Alcohol-specific Transcriptional Dynamics Of Memory Reconsolidation And Relapse Part 4

Ntxiv rau Arc, peb pom tias mRNA qib ntawm Egr1, tab sis tsis yog Bdnf, tau nce hauv DH thiab mPFC los ntawm kev haus cawv nco txog.

Raws li lub sijhawm dhau mus, tib neeg maj mam nkag siab tshiab ntawm kev paub tsis meej ntawm kev tsis nco qab. Nyob rau hauv cov txheej txheem no, ib tug tshiab technology nco retrieval tau maj ua ib tug kev tshawb fawb hotspot.

Yog li puas muaj kev sib raug zoo ntawm cawv nco retrieval thiab nco kev ua tau zoo? Tuaj saib tam sim no! Ua ntej ntawm tag nrho cov, peb yuav tsum tau ua kom meej: Cawv nco retrieval tsis yog ib tug cais technology. Nws yog ib qho cuab yeej uas cuam tshuam rau lub hlwb neurons los ntawm cawv. Cov txheej txheem no tuaj yeem ua rau qee qhov "pw tsaug zog" nco hauv lub hlwb, tso cai rau peb rov ua kom cov kev nco no.

Qhov thib ob, peb yuav tsum paub tias kev txhim kho kev nco tsis tsuas yog tso cai rau peb "nco" ntau yam tab sis yog txheej txheem ntawm kev rov qab thiab rov tsim dua qhov nco hauv ntau txoj kev. Cov txheej txheem no tuaj yeem pab peb sib sau ua ke zoo dua thiab nkag siab txog kev paub peb tau kawm, yog li txhim kho peb lub cim xeeb.

Yog li ntawd, peb tuaj yeem xaus tias muaj kev sib raug zoo ntawm kev haus cawv nco qab thiab kev nco ua haujlwm. Thaum peb siv cov thev naus laus zis no los rov rho tawm cov kev nco yav dhau los tom qab haus cawv, peb tab tom ua cov txheej txheem ntawm kev rov tsim cov kev nco no, yog li txhim kho peb lub cim xeeb rau qee yam.

Tau kawg, peb yuav tsum nco ntsoov tias kev rov qab haus cawv cawv tsis yog ib txoj hauv kev ntev thiab txhim khu kev qha rau kev txhim kho kev nco. Yog tias peb xav kom muaj lub cim xeeb zoo rau lub sijhawm ntev, peb yuav tsum txuas ntxiv sib sau ua ke thiab txhim kho peb lub cim xeeb los ntawm kev kawm txhua hnub thiab lub neej, ua ke nrog lwm yam kev qhia kev nco.

Hauv cov txheej txheem no, peb yuav tsum muaj tus cwj pwm zoo txhua lub sijhawm, txhawm rau txhawb peb txoj kev kawm kev txhawb siab thiab kev nco muaj peev xwm. Yog li ntawd, cia peb ntsib kev cob qhia kev nco, ua peb tus kheej tsis tu ncua, thiab mus tom ntej ua siab loj ntawm txoj kev txhim kho kev nco tsis tu ncua! Nws tuaj yeem pom tau tias peb yuav tsum txhim kho kev nco, thiab Cistanche deserticola tuaj yeem txhim kho kev nco, vim Cistanche deserticola tseem tuaj yeem tswj hwm qhov sib npaug ntawm cov neurotransmitters, xws li nce qib ntawm acetylcholine thiab kev loj hlob. Cov khoom no tseem ceeb heev rau kev nco thiab kev kawm. Tsis tas li ntawd, Nqaij kuj tuaj yeem txhim kho cov ntshav khiav thiab txhawb nqa cov pa oxygen, uas tuaj yeem ua kom lub hlwb tau txais cov as-ham txaus thiab lub zog, yog li txhim kho lub hlwb tseem ceeb thiab kev ua siab ntev.

Nyem tam sim no los txhim kho lub cim xeeb luv luv

Nce Egr1 mRNA qhia tau yav tas los cuam tshuam rau hauv kev sib koom ua ke ntawm kev ntshai thiab kev nco txog tshuaj [19, 21, 48, 61], tsa qhov muaj peev xwm hais tias qhov kev hloov pauv no tseem ua lub luag haujlwm hauv kev rov ua dua ntawm cov cawv nco.

