Karolo ea bohlokoa ea Histone Methyltransferase G9a Plastiki e entsoeng ka Cocaine (2010)

Liphetoho tse bakoang ke Cocaine ho polelo ea gene li baka phetoho ho morphology ea boitšoaro le boits'oaro tse ka bakang temallo ea koae. Re supile karolo ea bohlokoa bakeng sa dimethylation ea histone 3 lysine 9 (H3K9) le lysine dimethyltransferase G9a ho cocaine e susumetsang sebopeho sa semmuso le boits'oaro. Tsamaiso e pheta-phetoang ea koae e fokotsitse maemo a lefats'e a H3K9 dimethylation ho li-nucleus accumbens. Tphokotso ea hae ea histone methylation e kopantsoe le phetisetso ea G9a sebakeng sena sa boko, se neng se laoloa ke cocaine-ikiwa e hatisitsoeng factor criptionFosB. Re sebelisa mutageneis e nang le maemo le phetisetso ea liphatsa tsa lefutso e fumanehang ka bongata, re fumane hore G9a downregulation e eketsa dendritic spine plasticity ea nucleus e bokella li-neurons le ho ntlafatsa ho rata cocaine, ka ho etsa joalo e theha karolo ea bohlokoa ea histone methylation liketsong tsa nako e telele tsa cocaine.

Selelekela

Liphoofolo le kalafo

Ntle le ha ho boletsoe ka tsela e 'ngoe, litoeba li ne li beoa ka makhetlo a mane ho isa ho a mahlano ka konkreiti ka lebone la 12 la hora / lefifi (mabone ho tloha 7: 00 AM ho 7: 00 PM) ka mocheso o sa feleng (23 ° C) ad libitum phihlello ea metsi le lijo. Liprotheine tsohle tsa liphoofolo li amohetsoe ke IACUC ka bobeli UT Southwestern Medical Center le Mount Sinai School of Medicine.

Bakeng sa liteko tsa cocaine [li-immunohistochemitry, blotting bophirima, bongata ba PCR (qPCR), tlhahlobo ea microarray le chromatin immunoprecipitation (ChIP)], 8- ho 10-ea lilemo li 23 ea banna C57BL / 6J li sebelisitsoe. Liphoofolo li fumane liente ka mehla tsa 'saline' (7 kalafo ea letsoai, ip), 'acute' cocaine (phekolo ea 6 e sebelisa saline + kalafo e le 'ngoe 20 mg / kg cocaine-HCl, ip) kapa' e pheta-pheta 'koae (7 kalafo ea 20 mg / kg cocaine-HCl, ip). Lijana li ne li etsoa sehlabelo ka lihora tsa 1 kapa lihora tsa 24 kamora kalafo ea ho qetela. Bakeng sa lithuto tsa microarray, liphoofolo li ne li tšoaroa letsatsi le leng le le leng ka 'saline' (15 kalafo ea letsoai, ip), kalafo ea 'acute' cocaine (14 kalafo ea letsoai + 1 kalafo ea 20 mg / kg cocaine-HCl, ip), 'makhetlo a mangata a "acute" Litlhare tsa kalafo ea 7 tsa saline + 8 kalafo ea 20 mg / kg cocaine-HCl, ip) kapa ho 'khaotsa khafetsa + ka nakoana' cocaine (kalafo ea 7 20 mg / kg cocaine-HCl + 7 kalafo ea saline + 1 phephetso kalafo ea 20 mg / kg cocaine-HCl, ip) mme ba etsoa sehlabelo hora ea 1 kamora kalafo ea hoqetela. Litekong tsa boits'oaro, litoeba li ile tsa beoa ka kotloloho kamora ts'ebetso ea bongaka mme tsa phekoloa ka 10 mg / kg cocaine-HCl, ip joalo ka ha ho hlalositsoe ka tlase. Bakeng sa tlhahlobo ea mokokotlo oa dendritic le netefatso ea microarray e latelang ts'oaetso ea HSV-GFP le HSV-G9a-GFP, litoeba li ile tsa phekoloa ka 'saline' (kalafo ea 5 kalafo ea saline, ip) kapa 'khafetsa cocaine' (5 kalafo ea 20 mg / kg cocaine-HCl, ip ) nakong ea matsatsi a 3, ha protocol ena ea ente e kile ea bontšoa ho eketsa letsoalo la lesapo la mokokotlo ho li-neuron tsa li-neurus nakong ea nako ea polelo ea phetisetso ea Herpes simplex (HSV)E tlatselletsang Ref S1). Lice tse sebelisitsoeng bakeng sa tlhahlobo ea lesapo la dendritic li ile tsa etsoa mahlabelo lihora tsa 4 kamora kalafo ea ho qetela.

