Histone deacetylase inhibitors e thusa ho etsa moralo oa khetho ea molekane ho basali ba li-prairie voles (2013)

Nat Neurosci. Sengoloa se ngotsoeng; e fumaneha ho PMC Jan 1, 2014.

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inahaneloang

Kopanong e monate ea sechaba se le sengMicrotus ochrogaster), mating induces e mamella li-coupon tse qalileng ka ho thehoa ha khetho ea molekane le ho laoloa ke li-neurotransmitters tse fapaneng ho kenyelletsa oxytocin, vasopressin le dopamine. Mona re ile ra hlahloba mekhoa e ka sebelisang epigenetic e sebelisang matla a ho kopanya maqhama a mabeli. Re bontša hore nalane ea histone deacetylase inhibitors sodium butyrate le TrichoStatin A (TSA) e nolofalletsa khetho ea molekane nakong ea li-voes tsa basali tsa prairie kamora ho ba teng. Sena se ne se amahanngoa le taelo e ikhethileng ea li-oxytocin (OTR) le vasopressin V1a receptors (V1aR) ka har'a li-nucleus tsa li-nucleus, ka ho eketseha ha histone acetylation ho morekisi oa bona. Ho feta moo, khetho ea molekane ea tataisang ea TSA e neng e tataisoa ke TSA e ne e thibetsoe ke OTR kapa V1aR blockade ho li-nucleus accumbens. Habohlokoa le ho feta, ho ratoa ke balekane ho ile ha baka taolo e tšoanang ea li-epigenetic ea OTR le bahlahisi ba mofuta oa V1aR joalo ka TSA. Litlhokomeliso tsena li bonts'a hore TSA le ho nyalanya ho nolofalletsa khetho ea molekane ka liketsahalo tsa epigenetic, ho fana ka bopaki ba pele bo otlolohileng ba taolo ea epigenetic ea ho tlamahana.

Selelekela

Kopano ea sechaba ke tšobotsi ea bohlokoa ea boitšoaro ba motho sechabeng le bofokoli ba kelello sechabeng ke likarolo tse tloaelehileng ho mafu a mangata a amanang le neuropsychiatric ho kenyeletsa le lefu la kelello la schizophrenia le autism, hammoho le bokhoba le khatello ea maikutlo.1. Li-voles tse monate sechabengMicrotus ochrogaster) li hlahile e le mohlala o khahlisang haholo oa ho etsa lipatlisiso tsa methapo ea kutlo e amanang le kamano, kaha batho ka bobeli ba laboratori le ba sa pheleng ba theha litlamo tsa nako e telele.2-4, tse qaliloeng pele ka ho theha boits'oaro bo kopanyang ba setho se kopaneng sa molekane, se bitsoang khetho ea molekane5. Sebopeho sena sa khetho ea molekane se kenyeletsa mefuta e mengata ea li-neurotransmitters le lihormone, ho kenyelletsa le neuropeptides oxytocin le vasopressin (AVP) le mesolimbic dopamine5.

IKakaretso, sebopeho sa khetho ea molekane se kopantsoe ka methapo ea methapo ea methapo ea methapo ea methapo ea methapo (AVP neurotransuction) ho "ventral pallidum" le "seterum" ("LS)" ho banna, le oxytocin neurotransuction ho li-nucleus accumbens (NAcc) le li-cortex tsa prelimbic libakeng tsa basali tsa prairie.6-8.

Ka tloaelo e kenyelletsang moputsong oa tlhaho o kang ho tlolelana le dopamine, e boetse e sebetsa e le mokena-lipakeng oa bohlokoa oa khetho ea molekane ho li-voiri tsa prairie. Ho kenngwa tshebetsong ha dopamine D2-mofuta receptors (D2R) ho NAcc ho thusa ha ts'ebetso ea dopamine D1-mofuta receptors (D1R) e thibela ts'ebetso ea khetho ea molekane ho ba batona ba batona ba banna le ba basali.9-11.

Habohlokoa, ho feto-fetoha ha mofuta oa polelo ea oxytocin le AVP V1a receptors, OTR le V1aR, ka ho latellana, ka botsona li ka ama likhetho tsa molekane. Likopanong tsa basali tsa thoriso ea basali, ho etsa mohlala, overexpression ea OTR ho NAcc e nolofalletsa khetho ea molekane ha ho se na mathe12-14.

Ntle le taolo ea tlamahano ea maqhama a mabeli, oxytocin le AVP le tsona li na le tšusumetso mekhoeng e mengata e fapaneng ea boits'oaro, ho kenyeletsoa ho amoheloa sechabeng, mabifi le tlhokomelo ea bo-mme.15, 16. Haholo-holo, ho ferekana ha boitšoaro ba moraorao litšoantšong ho baka li-neuroadaptations tsa nako e telele ka mekhoa ea epigenetic, ho kenyelletsa le methapo ea methapo ea methapo ea methapo ea kutlo.17 le mefuta ea AVP18, hammoho le histone acetylation ea glucocorticoid receptor promoter19. Ho feta moo, li-inhibitors tsa histone deacetylase (HDAC), li ntlafatsa polelo ea liphatsa tsa lefutso ka ho eketseha ha histone acetylation bokong ba methapo20, e ka fetola liphetoho tsena19, hape e susumetse ka kotloloho boits'oaro sechabeng joalo ka ho etsa thobalano21. Habohlokoa, moleng oa sele oa kankere ea matšoafo, HDAC inhibitor trichostatin A (TSA) ka kotloloho e ntlafatsa mongolo oa OTR ka ho khothaletsa histone acetylation22.

Ka hona, ho ka khothaletsoa motheo oa epigenetic popehong ea khetho ea molekane libakeng tsa prairie. Ho etsa tlhahlobo ea maikutlo ana, re qalile re hlahloba litlamorao tsa li-inhibitors tsa HDAC tse peli, sodium butyrate (NaB) le TSA, ka sebopeho sa khetho ea molekane ho li-voles tsa basali ba baholo. Kamora moo, re ile ra etsa lipatlisiso ka mekhoa ea limolek'hule e buisanang le litlamorao tsa TSA ho kenya tšusumetso ea balekane ho li-voes tsa basali tsa basali. Kamora nako, re ile ra batla ho tseba hore na liphetoho tse tlisoang ke TSA nakong ea cohabitation le tsona li bakiloe ke ho tlolelana.

Results

Phekolo ea TSA e nolofalletsa khetho ea molekane

Likamano tsa botona le botšehali tsa basali ba basali ka mokhoa oa thobalano li ne li kenngoa kahare ho intracerebroventricularly (icv.) ka CSF kapa CSF e nang le 0.08, 0.4 kapa 4 ng of TSA hang-hang pele ho nako ea ho qeta lihora tsa 6 le monna ntle le ho nyaloa, mme khetho ea bona ea balekane e ile ea lekoa. Lihora tse tšeletseng tsa ho lula le monna ntle le ho nyalana ha li khothalletse hore ho khethoe ha molekane ho li-voes tsa basali ba basali4 ka hona mokhoa ona oa boitshwaro o sebelisitsoe haholo ho lekola litlamorao tsa lithethefatsi tse fapaneng ho tsamaiseng sebopeho sa khetho ea molekane5.