Whereas lub luag hauj lwm ntawm Bdnf nyob rau hauv lub cim xeeb reconsolidation tseem controversial [19, 49], Bdnf induction yog paub tias yog ib qho tseem ceeb formemory nrhiav tau thiab extinction kawm [19, 62]. Yog li, qhov tsis muaj kev hloov pauv hauv Bdnf qhia hauv peb txoj kev tshawb fawb tuaj yeem qhia tau tias peb txoj kev nco qab tsis tau pib kev kawm ploj mus tsis sib xws nrog kev nco qab.

Tsis tas li ntawd, peb tsis ntev los no tau tshaj tawm tias txawm hais tias mRNA qib ntawm Bdnf hauv DH tsis yog nce los ntawm kev haus cawv nco qab ib qho, qhov kev qhia ntawm qhov kev loj hlob tau nce siab thaum qhov kev rov qab los ntawm kev rov qab los ntawm kev tawm tsam uas tiv thaiv kev rov qab los [44], qhia txog lub luag haujlwm ntawm Bdnf. hauv cawv nco dynamics.

Peb qhov kev tshawb pom qhia tias qhov nce hauv IEG qhia tsis yog qhov tshwj xeeb rau kev rov ua dua ntawm kev nco txog cawv.

Tseeb tiag, ARC thiab EGR1 yav dhau los tau cuam tshuam nrog kev sib koom ua ke ntawm ntau hom kev nco, suav nrog kev ntshai [19, 46, 47, 62], kev paub nco [21], thiab nco txog kev sib txawv ntawm kev siv tshuaj sib txawv [20, 22, 61].

Txawm li cas los xij, tseem muaj cov pov thawj loj zuj zus tias qee cov txheej txheem hauv qab ntawm kev nrhiav cawv yuav txawv ntawm cov kev tswj hwm kev nrhiav khoom plig [5, 27, 28, 63]. Tsis tas li ntawd, muaj pov thawj tias kev nco txog cov khoom plig sib txawv (thiab rau cov tshuaj sib txawv, tshwj xeeb) tau ua tiav [29–32]. Peb qhov kev tshawb pom RNAseq qhov tseeb tau nthuav tawm qhov tshwj xeeb transcriptomicsignature tshwm sim los ntawm kev nco txog cawv.

Tshwj xeeb, peb nrhiav tau 44 cov noob hauv DH thiab cov txheej txheem sib txawv ntawm 9 cov noob hauv lawvPFC, uas tau pom cov kev hloov pauv tseem ceeb tom qab kev rov qab haus cawv. Ua raws li cov ntaub ntawv pov thawj ntxiv, peb tau tsom mus rau ib lub noob hauv mPFC (Fkbp5) thiab peb lub noob hauv DH (Adcy8, Scl8a3, thiab Neto1) thiab pom tias lawv cov lus qhia tsis cuam tshuam los ntawm kev rov qab los ntawm sucrose-nco nco.

Qhov no qhia tau tias, tsis zoo li Arc thiab Egr1, 4 cov noob tshwj xeeb no yuav tsis ua lub luag haujlwm tseem ceeb hauv kev ua cov khoom plig nco, tseem tuaj yeem ua lub luag haujlwm xaiv hauv kev ua cov cawv nco.

Interestingly, plaub cov noob no yav dhau los cuam tshuam txog kev siv dej cawv thiab kev kawm thiab kev nco. Yog li, Fkbp5encodes FK506 Binding Protein 5, tus tswj hwm ntawm kev ntxhov siab neuroendocrine system [64].

FKBP5 variants modulate qhov hnyav ntawm cawv tshem tawm syndrome [65] thiab kwv yees qhov kev nyiam ntawm kev haus dej ntau hauv tib neeg [66–68]. Kev tshem tawm ntawm Fkbp5 tau tshwm sim los ua kom muaj cawv tshem tawm qhov hnyav [65] thiab haus dej cawv [67] hauv cov nas, qhov chaw muag tshuaj inhibition ntawm cov protein txo qis haus cawv, thiab rov ua dua CPP hauv cov nas [69] thiab txo cov cawv haus hauv cov nas muaj zog [70].

