Anterior Basolateral Amygdala Neurons suav nrog Qhov Chaw Ntshai Ntshai Memory Engram Part 1
Dec 22, 2023
Taw qhia:
Kev hem thawj ib puag ncig cues feem ntau ua rau muaj kev ntshai tsis nco qab, tab sis yuav ua li cas cov no tau tsim thiab khaws cia tseem raug tshawb xyuas.
Ntshai nco yog ib hom kev nco hauv peb lub hlwb. Nws yog ib hom kev nco tshwj xeeb uas feem ntau ua rau peb ntshai, ntshai, thiab ntshai. Tab sis kev sib raug zoo ntawm lub cim xeeb ntawm kev ntshai thiab peb lub cim xeeb yog qhov nyuaj heev.
Ua ntej, kev ntshai kev nco tsis muaj dab tsi ua rau peb lub cim xeeb luv luv. Cov kev ntshai feem ntau yog khaws cia hauv peb lub txhab nyiaj nco mus ntev. Qhov no yog vim li cas qee zaum peb muaj kev nco los ntawm qhov ntev dhau los ntab nyob hauv peb lub siab.
Qhov thib ob, kev ntshai kev nco tsis tas cuam tshuam rau peb lub cim xeeb. Txawm hais tias nws yuav ua rau muaj kev hloov pauv hauv peb lub siab thiab lub siab, tsis muaj pov thawj tias nws ua rau nco tsis tau. Ntau tus neeg tseem tuaj yeem ua rau muaj kev nco zoo heev tom qab tau ntsib qee yam txaus ntshai.
Thaum kawg, peb yuav tsum tsis txhob cia tej kev nco ntshai dhau los ua lub ntsiab lus hauv peb lub neej. Peb yuav tsum ua qhov zoo los ua cov kev nco no thiab txav mus rau hauv txoj kev zoo dua. Qhov no suav nrog kev tau txais kev kho mob, ua kom muaj kev sib raug zoo, ua haujlwm ntawm koj lub cev thiab lub hlwb, thiab tswj lub siab zoo thiab kev noj qab haus huv ntawm lub hlwb.
Yog li, txawm hais tias kev nco ntshai yuav ua rau peb muaj kev ntxhov siab thiab tsis xis nyob, peb yuav tsum muaj kev ntseeg ntawm peb lub peev xwm thiab kev siv zog. Peb tuaj yeem ntsib cov kev nco txaus ntshai no, ntxiv dag zog rau peb lub cim xeeb, loj hlob, thiab ua neeg zoo dua, muaj kev ntseeg siab dua. Nws pom tau tias peb yuav tsum txhim kho peb lub cim xeeb. Cistanche deserticola tuaj yeem txhim kho kev nco zoo vim Cistanche deserticola yog cov khoom siv tshuaj suav tshuaj suav nrog ntau yam tshwj xeeb, ib qho ntawm kev txhim kho kev nco. Kev ua tau zoo ntawm cov nqaij minced los ntawm ntau yam khoom xyaw uas nws muaj, nrog rau cov kua qaub, polysaccharides, flavonoids, thiab lwm yam. Cov khoom xyaw no tuaj yeem txhawb lub hlwb kev noj qab haus huv ntau txoj hauv kev.
Nyem Paub Short-term Nco yuav ua li cas txhim kho
Rov qab nco txog qhov kev ntshai tsis ntev los no yog xav kom muaj kev cuam tshuam txog kev rov ua haujlwm ntawm cov neurons, hauv ntau lub hlwb hauv cheeb tsam, qhib thaum lub cim xeeb tsim, qhia tias kev faib tawm thiab kev sib cuam tshuam ntawm cov paj hlwb suav nrog kev ntshai kev nco.
Qhov ntev npaum li cas qhov kev ua kom lub cev tshwj xeeb reactivation engrams tseem nyob rau lub sijhawm ntev kev ntshai nco nco, txawm li cas los xij, tseem tsis tau tshawb nrhiav ntau. Peb xav tias tus thawj xib fwb neurons nyob rau hauv lub anterior basolateral amygdala (BLA), uas encode tsis zoo valence, acutelyreactivate nyob rau hauv tej thaj chaw deb ntshai nco nco rov los tsav ntshai cwj pwm.
