(ABANTU) Izimpendulo Zendlela Yokuziphatha Nezimo Ezihlobene Ne-Cocaine Ezingapheli Kudinga Umkhiqizo Ophuthumayo Okubandakanya i-APF neCalidum / Calmodulin-I-Protein Encike Kinase II ku-Nucleus Accumbens Shell (2013)

I-transcript factor ΔFosB kanye neproteinulin enolwazi olusezingeni eliphakeme lwe-calcium (i-CaMKIIcy) ifakwa kuma-nucleus accumbens (NAc) ngokuchayeka okungapheli kokusebenzisa kabi i-cocaine noma ezinye izidakamizwa ze-psychostimulant zokuhlukumeza, lapho amaphrotheni amabili elwela izimpendulo zemithi ezwela ukuqonda . Yize i-ΔFosB neCaMKIIα zombili zilawula ukuveza nokusebenza kwe-AMPA glutamate receptor kanye nomsebenzi ku-NAc, ukwakheka komgogodla nge-dendritic ku-NAc medium spiny neurons (MSNs), nokuzwela nge-locomotor ku-cocaine, akukho ukuxhumana okuqondile phakathi kwalawa ma-molecule kuze kube manje okuye kwahlolisiswa. Lapha, sibonisa ukuthi i-ΔFosB ifakwe phosphorylated yiCaMKIIcy eSer27 futhi isenza iCaMKII ukuthi iyadingeka ekuqongeleleni i-cocaine-mediated ye-ΔFosB ku-rat NAc.

Ngakolunye uhlangothi, sibonisa ukuthi i-ΔFosB iyadingeka futhi yanele ukuthola i-cocaine ye-CaMKIIcy gene expression ku-vivo, umphumela okhetha i-D₁-type MSNs in the subcion NAc igobolondo.

Ngaphezu kwalokho, ukwenziwa kwe-dendritic spines kuma-NAc MSN kanye nokwanda kokuphendula kokuziphatha kwe-cocaine ngemuva kwe-NAc overexpression ye-ΔFosB yomabili i-CaMKII.

Okubalulekile, sibonisa okokuqala ukutholwa kwe-ΔFosB ne-CaMKII ku-NAc ye imilutha yabantu ye-cocaine, okuphakamisa okuhlosiwe okungaba khona kokungenelela kokwelashwa okuzayo. Le mininingwane isungula ukuthi i-ΔFosB ne-CaMKII bazibandakanya ocansini oluhlobo lweseli- kanye nobuchopho obuqondene nobuchopho njengendlela ebalulekile yokulawula ukujikeleza kwengqondo kwengqondo ngenxa yecocaine engapheli.

Isingeniso

Ubufakazi obukhuphukayo buxhasa umbono wokuthi izinguquko ekushintsheni kwezakhi zofuzo ziba nomthelela ezinhlelweni zokuluthwa izidakamizwa (URobison noNestler, 2011). Umlamuli obalulekile walezi zinguquko yi-ΔFosB, into yomndeni ka-Fos (I-Nestler, i-2008). Ukuphathwa okungapheli kwanoma yisiphi isidakamizwa sokuhlukumeza kubangela ukuqongelela okuhlala isikhathi eside kwe-ΔFosB ku-nucleus accumbens (NAc), isifunda esinezimbambo ezibalulekile ekuziphatheni komvuzo. Si-uch induction ibonakala iqonde esigabeni se-NAc medium spiny neuron (MSN) esiveza i-D1 dopamine receptors. I-overexpression engafinyeleleki ye-ΔFosB kulezi zinhlobo zama-D1 zohlobo lwe-NAc zandisa i-locomotor nezimpendulo ezinomvuzo ku-cocaine ne-morphine (Kelz et al., 1999; UZachariou et al., 2006), kufaka phakathi ukuzikhulisa okwandayo kwe-cocaine (I-Colby et al., I-2003). Ngaphezu kwalokho, i-genetic noma i-viral blockade yomsebenzi we-ΔFosB wokuloba kunciphisa imiphumela evuza yale mishanguzo (UZachariou et al., 2006), okubonisa ukuthi lokhu kusungulwa okungaguquki kwe-osFosB kungumlamuleli obalulekile wezinguquko ezingapheli ezivezwe ku-NAc ngabaphathi bezidakamizwa ezingapheli.

Ukuqina okungajwayelekile kwe-ΔFosB (okuhlobene nawo wonke amanye amaprotheni omndeni we-Fos) kuyimpahla eyingqayizivele ye-molecule, ngenxa yokuncipha kwesizinda se-defon esikhona kubude obugcwele be-FosB (UCarle et al., 2007), nenqubo elawulwayo. I-ΔFosB ifakwe phosphorylated kwesibeletho futhi vivo kwaSer27, futhi lokhu kusabela kuqinisa ΔFosB, ~ 10-fold, kusiko leselula ne-NAc vivo (Ulery-Reynolds et al., 2009). Yize i-Ser27-ΔFosB ikhonjisiwe ukuthi iyindawo ephansi ye-casein kinase-2 kwesibeletho (Ulery et al., 2006), inqubo yayo ye vivo phosphorylation uhlala engaziwa.

I-calcium / mododulin-based protein proteinaseasease (CaMKII) yi-serine / threonine kinase ekhonjwe kakhulu futhi and isoforms yakhe i-dodecameric homo- kanye ne-hetero-holoenzymes vivo, futhi zibalulekile ezinhlotsheni eziningi ze-neuroplasticity (Lisman et al., 2002; UColbran noBrown, 2004). I-CaMKIIcy idonswa ngokungakhethi igobolondo le-NAc ngu-amphetamine (ULoweth et al., 2010), kanye ne-pharmacological blockade yomsebenzi we-CaMKII ku-NAc igobolondo kunciphisa ukuzwela kokuziphatha ku-amphetamine (ULoweth et al., 2008) kanye ne-cocaine (UPierce et al., 1998), ngenkathi i-viral overexpression ye-CaMKIIcy kule subcion ye-NAc ithuthukisa ukuzwakala kwendawo nokuzilawula kwe-amphetamine (ULoweth et al., 2010). I-CaMKIIcy ingathinta ukusebenza komvuzo ngokuguqulwa kwama-AMPA glutamate receptor subunits (UPierce et al., 1998), njengoba umsebenzi weCaMKIIα sekuyisikhathi eside uhlotshaniswa nomsebenzi we-AMPA receptor kanye nokuqondiswa kwe-synaptic ngezindlela eziningana ze-neuroplasticity (IMalinow neMalenka, 2002).

Lezi zincwadi zibonisa ukufana okuningana phakathi kwe-ΔFosB ne-CaMKII: zombili ziyadingeka futhi zinemiphumela eminingana yokuziphatha kwezidakamizwa zokuhlukumeza, zombili lezi zibalo ezilinganayo ezinhlobonhlobo zamaseli e-neuronal vivo (Uhambo et al., 2003; Maze et al., 2010), futhi bobabili basebenzisa eminye yemiphumela yabo yokuziphatha ngokuguqulwa kwama-receptors e-AMPA (Kelz et al., 1999; IMalinow neMalenka, 2002; I-Vialou et al., 2010). Ngaphandle kwalokhu kufana, akukho ukuxhumana okusebenzayo phakathi kwe-ΔFosB ne-CaMKII. Lapha, sisungula ukumiswa kokubuyisana phakathi kwe-ΔFosB ne-CaMKII, futhi sikhombise ukuthi lawo maprotheni amabili akha i-D1-hlobo MSN-othize feed-phambili loop ku-NAc igxathu elibangelwa yi-cocaine futhi lilawula uhla lwezimpendulo ze-cocaine vivo.

Iya ku:

Izimpahla nezindlela

I-1 Yesilingo: Ukuhlaziywa kweProteomic ye-iTRAQ kwe-NAc Shell neCore Ngemuva Kwe-Cocaine Ukwelashwa (I-Fig 1A)

Ama-Adult (8 weeks) amagundane abesilisa ayephathwa i-20 mg / kg cocaine noma i-IP ye-saline car kanye ngosuku ngezinsuku eziyisikhombisa. I-24 hr ngemuva komjovo wokugcina, igobolondo le-NAc kanye nomgogodla kwenziwa microdissected (I-Fig 1A) futhi kufrize iqhwa. Ukuhlaziywa kwe-iTRAQ kwenziwa njengoba kuchaziwe phambilini (URoss et al., 2004; UDavalos et al., 2010).

Umfanekiso we-1

Ukungeniswa kwe-Shell okuqondile kweCaMKII ku-NAc yi-cocaine

Isilingo 2: Quantifying Protein changes in Rat NAc Core and Shell Ngemuva Kwe-Cocaine Ukwelashwa (I-Fig 1B-D)

Ama-Adult (8 weeks) amagundane abesilisa ayephathwa i-10 mg / kg cocaine noma i-IP ye-saline IP kanye ngosuku izinsuku eziyisikhombisa kumagumbi okuqoshwa kwama-locomotor. Izimpendulo ze-locomotor kumjovo owodwa we-cocaine (i-5 mg / kg IP) kwaqoshwa kulezo zilwane ezaziphathwa ngaphambili nge-cocaine (ebizwa ngokuthi "okungapheli") kanye nengxenye yalabo abaphathwe nge-saline (ebizwa nge- "acute"), kanye nezimpendulo ze-locomotor ku-saline iyodwa yabhalwa ezilwaneni ezingama-saline ezingalapheki ezaziphathwa usawoti (ezibizwa nge- “saline”). Ukutholwa komsebenzi we-locomotor kwenziwa njengoba kuchaziwe (U-Hiroi et al., 1997). Kafushane nje, amagundane amadala abantu abadala afakwa emabhokisini okuqoshwa kwe-PAS evulekileyo (i-San Diego Instruments) ye-18 min ukuze ihlalwe, anikezwe umjovo owodwa we-IP kasawoti futhi waqashwa ngemizuzu eyengeziwe ye-24, futhi anikezwa umjovo owodwa we-IP we-30 mg / kg cocaine futhi ubhekelwe imizuzu ye-30.

I-24 hr kulandela lo mujovo wokugcina, amagundane anqunywa ngaphandle kwe-anesthesia ukugwema imiphumela yokubulala izinzwa emazingeni amaprotheni e-neuronal kanye nama-phospho-state. AmaBrains asikwa ngokulandelana nge-mathenx ye-1.2 mm (iBraintree Science) kanti izicubu eziqondisiwe zasuswa ku-phosphate buffered saline aqukethe ama-proteinase (Roche) kanye ne-phosphatase (Sigma Aldrich) inhibitors esebenzisa i-14 gauge punch ye-NAc core kanye ne-12 gauge punch of esele izicubu ze-NAc igobolondo (Bheka I-Fig 1A) futhi ngokushesha eqandisiwe eqhweni elomile. Amasampula aqanjwa yi-sonication ekhanyayo ku-RIPA buffer eguquliwe: Isisekelo se-10 mM Tris, i-150 mM sodium chloride, i-1 mM EDTA, i-0.1% i-sodium dodecyl sulfate, i-1% Triton X-100, i-1% sodium deoxycholate. njengoba ngenhla. Ngemuva kokungezelelwa kwe-Laemmli buffer, amaprotheni ahlukaniswe kuma-7.4-4% polyacrylamaide gradient gels (Criterion System, BioRad), futhi ukuqothuka kwaseNtshonalanga kwenziwa kusetshenziswa uhlelo lwe-Odyssey (Li-Cor) ngokusho kwemigomo yokusebenza yomakhi.

I-3 Yesilingo: Izilinganiso zokuguqula amaProtein ku-Rat NAc Core kanye ne-Shell Ngemuva kokuhoxiswa kwe-Cocaine (I-Fig 1E)

Ama-Adult (8 weeks) amagundane abesilisa ayephathwa i-10 mg / kg cocaine noma i-IP ye-saline car kanye ngosuku ngezinsuku eziyisikhombisa. Izinsuku ze-14 ngemuva komjovo wokugcina, izilwane eziphathwe nge-saline zanikezwa omunye umjovo we-saline (obizwa ngokuthi “usawoti), nezilwane ezaziphathwa nge-cocaine zanikezwa omunye umjovo we-saline (obizwa ngokuthi ukuhoxiswa kosuku lwe-14 noma“ i-14d WD ”) noma umjovo owodwa we-cocaine ( ibizwa nge- "14d WD Chal" ngenselelo). Ihora elilodwa ngemuva komjovo wokugcina, izilwane zabulawa futhi kwaqothulwa iNtshonalanga njengasekuqaleni Zama i-2.

Isilingo 4: Quantifying Protein changes in Rat NAc Core and Shell After Cocaine Ukuzilawula (I-Fig 2A-C)

Amagundane aqeqeshelwa ukuzilawula ngokwawo i-0.5 mg / kg / ukumnika i-cocaine esimisweni esisodwa se-hr ngaphansi kwesilinganiso se-1 esimisiwe sezinsuku eziyisishiyagalolunye. Ngemuva kweseshini eziyisishiyagalolunye eziyisisekelo, amagundane ahlukaniswe ngamaqembu amabili alinganiselwe ngokudla i-cocaine ezihlokweni ezimbili zokugcina. Iqembu elilodwa lamagundane lavunyelwa ukuzilawula i-cocaine (i-0.5 mg / kg / kg ukumnika) ngezikhathi ezithile ze-hr (ukufinyelela okufushane, i-ShA) ngenkathi elinye iqembu lamagundane elizenzela i-cocaine emihlanganweni eyisithupha-hr (ukufinyelela isikhathi eside, i-LgA ) izinsuku eziyishumi ezengeziwe (izikhathi zokukhuphuka).