Ho susumetsa ho hlakolwa ha G9a transcript ea Namibia bakeng sa NAc, re sebelisitse litoeba tsa mofuta oa homozygous bakeng sa G9a allele, e hlalositsoeng ka botlalo libakeng tse ling (S2). Ho kopants'oa hoa Cre-induction ho hlahisa XonUMX ho 22 ea ho tsoa lenaneong le isang boteng bo sa nahaneng le bohareng ba sengoloa se fetotsoeng. Re sebelisitse litoeba tsa G25a tse halikiloeng tse neng li khutlisetsoe ka botlalo ho litoeba tsa C9BL / 57J. Lits'ila li ile tsa kenngoa ka mokhoa o sa tsitsang ka har'a NAc e nang le livaerase tse amanang le kokoana-hloko ea Adeno (AAV) (serotype 6) tse hlalosang GFP kapa Cre-GFP pakeng tsa lilemo tsa 2 le 7 libeke. Tlhahlobo ea immunohistochemical e sebelisitsoe ho netefatsa ts'ebetso ea ho kopants'oa hoa motsoako oa Cre-medi (bona Tlatsetso ea feiga. S5). Re sebelisitse liphoofolo tse kenang tsa AAV ka matsatsi a 21 kamora ts'ebetso ea ngaka hobane recombination ho G9a litoeba tse peperaneng li ne li tsitsitse ebile li le ngata ka nako ena, li lumellana le litlaleho tse phatlalalitsoeng (S3-S4). Liteko tsa G9a le ΔJunD overexpression li ile tsa etsoa ka mokhoa o ts'oanang li sebelisa li-virus tsa vaerase ea HSV tse hlalosang mohlomong GFP, G9a-GFP, e shoeleng ka mokhoa o ts'oanelang kapa GΔNUMXK-GFP kapa ΔJunD-GFP (bona S2 bakeng sa lintlha tse mabapi le nts'etsopele ea G9a constructs). Litoeba tsa HSV tse sebelisang khatello ea kelello li sebelisitsoe matsatsi a 3 kamora ts'ebetso ea bongaka kamora ho qeta nako e ngata haholo joalokaha ho bonahala ka immunohistochemistry. Ka lebaka la sebopeho se potlakileng sa polelo ea HSV, le mofuta o tsitsitseng haholoanyane oa polelo ea AAV, li-veter tsa HSV li sebelisitsoe litekong tse hlokang polelo e potlakileng ea nako e khutšoane, athe lijana tsa AAV li ile tsa sebelisoa boitekong bo hlokang nako e telele ea polelo ea transgene. Lirapa tseo ka bobeli li bontšitsoe, lithutong tse ngata tsa pele, ho tšoaetsa methapo ea sele ea "neuronal" feela kahare ho sebaka sa boko se lemetseng, ntle le ts'oaetso ea li-neuron tse sebetsang.

Bakeng sa liteko tsa boits'oaro bo sebelisang pharmacological G9a / GLP inhibitor, BIX01294 (25 ng / μl), litoeba li kentsoe sterotaxically ka lipompo tse peli tse tsamaeang butle, hammoho le cannulae ea li-cannulae tse peli. Li-pump-Mini li ile tsa qaptjoa lihora tsa 12 pele ho ho kenngoa ho qala ts'ebetso e tsoelang pele (0.25 μl / hora) ea koloi kapa 5 hydroxypropyl β-cyclodextrin) kapa lithethefatsi bakeng sa matsatsi a 14, nakong eo liteko tsa boitšoaro li neng li etsoa.

Bakeng sa liteko tsa ΔFosB overexpression [bophirima ho qhibiliha, qPCR, le ChIP], banna ba batona ba banyenyane NSE-TTA X tetOP-ΔFosB ho ne ho sebelisoa litoeba (10-beke), moo ho neng ho se na tetracycline derivative doxecycline (libeke tsa 8 libeke tse tlohang doxycycline), liphoofolo li bonts'a polelo e matla ea "striatal" e lekantsoeng ea ΔFosB (S5). OFosB oxpxpression ho litoeba tsena e netefalitsoe ka qPCR. Ho netefatsa lintho tse fumanoeng li sebelisa NSE-TTA X tetOP-ΔFosB litoeba, hlaha haesale 8-beke ea beke C57BL / 6J litoeba tse tona li ile tsa kenngoa ka kotloloho ka intra-NAc ka methapo ea AAV e bontšang GFP kapa ΔFosB-GFP. Ho ile ha sebelisoa li-vector tsa AAV, maemong ana, ho netefatsa polelo e phahameng ea ΔFosB ho libeke tsa 8 kamora ho buuoa, ho lumella papiso e tobileng lipakeng tsa litoeba tse tšoaelitsoeng ke vaerase le bitransgenic ΔFosB overexpressing. Virtual overexpression e netefalitsoe ho sebelisa qPCR ho 8 libeke tse ka morao ho ts'ebetso (likotsi tsa 15-gauge NAc li ile tsa qhaloa tlasa sebaka sa ente). Litoeba tsa AAV-GFP le AAV-ΔFosB-GFP tse neng li sa sebelisoe bakeng sa qPCR li ne li alafshoa ka metsi a letsoai (14 kalafo ea letsoai, ip) kapa ka makhetlo a mangata cocaine (14 kalafo ea 30 mg / kg cocaine-HCl, ip) ho qala ka libeke tsa 6 ho buuoa. Matsatsi a 4 kamora kalafo ea ho qetela, likelello li ne li hlophisitsoe le 4% paraformaldehyde, e arotsoe ka vibrate mme e sebelisetsoa tlhahlobo ea mokokotlo oa dendritic.

