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Kwishyira hamwe kwingingo ngengabuzima byerekana ibyiringiro muri vitro platform yo kwerekana iterambere ryabantu nindwara. Nyamara, organoide ibura ihuza riri muri vivo, igabanya gukura kandi ikabuza kwishyira hamwe nizindi nzitizi zigenzura imyitwarire. Hano twerekana ko ingirabuzimafatizo ya kimuntu ikomoka kuri cortical organoide yatewe muri cortex ya somatosensory yimbeba ya neonatal yambaye ubusa ikura ubwoko bwingirabuzimafatizo zikuze zinjira mumyumvire yumutima. MRI yerekanye imikurire ya organoide nyuma yo guhindurwa mumirongo myinshi yingirabuzimafatizo hamwe ninyamaswa, mugihe isesengura ryibanze ryagaragaje iterambere rya corticogenez no kugaragara kwa gahunda yo kwandukura ibikorwa. Mubyukuri, cortical cortical neuron yatewe yerekana imiterere igoye ya morphologique, synaptic, na membrane imbere kuruta iyindi muri bagenzi babo ba vitro, bigatuma habaho gutahura inenge zifata ubwonko kubarwayi barwaye syndrome ya Timoteyo. Gukurikirana no gukora byerekanaga ko ingirangingo zatewe zakira thalamocortical na corticocortical inyongeramusaruro, kandi muri vivo gufata amajwi yibikorwa byubwonko byerekana ko ibyo byinjira bishobora gutanga ibisubizo byumviro mumasemburo yabantu. Hanyuma, cortical organoide yagura axon mubwonko bwimbeba, kandi gukora optogenetike biganisha kumyitwarire yo gushaka ibihembo. Rero, transplant ya cortex yumuntu ikuze kandi ikagira uruhare mukuzunguruka kwakiriye kugenzura imyitwarire. Turateganya ko ubu buryo bworoshya kumenya fenotipike yo murwego rwohejuru muri selile ikomoka kubarwayi idashobora gutahurwa nubundi buryo.
Ubwonko bwabantu butera imbere ninzira idasanzwe yo kwishyiriraho uburyo ingirabuzimafatizo zigwira, zigatandukanya, zikimuka, kandi zigahuza imikorere yimikorere ya neuronal ikora neza kurushaho kunonosorwa binyuze muburambe. Ikibazo cyingenzi mugusobanukirwa iterambere ryubwonko bwabantu, cyane cyane mubijyanye nindwara, nukutabona uburyo bwubwonko. Kwishyira hamwe kwingingo, harimo na cortex organoide yumuntu (hCO; izwi kandi nka cortex yumuntu), irashobora kubyara 2,3,4,5,6. Nubwo bimeze bityo ariko, imbogamizi nyinshi zigabanya uburyo bwagutse bwo gusobanukirwa iterambere nimikorere yimitsi itwara imitsi. By'umwihariko, ntibisobanutse niba gukura kwa hCO kugarukwaho no kubura ibinyabuzima bimwe na bimwe byangiza ibidukikije hamwe na sensory byinjira muri vivo. Byongeye kandi, kubera ko hCOs itinjijwe mumuzunguruko ushobora gutanga umusaruro wimyitwarire, akamaro kabo mukugaragaza imiterere ya genetique igoye kandi yimyitwarire mibi ya neuropsychiatricique.
Guhinduranya hCO mubwonko buzira umuze birashobora gutsinda izo mbogamizi. Ubushakashatsi bwibanze bwerekanye ko neuron zabantu zatewe muri cortex yimbeba zishobora kubaho, umushinga, no kuvugana ningirabuzimafatizo 7,8,9,10,11,12. Nyamara, ubu bushakashatsi busanzwe bukorerwa ku nyamaswa zikuze, zishobora kugabanya guhuza synaptic na axonal. Hano, turasobanura paradigima yo guhinduranya aho twahinduye 3D hCO ikomoka muri selile ya hiPS muri cortex yibanze ya somatosensory cortex (S1) yimbeba zidafite ubudahangarwa mugihe cyambere cyo gukura kwa plastike. Hron (t-hCO) yatewe muri neuron ikura cyane, yakira thalamocortical na cortical-cortical inyongeramusaruro itanga ibisubizo byumviro, kandi ikagura ibishushanyo mbonera mu bwonko bwimbeba kugirango itware imyitwarire ishaka ibihembo. Gukura kwinshi kwa t-hCO byagaragaje ubusembwa bw’imitsi ku barwayi barwaye syndrome ya Timoteyo (TS), indwara ikomeye ikomoka ku ihinduka ry’imihindagurikire y’imihindagurikire y’umuvuduko wa L-CaV1.2 ya calcium (ikoreshwa na CACNA1C).
Kugira ngo twige ubwonko bwa cortical neuron yabantu mumuzunguruko muri vivo, twimuye muburyo bwa 3D hCO muri S1 yimbeba za athymic zimaze kubyara (iminsi 3-7 nyuma yinyuma) (Ishusho 1a hamwe namakuru yagutse yishusho 1a-c). Kuri ubu, ibipimo bya thalamocortical na corticocortical axonal projection itararangiza S1 yimbere (reba 13). Rero, ubu buryo bwateguwe kugirango hongerwe t-hCO mugihe hagabanijwe ingaruka kumuzinga wa endogenous. Kugirango tumenye aho t-hCO iri mu nyamaswa nzima, twakoze ubwonko bwa T2 buremereye bwa MRI ubwonko bwimbeba nyuma y'amezi 2-33 nyuma yo guterwa (Ishusho 1b hamwe namakuru yagutse, Ishusho 1d). t-hCO byaragaragaye byoroshye kandi gupima ingano ya t-hCO byari bisa nibibarwa uhereye ku bice byagenwe (Data Yaguwe Ishusho 1d, e; P> 0.05). t-hCO byaragaragaye byoroshye kandi gupima ingano ya t-hCO byari bisa nibibarwa uhereye ku bice byagenwe (Data Yaguwe Ishusho 1d, e; P> 0.05). t-hCO легко наблюдались, а оемемнее измерения t-hCO были аналогичны рассчитанным для фик କାନованных срезов (расширенные псныее, рис. 1d, e; P> 0,05). t-hCO byagaragaye byoroshye, kandi ibipimo bya t-hCO byapimwe byari bisa nibibazwe kubice byagenwe (amakuru yagutse, Ishusho 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 ）。T-hCO ，并且 t-hCO t-hCO легко наблюдался, а оемемные измерения t-hCO были аналогичны рассчитанным для фик киованных срезов (расширенные псныее, рис. 1d, e; P> 0,05). t-hCO yagaragaye byoroshye, kandi ibipimo bya t-hCO byapimwe byari bisa nibibazwe kubice byagenwe (amakuru yagutse, Ishusho 1d, e; P> 0.05).Twahisemo t-hCO muri 81% yinyamaswa zatewe nyuma y'amezi 2 nyuma yo guterwa (n = inyamaswa 72; hCO kuva kumirongo 10 ya hiPS; imirongo ya hiPS mumurongo winyongera 1). Muri byo, 87% byari biherereye mu bwonko (Ishusho 1c). Mugukora scaneri ya MRI kumwanya munini mugihe cyimbeba imwe yatewe, twasanze kwiyongera inshuro icyenda mubunini bwa t-hCO mugihe cyamezi 3 (Igicapo 1d hamwe namakuru yagutse, Ishusho 1f). Amatungo yatewe yari afite igipimo kinini cyo kubaho (74%) mumezi 12 nyuma yo guhindurwa (amakuru yagutse, igishushanyo cya 1g hamwe nimbonerahamwe yinyongera 2), kandi nta moteri ifite moteri cyangwa yibuka, gliose, cyangwa electroencephalogramu (EEG) yabonetse. Amakuru Igishushanyo 1g hamwe nimbonerahamwe yinyongera 2). 1h - m na 3e).
a, Igishushanyo mbonera cyubushakashatsi. hCO ikomoka muri selile ya hiPS yatewe muri S1 yimbeba zambaye ubusa muminsi 30-60 yo gutandukana. b, T2 ifite uburemere bwa coronale na horizontal ya MRI yerekana t-hCO muri S1 amezi 2 nyuma yo guterwa. Umwanya munini, mm 2. c, Umubare wibipimo byatsinze byerekanwe kuri buri murongo wa selire ya hiPS (n = 108, imibare iri mu tubari yerekana umubare wa t-hCO kumurongo wa selire ya hIPS) hamwe na cortical cyangwa subcortical (n = 88). d, ishusho ya MRI yimitsi yumutima (ibumoso; umurongo wikigereranyo, mm 3) hamwe niyubaka rya 3D volumetric (igipimo cyumubyimba, mm 3) yerekana kwiyongera kwa t-hCO mumezi 3. e, Gusubiramo imiterere ya t-hCO muri cortex yubwonko. Umwanya munini, mm 1. f, Uhagarariye amashusho ya immunocytochemiki ya t-hCO yerekanwe kuva hejuru ibumoso ugana iburyo (mugihe cyo gutandukana): PPP1R17 (amezi 4), NeuN (amezi 8), SOX9 na GFAP (amezi 8), PDGFRα; (Amezi 8), MAP2 (amezi 8) na IBA1 (amezi 8). Umwanya munini, 20 µm. Guhuza imvugo ya HNA byerekana selile zikomoka kubantu. g, snRNA-seq: Guhuza ibipimo byinshi hamwe na projection (UMAP) kugabanya ibipimo byerekana amashusho yose yo mu rwego rwo hejuru t-hCO nuclei nyuma yo kwishyira hamwe kwa Seurat (n = 3 t-hCO ntangarugero, imirongo ya selile n = 2 hiPS). Astrocytes, selile z'umurongo wa astrocyte; cyc prog, kuzenguruka urubyaro; GluN DL, neuron yimbitse ya glutamatergic; GluN DL / SP, neuron yimbitse na sublamellar glutamatergic; GluN UL, hejuru ya glutamatergic neuron; oligodendrocytes, oligodendrocytes; OPC, oligodendrocyte progenitor selile; RELN, reelin neurons. h, isesengura rya Gene Ontology (GO) isesengura ryitunganyirizo rya gen ryagenzuwe cyane (ryahinduwe P <0.05, ihinduka ryikubye> 2, ryerekanwe byibuze 10% bya nuclei) muri t-hCO glutamatergic neuron ugereranije na neuron ya hCO glutamatergic. h, isesengura rya Gene Ontology (GO) isesengura ryitunganyirizo rya gen ryagenzuwe cyane (ryahinduwe P <0.05, ihinduka ryikubye> 2, ryerekanwe byibuze 10% bya nuclei) muri t-hCO glutamatergic neuron ugereranije na neuron ya hCO glutamatergic. h. . h, isesengura rya Gene Ontology (GO) isesengura ryitunganyirizwa rya gen hamwe nigikorwa gikomeye (cyahinduwe P <0.05, guhinduka inshuro> 2, imvugo byibuze nuclei 10%) muri t-hCO glutamatergic neuron ugereranije na neuron ya hCO glutamatergic. 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 ，倍数变化> 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

2 ， 至少 10% 的 核中 表达） 基因 基因 基因 GO GO GO GO GO GO GO , . h, ingirabuzimafatizo zagenzuwe cyane (zahinduwe P <0.05, guhindura inshuro> 2, zigaragara byibuze 10% ya nuclei) muri t-hCO glutamatergic neuron ugereranije na hCO glutamatergic neuron Ontologiya (GO) isesengura ryijambo ryo gutungisha.Umurongo utudomo werekana aq agaciro ka 0.05. i, UMAP ishusho yubwoko bwakagari ka GluN muri t-hCO ukoresheje label yoherejwe kuva kuri 22 snRNA-seq ikuze ya moteri cortex dataset. CT - selile corticothalamic, ET - selile zidasanzwe, IT - selile imbere ya telencephalic, NP - hafi ya projection.
