)) phantsi kwe-urethane (1.5 g / kg) i-anesthesia [njenge-9,21]. I-cannula yesikhokelo (i-Bioanalytical Systems, i-West Lafayette, i-IL) ibekwe ngaphezu kwe-ventral striatum (i-1.3 mm yangaphambili, i-1.5 mm esecaleni ukusuka kwi-bregma), intambo yesilivere eneklorini (i-Ag / AgCl) i-electrode yereferensi yafakwa kwi-cortex ye-contralateral kwaye zombini zaye zafakwa. zikhuselwe kukhakhayi ngezikrufu zentsimbi engatyiwayo kunye nesamente yamazinyo. I-micromanipulator equkethe i-carbon-fiber electrode (CFE) ifakwe kwi-cannula yesikhokelo kwaye i-electrode yathotywa kwi-ventral striatum. I-CFE kunye ne-electrode yereferensi zixhunywe kwi-headstage kwaye amandla e-CFE ahlaziywa ukusuka -0.4 ukuya ku- +1.3 V (vs. Ag / AgCl) kunye nomva (400 V / s; 10 Hz). I-electrode evuselela i-bipolar (i-Plastics One, i-Roanoke, i-VA) yabe iyancipha ngokuthe ngcembe kwindawo ye-ventral tegmental / substantia nigra pars compacta (VTA / SNpc; 5.2 mm ngasemva, i-1.0 mm ecaleni kwaye ekuqaleni i-7.0 mm ventral ukusuka kwi-bregma) kwi-0.2 mm ngokunyuka . Kunyuso ngalunye, uloliwe weepulses zangoku (60 pulses, 4 ms per pulse, 60 Hz, 400 µA) waziswa. Xa i-electrode evuselelayo ibekwe kwi-VTA/SNpc kwaye i-CFE ikwi-striatum, uvuselelo luvuselela ngokuthembekileyo ukukhutshwa kwe-dopamine-ikhutshwe kwidatha yevoltammetric kusetyenziswa uhlalutyo lwecandelo eliphambili. [9], [22]; kwaye iguqulelwe kugxininiso emva kokuba i-CFE nganye ilinganiswe kwinkqubo yokutofa ngokulandela umfuniselo ngamnye [23]. Isikhundla se-electrode evuselelayo silungiselelwe ukukhululwa okukhulu. I-CFE yavunyelwa ke ukuba ilingane i-10 min phambi kokuba iqalise uvavanyo. Ukukhutshwa kwe-Dopamine kuvuselelwe kukuvuselelwa kombane kweVTA/SNpc (iiparamitha ezifanayo nezingasentla), kwaye utshintsho oluye lwaphumela ekugxilweni kwe-dopamine lubalwe ukusuka kwi-−5 s ukuya kwi-10 s ngokunxulumene novuselelo. Ngokukhawuleza emva kokuvuselela, iigundane zafakwa nge-cocaine hydrochloride echithwe kwi-0.9% ye-saline (10 mg / kg ip) kwaye, i-10 min kamva, ukuvuselela kwaphindwa. Amandla ombane asetyenzisiweyo, ukufunyanwa kwedatha, kunye nohlalutyo lwenziwa kusetyenziswa isoftware ebhalwe kwiLabVIEW (Izixhobo zeSizwe, iAustin, TX, USA) [22].

I-Dopamine Reuptake

I-Dopamine iphinda isetyenziswe i-Demon Voltammetry Analysis Software (24; i-Wake Forest University, iWinston-Salem NC). Apha sinika ingxelo yokubola rhoqo njenge-tau yokulinganisa kwethu izinga lokuphinda kuthathwe i-dopamine. I-Tau ithathwe kwi-exponential curve fit equka uninzi lwe-dopamine clearance curve kwaye inxibelelene kakhulu (r = .9899) ne-K.m, ukunxulumana okubonakalayo kwe-dopamine ye-DAT [24]. Ukumisela isiphumo se-cocaine kwincopho yoxinaniso lwe-dopamine siye sathelekisa amaxabiso afunyenwe ngaphambi nasemva kolawulo (% utshintsho).

Histology

Emva kokurekhodwa ngalunye, i-electrode yensimbi engenasici (AM Systems #571500, Sequim, WA) yathotywa ukuya kubunzulu obufanayo ne-CFE kunye ne-lesion (10 µA, 4 s) yenziwe ukuphawula indawo yokurekhoda. Ubuchopho bususiwe kwaye bugcinwe kwi-10% ye-formalin. Imakroskopu ekhanyayo yasetyenziselwa ukuchonga indawo yesilonda kumacandelo e-coronal (50 µm) nge-striatum. Zonke ushicilelo oluxeliweyo apha lwenziwa kwi-ventral striatum [25].

