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Ƙungiyoyin jijiyoyi masu haɗa kai suna wakiltar dandamali mai ban sha'awa a cikin vitro don yin samfurin ci gaban ɗan adam da cututtuka. Koyaya, organoids ba su da haɗin kai da ke wanzuwa a cikin vivo, wanda ke iyakance balaga kuma yana hana haɗin kai tare da sauran da'irori waɗanda ke sarrafa ɗabi'a. Anan mun nuna cewa jikin mutum wanda aka samu cortical organoids wanda aka dasa a cikin somatosensory cortex na berayen tsiraicin jarirai suna haɓaka nau'ikan tantanin halitta masu girma waɗanda ke haɗuwa cikin da'irar hankali da kuzari. MRI ya bayyana ci gaban organoid bayan dasawa a cikin layukan sel da dabbobi da yawa, yayin da bincike-bincike guda ɗaya ya nuna ci gaban corticogenesis da fitowar shirin kwafi mai dogaro da aiki. Lallai, ƙwayoyin jijiyoyi da aka dasa su suna nuna ƙarin hadaddun sifofi, synaptik, da kaddarorin membrane na ciki fiye da takwarorinsu na in vitro, suna ba da damar gano lahani a cikin marasa lafiya da ciwon Timothawus. Binciken ilimin halitta da na aiki ya nuna cewa ƙwayoyin da aka dasa suna karɓar abubuwan thalamocortical da corticocortical, kuma a cikin vivo rikodin ayyukan jijiyoyi suna ba da shawarar cewa waɗannan bayanan na iya haifar da martani mai hankali a cikin ƙwayoyin ɗan adam. A ƙarshe, cortical organoids suna haɓaka axon a ko'ina cikin kwakwalwar bera, kuma kunnawar optogenetic su yana haifar da halayen neman lada. Don haka, jijiyoyi na jikin mutum da aka dasa su girma kuma suna shiga cikin da'irar mai masaukin da ke sarrafa ɗabi'a. Muna tsammanin wannan hanyar za ta sauƙaƙe gano nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan sel waɗanda ba za a iya gano su ta wasu hanyoyi ba.
Ƙwaƙwalwar ɗan adam mai tasowa wani tsari ne mai ban mamaki na tsarin kai wanda kwayoyin halitta ke yaduwa, bambanta, ƙaura, da kuma haɗawa don samar da da'irori na neuronal masu aiki waɗanda aka ƙara inganta su ta hanyar ƙwarewa. Matsala mai mahimmanci wajen fahimtar ci gaban kwakwalwar ɗan adam, musamman a cikin yanayin cututtuka, shine rashin samun damar shiga kwakwalwar kwakwalwa. Ƙungiyoyi masu tsara kansu, ciki har da cortex organoids (hCO; wanda kuma aka sani da yanayin cortex na mutum), na iya haifar da 2,3,4,5,6. Koyaya, iyakoki da yawa suna iyakance aikace-aikacen su mai fa'ida don fahimtar haɓakawa da aiki na da'irori. Musamman, ba a sani ba ko haɓakar hCO yana iyakance ta rashin wasu abubuwan microenvironmental da abubuwan da ke ji a cikin vivo. Bugu da ƙari, saboda hCOs ba a haɗa su cikin da'irori waɗanda za su iya haifar da sakamako na ɗabi'a, amfanin su a cikin ƙirar hadaddun kwayoyin halitta da cututtuka na neuropsychiatric a halin yanzu yana iyakance.
Dasawa na hCO zuwa cikin kwakwalwar da ba ta da kyau tana iya shawo kan waɗannan iyakoki. Binciken da aka yi a baya ya nuna cewa ƙwayoyin jikin ɗan adam da aka dasa a cikin ƙwayar rodent suna iya rayuwa, aiki, da sadarwa tare da ƙwayoyin rodent7,8,9,10,11,12. Koyaya, yawanci ana yin waɗannan gwaje-gwaje akan dabbobin manya, waɗanda zasu iya iyakance haɗin gwiwar synaptic da axonal. Anan, mun bayyana yanayin dasawa wanda a cikinsa muka dasa 3D hCO wanda aka samo daga ƙwayoyin hiPS zuwa farkon somatosensory cortex (S1) na berayen marasa ƙarfi a farkon matakin haɓaka filastik. HCO da aka dasa (t-hCO) neurons suna fuskantar matuƙar balaga, suna karɓar abubuwan thalamocortical da cortical-cortical abubuwan da ke haifar da amsoshi masu azanci, kuma suna faɗaɗa tsinkayar axonal cikin kwakwalwar bera don fitar da halayen neman lada. Extended maturation na t-hCO ya bayyana neuronal lahani a cikin marasa lafiya tare da Timothawus ciwo (TS), wani mummunan cuta cuta lalacewa ta hanyar maye gurbi a cikin ƙarfin lantarki-m irin L-type CaV1.2 calcium tashar (encoded by CACNA1C).
Don nazarin neurons cortical na ɗan adam a cikin da'irori a cikin vivo, mu stereotactically dasa m 3D hCO cikin S1 na farkon postnatal athymic berayen (kwanaki 3-7 postnatally) (Fig. 1a da kuma fadada bayanai na siffa 1a-c). A wannan lokaci, thalamocortical da corticocortical axonal tsinkaya ba tukuna kammala su S1 innervation (Ref. 13). Don haka, an ƙirƙiri wannan hanyar don haɓaka haɗin gwiwar t-hCO yayin da ake rage tasiri akan da'irori na endogenous. Don ganin yanayin wurin t-hCO a cikin dabbobi masu rai, mun yi T2-nauyin MRI kwakwalwa sake ginawa na berayen 2-3 watanni bayan dasawa (Fig. 1b da bayanan da aka kara, Fig. 1d). t-hCO an lura da sauri kuma ma'aunin ƙarar t-hCO sun kasance daidai da waɗanda aka ƙididdige su daga ƙayyadaddun yanka (Extended Data Figure 1d, e; P> 0.05). t-hCO an lura da sauri kuma ma'aunin ƙarar t-hCO sun kasance daidai da waɗanda aka ƙididdige su daga ƙayyadaddun yanka (Extended Data Figure 1d, e; P> 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO были аналогичны dani, 1d, e; t-hCO ana iya lura da su cikin sauƙi, kuma ma'aunin t-hCO na volumetric sun kasance daidai da waɗanda aka ƙididdigewa don ƙayyadaddun sassan (bayanan da aka fadada, 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（扩展数据图1d、e；P > 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 dani, 1d, e; t-hCO ya kasance mai sauƙin gani, kuma ma'auni na t-hCO masu girma sun kasance daidai da waɗanda aka ƙididdigewa don sassan gyarawa (bayanan da aka fadada, Fig. 1d, e; P> 0.05).Mun ƙaddara t-hCO a cikin 81% na dabbobin da aka dasa kamar watanni 2 bayan dasawa (n = 72 dabbobi; hCO daga 10 hiPS cell Lines; hiPS cell Lines a Ƙarin Table 1). Daga cikin waɗannan, 87% sun kasance a cikin kwakwalwar kwakwalwa (Fig. 1c). Ta hanyar yin gwaje-gwaje na MRI na serial a wurare masu yawa a cikin bera da aka dasa, mun sami karuwa sau tara a cikin ƙarar t-hCO a cikin watanni 3 (Fig. 1d da fadada bayanai, Fig. 1f). Dabbobin da aka dasa suna da ƙimar rayuwa mai girma (74%) a cikin watanni 12 bayan dasawa (faɗaɗɗen bayanai, Fig. 1g da Ƙarin Teburin 2), kuma ba a sami ɓarna a cikin mota ko nakasar ƙwaƙwalwar ajiya, gliosis, ko electroencephalogram (EEG). Bayanin siffa 1g da ƙarin tebur 2). 1h-m da 3e).
a, Tsarin ƙirar gwaji. An dasa hCO da aka samu daga ƙwayoyin hiPS zuwa cikin S1 na berayen tsirara waɗanda aka haifa a ranakun 30-60 na bambanta. b, T2-nauyin coronal da Hotunan MRI na kwance suna nuna t-hCO a cikin S1 2 watanni bayan dasawa. Tsawon sikelin, 2 mm. c, Ƙididdigar ƙimar nasarar aikin haɓakawa da aka nuna ga kowane layin salula na hiPS (n = 108, lambobi a cikin sanduna suna nuna adadin t-hCO kowane layin salula na hIPS) da wurin cortical ko subcortical wuri (n = 88). d, Hoton MRI na jijiya na jijiyoyin jini (hagu; ma'auni ma'auni, 3 mm) da kuma daidaitaccen gyare-gyare na 3D (ma'auni, 3 mm) yana nuna karuwa a t-hCO a kan watanni 3. e, Bitar tsarin t-hCO a cikin ɓangarorin bera. Tsawon sikelin, 1 mm. f, Wakilan Hotunan immunocytochemical na t-hCO da aka nuna daga sama zuwa hagu zuwa dama (a lokacin bambance-bambance): PPP1R17 (watanni 4), NeuN (watanni 8), SOX9 da GFAP (watanni 8), PDGFRα; (watanni 8), MAP2 (watanni 8) da IBA1 (watanni 8). Ma'auni, 20µm. Haɗin kai na HNA yana nuna ƙwayoyin asalin ɗan adam. g, snRNA-seq: Haɗe-haɗe da yawa da tsinkaya (UMAP) girman girman hoto na duk ingantattun t-hCO nuclei bayan haɗin Seurat (n = 3 t-hCO samfurori, n = 2 layin salula na hiPS). Astrocytes, sel na layin astrocyte; cyc prog, masu zagaya zuriya; GluN DL, zurfin glutamatergic neurons; GluN DL/SP, zurfin da sublamellar glutamatergic neurons; GluN UL, babban Layer glutamatergic neurons; oligodendrocytes, oligodendrocytes; OPC, oligodendrocyte progenitor sel; RELN, reelin neurons. h, Gene Ontology (GO) nazarin haɓakar haɓakar kwayoyin halitta yana haɓakawa sosai (daidaita P <0.05, canjin ninka> 2, wanda aka bayyana a cikin aƙalla 10% na nuclei) a cikin t-hCO glutamatergic neurons idan aka kwatanta da hCO glutamatergic neurons. h, Gene Ontology (GO) nazarin haɓakar haɓakar kwayoyin halitta yana haɓakawa sosai (daidaita P <0.05, canjin ninka> 2, wanda aka bayyana a cikin aƙalla 10% na nuclei) a cikin t-hCO glutamatergic neurons idan aka kwatanta da hCO glutamatergic neurons. h, Анализ обогащения терминов Gene Ontology (GO) для генов со значительной 2. hCO. h, Gene Ontology (GO) nazarin haɓakaccen lokaci don kwayoyin halitta tare da gagarumin kunnawa (daidaitacce P <0.05, canjin ninka> 2, magana a cikin akalla 10% nuclei) a cikin t-hCO glutamatergic neurons idan aka kwatanta da 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相比，t-hCO 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变化> 2术语富集分析。 h. ядер) в глутаматергических нейронах t-hCO по сравнению обогащения. h, kwayoyin halitta sun inganta sosai (daidaita P <0.05, canjin ninka> 2, wanda aka bayyana a cikin akalla 10% na nuclei) a cikin t-hCO glutamatergic neurons idan aka kwatanta da hCO glutamatergic neurons Ontological (GO) bincike na lokacin wadata.Layin dige-dige yana nuna ƙimar aq na 0.05. i, Hoto na UMAP na nau'ikan cellul GluN a cikin t-hCO ta amfani da canja wurin lakabi daga ma'anar 22 snRNA-seq manya manyan bayanan cortex. CT - Kwayoyin corticothalamic, ET - Kwayoyin extracerebral, IT - Kwayoyin telencephalic na ciki, NP - kusa da tsinkaya.
Sa'an nan kuma muka tantance cytoarchitecture da kuma gabaɗayan tsarin salula na t-hCO. Ƙwararren ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta ta IBA1 ta bayyana kasancewar microglia na bera a ko'ina cikin sassan (Fig. 1f da fadada bayanai, Fig. 3c, d). Immunostaining ya bayyana antigen na nukiliya na ɗan adam (HNA) tabbataccen sel waɗanda ke bayyana PPP1R17 (progenitors cortical), NeuN (neurons), SOX9 da GFAP (kwayoyin glial da aka samu) ko PDGFRα (progenitors oligodendrocyte) (Hoto 1f). Don nazarin tsarin salula na t-hCO a ƙudurin tantanin halitta, mun yi jerin abubuwan RNA guda ɗaya (snRNA-seq) bayan kusan watanni 8 na bambancewa. Yawan tacewa da kawar da ƙwayoyin berayen sun haifar da taswirar taswirar mononuclear masu inganci guda 21,500 (Fig. 1g da faɗaɗa bayanai, siffa 4a,b). Hanyoyin bayyanar da alamun nau'in nau'in kwayar halitta sun gano gungu na manyan nau'o'in kwayar halitta, ciki har da ƙananan ƙwayoyin glutamatergic neurons masu zurfi da na sama, masu rarraba progenitors, oligodendrocytes, da layin astrocyte (Fig. 1g, bayanan da aka fadada, Fig. 4c, da Ƙarin Table 3). Immunostaining don SATB2 da CTIP2 sun nuna cewa duk da kasancewar ƙananan ƙananan ƙwayoyin cuta, t-hCO ba ta nuna madaidaicin tsarin jiki ba (bayanan da aka fadada, Fig. 3a). snRNA-seq hCO mataki-matched ya samar da nau'ikan nau'ikan tantanin halitta iri ɗaya, tare da ƴan kaɗan, gami da rashin oligodendrocytes da kasancewar GABAergic neurons, wanda zai iya yin la'akari da yanayin da aka ruwaito a baya wanda ya dace a cikin vitro don ƙwayoyin zuriya na gefe15 (bayanai, Fig. 4f - i da Ƙarin Teburin 4). Bambance-bambancen maganganun maganganu ya nuna bambance-bambance masu mahimmanci a cikin glutamatergic neurons tsakanin t-hCO da hCO (Ƙarin Teburin 5), gami da kunna saitin kwayoyin halittar da ke da alaƙa da maturation na neuronal kamar siginar synaptic, yanki na dendritic, da ayyukan tashar wutar lantarki-gated (Hoto 1h da Ƙarin Teburin 5). tebur 6). Dangane da haka, ƙwayoyin cuta na cortical glutamatergic t-hCO neurons sun nuna saurin balaga.