Twahise dusuzuma cytoarchitecture hamwe na selile igizwe na t-hCO. Antibody yanduza ingirabuzimafatizo ya endoteliyale yerekanye imitsi hamwe na t-hCO, mugihe irangi rya IBA1 ryerekanaga ko hari microclia yimbeba mugihe cyose (Ishusho 1f hamwe namakuru yagutse, Ishusho 3c, d). Immunostaining yerekanye antigen ya kirimbuzi yumuntu (HNA) ingirabuzimafatizo nziza zerekana PPP1R17 (cortical progenitor), NeuN (neuron), SOX9 na GFAP (selile-glial selile) cyangwa PDGFRα (oligodendrocyte progenitor) (Ishusho 1f). Kugira ngo twige ibice bigize selile ya t-hCO kumurongo umwe umwe, twakoze urutonde rumwe rwa RNA rukurikirana (snRNA-seq) nyuma y amezi agera kuri 8 yo gutandukana. Kurungurura byinshi no kuvanaho nuclei yimbeba byatanze amakarita 21.500 yo mu rwego rwo hejuru yerekana ikarita ya kirimbuzi ya kimuntu (Ishusho 1g hamwe namakuru yagutse, Ishusho 4a, b). Uburyo bwo kwerekana ibimenyetso bisanzwe byerekana ubwoko bwama selile bwerekanye amatsinda yibyiciro byingenzi bya cortical selile, harimo na neuron yimbitse kandi itagaragara ya glutamatergic neuron, izenguruka progenitor, oligodendrocytes, hamwe numurongo wa astrocyte (Ishusho 1g, amakuru yagutse, Ishusho 4c, hamwe nimbonerahamwe 3). Ubudahangarwa bwa SATB2 na CTIP2 bwerekanye ko nubwo hariho ubwoko bwa cortical subtypes, t-hCO itagaragaje ibyiciro bisobanutse neza (amakuru yagutse, Ishusho 3a). icyiciro cyahujwe na snRNA-seq hCO yabyaye ibyiciro byingirabuzimafatizo bisa, usibye bake, harimo kuba nta oligodendrocytes idahari ndetse no kuba neurone ya GABAergic, ishobora kwerekana ibyatangajwe mbere mubihe byiza bya vitro kumasemburo ya progenitor15 (amakuru yagutse, Ishusho 4f - i hamwe nimbonerahamwe ya 4). Isesengura ryerekana imiterere ya gene ryagaragaje itandukaniro rinini muri neuron ya glutamatergic hagati ya t-hCO na hCO (Imbonerahamwe yinyongera 5), ​​harimo no gukora genes zifitanye isano no gukura kwa neuronal nko kwerekana ibimenyetso bya synaptic, dendritic localisation, hamwe nigikorwa cyumuyoboro wa voltage (Ishusho 1h nimbonerahamwe 5). imbonerahamwe 6). Kubera iyo mpamvu, cortical glutamatergic t-hCO neuron yerekanaga gukura kwihuse.
Kugira ngo tumenye niba izo mpinduka zanditswemo muri t-hCO zifitanye isano n’itandukaniro ry’imiterere hagati ya hCO muri vitro na t-hCO muri vivo, twongeye kubaka ibyiciro bihujwe na biocytine yuzuye hCO na hCO mu bice bikaze nyuma y’amezi 7-8 yo gutandukana. HCO neuron (Ishusho 2a). neuron ya t-hCO yari nini cyane, ifite inshuro 1.5 diametre ya soma, ikubye kabiri dendrite, kandi muri rusange kwiyongera inshuro esheshatu muburebure bwa dendritic ugereranije na vitro hCO (Ishusho 2b). Twongeyeho, twabonye ubwinshi bw’umugongo wa dendritic muri neurone ya t-hCO kuruta muri neurone ya hCO (Ishusho 2c). Ibi birerekana ko neurone ya t-hCO ihura cyane na dendritic no gushinga amashami, ibyo, hamwe no gukomeza gukwirakwiza ingirabuzimafatizo, bishobora kugira uruhare runini mu gukura kwa t-hCO nyuma yo guhindurwa (Ishusho 1d hamwe n’amakuru yagutse Ishusho 1f). Ibi byaduteye gukora iperereza kumiterere ya electrophysiologique. Ubushobozi bwa Membrane bwikubye inshuro umunani (amakuru yaguwe, Igicapo 8d), ubushobozi bwa reta ya reta ya membrane yarushijeho kuba hyperpolarize (hafi mV 20), kandi inshinge zubu zatumaga umuvuduko mwinshi mwinshi muri neurone ya t-hCO kuruta muri neurone ya hCO. muri vitro (Igishusho 2d), e), ijyanye nibintu binini kandi binini biranga morfologiya ya t-hCO. Byongeye kandi, inshuro zibyabaye kuri postynaptic yibyabaye (EPSC) byari hejuru cyane muri neuron ya t-hCO (Igishusho 2f), byerekana ko ubwiyongere bwubwinshi bwumugongo wa dendritic bwagaragaye muri neuron ya t-hCO bujyanye no gushimisha imikorere. gusambana. Twemeje imiterere idakuze ya neuron ya hCO muri vitro twandika glutamatergic neuron (amakuru yagutse, Ishusho 6a-c).
a, kwiyubaka 3D ya biocytine yuzuye hCO na t-hCO neuron nyuma y'amezi 8 itandukanye. b, Umubare wibintu biranga morphologie (n = 8 hCO neuron, n = 6 t-hCO neuron; ** P = 0.0084, * P = 0.0179 na *** P <0.0001). b, Umubare wibintu biranga morphologie (n = 8 hCO neuron, n = 6 t-hCO neuron; ** P = 0.0084, * P = 0.0179 na *** P <0.0001). б, количественная оценка морфологических признаков (n = 8 b, ingano yimiterere ya morfologiya (n = 8 neuron ya hCO, n = 6 t-hCO neuron; ** P = 0.0084, * P = 0.0179, na *** 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 *** 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 *** P <0.0001 ）。 б, количественная оценка морфологических признаков (n = 8 b, ingano yimiterere ya morfologiya (n = 8 neuron ya hCO, n = 6 t-hCO neuron; ** P = 0.0084, * P = 0.0179, na *** P <0.0001).c, kongera kubaka 3D amashami ya dendritic ya hCO na t-hCO nyuma y'amezi 8 atandukanye. Inyenyeri zitukura zerekana umugongo wa dendritic. Ubwinshi bwumugongo wa Dendritic (n = 8 hCO neuron, n = 6 t-hCO neuron; ** P = 0.0092). d, Umubare wibishobora kuruhuka (n = 25 hCO neuron, n = 16 t-hCO neuron; *** P <0.0001). d, Umubare wibishobora kuruhuka (n = 25 hCO neuron, n = 16 t-hCO neuron; *** P <0.0001). d. d, kuruhuka membrane ishobora kugereranywa (n = 25 hCO neuron, n = 16 t-hCO neuron; *** 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, kuruhuka membrane ishobora kugereranywa (n = 25 hCO neuron, n = 16 t-hCO neuron; *** P <0.0001). e. e. e. e, ubushobozi bwibikorwa byongeye kurasa muri hCO na t-hCO biterwa no kwiyongera kwubu no kugereranya igipimo ntarengwa cyo kurasa (n = 25 hCO neuron, n = 16 t-hCO neuron; *** 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 = 25 个 hCO 神经元， n = 16 个 t-hCO 神经元； *** P <0.0001 ）。 E ， 通过 增加 电流 注入 的 co hco 和 t-hco 重复 电位 放电 ， 以及 最 大 的 量化 （（ n = 25 个 hco 神经 ， n = 16 个 t-hco 神经 ； *** p <0.0001）。 e. e, kurasa inshuro nyinshi ibikorwa bya hCO na t-hCO biterwa no kongera amasoko agezweho no kugereranya igipimo ntarengwa cyo kurasa (n = 25 hCO neuron, n = 16 t-hCO neuron; *** P <0.0001). f. f. f, спонтанные EPSC (sEPSC) в. f. 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 神经元， n = 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 (sEPSCs) ，以及突触事件频率的量匼（ n = 25 hCO 神率的量匼（ n = 25 hCO 神率的量匼（ n = 25hCO P <0.0001）. f, спонтанные EPSC (sEPSC) в. f.Kuri bf, hCO na t-hCO kumurongo 1208-2 byakuwe mubice bimwe byo gutandukanya byakomezaga kubangikanya. g, Gene yashyizeho isesengura ryitunganyirizwa (ikizamini cya Fisher uruhande rumwe) rwa genes zagenzuwe cyane (zahinduwe P <0.05, impinduka zikubye> 2, zigaragara byibuze 10% bya nuclei) muri t-hCO glutamatergic neuron ugereranije na neuron ya hCO glutamatergic hamwe na gene ya gen-reaction ya mbere ya ERG hamwe nubushakashatsi bwakozwe na virusi ya LRG bwerekanwe na vivo. g, Gene yashyizeho isesengura ryitunganyirizwa (ikizamini cya Fisher uruhande rumwe) rwa genes zagenzuwe cyane (zahinduwe P <0.05, impinduka zikubye> 2, zigaragara byibuze 10% bya nuclei) muri t-hCO glutamatergic neuron ugereranije na neuron ya hCO glutamatergic hamwe na gene ya gen-reaction ya mbere ya ERG hamwe nubushakashatsi bwakozwe na virusi ya LRG bwerekanwe na vivo. گ сравнению с глута Муергическ минимронами hCO наб на башов как раннего (ERG), tak и позднего (LRG) яков, vitro17. g, isesengura ryogukungahaza gene (ikizamini kimwe cya Fisher umurizo umwe) wa gen hamwe nigikorwa gikomeye (cyahinduwe P <0.05, impinduka zikubye> 2, imvugo byibuze 10% ya nuclei) muri t-hCO glutamatergic neuron ugereranije na hCO glutamatergic neuron igizwe na genoside ya mbere ya ERG na nyuma ya LRG. 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 Umurobyi 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 后 p <0.05 ， 倍数> 2 ， 至少 至少 10% 的 细胞 核中 表达） 的 分析 isher g r r r r r r r基因组 16 和 神经元 神经元 17 中 中 17 中 17 的人类特异性 LRG。 گ Ти Фи Каа g, t-hCO glutamatergic neuron yagenzuwe cyane ugereranije na neuron ya hCO glutamatergic (yahinduwe P <0.05, ihinduka ryikubye> 2, byibuze 10% ibisubizo byambere (ERG) hamwe nisesengura ryitondewe rya gene (igeragezwa rimwe rya Fisher) ibisubizo biterwa nibikorwa bya gen (LRGs) byagaragaye mumbeba za vivo16 no muri vitro neuron.17 Umuntu wihariye LRGs.Umurongo utudomo werekana Bonferroni-yakosowe P agaciro ka 0.05. h, imvugo ya GluN (pseudo-pake no gupima buri gene) yagenzuwe cyane muri snRNA-seq yigana genes ya LRG muri neuron ya t-hCO glutamatergic. i, immunostaining yerekana imvugo ya SCG2 muri t-hCO (hejuru) na hCO (hepfo) neuron. Imyambi yera yerekana selile ya SCG2 +. Umwanya muto, 25 µm. Ibyatanzwe bigaragazwa nkibisobanuro iation gutandukana bisanzwe.
Hashingiwe ku bikorwa byiyongereye bya t-hCO byagaragaye mu bice bya vivo, snRNA-seq yerekanye ibikorwa bishingiye ku bikorwa byo kugenzura inyandiko-mvugo ya gene muri t-hCO ugereranije na hCO muri vitro. Glutamatergic t-hCO neuron yerekanaga urwego rwinshi rwa gen igenga ibikorwa bitinze (Ishusho 2g, h), wasangaga mubushakashatsi bwibanze ku mbeba na neurone yabantu 16,17. Kurugero, BDNF18, SCG2, na OSTN, gen-progaramu yihariye igenga ibikorwa, yerekanaga imvugo yiyongera muri neuron ya t-hCO ugereranije na neurone ya hCO (Ishusho 2g-i). Niyo mpamvu, neuron ya t-hCO yerekanaga imiterere yo gukura ugereranije na neurone ya hCO ukoresheje transcriptional, morphologie, hamwe nisesengura ryimikorere.
