Hanyoyin ΔFosB na nuna damuwa akan opioid da kuma cannabinoid mai karɓar sakonni mai karɓa a cikin ƙananan mahaifa (2011)

Neuropharmacology. 2011 Dec;61(8):1470-6. doi: 10.1016/j.neuropharm.2011.08.046.

Sim-Selley LJ, Cassidy MP, Sparta A, Zachariou V, Nestler EJ, Selley DE.

source

Department of Pharmacology and Toxicology and Institute for Drug and Alcohol Studies, Virginia Commonwealth Jami'ar Medicine, Richmond, VA 23298, Amurka.

Abstract

Kodayake ma'ajin rubutu ΔFosB an jawo shi a cikin ƙwayar ƙwayoyin cuta (NAc) ta hanyar daukan hoto da dama ga magungunan da ake amfani da su, da kuma bayyanar da ta fito daga ΔFosB a cikin striatum yana inganta kyawawan kaddarorin morphine da cocaine. Duk da haka, ba a fahimci ainihin abin da ake amfani dasu akan wadannan abubuwan ba. Mun yi amfani da samfurin motsi na bitransgenic tare da bayyanawa a cikin ΔFosB a cikin dopamine D (1) mai karɓa / dynorphin dauke da ƙananan ƙananan ƙarfe don sanin sakamakon bayyanar ΔFosB a kan alamar opioid da kuma cannabinoid a cikin NAc. Sakamako ya nuna cewa aikin G-protein da aka sanya wa opioid da kuma hanawa adenylyl cyclase an inganta su a cikin NAc na ƙuda da suka bayyana ΔFosB. Hakazalika, an kaddamar da hana yin amfani da adenylyl cyclase a jikin kappa a cikin ΔFosB.. Sabanin haka, siginar mai karɓa na karɓa na cannabinoid ba ya bambanta tsakanin ƙananan ƙwayar ΔFosB da sarrafawa ba. Tbinciken binciken da aka gano yana nuna cewa siginar opioid da kuma cannabinoid siginar ta karɓa ne daban-daban ta hanyar bayyanar ΔFosB, kuma sun nuna cewa bayyanar ΔFosB zai iya haifar da wasu daga cikin tasirinsa ta hanyar inganta alamar mu da kappa opioid sigina a cikin NAc.

keywords: G-protein, adenylyl cyclase, striatum

Ka tafi zuwa ga:

1. Gabatarwa

Opioid masu karɓa da kuma CB cannabinoid₁ masu karɓa (CB₁R) su ne manufofin neurobiological don maganin miyagun ƙwayoyi guda biyu da suka hada da morphine, heroin da opioids kwayoyi, da marijuana (Δ⁹-tetrahydrocannabinol (THC)), bi da bi. Sakamakon masu amfani da kwayoyin G-protein da guda biyu waɗanda suka hada da G_{i / o} sunadarai kuma samar da amsoshin gado mai zurfi irin su hanawa adenylyl cyclase (Childers, 1991, Childers, et al., 1992, Howlett, et al., 2002). Motar, ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ajiya da tasiri na Δ⁹-Wannan an samar da CB₁R (Huestis, et al., 2001, Zimmer, et al., 1999), wanda aka yadu a cikin kwakwalwa, tare da matakan da ke cikin basal ganglia, hippocampus da cerebellum (Herkenham, et al., 1991). Hanyoyin da ake amfani da su na cututtuka da kuma sakamako masu mahimmanci da magungunan maganin magungunan maganin magungunan maganin magungunan su ne magunguna ne masu magunguna (MOR)Matthes, et al., 1996), waxanda suke wadatar da su a cikin tsarin lalata da kwakwalwa (Mansour, et al., 1994). Tsarin mesolimbic, wanda ya hada da magungunan kwayoyin halitta daga yankin jiki (VTA) zuwa tsakiya (NAc), yana taka muhimmiyar rawa a sakamakon sakamako mai kyau na opioids da cannabinoids (Bozarth da Hikima, 1984, Vaccarino, et al., 1985, Zangen, et al., 2006), kazalika da sauran magunguna na zalunci (Koob da Volkow, 2010). Bugu da ƙari, ƙwayoyin opioid da cannabinoid da ke ciki suna da hannu a sakamakon sakamako mai yawa na nau'o'i masu yawa na kwayoyin psychoactive (Maldonado, et al., 2006, Trigo, et al., 2010). Saboda haka, yana da muhimmanci a samar da hanyoyin da ake amfani da ita wajen samar da hanyoyin sadarwa ta hanyar opioid da CB₁R alamar ana yin rajista a cikin NAc.

Tambaya ta tsakiya a cikin magungunan maganin miyagun ƙwayoyi shine ya gano sunadarin sunadarai wanda ke janyo hanzari daga matsananciyar cututtukan kwayoyin psychoactive. Takaddun rubutun AP-1 na ΔFosB yana da ban sha'awa sosai domin yana da wani barga mai sauƙi na samfurori na samfurin fosb jinsin da ke tarawa a kan maimaitawa zuwa magungunan ƙwayoyi ko ladabi na ladabi (McClung, et al., 2004, Nestler, 2008, Nestler, et al., 1999). Mun gano cewa an kwantar da ΔFosB a cikin kwakwalwa bayan an sake bayyanawa zuwa morphine, Δ⁹-THC, cocaine ko ethanol, tare da kowace miyagun ƙwayoyi da ke samar da wani yanki na musamman na yankin ΔFosB (Perrotti, et al., 2008). Sakamakon da aka gano a ko'ina cikin kwayoyi shi ne cewa ΔFosB da aka samu sosai a cikin striatum, inda dukkanin kwayoyi hudu suka haifar da ΔFosB a cikin NAC kuma duk sai dai Δ⁹-CKC da aka ƙaddamar da shi sosai a cikin nauyin NAC da kuma caudate-putamen.