Don bayyana ko waɗannan canje-canjen rubutun a cikin t-hCO suna da alaƙa da bambance-bambancen ilimin halittar jiki tsakanin hCO a cikin vitro da t-hCO a cikin vivo, mun sake gina matakan da suka dace da biocytin-cike hCO da hCO a cikin manyan sassan bayan 7-8 watanni na bambanci. hCO neurons (Fig. 2a). t-hCO neurons sun fi girma girma, suna da 1.5 sau diamita na soma, sau biyu na dendrites, da kuma yawan karuwar ninki shida a cikin jimlar dendritic tsawon idan aka kwatanta da in vitro hCO (Fig. 2b). Bugu da ƙari, mun lura da ƙananan ƙananan dendritic spines a cikin t-hCO neurons fiye da hCO neurons (Fig. 2c). Wannan yana nuna cewa t-hCO neurons suna yin tsayin daka na dendritic da kuma reshe, wanda, a hade tare da ci gaba da yaduwar kwayar halitta, na iya taimakawa wajen bunkasa girma na t-hCO bayan dasawa (Fig. 1d da Extended Data Fig. 1f). Wannan ya sa mu bincika abubuwan electrophysiological. Matsakaicin ƙarfin membrane ya kasance sau takwas mafi girma (faɗaɗɗen bayanai, siffa 8d), yuwuwar yuwuwar huta-jihar ta kasance mafi girma (kimanin 20 mV), kuma allurar ta yanzu ta haifar da matsakaicin matsakaicin tashin hankali a cikin t-hCO neurons fiye da na hCO neurons. in vitro (Fig. 2d), e), wanda ya dace da mafi girma da kuma hadaddun sifofi na t-hCO. Bugu da ƙari, yawancin abubuwan da suka faru na postsynaptic na yau da kullum (EPSC) sun kasance mafi girma a cikin t-hCO neurons (Fig. 2f), yana nuna cewa ƙara yawan ƙwayar dendritic spines da aka gani a cikin t-hCO neurons yana da alaƙa da haɓaka aiki. jima'i synapse. Mun tabbatar da halin rashin girma na hCO neurons a cikin vitro ta hanyar yin rikodi mai lakabin glutamatergic neurons (bayanan da aka fadada, Fig. 6a-c).
a, 3D sake ginawa na biocytin cike da hCO da t-hCO neurons bayan watanni 8 na bambancin. b, Ƙididdigar siffofi na ƙwayar cuta (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 da *** P <0.0001). b, Ƙididdigar siffofi na ƙwayar cuta (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 da *** P <0.0001). б. b, ƙayyadaddun siffofi na ƙwayoyin cuta (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, da *** P <0.0001). b. 形态学特征的量化（n = 8 b. 形态学特征的量化（n = 8 б. b, ƙayyadaddun siffofi na ƙwayoyin cuta (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, da *** P <0.0001).c, 3D sake ginawa na hCO da t-hCO dendritic rassan bayan watanni 8 na bambancin. Jajayen asterisks suna nuna kashin baya na dendritic. Ƙididdigar ƙididdige yawan kashin baya na dendritic (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0092). d, Ƙididdiga na yuwuwar membrane na hutawa (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). d, Ƙididdiga na yuwuwar membrane na hutawa (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). d. d, ƙwanƙwasawa mai yuwuwar membrane (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. d, ƙwanƙwasawa mai yuwuwar membrane (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, Maimaituwar yiwuwar harbe-harbe a cikin hCO da t-hCO da aka haifar ta hanyar haɓaka alluran yau da kullun, da ƙididdige ƙimar harbi mafi girma (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, Maimaituwar yiwuwar harbe-harbe a cikin hCO da t-hCO da aka haifar ta hanyar haɓaka alluran yau da kullun, da ƙididdige ƙimar harbi mafi girma (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, повторное возбуждение потенциала действия в hCO и t-hCO. скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, yiwuwar sake kunna wuta a cikin hCO da t-hCO da aka haifar da karuwa a halin yanzu da ƙididdige yawan adadin harbe-harbe (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e，通过加电流注入诱导的hCO 和t-hCO 重复动作电位放电，以及最大放电率的量媖神经元，n = 16 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, n = 16 个 t-hco 神经 ； *** p <0.0001） . e, повторяюющеся максимальной скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, maimaita harbe-harbe na hCO da t-hCO matakan aikin da aka haifar ta hanyar haɓakar haɓakawa na yanzu da ƙididdige ƙimar ƙima (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, EPSCs na gaggawa (sEPSCs) a cikin hCO da t-hCO neurons a cikin watanni 8 na bambance-bambance, da ƙididdige yawan abubuwan da suka faru na synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, EPSCs na gaggawa (sEPSCs) a cikin hCO da t-hCO neurons a cikin watanni 8 na bambance-bambance, da ƙididdige yawan abubuwan da suka faru na synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001) . f, спонтанные EPSC (sEPSC) в нейронах hCO и t-hCO через 8 месяцев дифференцировки событий (n = 25 нейронов hCO, n = 17 нейронов t-hCO; *** P <0,0001) . f, EPSCs na gaggawa (sEPSCs) a cikin hCO da t-hCO neurons a cikin watanni 8 na bambance-bambance da ƙididdige ƙimar abubuwan 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 17 t-hCO 神经元；***P <0.0001） . f，分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs神率的量匼（n = 25 hCO 神率的量匼（n = 25hCO P <0.0001） . f, спонтанные EPSC (sEPSC) в нейронах hCO и t-hCO через 8 месяцев дифференцировки событий (n = 25 нейронов hCO, n = 17 нейронов t-hCO; *** P <0,0001). f, EPSCs na gaggawa (sEPSCs) a cikin hCO da t-hCO neurons a cikin watanni 8 na bambance-bambance da ƙididdige ƙimar abubuwan synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001).Don bf, hCO da t-hCO a cikin layi na 1208-2 an ɗauke su daga nau'in bambance-bambancen da aka kiyaye a layi daya. g, Gene saitin haɓaka haɓakar haɓakawa (daidaitaccen gwajin Fisher na gefe ɗaya) na kwayoyin halitta sun haɓaka sosai (daidaita P <0.05, canjin ninka> 2, wanda aka bayyana a cikin aƙalla 10% na nuclei) a cikin t-hCO glutamatergic neurons idan aka kwatanta da hCO glutamatergic neurons tare da tsarin jigilar kwayoyin halitta na biyu-da-resa (RG) An gano shi daga binciken in vivo linzamin kwamfuta16 da takamaiman LRGs daga in vitro neurons17. g, Gene saitin haɓaka haɓakar haɓakawa (daidaitaccen gwajin Fisher na gefe ɗaya) na kwayoyin halitta sun haɓaka sosai (daidaita P <0.05, canjin ninka> 2, wanda aka bayyana a cikin aƙalla 10% na nuclei) a cikin t-hCO glutamatergic neurons idan aka kwatanta da hCO glutamatergic neurons tare da tsarin jigilar kwayoyin halitta na biyu-da-resa (RG) An gano shi daga binciken in vivo linzamin kwamfuta16 da takamaiman LRGs daga in vitro neurons17. g, анализ обогащения набора генов. P <0,05, kratnosti изменя> 2. глутаматергическими нейронами hCO наборы генов как раннего (ERG), так и позднего (LRG) генов, зависящихов, зависящих. идентифицированных в исследовании на мышах in vivo16, и специфических для человека LRG из нейронов in vitro17. g, nazarin haɓakar haɓakar haɓakar ƙwayoyin halitta (madaidaicin gwajin Fisher guda ɗaya) na kwayoyin halitta tare da kunnawa mai mahimmanci (daidaita P<0.05, canjin ninka> 2, magana a cikin aƙalla 10% na nuclei) a cikin t-hCO glutamatergic neurons idan aka kwatanta da hCO glutamatergic neurons sets na farkon (ERG) da aka gano a cikin aikin na farko (ERG). takamaiman LRGs daga neurons a cikin 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 上典倍数> 2 ， 至少 至少 10%的 细胞研究 中 的 早期 反应 反应 反应 和 晚期 反应 反应 (lrg) 活性 基因 1神经元 17 中 中 中 17 中 17的人类特异性LRG。 g, глутаматергические нейроны t-hCO были значительно ( скорректированный P<0,05, кратность изменения> 2, не менее 10% Анализ обогащения набора генов. Фишера) раннего ответа (ERG) и позднего гены, зависящие от активности ответа (LRG), m in vivo16 da нейронах in vitro17. g, t-hCO glutamatergic neurons an haɓaka su sosai idan aka kwatanta da hCO glutamatergic neurons (daidaita P <0.05, canjin ninka> 2, aƙalla 10% Amsar Farko (ERG) da kuma ƙididdigar haɓakar haɓakar amsawa ta ƙarshe (madaidaicin gwajin Fisher guda ɗaya) amsa ayyukan dogaro da kwayoyin halitta (LRGs) da aka gano a cikin 6. LRGs.Layin dige-dige yana nuna ƙimar P-Bonferroni da aka gyara na 0.05. h, GluN gene expression (pseudo-package and scaling of each gene) an inganta shi sosai a cikin snRNA-seq kwafin kwayoyin LRG a cikin t-hCO glutamatergic neurons. i, immunostaining nuna SCG2 magana a cikin t-hCO (babba) da hCO (ƙananan) neurons. Fararen kibau suna nuni ga sel SCG2+. Ma'aunin Sikeli, 25µm. Ana bayyana bayanai azaman ma'anar ± daidaitaccen karkacewa.
Dangane da karuwar ayyukan t-hCO da aka lura a cikin ex vivo yanka, snRNA-seq ya bayyana haɓakar dogaro da aiki na kwafin kwayoyin halitta a cikin t-hCO idan aka kwatanta da hCO a cikin vitro. Glutamatergic t-hCO neurons sun bayyana matakan mafi girma na kwayoyin halitta da ke daidaita ayyukan amsawa na ƙarshe (Fig. 2g, h), waɗanda aka samo a cikin binciken da suka gabata a cikin linzamin kwamfuta da ƙananan ƙwayoyin cuta na mutum16,17. Misali, BDNF18, SCG2, da OSTN, ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun tsarin aiki, sun nuna ƙarar magana a cikin ƙwayoyin t-hCO idan aka kwatanta da hCO neurons (Fig. 2g-i). Don haka, t-hCO neurons sun nuna ingantattun halaye na balaga idan aka kwatanta da hCO neurons ta hanyar kwafi, morphological, da nazarin aiki.