Kugirango turusheho gusuzuma ishyirahamwe ryo gukura kwa t-hCO hamwe niterambere ryubwonko bwabantu, twakoze igereranya ryerekana ubwoko bwimyanya ndangagitsina nubwoko bwabakuze 19,20 hamwe nabakuze 21,22 hamwe namakuru menshi yerekeye imiterere ya gene cortique 23 mugihe cyiterambere (amakuru yagutse, Ishusho 5). ). hamwe nakazi kashize 24, isi yose ya hCO na t-hCO imiterere yo gukura mumezi 7-8 yo gutandukana irahuza cyane mugihe cyiterambere rya vivo kandi ihwanye cyane nubuzima bwatinze (Data Data Data 5a). Ikigaragara ni uko twabonye ubwiyongere bw'inyandiko mvugo muri t-hCO ugereranije na hCO ihuye n'imyaka, hamwe no gukora transcriptome ifitanye isano na synaptogenezi, astrogenezi, na myelination (amakuru yagutse, Ishusho 5b-d). Ku rwego rwa selire, twabonye ibimenyetso byerekana ubwoko bworoshye bwa cortex muri t-hCO, hamwe na cluster ya glutamatergic neuron yuzuzanya na L2 / 3, L5, na L6 subtypes (Ishusho 1i). Ibinyuranye, cluster ihuza hagati ya glutamatergic t-hCO neuron na cortical cortical neuron yari mike cyane mugihe cyo gutwita (amakuru yagutse, Ishusho 5e-j). Kugirango tumenye niba neurone ya t-hCO isa na neurone ya nyuma ya muntu nyuma yo kubyara, twakoze amajwi ya electrophysiologique hamwe no kwiyubaka kwa anatomique ya neuron ya L2 / 3 ya piramide mu bice bikarishye bya cortex yumuntu (amakuru yagutse, Ishusho 7a). Imiterere ya electrophysiologique ya L2 / 3 ya piramide ya neuron yari imeze nka t-hCO pyramidal neuron (amakuru yagutse, Ishusho 7e). Morphologique, L2 / 3 neuron ziva mubyitegererezo byabantu nyuma yo kubyara zasa cyane na t-hCO kuruta hCO, nubwo selile L2 / 3 yari ndende, yarimo amashami menshi muri rusange, kandi ifite ubwinshi bwumugongo (Ishusho 3g hamwe namakuru yagutse, Ishusho 7b-). G).
a, guhinduranya hCO yakozwe no kugenzura hamwe na TS hiPS imirongo ya selile mu mbeba zivuka. b, 3D kwiyubaka kwa biocytine yuzuye t-hCO neuron nyuma y'amezi 8 yo gutandukana. c, ingano yuburebure bwa dendritic (n = 19 igenzura neuron, n = 21 TS neuron; ** P = 0.0041). d, amashami ya dendritic yubatswe na 3D kuva kugenzura na TS t-hCO mumezi 8 yo gutandukana, no kugereranya ubwinshi bwumugongo wa dendritic (n = 16 igenzura neuron, n = 21 TS neuron, *** P <0.0001). d, amashami ya dendritic yubatswe na 3D kuva kugenzura na TS t-hCO mumezi 8 yo gutandukana, no kugereranya ubwinshi bwumugongo wa dendritic (n = 16 igenzura neuron, n = 21 TS neuron, *** P <0.0001). d. D. d ，分化 8 个月时对照和 TS t-hCO 的 3D 重建树突分支，以及树突棘密度的量化（ n = 16 个对照神经元， n = 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 树突棘 密度 量化 （n = 16 个 神经 元 ， n = 21 个 ts 神经 ， *** p <0.0001 ）。 d. d, kwiyubaka kwa 3D kugenzura amashami ya dendritic na TS t-hCO mumezi 8 yo gutandukanya no kugereranya ubwinshi bwumugongo (n = 16 igenzura neuron, n = 21 TS neuron, *** P <0.0001).Inyenyeri zitukura zerekana umugongo wa dendritic. e, EPSCs ubwayo igenzura na TS t-hCO neuron nyuma y'amezi 8 yo gutandukana. f. g, Scholl isesengura rya TS no kugenzura neuron muri hCO na t-hCO. Imirongo ya dashe yerekana neuron yumuntu L2 / 3 nyuma yo kubyara pyramidal yo kugereranya (n = 24 igenzura t-hCO neuron, n = 21 TS t-hCO neuron, n = 8 igenzura neurone ya nCO, na n = 7 TS hCO neuron). Ibyatanzwe bigaragazwa nkuburyo ± gutandukana bisanzwe
Ubushobozi bwa t-hCO bwo kwigana imiterere yimikorere nimikorere ya neuron ya cortex yumuntu kurwego rwo hejuru byaduteye gushakisha niba t-hCO ishobora gukoreshwa mugutahura fenotipi yindwara. Twibanze kuri TS, indwara ikomeye ya neurodevelopmental yatewe no kunguka-imikorere-ihindagurika muri gene ikora CaV1.2, itangiza inyandiko-mvugo ishingiye kuri gene muri neuron. Twabonye hCO kubarwayi batatu ba TS bitwaje gusimburwa cyane (p.G406R) hamwe nubugenzuzi butatu (Ishusho 3a). Nyuma yo guhindurwa, twasanze morphologie dendritic yahinduwe muri neurone ya TS ugereranije nubugenzuzi (Igishusho 3b hamwe namakuru yagutse, ishusho ya 8a, b), hiyongereyeho inshuro ebyiri umubare wa dendrite yibanze ndetse no kwiyongera muri rusange kugabanuka no kugabanuka muri rusange uburebure bwa dendritic (Ishusho 3c namakuru yagutse, Ishusho 8c). Ibi byari bifitanye isano no kwiyongera k'uruti rw'umugongo no kwiyongera kwa EPSC zidasanzwe muri TS ugereranije no kugenzura neuron (Ishusho 3d - f hamwe namakuru yagutse, Ishusho 8g). Isesengura ryakozwe ryagaragaje uburyo ishami rya dendritic ridasanzwe muri t-hCO TS ugereranije n’ubugenzuzi, ariko ntabwo riri muri vitro TS hCO mu cyiciro kimwe cyo gutandukanya (Ishusho 3g). Ibi bihuye na raporo zacu zabanjirije zerekana ibikorwa biterwa no kugabanuka kwa dendritic muri TS kandi bikagaragaza ubushobozi bwuru rubuga rwo kwimura indwara ya vivo.
Twahise tubaza urugero selile t-hCO yinjizamo imikorere yimbeba S1. S1 mu nzoka yakira ibyinjira bikomeye bya synaptique biva mu mpande zombi za ventrale basal na inyuma ya thalamic nuclei, hamwe na moteri ya moteri hamwe na cortices ya somatosensory ya kabiri, hamwe na S1 itandukanijwe (Ishusho 4a). Kugirango tugarure imiterere yimbere, twanduye hCO virusi yibisazi-dG-GFP / AAV-G hanyuma twimura hCO mumbeba ya S1 nyuma yiminsi 3. Twabonye imvugo yuzuye ya GFP muri neuron ya S1 itabogamye S1 na ventrale basal ganglia nyuma yiminsi 7-14 nyuma yo guterwa (Ishusho 4b, c). Byongeye kandi, antibody yerekana marike ya thalamic netrin G1 yerekanaga ko t-hCO ihari (t. 4d, e). Kugirango dusuzume niba ibyo afferent projection ishobora gutanga ibisubizo bya synaptic muri selile t-hCO, twakoze selile zose zafashwe kuva selile zabantu mubice bikarishye bya thalamocortical. Gukangura amashanyarazi ya imbeba S1, capsule y'imbere, ibintu byera, fibre hafi ya t-hCO cyangwa optogenetike ya opsin-yerekana thalamic iherezo muri t-hCO itera EPSCs itinda ryihuse muri neuron ya t-hCO ihura na antagonist ya AMPA reseptor NBQX. (Igishusho 4f, g hamwe namakuru yagutse, Igicapo 9a - g). Aya makuru yerekana ko t-hCO yinjijwe muburyo bwubwonko bwimbeba kandi ikaba ishobora gukoreshwa ningingo zakira imbeba.
a, Igishushanyo mbonera cyibisazi bikurikirana. b, GFP n'imvugo yihariye ya STEM121 hagati ya t-hCO nimbeba yubwonko bwubwonko (panne yo hejuru). Herekanwe kandi imvugo ya GFP mumbeba itabogamye ya basal nucleus (VB) (ibumoso ibumoso) na S1 (iburyo hepfo). Umwanya munini, 50 µm. Ibibanza bitukura byerekana uduce twubwonko aho amashusho yafatiwe. c, ingano ya selile yerekana GFP (n = imbeba 4). d, e - Netrin G1 + thalamic terminal muri t-hCO. d yerekana igice cya coronale kirimo t-hCO na nuclei ya VB. Umwanya munini, mm 2. e yerekana Netrin G1 na STEM121 imvugo muri t-hCO (ibumoso) na VB (iburyo) neuron. Umwanya munini, 50 µm. Umurongo utudomo wa orange werekana umupaka wa t-hCO. f. EPSC amplitude hamwe na NBQX (n = 6 S1 neuron, * P = 0.0119; na n = 6 imbere ya capsule neuron, ** P = 0.0022) (hagati). Ijanisha rya neuron ya t-hCO yerekana EPSC mugusubiza ingufu z'amashanyarazi S1 (f) cyangwa capsule y'imbere (g) (iburyo). ACSF, ubwonko bwa cerebrospinal fluid. h, igishushanyo mbonera cyubushakashatsi bwa 2P (ibumoso). Kugaragaza GCaMP6s muri t-hCO (hagati). Umwanya munini, 100 µm. Fluorescence yatinze GCaMP6s (iburyo). i, Z-amanota yibikorwa bya fluorescence. j, igishushanyo mbonera cyo gukangura ubwanwa. k, z-amanota 2P fluorescence yinzira murugero rumwe, ihujwe no gutandukana na whisker mugihe cya zeru (umurongo ucagaguye) murugero selile. l, abaturage-bagereranije z-amanota y'ibisubizo by'utugari twose twahujwe no gutandukana na whisker mugihe cya zeru (umurongo ucagaguye) (umutuku) cyangwa ibyashizweho igihe cyagenwe (imvi). m. Igishushanyo mbonera cyubushakashatsi kuri marike optique. n, Umuyoboro wa voltage uva kurugero t-hCO selile mugihe ubururu bwa laser cyangwa ubururu bwa whisker. Imyambi itukura yerekana imitwe yambere iterwa numucyo (hejuru) cyangwa iterwa na whisker deflection (hepfo). Igicucu kijimye cyerekana ibihe byo gutandukana. o, Impinga yumucyo mwinshi hamwe na whisker deflection ibisubizo. p, imitwe yikigereranyo kimwe, ihujwe no gutandukana kwa whiskers muri selile yintangarugero. 0 yerekana gutandukana kwa whisker (umurongo ucagaguye). Q. r, Umubare wibice bifotora byahinduwe cyane na whisker deviation (n = imbeba 3) (ibumoso). Impinga z-amanota yatinze (n = imbeba 3; n = 5 (icyatsi kibisi), n = 4 (icyatsi kibisi cyijimye), na n = 4 (cyan) whisker deflection modulation modulation) (iburyo). Ibyatanzwe bigaragazwa nkibisobanuro iation gutandukana bisanzwe
Twahise tubaza niba t-hCO ishobora gukoreshwa nogukangura amarangamutima muri vivo. Twahinduye hCO yerekana ibimenyetso bya calcium ya genetike ya GCaMP6 mu mbeba za S1. Nyuma yiminsi 150, twakoze fibre fotometrie cyangwa amashusho abiri ya calcium ya calcium (Ishusho ya 4h hamwe namakuru yagutse, Ishusho 10a). Twabonye ko selile t-hCO yerekanaga ibikorwa bya rhythmic ibikorwa (Igicapo 4i, Amakuru Yaguwe, Ishusho 10b na Video yinyongera 1). Kugirango turangize ibikorwa bya t-hCO, twakoze amajwi adasanzwe ya electrophysiologique yafashwe mu mbeba zatewe aneste (imibare yagutse, Ishusho 10c-f). Twakoze stereotaxic coordinate kuva mumashusho ya MRI; bityo, ibi bice byanditse byerekana neuron yumuntu, nubwo electrophysiologiya yonyine itemerera ubwoko bwinkomoko kugenwa. Twabonye guhuza ibikorwa (guhuza amakuru, Ishusho 10d). Ibisasu byamaze ms 460 kandi bitandukanijwe nigihe cyo guceceka kingana na s 2 (amakuru yagutse, Ishusho 10d, e). Ibice byabantu barashe impuzandengo yikigereranyo cya gatatu kuri buri guturika, bingana na 73% byibice byanditse kuri buri guturika. Ibikorwa bya buri gice byari bifitanye isano cyane, kandi ayo masano yari hejuru ugereranije n’ibice byagaragaye mu nyamaswa zitakingiwe byanditswe mu bihe bimwe (amakuru yagutse, Ishusho 10f). Kugirango turusheho kuranga ibisubizo bya spike ya neuron yamenyekanye ikomoka kubantu, twakoze ubushakashatsi bwo kumurika urumuri ku mbeba zatewe aneste zatewe hamwe na hCO zerekana umuyoboro wa cation umuyoboro wa rhodopsin 2 (hChR2), unyuzamo t-hCO neuron imenyekana-bitinze (munsi ya ms 10) kugirango dusubize urumuri rwubururu (Ishusho 4m - o). neuron ya t-hCO yerekanaga ibikorwa byinshi bidatinze kuri radiyo isa nkiyagaragaye mu mashusho ya calcium, ndetse na electrophysiologique yafashwe muri t-hCO hatabayeho ibimenyetso byerekana urumuri (amakuru yagutse, Ishusho 10c-g). Nta gikorwa cyizana cyagaragaye mubyiciro bijyanye na hCO yanditswe muri vitro. Kugirango tumenye niba t-hCO ishobora gukoreshwa nogukangura ibyiyumvo, twahinduye muri make imbeba yimbeba kure ya t-hCO (Igishusho 4j, m hamwe namakuru yagutse, Ishusho 10h, k). Dukurikije ubushakashatsi bwabanjirije 8,10, agace kamwe ka selile t-hCO kagaragaje ibikorwa byiyongereye mugusubiza icyuho cya whisker, kitagaragaye mugihe amakuru yagereranijwe na kashe zidasanzwe (Ishusho 4k - q hamwe namakuru yagutse, Ishusho 10h - q). Mubyukuri, hafi 54% bya opto-yanditseho ibice bimwe byerekanaga umuvuduko wiyongereye cyane nyuma yo gukanguka, kugera kuri ms 650 (Ishusho 4r). Ufatiye hamwe, aya makuru yerekana ko t-hCO yakira inyongeramusaruro ikwiye kandi ishobora gukoreshwa nibidukikije.
