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Kuzvibatanidza neural organelles inomiririra inovimbisa in vitro chikuva yekuenzanisira kukura kwevanhu uye chirwere. Nekudaro, organoids inoshaya chinongedzo chiripo mu vivo, icho chinodzikamisa kukura uye chinodzivirira kubatanidzwa nemamwe maseketi anodzora maitiro. Pano isu tinoratidza kuti stem cell-yakatorwa cortical organoids yakasimwa mune somatosensory cortex yemakonzo ane mushutu anokura akura maseru marudzi anosanganisirwa mumasekete ane hukama uye anosimudzira. MRI yakaratidza kukura kwe-post-transplant organoid mune akati wandei stem cell mitsara nemhuka, nepo imwe-musimboti ongororo yakaratidza kufambira mberi kwecorticogenesis uye kubuda kwechirongwa-chinotsamira chirongwa chekunyora. Chokwadi, yakasimwa cortical neurons inoratidza zvakanyanya kuomarara morphological, synaptic, uye mukati me membrane zvimiro kupfuura yavo in vitro vamwe, zvichibvumira kuonekwa kwehurema neuronal muvarwere vane Timothy's syndrome. Anatomical uye functional tracing yakaratidza kuti organelles akasimwa anogashira thalamocortical uye corticocortical inputs, uye mu vivo kurekodha kweneural chiitiko chinoratidza kuti izvi zvinopinza zvinogona kuburitsa zvinonzwa mhinduro mumaseru evanhu. Pakupedzisira, cortical organoids inowedzera maaxon mukati mehuropi hwegonzo, uye yavo optogenetic activation inotungamira kune mubairo-wekutsvaga maitiro. Saka, yakasimwa cortex neurons inokura uye kutora chikamu mumaseketi emuenzi anodzora maitiro. Isu tinotarisira nzira iyi kuti iite kuti kuonekwa kwe-strand-level phenotypes mumasero anokonzerwa nemurwere asingagoni kuonekwa nedzimwe nzira.
Huropi hwemunhu huri kusimukira inzira inoshamisa yekuzvironga umo maseru anowanda, anosiyanisa, anotama, uye anobatana kuti aumbe anoshanda neuronal maseketi ayo anowedzera kunatswa kuburikidza neruzivo rwekunzwa. Dambudziko guru mukunzwisisa kukura kwehuropi hwevanhu, kunyanya mumamiriro ezvinhu echirwere, kushayikwa kwekuwana uropi. Kuzvironga organelles, kusanganisira human cortex organoids (hCO; inozivikanwawo seyemunhu cortex sphere), inogona kugadzira 2,3,4,5,6. Nekudaro, zvipingamupinyi zvakati wandei zvinodzikamisa yavo yakakura kushanda kuti vanzwisise kusimudzira uye kushanda kweneural maseketi. Kunyanya, hazvina kujeka kana hCO maturation inogumira nekusavapo kweimwe microenvironmental uye sensory inputs iripo mu vivo. Pamusoro pezvo, nekuti hCOs haina kubatanidzwa mumaseketi anogona kuburitsa maitiro ekuita, kushandiswa kwavo mukuenzanisa genetically yakaoma uye maitiro neuropsychiatric kusagadzikana parizvino kushoma.
Kuiswa kwehCO muhuropi hwehupenyu hwakasimba kunogona kukunda izvi zvisingakwanisi. Zvidzidzo zvakapfuura zvakaratidza kuti ma neuron evanhu akaiswa murodent cortex anokwanisa kurarama, kuronga, uye kutaurirana nemarodent cell7,8,9,10,11,12. Nekudaro, zviedzo izvi zvinowanzoitwa pamhuka dzakura, izvo zvinogona kudzikamisa synaptic uye axonal kubatanidzwa. Pano, isu tinotsanangura paradigm yekuisa paradigm yatakadyara 3D hCO yakatorwa kubva mumasero e-hiPS ichipinda mukutanga somatosensory cortex (S1) yemakonzo e immunodeficient padanho rekutanga rekukura kwepurasitiki. Yakasimwa hCO (t-hCO) neurons inokura zvakanyanya, inogamuchira thalamocortical uye cortical-cortical zvinopinza zvinounza mhinduro dzekunzwa, uye kuwedzera axonal fungidziro muuropi hwegonzo kutyaira maitiro ekutsvaga mubairo. Kuwedzera kukura kwe-t-hCO kwakaratidza kukanganisa neuronal kune varwere vane Timothy's syndrome (TS), chirwere chakakomba chemajini chinokonzerwa nekuchinja mu-voltage-sensitive L-type CaV1.2 calcium channel (encoded neCACNA1C).
Kuti tidzidze vanhu cortical neurons mumasekete mu vivo, isu takaisa stereotactically intact 3D hCO muS1 yemakonzo ekutanga mushure mekusununguka athymic (mazuva 3-7 mushure mekuberekwa) (Fig. 1a uye data yakawedzerwa yeFig. 1a-c). Panguva ino, thalamocortical uye corticocortical axonal projections haisati yapedza yavo S1 innervation (ref. 13). Nekudaro, nzira iyi yakagadzirirwa kukwirisa t-hCO kubatanidzwa uku ichideredza kukanganisa pamasekete endogenous. Kuti uone nzvimbo ye t-hCO mumhuka mhenyu, takaita T2-inorema MRI kugadziriswa kweuropi yemakonzo 2-3 mwedzi mushure mekutapurwa (Fig. 1b uye data yakawedzerwa, Fig. 1d). t-hCO yakaonekwa nyore nyore uye zviyero zvevhoriyamu zve t-hCO zvakanga zvakafanana kune zvakaverengwa kubva kune zvimedu zvakagadziriswa (Extended Data Fig. 1d, e; P> 0.05). t-hCO yakaonekwa nyore nyore uye zviyero zvevhoriyamu zve t-hCO zvakanga zvakafanana kune zvakaverengwa kubva kune zvimedu zvakagadziriswa (Extended Data Fig. 1d, e; P> 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированных срезов (расширенны, даных срезов (расширенные, данных срезов) 0,05). t-hCO yakaonekwa nyore nyore, uye volumetric t-hCO zviyero zvakafanana kune zvakaverengerwa zvikamu zvakagadziriswa (data yakawedzerwa, 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（扩（扩（扩（容易观察到t-hCO). 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 фиксированых срезов (расширенны, песни, данных срезов) 0,05). t-hCO yakaonekwa nyore nyore, uye volumetric t-hCO zviyero zvakafanana kune zvakaverengerwa zvikamu zvakagadziriswa (data yakawedzerwa, Fig. 1d, e; P> 0.05).Takasarudza t-hCO mu81% yemhuka dzakasimwa inenge mwedzi miviri mushure mekutapudza (n = 72 mhuka; hCO kubva ku10 hiPS cell lines; hiPS cell lines muSupplementary Table 1). Pakati peizvi, 87% yakanga iri mu cerebral cortex (Fig. 1c). Nokuita serial MRI scans panguva dzakawanda panguva imwechete yakasimwa rat, takawana kuwedzera kwepfumbamwe muhuwandu hwet-hCO mukati memwedzi ye3 (Fig. 1d uye data yakawedzerwa, Fig. 1f). Mhuka dzakasimwa dzaive nehuwandu hwehupenyu hwepamusoro (74%) pamwedzi we12 mushure mekutsvaga (data yakawedzerwa, Fig. 1g uye Supplementary Table 2), uye hapana yakanyanyisa motokari kana kukanganisa kwekuyeuka, gliosis, kana electroencephalogram (EEG) yakawanikwa. Data Fig. 1g uye supplementary table 2). 1h–m uye 3e).
a, Schematic of experimental design. hCO inotorwa kubva kumasero eHiPS yakasimwa muS1 yemakonzo achangozvarwa asina kupfeka pamazuva 30-60 ekusiyanisa. b, T2-inorema coronal uye yakatwasuka MRI mifananidzo inoratidza t-hCO muS1 2 mwedzi mushure mekusimwa. Scale bar, 2 mm. c, Quantification yeengraftment kubudirira mazinga anoratidzwa kune imwe neimwe yehiPS cell line (n = 108, nhamba mukati memabhawa dzinoratidza huwandu hwet-hCO pahIPS cell line) uye cortical kana subcortical nzvimbo (n = 88). d, MRI mufananidzo we coronary artery (kuruboshwe; scale bar, 3 mm) uye inowirirana 3D volumetric reconstruction (scale bar, 3 mm) inoratidza kuwedzera kwe t-hCO pamusoro pemwedzi ye3. e, Ongororo ye t-hCO mapatani mune rat cerebral cortex. Scale bar, 1 mm. f, Mumiririri immunocytochemical mifananidzo ye t-hCO inoratidzwa kubva kumusoro kuruboshwe kuenda kurudyi (panguva yekusiyanisa): PPP1R17 (4 mwedzi), NeuN (8 mwedzi), SOX9 uye GFAP (8 mwedzi), PDGFRα; (8 mwedzi), MAP2 (8 mwedzi) uye IBA1 (8 mwedzi). Scale bar, 20 µm. Co-kutaura kweHNA kunoratidza masero ekutanga kwevanhu. g. Astrocytes, masero emutsetse we astrocyte; cyc prog, kutenderera madzitateguru; GluN DL, yakadzika glutamatergic neurons; GluN DL / SP, yakadzika uye sublamellar glutamatergic neurons; GluN UL, yepamusoro-soro glutamatergic neurons; oligodendrocytes, oligodendrocytes; OPC, oligodendrocyte progenitor masero; RELN, reelin neurons. h, Gene Ontology (GO) izwi rekuvandudza kuongororwa kwemajini zvakanyanya kukwidziridzwa (yakagadziridzwa P <0.05, peta shanduko> 2, inoratidzirwa mune kanenge 10% ye nuclei) mu t-hCO glutamatergic neurons kana ichienzaniswa nehCO glutamatergic neurons. h, Gene Ontology (GO) izwi rekuvandudza kuongororwa kwemajini zvakanyanya kukwidziridzwa (yakagadziridzwa P <0.05, peta shanduko> 2, inoratidzirwa mune kanenge 10% ye nuclei) mu t-hCO glutamatergic neurons kana ichienzaniswa nehCO glutamatergic neurons. h, Анализ обогащения терминов Gene Ontology (GO) для генов со значительной активацией (скорректированный P <0,05, кратность изменекей применения > 2. в 10% ядер) в глутаматергических нейронах t-hCO по сравнению с глутаматергическими нейронами hCO. h, Gene Ontology (GO) izwi rekuvandudza kuongororwa kwemajini ane activation yakakosha (yakagadziriswa P <0.05, peta shanduko> 2, kutaura mune kanenge 10% nuclei) mu t-hCO glutamatergic neurons kana ichienzaniswa nehCO 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>倍发2,在至少10% 的细胞核中表达）的基因本体论(GO) 术语富集分析. h , 与 hco 谷氨酸 能 元 相比 , t-hco 谷氨酸 能 神经 元 基因 显着 上调 （后 嘎嘎（后 嘎 （后 嘎 （后 嘎 （后 嘎 （后 嘎） 2 , 至少 10% 的 核中 表达） 基因 基因 的 的 的 的 核中 表达） 基因 基因 h, гены значительно активизировались (скорректированный P <0,05, кратность изменения> 2, экспрессируется по крайней мере мере в 10% нейронах t-hCO по сравнению с глутаматергическими нейронами hCO Онтологический (GO) анализ термина обогащения. h, majini akanyatsogadziriswa (akagadziriswa P <0.05, kupeta shanduko> 2, inoratidzwa mune zvishoma 10% ye nuclei) mu t-hCO glutamatergic neurons kana ichienzaniswa nehCO glutamatergic neurons Ontological (GO) kuongororwa kweshoko rehupfumi.Mutsetse une doti unoratidza aq kukosha kwe0.05. ini, UMAP imaging yeGluN masero emhando mut-hCO uchishandisa label kutamisa kubva kune referensi 22 snRNA-seq mukuru mota cortex dataset. CT - corticothalamic masero, ET - extracerebral masero, IT - mukati telencephalic masero, NP - pedyo nekufungidzira.
