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Li-neural organelles tse ikemetseng li emela sethala se ts'episang sa in vitro bakeng sa ho etsa mohlala oa nts'etsopele ea batho le mafu. Leha ho le joalo, li-organoids ha li na khokahano e teng ho vivo, e fokotsang kholo le ho thibela ho hokahana le lipotoloho tse ling tse laolang boitšoaro. Mona re bonts'a hore stem cell-derived cortical organoids e fetiselitsoeng ka har'a cortex ea somatosensory ea likhoto tse hlobotseng tsa neonatal li hlahisa mefuta ea lisele tse holileng tse kopanyang lipotoloho tse amanang le kutlo le tšusumetso. MRI e ile ea senola kholo ea post-transplant organoid meleng e mengata ea stem cell le liphoofolo, ha tlhahlobo e le 'ngoe e senola tsoelo-pele ea corticogenesis le ho hlaha ha lenaneo la ho ngola le itšetlehileng ka mosebetsi. Ehlile, li-neuron tsa cortical tse kentsoeng li bonts'a litšobotsi tse rarahaneng tsa morphological, synaptic, le membrane ea ka hare ho feta li-in vitro tsa tsona, e leng se lumellang ho lemoha mefokolo ea methapo ho bakuli ba Timothy's syndrome. Ts'ebetso ea anatomical le ts'ebetso ea ts'ebetso e bonts'itse hore li-organelle tse fetiselitsoeng li fumana li-input tsa thalamocortical le corticocortical, 'me lirekoto tsa in vivo tsa ts'ebetso ea methapo li fana ka maikutlo a hore lintho tsena tse kenang li ka hlahisa likarabo tsa kutlo liseleng tsa motho. Qetellong, li-cortical organoids li atolosa li-axon ho pholletsa le boko ba likhoto, 'me ts'ebetso ea bona ea optogenetic e lebisa boitšoarong ba ho batla moputso. Ka hona, li-neuron tsa cortex tse fetisitsoeng lia hola 'me li nka karolo lipotolohong tsa moamoheli tse laolang boitšoaro. Re lebeletse hore mokhoa ona o be bonolo ho lemoha li-phenotypes tsa strand-level liseleng tse nkiloeng ke mokuli tse ke keng tsa fumanoa ka mekhoa e meng.
Boko ba motho bo ntseng bo hōla ke mokhoa o tsotehang oa ho itlhophisa oo lisele li ikatisang, li arohanang, li falla, 'me li hokahana ho etsa lipotoloho tse sebetsang tsa neuronal tse ntseng li ntlafatsoa ho feta ka phihlelo ea kutlo. Bothata bo ka sehloohong ba ho utloisisa tsoelo-pele ea boko ba motho, haholo-holo boemong ba lefu, ke ho se fumanehe ho lisele tsa boko. Li-organelle tse itlhophisang, tse kenyelletsang cortex organoids (hCO; e tsejoang hape e le cortex sphere ea motho), e ka hlahisa 2,3,4,5,6. Leha ho le joalo, mefokolo e mengata e fokotsa ts'ebeliso ea bona e pharaletseng ea ho utloisisa nts'etsopele le ts'ebetso ea li-circuits tsa neural. Haholo-holo, ha ho hlake hore na hCO maturation e lekanyelitsoe ke ho ba sieo ha lintho tse itseng tsa microenvironmental le kutlo tse teng ka vivo. Ho feta moo, hobane li-hCO ha lia kopanngoa le lipotoloho tse ka hlahisang liphello tsa boitšoaro, tšebeliso ea tsona ea ho etsa mohlala oa mathata a lefutso le a boits'oaro a neuropsychiatric e lekanyelitsoe.
Ho fetisoa ha hCO bokong bo phelang bo sa tsitsang ho ka hlola mefokolo ena. Liphuputso tse fetileng li bontšitse hore li-neurone tsa motho tse fetiselitsoeng ka har'a cortex ea rodent li khona ho phela, ho rera le ho buisana le lisele tsa litoeba7,8,9,10,11,12. Leha ho le joalo, liteko tsena hangata li etsoa ho liphoofolo tse kholo, tse ka fokotsang kopano ea synaptic le axonal. Mona, re hlalosa paradigm ea phetisetso eo ho eona re ileng ra fetisetsa 3D hCO e nkiloeng ho lisele tsa hiPS ho ea ho cortex ea mantlha ea somatosensory cortex (S1) ea likhoto tse immunodeficient qalong ea nts'etsopele ea polasetiki. Li-neurone tse fetisitsoeng tsa hCO (t-hCO) li hola haholo, li amohela li-thalamocortical le cortical-cortical tse fanang ka likarabo tsa maikutlo, 'me li atolose likhakanyo tsa axonal bokong ba likhoto ho tsamaisa boitšoaro ba ho batla moputso. Keketseho e atolositsoeng ea t-hCO e senotse bofokoli ba neuronal ho bakuli ba lefu la Timothy (TS), lefu le matla la liphatsa tsa lefutso le bakoang ke liphetoho tsa mofuta oa L-sensitive L-type CaV1.2 calcium channel (encoded by CACNA1C).
Ho ithuta li-neurons tsa cortical tsa batho lipotolohong tsa vivo, re ile ra fetisetsa 3D hCO e tsitsitseng ka mokhoa o tsitsitseng ho S1 ea likhoto tsa pele tsa pelehi tsa athymic (matsatsi a 3-7 ka mor'a ho tsoaloa) (Setšoantšo sa 1a le lintlha tse atolositsoeng tsa Fig. 1a-c). Nakong ena, likhakanyo tsa thalamocortical le corticocortical axonal ha li e-s'o phethe li-innervation tsa bona tsa S1 (ref. 13). Ka hona, mokhoa ona o etselitsoe ho eketsa kopanyo ea t-hCO ha o ntse o fokotsa tšusumetso ho lipotoloho tse sa feleng. Ho bona ka mahlo a kelello sebaka sa t-hCO liphoofolong tse phelang, re ile ra etsa li-reconstruction tsa boko ba MRI tse boima ba T2 ea likhoto 2-3 likhoeli ka mor'a ho fetisetsa (setšoantšo sa 1b le lintlha tse atolositsoeng, setšoantšo sa 1d). t-hCO e ne e hlokomeloa habonolo 'me litekanyo tsa molumo oa t-hCO li ne li tšoana le tse baloang ho tloha ho lilae tse tsitsitseng (Extended Data Fig. 1d, e; P> 0.05). t-hCO e ne e hlokomeloa habonolo 'me litekanyo tsa molumo oa t-hCO li ne li tšoana le tse baloang ho tloha ho lilae tse tsitsitseng (Extended Data Fig. 1d, e; P> 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированых срезов (расширенсные, песни, данных срезов) 0,05). t-hCO e ne e bonoa habonolo, 'me litekanyo tsa volumetric t-hCO li ne li tšoana le tse baloang bakeng sa likarolo tse tsitsitseng (data e atolositsoeng, setšoantšo sa 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， 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 (расширенсные, даных срезов) 0,05). t-hCO e ne e bonoa habonolo, 'me litekanyo tsa volumetric t-hCO li ne li tšoana le tse baloang bakeng sa likarolo tse tsitsitseng (lintlha tse atolositsoeng, setšoantšo sa 1d, e; P> 0.05).Re ile ra etsa qeto ea t-hCO ho 81% ea liphoofolo tse fetisitsoeng hoo e ka bang likhoeli tsa 2 ka mor'a hore li fetisetsoe (n = liphoofolo tse 72; hCO ho tloha meleng ea lisele tsa 10 tsa hiPS; mela ea lisele tsa hiPS ho Supplementary Table 1). Ho tsena, 87% e ne e le ka har'a cerebral cortex (setšoantšo sa 1c). Ka ho etsa litlhahlobo tsa serial MRI ka linako tse ngata ka rat e fetisitsoeng e tšoanang, re fumane keketseho ea makhetlo a robong ea molumo oa t-hCO nakong ea likhoeli tsa 3 (setšoantšo sa 1d le lintlha tse atolositsoeng, setšoantšo sa 1f). Liphoofolo tse fetisitsoeng li ne li e-na le tekanyo e phahameng ea ho phela (74%) likhoeling tsa 12 ka mor'a ho fetisetsa (lintlha tse atolositsoeng, Fig. 1g le Supplementary Table 2), 'me ha ho na ho senyeha ha motlakase kapa mohopolo, gliosis, kapa electroencephalogram (EEG) e fumanoeng. Lintlha Fig. 1g le tlatsetso tafole 2). 1h–m le 3e).
a, Sekema sa moralo oa liteko. hCO e nkiloeng ho lisele tsa hiPS e ile ea fetisetsoa ho S1 ea likhoto tse sa tsoa tsoaloa tse hlobotseng ka matsatsi a 30-60 a phapang. b, litšoantšo tsa MRI tse nang le boima ba T2 le tse rapameng tse bontšang t-hCO ka likhoeli tsa S1 2 ka mor'a ho fetisetsa. Sekhahla, 2 mm. c, Palo ea litekanyetso tsa katleho ea ho ngolisoa tse bontšitsoeng bakeng sa mohala o mong le o mong oa sele ea hiPS (n = 108, linomoro tse ka har'a mekoallo li bontša palo ea t-hCO ka hIPS cell line) le sebaka sa cortical kapa subcortical (n = 88). d, setšoantšo sa MRI sa methapo ea mali (ka ho le letšehali; tekanyo ea sekhahla, 3 mm) le ho tsosolosoa ha molumo oa 3D (scale bar, 3 mm) e bontšang keketseho ea t-hCO ho feta likhoeli tsa 3. e, Tlhahlobo ea mekhoa ea t-hCO ho rat cerebral cortex. Sekhahla, 1 mm. f, Moemeli oa litšoantšo tsa immunocytochemical tsa t-hCO tse bontšitsoeng ho tloha ka holimo ho le letšehali ho ea ho le letona (nakong ea phapang): PPP1R17 (likhoeli tse 4), NeuN (likhoeli tsa 8), SOX9 le GFAP (likhoeli tsa 8), PDGFRα; (likhoeli tse 8), MAP2 (likhoeli tse 8) le IBA1 (likhoeli tse 8). Scale bar, 20 µm. Tlhaloso e kopanetsoeng ea HNA e bontša lisele tsa tlhaho ea motho. g, snRNA-seq: Unified manifold and projection (UMAP) dimensionality reduction imaging of all high quality t-hCO nuclei after Seurat integration (n=3 t-hCO samples, n=2 hiPS cell line). Astrocyte, lisele tsa mola oa astrocyte; cyc prog, li-progenitors tse potolohang; GluN DL, methapo e tebileng ea glutamatergic; GluN DL / SP, li-neuron tse tebileng le tsa sublamellar glutamatergic; GluN UL, lera le ka holimo la glutamatergic neurons; li-oligodendrocyte, li-oligodendrocyte; OPC, lisele tsa pele tsa oligodendrocyte; RELN, li-neurone tsa reelin. h, Gene Ontology (GO) tlhahlobo ea ntlafatso ea nako ea liphatsa tsa lefutso e laoloang haholo (e fetotsoe P <0.05, phetoho ea makhetlo> 2, e hlalositsoeng bonyane 10% ea nuclei) ho t-hCO glutamatergic neurons ha e bapisoa le hCO glutamatergic neurons. h, Gene Ontology (GO) tlhahlobo ea ntlafatso ea nako ea liphatsa tsa lefutso e laoloang haholo (e fetotsoe P <0.05, phetoho ea makhetlo> 2, e hlalositsoeng bonyane 10% ea nuclei) ho t-hCO glutamatergic neurons ha e bapisoa le hCO glutamatergic neurons. h, Анализ обогащения терминов Gene Ontology (GO) для генов со значительной активацией в 10% ядер) в глутаматергических нейронах t-hCO по сравнению с глутаматергическими нейронами hCO. h, Gene Ontology (GO) tlhahlobo ea ntlafatso ea nako bakeng sa liphatsa tsa lefutso tse nang le ts'ebetso e kholo (e fetotsoeng P <0.05, phetoho ea makhetlo> 2, polelo ka bonyane 10% nuclei) ho t-hCO glutamatergic neurons ha e bapisoa le 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. h , 与 hco 谷氨酸 能 元 相比 , t-hco 谷氨酸 能 神经 元 基因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% в 10% нейронах t-hCO по сравнению с глутаматергическими нейронами hCO Онтологический (GO) анализ термина обогащения. h, liphatsa tsa lefutso li ne li laoloa haholo (ho lokisoa P <0.05, phetoho ea fold> 2, e hlalositsoeng bonyane 10% ea nuclei) ho t-hCO glutamatergic neurons ha e bapisoa le tlhahlobo ea hCO glutamatergic neurons Ontological (GO) ea nako ea ho rua.Mohala o nang le matheba o bontša boleng ba aq ea 0.05. i, ho nahana ka UMAP ea mefuta ea lisele tsa GluN ho t-hCO ho sebelisa phetisetso ea li-label ho tloha ho referense ea 22 snRNA-seq ea batho ba baholo ba koloi ea cortex dataset. CT - lisele tsa corticothalamic, ET - lisele tsa extracerebral, IT - lisele tse ka hare tsa telencephalic, NP - haufi le projeke.