Liphoofolo tse tšoaroang ke CSF li bonts'itse ts'ebelisano e sa khethang ka thoko le molekane kapa mojaki ka mor'a lihora tsa 6 tsa ho phomola ntle le ho nyalana (t15= 0.76, P = 0.46; Setšoantšo sa 1a). Le ha ho le joalo, liphoofolo tse tšoaroang ke TSA ho hang litekanyetso li lekiloe li qeta nako e ngata li ikhethile le molekane ho feta le motho osele (t8= 4.35, P = 0.002 ea TSA 0.08 ng sehlopha; t15= 3.63, P = 0.002 ea TSA 0.4 ng sehlopha; le t8= 2.58, P = 0.03 ea TSA 4 ng sehlopha). Ho bohlokoa, ha ho liphapang tse fumanoeng tsa sehlopha tse fumanoeng ts'ebetsong ea locomotor (F3,46 = 1.25, P= 0.30, Feiga. 1b) mme ha ho boits'oaro bo mabifi bo entsoeng ke mosali ea hlahlobang ho monna eo a sa mo tsebeng kapa balekane ba bonoeng, bo bonts'a hore litlamorao tsa TSA li ne li bua ka khethollo ea sechaba, ho fapana le ho ba phetoho ea phetoho ea ts'oants'o kapa moferefere sechabeng.

Setšoantšo sa 1  

Ente e kotsi ea trichostatin A (TSA) e thusa ho thehwa ha litlhophiso tsa balekane maemong a basali a botumo nakong ea ho ba sieo. (a) Mofuta oa Cerebrospinal fluid (CSF) - basali ba hlahelletseng ba pepesitsoe monna hore a be le lihora tse 6 kamora hore ho ba le ngoana a bonts'e ...

Ho etsa lipatlisiso tsa hore na TSA e ntlafatsa histone acetylation lits'ebetsong tsa boko tse amehang ho hlahiseng khetho ea molekane, karolo e arohaneng ea basali e ile ea kenngoa ka lethal dose e mahareng ea TSA (0.4 ng) mme o kopantsoe le e tona nakong ea ha e sa tlotsoe nakong ea metsotso ea 30, lihora tsa 2 kapa 9. Haholoholo, ha ho phapang e kholo maemong a lefats'e a H3 acetylation (Lys14) a ka bonoang ka nako efe kapa efe ntlheng ka bobeli ba NAcc le caudate putamen (P > 0.05 bakeng sa lihlopha tsohle, Setšoantšo sa 1c & d). Sena se bonts'a hore TSA e nolofalletsa sebopeho sa khethollo ea molekane ha ho se na mathoasoa, leha e sa ame nalane ea lefats'e ea H3 acetylation ho NAcc kapa caudate putamen.

Habohlokoa, sodium butyrate e boetse e nolofalitse khetho ea molekane ho li-voes tsa basali tsa basali ho latela ho lula le monna ka lihora tse 6 ntle le ho nyaloa, ho amanang le keketseho ea histone ea H3 acetylation (Lys14) ho NAcc (Setšoantšo sa tlatsetso 1). Litlamorao tsa TSA kamor'a khetho ea khetho ea molekane li ka hlahisoa hape ka inhibitor e 'ngoe ea HDAC, ho fana ka maikutlo a ho nka karolo ha thibelo ea HDAC, ho fapana le phello e seng e ikhethang ea TSA ts'ebetsong ea khetho ea molekane. Ho nka hore TSA ke sehlopha se ikhethileng hape se amanang le I / II HDAC inhibitor23, 24, le hore litlamorao tsa boits'oaro ba TSA li phatlalalitsoe haholo ho feta NaB, re ile ra khetha ho sebelisa TSA ka holim'a NaB bakeng sa ho etsa lipatlisiso ka likarolo tse ikhethileng tsa molek'hule likarolong tse latelang tsa thuto.

Litlatsetso tsa limolek'hule tsa khetho ea molekane ea tataisang-TSA

Joalo ka ha phapang ea maemo a polelo ea gene ho Vole NAcc e amahantsoe le maano a fapaneng a ho hanana pakeng tsa li-voogamous le non-monogamous voles, le phetoho ea sebopeho sa khetho ea molekane ho li-voles tsa prairie12, 13, 25, 26, re lekotse hore na sebopeho sa khetho ea balekane ba TSA se tataisoang ke TSA se amana le phetoho ea polelo ea gene ho NAcc.

Phekolo ea TSA (0.4 ng, icv.) e bakile keketseho ea maemo a OTR mRNA ho NAcc kamora lihora tsa 2 tsa cohabitation joalo ka ha li bapisoa le li-control tse tšoaroang ke CSF (t10 = 2.38, P = 0.038, Setšoantšo sa 2a), e neng e batla e ntlafatsoa kamora lihora tsa 9 tsa ho phomola (t9 = 2.17, P = 0.058, Feiga. 2b). Leha keketseho e nyane empa e seng ea bohlokoa ea V1aR mRNA e ka bonoa ho lihora tsa NAcc 2 kamora ts'ehetso ea TSA, ha ho liphapang tse ling tse fumanoeng ka nako e 'ngoe bakeng sa li-mRNA tse ling tse lekantsoeng, ho kenyelletsa D1R kapa D2R (P > 0.05, Feiga. 2a, b). Habohlokoa, ha ho phapang pakeng tsa sehlopha e ileng ea bonoa ho caudate putamen ka nako eohle ea nako le bakeng sa mRNA efe kapa efe e lekantsoeng (P > 0.05 bakeng sa lihlopha tsohle, Feiga. 2c, d), ho fana ka tlhahiso ea hore keketseho ea OTR mRNA e hlokometsoeng ho liphoofolo tse tšoaroang ke TSA e ne e ikhethile ho NAcc. Ntle le moo, tokiso e joalo e ne e le teng feela kamora ho kopana le monna, joalo ka maemo a OTR le V1aR mRNA ho NAcc a lula a sa fetohe lihora tsa 2 kamora ho ts'oaroa TSA ntle le cohabitation (OTR: 100.0% ± 11.70 ea sehlopha sa CSF, 86.7% ± 12.11 bakeng sa TSA sehlopha, t12 = 0.79, P = 0.444; V1aR: 100.0% ± 26.24 ea sehlopha sa CSF, 92.3% ± 13.75 ea sehlopha sa TSA, t9 = 0.27, P = 0.791).

Setšoantšo sa 2  

Phekolo ea TSA (0.4 ng) Melaetsa ea li-oxytocin (OTR) le li-vasopressin (V1aR) tse amohelang basali nakong ea ho lula le monna nakong ea ho kopanya le monna. Mehato ea OTR mRNA (a, b) le liprotheine (e, f) li ne li hlophisitsoe hantle ka mor'a lihora tsa 2 (a, e) le 9 ...