Izingxenye zobuchopho zalungiswa nge-immunohistochemistry njengoba kuchaziwe (I-Perrotti et al., I-2004). AmaBrains asetshenziswe i-18-24 hr ngemuva kokuchayeka okokugcina kwezidakamizwa, okuholele ekuwohlokeni kwanoma yiliphi iprotheni ye-FosB eseleyo yobude obugcwele ukuze konke ukugonywa okusele kubonise ΔFosB. Lokhu kulinyazwa kwaqinisekiswa ukuqothulwa kwaseNtshonalanga, okukhombisa ukungabi nalutho olubalulekile nge-antibody eqondiswe ethempelini C le-FosB yobude obugcwele engayiqapheli i-ΔFosB (idatha engakhonjisiwe). Ngemuva kokunamathisela ezingxenyeni ze-35 µm, inani lamaseli we-ΔFosB immunopositive labonwa ngumbukeli oyimpumputhe ezigabeni ezimbili nge-NAc yendawo ngayinye, futhi okusho amanani ngensimu ngayinye ye-40 × yabe ibalwa yisifunda isilwane ngasinye. Isilwane ngasinye sasibhekwa njengokubhekwa komuntu ngamunye kokuhlaziywa kwezibalo. Izifunda ezithintekayo zikhonjwe kusetshenziswa uPaxinos noWatson (I-Paxinos ne-Watson, i-2007).

Ukuqothulwa kwe-CaMKIIcy immunoreactivity kwenziwa ngokusebenzisa uhlelo lweLayisense njengoba kuchaziwe (UCovington et al., 2009). Amandla ahlanganisiwe weCaMKII ne-GAPDH anqunywa ngesoftware ye-Odyssey. Imiphumela yethulwa njengamanani wokuqina ahlanganisiwe mm² futhi kwethulwa njengezindlela ± sem (n = 4-10 ngeqembu ngalinye). Amanani we-GAPDH asetshenziswa njengokubhekiswa nokujwayeza ubukhulu be-CaMKII ubukhulu bokushuba kwesimo kanye nezimo.

Umfanekiso we-2

Ukungeniswa kweCaMKII ku-NAc igobolondo lamagundane azisebenzelayo kanye nemilutha yabantu ye-cocaine

Isilingo 5: Quantifying Protein Levels in Cocaine-Dependent Humans (I-Fig 2D)

Inqubo

Izicubu zobuchopho bomuntu wase-Postmortem zitholwe kwiQuebec Suicide Brain Bank (Douglas Mental Health University Institute, Montreal, Quebec, Canada). Ukulondolozwa kwezicubu kwenziwe ngokuyisisekelo njengoba kuchaziwe (I-Quirion et al., 1987). Kafushane nje, lapho selukhishwe, ubuchopho bubekwa eqhweni elimanzi ebhokisini leSkyrofoam bese liphuthuma ezikhungweni zeBhange iQuebec Suicide Brain Bank. AmaHemispheres ahlukaniswa ngokushesha ngumsiki we-sagittal phakathi kwengqondo, isiqu sobuchopho kanye ne-cerebellum. Imithambo yegazi, i-pineal gland, i-choroid plexus, uhhafu we-cerebellum, kanye nengxenye yesigaxa sobuchopho kuvame ukuhlakazwa kusuka endaweni engakwesobunxele, lapho-ke kusikwa kube yi-coronally ibe yizicucu ze-1 cm-thick ngaphambi kokubandwa. Isigaxa sokugcina se-cerebellum sinqunywa kube izingcezu ze-1cm-thick thick ngaphambi kokubanda. Amathishu aqandwe yi-flash ku-2-methylbutane ku-−40 ° C ye-X XUMUM sec. Zonke izicubu ezifriziwe zigcinwa zodwa ezikhwameni zepulasitiki ezise-−60 ° C kwisitoreji eside. Izindawo ezithize zobuchopho zihlukaniswe yizicucu zama-coron eqandisiwe epuletini yensimbi engenacala neqhwa elomile nxazonke ukulawula izinga lokushisa lemvelo. I-Western blotot yenziwa njengoba kuchaziwe ku Zama i-2.

UCohort

I-cohort yayiqhathaniswa nezifundo zabesilisa ze-37 nezifundo zabesifazane ze-3, ezineminyaka ephakathi kweminyaka engu-15-66. Zonke lezi zifundo zife kungazelelwe ngaphandle kwesimo se-agonal isikhathi eside noma ukugula kwezokwelapha okwatholakala isikhathi eside. Kuwona womabili la mazwe, imbangela yokufa yaqinisekiswa yihhovisi laseQuebec's Coroner, futhi kwenziwa isikrini esinobuthi ngesampuli yezicubu ukuthola imininingwane yezokwelapha nokusetshenziswa kwezidakamizwa ezingekho emthethweni ngesikhathi sokufa. Iqembu lezifundo laliqukethe abantu be-20 abahlangabezane nenqubo ye-SCID-I yokuxhomekeka kwe-cocaine. Iqembu lokulawula laliqukethe izifundo ze-20 ezingenawo umlando wokuxhomekeka kwe-cocaine futhi akukho ukuxilongwa okukhulu kwezifo zengqondo. Zonke izifundo zifa ngokuzumayo ngenxa yezimbangela ezazingenalo ithonya eliqondile kwezicubu zobuchopho. Amaqembu ayefaniswa iminyaka yobudala enganqunyelwe, ukubambezeleka kwesiqandisi, ne-pH. Kuzo zonke izifundo, ukucwengeka ngokwengqondo kwenziwa njengoba kuchaziwe ngaphambili (UDumais et al., 2005), kusivumela ukuthi sikwazi ukufinyelela kumininingwane yemininingwane enemininingwane emlandweni wezengqondo nezokwelapha, kanye neminye imininingwane efanelekile yezokwelapha kanye neyenhlalo. Kafushane nje, umuntu oxoxisana naye waqeqeshwa Uhlolokhono Olungisiwe Lomtholampilo we-DSM-IV Izinkinga Zokusebenza Kwezengqondo (i-PsIDchialric Disways (i-SCID-I) nomuntu oyedwa noma abaningi abazisa ngomufi. Iphaneli yodokotela ibukeze ukuhlolwa kwe-SCID-I, imibiko yamacala, amanothi we-coroner, kanye namarekhodi ezokwelashwa ukuthola ukutholwa kwezifo zengqondo okuvumelana.

Isilingo 6: Chromatin Immunoprecipation for Rat NAc (I-Fig 3A-C)

Ama-Adult (8 weeks) amagundane abesilisa ayephathwa i-10 mg / kg cocaine noma i-IP ye-saline car kanye ngosuku ngezinsuku eziyisikhombisa. I-24 hr ngemuva komjovo wokugcina, igobolondo le-NAc kanye nomgogodla kwenziwa microdissected. I-Chromatin immunoprecipitation (ChIP) yenziwa ngokudonswa amagobongo kwe-NAc igobolondo noma umongo kusuka kumagundane ayisikhombisa eqenjini ngalinye emaqenjini we-14 ingqikithi (isibalo sezilwane ze-98, amachibi we-cocaine we-7, amachibi we-7 saline). Amathayi axhunyaniswa ngesiphambano, ahlanzwa, futhi agcinwe ku-−80 ° C kuze kube yilapho kugawulwa i-chromatin. I-Sheared chromatin yashiswa ubusuku bonke ngama-antibodies ngaphambili aboshelwe kubuhlalu bozibuthe (Dynabeads M-280, Attitrogen). I-IgG engavimbeli mzimba yayisetshenziswa njengesilawuli. Ngemuva kokuxhuma okuxhunyaniswe kabusha nokuhlanzwa kwe-DNA, i-qPCR yayisetshenziselwa ukukala amazinga we-CaMKIIα Promoter DNA. Ama-primers aklanyelwe ukukhulisa isifunda esiqukethe ukulandelana kokuvumelana kwe-AP-1 etholakala ~ 450 bp ngaphambi kwesizinda sokuqalisa sokubhaliwe (Phambili: ACTGACTCAGGAAGAGGGATA; Buyisa emuva: TGTGCTCCTCAGAATCCACAA).

Umfanekiso we-3

Uhlobo lweseli- kanye ne-ΔFosB eqondene ne-CaMKIIα vivo

I-7 Yesilingo: Kukalwa i-CaMKII Transcript kanye ne-Protein expression nge-Cell-Type -pecific ΔFosB Overexpression (I-Fig 3D)

Amagundane abesilisa avela bitransgenic aqhamuka I-NSE-tTA (umugqa A) × I-TetOp-ΔfosB (umugqa 11) no I-NSE-tTA (umugqa B) × I-TetOp-FLAG-ΔfosB (umugqa 11) amagundane (Chen et al., 1998; Kelz et al., 1999; Werme et al., 2002; UZachariou et al., 2006) akhulelwa futhi akhuliswa ku-100 µg / ml doxycycline ukucindezela isisho se-ΔFosB ngesikhathi sokuthuthuka. Abalingani bahlukaniswa lapho belunyulwa: uhhafu wasala ku-doxycycline kwathi uhhafu washintshelwa emanzini, kanti nezilwane zasetshenziswa i-8 zaya kuma-11 amasonto kamuva lapho imiphumela ye-ΔFosB ebhaliwe ingaphezulu kakhuluKelz et al., 1999; UMcClung noNestler, i-2003). Ngokuhlaziya okubhalwe phansi, amagundane ayekhishwa ngokushesha, futhi izingqondo zisuswe bese zibekwa eqhweni. Ukuphazamiseka kwe-NAc kwathathwa ngenaliti yenaliti engu-14-gauge futhi kwaqandeka ngokushesha eqandeni elomile kuze kube yilapho kukhishwa i-RNA. Ukuhlukaniswa kwe-RNA, i-qPCR, kanye nokuhlaziywa kwedatha kwenziwa njengoba kuchaziwe ngaphambilini (I-LaPlant et al., 2009). Kafushane nje, i-RNA yahlukaniswa ne-TriZol reagent (i-Invitrogen), yahlanzwa futhi nge-RNAeasy micro kit evela eQiagen, yahlolwa ikhwalithi nge-Agilent's Bioanalyzer. Ukubhalwa emuva kubuye kwenziwa kusetshenziswa i-iScript (BioRad). I-qPCR yenziwa ngohlelo lwe-Applied Biosystems 7900HT RT PCR ngemingcele elandelayo yomjikelezo: Amaminithi we-10 e-95 ° C; Imijikelezo ye-40 ye-95 ° C ye-1 min, i-60 ° C ye-30 sec, i-72 ° C ye-30 sec; ukufudumeza okuhlanganisiwe ku-95 ° C ukukhiqiza ama-curve e-dissociation okuqinisekiswa kwemikhiqizo eyodwa ye-PCR. Ukuhlaziywa kwe-immunohistochemical kwe-ΔFosB ne-CaMKIIcy protocol kwenziwa ngendlela echazwe ku Zama i-4.

I-8 Yesilingo: Imiphumela ye-Intra-NAc D1 ne-D2 Dopamine Receptor Antagonists mayelana noshintsho lweProtein-Mediated Protein (I-Fig 3H)

Ama-Adult (8 weeks) amagundane besilisa aphathwa i-10 mg / kg cocaine noma i-carine saline ("imoto" iqembu) IP kanye ngosuku izinsuku eziyisikhombisa. Amaminithi we-30 ngaphambi komjovo ngamunye we-cocaine, amagundane ayephathwa yi-D1 receptor antagonist SCH 23390 (0.5 mg / kg, group "D1 Ant"), noma i-D2 receptor antagonist eticlopride (0.5 mg / kg, "D2 Ant" , noma umjovo wokulawula usawoti (iqembu le- “cocaine”). I-24 hr ngemuva komjovo wokugcina, izilwane zabulawa futhi amaprotheni anqunyelwa yi-Western blotting as per Zama i-2.

Isilingo 9: Imiphumela ye-AAV-Mediated ΔFosB Overexpression on expression of Protein (I-Fig 4 A-C)

Udokotela ohlinzayo we-Stereotaxic wenziwa kumagundane amaduna amadala (amaviki e-8) ukujova i-AAV-GFP (iprotheni eluhlaza okotshani) noma i-AAV-GFP-ΔFosB (Maze et al., 2010). Izinaliti ze-33 ze-gauge (Hamilton) zaqashwa kukho konke ukuhlinzwa, lapho i-0.5 µl yegciwane elihlanjululwe nge-titer ephezulu yafakwa khona ngaphakathi ngesikhathi sesikhathi se-5 min, yalandelwa isikhathi sokuphumula esingeziwe se-5 min. Wonke amabanga alinganiswa aphathelene ne-Bregma: 10 ° angle, AP = + 1.7 mm, Lat = 2.5 mm, DV = −6.7 mm. Izinsuku ze-14 ngemuva kokuhlinzwa, izilwane zinikezwe umjovo owodwa we-IP we-10 mg / kg cocaine emagumbini wokuqapha we-locomotor ukuhlola imiphumela yokuziphatha kwe-ΔFosB overexpression. I-24 hr ngemuva kwalomjovo wokugcina, amagundane anqunyelwa njengoba ngamunye Zama i-2, futhi izicubu ze-izicubu zenziwa ngaphansi kwesiqondisi se-fluorescence microscopic ukuthola i-GFP-positive NAc izicubu. Ukuqothulwa kwaseNtshonalanga lapho kwenziwa ngaleso sikhathi Isilingo 2.

Umfanekiso we-4

I-ΔFosB iyadingeka futhi yanele ekusetshenzisweni kweCocaMUMI yokwelashwa kwe-cocaine-Mediated receptor.

