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Self-assembling neural organelles sawv cev rau kev cog lus hauv vitro platform rau kev ua qauv rau tib neeg txoj kev loj hlob thiab kab mob. Txawm li cas los xij, organoids tsis muaj kev sib txuas uas muaj nyob hauv vivo, uas txwv kev loj hlob thiab tiv thaiv kev sib koom ua ke nrog lwm cov circuits uas tswj tus cwj pwm. Ntawm no peb qhia tau hais tias tib neeg qia cell-derived cortical organoids hloov mus rau hauv somatosensory cortex ntawm neonatal liab qab nas tsim cov hom cell loj uas koom ua ke rau hauv kev hnov ​​​​lus thiab kev txhawb siab ntsig txog circuits. MRI tau nthuav tawm tom qab hloov pauv organoid kev loj hlob nyob rau hauv ob peb kab cell kab thiab tsiaj, thaum ib leeg-ntxhais kev tshawb fawb qhia txog kev loj hlob ntawm corticogenesis thiab qhov tshwm sim ntawm kev ua haujlwm-nyob ntawm qhov kev hloov pauv. Tseeb tiag, transplanted cortical neurons nthuav tawm ntau txoj hauv kev morphological, synaptic, thiab cov ntaub so ntswg sab hauv ntau dua li lawv cov khoom siv hauv vitro, tso cai rau kev tshawb pom ntawm neuronal defects hauv cov neeg mob Timothy's syndrome. Anatomical thiab functional tracing tau qhia tias transplanted organelles tau txais thalamocortical thiab corticocortical inputs, thiab nyob rau hauv vivo recordings ntawm neural kev ua ub no qhia tau hais tias cov inputs muaj peev xwm tsim cov lus teb nyob rau hauv tib neeg lub hlwb. Thaum kawg, cortical organoids txuas axons thoob plaws hauv lub hlwb nas, thiab lawv cov optogenetic activation ua rau muaj nqi zog nrhiav tus cwj pwm. Yog li, transplanted tib neeg cortex neurons paub tab thiab koom nrog tus tswv tsev circuits uas tswj tus cwj pwm. Peb cia siab tias txoj hauv kev no yuav pab txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau txhawm rau strand-theem phenotypes
Txoj kev loj hlob ntawm tib neeg lub paj hlwb yog ib qho zoo kawg li tus kheej-organizing txheej txheem nyob rau hauv uas cov hlwb proliferate, sib txawv, migrate, thiab txuas mus rau lub functional neuronal circuits uas yog ntxiv refined los ntawm sensory kev. Ib qho teeb meem tseem ceeb hauv kev nkag siab txog tib neeg lub hlwb kev loj hlob, tshwj xeeb tshaj yog nyob rau hauv cov ntsiab lus ntawm cov kab mob, yog qhov tsis muaj kev nkag mus rau lub hlwb. Kev tswj tus kheej organelles, suav nrog tib neeg cortex organoids (hCO; tseem hu ua human cortex sphere), tuaj yeem tsim 2,3,4,5,6. Txawm li cas los xij, ntau qhov kev txwv txwv lawv txoj kev siv dav dua kom nkag siab txog kev txhim kho thiab kev ua haujlwm ntawm neural circuits. Tshwj xeeb, nws tsis paub meej tias hCO maturation raug txwv los ntawm qhov tsis muaj qee qhov microenvironmental thiab sensory inputs tam sim no hauv vivo. Tsis tas li ntawd, vim tias hCOs tsis tau koom ua ke rau hauv circuits uas tuaj yeem tsim cov txiaj ntsig kev coj tus cwj pwm, lawv cov txiaj ntsig hauv kev ua qauv ntawm cov caj ces nyuaj thiab kev coj tus cwj pwm tsis zoo hauv neuropsychiatric tam sim no txwv.
Kev hloov pauv ntawm hCO mus rau hauv lub hlwb tsis zoo tuaj yeem kov yeej cov kev txwv no. Cov kev tshawb fawb yav dhau los tau pom tias tib neeg cov neurons hloov mus rau hauv nas cortex muaj peev xwm ciaj sia, ua haujlwm, thiab sib txuas lus nrog nas hlwb7,8,9,10,11,12. Txawm li cas los xij, cov kev sim no feem ntau ua rau cov tsiaj laus, uas tuaj yeem txwv kev sib koom ua ke ntawm synaptic thiab axonal. Ntawm no, peb piav qhia txog kev hloov pauv hloov pauv uas peb hloov pauv 3D hCO tau los ntawm cov hlwb hiPS mus rau hauv thawj somatosensory cortex (S1) ntawm cov nas tiv thaiv kab mob thaum ntxov ntawm kev tsim cov yas. Transplanted hCO (t-hCO) neurons tau dhau los ua kom loj hlob, tau txais thalamocortical thiab cortical-cortical inputs uas elicit sensory teb, thiab txuas ntxiv axonal projections rau hauv nas hlwb los tsav nqi zog nrhiav tus cwj pwm. Kev loj hlob ntxiv ntawm t-hCO tau nthuav tawm cov teeb meem neuronal hauv cov neeg mob Timothy's syndrome (TS), kev mob caj ces hnyav tshwm sim los ntawm kev hloov pauv hauv qhov voltage-sensitive L-type CaV1.2 calcium channel (encoded los ntawm CACNA1C).
Txhawm rau kawm tib neeg cortical neurons hauv circuits hauv vivo, peb stereotactically hloov pauv tsis tu ncua 3D hCO rau hauv S1 ntawm cov nas thaum ntxov postnatal athymic (hnub 3-7 postnatally) (Daim duab 1a thiab nthuav cov ntaub ntawv ntawm Fig. 1a-c). Lub sijhawm no, thalamocortical thiab corticocortical axonal projections tseem tsis tau ua tiav lawv qhov S1 innervation (ref. 13). Yog li, txoj hauv kev no yog tsim los ua kom muaj kev sib koom ua ke ntawm t-hCO thaum txo kev cuam tshuam rau cov kab hluav taws xob endogenous. Txhawm rau pom qhov chaw ntawm t-hCO hauv cov tsiaj muaj sia, peb tau ua T2-hnyav MRI lub hlwb rov tsim kho cov nas 2-3 lub hlis tom qab hloov pauv (Daim duab 1b thiab cov ntaub ntawv txuas ntxiv, Fig. 1d). t-hCO tau yooj yim pom thiab ntsuas qhov ntim ntawm t-hCO tau zoo ib yam li cov kev suav los ntawm cov ntawv taag (Ntxiv Cov Ntaub Ntawv Fig. 1d,e; P> 0.05). t-hCO tau yooj yim pom thiab ntsuas qhov ntim ntawm t-hCO tau zoo ib yam li cov kev suav los ntawm cov ntawv taag (Ntxiv Cov Ntaub Ntawv Fig. 1d,e; P> 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированшер среннерованшер данные, рис. 1d, e; P> 0,05). t-hCO tau yooj yim pom, thiab kev ntsuas volumetric t-hCO zoo ib yam li cov kev suav rau cov seem ruaj khov (cov ntaub ntawv nthuav dav, Fig. 1d, e; P> 0.05).很就易观察到t-hCO，并且t-hCO 0.05).t-hCO, 并且t-hCO t-hCO легко наблюдался, а объемные измерения t-hCO были аналогичны рассчитанным для фиксировановых сраез данные, рис. 1d, e; P> 0,05). t-hCO tau yooj yim pom, thiab qhov ntsuas volumetric t-hCO tau zoo ib yam li cov kev suav rau cov seem ruaj khov (cov ntaub ntawv nthuav dav, Fig. 1d, e; P> 0.05).Peb txiav txim siab t-hCO hauv 81% ntawm cov tsiaj hloov pauv kwv yees li 2 lub hlis tom qab hloov pauv (n = 72 tsiaj; hCO los ntawm 10 hiPS cell kab; hiPS cell kab hauv Cov Lus Ntxiv 1). Ntawm cov no, 87% tau nyob hauv cerebral cortex (Fig. 1c). Los ntawm kev ua serial MRI scans ntawm ntau lub sij hawm cov ntsiab lus nyob rau hauv tib transplanted nas, peb pom ib tug cuaj-fold nce nyob rau hauv t-hCO ntim nyob rau hauv 3 lub hlis (Daim duab 1d thiab nthuav cov ntaub ntawv, Fig. 1f). Cov tsiaj hloov pauv tau muaj sia nyob siab (74%) ntawm 12 lub hlis tom qab hloov pauv (cov ntaub ntawv nthuav dav, daim duab 1g thiab Cov Lus Ntxiv 2), thiab tsis pom lub cev muaj zog lossis kev nco tsis zoo, gliosis, lossis electroencephalogram (EEG) tau pom. Cov ntaub ntawv Fig. 1g thiab cov lus ntxiv 2). 1 h-m 3e).
a, Schematic ntawm kev sim tsim. hCO muab tau los ntawm cov hlwb hiPS tau hloov mus rau hauv S1 ntawm cov nas liab liab qab nyob rau hnub 30-60 ntawm kev sib txawv. b, T2-hnyav coronal thiab kab rov tav MRI cov duab qhia t-hCO hauv S1 2 lub hlis tom qab hloov pauv. Scale bar, 2 mm. c, Quantification of engraftment success rates displayed for each hiPS cell line (n = 108, cov lej nyob rau hauv tuav qhia tus nqi ntawm t-hCO ib hIPS cell kab) thiab cortical los yog subcortical qhov chaw (n = 88). d, MRI duab ntawm cov hlab ntsha coronary (sab laug; scale bar, 3 mm) thiab coj 3D volumetric reconstruction (scale bar, 3 mm) qhia qhov nce ntawm t-hCO tshaj 3 lub hlis. e, Kev tshuaj xyuas ntawm t-hCO cov qauv hauv nas cerebral cortex. Scale bar, 1 mm. f, Tus neeg sawv cev immunocytochemical dluab ntawm t-hCO qhia los ntawm sab laug mus rau sab xis (thaum lub sij hawm sib txawv): PPP1R17 (4 hli), NeuN (8 hli), SOX9 thiab GFAP (8 hli), PDGFRα; (8 lub hlis), MAP2 (8 lub hlis) thiab IBA1 (8 lub hlis). Scale bar, 20 µm. Co-expression ntawm HNA qhia cov hlwb ntawm tib neeg keeb kwm. g, snRNA-seq: Unified manifold thiab projection (UMAP) dimensionality txo cov duab ntawm tag nrho cov high-zoo t-hCO nuclei tom qab Seurat kev koom ua ke (n = 3 t-hCO qauv, n = 2 hiPS cell kab). Astrocytes, hlwb ntawm kab astrocyte; cyc prog, circulating progenitors; GluN DL, sib sib zog nqus glutamatergic neurons; GluN DL / SP, sib sib zog nqus thiab sublamellar glutamatergic neurons; GluN UL, txheej txheej glutamatergic neurons; oligodendrocytes, oligodendrocytes; OPC, oligodendrocyte progenitor hlwb; RELN, reelin neurons. h, Gene Ontology (GO) lub sij hawm ua kom muaj txiaj ntsig zoo ntawm cov noob tau nce siab (hloov P <0.05, hloov pauv> 2, qhia tsawg kawg 10% ntawm nuclei) hauv t-hCO glutamatergic neurons piv nrog hCO glutamatergic neurons. h, Gene Ontology (GO) lub sij hawm ua kom muaj txiaj ntsig zoo ntawm cov noob tau nce siab (hloov P <0.05, hloov pauv> 2, qhia tsawg kawg 10% ntawm nuclei) hauv t-hCO glutamatergic neurons piv nrog hCO glutamatergic neurons. h, Анализ обогащения терминов Gene Ontology (GO) для генов со значительной активацией (скорректированный P <0,05, скорректированный P <0,05, 2, экспрессия по крайней мере в 10% ядер) в глутаматергических нейронах t-hCO по сравнению с глусагм nейронами hCO. h, Gene Ontology (GO) lub sij hawm ua kom muaj txiaj ntsig zoo rau cov noob nrog kev ua kom muaj zog (hloov P <0.05, hloov pauv> 2, qhia tsawg kawg 10% nuclei) hauv t-hCO glutamatergic neurons piv rau hCO glutamatergic neurons. h, 与hCO 谷氨酸能神经元相比, t-hCO 谷氨酸能神经元中基因显着上调（调整后P < 倍> 0.0. 2，在至少10%的细胞核中表达）的基因本体论(GO) 术语富集分析. h ， 与 hco 谷氨酸 能 元 相比 ， t-hco 谷氨酸 能 神经 元 基因 显着 上调（后 变匎 p <0 . 2 ， 至少 10% 的 核中 表达 ) 基因 基因 的 基因 的 本体论 基因 本体论 ( 寐子 本体论 本体论 寐 寐 子 本体论 寐 寐 子 ) 。 h, гены значительно активизировались (скорректированный P <0,05, кратность изменения> 2, экспрессирует скспрессирует 10% ядер) в глутаматергических нейронах t-hCO по сравнению с глутаматергическими нейронами hCO гинтолол термина обогащения. h, cov noob tau raug kho kom zoo (hloov P <0.05, hloov pauv> 2, qhia tsawg kawg 10% ntawm cov nuclei) hauv t-hCO glutamatergic neurons piv rau hCO glutamatergic neurons Ontological (GO) tsom xam ntawm lub sij hawm enrichment.Cov kab dotted qhia aq tus nqi ntawm 0.05. i, UMAP imaging ntawm GluN cell hom nyob rau hauv t-hCO siv daim ntawv hloov chaw los ntawm kev siv 22 snRNA-seq cov neeg laus lub cev muaj zog cortex dataset. CT - corticothalamic cells, ET - cov hlwb extracerebral, IT - internal telencephalic cells, NP - ze rau qhov projection.