Tumellanong le maemo a holimo a OTR mRNA, liphoofolo tse tšoaroang ke TSA le tsona li bonts'itse liprotheine tse phahameng tsa proteni ea OTR maemong ohle a nako ka li-NAcc (lihora tsa 2: t10 = 2.34, P = 0.041; Lihora tsa 9: t10 = 3.16, P = 0.01, Feiga. 2e, f), empa eseng caudate putamen (t10 = 0.41, P = 0.69, Feiga. 2g, h). Ho khahlisang ke hore, ha ho sena phetoho ea bohlokoa ea maemo a V1aR mRNA e ka fumanoang ho NAcc ka 2 kapa lihora tsa 9 kamora ho entoa TSA (Feiga. 2a, b), maemo a protheine ea V1aR a ile a eketseha haholo ka lihora tsa 9, ha a bapisoa le liphoofolo tse tšoaroang ke CSF, ho NAcc (t9 = 3.46, P = 0.007, Feiga. 2f) empa eseng caudate putamen (t10 = 0.98, P = 0.35, Feiga. 2h). Le ha ho na le phapang e itseng, maemo a protheine ea D1R le D2R ho NAcc le caudate putamen ha a anngoe haholo ke tsamaiso ea TSA (P > 0.05, Feiga. 2e-h).

TSA e thusa histone acetylation ea oxtr le avpr1a

Keketseho ea maemo a mabeli a mRNA le liprotheine bakeng sa OTR kamora ho phekoloa ha TSA ho fana ka tlhahiso ea hore mohlomong TSA e eketse tlhahiso ea oxtr, mofuta oa ho ngola mofuta oa OTR, ho fapana le ho fetola phetolelo kapa phetoho ea protheine. Ho feta moo, maemo a protheine ea V1aR a ne a phahame ho NAcc, a amahanngoa le keketseho e nyane empa e seng ea bohlokoa ea maemo a mRNA a latelang kalafo ea TSA (Feie. 2). Ho nka hore TSA ke sehlopha se matla sa I le II HDAC inhibitor23, 24, 27, re kholoa hore TSA e eketsetse nalane ea histone ho oxtr 'me avpr1a baphatlalatsi ho NAcc, kamora moo ba ntlafatsa sengoloa sa bona. Sehlopha se secha sa liphoofolo se amohetse icv. ente ea TSA (0.4 ng) hang-hang o kopantsoe le e tona ntle le ho tloheloa metsotso ea 30 pele a etsoa sehlabelo. Fensetere ea nako ea 30-min e ile ea khethoa ho latela mosebetsi o fetileng oa ho tlaleha keketseho e kholo ea histone acetylation kamora injente ea TSA ea lehae litsing le litoeba28, 29. H3K14 acetylation ha oxtr 'me avpr1a Barekisi ba ile ba hlahlobisoa ke chromatin immunoprecipitation.

Tumellanong le keketseho ea maemo a OTR mRNA le liprotheine tse shebiloeng pele, liphoofolo tse tšoaroang ke TSA li bontšitse keketseho e phahameng haholo (+ 460%) ho histone H3 acetylation ho oxtr Morekisi oa gene, joalo ka ha a bapisoa le taolo e tšoaroang ke CSF, ho NAcc (t10 = 5.88, P = 0.0002), empa eseng Caudate putamen (t9= 0.31, P = 0.76, Setšoantšo sa 3a). Ntle le moo, histone H3 acetylation ha avpr1a morekisi o ne a phahamisitsoe haholo 30 min kamora ho tsamaisoa ha TSA (+ 196%) ho NAcc (t10 = 3.12, P = 0.01) empa eseng caudate putamen (t9= 0.38, P = 0.71), bapisoa le li-control tse tšoaroang ke CSF (Feiga. 3b). Ka hona, TSA e ile ea eketsa sebaka sa nalane ea acostlation ka ho khetheha ho NAcc kamora metsotso ea 30 kamora ho qaleha ha kopano ea motho le monna.

Setšoantšo sa 3  

Phekolo ea TSA e ntlafatsa histone acetylation ea oxtr 'me avpr1a Banyatsi ha ba kopane le monna nakong eo ba sa kopaneng. Nalane H3 acetylation (Lys14) ho oxtr (a) le avpr1a (b) baphatlalatsi ba eketsehile ho li-nucleus accumbens (NAcc) empa ha ho joalo ...

TSA e nolofalletsa khetho ea molekane ka OTR le V1aR

Ho tsoa sethaleng se fetileng sa liteko, ho hlaha mofuta oa molek'hule, moo nakong ea cohabitation, TSA potentiates histone acetylation ho oxtr 'me avpr1a barekisi, kamora moo ba ntlafatsa mongolo oa bona mme ba baka maemo a liprotheine a OTR le V1aR e phahameng ho fihlela lihora tsa 9 kamora ho qaleha ha nako ea cohabitation. Taba ea bohlokoa ke hore phello ea TSA e hlophisitsoe ka mokhoa o ikhethileng joalo ka ha caudate putamen e sa sebetse. Mona re lekile hore na keketseho ena e susumetsoang ke TSA ho OTR le V1aR e amana le ho nolofalletsa sebopeho sa khetho ea molekane. Li-voes tsa prairie tsa basali li fumane ente ea intra-NAcc ea TSA (0.04ng lehlakoreng ka bophara kapa ka ente ea pele (30minutes pele ho ente ea TSA, 0.5ng lehlakoreng ka leng la CSF kapa CSF e nang le e 'ngoe ea bahanyetsi ba babeli ba fapaneng ba OTR, OTA (B) le OTA (T), kapa mohanyetsi oa V1aR (V1aRA). Hang-hang ka mor'a ente ea TSA, tse tšehali li ile tsa kopanngoa le e motona bakeng sa lihora tsa 6 ntle le ho nyaloa, tsa lateloa ke tlhahlobo ea khetho ea molekane.

Liphoofolo tse tšoaroang ka CSF ha lia ka tsa bontša khetho ea molekane (t5 = 0.17, P = 0.87, Setšoantšo sa 4a). Leha ho le joalo, liphoofolo tse tšoaroang ke TSA li qetile nako e ngata li buisana le "molekane" ho feta le "osele", ho fana ka tlhahiso ea hore ente e le 'ngoe ea TSA ka kotloloho ho NAcc e lekane ho tsamaisa khetho ea molekane ntle le ho nyalana (t5 = 7.04, P = 0.0009). Hoa thahasellisa hore ebe blockade of OTR kapa V1aR ka kalafo ea kalafo e fetileng le OTA (B), OTA (T), kapa V1aRA e thibetse litlamorao tsa TSA (P > 0.05 bakeng sa lihlopha tsohle). Kaha ha ho liphapang tsa sehlopha tse fumanoeng mesebetsing ea baetsi ba likoloiF4,32 = 1.89, P = 0.14, Feiga. 4b), datha tsena li fana ka maikutlo a hore TSA ho NAcc e thusa ho hlophisa meralo ea khetho ea molekane ka OTR- le mekhoa ea bohokela ea V1aR ka tsela e ikhethileng.