I-Subcellular Fractionation ye-Striatal Tissue

Iigundane (LFD-2 wk, HFD-2 wk, LFD-6 wk, kunye ne-HFD-6 wk; n = 10 / iqela; akukho mmahluko kubunzima bomzimba) babulawa ngokuchithwa. Ukuhlukaniswa kwe-biochemical kwenziwa ngokusebenzisa iprotocol echazwe kuyo [26], ngohlengahlengiso olungephi. Ubuchopho bususwe ngokukhawuleza, bufakwe kwi-isopentane kwaye bunqunywe kwi-cryostat (HM505E, iMicrom, iWalldorf, eJamani, -20 ° C) de ifike kwi-striatum. Amacala amabini 1-mm3 amanqindi kwi-ventral striatum (i-avareji yobunzima bethishu: 15.2 mg) yenziwe i-homogenized kwi-20 s kwi-0.8 ml ye-TEVP ebandayo yomkhenkce (10 mM Tris base, 5 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM EGTA, pH 7.4) +320 mM sucrose buffer. I-aliquot ye-100 µl ye-homogenate epheleleyo (H) yagcinwa. Intsalela ye-H yayiyi-centrifuged kwi-800×g ye-10 min kwi-4 ° C. I-pellet (P1, i-nuclei kunye ne-debris enkulu) yaphinda yamiswa kwi-0.2 ml ye-TEVP buffer kwaye igcinwe. I-supernatant (S1) yasuswa kwaye yafakwa kwityhubhu ecocekileyo emkhenkceni. I-S1 yayiyi-centrifuged kwi-9200 × g ye-15 min kwi-4 ° C ukuvelisa i-pellet (P2, i-crude synaptosomal membranes) kunye ne-supernatant (S2). I-P2 yahlanjululwa kanye kwi-TEVP + 35.6 mM i-sucrose buffer kwaye yaphinda yamiswa kwi-0.25 ml ye-TEVP + 35.6 mM i-sucrose buffer, i-vortexed ngobumnene kwi-3 s kunye ne-hypo-osmotically lysed ngokugcina isampuli kwi-ice 30 min. I-Supernatant (S2) yaqokelelwa kwaye yaphonswa kwi-165,000 × g ye-2 h ukuvelisa i-pellet (i-P3, i-membrane yokukhanya, i-endosomes yokubuyisela kwakhona) eyaphinda yamiswa kwi-TEVP (0.1 ml) kwaye igcinwe. Zonke iisampuli zagcinwa ku-−80°C de i-polyacrylamide gel electrophoresis.

I-Gel Electrophoresis kunye ne-Western Blotting

Umxholo weprotheyini unqunywe kusetyenziswa i-Bio-Rad DC Protein Assay kit (Hercules, CA), kwaye ukuxinwa kwesampulu nganye kulungiswe kwiprotheni ye-0.3 mg / ml. I-NuPAGE LDS (i-lithium dodecyl sulfate) i-buffer yesampula (Invitrogen, Carlsbad, CA) kunye ne-50 mM dithiothreitol yongezwa kwisampuli nganye ngaphambi kokufudumeza kwi-70 ° C kwi-10 min. Ukulayisha ixabiso elilinganayo leprotheyini kwiqhezu ngalinye, i-3 µg yesampuli nganye ilayishwe kwi-NuPAGE Novex 4-12% Bis-Tris gels (Invitrogen) yokwahlula nge-gel electrophoresis. Iiprotheyini zaye zatshintshelwa emva koko kwi-polyvinylidene fluoride membrane (PVDF) (PerkinElmer Life Sciences, Boston, MA). Iziza ezibophelelayo ezingachazwanga ziye zavalwa ngeyure ezi-2 kwiqondo lobushushu begumbi kwi-blocking buffer (5% nonfat ubisi olomileyo kwi-PBS kunye ne-0.02% Tween 20 [PBS-T]). Amablothi aye afakwa kwi-antibody yokuqala (1∶3000 mouse monoclonal anti-NR2B [#05–920, Millipore], 1∶5000 anti-DAT yomvundla [#AB2231, Millipore], kunye ne-1∶1000 monoclonal anti-receptor TfR) [#13–6800, i-Invitrogen]. Amablothi asikwa abe ngamacandelo ama-3: aphezulu (>97 kDa), aphakathi (46–97 kDa), kunye nobunzima obuphantsi (<46 kDa) kunye nelungu ngalinye lihluzwe nge-antibody eyaziwayo. Iprotheyini ephakathi kolu luhlu lobunzima Ubunzima obucacileyo bemolekyuli kwizilwa-buhlungu ezisetyenzisiweyo zezi: NR2B, 180 kDa, DAT, 75, 64, kunye ne-50 kDa, TrfR, 95 kDa. kunye ne-stripping buffer (62.5 mM Tris, 2% SDS, 100 mM β-mercaptoethanol, pH 6.8) kwimizuzu eyi-15 kwi-50 ° C. Amabhulothi emva koko aphinde avalwa kwaye aphenywa nge-anti-TfR. SeeBlue Plus 2 (Invitrogen) kwangaphambili- imigangatho enebala yaqhutywa kuqikelelo lobunzima bemolekyuli.

I-protein immunoblots yahlalutywa kusetyenziswa i-Carestream Molecular Imaging Software 5.0. Ubunzulu besambuku (isimbuku seepixels ngaphakathi kwebhendi yomdla thabatha isixa seepixels ezingasemva) zimiselwe kwibhendi nganye. Ukuvumela ukuthelekisa phakathi kwamablothi, idatha yayiqhelekile kulawulo lweLFD kwi-2 kunye ne-6 wks. Idatha ivakaliswa njenge-fold fold induction xa kuthelekiswa neLFD ± SEM.

Ubungakanani bexesha lokwenyani lokuReverse Transcriptase Polymerase Chain Reaction (qRT-PCR)