Txoj kev:
Siv cov neeg laus cov xeeb ntxwv ntawm TRAP2 thiab Ai14 nas, tsis tu ncua tdTomatoexpression tau siv rau "TRAP" aBLA neurons uas underwent Fos-activationduring contextual ntshai txias txias (hluav taws xob shocks) los yog cov ntsiab lus xwb conditioning (tsis muaj shocks) (n=5/pab pawg) . Peb lub lis piam tom qab, cov nas tau rov nthuav tawm rau tib lub ntsiab lus cues rau kev nco qab tej thaj chaw deb thiab tom qab ntawd txi rau Fosimmunohistochemistry.
Cov txiaj ntsig:
TRAPed (tdTomato +), Fos +, thiab reactivated (ob-labeled) neuronal ensembles tau loj nyob rau hauv kev ntshai tshaj cov ntsiab lus-conditioned nas, nrog rau nruab nrab sub-region thiab nruab nrab/caudal dorsomedial quadrants ntawm aBLAdisplaying qhov loj tshaj densities ntawm tag nrho peb pawg neeg.
WhereastdTomato + ensembles tau dominantly glutamatergic nyob rau hauv cov ntsiab lus teb thiab ntshai pawg, freezing tus cwj pwm thaum tej thaj chaw deb nco nco tsis correlated nrog ntau thiab tsawg pab pawg neeg.
Kev sib tham:
Peb txiav txim siab tias txawm hais tias aBLA-nrog kev ntshai memoryengram cov ntaub ntawv thiab tseem nyob ntawm lub sijhawm nyob deb, plasticity impactingelectrophysiological teb ntawm engram neurons, tsis yog lawv cov pejxeem loj, encodes ntshai nco thiab ua rau tus cwj pwm tshwm sim ntawm lub sij hawm ntev kev ntshai nco nco.
Taw qhia
Ntshai-eliciting stimuli feem ntau tsim muaj zog thiab ua kom muaj kev sib raug zoo nrog kev nco uas txuas ib puag ncig cues rau hem cov xwm txheej hauv lub neej.
Kev sib ntsib tom ntej nrog cov kev hem thawj zoo sib xws ua rau muaj kev tiv thaiv tus cwj pwm zoo dua, xav txog kev nco txog kev ntshai. Kev ua yuam kev hauv kev ntshai-kev koom tes ua haujlwm tuaj yeem ua rau qhov tsis zoo-valence puas hlwb, suav nrog kev ntxhov siab thiab kev nyuaj siab tom qab (PTSD) (Tovote li al., 2015).
Nkag siab txog qhov nyuaj ntawm cov txheej txheem hauv qab kev ntshai kev nco yog qhov tseem ceeb rau kev txheeb xyuas cov kev ua haujlwm tsis zoo hauv Circuit Court uas txhawb nqa kev ntshai txog kev puas siab puas ntsws kab mob thiab qhia cov hom phiaj kho tshiab kho kom rov zoo li qub kev ntshai.
Ntau xyoo dhau los, kev ntshai kev tshawb fawb txog kev nco tau tsom mus rau lub sijhawm luv luv ntawm kev nco, khaws cia, thiab nco qab (Kimand Jung, 2006; Kim and Cho, 2020; Lee et al., 2021).
Studieshave implicated dav faib interconnected CNS neuronalensembles raws li lub luag hauj lwm rau ntshai-nco tsim thiab cia (Ramirez li al., 2013; Liu li al., 2014; Roy li al., 2022). Kev ua kom cov pawg no los ntawm kev pom kev hem thawj ua rau muaj zog synaptictransmission uas sawv cev rau lub cev tsis tu ncua hu ua amemory engram (Yuan li al., 2011; Miyawaki thiab Mizuseki, 2022).
Kev nco qab lub sijhawm luv luv yog xav kom muaj kev cuam tshuam txog kev rov ua haujlwm ntawm tib lub paj hlwb uas tau qhib thaum lub sijhawm tsim lub cim xeeb, nrog cov pov thawj qhia tias txawm tias ib feem ntawm ib pawg neuronal tuaj yeem ua rau muaj kev ntshai txog kev coj cwj pwm, qhia txog kev nco qab (Roy li al., 2022. ). Whilstanatomically ruaj khov potentiated Circuit Court engrams yuav piav qhia luv luv-term ntshai nco, qhov uas lawv koom nyob rau hauv lub sij hawm ntev kev ntshai nco tsis meej.