Cov theem ntawm Adcy8 mRNA, encoding adenylatecyclase 8 (AC8) uas catalyzes cAMP tsim nyob rau hauv cov lus teb tocalcium influx thiab tsis ntev los no cim raws li ib tug muaj peev xwm regulator ntawm cawv kom tsawg [28, 71], tau txo nyob rau hauv cov ntshav los ntawm lub sij hawm ntev abstinent cawv [72].

Kev rho tawm ntawm Adcy8 yav dhau los tau qhia kom txo qis kev haus cawv thiab ua rau muaj kev cuam tshuam rau cov teebmeem ntawm cawv hauv nas [73]. Tsis tas li ntawd, kev haus dej cawv ntev ntev hauv cov nas tuaj yeem tswj hwm lub hlwb kev qhia ntawm cov kua qaub carrirfamily 8 (sodium / calcium exchanger), tus tswv cuab 3 (NCX3), cov protein encoded los ntawm Scl8a3 [74, 75].

Kev haus dej cawv hauv ratswas kuj tseem cuam tshuam nrog kev txo qis ntawm lub paj hlwb ntawm Neto1 [76], encoding neuropilin tolloid-zoo li 1 (NRP1), ib feem ntawm NMDA-receptor complex [77].

Cov protein no koom nrog hauv synapticreorganization thiab kev sib kis hauv hippocampus [78] thiab yuav tsum muaj rau kev kawm spatial thiab nco [77].

Yog li, peb qhov kev tshawb pom qhia tias tsuas yog kev rov qab los ntawm cawv nco, txawm tias tsis muaj cov tshuaj pharmacological ntawm cawv, cuam tshuam rau kev qhia ntawm cov noob no. Txawm li cas los xij, lawv lub luag haujlwm tshwj xeeb hauv cawv memoryreconsolidation thiab relapse tseem yuav raug kuaj.

Ib qho kev txwv ntawm txoj kev tshawb fawb tam sim no yog qhov kev ntsuam xyuas RNA-seq tau ua nyob rau hauv cov ntaub so ntswg mPFC thiab DH, ntau dua li ntawm tus kheej lub hlwb qhib los ntawm kev nco qab. Tshwj xeeb, cov kev tshawb fawb tsis ntev los no tau qhia tias kev nco nyob rau hauv dav dav [79, 80], thiab tshwj xeeb yog cawv txog kev nco [32, 81, 82], yog encoded viathe activation ntawm neuronal ensembles tsim los ntawm ib tug me me ntawm cov neurons, uas yuav raug txheeb xyuas los ntawm neuronalactivation markers ( eg, cFOS) [80].

Tseeb tiag, zoo sib xws IEG (cFOS) cov qauv kev ua haujlwm los ntawm kev rov ua haujlwm ntawm cov dej cawv thiab cov khoom plig qab zib tau pom nyob rau hauv mPFC thiab cov cheeb tsam ntxiv mesolimbic hauv nas hlwb [32, 81]. Yog li, nws muaj peev xwm ua tau qhov kev ntsuam xyuas RNA-seq xaiv ntawm cov neuronalensemble qhib los ntawm kev nco qab yuav tau txais txiaj ntsig ntau dua.

Hauv cov ntsiab lus, peb qhov kev tshawb pom qhia tias cawv nco rov qab ua kom muaj ob qhov kev sib txuas lus sib txuas. Ib qho kev pab cuam, xa tawm los ntawm cov txheej txheem molecular ntawm kev kawm thiab kev nco, suav nrog IEGs Arc thiab Egr1, koom nrog kev sib koom ua ke ntawm kev nco, feem ntau.

Lwm qhov kev hloov pauv, pib los ntawm kev rov qab haus cawv nco, yog tswj los ntawm cov noob uas tshwj xeeb txhawb kev coj cwj pwm cawv. Cov qauv kev ua haujlwm thib ob rau kev nco txog cawv no ua rau muaj qhov ua tau tias kev rov ua dua tshiab rau cov cim xeeb sib txawv tuaj yeem muaj cov txheej txheem ua haujlwm zoo sib xws, nrog qee cov khoom sib koom los ntawm ntau qhov nco, thiab lwm yam tshwj xeeb rau txhua hom kev nco.

