(HUMAN) Abubuwan da ke faruwa a cikin jiki da kuma Tsarin Tsarin Tsarin Harshen Cocaine na buƙatar Harkokin Gyara Tafiyar Abincin ΔFosB da Kwayoyin Halitta / Calmodulin-Dependent Protein Kinase II a cikin Harshen Accumbens Shell (2013)

Matsayin sakonnin ΔFosB da kwakwalwa mai kwakwalwa na kwakwalwa da kwantar da hankulan kwakwalwa da ke cikin kwakwalwa (CaMKIIa) ana haifar da shi a cikin ƙwayar cuta (NAc) ta hanyar kwantar da hankali ga cocaine ko sauran magungunan psychostimulant, wanda ma'anar sunadaran sunadaran sunadarai . Kodayake ΔFosB da CaMKIIa duka sun tsara fassarar mai karfin sutura ta AMPA da aiki a NAc, samfurin dindritic spin formation a kan nau'o'in spiny neo na MSC, da kuma haɓakar locomotor zuwa cocaine, babu hanyar haɗin kai tsaye tsakanin waɗannan kwayoyin da aka gano a yau. A nan, muna nuna cewa ΔFosB an samo phosphorylated ta hanyar CaMKII a a cikin Ser27 na gina jiki da kuma cewa CaMKII shine da ake buƙata don haɗin gwiwar cocaine na ΔFosB a rat Nac.

Hakanan, muna nuna cewa ΔFosB yana da mahimmanci kuma isasshen maganin cocaine na shigar da kalmar CaMKIIa a cikin vivo, wani zaɓi na zaɓi na D₁MSNs a cikin takaddamar gwaninta na NAc.

Bugu da ƙari, ƙaddamar da spendendic spines a kan NAc MSNs da kuma ƙara yawan halayyar halayyar maganin hawan cocaine bayan da NAc nunawa na ΔFosB ne duka CaMKII dogara.

Mafi mahimmanci, zamu nuna a karo na farko shigar da ΔFosB da CaMKII a cikin NAc na 'yan tawayen cocaine, bayar da shawarar yiwuwar da za a iya ci gaba da yin rigakafi. Wadannan bayanan sun tabbatar da cewa ΔFosB da CaMKII sun shiga cikin ƙwayoyin salula-da ƙwaƙwalwar ƙwaƙwalwar ajiya don ciyar da ladaran ladabi na kwakwalwa don mayar da martani ga cocaine na yau da kullum.

Gabatarwa

Ƙarin shaidun da ke goyan bayan ra'ayi wanda yake canzawa a furcin kalma yana taimakawa wajen maganin ƙwayar magunguna (Robison da Nestler, 2011). Ɗaya mai mahimmanci mai mahimmanci na waɗannan canje-canje shi ne ΔFosB, Fos family factor transcription factor (Nestler, 2008). Tsarin lokaci na kusan duk wani miyagun ƙwayoyi na zalunci ya haifar da jigilar ΔFosB a cikin ƙananan ƙwayoyin jiki (NAc), wani yanki na musamman wanda yake da muhimmanci ga halin halayen. Sƴan shigarwa yana nuna takamaiman ƙwayar NAC na spiny neuron (MSN) wanda ke nuna masu karɓa na D1 dopamine. Kuskuren ƙyama na ΔFosB a cikin wadannan nau'o'i na NAc MSX na D1 yana ƙaruwa locomotor da amsa martani ga cocaine da morphine (Kelz et al., 1999; Zachariou et al., 2006), ciki har da ci gaba da kula da hawan cocaine (Colby et al., 2003). Bugu da ƙari kuma, kwayoyin halitta ko maganin hoto na ΔFosB zai rage sakamakon sakamako na wadannan kwayoyi (Zachariou et al., 2006), yana nuna cewa wannan ci gaba na ΔFosB shi ne mai matsakanci mai mahimmanci na canje-canje na canjin da aka samu a cikin NAc ta hanyar ci gaba da maganin miyagun ƙwayoyi.

Kwancen da aka saba da shi na ΔFosB (dangantaka da sauran sauran sunadarai na Fos) shine duk wani abu ne mai mahimmanci na kwayoyin, saboda ƙaddarar yankunan degron da ke cikin FosB mai cikakke (Carle et al., 2007), da kuma tsarin da aka tsara. ΔFosB ne phosphorylated a vitro da kuma a vivo a Ser27, kuma wannan karfin ya kara karfafa ΔFosB, ~ 10-ninka, a cikin al'ada da NAc a vivo (Ulery-Reynolds et al., 2009). Kodayake Ser27-ΔFosB an nuna su zama madogara ga casin kinase-2 a vitro (Ulery et al., 2006), aikinta na a vivo phosphorylation ba a sani ba.

Kwayoyin calcium / proteodine-dependent protein kinase II (CaMKII) wani sashin / threonine kinase da aka bayyana sosai wanda bayyanar kwaikwayon α da β ya zama dodecameric homo- da hetero-holoenzymes a vivo, kuma suna da mahimmanci ga siffofin da yawa na neuroplasticity (Lisman et al., 2002; Colbran da Brown, 2004). Ana haifar da CaMKIIa a cikin NAc harsashi ta hanyar amphetamine na kullum (Loweth et al., 2010), da kuma maganin kariya na maganin CaMKII a gurbin NAc ya rage karfin hali na amphetamine (Loweth et al., 2008) da kuma cocaine (Pierce et al., 1998), yayin da maganganu na CaMKIIa a cikin wannan tsarin na NAc yana ƙarfafa fahimtar locomotor da kuma kula da kai na amphetamine (Loweth et al., 2010). CaMKIIa zai iya rinjayar halin halayen ta hanyar yin amfani da madogara na rukunin mai karɓa na AMPA (glutamate receptor subunits)Pierce et al., 1998), kamar yadda aikin CaMKIIa ya dade daɗe da dangantaka da aiki na mai karɓar AMPA da synaptic-ke niyya a wasu nau'i na neuroplasticity (Malinow da Malenka, 2002).

Wadannan wallafe-wallafen suna nuna alamu da yawa tsakanin ΔFosB da CaMKII: duka biyu suna da bukata kuma suna da isasshen nauyin halayen maganganu na magunguna, dukansu sunadaran dendritic spines a cikin daban-daban na sel neuronal a vivo (Jourdain et al., 2003; Maze et al., 2010), kuma duka biyu suna gwada wasu daga cikin halin halayen su ta hanyar sauya masu karɓa na AMPA (Kelz et al., 1999; Malinow da Malenka, 2002; Vialou et al., 2010). Duk da waɗannan daidaitattun, babu alamar haɗin aiki tsakanin ΔFosB da CaMKII da aka sani. A nan, mun kafa tsarin sulhu tsakanin ΔFosB da CaMKII, kuma sun nuna cewa sunadarai guda biyu suna samar da madaidaicin madaidaiciya na MSN-D1-nau'i na kwakwalwar NAc da aka jawo ta hanyar cocaine kuma tana tsara adadin maganin cocaine. a vivo.

Ka tafi zuwa ga:

Kaya da matakai

Gwajin 1: iTRAQ Analysis Analysis of Nac Shell da Core Bayan Cocaine Jiyya (Fig 1A)

Adult (8 makonni) an yi amfani da ratsin raunin 20 mg / kg cocaine ko kayan salin IP sau ɗaya a kowace rana har kwana bakwai. 24 hr bayan da allurar rigakafi, NAC harsashi da kuma mahimmanci sun kasance microdissected (Fig 1A) da kuma walƙiya. An gudanar da nazarin ILRAQ kamar yadda aka bayyana a baya (Ross et al., 2004; Davalos et al., 2010).

Figure 1

Sakamakon takaddama na Shell ta CaMKII a cikin NAc ta hanyar cocaine

Gwajin 2: Tada Girma Tsarin Protein a Ratin NAC Core da Shell Bayan Kwayar Ciki (Fig 1B-D)

Adult (8 makonni) an yi amfani da ratsin raunin 10 mg / kg ko kuma salin salin IP sau daya a kowace rana don kwana bakwai a cikin ɗakin rikodi na locomotor. Ana yin rikodin Locomotor zuwa wani allurar hawan cocaine (5 mg / kg IP) a cikin wadanda aka kula da su a baya tare da cocaine (wanda ake kira "na yau da kullum") da kuma wani ɓangare na waɗanda aka bi da saline (da ake kira "m"), da kuma maganin locomotor ga saline kadai an rubuta shi a sauran sauran dabbobin da ake kula da saline (wanda ake kira "saline"). Anyi amfani da gwajin aikin Locomotor kamar yadda aka bayyana (Hiroi et al., 1997). A takaice dai, an sanya ratsan jinsi maza a cikin 18 "× 24" KASA masu ba da izinin budewa (San Diego Instruments) don 30 min zuwa mazaunin, an ba da wani ingancin IP na saline kuma ana kulawa don ƙarin 30 min, kuma an ba su injection IP kawai na 5 mg / kg cocaine da kuma kula da 30 min.

24 Hr bayan wannan allurar rigakafi, an yi amfani da berayen ba tare da maganin rigakafi ba don kaucewa sakamakon ilimin ƙwayoyin cuta akan matakan gina jiki da kuma phospho-jiho. An cire sutura a cikin 1.2 mm matrix (Braintree Scientific) kuma an cire nama a cikin salumin da ke dauke da phosphate (Sigma Aldrich) tare da amfani da nau'in 14 ma'auni na ainihin NAC da kuma nau'in 12 na sauran nama don NAc harshe (Dubi Fig 1A) kuma nan da nan daskararre kan kankara. Samfurin RIPA ya kunshi samfurori tare da hasken haske a cikin buffer RIPA da aka gyara: 10 mM Tris tushe, 150 mM sodium chloride, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% sodium deoxycholate, pH 7.4, protease da phosphatase inhibitors kamar yadda a sama. Bayan dawowar Laemmli buffer, an raba sunadaran a kan 4-15% gels gradlamaide (Criterion System, BioRad), kuma an yi amfani da Tsarin Yamma ta amfani da tsarin Odyssey (Li-Cor) bisa ga ka'idodin masu sana'a.

Gwajin 3: Tattarawar Canjin Kwayoyin Protein a cikin Ratin NAC da Shell Bayan Ruwan Cocaine Gyara (Fig 1E)

Adult (8 makonni) an yi amfani da ratsin raunin 10 mg / kg cocaine ko kayan salin IP sau ɗaya a kowace rana har kwana bakwai. 14 kwanakin bayan da allurar rigakafi, an ba da dabbobi da saline wani allura salin (wanda ake kira "saline"), kuma an ba da sauran allurar salin da ake kira 14 day withdrawal ko "14d WD" ko kuma allurar cocaine ( da ake kira "14d WD Chal" don ƙalubalanci). Ɗaya daga cikin hr bayan da allurar rigakafi, an kwantar da dabbobi da kuma yin amfani da shi a Yamma Experiment 2.

Gwajin 4: Tadawa Canjin Canjin Kwayoyin Kwayoyin Kwayoyin Kwayoyin Kwayoyin Kwayoyin Kasuwanci NAc Core da Shell Bayan Bayanin Gudanar da Kai Kwanci (Fig 2A-C)

Rats an horar da su don gudanarwa ta 0.5 mg / kg / jiko na cocaine a cikin hutun guda daya a karkashin tsarin 1 na tsawon kwanaki tara. Bayan bayanai tara, an rabe ratsi zuwa ƙungiyoyi biyu da cin abinci na cocaine suke gudanarwa a cikin zamanni biyu na ƙarshe. Wata ƙungiya na berayen an yarda su yi amfani da cocaine mai gudanarwa (0.5 mg / kg / jiko) a cikin lokutan guda daya (damar shiga, ShA) yayin da sauran rukuni na hade cocaine a cikin huxu na shida (tsawon lokaci, LgA ) na tsawon kwanaki goma (haɓakawa zaman).