Txawm hais tias kev ntshai mus sij hawm ntev kev nco txog kev sib koom ua ke yog xav nyob ntawm qhov kev vam meej lossis kev txhawb nqa ntawm lub sijhawm luv luv, ob peb cov kev tshawb fawb tsis ntev los no qhia tias lub sijhawm luv luv nco txog neuronal ensembles undergo lub sij hawm-dependentreorganization, uas ua rau kev tsiv teb tsaws ntawm lub cim xeeb cia rau ib qho sib txawv neuronal pawg nyob rau hauv. tib lub hlwb (DeNardo li al., 2019) los yog ntau lub hlwb thaj tsam tag nrho (Frankland thiab Bontempi, 2005; Do Monte li al., 2016).
Lub basolateral amygdala (BLA) yog lub hlwb thaj tsam ib qho tseem ceeb rau kev ntshai thiab tau cuam tshuam ob qho tib si nyob rau hauv tsis ntev los no thiab tej thaj chaw deb ntshai nco nco (Maren li al., 1996; Gale li al., 2004; Kitamura et al., 2017; Liu et al. Ib., 2022).
Qhov tseem ceeb, cov ntawv ceeb toom tau piav qhia txog lub sijhawm luv luv ntshai-nco engram hauv BLA uas yog (1) qhib thaum lub cim xeeb tsim, (2) rov ua haujlwm thaum nco qab, thiab (3) tuaj yeem tsav tus cwj pwm zoo li kev ntshai hauv cov ntsiab lus tsis ntshai ( Royet al., 2022; Zaki et al., 2022). Txawm li cas los xij, qhov twg aBLA neuronal ensemble tau qhib thaum lub sij hawm tsim kev nco yog rov ua haujlwm thaum lub sij hawm nco txog tej thaj chaw deb tseem tsis tau daws.
Hauv txoj kev tshawb no, peb tau sim qhov kev xav tias kev rov ua haujlwm ntawm BLAfear lub cim xeeb yog pom ntawm lub sijhawm nyob deb ntawm 3 lub lis piam tom qab kev ntshai txias. Ntawm qhov tseem ceeb tshwj xeeb, BLA housescircuits pab txhawb ob qho tib si rau aversive thiab muab nqi zog-nrhiav tus cwj pwm (Hu, 2016), nrog rau qhov tsis zoo-valence neurons teb rau ntshaistimuli localized tshwj xeeb rau anterior BLA (aBLA) (Goosensand Maren, 2001; Kim li al., 2016, 2017; Zhang et al., 2020).
Tsom ntsoov rau aBLA, peb ua haujlwm thib ob tiam TargetedRecombination nyob rau hauv Activated Populations (TRAP2) transgenic nas (DeNardo li al., 2019), nyob rau hauv uas Cre-recombinase kev ua yog tiedto c-fos tam sim ntawd thaum ntxov gene txhawb nqa cov ntsiab lus los ntawm txoj kev txhim kho Cre. -estrogen receptor (ER) complex. Neuronsexpressing qhov complex no tuaj yeem ua rau tsis tu ncua Cre recombination, piv txwv li, tuaj yeem "TRAPed," los ntawm kev tswj hwm ntawm ER agonistamoxifen los yog nws luv luv-acting analog 4-hydroxytamoxifen (4-OHT), tso cai rau kev qhia tsis tu ncua ntawm Cre. -dependent effectoror reporter molecules.
Ntawm no, peb hla TRAP2 nas thiab Cre-dependent tdTomato reporter kab Ai14 (Madisen li al., 2010) thiab ntsuas qhov ntau npaum li cas aBLA neurons TRAPed (tdTomatopositive) thaum lub sij hawm contextual ntshai txias txias yog reactivated (express Fos immunorereactive protein) 3 lub lis piam tom qab.
Raws li nyob rau hauv lub sij hawm luv luv ntshai nco, peb qhov kev tshawb pom txhawb nqa lub sijhawm ntev glutamatergic ntshai memoryneuronal ensembles hauv aBLA.