Twakoze iperereza niba t-hCO ishobora gukora imirongo yimbeba kugirango igenzure imyitwarire. Twabanje gukora iperereza niba axon ya t-hCO neurons umushinga mubice bikikije imbeba. Twanduye hCO na lentivirus irimo kodegisi ya HChR2 ihujwe na EYFP (hChR2-EYFP). Nyuma yiminsi 110, twabonye imvugo ya EYFP mukarere ka cortique itabogamye, harimo cortices yunvikana, moteri, na somatosensory, ndetse no mubice byo munsi, harimo striatum, hippocampus, na thalamus (Ishusho 5a). Kugirango tumenye niba ibyo bishushanyo mbonera bishobora gutanga ibisubizo bya synaptic mu ngirabuzimafatizo, twahisemo gukora t-hCO ingirabuzimafatizo zerekana hChR2-EYFP twandika selile cerebral cortex selile mubice byubwonko bukaze. Gukora axon ya t-hCO hamwe nurumuri rwubururu byateje EPSCs mugihe gito cyimbeba ya pyramidal cortex neuron, zahagaritswe na NBQX (Ishusho 5b - g). Byongeye kandi, ibisubizo birashobora guhagarikwa na tetrodotoxine (TTX) hanyuma bigasubizwa na 4-aminopyridine (4-AP), byerekana ko byatewe no guhuza monosynaptike (Ishusho 5e).
a, Igishushanyo mbonera cya axon ikurikirana (ibumoso). imvugo ya t-hCO EYFP (iburyo). Umwanya munini, 100 µm. A1, cortex yo kumva, ACC, imbere ya cingulate cortex, d. striatum, striatum dorsal, HPC, imvubu; Diaphragm, septum kuruhande, mPFC, cortex yo hagati yambere, piri, cortex ya piriform, v. Ibibanza bitukura byerekana uduce twubwonko aho amashusho yafatiwe. b, Igishushanyo mbonera cyubushakashatsi bwo gukangura. c. e, f. g, ubukererwe bwibisubizo biterwa nurumuri rwubururu muri selile yimbeba (n = 16 selile); utubari dutambitse twerekana impuzandengo (7.13 ms) (ibumoso). Amplitude ya EPSCs iterwa n'umucyo yanditswe hamwe cyangwa idafite NBQX (n = 7 selile; *** P <0.0001) (hagati). Amplitude ya EPSCs iterwa n'umucyo yanditswe hamwe cyangwa idafite NBQX (n = 7 selile; *** P <0.0001) (hagati). Амплитуда в Канванных светом EPSC, марегстрированных с или ути NBQX (n = 7 клеток; *** P <0,0001) (в центре). Amplitude ya EPSCs iterwa numucyo yanditswe hamwe cyangwa idafite NBQX (n = 7 selile; *** P <0.0001) (hagati).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 в Канванных светом EPSC, марегстрированных с или ути NBQX (n = 7 клеток; *** P <0,0001) (в центре). Amplitude ya EPSCs iterwa numucyo yanditswe hamwe cyangwa idafite NBQX (n = 7 selile; *** P <0.0001) (hagati).Ijanisha rya selile yimbeba yerekana EPSCs isubiza urumuri rwubururu (iburyo). h, Igishushanyo mbonera cyibikorwa byimyitwarire. d0, umunsi 0. i. Imikorere yinyamaswa ntangarugero kumunsi wa 1 (ibumoso) cyangwa umunsi wa 15 (iburyo) wamahugurwa. Umubare ugereranije wa licks wakozwe kumunsi wa 1 (ibumoso) cyangwa kumunsi wa 15 (hagati iburyo) (n = ibigeragezo byumucyo wubururu 150, n = ibigeragezo bitukura 150; *** P <0.0001). Umubare ugereranije wa licks wakozwe kumunsi wa 1 (ibumoso) cyangwa kumunsi wa 15 (hagati iburyo) (n = ibigeragezo byumucyo wubururu 150, n = ibigeragezo bitukura 150; *** P <0.0001). Среднее количесто облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 испытаний с синим светом, n = 150 испытаний с красным светом; *** P <0,0001). Ikigereranyo cya licks zakozwe kumunsi wa 1 (ibumoso) cyangwa kumunsi wa 15 (hagati iburyo) (n = ibigeragezo byurumuri rwubururu 150, n = ibigeragezo bitukura 150; *** 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 количесто облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 испытаний с синим светом, n = 150 испытаний с красным светом; *** P <0,0001). Ikigereranyo cya licks zakozwe kumunsi wa 1 (ibumoso) cyangwa kumunsi wa 15 (hagati iburyo) (n = ibigeragezo byurumuri rwubururu 150, n = ibigeragezo bitukura 150; *** P <0.0001).Cumulative licks kubigeragezo bitukura nubururu kumunsi wa 1 (hagati ibumoso) cyangwa kumunsi wa 15 (iburyo). NS, ntabwo ari ngombwa. j, k, Ibiranga imyitwarire yinyamaswa zose zatewe na t-hCO zerekana hChR2-EYFP (j) cyangwa kugenzura fluorophore (k) kumunsi wa 1 cyangwa 15 (hChR2-EYFP: n = imbeba 9, ** P = 0.0049; kugenzura: n = 9, P = 0.1497). l, Ubwihindurize bw'amanota akunda (n = 9 hChR2, n = 9 kugenzura; ** P <0.001, *** P <0.0001). l, Ubwihindurize bw'amanota akunda (n = 9 hChR2, n = 9 kugenzura; ** P <0.001, *** P <0.0001). , l, Ubwihindurize bw'amanota akunda (n = 9 hChR2, n = 9 igenzura; ** 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, Ubwihindurize bw'amanota akunda (n = 9 hChR2, n = 9 igenzura; ** P <0.001, *** P <0.0001).m, imvugo ya FOS mugusubiza optogenetike ya t-hCO muri S1. Amashusho ya FOS imvugo (ibumoso), no kugereranya (n = 3 kuri buri tsinda; * P <0.05, ** P <0.01 na *** P <0.001) (iburyo) birerekanwa. Amashusho ya FOS imvugo (ibumoso), no kugereranya (n = 3 kuri buri tsinda; * P <0.05, ** P <0.01 na *** P <0.001) (iburyo) birerekanwa. Показны изображения Кресрессии FOS (слева) и количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 и *** P <0,001) (справа). Amashusho ya FOS imvugo (ibumoso) no kugereranya (n = 3 kuri buri tsinda; * P <0.05, ** P <0.01, na *** P <0.001) irerekanwa (iburyo).OS 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。OS 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。 Показны изображения Кресрессии FOS (слева) и количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 и *** P <0,001) (справа). Amashusho ya FOS imvugo (ibumoso) no kugereranya (n = 3 kuri buri tsinda; * P <0.05, ** P <0.01, na *** P <0.001) irerekanwa (iburyo).Umwanya munini, 100 µm. Ibyatanzwe bisobanurwa nkibisobanuro error ikosa risanzwe rya BLA, toni yibanze, MDT, dorsomedial thalamic nucleus, PAG, imvi zijimye.
Hanyuma, twabajije niba t-hCO ishobora guhindura imyitwarire yimbeba. Kugirango tugerageze ibi, twimuye hChR2-EYFP-yerekana HCO muri S1, hanyuma nyuma yiminsi 90, dushyira fibre optique muri t-hCO kugirango itange urumuri. Twahise tumenyereza imbeba hamwe na paradigima yahinduwe ikora (Ishusho 5h). Twashyize inyamanswa mucyumba cyipimisha imyitwarire hanyuma dushiraho uburyo butandukanye 5 ubururu bwa kabiri (473 nm) nubururu butukura (635 nm). Inyamaswa zabonye igihembo cyamazi iyo zirigata mugihe cyo gukurura urumuri rwubururu ariko ntirurigata mugihe cyumucyo utukura. Ku munsi wa mbere wamahugurwa, inyamaswa zerekanye itandukaniro ryo guswera iyo zishushanijwe numucyo wubururu cyangwa umutuku. Nyamara, kumunsi wa 15, inyamaswa zatewe hamwe na hCO zerekana hChR2-EYFP zerekanaga guswera cyane iyo zishushe numucyo wubururu ugereranije no gukurura urumuri rutukura. Izi mpinduka mu myitwarire yo guswera ntizagaragaye mu nyamaswa zigenzura zatewe na hCO zigaragaza fluorophore igenzura (igipimo cyo gutsinda: hChR2 89%, EYFP 0%, Ishusho 5i-1 na Video y'inyongera 2). Aya makuru yerekana ko selile t-hCO ishobora gukora imbeba ya neuron kugirango itere imyitwarire ishaka ibihembo. Kugirango tumenye imbeba ya t-hCO imitsi ishobora kugira uruhare muri izi mpinduka zimyitwarire, twahisemo gukora t-hCO mubikoko byatojwe hanyuma dusarura imyenda nyuma yiminota 90. Immunohistochemie yerekanye imvugo ya poroteyine FOS ishingiye ku bikorwa mu turere twinshi tw’ubwonko tugira uruhare mu myitwarire ishishikaje, harimo cortex yo hagati ya cortex, thalamus medial, na periaqueductal gray gray, ibyo bikaba byaragaragaye haba mu nyamaswa zidateganijwe cyangwa ku nyamaswa. umuceri. 5m). Ufatiye hamwe, aya makuru yerekana ko t-hCO ishobora guhindura imikorere yimbeba neuronal kugirango itware imyitwarire.
Neural organoide igereranya sisitemu itanga icyizere cyo kwiga iterambere ryabantu nindwara muri vitro, ariko bigarukira kubwo kubura amasano hagati yumuzunguruko uba muri vivo. Twateje imbere urubuga rushya aho twimuye hCO muri S1 yimbeba zidakingirwa hakiri kare imbeba nyuma yo kubyara kugirango twige iterambere ryimikorere yabantu nimikorere muri vivo. Twerekanye ko t-hCO itezimbere ubwoko bwakagari bukuze butagaragara muri vitro28 kandi ko t-hCO ihuriweho kandi ikora mubwonko bwimbeba. Kwishyira hamwe kwa t-hCO mu miyoboro y’imitsi y’imitsi byadushoboje gushyiraho isano hagati yimikorere ya selile yumuntu no kwiga imyitwarire yinyamaswa, byerekana ko t-hCO neuron ishobora guhindura imikorere yimbeba ya neuronal kugirango itware ibisubizo byimyitwarire.
Ihuriro dusobanura rifite ibyiza byinshi kurenza ubushakashatsi bwibanze ku kwimura ingirabuzimafatizo mu bwonko bwimbeba. Ubwa mbere, twimuye hCO muri cortex itera imbere yimbeba za nyuma yo kubyara, zishobora koroshya guhuza ibikorwa no gukora. Icya kabiri, gukurikirana t-hCO MRI byadushoboje kwiga ibihingwa no gukura kwinyamaswa nzima, bidufasha gukora ubushakashatsi bwigihe kirekire bwinyamanswa no kwerekana ubwizerwe bwimirongo myinshi ya hiPS. Hanyuma, twahinduye organoide idahwitse, aho guhagarika ingirabuzimafatizo imwe yonyine, idashobora kwangiza ingirabuzimafatizo z'umuntu kandi irashobora guteza imbere kwishyira hamwe no kubyara neurone ya cortex yabantu mubwonko bwimbeba.
Turemera ko nubwo iterambere ryakozwe muri uru rubuga, imbogamizi z’agateganyo, ahantu hatandukanye, ndetse n’ibinyabuzima byambukiranya imipaka bibuza ishyirwaho ry’imitsi y’imitsi y’abantu bafite ubudahemuka bukabije, ndetse na nyuma yo guterwa hakiri kare iterambere. Kurugero, ntibisobanutse niba ibikorwa byizana bigaragara muri t-hCO byerekana fenotipike yiterambere isa nigikorwa cyinjyana yagaragaye mugihe cyiterambere rya cortique, cyangwa niba biterwa no kubura ubwoko bwimikorere ya selile buboneka muri t-hCO. Mu buryo nk'ubwo, ntibisobanutse ku buryo kutagira lamination muri t-hCO bigira ingaruka ku guhuza urunigi30. Akazi kazoza kazibanda ku guhuza ubundi bwoko bwimikorere nka microglia yumuntu, selile endothelia yumuntu, hamwe nuburyo butandukanye bwa GABAergic interneurons nkuko bigaragara ukoresheje inteko 6 muri vitro, ndetse no gusobanukirwa uburyo guhuza imitekerereze no gutunganya bishobora kubaho muri t-hCO yahinduwe. urwego rwandikirwa, synaptic nimyitwarire murwego rwakuwe mubarwayi.