I-10 Yesilingo: Imiphumela ye-AAV-Mediated ΔJunD Overexpression kwi-Cocaine-Dependent Protein expression (I-Fig 4 D-F)

Ukufakwa umjovo kwe-Stereotaxic kwe-AAV-GFP noma i-AAV-GFP-unJunD kwenziwa ngakunye Isilingo 8. Izinsuku ze-14 ngemuva kokuhlinzwa, izilwane zaziphathwa i-10 mg / kg cocaine noma i-IP ye-saline IP kanye ngosuku izinsuku eziyisikhombisa kumagumbi okuqopha i-locomotor. Izimpendulo ze-locomotor kumjovo owodwa we-cocaine (5 mg / kg IP) noma i-saline yaqoshwa. I-24 hr ngemuva kwalomjovo wokugcina, amagundane anqunyelwa, kwavunwa izicubu, futhi kwaqhuma amabanga asentshonalanga njengase- Isilingo 9.

Isilingo 11: In Vitro Amaphrotheni Kinase Assays (I-Fig 5A-D)

I-Recombinant CaMKIIcy ne-ΔFosB bahlanziwe kusuka kumaseli ezinambuzane (I-Brickey et al., 1990; UJorissen et al., 2007), futhi kwenziwa kwe-protein kinase assows (I-Colbran, 1993), njengoba kuchaziwe ngaphambili. Kafushane nje, iCaMKII yenziwa kabusha eqhweni nge-2.5 µM ​​(noma ukuvezwa okuhlushwa) kwe-ΔFosB, 1 mM Ca²⁺, 40 mM Mg²⁺, I-15 µM ​​ye-mododulin, kanye ne-200 mM HEPES pH 7.5. I-Phosphorylation yaqalwa ngokungezwa kwe-200 µM ​​ATP nge noma ngaphandle [γ-³²P] I-ATP futhi ivunyelwe ukuqhubeka imizuzu engu-10 ngamazinga okushisa asekamelweni (Umdwebo 5A & B) noma i-2 amaminithi eqhweni (Umdwebo 5C & D). Imikhiqizo ixazululwe yi-Western blotting (Umdwebo 5A & B) noma nge-autoradiogram nokubalwa kwe-scintillation (Fig B-D).

Umfanekiso we-5

I-ΔFosB iyi-substrate enamandla yeCaMKIIα

Isilingo 12: Ukukhonjwa kweSer27 ΔFosB Phosphorylation (I-Fig 5E)

In vitro kinase assays zenziwa njengoba ngamunye Zama i-11, amaprotheni ahlukaniswe yi-SDS-PAGE, futhi amabhendi ahambelana ne-ΔFosB asikiwe futhi afakwa ngaphansi kwe-tandem mass spectrometry. Izabelo ze-m / z zezingcezu ezihambisanayo ze-ion kuwo wonke amapaneli zibhalwe ngaphezulu kweziqongo ze-ion. Akuzona zonke izingcezu ze-vipande ezilebulwe ngenxa yomkhawulo wesikhala. Ngokuvamile, umbhalo welebuli le-ion lezinto ezihlanganisiwe afakwa imibala emnyama ngaphandle kokuthi uma eqinisekisa ngokuqondile noma engeza ubufakazi ebukhoneni bezindawo zokuthakazelela, lapho kulapho kubhalwe khona obomvu. Ubufakazi bemikhiqizo yokuqhekeka emgogodleni kwethulwa ngokulandelana kokufundwa kwe-phosphopeptide nendawo etholakele yezinsalela ze-phosphorylation ekhonjiswe ngokubomvu ngenhlamvu eyodwa ye-amino acid. Incazelo yezinombolo ye-kugaw ecetshwayo yama-ion nayo imakwe ekulandelaneni kwe-peptide njenge-b and y ions. Izici zokusondeza kwezingxenye zama-m / z axis ukukhombisa ubuncane bezingcezu zama-ion zibhalwe ngenhla kwesikrini ngasinye sombukiso. I-ipes ye-fragment eboniswe kwipaneli H iqinisekisa ubukhona be-isXormUMX phosphorylated isoform, noma kunjalo, ngaphakathi kwengxubevangezinye isoforms ze-phosphorylated kumasayithi i-Ser27, Ser28, Ser31, ne-Thr34. Ukuba khona kwe-pa37, i-pa5-P, pb5, ne-pb5-P ions kuqinisekisa ngokuhlukile i-phosphorylation yokusala kweSer5.

I-13 yesilingo: Ukulinganiswa kwe-phosphorylation yeSer27 (I-Fig 5F)

Ama-peptides ajwayelekile aklanywa ukulingisa izinhlobo ze-phospho nezingelona i-phospho zeSer27 ΔFosB. Ngemuva kokuhlanganiswa nokuhlanzwa, i-peptide eyodwa enzima ye-idiotypic yachithwa endaweni ye-50 / 50 acetonitrile / buffer yamanzi futhi yathunyelwa ukuze kuhlaziywe i-amino acid ukuthola ukunikezwa okuphelele kwekhambi lezinto zokwenziwa ze-peptide. I-peptide ngayinye “esindayo” ibese ifakwa ngqo kwi-4000 QTRAP mass spectrometer (MS) ukunquma amandla angcono kakhulu okushayisana kokuqhekeka kwe-MS / MS kanye nezinguquko ezimbili kuya kwezine ze-MRM. Ngokulandelayo, ama-peptides acacile "asindayo" afakwa kwi-LCMS kwi-4000 QTRAP ukuqinisekisa ukuhlukaniswa kwe-peptide. Insimbi yayiqhutshwa ngemodi ephindwe kathathu ye-quadrupole, i-Q1 isethwe kunombolo ethile ye-m / z ye-precisor (i-Q1 ayisihloliwe), futhi i-Q3 isethwe kunani elithile le-m / z elihambelana nocezu oluthile lwaleyo peptide. Kwindlela ye-MRM, uchungechunge lokuphendula okukodwa (okwandulelayo / okuguqukayo ion okuguqukayo lapho amandla okushayisana akhiwa khona ukuze kukhuliswe ubukhulu bezithako zezinhlaka) kukalwa ngokulandelana, futhi umjikelezo (ngokuvamisile i-1-2 sec) wawudlule kulo lonke sonke isikhathi sokuhlukaniswa kwe-HPLC Ukuguqulwa kwe-MRM kunqunywe kusukela ku-MS / MS spectra yama-peptides akhona. Izinguqulo ezimbili nge-peptide ngayinye, ezihambisana nama-ion e-high compenseity, kwabe sekukhethiwe futhi amandla okushayisana enzelwa ukwandisa amandla esignali woshintsho lwe-MRM kusetshenziswa isoftware ezenzakalelayo. Iziqongo ezivela kuma-peptides ajwayelekile namasampula we-ΔFosB adalulwe ku-CaMKII noma ukulawulwa kwaqhathaniswa ukuqaphela inani eliphelele lefomu ngalinye le-peptide ekuphenduleni. Ukuhlaziywa kwedatha kudatha ye-LC-MRM kwenziwa kusetshenziswa isoftware ye-AB Multiquant 1.1.

Isilingo 14: Isingeniso se-ΔFosB ku-CaMKII Oreatxpressing Mice (Umdwebo 5G & H)

Amagundane eTransgenic overexpressing T286D CaMKII (UMayford et al., 1996; UKourrich et al., 2012) nabalingani bohlobo lwasendle bakhuliswe lapho bungekho i-doxycycline ukuvumela ukukhuluma kwe-transgene. Amagundane amadala aphathwa i-20 mg / kg cocaine noma i-saline IP kanye nsuku zonke ngezinsuku ze-14. I-24 hr ngemuva komjovo wokugcina, izilwane zachazwa futhi zagonywa futhi zalungiswa isisho se-ΔFosB njengasenziwa Zama i-4.

Isilingo 15: Imiphumela ye-HSV-Mediated ΔFosB Overexpression neCaMKII Inhibition ku-NAc Dendritic Spines (I-Fig 6A-E)

Amagundane amadala abantu abadala (amasonto e-8) afakwe i-NAc nge-HSV-GFP, HSV-GFP-ΔFosB (I-Olausson et al., I-2006), I-HSV-GFPAC3I, noma i-HSV-GFPAC3I-ΔFosB. Kulezi zakhi, i-AC3I, inhibitor esekwe peptide yomsebenzi weCaMKII, ifakwa ku-C-terminus ye-GFP. I-GFPAC3I iboshwe yi-PCR isebenzisa i-pMM400-vector equkethe i-GFPAC3I njengesifanekiso esinezigcawu ezilandelayo: GFP-AC3I-F: 5 'CC GCTAGC GCCGCCACC ATGGTGAGCAGUNDLITNLLNNLLNLNLNLNLNLNLNLNLNLNLNLNLNLLNLLNLLNLLNLLNLLLLLLLIPNLLNLLLNLLNLLLNLLLNLLNLLNLLNLLNLLNLLLLLLamp; I-GFP-AC3I-R: I-3 'CC TCCGGA TTACAGGCAGTCCACGGCCT 5' (clampBspEIstop). Umkhiqizo ophumele we-PCR wafakwa kwi-p3 + ne-p1005 + -Δ FosB veveni besebenzisa amasayithi we-NheI ne-BspEI. Ukwakhiwa kwaqinisekiswa ngokulandelana. Ukuxhumanisa kwe-Stereotaxic kwakungu: 1005 ° angle, AP = + 10 mm, Lat = + 1.6 mm, DV = −1.5 mm (Barrot et al., 4.4). I-Perfusion kanye nokuhlukaniswa kwengqondo kwenziwa njenge-per Zama i-4.

Ukuhlaziywa komgogodla kwenziwa njengoba kuchaziwe (UChristoffel et al., 2011). Kafushane nje, izingxenye ezi-dendritic 50-150 µm kude ne-soma zikhethwe ngokungahleliwe kumaseli angenwe yi-HSV aveza i-GFP. Izithombe zatholakala kwi-LSM 710 eyimfihlo (i-Carl Zeiss) yokuhlaziywa kwe-morphological kusetshenziswa i-NeuronStudio nge-rayburst algorithm. I-NeuronStudio ihlukanisa imisipha njengemincane, i-amakhowe, noma isigaxa ngokususelwa kumanani alandelayo: (1) ratio factor, (2) ikhanda kuya ku-ratio wentamo, kanye (3) yekhanda ububanzi. Izithelo ezinentamo zingahlukaniswa ngokuthi zizacile noma zi-amakhowe, futhi lezo ezingenawo umthambo obalulekile zihlukaniswa njengesiqu. Izithelo ezinentamo zibhalwe njengezincanyana noma i-mushroom ngokususelwa kububanzi bekhanda.

Umfanekiso we-6

I-blockade yomsebenzi we-CaMKII ivimbela imiphumela ye-morphological kanye nokuziphatha ye-ΔFosB ku-NAc

Isilingo 16: Imiphumela ye-HSV-Mediated ΔFosB Overexpression ne-CaMKII Inhibition kuma-Cocaine Responses (I-Fig 6F)

Amagundane amadala abantu abadala afakwa kanye namagciwane njengoba ngakunye Zama i-15, nezimpendulo ze-locomotor kumjovo owodwa we-5 mg / kg we-cocaine walinganiswa njengoba ngamunye Isilingo 9. Imininingwane ye-locomotor ivezwa njengokuqhekeka ngokuphelele kogongolo ngaphezulu kwamaminithi we-30 ngemuva komjovo we-cocaine.

Ulwazi Olwengeziwe

Ukuhlala Kwezilwane

Amagundane abesilisa bakwa-Sprague Dawley (250-275 g; Charles River Laboratories) ahlanganiswe nendlu eyodwa. Amagundane abesilisa ayisishiyagalombili i-C57BL / 6J (iJackson Laborator) ayehlanganiswe nezilwane ezinhlanu ngenhla yezinyoni. Zonke izilwane zahlaliswa esikhungweni sezilwane i-≥1 ngesonto ngaphambi kokukhohlisa futhi zagcinwa kumagumbi alawulwa isimo sezulu (i-23-25 ° C) kumjikelezo we-12 hr / umjikelezo omnyama (izibani eziku-7: 00 AM) ngokufinyelela kokudla namanzi isikhangiso. Ucwaningo lwenziwe ngokulandela imihlahlandlela ye-Society for Neuroscience kanye nekomidi lesikhungo sokunakekelwa kwezilwane nokusebenzisa (IACUC) eMount Sinai.

Izidakamizwa

Izidakamizwa zaziphathwa nge-IP futhi ziqedwe ku-saline oyinyumba, kufaka phakathi i-cocaine (5-20 mg / kg nge-10 µl yezimungumungwane, nge-1 ml yamagundane, i-NIDA) ne-SCH 23390 noma i-eticlopride hydrochloride (0.5 mg / kg nge-1 ml, Tocris) . Ngokuhlinzwa okwenziwa i-stereotaxic, amagundane afakwa i-“cocktail” ye-ketamine (100 mg / kg) ne-xylazine (10 mg / kg) (uHenry Schein) ku-saline oyinyumba.

Ama-Antibodies

I-CaMKIIα (isiyonke): I-05-532 ephezulu, i-1: I-5,000

I-CaMKII phospho-Thr286: I-Promega V111A, 1: 1,000

I-ΔFosB (isiyonke): I-Cell Signaling 5G4, 1: 250

I-ΔFosB phospho-Ser27: Phosphosolutions, 1: 500

I-GluA1 (isiyonke): Abcam, Ab31232, 1: 1,000

I-GluA1 phospho-Ser831: Millipore N453, 1: 1,000

I-GluA1 phospho-Ser845: I-Chemicon Ab5849, 1: 2,000

IGluA2: Millipore 07-598, 1: 2,000

I-NR2A: ISigma HPA004692, 1: 2,500

I-NR2B: Millipore Ab1557P, 1: 1,000

Ukuhlaziywa kwesitatimende

Lonke ucwaningo lwezibalo lwenziwa kusetshenziswa iphakethe le-software ye-Prism 6 (GraphPad). I-t-izivivinyo zabafundi zazisetshenziselwa ukuqhathanisa okuphathelene nobabili (kuboniswe kumiphumela lapho kunikezwa khona inani le-t), futhi ama-ANOVA asetshenziswa ngendlela eyodwa kuyo yonke ukuqhathanisa okuningi (kuboniswe esigabeni semiphumela lapho inani le-F linikezwa).

