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Maturation na ntinye nke akụkụ ahụ cortical mmadụ transplanted

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Akụkụ akwara ndị na-agbakọta onwe ha na-anọchite anya ikpo okwu in vitro na-ekwe nkwa maka imepụta mmepe na ọrịa mmadụ. Otú ọ dị, organoids enweghị njikọ nke dị na vivo, nke na-egbochi ntozu okè ma na-egbochi ịbanye na sekit ndị ọzọ na-achịkwa omume. N'ebe a, anyị na-egosi na cortical organoids sitere na mkpụrụ ndụ mmadụ na-atụgharị n'ime somatosensory cortex nke ụmụ oke nwa ọhụrụ na-etolite ụdị cell tozuru okè nke na-ejikọta na sekit metụtara mmetụta uche na mkpali. MRI kpughere uto organoid transplant post-transplant n'ọtụtụ ahịrị sel na anụmanụ, ebe nyocha otu-isi gosipụtara ọganihu nke corticogenesis na mpụta nke mmemme ndegharị dabere na ọrụ. N'ezie, neurons cortical transplanted na-egosipụta ihe mgbagwoju anya morphological, synapti na ime akpụkpọ ahụ karịa ndị ibe ha in vitro, na-enye ohere ịchọpụta ntụpọ neuronal na ndị ọrịa nwere ọrịa Timoti. Nchọgharị anatomical na arụ ọrụ egosila na akụkụ ahụ ndị a tụgharịrị na-enweta ntinye thalamocortical na corticocortical, na ndekọ vivo nke ọrụ akwara na-atụ aro na ntinye ndị a nwere ike iwepụta nzaghachi mmetụta na sel mmadụ. N'ikpeazụ, cortical organoids na-agbatị axon n'ime ụbụrụ oke, na ọrụ optogenetic ha na-eduga n'omume ịchọ ụgwọ ọrụ. Ya mere, neurons cortex mmadụ a tụgharịrị na-eto eto ma sonye na sekit onye ọbịa na-achịkwa omume. Anyị na-atụ anya na ụzọ a ga-eme ka nchọpụta nke strand-level phenotypes na mkpụrụ ndụ ndị ọrịa enwetara nke a na-apụghị ịchọta site n'ụzọ ndị ọzọ.
Ụbụrụ mmadụ na-emepe emepe bụ usoro nhazi onwe onye dị ịrịba ama nke mkpụrụ ndụ na-amụba, dị iche iche, ịkwaga, na jikọọ iji mepụta sekit neuronal na-arụ ọrụ nke na-emeziwanye site na ahụmahụ mmetụta. Nsogbu bụ isi n'ịghọta mmepe ụbụrụ mmadụ, karịsịa n'ihe gbasara ọrịa, bụ enweghị ohere ịnweta anụ ahụ ụbụrụ. Organelles na-ahazi onwe ya, gụnyere mmadụ cortex organoids (hCO; nke a makwaara dị ka akụkụ cortex mmadụ), nwere ike ịmepụta 2,3,4,5,6. Otú ọ dị, ọtụtụ njedebe na-ejedebe ngwa ha sara mbara iji ghọta mmepe na ịrụ ọrụ nke sekit akwara. Karịsịa, amabeghị ma hCO maturation na-ejedebe site na enweghị ụfọdụ ntinye microenvironmental na sensọ dị na vivo. Na mgbakwunye, n'ihi na hCO adịghị etinye n'ime sekit nke nwere ike ịmepụta nsonaazụ omume, uru ha na-emepụta ihe mgbagwoju anya na ọrịa neuropsychiatric omume dị ugbu a.
Ntugharị nke hCO n'ime ụbụrụ dị ndụ na-adịghị mma nwere ike imeri njedebe ndị a. Nnyocha ndị gara aga egosila na akwara mmadụ na-atụgharị n'ime cortex rodent na-enwe ike ịlanarị, rụọ ọrụ, na ikwurịta okwu na mkpụrụ ndụ òké7,8,9,10,11,12. Otú ọ dị, a na-emekarị nnwale ndị a na anụmanụ ndị toro eto, nke nwere ike igbochi njikọ synapti na axonal. N'ebe a, anyị na-akọwa usoro ntụgharị nke anyị tụgharịrị 3D hCO ewepụtara site na mkpụrụ ndụ hiPS n'ime cortex somatosensory bụ isi (S1) nke oke oke immunodeficient n'oge mmalite nke mmepe rọba. Neuron hCO (t-hCO) tụgharịrị na-enweta ntozu oke, na-enweta ntinye thalamocortical na cortical-cortical nke na-enweta nzaghachi mmetụta uche, na ịgbatị amụma axonal n'ime ụbụrụ oke iji kwalite omume ịchọ ụgwọ ọrụ. Mmụba nke t-hCO ekpughere ntụpọ neuronal na ndị ọrịa nwere ọrịa Timoti (TS), ọrịa mkpụrụ ndụ ihe nketa siri ike nke sitere na mmụgharị na chanel L-ụdị CaV1.2 calcium chanel (nke CACNA1C debere ya).
Iji mụọ neurons cortical mmadụ na sekit na vivo, anyị stereotactically transplanted 3D hCO emebibeghị n'ime S1 nke mmalite postnatal athymic oke (ụbọchị 3-7 postnatally) (Fig. 1a na gbasaa data nke Fig. 1a-c). N'ebe a, thalamocortical na corticocortical axonal projections emechabeghị S1 innervation ha (ref. 13). Ya mere, a na-eme usoro a iji mee ka ntinye t-hCO dịkwuo elu ma na-ebelata mmetụta na sekit endogenous. Iji jiri anya nke uche hụ ebe t-hCO dị na anụmanụ dị ndụ, anyị mere T2-weighted MRI ụbụrụ reconstructions nke oke 2-3 ọnwa mgbe transplantation (Fig. 1b na ogologo data, Fig. 1d). A na-ahụta t-hCO ngwa ngwa na nha olu nke t-hCO yiri nke a gbakọrọ site na mpekere edozi (Felụ Data Extended. 1d,e; P> 0.05). A na-ahụta t-hCO ngwa ngwa na nha olu nke t-hCO yiri nke a gbakọrọ site na mpekere edozi (Felụ Data Extended. 1d,e; P> 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO dannыe, 1d, e; A na-ahụta t-hCO n'ụzọ dị mfe, na nha t-hCO volumetric yiri nke a gbakọrọ maka ngalaba edozi (data gbasaa, Fig. 1d, e; P> 0.05).很容易观察到t-hCO，并且t-hCO的体积测量值与从固定切片计算的测量值相似（扩展数据图1d、e；P > 0.05）。很容易观察到t-hCO，并且t-hCO t-hCO легко наблюдался, а объемные измерения t-hCO dannыe, 1d, e; t-hCO dị mfe na-ahụ anya, na nha t-hCO volumetric yiri nke a gbakọrọ maka ngalaba edozi (data gbasaa, Fig. 1d, e; P> 0.05).Anyị kpebisiri ike t-hCO na 81% nke anụmanụ transplant ihe dị ka ọnwa 2 ka a tụgharịrị ya (n = anụmanụ 72; hCO sitere na ahịrị cell 10 hiPS; ahịrị cell hiPS na tebụl mgbakwunye 1). N'ime ndị a, 87% dị na ụbụrụ ụbụrụ (Fig 1c). Site n'ịme nyocha MRI na-enyocha n'ọtụtụ oge n'otu oke a na-atụgharị, anyị hụrụ mmụba itoolu na t-hCO olu n'ime ọnwa 3 (Fig 1d na gbasaa data, Fig. 1f). Anụmanụ ndị a na-atụgharị nwere nnukwu nlanarị (74%) na ọnwa 12 ka a na-atụgharị ya (data gbasasịrị, Fig. 1g na Tebụl mgbakwunye 2), na enweghị moto ma ọ bụ ihe nchekwa nchekwa, gliosis, ma ọ bụ electroencephalogram (EEG) hụrụ. Ihe oyiyi data 1g na tebụl mgbakwunye 2). 1h-m na 3e).
a, Nhazi nke nnwale. A tụgharịrị hCO sitere na sel hiPS n'ime S1 nke oke gba ọtọ mụrụ ọhụrụ na ụbọchị 30-60 nke ọdịiche. b, T2-dị arọ coronal na onyonyo MRI kwụ ọtọ na-egosi t-hCO na S1 2 ọnwa mgbe transplantation gasịrị. Ogwe ọnụ ọgụgụ, 2 mm. c, Ọnụ ọgụgụ nke ịga nke ọma nke engraftment egosiri maka ahịrị sel hiPS ọ bụla (n = 108, ọnụọgụ dị n'ime ụlọ mmanya na-egosi ọnụọgụ t-hCO kwa ahịrị sel hIPS) yana ọnọdụ cortical ma ọ bụ subcortical (n = 88). d, MRI oyiyi nke akwara akwara ọbara (n'aka ekpe; ọnụ ọgụgụ dị elu, 3 mm) na 3D volumetric reconstruction (ogwe ọnụ ọgụgụ, 3 mm) na-egosi mmụba na t-hCO karịa ọnwa 3. e, Nyochaa ụkpụrụ t-hCO na ụbụrụ ụbụrụ oke. Ogwe ọnụ ọgụgụ, 1 mm. f, Ihe nnọchianya immunocytochemical nke t-hCO gosiri site n'elu aka ekpe gaa n'aka nri (n'oge dị iche iche): PPP1R17 (4 ọnwa), NeuN (8 ọnwa), SOX9 na GFAP (8 ọnwa), PDGFRα; (ọnwa 8), MAP2 (ọnwa asatọ) na IBA1 (ọnwa asatọ). Ogwe ọnụ ọgụgụ, 20µm. Nkọwa ọnụ nke HNA na-egosi sel sitere na mmadụ. g, snRNA-seq: Njikọ dị n'otu na ntule (UMAP) nha nha nha nha nha nke t-hCO nuclei dị elu mgbe njikọta Seurat (n = 3 t-hCO samples, n = 2 ahịrị cell hiPS). Astrocytes, sel nke eriri astrocyte; cyc prog, na-ekesa progenitors; GluN DL, neurons glutamatergic miri emi; GluN DL/SP, neurons glutamatergic miri na sublamellar; GluN UL, akwara elu glutamatergic neurons; oligodendrocytes, oligodendrocytes; OPC, mkpụrụ ndụ progenitor oligodendrocyte; RELN, reelin neurons. h, Gene Ontology (GO) nyocha okwu ịba ụba nke mkpụrụ ndụ ihe nketa nke ukwuu (gbanwee P <0.05, mgbanwe mgbanwe> 2, gosipụtara na opekata mpe 10% nke nuclei) na t-hCO glutamatergic neurons ma e jiri ya tụnyere hCO glutamatergic neurons. h, Gene Ontology (GO) nyocha okwu ịba ụba nke mkpụrụ ndụ ihe nketa nke ukwuu (gbanwee P <0.05, mgbanwe mgbanwe> 2, gosipụtara na opekata mpe 10% nke nuclei) na t-hCO glutamatergic neurons ma e jiri ya tụnyere hCO glutamatergic neurons. h, Анализ обогащения терминов Gene Ontology (GO) для генов со значительной 2. hCO. h, Gene Ontology (GO) nyocha okwu ịba ụba maka mkpụrụ ndụ ihe nketa nwere ọrụ dị ịrịba ama (gbanwee P<0.05, mgbanwe mgbanwe> 2, nkwupụta na opekata mpe 10% nuclei) na t-hCO glutamatergic neurons ma e jiri ya tụnyere hCO glutamatergic neurons. h，与hCO 谷氨酸能神经元相比，t-hCO 谷氨酸能神经元中基因显着上调相比，t-hCO 谷氨酸能神经元中基因显着上调相比，t-hCO 2,在至少10% 的细胞核中表达）的基因本体论(GO) 术语富集分析。 h .变化> 2术语富集分析. h. ядер) в глутаматергических нейронах t-hCO по сравнению обогащения. h, a na-emezigharị mkpụrụ ndụ ihe nketa (gbanwee P <0.05, mgbanwe mgbanwe> 2, gosipụtara na opekata mpe 10% nke nuclei) na t-hCO glutamatergic neurons ma e jiri ya tụnyere hCO glutamatergic neurons Ontological (GO) nyocha nke okwu nkwalite.Ahịrị nwere ntụpọ na-egosi uru aq nke 0.05. i, onyonyo UMAP nke ụdị cell GluN dị na t-hCO site na iji mbufe labelụ site na ntụaka 22 snRNA-seq okenye cortex dataset. CT - mkpụrụ ndụ corticothalamic, ET - sel extracerebral, IT - cell telencephalic cell, NP - nso ntule.