Nazarin Pharmacological ya nuna cewa gwamnatin co-administration na dopamine D₁ Mai karɓa (D₁R) mai maganin SCH 23390 ya katange ΔFosB shigarwa a cikin NAc da kuma caudate-sakaamen bayan cocaine intermittent ko tsarin morphine, yana nuna muhimmancin D₁R-bayyana neurons (Muller da Unterwald, 2005, Nye, et al., 1995). An yi nazarin sakamako na ΔFosB akan halayen miyagun ƙwayoyin cuta ta amfani da mice bitransgenic wanda ke bayyana ΔFosB a wasu al'ummomin neuronal musamman na NAc da dorsal striatum (Chen, et al., 1998). Mice da ke bayyana ΔFosB a dynorphin / D₁R a cikin NAC da kuma dorsal striatum (layin 11A) sun nuna nuna martani ga magungunan ƙwayoyi, musamman kara ingantaccen tunani ga sakamakon sakamako na cocaine ko morphine (Colby, et al., 2003, Kelz, et al., 1999, Zachariou, et al., 2006). Wadannan canje-canje sun faru ne idan babu canje-canje a cikin matakan MOR ko ɗakunan G-protein. Kodayake, matakan mRNA na dynorphin sun rage a cikin NAc na ΔFosB suna nuna mice (Zachariou, et al., 2006), yana nuna cewa manufa daya na ΔFosB shine jigon halittar da ke kunshe da peptide opioid. ΔFosB shigarwa zai iya haifar da canje-canje ta hanyar daidaita tsarin alamar mai karɓa a cikin NAc, amma wannan yiwuwar ba a bincika ba. Saboda haka, binciken da aka yi a yanzu ya yi amfani da samfurin bitransgenic mouse don tantance ko bayyanar ΔFosB a dynorphin / D₁R dauke da ƙananan ƙananan ƙwayoyin suna canzawa da G-protein da ake kira MOR da MOR- da kuma KOR-adresylyl cyclase a cikin NAc. Sakamakon ΔFosB akan CB₁An kuma gwada aikin G-haɗin gwargwadon R na R don Δ⁹-Gashin kula da CPC ya jawo ΔFosB a cikin NAc (Perrotti, et al., 2008) da kuma tsarin endocannabinoid da aka sani don tsara tsarin layin kwakwalwa (Gardner, 2005, Maldonado, et al., 2006), amma ba a bincika sakamakon ΔFosB akan tsarin endocannabinoid ba.

Ka tafi zuwa ga:

2. Kaya da matakai

2.1. Maganin

[³⁵S] GTPγS (1250 Ci / mmol), [α-³²P] ATP (800 Ci / mmol) da kuma [³H] cAMP (26.4 Ci / mmol) an saya daga PerkinElmer (Shelton, CT). ATP, GTP, GDP, CAMP, celinine bovine, creatine phosphokinase, papaverine, imidazole da WIN-55212-2, an saya daga Sigma Aldrich (St. Louis, MO). An sayi GTPYS ne daga Kamfanin Roche Diagnosis (Chicago, IL). Hukumar Drug Supply na Cibiyar Kasa ta Kasa ta Drug (Rockville, MD) ta bayar da DAMGO. An samo ruwa na Econo-1 a cikin Fisher Scientific (Norcross, GA). An samo ruwa mai tsafta daga ICN (Costa Mesa, CA). Duk sauran sunadaran sun samo daga Sigma Aldrich ko Fisher Scientific.

2.2. Mice

Mace bitransgenic da aka samo daga NSE-tTA (layin A) × TetOp-ΔFosB (11 XNUMX) an samo asali a cikin Kelz et al. (Kelz, et al., 1999). An haife mahaukaci na Bitransgenic da kuma tashe su a kan doxycycline (100 μg a ruwa mai sha) don kawar da fadin transgene. A makonni na 8 yana da shekaru, an cire watsi da kwayar cutar daga ruwa don rabi na mice don ba da izini na fadi, yayin da sauran 'yan ƙuƙuka suka kasance a kan doxycycline don su rage transgene. An tattara kwakwalwa ta hanyar 8 makonni baya, lokacin da tasirin bayanan ΔFosB ne mafi girma (McClung da Nestler, 2003). An yi amfani da jigon linzamin na biyu a cikin abin da Δc-Jun, wanda ke da magungunan c-Jun, ya bayyana a cikin D₁R / dynorphin da D₂R / enkephalin sel na striatum, hippocampus da peietal cortex (Peakman, et al., 2003). C-Jun da kuma sauran sunadaran dangin Jun sun hada da fos din dangin iyali kuma sun danganta zuwa shafin AP-1 na tushen kwayoyin don tsara fassarar. Duk da haka, ƙaddamarwa na N-karshen na C-Jun (Δc-Jun) ya ba da hadarin da ba shi da aiki kuma yana iya hana haɗin DNA na ƙungiyar AP-1 mai aiki. Mace bitransgenic da aka samo daga NSE-tTA (layin A) × TetOp-FLAG-Δc-Jun (layin E) an yi kamar yadda aka bayyana a cikin Peakman et al. (Peakman, et al., 2003). An haife mahaukaci na Bitransgenic da kuma tashe su a kan doxycycline (100 μg a ruwa mai sha) don kawar da fadin transgene. An yaye Pups a cikin makonni 3, an samo su, kuma sun rabu cikin kungiyoyi, tare da rabi suna cike da ruwa da rabi a kan ruwan sha na yau da kullum don haifar da furcin FLAG-Δc-Jun. An tattara kwakwalwan ƙwayoyin 6 bayan haka, lokacin da aka auna girman matakan FLAG-Δc-Jun (Peakman, et al., 2003). Dukkan hanyoyin dabba an gudanar da su bisa ga Cibiyar Nazarin Kiwon Lafiyar Jama'a don Kulawa da Amfani da Dabbobin Laboratory.

