I-DeltaFosB ilawula ngokungaqhelekanga umsebenzi we-Cck promoter (2010)

Ubuchopho Res. 2010 Meyi 6; 1329: 10–20.

Ipapashwe kwi-intanethi ngo-2010 ngoMatshi 11. doi: 10.1016 / j.brainres.2010.02.081

PMCID: PMC2876727

UJohn F. Enwright, III,¹ UMegan Wald,¹ Madison Paddock,¹ Elizabeth Hoffman,¹ URachel Arey,² Scott Edwards,² Sade Spencer,² Eric J. Nestler,³ kwaye Colleen A. McClung^{2, *}

Ulwazi loMbhali ► Ulwazi lobunikazi kunye neelayisensi ►

Inguqulelo yokugqibela yomhleli yeli nqaku iyafumaneka apha Res Resin

Bona amanye amanqaku ku-PMC Wisdom nqaku epapashwe.

Abstract

Olunye lolona tshintsho lubalulekileyo lwe-biochemical, i-structural, kunye nokuziphatha olubangelwa kukuvezwa okungapheliyo kwiziyobisi zokuxhatshazwa kubonakala ngathi kulamlwa yinto ekhutshelweyo ezinzileyo kakhulu ΔFosB. Umsebenzi wangaphambili ubonise ukuba i-ΔFosB overexpression kwiimpuku kwiiveki ze-2 ikhokelela ekonyukeni kokubonakaliswa kohlobo oluninzi kwi-striatum, uninzi lwazo lwathi lwathotywa emva kweeveki ezisi-8 zokubonakaliswa kwe-ΔFosB. Okubangel 'umdla kukuba, inani elikhulu lezi ntlobo zofuzo ziye zalawulwa kwiimpuku ezigqithisa into ebhaliweyo ye-CREB. Bekungacaci kolu phononongo, nangona kunjalo, ukuba i-ΔFosB yexesha elifutshane ilawula ezi genes nge-CREB. Apha sifumanisa ukuba iiveki ezi-2 ze-ΔFosB overexpression yonyusa ukubonakaliswa kwe-CREB kwi-striatum, isiphumo esichithayo ngeeveki ezisi-8.. Ukungeniswa kwangethuba kunxulunyaniswa nokunyuka kweCREB yokubophelela kubakhuthazi abathile bemfuza ekujoliswe kuyo kulo mmandla wobuchopho. Okumangalisayo kukuba, enye yemfuza eyayicingelwa ekujoliswe kuyo yi-CREB ngokusekelwe kwiingxelo zangaphambili, cholecystokinin (Cck), yayingalawulwa yi-CREB kwi-striatum. Ukuphanda ngakumbi ngolawulo lwe Cck Ukulandela i-ΔFosB overexpression, siqinisekisile ukuba i-ΔFosB yexesha elifutshane i-overexpression yonyusa zombini Cck umsebenzi wokukhuthaza kunye nokubonakaliswa kofuzo. Ikwanyusa umsebenzi obophelelayo kwindawo yokubopha iCREB (CRE) kwindawo Cck umkhuthazi. Nangona kunjalo, ngelixa indawo ye-CRE iyimfuneko kwinkcazo eqhelekileyo ye-basal ye Cck, ayifuneki kwi-ΔFosB yokwenziwa kwe Cck. Zithathiwe kunye, ezi ziphumo zibonisa ukuba ngelixa ukufakwa kwe-ΔFosB kwexesha elifutshane kwandisa ukubonakaliswa kwe-CREB kunye nomsebenzi kubakhuthazi abathile bemfuza, le ayisiyiyo kuphela indlela yokulawula ufuzo phantsi kwezi meko.

Internet: i-nucleus accumbens, ushicilelo, umlutha

INTSHAYELELO

Ukuboniswa okungapheliyo kwiziyobisi zokusetyenziswa kakubi kubangela utshintsho lwebhayoloji kunye nolwakhiwo kwimimandla emininzi yobuchopho. Olu tshintsho lucingelwa ukuba lubandakanya utshintsho kwiiprofayili zokubonisa imfuza kwinkqubo ye-mesolimbic. Le nkqubo yenziwe nge-dopaminergic neurons eprojekthi ukusuka kwindawo ye-ventral tegmental (VTA) kwi-midbrain ukuya kwi-spiny neurons ephakathi kwi-nucleus accumbens (NAc) (ventral striatum) kunye nenani leminye imimandla yobuchopho. Utshintsho kumsebenzi wezinto ezimbini ezikhutshelweyo, impendulo ye-cAMP ebophelelayo iprotheni (CREB) kunye ne-ΔFosB (iprotheni yosapho lwe-Fos), iye yacetywa ukuba ilamle uninzi lwe-biochemical, i-structural, kunye neenguqu zokuziphatha ezibonwa kunye nokuvezwa okungapheliyo kwiziyobisi zokuxhatshazwa. iphononongwe ngu Nestler, 2005).

I-CREB yinto echazwe kwindawo yonke elilungu losapho lwe-CREB/ATF. I-CREB homodimers ibophelela kwiiyantlukwano zokulandelelana kwe-CRE (imvumelwano: TGACGTCA) efunyenwe kubakhuthazi bemfuza emininzi. Iphosphorylation ye-CREB (ikakhulu kwi-serine 133, nangona ezinye iisayithi zichongiwe) zinokuvuselelwa ziimpawu ezininzi ze-cascades ezibandakanya ezo zisezantsi ze-dopamine receptors.Cole et al., ngo-1995). Phezu kwe-phosphorylation kwi-serine 133, i-CREB iqesha i-co-activator i-CREB yokubopha iprotheni (CBP) okanye iiprotheni ezinxulumeneyo ezikhokelela ekwandisweni kwembonakalo yemfuza (ihlaziywe ngu. UJohannessen et al., 2004). Ukuvezwa okungapheliyo kwi-opiate okanye i-psychostimulant yeziyobisi zokuxhatshazwa kubangela ukonyuka okwethutyana kumsebenzi we-CREB kwi-nucleus accumbens. (Barrot et al., 2002; Shaw-Lutchman et al., 2002, 2003), Ingcamango yohlengahlengiso yokunciphisa iimpawu ezinomvuzo zala machiza kunye nokuba negalelo kwimeko embi yeemvakalelo yokuyeka iziyobisi (Nestler, 2005).

LUlungelelwaniso oluhlala ixesha elide kwiziyobisi zokuxhatshazwa kucingelwa ukuba lulamlwa ngokuyinxenye yi-ΔFosB, i-splice eyahlukileyo ye-FosB (Nestler et al., 1999; McClung et al., 2004; Nestler, 2008). Amalungu osapho lwe-Fos ayancipha kunye namalungu osapho lwakwaJun ukwenza iprotein 1 ye-activator 1 (AP-1). I-AP-1 transcription complexes ibophelela kwiiyantlukwano ze-AP-XNUMX impendulo element (imvumelwano: TGACTCA) ukulawula ushicilelo (ihlaziywe ngu Chinenov kunye neKerppola, ngo-2001). Ngelixa ukuvezwa ngokuqatha kwiziyobisi zokuxhatshazwa kukhokelela ekufakweni kwexesha elifutshane (iiyure) zamalungu osapho lwe-Fos (kunye nokonyuka komsebenzi we-CREB), ukubonakaliswa kweziyobisi okuphindaphindiweyo kukhokelela ekuqokelelweni kwe-ΔFosB ezinzile kakhulu.Hope et al., 1994; Perrotti et al., 2008), ukubonakaliswa kwayo kuqhubeka iiveki.

Iimpuku ze-Bi-transgenic ngokugqithiswa ngokugqithisileyo nokuba yi-ΔFosB okanye i-CREB kwi-striatum yabantu abadala ibonisa uninzi lwe-biochemical kunye ne-phenotypes yokuziphatha ebonwa kwizilwanyana ezivezwe ngokuphindaphindiweyo kwii-psychostimulants okanye ezinye iziyobisi zokuxhatshazwa. AUhlalutyo lwenguqu yembonakalo yemfuza kwezi zilwanyana zitshintshileyo zichonge iindidi ezininzi zofuzo ezilawulwa lixesha elifutshane (iiveki ezi-2) ukuxinana okugqithisileyo kwe-ΔFosB, utshintsho olunxulunyaniswa nokuhla okuhambelana nomvuzo weziyobisi. Utshintsho kwimbonakalo yemfuza kunye nempendulo yokuziphatha kunye nexesha elifutshane ΔFosB zifana ngokuphawulekayo nezo zibonwa kwizilwanyana ezigqithise i-CREB kwiiveki ezi-2 okanye ezi-8.McClung noNestler, 2003). Ngokwahlukileyo omangalisayo, ukugqithiswa kwexesha elide (iiveki ze-8) ze-ΔFosB yehlisile ukubonakaliswa kolu hlobo lofuzo kwaye ikhokelele ekonyukeni komvuzo weziyobisi.UKelz et al., 1999; McClung noNestler, 2003).

