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Ama-neural organelles azihlanganisayo amelela isiteji esithembisayo se-in vitro sokumodela ukuthuthukiswa komuntu kanye nezifo. Kodwa-ke, ama-organoid awanakho ukuxhumana okukhona ku-vivo, okukhawulela ukuvuthwa futhi kuvimbele ukuhlanganiswa namanye amasekethe alawula ukuziphatha. Lapha sibonisa ukuthi i-stem cell-derived cortical organoids itshalwe ku-somatosensory cortex yamagundane anqunu asanda kuzalwa athuthukisa izinhlobo zamaseli avuthiwe ahlanganisa amasekhethi ahlobene nezinzwa kanye nogqozi. I-MRI yembule ukukhula kwe-organoid yangemva kokufakelwa emigqeni ye-stem cell nezilwane, kuyilapho ukuhlaziya okuwumgogodla owodwa kwembula ukuqhubeka kwe-corticogenesis kanye nokuvela kohlelo lokuloba oluncike emsebenzini. Ngempela, ama-neurons e-cortical atshalwe kabusha abonisa izakhiwo eziyinkimbinkimbi ye-morphological, synaptic, kanye ne-membrane yangaphakathi kunozakwabo be-in vitro, okuvumela ukutholwa kokukhubazeka kwe-neuronal ezigulini ezine-Timothy's syndrome. Ukulandelela kwe-anatomical nokusebenza kubonise ukuthi ama-organelle atshalwe kabusha athola okokufaka kwe-thalamocortical kanye ne-corticortical, futhi ukurekhoda kwe-vivo komsebenzi we-neural kuphakamisa ukuthi lokhu okokufaka kungakhiqiza izimpendulo zezinzwa kumaseli omuntu. Ekugcineni, ama-cortical organoid andisa ama-axon kulo lonke ubuchopho begundane, futhi ukusebenza kwawo kwe-optogenetic kuholela ekuziphatheni kokufuna umvuzo. Ngakho-ke, ama-neurons e-cortex yabantu atshalwe kabusha ayavuthwa futhi abambe iqhaza kumasekhethi osokhaya alawula ukuziphatha. Silindele ukuthi le ndlela yokwenza kube lula ukutholwa kwama-phenotypes eleveli ye-strand kumaseli asuselwa esigulini angakwazi ukutholwa ngezinye izindlela.
Ubuchopho bomuntu obukhulayo buyinqubo ephawulekayo yokuzihlela lapho amangqamuzana anda, ahluke, afuduke, futhi axhume ukuze akhe amasekhethi asebenzayo e-neuronal abuye acwengwe ngokuqhubekayo ngezinzwa. Inkinga eyinhloko ekuqondeni ukuthuthukiswa kobuchopho bomuntu, ikakhulukazi kumongo wezifo, ukuntuleka kokufinyelela kwezicubu zobuchopho. Ama-organelles azihlelayo, okuhlanganisa nama-cortex organoids (hCO; eyaziwa nangokuthi i-human cortex sphere), angakhiqiza u-2,3,4,5,6. Kodwa-ke, imikhawulo eminingana ikhawulela ukusetshenziswa kwayo okubanzi ekuqondeni ukuthuthukiswa nokusebenza kwamasekhethi e-neural. Ikakhulukazi, akucaci ukuthi ukuvuthwa kwe-hCO kunqunyelwe yini ukungabi khona kokungenayo okuthile kwe-microenvironmental kanye nezinzwa ezikhona ku-vivo. Ukwengeza, ngenxa yokuthi ama-hCO awahlanganisiwe kumasekethe angaveza imiphumela yokuziphatha, usizo lwawo ekufanekiseni izifo ze-neuropsychiatric eziyinkimbinkimbi ngokofuzo kanye nokuziphatha lunqunyelwe.
Ukufakelwa kabusha kwe-hCO ebuchosheni obuphilayo obuphelele kunganqoba le mikhawulo. Ucwaningo lwangaphambilini lubonise ukuthi ama-neurons omuntu atshalwe ku-rodent cortex ayakwazi ukuphila, akhiqize, futhi axhumane namaseli wamagundane7,8,9,10,11,12. Kodwa-ke, lezi zivivinyo zivame ukwenziwa ezilwaneni ezindala, ezingakhawulela ukuhlanganiswa kwe-synaptic ne-axonal. Lapha, sichaza i-paradigm yokufakelwa lapho satshala khona i-3D hCO etholakala kumaseli e-hiPS yayisa ku-primary somatosensory cortex (S1) yamagundane avikela amasosha omzimba ekuqaleni kokukhula kwepulasitiki. Ama-neurons e-hCO (t-hCO) atshalwe kabusha avuthwa kakhulu, athole okokufaka kwe-thalamocortical kanye ne-cortical-cortical aletha izimpendulo zezinzwa, futhi andise ukuqagela kwe-axonal ebuchosheni begundane ukushayela ukuziphatha kokufuna umvuzo. Ukuvuthwa okunwetshiwe kwe-t-hCO kwembule ukukhubazeka kwe-neuronal ezigulini ezine-Timothy's syndrome (TS), ukuphazamiseka kofuzo okunzima okubangelwa ukuguqulwa kwe-voltage-sensitive L-type CaV1.2 channel calcium (efakwe ikhodi yi-CACNA1C).
Ukutadisha ama-neurons e-cortical human kumasekhethi ku-vivo, sifake i-3D hCO engashintshile ku-S1 yamagundane e-athymic angaphambi kokubeletha (izinsuku ezingu-3-7 ngemuva kokubeletha) (Umdwebo 1a nedatha enwetshiwe ye-Fig. 1a-c). Kuleli qophelo, ukuqagela kwe-thalamocortical kanye ne-corticortical axonal akukakaqedi ukugcinwa kwabo kwangaphakathi kwe-S1 (ref. 13). Ngakho-ke, le ndlela iklanyelwe ukukhulisa ukuhlanganiswa kwe-t-hCO ngenkathi inciphisa umthelela kumasekhethi angapheli. Ukuze ubone ngeso lengqondo indawo ye-t-hCO ezilwaneni eziphilayo, senze ukuvuselelwa kobuchopho be-MRI enesisindo se-T2 yamagundane izinyanga ezingu-2-3 ngemva kokufakelwa kabusha (I-Fig. 1b kanye nedatha enwetshiwe, i-Fig. 1d). I-t-hCO yaqashelwa kalula futhi izilinganiso zevolumu ye-t-hCO zazifana nalezo ezibalwe kusuka ezingcezu ezingaguquki (Idatha Enwetshiwe Fig. 1d, e; P > 0.05). I-t-hCO yaqashelwa kalula futhi izilinganiso zevolumu ye-t-hCO zazifana nalezo ezibalwe kusuka ezingcezu ezingaguquki (Idatha Enwetshiwe Fig. 1d, e; P > 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированных срезов (расширенные, данных срезов (расширенные, данных срезов) 0,05). I-t-hCO yabonwa kalula, futhi izilinganiso ze-volumetric t-hCO zazifana nalezo ezibalwe ezigabeni ezingaguquki (idatha enwetshiwe, i-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（扩宗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). I-t-hCO yabonwa kalula, futhi izilinganiso ze-volumetric t-hCO zazifana nalezo ezibalelwe izigaba ezingaguquki (idatha enwetshiwe, i-Fig. 1d, e; P > 0.05).Sinqume i-t-hCO ku-81% wezilwane ezitshalwe kabusha cishe izinyanga ezi-2 ngemva kokufakelwa kabusha (n = izilwane ezingama-72; i-hCO isuka emigqeni yeseli ye-hiPS engu-10; imigqa yamaseli e-hiPS kuThebula Lokwengeza 1). Kulawa, ama-87% atholakala ku-cerebral cortex (Fig. 1c). Ngokwenza ama-serial MRI scans at multiple time points in the same transplanted rat, sithole ukwanda okuyisishiyagalolunye kwevolumu ye-t-hCO phakathi nezinyanga ze-3 (Fig. 1d nedatha eyandisiwe, Fig. 1f). Izilwane ezitshaliwe zazinezinga eliphezulu lokusinda (74%) ezinyangeni ezingu-12 ngemva kokufakelwa kabusha (idatha eyandisiwe, i-Fig. 1g kanye neThebula le-Supplementary 2), futhi akukho ukukhubazeka okukhulu kwemoto noma inkumbulo, i-gliosis, noma i-electroencephalogram (EEG) etholakele. Idatha Fig. 1g kanye nethebula lokwengeza 2). 1h–m kanye no-3e).
a, Isikimu somklamo wokuhlola. I-hCO etholakala kumaseli e-hiPS yafakwa ku-S1 yamagundane anqunu asanda kuzalwa ngezinsuku ezingama-30-60 zokuhlukaniswa. b, izithombe ze-MRI ezinesisindo se-T2 nezivundlile ezibonisa i-t-hCO ku-S1 2 izinyanga ngemva kokufakelwa. Ibha yesikali, 2 mm. c, Ukulinganisa amanani okuphumelela okufakwayo aboniswa kulayini weseli ngalinye le-hiPS (n = 108, izinombolo ezingaphakathi kwamabha zibonisa inani le-t-hCO ngomugqa weseli we-hIPS) kanye nendawo yekhorotiki noma engaphansi (n = 88). d, isithombe se-MRI somthambo we-coronary (kwesokunxele; ibha yesikali, 3 mm) kanye nokwakhiwa kabusha kwevolumu ye-3D ehambisanayo (i-scale bar, 3 mm) ebonisa ukwanda kwe-t-hCO ngaphezu kwezinyanga ezingu-3. e, Ukubuyekezwa kwamaphethini we-t-hCO ku-rat cerebral cortex. Ibha yesikali, 1 mm. f, Izithombe ezimele ze-immunocytochemical ze-t-hCO eziboniswe kusukela phezulu kwesobunxele kuya kwesokudla (ngesikhathi sokuhlukaniswa): PPP1R17 (izinyanga ezingu-4 ubudala), i-NeuN (izinyanga ezingu-8 ubudala), i-SOX9 ne-GFAP (izinyanga ezingu-8 ubudala), i-PDGFRα; (izinyanga eziyi-8), i-MAP2 (izinyanga eziyi-8) kanye ne-IBA1 (izinyanga eziyi-8). Ibha yesikali, 20 µm. I-Co-expression ye-HNA ibonisa amaseli emvelaphi yomuntu. g, snRNA-seq: I-manifold ehlanganisiwe ne-projection (UMAP) yesithombe sokunciphisa ubukhulu bayo yonke i-nuclei yekhwalithi ephezulu ye-t-hCO ngemva kokuhlanganiswa kwe-Seurat (n=3 t-hCO amasampuli, n=2 imigqa yeseli ye-hiPS). Ama-astrocyte, amangqamuzana omugqa we-astrocyte; i-cycl prog, ama-progenitors ajikelezayo; I-GluN DL, ama-neurons ajulile e-glutamatergic; I-GluN DL/SP, i-deep and sublamellar glutamatergic neurons; I-GluN UL, ungqimba olungaphezulu lwe-glutamatergic neurons; ama-oligodendrocyte, ama-oligodendrocyte; I-OPC, amaseli e-oligodendrocyte progenitor; I-RELN, i-reelin neurons. h, I-Gene Ontology (GO) ukuhlaziywa kwethemu lokunothisa kwezakhi zofuzo ezibekwe phezulu kakhulu (i-P <0.05 elungisiwe, i-fold change > 2, evezwe okungenani ku-10% wama-nuclei) kuma-neurons we-t-hCO glutamatergic uma kuqhathaniswa ne-hCO glutamatergic neurons. h, I-Gene Ontology (GO) ukuhlaziywa kwethemu lokunothisa kwezakhi zofuzo ezibekwe phezulu kakhulu (i-P <0.05 elungisiwe, i-fold change > 2, evezwe okungenani ku-10% wama-nuclei) kuma-neurons we-t-hCO glutamatergic uma kuqhathaniswa ne-hCO glutamatergic neurons. h, Анализ обогащения терминов I-Gene Ontology (GO) для генов со значительной активацией (скорректированный P <0,05, кратность изменения 2ратность изменения ku-10% ядер) ku-глутаматергических нейронах t-hCO по сравнению с глутаматергическими нейронами hCO. h, I-Gene Ontology (GO) ukuhlaziywa kwethemu lokucebisa izakhi zofuzo ezinokuqaliswa okuphawulekayo (okulungisiwe P<0.05, ukugoqa ukuguqulwa >2, inkulumo okungenani engu-10% nuclei) kuma-neurons we-t-hCO glutamatergic uma kuqhathaniswa nama-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 显着 上调 （后 嘎受 嘎 5 . 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 вдертайский 10% нейронах t-hCO по сравнению с глутаматергическими нейронами hCO Онтологический (GO) анализ термина обогащения. h, izakhi zofuzo zazihlelwe kakhulu (i-P <0.05 elungisiwe, i-fold change> 2, evezwe okungenani ku-10% ye-nuclei) kuma-neurons we-t-hCO glutamatergic uma kuqhathaniswa nokuhlaziywa kwe-hCO glutamatergic neurons Ontological (GO) yethemu lokucebisa.Umugqa wamachashazi ubonisa inani le-aq lika-0.05. i, ukuthwebula kwe-UMAP kwezinhlobo zamaseli e-GluN ku-t-hCO kusetshenziswa ukudluliswa kwelebula kusukela kudathasethi yereferensi engu-22 snRNA-seq motor cortex yabantu abadala. I-CT - amaseli e-corticothalamic, ET - amaseli e-extracerebral, i-IT - amaseli e-telencephalic angaphakathi, i-NP - eduze nokuqagela.