Tom qab ntawd peb tau soj ntsuam cytoarchitecture thiab tag nrho cov cellular muaj pes tsawg leeg ntawm t-hCO. Antibody staining ntawm nas endothelial hlwb tau nthuav tawm vascularization nrog t-hCO, thaum IBA1 staining qhia pom cov nas microglia thoob plaws hauv cov graft (Fig. 1f thiab nthuav cov ntaub ntawv, Fig. 3c, d). Immunostaining qhia human nuclear antigen (HNA) zoo hlwb co-expressing PPP1R17 (cortical progenitors), NeuN (neurons), SOX9 thiab GFAP (glial-derived cells) los yog PDGFRα (oligodendrocyte progenitors) (Daim duab 1f). Txhawm rau kawm cov cellular muaj pes tsawg leeg ntawm t-hCO ntawm ib qho kev daws teeb meem ntawm tes, peb tau ua ib leeg-core RNA sequencing (snRNA-seq) tom qab kwv yees li 8 lub hlis ntawm kev sib txawv. Kev pom ntau thiab tshem tawm cov nas nuclei tau txais 21,500 daim duab qhia kev zoo tib neeg mononuclear (Fig. 1g thiab nthuav cov ntaub ntawv, Fig. 4a,b). Cov qauv kev nthuav qhia ntawm cov cim ntawm tes tau txheeb xyuas cov pawg ntawm cov chav kawm loj cortical cell, suav nrog cov kab mob sib sib zog nqus thiab sab nraud glutamatergic neurons, circulating progenitors, oligodendrocytes, thiab astrocyte kab (Fig. 1g, nthuav cov ntaub ntawv, Fig. 4c, thiab Supplementary Table 3). Kev txhaj tshuaj tiv thaiv kab mob rau SATB2 thiab CTIP2 tau pom tias txawm tias muaj cov kab mob cortical subtypes, t-hCO tsis qhia meej anatomical stratification (cov ntaub ntawv nthuav dav, daim duab 3a). theem-matched snRNA-seq hCO tsim cov chav kawm ntawm tes zoo sib xws, nrog rau qee qhov kev zam, suav nrog qhov tsis muaj oligodendrocytes thiab muaj GABAergic neurons, uas tuaj yeem cuam tshuam txog yav dhau los tau tshaj tawm qhov kev pom zoo hauv vitro rau lateral progenitor cells15 (cov ntaub ntawv nthuav dav, Fig. 4f - Table4 thiab Supplementary). Kev txheeb xyuas cov noob sib txawv tau qhia txog qhov sib txawv tseem ceeb hauv glutamatergic neurons ntawm t-hCO thiab hCO (Table 5), suav nrog kev ua kom cov kab mob cuam tshuam nrog kev loj hlob ntawm cov paj hlwb xws li synaptic signaling, dendritic localization, thiab voltage-gated channel activation (Daim duab 1h thiab Supplementary Table 5). tabl 6). Raws li, cortical glutamatergic t-hCO neurons nthuav tawm nrawm nrawm ntawm kev loj hlob.
Txhawm rau txheeb xyuas seb cov kev hloov pauv hauv t-hCO puas muaj feem cuam tshuam rau qhov sib txawv ntawm morphological ntawm hCO hauv vitro thiab t-hCO hauv vivo, peb rov tsim kho theem-matched biocytin-filled hCO thiab hCO hauv cov kab mob hnyav tom qab 7-8 lub hlis ntawm kev sib txawv. hCO neurons (Fig. 2a). t-hCO neurons tau loj dua, muaj 1.5 npaug ntawm txoj kab uas hla ntawm soma, ob zaug ntau dendrites, thiab tag nrho rau-fold nce nyob rau hauv tag nrho dendritic ntev piv rau hauv vitro hCO (Fig. 2b). Tsis tas li ntawd, peb tau pom ib qho siab dua siab dua ntawm cov nqaj qaum hauv T-HCO Neurons dua li hauv HCO Neurons (Daim duab 2C). Qhov no qhia tau hais tias t-hCO neurons tau txais kev nthuav dav dendritic elongation thiab branching, uas, ua ke nrog txuas ntxiv cell proliferation, tuaj yeem ua rau muaj kev loj hlob ntawm t-hCO tom qab hloov pauv (Daim duab 1d thiab Extended Data Fig. 1f). Qhov no ua rau peb tshawb xyuas cov khoom electrophysiological. Membrane capacitance yog yim npaug siab dua (cov ntaub ntawv nthuav dav, Fig. 8d), qhov chaw so ntawm lub xeev membrane muaj peev xwm ntau dua hyperpolarized (kwv yees li 20 mV), thiab kev txhaj tshuaj tam sim no ua rau muaj qhov siab tshaj plaws excitation hauv t-hCO neurons ntau dua li hauv hCO neurons. nyob rau hauv vitro (Fig. 2d), e), uas yog raws li qhov loj thiab complex morphological nta ntawm t-hCO. Tsis tas li ntawd, qhov zaus ntawm spontaneous excitatory postsynaptic tam sim no cov xwm txheej (EPSC) tau nce siab dua hauv t-hCO neurons (Fig. 2f), qhia tias qhov nce ceev ntawm dendritic spines pom hauv t-hCO neurons tau cuam tshuam nrog kev ua haujlwm excitability. kev sib deev synapse. Peb tau lees paub qhov tsis paub qab hau ntawm hCO neurons hauv vitro los ntawm kev sau npe glutamatergic neurons (cov ntaub ntawv nthuav dav, Fig. 6a-c).
a, 3D reconstruction ntawm biocytin-filled hCO thiab t-hCO neurons tom qab 8 lub hlis ntawm kev sib txawv. b, Quantification ntawm morphological nta (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 thiab *** P < 0.0001). b, Quantification ntawm morphological nta (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 thiab *** P < 0.0001). б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084,0 ***P 7 ). b, quantification of morphological nta (n=8 hCO neurons, n=6 t-hCO neurons; **P=0.0084, *P=0.0179, and ***P<0.0001). b, 形态学特征的量化（n = 8 个hCO 神经元，n = 6 个t-hCO 神经元；**P = 0.0084，*P = 0.0170 < 0. b, 形态学特征的量化（n = 8 个hCO 神经元，n = 6 个t-hCO 神经元；**P = 0.0084，*P = 0.0170 < 0. б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084,0 ***P 7 ). b, quantification of morphological nta (n=8 hCO neurons, n=6 t-hCO neurons; **P=0.0084, *P=0.0179, and ***P<0.0001).c, 3D reconstruction ntawm hCO thiab t-hCO dendritic ceg tom qab 8 lub hlis ntawm kev sib txawv. Liab asterisks qhia tias muaj cov kab mob dendritic. Dendritic spine density quantification (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0092). d, Quantification ntawm so membrane muaj peev xwm (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, Quantification ntawm so membrane muaj peev xwm (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, количественная оценка мембранного потенциала покоя (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,001). d, so membrane peev xwm quantification (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, 静息膜电位的量化(n = 25 hCO 神经元, n = 16 t-hCO 神经元；***P < 0.0001). d, 静息膜电位的量化(n = 25 hCO 神经元, n = 16 t-hCO 神经元；***P < 0.0001). d, количественная оценка мембранного потенциала покоя (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,001). d, so membrane peev xwm quantification (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Kev rov ua dua qhov muaj peev xwm tua hluav taws hauv hCO thiab t-hCO raug ntxias los ntawm kev txhaj tshuaj tam sim no, thiab kom muaj nuj nqis ntawm qhov kev tua hluav taws ntau tshaj plaws (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P < 0.0001). e, Kev rov ua dua qhov muaj peev xwm tua hluav taws hauv hCO thiab t-hCO raug ntxias los ntawm kev txhaj tshuaj tam sim no, thiab kom muaj nuj nqis ntawm qhov kev tua hluav taws ntau tshaj plaws (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P < 0.0001). e, повторное возбуждение потенциала действия в hCO и t-hCO, вызванное увеличением тока, и количонественная скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, kev ua kom muaj peev xwm rov tua hluav taws hauv hCO thiab t-hCO raug ntxias los ntawm kev nce tam sim no thiab qhov ntau ntawm qhov siab tshaj tawm hluav taws xob (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P < 0.0001). e, 通过增加电流注入诱导的hCO 和t-hCO 重复动作电位放电，以及最大放电率的量化 5 CO (n =神经元, n = 16 个t-hCO 神经元；***P < 0.0001). E, 通过增加 电流注入的的 hco 和 t-hco 重复 电位 放电，以及最大的量化（n = h 25 co个 t-hco 神经 ； *** p <0.0001). e, повторяющееся возбуждение потенциала действия hCO и t-hCO, вызванное увеличением подачи тока, и кятаце максимальной скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, rov rov ua dua ntawm hCO thiab t-hCO kev ua haujlwm muaj peev xwm tshwm sim los ntawm kev nce ntxiv tam sim no thiab kom muaj nuj nqis ntawm kev tua hluav taws ntau (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P < 0.0001). f, Spontaneous EPSCs (sEPSCs) hauv hCO thiab t-hCO neurons ntawm 8 lub hlis ntawm kev sib txawv, thiab kom muaj nuj nqis ntawm qhov zaus ntawm cov xwm txheej synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P < 0.0001). f, Spontaneous EPSCs (sEPSCs) hauv hCO thiab t-hCO neurons ntawm 8 lub hlis ntawm kev sib txawv, thiab kom muaj nuj nqis ntawm qhov zaus ntawm cov xwm txheej synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P < 0.0001). f, спонтанные EPSC (sEPSC) в нейронах hCO и t-hCO через 8 месяцев дифференцировки и количесоттвенная оциченка оциченка событий (n = 25 нейронов hCO, n = 17 нейронов t-hCO; *** P <0,0001). f, Spontaneous EPSCs (sEPSCs) nyob rau hauv hCO thiab t-hCO neurons ntawm 8 lub hlis ntawm kev sib txawv thiab kom muaj nuj nqis ntawm cov xwm txheej synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P<0.0001). f, 分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs (sEPSCs), 以及突触事事件频率的量化 hCO (n = 2 率的量化) t-hCO 神经元；***P <0.0001). f, 分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs (sEPSCs), 以及突触事件频率的量匼的 hCO n = 2 = 25 hCO 神率的量匼（n = 25hCO P < 0.0001） . f, спонтанные EPSC (sEPSC) в нейронах hCO и t-hCO через 8 месяцев дифференцировки и количесоттвенная оциченка оциченка событий(n = 25 нейронов hCO, n = 17 нейронов t-hCO; *** P <0,0001). f, Spontaneous EPSCs (sEPSCs) nyob rau hauv hCO thiab t-hCO neurons ntawm 8 lub hlis ntawm kev sib txawv thiab kom muaj nuj nqis ntawm cov xwm txheej synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P<0.0001).Rau bf, hCO thiab t-hCO nyob rau hauv kab 1208-2 raug coj los ntawm tib qhov sib txawv batch tswj nyob rau hauv parallel. g, Gene teem enrichment tsom xam (ib-sab Fisher qhov tseeb kuaj) ntawm cov noob ho nce kev tswj hwm (hloov P < 0.05, fold hloov> 2, qhia tsawg kawg 10% ntawm nuclei) hauv t-hCO glutamatergic neurons piv nrog hCO glutamatergic neurons nrog ponrese (Genes thaum ntxov) (LRG) kev ua haujlwm-nyob ntawm cov noob txheeb xyuas los ntawm kev tshawb fawb hauv vivo nas16 thiab tib neeg tshwj xeeb LRGs los ntawm hauv vitro neurons17. g, Gene teem enrichment tsom xam (ib-sab Fisher qhov tseeb kuaj) ntawm cov noob ho nce kev tswj hwm (hloov P < 0.05, fold hloov> 2, qhia tsawg kawg 10% ntawm nuclei) hauv t-hCO glutamatergic neurons piv nrog hCO glutamatergic neurons nrog ponrese (Genes thaum ntxov) (LRG) kev ua haujlwm-nyob ntawm cov noob txheeb xyuas los ntawm kev tshawb fawb hauv vivo nas16 thiab tib neeg tshwj xeeb LRGs los ntawm hauv vitro neurons17. g, анализ обогащения набора генов (односторонний точный критерий Фишера) генов со значительной активацией активацией <0,05, кратность изменения > 2, экспрессия по меньшей мере в 10% ядер) в глутаматергических нейрона в глутаматергических нейрона хнейрона глутаматергическими нейронами hCO наборы генов как раннего (ERG), так и позднего (LRG) генов, зависящих от ависящих от ависящих идентифицированных в исследовании на мышах in vivo16, и специфических для человека LRG из нейронов in vitro17. g, tsom xam cov noob txheej enrichment (ib-tailed Fisher qhov tseeb kuaj) ntawm cov noob nrog kev ua kom tseem ceeb (hloov P <0.05, hloov pauv> 2, qhia tsawg kawg yog 10% ntawm nuclei) hauv t-hCO glutamatergic neurons piv rau hCO glutamatergic neurons teeb tsa ntawm ob qho tib si thaum ntxov (ERD) pom nyob rau hauv vivo nas16 thiab tib neeg tshwj xeeb LRGs los ntawm cov neurons hauv vitro17. g,t-hCO谷氨酸能神经元与hCO谷氨酸能神经元相比，t -hCO谷氨酸能神经元显着上调（调整后P<0.05，倍数变化> 2, nyob rau hauv 10%的细胞核中表达）的基因集富集分析（单侧F Isher 精确检验) Cov ntaub ntawv ERG和晚期反应(LRG) 活性依赖性基因的基因组16 和体外神经元17 中的人类特异性LRG. g ， t-hco 谷氨酸 神经 元 与 hco 谷氨酸 神经 元 相比 ， t-hco 谷氨酸 神经 元 <0 上尃倍数> 2 ， 至少 至少 10%的细胞 核中 表达）的集富集分析（单侧 fisher 精确）丽富集分析（的单侧 fisher 精确）丽魔分析早期 反应 反应 和 晚期 反应 反应 (lrg) 活性基因的 基因组 16 和 神经中 17 中 17的人类特异性LRG. g, глутаматергические нейроны t-hCO были значительно активизированы по сравнению с глутаоматергический (скорректированный P<0,05, кратность изменения> 2, не менее 10% Анализ обогащения набора генов (одностйтонте Фишера) раннего ответа (ERG) и позднего гены, зависящие от активности ответа (LRG), идентифицировванныяша in vivo 16 thiab нейронах in vitro17. g, t-hCO glutamatergic neurons tau raug kho kom zoo dua piv rau hCO glutamatergic neurons (hloov P<0.05, hloov pauv> 2, yam tsawg kawg 10% Cov Lus Teb Thaum Ntxov (ERG) thiab lig teb gene enrichment tsom xam (ib-tailed Fisher's pes tsawg xeem) cov lus teb kev ua ub no nyob rau hauv mR 6 genes thiab voids (L. vitro neurons.17 Tib neeg tshwj xeeb LRGs.Cov kab dotted qhia txog Bonferroni-kho P tus nqi ntawm 0.05. h, GluN gene qhia (pseudo-pob thiab scaling ntawm txhua tus noob) tau nce qib hauv snRNA-seq replicas ntawm LRG noob hauv t-hCO glutamatergic neurons. i, immunostaining qhia SCG2 qhia hauv t-hCO (sab sauv) thiab hCO (qis) neurons. Cov xub dawb taw qhia rau SCG2+ hlwb. Scale bar, 25 µm. Cov ntaub ntawv yog qhia raws li txhais tau tias ± tus qauv sib txawv.
Raws li kev ua haujlwm nce ntxiv ntawm t-hCO pom hauv ex vivo hlais, snRNA-seq tau qhia txog kev ua haujlwm-raws li kev tswj hwm ntawm cov gene transcripts hauv t-hCO piv rau hCO hauv vitro. Glutamatergic t-hCO neurons qhia ntau dua ntawm cov noob tswj cov lus teb lig (Fig. 2g, h), uas tau pom nyob rau hauv cov kev tshawb fawb yav dhau los hauv nas thiab tib neeg neurons16,17. Piv txwv li, BDNF18, SCG2, thiab OSTN, primate-specific activating-regulating gene, pom tau hais tias muaj zog nyob rau hauv t-hCO neurons piv rau hCO neurons (Fig. 2g-i). Yog li, t-hCO neurons tau nthuav tawm cov yam ntxwv ntawm kev loj hlob zoo dua piv rau hCO neurons los ntawm kev hloov pauv, morphological, thiab kev ua haujlwm.