Ukulandela ukuqokelelwa kweeputshi zokubethelwa kuhlalutyo lwe-blot yasentshona, ubuchopho obunomkhenkce bahlulwa ngokwe-coronally kwi-microtome de bafike kwi-VTA/SN. Amacala amabini 1-mm3 iipuntshi ze-VTA kunye ne-SN tissue (i-avareji ye-tissue weight = 15.0 mg) yenziwe kwaye i-RNA ikhutshwe kusetyenziswa i-PureLink RNA Mini Kit (Invitrogen). Umgangatho we-RNA kunye nobuninzi bahlolwe kusetyenziswa i-RNA 6000 Nano Chip (Agilent, Santa Clara, CA) kwi-Agilent Bioanalyzer 2100. Inombolo yengqibelelo ye-RNA (RIN) idlule i-7 kuzo zonke iisampuli, ebonisa umgangatho ophezulu. I-microgram enye ye-RNA iyonke isetyenziselwe ukudibanisa i-cDNA kusetyenziswa i-iScript cDNA Synthesis Kit (BioRad) kwi-ThermoHybaid iCycler (i-Thermo Scientific). Iiprimers ezithe ngqo ze-DAT (Slc6a3; I-primer ephambili: GGAAGCTGGTCAGCCCCTGCTT, i-reverse primer: GAATTGGCGCACCTCCCCTCTG), β-actin (Nba; I-primer ephambili: AGGGAAATCGTGCGTGACAT; i-primer ebuyela umva: AAGGAAGGCTGCTGGAAGAGAnding ye-TATTCC; i-protein ye-TATTCCAGATGC; GTGCC; Ukubuyisela umva iprimer : GCTCCTGTGCACACCATTTTCCC) iijini (iinombolo zofikelelo lweGenbank NM_012694, NM_031144, kunye ne-NM_001004198) zayilwa kusetyenziswa iNCBI Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) yaza yathengwa kwi-Integrated DNA Technologies (Coralville, Iowa). Uhlalutyo lwe-Melt curve kunye ne-polyacrylamide gel electrophoresis luqinisekisile ukucaciswa kweeprimers. I-DAT, i-β-actin, kunye ne-Tbp amplicons ziyi-266, i-182, kunye ne-136 izibini ezisisiseko ubude, ngokulandelanayo.

Ikhithi ye-Q-PCR (iQ SybrGreen Supermix, i-BioRad) isetyenzisiwe. Ukusabela kwaqhutywa kwi-MyiQ eSingle Color ye-Real-Time PCR Detection System (BioRad) kumthamo we-20 µl, kunye ne-2 µL ye-3 µM phambili kunye ne-primers ebuyela umva kunye ne-4 µL cDNA isampulu exutywe 1∶10. Iimeko zebhayisikili ze-PCR zaziyi-95 ° C kwi-5 min; Imijikelezo ye-40 kwi-94 ° C kwi-15 s, i-60 ° ye-15 s, i-72 ° C ye-15 s. Idatha iqokelelwe kwiqondo lokushisa lokufunda le-84 ° C kwi-15 s ngokusekelwe kwiqondo lokushisa le-amplicon yokunyibilika. Iigophe ze-dilution ezisemgangathweni zenziwe kwi-primer nganye esetwe ngokuxutywa kwe-serial (1.00, 0.2, 0.04, kunye ne-0.008-fold) isitokhwe se-cDNA esiyinkosi esiquka umxube olinganayo we-cDNA kuwo onke amaqela onyango. Ilog10 amaxabiso odilution acwangciswe ngokuchasene nexabiso lomjikelo wemigangatho yeegophe eziqhelekileyo. I-MyiQ Optical System Software (BioRad) yayisetyenziselwa ukuhlalutya idatha. Iisampulu ezingenanto itemplate ye-cDNA kunye neesampulu ezivela kwiimpendulo ze-cDNA eziqulethe i-reverse transcriptase ziqhutywe njengolawulo lokungcoliseka kunye nokukhulisa i-genomic DNA, ngokulandelanayo. Amaxabiso axeliweyo aye aqhelaniswa nexabiso eliphakathi kwimigangatho yangaphakathi ye-ß-actin kunye ne-Tbp kwisampulu nganye. Idatha ichazwa njengemilinganiselo enxulumene ne-DAT/imigangatho yangaphakathi ye-mRNA±SEM.

Uhlalutyo lweSatisati

Inkcazo ye-DAT iyatshintsha ngokuguquguqukayo ngexesha lomjikelo wobomi kubo bobabini abantu [27] kunye neempuku [28], [29]. Ukongeza, i-dopamine kunye nendlela yokuziphatha kwi-cocaine nayo iyatshintsha njengoko iimpuku ezincinci zikhula [30]. Ngaloo ndlela, imilinganiselo ye-DAT inokwahluka kunye nobudala kwaye inqande uthelekiso olunentsingiselo phakathi kwe-2 wk kunye namaqela e-6 wk. Ke ngoko, iqela lithetha ukutya okutyiwayo, ubunzima bomzimba, incopho yoxinaniso lwe-dopamine, i-tau, utshintsho lwe-%, kunye nokubonakaliswa kofuzo oluzalanayo kwathelekiswa ngokwahlukeneyo kumaqela e-2 kunye ne-6 wk esebenzisa uvavanyo lwe-t olungabhangqwanga loMfundi. Kuhlalutyo lweblot yasentshona, iyantlukwano yeqela kubunzulu bebhanti yeDAT eqhelekileyo yathelekiswa ngokwahlukeneyo kumaqela e-2 kunye ne-6 wk esebenzisa iindlela ezimbini eziphindaphindiweyo-imilinganiselo ye-ANOVA (dietXfraction). Lonke uhlalutyo lwamanani lwenziwa kwiGraph Pad 5 (Prism Inc.).

iziphumo

I-HFD ikhuthaza ukuSetyenziswa kweFat

Ngaphambi kokuqala kokubonakaliswa kokutya kwakungekho nantlukwano kubunzima bomzimba bokuqala kwi-2 wk (LFD: 275.22 +/-4.1 g; HFD: 280.87 +/-4.8 g; p = 0.37), okanye 6 iiveki (LFD: 287.31+/−4.9 g; HFD: 289.44+/−5.1 g; 6 iiveki p = 0.97) amaqela. Ngaphandle kokutya ukutya okunokwakheka okwahlukileyo kakhulu, asifumananga mahluko kubunzima bomzimba phakathi kwamaqela okutya alandelayo okanye iiveki ezi-2 okanye ezi-6 (Isazobe 1a–b; zombini ns). Kwakungekho mahluko kwi-kcals iyonke esetyenzisiweyo phakathi kwamaqela alandela zombini ii-2 kunye ne-6 iiveki zokuvezwa kokutya (Isazobe 1c–d; ns). Nangona kunjalo, iimpuku ze-HFD zitye kakhulu kcals kumafutha.Umzobo 1e-f; Iiveki ezi-2: t(32) = 25.59; iiveki ezi-6: t(31) = 27.54; p<0.0001 kuzo zombini ixesha lokutya).