Ufuzo olunye oluchongiweyo kolu phando lu cholecystokinin (Cck), i-neuropeptide eveliswa kwiindawo ezininzi zobuchopho, kubandakanya i-striatum (Hokfelt et al., 1980). I-CCK inokulungelelanisa ukuhanjiswa kwe-dopaminergic (IVaccarino, ngo-1992), ubandakanyeka kumvuzo weziyobisi kunye nokuqiniswa (Josselyn et al., 1996; UJosselyn et al., ngo-1997; Hamilton, et al., 2000; Beinfeld et al., 2002; Rotzinger et al., 2002), kwaye ifakwe kwi-striatum yi-cocaine engapheliyo (UDing kunye noMocchetti, ngo-1992). Ukongeza i Cck umgqugquzeli ubonakaliswe ukuba uphendule kuzo zombini ii-CREB kunye ne-AP-1 complexes (UHaun noDixon, ngo-1990; Deavall, et al., 2000; UHansen, 2001). Ekubeni uninzi lwemfuza (kubandakanywa Cck) ebonise intetho eyongeziweyo ilandela zombini i-CREB kunye nexesha elifutshane lokuchazwa kwe-ΔFosB zaziwa okanye kurhanelwa ukuba yi-CREB ekujoliswe kuyo (McClung noNestler, 2003), besifuna ukufumanisa ukuba ibinzana lexesha elifutshane le-ΔFosB likhokelela kulawulo lwezi geni (ngokugxila Cck) ngolawulo lwe-CREB okanye ngeendlela ezintsonkothileyo zolawulo lofuzo.

IINKCUKACHA

ΔFosB overexpression overexpression inyusa amanqanaba eCREB

Sisebenzise i-Western blotting ukuphanda ukuba i-ΔFosB overexpression inyusa amanqanaba eprotheyini ye-CREB kwi-striatum yeempuku. Kolu phononongo, sisebenzise iimpuku ze-bi-transgenic (umgca we-11A) ezithwala zombini i-NSE-tTA transgene kunye neTetOp-ΔFosB transgene. Ukungabikho kwe-doxycycline, ezi iimpuku zibonisa ukonyuka okuqinileyo kwi-ΔFosB yokubonisa kwi-dynorphin positive neurons kwi-striatum (UKelz et al., 1999). Le ndlela yokubonisa kakhulu i-ΔFosB ibhalwe ngokubanzi kwaye ivumela ukugqithisa okuthe ngqo kwingingqi, ehambelana nokungeniswa kwe-ΔFosB ebonwa kwizilwanyana ezivezwe kwi-cocaine engapheliyo.Hope et al., 1994; UKelz et al., 1999). Sifumene ukuba kwi-11A iimpuku ezigqithise kakhulu i-ΔFosB, amanqanaba eprotheyini ye-CREB alawulwa kakhulu emva kweeveki ze-2 zokuthetha kwaye la manqanaba abuyele kwisiseko emva kweeveki ze-8 zokuthetha (Umzobo 1A, n=8). Ukubona ukuba ngaba utshintsho kumanqanaba e-CREB ngexesha elifutshane loxinzelelo lwe-ΔFosB lunokuphindaphindwa kwenye inkqubo, siye sabonisa i-ΔFosB okwexeshana kwiiseli ze-PC12 kwaye safumanisa ukuba amanqanaba e-CREB anyuke kakhulu (p <0.05) xa ethelekiswa nolawulo.Umzobo 1BN=4). Ezi ziphumo zibonisa ukuba intetho ye-ΔFosB yexesha elifutshane yonyusa amanqanaba eprotheyini ye-CREB.

Amanqanaba e-CREB anyuka nge-ΔFosB yoxinzelelo olugqithisileyo. Uhlalutyo lweblothi lwaseNtshona lwe-lysates ukusuka (A) mouse striata ukusuka 11A iimpuku off dox for 2 okanye 8 iiveki okanye (B) I-PC12 iiseli ezibonisa ngokugqithisileyo i-ΔFosB. Idatha ngaphakathi A ziboniswa njengepesenti yotshintsho kwi-dox xa kuthelekiswa ...

Zombini i-ΔFosB kunye ne-CREB zibophelela kubakhuthazi bemfuza ethile ekujoliswe kuyo kwi-striatum elandela i-FosB yexeshana elifutshane.

Sisebenzise i-ChIP (i-chromatin immunoprecipitation) iimvavanyo ze-striatal tissue ukufumanisa ukuba i-ΔFosB okanye i-CREB isibophelelo senzeke kubakhuthazi abathile bemfuza ekujoliswe kuyo kwaye ukuba oku kubotshwa kuye konyuswa kulandela ukugqithiswa kwexesha elifutshane kwe-ΔFosB. Sihlalutye ukubopha kwi BDNF kwaye Cck abakhuthazi, kuba zombini iimfuza zilawulwa kakhulu emva kwexesha elifutshane le-ΔFosB overexpression, i-CREB overexpression, okanye unyango olungapheliyo lwe-cocaine (McClung noNestler, 2003). Siphinde sahlalutya ukubophelela kwi CDK5 umkhuthazi, kuba iyindawo ekujoliswe kuyo ngqo ye-ΔFosB (Chen et al., 2000; Bibb et al., 2001; Kumar et al., 2005). Ekugqibeleni, silinganise ukubopha kwi prodynorphin umkhuthazi, ekubeni izifundo zangaphambili zifumene ukuba inokubotshwa zizo zombini i-ΔFosB kunye ne-CREB phantsi kweemeko ezahlukeneyo (Andersson et al., 2001; Zachariou et al., 2006).

Uhlalutyo lwe-ChIP lwenziwa kwi-striata ukusuka kwi-11A yeempuku ezigqithise kakhulu i-ΔFosB kwiiveki ezi-2 kusetyenziswa izilwa-buhlungu eziqaphela i-CREB okanye i-ΔFosB. Phantsi kweemeko eziqhelekileyo, sifumene ukuba i-ΔFosB ibophelela kwi CDK5 kwaye prodynorphin abagqugquzeli, ngelixa kungekho zibophelelo ezibonakalayo kwi BDNF or Cck abakhuthazi (1 Table). Ngaphaya koko, i-ΔFosB overexpression yonyusa ukubophelela kwe-ΔFosB kwindawo CDK5 umxhasi, kodwa hayi kwi prodynorphin umkhuthazi. Ngokulandelayo silinganise ukubophelela kweCREB kwaba bakhuthazi kwaye safumanisa ukuba iCREB ibophelela kwi CDK5, BDNF kwaye prodynorphin abakhuthazi, kodwa hayi kuba Cck umgqugquzeli, kwi-striatum eqhelekileyo, kunye nokuba i-ΔFosB overexpression yeeveki ezi-2 yonyusa ukubophelela kwe-CREB BDNF kwaye prodynorphin abakhuthazi, kodwa hayi CDK5 umkhuthazi. Zithathiwe kunye, ezi ziphumo zibonisa ukuba i-ΔFosB kunye ne-CREB zinokubophelela zombini kubakhuthazi abathile bemfuza abafana prodynorphin kwaye CDK5, nangona kunjalo, ukubopha kwe-CREB kukhethekileyo kwabanye abakhuthazi abafana BDNF. Ngaphaya koko, i-ΔFosB overexpression ikhokelela ekonyukeni kokubophelela kwe-ΔFosB kubakhuthazi abathile, njengoko bekuya kulindeleka, kodwa nakwi-CREB ibophelela kubakhuthazi abathile, ngokuhambelana nokungeniswa kwe-CREB ye-ΔFosB-mediated eqatshelwe ngeli xesha.

Ukubophelela iiproteni zokulawula ezibekayo kubakhuthazi abohlukeneyo kwiimpuku ezigqithise kakhulu kwi-ΔFosB iiveki ezimbini.