Txhawm rau ntsuas kev sib koom ua ke ntawm t-hCO maturation nrog tib neeg lub hlwb kev loj hlob, peb tau ua transcriptomic piv ntawm fetal thiab cov neeg laus cortical cell type19,20 thiab adult21,22 nrog rau cov ntaub ntawv dav dav ntawm cortical gene expression23 thaum lub sij hawm kev loj hlob (cov ntaub ntawv nthuav dav, Fig. 5). ). nrog rau yav dhau los ua hauj lwm 24, lub ntiaj teb no hCO thiab t-hCO t-hCO transcriptome maturation raws li txoj cai nyob rau hauv 7-8 lub hlis ntawm kev sib txawv yog broad raws li nyob rau hauv vivo txoj kev loj hlob lub sij hawm thiab yog feem ntau sib npaug rau fetal lub neej lig (Ntxiv cov ntaub ntawv Fig. 5a). Qhov tseem ceeb, peb tau pom qhov kev loj hlob ntawm kev loj hlob hauv t-hCO piv rau hnub nyoog sib txuam hCO, nrog rau kev ua kom cov ntaub ntawv cuam tshuam nrog synaptogenesis, astrogenesis, thiab myelination (cov ntaub ntawv nthuav dav, Fig. 5b-d). Nyob rau theem cellular, peb pom cov pov thawj ntawm thinner cortex subtypes hauv t-hCO, nrog pawg ntawm glutamatergic neurons overlapping nrog cov neeg laus L2/3, L5, thiab L6 neuron subtypes (Daim duab 1i). Hauv qhov sib piv, pawg sib tshooj ntawm glutamatergic t-hCO neurons thiab fetal cortical neurons tau txwv ntau dua hauv nruab nrab cev xeeb tub (cov ntaub ntawv nthuav dav, Daim duab 5e-j). Txhawm rau txiav txim siab seb t-hCO neurons puas ua haujlwm zoo ib yam li tib neeg postnatal neocortical neurons, peb tau ua electrophysiological recordings thiab anatomical reconstructions ntawm tib neeg L2/3 pyramidal neurons nyob rau hauv ntse seem ntawm tib neeg postnatal cortex (cov ntaub ntawv nthuav, Fig. 7a). Cov khoom ntawm electrophysiological ntawm L2/3 pyramidal neurons zoo ib yam li cov t-hCO pyramidal neurons (cov ntaub ntawv nthuav dav, Fig. 7e). Morphologically, L2 / 3 Neurons ntawm cov qauv Postnatal tau zoo ib yam li rau HCO, thiab muaj ntau cov ceg tawv taub hau, thiab muaj cov ntaub ntawv ntau dua (Daim duab, Daim Duab 7B-). G).
a, kev hloov pauv ntawm hCO uas tsim los ntawm kev tswj hwm thiab TS hiPS cell kab rau hauv cov nas me me. b, 3D reconstruction ntawm biocytin-filled t-hCO neurons tom qab 8 lub hlis ntawm kev sib txawv. c, quantification of mean dendritic length (n = 19 control neurons, n = 21 TS neurons; **P = 0.0041). d, 3D-reconstructed dendritic ceg los ntawm kev tswj thiab TS t-hCO ntawm 8 lub hlis ntawm kev sib txawv, thiab kom muaj nuj nqis ntawm dendritic spine ceev (n = 16 tswj neurons, n = 21 TS neurons, ***P <0.0001). d, 3D-reconstructed dendritic ceg los ntawm kev tswj thiab TS t-hCO ntawm 8 lub hlis ntawm kev sib txawv, thiab kom muaj nuj nqis ntawm dendritic spine ceev (n = 16 tswj neurons, n = 21 TS neurons, ***P <0.0001). d, 3D-реконструкция дендритных ветвей из контроля и TS t-hCO через 8 месяцев диффференцировки ка и колиез плотности дендритных шипов(n=16 контрольных нейронов, n=21 TS нейронов, ***P <0,0001). d, 3D reconstruction ntawm dendritic ceg los ntawm kev tswj thiab t-hCO TS ntawm 8 lub hlis ntawm kev sib txawv thiab dendritic spine density quantification (n=16 tswj neurons, n=21 TS neurons, ***P<0.0001). d, 分化8 个月时对照和TS t-hCO 的 3D 重建树突分支，以及树突棘密度的量化（n = 16 珸珊珊树突棘密度的量化） 21 个TS 神经元,***P < 0.0001). d ，分化 8 个时对照和 ts t-hco 的 3d 重建 分支 以及 树突棘 密度 重建 分支 以及 树突棘 密度 量化 量化元, n = 21 个 ts 神经, *** p <0.0001). d, 3D-реконструкция дендритных ветвей контроля и TS t-hCO через 8 месяцев дифференцировоки и нкаволичест плотности дендритных шипов(n=16 контрольных нейронов, n=21 TS нейронов, ***P <0,0001). d, 3D reconstruction of control dendritic branch and TS t-hCO at 8 months of differentiation and dendritic spine density quantification (n=16 tswj neurons, n=21 TS neurons, ***P<0.0001).Liab asterisks qhia tias muaj cov kab mob dendritic. e, tus kheej EPSCs hauv kev tswj hwm thiab TS t-hCO neurons tom qab 8 lub hlis ntawm kev sib txawv. f, cumulative zaus zajlus thiab kom muaj nuj nqis ntawm zaus thiab amplitude ntawm synaptic txheej xwm (n = 32 tswj neurons, n = 26 TS neurons; ** P = 0.0076 thiab P = 0.8102). g, Scholl tsom xam ntawm TS thiab tswj cov neurons hauv hCO thiab t-hCO. Dashed kab qhia tib neeg L2/3 postnatal pyramidal neurons rau kev sib piv (n = 24 tswj t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 tswj hCO neurons, thiab n = 7 TS hCO neurons). Cov ntaub ntawv raug qhia raws li qhov nruab nrab ± tus qauv sib txawv
Lub peev xwm ntawm t-hCO kom rov ua dua cov morphological thiab kev ua haujlwm ntawm tib neeg cortex neurons nyob rau theem siab ua rau peb tshawb nrhiav seb t-hCO puas tuaj yeem siv los kuaj cov kab mob phenotypes. Peb tsom mus rau TS, ib qho teeb meem loj ntawm kev loj hlob ntawm neurodevelopmental tshwm sim los ntawm kev nce-ntawm-function mutations hauv cov noob encoding CaV1.2, uas pib ua haujlwm-dependent gene transcription hauv neurons. Peb tau txais hCO los ntawm peb tus neeg mob TS nqa qhov kev hloov pauv ntau tshaj plaws (p.G406R) thiab peb tswj (Fig. 3a). Tom qab hloov pauv, peb pom tias dendritic morphology tau hloov pauv hauv TS neurons piv rau kev tswj hwm (Daim duab 3b thiab nthuav dav cov ntaub ntawv, Fig. 8a, b), nrog rau ob npaug ntxiv ntawm cov thawj dendrites thiab tag nrho qhov nce ntawm qhov nruab nrab thiab tag nrho cov dendritic ntev (Daim duab 3c thiab cov ntaub ntawv txuas ntxiv, Fig. 8c). Qhov no tau cuam tshuam nrog kev nce ceev ntawm cov txha nraub qaum thiab nce zaus ntawm EPSCs spontaneous hauv TS piv rau kev tswj cov neurons (Fig. 3d-f thiab nthuav cov ntaub ntawv, Fig. 8g). Kev soj ntsuam ntxiv tau nthuav tawm cov qauv ntawm qhov txawv txav dendritic branching hauv t-hCO TS piv rau kev tswj hwm, tab sis tsis nyob rau hauv vitro TS hCO nyob rau theem zoo sib xws (Fig. 3g). Qhov no yog ua raws li peb cov ntawv tshaj tawm dhau los ntawm kev ua haujlwm-dendritic shrinkage hauv TS thiab qhia txog lub peev xwm ntawm qhov kev hloov pauv no txhawm rau txheeb xyuas cov kab mob phenotypes hauv vivo.
Peb mam li nug txog seb t-hCO hlwb puas ua haujlwm rau hauv nas S1. S1 nyob rau hauv nas tau txais cov synaptic inputs muaj zog los ntawm ipsilateral ventral basal thiab posterior thalamic nuclei, nrog rau cov ipsilateral motor thiab secondary somatosensory cortices, thiab contralateral S1 (Fig. 4a). Txhawm rau kho cov qauv innervation, peb kis hCO nrog tus kab mob rabies-dG-GFP/AAV-G thiab hloov hCO rau hauv S1 nas 3 hnub tom qab. Peb pom cov GFP ntom ntom hauv cov neurons ntawm ipsilateral S1 thiab ventral basal ganglia 7-14 hnub tom qab hloov pauv (Fig. 4b, c). Tsis tas li ntawd, cov tshuaj tiv thaiv kab mob staining ntawm thalamic marker netrin G1 qhia pom muaj thalamic endings hauv t-hCO (Fig. 4d, e). Txhawm rau ntsuas seb qhov kev kwv yees afferent no tuaj yeem cuam tshuam cov lus teb synaptic hauv t-hCO hlwb, peb tau ua tag nrho cov xov tooj ntawm tes kaw los ntawm tib neeg lub hlwb hauv cov ntu ntse ntawm thalamocortical txheej. Hluav taws xob stimulation ntawm nas S1, sab hauv capsule, dawb teeb meem, fibers ze t-hCO los yog optogenetic activation ntawm opsin-expressing thalamic xaus nyob rau hauv t-hCO induced luv-latency EPSCs nyob rau hauv t-hCO neurons raug rau AMPA receptor antagonist NBQX. (Fig. 4f, g thiab txuas ntxiv cov ntaub ntawv, Fig. 9a–g). Cov ntaub ntawv no qhia tau hais tias t-hCO yog anatomically koom ua ke rau hauv nas hlwb thiab muaj peev xwm ua tau los ntawm nas cov ntaub so ntswg.
a, Schematic daim duab ntawm rabies nrhiav kev sim. b, GFP thiab tib neeg tshwj xeeb STEM121 qhia ntawm t-hCO thiab nas cerebral cortex (saum vaj huam sib luag). Kuj pom yog GFP qhia nyob rau hauv nas ipsilateral ventral basal nucleus (VB) (sab laug sab) thiab ipsilateral S1 (tso cai). Scale bar, 50 µm. Cov squares liab sawv cev rau thaj chaw ntawm lub paj hlwb uas cov duab raug coj mus. c, kom muaj nuj nqis ntawm cov hlwb qhia GFP (n = 4 nas). d, e — Netrin G1+ thalamic terminals hauv t-hCO. d qhia ib coronal seem uas muaj t-hCO thiab VB nuclei. Scale bar, 2 mm. e qhia Netrin G1 thiab STEM121 qhia hauv t-hCO (sab laug) thiab VB (txoj cai) neurons. Scale bar, 50 µm. Lub txiv kab ntxwv dotted kab qhia txog t-hCO ciam teb. f, g, Cov kab tam sim no ntawm t-hCO neurons tom qab hluav taws xob stimulation hauv S1 nas (f) lossis sab hauv capsule (g), nrog (ntsuab) lossis tsis muaj (dub) NBQX (sab laug). EPSC amplitudes nrog thiab tsis muaj NBQX (n = 6 S1 neurons, * P = 0.0119; thiab n = 6 internal capsule neurons, ** P = 0.0022) (center). Feem pua ​​​​ntawm t-hCO neurons qhia EPSC nyob rau hauv cov lus teb rau hluav taws xob stimulation ntawm nas S1 (f) los yog sab hauv capsule (g) (txoj cai). aCSF, dag cerebrospinal kua. h, schematic daim duab ntawm 2P duab sim (sab laug). Kev nthuav qhia ntawm GCaMP6s hauv t-hCO (nruab nrab). Scale bar, 100 µm. Fluorescence time lapse ntawm GCaMP6s (txoj cai). i, Z-cov qhab nia ntawm spontaneous kev ua fluorescence. j, schematic illustration ntawm mustache stimulation. k, z-scoreed 2P fluorescence trajectories nyob rau hauv ib qho kev sim, ua raws li kev sib txawv ntawm lub sij hawm xoom (dashed kab) hauv cov piv txwv. l, cov pej xeem-nruab nrab z-cov qhab nia cov lus teb ntawm txhua lub hlwb ua raws li qhov sib txawv ntawm qhov sib txawv ntawm lub sij hawm xoom (dashed kab) (liab) lossis randomly generated timestamps (grey). m. Schematic daim duab ntawm qhov kev sim ntawm optical marking. n, Raw voltage curves los ntawm ib qho piv txwv t-hCO cell thaum lub sij hawm xiav laser stimulation los yog whisker deflection. Cov xub liab qhia thawj qhov tshwm sim los ntawm lub teeb (sab saum toj) lossis tshwm sim los ntawm whisker deflection (hauv qab). Grey shading qhia lub sijhawm ntawm whisker deflection. o, Peak lub teeb waveforms thiab whisker deflection teb. p, spikes ntawm ib qho kev sim, ua raws li qhov sib txawv ntawm cov whiskers hauv cov hlwb ntawm qhov piv txwv. 0 qhia hais tias whisker sib txawv (dashed kab). q, pejxeem-nruab nrab z-score firing tus nqi rau tag nrho cov photosensitive hlwb, raws li zoo raws li whisker sib txawv ntawm lub sij hawm xoom (dashed kab) (liab) los yog randomly generated timestamps (grey). r, Tus nqi ntawm cov photosensitive units ho modulated los ntawm whisker deviation (n = 3 nas) (sab laug). Peak z-score latency (n = 3 nas; n = 5 (lub teeb ntsuab), n = 4 (tsub ntsuab), thiab n = 4 (cyan) whisker deflection modulation units ib nas) (txoj cai). Cov ntaub ntawv yog qhia raws li txhais tau tias ± tus qauv sib txawv
Peb mam li nug yog tias t-hCO tuaj yeem qhib los ntawm kev hnov ​​​​mob hauv vivo. Peb tau hloov pauv hCO qhia txog cov tshuaj ntsuam genetic encoded calcium GCaMP6 rau hauv S1 nas. Tom qab 150 hnub, peb ua fiber ntau photometry lossis ob-photon calcium imaging (Daim duab 4h thiab nthuav cov ntaub ntawv, Fig. 10a). Peb pom tias t-hCO hlwb nthuav tawm synchronized atherosclerosis (Daim duab 4i, Cov ntaub ntawv nthuav dav, daim duab 10b thiab Ntxiv Video 1). Txhawm rau ua tus cwj pwm ncov t-hCO kev ua si, peb tau ua extracellular electrophysiological recordings nyob rau hauv anesthetized transplant nas (cov ntaub ntawv nthuav, Fig. 10c-f). Peb tau tsim stereotaxic coordinates los ntawm MRI cov duab; Yog li, cov ntaub ntawv sau tseg no sawv cev rau tib neeg cov neurons, txawm hais tias electrophysiology ib leeg tsis tso cai rau hom keeb kwm los txiav txim. Peb pom synchronized bursts ntawm kev ua (ntau cov ntaub ntawv, Fig. 10d). Qhov tawg tau ntev li ntawm 460 ms thiab tau sib cais los ntawm lub sijhawm ntsiag to txog li 2 s (cov ntaub ntawv nthuav dav, Fig. 10d, e). Cov chav nyob ib leeg raug rho tawm qhov nruab nrab ntawm peb qhov kev sib tsoo ib zaug, uas yog kwv yees li 73% ntawm cov npe sau npe rau ib qho tawg. Cov kev ua ub no ntawm ib tus neeg muaj kev sib raug zoo heev, thiab cov kev sib raug zoo no siab dua cov chav nyob hauv cov tsiaj uas tsis tau txhaj tshuaj tau sau tseg raws li cov xwm txheej qub (cov ntaub ntawv nthuav dav, daim duab 10f). Txhawm rau ua kom pom cov lus teb ntxiv ntawm cov neeg tau txais txiaj ntsig neurons, peb tau ua qhov kev sim teeb pom kev ntawm cov nas tsuag tshuaj loog hloov nrog hCO qhia lub teeb-sensitive cation channel rhodopsin 2 (hChR2), los ntawm qhov uas t-hCO neurons luv-latency paub (tsawg dua 10 ms).i (tsawg dua 10 ms). t-hCO neurons tau nthuav tawm qhov tshwm sim ntawm kev ua haujlwm ntawm qhov zaus zoo ib yam li cov kev soj ntsuam hauv calcium imaging, nrog rau cov ntaub ntawv electrophysiological ua nyob rau hauv t-hCO thaum tsis muaj teeb pom kev zoo (cov ntaub ntawv nthuav dav, daim duab 10c-g). Tsis muaj kev tshwm sim tshwm sim nyob rau hauv cov theem sib thooj ntawm hCO sau tseg hauv vitro. Txhawm rau ntsuam xyuas seb t-hCO tuaj yeem ua haujlwm los ntawm kev hnov ​​​​qab, peb luv luv deflected nas whiskers deb ntawm t-hCO (Daim duab 4j, m thiab cov ntaub ntawv txuas ntxiv, Fig. 10h, k). Raws li cov kev tshawb fawb yav dhau los 8,10, ib pawg ntawm t-hCO hlwb pom tau tias muaj kev nce ntxiv hauv cov lus teb rau whisker deflection, uas tsis tau pom thaum cov ntaub ntawv raug muab piv nrog cov sij hawm random (Fig. 4k–q thiab nthuav cov ntaub ntawv, Fig. 10h–q). Tseeb tiag, txog 54% ntawm cov opto-labeled ib chav nyob tau pom tias muaj kev nce siab ntxiv tom qab kev txhawb nqa whisker, peaking ntawm txog 650 ms (Fig. 4r). Ua ke, cov ntaub ntawv no qhia tias t-hCO tau txais cov khoom siv tsim nyog thiab tuaj yeem ua haujlwm los ntawm ib puag ncig stimuli.