Sabe sesihlola i-cytoarchitecture kanye nokwakheka kwamaselula sekukonke kwe-t-hCO. I-antibody staining yamangqamuzana e-rat endothelial yembula i-vascularization nge-t-hCO, kuyilapho i-IBA1 staining yembula ukuba khona kwe-rat microglia kulo lonke ukuhlanganiswa (Fig. 1f nedatha enwetshiwe, Fig. 3c, d). I-Immunostaining yembule i-human nuclear nuclear antigen (HNA) amaseli amahle abonisa i-PPP1R17 (i-cortical progenitors), i-NeuN (neurons), i-SOX9 ne-GFAP (amaseli e-glial-derived) noma i-PDGFRα (i-oligodendrocyte progenitors) (Figure 1f). Ukutadisha ukwakheka kweselula kwe-t-hCO ekulungisweni kweseli eyodwa, senze ukulandelana kwe-RNA okukodwa (snRNA-seq) ngemva cishe kwezinyanga ezingu-8 zokuhlukanisa. Ukuhlungwa ngobuningi nokususwa kwama-nuclei amagundane kuveze amamephu e-mononuclear ekhwalithi ephezulu yabantu abangu-21,500 (Fig. 1g nedatha enwetshiwe, Fig. 4a,b). Amaphethini okuveza omaka ohlobo lwamaseli ajwayelekile ahlonze amaqoqo ezigaba ezinkulu zamaseli e-cortical, okuhlanganisa ama-neurons e-glutamatergic ajulile nangaphezulu, okhokho abajikelezayo, ama-oligodendrocyte, nohlu lwe-astrocyte (Fig. 1g, idatha enwetshiwe, i-Fig. 4c, kanye ne-Supplementary Table 3). I-Immunostaining ye-SATB2 ne-CTIP2 ibonise ukuthi naphezu kokuba khona kwe-cortical subtypes, i-t-hCO ayizange ibonise ukuhlukaniswa okucacile kwe-anatomical (idatha enwetshiwe, i-Fig. 3a). I-snRNA-seq hCO ehambisana nesiteji ikhiqize amakilasi amaseli afanayo ngobubanzi, ngaphandle kokumbalwa, okuhlanganisa ukungabi khona kwama-oligodendrocyte kanye nokuba khona kwe-GABAergic neurons, okungase kubonise izimo ezibikwe ngaphambili ezivumayo ze-in vitro kumaseli we-lateral progenitor cell15 (idatha eyandisiwe, i-Fig. 4f - i kanye ne-Supplementary Table 4). Ukuhlaziywa kokuchazwa kofuzo okuhlukile kwembula umehluko omkhulu kuma-neurons e-glutamatergic phakathi kwe-t-hCO ne-hCO (Ithebula Lesengezo 5), okuhlanganisa ukwenziwa kusebenze kwamasethi ofuzo ahlotshaniswa nokuvuthwa kwe-neuronal njengokusayina kwe-synaptic, indawo ye-dendritic, kanye nomsebenzi wesiteshi esine-voltage-gated (Umfanekiso 1h kanye neThebula Lokwengeza 5). ithebula 6). Ngokuvumelana nalokho, ama-neurons e-cortical glutamatergic t-hCO abonise ukuvuthwa kokubhala okusheshisiwe.
Ukuze sicacise ukuthi lezi zinguquko zokuloba ku-t-hCO zazihlobene yini nomehluko we-morphological phakathi kwe-hCO in vitro kanye ne-t-hCO ku-vivo, sakha kabusha i-hCO ne-hCO egcwaliswe nge-biocytin ehambisana nesiteji ezigabeni ezinzima ngemva kwezinyanga ezingu-7-8 zokuhlukaniswa. i-hCO neurons (Fig. 2a). I-t-hCO neurons yayinkulu kakhulu, yayinezikhathi ze-1.5 ubukhulu be-soma, ama-dendrites amaningi kabili, kanye nokwanda okuphindwe kasithupha kobude obuphelele be-dendritic uma kuqhathaniswa ne-in vitro hCO (Fig. 2b). Ukwengeza, sabona ukuminyana okuphezulu kakhulu kwe-dendritic spines kuma-neurons we-t-hCO kunama-hCO neurons (Fig. 2c). Lokhu kusikisela ukuthi i-t-hCO neurons idlula i-dendritic elongation eningi kanye ne-branching, okuthi, ngokuhambisana nokukhula kwamaseli okuqhubekayo, kungase kube nomthelela ekukhuleni okujulile kwe-t-hCO ngemva kokufakelwa (I-Fig. 1d ne-Extended Data Fig. 1f). Lokhu kusishukumisele ukuba siphenye izakhiwo ze-electrophysiological. I-Membrane capacitance yayiphakeme ngokuphindwe kasishiyagalombili (idatha eyandisiwe, i-Fig. 8d), amandla olwelwesi lwesimo sokuphumula ayenziwe nge-hyperpolarized (cishe i-20 mV), futhi umjovo wamanje udale izinga eliphezulu lokuthakazelisa eliphezulu kuma-neurons e-t-hCO kunama-hCO neurons. in vitro (Fig. 2d), e), ehambisana nezici ezinkulu neziyinkimbinkimbi ze-morphological ze-t-hCO. Ukwengeza, imvamisa yezenzakalo zamanje ze-postsynaptic ezizenzakalelayo (EPSC) zaziphakeme kakhulu kuma-neurons we-t-hCO (I-Fig. 2f), ephakamisa ukuthi ukuminyana okwandisiwe kwama-dendritic spines abonwa kuma-neurons we-t-hCO kwakuhlotshaniswa nokuthakazelisa kokusebenza. i-synapse yocansi. Siqinisekise uhlamvu olungavuthiwe lwama-hCO neurons in vitro ngokurekhoda okubhalwe ukuthi ama-glutamatergic neurons (idatha enwetshiwe, Fig. 6a-c).
a, ukwakhiwa kabusha kwe-3D kwe-hCO egcwele i-biocytin ne-t-hCO neurons ngemva kwezinyanga eziyi-8 zokuhlukaniswa. b, Ukulinganisa izici ze-morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0084, *P = 0.0179 kanye ne-***P <0.0001). b, Ukulinganisa izici ze-morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0084, *P = 0.0179 kanye ne-***P <0.0001). б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 futhi *** P <0,00). b, ukulinganisa izici ze-morphological (n=8 hCO neurons, n=6 t-hCO neurons; **P=0.0084, *P=0.0179, kanye ne-***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 <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 = 0.0179 和**00 <0 . б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 futhi *** P <0,00). b, ukulinganisa izici ze-morphological (n=8 hCO neurons, n=6 t-hCO neurons; **P=0.0084, *P=0.0179, kanye ne-***P<0.0001).c, ukwakhiwa kabusha kwe-3D kwamagatsha e-hCO kanye ne-t-hCO dendritic ngemva kwezinyanga eziyisi-8 zokuhlukaniswa. Izinkanyezi ezibomvu zibonisa i-puative dendritic spines. I-Dendritic spine density quantification (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0092). d, Ukulinganisa amandla e-membrane yokuphumula (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, Ukulinganisa amandla e-membrane yokuphumula (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, ulwelwesi oluphumuzayo ukulinganisa okungaba khona (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, ulwelwesi oluphumuzayo ukulinganisa okungaba khona (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Isenzo esiphindaphindayo ukudubula okungase kube khona ku-hCO naku-t-hCO okudalwe ukwanda kwemijovo yamanje, kanye nokulinganisa izinga eliphezulu lokudubula (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Isenzo esiphindaphindayo ukudubula okungase kube khona ku-hCO naku-t-hCO okudalwe ukwanda kwemijovo yamanje, kanye nokulinganisa izinga eliphezulu lokudubula (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, повторное возбуждение потенциала действия в hCO ne-t-hCO, вызванное увеличением тока, и количественная оценка максимальной скорости 2 возh (COY = 5) n = 16 нейронов t-hCO *** P <0,0001). e, ukudubula okungase kube khona kwesenzo ku-hCO naku-t-hCO okubangelwa ukwanda kwamanje kanye nomthamo wezinga eliphezulu lokudubula (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（n 神 = 25)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（5） 16 个 t-hco 神经 ; *** p <0.0001） . e, повторяющееся возбуждение потенциала действия hCO kanye ne-t-hCO, вызванное увеличением подачи тока, и количественная оценка максимальний максимальний максимальний максимальний 2015 нейронов hCO, n = 16 нейронов t-hCO *** P <0,0001). e, ukudubula okuphindaphindiwe kwamandla esenzo se-hCO kanye ne-t-hCO okudalwe ukuhlinzekwa kwamanje okwandisiwe kanye nokulinganisa izinga eliphezulu lokudubula (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, Ama-EPSC azenzekelayo (sEPSCs) ku-hCO kanye ne-t-hCO neurons ezinyangeni ze-8 zokuhlukaniswa, kanye nokulinganisa imvamisa yezenzakalo ze-synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; ***P <0.0001). f, Ama-EPSC azenzekelayo (sEPSCs) kuma-hCO kanye ne-t-hCO neurons ezinyangeni ezingu-8 zokuhlukaniswa, kanye nokulinganisa imvamisa yezenzakalo ze-synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; ***P <0.0001) . f, спонтанные EPSC (sEPSC) в нейронах hCO kanye t-hCO через 8 месяцев дифференцировки kanye количественная оценка частоты синаптический соб, 2 = 2 17 нейронов t-hCO *** P <0,0001) . f, Ama-EPSC azenzekelayo (sEPSCs) kuma-hCO kanye ne-t-hCO neurons ezinyangeni ezingu-8 zokuhlukaniswa kanye nokulinganisa kwamazinga omcimbi we-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） . e神率的量匼（n = 25hCO P <0.0001） . f, спонтанные EPSC (sEPSC) в нейронах hCO kanye t-hCO через 8 месяцев дифференцировки kanye количественная оценка частоты синаптический соб, 2 = 2 17 нейронов t-hCO *** P <0,0001). f, Ama-EPSC azenzakalelayo (sEPSCs) kuma-hCO kanye ne-t-hCO neurons ezinyangeni ze-8 zokuhlukaniswa kanye nokulinganisa kwamazinga we-synaptic umcimbi (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001).Ku-bf, hCO kanye ne-t-hCO emgqeni 1208-2 kuthathwe eqeqebeni elifanayo lokuhlukanisa eligcinwe ngokuhambisana. g, i-Gene isethi ukuhlaziya okunothisayo (ukuhlolwa okuqondile kwe-Fisher yohlangothi olulodwa) kwezakhi zofuzo ezibekwe phezulu kakhulu (i-P <0.05 elungisiwe, i-fold change > 2, evezwe okungenani nge-10% ye-nuclei) kuma-neuron e-t-hCO glutamatergic uma kuqhathaniswa ne-hCO glutamatergic neurons enamasethi ofuzo kokubili kokusebenza okuncibilikisiwe kwe-LRG (i-latenseresponseres) kanye ne-L. izakhi zofuzo ezikhonjwe ocwaningweni lwegundane le-vivo16 kanye nama-LRG aqondene nomuntu asuka ku-in vitro neurons17. g, i-Gene isethi ukuhlaziya okunothisayo (ukuhlolwa okuqondile kwe-Fisher yohlangothi olulodwa) kwezakhi zofuzo ezibekwe phezulu kakhulu (i-P <0.05 elungisiwe, i-fold change > 2, evezwe okungenani nge-10% ye-nuclei) kuma-neuron e-t-hCO glutamatergic uma kuqhathaniswa ne-hCO glutamatergic neurons enamasethi ofuzo kokubili kokusebenza okuncibilikisiwe kwe-LRG (i-latenseresponseres) kanye ne-L. izakhi zofuzo ezikhonjwe ocwaningweni lwegundane le-vivo16 kanye nama-LRG aqondene nomuntu asuka ku-in vitro neurons17. g, анализ обогащения набора генов (односторонний точный критерий Фишера) генов со значительной активацией (скорректированмены5й, P. 2, i- экспрессия по меньшей мере ku-10% ядер) ku-глутаматергических нейронах t-hCO mayelana ne-глутаматергическими нейгронами hCO набор позднего (LRG) генов, зависящих от активности, идентифицированных в исследовании на мышах in vivo16, futhi специфических для челозвека 15 LRG15. g, ukuhlaziywa kwesethi yokucebisa isakhi sofuzo (uhlolo olunembile lukaFisher olunomsila owodwa) lwezakhi zofuzo ezinokuvuselelwa okubalulekile (okulungisiwe P<0.05, ukuguqulwa okugoqiwe >2, ukubonakaliswa okungenani ku-10% wama-nuclei) kuma-neuron we-t-hCO glutamatergic uma kuqhathaniswa namasethi e-hCO glutamatergic neurons yakho kokubili ekuqaleni (i-ERG genes) ehlonziwe1-incike ngemuva kwesikhathi (ERG genes) kanye ne-inpendence1 kanye nama-LRG aqondene nomuntu asuka kuma-neurons in 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话 .倍数> 1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1 中 1 神经元 中1 17的人类特异性LRG. g, глутаматергические нейроны t-hCO были значительно активизированы по сравнению с глутаматергическими нейронами hCO (скорректированмены5, P. 2, hhayi 10% I-Анализ обогащения набора генов (односторонний точный тест Фишера) раннего ответа (ERG) futhi позднего гены, завися идентифицированные в исследованиях на мышах in vivo16 futhi нейронах in vitro17, специфичные для человека. g, ama-neuron e-t-hCO glutamatergic aye alawulwa kakhulu uma kuqhathaniswa nama-hCO glutamatergic neurons (okulungisiwe P<0.05, ukuguqulwa okugoqiwe >2, okungenani 10% Ukusabela Kwangaphambi kwesikhathi (ERG) kanye nokuhlaziywa kofuzo lokuphendula sekwephuzile (ukuhlolwa okuqondile kukaFisher okunomsila owodwa) izakhi zofuzo ezincike ku-vitro1 ku-vitro1 ku-vitro7 mice. Ama-LRGs.Ulayini onamachashazi ubonisa inani elingu-P elilungiswe yi-Bonferroni elingu-0.05. h, ukubonakaliswa kofuzo lwe-GluN (iphakheji-mbumbulu kanye nokukalwa kofuzo ngalunye) kwaqondiswa kakhulu kuma-snRNA-seq replicas wezakhi zofuzo ze-LRG kuma-neuron we-t-hCO glutamatergic. i, i-immunostaining ebonisa inkulumo ye-SCG2 ku-t-hCO (phezulu) kanye ne-hCO (ephansi) neurons. Imicibisholo emhlophe ikhomba kumaseli e-SCG2+. Ibha yesikali, 25 µm. Idatha ichazwa njengokushiwo ± ukuchezuka okujwayelekile.
Ngokusekelwe kumsebenzi owandisiwe we-t-hCO obonwe kuzingcezu ze-ex vivo, i-snRNA-seq iveze ukulawulwa okuncike kumsebenzi wemibhalo yofuzo ku-t-hCO uma kuqhathaniswa ne-hCO in vitro. I-Glutamatergic t-hCO neurons ibonise amazinga aphezulu ezakhi zofuzo ezilawula umsebenzi wokuphendula sekwephuzile (I-Fig. 2g, h), etholwe ezifundweni zangaphambilini kuma-mouse nama-neurons abantu16,17. Isibonelo, i-BDNF18, i-SCG2, ne-OSTN, isakhi sofuzo esilawula umsebenzi othize we-primate, sibonise ukukhuphuka kokukhuluma kuma-neurons we-t-hCO uma kuqhathaniswa nama-hCO neurons (Fig. 2g-i). Ngakho-ke, ama-neuron e-t-hCO abonise izici zokuvuthwa ezithuthukisiwe uma kuqhathaniswa nama-hCO neurons ngokuhlaziya okubhaliwe, kwe-morphological, nokusebenza.
