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I-self-assembling organelles ye-neural imele iqonga elithembisayo le-vitro lomzekelo wophuhliso lwabantu kunye nezifo. Nangona kunjalo, i-organoids ayinalunxibelelwano olukhoyo kwi-vivo, oluthintela ukuvuthwa kunye nokuthintela ukudityaniswa nezinye iisekethe ezilawula ukuziphatha. Apha sibonisa ukuba i-cell stem-derived cortical organoids ifakwe kwi-cortex ye-somatosensory ye-neonatal nude rats iphuhlisa iintlobo zeeseli ezikhulileyo ezidibanisa kwiisekethe ezinxulumene neemvakalelo kunye nenkuthazo. I-MRI ibonise ukukhula kwe-organoid emva kokutshintshwa kwimizila emininzi ye-stem cell kunye nezilwanyana, ngelixa uhlalutyo olulodwa lubonisa ukuqhubela phambili kwe-corticogenesis kunye nokuvela kwenkqubo yokushicilela exhomekeke kumsebenzi. Ewe, i-cortical neurons etshintshiweyo ibonakalisa i-morphological, i-synaptic, kunye nepropathi ye-membrane yangaphakathi eyinkimbinkimbi kune-in vitro ehambelana nayo, evumela ukubonwa kweziphene ze-neuronal kwizigulana ezine-Timothy's syndrome. Ukulandelwa kwe-anatomical kunye nokusebenza kubonise ukuba i-organelles etshintshiweyo ifumana i-thalamocortical kunye ne-corticocortical inputs, kwaye kwi-vivo ukurekhoda komsebenzi we-neural kubonisa ukuba la magalelo anokuvelisa iimpendulo zeemvakalelo kwiiseli zomntu. Okokugqibela, i-cortical organoids yandisa i-axons kuyo yonke ingqondo yegundane, kwaye ukusebenza kwabo kwe-optogenetic kukhokelela ekuziphatheni okufunwa ngumvuzo. Ke ngoko, i-cortex neurons etshintshwe ngokutsha iyakhula kwaye ithathe inxaxheba kwiisekethe zomamkeli ezilawula ukuziphatha. Silindele ukuba le ndlela iququzelele ukufunyanwa kwe-phenotypes ye-strand-level kwiiseli ezithathwe kwisigulane ezingenakufunyanwa ngezinye iindlela.
Ubuchopho bomntu obuphuhlayo yinkqubo ephawulekayo yokuzicwangcisa apho iiseli zanda, zahlula, zifuduka, kwaye ziqhagamshele ukwenza iisekethe ze-neuronal ezisebenzayo eziye zacokiswa ngakumbi ngamava eemvakalelo. Ingxaki ephambili ekuqondeni ukuphuhliswa kwengqondo yomntu, ngokukodwa kwimeko yesifo, kukungabikho kokufikelela kwizicubu zengqondo. Ii-organelles ezizilungelelanisayo, ezibandakanya i-cortex organoids (hCO; eyaziwa ngokuba yi-human cortex sphere), inokuvelisa i-2,3,4,5,6. Nangona kunjalo, imida eliqela inciphisa ukusetyenziswa kwabo okubanzi ekuqondeni uphuhliso kunye nokusebenza kweesekethe ze-neural. Ngokukodwa, akucaci ukuba ukuvuthwa kwe-hCO kukhawulelwe ngokungabikho kwezinto ezithile ze-microenvironmental kunye nezimvo zoluvo ezikhoyo kwi-vivo. Ukongeza, ngenxa yokuba ii-hCOs azidityaniswanga kwiisekethe ezinokuvelisa iziphumo zokuziphatha, ukusetyenziswa kwazo kwimodeli yofuzo oluntsonkothileyo kunye nokuziphatha kwe-neuropsychiatric ukuphazamiseka okwangoku kulinganiselwe.
Ukutshintshwa kwe-hCO kwingqondo ephilileyo ephilileyo kunokoyisa le mida. Uphononongo lwangaphambili lubonise ukuba ii-neuron zabantu ezifakwe kwi-rodent cortex ziyakwazi ukuphila, iprojekthi, kunye nokunxibelelana neeseli zeempuku7,8,9,10,11,12. Nangona kunjalo, olu vavanyo luhlala lwenziwa kwizilwanyana ezikhulileyo, ezinokunciphisa ukuhlanganiswa kwe-synaptic kunye ne-axonal. Apha, sichaza i-paradigm yokutshintshwa apho satshintshela i-3D hCO ephuma kwiiseli ze-hiPS ukuya kwi-primary somatosensory cortex (S1) yeegundane ze-immunodeficient kwinqanaba lokuqala lophuhliso lweplastiki. I-hCO (t-hCO) neurons etshintshiweyo ikhula kakhulu, ifumana i-thalamocortical kunye ne-cortical-cortical igalelo elifuna iimpendulo zeemvakalelo, kunye nokwandisa uqikelelo lwe-axonal kwingqondo yegundane ukuqhuba ukuziphatha kokufuna umvuzo. Ukuvuthwa okwandisiweyo kwe-t-hCO kuye kwatyhila iziphene ze-neuronal kwizigulane ezine-Timothy's syndrome (TS), isifo esinzima sofuzo esibangelwa ukuguqulwa kwe-voltage-sensitive L-type CaV1.2 channel ye-calcium (encoded yi-CACNA1C).
Ukufunda i-neurons ye-cortical yabantu kwiisekethe kwi-vivo, sitshintshe ngokunyanisekileyo i-3D hCO engaguqukiyo kwi-S1 yeegundane ze-athymic zasemva kokubeleka (iintsuku ze-3-7 emva kokubeleka) (Umfanekiso we-1a kunye nedatha eyandisiweyo ye-Fig. 1a-c). Ngeli xesha, i-thalamocortical kunye ne-corticocortical axonal projections ayikagqibi i-innervation yabo ye-S1 (ref. 13). Ke, le ndlela yenzelwe ukukhulisa ukuhlanganiswa kwe-t-hCO ngelixa kuncitshiswa impembelelo kwiisekethe ezingapheliyo. Ukujonga ngeso lengqondo indawo ye-t-hCO kwizilwanyana eziphilayo, senze i-T2-weighted MRI brain reconstructions of rats 2-3 months after transplantation (Fig. 1b kunye nedatha eyandisiweyo, iFig. 1d). I-t-hCO yabonwa ngokukhawuleza kwaye imilinganiselo yevolumu ye-t-hCO yayifana nezo zibalwe kwiinqununu ezisisigxina (iDatha eyandisiweyo Fig. 1d, e; P> 0.05). I-t-hCO yabonwa ngokukhawuleza kwaye imilinganiselo yevolumu ye-t-hCO yayifana nezo zibalwe kwiinqununu ezisisigxina (iDatha eyandisiweyo Fig. 1d, e; P> 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированных срезов (расширенсные; 0,05). I-t-hCO yabonwa ngokulula, kwaye imilinganiselo ye-volumetric t-hCO yayifana neyokubalwa kumacandelo asisigxina (idatha eyandisiweyo, i-Fig. 1d, e; P> 0.05).很容易观察到t-hCO，并且t-hCO 的体积测量值与从固定切片计算的测量值相似（扩似（扩似（扩似（扩似（扩（扩（扩容。 0.05.很容易观察到t-hCO，并且t-hCO t-hCO легко наблюдался, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированных срезов (расширенсны, расширенсные, друг, 1, 10; 0,05). I-t-hCO yabonwa ngokulula, kwaye imilinganiselo ye-volumetric t-hCO yayifana neyokubalwa kumacandelo asisigxina (idatha eyandisiweyo, i-Fig. 1d, e; P> 0.05).Siye samisela i-t-hCO kwi-81% yezilwanyana ezitshintshiweyo malunga neenyanga ezi-2 emva kokutshintshwa (n = izilwanyana ezingama-72; i-hCO ukusuka kwimigca yeseli ye-hiPS eyi-10; imigca yeeseli ze-hiPS kwiTheyibhile eyoNgezelelweyo 1). Kwezi, ama-87% abekwe kwi-cerebral cortex (Fig. 1c). Ngokwenza i-serial MRI scans ngamaxesha amaninzi kwi-rat yokutshintshwa okufanayo, sifumene ukunyuka okuphindwe ezilithoba kwivolumu ye-t-hCO kwiinyanga ze-3 (umzobo we-1d kunye nedatha eyandisiweyo, i-Fig. 1f). Izilwanyana ezitshintshiweyo zinezinga eliphezulu lokuphila (74%) kwiinyanga ze-12 emva kokutshintshwa (idatha eyandisiweyo, i-Fig. 1g kunye neThebhile ye-Supplementary 2), kwaye akukho kuphazamiseka kwemoto okanye imemori, i-gliosis, okanye i-electroencephalogram (EEG) yafunyanwa. Idatha Umfanekiso we-1g kunye netheyibhile eyongezelelweyo 2). 1h–m kunye no-3e).
a, Schematic yoyilo lokulinga. I-hCO ephuma kwiiseli ze-hiPS yatshintshwa kwi-S1 yeegundane ezisandul 'ukuzalwa zeentsuku ezingama-30-60 zokwahlula. b, imifanekiso ye-T2 ye-coronal kunye ne-horizontal ye-MRI ebonisa i-t-hCO kwi-S1 iinyanga ze-2 emva kokutshintshwa. Isikali bar, 2 mm. c, Ubungakanani bemilinganiselo yempumelelo yokungena eboniswe kumgca ngamnye weseli ye-hiPS (n = 108, amanani angaphakathi kwemivalo abonisa inani le-t-hCO ngomgca weseli we-hIPS) kunye ne-cortical okanye i-subcortical indawo (n = 88). d, umfanekiso we-MRI we-coronary artery (ngasekhohlo; ibha yesikali, i-3 mm) kunye ne-3D ehambelanayo yokwakhiwa kwakhona kwe-volumetric (i-scale bar, i-3 mm) ebonisa ukwanda kwe-t-hCO kwiinyanga ze-3. e, Uphononongo lweepateni ze-t-hCO kwi-rat cerebral cortex. Ibar yesikali, 1 mm. f, Ummeli wemifanekiso ye-immunocytochemical ye-t-hCO eboniswe ukusuka phezulu ukuya ngasekunene (ngexesha lokuhlukana): PPP1R17 (iinyanga ezi-4 ubudala), i-NeuN (ienyanga ezi-8 ubudala), i-SOX9 kunye ne-GFAP (iinyanga ze-8), i-PDGFRα; (iinyanga ezisi-8), MAP2 (iinyanga ezisi-8) kunye ne-IBA1 (iinyanga ezisi-8). Ibar yesikali, 20 µm. I-Co-expression ye-HNA ibonisa iiseli zemvelaphi yomntu. g, snRNA-seq: Ubuninzi obudityanisiweyo kunye noqikelelo (UMAP) ukuncitshiswa kwe-dimensionality imaging yazo zonke ii-nuclei ezikumgangatho ophezulu we-t-hCO emva kokudityaniswa kwe-Seurat (n=3 t-hCO iisampulu, n=2 imigca yeeseli ze-hiPS). I-Astrocytes, iiseli zomgca we-astrocyte; i-cyclic prog, i-progenitors ejikelezayo; I-GluN DL, i-neurons enzulu ye-glutamatergic; I-GluN DL / SP, i-deep and sublamellar glutamatergic neurons; I-GluN UL, umaleko ophezulu we-glutamatergic neurons; i-oligodendrocytes, i-oligodendrocytes; OPC, iiseli oligodendrocyte progenitor; I-RELN, i-reelin neurons. h, i-Gene Ontology (GO) uhlalutyo lwexesha lokutyebisa i-genes elawulwa kakhulu (i-P <0.05, i-fold change> 2, echazwe ubuncinane kwi-10% ye-nuclei) kwi-t-hCO glutamatergic neurons xa kuthelekiswa ne-hCO glutamatergic neurons. h, i-Gene Ontology (GO) uhlalutyo lwexesha lokutyebisa i-genes elawulwa kakhulu (i-P <0.05, i-fold change> 2, echazwe ubuncinane kwi-10% ye-nuclei) kwi-t-hCO glutamatergic neurons xa kuthelekiswa ne-hCO glutamatergic neurons. h, Анализ обогащения терминов Gene Ontology (GO) для генов со значительной активацией P <0,05, кратность изменекя в 10% ядер) в глутаматергических нейронах t-hCO по сравнению с глутаматергическими нейронами hCO. h, i-Gene Ontology (GO) uhlalutyo lwexesha lokutyebisa izakhi zofuzo kunye nokusebenza okubalulekileyo (ukulungelelaniswa kwe-P <0.05, ukuguqulwa kwe-fold> 2, ukubonakaliswa ubuncinane kwi-nuclei ye-10%) kwi-t-hCO glutamatergic neurons xa kuthelekiswa ne-hCO glutamatergic neurons. h, 与hCO 谷氨酸能神经元相比，t-hCO 谷氨酸能神经元中基因显着上调（调整后P <0.05>倍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. , 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 h, гены значительно активизировались (скорректированный P <0,05, кратность изменения> 2, экспрессируется по крайней мере вдертайский 10% нейронах t-hCO по сравнению с глутаматергическими нейронами hCO Онтологический (GO) анализ термина обогащения. h, izakhi zofuzo zilawulwa ngokuphawulekayo (ukulungelelaniswa kwe-P <0.05, i-fold change> 2, echazwe ubuncinane kwi-10% ye-nuclei) kwi-t-hCO glutamatergic neurons xa kuthelekiswa ne-hCO glutamatergic neurons Ontological (GO) uhlalutyo lwexesha lokutyebisa.Umgca onamachaphaza ubonisa ixabiso le-aq le-0.05. i, umfanekiso we-UMAP weentlobo zeeseli ze-GluN kwi-t-hCO usebenzisa ukuhanjiswa kwelebhile ukusuka kwireferensi ye-22 snRNA-seq yedatha yedatha ye-motor cortex yabantu abadala. I-CT - iiseli ze-corticothalamic, i-ET - iiseli ze-extracerebral, i-IT - iiseli ze-telencephalic zangaphakathi, i-NP - kufuphi neprojekthi.
Emva koko siye savavanya i-cytoarchitecture kunye nokwakheka kweeselula ngokubanzi kwe-t-hCO. I-Antibody staining yeeseli ze-rat endothelial ibonakalise i-vascularization nge-t-hCO, ngelixa i-IBA1 ibala ibonakalise ubukho be-rat microglia kulo lonke i-graft (Umfanekiso we-1f kunye nedatha eyandisiweyo, i-Fig. 3c, d). I-Immunostaining ibonakalise i-antigen ye-nyukliya yabantu (i-HNA) iiseli ezilungileyo ezibonisa i-PPP1R17 (i-cortical progenitors), i-NeuN (i-neurons), i-SOX9 kunye ne-GFAP (iiseli ze-glial-derived) okanye i-PDGFRα (i-oligodendrocyte progenitors) (Umfanekiso 1f). Ukufunda ukubunjwa kweeseli ze-t-hCO kwisisombululo seseli enye, senze i-single-core RNA sequencing (snRNA-seq) emva kweenyanga ezi-8 zokwahlula. Ukuhluzwa ngobuninzi kunye nokususwa kwee-nuclei zeegundane kuvelise i-21,500 i-high-quality yeemephu ze-mononuclear yomntu (Umfanekiso we-1g kunye nedatha eyandisiweyo, i-Fig. 4a, b). Iipateni zokubonisa iimpawu zohlobo lweeseli eziqhelekileyo ezichongiweyo amaqoqo eeklasi ezinkulu ze-cortical cell, kubandakanywa i-neurons ye-glutamatergic enzulu kunye ne-superficial, i-progenitors ejikelezayo, i-oligodendrocytes, kunye ne-astrocyte lineage (Umfanekiso we-1g, idatha eyandisiweyo, i-Fig. 4c, kunye ne-Supplementary Table 3). I-Immunostaining ye-SATB2 kunye ne-CTIP2 ibonise ukuba nangona ubukho be-cortical subtypes, i-t-hCO ayizange ibonise i-stratification ye-anatomical ecacileyo (idatha eyandisiweyo, i-Fig. 3a). I-stage-matched snRNA-seq hCO ivelise ngokubanzi iiklasi zeeseli ezifanayo, ngaphandle kwezinto ezimbalwa, kubandakanywa ukungabikho kwe-oligodendrocytes kunye nobukho be-GABAergic neurons, enokuthi ibonise iimeko ezichazwe ngaphambili ezifanelekileyo kwi-vitro ye-lateral progenitor cells15 (idatha eyandisiweyo, i-Fig. 4f - i kunye ne-Supplementary Table 4). Uhlalutyo oluhlukeneyo lwentetho yemfuza lubonakalise ukungafani okuphawulekayo kwi-neurons ye-glutamatergic phakathi kwe-t-hCO kunye ne-hCO (iTheyibhile eyoNgezelelweyo ye-5), kubandakanywa nokusebenza kweeseti zofuzo ezinxulumene nokuvuthwa kwe-neuronal ezifana nokubonakaliswa kwe-synaptic, indawo ye-dendritic, kunye nomsebenzi wesiteshi se-voltage-gated (Umfanekiso we-1h kunye neTheyibhile eyoNgezelelweyo ye-5). itafile 6). Ngokufanelekileyo, ii-neuron ze-cortical glutamatergic t-hCO zibonise ukukhula okukhawulezileyo kokubhaliweyo.