Setšoantšo sa 4  

Khetho ea molekane ea tataisoang ke TSA e hloka hore oxytocin- (OTR) le vasopressin (V1aR) li-receptors-mediated neurotransuction ho li-nucleus tsa basali li hlahise li-nucleus tsa basali. (a) TSA e nolofalletsa khetho ea molekane ha e kenngoa ka har'a li-nucleus accumbens (0.04 ng ka lehlakoreng), ...

Mating e etsa li-neuroadaptations tse tšoanang le TSA

Kamora maikutlo a rona a pejana, re ile ra tseba hore phello ea epigenetic ea oxetocin le vasopressin neurotransmission ho NAcc ea basali e ne e lekane ho tsamaisa mokhoa oa ho khetha oa molekane ntle le ho ruruha. Ho etsa lipatlisiso tsa hore na li-neuroadaptations tsena li etsahala le nakong ea kemiso ea tlhaho ea khetho ea balekane, li-voes tsa basali tsa basali li ile tsa kopanngoa le e motona nakong ea lihora tsa 24 ka pel'a ho tlolelana, e leng se hohelang khetho ea molekane.4, 'me oa nyeheloa. Re hlokometse keketseho ea maemo a bobeli a OTR le V1aR mRNA le liprotheine ho NAcc, ha ho bapisoa le basali ba botona le botšehali ba nang le thobalano (OTR: + 38%, t10 = 2.68, P = 0.02 bakeng sa mRNA, le + 58%, t8 = 3.05, P = 0.01 ea protheine; V1aR: + 89%, t14= 2.53, P = 0.02 bakeng sa mRNA, le + 26%, t20 = 2.23, P = 0.037 ea protheine, Feiga. 5a, b).

Setšoantšo sa 5  

Ho tloaelana le ho ruruha ho etsa hore ho be le ho eketseha ha li-oxytocin (OTR) le li-vasopressin (V1aR) tse fumanehang ka har'a li-nucleus accumbens (NAcc) tsa li-voes tsa basali tsa praemia. 24h Cohabitation le monna e nang le mating up-regates OTR (a) le V1aR (b) mRNA le protheine ...

Ha maemo a ka bobeli a mRNA le protheine bakeng sa OTR le V1aR li ntse li eketseha ka ho kopanngoa le ho kopanngoa, hamorao re tla etsa lipatlisiso hore na molao-motheo ona o amanang le ntlafatso ea epigenetic ea oxtr 'me avpr1a transcript ea liphatsa tsa lefutso. Sehlopha se secha sa basali se ile sa kopanngoa le e motona bakeng sa lihora tsa 6 ka nako ea mating le H3K14 acetylation oxtr 'me avpr1a baphatlalatsi ba lekantsoeng ke chromatin immunoprecipitation. Tumellanong le OTR le V1aR mRNA le maemo a liprotheine, li-voes tsa basali tsa praemia li bonts'a acetylation e phahameng ea H3K14 oxtr 'me avpr1a Barekisi ba NAcc, ha ba bapisoa le basali ba thobalano ba na sexuallyve (oxtr: t9 = 2.64, P = 0.02; avpr1a: t9 = 2.91, P = 0.017 Feiga. 5c, d). Lintlha tsena li fana ka maikutlo a hore ho sebetsa ka mokhoa o hlakileng ho susumetsang boikhethelo ba basali ho roriseng basali ho etsa hore ho be le melaoana ea OTR le V1aR polelo ea NAcc ka mekhoa ea epigenetic, joalo ka ha ho hlokometsoe kamora kalafo ea TSA.

Puisano

Thutong e teng hona joale, re tlaleha ka lekhetlo la pele molao oa ho hlaba ka epigenetic oa mofuta oa khetho ea molekane. Taba ea mantlha, re bonts'itse hore histone acetylation e ntseng e eketseha ho NAcc ka tsamaiso ea HDAC inhibitor e thusa ho hlophisa khetho ea molekane maemong a basali ba baholo nakong ea ho ba sieo. Then, re senotse bopaki bo tobileng ba hore khetho ea balekane ho basali e tsamaisoa ka sephethe-phethe, joalo ka ha ho kopane le ho ratana le monna ho eketseha oxtr 'me avpr1a polelo ea liphatsa tsa lefutso ka ho ntlafatsa histone acetylation ho NAcc. Tsamaiso ea TSA ho NAcc e entse khetho ea molekane 'me ea lebisa maemong a holimo a OTR mRNA le liprotheine ho NAcc. Ntle le moo, le ha histone ea lefats'e H3 acetylation e ne e sa sebetse ho basali ba tšoaroang ke TSA, ts'ebeliso e ntle ea histone acetylation ho oxtr papatso ho NAcc e ile ea bonoa e sa le qalong ea metsotso ea 30 ka mor'a tsamaiso ea TSA. Qetellong, ho thibela OTR ho NAcc ho ne ho lekane ho thibela khetho ea molekane ea tataisang TSA. Kaha liphetoho tse tšoanang tse tsamaeang le epigenetically li fumanoe kamora ho lula hammoho le batho ba bang, tlasa lits'ebetso tse tsejoang ho susumetsa khetho ea molekane, datha ea rona e beha mohlala molemong oa taolo ea boiphihlelo ba bophelo ba sechaba. Dho kopa ho kopanngoa le monna, TSA, kapa mating, ho potlakisa ho hlahisa nalane e khethehileng ea histone H3 ho oxtr ntšetso-pele ea NAcc e ntlafatsang phetisetso ea eona, e hlahise maemo a holimo a OTR mRNA le liprotheine, tseo kamora moo li nolofalletsang moratuoa oa khetho.

Likopanong tsa basali tsa basali tsa basali, lihora tsa 6 tsa ho phelisana le monna ntle le ho nyala ha li khothalletse mofuta oa khetho ea molekane.4, mme mokhoa ona oa boitshwaro o sebelisitsoe ho hlahloba litlamorao tsa ho qhekelloa hoa meriana mabapi le tšibollo ea khetho ea molekane.5. Thutong ea rona, le ha li-control tse tšoaroang ke letsoai- kapa CSF li sa ka tsa etsa khetho ea balekane, li-voes tsa basali tsa thoriso tsa basali tse tšoaroang ke NaB kapa TSA li entse. Joalo ka ha NaB kapa TSA e sa ametse tšusumetso e akaretsang, litlamorao tsa bona ho khetho ea balekane li bonahala e le litekanyetso ho fapana le litlamorao tsa pherekano. Tšusumetso e khethehileng ea TSA e ile ea tiisoa hape ke lipatlisiso tsa rona tsa molek'hule. Ho joalo, leha e tsamaisoa icv., re atlehile ho fumana phetoho e ikhethang ea polelo ea liphatsa tsa lefutso ho NAcc empa eseng ka sebopeho se haufi le eona, caudate putamen. Ntle le moo, esita le ka har'a NAcc, D1R le D2R mRNA le maemo a liprotheine a lula a sa angoe. Ho tsebahala ho joalo ho bonahala ho makatsa bakeng sa inhibitor e pharaletseng ea HDAC joalo ka TSA e amang sehlopha sa bobeli sa I le II HDAC. Leha ho le joalo, TSA e tlalehiloe e ama tlhahiso ea karoloana e nyane ea liphatsa tsa lefutso ho genome ea mammalian30-32, ho kenyelletsa le litoeba20.

