Kev tshuaj xyuas ntawm Diurnal Variation Thiab Kev Sib Deev Kev Sib Deev hauv Hippocampal Neurophysiology Thiab Spatial Memory Part 2

Tag nrho-cell thaj-clamp kaw

Tag nrho cov ntaub ntawv tau sau los ntawm coronal hippocampal slicesduring ZT 2-6 (hnub) los yog ZT 13-17 (hmo) ntawm 32 degree hauv ACSF tus qauv uas muaj cov nram qab no (hauv mM): 125 NaCl, 2.5KCl, 1.25 Na2PO4, 2 CaCl2, 1 MgCl2, 25 NaHCO3, thiab 25glucose, npuas nrog 95% O2 / 5% CO2.

Lub coronal hippocampus yog thaj chaw tseem ceeb ntawm lub hlwb uas ua lub luag haujlwm tseem ceeb hauv tib neeg kev nco.

Kev tshawb fawb qhia tau hais tias lub coronal hippocampus yog qhov tseem ceeb rau kev khaws cia thiab khaws cov kev nco mus ntev. Nws yog qhov uas tib neeg hu ua "tus neeg ncig tebchaws" uas tuaj yeem coj tib neeg rov qab mus rau qhov kev paub dhau los thiab kev paub dhau los. Lub coronal hippocampus plays lub luag haujlwm tseem ceeb hauv peb tus kheej, tswj kev xav, kev nkag siab tus kheej, thiab kev sib raug zoo ntawm tus kheej.

Peb lub cim xeeb tsis zoo nyob rau lub sijhawm, tab sis muaj ob peb yam uas peb tuaj yeem ua tau los txhim kho peb lub cim xeeb. Ua ntej tshaj plaws, nws yog ib qho tseem ceeb kom muaj tus cwj pwm zoo vim qhov no tuaj yeem txo kev ntxhov siab thiab txhim kho kev nco. Qhov thib ob, kev tawm dag zog ntau dua thiab noj zaub mov tuaj yeem pab txhawb kev ua haujlwm ntawm lub plawv hippocampus thiab muaj txiaj ntsig zoo rau lub cev noj qab haus huv. Thaum kawg, kev cob qhia nco thiab siv cov cuab yeej xws li notepads kuj tuaj yeem pab txhim kho kev nco.

Tag nrho hauv tag nrho, lub coronal hippocampus plays lub luag haujlwm hloov tsis tau hauv tib neeg kev nco thiab kev txawj ntse. Yog li ntawd, peb yuav tsum tau xyuam xim rau nws lub luag haujlwm thiab ua kom nws nyob hauv lub xeev noj qab haus huv ntau li ntau tau. Los ntawm kev ua cov kauj ruam ua tau zoo, peb tuaj yeem txhim kho peb lub cim xeeb thiab ua kom nws tsis zoo kom peb thiaj li xav tau kom meej meej thiab ua lub neej muaj txiaj ntsig. 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 paub txoj hauv kev los txhim kho lub hlwb

Tag nrho-cellpatch-clamp kaw ntawm CA1 pyramidal neurons tau txais los ntawm kev siv cov txheej txheem dig muag. Luv luv, patchpipettes tau muab tso rau ntawm qhov nruab nrab lossis sab nraud ntawm thaj chaw CA1 (nyob ntawm seb ib daim ntawv yog los ntawm sab laug lossis sab xis hemisphere) ntawm qhov tob ntawm; 50-150 mM, qhov zoo siab tau siv raws li cov pipette tau maj mam nce los ntawm medially lossis tom qab los ntawm lub pyramidal cell txheej kom txog rau thaum muaj kev nce sai hauv pipette kuj qhia tau hais tias kev sib cuag nrog ib tug neuron, nyob rau hauv lub point zoo siab raug tso tawm, ib tug nruj seal (.1 GX) tau txais, thiab slightnegative siab tau thov mus cuag tag nrho-cell patchconfiguration.

Cov ntaub ntawv tau txais los ntawm kev siv Multiclamp700B amplifier, Axon Digidata 1440A thiab 1550B digitizer, thiab pClamp10/11 software (Molecular Devices).Patch pipettes (BF150–086; Sutter Instruments) tau rub rau ntawm Sutter Phoriztals (7}} ) rau qhov tsis kam ntawm 2.5 thiab 5 MV. Cells tau dialyzed rau 5 feeb ua ntej kev sim kaw. Cells siv foranalysis muaj kev tiv thaiv, 30 MV uas tsis nce .20% rau lub sijhawm ntawm txhua qhov kev sim 5-min.