Tom qab ntawd peb tshawb xyuas seb t-hCO puas tuaj yeem qhib cov kab hluav taws xob hauv nas kom tswj tus cwj pwm. Peb thawj zaug tshawb xyuas seb lub axons ntawm t-hCO neurons project rau hauv cov ntaub so ntswg ntawm nas. Peb kis hCO nrog lentivirus encoding hChR2 fused rau EYFP (hChR2-EYFP). Tom qab 110 hnub, peb tau soj ntsuam EYFP cov lus qhia nyob rau hauv ipsilateral cortical cheeb tsam, nrog rau lub mloog pob ntseg, lub cev muaj zog, thiab somatosensory cortices, raws li zoo raws li nyob rau hauv subcortical cheeb tsam, nrog rau cov striatum, hippocampus, thiab thalamus (Fig. 5a). Txhawm rau txheeb xyuas seb qhov kev kwv yees zoo li no tuaj yeem cuam tshuam cov lus teb synaptic hauv cov hlwb nas, peb optically qhib t-hCO hlwb qhia hChR2-EYFP los ntawm kev sau cov nas cerebral cortex hlwb hauv cov hlwb ntse. Kev ua kom muaj t-hCO axons nrog lub teeb xiav induced luv-latency EPSCs hauv nas pyramidal cortex neurons, uas tau thaiv los ntawm NBQX (Figs. 5b–g). Tsis tas li ntawd, cov lus teb no tuaj yeem raug thaiv los ntawm tetrodotoxin (TTX) thiab rov qab los ntawm 4-aminopyridine (4-AP), qhia tias lawv tau tshwm sim los ntawm kev sib txuas monosynaptic (Fig. 5e).
a, Schematic daim duab ntawm axon nrhiav (sab laug). t-hCO EYFP kev qhia (txoj cai). Scale bar, 100 µm. A1, auditory cortex, ACC, anterior cingulate cortex, d. striatum, dorsal striatum, HPC, hippocampus; Diaphragm, lateral septum, mPFC, medial prefrontal cortex, piri, piriform cortex, v. striatum, ventral striatum, VPM, ventropostomedial nucleus of the thalamus, VTA, ventral tegmental region. Cov squares liab sawv cev rau thaj chaw ntawm lub paj hlwb uas cov duab raug coj mus. b, Schematic daim duab ntawm qhov kev sim stimulation. c, d, Piv txwv ntawm cov lus teb ntawm xiav lub teeb-induced photocurrent (sab saum toj) thiab voltage (hauv qab) hauv tib neeg (c) EYFP + t-hCO lossis nas (d) EYFP- hlwb. e, f, Cov kab tam sim no ntawm nas neurons tom qab lub teeb xiav stimulation ntawm t-hCO axons nrog TTX thiab 4-AR (ntsuab), TTX (grey) lossis aCSF (dub) (e), nrog (violet) lossis tsis muaj (dub)) NBQX (e). g, latency ntawm cov lus teb tshwm sim los ntawm lub teeb xiav hauv nas hlwb (n = 16 hlwb); kab rov tav bar qhia qhov nruab nrab latency (7.13 ms) (sab laug). Amplitude ntawm lub teeb-evoked EPSCs kaw nrog lossis tsis muaj NBQX (n = 7 hlwb; ***P <0.0001) (nruab nrab). Amplitude ntawm lub teeb-evoked EPSCs kaw nrog lossis tsis muaj NBQX (n = 7 hlwb; ***P <0.0001) (nruab nrab). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). Amplitude ntawm lub teeb-induced EPSCs kaw nrog los yog tsis muaj NBQX (n = 7 hlwb; ***P < 0.0001) (center).使用或不使用NBQX 记录的光诱发EPSC的振幅（n = 7 个细胞；***P < 0.0001）（中）.使用或不使用NBQX 记录的光诱发EPSC的振幅（n = 7 个细胞；***P < 0.0001）（中）. Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). Amplitude ntawm lub teeb-induced EPSCs kaw nrog los yog tsis muaj NBQX (n = 7 hlwb; ***P < 0.0001) (center).Feem pua ​​​​ntawm cov hlwb nas qhia EPSCs uas teb rau lub teeb xiav (txoj cai). h, Schematic daim duab ntawm ib tug cwj pwm ua hauj lwm. d 0,hnu 0. Kev ua yeeb yam ntawm cov tsiaj ua qauv rau hnub 1 (sab laug) lossis hnub 15 (sab xis) ntawm kev cob qhia. Qhov nruab nrab ntawm cov licks ua rau hnub 1 (sab laug) lossis hnub 15 (txoj cai centre) (n = 150 xiav teeb sim, n = 150 kev sim teeb liab; ***P <0.0001). Qhov nruab nrab ntawm cov licks ua rau hnub 1 (sab laug) lossis hnub 15 (txoj cai centre) (n = 150 xiav teeb sim, n = 150 kev sim teeb liab; ***P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 исний пыснима светом, n = 150 испытаний с красным светом; ***P <0,0001). Lub ntsiab lus ntawm licks ua rau hnub 1 (sab laug) lossis hnub 15 (nruab nrab sab xis) (n = 150 xiav teeb sim, n = 150 teeb liab sim; ***P <0.0001).第1 天（左）或第15 天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150 次红 0 ； 0 *** .第1 天（左）或第15天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150 次红 0 ｪ Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 исний пыснима светом, n = 150 испытаний с красным светом; ***P <0,0001). Lub ntsiab lus ntawm licks ua rau hnub 1 (sab laug) lossis hnub 15 (nruab nrab sab xis) (n = 150 xiav teeb sim, n = 150 teeb liab sim; ***P <0.0001).Nquag licks rau kev sim teeb liab thiab xiav rau hnub 1 (hauv nruab nrab sab laug) lossis hnub 15 (txoj cai). NS, tsis tseem ceeb. j,k, Kev coj cwj pwm ntawm txhua tus tsiaj hloov pauv nrog t-hCO qhia hChR2-EYFP (j) lossis tswj fluorophore (k) hnub 1 lossis 15 (hChR2-EYFP: n = 9 nas, ** P = 0.0049; tswj: n = 9, P = 0.1497). l, Evolution of preference score (n = 9 hChR2, n = 9 control; **P < 0.001, ***P < 0.0001). l, Evolution of preference score (n = 9 hChR2, n = 9 control; **P < 0.001, ***P < 0.0001). l, Эволюция показателя предпочтения (n = 9 hChR2, n = 9 контрольных; **P <0,001, ***P <0,0001). l, Evolution of preference score (n = 9 hChR2, n = 9 controls; **P < 0.001, ***P < 0.0001). l, 偏好评分的演变(n = 9 hChR2, n = 9 对照；**P < 0.001,***P < 0.0001). l, 偏好评分的演变(n = 9 hChR2, n = 9 对照；**P < 0.001,***P < 0.0001). l, Эволюция показателей предпочтения (n = 9 hChR2, n = 9 контролей; **P <0,001, ***P <0,0001). l, Evolution of preference scores (n = 9 hChR2, n = 9 controls; **P < 0.001, ***P < 0.0001).m, FOS qhia nyob rau hauv teb rau optogenetic activation ntawm t-hCO hauv S1. Cov duab ntawm FOS qhia (sab laug), thiab qhov ntau (n = 3 ib pab pawg; * P < 0.05, ** P < 0.01 thiab *** P < 0.001) (txoj cai) yog qhia. Cov duab ntawm FOS qhia (sab laug), thiab qhov ntau (n = 3 ib pab pawg; * P < 0.05, ** P < 0.01 thiab *** P < 0.001) (txoj cai) yog qhia. Показаны изображения экспрессии FOS (слева) и количественного определения (n = 3 на группу; * P <0,05, ** P <0,01,01. Cov duab ntawm FOS qhia (sab laug) thiab qhov ntau (n = 3 ib pab pawg; *P<0.05, **P<0.01, thiab ***P<0.001) yog qhia (txoj cai).显示了FOS 表达（左）和量化（每组n = 3；*P < 0.05、**P < 0.01 和***P < 0.001）（右）的图像.显示了FOS 表达（左）和量化（每组n = 3；*P < 0.05、**P < 0.01 和***P < 0.001）（右）的图像. Показаны изображения экспрессии FOS (слева) и количественного определения (n = 3 на группу; * P <0,05, ** P <0,01,01. Cov duab ntawm FOS qhia (sab laug) thiab qhov ntau (n = 3 ib pab pawg; *P<0.05, **P<0.01, thiab ***P<0.001) yog qhia (txoj cai).Scale bar, 100 µm. Cov ntaub ntawv raug qhia raws li qhov txhais tau tias ± tus qauv yuam kev ntawm BLA, basolateral tonsil, MDT, dorsomedial thalamic nucleus, PAG, periaqueductal grey.
Thaum kawg, peb nug seb t-hCO puas tuaj yeem hloov tus cwj pwm nas. Txhawm rau kuaj qhov no, peb tau hloov pauv hChR2-EYFP-qhia hCO rau hauv S1, thiab 90 hnub tom qab, peb cog cov fibers optical rau hauv t-hCO rau lub teeb xa khoom. Peb mam li cob qhia cov nas nrog kev hloov kho cov neeg ua haujlwm txias (daim duab 5h). Peb muab cov tsiaj tso rau hauv chav kuaj tus cwj pwm thiab randomly thov 5 thib ob xiav (473 nm) thiab liab (635 nm) laser stimuli. Tsiaj txhu tau txais nqi zog dej yog tias lawv licked thaum lub teeb xiav stimulation tab sis tsis yaim thaum lub teeb liab stimulation. Nyob rau thawj hnub ntawm kev cob qhia, cov tsiaj tsis pom qhov txawv ntawm licking thaum txhawb nqa nrog lub teeb xiav lossis liab. Txawm li cas los xij, nyob rau hnub 15, cov tsiaj hloov pauv nrog hCO qhia hChR2-EYFP tau pom ntau dua licking thaum txhawb nrog lub teeb xiav piv rau lub teeb liab stimulation. Cov kev hloov pauv ntawm licking tus cwj pwm no tsis tau pom nyob rau hauv kev tswj cov tsiaj hloov pauv nrog hCO qhia txog kev tswj fluorophore (kev kawm tau zoo: hChR2 89%, EYFP 0%, Daim duab 5i-1 thiab Cov Video Ntxiv 2). Cov ntaub ntawv no qhia tias t-hCO hlwb tuaj yeem ua rau nas neurons los txhawb tus cwj pwm nrhiav nqi zog. Txhawm rau kom paub seb cov nas t-hCO neural circuits twg tuaj yeem koom nrog hauv cov kev hloov pauv no, peb optogenetically qhib t-hCO hauv cov tsiaj uas tau kawm thiab sau cov ntaub so ntswg 90 feeb tom qab. Immunohistochemistry tau qhia txog kev ua haujlwm-nyob ntawm FOS protein nyob rau hauv ntau thaj tsam ntawm lub paj hlwb koom nrog kev txhawb nqa tus cwj pwm, suav nrog medial prefrontal cortex, medial thalamus, thiab periaqueductal grey teeb meem, uas tau hais tawm hauv cov tsiaj tsis muaj zog lossis hauv cov tsiaj. mov. 5 m). Ua ke, cov ntaub ntawv no qhia tias t-hCO tuaj yeem hloov kho nas neuronal kev ua haujlwm los tsav tus cwj pwm.
Neural organoids sawv cev rau kev cog lus rau kev kawm tib neeg txoj kev loj hlob thiab kab mob hauv vitro, tab sis lawv raug txwv los ntawm qhov tsis muaj kev sib txuas ntawm circuits uas muaj nyob hauv vivo. Peb tau tsim lub platform tshiab uas peb hloov pauv hCO rau hauv S1 ntawm cov tshuaj tiv thaiv kab mob thaum ntxov tom qab yug me nyuam los kawm txog tib neeg txoj kev loj hlob ntawm tes thiab kev ua haujlwm hauv vivo. Peb tau pom tias t-hCO tsim cov cell loj hlob yam tsis tau pom nyob rau hauv vitro28 thiab tias t-hCO yog anatomically thiab functionally integrated rau hauv nas hlwb. Kev sib koom ua ke ntawm t-hCO rau hauv nas neural circuits tau tso cai rau peb los tsim kom muaj kev sib txuas ntawm tib neeg kev ua haujlwm ntawm tes thiab kawm txog tus cwj pwm tsiaj, qhia tias t-hCO neurons tuaj yeem hloov kho nas neuronal kev ua haujlwm los tsav kev coj tus cwj pwm.