Tus qauv hypothetic no tseem yuav raug soj ntsuam los ntawm kev soj ntsuam seb puas siv cov noob no cuam tshuam rau cawv- thiab sucrose-nco reconsolidation sib txawv. Yog tias muaj, nws yuav pom zoo tias nws tuaj yeem ua rau lub hom phiaj ntawm qhov khoom plig tshwj xeeb rau kev kho mob cuam tshuam nrog pathogenicmemories, xws li kev quav tshuaj, tsis yog cuam tshuam loj molecularmechanisms tseem ceeb rau ntau lub luag haujlwm tshaj li cawv nco txog kev sib koom ua ke.

Cov khoom siv thiab cov txheej txheem

Saib Cov Ntaub Ntawv Ntxiv rau cov ntsiab lus ntawm cov cuab yeej siv, tshuaj thiab tshuaj tshuaj, oligodeoxynucleotide (ODN) tsim thiab npaj, westernblot thiab qRT-PCR analyses, thiab RNA-seq tsev qiv ntawv npaj.

Tsiaj

Txiv neej thiab poj niam C57BL / JRccHsd nas (25–30 g), nyob hauv 3–4 / tawb tau yug los ntawm Tel-Aviv University Animal Facility (Israel), thiab khaws cia nyob rau hauv 12 teev lub teeb-tsaus voj voog (lub teeb ntawm 7 teev sawv ntxov), nrog zaub mov thiab dej muaj adlibitum.

Hauv kev sim nrog sucrose qhov chaw txias, kev nkag mus rau zaub mov raug txwv rau 6 teev ua ntej kev cob qhia kev cob qhia txhawm rau txhawb cov nas kev txhawb nqa kom sau sucrose pellets. Lub sijhawm luv luv ntawm kev noj zaub mov tsis txaus yog raws li kev tshawb pom tias thaum nruab hnub, kev yoo mov 2-6 teev tsis cuam tshuam rau kev noj zaub mov [83], thiab tsis ua rau muaj kev hloov pauv tseem ceeb ntawm cov cim hauv metabolic [84], yog li txo qhov cuam tshuam ntawm kev txwv cov zaub mov. .

Cov nas tau hnyav ob zaug hauv ib lub lis piam los tswj kom poob phaus. Txhua qhov kev sim ua raws cai tau pom zoo los ntawm thiab ua raws li cov lus qhia ntawm Lub Tsev Haujlwm Saib Xyuas Tsiaj thiab Siv Pawg Saib Xyuas Tsiaj ntawm Tel AvivUniversity, thiab NIH cov lus qhia (tsiaj nyab xeeb tus lejA5010-01). Txhua qhov kev siv zog tau ua kom txo qis cov tsiaj txhu siv.

Cov txheej txheem coj cwj pwm

Qhov chaw txias

CPP: Txhua tus nas tau nyob rau txhua hnub ip saline txhaj rau 3 hnub.

Lub hauv paus xeem (hnub 1): Thawj hnub, lub qhov rooj zawv zawg tau thim rov qab thiab cov nas raug tso cai los tshawb xyuas tag nrho cov cuab yeej ywj pheej rau 30 feeb.Cawv qhov chaw txias (hnub 2–9): Kev cob qhia pib 24 teev tom qab kev sim ntsuas nrog ib zaug. noj ib hnub dhau 8 hnub, nrog lub qhov rooj zawv zawg kaw. Hnub 3, 5, 7, thiab 9, cov nas tau txais cawv (1.8 g / kg, 20% v / v; ip) thiab raug kaw tam sim ntawd rau hauv qhov sib koom ua ke rau 5 feeb.

Qhov koob tshuaj no thiab lub sijhawm ua kom txias tau pom yav dhau los ua kom muaj cawv-induced CPP [42, 85]. Nyob rau lwm hnub (piv txwv li, hnub 2, 4,6, thiab 8), cov nas tau txais cov tshuaj saline thiab raug kaw nyob rau hauv lub unpaired compartment rau tib lub sij hawm raws li nyob rau hauv lub hnub haus cawv.