Ukuze kuqhutshekwe nokuhlola ukuhlotshaniswa kokuvuthwa kwe-t-hCO nokuthuthukiswa kobuchopho bomuntu, senze ukuqhathanisa kwe-transcriptomic kwezinhlobo ze-fetus kanye ne-cortical cell yabantu abadala19,20 kanye ne-adult21,22 kanye nedatha ebanzi ekukhulumeni kofuzo lwe-cortical23 ngesikhathi sokuthuthukiswa (idatha eyandisiwe, Fig. 5). ). ngomsebenzi wangaphambilini we-24, isimo somhlaba wonke sokuvuthwa kwe-hCO kanye ne-t-hCO ye-transcriptome ezinyangeni ezingu-7-8 zokuhlukanisa ngokubanzi sihambisana nesikhathi sokuthuthukiswa kwe-vivo futhi silingana kakhulu nokuphila kwe-fetus sekwephuzile (Idatha Enwetshiwe Fig. 5a). Ngokuphawulekayo, sibone ukukhula okukhulayo kwe-transcriptome ku-t-hCO uma kuqhathaniswa ne-hCO ehambisana neminyaka yobudala, kanye nokusebenza kwe-transcriptome okuhlotshaniswa ne-synaptogenesis, i-astrogenesis, ne-myelination (idatha eyandisiwe, i-Fig. 5b-d). Ezingeni lamaselula, sithole ubufakazi be-cortex subtype ezacile ku-t-hCO, enamaqoqo e-glutamatergic neurons adlulana ne-L2/3, L5, kanye ne-L6 neuron subtypes yabantu abadala (Figure 1i). Ngokuphambene, ukunqwabelana phakathi kwe-glutamatergic t-hCO neurons kanye ne-fetus cortical neurons bekunomkhawulo kakhulu phakathi nokukhulelwa (idatha eyandisiwe, Umfanekiso 5e-j). Ukuze sinqume ukuthi ingabe ama-neuron e-t-hCO asebenza ngokufana nama-neocortical neurons angemva kokubeletha, senze ukurekhodwa kwe-electrophysiological kanye nokwakhiwa kabusha kwe-anatomical kwama-neurons e-L2/3 pyramidal neurons ezigabeni ezibukhali ze-cortex yomuntu yangemva kokubeletha (idatha enwetshiwe, Fig. 7a). Izici ze-electrophysiological ze-L2/3 pyramidal neurons zazifana nalezo ze-t-hCO pyramidal neurons (idatha eyandisiwe, Fig. 7e). I-Morphologically, i-L2 / 3 neurons evela kumasampuli omuntu wangemva kokubeletha yayifana kakhulu ne-t-hCO kune-hCO, nakuba amaseli e-L2 / 3 ayemade, equkethe amagatsha amaningi jikelele, futhi abe nokukhuluphala komgogodla (Fig. 3g nedatha enwetshiwe, Fig. 7b-). G).
a, ukufakelwa kabusha kwe-hCO ekhiqizwa ngokulawula kanye nemigqa yeseli ye-TS hiPS ibe amagundane asanda kuzalwa. b, ukwakhiwa kabusha kwe-3D kwama-neuron e-t-hCO agcwele i-biocytin ngemva kwezinyanga eziyi-8 zokuhlukaniswa. c, ukulinganisa ubude be-dendritic yesilinganiso (n = 19 control neurons, n = 21 TS neurons; **P = 0.0041). d, amagatsha e-dendritic akhiwe kabusha e-3D avela ekulawuleni kanye ne-TS t-hCO ezinyangeni ze-8 zokuhlukaniswa, kanye nomthamo we-dendritic spine density (n = 16 control neurons, n = 21 TS neurons, ***P <0.0001). d, amagatsha e-dendritic akhiwe kabusha e-3D avela ekulawuleni kanye ne-TS t-hCO ezinyangeni ze-8 zokuhlukaniswa, kanye nomthamo we-dendritic spine density (n = 16 control neurons, n = 21 TS neurons, ***P <0.0001). d, 3D-реконструкция дендритных ветвей из контроля и TS t-hCO через 8 месяцев дифференцировки и количественная оценка плотдисти 6 = 6 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, ukwakhiwa kabusha kwe-3D kwamagatsha e-dendritic kusukela ekulawuleni kanye ne-t-hCO TS ezinyangeni ezingu-8 zokuhlukaniswa kanye ne-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 重建树突分支，以及树突棘密度的量化（n = 16 內內个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 量化 1 神帅 （n 1 = 1 = 6 21 个 ts 神经 , *** p <0.0001 . d, 3D-реконструкция дендритных ветвей контроля и TS t-hCO через 8 месяцев дифференцировки kanye количественная оценка плотностиный плотностиный 1 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, ukwakhiwa kabusha kwe-3D kokulawula amagatsha e-dendritic kanye ne-TS t-hCO ezinyangeni ze-8 zokuhlukanisa kanye ne-dendritic spine density quantification (n=16 control neurons, n=21 TS neurons, ***P<0.0001).Izinkanyezi ezibomvu zibonisa i-puative dendritic spines. e, ama-EPSC azenzakalelayo alawulayo kanye nama-neurons e-TS t-hCO ngemva kwezinyanga eziyi-8 zokuhlukaniswa. f, i-cumulative frequency plot kanye ne-quantification ye-frequency kanye ne-amplitude yezenzakalo ze-synaptic (n=32 control neurons, n=26 TS neurons; **P=0.0076 kanye ne-P=0.8102). g, Ukuhlaziywa kwe-Scholl kwe-TS nokulawula ama-neurons ku-hCO ne-t-hCO. Imigqa edayishiwe ibonisa ama-neuron e-L2/3 ephiramidi yangemva kokubeletha ukuze kuqhathaniswe (n = 24 control t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 control hCO neurons, kanye n = 7 TS hCO neurons). Idatha ichazwa njengokuchezuka okujwayelekile okungu-±
Ikhono le-t-hCO lokuphindaphinda izici ze-morphological kanye nokusebenza kwe-cortex neurons yomuntu ezingeni eliphezulu lasishukumisela ukuba sihlole ukuthi i-t-hCO ingasetshenziswa yini ukuthola izifo ze-phenotypes. Sigxile ku-TS, ukuphazamiseka okunzima kwe-neurodevelopmental okubangelwa ukuguqulwa kokuzuzwa komsebenzi ku-gene encoding CaV1.2, eqala ukuloba okuncike kumsebenzi kuma-neurons. Sithole i-hCO ezigulini ezintathu ze-TS eziphethe ukushintshwa okuvame kakhulu (p.G406R) nezilawuli ezintathu (Fig. 3a). Ngemuva kokufakelwa kabusha, sithole ukuthi i-dendritic morphology yashintshwa kuma-neurons we-TS uma kuqhathaniswa nezilawuli (Fig. 3b kanye nedatha eyandisiwe, i-Fig. 8a, b), ngokunyuka okuphindwe kabili kwenani lama-dendrites ayinhloko kanye nokwanda okuphelele kwesilinganiso kanye nokuncipha okuphelele kobude be-dendritic (Fig. 3c kanye nedatha enwetshiwe, Fig. 8c). Lokhu kwakuhlotshaniswa nokuminyana okwandisiwe kwe-spines kanye nemvamisa eyengeziwe yama-EPSC azenzakalelayo ku-TS uma kuqhathaniswa nokulawula ama-neurons (Fig. 3d-f kanye nedatha eyandisiwe, i-Fig. 8g). Ukuhlaziya okwengeziwe kwembule amaphethini we-dendritic branching engavamile ku-t-hCO TS uma kuqhathaniswa nezilawuli, kodwa hhayi ku-in vitro TS hCO esigabeni esifanayo sokuhlukanisa (Fig. 3g). Lokhu kuhambisana nemibiko yethu yangaphambilini yokuncipha kwe-dendritic encike emsebenzini ku-TS futhi kugqamisa ikhono lale nkundla yokufakelwa ukuze ithole ama-phenotypes esifo ku-vivo.
Sibe sesibuza ukuthi amaseli e-t-hCO ahlanganiswa kangakanani ngokusebenza negundane i-S1. I-S1 kumagundane ithola okokufaka okuqinile kwe-synaptic kusuka ku-ipsilateral ventral basal ne-posterior thalamic nuclei, kanye ne-ipsilateral motor kanye ne-somatosensory cortices yesibili, kanye ne-contralateral S1 (Fig. 4a). Ukuze sibuyisele iphethini yokugcinwa kwangaphakathi, sathelela i-hCO nge-rabies virus-dG-GFP/AAV-G futhi satshala i-hCO kugundane elingu-S1 ezinsukwini ezi-3 kamuva. Sibone ukubonakaliswa kwe-GFP okuminyene kuma-neurons we-ipsilateral S1 kanye ne-ventral basal ganglia izinsuku ezingu-7-14 ngemva kokufakelwa kabusha (I-Fig. 4b, c). Ngaphezu kwalokho, ukungcoliswa kwe-antibody ye-thalamic marker netrin G1 yembula ukuba khona kweziphetho ze-thalamic ku-t-hCO (Fig. 4d, e). Ukuze sihlole ukuthi ingabe lokhu kuqagela okuhlukile kungathola yini izimpendulo ze-synaptic kumaseli e-t-hCO, senze ukuqoshwa kwamaseli wonke asuka kumaseli omuntu ezigabeni ezicijile zesendlalelo se-thalamocortical. Ukugqugquzelwa kukagesi kwegundane i-S1, i-capsule yangaphakathi, into emhlophe, imicu eseduze ne-t-hCO noma ukwenziwa kusebenze kwe-optogenetic kweziphetho ze-thalamic eziveza i-opsin kuma-EPSCs we-t-hCO abangele ukubambezeleka okufushane kwama-neurons e-t-hCO adalulwe kumphikisi we-AMPA receptor NBQX. (Fig. 4f, g kanye nedatha enwetshiwe, Fig. 9a–g). Le datha ibonisa ukuthi i-t-hCO ihlanganiswe ngokwemvelo ebuchosheni begundane futhi iyakwazi ukwenziwa isebenze izicubu zokusingatha amagundane.
a, Umdwebo ohleliwe wokuhlolwa kokulandela amarabi. b, i-GFP kanye nenkulumo ethi STEM121 yomuntu ethile phakathi kwe-t-hCO ne-rat cerebral cortex (iphaneli ephezulu). Kuphinde kuboniswe isisho se-GFP ku-rat ipsilateral ventral basal nucleus (VB) (phansi kwesokunxele) kanye ne-ipsilateral S1 (ezansi kwesokudla). Ibha yesikali, 50 µm. Izikwele ezibomvu zimelela izindawo zobuchopho lapho izithombe zithathwe khona. c, ukulinganisa kwamaseli aveza i-GFP (n = amagundane angu-4). d, e - Amatheminali e-Netrin G1+ thalamic ku-t-hCO. d ikhombisa ingxenye ye-coronal equkethe i-t-hCO ne-VB nuclei. Ibha yesikali, 2 mm. e ikhombisa i-Netrin G1 ne-STEM121 inkulumo ku-t-hCO (kwesokunxele) kanye ne-VB (kwesokudla) neurons. Ibha yesikali, 50 µm. Umugqa wamachashazi awolintshi ubonisa umngcele we-t-hCO. f, g, Izindlela zamanje zama-neurons e-t-hCO ngemva kokuvuselela ugesi ku-S1 rat (f) noma i-capsule yangaphakathi (g), eno (purple) noma ngaphandle (okumnyama) NBQX (kwesokunxele). Ama-amplitudes e-EPSC ane-NBQX nangenayo (n = 6 S1 neurons, *P = 0.0119; futhi n = 6 ama-capsule neurons angaphakathi, **P = 0.0022) (maphakathi). Iphesenti lama-neurons e-t-hCO abonisa i-EPSC ekuphenduleni ukukhuthazwa kagesi kwegundane i-S1 (f) noma i-capsule yangaphakathi (g) (kwesokudla). i-aCSF, uketshezi lwe-cerebrospinal yokwenziwa. h, umdwebo wohlelo lokuhlolwa kwesithombe kwe-2P (kwesokunxele). Ukuvezwa kwe-GCaMP6s ku-t-hCO (maphakathi). Ibha yesikali, 100 µm. Ukuphelelwa isikhathi kwe-Fluorescence kwe-GCaMP6s (kwesokudla). i, i-Z-score yomsebenzi ozenzakalelayo we-fluorescence. j, umfanekiso wohlelo lokukhuthazwa kwamadevu. k, ama-trajectories e-fluorescence angu-2P athole amaphuzu angu-2 esivivinyweni esisodwa, aqondaniswe nokuchezuka kwentshebe ngesikhathi uziro (umugqa wedeshi) kumaseli ayisibonelo. l, izimpendulo zesilinganiso sesibalo sika-z-score zawo wonke amaseli aqondaniswe nokuchezuka kwentshebe ngesikhathi uziro (umugqa oyideshi) (obomvu) noma izitembu zesikhathi ezikhiqizwa ngokungahleliwe (mpunga). m. Umdwebo wohlelo lokuhlolwa kokumaka okubonakalayo. n, Amajika kagesi we-Raw kusukela kusibonelo seseli ye-t-hCO ngesikhathi sokukhuthazwa kwe-laser eluhlaza okwesibhakabhaka noma ukuchezuka kwentshebe. Imicibisholo ebomvu ikhombisa izikhonkwane zokuqala ezibangelwa ukukhanya (phezulu) noma okubangelwa ukuchezuka kwentshebe (phansi). Ukufiphaza okumpunga kubonisa izikhathi zokuchezuka kwentshebe. o, Amagagasi okukhanya aphezulu kanye nezimpendulo zokuchezuka kwentshebe. p, ama-spikes omzamo owodwa, ohambisana nokuphambuka kwentshebe kumaseli esibonelo. 0 ukhombisa ukuchezuka kwentshebe (umugqa odeshi). q, isilinganiso sokudubula esiphakathi kwabantu esingu-z-score kuwo wonke amaseli azwelayo izithombe, aqondaniswe nokuchezuka kwewhisk ngesikhathi uziro (umugqa odeshisiwe) (obomvu) noma izitembu zesikhathi ezikhiqizwa ngokungahleliwe (mpunga). r, Ingxenye yamayunithi e-photosensitive ashintshwe kakhulu ngokuchezuka kwe-whisker (n = 3 amagundane) (kwesokunxele). Ukubambezeleka okuphezulu kwe-z-score (n = 3 amagundane; n = 5 (okuluhlaza okukhanyayo), n = 4 (okuluhlaza okumnyama), kanye no-n = 4 (cyan) amayunithi okuguquguquka kwe-whisker deflection per rat) (kwesokudla). Idatha ichazwa njengokushiwo ± ukuchezuka okujwayelekile
Sibe sesibuza ukuthi ingabe i-t-hCO ingenziwa isebenze ngokushukumisa izinzwa ku-vivo. Sitshale kabusha i-hCO eveza izinkomba ze-calcium efakwe ngokofuzo ku-GCaMP6 kumagundane e-S1. Ngemva kwezinsuku ezingu-150, senza i-fiber photometry noma i-imaging ye-calcium ye-photon ezimbili (Fig. 4h nedatha enwetshiwe, Fig. 10a). Sithole ukuthi amaseli e-t-hCO abonisa umsebenzi wesigqi ovumelanisiwe (Umfanekiso 4i, Idatha Enwetshiwe, Umfanekiso 10b kanye Nevidiyo Eyengeziwe 1). Ukuze sibonise umsebenzi omkhulu we-t-hCO, senze ukurekhodwa kwe-electrophysiological extracellular kumagundane okufakelwa afakwe izinzwa (idatha enwetshiwe, i-Fig. 10c-f). Senze izixhumanisi ze-stereotaxic ezithombeni ze-MRI; ngakho-ke, lawa mayunithi aqoshiwe amelela ama-neuron e-putive human, nakuba i-electrophysiology iyodwa ingavumeli uhlobo lwemvelaphi ukuthi lunqunywe. Sibone ukuqhuma okuvunyelanisiwe komsebenzi (idatha enwetshiwe, i-Fig. 10d). Ukuqhuma kuthathe cishe i-460 ms futhi kwahlukaniswa izikhathi zokuthula cishe ama-2 s (idatha enwetshiwe, Fig. 10d, e). Amayunithi angawodwana adubule isilinganiso esingangemizuliswano emithathu ngokuqhuma ngakunye, okungama-73% amayunithi abhalisiwe ngokuqhuma ngakunye. Imisebenzi yamayunithi ngamanye yayihlotshaniswa kakhulu, futhi lokhu kuhlobana kwakuphakeme kunaleyo yamayunithi ahlonzwe ezilwaneni ezingagonyiwe ezirekhodwe ngaphansi kwezimo ezifanayo (idatha eyandisiwe, Fig. 10f). Ukuze siqhubeke sibonise izimpendulo ze-spike zama-neurons atholwe ngumuntu, senze izivivinyo zokumaka ukukhanya kumagundane abulala izinzwa atshalwa kabusha nge-hCO eveza isiteshi se-cation esizwela ukukhanya i-rhodopsin 2 (hChR2), lapho i-t-hCO neurons ibonwa i-short-latency (ngaphansi kwe-10 ms-fimulimstimuli) ekuphenduleni kwe-blue-light. I-t-hCO neurons ibonise ukuqhuma komsebenzi ozenzakalelayo kumafrikhwensi afana nalawo abonwa ekuthwebuleni kwe-calcium, kanye nokurekhodwa kwe-electrophysiological okwenziwa ku-t-hCO ngaphandle kokumaka ukukhanya (idatha eyandisiwe, i-Fig. 10c-g). Awukho umsebenzi ozenzakalelayo owabonwa ezigabeni ezihambisanayo ze-hCO eqoshwe ku-vitro. Ukuhlola ukuthi ingabe i-t-hCO ingenziwa isebenze ngokushukumisa izinzwa, sichezukise kafushane amadevu egundane asuka ku-t-hCO (Fig. 4j,m kanye nedatha enwetshiwe, Fig. 10h,k). Ngokwezifundo zangaphambilini8,10, i-subset yamaseli e-t-hCO ibonise umsebenzi owandayo ekuphenduleni ukuphambuka kwe-whisker, engazange ibonwe lapho idatha iqhathaniswa nezitembu zesikhathi esingahleliwe (Fig. 4k-q kanye nedatha eyandisiwe, Fig. 10h-q). Ngempela, cishe i-54% yamayunithi angashadile ane-opto abonise izinga lokuvusa ngokuphawulekayo ngemva kokugqugquzelwa kwe-whisker, ukuphakama cishe ku-650 ms (Fig. 4r). Ihlanganiswe ndawonye, ​​le datha iphakamisa ukuthi i-t-hCO ithola okokufaka okusebenzayo okufanelekile futhi kungenziwa kusebenze izisusa zemvelo.