2.3. Shirin Shirin Membrane

An adana kwakwalwa a -80 ° C har zuwa ranar gwajin. Kafin gwaji, an kwantar da kwakwalwa, kuma an rarraba NAC akan kankara. Kowace samfurin an haɗa shi a cikin 50 mM Tris-HCl, 3 mM MgCl₂, 1 mM EGTA, pH 7.4 (buffer membrane) tare da kwakwalwan 20 daga homogenizer gilashin a 4 ° C. An sanya homogenate a 48,000 × g a 4 ° C don 10 min, wanda aka dakatar a cikin buffer membrane, sake mayar da shi a 48,000 × g a 4 ° C na 10 min da kuma sake dakatarwa a 50 mM Tris-HCl, 3 mM MgCl₂, 0.2 mM EGTA, 100 mM naCl, pH 7.4 (buffer buffer). Matakan protein sun ƙaddara ta Hanyar Bradford (Bradford, 1976) ta yin amfani da kwayar bovine albumin (BSA) a matsayin misali.

2.4. Agonist-Sanya [³⁵S] GTPYS Binding

An gabatar da lambobin zuwa minti na 10 a 30 ° C tare da adenosine deaminase (3 mU / ml) a cikin buffer. An hada da sunadarai (5-10 μg protein) don 2 hr a 30 ° C a cikin buffer da ke dauke da 0.1% (w / v) BSA, 0.1 nM [³⁵S] GTPγS, 30 μM GDP da adenosine deaminase (3 mU / ml) tare da ba tare da halayen dacewa na DAMGO ko WIN55,212-2 ba. An ƙaddamar da ƙayyadadden ƙaddamarwa tare da 20 μM GTPγS. An kare gurbin ta ta hanyar filtration ta hanyar GF / B gilashin fiber gilashi, sa'annan 3 ya wanke tare da 3 ml 50 mM Tris-HCl sanyi mai sanyi, pH 7.4. Rashin radiyo ta ƙayyadad da labarun bayanan bayanan bayanan da aka cire a cikin watsi na Econo-1.

2.5. Adenylyl Cyclase Assay

Membranes (5-25 μg protein) sun kasance sunadarai tare da adenosine deaminase kamar yadda aka bayyana a sama, sa'an nan kuma sun hada da 15 min a 30 ° C a gaban ko babu 1μM forskolin, tare da ko ba tare da DAMGO, U50,488H ko WIN55,212-2 ba 50 μM ATP, [α-³²P] ATP (1.5 μCi), 0.2 MM DTT, 0.1% (w / v) BSA, 50 μM AMP na cyclic, 50 μM GTP, 0.2 mM Papaverine, 5 mM phosphocreatine, 20 raka'a / ml creatine phosphokinase da adenosine deaminase (3 mU / ml) a cikin ƙarshe na 100 μl. A karkashin wadannan yanayi, jimlar [α-³²P'ACAMP da aka dawo dasu bai kasance ba fãce 1% na yawan adadin [α-³²P] ATP a kowane samfurin. An ƙare wannan motsi ta tafasa don 3 min da [³²P] Cyclic AMP an ware shi ta hanyar hanyar dualx (Dowex da alumina) na Salomon (Salomon, 1979). [³H] cAMP (10,000 dpm) an kara da shi a kowanne tube a gaban rubutun chromatography kamar yadda yake ciki. Rahoton samfurin scintillation na ruwa ya ƙaddamar da radiyo (45% ya dace don ³H) bayan an cire 4.5 ml na biyu a cikin 14.5 ml na Ecolite scintillation fluid.

2.6. Bayanin bayanai

Sai dai idan ba a nuna hakan ba, ana bayar da bayanai a matsayin mahimmanci ± SE na 4-8 gwaje-gwajen da aka raba, kowannensu an yi a cikin gajeren lokaci. Net-daɗa [³⁵S] GTPYS takaddama an lissafta shi azaman karamin motsi wanda ke da mahimmanci ƙananan basali. Net forskolin-daɗaɗɗa adenylyl cyclase aiki an bayyana shi azaman aikin forskolin-ƙaddara - aikin basal (amol / mg / min). Kisan kashi na aikin ƙaddamar da ƙwayar adenylyl cyclase donskolin-nema (net forskolin-stimulated activity in babu agonist - net forskolin-stimulated aiki a gaban agonist / net forskolin-stimulated aiki a cikin babu agonist) × 100. An gudanar da nazari na lissafi tare da yin amfani da Prism 4.0c (GraphPad Software, Inc., San Diego, CA). An yi nazari akan ƙididdigar ƙididdigar ta hanyar rikici da ba a layi ba don samun EC₅₀ da kuma E_max dabi'u. Ƙididdigar ilimin kididdigar bayanai ta ƙaddamarwa ya ƙaddara ta hanyar yin amfani da hanyoyi guda biyu (ANOVA), ta hanyar amfani da kwayar cutar da kuma haifar da kwayoyin (a kunne ko a kashe) a matsayin ainihin dalilai. Ƙididdigar lissafi na ƙididdigar ƙira (E_max ko EC₅₀) an ƙaddara ta t-test ɗin ɗalibin da ba a haɗa guda biyu ba, ta amfani da gyaran Welch ko canjin tushen murabba'in bayanan inda ya zama dole don gyara don bambancin da bai dace ba (wanda F-test ya gano) a cikin EC₅₀ dabi'u.