Ka mor'a moo re ile ra hlahloba cytoarchitecture le kakaretso ea cellular ea t-hCO. Antibody staining ea rat endothelial cells e senotse vascularization le t-hCO, ha IBA1 staining e senola boteng ba rat microglia ho pholletsa le graft (Fig. 1f le data e atolositsoeng, Fig. 3c, d). Immunostaining e senotse lisele tse ntle tsa nyutlelie tsa motho (HNA) tse hlahisang PPP1R17 (cortical progenitors), NeuN (neurons), SOX9 le GFAP (lisele tse tsoang ho glial) kapa PDGFRα (oligodendrocyte progenitors) (Setšoantšo sa 1f). Ho ithuta sebopeho sa cellular sa t-hCO ho qeto ea sele e le 'ngoe, re entse tatellano ea mantlha ea RNA (snRNA-seq) kamora likhoeli tse ka bang 8 tsa phapang. Ho hloekisa ka bongata le ho tlosoa ha li-nuclei tsa rat ho ile ha fana ka limmapa tsa 21,500 tsa boleng bo phahameng ba batho ba mononuclear (setšoantšo sa 1g le lintlha tse atolositsoeng, setšoantšo sa 4a, b). Mekhoa ea maikutlo ea matšoao a tloaelehileng a mofuta oa lisele tse khethollang lihlopha tsa lihlopha tse kholo tsa lisele tsa cortical, ho kenyelletsa le li-neuron tse tebileng le tse ka holimo tsa glutamatergic, li-progenitors tse potolohang, li-oligodendrocyte, le leloko la astrocyte (setšoantšo sa 1g, lintlha tse atolositsoeng, setšoantšo sa 4c le Supplementary Table 3). Immunostaining bakeng sa SATB2 le CTIP2 e bontšitse hore ho sa tsotellehe ho ba teng ha li-cortical subtypes, t-hCO ha ea ka ea bontša stratification e hlakileng ea anatomical (data e atolositsoeng, setšoantšo sa 3a). Stage-matched snRNA-seq hCO e hlahisitse lihlopha tsa lisele tse tšoanang ka mokhoa o pharaletseng, ntle le mekhelo e seng mekae, ho kenyelletsa le ho ba sieo ha li-oligodendrocyte le ho ba teng ha li-neuron tsa GABAergic, tse ka 'nang tsa bonahatsa maemo a pele a tlalehiloeng a le motle bakeng sa lisele tsa lateral progenitor cell15 (data e atolositsoeng, setšoantšo sa 4f - i le Supplementary Table 4). Tlhahlobo e fapaneng ea liphatsa tsa lefutso e senotse liphapang tse kholo ho li-neuron tsa glutamatergic pakeng tsa t-hCO le hCO (Letlapa la Tlatsetso ea 5), ​​ho kenyelletsa le ts'ebetso ea lihlopha tsa liphatsa tsa lefutso tse amanang le ho hōla ha neuronal tse kang synaptic signaling, dendritic localization, le voltage-gated channel activity (Figure 1h le Supplementary Table 5). tafole 6). Ka hona, li-neuron tsa cortical glutamatergic t-hCO li bonts'itse kholo e potlakileng ea mongolo.
Ho hlakisa hore na liphetoho tsena tsa transcriptional ho t-hCO li ne li amana le phapang ea morphological pakeng tsa hCO in vitro le t-hCO in vivo, re ile ra tsosolosa hCO e tletseng li-biocytin e tletseng sethala le hCO likarolong tse matla ka mor'a likhoeli tsa 7-8 tsa phapang. hCO neurons (setšoantšo sa 2a). Li-neuron tsa t-hCO li ne li le kholoanyane haholo, li ne li e-na le linako tse 1.5 tsa bophara ba soma, li-dendrites tse ngata ka makhetlo a mabeli, le keketseho ea makhetlo a tšeletseng ka kakaretso ea bolelele ba dendritic ha e bapisoa le in vitro hCO (Setšoantšo sa 2b). Ho phaella moo, re bone palo e phahameng haholo ea dendritic spines ka t-hCO neurons ho feta hCO neurons (Fig. 2c). Sena se fana ka maikutlo a hore li-neuron tsa t-hCO li na le elongation e pharaletseng ea dendritic le branching, eo, hammoho le ho tsoela pele ho ata ha lisele, ho ka tlatsetsa khōlong e matla ea t-hCO ka mor'a ho fetisoa (setšoantšo sa 1d le Boitsebiso bo Atolositsoeng Fig. 1f). Sena se ile sa re susumelletsa ho batlisisa thepa ea electrophysiological. Membrane capacitance e ne e phahame ka makhetlo a robeli (lintlha tse atolositsoeng, setšoantšo sa 8d), monyetla oa ho phomola oa membrane o ne o e-na le hyperpolarized (hoo e ka bang 20 mV), 'me ente ea hona joale e ile ea etsa hore ho be le sekhahla se phahameng sa ho tsosoa ho t-hCO neurons ho feta li-neuron tsa hCO. in vitro (setšoantšo sa 2d), e), e lumellanang le likarolo tse kholo le tse rarahaneng tsa morphological tsa t-hCO. Ho phaella moo, makhetlo a mangata a liketsahalo tsa morao-rao tse thabisang tsa postsynaptic (EPSC) li ne li phahame haholo ho li-neuron tsa t-hCO (Fig. 2f), e leng se bontšang hore ho eketseha ha sekhahla sa li-dendritic spines tse hlokometsoeng ho t-hCO neurons ho ne ho amahanngoa le thabo e sebetsang. synapse ea thobalano. Re tiisitse sebopeho se sa tsitsang sa hCO neurons in vitro ka ho rekota e ngotsoeng glutamatergic neurons (data e atolositsoeng, Fig. 6a-c).
a, 3D tsosoloso ea hCO e tletseng biocytin le t-hCO neurons ka mor'a likhoeli tsa 8 tsa phapang. b, Tekanyetso ea likarolo tsa morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0084, *P = 0.0179 le ***P <0.0001). b, Tekanyetso ea likarolo tsa morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0084, *P = 0.0179 le ***P <0.0001). б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,0179 le *** P <0,0). b, palo ea likarolo tsa morphological (n=8 hCO neurons, n=6 t-hCO neurons; **P=0.0084, *P=0.0179, le ***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 le *** P <0,0). b, palo ea likarolo tsa morphological (n=8 hCO neurons, n=6 t-hCO neurons; **P=0.0084, *P=0.0179, le ***P<0.0001).c, 3D tsosoloso ea makala a hCO le t-hCO dendritic ka mor'a likhoeli tse 8 tsa phapang. Linaleli tse khubelu li bontša li-spines tsa putative dendritic. Dendritic spine density quantification (n = 8 hCO neurons, n = 6 t-hCO neurons; **P = 0.0092). d, Tekanyetso ea bokhoni ba lera la ho phomola (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). d, Tekanyetso ea bokhoni ba lera la ho phomola (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, lera le phomotseng le ka bang teng 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, lera le phomotseng le ka bang teng quantification (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Ketso e pheta-phetoang e ka 'nang ea thunya ka hCO le t-hCO e bakoang ke ho eketseha ha liente tsa morao-rao, le quantification ea tekanyo e kholo ea ho thunya (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Ketso e pheta-phetoang e ka 'nang ea thunya ka hCO le t-hCO e bakoang ke ho eketseha ha liente tsa morao-rao, le quantification ea tekanyo e kholo ea ho thunya (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, повторное возбуждение потенциала действия в hCO и t-hCO, вызванное увеличением тока, и количественная оценка максимальной скорости 2 возбенка n = 16 нейронов t-hCO *** P <0,0001). e, ketso e ka 'nang ea boela ea thunya ka hCO le t-hCO e bakoang ke keketseho ea hona joale le palo ea tekanyo e phahameng ea ho thunya (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). +个t-hCO 神经元；***P <0.0001）. E , 通过 增加 电流 注入 的 hco 和 t-hco 重复 电位 放电 , 以及 最 大的 量化 （2 （5 （5 （5（5（5（5 16 个 t-hco 神经 ； *** p <0.0001） . e, повторяющееся возбуждение потенциала действия hCO и t-hCO, вызванное увеличением подачи тока, и количественная оценка максимальствия максимальний максимальдежи 2 зкой нейронов hCO, n = 16 нейронов t-hCO *** P <0,0001). e, ho thunngoa ho pheta-phetang ha menyetla ea liketso tsa hCO le t-hCO e bakoang ke phepelo e eketsehileng ea hona joale le palo ea tekanyo e phahameng ea ho thunya (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) ho hCO le t-hCO neurons likhoeling tsa 8 tsa phapang, le palo ea makhetlo a mangata a liketsahalo tsa synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; ***P <0.0001). f, Spontaneous EPSCs (sEPSCs) ho hCO le t-hCO neurons likhoeling tsa 8 tsa phapang, le palo ea makhetlo a mangata a liketsahalo tsa 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, n2 = 2 17 нейронов t-hCO *** P <0,0001) . f, Spontaneous EPSCs (sEPSCs) ho hCO le t-hCO neurons likhoeling tsa 8 tsa phapang le palo ea litekanyetso tsa liketsahalo tsa synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). e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 hCO 25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, n2 = 2 17 нейронов t-hCO *** P <0,0001). f, Spontaneous EPSCs (sEPSCs) ho hCO le t-hCO neurons likhoeling tsa 8 tsa phapang le palo ea litekanyetso tsa liketsahalo tsa synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001).Bakeng sa bf, hCO le t-hCO moleng oa 1208-2 li nkiloe sehlopheng se le seng sa phapang se bolokiloeng ka ho tšoana. g, Gene e beha tlhahlobo ea ntlafatso (teko e nepahetseng ea lehlakore le le leng la Fisher) ea liphatsa tsa lefutso tse hlophisitsoeng haholo (tse fetotsoeng P <0.05, phetoho ea fold> 2, e hlalositsoeng bonyane 10% ea nuclei) ho t-hCO glutamatergic neurons ha e bapisoa le hCO glutamatergic neurons e nang le lihlopha tsa liphatsa tsa lefutso tsa bobeli ba pele-pele (LRG) liphatsa tsa lefutso tse khethiloeng ho tsoa thutong ea in vivo mouse16 le li-LRG tse ikhethileng ho tsoa ho in vitro neurons17. g, Gene e beha tlhahlobo ea ntlafatso (teko e nepahetseng ea lehlakore le le leng la Fisher) ea liphatsa tsa lefutso tse hlophisitsoeng haholo (tse fetotsoeng P <0.05, phetoho ea fold> 2, e hlalositsoeng bonyane 10% ea nuclei) ho t-hCO glutamatergic neurons ha e bapisoa le hCO glutamatergic neurons e nang le lihlopha tsa liphatsa tsa lefutso tsa bobeli ba pele-pele (LRG) liphatsa tsa lefutso tse khethiloeng ho tsoa thutong ea in vivo mouse16 le li-LRG tse ikhethileng ho tsoa ho in vitro neurons17. g, анализ обогащения набора генов (односторонний точный критерий Фишера) генов со значительной активацией (скорректированмены5, песни, песни 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 in vivo16, и специфических для челозвеtro17 и пецифических для челозвеtro17 и пецифических для челозвеtro17 и для челозвеtro g, tlhahlobo ea ntlafatso ea liphatsa tsa lefutso (teko e nepahetseng ea Fisher ea mohatla o le mong) ea liphatsa tsa lefutso tse nang le ts'ebetso e kholo (e fetotsoeng P <0.05, phetoho ea makhetlo> 2, polelo bonyane 10% ea li-nuclei) ho t-hCO glutamatergic neurons ha e bapisoa le lihlopha tsa hCO glutamatergic neurons tsa bobeli ba pele (ERG genes) tse tsejoang ka morao-rao (ERG genes) le LRGs tse ikhethileng ka batho tse tsoang ho li-neuron 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 上调漎 0 连 5 .倍数> 2 至少 至少 10%的 细胞 核中 表达） 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, ha ho na 10% Leha ho le joalo, ha ho na 10% ea hore na ke hobane'ng ha ho na le lintho tse ngata tse amanang le tsamaiso ea 'mele ea ho itšireletsa mafung. идентифицированные в исследованиях на мышах in vivo16 and нейронах in vitro17, специфичные для человека. g, t-hCO glutamatergic neurons e ne e laoloa haholo ha e bapisoa le hCO glutamatergic neurons (e fetotsoeng P <0.05, phetoho ea makhetlo> 2, bonyane 10% Karabelo ea pele (ERG) le tlhahlobo ea morao-rao ea ntlafatso ea liphatsa tsa lefutso (teko e nepahetseng ea Fisher) e itšetlehileng ka liphatsa tsa lefutso tsa batho (LRG1 in vitro mice1) LRGs.Mohala o nang le matheba o bontša boleng ba P bo lokisitsoeng ke Bonferroni ba 0.05. h, GluN gene expression (pseudo-package le scaling ea mofuta o mong le o mong) e ne e laoloa haholo ho snRNA-seq replicas ea LRG liphatsa tsa lefutso ho t-hCO glutamatergic neurons. i, immunostaining e bonts'ang polelo ea SCG2 ho t-hCO (ka holimo) le hCO (tlase) neurons. Metsu e tšoeu e supa liseleng tsa SCG2+. Scale bar, 25 µm. Lintlha li hlahisoa ka mokhoa o bolelang ± ho kheloha ho tloaelehileng.
Ho ipapisitsoe le ts'ebetso e ntseng e eketseha ea t-hCO e hlokometsoeng lilae tsa ex vivo, snRNA-seq e senotse ts'ebetso e itšetlehileng ka ts'ebetso ea liphatsa tsa lefutso ho t-hCO ha e bapisoa le hCO in vitro. Glutamatergic t-hCO neurons e bontšitse litekanyetso tse phahameng tsa liphatsa tsa lefutso tse laolang mosebetsi oa ho arabela ka morao (setšoantšo sa 2g, h), se fumanoeng lithutong tse fetileng ho mouse le li-neuron tsa batho16,17. Ka mohlala, BDNF18, SCG2, le OSTN, mofuta o laolang mosebetsi o itseng oa primate, o bontšitse ho eketseha ha polelo ho t-hCO neurons ha e bapisoa le hCO neurons (Fig. 2g-i). Kahoo, li-neuron tsa t-hCO li bonts'itse litšobotsi tse ntlafalitsoeng tsa kholo ha li bapisoa le li-neuron tsa hCO ka litlhahlobo tsa transcriptal, morphological, le ts'ebetso.
