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Kudziphatika kwa neural organelles kumayimira nsanja yodalirika mu vitro yowonetsera chitukuko cha anthu ndi matenda. Komabe, ma organoids alibe kulumikizana komwe kuli mu vivo, komwe kumachepetsa kukhwima ndikuletsa kusakanikirana ndi mabwalo ena omwe amawongolera machitidwe. Apa tikuwonetsa kuti ma cell stem cell-derived cortical organoids amalowetsedwa mu somatosensory cortex ya neonatal maliseche makoswe amapanga maselo okhwima omwe amaphatikizana ndi mabwalo okhudzidwa ndi zolimbikitsa. MRI idavumbulutsa kukula kwa organoid pambuyo pomuika m'mizere ingapo yama cell ndi nyama, pomwe kusanthula kwapakatikati kunavumbulutsa kupita patsogolo kwa corticogenesis komanso kuwonekera kwa pulogalamu yolembera yodalira ntchito. Zowonadi, ma cortical neurons oyikidwa amawonetsa zovuta kwambiri za morphological, synaptic, and membrane yamkati kuposa ma in vitro, zomwe zimalola kuzindikira zolakwika za neuronal mwa odwala omwe ali ndi matenda a Timothy. Kufufuza kwa ma anatomical ndi magwiridwe antchito kwawonetsa kuti ma organelles obzalidwa amalandira zolowetsa za thalamocortical ndi corticocortical, ndipo zojambulira mu vivo za zochitika za neural zikuwonetsa kuti zolowetsazi zimatha kupanga mayankho omveka m'maselo amunthu. Pomaliza, ma cortical organoids amakulitsa ma axon muubongo wonse wa makoswe, ndipo kuyambitsa kwawo kwa optogenetic kumabweretsa kufunafuna mphotho. Chifukwa chake, ma cortex neurons osinthidwa amakhwima ndikuchita nawo mabwalo omwe amawongolera machitidwe. Tikuyembekeza njira iyi kuti iwonetsetse ma phenotypes a strand-level m'maselo opangidwa ndi odwala omwe sangathe kudziwika ndi njira zina.
Ubongo wamunthu womwe ukukula ndi njira yodzikonzekeretsa yodabwitsa momwe maselo amachulukira, kusiyanitsa, kusamuka, ndikulumikizana kuti apange ma neurons omwe amagwira ntchito bwino omwe amayengedwanso kudzera mu chidziwitso. Vuto lalikulu pakumvetsetsa kukula kwa ubongo waumunthu, makamaka pankhani ya matenda, ndi kusowa kwa minyewa yaubongo. Ma organelle odzipangira okha, kuphatikiza ma cortex organoids (hCO; amadziwikanso kuti human cortex sphere), amatha kupanga 2,3,4,5,6. Komabe, zolepheretsa zingapo zimalepheretsa kugwiritsa ntchito kwawo kwakukulu kumvetsetsa kakulidwe ndi kagwiritsidwe ntchito ka ma neural circuits. Makamaka, sizikudziwika ngati kusasitsa kwa hCO kumachepa chifukwa chosowa zinthu zina za microenvironmental ndi zomverera zomwe zilipo mu vivo. Kuphatikiza apo, chifukwa ma hCO sanaphatikizidwe m'mabwalo omwe angapangitse zotsatira zamakhalidwe, ntchito yawo popanga ma genetic ovuta komanso zovuta zama neuropsychiatric pakali pano ndizochepa.
Kuyika kwa hCO muubongo wamoyo wokhazikika kumatha kuthana ndi izi. Kafukufuku wam'mbuyomu awonetsa kuti ma neuron amunthu omwe adalowetsedwa mu rodent cortex amatha kukhala ndi moyo, projekiti, komanso kulumikizana ndi makoswe7,8,9,10,11,12. Komabe, zoyesererazi nthawi zambiri zimachitidwa pa nyama zazikulu, zomwe zimatha kuchepetsa kuphatikiza kwa synaptic ndi axonal. Apa, tikufotokozera paradigm yosinthira momwe tidasinthira 3D hCO yochokera ku ma cell a hiPS kupita ku somatosensory cortex (S1) ya makoswe a immunodeficient koyambirira kwa pulasitiki. Ma neurons osiyidwa a hCO (t-hCO) amakhwima kwambiri, amalandila zolowetsa za thalamocortical ndi cortical-cortical zomwe zimabweretsa mayankho amalingaliro, ndikukulitsa ma axonal muubongo wa makoswe kuti ayendetse khalidwe lofunafuna mphotho. Kukula kowonjezereka kwa t-hCO kwawonetsa kuwonongeka kwa neuronal kwa odwala omwe ali ndi matenda a Timothy (TS), vuto lalikulu la majini lomwe limayambitsidwa ndi masinthidwe amtundu wa L-sensitive L-mtundu wa CaV1.2 calcium channel (wosungidwa ndi CACNA1C).
Kuti tiphunzire ma neuroni amtundu wa anthu m'mabwalo mu vivo, tidasinthiratu 3D hCO yosasinthika mu S1 ya makoswe oyambirira a athymic (masiku 3-7 pambuyo pobereka) (Mkuyu 1a ndi deta yowonjezera ya Mkuyu 1a-c). Panthawiyi, thalamocortical ndi corticocortical axonal projections sanamalize S1 innervation yawo (ref. 13). Chifukwa chake, njira iyi idapangidwa kuti iwonjezere kuphatikiza kwa t-hCO ndikuchepetsa kukhudzidwa kwa mabwalo amkati. Kuti tiwone momwe malo a t-hCO alili mu zinyama zamoyo, tinapanga T2-zolemera za MRI zokonzanso ubongo wa makoswe 2-3 miyezi pambuyo pa kuikidwa (Mkuyu 1b ndi deta yowonjezereka, Mkuyu 1d). t-hCO inawonedwa mosavuta ndipo miyeso ya voliyumu ya t-hCO inali yofanana ndi yowerengedwa kuchokera ku magawo okhazikika (Extended Data Fig. 1d, e; P> 0.05). t-hCO inawonedwa mosavuta ndipo miyeso ya voliyumu ya t-hCO inali yofanana ndi yowerengedwa kuchokera ku magawo okhazikika (Extended Data Fig. 1d, e; P> 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированных срезов (расширенны, ризерены, 1, 1, 11; 0,05 ndime. t-hCO inkawoneka mosavuta, ndipo miyeso ya volumetric t-hCO inali yofanana ndi yowerengera magawo okhazikika (deta yowonjezera, Fig. 1d, e; P> 0.05).很容易观察到t-hCO，并且t-hCO 的体积测量值与从固定切片计算的测量值相似（扩扩（扩似（扩似（扩似（扩（扩（扩（ 0.05.很容易观察到t-hCO，并且t-hCO t-hCO легко наблюдался, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированных срезов (расширенны, песни 1, данных срезов) 0,05 ndime. t-hCO inkawoneka mosavuta, ndipo miyeso ya volumetric t-hCO inali yofanana ndi yowerengedwa pazigawo zokhazikika (deta yowonjezera, Fig. 1d, e; P> 0.05).Tidatsimikiza t-hCO mu 81% ya nyama zomwe zidasiyidwa pafupifupi miyezi iwiri mutatha kuziika (n = 72 nyama; hCO kuchokera ku mizere ya cell ya 10 hiPS; mizere ya cell ya hiPS mu Supplementary Table 1). Mwa awa, 87% anali mu cerebral cortex (mkuyu 1c). Pochita zojambula za MRI zowonongeka pazigawo zingapo mu makoswe omwe anaziika, tinapeza kuwonjezeka kasanu ndi kamodzi kwa voliyumu ya t-hCO mkati mwa miyezi ya 3 (mkuyu 1d ndi deta yowonjezera, Fig. 1f). Zinyama zomwe zinasinthidwa zinali ndi moyo wapamwamba (74%) pa miyezi ya 12 pambuyo pa kupatsirana (deta yowonjezera, Fig. 1g ndi Supplementary Table 2), ndipo palibe kuwonongeka kwa galimoto kapena kukumbukira kukumbukira, gliosis, kapena electroencephalogram (EEG) anapezeka. Deta Fig. 1g ndi tebulo lowonjezera 2). 1h–m ndi 3e).
a, Schematic of experimental design. hCO yochokera ku ma cell a hiPS idasiyidwa mu S1 ya makoswe obadwa kumene amaliseche pamasiku 30-60 osiyanitsa. b, zithunzi za T2 zolemetsa za coronal ndi zopingasa za MRI zosonyeza t-hCO mu S1 2 miyezi pambuyo pa kuikidwa. Mzere wapakati, 2 mm. c, Kuchulukitsidwa kwa ma engraftment opambana omwe amawonetsedwa pamzere uliwonse wa hiPS cell (n = 108, manambala mkati mwa mipiringidzo amawonetsa kuchuluka kwa t-hCO pamzere wa cell wa hIPS) ndi malo a cortical kapena subcortical (n = 88). d, chithunzi cha MRI cha mtsempha wamagazi (kumanzere; sikelo, 3 mm) ndi kukonzanso kwa 3D volumetric (scale bar, 3 mm) kusonyeza kuwonjezeka kwa t-hCO pa miyezi itatu. e, Kuunikanso kwa ma t-hCO mu makoswe a cerebral cortex. Mlingo wamba, 1 mm. f, Zithunzi zoimira immunocytochemical za t-hCO zosonyezedwa kuchokera pamwamba kumanzere kupita kumanja (pa nthawi yosiyana): PPP1R17 (miyezi 4), NeuN (miyezi 8), SOX9 ndi GFAP (miyezi 8), PDGFRα; (Miyezi 8), MAP2 (miyezi 8) ndi IBA1 (miyezi 8). Scale bar, 20 µm. Co-mafotokozedwe a HNA amasonyeza maselo chiyambi cha munthu. g. Astrocyte, maselo a mzere wa astrocyte; cycl prog, ozungulira makolo; GluN DL, mitsempha yakuya ya glutamatergic; GluN DL/SP, zakuya ndi sublamellar glutamatergic neurons; GluN UL, ma neurons apamwamba a glutamatergic; oligodendrocytes, oligodendrocytes; OPC, maselo a oligodendrocyte; RELN, reelin neurons. h, Gene Ontology (GO) kusanthula kwanthawi yayitali kwa majini kumayendetsedwa bwino (kusinthidwa P <0.05, pindani kusintha> 2, kuwonetsedwa osachepera 10% ya nuclei) mu t-hCO glutamatergic neurons poyerekeza ndi hCO glutamatergic neurons. h, Gene Ontology (GO) kusanthula kwanthawi yayitali kwa majini kumayendetsedwa bwino (kusinthidwa P <0.05, pindani kusintha> 2, kuwonetsedwa osachepera 10% ya nuclei) mu t-hCO glutamatergic neurons poyerekeza ndi hCO glutamatergic neurons. h. в 10% ядер) mu глутаматергических нейронах t-hCO pa глутаматергическими нейронами hCO. h, Gene Ontology (GO) kusanthula kwanthawi yayitali kwa majini omwe ali ndi kutsegulira kwakukulu (kusinthidwa P <0.05, pinda kusintha> 2, kufotokozera osachepera 10% nuclei) mu t-hCO glutamatergic neurons poyerekeza ndi 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 <0.05>倏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2 , 至少 10% 的 核中 表达） 基因 基因 h, гены значительно активизировались (скорректированный P <0,05, кратность изменения> 2, экспрессируется по крайней мере мере 10% нейронах t-hCO pa сравнению с глутаматергическими нейронами hCO Онтологический (GO) анализ термина обогащения. h, majini adasinthidwa kwambiri (kusinthidwa P <0.05, pindani kusintha> 2, kufotokozedwa osachepera 10% ya nuclei) mu t-hCO glutamatergic neurons poyerekeza ndi hCO glutamatergic neurons Ontological (GO) kusanthula kwa nthawi yopindulitsa.Mzere wamadontho ukuwonetsa mtengo wa aq wa 0.05. i, kuyerekezera kwa UMAP kwa mitundu ya ma cell a GluN mu t-hCO pogwiritsa ntchito kusamutsidwa kwa zilembo kuchokera ku referensi 22 snRNA-seq wamkulu motor cortex dataset. CT - maselo a corticothalamic, ET - maselo a extracerebral, IT - maselo a telencephalic amkati, NP - pafupi ndi pulojekiti.
Kenako tidawunika mawonekedwe a cytoarchitecture komanso ma cell a t-hCO. Kudetsa kwa antibody kwa makoswe endothelial maselo kunavumbulutsa vascularization ndi t-hCO, pamene IBA1 staining inasonyeza kukhalapo kwa makoswe a microglia muzitsulo zonse (Mkuyu 1f ndi deta yowonjezera, Mkuyu 3c, d). Immunostaining inavumbulutsa maselo abwino a nyukiliya (HNA) omwe amafotokozera PPP1R17 (cortical progenitors), NeuN (neurons), SOX9 ndi GFAP (maselo opangidwa ndi glial) kapena PDGFRα (oligodendrocyte progenitors) (Chithunzi 1f). Kuti tiphunzire kapangidwe ka ma cell a t-hCO pakusintha kwa cell imodzi, tidachita ma single-core RNA sequencing (snRNA-seq) patatha pafupifupi miyezi 8 yosiyana. Kusefedwa kochuluka ndi kuchotsedwa kwa makoswe kunapereka mapu a 21,500 apamwamba aumunthu a mononuclear (Mkuyu 1g ndi deta yowonjezera, Chithunzi 4a, b). Mawonekedwe a zolembera zamtundu wa ma cell amazindikiritsa magulu amagulu akuluakulu a cortical cell, kuphatikiza ma neuroni akuya komanso apamwamba kwambiri a glutamatergic, ma progenitor ozungulira, oligodendrocytes, ndi mzere wa astrocyte (mkuyu 1g, deta yowonjezereka, mkuyu 4c, ndi Supplementary Table 3). Immunostaining kwa SATB2 ndi CTIP2 inasonyeza kuti ngakhale kukhalapo kwa cortical subtypes, t-hCO sinasonyeze bwino stratification ya anatomical (deta yowonjezera, mkuyu 3a). siteji yofanana ndi snRNA-seq hCO inapanga magulu a maselo ofanana kwambiri, kupatulapo ochepa, kuphatikizapo kusowa kwa oligodendrocytes ndi kukhalapo kwa GABAergic neurons, zomwe zingasonyeze zomwe zinanenedwa kale kuti zili bwino mu m'galasi ma cell a lateral progenitor cells15 (deta yowonjezera, Mkuyu 4f - i ndi Supplementary Table 4). Kusanthula kwa ma jini osiyanasiyana kunawonetsa kusiyana kwakukulu kwa ma glutamatergic neurons pakati pa t-hCO ndi hCO (Supplementary Table 5), kuphatikiza kuyambitsa ma jini okhudzana ndi kusasitsa kwa neuronal monga ma synaptic signing, dendritic localization, and voltage-gated channel activity (Chithunzi 1h ndi Supplementary Table 5). tebulo 6). Chifukwa chake, ma cortical glutamatergic t-hCO neurons adawonetsa kukhwima kwa mawu olembedwa.