Sibe sesiphenya ukuthi i-t-hCO ingakwazi yini ukusebenzisa amasekhethi kumagundane ukuze silawule ukuziphatha. Siqale saphenya ukuthi ama-axon we-t-hCO neurons ayangena yini ezicutshini ezizungezile zegundane. Sathelela i-hCO nge-lentivirus efaka ikhodi engu-hChR2 ehlanganiswe ku-EYFP (hChR2-EYFP). Ngemva kwezinsuku ezingu-110, sabona ukubonakaliswa kwe-EYFP ezifundeni ze-ipsilateral cortical, okuhlanganisa i-auditory, motor, ne-somatosensory cortices, kanye nasezifundeni ezingaphansi kwe-subcortical, okuhlanganisa i-striatum, i-hippocampus, ne-thalamus (Fig. 5a). Ukuhlola ukuthi ingabe lokhu kuqagela okunamandla kungathola izimpendulo ze-synaptic kumaseli egundane, senze amaseli e-t-hCO asebenze aveza i-hChR2-EYFP ngokurekhoda amaseli e-rat cerebral cortex ezigabeni ezibukhali zobuchopho. Ukwenziwa kusebenze kwama-axon e-t-hCO anokukhanya okuluhlaza okwenzelwe ama-EPSC wesikhathi esifushane wokubambezeleka kuma-rat pyramidal cortex neurons, abevinjwe yi-NBQX (Figs. 5b–g). Ukwengeza, lezi zimpendulo zingavinjelwa yi-tetrodotoxin (TTX) futhi zibuyiselwe yi-4-aminopyridine (4-AP), ephakamisa ukuthi zibangelwa ukuxhumana kwe-monosynaptic (Fig. 5e).
a, Umdwebo wohlelo lokulandela i-axon (kwesokunxele). t-hCO EYFP expression (kwesokudla). Ibha yesikali, 100 µm. I-A1, i-auditory cortex, i-ACC, i-cingulate cortex yangaphambili, d. i-striatum, i-dorsal striatum, i-HPC, i-hippocampus; I-diaphragm, i-lateral septum, i-mPFC, i-medial prefrontal cortex, i-piri, i-piriform cortex, i-v. striatum, i-ventral striatum, i-VPM, i-ventropostomedial nucleus ye-thalamus, i-VTA, isifunda se-ventral tegmental. Izikwele ezibomvu zimelela izindawo zobuchopho lapho izithombe zithathwe khona. b, Umdwebo wesikimu wokuhlolwa kokuvuselela. c, d, Izibonelo zempendulo ye-photocurrent eyenziwe ukukhanya okuluhlaza okwesibhakabhaka (phezulu) kanye ne-voltage (ezansi) kumuntu (c) EYFP+ t-hCO noma igundane (d) EYFP- amaseli. e, f, Ukulandelana kwamanje kwama-neurons egundane ngemva kokuvuselela ukukhanya okuluhlaza okwesibhakabhaka kwama-axon e-t-hCO ane-TTX ne-4-AR (eluhlaza okotshani), i-TTX (empunga) noma i-aCSF (emnyama) (e), ene-(violet) noma ngaphandle (emnyama) ) ) I-NBQX (e). g, ukubambezeleka kwezimpendulo okubangelwa ukukhanya okuluhlaza okwesibhakabhaka kumaseli wamagundane (n = 16 amaseli); amabha avundlile akhombisa ukubambezeleka okumaphakathi (7.13 ms) (kwesokunxele). I-amplitude yama-EPSC akhishwe ukukhanya arekhodwe nge-NBQX noma ngaphandle kwayo (n = 7 amaseli; ***P <0.0001) (maphakathi). I-amplitude yama-EPSC akhishwe ukukhanya arekhodwe nge-NBQX noma ngaphandle kwayo (n = 7 amaseli; ***P <0.0001) (maphakathi). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). I-amplitude yama-EPSC enziwe ukukhanya arekhodwe nge-NBQX noma ngaphandle kwayo (n = 7 amaseli; ***P <0.0001) (maphakathi).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). I-amplitude yama-EPSC enziwe ukukhanya arekhodwe nge-NBQX noma ngaphandle kwayo (n = 7 amaseli; ***P <0.0001) (maphakathi).Amaphesenti amaseli egundane abonisa ama-EPSC aphendula ukukhanya okuluhlaza okwesibhakabhaka (kwesokudla). h, Umdwebo wohlelo lomsebenzi wokuziphatha. d0, usuku 0. i. Ukusebenza kwezilwane eziyisibonelo ngosuku 1 (kwesokunxele) noma usuku lwe-15 (kwesokudla) lokuqeqeshwa. Isilinganiso senani lokukhothwa okwenziwa ngosuku 1 (kwesokunxele) noma usuku 15 (maphakathi kwesokudla) (n = 150 ukukhanya okuluhlaza okwesibhakabhaka izilingo, n = 150 ukukhanya okubomvu izilingo; ***P <0.0001). Isilinganiso senani lokukhothwa okwenziwa ngosuku 1 (kwesokunxele) noma usuku 15 (maphakathi kwesokudla) (n = 150 ukukhanya okuluhlaza okwesibhakabhaka izilingo, n = 150 ukukhanya okubomvu izilingo; ***P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) noma день 15 (в центре справа) (n = 150 испытаний с синим светой, n = 150 светой, n = 150 светом; ***P <0,0001). Inani elisho inani lokukhothwa okwenziwa ngosuku 1 (kwesokunxele) noma usuku 15 (maphakathi kwesokudla) (n = 150 ukukhanya okuluhlaza okwesibhakabhaka, n = 150 ukukhanya okubomvu; ***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 试验，n = 150 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 右中）执行的平均舔次数) Среднее количество облизываний, выполненных в день 1 (слева) noma день 15 (в центре справа) (n = 150 испытаний с синим светой, n = 150 светой, n = 150 светом; ***P <0,0001). Inani elisho inani lokukhothwa okwenziwa ngosuku 1 (kwesokunxele) noma usuku 15 (maphakathi kwesokudla) (n = 150 ukukhanya okuluhlaza okwesibhakabhaka, n = 150 ukukhanya okubomvu; ***P <0.0001).Ukukhothwa okunqwabelanayo kwesivivinyo sokukhanya okubomvu nokuluhlaza okwesibhakabhaka ngosuku 1 (maphakathi kwesokunxele) noma usuku 15 (kwesokudla). I-NS, ayibalulekile. j,k, Izici zokuziphatha zazo zonke izilwane ezitshalwe kabusha nge-t-hCO eveza hChR2-EYFP (j) noma ilawula i-fluorophore (k) ngosuku 1 noma 15 (hChR2-EYFP: n = 9 amagundane, ** P = 0.0049; control: n = 9, P = 17). l, Ukuvela kwemiphumela ethandwayo (n = 9 hChR2, n = 9 control; **P <0.001, ***P <0.0001). l, Ukuvela kwemiphumela ethandwayo (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, Ukuvela kwemiphumela yokuthandwayo (n = 9 hChR2, n = 9 izilawuli; **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, Ukuvela kwezikolo ezithandwayo (n = 9 hChR2, n = 9 izilawuli; **P <0.001, ***P <0.0001).m, isisho se-FOS ekuphenduleni ukusebenza kwe-optogenetic ye-t-hCO ku-S1. Izithombe zenkulumo ye-FOS (kwesobunxele), nokulinganisa (n = 3 iqembu ngalinye; *P <0.05, **P <0.01 kanye ne-***P <0.001) (kwesokudla) ziyaboniswa. Izithombe zenkulumo ye-FOS (kwesobunxele), nokulinganisa (n = 3 iqembu ngalinye; *P <0.05, **P <0.01 kanye ne-***P <0.001) (kwesokudla) ziyaboniswa. Показаны изображения экспрессии FOS (слева) and количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 and *** P <0,001) (справа). Izithombe zenkulumo ye-FOS (kwesobunxele) nomthamo (n = 3 iqembu ngalinye; *P<0.05, **P<0.01, kanye ne-***P<0.001) ziyaboniswa (kwesokudla).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) and количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 and *** P <0,001) (справа). Izithombe zenkulumo ye-FOS (kwesobunxele) nomthamo (n = 3 iqembu ngalinye; *P<0.05, **P<0.01, kanye ne-***P<0.001) ziyaboniswa (kwesokudla).Ibha yesikali, 100 µm. Idatha ichazwa njengephutha ± elijwayelekile le-BLA, i-basolateral tonsil, i-MDT, i-dorsomedial thalamic nucleus, i-PAG, impunga ye-periaqueductal.
Ekugcineni, sibuze ukuthi i-t-hCO ingakwazi yini ukulinganisa ukuziphatha kwamagundane. Ukuhlola lokhu, satshala i-hChR2-EYFP-eveza i-hCO ku-S1, futhi ngemva kwezinsuku ezingu-90, safaka ama-optical fibers ku-t-hCO ukuze kuthunyelwe ukukhanya. Sabe sesiqeqesha amagundane nge-modified operant conditioning paradigm (Fig. 5h). Sibeke izilwane egumbini lokuhlola ukuziphatha futhi ngokungahleliwe sasebenzisa imizuzwana emi-5 eluhlaza okwesibhakabhaka (473 nm) kanye ne-laser ebomvu (635 nm). Izilwane zithole umvuzo wamanzi uma zikhotha ngesikhathi zivuselelwa ukukhanya okuluhlaza okwesibhakabhaka kodwa azikhothanga ngesikhathi zikhanyisa okubomvu. Ngosuku lokuqala lokuqeqeshwa, izilwane azizange zibonise umehluko ekukhotheni lapho zikhuthazwa ngokukhanya okuluhlaza okwesibhakabhaka noma okubomvu. Kodwa-ke, ngosuku lwe-15, izilwane ezitshalwe kabusha nge-hCO eveza i-hChR2-EYFP zibonise ukukhotha okusebenzayo uma kukhuthazwa ngokukhanya okuluhlaza okwesibhakabhaka uma kuqhathaniswa nokuvuselela ukukhanya okubomvu. Lezi zinguquko ekuziphatheni kokukhotha azizange zibonwe ezilwaneni ezilawulayo ezitshalwe kabusha nge-hCO eveza i-fluorophore yokulawula (izinga lempumelelo yokufunda: hChR2 89%, EYFP 0%, Umfanekiso 5i-1 kanye Nevidiyo Eyengeziwe 2). Le datha iphakamisa ukuthi amaseli e-t-hCO angasebenzisa ama-neurons egundane ukuze akhuthaze ukuziphatha kokufuna umvuzo. Ukuthola ukuthi iyiphi i-rat t-hCO neural circuits engase ihileleke kulezi zinguquko zokuziphatha, senze i-optogenetically isebenze i-t-hCO ezilwaneni eziqeqeshiwe kanye nezicubu ezivunwa ngemva kwemizuzu engu-90. I-Immunohistochemistry iveze ukubonakaliswa kwephrotheni ye-FOS encike emsebenzini ezindaweni ezimbalwa zobuchopho ezihilelekile ekuziphatheni okugqugquzelwayo, okuhlanganisa i-medial prefrontal cortex, i-medial thalamus, kanye ne-periaqueductal grey matter, evezwe ezilwaneni ezingalawulwa noma ezilwaneni. irayisi. 5m). Ihlanganiswe ndawonye, ​​le datha iphakamisa ukuthi i-t-hCO ingakwazi ukulinganisa umsebenzi we-rat neuronal ukushayela ukuziphatha.
Ama-Neural organoid amele uhlelo oluthembisayo lokufunda ukukhula komuntu kanye nezifo ku-vitro, kodwa anqunyelwe ukuntuleka kokuxhumana phakathi kwamasekethe akhona ku-vivo. Senze inkundla yenoveli lapho sifake khona i-hCO ku-S1 yamagundane asanda kuzalwa angenawo amandla okuzivikela ukuze sifunde ukuthuthukiswa kwamangqamuzana omuntu nokusebenza ku-vivo. Sibonise ukuthi i-t-hCO ithuthukisa izinhlobo zamaseli avuthiwe ezingabonwa ku-vitro28 nokuthi i-t-hCO ihlanganiswe ngokwemvelo nangokusebenza ebuchosheni begundane. Ukuhlanganiswa kwe-t-hCO kuma-rodent neural circuits kusivumele ukuba sisungule ukuxhumana phakathi komsebenzi wamaselula womuntu kanye nokuziphatha kwezilwane ezifundwayo, okubonisa ukuthi ama-neurons e-t-hCO angakwazi ukulinganisa umsebenzi we-rat neuronal ukushayela izimpendulo zokuziphatha.