Umsebenzi wangaphambili ubonise ukuba utshintsho kwimbonakalo yemfuza ebangelwa yi-ΔFosB yexesha elide yokuxinana inxulumene nohlengahlengiso lwe-chromatin, ngakumbi, i-acetylation ye-histone H3, kubakhuthazi abathile bemfuza.Kumar et al., 2005). Ukufumanisa ukuba oku kunokubangela utshintsho kwimbonakalo yofuzo kwixesha elifutshane le-ΔFosB lokuziphendulela ngokugqithisileyo, silinganise ukubophelela kwe-acetylated histone H3 (ulungiso lwe-histone olunxulunyaniswa nechromatin ebhaliweyo esebenzayo), okanye i-methylated histone H3 (Lys9, ukuguqulwa kwe-histone ehambelana nokubhaliweyo ngokubhaliweyo). ichromatin engasebenziyo). Sifumene ukuba ukugqithiswa kwe-ΔFosB kwiiveki ze-2 kubangele kungabikho lutshintsho ekubopheni i-acetylated H3 nakweyiphi na imfuza efundiswayo (1 Table). Siye safumana ukuncipha okukhulu ekubopheni i-methylated histone H3 kwi prodynorphin umgqugquzeli, kodwa akukho mbophelelo kwi Cck umgqugquzeli kwaye akukho tshintsho ekubopheni kwi CDK5 kwaye BDNF abakhuthazi, becebisa ukuba le ayisiyondlela eqhelekileyo apho i-ΔFosB, ngexesha elifutshane, ilawula ukubonakaliswa kofuzo. Sothukile kwaye sinomdla kwinto yokuba asifumananga mahluko kwiimvavanyo zethu ze-ChIP ezinokuphendula ngokunyuka Cck Inkcazo ye-mRNA kwiimpuku ze-11A ezilandela iiveki ezi-2 zokuchazwa kwe-ΔFosB kwaye yagqiba kwelokuba iphande le ndlela ngakumbi.

Ixesha elifutshane lokunyuka kweFosB Cck ukubonakaliswa kwiinkqubo ezininzi

Ukubonakaliswa kweMicroarray ye-bi-transgenic ye-11A yeempuku ezibonisa ngokugqithisileyo i-ΔFosB kwi-striatum yafumanisa ukuba Cck Amanqanaba enkcazo ayanda emva kweeveki ezi-2 zokuxinezeleka kakhulu kwaye emva koko anciphe ngokuthe ngcembe ngamaxesha amade e-ΔFosBUmzobo 2AN=3). Siqinisekise le siphumo Cck usebenzisa i-PCR yexesha langempela kwiqela elihlukeneyo lezilwanyana kwii-2 kunye neeveki ze-8 kwaye zifumene iziphumo kumaxesha amabini ukuba zifane nohlalutyo lwe-microarray (idatha engaboniswa).

Ukugqithisa kwexesha elifutshane lokunyuka kwe-ΔFosB Cck imbonakalo yofuzo. (A) Cck Inkcazo ye-mRNA kwi-striatum elandela i-ΔFosB overexpression kwi-11A iimpuku ze-1, 2, 4, okanye iiveki ezi-8. Uhlalutyo lweMicroarray lwenziwa kwiisampulu zokufa kunye Cck Amanqanaba ...

Ukuphanda ngakumbi ukubanakho kwe-ΔFosB ukulawula Cck intetho, sisebenzise iiseli ze-PC12 ezosulelwa okwethutyana nge Cck-luciferase reporter plasmid kunye nokuba yi-ΔFosB expression plasmid okanye inani elilinganayo le-pCDNA. Zombini ezimbini (n=5-9) kunye neentsuku ezintathu (n=11-13) ze-ΔFosB zokuchazwa ngokugqithisileyo kubangele ukonyuka okukhulu Cck-luciferase expression. Ukongeza, iintsuku ezintathu ze-ΔFosB overexpression ibangele kakhulu ngakumbi Cck-luciferase induction xa kuthelekiswa neentsuku ezimbini zoxinzelelo olukhulu (Umzobo 2B). Uxinzelelo olugqithisileyo lwe-ΔFosB alukhange lukhuthaze ulwakhiwo lwe-luciferase engenamxhasi (idatha ayiboniswanga). Ngaphantsi kweemeko zonyango kwakuncitshiswa Cck-umsebenzi we-luciferase uyabonakala. Ezi ziphumo zibonisa ukuba imbonakalo yexesha elifutshane ye-ΔFosB yonyusa umsebenzi we Cck umgqugquzeli, kwaye ushiya engaphendulwanga indlela apho i-ΔFosB yexesha elide icinezela ngokugqithisileyo Cck ibinzana.

Uxinzelelo olugqithisileyo lwe-ΔFosB yonyusa ukubophelela kwindawo ebophelelayo ye-CREB kwindawo Cck umkhuthazi

Uhlalutyo lwethu lufumanise oko Cck intetho yonyuka ilandela ibinzana lexesha elifutshane le-ΔFosB, nangona kunjalo, iimvavanyo zethu zeChIP zifumanise ukuba akukho CREB okanye ΔFosB ezibophelelayo Cck umkhuthazi. Ukuqinisekisa ukuba kukho naluphi na utshintsho kwiprotheyini ebophelelayo kwi Cck umgqugquzeli olandela i-ΔFosB overexpression, sisebenzise i-electrophoretic mobility assays (EMSA) kunye ne-probe equlethe indawo efana ne-CRE ekhoyo ngaphakathi Cck umkhuthazi. Sisebenzisa izicatshulwa zenyukliya ezivela kwi-striata ye-11A yeempuku ezigqithise kakhulu kwi-ΔFosB kwiiveki ezi-2, sifumene ukonyuka kokubophelela kwindawo ye-CRE yokubeka kwindawo Cck umxhasi (Isazobe 3 A,CN=4). Okubangela umdla kukuba, iimpuku ze-11A ezigqithise kakhulu i-ΔFosB kwiiveki ezisibhozo, ezibonisa ukuncipha Cck intetho, ibonise ukwanda kokubophelela kwesi siza (Isazobe 3B,CN=4). Njengothelekiso, siye savavanya ukubophelela kwindawo ye-CRE yemvumelwano kwiimpuku ezigqithise kakhulu i-ΔFosB kwiiveki ezi-2 kwaye safumana i-CRE eyomeleleyo ebophelelayo kwizicatshulwa ezibulalayo, kodwa akukho kunyuka kokubopha kulandela ukufakwa kwe-ΔFosB (Umzobo 3FN=4). Ke, ngaphandle kokunyuka okubangelwa yi-ΔFosB kumanqanaba e-CREB ewonke abonwe ngeli xesha, ezi ziphumo zihambelana novavanyo lwethu lwe-ChIP olufumanisa ukuba ukonyuka kwe-CREB kubopha kubakhuthazi abalandela i-ΔFosB overexpression ikhethekileyo kuphela kwimizila ethile kwaye ayisiyonto yehlabathi. . Ukumisela ukuba iCREB ibotshelelwe kwi Cck umkhuthazi kuhlalutyo lwethu lwe-EMSA, senze iimvavanyo eziphezulu nge-antibody ethile ye-CREB. Ngokuvumelana novavanyo lwethu lwe-ChIP, asifumananga nakuphi na ukubophelela kwe-CREB ku Cck uphononongo oluqulethe indawo ye-CRE yokubeka kwiimvavanyo ze-EMSA, ngelixa i-CREB iye yabopha kakhulu kwaye yatshintsha indawo ye-CRE yemvumelwano (Isazobe 3D,EN=4). Umsebenzi wokubopha i-DNA kwi Cck umkhuthazi naye akazange achatshazelwe li-antibody kwi-ΔFosB (idatha engaboniswanga), phantsi kweemeko eziboniswe kwizifundo ezininzi zangaphambili ukuvala i-ΔFosB ibophelela kwiindawo ezithembekileyo ze-AP-1 (Ithemba et al., 1994a, 1994b; Chen et al., 1995, Hiroi et al., 1998). Senze novavanyo lwe-ChIP kwi-striata yezilwanyana eziyi-11A kulandela iiveki ezisi-8 zokubonakaliswa kwe-ΔFosB kwaye khange sifumane ukubophelela kwe-CREB okanye i-ΔFosB Cck umkhuthazi (idatha ayiboniswanga). Ke, ukubonakaliswa kwe-ΔFosB kukhokelela ekonyukeni kokubopha iprotheni kwindawo Cck umgqugquzeli emva kweeveki zombini kunye ne-2 yokuvakalisa; nangona kunjalo, ukuchazwa kwezi zinto kuhlala kungaziwa.