Lub platform peb piav qhia muaj ntau qhov zoo dua li kev tshawb fawb yav dhau los ntawm kev hloov tib neeg lub hlwb rau hauv nas hlwb. Ua ntej, peb hloov hCO rau hauv kev tsim cov cortex ntawm cov nas thaum ntxov tom qab yug me nyuam, uas yuav pab txhawb kev sib koom ua ke ntawm anatomical thiab kev ua haujlwm. Qhov thib ob, t-hCO MRI kev soj ntsuam tau tso cai rau peb los kawm txog graft txoj hauj lwm thiab kev loj hlob ntawm cov tsiaj nyob, tso cai rau peb mus ua kev tshawb fawb ntau yam tsiaj txhu thiab tsim kom muaj kev ntseeg siab ntawm ob peb hiPS cell kab. Thaum kawg, peb tau hloov cov organoids tsis zoo, tsis yog cais ib leeg ntawm tes ncua, uas tsis tshua muaj kev puas tsuaj rau tib neeg lub hlwb thiab tuaj yeem txhawb kev sib koom ua ke thiab tsim cov tib neeg cortex neurons hauv nas hlwb.
Peb lees paub tias txawm tias muaj kev nce qib hauv lub platform no, lub cev nqaij daim tawv, qhov chaw, thiab cov kab mob sib kis tau txwv tsis pub tsim cov tib neeg neural circuits nrog kev ncaj ncees, txawm tias tom qab hloov pauv thaum ntxov ntawm txoj kev loj hlob. Piv txwv li, nws tsis paub meej tias qhov kev ua haujlwm ntawm tus kheej tau pom hauv t-hCO sawv cev rau kev loj hlob phenotype zoo ib yam li cov kev ua ub ua no tau pom thaum lub sij hawm kev loj hlob ntawm cortical, los yog seb nws yog vim tsis muaj kev txwv ntawm tes muaj nyob hauv t-hCO. Ib yam li ntawd, nws tsis paub meej tias qhov tsis muaj lamination hauv t-hCO cuam tshuam li cas rau kev sib txuas ntawm cov saw txuas 30. Kev ua haujlwm yav tom ntej yuav tsom mus rau kev sib koom ua ke ntawm lwm hom ntawm tes xws li tib neeg microglia, tib neeg cov kab mob endothelial, thiab qhov sib txawv ntawm GABAergic interneurons raws li qhia siv sib dhos 6 hauv vitro, nrog rau kev nkag siab txog kev sib koom ua ke ntawm neural thiab kev ua haujlwm tuaj yeem tshwm sim hauv t-hCO. transcriptional, synaptic thiab kev coj cwj pwm theem nyob rau hauv hlwb tau los ntawm cov neeg mob.
Zuag qhia tag nrho, qhov no hauv vivo platform sawv cev rau cov peev txheej muaj zog uas tuaj yeem ua tiav hauv vitro tib neeg lub hlwb kev loj hlob thiab kev tshawb fawb kab mob. Peb cia siab tias lub platform no yuav tso cai rau peb tshawb pom cov strand-theem phenotypes tshiab hauv cov neeg mob uas tau txais cov hlwb tsis zoo thiab sim cov tswv yim kho tshiab.
Peb tsim hCO2.5 los ntawm HiPS hlwb raws li tau piav qhia dhau los. Txhawm rau pib hCO ntau lawm los ntawm hiPS hlwb kab lis kev cai nyob rau hauv cov txheej txheem feeder, cov kab mob tsis zoo ntawm cov kab mob hiPS raug tshem tawm ntawm kab lis kev cai tais diav siv dispase (0.35 mg / mL) thiab xa mus rau cov kab lis kev cai qis qis uas muaj cov tais diav nrog hiPS cell kab lis kev cai nruab nrab. (Corning) ntxiv nrog ob SMAD inhibitors dorsomorphine (5 μM; P5499, Sigma-Aldrich) thiab SB-431542 (10 μM; 1614, Tocris) thiab ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Hauv thawj 5 hnub, qhov nruab nrab ntawm hiPS tau hloov pauv txhua hnub thiab dorsomorphine thiab SB-431542 tau ntxiv. Hnub thib rau hauv kev raug ncua, neural spheroids raug xa mus rau qhov nruab nrab nruab nrab uas muaj cov neurobasal-A (10888, Life Technologies), B-27 ntxiv yam tsis muaj vitamin A (12587, Lub Neej Technologies), GlutaMax (1: 100, Lub Neej Technologies), penicillin thiab 10 technologies ntxiv rau lub neej. epidermal growth factor (EGF; 20 ng ml−1; R&D Systems) thiab fibroblast growth factor 2 (FGF2; 20 ng ml−1; R&D Systems) mus txog hnub 24. Los ntawm hnub 25 mus rau hnub 42, qhov nruab nrab tau supplemented nrog lub hlwb-derived neurotrophic factor (BDNF; 1 trophin 3 ml) thiab 20 NT 3. ng ml-1; Peprotech) nrog kev hloov pauv nruab nrab txhua hnub. Hnub thib rau hauv kev raug ncua, neural spheroids raug xa mus rau qhov nruab nrab nruab nrab uas muaj cov neurobasal-A (10888, Life Technologies), B-27 ntxiv yam tsis muaj vitamin A (12587, Lub Neej Technologies), GlutaMax (1: 100, Lub Neej Technologies), penicillin thiab 10 technologies ntxiv rau lub neej. epidermal growth factor (EGF; 20 ng ml−1; R&D Systems) thiab fibroblast growth factor 2 (FGF2; 20 ng ml−1; R&D Systems) mus txog hnub 24. Los ntawm hnub 25 mus rau hnub 42, qhov nruab nrab tau supplemented nrog lub hlwb-derived neurotrophic factor (BDNF; 1 trophin 3 ml) thiab 20 NT 3. ng ml-1; Peprotech) nrog kev hloov pauv nruab nrab txhua hnub.Hnub thib rau hauv kev ncua, cov neural spheroids raug xa mus rau qhov nruab nrab neural uas muaj Neurobasal-A (10888, Life Technologies), B-27 ntxiv yam tsis muaj vitamin A (12587, Lub Neej Technologies), GlutaMax (1: 100, Life Technologies), penicillin.и стрептомицин (1:100, Life Technologies) thiab дополнены эпидермальным фактором роста (EGF; 20 нг/мл; R&D Systems) и факто фибробластов 2 (FGF2; 20 нг/мл; R&D Systems) до 24-го дня. thiab streptomycin (1:100, Lub Neej Technologies) thiab ntxiv nrog rau kev loj hlob ntawm epidermal (EGF; 20 ng / ml; R & D Systems) thiab fibroblast kev loj hlob factor 2 (FGF2; 20 ng / ml; R & D Systems) txog hnub 24.Los ntawm hnub 25 mus rau 42, lub hlwb-derived neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) thiab neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) tau ntxiv rau qhov nruab nrab, hloov nruab nrab txhua hnub.在悬浮的第6 天，将神经球体转移到含有neurobasal-A (10888,Life Technologies)、不含维生素A的B-27 (7,12fee 8 剅) Technologies), GlutaMax (1: 100, Lub Neej Technologies), 青霉素的神经培养基中和链霉素 (1: 100, Life Technologies) 并辅以表隿囮ml-1；R&D Systems) 和成纤维细胞生长因子2 (FGF2；20 ng ml-1；R&D Systems) 直至第24天.在悬浮的第第6天将神经 球体转移含有含有 neurobasal-a (10888，的 Life Technologies) 不含维兟素(12587, Lub Neej Technologies) Glutamax (1: 100, Life TechNOGIS青霉素的神经培养基 中 链霉素 (1: 100 , Life Technologies 忐) ((20 ng ml-1 ； r & d Systems) 成 纤维 细胞 生长 2 (fgf2 ； 20 ng ml- 1； R&D Systems) 直至第24天. На 6-й день суспензии нейросферы были переведены на добавку, содержащую нейробазал-А (10888, Life Technologies-2 задек), витамина А (12587, Life Technologies), GlutaMax (1:100, Lub Neej Technologies), пенициллин- нейтрализованный стрептомицин (1:100, Life Technologies) эпидермального фактора роста (EGF; 20 нг мл-1; R&D Systems) и фактора роста фибробластов 2 (FGF2; 20 нг); 1 мл-1 Hnub 6, neurosphere suspensions tau hloov mus rau ib qho ntxiv uas muaj neurobasal-A (10888, Lub Neej Technologies), B-27 ntxiv yam tsis muaj vitamin A (12587, Lub Neej Technologies), GlutaMax (1:100, Lub Neej Technologies), penicillin-neutralized streptomycin, ntxiv rau lub neej: (EGF; 20 ng ml-1; R&D Systems) thiab fibroblast kev loj hlob 2 (FGF2; 20 ng ml-1) 1; R&D Systems) rau 24 xyoo. R&D Systems) mus txog hnub 24.Los ntawm hnub 25 mus rau 42, lub hlwb-derived neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) thiab neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) tau ntxiv rau cov kab lis kev cai nruab nrab txhua hnub. Nruab nrab hloov ib zaug.Pib txij hnub 43, hCO tau khaws cia hauv qhov nruab nrab tsis muaj zog neurobasal-A (NM; 1088022, Thermo Fisher) nrog kev hloov nruab nrab txhua 4-6 hnub. Yuav kom tau txais hCO los ntawm cov hlwb hiPS kab lis kev cai nyob rau hauv cov kab mob tsis pub noj, cov hlwb hiPS tau tsim nrog Accutase (AT-104, Innovate Cell Technologies) ntawm 37 ° C rau 7 feeb, dissociated rau hauv ib lub hlwb, thiab plated ntawm AggreWell 800 daim hlau (34815, STEMCELL Technologies) ntawm ib lub hlwb zoo ib yam. 8 nruab nrab ntxiv nrog ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Tom qab 24 teev, cov xov xwm hauv qhov dej tau muab tso rau hauv cov xov xwm uas muaj Essential 6 media (A1516401, Life Technologies) ntxiv nrog dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) thiab SB-431542 (10 μM; 161 μM); , Tocrida ib). Los ntawm hnub 2 txog 6, Qhov Tseem Ceeb 6 nruab nrab tau hloov txhua hnub nrog dorsomorphine thiab ntxiv SB-431542. Txij hnub thib rau, cov kev ncua neurosphere raug xa mus rau qhov nruab nrab neurobasal thiab khaws cia raws li tau piav qhia saum toj no.
Txhua tus txheej txheem tsiaj tau ua raws li cov txheej txheem saib xyuas tsiaj pom zoo los ntawm Stanford University Laboratory Animal Care Administration Committee (APLAC). Cev xeeb tub euthymic RNU (rnu / +) nas tau yuav (Charles River Laboratories) lossis tsev. Tsiaj txhu tau khaws cia rau ntawm 12-teev lub teeb-dub voj voog nrog zaub mov thiab dej ad libitum. Nude (FOXN1–/–) cov menyuam nas hnub nyoog peb mus rau xya hnub tau txheeb xyuas los ntawm kev loj hlob ntawm cov tawv nqaij tsis paub tab ua ntej culling. Cov menyuam dev (txiv neej thiab poj niam) tau tshuaj loog nrog 2-3% isoflurane thiab muab tso rau ntawm stereotaxic thav duab. Ib qho trepanation ntawm pob txha taub hau nrog ib txoj kab uas hla ntawm kwv yees li 2-3 hli saum toj no S1 tau ua thaum tswj kev ncaj ncees ntawm dura mater. Tom qab ntawd siv 30-G koob (kwv yees li 0.3 hli) sab nraum lub craniotomy txhawm rau tho lub dura. Tom qab ntawd siv HCO rau nyias 3 × 3 cm parafilm thiab tshem tawm qhov nruab nrab. Siv Hamilton syringe txuas nrog 23 G, 45 ° koob, maj mam rub hCO rau hauv qhov kawg ntawm qhov kawg ntawm rab koob. Tom qab ntawd nruab lub syringe ntawm lub syringe twj tso kua mis txuas nrog lub stereotaxic ntaus ntawv. Tom qab ntawd tso lub taub ntawm rab koob hla lub qhov ua ntej ua 0.3 hli dav puncture qhov hauv dura (z = 0 mm) thiab nqaim lub koob txhaj tshuaj 1-2 mm (z = kwv yees li -1.5 mm) kom txog thaum lub koob nyob nruab nrab ntawm dura mater A. lub foob ntom ntom yog tsim. Tom qab ntawd tsa cov koob txhaj tshuaj mus rau qhov chaw ntawm qhov chaw cortical ntawm z = -0.5 hli thiab txhaj hCO ntawm tus nqi ntawm 1-2 µl ib feeb. Tom qab ua tiav ntawm kev txhaj tshuaj hCO, rab koob yog thim rov qab ntawm tus nqi ntawm 0.2-0.5 hli ib feeb, daim tawv nqaij yog sutured, thiab tus menyuam dev yog tam sim ntawd muab tso rau ntawm lub tshuab cua sov kom txog thaum rov ua tiav. Qee cov tsiaj tau hloov pauv ob sab.
Txhua tus txheej txheem tsiaj tau ua raws li Stanford University APLAC-pom zoo cov txheej txheem saib xyuas tsiaj. Cov nas tsuag (ntau dua 60 hnub tom qab hloov pauv) tau raug ntxias nrog 5% isoflurane tshuaj loog thiab tshuaj loog nrog 1-3% isoflurane thaum kuaj pom. Rau kev pom, 7 Tesla nquag tiv thaiv kab rov tav borehole scanner Bruker (Bruker Corp.) nrog rau International Electric Company (IECO) gradient tsav, shielded gradient insert nrog ib txoj kab uas hla ntawm 120 mm (600 mT / m, 1000 T / m / s) siv AVANCE. III, yim-channel multi-coil RF thiab ntau lub peev xwm, thiab nrog Paravision 6.0.1 platform. Cov ntaubntawv povthawj siv tau ua los ntawm kev siv lub zog decoupled volumetric RF coil nrog ib txoj kab uas hla ntawm 86 hli thiab plaub-channel cryo-txias RF coil rau tau txais xwb. Axial 2D Turbo-RARE (repetition time = 2500 ms, ncha time = 33 ms, 2 averages) nrog 16 daim captures, hlais thickness 0.6–0.8 mm, muaj 256 × 256 kuaj. Cov teeb liab tau txais siv lub quadrature transceiver volumetric RF coil nrog ib txoj kab uas hla ntawm 2 cm (Rapid MR International, LLC). Thaum kawg, siv lub built-in Imaris (BitPlane) nto kwv yees ua haujlwm rau 3D rendering thiab ntsuas ntim. Ib qho kev hloov pauv tau zoo tau txhais tau tias yog ib qho ntawm thaj chaw ntawm T2-hnyav MRI teeb liab tau tsim nyob rau hauv lub hemisphere transplanted. Graft rejection yog txhais raws li ib tug graft uas tsis tsim cov cheeb tsam ntawm tas li T2-hnyav MRI teeb liab nyob rau hauv lub transplanted hemisphere. Subcortical t-hCO raug tshem tawm los ntawm kev soj ntsuam tom qab.