Isu takazoongorora iyo cytoarchitecture uye yakazara cellular kuumbwa kwet-hCO. Antibody staining ye rat endothelial cells yakaratidza vascularization ne t-hCO, nepo IBA1 tsvina yakaratidza kuvapo kwekonzo microglia mukati megraft (Fig. 1f uye data yakawedzerwa, Fig. 3c, d). Immunostaining yakaratidza vanhu nuclear yenyu antigen (HNA) masero akanaka co-expressing PPP1R17 (cortical progenitors), NeuN (neurons), SOX9 uye GFAP (glial-derived cells) kana PDGFRα (oligodendrocyte progenitors) (Mufananidzo 1f). Kuti tidzidze kuumbwa kwesero ye t-hCO pane imwechete sero resolution, takaita single-core RNA sequencing (snRNA-seq) mushure memwedzi ingangoita 8 yekusiyanisa. Kusefa kwehuwandu uye kubviswa kwemakonzo nuclei kwakabereka 21,500 yepamusoro-soro yemamepu evanhu mononuclear (Fig. 1g uye data yakawedzerwa, Fig. 4a, b). Matauriro emhando dzemhando dzemasero akacherekedza masumbu emakirasi makuru ecortical cell, anosanganisira akadzika uye epamusoro glutamatergic neurons, anotenderera maprogenitors, oligodendrocytes, uye astrocyte mutsara (Fig. 1g, data yakawedzera, Fig. 4c, uye Supplementary Table 3). Immunostaining yeSATB2 uye CTIP2 yakaratidza kuti pasinei nekuvapo kwekortical subtypes, t-hCO haina kuratidza zvakajeka anatomical stratification (data yakawedzerwa, Fig. 3a). nhanho-inofananidzwa snRNA-seq hCO yakagadzira makirasi emasero akafanana, ane zvishoma, kusanganisira kusavapo kweoligodendrocytes uye kuvapo kweGABAergic neurons, iyo inogona kuratidza yakambotaurwa kuti yakanaka in vitro mamiriro e lateral progenitor cells15 (data yakawedzerwa, Fig. 4f - i uye Supplementary Table 4). Differential gene expression analysis yakaratidza misiyano yakakura mu glutamatergic neurons pakati pe t-hCO nehCO (Supplementary Table 5), kusanganisira activation yemaseti emajini ane chekuita neuronal maturation senge synaptic signing, dendritic localization, uye voltage-gated chiteshi chiitiko (Mufananidzo 1h uye Supplementary Tafura 5). tafura 6). Saizvozvo, cortical glutamatergic t-hCO neurons yakaratidza kukurumidza kunyorwa kwekunyora.
Kuti tijekese kana shanduko idzi dzechinyorwa mu t-hCO dzakabatana nekusiyana kwe morphological pakati pe hCO in vitro uye t-hCO mu vivo, takavakazve nhanho-inofananidzwa nebiocytin-yakazadza hCO uye hCO muzvikamu zvakakomba mushure memwedzi 7-8 yekusiyanisa. hCO neurons (Fig. 2a). t-hCO neurons yakanga yakakura zvikuru, yakanga ine 1.5 nguva ye soma dhayamita, kaviri yakawanda dendrites, uye kuwedzera kwekatanhatu kuwedzera kwehuwandu hwe dendritic hurefu huchienzaniswa ne in vitro hCO (Fig. 2b). Mukuwedzera, takaona kukwirira kwakanyanya kwedendritic spines mu t-hCO neurons pane hCO neurons (Fig. 2c). Izvi zvinoratidza kuti t-hCO neurons inopinda yakakura dendritic elongation uye branching, iyo, pamwe chete nekuenderera mberi kwekuwedzera kwesero, inogona kuita kuti kukura kwakasimba kwe t-hCO mushure mekusimwa (Fig. 1d uye Extended Data Fig. 1f). Izvi zvakaita kuti tiongorore maitiro e electrophysiological. Membrane capacitance yaive yakapetwa kasere (yakawedzerwa data, Fig. 8d), iyo yekuzorora-nyika membrane inogona kunge yakanyanya hyperpolarized (inenge 20 mV), uye jekiseni yemazuva ano yakakonzera huwandu hwepamusoro hwekunakidzwa mu t-hCO neurons kupfuura muhCO neurons. in vitro (Fig. 2d), e), iyo inopindirana nehukuru uye hwakaoma morphological features of t-hCO. Mukuwedzera, kuwanda kwezvinoitika zvinongoerekana zvaitika zve postsynaptic ikozvino zviitiko (EPSC) zvaive zvakanyanya kukwirira mu t-hCO neurons (Fig. 2f), zvichiratidza kuti kuwedzera kwehuwandu hwe dendritic spines yakaonekwa mu t-hCO neurons yakabatanidzwa nekunakidza kwekushanda. zvepabonde synapses. Takasimbisa hutsika husina kukura hwehCO neurons in vitro nekunyora kunonzi glutamatergic neurons (data yakawedzerwa, Fig. 6a-c).
a, 3D kuvakwazve kwebiocytin-yakazara hCO uye t-hCO neurons mushure memwedzi ye8 yekusiyanisa. b, Quantification yezvinyorwa zve morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 uye *** P <0.0001). b, Quantification yezvinyorwa zve morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 uye *** P <0.0001). б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 uye *** P <0,00). b, kuwanda kwemaitiro e-morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, uye *** 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 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 和**00 . б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 uye *** P <0,00). b, kuwanda kwemaitiro e-morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, uye *** P <0.0001).c, 3D kuvakwazve kwehCO uye t-hCO dendritic mapazi mushure me8 mwedzi yekusiyanisa. Nyeredzi dzvuku dzinoratidza putative dendritic spines. Dendritic musana density quantification (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0092). d, Quantification yekuzorora membrane inogona (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, Quantification yekuzorora membrane inogona (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, kuzorora membrane inogona 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, kuzorora membrane inogona quantification (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Kudzokorora chiito chinogona kupfura muhCO uye t-hCO inokonzerwa nekuwedzera majekiseni azvino, uye quantification yehuwandu hwekupfura (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Kudzokorora chiito chinogona kupfura muhCO uye t-hCO inokonzerwa nekuwedzera majekiseni azvino, uye quantification yehuwandu hwekupfura (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, повторное возбуждение потенциала действия в hCO uye t-hCO, вызванное увеличением тока, и количественная оценка максимальной скорости 2 воз, COней = 5. n = 16 нейронов t-hCO *** P <0,0001). e, chiito chinogona kupfuta zvakare mu hCO uye t-hCO inokonzerwa nekuwedzera kwemazuva ano uye kuwanda kwehuwandu hwehuwandu hwekupfura (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, 通过增加电流注入诱导的hCO 和t-hCO 重复动作电位放电，以及最大放电率的量化（n CO = 25 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 神 = 2 . 16 个 t-hco 神经 ; *** p <0.0001） . e, повторяющееся возбуждение потенциала действия hCO ne t-hCO, вызванное увеличением подачи тока, и количественная оценка максимальний максимальний 2005 = нейронов hCO, n = 16 нейронов t-hCO *** P <0,0001). e, kudzokorora kupfurwa kwehCO uye t-hCO maitiro echiito anokonzerwa nekuwedzera ikozvino kugovera uye kuwanda kwehuwandu hwekupfura chiyero (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) muhCO uye t-hCO neurons pamwedzi we8 wekusiyanisa, uye quantification yehuwandu hwezviitiko zve synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) muhCO uye t-hCO neurons pamwedzi we8 wekusiyanisa, uye uwandu hwehuwandu hwezviitiko zve synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, спонтанные EPSC (sEPSC) в нейронах hCO и t-hCO через 8 месяцев дифференцировки и количественная оценка частоты синаптический соб, 2 = 2 = 2 17 нейронов t-hCO *** P <0,0001) . f, Spontaneous EPSCs (sEPSCs) muhCO uye t-hCO neurons pamwedzi misere yekusiyanisa uye quantification ye synaptic chiitiko rates (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, 分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs (sEPSCs), 以及突触事件频率的量化（n = 25 hCO 7 神经 = 25 hCO 7 神神经元；***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 神n 25 hCO 神n神率的量匼（n = 25hCO P < 0.0001） . f, спонтанные EPSC (sEPSC) в нейронах hCO и t-hCO через 8 месяцев дифференцировки и количественная оценка частоты синаптический соб, 2 = 2 = 2 17 нейронов t-hCO *** P <0,0001). f, Spontaneous EPSCs (sEPSCs) mu hCO uye t-hCO neurons pamwedzi 8 yekusiyanisa uye quantification ye synaptic chiitiko rates (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001).Kune bf, hCO uye t-hCO mumutsara 1208-2 zvakatorwa kubva kune imwechete mutsauko batch inochengetedzwa yakafanana. g. majini akaonekwa kubva mu vivo mbeva kudzidza16 uye maLRG chaiwo evanhu kubva mu vitro neurons17. g. majini akaonekwa kubva mu vivo mbeva kudzidza16 uye maLRG chaiwo evanhu kubva mu vitro neurons17. g, анализ обогащения набора генов (односторонний точный критерий Фишера) генов со значительной активацией (скорректированменозий, песни, песни) 2, экспрессия по меньшей мере mu 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 in vivo16, и специфических для челозвеtroванных в исследовании на мышах in vivo16, и специфических для челозвеtro17 и пецифических для челозвеtro11 RG. g, kuongororwa kwejini set enrichment (one-tailed Fisher's exact test) yemajini ane akakosha activation (yakagadziriswa P <0.05, peta shanduko> 2, kutaura mune kanenge 10% nuclei) mu t-hCO glutamatergic neurons kana ichienzaniswa nehCO glutamatergic neurons sets yezvose zviri zviviri zvekutanga (ERG genes) zvakaonekwa mumashure mekutanga (ERG genes) uye kunonoka (ERG) uye maLRG chaiwo evanhu kubva kune neurons mu vitro17. g,t-hCO谷氨酸能神经元与hCO谷氨酸能神经元相比，t -hCO谷氨酸能神经元显着上调（调整后P<0.05,倍数变化>2，在至少10%的细胞核中表达）的基因集富集分析（单侧F isher精确检验）从体内小鼠研究中鉴定的早期反应(ERG)和晚期反应(LRG) 活性依赖性基因的基因组16 和体外神经元17 中的人类特异性LRG. g , t-hco 谷氨酸 神经 元 与 hco 谷氨酸 神经 元 相比 , t-hco 谷氨酸 神经 元 上调比 （0 诅 .倍数> 2 , 至少 至少 10%的 细胞 核中 表达） 集富集 分析 （单侧 fisher 精确你）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 神17的人类特异性LRG. g, глутаматергические нейроны t-hCO были значительно активизированы по сравнению с глутаматергическими нейронами hCO (скоррективизированы, 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, t-hCO glutamatergic neurons yakasimudzwa zvakanyanya kana ichienzaniswa nehCO glutamatergic neurons (yakagadziridzwa P <0.05, kupeta shanduko> 2, kanenge 10% Early response (ERG) uye kunonoka kupindura gene enrichment analysis (one-tailed Fisher's exact test) maitiro emhinduro anoenderana nemajini mu vitro1 mu vitro1 (LRGvos) LRGs.Mutsetse une doti unoratidza Bonferroni-yakagadziriswa P kukosha kwe0.05. h, GluN gene expression (pseudo-package uye scaling yejini rega rega) yakanyatsogadziriswa mu snRNA-seq replicas yeLRG genes mu t-hCO glutamatergic neurons. ini, immunostaining inoratidza SCG2 kutaura mu t-hCO (kumusoro) uye hCO (pasi) neurons. Miseve michena inonongedzera kumasero eSCG2+. Scale bar, 25 µm. Data inoratidzwa sezvinoreva ± chiyero chakatsauka.
Zvichienderana nekuwedzera kwekuita kwet-hCO inocherechedzwa mune ex vivo zvimedu, snRNA-seq yakaratidza chiitiko-chinoenderana nekusimudzira kwemajini zvinyorwa mu t-hCO zvichienzaniswa nehCO in vitro. Glutamatergic t-hCO neurons yakaratidza mazinga akakwirira emajini anodzora kunonoka kupindura basa (Fig. 2g, h), iyo yakawanikwa mune zvidzidzo zvekare mumakonzo uye neuroni dzevanhu16,17. Semuenzaniso, BDNF18, SCG2, uye OSTN, primate-specific activity-regulating gene, yakaratidza kuwedzera kutaura mu t-hCO neurons kana ichienzaniswa nehCO neurons (Fig. 2g-i). Nekudaro, t-hCO neurons yakaratidza kukwidziridzwa hunhu kana ichienzaniswa nehCO neurons nekunyora, morphological, uye mashandiro ekuongorora.
