Front Endocrinol (Lausanne). 2017; 8: 216.
E hatisitsoe Inthaneteng 2017 Aug 29. doi: 10.3389 / fendo.2017.00216
PMCID: PMC5581815
Julie Paradis,1 Pierre Boureau,1 Thomas Moyon,1 Sophie Nicklaus,2 Patricia Parnet,1,*† 'me Vincent Paillé1,*†
inahaneloang
Tšebeliso e mpe ea bo-'m'e ea lijo tse matlafatsang e eketsa kotsi ea ho ba le botenya ho bana. Sena se amahanngoa le ts'ebeliso e fetelletseng ea lijo tse matlafalitsoeng tse jetsoeng thepa ea tsona ea hedonic. Mokhoa o ka sehloohong o kopanyang lijo tsa 'mele oa bo-mme le ho rata ha bana mafura o ntse o sa utloisisehe hantle. Thutong ena, re ikemiselitse ho ithuta ka tšusumetso ea phepo e matla ea bo-'m'e / lijo tse nang le tsoekere e ngata [lijo tsa bophirima (WD)] nakong ea boimana le ho lactation litseleng tse nang le moputso tse laolang ho fepa ka popelong ho tloha tsoalong ho fihlela kholong ea thobalano. Re entse tlhahlobo ea nako e telele ea WD le taolo ea bana ka linako tse tharo tse mahlonoko (bongoaneng, bohlankana le ho ba motho e moholo) mme ra tsepamisa maikutlo ho fumaneng tšusumetso ea ho pepeseha hoa ponaletso ea lijo tse fumanehang ho (i) ho rata mafura, (ii) boemo ba polelo ea mofuta oa , le (iii) liphetoho tsa neuroanatomical / architike tsa mesolimbic dopaminergic network. Re bontšitse hore phepelo ea WD e lekanyelitsoeng nakong ea mehla ea peto e na le tšusumetso e hlakileng ea nako e telele mokhatlong oa lipotoloho tsa homeostatic le hedonic empa eseng ka khetho ea mafura. Re bonts'a nako e ikhethang ea phetoho ea mafura eo re e kopantseng le li-signature tse khethehileng tsa limolek'hule. Ho bana ho tsoa matamong a fepa a WD, re hlokometse nakong ea bongoana ho ba teng ha khetho ea mafura e amanang le polelo e phahameng ea liphatsa tsa bohlokoa tsa lefutso tse kenyelletsang litsamaisong tsa dopamine (DA); lilemong tsa bocha, khetho e nang le mafura a mangata bakeng sa lihlopha tsena ka bobeli, e fokotseha hanyane ka hanyane nakong ea teko ea matsatsi a 3 bakeng sa sehlopha sa WD mme e amana le polelo e fokotsehileng ea gene ea bohlokoa e kenelletseng lits'ebetsong tsa DA bakeng sa sehlopha sa WD se ka fanang ka tlhahiso ea mokhoa oa ho qobella ho ba sireletsa. ho tloha ho hlahisa mafura a mangata haholo; 'me qetellong ka ho ba motho e moholo, khetho ea mafura e neng e ts'oana le ho laola likhoto empa e amana le phetoho e kholo liphatseng tsa bohlokoa tse kenyellelitsoeng marang-rang a γ-aminobutyric acid, li-receptors tsa serotonin le polysialic acid-NCAM e itšetlehileng ka phetisetso ea hypothalamus. Ka kakaretso, datha tsena li senola hore bo-mme ba WD, bo lekanyelitsoeng nako ea bokhachane, ha bo na tšusumetso e tsitsitseng ho matla homeostasis le ho rata mafura hamorao bophelong leha ho bile le mokhoa o matla oa ho ntlafatsa mokhoa oa homeothostic le moputso o neng o kentse letsoho ts'ebetsong ea ho ja. Teko e eketsehileng ea ts'ebetso e tla hlokahala ho utloisisa bohlokoa ba lits'oants'o tsena.
Selelekela
Boemo ba bophelo ba bongoaneng le liketsahalo li se li tsebahala haholo ho kenya letsoho phepelong ea maloetse le mafu hamorao bophelong (1-3). Ho hlahisitsoe mohopolo oa ho ts'oaroa ha metabolic ho hlalosa hore na liphetoho tse teng tikolohong ea phepo e nepahetseng le li-hormone nakong ea koluoa li ka etsa hore ngoana a be le botenya le mekhoa e amanang le eona hamorao. Taba e kholo ea bophelo ba rona ba boloi ke ho se fepehe ka bongata ka lebaka la tšebeliso ea lijo tse matla haholo. Ka sebele, batho ba nang le monyetla oa ho ja lijo tsa basali ka bongata ba bona ba kotsing e kholo ea ho ba le botenya le metabolic syndrome.4, 5). Boithuto bo bongata bo bontšitse hore phepo e matla ea 'm'a (HFD) ka ho emara le ho anya e na le phello ea nako e telele ho metabolism ea bana.6-8). Ntle le lits'ebetso tse kenelletseng taolong ea metabolic, lits'ebetso tsa moputso oa boko le tsona li bapala karolo ea bohlokoa ho fepa boitšoaro (9, 10). Mesolimbic dopamine (DA) neurotransication, e ithutoang ka botebo molemong oa moputso le bokhoba, e fetoloa ho feteng lijo tse bakiloeng ke botena ho batho ka bobeli (11-13) le liphoofolo (14-16). Litefiso tsa DA li nts'etsapele karolo e kholo, kamora nako e telele (17), ka hona nts'etsopele ea bona e ka angoa ke lijo tsa pele. Lilemong tse 'maloa tse fetileng, liteko tsa litoeba li paka hore tšebeliso ea HFD ea bo-'m'e e ntlafatsa phepo ea hedonic ho bana (18, 19). Leha tlhokomeliso ena e kenyelletsa liphetoho tse ling tšebetsong ea tšebetso ea DA (20-22), data e na le moeli e fumanehang mabapi le ho kenella le ho nchafatsoa ha litsela tsa moputso nakong ea bophelo ba pele (21). Ho feta moo hore na karolo e sa anngoeng ke DA ea sistimi ea moputso joalo ka GABA (γ-aminobutyric acid) e ka angoa ke khatello ea maikutlo ea phepo e nepahetseng ha e tlalehoe. Ka sebele, li-neurons tsa GABA li bonahala li bapala karolo ea bohlokoa moputsong le ho setseng. Ventral tegmental Area (VTA) Gaba neurons e fumana mokhoa o ts'oanang oa ho tsoa libakeng tse fapaneng tsa boko (23), le lithuto tsa morao-rao tse amanang le boitšoaro tsa optogenetic li totobatsa karolo e kholo ea VTA GABA maemong a phephetso a sebaka (24) le ka moputso boitšoaro bo nyatsang (25). Li-nyuklea tsa nyutlelie (NAc) e hlahisoa haholo ke tšebetso ea methapo ea methapo ea methapo ea methapo ea methapo ea methapo ea methapo ea methapo 'me e sebetsa e le sebopeho sa maoto le maoto se kopanyang matšoao a tsoang tsamaisong ea methapo le matsoho le ho se etsa tšebetso. dessyecik tlhahiso ho "ventral pallidum" (VP) le tse ling tsa motlakase (26). Mme qetellong, hypothalamus e hlahisoang ke likhokahanyo tse ngata tsa GABA ho LH (27) le "arcuate nucleus", e kopanyang matšoao a tlala le ho iketla.10).
Boithuto bona bo ikemiselitse ho tsebahatsa tšusumetso ea ho ja lijo tsa botona kapa tsa botšehali ho tsoa ho setho sa botona ho tloha ho tsoalo ho ea ho thobalano (i) ho khethollo ea mafura (ii) ponts'ong ea mofuta oa tsamaiso ea DA, GABAergic system le polasetiki ea hypothalamus , le (iii) ho liphetoho tsa neuroanatomical / architic tsa mesolimbic dopaminergic network ka nako e ts'oanang. Ka hona re ile ra hlahloba, ho boithuto ba ho nka nako e telele (ho tloha ho khoesoa, P25, ho fihlela kholong ea thobalano, P45 le ho ba motho e moholo, P95), phello ea WD maternal hodima boima ba mmele le nts'etsopele ea tishu ea "adipose" ea bana e bolokiloeng tlasa kamehla chow kamora ho khoesoa. Ka mokhoa o tšoanang, re ile ra etsa tlhahlobo ea tlhaiso ea mafura e lateloang ke tlhahlobo e ikemetseng ea transcripttomic le tlhahlobo ea mantlha ea karolo ea mantlha (PCA) ea khetho ea mats'oao a ho ja lijo, khetho le litsamaiso tsa taolo ea tšusumetso. Liphetho tsa rona li ntlafalitse haholo litholoana tsa morao-rao tse shebileng lenaneo la phepo e nepahetseng tsamaisong ea DA.