Ukucacisa ukuba ngaba olu tshintsho lwe-transcriptional kwi-t-hCO lunxulumene nokwahlukana kwe-morphological phakathi kwe-hCO in vitro kunye ne-t-hCO kwi-vivo, sakha kwakhona i-hCO kunye ne-hCO ehambelana nesigaba esihambelana ne-biocytin kumacandelo abukhali emva kweenyanga ze-7-8 zokuhlukana. I-hCO neurons (Umfanekiso 2a). I-t-hCO neurons yayinkulu kakhulu, ibe namaxesha e-1.5 ubukhulu be-soma, ngokuphindwe kabini i-dendrites, kunye nokunyuka okuphindwe kathandathu kubude obupheleleyo be-dendritic xa kuthelekiswa ne-in vitro hCO (Fig. 2b). Ukongezelela, siye sabona ubuninzi obuphezulu be-dendritic spines kwi-t-hCO neurons kune-hCO neurons (Fig. 2c). Oku kuphakamisa ukuba i-t-hCO neurons idlula i-dendritic elongation elongation kunye ne-branching, leyo, ngokudibanisa nokunyuka kweseli eqhubekayo, inokuba negalelo ekukhuleni okukhulu kwe-t-hCO emva kokutshintshwa (Umfanekiso we-1d kunye nedatha eyandisiweyo ye-Fig. 1f). Oku kwasenza ukuba siphande iimpawu ze-electrophysiological. I-Membrane capacitance yayiphindwe ngokuphindwe kasibhozo (idatha eyandisiweyo, i-Fig. 8d), i-membrane yesimo sokuphumla inokuba yi-hyperpolarized (malunga ne-20 mV), kwaye i-injection yangoku ibangele izinga eliphezulu lokuvuselela kwi-neurons ye-t-hCO kune-neurons ye-hCO. in vitro (Umfanekiso 2d), e), ehambelanayo kunye neempawu ezinkulu kunye neziyinkimbinkimbi ze-morphological ze-t-hCO. Ukongezelela, i-frequency ye-excitatory excitatory current events (EPSC) yayiphezulu kakhulu kwi-neurons ye-t-hCO (Umfanekiso we-2f), ebonisa ukuba ukunyuka kwe-dendritic spines ebonwe kwi-neurons ye-t-hCO yayinxulumene ne-excitability yokusebenza. i-synapse yesondo. Siqinisekise umlingiswa ongekavuthwa we-hCO neurons kwi-vitro ngokurekhoda okubhalwe i-glutamatergic neurons (idatha eyandisiweyo, i-Fig. 6a-c).
a, ukwakhiwa kwakhona kwe-3D ye-hCO ezaliswe yi-biocytin kunye ne-t-hCO neurons emva kweenyanga ze-8 zokuhlukana. b, Ubungakanani beempawu ze-morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 kunye ne-***P <0.0001). b, Ubungakanani beempawu ze-morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 kunye ne-***P <0.0001). б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 kwaye *** P <0,00). b, ubungakanani beempawu ze-morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, kunye ne-**** P <0.0001). b, 形态学特征的量化（n = 8 个hCO 神经元，n = 6 个t-hCO 神经元；**P = 0.0084,*P = 0.0179 和**00 . b, 形态学特征的量化（n = 8 个hCO 神经元，n = 6 个t-hCO 神经元；**P = 0.0084,*P = 0.0179 和**00 . б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 kwaye *** P <0,00). b, ubungakanani beempawu ze-morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, kunye ne-**** P <0.0001).c, ukwakhiwa kwakhona kwe-3D kwamasebe e-hCO kunye ne-t-hCO dendritic emva kweenyanga ezi-8 zokwahlula. Iinkwenkwezi ezibomvu zibonisa i-puative dendritic spines. I-Dendritic spine density quantification (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0092). d, Ubungakanani be-membrane yokuphumla enokubakho (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, Ubungakanani be-membrane yokuphumla enokubakho (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, количественная оценка мембранного потенциала покоя (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). d, ukuphumla kwe-membrane enokubakho kwe-quantification (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d,静息膜电位的量化（n = 25 hCO 神经元,n = 16 t-hCO 神经元；***P <0.0001）. d,静息膜电位的量化（n = 25 hCO 神经元,n = 16 t-hCO 神经元；***P <0.0001）. d, количественная оценка мембранного потенциала покоя (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). d, ukuphumla kwe-membrane enokubakho kwe-quantification (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Isenzo esiphindaphindiweyo sokudubula okunokwenzeka kwi-hCO kunye ne-t-hCO eyenziwa ngokunyusa ii-injection zangoku, kunye nobungakanani bezinga eliphezulu lokudubula (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Isenzo esiphindaphindiweyo sokudubula okunokwenzeka kwi-hCO kunye ne-t-hCO eyenziwa ngokunyusa ii-injection zangoku, kunye nobungakanani bezinga eliphezulu lokudubula (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, повторное возбуждение потенциала действия в hCO kunye t-hCO, вызванное увеличением тока, и количественная оценка максимальной скорости 2возh, n = 16 нейронов t-hCO *** P <0,0001). e, isenzo esinokuphinda siqhume kwakhona kwi-hCO kunye ne-t-hCO ebangelwa ukunyuka kwangoku kunye nobungakanani bezinga eliphezulu lokudubula (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e,通过增加电流注入話导的hCO 和t-hCO 重复动作电位放电，以及最大放电率的量化（n 16 神 = 25个t-hCO 神经元；***P <0.0001）. E , 通过 增加 电流 注入 的 hco 和 t-hco 重复 电位 放电 , 以及 最 大 的 量化 （2 （5 （5 （5 16 个 t-hco 神经 ; *** p <0.0001） . e, повторяющееся возбуждение потенциала действия hCO kunye t-hCO, вызванное увеличением подачи тока, и количественная оценка максимальний 5 нейронов hCO, n = 16 нейронов t-hCO *** P <0,0001). e, ukudubula okuphindaphindiweyo kwe-hCO kunye ne-t-hCO yezenzo ezinokuthi zenziwe ngokunyuka kobonelelo lwangoku kunye nobungakanani bezinga eliphezulu lokudubula (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, ii-EPSCs ezizenzekelayo (sEPSCs) kwi-hCO kunye ne-t-hCO neurons kwiinyanga ze-8 zokwahlula, kunye nobungakanani bexesha leziganeko ze-synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, ii-EPSCs ezizenzekelayo (sEPSCs) kwi-hCO kunye ne-t-hCO neurons kwiinyanga ze-8 zokwahlula, kunye nobungakanani bezihlandlo zeziganeko ze-synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, спонтанные EPSC (sEPSC) в нейронах hCO kunye t-hCO через 8 месяцев дифференцировки и количественная оценка частоты синаптический соб =2 =2 17 нейронов t-hCO *** P <0,0001) . f, ii-EPSCs ezizenzekelayo (sEPSCs) kwi-hCO kunye ne-t-hCO neurons kwiinyanga ze-8 zokwahlula kunye nobungakanani bezinga leziganeko ze-synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, 分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs (sEPSCs), 以及突触事件频率的量化（n = 25 hCO 7 神元神经元；***P < 0.0001） . f,分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs (sEPSCs),以及突触事件频率的量匼（n = 25 hCO 25 hCO 25 神神率的量匼（n = 25hCO P <0.0001） . f, спонтанные EPSC (sEPSC) в нейронах hCO kunye t-hCO через 8 месяцев дифференцировки и количественная оценка частоты синаптический соб =2 =2 17 нейронов t-hCO *** P <0,0001). f, ii-EPSCs ezizenzekelayo (sEPSCs) kwi-hCO kunye ne-t-hCO neurons kwiinyanga ze-8 zokwahlula kunye nobungakanani bezinga leziganeko ze-synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001).I-bf, i-hCO kunye ne-t-hCO kumgca we-1208-2 zithathwe kwibhetshi efanayo yokwahlula egcinwe ngokufana. g, i-Gene ibeka uhlalutyo lokutyebisa (uvavanyo oluchanekileyo lwe-Fisher lwecala elinye) lwezakhi zofuzo eziphuculwe kakhulu (ukulungelelaniswa kwe-P <0.05, i-fold change> 2, echazwe ubuncinane kwi-10% ye-nuclei) kwi-t-hCO glutamatergic neurons xa kuthelekiswa ne-hCO glutamatergic neurons kunye ne-gene sets of bothresponseres (LPG) kunye ne-L. iijini ezichongiweyo kwi-vivo mouse study16 kunye nee-LRGs ezingqalileyo zomntu ezivela kwi-vitro neurons17. g, i-Gene ibeka uhlalutyo lokutyebisa (uvavanyo oluchanekileyo lwe-Fisher lwecala elinye) lwezakhi zofuzo eziphuculwe kakhulu (ukulungelelaniswa kwe-P <0.05, i-fold change> 2, echazwe ubuncinane kwi-10% ye-nuclei) kwi-t-hCO glutamatergic neurons xa kuthelekiswa ne-hCO glutamatergic neurons kunye ne-gene sets of bothresponseres (LPG) kunye ne-L. iijini ezichongiweyo kwi-vivo mouse study16 kunye nee-LRGs ezingqalileyo zomntu ezivela kwi-vitro neurons17. g, анализ обогащения набора генов (односторонний точный критерий Фишера) генов со значительной активацией (скорректированны5й, P) 2, экспрессия по меньшей мере в 10% ядер) в глутаматергических нейронах t-hCO по сравнению с глутаматергическими нейгронами hCO набор позднего (LRG) генов, зависящих от активности, идентифицированных в исследовании на мышах kwi vivo16, и специфических для челозвеtro11 и специфических для челозвеtro11 и специфических для челозвеtro110 LRG. g, uhlalutyo lwe-gene set enrichment (uvavanyo oluchanekileyo lwe-Fisher's one-tailed) lwemfuza ene-activation ephawulekayo (ukulungelelaniswa kwe-P <0.05, i-fold utshintsho> i-2, ibonakaliso ubuncinane kwi-10% ye-nuclei) kwi-t-hCO glutamatergic neurons xa kuthelekiswa ne-hCO glutamatergic neurons iiseti zombini ekuqaleni (ERG genes) ezichongiweyo kwi-inpendence yangaphambili (ERG genes) kunye ne-mipendence yangaphambili (ERG) kunye nee-LRGs ezikhethekileyo zomntu ezivela kwi-neurons in vitro17. g,t-hCO谷氨酸能神经元与hCO谷氨酸能神经元相比，t -hCO谷氨酸能神经元显着上调（调整后P<0.05,倍数变化>2,在至少10%的细胞核中表达）的基因集富集分析（单侧F isher精确检验）从体内小鼠研究中鉴定的早期反应(ERG)和晚期反应(LRG) 活性依赖性基因的基因组16 和体外神经元17 中的人类特异性LRG. g , t-hco 谷氨酸 神经 元 与 hco 谷氨酸 神经 元 相比 , t-hco 谷氨酸 神经 元 上调成 5 .倍数> 2 , 至少 至少 10%的 细胞 核中 表达） 的 集富集 分析中 的 早期 反应 反应 反应 和晚期 反应 反应 (lrg) 活性 基因的基因组 16 和 神经元 中1 中 1 神经元 中1 17的人类特异性LRG. g, глутаматергические нейроны t-hCO были значительно активизированы по сравнению с глутаматергическими нейронами hCO 2, не менее 10% Анализ обогащения набора генов (односторонний точный тест Фишера) раннего ответа (ERG) и позднего гены, завися идентифицированные в исследованиях на мышах in vivo16 и нейронах in vitro17, специфичные для человека. g, i-t-hCO i-neurons ye-glutamatergic yayilawulwa kakhulu xa kuthelekiswa ne-hCO glutamatergic neurons (ukulungiswa kwe-P <0.05, ukuguqulwa kwe-fold> 2, ubuncinane i-10% Ukuphendula kwangaphambili (ERG) kunye nohlalutyo lokuphucula i-gene yoluntu (uvavanyo oluchanekileyo lwe-Fisher) umsebenzi wokuphendula oxhomekeke kwiigenes kwi-vitro ye-vitro1 echongiweyo kwi-vitro1 mice neurons. iiLRGs.Umgca onamachaphaza ubonisa ixabiso le-P elilungiswe yi-Bonferroni lika-0.05. h, i-GluN gene expression (i-pseudo-package kunye nokulinganisa kwe-gene nganye) yayilawulwa kakhulu kwi-snRNA-seq replicas ye-LRG genes kwi-t-hCO glutamatergic neurons. i, i-immunostaining ebonisa ukubonakaliswa kwe-SCG2 kwi-t-hCO (phezulu) kunye ne-hCO (ephantsi) i-neurons. Iintolo ezimhlophe zikhomba kwi-SCG2+ iiseli. Ibar yesikali, 25 µm. Idatha ichazwa njengentsingiselo ± ukutenxa okusemgangathweni.
Ngokusekwe kumsebenzi owandisiweyo we-t-hCO oqatshelwe kwizilayi ze-ex vivo, i-snRNA-seq ibonise ukulawulwa okuxhomekeke kumsebenzi we-gene transcripts kwi-t-hCO xa kuthelekiswa ne-hCO in vitro. I-Glutamatergic t-hCO neurons ibonise amanqanaba aphezulu eejeni ezilawula umsebenzi wokuphendula emva kwexesha (Umfanekiso we-2g, h), ofunyenwe kwizifundo zangaphambili kwi-mouse kunye ne-neurons yabantu16,17. Ngokomzekelo, i-BDNF18, i-SCG2, kunye ne-OSTN, i-primate-specific activity-regulating gene, ibonise ukwanda kwentetho kwi-t-hCO neurons xa kuthelekiswa ne-hCO neurons (Fig. 2g-i). Ke, ii-neuron ze-t-hCO zibonise iimpawu zokuvuthwa eziphuculweyo xa kuthelekiswa ne-hCO neurons ngokubhala okubhaliweyo, kwe-morphological, kunye nohlalutyo olusebenzayo.