Qhov tseem ceeb, txawm li cas los xij, qhov loj ntawm aBLA neuronal ensembles, txawm tias qhib thaum lub caij txias lossis qhov chaw nco nco rov qab los yog ob qho tib si, tsis cuam tshuam nrog kev ntshai kev coj cwj pwm, qhia tias qhov xwm txheej ntawm kev ntshai kev kawm-pab plasticity thiab nws qhov kev cuam tshuam rau tej thaj chaw deb ntshai nco nco txog cov lus teb electrophysiological ntawm pawg neurons. , tsis yog qhov loj ntawm cov pej xeem pawg, feem ntau tsav kev coj cwj pwm kev ntshai nco txog tej thaj chaw deb.
Cov ntaub ntawv thiab cov txheej txheem
Kev pom zoo rau kev ncaj ncees
Cov txheej txheem sim tau pom zoo los ntawm InstitutionalAnimal Care and Use Committee ntawm University of Texas Health SanAntonio thiab ua raws li National Research Council Guide for the Care and Use of Laboratory Tsiaj.
Tsiaj
Kev yug me nyuam ntawm homozygous Fos2A−iCreER/2A-iCreER knockin (TRAP2, #030323) thiab R26Ai14/+ (Ai14, #007914) nas (Jackson Laboratories) tau hla los tsim cov txiv neej hemizygousTRAP2:Ai14 tawm (AiFigure). Tom qab kev phais, cov nas tau muab tso rau hauv lub tawb yas (29 × 18 × 13 cm) uas muaj cov ntaub pua plag (Sani-chips; Harlan Teklad, Madison, WI, USA).Lub vivarium yog ntsuas kub (24◦C) nrog rau 14 : 10 hlight-dub voj voog (lub teeb ntawm 0600 h). Khoom noj thiab dej twb muaj ad libitum. Kev sim tau pib thaum nas mus txog 3 lub hlis.
Kev coj cwj pwm
Ib yam li hauv kev tshawb fawb yav dhau los (DeNardo li al., 2019), cov nas tau txais tsib hnub sib law liag ntawm kev cob qhia kev coj tus cwj pwm tam sim ua ntej qhov kev ntshai ntawm qhov xwm txheej kom txo qis cfos induction nyob rau hauv cov lus teb rau kev sim tej yam kev mob uas tsis underinvestigation (xws li, tuav, tshiab ntsiab lus tshawb nrhiav, thiab lwm yam.)( Daim duab 1B).
Txhua qhov kev sib tham txhua hnub tau siv sijhawm li 3 feeb thiab muaj kev cuam tshuam rau chav txias (HabitestModular System, Coulbourn Instruments). Chamber sensory cuesconsisted of (1) 70% ethanol (olfactory cue), (2) metal grid floor (haptic cue), (3) patterned background (visual cue), thiab (4) visiblelight (visual cue).
Tsis tas li ntawd, txhua tus nas tau tuav tes rau ib feeb ua ntej muab tso rau hauv chav txias thiab maj mam "scruffed" rau 10 vib nas this thaum tawm ntawm lub chamber. Habituation sessions tau tsim los ua raws li kev tuav cov nas uas yuav muaj nyob rau hnub txias tshwj tsis yog tsis muaj kev txhaj tshuaj intraperitoneal (IP) tau ua.
Contextual ntshai conditioning
Cov nas tom ntej tau muab tso rau hauv qhov kev ntshai-qhib (kev poob siab) thiab cov ntsiab lus-txias (tsis muaj kev poob siab / cov ntsiab lus-tsuas yog) pawg.Conditioning pib rau Hnub 0 thiab muaj cov nas raug tso rau hauv chav txias thiab tso cai rau tshawb nrhiav 2 min. Tshaj li 4 feeb tom ntej, kev ntshai-txias micerceived ib tug series ntawm tsib-taw shocks ntawm 1 s ntev thiab 0.75 mAintensity.
Kev poob siab tau raug xa tawm ntawm lub sijhawm uas tsis tuaj yeem xav txog rau cov nas, tab sis zoo ib yam ntawm cov nas. Context-conditionedmice underwent ib tug zoo tib yam raws tu qauv tsis suav rau ko taw-shocks (Daim duab 1C). Thaum ua haujlwm tiav, txhua tus nas tau txhaj tshuaj ntawm 4-hydroxytamoxifen (4-OHT, 100 mg / kg, IP) dissolvedin 4: 1 cocktail ntawm sunflower thiab castor roj npaj raws li tau piav qhia dhau los (DeNardo li al., 2019).