Kuti uwedzere kuongorora kushamwaridzana kwet-hCO kukura nekukura kwehuropi hwevanhu, takaita transcriptomic kuenzanisa kwe fetal uye munhu mukuru cortical cell types19,20 uye vanhu vakuru21,22 pamwe chete nemashoko akawanda e cortical gene expression23 panguva yekukura (data yakawedzerwa, Fig. 5). ) nebasa rekare 24, pasi rose hCO uye t-hCO transcriptome maturation status pa 7-8 mwedzi yekusiyanisa inopindirana zvakanyanya munguva yekukura kwe vivo uye inonyanya kufanana nehupenyu hwekupedzisira hwe fetus (Extended Data Fig. 5a). Zvinonyanya kukosha, takaona kuwedzera kwekukura kwezvinyorwa mu t-hCO zvichienzaniswa nezera-rinofananidzwa hCO, pamwe chete nekushandura kushandura kunobatanidza ne synaptogenesis, astrogenesis, uye myelination (data yakawedzerwa, Fig. 5b-d). Padanho remasero, takawana humbowo hwehutete cortex subtype mu t-hCO, ine masumbu e glutamatergic neurons anopindirana nevakuru L2 / 3, L5, uye L6 neuron subtypes (Mufananidzo 1i). Kusiyana neizvi, kupindirana kwemasumbu pakati pe glutamatergic t-hCO neurons uye fetal cortical neurons yaive yakanyanya kuganhurirwa pakati pepamuviri (yakawedzera data, Mufananidzo 5e-j). Kuti tione kana t-hCO neurons inoshanda zvakafanana nemunhu mushure mekuberekwa neocortical neurons, takaita zvinyorwa zve electrophysiological uye anatomical reconstructions evanhu L2 / 3 pyramidal neurons muzvikamu zvakapinza zvekortex yemunhu mushure mekuberekwa (data yakawedzerwa, Fig. 7a). Iyo electrophysiological properties yeL2 / 3 pyramidal neurons yakanga yakafanana neye t-hCO pyramidal neurons (yakawedzerwa data, Fig. 7e). Morphologically, L2 / 3 neurons kubva mushure mekuberekwa kwevanhu samples yakanga yakafanana ne t-hCO kupfuura hCO, kunyange zvazvo masero eL2 / 3 akanga akareba, ane matavi akawanda, uye aiva nepamusoro-soro yemusana (Fig. 3g uye data yakawedzerwa, Fig. 7b-). G).
a, kuisirwa hCO inogadzirwa nekutonga uye TS hiPS cell mitsara kuita neonatal makonzo. b, 3D kuvakwazve kwebiocytin-yakazara t-hCO neurons mushure memwedzi ye8 yekusiyanisa. c, chiyero chezvinoreva dendritic kureba (n = 19 control neurons, n = 21 TS neurons; ** P = 0.0041). d, 3D-yakagadzirwazve matavi edendritic kubva pakutonga uye TS t-hCO pamwedzi we8 wekusiyanisa, uye quantification ye dendritic spine density (n = 16 control neurons, n = 21 TS neurons, ***P <0.0001). d, 3D-yakagadzirwazve matavi edendritic kubva pakutonga uye TS t-hCO pamwedzi we8 wekusiyanisa, uye quantification ye 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 оценка плотностин плотностин контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, 3D kuvakazve matavi edendritic kubva pakutonga uye t-hCO TS pamwedzi we8 wekusiyanisa uye 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 = 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 плотностиный = контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, 3D kugadziriswa kwekutonga dendritic matavi uye TS t-hCO pamwedzi we8 wekusiyanisa uye dendritic spine density quantification (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001).Nyeredzi dzvuku dzinoratidza putative dendritic spines. e, maEPSC anongoerekana aita mukutonga uye TS t-hCO neurons mushure memwedzi ye8 yekusiyanisa. f, cumulative frequency plot uye quantification yefrequency uye amplitude yezviitiko zve synaptic (n = 32 control neurons, n = 26 TS neurons; ** P = 0.0076 uye P = 0.8102). g, Scholl kuongororwa kweTS uye kudzora neurons muhCO uye t-hCO. Dashed mitsetse inoratidza vanhu L2/3 postnatal pyramidal neurons yekuenzanisa (n = 24 control t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 control hCO neurons, uye n = 7 TS hCO neurons). Data inoratidzirwa seyanoreva ± chiyero chakatsauka
Kugona kwe t-hCO kudzokorora maitiro e morphological uye anoshanda emunhu cortex neurons pahupamhi hwakaita kuti tiongorore kana t-hCO inogona kushandiswa kuona chirwere che phenotypes. Takaisa pfungwa paTS, chirwere chakakomba cheurourodevelopmental chinokonzerwa nekuwana-kwe-function mutations mune gene encoding CaV1.2, iyo inotanga basa-inotsamira gene transcription mumaneuroni. Takawana hCO kubva kune vatatu TS varwere vakatakura zvinonyanya kutsiva (p.G406R) uye zvidzoreso zvitatu (Fig. 3a). Mushure mokutama, takaona kuti dendritic morphology yakashandurwa muTS neurons kana ichienzaniswa nekutonga (Fig. 3b uye data yakawedzerwa, Fig. 8a, b), ine kaviri kuwedzera kwenhamba yeprimary dendrites uye kuwedzera kwese kwehuwandu uye kuderera kwehuwandu hwe dendritic urefu (Fig. 3c uye data yakawedzerwa, Fig. 8c). Izvi zvakabatanidzwa nekuwedzera kwehuwandu hwema spines uye kuwedzera kwemaitiro eEPSCs ega ega muTS zvichienzaniswa nekudzora neuroni (Fig. 3d-f uye data yakawedzerwa, Fig. 8g). Kumwe kuongorora kwakaratidza maitiro eabnormal dendritic branching mu t-hCO TS kana ichienzaniswa nekutonga, asi kwete in vitro TS hCO pane imwe nhanho yekusiyanisa (Fig. 3g). Izvi zvinoenderana nemishumo yedu yapfuura yebasa-inotsamira dendritic shrinkage muTS uye inosimbisa kugona kweiyi chikuva chekutapurirana kuona chirwere phenotypes in vivo.
Takazobvunza kuti t-hCO maseru anobatanidzwa sei mugonzo S1. S1 mumakonzo inogamuchira yakasimba synaptic inputs kubva kune ipsilateral ventral basal uye posterior thalamic nuclei, pamwe ne ipsilateral motokari uye yechipiri somatosensory cortices, uye contralateral S1 (Fig. 4a). Kuti tidzorere maitiro ekuchengetedza, takatapurira hCO nehutachiona hwechimbwamupengo-dG-GFP/AAV-G uye takaisa hCO muS1 rat mazuva matatu gare gare. Takaona dense GFP kutaura mu neurons ye ipsilateral S1 uye ventral basal ganglia 7-14 mazuva mushure mekusimwa (Fig. 4b, c). Pamusoro pezvo, kusviba kwemasoja emucherechedzo wethalamic netrin G1 kwakaratidza kuvepo kwemagumo ethalamic mu t-hCO (Fig. 4d, e). Kuti tiongorore kana aya mafungidziro anopesana achigona kuunza mhinduro dzesynaptic mumaseru e-t-hCO, takaita maseru ese akarekodha kubva kumasero emunhu muzvikamu zvakapinza zvethalamocortical layer. Kukurudzira kwemagetsi kwegonzo S1, mukati capsule, white matter, fibers pedyo ne t-hCO kana optogenetic activation ye opsin-inoratidza thalamic endings mu t-hCO induced short-latency EPSCs mu t-hCO neurons yakazarurirwa kune AMPA receptor antagonist NBQX. (Fig. 4f, g uye data yakawedzerwa, Fig. 9a–g). Idzi data dzinoratidza kuti t-hCO inosanganisirwa muhuropi hwegonzo uye inokwanisa kushandiswa nemakonzo.
a, Schematic diagram yekuyedza yekutevera chimbwamupengo. b, GFP uye kutaura kwevanhu-yakananga STEM121 pakati pe t-hCO uye rat cerebral cortex (upper panel). Zvakare zvinoratidzwa kutaura kweGFP mune rat ipsilateral ventral basal nucleus (VB) (pazasi kuruboshwe) uye ipsilateral S1 (pasi kurudyi). Scale bar, 50 µm. Zvikwere zvitsvuku zvinomiririra nzvimbo dzeuropi uko mifananidzo yakatorwa. c, kuwanda kwemasero anoratidza GFP (n = 4 rats). d, e - Netrin G1+ thalamic zviteshi mu t-hCO. d inoratidza coronal chikamu chine t-hCO uye VB nuclei. Scale bar, 2 mm. e inoratidza Netrin G1 uye STEM121 kutaura mu t-hCO (kuruboshwe) uye VB (kurudyi) neurons. Scale bar, 50 µm. Mutsetse weorenji une doti unoratidza muganho wet-hCO. f, g, Ikozvino mitsetse ye t-hCO neurons mushure mekusimudzira magetsi muS1 rat (f) kana mukati capsule (g), ine (yepepuru) kana isina (dema) NBQX (kuruboshwe). EPSC amplitudes ine uye isina NBQX (n = 6 S1 neurons, * P = 0.0119; uye n = 6 mukati capsule neurons, ** P = 0.0022) (pakati). Chikamu che t-hCO neurons chinoratidza EPSC mukupindura kukurudzirwa kwemagetsi egonzo S1 (f) kana mukati capsule (g) (kurudyi). aCSF, artificial cerebrospinal fluid. h, dhiyabhorosi dhayagiramu ye2P yekufungidzira kuyedza (kuruboshwe). Kutaura kweGCaMP6s mu t-hCO (pakati). Scale bar, 100 µm. Fluorescence nguva kupera kweGCaMP6s (kurudyi). ini, Z-score yezvinoitika zvechiito fluorescence. j, schematic mufananidzo wekukurudzira ndebvu. k, z-yakawana 2P fluorescence trajectories mune imwe kuyedza, inofambirana nekutsauka kwewhisk panguva zero (dashed line) mumuenzaniso maseru. l, avhareji yehuwandu-z-score mhinduro dzemasero ese anoenderana newhisker kutsauka panguva zero (dashed line) (dzvuku) kana randomly kugadzirwa timestamps (grey). m. Schematic dhayagiramu yekuedza pane optical marking. n, Raw voltage curves kubva kumuenzaniso t-hCO sero panguva yeblue laser kukurudzira kana whisker deflection. Miseve dzvuku inoratidza mapikisi ekutanga anokonzerwa nechiedza (pamusoro) kana kukonzerwa nekutsemuka kwendebvu (pasi). Grey shading inoratidza nguva dzekutsauka kwewhisker. o, Peak light waveforms uye whisker deflection mhinduro. p, spikes yekuedza kumwe chete, inopindirana nekutsauka kwendebvu mumasero emuenzaniso. 0 inoratidza whisker kutsauka (dashed line). q, avhareji yehuwandu z-chibodzwa kupfurwa kwemaseru ese ane photosensitive, anoenderana nekutsauka kwewhisk panguva zero (dashed line) (dzvuku) kana zvidhindo zvenguva zvisina tsarukano (grey). r, Chikamu chephotosensitive zvikamu zvakagadziridzwa zvakanyanya newhisker kutsauka (n = 3 makonzo) (kuruboshwe). Peak z-score latency (n = 3 rats; n = 5 (chiedza chegirini), n = 4 (yakasviba girini), uye n = 4 (cyan) whisker deflection modulation units per rat) (kurudyi). Data inoratidzwa sezvinoreva ± chiyero chakatsauka
Isu takazobvunza kana t-hCO yaigona kuvhurwa nekunzwa stimuli mu vivo. Isu takadyara hCO ichiratidza genetically encoded calcium zviratidzo GCaMP6 muS1 makonzo. Mushure memazuva 150, takaita fiber photometry kana maviri-photon calcium imaging (Fig. 4h uye data yakawedzerwa, Fig. 10a). Takaona kuti t-hCO masero anoratidza synchronized rhythmic chiitiko (Mufananidzo 4i, Yakawedzerwa Data, Figure 10b uye Supplementary Vhidhiyo 1). Kuratidzira peak t-hCO basa, takaita extracellular electrophysiological rekodhi mune anesthetized transplant rats (yakawedzera data, Fig. 10c-f). Takagadzira stereotaxic coordinates kubva kuMRI mifananidzo; saka, aya mayuniti akarekodhwa anomiririra putative human neurons, kunyange zvazvo electrophysiology yoga isingabvumiri rudzi rwemavambo kuti ruonekwe. Takacherechedza kuputika kwakafanana kwechiitiko (data yakawedzerwa, Fig. 10d). Kuputika kwakagara pamusoro pe460 ms uye kwakaparadzaniswa nenguva yekunyarara inenge 2 s (data yakawedzerwa, Fig. 10d, e). Mayuniti ega ega akadzinga avhareji yeanosvika matatu kutenderera pakuputika, inova ingangoita 73% yemayuniti akanyoreswa pakuputika. Mabasa ezvikamu zvega ega akabatanidzwa zvakanyanya, uye kuwirirana uku kwaive kwakakwirira kudarika iyo yezvikamu zvakaonekwa mumhuka dzisina kuvharwa dzakanyorwa pasi pemamiriro akafanana (data yakawedzerwa, Fig. 10f). Kuti tiwedzere kuratidza mhinduro dzespike dzema neuron akaonekwa nevanhu, takaita mwenje-tagging kuyedza pamakonzo anesthetized akasimwa nehCO ichiratidza chiedza-sensitive cation chiteshi rhodopsin 2 (hChR2), kuburikidza iyo t-hCO neurons kupfupika-latency kucherechedzwa (pasi pe 10 ms- 4 mwenje) mukupindura . t-hCO neurons yakaratidza kuputika kwebasa rinongoerekana raitika pamafafitera akafanana neaya anoonekwa mukufungidzira kwekarusiyamu, pamwe chete nemagetsi emagetsi akaitwa mu t-hCO pasina chiedza chekucherechedza ( data yakawedzerwa, Fig. 10c-g). Hapana chiitiko chekuzvimiririra chakaonekwa mumatanho anoenderana nehCO yakanyorwa mu vitro. Kuti tiongorore kana t-hCO inogona kushandiswa nemafungiro emanzwiro, takabvisa muchidimbu ndebvu dzekonzo kubva ku t-hCO (Fig. 4j, m uye data yakawedzerwa, Fig. 10h, k). Maererano nezvidzidzo zvekare8,10, chikamu che-t-hCO masero chakaratidza kuwedzera kwebasa mukupindura whisker deflection, iyo yakanga isina kuonekwa apo data yakaenzaniswa nematampu enguva (Fig. 4k-q uye data yakawedzerwa, Fig. 10h-q). Zvechokwadi, inenge 54% ye-opto-labeled single units yakaratidza kuwedzera kwakanyanya kwekukurudzira mushure mekusimudzira whisker, ichikwira pamusoro pe650 ms (Fig. 4r). Kutorwa pamwe chete, idzi data dzinoratidza kuti t-hCO inogamuchira yakakodzera maitiro ekushandisa uye inogona kushandiswa nekukurudzira kwezvakatipoteredza.