Iiprotheyini ezibophelela kwi Cck umkhuthazi. (A, B) Uvavanyo lokuhamba kwe-Electrophoretic usebenzisa i Cck Indawo efana ne-CRE enezicubu ezibulalayo ezivela kwizilwanyana ezigqithise kakhulu i-ΔFosB kwiiveki ezi-2 (A) okanye iiveki ezisi-8 (B). Ku-(A), ukhuphiswano kunye nomntu okhuphisanayo ongabhalwanga ngokugqithisileyo ...

Indawo yokubeka CRE kwindawo Cck umgqugquzeli akanalo uxanduva lokunyusa umsebenzi wokukhuthaza

Ekubeni siye safumana ukwanda kwiprotheyini ebophelelayo usebenzisa iqhekeza le Cck umgqugquzeli, oqulathe indawo ye-CRE yokubeka, elandela i-ΔFosB overexpression, besifuna ukufumanisa ukuba le ndawo iyimfuneko ekonyusweni. Cck intetho elandela i-ΔFosB yokubonisa ngokugqithisileyo. Ukuvavanya oku kunokwenzeka, sosulele iiseli zePC12 nge Cck-luciferase plasmid equlethe indawo yayo efana ne-CRE-efana ne-CRE okanye enye equlethe ukuguqulwa kwisayithi eya kuphelisa nayiphi na intsebenziswano kunye ne-CREB. Okubangela umdla kukuba, ukuguqulwa kwesiza esifana ne-CRE kwehlile i-basal Cck umsebenzi wokukhuthaza ngama-32% (Umzobo 4A, n=9), kodwa ayichaphazeli i Cck ukwaziswa komxhasi nge-ΔFosB overexpression (Umzobo 4B, n=11-13). Oku kuthetha ukuba, nangona kunjalo Cck umgqugquzeli ufuna indawo efana ne-CRE efana ne-CRE yomsebenzi opheleleyo we-basal, ukwanda komsebenzi wokukhuthaza okubangelwa yi-ΔFosB overexpression ayifuni ukulandelelana okufana ne-CRE.

The Cck-like CRE site akuyomfuneko ukuba Cck ukwenziwa kwe-ΔFosB. (A) Cck-umsebenzi we-luciferase ulinganiswe kwiintsuku ze-2 emva kokudluliselwa kunye nokuba yinto eqhelekileyo Cck-luciferase okanye enye apho indawo efana ne-CRE yatshintshwa. * p<0.05 (B) Cck-luciferase ...

cFos ibophelela kwi Cck umkhuthazi

Izifundo zangaphambili zifumanise ukuba ii-cFos I-mRNA yonyuka ngentetho yexesha elifutshane ye-ΔFosB, nangona kunjalo, emva kokuchazwa kwexesha elide le-ΔFosB, amandla onyango lwe-cocaine ukukhuthaza i-cFos kwi-striatum iyancipha.McClung noNestler, 2003; Renthal et al., 2008). Ke ngoko, kunokwenzeka ukuba i-cFos ibe negalelo ekwandeni kwe Cck intetho elandela ibinzana lexesha elifutshane ΔFosB. Senze uvavanyo lwe-ChIP nge-antibody ethile kwi-cFos kwaye silinganisa i-cFos ebophelelayo kwi Cck umkhuthazi kunye nangaphandle kwexesha elifutshane lokubonisa i-ΔFosB. Ngelixa sifumene ukuba i-cFos iyabophelela kwi Cck umkhuthazi, esi sibophelelo asizange sonyuke kakhulu emva koxinzelelo olugqithisileyo lwe-ΔFosB (Umzobo 5, n=5). Oku kuphakamisa ukuba njengoko i-cFos ibophelela i Cck umkhuthazi, inokuba negalelo kummiselo jikelele we Cck intetho, kodwa kusenokwenzeka ukuba ayibandakanyekanga kulawulo lwe Cck umxhasi we-ΔFosB.

cFos ibophelela kwi Cck umkhuthazi. Uvavanyo lwe-Chromatin immunoprecipitation lwenziwa nge-antibody ethile ye-cFos kusetyenziswa izicubu ezibulalayo ezivela kwi-ΔFosB ezigqithisa kakhulu iimpuku nokuba kukwi-dox, okanye emva kweeveki ezi-2 zokususwa kwe-dox. Ixesha lokwenene uhlalutyo lwe-PCR lwaluyiyo ...

UKUQALA

Olu phononongo luqinisekisa kwaye lwandisa kwiziphumo zangaphambili ezibonisa ukuba i-ΔFosB ilawula ukubonakaliswa kofuzo kwi-striatum kwaye sifumanisa ukuba ikwenza oko ngeendlela ezininzi emva kokuchazwa kwexesha elifutshane. Sibonisa ukuba, emva kweeveki ze-2 zoxinzelelo olugqithisileyo, i-ΔFosB ibophelela ngokuthe ngqo kubakhuthazi kwiimfuza ezithile ezikhokelela kutshintsho kwintetho (okt. CDK5). Ngaphaya koko, yonyusa amanqanaba eprotheyini ye-CREB, isiphumo esibonwa kwiiseli ezikhuliswayo kunye nakwi-striatum, ekhokelela ekonyukeni kokubopha kwe-CREB kwabanye abakhuthazi bemfuza (okt. dynorphin kwaye BDNF). Kuphononongo lwangaphambili, sifumanise ukuba ukugqithiswa kwexesha elifutshane kwe-ΔFosB kwi-striatum kukhokelela kwiinguqu ezininzi ezifanayo zegenesis ezifumaneka xa i-CREB igxininisekile, kwaye ikhokelela kwiimpendulo ezifanayo zokuziphatha kwimilinganiselo yokukhetha i-cocaine.McClung noNestler, 2003). Ke ngoko, ukufunyaniswa kwangoku ukuba i-ΔFosB ikhokelela ekufakweni kwe-CREB, kunye nokubophelela kwe-CREB kubakhuthazi abathile bemfuza, inceda ukucacisa ukuba kutheni uninzi lotshintsho lwembonakalo yemfuza kwabelwana ngalo ngezi zinto zimbini zokukhutshelwa.

Ukwenziwa kwe-CREB ngu-ΔFosB kunomdla, kuba kubonisiwe ukuba iziyobisi zokusetyenziswa kakubi zibangela utshintsho kumanqanaba e-serine 133-phosphorylated CREB.UMattson et al., 2005) kunye nokwandisa ushicilelo lwe-CRE-mediated (Barrot et al., 2002; Shaw-Lutchman et al., 2002, 2003), ngaphandle kokutshintsha amanqanaba ewonke e-CREB. Kungenzeka ukuba utshintsho kumanqanaba e-CREB lunokudlula, kwaye, ngoko ke, luphoswe ngokulula kwezinye izifundo. Ezandleni zethu, sikubonile ukonyuka okubangelwa yi-cocaine kwi-CREB mRNA kuvavanyo oluthile, nangona kunjalo, esi siphumo siguquguquka kakhulu (uqwalaselo olungapapashwanga). Kuba zombini iimpuku ze-11A transgenic kunye neeseli ze-PC-12 zibonisa ukwenziwa kwe-CREB kulandela ugqithiso lwexesha elifutshane lwe-ΔFosB, oku kubonisa ukuba i-CREB ngenene yenziwe yi-ΔFosB (okanye ekujoliswe kuyo yi-ΔFosB), ibonelela ngenye indlela yokuchaza utshintsho olwenziwe ngamachiza kumfuza. intetho.