Rau kev sim ntsuas hluav taws xob-clamp, tag nrho cov hlwb tau tuav ntawm 70 mV, thiab cov teeb liab raug lim ntawm 5 kHz thiab digitized ntawm 10 kHz. Cov kev sim IPSCs siv cov tshuaj patch pipette uas muaj (hauv mM): 140 CsCl, 10 EGTA, 5 MgCl2, 2 NaATP, 0.3 Na-GTP, 10 HEPES, 0.2% biocytin (pH 7.3, 290mm), thiab 5 QX-314 (sodium channel antagonist) ntxiv rau lub sijhawm siv. IPSCs tau muab tshuaj cais tawm nrog kev da dej ntawm 10 mM NBQX (AMPAR antagonist, HelloBio) thiab 5 mM CPP (NDMAR antagonist, Hello Bio).

EPSCsexperiments siv cov tshuaj patch pipette uas muaj (inmM): 100 CsOH, 100 gluconic acid (50%), 0.6 EGTA, 5MgCl2, 2 Na- ATP*3H2O, 0.3 Na-GTP, 40 HEPES, 7 phosphocreatine, biocytin (0.2%), thiab 5 QX-314 ntxiv thaum siv. EPSCs tau muab tshuaj cais tawm nrog kev da dej ntawm 10 mM gabazine (GABAAR antagonist, Hello Bio).Sib cais sim ntsuas me me IPSC thiab me me EPSCs (mIPSCs / mEPSCs) tau sau tseg raws li saum toj no nrog qhov sib ntxiv ntawm 0.5 mM tetrodotoxin (TTX; voltage-gatedsodium channel. inhibitor, Tocris).

Rau kev sim tam sim no-clamp, cov teeb liab tau lim ntawm 10 kHz thiab digitized ntawm 20 kHz. Patch pipette solutioncontained (in mM): 135 K-Gluconate, 2 MgCl2, 0.1 EGTA,10 HEPES, 4 KCl, 2 Mg-ATP, 0.5 Na-GTP, 10 phosphocreatine, thiab biocytin (0.2%; pH 7.3, 310 mOsm, thiab 2-4 MV).Neuronal excitability raug soj ntsuam los ntawm kev txhaj tshuaj nce qib ntawm depolarizing tam sim no los ntawm so (0-500 pA, D 20 pA) thiab suav cov naj npawb ntawm cov kev ua haujlwm muaj peev xwm raug rho tawm haujlwm thaum muaj 1000 ms tam sim no kauj ruam.

Cov lus teb nqes hav tau txais los ntawm kev suav cov kab sib raug zoo ntawm firingfrequency thiab txhaj tam sim no hla {{0}} rau 400-pAsteps. Qhov siab tshaj plaws uas muaj peev xwm (AP) tua ntau zaus (max) thiab tam sim no uas max tshwm sim (Imax) kuj tau ntsuas. Sag tau ntsuas raws li qhov amplitude (mV) ntawm qhov siab tshaj qhov hluav taws xob los ntawm kev txhaj tshuaj tam sim no hyperpolarizing uas tau ua tiav qhov ruaj khov-xeev daim nyias nyias muaj peev xwm ntawm 90-93 mV.Input resistance (MX) tau ntsuas raws li txoj kab nqes ntawm cov lus teb tam sim no mus rau ib qho ntawm hyperpolarizing tam sim no. txhaj tshuaj (150 rau 0 pA, D 50 pA).