An tsara sassan layi don immunohistochemistry kamar yadda aka bayyana (Perrotti et al., 2004). An yi amfani da ƙwayoyin cutar 18-24 hr bayan da aka kwashe su zuwa miyagun ƙwayoyi, wanda zai haifar da lalacewar duk wani furotin FosB mai cikakke cikakku kamar yadda duk abin da aka rage ya nuna ΔFosB. Wannan ƙasƙanci ya tabbatar da ta Yammacin Turai, wanda bai nuna matukar muhimmanci ba tare da wani wanda aka yi wa jagorancin C-FosB wanda ba ya san ΔFosB (bayanan ba a nuna ba). Bayan slicing zuwa cikin sassan 35 μm, yawan mai kwakwalwa na ΔFosB ya ƙididdige yawan adadin kwayoyin ΔFosB ta hanyar NAC na kowane ƙira, kuma ana kwatanta ma'auni ta hanyar 40 × filin ta kowace yankin. Kowace dabba an dauke shi kallon mutum don nazarin lissafi. An gano rukunin amfani da amfani da Paxinos da Watson (Paxinos da Watson, 2007).

Ƙaddamarwa na CaMKIIa rigakafi da aka yi ta amfani da tsarin Licor kamar yadda aka bayyana (Covington et al., 2009). Ƙaddamarwar ƙarfin CaMKII da GAPDH an ƙaddara su tare da software Odyssey. Sakamako an gabatar da shi azaman ƙananan dabi'u da mm² kuma an gabatar da su wajen nufin ± sem (n = 4-10 da ƙungiya). Ana amfani da dabi'un GAPDH don yin la'akari da daidaita tsarin CaMKII don rassan wuri da yanayi.

Figure 2

Kashi na CaMKII a cikin NAc da ke kula da ratsan ratsi da haɗin gwiwar mutane

Gwajin 5: Tattar da Matsayin Kwayoyin Halitta a Yankin Tsarin Harsashin Cocaine -Fig 2D)

hanya

An sami kwakwalwa daga cikin kwakwalwa daga cikin kwakwalwa daga kwakwalwa ta kwakwalwa ta Quebec (Douglas Mental Health University Cibiyar, Montreal, Quebec, Kanada). Da adana nama ya ci gaba sosai kamar yadda aka bayyana (Quirion et al., 1987). A taƙaice, sau ɗaya an cire shi, sai an sanya kwakwalwa kan kankarar rigar a cikin akwatin Styrofoam kuma ta gudu zuwa Ƙungiyar Binciken Ƙungiyar Cutar Cutar Ƙasar Quebec. Hakanan an raba wasu nau'o'i a cikin kwakwalwa, kwakwalwar kwakwalwa, da kuma cerebellum. Ruwan jini, glandal gira, choroid plexus, rabin cerebellum, da rabi kwakwalwar kwakwalwa ana rarraba su daga hagu na hagu wanda aka yanka a cikin nau'in 1 cm-mintuna kafin a daskarewa. A karshen rabin cerebellum an yanke sagittally cikin 1cm-mai farin ciki kafin a daskarewa. Tissues suna da haske a cikin 2-methylbutane a -40 ° C domin ~ 60 sec. Dukkanin takalmin da aka daskare suna ajiye su a cikin jaka a filayen filastik a -80 ° C don ajiya na dogon lokaci. Yankunan kwakwalwar kwakwalwa suna rarraba daga nau'in katako na daskararre a kan wani farantin karfe wanda aka yi da busassun kankara don sarrafa yawan zafin jiki na yanayin. An yi watsi da Yammacin Turai kamar yadda aka bayyana a Experiment 2.

Ƙungiyar

Ƙungiyar ta ƙunshi nau'ikan 37 maza da 3 mata masu juna biyu, waɗanda ke da shekaru a tsakanin shekaru 15-66. Dukkanin batutuwa sun mutu ba zato ba tsammani ba tare da jihohi ba, ko kuma rashin lafiyar likita. A kowane hali, ofishin Coroner na Quebec ya tabbatar da dalilin mutuwar, kuma an gudanar da allon mai ciki tare da samfurori na samfurori don samun bayani game da magani da rashin amfani da kayan abu a lokacin mutuwar. Ƙungiyar ta ƙunshi mutanen 20 waɗanda suka sadu da ka'idojin SCID-I don dogara da hawan cocaine. Ƙungiyar ta kunshi nau'o'in 20 ba tare da tarihin maganin cocaine ba kuma babu manyan cututtukan likita. Dukkan batutuwa sun mutu ba zato ba tsammani daga haddasawa wadanda basu da tasiri a kan kwakwalwa. Ƙungiyoyi sun dace da maƙasudin shekaru, jinkirin sanyi, da kuma pH. Ga dukan batutuwa, an yi kwaskwarima a cikin kwakwalwa kamar yadda aka bayyana a baya (Dumais et al., 2005), yana ba mu damar samun cikakkun bayanai game da ilmin likita da kuma tarihin kiwon lafiya, da kuma sauran bayanan asibiti da kuma bayanan zamantakewa. A takaitaccen bayani, mai jarrabawar da aka gudanar ya gudanar Jirgin Intanet na Kamfanin Clinical Interview for DSM-IV Cutar Psychiatric (SCID-I) tare da ɗaya ko fiye da sanarwa game da marigayin. Kungiyar likitoci sun gwada nazarin SCID-I, rahotanni, bayanin marubuta, da kuma rubutun likita don samo likitoci na likita.

Gwajin 6: Gidaran Chromatin na Ratti NAc (Fig 3A-C)

Adult (8 makonni) an yi amfani da ratsin raunin 10 mg / kg cocaine ko kayan salin IP sau ɗaya a kowace rana har kwana bakwai. 24 hr bayan da allurar rigakafi, da ma'anar NAc da kuma mahimmanci sun kasance microdissected. Chromatin immunoprecipitation (ChIP) an gudanar da yin wanka da nauyin nau'i nau'i nau'i na nau'i nau'i nau'i daga nau'i nau'i nau'i nau'i nau'i na nau'i nau'i na nau'in 14 (jinsunan 98 duka, koguna na cocaine 7, 7 saline pools). Tissues sun haɗu da juna, wanke su, kuma sun adana a -80 ° C har sai jinyar da ake ciki ta hanyar sonication. An haɗu da chromatin tare da dare tare da kwayoyin rigakafi da aka ɗaure da su a kwanan baya zuwa nau'ikan katako (Dynabeads M-280, Invitrogen). An yi amfani da IgG maras amfani da ita azaman iko. Bayan an yi watsi da haɗin giciye da kuma tsarkakewar DNA, an yi amfani da qPCR don auna matakan DNA na CaMKIIa. An tsara mahimmanci don fadada wani yanki wanda ke ƙunshe da jerin AP-1 da ke cikin ~ 450 bp kafin wurin farawa na karatun (Gyara: ACTGACTCAGGAAGAGGGATA; Kashi: TGTGCTCCTCAGAATCCACAA).

Figure 3

Yanayin salula- da yanki na musamman na ΔFosB na CaMKIIa a vivo

Gwaji 7: Daidaita CaMKII Kaya da Maganin Protein tare da Siffar-Siffar-ta musamman ΔFosB Overexpression (Fig 3D)

Mace bitransgenic an samu daga NSE-tTA (layin A) × TetOp-ΔfosB (layin 11) kuma NSE-tTA (layin B) × TetOp-FLAG-ΔfosB (layi na 11) mice (Chen et al., 1998; Kelz et al., 1999; Werme et al., 2002; Zachariou et al., 2006) an yi ciki kuma an tashe shi a kan 100 μg / ml doxycycline don rufe kalmar ΔFosB yayin ci gaban. An raba raguwa da juna a lokacin wankewa: rabi ya kasance a kan doxycycline kuma rabi sun sauya ruwa, kuma an yi amfani da dabbobi 8 zuwa 11 makonni bayan haka lokacin da sakamakon na ΔFosB ne mafi girma (Kelz et al., 1999; McClung da Nestler, 2003). Don nazarin ilimin lissafin rubutu, an kwantar da hanzari, kuma an cire kwakwalwa kuma an sanya shi kan kankara. An cire fassarar NAc tare da nau'in buƙata na injin 14 da sauri a daskararre a lokacin da aka cire RNA. RNA isowa, qPCR, da kuma bayanan bayanai sun kasance kamar yadda aka bayyana a baya (LaPlant et al., 2009). A taƙaice, an raba RNA tare da mahalarta TriZol (Invitrogen), an tsarkake shi da RNAeasy micro Kit daga Qiagen, kuma an duba shi don inganci tare da Agilent's Bioanalyzer. An yi amfani da bayanan da aka ƙi ta amfani da iScript (BioRad). An gudanar da qPCR tare da tsarin 7900HT RT PCR na Aikace-aikacen da aka Yi amfani da su tare da sigogi masu biyo baya: 10 min a 95 ° C; 40 cycles na 95 ° C na 1 min, 60 ° C don 30 sec, 72 ° C na 30 sec; Ƙararrawan da aka yi amfani da ita zuwa 95 ° C don samar da ƙananan hanyoyi don tabbatar da samfurorin PCR. Bayanan Immunohistochemical na ΔFosB da kuma maganin furotin na CaMKIIA an yi kamar yadda aka bayyana a Experiment 4.

Gwaji 8: Hanyoyin Intra-NAc D1 da D2 Dopamine Receptor Antagonists a kan Canjin Canji na Gwanar da Cocaine -Fig 3H)

Adult (8 makonni) an yi amfani da ratsan raunin 10 mg / kg cocaine ko kayan salin (ƙungiyar "motar") IP sau ɗaya a kowace rana har kwana bakwai. 30 min kafin kowace allurar hawan cocaine, ratsi an yi IP ne ko dai mai karɓar mai karɓa na D1 SCH 23390 (0.5 mg / kg, ƙungiyar "D1 Ant"), ko kuma mai kwakwalwa mai kwakwalwa na D2 eticlopride (0.5 mg / kg, kungiyar "D2 Ant") , ko kuma injin saline (ƙungiyar cocaine). 24 hr bayan da allurar rigakafi, an kwashe dabbobi da sunadarai wadanda aka sanya su ta hanyar yammaci kamar yadda Experiment 2.

Gwaji 9: Hanyoyin AAV-Mediated ΔFosB Overexpression akan Bayanin Protein (Fig 4 A-C)

An yi aikin tiyata a kan ƙananan rago mata (8 makonni) don yin amfani da AAV-GFP (furotin furotin mai fadi) ko AAV-GFP-ΔFosB (Maze et al., 2010). 33 ma'auni na ma'auni (Hamilton) an yi amfani da su don dukan tiyata, a lokacin da 0.5 μl na tsabta mai tsabta mai tsabta ya kasance a cikin wani lokaci na 5, sannan ya kara da ƙarin lokacin 5 min bayan jinkiri. Dukkan nisa da aka auna dangane da Bregma: 10 ° kusurwa, AP = + 1.7 mm, Lat = 2.5 mm, DV = -6.7 mm. 14 kwanaki bayan tiyata, an ba da dabbobi iri daya na IP na 10 mg / kg cocaine a cikin ɗakunan dubawa na locomotor don tantance halin da ake ciki na ΔFosB overexpression. 24 hr bayan wannan allurar rigakafi, an yi amfani da ratsi kamar yadda ta Experiment 2, kuma an yi amfani da microdissection a karkashin jagorancin binciken microscopic mai haske don samun GFP-tabbatacciyar fata na NAc. An yi amfani da shi a yammacin Turai kamar yadda Gwaji 2.

Figure 4

ΔFosB ya zama wajibi ne da isasshen ƙwayar CaMKIIa mai kwakwalwa ta hanyar kwakwalwa ta hanyar kwakwalwa ta hanyar kwakwalwa.

