(HUMAN) Nā pane kūpono a me nā hoʻoponopono i ka Hoʻoponopono Cocaine Manaʻo no ka Hoʻouluʻana i ka Loop Involving ΔFosB a me ka Calcium / Calmodulin-Protein Protein Kinase II ma ka Nucleus Accumbens Shell (2013)

ʻO ka transwr factor ΔFosB a me ka paʻakai o ka paʻakai o ka naʻau / kalona i ka ʻaumedulin me ka protein kinase II (CaMKIIα) i hoʻoiho ʻia i loko o ka nucleus accumbens (NAc) ma ka nānā mau i ka cocaine a i ʻole nā ​​psychostimulant o nā hōʻino o ka hōʻino. . ^ E Ha yM. ʻOiai ʻo ΔFosB lāua ʻo CaMKIIα e hoʻoponopono i ka hōʻike a me ka hana o ka mea hoʻopihapiha AMPA i ka NAc, i hoʻokumu ʻia ai ka spendritik spine ma nā NAc medium spiny neurons (MSNs), a me ka lokomaʻomaʻo lokomotor i ka cocaine, ʻaʻohe pilina pololei ma waena o kēia mau molekula i kēia manawa. Maanei, hōʻike mākou i ka ΔFosB ka phosphorylated e CaMKIIα i ka protein-stabilizing Ser27 a ʻo CaMKII ka koi no ka hōʻuluʻulu ʻana o ka cocaine-mediated o ΔFosB ma rat rat.

ʻO ka mea hoʻohuli, hōʻike mākou i nā ΔFosB e pono a lawa pono no ka hoʻokaʻina kokina o ka caMKIIα gen expression i nā vivo, kahi hopena koho no D₁-ʻaeʻo MSN i ka ʻāpana ʻili o NAc.

Eia kekahi, ʻo ka hoʻopiʻi ʻana o nā spines dendritic ma NAC MSNs a me ka hoʻonui ʻana i ka pane o ka hoʻonaninani i ka cocaine ma hope o ka hoʻomakeke ʻana o NA NA ΔFosB, ka hilinaʻi ʻo CaMKII.

ʻO ka mea nui, hōʻike mākou no ka manawa mua o ka manawa o ka ΔFosB a me CaMKII i loko o ka NAc o kanaka hoʻohui lawena kanaka, e kuhikuhi ana i nā mea hiki ke hiki no ka hoʻoikaika therapeutic i hope. Kūkulu kēia mau ʻike i ka ΔFosB a me CaMKII e hana ana i kahi pona-cell-type a me ka ʻona hoʻonaʻauao ʻano pono kūpono kūpono ma ke ʻano he mālamalama kumu nui no ka hoʻoponopono ʻana i ka noho pākuʻina o ka lolo i ka pane ʻana i ka kokā.

Introduction

Ke kākoʻo nei ka hoʻonui ʻana i nā hōʻike i ke ʻano o nā hoʻololi ʻana i ka huaʻōlelo gen i nā hana o ka hoʻohui lāʻau (ʻO Robison lāuaʻo Nestler, 2011). ʻO kekahi mea nūnū nui o kēia mau hoʻololi i ka ΔFosB, kahi kūlana transit ʻohana Fos (Nestler, 2008). ʻO kahi mālama hoʻomehana ʻoi loa i kekahi lāʻau o ka hōʻino e hoʻonāukiuki i ka hōʻea o ka lōʻihi o ka hōʻuluʻulu ʻana o ΔFosB i ka accumbens nucleus (NAc), kahi ʻāina kūloko e pono ai no nā hana uku. Such induction ka mea i hōʻike ʻia i ka papa o NAc medium spiny neuron (MSN) e hōʻike ana i nā kaila D1 dopamine. Ka hoʻonui ʻana i ka pā o ΔFosB i kēia mau pūʻulu NAC MSN o D1 i hoʻonui i ka locomotor a me nā pane uku i ka kokika a me ka morphine (Kelz et al., 1999; Zachariou et al., 2006), ka hoʻohui ʻana o ka mālama mālama kokua (Colby et al., 2003). Eia kekahi, hoʻowalewale genetic a viral blockade o ka hana transcriptional ΔFosB e hōʻemi i nā hopena pōmaikaʻi o kēia mau lāʻau (Zachariou et al., 2006), e hōʻike ana i kēia ka hoʻomau nei o ΔFosB he mea moho nui o ka hoʻololi mau loa i hoʻihoʻi ʻia i NAc e ka hoʻokō lāʻau mālama kino.

ʻO ka maʻamau kūlike o ΔFosB (pili i nā ʻohana ʻohana Fos āpau) ʻo ia hoʻi kahi waiwai o nā molekina, ma muli o ke kahe o nā kāhea degron e piha nei i ka FosB piha piha (Carle et al., 2007), a he kaʻina hana i hoʻoponopono ʻia. ʻO ka ΔFosB ka phosphorylated ma ka vitro a I loko o ka ola ma Ser27, a e hoʻomau hou ka pane a kēia ΔFosB, ~ 10-lip, i ka moʻomeheu cell a me NAc I loko o ka ola (Ulery-Reynolds et al., 2009). ʻOiai ua hōʻike ʻia ʻo Ser27-ΔFosB e lilo i substrate no ka casein kinase-2 ma ka vitro (Ulery et al., 2006), nā kāpena o ka I loko o ka ola ʻAʻole ʻike ʻia ka phosphorylation.

ʻO ka calcium / ka kalebona me ka protein kinase II (CaMKII) kahi serine / threonine kinase nona ka α a me ka β isoforms puka dodecameric homo- a hetero-holoenzymes I loko o ka ola, a he kūpono no nā ʻano he nui o ka neuroplasticity (Lisman et al., 2002; ʻO Colbran a me Brown, 2004). Ka CaMKIIα i hoʻoili ʻia i ka ʻili NAc ma ka amphetamine maʻi maʻi (Loweth et al., 2010), a me ka blockacological blockade o ka hana caMKII ma NAc shell ka hōʻemi ʻana i ka hoʻonaninani ʻana i ka hoʻomaʻamaʻa ʻana i nā amphetamine (Loweth et al., 2008) a me ka cocaine (Pierce et al., 1998), ʻoiai ka pōkole o ka pōʻino o CaMKIIα i kēia ʻōpū NAc e hoʻomaikaʻi ana i ka ʻike lokomaʻomaʻo a me ke kāohi ponoʻī o ka amphetamine (Loweth et al., 2010). Ka CaMKIIα e hoʻopilikia i ka hana o ka uku ma o ka loli ʻana o ka subunits receptor AMPA glutamate (Pierce et al., 1998), ʻo ka hana ʻo CaMKIIα ua lōʻihi loa a pili me ka hana receptor AMPA a me ka hoʻāʻo ʻia ʻo synaptic i kekahi mau ʻano o ka neuroplasticity (Malinow a me Malenka, 2002).

Hōʻike kēia ʻatikalaʻī he mau mea hoʻohālikelike ma waena o ΔFosB a me CaMKII: pono a lawa a lawa no ka nui o nā hopena ʻano o nā lāʻau hōʻino, ʻelua e hāpai ana i nā spines dendritic i nā ʻano cellonal cellally I loko o ka ola (Jourdain et al., 2003; Maze et al., 2010), a hoʻomaʻamaʻa ʻelua i kekahi mau hopena o kā lākou ʻano hana ma o ke ʻano o ka ʻĀina AMPA.Kelz et al., 1999; Malinow a me Malenka, 2002; Vialou a me., 2010). ʻOiai kēia mau ʻokoʻa, ʻaʻole ʻike kahi pilina ma waena o ΔFosB a me CaMKII. Maanei, ua hoʻokū mākou i ka hoʻoponopono kūmole ma waena o ΔFosB a me CaMKII, a ke hōʻike nei i nā ʻōmole ʻelua i hana i kahi hāmeʻa hoʻolaʻa hāmeʻa D1 type type ADN a me nā māka e hoʻoulu ʻia e ka cocaine a hoʻoponopono i ka nui o nā pane kokoleka. I loko o ka ola.

E hele:

Nā Pono a me nā Una

Ua hōʻike ʻo 1: Ka Manaʻo Proteomic ʻo iTRAQ o ka ʻoll Shell a me ka Core ma hope o ka mālama koko.X XUMUMA)

ʻO nā pākeke kāne (8) mau kāne kāne i lawelawe ʻia i ka ʻōmole kāne ma 20 mg / kg cocaine a i ʻole ka paʻakai IP IP hoʻokahi i hoʻokahi lā no nā lā ʻehiku. ʻO 24 hr ma hope o ka maʻi o ka hope hope loa, ua loaʻa nā moliki NAc a me nā kumu nui (X XUMUMA) a hoʻoiho uila. Ua hana ʻia ʻo ʻoRAʻO nā hana noiʻi ʻoʻAQ e like me ka mea i hōʻike mua ʻia (Ross et al., 2004; Davalos et al., 2010).

Hōʻike 1

Shell-spuction induction of CaMKII in NAc by cocaine

Ua hōʻike ʻo 2: Ka hoʻololi ʻana i ka loli o ka prototein ma ka rat NAc Core a me ka Shell ma hope o ka mālama koko.Kue 1B – D)

ʻO ka pākeke kāne (8 wiki) i mālama ʻia nā kāne kāne 10 mg / kg cocaine a i ʻole ka hale saline IP hoʻokahi i hoʻokahi lā no nā lā ʻehiku i loko o nā keʻena hoʻopaʻa inoa lokomaikaʻi. ʻO ka pane ʻana o Locomotor i kahi maʻi hoʻokahi o ka cocaine (5 mg / kg IP) i hoʻopaʻa ʻia i kēlā mau holoholona i mālama ʻia me ka kokā (kapa ʻia "ʻano maʻi") a me kahi hapa o ka poʻe i mālama ʻia me ka paʻakai (i kapa ʻia "ʻohu"), a me ka pane ʻana o ka locomotor i ka paʻakai. Ka mea wale nō i hoʻopaʻa ʻia ma ke koena o nā holoholona mālama ʻia lāʻau lapaʻau (i kapa ʻia ʻo "saline"). Mea hana ʻia nā hana lokomaʻi i hana ʻia e like me ka mea i hōʻike ʻia (Hiroi et al., 1997). ʻO ka mea pōkole, ua kau ʻia nā kiʻi kāne kāne ma 18 "× 24" PAS palapala i hoʻopaʻa ʻia nā pahu hoʻomalu (San Diego Instruments) no 30 min ke habituate, ua hāʻawi ʻia i ka inikua IP hoʻokahi o ka paʻakai a ua loilo ʻia ʻia no kekahi mau minima 30, a ua hāʻawi ʻia. Hoʻololi hoʻokahi IP o ka 5 mg / kg cocaine a hoʻopaʻa ʻia no 30 min.