Takazoongorora kana t-hCO inogona kumisa masekete mumakonzo kudzora maitiro. Isu takatanga taongorora kana maaxon et-hCO neurons ari mukati mematishu akatenderedza egonzo. Isu takatapurira hCO nelentivirus encoding hChR2 yakasanganiswa kuEYFP (hChR2-EYFP). Mushure memazuva 110, takaona EYFP kutaura munzvimbo ipsilateral cortical, kusanganisira auditory, motokari, uye somatosensory cortices, uyewo subcortical nzvimbo, kusanganisira striatum, hippocampus, uye thalamus (Fig. 5a). Kuti tiongorore kana aya efferent fungidziro anogona kuunza synaptic mhinduro mumakonzo maseru, isu optical activated t-hCO masero anoratidza hChR2-EYFP nekurekodha rat cerebral cortex maseru muzvikamu zvehuropi zvakapinza. Kushandiswa kwet-hCO axons nechiedza chebhuruu chakakonzera pfupi-latency EPSCs mune rat pyramidal cortex neurons, iyo yakavharwa neNBQX (Figs. 5b-g). Mukuwedzera, mhinduro idzi dzinogona kudziviswa ne tetrodotoxin (TTX) uye kudzorerwa ne4-aminopyridine (4-AP), zvichiratidza kuti dzakakonzerwa nekubatana kwe monosynaptic (Fig. 5e).
a, Schematic dhizaini yeaxon yekutevera (kuruboshwe). t-hCO EYFP kutaura (kurudyi). Scale bar, 100 µm. A1, auditory cortex, ACC, anterior cingulate cortex, d. striatum, dorsal striatum, HPC, hippocampus; Diaphragm, lateral septum, mPFC, medial prefrontal cortex, piri, piriform cortex, v. striatum, ventral striatum, VPM, ventropostomedial nucleus ye thalamus, VTA, ventral tegmental region. Zvikwere zvitsvuku zvinomiririra nzvimbo dzeuropi uko mifananidzo yakatorwa. b, Schematic dhayagiramu yekuedza kwekukurudzira. c, d, Mienzaniso yemhinduro yeblue light-induced photocurrent (yepamusoro) uye voltage (pazasi) mumunhu (c) EYFP+ t-hCO kana rat (d) EYFP- masero. e, f, Ikozvino kurongeka kwemakonzo neuroni mushure mechiedza chebhuruu kukurudzirwa kwet-hCO axons neTTX uye 4-AR (girini), TTX (grey) kana aCSF (dema) (e), ine (violet) kana isina (dema) ) ) NBQX (e). g, latency yemhinduro inokonzerwa nechiedza chebhuruu mumasero emakonzo (n = 16 masero); mbariro dzakachinjika dzinoratidza avhareji yekunonoka (7.13 ms) (kuruboshwe). Amplitude yeEPSC yakaburitswa nemwenje yakarekodhwa kana isina NBQX (n = 7 masero; ***P <0.0001) (pakati). Amplitude yeEPSC yakaburitswa nemwenje yakarekodhwa kana isina NBQX (n = 7 masero; ***P <0.0001) (pakati). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). Amplitude yechiedza-induced EPSCs yakanyorwa kana isina NBQX (n = 7 masero; *** P <0.0001) (pakati).使用或不使用NBQX 记录的光诱发EPSC 的振幅（n = 7 个细胞；***P <0.0001）（中）.使用或不使用NBQX 记录的光诱发EPSC 的振幅（n = 7 个细胞；***P <0.0001）（中）. Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). Amplitude yechiedza-induced EPSCs yakanyorwa kana isina NBQX (n = 7 masero; *** P <0.0001) (pakati).Mazana emasero emakonzo anoratidza maEPSC anopindura kuchiedza chebhuruu (kurudyi). h, Schematic diagiramu yebasa rekuzvibata. d0, zuva 0. i. Kuita kwemhuka dzemuenzaniso pazuva 1 (kuruboshwe) kana zuva rechi15 (kurudyi) rekudzidziswa. Iyo inorehwa nhamba yezvinwiwa zvakaitwa pazuva 1 (kuruboshwe) kana zuva 15 (kurudyi pakati) (n = 150 bhuruu chiedza miedzo, n = 150 chiedza chitsvuku miedzo; *** P <0.0001). Iyo inorehwa nhamba yezvinwiwa zvakaitwa pazuva 1 (kuruboshwe) kana zuva 15 (kurudyi pakati) (n = 150 bhuruu chiedza miedzo, n = 150 chiedza chitsvuku miedzo; *** P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) или день (в центре справа) (n = 150 испытаний с синим светой, n = 150 светом; ***P <0,0001). Zvinoreva nhamba yezvinanzva zvakaitwa pazuva 1 (kuruboshwe) kana zuva 15 (pakati kurudyi) (n = 150 mwenje webhuruu miedzo, n = 150 mwenje tsvuku miedzo; ***P <0.0001).第1 天（左）或第15 天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150 还0.0001.第1 天（左）或第15 天（右中) Среднее количество облизываний, выполненных в день 1 (слева) или день (в центре справа) (n = 150 испытаний с синим светой, n = 150 светом; ***P <0,0001). Zvinoreva nhamba yezvinanzva zvakaitwa pazuva 1 (kuruboshwe) kana zuva 15 (pakati kurudyi) (n = 150 mwenje webhuruu miedzo, n = 150 mwenje tsvuku miedzo; ***P <0.0001).Cumulative licks kune tsvuku uye yebhuruu chiedza miedzo pazuva 1 (pakati kuruboshwe) kana zuva 15 (kurudyi). NS, haina kukosha. j,k, Maitiro ehunhu hwemhuka dzese dzakasimwa ne t-hCO inotaura hChR2-EYFP (j) kana kudzora fluorophore (k) pazuva 1 kana 15 (hChR2-EYFP: n = 9 rats, ** P = 0.0049; control: n = 9, P = 0. l, Evolution yezvavanoda zvibodzwa (n = 9 hChR2, n = 9 control; **P <0.001, ***P <0.0001). l, Evolution yezvavanoda zvibodzwa (n = 9 hChR2, n = 9 control; **P <0.001, ***P <0.0001). l, Эволюция показателя предпочтения (n = 9 hChR2, n = 9 контрольных; **P <0,001, ***P <0,0001). l, Evolution yezvibodzwa zvibodzwa (n = 9 hChR2, n = 9 controls; **P <0.001, ***P <0.0001). l, 偏好评分的演变（n = 9 hChR2，n = 9 对照；**P < 0.001,***P < 0.0001）. l, 偏好评分的演变（n = 9 hChR2，n = 9 对照；**P < 0.001,***P < 0.0001）. l, Эволюция показателей предпочтения (n = 9 hChR2, n = 9 контролей; **P <0,001, ***P <0,0001). l, Evolution yezvibodzwa zvezvibodzwa (n = 9 hChR2, n = 9 controls; **P <0.001, ***P <0.0001).m, FOS kutaura mukupindura optogenetic activation ye t-hCO muS1. Mifananidzo yeFOS matauriro (kuruboshwe), uye quantification (n = 3 paboka; *P <0.05, **P <0.01 uye ***P <0.001) (kurudyi) inoratidzwa. Mifananidzo yeFOS matauriro (kuruboshwe), uye quantification (n = 3 paboka; *P <0.05, **P <0.01 uye ***P <0.001) (kurudyi) inoratidzwa. Показаны изображения экспрессии FOS (слева) и количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 and *** P <0,001) (справа). Mifananidzo yeFOS matauriro (kuruboshwe) uye quantification (n = 3 paboka; *P<0.05, **P<0.01, uye ***P<0.001) inoratidzwa (kurudyi).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 and *** P <0,001) (справа). Mifananidzo yeFOS matauriro (kuruboshwe) uye quantification (n = 3 paboka; *P<0.05, **P<0.01, uye ***P<0.001) inoratidzwa (kurudyi).Scale bar, 100 µm. Dhata inoratidzwa sezvinoreva ± yakajairwa kukanganisa kweBLA, basolateral tonsil, MDT, dorsomedial thalamic nucleus, PAG, periaqueductal grey.
Chekupedzisira, takabvunza kana t-hCO ichigona kuenzanisa maitiro emakonzo. Kuti tiedze izvi, takaisa hChR2-EYFP-inoratidza hCO muS1, uye mazuva makumi mapfumbamwe gare gare, takaisa optical fibers mu t-hCO kuti ibudise chiedza. Takabva tadzidzisa makonzo ane modified orant conditioning paradigm (Fig. 5h). Takaisa mhuka mukamuri yekuzvibata uye takaisa zvishanu sekondi yebhuruu (473 nm) uye tsvuku (635 nm) laser stimuli. Mhuka dzakawana mubairo wemvura kana dzakananzva panguva yechiedza cheblue stimulation asi hadzina kunanzva panguva yered light stimulation. Pazuva rekutanga rekudzidziswa, mhuka dzakaratidza kusave nemusiyano mukunanzva painokurudzirwa nebhuruu kana tsvuku mwenje. Nekudaro, pazuva regumi neshanu, mhuka dzakasimwa nehCO ichiratidza hChR2-EYFP yakaratidza kushingaira kunanzva kana yamutswa nechiedza chebhuruu zvichienzaniswa nekukurudzira kwechiedza chitsvuku. Kuchinja uku mukunanzva maitiro hakuna kucherechedzwa mukudzora mhuka dzakasimwa nehCO ichiratidza kudzora fluorophore (yekudzidza yekubudirira mwero: hChR2 89%, EYFP 0%, Mufananidzo 5i-1 uye Supplementary Video 2). Idzi data dzinoratidza kuti t-hCO masero anogona kumisa rat neurons kukurudzira maitiro ekutsvaga mubairo. Kuti tizive kuti ndeipi rat t-hCO neural circuits inogona kubatanidzwa mukushanduka kwemaitiro aya, isu optogenetically activated t-hCO mumhuka dzakadzidziswa uye kukohwa matishu 90 maminetsi gare gare. Immunohistochemistry yakaratidza kutaura kweiyo basa-inotsamira FOS protein munzvimbo dzinoverengeka dzehuropi dzakabatanidzwa muhunhu hunokurudzirwa, kusanganisira yepakati prefrontal cortex, medial thalamus, uye periaqueductal grey nyaya, iyo yairatidzwa mungave mumhuka dzisingadzoreki kana mhuka. mupunga. 5m). Zvakatorwa pamwechete, idzi data dzinoratidza kuti t-hCO inogona kuenzanisa rat neuronal chiitiko kutyaira maitiro.
Neural organoids inomiririra sisitimu inovimbisa yekudzidza kukura kwevanhu uye chirwere in vitro, asi inoganhurwa nekushaikwa kwekubatana pakati pemaseketi aripo mu vivo. Isu takagadzira nhanho yepuratifomu yatakaisa hCO muS1 yemakonzo ekutanga mushure mekusununguka kuti adzidze kukura kwesero remunhu uye kushanda mu vivo. Isu takaratidza kuti t-hCO inogadzira akakura maseru marudzi asina kucherechedzwa mu vitro28 uye kuti t-hCO ine anatomically uye inoshanda yakabatanidzwa muhuropi hwegonzo. Kubatanidzwa kwe t-hCO mu rodent neural circuits kwakatibvumira kuti titange hukama pakati pekuita kwemasero evanhu uye kudzidza maitiro emhuka, zvichiratidza kuti t-hCO neurons inogona kugadzirisa rat neuronal chiitiko kutyaira maitiro ekuita.
Iyo puratifomu yatinotsanangura ine akati wandei mabhenefiti pamusoro petsvakiridzo yapfuura pakuisa masero emunhu muhuropi hwegonzo. Chekutanga, isu takadyara hCO mune inokura cortex yemakonzo ekutanga ekuberekwa, izvo zvinogona kufambisa anatomical uye inoshanda kubatanidzwa. Chechipiri, t-hCO MRI monitoring yakatibvumira kudzidza nzvimbo yegraft uye kukura mumhuka mhenyu, zvichiita kuti tiite zvidzidzo zvenguva refu zvemhuka dzakawanda uye kusimbisa kuvimbika kwemitsara yakawanda yehiPS. Chekupedzisira, isu takadyara intact organoids, pane kugadzika sero rimwe chete kumiswa, izvo zvisinganyanye kuparadza kumasero emunhu uye zvinogona kukurudzira kubatanidzwa uye chizvarwa chevanhu cortex neurons muuropi hwemakonzo.