Muri rusange, iyi muri vivo platform yerekana umutungo ukomeye ushobora kuzuzanya mugutezimbere ubwonko bwabantu no mubushakashatsi bwindwara. Turateganya ko iyi platform izadufasha kuvumbura udushya twa fenotipike yo mu rwego rwo mu zindi ngirabuzimafatizo zikomoka ku barwayi no kugerageza ingamba nshya zo kuvura.
Twabyaye hCO2.5 muri selile ya HiPS nkuko byasobanuwe mbere. Kugirango utangire umusaruro wa hCO uturutse mu tugari twa hiPS utunzwe no kugaburira ibiryo, ubukoroni butagira ingano bwa selile hiPS bwakuwe mu biryo by’umuco hakoreshejwe disase (0.35 mg / mL) hanyuma byimurirwa mu mico ya plastike ya ultra-low attachment irimo ibyokurya bifite umuco wa hiPS. . Mugihe cyiminsi 5 yambere, hiPS selile ya selile yahinduwe burimunsi hanyuma hiyongeraho dorsomorphine na SB-431542. Ku munsi wa gatandatu mu guhagarikwa, spéroide neural yimuriwe mu mitsi irimo neurobasal-A (10888, Life Technologies), B-27 inyongera idafite vitamine A (12587, Ubuzima bwa Technologies), GlutaMax (1: 100, Ubuzima bwa Technologies), penisiline na streptomycine (1: 100, Ubuzima bwa tekinoloji); . Ku munsi wa gatandatu mu guhagarikwa, spéroide neural yimuriwe mu mitsi irimo neurobasal-A (10888, Life Technologies), B-27 inyongera idafite vitamine A (12587, Ubuzima bwa Technologies), GlutaMax (1: 100, Ubuzima bwa Technologies), penisiline na streptomycine (1: 100, Ubuzima bwa tekinoloji); .Ku munsi wa gatandatu mu guhagarikwa, spéroide neural yimuriwe mu mitsi irimo Neurobasal-A (10888, Life Technologies), B-27 inyongera idafite vitamine A (12587, Ubuzima bwa Technologies), GlutaMax (1: 100, Ubuzima bwa Technologies), penisiline.и ст кромомицин (1: 100, Ikoranabuhanga mu Buzima) и дополнены эпидер кинемигом роста (EGF; 20 нг / igitekerezo; R&D Sisitemu) и na streptomycine (1: 100, Ubuzima bwa Technologies) kandi hiyongeraho ibintu bikura byindwara (EGF; 20 ng / ml; R&D Sisitemu) hamwe na fibroblast yo gukura 2 (FGF2; 20 ng / ml; R&D Sisitemu) kugeza kumunsi wa 24.Kuva ku minsi 25 kugeza 42, ibintu bikomoka mu bwonko biva mu bwonko (BDNF; 20 ng ml-1, Peprotech) na neurotrophine 3 (NT3; 20 ng ml-1, Peprotech) byongewemo hagati, bihindura uburyo buri munsi.6在 悬浮 的 第 6 （（（ 20 ng ml-1 ； r & d Sisitemu） 成 纤维 细胞 生长 2 （fgf2 ； 20 ng ml- 1 Systems Sisitemu ya R&D ）直至第 24 天。 На-6 . Ku munsi wa 6, ihagarikwa rya neurosiporo ryahinduwe ku nyongera irimo neurobasal-A (10888, Ubuzima bwa Technologies), B-27 inyongera idafite vitamine A (12587, Ubuzima bwa Technologie), GlutaMax (1: 100, Ubuzima bwa Technologies), insimburangingo ya penisiline (1: 100, Ubuzima bwa Technologie) hamwe na fibrob 20; (FGF2; 20 ng ml-1) 1; Sisitemu ya R&D) до 24-г дня. Sisitemu ya R&D) kugeza kumunsi wa 24.Kuva ku minsi 25 kugeza 42, ubwonko bukomoka mu bwonko bukomoka mu bwonko (BDNF; 20 ng ml-1, Peprotech) na neurotrophique 3 (NT3; 20 ng ml-1, Peprotech) byongewe kumuco buri munsi. Guhindura hagati.Guhera kumunsi wa 43, hCO yagumishijwe muri neurobasal ituzuye-A igikoresho (NM; 1088022, Thermo Fisher) hamwe nimpinduka ziciriritse buri minsi 4-6. Kugirango ubone hCO mu ngirabuzimafatizo za hiPS zitezimbere mu bihe bitagaburiwe, selile ya hiPS yashyizwemo na Accutase (AT-104, Innovate Cell Technologies) kuri 37 ° C mu minota 7, igabanywa mu ngirabuzimafatizo imwe, hanyuma ishyirwa ku byapa bya AggreWell 800 (34815, STEMCELL Technologies) ku bucucike bwa 3 × 106 ingirabuzimafatizo kuri Y-8; Selleckchem). Nyuma yamasaha 24, itangazamakuru mu mariba ryashyizwe mu majwi no mu bitangazamakuru birimo itangazamakuru rya ngombwa 6 (A1516401, Ubuzima bwa Technologies) ryuzuzwa na dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) na SB-431542 (10 μM; 1614). , Tocrida). Kuva kumunsi wa 2 kugeza 6, Ibyingenzi 6 byingenzi byasimburwaga burimunsi na dorsomorphine no kuzuza SB-431542. Kuva ku munsi wa gatandatu, ihagarikwa rya neurosfera ryimuriwe mu cyerekezo cya neurobasal kandi rikomeza nkuko byasobanuwe haruguru.
Inzira zose z’inyamanswa zakozwe hakurikijwe amabwiriza yo kwita ku nyamaswa zemejwe na komite ishinzwe imiyoborere y’amatungo ya kaminuza ya kaminuza ya Stanford (APLAC). Imbeba zitwite za RNU (rnu / +) zaguzwe (Laboratoire ya Charles River) cyangwa ziracumbikirwa. Inyamaswa zabitswe kumasaha 12 yumucyo-mwijima hamwe nibiryo n'amazi ad libitum. Imbeba zambaye ubusa (FOXN1 - / -) imbeba yimbeba ifite imyaka itatu kugeza kuri irindwi yamenyekanye no gukura kwa whiskers idakuze mbere yo kwica. Ibibwana (igitsina gabo nigitsina gore) byatewe aneste hamwe na 2-3% isoflurane hanyuma bigashyirwa kumurongo wa stereotaxic. Gukandagira igihanga gifite umurambararo wa mm 2-3 hejuru ya S1 byakozwe mugukomeza ubusugire bwa mater mater. Noneho koresha urushinge rwa 30-G (hafi 0,3 mm) hanze ya craniotomy kugirango utobore dura. Noneho shyira HCO kuri parafilm yoroheje 3 × 3 hanyuma ukureho uburyo burenze. Ukoresheje inshinge ya Hamilton ifatanye nurushinge rwa 23 G, 45 °, shushanya witonze hCO mumutwe wa kure cyane. Noneho shyira seringe kuri pompe ya syringe ihujwe nigikoresho cya stereotaxic. Noneho shyira isonga y'urushinge hejuru ya 0.3 mm z'ubugari bwacumuye muri dura (z = 0 mm) hanyuma ugabanye inshinge ya mm2-22 (z = hafi -1,5 mm) kugeza urushinge ruri hagati ya dura mater A. hashyizweho kashe yuzuye. Noneho uzamure syringe hagati yubuso bwa cortical kuri z = -0.5 mm hanyuma utere hCO ku gipimo cya 1-2 µl kumunota. Nyuma yo guterwa inshinge za hCO, urushinge rusubirwamo ku gipimo cya mm 0.2-0.5 ku munota, uruhu ruradodo, kandi ikibwana gihita gishyirwa kumashanyarazi ashyushye kugeza gikize. Inyamaswa zimwe zatewe mu buryo bubiri.
Inzira zose zinyamanswa zakozwe hakurikijwe amabwiriza ya kaminuza ya Stanford yemewe na APLAC. Imbeba (zirenze iminsi 60 nyuma yo guhindurwa) zatewe na anesthesia ya isoflurane 5% hanyuma itera aneste hamwe na 1-3% isoflurane mugihe cyo gufata amashusho. Kugirango ubone amashusho, Tesla 7 yarinze cyane scaneri ya borehole scaneri Bruker (Bruker Corp.) hamwe na moteri mpuzamahanga ya sosiyete ikora amashanyarazi (IECO), icyuma gikingira icyuma cyinjizwamo diameter imbere ya mm 120 (600 mT / m, 1000 T / m / s) hakoreshejwe AVANCE. III, imiyoboro umunani-coil RF hamwe nubushobozi-bwibanze, hamwe na Paravision 6.0.1 iherekeza. Gufata amajwi byakozwe hifashishijwe igicapo cya RF gifite imbaraga zingana na diameter y'imbere ya mm 86 hamwe na bine ya cryo-yakonje ya RF coil kugirango yakire gusa. Axial 2D Turbo-NTIBISANZWE (igihe cyo gusubiramo = ms 2500 ms, echo igihe = 33 ms, impuzandengo 2) hamwe nuduce 16 twafashe, uburebure bwa 0,6-0.8 mm, burimo 256 × 256. Ibimenyetso byakiriwe hifashishijwe coadrature transceiver volumetric RF coil ifite diameter y'imbere ya cm 2 (Rapid MR International, LLC). Hanyuma, koresha ibyubatswe muri Imaris (BitPlane) igereranya ryimikorere yo kugereranya 3D no gusesengura amajwi. Guhinduranya neza byasobanuwe nkimwe aho ibimenyetso byerekana ibimenyetso bya T2 biremereye bya MRI byakorewe mu gice cyimuwe. Kwanga ibihano byasobanuwe nkigishushanyo kidatanga uduce twikimenyetso cya T2 gifite uburemere bwa MRI mu gice cyimuwe. Subcortical t-hCO yakuweho isesengura ryakurikiyeho.
Kugirango ugaragaze neza GCaMP6s muri hCO kumashusho ya calcium ya fotoni ebyiri, selile hiPS yanduye pLV [Exp] -EF1a :: GcaMP6s-WPRE-Puro hakurikiraho guhitamo antibiyotike. Muri make, selile zatandukanijwe na EDTA hanyuma zihagarikwa muri ml 1 ya Essential 8 iciriritse ku bucucike bwa selile zigera ku 300.000 imbere ya polybrene (5 μg / ml) na 15 μl za virusi. Ingirabuzimafatizo zahise zihagarikwa mu minota 60 hanyuma zitera imbuto zingana na selile 50.000 kuri buri riba. Nyuma yo guhurira hamwe, selile zavuwe hamwe na 5-10 μg ml-1 puromycine muminsi 5-10 cyangwa kugeza ubukoloni buhamye bugaragaye. Indwara ikaze ya hCO yakozwe nkuko byasobanuwe5 hamwe na bimwe byahinduwe. Muri make, ohereza umunsi 30-45 hCO muri 1.5 ml Eppendorf microcentrifuge irimo 100 µl yumutima. Noneho hafi 90 µl yikigereranyo ikuweho, 3-6 µl ya titer lentivirus yo hejuru (kuva 0.5 x 108 kugeza 1.2 x 109) yongewe kumuyoboro, hanyuma hCO yimurirwa muri incubator muminota 30. Noneho shyiramo 90-100 µl yo hagati kuri buri tube hanyuma usubize tebes muri incubator ijoro ryose. Umunsi ukurikira, ohereza hCO muburyo bushya bwimitsi mumasahani make. Nyuma yiminsi 7, hCO yimuriwe kumasahani 24 yibirahuri byo hasi kugirango ibone kandi isuzume ubuziranenge bwanduye. pLV [Exp] -SYN1 :: EYFP-WPRE na pLV [Exp] -SYN1 :: hChR2-EYFP-WPRE byakozwe na VectorBuilder. Lentivirus ikoreshwa mubushakashatsi bwinshi kuko yinjijwe muri genome yakira, bigatuma umunyamakuru agaragaza imvugo mumirongo yanduye. Kubakurikirana ibisazi, umunsi wa 30-45 hCO yanduye indwara yindwara-ΔG-eGFP na AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid # 67528, Addgene), yoza neza iminsi 3, hanyuma yinjizwa muri imbeba muri S1 kandi ikomeza muri vivo iminsi 7-14.