ʻO 24 hr ma hope o kēia hōʻki hope ʻana, ua kāpae ʻia nā mau i ka make ʻole o ka anesthesia e pale ai i nā hopena o ke anesthetics i nā pae o ka protein neuronal a me nā phospho-estado. Ua hōʻili ʻia ka huina ma ka 1.2 mm matrix (Braintree Scientific) a ua lawe ʻia ka pahuhopu kiko i nā wai paʻakai phosphate i loaʻa i ka paʻakai protease (Roche) a me phosphatase (Sigma Aldrich) e hoʻohana ana i kahi pāpaʻi 14 no ka uʻi o NAc a me kahi punika 12 kaʻi o nā koena o ke koena. u ka kiko no NAc shell (See X XUMUMA) a pipi koke i ka hau hau maloʻo. Kahi i nā hoʻohālikelike ʻia e ka sonication māmā ma ka pahu RIPA i hoʻololi ʻia: 10 mM Tris base, 150 mM sodium chloride, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% sodium deoxycholate, pH 7.4, pH 4, protease pH e like me luna. Ma hope o ka hoʻopili ʻana i ka pōʻaiʻai Laemmli, hoʻokaʻawale ʻia nā protein ma 15 – XNUMX% polyacrylamaide gradient gels (Criterion System, BioRad), a me ka hana ʻana i nā Western ke hana me ka ʻōnaehana Odyssey (Li-Cor) e like me nā protocol hana.

Ua hōʻike ʻo 3: Ka hoʻololi ʻana o ka hoʻololi o ka prototeine ​​i ka kiʻomau a NA NA AI a me nā Shell Ma hope o ka hopena o CocaineX XUMUME)

ʻO nā pākeke kāne (8) mau kāne kāne i lawelawe ʻia i ka ʻōmole kāne ma 10 mg / kg cocaine a i ʻole ka paʻakai IP IP hoʻokahi i hoʻokahi lā no nā lā ʻehiku. 14 mau lā ma hope o ka pau ʻana o ka make, ua hāʻawi ʻia nā holoholona i mālama ʻia me ka paʻakai (i kapa ʻia ʻo "saline), a me nā holoholona i mālama ʻia i ka cocaine i hāʻawi ʻia i kahi maʻi saline (i kapa ʻia ʻo 14 day withdrawal a i ʻole" 14d WD ") a i ʻole kahi lāʻau hou o ka cocaine ( ua kapa ʻia ʻo "14d WD Chal" no ka paʻakikī. I hoʻokahi hr ma hope o ka ʻōlelo hope loa, ua hoʻopau ʻia nā holoholona a hoʻomaʻamaʻa ke Komohana Hōʻike 2.

Ua hōʻike ʻo 4: Ka hoʻololi ʻana i ka loli o ka prototein i ka hana o ka NAc Core a me nā Shell Ma hope o ka lawelawe ʻana o Cocaine Iho (Kue 2A – C)

Ua hoʻomaʻamaʻa ʻo Rats e hoʻolako iā 0.5 mg / kg / infusion o ka niu i loko o hoʻokahi papa hoʻokahi i lalo o ka papa hoʻonohonoho 1 paʻa no nā lā ʻeiwa. Ma hope o ʻewalu mau papa hana palena, ua hoʻokaʻawale ʻia nā mauʻu i ʻelua mau pūʻulu i kaulua ʻia e ka cocaine intake ma nā papa ʻelua ʻelua. Kahi hui pūʻulu i ae ʻia i ka cocaine e hoʻokō iho i ka cocaine (0.5 mg / kg / infusion) i loko o hoʻokahi mau hola (komo koke ʻana, ShA) aʻo ka hui ʻē aʻe o ka mauʻu pōkole ponoʻī i loko o nā hālāwai ʻeono-hr (komo lōʻihi, LgA ) no nā lā he ʻumi (nā hālāwai escalation).

Ua hoʻōla ʻia nā ʻāpana o nā pala no ka immunohistochemistry e like me ka mea i hōʻike ʻia (Perrotti et al., 2004). Ua pakanala ʻia nā naʻau i ka 18-24 hr ma hope o ka hōʻike hope loa ʻana i ka lāʻau lapaʻau, e hōʻemi ana i nā koena o nā protein FosB piha i waiho ʻia e like me ka maʻi o ka immunoreactivity e hōʻike ana i ka ΔFosB. Ua hōʻoia ʻia kēia hōʻino ʻia e ke kelepona ʻana i ke Komohana, ʻaʻole i hōʻike i ka nui o ka noho ʻana me kahi antibody i kūʻē ʻia i ka hopena C o FosB holoʻokoʻa loa i ʻike ʻole i ka ΔFosB (nā hōʻike i hōʻike ʻole ʻia). Ma hope o ka kāwili ʻana i ka pauku 35 µm, ua helu ʻia ka nui o nā kelima unFosB immunopositive e ka mea ʻike makaʻala ma nā ʻāpana ʻelua i kahi o nā kiʻī a kēlā me kēia mea, a ʻo nā kumu waiwai o kēlā me kēia mahina 40 × e helu ʻia e ka wahi no kēlā me kēia holoholona. Ua noʻonoʻo ʻia kēlā me kēia holoholona kahi nānā i kēlā me kēia no ka helu helu helu. Ua ʻike ʻia nā mahele hoihoi e hoʻohana ana me Paxinos a me Watson (Paxinos a me Watson, 2007).

ʻO ka helu ʻana o CaMKIIα immunoreactivity i hoʻohana ʻia me ka ʻōnaehana Pūnaewele e like me ka mea i hōʻike ʻia (ʻO Covington et al., 2009). Ua hoʻoholo ʻia nā ikaika o CaMKII a me GAPDH me ka polokalamu Odyssey. Hōʻike ʻia nā hualoa e like me ka waiwai hoʻohui kino o kēlā me ka mm² a ua hōʻike ʻia ma nā ʻano ± sem (n = 4-10 no kēlā me kēia hui). Ua hoʻohana ʻia nā kumukūʻai no GAPDH e kuhikuhi i ka hoʻoponopono ʻana i ka nui o ka CaMKII no ka mānoino o ka mānoanoa a me nā kūlana.

Hōʻike 2

Ka hoʻohuihui o CaMKII ma NAc shell o nā kahe ʻona pilikino ponoʻī a me nā mea kanu koko

Ka Manaʻo 5: Ka Hoʻohui ʻana i ke kiʻekiʻe o ka Protein ma Cocaine-Dependent Humans (Pane 2D)

Ke Kaʻina hana

Loaʻa nā kōkopa olakino o ka postmortem i loaʻa mai ka Quebec Suicide Brain Bank (Douglas Mental Health University Institute, Montréal, Quebec, Kanata). Ke hoʻomau nei ka mālama ʻana i nā kiko i ka mea i hōʻike ʻia (Quirion et al., 1987). ʻO ka mea pōkole, i ka wā i ʻoki ʻia, ua kau ʻia ka lolo i ka hau hau i loko o kahi pahu Styrofoam a holo koke i nā keʻena ʻo Quebec Suicide Brain Bank. Hōʻola koke ʻia ka hemispheres e kahi ʻāpana sagittal i waenakonu o ka lolo, ka lolo o ka lolo, a me ka cerebellum. ʻO nā kīʻaha koko, nā lemal pineal, choroid plexus, hapalua cerebellum, a me ka hapalua o ka uakiea i hōʻike ʻia ʻole mai ka ʻolua hema a laila ʻokiʻoki ʻia i ka coronally i loko o 1 cm-mānoanoa ma mua o ka manuahi. ʻO ka hapalua cerebellum hope i hōʻoki ʻia i ka sagittally i 1cm-ʻalihi ʻekika ma mua o ka manuahi. Hoʻolaha nā kūleʻa i 2-methylbutane ma −40 ° C no ka ~ 60 sec. Mālama ʻia nā ʻōniʻa a pau i hoʻokaʻawale ʻia i loko o nā ʻeke polu i −80 ° C no ka mālama lōʻihi. Wehewehe nā ʻekikina ʻekekī āpau mai nā māka coronal hoʻokolo ma luna o ka moku keleawe kila me ka hau maloʻo a puni e puni nei i ka mālama ʻana i ke ʻano o ke kaiapuni. Ua hana ʻia ka hoʻopau ʻana ke komohana Hōʻike 2.

Cohort

Ua hui ʻia ʻo cohort ma nā kumuhana wahine 37 a me 3 wahine, e piʻi ana i loko o nā makahiki ma waena o 15-66 mau makahiki. Ua make koke nā poʻomanaʻo āpau me ka ʻole o kahi kūlana agonal a lōʻihi ʻole a he maʻi maʻi protracted. I kēlā me kēia kūlana, ua ʻike ʻia ke kumu o ka make e ka keʻena Coroner o Quebec, a ua ʻō ʻia kahi ʻaoʻao toxicological me nā kiʻi kiko e loaʻa ai ka ʻikepili i ka lāʻau lapaʻau a me ka hoʻohana pono ʻana i ka wā e make ai. ʻO ka hui kumuhana i pili i nā kānaka 20 i hālāwai me nā koina o ka SCID-I no ka hilinaʻi kokela. Ua hoʻonohonoho ʻia ka pūʻulu hui me nā kumuhana 20 me ka mōʻaukala o ka cocaine dependence a ʻaʻohe ʻano psychiatric koʻikoʻi. Ua make koke nā mea āpau mai nā kumu i ʻole he kumu no ka mālama ʻana i ka lolo. Ua hui ʻia nā hui no ke ʻano o ke kumu kumuhana, ke hoʻomaha ʻana a me ka pH. No nā kumuhana āpau, hana ʻia nā naʻau ʻalani e like me ka mea i hōʻike mua ʻia ma mua (Dumais et al., 2005), e ʻae iā mākou e loaʻa i nā ʻike kikoʻī kikoʻī e pili ana i ka psychiatric a me ka mōʻaukala olakino, a me nā ʻikepili ʻokoʻa a me ka ʻikepikopa ʻano. I ka pōkole, ua alakaʻi aku kahi mea noiʻi hoʻomaikaʻi i Nānā Kaukau Lila Maikaʻi no ke DSM-IV Nā maʻi psychiatric (SCID-I) me hoʻokahi a i ʻole nā ​​mea hōʻike o ka mea make. Ua loiloi kahi papa o nā mea lapaʻau i nā loiloi SCID-I, nā hōʻike hihia, nā leka a ka mea kākau, a me nā moʻolelo lapaʻau e loaʻa ai nā ʻomaʻomaʻomaʻo psychiatric diagnoses.

Ua hoʻokō ʻia ʻo 6: Hoʻomehu Chromatin no ka rat NA NA (Kue 3A – C)

ʻO nā pākeke kāne (8) mau kāne kāne i lawelawe ʻia i ka ʻōmole kāne ma 10 mg / kg cocaine a i ʻole ka paʻakai IP IP hoʻokahi i hoʻokahi lā no nā lā ʻehiku. ʻO 24 hr ma hope o ka maʻi o ka hope ma hope, ua microdissected a ʻoi a me ka inti. Ua hana ʻia ʻo Chromatin immunoprecipitation (ChIP) e kau ana i ka punc bilateral NAc punce o ka pulupulu a i ʻole inti mai ʻehiku mau puʻupuʻu ma kēlā me kēia hui o 14 ka nui o nā holoholona (98 ka nui, 7 inu kokoleka, 7 punawai wai). Ua hoʻopili ʻia ka ʻāwili, a holoi ʻia, a mālama ʻia ma −80 ° C a hiki i ka mālama chromatin ma ka sonication. Ua hoʻopili ʻia ka chromatin i ka pō me ka antibodies i hoʻopaʻa mua ʻia i nā makika magnetic (Dynabeads M-280, Invitrogen). Ua hoʻohana ʻia ʻo IgG i ka pale ole ma ke ʻano he kaohi. Ma hope o ka hoʻohuli ʻana i ka pili ʻana a me ka hoʻomaʻemaʻe ʻana iā DNA, ua hoʻohana ʻia ka qPCR e ana i ke ana o ka AD o ka mea hoʻolaha o CaMKIIα. Ua hana ʻia nā mea mua e hoʻonui i kahi wahi e loaʻa ana ka papa APensin XXUMX kahi i loaʻa iā 1 bp ma mua o ka pūnaewele transkripment (Forward: ACTGACTCAGGAAGAGGGATA; Huli: TGTGCTCCTCAGAATCCACAA).