Re bontšitse mona hore acetylation ea histone H3 e Lys14 ho oxtr papatso, phetoho e amanang le ho fetisoa hoa mofuta oa gene ho kenyelletsa le nakong ea "protein" ea "cerebral"33, 34, e theola maemo a holimo a OTR mRNA le liprotheine tse phahameng. Ho arabela TSA, histone acetylation ho oxtr papatso e eketsa le ho nolofalletsa ts'ebetso ea phetiso ea eona ho sele ea batho22, e ts'ehetsa ho fumana ha rona seo oxtr e ka laoloa ka epigenetically. Kaha thibelo ea lehae ea OTR ho NAcc e ne e lekane ho thibela litlamorao tsa TSA, datha ea rona e fana ka maikutlo a hore polelo ea khothaletso ea TSA ea OTR ho NAcc nakong ea lipuisano e ile ea thusa ho hlophisa sebopeho sa khetho ea molekane. Ntle le moo, lihora tsa 24 tsa ho ikopanya le ho ruruha, ts'ebetso e tsejoang ka mokhoa o ts'epahalang ho khetha khetho ea molekane li-voles tsa basali tsa basali, e bakile keketseho e ts'oanang ea polelo ea OTR ho NAcc ea basali. Sena se lumellana ka botlalo le tšebeliso e tsejoang ea oxytocin le receptor ea eona ho neurobiology ea sebopeho sa khetho ea molekane li-voles tsa basali. Ho ruruha ho etsa hore ho be le keketseho ea maemo a tsoang kantle ho oxytocin ho NAcc25, le infusion ea lehae ea oxytocin ho NAcc e nolofalletsa moralo oa khetho ea molekane ntle le hore o tlohelana8. Ntle le moo, bahanyetsi ba OTR ba thibela ho khethoa ha molekane ka ho susumetsoa ke tsamaiso ea oxytocin kapa mating8, 35. Habohlokoa le ho feta, botala bo pakeng tsa bongata ba OTR ho NAcc ea basali bo lekane ho tsamaisa sebopeho sa khetho ea molekane.12, 13.

Ntle le ho matlafatsa karolo ea OTR, liphetho tsa rona li boetse li fana ka bopaki ba ts'ebetso ea polelo ea mofuta oa OTR ka mekhoa ea epigenetic nakong ea ho ikopanya le monna nakong ea ho tlola. Ehlile, leha e sa lekane ho susumetsa khetho ea molekane, ho se kopane ntle le ho ts'oaroa ha nakoana ho hlahisa ts'ebetso ea methapo ea kutlo e bakang khetho ea molekane.. Mohlala, lihora tse peli tsa ho pepesehela mahala e tona ho monna e ts'oara maemo a nyane empa e seng a bohlokoa ho lokolloeng ha oxytocin ho NAcc ea basali25. Ka hona ho ka ananeloa hore TSA kapa NaB e khone ho hlahisa li-neuroadaptations tse susumetsoang ke ho kopana le monna, ho tsamaisa nts'etsopele ea khetho ea molekane. Ho khahlisang ke hore ts'ebeliso e joalo e se e tlalehiloe litoeba moo sehlopha sa I le II IIAC inhibitors, ho kenyelletsa NaB le TSA, li nolofalletsa ho kopanya ketsahalo ea ho ithuta e sa faneng ka tlhahiso ea memori ea nako e telele ho liphoofolo tse laoloang36, 37. Ho ts'ehetsa maikutlo ana, nako e teletsana ea ho lula ka khotso (mohlala., 48hours) e ka susumetsa khetho ea molekane leha e le sieo ea ho tlolelana4. Ho bohlokoa hape ho hlokomela hore ho lula hammoho ka ho nyanyana ho qalileng ka hara mosali oa NAcc tokiso ea ntlafatso ea OTR le V1aR ka mekhoa e ts'oanang ea epigenetic joalo ka e bonoang kamora ho ikopanya le kalafo ea TSA, e bonts'ang hore TSA le ho nyaloa ho ama mekhoa e ts'oanang ea ho nts'etsapele. sebopeho sa khetho ea molekane. Habohlokoa, TSA ha e khothalletse ho nyolloa ha OTR le V1aR ho NAcc ea basali ha ho se letho le kopanang le monna. Ka kakaretso, hona ho tšehetsa khopolo-taba ea hore TSA e thusa ho theha khetho ea molekane ka ts'ebeliso ea li-neuroadaptations tsa tlhaho ea tlhaho ka tlhaho e susumetsoang ke ho kopanngoa le monna, ho fapana le ho kenya ts'ebetso ea hae kapa tsena tse fapaneng tsa neuroadaptations.

OBoithuto ba Ur bo boetse bo totobatsa karolo ea bohlokoa ea NAcc V1aR ka sebopeho sa khetho ea molekane oa basali, ha liphoofolo tse tšoaroang ke TSA li bonts'a maemo a holimo a V1aR, eo blockade ea eona e neng e thibela ho theha molekane oa TSA.. Ntle le moo, litlamorao tsena li ne li amahanngoa le histone acetylation e phahameng ho avpr1a papatso, leha ho se na phallo ea bohlokoa ea V1aR mRNA mohlomong ka lebaka la nako e sa lokelang. Leha re ke ke ra khona ho laola tsamaiso ea botsitso ba protheine ke TSA ka acetylation ea protheine e seng histone38, sena se fana ka maikutlo a hore, ho tšoana le oxtr papatso, TSA e kanna ea khothaletsa avpr1a sengoloa ka nalane ea sebakeng sa khale ea histone. Le ha ho ntse ho hlalosoa monehelo oa AVP molemong oa tlamahano ea banna5, karolo ea eona boits'oarong ba mosali e ntse e le phehisano. Ka lehlakoreng le leng, a icv. Sesebelisoa sa AVP se thusa ho theha khetho ea molekane ho li-voles tsa banna le tsa basali, tse thibetsoeng ke blockade ea V1aR kapa OTR39. Ka lehlakoreng le leng, a icv. ente ea mohanyetsi oa V1aR e thibela khetho ea molekane ea emelang ho banna, empa eseng ho basali, liemahale tsa basali40. Leha ho le joalo, lithuto tsena kaofela li sebelisitsoe icv. ente, ho thibela temohisiso e ngoe hape mabapi le meaho e amehang. Mona, re fana ka bopaki ba pele ba hore methapo ea kutlo ea AVP kahare ho NAcc e ka nka karolo sebopeho sa khetho ea molekane li-voles tsa basali, ha lingoliloeng tse ngata li hlalosa ho nka karolo ha tsona libakeng tse fapaneng joalo ka ventral pallidum, lateral septum, senotlolo sa bethe stria terminalis, le amygdala ho banna5. Ka hona hoa thabisa ho hlokomela hore thibelo ea OTR kapa V1aR ho NAcc ea mosali e ne e lekane ho thibela sebopeho sa khetho ea molekane ka mor'a kalafo ea TSA, a fana ka maikutlo a hore sebopeho sa khetho ea molekane se hloka ts'ebetso ea bobeli ba V1aR le OTR. Tseo a se fumaneng se tsamaisana le, 'me sea ts'ehetsa, tlhokomeliso ea pejana ea libaka tsa basali tsa banna hore ho fihlella ka nako e tšoanang ho OTR le V1aR ho LS ho bohlokoa bakeng sa khetho ea molekane ea AVP.6. Ho feta moo, tlhokomeliso ea keketseho e khethehileng maemong ohle a OTR le V1aR ho liphoofolo tse tšoaroang ke TSA e boetse e ts'ehetsa tlhokahalo ea ts'ebetso e ts'oanang ea ts'ebetso ea AVP le oxytocin neurotransmissions bakeng sa bonding.