Ukuvavanya ngakumbi umbutho wokuvuthwa kwe-t-hCO kunye nophuhliso lobuchopho bomntu, senze uthelekiso lwe-transcriptomic lwe-fetus kunye neentlobo ze-cortical cell cell19,20 kunye nabantu abadala21,22 kunye nedatha ebanzi kwi-cortical gene expression23 ngexesha lophuhliso (idatha eyandisiweyo, i-Fig. 5). ). kunye nomsebenzi wangaphambili 24, i-hCO yehlabathi jikelele kunye ne-t-hCO isimo sokuvuthwa kwe-transcriptome kwiinyanga ze-7-8 zokuhlukana ngokubanzi zihambelana nexesha lophuhliso lwe-vivo kwaye lilingana kakhulu nobomi be-fetus kade (Idatha eyandisiweyo ye-Fig. 5a). Ngokuphawulekayo, siye sabona ukunyuka kwe-transcriptome ukukhula kwi-t-hCO xa kuthelekiswa ne-hCO ehambelana neminyaka yobudala, kunye nokusebenza kwe-transcriptome ehambelana ne-synaptogenesis, i-astrogenesis, kunye ne-myelination (idatha eyandisiweyo, i-Fig. 5b-d). Kwinqanaba leselula, sifumene ubungqina be-cortex subtype encinci kwi-t-hCO, kunye namaqela e-glutamatergic neurons agqithe kunye ne-L2 / 3 yabantu abadala, i-L5, kunye ne-L6 neuron subtypes (Umfanekiso 1i). Ngokwahlukileyo, i-cluster overlap phakathi kwe-glutamatergic t-hCO neurons kunye ne-fetal cortical neurons yayilinganiselwe kakhulu phakathi kokukhulelwa (idatha eyandisiweyo, i-Figure 5e-j). Ukufumanisa ukuba i-t-hCO neurons isebenza ngokufana ne-neocortical neurons emva kokubeleka komntu, senze ukurekhoda kwe-electrophysiological kunye nokuvuselelwa kwe-anatomical ye-L2 / 3 ye-pyramidal neurons kumacandelo abukhali e-cortex yomntu emva kokubeleka (idatha eyandisiweyo, i-Fig. 7a). Iimpawu ze-electrophysiological ze-L2 / 3 ze-pyramidal neurons zazifana nezo ze-t-hCO ze-pyramidal neurons (idatha eyandisiweyo, i-Fig. 7e). I-Morphologically, i-L2 / 3 neurons evela kwiisampuli zomntu emva kokubeleka yayifana kakhulu ne-t-hCO kune-hCO, nangona iiseli ze-L2 / 3 zazinde, ziqulethe amasebe amaninzi ngokubanzi, kwaye zinobunzima bomgudu ophezulu (Umfanekiso we-3g kunye nedatha eyandisiweyo, i-Fig. 7b-). G).
a, ukutshintshwa kwe-hCO eveliswe ngolawulo kunye nemigca yeseli ye-TS hiPS kwiimpuku ezisanda kuzalwa. b, ukwakhiwa kwakhona kwe-3D ye-biocytin-ezaliswe yi-t-hCO neurons emva kweenyanga ze-8 zokuhlukana. c, ubungakanani bentsingiselo yobude be-dendritic (n = 19 control neurons, n = 21 TS neurons; ** P = 0.0041). d, amasebe e-dendritic akhiwe ngokutsha avela kulawulo kunye ne-TS t-hCO kwiinyanga ze-8 zokwahlula, kunye nobungakanani be-dendritic spine density (n = 16 control neurons, n = 21 TS neurons, ***P <0.0001). d, amasebe e-dendritic akhiwe ngokutsha avela kulawulo kunye ne-TS t-hCO kwiinyanga ze-8 zokwahlula, kunye nobungakanani be-dendritic spine density (n = 16 control neurons, n = 21 TS neurons, ***P <0.0001). d, 3D-реконструкция дендритных ветвей из контроля и TS t-hCO через 8 месяцев дифференцировки и количественная оценка плотностинт6 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, ukwakhiwa kwakhona kwe-3D yamasebe e-dendritic ukusuka kulawulo kunye ne-t-hCO TS kwiinyanga ze-8 zokwahlula kunye ne-dendritic spine density quantification (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001). d,分化8 个月时对照和TS t-hCO的3D 重建树突分支，以及树突棘密度的量化（n = 16 內內个TS 神经元, ***P <0.0001）. d , 分化 8 个 时 对照 和 ts t-hco 的 3d 重建 分支 分支 以及 树突棘 密度 量化 1 （n 1 = 1 = 6 21 个 ts 神经 , *** p <0.0001. d, 3D-реконструкция дендритных ветвей контроля и TS t-hCO через 8 месяцев дифференцировки и количественная оценка плотностипены 1 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, ukwakhiwa kwakhona kwe-3D yokulawula amasebe e-dendritic kunye ne-TS t-hCO kwiinyanga ze-8 zokwahlula kunye ne-dendritic spine density quantification (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001).Iinkwenkwezi ezibomvu zibonisa i-puative dendritic spines. e, ii-EPSC ezizenzekelayo ekulawuleni kunye ne-TS t-hCO neurons emva kweenyanga ze-8 zokwahlula. f, iploti yefrequency eqokelelweyo kunye nobungakanani bezihlandlo kunye ne-amplitude yeziganeko ze-synaptic (n = 32 control neurons, n = 26 TS neurons; ** P = 0.0076 kunye ne-P = 0.8102). g, Uhlalutyo lwe-Scholl lwe-TS kunye nokulawula i-neurons kwi-hCO kunye ne-t-hCO. Imigca edawuniweyo ibonisa i-neurons ye-L2 / 3 yabantu emva kokubeleka kwepyramidal ukuthelekisa (n = 24 ukulawula i-t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 control hCO neurons, kunye n = 7 TS hCO neurons). Idatha ichazwa njengentsingiselo ± ukutenxa okusemgangathweni
Ikhono le-t-hCO ukuphindaphinda iimpawu ze-morphological and functional of human cortex neurons kwinqanaba eliphezulu lisishukumisele ukuba sihlolisise ukuba i-t-hCO ingasetyenziselwa ukufumanisa izifo ze-phenotypes. Sigxininise kwi-TS, ingxaki enzima ye-neurodevelopmental disorder ebangelwa ukuguqulwa kokufumana umsebenzi kwi-gene encoding CaV1.2, eqalisa umsebenzi oxhomekeke kwi-gene transcript kwi-neurons. Sifumene i-hCO kwizigulane ezintathu ze-TS ezithwele indawo eqhelekileyo (p.G406R) kunye nolawulo oluthathu (umzobo 3a). Emva kokutshintshwa, sifumene ukuba i-dendritic morphology yatshintshwa kwii-neurons ze-TS xa kuthelekiswa nokulawula (Umfanekiso we-3b kunye nedatha eyandisiweyo, i-Fig. 8a, b), kunye nokunyuka okuphindwe kabini kwinani le-dendrites eziphambili kunye nokunyuka ngokubanzi kwintsingiselo kunye nokunciphisa ubude be-dendritic ubude (Umfanekiso 3c kunye nedatha eyandisiweyo, i-Fig 8c). Oku kwakudityaniswa nokuxinana okwandisiweyo kwee-spines kunye nokunyuka kwamaxesha amaninzi e-EPSCs azenzekelayo kwi-TS xa kuthelekiswa nokulawula i-neurons (Umfanekiso we-3d-f kunye nedatha eyandisiweyo, i-Fig. 8g). Uhlalutyo olongezelelweyo luveze iipatheni ze-dendritic branching engaqhelekanga kwi-t-hCO TS xa kuthelekiswa nolawulo, kodwa kungekhona kwi-in vitro TS hCO kwinqanaba elifanayo lokwahlula (Fig. 3g). Oku kuhambelana neengxelo zethu zangaphambili ze-dendritic shrinkage exhomekeke kumsebenzi kwi-TS kwaye igxininisa amandla ale qonga lokutshintshela ukufumanisa izifo ze-phenotypes kwi-vivo.
Emva koko sibuze ukuba ingakanani i-t-hCO iiseli ezidityaniswe ngokusebenzayo kwi-rat S1. I-S1 kwiigundane ifumana igalelo elinamandla le-synaptic ukusuka kwi-ipsilateral ventral basal kunye ne-posterior thalamic nuclei, kunye ne-ipsilateral motor kunye ne-secondary somatosensory cortices, kunye ne-contralateral S1 (umzobo 4a). Ukubuyisela ipateni yangaphakathi, sosulela i-hCO nge-rabies virus-dG-GFP/AAV-G kwaye safakela i-hCO kwi-S1 ibuzi kwiintsuku ezi-3 kamva. Siqaphele ukubonakaliswa kwe-GFP exineneyo kwi-neurons ye-ipsilateral S1 kunye ne-ventral basal ganglia 7-14 iintsuku emva kokutshintshwa (Umfanekiso 4b, c). Ukongezelela, ukungcoliswa kwe-antibody ye-thalamic marker netrin G1 ibonise ubukho be-thalamic endings kwi-t-hCO (Fig. 4d, e). Ukuvavanya ukuba ngaba olu qikelelo oluhambelanayo lunokufumana iimpendulo ze-synaptic kwiiseli ze-t-hCO, senze ukurekhoda kweeseli ezipheleleyo kwiiseli zomntu kumacandelo abukhali we-thalamocortical layer. Ukukhuthazwa kombane kwi-rat S1, i-capsule yangaphakathi, i-white matter, i-fibers kufuphi ne-t-hCO okanye i-optogenetic activation ye-opsin-expressing endings thalamic in t-hCO ibangele i-EPSCs emfutshane-latency kwi-t-hCO neurons evezwe kwi-AMPA receptor antagonist NBQX. (Umfanekiso we-4f, g kunye nedatha eyandisiweyo, i-Fig. 9a-g). Ezi datha zibonisa ukuba i-t-hCO idityaniswe ngokwe-anatomically kwingqondo yegundane kwaye iyakwazi ukuqaliswa ngokusebenza kwezicubu zegundane.
a, Umzobo weSchematic welingo lokulandela umgada. b, i-GFP kunye nenkcazo ye-STEM121 ecacileyo yomntu phakathi kwe-t-hCO kunye ne-rat cortex ye-cerebral (iphaneli ephezulu). Kwakhona kuboniswe ukubonakaliswa kwe-GFP kwi-rat ipsilateral ventral basal nucleus (VB) (ezantsi ngasekhohlo) kunye ne-ipsilateral S1 (ezantsi ekunene). Ibar yesikali, 50 µm. Izikwere ezibomvu zimela iindawo zengqondo apho imifanekiso ithathwe khona. c, ubungakanani beeseli ezibonisa i-GFP (n = 4 iigundane). d, e - I-Netrin G1 + iitheminali zethalamic kwi-t-hCO. d ibonisa icandelo le-coronal equkethe i-t-hCO kunye ne-VB nuclei. Isikali bar, 2 mm. e ibonisa i-Netrin G1 kunye ne-STEM121 intetho kwi-t-hCO (ngasekhohlo) kunye ne-VB (ekunene) i-neurons. Ibar yesikali, 50 µm. Umgca wamachaphaza orenji ubonisa umda we-t-hCO. f, g, Iimpawu zangoku ze-t-hCO neurons emva kokuvuselela umbane kwi-S1 rat (f) okanye i-capsule yangaphakathi (g), kunye (emfusa) okanye ngaphandle (omnyama) NBQX (ngasekhohlo). I-EPSC amplitudes kunye nangaphandle kwe-NBQX (n = 6 S1 neurons, * P = 0.0119; kunye n = 6 i-capsule neurons yangaphakathi, ** P = 0.0022) (phakathi). Ipesenti ye-t-hCO neurons ebonisa i-EPSC ekuphenduleni ukuvuselela umbane we-rat S1 (f) okanye i-capsule yangaphakathi (g) (ekunene). i-aCSF, i-cerebrospinal fluid eyenziweyo. h, umzobo weskim somfuniselo womfanekiso we-2P (ekhohlo). Ukubonakaliswa kwe-GCaMP6s kwi-t-hCO (phakathi). Ibar yesikali, 100 µm. Ukuphela kwexesha le-Fluorescence ye-GCaMP6s (ekunene). i, Z-inqaku lomsebenzi ozenzekelayo we-fluorescence. j, umzobo weskim wokuvuselela amadevu. k, z-ifumene i-2P fluorescence trajectories kulingo olunye, ehambelana nokutenxa kwewhisk ngexesha elinguziro (umgca odayiweyo) kwimizekelo yeeseli. l, i-avareji yabemi-i-avareji ye-z-score iimpendulo zazo zonke iiseli ezihambelana nokutenxa ngewhisk ngexesha elinguziro (umgca odayiweyo) (obomvu) okanye izitampu zexesha ezenziwe ngokungenamkhethe (ngwevu). m. Umzobo weSchematic welinge lokumakishwa kwamehlo. n, Iijika zombane okrwada ukusuka kumzekelo we-t-hCO iseli ngexesha lokuvuselela i-laser eluhlaza okanye i-whisker deflection. Iintolo ezibomvu zibonisa izikhonkwane zokuqala ezibangelwa kukukhanya (phezulu) okanye okubangelwa kukuphambuka kwewhisk (ezantsi). I-Grey shading ibonisa amaxesha okuphambuka kwe-whisker. o, Incopho yokukhanya kwamaza kunye neempendulo zokuphambuka kwe-whisker. p, izikhonkwane zomzamo omnye, zihambelana nokuphambuka kwamabhovu kwiiseli zomzekelo. I-0 ibonisa ukutenxa kwe-whisker (umgca odayishiweyo). q, i-avareji yabemi-avareji ye-z-inqaku lokudubula kuzo zonke iiseli ze-photosensitive, ezilungelelaniswe ne-whisker enxaxhileyo ngexesha elinguziro (umgca odayiweyo) (obomvu) okanye izitampu zexesha ezenziwe ngokungakhethiyo (ezingwevu). r, Umyinge weeyunithi ze-photosensitive zihlengahlengiswe ngokuphawulekayo ngokunxaxha kwe-whisker (n = 3 iimpuku) (ekhohlo). Incopho ye-z-score latency (n = 3 iimpuku; n = 5 (luhlaza okhanyayo), n = 4 (luhlaza obumnyama), kunye n = 4 (cyan) iiyunithi zokumodareyitha iwhisker kwimpuku nganye) (ekunene). Idatha ichazwa njengentsingiselo ± ukutenxa okusemgangathweni
Emva koko sabuza ukuba i-t-hCO inokuthi isebenze nge-sensory stimuli kwi-vivo. Safakela i-hCO echaza izalathi zekhalsiyam ezifakwe ngokwemfuza kwi-GCaMP6 kwiimpuku ze-S1. Emva kweentsuku ze-150, senze i-fiber photometry okanye i-photon-photon imaging calcium (umzobo we-4h kunye nedatha eyandisiweyo, i-Fig. 10a). Sifumene ukuba iiseli ze-t-hCO zibonise umsebenzi wesingqisho ohambelanayo (Umfanekiso we-4i, iDatha eyandisiweyo, i-Figure 10b kunye neVidiyo eyoNgezelelweyo ye-1). Ukubonakalisa umsebenzi we-peak t-hCO, senze i-extracellular electrophysiological recordings kwi-anesthetized transplant rats (idatha eyandisiweyo, i-Fig. 10c-f). Siye savelisa ulungelelwaniso lwe-stereotaxic ukusuka kwimifanekiso ye-MRI; ngoko ke, ezi yunithi ezirekhodiweyo zimele putative human neurons, nangona i-electrophysiology iyodwa ayivumeli ukuba kumiselwe uhlobo lwemvelaphi. Siqaphele ukuqhuma komsebenzi ohambelanayo (idatha eyandisiweyo, i-Fig. 10d). Ukuqhuma kwahlala malunga ne-460 ms kwaye kwahlulwa ngamaxesha okuthula malunga ne-2 s (idatha eyandisiweyo, i-Fig. 10d, e). Iiyunithi zomntu ngamnye zidubule umyinge omalunga nemijikelo emithathu ngokugqabhuka ngakunye, okumalunga nama-73% eeyunithi ezibhalisiweyo ngokugqabhuka ngakunye. Imisebenzi yeeyunithi zomntu ngamnye yayinxulunyaniswa kakhulu, kwaye ezi zilungelelaniso zaziphezulu kunezo zeyunithi ezichongiweyo kwizilwanyana ezingagonywanga ezibhalwe phantsi kweemeko ezifanayo (idatha eyandisiweyo, i-Fig. 10f). Ukubonisa ngakumbi iimpendulo ze-spike ze-neurons ezichongiweyo ezithathwe ngabantu, senze imifuniselo yokubonisa ukukhanya kwiimpuku ezine-anesthetized ezifakwe kwi-hCO ebonisa ukukhanya kwe-cation channel i-rhodopsin 2 (hChR2), apho i-t-hCO neurons i-short-latency recognition (ngaphantsi kwe-10 ms-i-fifimulimlim) kwimpendulo ye-bluetoog. I-t-hCO neurons ibonise ukuqhuma komsebenzi ozenzekelayo kwiifrikhwensi ezifana nezo zibonwa kwi-calcium imaging, kunye nokurekhoda kwe-electrophysiological eyenziwa kwi-t-hCO ngokungabikho kokumakishwa kokukhanya (idatha eyandisiweyo, i-Fig. 10c-g). Akukho msebenzi ozenzekelayo wabonwa kwizigaba ezihambelanayo ze-hCO ezirekhodiweyo kwi-vitro. Ukuvavanya ukuba i-t-hCO inokuthi isebenze yi-sensory stimuli, sijike ngokufutshane i-whiskers ye-rat kude ne-t-hCO (Fig. 4j, m kunye nedatha eyandisiweyo, i-Fig. 10h, k). Ngokutsho kwezifundo zangaphambili8, i-10, i-subset ye-t-hCO iiseli zibonise umsebenzi owandisiweyo ekuphenduleni ukuchithwa kwe-whisker, engazange ibonwe xa idatha ifaniswa nezitampu zexesha elingahleliwe (umzobo 4k-q kunye nedatha eyandisiweyo, i-Fig. 10h-q). Enyanisweni, malunga ne-54% ye-opto-labeled single units ibonise ukunyuka okukhulu kwesantya sokuvusa emva kokuvuselela i-whisker, ukunyuka malunga ne-650 ms (Fig. 4r). Xa zidibene, ezi datha zibonisa ukuba i-t-hCO ifumana amagalelo asebenzayo afanelekileyo kwaye inokuthi isebenze ngokuvuselela indalo.