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Umzobo 1. Ukutya kunye nemilinganiselo yobunzima bomzimba.

Kwakungekho mahluko phakathi kwe-HFD kunye ne-LFD kubunzima bomzimba bokugqibela (a–bokanye iikilocalories zizonke ezisetyenzisiweyo (c–d) kulandela iiveki ezi-2 okanye ezi-6 zokuvezwa kokutya. (e–fIigundane ze-HFD zidle kakhulu i-kilocalories ukusuka kumanqatha kuneegundane ze-LFD kuzo zombini iiveki ze-2 kunye neemeko zeeveki ze-6 (***p<0.001).

http://dx.doi.org/10.1371/journal.pone.0058251.g001

Ixesha elide i-HFD Yehlisa ireyithi yokuphinda kuthathwe kwakhona iDA

Ukurekhodwa kweVoltammetric kwenziwa kwi-ventral striatum (Umzobo 2). Umzobo 3 ibonisa utshintsho olukhutshwe ngumbane kugxininiso lwe-dopamine olufunyenwe kwiimpuku ezilandela iiveki ezi-6 zokutya. Kwisiseko, ubukhulu be-dopamine ekhutshiweyo abuhlukanga phakathi kwamaqela okutya kunye nexesha lokutya lonke (Isazobe 4a–b, zombini ns). Nangona kunjalo, ukuhlolwa kwemizekelo yomntu ngamnye kucebise ukuba izinga lokubola kulandela incopho ye-dopamine yoxinaniso yahluke phakathi kwamaqela okutya emva kwe-6 wks yokuvezwa kokutya.Umzobo 3 a–b imizekelo). Izinga lokubola libangelwa ngokuyintloko kucoceko lwe-dopamine yi-DAT [31], esiye sayimodareyitha njengenqanaba elinye lokubonisa i-tau. Kwakungekho mahluko phakathi kwamaqela okutya alandelayo kwiiveki ezi-2 zokuvezwa kokutya (Umzobo 4c). Nangona kunjalo, emva kwee-6 iiveki zokuvezwa kokutya, i-tau yayinkulu kakhulu kwiigundane ze-HFD-6 wk ngokumalunga ne-LFD-6 wk (Umzobo 4d; t(11) = 2.668; p<0.05). Ke, ii-6 wks ze-HFD zehlisa inqanaba lokucaciswa kwe-dopamine kwi-ventral striatum xa kuthelekiswa nezilwanyana ezitya i-LFD.

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Umzobo 2. Ukuqinisekiswa kwe-Histological yeendawo zokurekhoda ukuhlalutya kwakhona.

Iindawo zokurekhodwa kweempuku ze-LFD zifakwe iikhowudi ngonxantathu ongwevu kunye neempuku ze-HFD ngezangqa ezimnyama. Amanani abonisa umgama kwi-mm ngaphambili ukuya eBregma. Umfanekiso uthatyathwe kwi-Paxinos kunye neWatson ngo-2006.

http://dx.doi.org/10.1371/journal.pone.0058251.g002

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Umzobo 3. Ukukhuthazwa kombane kweVTA/SNc kuvuselela i-phasic spike kugxininiso lwe-dopamine.

Imizekelo emele yedatha efunyenwe emva kweeveki ze-6 zokuvezwa kokutya. a) I-background-subtracted color color ibonisa utshintsho lwangoku kwiimpawu ezahlukeneyo ze-electrode ngaphambi (-5 ukuya kwi-0 s ngokumalunga nokuqala) nasemva (0.1 ukuya kwi-10 s ngokumalunga nokuqala) ukuvuselela kombane (STIM) ye-VTA / Snc. Ixesha liyi-abscissa, amandla e-electrode yi-ordination, kwaye utshintsho lwangoku lufakwe ngekhowudi kumbala wobuxoki. I-Dopamine [echongiwe nge-oxidation yayo (+0.6 V; eluhlaza) kunye nokunciphisa (-0.2 V; i-blue) iimpawu] zanda ngokukhawuleza ekuphenduleni ukuvuselela kule rat ye-LFD-6 wk. b) Kuyafana naku-a), ngaphandle kwe-HFD-6 wk rat. c) Ugxininiso lweDopamine njengomsebenzi wexesha lukhutshwa kwibala lombala kwi-a) kwaye i-tau ichongwa ngokulingana kwegophe. Amachaphaza amabini abomvu aphawula incopho kunye nokugxilwa kwe-dopamine ngexesha apho i-tau ifikelelwe. UTau uboniswe ekunene. d) Kuyafana naku-c) kodwa idatha itsalwa ku-b).

http://dx.doi.org/10.1371/journal.pone.0058251.g003

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Umzobo 4. Iiveki ezintandathu zokutya okunamafutha aphezulu kunciphisa izinga lokuphinda kuthathwe kwakhona i-dopamine kwaye kuthintele impendulo ye-dopamine kwi-cocaine.