Ha e kopantsoe le oxytocin le AVP, dopamine neurotransuction ho li-modulates tsa NAcc molemong oa khetho ea molekane lihlopheng tsa basali.9. Le ha ho ruruha ho etsa hore dopamine e lokolloe ho NAcc9, phapang mehatong ea li-receptor e shejoa feela kamora nako e telele-e telele ho feta 24h- ​​ea ho ts'oana ka ho tsofala le bohlokoa, ho bohlokoa ho lokiseng bonto10. Tumellanong le lipatlisiso tsena, li-voes tsa basali tsa basali tse tšoaroang ke TSA li bonts'itse khetho ea molekane ntle le phapang e kholo ho dopamine D1R le li-receptor tsa D2R. Ka hona, ho ba sieo hoa taolo ea dopamine receptor ho fana ka bopaki bo bong bakeng sa ho hlaka ha TSA.

Tlaleho ea rona ea data ka lekhetlo la pele karolo ea epigenetic ho neurobiology ea joal-bonding, mme re fana ka maikutlo a hore TSA e etsa "boemo bo lumelletsoeng" ho li-voes tsa basali tsa praemia, e hlahisang karabelo ea tlhaho ea limolek'hule molemong oa ho kopanya, le ho khothaletsa sebopeho sa tšebelisano e matla sechabeng. ho lebisa ho khethoe ke molekane. Ka hona, e se e le tekong ea ho nahana hore khetho ea molekane ea tataisoang ke TSA e ka matlafatsoa le ho lebisa tlamahanong e phehellang. Le ha li-HDAC tse amehang li ntse li tlameha ho tsebahala, ka hona e tla ba ho khahlisang ho tsoela pele ho etsa lipatlisiso ka litlamorao tsa TSA ho boits'oaro bo bong bo amanang le leano la bophelo bo bobe ho li-voes tsa praemia, joalo ka khethollo e mpe le tlhokomelo e ntle ea batsoali. Ha ho nahanoa bohlokoa ba maqhubu a sebaka sa thoriso molemong oa mehato ea methapo ea kutloano ea batho ba babeli5, le li-inhibitors tse tšepisang tsa HDAC tse seng li ntse li lekoa liteko tsa bongaka24, 41, 42, datha ea rona e bula tsela bakeng sa menyetla e mecha ea pharmacological ho susumetsa boitšoaro ba sechaba.

mekhoa

Lihlooho

Ka thobalano naïve basali prairie voles (Microtus ochrogaster) ho tsoa koloneng e tlisoang ka laboratoring ba ile ba khoesoa matsatsing a 21 mme ba lula ka likamoreng tse peli tsa ngoan'abo ngoanana a le mong ka mekotleng ea polasetiki (12 × 28 × 16 cm) ka metsi le lijo tse fanoeng ad libitum. Litsela tsohle li ne li bolokoa tlasa 14: 10 h khanya e lefifi, 'me mocheso o ne o batla o le 20 ° C. Liphoofolo tsohle li ne li abeloa ka mokhoa o hlophisitsoeng ka lihlopha tsa liteko ha li fihla matsatsing a 70-90 ea lilemo. Palo ea liphoofolo tse sebelisitsoeng e ne e ipapisitse le lithuto tse fetileng lebaleng ke sehlopha sa rona le ba bang, ho kopantsoe le tlhahlobo ea matla. Ts'ebetso ea liteko e amohetsoe ke Komiti ea Tlhokomelo ea Liphoofolo le Ts'ebeliso Univesithing ea Florida State.

lithethefatsi

Sodium butyrate (NaB), e qhibilihisitsoeng ka saline, le Trichostatin A (TSA), e qhibilihileng ka har'a mokelikeli oa mokelikeli oa likhoerekhoere (CSF, BioFluids, Rockville, MD) ka bobeli ba rekiloe ho Sigma-Aldrich (St Louis, MO). NaB e ile ea kenngoa ka sekhukhu (ip) ka tekanyetso ea 600 mg / kg, e tsejoang ho susumetsa histone acetylation mehahong e 'maloa ea boko ho litoeba43, 44. Ka mokhoa o ts'oanang, lethathamo la lethal dose le sebelisitsoeng bakeng sa TSA le ne le ipapisitse le mosebetsi o fetileng o bonts'ang katleho ea lona ea ho susumetsa liketsahalo tsa nalane ea histone le phapang lipuong tsa mofuta oa genices19, 20. V1aR e khethollang V1aRA d(CH2)5[Tyr (Me)] AVP, le OTR antagonist OTA (B), [d(CH2)5, Tyr (Me)2, Sup4, Tyr-NH29] -OVT), e fumanoe ho Bachem (Torrance, CA). Mohanyetsi oa bobeli oa OTR ea ikhethileng, OTA (T), dGly-NH2-d(CH2)5 [Tir (Me)2, Sup4] OVT45, e fanoe ka mosa ke Dr. Maurice Manning (University of Toledo, OH). Liphetoho tsena le litekanyetso li sebelisitsoe li khethiloe ho latela lithuto tse fetileng tse bonts'ang boikhethelo ba V1aR kapa OTR, ka ho latellana35, 39, 46-49.