Anyị wee nyochaa cytoarchitecture na mkpokọta cellular mejupụtara t-hCO. Mgbochi mgbochi nke mkpụrụ ndụ endothelial oke gosipụtara vascularization na t-hCO, ebe ntụpọ IBA1 gosipụtara ọnụnọ nke microglia oke n'oge graft (Fig 1f na data gbasaa, Fig. 3c, d). Immunostaining kpughere antigen nuklia mmadụ (HNA) dị mma na-egosipụta PPP1R17 (cortical progenitors), NeuN (neurons), SOX9 na GFAP (sel glial enwetara) ma ọ bụ PDGFRα (oligodendrocyte progenitors) (Nyocha 1f). Iji mụọ ihe mejupụtara cellular nke t-hCO na mkpebi otu cell, anyị rụrụ usoro RNA otu-isi (snRNA-seq) ka ọnwa 8 gachara nkewa. Nnukwu filtration na mwepụ nke oke oke nyere 21,500 maapụ mononuclear mmadụ dị elu (Fig 1g na data gbasaa, Fig. 4a,b). Usoro ngosipụta nke ihe nrịbama ụdị cell na-ahụkarị na-achọpụta ụyọkọ nke klaasị sel cortical, gụnyere akwara glutamatergic miri emi na nke elu, ndị nna na-ekesa, oligodendrocytes, na usoro ọmụmụ astrocyte (Fig. 1g, data gbasaa, Fig. 4c, na Tebụl mgbakwunye 3). Immunostaining maka SATB2 na CTIP2 gosiri na n'agbanyeghị na ọnụnọ nke cortical subtypes, t-hCO egosighi doro anya nke anatomical stratification (data gbasaa, Fig. 3a). snRNA-seq hCO na-ejikọta ọnụ na-emepụta klas sel ndị yiri ya nke ukwuu, na ihe ole na ole, gụnyere enweghị oligodendrocytes na ọnụnọ nke neurons GABAergic, nke nwere ike igosi ọnọdụ dị mma na vitro na mbụ maka cell progenitor sel15 (gbasaa data, Fig. 4f - i na Tebụl mgbakwunye 4). Nyocha okwu mkpụrụ ndụ dị iche iche gosipụtara ọdịiche dị ukwuu na neurons glutamatergic n'etiti t-hCO na hCO (Ntinye mgbakwunye 5), gụnyere ịgbalite usoro nke mkpụrụ ndụ ihe nketa jikọtara ya na maturation neuronal dị ka akara synapti, mpaghara dendritic, na ọrụ ọwa voltaji-gated (Nyocha 1h na Tebụl mgbakwunye 5). okpokoro 6). N'ihi nke a, neurons cortical glutamatergic t-hCO gosipụtara ntozu oke ederede.
Iji kọwapụta ma mgbanwe mgbanwe ndị a na t-hCO metụtara morphological ọdịiche dị n'etiti hCO in vitro na t-hCO na vivo, anyị wughachiri ogbo-dakọtara biocytin jupụtara hCO na hCO na nnukwu ngalaba mgbe ọnwa 7-8 gachara. hCO neurons (Fig 2a). t-hCO neurons buru ibu nke ukwuu, nwere 1.5 ugboro nke soma dayameta, okpukpu abụọ nke dendrites, yana mmụba okpukpu isii na ngụkọta ogologo dendritic ma e jiri ya tụnyere in vitro hCO (Fig 2b). Na mgbakwunye, anyị hụrụ njupụta dị elu nke spines dendritic na t-hCO neurons karịa na hCO neurons (Fig 2c). Nke a na-egosi na t-hCO neurons na-enweta nnukwu dendritic elongation na branching, nke, na-ejikọta ya na mgbasawanye cell na-aga n'ihu, nwere ike inye aka na uto siri ike nke t-hCO mgbe transplantation (Fig 1d na Extended Data Fig. 1f). Nke a mere ka anyị nyochaa njirimara electrophysiological. Ike nke membrane ji okpukpu asatọ dị elu (data gbasaa, Fig. 8d), ikike izu ike na-eme ka akpụkpọ ahụ dịkwuo elu (ihe dị ka 20 mV), na injections nke ugbu a na-ebute oke mkpali dị elu na t-hCO neurons karịa na hCO neurons. in vitro (Fig. 2d), e), nke kwekọrọ na nnukwu na mgbagwoju anya morphological atụmatụ nke t-hCO. Tụkwasị na nke ahụ, ugboro ole nke postsynaptic na-eme ihe na-akpali akpali (EPSC) dị elu nke ukwuu na t-hCO neurons (Fig. 2f), na-atụ aro na ụbara njupụta nke spines dendritic a na-ahụ na t-hCO neurons jikọtara ya na ọrụ excitability. mmekọahụ synapse. Anyị kwadoro àgwà akabeghị aka nke hCO neurons in vitro site na ịdekọ aha neurons glutamatergic (data gbasaa, Fig. 6a-c).
a, 3D nwughari nke biocytin jupụtara hCO na t-hCO neurons mgbe ọnwa 8 gasịrị. b, Ọnụ ọgụgụ nke njirimara morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 na *** P <0.0001). b, Ọnụ ọgụgụ nke njirimara morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 na *** P <0.0001). б. b, ọnụọgụ nke njirimara morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, na *** P <0.0001). b，形态学特征的量化（n = 8 b，形态学特征的量化（n = 8 б. b, ọnụọgụ nke njirimara morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, na *** P <0.0001).c, 3D nwughari nke hCO na t-hCO dendritic alaka mgbe ọnwa 8 nke iche. Asterisks na-acha uhie uhie na-egosi spines dendritic putative. Dendritic spine njupụta quantification (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0092). d, Ọnụ ọgụgụ nke akpụkpọ ahụ izu ike (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). d, Ọnụ ọgụgụ nke akpụkpọ ahụ izu ike (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). d. d, akpụkpọ ahụ izu ike nke nwere ike ịkọwapụta (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). d，静息膜电位的量化（n = 25 hCO 神经元，n = 16 t-hCO 神经元；***P <0.0001）. d，静息膜电位的量化（n = 25 hCO 神经元，n = 16 t-hCO 神经元；***P <0.0001）. d. d, akpụkpọ ahụ izu ike nke nwere ike ịkọwapụta (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, Mmeghachi omume ugboro ugboro nwere ike ịgbanye na hCO na t-hCO kpatara site na ịba ụba injections ugbu a, na ọnụọgụ nke ọnụọgụ ọkụ kachasị (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, Mmeghachi omume ugboro ugboro nwere ike ịgbanye na hCO na t-hCO kpatara site na ịba ụba injections ugbu a, na ọnụọgụ nke ọnụọgụ ọkụ kachasị (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, повторное возбуждение потенциала действия в hCO na t-hCO. скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, ihe nwere ike imegharị ọkụ na hCO na t-hCO kpatara site na mmụba ugbu a na ọnụọgụ nke ọnụọgụ ọkụ kachasị (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e，通过加电流注入诱导的hCO 和t-hCO 重复动作电位放电，以及最大放电率的量媾神经元，n = 16 个t-hCO 神经元；***P <0.0001）。 E 通过 增加 电流 注入 的 hco 和 t-hco 重复 电位 放电, n = 16 个 t-hco 神经 ； *** p <0.0001） . e. максимальной скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, mgbagha ugboro ugboro nke hCO na t-hCO nwere ike ime site na mmụba dị ugbu a na ọnụọgụ nke ọnụọgụ ọkụ kachasị (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, EPSC na-enweghị atụ (sEPSCs) na hCO na t-hCO neurons na ọnwa 8 nke ọdịiche, na ọnụọgụ nke ugboro nke ihe omume synaptik (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) na hCO na t-hCO neurons na 8 ọnwa nke ọdịiche, na quantification nke ugboro nke synaptik ihe omume (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001) . f, спонтанные EPSC (sEPSC) в нейронах hCO и t-hCO через 8 событий (n = 25 нейронов hCO, n = 17 нейронов t-hCO; *** P <0,0001) . f, EPSCs ozugbo (sEPSCs) na hCO na t-hCO neurons na ọnwa 8 nke ọdịiche na ọnụọgụ nke ọnụọgụ ihe omume synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f，分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs 17 t-hCO 神经元； *** P <0.0001）. f,分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs (sEPSCs)神率的量匼（n = 25 hCO 神率的量匼（n = 25hCO P <0.0001）. f, спонтанные EPSC (sEPSC) в нейронах hCO и t-hCO через 8 событий (n = 25 нейронов hCO, n = 17 нейронов t-hCO; *** P <0,0001). f, EPSC ozugbo (sEPSCs) na hCO na t-hCO neurons na ọnwa 8 nke ọdịiche na ọnụọgụ nke ọnụọgụ ihe omume synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001).Maka bf, hCO na t-hCO n'ahịrị 1208-2 ka ewepụtara site na otu ihe dị iche iche edobere n'otu. g, Gene set enrichment analysis (nnwale nke otu akụkụ Fisher kpọmkwem) nke mkpụrụ ndụ ihe nketa na-akwalite nke ọma (gbanwee P <0.05, mgbanwe mgbanwe> 2, gosipụtara na opekata mpe 10% nke nuclei) na t-hCO glutamatergic neurons ma e jiri ya tụnyere hCO glutamatergic neurons na usoro mkpụrụ ndụ ihe nketa nke ma ọ bụ nzaghachi mbụ (RG) Achọpụtara site na nyocha in vivo òké16 na mmadụ-kpọmkwem LRGs sitere na in vitro neurons17. g, Gene set enrichment analysis (nnwale nke otu akụkụ Fisher kpọmkwem) nke mkpụrụ ndụ ihe nketa na-akwalite nke ọma (gbanwee P <0.05, mgbanwe mgbanwe> 2, gosipụtara na opekata mpe 10% nke nuclei) na t-hCO glutamatergic neurons ma e jiri ya tụnyere hCO glutamatergic neurons na usoro mkpụrụ ndụ ihe nketa nke ma ọ bụ nzaghachi mbụ (RG) Achọpụtara site na nyocha in vivo òké16 na mmadụ-kpọmkwem LRGs sitere na in vitro neurons17. g, анализ обогащения набора генов. P <0,05, kratnost изmenenya > 2, ihe eji eme ihe na 10% nkeji) глутаматергическими нейронами hCO наборы генов как ранего (ERG), так и позднего (LRG) генов, зависящитки, идентифицированных в исследовании на мыshах in vivo16, и специфических для человека LRG из нейронов in vivo17. g, nyocha nke gene set enrichment (nnwale otu-tailed Fisher kpọmkwem) nke mkpụrụ ndụ ihe nketa nwere ọrụ dị ịrịba ama (gbanwee P<0.05, mgbanwe mgbanwe> 2, okwu na opekata mpe 10% nke nuclei) na t-hCO glutamatergic neurons ma e jiri ya tụnyere hCO glutamatergic neurons sets nke ma mmalite (ERG) na mbubreyo (ERG) 6 na ọrụ mbubreyo 6. LRG nke mmadụ kpọmkwem sitere na neurons na 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 上船， 倍数> 2 ， 至少 至少 10%研究 中 的 早期 反应 反应 反应 和 晚期 反应 反应 (lrg) 活性 基因 1神经元 17 中 中 17 中 17的人类特异性LRG。 