Umndilili wencopho ye-dopamine yoxinaniso ekhutshwe yi-VTA/SNpc yokuvuselela emva nokuba yi-2 (a) okanye iiveki ezi-6 (b) kokuvezwa kokutya phambi kokutofa kwecocaine. c–d) I-avareji yeTau elandelayo 2 (c) iiveki okanye iiveki ezi-6 (d) Ukuvezwa kokutya. I-Tau yayinkulu kakhulu kwi-HFD-6 wk iimpuku xa kuthelekiswa neempuku ze-LFD-6 wk (*p<0.05). e–fUtshintsho lwepesenti kwincopho luvuse ugxininiso lwe-dopamine emva kwenaliti ye-cocaine ye-2 (e) kunye ne6 (f) iiveki zokuvezwa kokutya. Utshintsho lwepesenti lwaluncinci kakhulu kwi-HFD-6 wk xa kuthelekiswa ne-LFD-6 wk rats (**p<0.01).

http://dx.doi.org/10.1371/journal.pone.0058251.g004

I-HFD ende Yehlisa iMpendulo yeDA kuCocaine

Ukuphonononga ngakumbi utshintsho olubangelwa kukutya kwi-DAT, satofa iimpuku nge-DAT blocker cocaine. Incopho ye-dopamine yoxinaniso olulandela uvuselelo lombane lubangelwa kukukhutshwa kwe-dopamine kodwa ikwathintelwa kukususwa ngaxeshanye kwe-dopamine yi-DAT. [21]. Sibonise ifuthe le-cocaine kukuhanjiswa kwe-dopamine ngokubala utshintsho kubungakanani be-dopamine ekhutshiweyo kumaxabiso angaphambi kweziyobisi (% utshintsho). Iiveki ezimbini ze-HFD azizange zichaphazele utshintsho lwe-% ngokunxulumene neLFD (Umzobo 4e; ns). Nangona kunjalo, ukulandela iiveki ezi-6 zokuvezwa kokutya, utshintsho lwe-% lwalungacacanga kakhulu kwi-HFD ngokumalunga ne-LFD (Umzobo 4f; t(10) = 4.014; p<0.01). Iziphumo zethu zibonisa ukuba i-6, kodwa hayi ii-2 wks, zokuvezwa kwe-HFD kunciphisa impendulo ye-dopamine kwi-cocaine.

Ukubonakaliswa kwexesha elide kwe-HFD kunciphisa iProtein ye-DAT ukubonakaliswa kwi-Synaptosomal Membranes

Ukufumanisa ukuba iziphumo ze-HFD ezinde zibangelwa utshintsho kwinani le-DAT, amanqanaba eprotheyini e-DAT aye alinganiswa kwii-homogenates ze-tissue zizonke (i-H fractional), i-synaptosomal membranes (i-P2 fraction) kunye ne-intracellular recycling endosomes (i-P3 iqhezu). DAT yi NI-glycoprotein edibeneyo kunye nobunzima obubonakalayo bemolekyuli phakathi kwe-50 kunye ne-80 kDa ngenxa yokunyuka kwamanqanaba e-glycosylation njengoko iprotheni ikhula. [32]. Ulwahlulo lwaqinisekiswa ngokubonakaliswa okutyetyisiweyo kwe-NR2B subunit ye-NMDA receptor kwiqhezu le-synaptosomal membrane kunye ne-transferrin receptor kwiqhezu le-endosomal (umzekelo blot bona Umzobo 5b). Asifumananga ntlukwano kwiprotheyini ye-DAT epheleleyo emva kwe-2 kunye ne-6 wks yokuvezwa kokutya (idatha engaboniswa). Ukuvavanya i-fraction-specific differences kwiprotein ye-DAT, sisebenzise iindlela ezimbini zokuphindaphinda i-ANOVA (dietXfraction). Ngokuhambelana novavanyo lwe-voltammetry, ii-2 wks zokuvezwa kokutya kwakunganelanga ukuguqula amanqanaba ayo nayiphi na i-isoforms ye-DAT kwi-P2 okanye i-P3 amaqhezu (Ikhiwane. 5. c,e,g; zonke ns). Nangona kunjalo, emva kwee-6 iiveki zokuvezwa kokutya, kwakukho i-dietXfraction interaction ebalulekileyo (F(1,18) = 8.361, p<0.01); Umzobo 5d) ye-50 kD isoform yeDAT. Ngaloo ndlela, i-HFD ende yanciphisa kakhulu i-isoform ye-50 kD ye-DAT kwi-fraction ye-P2 kwaye yabangela ukuthambekela ekunyuseni kwinqanaba le-P3. Asifumananga siphumo sokutya okanye iqhezu kwi-64 kD (Umzobo 5f; ns) okanye i-70 kD (Umzekeliso. 5h; ns) DAT isoforms.

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Umzobo 5. Ukusetyenziswa kokutya okunamafutha aphezulu kunciphisa i-membrane ehambelana neprotheni ye-DAT kwi-ventral striatum.

a) Umfanekiso omeleyo obonisa (2) 1 × 1 mm iipuntshi zethishu ezithathwe kwi-ventral striatum ezidityaniswe kuhlalutyo lweprotheyini ye-DAT. VStr = Ventral Striatum; DStr = I-Dorsal Striatum; cc = corpus callosum; ac = i-commissure yangaphambili. b) Abameli bamachatha asentshona edatha eboniswe kwi-c–h. L = LFD; H = HFD; TfR = i-transferrin receptor; I-NR2B = i-subunit ye-NR2B ye-NMDA receptor. c) Kwakungekho mahluko kwiprotheni ye-50 kD DAT nokuba yi-P2 okanye i-P3 yamaqhezu alandelayo kwiiveki ze-2 zokuvezwa kokutya. d) Iprotheni ye-50 kD DAT iyancipha kakhulu kwi-P2 (* = p<.05), kodwa hayi i-P3 iqhezu le-ventral striatal tissue kwi-HFD-6 wk ngokumalunga ne-LFD-6 wk rats. Kwakungekho mahluko kwiprotheni ye-64 kD DAT ilandela nokuba yi-2 (e) okanye iiveki ezi-6 (f) Ukuvezwa kokutya. Kwakungekho mahluko kwiprotheni ye-70 kD DAT elandela nokuba yi-2 (g) okanye iiveki ezi-6 (h) Ukuvezwa kokutya.