Inkundla esiyichazayo inezinzuzo ezimbalwa ngaphezu kocwaningo lwangaphambilini lokufakelwa amangqamuzana omuntu abe ubuchopho begundane. Okokuqala, sifake i-hCO ku-cortex ekhulayo yamagundane angaphambi kokubeletha, angase enze kube lula ukuhlanganiswa kwe-anatomical nokusebenza. Okwesibili, ukuqapha kwe-t-hCO MRI kwasivumela ukuba sifunde isikhundla sokuxhunyelelwa nokukhula kwezilwane eziphilayo, okusivumela ukuba senze izifundo zesikhathi eside zezilwane eziningi futhi siqinisekise ukwethembeka kwemigqa eminingana ye-hiPS cell. Ekugcineni, satshala ama-organoid angaguquki, esikhundleni sokumiswa kweseli eyodwa okukodwa, okungonakalisi kangako amangqamuzana omuntu futhi angakhuthaza ukuhlanganiswa nokukhiqizwa kwama-neurons e-cortex yabantu ebuchosheni bamagundane.
Siyavuma ukuthi naphezu kwentuthuko kule nkundla, izithiyo zesikhashana, zendawo, kanye nezinhlobo ezihlukahlukene zivimbela ukwakheka kwamasekhethi emizwa yomuntu ngokwethembeka okuphezulu, ngisho nangemva kokufakelwa kabusha ekuqaleni kwentuthuko. Isibonelo, akucaci ukuthi umsebenzi ozenzakalelayo obonwa ku-t-hCO umele i-phenotype ethuthukayo efana nomsebenzi wesigqi obonwa ngesikhathi sokuthuthukiswa kwe-cortical, noma ukuthi kungenxa yokungabikho kwezinhlobo zamaseli ezicindezelayo ezikhona ku-t-hCO. Ngokufanayo, akucaci ukuthi ukungabikho kwe-lamination ku-t-hCO kuthinta kangakanani ukuxhumeka kweketango30. Umsebenzi wesikhathi esizayo uzogxila ekuhlanganiseni ezinye izinhlobo zamangqamuzana njenge-microglia yomuntu, amangqamuzana e-endothelial omuntu, kanye nezilinganiso ezihlukahlukene ze-GABAergic interneurons njengoba kuboniswa kusetshenziswa i-assembly 6 in vitro, kanye nokuqonda ukuthi ukuhlanganiswa kwe-neural nokucubungula kungenzeka kanjani ku-t-hCO eguquliwe. Amazinga okuloba, ama-synaptic kanye nokuziphatha kumaseli atholwe ezigulini.
Sekukonke, le nkundla ye-vivo imele insiza enamandla engahambisana nokuthuthukiswa kobuchopho bomuntu ku-vitro kanye nocwaningo lwezifo. Silindele ukuthi le nkundla izosivumela ukuthi sithole ama-phenotypes anoveli ezingeni le-strand kumaseli atholakala esigulini angatholakali kalula futhi sihlole amasu amasha okwelapha.
Senze i-hCO2.5 kumaseli e-HiPS njengoba kuchazwe ngaphambilini. Ukuze kuqalwe ukukhiqizwa kwe-hCO kusuka kumaseli e-hiPS akhuliswe ezingqimbeni zokuphakelayo, amakholomu aqinile amaseli e-hiPS akhishwa ezitsheni zamasiko kusetshenziswa i-dispase (0.35 mg/mL) futhi adluliselwa kumasiko epulasitiki anamathiselwe kakhulu aphansi aqukethe izitsha ezine-hiPS cell culture medium. (Corning) engezwe ngama-SMAD inhibitor amabili i-dorsomorphine (5 μM; P5499, Sigma-Aldrich) kanye ne-SB-431542 (10 μM; 1614, Tocris) kanye ne-ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Ezinsukwini ezi-5 zokuqala, i-hiPS cell medium yayishintshwa nsuku zonke futhi kwengezwa i-dorsomorphine ne-SB-431542. Ngosuku lwesithupha lokumiswa, ama-neural spheroids adluliselwa ku-neural medium equkethe i-neurobasal-A (10888, Life Technologies), isithasiselo se-B-27 esingenayo i-vitamin A (12587, Life Technologies), i-GlutaMax (1:100, i-Life Technologies), i-penicillin ne-streptomy00, i-Lifeed0 kanye ne-Technologies yokukhula kwe-Lifeed0 (1:1) isici (EGF; 20 ng ml−1; R&D Systems) kanye ne-fibroblast growth factor 2 (FGF2; 20 ng ml−1; R&D Systems) kuze kube usuku lwama-24. Kusukela osukwini lwama-25 kuya osukwini lwama-42, okuphakathi kwakwenezelwa nge-neurotrophic factor ephuma ebuchosheni (BDNF; 20, trophin-3; 30 trophin; 3 trophin; 3 ml) 20 ng ml−1; Ngosuku lwesithupha lokumiswa, ama-neural spheroids adluliselwa ku-neural medium equkethe i-neurobasal-A (10888, Life Technologies), isithasiselo se-B-27 esingenayo i-vitamin A (12587, Life Technologies), i-GlutaMax (1:100, i-Life Technologies), i-penicillin ne-streptomy00, i-Lifeed0 kanye ne-Technologies yokukhula kwe-Lifeed0 (1:1) isici (EGF; 20 ng ml−1; R&D Systems) kanye ne-fibroblast growth factor 2 (FGF2; 20 ng ml−1; R&D Systems) kuze kube usuku lwama-24. Kusukela osukwini lwama-25 kuya osukwini lwama-42, okuphakathi kwakwenezelwa nge-neurotrophic factor ephuma ebuchosheni (BDNF; 20, trophin-3; 30 trophin; 3 trophin; 3 ml) 20 ng ml−1;Ngosuku lwesithupha lokumiswa, ama-neural spheroids adluliselwa endaweni ye-neural equkethe i-Neurobasal-A (10888, Life Technologies), i-B-27 engeza i-vitamin A (12587, i-Life Technologies), i-GlutaMax (1: 100, i-Life Technologies), i-penicillin.и стрептомицин (1:100, Life Technologies) и дополнены эпидермальным фактором роста (EGF; 20 нг/мл; R&D Systems) и фактором роста фибл20Fласто; R&D Systems) ukuya ku-24-го дня. kanye ne-streptomycin (1:100, Life Technologies) futhi yengezwe nge-epidermal growth factor (EGF; 20 ng/ml; R&D Systems) kanye ne-fibroblast growth factor 2 (FGF2; 20 ng/ml; R&D Systems) kuze kube usuku lwama-24.Kusukela ezinsukwini ezingu-25 kuya ku-42, i-neurotrophic factor etholakala ebuchosheni (BDNF; 20 ng ml-1, Peprotech) kanye ne-neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) yengezwe endaweni ephakathi, ukushintsha okuphakathi njalo ngosuku.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 kanye 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 维移 含有 含有 neurobasal-a (10888 , Life Technologies) (12587 , Life Technologies) Glutamax (1: 100 , Life TechNOGIS青霉素 的 神经 培养 基 中 链霉素 （（1:100” Life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 фактормального факторан (1:100, Life Technologies); I-R&D Systems) kanye ne-фактора роста фибробластов 2 (FGF2; 20 нг мл-1) 1; Ngosuku lwe-6, ukumiswa kwe-neurosphere kwashintshelwa kwesengezo esiqukethe i-neurobasal-A (10888, Life Technologies), isithasiselo se-B-27 esingenayo i-vitamin A (12587, Life Technologies), i-GlutaMax (1:100, i-Life Technologies), i-penicillin-neutralized factor streptomycin (1:100) (EGF; 20 ng ml-1; R&D Systems) kanye ne-fibroblast factor factor 2 (FGF2; 20 ng ml-1) 1; R&D Systems) ukuya ku-24-го дня. R&D Systems) kuze kube usuku lwama-24.Kusukela ezinsukwini ezingu-25 kuya kwezingu-42, i-neurotrophic factor etholakala ebuchosheni (BDNF; 20 ng ml-1, Peprotech) kanye ne-neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) yengezwe endaweni yesiko njalo ngemva kolunye usuku. Ushintsho olumaphakathi kanye.Kusukela osukwini lwama-43, i-hCO ibigcinwa ku-neurobasal-A medium engangeziwe (NM; 1088022, Thermo Fisher) ngoshintsho oluphakathi njalo ezinsukwini ezi-4-6. Ukuze uthole i-hCO kusuka kumaseli e-hiPS akhuliswe ngaphansi kwezimo ezingenasici, amaseli e-hiPS afakwe i-Accutase (AT-104, Innovate Cell Technologies) ku-37 ° C imizuzu engu-7, ahlukaniswe abe amaseli angashadile, futhi afakwe kumapuleti e-AggreWell 800 (34815, STEMCELL Amathuluzi angu-6 ku-single × 3 amaseli e-single × 3 cell dechnologies) I-Essential 8 medium ingezwe nge-ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Ngemva kwamahora angu-24, imidiya emithonjeni yafakwa ngamapayipi phezulu naphansi kwabezindaba eziqukethe i-Essential 6 media (A1516401, Life Technologies) ehlanganiswe ne-dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) kanye ne-SB-431542 (10 μM; 1614). , Tocrida). Kusukela ezinsukwini ezi-2 kuye kwezi-6, i-Essential 6 medium yashintshwa nsuku zonke nge-dorsomorphine kanye nesengezo se-SB-431542. Kusukela ngosuku lwesithupha, ukumiswa kwe-neurosphere kwadluliselwa ku-neurobasal medium futhi kwagcinwa njengoba kuchazwe ngenhla.
Zonke izinqubo zezilwane zenziwe ngokuhambisana neziqondiso zokunakekelwa kwezilwane ezigunyazwe yi-Stanford University Laboratory Animal Care Administrative Committee (APLAC). Amagundane e-euthymic RNU (rnu/+) akhulelwe athengiwe (Charles River Laboratories) noma ahlaliswa. Izilwane zazigcinwa emjikelezweni wokukhanya wobumnyama wamahora angu-12 ngokudla kanye ne-ad libitum yamanzi. Imidlwane yamagundane enqunu (i-FOXN1–/–) enezinsuku ezintathu kuya kweziyisikhombisa ibonakale ngokukhula kwamadevu angakakhuli ngaphambi kokuba akhishwe. Imidlwane (owesilisa nowesifazane) yalaliswa ngo-2-3% we-isoflurane futhi yabekwa kuhlaka lwe-stereotaxic. Ukunyakaziswa kogebhezi olunobubanzi obungu-2-3 mm ngaphezu kwe-S1 kwenziwa ngenkathi kugcinwa ubuqotho be-dura mater. Bese usebenzisa inaliti engu-30-G (cishe u-0.3 mm) ngaphandle nje kwe-craniotomy ukuze ubhoboze i-dura. Bese usebenzisa i-HCO ku-parafilm encane engu-3 × 3 cm bese ususa i-medium ngokweqile. Usebenzisa isirinji ye-Hamilton exhunywe enaliti engu-23 G, 45°, dweba ngobumnene i-hCO ekugcineni kwayo inaliti. Bese ufaka isirinji empompini yesirinji exhunywe kudivayisi ye-stereotaxic. Bese ubeka ichopho lonaliti phezu kwembobo yokubhoboza ebanzi eyenziwe ngaphambilini engu-0.3 mm ku-dura (z = 0 mm) futhi unciphise isirinji ngo-1-2 mm (z = cishe -1.5 mm) kuze inaliti ibe phakathi kwe-dura mater A. kwakheka uphawu oluminyene. Bese uphakamisa isirinji phakathi nendawo yekhohlo ku-z = -0.5 mm bese ujova i-hCO ngesilinganiso esingu-1-2 µl ngomzuzu. Ngemuva kokuphothulwa komjovo we-hCO, inaliti ihoxiswa ngenani lika-0.2-0.5 mm ngomzuzu, isikhumba siyathungwa, futhi umdlwane ufakwa ngokushesha endaweni yokushisa efudumele kuze kube yilapho ululama ngokuphelele. Ezinye izilwane zatshalwa kabusha ngokuhlanganyela.
Zonke izinqubo zezilwane zenziwe ngokuhambisana neziqondiso zokunakekelwa kwezilwane ezigunyazwe yi-APLAC yaseStanford University. Amagundane (amakhudlwana kunezinsuku ezingu-60 ngemva kokufakelwa kabusha) ayengelwa nge-5% isoflurane anesthesia futhi afakwe izinzwa nge-1-3% isoflurane ngesikhathi sokuthwebula. Ukuze ubone ngeso lengqondo, isithwebuli se-borehole esingu-7 se-Tesla esivikelwe ngenkuthalo i-Bruker (Bruker Corp.) esinedrayivu yegradient ye-International Electric Company (IECO), ukufakwa okuvikelekile okunobubanzi bangaphakathi obungu-120 mm (600 mT/m, 1000 T/m/s) kusetshenziswa i-AVANCE. III, iziteshi eziyisishagalombili ezinamakhoyili amaningi i-RF kanye namakhono amakhoyili amaningi, kanye nengxenyekazi ehambisana nayo yeParavision 6.0.1. Ukuqopha kwenziwa kusetshenziswa ikhoyili ye-RF yevolumetric ehlukanisiwe enobubanzi bangaphakathi obungu-86 mm kanye nekhoyili ye-RF eneziteshi ezine epholile ukuze kwamukelwe kuphela. I-Axial 2D Turbo-RARE (isikhathi sokuphindaphinda = 2500 ms, isikhathi se-echo = 33 ms, isilinganiso esingu-2) ngokuthwebula izingcezu ezingu-16, ukujiya kocezu okungu-0.6–0.8 mm, okuqukethe amasampula angu-256 × 256. Amasignali atholwe kusetshenziswa ikhoyili ye-quadrature transceiver volumetric RF enobubanzi bangaphakathi obungu-2 cm (Rapid MR International, LLC). Okokugcina, sebenzisa imisebenzi yokulinganisa indawo eyakhelwe ngaphakathi ye-Imaris (BitPlane) ukuze unikeze i-3D nokuhlaziya ivolumu. Ukufakelwa okuphumelelayo kwachazwa njengendawo lapho izindawo zesignali ye-MRI enesisindo esiqhubekayo se-T2 zakhiwa khona ku-hemisphere etshaliwe. Ukwenqatshwa kwe-graft kwachazwa njenge-graft engazange ikhiqize izindawo zesignali ye-MRI enesisindo esiqhubekayo se-T2 ku-hemisphere etshaliwe. I-Subcortical t-hCO ayifakwanga ekuhlaziyweni okwalandela.