Hōʻike 3

Pūnaewele a me nā ʻona kelepona ΔFosB o ka CaMKIIα I loko o ka ola

Hoʻolaha Ka Hōʻailona 7: Ka Waʻo CaMKII Nānā a me Ka Hoʻolaha Protein me ka Cell-Type-Spectric ΔFosB Overexpression (Pane 3D)

ʻO nā kiʻi kāne bitransgenic i loaʻa NSE-tTA (laina A) × TetOp-ΔfosB (laina 11) a NSE-tTA (nā laina B) × TetOp-FLAG-ΔfosB (ka laina 11) nā kiole (Chen et al., 1998; Kelz et al., 1999; Werme et al., 2002; Zachariou et al., 2006) ua hāpai ʻia a hoʻokiʻekiʻe ʻia ma 100 µg / ml doxycycline e kāohi i ka haʻi ΔFosB i ka wā o ke kūkulu ʻana. Ua hoʻokaʻawale ʻia Littermates ma ka weaning: ʻoi ka mea i koe ma ka doxycycline a ua hoʻoneʻe ʻia ka hapa i ka wai, a ua hoʻohana ʻia nā holoholona ma 8 a 11 mau pule hope mai ka wā i hoʻonui ʻia ai nā hopena transwral o ΔFosB (Kelz et al., 1999; ʻO McClung a me Nestler, 2003). No nā hoʻokolokolo ninaninau, ua hoʻopau wikiwiki nā kiʻi, a ua lawe ʻia nā pelekikena a kau ʻia ma luna o ka hau. Ua lawe ʻia nā pōpeku o NAc me kahi pā kui ʻona a 14-gauge a wikiwiki hoʻi i ka hau hau a hiki i ka lawe ʻia ʻana o RNA. Wehewehe RNA, qPCR, a me ka ʻike ikepili e like me ka mea i hōʻike mua ʻia (LaPlant et al., 2009). ʻO ka pōkole, ua hoʻokaʻawale ʻia ʻo RNA me TriZol reagent (Invitrogen), hoʻomaʻemaʻe hou ʻia me ka kit microNA RNAeasy mai Qiagen, a loiloi no ka maikaʻi me ka ʻoihana Agilent's Bioanalyzer. Ua hana ʻia ka palapala transpending me ka hoʻohana ʻana i ialea (BioRad) ua lawe ʻia ʻo qPCR me kahi ʻōnaehana Biosystem Applied 7900HT RT PCR me nā ʻāpana helu e kū nei: 10 min ma 95 ° C; 40 sikina o 95 ° C no ke 1 min, 60 ° C no ka 30 sec, 72 ° C no 30 sec; ka hoʻomoʻa ʻana i ka lua i ka 95 ° C e hoʻokumu i nā pihi dissociation no ka hōʻoia ʻana o nā huahana PCR hoʻokahi. ʻO nā loiloi Immunohistochemical o ΔFosB a me CaMKIIα e hōʻike ʻia e ka mea i hōʻike ʻia e like me ka mea i hōʻike ʻia ma Hōʻike 4.

Ua hoʻokolohua ʻo 8: nā hopena o Intra-NAc D1 a me D2 Dopamine Receptor Antagonists ma ka Cocaine-Mediated Protein ChangeKue 3H)

ʻO ka pākeke kāne (8 mau pule) i lawelawe ʻia nā kāne he kāne 10 mg / kg cocaine a i ʻole ka paʻakai (“kaʻa” pūʻulu) IP hoʻokahi i ka lā no nā lā ʻehiku. ʻO 30 min ma mua o kēlā me kēia hae kokoti, ua mālama ʻia nā kiʻina IP ma o ka D1 receptor antagonist SCH 23390 (0.5 mg / kg, "D1 Ant" pūʻulu), a i ʻole D2 receptor antagonist eticlopride (0.5 mg / kg, "D2 Ant" pūʻulu) , a i ʻole hui saline control injection ("cocaine" hui). 24 hr ma hope o ka hopena o ka pau loa, ua hoʻoholo ʻia nā holoholona a hoʻomoʻa ʻia nā protein e ke kuhi ʻana o ke Kanaka ma kēlā me kēia Hōʻike 2.

Laulina 9: hopena o ka AAV-Mediated ΔFosB Overexpression ma Protein Expression (Hōʻoia 4 A – C)

Ua mālama ʻia ka hoʻokele Stereotaxic ma nā kiʻi kāne makua (wiki 8) e hoʻoiho ai i kahi AAV-GFP (ʻōmole fluorescent ʻōpiopio) a i ʻole AAV-GFP-ΔFosB (Maze et al., 2010). Ua hoʻohana ʻia nā jarami 33 (Hamilton) no ka hoʻopiʻi ʻana i nā manawa āpau, a ʻo 0.5 virusl i hoʻomaʻemaʻe ʻia i nā maʻi hemolele hemolele i hoʻopili ʻia ma kahi o ka manawa minima 5, a ukali ʻia e 5 min aʻinā hope o ka wā hoʻomaha hoʻomaha. He ana ke kikowaena āpau i ka pili i Bregma: 10 ° kihi, AP = + 1.7 mm, Lat = 2.5 mm, DV = −6.7 mm. 14 lā ma hope o ka hana ʻana, ua hāʻawi ʻia nā holoholona i hoʻokahi IP o ka paʻi o ka 10 mg / kg cocaine ma nā keʻena mālama mālama lokomotor e loiloi i ka hopena pili hewa o ka overexpression ΔFosB. 24 hr ma hope o kēia hōʻiliʻili hope ʻana, ua koho ʻia nā kiʻī e like me kēlā me kēia Hōʻike 2a, ua hana ʻia me ka hoʻopā ʻana o ka microdissection ma lalo o ke alakaʻi ʻana i ka mikroscopic fluores e loaʻa ai ka ʻōnaehana NAc-maikaʻi. Hana ʻia ke kipi komohana ma ke ʻano he Hōʻoia 2.

Hōʻike 4

NecessaryFosB ka mea pono a lawa pono no ka cocaine-mediated D1 receptor-dependence CaMKIIα induction ma NAc shell

Laulina 10: hopena o ka AAV-Mediated ΔJunD Overexpression ma Cocaine-Dependent Protein Expression (Kue 4 D – F)

Hoʻokomo ʻia ka maʻi Stereotaxic o AAV-GFP a i ʻole AAV-GFP-ΔJunD e like me kēlā Hōʻoia 8. 14 mau lā ma hope o ka hana ʻana, ua mālama ʻia nā holoholona ma ka 10 mg / kg cocaine a i ʻole ka hale paʻakai IP i hoʻokahi lā i hoʻokahi lā no nā lā ʻehiku i loko o nā keʻena hoʻopaʻa ʻenehana lokomaikaʻi. ʻO ka pane ʻana o Locomotor i kahi hōʻeha o hoʻokahi kokoti (5 mg / kg IP) a i ʻole ka saline ua hoʻopaʻa ʻia. 24 hr ma hope o kēia hōʻiliʻili hope ʻana, hoʻokaʻawale nā ​​nui, ke ʻokiʻoki ana, a me nā pōpoki Komohana e like me Hōʻoia 9.

Laulana 11: I Vitro Manaʻo ʻAka Kinase (Kue 5A – D)

Ua huikala ʻia ka Recombinant CaMKIIα a me ΔFosB mai nā pūnaka pepeke (Brickey et al., 1990; Jorissen et al., 2007), ʻo ka protein kinase assade ua hana (Colbran, 1993), e like me ka mea i wehewehe mua ʻia. ʻO ka pōkole, ua preincubated ʻo CaMKII i ka hau me 2.5 µM ​​(a i ʻole hōʻike ʻia) o ΔFosB, 1 mM Ca²⁺, 40 mM Mg²⁺, Iona 15 µM ​​kahakalula, a 200 mM HEPES pH 7.5. Hoʻomaka ka phosphorylation e ka hoʻohui ʻana o 200 µM ​​ATP me i ʻole a i ʻole [γ-³²P] ATP a ʻae ʻia e hele no 10 min ma ke keʻena lumi (Kiʻi 5A & B) ai ʻole 2 min i ka hau (Kiʻi 5C & D.). Ua hoʻoholo ʻia nā huahana e nā Blotting Western (Kiʻi 5A & B) a i ka autoradiogram a me ke helu scintillation (Fig B – D).

Hōʻike 5

ΔFosB kahi hana papa hana no ka caMKIIα

Hoʻolaha 12: Ka ʻike o Ser27 ΔFosB Phosphorylation (X XUMUME)

In vitro hana ʻia nā assase kinase e like me kēlā Hōʻike 11, ua hoʻokaʻawale ʻia nā ʻōmole e SDS-PAGE, a ua ʻoki ʻia nā kaula e like me ΔFosB a hoʻopaʻa ʻia i kahi kiko spectrometry papaha. Nā mākuhi m / z i nā ʻāpana hāleʻa like ʻole i nā panela āpau i kāhea ʻia ma luna o nā kī ʻana o ke koʻo. ʻAʻole hōʻailona ʻia nā ʻoneki āpau no ka palena o ka nui. ʻO ka maʻamau, ʻokoʻa ka huaʻōlelo no ka ʻāpana hāpana hāpana i ka ʻeleʻele inā ke kū pololei nei a i ʻole e hoʻohui i nā hōʻike i ka hele ʻana o nā puna o ka phosphorylation, ma laila lākou i hōʻailona ʻia i ʻulaʻula. Hōʻike ʻia nā hōʻike no nā huahana fragmentation hope i ka pauku e heluhelu ana i ka phosphopeptide me ka wahi i ʻike ʻia o ke koena o ka phosphorylation i hōʻike ʻia me kahi ʻulaʻula me kahi hoʻolālā palapala hoʻokahi amino acid. Hōʻailona ʻia ka helu helu i nā ʻāpana i nā peptide e like me ka b a me nā mea y. Hōʻikeʻike nā mea kūpono no nā ʻāpana o ka m / z axis e hōʻike i ka haʻahaʻa hālima haʻahaʻa i ʻike ʻia ma ka piko o kēlā me kēia kiko laha. ʻO nā hua hāpana i hōʻike ʻia ma ka papa H i hōʻoia i ka hiki ʻana o ka isoform Ser27 phosphorylated, eia naʻe, i loko o kahi hui o nā isoforms phosphorylated i nā pae Ser28, Ser31, Ser34, a me Thr37. ʻO ka noho ʻana o ka pa5, pa5-P, pb5, a me nā pahola pb5-P i hōʻoia i ka phosphorylation o ke koena Ser27.