http://dx.doi.org/10.1371/journal.pone.0058251.g005

Ukufumanisa ukuba amanqanaba anciphayo eprotheni ye-DAT kwi-P2 iqhezu lalibangelwa, ngokuyinxenye, ekunciphiseni ukubhalwa kwe-DAT, i-VTA / SNc DAT mRNA amanqanaba alinganiswa kwiigundane ezingentla.Umzobo 6a umzekelo). Asibonanga mahluko phakathi kwamaqela okutya kwi-midbrain DAT mRNA emva kokuba i-2 okanye i-6 wks yokuvezwa kokutya.Isazobe 6b–c; zombini ns). Ke ngoko, umahluko kumanqanaba eprotheyini ye-DAT ngaphakathi kwe-ventral striatum ayinakwenzeka ukuba ibe ngenxa yokusilela kwimveliso ye-DAT.

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Umzobo 6. Ukusetyenziswa kokutya okunamafutha amaninzi akutshintshi amanqanaba e-DAT mRNA. a)

Umfanekiso omeleyo obonisa i-1 × 1 mm iipuntshi ze-tissue ezithathwe kwi-VTA / SN kwaye zidibene nohlalutyo lwe-DAT mRNA. cp = i-penduncle yobuchopho; pc = i-posterior commissure; MM = i-nucleus ye-mammillary ephakathi. Kwakungekho mahluko kumanqanaba e-DAT mRNA alandelayo emva kweeveki ezi-2 (b) okanye iiveki ze-6 zokuvezwa kokutya (c).

http://dx.doi.org/10.1371/journal.pone.0058251.g006

ingxoxo

Ukusetyenziswa kwexesha elide le-HFD kunokukhokelela kwi-DIO kunye neplastiki ngaphakathi kwenkqubo ye-nervous central. I-Dopamine neurons kunye ne-striatal dopamine receptors zibonakala njengeseti enye yeethagethi ze-CNS ezichatshazelwa yi-HFD kunye nakubantu abatyebe kakhulu. [11], [13], [33]. Apha sinika ingxelo yokuba i-HFD yanciphisa inqanaba lokuphinda kuthathwe i-dopamine kwi-ventral striatum kwaye esi siphumo sasixhomekeke kwixesha lokuvezwa. Okubalulekileyo, umphumo we-HFD kumsebenzi we-DAT wenzeka ngokungabikho kwe-DIO. Ngelixa singakhange silinganise ngokuthe ngqo iimpawu zobunzima bomzimba kolu phononongo, izilwanyana ziye zahlelwa ngokwesiko njenge-DIO okanye ukunganyangeki kokutya okusekwe ekufumaneni ubunzima bomzimba emva kokuvezwa kwi-HFD. [34]. I-HFD yexesha elide yanciphisa kakhulu amandla e-cocaine, ephazamisana ne-DAT, ukwenza ubukhulu bokukhutshwa kwe-dopamine. Silinganise amanqanaba eprotheyini ye-DAT kwi-ventral striatum sisebenzisa uhlalutyo lwe-Western blot-ukwahlula phakathi kwe-DAT yendawo ngaphakathi kwamaqhezu asezantsi aphuculwe nokuba yi-plasma membrane okanye i-endosomes yokuhlaziya. Sifumene ukunciphisa okubalulekileyo kwi-isoform engakafiki ye-DAT ehambelana ne-plasma membrane. Ke, ixesha elide i-HFD ibonakala ngathi yehlisa ireyithi yokuphinda ithathelwe ingqalelo nge-DAT ngokuphazamisana nokurhweba nge-DAT okanye mhlawumbi ukuvuthwa kodwa hayi ngokunciphisa ukubonakaliswa kofuzo lwe-DAT okanye uzinzo lwe-DAT mRNA. Ngaphezu koko, ixesha eliphakathi kweeveki ezimbini ukuya kwezintandathu zokuvezwa kwi-HFD libonakala lilelona xesha liphambili lokunika iplastiki ye-diet-induced plasticity ngokubhekiselele kwi-DAT.

Ukutyeba kakhulu kunxulunyaniswa nemiba emininzi yokubonakaliswa kwe-striatal dopamine, kubandakanya nokufumaneka kwe-DAT kubo bobabini abantu. [18] kunye neempuku [19]. Nangona kunjalo, kutsha nje kuboniswe ukuba uphuhliso lwe-DIO luguqula inqanaba lokuphinda kuthathwe i-dopamine kwiimpuku. [20]. Ngelixa olu phononongo lubonise ukonakaliswa kwakhona kwe-dopamine kulandela ukusetyenziswa kwe-dopamine exogenously emva kweeveki ezi-4 kuphela ze-HFD, izilwanyana ezazigcinwe kwi-HFD zakhethwa ngokusekwe kwinzuzo yokuqala yobunzima kwaye ke zinokumela abantu abahlukileyo. Ngokuhambelana nalo mbono, izilwanyana ze-HFD zaqhubeka zisitya iikhalori ezininzi kwaye zifumana ubunzima obuninzi xa kuthelekiswa nolawulo lwe-LFD. Olunye uphononongo lwakutsha nje luchaze ukusilela kwakhona kwe-dopamine emva kweeveki ezili-12 ze-HFD kwiimpuku ezikhuliswe ngaphandle. [35]. Nangona kunjalo, kukho ukungafani okuphawulekayo kubunzima bomzimba phakathi kwezilwanyana ezondla i-HFD ngokuchasene ne-standard lab chow diet xa imilinganiselo yokubuyisela yenziwe. Ke ngoko, kwahlala kungacacanga ukuba ngaba ukuthomalalisa kwi-dopamine reuptake kuvela njengesiphumo esithe ngqo, okanye sandulele, uphuhliso lwe-DIO. Ngokuchasene nezi ngxelo zamva nje, asifumananga mahluko kubunzima bomzimba okanye ukusetyenziswa kwe-kcal iyonke phakathi kwamaqela ethu okutya xa imilinganiselo yokuphinda yenziwe kwakhona. Ukuba sifumene iyantlukwano kwi-dopamine reuptake emva kwe-6, kodwa hayi i-2, iiveki ze-HFD zibonisa ukuba utshintsho olubangelwa kukutya kwi-dopamine reuptake luyimpendulo engapheliyo, kodwa hayi ebukhali, utshintsho kwindlela yokutya. Ukongezelela, iziphumo zethu zibonisa ukuba endaweni yokuba ngumphumo wokutyeba, ukuguqulwa kokutya kwi-DAT kunokufaka isandla ekuphuhliseni isifo. Uphononongo lwexesha elizayo luya kufuna ukujongana nokuba ingaba izilwanyana ezinokuthi zichaphazeleke ngokwahlukileyo kwi-DIO [34] zineeyantlukwano preexisting in DAT intetho/umsebenzi okanye ngokwahlukileyo zichaphazeleka ngokwahlukileyo kutshintsho ukutya-eyenziwe kwi-DAT.