Siye saphanda ukuba i-t-hCO inokusebenzisa iisekethe kwiimpuku ukulawula ukuziphatha. Siqale saphanda ukuba ngaba ii-axon ze-t-hCO neurons ziprojekthi kwiithishu ezijikelezileyo zerat. Sosulela i-hCO nge-lentivirus ekhowudayo i-hChR2 edityaniswe kwi-EYFP (hChR2-EYFP). Emva kweentsuku ze-110, siye saqwalasela ukubonakaliswa kwe-EYFP kwimimandla ye-cortical ipsilateral, kuquka i-auditory, i-motor, kunye ne-somatosensory cortices, kunye nemimandla ye-subcortical, kuquka i-striatum, i-hippocampus, kunye ne-thalamus (umzobo 5a). Ukuvavanya ukuba ingaba olu qikelelo lunamandla na lunokubangela iimpendulo ze-synaptic kwiiseli zeempuku, siye sasebenza ngokubonakalayo iiseli ze-t-hCO ezibonisa i-hChR2-EYFP ngokurekhoda iiseli ze-cerebral cortex kwiindawo ezibukhali zobuchopho. Ukusebenza kwee-axons ze-t-hCO ezinokukhanya okuluhlaza okwesibhakabhaka kubangele ii-EPSC ze-latency ezimfutshane kwi-rat pyramidal cortex neurons, ezazivalwe yi-NBQX (Imifanekiso 5b-g). Ukongezelela, ezi mpendulo zinokuvalwa yi-tetrodotoxin (TTX) kwaye zibuyiselwe yi-4-aminopyridine (4-AP), ebonisa ukuba zibangelwa uxhulumaniso lwe-monosynaptic (Umfanekiso 5e).
a, Umzobo weSchematic of axon tracking (ekhohlo). t-hCO EYFP intetho (ekunene). Ibar yesikali, 100 µm. I-A1, i-cortex yokuva, i-ACC, i-cingulate cortex yangaphambili, d. i-striatum, i-dorsal striatum, i-HPC, i-hippocampus; I-diaphragm, i-lateral septum, i-mPFC, i-cortex ye-prefrontal ye-medial, i-piri, i-piriform cortex, i-v. striatum, i-ventral striatum, i-VPM, i-nucleus ye-ventropostomedial ye-thalamus, i-VTA, i-ventral tegmental region. Izikwere ezibomvu zimela iindawo zengqondo apho imifanekiso ithathwe khona. b, Umzobo weSchematic wovavanyo lokuvuselela. c, d, Imizekelo yempendulo ye-photocurrent eyenziwe ngokukhanya eluhlaza okwesibhakabhaka (phezulu) kunye ne-voltage (ezantsi) kumntu (c) EYFP + t-hCO okanye i-rat (d) EYFP- iiseli. e, f, Iimpawu zangoku ze-rat neurons emva kokuvuselela ukukhanya okuluhlaza okwesibhakabhaka kwe-t-hCO axons kunye ne-TTX kunye ne-4-AR (eluhlaza), i-TTX (grey) okanye i-aCSF (emnyama) (e), kunye (i-violet) okanye ngaphandle (emnyama) ) I-NBQX (e). g, i-latency yeempendulo ezibangelwa ukukhanya okwesibhakabhaka kwiiseli zegundane (n = 16 iiseli); iibar ezithe tyaba zibonisa umndilili wokulibaziseka (7.13 ms) (ekhohlo). I-amplitude yee-EPSC ezikhutshwe ngokukhanya ezirekhodwe kunye okanye ngaphandle kwe-NBQX (n = iiseli ezi-7; *** P <0.0001) (phakathi). I-amplitude yee-EPSC ezikhutshwe ngokukhanya ezirekhodwe kunye okanye ngaphandle kwe-NBQX (n = iiseli ezi-7; *** P <0.0001) (phakathi). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). I-Amplitude yee-EPSC ezenziwe ngokukhanya ezirekhodiweyo okanye ngaphandle kwe-NBQX (n = iiseli ezi-7; *** P <0.0001) (phakathi).使用或不使用NBQX 记录的光诱发EPSC 的振幅（n = 7 个细胞；***P <0.0001）（中）.使用或不使用NBQX 记录的光诱发EPSC 的振幅（n = 7 个细胞；***P <0.0001）（中）. Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). I-Amplitude yee-EPSC ezenziwe ngokukhanya ezirekhodiweyo okanye ngaphandle kwe-NBQX (n = iiseli ezi-7; *** P <0.0001) (phakathi).Ipesenti yeeseli zeempuku ezibonisa ii-EPSCs eziphendula ukukhanya okuluhlaza okwesibhakabhaka (ekunene). h, Umzobo weSchematic womsebenzi wokuziphatha. d0, usuku 0. i. Ukusebenza kwezilwanyana ezingumzekelo ngosuku loku-1 (ekhohlo) okanye usuku lwe-15 (ekunene) loqeqesho. Inani eliqhelekileyo lokukhothwa okwenziwe ngosuku 1 (ekhohlo) okanye usuku lwe-15 (iziko lasekunene) (n = 150 izilingo zokukhanya okuluhlaza okwesibhakabhaka, n = 150 izilingo zokukhanya okubomvu; ***P <0.0001). Inani eliqhelekileyo lokukhothwa okwenziwe ngosuku 1 (ekhohlo) okanye usuku lwe-15 (iziko lasekunene) (n = 150 izilingo zokukhanya okuluhlaza okwesibhakabhaka, n = 150 izilingo zokukhanya okubomvu; ***P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 испытаний с синим светойм, nысный = 150 светом ***P <0,0001). Mean inani lokukhothwa okwenziwe ngosuku 1 (ekhohlo) okanye usuku lwe-15 (embindini ekunene) (n = 150 izilingo zokukhanya okuluhlaza okwesibhakabhaka, n = 150 izilingo zokukhanya okubomvu; ***P <0.0001).第1 天（左）或第15 天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150 试验，n = 150 0.0001).第1 天（左）或第15 天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150 次纕0P*0 次0 Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 испытаний с синим светойм, nысный = 150 светом ***P <0,0001). Mean inani lokukhothwa okwenziwe ngosuku 1 (ekhohlo) okanye usuku lwe-15 (embindini ekunene) (n = 150 izilingo zokukhanya okuluhlaza okwesibhakabhaka, n = 150 izilingo zokukhanya okubomvu; ***P <0.0001).Izikhothwa ezongezelekayo zovavanyo lokukhanya obomvu noluhlaza ngosuku 1 (embindini ekhohlo) okanye usuku 15 (ekunene). NS, ayibalulekanga. j, k, Iimpawu zokuziphatha kuzo zonke izilwanyana ezitshintshwe nge-t-hCO evakalisa i-hChR2-EYFP (j) okanye ukulawula i-fluorophore (k) ngosuku lwe-1 okanye i-15 (hChR2-EYFP: n = 9 iigundane, ** P = 0.0049; ulawulo: n = 9, P9 = 0. l, I-Evolution yamanqaku akhethiweyo (n = 9 hChR2, n = 9 ulawulo; ** P <0.001, ***P <0.0001). l, I-Evolution yamanqaku akhethiweyo (n = 9 hChR2, n = 9 ulawulo; ** P <0.001, ***P <0.0001). l, Эволюция показателя предпочтения (n = 9 hChR2, n = 9 контрольных; **P <0,001, ***P <0,0001). l, I-Evolution yamanqaku akhethwayo (n = 9 hChR2, n = 9 ulawulo; ** P <0.001, ***P <0.0001). 1 1 l, Эволюция показателей предпочтения (n = 9 hChR2, n = 9 контролей; **P <0,001, ***P <0,0001). l, I-Evolution yamanqaku akhethwayo (n = 9 hChR2, n = 9 ulawulo; ** P <0.001, ***P <0.0001).m, inkcazo ye-FOS ekuphenduleni i-optogenetic activation ye-t-hCO kwi-S1. Imifanekiso yentetho ye-FOS (ekhohlo), kunye nobungakanani (n = 3 kwiqela ngalinye; *P <0.05, **P <0.01 kunye ne-***P <0.001) (ekunene) ziyaboniswa. Imifanekiso yentetho ye-FOS (ekhohlo), kunye nobungakanani (n = 3 kwiqela ngalinye; *P <0.05, **P <0.01 kunye ne-***P <0.001) (ekunene) ziyaboniswa. Показаны изображения экспрессии FOS (слева) и количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 kunye *** P <0,001) (справа). Imifanekiso yentetho ye-FOS (ekhohlo) kunye nobungakanani (n = 3 kwiqela ngalinye; *P<0.05, **P<0.01, kunye ne-***P<0.001) ibonisiwe (ekunene).显示了FOS 表达（左）和量化（每组n = 3;*P <0.05,**P <0.01 和***P <0.001）（右）的图像。显示了FOS 表达（左）和量化（每组n = 3;*P <0.05,**P <0.01 和***P <0.001）（右）的图像。 Показаны изображения экспрессии FOS (слева) и количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 kunye *** P <0,001) (справа). Imifanekiso yentetho ye-FOS (ekhohlo) kunye nobungakanani (n = 3 kwiqela ngalinye; *P<0.05, **P<0.01, kunye ne-***P<0.001) ibonisiwe (ekunene).Ibar yesikali, 100 µm. Idatha ichazwa njengentsingiselo ± impazamo eqhelekileyo ye-BLA, i-basolateral tonsil, i-MDT, i-dorsomedial thalamic nucleus, i-PAG, i-periaqueductal grey.
Okokugqibela, sibuze ukuba i-t-hCO iyakwazi ukulungelelanisa impatho yeempuku. Ukuvavanya oku, safakela i-hChR2-EYFP-ebonisa i-hCO kwi-S1, kwaye emva kweentsuku ze-90, safakela iifibers ze-optical kwi-t-hCO yokuhanjiswa kokukhanya. Emva koko siqeqeshe iigundane nge-paradigm ye-operant conditioning modified (Umfanekiso we-5h). Sibeke izilwanyana kwigumbi lokuvavanya ukuziphatha kwaye sisebenzise ngokungaqhelekanga i-5 yesibini eluhlaza okwesibhakabhaka (473 nm) kunye nobomvu (635 nm) i-laser stimuli. Izilwanyana zafumana umvuzo wamanzi ukuba zikhothe ngexesha lokuvuselela ukukhanya okuluhlaza kodwa azizange zikhothe ngexesha lokuvuselela ukukhanya okubomvu. Ngosuku lokuqala loqeqesho, izilwanyana azizange zibonise umehluko ekukhotheni xa zikhuthazwa ngokukhanya okuluhlaza okwesibhakabhaka okanye obomvu. Nangona kunjalo, ngomhla we-15, izilwanyana ezitshintshwe nge-hCO ebonisa i-hChR2-EYFP yabonisa ukukhothwa okusebenzayo ngakumbi xa kukhuthazwa ngokukhanya okuluhlaza okwesibhakabhaka xa kuthelekiswa nokuvuselela ukukhanya okubomvu. Olu tshintsho ekuziphatheni kokukhotha aluzange lubonwe kwizilwanyana zokulawula ezitshintshwe kunye ne-hCO evakalisa ulawulo lwe-fluorophore (izinga lokuphumelela lokufunda: hChR2 89%, EYFP 0%, Figure 5i-1 kunye neVidiyo ye-Supplementary 2). Ezi datha zibonisa ukuba iiseli ze-t-hCO zinokuvula ii-neuron zegundane ukuvuselela isimilo sokufuna umvuzo. Ukufumana ukuba yiyiphi i-rat t-hCO ye-neural circuits enokuthi ibandakanyeke kolu tshintsho lokuziphatha, thina i-optogenetically activated t-hCO kwizilwanyana eziqeqeshwe kunye nezicubu ezivuniweyo emva kwemizuzu ye-90. I-Immunohistochemistry iveze imbonakalo yeprotein ye-FOS exhomekeke kumsebenzi kwimimandla emininzi yobuchopho ebandakanyeka kwindlela yokuziphatha ekhuthazayo, kubandakanya i-medial prefrontal cortex, i-medial thalamus, kunye ne-periaqueductal grey, echazwe nokuba kukulawulwa kwezilwanyana okanye kwizilwanyana. irayisi. 5m). Kuthatyathwe kunye, ezi datha zibonisa ukuba i-t-hCO inokulungelelanisa umsebenzi we-rat neuronal ukuqhuba ukuziphatha.
I-Neural organoids imele inkqubo ethembisayo yokufunda uphuhliso lomntu kunye nezifo kwi-vitro, kodwa zisikelwe umda kukungabikho konxibelelwano phakathi kweesekethe ezikhoyo kwi-vivo. Siye saphuhlisa iqonga elinoveli apho safakela i-hCO kwi-S1 yeempuku zasemva kokubeleka ezibuthathaka ukuze sifunde ukukhula kweeseli zabantu kunye nokusebenza kwi-vivo. Siye sabonisa ukuba i-t-hCO iphuhlisa iintlobo zeeseli ezivuthiweyo ezingabonwanga kwi-vitro28 kwaye i-t-hCO i-anatomically kwaye isebenza ngokudibeneyo kwingqondo ye-rodent. Ukudityaniswa kwe-t-hCO kwii-rodent neural circuits kusivumele ukuba siseke ikhonkco phakathi komsebenzi weselula kunye nokuziphatha kwezilwanyana ezifundwayo, ebonisa ukuba i-t-hCO neurons inokulungelelanisa umsebenzi we-rat neuronal ukuqhuba iimpendulo zokuziphatha.
Iqonga esilichazayo lineengenelo ezininzi ngaphezulu kophando lwangaphambili malunga nokufakela iiseli zomntu kwiingqondo zeempuku. Okokuqala, safakela i-hCO kwi-cortex ekhulayo yeempuku zasemva kokubeleka, ezinokuthi ziququzelele ukuhlanganiswa kwe-anatomical kunye nokusebenza. Okwesibini, ukubeka iliso kwe-t-hCO MRI kwasivumela ukuba sifunde indawo ye-graft kunye nokukhula kwezilwanyana eziphilayo, okusivumela ukuba siqhube izifundo zexesha elide zezilwanyana kunye nokuseka ukuthembeka kwemigca yeeseli ezininzi ze-hiPS. Okokugqibela, safakela i-organoids ezinzileyo, endaweni yokumiswa kweseli enye, engonakalisi kangako iiseli zomntu kwaye inokukhuthaza ukudityaniswa kunye nokuveliswa kwee-cortex neurons kubuchopho begundane.