Stereotaxic cannulation le microinjection

Basali ba ne ba sa anngoe ka sodium pentobarbital (1mg / 10g boima ba 'mele), le 26 gauge stainless iron guide cannulae (Plastics One, Roanoke, VA) ba ne ba kentse li-stereotaxically, li reretsoe ho ea morao-morao ventricle (unilaterally; pua bar ho −2.5 mm. rostral, 0.6 mm lateral, le 1.0 mm ventral to bregma) kapa saete-ka kotloloho ho NAcc (biliveally; bar bar ea nose ho −2.6 mm, 2.5 mm rostral, ± 1.7 mm bilralal, and 1.0 mm ventral to bregma). Kamora matsatsi a 4.5 a ho hlaphoheloa, lithuto li fumane li-microinjection tsa CSF kapa CSF tse nang le likhaohano tse fapaneng tsa TSA. Ha ho ne ho sebelisoa bahanyetsi ba khethang OTR kapa V3aR, ba ile ba kenngoa metsotso ea 1 pele ho TSA. Lisebelisoa li entsoe ka nale ea 30 gauge eo e atolositseng 33 mm ka tlase ho cannula ea tataiso sebakeng sa sepheo, ka bongata ba ente ea 1 nL ho "voal" ea morao.icv.) kapa 200 nL ka lehlakoreng lehlakoreng la NAcc. Lenalano le ne le hokahane le Hamilton Syringe (Hamilton, Reno, NV) ka polingthylene-20 tubing le khatello ea maikutlo ea plunger li ne li etsoa butle, li hloka motsotso oa 1 ka jekete. Qetellong ea tlhahlobo, lithuto tsohle li ile tsa etsoa sehlabelo ka ho fafatsa kapele le masapo a huliloeng ho netefatsa ho beoa ha cannulae ke motho ea sa boneng a sa boneng maemo a liteko. Lintho tse nang le likanono tse sa sebetsoang hantle li ile tsa behelloa ka thoko ho tlhaiso-leseling.

Teko ea tlhahlobo le boikhethelo ba molekane

Hang-hang latela ip., icv., kapa intra-NAcc liente tsa lithethefatsi, tse tšehali li ne li kopantsoe le e motona bakeng sa lihora tsa 6 ntle le ho nyaloa. Ho ba sieo ha mating ho netefalitsoe ke ho hlahloba boits'oaro ba videot. Bakeng sa lipatlisiso tsa li-neuroadaptations tse bakiloeng ke ho kopana le batho ba sa tsubang, ba basali ba phello ea estrogen (2µg ka letsatsi, ip., bakeng sa matsatsi a 3) li ne li kopantsoe le e tona nakong ea lihora tsa 6 kapa 24, 'me boteng ba mating bo netefalitsoe e posteriori videotape (ho tloha ho 6 ho ea ho 11 bouts nakong ea lihora tsa pele tsa 6 tsa cohabitation).

Teko ea khetho ea molekane e entsoe hang-hang kamora ho qeta lihora tse 6, joalo ka ha ho hlalositsoe pele11. Ka bokhutšoanyane, lisebelisoa tsa liteko tsa likamore tse tharo li ne li e-na le lesaka le sa nke lehlakore le hokahantsoeng le masaka a mabeli a tšoanang, ntlo ka 'ngoe e na le phoofolo e hlasimollang - "mojaki" ea sa tloaelehang oa monna kapa "molekane" ea tloaelehileng oa monna ea sebelisitsoeng nakong ea ho lula hammoho. Lihlooho tsa basali li ne li lokolohile ho tsamaea ka har'a lisebelisoa nakong ea liteko tsa lihora tsa 3, 'me banna ba khothatsang ba ne ba ts'oeroe ka har'a masaka a bona, ba sa lumellane ka kotloloho. Lenaneo lohle le ile la hatisoa ka video mme nako ea ho ikopanya le lehlakore le lehlakore le molekane kapa motho eo a sa mo tsebeng hamorao e ile ea fetoleloa ke mofuputsi ea koetlisitsoeng ea sa tsebeng lihlopha tsa baeloji. Khetho ea molekane e hlalosoa e le lithuto tse sebelisang nako e ngata ho ama 'mele le molekane ho fapana le motho eo u sa mo tsebeng, joalo ka ha ho khethiloe ke ba babeli, ba mehatla e' meli t-etona. Ntle le moo, lisebelisoa tsa likamore tse tharo li ne li e-na le li-sensor tsa Photobeam-sensors, e lumellang boikemisetso ba ts'ebetso ea locomotor e bontšitsoeng ke palo ea ho kenella ha mosali ka likamoreng tsa khothatso. Morero ona oa locomotor o re lumella ho laola litlamorao tse tlisoang ke lithethefatsi ts'ebetsong ea basali, joalo ka tšebetso e akaretsang, matšoenyeho, kapa ho fetoloa hoa tikoloho e ncha, joalo ka ha e sebelisoa ke sehlopha sa rona le batho ba bang.12.

RNA le liprotheine li ntšoa

Tse tšehali li ne li etsoa sehlabelo ka ho fafatsa kapele, 'me kapele masapo a moea a ne a ntšoa' me a hoamisoa leqhoeng le omileng. Likarolo tsa Coronal (200 µm) li ile tsa rengoa ka sekontiri 'me serame sa kenngoa holim'a li-slide tsa microscope. Lithapo tsa "bilateral tisches" tse nang le bophara ba 1 mm li nkuoe ho NAcc le caudate putamen, sebaka sa morao e le sebaka sa taolo, 'me se bolokiloe ho −80 ° C ho fihlela e sebetsoa. Kakaretso ea RNA le liprotheine li ile tsa nkuoa ho sebelisoa protocol ea TRI-Reagent ho latela litaelo tsa moetsi (Setsi sa Lipatlisiso sa Limolek'hule, Cincinnati, OH).

Tlhatlhobo ea polelo ea protheine ke Western-blot

Kamora ho arohana ho 10% polyacrylamide gel (15% bakeng sa histones), liprotheine li fetiselitsoe ho membrane ea nitrocellulose mme li kenngoa ka li-antibodies tse latelang: anti-OTR (sc-8102, 1: 1000), -V1aR (sc-18096, 1: 500), -D1R (sc-33660, 1: 1000), -D2R (sc-9113, 1: 1000, Santa Cruz Biotechnology, Santa Cruz, CA), -actin (A2066, 1: 1000: 3: 14: 06: 911: 1: 1000: 3: 05: 928: 1: 1000: 81: 96: 6.0: 3: 3: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX: XNUMX) St Louis, MO), kapa anti-acetyl histone HXNUMX (LysXNUMX, # XNUMX-XNUMX, XNUMX: XNUMX) le HXNUMX kaofela (# XNUMX-XNUMX, XNUMX: XNUMX, Millipore, Temecula, CA). Li-antibodies tsohle li netefalitsoe bakeng sa ts'ebeliso ea tsona ho batho, likhoto, le litoeba, tseo mabotho a tumisang a arolelanang liperesente tse phahameng tsa thuto ea mahlale (ho tloha ho XNUMX ho isa XNUMX%). Kamora ho nyalisoa le motsoako oa sekhukhu oa HRP o kopantsoeng, li ile tsa senoloa ka ECL (ECL SuperSignal West Dura substrate, Pierce Biotechnologies, Rockford, IL) mme ea hlahisoa ka filimi ea Fuji XAR (Filimi ea Fuji, Tokyo, Japan). Quantification e ile ea etsoa ho sebelisoa software ea AIS XNUMX ea Setšoantšo (Imaging Research, St. Catharines, Ontario, Canada), 'me matšoao' ohle a ne a etselitsoe kahare ho membrane e le 'ngoe bakeng sa actin, ntle le pontso ea acetyl-HXNUMX e neng e tloaelehile ho letšoao la histone HXNUMX kaofela. Lintlha tse tloaelehileng li hlahisoa e le liperesente tsa liphoofolo tse tšoaroang ke CSF.