Txhawm rau nthuav qhia GCaMP6s hauv hCO rau ob-photon calcium imaging, hiPS hlwb tau kis tus kab mob pLV[Exp]-EF1a::GcaMP6s-WPRE-Puro tom qab xaiv cov tshuaj tua kab mob. Luv luv, cov hlwb raug cuam tshuam nrog EDTA thiab raug tshem tawm hauv 1 ml ntawm Essential 8 nruab nrab ntawm qhov ntom ntawm kwv yees li 300,000 hlwb nyob rau hauv muaj polybene (5 μg / ml) thiab 15 μl ntawm tus kab mob. Tom qab ntawd cov hlwb tau muab tso rau hauv kev ncua rau 60 feeb thiab noob ntawm qhov ntom ntawm 50,000 hlwb ntawm ib qhov dej. Tom qab kev sib txuam, cov hlwb raug kho nrog 5-10 μg ml-1 puromycin rau 5-10 hnub los yog kom txog thaum cov cheeb tsam ruaj khov tshwm sim. Tus kab mob hCO mob hnyav tau ua raws li tau piav qhia dhau los 5 nrog qee qhov kev hloov kho. Luv luv, hloov hnub 30-45 hCO rau hauv 1.5 ml Eppendorf microcentrifuge hlab uas muaj 100 µl ntawm paj hlwb nruab nrab. Tom qab ntawd kwv yees li 90 µl ntawm qhov nruab nrab raug tshem tawm, 3-6 µl ntawm high titer lentivirus (los ntawm 0.5 x 108 mus rau 1.2 x 109) yog ntxiv rau lub raj, thiab hCO raug xa mus rau qhov chaw tsim khoom rau 30 feeb. Tom qab ntawd ntxiv 90-100 µl ntawm qhov nruab nrab rau txhua lub raj thiab xa cov raj xa mus rau lub tshuab nqus tsev thaum hmo ntuj. Hnub tom qab, hloov hCO mus rau cov paj hlwb tshiab hauv cov ntawv txuas qis. Tom qab 7 hnub, hCO raug xa mus rau 24-zoo iav hauv qab daim hlau rau kev pom thiab kev ntsuam xyuas ntawm cov kab mob zoo. pLV[Exp]-SYN1::EYFP-WPRE thiab pLV[Exp]-SYN1::hChR2-EYFP-WPRE tau tsim los ntawm VectorBuilder. Lentivirus yog siv nyob rau hauv feem ntau cov kev sim vim hais tias nws yog kev koom ua ke rau hauv lub host genome, tso cai rau reporter gene qhia nyob rau hauv cov kab mob ntawm tes. Rau kev soj ntsuam kab mob vwm, hnub 30-45 hCO tau kis tus kab mob rabies-ΔG-eGFP thiab AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), ntxuav kom huv si rau 3 hnub, thiab hloov mus rau nas hauv S1 thiab khaws cia hauv vivo rau 7-14 hnub.
Rau immunocytochemistry, tsiaj tau tshuaj loog thiab transcardially perfused nrog PBS ua raws li 4% paraformaldehyde (PFA hauv PBS; Electron Microscopy Sciences). Lub hlwb raug kho hauv 4% PFA rau 2 teev lossis ib hmos ntawm 4 ° C, cryopreserved hauv 30% sucrose hauv PBS rau 48-72 teev, thiab embedded hauv 1: 1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583, Sakura Finetek 3) thiab statm ntawm coronal. (Leica). Rau immunohistochemistry ntawm cov ntu tuab, cov tsiaj tau perfused nrog PBS, thiab lub paj hlwb tau muab faib ua coronally ntawm 300-400 µm siv vibratome (Leica) thiab cov seem tau kho nrog 4% PFA rau 30 feeb. Tom qab ntawd cov khoom siv hluav taws xob los yog cov ntu tuab tau muab ntxuav nrog PBS, thaiv rau 1 teev ntawm chav tsev kub (10% ib txwm muaj ntshav qab zib (NDS) thiab 0.3% Triton X-100 diluted hauv PBS) thiab thaiv nrog kev thaiv kev daws ntawm 4 ° C. - Incubation Cryosections tau incubated ib hmos thiab tuab seem tau incubated rau 5 hnub. Thawj cov tshuaj tiv thaiv uas siv tau yog: anti-NeuN (nas, 1: 500; ab104224, abcam) anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (luav, 1: 1,000; Z0334, Dako), anti-en, 00ch (: GFP; GTX13970, GeneTex), anti-HNA (nas, 1: 200; ab191181, abcam), anti-NeuN (luav, 1: 500; ABN78, Millipore), anti-PDGFRA (luav, 1: 200; sc-338, Santa Cruz), anti-PP: 2 HPA047819, Atlas Antibodies), anti-RECA-1 (nas, 1:50; ab9774, abcam), anti-SCG2 (Lavbit, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (tshis, 1:500; AF&rinat Systems), R. 1:100; AF1166, R&D Systems), anti-STEM121 (nas, 1:200; Y40410, Takara Bio), anti-SATB2 (nas, 1:50; ab51502, abcam), anti-GAD65/67 (liab, 1:4900, anti-ABNAA) thiab 1:100; ab 5076, abcam). Thawj cov tshuaj tiv thaiv uas siv tau yog: anti-NeuN (nas, 1: 500; ab104224, abcam) anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (luav, 1: 1,000; Z0334, Dako), anti-en,00ch (: GFP; GTX13970, GeneTex), anti-HNA (nas, 1: 200; ab191181, abcam), anti-NeuN (luav, 1: 500; ABN78, Millipore), anti-PDGFRA (luav, 1: 200; sc-338, Santa Cruz), anti-PP: 2 HPA047819, Atlas Antibodies), anti-RECA-1 (nas, 1:50; ab9774, abcam), anti-SCG2 (Lavbit, 1: 100; 20357-1-AP, Proteintech), anti-SOX9 (tshis, 1: 500; AF & NetSystema), 1:100; AF1166, R&D Systems), anti-STEM121 (nas , 1:200; Y40410, Takara Bio), anti-SATB2 (nas, 1:50; ab51502, abcam), anti-GAD65/67 (liab, IB-ABN1900); 1:100; ab 5076, abcam). Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2 (кры; abcam), анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA (мышь, 81:19ab10), 1:20 анти-NeuN (кролик, 1:500; ABN78, Millipore), анти-PDGFRA (кролик, 1:200; sc-338, Санта-Круз), анти-PPP1R17 (кролик, 1:200; Antibodies), анти-RECA-1 (мышь, 1:50; ab9774, abcam), анти-SCG2 (кролик , 1:100; 20357-1-AP, Proteintech), анти-SOX9 (козий, 1:357, AF&System) нетрин G1a (козий, 1:100; AF1166, R&D Systems), анти-STEM121 (мышиный , 1:200; Y40410, Takara Bio), анти-SATB2 (мышь, 1:150), 1:50; анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) и анти-IBA1 (коза, 1:100; аб5076, абкам). Thawj cov tshuaj tiv thaiv uas siv tau yog: anti-NeuN (nas, 1: 500; ab104224, abcam), anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (Lavbit, 1: 1000; Z0334, Dako), anti-GFP1: 100, anti-GFP; GeneTex), anti-HNA (nas, 1: 200; ab191181, abcam), anti-NeuN (luav, 1: 500; ABN78, Millipore), anti-PDGFRA ( luav, 1: 200; sc-338, Santa Cruz), anti-PPP1R17: 200las, Atlanta; Antibodies), anti-RECA-1 (nas, 1:50; ab9774, abcam), anti- SCG2 (luav, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (tshis, 1: 500; AF3075, R&D Systems: 1goatrin G1a; R&D Systems), anti- STEM121 (nas, 1:200; Y40410, Takara Bio), anti-SATB2 (nas, 1:50; ab51502, abcam), anti-GAD65/67 (Lavbit, 1:400; ABN904, Millipore: musat, 710; abkam).使用的一抗是：抗NeuN（小鼠，1:500；ab104224，abcam）抗CTIP2（大鼠，1:3 00；ab18465，abcam），抗GFAP（兔，1:1,000；Z0334，Dako），抗-GF P(1:1,000;GTX13970,GeneTex),抗HNA(小鼠,1:200；ab191181,abcam),抗NeuN(兔,1:500；ABN78,Millipore),抗PDG 1:200；sc-338，Santa Cruz），抗PPP1R17（兔，1:200；HPA047819，Atlas抗体），抗RECA-1（小鼠，1:50；ab9774，SC） 1: 100; 20357-1-AP, Proteintech), 抗SOX9 (山羊, 1: 500; AF3075, R&D Systems), Netrin G1a (山羊, 1: 100； AF1166, R&D Systems 1: 1, 10, ST;使用的一抗是：抗NeuN（小鼠，1:500；ab104224，abcam）抗CTIP2（大鼠，1:300；ab18465，abcam），抗GFAP兔，104224，00； -GFP (1: 1,000; GTX 13970, GeneTex), 抗HNA (小鼠, 1: 200; ab191181, abcam), 抗NeuN (兔, 1: 500； ABN78, Millipore, 1: 4. 200；sc-338，Santa Cruz），抗PPP1R17（兔，1:200；HPA047819，Atlas 抗体），抗RECA-1（小鼠，1:50；ab9774，Abcam）， 100; 20357-1-AP, Proteintech), 抗SOX9 (山羊, 1:500; AF3075, R&D Systems), Netrin G1a (1: 100； AF1166, R&D Systems), 1: 20;Y40410, Takara Bio), anti-SATB2 (nas, 1:50; ab51502, abcam), anti-GAD65/67 (Lavbit, 1:400; ABN904, Millipore) thiab anti-IBA1 (tshis, 1:100; ab5076, abcam).Thawj cov tshuaj tiv thaiv siv tau yog: anti-NeuN (nas, 1: 500; ab104224, abcam), anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (Lavbit, 1: 1000; Z0334, Dako). , anti-GFP (nqaij qaib, 1:1000; GTX13970, GeneTex), anti-HNA (nas, 1:200; ab191181, abcam), anti-NeuN (Lavbit, 1:500; ABN78, Millipore), anti-PDGFRA (20 luav, 1: 0. anti-PPP1R17 (luav, 1:200; HPA047819, Atlas antibody), anti-RECA-1 (nas, 1:50; ab9774, abcam), anti- SCG2 (Lavbit), 1:100;20357-1-AP, Proteintech), анти-SOX9 (коза, 1:500; AF3075, R&D Systems), Нетрин G1a (коза, 1:100; AF1166, R&D Systems), ш, 10 (1:20m) Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) и анти-IBA70; 1,ко pab). 20357-1-AP, Proteintech), anti-SOX9 (tshis, 1: 500; AF3075, R&D Systems), Netrin G1a (tshis, 1: 100; AF1166, R&D Systems), anti-STEM121 (nas, 1: 200; anti-mouse, Takara Bio) 1:50; ab51502, abcam), anti-GAD65/67 (Lavbit, 1:400; ABN904, Millipore), thiab anti-IBA1 (tshis, 1:100; ab5076, abkam).Tom qab ntawd cov seem raug ntxuav nrog PBS thiab incubated nrog lwm cov tshuaj tiv thaiv rau 1 teev nyob rau hauv chav tsev kub (cov seem khov) los yog thaum hmo ntuj ntawm 4 ° C (thick sections). Alexa Fluor Secondary Antibody (Lub Neej Technologies) diluted 1: 1000 hauv kev daws teeb meem tau siv. Tom qab ntxuav nrog PBS, lub nuclei tau pom nrog Hoechst 33258 (Lub Neej Technologies). Thaum kawg, cov slides tau muab tso rau ntawm lub tshuab tsom iav nrog cov npog npog (Fisher Scientific) siv Aquamount (Polysciences) thiab tshuaj xyuas ntawm Keyence fluorescent microscope (BZ-X analyzer) lossis Leica TCS SP8 confocal microscope (Las-X) ntawm daim duab. Cov duab tau ua tiav siv ImageJ program (Fiji). Txhawm rau kom muaj nuj nqis ntawm tib neeg cov neurons hauv t-hCO thiab cov nas cortex, 387.5 μm dav duab plaub raug coj los ntawm qhov chaw ntawm t-hCO, ntawm lossis ze ntawm ntug ntawm nas cortex. Graft margins tau txiav txim siab los ntawm kev ntsuas cov kev hloov pauv ntawm cov ntaub so ntswg pob tshab, HNA + nuclei, thiab / lossis muaj cov ntaub so ntswg autofluorescence. Hauv txhua daim duab, tag nrho cov NeuN + thiab HNA + cov hlwb tau muab faib los ntawm tag nrho cov NeuN + cov hlwb hauv tib cheeb tsam. Txhawm rau kom ntseeg tau tias tsuas yog cov hlwb nrog nuclei hauv daim duab dav hlau raug suav, tsuas yog cov hlwb uas tseem Hoechst + suav nrog hauv kev suav. Ob daim duab sib cais los ntawm tsawg kawg yog 1 hli tau nruab nrab kom txo tau qhov kev ua yuam kev.
Ib lub lim tiam ua ntej sau cov qauv, tso hCO cov tsiaj hloov pauv (kwv yees li 8 lub hlis ntawm kev sib txawv) hauv chav tsaus nti nrog cov tshuaj txhuam hniav kom txo qis kev hnov ​​​​mob. Kev rho tawm ntawm nuclei tau ua raws li tau piav qhia dhau los, nrog qee qhov kev hloov kho. Luv luv, t-hCO thiab hCO raug rhuav tshem siv cov tshuaj ntxuav tes-mechanical cell lysis thiab 2 ml iav cov ntaub so ntswg grinder (D8938, Sigma-Aldrich/KIMBLE). Cov nuclei crude tau lim tawm siv lub lim 40 µm thiab centrifuged ntawm 320 g rau 10 feeb ntawm 4 ° C ua ntej ua qhov sucrose density gradient. Tom qab cov kauj ruam centrifugation (320 g rau 20 min ntawm 4 ° C), cov qauv tau rov ua dua hauv 0.04% BSA / PBS nrog ntxiv ntawm 0.2 units ntawm µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) thiab dhau los ntawm 40 µm ntws lim. Cov nuclei dissociated tau rov qab muab tso rau hauv PBS uas muaj 0.02% BSA thiab thauj mus rau Chromium Single Cell 3′ nti (kwv yees rov qab ntawm 8,000 hlwb ib txoj kab). snRNA-seq cov tsev qiv ntawv tau npaj nrog Chromium Ib leeg cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). snRNA-seq cov tsev qiv ntawv tau npaj nrog Chromium Ib leeg cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Библиотеки snRNA-seq были приготовлены с помощью Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Cov tsev qiv ntawv snRNA-seq tau npaj siv Chromium Ib leeg ntawm tes 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). snRNA-seq 文库是使用Chromium Ib leeg cell 3′ GEM 、Library & Gel Bead Kit v3 (10x Genomics) 制备的. snRNA-seq 文库是使用Chromium Ib leeg cell 3′ GEM 、Library & Gel Bead Kit v3 (10x Genomics) 制备的. Библиотеку snRNA-seq готовили с использованием Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Lub tsev qiv ntawv snRNA-seq tau npaj siv Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics).Cov tsev qiv ntawv los ntawm cov qauv sib txawv tau sib sau ua ke thiab ua raws los ntawm Admera Health ntawm NovaSeq S4 (Illumina).