Iya ku:

Imiphumela

I-Chronic Cocaine Induces CaMKII ku-NAc Shell

Ucwaningo oluningi luveze ukuthi ama-MSN asemgobeni we-NAc kanye nomongo unezimpendulo ezingefani ze-biochemical nezomzimba zokuvezwa ngokuphelele kwezidakamizwa zokuhlukumezeka (UKourrich noThomas, 2009; ULoweth et al., 2010) nokuthi lezi zigatshana ezimbili zilawula ngokwehlukile ukuziphatha okufuna izidakamizwa (Ito et al., 2004). Ukunquma imiphumela ehlukile ye-cocaine kwizakhi zamaprotheni ze-NAc igobolondo vs. umnyombo, sisebenzise i-Multiplexed Isobaric Tagging (iTRAQ) kanye ne-tandem mass spectroscopy (MS / MS). Amagundane amadala abantu abadala afakwa i-IP nge-cocaine (20 mg / kg) noma usawoti nsuku zonke ngezinsuku ze-7; I-24 hr ngemuva komjovo wokugcina, igobolondo le-NAc kanye nomgogodla kwenziwa microdissected (I-Fig 1A) futhi kufrize iqhwa. Amaprotheni kulezi zinhlayiya abese enqanyulwa kusetshenziswa iTRAQ. Womane ama-isoforms womane we-CaMKII abonise ukwanda okukhulu kokubonisa ngemuva kokuphathwa kwe-cocaine ebekacacisiwe egobolondweni le-NAc ngokuqhathaniswa nomongo. Ama-phosphatases amaningana, kufaka phakathi i-PP1 catalytic and subunits yokulawula kanye ne-PP2A, ebikade ihlotshaniswa nemikhakha ehlukahlukene ye-CaMKII kwezinye izinhlelo (I-Colbran, 2004), kulandele iphethini efanayo. Lokhu okutholakele kunikeze inoveli, ubufakazi obungakhethi bokuthi indlela yokusayina yeCaMKII ilawulwa ngokugqamile yi-cocaine e-NAc ngendlela eqondene negobolondo.

Ukuqinisekisa lokhu kutholakala ngokwezinga elingaphezulu, siphathe amagundane njengangenhla nge-cocaine (kumithamo ehlukahlukene) noma nge-saline futhi sathola izimpendulo ze-locomotor ku-cocaine (5 mg / kg) noma ipayipi lenselelo ye-saline. Ukuchayeka okuphindaphindwe ku-10 mg / kg cocaine kuholele kuphethini ejwayelekile yokuzwela i-locomotor (I-Fig 1B). Ukuqhubeka kokufunda ngale ndlela ye-dosing, ngokusebenzisa i-Western blotting, okuphindaphindwe kabili kwe-cocaine kubangela i-CaMKIIα ngokungakhethi ku-NAc igobolondo 24 hr ngemuva komjovo wokugcina we-cocaine (I-Fig 1C ne-D; p = 0.0019; F = 7.943; df = 29). Ngaphezu kwalokho, i-phosphorylation ye-canonical CaMKII engaphansi kweSer831 ye-GluA1 subunit ye-AMPA receptor yanda kakhulu ngegobolondo le-NAc futhi hhayi umgogodla (p = 0.0261; F = 4.208; df = 28), ngenkathi i-CaMKIIα Thr286 Autophosph kodwa i-notophrophicph. umkhuba obalulekile wokungeniswa egobolondweni kuphela (I-Fig 1D). Amanye ama-receptors ama-glutamate ambalwa awatholakalanga. Ngokuphikisana nalezi zinyathelo zeCaMKII, lawo masampula wezicubu afanayo akhombisa ukwenziwa kwe-ΔFosB kuwo womabili amagobolondo (p = 0.0260; F = 4.189; df = 29) kanye nomongo (p = 0.0350; F = 3.807; df = 29) ye-NAc (I-Fig 1C ne-D), kuyahambisana nokutholakele kwangaphambilini (I-Perrotti et al., I-2008).

Njengoba izifundo eziningana zangaphambili zokulawulwa kwe-cocaine kwama-AMPA receptors zahlaziya izilwane ngemuva kwezinsuku eziyi-14 zokuhoxa ku-cocaine engapheli (bheka Ingxoxo), siphinde saphinda lokhu kuhlaziya kwe-biochemical ngalesi sikhathi. Sithole ukuthi, izinsuku ezingama-14 ngemuva komjovo wokugcina we-cocaine, i-ΔFosB isalokhu iphakanyisiwe ku-NAc (p = 0.0288; F = 4.258; df = 22), ngenkathi kungekho CaMKII noma phosphorylation yeGluA1 Ser831 esakhuphukile (I-Fig 1E). Kodwa-ke, i-1 hr ngemuva kwethamo elilodwa le-cocaine le-10 mg / kg ye-cocaine, amazinga e-CaMKII (p = 0.0330; F = 3.947; df = 26) ne-GluA1 Ser831 (p = 0.0213; F = 4.509; df = 27) I-phosphorylation yomabili iphakanyiselwe ezingeni elifana nalelo elitholwe ngemuva kokuvezwa kwe-cocaine yokuqala (I-Fig 1E). Le mininingwane ikhombisa ukuthi i-NAc shell neurons yenzelwe ukungeniswa kwe-CaMKII ngezikhathi ezengeziwe zokuzila, mhlawumbe nge-priming eqondile ye-CaMKII gene promoter (bheka Ingxoxo). Ngaphezu kwalokho, iqiniso lokuthi i-ΔFosB induction iphikelela kakhulu kunokuqalwa kweCaMKII iphakamisa ukuba khona kwezindlela ezingezelelweyo, noma ngabe zise-chromatin noma ngenye indlela, ezifaka “ukubopha” emthethweni weCaMKII, njengoba kumbozwe engxoxweni.

Ukuqhubeka nokuqinisa lokhu kucashunwa, sibheke izinhlobo zokuzilawula ze-cocaine, ezibandakanya ukuphuza izidakamizwa okwejwayelekile. Amagundane amadala abantu abadala anikezwa ukufinyelela okufushane noma okude kwe-cocaine; njengoba kulindeleke (U-Ahmed no-Koob, i-1998), kuphela izimo zokufinyelela ezinde eziholele ekwehlekeni kokuzibusa okwandayo komuthi (I-Fig 2A). I-ΔFosB yancengwa ngezinga elikhulu isikhathi eside vs. ukufinyelela okufushane kwe-cocaine kuwo womabili amagobolondo e-NAc (p = 0.0011; F = 11.12; df = 17) kanye nomongo (p = 0.0004; F = 13.86; df = 17). Ngokuphambene, i-CaMKIIα yenziwa igobolondo le-NAc kuphela ngokufinyelela isikhathi eside ku-cocaine (I-Fig 2B ne-C; p = 0.0236; F = 4.957; df = 16). Kuyathakazelisa ukuqhathanisa umthamo we-cocaine ojwayelekile wosuku zonke ezilwaneni ezifinyelela ngokufushane (~ 12 mg / kg IV), izilwane ezifinyelela isikhathi eside (~ 70 mg / kg IV), nezilwane ezilawulwa ngokuhlolwa (i-10 mg / kg), kanye buza ukuthi kungani lokhu kungeniswa kokugcina kwe-ΔFosB neCaMKII kanti ukufinyelela okufushane kungavumeli. Lokhu kungafani kungenzeka kungenxa yokuhlukahluka kwamazinga aphezulu we-cocaine (i-cocaine ephethwe ngokuhlola inikezwa njenge-IP eyodwa ye-bolus, kuyilapho i-cocaine elawulwa yona idluliswa ngemithamo eminingi ye-IV), noma umehluko ngobude bokuvezwa kwezidakamizwa (izinsuku ze-7 zokuhlola ukuphatha, izinsuku ze-19 zokuzilawula).

Ngaphandle kwezincwadi ezinkulu ze-ΔFosB neCaMKII ekusebenzeni kwe-cocaine, azikho izifundo zalawa maprotheni kubasebenzisi be-cocaine. Lapha, sethula ubufakazi bokuqala bokuthi amazinga we-ΔFosB (p = 0.0316; t = 1.921; df = 34) kanye neCaMKII (p = 0.0444; t = 1.755; df = 32) andiswa ku-NAc yabantu abaxhomekeke ku-cocaine (I-Fig 2D, Ithebula 1). Le mininingwane ikhombisa ukuthi ukuhlolwa kwethu kwe-ΔFosB ne-CaMKII lokungeniswa yi-cocaine ku-rodent NAc kuhambelana nomlutha womuntu we-cocaine.

Ithebula 1

Ukubonakaliswa kwamasampula avela kumlutha we-cocaine wabantu kanye neqembu lokulawula elifanisiwe

I-ΔFosB ilawula Ukubhalwa kwe-CaMKII Ngokukhethekile ku-D1-Type MSNs of NAc Shell

Ukuthola ukuthi i-CaMKII ne-ΔFosB kubhalwe kabusha yi-cocaine ku-rodc ye-NAc kwaholela ekutholeni ukuthi i-ΔFosB ingalawula yini ukubhalwa kohlobo lwe-CaMKII. Sasibike i-CaMKIIα phambilini njengethagethi engenzeka ye-ΔFosB ekuhlaziyeni okungachemi kwe-NAc ye-NAc (UMcClung noNestler, i-2003), kepha lokhu okutholakele bekungakaqinisekiswa kulolo cwaningo. Siqale sasebenzisa i-ChIP ye-quantitative (qChIP-ChIP elandelwa yi-PCR eningi) ukuthola ukuthi ngabe i-ΔFosB iyabopha yini kumkhuthazi wezinhlobo ze-CaMKIIcy ku-NAc yamagundane amadala abantu besilisa, futhi sathola ngokumangazayo ukuthi lokhu kuhlangana kukhula kakhulu, ngokuphathwa kwe-cocaine engapheli, egobolondweni ( p = 0.0133; t = 2.901; df = 12), kepha hhayi umnyombo, ukuzithoba (I-Fig 3A). Ukuqhubeka ukuqonda izindlela ezihlobene nalokhu umehluko oqondile we-ΔFosB ekubophisweni kwe-CaMKIIα, sisebenzise i-qChIP ukwenza isimo se-histone modified kulesifunda se-genomic. Ucwaningo lwangaphambilini lubonise ukwenziwa kwe-cocaine kwe-H3 acetylation esikhuthazweni seCaMKIIcy ku-NAc yegundane ephelele (U-Wang et al., I-2010). Ngokuphambene nalokho, sithole ukuthi i-cocaine inciphisa i-acetylation ye-H3 endaweni yokuphromotha ye-CaMKIIα ngokukhetha ku-NAc core (I-Fig 3B; p = 0.0213; t = 2.726; df = 10), ngaphandle koshintsho olubonakalayo egobolondweni, elihambisana nokuguqulwa okuqondile kwe-chromatin engaphezulu kwe-ΔFosB. I-qChIP yamamaki acindezelayo, i-dimethylated H3 lysine 9 (H3K9me2), iveze ukuthambekela kokuncipha kokubekelwa phansi kwegobolondo ne-core (I-Fig 3C).

Ukunquma ukuthi ngabe i-ΔFosB ilawula okubhaliwe kwe-CaMKIIcy vivo, sisebenzise imigqa emibili yegundane ye-bitransgenic ephatha ngokungaphezi i-ΔFosB ngokuqondile ku-D1 vs. Ama-DNNUMX-Type MSNs ngendlela elawulwa ukuphathwa kwe-doxycycline emanzini okuphuza (Chen et al., 1998; Kelz et al., 1999; Werme et al., 2002). Amagundane amadala abantu abadala i-ΔFosB kuphela kuhlobo lwe-D1-uhlobo lwama-MSN ayekhuphule kakhulu amazinga we-CaMKIIcy mRNA ku-NAc (p = 0.0337; t = 1.996; df = 13), umphumela ongabonwa kumagundane aphindelela ΔFosB ikakhulukazi kuhlobo lwe-D2I-Fig 3D). Ukwanda kweCaMKIIα mRNA, okubangelwa isincazelo se-ΔFosB kuhlobo lwe-D1-uhlobo, ihambisane nokwenyuka okufana nako kwamaphrotheni weCaMKIIcy kuwo womabili ama-NAc igobolondo (p = 0.0030; t = 3.578; df = 14) kanye nomongo (p = 0.0392; t = 2.275; df = 14; Amakhiwane 3E no-F). Le mininingwane ikhombisa ukuthi i-ΔFosB iyakwazi ukushayela i-CaMKIIα gene expression ku-D1-hlobo MSNs kuzo zombili izifunda Umfanekiso we-3B kuphakamisa ukuthi izinguquko ze-cocaine-mediated chromatin ku-CaMKIIα Promoter (isb., ukunciphisa i-acetylation) zivimbela i-ΔFosB ekukhupheni i-CaMKII ekuvumelaneni okuyinhloko ngemuva kwe-cocaine.