Lisebelisoa le mekhoa
Boitšoaro ba Melao
Liteko tsohle li entsoe ho latela tataiso ea komiti ea tikoloho ea paballo ea liphoofolo, EU (taelo 2010 / 63 / EU), Institut National de la Recherche Agronomique (Paris, France), le Lefapha la Bongaka ba liphoofoloA44276). Protocol ea liteko e amohetsoe ke komiti ea melao ea boits'oaro mme e ngolisitsoe tlasa boits'oaro APAFIS 8666. Ho ne ho nkuoa tlhokomelo e 'ngoe le e' ngoe ho fokotsa khatello ea maikutlo le palo ea liphoofolo tse sebelisitsoeng lethathamong ka leng la liteko.
Liphoofolo le lijo
Liphoofolo li ne li bolokiloe mochineng oa 12 h / 12 h o bobebe / o lefifi ka 22 ± 2 ° C ka lijo le metsi. ad libitum. Likhoto tse mashome a mararo a metso e 'meli tsa basali tsa Sprague-Dawley (boima ba' mele: 240-290 g) ka letsatsi la ho emara 1 (G1) li ile tsa rekoa ka kotloloho ho Janvier (Le Genest Saint Isle, France). Li ne li beoa ka bonngoe 'me li fepiloe ka mokhoa oa ho ja (CD) (5% nama ea khomo le 0% sucrose) bakeng sa 16 ea tsona kapa WD (21% nama ea khomo ea nama le 30% sucrose ea 16 ea tsona nakong ea kemaro le lebelo (bona Lethathamo Tafole1: 1: Karolo ea lijo ka liperesente tse kcal ho tsoa ho ABdiet Woerden, The Netherlands). Ha a hlaha, boholo ba litter bo ile ba fetoloa hore e be malinyane a robeli kahare ho 1: 1 e motona le e 'ngoe ea basali. Re ne re boloka 12 e le ntle ho matamo a 16 a nang le litšila tse entsoe ka banna ba 4 le basali ba 4 bakeng sa sehlopha ka seng. Ha a lema (P21), malinyane a hlahileng ho CD le matamo a WD a ne a bolokiloe ho pop e tloaelehileng ho fihlela qetellong ea liteko. (Figures1A, B) .1A, B). Boima ba 'mele oa Pup bo ne bo tlalehiloe ha a hlaha mme ka mor'a moo letsatsi le leng le le leng ho 10: 00 hoseng ho fihlela P21 (weaning). Kamora ho khoesoa le ho fihlela qetellong ea tlhahlobo, likhoto li ne li beoa boima ka matsatsi ohle a 3. Re hlahisa tlhahisoleseling ho bana ba banna feela. Likhoto tsa basali li ile tsa sebelisoa bakeng sa boithuto bo bong (Setšoantšo (Figure11).
Boitšoaro (Tlhahlobo e 'meli ea Botlolo)
Linako tse tharo tsa bohlokoa tsa ntlafatso li ithutile (P21 ho P25: mocha, P41 ho P45: bocha le P91 ho P95: batho ba baholo ba baholo). 24 malinyane a banna (n = 12 ka sehlopha) li ile tsa khethoa ka mokhoa o sa reroang 'me tsa beoa ka mokoting ka bomong ho etsa tlhahlobo ea mahala ea libotlolo tse peli (Litšoantšo (Figures1A, B) 1A, B) (28-30). Teko ena e ne e sebelisetsoa ho ithuta ka ho khetheha ho hoheleha hoa tatso ea mafura ka ho e khetholla ho tsoa ho tatso e monate le ka hohle kamoo ho ka khonehang ho tloha tšusumetsong ea metabolism ea likhalori. Ka sebele, tšebeliso ea tharollo ea oli ea poone ea 1% e amana le ho ja ha 0.09 kcal / ml feela. Kamora letsatsi le le leng la ho lula boteng ba libotlolo tse peli, tlhahlobo e ile ea etsoa matsatsing a 2 ho P25 le ka matsatsi a 4 ho P41 le P91 (Setšoantšo. (Figure1A) .1A). Lintlha ka botlalo, ho weaning (P21), malinyane a 24 a ne a lula ka bonngoe bakeng sa matsatsi a 2 (Setšoantšo. (Figure1A): 1A): letsatsi la 1, karolo ea bolulo, letsatsi la 2, likhoto li fuoe khetho ea mahala ea botlolo tse peli pakeng tsa emulsion ea 1% oli ea poone ho 0.3% xanthan gum (Sigma Aldrich, St. Quentin Fallavier, France) le tharollo ea ganthan gum ( 0.3%). Ho P41 le P91, ho ile ha sebelisoa lipopa tsa 24 mme khetho ea mahala ea libotlolo tse peli e ile ea buoa ka matsatsi a mararo a latellanang. Tšebeliso ea tharollo ea xanthan gumthan le tharollo ea tatso (oli ea poone 1%) e ne e rekisoa letsatsi le letsatsi ho 11: 00 am bakeng sa matsatsi a 3 (P45 le P95). Boemo ba libotlolo tsena tse peli bo ne bo khelosoa letsatsi le letsatsi ho thibela khethollo. Lenane la khetho ea mafura le lekantsoe e le karo-karolelano ea "tharollo ea mafura" e sebelisoang ho kakaretso e sebelisitsoeng ho 24 h. Likhoto tsohle li ile tsa bolokoa tlasa lijo tse tloaelehileng tsa chow ho pholletsa le tlhahlobo ea boitšoaro.
Ho bokelloa ha lithapo le sampole ea mali
Letsatsi kamora letsatsi la ho qetela la tlhahlobo ea likhetho tsa libotlolo tse peli ntle le halofo ea likhoto (n = 6 ka sehlopha) li ile tsa matlafatsoa ka potlako lipakeng tsa 09:00 le 12:00 am ke CO2 ho hema. Mali a ne a bokelloa ka li-tubes tse nang le EDTA (Laboratoires Léo SA, St Quentin en Yvelines, France) le centrifuged ho 2,500 g bakeng sa 15 min ho 4 ° C. Plasma e ile ea angoa ke −20 ° C. Organs le depotiti e le 'ngoe ea mafura e ne e sa tlotsoe ebile e le boima. Boko bo tlositsoe kapele 'me bo behiloe ka har'a matrix ea boko (WPI, Sarasota, FL, USA rat 300-600 g). Pele hypothalamus e ile ea qhaloa [ho ea ka likhokahano tsa atlas tsa Paxinos: −1.0 ho −4.5 mm ho tloha Bregma (31)] joale, bakeng sa rat e 'ngoe le e' ngoe, likhalase tse peli tsa coron tsa botenya ba 2 mm ka tekanyo ea NAc le e 'ngoe e maemong a VTA li fumanoe. Mehlala ea letsoho le letona le le letšehali la NAc le le letona le le letšehali (li-sampuli tse 'ne ka kakaretso phoofolo ka' ngoe) li ile tsa fumanoa ka potlako li sebelisa li-punop tse peli tse fapaneng (Stiefel Laboratories, Nanterre, France) (bophara ba 4 mm bakeng sa NAc le 3 mm bakeng sa "cyb midbrain". Mehlala e ne e phuthoe ka hoetla nitrogen e nang le metsi ebe e bolokoa ho −80 ° C bakeng sa qeto ea morao-rao ea polelo ea mofuta oa TaqMan low-density array (TLDA).
Liroto tse ling (n = 6 sehlopheng ka seng) ba ne ba thethefalitsoe ka ho teba ke pentobarbital (150 mg / kg ip) mme ba tšeloa ka monko o monate oa 'mele oa letsoai o lateloang ke leqhoa le batang la 4% paraformaldehyde ka phosphate buffer (PB), pH 7.4. Boko bo ile ba tlosoa ka potlako, ba qoelisoa ka mokhoa o tšoanang bakeng sa 1 h ho 4 ° C, mme qetellong ba bolokoa ho 25% PB sucrose bakeng sa 24-48 h. Boko bo ile ba hoamisoa ka isopentane ho -60 ° C, mme qetellong ba bolokoa ho -80 ° C ho fihlela e sebelisoa. NAc, hypothalamus le VTA li ile tsa khaoloa likarolo tsa 20 serialm serial coronal ka cryostat (Microm, Microtech, Francheville, France). Li-slide tse peli kapa tse tharo tsa li-slide tsa khalase tse 10 tse nang le likarolo tsa 4-6 li ile tsa etsoa sebakeng ka seng sa boko. Bakeng sa selae se seng le se seng, likarolo tsa serial li arotsoe ka 200 (m (Setšoantšo (Figure66).