Okothusayo kukuba, siye safumanisa ukuba akukho CREB okanye ΔFosB ebophelelayo kwi Cck umkhuthazi nangona Cck intetho ilawulwa ngokucacileyo kulandela ibinzana lexesha elifutshane le-ΔFosB. I-Cck yi-neuropeptide eninzi echazwe kuzo zombini i-VTA kunye ne-NAc (Hokfelt et al., 1980) kwaye mhlawumbi ubandakanyeka kwiimpendulo zokuziphatha kwiziyobisi zokuxhatshazwa (Josselyn et al., 1996; UJosselyn et al., ngo-1997; Hamilton, et al., 2000; Beinfeld et al., 2002; Rotzinger et al., 2002). Kwiimvavanyo zenkcubeko yeeseli, i Cck umgqugquzeli ubonakaliswe ngokubanzi kwaye ubonakaliswe njengempendulo kwi-CREB kunye namalungu osapho lwe-AP-1 (ihlaziywe ngu UHansen, 2001). UHaun kunye noDixon (1990) wabonisa ukuba izakhiwo ze-AP-1 zinokubophelela kwi- Cck Indawo efana neCRE in vitro, kwaye kamva yaboniswa, isebenzisa iiseli ze-SK-N-MC ze-neuroblastoma, ukuba ukuguqulwa kwendawo efana ne-CRE kunciphise ukuphendula komgqugquzeli kwi-cFos / cJun egqithisileyo (Rourke et al., 1999). Ngokwenene, sikwafumana ukwanda Cck umsebenzi womkhuthazi (Umzobo 2) kunye nokubophelela kwindawo okanye ejikeleze into efana ne-CRE (Umzobo 3) phezu koxinzelelo olugqithisileyo lwe-ΔFosB, elinye ilungu losapho le-AP-1, kodwa asifumani mbophelelo ngqo ye-ΔFosB kwi Cck umkhuthazi kwi vivo or in vitro, kwanaxa ichazwa kakhulu.

Umsebenzi omninzi ubonise indima ye-CREB kulawulo lwe Cck umsebenzi wokukhuthaza. I Cck Indawo efana ne-CRE igcinwe kwizilwanyana ezinethambo lomqolo (UHansen, 2001) kwaye, kwezinye iimvavanyo zenkcubeko yeeseli, zombini i-CREB kunye ne-AP-1 iikhompleksi zibophelela kwesi siza kwaye ziyimfuneko Cck umsebenzi womkhuthazi (UHaun noDixon, ngo-1990; Deavall, et al., 2000; UHansen, 2001). Ukongeza, ii-activators ezininzi ezaziwayo ze Cck intetho (kubandakanywa i-bFGF, i-PACAP, i-peptones, kunye ne-depolarization) iboniswe ukuba isebenze nge-CREB (Hansen, et al., 1999; Deavall, et al, 2000; UBernard, et al., 2001; Gevrey, et al., 2002; Hansen, et al., 2004). Yethu Cck-luciferase ingxelo yedatha yofuzo ixhasa indima ebalulekileyo kwisiza esifana ne-CRE ekulawuleni Cck umsebenzi wokukhuthaza, kuba ukuguqulwa kwesi siza kunciphisa i-basal Cck umsebenzi wokukhuthaza kunye Cck-I-luciferase ibonakaliso eyenziwa yi-VP16-CREB, ifom ye-CREB esebenzayo, ilahlekile xa le ndawo iguqulwa (ukubonwa okungashicilelwanga). Ke, sothuswa kukufumanisa ukuba i-CREB ayibonakali ngathi ibophelela kwi Cck umgqugquzeli kwizicatshulwa zokubethelwa mhlawumbi kwisiseko okanye kwixesha elifutshane le-ΔFosB yoxinzelelo olugqithisileyo xa amanqanaba e-CREB esondiswa. Oku kuthetha ukuba amanqanaba e-CREB ngomntu ngamnye ayisiyiyo yodwa into ekumiseleni ubungakanani bokubopha kwesi siza, kwaye oku kuxhaswa ngumsebenzi wabanye (Cha-Molstad, et al., 2004). Ekubeni abagqugquzeli bezinye, ezichongiweyo ngaphambili i-CREB yemfuza ekujoliswe kuyo, njenge BDNF kwaye prodynorphin, ibophe i-CREB, siqinisekile ekufumaneni kwethu ukuba i-CREB ayibopheleli kwi Cck umxhasi kwiincindi zenyukliya ze-striatal. Ngaphaya koko, ukwenziwa kwe Cck-umsebenzi we-luciferase ngu-ΔFosB wawungaxhomekekanga kwindawo efana ne-CRE, ebonisa ukuba i-ΔFosB ayilawuli Cck intetho ngokulawula ubophelelo oluthe ngqo lwe-CREB kwi Cck umkhuthazi.

Ukungakwazi kwethu ukubona iCREB inxibelelana ne Cck umkhuthazi uxhaswa ngu Renthal et al. (2009), osebenzise indlela ye-Chip-chip ukujonga utshintsho lwehlabathi kwi-phosphorylated CREB (pCREB) kunye ne-ΔFosB ebophelela kwi-striatum emva kokuvezwa okungapheliyo kwe-cocaine. Kolu vavanyo, i-DNA-protein complexes yafakwa i-immunoprecipitated nge-ΔFosB okanye i-pCREB antibodies kunye ne-DNA enciphayo, emva kokulebula, yaxutywa kwi-microarray yokukhuthaza. Ngelixa uninzi lweendlela zofuzo ekujoliswe kuzo zeCREB ngaphambili zachongwa ngale ndlela (umz., BDNF, prodynorphin), Cck ayizange ichongwe. Ukongeza, kubonakaliswe ukuba ukubophelela kwe-CREB kwisiza se-CRE esivumelanayo kuyahluka kakhulu phakathi kwemigca yeeseli ekhulisiwe (Cha-Molstad, et al., 2004). Zonke izifundo zangaphambili ezifumene intsebenziswano yeCREB kunye ne Cck umgqugquzeli wenziwa kwinkcubeko yeseli (kungekhona ingqondo apho i-EMSA yethu kunye neesampuli ze-ChIP zithathwe khona) kwaye zisebenzisa i-gel shift assays ukuphanda i-protein-DNA interactions. Ngelixa i-EMSA inokuvavanya amandla okuba yinto enokuthi ibophe ukulandelelana kwe-DNA, iimvavanyo ze-ChIP zibonelela ngokujonga inoveli kule ntsebenziswano. kwi vivo. Ngaphaya koko, uninzi lwedatha ebonisa nokuba ukufakwa kwe Cck umkhuthazi umsebenzi okanye CREB ukubophelela kwi Cck umgqugquzeli wafunyanwa kwiiseli eziye zakhuthazwa zizinto ezifana neepeptones (UBernard et al., 2001), depolarization (Hansen, et al., 2004), okanye iindidi ze-activators ze-intracellular signaling cascades ezibandakanya i-cAMP kunye ne-ERK (UHansen et al., 1999). Kuvavanyo lwethu, kuphela "isivuseleli" esisetyenzisiweyo kukugqithiswa kwe-ΔFosB, okwaneleyo ukuphembelela Cck intetho. Xa kuthatyathwe kunye, oku kuphakamisa ukuba ukukwazi kwe-CREB ukuzibophelela (kunye nokuba nokulawula) kwi Cck umgqugquzeli uxhomekeke kakhulu kuhlobo lweeseli kunye nokusetyenziswa kweendlela ezithile zokubonisa. Ukongeza, i Cck Into yokukhuthaza efana ne-CRE (ubuncinci kwiiseli ze-PC12 ezingafakwanga nakwi-striatum yempuku) ayijoli ngqo nokuba yi-CREB okanye i-ΔFosB. Okubangela umdla kukuba, ummiselo we FosB uvakaliso lwe-CREB lukhethekileyo kuhlobo lweeseli kunye nodidi lovuselelo. Uphononongo olwenziwe nguAndersson okqhubekayo. yafumanisa ukuba inaliti ye-CREB antisense oligonucleotides kwi-striatum ye mouse ithintele ngokuyinxenye ukwenziwa kwe FosB kulandela ulawulo lwecocaine (Andersson et al., 2001). Nangona kunjalo, baye bafumanisa ukuba ukukwazi kwe-L-Dopa ukukhuthaza FosB intetho kwi-6-OHDA-lesioned striatum ayizange iphenjelelwe bubukho be-CREB antisense oligonucleotides.