Ho tsoela pele ho hlahloba kamano ea t-hCO le tsoelo-pele ea boko ba motho, re ile ra etsa lipapiso tsa transcriptomic tsa fetal le batho ba baholo ba mefuta ea cortical cell19,20 le batho ba baholo21,22 hammoho le lintlha tse ngata tsa cortical gene expression23 nakong ea tsoelo-pele (data e atolositsoeng, setšoantšo sa 5). ). ka mosebetsi o fetileng oa 24, boemo ba lefats'e ba hCO le t-hCO transcriptome maturation ka likhoeli tse 7-8 tsa phapang e lumellana ka ho pharaletseng le nako ea tsoelo-pele ea vivo 'me e lekana haholo le bophelo ba lesea la morao (Extended Data Fig. 5a). Ka ho hlakileng, re bone khōlo e ntseng e eketseha ea transcriptome ho t-hCO ha e bapisoa le hCO e lekanang le lilemo, hammoho le ts'ebetso ea transcriptome e amanang le synaptogenesis, astrogenesis, le myelination (data e atolositsoeng, setšoantšo sa 5b-d). Boemong ba cellular, re fumane bopaki ba thinner cortex subtype ho t-hCO, e nang le lihlopha tsa glutamatergic neurons tse koahelang batho ba baholo L2 / 3, L5, le L6 neuron subtypes (Setšoantšo sa 1i). Ka lehlakoreng le leng, ho kopana ha sehlopha pakeng tsa li-neuron tsa glutamatergic t-hCO le fetal cortical neurons ho ne ho lekanyelitsoe bohareng ba bokhachane (data e atolositsoeng, Figure 5e-j). Ho fumana hore na li-neuron tsa t-hCO li sebetsa ka mokhoa o ts'oanang le li-neocortical neurons tsa motho, re entse lirekoto tsa electrophysiological le reconstruction ea anatomical ea L2 / 3 pyramidal neurons likarolong tse bohale tsa cortex ea postnatal (data e atolositsoeng, setšoantšo sa 7a). Lintho tsa electrophysiological tsa L2 / 3 pyramidal neurons li ne li tšoana le tsa t-hCO pyramidal neurons (lintlha tse atolositsoeng, setšoantšo sa 7e). Morphologically, L2 / 3 neurons ho tloha ka mor'a ho beleha sampuli ea batho e ne e tšoana haholo le t-hCO ho feta hCO, le hoja lisele tsa L2 / 3 li ne li le telele, li na le makala a mangata ka kakaretso, 'me li na le sekhahla se phahameng sa mokokotlo (setšoantšo sa 3g le data e atolositsoeng, setšoantšo sa 7b-). G).
a, transplantation ea hCO e hlahisoang ke taolo le mela ea lisele tsa TS hiPS ho likhoto tse sa tsoa tsoaloa. b, 3D tsosoloso ea biocytin e tletseng t-hCO neurons ka mor'a likhoeli tsa 8 tsa phapang. c, quantification ea bolelele ba dendritic bolelele (n = 19 control neurons, n = 21 TS neurons; ** P = 0.0041). d, makala a dendritic a tsosolositsoeng a 3D ho tloha taolong le TS t-hCO likhoeling tsa 8 tsa phapang, le quantification ea dendritic spine density (n = 16 control neurons, n = 21 TS neurons, ***P <0.0001). d, makala a dendritic a tsosolositsoeng a 3D ho tloha taolong le TS t-hCO likhoeling tsa 8 tsa phapang, le quantification ea 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 плотностин плотности1 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, 3D tsosoloso ea makala a dendritic ho tloha taolong le t-hCO TS likhoeling tsa 8 tsa phapang le 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 1 神个TS 神经元, ***P <0.0001）. d , 分化 8 个 时 对照 和 ts t-hco 的 3d 重建 分支 分支 以及 树突棘 密度 量化 量化 1 = 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 плотностипны плотностины контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, 3D tsosoloso ea makala a taolo ea dendritic le TS t-hCO likhoeling tsa 8 tsa phapang le dendritic spine density quantification (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001).Linaleli tse khubelu li bontša li-spines tsa putative dendritic. e, li-EPSC tse ikemetseng tse laolang le li-neurons tsa TS t-hCO ka mor'a likhoeli tsa 8 tsa phapang. f, palo ea makhetlo a mangata le palo ea makhetlo a mangata le amplitude ea liketsahalo tsa synaptic (n = 32 control neurons, n = 26 TS neurons; ** P = 0.0076 le P = 0.8102). g, tlhahlobo ea Scholl ea TS le ho laola li-neurone ho hCO le t-hCO. Methapo e phunyeletsoeng e bontša li-neuron tsa L2 / 3 tsa postnatal pyramidal neurons bakeng sa ho bapisa (n = 24 control t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 laola hCO neurons, le n = 7 TS hCO neurons). Lintlha li hlahisoa e le moelelo ± kheloha e tloaelehileng
Bokhoni ba t-hCO ho pheta-pheta likarolo tsa morphological le tse sebetsang tsa cortex neurons ea motho boemong bo phahameng bo ile ba re susumelletsa ho hlahloba hore na t-hCO e ka sebelisoa ho lemoha phenotypes ea mafu. Re tsepamisitse maikutlo ho TS, lefu le matla la neurodevelopmental le bakoang ke liphetoho tsa ho fumana mosebetsi ho gene encoding CaV1.2, e qalang ho ngoloa ha liphatsa tsa lefutso ho li-neurone tse itšetlehileng ka mosebetsi. Re fumane hCO ho bakuli ba bararo ba TS ba jereng sebaka se tloaelehileng ka ho fetisisa (p.G406R) le li-control tse tharo (setšoantšo sa 3a). Ka mor'a ho fetisoa, re fumane hore dendritic morphology e fetotsoe ho li-neurons tsa TS ha li bapisoa le li-control (setšoantšo sa 3b le data e atolositsoeng, setšoantšo sa 8a, b), ka keketseho ea makhetlo a mabeli palo ea li-dendrites tse ka sehloohong le ho eketseha ka kakaretso ho bolela le ho fokotseha ka kakaretso ka bolelele ba dendritic (setšoantšo sa 3c le data e atolositsoeng, Setšoantšo sa 8c). Sena se ne se amahanngoa le ho eketseha ha sekhahla sa li-spines le ho eketseha ha makhetlo a mangata a li-EPSC tse ikemetseng ho TS ha li bapisoa le li-neurons tse laolang (setšoantšo sa 3d-f le data e atolositsoeng, setšoantšo sa 8g). Tlhahlobo e eketsehileng e ile ea senola mekhoa e sa tloaelehang ea dendritic branching ho t-hCO TS ha e bapisoa le litsamaiso, empa eseng ka in vitro TS hCO ka nako e tšoanang ea phapang (setšoantšo sa 3g). Sena se lumellana le litlaleho tsa rona tse fetileng tsa ho fokotseha ha dendritic ho itšetlehileng ka mosebetsi ho TS le ho totobatsa bokhoni ba sethala sena sa transplant ho lemoha phenotypes ea mafu ka vivo.
Ka mor'a moo re ile ra botsa hore na lisele tsa t-hCO li kenyelelitsoe hakae ho rat S1. S1 ka litoeba e fumana li-synaptic tse matla tse tsoang ho ipsilateral ventral basal le posterior thalamic nuclei, hammoho le ipsilateral motor le secondary somatosensory cortices, le contralateral S1 (Fig. 4a). E le ho tsosolosa mokhoa oa ho itlhokomela, re ile ra tšoaetsa hCO ka kokoana-hloko ea rabies-dG-GFP/AAV-G 'me ra fetisetsa hCO ho S1 rat matsatsi a 3 hamorao. Re hlokometse polelo e teteaneng ea GFP ka li-neuron tsa ipsilateral S1 le ventral basal ganglia matsatsi a 7-14 ka mor'a ho fetisetsa (setšoantšo sa 4b, c). Ho phaella moo, ho silafala ha li-antibody tsa thalamic marker netrin G1 ho senotse ho ba teng ha lipheletso tsa thalamic ho t-hCO (Fig. 4d, e). Ho lekola hore na likhakanyo tsena tse fapaneng li ka hlahisa likarabo tsa synaptic liseleng tsa t-hCO, re entse lirekoto tsa lisele tse felletseng ho tsoa liseleng tsa motho likarolong tse bohale tsa lera la thalamocortical. Ho tsosoa ha motlakase oa rat S1, capsule ea ka hare, taba e tšoeu, likhoele tse haufi le t-hCO kapa ts'ebetso ea optogenetic ea liphetho tsa thalamic tse hlahisang opsin ho t-hCO e bakileng nako e khuts'oane ea latency EPSC ho t-hCO neurons e pepesehileng ho mohanyetsi oa AMPA receptor NBQX. (Setšoantšo sa 4f, g le lintlha tse atolositsoeng, setšoantšo sa 9a-g). Lintlha tsena li bonts'a hore t-hCO e kenyelelitsoe bokong ba likhoto mme e khona ho kengoa tšebetsong ke lisele tse amohelang likhoto.
a, Sets'oants'o sa moralo oa teko ea ho latella lefu la rabies. b, GFP le polelo e tobileng ea STEM121 pakeng tsa t-hCO le rat cerebral cortex (phanele e ka holimo). Hape ho bontšitsoe polelo ea GFP ho rat ipsilateral ventral basal nucleus (VB) (ka tlaase ka ho le letšehali) le ipsilateral S1 (ka tlaase ho le letona). Scale bar, 50 µm. Likarolo tse khubelu li emela libaka tsa boko moo litšoantšo li nkiloeng teng. c, quantification ea lisele tse hlalosang GFP (n = 4 likhoto). d, e - Netrin G1+ thalamic terminals ho t-hCO. d e bonts'a karolo ea coronal e nang le t-hCO le VB nuclei. Sekhahla, 2 mm. e bonts'a polelo ea Netrin G1 le STEM121 ho t-hCO (ka ho le letšehali) le VB (ka ho le letona) li-neurons. Scale bar, 50 µm. Mohala oa matheba a lamunu o bontša moeli oa t-hCO. f, g, Mesaletsa ea hona joale ea t-hCO neurons ka mor'a ho tsosoa ha motlakase ho S1 rat (f) kapa capsule e ka hare (g), e nang le (pherese) kapa ntle le (e ntšo) NBQX (ka ho le letšehali). EPSC amplitudes le ntle le NBQX (n = 6 S1 neurons, * P = 0.0119; le n = 6 ka hare capsule neurons, ** P = 0.0022) (bohareng). Peresente ea li-neuron tsa t-hCO tse bontšang EPSC ho arabela ho tsosoa ha motlakase oa rat S1 (f) kapa capsule ea ka hare (g) (ka ho le letona). aCSF, mokelikeli oa cerebrospinal oa maiketsetso. h, setšoantšo sa moralo oa teko ea ho nka litšoantšo ea 2P (ka ho le letšehali). Polelo ea GCaMP6s ho t-hCO (bohareng). Scale bar, 100 µm. Nako ea Fluorescence e fela ea GCaMP6s (ka ho le letona). i, lintlha tsa Z tsa tšebetso e iketsang ea fluorescence. j, papiso e hlophisitsoeng ea ho susumetsa litelu. k, z-shed 2P fluorescence trajectories tekong e le 'ngoe, e tsamaellanang le ho kheloha ha litelu ka nako zero (mohala o khotliloeng) ho lisele tsa mohlala. l, likarabelo tsa palo-karolelano ea palo ea z-score tsa lisele tsohle tse tsamaellanang le ho kheloha ha litelu ka nako zero (mothalo o mokhubelu) (o khubelu) kapa litempe tsa linako tse hlahisoang ka tšohanyetso (tse bohlooho). m. Sets'oants'o sa moralo oa teko ea ho tšoaea optical. n, Raw voltage curves ho tloha mohlala oa t-hCO sele nakong ea putsoa ea laser stimulation kapa whisker deflection. Metsu e khubelu e bontša li-spikes tsa pele tse bakoang ke khanya (holimo) kapa tse bakoang ke ho kheloha ha litelu (ka tlase). Moriti o mosoeu o bontša linako tsa ho kheloha ha litelu. o, Maqhubu a maqhubu a mabone a holimo-limo le likarabo tsa ho kheloha litelu. p, li-spikes tsa teko e le 'ngoe, tse tsamaellanang le ho kheloha ha litelu liseleng tsa mohlala. 0 bontsha ho kgeloha ha ditedu (mothalo o mothalo). q, palo-hare ea palo ea batho ba z-score sekhahla sa ho thunya liseleng tsohle tse utloang bohloko, tse tsamaellanang le ho kheloha ha litelu ka nako zero (mothalo o mokhubelu) (khubelu) kapa litempe tsa linako tse entsoeng ka tšohanyetso (tse thokoa). r, Karolo ea li-unit tsa li-photosensitive li fetotsoe haholo ke ho kheloha ha litelu (n = litoeba tse 3) (ka ho le letšehali). Tlhōrō ea z-score latency (n = 3 likhoto; n = 5 (botala bo khanyang), n = 4 (botala bo lefifi), le n = 4 (cyan) li-modulation tsa whisker deflection units per rat) (ka ho le letona). Lintlha li hlahisoa ka mokhoa o bolelang ± ho kheloha ho tloaelehileng
Ka mor'a moo re ile ra botsa hore na t-hCO e ka kenngoa ts'ebetsong ka ho susumetsa maikutlo a vivo. Re jalelitse hCO e bonts'ang matšoao a calcium a kentsoeng ka liphatsa tsa lefutso GCaMP6 ho likhoto tsa S1. Ka mor'a matsatsi a 150, re ile ra etsa fiber photometry kapa litšoantšo tse peli tsa photon calcium (setšoantšo sa 4h le data e atolositsoeng, setšoantšo sa 10a). Re fumane hore lisele tsa t-hCO li bonts'a ts'ebetso ea morethetho e lumellanang (Setšoantšo sa 4i, Boitsebiso bo Atolositsoeng, Setšoantšo sa 10b le Video ea Tlatsetso ea 1). E le ho khetholla tlhōrō ea ts'ebetso ea t-hCO, re ile ra etsa lirekoto tsa extracellular electrophysiological ka likhoto tsa transplant tse nang le anesthetized (data e atolositsoeng, setšoantšo sa 10c-f). Re hlahisitse li-coordinate tsa stereotaxic ho tloha litšoantšong tsa MRI; ka hona, likarolo tsena tse rekotiloeng li emela methapo ea kutlo ea motho, leha electrophysiology e le 'ngoe e sa lumelle mofuta oa tlhaho ho tsebahatsoa. Re hlokometse ho phatloha ho hokahaneng ha ts'ebetso (data e atolositsoeng, setšoantšo sa 10d). Ho phatloha ho ile ha nka hoo e ka bang 460 ms 'me ho arohanngoa ke nako ea khutso ea hoo e ka bang 2 s (data e atolositsoeng, setšoantšo sa 10d, e). Diyuniti ka bomong di ile tsa thunya palohare ya dikgato tse ka bang tharo ha ho qhoma, e leng hoo e ka bang 73% ya diyuniti tse ngodisitsweng ka ho phatloha ho hong. Mesebetsi ea lihlopha ka bomong e ne e amana haholo, 'me likamano tsena li ne li phahame ho feta tsa lihlopha tse khetholloang liphoofolong tse sa entoa tse tlalehiloeng tlas'a maemo a tšoanang (data e atolositsoeng, setšoantšo sa 10f). Ho tsoela pele ho bontša likarabo tsa spike tsa li-neurone tse nkiloeng ke batho, re ile ra etsa liteko tsa ho fumana leseli ho likhoto tse hlabosang tse fetisitsoeng ka hCO e bonts'ang mocha o bobebe oa cation rhodopsin 2 (hChR2), eo ka eona t-hCO neurons e lemohang nako e khuts'oane (tlase ho 10 ms-4) karabelong ea leseli le leputsoa. Li-neurons tsa t-hCO li bonts'itse ho phatloha ha mesebetsi e ikemetseng ka maqhubu a tšoanang le a bonoang litšoantšong tsa khalsiamo, hammoho le lirekoto tsa electrophysiological tse entsoeng ho t-hCO ha ho se na letšoao la leseli (data e atolositsoeng, setšoantšo sa 10c-g). Ha ho ts'ebetso e ikemetseng e ileng ea bonoa mekhahlelong e lumellanang ea hCO e tlalehiloeng ka har'a vitro. E le ho hlahloba hore na t-hCO e ka sebelisoa ke tšusumetso ea maikutlo, re ile ra khelosa ka bokhutšoanyane litelu tsa rat ho tloha t-hCO (setšoantšo sa 4j, m le data e atolositsoeng, setšoantšo sa 10h, k). Ho ea ka liphuputso tse fetileng8,10, karolo e nyenyane ea lisele tsa t-hCO e bontšitse mosebetsi o eketsehileng ka lebaka la ho kheloha ha whisker, e neng e sa hlokomeloe ha data e bapisoa le litempe tsa nako e sa reroang (setšoantšo sa 4k-q le lintlha tse atolositsoeng, setšoantšo sa 10h-q). Ha e le hantle, hoo e ka bang 54% ea li-unit tsa opto-labeled single li bontšitse hore ho na le sekhahla se matla sa ho tsosoa ka mor'a ho tsosoa ha whisker, ho phahamisa hoo e ka bang 650 ms (setšoantšo sa 4r). Ha li kopane, lintlha tsena li fana ka maikutlo a hore t-hCO e fumana mekhoa e nepahetseng ea ts'ebetso 'me e ka sebelisoa ke tšusumetso ea tikoloho.