Cov nas raug xa rov qab mus rau lawv lub tsev tawb uas lawv tau tso tseg tsis muaj kev cuam tshuam hauv chav nyob ib sab ntawm chav coj tus cwj pwm tsawg kawg 72 teev ua ntej rov ua haujlwm li qub.
Peb lub lis piam tom qab kev ntshai txias (Hnub 21), nas nyob rau hauv qhov kev ntsuam xyuas nco qab (Daim duab 1D) suav nrog kev rov qab mus rau chav kuaj nrog kev hnov lus zoo ib yam li cov neeg tam sim no thaum lub caij txias. Cov nas raug tso cai los tshawb xyuas lub chamber rau 5 min thaum lub sijhawm ntshai tus cwj pwm (posturalfreezing) tau qhab nia siv FreezeFrame 4 video nrhiav software (ActiMetrics, Wilmette IL, USA).
Lub hlwb fixation thiab histology
cuaj caum feeb tom qab kev sim nco qab, nas tau muab tshuaj loog nrog isoflurane (5% hauv oxygen) thiab tom qab ntawd ua rau lub plawv dhia nrog 30 mL ntawm heparinized (100 U / mL) isotonic saline tom qab 100 mL ntawm 4% paraformaldehyde (PFA ) nyob rau hauv 0.1 M phosphate tsis.
Lub hlwb raug tshem tawm, tom qab kho hauv 4% PFA rau 6 teev ntawm chav tsev kub (∼22◦C), thiab cryoprotectedin 30% sucrose-0.01 M phosphate buffer saline (PBS) tsawg kawg yog 2 hnub.
Lub hlwb raug muab txiav rau hauv 30 µm-thick coronal seem ntawm cov microtome khov (Leica Microsystems, Wetzlar, Lub teb chaws Yelemees) thiab ntu uas muaj cov aBLA (Paxinos thiab Franklin, 2019), tau muab khaws cia hauv polyvinylpyrrolidone (PVP) cryoprotectant ntawm −20◦C.
Immunohistochemistry
Kev txhaj tshuaj tiv thaiv kab mob tau ua raws li tau piav qhia dhau los (Mitchell li al., 2018; Maruyama li al., 2019).
Luv luv, ntu tau raug ntxuav hauv PBS kom tshem tawm PVP cryoprotectant thiab incubated rau 30 min hauv PBS uas muaj sodium borohydride (0.5%) kom tshem tawm auto-fluorescent aldehydes tsim thaum kho. Tom qab ntxiv PBS ntxuav, cov seem tau incubated hauv thaiv tsis pub (3% tshis serum, 0.05% Triton-X-100 hauv PBS) rau 2 h ntawm ∼22◦C raws li incubation ntawm 4◦C hauv thaiv tsis muaj apolyclonal luav -Fos thawj antibody (Ab) rau 72 h (1: 1,500, synaptic systems #226 003) lossis monoclonal nas CaMKII primaryAb rau 24 h (1: 500, Enzo Life Sciences, #ADI-KAM-CA002).
Tom qab ntxuav ntxiv, Fos thiab CaMKII ntu tau cais tawm rau 2 teev ntawm ∼22◦C hauv biotinylated tshis anti-rabbitIgG theem nrab Ab (1:250 EMD Millipore #AP132B) ua raws li kev ntxhua khaub ncaws hauv 0. 05 M tris-buffered saline (TBS) thiab 0.1 Msodium acetate ces raug rau streptavidin-Alexa Fluor 488 (1:250, Invitrogen #S11223) rau 1 h hauv TBS-raws li thaiv kev daws.
Thaum kawg, ntu tau muab ntxuav hauv Tris tsis thiab mounted ntawm slides nrog Fluoromount-G (Invitrogen, #00-4958-02).