Kuti tidziwe ngati kusintha kwa zilembo za t-hCO kunali kogwirizana ndi kusiyana kwa morphological pakati pa hCO mu vitro ndi t-hCO mu vivo, tinamanganso hCO yodzaza ndi biocytin yodzaza ndi hCO ndi hCO m'magawo ovuta pambuyo pa miyezi 7-8 yosiyana. hCO neurons (mkuyu 2a). T-hCO neurons inali yaikulu kwambiri, inali ndi 1.5 nthawi ya soma m'mimba mwake, kawiri kawiri dendrites, ndi kuwonjezeka kasanu ndi kamodzi mu kutalika kwa dendritic poyerekeza ndi in vitro hCO (Mkuyu 2b). Kuonjezera apo, tinawona kuchuluka kwakukulu kwa dendritic spines mu t-hCO neurons kusiyana ndi hCO neurons (Mkuyu 2c). Izi zikusonyeza kuti t-hCO neurons imadutsa kwambiri dendritic elongation ndi nthambi, zomwe, kuphatikizapo kupitiriza kufalikira kwa maselo, zingathandize kuti t-hCO ikhale yowonjezereka pambuyo pa kuikidwa (Fig. 1d ndi Extended Data Fig. 1f). Izi zidatipangitsa kuti tifufuze ma electrophysiological properties. Kuthekera kwa Membrane kunali kuwirikiza kasanu ndi katatu (deta yowonjezera, mkuyu 8d), mphamvu ya membrane yopuma inali yowonjezereka kwambiri (pafupifupi 20 mV), ndipo jekeseni yamakono inachititsa kuti pakhale chisangalalo chapamwamba kwambiri mu t-hCO neurons kusiyana ndi hCO neurons. mu vitro (mkuyu 2d), e), zomwe zimagwirizana ndi zazikulu komanso zovuta kwambiri za morphological t-hCO. Kuonjezera apo, mafupipafupi a zochitika zowonongeka za postynaptic zamakono (EPSC) zinali zapamwamba kwambiri mu t-hCO neurons (mkuyu 2f), kutanthauza kuti kuchulukitsidwa kwa dendritic spines komwe kumawonedwa mu t-hCO neurons kumagwirizanitsidwa ndi chisangalalo chogwira ntchito. kugonana synapses. Tinatsimikizira khalidwe losakhwima la ma hCO neurons mu vitro polemba otchedwa glutamatergic neurons (deta yowonjezera, Fig. 6a-c).
a, kukonzanso kwa 3D kwa hCO yodzaza ndi biocytin ndi t-hCO neurons pambuyo pa miyezi 8 yosiyana. b, Kuchuluka kwa mawonekedwe a morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0084, *P = 0.0179 ndi ***P <0.0001). b, Kuchuluka kwa mawonekedwe a morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0084, *P = 0.0179 ndi ***P <0.0001). б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 ndi *** P <0,00). b, kuchuluka kwa mawonekedwe a morphological (n=8 hCO neurons, n=6 t-hCO neurons; **P=0.0084, *P=0.0179, ndi ***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 = 0.0179 和**00 . 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 = 0.0179 和**00 . б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 ndi *** P <0,00). b, kuchuluka kwa mawonekedwe a morphological (n=8 hCO neurons, n=6 t-hCO neurons; **P=0.0084, *P=0.0179, ndi ***P<0.0001).c, kukonzanso kwa 3D kwa nthambi za hCO ndi t-hCO dendritic pambuyo pa miyezi 8 yosiyana. Nyenyezi zofiira zimawonetsa misana ya putative dendritic. Dendritic spine density quantification (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0092). d, Kuwerengera mphamvu ya nembanemba yopumula (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, Kuwerengera mphamvu ya nembanemba yopumula (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 <0,0001). d, kupumula kwa nembanemba komwe kungatheke (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 <0,0001). d, kupumula kwa nembanemba komwe kungatheke (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Kubwerezabwereza zomwe zingatheke kuwombera mu hCO ndi t-hCO chifukwa cha kuchuluka kwa jakisoni wamakono, ndi kuchuluka kwa kuwombera kwakukulu (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Kubwerezabwereza zomwe zingatheke kuwombera mu hCO ndi t-hCO chifukwa cha kuchuluka kwa jakisoni wamakono, ndi kuchuluka kwa kuwombera kwakukulu (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, повторное возбуждение потенциала действия в hCO ndi t-hCO, вызванное увеличением тока, и количественная оценка максимальной скорости 2 возh, CONTENTI 2 возh; n = 16 нейронов t-hCO *** P <0,0001). e, zomwe zingatheke kuwomberanso mu hCO ndi t-hCO chifukwa cha kuwonjezeka kwaposachedwa komanso kuchuluka kwa kuwombera kwakukulu (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). +个t-hCO 神经元；***P <0.0001）. E , 通过 增加 电流 注入 的 hco 和 t-hco 重复 电位 放电 , 以及 最 大的 量化 （ 2 （5 （5 （5 16 个 t-hco 神经 ; *** p <0.0001). e, повторяющееся возбуждение потенциала действия hCO ndi t-hCO, вызванное увеличением подачи тока, и количественная оценка максимальстевия максимальний 2005 = нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, kuwombera mobwerezabwereza kwa hCO ndi t-hCO zomwe zingatheke chifukwa cha kuwonjezeka kwaposachedwa komanso kuchuluka kwa kuwombera kwakukulu (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) mu hCO ndi t-hCO neurons pa miyezi 8 yosiyana, ndi kuchuluka kwa mafupipafupi a zochitika za synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; ***P <0.0001). f, Spontaneous EPSCs (sEPSCs) mu hCO ndi t-hCO neurons pa miyezi 8 yosiyana, ndi kuchuluka kwa mafupipafupi a zochitika za synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; ***P <0.0001). f, спонтанные EPSC (sEPSC) в нейронах hCO ndi t-hCO через 8 месяцев дифференцировки ndi количественная оценка частоты синаптический соб, n2 = 2. 17 нейронов t-hCO *** P <0,0001) . f, Spontaneous EPSCs (sEPSCs) mu hCO ndi t-hCO neurons pa miyezi 8 yosiyanitsa ndi kuchuluka kwa zochitika za 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), 以及突触事件频率的量化（n = 25 hCO 7 神经神经元；***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（n = 25 hCO 25 hCO 2 神神率的量匼（n = 25hCO P <0.0001） . f, спонтанные EPSC (sEPSC) в нейронах hCO ndi t-hCO через 8 месяцев дифференцировки ndi количественная оценка частоты синаптический соб, n2 = 2. 17 нейронов t-hCO *** P <0,0001). f, Spontaneous EPSCs (sEPSCs) mu hCO ndi t-hCO neurons pa miyezi 8 yosiyana ndi kuchuluka kwa zochitika za synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001).Kwa bf, hCO ndi t-hCO mu mzere 1208-2 adatengedwa kuchokera ku gulu lomwelo losiyanitsira lomwe limasungidwa molumikizana. g, Gene adakhazikitsa kusanthula kopindulitsa (kuyesa kwa mbali imodzi ya Fisher) kwa majini omwe amawongolera kwambiri (osinthidwa P <0.05, kusintha kosintha> 2, owonetsedwa osachepera 10% ya nuclei) mu t-hCO glutamatergic neurons poyerekeza ndi hCO glutamatergic neurons yokhala ndi jini ya onse otsalira-posachedwa) majini odziwika kuchokera mu kafukufuku wa mbewa mu vivo16 ndi ma LRG enieni a anthu ochokera ku vitro neurons17. g, Gene adakhazikitsa kusanthula kopindulitsa (kuyesa kwa mbali imodzi ya Fisher) kwa majini omwe amawongolera kwambiri (osinthidwa P <0.05, kusintha kosintha> 2, owonetsedwa osachepera 10% ya nuclei) mu t-hCO glutamatergic neurons poyerekeza ndi hCO glutamatergic neurons yokhala ndi jini ya onse otsalira-posachedwa) majini odziwika kuchokera mu kafukufuku wa mbewa mu vivo16 ndi ma LRG enieni a anthu ochokera ku vitro neurons17. g, , ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,. 2, kuwerengera ndalama mu 10% ya ndalama) mu глутаматергических нейронах t-hCO pa глутаматергическими нейгронами hCO набор позднего (LRG) генов, зависящих от активности, идентифицированных в исследовании на мышах mu vivo16, ndi специфических для челозвека 17 и пецифических для челозвека 17 и пецифических для челозвека 17 в пецифических для челозвека 17 и пецифических для челозвека 17 в пецифических для челозвека 17. g, kusanthula kwa jini yolemeretsa (mayeso enieni a Fisher amtundu umodzi) wa majini omwe ali ndi kutsegulira kwakukulu (kusinthidwa P <0.05, kusintha kosintha> 2, kufotokozera osachepera 10% ya nuclei) mu t-hCO glutamatergic neurons poyerekeza ndi hCO glutamatergic neurons seti za onse oyambirira (ERG genes) omwe amadziwika kale (ERG genes) ndi ntchito yochedwa (ERG) ndi ma LRG enieni amunthu kuchokera ku ma neuron mu 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，在至少10%的细胞核中表达）的基因集富集分析（单侧F isher精确检验）从体内小鼠研究中鉴定的早期反应(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 上调比 （0诅 .倍数> 2 , 至少 至少 10%的 细胞 核中 表达） 集富集 分析中 的 早期 反应 反应 反应 和晚期 反应 反应 (lrg) 活性 基因的基因组 16 和 神经元 中7 中 1 中17的人类特异性LRG. g, глутаматергические нейроны t-hCO были значительно активизированы по сравнению с глутаматергическими нейронами hCO 2, osati 10% Kuchepetsa kwapang'onopang'ono (kuchokera pagulu lazopangapanga) раннего ответа (ERG) ndi pozdнего гены, завися ndi нейронах mu vitro 17, специфичные для человека. g, t-hCO glutamatergic neurons inali yoyendetsedwa kwambiri poyerekeza ndi hCO glutamatergic neurons (zosinthidwa P <0.05, pindani kusintha> 2, osachepera 10% Early response (ERG) ndi kusanthula kwamtundu wa jini mochedwa (mayeso enieni a Fisher) kuyankha kwa majini odalira jini (LRG6 mu vitro 7 mice). Zithunzi za LRG.Mzere wamadontho ukuwonetsa mtengo wa P wokonzedwa ndi Bonferroni wa 0.05. h, GluN gene expression (pseudo-phukusi ndi makulitsidwe a jini iliyonse) idasinthidwa kwambiri mu snRNA-seq replicas ya majini a LRG mu t-hCO glutamatergic neurons. i, immunostaining kusonyeza SCG2 mawu mu t-hCO (chapamwamba) ndi hCO (otsika) neurons. Mivi yoyera imaloza ku maselo a SCG2+. Scale bar, 25 µm. Deta imawonetsedwa ngati ± kupatuka kokhazikika.
Kutengera kuchulukirachulukira kwa t-hCO komwe kumawonedwa m'magawo a ex vivo, snRNA-seq idavumbulutsa kutsogola kotengera zochitika zamtundu wa t-hCO poyerekeza ndi hCO in vitro. Glutamatergic t-hCO neurons inasonyeza milingo yapamwamba ya majini yomwe imayang'anira ntchito yoyankha mochedwa (Mkuyu 2g, h), yomwe inapezeka m'maphunziro apitalo mu mbewa ndi ma neuroni aumunthu16,17. Mwachitsanzo, BDNF18, SCG2, ndi OSTN, jini yoyendetsera ntchito yeniyeni ya primate, inasonyeza kuwonjezeka kwa t-hCO neurons poyerekeza ndi hCO neurons (Mkuyu 2g-i). Chifukwa chake, ma neuron a t-hCO adawonetsa kukhwima kosinthika poyerekeza ndi ma hCO neurons mwa kusanthula kolemba, morphological, ndi magwiridwe antchito.