Hoʻolaha 13: Ka hoʻohuhinu ʻana o ka phosphorylation Ser27 (Keia 5F)

Hoʻonohonoho iki nā peptides maʻamau i ke kāʻei i nā phospho a me nā ʻano phospho o Ser27 ΔFosB. Ma hope o ka hoʻohuihui a me ka hoʻomaʻemaʻe, ua hoʻoneʻe ʻia kēlā me kēia "pede" idiotypic peptide i kahi 50 / 50 acetonitrile / wai kai a hoʻouna ʻia no ka loiloi waikawa amino e hoʻoholo i ke kūpaʻa piha ʻana i ka syntept peptide stock solution. Ua hoʻopili pololei ʻia kēlā peptide i kēlā me kēia "peptide" 4000 QTRAP papaha nui (MS) no ka hoʻoholo ʻana i ka ikehu olakino maikaʻi loa no ka fragmentation MS / MS a he ʻelua a ʻehā mau hoʻololi MRM. A laila, ua hoʻopili ʻia nā peptides "neat" i nā puke maha ma ka 4000 QTRAP e maopopo ai ka hoʻokaʻawale peptide. Hū ʻia ka mea hana i ka mode quadrupole triple, me ka hoʻonohonoho ʻana Q1 ma ka waiwai o ka pore m / z iʻike mua ʻole (Q1 ʻaʻole ia e nānā), a hoʻonohonoho ʻia ʻo Q3 i ka waiwai m / z kūpono i pili i kahi kikoʻī o kēlā peptide. Ma ka ʻano moʻo MRM, kahi hui ʻana i hoʻokahi mau pane (neʻe i ka pāʻina a me ke neʻe a ka mea i hoʻoulu ʻia ai ke kūleʻa o ke kiʻina i ke kūleʻa i nā ikaika o nā mea i makemake ʻia), a me ke alapine (me ka maʻamau 1 – 2 sec) ka manawa holoʻokoʻa o ka hoʻokaʻawale ʻana o HPLC. Ua hoʻoholo ʻia nā loli MRM mai ka mākaʻikaʻi MS / MS o nā peptides i loaʻa. Ua koho ʻia nā alua he ʻelua o nā peptide, e pili ana i nā kiʻekiʻena hāhā i kaha nui, a ua hoʻoneʻe ʻia ka ikehu pakenehi i hiki ke hoʻonui i ka ikaika o ka hana MRM ma o ka polokalamu loea. ʻO nā kihi i loaʻa mai nā peptides maʻamau a me nā ΔFosB hōʻailona i hōʻike ʻia i ka CaMKII a i ʻole ka hoʻokalakupua, a laila e hoʻohālikelike ʻia e ʻike i ka nui o nā peptide i kēlā me kēia hopena. Hana ʻia ka ʻikeʻikepili ma ka ʻikepili LC-MRM me ka hoʻohana ʻana iā AB Multiquant 1.1 lako polokalamu.

Ua hoʻokō ʻO 14: Ka hōʻaha ʻana o ΔFosB i CaMKII Mea Mīkini Kaʻeha Kuhi (Kiʻi 5G & H)

ʻO ka kiʻi keu transgenic e pā aku nei i ka helu T286D CaMKII (Mayford et al., 1996; Kourrich et al., 2012) a ua hānai ʻia nā mea momona manu maoli i ka loaʻa ʻole o ka doxycycline e ʻae i ka hōʻike transgene. Hoʻokomo ʻia nā kiʻomo pākahi ma 20 mg / kg cocaine a i ʻole ka paʻakai IP hoʻokahi i kēlā me kēia lā no 14 lā. 24 hr ma hope o ka pau ʻana o ka make, ua pau nā holoholona i ka immunohistochemistry a me ka helu ʻana o ka hōʻike ΔFosB e like me ka hana ma Hōʻike 4.

Hoʻolālā 15: Nā hopena o ka HSV-Mediated ΔFosB Overexpression a me CaMKII Inhibition ma NAc Dendritic Spines (Kaul 6A – E)

ʻO nā kiele kāne ʻelemakule (8 wiki) i hōʻeha ʻia i NAc me HSV-GFP, HSV-GFP-ΔFosB (Olausson et al., 2006), HSV-GFPAC3I, a i ʻole HSV-GFPAC3I-ΔFosB. I loko o kēia mau mea hana, ʻo AC3I, kahi mea pale peptide pili i ka hana CaMKII, ua hoʻopili ʻia i ka C-terminus o GFP. Ua hui ʻia ʻo GFPAC3I e PCR me ka hoʻohana ʻana i ka pMM400-vector e loaʻa ana iā GFPAC3I ma ke ʻano he kumu hoʻohālikelike me kēia: GFP-AC3I-R: 5 'CC TCCGGA TTACAGGCAGTCCACGGCCT 3' (clampBspEIstop). ʻO ka hua hua PCR i hoʻokomo ʻia i ka p3 + a me ka p5 + -Δ FosB vectors e hoʻohana ana i nā pūnaewele NheI a me BspEI. Hana ʻia ka hana kahi e ka male ʻana. Nā koikoi Stereotaxic i: 3 ° ʻoki, AP = + 1005 mm, Lat = + 1005 mm, DV = −10 mm (Barrot et al., 1.6). Hana ʻia ka hemolele a me ka hoʻokaʻawale ʻula e like me kēlā Hōʻike 4.

Ua hoʻāʻo ʻia ka spine anal ʻana.Christoffel et al., 2011). ʻO ka mea pōkole, nā māhele dendritic 50-150 µm kahi mamao aku i ka soma i koho maʻalahi ʻia mai nā ʻōhua maʻi HSV e hōʻike ana i ka GFP. Loaʻa nā kiʻi i ka LSM 710 a (conf. Carl Zeiss) no ka loiloi morphological me ka hoʻohana ʻana iā NeuronStudio me ka algorithm rayburst. Ua papahele ʻo NeuronStudio i nā kiʻina e like me ka ipo, ka ʻūhā, a i ʻole ka ʻōpala e pili ana i nā kumumanaʻo: (1) pae, a (2) poʻo i ka ʻāʻī o ka ʻāʻī, a (3) poʻomaka poʻo. ʻO nā kuʻi ʻāʻī me ke ʻāʻī ke hiki ke hoʻopaʻa ʻia me ka ʻūpiki a i ʻole ka lolo, a ʻo ka poʻe me ke ʻāʻī nui ʻole e helu ʻia ʻo ia he mau mea he nui. Hoʻokaʻawale ʻia nā mea kuʻi me ka ʻāʻī e like me ka mea ʻala a pulu paha e pili ana i ka diameter o ke poʻo.

Hōʻike 6

Hoʻokaʻawale i ka hana CaMKII e pale i ka hopena morphological a me ka hopena o ka ΔFosB ma NAc

Hoʻolauleʻa 16: Nā hopena o ka HSV-Mediated ΔFosB Overexpression a me CaMKII hōʻike ʻana ma ka pane o Cocaine (Keia 6F)

Hoʻokomo ʻia nā ʻiole kāne makua ʻia me nā maʻi āpau e like me kēlā Hōʻike 15, a me nā pane ʻana o locomotor i kahi 5 mg / kg kahi o ka cocaine i ana e like me kēlā me kēia Hōʻoia 9. Hōʻike ʻia ka ʻikepili Locomotor ma ke kaʻe o ka beam pahiki ma 30 min ma hope o ke kau ʻana i ka cocaine.

Additional Information

Nā hale holoholona

ʻO nā kihe kāne ʻo Sprague Dawley (250-275 g; Charles River Laboratories) ʻelua mau hale. ʻO C57BL / 6J kāne heʻe kāne he ʻona wikiō ʻokoʻa (The Jackson Laboratory) i hui pū ʻia me ka nui o nā holoholona ʻelima no kēlā me kēia kahu. Ua noho ʻia nā holoholona āpau ma ka hale holoholona no ≥1 pule ma mua o ka hoʻāʻo ʻana a hoʻokolohua ʻia i loko o nā keʻena mālama hau (23 – 25 ° C) ma kahi mālamalama 12 hr / pōʻeleʻele (mau kukui ma 7: 00 AM) me ka komo ʻana i ka meaʻai a me ka wai ponoʻole. ^ E Ha yM. Ua hana ʻia nā hoʻokolohua e like me nā kulekele o ka Society for Neuroscience a me ke komite mālama holoholona mālama a me ka hoʻohana komite (IACUC) ma ka mauna Sinai.

ai '

Hoʻohana ʻia nā lāʻau ma IP a hoʻohemo ʻia i ka paʻakai paʻakai, me ka cocaine (5-20 mg / kg ma 10 µl no nā kiʻikima, ma 1 ml no nā kīlio, NIDA) a me SCH 23390 a i eticlopride hydrochloride (0.5 mg / kg ma 1 ml, Tocris) . ^ E Ha yM. No ka hana o ka stereotaxic, ua hana ʻia nā kiʻi me kahi "cocktail" o ka ketamine (100 mg / kg) a me xylazine (10 mg / kg) (Henry Schein) i ka paʻakai laue.

Nā Antibodies

CaMKIIα (ʻĀpana): Upstate 05 – 532, 1: 5,000

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

ΔFosB (āpau): Pūʻali Pūnaewele 5G4, 1: 250

ΔFosB phospho-Ser27: Phosphosolutions, 1: 500

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

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

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

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

NR2A: Sigma HPA004692, 1: 2,500

NR2B: Millipore Ab1557P, 1: 1,000

NāʻIke Heluhelu

Ua nānā ʻia nā helu helu helu helu āpau me ka hoʻohana ʻana i ka polokalamu hoʻopihapiha Prism 6 (GraphPad). Ua hoʻohana ʻia nā kauā hōʻike t a nā haumāna no nā hoʻohālikelike pālua ʻelua (hōʻike ʻia i nā hopena kahi i hāʻawi ʻia ai ka waiwai u), a ua hoʻohana ʻia nā ANOVA i hoʻokahi mau hoʻohālikelike i hoʻohālikelike ʻia.

E hele:

Results

Ka Hoʻōla Cocole Kīwī ʻO CaMKII i loko o ka NAc Shell

Ua hōʻike nui nā haʻawina he nui nā MSN i loko o ka NAc a me ka nui i loaʻa nā pane biochemical a me ka physiological pane i ka hopena maʻi i nā lāʻau hōʻino (Kourrich lāua ʻo Thomas, 2009; Loweth et al., 2010) a i ka ʻāpono o nā subregions i hiki ʻole ke hoʻoponopono i ka lawaiʻa e ʻimi ai i ka lāʻau (Ito et al., 2004). E hoʻoholo i nā hopena ʻokoʻa o ka cocaine ma ka pūnaehana protein o nā pū o NAc vs. ʻO ka hana nui, ua hoʻohana mākou i Nui Isobaric Tagging (iTRAQ) a me tandem mass spectroscopy (MS / MS). Kuhi ʻia nā ʻōpiopio kāne iā IP me ka cocaine (20 mg / kg) a i ʻole ka paʻakai i kēlā me kēia lā no 7 lā; ʻO 24 hr ma hope o ka maʻi o ka hope hope loa, ua loaʻa nā moliki NAc a me nā kumu nui (X XUMUMA) a hoʻoiho uila. Kuhi ʻia nā protein i loko o kēia mau ʻōhua me ka hoʻohana ʻana iā iTRAQ. Hōʻike nui ʻia nā isoform i ʻehā CaMKII i ka nui ma hope o ka mālama ʻana i ka kokā ma ke ʻano like ʻole o ka pūpū NAc. ʻO ka nui o nā phosphatases protein, me ka PP1 catalytic a me nā subunits o nā hoʻoponopono a me PP2A, ka mea i hoʻopili mua ʻia me nā ʻano kaila CaMKII i nā ʻōnaehana ʻē aʻe (Colbran, 2004), hahai i nā ʻano like like. Ua loaʻa kēia mau ʻike i ka moʻolelo, a i ʻole hōʻike ʻoi loa i ke ala hōʻailona o ka caMKII e kuhi ʻia nei e ka cocaine i NAc i kahi hana kikoʻī.