Tlhahlobo ea blot Bophirimela

Li-punches tsa 14-gauge NAc li nkuoe ho tsoa likarolo tsa 1 mm coronal tse fumanoeng li sebelisoa ka lesela le sa sebetseng la panya ea boko mme tsa hlahisoa ka 1 M HEPES lysis buffer (1% SDS) e nang le proteinase le phosphatase inhibitors. 10-Li-sampuli tsa 30 μg tsa proteni e felletseng li ile tsa khethoa li-electrophores ho li-gels tsa 18% SDS. Liprotheine li fetiselitsoe ho li-membrane tsa PVDF 'me li kenelletse ho anti-H3K9me2 (mouse monoclonal, 1: 500), anti-β-tubulin (mouse monoclonal, 1: 60,000), anti-Jumla histone H3: 1, 5,000 anti-GFP (e sebelisitsoeng bakeng sa netefatso ea polelo e lekanang ea vaerase ho lithane tse otliloeng) (rabi polyclonal, 1: 1000), anti-H3K27me3 (rabi polyclonal, 1: 500) kapa li-anti -odin antibodies (mouse monoclonal, 1) 60,000 4 ° C (litho tsohle li ne li koetsoe lebese la 5% kapa 5% bovine serum albin). Membranes ka nako eo e ne e kentsoe li-antioxodies tsa morao-rao tsa peroxidase (1: 15,000-1: 60,000 ho latela mohala o ka sehloohong o sebelisitsoeng) mme lihlopha li ne li bonoa li sebelisa subSete ea WestSignal West Dura. Mabanta a ne a hlakotsoe ka NIH Image J Software le lihlopha tsa H3K9me2 li ne li etselitsoe maemo a tloaelo ho li-actin kapa β-tubulin le ho ngola histone H3 ka botlalo bakeng sa ho laolla ka ho lekana. K'hok'heine e phetoang hangata e ne e se na litlamorao ho litekanyetso tsa Actin (Setšoantšo sa S8) kapa histone eohle ea 3 (Setšoantšo sa S1) ho NAc. Ntle le moo, ts'oaetso ea HSV-G9a-GFP le HSV-G9aH1093K-GFP ha ea ka ea ba le litlamorao ho levels-tubulin ho NAc (Setšoantšo sa S8).

Immunohistochemistry

Lintsu li ne li kengoe ka lethal e bolaeang ea chloral hydrate mme li entsoe ka 4% paraformaldehyde pele ho hlahlojoa ke immunohistochemistry e le 'ngoe kapa habeli joalo ka ha ho hlalositsoe pele (S6). Ka bokhuts'oane, li-brains tse kentsoeng ka morao-rao li ile tsa kenngoa mochesong oa kamore bosiu ho 30% sucrose pele e kenngoa ho 35 μm (likelello tse sebelisitsoeng bakeng sa tlhahlobo ea mokokotlo oa dendritic li ne li behiloe ka vibrate ho likarolo tsa 100 μm ka ho hloka 30% sucrose). Likarolo tse sa thelliseng tsa NAc li ile tsa hlatsuoa ka 1 PBS, e koetsoe (3% serum e tloaelehileng ea esele, 0.1% tritonX, 1X PBS) mme hamorao ea kenella ka anti-GFP (kuku polyclonal, 1: 8000) le / kapa anti-G9a (rabi polyclonal , 1: 500) li-antibodies ka tharollo ea tharollo. Likarolo tse hlahlojoang bakeng sa methapo ea dendritic li ile tsa kenngoa ka motsoako oa 'mele oa anti-GFP oa mmutla ho 1: 200. Kamora ho kenella ka nako e sa lekanyetsoang, likarolo tsa NAc li ile tsa hlakoloa makhetlo a 3 bakeng sa metsotso ea 10 ka 1X PBS, ea lateloa ke ho incubation le cyXNNUMX le / kapa cy2 fluorescent-e kopantsoeng le li-antibodies tsa morao-rao ka 3X PBS tharollo ea thibelo bakeng sa lihora tsa 1. Likarolo tse sebelisitsoeng bakeng sa lithuto tsa morphology li ile tsa kenngoa kahare ho motsoako oa bobeli bosiu ka mocheso oa kamore. Tšebelisano ea matla a nyutlelie e fihletsoe ke likarolo tse hohelang tsa 2X PBS tse nang le DAPI (1: 1) bakeng sa metsotso ea 50,000. Likarolo li ile tsa hlatsuoa hape, tsa lateloa ke ho nyekeloa ke metsi le ho nyoloha le DPX. Likarolo tsohle li ne li etsoa ka setšoantšo sa microscopy ea sephiri.