Don ƙarin kimanta ƙungiyar t-hCO maturation tare da ci gaban kwakwalwar ɗan adam, mun yi kwatancen kwatancen kwatancen fetal da manya nau'ikan sel cortical 19,20 da manya21,22 da kuma cikakkun bayanai game da maganganun kwayar halitta na cortical23 yayin haɓakawa (faɗaɗɗen bayanai, Fig. 5). ). tare da aikin da ya gabata 24 , yanayin hCO na duniya da t-hCO na bayanan balagagge a cikin watanni 7-8 na bambance-bambancen ya dace daidai da lokacin ci gaba na vivo kuma ya fi dacewa da marigayi tayin (Extended Data Fig. 5a). Musamman ma, mun lura da karuwar balagagge a cikin t-hCO idan aka kwatanta da shekarun da suka dace da hCO, da kuma kunnawa da aka haɗa da synaptogenesis, astrogenesis, da myelination (bayanan da aka fadada, Fig. 5b-d). A matakin salon salula, mun sami shaida na wani nau'in cortex na bakin ciki a cikin t-hCO, tare da gungu na glutamatergic neurons da ke hade da manya L2/3, L5, da L6 neuron subtypes (Hoto 1i). Sabanin haka, tari tsakanin glutamatergic t-hCO neurons da ƙoƙon jijiyoyi na tayi ya fi iyakancewa a tsakiyar ciki (bayanan da aka fadada, Hoto 5e-j). Don sanin ko ƙananan ƙwayoyin t-hCO suna aiki kama da ƙananan ƙwayoyin neocortical neocortical na mutum, mun yi rikodin electrophysiological da kuma sake gina jiki na L2 / 3 pyramidal neurons a cikin sassan sassan jikin mutum na postnatal cortex (bayanai, Fig. 7a). Abubuwan electrophysiological na L2 / 3 pyramidal neurons sun kasance daidai da na t-hCO pyramidal neurons (bayanan da aka fadada, Fig. 7e). Halin dabi'a, L2 / 3 neurons daga samfuran ɗan adam bayan haihuwa sun fi kama da t-hCO fiye da hCO, kodayake ƙwayoyin L2 / 3 sun fi tsayi, sun ƙunshi ƙarin rassa gabaɗaya, kuma suna da ƙarancin kashin baya (Fig. 3g da fadada bayanai, Fig. 7b-). G).
a, dasawa na hCO da aka samar ta hanyar sarrafawa da kuma layin salula na TS hiPS cikin berayen jariri. b, 3D sake ginawa na biocytin-cikakken t-hCO neurons bayan watanni 8 na bambancin. c, ƙididdige ma'anar tsawon dendritic (n = 19 neurons sarrafawa, n = 21 TS neurons; ** P = 0.0041). d, 3D-sake gina rassan dendritic daga sarrafawa da TS t-hCO a watanni 8 na bambance-bambance, da kuma ƙididdige yawan dendritic spine density (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001). d, 3D-sake gina rassan dendritic daga sarrafawa da TS t-hCO a watanni 8 na bambance-bambance, da kuma ƙididdige yawan 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 плотности дендритных шипов (n = 16 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, 3D sake gina rassan dendritic daga sarrafawa da t-hCO TS a watanni 8 na bambance-bambancen da 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 t-hCO 21 个TS 神经元，***P <0.0001）。 d 分化 8 个 时 对照 和 ts t-hco 的 3d 重建 分支 分支 以及元 , n = 21 个 ts 神经 ， *** p <0.0001 ）。 d, 3D-реконструкция дендритных ветвей контроля и TS t-hCO плотности дендритных шипов (n = 16 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, 3D sake gina rassan dendritic na sarrafawa da TS t-hCO a watanni 8 na bambance-bambancen da dendritic spine density quantification (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001).Jajayen asterisks suna nuna kashin baya na dendritic. e, EPSCs na kwatsam a cikin sarrafawa da TS t-hCO neurons bayan watanni 8 na bambance-bambance. f, ƙididdigar mitar mitar da ƙididdige mitar da girman abubuwan da suka faru na synaptic (n = 32 sarrafa neurons, n = 26 TS neurons; ** P = 0.0076 da P = 0.8102). g, Binciken Scholl na TS da sarrafa ƙwayoyin cuta a cikin hCO da t-hCO. Layukan da aka lalata suna nuna ɗan adam L2 / 3 ƙwayoyin pyramidal na bayan haihuwa don kwatanta (n = 24 sarrafa t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 sarrafa hCO neurons, da n = 7 TS hCO neurons). Ana bayyana bayanai azaman ma'anar ± daidaitaccen karkata
Ƙarfin t-hCO don yin kwafi da sifofi da ayyuka na ƙwayoyin cuta na ƙwayoyin cuta na ɗan adam a babban matakin ya sa mu bincika ko za a iya amfani da t-hCO don gano cututtukan phenotypes. Mun mayar da hankali kan TS, rashin lafiyar neurodevelopment mai tsanani wanda ya haifar da maye gurbin-aiki a cikin kwayar halittar CaV1.2, wanda ke fara rubutawa na dogara da aiki a cikin neurons. Mun sami hCO daga marasa lafiya na TS guda uku dauke da maye gurbin da aka fi sani da (p.G406R) da kuma sarrafawa guda uku (Fig. 3a). Bayan dasawa, mun gano cewa an canza yanayin halittar dendritic a cikin TS neurons idan aka kwatanta da sarrafawa (Fig. 3b da kuma fadada bayanai, Fig. 8a,b), tare da karuwa sau biyu a yawan adadin dendrites na farko da kuma yawan karuwa a cikin ma'ana da kuma raguwa a tsayin dendritic (Fig. 3c da tsawo bayanai, Fig. 8c). Wannan yana da alaƙa da haɓakar ƙayyadaddun kashin baya da kuma ƙara yawan adadin EPSCs ba tare da bata lokaci ba a cikin TS idan aka kwatanta da sarrafa ƙwayoyin cuta (Fig. 3d-f da bayanan fadada, Fig. 8g). Ƙarin bincike ya nuna alamu na reshe na dendritic mara kyau a cikin t-hCO TS idan aka kwatanta da sarrafawa, amma ba a cikin in vitro TS hCO a irin wannan mataki na bambanci (Fig. 3g). Wannan ya yi daidai da rahotanninmu na baya na raguwar dendritic da ke dogara da ayyuka a cikin TS kuma yana nuna ikon wannan dandalin dasawa don gano cututtukan phenotypes a cikin vivo.
Sai muka tambaya zuwa wane irin nau'in ƙwayoyin t-hCO ke haɗa su cikin aikin bera S1. S1 a cikin rodents yana karɓar shigarwar synaptic mai ƙarfi daga basal na ventral na ipsilateral da na baya na thalamic nuclei, da kuma motar ipsilateral da kuma na biyu na somatosensory cortices, da S1 contralateral (Fig. 4a). Don dawo da tsarin innervation, mun kamu da hCO tare da cutar rabies-dG-GFP/AAV-G kuma mun dasa hCO cikin bera S1 bayan kwanaki 3. Mun lura da ma'anar GFP mai yawa a cikin neurons na ipsilateral S1 da ventral basal ganglia 7-14 kwanaki bayan dasawa (Fig. 4b, c). Bugu da kari, maganin antibody tabo na thalamic alamar netrin G1 ya bayyana kasancewar ƙarshen thalamic a cikin t-hCO (Fig. 4d, e). Don kimanta ko waɗannan tsinkaya na iya haifar da martanin synaptic a cikin ƙwayoyin t-hCO, mun yi rikodin tantanin halitta gabaɗaya daga sel ɗan adam a cikin sassan kaifi na thalamocortical Layer. Ƙarfafa wutar lantarki na bera S1, capsule na ciki, fararen fata, filaye kusa da t-hCO ko kunnawa optogenetic na opsin-bayyana ƙarshen thalamic a cikin t-hCO haifar da gajeriyar EPSCs a cikin t-hCO neurons wanda aka fallasa ga mai karɓar mai karɓar AMPA NBQX. (Fig. 4f, g da bayanan da aka fadada, Fig. 9a-g). Waɗannan bayanan sun nuna cewa t-hCO an haɗa shi ta jiki cikin kwakwalwar bera kuma ana iya kunna shi ta hanyar nama mai masaukin bera.
a, Tsarin tsari na gwaji na bin diddigin rabies. b, GFP da STEM121 na musamman na mutum tsakanin t-hCO da rat cerebral cortex (babban panel). Hakanan ana nuna GFP magana a cikin ɓangarorin ɓangarorin ɓacin rai na bera (VB) (ƙananan hagu) da ipsilateral S1 (ƙananan dama). Ma'auni, 50 µm. Jajayen murabba'i suna wakiltar wuraren kwakwalwa inda aka ɗauki hotunan. c, ƙididdige sel masu bayyana GFP (n = 4 berayen). d, e - Netrin G1+ thalamic tashoshi a cikin t-hCO. d yana nuna ɓangaren coronal mai ɗauke da t-hCO da VB nuclei. Tsawon sikelin, 2 mm. e yana nuna Netrin G1 da STEM121 magana a cikin t-hCO (hagu) da VB (dama) neurons. Ma'auni, 50 µm. Layin mai digon orange yana nuna iyakar t-hCO. f, g, Halin halin yanzu na t-hCO neurons bayan kuzarin lantarki a cikin S1 bera (f) ko capsule na ciki (g), tare da (purple) ko ba tare da (baki) NBQX (hagu). EPSC amplitudes tare da kuma ba tare da NBQX (n = 6 S1 neurons, * P = 0.0119; da n = 6 na ciki capsule neurons, ** P = 0.0022) (tsakiya). Kashi na t-hCO neurons da ke nuna EPSC don mayar da martani ga kuzarin lantarki na bera S1 (f) ko capsule na ciki (g) (dama). aCSF, ruwa na cerebrospinal na wucin gadi. h, zane-zane na gwajin hoto na 2P (hagu). Bayanin GCaMP6s a t-hCO (tsakiyar). Ma'auni, 100µm. GCaMP6s ya ƙare (dama). i, Z-maki na kyalli na kwatsam. j, kwatancin ƙira na ƙarfafa gashin baki. k, z-sized 2P fluorescence trajectories a cikin gwaji ɗaya, daidaitacce tare da karkatar da whisker a lokacin sifili (layin tsinke) a cikin misali sel. l, matsakaicin adadin z-maki na duk sel masu daidaitawa tare da karkatar da whisker a lokacin sifili (layin da aka tsinke) (ja) ko tamburan lokutan da aka ƙirƙira bazuwar (launin toka). m. Tsarin tsari na gwaji akan alamar gani. n, Raw ƙarfin lantarki mai lankwasa daga misali t-hCO cell yayin da blue Laser stimulating ko whisker karkata. Jajayen kibau suna nuna karukan farko da haske (saman) ya haifar ko kuma ya haifar da karkatar da whisker (kasa). Launin launin toka yana nuna lokutan jujjuyawa. o, Kololuwar hasken kalaman kalamai da martanin juyar da kai. p, spikes na ƙoƙari guda ɗaya, masu daidaitawa tare da karkatar da barasa a cikin sel na misalin. 0 yana nuna karkatawar whisker (layin da aka kaɗe). q, matsakaicin adadin yawan harbe-harbe z-maki ga duk sel masu ɗaukar hoto, mai daidaitawa tare da karkatar da wuski a lokacin sifili (layin da aka kaɗe) (ja) ko tamburan lokutan da aka ƙirƙira dazu (launin toka). r, Matsakaicin raka'o'in hotuna masu mahimmanci waɗanda aka daidaita su ta hanyar karkatar da whisker (n = 3 beraye) (hagu). Latency mafi girman z-score (n = 3 berayen; n = 5 (kore mai haske), n = 4 (kore mai duhu), da n = 4 (cyan) juzu'i na jujjuya juzu'i a kowane bera) (dama). Ana bayyana bayanai azaman ma'anar ± daidaitaccen karkacewa
Sai muka tambaya ko za a iya kunna t-hCO ta hanyar motsa jiki a cikin vivo. Mun dasa hCO yana bayyana alamomin calcium ɗin GCaMP6 da aka sanya su a cikin berayen S1. Bayan kwanaki 150, mun yi fiber photometry ko hotunan calcium mai hoto biyu (Fig. 4h da fadada bayanai, Fig. 10a). Mun gano cewa ƙwayoyin t-hCO sun nuna ayyukan rhythmic aiki tare (Hoto 4i, Faɗaɗɗen Bayanai, Hoto 10b da Ƙarin Bidiyo 1). Don kwatanta aikin t-hCO na kololuwa, mun yi rikodin electrophysiological na waje a cikin berayen dasawa da aka yi amfani da su (faɗaɗɗen bayanai, siffa 10c-f). Mun haifar da haɗin gwiwar stereotaxic daga hotunan MRI; don haka, waɗannan raka'o'in da aka yi rikodin suna wakiltar jijiya na ɗan adam, kodayake electrophysiology kaɗai ba ya ƙyale a tantance nau'in asali. Mun lura da fashe ayyukan aiki tare (faɗaɗɗen bayanai, siffa 10d). Fashewar ta dau kusan 460 ms kuma an raba su da lokutan shiru na kusan 2 s (faɗaɗɗen bayanai, siffa 10d, e). Raka'a ɗaya ɗaya sun yi harbi kusan kusan zagaye uku a kowane fashe, wanda shine kusan kashi 73% na raka'o'in da aka yiwa rajista kowane fashe. Ayyukan raka'a guda ɗaya sun kasance masu alaƙa sosai, kuma waɗannan alaƙa sun fi na raka'a da aka gano a cikin dabbobin da ba a yi musu rigakafi ba da aka rubuta a ƙarƙashin yanayi guda (faɗaɗɗen bayanai, siffa 10f). Don ƙarin fayyace martanin karu na ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙwayoyin cuta na ɗan adam, mun yi gwaje-gwajen alamar haske akan berayen da aka dasa tare da hCO suna bayyana tashar tashar cation rhodopsin 2 (hChR2), ta hanyar da t-hCO neurons na gajeriyar jinkiri (kasa da 10 ms) don amsawa ga hasken shuɗi. stimu 4. t-hCO neurons sun nuna fashewar ayyukan da ba ta dace ba a mitoci masu kama da waɗanda aka lura a cikin hotunan calcium, da kuma rikodin electrophysiological da aka yi a t-hCO a cikin rashin alamar haske (bayanan da aka fadada, Fig. 10c-g). Ba a lura da wani aiki na kwatsam ba a cikin matakan da suka dace na hCO da aka rubuta a cikin vitro. Don tantance ko za a iya kunna t-hCO ta hanyar motsa jiki, mun ɗan karkatar da raɗaɗin bera daga t-hCO (Fig. 4j, m da ƙarin bayanai, siffa 10h,k). Bisa ga binciken da aka yi a baya8,10, wani nau'i na ƙwayoyin t-hCO sun nuna ƙara yawan aiki don mayar da martani ga ɓarna na whisker, wanda ba a lura da shi ba lokacin da aka kwatanta bayanan da bazuwar lokaci (Fig. 4k-q da kuma fadada bayanai, Fig. 10h-q). Lallai, kusan kashi 54% na raka'o'i guda ɗaya da aka yi wa lakabin opto sun nuna haɓakar haɓakar haɓakar haɓakawa sosai bayan haɓakar whisker, wanda ya kai kusan 650 ms (Fig. 4r). Haɗe tare, waɗannan bayanan suna ba da shawarar cewa t-hCO yana karɓar abubuwan da suka dace na aiki kuma ana iya kunna su ta hanyar motsa jiki.
