(BOPHELO) Likarabo tsa Boits'oaro le Mekhoa ea Boipheliso ea Cocaine e Hlokehang ho Hlokahala Mokhabo o Ntseng o Fumella ho Akarelletsa le Protheine ea DFFB le ea Calcium / Calmodulin-e theolelang Kinase II Nucleus Accumbens Shell (2013)

J Neurosci. 2013 Mar 6;33(10):4295-4307.

Robison AJ, Vialou V, Mazei-Robison M, Feng J, Kourrich S, Collins M, Wee S, Koob G, Turecki G, Neve R, Thomas M, Nestler EJ.

mohloli o moholo

Lefapha la Fishberg la Neuroscience le Friedman Brain Institute, Mount Sinai School of Medicine, New York, New York, 10029, Mekhatlo ea Neuroscience le Psychology, Institute of Human Genetics, Univesithi ea Minnesota, Minneapolis, Minnesota 55455, Komiti ea Neurobiology ea Mathata a Addictive , The Scripps Research Institute, La Jolla, Mokhatlo oa California oa khatello ea maikutlo oa khatello ea kelello, Institute of University of Douglas Mental Health le Univesithi ea McGill, Montréal, Quebec, Canada, H92037H 4R1, le Lefapha la Bohlale ba Setsebi le Tsebo ea Bohlale, Massachusetts Institute of Technology 3 .

inahaneloang

Ntho e ngotsoeng ka mokhoa o hlalosang ΔFosB le protein kinase II e nang le boko bo nang le bokhoni bo nang le boko ba pelo (CaMKIIA) e kenyelletsoa ka nucleus accumbens (NAc) ka ho hlaseloa ha cocaine kapa lithethefatsi tse ling tsa ho hlekefetsoa, ​​tseo liprotheine tsena tse peli li kopanelang li arabisang likarabo tsa lithethefatsi . Le hoja ΔFosB le CaMKIIa li laola tsela ea AMP glutamate receptor le ho sebetsa ka NAc, sebopeho sa mokokotlo oa mokokotlo oa li-NAC medium spiny neurons (MSNs), le ho khothaletsoa ha li-coconine, ho se hokae ho hokahanngoa ka ho toba pakeng tsa limolek'hule tsena. Mona, re bontša hore ΔFosB ke phosphorylated ke CaMKIIa ho Ser27 ea protheine-stabilizing le hore CaMKII ke e hlokehang bakeng sa pokello ea cocaine-mediated accumulated of ΔFosB ka rat NAc.

Ka lehlakoreng le leng, re bontša hore ΔFosB e hlokahala ebile e lekaneng bakeng sa tlhahiso ea k'hok'haine ea CaMKIIa lipolelo tsa liphatsa tsa lefutso ka vivo, phello e khethollang D1Metsing ea MSN sebakeng sa NAc shell.

Ho feta moo, ho kenngoa ha metsoako ea dendritic ho NAc MSNs le ho eketseha ha boitšoaro ho cocaine ka mor'a hore NAc e fetelle ho feta ea DFFB ke bobeli ba CaMKII.

Habohlokoa, re bonts'a ka lekhetlo la pele tšebetso ea ΔFosB le CaMKII ho NAc ea batho ba noang li-cocaine, ho fana ka maikutlo a hore ho ka etsahala hore ho be le mekhoa ea ho phekoloa nakong e tlang. Litlaleho tsena li theha hore ΔFosB le CaMKII ba kene ka selefouno ea mofuta oa sepetlele-le boko bo nepahetseng e le mokhoa o ka sehloohong oa ho laola potoloho ea moputso oa boko bo sebetsanang le cocaine e sa feleng.

Selelekela

Bopaki bo ntseng bo eketseha bo tšehetsa maikutlo a hore liphetoho tsa liphatsa tsa lefutso li kenya letsoho litabeng tsa ho lemalla lithethefatsi (Robison le Nestler, 2011). Mohokahanyi e mong oa bohlokoa oa liphetoho tsena ke ΔFosB, motsoako oa phetolelo ea lelapa la Fos (Nestler, 2008). Tsamaiso e tloaelehileng ea hoo e batlang e le lithethefatsi leha e le efe ea tlhekefetso e etsa hore ho be le nako e telele ea ho bokella DFBB nucleus accumbens (NAc), sebaka sa maiketsetso se hlokahalang bakeng sa boitšoaro ba moputso. Sho kenyelletsoa ha ho hlaha ho hlaha ka ho toba ho sehlopha sa NAc sepheo sa spiny neuron (MSN) se hlahisang D1 dopamine receptors. Ho se ts'oenyehe ho sa khoneheng ha ΔFosB ho tsena tsa D1-mofuta oa MSN MSN ho eketsa mohloli le likarabo tse khotsofatsang ho cocaine le morphine (Kelz et al., 1999; Zachariou et al., 2006), ho kenyeletsa le ho eketseha ha k'hok'heine ea boipheliso (Colby et al., 2003). Ho feta moo, ho thibela liphatsa tsa lefutso kapa tšoaetso ea kokoana-hloko ea ΔFosB ho etsa liphetoho ho fokotsa liphello tse putsang tsa lithethefatsi tsena (Zachariou et al., 2006), e bontšang hore ΔFosB e tsitsitseng pele e le moemeli oa bohlokoa oa liphetoho tsa nako e telele tse entsoeng ka NAc ke tsamaiso e sa foleng ea lithethefatsi.

Boikutlo bo sa tloaelehang ba DFFB (bo amanang le mefuta eohle ea liprotheine tsa lelapa la Fos) ke thepa ea tlhaho ea molek'hule, ka lebaka la libaka tsa marang-rang tse hlahisitsoeng FosB e telele ka ho feletseng (Carle et al., 2007), le mokhoa o laoloang. DFFB ke phosphorylated a vitro 'me Ka vivo ho Ser27, 'me karabelo ena e tsoela pele ho tsitsisa ΔFosB, ~ 10, ka seleng ea setso le NAc Ka vivo (Ulery-Reynolds et al., 2009). Le hoja Ser27-ΔFosB e 'nile ea bontšoa hore ke substrate bakeng sa casein kinase-2 a vitro (Ulery et al., 2006), mokhoa oa eona Ka vivo phosphorylation e ntse e sa tsejoe.

Calcium / calmodulin-e itšetlehileng ka protheine kinase II (CaMKII) ke serine / threonine kinase e hlalositsoeng haholo-holo eo α le β isoforms e etsang dodecameric homo-hetero-holoenzymes Ka vivo, 'me ke tsa bohlokoa bakeng sa mefuta e mengata ea phekolo ea mali (Lisman et al., 2002; Colbran le Brown, 2004). CaMKIIA e bakoa ke khetho ea NAc shell ka amphetamine e sa foleng (Loweth et al., 2010), 'me thibelo ea bongaka ea mesebetsi ea CaMKII ka NAc shell e fokotsa boits'oaro bo matla ho amphetamine (Loweth et al., 2008) le cocaine (Pierce et al., 1998), ha ts'oaetso ea kokoana-hloko ea CaMKIIA ho mona e na le matla a ho khothaletsa ho khothaletsoa ke li-amphetamine (Loweth et al., 2010). CaMKIIA e ka ama boitšoaro ba moputso ka mokhoa oa ho feto-fetoha ha lihlopha tsa AMPA glutamate receptor (Pierce et al., 1998), kaha mosebetsi oa CaMKIIa o 'nile oa amahanngoa le mosebetsi oa AMPA le ho hlasela-synaptic ka mekhoa e sa tšoaneng ea methapo ea pelo (Malinow le Malenka, 2002).

Lingoliloeng tsena li bontša phapang e 'maloa pakeng tsa ΔFosB le CaMKII: ka bobeli li hlokahala ebile li lekaneng bakeng sa liphello tse ngata tsa boitšoaro ba lithethefatsi tsa tlhekefetso, bobeli ba hlophisa likhahla tsa dendritic mefuta e sa tšoaneng ea neuronal cell Ka vivo (Jourdain et al., 2003; Maze et al., 2010), 'me ka bobeli ba etsa liphello tse ling tsa boitšoaro ba bona ka ho feto-fetoha ha methapo ea AMPA (Kelz et al., 1999; Malinow le Malenka, 2002; Vialou et al., 2010). Ho sa tsotellehe lipapiso tsena, ha ho letho le sebetsang pakeng tsa ΔFosB le CaMKII. Ha re le mona, re etsa molao oa tsamaiso pakeng tsa ΔFosB le CaMKII, mme re bonts'a hore liprotheine tse peli li theha sejo se ts'ebelisoang sa phepo sa MSN sa D1 sekhethong sa NAc se susumelitsoeng ke cocaine mme se laola likarabo tse ngata tsa cocaine Ka vivo.

Eya ho:

Lisebelisoa le mekhoa

Hlahloba 1: iTRAQ Proteomic Analysis ea NAc Shell le Core After Cocaine Treatment (Setšoantšo sa 1A)

Batho ba baholo (libeke tsa 8) likokoanyana tsa banna li ne li fanoa ka 20 mg / kg cocaine kapa koloi ea saline IP hang ka letsatsi ka matsatsi a supileng. 24 hr ka mor'a hore e entsoe ka lekhetlo la ho qetela, NAc shell le mantlha li ne li nyenyane haholo (Setšoantšo sa 1A) 'me e khanyelitsoeng leqhoa. lintlha tsa iTRAQ li ile tsa etsoa joalokaha ho hlalositsoe pele (Ross et al., 2004; Davalos et al., 2010).

Setšoantšo sa 1

Setšoantšo sa 1

Ho kenyelletsoa ha li-Shell ka CaLKII ka NAC ka cocaine

Hlahloba 2: Ho qaqisa liprotheine tsa phetoho Pulong ea NAc Core le Shell ka mor'a Kalafo ea Kalaine (Setšoantšo sa 1B-D)

Batho ba baholo (li-8 libeke) li-rats li ne li fanoa ka 10 mg / kg k'hak'haine kapa koloi ea saline IP hang ka letsatsi ka matsatsi a supileng ka ho lokisa likamore. Likarabo tsa li-locomotor ho lejoe le le leng la cocaine (5 mg / kg IP) li ile tsa tlalehoa liphoofolong tseo tse tšoaroang pejana ka cocaine (e bitsoang "e sa foleng") le karolo ea ba phekoloang ka saline (e bitsoang "a hlobaetsang"), le likarabo tsa motlakase ho saline e le 'ngoe e tlalehiloe liphoofolong tse sa foleng tse tšoaroang ka letsoai (tse bitsoang "saline"). Litlhahlobo tsa ho etsa lintho tse tloaelehileng li ile tsa etsoa joalokaha ho hlalositsoe (Hiroi et al., 1997). Ka bokhutšoanyane, likhoto tse kholo tsa banna li ile tsa kenngoa ka lebokose la 18 "× 24" ha ho na mabokose a rekisang mabala a bulehileng (San Diego Instruments) bakeng sa 30 min ho tloaela, ba fuoa sehlabelo se le seng sa IP sa saline le ho hlahlojoa ka 30 min, hape ba fuoa injection e le 'ngoe ea IP ea 5 mg / kg cocaine le ho hlahlojoa bakeng sa 30 min.