Ka tafi zuwa ga:

3. Sakamako

3.1. Hanyoyin ΔFosB a kan maganin G-protein wanda aka ƙaddamar da shi ta hanyar opioid da cannabinoid

Don sanin ko MOR- ko CB₁An sauya haɗin G-haɗin gwargwadon R da aka ƙaddara ta hanyar fassarar fassarar ΔFosB a cikin NAc,³⁵S] GTPYS an ɗaure shi a cikin tsararru wanda aka shirya daga wannan yankin na bitransgenic mice yanayin da yake nuna (ΔFosB on) ko ba bayyana (ΔFosB kashe) ΔFosB transgene ba. Ana amfani da DAMGO mai analog na MOR-mai amfani mai amfani don kunna MOR da kuma WIN55,212-2 na aminoalkylindole cannabinoid don amfani da CB₁R. Wadannan alamu sun nuna cewa sun kasance cikakke ne a MOR da CB₁R, bi da bi (Breivogel, et al., 1998, Selley, et al., 1997). Ba'a yiwuwa a bincika aikin G-haɗin gwargwadon rahoto na KOR ba saboda siginar ya yi ƙasa da ƙananan kwakwalwa (Childers, et al., 1998). Sakamakon ya nuna ƙarfin haɗin gwargwadon aikin G-gina jiki ta DAMGO da WIN55,122-2 a cikin NAc daga ΔFosB da kuma ΔFosB a kan mice (Figure 1). Don DAMGO-ta daɗaɗa aikin (Hoto 1A), hanyoyi guda biyu ANOVA na bayanan tasirin tasiri sun nuna mahimman tasirin tasirin ΔFosB (p <0.0001, F = 22.12, df = 1) da ƙaddamar da DAMGO (p <0.0001, F = 29.65, df = 5) ba tare da babu Mu'amala mai mahimmanci (p = 0.857, F = 0.387, df = 5). Binciken da ba a kan layi ba game da raunin tasirin-tasiri ya bayyana mafi girma DAMGO E_max darajar a ΔFosB a kan ƙananan (E_max = 73 ± 5.2% ƙarfafa) dangane da ƴan ΔFosB kashe ƙananan yara (E_max = 56 ± 4.1% kara kuzari; p <0.05 ya bambanta da ΔFosB akan beraye ta t-gwajin dalibi). DAMGO EC₅₀ ƙididdiga ba su bambanta tsakanin ΔFosB da kuma 'yan ƙananan ΔFosB ba (302 ± 72 nM da 212 ± 56 nM, daidai da haka, p = 0.346).

Sakamakon ΔFosB magana a kan agonist-stimulated [³⁵S] GTPYS ke ɗaure a cikin NAc. Membranes daga ΔFosB-bayyana (ΔFosB a kan) ko iko (ΔFosB kashe) ƙwararrun ƙwararriya kamar yadda aka bayyana a cikin hanyoyin ta amfani da ƙananan yawa ...

Ya bambanta da sakamakon da aka samu tare da DAMGO agonist MOR, ba a lura da bambance-bambancen hali na ΔFosB a G-protein activation tare da WIN55,212-2 mai maganin cannabinoid (Hoto na 1B). Hanya-biyu ANOVA na bayanan ƙarfin tasirin WIN55,212-2 ya bayyana babban tasirin tasirin WIN55,212-2 (p <0.0001, F = 112.4, df = 7), amma ba na ΔFosB ba (p = 0.172 , F = 1.90, df = 1) kuma babu ma'amala (p = 0.930, F = 0.346, df = 7). Hakanan, babu tasirin matsayin osFosB akan WIN55,212-2 E_max dabi'u (103 ± 6% da 108 ± 8% ƙarfafawa a ΔFosB akan kashewa da kashe ƙananan, p = 0.813 ta T-gwaji na Student) ko EC₅₀ dabi'u (103 ± 20 nM da 170 ± 23 nM a cikin ΔFosB a kunne da kashe ƙananan yara, p = 0.123).

Bisa ga siffar ɗigin hanyoyi da gaskiyar cewa bincikenmu na baya ya nuna alamar WIN55,212-2 mai kwakwalwa a cikin kwakwalwa (Breivogel, et al., 1999, Breivogel, et al., 1998), an kuma gwada hanyoyi na WIN55,212-2 ta hanyar amfani da samfuri biyu. Tattaunawa game da bayanan da aka saukar ya nuna wani ɗan ƙaramin cigaban ingantacciyar fitarwa ta hanyar amfani da samfurin biyu (R² = 0.933 da 0.914, jimlar murabba'in = 3644 da 5463 a ΔFosB akan kashewa da kashe su) idan aka kwatanta da samfurin guda-rukunin (R² = 0.891 da 0.879, jimlar murabba'i = 6561 da 6628 a ΔFosB akan kashewa da kashe su). Duk da haka, babu wani bambance-bambance mai banbanci tsakanin ΔFosB akan kashewa da kuma kashe su a ko dai E_max ko EC₅₀ ƙididdiga na manyan wurare masu mahimmanci (Ƙarin Shafi na 1), kodayake akwai tasowa zuwa ƙananan EC₅₀ darajar a tashar mai girma a cikin ƙananan yara tare da ΔFosB akan (EC₅₀high = 28.0 ± 10.6 nM) idan aka kwatanta da wadanda suke tare da ΔFosB kashe (EC₅₀high = 71.5 ± 20.2 nM; p = 0.094). Bugu da ƙari, babu wani sakamako na ΔFosB akan basal [³⁵S] GTPYS ke ɗaure a cikin membranes na NAc (253 ± 14 da 226 ± 14 fmol / MG a ΔFosB a kan kuma kashe ƙuda, p = 0.188). Wadannan bayanan sun nuna cewa maganganun da ke cikin ΔFosB a cikin NAC na ƙuda sun ƙãra ƙarfin G-protein wanda aka ƙaddara ta MOR ba tare da ya shafi CB ba₁R-rikice-rikicecen G ko haɗin G-gina jiki.