Ke hōʻoia i kēia ʻike nui ʻana, ua mālama mākou i nā lālani i luna me ka cocaine (ma nā ʻano like ʻole) a i ʻole ka paʻakai a i ʻole ʻia nā pane ʻomomotor a i kahi koka (5 mg / kg) a i ʻole ka waiʻu mina a paʻakai. Hōʻike pinepine ʻia i ka 10 mg / kg cocaine i hopena i ka hoʻohālike maʻamau o ka lokomaʻomaʻo lokomotor (Fig 1B). Ka hōʻike hou ʻana me kēia regimen dose i hōʻike ʻia, ma ka hoʻohana ʻana i ke kelepona ʻana i ke Komohana, ua hoʻihoʻi ka cocaine hou i ka caMKIIα koho i NAc shell 24 hr ma hope o ka pau ʻana o ka kokoli (ʻO X XUMUMXC a me D; p = 0.0019; F = 7.943; df = 29). Eia kekahi, ʻo ka phosphorylation o ka canalical CaMKII substrate Ser831 o nā sublu GluA1 o ka ʻĀnike resap AMPA ua hoʻonui nui ʻia i ka pōʻai NAc a ʻaʻole ia o inti (p = 0.0261; F = 4.208; df = 28), aʻo ka caMKIIα Thr286 autophosphorylation he nui akā ʻaʻole ia. hiʻona nui i ka induction i ka pōʻai wale (Pane 1D). ʻO nā mea ʻē aʻe he glutamate i hoʻopili ʻia. Ke hoʻohālikelike nei i kēia mau hana o CaMKII, nā hōʻike like like o ka hōʻike o ka ΔFosB ma nā pūʻe ʻelua (p = 0.0260; F = 4.189; df = 29) a me ka mana (p = 0.0350; F = 3.807; df = 29) o nā NAc; ((ʻO X XUMUMXC a me D), kūlike me nā makana ma mua (Perrotti et al., 2008).

Mai nā haʻawina mua o ka cocaine regulation o AMPA receptors i nānā aku i nā holoholona ma hope o ~ 14 lā no ka haʻalele ʻana i ka cocaine maʻi (ʻike Nīnau), ua hana hou mākou i kēia mau loiloi biochemical i kēia manawa. ʻIke mākou i kēlā lā, ʻo 14 ma hope o ka inikua hope loa o ka niu, ua noho ʻia ʻo ΔFosB ma NAc (p = 0.0288; F = 4.258; df = 22), aʻo ka CaMKII a i ʻole ka phosphorylation o GluA1 Ser831 e hoʻonui mau ana (X XUMUME). Eia naʻe, 1 hr ma hope o hoʻokahi 10 mg / kg wailiki o ka cocaine, pae o ka nui CaMKII (p = 0.0330; F = 3.947; df = 26) a o GluA1 Ser831 (p = 0.0213; F = 4.509; df = 27) ʻO ka phosphorylation i hoʻokiʻekiʻe ʻia i kahi kiʻekiʻe e like me ka mea i loaʻa ma hope o ka loaʻa ʻana o ka niu kokoleka.X XUMUME). Hōʻike ʻia kēia mau haʻawina ʻo ka NAc shell neurons ua hoʻomaka ʻia no ka CaMKII induction i ka manawa lōʻihi o ka haʻalele ʻana, aia paha ma o ke kuhikuhi mua ʻana i ka hoʻolaha mea hoʻonā CaMKII. Eia kekahi, ʻoi aku ka hoʻomau ʻia o ka hoʻouka ʻana o thanFosB ma mua o ka hoʻopalike ʻana CaMKII i ka noho ʻana o nā mea hana hou, a i ka chromatin a i ʻole a he mea ia e hana i ka "brek" ma ke kānāwai CaMKII, e like me ka uhi ʻia i loko o ka Paʻaina.

No ka hoʻoikaika hou ʻana i kēia mau mea nānā, ua ʻimi mākou i nā hiʻohiʻona o ka hoʻokele cocaine, kahi e komo ai ka nui o ka lāʻau laʻa. Ua hāʻawi ʻia i nā pōpoki kāne ʻelemakule a i ʻole ka loaʻa ʻana i ka cocaine; ʻo ka manaʻo (ʻO Ahmed a me Koob, 1998), aia nā kūlana lōʻihi wale nō i alakaʻi i ka lawelawe pono ʻana o ka lāʻau lapaʻau (X XUMUMA). Ua hoʻoiho ʻia a ΔFosB i ka nui e ka lōʻihi vs. komo pōkole i ka kokā ma nā kinana NAc (p = 0.0011; F = 11.12; df = 17) a me ka core (p = 0.0004; F = 13.86; df = 17). Ke hoʻohālikelike nei, ua lawe ʻia ʻo CaMKIIα i loko o ka ʻoki NAc ma o ke ala wale nō ia i ka cocaine (ʻO X XUMUMXB a me C; p = 0.0236; F = 4.957; df = 16). He mea hoʻohiwahiwa e hoʻohālikelike i ka maʻamau o ka cocaine intake maʻamau i loko o nā holoholona loea pōkole (~ 12 mg / kg IV), nā holoholona lōʻihi loa (~ 70 mg / kg IV), a me nā holoholona hoʻokele waiwai (10 mg / kg), a me e nīnau i ke kumu o ka hoʻopiʻi hope ʻana o ka hoʻokomo o ΔFosB a me CaMKII, akā ʻaʻole i loaʻa ka pōkole. Hōʻike ʻia kēia ʻano ʻokoʻa ma muli o nā ʻokoʻa i nā kiʻekiʻe o ka kokela kokela (ua hāʻawi ʻia nā kokela hoʻākāka hoʻokūkū me ka bolus IP hoʻokahi, ʻoiai ua hāʻawi ʻia ka cocaine hoʻoliʻina iā ia iho ma nā helu he IV), a i ʻole nā ​​ʻokoʻa o ka lōʻihi o ka hōʻemi ʻana i nā lāʻau lapaʻau (7 mau lā no ka mea hōʻike. hoʻokō, 19 lā no ka hoʻokele pilikino).

ʻOiai ka ʻikepili nui ma ΔFosB a me CaMKII i ka hana cocaine, ʻaʻohe ʻike o kēia mau palama i loko o nā mea hoʻohana kokua. Maanei, ke hōʻike nei mākou i nā hōʻike mua e pili ana nā pae o ʻelua ΔFosB (p = 0.0316; t = 1.921; df = 34) a me CaMKII (p = 0.0444; t = 1.755; df = 32) e hoʻonui nui ʻia i NAc o nā kānaka i hilinaʻi i ka kokela.Pane 2D, 1 Pūnaewele). Hōʻike kēia mau hōʻike i kā mākou hōʻike ʻana o ka ΔFosB a me ka caMKII induction e ka cocaine ma rodent NAc e pili pono ana i ka pili koko.

1 Pūnaewele

Ka hōʻike ʻana o nā hiʻohiʻona mai nā mea ulu a kokela a me nā pūʻulu mana o ka hui

UlatesFosB Ka Hoʻoponopono i ka Palapala CaMKII Ma kahi koho i D1-Type MSNs o NAc Shell

ʻO ka ʻike i ka mālama ʻia ʻana o CaMKII a me areFosB ka mākeke i ka cocent NAc i alakaʻi iā mākou e hoʻoholo inā pili paha ʻo ΔFosB i ka hoʻoponopono ʻana i ka hua CaMKII. Ua hōʻike mua mākou iā CaMKIIα i kahi hiki hiki ke ΔFosB i loko o ka nānā ʻana i ka microarray e hoʻohālikelike ai i ka NAc (ʻO McClung a me Nestler, 2003) akā, ʻaʻole kūpono ka loaʻa ʻana o kēia ʻike. Ua hoʻohana mua mākou i ka ChIP kuʻuna (qChIP-ChIP e ukali ʻia e PCR kuʻikuʻi) e hoʻoholo ai inā hoʻopaʻa ʻia ʻo ΔFosB i ka mea hoʻomāhuahua i ka CaMKIIα i loko o nā koki kāne kāne makua, a ʻike nui ʻia ka hoʻonui ʻia ʻana o kēia hōʻaia, e ka hoʻomalu ʻo kokela, ma ka ʻili ( p = 0.0133; t = 2.901; df = 12) akā, ʻaʻole pili ka waihona.X XUMUMA). I mea e maopopo hou ai i nā hana pili i kēia ʻokoʻa-kūʻokoʻa ʻānō i loko o ka ΔFosB e hoʻopili ai i ka mea hoʻolaha CaMKIIα, ua hoʻohana mākou i qChIP e hōʻike i ka ʻano o nā loli o ka histone ma kēia kuʻina genomic. Ua hōʻike mua nā haʻawina mua i ka hoʻoilo ʻana i ka cocaine o ka acetylation H3 i ka mea hoʻolaha CaMKIIα ma ka kiʻi nui o nā Kic (Wiki a me., 2010). Ma kahi hoʻohālikelike, ʻike mākou i ka hoʻemi ʻana o ka cocaine i ka acetylation H3 ma ka CaMKIIα promoterly sellyly ma NAc core (Fig 3B; p = 0.0213; t = 2.726; df = 10), me ka loli ʻole i ka ʻike, kūlike me ka loli o ka chromatin subregion i ʻokoʻa ma o ΔFosB aʻa. ʻO QChIP no ka hōʻailona repressive, dimethylated H3 lysine 9 (H3K9me2), hōʻike i nā kuʻuna no ka hoʻohaʻahaʻa i loko o nā ʻāpana a me nā ʻili o nā ʻae ʻana nui (Pane 3C).