Sannan mun bincika ko t-hCO na iya kunna da'irori a cikin berayen don sarrafa ɗabi'a. Mun fara bincika ko axon na t-hCO neurons suna aiki a cikin sassan da ke kewaye da bera. Mun kamu da hCO tare da lentivirus mai ɓoye hChR2 haɗe zuwa EYFP (hChR2-EYFP). Bayan kwanaki 110, mun lura da maganganun EYFP a cikin yankuna na ipsilateral cortical, ciki har da masu sauraro, mota, da kuma somatosensory cortices, da kuma a cikin yankunan subcortical, ciki har da striatum, hippocampus, da thalamus (Fig. 5a). Don tantance ko waɗannan tsinkaya masu ƙarfi na iya haifar da martanin synaptic a cikin ƙwayoyin bera, mun kunna ƙwararrun t-hCO da ke bayyana hChR2-EYFP ta hanyar rikodin sel cortex na bera a cikin sassan kwakwalwa masu kaifi. Kunna t-hCO axon tare da shuɗi mai haske ya haifar da gajeriyar EPSCs a cikin ratsan pyramidal cortex neurons, wanda NBQX ya toshe (Figs. 5b-g). Bugu da ƙari, waɗannan martani za a iya toshe su ta hanyar tetrodotoxin (TTX) da kuma mayar da su ta hanyar 4-aminopyridine (4-AP), suna nuna cewa an haifar da su ta hanyar haɗin gwiwar monosynaptic (Fig. 5e).
a, Tsarin tsari na bin diddigin axon (hagu). t-hCO EYFP magana (dama). Ma'auni, 100µm. A1, bawo mai ji, ACC, cingulate cortex na gaba, d. striatum, dorsal striatum, HPC, hippocampus; Diaphragm, septum na gefe, mPFC, medial prefrontal cortex, piri, piriform cortex, v. striatum, ventral striatum, VPM, ventropostomedial nucleus na thalamus, VTA, ventral tegmental area. Jajayen murabba'i suna wakiltar wuraren kwakwalwa inda aka ɗauki hotunan. b, Zane-zane na gwajin ƙarfafawa. c, d, Misalai na martani na shuɗi haske-jawo photocurrent (saman) da ƙarfin lantarki (kasa) a cikin mutum (c) EYFP+ t-hCO ko bera (d) EYFP- Kwayoyin. e.f g, jinkirin martanin da hasken shuɗi ya jawo a cikin ƙwayoyin bera (n = sel 16); sandunan kwance suna nuna matsakaicin jinkiri (7.13 ms) (hagu). Girman EPSCs masu haske da aka rubuta tare da ko ba tare da NBQX (n = sel 7; *** P <0.0001) (tsakiyar). Girman EPSCs masu haske da aka rubuta tare da ko ba tare da NBQX (n = sel 7; *** P <0.0001) (tsakiyar). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; *** P <0,0001) (в центре). Girman EPSCs masu haske da aka rubuta tare da ko ba tare da NBQX (n = 7 sel; *** P <0.0001) (tsakiya).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). Girman EPSCs masu haske da aka rubuta tare da ko ba tare da NBQX (n = 7 sel; *** P <0.0001) (tsakiya).Kashi na ƙwayoyin bera suna nuna EPSCs waɗanda ke amsa haske mai shuɗi (dama). h, Tsarin tsari na aikin ɗabi'a. d0, rana 0. i. Ayyukan dabbobi masu kyau a ranar 1 (hagu) ko ranar 15 (dama) na horo. Matsakaicin adadin lasa da aka yi a ranar 1 (hagu) ko ranar 15 (cibiyar dama) (n = 150 gwajin haske shuɗi, n = 150 gwajin haske ja; *** P <0.0001). Matsakaicin adadin lasa da aka yi a ranar 1 (hagu) ko ranar 15 (cibiyar dama) (n = 150 gwajin haske shuɗi, n = 150 gwajin haske ja; *** P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 n = 150 испытаний с красным светом; *** P <0,0001). Matsakaicin adadin lasa da aka yi a rana ta 1 (hagu) ko ranar 15 (tsakiyar dama) (n = 150 gwajin haske blue, n = 150 gwajin haske ja; *** P <0.0001).第1 天（左）或第15 天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150次红光试验；***P <0.0001）。第1 天（左）或第15 天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150次红光试验；***P < 0.001 Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 n = 150 испытаний с красным светом; *** P <0,0001). Matsakaicin adadin lasa da aka yi a rana ta 1 (hagu) ko ranar 15 (tsakiyar dama) (n = 150 gwajin haske blue, n = 150 gwajin haske ja; *** P <0.0001).Tarin lasa don gwajin haske na ja da shuɗi a rana ta 1 (hagu ta tsakiya) ko rana ta 15 (dama). NS, ba mahimmanci ba. j,k, Halayen halayen duk dabbobin da aka dasa tare da t-hCO suna bayyana hChR2-EYFP (j) ko sarrafa fluorophore (k) a ranar 1 ko 15 (hChR2-EYFP: n = 9 berayen, ** P = 0.0049; sarrafawa: n = 9, P = 0.1497). l, Juyin Halin fifiko (n = 9 hChR2, n = 9 sarrafawa; ** P <0.001, *** P <0.0001). l, Juyin Halin fifiko (n = 9 hChR2, n = 9 sarrafawa; ** P <0.001, *** P <0.0001). l, Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwararren Ƙwaƙwalwa na Ƙaƙwalwa na Ƙaƙwalwa na Ƙaƙa ) (n = 9 hChR2 , n = 9 , * P <0,001 , *** P <0,0001 ). l, Juyin Halin fifiko (n = 9 hChR2, n = 9 sarrafawa; ** P <0.001, *** P <0.0001). l. l. l, Ƙwararren Ƙwararrun Ƙwararrun Ƙwararru (n = 9 hChR2, n = 9 kontroley; ** P <0,001, *** P <0,0001). l, Juyin Halitta na fifiko (n = 9 hChR2, n = 9 sarrafawa; ** P <0.001, *** P <0.0001).m, FOS magana a mayar da martani ga optogenetic kunna t-hCO a cikin S1. Hotunan furcin FOS (hagu), da ƙididdigewa (n = 3 kowace ƙungiya; * P <0.05, ** P <0.01 da *** P <0.001) (dama) ana nunawa. Hotunan furcin FOS (hagu), da ƙididdigewa (n = 3 kowace ƙungiya; * P <0.05, ** P <0.01 da *** P <0.001) (dama) ana nunawa. Показаны изображения эkspressyy FOS (sleva) da kuma kolichestvennoho opredelenyya (n = 3 на групу; * P <0,05, ** P <0,01 , p <0,01 , p <0,01 ). Hotunan furcin FOS (hagu) da ƙididdigewa (n = 3 kowace ƙungiya; * P <0.05, ** P <0.01, da *** P <0.001) ana nunawa (dama).显示了FOS 表达（左）和量化（每组n = 3；*P <0.05、**P <0.01 和***P < 0.001）显示了FOS 表达（左）和量化（每组n = 3；*P <0.05、**P <0.01 和***P < 0.001） Показаны изображения эkspressyy FOS (sleva) da kuma kolichestvennoho opredelenyya (n = 3 на групу; * P <0,05, ** P <0,01 , p <0,01 , p <0,01 ). Hotunan furcin FOS (hagu) da ƙididdigewa (n = 3 kowace ƙungiya; * P <0.05, ** P <0.01, da *** P <0.001) ana nunawa (dama).Ma'auni, 100µm. An bayyana bayanai azaman ma'anar ± daidaitaccen kuskure na BLA, tonsil basolateral, MDT, dorsomedial thalamic nucleus, PAG, periaqueductal launin toka.
A ƙarshe, mun tambayi ko t-hCO na iya daidaita halayen bera. Don gwada wannan, mun dasa hChR2-EYFP-bayyana hCO cikin S1, kuma kwanaki 90 bayan haka, mun dasa filaye masu gani a cikin t-hCO don isar da haske. Daga nan mun horar da berayen tare da ingantaccen yanayin yanayin aiki (Fig. 5h). Mun sanya dabbobin a cikin ɗakin gwajin ɗabi'a kuma mun yi amfani da bazuwar 5 shuɗi na biyu (473 nm) da ja (635 nm) abubuwan motsa jiki. Dabbobi sun sami ladan ruwa idan sun lasa a lokacin motsa hasken shuɗi amma ba su lasa ba yayin jan haske. A ranar farko ta horo, dabbobin ba su nuna bambanci a cikin lasa ba lokacin da aka motsa su da haske mai launin shuɗi ko ja. Duk da haka, a ranar 15, dabbobin da aka dasa tare da hCO suna bayyana hChR2-EYFP sun nuna yawan lasa lokacin da aka motsa su tare da haske mai launin shuɗi idan aka kwatanta da jan haske. Ba a lura da waɗannan canje-canje a cikin halayen lasa ba a cikin dabbobi masu sarrafawa da aka dasa tare da hCO suna bayyana fluorophore mai sarrafawa (yawan nasarar koyo: hChR2 89%, EYFP 0%, Hoto 5i-1 da Ƙarin Bidiyo 2). Waɗannan bayanan sun ba da shawarar cewa ƙwayoyin t-hCO na iya kunna ƙwayoyin bera don haɓaka halayen neman lada. Don gano wane nau'ikan da'irori na bera na t-hCO na iya shiga cikin waɗannan canje-canjen halayen, mun kunna t-hCO ta hanyar optogenetically a cikin horarrun dabbobi da kayan girbe mintuna 90 daga baya. Immunohistochemistry ya bayyana bayanin furotin FOS da ke dogaro da ayyuka a cikin yankuna da yawa na kwakwalwa da ke da hannu cikin ɗabi'a mai motsa rai, gami da medial prefrontal cortex, medial thalamus, da ƙwayar launin toka na periaqueductal, wanda aka bayyana ko dai a cikin dabbobi marasa ƙarfi ko cikin dabbobi. shinkafa. 5m ku). Haɗe tare, waɗannan bayanan suna ba da shawarar cewa t-hCO na iya daidaita ayyukan bera don motsa ɗabi'a.
Neural organoids suna wakiltar tsari mai ban sha'awa don nazarin ci gaban ɗan adam da cututtuka a cikin vitro, amma an iyakance su ta hanyar rashin haɗin kai tsakanin da'irori da ke cikin vivo. Mun ƙirƙira wani dandalin labari wanda a cikinsa muka dasa hCO zuwa cikin S1 na berayen da ba su da lafiya a farkon haihuwa don nazarin haɓakar ƙwayoyin ɗan adam da aiki a cikin vivo. Mun nuna cewa t-hCO yana haɓaka nau'ikan tantanin halitta balagagge ba a lura da su a cikin vitro28 kuma cewa t-hCO an haɗa shi ta jiki da aiki cikin kwakwalwar rodent. Haɗin t-hCO cikin da'irar jijiyoyi na rodent ya ba mu damar kafa hanyar haɗi tsakanin ayyukan salula na ɗan adam da kuma nazarin halayen dabba, yana nuna cewa t-hCO neurons na iya daidaita ayyukan ɓacin rai don fitar da martanin ɗabi'a.
Dandalin da muka bayyana yana da fa'idodi da yawa akan binciken da aka yi a baya kan dashen kwayoyin halittar dan adam zuwa kwakwalwar rowan. Da farko, mun dasa hCO cikin haɓakar bawo na berayen bayan haihuwa, wanda zai iya sauƙaƙe haɗin jiki da aiki. Na biyu, t-hCO MRI saka idanu ya ba mu damar yin nazarin matsayi da girma a cikin dabbobi masu rai, yana ba mu damar gudanar da nazarin dabbobi da yawa na dogon lokaci da kuma tabbatar da amincin yawancin layin salula na hiPS. A ƙarshe, mun dasa ingantattun ƙwayoyin cuta, maimakon keɓantaccen dakatarwar tantanin halitta guda ɗaya, waɗanda ba su da lahani ga ƙwayoyin ɗan adam kuma suna iya haɓaka haɓakawa da haɓaka ƙwayoyin ƙwayoyin cuta na ɗan adam a cikin kwakwalwar bera.