3.2. Sakamakon ΔFosB akan opioid da cannabinoid haɓakar mai karɓa na adenylyl cycse

Don kimanta sakamakon sakamako mai sauƙi na ΔFosB a kan tasirin aikin ƙwaƙwalwar ƙasa ta hanyar MOR da CB₁R, haramtawa na 1 μM forskolin-daɗaɗɗen adenylyl cyclase aiki aka bincika a cikin NAc membranes. Baya ga MOR- da CB₁An hana rukunin R-mediated na ayyukan adenylyl cyclase, sakamakon aikin KOR wanda aka yi amfani da shi ta amfani da cikakken zauren agonist U50,488 (KOR-selective agonist)Zhu, et al., 1997), saboda sakamakon da ya gabata ya nuna cewa mRNA dynorphin shine manufa ta ΔFosB a cikin bitransgenic model (Zachariou, et al., 2006). Sakamako ya nuna cewa DAMGO, U50,488 da WIN55,212-2 kowannensu ya samar da ƙyama ga aikin adenylyl cyclase a duka biyu na ΔFosB da ΔFosB a kan mice (Figure 2). Hanyar guda biyu ANOVA na DAMGO maida hankali-bayanan bayanai (Hoto 2A) ya bayyana babban tasirin tasirin ΔFosB (p = 0.0012, F = 11.34, df = 1) da ƙaddamar da DAMGO (p <0.0001, F = 29.61, df = 6), amma babu wata ma'amala mai mahimmanci (p = 0.441, F = 0.986 , df = 6). Binciken da ba a kan layi ba na rayayyun tasirin tasirin DAMGO ya bayyana ƙaramar DAMGO EC₅₀ darajar a cikin ΔFosB akan beraye (101 ± 11 nM) idan aka kwatanta da ΔFosB daga beraye (510 ± 182 nM, p <0.05 ta t-gwajin dalibi). Koyaya, babu wani bambanci mai mahimmanci a cikin DAMGO E_max dabi'u (20.9 ± 1.26% tare da 19.8 ± 1.27% hani a ΔFosB akan kashewa, kashewa, p = 0.534).

Hanyoyin ΔFosB akan hana hana adenylyl cyclase a cikin NAc. Membranes daga ΔFosB-bayyana (ΔFosB a kan) ko iko (ΔFosB kashe) anyi kyan ƙuda kamar yadda aka bayyana a cikin hanyoyin a gaban 1 μM ...

Hanya na adenylyl cyclase marar kyau ta KOR ya bambanta a matsayin aikin fassarar transgenic maras kyau na ΔFosB (Hoto na 2B). Hanya-biyu ANOVA na U50,488 tattara sakamako-sakamako sun nuna manyan mahimmancin tasirin ΔFosB (p = 0.0006, F = 14.53, df = 1) da U50,488 taro (p <0.0001, F = 26.48, df = 3) , ba tare da wata ma'amala mai ma'ana ba (p = 0.833, F = 0.289, df = 3). Binciken rashin daidaituwa game da raƙuman tasirin-tasiri ya bayyana mafi girman U50,488 E_max darajar a cikin ΔFosB akan ƙananan yara (18.3 ± 1.14% hanawa) idan aka kwatanta da ΔFosB daga ƙananan yara (12.5 ± 2.03% hanawa; p <0.05 daban-daban daga ΔFosB akan ta t-gwajin dalibi), ba tare da wani bambanci mai mahimmanci ba a cikin U50,488 EC₅₀ dabi'u (310 ± 172 nM da 225 ± 48 nM a cikin ΔFosB a kunne da kashe ƙananan yara, p = 0.324).

Ya bambanta da tasirin da aka gani tare da MOR da KOR, babu wani sakamako mai mahimmanci na maganganu na ΔFosB wanda ba zai iya haifuwa ba akan hanawa adenylyl cyclase ta hanyar WIN55212-2 mai maganin cannabinoid (Hoto 2C). Hanya-biyu ANOVA na bayanan tasirin tasirin WIN55,212-2 sun nuna mahimmancin tasirin maganin ƙwayoyi (p <0.0001, F = 23.6, df = 2), amma ba halin statusFosB ba (p = 0.735, F = 0.118, df = 1) kuma babu wata ma'amala mai mahimmanci (p = 0.714, F = 0.343, df = 2). Bugu da ƙari kuma, babu tasirin matsayin osFosB a kan basal ko forskolin-daɗa ƙwazo adenylyl cyclase ba tare da wani agonist ba. Ayyukan basal adenylyl cyclase shine 491 ± 35 pmol / mg / min a cikin osFosB akan bera idan aka kwatanta da 546 ± 44 a cikin ΔFosB daga mice (p = 0.346 ta t-test na Student). Hakanan, aikin adenylyl cyclase a gaban 1 µM forskolin shine 2244 ± 163 pmol / mg / min a cikin osFosB akan beraye akan 2372 ± 138 pmol / mg / min a cikin ΔFosB daga mice (p = 0.555).