Semi-quantitative ea nako ea 'nete ea li-polymerase tsa li-reaction (RT-PCR)

0.5 µg ea RNA e felletseng e ile ea sebetsoa molemong oa tlatsetso ea DNA, 'me ea hlahlobisisoa joalo ka ha ho hlalositsoe pele50 ka ho tloaela tlhaho ho gene ea nicotinamide adenine dinucleotide dehydrogenase (NADH). Liphetoho tsohle li entsoe ka makhetlo a mararo mme ho hlaka ha tsona ho netefalitsoe ke ho qhibiliha ha li-curve le karohano ho 2% Agarose gel. The primers tatellanong sebediswa ne tjena: 5'-TCCAAGGCCAAAATCCGCACGG-3 '(Fwd) le 5'-GGCAGAAGCTTCCTTGGGCGC-3' (Tšen) bakeng sa OTR, 5'-GAGGTGAACAATGGCACTAAAACC-3 '(Ka) le 5'-CCAGATGTGGTAGCAGATGAAGC-3' (Rev) bakeng sa AVP1aR, 5'- TTAACAACAATGGGGGGGG-3 '(Bakeng sa) le 5'-GGCATGAGGGATCTAGGAA-3' (Rev) bakeng sa D1R, 5'-GTGGGGTGGGTGGGTGGNGGGGGGGGGGGGGGGGGGGGGGGGGG.GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG " ) bakeng sa D3R, le 5'-CTATTAATCCCCGCCTGACC-3 '(Bakeng sa) le 2'-GGAGCTCGATTTGTTTGGC-5' (Rev) bakeng sa NADH. Lintlha tse tloaelehileng li hlahisoa e le karolo ea liphoofolo tse tšoaroang ke CSF.

Chromatin Immunoprecipitation

Histone H3 acetylation (Lys14) ho NAcc le punudate putamen punches li ile tsa hlahlojoa ka ho sebelisa protheine ea Magna ChIP G Tissue Kit (Millipore, Temecula, CA) kamora litaelo tsa moetsi. Ka bokhutšoanyane, kamora ho qhekella ka 1% formaldehyde, chromatin e ile ea khaoloa ho sebelisoa Misonix XL-2000 ho likotoana tsa 200-600 bp. Immunoprecipitation of acetylated histone H3 (Lys14) e ile ea lemohuoa ka 10µg ea anti acetyl-H3 (Lys14) antibody (Millipore) bosiu bo le bong ho 4 ° C. Kamora ho hlatsuoa, ho hlakoloa ka lieta le ho khutlisetsa marang-rang, li-CD tsa immunopreciplect li ile tsa hloekisoa le ho hlahlojoa ka makhetlo a mararo ke RT-PCR sethaleng sa iCyste (bona kaholimo) ka khalase e tloaelehileng ea kahare e entsoeng ka samples tse kentsoeng tsa INPUT. Li-primers li ne li etselitsoe ho holisa sebaka sa bolelele ba 236 bp se 128 bp e nyolohang mohloling oa pele oa "exon" bakeng sa prairie vole OTR (oxtr, GenbankAF079980), kapa 192 sebaka se telele sa bp se fumanehang 141 bp e holimo mohloling oa pele oa likh'outu tsa lebitso la V1aR (avpr1a, GenbankAF069304). Mekhahlelo ena e ne e le e latelang: 5'-CTCCGGAGCCGGGGAATT-3 '(Fwd) le 5'-ACCGCTTCCCCGAGAGTAGGG-3' (Rev) bakeng sa oxtr, le 5'-GGTGGACCAGCCAGACCC-3 '(Fwd) le 5'-TGCAGAGCCAGGCGCTTTCC-3' (Rev) bakeng sa avpr1a. Mohlala o mong le o mong o ne o lekantsoe ka boleng ba INPUT, 'me lintlha li hlahisoa e le liperesente tsa liphoofolo tse tšoaroang ke CSF.

Ts'ebetso ea lipatlisiso le ts'ebetso ea data

Bakeng sa tlhahlobo ea khetho ea balekane, liphoofolo tse ileng tsa bonts'a boits'oaro ba nako ea ho hola nakong ea ho phomola kapa ka sebatli se sa sebetseng hantle li ile tsa behelloa ka thoko. Bakeng sa litlhahlobo tse ling tsohle tsa limolek'hule, lintlha tse ngata tsa palo e le 'ngoe ka sehlopha sa baeloji li ile tsa qheleloa ka thoko ha ho tsejoa hore ke tsa thekiso ea thepa. Boholo ba liteko li entsoe ntle le ha litholoana li ne li hlakile haholo. Nako e sebelisitsoeng ho buisana le phoofolo efe kapa efe ea tšusumetso nakong ea teko ea khetho ea molekane e ile ea hlahlojoa ka lipara tse peli tse marang-rang. t-etona. Likarolo tsa locomotion li ile tsa hlahlojoa ho sebelisoa mehele e 'meli t-Test (bakeng sa lihlopha tse peli) kapa ANOVA e le 'ngoe (bakeng sa lihlopha tse fetang tse peli),' me ha ho loketse, Fischer's PLSD post-hoc liteko li entsoe ka mokhoa oa bohlokoa oa P <0.05. Kamora netefatso ea tloaelo, tlhaiso-leseling e ngoe kaofela e ile ea hlahlojoa ka mehatla e 'meli t-Tlhahlobo e lekanang phapang e lekantsoeng kapa e sa lekanang pele e hlahlojoa pele. Litlhahlobo tsohle tsa lipalo li ile tsa etsoa ho sebelisoa software ea StatView (SAS Institute). Ha datha e lekantsoe ho li-taolo tsa tsona tse fapaneng (% ea CSF, Saline kapa lihlopha tsa Mating), tlhahlobo ea lipalopalo e ile ea etsoa mabapi le data e tala.

Boitsebiso bo Eketsehileng

lumela hore baa fokola

Mosebetsi ona o tšehelitsoe ke National Institute of Mental Health (NIMH) ea lithuso MHR21-083128 ho MK le ZW, le MHR01-058616 ho ZW Re boetse re leboha Dr. Maurice Manning ka mpho e fanoeng ke mohanyetsi oa OTR Oag (T).

Mongolo o botlaaseng ba leqephe

 

Khohlano ea thahasello: Bangoli ba phatlalatsa hore ha ho na likhohlano tsa thahasello.

Menehelo ea Mongoli

HW, FD le YL ba entse liteko. HW le FD ba hlahlobile data. HW, FD, ZW le MK ba qapile thuto. FD, ZW le MK ba ngotse pampiri ena. Bangoli bohle ba ile ba buisana ka liphetho 'me ba fana ka maikutlo a bona.

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