Kuti tipitirize kuunika kuyanjana kwa kusasitsa kwa t-hCO ndi kukula kwa ubongo waumunthu, tinapanga mafananidwe a transcriptomic a fetal ndi akuluakulu amtundu wa cortical cell19,20 ndi wamkulu21,22 komanso deta yochuluka pa cortical gene expression23 panthawi ya chitukuko (deta yowonjezera, Mkuyu 5). ). ndi ntchito yapitayi 24, hCO ndi t-hCO transcriptome maturation maturation status at 7-8 months of differentiation imagwirizana kwambiri ndi nthawi ya chitukuko cha vivo ndipo imakhala yofanana kwambiri ndi moyo wa fetal (Extended Data Fig. 5a). Mwachidziwitso, tinawona kukula kwa transcriptome mu t-hCO poyerekeza ndi zaka zofananira hCO, komanso transcriptome activation yogwirizana ndi synaptogenesis, astrogenesis, ndi myelination (deta yowonjezera, Fig. 5b-d). Pamlingo wa ma cell, tidapeza umboni wa cortex subtype yocheperako mu t-hCO, yokhala ndi magulu a glutamatergic neurons omwe amalumikizana ndi akulu L2 / 3, L5, ndi L6 neuron subtypes (Chithunzi 1i). Mosiyana ndi izi, kuphatikizika kwamagulu pakati pa glutamatergic t-hCO neurons ndi fetal cortical neurons kunali kochepa pakati papakati (zowonjezera zambiri, Chithunzi 5e-j). Kuti tidziwe ngati t-hCO neurons imagwira ntchito mofanana ndi ma neuroni amtundu wa postnatal neocortical neurons, tinajambula ma electrophysiological reconstruction and anatomical reconstruction of human L2/3 pyramidal neurons m'zigawo zakuthwa za khosi la postnatal cortex (deta yowonjezera, Mkuyu 7a). Ma electrophysiological properties a L2 / 3 pyramidal neurons anali ofanana ndi a t-hCO pyramidal neurons (deta yowonjezera, Fig. 7e). Morphologically, L2 / 3 neurons kuchokera ku zitsanzo za anthu obadwa pambuyo pobereka anali ofanana kwambiri ndi t-hCO kusiyana ndi hCO, ngakhale kuti maselo a L2 / 3 anali aatali, anali ndi nthambi zambiri, ndipo anali ndi mitsempha yambiri ya msana (mkuyu 3g ndi deta yowonjezera, Mkuyu 7b-). G).
a, transplantation wa hCO wopangidwa ndi control ndi TS hiPS cell mizere kukhala makoswe akhanda. b, kukonzanso kwa 3D kwa ma neurocytin odzazidwa ndi t-hCO pambuyo pa miyezi 8 yosiyana. c, kuchuluka kwa kutalika kwa dendritic (n = 19 control neurons, n = 21 TS neurons; **P = 0.0041). d, nthambi za dendritic zomangidwanso za 3D kuchokera ku ulamuliro ndi TS t-hCO pa miyezi ya 8 yosiyana, ndi kuchuluka kwa dendritic spine density (n = 16 control neurons, n = 21 TS neurons, ***P <0.0001). d, nthambi za dendritic zomangidwanso za 3D kuchokera ku ulamuliro ndi TS t-hCO pa miyezi ya 8 yosiyana, ndi kuchuluka kwa dendritic spine density (n = 16 control neurons, n = 21 TS neurons, ***P <0.0001). d, 3D-реконструкция дендритных ветвей из контроля ndi TS t-hCO через 8 месяцев дифференцировки ndi количественная оценка оценка плотностин 6 (6) контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, kukonzanso kwa 3D kwa nthambi za dendritic kuchokera ku ulamuliro ndi t-hCO TS pa miyezi ya 8 yosiyanitsa ndi dendritic spine density quantification (n = 16 control 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 內 1 神个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 分支 以及 树突棘 密度 量化 1 = 6 化 1 = 6 21 ts 神经 , *** p <0.0001 . d, 3D-реконструкция дендритных ветвей контроля ndi TS t-hCO через 8 месяцев дифференцировки ndi количественная оценка плотностиный = 1 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, kukonzanso kwa 3D kwa nthambi zowongolera za dendritic ndi TS t-hCO pamiyezi ya 8 yosiyanitsa ndi dendritic spine density quantification (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001).Nyenyezi zofiira zimawonetsa misana ya putative dendritic. e, ma EPSC odzilamulira okha ndi ma TS t-hCO neurons pambuyo pa miyezi 8 yosiyana. f, kuchuluka kwa ma frequency ndi kuchuluka kwa ma frequency ndi matalikidwe a zochitika za synaptic (n = 32 control neurons, n = 26 TS neurons; ** P = 0.0076 ndi P = 0.8102). g, Scholl kusanthula kwa TS ndi kuwongolera ma neurons mu hCO ndi t-hCO. Mizere yokhotakhota imasonyeza ma neurons a L2 / 3 pambuyo pobereka piramidi poyerekeza (n = 24 control t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 control hCO neurons, ndi n = 7 TS hCO neurons). Deta imawonetsedwa ngati ± kupatuka kokhazikika
Kuthekera kwa t-hCO kubwereza mawonekedwe a morphological ndi magwiridwe antchito a ma cortex neurons amunthu pamlingo wapamwamba adatipangitsa kuti tifufuze ngati t-hCO ingagwiritsidwe ntchito pozindikira phenotypes ya matenda. Tidayang'ana kwambiri pa TS, vuto lalikulu la neurodevelopmental lomwe limayamba chifukwa cha kusintha kwa jini mu encoding CaV1.2, yomwe imayambitsa kusindikiza kwa majini odalira ntchito mu ma neuron. Tinalandira hCO kuchokera kwa odwala atatu a TS onyamula m'malo ambiri (p.G406R) ndi maulamuliro atatu (mkuyu 3a). Pambuyo pa kuikidwa, tinapeza kuti dendritic morphology inasinthidwa mu TS neurons poyerekeza ndi maulamuliro (mkuyu 3b ndi deta yowonjezereka, Mkuyu 8a, b), ndi kuwonjezereka kwawiri kwa chiwerengero cha dendrites oyambirira ndi kuwonjezeka kwapakati ndi kuchepa kwa chiwerengero cha dendritic kutalika (Mkuyu 3c ndi deta yowonjezera, Mkuyu 8c). Izi zimagwirizanitsidwa ndi kuchulukitsidwa kwa misana ndi kuwonjezereka kwafupipafupi kwa EPSCs zodziwikiratu mu TS poyerekeza ndi ma neuroni olamulira (Fig. 3d-f ndi deta yowonjezera, Mkuyu 8g). Kusanthula kwina kunavumbulutsa machitidwe a matenda a dendritic nthambi mu t-hCO TS poyerekeza ndi maulamuliro, koma osati mu m'galasi TS hCO pa siteji yofanana ya kusiyana (mkuyu. 3g). Izi zikugwirizana ndi malipoti athu am'mbuyomu a dendritic shrinkage yodalira ntchito mu TS ndipo ikuwonetsa kuthekera kwa nsanja yoyika iyi kuti izindikire ma phenotypes mu vivo.
Kenako tidafunsa kuti ma cell a t-hCO amaphatikizidwa bwanji mu makoswe S1. S1 mu makoswe amalandira zolowetsa zamphamvu za synaptic kuchokera ku ipsilateral ventral basal ndi posterior thalamic nuclei, komanso ipsilateral motor ndi secondary somatosensory cortices, ndi contralateral S1 (mkuyu 4a). Kuti tibwezeretse njira yosungiramo, tidayambukira hCO ndi chiwewe virus-dG-GFP/AAV-G ndikuyika hCO mu khoswe wa S1 patatha masiku atatu. Tinawona kufotokozera kwa GFP mu neurons ya ipsilateral S1 ndi ventral basal ganglia masiku 7-14 pambuyo pa kuikidwa (Mkuyu 4b, c). Kuphatikiza apo, kuipitsidwa kwa antibody kwa thalamic marker netrin G1 kunawonetsa kukhalapo kwa mathero a thalamic mu t-hCO (Mkuyu 4d, e). Kuti tiwone ngati zoyerekeza izi zitha kubweretsa mayankho a synaptic m'maselo a t-hCO, tidajambula ma cell athunthu kuchokera kuma cell amunthu m'magawo akuthwa a thalamocortical layer. Kukondoweza kwamagetsi kwa makoswe S1, kapisozi wamkati, zinthu zoyera, ulusi pafupi ndi t-hCO kapena optogenetic activation ya opsin-yowonetsa mathero a thalamic mu t-hCO inachititsa ma EPSC afupiafupi a latency mu t-hCO neurons zowululidwa ndi AMPA receptor antagonist NBQX. (Mkuyu 4f, g ndi deta yowonjezera, Mkuyu 9a-g). Deta iyi ikuwonetsa kuti t-hCO imaphatikizidwa mu ubongo wa makoswe ndipo imatha kuyendetsedwa ndi minofu ya makoswe.
a, Schematic chithunzi cha kuyesa kutsatira chiwewe. b, GFP ndi mawu enieni a STEM121 pakati pa t-hCO ndi rat cerebral cortex (pamwamba). Zomwe zikuwonetsedwa ndi mawu a GFP mu rat ipsilateral ventral basal nucleus (VB) (m'munsi kumanzere) ndi ipsilateral S1 (pansi kumanja). Scale bar, 50 µm. Mabwalo ofiira amaimira madera a ubongo kumene zithunzizo zinatengedwa. c, kuchuluka kwa maselo akuwonetsa GFP (n = makoswe 4). d, e - Netrin G1+ thalamic terminals mu t-hCO. d ikuwonetsa gawo la coronal lomwe lili ndi t-hCO ndi VB nuclei. Mzere wapakati, 2 mm. e akuwonetsa Netrin G1 ndi STEM121 mawu mu t-hCO (kumanzere) ndi VB (kumanja) neurons. Scale bar, 50 µm. Mzere wa madontho a lalanje umasonyeza malire a t-hCO. f, g, Zomwe zikuchitika pano za t-hCO neurons pambuyo pa kukondoweza kwamagetsi mu S1 rat (f) kapena kapisozi wamkati (g), ndi (wofiirira) kapena wopanda (wakuda) NBQX (kumanzere). EPSC amplitudes ndi popanda NBQX (n = 6 S1 neurons, * P = 0.0119; ndi n = 6 mkati kapisozi neurons, ** P = 0.0022) (pakati). Maperesenti a t-hCO neurons akuwonetsa EPSC poyankha kukondoweza kwamagetsi kwa makoswe S1 (f) kapena kapisozi wamkati (g) (kumanja). aCSF, artificial cerebrospinal fluid. h, chithunzi chojambula cha kuyesa kwa kujambula kwa 2P (kumanzere). Kufotokozera kwa GCaMP6s mu t-hCO (pakati). Scale bar, 100 µm. Kutha kwa nthawi ya Fluorescence kwa GCaMP6s (kumanja). i, Z-score of mowiriza ntchito fluorescence. j, fanizo lokhazikika la kukondoweza kwa masharubu. k, z-zigoli 2P fluorescence trajectories mu yesero limodzi, lolumikizidwa ndi mandevu apatuka pa nthawi ziro (mzere woduka) mumaselo achitsanzo. l, mayankho owerengera z-chiwerengero cha ma cell onse olumikizidwa ndi kupotoza kwa ndevu pa nthawi ziro (mzere wodukaduka) (wofiira) kapena masitampu opangidwa mwachisawawa (imvi). m. Chithunzi chojambula cha kuyesa pazolemba za kuwala. n, Ma volteji opindika kuchokera ku chitsanzo cha t-hCO cell pakukondoweza kwa laser buluu kapena kupatuka kwa ndevu. Mivi yofiyira imawonetsa mapiko oyamba chifukwa cha kuwala (pamwamba) kapena chifukwa cha kupotoza kwa ndevu (pansi). Mthunzi wotuwa umasonyeza nthawi ya masharubu. o, Mawonekedwe opepuka kwambiri komanso mayankho a ndevu. p, ma spikes a kuyesa kamodzi, kogwirizana ndi kupatuka kwa ndevu m'maselo a chitsanzo. 0 ikuwonetsa kupatuka kwa ndevu (mzere woduka). q, chiŵerengero cha z-chiwerengero cha kuwombera kwa ma cell onse omwe ali ndi zithunzi, zolumikizidwa ndi kupatuka kwa ndevu panthawi ziro (mzere woduka) (wofiira) kapena masitampu opangidwa mwachisawawa (imvi). r, Gawo la mayunitsi owoneka bwino omwe amasinthidwa kwambiri ndi kupotoza kwa ndevu (n = makoswe 3) (kumanzere). Peak z-score latency (n = 3 makoswe; n = 5 (wobiriwira wobiriwira), n = 4 (wobiriwira wakuda), ndi n = 4 (cyan) mayunitsi osinthira ma whisker pa khoswe) (kumanja). Deta imawonetsedwa ngati ± kupatuka kokhazikika
Kenako tidafunsa ngati t-hCO ingayambitsidwe ndi zokondoweza mu vivo. Tidasinthitsa hCO yosonyeza zizindikiro za calcium GCaMP6 mu makoswe a S1. Pambuyo pa masiku a 150, tinachita fiber photometry kapena zithunzi ziwiri za kashiamu (mkuyu 4h ndi deta yowonjezera, mkuyu 10a). Tidapeza kuti ma cell a t-hCO amawonetsa zochitika zofananira (Chithunzi 4i, Expanded Data, Figure 10b ndi Supplementary Video 1). Kuti tiwonetsere nsonga ya t-hCO, tinachita zojambula za extracellular electrophysiological mu makoswe opangidwa ndi anesthetized (deta yowonjezera, Mkuyu 10c-f). Tapanga ma stereotaxic coordinates kuchokera ku zithunzi za MRI; motero, mayunitsi olembedwawa akuyimira ma neuroni amunthu omwe amaika, ngakhale kuti electrophysiology yokha siyilola kuti mtundu wachilengedwe udziwike. Tinawona kuphulika kofananira kwa zochitika (zowonjezera deta, mkuyu 10d). Kuphulika kunachitika pafupi ndi 460 ms ndipo kunasiyanitsidwa ndi nthawi za chete za 2 s (deta yowonjezera, Fig. 10d, e). Magawo a munthu aliyense amawombera pafupifupi maulendo atatu pakuphulika kulikonse, komwe ndi pafupifupi 73% ya mayunitsi olembetsedwa pakuphulika kulikonse. Zochita zamagulu amtundu uliwonse zinali zogwirizana kwambiri, ndipo kugwirizanitsa kumeneku kunali kwakukulu kusiyana ndi mayunitsi omwe amazindikiridwa mu zinyama zopanda katemera zolembedwa pansi pa zikhalidwe zomwezo (deta yowonjezera, mkuyu 10f). Kuti tiwonetserenso mayankho amtundu wa ma neuron opangidwa ndi anthu, tidayesa zowunikira makoswe omwe adayikidwa ndi hCO akuwonetsa njira yowoneka bwino ya rhodopsin 2 (hChR2), yomwe t-hCO neurons imazindikira mwachidule (osakwana 10 ms-fimulimsti) poyankha. T-hCO neurons inawonetsa kuphulika kwa zochitika zodziwikiratu pafupipafupi zofanana ndi zomwe zimawonedwa mu kujambula kwa kashiamu, komanso zojambula za electrophysiological zomwe zimachitika mu t-hCO popanda chizindikiro cha kuwala (deta yowonjezera, Fig. 10c-g). Palibe zochitika zodziwikiratu zomwe zidawonedwa mu magawo ofanana a hCO olembedwa mu vitro. Kuti tiwone ngati t-hCO ikhoza kuyendetsedwa ndi zokopa zamaganizo, tinasintha mwachidule ndevu za makoswe kutali ndi t-hCO (Mkuyu 4j, m ndi deta yowonjezera, Mkuyu 10h, k). Malingana ndi maphunziro apitalo8,10, kagawo kakang'ono ka maselo a t-hCO anasonyeza ntchito yowonjezereka chifukwa cha kusokonezeka kwa whisker, zomwe sizinawonedwe pamene deta imafananizidwa ndi masitampu a nthawi (Fig. 4k-q ndi deta yowonjezera, Fig. 10h-q). Zoonadi, pafupifupi 54% ya opto-labeled single units inasonyeza kuwonjezeka kwakukulu kwa kudzutsidwa pambuyo pa kukondoweza kwa whisker, kukwera pafupi ndi 650 ms (mkuyu 4r). Kuphatikizidwa pamodzi, deta iyi imasonyeza kuti t-hCO imalandira zofunikira zogwirira ntchito ndipo ikhoza kuyambitsidwa ndi zolimbikitsa zachilengedwe.