Rheobase tau txhais tias qhov tsawg kawg nkaus tam sim no yuav tsum tau rub tawm ib qho AP. Ib qho APselicited los ntawm rheobase tau siv los txheeb xyuas qhov ua tau zoo (Tables 1, 2). AP amplitude tau txhais tias yog qhov sib txawv ntawm qhov AP qhov pib thiab nws qhov siab tshaj. Qhov chaw pib tau txhais raws li qhov voltage (mV) ntawm qhov AP thawj derivative (dV / dt) tshaj 20 mV / s. AP nce lub sij hawm yog lub sij hawm (ms) rau AP kom ncav cuag 90% ntawm nws qhov siab tshaj plaws amplitudefrom 10% ntawm nws lub ncov. Lub sijhawm lwj yog lub sijhawm nruab nrab ntawm 90% thiab 10% ntawm AP peak amplitude. Ib nrab-dav yog lub sijhawm (ms) nruab nrab ntawm ib nrab amplitudes ntawm qhov nce thiab lwj ntawm AP waveform. Afterhyperpolarization (AHP) yog qhov sib txawv ntawm lub hauv paus thiab qhov feem ntau hyperpolarizedpoint tshwm sim nyob rau hauv 3 ms tom qab AP qhov pib rau ceev-AHP(fAHP) thiab 10-50 ms tom qab AP pib rau nruab nrab-AHP(mAHP).

Peak AP nce thiab poob tau txhais tias yog qhov siab tshaj (DmV/Dms) rau AP nce thiab lwj, raws li.Baseline membrane peev xwm raug xam raws li qhov nruab nrab ntawm 1400-ms cheb thaum lub sij hawm 0-pA kauj ruam. Cov kev sim pib tau ua tiav thaum tsis muaj synaptic blockers los txiav txim siab seb kev sib deev thiab lub sijhawm ntawm hnub ua rau CA1 pyramidal neuron excitability nyob rau hauv lub voj voog tsis zoo. Txhawm rau pib ntsuas qhov cuam tshuam ntawm kev sib kis ntawm synaptic ntawm kev ua kom muaj zog thaum hmo ntuj, kev sib cais, kev sim ua raws li tau ua nyob rau hauv lub xub ntiag ntawm GABAA antagonist, gabazine (10 mM), thiab glutamatergic antagonists, NBQX (10mM) thiab CPP (5 mM) .

Immunohistochemistry

Txhawm rau kom paub meej tias cov cell kaw los ntsuas postsynapticcurrents yog CA1 pyramidal hlwb, txhua lub hlwb tau ntim nrog biocytin tsawg kawg 20 min. Slices uas muaj cov cell puv tau kho nyob rau hauv 4% paraformaldehyde rau tsawg kawg 24 h, ces ntxuav rau 3 10 min hauv PBS, thiab incubated rau 2-3h ntawm RT nyob rau hauv ib tug TBS tov muaj 10% NDS, 3% BSA, 1% glycine, 0.4 % Triton X-100, thiab streptavidin-488(1:1000).

Cov hlais raug ntxuav rau 3 10 min hauv PBSand mounted rau iav slides thiab npog nrog ProLongGold Antifade mounting media uas muaj DAPI. Slideswere pom ntawm BZ-X700 fluorescence microscope (Keyence). Txhua lub hlwb uas tsis tuaj yeem muab cais ua CA1pyramidal hlwb raws li qhov chaw thiab morphology raug tshem tawm los ntawm kev tshuaj xyuas.

Kev txheeb xyuas thiab txheeb cais
Cov ntaub ntawv raug txheeb xyuas thiab pom tau siv SPSS (version27/28) thiab Prism-GraphPad software. Cov kev xav ntawm cov kev ntsuas parametric, suav nrog qhov qub thiab homogeneity ntawm qhov sib txawv tau raug soj ntsuam, thiab yog tias ua txhaum, cov ntaub ntawv tau hloov pauv, lossis cov kev ntsuas tsis sib xws tau siv. Txwv tsis pub hais, qhov tseem ceeb tau hais nyob rau ntawm p, 0.05.Ib qho ntsiab lus ntawm txhua qhov kev ntsuam xyuas muaj nyob rau hauv ExtendedData Table 1-1.

Yam khoom qhov chaw nco

OLM cov ntaub ntawv tau txheeb xyuas los ntawm kev ywj pheej ntawm ob txoj kev ANOVA nrog lub sijhawm-ntawm-hnub thiab poj niam txiv neej raws li kev ywj pheej thiab kev ntxub ntxaug raws li qhov sib txawv ntawm qhov sib txawv (Fig. 1D). Ib qho kev sib raug zoo ntawm Pearson tau siv los ntsuas qhov kev sib raug zoo ntawm tag nrho cov sij hawm tshawb nrhiav thiab cov qhab nia ntawm kev ntxub ntxaug thiab kev tshawb xyuas qhov xwm txheej tau siv los txiav txim siab qhov kev faib tawm ntawm cov sij hawm tshawb nrhiav qis dua thoob plaws poj niam txiv neej thiab lub sij hawm ntawm hnub (ExtendedData Fig. 1-1B) .