Siyavuma ukuba nangona ukuqhubela phambili kweli qonga, izithintelo zexesha, zendawo, kunye neentlobo ezahlukeneyo zithintela ukubunjwa kwee-neural circuits ngokuthembeka okuphezulu, nasemva kokutshintshwa kwinqanaba lokuqala lophuhliso. Ngokomzekelo, akucaci ukuba umsebenzi ozenzekelayo obonwa kwi-t-hCO ubonisa i-phenotype yophuhliso efana nomsebenzi wesingqisho obonwa ngexesha lophuhliso lwe-cortical, okanye nokuba kungenxa yokungabikho kweentlobo zeeseli ezicinezelayo ezikhoyo kwi-t-hCO. Ngokufanayo, akucaci ukuba ukungabikho kwe-lamination kwi-t-hCO kuchaphazela kangakanani uxhumano lwe-chain30. Umsebenzi wexesha elizayo uya kugxila ekudibaniseni ezinye iintlobo zeeseli ezifana ne-microglia yomntu, iiseli ze-endothelial zabantu, kunye nemilinganiselo eyahlukeneyo ye-GABAergic interneurons njengoko kubonisiwe kusetyenziswa i-assembly 6 in vitro, kunye nokuqonda indlela ukuhlanganiswa kwe-neural kunye nokucubungula okwenzekayo kwi-t-hCO etshintshileyo. amanqanaba e-transcriptional, synaptic kunye nokuziphatha kwiiseli ezifunyenwe kwizigulane.
Ngokubanzi, eli qonga le-vivo limele isixhobo esinamandla esinokuncedisana nophuhliso lobuchopho bomntu kwi-vitro kunye nophando lwezifo. Silindele ukuba eli qonga liza kusivumela ukuba sifumane i-phenotypes ye-novel strand-level phenotypes kwezinye iiseli eziphuma kwisigulane kunye nokuvavanya iindlela ezintsha zonyango.
Senze i-hCO2.5 kwiiseli ze-HiPS njengoko kuchaziwe ngaphambili. Ukuqalisa imveliso ye-hCO esuka kwiiseli ze-hiPS ezikhuliswe kwiileyiti ze-feeder, iikholoni ezingaguqukiyo zeeseli ze-hiPS zaye zasuswa kwizitya zenkcubeko kusetyenziswa i-dispase (0.35 mg / mL) kwaye idluliselwe kwiinkcubeko zeplastiki ezinamathele kwi-ultra-low ezinezitya ezine-hiPS cell culture medium. (I-Corning) yongezwa ezimbini ze-SMAD inhibitors dorsomorphine (5 μM; P5499, Sigma-Aldrich) kunye ne-SB-431542 (10 μM; 1614, Tocris) kunye ne-ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Ngethuba leentsuku zokuqala ze-5, i-cell medium ye-hiPS yatshintshwa imihla ngemihla kwaye i-dorsomorphine kunye ne-SB-431542 yongezwa. Ngosuku lwesithandathu ekunqunyanyisweni, i-neural spheroids idluliselwe kwi-neural medium equkethe i-neurobasal-A (10888, i-Life Technologies), i-B-27 yongezelela ngaphandle kwe-vitamin A (12587, i-Life Technologies), i-GlutaMax (1: 100, i-Life Technologies), i-penicillin kunye ne-streptomy0, i-Lifeed0 kunye ne-Technologies yokukhula kunye ne-Lifeed0 kunye ne-Technologies ye-Technologies (1:1) factor (EGF; 20 ng ml-1; R & D Systems) kunye ne-fibroblast factor factor 2 (FGF2; 20 ng ml-1; R&D Systems) kude kube yimini ye-24. Ukususela kwimini ye-25 ukuya kwi-42, i-medium yongezwa nge-brain-derived neurotrophic factor (BDNF; 20 trophin; 20 trophin; 20 trophin; 3 trophin; 20 ng ml−1; Ngosuku lwesithandathu ekunqunyanyisweni, i-neural spheroids idluliselwe kwi-neural medium equkethe i-neurobasal-A (10888, i-Life Technologies), i-B-27 yongezelela ngaphandle kwe-vitamin A (12587, i-Life Technologies), i-GlutaMax (1: 100, i-Life Technologies), i-penicillin kunye ne-streptomy0, i-Lifeed0 kunye ne-Technologies yokukhula kunye ne-Lifeed0 kunye ne-Technologies ye-Technologies (1:1) factor (EGF; 20 ng ml-1; R & D Systems) kunye ne-fibroblast factor factor 2 (FGF2; 20 ng ml-1; R&D Systems) kude kube yimini ye-24. Ukususela kwimini ye-25 ukuya kwi-42, i-medium yongezwa nge-brain-derived neurotrophic factor (BDNF; 20 trophin; 20 trophin; 20 trophin; 3 trophin; 20 ng ml−1;Ngomhla wesithandathu ekumisweni, i-neural spheroids idluliselwe kwi-neural medium equkethe i-Neurobasal-A (10888, i-Life Technologies), i-B-27 yongezelela ngaphandle kwe-vitamin A (12587, i-Life Technologies), i-GlutaMax (1: 100, i-Life Technologies), i-penicillin.и стрептомицин (1:100, Life Technologies) и дополнены эпидермальным фактором роста (EGF; 20 нг/мл; IiNkqubo zeR&D) и фактором роста фибл20Fласто фибл20Gсто фибл20Gсто; IiNkqubo zeR&D) до 24-го дня. kunye ne-streptomycin (1: 100, i-Life Technologies) kunye nokuxhaswa nge-epidermal factor factor (EGF; 20 ng / ml; Iinkqubo ze-R & D) kunye ne-fibroblast factor factor 2 (FGF2; 20 ng / ml; Iinkqubo ze-R & D) kude kube yimini ye-24.Ukususela kwiintsuku ze-25 ukuya kwi-42, i-brain-derived neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) kunye neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) yongezwa kwi-medium, iguqula i-medium yonke imihla.在悬浮的第6 天，将神经球体转移到含有neurobasal-A(10888，Life Technologies) Technologies, GlutaMax (1:100, Life Technologies), 青霉素的神经培养基中和链霉素 (1:100, Life Technologies) ml-1;R&D Systems）和成纤维细胞生长因子2（FGF2；20 ng ml-1;R&D Systems）直至第24 天.在 悬浮 的第 第 6 天 将 神经 球体 转移 含有 含有 neurobasal-a (10888 , Life Technologies） 不 含 维移 含有(12587 , Life Technologies) Glutamax (1: 100 , Life TechNOGIS青霉素的 神经 培养 基生长 因子 （（20 ng ml-1 ; r & d Systems） 成 纤维 细胞 生长 2（fgf2; 20 ng ml- 1；R&D Systems）笴2） На 6-й день суспензии нейросферы были переведены на добавку, содержащую нейробазал-А (10888, Life Technologies), добавку 5 В-7 Life Technologies), GlutaMax (1:100, Life Technologies), пенициллин- нейтрализованный стрептомицин (1:100, Life Technologies) с добавлением эпидермальго факторангей факторан (1:100, Life Technologies) ; IiNkqubo ze-R&D) kunye ne-фактора роста фибробластов 2 (FGF2; 20 нг мл-1) 1; Ngosuku olu-6, ukumiswa kwe-neurofricare kwatshintshelwa kwisongezelelo esine-neurobasal-o (1888, itekhnoloji yobomi), i-Glutamax (1: 27, itekhnoloji yobomi), itekhnoloji yobomi) ye-vitamidel atwermal (EGF; 20 ng ml-1; R & D Systems) kunye ne-fibroblast factor factor 2 (FGF2; 20 ng ml-1) 1; IiNkqubo zeR&D) до 24-го дня. IiNkqubo zeR&D) kude kube ngumhla wama-24.Ukususela kwiintsuku ze-25 ukuya kwi-42, i-brain-derived neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) kunye ne-neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) yongezwa kwinkcubeko yenkcubeko yonke imihla. Utshintsho oluphakathi kanye.Ukususela ngomhla we-43, i-hCO yagcinwa kwi-neurobasal-A medium engaxhaswanga (NM; 1088022, Thermo Fisher) kunye noshintsho oluphakathi rhoqo kwiintsuku ze-4-6. Ukufumana i-hCO kwiiseli ze-hiPS ezikhuliswe phantsi kweemeko ezingenasondlo, iiseli ze-hiPS zifakwe kwi-Accutase (AT-104, i-Innovate Cell Technologies) kwi-37 ° C kwimizuzu ye-7, idityaniswe kwiiseli ezizimeleyo, kwaye ifakwe kwiiplate ze-AggreWell 800 (34815, STEMCELL i-Technologies ye-6 ye-cell × i-6 ye-cell × i-3 ye-cell) I-Essential 8 medium yongezwa nge-ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Emva kweeyure ze-24, amajelo eendaba emigodini afakwe umbhobho phezulu nangaphantsi kwimidiya equlethe i-Essential 6 media (A1516401, i-Life Technologies) eyongezwa nge-dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) kunye ne-SB-431542 (10 μM; 1614). , Tocrida). Ukususela kwiintsuku ze-2 ukuya kwi-6, i-Essential 6 medium yatshintshwa imihla ngemihla nge-dorsomorphine kunye nokuncedisa i-SB-431542. Ukususela ngomhla wesithandathu, ukumiswa kwe-neurosphere kwadluliselwa kwi-neurobasal medium kwaye igcinwe njengoko kuchazwe ngasentla.
Zonke iinkqubo zezilwanyana zenziwe ngokuhambelana nezikhokelo zokhathalelo lwezilwanyana ezivunyiweyo yiKomiti yoLawulo lweZilwanyana zeYunivesithi yaseStanford University (APLAC). Iimpuku ze-euthymic RNU (rnu/+) ezikhulelweyo zathengwa (Charles River Laboratories) okanye zahlaliswa. Izilwanyana zagcinwa kwi-12 yeyure yokukhanya-ubumnyama bomjikelo kunye nokutya kunye namanzi ad libitum. Amantshontsho eempuku anqunu (FOXN1–/–) aneminyaka emithathu ukuya kwesixhenxe ubudala aye achongwa ngokukhula kwamabhovu angekavuthwa phambi kokuba anqunyulwe. Amantshontsho (amadoda nabasetyhini) athotywa i-anesthetized nge-2-3% isoflurane kwaye abekwe kwifreyimu ye-stereotaxic. I-trepanation ye-skull ene-diameter emalunga ne-2-3 mm ngaphezu kwe-S1 yenziwa ngelixa igcina ingqibelelo ye-dura mater. Emva koko sebenzisa inaliti ye-30-G (malunga ne-0.3 mm) ngaphandle nje kwe-craniotomy ukugqobhoza i-dura. Emva koko sebenzisa i-HCO kwi-parafilm encinci ye-3 × 3 cm kwaye ususe okuphakathi. Ukusebenzisa i-syringe ye-Hamilton efakwe kwi-23 G, i-45 ° inaliti, udwebe ngobumnene i-hCO kwindawo ekude kakhulu yenaliti. Emva koko fakela isirinji kwimpompo yesirinji eqhagamshelwe kwisixhobo se-stereotaxic. Emva koko beka incam yenaliti phezu kwangaphambili eyenziwe 0.3 mm umngxuma wokugqobhoza ububanzi kwi dura (z = 0 mm) kwaye ucuthe isirinji 1-2 mm (z = malunga -1.5 mm) de inaliti ibe phakathi dura mater A. a dense seal yenziwe. Emva koko phakamisa isirinji kumbindi we-cortical surface kwi-z = -0.5 mm kwaye utofe i-hCO ngesantya se-1-2 µl ngomzuzu. Emva kokugqitywa kwenaliti ye-hCO, inaliti ihoxiswa ngesantya se-0.2-0.5 mm ngomzuzu, ulusu lufakwe kwi-sutured, kwaye i-puppy ifakwe ngokukhawuleza kwi-pad yokufudumala efudumeleyo kuze kube yilapho ibuyiselwa ngokupheleleyo. Ezinye izilwanyana zaye zafakelwa kwezinye iindawo.
Zonke iinkqubo zezilwanyana zenziwa ngokuhambelana neYunivesithi yaseStanford APLAC evunyiweyo yezikhokelo zokunakekelwa kwezilwanyana. Iigundane (ezinkulu kuneentsuku ze-60 emva kokutshintshwa) zenziwe nge-5% isoflurane anesthesia kunye ne-anesthetized nge-1-3% isoflurane ngexesha lokucinga. Ukujonga, i-7 Tesla ikhusele ngokukhutheleyo i-horizontal borehole scanner i-Bruker (i-Bruker Corp.) kunye ne-International Electric Company (IECO) i-gradient drive, ukufakwa kwe-gradient ekhuselekileyo kunye ne-diameter yangaphakathi ye-120 mm (600 mT / m, 1000 T / m / s) isetyenziswe ngokusebenzisa i-AVANCE. III, iitshaneli ezisibhozo ze-coil ezininzi ze-RF kunye nezakhono ezininzi ezingundoqo, kunye neqonga elikhaphayo leParavision 6.0.1. Urekhodisho lwenziwa kusetyenziswa ikhoyili yeRF yevolumetric edityanisiweyo enedayamitha yangaphakathi eyi-86 mm kunye nekhoyili yeRF enezitshaneli ezine ezipholileyo zokwamkela kuphela. I-Axial 2D Turbo-RARE (ixesha lokuphindaphinda = 2500 ms, ixesha le-echo = 33 ms, i-2 i-avareji) kunye ne-16 slice captures, ubukhulu be-slice 0.6-0.8 mm, equkethe i-256 × 256 iisampuli. Imiqondiso yafunyanwa kusetyenziswa ikhoyili yeRF ye-quadrature transceiver volumetric RF enedayamitha yangaphakathi eyi-2 cm (Rapid MR International, LLC). Ekugqibeleni, sebenzisa i-Imaris eyakhelwe ngaphakathi (BitPlane) imisebenzi yoqikelelo lomphezulu wonikezelo lwe-3D kunye nohlalutyo lwevolumu. Ukutshintshwa okuyimpumelelo kwachazwa njengenye apho iindawo ze-MRI eziqhubekayo ze-T2-weighted signal zenziwa kwi-hemisphere etshintshwayo. Ukugatywa kwe-graft kwachazwa njenge-graft engazange ivelise imimandla ye-T2-weighted MRI yesiginali kwi-hemisphere yokutshintshwa. I-Subcortical t-hCO ayizange ibandakanywe kuhlalutyo olulandelayo.