Ho khetholoha RNA le qPCR

Likotlo tse 14 tsa gauge tsa NAc li ile tsa ntlafatsoa homogenized ho Trizol mme tsa sebetsoa ho latela litaelo tsa moetsi. RNA e ile ea hloekisoa ka likholomo tsa RNAesy Micro le spectroscopy e netefalitse hore likabelo tsa RNA 260/280 le 260/230 e ne e le> 1.8. Ka mor'a moo RNA e ile ea fetoloa e ngotsoe ka mokhoa oa Bio-Rad iScript Kit. cDNA e lekantsoe ke qPCR e sebelisa SYBR Green. Karabelo e 'ngoe le e' ngoe e ne e kopitsoa habeli kapa e entsoe habeli ebe e ile ea hlahlojoa ho latela mokhoa oa ΔΔCt joalokaha ho hlalositsoe pejana ho sebelisoa glyceraldehyde-3-phosphate dehydrogenase (GAPDH) joalo ka taolo ea tloaelo (S7). Bona Tafole e tlatselletsang S5 bakeng sa tatelano ea li-mRNA tsa pele.

Tlhahlobo ea microarray ea DNA

Lihlopha tse 'ne (li-replicates tse ikemetseng tsa 3 ka sehlopha ka seng) li ile tsa sebelisoa bakeng sa boithuto ba microarray, kakaretso ea 12 microarrays. Hora ea 1 kamora ho ente ea cocaine ea ho qetela, liphoofolo li ile tsa bolaoa ka potlako mme masapo a tlosoa mme a beoa leqhoeng. Mathata a NAc a ile a nkuoa a sebelisa punch ea nale ea 15-geji 'me a hoamisoa kapele ka leqhoa le omileng ho fihlela RNA e ntšoa. Likotlo tse matla li ile tsa khangoa ho tsoa ho liphoofolo tse 'ne bakeng sa papiso e' ngoe le e 'ngoe, litoeba tsa 12 ka kakaretso sehlopha ka seng. Ho khetholloa ha RNA, ts'ebetso ea microarray, le tlhahlobo ea data li entsoe joalo ka ha ho hlalositsoe pele (S8). Ka bokhutšoanyane, RNA e ile ea arohanngoa mme ea hloekisoa joalo ka ha ho hlalositsoe kaholimo mme ea hlahlojoa hore na ea boleng e sebelisoa ke Agilent's Bioanalyzer. Ho pheta-pheta phetolelo, ho matlafatsa, ho ngola mabitso le ho nyalanya lihlahisoa tsa Illumina MouseWG-6 v2.0 li ile tsa etsoa ho sebelisoa lits'ebetso tse tloaelehileng ke konokono ea UT Southwestern's microarray. Lintlha tse sa hlahisoeng li ile tsa ntšoa ka morao 'me tsa hlophisoa ka mokhoa o tloaelehileng ho sebelisoa software ea Beadstudio. Lintlha tse tloaelehileng li ile tsa hlahlojoa ho sebelisoa software ea GeneSpring mme li-genelists li hlahisitsoe ho sebelisoa lintlha tsa bohlokoa tsa 1.3 cut change cutoff hammoho le p-value cutoff ea p <0.05 e seng thata.

Re boloka tshepo e phahameng ea datha tsena ka mabaka a 'maloa. Taba ea pele, liphoofolo tsohle li ne li tšoaroa ka letsoho, li phekoloa le ho bolaoa ka nako e le 'ngoe, tlasa maemo a ts'oanang. Hape, ts'ebetso ea RNA kaofela le e hlophisitsoeng e entsoe ka nako e le 'ngoe. Taba ea bobeli, re sebelisitse menyetla e meraro ebe re kenya liphoofolo tse 'maloa ka sampole e le' ngoe, ka tsela eo re fokotsa phapang ka lebaka la phapang e fapaneng le matla a lipalo a eketsehang (S9). Taba ea boraro, mekhoa ea tlhahlobo ea data e sebelisitsoeng thutong ea rona e khothalelitsoe ke Morero oa Taolo ea Boleng ba MicroArray, kaha lintlha tsena li netefalitsoe ho fana ka tlhahiso e phahameng ea tlhahiso ea maiketsetso le ho kenella hape ka hare ho intraplatform (S10-S11).