Cov ntaub ntawv hauv teb

Cov ntaub ntawv tawm tswv yim-tso tawm tau raug tshuaj xyuas siv cov qauv sib xyaw ua ke nrog fEPSP txoj kab nqes raws li kev ua haujlwm ntawm Lub Sijhawm-ntawm-hnub, Kev Sib Deev, thiab Kev Tawm Tsam Tawm Tsam. Rau cov kev sim LTP, cov nqes fEPSP tau ua raws li cov lus teb hauv paus, thiab cov lus teb tau txais nyob rau hauv 10 feeb kawg ntawm 40-minutepost-HFS lub sij hawm kaw tau raug tshuaj xyuas siv peb txoj kev ANOVA nrog rov ntsuas dua (RM-ANOVA).

Tag nrho-cell thaj-clamp electrophysiology

Postsynaptic tam sim no (inhibitory thiab excitatory) tau kuaj pom tsis siv neeg los ntawm 5-feeb kaw lus sivpClamp qhov kev tshwm sim template tshawb nrhiav thiab tom qab ntawd manually tshuaj xyuas qhov kev tshawb pom tsis tseeb. Lub amplitudes thiab intereventinterval (IEI) tau soj ntsuam siv qhov kev kwv yees dav dav (GEE) uas tso cai rau kev kwv yees kwv yees nrog cov pej xeem-nruab nrab cov qauv thaum coj mus rau hauv kev sib raug zoo ntawm kev ntsuas rov qab hauv cov ntsiab lus (Reed thiab Kaas, 2010; Cook et al., 2016).

Cov qauv GEE tau qhia txog kev ua haujlwm tsis sib xws ntawm matrixstructure, cov ntsiab lus ntawm lub xov tooj ntawm tes, thiab qhov cuam tshuam hauv cov ntsiab lus ntawm cov xwm txheej postsynaptic. Cov ntaub ntawv nyoos tau muaj qhov cuam tshuam zoo nrog qhov muaj nuj nqis heev thiab yog li, raug txiav tawm ntawm sab sauv thiab sab qis (10%) ua raws li los ntawm kev hloov pauv hauv cov ntaub ntawv ntawm amplitudedata lossis lub cav 1 1 hloov pauv hauv rooj plaub ntawm IEI cov ntaub ntawv , tomeet assumptions ntawm ib txwm faib ua ntej kev soj ntsuam.

Tag nrho cov ntaub ntawv tam sim no tau txheeb xyuas nrog EasyElectrophysiology (Easy Electrophysiology, RRID: SCR_021190), ib pob software uas siv Neo (Garcia etal., 2014). Cov peev xwm ua tau raug suav nrog siv Action Potential Counting module nrog lub neej ntawd, AutoThreshold Spike algorithm. Ib qho RM-ANOVA tau siv los txheeb xyuas qhov muaj peev xwm ua tau nyob thoob plaws cov kauj ruam tam sim no uas cov ntaub ntawv tsis ua txhaum cov kev xav ntawm linearity thiab normality: 160-400 pA. Tag nrho lwm cov khoom siv membrane (Tables 1, 2) tau soj ntsuam siv ob txoj kev ANOVA nrog kev ywj pheej hloov pauv ntawm lub sijhawm nruab hnub thiab kev sib deev. Tag nrho cov ntaub ntawv tam sim no-clamp raug stratified los ntawm txoj hauj lwm nyob rau hauv lub anterior-posterior axis ua ntej kawg statistical tsom xam.

Cov txiaj ntsig

Qhov sib txawv nruab hnub hmo ntuj hauv OLM kev ua tau zoo nyob ntawm kev sib deev

Txhawm rau tshuaj xyuas qhov cuam tshuam ntawm kev sib deev ntawm circadian rhythms oflearning thiab nco, peb siv qhov khoom qhov chaw nco (OLM) kev soj ntsuam, uas tso siab rau tus nas txoj kev nyiam los tshawb txog cov khoom hauv qhov chaw tshiab, ntsuas hippocampalspatial nco (Barker thiab Warburton, 2011; Takahashiet al. ., 2013; Chao et al., 2016). Txawm hais tias circadian thiab diurnal sib txawv hauv kev ua tau zoo ntawm OLM tau raug tshaj tawm (Takahashi li al., 2013; Snider et al., 2016), qhov cuam tshuam ntawm kev sib deev ntawm kev sib txawv ntawm lub sijhawm ntev hauv OLM kev ua tau zoo tseem to taub tsis zoo.