Kulwazi lwethu, esi sisifundo sokuqala esibonisa ukuba i-HFD iyanciphisa impendulo ye-dopamine kwi-cocaine. Ngenxa yendima ye-dopamine kumvuzo weziyobisi, iziphumo zethu ziyahambelana nomsebenzi wangaphambili obonisa ukuba iimpuku ezondla i-HFD kangangeeveki ezi-6 ziyacotha ukufumana ukuzilawula kwe-cocaine kunezilwanyana ezondla ukutya okulawulayo. [36]. Okubalulekileyo, esi siphumo sasizimele kuphuhliso lwe-DIO. Ukongeza, iimpuku ezikhethwa ngokukhetha ukuchaphazeleka kwi-DIO zibonisa ukhetho oluncitshisiweyo lwendawo ye-cocaine, ecebisa ukuba iipropathi ezinomvuzo ze-cocaine zibhuqiwe kwezi zilwanyana. [37]. Ukuhla kwempendulo kwi-cocaine esiyibonile kwiimpuku ze-HFD-6 wk inokuba ngenxa yokuncipha kokufumaneka kwe-DAT. Nangona kunjalo, i-cocaine ikwanyusa ukubonakaliswa kwe-dopamine ngeendlela ezingaxhomekekanga kwi-DAT. Ngokukodwa, i-HFD yayinokuthi iphazamise ukuhlanganisana kwe-cocaine ye-reserve dopamine vesicles. [38]. I-Cocaine ikwanciphisa usasazo lwe-GABA kwi-dopamine neurons ngaphakathi kwe-VTA [39] kwaye yenza i-oscillations kwisantya sokudubula semizimba yeeseli ze-dopamine [40]. Naziphi na okanye zonke ezi nkqubo zinokuchatshazelwa yi-HFD. Uphando lwexesha elizayo luya kufuna ukujongana neendlela eziphantsi kwendlela i-HFD eyiguqula ngayo imiba enomvuzo ye-cocaine kunye / okanye ukubanakho ukulungelelaniswa kwe-neural eyenziwe ngeziyobisi. [18]. Ukusetyenziswa kwe-HFD kuthoba zombini iindlela zokuziphatha [41] kunye nempendulo ye-dopamine [20], [42] kwi-amphetamine, ekwaphazamisana ne-DAT. Okubalulekileyo, iimpuku ezithatha i-HFD zihambelana nekhalori kunye neempuku ezondliwa ngokutya okulawulwayo aziphuhlisi i-DIO kodwa ziyasilela ukuphuhlisa indawo ekhethwa yi-amphetamine. [41]. Kunye nedatha eboniswe apha, kubonakala ngathi ukusetyenziswa kwe-HFD kuphazamisa impendulo kwii-psychostimulants. Zonke iziyobisi zokusetyenziswa kakubi zinempembelelo kwinkqubo ye-dopamine, kwaye ukongezwa kweziyobisi okubangelwa yi-dopamine kucingelwa ukuba kubaluleke kakhulu kuphuhliso lokulutha. [43]. Ke, impendulo encitshisiweyo ye-cocaine kwiimpuku ze-HFD iyahambelana neengxelo zokuba abantu abatyebe kakhulu banomngcipheko ophantsi kakhulu wokufumana ingxaki yokusetyenziswa gwenxa kweziyobisi. [44]. Umsebenzi wexesha elizayo kuya kufuneka ujongane nokuba ireyithingi yomvuzo we-cocaine iyahluka kubantu abatyebe kakhulu xa kuthelekiswa nolawulo lobunzima obuqhelekileyo.