E hoʻoholo ai inā hoʻoponopono ʻo ΔFosB i ka palapala kākau CaMKIIα I loko o ka ola, ua hoʻohana mākou i ʻelua laina o nā kiole bitransgenic e hiki ʻole i ke pani overe ΔFosB kūikawā ma D1 vs. ʻO ka Mana D2 type-9N i ke ʻano e hoʻomālamalama ʻia e ka doxycycline e inu ana i ka wai inu (Chen et al., 1998; Kelz et al., 1999; Werme et al., 2002). Ka nui o nā kiʻoki kāne aʻelemakule ʻo ΔFosB i loko o ka D1 type type MSN nui i hoʻonui i ke kiʻekiʻe o ka caMKIIα mRNA i NAc (p = 0.0337; t = 1.996; df = 13), he hopena i ʻike ʻole ʻia i loko o nā kiole ka nui exFosB mau nui i loko o D2-type MSNsPane 3D). ʻO ka piʻi ʻana o CaMKIIα mRNA, hoʻihoʻi ʻia e ka ΔFosB expression i ka D1 type type MSN, i ukali ʻia me ka hoʻonui pū ʻana o ka protein CaMKIIα ma nā pūloko NAc ʻelua (p = 0.0030; t = 3.578; df = 14) a me ka waiwai (p = 0.0392; t = 2.275; df = 14; Figs 3E a me F). Hōʻike kēia mau ʻikepili i hiki ke ΔFosB hiki ke ka hoʻokele aku i ka hōʻike o ka wai CaMKIIα ma D1 type type MSN i nā ʻāpana ʻelua, ʻoiai Hōʻike 3B manaʻo e hoʻololi i ka hoʻololi i ka chromatin cocaine ma ka mea hoʻolaha CaMKIIα (e hoʻohālike, ka hoʻohaʻahaʻa acetylation) pale aku i ka ΔFosB mai ka hoʻomakeʻa ʻana i ka CaMKII ma ka hui subiion ma hope o ka cocaine.

Ma muli o ka hōʻike ʻana o kā mākou ʻikepili iole transgenic i ka hoʻokomo ΔFosB o ke ʻano gen CaMKII i ka D1-type MSNs ma NAc, ua ʻimi hou mākou e ʻike inā e koi ana ka hoʻowalewale ʻana i ka cocaine o CaMKII i ka hoʻāpono o ka D1 dopamine receptor. Ua lawelawe ʻia nā ʻiole kāne kāne makua i ka cocaine mau a i ʻole ka paʻakai e like me ma mua, akā 30 mau min ma mua o kēlā me kēia ʻoki, hāʻawi ʻia nā ʻiole i ka hui cocaine i ka IP injection o saline, ka D1 antagonist SCH 23390 (0.5 mg / kg), a i ʻole ka D2 receptor antagonist eticlopride (0.5 mg / kg). Nānā ʻia nā holoholona i 24 mau hola ma hope o ka ʻoki hope loa ʻana o ka cocaine. Ua hōʻike ka blotting komohana i ka D1, akā ʻaʻole ka D2, ua pale loa ka antagonist i ka hoʻonui ʻana o ka cocaine ma ΔFosB (p <0.0001; F = 18.96; df = 18), e like me ka mea i hōʻike mua ʻiaNye a me., 1995), penei ma CaMKII (p = 0.0005; F = 10.99; df = 18; Hōʻoia 3G lāua ʻo H). Kākoʻo kēia mau ʻikepili i ka hypothesis e hoʻopaʻa i ka cocaine i ka hoʻonui ΔFosB-mediated i loko o ka hōʻike caMMKII kiko kikoʻī ma D1-type MSNs of NAc shell. He mea nui ia i nā haʻawina o hope e hōʻike pololei i kēia ʻano neʻe i ke ʻano o ka cocaine ma ka hōʻike ʻo CaMKII i loko o kēia ʻāpana o ka lolo.

ΔFosB ka Pono a he ʻokoʻa kūpono no ka kokua Cocaine no ka caMKII ma NAc Shell

No ka hoʻohui i ka hoʻohana ʻana o nā kiʻi bitransgenic, ua aʻo hou mākou i ke kuleana o ΔFosB i ka hoʻomohala i ka hoʻokaʻina o ka cocaine o ka caMKIIα ma ka hoʻohana ʻana i ka ʻōnaehana hoʻololi virus-mediated i loko o nā kahe nui. Hoʻopili mākou i nā pālule manuahi e pili ana i ka pehu a adeno e pili ana i ka pehu a nā kāne kāne (kahi e hiki ke koho ʻohi ʻia ai ka pale) i mea e kau ʻole ai i ka ΔFosB me GFP a i ʻole GFP wale nō. Hāʻawi ʻia nā holoholona i hoʻokahi pūpū IP hoʻokahi o 10 mg / kg cocaine. Ua hōʻike ka holoholona i ke nānā aku i ka ΔFosB / GFP i ka pane ʻana i ka pane lokomotor e hoʻohālikelike ʻia me nā holoholona nui o ka hoʻohālikelike ʻana i ka GFP.X XUMUMA). Ma ke ʻano o 24 hr ma hope o ka maʻi ʻaina o ka kokela, hoʻōla wale ʻia ka NAc kūloko o ka NAF mai kēia mau holoholona ma o ka ʻae ʻana ma lalo o kahi kumu wai māmā. ʻO ka ʻaihui ʻana o kēia ʻaoʻao.ʻO X XUMUMXB a me C) hōʻike i ka overexpression ΔFosB ikaika a me ka nui o ka hoʻonui nui ʻana i ka protein CaMKIIα i hoʻohālikelike ʻia me nā holoholona GFP (p = 0.0070; t = 2.894; df = 30), like me ka hoʻopiʻi ʻike ʻia me ka hoʻokō kokela. Eia kekahi, ʻo CaMKIIα autophosphorylation ma Thr286 (hōʻike i ka hoʻōla o ka hoʻouluulu) ua hoʻonui ʻia e ka ΔFosB overexpression (p = 0.0330; t = 2.243; df = 28), e like me ka phosphorylation o ke kumu CaMKII, Ser831 o GluA1 (p = 0.0540); 2.012; df = 28), hana hou aku i nā hana a nā maʻi kokoleka (ʻO X XUMUMXC a me D). TʻO ka aken, ʻo kēia mau ikepili e hāʻawi hou ana i nā hōʻike e hōʻike nei ka ΔFosB expression i ka ʻc o NAc i lawa ka hoʻonaʻauao ʻana i ka cocaine a no ka ind ka CaMKII a hoʻōla ʻia i kēia subregion.

Ua hoʻohana mākou i kahi ala like e hoʻoholo ai he pono hoʻi ʻo ΔFosB no ka cocaine-mediated induction o CaMKIIα i loko o ka NAc shell. Ua hoʻohana ʻia ʻo AAV e hoʻolauna i kahi protein JunD i kālai ʻia, i kapa ʻia ʻo ΔJunD, ʻo ia ka hoʻoponopono hewa kino o ka hana criptionFosB transcriptional (Winstanley et al., 2007) me ka GFP a i ʻole GFP wale nō. ʻElua mau pule ma hope aku, i ka nui o ka hōʻike transgene, he nui ka holoholona i hāʻawi ʻia i ka cocaine (10 mg / kg) a i ʻole ka paʻakai i kēlā me kēia lā no 7 mau lā, a hoʻāʻo ʻia no ka pane ʻana o ka lokomaʻomaʻo i kahi hopohopo cocaine (5 mg / kg) 24 hr ma hope o ka maʻi ʻomaʻomaʻi hope loa (Pane 4D). PreventJunD overexpression i pale aku i ka nānā ʻana i ka lokomotor i ka cocaine, a ua pale ʻia hoʻi i ka CaMKIIα induction a me ka hana ʻana i ka NAc shell (ʻO X XUMUMXE a me F; p = 0.0437; F = 2.997; huina df = 38), e hōʻike ana he pono pono ka hana transSLal ΔFosB no ka hoʻomo a kokua o ka caMKIIα ma kēia ʻokoʻa. ʻO ka mea mahalo, ua ʻike mākou i ka hōʻemi o ΔJunD i ka pae o ka ΔFosB ma lalo o nā ʻano wai paʻakai a me ka kokela (p = 0.0004; F = 8.110; df = 35), e hāpai ana i ka mana o ka moʻolelo e pili ana ka ΔFosB i ka hana AP-1 no kāna mau kūlana hōʻike.

CaMKII Phosphorylates ΔFosB ma Ser27

E ho ohana ma ka vitro Ke ʻōlelo nei ka protein kinase, ua hoʻoholo mākou ua hoʻomaʻemaʻe ʻia ka ʻae ʻo isFosB i kahi palaka maikaʻi loa no ka caMKIIα. Hoʻohālikelike o kāna₆-ΔFosB me CaMKIIα a me ATP i hoʻoili ai i kahi hoʻololi kiʻekiʻe i ka pehu elektroforetic ʻo ΔFosB (X XUMUMA); ua hoʻolālā ʻia e nā mau hua helu he nui i nā pūnaewele o ka phosphorylation. Kūlike ma ka vitro hana kinase e hoʻohana ana i [γ-³²P] ATP i hōʻike i ka hoʻokomo ʻana i ka phosphate o radiolabeled i loko o nā piʻina ΔFosB i hoʻololi ʻia (Fig 5B), ke hōʻike nei i nā phosphorylation pololei o ka protein. Hoʻokumu mākou i kahi antibody kikoʻīpeke hemropho i ka Ser27 i kapa mua ʻia o osFosB (Ulery et al., 2006). ʻOiai ʻaʻole kēia huahana i hāʻawi i kahi hōʻailona e kū'ē i nā ʻili o nā lolo e loaʻa iā Ser27-phosphorylated ΔFosB (nā hōʻike ʻole i hōʻike ʻia), hiki iā mākou ke ʻike i ka phosphorylation Ser27 i loko o ka ma ka vitro kinase assay using CaMKII (Fig 5B). Ka hoʻāʻo ʻana o Kinetic o ka phosphorylation CaMKII o ΔFosB e hōʻike ana he potent substrate no ka kinase (Pane 5C), me Kiska ikea_M o 5.7 ± 2.0µM ame K_Kuhi o 2.3 ± 0.3min^-1. ^ E Ha yM. Hoʻohālikelike kēia mau hopena i nā hiʻohiʻona maikaʻi I loko o ka ola Nā Mana ʻo Nā CaMKII (ʻO Colbran a me Brown, 2004). Eia kekahi, hoʻoholo mākou i ka mea cory o CaMKII ΔFosB me kahi stoichiometry o 2.27 ± 0.07 mol / mol (Pane 5D), e hōʻike ana ma ka liʻiliʻi he ʻekolu mau wahi no ka hoʻokaʻawale ʻana o CaMKII ma loko o kāna₆-ΔΔΔos protein protein protein protein protein protein protein protein protein protein protein protein X XUMUMA.

E noiʻi i nā pūnaewele pilikino o ka phosphorylation, hana mākou i nā loiloi MS no nā ʻāpana mai kā mākou ma ka vitro kamaʻilio kinase. X XUMUME hōʻike i ka phosphorylation ΔFosB ma ka hanana mua Ser27 a ma kekahi mau pūnaewele hou (ʻaʻole i hōʻike ʻia nā hōʻike). Hāʻawi ʻia i ka ʻano hana mua o Ser27, ua noʻonoʻo mākou i kēia pūnaewele ma ka hoʻomohala ʻana i nā peptides synthetic synted e hoʻohālike i ka phospho- a me nā wahi kīnā ʻole o Ser27, a laila ua ʻike ʻia ka nui o kēia mau peptides e like me nā kūlana ma ka loiloi MRM o ΔFosB ma mua a ma hope ma ka vitro ka phosphorylation e CaMKII. Ke kiʻi ʻokoʻa (Keia 5F) e hōʻoia ana i ka Ser27 kahi pākahi potent no CaMKII. Hōʻike kēia mau hopena i waena o nā koena i ka phosphorylated i loko o ΔFosB, ʻo Ser27 kahi papa hana maikaʻi loa no CaMKII.