g, глутаматергические нейроны t-hCO были значительно активизированы ( скоррректированный P<0,05, кратность изменения> 2, na 10% Анализ обогащения набора генов Фишера) раннего ответа (ERG) и позднего гены, зависящие от активности ответа (LRG), мыshах na vivo16 na нейронах in vitro17. g, t-hCO glutamatergic neurons na-emeziwanye nke ọma ma e jiri ya tụnyere hCO glutamatergic neurons (gbanwee P<0.05, mgbanwe mgbanwe> 2, ma ọ dịkarịa ala 10% nzaghachi mbụ (ERG) na nzaghachi nzaghachi mbubreyo mkpụrụ ndụ mkpụrụ ndụ (nnwale nke otu ọdụ Fisher) mmeghachi omume na-adabere na mkpụrụ ndụ ihe nketa (LRGs) nke a chọpụtara na vitro 6. Ndị LRG.Ahịrị nwere ntụpọ na-egosi uru Bonferroni emeziri P nke 0.05. h, GluN mkpụrụ ndụ ihe nketa (pseudo-package na scaling nke mkpụrụ ndụ ihe nketa ọ bụla) ka edoziri nke ọma na snRNA-seq nke mkpụrụ ndụ ihe nketa LRG na t-hCO glutamatergic neurons. i, immunostaining na-egosi SCG2 okwu na t-hCO (elu) na hCO (ala) neurons. Àkụ́ ọcha na-atụ aka na sel SCG2+. Ogwe ọnụ ọgụgụ, 25µm. A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ.
Dabere na mmụba nke t-hCO a hụrụ na mpekere ex vivo, snRNA-seq kpughere ngbanwe nke mkpụrụ ndụ ihe nketa dabere na ọrụ na t-hCO ma e jiri ya tụnyere hCO in vitro. Glutamatergic t-hCO neurons gosipụtara ọkwa dị elu nke mkpụrụ ndụ ihe nketa na-achịkwa ọrụ nzaghachi mbubreyo (Fig 2g, h), nke ahụrụ na ọmụmụ gara aga na òké na neurons mmadụ16,17. Dịka ọmụmaatụ, BDNF18, SCG2, na OSTN, mkpụrụ ndụ ihe nketa na-achịkwa ọrụ primate, gosipụtara mmụba dị ukwuu na t-hCO neurons ma e jiri ya tụnyere hCO neurons (Fig. 2g-i). Ya mere, neurons t-hCO gosipụtara njiri mara ntozu ma e jiri ya tụnyere akwara hCO site na nyocha ederede, morphological, na arụmọrụ.
Iji nyochaa njikọ nke t-hCO maturation na ụbụrụ ụbụrụ mmadụ, anyị mere ntụnyere transcriptomic nke ụdị nwa ebu n'afọ na ndị okenye cortical cell19,20 na okenye21,22 yana nnukwu data na okwu cortical gene expression23 n'oge mmepe (gbasaa data, Fig. 5). ). na ọrụ gara aga 24, ọnọdụ hCO zuru ụwa ọnụ na t-hCO transcriptome maturation na ọnwa 7-8 nke ọdịiche dị n'ụzọ sara mbara na-adabere na oge mmepe nke vivo ma bụrụ nke kachasị na ndụ nwa ebu n'afọ (Extended Data Fig. 5a). N'ụzọ doro anya, anyị hụrụ mmụba transcriptome ntozu na t-hCO ma e jiri ya tụnyere afọ hCO dakọtara, yana ịgbalite transcriptome metụtara synaptogenesis, astrogenesis, na myelination (data gbasaa, Fig. 5b-d). N'ọkwa cellular, anyị chọtara ihe akaebe nke subtype cortex dị gịrịgịrị na t-hCO, yana ụyọkọ nke neurons glutamatergic jikọtara ya na ndị okenye L2/3, L5, na L6 neuron subtypes (Nyocha 1i). N'ụzọ dị iche, nchịkọta ụyọkọ n'etiti glutamatergic t-hCO neurons na ụbụrụ cortical nwa ebu n'afọ bụ nke nwere oke n'etiti ime ime (data gbasasịrị, foto 5e-j). Iji chọpụta ma t-hCO neurons na-arụ ọrụ yiri nke mmadụ postnatal neocortical neurons, anyị mere ndekọ electrophysiological na nhazigharị nke mmadụ L2 / 3 pyramidal neurons na akụkụ dị nkọ nke cortex postnatal mmadụ (gbasaa data, Fig. 7a). Njirimara electrophysiological nke L2 / 3 pyramidal neurons yiri nke t-hCO pyramidal neurons (data gbasaa, Fig. 7e). Morphologically, L2 / 3 neurons si na postnatal mmadụ samples yiri t-hCO karịa hCO, ọ bụ ezie na L2 / 3 mkpụrụ ndụ dị ogologo, nwere ọtụtụ alaka n'ozuzu, ma nwee nnukwu ọkpụkpụ azụ (Fig 3g na data gbasaa, Fig. 7b-). G).
a, ntụgharị nke hCO mepụtara site na njikwa yana ahịrị cell TS hiPS n'ime oke ụmụ ọhụrụ. b, Nrụgharị 3D nke neurons t-hCO jupụtara biocytin mgbe ọnwa 8 gasịrị. c, quantification nke ogologo dendritic pụtara (n = 19 nchịkwa nchịkwa, n = 21 TS neurons; ** P = 0.0041). d, 3D-arụzigharị alaka dendritic site na njikwa na TS t-hCO na ọnwa 8 nke ọdịiche, na ọnụọgụ nke dendritic spine density (n = 16 akara neurons, n = 21 TS neurons, *** P <0.0001). d, 3D-arụzigharị alaka dendritic site na njikwa na TS t-hCO na ọnwa 8 nke ọdịiche, na ọnụọgụ nke dendritic spine density (n = 16 akara neurons, n = 21 TS neurons, *** P <0.0001). d. плотности дендритных шипов (n = 16 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, 3D nwughari nke alaka dendritic site na njikwa na t-hCO TS na ọnwa 8 nke ọdịiche na dendritic spine density quantification (n = 16 akara neurons, n = 21 TS neurons, *** P <0.0001). d，分化8 个月时对照和TS t-hCO 21 个TS 神经元，***P <0.0001）。 d 分化 8 个 时 对照 和 ts t-hco 的 3d 重建 分支 分支 以及元 , n = 21 个 ts 神经 ， *** p <0.0001 ）. d. плотности дендритных шипов (n = 16 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, 3D nwughari nke ngalaba dendritic njikwa na TS t-hCO na ọnwa 8 nke ọdịiche na dendritic spine density quantification (n = 16 akara neurons, n = 21 TS neurons, *** P <0.0001).Asterisks na-acha uhie uhie na-egosi spines dendritic putative. e, EPSC na-akpaghị aka na njikwa na TS t-hCO neurons mgbe ọnwa 8 gasịrị. f, nchịkọta ugboro ugboro na ọnụọgụ nke ugboro na njupụta nke ihe omume synapti (n = 32 nchịkwa neurons, n = 26 TS neurons; ** P = 0.0076 na P=0.8102). g, Nyocha Scholl nke TS na nchịkwa nchịkwa na hCO na t-hCO. Ahịrị ndị gbajiri agbaji na-egosi neurons pyramidal L2/3 postnatal maka ntụnyere (n = 24 akara t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 akara hCO neurons, na n = 7 TS hCO neurons). A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ
Ikike nke t-hCO iji megharịa ọdịdị ọdịdị na arụ ọrụ nke neurons cortex mmadụ na ọkwa dị elu kpaliri anyị ịchọpụta ma enwere ike iji t-hCO chọpụta ọrịa phenotypes. Anyị lekwasịrị anya na TS, ọrịa neurodevelopmental siri ike kpatara site na mmụgharị uru na-arụ ọrụ na mkpụrụ ndụ ihe nketa na-edobe CaV1.2, nke na-amalite ndegharị mkpụrụ ndụ dabere na ọrụ na neurons. Anyị nwetara hCO site n'aka ndị ọrịa TS atọ na-ebu ihe nnọchi anya (p.G406R) na njikwa atọ (Fig 3a). Mgbe transplant, anyị chọpụtara na morphology dendritic gbanwere na TS neurons ma e jiri ya tụnyere njikwa (Fig. 3b na gbasaa data, Fig. 8a,b), na-amụba okpukpu abụọ na ọnụ ọgụgụ nke dendrites bụ isi na mmụba n'ozuzu ya na mbelata ogologo ogologo dendritic (Fig 3c na ogologo data, Fig. 8c). Ejikọtara nke a na njupụta nke spines na ụbara ugboro ugboro nke EPSC na-apụtaghị ìhè na TS ma e jiri ya tụnyere nchịkwa nchịkwa (Fig 3d-f na data gbasaa, Fig. 8g). Ntụle ọzọ gosipụtara usoro nke dendritic branching na-adịghị mma na t-hCO TS ma e jiri ya tụnyere njikwa, ma ọ bụghị na vitro TS hCO na ọkwa yiri nke dị iche iche (Fig 3g). Nke a kwekọrọ na akụkọ anyị gara aga nke mbelata dendritic na-adabere na ọrụ na TS ma gosipụta ike nke ikpo okwu a na-atụgharị iji chọpụta ọrịa phenotypes na vivo.