Kaho ea livaerase tsa vaerase

Ka lebaka la mathata a kenyelletso ea boholo ba veteral vector, tatellano ea likh'ara tsa G9a (G9a) kapa lefu le bolaeang G9a (G9aH1093K) li ile tsa fallisoa ka har'a bicistronic p1005 + HSV plasmid e bontšang GFP ea GMP kapele ea taolo ea CMP boholo ba ho kenyelletsa e ne e le ~ 9 kb, e fetang boholo bo boholo ba ho kenngoa bakeng sa lijana tsa AAV-3.96). Morekisi oa IE2 / 4 o khanna polelo ea G5a. Likhechana li ile tsa kenngoa ka har'a bicistronic p9 + HSV plasmid ka tsela e sa tsitsang ea sefahleho ka Klenow ea tšoaroa ke PmeI le EcoRI e silafalitsoeng G1005a (ho tloha pcDNA9) le CIP ea alafshoa p3.1 + kamora ho qoelisoa ha EcoRI. Bakeng sa tlhahiso ea HSV-ΔJunD-GFP, tatellano ea likhoutu ea ΔJunD e ts'oeroeng ke libaka tsa thibelo ea EcoRI e hlahisitsoe ke PCR e sebelisa li-primer oligonucleotides tse nang le sebaka sa EcoRI. Sehlahisoa sa PCR se ile sa kenngoa mokokotlong sebakeng sa EcoRI sa p1005 + vector. Polelo ea lehae ea Cre recombinase ho li-neuron tsa NAc e fihlelitsoe ka ho tsamaisoa ha gene-mediated gene ho sebelisoa veter ea AAV joalo ka ha ho hlalositsoe (S12). GFP kapa N-terminal fusion ea GFP ho Cre e ile ea kenngoa mochining o kopanyang oa AAV-2 o nang le papatso ea CMV e nang le tatellano ea kamohelo ea mofani oa splice le letšoao la polyadenylation. Lintho tsohle tse kentsoeng vector li netefalitsoe ke ho latela tatellano ea litaba. Lirosa tsa vaerase li ile tsa hlahisoa ho sebelisoa mokhoa oa ho fetisetsa makhetlo a mararo, o sa thuseng, joalo ka ha ho hlalositsoe pele (S13). Kokoana-hloko e hloekisitsoeng e ne e bolokiloe ho −80 ° C. Boleng ba vaerase bo ile ba hlahlojoa ke titer ea tšoaetso e hlahlojoang liseleng tsa HEK293. Vaerase ea AAV-ΔFosB-GFP le eona e ne e lokiselitsoe ka mokhoa o tšoanang. Bakeng sa HSV-Cre, polelo ea Cre e ne e tsamaisoa ke morekisi oa IRES, ho fapana le morekisi oa IE4 / 5, e le ho fokotsa polelo ea Cre le ho thibela chefo ea neuronal (bona S14 bakeng sa kaho ea vaerase). Maemong ohle, ho ba le tšebeliso ea botona kapa botšehali ho netefalitsoe, ka bobeli a vitro 'me ka vivo, ka qPCR, le livaerase li ile tsa netefatsoa immunohistochemically ho bonts'a polelo e thibetsoeng ea NAc kamora ho buoa.

Ho buuoa ka thipa

Tlas'a ketamine (100 mg / kg) / xylazine (10 mg / kg) anesthesia, litoeba li ne li behiloe ka har'a sesebelisoa se bobebe sa liphoofolo tse nyane, mme bokaholimo ba Craneal bo ile ba pepesoa. Ho ne ho sebelisoa li-sinding tsa mashome a mararo a metso e meraro bakeng sa ho kenya 0.5 μl ea vaerase ho NAc maemong a 10 ° (AP + 1.6; ML + 1.5; DV - 4.4) ka sekhahla sa 0.1 μl / min. Liphoofolo tse amohelang liente tsa HSV li ile tsa lumelloa ho hlaphoheloa bakeng sa matsatsi a 2 kamora ho buuoa, ha litoeba tse sebelisetsoang tlhahlobo ea boitšoaro tse amohelang lingaka tsa AAV li ile tsa lumelloa ho fola matsatsi a 20 pele li beoa maemong. Linako tsena li tsamaisana le linako tsa polelo e fetisisang ea viral-Mediated transgene bakeng sa lierekisi tse peli. Bakeng sa infusions ea BIX01294, e 'ngoe le e' ngoe ea lipompo tse peli tsa mini e ne e behiloe ka mokhoa o ikhethileng mokokotlong oa panya. Ho beoa ha Cannulae ho fumanoe ka ho cheka likoti tse nyane tsa craneal kaholimo ho NAc, le ka ho tsamaisoa ha cannula e tsoang bregma (AP + 1.5; ML + 1.0; DV - 5.4). Litsuane li ile tsa lumelloa ho fola ho tsoa opereisheneng bakeng sa matsatsi a 4 ho isa ho 5 pele ho qala ts'ebetso ea maemo a sebaka ho cocaine joalokaha ho hlalositsoe ka tlase.