Peb pom tias OLM kev ua tau zoo sib txawv nyob rau lub sijhawm nruab hnub; Txawm li cas los xij, tus qauv ntawm qhov sib txawv ntawm qhov sib txawv hauv kev ua tau zoo sib txawv ntawm kev sib deev (p=0.023, ob txoj kev ANOVA kev sib cuam tshuam).

Thaum cov txiv neej ua tau zoo dua thaum hmo ntuj piv nrog rau nruab hnub, raws li xav tau (p {{0}}}.028, cov teebmeem tseem ceeb piv rau nruab hnub thiab hmo ntuj hauv cov txiv neej; Fig. 1D), poj niam nas ua tau zoo dua thaum nruab hnub piv nrog hmo ntuj (p=0.004, yooj yim lub ntsiab cuam tshuam piv hnub vs hmo ntuj hauv poj niam; Daim duab 1D). Tsis muaj kev cuam tshuam ntawm lub sijhawm-ntawm-hnub lossis poj niam txiv neej rau lub sijhawm tshawb nrhiav tag nrho (p=0.926 thiab 0.936, ob-wayANOVA cov teebmeem tseem ceeb; Extended Data Fig. 1-1A). Tsis muaj kev sib raug zoo ntawm tag nrho cov kev tshawb nrhiav thiab DIscores (r(52)=0.053, ns p=0.704, Pearson's correlation; Extended Data Fig. 1-1B).

LTP magnitude thaum hmo ntuj yog ntau dua nruab hnub, tsis hais poj niam txiv neej
Long-term potentiation (LTP) yog suav hais tias yog cellularcorrelate ntawm kev kawm thiab nco. LTP ntawm CA3-CA1 synapses siab dua thaum hmo ntuj piv nrog hnub hauv txiv neej (Chaudhury li al., 2005; Besing et al., 2017; Davis etal., 2020), tab sis rau peb txoj kev paub, tsis muaj luam tawm cov lus ceeb toom ntawm qhov cuam tshuam ntawm lub sijhawm-ntawm-hnub ntawm LTP qhov loj ntawm cov poj niam nas. Muab peb qhov kev tshawb pom tias qhov sib txawv diurnal hauv kev ua tau zoo ntawm hippocampal-dependentmemory assay yog nyob ntawm poj niam txiv neej, peb nrhiav tom ntej los txiav txim seb kev sib deev puas cuam tshuam rau diurnal sib txawv hauv LTP.

Ua ntej, txhawm rau ntsuas lub zog ntawm basal synaptic kis tau tus mob ntawm CA{{0}}CA1 synapses, peb tsim I/O nkhaus los ntawm kev ntsuas qhov fEPSP txoj kab nqes los ntawm CA1 stratum radiatum insponse rau Schaffer collateral stimulation tshaj ib tug series ntawm nce stimulation intensities ( 0.2–200 mA, D 10 mA) thaum nruab hnub thiab hmo ntuj ntawm cov nas txiv neej thiab poj niam (Fig. 2A,B). Txawm hais tias tsis muaj kev sib deev los yog lub sij hawm ntawm-hnub muaj qhov cuam tshuam tseem ceeb ntawm kev sib kis ntawm basal synaptic tshaj qhov stimulusrange kuaj (p=0.552 thiab 0.981, raws li, LMMmain effect), muaj kev sib deev-los ntawm kev siv zog sib deev ( p, 0.001, LMM; Fig. 2A, B).

Cov txiv neej muaj qhov siab tshaj EPSP piv nrog cov poj niam tsuas yog ntawm 180,190, thiab 200mA, tsis hais lub sij hawm-ntawm-hnub (p =0.041, 0.043, thiab 0.035, feem; Cov teebmeem tseem ceeb yooj yim piv rau txiv neej thiab poj niam thoob plaws txhua qhov kev siv zog; Fig. 2A). Zuag qhia tag nrho, cov txiaj ntsig no qhia tau hais tias lub sijhawm-ntawm-hnub tsis cuam tshuam rau kev sib kis ntawm basal synaptic thiab tias kev sib deev cuam tshuam rau cov lus teb tsuas yog ntawm qhov siab tshaj plaws stimulation.