Placma ea biochemical
Plasma ea EDTA e bokelletsoeng ho likhakanyo tsa P25, P45, le P95 li ne li sebelisoa ho lekanya tsoekere ea plasma, NEFA (mafura a seng mafura a mangata), insulin le leptin. Glucose le NEFA li ne li lekantsoe li sebelisa li-colorimetric enzymatic reaction ka li-kits tse khethehileng (glucose le NEFA PAP 150 kits, BioMérieux, Marcy-l'Etoile, France). Lihormone li ile tsa ts'oaroa ka lisebelisoa tse khethehileng tsa ELISA kamora litaelo tsa moetsi bakeng sa insulin le leptin (rat / mouse insulin ELISA kit, rat leptin ELISA kit, Pato Research, St. Charles, MO, USA).
Immunohistochemistry
Li-slide tsa khalase tse nang le likarolo tsa serial VTA le NAc li ile tsa thibeloa pele bakeng sa 3-4 h ebe li kenngoa bosiu bosiu ho 4 ° C ka motsoako oa li-antibodies tse latelang: mouse anti-NeuN (1: 500; IgM; Millipore Bioscience Research Reagents, Merk, USA) le bo-anti anti-TH (1: 1,000; Millipore Bioscience Research Reagents, Merk, USA). Kamora ho kenella ka li-antibodies tsa mantlha le ho hlatsoa kamora nako le PB, likarolo li ile tsa kenngoa ka har'a motsoako oa li-antibodies tsa bobeli: Alexa 488 e kopantseng esele anti-mouse IgM le Alexa 568-conjugated esele anti-rabbi IgG (1: 500; Attitrogen, ThermoFisher Science science, Waltham , MA, USA) bakeng sa 2 h. Likarolo li ne li behiloe ka har'a li-slide tsa khauta tse ntle haholo (ThermoFisher Science science, Waltham, MA, USA), li omisitsoe ka moea, li koaetsoe ka ProLong ™ Gold antifade reagent (Attitrogen, ThermoFisher Science science, Waltham, MA, USA).
Palo ea Neurons ho VTA
Bakeng sa rat e 'ngoe le e' ngoe, lisele tse ntle tsa TH li ile tsa ba le lipalo tse hlalositsoeng pele32) maemong a mararo a fapaneng a rostrocaudal ea VTA: boemong ba ho tsoa ha methapo ea boraro (sebaka se haufi le Bregma: −5.3 mm), 200 µm rostral le 200 µm caudal ho boemo bona (Litšoantšo. (Figures6A) .6A). Ka lehlakoreng le letšehali le ka lehlakoreng le letona, setšoantšo se nang le dijiti se kenyelletsang VTA eohle ho tloha pampiring ea "terminal" e fumanehang kahare ho isa moeling oa mesencephalon se fumanoe se sebelisa X 40 ho phahamisa senotlolo sa li-slide tsa NanoZoomer-XR C12000 (Hamamatsu, Japane). Ho ne ho huloa mola ho potoloha karolo ea VTA bakeng sa karolo ka 'ngoe. Meeli e ile ea khethoa ka ho hlahloba sebopeho sa lisele mme ba bua ka sethala sa Paxinos le Watson. Dopaminergic neuron e ne e hlalosoa e le mmele oa sele oa NeuN (+) / TH (+) o nang le li-nucleus tse hlakileng tse hlakileng. U sebelisa software ea NIH Image J (cell counter plugin), lisele tsa NeuN (+) / TH (+) li ile tsa baloa ke batho ba babeli ba fapaneng ba neng ba sena tsebo ea lihlopha tsa liphoofolo. Liphoso tsa ho arola lisele li ile tsa lokisoa ho sebelisoa foromo ea Abercrombie (32), kae N = n[t/(t + d)] ((N = kakaretso ea lisele; n = palo ea lisele tse baloang; t = botenya ba karolo; le d = bophara ba sele), 'me ntlha ena ea tokiso e ne e le 0.65. Lintlha li hlahisoa li le mpe [NeuN (+) / TH (+) ho VTA ea leqeleng le ho le letona] ± SEM.
Tekanyetso ea fiber ea Ford ho NAc
Litaba tsa prothepa tsa TH tse maemong a methapo ea kutlo ea dopaminergic ea NAc li hakanngoa ka tlhahlobo ea densitometric ea anatomical ea likarolo tsa TH immunolabeled. Sekhahla sa TH nyuzi se ile sa hlakoloa maemong a mararo a hanyetsanang haufi le axostrocaudal axis ea NAc (Bregma 2.20, 1.70, le 1.20 mm) (Setšoantšo. (Figure6B) .6B). Ka bokhutšoanyane, setšoantšo se nang le linepe tse kenyelletsang striatum eohle le NAc e fumanoeng e sebelisa x 40 ntlafatso ea senepe sa NanoZoomer-XR Digital slide C12000 (Hamamatsu, Japane) li fumanoe. Bakeng sa NAc e fanoeng, mohala o ne o huloa ho potoloha konopo eohle ho hlalosa sebaka sa litekanyo tsa optical density (OD) (Setšoantšo. (Figure6B) .6B). Boleng bo fumanoeng bo ne bo lekantsoe le boleng ba OD bo lekantsoeng ho tloha sebakeng se chitja se huleloang ho corpus callosum (sebaka se sa thijoeng bakeng sa immunochemistry ea TH) sa karolo e le 'ngoe se sebelisa software ea NIH Image J. Lintlha li hlahisoa e le moelelo oa karolelano ea OD (boleng ba OD ho boleng ba NAc / OD ho Corpus callosum ea likarolo tse tharo) ± SEM.
Tlhatlhobo ea Gene ka TLDA le TaqMan
RNA e ne e qheletsoe ka thoko ho leqhoa le nang le lehloa la li-NAc, li-sampuli tse ntlafalitsoeng tsa VTA, le hypothalamus, e sebelisa setsi sa NucleoSpin RNA / protheine (Macherey-Nagel, Hoerdt, France). Kakaretso ea RNA e ile ea kenngoa ho daoa ea DNase kamora litaelo tsa moetsi, palo e lekantsoe ke monko oa 260 / 280 nm UV, mme boleng bo ile ba hlahlojoa ho sebelisoa Agilent 2100 Bioanalyzer System, nomoro ea botšepehi ba RNA (RIN) e ile ea baloa ka nako eo. Mehlala e nang le RIN ka tlase ho 8 e ile ea lahloa. K'hamphani e le 'ngoe ea RNA e neng e sebelisitsoe RNA e ile ea fetisetsoa ho CDNA e sebelisa tekanyetso e phahameng ea RT (E sebelisoa ke Biosystems, Foster City, CA, USA) ka kakaretso ea 10 µl.
Joalokaha ho hlalositsoe pele (33), TLDA ke karete ea 384-hantle-e nyane eo ka eona li-PCR li ka etsisoang ka nako e tšoanang (Li-Biosystems, Foster City, CA, USA). Re sebelisitse TPLA e hlophisitsoeng ka kotloloho ho koahela malapa a mefuta e fapaneng a amanang le polasetiki le taolo ea lijo. Karete e 'ngoe le e' ngoe e tloaetsoeng e ne e hlophisitsoe joalo ka mela ea ho tsamaisa sampole ea 384 × 2-XMUMX × 4 (reference: 2a). Sesebelisoa sa 48-gene (Tafole S1 ho Supplementary Material) le mefuta e mene ea ho boloka matlo (18S, Gapdh, Polr2a, le Ppia) ba ithutile. PCR ea nako ea nnete e ile ea etsoa ho sebelisoa Life Technologies TaqMan reagents mme e tsamaisoa ho ABI Prism 7900HT sequence diagnost system (Applied Biosystems, Foster City, CA, USA). Lintlha tse tala tsa fluorescence li ile tsa bokelloa ka PCR e sebelisa software ea SDS 2.3 (Applied Biosystems, Foster City, CA, USA), e ileng ea nts'etsapele lipokello tsa Ct ka boiketsetso ba hang-hang le ts'ebetso. Kamora ho sefa ka ho sebelisa ThermoFisher leru App (ThermoFisher, USA) ho khetholla li-PCR tse tsamaeang, likhakanyo tsa sampole e ne e le n = 6 (n = 5 bakeng sa sehlopha sa WD ho P25). Lintlha li ile tsa hlahlojoa ka ThermoFisher Cloud App (ThermoFisher, USA) bakeng sa palo e lekanyelitsoeng. Palo e lekanyelitsoeng ea polelo ea liphatsa tsa lefutso (RQ) e ne e ipapisitse le mokhoa oa ho bapisa Ct o sebelisa equation RQ = 2−ΔΔCt, moo ΔΔCt bakeng sa sepheo se le seng sa gene e ne e tlositsoe ho phallo ea Ct ho sampole ea calibrator mme e lekantsoe ka taolo ea tlhaho. Ka nepahalo, re fumane mofuta o tsitsitseng oa ho boloka matlo o sebelisang geNorm algorithm (ThermoFisher Cloud App RQ, ThermoFisher, USA). Har'a mefuta e mene ea ho boloka matlo, Gapdh e hlalositsoe e le taolo ea tlhaho ea NAc le hypothalamus, le Ppia bakeng sa VTA 'me sena e ne e le' nete bakeng sa sampole tsohle ho tloha nakong tse tharo tse hlahlobiloeng. Pontšo ea setšoantšo sa liphatsa tsa lefutso e entsoe ka letsoho ho abela 'mala o le mong bakeng sa keketseho ea 10% ea polelo ea liphatsa tsa lefutso ho sehlopha sa CD. Phapang ea bohlokoa, e sebelisang tlhahlobo e saenneng ea parametric Wilcoxon, e bonoe ka asterisk.