Kenako tidafufuza ngati t-hCO ikhoza kuyambitsa mabwalo mu makoswe kuti azitha kuwongolera machitidwe. Tidafufuza kaye ngati ma axon a t-hCO neurons amalowa mu minofu yozungulira makoswe. Tidapatsira hCO ndi lentivirus encoding hChR2 yolumikizidwa ku EYFP (hChR2-EYFP). Pambuyo pa masiku a 110, tinawona mafotokozedwe a EYFP m'madera a cortical ipsilateral, kuphatikizapo makutu, motor, ndi somatosensory cortices, komanso m'madera a subcortical, kuphatikizapo striatum, hippocampus, ndi thalamus (Mkuyu 5a). Kuti tiwone ngati zoyerekeza izi zitha kubweretsa mayankho a synaptic m'maselo a makoswe, tidayatsa ma cell a t-hCO owonetsa hChR2-EYFP pojambula ma cell a cerebral cortex m'magawo akuthwa aubongo. Kutsegula kwa ma axon a t-hCO okhala ndi kuwala kwa buluu kunapangitsa ma EPSC afupipafupi a latency mu makoswe a pyramidal cortex neurons, omwe anali otsekedwa ndi NBQX (Figs. 5b-g). Kuonjezera apo, mayankhowa akhoza kutsekedwa ndi tetrodotoxin (TTX) ndi kubwezeretsedwa ndi 4-aminopyridine (4-AP), kutanthauza kuti amayamba chifukwa cha kugwirizana kwa monosynaptic (Mkuyu 5e).
a, Schematic chithunzi cha kutsatira axon (kumanzere). t-hCO EYFP mawu (kumanja). Scale bar, 100 µm. A1, cortex yomveka, 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 ya thalamus, VTA, ventral tegmental dera. Mabwalo ofiira amaimira madera a ubongo kumene zithunzizo zinatengedwa. b, Chithunzi cha Schematic cha kuyesa kolimbikitsa. c, d, Zitsanzo za kuyankhidwa kwa blue light-induced photocurrent (pamwamba) ndi voteji (pansi) mwa anthu (c) EYFP+ t-hCO kapena makoswe (d) EYFP- maselo. e, f, Ma neuroni amakono a makoswe pambuyo pa kukondoweza kwa buluu kwa t-hCO axon ndi TTX ndi 4-AR (wobiriwira), TTX (imvi) kapena aCSF (wakuda) (e), ndi (violet) kapena opanda (wakuda) ) ) NBQX (e). g, kuchedwa kwa mayankho opangidwa ndi kuwala kwa buluu m'maselo a makoswe (n = 16 maselo); mipiringidzo yopingasa imasonyeza kuchedwa kwapakati (7.13 ms) (kumanzere). Kukula kwa ma EPSC opangidwa ndi kuwala ojambulidwa ndi kapena opanda NBQX (n = 7 maselo; ***P <0.0001) (pakati). Kukula kwa ma EPSC opangidwa ndi kuwala ojambulidwa ndi kapena opanda NBQX (n = 7 maselo; ***P <0.0001) (pakati). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). Kukula kwa ma EPSC opangidwa ndi kuwala ojambulidwa ndi kapena opanda NBQX (n = 7 maselo; ***P <0.0001) (pakati).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). Kukula kwa ma EPSC opangidwa ndi kuwala ojambulidwa ndi kapena opanda NBQX (n = 7 maselo; ***P <0.0001) (pakati).Maperesenti a makoswe akuwonetsa ma EPSC omwe amayankha kuwala kwa buluu (kumanja). h, schematic chithunzi cha khalidwe khalidwe. d0, tsiku 0. i. Kuchita kwa nyama zabwino pa tsiku 1 (kumanzere) kapena tsiku 15 (kumanja) kwa maphunziro. Chiwerengero cha malawi omwe amachitidwa pa tsiku 1 (kumanzere) kapena tsiku 15 (pakati kumanja) (n = 150 mayesero a buluu, n = 150 mayesero ofiira ofiira; *** P <0.0001). Chiwerengero cha malawi omwe amachitidwa pa tsiku 1 (kumanzere) kapena tsiku 15 (pakati kumanja) (n = 150 mayesero a buluu, n = 150 mayesero ofiira ofiira; *** P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (n = 150 испытаний с синим светой, n = 150 светом, n = 150 chithunzi; ***P <0,0001). Tanthauzo la malawi omwe amachitidwa pa tsiku 1 (kumanzere) kapena tsiku 15 (pakati kumanja) (n = 150 mayesero a buluu, n = 150 mayesero ofiira ofiira; *** 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 还0.0001).第1 天（左）或第15 天（右中) Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (n = 150 испытаний с синим светой, n = 150 светом, n = 150 chithunzi; ***P <0,0001). Tanthauzo la malawi omwe amachitidwa pa tsiku 1 (kumanzere) kapena tsiku 15 (pakati kumanja) (n = 150 mayesero a buluu, n = 150 mayesero ofiira ofiira; *** P <0.0001).Nyambita zochulukira poyesa kuwala kofiira ndi buluu pa tsiku loyamba (pakati kumanzere) kapena tsiku 15 (kumanja). NS, osati yofunika. j, k, Makhalidwe a zinyama zonse zomwe zimayikidwa ndi t-hCO kufotokoza hChR2-EYFP (j) kapena kulamulira fluorophore (k) pa tsiku 1 kapena 15 (hChR2-EYFP: n = 9 makoswe, ** P = 0.0049; kulamulira: n = 9, P = 0. l, Chisinthiko chazokonda (n = 9 hChR2, n = 9 control; **P <0.001, ***P <0.0001). l, Chisinthiko chazokonda (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, Chisinthiko chazokonda (n = 9 hChR2, n = zowongolera 9; **P <0.001, ***P <0.0001). l, 偏好评分的演变（n = 9 hChR2,n = 9 照；**P <0.001,***P <0.0001 l, 偏好评分的演变（n = 9 hChR2,n = 9 照；**P <0.001,***P <0.0001 l, Эволюция показателей предпочтения (n = 9 hChR2, n = 9 контролей; **P <0,001, ***P <0,0001). l, Kusintha kwa zikondamoyo (n = 9 hChR2, n = 9 zowongolera; **P <0.001, ***P <0.0001).m, mawu a FOS poyankha optogenetic activation ya t-hCO mu S1. Zithunzi za mawu a FOS (kumanzere), ndi kuchuluka (n = 3 pagulu; *P <0.05, **P <0.01 ndi ***P <0.001) (kumanja) akuwonetsedwa. Zithunzi za mawu a FOS (kumanzere), ndi kuchuluka (n = 3 pagulu; *P <0.05, **P <0.01 ndi ***P <0.001) (kumanja) akuwonetsedwa. Показаны изображения экспрессии FOS (слева) ndi количественного определения (n = 3 pa группу; * P <0,05, ** P <0,01 ndi *** P <0,001) (справа). Zithunzi za mawu a FOS (kumanzere) ndi kuchuluka (n = 3 pagulu; *P<0.05, **P<0.01, ndi ***P<0.001) akuwonetsedwa (kumanja).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) ndi количественного определения (n = 3 pa группу; * P <0,05, ** P <0,01 ndi *** P <0,001) (справа). Zithunzi za mawu a FOS (kumanzere) ndi kuchuluka (n = 3 pagulu; *P<0.05, **P<0.01, ndi ***P<0.001) akuwonetsedwa (kumanja).Scale bar, 100 µm. Deta imawonetsedwa ngati ± zolakwika zokhazikika za BLA, tonsil ya basolateral, MDT, dorsomedial thalamic nucleus, PAG, periaqueductal grey.
Pomaliza, tidafunsa ngati t-hCO ingasinthe machitidwe a makoswe. Kuti tiyese izi, tidayika hChR2-EYFP-yowonetsa hCO mu S1, ndipo patatha masiku 90, tidayika ulusi wa kuwala mu t-hCO kuti upereke kuwala. Kenako tinaphunzitsa makoswe ndi paradigm yosinthidwa yogwiritsira ntchito (mkuyu 5h). Tidayika nyamazo m'chipinda choyesera zamakhalidwe ndikuyika mwachisawawa 5 second blue (473 nm) ndi red (635 nm) laser stimuli. Nyama zimalandira mphotho yamadzi ngati zinyambita pakukondoweza kwa buluu koma sizinanyambire pakukondoweza kwa kuwala kofiira. Patsiku loyamba la maphunziro, nyamazo sizinawonetse kusiyana pakunyambita pamene zimalimbikitsidwa ndi kuwala kwa buluu kapena kofiira. Komabe, pa tsiku la 15, nyama zomwe zidasiyidwa ndi hCO zosonyeza hChR2-EYFP zidawonetsa kunyambita kogwira mtima zikalimbikitsidwa ndi kuwala kwa buluu poyerekeza ndi kukondoweza kwa kuwala kofiira. Kusintha kumeneku kwa khalidwe lakunyambita sikunawonedwe poyang'anira zinyama zomwe zimayikidwa ndi hCO zomwe zimasonyeza kulamulira kwa fluorophore (kupambana kwa kuphunzira: hChR2 89%, EYFP 0%, Chithunzi 5i-1 ndi Supplementary Video 2). Izi zikuwonetsa kuti ma cell a t-hCO amatha kuyambitsa ma neuron a makoswe kuti alimbikitse kufunafuna mphotho. Kuti tidziwe kuti rat t-hCO neural circuits iti yomwe ingakhudzidwe ndi kusintha kwa khalidweli, tinayambitsa optogenetically t-hCO mu nyama zophunzitsidwa ndi kukolola minofu pambuyo pa mphindi 90. Immunohistochemistry idawulula mafotokozedwe a mapuloteni a FOS omwe amadalira zochita m'magawo angapo aubongo omwe amakhudzidwa ndi machitidwe olimbikitsa, kuphatikiza medial prefrontal cortex, medial thalamus, ndi periaqueductal gray matter, zomwe zidawonetsedwa munyama kapena nyama. mpunga. 5m pa). Kutengera pamodzi, izi zikuwonetsa kuti t-hCO imatha kusintha machitidwe a rat neuronal kuyendetsa khalidwe.
Neural organoids imayimira njira yodalirika yophunzirira chitukuko cha anthu ndi matenda mu vitro, koma amachepetsedwa ndi kusowa kwa kulumikizana pakati pa mabwalo omwe amapezeka mu vivo. Tapanga nsanja yatsopano momwe tidasinthira hCO mu S1 ya makoswe obadwa kale osakwanira kuti tiphunzire kakulidwe ka maselo amunthu ndikugwira ntchito mu vivo. Tawonetsa kuti t-hCO imapanga mitundu ya maselo okhwima omwe sanawonedwe mu vitro28 komanso kuti t-hCO imaphatikizidwa mu ubongo wa makoswe. Kuphatikizika kwa t-hCO m'mabwalo a rodent neural kunatilola kukhazikitsa ulalo pakati pa zochitika zama cell a anthu ndikuphunzira zamakhalidwe a nyama, kuwonetsa kuti ma t-hCO neurons amatha kusintha machitidwe a makoswe kuti ayendetse mayankho amakhalidwe.
Pulatifomu yomwe timafotokoza ili ndi maubwino angapo kuposa kafukufuku wam'mbuyomu wosinthira ma cell amunthu muubongo wa makoswe. Choyamba, tidayika hCO mu cortex yomwe ikukula ya makoswe obadwa kumene, zomwe zingathandize kuphatikizika kwa anatomical ndi magwiridwe antchito. Chachiwiri, kuyang'anira kwa t-hCO MRI kunatilola kuphunzira malo a graft ndi kukula kwa nyama zamoyo, zomwe zimatilola kuchita maphunziro a nthawi yaitali a zinyama zambiri ndikukhazikitsa kudalirika kwa mizere yambiri ya hiPS. Pomaliza, tidayika ma organoid osakhazikika, m'malo mongoyimitsa ma cell amodzi, omwe sawononga kwambiri maselo amunthu ndipo amatha kulimbikitsa kuphatikiza ndi kupanga ma neurons amunthu muubongo wa makoswe.