Tinobvuma kuti pasinei nekufambira mberi kwepuratifomu iyi, zvipingamupinyi zvenguva, nzvimbo, uye zvakasiyana-siyana zvinodzivirira kuumbwa kwevanhu neural circuits nekutendeseka kwepamusoro, kunyange mushure mekusimwa panguva yekutanga yekuvandudzwa. Semuenzaniso, hazvina kujeka kana basa rinongoerekana raonekwa mu t-hCO rinomiririra phenotype yekuvandudza yakafanana neyo rhythmic chiitiko chinoonekwa panguva yekukura kwecortical, kana kuti imhaka yekushayikwa kwemhando dzemasero anodzvinyirira aripo mu t-hCO. Saizvozvo, hazvisi pachena kuti kusavapo kwelamination mu t-hCO kunokanganisa sei cheni yekubatanidza30. Basa remangwana richatarisa pakubatanidza mamwe marudzi emasero akadai seanhu microglia, endothelial masero evanhu, uye zvakasiyana-siyana zveGABAergic interneurons sezvinoratidzwa kushandisa assembly 6 in vitro, pamwe nekunzwisisa kuti neural integration uye kugadzirisa zvinogona kuitika sei mukushandurwa t-hCO. transcriptal, synaptic uye maitiro ekuzvibata mumasero akawanikwa kubva kuvarwere.
Pakazere, iyi in vivo chikuva inomiririra sosi ine simba iyo inogona kutsigira mu vitro huropi hwemunhu kukura uye kutsvagisa chirwere. Isu tinotarisira kuti iyi puratifomu ichatitendera kuti tiwane novel strand-level phenotypes mune imwe nzira isinganzwisisike-yakatorwa nemurwere maseru uye kuyedza nzira nyowani dzekurapa.
Isu takagadzira hCO2.5 kubva kumaseru eHiPS sezvakatsanangurwa kare. Kuti utange hCO kugadzirwa kubva kumaseru eHiPS akasimwa pazvikamu zvekudyisa, makoroni emasero eHiPS akabviswa mundiro dzetsika achishandisa dispase (0.35 mg/mL) ndokuendeswa kune ekupedzisira-yakaderera akabatanidzwa epurasitiki tsika ane ndiro dzine hiPS cell tsika yepakati. (Corning) yakawedzerwa maviri SMAD inhibitors dorsomorphine (5 μM; P5499, Sigma-Aldrich) uye SB-431542 (10 μM; 1614, Tocris) uye ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Mumazuva ekutanga e5, iyo hiPS cell medium yakashandurwa zuva nezuva uye dorsomorphine uye SB-431542 yakawedzerwa. Pazuva rechitanhatu mukumiswa, neural spheroids yakaendeswa kune neural medium ine neurobasal-A (10888, Life Technologies), B-27 supplement isina vhitamini A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin uye streptomy00, Lifepichnologies yekuwedzera uye 1:1: chinhu (EGF; 20 ng ml-1; R & D Systems) uye fibroblast growth factor 2 (FGF2; 20 ng ml-1; R & D Systems) kusvikira zuva 24. Kubva pazuva 25 kusvika kuzuva 42, iyo yepakati yakawedzerwa neuropi-derived neurotrophic factor (BDNF; 20 trophin; 3 trophin; 3 mln trophin) (3 trophin trophic); 20 ng ml−1; Peprotech) ine shanduko yepakati zuva rega rega. Pazuva rechitanhatu mukumiswa, neural spheroids yakaendeswa kune neural medium ine neurobasal-A (10888, Life Technologies), B-27 supplement isina vhitamini A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin uye streptomy00, Lifepichnologies yekuwedzera uye 1:1: chinhu (EGF; 20 ng ml-1; R & D Systems) uye fibroblast growth factor 2 (FGF2; 20 ng ml-1; R & D Systems) kusvikira zuva 24. Kubva pazuva 25 kusvika kuzuva 42, iyo yepakati yakawedzerwa neuropi-derived neurotrophic factor (BDNF; 20 trophin; 3 trophin; 3 mln trophin) (3 trophin trophic); 20 ng ml−1; Peprotech) ine shanduko yepakati zuva rega rega.Pazuva rechitanhatu mukumiswa, neural spheroids yakaendeswa kune neural medium ine Neurobasal-A (10888, Life Technologies), B-27 supplement isina vitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin.и стрептомицин (1:100, Life Technologies) и дополнены эпидермальным фактором роста (EGF; 20 нг/мл; R&D Systems) и фактором роста фибл20FLAF; R&D Systems) до 24-го дня. uye streptomycin (1:100, Life Technologies) uye yakawedzerwa ne epidermal growth factor (EGF; 20 ng/ml; R&D Systems) uye fibroblast growth factor 2 (FGF2; 20 ng/ml; R&D Systems) kusvika zuva rechi24.Kubva pamazuva 25 kusvika ku42, brain-derived neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) uye neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) yakawedzerwa kune svikiro, kuchinja svikiro rimwe nerimwe zuva.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 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）第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 сстрептомицин (1:100, Life Technologies) с добавлением эпидермальго факторангей факторангей факторага; R&D Systems) и фактора роста фибробластов 2 (FGF2; 20 нг мл-1) 1; Pazuva rechitanhatu, neurosphere suspensions yakachinjirwa kune yekuwedzera ine neurobasal-A (10888, Life Technologies), B-27 yekuwedzera isina vhitamini A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin-neutralized factor streptomycin (1: 1000 Life supplement) (EGF; 20 ng ml-1; R&D Systems) uye fibroblast kukura factor 2 (FGF2; 20 ng ml-1) 1; R&D Systems) до 24-го дня. R&D Systems) kusvika zuva 24.Kubva pamazuva 25 kusvika ku42, brain-derived neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) uye neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) yakawedzerwa kune tsika yemagariro zuva rimwe nerimwe. Kuchinja kwepakati kamwe chete.Kutanga kubva pazuva rechimakumi mana nematatu, hCO yakachengetedzwa mune isina kuwedzererwa neurobasal-A svikiro (NM; 1088022, Thermo Fisher) ine yepakati shanduko mazuva ese 4-6. Kuti uwane hCO kubva kumasero e-hiPS akasimwa pasi pemamiriro asina kudya, masero e-hiPS akaiswa neAccutase (AT-104, Innovate Cell Technologies) pa 37 ° C kwemaminitsi manomwe, akaparadzaniswa kuva masero mamwechete, uye akaputirwa paAggreWell 800 plates (34815, STEMCELL a well 6 × 3 masero) Yakakosha 8 yepakati yakawedzerwa neROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Mushure memaawa makumi maviri nemana, midhiya mumatsime yakapfapfaidzwa kumusoro nekudzika munhau ine Essential 6 media (A1516401, Life Technologies) yakawedzerwa nedorsomorphine (2.5 μM; P5499, Sigma-Aldrich) uye SB-431542 (10 μM; 1614). , Tocrida). Kubva pamazuva 2 kusvika ku6, Essential 6 yepakati yakatsiviwa zuva nezuva nedorsomorphine uye kuwedzera SB-431542. Kubva pazuva rechitanhatu, iyo neurosphere suspensions yakaendeswa kune neurobasal medium uye yakachengetedzwa sezvakatsanangurwa pamusoro.
Maitirwo ese emhuka akaitwa zvinoenderana negwara rekuchengeta mhuka rakabvumidzwa neStanford University Laboratory Animal Care Administrative Committee (APLAC). Ane nhumbu euthymic RNU (rnu/+) makonzo akatengwa (Charles River Laboratories) kana kugara. Mhuka dzaichengetwa pa 12-hour light-dark cycle ine chikafu nemvura ad libitum. Mushwi (FOXN1–/–) vana vemakonzo vane mazuva matatu kusvika manomwe vaionekwa nekukura kwendebvu dzisati dzakura dzisati dzatemwa. Mhuka (chirume nehadzi) dzakabatwa ne 2-3% isoflurane uye yakaiswa pane stereotaxic furemu. Kubvunda kwedehenya rine dhayamita ingangoita 2-3 mm pamusoro peS1 yakaitwa ichichengetedza kuvimbika kwedura mater. Zvadaro shandisa tsono ye30-G (inenge 0.3 mm) kunze kwecraniotomy kuboora dura. Wobva waisa HCO kune yakaonda 3 × 3 cm parafilm uye bvisa yakawandisa yepakati. Uchishandisa sirinji yeHamilton yakasungirirwa pane 23 G, 45 ° tsono, zvinyoronyoro dhonza hCO kumucheto wakareba kwazvo wetsono. Wobva waisa sirinji papombi yesirinji yakabatana neiyo stereotaxic mudziyo. Woisa muromo wetsono pamusoro peyakamboitwa 0.3 mm yakafara puncture gomba mudura (z = 0 mm) uye kutetepa sirinji 1-2 mm (z = anenge -1.5 mm) kusvikira tsono iri pakati pe dura mater A. dense seal inoumbwa. Zvadaro simudza sirinji kusvika pakati pecortical surface pa z = -0.5 mm uye jekiseni hCO nechiyero che 1-2 µl paminiti. Mushure mekupedzwa kwejekiseni reHCO, tsono inodzoserwa pamwero we 0.2-0.5 mm paminiti, ganda rinoiswa sutured, uye mwana wembwa anobva angoiswa pane inodziya yekudziya padhi kusvika yapora zvachose. Dzimwe mhuka dzakasimwa pairi.
Maitirwo ese emhuka akaitwa zvinoenderana neStanford University APLAC-inotenderwa miitiro yekuchengetedza mhuka. Makonzo (akakura kupfuura mazuva makumi matanhatu mushure mekusimwa) akakurudzirwa ne5% isoflurane anesthesia uye anesthetized ne 1-3% isoflurane panguva yekufungidzira. Kuti utarise, 7 Tesla yakanyatso dzivirira yakachinjika borehole scanner Bruker (Bruker Corp.) ine International Electric Company (IECO) gradient drive, yakadzivirirwa gradient inopinza ine dhayamita yemukati ye120 mm (600 mT/m, 1000 T/m/s) yakashandiswa uchishandisa AVANCE. III, sere-chiteshi akawanda-coil RF uye akawanda-musimboti kugona, uye inoperekedza Paravision 6.0.1 chikuva. Kurekodha kwakaitwa pachishandiswa inoshingairira decoupled volumetric RF coil ine dhayamita yemukati ye86 mm uye ina-channel cryo-yakatonhorera RF coil yekugamuchira chete. Axial 2D Turbo-RARE (kudzokorodza nguva = 2500 ms, echo nguva = 33 ms, 2 mavhareji) ne 16 zvidimbu zvakatorwa, chidimbu chechidimbu 0.6-0.8 mm, chine 256 × 256 samples. Iwo masaini akagamuchirwa uchishandisa quadrature transceiver volumetric RF coil ine dhayamita yemukati ye2 cm (Rapid MR International, LLC). Chekupedzisira, shandisa yakavakirwa-mukati Imaris (BitPlane) pamusoro estimation mabasa kune 3D kupa uye kuongorora vhoriyamu. Kusimwa kwakabudirira kwakatsanangurwa seimwe inzvimbo dzekuenderera mberi T2-inorema MRI chiratidzo chakaumbwa mune yakasimwa hemisphere. Kurambwa kwegraft kwakatsanangurwa segraft iyo isina kubudisa nzvimbo dzekuenderera mberi T2-inorema MRI chiratidzo mune yakasimwa hemisphere. Subcortical t-hCO yakanga isingabatanidzwe kubva pakuongorora kwakatevera.
Kuti ataure zvakadzikama GCaMP6s muhCO ye-two-photon calcium imaging, masero eHiPS akabatwa nepLV[Exp] -EF1a::GcaMP6s-WPRE-Puro ichiteverwa nekusarudzwa kwemishonga inorwisa mabhakitiriya. Nenguva pfupi, maseru akaparadzaniswa neEDTA uye akamiswa mu1 ml yeEssential 8 yepakati pahuwandu hwemaseru mazana matatu pamberi pe polybrene (5 μg/ml) uye 15 μl yehutachiona. Masero acho akaiswa mukumiswa kwemaminetsi makumi matanhatu uye akadyarwa pahuwandu hwemasero makumi mashanu ezviuru patsime. Mushure mekubatana, maseru aibatwa ne 5-10 μg ml-1 puromycin kwemazuva 5-10 kana kusvika makoroni akagadzikana aoneka. Acute hCO chirwere chakaitwa sezvakambotsanangurwa 5 pamwe nekumwe kugadziridzwa. Muchidimbu, shandura zuva 30-45 hCO mu 1.5 ml Eppendorf microcentrifuge tubes ine 100 μl ye nerve medium. Ipapo inenge 90 µl yepakati inobviswa, 3-6 µl ye high titer lentivirus (kubva pa 0.5 x 108 kusvika 1.2 x 109) inowedzerwa kuchubhu, uye hCO inotamiswa kune incubator kwemaminitsi makumi matatu. Wobva wawedzera 90–100 µl yepakati pachubhu yega yega wodzosera machubhu acho ku incubator husiku hwose. Zuva rinotevera, endesa hCO kune nyowani nerve yepakati mumapureti akaderera. Mushure memazuva manomwe, hCO yakaendeswa kune makumi maviri nemana-magirazi epasi mahwendefa ekuona uye kuongororwa kwehutano hwehutachiona. pLV[Exp]-SYN1::EYFP-WPRE uye pLV[Exp]-SYN1::hChR2-EYFP-WPRE yakagadzirwa neVectorBuilder. Lentivirus inoshandiswa mukuedza kwakawanda nekuti inosanganiswa mune inotambira genome, ichibvumira mutori wenhau gene kutaura mumitsara ine hutachiona. Nekuda kwechimbwamupengo chekutevera, zuva 30-45 hCO yakabatwa nerabies-ΔG-eGFP uye AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), yakashambidzwa zvakakwana kwemazuva matatu, uye yakasimwa mumakonzo muS1 uye yakachengetedzwa mumazuva 7-1.