Ukuze kuvezwe ngokuzinzile i-GCaMP6s ku-hCO yezithombe ze-calcium ezinezithombe ezimbili, amaseli e-hiPS angenwe yi-pLV[Exp]-EF1a::GcaMP6s-WPRE-Puro elandelwa ukukhethwa kwemithi elwa namagciwane. Kafushane, amaseli ahlukanisiwe ne-EDTA futhi amiswa ku-1 ml we-Essential 8 medium ekushubeni kwamaseli cishe angama-300,000 lapho kukhona i-polybrene (5 μg/ml) kanye ne-15 μl yegciwane. Amangqamuzana abe esefukanyelwa ekumisweni imizuzu engama-60 futhi ahlwanyelwa ekushubeni kwamaseli angama-50,000 emthonjeni ngamunye. Ngemva kokuhlangana, amaseli aphathwe nge-5-10 μg ml-1 puromycin izinsuku ezingu-5-10 noma kuze kuvele amakoloni azinzile. Ukutheleleka kwe-hCO okunamandla kwenziwa njengoba kuchazwe ngaphambilini5 ngokulungiswa okuthile. Kafushane, dlulisela usuku 30-45 hCO kumashubhu e-Eppendorf microcentrifuge angu-1.5 aqukethe u-100 µl we-nerve medium. Bese kuthi cishe ama-90 µl we-medium akhishwe, u-3-6 µl we-lentivirus ye-titer ephezulu (kusuka ku-0.5 x 108 kuya ku-1.2 x 109) yengezwa eshubhuni, bese i-hCO idluliselwa ku-incubator imizuzu engu-30. Bese wengeza u-90–100 µl wesilinganiso esimaphakathi eshubhuni ngalinye bese ubuyisela amashubhu ku-incubator ubusuku bonke. Ngosuku olulandelayo, dlulisela i-hCO ku-nerve medium kumapuleti anamathiselwe aphansi. Ngemva kwezinsuku ezingu-7, i-hCO yadluliselwa kumapuleti angaphansi engilazi anemithombo engama-24 ukuze kubonwe ngeso lengqondo futhi kuhlolwe izinga lokutheleleka. I-pLV[Exp]-SYN1::EYFP-WPRE kanye ne-pLV[Exp]-SYN1::hChR2-EYFP-WPRE zenziwe yi-VectorBuilder. I-Lentivirus isetshenziswa ekuhloleni okuningi ngoba ihlanganiswe ku-genome yomsingathi, okuvumela ukubonakaliswa kofuzo lwentatheli emigqeni yeseli ethelelekile. Ukuze kulandelelwe amarabi, usuku lwama-30-45 hCO yatheleleka kanye namarabi-ΔG-eGFP kanye ne-AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), yagezwa kahle izinsuku ezingu-3, ​​futhi yafakwa kumagundane ku-S1 futhi igcinwe izinsuku ezingu-7-1 nge-vivo.
Ku-immunocytochemistry, izilwane zalaliswa izinzwa futhi zagcotshwa nge-PBS zilandelwa yi-4% paraformaldehyde (PFA ku-PBS; i-Electron Microscopy Sciences). Ubuchopho bulungiswe ku-4% PFA amahora angu-2 noma ubusuku bonke ku-4°C, bugcinwe ku-30% sucrose ku-PBS amahora angu-48-72, futhi bushumekwe kokuthi 1:1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583 , Sakura Finetes0rsection at ccoronat 3) (Leica). Ku-immunohistochemistry yezingxenye eziwugqinsi, izilwane zagcotshwa nge-PBS, futhi ubuchopho bahlukaniswa futhi bahlukaniswa nge-coronally ku-300-400 µm kusetshenziswa i-vibratome (Leica) futhi izigaba zalungiswa nge-4% PFA imizuzu engu-30. Khona-ke ama-cryosections noma izingxenye eziwugqinsi zagezwa nge-PBS, zavinjwa ihora elingu-1 ekamelweni lokushisa (10% i-serum evamile yembongolo (NDS) kanye ne-0.3% ye-Triton X-100 ehlanjululwe ku-PBS) futhi ivinjwe ngesisombululo sokuvimba ku-4 ° C. - I-Incubation Cryosections yafukanyelwa ubusuku bonke futhi izingxenye eziwugqinsi zafukanyelwa izinsuku ezi-5. Amasosha omzimba ayinhloko ayesetshenziswa: i-anti-NeuN (igundane, 1:500; ab104224, abcam) anti-CTIP2 (igundane, 1:300; ab18465, abcam), i-anti-GFAP (unogwaja, 1:1,000; Z0334, Dako), i-anti-GFP0, GTX7, i-3,00 yenkukhu, i-GFP0; I-GeneTex), i-anti-HNA (igundane, 1:200; ab191181, abcam), i-anti-NeuN (unogwaja, 1:500; ABN78, Millipore), i-anti-PDGFRA (unogwaja, 1:200; sc-338, Santa Cruz), i-anti-PPP20, 7; I-HPA047819, i-Atlas Antibodies), i-anti-RECA-1 (igundane, 1:50; ab9774, abcam), i-anti-SCG2 (unogwaja, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (imbuzi, 1:500; NetgorinD1 Systema, G30, G&30 1:100; AF1166, R&D Systems), anti-STEM121 (igundane, 1:200; Y40410, Takara Bio), anti-SATB2 (igundane, 1:50; ab51502, abcam), anti-GAD65/67 (unogwaja, 1:490, anti-ABIN00; (imbuzi, 1:100; ab5076, abcam). Amasosha omzimba ayinhloko ayesetshenziswa: i-anti-NeuN (igundane, 1:500; ab104224, abcam) anti-CTIP2 (igundane, 1:300; ab18465, abcam), i-anti-GFAP (unogwaja, 1:1,000; Z0334, Dako), i-anti -GFP0, GTX7, i-3 yenkukhu, i-GTX0; I-GeneTex), i-anti-HNA (igundane, 1:200; ab191181, abcam), i-anti-NeuN (unogwaja, 1:500; ABN78, Millipore), i-anti-PDGFRA (unogwaja, 1:200; sc-338, Santa Cruz), i-anti-PPP20, 7; I-HPA047819, i-Atlas Antibodies), i-anti-RECA-1 (igundane, 1:50; ab9774, abcam), anti-SCG2 (unogwaja , 1:100; 20357-1-AP, Proteintech), anti-SOX9 (imbuzi, 1:500; Neta1 Systems), Neta10D&30 1:100; AF1166, R&D Systems), anti-STEM121 (igundane , 1:200; Y40410, Takara Bio), anti-SATB2 (igundane, 1:50; ab51502, abcam), anti-GAD65/67 (unogwaja, 1:400; (imbuzi, 1:100; ab5076, abcam). Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2 (крысиные, 1,5b840); анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA (мышь, 1:200; ab1911181, ab1911181, ab1911181, 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; I-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). Amasosha omzimba ayinhloko ayesetshenziswa: i-anti-NeuN (igundane, 1:500; ab104224, abcam), i-anti-CTIP2 (igundane, 1:300; ab18465, abcam), i-anti-GFAP (unogwaja, 1:1000; Z0334, Dako), i-anti-GFP0; i-anti-HNA (igundane, 1:200; ab191181, abcam), i-anti-NeuN (unogwaja, 1:500; ABN78, Millipore), anti-PDGFRA ( unogwaja, 1:200; sc-338, Santa Cruz), anti-PPP1R17, 1:03 HPAb4; Ama-anti-RECA-1 (igundane, 1:50; ab9774, abcam), anti- SCG2 (unogwaja, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (imbuzi, 1:500; AF3075, R&D Systems), netrin 1:10, G1, netrin 10; I-R&D Systems), i-anti- STEM121 (igundane, 1:200; Y40410, Takara Bio), i-anti-SATB2 (igundane, 1:50; ab51502, abcam), anti-GAD65/67 (unogwaja, 1:400; ABN904, Millipore 1:0) kanye ne-goat-anti, 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,GRAP）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，1:500；ABN78％Millipo％Millipo） I-200 100;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 (igundane, 1:50; ab51502, abcam), anti-GAD65/67 (unogwaja, 1:400; ABN904, Millipore) kanye ne-anti-IBA1 (imbuzi, 1:100; ab5076, abcam).Amasosha omzimba ayinhloko ayesetshenziswa: i-anti-NeuN (igundane, 1:500; ab104224, abcam), i-anti-CTIP2 (igundane, 1:300; ab18465, abcam), i-anti-GFAP (unogwaja, 1:1000; Z0334, Dako) . , i-anti-GFP (inkukhu, 1:1000; GTX13970, GeneTex), anti-HNA (igundane, 1:200; ab191181, abcam), anti-NeuN (unogwaja, 1:500; ABN78, Millipore), anti-PDGFRA (rab303, 108; anti-PPP1R17 (unogwaja, 1:200; HPA047819, Atlas antibody), anti-RECA-1 (igundane, 1:50; ab9774, abcam), anti- SCG2 (unogwaja), 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, Taka), 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 (imbuzi, 1:500; AF3075, R&D Systems), Netrin G1a (imbuzi, 1:100; AF1166, R&D Systems), anti-STEM121 (igundane, 1:200; i-anti-SATBra, Y404), i-Takamo1 1:50; ab51502, abcam), anti-GAD65/67 (unogwaja, 1:400; ABN904, Millipore), kanye ne-anti-IBA1 (imbuzi, 1:100; ab5076, abkam).Izingxenye zazibe sezigezwa nge-PBS futhi zifakwe amasosha omzimba esibili ihora elingu-1 ekamelweni lokushisa (izingxenye eziqandisiwe) noma ngobusuku obungu-4°C (izingxenye eziwugqinsi). I-Alexa Fluor antibody yesibili (Life Technologies) ehlanjululwe 1:1000 kusixazululo sokuvimba isetshenzisiwe. Ngemva kokugeza nge-PBS, i-nuclei yabonwa nge-Hoechst 33258 (Life Technologies). Ekugcineni, amaslayidi abekwa kusibonakhulu anamakhava (Fisher Scientific) kusetshenziswa i-Aquamount (Polysciences) futhi ahlaziywa ngesibonakhulu se-Keyence fluorescent (BZ-X analyzer) noma isibonakhulu se-Leica TCS SP8 confocal (Las-X) esithombeni. Izithombe zicutshungulwe kusetshenziswa uhlelo lwe-ImageJ (Fiji). Ukuze kulinganiswe ingxenye yama-neurons omuntu ku-t-hCO kanye ne-rat cortex, izithombe eziwunxande ezinobubanzi obungu-387.5 μm zithathwe maphakathi ne-t-hCO, emaphethelweni noma eduze konqenqema lwe-rat cortex. Amamajini egraft anqunywa ngokuhlola izinguquko ekukhanyeni kwezicubu, i-HNA+ nuclei, kanye/noma ukuba khona kwezicubu ezizenzakalelayo. Esithombeni ngasinye, ingqikithi yenani lamaseli e-NeuN+ ne-HNA+ ahlukaniswe ngesamba senani lamaseli e-NeuN+ endaweni efanayo. Ukuqinisekisa ukuthi kubalwa kuphela amaseli anama-nuclei endizeni yesithombe, amaseli kuphela nawo ayi-Hoechst+ afakiwe esibalweni. Izithombe ezimbili ezihlukaniswe okungenani ngo-1 mm zilinganiselwe ukuze kuncishiswe iphutha lezibalo.
Isonto elilodwa ngaphambi kokuqoqwa kwesampula, beka izilwane zokufakelwa i-hCO (izinyanga ezingaba ngu-8 zokuhlukaniswa) ekamelweni elimnyama elinentshebe esikiwe ukuze kuncishiswe ukukhuthazwa kwezinzwa. Ukuhlukaniswa kwama-nuclei kwenziwa njengoba kuchaziwe ngaphambili, nokulungiswa okuthile. Kafushane, i-t-hCO ne-hCO kucekelwe phansi kusetshenziswa i-detergent-mechanical cell lysis kanye ne-2 ml yengilazi yokugaya ithishu (D8938, Sigma-Aldrich/KIMBLE). Ama-nuclei aluhlaza abe esehlungwa kusetshenziswa isihlungi esingu-40 µm futhi safakwa phakathi ku-320 g imizuzu eyi-10 ku-4 °C ngaphambi kokwenza ukuminyana kwe-sucrose. Ngemva kwesinyathelo se-centrifugation (320 g imizuzu engu-20 ku-4°C), amasampuli aphinde amiswa ngo-0.04% BSA/PBS ngokwengezwa kwamayunithi angu-0.2 we-µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) futhi adlula kwisihlungi esingu-40 µ. Ama-nuclei ahlukanisiwe abe esemiswa kabusha ku-PBS equkethe u-0.02% BSA futhi alayishwa ku-chip ye-Chromium Single Cell 3′ (isilinganiso sokutholwa kwamaseli angu-8,000 ngomzila ngamunye). Imitapo yolwazi ye-snRNA-seq yalungiswa nge-Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Imitapo yolwazi ye-snRNA-seq yalungiswa nge-Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Библиотеки snRNA-seq были приготовлены с помощью Iseli Ye-Chromium Single 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Imitapo yolwazi ye-snRNA-seq ilungiswe kusetshenziswa i-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 I-Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Umtapo wezincwadi we-snRNA-seq walungiswa kusetshenziswa i-Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics).Imitapo yolwazi evela kumasampuli ahlukene yahlanganiswa futhi yalandelwa yi-Admera Health ku-NovaSeq S4 (Illumina).
Amazinga enkulumo yofuzo yebhakhodi yenuzi ebekayo ngayinye alinganiswa kusetshenziswa iphakheji yesofthiwe yokuhlaziya ye-10x Genomics CellRanger (inguqulo 6.1.2). Ngokucacile, ukufundwa kwafaniswa nenhlanganisela yomuntu (GRCh38, Ensemble, version 98) kanye nereferensi yegundane (Rnor_6.0, Ensemble, version 100) edalwe ngomyalo we-mkref nokusebenzisa ukubala ngomyalo othi -include-introns=TRUE wokulinganisa kufaka phakathi ukufundwa okufakwe kumephu ukuze kwethulwe izifunda. Kumasampula e-t-hCO, ama-nuclei omuntu ahlonzwe ngokusekelwe kwimfuneko elondolozayo yokuthi okungenani u-95% wakho konke ukufundwa kwemephu kufane ne-genome yomuntu. Konke ukuhlaziya okwalandela kwenziwa ekukhishweni kohlu lwebhakhodi ehlungiwe kusukela ku-CellRanger kusetshenziswa iphakheji ye-R (inguqulo 4.1.2) Seurat (inguqulo 4.1.1)32.