Ukuvakalisa ngokuzinzileyo i-GCaMP6s kwi-hCO kwimifanekiso ye-calcium yeefotoni ezimbini, iiseli ze-hiPS zosulelwe yi-pLV[Exp] -EF1a::GcaMP6s-WPRE-Puro elandelwa kukukhethwa kwamayeza okubulala iintsholongwane. Ngokufutshane, iiseli zaye zahlulwa kunye ne-EDTA kwaye zanqunyanyiswa kwi-1 ml ye-Essential 8 medium kubuninzi beeseli ezimalunga ne-300,000 phambi kwe-polybrene (5 μg / ml) kunye ne-15 μl yentsholongwane. Emva koko iiseli zifakwe kwi-suspension imizuzu engama-60 kwaye zihlwayelwe kubuninzi beeseli ezingama-50,000 kwiqula ngalinye. Emva kokudibana, iiseli zaphathwa nge-5-10 μg ml-1 puromycin iintsuku ezi-5-10 okanye de kwavela amakholoni azinzileyo. Usulelo olubukhali lwe-hCO lwenziwa njengoko kuchaziwe ngaphambili5 kunye nohlengahlengiso oluthile. Ngokufutshane, tshintshela usuku lwe-30-45 hCO kwi-1.5 ml ye-Eppendorf microcentrifuge tubes equlethe i-100 µl ye-nerve medium. Emva koko malunga ne-90 µl yesiphakathi siyasuswa, i-3-6 µl ye-high titer lentivirus (ukusuka ku-0.5 x 108 ukuya kwi-1.2 x 109) yongezwa kwityhubhu, kwaye i-hCO igqithiselwa kwi-incubator imizuzu engama-30. Emva koko yongeza i-90–100 µl ephakathi kwityhubhu nganye kwaye ubuyisele imibhobho kwi-incubator ngobusuku. Ngosuku olulandelayo, tshintshela i-hCO kwi-nerve medium medium kwiiplate zokuncamathela eziphantsi. Emva kweentsuku ezisi-7, i-hCO yatshintshelwa kwiipleyiti ezisezantsi zeglasi ezingama-24 ukuze zibonwe kwaye zivavanywe umgangatho wosulelo. i-pLV[Exp]-SYN1::EYFP-WPRE kunye ne-plV[Exp]-SYN1::hChR2-EYFP-WPRE zenziwe yiVectorBuilder. I-Lentivirus isetyenziswa kwiimvavanyo ezininzi kuba idityaniswe kwijenome yomkhosi, ivumela ukubonakaliswa kofuzo lwentatheli kwimigca yeeseli ezosulelekileyo. Ukulandelwa kwe-rabies, imini ye-30-45 hCO idibene ne-rabies-ΔG-eGFP kunye ne-AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), ihlanjwe ngokucokisekileyo iintsuku ze-3, kwaye ifakwe kwiigundane kwi-S1 kwaye igcinwe kwiintsuku ze-7-14.
Kwi-immunocytochemistry, izilwanyana zahlanjululwa kwaye zixutywe nge-PBS zilandelwa yi-4% ye-paraformaldehyde (PFA kwi-PBS; i-Electron Microscopy Sciences). Iingqondo zilungiswe kwi-4% PFA kwiiyure ze-2 okanye ubusuku bonke kwi-4 ° C, i-cryopreserved kwi-30% ye-sucrose kwi-PBS kwiiyure ze-48-72, kwaye ifakwe kwi-1: 1, i-30% ye-sucrose: i-OCT (I-Tissue-Tek OCT Compound 4583, i-Sakura Finetes 3 yecandelo le-coronat0 yenziwe nge-coronat3 ye-coronat (Leica). Kwi-immunohistochemistry yamacandelo angqingqwa, izilwanyana zaxutywa nge-PBS, kwaye ingqondo yachithwa kwaye yahlulwa ngokwe-coronally kwi-300-400 µm kusetyenziswa i-vibratome (i-Leica) kwaye amacandelo ayenziwe nge-4% PFA kwimizuzu ye-30. Emva koko i-cryosections okanye amacandelo amaninzi ahlanjwe nge-PBS, ivalwe ngeyure eli-1 kwindawo yokushisa kwegumbi (i-10% ye-donkey serum eqhelekileyo (NDS) kunye ne-0.3% ye-Triton X-100 ehlanjululwe kwi-PBS) kwaye ivalwe ngesisombululo sokuthintela kwi-4 ° C. – I-Incubation Cryosections yafukanywa ngobusuku kwaye amacandelo ashinyeneyo afukanywa iintsuku ezi-5. I-antibodies ephambili esetyenzisiweyo yayiyi: i-anti-NeuN (i-mouse, 1: 500; ab104224, abcam) i-anti-CTIP2 (i-rat, i-1: 300; i-ab18465, i-abcam), i-anti-GFAP (umvundla, 1: 1,000; Z0334, i-Dako), i-anti-GFP0, i-GTX0, i-3, i-1, i-0, i-anti-GFP0; GeneTex), anti-HNA (mouse, 1:200; ab191181, abcam), anti-NeuN (umvundla, 1:500; ABN78, Millipore), anti-PDGFRA (umvundla, 1:200; sc-338, Santa Cruz), anti-PPP201R; I-HPA047819, i-Atlas Antibodies), i-anti-RECA-1 (impuku, 1:50; ab9774, abcam), i-anti-SCG2 (umvundla, 1:100; 20357-1-AP, Proteintech), i-anti-SOX9 (ibhokhwe, 1:500; i-RA&30D Systema7), i-R&30D1 1:100 AF1166, R & D Systems), anti-STEM121 (mouse, 1:200; Y40410, Takara Bio), anti-SATB2 (mouse, 1:50; ab51502, abcam), anti-GAD65/67 (umvundla, 1:400; (ibhokhwe, 1:100; ab5076, abcam). I-antibodies ephambili esetyenzisiweyo yayiyi: anti-NeuN (mouse, 1:500; ab104224, abcam) anti-CTIP2 (i-rat, 1: 300; ab18465, abcam), i-anti-GFAP (umvundla, 1: 1,000; Z0334, Dako), i-anti-GFP0, i-GTX0, i-3 GTX 0; GeneTex), anti-HNA (mouse, 1:200; ab191181, abcam), anti-NeuN (umvundla, 1:500; ABN78, Millipore), anti-PDGFRA (umvundla, 1:200; sc-338, Santa Cruz), anti-PPP201R; HPA047819, Atlas Antibodies), anti-RECA-1 (mouse, 1:50; ab9774, abcam), anti-SCG2 (umvundla, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (ibhokhwe, 1:500; Neta1 Systems, Neta, 30D , RAFgorin); 1:100; AF1166, R & D Systems), anti-STEM121 (mouse, 1:200; Y40410, Takara Bio), anti-SATB2 (mouse, 1:50; ab51502, abcam), anti-GAD65/67 (umvundla, 1:400, anti-ABIN90) (ibhokhwe, 1:100; ab5076, abcam). Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2 (крысиные, 1:5840); анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA (мышь, 1:200; ab1911181, ab1911181, ab1911181, 1:500; ABN78, Millipore), анти-PDGFRA (кролик, 1:200; sc-338, Санта-Круз), анти-PPP1R17 (кролик, 1:200; HPA047819, Atlas, Atlas, Antibodies) 1:50; ab9774, abcam), анти-SCG2 (кролик , 1:100; 20357-1-AP, Proteintech), анти-SOX9 (козий, 1:500; AF3075, R&D Systems), нетриз10, G61; IiNkqubo ze-R&D), анти-STEM121 (мышиный , 1:200; Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кроли00, AB9, ABN; анти-IBA1 (коза, 1 :100; аб5076, абкам). Ii-antibodies eziphambili ezisetyenzisiweyo zaziyi: anti-NeuN (mouse, 1:500; ab104224, abcam), i-anti-CTIP2 (i-rat, 1: 300; ab18465, abcam), i-anti-GFAP (umvundla, 1: 1000; Z0334, Dako), i-anti-GFP0, i-GTX7, i-GTX9, i-anti-GFP, i-GTX 0, i-GTX 0, i-anti-GFP, i-GTX00, i-300 ye-anti anti-HNA (mouse, 1:200; ab191181, abcam), anti-NeuN (umvundla, 1:500; ABN78, Millipore), anti-PDGFRA ( umvundla, 1: 200; sc-338, Santa Cruz), anti-PPP1R17, 1: 1, HPAb4, 17 HPAb4; Antibodies), anti-RECA-1 (impuku, 1:50; ab9774, abcam), anti- SCG2 (umvundla, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (ibhokhwe, 1:500; AF3075, R&D Systems1), netrin 10; R & D Systems), anti- STEM121 (mouse, 1:200; Y40410, Takara Bio), anti-SATB2 (mouse, 1:50; ab51502, abcam), anti-GAD65/67 (umvundla, 1:400; ABN904, Millipore 1:0; hamba anti, 0; hamba-anti, 0); ab5076, abkam).使用的一抗是：抗NeuN（小鼠，1:500；ab104224，abcam）抗CTIP2（大鼠，1:3 00；ab18465,abcam），抗GFAP（兔，1:1,000;Z0334，Dako），抗-GF P（鸡，1:1,000;GTX13970，GeneTex），抗HNA（小鼠，1:200；ab191181,abcam），抗NeuN（兔，1:500;ABN78,GPARE） 1:200;sc-338，Santa Cruz），抗PPP1R17（兔，1:200;HPA047819，Atlas 抗体），抗RECA-1（小鼠，1:50;ab9774ab9774ab9774ab , 1:100;20357-1-AP，Proteintech) 1:200;使用的一抗是：抗NeuN（小鼠，1:500；ab104224，abcam）抗CTIP2（大鼠，1 :300；ab18465,abcam），抗GFAP（兔，1:1,000;Z0334，Dako），抗-GFP（鸡，1:1,000;GTX13970，GeneTex），抗HNA（小鼠，1:200；ab191181,abcam），抗NeuN（兔，1:500；ABN78＊Millipo％Millipo） 200；sc-338，Santa Cruz），抗PPP1R17（兔，1:200;HPA047819，Atlas 抗体），抗RECA-1（小鼠，1:50;ab9774）ab9774，abcam 100;20357-1-AP，Proteintech），抗SOX9（山羊，1:500；AF3075,R&D Systems），Netrin G1a（山羊,1:100；AF1166,R&D Systems1:20,Y40410, Takara Bio), anti-SATB2 (mouse, 1:50; ab51502, abcam), anti-GAD65/67 (umvundla, 1:400; ABN904, Millipore) kunye anti-IBA1 (ibhokhwe, 1:100; ab5076, abcam).Ii-antibodies eziphambili ezisetyenzisiweyo zaziyi: anti-NeuN (mouse, 1:500; ab104224, abcam), anti-CTIP2 (rat, 1:300; ab18465, abcam), anti-GFAP (umvundla, 1: 1000; Z0334, Dako). , i-anti-GFP (inkukhu, 1: 1000; GTX13970, GeneTex), anti-HNA (mouse, 1:200; ab191181, abcam), anti-NeuN (umvundla, 1:500; ABN78, Millipore), i-anti-PDGFRA (i-rab303, i-100; i-anti-PPP1R17 (umvundla, i-1: 200; i-HPA047819, i-Atlas antibody), i-anti-RECA-1 (imouse, 1:50; ab9774, abcam), i-anti- SCG2 (umvundla), 1: 100;20357-1-AP, Proteintech), анти-SOX9 (коза, 1:500; AF3075, R&D Systems), Нетрин G1a (коза, 1:100; AF1166, R&D Systems), анти -STEM121 (Y4:040, Bios анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) и анти-IBA1 (коза, 1:100; аб5076, аб5076). 20357-1-AP, Proteintech), anti-SOX9 (ibhokhwe, 1:500; AF3075, R&D Systems), Netrin G1a (ibhokhwe, 1:100; AF1166, R&D Systems), anti-STEM121 (impuku, 1:200; i-anti-sat-SATA2), i-Bio-Tkamo1 1:50; ab51502, abcam), anti-GAD65/67 (umvundla, 1:400; ABN904, Millipore), kunye ne-anti-IBA1 (ibhokhwe, 1:100; ab5076, abkam).Amacandelo ahlanjwe nge-PBS kwaye afakwe kwi-antibody yesibini kwiyure ye-1 kwindawo yokushisa (amacandelo afriziwe) okanye ubusuku bonke kwi-4 ° C (amacandelo atyebileyo). I-Alexa Fluor yesibini ye-antibody (i-Life Technologies) ihlanjululwe i-1: 1000 kwisisombululo sokuthintela sasetyenziswa. Emva kokuhlamba nge-PBS, i-nuclei yabonwa nge-Hoechst 33258 (i-Life Technologies). Ekugqibeleni, iislayidi zafakwa kwi-microscope kunye ne-coverlips (Fisher Scientific) usebenzisa i-Aquamount (Polysciences) kwaye ihlalutye kwi-microscope ye-Keyence fluorescent (i-BZ-X analyzer) okanye i-Leica TCS SP8 i-microscope ye-confocal (Las-X) kumfanekiso. Imifanekiso iqhutywe kusetyenziswa inkqubo ye-ImageJ (Fiji). Ukulinganisa umlinganiselo we-neurons yabantu kwi-t-hCO kunye ne-rat cortex, i-387.5 μm ububanzi bemifanekiso ye-rectangular ithathwe kumbindi we-t-hCO, okanye kufuphi nomda we-rat cortex. Imida ye-Graft yamiselwa ngokuvavanya utshintsho kwi-transparency yezicubu, i-HNA + nuclei, kunye / okanye ubukho be-tissue autofluorescence. Kumfanekiso ngamnye, inani elipheleleyo leeseli ze-NeuN+ kunye ne-HNA+ zahlulwe ngenani elipheleleyo leeseli ze-NeuN+ kwindawo enye. Ukuqinisekisa ukuba kuphela iiseli ezine-nuclei kwinqwelo-moya yomfanekiso zibaliwe, kuphela iiseli ezikwa-Hoechst+ zibandakanyiweyo ekubaleni. Imifanekiso emibini eyahlulwe ubuncinci be-1 mm yalinganiselwa ukunciphisa impazamo yamanani.
Kwiveki enye ngaphambi kokuqokelela isampuli, beka izilwanyana zokutshintshwa kwe-hCO (malunga neenyanga ezi-8 zokwahlula) kwigumbi elimnyama elinamabhovu alungisiweyo ukuze kuncitshiswe ukuvuselela ingqondo. Ukwahlukaniswa kwee-nuclei kwenziwa njengoko kuchaziwe ngaphambili, kunye nohlengahlengiso oluthile. Ngokufutshane, i-t-hCO kunye ne-hCO zatshatyalaliswa kusetyenziswa i-detergent-mechanical cell lysis kunye ne-2 ml ye-glass tissue grinder (D8938, Sigma-Aldrich / KIMBLE). Iinuclei ezikrwada ke zahluzwa kusetyenziswa isihluzo esingama-40 µm saze safakwa phakathi kwi-320 g kangangemizuzu eli-10 kwi-4 °C phambi kokwenza ingxinano ye-sucrose. Emva kwesinyathelo se-centrifugation (i-320 g ye-20 min kwi-4 ° C), iisampuli zabuyiselwa kwi-0.04% ye-BSA / PBS kunye nokongezwa kweeyunithi ze-0.2 ze-µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) kwaye zadlula nge-40 µm filter. I-nuclei ehlukanisiweyo yaphinda yamiswa kwakhona kwi-PBS equkethe i-0.02% ye-BSA kwaye ilayishwe kwi-Chromium Single Cell 3′ chip (uqikelelo lokubuyiswa kweeseli ze-8,000 ngomzila ngamnye). Iilayibrari ze-snRNA-seq zalungiswa nge-Chromium Single cell 3 'GEM, Library & Gel Bead Kit v3 (10x Genomics). Iilayibrari ze-snRNA-seq zalungiswa nge-Chromium Single cell 3 'GEM, Library & Gel Bead Kit v3 (10x Genomics). Библиотеки snRNA-seq были приготовлены с помощью Iseli eNye yeChromium 3′ GEM, iThala leencwadi kunye neGel Bead Kit v3 (10x Genomics). Iilayibrari ze-snRNA-seq zalungiswa kusetyenziswa i-Chromium Single cell 3'GEM, Library & Gel Bead Kit v3 (10x Genomics). snRNA-seq 文库是使用Chromium Iseli enye 3′ GEM、Thala leencwadi & neGel Bead Kit v3 (10x Genomics) 制备的. snRNA-seq 文库是使用Chromium Iseli enye 3′ GEM、Thala leencwadi & neGel Bead Kit v3 (10x Genomics) 制备的. Библиотеку snRNA-seq готовили с использованием Iseli Eyodwa yeChromium 3′ GEM, Ithala leencwadi & neGel Bead Kit v3 (10x Genomics). Ithala leencwadi le-snRNA-seq lalungiswa kusetyenziswa i-Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics).Amathala eencwadi asuka kwiisampulu ezahlukeneyo adityaniswa kwaye alandelelwaniswa yi-Admera Health kwiNovaSeq S4 (Illumina).