Mun yarda cewa duk da ci gaban da aka samu a wannan dandali, na ɗan lokaci, sararin samaniya, da kuma nau'ikan nau'ikan nau'ikan nau'ikan ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun nau'ikan sun hana samuwar da'irar jijiyoyin ɗan adam tare da aminci mai ƙarfi, koda bayan dasawa a farkon matakin haɓakawa. Alal misali, ba a bayyana ko aikin da aka yi ba a cikin t-hCO yana wakiltar wani nau'i na ci gaba mai kama da aikin rhythmic da aka lura a yayin ci gaba na cortical, ko kuma saboda rashin nau'in kwayar halitta da ke cikin t-hCO. Hakazalika, ba a bayyana ko menene rashin lamination a cikin t-hCO ke shafar haɗin sarkar30 ba. Ayyukan da ke gaba za su mayar da hankali kan haɗa wasu nau'o'in tantanin halitta irin su microglia na mutum, ƙwayoyin endothelial na mutum, da nau'i daban-daban na GABAergic interneurons kamar yadda aka nuna ta yin amfani da taro 6 a cikin vitro, da kuma fahimtar yadda haɗin kai da aiki na iya faruwa a canza t-hCO. rubuce-rubucen, synaptic da matakan hali a cikin sel da aka samu daga marasa lafiya.
Gabaɗaya, wannan dandamali a cikin vivo yana wakiltar albarkatu mai ƙarfi wanda zai iya haɗawa cikin ci gaban kwakwalwar ɗan adam da binciken cututtuka. Muna tsammanin wannan dandali zai ba mu damar gano sabon salo-matakin phenotypes a cikin in ba haka ba sel marasa lafiya da aka samu da kuma gwada sabbin dabarun warkewa.
Mun haifar da hCO2.5 daga ƙwayoyin HiPS kamar yadda aka bayyana a baya. Don fara samar da hCO daga ƙwayoyin hiPS waɗanda aka haɓaka akan yadudduka masu ciyarwa, an cire ƙaƙƙarfan mazaunan sel na hiPS daga jita-jita na al'ada ta amfani da zubar (0.35 mg/mL) kuma an tura su zuwa al'adun filastik masu ƙarancin rahusa waɗanda ke ɗauke da jita-jita tare da matsakaicin al'adun cell na hiPS. (Corning) wanda aka haɓaka tare da masu hana SMAD guda biyu dorsomorphine (5 μM; P5499, Sigma-Aldrich) da SB-431542 (10 μM; 1614, Tocris) da ROCK inhibitor Y-27632 (10 μM; S1049, Selleck). A cikin kwanakin 5 na farko, an canza matsakaicin kwayar halitta na hiPS kowace rana kuma an ƙara dorsomorphine da SB-431542. A rana ta shida a cikin dakatarwa, an canza spheroids neural zuwa matsakaici na neurobasal-A (10888, Life Technologies), B-27 kari ba tare da bitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin da streptomycin (1: 100, Life Technologies); 1 ml na haɓaka tare da EGF. R & D Systems) da kuma fibroblast girma factor 2 (FGF2; 20 ng ml-1; R & D Systems) har zuwa ranar 24. Daga ranar 25 zuwa rana ta 42, an ƙaddamar da matsakaici tare da ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwa (BDNF; 20 ng ml-1, Peprotech) da neurotrophin 3 (NT3); - 20 kowace rana tare da canje-canje na neurotrophin. A rana ta shida a cikin dakatarwa, an canza spheroids neural zuwa matsakaici na neurobasal-A (10888, Life Technologies), B-27 kari ba tare da bitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin da streptomycin (1: 100, Life Technologies); 1 ml na haɓaka tare da EGF. R & D Systems) da kuma fibroblast girma factor 2 (FGF2; 20 ng ml-1; R & D Systems) har zuwa ranar 24. Daga ranar 25 zuwa rana ta 42, an ƙaddamar da matsakaici tare da ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwa (BDNF; 20 ng ml-1, Peprotech) da neurotrophin 3 (NT3); - 20 kowace rana tare da canje-canje na neurotrophin.A rana ta shida a cikin dakatarwa, an tura spheroids na jijiyoyi zuwa matsakaicin jijiyar da ke dauke da Neurobasal-A (10888, Life Technologies), B-27 kari ba tare da bitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin.da streptomycin (1: 100, Life Technologies) da kuma dopolnenы эpydermalnыm faktorom rosta (EGF; 20 ng/ml; R&D Systems) da kuma Faktorom 2 (FGF2; 20 нг/мл; R&D Systems) zuwa 24-го дня. da streptomycin (1: 100, Life Technologies) da kuma haɓaka tare da haɓakar haɓakar epidermal (EGF; 20 ng / ml; R & D Systems) da kuma fibroblast girma factor 2 (FGF2; 20 ng / ml; R & D Systems) har zuwa ranar 24.Daga kwanakin 25 zuwa 42, ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwa (BDNF; 20 ng ml-1, Peprotech) da neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) an ƙara su zuwa matsakaici, canza matsakaici kowace rana.在悬浮的第6 天。 Fasaha) , GlutaMax (1:100, Life Technologies) Technologies）并辅以表皮生长因子（EGF；20 ng ml-1；R&D Systems Systems）直至第24 天。在 浮 的 第第 6 天 将 神经 球体 转移 含有 含有 neurobasal-a （10888 ， Life Technologies４ 不 含 約补充剂 (12587 ， Life Technologies） Glutamax (1: 100生长 因子 （； (20 ng ml-1 ； r & d Systems） На 6-й день суспензии нейросферы были переведены на добавку, содержащую нейробазазал-А (10888), 7 Life Technology витамина А (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), пеницилинный стрептомины (1:100, Life Technologies) za a iya gani фактора роста (EGF; 20 нг мл-1; R&D Systems) da фактора роста фибробластов 2 (FGF2; 20 нг мл-1) 1; A ranar 6, an canza dakatarwar neurosphere zuwa kari wanda ke dauke da neurobasal-A (10888, Life Technologies), B-27 kari ba tare da bitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin-neutralized streptomycin (1: 100, Life Technologies) ci gaban EGF Systems) da kuma fibroblast girma factor 2 (FGF2; 20 ng ml-1) 1; R&D Systems) zuwa 24-го дня. R&D Systems) har zuwa ranar 24.Daga kwanakin 25 zuwa 42, ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwar ƙwaƙwalwa (BDNF; 20 ng ml-1, Peprotech) da kuma neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) an ƙara su zuwa matsakaicin al'ada kowace rana. Matsakaicin canji sau ɗaya.An fara daga ranar 43, hCO an kiyaye shi a cikin neurobasal-A matsakaici (NM; 1088022, Thermo Fisher) tare da matsakaicin canji kowane kwanaki 4-6. Don samun hCO daga ƙwayoyin hiPS waɗanda aka haɓaka a ƙarƙashin yanayin rashin abinci, ƙwayoyin hiPS an haɗa su tare da Accutase (AT-104, Innovate Cell Technologies) a 37 ° C na mintuna 7, an raba su cikin sel guda ɗaya, kuma an sanya su a kan faranti na AggreWell 800 (34815, STEMCELL Technologies) a cikin mafi ƙarancin ƙarin sel guda 3 × 3. Mai hana ROCK Y-27632 (10 μM; S1049, Selleckchem). Bayan sa'o'i 24, kafofin watsa labaru a cikin rijiyoyin sun kasance suna bututu sama da ƙasa zuwa kafofin watsa labaru masu ɗauke da mahimmancin 6 kafofin watsa labarai (A1516401, Life Technologies) wanda aka haɓaka tare da dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) da SB-431542 (10 μM; 1614). , Tocrida). Daga kwanaki 2 zuwa 6, An maye gurbin Mahimman Mahimmancin 6 kowace rana tare da dorsomorphine da ƙarin SB-431542. Tun daga rana ta shida, an dakatar da dakatarwar neurosphere zuwa matsakaicin neurobasal kuma ana kiyaye su kamar yadda aka bayyana a sama.
An yi duk hanyoyin dabba bisa ka'idojin kula da dabbobi da Kwamitin Gudanar da Kula da Dabbobi na Jami'ar Stanford (APLAC) ya amince da su. An sayi berayen euthymic RNU (rnu/+) masu ciki (Charles River Laboratories) ko kuma a ajiye su. An ajiye dabbobi a kan zagayowar haske na sa'o'i 12 tare da abinci da ruwa ad libitum. Tsirara (FOXN1–/-) ƴan bera masu shekaru uku zuwa kwana bakwai an gano su ta hanyar haɓakar wasiƙar da ba ta girma ba kafin yankewa. Ƙwararru (namiji da mata) an yi musu wariyar launin fata tare da 2-3% isoflurane kuma an sanya su a kan firam na stereotaxic. An yi maƙalar kwanyar tare da diamita na kusan 2-3 mm sama da S1 yayin kiyaye amincin dura mater. Sannan yi amfani da allurar 30-G (kimanin 0.3 mm) kusa da craniotomy don huda dura. Sa'an nan kuma shafa HCO zuwa wani siriri 3 × 3 parafilm da kuma cire wuce haddi matsakaici. Yin amfani da sirinji na Hamilton da ke haɗe zuwa allura 23 G, 45°, a hankali zana hCO zuwa ƙarshen allurar. Sannan shigar da sirinji akan famfon sirinji da aka haɗa da na'urar stereotaxic. Sa'an nan kuma sanya titin allura a kan rami mai faɗi mai faɗin 0.3 mm a baya a cikin dura (z = 0 mm) kuma kunkuntar sirinji 1-2 mm (z = kusan -1.5 mm) har allurar ta kasance tsakanin dura mater A. an kafa hatimi mai yawa. Sa'an nan kuma ɗaga sirinji zuwa tsakiyar farfajiyar cortical a z = -0.5 mm kuma a yi wa hCO allurar a cikin adadin 1-2 µl a minti daya. Bayan kammala allurar hCO, ana cire allurar a cikin adadin 0.2-0.5 mm a minti daya, fata yana sutured, kuma an sanya kwikwiyo nan da nan a kan kushin dumi mai dumi har sai an dawo da shi sosai. An dasa wasu dabbobin bi-biyu.
Dukkan hanyoyin dabba an yi su daidai da jagororin kula da dabbobi na Jami'ar Stanford APLAC. Rats (fiye da kwanaki 60 bayan dasawa) an jawo su tare da 5% isoflurane anesthesia da anesthetized tare da 1-3% isoflurane yayin hoto. Don gani, an yi amfani da na'urar daukar hoto ta Tesla ta 7 Tesla da ta yi amfani da na'urar daukar hoto ta kwancen burtsatse Bruker (Bruker Corp.) tare da faifan Kamfanin Wutar Lantarki na Duniya (IECO), abin sa mai kariya da diamita na ciki na 120 mm (600 mT/m, 1000 T/m/s) ta amfani da AVANCE. III, tashoshi takwas mai yawa na RF da damar iyakoki da yawa, da dandamali na Paravision 6.0.1. Anyi rikodi ta amfani da na'urar RF mai ƙarfi da aka lalatar da ita tare da diamita na ciki na 86 mm da tashoshi huɗu na cryo-sanyi RF nada don karɓa kawai. Axial 2D Turbo-RARE (lokacin maimaitawa = 2500 ms, lokacin amsawa = 33 ms, matsakaicin 2) tare da ɗaukar yanki na 16, kauri yanki 0.6-0.8 mm, yana ɗauke da samfuran 256 × 256. An karɓi siginar ta amfani da na'ura mai jujjuyawar juzu'i na RF na'ura tare da diamita na ciki na 2 cm (Rapid MR International, LLC). A ƙarshe, yi amfani da ginanniyar ayyukan kimanta saman Imaris (BitPlane) don ma'anar 3D da ƙididdigar girma. An bayyana nasarar dasawa a matsayin wanda aka kafa wuraren ci gaba da siginar MRI mai nauyin T2 a cikin dashen da aka dasa. An bayyana ƙin yarda da alƙawarin a matsayin datti wanda bai samar da wuraren ci gaba da siginar MRI mai nauyin T2 a cikin dashen da aka dasa ba. Subcortical t-hCO an cire shi daga bincike na gaba.
Don bayyana GCaMP6s a tsaye a cikin hCO don hotunan calcium mai hoto biyu, ƙwayoyin hiPS sun kamu da pLV[Exp] -EF1a :: GcaMP6s-WPRE-Puro sannan zaɓin maganin rigakafi. A taƙaice, an raba sel tare da EDTA kuma an dakatar da su a cikin 1 ml na Mahimmancin 8 matsakaici a yawan kusan ƙwayoyin 300,000 a gaban polybrene (5 μg / ml) da 15 μl na ƙwayar cuta. Sa'an nan kuma an sanya ƙwayoyin sel a cikin dakatarwa na tsawon mintuna 60 kuma an shuka su a yawan sel 50,000 kowace rijiya. Bayan haɗuwa, an yi amfani da sel tare da 5-10 μg ml-1 puromycin na kwanaki 5-10 ko har sai an sami kwanciyar hankali. An yi mummunan kamuwa da cutar hCO kamar yadda aka bayyana a baya5 tare da wasu gyare-gyare. A taƙaice, canja wurin rana 30-45 hCO cikin 1.5 ml Eppendorf microcentrifuge tubes dauke da 100 µl na matsakaicin jijiya. Sannan an cire kusan 90 µl na matsakaici, 3-6 µl na lentivirus mai girma titer (daga 0.5 x 108 zuwa 1.2 x 109) an ƙara zuwa bututu, kuma ana tura hCO zuwa incubator na minti 30. Sa'an nan kuma ƙara 90-100 µl na matsakaici zuwa kowane bututu kuma mayar da bututun zuwa incubator na dare. Kashegari, canja wurin hCO zuwa sabon matsakaicin jijiya a cikin ƙananan faranti. Bayan kwanaki 7, an tura hCO zuwa faranti na ƙasan gilashin rijiyar 24 don gani da kimanta ingancin kamuwa da cuta. pLV[Exp] -SYN1::EYFP-WPRE da pLV[Exp] -SYN1:: hChR2-EYFP-WPRE ne suka samar da VectorBuilder. Ana amfani da Lentivirus a yawancin gwaje-gwaje saboda an haɗa shi a cikin kwayoyin halitta, yana ba da damar bayyanar da kwayar cutar ta hanyar ƙwayoyin cuta a cikin layin salula. Don bin rabies, ranar 30-45 hCO an haɗa shi tare da rabies-ΔG-eGFP da AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), an wanke shi sosai don kwanaki 3, kuma an dasa shi cikin berayen a cikin S1 kuma ana kiyaye su a cikin vivo don kwanaki 7-14.