Gwajin 10: Hanyoyin AAV-Mediated ΔJunD Ƙinƙwasawa akan Maganin Protein Cocaine-Dependent (Fig 4 D-F)

An yi amfani da allurar mai amfani na AAV-GFP ko AAV-GFP-ΔJunD kamar yadda Gwaji 8. 14 kwanaki bayan tiyata, ana amfani da dabbobi 10 mg / kg cocaine ko kayan salin IP sau ɗaya a kowace rana don kwana bakwai a cikin ɗakin ɗakin rikodi na locomotor. Amfani da Locomotor zuwa wata allurar cocaine (5 mg / kg IP) ko saline. 24 hr bayan wannan allurar rigakafi, an yi amfani da ratsi, da kayan da aka girbe, da kuma Western blots yi kamar yadda a cikin Gwaji 9.

Gwajin 11: a vitro Amfanin Tsaro na Kariya (Fig 5A-D)

Mafarin CaMKIIa da ΔFosB sun tsarkake daga kwayoyin kwari (Brickey et al., 1990; Jorissen et al., 2007), kuma an yi gwajin kinase kinase (Colbran, 1993), kamar yadda aka bayyana a baya. A takaice dai, CaMKII ya kasance a kan kankara tare da 2.5 μM (ko nuna maida hankali) na ΔFosB, 1 mM Ca²⁺, 40 MM Mg²⁺, 15 μM calmodulin, da kuma 200 mM HEPES pH 7.5. An fara samfurin phosphorylation ta kari da 200 μM ATP tare da ko ba tare da [γ-³²P] ATP kuma an yarda ta ci gaba don 10 min a ɗakin zafin jiki (Siffa 5A & B) ko 2 min akan kankara (Siffa 5C & D.). Abubuwan da aka sanya ta Yammacin Yamma (Siffa 5A & B) ko ta autoradiogram da ƙididdigar scintillation (Fig B-D).

Figure 5

ΔFosB shine matashi mai mahimmanci ga CaMKIIa

Gwajin 12: Bayyana Ser27 ΔFosB Phosphorylation (Fig 5E)

a vitro An yi gwajin kinase a matsayin ta Experiment 11, SDS-PAGE ya rabu da sunadarai, kuma an ware sassan da aka dace da ΔFosB kuma sun kasance sunyi amfani da jimla. Ayyukan m / z na gishiri na daskararru masu jituwa a cikin dukkanin bangarori suna lakafta a saman ginshiƙan dutsen. Ba dukkanin ions da aka lakaba ba saboda iyakokin sararin samaniya. Yawanci, rubutu don alamomin jeri na launin launin launin baki ne a baki sai dai lokacin da suke tabbatarwa ko ƙara shaida akan kasancewar alamomin phosphorylation na sha'awa, a cikin wannan hali ana alama su a ja. An tabbatar da shaidar samfurori na samfurori a cikin jerin rubutun na phosphoreptide tare da shafin da aka gano na phosphorylation sauran da aka nuna a cikin ja tare da amino acid rubutun wasika. Ana kwatanta fasalin lambobi na ions da aka lura da shi akan jerin peptide a matsayin b da y ions. Abubuwan zuƙowa don sassan sashen m / z don nuna alamun ions mai ƙananan ƙananan suna alama a saman kowane ɓangaren jeri. Kwanan da aka nuna a cikin rukunin H ya tabbatar da kasancewar Ser27 phosphorylated isoform, duk da haka, a cikin wani cakuda sauran matakan phosphorylated a shafukan yanar gizo Ser28, Ser31, Ser34, da Thr37. Gabatarwar pa5, pa5-P, pb5, da kuma pb5-P da keɓaɓɓun tabbatar da phosphorylation na Ser27 sauran.

Gwaji 13: Bayani na Ser27 phosphorylation (Fig 5F)

An tsara nau'i-nau'i masu daidaitattun siffofin phospho da wadanda ba phospho na Ser27 ΔFosB. Bayan kira da kuma tsarkakewa, an cire nau'in peptide na "nauyi" a cikin wani nau'in 50 / 50 acetonitrile / ruwa da kuma aikawa da amino acid don tantance cikakkiyar maida hankali kan maganin maganin peptide. Kowane "peptide" mai nauyi "a yanzu ya shiga cikin 4000 QTRAP mashigin (MS) domin sanin ƙayyadadden makamashi na haɗakarwa ga musayar MS / MS da sau biyu zuwa hudu MRM. Bayan haka, an yi amfani da peptides masu nauyi "masu nauyi" a LCMS akan 4000 QTRAP don tabbatar da rabuwa peptide. An yi amfani da kayan aiki a cikin yanayin sau uku, tare da Q1 da aka ƙayyade a kan takamaiman m / z darajar (Q1 ba a dubawa ba), kuma Q3 ya daidaita zuwa m / z darajar daidai da wani ɓangaren wannan peptide. A cikin yanayin MRM, jerin jerin halayen guda (haɓakaccen ƙwayoyin jigilar iska inda ake amfani da makamashi na haɗuwa don inganta ƙarfin ions na sha'awa) an auna su, kuma sake zagayowar (kamar 1-2 sec) duk tsawon lokacin rarraba HPLC. MRM fassarar an ƙaddara daga MS / MS spectra na peptides na yanzu. Hanya guda biyu da peptide, daidai da ions mai ƙarfi, da aka zaɓa kuma ƙarfin haɗakarwa da aka ƙaddara don ƙarfafa ƙarfin siginar MRM fassarar ta amfani da software ta atomatik. Mahimmanci daga sakamakon peptides da kuma samfurori na ΔFosB da aka fallasa zuwa CaMKII ko iko sun kasance idan aka kwatanta su don ƙayyade cikakkiyar nauyin kowane nau'i na peptide a cikin dauki. Ana yin nazarin bayanai a kan LC-MRM bayanai ta amfani da software AB Multiquant 1.1.

Gwajin 14: Ƙaddamar da ΔFosB a CaMKII Overexpressing Mice (Siffa 5G & H)

Mice masu juyayi na tsammanin T286D CaMKII (Mayford et al., 1996; Kourrich et al., 2012) da kuma irin wadanda suka hada da dabbobin daji sun tashi a cikin rashin yin dogoncycline don ba da iznin transgene. An yi amfani da ƙananan ƙwararruwan 20 mg / kg cocaine ko saline IP sau ɗaya kowace rana don kwanaki 14. 24 hr bayan da allurar rigakafi, an kwashe dabbobi da kuma immunohistochemistry da kuma rarraba maganar ΔFosB kamar yadda a cikin Experiment 4.

Gwaji 15: Hanyoyin HSV-Mediated ΔFosB Overexpression da CaMKII Inhibition a kan NAc Dendritic Spines (Fig 6A-E)

Mice maza marayu (8 makonni) ana amfani da shi a cikin NAc tare da HSV-GFP, HSV-GFP-ΔFosB (Olausson et al., 2006), HSV-GFPAC3I, ko HSV-GFPAC3I-ΔFosB. A cikin wadannan haruffa, AC3I, mai maganin peptide na ayyukan CaMKII, an haɗa shi zuwa C-finus na GFP. GFPAC3I an rufe shi ta PCR ta yin amfani da pMM400-vector dauke dauke da GFPAC3I a matsayin samfuri tare da masu saiti na gaba: GFP-AC3I-F: 5 'CC GCTAGC GCCGCCACC ATGGTGAGCAAGGGCGAGGAGCTGT 3' (clampNheIKozakmet); GFP-AC3I-R: 5 'CC TCCGGA TTACAGGCAGTCCACGGCCT 3' (clampBspEIstop). An saka samfurin PCR a cikin shafukan p1005 + da p1005 + -Δ FosB ta amfani da shafukan NheI da BspEI. An gina wannan ginin ta hanyar zane-zane. Tsarin maganganu sune: 10 ° kusurwa, AP = + 1.6 mm, Lat = + 1.5 mm, DV = -4.4 mm (Barrot et al., 2002). Furofikan da sashin kwakwalwa ya kasance kamar yadda Experiment 4.

An yi nazari kan spine kamar yadda aka bayyana (Christoffel et al., 2011). A takaice dai, an cire sassan 50-150 μm daga soma daga cikin soma daga kamfanonin HSV wadanda ke nuna GFP. An samo hotunan a kan LSM 710 (Carl Zeiss) don nazarin morphological ta yin amfani da NeuronStudio tare da algorithm rayburst. NeuronStudio yayi la'akari da spines a matsayin mai mahimmanci, naman kaza, ko makirci bisa ga dabi'un da suka biyo baya: (1) rabo mai siffar, (2) kai zuwa rukuni na rukuni, da (3) shugaban diamita. Za'a iya rarraba spines tare da wuyansa a matsayin ko dai na bakin ciki ko naman kaza, kuma wadanda ba tare da wani gagarumar wuyansa ba an lasafta su ne. Spines tare da wuyansa ana lakafta kamar bakin ciki ko naman kaza bisa tushen diamita.

Figure 6

Blockade na CaMKII aiki ya hana ilimin halittar jiki da na hali na ΔFosB a NAc

Gwajin 16: Hanyoyin HSV-Mediated ΔFosB Cigaba da Mawuyacin CaMKII akan Maganin Cocaine Responses (Fig 6F)

An yi wa ƙwayar mata maza da ƙwayoyin cuta ƙwayar cuta kamar yadda Experiment 15, da kuma maganin locomotor zuwa guda 5 mg / kg inji na cocaine aka auna kamar yadda Gwaji 9. Ana bayyana bayanan Locomotor a matsayin ƙwaƙwalwar katako a kan 30 min bayan allurar cocaine.

ƙarin Bayani

Gidajen dabbobi

Dan rawan Sprague Dawley (250-275 da Charles Laboratories) sun kasance biyu. C57BL / 6J 'yan mata maza takwas (Laboratory Jackson) sun kasance da ƙananan dabbobi guda biyar a gidan. Duk dabbobin da aka saba wa dabbobin dabba don ≥Nwanin 1 kafin gwajin gwagwarmaya da kuma zama a cikin dakunan da ake sarrafawa (23-25 ° C) a kan 12 hr haske / duhu (hasken wuta a 7: 00 AM) tare da samun damar abinci da ruwa ad libitum. An gudanar da gwaje-gwaje bisa ga jagorancin kamfanin na Neuroscience da kuma kula da kula da dabbobin kula da dabba (IACUC) a Dutsen Sinai.

kwayoyi

An yi amfani da kwayoyi a cikin salin bakararre, ciki har da cocaine (5-20 mg / kg ta 10 μl ga ƙuda, ta 1 ml ga ratsi, NIDA) da kuma SCH 23390 ko eticlopride hydrochloride (0.5 mg / kg ta 1 ml, Tocris) . Don aikin tiyata, an yi amfani da mice tare da "cocktail" na ketamine (100 mg / kg) da kuma xylazine (10 mg / kg) (Henry Schein) a saline salin.

antibodies

Kayan (duka): Upstate 05-532, 1: 5,000

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

ΔFosB (duka): Alamar Sigina ta 5G4, 1: 250

ΔFosB phospho-Ser27: Phosphosolutions, 1: 500

GluA1 (duka): Abcam, Ab31232, 1: 1,000

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

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

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

NR2A: Sigma HPA004692, 1: 2,500

NR2B: Abokiyar Ab1557P, 1: 1,000

Nazarin ilimin lissafi

An gudanar da nazarin ilimin lissafi ta amfani da software na Prism 6 (GraphPad). An yi amfani da t-gwaji a kowane ɗan jarrabawa (wanda aka nuna a cikin Sakamakon inda aka ba t darajar), kuma an yi amfani da ANOVA guda daya don kwatanta jimloli (aka nuna a sashen binciken inda aka ba F darajar).