24 hr ho latela lenane lena la ho qetela, likhoho li ile tsa senyeha ntle le ho thibela anesthesia ho qoba liphello tsa anesthetics ho li-protein tsa neuronal le liphospho-states. Bokooa bo ne bo fokotsoe ka makhetlo a mararo a 1.2 mm (Braintree Scientific) le lihlahisoa tse hlaselitsoeng li ile tsa tlosoa ka saline e nooang ea phosphate e nang le protease (Roche) le phosphatase (Sigma Aldrich) inhibitors e sebelisang pina ea 14 e lekaneng bakeng sa melatsoana ea NAC le pene ea 12 ea setseng. lisele tsa NAC shell (Bona Setšoantšo sa 1A) 'me hang-hang ba qhibililoe ka leqhoa le omileng. Lisebelisoa li ne li nkoa e le homogenized ka morao o khanyang ka botlolo ba RIPA: 10 mM Tris base, 150 mM sodium chloride, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% deoxycholate sodium, pH 7.4, protease le phosphatase inhibitors joalo ka holimo. Ka mor'a hore ho kenyeletsoe sehlahisoa sa Laemmli, liprotheine li ne li arohane ka li-gels tsa 4-15% tsa polyacrylamaide (System ea Criterion, BioRad), le ho thibela Bophirimela ho ne ho etsoa ka mokhoa oa Odyssey (Li-Cor) ho latela liprotheke tsa moetsi.

Hlahloba 3: Ho koetlisetsa liprotheine tsa phetoho Pulong ea NAc Core le Shell ka mor'a hore ho tlosoe Cocaine (1E ea setšoantšo)

Batho ba baholo (libeke tsa 8) likokoanyana tsa banna li ne li fanoa ka 10 mg / kg cocaine kapa koloi ea saline IP hang ka letsatsi ka matsatsi a supileng. Matsatsi a 14 ka mor'a hore e hlajoe ka lekhetlo la ho qetela, liphoofolo tse phekoloang ka saline li ile tsa fuoa ente e 'ngoe ea saline (e bitsoang "saline),' me liphoofolo tse tšoaroang ka cocaine li ile tsa fuoa ente e 'ngoe ea saline (e bitsoang 14 letsatsi la ho tlohela kapa" 14d WD ") e bitsoang "14d WD Chal" bakeng sa phephetso). Nako e le 'ngoe ka mor'a ho kenngoa ha ho qetela, liphoofolo li ile tsa senyeha' me ho thijoa ha Bophirimela ho etsa joalo Leka 2.

Hlahloba 4: Ho qaqisa liprotheine tsa liphetoho Pulong ea NAc Core le Shell ka mor'a hore ho be le boipheliso ba Cocaine (Setšoantšo sa 2A-C)

Li-rats li ile tsa koetlisetsoa ho itlhokomela 0.5 mg / kg / infusion ea cocaine ka mananeo a le mong hr tlas'a lenane le tsitsitseng la 1 bakeng sa matsatsi a robong. Ka mor'a likarolo tse robong tsa motheo, lipotlolo li arotsoe ka lihlopha tse peli tse lekantsoeng le ho noa ha cocaine likopanong tse peli tse fetileng. Sehlopha se seng sa likhoto se ile sa lumelloa ho itlhahisa ka cocaine (0.5 mg / kg / infusion) mananeo a le mong (hanyane ho fihlela, ShA) ha sehlopha se seng sa likhoto se e-na le cocaine e ikemetseng ka linako tse tšeletseng (ho fihlela nako e telele, LgA ) ka matsatsi a leshome (matsatsi a eketsehileng).

Likarolo tsa boko li ile tsa sebelisoa bakeng sa immunohistochemistry joalokaha ho hlalositsoe (Perrotti et al., 2004). Bokooa bo ile ba sebelisoa hampe 18-24 hr ka mor'a ho pepesa ho qetela ho lithethefatsi, e leng se bakang ho senyeha ha protheine ea FosB e feletseng e feletseng e le hore tsohle tse setseng li sitoe ho bontša ΔFosB. Phoso ena e tiisitsoe ke ho thibela Bophirimela, e neng e sa bontše litšila tse matla ka antibody e lebisitsoeng khahlanong le terminus ea C ea bolelele ba FosB e sa tsebeng DFB (data e sa bonts'eng). Ka mor'a ho tšela likaroloana tsa 35 μm, palo ea lisele tsa DFB tse sa sebetsaneng le metsi li ne li hlahlojoa ke motho ea foufetseng likarolong tse peli ka NAC ea 'ngoe le e' ngoe, 'me ho boleloa hore litekanyetso tsa 40 × li ne li baloa ka sebaka sa phoofolo ka' ngoe. Phoofolo e 'ngoe le e' ngoe e ne e nkoa e le pono ea motho ka mong bakeng sa tlhahlobo ea lipalo Libaka tsa thahasello li ile tsa khetholloa ho sebelisa Paxinos le Watson (Paxinos le Watson, 2007).

Quantification ea CaMKIIa e sa sebetsane le boits'oaro e ne e etsoa ho sebelisa tsamaiso ea License joalokaha ho hlalosoa (Covington et al., 2009). Matla a kopaneng a CaMKII le GAPDH a ne a khethiloe ka software ea Odyssey. Liphetho li hlahisoa e le matla a kopantsoeng litekanyetso ka mm2 mme li hlahisoa ka mokhoa oa ± sem (n = 4-10 ka sehlopha). Melao ea GAPDH e ne e sebelisoa ha ho buuoa ka ho tiisa matla a CaMKII bakeng sa sekhahla le maemo.

Setšoantšo sa 2

Setšoantšo sa 2

Ho kenyelletsa CaMKII ho NAc shell ea likhopo tsa ho itlhokomela le ho lemalla batho ba nang le cocaine

Hlahloba 5: Ho koetlisa Maemo a Protheine ho Batho ba Tšepahalang ba Cocaine (Setšoantšo sa 2D)

Tsamaiso

Li-tissue tsa boko ba motho li ile tsa fumanoa baneng ea Quebec Suicide Brain Bank (Setsi sa Douglas Mental Health University, Montreal, Quebec, Canada). Ho boloka mahlaseli ho ile ha qala ka mokhoa o hlalositsoeng (Quirion et al., 1987). Ka bokhutšoanyane, hang ha e ntšoa, boko bo kenngoa ka leqhoa le nang le metsi ka lebokose la Styrofoam ebe le potlakela ho ea liofising tsa Quebec Suicide Brain Bank. Hang-hang li-hémitephe li arotsoe ke sekhahla sa sagittal bohareng ba boko, boko ba boko le cerebellum. Lijana tsa mali, sekhahla sa phaena, plexus ea choroid, halofo ea cerebellum, le boko ba boko ba bokooa hangata li aroloa ho tloha karolong e ka letsohong le letšehali eo ka nako eo e khaotsoang ka likaroloana tse mashome a mararo tsa 1 pele ho na mali. Halofo ea halofo ea cerebellum e khaola ka makhetlo a mararo ho 1cm-teteaneng pele ho serame. Mahlaseli a khanyang a leqhoa ka 2-methylbutane ho -40 ° C bakeng sa 'metsotso ea 60. Li-tissue tsohle tse nang le serame li bolokiloe ka thōko ka mekotla ea polasetiki -XNUM ° C bakeng sa polokelo ea nako e telele. Libaka tse khethehileng tsa boko li aroloa likarolong tse nang le serame tse nang le serame se nang le leqhoa le nang le leqhoa le omileng ho potoloha mocheso oa tikoloho. Ho thibela Bophirimela ho ne ho etsoa joalokaha ho hlalositsoe Leka 2.

Sehlopha

Sehlopha se ne se entsoe ka banna ba 37 le ba basali ba 3, ho tloha lilemong tsa lilemo tsa 15-66. Bafosi bohle ba ile ba shoa ka tšohanyetso ntle le naha ea agonal e telele kapa boloetse ba nako e telele ea bongaka. Boemong bo bong le bo bong, sesosa sa lefu se ile sa fumanoa ke ofisi ea Quebec Coroner, 'me setsi sa likokoana-hloko se etsoa ka lisebelisoa tsa lisele ho fumana lintlha tsa meriana le tšebeliso e mpe ea lithethefatsi nakong ea lefu. Sehlopha sena se ne se entsoe ka batho ba 20 ba ileng ba kopana le melao ea SCID-I bakeng sa ho itšetleha ka cocaine. Sehlopha sa taolo se ne se e-na le lithuto tsa 20 tse se nang histori ea ho itšetleha ka cocaine ebile ha ho na mafu a mangata a ho hlahloba mafu a kelello. Bafosi bohle ba ile ba shoa ka tšohanyetso linthong tse neng li se na tšusumetso e tobileng liphateng tsa boko. Lihlopha li ne li lekanngoa bakeng sa lilemo tse boima, ho lieha ha sehatsetsing, le pH. Bakeng sa lihlooho tsohle, li-autopsies tsa kelello li ile tsa etsoa joalokaha ho hlalositsoe pele (Dumais et al., 2005), ho re fa monyetla oa ho fumana lintlha tse qaqileng tsa boitsebiso litabeng tsa kelello le tsa bongaka, hammoho le lintlha tse ling tse amanang le tsalaki le tsa sechaba. Ka bokhutšoanyane, moqoqo o koetlisitsoeng o entse joalo Tlhahlobo ea Kliniki e hlophisitsoeng bakeng sa DSM-IV Mathata a kelello (SCID-I) a nang le e le 'ngoe kapa tse ling tse ngata tsa mofu. Lekala la lingaka li ile tsa hlahloba litlhahlobo tsa SCID-I, litlaleho tsa liketsahalo, lintlha tsa coroner, le litlaleho tsa bongaka bakeng sa ho fumana tumellano ea mafu a mafu a kelello.