3.3. Sakamakon ΔcJun akan kangewa da kuma cannabinoid haɓakar mai karɓa na adenylyl cyclase

Saboda faɗakarwar sakonnin ΔFosB na ingantaccen siginar sigina daga MOR da KOR zuwa adenylyl cyclase a cikin NAc, yana da sha'awar ƙayyade ko mai rinjayar magungunan na ΔFosB-transcriptive rubutun zai canza tsarin sigina na opioid a wata hanya dabam. Don magance wannan tambaya, ba da izinin ayyukan DAMGO da U50,488 ba, sun kaddamar da su ne daga shirin NAC na bitransgenic mice wanda ke nuna ΔcJun. Sakamakon ya nuna babu wani sakamako mai mahimmanci akan maganganun ΔcJun game da hana aikin adenylyl cyclase na MOR ko KOR (Figure 3). Hanya-biyu ANOVA na masu lankwasa tasirin-DAMGO sun nuna babban tasirin tasirin DAMGO (p <0.0001, F = 20.26, df = 6), amma ba halin ΔcJun ba (p = 0.840, F = 0.041, df = 1) kuma babu wata ma'amala mai mahimmanci (p = 0.982, F = 0.176, df = 6). Hakanan, babu wani bambanci mai mahimmanci a cikin E_max ko EC₅₀ dabi'u tsakanin mice tare da ΔcJun a kan (E_max = 23.6 ± 2.6%; EC₅₀ = 304 ± 43 nM) ko ΔcJun kashe (E_max = 26.1 ± 2.5%, p = 0.508; EC₅₀ = 611 ± 176 nM, p = 0.129). An ga irin wannan sakamakon tare da U50,488, irin wannan hanyar ANOVA ta hanyoyi biyu masu lankwasa-sakamakon tasiri ya nuna mahimmin sakamako na maida hankali (p <0.0001, F = 11.94, df = 6), amma ba na ΔcJun status (p = 0.127 , F = 2.391, df = 1) kuma babu wata ma'amala mai mahimmanci (p = 0.978, F = 0.190, df = 6). Hakanan, babu wani bambanci mai mahimmanci a cikin E_max ko EC₅₀ dabi'u tsakanin mice tare da ΔcJun a kan (E_max = 14.8 ± 2.9%; EC₅₀ = 211 ± 81 nM) ko kashe (E_max = 16.7 ± 1.8%, p = 0.597; EC₅₀ = 360 ± 151 nM, p = 0.411).

Sakamakon magana ta ΔcJun game da hana hana adenylyl cyclase a cikin NAc. Membranes daga ΔcJun-bayyana (ΔcJun on) ko kuma iko (ΔcJun kashe) an haɗu da mice a gaban DAMGO (A), U50,488H (B) ko WIN55,212-2 ...

Maganar ΔcJun kuma ba ta shafi tasirin hana adenylyl cyclase a cikin NAc ba ta hanyar agonist na cannabinoid. Hanya-biyu ANOVA na masu lankar WIN55,212-2 sun nuna babban tasirin tasirin WIN55,212-2 (p <0.0001, F = 15.53, df = 6), amma ba na jinsi ba (p = 0.066, F = 3.472, df = 1) kuma babu wata ma'amala mai mahimmanci (p = 0.973, F = 0.208, df = 6). Hakanan, babu manyan bambance-bambance a cikin WIN55,212-2 E_max dabi'u (13.0 ± 2.3% da 13.6 ± 0.9% hani a cikin ΔcJun a kan ƙananan ƙwayoyi, kamar yadda p = 0.821) da kuma EC₅₀ dabi'u (208 ± 120 nM da 417 ± 130 nM a ΔcJun a kan ƙananan ƙwayoyin, bi da bi, p = 0.270). Sabili da haka, ko da yake akwai yanayin da ke faruwa a cikin WIN55,212-2 a cikin mice da ke nuna ΔcJun, transgene ba ya canza cannabinoid hanawa na adenylyl cyclase. Bugu da ƙari, babu wani sakamako na ΔcJun a kan basal ko forskolin-daɗaɗɗa adenylyl cyclase aiki. Hanyar adenylyl cyclase aiki shine 1095 ± 71 pmol / MG / min da 1007 ± 77 pmol / MG / min (p = 0.403) a cikin ƙananan mata tare da ΔcJun a kan ko kashe, bi da bi. Ayyukan Adenylyl cyclase na 1 μM forskolin sune 4185 ± 293 pmol / MG / min da 4032 ± 273 pmol / MG / min (p = 0.706) a cikin yara tare da ΔcJun kan ko kashe, daidai da haka.

3.4. Tattaunawa

Sakamakon wannan binciken ya nuna ingantaccen G-protein da aka yi wa MOR da kuma ƙin adenylyl cyclase a cikin NAc na ƙuda da sakonnin transzinic induci na ΔFosB a dynorphin / D₁R dauke da ƙwayoyin hannu. An ƙaddamar da hana ƙaddamar da aikin adenylyl cyclase ta KOR a cikin NAc na ΔFosB yana nuna ƙwayar mice, yana nuna cewa ΔFosB yana sarrafa tsarin opioid na ƙarshe a cikin NAc. DAMGO E_max darajar ita ce mafi girma ga MOR-zuga [³⁵S] GTPYS da kuma EC₅₀ darajar da aka ƙananan don ƙin adenylyl cyclase, a cikin ΔFosB akan nuna ƙananan mice idan aka kwatanta da ƙwayoyin sarrafawa. Wadannan binciken sun bada yiwuwar yiwuwar ajiyar mai karɓa don yanayin haɓaka amma ba G-protein kunnawa a ƙarƙashin yanayin gwaji da aka bincika. Sakamakon gano cewa ƙin adenylyl cyclase mafi girma ya kasance da maganin ΔFosB ya shafa ya nuna cewa bashin ajiyar mai karɓa don amsawa ta KOR, daidai da ƙananan matakan KOR shafukan yanar gizo a cikin ƙwaƙwalwar ƙwaƙwalwa.Unterwald, et al., 1991). Ya bambanta, CB₁G-haɗin g-haɗin g R da kuma ƙin adenylyl cyclase ba su da alamar ΔFosB magana, suna nuna cewa tsarin opioid da cannabinoid sun bambanta a cikin amsawarsu ga ΔFosB a cikin wadannan naurorin ne na NAc.