Amanqanaba embonakalo yeGene kwibhakhowudi nganye yenyukliya ebekiweyo aye alinganiswa kusetyenziswa iphakheji yesoftware yohlalutyo ye-10x ye-Genomics CellRanger (uguqulelo 6.1.2). Ngokukodwa, ukufundwa kwadityaniswa ngokuchasene nendibaniselwano yomntu (GRCh38, Ensemble, version 98) kunye nerat (Rnor_6.0, Ensemble, version 100) ireferensi yejenomes edalwe ngomyalelo we-mkref kunye nokusebenzisa ukubala kunye no -include-introns=TRUE umyalelo wobungakanani kubandakanya ukufundwa kwemephu kwingingqi yeintron. Kwiisampuli ze-t-hCO, ii-nuclei zomntu zichongiwe ngokusekelwe kwimfuno yolondolozo yokuba ubuncinane i-95% yazo zonke iimephu ezifundwayo zihambelana ne-genome yomntu. Zonke iimvavanyo ezilandelayo zenziwa kwi-barcode ehluziweyo yoluhlu oluphuma kwi-CellRanger usebenzisa iphakheji ye-R (uguqulelo 4.1.2) Seurat (uguqulelo 4.1.1)32.
Ukuqinisekisa ukuba kuphela i-nuclei yomgangatho ophezulu ifakiwe kuhlalutyo olulandelayo, inkqubo yokucoca ngokuphindaphindiweyo yaphunyezwa kwisampuli nganye. Okokuqala, i-nuclei esemgangathweni ephantsi kunye ne-1000 yemfuza ekhethekileyo efunyenweyo kwaye ngaphezu kwe-20% ye-mitochondria iyonke ichongiwe kwaye isuswe. Emva koko, i-matrix yenombolo yemfuza ekrwada yenziwa yesiqhelo ngokuhlehliswa kwe-binomial okuqhelekileyo kusetyenziswa i-sctransform(vst.flavor=”v2″) umsebenzi, okwachonge i-3000 yemfuza eguquguqukayo kakhulu kusetyenziswa iparameters.Ukuncipha komda kwenziwa kuhlobo oluguquguqukayo oluphezulu kusetyenziswa iPrincipal Component Analysis (PCA dimdim0 of the default Component Analysis) kunye ne-3000 yedatha I-30 yakhethwa ngokusekelwe ekuhlolweni okubonakalayo kweendawo zamadolo kwaye zisetyenziselwa zonke iisampuli kunye nohlalutyo oludibeneyo (i-resolution = 1) ukuze sihlele i-gene ephantsi ngokungaqhelekanga (i-median engaphantsi kwe-10th percentile), ngokungaqhelekanga i-mitochondrial ephezulu ngaphezu kwe-9 yokususa i-cell genelu kunye/okanye umlinganiselo ophezulu wamawele akrokrelekayo achongiwe kusetyenziswa iphakheji ye-DoubletFinder33 (ithetha ukuba amanqaku e-DoubletFinder ngaphezu kwe-95th percentile iisampulu ze-t-hCO (n=3) kunye ne-hCO (n=3) zidityaniswe ngokwahlukeneyo kusetyenziswa umsebenzi we-IntegrateData kunye nemilinganiselo engasentla.
Emva kokususa ii-kernels zekhwalithi ephantsi, i-dataset edibeneyo yahlulwa (isisombululo = 0.5) kwaye ifakwe kwiinjongo zokujonga i-UMAP34. Ijene zomakishi kwiqela ngalinye ziye zamiselwa kusetyenziswa umsebenzi weFindMarkers oneparameters engagqibekanga ebalwa ukusuka kwidatha yesiqhelo yokuchazwa kwemfuza. Sichonga kwaye sihlele iiklasi eziphambili zeeseli ngokudibanisa iiseti zedatha yereferensi ye-fetal kunye necortical yabantu abadala kunye ne-marker gene expression 19,20,21,35 kunye ne-annotation. Ngokukodwa, ii-precursors ezijikelezayo zichongiwe ngokuchazwa kwe-MKI67 kunye ne-TOP2A. Amaqela e-Progenitor achazwe ngokungabikho kokubhaliweyo kwe-mitotic, ukugqithwa okuphezulu kunye ne-multipotent glial progenitor clusters echazwe kwi-cortex ye-fetal metaphase emva kwexesha, kunye ne-EGFR kunye ne-OLIG1 yokubonisa. Sisebenzisa igama elithi astrocyte ukuquka amazwe amaninzi omahluko we-astrocyte, ukusuka kwi-radial glia kade ukuya ekuvuthweni kwee-astrocyte. Amaqela e-Astrocyte abonakalisa amanqanaba aphezulu e-SLC1A3 kunye ne-AQP4 kwaye abonisiwe ukwenza imephu eneentlobo ezincinci ze-fetal radial glia kunye/okanye ii-astrocyte zabantu abadala. Ii-OPCs zivakalisa i-PDGFRA kunye ne-SOX10 ngelixa i-oligodendrocytes ibonisa iimpawu ze-myelination (MOG kunye ne-MYRF). I-Glutamatergic neurons ichongiwe kubukho be-neuronal transcripts (SYT1 kunye ne-SNAP25), ukungabikho kwamanqaku e-GABAergic (GAD2), kunye nokubonakaliswa kwe-NEUROD6, SLC17A7, BCL11B, okanye i-SATB2. I-GluN neurons yahlulwe ngakumbi yaba phezulu (i-SATB2 intetho kunye nokulahlekelwa kwe-BCL11B) kunye ne-deep (BCL11B expression) subclasses. I-Pututive subplate (SP) i-neurons ivakalisa iimpawu ze-SP18 ezaziwa njenge-ST18 kunye ne-SORCS1 ukongeza kwiimpawu ezinzulu ze-GluN. Iiseli ze-Choroid plexus-like zachongwa ngokuchazwa kwe-TTR, kunye neeseli ezifana ne-meningeal zivakalisa iijene ezihambelana ne-fibroblast kunye neeseli zepiyali ezifakwe kwiimephu zeseti yedatha yereferensi.
Uhlalutyo olwahlukileyo lwe-gene expression phakathi kwe-t-hCO kunye ne-hCO subclasss lwenziwa kusetyenziswa indlela entsha ye-pseudo-batch eveliswe kwakhona kwiisampuli eziphunyeziweyo kusetyenziswa iphakheji ye-Libra R (uguqulelo 1.0.0). Ngokukodwa, iimvavanyo ze-edgeR log-ukubakho (uguqulelo 3.36.0, ipakethe R) zenzelwe amaqela ngokushwankathela inani leejene kwiiseli zeklasi yeseli enikiweyo kwisampulu nganye yokuphindaphinda. Ukubonwa kwemephu yobushushu, amaxabiso aqhelekileyo ngokwesigidi (CPM) ahlanganiswa kusetyenziswa i-edgeR (cpm () umsebenzi) kwaye ilinganiswe (ukufezekisa intsingiselo = 0, ukutenxa okusemgangathweni = 1). Uhlalutyo lwe-Gene Ontology (GO) lwe-erichment ye-t-hCO GluN ye-genes ilawulwa kakhulu (i-Benjamini-Hochberg ilungiswe ixabiso le-P elingaphantsi kwe-0.05 echazwe ubuncinane kwi-10% yeeseli ze-t-hCO GluN kunye nokunyuka kwe-fold fold in change of at least 2 times). eyenziwa kusetyenziswa iToppGene Suite (https://toppgene.cchmc.org/)37. Sisebenzisa i-app ye-ToppFun eneparameters engagqibekanga kwaye sixela amaxabiso e-P kaBenjamin-Hochberg abalwe kwiimvavanyo ze-GO-annotated hypergeometric.
Ukuhambelana namaqela ethu e-snRNA-seq kunye namaqela eeseli ezichasiweyo ukusuka kwizifundo zereferensi ye-primary single-cell RNA-seq okanye i-snRNA-seq19,20,21,22 yabantu abadala, sisebenzise indlela yokudibanisa idatha edibeneyo. Sisebenzise i-SCTransform (v2) ye-normalization workflow kwi-Seurat ukudibanisa nokuthelekisa ukugqithwa kweqela phakathi kwedatha (usebenzisa iiparamitha ezifanayo ezingentla). Iiseti zedatha zomntu ngamnye ziye zamiselwa ngokungenamkhethe ukuya kutsho kwiiseli ezingama-500 okanye ii-cores ngokweqela lokuqala lobuchule bokubala. Ukusebenzisa indlela efanayo njengoko kuchaziwe ngaphambili, ukugqithelana kweqela kwachazwa njengomlinganiselo weeseli okanye ii-nuclei kwiqela ngalinye elidityanisiweyo eliye lawelana neleyibhile yeqela lereferensi. Ukwahlula ngakumbi ii-GluNs, sisebenzise i-Seurat's TransferData workflow ye-GluN subset data ukunika iilebhile zereferensi yedataset kwiiseli zethu ze-GluN.
Ukuvavanya imeko yokuvuthwa kwe-transcriptome yehlabathi jikelele ye-t-hCO kunye neesampuli ze-hCO, sithelekise iisampulu zethu ze-pseudo-bulk kunye ne-BrainSpan/psychENCODE23, equka ulandelelwano olukhulu lwe-RNA oluthatha uphuhliso lobuchopho bomntu. Senze i-PCA kwi-pattern-normalized gene expression matrix ukusuka kwiisampulu ze-cortical iiveki ze-10 emva kokukhulelwa kwaye kamva, kwii-genes ze-5567 (kunye neenkcukacha zethu) ezazichongiwe ngaphambili njengezisebenzayo kwi-BrainSpan iisampulu ze-cortical (echazwe njengenkulu kune-50% kwintlukwano yophuhliso echazwe ngobudala usebenzisa imodeli ye-cubic)38. Ukongeza, siye safumana iijini ezinxulunyaniswa nokutyikitywa okukhulu kwe-neurodevelopment kusetyenziswa i-non-negative matrix factorization njengoko bekuchaziwe ngaphambili. Izisindo zesampulu ezibalwe kusetyenziswa inkqubo ye-matrix factorization ye-non-negative icwangciswe kwiiFigs. I-5b kunye nedatha eyandisiweyo nganye kwezintlanu ezisayinwe zichazwe nguZhu et al.38. Kwakhona, iimpawu zokukhutshelwa ezixhomekeke kumsebenzi zithathwe kwizifundo ezipapashwe ngaphambili. Ngokukodwa, i-ERG kunye ne-LRG zilawulwa kakhulu kwi-neurons ye-glutamatergic echongwe yi-mouse visual cortex snRNA-seq collection emva kokuvuselela okubonakalayo kwi-Supplementary Table 3 Hrvatin et al.16. Ii-LRGs ezityetyisiweyo ngabantu zafunyanwa kwi-KCl-eye yasebenza kwiinkcubeko zobuchopho bomntwana ongekazalwa kwaye zavunwa iiyure ezi-6 emva kokuvuselela, kwaye imfuza ehluziweyo yayilawulwa kakhulu ebantwini kodwa kungekhona kwiimpuku (Itheyibhile eyoNgezelelweyo yesi-4). Uhlalutyo lweseti yemfuza yokutyebisa kusetyenziswa ezi seti zofuzo lwenziwa kusetyenziswa indlela enye yovavanyo oluchanekileyo lukaFisher.
Iigundane ze-Anesthetize nge-isoflurane, susa ubuchopho kunye nendawo ebandayo (malunga ne-4 ° C) i-oxygen (95% O2 kunye ne-5% CO2) isisombululo se-sucrose kumacandelo aqukethe: 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM. I-NaH2PO4, 10 mM MgSO4 kunye ne-0.5 mM CaCl2 (malunga ne-310 mOsm). Amacandelo e-coronal ye-rat brain (300-400 µm) equlethe i-t-hCO yenziwe kusetyenziswa i-Leica VT1200 vibratome njengoko kuchazwe ngaphambili39. Emva koko amacandelo adluliselwe kwigumbi lecandelo kunye ne-oksijini yeqondo lokushisa eliqhubekayo eliqukethe i-aCSF elungiselelwe ukusuka: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 kunye ne-126 mM MOsm29 (29). ubuncinane imizuzu engama-45 phambi kokurekhoda. Amacandelo arekhodwa kwigumbi lokucwiliswa apho ahlala exutywe nge-aCSF (95% O2 kunye ne-5% CO2 vial). Yonke idatha yarekhodwa kwiqondo lokushisa. I-t-hCO neurons yapheliswa nge-borosilicate glass pipette egcwele isisombululo esine-127 mM potassium gluconate, i-8 mM NaCl, i-4 mM magnesium ATP, i-0.3 mM ye-sodium GTP, i-10 mM HEPES, kunye ne-0.6 mM EGTA, i-pH 7.2, isisombululo sangaphakathi esilungiswa nge-KOO m29m. Ukuze kubuyiselwe, i-biocytin (0.2%) yongezwa kwisisombululo sokurekhoda.
Idatha yafunyanwa kusetyenziswa i-MultiClamp 700B amplifier (i-Molecular Devices) kunye ne-Digidata 1550B digitizer (i-Molecular Devices), i-low-pass filtered kwi-2 kHz, ifakwe kwidijithali kwi-20 kHz, kwaye ihlalutye usebenzisa i-Clampfit (i-Molecular Devices), i-Origin (OriginPro). 2021b, OriginLab). kunye nemisebenzi ye-MATLAB yesiko (iMathematika). Ikhono lokuhlangana libalwe kusetyenziswa i-JPCalc kwaye amangenelo ahlengahlengiswa kwixabiso elibaliweyo le--14 mV. I-Operation IV iquka uchungechunge lwamanyathelo angoku kwi-10-25 pA amanyathelo, ukusuka -250 ukuya kwi-750 pA.
Ithalamus, into emhlophe, kunye ne-S1 afferents zavuselelwa ngombane kwizilayi ze-thalamocortical ngexesha lokurekhodwa kwe-patch-clamp ye-hCO neurons, njengoko kuchaziwe ngaphambili. Ngokufutshane, ingqondo yabekwa kwitafile yoshicilelo ye-3D ethambekele kwi-engile ye-10 °, kwaye umphambili wengqondo wanqunyulwa nge-engile ye-35 °. Ingqondo ke yancanyathiselwa kumphezulu osikiweyo kwaye yahlulwa, igcina i-thalamocortical protruding axons. I-bipolar tungsten electrodes (0.5 MΩ) ifakwe kwi-micromanipulator yesibini kwaye ibekwe ngokucwangcisekileyo ukuvuselela imimandla emine ngeseli (i-capsule yangaphakathi, i-white matter, i-S1 kunye ne-hCO). Rekhoda iimpendulo ze-synaptic emva kwe-300 µA yokuvuselela i-phasic kwi-0.03-0.1 Hz.
I-hChR2-ebonisa i-hCO neurons yenziwe yasebenza kwi-480 nm kunye neepulses ezikhanyayo ezenziwe yi-LED (Prizmatix) zisetyenziswe ngenjongo ye-× 40 (0.9 NA; Olympus) ukurekhoda ukubonakaliswa kwe-hChR2 kufuphi neeseli. Idayamitha yentsimi ekhanyisiweyo imalunga ne-0.5 mm kwaye amandla ewonke yi-10-20 mW. Ububanzi be-pulse bubekwe kwi-10 ms, ehambelana ne-pulse enikwe ngexesha lovavanyo lokufunda ukuziphatha. Iintlobo ezahlukeneyo zokuvuselela zisetyenzisiwe, ukusuka kwi-1 ukuya kwi-20 Hz, kodwa kuphela i-pulse yokuqala yochungechunge yayisetyenziselwa ubungakanani. Izithuba phakathi koololiwe zikholisa ukuba kude kune-30 s ukunciphisa isiphumo kwi-synaptic inhibitory okanye iindlela eziququzelelayo. Ukuvavanya ukuba impendulo ye-hChR2 yayiyi-monosynaptic, safaka i-TTX (1 μM) kwindawo yokuhlambela de i-reactivity ye-EPSC yanyamalala, kwaye yasebenzisa i-4-aminopyridine (4-AP; 100 μM). Ngokwesiqhelo, impendulo ibuyiswa ngaphakathi kwemizuzu embalwa, kunye nokulibaziseka okude kancinci phakathi kokudubula kwe-LED kunye nesizukulwana se-EPSC. I-NBQX (i-10 μM) yayisetyenziselwa ukuvavanya ukuba impendulo iqhutywa ngabamkeli be-AMPA.