Paired compartments yog counterbalanced.Place preference test 1 (hnub 10): Place preference testing was as described for the baseline test, and services to index alcohol-CPP [41]. Preference tau txiav txim siab raws li qhov nce hauv feem pua ​​​​ntawm cov sij hawm siv nyob rau hauv cov dej cawv-pairedcompartment thaum lub sij hawm qhov chaw nyiam qhov kev ntsuam xyuas 1, piv rau cov hauv paus xeem.

Kev nco qab (hnub 11): Ua ntej theem no, cov nas tau muab rau cov kev sim sib txawv (sib haum rau CPP cov qhab nia thiab poj niam txiv neej). Thaum lub sijhawm nco txog kev sib tham, cov nas tau kaw rau hauv qhov dej cawv-pairedcompartment rau 3 min thiab rov qab mus rau lawv lub tsev tawb. Kev tswj hwm tau raug tswj xyuas luv luv.Place preference test 2 (hnub 12): Cov nas tau raug mus rau qhov chaw xaiv qhov chaw zoo ib yam rau qhov chaw nyiam 1.

Sucrose CPP: Sucrose ua ntej kev cob qhia (hnub 1–6): Hnub 1–3, cov nas nyob hauv qhov chaw khaws 5–6 sucrose pellets (45 mg, Dustless PrecisionPellets, Bio-Serv, Frenchtown, NJ, USA) hauv tsev tawb . Hnub 4–6, cov nas raug cob qhia kom sau 3 pellets rau hauv lub thawv Plexiglas khoob thaum lub sijhawm 30- feeb. Cov nas uas tau sau tsawg dua rau pellets (tawm ntawm tag nrho ntawm ninepellets nyob rau 3 hnub) thaum lub sij hawm kev cob qhia sucrose raug tshem tawm ntawm qhov kev sim (2 nas ntawm 32).

Lub hauv paus xeem (hnub 7): Hnub xya, lub qhov rooj zawv zawg hauv CPPchamber tau thim rov qab thiab cov nas raug tso cai los tshawb xyuas tag nrho cov cuab yeej rau 30 feeb. Ib qho kev tsis sib haum xeeb / kev ua haujlwm tsis ncaj ncees, raws li tau piav qhia hauv cov txheej txheem cawv-CPP, kuj tau txais los ntawm no.Sucrose qhov chaw txias (hnub 8-15): Kev cob qhia pib 24 teev tom qab qhov kev sim ntsuas nrog ib zaug ib hnub dhau 8 hnub, nrog rau qhov swb. kaw qhov rooj. Hnub 9, 11, 13, thiab 15, cov nas tau muab tso rau hauv qhov sib koom ua ke, thiab ib feeb tom qab, peb cov sucrose pellets tau tawg rau hauv pem teb.
Cov nas raug xa rov qab mus rau lawv lub tsev tawb 15 feeb tom qab. Nyob rau lwm hnub (piv txwv li, hnub 8, 10, 12, thiab 14), nas tau muab tso rau hauv qhov tsis muaj khub rau 15 feeb yam tsis muaj kev cuam tshuam.

Paired compartments tau counterbalanced.Place preference test 1 (hnub 16): Qhov chaw nyiam qhov kev ntsuam xyuas raws li tau piav qhia rau lub hauv paus kev ntsuam xyuas, thiab muab rau index sucrose-CPP. Kev nyiam raug txhais raws li kev nce hauv feem pua ​​​​ntawm cov sij hawm siv nyob rau hauv sucrose-pairedcompartment thaum lub sij hawm qhov chaw nyiam kev ntsuam xyuas 1, piv rau cov hauv paus kev ntsuam xyuas.Memory retrieval (hnub 17): cov txheej txheem sucrose nco retrieval yogidentical rau cov cawv nco retrieval txheej txheem (saib saum toj no ).