Anyị jụrụ ruo ókè sel t-hCO na-arụ ọrụ n'ime oke S1. S1 na rodents na-enweta ntinye synaptik siri ike site na ipsilateral ventral basal na posterior thalamic nuclei, yana moto ipsilateral na nke abụọ somatosensory cortices, na contralateral S1 (Fig. 4a). Iji weghachi ụkpụrụ innervation, anyị butere hCO ọrịa rabies virus-dG-GFP/AAV-G wee tụnye hCO n'ime oke S1 ụbọchị 3 ka e mesịrị. Anyị hụrụ nnukwu okwu GFP na neurons nke ipsilateral S1 na ventral basal ganglia 7-14 ụbọchị mgbe transplantation (Fig. 4b, c). Na mgbakwunye, mgbochi mgbochi nke thalamic markker netrin G1 kpughere ọnụnọ nke njedebe thalamic na t-hCO (Fig. 4d, e). Iji nyochaa ma amụma afferent ndị a nwere ike iwepụta nzaghachi synaptik na sel t-hCO, anyị mere ndekọ sel niile sitere na sel mmadụ n'akụkụ dị nkọ nke oyi akwa thalamocortical. Mkpali eletriki nke oke S1, capsule dị n'ime, ihe ọcha, eriri dị nso t-hCO ma ọ bụ mmalite optogenetic nke opsin na-egosipụta njedebe thalamic na t-hCO butere EPSC dị mkpụmkpụ na t-hCO neurons kpughere AMPA antagonist NBQX. (Fig 4f, g na data agbatị, Fig. 9a-g). Data ndị a na-egosi na t-hCO na-ejikọta ya n'ụzọ anụ ahụ n'ime ụbụrụ oke ma nwee ike ịgbalite ya site na anụ ahụ ndị ọbịa oke.
a, Eserese eserese nke nnwale nsuso rabies. b, GFP na okwu STEM121 nke mmadụ kpọmkwem n'etiti t-hCO na oke ụbụrụ ụbụrụ (panel elu). Egosikwara bụ okwu GFP n'ime rat ipsilateral ventral basal nucleus (VB) (nke dị n'aka ekpe) na ipsilateral S1 (aka nri ala). Ogwe ọnụ ọgụgụ, 50µm. Oghere uhie na-anọchi anya akụkụ ụbụrụ ebe e sere foto. c, ọnụọgụ mkpụrụ ndụ na-egosipụta GFP (n = 4 oke). d, e - Netrin G1+ thalamic ọnụ na t-hCO. d na-egosi ngalaba coronal nwere t-hCO na VB nuclei. Ogwe ọnụ ọgụgụ, 2 mm. e na-egosi Netrin G1 na STEM121 okwu na t-hCO (aka ekpe) na VB (n'aka nri) neurons. Ogwe ọnụ ọgụgụ, 50µm. Ahịrị nwere ntụpọ oroma na-egosi oke t-hCO. f, g, Ihe ndị dị ugbu a nke t-hCO neurons mgbe mkpali eletriki dị na S1 rat (f) ma ọ bụ capsule n'ime (g), na (acha odo odo) ma ọ bụ na-enweghị (nwa) NBQX (aka ekpe). EPSC amplitudes na na enweghị NBQX (n = 6 S1 neurons, * P = 0.0119; na n = 6 n'ime akwara capsule neurons, ** P = 0.0022) (etiti). Pasent t-hCO neurons na-egosi EPSC na nzaghachi na mkpali eletriki nke oke S1 (f) ma ọ bụ capsule ime (g) (n'aka nri). aCSF, mmiri mmiri cerebrospinal artificial. h, eserese schematic nke nnwale onyonyo 2P (aka ekpe). Nkwupụta nke GCaMP6s na t-hCO (n'etiti). Ogwe ọnụ ọgụgụ, 100µm. Ogologo oge fluorescence nke GCaMP6s (n'aka nri). i, Z-akara nke fluorescence ọrụ akpaghị aka. j, ihe atụ schematic nke mkpali afụ ọnụ. k, trajectories 2P fluorescence nke z-akara n'otu nnwale, kwekọọ na ngbanwe whisker n'oge efu (akara dashed) na mkpụrụ ndụ ihe atụ. l, nzaghachi z-score nke ọnụ ọgụgụ ndị bi na nke sel niile dabara na nhụsianya whisker n'oge efu (akara dashed) (acha uhie uhie) ma ọ bụ oge emepụtara na-enweghị usoro (isi awọ). m. Eserese eserese nke nnwale na akara anya. n, Ntụgharị voltaji ọkụ sitere na ihe atụ t-hCO cell n'oge mkpali laser na-acha anụnụ anụnụ ma ọ bụ nhụsianya whisker. Àkụ na-acha uhie uhie na-egosi spikes mbụ nke ìhè (n'elu) kpatara ma ọ bụ kpatara ntugharị whisker (ala). Ndo isi awọ na-egosi oge ngbanwe afụ ọnụ. o, Nzaghachi nzagharị ọkụ kacha elu na ọzịza. p, spikes nke otu mgbalị, kwekọọ na ngbanwe nke whiskers na sel nke ihe atụ. 0 na-egosi ngbanwe whisker (akara akara). q, ọnụ ọgụgụ ịgba egbe z-akara ọnụ ọgụgụ ndị mmadụ maka sel niile na-ahụ anya, dabara na nhụsianya whisker n'oge efu (akara dash) (acha uhie uhie) ma ọ bụ oge emepụtara na-enweghị usoro (isi awọ). r, Oke nkeji foto na-emetụ n'ahụ nke ukwuu gbanwetụrụ site na nhụsianya whisker (n = 3 oke) (aka ekpe). Ọnụ ọgụgụ kacha elu z-score latency (n = 3 oke; n = 5 (ọkụ ndụ ndụ ndụ), n = 4 (akwụkwọ ndụ akwụkwọ ndụ gbara ọchịchịrị), na n = 4 (cyan) whisker deflection modulation units per rat) (nri). A na-akọwapụta data dị ka ihe pụtara ± ọkọlọtọ
Anyị jụrụ ma enwere ike ịgbalite t-hCO site na mkpali mmetụta na vivo. Anyị tụgharịrị hCO na-egosipụta ihe ngosi calcium agbakwunyere na mkpụrụ ndụ ihe nketa GCaMP6 n'ime oke S1. Mgbe ụbọchị 150 gasịrị, anyị mere fiber photometry ma ọ bụ foto calcium foto abụọ (Fig 4h na gbasaa data, Fig. 10a). Anyị chọpụtara na mkpụrụ ndụ t-hCO gosipụtara ọrụ rhythmic mekọrịtara ọnụ (Nyocha 4i, Data gbasachara, eserese 10b na vidiyo mgbakwunye 1). Iji mara ọnụ ọgụgụ kasị elu t-hCO ọrụ, anyị mere extracellular electrophysiological ndekọ na anesthetized transplant oke (gbasaa data, Fig. 10c-f). Anyị ewepụtala nhazi stereotaxic site na onyonyo MRI; ya mere, nkeji ndị a e dekọrọ na-anọchi anya neurons nke mmadụ na-etinye, ọ bụ ezie na electrophysiology naanị anaghị ekwe ka a chọpụta ụdị sitere. Anyị hụrụ mgbawa ọrụ mekọrịtara ọnụ (data gbasaara, Fig. 10d). Ihe mgbawa ahụ mere ihe dịka 460 ms wee kewapụ ya site na oge ịgbachi nkịtị nke ihe dịka 2 s (data gbasaa, Fig. 10d, e). Ndị otu n'otu n'otu gbara ihe dị ka agba atọ n'otu mgbawa, nke bụ ihe dịka 73% nke nkeji edebanyere aha n'otu mgbawa. A na-ejikọta ọrụ nke otu n'otu n'otu nke ọma, na njikọ ndị a dị elu karịa nke ndị a na-achọpụta n'ime anụmanụ ndị a na-edeghị ọgwụ na-edekọ n'okpuru otu ọnọdụ (data gbasaa, Fig. 10f). Iji kọwakwuo nzaghachi spike nke neurons ndị mmadụ na-enweta, anyị mere nnwale mkpado ọkụ na oke anesthetized transplanted na hCO na-egosipụta ọkụ nwere mmetụta cation channel rhodopsin 2 (hChR2), site na nke t-hCO neurons njirimara mkpụmkpụ-latency (ihe na-erughị 10 ms) na nzaghachi na-acha anụnụ anụnụ). t-hCO neurons gosipụtara mgbawa nke ọrụ mberede na ugboro ugboro yiri nke a na-ahụ na foto calcium, yana ndekọ electrophysiological mere na t-hCO na-enweghị akara ìhè (data gbasaa, Fig. 10c-g). Ọ dịghị ọrụ a hụrụ n'otu ntabi anya n'ime usoro hCO kwekọrọ na nke e dekọrọ na vitro. Iji chọpụta ma enwere ike ịmegharị t-hCO site na mkpali mmetụta, anyị na-ewepụ obere anụ ọhịa oke na t-hCO (Fig. 4j, m na data agbatị, Fig. 10h,k). Dị ka ọmụmụ ndị gara aga8,10 si kwuo, akụkụ nke mkpụrụ ndụ t-hCO gosipụtara mmụba na-arụ ọrụ na nzaghachi na ntụgharị nke whisker, nke a na-ahụghị mgbe e jiri data tụnyere oge stampụ (Fig. 4k-q na gbasaa data, Fig. 10h-q). N'ezie, ihe dị ka 54% nke otu nkeji opto-akara ngosi gosipụtara mmụba dị ukwuu nke mkpali mgbe mkpali whisker gasịrị, na-arị elu na ihe dịka 650 ms (Fig 4r). Ejikọtara ọnụ, data ndị a na-atụ aro na t-hCO na-enweta ntinye ọrụ kwesịrị ekwesị ma nwee ike ịme ya site na mkpali gburugburu ebe obibi.