Kev ntsuas thiab ntsuas
Ib qho A{{0}}ntsis photomultiplier raj cuam tshuam nrog Zeiss LSM710laser scanning confocal microscope, nruab nrog cov kab tsim nyog, tau siv los ntes aBLA cov duab siv lub hom phiaj 20 × (NA 0.8) thiab scan taub hau pinhole loj ntawm 4 7,5m. Rau txhua daim duab, ImageJ software (NIH, Bethesda, MD) tau siv los tsim cov duab kos duab sib cais ntawm tdTomato (ie, TRAPed, liab) thiab Alexa-488(Fos, ntsuab) fluorescence.
Pixel threshold siv thiab wavelengthoverlap nrhiav tau rau ob daim ntawv lo tau txiav txim siab los ntawm kev sib piv cov ntu ua tiav nrog thiab tsis muaj Fo tus thawj Ab tau piav qhia (Mitchell li al., 2018; Maruyama li al., 2019).Cell-counting thiab co-localization cov ntaub ntawv tau txais thesemi-autonomous ImageJ plugin EzColocalization (Stauffer li al., 2018).
Cov duab ntawm aBLA tau muab faib ua peb rostro-caudal subregions ntawm ze li ntawm qhov sib npaug, nrog rau txhua cheeb tsam sub-hais los ntawm aspecific ntau yam ntawm rostral-caudal stereotaxic coordinates txheeb ze tobregma: rostral, −0.8 txog −1.1 hli; nruab nrab, -1.2 txog -1.5 hli; andcaudal, −1.6 txog −1.9 mm (Paxinos and Franklin, 2019). Region of Interest (ROI) tus thawj coj hauv ImageJ tau siv los faib theaBLA raws nws cov dorsal-ventral thiab medial-lateral quadrants txheeb ze rau nws thaj tsam. Cov qauv no tau siv tom qab siv rau txhua qhov chaw nyob hauv txhua qhov teev tseg rostral-caudal sub-region.
Lub aBLAperimeter demarcation tau siv nrog ImageJ EzColocalizationplug-in los tsim ib qho 2-D spatial zajlus ntawm tag nrho cov neuronal suav ineach duab. Spatial plots nyob rau hauv lub rostral, nruab nrab, thiab caudal subregions ces cais overlayed. Qhov no tau ua rau 10 kev ntshai thiab 10 lub ntsiab lus hauv cheeb tsam rau lub cheeb tsam rostral, 10 ntshai thiab 13 lub ntsiab lus rau thaj tsam nruab nrab, thiab 12 ntshai thiab 13 lub ntsiab lus hauv cheeb tsam caudal.
Txhawm rau kho kom muaj ntau dua ntawm cov duab hauv cheeb tsam hauv cheeb tsam hauv pawg ntsiab lus, kev kho qhov tseem ceeb ntawm 1.0(10/10), 0.77 (10/13), thiab 0.92 (12/13) tau siv, feem, rau cov rostral, nruab nrab, thiab caudal sub-region thaj tsam ntawm pawg contextgroup. Txhawm rau zam kev tsis ncaj ncees rau qhov kev faib tawm saum toj kawg nkaus ntawm suav hauv cov ntsiab lus cov ntaub ntawv, lub tshuab hluav taws xob random hauv MS Excel tau siv los txheeb xyuas cov suav tshwj xeeb kom tshem tawm ntawm txhua thaj tsam aBLAsub.
Tom ntej no, peb tau muab faib ua BLA sub-regions rau hauv quadrants, xaiv 1–4 (saib daim duab 2D). Txhawm rau ua li ntawd, qhov kev sib tshuam ntawm kev sib tshuam (0,0) tau txiav txim siab hauv MS-Excel raws li qhov taw qhia hauv 2-D dav hlau uas thaj tsam ntawm txhua lub quadrant yog kwv yees li ntawm ib feem plaub. ntawm tag nrho cheeb tsam ntawm txhua rostral-caudal subregion. Quadrant axes thiab xy coordinates ntawm txhua tus suav tau txiav txim siab thiab tig 30◦clockwise, yog li muab lawv mus rau theanatomical axis ntawm aBLA.
Txhawm rau suav cov kev hloov pauv me me hauv qhov loj ntawm cov cheeb tsam hauv cheeb tsam thiab lawv cov quadrants, suav cov ntaub ntawv tau normalized rau thaj tsam ntawm thaj chaw (suav / mm2) kom cov txiaj ntsig tau tshaj tawm sawv cev qhov ntom ntawm cov suav hauv ib cheeb tsam lossis quadrant.