Hlahloba 6: Chromatin Immunoprecipitation bakeng sa Rat NAc (Setšoantšo sa 3A-C)

Batho ba baholo (libeke tsa 8) likokoanyana tsa banna li ne li fanoa ka 10 mg / kg cocaine kapa koloi ea saline IP hang ka letsatsi ka matsatsi a supileng. 24 hr ka mor'a hore e entsoe ka lekhetlo la ho qetela, NAc shell le mantlha li ne li nyenyane haholo. Chromatin immunoprecipitation (ChIP) e ne e etsoa ka mahlakoreng a mabeli a NAC majoe a khetla kapa a mantlha ho tloha ho likhoto tse supileng ka sehlopha se le sehlopha sa 14 (liphoofolo tsa 98 kaofela, matamo a 7, li-saline tsa 7). Lihlahisoa li ne li kopantsoe, li hlatsuoa li bile li bolokoa ho -80 ° C ho fihlela ho khabisoa ha chromatin ka sonication. Chromatin ea letsoai e ne e koahetsoe ka bosiu bo bong ka li-antibodies tse neng li e-na le lihlahisoa tsa magnetic (Dynabeads M-280, Invitrogen). IgG e se nang sesole sa 'mele e sebelisitsoe e le taolo. Ka morao ho hokahanya le ho hloekisa DNA, qPCR e ne e sebelisetsoa ho lekanya maemo a CaMKIIa promoter DNA. Litsebi li ne li etselitsoe ho matlafatsa sebaka se nang le sephetho sa tumellano sa AP-1 se fumanehang ~ 450 bp pele ho sebaka sa ho qala ho ngolisa (Tsoela pele: ACTGACTCAGGAAGAGGGATA; Ho fapana le: TGTGCTCCTCAGAATCCACAA).

Setšoantšo sa 3

Setšoantšo sa 3

Sehlopha sa sele-le sebaka se khethehileng sa DFFBB sa CaMKIIA Ka vivo

Hlahloba 7: Ho lekanya CaMKII Transcription le Protheine Expression ka Cell-Type-Specific ΔFosB Ho tlōla tekano (Setšoantšo sa 3D)

Litoeba tsa bitransgenic tsa banna tse tsoang ho NSE-tTA (mohala A) × TetOp-ΔfosB (line 11) le NSE-tTA (line B) × TetOp-FLAG-ΔfosB (line 11) litoeba (Chen le al., 1998; Kelz et al., 1999; Werme et al., 2002; Zachariou et al., 2006) li ile tsa emoloa le ho hōlisetsoa 100 μg / ml doxycycline ho thibela polelo ea ΔFosB nakong ea ntshetsopele. Littermates li arotsoe ka ho roka: halofo e sala e le doxycycline 'me halofo e fetotsoe metsing,' me liphoofolo li sebelisitsoe 8 libeke tse 11 hamorao ha liphello tsa ho ngolisoa ha DFB li lekanngoa haholo (Kelz et al., 1999; McClung le Nestler, 2003). Bakeng sa lipatlisiso tsa tatellano, litoeba li ile tsa senyeha ka potlako, 'me boko ba tlosoa' me ba behoa holim'a leqhoa. Liphetoho tsa NAc li nkiloe ka kotlolo ea nale ea 14-gauge 'me ka potlako li nahane ka leqhoa le omeletseng ho fihlela RNA e ntšoa. RNA ho itšehla thajana, qPCR, le tlhahlobo ea lits'ebeletso li ile tsa etsoa joalokaha ho hlalositsoe pele (LaPlant et al., 2009). Ka bokhutšoanyane, RNA e ne e arohane le TriZol reagent (Invitrogen), e hloekisitsoeng le RNAeasy micro kit ho Qiagen, mme e hlahloba boleng le Agilent's Bioanalyzer. Phetolelo e fapaneng e ne e etsoa ka Script (BioRad). qPCR e ne e etsoa ka mokhoa oa Applied Biosystems 7900HT RT PCR ka mekhahlelo e latelang: 10 min ho 95 ° C; Metsotso ea 40 ea 95 ° C bakeng sa 1 min, 60 ° C bakeng sa secane ea 30, 72 ° C bakeng sa secane ea 30; mocheso o nang le marang-rang oa 95 ° C ho hlahisa mahlakore a khethollang bakeng sa tiiso ea lihlahisoa tsa PCR tse se nang molekane. Lipatlisiso tsa immunohistochemical tsa DFFB le CaMKIIa protheine li ne li etsoa joalokaha ho hlalositsoe Leka 4.

Hlahloba 8: Litholoana tsa Intra-NAc D1 le D2 Dopamine Receptor Antagonists ho liphetoho tsa protheine tsa Cocaine-Mediated (Setšoantšo sa 3H)

Batho ba baholo (libeke tsa 8) likokoanyana tsa banna li ne li fanoa ka 10 mg / kg cocaine kapa koloi ea saline (sehlopha "koloi") IP hang ka letsatsi ka matsatsi a supileng. Metsotso ea 30 pele ho ente e 'ngoe le e' ngoe ea cocaine, likhofu e ne e le IP e tsamaisoang ke mohanyetsi oa D1 ea amohelang mofuthu SCH 23390 (0.5 mg / kg, "sehlopha sa D1"), kapa mohanyetsi oa D2 eticlopride (0.5 mg / kg, "D2 Ant" sehlopha) , kapa jenete ea ho laola saline (sehlopha sa "cocaine"). 24 hr ka mor'a ho hlajoa ha ho qetela, liphoofolo li ile tsa senyeha 'me liprotheine li lekanyelitsoe ke ho thiba Bophirimela ho latela Leka 2.

Hlahloba 9: Litholoana tsa AAV-Mediated ΔFosB e fetisisang tlhaloso e hlalosang mantsoe a Protheine (Setšoantšo sa 4 A-C)

Ho buuoa ke stereotaxic ho ne ho etsoa ka likhoto tse kholo tsa banna (libeke tse 8) ho kenya AAV-GFP (protheine e tala ea fluorescent) kapa AAV-GFP-ΔFosB (Maze et al., 2010). Li-needle tsa 33 (Hamilton) li ne li sebelisoa bakeng sa ho buuoa hohle, nakong eo 0.5 μl ea kokoana-hloko e hloekisitsoeng e phahameng e neng e hloekisitsoe ka makhetlo a mararo ka nako ea 5, e lateloa ke nako e eketsehileng ea phomolo ea 5 ka morao. Libaka tsohle li lekantsoe ho latela Bregma: angle ea 10, AP = + 1.7 mm, Lat = 2.5 mm, DV = -6.7 mm. Matsatsi a 14 ka mor'a ho buuoa, liphoofolo li ile tsa fuoa eng e le 'ngoe ea IP e entsoeng ka 10 mg / kg cocaine ka likamore tsa ho lekola ho lokisa liketso tsa boitšoaro ba DFF. 24 hr ka mor'a ho kenya koana ena ea ho qetela, likhofu li ile tsa senngoa ka ho feletseng Leka 2, le microdissection ea maiketsetso e ne e etsoa tlas'a tataiso e nyenyane ea fluorescence ho fumana GFP-positive NAc lisele. Tloso ea Bophirimela e ne e sebelisoa ka nako e le 'ngoe Leka 2.

Setšoantšo sa 4

Setšoantšo sa 4

DFFB e hlokahala ebile e lekaneng bakeng sa tlhahiso ea CaMKIIa e amohelehang ea coxine e fumanehang ka cocaine ho shell ea NAc

Hlahloba 10: Litla-morao tsa AAV-Mediated OJunD Overexpress on Expression ea Protheine e Tšepahalang ea Cocaine (Setšoantšo sa 4 D-F)

Injection ea Stereotaxic ea AAV-GFP kapa AAV-GFP-ΔJunD e ne e etsoa ka ho ea ka Leka 8. Matsatsi a 14 ka mor'a ho buuoa, liphoofolo li ne li tsamaisoa 10 mg / kg cocaine kapa koloi ea saline IP hanngoe ka letsatsi ka matsatsi a supileng ka ho lokisa likamore. Likarabo tsa libaka tsa tlhaho li hlahisa li-cocaine (5 mg / kg IP) kapa saline e tlalehiloe. 24 hr ka mor'a ho kenya koana ena ea ho qetela, likhoto li ile tsa senyeha, ho kotuloa lisele le Bophirimela tsa Bophirimela tse kang Leka 9.

Leka 11: In Vitro Protein Kinase Assays (Setšoantšo sa 5A-D)

Recombinant CaMKIIa le DFFB li ile tsa hloekisoa liseleng tsa likokoanyana (Brickey et al., 1990; Jorissen et al., 2007), le liteko tsa protheine kinase li ile tsa etsoa (Colbran, 1993), joalokaha ho hlalositsoe pele. Ka bokhutšoaane, CaMKII e ne e etsoa ka leqhoa ka leqhoa le 2.5 μM (kapa mohopolo o bontšitsoeng) oa DFFB, 1 mM Ca2+, 40 mM Mg2+, 15 μM khutsufatso, le 200 mM HEPES pH 7.5. Phosphorylation e qalile ka ho phaella ha 200 μM ATP kapa ntle le [γ-32P] ATP mme a lumelloa ho tsoela pele bakeng sa minara ea 10 ka mocheso oa kamore (Setšoantšo sa 5A & B) kapa 2 min ka leqhoa (Setšoantšo sa 5C & D.). Mesebetsi e ile ea rarolloa ke ho thibela Bophirimela (Setšoantšo sa 5A & B) kapa ka ho bala 'meleng oa li-autoradiogram (Fig. B-D).

Setšoantšo sa 5

Setšoantšo sa 5

DFFB ke karolo e matla ea CaMKIIA

Leka 12: Boitsebiso ba Ser27 ΔFosB Phosphorylation (5E ea setšoantšo)

In vitro litase tsa kinase li ne li etsoa ka nako Leka 11, liprotheine li ne li arohanngoa ke SDS-PAGE, 'me lihlopha tse lekanang le DFBB li ne li khaoloa' me li ne li lekanngoa liphatlalatso tse ngata. Likabelo tsa m / z tsa likaroloana tsa ion tse tsamaisanang le likarolo tsohle li ngotsoe ka holim'a litlhōrō tsa ion. Hase litšobotsi tsohle tse ngotsoeng ka lebaka la meeli ea sebaka. Ka kakaretso, litemana tsa li-ion tsa fragment li na le mebala e lefifi ntle le ha li tiisa ka ho toba kapa li eketsa bopaki bo teng ho libaka tsa phosphorylation tsa thahasello, mohlomong li tšoauoa ka khubelu. Bopaki ba lihlahisoa tsa ho arohana ha masapo li hlahisoa ho latellana ho bala phosphopeptide le sebaka se fumanoeng sa libaka tsa phosphorylation tse bontšitsoeng ka khubelu ka lebitso le le leng la amino acid. Tlhaloso ea linomoro tsa ion ea fragment e hlokometsoeng e boetse e tšoauoa ka tatellano ea peptide e le b le y ions. Lintlha tsa ho hlahisa likarolo tsa m / z lihlahisoa tsa ho bontša li-ion tse fokolang tse matla li tšoauoa ka holim'a setho ka seng sa masapo a maholo. Li-ions tsa fragment tse bontšitsoeng ka lekhahla H li tiisa boteng ba serform ea ser27 phosphorylated, leha ho le joalo, ka motsoako oa li-isoform tse ling tsa phosphorylated libakeng tsa Ser28, Ser31, Ser34 le Thr37. Ho ba teng ka pa5, pa5-P, pb5, le pb5-P ions ka ho khetheha ho netefatsa phosphorylation ea masala a Ser27.