Sebaka se khethiloeng sa sebaka

Ts'ebetso ea maemo a sebaka e entsoe joalo ka ha ho hlalositsoe pele, ka liphetoho tse latelang (S7). Ka bokhutšoanyane, matsatsi a 3 kamora ho tšoaetsoa ha intra-NAc ea HSV-G9a-GFP, HSV-G9aH1093K-GFP kapa HSV-GFP, litoeba li ile tsa beoa likamoreng tsa maemo, tse nang le libaka tse tharo tse fapaneng. Litoeba tse bonts'itseng khetho e kholo bakeng sa likamore tse peli tsa maemo li khelositsoe thutong (<10% ea liphoofolo tsohle). Lihlopha tsa maemo li ne li lekanngoa ka nako eo ho ikamahanya le khethollo efe kapa efe ea kamore eo e ka bang teng. Matsatsing a latelang, liphoofolo li ne li entoa ka letsoai ebe li koalloa kamoreng e le ngoe thapama metsotso e 30 ebe li tšeloa k'hok'heine (10 mg / kg, ip) ebe li koalloa metsotso e 30 kamoreng e 'ngoe mantsiboea matsatsi a 2 (tse peli saline le lipara tse peli tsa k'hok'heine). Letsatsing la liteko, litoeba li ile tsa khutlisetsoa ka har'a lisebelisoa ntle le kalafo bakeng sa metsotso ea 20 mme tsa lekoa ho lekola hore na li khetha lehlakore lefe. Likarabo tsa locomotor ho cocaine li ile tsa hlahlojoa ka likheo tsa boroko likamoreng tse nang le koae ho netefatsa katleho ea kalafo ea lithethefatsi. Bakeng sa liteko tsa AAV le BIX01294 CPP, ho ile ha sebelisoa protocol e fetotsoeng hanyane. Liphoofolo li ile tsa kenoa hape ka letsoai kapa k'hok'heine (10 mg / kg, ip) mme ea koalloa likamoreng tse ikhethileng bakeng sa linako tsa metsotso ea 30, empa e ne e beoa hantle feela hang ka letsatsi matsatsi a 4, e lateloe ke teko ka letsatsi la 5 (liphoofolo li ne li le maemong a hantle. mantsiboea le kalafo ea maemo a ile a chencha). Bakeng sa lihlopha tsohle, mochini oa mantlha oa karabelo ea letsoai o ile oa hlahlojoa ho netefatsa hore locomotion ha e amehe ke kalafo ea vaerase kapa inhibitor.

Intravenous cocaine e itaolang

Likhoto tsa lilemong tsa bocha tsa Long-Evans tsa banna, tse boima ba 230-250 g qalong ea liteko, li fumanoe. Li ne li kentsoe sebakeng se mongobo- le se laoloang ke mocheso tikolohong e khutliselitsoeng ka nako ea 12 hora lebone / lefifi (mabone a ile la 9: 00 am) ad libitum phihlello ea lijo le metsi. Likhoto li ile tsa lumelloa ho hlaha tikolohong ea tsona e ncha 'me li ne li tšoaroa letsatsi le leng le le leng bakeng sa beke ea 1 pele ho qala teko. Ts'ebetso tsohle li entsoe ho latela tataiso ea Setsi sa Naha sa Bophelo bakeng sa Tlhokomelo le Ts'ebeliso ea Liphoofolo tsa Laboraro mme se amohetsoe ke Komiti ea Tlhokomelo ea liphoofolo le tšebeliso ea Mount Sinai. Sesebelisoa sa ho iphelisa se ne se entsoe ka maballo a infrared ho lekanya boitšoaro ba locomotor. Boitšoaro bo ile ba etsoa kamoo ho hlalositsoeng pele (S15-S16) ka li-catheters tse kenngoeng mothapong o nepahetseng oa jugular tlasa isoflurane (2.4-2.7%) anesthesia. Li-catheters li ne li fafatsoa ka 0.1 ml ea metsi a letsoai a nang le 10 U heparin le ampicillin (50 mg / kg). Kamora beke e le 'ngoe ea ho hlaphoheloa opereisheneng, koetliso ea boipheliso e qalile nakong ea lefifi la potoloho ea leseli / lefifi. Liphoofolo li ne li lumelloa ho fihlella hora ea 3 ka letsatsi ea k'hok'heine (0.75 mg / kg / infusion) tlasa kemiso e sa fetoheng ea matlafatso-1 (FR1), moo mochini oa khatiso o sebetsang oa lever o hlahisitseng ts'oaetso e le 'ngoe ea sethethefatsi. Likhoto li tsitsisitse ts'ebeliso ea bona ea cocaine kamora matsatsi a 1 (<6% phapang ea sekhahla sa karabelo matsatsing a 15 a latellanang, bonyane 3% e arabela ho lever e matlafalitsoeng. Lihora tse 75 kamora seboka sa ho qetela sa boitaolo, likhoto li ile tsa khaoloa hlooho kapele, boko ba tlosoa kapele mme ba sebetsoa bakeng sa ho itšehla thajana ka RNA le qPCR.