Txhawm rau ntsuas qhov excitatory phenotype ntawm neurons ntes los ntawm peb lub cev TRAP2, kev sib koom ua ke ntawm kev txias-TRAPed tdTomatoexpression nrog calcium-calmodulin-dependent protein kinaseII (CaMKII), ib qho cim tsim ntawm glutamatergic neurons (McDonald li al., 2002quantified). dluab ntawm tsawg kawg peb ntuaBLA ib tus nas siv ImageJ cell counter plug-in.
Kev txheeb cais
Kev ntsuas kev txheeb xyuas tau ua nrog Prism 9.5.1 software (GraphPad, San Diego, CA, USA) thiab suav nrog kev sim rau kev faib tawm ib txwm muaj ntawm txhua cov ntaub ntawv siv Shapiro-Wilk test.Pob piv rau cov ntaub ntawv txuas ntxiv tau ua nrog ob-tailed unpaired tub ntxhais kawm. t-tests los yog los ntawm ob-los yog peb-txoj kev ANOVA ua raws li Sidak's thiab Tukey ntau qhov kev sib piv sib piv, raws li, rau kev sib piv ntawm khub tom qab kev sib cuam tshuam ANOVA tseem ceeb.
Nyob rau hauv-pab pawg correlation tsom xam tau ua los txiav txim Pearson tus r-coefficient thiab coj P-tus nqi. Kev sib raug zoo tau muab piv rau thoob plaws pab pawg siv Fisher'sZ transformation (Table 1). Cov ntaub ntawv pab pawg tau nthuav tawm raws li txhais tau tias ± SEM. Kev txheeb xyuas qhov tseem ceeb tau teeb tsa ntawm P < 0.05.
Cov txiaj ntsig
Ntshai txias induces ib tug remotefear-nco engram
Txhawm rau tsim kom muaj qhov twg aBLA neurons tau qhib thaum lub sijhawm kev ntshai kev tsim kho tau rov ua haujlwm thaum lub sijhawm nco txog tej thaj chaw deb, nas tau ntshai (n=5), lossis cov ntsiab lus (n=5) txias ua raws li kev txhaj tshuaj ntawm 4-OHT (Daim duab 1A–C).
Kev tshuaj xyuas kev tsim kev ntshai (piv txwv li, kev kawm ntshai), kev sib cuam tshuam ntawm kev kho mob (shock vs. tsis muaj kev poob siab) thiab lub sij hawm rau hauv (30 s intervalafter txhua qhov poob siab) ntawm kev coj tus cwj pwm khov tau pom [ob txoj kev ANOVA, F(5, 40)=6.733, P=0 0001]. Fear-conditioned miceshowed nce khov lub sij hawm txheeb ze rau lub hauv paus tom qab poob siab 3 (275 ± 68% ntawm BL, P=0.0203), 4 (486 ± 76% ntawm BL, P < 0.0001) thiab 5 (402 ± 26). % ntawm BL, P <0.0001), thiab txheeb ze rau cov ntsiab lus mob nas tom qab poob siab 4 (251 ± 38% ntawm cov ntsiab lus, p < 0.0001) thiab 5 (375 ± 25% ntawm cov ntsiab lus, p < 0.0001).
Raws li qhov xav tau, rov ua rau lub tshuab txias txias rau cov chaw taws teeb nco rov qab hnub no 21 tom qab txias txias tau pom cov nas ntshai-kho tau nthuav tawm ntau lub sijhawm siv khov rau piv rau cov ntsiab lus tswj [346 ± 18%, unpaired t-test, t(8) {{7} }.16, P < 0.0001] (Daim duab 1D).
Sib sau ua ke, cov ntaub ntawv hauv daim duab 1 qhia tau hais tias peb qhov kev ntshai kho mob raws tu qauv ua rau muaj kev ntshai kev nco thiab kev nco txog tej thaj chaw deb ntshai.