Timavomereza kuti ngakhale kupita patsogolo kwa nsanjayi, zovuta zanthawi, malo, ndi mitundu yosiyanasiyana zimalepheretsa mapangidwe a mitsempha yaumunthu ndi kukhulupirika kwakukulu, ngakhale pambuyo pa kuikidwa pa chiyambi cha chitukuko. Mwachitsanzo, sizikudziwika ngati ntchito yodziwikiratu yomwe imawonedwa mu t-hCO ikuyimira phenotype yachitukuko yofanana ndi ntchito ya rhythmic yomwe imawonedwa panthawi ya chitukuko cha cortical, kapena chifukwa cha kusakhalapo kwa maselo opondereza omwe alipo mu t-hCO. Mofananamo, sizikudziwikiratu kuti kusakhalapo kwa lamination mu t-hCO kumakhudza bwanji kulumikizidwa kwa unyolo30. Ntchito yamtsogolo idzayang'ana pakuphatikiza mitundu ina yama cell monga ma microglia amunthu, ma cell endothelial amunthu, ndi magawo osiyanasiyana a GABAergic interneurons monga awonetsedwera pogwiritsa ntchito assembly 6 in vitro, komanso kumvetsetsa momwe kuphatikizika kwa neural ndi kukonza kungachitike mu t-hCO yosinthidwa. Ma transcriptal, synaptic and behaviour level m'maselo otengedwa kuchokera kwa odwala.
Ponseponse, nsanja iyi ya vivo ikuyimira gwero lamphamvu lomwe lingathandizire kukula kwaubongo wamunthu mu vitro komanso kafukufuku wamatenda. Tikuyembekeza kuti nsanjayi itilola kupeza ma phenotypes amtundu wa strand-level m'maselo osowa odwala ndikuyesa njira zatsopano zochiritsira.
Tinapanga hCO2.5 kuchokera ku maselo a HiPS monga tafotokozera kale. Kuti ayambitse kupanga hCO kuchokera ku ma cell a hiPS opangidwa pazigawo zodyetsa, magulu osasinthika a ma cell a hiPS adachotsedwa ku mbale zachikhalidwe pogwiritsa ntchito dispase (0.35 mg/mL) ndikusamutsira ku zikhalidwe zapulasitiki zotsika kwambiri zomwe zimakhala ndi mbale zokhala ndi ma cell a hiPS. (Corning) yowonjezeredwa ndi ma SMAD inhibitors dorsomorphine (5 μM; P5499, Sigma-Aldrich) ndi SB-431542 (10 μM; 1614, Tocris) ndi ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). M'masiku oyamba a 5, sing'anga yama cell a hiPS idasinthidwa tsiku lililonse ndipo dorsomorphine ndi SB-431542 zidawonjezeredwa. Patsiku lachisanu ndi chimodzi loyimitsidwa, ma neural spheroids adasamutsidwa kupita ku neural sing'anga yomwe ili ndi neurobasal-A (10888, Life Technologies), B-27 supplement yopanda vitamini A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin ndi streptomy0cin, Life Technologies ndi streptomy0cin (1:1) ndi Life Technologies factor (EGF; 20 ng ml-1; R & D Systems) ndi fibroblast kukula factor 2 (FGF2; 20 ng ml-1; R & D Systems) mpaka tsiku la 24. Kuyambira tsiku la 25 mpaka tsiku la 42, sing'anga inawonjezeredwa ndi ubongo-derived neurotrophic factor (BDNF; 20 trophin; 3 trophin; 3 mln trophin; 3 trophin) 20 ng ml−1; Peprotech) ndi zosintha zapakatikati tsiku lililonse. Patsiku lachisanu ndi chimodzi loyimitsidwa, ma neural spheroids adasamutsidwa kupita ku neural sing'anga yomwe ili ndi neurobasal-A (10888, Life Technologies), B-27 supplement yopanda vitamini A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin ndi streptomy0cin, Life Technologies ndi streptomy0cin (1:1) ndi Life Technologies factor (EGF; 20 ng ml-1; R & D Systems) ndi fibroblast kukula factor 2 (FGF2; 20 ng ml-1; R & D Systems) mpaka tsiku la 24. Kuyambira tsiku la 25 mpaka tsiku la 42, sing'anga inawonjezeredwa ndi ubongo-derived neurotrophic factor (BDNF; 20 trophin; 3 trophin; 3 mln trophin; 3 trophin) 20 ng ml−1; Peprotech) ndi zosintha zapakatikati tsiku lililonse.Patsiku lachisanu ndi chimodzi loyimitsidwa, ma neural spheroids adasamutsidwa ku neural medium yomwe ili ndi Neurobasal-A (10888, Life Technologies), B-27 supplement popanda vitamini A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin.ndi стрептомицин (1:100, Life Technologies) ndi дополнены эпидермальным фактором роста (EGF; 20 нг/мл; R&D Systems) ndi фактором роста фибл20FLAF; R&D Systems) mpaka 24-го дня. ndi streptomycin (1:100, Life Technologies) ndi kuwonjezeredwa ndi epidermal growth factor (EGF; 20 ng/ml; R&D Systems) ndi fibroblast growth factor 2 (FGF2; 20 ng/ml; R&D Systems) mpaka tsiku la 24.Kuyambira masiku 25 mpaka 42, ubongo wochokera ku ubongo wa neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) ndi neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) adawonjezeredwa pakatikati, kusintha sing'anga tsiku lililonse.在悬浮的第6 天，将神经球体转移到含有neurobasal-A(10888，Life Technologies) Technologies, GlutaMax(1:100, Life Technologies),青霉素的神经培养基中和链霉素 (1:100, Life Technologies) 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 , Life Technologies) 不 含 维维 生 7 a (12587 , Life Technologies) Glutamax (1: 100 , Life TechNOGIS青霉素 的 神经 培养 基生长 因子 （(20 ng ml-1 ; r & d Systems） 成 纤维 细胞 生长 2 （fgf2 ; 20 ng ml- 1 ;R&D Systems 至笴2 . На 6-й день суспензии нейросферы были переведены на добавку, содержащую нейробазал-А (10888, Life Technologies), добавку 5 В-7 Life Technologies), GlutaMax (1:100, Life Technologies), пенициллин- нейтрализованный стрептомицин (1:100, Life Technologies) с добавлением эпидермальго факторангей факто (1000, Life Technologies); R&D Systems) ndi фактора роста фибробластов 2 (FGF2; 20 нг мл-1) 1; Patsiku la 6, kuyimitsidwa kwa neurosphere kudasinthidwa kukhala chowonjezera chokhala ndi neurobasal-A (10888, Life Technologies), B-27 supplement popanda vitamini A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin-neutralized factor streptomycin (1:100) Life supplement with Technologies (EGF; 20 ng ml-1; R & D Systems) ndi fibroblast kukula factor 2 (FGF2; 20 ng ml-1) 1; R&D Systems) mpaka 24-го дня. R&D Systems) mpaka tsiku la 24.Kuyambira masiku 25 mpaka 42, neurotrophic factor yochokera ku ubongo (BDNF; 20 ng ml-1, Peprotech) ndi neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) inawonjezeredwa ku chikhalidwe cha chikhalidwe tsiku lililonse. Kusintha kwapakatikati kamodzi.Kuyambira tsiku la 43, hCO idasungidwa mu neurobasal-A sing'anga (NM; 1088022, Thermo Fisher) ndi kusintha kwapakatikati masiku 4-6 aliwonse. Kuti mupeze hCO kuchokera ku ma cell a hiPS otukuka pansi pa zinthu zopanda chakudya, maselo a hiPS adalumikizidwa ndi Accutase (AT-104, Innovate Cell Technologies) pa 37 ° C kwa mphindi 7, osagwirizana m'maselo amodzi, ndikuyika pa mbale za AggreWell 800 (34815, STEMCELL Technologies 6 pa ma cell 6 pa dechnologies 1) Essential 8 sing'anga yowonjezeredwa ndi ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Pambuyo pa maola 24, zofalitsa zomwe zinali m'zitsimezo zidayikidwa mmwamba ndi pansi muzofalitsa zomwe zili ndi Essential 6 media (A1516401, Life Technologies) yowonjezeredwa ndi dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) ndi SB-431542 (10 μM; 1614). , Tocrida). Kuyambira masiku 2 mpaka 6, Essential 6 sing'anga idasinthidwa tsiku lililonse ndi dorsomorphine ndikuwonjezera SB-431542. Kuyambira tsiku lachisanu ndi chimodzi, kuyimitsidwa kwa neurosphere kudasamutsidwa kupita ku neurobasal sing'anga ndikusungidwa monga tafotokozera pamwambapa.
Njira zonse zoyendetsera nyama zidachitika motsatira malangizo osamalira nyama omwe avomerezedwa ndi Stanford University Laboratory Animal Care Administrative Committee (APLAC). Makoswe apakati a euthymic RNU (rnu/+) anagulidwa (Charles River Laboratories) kapena kusungidwa. Nyama zinkasungidwa pa 12 maola kuwala-mdima kuzungulira ndi chakudya ndi madzi ad libitum. Ana amaliseche (FOXN1–/–) a makoswe azaka zamasiku atatu mpaka asanu ndi awiri ankazindikiridwa ndi kukula kwa ndevu zosakhwima asanadulidwe. Ana agalu (aamuna ndi aakazi) adagonekedwa ndi 2-3% isoflurane ndikuyikidwa pa stereotaxic frame. Kugwedeza kwa chigaza chokhala ndi m'mimba mwake pafupifupi 2-3 mm pamwamba pa S1 kunkachitika posunga kukhulupirika kwa dura mater. Kenako gwiritsani ntchito singano ya 30-G (pafupifupi 0.3 mm) kunja kwa craniotomy kuboola dura. Kenako gwiritsani ntchito HCO ku parafilm yopyapyala ya 3 × 3 cm ndikuchotsa sing'anga mopitilira muyeso. Pogwiritsa ntchito syringe ya Hamilton yolumikizidwa ku singano ya 23 G, 45 °, jambulani pang'onopang'ono hCO kumapeto kwenikweni kwa singano. Kenako yikani syringe papampu ya syringe yolumikizidwa ndi chipangizo cha stereotaxic. Kenako ikani nsonga ya singano pa dzenje lokhala ndi 0,3 mm m'lifupi lobowola mu dura (z = 0 mm) ndikuchepetsa syringe 1-2 mm (z = pafupifupi -1.5 mm) mpaka singano ili pakati pa dura mater A. chisindikizo cholimba chimapangidwa. Kenako kwezani syringe pakati pa kortical pamwamba pa z = -0.5 mm ndikubaya hCO pamlingo wa 1-2 µl pamphindi. Akamaliza jekeseni wa hCO, singano imachotsedwa pamlingo wa 0.2-0.5 mm pamphindi, khungu limapangidwa ndi sutured, ndipo mwana wagaluyo nthawi yomweyo amaikidwa pa kutentha kwa kutentha mpaka kuchira kwathunthu. Nyama zina anaziika m’zigawo ziŵiri.
Njira zonse zoyendetsera nyama zidachitika motsatira malangizo a ku Stanford University APLAC ovomerezeka osamalira nyama. Makoswe (oposa masiku 60 pambuyo pa kuikidwa) adanyengedwa ndi 5% isoflurane anesthesia ndi anesthetized ndi 1-3% isoflurane panthawi yojambula. Kuti muwone, 7 Tesla yotchinga mwamphamvu yopingasa borehole scanner Bruker (Bruker Corp.) yokhala ndi International Electric Company (IECO) gradient drive, chotchinga chotchinga chokhala ndi mainchesi 120 mm (600 mT/m, 1000 T/m/s) chinagwiritsidwa ntchito pogwiritsa ntchito AVANCE. III, njira zisanu ndi zitatu za ma coil RF amitundu yambiri ndi kuthekera kwamitundu yambiri, ndi nsanja yotsagana nayo ya Paravision 6.0.1. Kujambulitsa kudachitika pogwiritsa ntchito koyilo ya RF yolumikizidwa mwachangu yokhala ndi mainchesi 86 mm ndi koyilo ya RF yanjira zinayi yoziziritsa kuti ingolandira. Axial 2D Turbo-RARE (nthawi yobwereza = 2500 ms, echo time = 33 ms, 2 average) yokhala ndi magawo 16, makulidwe a magawo 0.6-0.8 mm, okhala ndi zitsanzo za 256 × 256. Zizindikirozi zidalandiridwa pogwiritsa ntchito koyilo ya quadrature transceiver volumetric RF yokhala ndi mainchesi amkati a 2 cm (Rapid MR International, LLC). Pomaliza, gwiritsani ntchito zowerengera za Imaris (BitPlane) zomangidwira popereka 3D ndi kusanthula voliyumu. Kupititsa patsogolo kopambana kunatanthauzidwa ngati komwe madera a T2-weighted MRI chizindikiro adapangidwa mu hemisphere yosinthidwa. Kukanidwa kwa graft kumatanthauzidwa ngati kumezanitsa komwe sikunapange madera opitilira T2-zolemera chizindikiro cha MRI mu hemisphere yosinthidwa. Subcortical t-hCO idachotsedwa pakuwunika kotsatira.
Kuti afotokoze mokhazikika GCaMP6s mu hCO kwa kujambula kwa kashiamu wa ma photon awiri, maselo a hiPS anali ndi pLV[Exp] -EF1a::GcaMP6s-WPRE-Puro kutsatiridwa ndi kusankha kwa maantibayotiki. Mwachidule, ma cell adasiyanitsidwa ndi EDTA ndikuyimitsidwa mu 1 ml ya Essential 8 sing'anga pa kachulukidwe ka maselo pafupifupi 300,000 pamaso pa polybrene (5 μg/ml) ndi 15 μl ya virus. Maselowo adakulungidwa ndikuyimitsidwa kwa mphindi 60 ndikuyika mbewu pamtunda wa ma cell 50,000 pachitsime chilichonse. Pambuyo polumikizana, maselo amathandizidwa ndi 5-10 μg ml-1 puromycin kwa masiku 5-10 kapena mpaka madera okhazikika atawonekera. Matenda owopsa a hCO adachitidwa monga momwe tafotokozera kale5 ndi zosintha zina. Mwachidule, sinthani tsiku la 30-45 hCO mu 1.5 ml Eppendorf microcentrifuge machubu okhala ndi 100 μl ya sing'anga ya mitsempha. Kenako pafupifupi 90 µl ya sing'anga imachotsedwa, 3-6 µl ya lentivirus yapamwamba (kuchokera 0.5 x 108 mpaka 1.2 x 109) imawonjezeredwa ku chubu, ndipo hCO imasamutsidwa ku chofungatira kwa mphindi 30. Kenaka onjezerani 90-100 µl zapakati pa chubu chilichonse ndikubwezera machubu ku chofungatira usiku wonse. Tsiku lotsatira, tumizani hCO kupita ku sing'anga yatsopano m'ma mbale otsika. Pambuyo pa masiku 7, hCO idasamutsidwa ku mbale zapansi zagalasi 24 kuti ziwonedwe ndikuwunika momwe matenda alili. pLV[Exp] -SYN1::EYFP-WPRE ndi pLV[Exp]-SYN1::hChR2-EYFP-WPRE adapangidwa ndi VectorBuilder. Lentivirus imagwiritsidwa ntchito poyesa zambiri chifukwa imaphatikizidwa mumtundu wamtundu womwe umakhala nawo, kulola kufotokozera kwa jini ya mtolankhani m'maselo omwe ali ndi kachilombo. Pakutsata kwachiwewe, tsiku la 30-45 hCO lidakhudzidwa ndi chiwewe-ΔG-eGFP ndi AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), osambitsidwa bwino kwa masiku atatu, ndikuyika makoswe mu S1 ndikusungidwa kwa masiku 7-4.