Hlahloba 13: Quantification ea serxNUMX phosphorylation (Setšoantšo sa 5F)

Li-peptide tse tloaelehileng li ne li etselitsoe ho etsisa mefuta ea phospho le e seng phospho ea Ser27 ΔFosB. Ka mor'a ho qaptjoa le ho hloeka, peptide e 'ngoe le e' ngoe e "boima" idiotypic e ile ea qhibiliha ka har'a mochine oa metsi a 50 / 50 e leng acetonitrile / metsi 'me ea romeloa bakeng sa tlhahlobo ea amino acid ho fumana hore na ho na le tharollo e feletseng holim'a sesebelisoa sa peptide ea sesebelisoa. Peptide e 'ngoe le e' ngoe e boima 'e ne e kenngoa ka ho toba ka sesebelisoa sa masole a 4000 QTRAP (MS) ho fumana matla a ho qhibiliha ka matla bakeng sa ho arohana ha MS / MS le liphetoho tse peli ho isa ho tse' nè tsa MRM. Ka mor'a moo, li-peptide tse "mahetla" tse makhethe li ne li behoa LCMS ho 4000 QTRAP ho tiisa hore peptide e arohane. Sesebelisoa se ne se tsamaisoa mocheng oa boraro oa quadrupole, ka Q1 e behiloeng ho bohlokoa ba pele-pele m / z (Q1 ha e hlahlobe), 'me Q3 e behiloe ho bohlokoa ba m / z e lekanang le sekhechana se itseng sa peptide. Mokhoa oa MRM, letoto la likarolo tse se nang moeli (liphetoho tsa pele-pele / likhechana moo matla a ho thulana a sebetsanang le ho matlafatsa matla a sekhechana sa thahasello) a lekantsoe ka sequentially, 'me potoloho (ka tloaelo ea 1-2 sec sec) e ne e lokolotsoe hohle nako eohle ea ho arohana ha HPLC. Liphetoho tsa MRM li ile tsa khethoa ho tloha ho MS / MS palo ea li-peptide tse teng. Liphetoho tse peli bakeng sa peptide, tse lumellanang le li-ions tse matla haholo, li ile tsa khethoa 'me matla a ho thulana a ntlafatsoa ho matlafatsa matla a pontšo ea liphetoho tsa MRM ho sebelisa software e iketsang. Litlhōrō tse bakoang ke li-peptide tse tloaelehileng le litekanyetso tsa ΔFosB tse pepesehileng ho CaMKII kapa ho laola li ile tsa bapisoa le ho lekanyetsa boholo ba palo e 'ngoe le e' ngoe ea peptide ka mokhoa oo. Tlhahlobo ea lintlha mabapi le LC-MRM data e etsoa ho sebelisa AB Multiquant 1.1 software.

Hlahloba 14: Khothaletso ea DFFB ho Lik'hamphani tsa Manonyeletso tsa CaMKII (Setšoantšo sa 5G & H.)

Manyolo a Transgenic a hlokomolohang T286D CaMKII (Mayford et al., 1996; Kourrich et al., 2012) le littermates tsa mofuta o hlaha li ile tsa hōlisoa ka ho ba sieo ha doxycycline ho lumella tlhaloso ea bohata. Litoeba tsa batho ba baholo li ne li tsamaisoa ka 20 mg / kg cocaine kapa saline IP hang ka letsatsi bakeng sa matsatsi a 14. 24 hr ka mor'a ho hlajoa ha ho qetela, liphoofolo li ile tsa senyeha le immunohistochemistry le quantification ea polelo ea ΔFosB e ne e etsoa ka Leka 4.

Hlahloba 15: Tlhahisoleseding ea HSV-Mediated ΔFosB Ho tlosoa ho tlōla le ho thibela CaMKII ho NAC Dendritic Spines (Setšoantšo sa 6A-E)

Litoeba tsa banna ba baholo (libeke tsa 8) li ne li kenngoa ka stereotaxically ka NAc le HSV-GFP, HSV-GFP-ΔFosB (Olausson et al., 2006), HSV-GFPAC3I, kapa HSV-GFPAC3I -ΔFosB. Ha li hahiloe, AC3I, e nang le inhibitor e thehiloeng ho peptide ea mosebetsi oa CaMKII, e kopanngoa le C-terminus ea GFP. GFPAC3I e ne e khethoa ke PCR e sebelisa pMM400-vector e nang le GFPAC3I e le setšoantšo se nang le lintlha tse latelang: GFP-AC3I-F: 5 'CC GCTAGC GCCGCCACC ATGGTGAGCAAGGGCGAGGAGCTGT 3' (clampNheIKozakmet); GFP-AC3I-R: 5 'CC TCCGGA TTACAGGCAGTCCACGGCCT 3' (clampBspEIstop). Sehlahisoa se entsoeng ka PCR se kenngoa litsing tsa p1005 + le p1005 + -Δ FosB tse sebelisa li-NheI le BspEI. Mohaho ona o tiisitsoe ka ho hlophisa. Li-coordinate tsa Stereotaxic e ne e le: 10 ° angle, AP = + 1.6 mm, Lat = + 1.5 mm, DV = -4.4 mm (Barrot et al., 2002). Ho senyeha ha maikutlo le ho aroloa ha boko ho ne ho etsoa ka nako Leka 4.

Tlhahlobo ea lesapo e ne e etsoa joalokaha e hlalositsoe (Christoffel et al., 2011). Ka bokhutšoanyane, likarolo tsa dendritic 50-150 μm hole le soma li khethiloe ka mokhoa o khethollang lisele tse nang le tšoaetso ea HSV tse hlalosang GFP. Litšoantšo li ile tsa fumanoa ka LSM 710 (Carl Zeiss) ea confocal bakeng sa tlhahlobo ea morphological sebelisa NeuronStudio le rayburst algorithm. NeuronStudio e khetholla li-spines e le tse tšesaane, li-mushroom kapa litlhaka ho latela litekanyetso tse latelang: (1) sekhahla sa karolo, (2) sekhahla sa hlooho le molala, le (3) hlooho ea bophara. Ho hlajoa ka molala ho ka hlalosoa e le bofubelu kapa bo-mushroom, 'me ba se nang molala o boima ba khetholloa e le likoti. Ho hlajoa ka molala ho thoe ke tšesaane kapa li-mushroom tse thehiloeng hloohong.

Setšoantšo sa 6

Setšoantšo sa 6

Thibelo ea mosebetsi oa CaMKII e thibela liphello tsa morphological le boitšoaro ba DFFB ka NAC

Hlahloba 16: Litla-morao tsa HSV-Mediated ΔFosB Ho tlosoa ho feteletseng le CaMKII Tšitiso ea Likarabo tsa Cocaine (Setšoantšo sa 6F)

Litoeba tsa banna ba baholo li ne li kenngoa ka likokoana-hloko ka holimo Leka 15, le likarabo tsa motlakase ho lejoe le le leng la 5 mg / kg la cocaine le ne le lekanyetsoa ka ho lekaneng Leka 9. Lintlha tsa boitsebiso ba libaka li hlahisoa e le likhetho tse fokolang ka ho fetisisa ho feta 30 min ka mor'a ente ea cocaine.

Tlhahisoleseling e 'Ngoe

Mehaho ea Bolulo

Masapo a banna ba Sprague Dawley (250-275 g; Charles River Laboratories) a ne a lula ka bobeli. Mantsoe a banna ba robeli a libeke tse robeli a C57BL / 6J (The Jackson Laboratory) e ne e le sehlopheng se nang le liphoofolo tse fetang tse hlano ka cage. Liphoofolo tsohle li ne li tloaetse ho ea setsing sa liphoofolo bakeng sa ≥1 beke pele ho litekanyetso tsa liteko le ho lula likamoreng tse laoloang ke mocheso (23-25 ° C) ka 12 hr leseli / lefifi (li khantša 7: 00 AM) le ho fumana lijo le metsi ad libitum. Liteko li ile tsa etsoa ho latela tataiso ea Mokhatlo oa Neuroscience le komiti ea tlhokomelo ea liphoofolo le ea ho sebelisa (IACUC) Thabeng ea Sinai.

lithethefatsi

Lithethefatsi li ne li tsamaisoa IP le ho qhibiliha ka saline e nyopa, ho akarelletsa cocaine (5-20 mg / kg ka 10 μl bakeng sa litoeba, ka 1 ml bakeng sa likhoba, NIDA) le SCH 23390 kapa eticlopride hydrochloride (0.5 mg / kg ka 1 ml, Tocris) . Bakeng sa opereishene ea stereotaxic, litoeba li ne li haelloa ke "lijo" tsa ketamine (100 mg / kg) le xylazine (10 mg / kg) (Henry Schein) ka saline e nyopa.

Li-antibodies

CaMKIIa (kakaretso): Ka holimo ho 05-532, 1: 5,000

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

DFFB (kakaretso): Pontšo ea seleng 5G4, 1: 250

ΔFosB phospho-Ser27: Phosphosolutions, 1: 500

GluA1 (kakaretso): Moekameli, AB31232, 1: 1,000

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

GluA1 phospho-Ser845: Chemicon AB5849, 1: 2,000

GluA2: Millipore 07-598, 1: 2,000

NR2A: Sigma HPA004692, 1: 2,500

NR2B: Millipore AB1557P, 1: 1,000

Lipatlisiso tsa Statistical

Lipalo-palo tsohle li ne li etsoa ho sebelisa sephutheloana sa software ea Prism 6 (GraphPad). Liteko tsa liithuti li ne li sebelisetsoa ho bapisa lintho tse peli tse bonts'itsoeng (ho bontšoa Liphello moo ho bohlokoa ho fanoang), 'me tsela e le' ngoe li-ANOVA li ne li sebelisetsoa ho bapisa lintho tse ngata (ho bontšitsoe likarolong tsa liphello moo ho fumanoa phaello ea F).