Tlhahlobo ea Litekanyetso
Liphetho li hlahisoa e le bokhopo ± SEM litafoleng le lipalo. Tlhahlobo ea Mann-Whitney eo e seng ea parametric e sebelisitsoe ho hlahlobisisa boima ba 'mele ka linako tse fapaneng, likhetho tsa mafura, le karolelano ea OD e fumanoang ho immunohistochemistry.
Ho lekola bohlokoa ba likhetho tsa mafura tsa 3 tsa matsatsi, re ile ra etsa tlhahlobo ea lipalo bakeng sa letsatsi ka leng. Bakeng sa sehlopha se seng le se seng, tšebeliso ea tharollo ea mafura le tharollo ea taolo e ile ea lekoa ho sebelisoa tlhahlobo e saennoeng ea Parametric Wilcoxon. Re bapisitse boleng ba bohlokoa ba se ratoang le boleng ba boikaketsi ba 50% (mola o mofubelu o nang le matšoao). Ho fapana ho fapaneng ho ile ha bonoa ka asterisk e khubelu. Re sebelisitse tlhahlobo e ts'oanang ea tlhahlobo ea boleng ba qPCR RQ; re bapisitse boleng ba RQ le boleng ba 1 bo bongata. Phapang ea bohlokoa e bonts'itsoe ka asterisk (Setšoantšo (Figure44).
Bakeng sa tlhahlobo ea sampole ea plasma, re ile ra etsa tlhahlobo ea Mann eo e seng ea parametric le Whitney. Palo ea lisele tse nang le boleng bo botle e ile ea hlahlojoa ka mokhoa oa mekhoa e 'meli ea ANOVA le ea p boleng bo lekantsoe. Ka lebaka la makhetlo a mangata a liteko tse sebelisitsoeng, Bonferroni post hoc khalemelo e sebelisitsoe feela kamora tlhahlobo ena. Tlhahlobo ea lipalopalo e entsoe ka software ea Prism 6.0 (GraphPad Software Inc., La Jolla, CA, USA).
PCA e sa laoloang e ile ea etsoa ka lekhetlo la pele ho li-parameter tsa 130 (TLDA, boits'oaro, le data ea plasma) ka nako e fapaneng bakeng sa punctuop e 'ngoe le e' ngoe ea biopsy (VTA, NAc, le hypothalamus) ho bona sebopeho se akaretsang sa data e behiloeng (ke hore, PCA e meraro ea lefatše ka bophara ka ntlha ea nako). PCA e ka hlalosoa e le khakanyo ea orthogonal ea data ho sebaka se tlase sa bokahare, e le hore phapang ea lintlha tse lekantsoeng e matlafalitsoe polokelong. Re qalile ka ho tsoa liphatsa tsa lefutso tse sa hlahisoang kapa tse sa hlahisoang hanyane (setšoantšo (Figure5) .5). Litaelo tsa bana ho tsoa matamong a fepa ka CD le matamo a filoeng ke WD li hlahile ka mebala e fapaneng meriring ea PCA ho bona ka mahlo haeba lihlopha tsena tse peli tsa liteko li arotsoe hantle ke likarolo tsa PCA tse sa laoloang. Tlhahlobo ena e arola lihlopha tsa liphatsa tsa lefutso tse hlalosoang ka mokhoa o fapaneng lipakeng tsa lihlopha tsena tse peli tsa bana. Kamora nako, li-PCA tse shebaneng li ile tsa etsoa lihlopheng tse fapaneng tsa matšoao a mRNA: polasetiki (khokahano ea sele, cytoskeleton, neurotrophic factor, synaptogenesis, le transcript registry), tsela ea DA, GABAergic tsela, li-modulators tsa epigenetic (histone deacetylase le histone acetyl transase). Li-PCA tsena tse ikemetseng li lumella motho ho bona ka nako e ts'oanang ho kopanya lipakeng tsa lijo tsa bo-mme le matšoao a itseng le khokahano lipakeng tsa mefuta e meng ea malapa. Sekala sa boleng se sebelisitsoe bakeng sa tlhahlobo ea PCA le PCA e tsepamisitsoeng: +++: karohano e ntle haholo; ++: karohano e ntle le rat e le 'ngoe ka lehlakoreng le fosahetseng la karohano ea PCA; +: karohano e ntle haholo le likhoto tse peli (e 'ngoe ea sehlopha se seng le se seng) ka lehlakoreng le fosahetseng, -: ha ho karohano e hlakileng.
Results
Boima ba 'mele le Khōlo
Tšebeliso ea 'mele ea Maternal nakong ea boitšisinyo ba mmele (ho tloha G1 ho ea G21) ha ea ka ea ama boima ba' mele nakong ea tlhaho. (Figure2) 2) (CD: 6.55 ± 0.07 g vs WD: 6.54 ± 0.05 g p = 0.9232) (Litšoantšo (Figures2A, B) .2A, B). Keketseho ea boima ba 'mele ho tloha ha a hlaha ho fihlela a khoesoa e ne e le 21% liputsong tse tsoetsoeng ke matamo a WD ho feta bana ba matamo a CD ba nang le boima ba' mele bo phahameng haholo ha ba khoesoa ka masea a tsoetsoeng ke matamo a WD (36.19 ± 0.90 g vs 47.32 g p <Setšoantšo sa 0.001) (Figure2C) .2C). Ho tloha ha a khoesoa ho fihlela qetellong ea teko (P95), likhoto li ne li feptjoa ka lijo tse tloaelehileng le boima ba 'mele bo lula bo le phahameng bakeng sa bana ho tloha matamong a WD ho feta ho tsoa ho matamo a CD. Lintlha, nakong ea bocha (P39) (Litšoantšo (Figures2A, D), 2A, D), CD: 176.8 ± 3.3 g vs WD: 192.2 ± 3.3 g p = 0.0016 le ho P93 (motho e moholo e monyane) (Figures (Figures2A, E) 2A, E) CD: 478 ± 9.9 g vs WD: 508.6 ± 10.3 g p = 0.0452.
Li-Hormones le Matšoao a Metabolic ka Nako e fapaneng
Plasma leptin, insulin, glucose, le mohopolo oa NEFA li ile tsa lekanngoa ho P25, P45, le P95. Ka lilemo tsohle, tsoekere ea plasma, NEFA le leptin ea litloholo tsa WD li ne li sa fapana ka lipalo le bana ba CD (Lethathamo (Tafole2,2, n = 6 ka sehlopha). Re bone keketseho e kholo ea phepelo ea mafura (karolelano ea boima ba mafura) ho bana ba tsoang matamong a fepiloeng ke WD ka P25 feela (p = 0.0327, teko ea Mann le Whitney).
Kameho ea Perinatal WD ho Khetho ea Mafura ho tloha ho Weaning ho fihlela ho Adult
Ho hlahloba tšusumetso ea WD ho khethollo ea mafura, re sebelisitse paradigm ea likhetho tse peli ka nako tse tharo tse fapaneng nakong ea kholo. Teko ena e ile ea sebelisetsoa ho ithuta ka ho khetheha khetho ea mafura ka ho qoba matla a metabolism ea ho kenella ha eona Re bonts'itse hore phapang ea "keketso" e eketsehileng ea khalori e tsoang botlolong (ka P25, P45, le P95) ha e bohlokoa lipalo-palo. lipakeng tsa lihlopha (Litšoantšo S1A-C ho thepa e tlatsetsang). Ho feta moo phapang ea tšebeliso ea 1% tharollo ea oli ea poone e fella ka keketseho ea khalori ka 1% bakeng sa likhoto tsa WD ho P25 (WD: 4.9% vs CD: 3.9% ea likhalori tse kenyellelitsoeng) le 0.5% bakeng sa likhoto tsa CD ho P45 (WD: 2% vs CD: 2.5% ea likhalori li entsoe) (Litšoantšo S1D-F ho Litaba tse tlatsetsang). Ho P25, malinyane a matamo a CD ha a rate mafura (44.87 ± 9.8%, p = 0.339); ka likhoto tse fapaneng tsa WD li hlahisa khetho ea mafura (75.12 ± 8.04%, p = 0.039 e latelang tlhahlobo ea boemo bo saennweng ba Wilcoxon, asterisk e khubelu). Ho feta moo ho na le phapang ea lipalo lipakeng tsa lihlopha tsena tse peli le p = 0.0347 (Teko ea Mann le Whitney, letšoao le letšo la hash) (Setšoantšo (Figure33A).