Anyị wee nyochaa ma t-hCO nwere ike ịgbalite sekit na oke iji chịkwaa omume. Anyị buru ụzọ nyochaa ma axon nke t-hCO neurons na-arụ ọrụ n'ime anụ ahụ gbara oke oke. Anyị butere hCO jiri lentivirus na-etinye koodu hChR2 jikọtara na EYFP (hChR2-EYFP). Mgbe ụbọchị 110 gasịrị, anyị hụrụ okwu EYFP na mpaghara ipsilateral cortical, gụnyere auditory, moto, na somatosensory cortices, yana mpaghara subcortical, gụnyere striatum, hippocampus, na thalamus (Fig 5a). Iji chọpụta ma amụma ndị a na-eme ka ọ pụta ìhè na nzaghachi synaptik na mkpụrụ ndụ oke, anyị na-arụ ọrụ sel t-hCO na-egosipụta hChR2-EYFP site na ịdekọ mkpụrụ ndụ ụbụrụ ụbụrụ ụbụrụ na akụkụ ụbụrụ dị nkọ. Ịkwalite axon t-hCO nwere ọkụ na-acha anụnụ anụnụ na-ebute EPSCs mkpụmkpụ mkpụmkpụ na rat pyramidal cortex neurons, nke NBQX gbochiri (Figs. 5b-g). Tụkwasị na nke a, nzaghachi ndị a nwere ike igbochi tetrodotoxin (TTX) ma weghachi ya site na 4-aminopyridine (4-AP), na-atụ aro na ọ bụ njikọ monosynaptic kpatara ya (Fig 5e).
a, Eserese atụmatụ nke nsuso axon (aka ekpe). t-hCO EYFP okwu (n'aka nri). Ogwe ọnụ ọgụgụ, 100µm. A1, cortex auditory, ACC, ihu cingulate cortex, d. striatum, dorsal striatum, HPC, hippocampus; Diaphragm, mpụta septum, mPFC, medial prefrontal cortex, piri, piriform cortex, v. striatum, ventral striatum, VPM, ventropostomedial nucleus nke thalamus, VTA, ventral tegmental region. Oghere uhie na-anọchi anya akụkụ ụbụrụ ebe e sere foto. b, Eserese eserese nke nnwale mkpali. c, d, Ihe atụ nke nzaghachi nke ìhè-acha anụnụ anụnụ na-ebute fotocurrent (n'elu) na voltaji (ala) n'ime mmadụ (c) EYFP+ t-hCO ma ọ bụ oke (d) EYFP-sel. e, f, Ihe ndị dị ugbu a nke neurons oke mgbe mkpali ọkụ na-acha anụnụ anụnụ nke t-hCO axon na TTX na 4-AR (green), TTX (agba ntụ) ma ọ bụ aCSF (nwa) (e), na (violet) ma ọ bụ na-enweghị (nwa) )) NBQX (e). g, nkwụsị nke nzaghachi nke ọkụ na-acha anụnụ anụnụ na-ebute na mkpụrụ ndụ oke (n = sel 16); Ogwe kehoraizin na-egosi nkezi nkwụsị (7.13 ms) (aka ekpe). Ọnụ ọgụgụ nke EPSC na-enwu ọkụ na-edekọ ma ọ bụ na-enweghị NBQX (n = 7 mkpụrụ ndụ; *** P <0.0001) (n'etiti). Ọnụ ọgụgụ nke EPSC na-enwu ọkụ na-edekọ ma ọ bụ na-enweghị NBQX (n = 7 mkpụrụ ndụ; *** P <0.0001) (n'etiti). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; *** P <0,0001) (в центре). Ọnụ ọgụgụ nke EPSC na-eme ka ọkụ na-edekọ ma ọ bụ na-enweghị NBQX (n = 7 cell; *** P <0.0001) (etiti).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). Ọnụ ọgụgụ nke EPSC na-eme ka ọkụ na-edekọ ma ọ bụ na-enweghị NBQX (n = 7 cell; *** P <0.0001) (etiti).Pasent nke mkpụrụ ndụ oke na-egosi EPSC na-anabata ìhè anụnụ anụnụ (n'aka nri). h, Eserese atụmatụ nke ọrụ omume. d0, ụbọchị 0.i. Ịrụ anụ ezi ihe nlereanya na ụbọchị 1 (aka ekpe) ma ọ bụ ụbọchị 15 (n'aka nri) nke ọzụzụ. Ọnụ ọgụgụ licks pụtara n'ụbọchị 1 (aka ekpe) ma ọ bụ ụbọchị 15 (n'etiti aka nri) (n = 150 ule ọkụ na-acha anụnụ anụnụ, n = 150 ule ọkụ uhie; *** P <0.0001). Ọnụ ọgụgụ licks pụtara n'ụbọchị 1 (aka ekpe) ma ọ bụ ụbọchị 15 (n'etiti aka nri) (n = 150 ule ọkụ na-acha anụnụ anụnụ, n = 150 ule ọkụ uhie; *** P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) или день 15 n = 150 испытаний с красным светом; *** P <0,0001). Ọnụ ọgụgụ licks pụtara n'ụbọchị 1 (aka ekpe) ma ọ bụ ụbọchị 15 (n'etiti aka nri) (n = 150 ule ọkụ na-acha anụnụ anụnụ, n = 150 nnwale ọkụ uhie; *** P <0.0001).第1 天（左）或第15 天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150次红光试验； *** P <0.0001）。第1 天（左）或第15 天（右中）执行的平均舔次数（n = 150 次蓝光试验，n = 150次红光试验； *** P < 0.001 Среднее количество облизываний, выполненных в день 1 (слева) или день 15 n = 150 испытаний с красным светом; *** P <0,0001). Ọnụ ọgụgụ licks pụtara n'ụbọchị 1 (aka ekpe) ma ọ bụ ụbọchị 15 (n'etiti aka nri) (n = 150 ule ọkụ na-acha anụnụ anụnụ, n = 150 nnwale ọkụ uhie; *** P <0.0001).Nchịkọta licks maka nnwale ọbara ọbara na-acha anụnụ anụnụ na ụbọchị 1 (etiti aka ekpe) ma ọ bụ ụbọchị 15 (n'aka nri). NS, ọ dịghị mkpa. j,k, Àgwà omume nke anụmanụ niile transplanted na t-hCO na-egosipụta hChR2-EYFP (j) ma ọ bụ akara fluorophore (k) na ụbọchị 1 ma ọ bụ 15 (hChR2-EYFP: n = 9 oke, ** P = 0.0049; njikwa: n = 9, P = 0.1497). l, Evolution nke akara mmasị (n = 9 hChR2, n = 9 njikwa; ** P <0.001, *** P <0.0001). l, Evolution nke akara mmasị (n = 9 hChR2, n = 9 njikwa; ** P <0.001, *** P <0.0001). l, Эvolyutsyya pokzatelya PREDPOCHENYA (n = 9 hChR2, n = 9 kọntrolnыh; ** P <0,001, *** P <0,0001). l, Evolution nke akara mmasị (n = 9 hChR2, n = 9 njikwa; ** P <0.001, *** P <0.0001). l，偏好评分的演变（n = 9 hChR2，n = 9 对照；**P <0.001，***P <0.0001）. l，偏好评分的演变（n = 9 hChR2，n = 9 对照；**P <0.001，***P <0.0001）. l, Эvolyutsyya pokzateley PREDPOCHENYA (n = 9 hChR2, n = 9 krone; ** P <0,001, *** P <0,0001). l, Evolution nke akara mmasị (n = 9 hChR2, n = 9 njikwa; ** P <0.001, *** P <0.0001).m, FOS okwu na nzaghachi na optogenetic activation nke t-hCO na S1. Egosiputa onyonyo nke okwu FOS (aka ekpe), na quantification (n = 3 kwa otu; * P <0.05, ** P <0.01 na ***P <0.001) (n'aka nri). Egosiputa onyonyo nke okwu FOS (aka ekpe), na quantification (n = 3 kwa otu; * P <0.05, ** P <0.01 na ***P <0.001) (n'aka nri). Показаны изображения эkspressy FOS (sleva) na kọlụm opredelennya (n = 3 na групу; * P <0,05, ** P <0,01 , p <0,01 , p <0,01 ,p. Ihe onyonyo nke okwu FOS (aka ekpe) na ọnụọgụ (n = 3 kwa otu; * P<0.05, ** P<0.01, na ***P <0.001) ka egosiri (n'aka nri).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）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） Показаны изображения эkspressy FOS (sleva) na kọlụm opredelennya (n = 3 na групу; * P <0,05, ** P <0,01 , p <0,01 , p <0,01 ,p. Ihe onyonyo nke okwu FOS (aka ekpe) na ọnụọgụ (n = 3 kwa otu; * P<0.05, ** P<0.01, na ***P <0.001) ka egosiri (n'aka nri).Ogwe ọnụ ọgụgụ, 100µm. Ekwuputara data dị ka pụtara ± njehie ọkọlọtọ nke BLA, tonsil basolateral, MDT, dorsomedial thalamic nucleus, PAG, periaqueductal isi awọ.