Sakamakon ΔFosB akan siginar mai karɓa na mai samfurin opioid daidai ne da rahotonmu na baya cewa ΔFosB maganganun a cikin harsashi sun canza mummunar cututtuka na morphine (Zachariou, et al., 2006). Ɗaya daga cikin binciken wannan binciken shi ne cewa ƙuƙwalwa tare da maganganu na ΔFosB a dynorphin / D₁Ruƙan ƙwayar ƙarfe na R sun fi kulawa da morphine a cikin yanayin kwandon ruwa fiye da sarrafawa. Bugu da ƙari kuma, wannan tasirin ya kasance mai ladabi ta hanyar maganganu na ΔFosB ta hanyar rigakafi ta hanyar shafin a cikin NAc. Wadannan bayanan sunyi daidai da sakamakon yanzu da ke nuna alamar MOR da aka inganta a cikin NAc.

Mun riga mun gano jigilar halittar dynorphin a matsayin manufa ta ΔFosB, kuma ya bada shawara cewa rage dynorphin zai kasance daidai da ingantaccen kaddarorin morphine a cikin ΔFosB bitransgenic mice (Zachariou, et al., 2006). Sakamakon da ke yanzu ya nuna cewa an haramta ƙin adenylyl cycse na KOR a cikin NAc akan ΔFosB yana nuna ƙwayar mice, wanda zai iya nuna yawan karuwar yawancin jiki a cikin jiki mai kyau bayan rage dynorphin. Binciken da ya gabata ya nuna cewa KOR da aka ƙaddara a wasu yankuna na kwakwalwa na ƙananan ƙwararrakin prodynorphin, ciki har da NAc (Clarke, et al., 2003).

Ya bambanta da ΔFosB, furci mai lalacewa ta hanyar ΔcJun, mahalarci mai ƙananan ƙwararrun abokin tarayya na ΔFosB cJun abokin tarayya, bai canza musanya adenylyl cyclase ta MOR ko KOR agonists ba. Wadannan sakamakon sun nuna cewa matakan basal na ΔFosB, wanda basu da mahimmanci, ba su taka muhimmiyar rawa wajen rike alamar siginar mai karɓa a wannan matakin siginar a cikin NAc. Gaskiyar cewa yanayin rashin lafiya na morphine ya rage ta hanyar ΔcJun a cikin bincikenmu na baya (Zachariou, et al., 2006) ya nuna ko dai cewa shigarwa na morpine na ΔFosB a lokacin aikin kwantar da hankali yana da mahimmanci wajen gyaran halayen halayyar halayyar maganin miyagun ƙwayoyi ko sakamakon rikodin ΔFosB banda wadanda ke nuna alamar sakonni ta hanyar masu karɓa na opioid zasu iya tasiri sakamakon sakamako. A kowane hali, sakamakon binciken na yanzu ya nuna a sarari cewa, lokacin da ake nuna ΔFosB magana a sama da matakan basal a dynorphin / D₁R-bayyana ƙananan ƙarfe, akwai karuwa mai ƙarfi a cikin haɗin gwiwar MOR da KOR don hanawa adenylyl cyclase a cikin NAc.

Hanyoyin da MOR- da KOR-mediated signaling suka inganta ta ΔFosB overexpression ne m, amma mun nuna a baya cewa matakan MOR, kimantawa ta [³H] naloxone ɗaure, ba su da bambanci a cikin NAc na ΔFosB a kan ƙananan mice (Zachariou, et al., 2006). Haka binciken ya gano cewa Ga_i1 da 2 ba matsala ba a cikin yankin nan ta hanyar ΔFosB. Duk da haka, bayanan maganganun nunawa na baya ya nuna cewa Ga_o MRNA ba shi da lalata a NAc na ΔFosB a kan ƙuda (McClung da Nestler, 2003). Zai yi amfani da nazarin da za a yi a gaba don bincika tasiri na bayyanar ΔFosB akan maganganun G-protein a fannin gina jiki da kuma nuna yawancin sunadaran G-protein.

Yana da ban sha'awa cewa bayanin ΔFosB bai inganta CB₁R-sanarwa mai sakonni a cikin NAc. Yana yiwuwa yiwuwar canje-canje a CB₁R alamar alama tana faruwa ne a cikin yawan mutane da yawa waɗanda aka ɓoye a cikin dukan shirin NAc. Alal misali, gwamnatin Δ⁹- THC ya haifar da ΔFosB sosai a cikin mahimmanci, amma ba harsashi, na NAc baPerrotti, et al., 2008). NiMuddin, an nuna cewa kalubale tare da Δ⁹-THC bayan ci gaba da gwamnatin Δ⁹-THC ta ƙãra sanarwa dopamine a cikin NAc, amma rage rage a cikin harsashi (Cadoni, et al., 2008). Yana da mahimmanci a lura cewa layin 11A na mice bitransgenic ya bayyana ΔFosB kawai a dynorphin / D₁R na ainihin matsakaici na ƙananan igiyoyi na striatum, amma CB₁R aka bayyana a duka dynorphin / D₁R da enkephalin / D₂R tabbatacciyar sifofin ne (Hohmann da Herkenham, 2000), kazalika da a kan magunguna na afferents cortical (Robbe, et al., 2001). Magana game da mai mahimmanci mai kula da fassarar ΔFosB, ΔcJun, kuma ba shi da wani tasiri mai muhimmanci a kan siginar mai karɓa na cannabinoid, kodayake ΔcJun ya bayyana a cikin D biyu duka₁ da kuma D₂-nana ci gaba da yawancin ƙananan ƙwayoyin maƙalaƙi a cikin ƙananan ƙwayoyin (Peakman, et al., 2003). Zai yiwu, duk da haka, wannan maganganun ΔFosB basal yana da ƙananan ƙananan cewa ΔcJun ba zai shafar alamar mai karɓar ba, kamar yadda aka ba da shawara tare da MOR da KOR. Haka kuma yana yiwuwa CB₁R alamar haɓaka ta haɓaka ta hanyar ingantaccen maganganu na ΔFosB, wanda hakan ya kara kara yawan bayyanar ΔFosB ko hana ayyukansa tare da ΔcJun yana da ƙananan sakamako wanda bai isa matakin mahimmanci ba. Taimakon kai tsaye don wannan fassarar za a iya gani ta gwada WIN55,212-2 EC₅₀ Ƙididdiga tsakanin mice suna bayyana ΔcJun zuwa ΔFosB. Yanayin WIN55,212-2 EC₅₀ darajar adinylyl cyclase hanawa a cikin mice tare da furtaccen ΔcJun zuwa EC₅₀ darajar ginin G-ginawa a cikin ƙananan yara tare da furtaccen ΔFosB shine 4.0, yayin da rabo guda a cikin mice ba tare da shigarwa ko dai transgene ba ne 1.2.