Ho P45 le P95, lihlopha tsena ka bobeli li na le khetho e kholo ea mafura, ke hore, li fapane haholo le boleng ba theory ba 50% (ho P45, CD: 80.68 ± 2.2% p = 0.0005 le WD: 78.07 ± 3.25% p = 0.0005; ho P95, CD: 74.84 ± 8.4% p = 0.0425 le WD: 69.42 ± 8.9% p = 0.109 ho latela tlhahlobo ea boemo bo saennweng ba Wilcoxon, asterisk e khubelu) (Setšoantšo (Figure3A) .3A). Melemo ea lihlopha tsena tse peli e ne e sa bonahale ka mor'a letsatsi le le leng la tatso (ho P45) p = 0.7857 le ho P95 p = Teko ea 0.9171 ea Mann-Whitney) (Setšoantšo (Figure33A).
Ho tseba hore na likhoto li laola tšebeliso ea tsona ea mafura joang ha nako e ntse e tsamaea, re phethile tlhahiso ea mafura ka matsatsi a mararo a latellanang ho P45 le P95 (Litšoantšo. (Figures3B, C) .3B, C). Ho khahlisang ho P45, ke ba batona feela ba matamo a WD bao butle-butle ba ileng ba lahleheloa ke khetho ea tharollo ea mafura (Setšoantšo. (Figure3B) 3B) (letsatsi la boraro: 53.12 ± 8.36% p = 0.851 ho latela tlhahlobo ea boemo bo saennweng ea Wilcoxon). Leha ho le joalo, ho P95 (lilemo tsa batho ba baholo) liphoofolo tsohle li khethile mafura a se nang tlhabollo nakong ea tlhahlobo ea matsatsi a 3 (Setšoantšo (Figure33C).
Ka bokhutšoanyane, mohlaleng ona, re hlokometse, qalong ea bongoaneng (bongoaneng), khetho ea mafura ho rat e fepiloeng ke matamo a WD ka ho se tsotelle ho fetelletseng ka nako nakong ea bohlankana. Ha rea bona phapang lipakeng tsa lihlopha tse peli tsa likhoto ha li se li le batho ba baholo.
Signature ea Molek'hule ea Brain Plasticity le GABA Circuits Reodeling ho Hypothalamus le Reward Pathways
Ho fumana hore na matšoao a ho kula a basali nakong ea ho emara le ho pepa ho na le tšusumetso ho methapo ea kutlo le moputso oa litloholo, re lekantse polelo ea lintlha tse 'maloa tsa bohlokoa tsa polasetiki ea boko, mohlala oa boko, le matšoao a potoloho ea methapo e amanang le tšebeliso ea lijo le li-epigenetic. batsamaisi. Re sebelisitse TLDA ho sekaseka bongata ba bona sebakeng se fapaneng sa boko (ke hore, hypothalamus, VTA, le NAc) (Lethathamo S1 ho Tlatsetso ea Litaba) ka linako tse tharo. Screening e ile ea etsoa kamora liteko tsa khetho ea libotlolo tse peli ho P25, P45, le P95 (Setšoantšo. (Figure1) 1) ho banna ba tšeletseng ba tsoetsoeng ke matamo a WD le ba batona ba tšeletseng ba tsoetsoeng ke matamo a CD.
Ho P25 ho hypothalamus, mefuta e mehlano ho tsoa mekhatlong e leshome le metso e meraro e bonts'a boemo bo tlase ba polelo ea MRNA haholo makaleng a matšoao a polasetiki le matšoao a GABA a pakeng tsa −20% (Gfap) le −40% (Gabra5) ho malinyane a tsoang matomong a fepa a WD CD li filoe matamo. Ho moputso wa tseleng ya moputso biopsies (VTA le NAc), mefuta e mmedi e bontshitse maemo a phahameng a polelo ya mRNA (D2R le Gabra1), ke ho re, DA signaling le GABA receptors le mofuta o le mong polelo e tlase (Hcrtr2) (ke hore, orexin 2 receptor) ho NAc , athe mefuta e mene e bonts'itse boemo ba polelo ba mRNA bo phahameng haholo (Map2, Gabara1, Hcrtr1, and Hcrtr2) (ke hore, letshwao la polasetiki, li-receptors tsa GABA le li-serotoninergic receptors ho VTA (Setšoantšo. (Figure44).
Ho P45 ho hypothalamus, mefuta e mehlano ho tsoa mekhatlong e leshome le metso e meraro e fapaneng e bontšitse boemo bo tlase ba polelo ea mRNA e pakeng tsa −20% (Fos) le −50% (FosB) ho malinyane a tsoang matamong a fepa a WD ha a bapisoa le likhoto tse tsoang ho matamo a fepa a CD. Ho P45 ho moputso tseleng ea biopsies, mefuta e mene e bonts'itse boemo bo phahameng ba polelo ea MRNA (Gfap, Dat, Cck2r, le Kat5) le mefuta e 'meli polelo e tlaase (Fos le FosB) ho NAc athe mefuta e meraro e bonts'itse boemo bo tlase ba polelo ea MRNA (Arc, FosB, le Th) le mofuta o le mong oa boemo bo holimo (Gabrg2) ho VTA.
Ho P95 ho hypothalamus, mefuta ea 20 ho tsoa mekhatlong e leshome le metso e meraro e fapaneng e bonts'itse boemo bo phahameng ba polelo ea mRNA e pakeng tsa + 20 le + 40% (mefuta ea Syt4 ho Gjd2) le 3 e bonts'itse polelo e tlase ea MRNA (FosB, D1r, le Gabarb1 ho tsoa ho tsoa ho GarbXNX matamo a fepisitsoeng ha a bapisoa le likhoto tse tsoang ho matamo a fepa CD. Ho P95 ho li-wayway tsa tseleng ea moputso, mefuta ea 12 e bontšitse boemo bo phahameng ba polelo ea mRNA e pakeng tsa + 20 le + 40% (Syn1 ho Hcrt1) le 1 gene polelo e tlase (Th) ho NAc, 6 genes e bonts'itse boemo bo phahameng ba polelo ea MRNA (Ncam1 , Gja1, Gjd2, Gabra5, Htr1a, le Htr1b), le mefuta ea 6 e bonts'itse boemo ba polelo ea tlase ba MRNA (Cntf, Igf1, Fos, Socs3, Gabrb2, le Hdac3) ho V.
Re ntan'o etsa PCA tse tharo tse sa hlokeng ho tsamaellana le li-biopsies tse tharo tsa boko re sebelisa likarolo tsohle tse hlalositsoeng (ke hore, litekanyetso tsa plasma, datha ea boits'oaro le mefuta ea polelo ea mRNA). Karohano e hlakileng ea lihlopha tsena tse peli e fumanoe feela ho P95 ea NAc le VTA (Tafole (Tafole33).
Ho ea ka potoloho ea khokahano ea PCA le data ea TLDA (e emelang boholo ba lintho tse kenyellelitsoeng ho PCA ena), re hlalositse malapa a neng a ka ikarabella ho karohano mme a etsa PCA e shebaneng (Litšoantšo. (Litšoantšo5A, B, 5A, B, mohlala). PCA e tsepamisitsoeng e senoletse hore libakeng tsa "P25 DA" tsa NAc le matšoao a polasetiki a hypothalamus a ka arola lihlopha tse peli tsa bana (Lethathamo (Tafole33 bakeng sa kakaretso). Ha ho khethollo e joalo e neng e fumanoe ka nako eo ho P45. Leha ho le joalo, tlhahlobo e ts'oanang ho P95 e senoletse hore matšoao a fapaneng a tsamaiso ea GABA ho NAc le hypothalamus, le matšoao a polasetiki (ho hypothalamus, NAc le VTA) le balaoli ba epigenetic (feela ho NAc) ba kenya letsoho ho arola lihlopha tsena tse peli tsa liphoofolo ( Setšoantšo (Figure5; 5; Tafole Lethathamo33).
Tlhahlobo ena e senola tšusumetso ea nako e telele ea lijo tsa perinatal ho matšoao a GABAergic le matšoao a polasetiki le a epigenetic kahare ho homeostatic le tseleng ea moputso e kenyellelitsoeng boits'oarong.