Gene qhia theem rau txhua qhov putative nuclear barcode tau suav nrog siv 10x Genomics CellRanger tsom xam software pob (version 6.1.2). Tshwj xeeb, cov ntawv nyeem tau raug sib koom ua ke ntawm tib neeg (GRCh38, Ensemble, version 98) thiab nas (Rnor_6.0, Ensemble, version 100) siv cov genomes tsim nrog mkref hais kom ua thiab siv suav nrog -include-introns = TRUE cov lus txib kom muaj nuj nqis suav nrog kev nyeem ntawv mapped rau thaj tsam intron. Rau cov qauv t-hCO, tib neeg nuclei raug txheeb xyuas raws li qhov xav tau ntawm kev saib xyuas uas tsawg kawg 95% ntawm tag nrho cov ntawv nyeem tau phim tib neeg genome. Txhua qhov kev soj ntsuam tom qab tau ua tiav ntawm qhov lim barcode array tso tawm los ntawm CellRanger siv R pob (version 4.1.2) Seurat (version 4.1.1)32.
Txhawm rau kom ntseeg tau tias tsuas yog cov nuclei zoo tau suav nrog hauv kev tshuaj ntsuam tom ntej, cov txheej txheem lim dej rov qab tau siv rau txhua tus qauv. Ua ntej, cov nuclei tsis zoo nrog tsawg dua 1000 cov noob caj noob ces pom thiab ntau dua 20% ntawm tag nrho cov mitochondria raug txheeb xyuas thiab tshem tawm. Tom qab ntawd, cov noob qoob loo cov lej matrix tau zoo li qub los ntawm kev ua tsis zoo binomial regression siv sctransform(vst.flavor = "v2") muaj nuj nqi, uas kuj tau txheeb xyuas 3000 cov noob hloov pauv feem ntau siv cov khoom tsis raug. Dimension txo tau ua nyob rau sab sauv sib txawv ntawm cov noob siv Analysis (Principal default) cov ntaub ntawv piv txwv. 30 (dims = 30 raug xaiv raws li kev soj ntsuam ntawm lub hauv caug qhov chaw thiab siv rau tag nrho cov qauv thiab pawg analyses Peb mam li ua ob peb rounds ntawm iterative pawg (kev daws teeb meem = 1) los faib cov noob raws li qhov txawv txav ntawm cov noob suav (qhov nruab nrab qis dua 10th feem pua), abnormally 9 feem pua ​​​​ntawm cov noob caj noob ces. txheeb xyuas thiab tshem tawm cov cell uas tsis zoo thiab/lossis feem ntau ntawm cov menyuam ntxaib uas xav tias yog siv cov DoubletFinder33 pob (txhais tau tias DoubletFinder qhab nia saum toj no 95 feem pua ​​​​t-hCO cov qauv (n=3) thiab hCO cov qauv (n=3) tau sib xyaw ua ke sib cais siv cov ntaub ntawv sib xyaw ua ke ntawm lwm qhov kev sib xyaw ua ke ntawm cov khoom siv lim dej saum toj no ua raws li tau piav saum toj no.
Tom qab tshem tawm cov kernels tsis zoo, cov ntaub ntawv sib xyaw ua ke tau muab faib ua pawg (kev daws teeb meem = 0.5) thiab kos rau UMAP34 lub hom phiaj kev pom. Cov noob cim cim rau txhua pawg tau txiav txim siab siv FindMarkers muaj nuj nqi nrog rau qhov tsis zoo suav los ntawm cov ntaub ntawv qhia cov noob caj ces. Peb txheeb xyuas thiab faib cov chav kawm loj ntawm tes los ntawm kev sib txuas cov ntaub ntawv ntawm tus menyuam hauv plab thiab cov neeg laus cortical reference datasets nrog cov cim noob qhia 19,20,21,35 thiab annotation. Hauv tshwj xeeb, cov khoom siv ua ntej tau txheeb xyuas los ntawm kev qhia ntawm MKI67 thiab TOP2A. Cov pawg Progenitor tau txhais los ntawm qhov tsis muaj mitotic transcripts, siab sib tshooj nrog ntau lub zog glial progenitor pawg tau piav qhia nyob rau hauv metaphase fetal cortex, thiab EGFR thiab OLIG1 qhia. Peb siv lub sij hawm astrocyte los encompass ntau lub xeev ntawm astrocyte sib txawv, los ntawm lig radial glia mus rau maturation ntawm astrocytes. Astrocyte pawg qhia cov qib siab ntawm SLC1A3 thiab AQP4 thiab tau pom los ua daim ntawv qhia nrog subtypes ntawm fetal radial glia thiab / lossis cov neeg laus astrocytes. OPCs qhia PDGFRA thiab SOX10 thaum oligodendrocytes qhia cov cim myelination (MOG thiab MYRF). Glutamatergic neurons tau txheeb xyuas los ntawm qhov muaj cov ntaub ntawv pov thawj neuronal (SYT1 thiab SNAP25), qhov tsis muaj GABAergic cov cim (GAD2), thiab kev qhia ntawm NEUROD6, SLC17A7, BCL11B, lossis SATB2. GluN neurons tau muab faib ntxiv rau sab sauv (SATB2 qhia thiab poob ntawm BCL11B) thiab tob (BCL11B qhia) subclasses. Putative subplate (SP) neurons qhia paub cov cim SP18 xws li ST18 thiab SORCS1 ntxiv rau qhov sib sib zog nqus GluN cov cim. Choroid plexus-zoo li cov hlwb raug txheeb xyuas los ntawm TTR qhia, thiab cov hlwb zoo li meningeal qhia cov noob caj noob ces fibroblast thiab mapped pial / vascular hlwb ntawm cov ntaub ntawv siv.
Kev tsom xam qhov sib txawv ntawm cov noob qhia ntawm t-hCO thiab hCO subclasses tau ua los ntawm kev siv cov txheej txheem pseudo-batch tshiab tsim tawm hauv cov qauv siv los ntawm Libra R pob (version 1.0.0). Tshwj xeeb, edgeR log-likelihood tests (version 3.36.0, package R) tau ua rau cov pab pawg los ntawm kev suav cov noob hauv cov hlwb rau cov chav kawm cell rau txhua tus qauv replication. Rau heatmap visualization, normalized ib lab (CPM) qhov tseem ceeb yog xam siv edgeR (cpm() muaj nuj nqi) thiab scaled (kom ua tiav txhais tau tias = 0, tus qauv sib txawv = 1). Gene Ontology (GO) enrichment tsom xam ntawm ho upregulated t-hCO GluN noob tau ua (Benjamini-Hochberg kho P tus nqi tsawg dua 0.05 qhia nyob rau hauv tsawg kawg yog 10% ntawm t-hCO GluN hlwb thiab ib tug fold nce nyob rau hauv kev hloov ntawm tsawg kawg yog 2 zaug). ua siv TopGene Suite (https://toppgene.cchmc.org/)37. Peb siv ToppFun app nrog lub neej ntawd tsis muaj thiab tshaj tawm Benjamini-Hochberg-kho P-tus nqi xam los ntawm GO-annotated hypergeometric tests.
Txhawm rau kom phim peb snRNA-seq pawg nrog cov lus piav qhia ntawm tes los ntawm kev tshawb fawb ntawm thawj ib leeg-hlwb RNA-seq lossis cov neeg laus snRNA-seq19,20,21,22, peb siv cov ntaub ntawv sib koom ua ke. Peb tau siv SCTransform (v2) normalization workflow nyob rau hauv Seurat los integrate thiab sib piv pawg overlaps ntawm datasets (siv tib yam tsis raws li saum toj no). Cov ntaub ntawv tus kheej tau muab faib ua ntu zus mus txog 500 lub hlwb lossis cov cores ib pawg thawj coj rau kev suav ua haujlwm. Siv ib txoj hauv kev zoo ib yam li tau piav qhia yav dhau los, pawg sib tshooj tau txhais tias yog qhov feem pua ​​​​ntawm cov hlwb lossis cov nuclei hauv txhua pawg pawg uas sib tshooj nrog daim ntawv lo ntawm pawg siv. Txhawm rau faib GluNs ntxiv, peb siv Seurat's TransferData workflow rau GluN cov ntaub ntawv subset los muab cov ntaub ntawv siv cov ntawv teev npe rau peb GluN hlwb.
Txhawm rau ntsuas qhov kev loj hlob ntawm lub ntiaj teb transcriptome ntawm t-hCO thiab hCO cov qauv, peb muab piv rau peb cov pseudo-bulk qauv nrog BrainSpan / psychENCODE23, uas muaj xws li ib tug loj RNA ib theem zuj zus ntawm tib neeg lub hlwb kev loj hlob. Peb tau ua PCA ntawm cov qauv sib xyaw ua ke ntawm cov qauv sib xyaw ua ke los ntawm cov qauv cortical 10 lub lis piam tom qab kev xeeb tub thiab tom qab ntawd, hauv 5567 cov noob (ua ke nrog peb cov ntaub ntawv) uas tau txheeb xyuas yav dhau los ua haujlwm hauv BrainSpan cortical qauv (txhais tias ntau dua 50% hauv kev loj hlob sib txawv piav qhia los ntawm hnub nyoog 38. Tsis tas li ntawd, peb tau txais cov noob uas cuam tshuam nrog cov ntawv sau npe tseem ceeb ntawm kev tsim kho neurodevelopment uas tsis yog-negative matrix factorization raws li tau piav qhia dhau los. Cov qauv hnyav suav nrog cov txheej txheem tsis zoo matrix factorization yog npaj hauv Fig. 5b nrog cov ntaub ntawv nthuav dav rau txhua ntawm tsib lub npe kos npe piav qhia los ntawm Zhu li al.38. Ntxiv dua thiab, kev ua haujlwm nyob ntawm cov cim kev hloov pauv tau muab los ntawm cov kev tshawb fawb yav dhau los. Tshwj xeeb, ERG thiab LRG tau nce qib hauv glutamatergic neurons txheeb xyuas los ntawm nas pom cortex snRNA-seq sau tom qab kev pom kev pom los ntawm Cov Lus Ntxiv 3 Hrvatin li al.16. Tib neeg-enriched LRGs tau txais los ntawm KCl-activated human fetal brain cultures and harvested 6 hours post-stimulation, and the filtered genes were significantly upregulated in humans but not in rats (Supplementary Table 4). Kev soj ntsuam ntawm cov noob txheej enrichment siv cov noob caj noob ces tau ua los ntawm ib txoj kev Fisher qhov tseeb.
Tshuaj loog nas nrog isoflurane, tshem tawm lub hlwb thiab tso rau hauv txias (kwv yees li 4 ° C) oxygenated (95% O2 thiab 5% CO2) sucrose tov rau cov seem uas muaj: 234 mM sucrose, 11 mM qabzib, 26 mM NaHCO3, 2.5 mM KCl, 1. NaH2PO4, 10 mM MgSO4 thiab 0.5 mM CaCl2 (txog 310 mOsm). Rat hlwb coronal seem (300–400 µm) uas muaj t-hCO tau tsim los siv Leica VT1200 vibratome raws li tau piav qhia yav dhau los39. Tom qab ntawd cov seem raug xa mus rau ib chav tsev nrog rau chav tsev kub tsis tu ncua oxygenation uas muaj aCSF npaj los ntawm: 10 mM qabzib, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 thiab 126 mM NaCl (298 mOsm). tsawg kawg yog 45 feeb ua ntej kaw. Cov seem tau sau tseg rau hauv ib chav immersed uas lawv tau txuas ntxiv perfused nrog aCSF (95% O2 thiab 5% CO2 vial). Tag nrho cov ntaub ntawv raug kaw nyob rau hauv chav tsev kub. t-hCO neurons raug txiav nrog borosilicate iav pipette uas muaj cov tshuaj uas muaj 127 mM poov tshuaj gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, thiab 0.6 mM EGTA, pH 7.2, cov tshuaj sab hauv kho nrog 20 mKO (mKO sm). Txhawm rau kom rov zoo, biocytin (0.2%) tau ntxiv rau cov tshuaj kaw.
Cov ntaub ntawv tau txais los ntawm MultiClamp 700B amplifier (Molecular Devices) thiab Digidata 1550B digitizer (Molecular Devices), low-pass filtered at 2 kHz, digitized at 20 kHz, and analyzed using Clampfit (Molecular Devices), Origin (OriginPro). 2021b, OriginLab). thiab kev cai MATLAB functions (Mathworks). Lub peev xwm sib txuas tau suav nrog JPCalc thiab cov ntawv nkag tau hloov kho rau tus nqi suav ntawm -14 mV. Kev Ua Haujlwm IV muaj cov kauj ruam tam sim no hauv 10-25 pA cov kauj ruam, los ntawm -250 mus rau 750 pA.
Cov thalamus, cov teeb meem dawb, thiab S1 afferents tau hluav taws xob txhawb nqa hauv thalamocortical slices thaum lub sij hawm patch-clamp kaw ntawm hCO neurons, raws li tau piav qhia yav dhau los. Luv luv, lub hlwb tau muab tso rau ntawm lub rooj 3D luam ntawv tilted ntawm lub kaum sab xis 10 °, thiab sab xub ntiag ntawm lub hlwb raug txiav ntawm lub kaum sab xis 35 °. Tom qab ntawd lub hlwb raug muab khi rau ntawm qhov chaw txiav thiab txiav, khaws cia cov thalamocortical protruding axons. Bipolar tungsten electrodes (0.5 MΩ) tau teeb tsa rau ntawm lub micromanipulator thib ob thiab tau tsim cov tswv yim los txhawb plaub thaj tsam ntawm ib lub xov tooj (cov tshuaj ntsiav, cov teeb meem dawb, S1 thiab hCO). Sau cov lus teb synaptic tom qab 300 µA phasic stimulation ntawm 0.03-0.1 Hz.
hChR2-expressing hCO neurons tau qhib rau ntawm 480 nm thiab lub teeb pulses tsim los ntawm LED (Prizmatix) tau siv los ntawm lub hom phiaj × 40 (0.9 NA; Olympus) los sau hChR2 qhia nyob ze ntawm lub hlwb. Lub illuminated teb txoj kab uas hla yog kwv yees li 0.5 hli thiab tag nrho lub hwj chim yog 10-20 mW. Cov mem tes dav tau teem rau 10 ms, uas sib haum rau cov mem tes muab thaum lub sij hawm xyaum coj cwj pwm. Ntau yam stimulation zaus tau siv, los ntawm 1 mus rau 20 Hz, tab sis tsuas yog thawj lub mem tes ntawm cov koob tau siv rau kom muaj nuj nqis. Lub sijhawm nruab nrab ntawm cov tsheb ciav hlau feem ntau yog ntev dua 30 s kom txo tau qhov cuam tshuam ntawm synaptic inhibitory lossis pab txoj hauv kev. Txhawm rau kuaj seb cov lus teb hChR2 yog monosynaptic, peb thov TTX (1 μM) rau da dej kom txog thaum cov tshuaj tiv thaiv EPSC ploj, thiab tom qab ntawd thov 4-aminopyridine (4-AP; 100 μM). Feem ntau, cov lus teb tau xa rov qab li ntawm ob peb feeb, nrog kev ncua ntev me ntsis ntawm LED firing thiab EPSC tiam. NBQX (10 μM) tau siv los ntsuas seb cov lus teb puas tau tsav los ntawm AMPA receptors.