N'ikpeazụ, anyị jụrụ ma t-hCO nwere ike gbanwee omume oke. Iji nwalee nke a, anyị tụgharịrị hChR2-EYFP-na-egosipụta hCO n'ime S1, na ụbọchị 90 ka e mesịrị, anyị kụnyere eriri anya n'ime t-hCO maka nnyefe ọkụ. Anyị wee zụọ oke ndị ahụ site n'iji ihe nrụnye arụrụ arụ arụrụ arụ (Fig. 5h). Anyị debere ụmụ anụmanụ n'ime ụlọ nyocha omume wee tinye ihe mkpali laser 5 nke abụọ na-acha anụnụ anụnụ (473 nm) na uhie (635 nm). Ụmụ anụmanụ nwetara ụgwọ ọrụ mmiri ma ọ bụrụ na ha rachara n'oge mkpali ọkụ na-acha anụnụ anụnụ mana ha arachaghị n'oge mkpali ọkụ uhie. N'ụbọchị mbụ nke ọzụzụ, ụmụ anụmanụ egosighi ihe dị iche na ịcha mgbe a na-akpali ya na-acha anụnụ anụnụ ma ọ bụ na-acha uhie uhie. Otú ọ dị, n'ụbọchị 15, ụmụ anụmanụ ndị a tụgharịrị na hCO na-egosipụta hChR2-EYFP gosipụtara na-arụsi ọrụ ike karị mgbe ọkụ na-acha anụnụ anụnụ na-akpali ya ma e jiri ya tụnyere mkpali ọkụ na-acha uhie uhie. A hụghị mgbanwe ndị a na omume ịra ihe na ụmụ anụmanụ na-achịkwa nke a tụgharịrị na hCO na-egosipụta fluorophore nchịkwa (ọnụego mmụta mmụta: hChR2 89%, EYFP 0%, Figure 5i-1 na Video 2 mgbakwunye). Ihe data ndị a na-egosi na mkpụrụ ndụ t-hCO nwere ike rụọ ọrụ neurons iji kpalite omume ịchọ ụgwọ ọrụ. Iji chọpụta nke sekit akwara t-hCO nke oke nwere ike itinye aka na mgbanwe omume ndị a, anyị na-arụ ọrụ t-hCO nke ọma n'ime anụmanụ a zụrụ azụ na anụ ahụ ewepụtara 90 nkeji ka e mesịrị. Immunohistochemistry kpughere ngosipụta nke protein FOS dabere na ọrụ n'ọtụtụ mpaghara ụbụrụ na-etinye aka na omume mkpali, gụnyere medial prefrontal cortex, medial thalamus, na ihe isi awọ periaqueductal, nke egosipụtara n'ime anụmanụ na-achịkwaghị achịkwa ma ọ bụ n'ime anụmanụ. osikapa. 5m). N'ịchịkọta ọnụ, data ndị a na-egosi na t-hCO nwere ike gbanwee ọrụ neuronal oke iji chụpụ omume.
Neural organoids na-anọchite anya usoro na-ekwe nkwa maka ịmụ mmepe mmadụ na ọrịa na vitro, mana ha na-ejedebe site na enweghị njikọ dị n'etiti sekit dị na vivo. Anyị ewepụtala ikpo okwu ọhụụ nke anyị tụgharịrị hCO n'ime S1 nke ụmụ oke ụmụ oke na-ebute ọrịa mgbochi iji mụọ mmepe cell mmadụ na ịrụ ọrụ na vivo. Anyị egosila na t-hCO na-etolite ụdị mkpụrụ ndụ tozuru oke anaghị ahụ ya na vitro28 yana t-hCO bụ nke anatomically na arụ ọrụ n'ime ụbụrụ òké. Ntinye nke t-hCO n'ime sekit akwara na-ekwe ka anyị guzobe njikọ dị n'etiti ọrụ cellular mmadụ na omume anụmanụ na-amụ, na-egosi na t-hCO neurons nwere ike gbanwee ọrụ neuronal nke oke iji kpalite nzaghachi omume.
Ikpo okwu anyị na-akọwa nwere ọtụtụ uru karịa nyocha gara aga gbasara ịtụgharị mkpụrụ ndụ mmadụ n'ime ụbụrụ òké. Nke mbụ, anyị tụgharịrị hCO n'ime cortex na-emepe emepe nke oke ụmụ oke na-amụ nwa, nke nwere ike ịkwado anatomical na ntinye ọrụ. Nke abụọ, t-hCO MRI nlekota mere ka anyị mụọ ọnọdụ graft na uto na ụmụ anụmanụ dị ndụ, na-ekwe ka anyị na-eduzi ọmụmụ ọtụtụ anụmanụ ogologo oge ma guzobe ntụkwasị obi nke ọtụtụ ahịrị sel hiPS. N'ikpeazụ, anyị tụgharịrị organoids na-adịghị emebi emebi, kama ịnọpụ iche nkwusioru otu cell, nke na-adịchaghị emebi emebi na mkpụrụ ndụ mmadụ ma nwee ike ịkwalite njikọta na ọgbọ nke neurons cortex mmadụ na ụbụrụ oke.
Anyị na-ekweta na n'agbanyeghị ọganihu ndị a na-enwe na ikpo okwu a, ihe mgbochi oge, ohere, na ihe ndị dị iche iche na-egbochi ịmepụta sekit neural nke mmadụ na ntụkwasị obi dị elu, ọbụlagodi mgbe a gbanyechara ya n'oge mmalite nke mmepe. Dịka ọmụmaatụ, ọ bụghị ihe doro anya ma ọrụ a na-ahụ anya na t-hCO na-anọchite anya phenotype mmepe nke yiri ọrụ rhythmic a na-ahụ n'oge mmepe cortical, ma ọ bụ na ọ bụ n'ihi enweghị ụdị cell na-egbochi ndị dị na t-hCO. N'otu aka ahụ, o doghị anya ruo ókè enweghị lamination na t-hCO na-emetụta njikọ njikọ 30. Ọrụ n'ọdịnihu ga-elekwasị anya na ijikọta ụdị sel ndị ọzọ dị ka microglia mmadụ, mkpụrụ ndụ endothelial mmadụ, na nha dịgasị iche iche nke GABAergic interneurons dị ka egosipụtara site na iji mgbakọ 6 na vitro, yana ịghọta otú ntinye na nhazi nke neural nwere ike isi mee na t-hCO gbanwere. transcriptional, synaptik na omume ọkwa na sel enwetara n'aka ndị ọrịa.
N'ozuzu, nke a na vivo ikpo okwu na-anọchi anya ihe onwunwe dị ike nke nwere ike ịkwado na vitro ụbụrụ mmadụ na nchọpụta ọrịa. Anyị na-atụ anya na ikpo okwu a ga-enye anyị ohere ịchọpụta phenotypes strand-level phenotypes n'ime mkpụrụ ndụ ndị ọrịa na-enweta ma ọ bụ nwalee usoro ọgwụgwọ ọhụrụ.
Anyị mepụtara hCO2.5 site na sel HiPS dị ka akọwara na mbụ. Iji bido mmepụta hCO sitere na mkpụrụ ndụ hiPS emebere n'ọkwa nri, ewepụrụ mpaghara sel hiPS na-emebibeghị na efere omenala site na iji mkpofu (0.35 mg/mL) wee bufee ya na omenala rọba na-agbakwunye dị ala nke nwere efere nwere usoro omenala hiPS cell. (Corning) agbakwunyere na ndị na-emechi SMAD abụọ dorsomorphine (5 μM; P5499, Sigma-Aldrich) na SB-431542 (10 μM; 1614, Tocris) na ROCK inhibitor Y-27632 (10 μM; S1049), Selleck. N'ime ụbọchị 5 mbụ, a gbanwere usoro cell hiPS kwa ụbọchị yana dorsomorphine na SB-431542 gbakwụnye. N'ụbọchị nke isii na nkwusioru, a na-ebufe neural spheroids na neural media nwere neurobasal-A (10888, Life Technologies), B-27 mgbakwunye na-enweghị vitamin A (12587, Life Teknụzụ), GlutaMax (1: 100, Life Teknụzụ), penicillin na streptomycin (1: 100, Life Teknụzụ) na gbakwunyere na EGF 1. R & D Systems) na ihe na-eto eto fibroblast 2 (FGF2; 20 ng ml-1; R & D Systems) ruo ụbọchị 24. Site na ụbọchị 25 ruo ụbọchị 42, a na-agbakwunye usoro ahụ na ụbụrụ neurotrophic nke ụbụrụ na-enweta (BDNF; 20 ng ml-1, Peprotech) na neurotrophin 3 (NT3) - 1 ml na-agbanwe kwa ụbọchị. N'ụbọchị nke isii na nkwusioru, a na-ebufe neural spheroids na neural media nwere neurobasal-A (10888, Life Technologies), B-27 mgbakwunye na-enweghị vitamin A (12587, Life Teknụzụ), GlutaMax (1: 100, Life Teknụzụ), penicillin na streptomycin (1: 100, Life Teknụzụ) na gbakwunyere na EGF 1. R & D Systems) na ihe na-eto eto fibroblast 2 (FGF2; 20 ng ml-1; R & D Systems) ruo ụbọchị 24. Site na ụbọchị 25 ruo ụbọchị 42, a na-agbakwunye usoro ahụ na ụbụrụ neurotrophic nke ụbụrụ na-enweta (BDNF; 20 ng ml-1, Peprotech) na neurotrophin 3 (NT3) - 1 ml na-agbanwe kwa ụbọchị.N'ụbọchị nke isii na nkwusioru, a na-ebugharị spheroids neural na ntanetị nke nwere Neurobasal-A (10888, Life Technologies), mgbakwunye B-27 na-enweghị vitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin.na streptomycin (1: 100, Life Teknụzụ) na dopolnenы эpydermalnыm phaktorom rosta (EGF; 20 nng/ml; R&D Systems) na faktorom 2. (FGF2; 20 нг/мл; R&D Sistemụ) na 24-го дня. na streptomycin (1: 100, Life Technologies) ma gbakwunye ya na ihe na-eto eto epidermal (EGF; 20 ng / ml; R & D Systems) na fibroblast growth factor 2 (FGF2; 20 ng / ml; R & D Systems) ruo ụbọchị 24.Site na ụbọchị 25 ruo 42, a na-agbakwunye ụbụrụ ụbụrụ neurotrophic (BDNF; 20 ng ml-1, Peprotech) na neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) na ọkara, na-agbanwe usoro ahụ kwa ụbọchị ọ bụla.在悬浮的第6 天，将神经球体转移到含有neurobasal-A（10888፣Life Teknụzụ Teknụzụ) Teknụzụ）并辅以表皮生长因子（EGF；20 ng ml-1；R&D Systems）和成纤维细胞生长因子2（FGF2；子2（FGF2； Sistemụ) 直至第24在 浮 的 第 第 6 天 将 神经 球体 转移 含有 含有 neurobasal-a (10888 ， Life Technologies４ 不 含 维补充剂 (12587 ， Life Teknụzụ) Glutamax (1: 100 ， Life TechNOGIS青霉素 的生长 因子 (； 20 ng ml-1 ； r & d Systems） На 6-й день суспензии нейросферы были переведены на добавку, содержащую нейробазазал-А (10888), Life Technology 7. витамина А (12587, Life Teknụzụ), GlutaMax (1: 100, Life Teknụzụ), ndị na-emepụta ihe na-emepụta ihe na-emepụta ihe (1: 100, Life Teknụzụ) фактора роста (EGF; 20 нг мл-1; R&D Systems) na фактора роста фибробластов 2 (FGF2; 20 нг мл-1) 1; N'ụbọchị 6, a gbanwere nkwusioru neurosphere na mgbakwunye nwere neurobasal-A (10888, Life Technologies), B-27 na-enweghị vitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin-neutralized streptomycin (1: 100, Life Technologies) gbakwunyere na epider R&ml. Sistemu) na ihe na-eto eto fibroblast 2 (FGF2; 20 ng ml-1) 1; R&D Systems) maka afọ 24. R&D Systems) ruo ụbọchị 24.Site na ụbọchị 25 ruo 42, a na-agbakwunye ụbụrụ ụbụrụ neurotrophic (BDNF; 20 ng ml-1, Peprotech) na neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) na omenala omenala kwa ụbọchị ọ bụla. Ọkara mgbanwe otu ugboro.Malite na ụbọchị 43, a na-edobe hCO na neurobasal-A ọkara (NM; 1088022, Thermo Fisher) na-agbanwe mgbanwe kwa ụbọchị 4-6. Iji nweta hCO sitere na sel hiPS nke emebere n'okpuru ọnọdụ enweghị nri, mkpụrụ ndụ hiPS ka etinyere ya na Accutase (AT-104, Innovate Cell Technologies) na 37 Celsius C maka nkeji 7, kewapụrụ ya n'otu sel, ma tinye ya na AggreWell 800 plates (34815, STEMCELL Teknụzụ) na njupụta nke otu sel nke 3 × 3. ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Mgbe awa 24 gachara, a na-agbanye mgbasa ozi na olulu mmiri n'elu na ala n'ime mgbasa ozi nwere 6 mgbasa ozi dị mkpa (A1516401, Life Technologies) gbakwunyere na dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) na SB-431542 (10 μM; 1614). , Tocrida). Site na ụbọchị 2 ruo 6, a na-eji dorsomorphine dochie ihe dị mkpa 6 kwa ụbọchị yana mgbakwunye SB-431542. Site na ụbọchị nke isii, a na-ebufe nkwusioru neurosphere na neurobasal media ma na-echekwa ya dị ka akọwara n'elu.