Immunohistochemistry ea TH Cell Tse netefalitsoeng Tlhahlobo ea Transcript
Hobane re hlokometse phapang e ngoe ho TH mRNA ho NAc le VTA ka linako tse fapaneng tsa nts'etsopele, re ikemiselitse ho hokahanya liphetho tsena ka mokhoa oa TH. Palo ea lisele tse ntle tsa TH / NeuN e ile ea hlahlojoa ho VTA moo litopo tsa dopaminergic cell li leng teng mme OD ea TH immunolabeling e ne e lekantsoe pheletsong ea methapo e ho NAc. Lisele tsa TH (+) li ne li se na bongata ho VTA ea WD ha li bapisoa le likhoto tsa CD ho P45 feela (Litšoantšo. (Litšoantšo6A, C, E; 6A, C, E; Setšoantšo S2E na le thepa e tlatsetsang. Ho ne ho se na tšebelisano ea bohlokoa lipakeng tsa karolo ea karolo le quanution ea TH / NeuN ka linako tse tharo (P25 p = 0.9991, P45 p = 0.9026, le P95 p = 0.9170). Ho P45 feela, phapang ea lipalo e fumanoe lipakeng tsa lihlopha tse peli tsa bana (p = 0.0002) (Setšoantšo (Figure6E) .6E). Ntle le moo, ha re bone phapang ho OD ea TH immunostain ho NAc ho P25 le P45 pakeng tsa lihlopha tsena tse peli (boleng ba OD ratio ho P25: 1.314 ± 0.022 ho CD vs 1.351 ± 0.026 ho WD, p = 0.2681; Litekanyetso tsa karolelano ea OD ho P45: 1.589 ± 0.033 ho CD vs 1.651 ± 0.027 ho WD, p = 0.1542). Leha ho le joalo, ho fokotseha ho hoholo ha OD ea methapo ea methapo ea TH ho fumanoe NAc ho tsoa sehlopheng sa WD ho P95 (litekanyetso tsa karolelano ea OD ho p95: 1.752 ± 0.041 ho CD vs 1.550 ± 0.046 ho WD, p = 0.0037) (Litšoantšo (Figures6B, D, F; 6B, D, F; Setšoantšo S2B ho thepa e tlatsetsang).
Puisano
Thutong ena, re fane ka maikutlo a hore ts'oaetso ea phepo e nepahetseng ea bo-mme e tla ba le tšusumetso lenaneong la nts'etsopele ea meputso e amanang le matla homeostasis, khetho ea lijo, le lijo tseo bana ba li jang. Re ile ra hlahloba ka botlalo litlamorao tsa phepelo ea bokhachane ea WD ho tloha popelong ho isa ho khoesoa ho GABA, serotonin, le litseleng tsa DA tsa libaka tse itseng tsa bokong (VTA, NAc, le hypothalamus) ho tloha ka bongoaneng ho fihlela ho ba motho e moholo. Liphetho tsa rona li fana ka maikutlo a hore tšebeliso ea lijo, e nang le mafura a mangata ebile e monate, e tsitsitseng nako ea pherekano e na le tšusumetso ho khethollo ea mafura a mantlha (bongoaneng) ho bana ba amanang le phetoho ea mofuta oa polelo ea liphatsa tsa lefutso le liphetoho tsa kaho ea mesolimbic marang-rang a dopaminergic. Leha ho le joalo, ha peo e ne e bolokiloe e le phepo e nepahetseng, re hlokometse ho WD ea lilemong tsa bocha ea fepa tahlehelo e tsoelang pele ea ho hohela mafura e neng e kopantsoe le polelo e fokotsehileng ea mefuta ea tsamaiso ea DA le phokotso e nyane ea li-neurons tsa TH tse ntle ho VTA . Hamorao bophelong ho rata ha mafura ho ne ho sa fapana lipakeng tsa lihlopha le ha e ne e le polasetiki ea bohlokoa ea marang-rang a GABAergic le marang-rang a matla a homeostasis ea hypothalamus a fumanoeng ka rat ho tsoa ho matamo a WD. (Figure77).
Tšusumetso ea pele ea tšebeliso ea "perinatal-WD" eo re e boneng thutong ena ke boima bo eketsehileng ba 'mele ba ngoana ha ba khoesoa empa ha ho na phapang ka nako ea tlhaho. Ehlile, liphoofolo tsa sehlopha sa WD li fumana boima ba 21% ho feta CD qetellong ea nako ea ho anyesa. Boithuto ba pejana bo fane ka litholoana tse hanyetsanang mabapi le phetoho ea boima ba tsoalo ho bana ho tsoa matamong a fepa a WD: boima bo phahameng ba 'mele (19, 34), boima bo tlase ba 'mele (18, 21, 35) kapa ha ho na phapang (6, 22). Lintlha tsa rona li tsamaisana le tlhahlobo ea morao-rao ea meta-regression (36) e entsoeng ho lingoliloeng tsa liteko tsa 171 tse ileng tsa fihlela qeto ea hore ho pepesetsoa ha HFD ea bo-mme ha ho ame boima ba tsoalo empa ho bakile boima ba 'mele qetellong ea nako ea ho beleha. Boima bo phahameng ba 'mele ba bana ba WD mohlomong bo bonts'a phetoho sebopeho sa lebese le / kapa tlhahiso ea lebese e bonts'itsoeng liphatlalatsong tse fetileng (37, 38). Ho latela boima ba 'mele ba bona bo phahameng, palo ea mafura a khutlisetsang a bana ba WD e ne e phahame haholo ho feta ea bana ba CD qetellong ea nako ea ho anyesa (P25, Tafole. Tafole2), 2), e lumellanang le lithuto tse fetileng (18, 21). Leha ho le joalo, ho iphahamisa ho phahameng ha hoa ka ha phehella ho P45 le P95, mme likarolo tse ling tsa metabolic tse kang insulin, NEFA, le plasma ea glucose li ne li sa fapana lipakeng tsa lihlopha. Liphetho tsa rona li bonts'a hore ntle le botena bo hlakileng ba 'mele nakong ea ho emara le ho beleha, lijo ka botsona ha lia lekana ho kenya litlamorao tsa metabolic ho bana (22, 39, 40).
Ho tlalehiloe hore karolo ea HFD ea perinatal e lumellana hantle le khetho ea bana bakeng sa lijo tse fumanehang habonolo (41). Thutong ea rona, re ile ra etsa thuto ea khale ea sepheo sa ho lekola ho rata mafura ho bana ba khoesitsoeng ho chow.
Kameho ea Perinatal WD ho bongoaneng (kamora ho khoesoa)
Malinyane a maiketsetso a ja lijo tse tiileng tsa 19-20 matsatsi a kamora ho hlaha (42) ha litsela tsa bona tsa moputso oa likomkomere li e-so tsofale (17). Ka hona ho ne ho le monate haholo ho ithuta khetho ea bona ea pele haholo bakeng sa mafura le ho tsamaisa khetho ea bona ea pele ka tlhaiso ea lingoloa tsa boko. Nakoana kamora ho khoesoa, re bone ho rata mafura ho bana ba WD ho neng ho sa bonahale ho litoeba tsa CD. Sena se tumellanong le litlaleho tse ling tse bonts'ang khokahano lipakeng tsa khaello ea phepo e nepahetseng le khetho ea lijo tse ratehang le khetho e tlase ea mafura a sa le monyane bakeng sa likhoto tsa taolo (43).
PCA ea lefatše ka bophara ha ea ka ea lumella khethollo ea sehlopha sa malinyane mabapi le lijo tsa bo-mme ka nako eo. Leha ho le joalo, ha PCA e lebisitsoeng, e lekantsoeng ho matšoao a DA feela, e etsoa, re ile ra fumana karohano e ntle ea lihlopha. Ehlile, ho na le keketseho e tšoaeang pontšong ea li-mRNA tsa D2 receptor mRNA ho NAc ho bana ba WD. Ena oxtxpapt D2 overexpression ho NAc e ka ba karolo e 'ngoe ea khothatso e phahameng ea mafura (44). Ho na le lingoliloeng tse ling tse fokolang tse fetotsoeng ho li-WD pups ha li bapisoa le li-CD, tse kang ho eketseha ha alpha 1 GABAA subunit ho NAc le VTA le ho fokotseha ha alpha 5 GABAA subunit ho hypothalamus e fanang ka tlhahiso ea ho hlophisoa ha li-receptor tsa GABAA ho li-nuclei tsena.
Kameho ea Perinatal WD ho Adolescence
Ho P45, re hlokometse mofuta o tšoanang o nang le mafura a mangata ka letsatsi la pele la tlhahiso empa, ho khahlisang, litoeba tsa WD li ile tsa tsoela pele ho lahleheloa ke thahasello ea tsona ea mafura kamora ho hlahisa khafetsa. Nako ea bocha ke nako e mahlonoko ea ho hlophisa bocha sebopeho se hlokahalang bakeng sa ts'ebetso ea kelello ea nako e telele (45), 'me lithuto tse fapaneng li bonts'itse tšoaetso e kotsi ea phello e mpe ea boikoetliso ba mafura (46-48). Sephetho sena se lumellana le mosebetsi oa nakong e fetileng oa sehlopha sa Muhlhausler (21, 35) moo likhoto tsa bana (libeke tsa 6) li bonts'itseng khetho e hlakileng ea lijo tse se nang phepo. Leha ho le joalo, ka har'a lingoliloeng tsa bona mokhoa oa teko oa liteko o ne o fapane hobane likhoto li ne li na le phihlello ea mahala ho lijo tse tloaelehileng le tse se nang phepo ho tloha ho khoesoa sehlabelong (libeke tsa 6).