Kwa immunocytochemistry, nyama zidazimiririka ndikuphatikizidwa ndi PBS ndikutsatiridwa ndi 4% paraformaldehyde (PFA mu PBS; Electron Microscopy Sciences). Ubongo unakhazikitsidwa mu 4% PFA kwa maola 2 kapena usiku wonse pa 4 ° C, cryopreserved mu 30% sucrose mu PBS kwa maola 48-72, ndipo ophatikizidwa mu 1: 1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583 , Sakura Finetes03 gawo accoronat 3) (Leica). Kwa immunohistochemistry ya zigawo zokhuthala, nyama zidapangidwa ndi PBS, ndipo ubongo udagawika ndikugawidwa m'magawo a 300-400 µm pogwiritsa ntchito vibratome (Leica) ndipo magawowo adakhazikitsidwa ndi 4% PFA kwa mphindi 30. Kenako ma cryosections kapena magawo okhuthala adatsukidwa ndi PBS, otsekedwa kwa ola la 1 kutentha kwachipinda (10% seramu yachibadwa ya abulu (NDS) ndi 0.3% Triton X-100 kuchepetsedwa mu PBS) ndikutsekedwa ndi njira yotsekereza pa 4 ° C. - Ma Incubation Cryosections adakulungidwa usiku wonse ndipo zigawo zokhuthala zidakulungidwa kwa masiku asanu. Ma antibodies oyambirira omwe amagwiritsidwa ntchito anali: anti-NeuN (mbewa, 1:500; ab104224, abcam) anti-CTIP2 (khoswe, 1:300; ab18465, abcam), anti-GFAP (kalulu, 1: 1,000; Z0334, Dako), anti-GFP0 1 GTX, 3,000 GFP0; GeneTex), anti-HNA (mbewa, 1:200; ab191181, abcam), anti-NeuN (kalulu, 1:500; ABN78, Millipore), anti-PDGFRA (kalulu, 1:200; sc-338, Santa Cruz), anti-PPP201R; HPA047819, Atlas Antibodies), anti-RECA-1 (mbewa, 1:50; ab9774, abcam), anti-SCG2 (kalulu, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (mbuzi, 1:500; Netgoat Systema, G30D Systema7 1:100; AF1166, R&D Systems), anti-STEM121 (mbewa, 1:200; Y40410, Takara Bio), anti-SATB2 (mbewa, 1:50; ab51502, abcam), anti-GAD65/67 (kalulu, 1:400, anti-ABIN90; (mbuzi, 1:100; ab5076, abcam). Ma antibodies oyambirira omwe anagwiritsidwa ntchito anali: anti-NeuN (mbewa, 1: 500; ab104224, abcam) anti-CTIP2 (rat, 1:300; ab18465, abcam), anti-GFAP (kalulu, 1: 1,000; Z0334, Dako), anti -GFP0 1 GTX 1,000; GeneTex), anti-HNA (mbewa, 1:200; ab191181, abcam), anti-NeuN (kalulu, 1:500; ABN78, Millipore), anti-PDGFRA (kalulu, 1:200; sc-338, Santa Cruz), anti-PPP201R; HPA047819, Atlas Antibodies), anti-RECA-1 (mbewa, 1:50; ab9774, abcam), anti-SCG2 (kalulu , 1:100; 20357-1-AP, Proteintech), anti-SOX9 (mbuzi, 1:500; Neta1 Systems), Neta1D & 30D; 1:100; AF1166, R&D Systems), anti-STEM121 (mbewa , 1:200; Y40410, Takara Bio), anti-SATB2 (mbewa, 1:50; ab51502, abcam), anti-GAD65/67 (kalulu, anti-ABIN90; (mbuzi, 1:100; ab5076, abcam). Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2 (крысиные, 1:506); анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA (мышь, 1:200; ab1911181, Nтиклик, анкликс 1:500; ABN78, Millipore), анти-PDGFRA (кролик, 1:200; sc-338, Санта-Круз), анти-PPP1R17 (кролик, 1:200; HPA047819, Atlas-Atlas, Antibodies) 1:50; ab9774, abcam), анти-SCG2 (кролик , 1:100; 20357-1-AP, Proteintech), анти-SOX9 (козий, 1:500; AF3075, R&D Systems), нетризн 6, 6 G10; R&D Systems), анти-STEM121 (мышиный , 1:200; Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кроли00, AB9, ABN; анти-IBA1 (коза, 1 :100; аб5076, абкам). Ma antibodies oyambirira omwe amagwiritsidwa ntchito anali: anti-NeuN (mbewa, 1: 500; ab104224, abcam), anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (kalulu, 1: 1000; Z0334, Dako), anti-GFP0; GTX7 XX00; anti-HNA (mbewa, 1:200; ab191181, abcam), anti-NeuN (kalulu, 1:500; ABN78, Millipore), anti-PDGFRA ( kalulu, 1:200; sc-338, Santa Cruz), anti-PPP1R17, 1:00 HPAb4; Antibodies), anti-RECA-1 (mbewa, 1:50; ab9774, abcam), anti- SCG2 (kalulu, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (mbuzi, 1:500; AF3075, R&D Systems1), netrin 10, G1, netrin 10; R&D Systems), anti- STEM121 (mbewa, 1:200; Y40410, Takara Bio), anti-SATB2 (mbewa, 1:50; ab51502, abcam), anti-GAD65/67 (kalulu, 1:400; ABN904, Millipore 1:0; ab5076, 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（鸡，1:1,000;GTX13970，GeneTex），抗HNA（小鼠，1:200;ab191181,abcam），抗NeuN（兔，1:500;ABN78）Millipore）Millipore） 1:200;sc-338，Santa Cruz），抗PPP1R17（兔，1:200;HPA047819，Atlas 抗体），抗RECA-1（小鼠，1:50;ab9774ab9774ab9774ab 1 1:200;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，1:1,000;Z0334，Dako），抗-GFP（鸡，1:1,000;GTX13970，GeneTex），抗HNA（小鼠，1:200;ab191181,abcam），抗NeuN（兔（兔，1:500;ABN78％Millipo％ABN78， 200;sc-338，Santa Cruz），抗PPP1R17（兔，1:200;HPA047819，Atlas 抗体），抗RECA-1（小鼠，1:50;ab9774）ab9774，abcam 100;20357-1-AP，Proteintech），抗SOX9（山羊，1:500;AF3075，R&D Systems），Netrin G1a（山羊，1:100;AF1166,R&D Systems1:20,Y40410, Takara Bio), anti-SATB2 (mbewa, 1:50; ab51502, abcam), anti-GAD65/67 (kalulu, 1:400; ABN904, Millipore) ndi anti-IBA1 (mbuzi, 1:100; ab5076, abcam).Ma antibodies oyambirira omwe amagwiritsidwa ntchito anali: anti-NeuN (mbewa, 1: 500; ab104224, abcam), anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (kalulu, 1: 1000; Z0334, Dako). , anti-GFP (nkhuku, 1:1000; GTX13970, GeneTex), anti-HNA (mbewa, 1:200; ab191181, abcam), anti-NeuN (kalulu, 1:500; ABN78, Millipore), anti-PDGFRA (kalulu C308, 108; anti-PPP1R17 (kalulu, 1: 200; HPA047819, Atlas antibody), anti-RECA-1 (mbewa, 1:50; ab9774, abcam), anti- SCG2 (kalulu), 1: 100;20357-1-AP, Proteintech), анти-SOX9 (коза, 1:500; AF3075, R&D Systems), Нетрин G1a (коза, 1:100; AF1166, R&D Systems), анти -STEM121 (Y4:040, Takaka анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) и анти-IBA1 (коза, 1:100; аб5076, аб5076,). 20357-1-AP, Proteintech), anti-SOX9 (mbuzi, 1:500; AF3075, R&D Systems), Netrin G1a (mbuzi, 1:100; AF1166, R&D Systems), anti-STEM121 (mbewa, 1:200; anti-SATBra2), Y404 1:50; ab51502, abcam), anti-GAD65/67 (kalulu, 1:400; ABN904, Millipore), ndi anti-IBA1 (mbuzi, 1:100; ab5076, abkam).Magawo adatsukidwa ndi PBS ndikuyikidwa ndi antibody yachiwiri kwa ola la 1 kutentha kwa chipinda (zigawo zachisanu) kapena usiku wonse pa 4 ° C (zigawo zokhuthala). Alexa Fluor secondary antibody (Life Technologies) yosungunuka 1: 1000 mu njira yotsekera idagwiritsidwa ntchito. Pambuyo kutsuka ndi PBS, ma nuclei adawonetsedwa ndi Hoechst 33258 (Life Technologies). Pomaliza, zithunzizo zinayikidwa pa microscope yokhala ndi zophimba (Fisher Scientific) pogwiritsa ntchito Aquamount (Polysciences) ndikuwunikidwa pa microscope ya Keyence fluorescent (BZ-X analyzer) kapena Leica TCS SP8 confocal microscope (Las-X) pachithunzichi. Zithunzizi zidakonzedwa pogwiritsa ntchito pulogalamu ya ImageJ (Fiji). Kuti ayese kuchuluka kwa ma neuron aumunthu mu t-hCO ndi kotekisi ya makoswe, zithunzi za 387.5 μm zazikulu zamakona anayi zidatengedwa pakatikati pa t-hCO, m'mphepete mwa makoswe kapena pafupi ndi kotekisi. Mitsinje ya graft idatsimikiziridwa ndikuwunika kusintha kwa kuwonekera kwa minofu, ma nuclei a HNA +, ndi / kapena kukhalapo kwa minofu autofluorescence. Pa chithunzi chilichonse, chiwerengero chonse cha maselo a NeuN+ ndi HNA+ anagawidwa ndi chiwerengero chonse cha maselo a NeuN+ m'dera lomwelo. Kuonetsetsa kuti maselo okhawo omwe ali ndi ma nuclei mu ndege ya fano amawerengedwa, maselo okhawo omwe ali ndi Hoechst + amaphatikizidwa mu kuwerengetsa. Zithunzi ziwiri zolekanitsidwa ndi osachepera 1 mm zidawerengedwa kuti zichepetse zolakwika za chiwerengero.
Patangotha ​​​​sabata imodzi kuti mutenge zitsanzo, ikani nyama zopatsira hCO (pafupifupi miyezi 8 yosiyana) m'chipinda chamdima chokhala ndi ndevu zokonzedwa kuti muchepetse kukondoweza. Kudzipatula kwa ma nuclei kunachitika monga tafotokozera kale, ndikusinthidwa. Mwachidule, t-hCO ndi hCO anawonongedwa pogwiritsa ntchito detergent-mechanical cell lysis ndi 2 ml galasi minofu chopukusira (D8938, Sigma-Aldrich/KIMBLE). Mitsemphayi idasefedwa pogwiritsa ntchito fyuluta ya 40 µm ndi centrifuged pa 320 g kwa mphindi 10 pa 4 ° C isanapange sucrose density gradient. Pambuyo pa sitepe ya centrifugation (320 g kwa mphindi 20 pa 4 ° C), zitsanzozo zinayimitsidwanso mu 0.04% BSA/PBS ndikuwonjezera mayunitsi 0,2 a µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) ndikudutsa mu fyuluta ya 40 µm. Ma nuclei olekanitsidwa adayimitsidwanso mu PBS yomwe ili ndi 0.02% BSA ndikuyika pa Chromium Single Cell 3′ chip (kuyerekeza kuchira kwa maselo 8,000 pamzere uliwonse). malaibulale a snRNA-seq adakonzedwa ndi Chromium Single cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics). malaibulale a snRNA-seq adakonzedwa ndi Chromium Single 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). Ma library a snRNA-seq adakonzedwa pogwiritsa ntchito Chromium Single cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics). snRNA-seq 文库是使用Chromium Single cell 3′ GEM、Library & Gel Bead Kit v3 (10x Genomics) 制备的. snRNA-seq 文库是使用Chromium Single 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). Laibulale ya snRNA-seq idakonzedwa pogwiritsa ntchito Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics).Ma library ochokera ku zitsanzo zosiyanasiyana adaphatikizidwa ndikutsatiridwa ndi Admera Health pa NovaSeq S4 (Illumina).
Miyezo ya ma gene pamtundu uliwonse wa nyukiliya woyika idayesedwa pogwiritsa ntchito phukusi la pulogalamu ya 10x Genomics CellRanger (mtundu 6.1.2). Mwachindunji, zowerengerazo zidafananizidwa ndi kuphatikiza kwa anthu (GRCh38, Ensemble, version 98) ndi makoswe (Rnor_6.0, Ensemble, version 100) ma genomes omwe adapangidwa ndi lamulo la mkref ndikugwiritsa ntchito kuwerengera ndi -include-introns=TRUE kuwerengera kumaphatikizapo kuwerengera komwe kumapangidwira madera. Pazitsanzo za t-hCO, ma nuclei aumunthu adazindikirika potengera kufunikira kosunga kuti osachepera 95% ya zowerengera zonse zomwe zajambulidwa zigwirizane ndi ma genome amunthu. Zofufuza zonse zotsatila zinachitidwa pazitsulo zosefedwa za barcode kuchokera ku CellRanger pogwiritsa ntchito phukusi la R (mtundu 4.1.2) Seurat (mtundu 4.1.1)32.