CaMKII Hoʻolālā o ka Cocaine Accumulation o ΔFosB i loko o ka NAc Shell

No ka mea hiki iā CaMKII hiki i ka phosphorylate ΔFosB ma ka vitro ma kahi pūnaewele e hoʻoikaika maikaʻi ana i kona kūpaʻa ma ka vitro a I loko o ka ola (Ulery et al., 2006; Ulery-Reynolds et al., 2009), ua hoʻoholo mākou inā loaʻa ka hana CaMKII i nā pae ΔFosB ma NAc I loko o ka ola. ^ E Ha yM. No ka pane ʻana i kēia nīnau, ua hoʻohana mua mākou i kahi laina kiʻīwī e overexpressing i kahi kūʻokoʻa kūʻokoʻa kūʻokoʻa o ka caMKIIα (T286D) i loko o nā wahi kīkahi o ka lolo me ka NAc (Mayford et al., 1996; Kourrich et al., 2012). Ua hoʻopiʻi mākou i kahi maʻi kāne makua kāne kiʻekiʻe a me kahi mea hoʻopukapuka wildtype me 20 mg / kg cocaine a i ʻole paʻakai i kēlā me kēia lā no 14 mau lā, a laila e kāʻei i nā holoholona i hoʻokahi lā ma hope o ka pau loa. ʻIke mākou ua hoʻonui ʻia nā pae basal o ΔFosB i nā holoholona mutant i ka NAc shell (p = 0.0001; F = 9.207; df = 37), akā ʻaʻole ka lula (Hōʻoia 5G lāua ʻo H). ʻOiai ka pīhoihoi, ua hoʻopaʻa ʻia ka niu o ka ΔFosB i ka holoholona mutant ma ka pūpū a me ka nui, e hōʻike ana nō, ʻoiai ʻo ka CaMKII hiki ke hoʻoponopono pololei i ka noho ΔFosB ma ka pōʻai NAc, hiki nō paha i ka kahawai o ΔFosB nā ala o ka cocaine i hoʻōla ʻia ma nā ʻōpū o ʻelua. . ^ E Ha yM.

Manaʻo ʻia ka hana caMKII no ka ΔFosB-Mediated Structural a me ke ʻano

ʻO Cocaine induction o dendritic spines i NAc MSNs ʻo ia kekahi o nā hana hoʻokūkū-i loaʻa i ka lāʻau ma kēia ʻāina o ka lolo, a ua hoʻopili ʻia ka spine induction i ke ʻano o ka pane ʻana i ka hana i ka lāʻau lapaʻau (ʻO Robinson a me Kolb, 2004; Russo et al., 2010) a hōʻike ʻia i koho ʻia no ka D1 type MSNs (Lee et al., 2006). Ua hōʻike hou mākou i ka hoʻomau ʻana o ka cocaine i nā spines dendritic ma NAc i hilinaʻi ʻia i ΔFosB a me kāna papahana transwriter hilaki.Maze et al., 2010). ʻOiai he nui nā palapala pili e pili ana i ka hoʻopili ʻana iā CaMKII i loko o ka morphology spine dendritic a me ka hoʻoili ʻia ma nā wahi ulu o ka lolo a me nā ʻōnaehana hoʻokolohua (Jourdain et al., 2003; Penzes et al., 2008; Okamoto et al., 2009), ʻo kāna hana ma NAc MSN spine form i aʻo ʻole ʻia. No laila, ua hoʻoholo mākou inā makemake ʻia ka hana CaMKII no ka ΔFosB-mediated induction o MSN dendritic spines ma ka hoʻohana ʻana i ka HSV-mediated overexpression o ka caMKII inhibitor peptide AC3I i hoʻopiʻi ʻia ma GFP, kahi hana i hōʻike mua ʻia e hoʻoweliweli i ka hana CaMKII I loko o ka ola (Zhang et al., 2005; Klug et al., 2012). ʻO ka Viral overexpression o ΔFosB i ka pūpū NAc o nāʻiole mākua i hoʻoulu i ka piʻi nui o ka MSN dendritic spine density (p <0.0001; F = 8.558; df = 59; ʻO X XUMUMXA a me B) e like me ka hōʻike ma mua (Maze et al., 2010), a hoʻonui ʻia ka hoʻonui ʻana e nā manawaleʻa (p = 0.0027; F = 5.319; df = 59) a me stubby (p = 0.0378; F = 2.988; df = 59) nā momo spine (ua manaʻo ʻia nā ʻano kīnā ʻole)Kue 6C – E). ʻAʻohe hopena i ʻike ʻia i nā hua loea pākoki. Eia nō naʻe, i ka hoʻopā ʻia ʻana o GFP-AC3I, ua hoʻopau ʻia ka waihona ΔFosB i ka nui o nā paio (Kaul 6A – E), e hōʻike ana i ka hana no CaMKII no ka uctionFosB induction o ka dendritic spines in NAc shell.

Ua hoʻohana mākou i nā pahuhū na mea like ʻole e hoʻoholo ai inā makemake ʻia ka hana CaMKII no ka hopena o ΔFosB ma ka hoʻoikaika ʻana i ka cocaine. ʻO 72 hr ma hope o ka hōʻili ʻia o ka maʻi i ka shell NAc, ua hāʻawi ʻia nā holoholona i hoʻokahi manawa o ka 5 mg / kg cocaine a me kā lākou hana lokomotor. E like me ka hōʻike mua o ka AAV overexpression o ΔFosB (X XUMUMA), HSV-mediated overexpression o ΔFosB i hoʻonui i ka lokomaʻomaʻi hakanui i ka cocaine (p = 0.0002; F = 8.823; df = 37; Keia 6F). E like me ka hoʻopiʻi ʻana o nā kuikahi dendritic, hoʻōki ʻia i ka hana CaMKII e ka hoʻopiʻi ʻana o ka hōʻea ʻana o GFP-AC3I i kāohi ʻia ai ka hoʻonui ʻana o ka ΔFosB ma ka cocaine sensitivity, e hōʻike ana i ka hana CaMKII e koi ʻia no ka hoʻololi ΔFosB-hoʻohuihui i loko o ka hopena o ka hana mālama kokoleka.

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kūkākūkā

Ua hoʻoponopono ka haʻawina i kēia manawa i kahi hana hoʻomohala hou a kahi e hoʻokaʻawale i ka kokoleka ΔFosB ma NAc, e hāpai ana i ka unuhi ʻana o ka hua kaʻi CaMKIIα ma NAc shell. CaMKII a laila hoʻohuli ʻia ka phosphory template a hoʻopaʻa hou i ka ΔFosB e alakaʻi ana i ka hōʻuluʻulu ΔFosB nui loa a hiki i ka houluulu CaMKIIα (Keika 6G). Mālama ka pae o ka lua o ka protein i ka wā o ka hele ʻana i ka cocaine a laila e hāʻawi i nā hana nui e hoʻoiho i ka pane ʻana i nā pane ʻana i ka lāʻau lapaʻau. He hōʻike ʻoluʻolu kēia e like me ʻelua ΔFosB a me CaMKII i hōʻike ʻia i kēlā me kēia e pono ai no ka hoʻonui ʻia ʻana o ka pane i ka cocaine (Pierce et al., 1998; Peakman et al., 2003), a kāhea mākou i kēia ʻike no ka ΔFosB ma kaʻi NAc me ka hoʻohana ʻana i kahi ala viral (Nā Huahelu 4 a A me66).

ʻOiai ʻo ka overexpression transgenic ΔFosB ma ka D1 type type MSNs hiki ke hoʻoheheʻe i ka wai CaMKII ma nā pū o NAc a me ka holoholona o ka holoholona kokahāʻiwa, ma ke ʻano o ka cocaine, ka hōʻiliʻili o ka ogenFosB endogenous, ka mea i ka lua o nā subregions, ka lawe ʻana i ka hōʻuluʻulu ʻana o ka caMKII i loko o nā ʻae a NAc . ^ E Ha yM. Hiki i kēia ʻokoʻa ke pili i nā kiʻekiʻe kiʻekiʻe o ka ʻΔFosB i hoʻoili ʻia i loko o kā mākou hiʻohiʻona bitransgenic, eia nō naʻe, e hoʻohuli ʻia paha ka hiki o nā cocaine e hoʻohuli i ka hoʻololi ʻana i ka mea hoʻolaha CaMKIIα ma ka ʻili vs. core MSNs e hoʻomaikaʻi paha i ka ΔFosB hoʻopaʻa i ka mea i ma mua a haʻalele paha iā ia ma ka ʻāpana o ka hope. ʻO ka mea ʻike, kā mākou data ChIP, ka mea e hōʻike ana i ka deacetylation o ka cocaine-mediated o nā histones ma ka mea hoʻolaha nui o ka caMKIIα ma NAc core wale nō, e kākoʻo i ka hiki ke komo i kahi hana chromatin. I ka mālama ʻana i kēia hypothesis, ua hiki i ka nui o exFosB overdpression ma D1 type type MSNs ke hiki i ka pahu o ka CaMKIIα i loko o ka NAc kinohi ma ka ʻole o ka cocaine (Keia 3F), e kuhikuhi ana ana i nā loli mana o ka mea hoʻolaha CaMKIIα e pale ai i kēia ʻauʻau i ka wā o ka ʻike kokoleka. ʻO ka hoʻoponopono ʻana i ka hiʻona chromatin ma ka mea hoʻolaha CaMKII e wehewehe pono paha i ke kumu e hoʻoiho ʻia ai ka CaMKII e ka loaʻa ʻana o ka huehue i loko o nā kinipoka ʻo NAc o nā kukuna ʻoi kino hoʻowahāwahā (X XUMUME) ʻaʻole nā ​​holoholona-naïve holoholona (Pane 1D). Hiki i kēia ke kuhikuhi i ka epigenetic "gen priming" hopena o ΔFosB (ʻO Robison lāuaʻo Nestler, 2011), a no laila kekahi mea molekele o ka incubation o ka cocaine craving (Pickens et al., 2011). Akā, no kēia hoʻololi chromatin e hoʻopili ʻia me ka hoʻopili ʻana i ka makemake makemake nui ʻia, e hoʻonui ʻia ana ka wā. E pōleʻa ia a hoʻoholo inā ʻo ia ka hihia, a loea e hōʻike ʻia inā nā ʻano ʻē aʻe i hōʻike i ka ΔFosB-hilinaʻi, subregion-spula nā cocaine. He mea nui hoʻi ia e hoʻomaopopo he ka loulou o ka hānai hoʻāla a mākou e wehewehe nei i ke alakaʻi ʻole ʻana i ka hopena o CaMKII a i ʻole ΔFosB (X XUMUME); ka wehe 'ana i ka molika "break" kuleana no kēia ka pahuhopu nui o nā noi mua.