Domin immunocytochemistry, dabbobi an anesthetized da transcardially transcardially tare da PBS biye da 4% paraformaldehyde (PFA a PBS; Electron Microscope Sciences). An gyara kwakwalwa a cikin 4% PFA na sa'o'i 2 ko na dare a 4 ° C, an adana cryopreserved a cikin 30% sucrose a cikin PBS na sa'o'i 48-72, kuma an saka shi cikin 1: 1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583, Sakura Finetek ta amfani da kuka) da coronal 3. (Leka). Don immunohistochemistry na sassan lokacin farin ciki, an shafe dabbobi tare da PBS, kuma an rarraba kwakwalwa kuma an raba su a cikin 300-400 µm ta amfani da vibratome (Leica) kuma an gyara sassan tare da 4% PFA na minti 30. Sa'an nan kuma an wanke sassan cryosections ko lokacin farin ciki tare da PBS, an toshe shi na awa 1 a zafin jiki (10% al'ada na jaki (NDS) da 0.3% Triton X-100 a cikin PBS) kuma an toshe shi tare da maganin toshewa a 4 ° C. – An shirya Cryosections incubation na dare kuma an sanya sassan masu kauri na kwanaki 5. Magungunan rigakafi na farko da aka yi amfani da su sune: anti-NeuN ( linzamin kwamfuta, 1: 500; ab104224, abcam) anti-CTIP2 (bera, 1:300; ab18465, abcam), anti-GFAP (zomo, 1:1,000; Z0334, Dako), anti-GFP:11ex, GTX 0 (kaza, GTX 0), GFP: 0334, GTX 0, GFP, GTX 0, 00, GFP, 0, 0, 0, 0, 1, 0, 0, GFP, 1, 0, 0, 0, 11, 0, GFP. anti-HNA ( linzamin kwamfuta, 1:200; ab191181, abcam), anti-NeuN (zomo, 1:500; ABN78, Millipore), anti-PDGFRA (zomo, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (zomo, 1:0408); HPAdies anti-RECA-1 ( linzamin kwamfuta, 1:50; ab9774, abcam), anti-SCG2 (zomo, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (akuya, 1:500; AF3075, R&D Systems), Netrin G1a (awaki, 6; RAF0, 1:11), anti-STEM121 ( linzamin kwamfuta, 1:200; Y40410, Takara Bio), anti-SATB2 ( linzamin kwamfuta, 1:50; ab51502, abcam), anti-GAD65/67 (zomo, 1:400; ABN904, Millipore) da kuma anti-IBA1 (akuya, 1:000, 1:000, abcam). Magungunan rigakafi na farko da aka yi amfani da su sune: anti-NeuN ( linzamin kwamfuta, 1: 500; ab104224, abcam) anti-CTIP2 (bera, 1:300; ab18465, abcam), anti-GFAP (zomo, 1:1,000; Z0334, Dako), anti-GFP:01, GTX 0 (kaza, GTX 0), anti-HNA ( linzamin kwamfuta, 1:200; ab191181, abcam), anti-NeuN (zomo, 1:500; ABN78, Millipore), anti-PDGFRA (zomo, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (zomo, 1:0408); HPAdies anti-RECA-1 ( linzamin kwamfuta, 1:50; ab9774, abcam), anti-SCG2 (zomo, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (akuya, 1:500; AF3075, R & D Systems), Netrin G1a (akuya, 106; 100, AF&D Systems), anti-STEM121 ( linzamin kwamfuta , 1: 200; Y40410, Takara Bio), anti-SATB2 ( linzamin kwamfuta, 1:50; ab51502, abcam), anti-GAD65/67 (zomo, 1:400; ABN904, Millipore) da kuma anti-IBA1: abkuwa 176, 1 cam. Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2: 3кыры; ab18465, abcam), анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA ( ab19cam 1), анти-NeuN (кролик, 1:500; ABN78, Millipore), анти-PDGFRA (кролик, 1:200; sc-338, Санта-Круз), анти-PPP1R17 (kр10240, HP; Antibodies), анти-RECA-1 (мышь, 1:50; ab9774, abcam), анти-SCG2 (кролик , 1:100; 20357-1-AP, Proteintech), анти-SOX9 (кози0; R&AFS Systems), 1:50 нетрин G1a (козий, 1:100; AF1166, R&D Systems), анти-STEM121 (мышиный , 1:200; Y40410, Takara Bio), анти-SATB2 (мышь, ab51), 502; ab51:50; анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) da анти-IBA1 (коза, 1:100; аб5076, абкам). Magungunan rigakafi na farko da aka yi amfani da su sune: anti-NeuN ( linzamin kwamfuta, 1: 500; ab104224, abcam), anti-CTIP2 (bera, 1:300; ab18465, abcam), anti-GFAP (zomo, 1:1000; Z0334, Dako), anti-GFP (kaza, 10300; GTX 10, GTX, 1030, GTX, 10300T), GFP anti-HNA ( linzamin kwamfuta, 1:200; ab191181, abcam), anti-NeuN (zomo, 1:500; ABN78, Millipore), anti-PDGFRA (zomo, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (zomo, 1:0408); HPAdies anti-RECA-1 ( linzamin kwamfuta, 1:50; ab9774, abcam), anti-SCG2 (zomo, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (akuya, 1:500; AF3075, R&D Systems), netrin G1a (akuya, 1:100, R&D Systems), STEM121 ( linzamin kwamfuta, 1:200; Y40410, Takara Bio), anti-SATB2 ( linzamin kwamfuta, 1:50; ab51502, abcam), anti-GAD65/67 (zomo, 1:400; ABN904, Millipore) da anti-IBA1 (awaki, ab51500).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 81,abcam), 抗NeuN (兔, 1:500;ABN78, Millipore), 抗PDGFRA（兔, 1: 200;sc-338, Santa Cruz), 抗PPP1R17 (兔, 1:200; HPA047819, Atlas抗体） 抗RECA-1（小鼠，1:50；ab9774） abcam）） 抗SCG2（兔） , 1:100; 20357-1-AP Systems： 抗STEM121（小鼠, 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））NeuN（兔，1:500；兔，1:500；兔，1:500；兔，1:500；兔；1；弛200;sc-338, Santa Cruz), 抗PPP1R17 (兔, 1:200；HPA047819, Atlas 抗体），抗RECA-1（小鼠，1:50（小鼠，1:50；abcam97； 100; 20357-1-APY40410, Takara Bio), anti-SATB2 ( linzamin kwamfuta, 1:50; ab51502, abcam), anti-GAD65/67 (zomo, 1:400; ABN904, Millipore) da kuma anti-IBA1 (akuya, 1:100; ab5076, abcam).Magungunan rigakafi na farko da aka yi amfani da su sune: anti-NeuN ( linzamin kwamfuta, 1: 500; ab104224, abcam), anti-CTIP2 (rat, 1:300; ab18465, abcam), anti-GFAP (zomo, 1:1000; Z0334, Dako) . , anti-GFP (kaza, 1: 1000; GTX13970, GeneTex), anti-HNA ( linzamin kwamfuta , 1: 200; ab191181, abcam), anti-NeuN (zomo, 1: 500; ABN78, Millipore), anti-PDGFRA (zomo, 1: 3300), 1 anti-Centro, Santa Claus (Zomo, 1: 3300), anti-NeuN. (zomo, 1:200; HPA047819, Atlas antibody), anti-RECA-1 ( linzamin kwamfuta, 1:50; ab9774, abcam), anti- SCG2 (zomo), 1:100;20357-1-AP. Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) da анти-IBA1 (коза0); abin). 20357-1-AP, Proteintech), anti-SOX9 (akuya, 1:500; AF3075, R&D Systems), Netrin G1a (awaki, 1:100; AF1166, R&D Systems), anti-STEM121 ( linzamin kwamfuta, 1: 200; Y40410; 1: 2000; Y40410 anti-mouse, Takara TB, 50, 1 TB, 50-mouse, Takara 1 TB, 50-Mouse ab51502, abcam), anti-GAD65/67 (zomo, 1:400; ABN904, Millipore), da kuma anti-IBA1 (akuya, 1:100; ab5076, abkam).Sa'an nan kuma an wanke sassan da PBS kuma an sanya su tare da antibody na biyu na awa 1 a dakin da zafin jiki (daskararre sassan) ko na dare a 4 ° C (sashe masu kauri). Alexa Fluor sakandare antibody (Life Technologies) diluted 1:1000 a tarewa bayani da aka yi amfani. Bayan wankewa tare da PBS, an hango nuclei tare da Hoechst 33258 (Fasahar Rayuwa). A ƙarshe, an sanya nunin faifai a kan na'ura mai ma'ana tare da murfin rufewa (Fisher Scientific) ta amfani da Aquamount (Polysciences) kuma an yi nazari akan microscope na Keyence fluorescent (BZ-X analyzer) ko Leica TCS SP8 microscope confocal (Las-X) akan hoton. An sarrafa hotunan ta amfani da shirin ImageJ (Fiji). Don ƙididdige adadin ƙwayoyin jikin ɗan adam a cikin t-hCO da ƙwayar bera, an ɗauki hotuna masu faɗin 387.5 μm a tsakiyar t-hCO, a ko kusa da ƙarshen bawo. An ƙididdige ɓangarorin alƙawarin ta hanyar tantance canje-canje a cikin fayyace nama, HNA+ nuclei, da/ko kasancewar nama autofluorescence. A cikin kowane hoto, jimlar adadin sel NeuN+ da HNA+ an raba su ta jimlar adadin sel NeuN+ a cikin yanki ɗaya. Don tabbatar da cewa kawai ƙwayoyin da ke da nuclei a cikin hoton hoton an ƙidaya su, sel waɗanda su ma Hoechst + ne kawai aka haɗa cikin lissafin. Hotuna biyu da suka rabu da aƙalla mm 1 an daidaita su don rage kuskuren ƙididdiga.
Mako daya kafin tarin samfurin, sanya dabbobin dashen hCO (kimanin watanni 8 na banbanta) a cikin wani daki mai duhu tare da tarkace barasa don rage karfin kuzari. An yi wariyar ƙwayoyin cuta kamar yadda aka bayyana a baya, tare da wasu gyare-gyare. A taƙaice, an lalata t-hCO da hCO ta yin amfani da lysis-mechanical cell lysis da 2 ml gilashin nama grinder (D8938, Sigma-Aldrich/KIMBLE). Sannan an tace danyen nuclei ta hanyar amfani da tacewa 40 µm kuma an sanya shi a 320 g na mintuna 10 a 4 ° C kafin yin gradient mai yawa. Bayan matakin centrifugation (320 g na 20 min a 4 ° C), an sake dawo da samfurori a cikin 0.04% BSA / PBS tare da ƙari na 0.2 raka'a na µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) kuma sun wuce ta hanyar 40.µ tacewa. An sake dakatar da ɓangarorin ɓangarorin a cikin PBS mai ɗauke da 0.02% BSA kuma an ɗora su akan guntun Chromium Single Cell 3′ (ƙididdigar dawo da ƙwayoyin 8,000 a kowane layi). An shirya ɗakunan karatu na snRNA-seq tare da Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). An shirya ɗakunan karatu na snRNA-seq tare da Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Библиотеки snRNA-seq были приготовлены с помощью Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). An shirya ɗakunan karatu na snRNA-seq ta amfani da Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). snRNA-seq 文库是使用Chromium Single cell 3′ GEM、Library & Gel Bead Kit v3 (10x Genomics) 制备的。 snRNA-seq 文库是使用Chromium Single cell 3′ GEM、Library & Gel Bead Kit v3 (10x Genomics) 制备的。 Библиотеку snRNA-seq готовили с использованием Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). An shirya ɗakin karatu na snRNA-seq ta amfani da Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics).An tattara ɗakunan karatu daga samfurori daban-daban kuma an tsara su ta Lafiya ta Admera akan NovaSeq S4 (Illumina).
An ƙididdige matakan furci na Gene na kowane lambar lambar ɓoye ta amfani da fakitin software na bincike na 10x Genomics CellRanger (sigar 6.1.2). Musamman, karatun an daidaita su da haɗin ɗan adam (GRCh38, Rukunin, sigar 98) da bera (Rnor_6.0, Rukunin, sigar 100) nau'ikan abubuwan da aka kirkira tare da umarnin mkref da yin amfani da ƙidaya tare da -include-introns = TRUE umarni don ƙididdigewa sun haɗa da karatun da aka tsara zuwa yankuna masu shigowa. Don samfuran t-hCO, an gano nuclei na ɗan adam dangane da buƙatun ra'ayin mazan jiya cewa aƙalla 95% na duk karatun taswira sun dace da kwayoyin halittar ɗan adam. An yi duk nazarce-nazarcen da suka biyo baya akan fitowar lambar lamba da aka tace daga CellRanger ta amfani da fakitin R (sigar 4.1.2) Seurat (sigar 4.1.1)32.