Ka tafi zuwa ga:

results

Cocaine na yau da kullum yana nuna CaMKII a cikin NAc Shell

Yawancin bincike sun nuna cewa MSN a cikin nau'in NAC da kuma mahimmanci suna da nau'o'in biochemical da kuma martani na nakasassu ga yawan maganin da ake amfani da su (Kourrich da Thomas, 2009; Loweth et al., 2010) da kuma cewa ƙungiyoyi biyu sun tsara tsarin da ake amfani da miyagun ƙwayoyi (Ito et al., 2004). Don ƙayyade abubuwan da ke tattare da hawan cocaine akan gine-ginen gina jiki NAC vs. ainihin, mun yi amfani da Isobaric Tagging (iTRAQ) da Multiemple Mass spectroscopy (MS / MS). An yi amfani da berayen maza da mata a cikin IP tare da maganin cocaine (20 mg / kg) ko salin yau da kullum don 7 kwanakin; 24 hr bayan da allurar rigakafi, NAC harsashi da kuma mahimmanci sun kasance microdissected (Fig 1A) da kuma walƙiya. Ana amfani da sunadarai a cikin waɗannan samfurori ta amfani da iTRAQ. Dukkan batutuwan CaMKII guda hudu sun nuna yawan karuwar bayanan bayan maganin maganin cocaine da aka ba da takaddama na NAc idan aka kwatanta da ainihin. Da dama sunadarai phosphatases, ciki har da PP1 catalytic da dokoki subunits da PP2A, wanda aka baya dangantaka da daban-daban CaMKII substrates a wasu tsarin (Colbran, 2004), ya bi irin wannan tsari. Wadannan binciken sun ba da labari, shaidar ba tare da bambance-bambance cewa hanya ta alama ta CaMKII an tsara ta da kyau ta hanyar cocaine a cikin NAc ba.

Don inganta wannan binciken fiye da yawanci, mun bi da berayen sama kamar cocaine (a cikin daban-daban) ko saline kuma auna sakamakon maganin locomotor zuwa cocaine (5 mg / kg) ko kalubalar saline. Magancewa mai maimaitawa zuwa 10 mg / kg cocaine ya haifar da yanayin halayen locomotor (Fig 1B). Bugu da ƙari da nazarin binciken da aka tsara da wannan tsari, ta hanyar yin amfani da Tsarin Yamma, wannan cocaine wanda ya ci gaba da haifar da CaMKII a cikin maɓallin NAC 24 hr bayan ingancin karshe na cocaine (Fig 1C da D; p = 0.0019; F = 7.943; df = 29). Bugu da ƙari, phosphorylation na magungunan CaMKII na maye gurbin Ser831 na GluA1 ƙarƙashin mai karɓa na AMPA ya karu sosai a cikin nau'in NAc kuma ba ainihin ba (p = 0.0261; F = 4.208; df = 28), yayin da CaMKIIa Thr286 autophosphorylation yana da ƙarfi amma ba babbar mahimmanci ga shigarwa a harsashi kawai (Fig 1D). Yawancin masu karɓar mahaukaci ba su da alamun. Ya bambanta da waɗannan matakan CaMKII, samfuran samfurori iri guda da aka nuna an shigar da ΔFosB a duka harsashi (p = 0.0260; F = 4.189; df = 29) da kuma ainihin (p = 0.0350; F = 3.807; df = 29) na NAC (Fig 1C da D), daidai da binciken da suka gabata (Perrotti et al., 2008).

Tun da yawa daga cikin nazarin ka'idar cocaine na masu sauraron AMPA sun binciki dabbobin bayan ~ 14 kwanakin janyewa daga cocaine na yau da kullum (duba Tattaunawa), mun sake maimaita wadannan nazarin halittun a wannan lokaci. Mun gano cewa, kwanakin 14 bayan an gama ingancin cocaine, ΔFosB ya kasance a cikin NAc (p = 0.0288; F = 4.258; df = 22), yayin da CaMKII ko phosphorylation na GluA1 Ser831 ya karu (Fig 1E). Duk da haka, 1 hr bayan nauyin nauyin kaya guda 10 na mg / kg na cocaine, matakan matakin Kayan Kaya (p = 0.0330; F = 3.947; df = 26) da na GluA1 Ser831 (p = 0.0213; F = 4.509; df = 27) phosphorylation suna dauke da su zuwa wani digiri kwatankwacin da aka gano bayan an fara hawan cocaine na kullum (Fig 1E). Wadannan bayanan sun nuna cewa nau'ikan kwalliya na NAc suna farawa ne don shigar da CaMKII a lokacin karin lokaci na abstinence, watakila ta hanyar yin amfani da kai tsaye na kamfani na CaMKII (duba Tattaunawa). Bugu da ƙari, gaskiyar cewa shigarwar ΔFosB ya fi tsayuwa fiye da shigarwar CaMKII yana nuna kasancewar wasu matakan, ko na tushen chromatin ko in ba haka ba, cewa yana yin "raguwa" akan ka'idar CaMKII, kamar yadda aka rufe a Tattaunawa.

Don kara ƙarfafa wannan ra'ayi, mun bincika samfurori na gwamnati da ke dauke da cutar cocaine, wanda ya haɗa da ciwon magunguna. An ba da berayen maza da mata ko kaɗan don samun damar cocaine; kamar yadda aka sa ran (Ahmed da Koob, 1998), hanyoyi masu tsawo ne kawai suka haifar da haɓaka mulkin gwamnati na miyagun ƙwayoyi (Fig 2A). ΔFosB ya haifar da mafi girma ta tsawon lokaci vs. Hanyar samun dama ga cocaine a duka nau'in NAc (p = 0.0011; F = 11.12; df = 17) da kuma ainihin (p = 0.0004; F = 13.86; df = 17). Sabanin haka, CaMKIIA ya samo asali a cikin NAc kawai ne kawai ta hanyar dogon lokaci zuwa cocaine (Fig 2B da C; p = 0.0236; F = 4.957; df = 16). Yana da ban sha'awa don kwatanta ciwon hawan cocaine na yau da kullum a cikin dabbobin da ba su da damar samun damar shiga (~ 12 mg / kg IV), dabbobi masu nisa da sauri (~ 70 mg / kg IV), da dabbobi masu sarrafa gwaji (10 mg / kg), da kuma tambayi dalilin da yasa hakan ya haifar dashi sosai na ΔFosB da CaMKII yayin da ba dama damar shiga ba. Wannan bambance-bambance yana iya haifar da bambance-bambance a cikin matakan hawan cocaine (cocaine mai aikin gwaji an ba shi a matsayin guda ɗaya na IP, yayin da ake amfani da cocaine wanda ake sarrafawa ta hanyar jinsin IV), ko kuma ta hanyar bambance-bambance a tsawon lokacin shan magani (kwanaki 7 don gwaji Gudanarwa, kwanakin 19 don kula da kai).

Duk da manyan wallafe-wallafe a kan ΔFosB da CaMKII a cikin aikin cocaine, babu wani bincike game da wadannan sunadarai a masu amfani da cocaine. A nan, mun gabatar da shaidar farko cewa matakan duka ΔFosB (p = 0.0316; t = 1.921; df = 34) da CaMKII (p = 0.0444; t = 1.755; df = 32) suna karuwa a NAc na mutane masu dogara da hawan cocaine (Fig 2D, Table 1). Wadannan bayanai sun nuna cewa jarrabawar ta ΔFosB da CaMKII ta hanyar cocaine a cikin rodent NAc yana dacewa da jaraba da hawan cocaine.

Table 1

Nuna samfurori daga samfurori na hawan cocaine da kuma ƙungiyar kulawa da ta dace

ΔFosB Yana Sarrafa Bayanan CaMKII Dalili a D1-Type MSN na NAc Shell

Sakamakon cewa duka CaMKII da ΔFosB sunadaita ta hanyar cocaine a cikin rodent NAc sun jagoranci mu don sanin ko ΔFosB zai iya tsara rubutun Kayan CaMKII. Mun riga mun yi rahoton CaMKII a matsayin mai yiwuwa ga ΔFosB a cikin wani bincike mai ban sha'awa na NAC (McClung da Nestler, 2003), amma wannan binciken bai ƙara ingantawa a wannan binciken ba. Mun fara amfani da Chitimitimitim (QChIP-ChIP ya biyo bayan PCR) don sanin ko ΔFosB yana daura ga mai gabatarwa na CaMKIIa a cikin NAc na ratsan rago mata, kuma ya gano cewa wannan nauyin ya karu sosai, ta hanyar cocaine na cocaine, a cikin harsashi ( p = 0.0133; t = 2.901; df = 12), amma ba ainihin ba, rukunin karkashin kasa (Fig 3A). Don ƙarin fahimtar hanyoyin da suka danganci wannan bambancin da ke ƙarƙashin tsarin mulki a cikin ΔFosB dauri ga mai amfani na CaMKIIa, mun yi amfani da qChIP don faɗar yanayin tarihin histone a wannan yanki. Binciken da aka gabatar na farko ya nuna kwantar da hankalin cocaine na H3 acetylation a cikin kasuwa na CaMKIIa a cikin dukkan nau'i nau'i NAC (Wang et al., 2010). Ya bambanta, mun gano cewa cocaine yana rage H3 acetylation a mai daukar hoto na CaMKIIa a cikin NAc core (Fig 3B; p = 0.0213; t = 2.726; df = 10), ba tare da canji da aka nuna a harsashi ba, daidai da gyare-gyare na chromatin da ke ƙarƙashin sauyin sauƙi fiye da ΔFosB. qChIP don alamar farfadowa, H3 lysine 9 dimethylated (H3K9me2), ya bayyana yanayin don ragewa a cikin harsashi da kuma manyan yankuna (Fig 3C).

Don ƙayyade ko ΔFosB ke sarrafa takardun CaMKIIa a vivo, mun yi amfani da layin linzamin kwamfuta guda biyu wanda ya fi dacewa da ΔFosB na musamman a cikin D1 vs. DNNUMX-type MSN a cikin hanyar sarrafawa ta hanyar doxycycline mulki a cikin ruwan sha (Chen et al., 1998; Kelz et al., 1999; Werme et al., 2002). Yarinya maza da mata masu tsinkaya ΔFosB kawai a cikin MSNs na D1 sun karu da matakan karfin mRNA na CaMKII a NAc (p = 0.0337; t = 1.996; df = 13), wani sakamako da ba a gani ba a cikin ƙwayoyin ΔFosB yawanci a cikin MSNs na D2 (Fig 3D). Ƙarawa a cikin mRNA CaMKII, wanda ΔFosB ya gabatar a cikin MSNs na D1, an haɗa shi tare da karuwa mai karuwa a cikin protein na CaMKIIa a cikin duka nau'in NAc (p = 0.0030; t = 3.578; df = 14) da kuma ainihin (p = 0.0392; t = 2.275; df = 14; Figs 3E da F). Wadannan bayanan sun nuna cewa ΔFosB na iya motsa kallon CaMKIIa a cikin DNNUMX-type MSNs a cikin bangarorin biyu, ko da yake Hoto na 3B ya nuna cewa sauye-sauye na chromatin da aka sanya a cocaine a cikin mai daukar hoto na CaMKIIa (misali, rage acetylation) ya hana ΔFosB daga karɓan CaMKII a cikin magunguna na tsakiya bayan cocaine.

Saboda bayanan linzamin mu na transgenic ya nuna cewa shigarwar ΔFosB na bayyanar CaMKII jigon halittar takamaiman D1-type MSNs a NAc, muna gaba don bincika ko halayyar cocaine dogaro da CaMKII yana buƙatar kunna mai karɓar D1 dopamine. An ba da berayen maza manya na cocaine ko saline kamar yadda suke a da, amma minti 30 kafin kowane allura, berayen da ke cikin cocaine an ba su allurar IP ta salin, D1 antagonist SCH 23390 (0.5 mg / kg), ko kuma mai karɓar mai karɓar D2 eticlopride (0.5 mg / kg). An bincika dabbobi 24 hr bayan allurar ƙarshe na hodar iblis. Yammacin Yammacin ya nuna cewa D1, amma ba D2 ba, mai adawa ya hana haɓakar cocaine a cikin ΔFosB (p <0.0001; F = 18.96; df = 18), kamar yadda aka ruwaito a baya (Nye et al., 1995), da kuma a cikin CaMKII (p = 0.0005; F = 10.99; df = 18; Fig 3G da H). Wadannan bayanan sun goyi bayan hypothesis cewa cocaine yana ƙaddamar da karuwar ΔFosB a cikin karfin CaMKII musamman a cikin MSNs na MSN na na NAc. Zai zama mahimmanci a binciken da za a yi a nan gaba don nuna kai tsaye wannan nau'i na musamman na kwayar cutar cocaine akan maganganun CaMKII a cikin wannan kwakwalwa.