Ngoba idatha yethu yegundane ye-transgenic ikhombisile ukuthi ukufakwa kwe-ΔFosB kwe-CaMKII expression expression kucacisiwe kuma-D1-uhlobo ama-MSNs ku-NAc, ngokulandelayo safuna ukuthola ukuthi ngabe ukulawulwa kwe-cocaine okuxhomeke ku-CaMKII kudinga ukwenziwa kwe-D1 dopamine receptor. Amagundane amadala amadala anikezwa i-cocaine engapheli noma i-saline njengaphambilini, kepha ama-30 min ngaphambi komjovo ngamunye, amagundane eqenjini le-cocaine anikezwa umjovo we-IP ka-saline, umphikisi we-D1 SCH 23390 (0.5 mg / kg), noma i-D2 receptor antagonist eticlopride (0.5 mg / kg). Izilwane zahlaziywa i-24 hr ngemuva komjovo wokugcina we-cocaine. Ukuqothulwa kweNtshonalanga kuveze ukuthi i-D1, kepha hhayi i-D2, umphikisi uvimbe ngokuphelele ukwanda okuxhumene ne-cocaine ku-ΔFosB (p <0.0001; F = 18.96; df = 18), njengoba kwabikwa ngaphambilini (Nye et al., 1995), kanye naku-CaMKII (p = 0.0005; F = 10.99; df = 18; I-Fig 3G ne-H). Le mininingwane isekela i-hypothesis yokuthi i-cocaine ifaka ukwanda okungaphakathi kwe-ΔFosB-in-gene expression ku-CaMKII gene expression ku-D1-hlobo MSNs ye-NAc igobolondo. Kungakuhle ezifundweni zesikhathi esizayo ukukhombisa ngqo lo mphumela wohlobo lweseli lwe-cocaine esenzweni seCaMKII kulesi sifunda sobuchopho.

I-ΔFosB Kokubili iyadingeka futhi Yanele ukusetjenziswa kweCocaine yeCaMKII ku-NAc Shell

Ukuphelelisa ukusetshenziswa kwamagundane we-bitransgenic, ngokulandelayo safunda indima ye-ΔFosB ekulameni lokungeniswa kwe-cocaine yeCaMKIIcy ngokusebenzisa ukudluliselwa kofuzo okuphathelene nogciwane kumagundane. Sifaka izinhlayiya zegciwane elihlotshaniswa ne-adeno ehlobene ne-vireno ku-NAc igobolondo lamagundane amadala abantu abadala (lapho igobolondo lingaqondiswa khona ngokukhethekile) ku-overexpress ΔFosB kanye ne-GFP noma i-GFP kuphela. Izilwane zabe zinikezwa umjovo owodwa we-IP we-10 mg / kg cocaine. Izilwane zidonsa ngokweqile i-ΔFosB / GFP zibonise impendulo ekhuphukayo yokuqhathanisa nezilwane uma kuqhathaniswa nezilwane ezedlula i-GFP kuphela (I-Fig 4A). I-24 hr ngemuva komjovo owodwa we-cocaine, izicubu ze-NAF ze-GFP-positive zaxoshwa kulezi zilwane ngokuhlukaniswa ngaphansi komthombo wokukhanya we-fluorescent. Ukuqothulwa kwale ntuthu (I-Fig 4B ne-C) iveze i-ΔFosB overexpression enamandla kanye nokwanda okukhulu kwamaprotheni e-CaMKIIα aqhathaniswa nezilwane ze-GFP (p = 0.0070; t = 2.894; df = 30), efana nokungeniswa okubonwe nokuphathwa kwe-cocaine okungamahlalakhona. Ngaphezu kwalokho, i-CaMKIIα autophosphorylation ku-Thr286 (ekhombisa ukusebenza kwe-enzyme) inyuswe yi-ΔFosB overexpression (p = 0.0330; t = 2.243; df = 28), njengoba bekunjalo phosphorylation we-CaMKII substrate, Ser831 of GluAX = = = I-1; df = 0.0540), iphinda ilingise isenzo se-cocaine engapheli (I-Fig 1C ne-D). Tngokuhlangana, le mininingwane ihlinzeka obunye ubufakazi bokuthi i-ΔFosB expression ku-NAc igobolondo lanele ekuzwisiseni i-locomotor ku-cocaine naku-CaMKII lokungeniswa kanye nokwenza kusebenze kulokhu kukhonjelwa.

Sisebenzise indlela efanayo ukuthola ukuthi i-ΔFosB iyadingeka futhi yini ekuzisweni kwe-cocaine-mediated yeCaMKIIcy kugobolondo le-NAc. I-AAV yayisetshenziselwa ukwedlula iprotheni ye-JunD eyisicefe, ebizwa ngokuthi yi-ΔJunD, okuyisilawuli esibi sokusebenza kwe-ΔFosB transcriptal activation (I-Winstanley et al., I-2007) kanye ne-GFP noma i-GFP kuphela. Emavikini amabili kamuva, lapho i-transgene expression is maximal, izilwane zanikwa i-cocaine (10 mg / kg) noma i-saline nsuku zonke izinsuku ze-7, futhi zahlolwa izimpendulo ze-locomotor kwinselelo ye-cocaine (5 mg / kg) i-24 hr ngemuva komjovo wokugcina (I-Fig 4D). I-vereJunD overexpression ivikele ukuzwela kwe-locomotor ku-cocaine, futhi futhi yavimbela ukwenziwa kweCaMKIIcy nokwenza kusebenze kugobolondo le-NAcI-Fig 4E ne-F; p = 0.0437; F = 2.997; isamba se-df = 38), okubonisa ukuthi umsebenzi wokuqoshwa we-ΔFosB uyadingeka ukuze kufakwe i-cocaine-Mediated yeCaMKIIcy kule sub subion. Ngokuthabisayo, sithole ukuthi i-ΔJunD yehlise amazinga we-ΔFosB ngaphansi kwezimo zombili eziphathwa nge-saline ne-cocaine (p = 0.0004; F = 8.110; df = 35), ikhulisa amathuba okuba i-ΔFosB incike emsebenzini we-AP-1 wamazinga wayo wokubonisa.

I-CaMKII Phosphorylates ΔFosB eSer27

Ukusebenzisa kwesibeletho amaprotheni kinase assows, sanquma ukuthi i-ΔFosB ehlanziwe iyisigcawu esingaphansi seCaMKIIcy. Incubation of Yakhe₆-FFB ngeCaMKIIcy ne-ATP kudale ukuguquguquka okungaphezulu kokuhamba kwe-electrophoretic kwe-ΔFosB (I-Fig 5A); amabhendi ambalwa aphumelekayo aphakamise amasayithi amaningi we-phosphorylation. Okufanayo kwesibeletho kinase assays usebenzisa [γ-³²I-P] i-ATP ikhombise ukufaka i-phosphate eyi-radiolabeled kuma-ΔFosB band aguquliwe (I-Fig 5B), ekhombisa i-phosphorylation eqondile yeprotheni. Senze i-anti-phospho eqondene ngqo ne-Ser27 ebiboniswe ngaphambili ye-ΔFosB (Ulery et al., 2006). Ngenkathi lo anti anti engakhiqali isinali emelana nokukhishwa kobuchopho okuqukethe i-Ser27-phosphorylated ΔFosB (idatha engakhonjisiwe), sikwazile ukuthola ifosphorylation yeSer27 kwesibeletho kinase assay using CaMKII (I-Fig 5B). Ukuhlaziywa kweKinetic kwe-phosphorylation ye-CaMKII ye-ΔFosB kukhombisa ukuthi i-substrate enamandla ye-kinase (I-Fig 5C), ene-K_M kwe-5.7 ± 2.0µM no-K_CAT kwe-2.3 ± 0.3min^-1. Le miphumela iqhathaniswa neyodwa enezimpawu ezinhle vivo izingxenyana zeCaMKII (UColbran noBrown, 2004). Ngaphezu kwalokho, sinqume ukuthi i-phosphorylates ye-CaMKII ΔFosB nge-stoichiometry ye-2.27 ± 0.07 mol / mol (I-Fig 5D), okukhombisa ukuthi okungenani kuneziza ezintathu ze-CaMKII phosphorylation ngaphakathi kwaKhe₆-ΔFosB amaprotheni, okuvumelana nawo I-Fig 5A.

Ukuphenya amasayithi ngazinye we-phosphorylation, sisebenzise ama-MS ahlaziya amasampula awakithi kwesibeletho kinase assays. I-Fig 5E ikhombisa i-phosphorylation ye-ΔFosB eSer27 eyayiboniswe ngaphambili nakwamasayithi athile angeziwe (idatha engakhonjisiwe). Njengoba kunikezwe ukusebenza kokusebenza kwangaphambili kweSer27, sigxile kulesi siza ngokukhiqiza ama-peptides anelebula elenziwa abalingisa izifunda ze-phospho- kanye nezama-nonphospho-zaseSer27, sabe sisebenzisa amanani aziwayo ala peptides njengezindinganiso ku-MRM ukuhlaziywa kwe-osFosB ngaphambili nangemuva kwesibeletho phosphorylation nguCaMKII. Ukufakwa kwenombolo okulandelayoI-Fig 5F) iqinisekisa ukuthi iSer27 ingxenye encane enamandla yeCaMKII. Le miphumela ikhombisa ukuthi, phakathi kwezinsalela eziningi ze-phosphorylated ngaphakathi kwe-ΔFosB, iSer27 iyindawo esebenza ngempumelelo ikakhulukazi yeCaMKII.

I-CaMKII Mediates Cocumine Ukuqongelelwa kwe-ΔFosB ku-NAc Shell

Njengoba i-CaMKII ingakwazi ukufaka ifosphorylate ΔFosB kwesibeletho esizeni esikhulisa ngokuqinile ukuqina kwaso kwesibeletho futhi vivo (Ulery et al., 2006; Ulery-Reynolds et al., 2009), sithole ukuthi ngabe imisebenzi ye-CaMKII ilawula amazinga we-ΔFosB ku-NAc vivo. Ukuze siphendule lo mbuzo, siqale sasebenzisa umugqa wegundane ngokweqile kwe-mutant ngokuzimela kwe-calcium yeCaMKIIcy (T286D) ezifundeni eziningi zobuchopho kufaka i-NAc (UMayford et al., 1996; UKourrich et al., 2012). Silimale abantu abadala abaneminyaka yobudala efana ne-mutant kanye nama-ptytype asebenzisa i-20 mg / kg cocaine noma usawoti kanye nsuku zonke ngezinsuku ze-14, sabe sesihlaziya izilwane ngolunye usuku ngemuva komjovo wokugcina. Sithole ukuthi amazinga we-basal we-ΔFosB andisiwe ezilwaneni eziguqukayo ku-NAc igobolondo (p = 0.0001; F = 9.207; df = 37), kodwa hhayi umgogodla (I-Fig 5G ne-H). Ngokumangazayo, ukungena kwe-cocaine kuncike ku-ΔFosB kwakuvinjelwe ezilwaneni eziguqukayo kuzo zombili igobolondo nomongo, kuphakamisa ukuthi, yize iCaMKII ingalawula ngokuqondile ukuqina kwe-stabilityFosB egobolondweni le-NAc, nayo ingahle ibe phezulu komfula ΔFosB ezindleleni ezisetshenziswe yi-cocaine ku-subcions ngayinye ye-NAc .

Umsebenzi weCaMKII Uyadingeka ku-ΔFosB-Mediated Stologicalural and Behavial Plasticity

Ukuqalwa kwe-cocaine kwezindawo ezinjenge-dendritic spine kuma-NAc MSNs kungenye yezindlela ezinhle kakhulu ezisungulwe izidakamizwa esifundeni sobuchopho, futhi lokho kufakwa komgogodla kuhlobene nezimpendulo zokuziphatha ezizwelayo kulo muthi (URobinson noKolb, 2004; Russo et al., 2010) futhi kubikwa ukuthi ukhetha ama-D1-Type MSNs (U-Lee et al., 2006). Sikhombisile muva nje ukuthi i-cocaine induction ye-dendritic spines e-NAc incike ku-ΔFosB nohlelo lwayo lokuloba olusezansi (Maze et al., 2010). Noma kukhona izincwadi ezibanzi eziphathelene nokubandakanyeka kweCaMKII ku-dendritic spine morphology kanye nokungeniswa kwezinye izindawo zobuchopho kanye nezinqubo zokuhlola (Uhambo et al., 2003; Ama-Penzes et al., 2008; I-Okamoto et al., 2009), iqhaza layo kwe-NAc MSN ukwakheka komgogodla alifundwanga. Ngakho-ke, sinqume ukuthi ngabe umsebenzi we-CaMKII uyadingeka yini ukuze kufakwe i-ΔFosB-mediated spores ye-MSN ngokusebenzisa i-HSV-mediated overexpression ye-CaSKII inhibitor peptide AC3I fuse ku-GFP, ukwakhiwa okuboniswe ngaphambili ukuvimbela umsebenzi we-CaMKII vivo (Zhang et al., 2005; UKlug et al., 2012). Ukuchazwa ngokweqile kwe-alFosB kugobolondo le-NAc lamagundane amadala kubangele ukwanda okukhulu kwe-MSN dendritic spine density (p <0.0001; F = 8.558; df = 59; I-Fig 6A ne-B) njengoba kubikwe ngaphambili (Maze et al., 2010), futhi lokhu kunyuka kuqhutshwa ngokuyinhloko ngomzimba omncane (p = 0.0027; F = 5.319; df = 59) kanye ne-nestby (p = 0.0378; F = 2.988; df = 59) izinhlobo zomgogodla (zombili ezacatshangwa ukuthi zingama-spature infature) (I-Fig 6C-E). Awukho umphumela owabonwa ezinhlotsheni ezivuthwe ngokwengeziwe ezinjengekhowe. Kodwa-ke, lapho i-GFP-AC3I ihlanganiswa, i-ΔFosB yokungeniswa komgogodla yachithwa ngokuphelele (I-Fig 6A-E), okubonisa ukuthi umsebenzi we-CaMKII uyadingeka ukuze kuhlanganiswe i-ΔFosB yemikhompetha eyimpinda egobolondweni le-NAc.