Eya ho:

Results

K'homphieutha e sa foleng ea Cocaine CaMKII ho NAc Shell

Liphuputso tse ngata li bontšitse hore li-MSN li-shell tsa NAc le tsa mantlha li na le likarabo tse fapaneng tsa tlhaho ea likokoana-hloko le tsa tlhaho ho pepesehela lithethefatsi tsa tlhekefetso (Kourrich le Thomas, 2009; Loweth et al., 2010) le hore lihlopha tse peli tsa metse e meholo li laola mekhoa ea ho batla lithethefatsi (Ito et al., 2004). Ho khetholla liphello tse fapaneng tsa cocaine ho liprotheine tsa NAc shell vs. mantlha, re sebelisitse Matšoao a Isobaric (Multiplexed Tagging Tagging) (iTRAQ) le setšoantšo sa maqhubu a mangata (MS / MS). Li-rats tse kholo li ne li kenngoa ka IP le cocaine (20 mg / kg) kapa letsatsi le letsatsi bakeng sa matsatsi a 7; 24 hr ka mor'a hore e entsoe ka lekhetlo la ho qetela, NAc shell le mantlha li ne li nyenyane haholo (Setšoantšo sa 1A) 'me e khanyelitsoeng ka serame. Liprotheine likhethong tsena li ne li sebelisoa ka ho sebelisa iTRAQ. Li-CaMKII tse 'nè li-isoform li bonts'a keketseho e kholo ea polelo ka mor'a phekolo ea cocaine e neng e totobetse ho shell ea NAc ha e bapisoa le meholo. Liprotheine phosphatase tse ngata, tse kenyelletsoang li-subunits tsa PP1 le tsa tsamaiso le PP2A, tse neng li kile tsa amahanngoa le litšebeletso tse sa tšoaneng tsa CaMKII mekhoeng e meng (Colbran, 2004), o latela mohlala o tšoanang. Liphuputso tsena li fane ka bopaki bo sa nepahalang bo bontšang hore tsela ea ho tiisa ka CaMKII e laoloa haholo ke cocaine e NAc ka tsela e khethehileng ea khetla.

E le ho tiisa hore sena se fumanoe haholo, re ne re tšoara likhoho tse boletsoeng ka holimo ka cocaine (ka tekanyo e fapaneng) kapa likarabo tsa mokokotlo oa lik'hemik'hale (5 mg / kg) kapa tekanyo ea phephetso ea saline. Ho pepeseha ha 10 mg / kg cocaine ho ile ha fella ka mokhoa o tloaelehileng oa ho hlohlelletsa batho ba nang le lisebelisoa (Setšoantšo sa 1B). Liphuputso tse ling le tsamaiso ena ea li-dosing li senoletsoe, ka tšebeliso ea ho thibela Bophirimela, hore cocaine e pheta-phetoang e etsa hore CaMKIIa e khethoe ka NAc shell 24 hr ka mor'a hore ho hlajoe kocaine ea ho qetela (Setšoantšo sa 1C le D;; p = 0.0019; F = 7.943; df = 29). Ho phaella moo, phosphorylation ea submonate ea CaMKII ea SerxNUMX ea seboka sa GluA831 ea receptor ea AMPA e ile ea eketseha ka NAc shell 'me e se ea bohlokoa (p = 1; F = 0.0261; df = 4.208), ha CaMKIIA Thr28 autophosphorylation e na le matla empa e sa matla mekhoa ea bohlokoa ho kenngoa ka har'a shell feela (Setšoantšo sa 1D). Li-receptor tse ling tse 'maloa li ne li sa amehe. Ho fapana le litekanyo tsena tsa CaMKII, lisebelisoa tse tšoanang tsa lisele li bontsitsoe ho hlahisoa ha ΔFosB ka khetla ka bobeli (p = 0.0260; F = 4.189; df = 29) le mantlha (p = 0.0350; F = 3.807; df = 29) ea NAc (Setšoantšo sa 1C le D), tumellanong le liphuputso tse fetileng (Perrotti et al., 2008).

Kaha lipatlisiso tse ngata tsa pele tsa phekolo ea cocaine ea li-receptors tsa AMPA li hlahlobisisa liphoofolo ka mor'a hore ~ ​​matsatsi a 14 a tlohe ho cocaine e sa foleng (bona Tlhaloso), re pheta lintlha tsa tlhahlobo ea likokoana-hloko tsena nakong ena. Re fumane hore, matsatsi a 14 ka mor'a ho kenngoa ha cocaine ka morao, ΔFosB e ntse e phahame ho NAc (p = 0.0288; F = 4.258; df = 22), athe ha ho na CaMKII kapa phosphorylation ea GluA1 Ser831 e ntseng e eketseha (1E ea setšoantšo). Leha ho le joalo, 1 hr ka mor'a tekanyo e le 'ngoe ea 10 mg / kg tekanyetso ea cocaine, litekanyetso tsa CaMKII eohle (p = 0.0330; F = 3.947; df = 26) le GluA1 Ser831 (p = 0.0213; F = 4.509; df = 27) phosphorylation ka bobeli e phahame ho isa tekanyong e tšoanang le e fumanoeng ka mor'a ho pepeseha ha cocaine ea pele e sa foleng (1E ea setšoantšo). Litlaleho tsena li bontša hore NAC shell neurons e amoheloa bakeng sa tlhahiso ea CaMKII nakong ea nako e telele ea ho ikhula, mohlomong ka tsela e tobileng ea mofani oa liphatsa tsa lefutso tsa CaMKII (bona Puisano). Ho feta moo, taba ea hore ΔFosB ho qobelloa ho phehella ho feta phetolelo ea CaMKII e fana ka maikutlo a hore ho na le mekhoa e eketsehileng, ebang ke ea chromatin e thehiloeng kapa e seng joalo, e etsang "ho senya" molaong oa CaMKII, joalokaha o koahetsoe Puisanong.

E le hore re matlafatse litlhaloso tsena, re ile ra hlahloba mefuta e meng ea k'hok'heine ea boipheliso, e akarelletsang ho kenella lithethefatsi ka boithaopo. Li-rats tsa banna ba baholo li ile tsa fanoa ka nako e telele kapa e telele ho fumana cocaine; joalokaha ho lebeletsoe (Ahmed le Koob, 1998), maemo a mang a nako e telele a fumanehang a lebisitse ho eketseha ha botho ba lithethefatsi (Setšoantšo sa 2A). DFF e ile ea susumelletseha ho feta nako e telele vs. ho kena ha cocaine ka nakoana ho NAc shell (p = 0.0011; F = 11.12; df = 17) le mantlha (p = 0.0004; F = 13.86; df = 17). Ka lehlakoreng le leng, CaMKIIA e ile ea kenngoa ho NAc shell feela ka ho fumana nako e telele ea cocaine (Setšoantšo sa 2B le C;; p = 0.0236; F = 4.957; df = 16). Hoa thahasellisa ho bapisa ka kakaretso tlhahiso ea cocaine letsatsi le leng le le leng ho liphoofolo tse fokolang ho fihlela (~ 12 mg / kg IV), liphoofolo tse fihlang nako e telele (~ 70 mg / kg IV), le liphoofolo tse tsamaisoang ke experimenter (10 mg / kg), le botsa hore na ke hobane'ng ha ho qetela ho etsa hore DFFB le CaMKII e kenelle ka matla, athe ho fokotsa nako e khutšoanyane ha ho joalo. Phapang ena e ka 'na ea bakoa ke ho fapana ha tlhōrō ea cocaine (tekanyetso ea cocaine ea experimenter e fanoa e le mofuta o le mong oa IP, athe cocaine e fanoang ka boeena e fanoa ka mefuta e mengata ea lithethefatsi), kapa ka ho fapana ha bolelele ba lithethefatsi (matsatsi a 7 bakeng sa experimenter tsamaiso, matsatsi a 19 bakeng sa boithati).

Ho sa tsotellehe lingoliloeng tse kholo tsa DFFB le CaMKII ka khato ea cocaine, ha ho na liphuputso tsa liprotheine tsena ho basebelisi ba batho ba cocaine. Mona, re fana ka bopaki ba pele ba hore ΔFosB (p = 0.0316; t = 1.921; df = 34) le CaMKII (p = 0.0444; t = 1.755; df = 32) li eketseha ho NAc ea batho ba itšetlehileng ka cocaine (Setšoantšo sa 2D, Lethathamo 1). Litlaleho tsena li bontša hore tlhahlobo ea rona ea ΔFosB le CaMKII tlhahiso ea k'hok'heine e nang le likhohlopo tsa NAc ke ea bohlokoa bakeng sa ho lemalla k'hok'heine ea batho.

Lethathamo 1

Lethathamo 1

Ho khetholla likarolo tse tsoang ho batho ba lemaletseng k'hok'heine le ho tšoana le sehlopha sa taolo

ΔFosB e Laola K'homphieutha ea CaMKII ka Sekhetho sa D1-MSN ea NAC Shell

Ho fumanoa hore ka bobeli CaMKII le ΔFosB ba hlophisitsoe ke cocaine ho NAC ea molamu o re lebisitse ho bona hore na ΔFosB e ka 'na ea laola ho ngoloa ha liphatsa tsa lefutso tsa CaMKII. Re ne re kile ra tlaleha CaMKIIA e le sepheo se ka khonehang bakeng sa ΔFosB tlhahlobo ea microcaroud e sa lebelloang ea NAC (McClung le Nestler, 2003), empa ho fumanoa ha sena ha hoa ka ha hlokomeloa le ho feta thutong eo. Re qalile ho sebelisa ChIP e ngata (qChIP-ChIP e lateloang ke PCR e ngata) ho fumana hore na ΔFosB e tlamella mofani oa liphatsa tsa lefutso la CaMKIIa ho NAc ea likhoto tse kholo tsa banna, 'me o fumane ka mokhoa o tsotehang hore ho tlamisoa hona haholo, ka tsamaiso e sa foleng ea cocaine, p = 0.0133; t = 2.901; df = 12), empa ha se motheo, metse (Setšoantšo sa 3A). Ho tsoela pele ho utloisisa mekhoa e amanang le phapang ena e fapaneng le e fapaneng le DFFB e tlamella mohlophisi oa CaMKIIa, re sebelisitse qChIP ho hlalosa maemo a histone sebakeng sena sa genomic. Liphuputso tse qalileng li bontšitse tlhahiso ea cocaine e entsoeng ka H3 acetylation ho CaMKIIa mothatsong ka ho feletseng mouse mouse NAc (Wang et al., 2010). Ka lehlakoreng le leng, re fumane hore cocaine e fokotsa H3 acetylation ho CaMKIIa moetlisisi ka khetho ea NAC (Setšoantšo sa 3B;; p = 0.0213; T = 2.726; df = 10), ho se na phetoho e totobetseng ka khetla, e lumellanang le liphetoho tse itseng tsa chromatin tse ka tlaase ho ΔFosB tse tlamang. qChIP bakeng sa letšoao le khathatsang, dimethylated H3 lysine 9 (H3K9me2), mekhoa e senotsoeng ea ho fokotseha ho khetla le lihlopha tse ka sehloohong (Setšoantšo sa 3C).