Kuba i-CREB kunye ne-ΔFosB zibonakala ngathi zilawula ngokungathanga ngqo Cck umgqugquzeli, kunye notshintsho kwisakhiwo sechromatin zibhalwe ngokuphendula kwiintlobo ezahlukeneyo zempembelelo eyenza i-ΔFosB (I-Tsankova et al., 2004; Kumar et al., 2005; Renthal et al., 2008), siqiqe ngelithi i-ΔFosB inokumodareyitha ngokungathanga ngqo umsebenzi womkhuthazi ngokuguqula ubume bechromatin. Nangona kunjalo, kwiimpuku ezigqithise kakhulu i-ΔFosB kwiiveki ze-2, akukho tshintsho kwi-histone H3 acetylation Cck umxhasi (1 Table). Oku kuxhaswa yidatha yeChip-chip ye Renthal et al. (2009), owabika ukuba akukho tshintsho kwi-acetylated H3 yokubopha kwi- Cck umxhasi kwiimpuku ezivezwe kwi-cocaine engapheliyo. Ukususela kwi Cck umgqugquzeli uyasebenza kwi-striatum, akukho bophelelo be-methylated histone H3 bekulindelekile okanye babonwa. Okubangela umdla kukuba, asibonanga tshintsho ngenxa ye-ΔFosB yoxinzelelo olugqithisileyo kwi-acetylated H3 ebophayo BDNF umkhuthazi (obonise ukonyuka kokubophelela kwe-CREB) okanye kwi CDK5 kwaye prodynorphin abakhuthazi (ababonise ukonyuka kwe-ΔFosB). Kuba kukho intaphane yeenguqulelo ze-histone ezinxulumene notshintsho kumsebenzi wokukhuthaza (uhlaziywe ngu Rando kunye Chang, 2009), kusenokwenzeka ukuba kukho ezinye iinguqulelo zechromatin ezinxulumene nokuqaliswa kwezi genes. Naluphi na ukuguqulwa kwechromatin enye, nangona ngokuqhelekileyo kuqikelelwa kwinqanaba lomsebenzi womgqugquzeli, akunakutshintsha ngexesha lokusebenza kohlobo oluthile. Kumsebenzi wexesha elizayo, kuya kuba nomdla ukujonga ezinye iinguqu ezinokuthi zibekho kwisakhiwo sechromatin ngeenxa zonke Cck umgqugquzeli olandela i-ΔFosB overexpression. Okubangela umdla, i-cocaine engapheliyo (enokwenzeka ngokusebenzisa I-ΔFosB induction) ibonakaliswe ukuba ikhuthaze ukubonakaliswa kwe-sirtuin 1 kunye ne-2, iklasi ye-III ye-histone deacetylases ebonakala ngathi iguqula i-neuronal physiology, ukubonakaliswa kwe-ERK, kunye neempendulo zokuziphatha kwi-cocaine.Renthal et al., 2009). Ukwenziwa kwe-histone deacetylase kuya kuba nako ukulawula kwangaxeshanye ukubonakaliswa kwenani elikhulu lemizila yemfuza. ngokusebenzisa utshintsho lwehlabathi jikelele kwisakhiwo sechromatin.

Omnye onokuba ngumviwa obandakanyekayo Cck Ummiselo olandela i-ΔFosB overexpression yayililungu le-AP-1 losapho cFos. Uvakaliso lwe-cFos lulawulwa yi-CREB (Sheng et al., 1990; Impey et al., 2004), kunye ne-cFos overexpression (ngokuhambelana nokuchazwa okugqithisileyo kweqabane layo elibophelelayo cJun) liyenyuka Cck umsebenzi womkhuthazi (Rourke et al., 1999). Ke ngoko, ukonyuka kwamanqanaba e-CREB ngenxa yexesha elifutshane ΔFosB overexpression inokunyusa amanqanaba e-cFos kwaye ibangele ukonyuka kokubophelela Cck Indawo efana neCRE. cfos I-mRNA yenziwa kwiimpuku ezilandela iiveki ezimbini ze-ΔFosB overexpression (McClung noNestler, 2003) kwaye yehla kwiimpuku ezivezwe kwi-cocaine engapheliyo okanye ixesha elide le-ΔFosB overexpression (Renthal et al., 2008). Apha sifumanisa ukuba i-cFos ibophelela ngqo kwi Cck umkhuthazi kwizicubu ezibulalayo, nangona kunjalo, i-ΔFosB overexpression ayonyusi kakhulu ukubopha. Oku kucebisa ukuba ngelixa ii-cFos zinokubandakanyeka kulawulo Cck ukubonakaliswa ngokubanzi, utshintsho ekubopheni i-cFos iyodwa ayinakwenzeka indlela elawula ngayo i-ΔFosB. Cck intetho. Kuyenzeka, nangona kunjalo, ukuba i-ΔFosB isenokuthi ibangele, emva koguqulo utshintsho kwi-cFos (umz. i-phosphorylation etshintshiweyo) okanye ikhuthaze intetho yeqabane elibophelelayo (elifana ne-cJun) okanye iprotein ye-co-activator. Nangona kunjalo, ukusukela kwisiza esifana ne-CRE (esibonakaliswe ngaphambili njengesiza esibophelelayo sezakhiwo ze-AP-1, bona UHaun noDixon, ngo-1990) ayidingeki xa kunyuswa Cck Umsebenzi womkhuthazi obonwa nge-ΔFosB overexpression (njengoko ivavanyiwe kuvavanyo lwethu lofuzo lwentatheli), kuyavakala ukuba ezinye izinto ezenziwayo zilawulwa yi-ΔFosB.

The Cck isiqwenga somgqugquzeli esisetyenziswe kwimifuniselo yethu yofuzo ye-luciferase iqulethe indawo ebophelelayo ye-Sp1 kunye ne-E-box (ihlaziywe nguHansen, 2002). Ngokukodwa, ulandelelwano lwe-E-box lubonakaliswe ukubophelela izinto ezininzi ezikhutshelweyo (zihlaziywe ngu Forrest kunye noMcNamara, ngo-2004). Ukusebenzisa iiseli ze-PC12, sibonile ukuba ukuguqulwa kwebhokisi ye-E kuncipha Cck umsebenzi womkhuthazi, kodwa ayitshintshi impendulo yomgqugquzeli kwi-ΔFosB (idatha ayiboniswanga). Okubangela umdla kukuba, a Cck-intatheli ye-luciferase equlethe ukuguqulwa kwe-CRE indawo kunye ne-E-box ayinayo into ebonakalayo ebonakalayo kwaye ayiphenduli kwi-ΔFosB overexpression (ukubonwa okungapapashwa). Omnye umlamli onokubakho we-ΔFosB isenzo kwi Cck abagqugquzeli zii-ATFs, iindlela ezithile ezaziwa ngokuba zinyanzelwa kwi-striatum kukuvezwa okungapheliyo kwengqondo (psychostimulant exposure).Green et al., 2008). Nangona kunjalo, asifumananga bungqina bokungeniswa kwe-ΔFosB kwezi ATFs (idatha engaboniswanga), kwaye ii-ATF azinakulindeleka ukuba zibophelele kwindawo eguqulweyo efana ne-CRE Cck umkhuthazi.

Esinye isilumkiso solu phononongo kukuba sisebenzisa inkqubo ye-bi-transgenic ukugqithisela i-ΔFosB kwaye ke umntu kufuneka abe nolondolozo ekuzobeni ukufana phakathi kwale paradigm kunye nolawulo lwe-cocaine engapheliyo. Nangona kunjalo, iimpuku ze-11A ze-transgenic zinika ithuba elikhethekileyo lokujonga iziphumo ezithile ze-ΔFosB kwi-striatum, kuba ukubonakaliswa okugqithisileyo kulinganiselwe kulo mmandla wobuchopho (Chen et al., 1998), ngelixa ulawulo lwe-cocaine lubangela utshintsho kwiindawo ezahlukeneyo zobuchopho ezinokuthi emva koko zichaphazele ngokungathanga ngqo i-striatum. Ngaphaya koko, izifundo ezininzi zibhale i-phenotypes zokuziphatha ezifanayo kunye ne-molecular phenotypes kwiimpuku ze-11A xa zithelekiswa nezilwanyana ezingatshintshiyo eziphathwe nge-cocaine engapheliyo (UKelz et al., 1999; McClung noNestler, 2003; Renthal et al., 2009). Ukongezelela, Bibb et al. (2001) ichaze amanqanaba afanayo okubulala Cdk5 I-mRNA kunye nokungeniswa kweprotheyini kolu hlobo lufanayo lwe-11A xa kuthelekiswa ne-cocaine-treatated, non-transgenic littermates, kunye neenguqu ezifanayo kwiithagethi ze-CDK5, i-p35 kunye ne-DARPP-32.