Eaba re etsa lipatlisiso hore na t-hCO e ka kenya li-circuits ka likhoto ho laola boitšoaro. Re ile ra etsa lipatlisiso pele hore na li-axon tsa t-hCO neurons li kenella ka har'a lisele tse haufi tsa rat. Re tšoaelitse hCO ka lentivirus encoding hChR2 e kopantsoeng le EYFP (hChR2-EYFP). Ka mor'a matsatsi a 110, re ile ra hlokomela polelo ea EYFP libakeng tsa ipsilateral cortical, ho kenyelletsa le li-cortices tsa ho utloa, tsa motlakase le tsa somatosensory, hammoho le libakeng tse nyenyane, ho akarelletsa le striatum, hippocampus le thalamus (Setšoantšo sa 5a). Ho lekola hore na likhakanyo tsena tse hlakileng li ka hlahisa likarabo tsa synaptic liseleng tsa rat, re ile ra sebelisa lisele tsa t-hCO tse hlalosang hChR2-EYFP ka ho rekota lisele tsa cerebral cortex likarolong tse bohale tsa boko. Ts'ebetso ea li-axon tsa t-hCO tse nang le leseli le leputsoa li hlahisitse li-EPSC tsa nako e khuts'oane tsa rat pyramidal cortex neurons, tse neng li koetsoe ke NBQX (Figs. 5b–g). Ho phaella moo, likarabo tsena li ka thibeloa ke tetrodotoxin (TTX) 'me tsa tsosolosoa ke 4-aminopyridine (4-AP), tse bontšang hore li bakoa ke ho kopana ha monosynaptic (Fig. 5e).
a, Sets'oants'o sa moralo oa ho latela axon (ka ho le letšehali). t-hCO EYFP polelo (ka ho le letona). Scale bar, 100 µm. A1, cortex ea kutlo, 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, nucleus ea ventropostomedial ea thalamus, VTA, sebaka sa ventral tegmental. Likarolo tse khubelu li emela libaka tsa boko moo litšoantšo li nkiloeng teng. b, Sets'oants'o sa moralo oa teko ea ts'usumetso. c, d, Mehlala ea karabelo ea khanya e putsoa e hlahisoang ke khanya (top) le voltage (tlase) ho motho (c) EYFP+ t-hCO kapa rat (d) EYFP- lisele. e, f, Mekhoa ea morao-rao ea li-neurons tsa rat ka mor'a hore khanya e putsoa ea t-hCO axon e na le TTX le 4-AR (e tala), TTX (e bohlooho) kapa aCSF (e ntšo) (e), e nang le (violet) kapa ntle le (e ntšo) ) ) NBQX (e). g, latency ea likarabo tse bakoang ke leseli le leputsoa liseleng tsa rat (n = 16 lisele); litšepe tse tšekaletseng li bonts'a latency e tloaelehileng (7.13 ms) (ka ho le letšehali). Boholo ba li-EPSC tse hlahisitsoeng ke leseli tse tlalehiloeng ka NBQX kapa ntle le eona (n = 7 lisele; ***P <0.0001) (bohareng). Boholo ba li-EPSC tse hlahisitsoeng ke leseli tse tlalehiloeng ka NBQX kapa ntle le eona (n = 7 lisele; ***P <0.0001) (bohareng). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). Boholo ba li-EPSC tse hlahisoang ke leseli tse tlalehiloeng ka NBQX kapa ntle le tsona (n = 7 lisele; ***P <0.0001) (bohareng).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). Boholo ba li-EPSC tse hlahisoang ke leseli tse tlalehiloeng ka NBQX kapa ntle le tsona (n = 7 lisele; ***P <0.0001) (bohareng).Peresente ea lisele tsa likhoto tse bontšang li-EPSC tse arabelang khanya e putsoa (ka ho le letona). h, Setšoantšo sa moralo oa mosebetsi oa boitšoaro. d0, letsatsi 0. i. Ts'ebetso ea liphoofolo tse behang mohlala ka letsatsi la 1 (ka ho le letšehali) kapa letsatsi la 15 (ka ho le letona) la koetliso. Palo e bolelang ea lick e entsoeng ka letsatsi la 1 (ka ho le letšehali) kapa letsatsi la 15 (ka ho le letona bohareng) (n = liteko tse 150 tsa leseli le leputsoa, ​​n = liteko tse 150 tse khubelu; ***P <0.0001). Palo e bolelang ea lick e entsoeng ka letsatsi la 1 (ka ho le letšehali) kapa letsatsi la 15 (ka ho le letona bohareng) (n = liteko tse 150 tsa leseli le leputsoa, ​​n = liteko tse 150 tse khubelu; ***P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 испытаний с синим светой, n = 150 светом; ***P <0,0001). Bolela palo ea lick tse entsoeng ka letsatsi 1 (ka ho le letšehali) kapa letsatsi la 15 (bohareng ka ho le letona) (n = 150 liteko tse khanyang tse putsoa, ​​n = liteko tse 150 tse khubelu; ***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 0.0001).第1 天（左）或第15 天（右中) Среднее количество облизываний, выполненных в день 1 (слева) или день 15 (в центре справа) (n = 150 испытаний с синим светой, n = 150 светом; ***P <0,0001). Bolela palo ea lick tse entsoeng ka letsatsi 1 (ka ho le letšehali) kapa letsatsi la 15 (bohareng ka ho le letona) (n = 150 liteko tse khanyang tse putsoa, ​​n = liteko tse 150 tse khubelu; ***P <0.0001).Likhoeletso tse akaretsang bakeng sa liteko tse khubelu le tse putsoa ka letsatsi la 1 (bohareng ka ho le letšehali) kapa letsatsi la 15 (ka ho le letona). NS, ha e bohlokoa. j,k, Litšobotsi tsa boitšoaro ba liphoofolo tsohle tse fetiselitsoeng ka t-hCO e hlalosang hChR2-EYFP (j) kapa ho laola fluorophore (k) ka letsatsi la 1 kapa 15 (hChR2-EYFP: n = 9 likhoto, ** P = 0.0049; taolo: n = 9, P9 = 0). l, Phetoho ea lintlha tsa khetho (n = 9 hChR2, n = 9 taolo; **P <0.001, ***P <0.0001). l, Phetoho ea lintlha tsa khetho (n = 9 hChR2, n = 9 taolo; **P <0.001, ***P <0.0001). l, Эволюция показателя предпочтения (n = 9 hChR2, n = 9 контрольных; **P <0,001, ***P <0,0001). l, Phetoho ea lintlha tsa khetho (n = 9 hChR2, n = li-control tse 9; **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, Phetoho ea lintlha tsa khetho (n = 9 hChR2, n = li-control tse 9; **P <0.001, ***P <0.0001).m, polelo ea FOS ho arabela ts'ebetsong ea optogenetic ea t-hCO ho S1. Litšoantšo tsa polelo ea FOS (ka ho le letšehali), le palo (n = 3 ka sehlopha; *P <0.05, **P <0.01 le ***P <0.001) (ka ho le letona) lia bontšoa. Litšoantšo tsa polelo ea FOS (ka ho le letšehali), le palo (n = 3 ka sehlopha; *P <0.05, **P <0.01 le ***P <0.001) (ka ho le letona) lia bontšoa. Показаны изображения экспрессии FOS (слева) и количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 and *** P <0,001) (справа). Litšoantšo tsa polelo ea FOS (ka ho le letšehali) le palo (n = 3 ka sehlopha; *P<0.05, **P<0.01, le ***P<0.001) lia bontšoa (ka ho le letona).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). Litšoantšo tsa polelo ea FOS (ka ho le letšehali) le palo (n = 3 ka sehlopha; *P<0.05, **P<0.01, le ***P<0.001) lia bontšoa (ka ho le letona).Scale bar, 100 µm. Lintlha li hlahisoa ka mokhoa o bolelang ± phoso e tloaelehileng ea BLA, basolateral tonsil, MDT, dorsomedial thalamic nucleus, PAG, periaqueductal grey.
Qetellong, re ile ra botsa hore na t-hCO e ka fetola boitšoaro ba likhoto. Ho leka sena, re ile ra fetisetsa hChR2-EYFP-expressing hCO ho S1, 'me matsatsi a 90 hamorao, ra kenya likhoele tsa optical ho t-hCO bakeng sa ho fana ka leseli. Ka mor'a moo re ile ra koetlisa likhoto ka paradigm e fetotsoeng ea opereishene (setšoantšo sa 5h). Re ile ra kenya liphoofolo ka phaposing ea teko ea boitšoaro 'me ra sebelisa ka mokhoa o sa reroang metsotso e 5 e putsoa (473 nm) le e khubelu (635 nm) laser stimuli. Liphoofolo li ile tsa fumana moputso oa metsi haeba li ne li nyeka nakong ea ho hlasimoloha ha leseli le leputsoa empa ha lia ka tsa nyeka nakong ea ho tsosoa ha khanya e khubelu. Letsatsing la pele la koetliso, liphoofolo li ne li sa bontše phapang ea ho nyeka ha li susumetsoa ka lebone le leputsoa kapa le lefubelu. Leha ho le joalo, ka letsatsi la 15, liphoofolo tse fetiselitsoeng ka hCO e hlalosang hChR2-EYFP li bontšitse ho nyeka ka mafolofolo ha ho tsosoa ka leseli le leputsoa ha ho bapisoa le ho tsosoa ha leseli le khubelu. Liphetoho tsena tsa boitšoaro ba ho nyeka ha lia ka tsa hlokomeloa ho liphoofolo tse laolang tse fetisitsoeng ka hCO e hlalosang fluorophore ea taolo (sekhahla sa katleho ea ho ithuta: hChR2 89%, EYFP 0%, Figure 5i-1 le Supplementary Video 2). Lintlha tsena li fana ka maikutlo a hore lisele tsa t-hCO li ka kenya tšebetsong li-neurone tsa likhoto ho khothaletsa boits'oaro ba ho batla meputso. Ho fumana hore na ke lipotoloho life tsa rat t-hCO neural tse ka 'nang tsa ameha liphetohong tsena tsa boitšoaro, re ile ra sebelisa optogenetically t-hCO ho liphoofolo tse koetlisitsoeng le ho kotula lisele tse 90 metsotso hamorao. Immunohistochemistry e senotse polelo ea protheine ea FOS e itšetlehileng ka ts'ebetso libakeng tse 'maloa tsa boko tse amehang boitšoarong bo susumetsang, ho kenyeletsoa medial prefrontal cortex, medial thalamus, le periaqueductal grey, e hlahisitsoeng liphoofolong tse sa laoleheng kapa liphoofolong. raese. 5m). Ha li kopane, lintlha tsena li fana ka maikutlo a hore t-hCO e ka fetola ts'ebetso ea rat neuronal ho khanna boitšoaro.
Neural organoids e emela sistimi e ts'episang ea ho ithuta nts'etsopele ea motho le lefu la in vitro, empa e fokotsoa ke khaello ea likhokahano lipakeng tsa lipotoloho tse teng ka har'a vivo. Re thehile sethala sa morao-rao seo ho sona re ileng ra fetisetsa hCO ho S1 ea likhoto tsa pele tsa pelehi tse sa khoneng ho itšireletsa mafung ho ithuta nts'etsopele ea lisele tsa motho le ts'ebetso ea eona ka har'a vivo. Re bonts'itse hore t-hCO e hlahisa mefuta ea lisele tse holileng tse sa bonoeng ka har'a vitro28 le hore t-hCO e kopantsoe le boko ba litoeba ka mokhoa oa tlhaho le ka mokhoa o sebetsang. Ho kopanngoa ha t-hCO ho li-circuits tsa neural tsa rodent ho ile ha re lumella ho theha kamano pakeng tsa ts'ebetso ea lisele tsa motho le ho ithuta boitšoaro ba liphoofolo, ho bontša hore li-neuron tsa t-hCO li ka fetola tšebetso ea rat neuronal ho khanna likarabo tsa boitšoaro.
Sethala seo re se hlalosang se na le melemo e mengata ho feta lipatlisiso tse fetileng mabapi le ho fetisetsa lisele tsa motho bokong ba litoeba. Taba ea pele, re fetiselitse hCO ka har'a cortex e ntseng e tsoela pele ea likhoto tsa pele tsa pelehi, tse ka thusang ho kopanya anatomical le ts'ebetso. Ntlha ea bobeli, ho hlahloba t-hCO MRI ho ile ha re lumella ho ithuta boemo ba graft le ho hōla ha liphoofolo tse phelang, ho re lumella ho etsa lithuto tsa nako e telele tsa liphoofolo tse ngata le ho theha botšepehi ba lisele tse 'maloa tsa hiPS. Qetellong, re ile ra fetisetsa li-organoid tse sa fetoheng, ho fapana le ho emisoa ha sele e le 'ngoe, tse sa senyeng lisele tsa motho mme li ka khothaletsa kopanyo le tlhahiso ea li-neurons tsa cortex bokong ba likhoto.