A madadin haka, cannabinoids na iya haifar da magana ta ΔFosB ba tare da wani sakamako na tsaye a kan CB ba₁R alamar. A cikin wannan labari, cannabinoids na iya canza yanayin da ake amfani da kwayoyin kwayoyin kwayoyin cutar ta hanyar tsarin ta hanyar kwaskwarimar ΔFosB. Nin gaskiya, gwamnati na Δ⁹-CK na samar da haɗin giciye ga opioids da amphetamine (Cadoni, et al., 2001, Lamarque, et al., 2001), daidai da wannan magana. Bugu da ƙari kuma, an ba da rahoton gwamna na CPNNUMX mai yawan cannabinoid don kara yawan aikin G-protein wanda aka sanya shi a cikin NAc, kamar mawakan da ke nuna ΔFosB a cikin binciken da ake ciki yanzu (Vigano, et al., 2005). Sakamakon magana ΔFosB akan Δ⁹-Da halin kirkirar da aka yi wa CRC ba a ƙayyade ba, amma sakamakon yanzu ba ya hana wani hulɗa. Sakamakon wannan da bincikenmu na baya (Zachariou, et al., 2006) nuna gyare-gyaren ΔFosB a cikin MOR da KOR / dynorphin a cikin striatum. Sakamakon sakamako na Δ⁹-THC, kamar yadda aka auna ta wurin zaɓi, an soke shi a Mice null mice, yayin da sharewa na KOR ƙaddamar Δ⁹-THC wuri mai juyayi kuma ya saukar Δ⁹-Wannan zaɓi na zaɓi na SSC (Ghozland, et al., 2002). Bugu da ƙari, yanayin sharaɗi ya yi watsi da Δ⁹-THC ba ya nan a cikin pro-dynorphin knockout idan aka kwatanta da nau'i-nau'i-nau'i-nau'i (Zimmer, et al., 2001). Wadannan bayanan sun bada shawarar cewa Δ⁹-Wannan zai iya samun karin sakamako bayan an shigar da ΔFosB da kuma shigar da alamar MOR tare da raguwa a cikin bayanin dynorphin.

A cikin taƙaitaccen bayaniy, sakamakon wannan binciken ya nuna cewa bayyanar ΔFosB a D₁R / dynorphin tabbatacciyar ƙwararrun ƙwayoyin ƙarfe sun ƙarfafa MOR- da kuma KOR-sasantawa a cikin matakin G-protein wanda aka hana shi izinin adenylyl cyclase a cikin NAc. Wannan binciken yana daidai da binciken da suka nuna muhimmancin tsarin tsarin opioid na ƙarshe (sakamakon sakamako)Trigo, et al., 2010), da kuma samar da wata matsala mai amfani ga sakamakon ΔFosB a kan sakamako. Ya bambanta, CB₁Alamar R da aka sanyawa a cikin NAC ba ta da tasiri sosai game da bayyanar ΔFosB bayyanar a ƙarƙashin yanayin da aka bincika, kodayake ƙarin binciken yana da amfani don ƙayyade tasirin ΔFosB akan tsarin endocannabinoid.

Binciken bincike

Alamar MOR an inganta shi a cikin ƙananan ƙwayoyin mice da ke bayyana ΔFosB
KASAN abin da aka haramta na adenylyl cyclase an inganta shi a cikin mice suna bayyana ΔFosB
Magana daga ΔFosB ba ya canza CB₁R alamar alama a cikin ƙananan ƙwayar

Ka tafi zuwa ga:

Karin kayan

01

Danna nan don dubawa.^{(45K, pdf)}

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Godiya

Masu marubuta sun gode wa Hengjun He, Jordan Cox da Aaron Tomarchio don taimakon fasaha tare da [³⁵S] GTPYS sharaɗɗa jayayya. Wannan binciken ya goyan bayan DA014277 (LJS), DA10770 (DES) da P01 DA08227 (EJN).

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Bayanan kalmomi

Bayanin Mai Bugawa: Wannan fayil ɗin PDF ne na rubutattun takardun da ba a yarda da su ba. A matsayin sabis ga abokan cinikinmu muna samar da wannan farkon farkon rubutun. Rubutun za su shawo kan gurbatawa, gyare-gyare, da kuma nazarin tabbacin da aka samo kafin a buga shi a karshe. Lura cewa a yayin da ake samar da kurakurai za a iya gano abin da zai iya shafar abubuwan ciki, kuma duk abin da doka ta yanke game da wannan littafin ya shafi.

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