Ka mokhoa o ts'oanang, re lekantse keketseho ea Dat mRNA ho NAc le ho fokotseha ha Th mRNA ho VTA e netefalitsoeng ke immunohistochemistry e bonts'ang palo ea lisele tse fokolitsoeng tsa TH (+) ho VTA ea likhoto tsa WD. Kamora ts'ebetso e phahameng ea transcripttomic bakeng sa sistimi ea DA ho khoesoa, ts'ebetso e fokotsehileng ho P45 e ka hlalosa phaello e tlase bakeng sa lijo tse fumanehang habonolo tse fumanehang litulong tsa rona tsa WD. Hape ho lokela ho hopoloa hore ho fokotseha ho hlophisehileng ha polelo ea Fos le FosB mRNA ka har'a li-nuclei tse fapaneng tseo re li hlahlobileng e ka ba letšoao la ketsahalo e fokotsehileng ea likhoerese kamora ho pepesetsoa ha Mme.
Likhoto tsa lilemong tsa bocha tsa WD li bontsitse ho se tsotelle ha mafura kapele hoo ho leng khahlanong le boitšoaro ba bona ba pele. Ho sebelisa lijo tse "tloaelehileng" nakong ea bongoana ho bonahala ho li "sireletsa" ho latela khetho e mafura e fetelletseng lilemong tsa bocha. Ho fapana le moo, likhoto li na le mokhoa oa ho fumana lijo tsa junk ka mor'a ho khoesoa, joalo ka Ref. (21, 35), li bontša lilemong tsa bocha batho ba rata mafura haholo. Sephetho sena se fana ka maikutlo a hore ho ja ka mor'a libeke tsa 3 kamora ho khoesoa ho ka be ho hlophisitse li-circuits le ho etsa hore ngoana ea lilemong tsa bocha a seke a tsotella phephetso e matla ea mafura.
Kameho ea Perinatal WD ho Batho ba baholo
Likhoto tsa batho ba baholo ha li sa bonts'a phapang ea ho rata mafura, leha e se e hlahisitsoe ka makhetlo-khetlo joalokaha ho se ho hlalositsoe (22, 35). Ka mokhoa o ts'oanang, re bone ho fokotseha ha Th mRNA le protheine ho NAc, le tšekamelo ea polelo e fokotsehileng ea Dat mRNA ho VTA. Naef le mosebetsi-'moho (20) e kile ea tlaleha tšebetso e tlase ea sistimi ea DA ho likhoto tsa batho ba baholo tse fepeloang ka nako ea phello le HFD, karabelo ea DA e se nang molato ea amphetamine e lekantsoeng ka micodialysis le khothaletso ea moputso oa mafura (bona tafole e ileng ea akaretsa data ea qPCR ea morao-rao mabapi le mohlala ona. Tafole S2 ho Tlatsetso ea Litaba). Tekanyetso e le 'ngoe ea qu quaranto ea TH (mRNA le immunohistochemistry) ho NAc e tsoa ho taba ea hore lisele tsa NAc li ka hlahisa Th mRNA le protheine mme ka nako eo e ka khetha leeme la DA nyuzi49, 50). Le ha ho le joalo, ts'ebeliso ea thibelo ea TH ka har'a NAc e senotse haholo li-terminals tse letsoalo tsa axon tse tsoang li-neurbrain DA neurons (VTA le SNc). Ka tloaelo, li-neuron tse hlahisang mofuta oa "striatum" le NAc li ka lemohuoa feela ho liphoofolo tse lemalitsoeng haholo ke DA (51) ka hona re ka se bonoe likarolong tsa rona tsa immuno. Phuputsong ena, re bone le keketseho e matla ea li-mu opioid receptor ho NAc ha lihlopha tse ling, tse nang le mefuta e fapaneng, li bonts'itse polelo e fokotsehileng ho boemo ba "rat" ba pelehi bo pepesitsoeng HFD (nakong ea ho nyeka le mpa) (19, 21) kapa ha ho na phetoho (35). Liphetoho tsena, tse lekantsoeng boemong ba mRNA feela, li ka bonts'a ts'ebetso e fokolang ea li-circuits tsa DA tse amanang le maikutlo a phahameng a opioid (52) tseo mohlomong li sa lekanang ho ba le tšusumetso tlhahlobo ea boitšoaro eo re e entseng. Menahano ena e hloka ho netefatsoa ka ho sebelisa mekhoa e sebetsang. Tokomaneng ea morao-rao, e nang le mohlala o tšoanang, Romani-Perez et al. Ha baa khona ho bona keketseho e kholo ea li-boxbox tsa maemo a ts'ebetso bakeng sa bana ba HFD empa ba hlokometse molumo o mokhuts'oanyane oa ho fihlela lebokose la sepheo ho paradigm ea testway (22). Leha ho na le ho ba sieo ha khetho ea mafura a nako e telele maemong a rona a liteko, re fumane hore tšebeliso ea "perinatal maternal WD" e na le phello e tšoarellang lits'ebetsong tse ling tsa likhoerekhoere tse atisang ho buuoa haholo ke GABA reodeling ho NAc le Hypothalamus. NAc e nkuoa e le "sensor sentinel" bakeng sa boits'oaro bo kopanetsoeng (52). Boithuto ba morao-rao bo bontšitse hore tšebeliso ea lijo e matlafalitsoe ke thibelo ea li-neuron tsa GH-depting LH (53). O'Connor et al. e bonts'itse hore NAc D1R neurons (GABAergic projeks neurons) ka mokhoa o ikhethileng o thibela li-neurons tsa LH VGAT ho emisa ho kenella ha lijo (54). Liteko tsena li senola potoloho ea GABA (NAc / Hypothalamus) e kanna ea ikarabella ho laola karabelo ea boitšoaro. Sistimi ena ea ventral striatum-hypothalamic e sebetsa potoloho e 'ngoe e kenyelletsa boroko ba methapo ea methapo ea methapo ea methapo ea methapo ea methapo ea methapo ea kutlo.55). Karolo e 'ngoe ea bohlokoa ea potoloho e laolang takatso ea lijo e kenyeletsang le khetla ea NAc ke polelo ea Gaba-e tlosang phumano ho VP (56). Lintlha tsena li bonts'a karolo ea bohlokoa ea ho supa ha GABA lipuisanong pakeng tsa hypothalamus le NAc ho khothaletsa ho fepa. Thutong ea rona, ha re a khona ho khetholla palo ea li-neuron tse kentsoeng tšebetsong ea GABA le hore na liphetoho tsena li ka fetola marang-rang joang. Leha ho le joalo, karolo e bohareng ea lipotoloho tsa GABA e lokeloa ke thahasello e kholo. Haholo-holo ho ne ho tla ba monate haholo ho etsa liteko tse ling tse sebetsang tsa potoloho ena ea GABA u sebelisa mekhoa ea motlakase (57). Re boetse re hlokometse ntlafatso ea lefats'e ea mRNA transcript bakeng sa li-5HT1a le li-5HT1b receptors ho li-nuclei tse tharo tse ithutileng. Boholo ba li-fiber tsa serotonin tse sebelisoang li tsoa ho dorsal raphe nucleus (DRN) le median raphe nucleus (MRN). Lintlha tsa morao-rao tse tsoang Ka vivo lithuto tsa ho rekota le ho nahana ka litšoantšo li bonts'a karolo e ntle ea 5HT moputsong (58). Li-fiber tsa 5HT tse tsoang DRN li kentse letsoho taolong ea li-impulsivity (59). Ho eketsa 5HT1a ho VTA le NAc e ka ba mochine o qobelloang o ka laolang ho tsitsipana. Ho hypothalamus, lithuto tsa meriana li bontša hore 5HT1a receptor subtypes e ka hatella boitšoaro ba ho fepa bo susumetsoang ke ts'usumetso ea serotonin (60, 61). Ho eketsa 5HT1a le b receptors ho hypothalamus ho ka baka ts'ebetso e matlafatsang ea serotonin ka hona ho ka theha mochine o qobellang. Menahano ena e hloka ho netefatsoa ka ho etsa liteko tse sebetsang tse ntle.
Liphetoho tsena tsa marang-rang li amana le phetoho ea matšoao a polasetiki joaloka Ncam mRNA. Ho hypothalamus ea likhoto tsa batho ba baholo, re bone ho eketseha ha lingoliloeng tsa Ncam1 le St8sia4 tse fanang ka maikutlo le keketseho ea ho saena ha polysialic acid (PSA). PSA ke glycan e ka holim'a sele e fetohang litšebelisano tsa sele le sele. Polysialylation ea liprotheine tsa khomphutha ea lisele e kenella lits'ebetsong tse fapaneng tse tsamaeang le polasetiki ka har'a ts'ebetso ea methapo ea methapo mme ho tlalehiloe hore ho hlokahala polasetiki ea synaptic e lumellanang ea ho fepa lipotoloho nakong ea matla a leka-lekaneng a matla (33, 62). Ntle le moo, batsamaisi ba bang ba ho sebelisana ha lisele le li-synaptogeneis ba ka ameha ho sena polasetiki ea hypothalamic.