Rea lumela hore ho sa tsotellehe tsoelo-pele sethaleng sena, lithibelo tsa nakoana, sebaka, le mefuta-futa li thibela ho thehoa ha li-circuits tsa methapo ea motho ka botšepehi bo phahameng, esita le ka mor'a ho fetisoa qalong ea tsoelo-pele. Ka mohlala, ha ho hlake hore na mosebetsi o ikemetseng o hlokometsoeng ho t-hCO o emela phenotype ea tsoelo-pele e tšoanang le mosebetsi oa morethetho o hlokometsoeng nakong ea tsoelo-pele ea cortical, kapa hore na ke ka lebaka la ho se be teng ha mefuta ea lisele tse hatellang tse teng ho t-hCO. Ka mokhoa o ts'oanang, ha ho hlake hore na ho ba sieo ha lamination ho t-hCO ho ama ketane ea ketane hakae30. Mosebetsi oa nakong e tlang o tla tsepamisa maikutlo ho kopanyeng mefuta e meng ea lisele tse kang microglia ea motho, lisele tsa endothelial tsa motho, le likarolo tse sa tšoaneng tsa GABAergic interneurons joalokaha ho bontšitsoe ho sebelisa kopano ea 6 in vitro, hammoho le ho utloisisa hore na neural integration le ts'ebetso li ka etsahala joang ho t-hCO e fetotsoeng. maemo a transcriptional, synaptic le boitšoaro ka liseleng tse fumanoang ho bakuli.
Ka kakaretso, sethala sena sa vivo se emela mohloli o matla o ka tlatselletsang nts'etsopele ea boko ba motho ea in vitro le lipatlisiso tsa mafu. Re lebelletse hore sethala sena se tla re lumella ho fumana li-phenotypes tsa "strand-level" liseleng tse sa fumaneheng tse tsoang ho bakuli le ho leka maano a macha a kalafo.
Re hlahisitse hCO2.5 ho tloha liseleng tsa HiPS joalokaha ho hlalositsoe pejana. Ho qala tlhahiso ea hCO ho tsoa liseleng tsa hiPS tse hlahisitsoeng ka har'a li-feeder layers, li-colone tse sa fetoheng tsa lisele tsa hiPS li ile tsa ntšoa lijana tsa setso ho sebelisoa dispase (0.35 mg/mL) mme tsa fetisetsoa ho litloaelo tse tlase tsa polasetiki tse nang le lijana tse nang le setso sa setso sa hiPS. (Corning) e tlatsitsoe ka li-inhibitors tse peli tsa SMAD dorsomorphine (5 μM; P5499, Sigma-Aldrich) le SB-431542 (10 μM; 1614, Tocris) le ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Matsatsing a pele a 5, sebaka sa lisele tsa hiPS se ile sa fetoloa letsatsi le letsatsi mme dorsomorphine le SB-431542 li ile tsa eketsoa. Ka letsatsi la botšelela la ho emisoa, li-neural spheroids li ile tsa fetisetsoa ho neural medium e nang le neurobasal-A (10888, Life Technologies), tlatsetso ea B-27 ntle le vithamine A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin le streptomy0cin, epichnologies le streptomy0cin (1: 1) ntlha (EGF; 20 ng ml-1; R & D Systems) le fibroblast growth factor 2 (FGF2; 20 ng ml-1; R & D Systems) ho fihlela letsatsing la 24. Ho tloha letsatsing la 25 ho fihlela letsatsing la 42, seaplane se ile sa tlatsetsoa ka neurotrophic factor e tsoang bokong (BDNF; 20 trophin; 20 trophin; 3 mln trophin) le Peprotechn 20 ng ml−1; Peprotech) ka liphetoho tse mahareng letsatsi le leng le le leng. Ka letsatsi la botšelela la ho emisoa, li-neural spheroids li ile tsa fetisetsoa ho neural medium e nang le neurobasal-A (10888, Life Technologies), tlatsetso ea B-27 ntle le vithamine A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin le streptomy0cin, epichnologies le streptomy0cin (1: 1) ntlha (EGF; 20 ng ml-1; R & D Systems) le fibroblast growth factor 2 (FGF2; 20 ng ml-1; R & D Systems) ho fihlela letsatsing la 24. Ho tloha letsatsing la 25 ho fihlela letsatsing la 42, seaplane se ile sa tlatsetsoa ka neurotrophic factor e tsoang bokong (BDNF; 20 trophin; 20 trophin; 3 mln trophin) le Peprotechn 20 ng ml−1; Peprotech) ka liphetoho tse mahareng letsatsi le leng le le leng.Ka letsatsi la botšelela ha ho emisoa, li-neural spheroids li ile tsa fetisetsoa ho neural medium e nang le Neurobasal-A (10888, Life Technologies), B-27 tlatsetso ntle le vithamine 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 фибл 20; R&D Systems) до 24-го дня. le streptomycin (1: 100, Life Technologies) 'me e tlatsitsoe ka epidermal growth factor (EGF; 20 ng / ml; R & D Systems) le fibroblast growth factor 2 (FGF2; 20 ng / ml; R & D Systems) ho fihlela letsatsing la 24.Ho tloha matsatsing a 25 ho ea ho 42, boko bo entsoeng ke neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) le neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) li ile tsa kenngoa ho seaplane, ho fetola seaplane letsatsi le leng le le leng.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) EG并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 含有 neurobasal-a (12587 , Life Technologies) Glutamax (1: 100 , Life TechNOGIS青霉素 的 神经 培养 基 中 链霉素生长 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 эпидермальга факторангей факторан (1000, Life Technologies); R&D Systems) le фактора роста фибробластов 2 (FGF2; 20 нг мл-1) 1; Ka letsatsi la 6, li-neurosphere suspensions li ile tsa fetisetsoa ho tlatsetso e nang le neurobasal-A (10888, Life Technologies), tlatsetso ea B-27 ntle le vithamine A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin-neutralized factor streptomycin (1: 10000 Life supplement) (EGF; 20 ng ml-1; R & D Systems) le fibroblast growth factor 2 (FGF2; 20 ng ml-1) 1; R&D Systems) до 24-го дня. R&D Systems) ho fihlela letsatsi la 24.Ho tloha matsatsing a 25 ho isa ho a 42, boko bo entsoeng ke neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) le neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) li ile tsa kenngoa moetlong oa setso letsatsi le leng le le leng. Phetoho e mahareng hang.Ho tloha ka letsatsi la 43, hCO e ne e bolokiloe ka mokhoa o sa tlatsoang oa neurobasal-A medium (NM; 1088022, Thermo Fisher) ka phetoho e mahareng matsatsi a mang le a mang a 4-6. Ho fumana hCO ho tloha liseleng tsa hiPS tse hlahisitsoeng tlas'a maemo a se nang phepo, lisele tsa hiPS li ne li kenngoa ka Accutase (AT-104, Innovate Cell Technologies) ka 37 ° C bakeng sa metsotso ea 7, e arotsoe ka lisele tse le 'ngoe,' me e pentiloe holim'a lipoleiti tsa AggreWell 800 (34815, STEMCELL ka 6 liseleng tsa 6 ka lisele tse le 'ngoe tsa 6 × 1 ka lisele tse 1) Essential 8 medium e tlatsitsoeng ka ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Ka mor'a lihora tse 24, mecha ea litaba ka lilibeng e ile ea kenngoa ka liphaephe ho ea holimo le tlaase ho mecha ea litaba e nang le Essential 6 media (A1516401, Life Technologies) e tlatsitsoeng ka dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) le SB-431542 (10 μM; 1614). , Tocrida). Ho tloha matsatsing a 2 ho isa ho a 6, Essential 6 medium e ile ea nkeloa sebaka letsatsi le letsatsi ka dorsomorphine le tlatsetso SB-431542. Ho tloha ka letsatsi la botšelela, ho emisoa ha neurosphere ho ile ha fetisetsoa sebakeng sa neurobasal mme sa bolokoa joalo ka ha ho hlalositsoe ka holimo.
Mekhoa eohle ea ho sebetsana le liphoofolo e entsoe ho latela litataiso tsa tlhokomelo ea liphoofolo tse amohetsoeng ke Stanford University Laboratory Animal Care Administrative Committee (APLAC). Likhoto tsa euthymic RNU (rnu/+) tse emereng li ile tsa rekoa (Charles River Laboratories) kapa tsa behoa ka tlung. Liphoofolo li ne li bolokoa lihora tse 12 tsa potoloho ea lefifi le lefifi ka lijo le metsi ad libitum. Madinyane a hlobotseng (FOXN1–/–) a dilemong tsa matsatsi a mararo ho isa ho a supileng a ile a hlwauwa ka ho hola ha ditedu tse sa holang pele di hlangwa. Lintjanyana (tse tona le tse tšehali) li ile tsa koaheloa ka 2-3% ea isoflurane 'me tsa behoa holim'a foreimi ea stereotaxic. Ho ts'oaroa ha lehata ka bophara ba hoo e ka bang 2-3 mm ka holimo ho S1 ho ile ha etsoa ha ho ntse ho bolokoa botšepehi ba dura mater. Ebe u sebelisa nale ea 30-G (hoo e ka bang 0.3 mm) ka ntle ho craniotomy ho phunya dura. Ebe u sebelisa HCO ho parafil e tšesaane ea 3 × 3 cm 'me u tlose seaplane se feteletseng. U sebelisa syringe ea Hamilton e khomaretsoeng ho nale ea 23 G, 45 °, ka bonolo hula hCO karolong e hōle ka ho fetisisa ea nale. Ebe u kenya serinji pompong ea sering e hokahaneng le sesebelisoa sa stereotaxic. Ebe u beha ntlha ea nale holim'a lesoba la ho phunya ka bophara ba 0.3 mm ka dura (z = 0 mm) 'me u tšesaane serinji 1-2 mm (z = hoo e ka bang -1.5 mm) ho fihlela nale e pakeng tsa dura mater A. ho thehoa tiiso e teteaneng. Ebe u phahamisa serinji bohareng ba bokaholimo ba cortical ho z = -0.5 mm 'me u ente hCO ka sekhahla sa 1-2 µl ka motsotso. Ka mor'a ho phethoa ha ente ea hCO, nale e khutlisetsoa morao ka tekanyo ea 0.2-0.5 mm ka motsotso, letlalo le sutured, 'me lesea le behoa hang-hang holim'a mocheso o futhumetseng oa mocheso ho fihlela o hlaphoheloa ka ho feletseng. Liphoofolo tse ling li ile tsa jalleloa ka mahlakore ka bobeli.
Mekhoa eohle ea ho sebetsana le liphoofolo e entsoe ho latela litataiso tsa tlhokomelo ea liphoofolo tse amohetsoeng ke Univesithi ea Stanford APLAC. Likhoto (tse kholo ho feta matsatsi a 60 ka mor'a ho fetisoa) li ile tsa kenngoa ka 5% ea anesthesia ea isoflurane 'me ea e-na le anesthetized ka 1-3% isoflurane nakong ea litšoantšo. Bakeng sa pono, mochine oa 7 oa Tesla o sirelelitsoeng ka mafolofolo Bruker (Bruker Corp.) o nang le gradient drive ea International Electric Company (IECO), e sirelelitsoeng e kentsoeng ka bophara ba 120 mm (600 mT / m, 1000 T / m / s) e sebelisitsoe ho sebelisa AVANCE. III, likanale tse robeli tsa li-coil tse ngata tsa RF le bokhoni ba li-multi-core, le sethala se tsamaeang le Paravision 6.0.1. Ho rekota ho ile ha etsoa ho sebelisoa khoele ea RF ea volumetric e butsoitseng ka bophara e bophara ba 86 mm le khoele ea RF e pholileng ea likanale tse 'ne bakeng sa ho amohela feela. Axial 2D Turbo-RARE (nako ea ho pheta-pheta = 2500 ms, nako ea echo = 33 ms, 2 karolelano) e nang le likarolo tse 16 tse hapiloeng, botenya ba selae 0.6-0.8 mm, e nang le 256 × 256 sampuli. Lipontšo li ile tsa amoheloa ho sebelisoa quadrature transceiver volumetric RF coil e nang le bophara ba ka hare ba 2 cm (Rapid MR International, LLC). Qetellong, sebelisa mesebetsi ea likhakanyo tsa bokaholimo ba Imaris (BitPlane) bakeng sa tlhahiso ea 3D le tlhahlobo ea molumo. Phetisetso e atlehileng e ile ea hlalosoa e le sebaka seo ho sona ho ileng ha thehoa lets'oao le tsoelang pele la T2-weighted MRI ho hemisphere e fetisitsoeng. Ho lahloa hoa graft ho ne ho hlalosoa e le sethopo se sa kang sa hlahisa libaka tsa lets'oao le tsoelang pele la T2-weighted MRI sebakeng sa hemisphere se fetisitsoeng. Subcortical t-hCO e ne e sa kenyelletsoa tlhahlobong e latelang.