Kuonetsetsa kuti ma nuclei apamwamba okha akuphatikizidwa mu kusanthula kotsatira, ndondomeko yowonongeka yobwerezabwereza inakhazikitsidwa pa chitsanzo chilichonse. Choyamba, ma nuclei otsika kwambiri omwe ali ndi majini osakwana 1000 omwe amapezeka ndipo oposa 20% a mitochondria yonse amadziwika ndikuchotsedwa. Pambuyo pake, matrix a jini yaiwisi adasinthidwa ndi kusintha kosasintha kwa binomial pogwiritsa ntchito ntchito ya sctransform(vst.flavor=”v2″), yomwe idazindikiritsanso majini 3000 osinthika kwambiri pogwiritsa ntchito magawo osasinthika. 30 idasankhidwa potengera kuyang'ana kwamasamba a mawondo ndikugwiritsidwa ntchito pazitsanzo zonse ndi kusanthula kwamagulu angapo (resolution = 1) kuyika ma jini otsika kwambiri (wapakatikati pansi pa 10th percentile), mosadziwika bwino mitochondrial pamwamba pa jini yotsika 9 ndi ma cell apakati ndi/kapena kuchuluka kwa mapasa oganiziridwa kuti azindikiridwa pogwiritsa ntchito phukusi la DoubletFinder33 (kutanthauza mphambu ya DoubletFinder pamwamba pa zitsanzo za t-hCO (n=3) ndi hCO (n=3) zidaphatikizidwa mosiyana pogwiritsa ntchito ntchito ya IntegrateData yokhala ndi magawo omwe ali pamwambapa.
Pambuyo pochotsa maso otsika, deta yophatikizika idayikidwa m'magulu (resolution = 0.5) ndikuphatikizidwa pazolinga zowonera za UMAP34. Majini olembera gulu lililonse adatsimikiziridwa pogwiritsa ntchito ntchito ya FindMarkers yokhala ndi magawo osasinthika owerengedwa kuchokera ku data yodziwika bwino ya jini. Timazindikira ndi kugawa magulu akuluakulu a maselo pophatikiza ma dataseti a mwana wosabadwayo ndi achikulire okhala ndi jini 19,20,21,35 ndi mawu ofotokozera. Makamaka, zoyambira zozungulira zidadziwika ndi mawu a MKI67 ndi TOP2A. Magulu a progenitor amatanthauzidwa ndi kusakhalapo kwa zolembedwa za mitotic, kuphatikizika kwakukulu ndi magulu amtundu wa multipotent glial progenitor ofotokozedwa kumapeto kwa metaphase fetal cortex, ndi EGFR ndi OLIG1 mawu. Timagwiritsa ntchito mawu oti astrocyte kuphatikizira zigawo zingapo zakusiyanitsa kwa nyenyezi, kuchokera kumapeto kwa radial glia mpaka kukhwima kwa ma astrocyte. Magulu a astrocyte amawonetsa kuchuluka kwa SLC1A3 ndi AQP4 ndipo awonetsedwa mapu okhala ndi mitundu yaying'ono ya fetal radial glia ndi/kapena ma astrocyte akulu. Ma OPC amawonetsa PDGFRA ndi SOX10 pomwe oligodendrocyte amawonetsa zolembera za myelination (MOG ndi MYRF). Glutamatergic neurons adadziwika ndi kupezeka kwa zolembera za neuronal (SYT1 ndi SNAP25), kusowa kwa zolembera za GABAergic (GAD2), komanso mawu a NEUROD6, SLC17A7, BCL11B, kapena SATB2. GluN neurons adagawidwanso kukhala apamwamba (SATB2 mawu ndi kutayika kwa BCL11B) ndi kuya (BCL11B expression) subclasses. Ma neurons a Putative subplate (SP) amawonetsa zolembera zodziwika za SP18 monga ST18 ndi SORCS1 kuphatikiza zolembera zakuya za GluN. Maselo ngati plexus a choroid adadziwika ndi mawu a TTR, ndipo ma cell ngati meningeal amawonetsa majini okhudzana ndi fibroblast ndi ma cell a pial/vascular cell of the reference data set.
Kusanthula kosiyana kwa mafotokozedwe a majini pakati pa t-hCO ndi hCO subclass kunachitika pogwiritsa ntchito njira yongopeka kumene yopangidwanso muzitsanzo zomwe zakhazikitsidwa pogwiritsa ntchito phukusi la Libra R (mtundu 1.0.0). Mwachindunji, kuyesa kwa kuthekera kwa chipika cha edgeR (mtundu 3.36.0, phukusi R) kunachitika m'magulu pofotokoza mwachidule kuchuluka kwa majini m'maselo a kalasi ya cell yomwe yapatsidwa pachitsanzo chilichonse. Pakuwona kwa mapu akutentha, milingo yokhazikika pa miliyoni (CPM) imawerengedwa pogwiritsa ntchito edgeR (cpm() ntchito) ndikusinthidwa (kuti mukwaniritse tanthauzo = 0, kupatuka kokhazikika = 1). Gene Ontology (GO) kusanthula kwabwino kwa t-hCO GluN gene kunachitika (Benjamini-Hochberg adakonza P mtengo wochepera 0.05 wowonetsedwa osachepera 10% ya ma cell a t-hCO GluN ndikuwonjezera kosintha kosachepera ka 2). amachitidwa pogwiritsa ntchito ToppGene Suite (https://toppgene.cchmc.org/)37. Timagwiritsa ntchito pulogalamu ya ToppFun yokhala ndi magawo osasinthika ndikuwonetsa ma P-values ​​a Benjamini-Hochberg omwe adawerengedwa kuchokera ku GO-annotated hypergeometric test.
Kuti tifanane ndi magulu athu a snRNA-seq okhala ndi magulu ofotokozera kuchokera kumaphunziro oyambira a cell single-cell RNA-seq kapena wamkulu snRNA-seq19,20,21,22, tinagwiritsa ntchito njira yophatikizira dataset. Tinagwiritsa ntchito SCTransform (v2) mayendedwe okhazikika ku Seurat kuti aphatikizire ndikuyerekeza kuphatikizika kwamagulu pakati pa ma dataset (pogwiritsa ntchito magawo omwe ali pamwambapa). Zolemba zamunthu payekha zidasinthidwa mwachisawawa mpaka ma cell 500 kapena ma cores pagulu loyambirira kuti azitha kuwerengera bwino. Pogwiritsa ntchito njira yofanana ndi yomwe tafotokozera kale, kuphatikizika kwa masango kunatanthauzidwa ngati gawo la maselo kapena ma nuclei m'gulu lililonse lophatikizidwa lomwe limadutsana ndi chizindikiro cha gulu lolozera. Kuti tipitirize kuyika ma GluN, tidagwiritsa ntchito kayendedwe ka Seurat's TransferData pa data ya GluN kuti tigawire zilembo zama dataset kuma cell athu a GluN.
Kuti tiwone momwe kukhwima kwa zolemba zapadziko lonse lapansi za t-hCO ndi hCO zitsanzo, tidafanizira zitsanzo zathu zachinyengo ndi BrainSpan/psychENCODE23, zomwe zimakhala ndi mndandanda wawukulu wa RNA womwe umatengera kukula kwaubongo wamunthu. Tinachita PCA pamtundu wophatikizika-wodziwika bwino wa gene expression matrix kuchokera ku cortical samples masabata a 10 pambuyo pa kutenga pakati komanso pambuyo pake, mu majini a 5567 (pamodzi ndi deta yathu) omwe adadziwika kuti akugwira ntchito mu BrainSpan cortical samples (yomwe imatanthauzidwa kuti ndi yaikulu kuposa 50% mu kusiyana kwachitukuko komwe kumafotokozedwa ndi zaka pogwiritsa ntchito chitsanzo cha cubic)38. Kuphatikiza apo, tidapeza majini okhudzana ndi ma signature akuluakulu a neurodevelopment pogwiritsa ntchito non-negative matrix factorization monga tafotokozera kale. Miyezo yachitsanzo yowerengedwera pogwiritsa ntchito njira yosagwirizana ndi matrix factorization imayikidwa mu Mkuyu. 5b yokhala ndi deta yowonjezereka ya siginecha iliyonse mwa zisanu zomwe zafotokozedwa ndi Zhu et al.38. Apanso, zolembera zodalira zolembera zidachokera ku maphunziro omwe adasindikizidwa kale. Makamaka, ERG ndi LRG zidakwezedwa kwambiri mu glutamatergic neurons zozindikirika ndi mbewa zowoneka bwino za snRNA-seq zosonkhanitsira pambuyo pokondoweza kowoneka kuchokera ku Supplementary Table 3 Hrvatin et al.16. Ma LRG olemeretsedwa ndi anthu adapezedwa kuchokera ku zikhalidwe zaubongo wa mwana wakhanda wa KCl ndipo adakololedwa maola 6 atatha kukondoweza, ndipo majini osefedwa anali oyendetsedwa kwambiri mwa anthu koma osati makoswe (Supplementary Table 4). Kuwunika kwa kuchulukitsa kwa ma jini pogwiritsa ntchito ma jiniwa kudachitika pogwiritsa ntchito njira imodzi yoyesera ya Fisher.
Anesthetize makoswe ndi isoflurane, chotsani ubongo ndi malo ozizira (pafupifupi 4 ° C) okosijeni (95% O2 ndi 5% CO2) yankho la sucrose la magawo omwe ali ndi: 234 mM sucrose, 11 mM shuga, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM. NaH2PO4, 10 mM MgSO4 ndi 0.5 mM CaCl2 (pafupifupi 310 mOsm). Magawo a ubongo wa makoswe (300-400 µm) okhala ndi t-hCO adapangidwa pogwiritsa ntchito Leica VT1200 vibratome monga tafotokozera kale39. Magawo adasamutsidwira kuchipinda chokhala ndi kutentha kosalekeza kwa chipinda chokhala ndi oxygenation yokhala ndi aCSF yokonzedwa kuchokera ku: 10 mM shuga, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 ndi 126 mM NaClm8 mOsm29. osachepera mphindi 45 musanayambe kujambula. Magawo adalembedwa m'chipinda chomizidwa momwe amapitirizira kusakaniza ndi aCSF (95% O2 ndi 5% CO2 vial). Deta yonse inalembedwa kutentha kutentha. T-hCO neurons inathetsedwa ndi pipette ya galasi ya borosilicate yodzazidwa ndi yankho lomwe lili ndi 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, ndi 0.6 mM EGTA, pH 7.2, njira yamkati yosinthidwa ndi KOM290 mH. Kuti achire, biocytin (0.2%) anawonjezedwa pa kujambula njira.
Deta inapezedwa pogwiritsa ntchito MultiClamp 700B amplifier (Molecular Devices) ndi Digidata 1550B digitizer (Molecular Devices), otsika-pass kusefedwa pa 2 kHz, digitized pa 20 kHz, ndi kusanthula pogwiritsa ntchito Clampfit (Molecular Devices), Origin (OriginPro). 2021b, OriginLab). ndi machitidwe a MATLAB (Mathworks). Kuthekera kophatikizikako kudawerengedwa pogwiritsa ntchito JPCalc ndipo zolembedwazo zidasinthidwa kukhala mtengo wowerengeka wa -14 mV. Operation IV imakhala ndi masitepe angapo apano mu masitepe 10-25 pA, kuchokera -250 mpaka 750 pA.
Ma thalamus, white matter, ndi S1 ​​afferents adalimbikitsidwa ndi magetsi mu magawo a thalamocortical panthawi yojambulira chigamba cha ma hCO neurons, monga tafotokozera kale. Mwachidule, ubongo unayikidwa pa tebulo losindikizira la 3D lopendekeka pamtunda wa 10 °, ndipo kutsogolo kwa ubongo kunadulidwa ndi 35 °. Kenako ubongo umamatiridwa pamalo odulidwawo ndikumagawa, kusunga ma axon otuluka a thalamocortical. Ma electrodes a bipolar tungsten (0.5 MΩ) adayikidwa pa micromanipulator yachiwiri ndipo adayikidwa bwino kuti alimbikitse zigawo zinayi pa selo (kapisozi wamkati, chinthu choyera, S1 ndi hCO). Lembani mayankho a synaptic pambuyo pa kukondoweza kwa 300 µA phasic pa 0.03-0.1 Hz.
hChR2-expressing hCO neurons idatsegulidwa pa 480 nm ndi ma pulses opepuka opangidwa ndi LED (Prizmatix) adagwiritsidwa ntchito kudzera mu × 40 cholinga (0.9 NA; Olympus) kuti alembe mawu a hChR2 pafupi ndi ma cell. Kutalika kwa gawo lowala ndi pafupifupi 0.5 mm ndipo mphamvu yonse ndi 10-20 mW. Kutalika kwa pulse kunayikidwa ku 10 ms, komwe kumafanana ndi kugunda komwe kumaperekedwa panthawi yoyesera kuphunzira khalidwe. Maulendo osiyanasiyana olimbikitsira adagwiritsidwa ntchito, kuyambira 1 mpaka 20 Hz, koma kugunda koyambirira kwa mndandanda ndikomwe kunagwiritsidwa ntchito powerengera. Mipata pakati pa masitima apamtunda nthawi zambiri imakhala yotalikirapo kuposa 30s kuti muchepetse mphamvu ya synaptic inhibitory kapena njira zowongolera. Kuti tiyese ngati yankho la hChR2 linali monosynaptic, tinagwiritsa ntchito TTX (1 μM) posamba mpaka EPSC itatha, kenako tinagwiritsa ntchito 4-aminopyridine (4-AP; 100 μM). Nthawi zambiri, yankho limabwezedwa pakangopita mphindi zochepa, ndikuchedwa pang'ono pakati pa kuwombera kwa LED ndi m'badwo wa EPSC. NBQX (10 μM) idagwiritsidwa ntchito kuyesa ngati kuyankha kumayendetsedwa ndi ma receptors a AMPA.