Ukuqinisekisa ukuthi ama-nuclei ekhwalithi ephezulu kuphela afakiwe ekuhlaziyweni okulandelayo, inqubo yokuhlunga ephindaphindwayo yenziwa isampula ngayinye. Okokuqala, ama-nuclei ekhwalithi ephansi anezakhi zofuzo eziyingqayizivele ezingaphansi kwe-1000 ezitholiwe futhi ngaphezu kwe-20% yengqikithi ye-mitochondria iyabonakala futhi isusiwe. Ngokulandelayo, i-matrix yenombolo yofuzo eluhlaza yenziwa yajwayelekile ngokuhlehla okunegethivu kwe-binomial okuvamile kusetshenziswa umsebenzi we-sctransform(vst.flavor=”v2″), ophinde wahlonza izinhlobo zofuzo eziguquguqukayo kakhulu ezingu-3000 kusetshenziswa amapharamitha amisiwe. Ukwehliswa kobukhulu kwenziwa ezakhini zofuzo eziphezulu kusetshenziswa i-Principal Component Analysis (PCA dimension of the default Component Analysis (PCA) enedatha emisiwe = i-PCA enedatha ezenzakalelayo 30 yakhethwa ngokusekelwe ekuhlolweni okubonakalayo kwamasayithi amadolo futhi yasetshenziselwa wonke amasampuli nokuhlaziya okuhlanganisiwe (i-resolution = 1) ukuze sihlukanise izakhi zofuzo ezisekelwe ekubalweni kwezakhi zofuzo eziphansi ngokungavamile (i-median engaphansi kwe-10th percentile), ephezulu ngokungavamile ye-mitochondrial gene phezu kwesibalo esiphansi se-mitochondrial 5 kanye/noma ingxenye enkulu yamawele asolwayo ahlonzwe kusetshenziswa iphakheji ye-DoubletFinder33 (isho amaphuzu e-DoubletFinder ngaphezu kwamasampula e-t-hCO (n=3) kanye ne-hCO (n=3) ahlanganiswe ngokuhlukana kusetshenziswa umsebenzi we-IntegrateData namapharamitha angenhla.
Ngemva kokukhipha ama-kernel ekhwalithi ephansi, idathasethi ehlanganisiwe yahlanganiswa (ukulungiswa = 0.5) futhi yashumezelwa izinjongo zokuboniswa kwe-UMAP34. Izakhi zofuzo zomaka zeqoqo ngalinye ziye zanqunywa kusetshenziswa umsebenzi we-FindMarkers namapharamitha azenzakalelayo abalwe kusukela kudatha yesisho sofuzo esivamile. Sihlonza futhi sihlukanise izigaba zamaseli amakhulu ngokuhlanganisa amasethi edatha ereferensi ekhohlo yengane kanye neyabantu abadala nesisho sofuzo somaka 19,20,21,35 kanye nesichasiselo. Ikakhulukazi, izandulela ezijikelezayo zikhonjwe ngenkulumo ye-MKI67 ne-TOP2A. Amaqoqo okhokho achazwe ukungabikho kokulotshiweyo kwe-mitotic, ukugqagqana okuphezulu namaqoqo e-glial progenitor amaningi achazwe ku-metaphase fetal cortex yakamuva, kanye nenkulumo ye-EGFR ne-OLIG1. Sisebenzisa igama elithi astrocyte ukuze lihlanganise izifunda ezimbalwa zokuhlukaniswa kwe-astrocyte, kusukela ku-radial glia sekwephuzile ukuya ekuvuthweni kwama-astrocyte. Amaqoqo e-astrocyte aveza amazinga aphezulu e-SLC1A3 ne-AQP4 futhi akhonjiswe kumephu enezinhlobo ezincane ze-fetal radial glia kanye/noma ama-astrocyte amadala. Ama-OPC aveza i-PDGFRA ne-SOX10 kuyilapho ama-oligodendrocyte eveza omaka be-myelination (MOG ne-MYRF). Ama-neurons e-Glutamatergic akhonjwe ngokuba khona kwemibhalo ye-neuronal (SYT1 ne-SNAP25), ukungabi khona kwezimaka ze-GABAergic (GAD2), kanye nokuvezwa kwe-NEUROD6, SLC17A7, BCL11B, noma i-SATB2. Ama-GluN neurons aphinde ahlukaniswa aba ama-subclasses aphezulu (i-SATB2 kanye nokulahlekelwa kwe-BCL11B) kanye ne-deep (BCL11B expression). I-Pututive subplate (SP) neurons iveza omaka be-SP18 abaziwayo njenge-ST18 ne-SORCS1 ngaphezu komaka be-GluN abajulile. Amaseli afana ne-Choroid plexus akhonjwe ngokuveza kwe-TTR, futhi amaseli afana ne-meningeal aveza ufuzo oluhlobene ne-fibroblast kanye namaseli afakwe kumephu wepiyali/emithambo yesethi yedatha yesithenjwa.
Ukuhlaziywa okuhlukile kokubonakaliswa kofuzo phakathi kwezigaba ezingaphansi kwe-t-hCO ne-hCO kwenziwe kusetshenziswa indlela yeqoqo mbumbulu esanda kuthuthukiswa ekhiqizwe kabusha kumasampuli asetshenziswe kusetshenziswa iphakheji ye-Libra R (inguqulo 1.0.0). Ngokukhethekile, ukuhlolwa kokungenzeka kwelogi ye-edgeR (inguqulo 3.36.0, iphakheji R) yenzelwe amaqembu ngokufingqa inani lezakhi zofuzo kumaseli ekilasi leseli elinikeziwe lesampula ngalinye lokuphindaphinda. Ngokubukwa kwemephu yokushisa, amanani ajwayelekile ngesigidi (CPM) ahlanganiswa kusetshenziswa umsebenzi we-edgeR (cpm()) futhi akalwe (ukuthola incazelo = 0, ukuchezuka okujwayelekile = 1). I-Gene Ontology (GO) ukuhlaziywa okunothisayo kwezakhi zofuzo ze-t-hCO GluN ezilawuleke kakhulu zenziwe (i-Benjamini-Hochberg ilungise inani le-P elingaphansi kuka-0.05 elivezwe okungenani ku-10% wamaseli e-t-hCO GluN kanye nokwenyuka koshintsho okungenani izikhathi ezingu-2). kwenziwa kusetshenziswa i-ToppGene Suite (https://toppgene.cchmc.org/)37. Sisebenzisa uhlelo lokusebenza lwe-ToppFun olunamapharamitha azenzakalelayo futhi sibike amanani e-P alungiswe u-Benjamin-Hochberg abalwe kusukela kuhlolo lwe-GO-annotated hypergeometric.
Ukufanisa amaqoqo ethu e-snRNA-seq namaqoqo amaseli anezichasiselo kusukela ezifundweni eziyisithenjwa ze-primary single-cell RNA-seq noma i-snRNA-seq19,20,21,22 yabantu abadala, sisebenzise indlela yokuhlanganisa yedathasethi ebhanqiwe. Sisebenzise ukuhamba komsebenzi kwe-SCTransform (v2) kokujwayelekile ku-Seurat ukuze sihlanganise futhi siqhathanise ukugqagqana kweqoqo phakathi kwamasethi edatha (sisebenzisa amapharamitha afanayo njengenhla). Amasethi edatha angawodwana ahlelwa ngokungahleliwe afika kumaseli angu-500 noma ama-cores ngeqoqo langempela ngalinye ukuze kube nokusebenza kahle kwekhompyutha. Kusetshenziswa indlela efanayo njengoba kuchazwe ngaphambilini, ukugqagqana kweqoqo kwachazwa njengengxenye yamaseli noma ama-nuclei kuqoqo ngalinye elihlanganisiwe elidlula ilebula yeqoqo lesithenjwa. Ukuze siqhubeke sihlukanise ama-GluN ngezigaba, sisebenzise ukuhamba komsebenzi kwe-Seurat's TransferData kudatha yesethi engaphansi ye-GluN ukuze sinike amalebula edathasethi ayizethenjwa kumaseli ethu e-GluN.
Ukuhlola isimo sokuvuthwa se-transcriptome yomhlaba wonke yamasampuli e-t-hCO ne-hCO, siqhathanise amasampuli ethu enqwaba ye-BrainSpan/psychENCODE23, equkethe ukulandelana okukhulu kwe-RNA okuhlanganisa ukuthuthukiswa kobuchopho bomuntu. Senze i-PCA ku-matrix ye-matrix yokukhuluma yofuzo ehlanganisiwe yephethini ejwayeleke kusukela kumasampula ekhorali amaviki angu-10 ngemva kokukhulelwa futhi kamuva, kuzakhi zofuzo ezingu-5567 (kanye nedatha yethu) ezihlonzwe ngaphambilini njengezisebenzayo kumasampula e-BrainSpan cortical (okuchazwe njengokukhulu kunokungu-50% ekuhlukeni kokuthuthuka okuchazwe ngobudala kusetshenziswa imodeli ye-cubic)38. Ngaphezu kwalokho, sithole izakhi zofuzo ezihlotshaniswa namasiginesha amakhulu we-transcriptome we-neurodevelopment sisebenzisa i-non-negative matrix factorization njengoba kuchazwe ngaphambilini. Izisindo zesampula ezibalwe kusetshenziswa inqubo ye-factorization ye-matrix engeyona inegethivu zihlelwe ku-Fig. 5b enedatha enwetshiwe yesiginesha ngayinye kwamahlanu achazwe u-Zhu et al.38. Futhi, izimpawu zokuloba ezincike emsebenzini zithathwe ezifundweni ezishicilelwe ngaphambilini. Ikakhulukazi, i-ERG kanye ne-LRG zilawulwa kakhulu kuma-neurons e-glutamatergic akhonjwe iqoqo le-mouse visual cortex snRNA-seq ngemva kokukhuthazwa okubukwayo kusuka ku-Supplementary Table 3 Hrvatin et al.16. Ama-LRG anothiswe ngabantu atholwe emasikweni obuchopho bomntwana owenziwe wasebenza we-KCl futhi avunwa amahora angu-6 ngemva kokuvuselela, futhi izakhi zofuzo ezihlungiwe zalawulwa kakhulu kubantu kodwa hhayi kumagundane (Ithebula Lokwengeza 4). Ukuhlaziywa kokunothisa isethi yofuzo kusetshenziswa la maqoqo ofuzo kwenziwa kusetshenziswa indlela eyodwa yokuhlola enembile kaFisher.
Abulala amagundane ane-isoflurane, susa ubuchopho futhi ubeke endaweni ebandayo (cishe u-4°C) ene-oxygenated (95% O2 kanye no-5% CO2) isisombululo se-sucrose sezigaba eziqukethe: 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 15 mM. I-NaH2PO4, 10 mM MgSO4 kanye ne-0.5 mM CaCl2 (cishe 310 mOsm). Izingxenye zobuchopho begundane (300–400 µm) eziqukethe i-t-hCO zenziwe kusetshenziswa i-Leica VT1200 vibratome njengoba kuchazwe ngaphambilini39. Izigaba zibe sezidluliselwa egumbini elihlukanisayo elinezinga lokushisa eliqhubekayo le-oxygenation equkethe i-aCSF elungiselelwe kusukela: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 kanye 126 mM MOsm8 (29). okungenani imizuzu engama-45 ngaphambi kokurekhoda. Izigaba zarekhodwa ekamelweni elicwilisiwe lapho zazihlale zenziwe amakha nge-aCSF (95% O2 kanye ne-5% CO2 vial). Yonke idatha irekhodwe ngezinga lokushisa legumbi. I-t-hCO neurons yaqedwa nge-borosilicate glass pipette egcwele isixazululo esiqukethe 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, kanye 0.6 mM EGTA, pH 7.2, isixazululo sangaphakathi esilungiswe nge-KOOs290 mH. Ukuze ululame, i-biocytin (0.2%) yengezwe kwisixazululo sokurekhoda.
Idatha itholwe kusetshenziswa i-Amplifier ye-MultiClamp 700B (Amadivayisi Emangqamuzana) kanye ne-Digidata 1550B digitizer (Amadivayisi Emangqamuzana), i-low-pass yahlungwa ngo-2 kHz, yafakwa kudijithali ku-20 kHz, futhi yahlaziywa kusetshenziswa i-Clampfit (Amadivayisi Emangqamuzana), Umsuka (OriginPro). 2021b, i-OriginLab). kanye nemisebenzi ye-MATLAB yangokwezifiso (Izibalo). Amandla okuhlangana abalwe kusetshenziswa i-JPCalc futhi okufakiwe kwalungiselelwa inani elibaliwe elingu -14 mV. I-Operation IV iqukethe uchungechunge lwezinyathelo zamanje ezinyathelweni ezingu-10-25 pA, ukusuka -250 kuya ku-750 pA.
I-thalamus, i-white matter, nama-afferents e-S1 avuselelwe ngogesi ezincekwini ze-thalamocortical ngesikhathi sokuqoshwa kwe-patch-clamp yama-hCO neurons, njengoba kuchazwe ngaphambilini. Kafushane, ubuchopho bubekwe etafuleni lokuphrinta le-3D elitshekiswe nge-engeli engu-10°, futhi ingaphambili lobuchopho lasikwa nge-engeli engu-35°. Ubuchopho bese bunamathiselwe endaweni esikiwe futhi bahlukaniswe, kugcinwe ama-axon aphumayo e-thalamocortical. Ama-electrode e-bipolar tungsten (0.5 MΩ) afakwe kumshini omncane wesibili futhi abekwe kahle ukuze akhuthaze izifunda ezine ngeseli ngalinye (ikhephusuli yangaphakathi, i-white matter, i-S1 ne-hCO). Rekhoda izimpendulo ze-synaptic ngemva kokukhuthazwa kwe-phasic okungu-300 µA ku-0.03–0.1 Hz.
I-hChR2-expressing hCO neurons yenziwe yasebenza ku-480 nm futhi ama-pulses alula akhiqizwe i-LED (Prizmatix) asetshenziswe ngenjongo ye-×40 (0.9 NA; Olympus) ukurekhoda inkulumo ye-hChR2 eduze kwamaseli. Ububanzi bensimu ekhanyisiwe bucishe bube ngu-0.5 mm futhi amandla esewonke angu-10-20 mW. Ububanzi bokushaya kwenhliziyo bebusethwe ukuze bube ngu-10 ms, obuhambisana neshayela elinikezwe ngesikhathi sokuhlolwa kokuziphatha. Kusetshenziswe amaza okuvuselela ahlukahlukene, ukusuka ku-1 kuye ku-20 Hz, kodwa kuphela ukushaya kokuqala kochungechunge okusetshenziselwa ukulinganisa. Izikhawu phakathi kwezitimela ngokuvamile ziba zinde kune-30 s ukunciphisa umthelela ku-synaptic inhibitory noma izindlela ezilula. Ukuhlola ukuthi ingabe impendulo ye-hChR2 yayiyi-monosynaptic, sisebenzise i-TTX (1 μM) ekugezeni kuze kube yilapho ukusabela kwe-EPSC kunyamalala, bese sifaka i-4-aminopyridine (4-AP; 100 μM). Ngokuvamile, impendulo ibuyiselwa phakathi kwamaminithi ambalwa, ngokulibaziseka okude kancane phakathi kokudubula kwe-LED nokukhiqizwa kwe-EPSC. I-NBQX (10 μM) isetshenziselwe ukuhlola ukuthi impendulo iqhutshwa ama-AMPA receptors.