Don tabbatar da cewa ƙananan ƙwayoyin cuta masu inganci ne kawai aka haɗa a cikin bincike na gaba, an aiwatar da tsarin tacewa ga kowane samfurin. Na farko, ƙananan ƙananan ƙwayoyin cuta waɗanda ke da ƙasa da 1000 na musamman da aka samo kuma an gano fiye da 20% na jimlar mitochondria kuma an cire su. Daga baya, matrix ɗin matrix mai ɗanɗano mai ɗanɗano an daidaita shi ta hanyar koma baya mara kyau na binomial ta hanyar amfani da aikin sctransform (vst.flavor=”v2″), wanda kuma ya gano 3000 mafi yawan canjin kwayoyin halitta ta amfani da sigogin tsoho. An yi raguwar girma akan manyan kwayoyin halitta masu canzawa ta amfani da Babban Mahimman Abubuwan Halittu (PCA) tare da tsoho sigogi ta amfani da madaidaicin saiti na gani na 3 (dimension 3 bisa ga ma'aunin gani) Shafukan da aka yi amfani da su don duk samfurori da kuma nazarin gungu). Daga nan mun yi zagaye da yawa na tari mai maimaitawa (ƙuduri = 1) don rarraba kwayoyin halitta bisa ga ƙananan ƙananan ƙwayoyin cuta (matsakaici a ƙasa da kashi 10), ƙananan ƙwayoyin mitochondrial (matsakaici a sama da 95th percentile). Kunshin DoubletFinder33 (ma'ana DoubletFinder maki sama da kashi 95) samfuran t-hCO (n=3) da samfuran hCO (n=3) an haɗa su daban ta amfani da aikin IntegrateData tare da sigogin da ke sama.
Bayan cire ƙananan kernels masu inganci, an haɗa haɗaɗɗiyar saitin bayanai (ƙuduri = 0.5) kuma an saka shi don dalilai na gani na UMAP34. An ƙaddara kwayoyin halitta masu alamar ga kowane tari ta amfani da aikin FindMarkers tare da tsoho sigogi da aka ƙididdige su daga daidaitattun bayanan maganganun kwayoyin halitta. Muna ganowa da rarrabuwa manyan azuzuwan tantanin halitta ta hanyar haɗa bayanan ƙididdiga na ƙididdiga na tayi da manya tare da bayanin jigon alamar 19,20,21,35 da annotation. Musamman ma, an gano abubuwan da ke gudana ta hanyar bayyana MKI67 da TOP2A. An bayyana gungu na gaba ta hanyar rashi bayanan mitotic, babban jeri tare da manyan rukunonin glial masu girma da yawa da aka bayyana a cikin ƙarshen metaphase fetal cortex, da EGFR da OLIG1 magana. Muna amfani da kalmar astrocyte don haɗa jihohi da yawa na bambancin astrocyte, daga ƙarshen radial glia zuwa maturation na astrocytes. Tarin Astrocyte suna bayyana manyan matakan SLC1A3 da AQP4 kuma an nuna su don taswira tare da nau'ikan nau'ikan radial glia na tayin da/ko manyan taurarin taurari. OPCs suna bayyana PDGFRA da SOX10 yayin da oligodendrocytes ke bayyana alamun myelination (MOG da MYRF). Glutamatergic neurons an gano su ta hanyar kasancewar rubutun neuronal (SYT1 da SNAP25), rashin alamun GABAergic (GAD2), da kuma bayanin NEUROD6, SLC17A7, BCL11B, ko SATB2. GluN neurons an kara raba su zuwa babba (SATB2 magana da asarar BCL11B) da zurfi (BCL11B magana). Ƙunƙasar ƙanƙara (SP) neurons suna bayyana sanannun alamun SP18 kamar ST18 da SORCS1 ban da alamun GluN mai zurfi. Kwayoyin plexus-kamar Choroid an gano su ta hanyar magana ta TTR, kuma sel-kamar meningeal sun bayyana kwayoyin da ke hade da fibroblast da taswirar pial / jijiyoyi na bayanan bayanan bayanan.
Bambance-banci na maganganun kwayoyin halitta tsakanin t-hCO da hCO subclasses an yi su ta amfani da sabuwar hanyar pseudo-batch da aka sake bugawa a cikin samfurori da aka aiwatar ta amfani da kunshin Libra R (sigar 1.0.0). Musamman, gwaje-gwajen alamar log-R (sigar 3.36.0, kunshin R) an yi don ƙungiyoyi ta hanyar taƙaita adadin kwayoyin halitta a cikin sel don wani nau'in tantanin halitta don kowane samfurin samfurin. Don ganin taswirar zafi, ana ƙididdige ƙimar ƙimar kowane miliyan (CPM) ta amfani da EdgeR (cpm () aiki) da kuma daidaitawa (don cimma ma'ana = 0, daidaitaccen karkata = 1). Gene Ontology (GO) nazarin haɓakar haɓakar ƙwayoyin halittar t-hCO GluN da aka haɓaka (Benjamini-Hochberg ya gyara ƙimar P ƙasa da 0.05 wanda aka bayyana a cikin aƙalla 10% na ƙwayoyin t-hCO GluN da ninka karuwa a cikin canji aƙalla sau 2). Anyi amfani da ToppGene Suite (https://toppgene.cchmc.org/)37. Muna amfani da aikace-aikacen ToppFun tare da sigogin tsoho kuma muna ba da rahoton ingantattun ƙimar P-darajar Benjamini-Hochberg daga gwaje-gwajen hypergeometric na GO.
Don dacewa da gungu na snRNA-seq ɗin mu tare da rukunin tantanin halitta bayanai daga binciken bincike na farko-cell guda RNA-seq ko babba snRNA-seq19,20,21,22, mun yi amfani da tsarin haɗa bayanai guda biyu. Mun yi amfani da SCTransform (v2) daidaitaccen aikin aiki a cikin Seurat don haɗawa da kwatanta tari mai ruɗi tsakanin bayanan bayanai (ta amfani da sigogi iri ɗaya kamar na sama). Rukunin bayanan daidaikun mutane an tsara su ba da gangan ba har zuwa sel 500 ko ma'auni a kowane gungu na asali don dacewar lissafi. Yin amfani da irin wannan hanya kamar yadda aka bayyana a baya, an bayyana ma'anar tari a matsayin rabon sel ko nuclei a cikin kowane gungu wanda ya mamaye da alamar gunkin tunani. Don ƙarin rarrabuwa GluNs, mun yi amfani da Seurat's TransferData workflow for GluN subset data to sanya alamun saitin bayanai zuwa ƙwayoyin GluN ɗin mu.
Don tantance matsayin balagagge na kwafin duniya na t-hCO da samfuran hCO, mun kwatanta samfuran mu mai girma tare da BrainSpan/psychENCODE23, wanda ya ƙunshi babban jerin RNA wanda ya shafi ci gaban kwakwalwar ɗan adam. Mun yi PCA akan matrix ɗin ƙirar ƙirar al'ada na al'ada daga samfuran cortical makonni 10 bayan ɗaukar ciki kuma daga baya, a cikin kwayoyin halittar 5567 (tare da bayananmu) waɗanda a baya aka gano suna aiki a samfuran cortical BrainSpan (wanda aka bayyana a matsayin mafi girma fiye da 50% a cikin bambance-bambancen ci gaba da aka bayyana ta shekaru ta amfani da ƙirar cubic)38. Bugu da ƙari, mun samo kwayoyin halitta masu alaƙa da manyan sa hannu na ci gaban neurodevelopment ta amfani da matrix factorization mara kyau kamar yadda aka bayyana a baya. Ana ƙididdige ma'aunin samfurin da aka ƙididdige ta amfani da tsarin ƙididdiga mara kyau na matrix a cikin figs. 5b tare da faɗaɗa bayanai don kowane sa hannu guda biyar da Zhu et al.38 ya kwatanta. Bugu da ƙari, an samo alamun rubutun da suka dogara da ayyuka daga binciken da aka buga a baya. Musamman, ERG da LRG sun kasance masu haɓakawa sosai a cikin ƙwayoyin glutamatergic neurons da aka gano ta hanyar tarin gani na linzamin kwamfuta na snRNA-seq bayan haɓakar gani daga Ƙarin Teburin 3 Hrvatin et al.16. An samo LRGs masu wadatar ɗan adam daga al'adun kwakwalwar ɗan adam mai kunna KCl kuma an girbe sa'o'i 6 bayan ƙarfafawa, kuma ƙwayoyin da aka tace sun inganta sosai a cikin mutane amma ba a cikin rodents ba (Ƙarin Teburin 4). An yi nazarin haɓakar saitin kwayoyin halitta ta amfani da waɗannan saitin kwayoyin halitta ta amfani da ainihin gwajin Fisher ta hanya ɗaya.
Anesthetize berayen tare da isoflurane, cire kwakwalwa da sanya a cikin sanyi (kimanin 4 ° C) oxygenated (95% O2 da 5% CO2) maganin sucrose don sassan da ke dauke da: 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM MgSO4 da 0.5 mM CaCl2 (kimanin 310 mOsm). Sassan ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta (300-400 µm) masu ɗauke da t-hCO an yi su ta amfani da Leica VT1200 vibratome kamar yadda aka bayyana a baya39. Sa'an nan kuma an tura sassan zuwa ɗakin da aka raba tare da ci gaba da zafin jiki na oxygenation dauke da aCSF da aka shirya daga: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 da 126 mM NaCl (298 mM) . akalla mintuna 45 kafin yin rikodi. An rubuta sassan a cikin ɗakin da aka nutsar da su inda aka ci gaba da shafe su da aCSF (95% O2 da 5% CO2 vial). An yi rikodin duk bayanai a cikin zafin jiki. t-hCO neurons an ƙare tare da bututun gilashin borosilicate wanda aka cika da wani bayani wanda ke dauke da 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, da 0.6 mM EGTA, pH 7.2, bayani na ciki wanda aka gyara tare da KOHs2.9. Domin murmurewa, an ƙara biocytin (0.2%) zuwa maganin rikodi.
An samo bayanai ta amfani da MultiClamp 700B amplifier (Na'urorin Molecular) da Digidata 1550B digitizer (Molecular Devices), ƙarancin wucewa a 2 kHz, digitized a 20 kHz, kuma an bincika ta amfani da Clampfit (Na'urorin Molecular), Origin (OriginPro). 2021b, OriginLab). da ayyukan MATLAB na al'ada (Mathworks). An ƙididdige yuwuwar haɗuwa ta amfani da JPCalc kuma an daidaita abubuwan shigarwa zuwa ƙimar ƙididdigewa na -14 mV. Operation IV ya ƙunshi jerin matakai na yanzu a cikin matakan 10-25 pA, daga -250 zuwa 750 pA.
thalamus, farin kwayoyin halitta, da S1 afferents an motsa su ta hanyar lantarki a cikin sassan thalamocortical yayin rikodin faci-clamp na hCO neurons, kamar yadda aka bayyana a baya. A taƙaice, an ɗora kwakwalwa a kan tebur ɗin bugawa na 3D wanda aka karkata a kusurwa 10 °, kuma an yanke gaban kwakwalwa a kusurwa 35 °. Daga nan sai aka manne kwakwalwar a saman da aka yanke kuma aka raba shi, yana kiyaye thalamocortical protruding axon. Bipolar tungsten electrodes (0.5 MΩ) an ɗora su akan micromanipulator na biyu kuma an sanya su cikin dabara don tada yankuna huɗu a kowace tantanin halitta (capsule na ciki, fararen fata, S1 da hCO). Yi rikodin martanin synaptic bayan 300 µA haɓakar phasic a 0.03-0.1 Hz.
hChR2-bayyana hCO neurons an kunna su a 480 nm kuma an yi amfani da bugun haske da aka samar ta hanyar LED (Prizmatix) ta hanyar maƙasudin × 40 (0.9 NA; Olympus) don yin rikodin maganganun hChR2 kusa da sel. Diamita mai haske yana kusan 0.5 mm kuma jimlar ƙarfin shine 10-20mW. An saita faɗin bugun bugun zuwa 10 ms, wanda yayi daidai da bugun bugun da aka bayar yayin gwajin koyan ɗabi'a. An yi amfani da mitoci daban-daban na ƙarfafawa, daga 1 zuwa 20 Hz, amma kawai bugun farko na jerin an yi amfani dashi don ƙididdigewa. Matsakaicin tsakanin jiragen kasa yawanci ya fi 30s don rage tasirin hanawa na synaptic ko hanyoyin sauƙaƙewa. Don gwada idan amsawar hChR2 ta kasance monosynaptic, mun yi amfani da TTX (1 μM) zuwa wanka har sai lokacin da EPSC ya ɓace, sa'an nan kuma amfani da 4-aminopyridine (4-AP; 100 μM). Yawanci, ana mayar da martani a cikin ƴan mintuna kaɗan, tare da ɗan ɗan jinkiri tsakanin harbe-harben LED da tsarar EPSC. An yi amfani da NBQX (10 μM) don gwada ko masu karɓar AMPA ne ke jagorantar amsa.