Kuri immunocytochemie, inyamaswa zatewe aneste kandi zanduzwa na karubasi hamwe na PBS zikurikirwa na 4% paraformaldehyde (PFA muri PBS; Ubumenyi bwa Electron Microscopy). Ubwonko bwakosowe muri 4% PFA mumasaha 2 cyangwa ijoro ryose kuri 4 ° C, kode yabitswe muri 30% ya sucrose muri PBS mumasaha 48-72, hanyuma ishyirwa muri 1: 1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583, Sakura Finetek) nibice bya coronale byakozwe kuri 30 µm ukoresheje kristu (Leica). Kuri immunohistochemie yibice byimbitse, inyamanswa zashyizwemo PBS, maze ubwonko buratandukanywa kandi bugabanywa kuri 300-400 µm ukoresheje vibratome (Leica) hanyuma ibice bikosorwa hamwe na 4% PFA muminota 30. Hanyuma koroze cyangwa ibice byimbitse byogejwe hamwe na PBS, bifungwa kumasaha 1 mubushyuhe bwicyumba (10% serumu yindogobe isanzwe (NDS) na 0.3% Triton X-100 ivangwa muri PBS) hanyuma ihagarikwa hamwe no gukumira igisubizo kuri 4 ° C. - Incubation Cryosections yashizwemo ijoro ryose kandi ibice byimbitse byashyizwemo iminsi 5. Antibodies yibanze yakoreshejwe ni: anti-NeuN (imbeba, 1: 500; ab104224, abcam) anti-CTIP2 (imbeba, 1: 300; ab18465, abcam), anti-GFAP (urukwavu, 1: 1.000; Z0334, Dako), anti-GFP (inkoko, 1: 1000; GTX13970, GeneTNA), anti-NeuN (urukwavu, 1: 500; ABN78, Millipore), anti-PDGFRA (urukwavu, 1: 200; sc-338, Santa Cruz), anti-PPP1R17 (urukwavu, 1: 200; HPA047819, Atlas Antibodies), anti-RECA-1 (imbeba, 1: 2) 20357-1-AP, Proteintech), anti-SOX9 (ihene, 1: 500; AF3075, R&D Sisitemu), Netrin G1a (ihene, 1: 100; AF1166, R&D Sisitemu), anti-STEM121 (imbeba, 1: 200; Y40410, Takara Bio), anti-SATB2 (imbeba, 1:50 1: 400; ABN904, Millipore) na anti-IBA1 (ihene, 1: 100; ab5076, abcam). Antibodies yibanze yakoreshejwe ni: anti-NeuN (imbeba, 1: 500; ab104224, abcam) anti-CTIP2 (imbeba, 1: 300; ab18465, abcam), anti-GFAP (urukwavu, 1: 1.000; Z0334, Dako), anti -GFP (inkoko, 1: 1.000; GTX13970, GeneTNA); anti-NeuN (urukwavu, 1: 500; ABN78, Millipore), anti-PDGFRA (urukwavu, 1: 200; sc-338, Santa Cruz), anti-PPP1R17 (urukwavu, 1: 200; HPA047819, Atlas Antibodies), anti-RECA-1 (imbeba, 1: 2) 20357-1-AP, Proteintech), anti-SOX9 (ihene, 1: 500; AF3075, R&D Sisitemu), Netrin G1a (ihene, 1: 100; AF1166, R&D Sisitemu), anti-STEM121 (imbeba, 1: 200; Y40410, Takara Bio), anti-SATB2 (imbeba, 1:50 1: 400; ABN904, Millipore) na anti-IBA1 (ihene, 1: 100; ab5076, abcam). Использовались следующие первичные антитела: କାନ-NeuN (мшиные, 1: 500; ab104224, abcam), 1: 1000; Antibodies), һәр-RECA-1 (мышь, 1:50; ab9774, abcam), yin-SCG2 (кролик, 1: 100; 20357-1-AP, Proteintech), 1: 200. Antibodiyite zambere zakoreshejwe ni: anti-NeuN (imbeba, 1: 500; ab104224, abcam), anti-CTIP2 (imbeba, 1: 300; ab18465, abcam), anti-GFAP (urukwavu, 1: 1000; Z0334, Dako), anti-GFP (inkoko, 1: 1000; GTX13970, GeneTNA); abcam), anti-NeuN (urukwavu, 1: 500; ABN78, Millipore), anti-PDGFRA (urukwavu, 1: 200; sc-338, Santa Cruz), anti-PPP1R17 (urukwavu, 1: 200; HPA047819, Antlas Antibodies), anti-RECA-1 (ab 20357-1-AP, Proteintech), anti-SOX9 (ihene, 1: 500; AF3075, R&D Sisitemu), netrin G1a (ihene, 1: 100; AF1166, R&D Sisitemu), anti-STEM121 (imbeba, 1: 200; Y40410, Takara Bio), anti-SATB2 (imbeba, 1:50 1: 400; ABN904, Millipore) na anti-IBA1 (ihene, 1: 100; ab5076, abkam).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 -GF P （鸡， 1: 1.000 ； GTX13970 ， GeneTex ），抗 HNA （小鼠， 1: 200 ； ab191181 ， 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 ， Proteintech ），抗 SOX9 （山羊， 1: 500 ）， G1a （山羊， 1: 100 ； AF1166 ， R & D Sisitemu ），抗 STEM121 （小鼠, 1: 200;U 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 ， Millipore ％弔，抗 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 100 ； 20357-1-AP ， Proteintech ），抗 SOX9 （山羊， 1: 500 ； G1a （山羊， 1: 100 ； AF1166 ， R & D Sisitemu ）頏 1:20 ,;Y40410, Takara Bio), kurwanya SATB2 (imbeba, 1:50; ab51502, abcam), kurwanya GAD65 / 67 (urukwavu, 1: 400; ABN904, Millipore) na anti-IBA1 (ihene, 1: 100; ab5076, abcam)。Antibodies zambere zakoreshejwe ni: anti-NeuN (imbeba, 1: 500; ab104224, abcam), anti-CTIP2 (imbeba, 1: 300; ab18465, abcam), anti-GFAP (urukwavu, 1: 1000; Z0334, Dako). , kurwanya GFP (inkoko, 1: 1000; GTX13970, GeneTex), anti-HNA (imbeba, 1: 200; ab191181, abcam), anti-NeuN (urukwavu, 1: 500; ABN78, Millipore), kurwanya PDGFRA (urukwavu, 1: 200; sc-338, Santa Cruz), 200 anti-PPP1; antibody), anti-RECA-1 (imbeba, 1:50; ab9774, abcam), anti-SCG2 (urukwavu), 1: 100;20357-1-AP, Proteintech, . 20357-1-AP, Proteintech), anti-SOX9 (ihene, 1: 500; AF3075, R&D Sisitemu), Netrin G1a (ihene, 1: 100; AF1166, R&D Sisitemu), anti-STEM121 (imbeba, 1: 200; Y40410, Takara Bio), anti-SATB2 (imbeba, 1:50 1: 400; ABN904, Millipore), na anti-IBA1 (ihene, 1: 100; ab5076, abkam).Ibice byogejwe hamwe na PBS hanyuma bishyiramo antibody ya kabiri kumasaha 1 mubushyuhe bwicyumba (ibice bikonje) cyangwa ijoro ryose kuri 4 ° C (ibice byimbitse). Alexa Fluor antibody ya kabiri (Ubuzima bwa Technologies) yavanze 1: 1000 muguhagarika igisubizo yakoreshejwe. Nyuma yo gukaraba hamwe na PBS, nuclei yagaragaye hamwe na Hoechst 33258 (Ubuzima bwa Technologies). Hanyuma, amashusho yashyizwe kuri microscope ifite ibifuniko (Fisher Scientific) ukoresheje Aquamount (Polysciences) hanyuma isesengurwa kuri microscope ya Keyence fluorescent (BZ-X isesengura) cyangwa microscope ya Leica TCS SP8 (Las-X) kuri iyo shusho. Amashusho yatunganijwe hifashishijwe porogaramu ya ImageJ (Fiji). Kugirango ugereranye igipimo cya neurone zabantu muri t-hCO na cortex yimbeba, 387.5 mkm z'ubugari amashusho y'urukiramende yafatiwe hagati ya t-hCO, cyangwa hafi yinkombe ya cortex. Impapuro zashizweho zagenwe no gusuzuma impinduka mu mucyo, HNA + nuclei, na / cyangwa kuba hari autofluorescence. Muri buri shusho, umubare rusange wa NeuN + na HNA + selile wagabanijwe numubare rusange wa selile NeuN + mukarere kamwe. Kugirango umenye neza ko selile zifite nuclei gusa mumashusho yindege zibarwa, gusa selile nazo Hoechst + zashyizwe mubarwa. Amashusho abiri yatandukanijwe byibura mm 1 yagereranijwe kugirango agabanye amakosa yibarurishamibare.
Icyumweru kimwe mbere yikusanyamakuru, shyira inyamaswa zatewe muri HCO (hafi amezi 8 yo gutandukana) mucyumba cyijimye hamwe na whiskers zagabanijwe kugirango ugabanye ibyiyumvo. Kwigunga kwa nuclei byakozwe nkuko byasobanuwe mbere, hamwe na bimwe byahinduwe. Muri make, t-hCO na hCO zarasenyutse hakoreshejwe lysis ya detergent-mashini na lisansi ya miriyoni 2 (D8938, Sigma-Aldrich / KIMBLE). Nuclei ya kijyambere noneho yarayunguruwe hifashishijwe akayunguruzo ka 40 µm hanyuma igashyirwa kuri 320 g mu minota 10 kuri 4 ° C mbere yo gukora igipimo cyiza cya sucrose. Nyuma yintambwe ya centrifugation (320 g kuminota 20 kuri 4 ° C), ingero zongeye guhagarikwa muri 0.04% BSA / PBS hiyongeraho ibice 0.2 bya µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) hanyuma inyura mumashanyarazi ya 40 µm. Nuclei yatandukanijwe noneho ihagarikwa muri PBS irimo 0,02% BSA hanyuma ishyirwa kuri Chromium Single Cell 3 ′ chip (bivugwa ko izakira 8000 selile kumurongo). amasomero ya snRNA-seq yateguwe hamwe na Chromium Single selile 3 ′ GEM, Isomero & Gel Bead Kit v3 (10x Genomika). amasomero ya snRNA-seq yateguwe hamwe na Chromium Single selile 3 ′ GEM, Isomero & Gel Bead Kit v3 (10x Genomika). Библиотеки snRNA-seq были приготовлены с помощью Chromium Akagari kamwe 3 ′ GEM, Isomero & Gel Bead Kit v3 (10x Genomika). Amasomero ya snRNA-seq yateguwe hifashishijwe Chromium Single selile 3 ′ GEM, Isomero & Gel Bead Kit v3 (10x Genomika). snRNA-seq 文库是使用 Chromium Akagari kamwe 3 ′ GEM 、 Isomero & Gel Bead Kit v3 (10x Genomika) 制备的。 snRNA-seq 文库是使用 Chromium Akagari kamwe 3 ′ GEM 、 Isomero & Gel Bead Kit v3 (10x Genomika) 制备的。 Библиотеку snRNA-seq готовили с использованием Chromium Akagari kamwe 3 ′ GEM, Isomero & Gel Bead Kit v3 (10x Genomika). Isomero rya snRNA-seq ryateguwe hifashishijwe Chromium Akagari kamwe 3 ′ GEM, Isomero & Gel Bead Kit v3 (10x Genomika).Amasomero yo mu byitegererezo bitandukanye yahujwe kandi akurikiranwa nubuzima bwa Admera kuri NovaSeq S4 (Illumina).
Urwego rwerekana imiterere ya buri barcode ya kirimbuzi yashizwemo hifashishijwe porogaramu ya 10x ya Genomika CellRanger isesengura porogaramu (verisiyo 6.1.2). By'umwihariko, ibyasomwe byahujwe no guhuza abantu (GRCh38, Ensemble, verisiyo ya 98) nimbeba (Rnor_6.0, Ensemble, verisiyo 100) genome yerekanwe hamwe na mkref no gukoresha kubara hamwe na –include-introns = itegeko NYAKURI kubara harimo gusoma byashizwe mukarere ka intron. Ku byitegererezo bya t-hCO, nuclei yabantu yamenyekanye hashingiwe ku cyifuzo cyo guharanira ko byibuze 95% by’ibisomwa byose byashushanyije bihuye na genomuntu. Isesengura ryose ryakurikiyeho ryakozwe kuri filteri ya barcode yasohotse ivuye muri CellRanger ikoresheje pake ya R (verisiyo 4.1.2) Seurat (verisiyo 4.1.1) 32.