Ho fumana hore na ΔFosB e laola ho ngolisoa ha CaMKIIa Ka vivo, re sebelisitse mela e 'meli ea bitransgenic mouse e inducibly overexpress ΔFosB ka ho toba D1 vs. Li-MSN tsa mofuta oa D2 ka tsela e laoloang ke tsamaiso ea doxycycline ka metsi a nooang (Chen le al., 1998; Kelz et al., 1999; Werme et al., 2002). Litlhapi tse kholo tsa banna tse hlahisang maikutlo ΔFosB feela li-MSNs tsa mofuta oa D1 li ne li ekelitse haholo likarolo tsa CaMKIIA mRNA ka NAc (p = 0.0337; t = 1.996; df = 13), phello e sa bonngoeng litlameng tse ngata ΔFosB haholo-holo li-MSNs tsa D2 (Setšoantšo sa 3D). Ho eketseha ha CaMKIIA mRNA, e bakoang ke polelo ea ΔFosB ka MSNs ea mofuta oa D1, e ne e tsamaisana le keketseho e khotsofatsang ea protheine ea CaMKIIa ka bobeli NAc shell (p = 0.0030; t = 3.578; df = 14) le meholo (p = 0.0392; t = 2.275; df = 14; Lifeiga tsa 3E le F). Litlaleho tsena li bonts'a hore ΔFosB e khona ho khanna CaMKIIa liphatsa tsa lefutso ho li-MSNs tsa D1 lihlopheng tse peli, le hoja Setšoantšo sa 3B e fana ka maikutlo a hore chromatin e sebelisanang le k'hok'heine e fetoha moetlisisi oa CaMKIIa (mohlala, ho fokotsa acetylation) ho thibela ΔFosB ho phahamisa CaMKII mohahong o ka sehloohong kamora cocaine.

Hobane tlhaiso-leseling ea rona ea mouse ea transgenic e bonts'a hore ΔFosB e kenyellelitsoeng ka polelo ea liphatsa tsa lefutso ea CaMKII e totobetse ho li-MSN tsa mofuta oa D1 ho NAc, ka mor'a moo re ile ra batla ho tseba hore na ts'ebeliso e ts'ehetsoeng ke cocaine ea CaMKII e hloka ts'ebetso ea D1 dopamine receptor. Likhoto tsa banna ba baholo li ne li fuoa k'hok'heine e sa foleng kapa letsoai joaloka pele, empa metsotso e 30 pele ho ente e 'ngoe le e' ngoe, likhoto sehlopheng sa cocaine li ile tsa fuoa ente ea IP ea saline, mohanyetsi oa D1 SCH 23390 (0.5 mg / kg), kapa mohanyetsi oa D2 ea amohelang eticlopride (0.5 mg / kg). Liphoofolo li ile tsa hlahlojoa 24 hr kamora ente ea ho qetela ea cocaine. Ho hlakola Bophirimela ho senotse hore D1, empa eseng D2, mohanyetsi o thibetse ka botlalo keketseho e kopantsoeng le cocaine ho ΔFosB (p <0.0001; F = 18.96; df = 18), joalo ka ha ho tlalehiloe pejana (Nye et al., 1995), hammoho le CaMKII (p = 0.0005; F = 10.99; df = 18; Setšoantšo sa 3G le H). Ts'ehetso ea data ena khopolo-taba ea hore cocaine e kenyelletsa ho eketsa mohopolo oa ΔFosB mofuteng oa liphatsa tsa CaMKII ka ho khetheha ho MSNs ea D1 ea mofuta oa NAc shell. Ho tla ba bohlokoa nakong ea lithuto tsa morao-rao ho bontša ka mokhoa o hlakileng mokhoa ona o khethehileng oa cocaine ka polelo ea CaMKII ka har'a sebaka sena sa boko.

DFFB ke Nako ea Bohlokoa le e Lekaneng Bakeng sa ho Koaheloa ke Cocaine ea CaMKII ka NAC Shell

Ho tlatsetsa tšebeliso ea litoeba tsa bitransgenic, re ile ra latela karolo ea DFFB ho kenya letsoho ho kenya li-cocaine tlhahiso ea CaMKIIa ka tšebeliso ea liphatsa tsa lefutso tsa kokoana-hloko ea likokoana-hloko ka likhoto. Re na le likaroloana tse peli tsa adeno-associated likokoana-hloko (AAV) ho NAc shell ea liphoofolo tse kholo tsa banna (moo khetla e ka khetholloang ka ho khetheha) ho feta tekano ΔFosB hammoho le GFP kapa GFP feela. Ka nako eo liphoofolo li ile tsa fuoa sejo se le seng sa IP sa 10 mg / kg cocaine. Liphoofolo tse ngata ho hlahisa maikutlo ΔFosB / GFP li bontšitse karabo e ntseng e eketseha ea likarolo ha li bapisoa le liphoofolo tse hlahisang GFP feela (Setšoantšo sa 4A). 24 hr ka mor'a enta e le 'ngoe ea cocaine, lihlahisoa tsa GNP tse nang le GFP li ne li khahlisoa ke liphoofolo tsena ka ho qhalana mohloli oa mohloli o khanyang oa fluorescent. Bophahamo ba Bophirimela ba lisele tsena (Setšoantšo sa 4B le C) e ile ea senola matla a DFFB le ho eketseha ha kakaretso ea CaMKIIa protheine ho bapisoa le liphoofolo tsa GFP (p = 0.0070; t = 2.894; df = 30), e tšoanang le ho kenngoa ha li-cocaine tse sa foleng. Ho phaella moo, CaMKIIA autophosphorylation ho Thr286 (e bontšang ho sebelisoa ha enzyme) e ekelitsoe ke ΔFosB ho feta maikutlo (p = 0.0330; t = 2.243; df = 28), joalo ka phosphorylation ea substrate ea CaMKII, Ser831 ea GluA1 (p = 0.0540; t = 2.012; df = 28), hape e etsisa liketso tsa cocaine e sa foleng (Setšoantšo sa 1C le D). Tho bokella hammoho, lintlha tsena li fana ka bopaki ba hore tlhahiso ea ΔFosB ka NAc shell e lekaneng bakeng sa ho khothaletsa ho khothaletsa khokahano le k'hamphani ea CaMKII le ho kenya ts'ebetsong sebakeng sena.

Re sebelisitse mokhoa o tšoanang ho netefatsa hore na ΔFosB e boetse e hlokahala bakeng sa ho kenngoa ha cocaine ka har'a cocaine ka shell ea NAc. AAV e ne e sebelisetsoa ho fokotsa protheine ea JunD e nang le truncated, e bitsoang DO, e leng taolo e fosahetseng ea ts'ebetso ea phetolelo ea ΔFosB (Winstanley et al., 2007) hammoho le GFP kapa GFP feela. Libeke tse peli hamorao, ha polelo ea litloaelo e le khōlō, liphoofolo li ile tsa fanoa ka cocaine (10 mg / kg) kapa letsatsi le letsatsi bakeng sa matsatsi a 7, 'me li lekoa bakeng sa likarabo tsa likhoheli ho phephetso ea cocaine (5 mg / kg) 24 hr kamora'Setšoantšo sa 4D). Ho se ts'oanehe ha maikutlo ho thibela ho hlohlelletsoa ke locomotor ho cocaine, hape ho thibetsoe ho kenyelletsa CaMKIIa le ho kenya ts'ebetsong ka shell ea NAC (Setšoantšo sa 4E le F;; p = 0.0437; F = 2.997; kakaretso df = 38), e bontšang hore ΔFosB ts'ebetso ea phetolo e hlokahalang bakeng sa ho kenya li-cocaine-mediated induction of CaMKIIα sebakeng sena. Ho thahasellisang ke hore re fumane hore mefuta e fokolitsoeng ea DFBB tlas'a maemo a mabeli a sebelisoang ka saline le ea cocaine (p = 0.0004; F = 8.110; df = 35), e phahamisa monyetla oa hore "DFosB" e itšetlehile ka mosebetsi oa AP-1 bakeng sa maemo a eona.

CaMKII Phosphorylates DFFB ho Ser27

U sebelisa a vitro protein kinase testays, re ile ra etsa qeto ea hore DFFB e hloekisitsoeng ke karolo e matla ea CaMKIIa. Ho kenyelletsoa ha Hae6-FFBB le CaMKIIA le ATP li bakile ho fetoha holimo ho tsamaiso ea electrophoretic ea DFF (Setšoantšo sa 5A); lihlopha tse 'maloa tse hlahisoang li fana ka libaka tse ngata tsa phosphorylation. E tšoanang a vitro litase tsa kinase ho sebelisa [γ-32P] ATP e bontšitse ho kenngoa ha radiolabeled phosphate ka lihlopha tsa DFFB tse fetohileng (Setšoantšo sa 5B), ho bontša phosphorylation e tobileng ea protheine. Re hlahisitse anti-phospho-specific antibody ho Ser27 eo pele e neng e tsebahala ea ΔFosB (Ulery et al., 2006). Ha anti-opione ena e sa hlahise pontšo khahlanong le lihlahisoa tsa boko tse nang le ΔFosB ea Ser27-phosphorylated (data e sa bontšoang), re ile ra khona ho lemoha phosphorylation ea Ser27 a vitro tlhahlobo ea kinase e sebelisang CaMKII (Setšoantšo sa 5B). Tlhahlobo ea lik'hemik'hale tsa CaMKII phosphorylation ea ΔFosB e bontša hore ke substrate e matla bakeng sa kinase (Setšoantšo sa 5C), ka ho bonahala eka KM ea 5.7 ± 2.0μM le KCAT ea 2.3 ± 0.3min-1. Liphetho tsena li tšoana le tse ngata tse khethiloeng hantle Ka vivo substrate tsa CaMKII (Colbran le Brown, 2004). Ho phaella moo, re ile ra etsa qeto ea hore CaMKII phosphorylates ΔFosB e na le stoichiometry ea 2.27 ± 0.07 mol / mol (Setšoantšo sa 5D), ho bontšang hore bonyane ho na le libaka tse tharo tsa CaMKII phosphorylation ka hare ho Hae6-FFBB protein, e lumellanang le eona Setšoantšo sa 5A.

Ho etsa lipatlisiso ka libaka tsa batho ka bomong tsa phosphorylation, re sebelisitse lisebelisoa tsa MS ho rona a vitro tlhahiso ea kinase. 5E ea setšoantšo e bontša DFFB phosphorylation ho Ser27 e kileng ea tsebahala le libakeng tse 'maloa tse eketsehileng (data e sa bontšoang). Kaha re ne re khetholloa ke Ser27, re ile ra tsepamisa mohopolo ona setšeng sena ka ho hlahisa li-peptide tse entsoeng ka maiketsetso tse etsisang li-porose tsa serxNUMX tsa phospho- le nonphospho, ebe li sebelisitse boholo ba li-peptide tsena e le litekanyetso tsa MRM pele ho morao le ka morao a vitro phosphorylation ke CaMKII. Ka mor'a moo ho haelloa (Setšoantšo sa 5F) e tiisa hore Ser27 ke substrate e matla bakeng sa CaMKII. Liphello tsena li bontša hore, har'a lihlahisoa tse ngata tsa phosphorylated ka hare ho ΔFosB, Ser27 ke substrate e sebetsang haholo bakeng sa CaMKII.