Emere usoro anụmanụ niile dabere na ụkpụrụ nlekọta anụ ụlọ nke Stanford University Laboratory Animal Care Administrative Committee (APLAC) kwadoro. A zụtara oke euthymic RNU (rnu/+) dị ime (Charles River Laboratories) ma ọ bụ dobe ya. A na-edobe anụmanụ n'ime okirikiri ọkụ nke awa 12 nwere nri na mmiri ad libitum. Agba ọtọ (FOXN1–/-) ụmụ oke oke dị n'agbata ụbọchị atọ ruo ụbọchị asaa ka achọpụtara site na uto nke afụ ọnụ na-etobeghị tupu a gbuo ya. A na-eji 2-3% isoflurane mee ka nwa nkita (nwoke na nwanyị) na-egbu egbu ma tinye ya na etiti stereotaxic. A na-eme trepanation nke okpokoro isi nke nwere dayameta nke ihe dịka 2-3 mm n'elu S1 ka a na-ejigide iguzosi ike n'ezi ihe nke dura mater. Mgbe ahụ jiri agịga 30-G (ihe dị ka 0.3 mm) dị na mpụga craniotomy iji maa dura ahụ. Wee tinye HCO na parafilm 3 × 3 cm dị mkpa ma wepụ oke ọkara. Iji sirinji Hamilton nke etinyere na agịga 23 G, 45°, jiri nwayọọ dọpụta hCO n'ime ebe kacha dị anya nke agịga ahụ. Wee tinye sirinji na mgbapụta sirinji ejikọrọ na ngwaọrụ stereotaxic. Mgbe ahụ, tinye n'ọnụ nke agịga n'elu a na mbụ mere 0,3 mm n'obosara oghere oghere na dura (z = 0 mm) na warara sirinji 1-2 mm (z = ihe dị ka -1.5 mm) ruo mgbe agịga dị n'etiti dura mater A. a ok akara e guzobere. Wee bulite sirinji ahụ n'etiti etiti cortical na z = -0.5 mm ma gbanye hCO na ọnụego 1-2 µl kwa nkeji. Mgbe emechara ntụtụ hCO, a na-eweghachi agịga ahụ n'ogo nke 0.2-0.5 mm kwa nkeji, akpụkpọ ahụ na-edozi ya, a na-etinye nwa nkita ozugbo n'elu ihe mkpuchi ọkụ na-ekpo ọkụ ruo mgbe mgbake zuru ezu. A tụgharịrị ụfọdụ anụmanụ n'akụkụ abụọ.
Emere usoro anụmanụ niile dabere na ụkpụrụ nlekọta anụmanụ na Mahadum Stanford APLAC kwadoro. Ejiri 5% isoflurane anesthesia butere oke oke (ihe karịrị ụbọchị 60 ka a tụgharịrị ya) wee were 1-3% isoflurane were gbuo ya n'oge eserese. Maka ikiri anya, 7 Tesla na-arụsi ọrụ ike na-echebe ihe nyocha borehole kwụ ọtọ Bruker (Bruker Corp.) ya na draịva gradient International Electric Company (IECO), ntinye gradient mkpuchi nke nwere dayameta dị n'ime nke 120 mm (600 mT/m, 1000 T/m/s) jiri AVANCE. III, ọwa asatọ multi-coil RF na ikike multi-core, yana usoro Paravision 6.0.1 na-esote. Emere ndekọ site na iji eriri RF volumetric emebiela nke ọma nwere dayameta dị n'ime nke 86 mm yana igwe mkpuchi RF dị jụụ nke chawa anọ maka ịnata naanị. Axial 2D Turbo-RARE (oge nkwugharị = 2500 ms, oge nkwughachi = 33 ms, 2 nkezi) na njide 16 ibe, nha nha 0.6-0.8 mm, nwere ihe atụ 256 × 256. Enwetara akara ndị ahụ site na iji eriri RF quadrature transceiver volumetric nke nwere dayameta dị n'ime nke 2 cm (Rapid MR International, LLC). N'ikpeazụ, jiri ọrụ ntule elu Imaris (BitPlane) arụnyere arụnyere maka ntụgharị 3D na nyocha olu. A kọwapụtara ntụgharị na-aga nke ọma dị ka ebe a na-etolite ebe mgbaàmà MRI na-aga n'ihu nke T2 na-aga n'ihu na mbara ala a tụgharịrị. A kọwapụtara njụta ​​graft dị ka ntanye nke na-emepụtaghị ebe mgbama MRI na-aga n'ihu na T2 na-aga n'ihu na mbara ala a tụgharịrị. Ewepụrụ subcortical t-hCO na nyocha na-esote.
Iji kwupụta nke ọma GCaMP6s na hCO maka foto calcium foto abụọ, mkpụrụ ndụ hiPS butere pLV[Exp] -EF1a:: GcaMP6s-WPRE-Puro sochiri nhọrọ nke ọgwụ nje. Na nkenke, e kewapụrụ mkpụrụ ndụ na EDTA ma kwụsịtụrụ na 1 ml nke mkpa 8 dị mkpa na njupụta nke ihe dịka sel 300,000 na ọnụnọ polybrene (5 μg / ml) na 15 μl nke nje. A na-etinyezi mkpụrụ ndụ ndị ahụ na nkwusioru maka nkeji 60 wee kụọ mkpụrụ na njupụta nke sel 50,000 kwa olulu mmiri. Mgbe mkparịta ụka gasịrị, a na-emeso mkpụrụ ndụ na 5-10 μg ml-1 puromycin maka ụbọchị 5-10 ma ọ bụ ruo mgbe ndị ọchịchị kwụsiri ike pụtara. Emere nnukwu ọrịa hCO dị ka akọwara na mbụ5 yana mgbanwe ụfọdụ. Na nkenke, nyefee ụbọchị 30-45 hCO n'ime 1.5 ml Eppendorf microcentrifuge tubes nwere 100 µl nke akwara akwara. Mgbe ahụ, a na-ewepụ ihe dịka 90 µl nke ọkara, 3-6 µl nke lentivirus dị elu (site na 0.5 x 108 ruo 1.2 x 109) na-agbakwunye na tube, na-ebufe hCO na incubator maka nkeji 30. Mgbe ahụ, tinye 90-100 µl nke ọkara na tube ọ bụla ma weghachite tubes na incubator n'abali. N'echi ya, nyefee hCO gaa na akwara akwara ọhụrụ na efere mgbakwunye dị ala. Mgbe ụbọchị 7 gachara, e bufere hCO na efere ala iko 24 nke ọma maka nleba anya na nyocha nke ogo ọrịa. pLV[Exp] -SYN1::EYFP-WPRE na pLV[Exp] -SYN1::hChR2-EYFP-WPRE bụ VectorBuilder mebere ya. A na-eji Lentivirus eme ihe n'ọtụtụ nnwale n'ihi na etinyere ya n'ime genome nke ụlọ ọrụ, na-enye ohere ka ndị na-ede akụkọ na-egosipụta mkpụrụ ndụ n'ime ahịrị sel ndị nwere nje. Maka nyocha nke rabies, ụbọchị 30-45 hCO jikọtara ya na rabies-ΔG-eGFP na AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), sachara nke ọma maka ụbọchị 3, ma gbanye ya n'ime oke na S1 ma nọgide na-enwe na vivo maka ụbọchị 7-14.