Kune immunocytochemistry, mhuka dzaive anesthetized uye transcardially perfused nePBS ichiteverwa ne4% paraformaldehyde (PFA muPBS; Electron Microscopy Sayenzi). Uropi hwakagadziriswa mu4% PFA kwemaawa maviri kana husiku humwe pa4 ° C, cryopreserved mu30% sucrose muPBS kwemaawa 48-72, uye yakabatanidzwa mu1: 1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583, Sakura Finetes0 akaitwa ccoronat 3 chikamu akaitwa ccoronat3) (Leica). Kune immunohistochemistry yezvikamu zvakakora, mhuka dzakasvibiswa nePBS, uye huropi hwakaparadzaniswa uye hwakakamurwa coronally pa300-400 µm vachishandisa vibratome (Leica) uye zvikamu zvakagadziriswa ne4% PFA kwemaminetsi makumi matatu. Zvadaro mazamu kana zvikamu zvakakora zvakashambidzwa nePBS, zvakavharwa kweawa imwe patembiricha yekamuri (10% yakajairika dhongi serum (NDS) uye 0.3% Triton X-100 yakasvibiswa muPBS) uye yakavharwa nekuvhara mhinduro pa4 ° C. -Incubation Cryosections akaiswa mukati mehusiku humwe uye zvikamu zvakakora zvakaiswa kwemazuva mashanu. Masoja ekudzivirira ekutanga aishandiswa aive: anti-NeuN (mbeva, 1:500; ab104224, abcam) anti-CTIP2 (gonzo, 1:300; ab18465, abcam), anti-GFAP (tsuro, 1:1,000; Z0334, Dako), anti-GFP0 1 GTX0, 1,0, anti-GFP0; GeneTex), anti-HNA (mbeva, 1:200; ab191181, abcam), anti-NeuN (tsuro, 1:500; ABN78, Millipore), anti-PDGFRA (tsuro, 1:200; sc-338, Santa Cruz), anti-PPP20:1R; HPA047819, Atlas Antibodies), anti-RECA-1 (mbeva, 1:50; ab9774, abcam), anti-SCG2 (tsuro, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (mbudzi, 1:500; NetgorinD5 Systema, G30D5 1:100; AF1166, R&D Systems), anti-STEM121 (mbeva, 1:200; Y40410, Takara Bio), anti-SATB2 (mbeva, 1:50; ab51502, abcam), anti-GAD65/67 (tsuro, 1:400, anti-ABIN00; (mbudzi, 1:100; ab5076, abcam). Masoja ekudzivirira ekutanga aishandiswa aive: anti-NeuN (mbeva, 1:500; ab104224, abcam) anti-CTIP2 (gonzo, 1:300; ab18465, abcam), anti-GFAP (tsuro, 1: 1,000; Z0334, Dako), anti-GFP0 1 GTX0, 3, 0, anti-GFP0; GeneTex), anti-HNA (mbeva, 1:200; ab191181, abcam), anti-NeuN (tsuro, 1:500; ABN78, Millipore), anti-PDGFRA (tsuro, 1:200; sc-338, Santa Cruz), anti-PPP20:1R; HPA047819, Atlas Antibodies), anti-RECA-1 (mbeva, 1:50; ab9774, abcam), anti-SCG2 (tsuro , 1:100; 20357-1-AP, Proteintech), anti-SOX9 (mbudzi, 1:500; Neta, R&30D Systems) 1:100; AF1166, R&D Systems), anti-STEM121 (mbeva , 1:200; Y40410, Takara Bio), anti-SATB2 (mbeva, 1:50; ab51502, abcam), anti-GAD65/67 (tsuro, 1:400, anti-ABIN00) (mbudzi, 1:100; ab5076, abcam). Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2 (крысиные, 1:506); анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA (мышь, 1:200; ab1911181, ab1911181, ab1911181, 1:500; ABN78, Millipore), анти-PDGFRA (кролик, 1:200; sc-338, Санта-Круз), анти-PPP1R17 (кролик, 1:200; HPA047819, Atlas-Antibodies), Atlas, Antibodies 1:50; ab9774, abcam), анти-SCG2 (кролик , 1:100; 20357-1-AP, Proteintech), анти-SOX9 (козий, 1:500; AF3075, R&D Systems), нетризи10, 6 (1:10; R&D Systems), анти-STEM121 (мышиный , 1:200; Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кроли00, AB9, ABN; анти-IBA1 (коза, 1 :100; аб5076, абкам). Masoja ekudzivirira ekutanga aishandiswa aive: anti-NeuN (mbeva, 1:500; ab104224, abcam), anti-CTIP2 (gonzo, 1:300; ab18465, abcam), anti-GFAP (tsuro, 1:1000; Z0334, Dako), anti-GFP0 Gene; anti-HNA (mbeva, 1:200; ab191181, abcam), anti-NeuN (tsuro, 1:500; ABN78, Millipore), anti-PDGFRA ( tsuro, 1:200; sc-338, Santa Cruz), anti-PPP1R17, 1:03 HPAb4; Antibodies), anti-RECA-1 (mbeva, 1:50; ab9774, abcam), anti- SCG2 (tsuro, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (mbudzi, 1:500; AF3075, R&D Systems), netria (100 G1; R&D Systems), anti- STEM121 (mbeva, 1:200; Y40410, Takara Bio), anti-SATB2 (mbeva, 1:50; ab51502, abcam), anti-GAD65/67 (tsuro, 1:400; ABN904, Millipore 1: 0 goat, anti-0; ab5076, abkam).使用的一抗是：抗NeuN（小鼠，1:500；ab104224，abcam）抗CTIP2（大鼠，1:3 00;ab18465,abcam），抗GFAP（兔，1:1,000;Z0334，Dako），抗-GF P（鸡，1:1,000;GTX13970，GeneTex），抗HNA（小鼠，1:200;ab191181，abcam），抗NeuN（兔，1:500;ABN78,Millipore）Millipore）Millipore） 1:200;sc-338，Santa Cruz），抗PPP1R17（兔，1:200;HPA047819，Atlas 抗体），抗RECA-1（小鼠，1:50;ab9774ab9774ab9774ab9774ab 1 1:200;使用的一抗是：抗NeuN（小鼠，1:500；ab104224，abcam）抗CTIP2（大鼠，1 :300;ab18465,abcam），抗GFAP（兔，1:1,000;Z0334，Dako），抗-GFP（鸡，1:1,000;GTX13970，GeneTex），抗HNA（小鼠，1:200;ab191181,abcam），抗NeuN（兔，1:500;ABN78＊Millipo＊ABN78， 200;sc-338，Santa Cruz），抗PPP1R17（兔，1:200;HPA047819，Atlas 抗体），抗RECA-1（小鼠，1:50;ab9774）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 (mbeva, 1:50; ab51502, abcam), anti-GAD65/67 (tsuro, 1:400; ABN904, Millipore) uye anti-IBA1 (mbudzi, 1:100; ab5076, abcam).Masoja ekudzivirira chirwere akanyanya kushandiswa aive: anti-NeuN (mouse, 1:500; ab104224, abcam), anti-CTIP2 (rat, 1:300; ab18465, abcam), anti-GFAP (tsuro, 1:1000; Z0334, Dako). , anti-GFP (huku, 1:1000; GTX13970, GeneTex), anti-HNA (mouse, 1:200; ab191181, abcam), anti-NeuN (tsuro, 1:500; ABN78, Millipore), anti-PDGFRA (rab3scru C30, 108; anti-PPP1R17 (tsuro, 1:200; HPA047819, Atlas antibody), anti-RECA-1 (mbeva, 1:50; ab9774, abcam), anti- SCG2 (tsuro), 1:100;20357-1-AP, Proteintech), анти-SOX9 (коза, 1:500; AF3075, R&D Systems), Нетрин G1a (коза, 1:100; AF1166, R&D Systems), анти -STEM121 (Y4:040, Taka); анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) и анти-IBA1 (коза, 1:100; аб5076, аб5076). 20357-1-AP, Proteintech), anti-SOX9 (mbudzi, 1:500; AF3075, R&D Systems), Netrin G1a (mbudzi, 1:100; AF1166, R&D Systems), anti-STEM121 (mbeva, 1:200; Y400; 1:50; ab51502, abcam), anti-GAD65/67 (tsuro, 1:400; ABN904, Millipore), uye anti-IBA1 (mbudzi, 1:100; ab5076, abkam).Zvikamu zvakabva zvashambidzwa nePBS uye zvakaiswa nesecondary antibody kweawa imwe patembiricha yekamuri (zvikamu zvakaomeswa nechando) kana husiku hwese pa4°C (zvikamu zvakakora). Alexa Fluor secondary antibody (Life Technologies) yakanyungudutswa 1:1000 mukuvharisa mhinduro yakashandiswa. Mushure mekugeza nePBS, nuclei yakaonekwa neHoechst 33258 (Life Technologies). Pakupedzisira, masiraidhi akaiswa pamaikorosikopu ane anovhara (Fisher Scientific) achishandisa Aquamount (Polysciences) uye akaongororwa paKeyence fluorescent microscope (BZ-X analyzer) kana Leica TCS SP8 confocal microscope (Las-X) pamufananidzo. Mifananidzo yakagadziriswa pachishandiswa ImageJ chirongwa (Fiji). Kuyera chikamu chetsinga dzevanhu mu t-hCO uye rat cortex, 387.5 μm yakafara rectangular mifananidzo yakatorwa pakati pe t-hCO, padyo kana kumucheto kwegonzo cortex. Graft margins akatemerwa nekuongorora shanduko mukujeka kwetishu, HNA + nuclei, uye / kana kuvapo kwetishu autofluorescence. Mumufananidzo wega wega, huwandu hwese hweNeuN+ neHNA+ masero akakamurwa nehuwandu hwese hweNeuN+ masero munzvimbo imwechete. Kuve nechokwadi chekuti maseru ane nuclei mundege yemufananidzo anoverengwa, maseru chete ariwo Hoechst + anosanganisirwa mukuverenga. Mifananidzo miviri yakapatsanurwa neinenge 1 mm yakaverengerwa kudzikisa chikanganiso chehuwandu.
Vhiki imwe kusati kwaitwa sampuli yekuunganidza, isa hCO kuisirwa mhuka (inenge 8 mwedzi yekusiyanisa) mukamuri ine rima ine ndebvu dzakachekwa kuti kuderedze kurudziro yekunzwa. Isolation of nuclei yakaitwa sezvakatsanangurwa kare, nekumwe kugadziridzwa. Muchidimbu, t-hCO nehCO zvakaparadzwa pachishandiswa detergent-mechanical cell lysis uye 2 ml yegirazi tishu grinder (D8938, Sigma-Aldrich/KIMBLE). Manyuclei asina kuomeswa akabva apepetwa pachishandiswa 40 µm sefa uye centrifuged pa 320 g kwemaminetsi gumi pa 4 ° C asati aita sucrose density gradient. Mushure me centrifugation nhanho (320 g ye 20 min pa 4 ° C), samples dzakamiswa zvakare mu 0.04% BSA/PBS pamwe nekuwedzera 0.2 mayunitsi e µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) uye akapfuura nepakati pe40 µm sefa. Iwo akapatsanurwa nuclei akabva amiswazve muPBS ine 0.02% BSA uye akatakurwa paChromium Single Cell 3′ chip (inofungidzirwa kupora kwe8,000 masero pamukoto). snRNA-seq raibhurari dzakagadzirwa neChromium Single cell 3'GEM, Library & Gel Bead Kit v3 (10x Genomics). snRNA-seq raibhurari dzakagadzirwa neChromium Single cell 3'GEM, Library & Gel Bead Kit v3 (10x Genomics). Библиотеки snRNA-seq были приготовлены с помощью Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Iwo snRNA-seq raibhurari akagadzirwa achishandisa Chromium Single cell 3'GEM, Library & Gel Bead Kit v3 (10x Genomics). snRNA-seq 文库是使用Chromium Single cell 3′ GEM、Library & Gel Bead Kit v3 (10x Genomics) 制备的. snRNA-seq 文库是使用Chromium Single cell 3′ GEM、Library & Gel Bead Kit v3 (10x Genomics) 制备的. Библиотеку snRNA-seq готовили с использованием Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Iyo snRNA-seq raibhurari yakagadzirwa uchishandisa Chromium Single Cell 3'GEM, Raibhurari & Gel Bead Kit v3 (10x Genomics).Maraibhurari kubva kumasampuli akasiyana akaiswa pamwe nekuteedzerwa neAdmera Health paNovaSeq S4 (Illumina).