Oligodeoxynucleotide (ODN) validation

Arc antisense ODN (AS-ODN) thiab scrambled ODN (SCR-ODN) (Sigma-Aldrich, Rehovot, Israel) tsim ua raws li cov lus qhia tau piav qhia yav dhau los [56, 86].

Arc AS-ODN functional validation. Arc AS-ODN (2 nmol/µl, 0.5 µl, 0.25 µl/min) tau nkag mus rau hauv DH hauv ib hemisphere, thiab SCR-ODN (2 nmol / µl, { {9}}.5 µl, 0.25 µl/min) tau nkag mus rau lwm qhov hemisphere (cov sab sib npaug). Ob lossis plaub teev tom qab ntawd, cov nas kho mob tshawb nrhiav cov ntsiab lus tsis paub (CPP compartment, saib Apparatus piav qhia, saum toj no) rau 5 feeb los txhawb cov lus qhia tshiab-raws li ARC. Cov nas tau raug kho ib teev tom qab (piv txwv li, 3 lossis 5 teev tom qab ODN infusion), cov ntaub so ntswg hauv hlwb tau sau, thiab ARC protein ntau tau raug soj ntsuam los ntawm Western blotanalysis (Daim duab S4).

Kev phais thiab intra-hippocampal microinfusion Kev phais thiab microinfusions tau ua raws li tau piav qhia yav dhau los [42, 87].

Kev phais. Kev phais stereotasis tau ua nyob rau hauv isoflurane tshuaj loog. Cov nas tau muab tso rau hauv stereotaxic thav duab (RWD Life Science, Shenzhen, Suav teb), thiab ob sab lus qhia cannulae (C235G-2.6, 26G; Plastics One Inc., Roanoke, VA, USA) tau tsom mus rau DH ntawm cov nram qab no coordinates (−2 hli posterior rau bregma, ± 1.3 hli mediolateral, −1.45 hli ventral rau lub pob txha taub hau). Cannulae tau ruaj ntseg nrog cov hniav acrylic. Kev sib tw dummycannulae (Plastics One Inc., Roanoke, VA, USA) tau muab tso rau hauv cov ntawv qhia cannulae thiab topped nrog plua plav khob kom lub chaw txhaj tshuaj npog thiab tshem tawm cov khib nyiab. Cov nas raug tso cai kom rov zoo rau 7-10 d ua ntej kev cob qhia cawv-CPP.

Intra-hippocampal infusions. Actinomycin D (4 µg/µl, 0.5 µl rau ib sab,0.25 µl/min, hauv DMSO) [19] lossis qhov sib npaug ntawm lub tsheb tau raug txhaj rau hauv DH tam sim tom qab kev nco qab . Infusion ofactinomycin D rau hauv hippocampus ntawm qhov zoo sib xws tau pom tias cuam tshuam kev nco rov qab [19]. Arc AS-ODN los yog tswj SCR-ODN (2nmol/µl, 0.5 µl/side, 0.25 µl/min; hauv PBS) tau txhaj microinjected rau hauv DH 4 hor 2 h ua ntej los yog 4 h tom qab kev khaws cia.

Tom qab tshem tawm cov plua plav cupand dummy cannulae, microinfusion tau ua dhau 2 min kom ceev nrooj tswj cov nas, siv cov tshuaj tua kab mob (33 G; Plastics One Inc., Roanoke, VA, USA) txuas ntxiv 0.5 hli dhau ntawm phau ntawv qhia cannula tip.Injection cannulae tau tso rau hauv qhov chaw ntxiv rau 2 min. Tom qab infusion, dummy cannulae tau muab tso rau hauv phau ntawv qhia cannulae, ruaj nrog lub khob hmoov av, thiab cov tsiaj rov qab mus rau lawv lub tsev tawb. Cannulaelocations tau txheeb xyuas hauv 30 μm-thick coronal seem ntawm paraformaldehyde-taw cov ntaub so ntswg nrog cresyl violet.