Tom ntej no, peb tau soj ntsuam synaptic plasticity ntawm CA3-CA1synapse los ntawm kev ntsuas LTP hauv cov lus teb rau luv luv, siab zaus stimulation (HFS; Fig. 2C, D). Raws li yav dhau los tau tshaj tawm, qhov loj ntawm LTP tau ntau dua thaum hmo ntuj piv nrog hnub ntawm cov nas txiv neej thiab poj niam (p =0.003, peb-txoj kev RM-ANOVA; Fig. 2C, D); Txawm li cas los xij, tsis muaj qhov cuam tshuam loj ntawm lossis cuam tshuam nrog kev sib deev. Ua ke, cov kev tshawb pom no qhia tias lub sij hawm-ntawm-hnub cuam tshuam rau kev ua kom lub cev muaj zog hauv cov txiv neej thiab poj niam nas, tsis muaj kev cuam tshuam lub zog basal synaptic.

Synaptic inhibition mus rau CA1 pyramidal hlwb thaum nruab hnub yog ntau dua thaum hmo ntuj, tsis hais poj niam txiv neej.

Kev hloov pauv hauv LTP tuaj yeem raug ntaus nqi los ntawm cov txheej txheem synaptic thiab / lossis kev hloov pauv hauv qhov kev xav tau. Yog li ntawd, peb xub nrhiav los txiav txim seb lub sij hawm ntawm hnub thiab kev sib deev inhibitory thiab excitatory synaptic kis mus rau CA1 pyramidal neurons. Txhawm rau tshuaj xyuas cov synaptic inhibitiononto CA1 pyramidal hlwb, peb ntsuas qhov amplitude thiab zaus ntawm spontaneous IPSCs (sIPSCs) siv tag nrho-cell voltage clamp rau txiv neej thiab poj niam nas thaum nruab hnub thiab hmo ntuj (Fig. 3A). Peb pom tias sIPSC cuam tshuam lub sijhawm (IEI) thaum nruab hnub luv dua thaum hmo ntuj, tsis hais poj niam txiv neej (sijhawm-ntawm-hnub: p=0.033, GEE; Fig. 3A, C), qhia qhov zaus ntau dua ntawm inhibitory txheej xwm thaum nruab hnub.

Lub amplitude ntawm sIPSCs thaum nruab hnub yog loj dua thaum hmo ntuj ntawm ob leeg txiv neej thiab poj niam (lub sij hawm-ntawm-hnub: p=0.008, GEE; Fig. 3A, E). Qhov no nce hnub-sijhawm zaus thiab amplitude ntawm sIPSCs qhia muaj zog inhibition ntawm CA1 pyramidal neurons thaum nruab hnub piv nrog hmo ntuj.

Muaj zog synaptic inhibition thaum nruab hnub tuaj yeem tshwm sim los ntawm kev nce hauv presynaptic GABA tso tawm, lossis los ntawm kev nce postsynaptic GABAAR muaj nuj nqi. Txhawm rau kom paub qhov txawv ntawm qhov muaj peev xwm no, peb ntsuas me me IPSCS (mIPSCs) nyob rau hauv lub xub ntiag ntawm voltage-gated sodium channel blocker tetrodotoxin (TTX) nyob rau hauv ob leeg txiv neej thiab poj niam nas thaum nruab hnub thiab hmo ntuj (Fig. 4A). Thaum peb pom tias tsis yog poj niam txiv neej (p=0.392,GEE) lossis lub sijhawm-ntawm-hnub (p=0.760, GEE) muaj cov txiaj ntsig tseem ceeb ntawm mIPSC IEI, cov xwm txheej los ntawm cov txiv neej trendedtoward nthuav tawm ib nruab hnub hmo ntuj sib txawv (p= 0.068, kev sib deev lub sij hawm-ntawm-hnub, GEE; Fig. 3C).