Ngokulandelayo sisebenzise amathuluzi wegciwane afanayo ukuthola ukuthi umsebenzi weCaMKII uyadingeka yini emiphumeleni ye-ΔFosB ekuzwelweni kokuziphatha kwe-cocaine. I-72 hr ngemuva kokujova ngegciwane kugobolondo le-NAc, izilwane zanikwa umjovo owodwa we-5 mg / kg cocaine nomsebenzi wazo we-locomotor uqoshwa. Njengoba kukhonjiswe ngaphambilini nge-ovexpression ye-AAV enwetshiwe ye-ΔFosB (I-Fig 4A), I-HSV-mediated overexpression ye-ΔFosB ikhulisa ukuzwela kwendawo nge-cocaine (p = 0.0002; F = 8.823; df = 37; I-Fig 6F). Njengasendalini yokungena kokuphindaphindeka kwe-dendritic spines, inhibition of CaMKII function by coexpression of GFP-AC3I ukuvimbe ngokuphelele ukwanda okungaphakathi kwe-ΔFosB, okubonisa ukuthi umsebenzi we-CaMKII uyadingeka ekuguqulweni okwenziwe yi-ΔFosB emiphumeleni yokuziphatha kwe-cocaine.

Iya ku:

Ingxoxo

Ucwaningo olukhona lusebenzisa inqubo esetshenziswayo yokudlulisela inoveli lapho i-cocaine igxilisa i-ΔFosB ku-NAc, ekhulisa ukubhalwa kohlobo lwe-CaMKIIα ngokuzikhethela egobolondweni le-NAc. I-CaMKIIα ngokulandelayo iphosphorylates futhi izinze i-ΔFosB eholela ekuqokeleleni okukhulu kwe-ΔFosB futhi kuqhutshelwe ukwenziwa kweCaMKIIcy (I-Fig 6G). Amazinga akhuphukayo wamaprotheni amabili ngesikhathi sokuchayeka okungapheli kwe-cocaine abese esiza ngezindlela ezibalulekile zokuthola izimpendulo zokuziphatha nomuthi. Lesi isithonjana esithandekayo ikakhulukazi njengoba i-ΔFosB ne-CaMKII ngayinye ikhonjisiwe ngaphambili ukuthi iyadingeka ekuphenduleni okukhona kokuziphatha ku-cocaine (UPierce et al., 1998; I-Peakman et al., 2003), futhi sikuphindaphinda lokhu kutholwa kwe-ΔFosB kugobolondo lwe-NAc sisebenzisa indlela yegciwane (Amakhiwane 4 futhi And66).

Yize i-transgenic ΔFosB overexpression ku-D1-Type MSNs ingashayela lokungeniswa kwe-CaMKII kuzo zombili izinqola ze-NAc kanye nomongo wezilwane ze-cocaine-naïve, ngokwesimo se-cocaine, ukunqwabelana kwe-endo native ΔFosB, okwenzeka kuzo zombili lezi zigatshana, kuqhutshwa ukuhanjiswa kwe-CaMKII ngqo ku-NAc igobolondo . Lo mehluko ungaqondana namazinga aphezulu we-ΔFosB afakwe kumodeli yethu ye-bitransgenic, noma kunjalo, kungahle kubonise nekhono le-cocaine lokuguqula ngokwehlukile umgqugquzeli we-CaMKIIcy egobolondweni vs. ama-MSN ayisisekelo wokukhuthaza ukubopha i-ΔFosB kwangaphambilini noma ukuyikhipha ekubandakanyeni kwamuva. Eqinisweni, idatha yethu ye-ChIP, eyembula isiphikisi se-cocaine-Mediated gene ku-CaMKIIcy gene promoter ku-NAc core kuphela, isekela ukubandakanyeka komshini we-chromatin. Ngokuhambisana nale nkulumo-mbumbulu, ama-ΔFosB overexpression ku-D1-Type MSNs akwazi ukushayela i-CaMKIIcy ku-NAc core lapho kungekho cocaine (I-Fig 3F), kusikisela ukuthi kukhona ukuguqulwa okusebenzayo kwesiphakamisi se-CaMKIIα esivikela lokhu kungeniswa ngesikhathi sokuvezwa kwe-cocaine engalapheki. Ukulawulwa kokubukeka komhlaba we-chromatin esikhuthazeni seCaMKII kungahle futhi kuchaze ukuthi kungani i-CaMKII idonswa ngumthamo wensangu we-cocaine ku-NAc igobolondo le-cocaine lokuhoxisa amagundane (I-Fig 1E) kepha hhayi wezilwane ezine-drug-naïve (I-Fig 1D). Lokhu kungamelela umphumela we-epigenetic "gene priming" we-ΔFosB (URobison noNestler, 2011), futhi ngakho-ke kungaba yindlela eyodwa yokuqalwa kokufiswa kwe-cocaine (UPickens et al., 2011). Kodwa-ke, ukuze lolu shintsho lwe-chromatin luxhumeke kahle ekubangeleni kokufisa, kuzodingeka lukhule ngokuhamba kwesikhathi. Kungakujabulisa ukubona ukuthi ngabe lokhu kunjalo, futhi ubheke ukuthi ezinye izinhlobo zibonisa yini ukuthembela ku-ΔFosB, okulawulwa yi-cocaine. Kubalulekile futhi ukuqaphela ukuthi loop-phambili loop esiyichazayo ayiholeli ekuqongeleleni okungapheli kweCaMKII noma i-ΔFosB (I-Fig 1E); ukuthola “ukubhuleka” kwamangqamuzana okubhekele lokhu kuyinjongo ebalulekile yezifundo zesikhathi esizayo.

Imisebenzi eyaziwayo ye-ΔFosB ne-CaMKII ezinhlelweni eziningana zokuhlola kanye nezifunda zobuchopho ziyahlangana emazingeni amaningi (I-Fig 6F). Womabili la mamolekyulu ahlobene kakhulu nokukhula komgogodla we-dendritic: I-CaMKII ixhumana ne-actin cytoskeleton (I-Okamoto et al., 2009), ilawula usayizi wekhanda lomgogodla (Matsuzaki et al., 2004), futhi kuyadingeka futhi kwanele ngokwanda okwenziwe ngeplastiki ku-filopodia nenombolo yokuvumelanisa kumasiko we-hippocampal organotypic slice (Uhambo et al., 2003), wI-hile ΔFosB iyadingeka futhi yanele ekwakhekeni komgogodla we-cocaine-indened dendritic spine ku-NAc MSNs (Maze et al., 2010). Ngaphezu kwalokho, womabili ama-molecule ahlotshaniswa nokulawulwa kwe-AMPA glutamate receptors. I-CaMKII ayilawuli amanani aphelele we-AMPA receptor subunits, kepha idonsa ukufakwa kwama-receptors we-AMPA kuma-synapses futhi ikhulisa ukusebenza kwesiteshi se-AMPA ngokufaka i-phosphorylating GluA1 eSer831 kuma-hippocampal pyramidal neurons kusiko naku- vivo (kubuyekezwe ku- (IMalinow neMalenka, 2002; UColbran noBrown, 2004)). Ukushushumbisa okunjalo kwe-GluA1 kuya ku-synapse kuye kwaba nomthelela kwisenzo se-cocaine esingapheli (I-Boudreau ne-Wolf, i-2005). Ngaphezu kwalokho, izimpendulo zokuziphatha ku-AMPA receptor activation ku-NAc zithuthukiswa yi-CaMKIIα overexpression ngendlela ye-D1 dopamine receptor-receptor-way (Isangoma et al., 2010). I-overexpression yesikhathi eside ye-D1 eqondile ye-ΔFosB iboniswe ukuthi ifake umbhalo we-GluA2 ku-NAc (Kelz et al., 1999), okuthikameza izimpendulo ze-AMPA zixhumeke nge-GluA1, ngenkathi sibonisa lapha ukuthi i-ΔFosB ye-expxpression yesikhathi esifushane-kanye nokuvezwa kwe-cocaine yesikhathi esifushane — akunamphumela kulokhu kunqotshwa (I-Fig 1). Noma kunjalo, kamuva nje sithole ukuthi i-ΔFosB overexpression yesikhashana nje nokho inciphisa izimpendulo ze-AMPA kuma-D1-Type MSNs in NAc (UGrueter et al., 2013). Le mininingwane iphakamisa izindlela ezihlukile zesikhashana ezingakha uchungechunge olususelwa ku-neuroadaptations ku-cocaine olungaphansi kwezici ezihlukile zokuqhubekela phambili komlutha ezingakaqondakali kahle. Ezingeni lokuziphatha, zombili i-CaMKII ne-ΔFosB ziyadingeka ekuzwisiseni i-locomotor ku-cocaine (bona ngenhla), futhi zombili ziyadingeka ekuzilawuleni kwe-cocaine okwenziwe ngalo ezindongeni (I-Colby et al., I-2003; U-Wang et al., I-2010), kuphakamisa ukuthi la maprotheni amabili abalulekile ekuziphatheni kwezikhathi esifushane nokwesikhathi eside ekuvezweni kwezidakamizwa, yize esebenzisa izindlela ezithile ezingafani. Ngokunokwenzeka, i-ΔFosB ne-CaMKII balawula ukuzivumelanisa okunzima kokuziphatha ngalezi zinguquko emsebenzini we-NAc synaptic, yize umsebenzi omningi usudingeka ukuxhumanisa ngokuqondile izehlakalo ze-synaptic nokushintsha kokuziphatha.

I-CaMKII holoenzyme ngasikhathi sinye ixhumana ngamaprotheni ahlukahlukene ahlobene ne-synapse (URobison et al., 2005) okucatshangwa ukuthi kulawulwa ukuqondiswa kwalo kubuningi be-postynaptic density (PSD), into ephakanyisiwe njengebalulekile kwi-plastiki ye-synaptic. Ikakhulu, ukuxhumana kweCaMKII ne-GluN2B subunit ye-NMDA-hlobo glutamate receptor kusanda kukhonjiswa ukuthi kulawulwe zombili i-synaptic plasticity kanye nokufunda (UHalt et al., 2012). Ngenkathi i-peptide ye-AC3I ilingisa isizinda se-autoinhibitory yeCaMKII, futhi ngaleyo ndlela ivimbela umsebenzi wokuqinisa we-enzyme, ibuye ivimbele ukusebenzisana kwamaprotheni amaningi (Strack et al., 2000; URobison et al., 2005). Ngakho-ke, imiphumela yokuziphatha neyokuziphatha kwe-HSV-GFP-AC3I ebikwe lapha ingenzeka ngokuncipha kwama-phosphorylation wamaprotheni okuhlosiwe kwe-CaMKII, ushintsho ekuqondisweni kwe-CaMKII, noma ushintsho endimeni ehlongozwayo ye-CaMKII yesakhiwo esakhiwe kuma-synapses (Lisman et al., 2002).

Ukuvinjezelwa kwedatha ehlongozwayo ye-ΔFosB-CaMKII egobolondweni le-NAc kuyinto eqaphelekile, njengoba umsebenzi wakamuva ukhombise ukwehluka okuningana komzimba phakathi kwegobolondo le-NAc kanye nomongo ekuphenduleni ekuphathweni kwe-cocaine, umbono oqinisekiswe idatha yethu ye-iTRAQ engacashuniwe (Ithebula S1) . Ama-Microsoft e-NAc igobolondo akhombisa ukungakhululeki kumandla okudubula ngemuva kwe-cocaine engapheli esekelwa amaviki, ngenkathi ama-MSNs aqhamuka ezilwaneni ezifanayo abonisa ukwehla (i-1-3 usuku) ukukhuphuka kwamandla okudubula abuyela kumazinga we-basal ngaphakathi kwamaviki we-2 (UKourrich noThomas, 2009). Ngaphezu kwalokho, amaprotheni amaningi e-synaptic alawulwa ngokuhlukile egobolondweni le-NAc vs. umnyombo wezilwane ezivezwe ku-cocaine engapheli, kufaka phakathi i-GluA2 (Knackstedt et al., 2010). Njengoba i-amphetamine engapheli igxilisa iCaMKIIcy ngokukhethekile kugobolondo le-NAc (ULoweth et al., 2010), akumangazi ukuthi sithola umphumela ofanayo ne-cocaine. Kodwa-ke, njengoba i-ΔFosB ifakwa kuzo zombili igobolondo le-NAc nomongo yi-cocaine engapheli (I-Perrotti et al., I-2008), futhi njengoba sibonisa ukuthi ukungena kweCaMKIIα igobolondo kuncike ku-ΔFosB, okutholakele kunikeza ubufakazi obusha bezindlela ezihlukile zokubhaliwe endaweni yokuphromotha yeCaMKIIcy phakathi kwalezi zinto ezimbili, ezibhekene nokungeniswa okukhethiwe kweCaMKIIα egobolondweni.

Umsebenzi omkhulu wakamuva ubheke ukucacisa umehluko phakathi kwe-D1- ne-D2-hlobo NAc MSNs. Yize bobabili i-D1 ne-D2 receptors bebambe iqhaza emiphumeleni evuza ye-cocaine (I-Self, i-2010), umsebenzi wakamuva ukhombisa ukuthi kusebenze i-optogenetic ye-D1-Type MSNs kuongezeka kwempendulo yokuziphatha ku-cocaine, kuyilapho ukusebenza kwe-D2-uhlobo lwe-MSN kunomthelela ophambene (Lobo et al., 2010). Ngokuhambisana nalokhu okutholakele, amagundane e-D1-receptor Knoutout anele ekutholeni ukuzilawula kwe-cocaine (UCaine et al., 2007), ngenkathi i-D2 knockouts ingekho (UCaine et al., 2002). Ukuphathwa kwe-agonist ye-D1 ngqo ku-NAc kubangela ukuziphatha okufuna i-cocaine ku-paradigms yokubuyiselwa (I-Self, i-2010). Ngokuthokozisayo, lo mphumela udinga ukwanda okuncike ku-D1-receptor -misebenzi yomsebenzi we-CaMKII kwigobolondo le-NAc, kodwa hhayi umongo (U-Anderson et al., 2008), umphumela ovela ngokuqondile nge-D1- ne--FosB-CaMKII loop ehlongozwayo lapha.