CaMKII e kopana le Cocaine Ho bokella ΔFosB ho NAc Shell

Kaha CaMKII e na le phosphorylate ea DFFB a vitro sebakeng se ts'oanang se matlafatsang botsitso ba sona a vitro 'me Ka vivo (Ulery et al., 2006; Ulery-Reynolds et al., 2009), re ile ra etsa qeto ea hore na mosebetsi oa CaMKII o laola likarolo tsa FFBB ka NAc Ka vivo. Ho rarolla potso ena, re ile ra qala ka ho sebelisa moriri oa mouse ho fetisa molumo o ikemetseng oa khalmiamo oa CaMKIIA (T286D) libakeng tse ngata tsa boko bo akarelletsang NAc (Mayford et al., 1996; Kourrich et al., 2012). Re kentse likokoana-hloko tsa banna ba baholo le li-wildtype littermates tse nang le lilemo tse ngata letsatsi le leng le le leng bakeng sa matsatsi a 20, ebe re hlahloba liphoofolo letsatsi le leng le le leng ka mor'a ho kenngoa ha 'mele. Re fumane hore litekanyetso tsa basal tsa DFBB li ile tsa eketseha liphoofolong tse nang le mofuthu ka shell ea NAc (p = 14; F = 0.0001; df = 9.207), empa e seng ea motheo (Setšoantšo sa 5G le H). Ka mokhoa o makatsang, ho itšireletsa ha li-cocaine ho ΔFosB ho ne ho koetsoe liphoofolong tse nang le mofuthu ka bobeli le libokeng, ho fana ka maikutlo a hore, le hoja CaMKII e ka laola ka ho toba ΔFosB botsitso ho NAc shell, e ka 'na ea boela ea phahama le ΔFosB litseleng tse sebelisoang ke cocaine lihlopheng tse peli tsa NAc. .

Mosebetsi oa CaMKII o hlokahalang bakeng sa PFP-Mediated Structure and Behavioral Plasticity

Ho kenyelletsoa ha li-cocaine tsa li-spit tsa dendritic ho NAc MSNs ke e 'ngoe ea liphetoho tse ntlafalitsoeng tse entsoeng ke lithethefatsi sebakeng sena sa boko,' me tlhahiso eo ea mokokotlo e tsamaisane le likarabo tsa boitšoaro tse matlafatsang meriana (Robinson le Kolb, 2004; Russo et al., 2010) mme ba tlalehoa hore ba khethile li-MSNs tsa D1 (mofuta oa DXNUMX)Lee le al., 2006). Re bonts'a morao tjena hore tlhahiso ea k'hok'heine ea li-spin dendritic e NAc e itšetlehile ka ΔFosB le lenaneo la eona la ho ngolisa ka tlaase (Maze et al., 2010). Le hoja ho na le lingoliloeng tse ngata tse amanang le ho ameha ha CaMKII ka dendritic mokokotlo oa moralo oa mokokotlo le ho kenngoa ha libaka tse ling tsa boko le mekhoa ea liteko (Jourdain et al., 2003; Penzes et al., 2008; Okamoto et al., 2009), karolo ea eona ho NAc MSN sebopeho sa mokokotlo ha e e-s'o ithutiloe. Ka lebaka leo, re ile ra etsa qeto ea hore na mosebetsi oa CaMKII o hlokeha bakeng sa ho fokotsa li-spin MSN dendritic spines ka ho sebelisoa ha HSV-ho fetisetsoa ha HSV ho peptide ea CaMKII peptide AC3I e kopantsoeng le GFP, e hahiloeng pele e bontšitsoe ho thibela mosebetsi oa CaMKII Ka vivo (Zhang et al., 2005; Klug et al., 2012). Tlhaloso e feteletseng ea alFosB ho NAc shell ea litoeba tse kholo e entse hore ho be le keketseho e kholo ea boima ba mokokotlo oa MSN dendritic (p <0.0001; F = 8.558; df = 59; Setšoantšo sa 6A le B) joalokaha ho tlalehiloe pele (Maze et al., 2010), 'me keketseho ena e ne e susumelitsoe haholo-holo ke tšesaane (p = 0.0027; F = 5.319; df = 59) le ho foqoha (p = 0.0378; F = 2.988; df = 59) mefuta ea mokokotlo (bobeli ba nahanoang e le li-spin spines) (Setšoantšo sa 6C-E). Ha ho na phello e bonoeng likokotlong tse nang le mefuta e meholoanyane ea li-mushroom. Leha ho le joalo, ha GFP-AC3I e e-na le tšebetso e nyenyefalitsoeng, ΔFosB ho kenngoa ha li-spines e ile ea felisoa ka ho feletseng (Setšoantšo sa 6A-E), e leng se bontšang hore mosebetsi oa CaMKII o hlokeha bakeng sa ho fokotsa marotholi a DFFB ho DNA.

Re ile ra sebelisa lihlahisoa tse tšoanang tsa likokoana-hloko ho fumana hore na mosebetsi oa CaMKII o hlokahalang bakeng sa liphello tsa DFFB mabapi le ho ba le kutloelo-bohloko ea boitšoaro ho cocaine. 72 hr kamora 'mele oa kokoana-hloko ea NAC shell, liphoofolo li ne li fuoa joko e le' ngoe ea 5 mg / kg cocaine mme mosebetsi oa bona oa ho lokisa o ile oa ngoloa. Joalokaha ho bontšitsoe pejana ka ho eketseha ho eketsehileng ha AAV ea ΔFosB (Setšoantšo sa 4A), Tlhaloso e kholo ea HSV ea ΔFosB e ile ea eketsa botumo bo matla ba cocaine (p = 0.0002; F = 8.823; df = 37; Setšoantšo sa 6F). Joalo ka ho kenngoa ha methapo ea dendritic, ho thibela mosebetsi oa CaMKII ka khatello ea maikutlo ea GFP-AC3I ka ho feletseng ho thibetsoe ho eketseha ha khatello ea cocaine ho ΔFosB, e bontšang hore ts'ebetso ea CaMKII e hlokahalang bakeng sa liphetoho tsa Caosine tse bakoang ke liphello tsa lik'hemik'hale tsa cocaine.

Eya ho:

Puisano

Thuto ea morao tjena e hlahisa mokhoa o ts'oanang oa ho fepa lihlahisoa tsa cocaine tse hlahisang ΔFosB ka NAC, e leng ho kenyelletsa ho ngolisoa ha liphatsa tsa CaMKIIa ka khetho ea NAC. CaMKIIA e ntan'o latela phosphorylates le ho tsitsisa DFF e lebisang ho e kholo ho FosB ho bokella le ho eketsa CaMKIIa tlhahiso (Setšoantšo sa 6G). Mekhahlelo e ntseng e eketseha ea liprotheine tse peli nakong ea ho pepesehela k'hok'heine e sa feleng e tlatsetsa ka litsela tsa bohlokoa tsa ho matlafatsa likarabo tsa boitšoaro ho lithethefatsi. Ena ke khopolo-taba e ipiletsang ka ho fetisisa e nang le DFFB le CaMKII e 'ngoe le e' ngoe e bontšitsoeng pele e hlokahalang bakeng sa likarabo tse eketsehileng tsa lik'hok'heine (Pierce et al., 1998; Peakman et al., 2003), 'me re pheta phetoho ena bakeng sa ΔFosB ho NAc shell ka ho khetheha ho sebelisa mokhoa oa likokoana-hloko (Lifeiga tsa 4 'me Le66).

Le hoja ho tloaelehile ha DFNB ho hlahisa DNA ka D1 mofuta oa DNNUMX ho ka kenya koloi ea CaMKII ka bobeli ba NAC le motsoako oa liphoofolo tsa cocaine-naïve, ho latela cocaine, ho bokellana ha DFFB e tsitsitseng, e hlahang lihlopha tse peli tsa li-subregion, ho kenya tlhahiso ea CaMKII ka ho khetheha ho shell ea NAc . Phapang ena e ka amana le maemo a phahameng a DFBB a susumelitsoeng ka mohlala oa bitransgenic, leha ho le joalo, e ka boela ea bonahatsa bokhoni ba cocaine ho fetola mohlophisi oa CaMKIIa ka khethollo vs. MSNs tsa mantlha bakeng sa ho khothalletsa ΔFosB ho tlamahanya kapa ho e kenyelletsa sebakeng sa morao-rao. Ha e le hantle, litlaleho tsa rona tsa ChIP, tse senola diacetylation e tsoetseng pele ea cocaine ea histones ho CaMKIIa lihlahisoa tsa liphatsa tsa lefutso ka NAC, feela ho tšehetsa ho kenyelletsa mochine oa chromatin. Tumellanong le khopolo-taba ena, ΔFosB e fetisisang maikutlo ka MSNs ea mofuta oa D1 e ne e khona ho khanna lik'hamphani tsa CaMKIIa mofuteng oa NAc ka ho ba sieo ha cocaine (Setšoantšo sa 3F), ho fana ka maikutlo a hore ho na le liphetoho tse sebetsang tsa mothetsi oa CaMKIIa e thibelang ho kenngoa ha eona nakong ea ho hlahisa k'hok'heine e sa tloaelehang. Tlhophiso ea naha ea chromatin ho moetlisisi oa CaMKII e ka boela ea hlalosa hore na ke hobane'ng ha CaMKII e bakoa ke phephetso ea tekanyo ea k'hok'heine ka shell ea NAc ea likhopo tse sa foleng tsa cocaine (1E ea setšoantšo) empa eseng ka liphoofolo tse nang le lithethefatsi tse nang le lithethefatsi (Setšoantšo sa 1D). Sena se ka emela phello ea epigenetic ea "gene priming" ea DFF (Robison le Nestler, 2011), 'me ka tsela eo e ka' na ea e-ba mochine o le mong oa limolek'hule oa ho tšeloa ke takatso ea cocaine (Pickens et al., 2011). Leha ho le joalo, kaha chromatin ena e fetoloa e le hore e ka kopanngoa le ho kenngoa ha takatso ea takatso, e tla lokela ho eketseha ho feta nako. Ho tla ba monate ho fumana hore na ho joalo, le ho ithuta hore na liphatsa tse ling tsa liphatsa tsa lefutso li bontša joang DFB-e itšetlehile ka melao-motheo e ikemetseng ea cocaine. Hape ke habohlokoa hore re hlokomele hore sefate se tsoelang pele se hlalosang ha se lebise ho bokella ho sa feleng ha CaMKII kapa ΔFosB (1E ea setšoantšo); ho senola limolek'hule "tse senyehileng" tse ikarabellang bakeng sa sena ke pakane ea bohlokoa ea lithuto tsa nakong e tlang.