Kugirango hamenyekane neza ko nuclei yo mu rwego rwo hejuru yonyine ishyirwa mu isesengura ryakurikiyeho, inzira yo kuyungurura yashyizwe mu bikorwa kuri buri cyitegererezo. Ubwa mbere, nuclei yo mu rwego rwo hasi ifite genes zidasanzwe ziri munsi ya 1000 zabonetse kandi hejuru ya 20% ya mitochondriya yose iramenyekana kandi ikurwaho. Icyakurikiyeho, materix ya gene mbisi yari isanzwe muburyo busanzwe bwo gusubira inyuma hifashishijwe imikorere ya sctransform (vst.flavor = ”v2 ″), yanagaragaje genes 3000 zihinduka cyane ukoresheje ibipimo bidasubirwaho. Kugabanya ibipimo byakorewe kuri genes zo hejuru zahinduwe hakoreshejwe isesengura ryibanze ryibanze (PCA) hamwe nibipimo byakoreshwaga kugirango dusuzume ibipimo byerekana ibipimo bya 30 (dims = 30) twatoranijwe dukoresheje igenzura ryerekana ibipimo bya 30. iterateri itera (imyanzuro = 1) kugirango itondekanye genes zishingiye ku mibare idasanzwe ya gene (median iri munsi ya 10 ku ijana), kubara gene mitochondrial idasanzwe (median iri hejuru ya 95%) kugirango tumenye kandi dukureho uturemangingo dushyira mu gaciro kandi / cyangwa umubare munini wimpanga zikekwa zamenyekanye ukoresheje urugero rwa DoubletFinder33 (nincuro ya 3 = n) ukoresheje imikorere ya IntegrateData hamwe nibipimo byavuzwe haruguru.
Nyuma yo gukuraho intungamubiri zidafite ubuziranenge, dataset ihuriweho hamwe yashyizwe hamwe (imyanzuro = 0.5) hanyuma ishyirwa mubikorwa bya UMAP34. Imirasire ya gen kuri buri cluster yagenwe hakoreshejwe imikorere ya FindMarkers hamwe nibipimo bisanzwe bibarwa uhereye kubisanzwe byerekana imiterere ya gene. Tumenye kandi dushyire mubyiciro byingenzi byingirabuzimafatizo duhuza uruhinja hamwe nabakuze cortical reference datasets hamwe na marike gene imvugo 19,20,21,35 na annotation. By'umwihariko, kuzenguruka ibanzirizasuzuma byagaragajwe n'imvugo ya MKI67 na TOP2A. Amasoko ya progenitor yasobanuwe no kutagira inyandiko-mvugo ya mitoto, guhuzagurika cyane hamwe na glial progenitor ya classe yasobanuwe muri cortex ya metafase yatinze, hamwe na EGFR na OLIG1. Twifashishije ijambo astrocyte kugirango dukubiyemo leta zitandukanye zo gutandukanya astrocyte, kuva glia itinze ya radiyo kugeza gukura kwa astrocytes. Ihuriro rya Astrocyte ryerekana urwego rwo hejuru rwa SLC1A3 na AQP4 kandi byerekanwe ku ikarita hamwe na subtypes ya radiyo glia na / cyangwa astrocytes ikuze. OPCs igaragaza PDGFRA na SOX10 mugihe oligodendrocytes yerekana ibimenyetso bya myelination (MOG na MYRF). Glutamatergic neuron yamenyekanye no kuba hari inyandiko-mvugo ya neuronal (SYT1 na SNAP25), kutagira ibimenyetso bya GABAergic (GAD2), no kwerekana NEUROD6, SLC17A7, BCL11B, cyangwa SATB2. GluN neuron yagabanijwemo kabiri (imvugo ya SATB2 no gutakaza BCL11B) hamwe nimbaraga zimbitse (BCL11B imvugo). Neuron ya putative subplate (SP) yerekana ibimenyetso bizwi bya SP18 nka ST18 na SORCS1 hiyongereyeho ibimenyetso byimbitse bya GluN. Ingirabuzimafatizo zimeze nka Choroid zamenyekanye ku mvugo ya TTR, naho selile zisa na meningeal zagaragaje ingirabuzimafatizo zifitanye isano na fibroblast hamwe na mape pial / vascular selile zerekana amakuru yashyizweho.
Isesengura ritandukanye ryerekana imiterere ya gene hagati ya t-hCO na hCO subassassass yakozwe hakoreshejwe uburyo bushya bwa pseudo-batch bwerekanwe mubitegererezo byashyizwe mubikorwa hakoreshejwe pake ya Libra R (verisiyo 1.0.0). By'umwihariko, edgeR log-likibishoboka (verisiyo 3.36.0, pake R) yakorewe mumatsinda muguhuza umubare wa gen muri selile kumurongo watanzwe kuri buri cyitegererezo. Kuri hotmap visualisation, isanzwe kuri miriyoni (CPM) indangagaciro zibarwa ukoresheje edgeR (cpm () imikorere) kandi igapimwa (kugirango igere kubisobanuro = 0, gutandukana bisanzwe = 1). Isesengura ryitunganyirizo rya Gene Ontology (GO) ryakozwe ku buryo bugaragara gen-t-hCO GluN yagenzuwe (Benjamini-Hochberg yakosoye P agaciro kari munsi ya 0.05 byagaragaye byibuze 10% bya selile t-hCO GluN no kwiyongera kwikubye inshuro 2). byakozwe ukoresheje ToppGene Suite (https://toppgene.cchmc.org/)37. Dukoresha porogaramu ya ToppFun ifite ibipimo bisanzwe kandi dutanga raporo Benjamini-Hochberg yakosowe na P-gaciro yabazwe uhereye GO-annotated hypergeometric test.
Kugira ngo duhuze classe yacu ya snRNA-seq hamwe na classe itondekanye kuva mubushakashatsi bwibanze bwa selile imwe RNA-seq cyangwa abakuze snRNA-seq19,20,21,22, twakoresheje uburyo bwo guhuza dataset. Twakoresheje uburyo bwa SCTransform (v2) busanzwe bwo gukora muri Seurat kugirango duhuze kandi tugereranye guhuza cluster hagati ya datasets (dukoresheje ibipimo bimwe nkuko byavuzwe haruguru). Imibare yumuntu ku giti cye yashyizwe ku bushake ingirabuzimafatizo zigera kuri 500 cyangwa cores kuri cluster yumwimerere kugirango ikore neza. Ukoresheje uburyo busa nkuko byasobanuwe mbere, guhuzagurika byasobanuwe nkigipimo cya selile cyangwa nuclei muri buri cluster ihujwe ihujwe na label ya cluster yerekanwe. Kugirango turusheho gutondekanya GluNs, twakoresheje ibikorwa bya Seurat's TransferData kumurimo wa GluN subet data kugirango dushyireho ibimenyetso bya dataset kuri selile zacu GluN.
Kugirango dusuzume uko imiterere yimiterere yimiterere yisi yose ya t-hCO na hCO, twagereranije urugero rwa pseudo-bulk hamwe na BrainSpan / psychENCODE23, igizwe nurwego runini rwa RNA rukurikirana ubwonko bwabantu. Twakoze PCA ku buryo bwo guhuza imiterere ya gene imvugo isanzwe kuva cortical sample nyuma yibyumweru 10 nyuma yo gusama hanyuma nyuma, muri gen 5567 (hamwe namakuru yacu) byagaragaye mbere nkibikorwa muri sample cortical BrainSpan (bisobanurwa ko birenga 50% muburyo butandukanye bwiterambere byasobanuwe nimyaka ukoresheje moderi ya cubic) 38. Twongeyeho, twakuye genes zifitanye isano na signature nini ya transcriptque ya neurodevelopment dukoresheje matrix itari nziza nkuko byasobanuwe mbere. Ibipimo by'icyitegererezo bibarwa ukoresheje uburyo butari bwiza bwo kugereranya ibintu byateguwe mu mashusho. 5b hamwe namakuru yagutse kuri buri mukono wa gatanu wasobanuwe na Zhu et.38. Na none kandi, ibikorwa bishingiye ku nyandiko-mvugo yavuye mu bushakashatsi bwatangajwe mbere. By'umwihariko, ERG na LRG byagenzuwe cyane muri neuron ya glutamatergic yagaragajwe nimbeba yerekana cortex snRNA-seq nyuma yo gukanguka kugaragara kuva kumeza yinyongera 3 Hrvatin nabandi.16. LRG ikungahaye ku bantu yakuwe mu mico y'ubwonko ya KCl ikora kandi isarurwa nyuma y'amasaha 6 nyuma yo gukanguka, kandi ingirabuzimafatizo zashizwe mu buryo bugaragara mu bantu ariko ntabwo zari mu nzoka (Imbonerahamwe y'inyongera 4). Isesengura ryogukungahaza gene ukoresheje aya maseti ya gene ryakozwe hakoreshejwe ikizamini kimwe cya Fisher.
Anesthetize imbeba hamwe na isoflurane, ikureho ubwonko hanyuma ushire mubukonje (hafi 4 ° C) okisijene (95% O2 na 5% CO2) igisubizo cya sucrose kubice birimo: 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2,5 mM KCl, 1,25 mM. NaH2PO4, 10 mM MgSO4 na 0.5 mM CaCl2 (hafi 310 mOsm). Imbeba ubwonko bwa coronale (300-400 µm) irimo t-hCO yakozwe hakoreshejwe vibratome ya Leica VT1200 nkuko byasobanuwe mbere39. Ibice byahise bimurirwa mucyumba cyigabanyamo ubushyuhe bwa ogisijeni yubushyuhe bwicyumba burimo ACSF yateguwe kuva: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 na 126 mM NaCl (298 mOsm). byibura iminota 45 mbere yo gufata amajwi. Ibice byanditswe mu cyumba cyacengewemo aho byahoraga bitunganyirizwa hamwe na ACSF (95% O2 na 5% ya CO2). Amakuru yose yanditswe mubushyuhe bwicyumba. neuron ya t-hCO yarangiye hamwe na pisine ya borosilicate yuzuye yuzuyemo igisubizo kirimo gluconate ya mM 127 mM, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, na 0,6 mM EGTA, pH 7.2, igisubizo cyimbere cyahinduwe na KOH (290 mOsm). Kugirango ukire, biocytine (0.2%) yongewe kumuti wafashwe amajwi.
Amakuru yabonetse hakoreshejwe MultiClamp 700B amplifier (Ibikoresho bya Molecular) hamwe na Digidata 1550B ya digitifike (ibikoresho bya Molecular), pass-pass yungurujwe kuri 2 kHz, yandikwa kuri 20 kHz, hanyuma isesengurwa ukoresheje Clampfit (Ibikoresho bya Molecular), Inkomoko (OriginPro). 2021b, InkomokoLab). n'imikorere yihariye ya MATLAB (Imibare). Ubushobozi bwo guhuza bwabazwe hakoreshejwe JPCalc kandi ibyanditswe byahinduwe kubiciro byabazwe -14 mV. Igikorwa cya IV kigizwe nurukurikirane rw'intambwe zigezweho muri 10-25 pA intambwe, kuva -250 kugeza 750 pA.
Thalamus, ibintu byera, na S1 byatewe n'amashanyarazi mu bice bya thalamocortical mugihe cyo gufata amashusho ya neuron ya hCO, nkuko byasobanuwe mbere. Muri make, ubwonko bwashyizwe kumeza ya 3D icapa yegamye kuri 10 °, kandi imbere yubwonko yaciwe kuri 35 °. Ubwonko bwahise bufatirwa hejuru yaciwe hanyuma buragabanywa, burinda axon ya thalamocortique. Bipolar tungsten electrode (0.5 MΩ) yashyizwe kuri micromanipulator ya kabiri hanyuma ishyirwa mubikorwa kugirango itere uturere tune kuri selile (capsule y'imbere, ibintu byera, S1 na hCO). Andika ibisubizo bya synaptic nyuma ya 300 µA fasic stimulation kuri 0.03–0.1 Hz.
HChR2 yerekana neurone ya hCO yakoreshwaga kuri 480 nm kandi impiswi zoroheje zakozwe na LED (Prizmatix) zashyizwe mubikorwa × 40 (0.9 NA; Olympus) kugirango zandike imvugo ya hChR2 hafi ya selile. Umurambararo wumurima wa diametre ni mm 0,5 naho imbaraga zose ni 10-20 mW. Ubugari bwa pulse bwashyizwe kuri ms 10, bihuye na pulse yatanzwe mugihe cyo kwiga imyitwarire. Imirongo itandukanye yo gukangura yakoreshejwe, kuva 1 kugeza 20 Hz, ariko impiswi yambere yuruhererekane yakoreshejwe mukugereranya. Intera iri hagati ya gari ya moshi isanzwe irenze 30 s kugirango igabanye ingaruka kuri synaptic inhibitory cyangwa koroshya inzira. Kugirango tumenye niba igisubizo cya hChR2 cyari monosynaptic, twakoresheje TTX (1 μM) mu bwogero kugeza ubwo EPSC yabuze, hanyuma dushyira 4-aminopyridine (4-AP; 100 μM). Mubisanzwe, igisubizo gisubizwa muminota mike, hamwe no gutinda gato gato hagati yo kurasa LED hamwe na EPSC. NBQX (10 μM) yakoreshejwe mugupima niba igisubizo gitwarwa na reseptor ya AMPA.