Ukuqukumbela, sifumanisa ukuba intetho ye-ΔFosB yexesha elifutshane ikhokelela ekwenziweni kofuzo kwi-striatum ngeendlela ezininzi. Ezi ziquka ukubopha ngokuthe ngqo umgqugquzeli, ukufakwa kweprotheni ye-CREB kunye nomsebenzi, ukuguqulwa kwe-chromatin, ngaphezu kweendlela ezisafuneka zigqitywe.

IINKQUBO ZEMISEBENZI

izilwanyana

Izilwanyana ze-bi-transgenic ze-11A eziyindoda (NSE-tTA x TetOP-ΔFosB) zisetyenziswe kolu phononongo kwaye zibonakaliswe UKelz et al., 1999. Ukugqithisa i-ΔFosB, iigundane zasuswa kwi-doxycycline phakathi kwe-3 kunye ne-6 yeeveki ubudala, ngelixa iigundane zokulawula zigcinwe kwi-doxycycline. Zonke iimpuku zazigcinwe kwiqela kwaye zigcinwe kumjikelezo we-12:12 wokukhanya / omnyama, izibane zikhanyisa ngo-7 am kunye nezibane zicinywe ngo-7pm, kunye i-ad lib ukufikelela ekutyeni nasemanzini. Zonke iimvavanyo zempuku bezihambelana nemigaqo evunyiweyo yikomiti yokhathalelo lwezilwanyana kunye nokusetyenziswa kweYunivesithi yaseTexas Southwestern Medical Centre eDallas.

Intatheli kunye neeplasmids zokubonisa

Uhlobo lwasendle (WT) Cck umgqugquzeli-luciferase intatheli yalungiswa ngokufaka malunga ne-200 bp PCR fragment kwi-pGL3-luc vector (Promega). Esi siqwenga sifunyenwe kwi-mouse genomic DNA (ii-primers: 5 'TATCCTCATTCACTGGGACGC 3' phezulu, kunye ne-5' TACCTTTTGGATGGGGAAATCG 3' ezantsi) kwaye yaqala yafakwa kwi-pGEM-T Vector elula (Promega, #A1360). Isiqwenga somgqugquzeli saye sahlanganiswa kwiindawo ze-enzyme ze-Kpn1/Xho1 ze-pGL3-luc.

Ukwenza inqaku le-CRE utshintsho kwi Cck umgqugquzeli, i-mutagenic primer ejoliswe kwindawo echazwe ngaphambili efana ne-CRE (i-primer yoluvo: 5'CGTGTCCTGCTGGACTGAGCTCGCACTGGGTAAACA 3', i-antisense primer: 5'CTGTTTACCCAGTGCGCGCTGAGTCCAGCAGGACACG 3') isetyenziswe ngokudibanisa ne-Stratagenesis Quotstructions (IIIII) isetyenziswe ngokudibanisa ne-Stratagenesis . Oku kuguqula isayithi exeliweyo efana ne-CRE (ACTGCGTCAGC) ukuya kwi-ACTGAGCTCC. Zonke iiplasmids zentatheli ziqinisekiswe ngokulandelelana kweDNA. I-plasmid ye-ΔFosB yokubonisa iqulethe ubude obugcweleyo be-ΔFosB ulandelelwano olufakwe kwindawo eninzi ye-cloning ye-pCDNA 3.1 kwaye ichazwe ngaphambili (Ulery noNestler, ngo-2007).

Inkcubeko yeeseli kunye nokutshintshwa kwe-DNA

Iiseli zempuku ze-pheochromocytoma (PC12) zigcinwe kwi-Dulbecco's modified Eagle's medium F-12, zongezwa nge-10% ye-serum yehashe, i-5% ye-fetal bovine serum, kunye ne-1% ye-penicillin / streptomycin ku-37 ° C kunye ne-5% CO.₂. Iiseli zatshintshwa yi-electroporation kusetyenziswa i-electroporator ye-BTX 360 (350V, 0 ohms, kunye ne-850 μF) kwi-800 μL ye-phosphate buffered saline ye-Dulbecco kwi-cuvettes ye-gap eyi-4 mm kunye ne-10 μg yentatheli kunye ne-5 μg yentetho yokwakha. I-plasmid ye-vector engenanto (pCDNA) yayisetyenziselwa ukulungelelanisa izixa ezipheleleyo ze-DNA. Emva kosulelo, iiseli zakhuliswa kwi-35 mm ye-collagen-coated izitya ngexesha elibonisiweyo.

Iimvavanyo zeLuciferase

Kwiintsuku ezimbini okanye ezintathu emva kokudluliselwa, iiseli zahlanjwa amaxesha e-3 kwi-saline ye-phosphate buffered ye-Dulbecco, i-lysed (isebenzisa i-25 mM glycylglycine, 15 mM MgSO₄, 4 mM EGTA, 1% Triton X-100, pH 7.8, 1 mM DTT), iqokelelwe, kwaye icocwe nge-centrifugation. I-30 μL ye-lysate idityaniswe ne-140 μL i-luciferase assay buffer (25 mM glycylglycine, 15 mM MgSO₄, 4 mM EGTA, 1 mM DTT, 1 mM ATP, 1 mM potassium phosphate, 1 mM coenzyme A, pH 7.8). Umsebenzi weLuminescence ulinganiswe kusetyenziswa i-FLx-800 microplate fluorescence reader emva kwenaliti ezenzekelayo ye-40 μL 1 mM luciferin kwiqula ngalinye. Umsebenzi we-Luciferase ulungelelaniswe kumxholo weprotheyini iyonke njengoko kumiselwe luvavanyo lweprotheyini ye-BioRad.

Electrophoretic mobility shift assay

Izicatshulwa zeNyukliya ezivela kwizilayi zamacala amabini e-striatum ukusuka kwi-bi-transgenic 11A iimpuku (apho zigcinwe okanye zivaliwe i-doxycycline kangangeeveki ezi-2 okanye ezi-8) (McClung noNestler, 2003) zalungiswa ngokwe UHuang noWalters (1996). ³²Iiprobes ze-oligonucleotide ezibhalwe ngokuphindwe kabini zalungiswa kusetyenziswa iPromega Gel Shift Assay system protocol (#E3300) kwaye iiprobe zahlanjululwa kusetyenziswa iRoche Quick Spin Columns. Imvumelwano ye-CRE kunye ne-AP-1 yolandelelwano lophando yayisuka kwi-Promega (#E328A kunye ne-E320B, ngokulandelelanayo) kunye Cck Ulandelelwano lwe-CRE lwalu (Cck-CRE sense: CTAGCGAGCTCTGGACTGCGTCAGCACTGGGTGCA; Cck-CRE antisense: CCCAGTGCTGACGCAGTCCAGAGCTCGCTAGCTTT).

Iimpendulo ezibophezelayo kunye ne-electrophoresis zenziwe kusetyenziswa ukuguqulwa kwePromega Gel Shift Assay inkqubo inkqubo (#E3300). I-50,000 CPM yeprobe ebhaliweyo yadityaniswa ne-10 μg isicatshulwa senyukliya esibulalayo. I-DNA ekhuphisana ne-Cold okanye i-antibodies yongezwa ngaphambi kokuqaliswa kweeprobes ze-radiolabeled. Kuvavanyo lwe-supershift, i-2 μg ye-antibody ye-CREB (i-Upstate Biotechnology #06-083) yasetyenziswa. Iimpendulo ze-electrophoresed kwi-4% ye-polyacrylamide gels, yomisiwe, kwaye ibonakaliswe kwifilimu (usebenzisa izikrini zokuqinisa i-1 iyure ukuya kwiintsuku ze-2).