An ƙirƙiri sassan hCO masu kaifi kamar yadda aka bayyana a baya. A taƙaice, an shigar da sassan hCO a cikin 4% agarose kuma an tura su zuwa sel dauke da 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 da 10 mM NaHCO3 da 10 mM dse (+ 0) an yanke su a cikin Sashe (+ 0) µm a zazzabi na ɗaki ta amfani da jijjiga Leica VT1200 kuma an adana shi a cikin ASF a zazzabi na ɗaki. Bayan haka, an yi rikodin faci-sansanin sel gabaɗayan akan sassan hCO ƙarƙashin madaidaicin SliceScope microscope (Scientifica). An lulluɓe sassan da aCSF (95% O2 da 5% CO2) kuma an yi rikodin siginonin tantanin halitta a zafin daki. An yi amfani da ƙananan ƙwayoyin hCO ta amfani da pipette gilashin borosilicate wanda aka cika da bayani mai dauke da 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, da 0.6 mM EGTA, pH na ciki 7, 2, gyara tare da KOH (osmolarity). Don dalilai na dawowa, ƙara 0.2% Biocytin zuwa bayani na ciki.
Clampex (Clampex 11.1, Molecular Devices) ya samo bayanai ta hanyar amfani da MultiClamp 700B amplifier (Na'urorin Molecular) da Digidata 1550B digitizer (Na'urorin Molecular), ƙananan wucewar da aka tace a 2 kHz, digitized a 20 kHz, kuma an bincika ta amfani da na'urorin Clampfit.6. Ayyuka (MATLAB 2019b, Mathworks). An ƙididdige yuwuwar haɗuwa ta amfani da JPCalc kuma an daidaita abubuwan shigarwa zuwa yuwuwar junction ɗin -14 mV. Operation IV ya ƙunshi jerin matakai na yanzu a cikin matakan 5-10 pA daga -50 zuwa 250 pA.
Don sake gina ƙwayoyin cuta na ƙwayoyin cuta, an ƙara 0.2% biocytin (Sigma-Aldrich) zuwa maganin ciki. Ana cire ƙwayoyin sel na aƙalla mintuna 15 bayan hacking. Daga nan ana jawo pipette a hankali don 1-2 min har sai an rufe murfin da aka yi rajista gaba ɗaya. Bayan bin tsarin ilimin lissafi na sashe, an gyara sassan dare a 4 ° C. a cikin 4% PFA, an wanke tare da PBS X3, kuma an diluted 1: 1000 tare da streptavidin-conjugated DyLight 549 ko DyLight 405 (Vector Labs). Kwayoyin da ke cike da biocytin (2%; Sigma-Aldrich) an yi musu lakabi yayin rikodin manne a cikin zafin jiki na awanni 2. Sa'an nan kuma an ɗora sassan a kan faifan microscopy ta amfani da Aquamount (Thermo Scientific) kuma an gani a rana mai zuwa a kan Leica TCS SP8 microscope confocal ta amfani da maƙasudin nutsar mai tare da lamba ta lamba ×40 1.3, haɓakawa ×0.9-1.0, xy. Matsakaicin samfurin shine kusan pixels 7 akan micron. Z-stacks a 1 µm tazara an samu a jere, kuma z-stack mosaics da auto-stitching na tushen Leica an yi su don rufe dukan bishiyar dendritic na kowane neuron. Daga nan aka bibiyar ƙwayoyin jijiya da hannu ta hanyar amfani da neuTube 40 dubawa kuma an samar da fayilolin SWC. Sannan an ɗora fayilolin zuwa kayan aikin SimpleNeuriteTracer41 Fiji (ImageJ, sigar 2.1.0; NIH).
An samu nama na jikin ɗan adam tare da sanarwar izini bisa ga ƙa'idar da Hukumar Binciken Cibiyoyi ta Jami'ar Stanford ta amince. Samfurin guda biyu na jikin ɗan adam bayan haihuwa (shekaru 3 da 18) an samo su ta hanyar reshewar cortex na gaba (tsakiyar gyrus na gaba) a matsayin wani ɓangare na tiyata don refractory epilepsy. Bayan resection, girbi nama a cikin kankara-sanyi NMDG-aCSF dauke da: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM thiourea, 5 mMpy 5 mM sodium, sodium ascorvate. CaCl2 4H2O da 10 mM MgSO4 7H2O. Titrate zuwa pH 7.3-7.4 tare da maida hankali hydrochloric acid. An kai kayan nama zuwa dakin gwaje-gwaje a cikin mintuna 30 kuma an dauki sassan na jijiyoyin jini bisa ga tsarin da aka bayyana a sama.
Dukkan hanyoyin dabba an yi su daidai da jagororin kula da dabbobi na Jami'ar Stanford APLAC. Berayen (fiye da kwanaki 140 bayan dasawa) an jawo su tare da maganin sa barci na isoflurane 5% da anesthetized tare da 1-3% isoflurane intraoperatively. An sanya dabbobi a cikin firam ɗin stereotaxic (Kopf) kuma an yi wa ci gaba da sakin buprenorphine (SR) allurar subcutaneously. An fallasa kwanyar, an tsaftace shi kuma an saka sukurori 3-5. Don ƙaddamar da t-hCO, mun ƙirƙiri daidaitawar stereotaxic daga hotunan MRI. An haƙa rami mai burr a wurin sha'awa da zaruruwa (diamita 400 µm, NA 0.48, Doric) an saukar da 100 µm ƙasa da saman hCO kuma an kiyaye shi zuwa kwanyar tare da ciminti mai warkewa UV (Relyx).
An yi rikodin hoto na Fiber kamar yadda aka bayyana a baya42. Don yin rikodin ayyukan da ba zato ba tsammani, an sanya berayen a cikin keji mai tsabta kuma an haɗa kebul na fiber optic facin diamita na 400 µm (Doric) da aka haɗa da tsarin sayan bayanan fiber optic photometric zuwa igiyar fiber na gani da aka dasa. A lokacin rikodin minti 10 na ayyukan motsa jiki, dabbobin sun sami 'yanci don bincika keji. Don yin rikodin ayyukan da aka kora, berayen (fiye da kwanaki 140 bayan dasawa) an yi musu maganin 5% isoflurane don ƙaddamarwa da 1-3% isoflurane don kulawa. Sanya dabbar a cikin firam ɗin stereotactic (Kopf) kuma an gyara whiskers a gefe na t-hCO zuwa kusan 2 cm kuma an wuce ta hanyar raga da aka haɗa zuwa piezoelectric actuator (PI). An haɗa kebul na fiber optic patch na 400 µm (Doric) zuwa fiber da aka dasa kuma an haɗa shi da tsarin sayan bayanai. Daga nan an karkatar da barasar da ke gefe na t-hCO sau 50 (2 mm a 20 Hz, 2 s a kowace gabatarwa) a lokuta bazuwar ta hanyar injin piezoelectric sama da lokacin rikodi na mintuna 20. Yi amfani da Kunshin Tallafi na Arduino MATLAB don sarrafa lokacin karkatarwa tare da lambar MATLAB ta al'ada. Abubuwan da suka faru suna aiki tare da software na sayan bayanai ta amfani da transistor-transistor Logic (TTL).
Berayen (fiye da kwanaki 140 bayan dasawa) an jawo su tare da maganin sa barci na isoflurane 5% da anesthetized tare da 1-3% isoflurane intraoperatively. An sanya dabbobi a cikin firam na stereotaxic (Kopf) da buprenorphine SR da dexamethasone an yi musu allurar subcutaneously. An fallasa kwanyar, an tsaftace shi kuma an saka sukurori 3-5. Don ƙaddamar da t-hCO, mun ƙirƙiri daidaitawar stereotaxic daga hotunan MRI. An yi craniotomy madauwari (kimanin 1 cm a diamita) tare da rawar soja mai tsayi kai tsaye akan hCO da aka dasa. Da zarar kashi ya yi bakin ciki kamar yadda zai yiwu, amma kafin a yi hakowa ta cikin duka kashi, yi amfani da karfi don cire ragowar ƙwanƙwasa ƙwanƙwasa don bayyana ainihin t-hCO. An cika craniotomy da salin bakararre, kuma an makala abin rufe fuska da fil ɗin kai na musamman a kan kwanyar tare da simintin haƙora da aka warke (Relyx).
An yi hoton hoto guda biyu ta amfani da na'urar gani da ido na Bruker tare da manufar Nikon LWD (×16, 0.8 NA). An yi hoton GCaMP6 a 920 nm tare da haɓakar jirgin sama guda 1.4x da matsakaicin 8x na 7.5fps. An jawo berayen tare da 5% isoflurane anesthesia kuma ana kiyaye su tare da 1-3% isoflurane. An sanya berayen a cikin al'ada da aka yi da kai kuma an sanya su a ƙarƙashin ruwan tabarau. An sami rikodin bayanan bayan mintuna 3 na ayyukan motar. A cikin tsawon mintuna 20 na yin rikodi, 50 puffs (kowace gabatarwa mai tsayi 100 ms) an kai su ba da gangan ba ga kushin whisker da ke gaban t-hCO ta amfani da picospricer. Yi amfani da Kunshin Tallafi na Arduino MATLAB don sarrafa lokacin fashewa tare da lambar MATLAB na al'ada. Haɗa abubuwan da suka faru tare da software na sayan bayanai (PrairieView 5.5) ta amfani da bugun jini na TTL. Don bincike, an gyara hotunan don motsin xy ta amfani da gyaran affine a cikin shirin MoCo da aka ƙaddamar a Fiji. Cire alamun kyalli daga sel guda ta amfani da CNMF-E43. An fitar da fluorescence don kowane yanki na sha'awa, an canza shi zuwa masu lanƙwasa dF/F, sa'an nan kuma ya juya zuwa z-scores.
Berayen (fiye da kwanaki 140 bayan dasawa) an jawo su tare da maganin sa barci na isoflurane 5% da anesthetized tare da 1-3% isoflurane intraoperatively. An sanya dabbobi a cikin firam na stereotaxic (Kopf) da buprenorphine SR da dexamethasone an yi musu allurar subcutaneously. An yanke wuka a gefe na t-hCO zuwa kusan 2 cm kuma an zare ta hanyar raga da aka haɗa da mai kunna wuta ta piezoelectric. An fallasa kwanyar kuma an tsaftace shi. An makala bakin karfen ƙasa dunƙule a kan kwanyar. Don ƙaddamar da t-hCO, mun ƙirƙiri daidaitawar stereotaxic daga hotunan MRI. Yi craniotomy madauwari (kimanin 1 cm a diamita) tare da rawar soja mai tsayi sama da t-hCO. Da zarar kashi ya yi bakin ciki kamar yadda zai yiwu, amma kafin a yi hakowa ta cikin duka kashi, yi amfani da karfi don cire ragowar ƙwanƙwasa ƙwanƙwasa don bayyana ainihin t-hCO. An yi rikodin sel guda ɗaya ta amfani da tashoshi 32 ko tashoshi 64-tashar siliki mai girma na siliki (Cambridge Neurotech) wanda aka kafa zuwa screws na ƙasa kuma an inganta su da RHD amplifiers (Intan). Yi amfani da manipulator don saukar da na'urorin lantarki zuwa wurin da aka yi niyya ta hanyar craniotomy, wanda ke cike da saline mara kyau. An yi tarin bayanai a mitar 30 kHz ta amfani da tsarin sayan bayanan Buɗe Ephys. Rikodin ya ci gaba ne kawai lokacin da muka gano ayyukan rhythmic da ke da alaƙa sosai a cikin tashoshi sama da 10, yana nuna cewa ana amfani da na'urorin lantarki a cikin graft (dangane da bayanan hotunan calcium mai hoto biyu). An sami rikodin bayanan bayan mintuna 10 na ayyukan mota. Daga nan an karkatar da barasar da ke gefe na t-hCO sau 50 (2 mm a 20 Hz, 2 s a kowace gabatarwa) a lokuta bazuwar ta hanyar injin piezoelectric sama da lokacin rikodi na mintuna 20. Amfani da Kunshin Tallafi na MATLAB don Arduino (MATLAB 2019b), sarrafa lokacin juyewa tare da lambar MATLAB na al'ada. Yi amfani da bugun jini na TTL don daidaita abubuwan da ke faruwa tare da software na sayan bayanai.
Don gwaje-gwajen alamar gani, 200 µm na'urar faci na gani (Doric) da aka haɗa da Laser 473 nm (Omicron) an haɗa shi zuwa 200 µm fiber na gani da aka sanya akan craniotomy. Nan da nan kafin wannan, daidaita ikon jumper zuwa 20mW. Yi amfani da manipulator don saukar da na'urorin lantarki zuwa wurin da aka yi niyya ta hanyar craniotomy, wanda ke cike da saline mara kyau. A farkon rikodi, an fitar da fitulu goma na haske 473 nm (yawanci 2 Hz, tsawon bugun bugun jini 10 ms). An bayyana sel masu ɗaukar hoto azaman sel waɗanda ke nuna amsa mai girma a cikin 10 ms na haske a cikin 70% ko fiye na gwaji.