Chromatin immunoprecipitation (ChIP)

Lithapo tse ncha tsa NAc li ne li hokahane ka mokhoa o hloahloa 'me li lokiselitsoe ChIP joalo ka ha ho hlalositsoe pele (S17-S18) le liphetoho tse nyane. Ka bokhutšoanyane, likhakanyo tsa 4 14-gauge NAc ka phoofolo e ngoe (liphoofolo tsa 5 tse kentsoeng sampole) li ile tsa bokelloa, tsa hokahanngoa le 1% formaldehyde 'me tsa qhaloa ka 2 M glycine pele e kenella ho −80 ° C. Letsatsi la 1 pele ho sampole sonication, anti-mmutla / linku (ho latela moriana o thibelang pula) Lieta tsa makenete tsa IgG li ne li hlophisitsoe ka ho kenya lieta tse nepahetseng tsa motlakase ka li-anti-G9a (kalaka ea polyclonal ChIP) kapa anti-H3K9me2 (grade monoclonal ChIP grade) li-antibodies bosiu bo le bong ho 4 ° C tlasa ho potoloha khafetsa tharollo ea block. Ho sesa ka tissue le ho kuta ha chromatin ho entsoe joalo ka ha ho hlalositsoe pele (S17). Kamora sonication, likhakanyo tse lekanang tsa chromatin li fetiselitsoe ho li-tubes tse ncha mme ~ 5% ea lihlahisoa tsa ho qetela li ne li bolokiloe ho sebetsa e le taolo ea 'ho kenya'. Kamora ho hlatsoa ka botlalo le motsoako o kopantsoeng oa bead / antibody, metsoako e lekanang ea li-antibody / bead (~ 7.5 μg antibody / sampole) li ile tsa eketsoa sampole e 'ngoe le e' ngoe ea chromatin 'me ea incubated bakeng sa lihora tsa 16 tlas'a ho potoloha khafetsa ho 4 ° C. Lisampole li ile tsa hlatsuoa hape tsa kopanngoa ka sefofane ho 65 ° C bosiu bo le bong pele ho tlhoekiso ea DNA ho sebelisa kititi ea tlhoekiso ea PCR. Kamora ho hloekisoa ha DNA, disampole li ile tsa beoa ka tlase ho qPCR 'me tsa fetoloa ka tsela e tšoanang maemong a tsona a loketseng a "ho kenya" joalo ka ha ho hlalositsoe pele (S17). Katleho e tloaelehileng ea IgG immunoprecipitations e sebelisang antig ea polyGonal anti-IgG le eona e ile ea etsoa bakeng sa taolo bakeng sa ntlafatso e nepahetseng ea ntlafatso ea matšoao. Adenine phosphoribosyltransferase (APRT) e sebelisitsoe e le taolo e mpe bakeng sa liteko tsa cocaine le ΔFosB overexpression. Bona Letlapa la Tlatsetso S5 bakeng sa tatellano ea li-primer tsa papatso.