Sike sabika ukuthi iSer27 ku-ΔFosB ingafakwa phosphorylated yi-casein kinase-2 (Ulery et al., 2006), kepha-ke, simisa lapha ukuthi iCosMKII phosphorylates ΔFosB kulokhu nakwamanye amasayithi ane-kinetics enkulu ne-stoichiometry futhi ingaphindaphinda okuphakeme okubonakala ku-M_r kubhekelwe i-ΔFosB (I-Fig 5A) ngokuchayeka kwe-cocaine vivo (I-Nestler, i-2008). Sesivele sazi ukuthi i-phosphorylation yeSer27 inyusa ukuqina kwe-ΔFosB nomsebenzi wokuloba (Ulery et al., 2006; I-Ulery neNestler, 2007; Ulery-Reynolds et al., 2009). Umsebenzi wekusasa manje uzogxila ekuboneni nasemiphumeleni yokusebenza kwamanoveli ezingosi ze-ΔFosB phosphorylation ekhonjiswe esifundweni samanje.

Loop-phambili loop echazwe lapha ihlinzeka ngomshini omusha obonakalayo lapho ukuphathwa okuphindaphindiwe kwe-cocaine kushayela khona okulimazayo okuqhubekayo e-NAc. Njengoba kunje, lendlela ye-biochemical ingahlinzeka ngokuqondisa okubalulekile kokungenelela kokwelashwa esikhathini esizayo ekuphazamiseni umlutha. Ngoba i-CaMKII igcwele futhi iyadingeka emisebenzini eminingi ye-basal neuronal kanye nokuziphatha, ukusetshenziswa okuqondile kwe-CaMKII inhibitors kugwenywe njengendlela yokwelapha umlutha. Idatha yethu iphakamisa ukuthi ukuqondiswa okucashile okwengeziwe komshini we-CaMKII lokungeniswa, okucaciswe kuhlobo lweseli ngalinye kanye nokuzithoba kokujikeleza komvuzo wobuchopho, kunganikeza ithagethi yokwelapha engavikela ubunzima bezithiyo ze-systemic CaMKII.

Iya ku:

Ukubonga

Lo msebenzi wesekwa izibonelelo ezivela eNational Institute on Drug Abuse (EJN), NIDA-Yale Proteomics Center DA018343 (AJR ne-EJN), kanye neHartwell Foundation (AJR). Ababhali bangathanda ukubonga uGabby Rundenko ngesipho esivulekile se-ΔFosB noRoger Colbran ngesipho esivulekile seCaMKIIα.

Iya ku:

Okubhekwayo

1. U-Ahmed SH, uKoob GF. Ukushintshwa kusuka kokulinganiselayo kuya ekuvinjelweni ngokweqile kwezidakamizwa: ushintsho endaweni yephuzu le-hedonic Isayensi. I-1998; 282: 298-300. [I-PubMed]
2. U-Anderson SM, uKR odumile, uSadri-Vakili G, uKumaresan V, uSchmidt HD, uBass CE, uTerwilliger EF, uCha JH, uPierce RC. I-CaMKII: ibhuloho le-biochemical elixhumanisa ama-dopamine nezinhlelo ze-glutamate ekufuneni i-cocaine. Nat Neurosci. I-2008; 11: 344-353. [I-PubMed]
3. Boudreau AC, Wolf ME. Ukuzwela kokuziphatha kwe-cocaine kuhlotshaniswa nokwanda kwe-AMPA receptor surface expression kuma-nucleus accumbens. J Neurosci. I-2005; 25: 9144-9151. [I-PubMed]
4. Brickey DA, Colbran RJ, Fong YL, Soderling TR. Ukuvezwa kanye nokulinganiswa kwe-alpha-subunit yeCa2 + / protodulin-based protein proteinaseasease isebenzisa uhlelo lokubonisa lwe-baculovirus. I-Biochem Biophys Res Commun. I-1990; 173: 578-584. [I-PubMed]
5. I-Caine SB, i-Negus SS, iMello NK, Patel S, Bristow L, Kulagowski J, Vallone D, Saiardi A, Borrelli E. Indima ye-dopamine D2-like receptors in cocaine self-management: izifundo nge-D2 receptor mutant mice kanye ne-inoveli ye-D2 receptor Abaphikisi. J Neurosci. I-2002; 22: 2977-2988. [I-PubMed]
6. ICaine SB, iThomsen M, uGabriel KI, iBergkowitz JS, iGolide LH, uKoob GF, iTonegawa S, uZhang J, u-Xu M. Ukuntuleka kokuzibamba kwe-cocaine ku-dopamine D1 receptor-out mbeva. J Neurosci. I-2007; 27: 13140-13150. [Isihloko samahhala se-PMC] [I-PubMed]
7. Carle TL, Ohnishi YN, Ohnishi YH, Alibhai IN, Wilkinson MB, Kumar A, Nestler EJ. Izindlela ezixhomeke ku-proteasome futhi-ezingalindelekile ze-FosB ukuhlaziywa: ukuhlonza izizinda ze-FosB zezinkomba kanye nemiphumela yokuzinza kwe-DeltaFosB. U-Eur J Neurosci. 2007; 25: 3009-3019. [I-PubMed]
8. UCan J, uKelz MB, uZeng G, uSoyii N, uSteffen C, uShockett PE, uPicciotto MR, uDuman RS, uNestler EJ. Izilwane zeTransgenic ezinezinhlobo zesizalo esingaqondakali, ezihlosiwe ebuchosheni. I-Mol Pharmacol. I-1998; 54: 495-503. [I-PubMed]
9. UChristoffel DJ, uGolden SA, uDumitriu D, uRobison AJ, uJanssen WG, u-Ahn HF, uKrishnan V, uReyes CM, uHan MH, u-Ables JL, u-Eisch AJ, uDietz DM, uFerguson D, uNever RL, uGreengard P, uKim Y, uMorrison JH , URusso SJ. I-IkappaB kinase ilawula ukunqotshwa kwengcindezi okubandakanya ukucindezelwa nokuziphatha kwe-plastiki. J Neurosci. I-2011; 31: 314-321. [Isihloko samahhala se-PMC] [I-PubMed]
10. I-Colbran RJ. Ukuqalwa kwe-Ca2 + / proteinod-based protein proteinaseasease by basal autophosphorylation. J Biol Chem. I-1993; 268: 7163-7170. [I-PubMed]
11. I-Colbran RJ. Ama-phosphatases ama-protein kanye ne-calcium / mododulin-exhomeke ngamaprotheni kinase II. J Neurosci. I-2004; 24: 8404-8409. [I-PubMed]
12. UColbran RJ, uBrown AM. I-calcium / mododulin-based protein protein kinase II kanye neplaticicityity. UCrr Opin Neurobiol. I-2004; 14: 318-327. [I-PubMed]
13. U-Colby CR, uWhisler K, uSteffen C, uNestler EJ, u-Self DW. Ukwehliswa kwe-DeltaFosB ngokwe-cell striatal kukhuthaza ukugqugquzela i-cocaine. J Neurosci. 2003; 23: 2488-2493. [I-PubMed]
14. UCovington HE, 3rd, Maze I, LaPlant QC, Vialou VF, u-Ohnishi YN, uBerton O, uFass DM, uRenthal W, uRush AJ, u-3rd, u-Wu EY, uGhose S, uKrishnan V, uRorso SJ, uTamminga C, uHaggarty SJ, uNestler I-EJ. Izenzo eziphikisayo ze-histone deacetylase inhibitors. J Neurosci. I-2009; 29: 11451-11460. [Isihloko samahhala se-PMC] [I-PubMed]
15. UDavalos A, uFernandez-Hernando C, uSowa G, uDerakhshan B, uLin MI, uLee JY, uZhao H, uLuo R, uColangelo C, uSessa WC. Amaprotheni ama-proteinet we-caveolin-1 alawulwa ngamaphrotheyini: ukubonwa kwe-polymerase i kanye ne-transcript factor factor / CAVIN-1 kumaseli we-endothelial. Ama-Mol Cell Proteomics. I-2010; 9: 2109-2124. [Isihloko samahhala se-PMC] [I-PubMed]
16. UDumais A, Lesage AD, Alda M, Rouleau G, Dumont M, Chawky N, Roy M, Mann JJ, Benkelfat C, Turecki G. Ubungozi bokuqedwa kokuzibulala ekucindezelekeni okukhulu: ucwaningo lokulawulwa kwecala lokuziphatha okungahambisani nolaka ku amadoda. NginguJ Psychiatry. I-2005; 162: 2116-2124. [I-PubMed]
17. IGrueter BA, Robison AJ, Neve RL, Nestler EJ, Malenka RC. I-ΔFosB iguquguqula ngokwehlukile i-nucleus ibutha umsebenzi wendlela eqondile neqondile. Proc Natl Acad Sci USA. I-2013 emaphephandabeni. [Isihloko samahhala se-PMC] [I-PubMed]
18. IHalt AR, Dallapiazza RF, Zhou Y, Stein IS, Qian H, Juntti S, Wojcik S, Brose N, Silva AJ, Hell JW. Ukuhlanganiswa kwe-CaMKII ku-GluN2B kubalulekile ngesikhathi sokuhlanganiswa kwememori. I-EMBO J. 2012; 31: 1203-1216. [Isihloko samahhala se-PMC] [I-PubMed]
19. UHiroi N, uBrown JR, uHaile CN, uYe H, uGreenberg ME, uNestler EJ. Amagundane aguqukayo e-FosB: ukulahleka kokungeniswa okungapheli kwe-cocaine kwamaprotheni ahlobene ne-Fos nokuzwela okukhulu ku-psychomotor ye-cocaine nemiphumela emihle. I-Proc Natl Acad Sci US A. 1997; 94: 10397-10402. [Isihloko samahhala se-PMC] [I-PubMed]
20. Ito R, Robbins TW, Everitt BJ. Ukulawulwa okuhlukile kokuziphatha okufuna i-cocaine yi-nusus bokiza isisekelo kanye negobolondo. Nat Neurosci. I-2004; 7: 389-397. [I-PubMed]
21. UJorissen HJ, u-Ulery PG, uHenry L, uGourneni S, uNestler EJ, uRudenko G. Dimerization kanye nezindawo ezibopha iDNA zento ebhaliwe ye-DeltaFosB. I-Biochemistry. I-2007; 46: 8360-8372. [I-PubMed]
22. I-Jourdain P, i-Fukunaga K, iMuller D. I-calcium / i-proteinodase-encike i-protein casease inomthelela ekukhuleni kwe-filopodia kuncike emisebenzini. J Neurosci. I-2003; 23: 10645-10649. [I-PubMed]
23. U-Kelz MB, u-Chen J, u-Carlezon WA, uJr, u-Whisler K, u-Gilden L, u-Beckmann AM, uSteffen C, u-Zhang YJ, u-Marotti L, u-Self DW, u-Tkatch T, u-Baranauskas G, u-Surmeier DJ, u-Neve RL, u-Duman RS, u-Picciotto MR, I-Nestler EJ. Ukuchaza kwe-facta factor deltaFosB ebuchosheni ilawula ubuzwe be-cocaine. Imvelo. 1999; 401: 272-276. [I-PubMed]
24. UKlug JR, Mathur BN, Kash TL, Wang HD, Matthews RT, Robison AJ, Anderson ME, Deutch AY, Lovinger DM, Colbran RJ, Winder DG. I-genetic Inhibition yeCaMKII kuDorsal Striatal Medium Spiny Neurons Inciphisa Ukusebenza Kokuvumelana Kwezinqubo Ezigqamile kanye Ne-Enhances Intrinsic Excitability. I-PLoS One. I-2012; 7: e45323. [Isihloko samahhala se-PMC] [I-PubMed]
25. Knackstedt LA, Moussawi K, Lalumiere R, Schwendt M, Klugmann M, Kalivas PW. Ukuqeqeshwa kokuqothula ngemuva kokuzilawula kwe-cocaine kubangela ukuba ne-glutamatergic plasticity ukuvimbela ukufuna kwe-cocaine. J Neurosci. I-2010; 30: 7984-7992. [Isihloko samahhala se-PMC] [I-PubMed]
26. UKourrich S, uThomas MJ. Ama-neurons afanayo, ukuzivumelanisa okuphambene nalokho: Isipiliyoni se-psychostimulant siguqula umehluko ngokuqondile ngokudubula izakhiwo ku-accumbens core dhidi yegobolondo. J Neurosci. I-2009; 29: 12275-12283. [Isihloko samahhala se-PMC] [I-PubMed]
27. UKourrich S, Klug JR, Mayford M, Thomas MJ. Umphumela Ozimele we-AMPAR we-Striatal alphaCaMKII Ukhuthaza Ukuzwela Kwemvuzo WeCocaine. J Neurosci. I-2012; 32: 6578-6586. [Isihloko samahhala se-PMC] [I-PubMed]
28. LaPlant Q, Chakravarty S, Vialou V, Mukherjee S, Koo JW, Kalahasti G, Bradbury KR, Taylor SV, Maze I, Kumar A, Graham A, Birnbaum SG, Krishnan V, Truong HT, Neve RL, Nestler EJ, Russo SJ . Iqhaza le-factor factor ye-kappaB ku-ovarian hormone-Mediated stress hypersensitivity kumagundane abesifazane. I-Biol Psychiatry. I-2009; 65: 874-880. [Isihloko samahhala se-PMC] [I-PubMed]
29. U-Lee KW, u-Kim Y, u-Kim AM, u-Helmin K, uNairn AC, uGreengard P. Cocaine-indied dendritic spine ukwakheka kwe-D1 ne-D2 dopamine recopor-aqukethe i-kati spiny neurons kuma-nucleus accumbens. I-Proc Natl Acad Sci US A. 2006; 103: 3399-3404. [Isihloko samahhala se-PMC] [I-PubMed]
30. U-Lisman J, Schulman H, Cline H. Isisekelo samangqamuzana weCaMKII isebenza kwimemori e-synaptic ne-yokuziphatha. Nat Rev Neurosci. I-2002; 3: 175-190. [I-PubMed]
31. Lobo MK, Covington HE, 3rd, Chaudhury D, Friedman AK, ILanga H, Damez-Werno D, Dietz DM, Zaman S, Koo JW, Kennedy PJ, Mouzon E, Mogri M, Neve RL, Deisseroth K, Han MH, Nestler I-EJ. Ukulahleka kohlobo oluthile lweseli kwe-BDNF signaling mimics optogenetic yokulawulwa komvuzo we-cocaine. Isayensi. I-2010; 330: 385-390. [Isihloko samahhala se-PMC] [I-PubMed]
32. I-Loweth JA, i-Baker LK, i-Guptaa T, i-Guillory AM, i-Vezina P. Inhibition ye-CaMKII egobolondweni le-nucleus accumbens lehla ukunwebeka okuthuthukisiwe kwe-amphetamine kumagundane azwisisiwe. Neurosci Lett. I-2008; 444: 157-160. [Isihloko samahhala se-PMC] [I-PubMed]
33. I-Loweth JA, i-Singer BF, u-Baker LK, u-Wilke G, u-Inamine H, u-Bubula N, u-Alexander JK, uCarlezon WA, uJr, uNoe RL, uVezina P. Olexpression ngokweqile kwe-alpha-Ca2 + / ye-calmodulin-protein protini kinase II. ithuthukisa ukuphendula kokuziphatha ku-amphetamine. J Neurosci. I-2010; 30: 939-949. [Isihloko samahhala se-PMC] [I-PubMed]
34. Malinow R, Malenka RC. Ukuthengiswa kwe-AMPA receptor kanye ne-synaptic plasticity. Annu Rev Neurosci. I-2002; 25: 103-126. [I-PubMed]
35. I-Matsuzaki M, i-Honkura N, i-Ellis-Davies GC, i-Kasai H. Isisekelo sesisekelo sethonya elide lesikhathi eside emigqeni eyodwa ye-dendritic. Imvelo. I-2004; 429: 761-766. [I-PubMed]
36. UMayford M, Bach ME, Huang YY, uWang L, Hawkins RD, uKandel ER. Ukulawulwa kokwakhiwa kwememori ngenkulumo elawulwayo ye-CaMKII transgene. Isayensi. I-1996; 274: 1678-1683. [I-PubMed]
37. I-I, Covington HE, 3rd, Dietz DM, LaPlant Q, Renthal W, Russo SJ, Mechanic M, Mouzon E, Neve RL, Haggarty SJ, Ren Y, Sampath SC, Hurd YL, Greengard P, Tarakhovsky A, Schaefer A, I-Nestler EJ. Inselelo ebalulekile ye-histone methyltransferase G9a ku-plasticity eyenziwe nge-cocaine. Isayensi. 2010; 327: 213-216. [Isihloko samahhala se-PMC] [I-PubMed]
38. UMcClung CA, uNestler EJ. Umthethonqubo we-gene expression kanye ne-cocaine umvuzo ngu-CREB no-DeltaFosB. Nat Neurosci. 2003; 6: 1208-1215. [I-PubMed]
39. I-Nestler EJ. Buyekeza. Izindlela zokudlulisa izidakamizwa: indima yeDeltaFosB. I-Philos Trans R Soc Lond B Biol Sci. 2008; 363: 3245-3255. [Isihloko samahhala se-PMC] [I-PubMed]
40. Nye HE, Hope BT, Kelz MB, Iadarola M, Nestler EJ. Ucwaningo lwezemvelo lokulawulwa kwe-antigen ehlobene ne-FOS ehlobene ne-cocaine ku-striatum nase-nucleus accumbens. J Pharmacol Exp Ther. 1995; 275: 1671-1680. [I-PubMed]
41. I-Okamoto K, i-Bosch M, iHayashi Y. Izindima zeCaMKII ne-F-actin ku-plasticity ehlelekile ye-dendritic spines: ukuhlonza okungamakhompiyutha okungaba yithegi ye-synaptic? I-Physiology (Bethesda) 2009; 24: 357-366. [I-PubMed]
42. Olausson P, Jentsch JD, Tronson N, Neve RL, Nestler EJ, Taylor JR. I-DeltaFosB ku-nucleus accumbens ilawula ukuziphatha kwe-instrumental ukudla nokugqugquzela. J Neurosci. 2006; 26: 9196-9204. [I-PubMed]
43. I-Paxinos G, Watson C. Ingqondo yamagundwane ekuxhumaneni kwe-stereotaxic. I-6th Edition. I-Amsterdam; IBoston: I-Academic Press / i-Elsevier; I-2007.
44. I-Peakman MC, uColby C, i-Perrotti LI, i-Tekumalla P, i-Carle T, i-Ulery P, i-Chao J, i-Duman C, i- Steffen C, i-Monteggia L, i-Allen MR, i-Stock JL, i-Duman RS, i-McNeish JD, i-Barrot M, i-Self DW, i-Nestler EJ , I-Schaeffer E. Ukungatholakali, ukukhuluma kwesifunda somqondo we-mutant of mutant negative of c-Jun kumagundane e-transgenic kunciphisa ukuzwela ku-cocaine. I-Brain Res. 2003; 970: 73-86. [I-PubMed]
45. Ama-Penzes P, uCahill ME, uJones KA, uSrivastava DP. I-ConMgent CaMK ne-RacGEF amasignali alawula ukwakheka kokudwetshwa nomsebenzi. Amathrekhi e-Cell Biol. I-2008; 18: 405-413. [I-PubMed]
46. I-Perrotti LI, uHadeishi Y, Ulery PG, i-Barrot M, i-Monteggia L, i-Duman RS, i-Nestler EJ. Ukukhishwa kwe-deltaFosB ezinkambisweni zobuchopho ezihlobene nomvuzo ngemva kokucindezeleka okungapheli. J Neurosci. 2004; 24: 10594-10602. [I-PubMed]
47. Perrotti LI, Weaver RR, Robison B, Renthal W, Maze I, Yazdani S, Elmore RG, DJ Knapp, Selley DE, Martin BR, Sim-Selley L, Bachtell RK, Self DW, Nestler EJ. Amaphethini ahlukumezekile eDeltaFosB ekufakweni kobuchopho ngezidakamizwa zokuhlukunyezwa. Kuyavumelana. 2008; 62: 358-369. [Isihloko samahhala se-PMC] [I-PubMed]
48. UPickens CL, Airavaara M, Theberge F, Fanous S, Ithemba BT, Shaham Y. Neurobiology yokufakwa kwezifiso kwezidakamizwa. Ithrend Neurosci. I-2011; 34: 411-420. [Isihloko samahhala se-PMC] [I-PubMed]
49. UPierce RC, I-EA esheshayo, uReeder DC, uMorgan ZR, uKalivas PW. Izithunywa zesibili ze-calcium-Mediated module expression of sensalization sensitization to cocaine. J Pharmacol Exp Ther. I-1998; 286: 1171-1176. [I-PubMed]
50. I-Quirion R, i-Robitaille Y, iMartha J, i-Chabot JG, i-Lemoine P, i-Pilapil C, i-Dalpe M. I-human receptor autoradiography isebenzisa izingxenye ze-hemisphere: indlela ejwayelekile enciphisa izicubu zobuchopho. Synapse. I-1987; 1: 446-454. [I-PubMed]
51. I-Robinson TE, i-Kolb B. I-plasticity yesakhiwo ehambisana nokuchayeka kwezidakamizwa zokuhlukunyezwa. I-Neuropharmacology. 2004; 47 (Suppl 1): 33-46. [I-PubMed]
52. URobison AJ, uNestler EJ. Izindlela zokuloba kanye ne-epigenetic zomlutha. Nat Rev Neurosci. I-2011; 12: 623-637. [Isihloko samahhala se-PMC] [I-PubMed]
53. URobison AJ, uBass MA, uJiao Y, MacMillan LB, uCarmody LC, uBartlett RK, uColbran RJ. Ukusebenzisana okuningi kwe-calcium / mododulin-protein proteinasease-protein ine-proteinynensic density protein NR2B, densin-180, kanye ne-alpha-Actinin-2. J Biol Chem. I-2005; 280: 35329-35336. [I-PubMed]
54. URoss PL, Huang YN, Marchese JN, Williamson B, Parker K, Hattan S, Khainovski N, Pillai S, Dey S, Daniels S, Purkayastha S, Juhasz P, Martin S, Bartlet-Jones M, He F, Jacobson A, UPappin DJ. Ukuphindaphindwa kwamaprotheni amaningi ku-Saccharomyces cerevisiae kusetshenziswa ama-reagents we-amine-reactive is taging. Ama-Mol Cell Proteomics. I-2004; 3: 1154-1169. [I-PubMed]
55. URusso SJ, Dietz DM, Dumitriu D, Morrison JH, Malenka RC, Nestler EJ. I-synapse yomlutha: izindlela ze-synaptic kanye ne-organisation plasticity kuma-nucleus accumbens. Ithrend Neurosci. I-2010; 33: 267-276. [Isihloko samahhala se-PMC] [I-PubMed]
56. I-Self DW. Ku: Ama-Dopamine Receptors. Neve KA, mhleli. ENew York: IHumana Press; I-2010. I-pp. 479-524.
57. I-Singer BF, i-Loweth JA, i-Neve RL, i-Vezina P. iTransenterate ye-viral-Mediated overexpression ye-alpha-calcium / ye-mododulin-encike i-protein kinase II kugobolondo lwe-nucleus accumbens kuholela ekuphakanyisweni kokusebenza okuhlala isikhathi eside kwe-alpha-amino-3-hydroxyl-5 -methyl-4-isoxazole-propionate receptors: dopamine hlobo-1 receptor kanye ne-protein kinase Ukuxhomekeka. I-Eur J Neurosci. I-2010; 31: 1243-1251. [Isihloko samahhala se-PMC] [I-PubMed]
58. Strack S, McNeill RB, Colbran RJ. I-Mechanism kanye nokulawulwa kwe-calcium / mododulin-based protein protein kinase II ebhekiswe kwi-NR2B subunit ye-N-methyl-D-aspartate receptor. J Biol Chem. I-2000; 275: 23798-23806. [I-PubMed]
59. Ulery-Reynolds PG, uCastillo MA, Vialou V, uRusso SJ, uNestler EJ. I-Phosphorylation ye-DeltaFosB ixhumanisa ukuzinza kwayo ku-vivo. I-Neuroscience. I-2009; 158: 369-372. [Isihloko samahhala se-PMC] [I-PubMed]
60. Ulery PG, Nestler EJ. Ukulawulwa komsebenzi wokuqanjwa kwe-DeltaFosB nguSer27 phosphorylation. I-Eur J Neurosci. I-2007; 25: 224-230. [I-PubMed]
61. Ulery PG, Rudenko G, Nestler EJ. Ukulawulwa kweDeltaFosB ukuzinza nge-phosphorylation. J Neurosci. 2006; 26: 5131-5142. [I-PubMed]
62. Vialou V, et al. I-DeltaFosB emibuthanweni yomvuzo wobuchopho ivumelana nokuqina kokuxineka nezimpendulo zokulwa nokucindezela. Nat Neurosci. I-2010; 13: 745-752. [Isihloko samahhala se-PMC] [I-PubMed]
63. I-Wang L, Lv Z, Hu Z, Sheng J, Hui B, uSun J, Ma L. Ongapheli we-cocaine we-cocaine we-H3 ne-activation activation ye-CaMKIIalpha ku-nucleus accumbens kubalulekile ukuze kugqugquzelwe ukuqiniswa kwezidakamizwa. I-Neuropsychopharmacology. I-2010; 35: 913-928. [Isihloko samahhala se-PMC] [I-PubMed]
64. Werme M, Messer C, Olson L, Gilden L, Thoren P, Nestler EJ, Brene S. Delta FosB ilawula ukugijima kwesondo. J Neurosci. 2002; 22: 8133-8138. [I-PubMed]
65. Winstanley CA, LaPlant Q, Theobald DE, Green TA, Bachtell RK, Perrotti LI, DiLeone RJ, Russo SJ, Garth WJ, Self DW, Nestler EJ. I-deltaFosB ukungeniswa kwe-cortex ye-orbitofrontal iphikisana nokubekezelelana nokucindezeleka okubangelwa ukukhathazeka kwe-cocaine. J Neurosci. 2007; 27: 10497-10507. [I-PubMed]
66. Zachariou V, Bolanos CA, Selley DE, Theobald D, MP MPs, Kelz MB, Shaw-Lutchman T, Berton O, Sim-Selley LJ, Dileone RJ, Kumar A, Nestler EJ. Indima ebalulekile kuDeltaFosB ku-nucleus accumbens ekwenzeni i-morphine. Nat Neurosci. 2006; 9: 205-211. [I-PubMed]
67. UZhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Intengo EE, Jr, Thiel W, Guatimosim S, Iculo LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, Anderson ME. ICalmodulin kinase II inhibition ivikela izifo zenhliziyo ezihlelekile. Nat Med. I-2005; 11: 409-417. [I-PubMed]