Magawo a Sharp hCO adapangidwa monga momwe tafotokozera kale. Mwachidule, zigawo za hCO zinayikidwa mu 4% agarose ndikusamutsira ku maselo omwe ali ndi 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 ndi 10 mM -glucose d-0 pa 0 + 0 (0) µm kutentha kwa firiji pogwiritsa ntchito vibrator ya Leica VT1200 ndikusungidwa mu ASF kutentha. Kenako, kujambula pama cell athunthu kunachitika pazigawo za hCO pansi pa maikulosikopu ya SliceScope (Scientifica). Zigawo zinapangidwa ndi aCSF (95% O2 ndi 5% CO2) ndipo zizindikiro za selo zinalembedwa kutentha. Ma neuroni a hCO anagwiritsidwa ntchito pogwiritsa ntchito pipette ya galasi ya borosilicate yodzazidwa ndi yankho lomwe lili ndi 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, ndi 0.6 mM EGTA, pH ya mkati 7, 2, yosinthidwa ndi KOH29 (osmo). Pofuna kuchira, onjezani 0,2% Biocytin ku yankho lamkati.
Deta inapezedwa ndi Clampex (Clampex 11.1, Molecular Devices) pogwiritsa ntchito MultiClamp 700B amplifier (Molecular Devices) ndi Digidata 1550B digitizer (Molecular Devices), otsika-pass kusefedwa pa 2 kHz, digitized pa 20 kHz, kusanthula ma molekyulu, ndi kusanthula 100. zipangizo) ndi machitidwe a MATLAB (MATLAB 2019b, Mathworks). Kuthekera kwa mphambano kunawerengedwa pogwiritsa ntchito JPCalc ndipo zolemberazo zinasinthidwa kuti zikhale zowerengeka za -14 mV. Operation IV imakhala ndi masitepe angapo apano mu 5-10 pA masitepe kuchokera -50 mpaka 250 pA.
Pomanganso ma morphological a pinched neurons, 0.2% biocytin (Sigma-Aldrich) idawonjezedwa ku yankho lamkati. Maselo amasungidwa kwa mphindi zosachepera 15 mutabera. Pipette imakokedwa pang'onopang'ono kwa 1-2 min mpaka nembanemba yolembedwa itasindikizidwa kwathunthu. Kutsatira ndondomeko ya physiology ya gawo, zigawo zinakhazikitsidwa usiku wonse pa 4 ° C. mu 4% PFA, kutsukidwa ndi PBS X3, ndi kuchepetsedwa 1: 1000 ndi streptavidin-conjugated DyLight 549 kapena DyLight 405 (Vector Labs). Maselo odzazidwa ndi biocytin (2%; Sigma-Aldrich) adalembedwa panthawi yojambula patch clamp kutentha kwa maola awiri. Magawowo adayikidwa pazithunzi zapa microscopy pogwiritsa ntchito Aquamount (Thermo Scientific) ndipo adawonetsedwa tsiku lotsatira pa microscope ya Leica TCS SP8 pogwiritsa ntchito cholinga chomiza mafuta chokhala ndi kabowo ka manambala × 40 1.3, kukulitsa × 0.9-1.0, xy. Chiwerengero cha zitsanzo ndi pafupifupi ma pixel 7 pa micron. Z-stacks pa 1 µm intervals zinapezedwa mosalekeza, ndipo z-stack mosaics ndi Leica-based auto-stitching anachitidwa kuphimba mtengo wonse wa dendritic wa neuron iliyonse. Neuron ndiye adatsatiridwa theka-pamanja pogwiritsa ntchito mawonekedwe a neuTube 40 ndipo mafayilo a SWC adapangidwa. Mafayilowo adakwezedwa ku SimpleNeuriteTracer41 Fiji plugin (ImageJ, version 2.1.0; NIH).
Minofu ya cortical yaumunthu inapezedwa ndi chilolezo chodziwitsidwa malinga ndi ndondomeko yovomerezedwa ndi Institutional Review Board ya Stanford University. Zitsanzo ziwiri za minofu ya pambuyo pobereka (wazaka 3 ndi 18) adapezedwa ndikuchotsa kotekisi yakutsogolo (pakatikati yakutsogolo kwa gyrus) ngati gawo la opaleshoni ya khunyu. Mukatha kukonzanso, kololani minofu mu NMDG-aCSF yozizira kwambiri yomwe ili ndi: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM MCruvateM sodium ascorbate 0 mpy3 sodium ascorbate 0 mpyl2. 4H2O ndi 10 mM MgSO4 7H2O. Titrate to pH 7.3-7.4 ndi concentrated hydrochloric acid. Minofu inaperekedwa ku labotale mkati mwa mphindi 30 ndipo magawo a coronal adatengedwa molingana ndi ndondomeko yomwe tafotokozayi.
Njira zonse zoyendetsera nyama zidachitika motsatira malangizo a ku Stanford University APLAC ovomerezeka osamalira nyama. Makoswe (oposa masiku 140 atatha kupatsirana) adanyengedwa ndi 5% isoflurane anesthesia ndi anesthetized ndi 1-3% isoflurane intraoperatively. Nyama zinayikidwa mu stereotaxic chimango (Kopf) ndi kumasulidwa buprenorphine (SR) jekeseni subcutaneously. Chigaza chimawululidwa, kutsukidwa ndikuyika zomangira za 3-5. Kuti tikwaniritse t-hCO, tinapanga ma stereotaxic coordinates kuchokera ku zithunzi za MRI. Bowo la burr linabowoleredwa pamalo osangalatsa ndi ulusi (400 µm m'mimba mwake, NA 0.48, Doric) adatsitsidwa 100 µm pansi pa hCO pamwamba ndikutetezedwa ku chigaza ndi simenti ya mano yochiritsika ndi UV (Relyx).
Zojambula za Fiber Photometric zidachitidwa monga tafotokozera kale42. Kuti alembe zochitika zokha, makoswe anaikidwa mu khola loyera ndipo chingwe cha 400 µm cha fiber optic patch (Doric) cholumikizidwa ndi fiber optic photometric data acquisition system chinali cholumikizidwa ku chingwe choyikidwa cha fiber optic. Pa kujambula kwa mphindi 10 za zochitika zamagalimoto, nyamazo zinali zomasuka kufufuza khola. Kuti alembe zomwe zachitika, makoswe (opitilira masiku 140 atawaika) adagwidwa ndi 5% isoflurane kuti alowetsedwe ndi 1-3% isoflurane yokonza. Ikani nyamayo mu stereotactic frame (Kopf) ndipo ndevu kumbali ina ya t-hCO zimadulidwa mpaka pafupifupi 2 cm ndikudutsa mumtambo wolumikizidwa ndi piezoelectric actuator (PI). Chingwe cha 400 µm fiber optic patch (Doric) chinalumikizidwa ku minyewa yolumikizidwa ndikulumikizidwa kunjira yopezera deta. Ndevu za mbali ina ya t-hCO zinapatutsidwa nthawi 50 (2 mm pa 20 Hz, 2 s pa ulaliki) mwachisawawa ndi piezoelectric drive pa 20 mphindi kujambula. Gwiritsani ntchito Phukusi Lothandizira la Arduino MATLAB kuti muwongolere nthawi yopatuka ndi kachidindo ka MATLAB. Zochitika zimagwirizanitsidwa ndi pulogalamu yopezera deta pogwiritsa ntchito transistor-transistor logic (TTL) pulses.
Makoswe (oposa masiku 140 atatha kupatsirana) adanyengedwa ndi 5% isoflurane anesthesia ndi anesthetized ndi 1-3% isoflurane intraoperatively. Nyama zinayikidwa mu stereotaxic chimango (Kopf) ndi buprenorphine SR ndi dexamethasone anabayidwa subcutaneously. Chigaza chimawululidwa, kutsukidwa ndikuyika zomangira za 3-5. Kuti tikwaniritse t-hCO, tinapanga ma stereotaxic coordinates kuchokera ku zithunzi za MRI. Craniotomy yozungulira (pafupifupi 1 cm m'mimba mwake) inkachitidwa ndi kubowola kothamanga kwambiri molunjika pa hCO yobzalidwa. Fupa likangokhala lochepa thupi momwe mungathere, koma musanabowole fupa lonselo, gwiritsani ntchito forceps kuchotsa fupa la m'chiuno lotsalira kuti muwulule t-hCO. Craniotomy inali yodzazidwa ndi saline wosabala, ndipo chofunda ndi chikhomo chapadera chamutu zidalumikizidwa ku chigaza ndi simenti ya mano yopangidwa ndi UV (Relyx).
Kujambula kwazithunzi ziwiri kunachitika pogwiritsa ntchito maikulosikopu ya Bruker multiphoton yokhala ndi cholinga cha Nikon LWD (×16, 0.8 NA). Kujambula kwa GCaMP6 kunachitika pa 920 nm ndi kukula kwa 1.4x single z-ndege ndi 8x avareji ya 7.5 fps. Makoswe adakopeka ndi 5% isoflurane anesthesia ndikusungidwa ndi 1-3% isoflurane. Makoswewo anaikidwa pamutu wopangidwa mwachizolowezi ndikuyikidwa pansi pa mandala. Kujambula kumbuyo kwa mphindi 3 kwa zochitika zamagalimoto kunapezedwa. Pakadutsa mphindi 20 zojambulira, zofusa 50 (chiwonetsero chilichonse cha 100 ms kutalika) zidaperekedwa mwachisawawa ku whisker pad moyang'anizana ndi t-hCO pogwiritsa ntchito picospricer. Gwiritsani ntchito Phukusi Lothandizira la Arduino MATLAB kuti muwongolere nthawi yophulika ndi khodi ya MATLAB. Gwirizanitsani zochitika ndi pulogalamu yopezera deta (PrairieView 5.5) pogwiritsa ntchito ma pulses a TTL. Kuti tiwunikenso, zithunzizo zidakonzedwa kuti ziziyenda bwino pogwiritsa ntchito kukonza kwa affine mu pulogalamu ya MoCo yomwe idakhazikitsidwa ku Fiji. M'zigawo za fulorosenti m'maselo pogwiritsa ntchito CNMF-E43. Fluorescence idachotsedwa kudera lililonse lokonda, kusinthidwa kukhala ma curve a dF/F, kenako ndikusinthidwa kukhala z-scores.
Makoswe (oposa masiku 140 atatha kupatsirana) adanyengedwa ndi 5% isoflurane anesthesia ndi anesthetized ndi 1-3% isoflurane intraoperatively. Nyama zinayikidwa mu stereotaxic chimango (Kopf) ndi buprenorphine SR ndi dexamethasone anabayidwa subcutaneously. Ndevu zomwe zili mbali ina ya t-hCO zinadulidwa pafupifupi 2 cm ndikumangirira kudzera mu mesh yolumikizidwa ndi piezoelectric actuator. Chigazacho chimawonekera ndikutsukidwa. Chigaza chachitsulo chosapanga dzimbiri chimamangiriridwa ku chigaza. Kuti tikwaniritse t-hCO, tinapanga ma stereotaxic coordinates kuchokera ku zithunzi za MRI. Pangani craniotomy yozungulira (pafupifupi 1 cm m'mimba mwake) ndi kubowola kothamanga kwambiri pamwamba pa t-hCO. Fupa likangokhala lochepa thupi momwe mungathere, koma musanabowole fupa lonselo, gwiritsani ntchito forceps kuchotsa fupa la m'chiuno lotsalira kuti muwulule t-hCO. Maselo amodzi adajambulidwa pogwiritsa ntchito 32-channel kapena 64-channel high-density silicon probes (Cambridge Neurotech) okhazikika pansi ndi zomangirapo ndi zokulitsa za RHD (Intan). Gwiritsani ntchito manipulator kuti mutsitse ma elekitirodi kumalo omwe mukufuna kulowera kudzera mu craniotomy, yomwe ili ndi saline wosabala. Kusonkhanitsa deta kunachitika pafupipafupi 30 kHz pogwiritsa ntchito Open Ephys data acquisition system. Kujambulirako kudapitilira pomwe tidazindikira zochitika zongolumikizana kwambiri m'mayendedwe opitilira 10, kuwonetsa kuti ma elekitirodi anali mu graft (kutengera data ya zithunzi ziwiri za calcium imaging). Kujambula kumbuyo kwa mphindi 10 kwa zochitika zamagalimoto kunapezedwa. Ndevu za mbali ina ya t-hCO zinapatutsidwa nthawi 50 (2 mm pa 20 Hz, 2 s pa ulaliki) mwachisawawa ndi piezoelectric drive pa 20 mphindi kujambula. Pogwiritsa ntchito Phukusi Lothandizira la MATLAB la Arduino (MATLAB 2019b), wongolerani nthawi yopatuka ndi kachidindo ka MATLAB. Gwiritsani ntchito ma pulses a TTL kuti mulunzanitse zochitika ndi pulogalamu yopezera deta.
Pazoyesa zowunikira, 200 µm optical patch cord (Doric) yolumikizidwa ku 473 nm laser (Omicron) idalumikizidwa ku 200 µm optical fiber yoyikidwa pamwamba pa craniotomy. Izi zisanachitike, sinthani mphamvu ya jumper kukhala 20 mW. Gwiritsani ntchito manipulator kuti mutsitse ma elekitirodi kumalo omwe mukufuna kulowera kudzera mu craniotomy, yomwe ili ndi saline wosabala. Kumayambiriro kwa kujambula, ma pulses khumi a kuwala kwa 473 nm (mafupipafupi 2 Hz, pulse duration 10 ms) adatulutsidwa. Maselo a Photosensitive amatanthauzidwa ngati ma cell omwe amawonetsa kuyankha kwakanthawi mkati mwa 10 ms ya kuwala mu 70% kapena kupitilira apo.