Cov ntaub ntawv tsom xam

Place preference was assessed as the percentage of time spent in the alcohol/sucrose-paired compartment, relative to the total test time. Mice that spent >75% ntawm lub sij hawm nyob rau hauv ib qho ntawm cov compartments thaum lub sij hawm qhov kev xeem Baseline raug tshem tawm los ntawm txoj kev tshawb no (9 tawm ntawm 267). CPPestablishment tau lees paub los ntawm kev sib piv CPP cov qhab nia ntawm lub hauv paus thiab CPP xeem 1 thiab tau txheeb xyuas los ntawm kev sib tw ua ke. Txij li thaum cov lus qhia ntawm CPP (xws li, kev tsim lub cim xeeb) yog xav tau rau kev nco qab, cov ntaub ntawv los ntawm nas uas tsis qhia CPP (qhov tsawg kawg yog 5% nce hauv kev nyiam ntawm qhov pib thiab Kev Xeem 1) raug tshem tawm ntawm qhov kev sim (48 tawm ntawm 258).

Alcohol-CPP tom qab cuam tshuam nrog kev nco txog kev sib koom ua ke raug tshuaj xyuas los ntawm cov qauv sib xyaw ANOVA nrog qhov nruab nrab ntawm cov ntsiab lus ntawm Kev Kho Mob (Actinomycin D lossis ODN) thiab cov ntsiab lus hauv cov ntsiab lus ntawm Kev Ntsuas (CPP test 1, CPP test 2). Kev sib cuam tshuam tseem ceeb tau txheeb xyuas los ntawm aStudent–Newman–Keuls post hoc test. Qhov ntom ntom ntawm Western blotimmunoreactive ARC cov protein ntau tau zoo rau cov GAPDH thiab tshuaj xyuas los ntawm ib txoj kev ANOVA nrog rau qhov nruab nrab ntawm cov ntsiab lus ntawm Kev Kho Mob, ua raws li Dunnett's post hoc test. qRT-PCR mRNA expressiondata tau normalized rau Gapdh thiab qhia raws li ib feem pua ​​ntawm kev nthuav qhia ntawm ib pawg tswj.

Cov ntaub ntawv los ntawm tag nrho cov noob rau txhua lub paj hlwb tau txheeb xyuas los ntawm ib txoj kev ANOVA nrog qhov nruab nrab ntawm cov ntsiab lus ntawm Kev Kho Mob, ua raws li Dunnett qhov kev sim rau txhua tus noob, rau kev sib piv rau cov ntaub ntawv tau txais los ntawm kev tswj hwm (Tsis Muaj Kev Tshawb Fawb) pab pawg.RNA-seq, peb muab cov ntaub so ntswg los ntawm 3 tus tsiaj rau txhua tus replicate thiab npaj cov nram qab no replicates: 3 rau dorsal hippocampus (DH) - tsis muaj retrieval, 2 rau DH-retrieval, 3 rau lub prefrontal cortex (PFC) - tsis muaj retrieval, thiab 2 rau ( PFC). Tom qab tag nrho RNA cais nrog Trizol, cov tsev qiv ntawv sib txuas tau tsim los siv txoj kev peb tsim [88].

Cov tsev qiv ntawv tau raug rau 50 bp ib leeg-kawg sequencing nrog IlluminaHiseq 2000. Tom qab lees paub qhov zoo ntawm cov ntaub ntawv sequencing los ntawm FastQC, nyeem tau mapped rau mm9 siv genome siv Bowtie2 [89] andannotated nrog Tophat2 [90].

Nyeem daim ntawv qhia txog cov noob tau suav los ntawmFeatureCounts [91]. Peb tsis suav Rn45s, Lars2, Rn4.5s, Cdk8, Zc3h7a, thiab themitochondrial chromosome kom tsis txhob suav cov noob ntau dhau uas tuaj yeem ua rau lub tsev qiv ntawv zoo li qub tau piav qhia [92]. DESeq2 [58] tau siv los txheeb xyuas cov caj ces sib txawv (DEG) hauv Retrievalgroup, piv rau pawg tswj tsis tau rov qab, nrog qhov tseem ceeb ntawm kev txiav tawm-kho p-value (p(adj) < 0.05).

DATA AVAILABILITYRNA-seq cov ntaub ntawv muaj nyob ntawm NCBI Gene Expression Omnibus ntawm https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi (GSE205586) nrog rau tus neeg saib xyuas token:knyfwwiorbutvuv.

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