Amacandelo e-hCO abukhali adalwe njengoko kuchaziwe ngaphambili. Ngokufutshane, amacandelo e-hCO afakwe kwi-4% ye-agarose kwaye idluliselwe kwiiseli eziqukethe i-126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 kunye ne-10 mM -glucose d-0 kwiCandelo + 0 (0) µm kwiqondo lobushushu begumbi usebenzisa i-Leica VT1200 vibrator kwaye igcinwe kwi-ASF kwiqondo lobushushu begumbi. Emva koko, ukurekhodwa kwekampu yeeseli ezipheleleyo kwenziwa kumacandelo e-hCO phantsi kwe-microscope ethe ngqo ye-SliceScope (iScientifica). Amacandelo agxininiswe nge-aCSF (95% O2 kunye ne-5% CO2) kunye neempawu zeseli zirekhodwa kwiqondo lokushisa. I-hCO neurons isetyenziswe ngokusebenzisa i-borosilicate glass pipette ezaliswe isisombululo esine-127 mM potassium gluconate, i-8 mM NaCl, i-4 mM magnesium ATP, i-0.3 mM ye-sodium GTP, i-10 mM HEPES, kunye ne-0.6 mM EGTA, i-pH yangaphakathi 7, i-2, ilungiswe ne-KOH20 mos. Ngeenjongo zokubuyisela, yongeza i-0.2% ye-Biocytin kwisisombululo sangaphakathi.
Idatha ifunyenwe nguClampex (i-Clampex 11.1, i-Molecular Devices) isebenzisa i-MultiClamp 700B amplifier (i-Molecular Devices) kunye ne-Digidata 1550B digitizer (i-Molecular Devices), i-low-pass filtered kwi-2 kHz, ifakwe kwi-digitized kwi-20 kHz, kunye nohlalutyo lwe-molecular 10. izixhobo) kunye nemisebenzi ye-MATLAB yesiko (MATLAB 2019b, Mathworks). Amandla okudibana kwabalwa kusetyenziswa i-JPCalc kwaye amangenelo alungelelaniswa kwisakhono esibaliweyo sesiphambuka se -14 mV. I-Operation IV iquka uchungechunge lwamanyathelo angoku kwi-5-10 pA amanyathelo ukusuka -50 ukuya kwi-250 pA.
Ukwakhiwa ngokutsha kwe-morphological ye-pinched neurons, i-0.2% ye-biocytin (i-Sigma-Aldrich) yongezwa kwisisombululo sangaphakathi. Iiseli zilungiselelwe ubuncinci imizuzu eyi-15 emva kokuqhekezwa. I-pipette ifakwe ngokukhawuleza kwi-1-2 min de i-membrane ebhalisiweyo ivalwe ngokupheleleyo. Ukulandela inkqubo ye-physiology yecandelo, amacandelo ayenziwe ngobusuku kwi-4 ° C. kwi-4% PFA, ihlanjwe nge-PBS X3, kwaye ihlanjululwe i-1: 1000 nge-streptavidin-conjugated DyLight 549 okanye i-DyLight 405 (i-Vector Labs). Iiseli ezizaliswe nge-biocytin (2%; Sigma-Aldrich) zibhalwe ngexesha lokurekhoda kwe-patch clamp kwindawo yokushisa kweeyure ze-2. Amacandelo ke axhonywa kwi-microscopy izilayidi kusetyenziswa i-Aquamount (i-Thermo Scientific) kwaye yabonwa ngosuku olulandelayo kwi-microscope ye-Leica TCS SP8 edibeneyo kusetyenziswa injongo yokuntywiliselwa kwe-oyile kunye ne-aperture yamanani × 40 1.3, ukukhulisa × 0.9-1.0, xy. Ireyithi yesampulu imalunga ne-7 pixels ngemicron nganye. I-Z-stacks kwizithuba ze-1 µm zafunyanwa ngokulandelelana, kunye ne-z-stack mosaics kunye nokuthungwa okuzenzekelayo okusekwe kuLeica kwenziwa ukugubungela wonke umthi we-dendritic we-neuron nganye. IiNeurons zaye zalandelelwa ngesiqingatha-ngesandla kusetyenziswa i-neuTube 40 interface kunye neefayile zeSWC zenziwa. Iifayile zaye zalayishwa kwi-plugin ye-SimpleNeuriteTracer41 Fiji (UmfanekisoJ, inguqulo 2.1.0; NIH).
Izicubu ze-cortical yomntu zafunyanwa ngemvume enolwazi ngokwemigaqo evunyiweyo yiBhodi yokuHlola yeZiko leYunivesithi yaseStanford. Iisampulu ezimbini zezicubu zomntu zasemva kokubeleka (i-3 kunye ne-18 ubudala ubudala) zifunyenwe ngokwenziwa ngokutsha kwe-cortex yangaphambili (i-gyrus yangaphambili ephakathi) njengenxalenye yotyando lwe-refractory epilepsy. Emva kokususwa, vuna izicubu kwi-NMDG-aCSF ebandayo ebandayo equlethe: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mMruvate sodium ascorbate 0 mpy3 mpyl 2 isodium ascorbate 0. 4H2O kunye ne-10 mM MgSO4 7H2O. I-Titrate kwi-pH 7.3-7.4 kunye ne-hydrochloric acid egxininisiweyo. Izicubu zihanjiswe kwibhubhoratri ngaphakathi kwemizuzu engama-30 kwaye amacandelo e-coronal athathwe ngokwenkqubo echazwe ngasentla.
Zonke iinkqubo zezilwanyana zenziwa ngokuhambelana neYunivesithi yaseStanford APLAC evunyiweyo yezikhokelo zokunakekelwa kwezilwanyana. Iigundane (ezinkulu kuneentsuku ze-140 emva kokutshintshwa) zenziwe nge-5% isoflurane anesthesia kunye ne-anesthetized nge-1-3% isoflurane intraoperatively. Izilwanyana zafakwa kwisakhelo se-stereotaxic (Kopf) kunye nokukhululwa okuqhubekayo kwe-buprenorphine (SR) kwajojowe ngaphantsi kwe-subcutaneously. I-skull ibonakaliswe, icociwe kwaye i-3-5 i-screws yamathambo ifakwe. Ukujolisa kwi-t-hCO, senze ulungelelwaniso lwe-stereotaxic ukusuka kwimifanekiso ye-MRI. Umngxuma we-burr wagrunjwa kwindawo yomdla kunye neentsinga (400 µm idayamitha, NA 0.48, Doric) zathotywa nge-100 µm ngaphantsi komphezulu we-hCO kwaye zakhuselwa kukhakhayi ngesamente yamazinyo enyangekayo yi-UV (Relyx).
Ukurekhodwa kweFiber photometric kwenziwa njengoko bekuchaziwe ngaphambili42. Ukurekhoda umsebenzi ozenzekelayo, iimpuku zafakwa kwikheji ecocekileyo kunye nentambo ye-fiber optic patch ye-400 µm yedayamitha yedayamitha (Doric) eqhagamshelwe kwi-fiber optic photometric data acquisition system yadityaniswa kwintambo yefiber optic emiliselweyo. Ngethuba lokurekhodwa kwemizuzu eyi-10 yomsebenzi wemoto, izilwanyana zazikhululekile ukuba zihlolisise i-cage. Ukurekhoda umsebenzi okhutshiweyo, iigundane (ngaphezu kweentsuku ze-140 emva kokutshintshwa) zifakwe i-anesthetized nge-5% isoflurane yokungeniswa kunye ne-1-3% isoflurane yokugcina. Beka isilwanyana kwisakhelo se-stereotactic (i-Kopf) kunye ne-whiskers kwicala eliphambene ne-t-hCO ihlanjululwe malunga ne-2 cm kwaye idlule kwi-mesh exhunywe kwi-piezoelectric actuator (PI). Intambo ye-fiber optic ye-400 µm (i-Doric) yadityaniswa kwifayibha emiliselweyo yaza yaqhagamshelwa kwisixokelelwano sokufumana idatha. I-whiskers kwicala elichasene ne-t-hCO zaye zachithwa ngamaxesha angama-50 (2 mm kwi-20 Hz, i-2 s ngentetho nganye) ngamaxesha angaqhelekanga nge-piezoelectric drive kwimizuzu engama-20 yokurekhoda. Sebenzisa iPhakheji yeNkxaso ye-Arduino MATLAB ukulawula ixesha lokuphambuka ngekhowudi ye-MATLAB yesiko. Iziganeko zilungelelaniswa kwisoftware yokufumana idatha kusetyenziswa i-transistor-transistor logic (TTL) pulses.
Iigundane (ezinkulu kuneentsuku ze-140 emva kokutshintshwa) zenziwe nge-5% isoflurane anesthesia kunye ne-anesthetized nge-1-3% isoflurane intraoperatively. Izilwanyana zifakwe kwisakhelo se-stereotaxic (i-Kopf) kunye ne-buprenorphine SR kunye ne-dexamethasone zifakwe ngaphantsi kwe-subcutaneous. I-skull ibonakaliswe, icociwe kwaye i-3-5 i-screws yamathambo ifakwe. Ukujolisa kwi-t-hCO, senze ulungelelwaniso lwe-stereotaxic ukusuka kwimifanekiso ye-MRI. I-craniotomy ejikelezayo (malunga ne-1 cm ububanzi) yenziwa nge-drill yesantya esiphezulu ngokuthe ngqo kwi-hCO etshintshiweyo. Nje ukuba ithambo libe lincinci kangangoko, kodwa ngaphambi kokuba ubhoboze ithambo lonke, sebenzisa iforceps ukususa idiski ye-pelvic eseleyo ukuze uveze i-t-hCO engaphantsi. I-craniotomy yayizaliswa nge-saline eyinyumba, kunye ne-coverlip kunye ne-pin ekhethekileyo yentloko yayifakwe kwi-skull kunye ne-UV-cured dental cement (Relyx).
Imifanekiso yeefotoni ezimbini zenziwa kusetyenziswa i-Bruker multiphoton microscope ene-Nikon LWD (× 16, 0.8 NA) injongo. Umfanekiso we-GCaMP6 wenziwa kwi-920 nm kunye ne-1.4x enye yokukhulisa i-z-plane kunye ne-8x ye-avareji ye-7.5 fps. Iigundane zenziwe nge-5% isoflurane anesthesia kwaye zigcinwe nge-1-3% isoflurane. Iimpuku zafakwa kwintloko eyenziwe ngokwezifiso kwaye zibekwe phantsi kwelensi. Kufunyenwe ukurekhoda okungasemva kwemizuzu emi-3 yomsebenzi wemoto. Ngethuba lemizuzu engama-20 yokurekhoda, i-puffs ye-50 (inkcazo nganye yi-100 ms ubude) ihanjiswe ngokungenamkhethe kwi-whisker pad ejongene ne-t-hCO kusetyenziswa i-picospricer. Sebenzisa iPhakeji yeNkxaso ye-Arduino MATLAB ukulawula ixesha lokuqhuma ngekhowudi ye-MATLAB yesiko. Vumelanisa imicimbi kunye nesoftware yokufumana idatha (PrairieView 5.5) usebenzisa i-TTL pulses. Ukuhlalutya, imifanekiso yalungiswa kwi-xy motion usebenzisa ukulungiswa kwe-affine kwinkqubo ye-MoCo eyasungulwa eFiji. Ukutsalwa kweempawu ze-fluorescent kwiiseli zomntu ngamnye usebenzisa i-CNMF-E43. I-Fluorescence yakhutshwa kwingingqi nganye enomdla, yaguqulelwa kwi-dF/F curves, yaze yaguqulwa yaba ngu-z-score.
Iigundane (ezinkulu kuneentsuku ze-140 emva kokutshintshwa) zenziwe nge-5% isoflurane anesthesia kunye ne-anesthetized nge-1-3% isoflurane intraoperatively. Izilwanyana zifakwe kwisakhelo se-stereotaxic (i-Kopf) kunye ne-buprenorphine SR kunye ne-dexamethasone zifakwe ngaphantsi kwe-subcutaneous. I-whiskers kwicala eliphambene ne-t-hCO yayinqunywe malunga ne-2 cm kwaye ifakwe kwi-mesh edibeneyo kwi-actuator ye-piezoelectric. Ukhakhayi luvezwe kwaye lucocwe. Isikrufu somhlaba esingenastainless sincanyathiselwe kukhakhayi. Ukujolisa kwi-t-hCO, senze ulungelelwaniso lwe-stereotaxic ukusuka kwimifanekiso ye-MRI. Yenza i-craniotomy yesetyhula (malunga ne-1 cm ububanzi) kunye ne-speed drill drill ngaphezu kwe-t-hCO. Nje ukuba ithambo libe lincinci kangangoko, kodwa ngaphambi kokuba ubhoboze ithambo lonke, sebenzisa iforceps ukususa idiski ye-pelvic eseleyo ukuze uveze i-t-hCO engaphantsi. Iiseli zomntu ngamnye zirekhodwe zisebenzisa i-32-channel okanye i-64-channel high-density silicon probes (i-Cambridge Neurotech) esekelwe kwizikrufu zomhlaba kwaye zandiswe kwangaphambili nge-RHD amplifiers (Intan). Sebenzisa i-manipulator ukuthoba i-electrodes kwindawo ekujoliswe kuyo ngokusebenzisa i-craniotomy, egcwele i-saline eyinyumba. Ukuqokelelwa kwedatha kwenziwa kwi-frequency ye-30 kHz kusetyenziswa inkqubo yokufumana idatha ye-Open Ephys. Ukurekhoda kwaqhubeka kuphela xa sifumene umsebenzi ohambelana kakhulu wesingqisho ozenzekelayo kwiitshaneli ezingaphezu kwe-10, ebonisa ukuba i-electrodes ibekwe kwi-graft (esekelwe kwidatha ye-imaging ye-photon ye-calcium). Kufunyenwe ukurekhoda okungasemva kwemizuzu eli-10 yomsebenzi wemoto. I-whiskers kwicala elichasene ne-t-hCO zaye zachithwa ngamaxesha angama-50 (2 mm kwi-20 Hz, i-2 s ngentetho nganye) ngamaxesha angaqhelekanga nge-piezoelectric drive kwimizuzu engama-20 yokurekhoda. Ukusebenzisa iPhakeji yeNkxaso yeMATLAB ye-Arduino (MATLAB 2019b), lawula ixesha lokuphambuka kunye nekhowudi ye-MATLAB yesiko. Sebenzisa i-TTL pulses ukulungelelanisa imicimbi kunye nesoftware yokufumana idatha.
Kwimifuniselo yokumakisha yabona, i-200 µm patch cord (Doric) eqhagamshelwe kwi-473 nm laser (Omicron) yadityaniswa ne-200 µm ifayibha ebonakalayo ebekwe phezu kwecraniotomy. Kwangoko phambi koku, lungisa amandla e-jumper ukuya kuma-20 mW. Sebenzisa i-manipulator ukuthoba i-electrodes kwindawo ekujoliswe kuyo ngokusebenzisa i-craniotomy, egcwele i-saline eyinyumba. Ekuqaleni kokurekhoda, ii-pulses ezilishumi zokukhanya kwe-473 nm (i-frequency 2 Hz, ubude be-pulse 10 ms) zakhutshwa. Iiseli ze-Photosensitive zichazwe njengeeseli ezibonisa impendulo ye-spike ngaphakathi kwe-10 ms yokukhanya kwi-70% okanye ngaphezulu kwezilingo.