ΔFosB yana da mahimmanci da isasshen abincin Cocaine na CaMKII a NAc Shell

Don yin amfani da mice bitransgenic, zamu ƙara nazarin aikin na ΔFosB a yunkurin maganin cocaine na CaMKIIa ta hanyar amfani da kwayar cutar ta hanyar sarrafawa ta kyama. Muna bilantattun kwayoyin cututtukan hoto (AAV) masu adeno da adadin adana mai kwakwalwa a cikin kwamincin NAc na ratsan rago mata (inda za'a iya ƙaddamar da harsashi) zuwa ga ΔFosB tare da GFP ko GFP kawai. An ba da dabbobi a asibiti guda daya na 10 mg / kg cocaine. Dabbobin da suka shawo kan ΔFosB / GFP sun nuna yawan karuwar locomotor da aka kwatanta da dabbobi na GFP kawai da ke nunawa (Fig 4A). 24 hr bayan ingancin cocaine guda daya, GFP-tabbatacce Naman nauyin NC ya karɓo daga waɗannan dabbobi ta hanyar rarrabawa a ƙarƙashin haske mai haske. Western blotting wannan nama (Fig 4B da C) ya nuna karfi mai karfi na ΔFosB da kuma karuwa mai yawa a cikin dukkanin kodin CaMKIIA idan aka kwatanta da dabbobin GFP (p = 0.0070; t = 2.894; df = 30), kamar kamawar da aka gani tare da gwamnatin cocaine na yau da kullum. Bugu da ƙari, an ƙarfafa magunguna ta Thr286 (nuna alamar amsawar enzyme) ta ΔFosB overexpression (p = 0.0330; t = 2.243; df = 28), kamar yadda phosphorylation na CaMKII substrate, Ser831 na GluA1 (p = 0.0540; t = 2.012; df = 28), sa'an nan maimaita ayyukan da cocaine na yau da kullum (Fig 1C da D). TA haɗe tare, waɗannan bayanan sun bada ƙarin shaida cewa bayyanar ΔFosB a harsashin NAc shine isasshen fahimtar locomotor zuwa cocaine kuma don shigar da CaMKII da kunnawa a cikin wannan rukunin karkashin kasa.

Mun yi amfani da irin wannan tsari don sanin ko ΔFosB ma wajibi ne don shigar da cacaine a cikin CaMKII a cikin harsashin NAc. AAV an yi amfani da shi don nuna nauyin sunadarai JunD, wanda ake kira ΔJunD, wanda shine mai kula da ΔFosB izinin shiga (Winstanley et al., 2007) tare da GFP ko GFP kadai. Makonni biyu bayan haka, lokacin da kalma mai karfin jini ya fi iyaka, ana ba da dabbobi ga cocaine (10 mg / kg) kowace rana don 7 kwanakin, kuma an gwada su don maganin locomotor zuwa kalubalen cocaine (5 mg / kg) 24 hr bayan daji na karshe (Fig 4D). ΔJunD rashin wahalar da aka hana ya hana fahimtar locomotor zuwa cocaine, kuma ya hana CaMKIIa shigarwa da kunnawa a harsashi na NAc (Fig 4E da F; p = 0.0437; F = 2.997; total df = 38), yana nuna cewa aiki na ΔFosB ya zama dole don shigar da cacaine a cikin CaMKII a cikin wannan rukunin ƙasa. Abin sha'awa ne, mun gano cewa ΔJunD ya rage ragowar ΔFosB a ƙarƙashin yanayin salin da yanayin hawan cocaine (p = 0.0004; F = 8.110; df = 35), ƙaddara yiwuwar yiwuwar cewa ΔFosB ya dogara da aikin ap-1 don matakan nuna kansa.

CaMKII Csphorylates ΔFosB a Ser27

Amfani a vitro sunadarai kinase, mun ƙaddara cewa ΔFosB mai tsabta shi ne matsakaici mai karfi ga CaMKII. Tsayar da shi₆-ΔFosB tare da CaMKIIa da ATP sun haifar da ƙaura zuwa sama a cikin motsi na electromagnetic na ΔFosB (Fig 5A); da yawa sakamakon kuri'a nuna shafukan yanar gizo na phosphorylation. Haka ma a vitro yunkurin kinase ta amfani da [γ-³²P] ATP ya nuna kamfanonin phosphate da aka yiwa radiolabeled cikin ƙungiyar ΔFosB da aka canza (Fig 5B), kwatanta phosphorylation ta tsakiya na gina jiki. Mun kirkiro wani antibody-specific antibody zuwa a halin yanzu halin Ser27 na ΔFosB (Ulery et al., 2006). Duk da yake wannan mai cutar ba ya samar da siginar ta hanyar kwakwalwar ƙwaƙwalwar kwakwalwa wanda ya ƙunshi Ser27-phosphorylated ΔFosB (bayanan ba a nuna ba), mun sami damar gano Ser27 phosphorylation a cikin a vitro Kwanin kinase ta amfani da CaMKII (Fig 5B). Nazarin kwayoyin halitta na CaMKII phosphorylation na ΔFosB ya nuna cewa abu ne mai matukar muhimmanci ga kinase (Fig 5C), tare da wani fili K_M na 5.7 ± 2.0μM da K_CAT na 2.3 ± 0.3min^-1. Wadannan sakamakon sunyi kama da mutane da yawa a vivo substrates na CaMKII (Colbran da Brown, 2004). Bugu da kari, mun ƙaddara cewa CaHKII phosphorylates ΔFosB tare da stoichiometry na 2.27 ± 0.07 mol / mol (Fig 5D), yana nuna cewa akwai akalla shafuka uku na CaMKII phosphorylation a cikin sa₆-NFFB, a cikin yarjejeniya da Fig 5A.

Don bincika wuraren yanar gizo na phosphorylation, muna amfani da bincike na MS na samfurori daga mu a vitro Kwanin kinase. Fig 5E yana nuna ΔFosB phosphorylation a baya halin Ser27 da kuma a wasu ƙarin shafukan yanar gizo (bayanan ba a nuna) ba. Dangane da halayyar aiki na Ser27, mun mayar da hankalinmu a kan wannan shafin ta hanyar samar da peptides na kirji mai suna rubutun phospho- da nonphospho-jihohin Ser27, sa'an nan kuma amfani da yawancin waɗannan peptides a matsayin ka'idodin bincike na MRM na ΔFosB kafin da bayan a vitro phosphorylation da CaMKII. Ƙayyadewa na ƙarshe (Fig 5F) ya tabbatar da cewa Ser27 wani abu mai mahimmanci ne ga CaMKII. Wadannan sakamakon sun nuna cewa, a tsakanin maɓuɓɓuka maras amfani da phosphorylated a cikin ΔFosB, Ser27 shine tasiri mai mahimmanci ga CaMKII.

Cibiyar Cocaine ta Harkokin Cocaine na ΔFosB a cikin NAc Shell

Tunda CaMKII zai iya samfurin ΔFosB a vitro a wani shafin da ke bunkasa zaman lafiyarta sosai a vitro da kuma a vivo (Ulery et al., 2006; Ulery-Reynolds et al., 2009), mun ƙaddara ko ayyukan CaMKII na sarrafa ΔFosB a cikin NAc a vivo. Don magance wannan tambaya, mun fara yin amfani da layin linzamin kwamfuta wanda ke nuna maƙasudin mutuncin CaMKIIa (T286D) a cikin ƙwayoyin kwakwalwa masu yawa ciki har da NAc (Mayford et al., 1996; Kourrich et al., 2012). Mun yi allurar shekaru-wanda ya dace da mutun namiji da yaran da ke dauke da 20 mg / kg cocaine ko saline sau ɗaya a kowace rana don 14 kwanakin, sa'annan ya binciki dabbobi a rana daya bayan ingancin karshe. Mun gano cewa an kara yawan matakan ΔFosB a dabbobin mutun a cikin nau'in NAc (p = 0.0001; F = 9.207; df = 37), amma ba ainihin (Fig 5G da H). Abin mamaki shine, an katange kwakwalwa na ΔFosB a cikin dabbobin mutun a cikin harsashi da kuma mahimmanci, yana nuna cewa, kodayake CaMKII zai iya daidaita tsarin kwastar ΔFosB a cikin harsashin NAc, yana iya kasancewa kusa da ΔFosB a cikin hanyoyin da aka kunna a cocaine a cikin dukkanin hukumomi na NAc .

Kayan aiki na buƙatar aiki ne don ΔFosB-Mediated Structural and Behavioral Plasticity

Yin maganin cocaine na kwaskwarima a kan NAc MSN shine daya daga cikin maganganun da aka samo asali a cikin wannan kwakwalwa, kuma an haɗa su tare da fahimtar halayen halayyar maganin miyagun ƙwayoyi (Robinson da Kolb, 2004; Russo et al., 2010) kuma sun yi rahoton cewa za su zabi ga MSNs na D1 (Lee et al., 2006). Mun nuna a kwanan nan cewa ingancin cocaine na shinge na dendritic a cikin NAc yana dogara ne akan ΔFosB da tsarin sa na gaba (Maze et al., 2010). Ko da yake akwai littattafai masu yawa game da shigar da CaMKII a cikin ilimin lissafi na dindritic da kuma shigarwa a wasu sassan kwakwalwa da tsarin gwaji (Jourdain et al., 2003; Penzes et al., 2008; Okamoto et al., 2009), aikinsa na NAC MSIN ba a binciken shi ba. Sabili da haka, mun ƙaddara ko Ana bukatar CaMKII don ƴancin ΔFosB na sakawa na MSN dendritic ta hanyar yin amfani da HSV ta hanyar yin amfani da HSV ta captic peptide AC3I wanda aka sanya shi zuwa GFP, wani aikin da aka nuna a baya ya hana aikin CaMKII a vivo (Zhang et al., 2005; Klug et al., 2012). Ralwayar hoto ta ΔFosB a cikin NAc harsashi na ƙananan beraye ya haifar da ƙaruwa mai yawa a cikin ƙimar ƙwanƙwashin ƙwayar MSN (p <0.0001; F = 8.558; df = 59; Fig 6A da B) kamar yadda a baya ya ruwaito (Maze et al., 2010), kuma wannan ƙimar ta ƙaddamar da ƙananan ta bakin ciki (p = 0.0027; F = 5.319; df = 59) da kuma stubby (p = 0.0378; F = 2.988; df = 59) nau'in launi (duka zaton su zama marasa tsabta)Fig 6C-E). Babu wani sakamako da aka gani a kan mafi girma, naman kaza-dimbin yawa spines. Duk da haka, a lokacin da aka kwashe GFP-AC3I, an cire shafe ΔFosB na spines gaba ɗaya (Fig 6A-E), yana nuna cewa ana buƙatar aikin CaMKII don ΔFosB shigar da spendendic spines a cikin NAc harsashi.