Qhov tsis muaj qhov sib txawv nruab hnub hmo ntuj hauv cov poj niam qhia tau hais tias lub sij hawm-ntawm-hnub cuam tshuam rau sIPSCs yuav raug tsav los ntawm kev ua haujlwm hauv zos interneuron muaj peev xwm tua. Hauv cov txiv neej, qhov nruab nrab ntawm nruab hnub thiab hmo ntuj sib txawv los ntawm; 12 ms (txhais tau tias thiab SEM: txiv neej hnub, 98.24 61.05 ms; txiv neej hmo ntuj, 85.78 6 1.04 ms), tawm tswv yim hais tias muaj peev xwm-yooj yim inhibitory vesicle tso tawm yuav ntau zaus thaum hmo ntuj (Fig. 3C). Thaum peb soj ntsuam mIPSC amplitude, peb poob nthav pom kev sib cuam tshuam tseem ceeb ntawm lub sij hawm-ntawm-hnub thiab poj niam txiv neej (p=0.038,GEE), nrog amplitudes ntawm cov poj niam loj dua li txiv neej thaum nruab hnub (p {{16}) }.006, Wald x2 pairwise kev sib piv; Fig. 4E); Txawm li cas los xij, qhov no;2-pA qhov sib txawv yuav tsis cuam tshuam txog kev lom neeg (poj niam-hnub: 34.68 6 1.01 pA; txiv neej-hnub: 32.67 6 1.02 pA, txhais tau tias 6 SEM) .

Muab ua ke, cov spontaneous thiab me me IPSCdata qhia tias kev ua haujlwm muaj peev xwm-nyob ntawm qhov inhibition, tab sis tsis ua rau lub cev vesicle fusion, mus rau CA1 pyramidalcells ntau dua thaum nruab hnub piv nrog rau hmo ntuj ntawm ob leeg txiv neej thiab poj niam.

Synaptic excitation mus rau CA1 pyramidal hlwb nyob ntawm kev sib deev

Peb tom ntej no xav txiav txim siab seb puas muaj kev sib deev spontaneousexcitatory synaptic input rau CA1 pyramidal neuronswas cuam tshuam los ntawm kev sib deev thiab lub sijhawm ntawm hnub. Ua ntej, peb ntsuas tus kheej EPSCs (sEPSCs) siv tag nrho-cellvoltage-clamp kaw (Fig. 5A). Txawm hais tias tsis muaj qhov cuam tshuam tseem ceeb ntawm lub sijhawm-ntawm-hnub ntawm sEPSC amplitude (Fig. 5E), tsis hais poj niam txiv neej (p=0.371, GEE), statistical sib piv rau lub ntsiab tseem ceeb ntawm lub sij hawm-ntawm- hnub qhia tias sEPSC IEI kaw thaum nruab hnub yuav ntau dua li cov ntaub ntawv kaw thaum hmo ntuj (p=0.052,GEE), qhia txog qhov ntau dua ntawm kev ua yeeb yam thaum hmo ntuj (Fig. 5C). Zuag qhia tag nrho, peb pom tias poj niam muaj ntau qhov kev tawm tswv yim zoo tshaj plaws, nrog cov sEPSCamplitudes loj dua thiab luv dua IEIs piv nrog cov txiv neej (p=0.022 thiab 0.020, raws li, qhov tseem ceeb ntawm kev sib deev, GEE; Fig. 5C, E).

Tom ntej no, peb rov ua cov kev sim no nyob rau hauv lub xub ntiag ntawm voltage-gated sodium channel blocker tetrodotoxin (TTX) thiab ntsuas me me spontaneous excitatory synapticcurrents (mEPSCs) mus rau CA1 pyramidal neurons (Fig. 6A).Amplitude ntawm mEPSCs tsis txawv ntawm kev sib deev los yog lub sij hawm- ntawm-hnub(p= 0.227 thiab p= 0.150, qhov tseem ceeb ntawm kev sib deev thiab lub sij hawm ntawm-hnub, raws li, GEE; Fig. 6E); Txawm li cas los xij, kev hloov pauv nruab hnub hmo ntuj inmEPSC IEIs yog nyob ntawm kev sib deev (p= 0.021, kev sib deev raws sijhawm, GEE; Fig. 6C). Hauv cov txiv neej, mEPSC IEIs tau luv dua thaum hmo ntuj tshaj thaum nruab hnub (p= 0.002, Wald x2pairwise kev sib piv), qhia txog qhov tshwm sim ntau dua thaum hmo ntuj hauv cov txiv neej nas thiab yog li qhov yuav muaj qhov tshwm sim ntawm presynaptic tso tawm; Txawm li cas los xij, tsis muaj qhov sib txawv nruab hnub hmo ntuj hauv cov poj niam (p= 0.765,Wald x2 pairwise kev sib piv; Fig. 6C).