Cov ntu ntse hCO tau tsim raws li tau piav qhia dhau los. Luv luv, hCO seem tau muab tso rau hauv 4% agarose thiab xa mus rau cov hlwb uas muaj 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 thiab 10 mM d-ose (0+ 0µg) ntawm ntu 0µg. ntawm chav tsev kub uas siv lub vibrator Leica VT1200 thiab khaws cia hauv ASF ntawm chav tsev kub. Tom qab ntawd, thaj chaw kaw cia ntawm tag nrho cov hlwb tau ua tiav ntawm cov ntu hCO raws li SliceScope microscope (Scientifica). Cov seem tau perfused nrog aCSF (95% O2 thiab 5% CO2) thiab cell signals tau kaw nyob rau hauv chav tsev kub. hCO neurons tau siv los ntawm borosilicate iav pipette uas muaj cov tshuaj uas muaj 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, thiab 0.6 mM EGTA, sab hauv pH 7, 2, kho nrog KOH (osmolarity). Rau lub hom phiaj rov qab, ntxiv 0.2% Biocytin rau cov tshuaj sab hauv.
Cov ntaub ntawv tau txais los ntawm Clampex (Clampex 11.1, Molecular Devices) siv MultiClamp 700B amplifier (Molecular Devices) thiab Digidata 1550B digitizer (Molecular Devices), low-pass filtered at 2 kHz, digitized at 20 kHz., thiab haum rau kev soj ntsuam siv 1. custom MATLAB functions (MATLAB 2019b, Mathworks). Lub peev xwm sib txuas tau suav nrog JPCalc thiab cov kev nkag tau raug hloov kho rau kev suav qhov sib txuas ntawm -14 mV. Kev Ua Haujlwm IV muaj cov kauj ruam tam sim no hauv 5-10 pA cov kauj ruam ntawm -50 txog 250 pA.
Rau kev tsim kho morphological ntawm pinched neurons, 0.2% biocytin (Sigma-Aldrich) tau ntxiv rau cov tshuaj sab hauv. Cov hlwb yog primed rau yam tsawg kawg 15 feeb tom qab hacking. Lub pipette yog maj mam rub rau hauv 1-2 min kom txog rau thaum cov ntawv sau npe tau kaw tag. Ua raws li cov txheej txheem physiology, ntu tau kho thaum hmo ntuj ntawm 4 ° C. hauv 4% PFA, ntxuav nrog PBS X3, thiab diluted 1: 1000 nrog streptavidin-conjugated DyLight 549 lossis DyLight 405 (Vector Labs). Cells uas muaj biocytin (2%; Sigma-Aldrich) tau sau npe thaum lub sij hawm patch clamp kaw ntawm chav tsev kub rau 2 teev. Cov ntu tom qab ntawd tau muab tso rau ntawm cov duab me me uas siv lub Aquamount (Thermo Scientific) thiab pom cov hnub tom qab ntawm Leica TCS SP8 confocal microscope siv lub hom phiaj roj immersion nrog cov lej aperture × 40 1.3, magnification × 0.9–1.0, xy. Tus nqi piv txwv yog kwv yees li 7 pixels ib micron. Z-stacks ntawm 1 µm ncua sij hawm tau txais serially, thiab z-stack mosaics thiab Leica-raws li nws pib-stitching tau ua los npog tag nrho cov ntoo dendritic ntawm txhua neuron. Neurons tau taug qab ib nrab ntawm kev siv lub neuTube 40 interface thiab SWC cov ntaub ntawv raug tsim tawm. Cov ntaub ntawv tau raug xa mus rau SimpleNeuriteTracer41 Fiji plugin (ImageJ, version 2.1.0; NIH).
Tib neeg cov ntaub so ntswg cortical tau txais nrog kev pom zoo raws li kev pom zoo los ntawm Pawg Saib Xyuas Kev Tshawb Fawb ntawm Stanford University. Ob qho piv txwv ntawm tib neeg cov ntaub so ntswg tom qab yug me nyuam (3 thiab 18 xyoo) tau txais los ntawm kev tshem tawm ntawm lub ntsej muag cortex (middle frontal gyrus) ua ib feem ntawm kev phais rau refractory epilepsy. Tom qab tshem tawm, sau cov ntaub so ntswg hauv cov dej khov nab kuab NMDG-aCSF uas muaj: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM qabzib, 2 mM thiourea, 5 mM sodiumate mM, ascorb 5 mM. CaCl2 4H2O thiab 10 mM MgSO4 7H2O. Titrate rau pH 7.3-7.4 nrog concentrated hydrochloric acid. Cov ntaub so ntswg raug xa mus rau chav kuaj hauv 30 feeb thiab cov kab mob coronal raug coj raws li cov txheej txheem tau piav qhia saum toj no.
Txhua tus txheej txheem tsiaj tau ua raws li Stanford University APLAC-pom zoo cov txheej txheem saib xyuas tsiaj. Cov nas tsuag (ntau dua 140 hnub tom qab hloov pauv) tau raug ntxias nrog 5% isoflurane tshuaj loog thiab tshuaj loog nrog 1-3% isoflurane intraoperatively. Tsiaj txhu tau muab tso rau hauv stereotaxic thav duab (Kopf) thiab kev tso tawm buprenorphine (SR) tau txhaj tshuaj subcutaneously. Lub pob txha taub hau raug nthuav tawm, ntxuav thiab 3-5 cov pob txha pob txha raug tso. Txhawm rau tsom t-hCO, peb tsim cov kev tswj hwm stereotaxic los ntawm MRI cov duab. Lub qhov burr tau drilled ntawm qhov chaw txaus siab thiab fibers (400 µm txoj kab uas hla, NA 0.48, Doric) tau qis dua 100 µm hauv qab hCO nto thiab ruaj ntseg rau pob txha taub hau nrog UV-kho hniav cement (Relyx).
Fiber photometric kaw tau ua raws li tau piav qhia dhau los42. Txhawm rau sau cov haujlwm tsis tu ncua, nas tau muab tso rau hauv lub tawb huv si thiab 400 µm txoj kab uas hla fiber ntau optic thaj cable (Doric) txuas nrog fiber ntau optic photometric cov ntaub ntawv nrhiav tau qhov system txuas nrog rau cov khoom siv fiber ntau optic cable. Thaum lub sijhawm 10-feeb kaw ntawm lub cev muaj zog, cov tsiaj tuaj yeem tshawb xyuas lub tawb. Txhawm rau sau cov haujlwm evoked, nas (ntau dua 140 hnub tom qab hloov pauv) tau tshuaj loog nrog 5% isoflurane rau induction thiab 1-3% isoflurane rau kev kho. Muab cov tsiaj tso rau hauv ib qho stereotactic thav duab (Kopf) thiab cov whiskers ntawm sab nraud ntawm t-hCO yog txiav mus rau qhov ntev ntawm 2 cm thiab dhau los ntawm ib lub mesh txuas nrog lub piezoelectric actuator (PI). Ib qho 400 µm fiber ntau optic thaj cable (Doric) tau txuas nrog cov cog fiber ntau thiab txuas nrog cov ntaub ntawv tau txais. Cov whiskers nyob rau sab nraud ntawm t-hCO ces deflected 50 zaug (2 hli ntawm 20 Hz, 2 s ib qho kev nthuav qhia) ntawm random lub sij hawm los ntawm ib tug piezoelectric tsav tshaj 20 feeb teev. Siv Arduino MATLAB Support Package los tswj lub sij hawm deflection nrog kev cai MATLAB code. Cov xwm txheej tau synchronized rau cov ntaub ntawv tau txais software siv transistor-transistor logic (TTL) pulses.
Cov nas tsuag (ntau dua 140 hnub tom qab hloov pauv) tau raug ntxias nrog 5% isoflurane tshuaj loog thiab tshuaj loog nrog 1-3% isoflurane intraoperatively. Tsiaj txhu tau muab tso rau hauv stereotaxic thav duab (Kopf) thiab buprenorphine SR thiab dexamethasone tau txhaj tshuaj subcutaneously. Lub pob txha taub hau raug nthuav tawm, ntxuav thiab 3-5 cov pob txha pob txha raug tso. Txhawm rau tsom t-hCO, peb tsim cov kev tswj hwm stereotaxic los ntawm MRI cov duab. Ib ncig craniotomy (kwv yees li 1 cm nyob rau hauv txoj kab uas hla) tau ua nrog kev kub ceev xyaum ncaj qha hla lub hCO hloov. Thaum cov pob txha nyias nyias li ua tau, tab sis ua ntej drilling los ntawm tag nrho cov pob txha, siv forceps tshem tawm cov seem hauv plab pelvic disc kom pom cov hauv paus t-hCO. Lub craniotomy tau ntim nrog cov kua qaub tsis muaj kuab lom, thiab daim npog npog thiab lub taub hau tshwj xeeb tau txuas rau pob txha taub hau nrog UV-kho kho hniav cement (Relyx).
Ob-photon duab tau ua tiav siv Bruker multiphoton microscope nrog Nikon LWD (× 16, 0.8 NA) lub hom phiaj. GCaMP6 imaging tau ua tiav ntawm 920 nm nrog 1.4x ib zaug z-plane magnification thiab 8x nruab nrab ntawm 7.5 fps. Cov nas raug ntxias nrog 5% isoflurane tshuaj loog thiab tswj nrog 1-3% isoflurane. Cov nas tsuag tau muab tso rau hauv ib qho kev cai ua lub taub hau thiab muab tso rau hauv qab lub lens. 3-feeb tom qab kaw ntawm lub cev muaj zog tau txais. Tshaj li ntawm 20 feeb ntawm kev kaw, 50 puffs (txhua qhov kev nthuav qhia 100 ms ntev) tau muab xa mus rau lub ntsej muag whisker rov qab t-hCO siv lub picospricer. Siv Arduino MATLAB Support Package los tswj lub sijhawm tawg nrog kev cai MATLAB code. Synchronize cov xwm txheej nrog cov ntaub ntawv tau txais software (PrairieView 5.5) siv TTL pulses. Rau kev tsom xam, cov duab tau raug kho rau xyov kev siv affine kho nyob rau hauv MoCo qhov kev pab cuam launched hauv Fiji. Kev tshem tawm cov kab mob fluorescent los ntawm ib tus neeg lub hlwb siv CNMF-E43. Fluorescence tau muab rho tawm rau txhua cheeb tsam ntawm kev txaus siab, hloov mus rau dF / F nkhaus, thiab tom qab ntawd hloov mus rau z-cov qhab nia.
Cov nas tsuag (ntau dua 140 hnub tom qab hloov pauv) tau raug ntxias nrog 5% isoflurane tshuaj loog thiab tshuaj loog nrog 1-3% isoflurane intraoperatively. Tsiaj txhu tau muab tso rau hauv stereotaxic thav duab (Kopf) thiab buprenorphine SR thiab dexamethasone tau txhaj tshuaj subcutaneously. Lub whiskers nyob rau sab nraud ntawm t-hCO raug txiav mus txog 2 cm thiab threaded los ntawm ib tug mesh txuas nrog lub piezoelectric actuator. Lub pob txha taub hau raug nthuav tawm thiab ntxuav. Ib qho stainless hlau hauv av ntsia hlau yog txuas rau pob txha taub hau. Txhawm rau tsom t-hCO, peb tsim cov kev tswj hwm stereotaxic los ntawm MRI cov duab. Ua ib ncig craniotomy (kwv yees li 1 cm hauv txoj kab uas hla) nrog rau kev xyaum siab ceev tshaj li t-hCO. Thaum cov pob txha nyias nyias li ua tau, tab sis ua ntej drilling los ntawm tag nrho cov pob txha, siv forceps tshem tawm cov seem hauv plab pelvic disc kom pom cov hauv paus t-hCO. Cov hlwb raug kaw siv 32-channel lossis 64-channel high-density silicon probes (Cambridge Neurotech) grounded rau hauv av screws thiab pre-amplified nrog RHD amplifiers (Intan). Siv tus tswj xyuas kom txo cov electrodes mus rau qhov chaw ntawm lub hom phiaj los ntawm craniotomy, uas tau ntim nrog cov kua qaub tsis huv. Kev sau cov ntaub ntawv tau ua ntawm qhov zaus ntawm 30 kHz siv Open Ephys cov ntaub ntawv nrhiav tau. Cov ntaub ntawv txuas ntxiv tsuas yog thaum peb kuaj pom muaj kev sib raug zoo nrog kev ua haujlwm ntawm lub cev hauv ntau tshaj 10 txoj kev, qhia tias cov electrodes tau nyob rau hauv cov graft (raws li ob-photon calcium imaging data). Tau txais 10-feeb tom qab kaw ntawm lub cev muaj zog. Cov whiskers nyob rau sab nraud ntawm t-hCO ces deflected 50 zaug (2 hli ntawm 20 Hz, 2 s ib qho kev nthuav qhia) ntawm random lub sij hawm los ntawm ib tug piezoelectric tsav tshaj 20 feeb teev. Siv MATLAB Support Package for Arduino (MATLAB 2019b), tswj lub sij hawm deflection nrog kev cai MATLAB code. Siv TTL pulses los synchronize cov xwm txheej nrog cov ntaub ntawv nrhiav software.
Rau kev sim ntsuas qhov muag pom, 200 µm optical thaj qaum (Doric) txuas nrog 473 nm laser (Omicron) tau txuas nrog 200 µm kho qhov muag fiber ntau tso rau ntawm craniotomy. Tam sim ntawd ua ntej no, kho lub zog jumper rau 20 mW. Siv tus tswj xyuas kom txo cov electrodes mus rau qhov chaw ntawm lub hom phiaj los ntawm craniotomy, uas tau ntim nrog cov kua qaub tsis huv. Thaum pib ntawm kev kaw, kaum pulses ntawm lub teeb 473 nm (frequency 2 Hz, mem tes ntev 10 ms) tau tawm. Cov hlwb photosensitive tau raug txhais tias yog cov hlwb uas nthuav tawm cov lus teb dav dav hauv 10 ms ntawm lub teeb hauv 70% lossis ntau dua ntawm kev sim.