Mesebetsi e tsejoang ea DFFB le CaMKII ka mekhoa e 'maloa ea liteko le libaka tsa boko li fetoloa maemong a mangata (Setšoantšo sa 6F). Limolek'hule tsena ka bobeli li amana haufi le ho hōla ha mokokotlo oa mokokotlo: CaMKII e sebelisana le cytoskeleton ea actin (Okamoto et al., 2009), e laola boholo ba hlooho ea mokokotlo (Matsuzaki et al., 2004), 'me e le e hlokahalang le e lekaneng bakeng sa polasetiki-keketseho e kenyelletsoang palo ea filopodia le synapse meleng ea likotopo tsa hippocampal (Jourdain et al., 2003), wTlhompho ea DFFB e hlokahala ebile e lekaneng bakeng sa sebopeho sa mokokotlo oa mokokotlo oa li-cocaine ka NAC MSNs (Maze et al., 2010). Ho phaella moo, limolek'hule tse peli li 'nile tsa amahanngoa le taolo ea li-receptor tsa AMPA. CaMKII ha e laole likarolo tse felletseng tsa lihlopha tsa methapo ea AMPA, empa e khanna ho kenngoa ha li-receptor tsa AMPA li-synapses le ho eketsa boitšoaro ba mocha oa AMPA ka phosphorylating GluA1 ho Ser831 ho hippocampal pyramidal neurons moetlong le Ka vivo (e hlahlobisitsoe ho (Malinow le Malenka, 2002; Colbran le Brown, 2004)). Ts'ebetso ena e eketsehile ka ho rekisoa ha GluA1 ho synapse e entsoeng ka khato e sa foleng ea cocaine hape (Boudreau le Wolf, 2005). Ho phaella moo, likarabo tsa boitšoaro ho ts'ebetsong ea AMPA mofuteng oa NAc li matlafatsoa ke ho hatelloa ha CaMKIIa ka mokhoa o itšetlehileng ka mokhathala oa D1 dopamine (Singer le al., 2010). Nako e telele e hlalosang maikutlo a D1 ea DFFBB e bontšitsoe hore e kenyelle phetolelo ea GluA2 ka NAc (Kelz et al., 1999), e fokotsang likarabo tsa AMPA tse sebelisitsoeng ka har'a GluA1, ha re ntse re bontša mona hore nako e khutšoanyane ea DFFB-le ha e se e le khutšoanyane ea k'hok'heine-ha e na phello holim'a karoloana ena (Setšoantšo sa 1). Leha ho le joalo, re fumane morao tjena hore maikutlo a nakoana a ΔFosB a fokolang leha ho le joalo a fokotsa likarabo tsa AMPA mefuteng ea MSN ea D1 ka NAc (Grueter et al., 2013). Lintlha tsena li fana ka litsela tse khethollang nakoana tse ka 'nang tsa etsa hore ho be le lihlopha tse ngata tse sebelisoang ke nako ea ho noa k'hok'heine tse nang le likarolo tse sa tšoaneng tsa ho lemalla ho lemalla ho lemala eseng ho utloisisoa hantle. Boemong ba boitšoaro, bobeli ba CaMKII le ΔFosB ba hlokoa bakeng sa ho khothaletsoa ho hohela k'hok'heine (bona ka holimo), 'me ka bobeli li hlokeha bakeng sa ho itšireletsa ka cocaine e tsitsitseng ka litoeba (Colby et al., 2003; Wang et al., 2010), ho fana ka maikutlo a hore liprotheine tse peli ke tsa bohlokoa bakeng sa maemo a khutšoanyane le a nako e telele a ho itšoara ka lithethefatsi, ho sa tsotellehe mekhoa e fapaneng e khethollang lithethefatsi. Ho ka etsahala hore, DFFB le CaMKII li laola mokhoa o joalo o rarahaneng oa boitšoaro ka liphetoho tsa NAc synaptic function, le hoja ho hlokahala mosebetsi o mongata o hokahanyang ka ho toba li-synaptic tse etsahalang phetohong ea boitšoaro.

Holo ya CaMKII holoenzyme ka nako e le 'ngoe e sebelisana le liprotheine tse ngata tse amanang le synapse (Robison et al., 2005) tseo ho nahanoang hore li laoloa ke sekhahla sa postsynaptic (PSD), ntho e ts'oanelang ho ba ea bohlokoa bakeng sa polasetiki ea synaptic. Ka ho khetheha, ho sebelisana ha CaMKII le sehlopha sa GluN2B sa mokhatlo oa NMDA-mofuta oa glutamate receptor se sa tsoa bontšoa ho laola bobeli ba polasetiki le thuto (Halt et al., 2012). Le hoja peptide ea AC3I e etsisa sebaka sa autoinhibitory ea CaMKII, 'me kahoo e thibela tšebetso ea li-catalytic ea enzyme, e boetse e thibela li-protein tse ngata tsa protheine-protein (Strack et al., 2000; Robison et al., 2005). Ka hona, liphello tsa boitšoaro le tsa morphological tsa HSV-GFP-AC3I tse tlalehiloeng mona li ka hlaha ka phosphorylation e fokotsehileng ea liprotheine tse nang le sepheo sa CaMKII, liphetoho tse lebisong la CaMKII, kapa phetoho ea karolo ea morero ea CaMKII ho synapses (Lisman et al., 2002).

Thibelo ea sefapano sa ΔFosB-CaMKII sechabeng sa NAc ke sa bohlokoa, kaha mosebetsi oa morao-rao o bontšitse phapang e mengata ea liphatlalatso pakeng tsa khetla ea NAc le motheo oa ho tsamaisa tsamaiso ea cocaine, khopolo e netefalitsoeng ke lintlha tsa rona tsa iTRAQ (Table S1) e sa hlokahaleng . Li-MSN tsa NAc shell li bontša ho tepella ha matla ka mor'a ho qeta libeke tse ngata, ha li-MSN tsa liphoofolo tse tšoanang li bontša ho eketseha ha letsatsi (1-3) ka matla a ho thunngoa a khutlelang litsing tsa motheo nakong ea libeke tsa 2 (Kourrich le Thomas, 2009). Ho phaella moo, li-protein tse ngata tsa synaptic li laoloa ka tsela e sa tšoaneng ho NAc shell vs. motsoako oa liphoofolo tse pepesehileng ho cocaine e sa feleng, ho akarelletsa le GluA2 (Knackstedt le al., 2010). Joalokaha e sa foleng amphetamine e kenyelletsa CaMKIIa haholo-holo ho NAc shell (Loweth et al., 2010), ha ho makatse hore ebe re fumana phello e tšoanang le cocaine. Leha ho le joalo, kaha ΔFosB e bakoa ke khetla ea NAc le mantlha ka cocaine e sa foleng (Perrotti et al., 2008), 'me kaha re bontša hore CaMKIIA ho kenya likhetla ka khetla ke ΔFosB e itšetlehileng ka eona, liphihlelo tsa rona li fana ka bopaki bo bocha bakeng sa mekhoa e khethollang ea transcription ho mofani oa CaMKIIa pakeng tsa lihlopha tsena tse peli, tse ikarabellang bakeng sa ho kenyelletsoa ha CaMKIIa ka khetla.

Mosebetsi o mongata oa morao-rao o tsepamisitse maikutlo ho hlahisa phapang pakeng tsa D1- le D2-mofuta wa MSN MSNs. Le hoja li-receptor tse peli tsa D1 le D2 li ameha liphello tse putsang tsa cocaine (Boithati, 2010), Mosebetsi oa morao tjena o bontša hore ts'ebetso ea optogenetic ea MSNs ea D1-e eketsa likarabo tsa boitšoaro ho cocaine, ha ts'ebetso ea D2-mofuta oa MSN e na le phello e fapaneng (Lobo et al., 2010). Tumellanong le liphuputso tsena, litoeba tsa kokotlo tsa D1-receptor li haelloa ke ho fumana k'hok'heine ea botho (Caine et al., 2007), ha li-knockouts tsa D2 li se (Caine et al., 2002). D1 tsamaiso ea bo-agonist ka ho toba ka NAC e etsa hore boitšoaro bo batle ba k'hok'heine bo khutlisetsoe maemong a tsosoloso (Boithati, 2010). Hoa thahasellisa hore phello ena e hloka keketseho ea D1-receptor e amohelehang mesebetsing ea CaMKII ka shell ea NAc, empa e se ea motheo (Anderson et al., 2008), e leng sephetho se hlahisang hantle ka D1- le sekheo se khethehileng sa ΔFosB-CaMKII se hlahisitsoeng mona.

Re kile ra tlaleha hore Ser27 ho ΔFosB e ka ba phosphorylated ke casein kinase-2 (Ulery et al., 2006), leha ho le joalo, re theha mona hore CaMKII phosphorylates ΔFosB sebakeng sena le libakeng tse ling tse nang le kinetics e kholo haholo le stoichiometry 'me e ka pheta holimo ho bonahala Mr e hlokometse DFFB (Setšoantšo sa 5A) ka ho hlahisa k'hok'heine Ka vivo (Nestler, 2008). Re se re ntse re tseba hore phosphorylation ea Ser27 e eketsa botsitso ba DFFB le mosebetsi oa transcriptional (Ulery et al., 2006; Ulery le Nestler, 2007; Ulery-Reynolds et al., 2009). Mosebetsi oa nakong e tlang o tla lebisa tlhokomelo ho khethollo le liphello tsa ts'ebeliso ea libaka tse ncha tsa ΔFosB phosphorylation e bontšitsoeng ke thuto ea hona joale.

Tsela e hlalositsoeng mona e fana ka mokhoa o mocha oo ho oona ho tsamaisoang ha cocaine ho khanna ho sa tloaelehang ho NAc. Ka hona, tsela ena ea likokoana-hloko e ka 'na ea fana ka sepheo sa bohlokoa bakeng sa ho kenella kalafo ea meriana nakong ea mathata a meriana. Ka lebaka la hore CaMKII e na le maemo a mangata 'me e hlokahalang bakeng sa mesebetsi e mengata ea basal neuronal le ea boitšoaro, tšebeliso e tobileng ea li-inhibitor tsa CaMKII e' nile ea qojoa e le phekolo ea lithethefatsi. Dintlha tsa rona li fana ka maikutlo a hore ho kenoa ka mokhoa o poteletseng oa mochine oa CaMKII, e leng ho khethehileng ho mofuta o mong le o mong oa sele le moeli oa moputso oa boko, o ka fana ka sepheo sa phekolo se tla qoba mathata a tsamaiso ea CaMKII.

Eya ho:

Liteboho

Mosebetsi ona o ne o tšehetsoa ke lichelete tse tsoang Mokhatlo oa Sechaba oa Tlhekefetso ea Meriana (EJN), Mokhatlo oa NIDA-Yale Proteomics DA018343 (AJR le EJN), le Hartwell Foundation (AJR). Bangoli ba ka rata ho leboha Gabby Rundenko ka mpho e fanoeng ka seatla se bulehileng ea DFFB le Roger Colbran bakeng sa mpho e seatla se bulehileng ea CaMKIIa.

Eya ho:

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