Ua ke, cov txiaj ntsig no qhia tau hais tias qhov kev hloov pauv mus rau yav tsaus ntuj sEPSC zaus (tshwj xeeb tshaj yog cov poj niam) yog qhov muaj peev xwm ua tau. Txawm li cas los xij, nyob rau hauv cov txiv neej, thaiv kev ua haujlwm muaj peev xwm nthuav tawm thaum hmo ntuj nce ntau zaus uas tsis tau pom hauv cov EPSCs.

CA1 pyramidal neurons muaj kev zoo siab dua thaum hmo ntuj

Feem ntau, peb cov kev soj ntsuam pom tau hais tias synaptic inhibition ntau dua thaum hmo ntuj thiab synaptic excitation ntau dua thaum nruab hnub; Yog li, peb xav tau los txiav txim seb qhov kev tawm tsam diurnal variation hauv synaptic excitatory thiab inhibitory input ua rau diurnal variation hauv CA1 pyramidalneuron excitability. Txog rau qhov kawg no, peb patched CA1 pyramidalcells nyob rau hauv tam sim no clamp hom nrog lub voj voog tsis zoo (piv txwv li, tsis muaj synaptic antagonists) thiab tsis muaj clampingcell membrane peev xwm. Peb tau txhaj tshuaj ntau ntxiv ntawm depolarizing tam sim no (0-500 pA, D 20 pA, 1000-ms ntev) rau hauv pyramidal neurons thiab ntsuas tus naj npawb ntawm kev muaj peev xwm elicited.

Cov ntaub ntawv tau sau los ntawm cov neurons thoob plaws hauv anterior-posterior axis ntawm hippocampus. Cov kev tshawb fawb yav dhau los pom pom muaj ntau hom electrophysiological hauv CA1 pyramidal neurons uas nyob ntawm txoj hauj lwm hla axes (Spruston, 2008; Marcelin li al., 2012; Dougherty et al., 2012, 2013; Hönigsperger et al., 2013, 2013; Hönigsperger li al. Malik thiab al., 2016; Milior et al., 2016); Yog li, peb tau xaiv tus account rau qhov tseem ceeb no los ntawm kev faib tag nrho cov neurons ua "anterior" lossis "posterior" raws li cov kab mob ntawm lub cev (Allen Reference Atlas los ntawm https://atlas.brain-map.org/; Fig. 7A).

Thaum peb suav nrog anterior-posterior axisas ib qho tseem ceeb hauv peb qhov qauv ANOVA thawj zaug ntsuas qhov muaj peev xwm ua tau ntau yam hauv cov kauj ruam tam sim no, peb pom tias qhov loj tshaj plaws txhawb nqa yog thaj tsam (p= 0.{11}}05, cov nyhuv tseem ceeb, plaub-txoj kev RM-ANOVA). Tsis tas li ntawd, qhov sib txawv hauv cheeb tsam tseem ceeb tau pom rau kev tawm tsam (A: 64.27 6 1.95 MX, P: 75.90 6 2.47 MX, p, 0.001, peb txoj kev ANOVA), rheobase(A : 146. {23}}.29 pA. P: 386.90 610.82 pA, p= 0.047, peb-txoj kev ANOVA).

Cov kev sib txawv ntawm cov anterior thiab posterior neurons dlhos nrog yav dhau los cov kev tshawb fawb uas pom muaj ntau haiv neeg ntawm dorsal thiab ventral CA1 pyramidal neurons.Thaum peb coronal slice npaj tsis tau tso cai rau peb totruly cais ventral CA1, posterior seem feem ntau yuav muaj xws li ib co ventral CA1 pyramidal wens. pom tias posterior neurons muaj cov khoom zoo sib xws nrog cov ntaub ntawv luam tawm yav dhau los hauv ventralCA1 pyramidal neurons, thaum cov neurons anterior zoo ib yam li dorsal pyramidal neurons (Dougherty li al.,2012; Malik li al., 2016).

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