Uhlalutyo lwethu lwe-blot yasentshona lucebisa ukuba ukusetyenziswa ixesha elide kwe-HFD akuchaphazeli iprotein ye-DAT yokubulala, kodwa endaweni yoko kunciphisa ukudityaniswa kwe-non-glycosylated 50 kDa ye-DAT isoform kwi-synaptosomal membranes. Ngelixa i-DAT glycosylation iphucula isantya sothutho lwe-dopamine kwaye inyusa uzinzo lomphezulu we-membrane [45], [46], [47], non-glycosylated DAT evela ebantwini [45], [46] kwakunye neempuku [47] ihambisa i-dopamine ngokulula. Ukongeza, iimvavanyo ze-immunolabeling zityhila ukuba amanqanaba e-DAT engekho-glycosylated aphezulu kwi-ventral xa kuthelekiswa ne-dorsal striatum kuzo zombini iinkawu nakubantu. [47]. Kuthatyathwe kunye, ezi zifundo zibonisa ukuba amanqanaba e-membrane anciphileyo we-50 kDa DAT anokuba negalelo kwintsilelo yokuphinda siyibone kwi-6 wk HFD rats. Idatha yethu iyahambelana nophononongo lwangaphambili olubonisa ukusetyenziswa kwe-HFD kunciphisa ukufumaneka kwe-DAT kwi-ventral striatum yeempuku. [19]. Nangona kunjalo, olu phononongo aluzange lulinganise indawo ye-DAT kwiindawo ezahlukeneyo ze-intracellular. Ukongeza, iziphumo zethu ziyahambelana nophononongo olubonisa ukuncitshiswa komphezulu weseli ye-DAT kwi-striatum yeempuku ze-DIO. [20]. Olu phononongo luphinde lwaxela ukuba amanqanaba eprotheyini e-DAT ewonke ayengachaphazeleki kukutya kwimodeli ye-DIO. Sandisa oku kufunyanisiweyo ukubonisa ukuba iprotein ye-DAT iyonke ayichatshazelwa yi-HFD kwiimpuku eziphuma ngaphandle. Ke ngoko, ukusetyenziswa ixesha elide kwe-HFD ayitshintshi imbonakalo ye-DAT, kodwa inokuphazamisana nokurhweba nge-DAT okanye ukuvuthwa.

Ukungabikho kweyantlukwano kumanqanaba e-VTA/SNpc DAT mRNA emva kokuba i-2 okanye i-6 yeeveki zokuvezwa kwe-HFD ixhasa ngakumbi uluvo lokuba amanqanaba e-DAT ewonke ayengachatshazelwa bubuchule bethu bokutya. Esi siphumo sichasene nengxelo yangaphambili ebonisa ukunciphisa i-DAT mRNA kwi-VTA ye-mouse emva kweeveki ze-17 zokusetyenziswa kwe-HFD. [12]. Nangona kunjalo, kolu phononongo amanqanaba e-DAT mRNA alinganiswa emva kokuba amaqela okutya ahluke kubunzima bomzimba kwiiveki ze-12. Ke, iziphumo zabo zinokubonisa uhlengahlengiso lwasemva kwexesha kwi-DIO. Isishwankathelo, idatha yethu ibonelela ngobungqina obunamandla bokuba ukuvezwa kwe-HFD kukhokelela kutshintsho olusebenzayo kwi-striatal dopamine reuptake ngokuncipha kwe-DATs ehambelana ne-membrane ngaphandle kokuguqula inkcazo epheleleyo ye-DAT. Okubalulekileyo, sixela ukuba ukuphazamiseka okubangelwa ukutya kwi-DAT kunokwenzeka ngaphambi kokuqala kwe-DIO, ebonisa ukuba olu tshintsho lunokuba negalelo ekuphuhliseni ukukhuluphala.

Idatha yethu yongeza kuncwadi olukhulayo olubandakanya ukutya kulawulo lomsebenzi we-dopamine, kwaye ibonelele ngobungqina obongezelelweyo bokuba utshintsho oluye lwenziwa kukutya kwintetho ye-DAT lukhokelela kutshintsho olusebenzayo kumqondiso we-dopamine. Utshintsho oluye lwenziwa kukutya kutshintsho lwe-striatal dopamine signing nge-DAT lunokuba neziphumo zokuziphatha kokutyisa. Izivuseleli ezinxulumene nokutya zivusa ukonyuka kwe-phasic kwi-striatal dopamine [9], [48], [49], ezinokuthi zibethelele kwaye zomeleze izenzo ezijoliswe ekutyeni [50]. Apha sibonisa ukuba iiveki ezi-6 zokusetyenziswa kwe-HFD zandisa ixesha lokukhutshwa kwe-phasic dopamine ngokuncipha kwe-membrane ehambelana ne-DATs kwindawo ye-striatum apho umsebenzi we-dopamine ubalulekile ekutyeni. [51]. Utshintsho oluxhomekeke ekutyeni kwi-DAT lunokukhuthaza indlela yokuya phambili apho imiqondiso ye-dopamine eyandisiweyo ekhutshwe sisishukumiso sokutya yonyusa ukusebenza kwe-low affinity striatal dopamine D1 receptors, ezibaluleke kakhulu kwindlela yokuziphatha. [52], [53], [54]. Ngokuhamba kwexesha, ukuphakama kwexesha elide kwe-striatal dopamine kunokukhuthaza uhlengahlengiso, njengokuthotywa kwe-dopamine D2 receptors (D2R), ebonakaliswe kuzo zombini iimodeli zabantu kunye neempuku zokutyeba. [11], [33]. Uphononongo lwethu lucebisa ukuba ukukhula kokutyeba ayisiyomfuneko yokutshintsha i-dopamine reuptake. Ke, ukuncipha okunxulumene nokutya kwimembrane ye-DAT kunokwandulela kwaye kube negalelo ekuqalekeni kwe-D2R ephantsi, ukutyeba, kunye nokuziphatha okunyanzelekileyo kokutya okukhula ngexesha lokusetyenziswa kwe-HFD. [11].

Imibulelo

Sinqwenela ukubulela uGqr. UJamie D. Roitman noJames E. McCutcheon ngamagqabantshintshi aluncedo kwiinguqulelo zangaphambili zombhalo-ngqangi. Umxholo weli phepha kuphela uxanduva lwababhali kwaye alumeli iimbono ezisemthethweni ze-NIH okanye iChicago Biomedical Consortium.

Umbhali Wemivuzo

Kuqulunqwe kwaye kuyilwe imifuniselo: JJC EHC MFR. Yenze imifuniselo: JJC DNP SRE. Uhlalutye idatha: JJC EHC SRE MFR. Libhale iphepha: JJC EHC MFR.

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