Gene kutaura mazinga ega yega kodhi yenyukireya bhakodhi akaverengerwa pachishandiswa 10x Genomics CellRanger yekuongorora software package (vhezheni 6.1.2). Kunyanya, kuverenga kwaifananidzwa nemusanganiswa wevanhu (GRCh38, Ensemble, vhezheni 98) uye rat (Rnor_6.0, Ensemble, vhezheni 100) mareferensi genomes akagadzirwa nemkref command uye kushandisa kuverenga ne--include-introns=TRUE kuraira kuhuwandu kunosanganisira kuverenga kwakamisikidzwa kusuma dunhu. Kune t-hCO samples, nuclei yemunhu yakaonekwa zvichibva pane chinodiwa chekuchengetedza kuti inosvika makumi mapfumbamwe neshanu muzana ezvese zvinoverengwa pamepu zvinoenderana nejenome remunhu. Ongororo dzese dzakazotevera dzakaitwa pane yakasefa barcode array kubuda kubva kuCellRanger uchishandisa R package (version 4.1.2) Seurat (version 4.1.1)32.
Kuti ive nechokwadi chekuti nuclei yemhando yepamusoro chete inosanganisirwa mukuwongorora kunotevera, nzira yekusefa yakadzokororwa yakaitwa kune imwe neimwe sampuli. Chekutanga, yakaderera-mhando nuclei ine isingasviki 1000 genes akasiyana akawanikwa uye anopfuura makumi maviri muzana eiyo mitochondria yakazara inotaridzwa nekubviswa. Mushure mezvo, iyo mbishi gene number matrix yakagadziridzwa neyakajairwa negative binomial regression pachishandiswa sctransform(vst.flavor=”v2″) basa, iro rakaratidzawo 3000 emajini akawanda anosiyana-siyana achishandisa maparameter akasarudzika. 30 yakasarudzwa zvichienderana nekuongororwa kwekuona kwenzvimbo dzemabvi uye yakashandiswa kune ese masampuli uye ensemble analyses takaita denderedzwa rinoverengeka reiterative clustering (resolution = 1) kurongedza majini zvichienderana neasingawanzo yakaderera gene count (yepakati pazasi 10th percentile), abnormally high mitochondrial gene pazasi bvisa 9th percentile uye/kana chikamu chepamusoro chevanofungidzirwa kuti mapatya vakaonekwa pachishandiswa DoubletFinder33 pasuru (zvichireva kuti DoubletFinder zvibodzwa zviri pamusoro pe95th percentile t-hCO samples (n=3) nehCO samples (n=3) zvakabatanidzwa zvakaparadzana pachishandiswa IntegrateData maparameters ari pamusoro apa.
Mushure mekubvisa kernels yemhando yakaderera, dataset yakabatanidzwa yakaiswa mumapoka (resolution = 0.5) uye yakamisikidzwa yeUMAP34 yekuona zvinangwa. Makoni emajini esumbu rega rega akatemerwa kushandisa iyo FindMarkers basa ine default paramita akaverengerwa kubva yakajairika gene kutaura data. Isu tinocherekedza uye nekuisa makirasi makuru emasero nekubatanidza fetal uye evakuru cortical referensi datasets ane marker gene expression 19,20,21,35 uye annotation. Kunyanya, kutenderera kwekutanga kwakaonekwa nekutaura kweMKI67 uye TOP2A. Masumbu eprogenitor akatsanangurwa nekusavapo kwezvinyorwa zvemitotic, kupindirana kwepamusoro neakawanda glial progenitor masumbu anotsanangurwa mukupedzisira metaphase fetal cortex, uye EGFR uye OLIG1 kutaura. Isu tinoshandisa izwi rekuti astrocyte kutenderedza akati wandei nyika dzeastrocyte musiyano, kubva kunonoka radial glia kusvika kukukura kweastrocyte. Masumbu eAstrocyte anoratidza mazinga epamusoro eSLC1A3 neAQP4 uye akaratidzwa kumepu ine subtypes ye fetal radial glia uye/kana vakuru venyeredzi. OPCs inoratidza PDGFRA uye SOX10 nepo oligodendrocytes inoratidzira miyelination mamaki (MOG uye MYRF). Glutamatergic neurons yakaonekwa nekuvapo kweiyo neuronal transcripts (SYT1 uye SNAP25), kusavapo kweGABAergic mamaki (GAD2), uye kutaura kweNEUROD6, SLC17A7, BCL11B, kana SATB2. GluN neurons yakawedzera kukamurwa kuva kumusoro (SATB2 kutaura uye kurasikirwa kweBCL11B) uye yakadzika (BCL11B kutaura) madiki. Putative subplate (SP) neurons inoratidzira inozivikanwa SP18 mamaki senge ST18 uye SORCS1 mukuwedzera kune yakadzika maGluN mamaki. Choroid plexus-like masero akaonekwa neTTR kutaura, uye meningeal-like masero airatidza fibroblast-akabatanidzwa majini uye mamepu pial/vascular masero ereferenzi data set.
Kuongorora kwakasiyana kwekutaura kwemajini pakati pe t-hCO nehCO madiki akaitwa pachishandiswa nzira ichangoburwa yepseudo-batch yakaburitswa mumasampuli akashandiswa uchishandisa Libra R package (version 1.0.0). Kunyanya, edgeR log-inogona bvunzo (vhezheni 3.36.0, package R) yakaitwa kumapoka nekupfupikisa huwandu hwemajini mumaseru ekirasi yakapihwa sero yemuenzaniso wega wega kudzokorora. Kuti uone kupisa kwemepu, yakajairwa pamiriyoni (CPM) kukosha kunoverengerwa uchishandisa edgeR (cpm () basa) uye yakayerwa (kuzadzisa zvinoreva = 0, yakajairwa kutsauka = ​​1). Gene Ontology (GO) ongororo yekuvandudza yakanyatsogadziriswa t-hCO GluN majini yakaitwa (Benjamini-Hochberg yakaruramisa P kukosha kwepasi pe0.05 inoratidzwa mune inokwana 10% ye t-hCO GluN masero uye kupeta kuwedzera kwekuchinja kanenge ka2). inoitwa uchishandisa ToppGene Suite (https://toppgene.cchmc.org/)37. Isu tinoshandisa iyo ToppFun app ine default paramita uye tinoshuma Benjamini-Hochberg-yakagadziriswa P-tsika dzakaverengerwa kubva kuGO-annotated hypergeometric bvunzo.
Kufananidza masumbu edu e-snRNA-seq ane annotated cell clusters kubva kuongororo yezvidzidzo zveprimary single-cell RNA-seq kana yevakuru snRNA-seq19,20,21,22, takashandisa nzira yekubatanidza dataset. Isu takashandisa iyo SCTransform (v2) yakajairwa kufambiswa kwekushanda muSeurat kubatanidza uye kuenzanisa kupindirana kwemasumbu pakati pemadataset (uchishandisa iwo akafanana paramita sepamusoro). Maseti ega ega akaiswa pasi pesero kusvika ku500 maseru kana macores pachikwata chepakutanga chekombuta inoshanda. Kushandisa nzira yakafanana sezvakatsanangurwa kare, kupindirana kwesumbu kwakatsanangurwa sechikamu chemasero kana nuclei musumbu rega rega rakabatanidzwa iro rakapfuura nereferenzi yereferensi cluster. Kuti tienderere mberi nekuisa maGluNs, takashandisa Seurat's TransferData workflow yeGluN subset data kuti tipe referensi dataset zvinyorwa kumasero edu eGluN.
Kuti tiongorore mamiriro ekukura kweiyo pasi rose zvinyorwa zvet-hCO uye hCO samples, takafananidza edu emanyepo-akawanda samples neBrainSpan/psychENCODE23, iyo ine yakakura RNA kutevedzana inotora kukura kwehuropi hwemunhu. Takaita PCA pane yakasanganiswa pateni-yakajairwa gene expression matrix kubva cortical samples 10 masvondo mushure mekubata pamuviri uye gare gare, mu5567 majini (pamwe chete nedata redu) ayo akamboonekwa seanoshanda muBrainSpan cortical samples (inotsanangurwa seyakakura kupfuura 50% mukusiyana kwekusimudzira kwakatsanangurwa nezera uchishandisa cubic modhi)38. Pamusoro pezvo, isu takawana majini ane hukama nemasiginecha makuru ezvinyorwa zveurourodevelopment tichishandisa isiri-negative matrix factorization sezvakambotsanangurwa. Sample uremu akaverengerwa uchishandisa isiri-negative matrix factorization maitiro akarongwa muFig. 5b ine data yakawedzerwa kune imwe neimwe yemasiginecha mashanu anotsanangurwa naZhu et al.38. Zvekare, zviitwa zvinotsamira pakunyorerwa mamaki zvakatorwa kubva kuzvidzidzo zvakamboburitswa. Kunyanya, ERG neLRG zvakagadziriswa zvakanyanya mu glutamatergic neurons yakaonekwa ne mouse visual cortex snRNA-seq collection mushure mekuona kukurudzirwa kubva kune Supplementary Table 3 Hrvatin et al.16. MaLRG akafumiswa nevanhu akawanikwa kubva kuKCl-activated human fetal brain cultures uye akakohwa 6 maawa mushure mekusimudzira, uye majini akacheneswa akaiswa zvakanyanya muvanhu asi kwete mumakonzo (Supplementary Table 4). Ongororo yemajini set hupfumi uchishandisa aya majene seti akaitwa pachishandiswa nzira imwe chete yeFisher's chaiyo bvunzo.
Anesthetize rats ne isoflurane, bvisa huropi uye nzvimbo inotonhora (inenge 4 ° C) oxygenated (95% O2 uye 5% CO2) sucrose solution yezvikamu zvine: 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM. NaH2PO4, 10 mM MgSO4 uye 0.5 mM CaCl2 (inenge 310 mOsm). Rat brain coronal zvikamu (300-400 µm) ine t-hCO yakagadzirwa uchishandisa Leica VT1200 vibratome sezvakatsanangurwa kare39. Zvikamu zvakabva zvaendeswa kune imwe kamuri yekamuri ine inoenderera yekamuri tembiricha oxygenation ine aCSF yakagadzirwa kubva: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 uye 126 mM MOsm29 (29). kanenge 45 maminetsi asati kurekodha. Zvikamu zvakarekodhwa mukamuri yakanyudzwa mavairamba vachifemerwa ne aCSF (95% O2 uye 5% CO2 vial). Yese data yakanyorwa pakamuri tembiricha. t-hCO neurons yakapedzwa neborosilicate glass pipette yakazadzwa nemushonga une 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, uye 0.6 mM EGTA, pH 7.2, mukati memhinduro yakagadziriswa neKOOm29 mH. Kuti uwanezve, biocytin (0.2%) yakawedzerwa kune mhinduro yekurekodha.
Dhata rakatorwa pachishandiswa MultiClamp 700B amplifier (Molecular Devices) uye Digidata 1550B digitizer (Molecular Devices), yakaderera-pass yakasefa pa2 kHz, yakadhijitwa pa20 kHz, uye yakaongororwa uchishandisa Clampfit (Molecular Devices), Origin (OriginPro). 2021b, OriginLab). uye tsika MATLAB mabasa (Mathworks). Iyo junction inogona kuverengerwa uchishandisa JPCalc uye zvinyorwa zvakagadziridzwa kune yakaverengerwa kukosha kwe -14 mV. Operation IV ine akatevedzana matanho azvino mu10-25 pA nhanho, kubva -250 kusvika 750 pA.
Iyo thalamus, chena nyaya, uye S1 afferents akasimudzwa nemagetsi muthalamocortical zvimedu panguva yechigamba-clamp kurekodha yehCO neurons, sezvakatsanangurwa kare. Muchidimbu, uropi hwakaiswa patafura yekudhinda ye3D yakarereka pakona ye10°, uye kumberi kweuropi kwakachekwa pakona ye35°. Huropi hwakabva hwanamirwa kunzvimbo yakachekwa ndokuiswa muzvikamu, kuchengetedza thalamocortical protruding axon. Bipolar tungsten electrodes (0.5 MΩ) yakaiswa pane yechipiri micromanipulator uye yakamisikidzwa zvine hungwaru kuti ikurudzire matunhu mana pasero (mukati capsule, chena nyaya, S1 uye hCO). Rekodha mhinduro dzesynaptic mushure me300 µA phasic stimulation pa0.03–0.1 Hz.
hChR2-inoratidza hCO neurons yakashandiswa pa 480 nm uye chiedza chemhepo chakagadzirwa ne LED (Prizmatix) chakashandiswa kuburikidza ne × 40 chinangwa (0.9 NA; Olympus) kunyora hChR2 kutaura pedyo nemasero. Iyo yakavhenekerwa munda dhayamita ingangoita 0.5 mm uye iyo yakazara simba i10-20 mW. Iyo pulse wide yakaiswa kune 10 ms, iyo inoenderana nekupuruzira kwakapihwa panguva yekuzvibata kudzidza kuedza. Yakasiyana-siyana yekukurudzira ma frequency akashandiswa, kubva pa1 kusvika ku20 Hz, asi chete yekutanga kupuruzira kweiyo nhevedzano yakashandiswa quantification. Nguva dziri pakati pezvitima dzinowanzoreba kupfuura 30s kuderedza mhedzisiro pa synaptic inhibitory kana kufambisa nzira. Kuti tiedze kana iyo hChR2 mhinduro yaive monosynaptic, takaisa TTX (1 μM) kubhati kusvika iyo EPSC reaction yanyangarika, ndokuzoisa 4-aminopyridine (4-AP; 100 μM). Kazhinji, mhinduro inodzoserwa mukati memaminitsi mashoma, nekunonoka zvishoma pakati pe LED kupfura uye EPSC chizvarwa. NBQX (10 μM) yakashandiswa kuyedza kuti mhinduro yacho inofambiswa ne AMPA receptors.