Tlhahlobo ea lesapo la mokokotlo

Ho ithuta karolo ea G9a taolong ea neuronal morphology ho vivo, re sebelisitse mekhoa e hlalositsoeng pejana le liphetoho tse latelang (S1). Matsatsi a mararo kamora ho kenoa ka ente ea HSV-GFP, HSV-G9a-GFP, HSV-ΔJunD-GFP (li-virus tsohle li ne li sebelisoa ho liphoofolo tse hlaha tsa feberu ea C57Bl / 6J), kapa HSV-Cre-GFP (e sebelisitsoeng G9a^{fl / Fl} litoeba), ha polelo ea bongata ka bongata e ne e le boholo bo phahameng, litoeba li tlatsitsoe, likelello li ne li llisoa ebe li aroloa hamorao ho 100 μm hodima vibratome. Likarolo li ne li ts'oaroa ka litlhare ho sebelisa antibody khahlanong le GFP joalo ka ha ho hlalositsoe kaholimo (bona Immunohistochemistry). Ho lekola litlamorao tsa G9a overexpression le ho kokota hoa lipalo tsa lesapo la mokokotlo, hammoho le phello ea ΔJunD overexpression, re lekantse palo ea methapo ho li-neurites tse ka bang 1-2 ka neuron e lekanang bonyane 299 μm ea li-dendrites tsa bobeli ho GFP-expression NAc medium spiny neurons (MSNs). Ha ho fanoe ka hore li-MSN li arohane ka morphological ho tsoa ho lipalo tse ling tsa neuronal ho NAc, hammoho le litlaleho tse fetileng tse bonts'ang hore HSV e tšoaetsa li-neuron tsa DARPP-32 tse hlalosang li-neuron sebakeng sena sa boko (S19), re na le ts'epo ea hore li-MSN li ile tsa hlahlojoa feela lithutong tsena. Bakeng sa phoofolo e 'ngoe le e' ngoe, re ile ra hlahloba li-neuron tsa 6-8 ho liphoofolo tsa 3-4 ka sehlopha (lihlopha tsa 7), ka mor'a moo ho fumanoe boleng bo lekanang bakeng sa phoofolo e ngoe le e ngoe bakeng sa tlhahlobo ea lipalo. Liteko tse reretsoeng ho lekola litlamorao tsa BFosB overexpression ho lephoka la lesapo la mokokotlo la NAc li ne li tsamaisoa ka mokhoa o ts'oanang le se hlalositsoeng kaholimo, ntle le hore li-veA tsa AAV li sebelisitsoe ho hlahisa GFP kapa ΔFosB-GFP bakeng sa nako e telele (libeke tsa 8). Litšoantšo tsohle tsa HSV li hapiloe li sebelisa microscope e poteletseng e nang le sepheo sa ho kenya oli ea 100X (litšoantšo tsa AAV li hapiloe ka sepheo sa ho kenya oli ea 63X). Litšoantšo li ile tsa fumanoa ka phaephe e neng e behiloe sebakeng sa 1 sa maikemisetso le boholo ba sebopeho sa 1024 × 1024. Bolelele ba dendritic bo lekantsoe ho sebelisoa software ea NIH Image J, 'me lipalo tsa lesapo la mokokotlo li ne li baloa e le sefofu ke mohlahlobi ea ka sehloohong, joalo ka ha li-slide li ne li ngotsoe pele ho tlhahlobo ea liteko. Palo e tloaelehileng ea li-spines ka 10 μm ea dendrite e baliloe.

Tlhahlobo ea statistical

ANOVA e le 'ngoe le tse peli e entsoe ka sepheo sa ho bona bohlokoa ba mofuta oa boikhethelo ba sebaka le tlhahlobo ea lesapo la mokokotlo ka lihlopha tse fetang tse peli. Liteko tsa moithuti li sebeliselitsoe lipapiso tse ling ho kenyelletsa qPCR, blotting bophirima, tlhahlobo ea lesapo la mokokotlo ho bapisa HSV-GFP le HSV-Cre ho G9a^{fl / Fl} litoeba, litlhahlobo tsa microarray (bona kaholimo) le liteko tsa chromatin immunoprecipitation. Liteko tsa moithuti ea reriloeng li ile tsa sebelisoa kamora tlhahlobo ea ANOVA ea mekhoa e 'meli ea ts'oaetso ea mokokotlo oa mokokotlo oa mokokotlo kamora ΔFosB overexpression ka netefatso ea litlamorao tse kholo tsa kalafo ea lithethefatsi le vaerase. Litekanyetso tsohle tse kenyellelitsoeng lipaleng tsa lipale li emela ± SEM (* p ≤ 0.05; ** p <0.001). Litlhahlobo tse qaqileng tsa lipalo tsa Lifeiga. 1-3 mangolong a sehlooho a fanoa ho: Lipalo-leseling tse Hlakileng tsa Lipalo li kenyelletsa Lipalo.

Ke netefatsa hore ha ho na lisebelisoa tse kenyellelitsoeng ka har'a sengolo se ngotsoeng ka letsoho Karolo ea Bohlokoa ea Histone Methyltransferase G9a ho Cocaine e kentsoeng ke Cocaine e kile ea phatlalatsoa kapa e ntse e hlahlojoa libakeng tse ling, ho kenyeletsoa le inthaneteng.

Mosebetsi oohle o kenyelletsang tšebeliso ea liphoofolo o entsoe ho ipapisitsoe le litataiso tsa litsi le tsa IACUC ho Univesithi ea Texas Southwestern Medical Center le Mount Sinai School of Medicine.