Immunoblotting

Kwiiseli ze-PC12, iipleyiti ze-35 mm zeeseli ezosulelekileyo zahlanjwa kumkhenkce obandayo we-Dulbecco's phosphate-buffered saline kunye ne-lysates zalungiswa kumkhenkce obandayo we-RIPA lysis buffer (50 mM Tris pH 7.4, 5 mM NaCl, 5 mM EDTA, 1% deoxycholate, 1 I-% Triton X-100, i-0.1% ye-sodium dodecyl sulfate) equkethe i-protease inhibitors. Emva kwe-sonication, ukucoca, kunye ne-Bradford protein assay, i-lysates yachithwa ngokupheleleyo kwaye i-50 μg yesampuli nganye i-electrophoresed kwi-10% ye-SDS polyacrylamide gels. Iiprotheyini zidluliselwe kwi-membrane ye-PVDF, ivalwe ngeyure le-1 kwi-3% yobisi olomileyo olungenawo amafutha kwi-saline ye-Tris-buffered equkethe i-0.1% Tween-20 (ubisi lwe-TBS-T), kwaye i-probed ubusuku bonke kwi-4 ° C kunye ne-antibodies eziphambili (CREB- I-Upstate Biotechnology #06-083, isetyenziswe kwi-1: 1,000; i-GAPDH- Sigma #G8795, isetyenziswe kwi-1: 80,000) ixutywe kubisi lwe-TBS-T. Emva kokuhlamba kaninzi kwi-TBS-T, ama-blots ahlolwe iyure enye kwindawo yokushisa kwegumbi kusetyenziswa i-alkaline phosphatase-conjugated secondary antibodies (Sigma) diluted 1: 5,000 kubisi lwe-TBS-T. Emva kokuhlamba okuninzi kwi-Tris-buffered saline, ukusabela kombala kwenziwa ngokwe-BioRad (#170-6432) imiyalelo. Iimembranes zomiswa ngobusuku, ziskenwe kwi-scanner ye-flatbed kunye ne-densitometry eyenziwa kusetyenziswa i-ImageJ (jonga ngezantsi).

Kwizicatshulwa zokubulala, iimvavanyo zeBlot zaseNtshona zaqhutywa njengoko zipapashwe ngaphambili (Hope et al., 1994). I-tissue yasuswa kwiigundane ezinqunyulwe iintloko, zabekwa phezu komkhenkce kwaye zahlulwe kwi-matrix yengqondo kubukhulu be-1 mm. I-tissue punches zaye zathathwa kwaye zafakwa emkhenkceni kwi -80 ° C de zisetyenziswe. I-tissue yayifakwe kwi-ice kwi-detergent esekelwe kwi-buffer ene-phosphatase kunye ne-protease inhibitors (i-Roche, i-Sigma). Emva kwe-sonication, iisampuli zahlanjululwa emanzini abilayo kunye ne-centrifuged kwi-15,000xg yemizuzu ye-15; amandla amakhulu emva koko aqokelelwa aze aqhutywe; Izixa zeprotheyini zoxinaniso zaye zalinganiswa kusetyenziswa i-Bradford assay (i-Bio-Rad). Iisampulu ziqhutywe kwi-gel ye-10% ye-acrylamide / bisacrylamide, idluliselwe kwi-membrane ye-PVDF, ivalwe kwi-5% yobisi kwaye ifakwe kwii-antibodies eziphambili (i-Anti-CREB, i-Upstate, i-Lake Placid, i-NY). Amabhulothi emva koko abonwa kusetyenziswa inkqubo ye-chemiluminescence (Pierce). Zonke iisampulu zaye zenziwa zesiqhelo kwiGAPDH (Fitzgerald, Concord, MA). Iigophe ezisemgangathweni ziye zaqhutywa ukuze kuqinisekiswe ukuba sikuluhlu lomgca wovavanyo.

Uhlalutyo lwe-Densitometry

Kwi-PC12 immunoblots, uhlalutyo lwe-densitometry lwenziwa ngokusebenzisa i-ImageJ kunye ne-rodbard calibration. I-avareji yesignali yangasemva yathatyathwa kumlinganiselo ngamnye kwaye umlinganiselo we-CREB ukuya kumqondiso we-GAPDH ubalwe kwisampulu nganye. Kwi-striatal immunoblots kunye nohlalutyo lwe-EMSA, iScion Image 1.62c isetyenziswe ngokususwa kwemvelaphi.

I-Chromatin immunoprecipitation (ChIP)

Uvavanyo lwe-CIP lwenziwa ngokweendlela UTsankova et al. (2004) kwaye Kumar et al. (2005). Ngokufutshane, iisampulu ze-bilateral striatal ezivela kwi-11A iigundane ezigcinwe okanye zivaliwe i-doxycycline zidibene kunye ne-1% ye-formaldehyde kunye ne-crosslinking yacinywa nge-glycine (i-concentration yokugqibela ye-0.125 M). Ezi sampuli beziphuma kuzo zonke izilayi zobuchopho ezithathwe kwinqanaba le-nucleus accumbens kunye ne-cortex esusiwe. I-Chromatin yachetywa malunga ne-0.2 ukuya kwi-1 kb amaqhekeza nge-sonication, yacinywa ngamaso eProtein G (Thermo Scientific #22852), kunye neesampulu ezifakwe emkhenkceni ku -80°C. Phakathi kwe-60 kunye ne-100 μg yechromatin isetyenziswe kwimvula nganye. I-5-10 μg ye-antibody nganye ephambili isetyenzisiwe (CREB: Upstate Biotechnology #06-863, ΔFosB: Santa Cruz Biotechnology #SC-48x, acetylated H3: Upstate Biotechnology #06-599, methylated H3 (LYS9): ITekhnoloji yokuSawula iSeli, cFos: Santa Cruz Biotechnology #SC-7202x). I-Antibody-chromatin complexes yafakwa i-immunoprecipitated ngeProtein G kunye namaso ngokwemiyalelo yomenzi (Thermo Scientific #22852). Ukulandela i-reverse crosslinking ye-input kunye neesampuli ze-precipitated, isampuli nganye yayiphantsi kwe-PCR yobuninzi (qPCR). Ukusetyenziswa kwazo zonke ezi zithinteli-zifo kwi-ChIP kuye kwaqinisekiswa ngokubanzi (I-Tsankova et al., 2004; Kumar et al., 2005; Renthal et al., 2009).

Amanqanaba eprotheyini ebophelelayo kumxhasi ngamnye wemfuza womdla amiselwe ngokulinganisa isixa se-DNA enxulumeneyo yi-qPCR (Applied Biosystems (ABI) Prism 7700, Foster City, CA). Igalelo okanye i-DNA iyonke (i-nonimmunoprecipitated) kunye ne-immunoprecipitated DNA ziye zandiswa ngokuphindwe kathathu phambi kwe-SYBR Green (i-Applied Biosystems, CA). Amaxabiso e-CT kwisampulu nganye afunyenwe kusetyenziswa i-Sequence Detector 1.1 software. Ubungakanani obunxulumeneyo betemplate yeDNA yenziwa kusetyenziswa indlela ye-ΔΔCt (I-Tsankova et al., 2004). Iiprimers ezisetyenzisiweyo: Umgqugquzeli we-BDNF 4: CTTCTGTGTGCGTGAATTTGCT; AGTCCACGAGAGGGCTCCA CDK5 umxhasi: GCTGAAGCTGTCAGGAGGGTC; I-GTGCCCCGCTCTTGTTATTA Umxhasi we-Cck: CTTGGGCTAGCCTCATTCACTG; TTAAATAGCTCCTCCCGGTTCG Umxhasi weProdynorphin: GGCTTCCTTGTGCTTCAGAC; GCGCTGTTTGTCACTTTCAA.

Uhlalutyo lwesatisatisti

Zonke iinkcukacha zinikezelwe njengeendlela ± impazamo eqhelekileyo yentsingiselo. Umahluko ngokwamanani wamiselwa luvavanyo loMfundi olunemisila emibini (p<0.05). Xa uthelekiso oluninzi lwenziwa, amaxabiso e-p ahlengahlengiswa kusetyenziswa ulungiso lweBonferroni.

Imibulelo

Sithanda ukubulela u-Will Renthal kunye no-Arvind Kumar ngeengxoxo eziluncedo. Sikwathanda ukubulela i-NIDA ngokuxhasa ngemali le mifuniselo.

Imihlathi

Iphepha elichazayo ukuba awusenanto oyifunayo: Le fayili yeFayile yombhalo wesandla ongabhalwanga owamkelwe ukushicilelwa. Njengenkonzo kumakhasimende ethu sinika le ngcaciso yokuqala kwincwadi yesandla. Umbhalo wesandla uza kufumana ukukopishwa, ukufakela, nokuphonononga ubungqina obunokubakho ngaphambi kokuba kukhutshwe kwifomu yayo yokugqibela. Nceda uqaphele ukuba ngexesha lokuveliswa kweeprogram ezinokuthi zifumaneke ezinokuthi ziphazamise umxholo, kunye nazo zonke izisemthethweni ezichasayo ezisetyenziswa kwiphephancwadi.

Amagama ohlelo:

Icandelo: #1 I-Cellular kunye ne-Molecular Biology ye-Nervous systems

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