Qetellong, (Setšoantšo (Figure7), 7), ho ja oa basali oa WD ho na le tšusumetso ea nako e telele ho mokhatlo oa li-circuits tsa homeostatic le hedonic tse tsamaisang boitšoaro ba ho ja ka hara bana. Ka tlhahlobo ea linako tse mahlonoko tse tharo, re ile ra khona ho bonts'a phetoho e hlakileng ea khetho ea mafura e amanang le li-signature tse ikhethang tsa boko. Nakong ea bongoana, khetho ea mafura e ka tsamaellana le ts'ebetso e phahameng ea sistimi ea DA. Bokhachane, bo khetholloang ke ho kheloha khetho ea mafura, bo ne bo amahanngoa le polelo e tlase ea bangoli ba tsamaiso ea DA ba fanang ka maikutlo a hore ke mokhoa oa ho qobella. Ntlha e khahlisang haholo ho e tsebisa ke hore, ka mohlala ona, ho ja ka mokhoa o leka-lekaneng ka mor'a ho khoesoa ho ne ho ka sireletsa rat ea lilemong tsa bocha mokhoeng oa ho fepa ka ho fokotsa takatso ea bona ea mafura. Le ha e le batho ba baholo lihlopha tsena tse peli li na le mokhoa o tšoanang o phahameng oa ho rata mafura, litoeba tse tsoang matamong a fepa a WD li bonts'itse ntlafatso e kholo ea lipotoloho tsa GABA. Litlamorao tsa polasetiki ee e sa feleng ke life? Na ho ja ka mokhoa o fetelletseng oa nako ea bohloeki nakong ea bocha ho tla etsa hore mokhoa oo o lefelloang o sa bonahale o atleha? Lipotso tse joalo li kanna tsa ba le tšebeliso e nepahetseng ea tlhaiso ea masea a macha le bana ba tsohileng linaheng tse bophirima.
Boitšoaro ba Melao
Liteko tsohle li entsoe ho latela tataiso ea komiti ea tikoloho ea paballo ea liphoofolo, EU (taelo 2010 / 63 / EU), Institut National de la Recherche Agronomique (Paris, France), le Lefapha la Bongaka ba liphoofoloA44276). Protocol ea liteko e amohetsoe ke komiti ea melao ea boits'oaro mme e ngolisitsoe tlasa boits'oaro APAFIS 8666. Ho ne ho nkuoa tlhokomelo e 'ngoe le e' ngoe ho fokotsa khatello ea maikutlo le palo ea liphoofolo tse sebelisitsoeng lethathamong ka leng la liteko.
Menehelo ea Mongoli
JP le PB ba ile ba etsa liteko le ho nka karolo lipuisanong le ho ngola. TM e ile ea etsa PCA mme ea kenya letsoho lipuisanong le ho ngola. SN e kentse letsoho popehong ea liteko mme e nkile karolo lipuisanong. PP e kentse letsoho ho theheng teko, a nka karolo lipuisanong, 'me a ngola mongolo. VP e qapile le ho etsa liteko, ea sekaseka tlhaiso-leseling, 'me ea ngola sengoloa.
Tlhōlisano ea Tlhaloso
Bangoli ba bolela hore lipatlisiso li ne li etsoa ka ho se be le likamano leha e le life tsa khoebo kapa tsa lichelete tse ka nkoang e le khohlano e ka 'nang ea e-ba le thahasello.
lumela hore baa fokola
Bangoli ba ka thabela ho amohela Guillaume Poupeau le Blandine Castellano bakeng sa ho hlokomela liphoofolo nakong eohle ea lipatlisiso, Anthony Pagniez bakeng sa thuso ea hae ho li-mRNA extraction le TLDA, Isabelle Grit bakeng sa thuso ea hae tlhahlobisong ea samples ea plasma, le Alexandre Benani le Marie-Chantal Canivenc bakeng sa puisano ea bona e thusang le moralo oa TLDA.
Mongolo o botlaaseng ba leqephe
Lithuso. Patlisiso ena e ne e tšehelitsoe ke tikoloho des pays de la Loire Aid PARIMAD (VP), lithuso tsa motheo tsa LCL (VP le PP), SanteDige motheo (VP) le INRA Metaprogram DIDIT (SN, VP, PP).
Boitsebiso bo Eketsehileng
Lintho tse tlatsetsang bakeng sa sengoloa sena li ka fumaneha inthaneteng ho http://journal.frontiersin.org/article/10.3389/fendo.2017.00216/full#supplementary-material.
Setšoantšo sa S1
Kakaretso ea matla a mangata a tsoang ho oli ea poone e nang le botlolo. (A) Likhalori tse tsoang ho botlolo ea oli ea poone bakeng sa 24 h ho P25 ho malinyane a tsoang lijong tsa bophirima (WD) a fepa matamo le malinyane ho tsoa ho matamo a taolo (CD) a fedisitsoeng. (B) Likhalori tse tsoang ho botlolo ea oli ea poone bakeng sa 24 h ho P45 (letsatsi la boraro la tlhahlobo ea botlolo). (C) Likhalori tse tsoang ho botlolo ea oli ea poone bakeng sa 24 h ho P95 (letsatsi la boraro la tlhahlobo ea botlolo). Bakeng sa liphanele (A-C), datha li hlahisoa e le tsa bohlokoa ± SEM, ha ho phapang ea lipalo (p > 0.05) ho ile ha bonoa, kamora teko ea Mann le Whitney eo eseng ea parametric, lilemong tsohle. (D) Peresente ea likhalase tsa khalori tse tsoang botlolo ea oli ea poone e bapisoa le karolelano ea likhalori tse felletseng (botlolo ea oli ea poone + tloaelehileng chow lijo) bakeng sa 24 h ho P25 ho bana ba WD le lipopae tsa CD. (E) Peresente ea likhalase tsa khalori tse tsoang botlolo ea oli ea poone e bapisoa le karolelano ea likhalori tse felletseng (botlolo ea oli ea poone + tloaelehileng chow lijo) bakeng sa 24 h ho P45 (letsatsi la boraro la tlhahlobo ea botlolo) ho lipopao tsa WD le lipopae tsa CD. (F) Peresente ea likhalase tsa khalori tse tsoang botlolong ea oli ea poone e bapisoa le karolelano ea likhalori tse ngata (botlolo ea oli ea poone + tloaelo ea ho ja) bakeng sa 24 h ho P95 (letsatsi la boraro la tlhahlobo ea botlolo) ho lipopao tsa WD le lipopae tsa CD. Bakeng sa liphanele (D, E), datha li hlahisoa ka liperesente tsa palo eohle ea likhalori tse sa fumaneng phapang ea lipalo (p > 0.05) ho ile ha bonoa, ho latela chi-square ka khalemelo ea Yates, ka lilemo tsohle.
Setšoantšo sa S2
Li-photomicrographs tsa moemeli oa TH entunostunse ea li-nucleus accumbens (NAc) le sebaka sa ventral tegmental (VTA) ka linako tse tharo tse fapaneng. (A) Photomicrograph of TH / NeuN immunostuner at the VTA, −5.30 mm from Bregma. Re-label e khubelu ke ea NeuN, 'me e putsoa e le T. Motsu o mosoeu o bontša ho tsoa ha mothapo oa boraro. (B) Photomicrograph of TH entunosting maemong a NAc, + 1.70 mm ho tloha Bregma. Ho ngolla botala ke bakeng sa TH. Motsu o mosoeu o bonts'a selallo sa ka ntle.
Letlapa la S1
Lenane la liphatsa tsa lefutso tsa TaqMan le litekanyetso tse tsamaellanang tsa theknoloji li qopile likhoutu.
Letlapa la S2
Kakaretso ea tlhaiso-leseling e phatlalalitsoeng mabapi le polelo ea litlhaloso tsa dopamine. Litlhaku tse khubelu li tsamaisana le nako ea bongoana, tse putsoa ho ea bohlankana, le tse ntšo ho tsa batho ba baholo. =: E lumellana le polelo e tšoanang ea transcript pakeng tsa lihlopha, +: e tsamaellana le polelo e phahameng ea mongolo ho malinyane ho tsoa ho lijo tse phahameng tsa caloric [lijo tse se nang phepo e ntle, lijo tsa bophirima (WD), kapa lijo tse nang le mafura a mangata (HFD)], - e tsamaellana le polelo e tlase ea transcript ho malinyane a tsoang ho lijo tse phahameng tsa caloric (lijo tse se nang phepo, WD kapa HFD) matamo a phehiloeng.
References
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