Izingxenye ezibukhali ze-hCO zidalwe njengoba kuchazwe ngaphambilini. Kafushane, izingxenye ze-hCO zishumekwe ku-4% agarose futhi zadluliselwa kumaseli aqukethe i-126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 kanye no-10 mM -glucose 0 ku-0 (0) kusikwa ku-0 (0) µm kuthempelesha yegumbi kusetshenziswa isidlidlizi se-Leica VT1200 futhi agcinwe ku-ASF ekamelweni lokushisa. Bese, ukurekhodwa kwe-patch-camp kwamaseli wonke kwenziwa ezigabeni ze-hCO ngaphansi kwesibonakhulu esiqondile se-SliceScope (Scientifica). Izigaba zahlanganiswa nge-aCSF (95% O2 kanye ne-5% CO2) futhi amasignali amaseli aqoshwa ekamelweni lokushisa. Ama-hCO neurons asetshenziswa kusetshenziswa i-borosilicate glass pipette egcwele isixazululo esiqukethe 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, kanye 0.6 mM EGTA, i-pH yangaphakathi 7, 2, elungiswe nge-KOH20 mo. Ngezinjongo zokuthola kabusha, engeza u-0.2% we-Biocytin esixazululweni sangaphakathi.
Idatha itholwe yi-Clampex (Clampex 11.1, Molecular Devices) kusetshenziswa i-MultiClamp 700B amplifier (Molecular Devices) kanye ne-Digidata 1550B digitizer (Amadivaysi Emangqamuzana), i-low-pass yahlungwa ngo-2 kHz, yenziwe kudijithali ku-20 kHz, ihlaziywe inguqulo ye-Clamp6. amadivaysi) nemisebenzi ye-MATLAB yangokwezifiso (MATLAB 2019b, Mathworks). Amandla okuhlangana abalwe kusetshenziswa i-JPCalc futhi okufakiwe kwalungiswa ukuze abalwe amandla okuhlanganisa angu- -14 mV. I-Operation IV iqukethe uchungechunge lwezinyathelo zamanje ku-5-10 pA izinyathelo ukusuka -50 kuya ku-250 pA.
Ukuze kwakhiwe kabusha i-morphological of pinched neuron, i-0.2% biocytin (Sigma-Aldrich) yengezwe kwisixazululo sangaphakathi. Amaseli asetshenziswa okungenani imizuzu eyi-15 ngemuva kokugetshengwa. I-pipette idonswa kancane kancane imizuzu engu-1-2 kuze kube yilapho i-membrane ebhalisiwe ivalwe ngokuphelele. Ngokulandela inqubo ye-physiology yesigaba, izigaba zalungiswa ngobusuku obubodwa ku-4° C. ku-4% PFA, zagezwa nge-PBS X3, futhi zahlanjululwa ngu-1:1000 nge-streptavidin-conjugated DyLight 549 noma i-DyLight 405 (Vector Labs). Amaseli agcwaliswe nge-biocytin (2%; Sigma-Aldrich) alebulwe ngesikhathi sokuqoshwa kwe-patch clamp ekamelweni lokushisa amahora angu-2. Izigaba zabe sezigxunyekwa kumaslayidi e-microscope kusetshenziswa i-Aquamount (Thermo Scientific) futhi zabonwa ngosuku olulandelayo kusibonakhulu se-Leica TCS SP8 confocal kusetshenziswa umgomo wokucwiliswa kawoyela onembobo yenombolo ×40 1.3, ukukhulisa × 0.9–1.0, xy. Izinga lamasampula cishe amaphikseli angu-7 imakroni ngayinye. Izitaki ezingu-Z ngezikhathi ezingu-1 µm zitholwe ngokulandelana, futhi ama-mosaics we-z-stack kanye nokuthungwa okuzenzakalelayo okusekelwe ku-Leica kwenziwa ukumboza sonke isihlahla se-dendritic se-neuron ngayinye. Ama-Neurons abe eselandelwa kancane kusetshenziswa isixhumi esibonakalayo se-neuTube 40 futhi amafayela e-SWC akhiqizwa. Amafayela abe eselayishwa ku-plugin ye-SimpleNeuriteTracer41 Fiji (ImageJ, inguqulo 2.1.0; NIH).
Izicubu ze-cortical yomuntu zitholwe ngemvume enolwazi ngokusho kwephrothokholi egunyazwe i-Institutional Review Board yase-Stanford University. Amasampula amabili esicubu somuntu sangemva kokubeletha (iminyaka emi-3 neyi-18) atholwe ngokukhishwa kabusha kwe-frontal cortex (i-middle frontal gyrus) njengengxenye yokuhlinzwa kwesithuthwane esingalawuleki. Ngemva kokukhishwa kabusha, vuna izicubu ku-NMDG-aCSF ebandayo equkethe: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mMruvate sodium ascorbate 0 mpy3 sodium ascorbate 0 mpyl 2. 4H2O kanye no-10 mM MgSO4 7H2O. Titrate ku-pH 7.3-7.4 nge-concentrated hydrochloric acid. Izicubu zalethwa elabhorethri phakathi nemizuzu engu-30 futhi izingxenye ze-coronal zathathwa ngokuvumelana nenqubo echazwe ngenhla.
Zonke izinqubo zezilwane zenziwe ngokuhambisana neziqondiso zokunakekelwa kwezilwane ezigunyazwe yi-APLAC yaseStanford University. Amagundane (okungaphezu kwezinsuku ezingu-140 ngemuva kokufakelwa kabusha) afakwe nge-5% isoflurane anesthesia futhi afakwe izinzwa nge-1-3% isoflurane intraoperatively. Izilwane zafakwa kuhlaka lwe-stereotaxic (Kopf) futhi ukukhululwa okuqhubekayo kwe-buprenorphine (SR) kwajovwa ngaphansi kwesikhumba. Ukhakhayi luyavezwa, luhlanzwe bese kufakwa izikulufu zamathambo ezi-3-5. Ukuze siqondise i-t-hCO, senze izixhumanisi ze-stereotaxic ezivela ezithombeni ze-MRI. Imbobo ye-burr yabhojwa endaweni yentshisekelo kanye namafayibha (ububanzi obungu-400 µm, NA 0.48, Doric) yehliswa ngo-100 µm ngaphansi kwendawo ye-hCO futhi yavikelwa ogebhezini ngosimende wamazinyo owelapheka nge-UV (Relyx).
Ukuqoshwa kwe-fiber photometric kwenziwa njengoba kuchazwe ngaphambilini42. Ukuze urekhode umsebenzi ozenzakalelayo, amagundane abekwe ekhejini elihlanzekile kanye nekhebuli ye-fiber optic patch engu-400 µm ububanzi exhunywe kusistimu yokutholwa kwedatha ye-fiber optic photometric yayixhunywe kukhebuli ye-fiber optic egxiliwe. Ngesikhathi sokuqoshwa kwemizuzu eyi-10 yomsebenzi wezimoto, izilwane zazikhululekile ukuhlola ikheji. Ukuze urekhode umsebenzi ovusiwe, amagundane (izinsuku ezingaphezu kwezingu-140 ngemva kokufakelwa kabusha) abulawa izinzwa nge-5% isoflurane yokungeniswa kanye ne-1-3% isoflurane yokugcinwa. Beka isilwane ohlakeni lwe-stereotactic (i-Kopf) kanti amadevu ngakolunye uhlangothi lwe-t-hCO anqunywa abe ngu-2 cm futhi adlule ngemeshi exhunywe ku-actuator ye-piezoelectric (PI). Ikhebuli ye-fiber optic patch engu-400 µm (i-Doric) yayixhunywe ku-fiber egxiliwe futhi yaxhunywa kusistimu yokutholwa kwedatha. Amadevu asohlangothini oluphambene lwe-t-hCO abe esephambukiswa izikhathi ezingu-50 (2 mm ku-20 Hz, 2 s isethulo ngasinye) ngezikhathi ezingahleliwe ngedrayivu ye-piezoelectric esikhathini sokurekhoda esiyimizuzu engama-20. Sebenzisa iPhakheji Lokusekela le-Arduino MATLAB ukuze ulawule isikhathi sokuphambuka ngekhodi ye-MATLAB yangokwezifiso. Imicimbi ivunyelaniswa nesofthiwe yokutholwa kwedatha kusetshenziswa i-transistor-transistor logic (TTL) pulses.
Amagundane (okungaphezu kwezinsuku ezingu-140 ngemuva kokufakelwa kabusha) afakwe nge-5% isoflurane anesthesia futhi afakwe izinzwa nge-1-3% isoflurane intraoperatively. Izilwane zafakwa kuhlaka lwe-stereotaxic (Kopf) kanye ne-buprenorphine SR ne-dexamethasone zajovwa ngaphansi kwesikhumba. Ukhakhayi luyavezwa, luhlanzwe bese kufakwa izikulufu zamathambo ezi-3-5. Ukuze siqondise i-t-hCO, senze izixhumanisi ze-stereotaxic ezivela ezithombeni ze-MRI. I-craniotomy eyindilinga (ecishe ibe ngu-1 cm ububanzi) yenziwa ngokubhola ngesivinini esiphezulu ngqo phezu kwe-hCO etshaliwe. Uma ithambo selincane ngangokunokwenzeka, kodwa ngaphambi kokubhoboza lonke ithambo, sebenzisa ama-forceps ukuze ususe i-pelvic disc esele eqinile ukuze uveze i-t-hCO engaphansi. I-craniotomy yayigcwele usawoti oyinyumba, futhi isembozo kanye nephinikhodi ekhethekile kwaxhunywa ogebheni ngosimende wamazinyo we-UV-cured (Relyx).
Ukuthwebula izithombe ezinezithombe ezimbili kwenziwa kusetshenziswa isibonakhulu se-Bruker multiphoton enomgomo we-Nikon LWD (×16, 0.8 NA). Ukuthwebula kwe-GCaMP6 kwenziwa ku-920 nm nge-1.4x single z-plane magnification kanye nesilinganiso esingu-8x esingu-7.5 fps. Amagundane ayenziwe nge-5% isoflurane anesthesia futhi agcinwe nge-1-3% isoflurane. Amagundane abekwe endaweni yekhanda eyenziwe ngokwezifiso futhi abekwe ngaphansi kwelensi. Kutholwe ukurekhodwa kwangemuva kwemizuzu emi-3 komsebenzi wezimoto. Ngokuhamba kwemizuzu engu-20 yokurekhoda, ukukhukhumeza okungu-50 (isethulo ngasinye esingu-100 ms ubude) kwalethwa ngokungahleliwe ku-whisker pad ebhekene ne-t-hCO kusetshenziswa i-picospricer. Sebenzisa Iphakheji Lokusekela le-Arduino MATLAB ukuze ulawule isikhathi sokuqhuma ngekhodi ye-MATLAB yangokwezifiso. Vumelanisa imicimbi nesofthiwe yokutholwa kwedatha (PrairieView 5.5) usebenzisa ama-TTL pulses. Ukuze kuhlaziywe, izithombe zalungiselelwa i-xy motion kusetshenziswa ukulungiswa kwe-affine kuhlelo lwe-MoCo oluqaliswe e-Fiji. Ukukhipha imikhondo ye-fluorescent kumaseli ngamanye kusetshenziswa i-CNMF-E43. I-fluorescence yakhishelwa isifunda ngasinye sentshisakalo, yaguqulelwa kumajika e-dF/F, yase iguqulelwa kuzikolo ezingu-z.
Amagundane (okungaphezu kwezinsuku ezingu-140 ngemuva kokufakelwa kabusha) afakwe nge-5% isoflurane anesthesia futhi afakwe izinzwa nge-1-3% isoflurane intraoperatively. Izilwane zafakwa kuhlaka lwe-stereotaxic (Kopf) kanye ne-buprenorphine SR ne-dexamethasone zajovwa ngaphansi kwesikhumba. Amadevu ngakolunye uhlangothi lwe-t-hCO asikwe afinyelela ku-2 cm futhi axhunywe nge-mesh exhunywe ku-actuator ye-piezoelectric. Ugebhezi luyavezwa futhi luhlanzwe. Isikulufu sensimbi engenasici sinamathiselwe ogebheni. Ukuze siqondise i-t-hCO, senze izixhumanisi ze-stereotaxic ezivela ezithombeni ze-MRI. Yenza i-craniotomy eyindilinga (ecishe ibe ngu-1 cm ububanzi) ngokubhoboza ngesivinini esiphezulu ngenhla nje kwe-t-hCO. Uma ithambo selincane ngangokunokwenzeka, kodwa ngaphambi kokubhoboza lonke ithambo, sebenzisa ama-forceps ukuze ususe i-pelvic disc esele eqinile ukuze uveze i-t-hCO engaphansi. Amaseli ngamanye aqoshwa kusetshenziswa ama-silicon probes angama-32-channel noma angu-64-high-density high-density (i-Cambridge Neurotech) asekelwe kuzikulufu eziphansi futhi akhuliswe ngaphambili ngezikhulisamandla ze-RHD (Intan). Sebenzisa i-manipulator ukwehlisa ama-electrode endaweni okuqondiswe kuyo ngokusebenzisa i-craniotomy, egcwele usawoti oyinyumba. Ukuqoqwa kwedatha kwenziwa ngemvamisa engu-30 kHz kusetshenziswa isistimu yokutholwa kwedatha ye-Open Ephys. Ukurekhoda kwaqhubeka kuphela lapho sithola umsebenzi ozenzekelayo ohlobene kakhulu oyisigqi eziteshini ezingaphezu kwezingu-10, okuphakamisa ukuthi ama-electrode abekwe kugraft (ngokusekelwe kudatha yesithombe se-calcium enezithombe ezimbili). Kutholwe ukurekhodwa kwangemuva kwemizuzu eyi-10 komsebenzi wezimoto. Amadevu asohlangothini oluphambene lwe-t-hCO abe esephambukiswa izikhathi ezingu-50 (2 mm ku-20 Hz, 2 s isethulo ngasinye) ngezikhathi ezingahleliwe ngedrayivu ye-piezoelectric esikhathini sokurekhoda esiyimizuzu engama-20. Usebenzisa Iphakheji Lokusekela le-MATLAB le-Arduino (MATLAB 2019b), lawula isikhathi sokuchezuka ngekhodi ye-MATLAB yangokwezifiso. Sebenzisa ama-TTL pulses ukuvumelanisa imicimbi nesofthiwe yokutholwa kwedatha.
Ngokuhlolwa kokumaka kokubona, intambo ye-optical patch engu-200 µm (Doric) exhunywe kulaser engu-473 nm (Omicron) ixhunywe ku-200 µm optical fiber ebekwe phezu kwe-craniotomy. Ngokushesha ngaphambi kwalokhu, lungisa amandla e-jumper abe ngu-20 mW. Sebenzisa i-manipulator ukwehlisa ama-electrode endaweni okuqondiswe kuyo ngokusebenzisa i-craniotomy, egcwele usawoti oyinyumba. Ekuqaleni kokurekhoda, ama-pulses ayishumi okukhanya okungu-473 nm (imvamisa 2 Hz, ubude be-pulse 10 ms) akhishwe. Amaseli e-photosensitive achazwe njengamaseli abonise impendulo esheshayo phakathi kuka-10 ms wokukhanya kokungu-70% noma ngaphezulu kwezivivinyo.