ʻO nā hana e ʻike ʻia o ka ΔFosB a me CaMKII i loko o nā ʻōnaehana hoʻokolohua a me nā ʻili lolo e hui pū ai i nā pae he nui (Keia 6F). Ua hoʻopili ʻia nā molekela ʻelua i nā ulu spine dendritic: pili ʻo CaMKII me ka actin cytoskeleton (Okamoto et al., 2009), kau hoʻoponopono i ka nui o ka poʻo o ka mokulele (Matsuzaki et al., 2004), a he mea kūpono a lawa ia no ka hoʻonui ʻia ʻana o ka plasticity-indicated i ka filopodia a me ka helu synaps ma nā hippocampal organotypic slice culture (Jourdain et al., 2003), while ΔFosB pono a lawa pono no ka cocine-induced dendritic spine form ma NAc MSNs (Maze et al., 2010). Hoʻohui maikaʻi, ua pili nā nūpepa āpau i ka hoʻoponopono ʻana o nā mea kūlike o ka ʻĀina huakaʻi AMPA. ʻAʻole hoʻoponopono Ka CaMKII i ka nui o ka pae o nā subplu resA AMPA, akā hoʻokomo i ka hoʻokomo o nā mea hoʻoponopono o ka AMPA i loko o ka synapses a hoʻonui i ka alawai saluran AMPA e ka phosphorylating GluA1 ma Ser831 i nā hippocampal pyramidal neurons i ka moʻomeheu a me ke ʻano I loko o ka ola (loiloi (Malinow a me Malenka, 2002; ʻO Colbran a me Brown, 2004)). Ua hoʻonui ʻia kēlā me ke kalaiwa ʻo GluA1 i ka lihilihi.Boudreau a me Wolf, 2005). Eia kekahi, pane nā pane hana i ka hoʻonoho AMPA receptor i ka NAc e ka CaMKIIα overexpression i kahi D1 dopamine receptor-dependance kūlana (ʻO ka mele mele al., 2010). Ua hōʻike ʻia ka lōʻihi o ka lōʻihi o ka holo ʻana o ka D1-ΔFosB e hoʻonāukiuki iā GluA2 palapala i ka NAc (Kelz et al., 1999), ka mea e hopohopo nei i nā pane AMPA ma waena o GluA1, ʻoiai mākou e hōʻike ai ma ʻaneʻi ʻo overexpression pōkole ʻoʻΔ pōkole asFosB - me ka hōʻike pōkole ʻoi pōkole manawa - ʻaʻohe hopena o kēia subunit (X XUMUM). Eia nō naʻe, ua ʻike i kēlā me kēia manawa pōkole ʻo ΔFosB overexpression pōkole akā hōʻemi i nā pane AMPA ma D1-type MSNs i NAc (Grueter et al., 2013). Ke hōʻike nei kēia mau ʻāpana i nā hana kikowaena ʻokoʻa no ke ʻano o nā neuroadaptations manawa e pili ana i ka cocaine e kū nei i nā ʻano like ʻole o ka holomua hoʻomau i ʻike ʻole ʻia. Ma ka pae ʻai, ʻelua CaMKII a me ΔFosB e koi ʻia no ka hoʻonaninani ʻona i ka cocaine (e ʻike i luna), a e koi ʻia nā mea ʻelua no ka hoʻokō ʻana i nā mālama kokoti ma nā rodents (Colby et al., 2003; Wiki a me., 2010), e kuhikuhi ana i nā mea ʻelua i nā mea nui no ka hoʻohālikelike o ka hana lōʻihi i ka manawa lōʻihi a me ka lōʻihi o ka hoʻohālikelike ʻana i nā lāʻau lapaʻau, kū kekahi ma waena o nā mea i lalo. ʻO kēia, mālama ʻo ΔFosB a me CaMKII i kēlā mau ʻanoʻano paʻakikī i waena o nā loli ma NAc synaptic function, ʻoiai ʻoi aku ka hana e pono ai e hoʻopili i nā mea like a synaptic e pili ana i ka loli ʻano.

Hoʻopili ka CaMKII holoenzyme i kekahi ʻano like ʻole o ka protein synapseʻO Robison et al., 2005) ka mea i manaʻo e hoʻoponopono i kona nānā i nā pouynaptic density (PSD), kahi hanana i manaʻo ʻia e lilo i mea nui no ka synaptic plasticity. No ka hōʻike, ʻo ka pilina o CaMKII me nā pūʻali GluN2B o ka ʻohu glutamate NMDA-type ua hōʻike hou ʻia e hoʻoponopono pono i ka plasticapt synity me nā aʻo (Halt et al., 2012). ʻOiai ʻo ka ACPNNXX peptide ka mea e kaomi ai i ka domain autoinhibitory o CaMKII, a no laila, ke pale aku nei i ka hana catalytic enzyme, ke pale aku nei hoʻi i ka nui o nā pūmua protein-protein (Strack et al., 2000; ʻO Robison et al., 2005). No laila, ʻo ka hopena pili hewa a me ka morphological o HSV-GFP-AC3I i hōʻike ʻia ma aneʻi ma o ka hōʻemi ʻana o ka phosphorylation o nā hua target target CaMKII, nā hoʻololi i ka huli ʻana o CaMKII, a i ʻole ka hoʻololi ʻana i ka hana hoʻomohala a CaMKII i nā synapses (Lisman et al., 2002).

ʻO ke kaupalena i ka pahu ΔFosB-CaMKII i ʻōlelo ʻia i nā kinikini NAc he kikoʻī kūikawā, no ka mea ua hōʻike hou nei ka hana hou ʻana i nā ʻokoʻa o ka physiological ma waena o ka pūliki NAc a me ka pane ʻana i ka kulina kokoliko, he manaʻo manaʻo i hōʻoia ʻia e kā mākou ʻae ʻUa Aila (ANU S1) i hōʻike. . ^ E Ha yM. Hōʻike ka MSN i loko o nā NAc i ke kaumaha i ka puhi ahi ma hope o ka cocaine mau i mālama ʻia no nā hebedoma, ʻoiai ʻo ka MSNs mai nā holoholona like ʻole e hōʻike ana i kahi lā transient (1-3 lā) e hoʻonui i ke kaha ahi e hoʻi ana i ka pae basal i loko o nā wiki 2 (Kourrich lāua ʻo Thomas, 2009). Eia kekahi, nui nā protein synaptic i hoʻohālikelike ʻia i ka pūʻe NAc vs. ka kuhi o nā holoholona i hōʻike i ka kokolā e pili ana i ke kō, me GluA2 (Knackstedt et al., 2010). E like me ka maʻi nui o ka amphetamine e koi aku iā CaMKIIα i loko o NAc shell (Loweth et al., 2010), ʻaʻole he mea kupanaha i loaʻa iā mākou kahi hopena like me nā cocaine. Eia nō naʻe, e like me ka ΔFosB i hoʻoili ʻia i ka pūpū NAc a me ka kumu nui e ka kokoli (Perrotti et al., 2008), a i ka hōʻike ʻana o ka CaMKIIα induction i ke kinipona he ΔFosB-hilinaʻi, ʻike kā mākou mau ʻike i nā hōʻike hou no nā hana transcriptional ʻokoʻa ma ka mea hoʻomohala i ka CaMKIIα ma waena o kēia mau manawaleʻa ʻelua, ka mea pili i ka hoʻopili paʻa ʻana o CaMKIIα ma ka ʻili.

Nui ka hana nui o ka hana hou ʻana i pili i ka hoʻokaʻawale ʻana i waena o D1- a me D2-type NAc MSN. ʻOiai nā mea āpau a D1 a me D2 e komo pū ana i nā hopena pōmaikaʻi o ka cocaine (ʻOu iho, 2010), hōʻike i ka hana hou i ka hoʻonui ʻia o ka hana optogenetic o ka D1 type-type MSNs i ka pane ʻana i ka cocaine, aʻo D2 ka mea MSN ka hopena i ka hopena ʻē aʻe.Lobo et al., 2010). I ka laulā me kēia mau ʻike, ua nele nā ​​D1-receptor i nā kahe ʻeke kūwaho.Caine et al., 2007), ʻoiai ma kahi o D2 kikowaena (Caine et al., 2002). ʻO ka alakaʻi agonist D1 pololei i loko o NAc e hoʻomakei i ka loaʻa ʻana o ka cocaine e ʻimi nei i ka hana hou ʻana i nā paradigms (ʻOu iho, 2010). ʻO ka mea e mahalo ai, ʻo kēia hopena e pono ai i ka hoʻonui ʻana o ko D1-receptor hoʻonui i ka hana CaMKII i ka lāʻau NAc, akā ʻaʻole ia o inti (Anderson et al., 2008), he hopena e kūleʻa maikaʻi ana nā dovetail me ka D1- a me ka hana pūpū ΔFosB-CaMKII i ʻōlelo ʻia ma ʻaneʻi.

Ua hōʻike ma mua mākou ua hiki ʻo Ser27 i ΔFosB hiki i ka posoryory e ka casein kinase-2 (Ulery et al., 2006), akā, hoʻokumu mākou i ʻaneʻi ʻo CaMKII phosphory template ΔFosB ma kēia a me nā pūnaewele ʻē aʻe me nā kinetics nui loa a me stoichiometry a hiki ke hoʻohuli i ke kiʻekiʻe kiʻekiʻe o ka M_r nānā no ΔFosB (X XUMUMA) me ka hōʻike ʻana o ka cocaine I loko o ka ola (Nestler, 2008). Ua ʻike mua mākou i ka hoʻonui ʻia o ka phosphorylation Ser27 ΔFosB a me ka hana transcriptional (Ulery et al., 2006; ʻIli ʻia me Nestler, 2007; Ulery-Reynolds et al., 2009). E nānā mua kēia hana i ka wā e ʻike nei a me ka hopena o ka hopena o nā pūnaewele moʻolelo o ka oryFosB phosphorylation i hōʻike ʻia e kēia haʻawina.

Hāʻawi ʻia ka hānai hoʻākāka o ka hānai i hāʻawi ʻia i kahi hana hou e hoʻomohala ai i nā mana o ka kokela ma ke ʻano he mau hanana pono ʻole o ka NAc. E like me, hiki i kēia ala biochemical ke hāʻawi i kahi koʻikoʻi koʻikoʻi no ka interapeutic intervenative e hiki mai ana i loko o nā pilikia hoʻomohala. No ka mea he ʻano nui ʻo CaMKII no ka hana pono ʻana i ke ʻano neuronal basal a me nā hana kūpono, hoʻohana ʻole ʻia ka hoʻohana ʻana o CaMKII ʻohi ʻia he mea pili i ka mālama ʻana. Ke hōʻike nei kā mākou ʻikepili i kahi ʻoi aku ka ʻike hou ʻana i ka mīkini o ka hōʻemi ʻana o CaMKII, kahi ʻano ʻokoʻa o kēlā me kēia cell cell a subregion o ka circuitry uku ponoʻī, hiki ke hāʻawi i ka hopena therapeutic e pale ai i nā hoʻopiʻi o ka ʻōnaehana CaMKII.

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'Ōlelo hōʻeia

Ua kākoʻo ʻia kēia hana e nā loiloi mai ka National Institute on Drug Abuse (EJN), NIDA-Yale Proteomics Center DA018343 (AJR a me EJN), a me Hartwell Foundation (AJR). Makemake nui nā mea kākau e hoʻomaikaʻi iā Gabby Rundenko no ka makana lokomaikaʻi o ΔFosB lāua ʻo Roger Colbran no ka makana nui o ka caMKIIα.

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E hoʻomaopopo '

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