Ho hlahisa li-GCaMP6 ka mokhoa o tsitsitseng ho hCO bakeng sa litšoantšo tsa calcium tse photon tse peli, lisele tsa hiPS li ile tsa tšoaetsoa ke pLV[Exp] -EF1a::GcaMP6s-WPRE-Puro e lateloa ke khetho ea lithibela-mafu. Ka bokhutšoanyane, lisele li ile tsa aroloa le EDTA 'me tsa emisoa ka 1 ml ea Essential 8 medium ka bongata ba lisele tse ka bang 300,000 ka pel'a polybrene (5 μg/ml) le 15 μl ea kokoana-hloko. Ka mor'a moo, lisele li ne li kenngoa ka ho emisoa ka metsotso e 60 'me li lengoa ka bongata ba lisele tse 50,000 selibeng se seng le se seng. Kamora ho kopana, lisele li ne li phekoloa ka 5-10 μg ml-1 puromycin ka matsatsi a 5-10 kapa ho fihlela likolone tse tsitsitseng li hlaha. Ts'oaetso e matla ea hCO e entsoe joalo ka ha ho hlalositsoe pele5 ka liphetoho tse ling. Ka bokhutšoanyane, fetisetsa letsatsi la 30-45 hCO ho 1.5 ml Eppendorf microcentrifuge tubes e nang le 100 μl ea methapo ea kutlo. Ebe hoo e ka bang 90 µl ea mahareng e tlosoa, 3-6 µl ea high titer lentivirus (ho tloha 0.5 x 108 ho 1.2 x 109) e kenngoa ka har'a tube, 'me hCO e fetisetsoa ho incubator metsotso e 30. Ebe u eketsa 90-100 µl ea mahareng ho phaephe e 'ngoe le e 'ngoe ebe u khutlisetsa liphaephe ho incubator bosiu bo le bong. Letsatsing le hlahlamang, fetisetsa hCO sebakeng se secha sa methapo ka lipoleiti tse tlaase tsa li-attachment. Ka mor'a matsatsi a 7, hCO e ile ea fetisetsoa lipoleiti tse ka tlaase tsa likhalase tse 24 bakeng sa pono le tlhahlobo ea boleng ba tšoaetso. pLV[Exp]-SYN1::EYFP-WPRE le pLV[Exp]-SYN1::hChR2-EYFP-WPRE li entsoe ke VectorBuilder. Lentivirus e sebelisoa litekong tse ngata hobane e kopantsoe le genome e amohelang, e lumellang moqolotsi oa litaba oa liphatsa tsa lefutso meleng ea lisele tse nang le tšoaetso. Bakeng sa ho latela lefu la rabies, letsatsi la 30-45 hCO le tšoaelitsoe ke lefu la rabies-ΔG-eGFP le AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), e hlatsoitsoeng hantle ka matsatsi a 3, 'me ea fetisetsoa ho likhoto ka S1 le ho bolokoa ka matsatsi a 7-1.
Bakeng sa immunocytochemistry, liphoofolo li ne li e-na le anesthetized le transcardially perfusion ka PBS e lateloa ke 4% paraformaldehyde (PFA ho PBS; Electron Microscopy Sciences). Boko bo ne bo tsitsitse ho 4% PFA bakeng sa lihora tsa 2 kapa bosiu bo le bong ho 4 ° C, cryopreserved ka 30% sucrose ho PBS bakeng sa lihora tse 48-72, 'me e kenngoa ka 1: 1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583, Sakura Finetes0 e entsoe ka karolo ea 30 ea "corona" le "corona". (Leica). Bakeng sa immunohistochemistry ea likarolo tse teteaneng, liphoofolo li ne li tšeloa PBS, 'me boko bo ile ba aroloa le ho aroloa ka coronally ho 300-400 µm ho sebelisa vibratome (Leica)' me likarolo li ne li behiloe ka 4% PFA bakeng sa metsotso ea 30. Joale li-cryosections kapa likarolo tse teteaneng li ne li hlatsuoa ka PBS, li koetsoe ka hora ea 1 mocheso oa kamore (10% e tloaelehileng ea tonki serum (NDS) le 0.3% Triton X-100 e hlapollotsoeng ka PBS) 'me e koetsoe ka tharollo e thibelang ho 4 ° C. - Incubation Cryosections e ile ea eloa ka bosiu bo le bong, 'me likarolo tse teteaneng li ile tsa kenngoa matsatsi a 5. Li-antibodies tsa mantlha tse neng li sebelisoa e ne e le: anti-NeuN (toeba, 1: 500; ab104224, abcam) anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (mmutla, 1: 1,000; Z0334, Dako), anti-GFP0 1 GTX, 3, 0, anti-GFP0, GTX 1, 3,000; GeneTex), anti-HNA (toeba, 1:200; ab191181, abcam), anti-NeuN (mmutla, 1:500; ABN78, Millipore), anti-PDGFRA (mmutla, 1:200; sc-338, Santa Cruz), anti-PPP20, 7; HPA047819, Atlas Antibodies), anti-RECA-1 (toeba, 1:50; ab9774, abcam), anti-SCG2 (mmutla, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (poli, 1:500; Netgorins Systema, G30D5, R&30 1:100; AF1166, R&D Systems), anti-STEM121 (toeba, 1:200; Y40410, Takara Bio), anti-SATB2 (toeba, 1:50; ab51502, abcam), anti-GAD65/67 (mmutla, 1:400; (pōli, 1:100; ab5076, abcam). Li-antibodies tsa mantlha tse neng li sebelisoa e ne e le: anti-NeuN (toeba, 1: 500; ab104224, abcam) anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (mmutla, 1: 1,000; Z0334, Dako), anti-GFP0 1 GTX, 3, 0, anti-GFP0, GTX 1, 0, 0, 3 GFP0; GeneTex), anti-HNA (toeba, 1:200; ab191181, abcam), anti-NeuN (mmutla, 1:500; ABN78, Millipore), anti-PDGFRA (mmutla, 1:200; sc-338, Santa Cruz), anti-PPP20, 7; HPA047819, Atlas Antibodies), anti-RECA-1 (toeba, 1:50; ab9774, abcam), anti-SCG2 (mmutla , 1:100; 20357-1-AP, Proteintech), anti-SOX9 (podi, 1:500; Neta1 Systems, AFgorins), Neta1D&30D 1:100; AF1166, R&D Systems), anti-STEM121 (toeba , 1:200; Y40410, Takara Bio), anti-SATB2 (toeba, 1:50; ab51502, abcam), anti-GAD65/67 (mmutla, 1:400; (pōli, 1:100; ab5076, abcam). Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2 (крысиные, 1:1b840); анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA (мышь, 1:200; ab191camuNex), 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), нетризн10, нетриз10, 6 G10; R&D Systems), анти-STEM121 (мышиный , 1:200; Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кроли00, ABN90; ABN, 1, 20; анти-IBA1 (коза, 1 :100; аб5076, абкам). Li-antibodies tse ka sehloohong tse neng li sebelisoa e ne e le: anti-NeuN (mouse, 1: 500; ab104224, abcam), anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (mmutla, 1: 1000; Z0334, Dako), anti-GFP0, GTX7, GTX9, 3chicken, 3 Chicken, 1, X00, anti-GFAP anti-HNA (toeba, 1: 200; ab191181, abcam), anti-NeuN (mmutla, 1: 500; ABN78, Millipore), anti-PDGFRA ( mmutla, 1: 200; sc-338, Santa Cruz), anti-PPP1R17, 1: 9 HPAb4; Likokoana-hloko), anti-RECA-1 (toeba, 1:50; ab9774, abcam), anti- SCG2 (mmutla, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (poli, 1:500; AF3075, R&D Systems), netrin 1:10, G1, netrin 1, 10 G1 R&D Systems), anti- STEM121 (toeba, 1:200; Y40410, Takara Bio), anti-SATB2 (toeba, 1:50; ab51502, abcam), anti-GAD65/67 (mmutla, 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;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＊ 200;sc-338，Santa Cruz) 100;20357-1-AP，Proteintech)Y40410, Takara Bio), anti-SATB2 (toeba, 1:50; ab51502, abcam), anti-GAD65/67 (mmutla, 1:400; ABN904, Millipore) le anti-IBA1 (poli, 1:100; ab5076) abcam.Li-antibodies tse ka sehloohong tse sebelisoang e ne e le: anti-NeuN (mouse, 1: 500; ab104224, abcam), anti-CTIP2 (rat, 1: 300; ab18465, abcam), anti-GFAP (mmutla, 1: 1000; Z0334, Dako) . , anti-GFP (khoho, 1: 1000; GTX13970, GeneTex), anti-HNA (toeba, 1:200; ab191181, abcam), anti-NeuN (mmutla, 1:500; ABN78, Millipore), anti-PDGFRA (mmutla-208; anti-PPP1R17 (mmutla, 1: 200; HPA047819, Atlas antibody), anti-RECA-1 (toeba, 1:50; ab9774, abcam), anti- SCG2 (mmutla), 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 (Y4:0raш10; 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 (poli, 1:500; AF3075, R&D Systems), Netrin G1a (poli, 1:100; AF1166, R&D Systems), anti-STEM121 (toeba, 1:200; anti-SATBARA), Y404 1:50; ab51502, abcam), anti-GAD65/67 (mmutla, 1:400; ABN904, Millipore), le anti-IBA1 (poli, 1:100; ab5076, abkam).Likarolo li ne li hlatsuoa ka PBS 'me li kenngoa ka antibody ea bobeli bakeng sa hora ea 1 mocheso oa kamore (likarolo tse leqhoa) kapa bosiu bo le bong ho 4 ° C (likarolo tse teteaneng). Alexa Fluor secondary antibody (Life Technologies) e hlapollotsoeng 1:1000 ka tharollo e thibelang e sebelisitsoe. Ka mor'a ho hlatsoa ka PBS, li-nuclei li ile tsa bonoa ka Hoechst 33258 (Life Technologies). Qetellong, li-slide li ile tsa behoa holim'a microscope e nang le likoahelo (Fisher Scientific) ka ho sebelisa Aquamount (Polysciences) 'me ea hlahlojoa ka microscope ea Keyence fluorescent (BZ-X analyzer) kapa Leica TCS SP8 confocal microscope (Las-X) setšoantšong. Litšoantšo li ile tsa sebetsoa ho sebelisoa lenaneo la ImageJ (Fiji). Ho lekanya karolo ea li-neurone tsa batho ho t-hCO le rat cortex, litšoantšo tse 387.5 μm tse bophara ba likhutlo li nkiloe bohareng ba t-hCO, moeling kapa haufi le moeli oa rat cortex. Margins a graft a ne a khethoa ka ho lekola liphetoho tsa ponaletso ea lisele, HNA + nuclei, le/kapa boteng ba li-tissue autofluorescence. Setšoantšong ka seng, palo eohle ea lisele tsa NeuN+ le HNA+ li arotsoe ka palo ea lisele tsa NeuN+ sebakeng se le seng. Ho etsa bonnete ba hore ho baloa feela lisele tse nang le li-nuclei sefofaneng sa setšoantšo, ke lisele feela tse nang le Hoechst+ tse kenyellelitsoeng palong. Litšoantšo tse peli tse arohaneng ka bonyane 1 mm li ile tsa lekanngoa ho fokotsa phoso ea lipalo.
Beke pele ho pokello ea sampole, beha liphoofolo tse fetisetsoang hCO (hoo e ka bang likhoeli tse 8 tsa phapang) ka phaposing e lefifi e nang le litelu tse lokisitsoeng ho fokotsa ts'usumetso ea kutlo. Ho aroloa ha li-nuclei ho entsoe joalo ka ha ho hlalositsoe pejana, ka liphetoho tse ling. Ka bokhutšoane, t-hCO le hCO li ile tsa senngoa ho sebelisoa detergent-mechanical cell lysis le 2 ml ea khalase ea grinder ea lisele (D8938, Sigma-Aldrich/KIMBLE). Li-nuclei tse tala li ile tsa tlhotloa ho sebelisoa sefene sa 40 µm le centrifuged ho 320 g metsotso e 10 ho 4 ° C pele ho etsoa sucrose density gradient. Ka mor'a mohato oa centrifugation (320 g bakeng sa metsotso e 20 ho 4 ° C), lisampole li ile tsa khutlisetsoa morao ho 0.04% BSA/PBS ka ho eketsoa ha li-unit tse 0.2 tsa µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) 'me tsa feta µm filthara ea 40. Li-nuclei tse arohaneng li ile tsa khutlisetsoa morao ho PBS e nang le 0.02% BSA 'me tsa kenngoa ho Chromium Single Cell 3′ chip (e hakantsoeng ea ho hlaphoheloa ha lisele tse 8,000 ka tsela). lilaebrari tsa snRNA-seq li ile tsa lokisoa ka sele ea Chromium Single 3' GEM, Library & Gel Bead Kit v3 (10x Genomics). lilaebrari tsa snRNA-seq li ile tsa lokisoa ka sele ea Chromium Single 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). Lilaebrari tsa snRNA-seq li ile tsa lokisoa ho sebelisoa 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). Laebrari ea snRNA-seq e ile ea lokisoa ho sebelisoa Chromium Single Cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics).Lilaebrari tse tsoang lisampoleng tse fapaneng li ile tsa kopanngoa le ho hlophisoa ke Admera Health ho NovaSeq S4 (Illumina).
Maemo a polelo ea liphatsa tsa lefutso bakeng sa "barcode" e 'ngoe le e 'ngoe ea nyutlelie a ile a lekanngoa ho sebelisoa 10x Genomics CellRanger analysis software package (version 6.1.2). Haholo-holo, lipalo li ne li bapisitsoe le motsoako oa batho (GRCh38, Ensemble, version 98) le rat (Rnor_6.0, Ensemble, version 100) liphatsa tsa lefutso tse entsoeng ka taelo ea mkref le ho sebelisa ho bala ka taelo ea -include-introns=TRUE ho palo e kenyelletsa ho bala ho kenyelletsa libaka. Bakeng sa disampole tsa t-hCO, li-nuclei tsa motho li ile tsa khetholloa ho ipapisitsoe le tlhokahalo e hlokolosi ea hore bonyane 95% ea limmapa tsohle tse baloang li tsamaisane le genome ea motho. Litlhahlobo tsohle tse latelang li ile tsa etsoa holim'a sehlahisoa sa barcode se tlhotliloeng se tsoang ho CellRanger ho sebelisa sephutheloana sa R ​​(version 4.1.2) Seurat (version 4.1.1)32.
Ho netefatsa hore li-nuclei tsa boleng bo phahameng feela li kenyelelitsoe tlhahlobisong e latelang, ts'ebetso ea ho sefa e pheta-phetoang e ile ea sebelisoa bakeng sa sampole ka 'ngoe. Ntlha ea pele, li-nuclei tsa boleng bo tlaase tse nang le liphatsa tsa lefutso tse ka tlaase ho 1000 tse fumanoeng le ho feta 20% ea kakaretso ea mitochondria e khetholloa le ho tlosoa. Ka mor'a moo, palo e tala ea gene number matrix e ile ea tloaeleha ka "binomial regression" e tloaelehileng ho sebelisoa ts'ebetso ea sctransform(vst.flavor=”v2″), e ileng ea boela ea khetholla mefuta e 3000 ea liphatsa tsa lefutso ka ho fetisisa ho sebelisa li-parameter tsa kamehla. 30 e khethiloe ho ipapisitse le tlhahlobo ea pono ea libaka tsa mangole le ho sebelisoa bakeng sa lisampole tsohle le litlhahlobo tse kopaneng (re ile ra etsa mekhahlelo e mengata ea lihlopha tse pheta-phetoang (qeto = 1) ho arola liphatsa tsa lefutso ho latela palo e sa tloaelehang ea liphatsa tsa lefutso (bohareng bo ka tlase ho 10th percentile), ka tsela e sa tloaelehang e phahameng ea mitochondrial ka holim'a liphatsa tsa lefutso tsa 9 ho tlosa liphatsa tsa lefutso le/kapa karolo e phahameng ya mafahla a belaellwang a hlwauweng ho sebediswa sephutheloana sa DoubletFinder33 (ho bolela lintlha tsa DoubletFinder tse ka holimo ho disampole tsa t-hCO (n=3) le hCO (n=3) li ile tsa kopanngoa ka thoko ho sebelisoa tšebetso ea IntegrateData le lintlha tse ka holimo.