Ntshai kawm induces robustsub-region-specific aBLA rau pawg
Kev soj ntsuam ntawm TRAPed (tdTomato +) suav tau qhia qhov loj dua fos-activated aBLA cov pej xeem hauv kev ntshai-conditioned (110 ± 6.2) dua li cov ntsiab lus-conditioned (77 ± 3.1) nas[unpaired t-test, t(8)=4 .782, P=0.0014] (Table 1). Raws li kev cia siab, ∼70% ntawm cov ntsiab lus- thiab ∼80% ntawm kev ntshai-TRAPed neurons (n = 6 aBLAsections/pab pawg) yog CaMKII + (Cov duab ntxiv 1A, B), qhia tias lawv feem ntau yog glutamatergic projection neurons.
Zoo ib yam li cov ntawv tshaj tawm yav dhau los (Kim li al., 2016), peb faib theaBLA rau hauv peb thaj tsam rostrocaudal: rostral, nruab nrab, thiab caudal (Figures 2A–C) thiab piv TRAPed count densities (counts/mm2) hla sub-regions hauv thiab nruab nrab ntawm pab pawg kho mob (Daim duab 2D). Ob-txoj kev ANOVA tau qhia txog kev cuam tshuam tseem ceeb ntawm cheeb tsam hauv cheeb tsam thiab kev kho mob [F(2,24)=7.7099,P=0.0038].
Nyob rau hauv-pab pawg soj ntsuam tau qhia tias TRAPed countdensity ntau dua nyob rau hauv nruab nrab aBLA sub-region hauv kev ntshai (239 ± 9) thiab cov ntsiab lus (144 ± 12) pawg txheeb ze rau rostral (ntshai: 86 ± 6,P < { {8}}. }}.0132) sub-regions.
Nruab nrab ntawm-pab pawg txheeb xyuas qhia tawm uas traped suav ntom ntom cuab yeej ntau dua nyob rau hauv nruab nrab ntawm thaj chaw ntawm thaj av ntawm thaj av
Txhawm rau nthuav dav ntxiv qhov chaw ntawm TRAPed neurons, wesubdivided rostral, nruab nrab, thiab caudal aBLA sub-regions rau hauv plaub lub quadrants txhua (Daim duab 2E). Nyob rau hauv nruab nrab cheeb tsam, kev kho mob tseem ceeb ntawm kev suav qhov ceev tau pom [peb-txoj kev ANOVA, F(1,32)=17.44, P=0.0002] nrog siab dua TRAPedcount ceev nyob rau hauv qhov kev ntshai tshaj cov ntsiab lus pawg hauv quadrants 3 (218 ± 29 vs. 107 ± 17, P=0.0031), thiab 4 (240 ± 16 vs.101 ± 12, P < 0.0001).
Peb tau soj ntsuam ntxiv nyob rau hauv-pab pawg thiab hauv-quadrant TRAPed suav qhov ntom ntom ntawm ib cheeb tsam ntawm BLA (Daim duab 2E thiab Cov Lus Ntxiv 2). Qhov tseem ceeb, thaum piv rau rostral sub-region (59 ± 8), quadrant 2 ntawm pawg ntshai muaj siab dua TRAPed ntom nyob rau hauv nruab nrab (245 ± 21, P < 0.0001) thiab caudal (172 ± 23, P=0.0040) sub-region.
Peb tom ntej no tau soj ntsuam kev sib raug zoo ntawm kev coj tus cwj pwm khov thiab qhov loj ntawm TRAPed neuronal ensemble nyob rau hauv tag nrho aBLA, nyob rau hauv nws nruab nrab sub-region, thiab nyob rau hauv quadrants ntawm nruab nrab thiab caudal subregions uas pawg count densities txawv ntawm contextand ntshai-conditioned pab pawg. Tsis muaj kev sib raug zoo tseem ceeb hauv pawg ntshai lossis cov ntsiab lus (Daim duab 2F), qhia tias kev ntshai tus cwj pwm tsis cuam tshuam qhov loj ntawm qhov kev ntshai-kev kawm fosensemble hauv aBLA sub-regions.
Sib sau ua ke, kev tshawb pom hauv daim duab 2 qhia tias kev ntshai ua rau muaj kev cuam tshuam loj dua hauv aBLA ntau dua li qhov xwm txheej. Ensemble neurons feem ntau nyob hauv cheeb tsam mus rau nruab nrab aBLA sub-region, tab sis kev ntshai tus cwj pwm tsis cuam tshuam nrog cov neeg coob coob.
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