Mu na gaba sunyi amfani da kayan aikin hoto na musamman domin sanin ko ana bukatar CaMKII aikin azabar ΔFosB akan halayyar halayyar hawan cocaine. 72 Hr bayan ankarar kwayar cutar a cikin kwamincin NAc, ana ba da dabbobi iri ɗaya na 5 mg / kg cocaine kuma an rubuta aikin aikinsu na locomotor. Kamar yadda aka nuna a baya tare da karin kallo na AAV na ΔFosB (Fig 4A), HSV-mediated overexpression na ΔFosB ƙara yawan locomotor hankali ga cocaine (p = 0.0002; F = 8.823; df = 37; Fig 6F). Kamar yadda yake shigar da shinge na dendritic, hanawa na CaMKII ta hanyar maganin GFP-AC3I ya katange karuwar karuwar ΔFosB a sanadin hawan cocaine, yana nuna cewa ana buƙatar aiki na CaMKII don sauya ΔFosB a cikin halayen halayyar hawan cocaine.

Ka tafi zuwa ga:

tattaunawa

Binciken da ke cikin yanzu ya tsara wani nau'in kayan aiki na yau da kullum inda cocaine ke haifar da ΔFosB a NAc, wanda ya sauke rubuce-rubuce na Kayan CaMKIIa a cikin nauyin NAc. CaMKIIa daga bisani phosphorylates da kuma tabbatar da ΔFosB da ke haifar da girma ΔFosB ƙaddara kuma don ƙara CaMKIIa shigarwa (Fig 6G). Matakan haɓakawa na sunadarai guda biyu a yayin da ake nunawa ga cocaine sa'an nan kuma taimakawa cikin hanyoyi masu kyau don fahimtar halayen halayyar halayyar maganin miyagun ƙwayoyi. Wannan ambato ne mai ban sha'awa kamar yadda duka ΔFosB da CaMKII sun nuna a baya don an buƙata domin ƙara yawan halayen halayyar halayyar hakar haɗi (Pierce et al., 1998; Peakman et al., 2003), kuma zamu sake gwada wannan binciken na ΔFosB a cikin NAc harshe musamman ta amfani da magungunan hoto (Figs 4 da kuma Da kuma 66).

Kodayake siffofin ΔFosB a cikin D1-type MSN zasu iya fitar da caMKII a duka nau'in NAc da kuma ainihin dabbobin cocaine-naïve, a cikin cocaine, haɗuwa da ΔFosB na ƙarshe, wanda ke faruwa a cikin bangarorin biyu, ƙaddamar da CaMKII musamman a cikin harsashin NAc . Wannan bambanci zai iya danganta da matakai mafi girma na ΔFosB a cikin tsarin bitransgenicmu, duk da haka, zai iya nuna yiwuwar cocaine don canza bambancin CaMKIIa a kwasfa vs. ƙananan MSN don inganta korar ΔFosB a cikin tsohon ko kuma ware shi a cikin rukunin karkashin kasa. A gaskiya ma, bayanan ChIP, wanda ya nuna rikice-rikicen maganin cocaine na tarihi a masarautar CaMKIIa a cikin NAc kawai, yana goyon bayan yiwuwar yiwuwar tsarin tsarin chromatin. Dangane da wannan tsinkaya, ΔFosB overexpression a cikin MSNs na D1 ya iya fitar da CaMKIIa a cikin motsin NAc idan babu cocaine (Fig 3F), suna nuna cewa akwai gyare-gyaren da aka yi na Caotariya mai talla wanda zai hana wannan ƙaddamar yayin yaduwar cutar cocaine. Dokar tsarin wuri na chromatin a mai gabatarwa na CaMKII zai iya bayyana dalilin da ya sa CaMKII ya jawo shi ta hanyar kalubalanci na cocaine a cikin nauyin NAc na kwakwalwa na cocaine na yau da kullum -Fig 1E) amma ba na dabba-miyagun kwayoyi ba (Fig 1D). Wannan zai iya wakiltar sakamako na "samfurin" na ΔFosB (epigenetic)Robison da Nestler, 2011), kuma zai iya kasancewa ɗaya daga cikin kwayoyin halitta na incubation na cocaine craving (Pickens et al., 2011). Duk da haka, saboda wannan canzawar chromatin ya zama abin haɗari da haɗuwa da shiryawa da sha'awar sha'awa, to yana da ƙaruwa a tsawon lokaci. Zai zama mai ban sha'awa don sanin ko wannan shi ne yanayin, kuma don nazarin ko wasu kwayoyin suna nuna ΔFosB-dogara, ka'idoji na ƙaddamarwa ta hanyar cocaine. Yana da mahimmanci a lura cewa ƙaddamar madauki da muke bayyana baya haifar da ƙarancin tara na CaMKII ko ΔFosB (Fig 1E); Rashin kwalliyar "ƙaddamar" kwayoyin "wannan" muhimmin mahimmanci ne na bincike na gaba.

Ayyukan da aka sani na ΔFosB da CaMKII a cikin tsarin gwaje-gwajen da yawa da kuma ƙwararrun kwakwalwa sun haɗa zuwa matakan da dama (Fig 6F). Dukkan kwayoyin biyu suna da nasaba da haɓaka spine dendritic: CaMKII tana hulɗa da cytoskeleton actin (Okamoto et al., 2009), yana ƙaddamar da girman girman goshi (Matsuzaki et al., 2004), kuma yana da mahimmanci kuma ya isa don haɓakar filastik-haɓaka ƙãra a cikin ƙaura da kuma ɓarna a cikin tsarin kwayoyin halitta na hippocampal (Jourdain et al., 2003), wƁoye ΔFosB yana da mahimmanci kuma ya isa don samfurori na dendritic kafa a cikin NAC MSNs (Maze et al., 2010). Bugu da ƙari, dukkanin kwayoyin sun hade da tsari na masu karɓa na AMPA glutamate. CaMKII ba ya tsara jimillar matakan AMAP masu karɓa ba, amma yana motsa shigar da masu karɓa na AMPA a cikin synapses kuma yana kara tashar tashar AMPA ta phosphorylating GluA1 a Ser831 a cikin ƙananan igiya na hippocampal a al'ada da a vivo (sake dubawa (Malinow da Malenka, 2002; Colbran da Brown, 2004)). Irin wannan fataucin da ake samu na GluA1 zuwa gawarwakin ya shafi aikin cocaine na yau da kullum (Boudreau da Wolf, 2005). Bugu da ƙari, haɓakar haɓaka ta AMPA a activation a cikin NAc an inganta shi ta hanyar ƙari na caMKIIa a cikin hanyar D1 dopamine-dependent (Singer et al., 2010). D1-musamman takaddama na ΔFosB an nuna su haifar da rubutun GluA2 a NAc (Kelz et al., 1999), wanda ya shafe amfanar AMPA ta hanyar GluA1, yayin da muka nuna a nan cewa gajeren lokacin ΔFosB na nunawa-da kuma cin zarafin cocaine gajarta-ba su da wani tasiri kan wannan subunit (Fig 1). Duk da haka, mun sami kwanan nan cewa ɗan gajeren lokacin ΔFosB rashin nasara duk da haka rage martani AMPA a cikin MSNs na MSN a cikin NAc (Grueter et al., 2013). Wadannan bayanan sun bada ra'ayi na musamman na jiki wanda zai iya kasancewa jerin lokuttan neuroadaptations zuwa cocaine wanda ke haifar da hanyoyi daban-daban na cigaba da cigaba ba a fahimta ba. A matakin halayen, ana buƙatar CaMKII da ΔFosB don haɓakar locomotor zuwa cocaine (duba sama), kuma ana buƙatar duka biyu don ci gaba da kula da hawan cocaine a cikin rodents (Colby et al., 2003; Wang et al., 2010), yana nuna cewa sunadarai guda biyu suna da muhimmanci ga halayyar halayyar da ke cikin gajeren lokaci da kuma dogon lokacin da ake yin amfani da miyagun ƙwayoyi, duk da haka ta hanyar bangarori daban-daban. Mai yiwuwa, ΔFosB da CaMKII suna tsara irin wannan halayyar halayyar halayyar ta hanyar canje-canje a aikin NAC na synaptic, kodayake ana bukatar ƙarin aiki don danganta haɗin synaptic zuwa halayyar hali.

Hakanan na CaMKII holoenzyme yayi hulɗa da juna tare da wasu nau'o'in sunadarai masu haɗuwa da juna (Robison et al., 2005) wanda ake tsammani ya tsara dabarunsa zuwa ƙananan postsynaptic (PSD), wani abu da aka ba da shawara don zama da muhimmanci ga filastan synaptic. Musamman ma, hulɗar da CaMKII tare da gluN2B subunit na mai karɓa na glutamate na NMDA an nuna shi a kwanan nan don daidaita duka nauyin synaptic da ilmantarwa (Halt et al., 2012). Yayin da peptide na AC3I yana ƙaddamar da yankin na CaMKII, kuma ta haka ne ya hana aikin haɗari na enzyme, shi ma yana ƙulla dangantaka da haɓakar furotin-furotin da yawa (Strack et al., 2000; Robison et al., 2005). Saboda haka, sakamakon halayyar HSV-GFP-AC3I da aka gano a nan zai iya faruwa ta hanyar rage phosphorylation daga proteins masu ci gaba na CaMKII, canje-canje a cikin shirin CaMKII, ko canji a cikin tasirin da aka tsara na CaMKII a synapses (Lisman et al., 2002).

Hanyar ƙaddamarwa na ΔFosB-CaMKII zuwa ma'anar NAC na da ƙwarewa ta musamman, kamar yadda aikin kwanan nan ya nuna yawancin bambance-bambancen physiological a tsakanin ginin NAc da kuma ainihi saboda amsawar cocaine, wani ra'ayi ya tabbatar da bayanan iTRAQ (Table S1) ba tare da bambanci ba. . MSNs a cikin kwamincin na NAc suna nuna damuwa a cikin ƙarfin kisa bayan hawan cocaine na yau da kullum wanda aka dakatar da makonni, yayin da MSNs na asali daga cikin dabbobin guda suna nuna wani lokaci (1-3 day) karuwa a cikin ƙarfin kullun da ya dawo zuwa matakan basal a cikin 2 makonni (Kourrich da Thomas, 2009). Bugu da ƙari, yawancin sunadaran synaptic an tsara su ne a tsarin NAc vs. magungunan dabbobi da ke nuna wa cocaine na yau da kullum, ciki har da GluA2 (Knackstedt et al., 2010). Kamar yadda amphetamine na yau da kullum ya haifar da CaMKIIa musamman a NAc shell (Loweth et al., 2010), ba abin mamaki ba ne cewa muna samun sakamako irin wannan tare da cocaine. Duk da haka, kamar yadda ΔFosB ke haifar da duka nauyin NAC da kuma mahimmancin cocaine na yau da kullum (Perrotti et al., 2008), kuma tun lokacin da muka nuna cewa ƙaddamarwa ta CaMKIIa shi ne ΔFosB-dogara, bincikenmu yana samar da sabon shaidun da ke tattare da hanyoyin sadarwa ta hanyar CaMKIIa tsakanin waɗannan bangarorin biyu, wanda ke da alhakin zaɓin shigarwa na CaMKIIa a harsashi.

Ayyukan da suka shafi kwanan nan sun mayar da hankalin akan bambance-bambancen da ke tsakanin D1- da D2-type NAc MSNs. Ko da yake duka masu karɓa na D1 da D2 suna da hannu a sakamakon sakamako na cocaine (Kai, 2010), Ayyuka na baya-bayan nan sun nuna cewa samfurin MSP na D1 yana ƙara haɓakar halayyar halayyar hawan cocaine, yayin da D2-type MSN kunnawa yana da ƙananan sakamako (Lobo et al., 2010). Dangane da wadannan binciken, ƙananan ƙwararrun ƙwayoyin D1-receptor ba su da karfin samun karfin gwanin cocaine (Caine et al., 2007), yayin da D2 knockouts ba (Caine et al., 2002). Gwamnatin ta D1 ta kai tsaye a cikin NAc tana haifar da halayyar hawan cocaine a dabi'ar sake dawowa (Kai, 2010). Abin sha'awa, wannan sakamako yana bukatar ƙimar D1-receptor-dogara a cikin aikin CaMKII a cikin harsashin NAc, amma ba ainihin ba (Anderson et al., 2008), sakamakon da ya yi amfani da shi sosai tare da ƙaddamar da ΔFosB-CaMKII na musamman na D1- da harsashi.