Ka mor'a ho tlosa li-kernels tsa boleng bo tlaase, dataset e kopantsoeng e ile ea hlophisoa (qeto = 0.5) 'me ea kenngoa bakeng sa merero ea pono ea UMAP34. Liphatsa tsa lefutso tsa sehlopha se seng le se seng li ile tsa khethoa ho sebelisoa ts'ebetso ea FindMarkers ka li-parameter tsa kamehla tse baloang ho tsoa ho data e tloaelehileng ea polelo ea lefutso. Re hlwaya le ho arola dihlopha tse kgolo tsa disele ka ho kopanya disete tsa ditshupetso tsa lesea le la batho ba baholo le letshwao la gene 19,20,21,35 le tlhaloso. Ka ho khetheha, li-precursors tse potolohang li ile tsa khetholloa ka polelo ea MKI67 le TOP2A. Lihlopha tsa li-progenitor li ne li hlalosoa ke ho ba sieo ha lingoliloeng tsa mitotic, ho kopana ho phahameng le lihlopha tse ngata tsa glial progenitor tse hlalositsoeng ka morao ho metaphase fetal cortex, le polelo ea EGFR le OLIG1. Re sebelisa lentsoe astrocyte ho akaretsa linaha tse 'maloa tsa phapang ea astrocyte, ho tloha ho radial glia ho isa ho maturation of astrocyte. Lihlopha tsa Astrocyte li hlahisa maemo a holimo a SLC1A3 le AQP4 'me li bontšitsoe 'mapeng ka mefuta e nyane ea fetal radial glia le/kapa li-astrocyte tsa batho ba baholo. Li-OPC li hlahisa PDGFRA le SOX10 ha li-oligodendrocyte li hlahisa matšoao a myelination (MOG le MYRF). Li-neurons tsa Glutamatergic li ile tsa khetholloa ka ho ba teng ha li-neuronal transcripts (SYT1 le SNAP25), ho ba sieo ha matšoao a GABAergic (GAD2), le polelo ea NEUROD6, SLC17A7, BCL11B, kapa SATB2. Li-neuron tsa GluN li ile tsa aroloa hape ka li-subclasses tse ka holimo (SATB2 le tahlehelo ea BCL11B) le tse tebileng (BCL11B expression). Li-neuron tsa putative subplate (SP) li hlahisa matšoao a tsebahalang a SP18 joalo ka ST18 le SORCS1 ho kenyelletsa le matšoao a tebileng a GluN. Lisele tse kang choroid-like plexus li ile tsa khetholloa ka polelo ea TTR, 'me lisele tse kang meningeal li hlahisa liphatsa tsa lefutso tse amanang le fibroblast le lisele tsa 'mapa tsa pial/vascular tsa sete ea boitsebiso ba litšupiso.
Tlhahlobo e fapaneng ea polelo ea liphatsa tsa lefutso lipakeng tsa t-hCO le hCO subclass e entsoe ho sebelisoa mokhoa o mocha oa pseudo-batch o hlahisitsoeng ka lisampole tse kentsoeng ts'ebetsong ho sebelisoa sephutheloana sa Libra R (version 1.0.0). Haholo-holo, liteko tsa monyetla oa ho kena ho EdgeR (mofuta oa 3.36.0, sephutheloana sa R) li entsoe bakeng sa lihlopha ka ho akaretsa palo ea liphatsa tsa lefutso liseleng bakeng sa sehlopha se fanoeng sa sele bakeng sa mohlala o mong le o mong oa ho ikatisa. Bakeng sa pono ea 'mapa oa mocheso, litekanyetso tse tloaelehileng ho milione (CPM) li sebelisoa ho sebelisoa edgeR (cpm() ts'ebetso) le ho lekanngoa (ho fihlela moelelo = 0, ho kheloha ho tloaelehileng = 1). Tlhahlobo ea ntlafatso ea Gene Ontology (GO) ea liphatsa tsa lefutso tsa t-hCO GluN e laoloang haholo e ile ea etsoa (Benjamini-Hochberg e lokiselitse boleng ba P bo ka tlase ho 0.05 bo hlalositsoeng bonyane 10% ea lisele tsa t-hCO GluN le ho eketseha ha makhetlo a phetoho bonyane makhetlo a 2). e etsoa ho sebelisoa ToppGene Suite (https://toppgene.cchmc.org/)37. Re sebelisa sesebelisoa sa ToppFun se nang le li-parameter tsa kamehla, 'me re tlaleha litekanyetso tsa P tse lokisitsoeng tsa Benjamini-Hochberg tse baloang ho tsoa litekong tsa GO-annotated hypergeometric.
Ho bapisa lihlopha tsa rona tsa snRNA-seq le lihlopha tsa lisele tse hlalositsoeng ho tsoa liphuputsong tsa litšupiso tsa li-primary single-cell RNA-seq kapa snRNA-seq19,20,21,22 ea batho ba baholo, re sebelisitse mokhoa oa ho kopanya dataset. Re sebelisitse SCTransform (v2) ho tloaeleha ha mosebetsi oa Seurat ho kopanya le ho bapisa lihlopha tse fetang pakeng tsa li-datasets (ho sebelisa li-parameter tse tšoanang le tse ka holimo). Li-dataset tsa motho ka mong li ile tsa hlophisoa ka mokhoa o sa reroang ho fihla ho lisele tse 500 kapa li-cores ka sehlopha sa mantlha bakeng sa katleho ea khomphutha. Ho sebelisoa mokhoa o ts'oanang joalo ka ha ho hlalositsoe pejana, ho kopana ha lihlopha ho ile ha hlalosoa e le karolo ea lisele kapa nuclei sehlopheng se seng le se seng se kopantsoeng se fetang le leibole ea sehlopha sa litšupiso. Ho tsoela pele ho arola li-GluN, re sebelisitse Seurat's TransferData workflow bakeng sa data e nyane ea GluN ho abela li-label tsa dataset ho lisele tsa rona tsa GluN.
Ho hlahloba boemo ba khōlo ea transcriptome ea lefats'e ea t-hCO le hCO disampole, re bapisitse lisampole tsa rona tsa pseudo-bulk le BrainSpan/psychENCODE23, e nang le tatelano e kholo ea RNA e amang tsoelo-pele ea boko ba motho. Re ile ra etsa PCA ka mokhoa o kopantsoeng oa mokhoa o tloaelehileng oa liphatsa tsa lefutso ho tloha lisampole tsa cortical libeke tsa 10 ka mor'a kemolo le hamorao, ka liphatsa tsa lefutso tsa 5567 (hammoho le data ea rona) tse neng li tsejoa e le tse sebetsang ho BrainSpan sampuli ea cortical (e hlalosoang e le kholo ho feta 50% ka phapang ea tsoelo-pele e hlalositsoeng ke lilemo tse sebelisang mohlala oa cubic)38. Ntle le moo, re ile ra fumana liphatsa tsa lefutso tse amanang le li-signature tse kholo tsa transcriptome tsa neurodevelopment re sebelisa non-negative matrix factorization joalo ka ha ho hlalositsoe pejana. Mehlala ea boima ba 'mele e baloang ho sebelisoa mokhoa o se nang negative matrix factorization o reriloe ho Feiga. 5b e nang le lintlha tse atolositsoeng bakeng sa li-signature tse hlano tse hlalositsoeng ke Zhu et al.38. Hape, matšoao a ngotsoeng a itšetlehileng ka mosebetsi a nkiloe lithutong tse hatisitsoeng pele. Haholo-holo, ERG le LRG li ne li laoloa haholo ho li-neurons tsa glutamatergic tse khetholloang ke mouse visual cortex snRNA-seq pokello ka mor'a ho tsosoa ha pono ho tloha Supplementary Table 3 Hrvatin et al.16. Li-LRG tse ruisitsoeng ke batho li ile tsa fumanoa litloaelong tsa boko ba fetal tse entsoeng ke KCl 'me tsa kotuloa lihora tse 6 ka mor'a ho tsosoa,' me liphatsa tsa lefutso tse tlhotliloeng li ne li laoloa haholo ho batho empa eseng ho litoeba (Tlaleho ea Tlatsetso ea 4). Tlhahlobo ea ntlafatso ea liphatsa tsa lefutso e sebelisang lihlopha tsena tsa liphatsa tsa lefutso e entsoe ka mokhoa o le mong oa tlhahlobo ea Fisher.
Anesthetize likhoto tse nang le isoflurane, tlosa boko le sebaka se batang (hoo e ka bang 4 ° C) se nang le oksijene (95% O2 le 5% CO2) tharollo ea sucrose bakeng sa likarolo tse nang le: 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 15 m.25 m. NaH2PO4, 10 mM MgSO4 le 0.5 mM CaCl2 (e ka bang 310 mOsm). Likarolo tsa coronal boko ba rat (300-400 µm) tse nang le t-hCO li entsoe ka Leica VT1200 vibratome joalokaha ho hlalositsoe pele39. Likarolo li ile tsa fetisetsoa kamoreng ea ho arola e nang le oksijene ea mocheso oa kamore e tsoelang pele e nang le aCSF e lokiselitsoeng ho tloha: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 le 126 mM MOsm29 (29). bonyane metsotso e 45 pele o rekota. Likarolo li ne li ngotsoe ka kamoreng e qoelitsoeng moo li neng li lula li hloekisoa ka aCSF (95% O2 le 5% CO2 vial). Lintlha tsohle li ngotsoe ka mocheso oa kamore. Li-neurone tsa t-hCO li ile tsa felisoa ka pipette ea khalase ea borosilicate e tlatsitsoeng ka tharollo e nang le 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, le 0.6 mM EGTA, pH 7.2, tharollo ea ka hare e lokisoang ka KOOs 29 mH. E le hore u hlaphoheloe, biocytin (0.2%) e kenyelelitsoe tharollo ea ho rekota.
Lintlha li ile tsa fumanoa ho sebelisoa MultiClamp 700B amplifier (Molecular Devices) le Digidata 1550B digitizer (Molecular Devices), low-pass e tlhotliloeng ho 2 kHz, digitized ho 20 kHz, le ho hlahlojoa ho sebelisoa Clampfit (Molecular Devices), Origin (OriginPro). 2021b, OriginLab). le mesebetsi e tloaelehileng ea MATLAB (Lipalo). Monyetla oa ho kopana o ile oa baloa ho sebelisoa JPCalc mme likeno li fetotsoe ho boleng bo baliloeng ba -14 mV. Operation IV e na le letoto la mehato ea morao-rao ka mehato ea 10-25 pA, ho tloha -250 ho ea ho 750 pA.
Thalamus, taba e tšoeu, le li-afferent tsa S1 li ile tsa hlohlelletsoa ka motlakase ka lilae tsa thalamocortical nakong ea ho rekota li-hCO neurons, joalo ka ha ho hlalositsoe pejana. Ka bokhutšoanyane, boko bo ile ba behoa tafoleng ea khatiso ea 3D e sekame ka lehlakoreng la 10 °, 'me karolo e ka pele ea boko e ile ea khaoloa ka 35 ° angle. Joale boko bo ne bo khomaretsoa sebakeng se sehiloeng ebe se aroloa, ho boloka li-axon tse hlahelletseng tsa thalamocortical. Li-electrode tsa bipolar tungsten (0.5 MΩ) li ne li behiloe holim'a micromanipulator ea bobeli 'me li behiloe ka mokhoa o hlophisitsoeng ho susumetsa libaka tse' nè ka sele (capsule e ka hare, taba e tšoeu, S1 le hCO). Rekota likarabo tsa synaptic kamora 300 µA phasic stimulation ho 0.03–0.1 Hz.
hChR2-expressing hCO neurons e ile ea sebelisoa ho 480 nm le li-pulses tse khanyang tse hlahisoang ke LED (Prizmatix) li ile tsa sebelisoa ka sepheo sa × 40 (0.9 NA; Olympus) ho rekota polelo ea hChR2 haufi le lisele. Bophara ba tšimo e bonesitsoeng ke hoo e ka bang 0.5 mm mme kakaretso ea matla ke 10-20 mW. Bophara ba pulse bo ne bo behiloe ho 10 ms, e lumellanang le sekhahla se fanoeng nakong ea teko ea ho ithuta boitšoaro. Ho ile ha sebelisoa maqhubu a fapaneng a susumetsang, ho tloha ho 1 ho isa ho 20 Hz, empa ho ile ha sebelisoa phallo ea pele feela ea letoto bakeng sa quantification. Mekhahlelo e lipakeng tsa literene hangata e telele ho feta 30s ho fokotsa phello ea li-synaptic inhibitory kapa litsela tse bonolo. Ho hlahloba hore na karabo ea hChR2 e ne e le monosynaptic, re ile ra sebelisa TTX (1 μM) ho hlapa ho fihlela karabelo ea EPSC e nyamela, ebe re sebelisa 4-aminopyridine (4-AP; 100 μM). Ka tloaelo, karabo e khutlisetsoa ka mor'a metsotso e seng mekae, ka tieho e telele hanyane lipakeng tsa ho thunya ha LED le tlhahiso ea EPSC. NBQX (10 μM) e ne e sebelisetsoa ho lekola hore na karabelo e tsamaisoa ke li-receptor tsa AMPA.