Yakapinza hCO zvikamu zvakagadzirwa sezvakatsanangurwa kare. Muchidimbu, zvikamu zvehCO zvakaiswa mu4% agarose uye zvakaendeswa kune masero ane 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 uye 10 mM -glucose 0-0 0 (0) akachekwa muSection 0 + µm pane tembiricha yekamuri uchishandisa Leica VT1200 vibrator uye yakachengetwa muASF pakamuri tembiricha. Zvadaro, chigamba-kamba kurekodha kwemasero akazara chakaitwa pazvikamu zvehCO pasi peyakananga SliceScope maikorosikopu (Scientifica). Zvikamu zvakasvibiswa ne aCSF (95% O2 uye 5% CO2) uye zviratidzo zvemasero zvakanyorwa pakupisa kwekamuri. hCO neurons yakashandiswa uchishandisa borosilicate glass pipette yakazadzwa nemhinduro ine 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, uye 0.6 mM EGTA, yemukati pH 7, 2, yakagadziriswa neKOH20 mosmo. Nezvinangwa zvekudzorera, wedzera 0.2% Biocytin kune mhinduro yemukati.
Data yakawanikwa neClampex (Clampex 11.1, Molecular Devices) vachishandisa MultiClamp 700B amplifier (Molecular Devices) uye Digidata 1550B digitizer (Molecular Devices), yakaderera-pass yakasefa pa2 kHz, digitized pa 20 kHz, kuongorora molecular , uye 160 kuongorora. zvishandiso) uye tsika MATLAB mabasa (MATLAB 2019b, Mathworks). Iyo junction inogona kuverengerwa uchishandisa JPCalc uye zvinyorwa zvakagadziridzwa kune yakaverengerwa junction inogona ye -14 mV. Operation IV ine nhevedzano yematanho azvino mu5-10 pA nhanho kubva -50 kusvika 250 pA.
Nezve morphological reconstruction yepinched neurons, 0.2% biocytin (Sigma-Aldrich) yakawedzerwa kune mhinduro yemukati. Iwo maseru anokwiridzirwa kweanenge maminetsi gumi nemashanu mushure mekubira. Iyo pipette inobva yakweverwa zvishoma nezvishoma mukati me 1-2 min kusvikira membrane yakanyoreswa yakavharwa zvachose. Kutevera chikamu che physiology maitiro, zvikamu zvakagadziriswa usiku hwose pa 4 ° C. mu 4% PFA, yakashambidzwa nePBS X3, uye yakasvibiswa 1: 1000 ne streptavidin-conjugated DyLight 549 kana DyLight 405 (Vector Labs). Masero akazadzwa nebiocytin (2%; Sigma-Aldrich) akanyorwa panguva yekurekodha chigamba chekurekodha pakamuri tembiricha kwemaawa maviri. Zvikamu zvakabva zvakaiswa pamasiraidhi emaikorosikopu pachishandiswa Aquamount (Thermo Scientific) ndokuonekwa zuva raitevera paLeica TCS SP8 confocal microscope vachishandisa chinangwa chekunyudza mafuta chine aperture yenhamba ×40 1.3, magnification ×0.9–1.0, xy. Sampling rate ingangoita 7 pixels pa micron. Z-stacks pa1 µm nguva dzakawanikwa dzakateedzana, uye z-stack mosaics uye Leica-based auto-stitching yakaitwa kuvhara iyo yose dendritic muti weimwe neuron. Neurons dzakazoteverwa semi-manual vachishandisa neuTube 40 interface uye mafaira eSWC akagadzirwa. Mafaira akabva aiswa kuSimpleNeuriteTracer41 Fiji plugin (ImageJ, version 2.1.0; NIH).
Cortical tissue yemunhu yakawanikwa nemvumo ine ruzivo maererano neprotocol yakabvumidzwa neInstitutional Review Board yeStanford University. Mienzaniso miviri yezvinyama zvepashure zvemunhu (3 uye 18 yemakore) zvakawanikwa nekubviswa kweiyo frontal cortex (yepakati frontal gyrus) sechikamu chekuvhiyiwa kwerefractory pfari. Mushure mekuvhiyiwa, kohwa nyama muchando-inotonhora NMDG-aCSF ine: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mM sodium mCruvate mMpyM 0 sodium ascorbate, mpyruvate 0 sodium mpyl 2. 4H2O uye 10 mM MgSO4 7H2O. Titrate kusvika pH 7.3-7.4 ine concentrated hydrochloric acid. Mishonga yakaendeswa ku laboratori mukati memaminitsi makumi matatu uye zvikamu zve coronal zvakatorwa maererano nemaitiro anotsanangurwa pamusoro apa.
Maitirwo ese emhuka akaitwa zvinoenderana neStanford University APLAC-inotenderwa miitiro yekuchengetedza mhuka. Makonzo (anopfuura mazuva 140 mushure mekutapurwa) akaitwa ne5% isoflurane anesthesia uye anesthetized ne 1-3% isoflurane intraoperatively. Mhuka dzakaiswa mune stereotaxic furemu (Kopf) uye yakaramba ichiburitswa buprenorphine (SR) yakajairwa pasi pecutaneously. Dehenya rinoburitswa pachena, rinocheneswa uye 3-5 bhonzo screws inoiswa. Kuti titarise t-hCO, takagadzira stereotaxic coordinates kubva kuMRI mifananidzo. Gomba rakacherwa panzvimbo yekufarira uye fiber (400 µm dhayamita, NA 0.48, Doric) yakadzikiswa 100 µm pazasi pehCO pamusoro uye yakachengetedzwa kudehenya neUV-inorapika simende yemazino (Relyx).
Fiber photometric rekodhi zvakaitwa sezvakambotsanangurwa42. Kurekodha chiitiko chinongoerekana chaitika, makonzo akaiswa mukeji yakachena uye 400 µm dhayamita fiber optic patch cable (Doric) yakabatana nefiber optic photometric data acquisition system yakabatanidzwa kune yakasimwa fiber optic tambo. Munguva yekurekodhwa kwemaminetsi gumi emabasa emotokari, mhuka dzaive dzakasununguka kuongorora keji. Kunyora chiitiko chakamutswa, makonzo (kupfuura mazuva 140 mushure mekusimwa) akaiswa anesthetized ne5% isoflurane yekupinza uye 1-3% isoflurane yekuchengetedza. Isa mhuka mu stereotactic frame (Kopf) uye ndebvu dziri mhiri kwe t-hCO dzakachekwa kusvika masendimita maviri uye dzinopfuura nemu mesh yakabatana nepiezoelectric actuator (PI). A 400 µm fiber optic patch cable (Doric) yakanga yakabatana nefiber yakasimwa uye yakabatanidzwa kune data acquisition system. Ndebvu dziri mhiri kwe t-hCO dzakabva dzatsauswa ka50 (2 mm pa 20 Hz, 2 s per presentation) panguva dzisina kujairika nepiezoelectric drive pamusoro pemaminetsi makumi maviri ekurekodha nguva. Shandisa iyo Arduino MATLAB Tsigiro Package kudzora nguva yekutsauka netsika MATLAB kodhi. Zviitiko zvinowiriraniswa kune software yekutora data uchishandisa transistor-transistor logic (TTL) pulses.
Makonzo (anopfuura mazuva 140 mushure mekutapurwa) akaitwa ne5% isoflurane anesthesia uye anesthetized ne 1-3% isoflurane intraoperatively. Mhuka dzakaiswa mu stereotaxic frame (Kopf) uye buprenorphine SR uye dexamethasone dzakaiswa pasi pecutaneously. Dehenya rinoburitswa pachena, rinocheneswa uye 3-5 bhonzo screws inoiswa. Kuti titarise t-hCO, takagadzira stereotaxic coordinates kubva kuMRI mifananidzo. A circular craniotomy (inenge 1 cm mudhayamita) yakaitwa nekumhanya kwepamusoro zvakananga pamusoro peiyo yakasimwa hCO. Kana bhonzo raita mutete sezvinobvira, asi usati waboora bhonzo rose, shandisa forceps kubvisa yasara intact pelvic disc kuratidza zviri pasi pe t-hCO. Iyo craniotomy yakazadzwa nesaline isina utachiona, uye chivharo nepini yemusoro yakakosha zvakanamirwa padehenya neUV-yakaporeswa simende yemazino (Relyx).
Kufungidzira kwemafotoni maviri kwakaitwa pachishandiswa Bruker multiphoton microscope ine Nikon LWD (×16, 0.8 NA) chinangwa. GCaMP6 imaging yakaitwa pa920 nm ine 1.4x imwe chete z-ndege magnification uye 8x avhareji ye7.5 fps. Makonzo akaitwa ne5% isoflurane anesthesia uye akachengetwa ne1-3% isoflurane. Makonzo akaiswa mune yakasarudzika yakagadzirwa musoro uye akaiswa pasi pe lens. A 3-maminetsi kumashure kurekodha kwekuita kwemotokari kwakawanikwa. Mukufamba kwemaminetsi makumi maviri ekurekodha, 50 puffs (yega yega mharidzo 100 ms kureba) yakaunzwa zvisina tsarukano kune whisker pad yakatarisana net-hCO uchishandisa picospricer. Shandisa iyo Arduino MATLAB Tsigiro Package kudzora nguva yekuputika neyakajairwa MATLAB kodhi. Batanidza zviitiko nesoftware yekutora data (PrairieView 5.5) uchishandisa TTL pulses. Kuti uongororwe, iyo mifananidzo yakagadziridzwa kuitira xy motion uchishandisa affine kururamisa muchirongwa cheMoCo chakatangwa muFiji. Kubviswa kwemafuta efluorescent kubva kumasero ega ega achishandisa CNMF-E43. Fluorescence yakatorwa kudunhu rega rega rekufarira, rakashandurwa kuita dF/F curves, ndokuzoshandurwa kuita z-zvibodzwa.
Makonzo (anopfuura mazuva 140 mushure mekutapurwa) akaitwa ne5% isoflurane anesthesia uye anesthetized ne 1-3% isoflurane intraoperatively. Mhuka dzakaiswa mu stereotaxic frame (Kopf) uye buprenorphine SR uye dexamethasone dzakaiswa pasi pecutaneously. Ndebvu dziri mhiri kwe t-hCO dzakachekwa kusvika masendimita maviri uye dzakarukwa kuburikidza nemambure akabatana nepiezoelectric actuator. Dehenya rinoburitswa pachena uye rinocheneswa. Stainless steel ground screw inonamirwa padehenya. Kuti titarise t-hCO, takagadzira stereotaxic coordinates kubva kuMRI mifananidzo. Ita denderedzwa craniotomy (inenge 1 cm mudhayamita) ine chibooreso chepamusoro chiri pamusoro pe t-hCO. Kana bhonzo raita mutete sezvinobvira, asi usati waboora bhonzo rose, shandisa forceps kubvisa yasara intact pelvic disc kuratidza zviri pasi pe t-hCO. Masero ega ega akarekodhwa achishandisa 32-channel kana 64-channel high-density silicon probes (Cambridge Neurotech) yakadzikwa kusvika pasi screws uye pre-amplified neRHD amplifiers (Intan). Shandisa manipulator kudzikisa ma electrode kunzvimbo yakanangwa kuburikidza ne craniotomy, iyo izere ne sterile saline. Kuunganidzwa kwedata kwakaitwa pafrequency ye30 kHz uchishandisa Open Ephys data acquisition system. Kurekodha kwakaramba kuchienderera mberi chete patakaona chiitiko chakanyanya kurongeka chinongoitika mumatanho anopfuura gumi, zvichiratidza kuti maelectrode aive mugraft (yakavakirwa pambiri-photon calcium imaging data). A 10-maminiti kumashure kurekodha kwekuita kwemotokari kwakawanikwa. Ndebvu dziri mhiri kwe t-hCO dzakabva dzatsauswa ka50 (2 mm pa 20 Hz, 2 s per presentation) panguva dzisina kujairika nepiezoelectric drive pamusoro pemaminetsi makumi maviri ekurekodha nguva. Uchishandisa iyo MATLAB Tsigiro Package yeArduino (MATLAB 2019b), dzora nguva yekutsauka netsika MATLAB kodhi. Shandisa TTL pulses kuwiriranisa zviitiko nesoftware yekutora data.
Pakuedza kwekumaka kuyedza, 200 µm optical patch cord (Doric) yakabatana ne473 nm laser (Omicron) yakabatana ne200 µm optical fiber yakaiswa pamusoro pecraniotomy. Pakarepo izvi zvisati zvaitika, gadzirisa simba rejumper kusvika ku20 mW. Shandisa manipulator kudzikisa ma electrode kunzvimbo yakanangwa kuburikidza ne craniotomy, iyo izere ne sterile saline. Pakutanga kwekurekodha, gumi pulses yechiedza 473 nm (frequency 2 Hz, pulse duration 10 ms) yakabudiswa. Photosensitive masero akatsanangurwa semasero airatidza mhinduro yespike mukati me10 ms yechiedza mu70% kana kupfuura yemiedzo.