Ibice bikaze bya HCO byakozwe nkuko byasobanuwe mbere. Muri make, ibice bya hCO byinjijwe muri 4% agarose hanyuma bimurirwa muri selile zirimo 126 mM NaCl, 2,5 mM KCl, 1,25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 na 10 mM d - (+) -glucose mu bice byashyizwe mu cyumba cya VT kuri 2001200. Hanyuma, patch-camp yandika ingirabuzimafatizo zose zakozwe ku bice bya hCO munsi ya microscope ya SliceScope itaziguye (Scientifica). Ibice byatunganijwe hamwe na ACSF (95% O2 na 5% CO2) kandi ibimenyetso bya selile byanditswe mubushyuhe bwicyumba. HCO neuron yakoreshejwe hifashishijwe ikirahuri cya borosilicate ikirahuri cyuzuyemo igisubizo kirimo 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, na 0,6 mM EGTA, imbere pH 7, 2, ihujwe na KOH (osmolarity 290). Kugirango ugarure, ongeramo 0.2% Biocytine mubisubizo byimbere.
Amakuru yaguzwe na Clampex (Clampex 11.1, Ibikoresho bya Molecular) ukoresheje amplifier ya MultiClamp 700B (ibikoresho bya Molecular) hamwe na Digidata 1550B ya digitifike (ibikoresho bya Molecular), pass-pass yungurujwe kuri 2 kHz, yandikwa kuri 20 kHz, kandi isesengurwa ukoresheje Clampfit (verisiyo ya 10.6) kugirango isesengurwe, ibikoresho bya MATBB). Ubushobozi bwo guhuza bwabazwe ukoresheje JPCalc kandi ibyanditswe byahinduwe kubishobora kubarwa bya -14 mV. Igikorwa cya IV kigizwe nurukurikirane rw'intambwe ziriho muri 5-10 pA intambwe kuva -50 kugeza 250 pA.
Kugirango morfologiya yongere yubake neuron yometse, 0.2% biocytine (Sigma-Aldrich) yongewe kumuti wimbere. Ingirabuzimafatizo byibuze byibuze iminota 15 nyuma yo kwiba. Umuyoboro uhita ushushanya buhoro buhoro muminota 1-2 kugeza igihe ibyanditswe byanditse bifunze burundu. Gukurikira igice cya physiologiya, ibice byakosowe ijoro ryose kuri 4 ° C. muri 4% PFA, byogejwe na PBS X3, hanyuma bivangwa na 1: 1000 hamwe na streptavidin-conjugated DyLight 549 cyangwa DyLight 405 (Laboratoire ya Vector). Ingirabuzimafatizo zuzuyemo biocytine (2%; Sigma-Aldrich) zashyizweho ikimenyetso mugihe cyafashwe amajwi ku bushyuhe bwicyumba cyamasaha 2. Ibice byahise bishyirwa kumurongo wa microscopi ukoresheje Aquamount (Thermo Scientific) hanyuma ugaragara ejobundi kuri microscope ya Leica TCS SP8 ukoresheje intego yo kwibiza amavuta hamwe na aperture numero × 40 1.3, gukuza × 0.9-1.0, xy. Igipimo cyicyitegererezo ni pigiseli 7 kuri micron. Z-stack kuri 1 µm intera yabonetse muburyo bukurikiranye, hanyuma z-stack mosaika hamwe na Leica ishingiye kumodoka-idoda byakozwe kugirango bitwikire igiti cyose cya dendritic ya buri neuron. Neurons yakurikiranwe igice cyintoki ikoresheje interineti ya neuTube 40 hanyuma dosiye za SWC zirakorwa. Amadosiye yahise yoherezwa kuri plugin ya SimpleNeuriteTracer41 Fiji (ImageJ, verisiyo 2.1.0; NIH).
Tortue yumuntu yabonetse yabiherewe uruhushya hakurikijwe protocole yemejwe ninama ishinzwe isuzuma rya kaminuza ya Stanford. Ingero ebyiri z'umubiri wumuntu nyuma yo kubyara (imyaka 3 na 18) zabonetse mugukata cortex y'imbere (girus yo hagati imbere) murwego rwo kubaga igicuri kitavunika. Nyuma yo kwangwa, gusarura tissue muri NMDG-aCSF ikonje irimo urubura rurimo: 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 sodium ascorbate, 3 mM sodium pyruvate, mMH 2 Titrate kuri pH 7.3-7.4 hamwe na aside hydrochloric yibanze. Tissue zagejejwe muri laboratoire mu minota 30 hanyuma ibice bya coronale bifatwa hakurikijwe inzira yasobanuwe haruguru.
Inzira zose zinyamanswa zakozwe hakurikijwe amabwiriza ya kaminuza ya Stanford yemewe na APLAC. Imbeba (zirenze iminsi 140 nyuma yo guhindurwa) zatewe na 5% isoflurane anesthesia hanyuma itera aneste hamwe na 1-3% isoflurane idakorana. Inyamaswa zashyizwe muburyo bwa stereotaxic (Kopf) hanyuma buprenorphine irekura (SR) yatewe inshinge. Igihanga kiragaragara, gisukuye kandi hashyizwemo imigozi 3-5. Kugirango tumenye t-hCO, twabyaye stereotaxic coordinateur kuva mumashusho ya MRI. Hacukuwe umwobo wa burr ahashimishijwe kandi fibre (400 µm diametre, NA 0.48, Doric) yamanuwe 100 µm munsi yubuso bwa hCO hanyuma igashyirwa ku gihanga hamwe na sima y amenyo ya UV (Relyx).
Fibre Photometric yafashwe amajwi nkuko byasobanuwe mbere42. Kugira ngo wandike ibikorwa byizana, imbeba zashyizwe mu kato gasukuye kandi insinga ya 400 µm ya fibre optique yamashanyarazi (Doric) ihujwe na sisitemu yo gukusanya amakuru ya fibre optique yahujwe na fibre optique. Mugihe cyo gufata iminota 10 yerekana ibikorwa bya moteri, inyamaswa zidegembya gushakisha akazu. Kwandika ibikorwa byabyutswe, imbeba (nyuma yiminsi irenga 140 nyuma yo guterwa) zatewe aneste hamwe na 5% isoflurane kugirango yinjizwe na 1-3% isoflurane kugirango ibungabunge. Shira inyamanswa muburyo bwa stereotactique (Kopf) hanyuma whiskers kuruhande rwa t-hCO igabanijwe kugeza kuri cm 2 hanyuma unyuze mumashanyarazi ihujwe na piezoelectric actuator (PI). 400 µm fibre optique yamashanyarazi (Doric) yahujwe na fibre yatewe kandi ihujwe na sisitemu yo gushaka amakuru. Inkongoro ku rundi ruhande rwa t-hCO yahise ihindurwa inshuro 50 (mm 2 kuri 20 Hz, 2 s kuri buri cyerekezo) mugihe cyateganijwe na disiki ya piezoelectric mugihe cyiminota 20 yo gufata amajwi. Koresha Package ya Arduino MATLAB kugirango ugenzure igihe cyo gutandukana hamwe na kode ya MATLAB. Ibyabaye bihujwe na software yo gukusanya amakuru ukoresheje transistor-transistor logic (TTL) pulses.
Imbeba (zirenze iminsi 140 nyuma yo guhindurwa) zatewe na 5% isoflurane anesthesia hanyuma itera aneste hamwe na 1-3% isoflurane idakorana. Inyamaswa zashyizwe muburyo bwa stereotaxic (Kopf) hanyuma buprenorphine SR na dexamethasone batewe inshinge. Igihanga kiragaragara, gisukuye kandi hashyizwemo imigozi 3-5. Kugirango tumenye t-hCO, twabyaye stereotaxic coordinateur kuva mumashusho ya MRI. Craniotomy izenguruka (hafi cm 1 z'umurambararo) yakorewe imyitozo yihuta cyane hejuru ya hCO yatewe. Amagufwa amaze kuba mato ashoboka, ariko mbere yo gucukura mumagufwa yose, koresha imbaraga kugirango ukureho disiki ya pelvic isigaye kugirango ugaragaze t-hCO. Craniotomy yari yuzuyemo saline sterile, hanyuma igipfundikizo hamwe nigitereko cyihariye cyomutwe ku gihanga hamwe na sima y amenyo ya UV yakize (Relyx).
Kwerekana amashusho abiri yakozwe hakoreshejwe microscope ya Bruker ifite moteri ya Nikon LWD (× 16, 0.8 NA). Ishusho ya GCaMP6 yakorewe kuri 920 nm hamwe na 1.4x imwe ya z-indege ikuza na 8x ikigereranyo cya 7.5 fps. Imbeba zatewe na anesthesia ya 5% isoflurane kandi ikomeza hamwe na 1-3% isoflurane. Imbeba zashyizwe mubikorwa byabugenewe byakozwe mumutwe hanyuma bigashyirwa munsi yinzira. Habonetse iminota 3 yerekana ibikorwa bya moteri byabonetse. Mugihe cyiminota 20 yo gufata amajwi, puffs 50 (buri cyerekezo 100 ms z'uburebure) zagejejwe kubushake bwa pisine ya whisker ahateganye na t-hCO hakoreshejwe picospricer. Koresha Package ya Arduino MATLAB kugirango ugenzure igihe giturika hamwe na kode ya MATLAB. Guhuza ibyabaye hamwe na software yo gukusanya amakuru (PrairieView 5.5) ukoresheje TTL pulses. Kubisesengura, amashusho yakosowe kuri xy icyerekezo akoresheje gukosora affine muri gahunda ya MoCo yatangiriye muri Fiji. Gukuramo ibimenyetso bya fluorescent kuva selile zitandukanye ukoresheje CNMF-E43. Fluorescence yakuwe kuri buri karere k'inyungu, ihindurwamo umurongo wa dF / F, hanyuma ihinduka z-amanota.
Imbeba (zirenze iminsi 140 nyuma yo guhindurwa) zatewe na 5% isoflurane anesthesia hanyuma itera aneste hamwe na 1-3% isoflurane idakorana. Inyamaswa zashyizwe muburyo bwa stereotaxic (Kopf) hanyuma buprenorphine SR na dexamethasone batewe inshinge. Inkongoro ku rundi ruhande rwa t-hCO yaciwe kugeza kuri cm 2 hanyuma ihambirizwa mu rushundura ruhuza amashanyarazi ya piezoelectric. Igihanga kiragaragara kandi gisukuye. Icyuma cyubutaka kitagira ingese gifatanye na gihanga. Kugirango tumenye t-hCO, twabyaye stereotaxic coordinateur kuva mumashusho ya MRI. Kora uruziga ruzengurutse (hafi cm 1 z'umurambararo) hamwe n'umuvuduko mwinshi hejuru ya t-hCO. Amagufwa amaze kuba mato ashoboka, ariko mbere yo gucukura mumagufwa yose, koresha imbaraga kugirango ukureho disiki ya pelvic isigaye kugirango ugaragaze t-hCO. Ingirabuzimafatizo ku giti cye zafashwe amajwi hakoreshejwe umuyoboro wa 32 cyangwa umuyoboro wa 64 wa silikoni nini cyane (Cambridge Neurotech) wajugunywe ku butaka hanyuma ukongerwaho mbere na RHD (Intan). Koresha manipulator kugirango umanure electrode kurubuga ugenewe unyuze muri craniotomy, yuzuye saline sterile. Ikusanyamakuru ryakorewe kuri frequence ya 30 kHz ukoresheje sisitemu yo gufungura amakuru ya Ephys. Gufata amajwi byakomeje gusa igihe twasangaga ibikorwa bifitanye isano cyane nigitekerezo cyizana mu miyoboro irenga 10, byerekana ko electrode yari iherereye (hashingiwe ku makuru yerekana amashusho abiri ya calcium ya calcium). Habonetse iminota 10 yerekana ibikorwa bya moteri byabonetse. Inkongoro ku rundi ruhande rwa t-hCO yahise ihindurwa inshuro 50 (mm 2 kuri 20 Hz, 2 s kuri buri cyerekezo) mugihe cyateganijwe na disiki ya piezoelectric mugihe cyiminota 20 yo gufata amajwi. Ukoresheje ibikoresho bya MATLAB bifasha Arduino (MATLAB 2019b), igenzura igihe cyo gutandukana hamwe na kode ya MATLAB. Koresha TTL pulses kugirango uhuze ibyabaye na software yo gushaka amakuru.
Kubushakashatsi bwa optique, 200 µm optique yamashanyarazi (Doric) ihujwe na lazeri 473 nm (Omicron) yahujwe na fibre optique ya 200 µm yashyizwe hejuru ya craniotomy. Ako kanya mbere yibi, hindura imbaraga zo gusimbuka kuri 20 mW. Koresha manipulator kugirango umanure electrode kurubuga ugenewe unyuze muri craniotomy, yuzuye saline sterile. Mu gutangira gufata amajwi, hasohotse impiswi icumi zumucyo 473 nm (inshuro 2 Hz, impiswi zimara ms 10). Ingirabuzimafatizo zifotora zasobanuwe nkutugingo twerekanaga igisubizo muri ms 10 yumucyo muri 70% cyangwa byinshi mubigeragezo.