Likarolo tsa Sharp hCO li entsoe joalo ka ha ho hlalositsoe pejana. Ka bokhutšoanyane, likarolo tsa hCO li ile tsa kenngoa ka har'a 4% agarose 'me tsa fetisetsoa liseleng tse nang le 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 le 10 mM -glucose d-0 ho 0 (0) ho 0 (0) µm mocheso oa kamore o sebelisa vibrator ea Leica VT1200 mme e bolokiloe ho ASF mocheso oa kamore. Joale, ho rekota kampong ea lisele tse felletseng ho ile ha etsoa likarolong tsa hCO tlas'a microscope e tobileng ea SliceScope (Scientifica). Likarolo li ne li tšeloa ka aCSF (95% O2 le 5% CO2) le lipontšo tsa sele li ngotsoe mocheso oa kamore. Ho ile ha sebelisoa li-neurone tsa hCO ho sebelisoa pipette ea khalase ea borosilicate e tletseng tharollo e nang le 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, le 0.6 mM EGTA, pH ea ka hare 7, 2, e hlophisitsoeng ka KOH29 mo. Bakeng sa merero ea ho hlaphoheloa, eketsa 0.2% Biocytin ho tharollo ea ka hare.
Lintlha li ile tsa fumanoa ke Clampex (Clampex 11.1, Molecular Devices) ho sebelisoa MultiClamp 700B amplifier (Molecular Devices) le Digidata 1550B digitizer (Molecular Devices), low-pass filtered at 2 kHz, digitized at 20 kHz, and analysis 16 lisebelisoa) le mesebetsi e tloaelehileng ea MATLAB (MATLAB 2019b, Mathworks). Monyetla oa matekoane o ile oa baloa ho sebelisoa JPCalc mme likeno li ile tsa lokisoa ho ea ho bokhoni bo baliloeng ba mateano a -14 mV. Ts'ebetso ea IV e na le letoto la mehato ea hajoale ka mehato ea 5-10 pA ho tloha ho -50 ho isa ho 250 pA.
Bakeng sa tsosoloso ea morphological ea pinched neuron, 0.2% biocytin (Sigma-Aldrich) e ile ea eketsoa tharollong ea ka hare. Lisele li kenngoa bonyane metsotso e 15 ka mor'a ho hacking. Joale pipette e huloa butle-butle bakeng sa 1-2 min ho fihlela lesela le ngolisitsoeng le tiisitsoe ka ho feletseng. Ho latela ts'ebetso ea physiology ea karolo, likarolo li ne li behiloe bosiu bo le bong ho 4 ° C. ho 4% PFA, li hlatsuoa ka PBS X3, 'me li hlapolotsoe 1: 1000 ka streptavidin-conjugated DyLight 549 kapa DyLight 405 (Vector Labs). Lisele tse tlatsitsoeng ka biocytin (2%; Sigma-Aldrich) li ne li ngotsoe nakong ea ho rekota li-patch clamp mocheso oa kamore bakeng sa lihora tse peli. Ka mor'a moo, likarolo li ile tsa behoa holim'a li-slide tsa microscopy tse sebelisang Aquamount (Thermo Scientific) 'me tsa bonoa letsatsing le hlahlamang ka microscope ea Leica TCS SP8 confocal ho sebelisa sepheo sa ho qoelisoa ka oli se nang le sebaka sa linomoro ×40 1.3, magnification ×0.9–1.0, xy. Sekhahla sa sampole ke hoo e ka bang 7 pixels ka micron. Z-stack ka nako ea 1 µm li ile tsa fumanoa ka mokhoa o latellanang, 'me litšoantšo tsa mosaic tsa z-stack le li-auto-stitching li ile tsa etsoa ho koahela sefate sohle sa dendritic sa neuron e' ngoe le e 'ngoe. Li-neurons li ile tsa lateloa ka letsoho ka letsoho ho sebelisa sebopeho sa neuTube 40 'me lifaele tsa SWC li ile tsa hlahisoa. Lifaele li ile tsa kenngoa ho SimpleNeuriteTracer41 Fiji plugin (ImageJ, version 2.1.0; NIH).
Lithipa tsa cortical tsa motho li ile tsa fumanoa ka tumello e nang le tsebo ho latela protocol e amohetsoeng ke Boto ea Tlhahlobo ea Setsi ea Univesithi ea Stanford. Mehlala e 'meli ea lisele tsa motho ka mor'a ho pepa (lilemo tse 3 le tse 18) li ile tsa fumanoa ka ho tlosoa ha cortex e ka pele (bohareng ba gyrus e ka pele) e le karolo ea ho buuoa bakeng sa lefu la sethoathoa se refractory. Ka mor'a ho tsosolosoa, kotula linama ka har'a NMDG-aCSF e batang ka leqhoa e nang le: 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 MM sodium ascorbate 0 mpy3 sodium ascorbate 0 mpy3 sodium ascorbate. 4H2O le 10 mM MgSO4 7H2O. Titrate ho pH 7.3-7.4 le concentrated hydrochloric acid. Li-tissues li ile tsa isoa laboratoring nakong ea metsotso ea 30 'me likarolo tsa coronal li nkoa ho latela mokhoa o hlalositsoeng ka holimo.
Mekhoa eohle ea ho sebetsana le liphoofolo e entsoe ho latela litataiso tsa tlhokomelo ea liphoofolo tse amohetsoeng ke Univesithi ea Stanford APLAC. Likhoto (tse kholo ho feta matsatsi a 140 ka mor'a ho fetisoa) li ile tsa kenngoa ka 5% ea anesthesia ea isoflurane 'me ea e-na le anesthetized ka 1-3% isoflurane intraoperatively. Liphoofolo li ne li kenngoa ka foreimi ea stereotaxic (Kopf) 'me buprenorphine e tsitsitseng (SR) e ne e kenngoa ka tlas'a letlalo. Lehata le pepesitsoe, le hloekisoa 'me ho kenngoa li-screws tsa masapo tse 3-5. Ho lebisa t-hCO, re ile ra hlahisa li-coordinate tsa stereotaxic ho tloha litšoantšong tsa MRI. Ho ile ha chekoa lesoba sebakeng sa thahasello le likhoele (400 µm bophara, NA 0.48, Doric) li ile tsa theoleloa 100 µm ka tlase ho bokaholimo ba hCO 'me tsa tiisoa lehata ka samente ea meno e phekolehang ka UV (Relyx).
Lirekoto tsa fiber photometric li entsoe joalo ka ha ho hlalositsoe pele42. Ho tlaleha liketsahalo tse iketsahallang feela, likhoto li ne li behoa ka har'a lesaka le hloekileng 'me thapo ea 400 µm bophara ba fiber optic patch (Doric) e hoketsoeng ho sistimi ea ho fumana data ea fiber optic photometric e ne e hokahantsoe le thapo e kentsoeng ea fiber optic. Nakong ea ho rekota metsotso e 10 ea ts'ebetso ea likoloi, liphoofolo li ne li lokolohile ho hlahloba lesaka. Ho tlaleha ts'ebetso e tsositsoeng, likhoto (matsatsi a fetang 140 ka mor'a ho fetisoa) li ile tsa hlajoa ka 5% isoflurane bakeng sa induction le 1-3% isoflurane bakeng sa tlhokomelo. Beha phoofolo ka foreimi ea stereotactic (Kopf) 'me litelu ka lehlakoreng le leng la t-hCO li fokotsoe hoo e ka bang 2 cm' me li fetisitsoe ka letlooeng le amanang le piezoelectric actuator (PI). Mohala oa 400 µm fiber optic patch cable (Doric) o ne o hoketsoe ho faeba e kentsoeng 'me o hoketsoe ho sistimi ea ho fumana data. Litelu tse ka lehlakoreng le leng la t-hCO li ile tsa khelosoa ka makhetlo a 50 (2 mm ho 20 Hz, 2 s ka tlhahiso) ka linako tse ling ka koloi ea piezoelectric nakong ea ho rekota metsotso e 20. Sebelisa Sephutheloana sa Tšehetso sa Arduino MATLAB ho laola nako ea ho kheloha ka khoutu ea MATLAB e tloaelehileng. Liketsahalo li amahanngoa le software ea ho fumana data ho sebelisoa transistor-transistor logic (TTL) pulses.
Likhoto (tse kholo ho feta matsatsi a 140 ka mor'a ho fetisoa) li ile tsa kenngoa ka 5% ea anesthesia ea isoflurane 'me ea e-na le anesthetized ka 1-3% isoflurane intraoperatively. Liphoofolo li ne li kenngoa ka har'a foreimi ea stereotaxic (Kopf) le buprenorphine SR le dexamethasone li ne li kenngoa ka tlas'a letlalo. Lehata le pepesitsoe, le hloekisoa 'me ho kenngoa li-screws tsa masapo tse 3-5. Ho lebisa t-hCO, re ile ra hlahisa li-coordinate tsa stereotaxic ho tloha litšoantšong tsa MRI. Craniotomy e chitja (e ka bang bophara ba 1 cm) e entsoe ka ho phunya ka lebelo le phahameng ka kotloloho holim'a hCO e kentsoeng. Hang ha lesapo le le lesesaane ka hohle kamoo ho ka khonehang, empa pele le phunya lesapo lohle, sebelisa forceps ho tlosa lesapo le setseng la pelvic ho senola motheo oa t-hCO. Craniotomy e ne e tletse letsoai le hloekisitsoeng, 'me sekoahelo le phini e khethehileng ea hlooho li ile tsa kenngoa lehatang ka samente ea meno e phekotsoeng ke UV (Relyx).
Ho ile ha etsoa litšoantšo tse nang le lifoto tse peli ho sebelisoa maekorosekopo ea Bruker multiphoton e nang le sepheo sa Nikon LWD (×16, 0.8 NA). Litšoantšo tsa GCaMP6 li entsoe ho 920 nm ka 1.4x e le 'ngoe ea z-plane magnification le 8x karolelano ea 7.5fps. Likhoto li ile tsa hlohlelletsoa ka 5% ea isoflurane anesthesia le ho bolokoa ka 1-3% isoflurane. Likhoto li ne li behiloe ka har'a hlooho e entsoeng ka tloaelo 'me li behoa tlas'a lense. Ho ile ha fumanoa rekoto ea metsotso e 3 ea tšebetso ea makoloi. Nakong ea metsotso e 20 ea ho rekota, likhahla tse 50 (pontšo ka 'ngoe e bolelele ba 100 ms) li ile tsa isoa ka mokhoa o sa reroang ho whisker pad e shebaneng le t-hCO ho sebelisoa picospricer. Sebelisa sephutheloana sa tšehetso sa Arduino MATLAB ho laola nako ea ho phatloha ka khoutu ea MATLAB e tloaelehileng. Amahanya liketsahalo le software ea ho fumana data (PrairieView 5.5) u sebelisa TTL pulses. Bakeng sa tlhahlobo, litšoantšo li ile tsa lokisoa bakeng sa xy motion ho sebelisoa tokiso ea affine lenaneong la MoCo le qalileng Fiji. Ho ntšoa ha mesaletsa ea fluorescent ho tsoa liseleng ka bomong ho sebelisa CNMF-E43. Fluorescence e ile ea ntšoa sebakeng se seng le se seng sa thahasello, ea fetoleloa ho li-curve tsa dF/F, 'me ea fetoleloa ho lintlha tse z.
Likhoto (tse kholo ho feta matsatsi a 140 ka mor'a ho fetisoa) li ile tsa kenngoa ka 5% ea anesthesia ea isoflurane 'me ea e-na le anesthetized ka 1-3% isoflurane intraoperatively. Liphoofolo li ne li kenngoa ka har'a foreimi ea stereotaxic (Kopf) le buprenorphine SR le dexamethasone li ne li kenngoa ka tlas'a letlalo. Litelu tse ka lehlakoreng le leng la t-hCO li ne li khaotsoe hoo e ka bang 2 cm 'me li kenngoa ka har'a letlooeng le amanang le motlakase oa piezoelectric. Lehata le a pepeswa mme le hlwekiswe. Sekurufu sa fatše sa tšepe e sa hloekang se hokeletsoe lehatang. Ho lebisa t-hCO, re ile ra hlahisa li-coordinate tsa stereotaxic ho tloha litšoantšong tsa MRI. Etsa craniotomy e chitja (hoo e ka bang 1 cm ka bophara) ka ho cheka ka lebelo le holimo ka holimo ho t-hCO. Hang ha lesapo le le lesesaane ka hohle kamoo ho ka khonehang, empa pele le phunya lesapo lohle, sebelisa forceps ho tlosa lesapo le setseng la pelvic ho senola motheo oa t-hCO. Lisele ka bomong li ile tsa rekotoa ho sebelisoa likanale tse 32 kapa 64-channel high-density silicon probes (Cambridge Neurotech) tse theiloeng holim'a likurufu tsa fatše le ho holisoa pele ka li-amplifiers tsa RHD (Intan). Sebelisa manipulator ho theola li-electrode ho ea sebakeng se lebisitsoeng ka craniotomy, e tletseng letsoai le nyopa. Pokello ea lintlha e entsoe ka makhetlo a 30 kHz ho sebelisoa mokhoa oa ho fumana data oa Open Ephys. Ho rekota ho ile ha tsoela pele ha feela re bona tšebetso e amanang haholo le morethetho oa boithaopo ho likanale tse fetang 10, ho fana ka maikutlo a hore li-electrode li ne li le ka har'a graft (ho ipapisitse le data ea litšoantšo tsa calcium ea photon tse peli). Ho ile ha fumanoa rekoto ea metsotso e 10 e mabapi le tšebetso ea makoloi. Litelu tse ka lehlakoreng le leng la t-hCO li ile tsa khelosoa ka makhetlo a 50 (2 mm ho 20 Hz, 2 s ka tlhahiso) ka linako tse ling ka koloi ea piezoelectric nakong ea ho rekota metsotso e 20. U sebelisa Sephutheloana sa Ts'ehetso sa MATLAB bakeng sa Arduino (MATLAB 2019b), laola nako ea ho kheloha ka khoutu e tloaelehileng ea MATLAB. Sebelisa li-pulses tsa TTL ho hokahanya liketsahalo le software ea ho fumana data.
Bakeng sa liteko tsa ho tšoaea mahlo, mohala oa 200 µm optical patch (Doric) o hokahaneng le laser ea 473 nm (Omicron) o ne o hoketsoe ho 200 µm optical fiber e behiloeng holim'a craniotomy. Hang-hang pele ho sena, fetola matla a jumper ho 20 mW. Sebelisa manipulator ho theola li-electrode ho ea sebakeng se lebisitsoeng ka craniotomy, e tletseng letsoai le nyopa. Qalong ea ho rekota, ho ile ha ntšoa likhahla tse leshome tsa leseli la 473 nm (khafetsa 2 Hz, nako ea pulse 10 ms). Lisele tsa photosensitive li ne li hlalosoa e le lisele tse bonts'ang karabelo ea spike ka har'a 10 ms ea leseli ho 70% kapa ho feta ea liteko.