Mun riga mun ruwaito cewa Ser27 a ΔFosB za a iya samuwa ta hanyar casein kinase-2 (phosphorylated)Ulery et al., 2006), duk da haka, mun kafa a nan cewa CaMKII phosphorylates ΔFosB a wannan kuma wasu shafuka tare da mafi girma daga cikin abubuwan da ke ciki da kuma stoichiometry kuma za su iya yin kama da mafi girma M m_r An lura da ΔFosB (Fig 5A) tare da haɗin gwaninta a vivo (Nestler, 2008). Mun riga mun san cewa Ser27 phosphorylation yana ƙarfafa zaman lafiyar ΔFosB da aiki na rubutu (Ulery et al., 2006; Ulery da Nestler, 2007; Ulery-Reynolds et al., 2009). Ayyukan gaba za su mayar da hankali a kan ganewa da kuma sakamakon aiki na shafukan intanet na ΔFosB phosphorylation wanda binciken yanzu ya nuna.

Hanyar da aka tanadar da abinci wanda aka kwatanta a nan ya samar da sabon tsarin da wanda ke da magungunan cocaine ke tafiyar da ciwo mai mahimmanci a cikin NAc. Saboda haka, wannan hanya na biochemical zai iya samar da wata muhimmiyar mahimmanci ga magungunan warkewa a cikin magungunan addictive. Saboda CaMKII yana da cikakke da ake buƙata don ƙananan ƙananan neuronal da ayyuka na hali, amfani da kai tsaye na masu hanawa na CaMKII a matsayin magani. Bayananmu suna nuna cewa mafi mahimmanci da aka tsara game da tsarin aikin CaMKII, wanda ya kebanta da nau'in tantanin halitta da subregion na zagaye na ladaran ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwa, zai iya samar da magungunan warkewa wanda zai guje wa rikitarwa na hanawa ta CaMKII.

Ka tafi zuwa ga:

Godiya

Wannan aikin ya goyan bayan taimakon da Cibiyar Kasa ta Kasa ta Kasa (EJN), NIDA-Yale Proteomics Centre DA018343 (AJR da EJN), da Hartwell Foundation (AJR). Masu marubuta suna so su gode Gabby Rundenko don kyautar kyautar kyautar ΔFosB da Roger Colbran don kyautar kyautar CaMKIIa mai tsabta.

Ka tafi zuwa ga:

References

1. Ahmed SH, Koob GF. Tsarin daga matsakaici zuwa ciwon magungunan miyagun ƙwayoyi: canzawa a yanayin da yake tsaye. Kimiyya. 1998; 282: 298-300. [PubMed]
2. Anderson SM, Shahararren KR, Sadri-Vakili G, Daresan V, Schmidt HD, Bass CE, Terushiger EF, Cha JH, Pierce RC. CaMKII: wani gadar biochemical da ke haɓaka dopamine da tsarin glutamate a cikin binciken cocaine. Nat Neurosci. 2008; 11: 344-353. [PubMed]
3. Boudreau AC, Wolf ME. Hanyar fahimtar juna ga cocaine yana haɗuwa da ƙarar adadi mai karɓa ta AMPA a cikin ƙwayar mahaifa. J Neurosci. 2005; 25: 9144-9151. [PubMed]
4. Brickey DA, Colbran RJ, Fong YL, Soderling TR. Magana da halayyar alpha-subunit na Ca2 + / amincin sunadarin protease na II da amfani da tsarin maganin baculovirus. Biochem Biophys Res Commun. 1990; 173: 578-584. [PubMed]
5. Caine SB, Negus SS, Mello NK, Patel S, Bristow L, Kulagowski J, Vallone D, Saiardi A, Borrelli E. Matsayi na dopamine D2-kamar masu karɓa a cocaine kai kai mulki: nazarin tare da D2 mutant mice mutant da kuma littafin D2 receptor antagonists. J Neurosci. 2002; 22: 2977-2988. [PubMed]
6. Caine SB, Thomsen M, Gabriel KI, Berkowitz JS, Gold LH, Koob GF, Tonegawa S, Zhang J, Xu M. Ba tare da kula da kansu na cocaine ba a cikin kwayar ƙwaƙwalwar ƙwararrawa ta dopamine D1. J Neurosci. 2007; 27: 13140-13150. [PMC free article] [PubMed]
7. Carle TL, Ohnishi YN, Ohnishi YH, Alibhai IN, Wilkinson MB, Kumar A, Nestler EJ. Abubuwan da suka dace da kare lafiyar-da-da-kai ga FosB: samowa na yankunan FosB degron da kuma abubuwan da suka shafi zaman lafiyar DeltaFosB. Eur J Neurosci. 2007; 25: 3009-3019. [PubMed]
8. Chen J, Kelz MB, Zeng G, Sakai N, Steffen C, Shockett PE, Picciotto MR, Duman RS, Nestler EJ. Dabbobi masu dauke da kwayar halitta tare da ƙaddamarwa a cikin kwakwalwa. Mol Pharmacol. 1998; 54: 495-503. [PubMed]
9. Christoffel DJ, Golden SA, Dumitriu D, Robison AJ, Janssen WG, Ahn HF, Krishnan V, Reyes CM, Han MH, Manyan JL, Eisch AJ, Dietz DM, Ferguson D, Neve RL, Greengard P, Kim Y, Morrison JH , Russo SJ. IkappaB kinase yana jagorancin matsalolin zamantakewar al'umma da aka haifar da synaptic da halayyar hali. J Neurosci. 2011; 31: 314-321. [PMC free article] [PubMed]
10. Colbran RJ. Mactivation na Ca2 + / amincin sunadaran gina jiki ta jiki daga basal autophosphorylation. J Biol Chem. 1993; 268: 7163-7170. [PubMed]
11. Colbran RJ. Protein phosphatases da alli / calcium / calcium-dependent protein kinase II-dogara synaptic plasticity. J Neurosci. 2004; 24: 8404-8409. [PubMed]
12. Colbran RJ, Brown AM. Kwayoyin calcium / amincin sunadarin protease da kuma maganin synaptic. Curr Opin Neurobiol. 2004; 14: 318-327. [PubMed]
13. Colby CR, Whisler K, Steffen C, Nestler EJ, Kai DW. Ƙararren ƙwayoyin sirri na musamman na DeltaFosB yana ƙarfafa haɗin cocaine. J Neurosci. 2003; 23: 2488-2493. [PubMed]
14. Covington HE, 3rd, Maze I, LaPlant QC, Vialou VF, Ohnishi YN, Berton O, Fass DM, Muryar W, Rush AJ, 3rd, Wu EY, Ghose S, Krishnan V, Russo SJ, Tamminga C, Haggarty SJ, Nestler EJ. Ayyukan antidepressant na masu tarihi na histone deacetylase. J Neurosci. 2009; 29: 11451-11460. [PMC free article] [PubMed]
15. Davalos A, Fernandez-Hernando C, Sowa G, Derakhshan B, Lin MI, Lee JY, Zhao H, Luo R, Colangelo C, Sessa WC. Tsare-tsaren masana'antun sunadaran sunadarai-1-sunadarai: halayyar polymerase i da kundin bayanan wallafe-wallafen / CAVIN-1 IN cikin kwayoyin endothelial. Mol Cell Proteomics. 2010; 9: 2109-2124. [PMC free article] [PubMed]
16. Dumais A, Tatsun AD, Alda M, Rouleau G, Dumont M, Chawky N, Roy M, Mann JJ, Benkelfat C, Turecki G. Yanayin haɗari don kashe kansa a cikin manyan matsaloli: nazari game da rikici da rikice-rikice a cikin maza. Am J Zuciyar. 2005; 162: 2116-2124. [PubMed]
17. Grueter BA, Robison AJ, Neve RL, Nestler EJ, Malenka RC. ΔFosB na sha'ani daban-daban na tsakiya accumbens ta hanyar kai tsaye da kai tsaye. Sanar Natl Acad Sci Amurka. 2013 a latsa. [PMC free article] [PubMed]
18. Halt AR, Dallapiazza RF, Zhou Y, Stein IS, Qian H, Juntti S, Wojcik S, Brose N, Silva AJ, Jahannama JW. CaMKII dauri ga GluN2B yana da mahimmancin lokacin ƙarfafawa na ƙwaƙwalwa. EMBO J. 2012; 31: 1203-1216. [PMC free article] [PubMed]
19. Hiroi N, Brown JR, Haile CN, Ye H, Greenberg ME, Nestler EJ. FosB mutant mice: asarar cocaine na yau da kullum shigar da sunadarai masu dangantaka da Fos da haɓaka ƙwarewa ga psychomotor na cocaine da sakamakon sakamako. Kamfanin Natl Acad Sci US A. 1997; 94: 10397-10402. [PMC free article] [PubMed]
20. Ito R, Robbins TW, Everitt BJ. Gudanar da bambanci game da halayyar hawan cocaine ta hanyar haɓakacciyar zuciya da harsashi. Nat Neurosci. 2004; 7: 389-397. [PubMed]
21. Jorissen HJ, Ulery PG, Henry L, Gourneni S, Nestler EJ, Rudenko G. Dimerization da kuma DNA-dauri kaddarorin na takardu factor DeltaFosB. Biochemistry. 2007; 46: 8360-8372. [PubMed]
22. Jourdain P, Fukunaga K, Muller D. Calcium / amincin sunadaran protein din din II yana taimakawa wajen bunkasa ci gaba da aikin gyaran ƙwayar cuta. J Neurosci. 2003; 23: 10645-10649. [PubMed]
23. Kelz MB, Chen J, Carlezon WA, Jr, Whisler K, Gilden L, Beckmann AM, Steffen C, Zhang YJ, Marotti L, Kai DW, Tkatch T, Baranauskas G, Surmeier DJ, Neve RL, Duman RS, Picciotto MR, Nestler EJ. Magana game da batun sakonnin deltaFosB a cikin kwakwalwa yana kula da hankali ga cocaine. Yanayi. 1999; 401: 272-276. [PubMed]
24. Klug JR, Mathur BN, Kash TL, Wang HD, Matthews RT, Robison AJ, Anderson ME, Deutch AY, Ƙaunar DM, Colbran RJ, Winder DG. Tsarin Halitta na Kayan Kayan Kayan Kayan Kayan Kayan Kwaƙwalwar Kasuwanci Na Ƙasa Kasa Ƙarƙashin Ƙaƙwalwar Ayyukan Kasuwanci da Ƙarfafa Ƙarƙashin Ƙari. PLoS Daya. 2012; 7: e45323. [PMC free article] [PubMed]
25. Knackstedt LA, Moussawi K, Lalumiere R, Schwendt M, Klugmann M, Kalivas PW. Ƙararruwar horo bayan ginin gine-gine yana haifar da filastik filaye don hana karfin cocaine. J Neurosci. 2010; 30: 7984-7992. [PMC free article] [PubMed]
26. Kourrich S, Thomas MJ. Mudduka masu kama da juna, ba tare da sauye-sauye ba: kwarewa ta psychostimulant ya bambanta da kullun kaya a cikin ginshiƙan da ke cikin harsashi. J Neurosci. 2009; 29: 12275-12283. [PMC free article] [PubMed]
27. Kourrich S, Klug JR, Mayford M, Thomas MJ. AMPAR-Sakamakon Independent AlphaCaMKII na Gabatar da Ƙaddamar da Gwanin Cocaine. J Neurosci. 2012; 32: 6578-6586. [PMC free article] [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 . Matsayi na makaman nukiliya factor kappaB a cikin ganyayyaki na mace-mace-mai rikon kwakwalwa a cikin ƙananan mata. Biol Babban ilimin zuciya. 2009; 65: 874-880. [PMC free article] [PubMed]
29. Lee KW, Kim Y, Kim AM, Helmin K, Nairn AC, Greengard P. Cocaine sunyi samuwa a cikin D1 da kuma D2 wanda ke dauke da matsakaici na ƙwayoyin spiny a tsakiya na accumbens. Kamfanin Natl Acad Sci US A. 2006; 103: 3399-3404. [PMC free article] [PubMed]
30. Lisman J, Schulman H, Cline H. Tsarin kwayoyin halitta na CaMKII a cikin synaptic da kuma ƙwaƙwalwar hali. Nat Rev Neurosci. 2002; 3: 175-190. [PubMed]
31. Lobo MK, Covington HE, 3rd, Chaudhury D, Friedman AK, Sun H, Damez-Werno D, Dietz DM, Zaman S, Koo JW, Kennedy PJ, Mouzon E, Mogri M, Neve RL, Deisserot K, Han MH, Nestler EJ. Sashin ƙwayar salula na BDNF yana nuna mimics optogenetic iko na hawan cocaine sakamako. Kimiyya. 2010; 330: 385-390. [PMC free article] [PubMed]
32. Loweth JA, Baker LK, Guptaa T, Guillory AM, Vezina P. Nunawa na CaMKII a cikin tsakiya na ƙaddara harsashi yana rage karuwar amphetamine da aka samu a cikin ratsi masu mahimmanci. Neurosci Lett. 2008; 444: 157-160. [PMC free article] [PubMed]
33. Loweth JA, Singer BF, Baker LK, Wilke G, Inamine H, Bubula N, Alexander JK, Carlezon WA, Jr, Neve RL, Vezina P. Hanyar da ke ciki na Ca2 + yana inganta halayyar amphetamine. J Neurosci. 2010; 30: 939-949. [PMC free article] [PubMed]
34. Malinow R, Malenka RC. Fataucin masu karɓar ragamar AMPA da kuma filastan synaptic. Annu Rev Neurosci. 2002; 25: 103-126. [PubMed]
35. Matsuzaki M, Honatana N, Ellis-Davies GC, Kasai H. Tsarin gine-gine mai tsayi a dalsritic spines. Yanayi. 2004; 429: 761-766. [PubMed]
36. Mayford M, Bach ME, Huang YY, Wang L, Hawkins RD, Kandel ER. Sarrafa ƙwaƙwalwar ajiyar ƙwaƙwalwar ajiya ta hanyar ƙayyadaddun kalma na tasirin CaMKII transgene. Kimiyya. 1996; 274: 1678-1683. [PubMed]
37. Maze I, Covington HE, 3rd, Dietz DM, LaPlant Q, Muryar W, Russo SJ, Mikiyar M, Mouzon E, Neve RL, Haggarty SJ, Ren Y, Sampath SC, Hurd YL, Greengard P, Tarakhovsky A, Schaefer A, Nestler EJ. Muhimmanci na tarihi na histone methyltransferase G9A a cikin nau'in halayyar kwakwalwa ta hanyar kwakwalwa. Kimiyya. 2010; 327: 213-216. [PMC free article] [PubMed]
38. McClung CA, Nestler EJ. Ƙaddamar da ladabi da ladabi ta hanyar CREB da DeltaFosB. Nat Neurosci. 2003; 6: 1208-1215. [PubMed]
39. Nestler EJ. Review. Tsarin sakonnin jaraba: tasirin DeltaFosB. Philos Sanya Sanya. 2008; 363: 3245-3255. [PMC free article] [PubMed]
40. Ka ba HE, Fata BT, Kelz MB, Iadarola M, Nestler EJ. Nazarin Pharmacological game da tsari na maganin maganin antigen da ke dauke da FOS na cocaine a cikin striatum da tsakiya. J Pharmacol Exp Ther. 1995; 275: 1671-1680. [PubMed]
41. Okamoto K, Bosch M, Hayashi Y. Matsayi na CaMKII da F-actin a cikin nau'i na tsarin rubutun dendritic: ainihin kwayoyin halitta na alama na synaptic? Jiki na jiki (Bethesda) 2009; 24: 357-366. [PubMed]
42. Olausson P, Jentsch JD, Tronson N, Neve RL, Nestler EJ, Taylor JR. DeltaFosB a cikin ƙananan haɓaka yana ƙayyade dabi'un kayan aiki da karfafawa. J Neurosci. 2006; 26: 9196-9204. [PubMed]
43. Paxinos G, Watson C. Cikin kwakwalwa a kwakwalwa. 6th Edition. Amsterdam; Boston: Cibiyar Nazari / Elsevier; 2007.
44. Peakman MC, Colby C, Perrotti LI, Tekumalla P, Carle T, Ulery P, Chao J, Duman C, Steffen C, Monteggia L, Allen MR, Stock JL, Duman RS, McNeish JD, Barrot M, Self DW, Nestler EJ , Schaeffer E. Inducible, ƙwaƙwalwar kwakwalwa ƙananan yanki na ƙananan ƙwayoyin cuta na C-Jun a cikin ƙananan ƙwayoyin cuta yana rage yawan hankali ga cocaine. Brain Res. 2003; 970: 73-86. [PubMed]
45. Penzes P, Cahill ME, Jones KA, DP Srivastava. Kalmar CaMK mai haɗawa da kuma RacGEF sigina suna sarrafa tsarin dendritic da aiki. Trends Cell Biol. 2008; 18: 405-413. [PubMed]
46. Perrotti LI, Hadeishi Y, Ulery PG, Barrot M, Monteggia L, Duman RS, Nestler EJ. Ƙaddamar da deltaFosB a cikin ladabi da aka danganci kwakwalwa bayan an ƙarfafa danniya. J Neurosci. 2004; 24: 10594-10602. [PubMed]
47. Perrotti LI, Weaver RR, Robison B, Muryar W, Maze I, Yazdani S, Elmore RG, Knapp DJ, Selley DE, Martin BR, Sim-Selley L, Bachtell RK, Kai DW, Nestler EJ. Sakamakon bambanci na DeltaFosB shigarwa cikin kwakwalwa ta hanyar maganin zalunci. Synapse. 2008; 62: 358-369. [PMC free article] [PubMed]
48. Pickens CL, Airavaara M, Theberge F, Fanous S, Fata BT, Shaham Y. Neurobiology na incubation na miyagun ƙwayoyi craving. Trends Neurosci. 2011; 34: 411-420. [PMC free article] [PubMed]
49. Pierce RC, Quick EA, Reeder DC, Morgan ZR, Kalivas PW. Ma'aikatan manzannin biyu na kwayoyin Calcium sun tsara bayanin nuna halayen halayyar halayyar hawan cocaine. J Pharmacol Exp Ther. 1998; 286: 1171-1176. [PubMed]
50. Quirion R, Robitaille Y, Martial J, Chabot JG, Lemoine P, Pilapil C, Dalpe M. Sakon kwakwalwa na kwakwalwa ta hanyar amfani da sassan jikin mutum: hanyar da za ta rage yawan kayan tarihi. Synapse. 1987; 1: 446-454. [PubMed]
51. Robinson TE, Kolb B. Tsarin gine-gine masu haɗin gwiwar da ke dauke da kwayoyi masu cin zarafi. Neuropharmacology. 2004; 47 (Gudanar 1): 33-46. [PubMed]
52. Robison AJ, Nestler EJ. Hanyoyin sassaucin ra'ayi da jinsin halittu na jaraba. Nat Rev Neurosci. 2011; 12: 623-637. [PMC free article] [PubMed]
53. Robison AJ, Bass MA, Jiao Y, MacMillan LB, Carmody LC, Bartlett RK, Colbran RJ. Hanyoyin hulda da yawa na sinadarin calcium / proteinodulin-protease na biyu tare da sunadaran sunadarai masu tsohuwar jiki NR2B, densin-180, da alpha-actinin-2. J Biol Chem. 2005; 280: 35329-35336. [PubMed]
54. Ross 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, Ya F, Jacobson A, Pappin DJ. Ƙididdigar sunadarai masu yawa a cikin Saccharomyces cerevisiae ta amfani da isobaric tagging reagents amine-reactive. Mol Cell Proteomics. 2004; 3: 1154-1169. [PubMed]
55. Russo SJ, Dietz DM, Dumitriu D, Morrison JH, Malenka RC, Nestler EJ. Halin da ya kamu da jiki: sassan hanyoyin synaptic da kuma nau'in kayan aiki a cikin tsakiya. Trends Neurosci. 2010; 33: 267-276. [PMC free article] [PubMed]
56. Kai DW. A cikin: Masu karɓin Dopamine. Neve KA, edita. New York: Humana Press; 2010. shafi na 479-524.
57. Singer BF, Loweth JA, Neve RL, Vezina P. Magungunan ƙwayar maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin maganin kare jiki mai gina jiki mai gina jiki a cikin harsashin tsakiya na alpha-amino-3-hydroxyl-5. -emethyl-4-isoxazole-propionate masu karɓa: mai karfin dopamine-1 da protein kinase A dogara. Eur J Neurosci. 2010; 31: 1243-1251. [PMC free article] [PubMed]
58. Strack S, McNeill RB, Colbran RJ. Kayan aiki da tsari na alliyoyin calcium / aminodulin-protein kinase II wanda aka kebanta da ƙananan NR2B na mai karɓar N-methyl-D-aspartate. J Biol Chem. 2000; 275: 23798-23806. [PubMed]
59. Ulery-Reynolds PG, Castillo MA, Vialou V, Russo SJ, Nestler EJ. Harkokin samfuri na DeltaFosB yana tabbatar da kwanciyar hankali a vivo. Neuroscience. 2009; 158: 369-372. [PMC free article] [PubMed]
60. Ulery PG, Nestler EJ. Dokar aikin DeltaFosB ta hanyar Ser27 phosphorylation. Eur J Neurosci. 2007; 25: 224-230. [PubMed]
61. Ulery PG, Rudenko G, Nestler EJ. Dokar DeltaFosB kwanciyar hankali ta hanyar phosphorylation. J Neurosci. 2006; 26: 5131-5142. [PubMed]
62. Vialou V, et al. DeltaFosB a cikin kwakwalwa alamun ladabi sunyi matukar damuwa ga damuwa da maganin antidepressant. Nat Neurosci. 2010; 13: 745-752. [PMC free article] [PubMed]
63. Wang L, Lv Z, Hu Z, Sheng J, Hui B, Sun J, Ma Chronic Cocaine hade H3 acetylation da kuma rikodin rubutu na CaMKIIalpha a cikin ƙananan ƙwayoyin cuta yana da mahimmanci don motsawa don karfafa maganin miyagun ƙwayoyi. Neuropsychopharmacology. 2010; 35: 913-928. [PMC free article] [PubMed]
64. Werme M, Messer C, Olson L, Gilden L, Thoren P, Nestler EJ, Brene S. Delta FosB yana tsara motoci suna gudana. J Neurosci. 2002; 22: 8133-8138. [PubMed]
65. Winstanley CA, LaPlant Q, Theobald DE, Green TA, Bachtell RK, Perrotti LI, DiLeone RJ, Russo SJ, Garth WJ, Kai DW, Nestler EJ. Shigar da DeltaFosB a cikin asibiti na orbitofrontal na nuna haƙuri ga cocaine-induced dysfunction bincike. J Neurosci. 2007; 27: 10497-10507. [PubMed]
66. Zachariou V, Bolanos CA, Selley DE, Theobald D, Cassidy MP, Kelz MB, Shaw-Lutchman T, Berton O, Sim-Selley LJ, Dileone RJ, Kumar A, Nestler EJ. Matsayi mai mahimmanci ga DeltaFosB a cikin tsakiya yana karawa cikin aikin morphine. Nat Neurosci. 2006; 9: 205-211. [PubMed]
67. Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Farashin EE, Jr, Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, Anderson ME. Calmodulin kinase II na hana kare cutar cututtuka. Nat Med. 2005; 11: 409-417. [PubMed]