Maka immunocytochemistry, anesthetized anesthetized ụmụ anụmanụ na transcardially juputara na PBS sochiri 4% paraformaldehyde (PFA na PBS; Electron Microscope Sciences). A na-edozi ụbụrụ na 4% PFA maka awa 2 ma ọ bụ n'abali na 4 Celsius C, nke echekwara na 30% sucrose na PBS maka awa 48-72, ma tinye ya na 1: 1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583, Sakura Finetek na-eji akwa akwa 3) na coronal. (Leica). Maka immunohistochemistry nke akụkụ buru ibu, a na-eju anụmanụ na PBS, na ụbụrụ na-ekewa ma kewaa ya na 300-400 µm site na iji vibratome (Leica) na akụkụ ndị ahụ na-edozi ya na 4% PFA maka nkeji 30. Mgbe ahụ, a na-asacha cryosections ma ọ bụ akụkụ buru ibu na PBS, gbochie maka awa 1 na ụlọ okpomọkụ (10% nkịtị serum ịnyịnya ibu (NDS) na 0.3% Triton X-100 diluted na PBS) ma gbochie ya na ngwọta mgbochi na 4 ° C. - A na-etinye Cryosections incubation n'otu abalị ma tinye akụkụ gbara ọkpụrụkpụ maka ụbọchị 5. Ihe mgbochi izizi ejiri mee ihe bụ: mgbochi NeuN (òké, 1:500; ab104224, abcam) mgbochi CTIP2 (oke, 1:300; ab18465, abcam), mgbochi GFAP (oke bekee, 1:1,000; Z0334, Dako), mgbochi GFP:11ex mgbochi HNA (òké, 1:200; ab191181, abcam), mgbochi NeuN (oke bekee, 1:500; ABN78, Millipore), mgbochi PDGFRA (oke bekee, 1:200; sc-338, Santa Cruz), mgbochi PPP1R17 (oke oyibo, 1:0478), HPAdies. mgbochi RECA-1 (òké, 1:50; ab9774, abcam), mgbochi SCG2 (oke bekee, 1:100; 20357-1-AP, Proteintech), mgbochi SOX9 (ewu, 1:500; AF3075, R&D Systems), Netrin G1a (ewu, 6; RAF0, 1:11 Systems), mgbochi STEM121 (òké, 1:200; Y40410, Takara Bio), mgbochi SATB2 (òké, 1:50; ab51502, abcam), mgbochi GAD65/67 (oke bekee, 1:400; ABN904, Millipore) na mgbochi IBA1 (ewu, 1:000, ab5106; abcam). Ihe mgbochi izizi ejiri mee ihe bụ: mgbochi NeuN (òké, 1:500; ab104224, abcam) mgbochi CTIP2 (oke, 1:300; ab18465, abcam), mgbochi GFAP (oke bekee, 1:1,000; Z0334, Dako), mgbochi -GFP:11ex mgbochi HNA (òké, 1:200; ab191181, abcam), mgbochi NeuN (oke bekee, 1:500; ABN78, Millipore), mgbochi PDGFRA (oke bekee, 1:200; sc-338, Santa Cruz), mgbochi PPP1R17 (oke oyibo, 1:0478), HPAdies. mgbochi RECA-1 (òké, 1:50; ab9774, abcam), mgbochi SCG2 (oke bekee, 1:100; 20357-1-AP, Proteintech), mgbochi SOX9 (ewu, 1:500; AF3075, R&D Systems), Netrin G1a (ewu, 106; 10, AF & D Systems), mgbochi STEM121 (òké , 1:200; Y40410, Takara Bio), mgbochi SATB2 (òké, 1:50; ab51502, abcam), mgbochi GAD65/67 (oke bekee, 1:400; ABN904, Millipore) na mgbochi IBA1: 070, cam abcam . Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2: 3кыры; ab18465, abcam), анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA ( ab1900 ), 19cam 1 , ab19 1 , ab19 0 , 19cam 1 , ab19 1 , ab19 1 , ab19 1 , ab19 0 , ab19 0 1 , ab19 1 , ab19 0 , 19cam 1 . анти-NeuN (кролик, 1:500; ABN78, Millipore), анти-PDGFRA (кролик, 1:200; sc-338, Санта-Круз), анти-PPP1R17 (Kролик, 1:240, HPAAT) Antibodies), анти-RECA-1 (mышь, 1:50; ab9774, abcam), анти-SCG2 (кролик , 1:100; 20357-1-AP, Proteintech), анти-SOX9), (козий, 1:507) нетрин G1a (козий, 1:100; AF1166, R&D Systems), анти-STEM121 (мышиный , 1:200; Y40410, Takara Bio), анти-SATB2 (mышь, ab51), 502; ab 1:50; анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) na анти-IBA1 (коза, 1:100; аб5076, бкам). Ihe mgbochi izizi ejiri mee ihe bụ: mgbochi NeuN (òké, 1:500; ab104224, abcam), mgbochi CTIP2 (oke, 1:300; ab18465, abcam), mgbochi GFAP (oke bekee, 1:1000; Z0334, Dako), mgbochi GFP (ọkụkọ, 10300; GTX 1: 10exT), GTX mgbochi HNA (òké, 1:200; ab191181, abcam), mgbochi NeuN (oke bekee, 1:500; ABN78, Millipore), mgbochi PDGFRA (oke bekee, 1:200; sc-338, Santa Cruz), mgbochi PPP1R17 (oke oyibo, 1:0478), HPAdies. mgbochi RECA-1 (òké, 1:50; ab9774, abcam), mgbochi SCG2 (oke bekee, 1:100; 20357-1-AP, Proteintech), mgbochi SOX9 (ewu, 1:500; AF3075, R&D Sistemu), netrin G1a (mkpi, 106; 1:10, R&D Systems), & D. STEM121 (òké, 1:200; Y40410, Takara Bio), mgbochi-SATB2 (òké, 1:50; ab51502, abcam), mgbochi GAD65/67 (oke bekee, 1:400; ABN904, Milipore) na mgbochi IBA1 (ewu, ab51502; 1:70).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；ab1911 81, abcam), 抗NeuN (兔, 1:500;ABN78, Millipore), 抗PDGFRA（兔, 1: 200;sc-338, Santa Cruz), 抗PPP1R17 (兔, 1: 200; HPA047819, Atlas抗体） 抗RECA-1（小鼠：1:50；ab9774） abcam））抗SCG2（兔） , 1: 100; 20357-1-AP, Proteintech, 抗SOX9 (山羊, 1:500; AF3075, R&D Systems), Netrin G1a (1:100), AF1166, R&D Sistemu） 抗STEM121（小鼠, 1:200;使用的一抗是：抗NeuN（小鼠，1:500；ab104224，abcam）抗CTIP2（大鼠，1 :300;ab18465,abcam),抗GFAP（兔,1:1,000;Z0334,Dako），抗-GFP（鸡：1:1,000：GTX13970، GeneTex）））HNA（小鼠，1:200；ab191181：abcam））NeuN（兔，1:500；兔，1:500；兔，1:500；兔，1:500；小鼠，1:200 200；sc-338، Santa Cruz））抗PPP1R17（兔：1:200；HPA047819） Atlas 抗体））抗RECA-1（小都＠，1:50（小鼠，1:50，abcam97；abcam 100;20357-1-AP, Proteintech, 抗SOX9（山羊，1:500；AF3075：R&D Systems）Netrin G1a（山羊）1:100；AF1166，R&D Systems：1.Y40410, Takara Bio), mgbochi SATB2 (òké, 1:50; ab51502, abcam), mgbochi GAD65/67 (oke bekee, 1:400; ABN904, Millipore) na mgbochi IBA1 (ewu, 1:100; ab5076, abcam).Ihe mgbochi izizi ejiri mee ihe bụ: mgbochi NeuN (òké, 1:500; ab104224, abcam), mgbochi CTIP2 (oke, 1:300; ab18465, abcam), mgbochi GFAP (oke bekee, 1:1000; Z0334, Dako) . , mgbochi GFP (ọkụkọ, 1: 1000; GTX13970, GeneTex), mgbochi HNA (òké, 1:200; ab191181, abcam), mgbochi NeuN (oke oyibo, 1:500; ABN78, Millipore), mgbochi PDGFRA (oke bekee, 1-3300), Santa Claus, 1-3300 anti-NeuN. (oke bekee, 1:200; HPA047819, Atlas mgbochi), mgbochi RECA-1 (òké, 1:50; ab9774, abcam), mgbochi- SCG2 (oke bekee), 1:100;20357-1-AP, Proteintech), анти-SOX9 (коза, 1:500; AF3075, R&D Systems), Нетрин G1a (коза, 1:100; AF1166, R & D Sistemu), na -STEM: 1201; Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) na анти-IBA1 (коза0; 6:50; ebe). 20357-1-AP, Proteintech), mgbochi SOX9 (ewu, 1:500; AF3075, R&D Systems), Netrin G1a (ewu, 1:100; AF1166, R&D Systems), mgbochi STEM121 (òké, 1:200; Y40410; Mgbochi-SA 1:200; Y40410; Mgbochi-SA 1:100; Mkpi, 1:100). ab51502, abcam), mgbochi GAD65/67 (oke bekee, 1:400; ABN904, Millipore), na mgbochi IBA1 (ewu, 1:100; ab5076, abkam).A na-asacha akụkụ ahụ na PBS ma tinye ya na mgbochi nke abụọ maka awa 1 n'ime ụlọ okpomọkụ (akụkụ oyi kpọnwụrụ) ma ọ bụ n'abali na 4 ° C (ngalaba oke). Alexa Fluor secondary antibody (Life Technologies) gbazere 1:1000 na ngwọta mgbochi ejiri. Mgbe a sachara ya na PBS, a na-eji Hoechst 33258 (Life Teknụzụ) jiri anya ya hụ oghere ndị ahụ. N'ikpeazụ, etinyere ihe mmịfe ahụ na microscope nwere mkpuchi mkpuchi (Fisher Scientific) na-eji Aquamount (Polysciences) wee nyochaa ya na Keyence fluorescent microscope (BZ-X analyzer) ma ọ bụ Leica TCS SP8 confocal microscope (Las-X) na onyonyo a. Ejiri mmemme ImageJ (Fiji) hazie ihe onyonyo a. Iji kọwapụta ọnụọgụ nke neurons mmadụ na t-hCO na cortex oke, 387.5 μm n'obosara akụkụ akụkụ anọ ka ewepụtara na etiti t-hCO, na ma ọ bụ n'akụkụ nsọtụ nke oke oke. Ekpebiri akụkụ aka aka site n'ịtụle mgbanwe na nghọta anụ ahụ, HNA + nuclei, na / ma ọ bụ ọnụnọ nke autofluorescence anụ ahụ. N'ihe onyonyo ọ bụla, e kewara ọnụ ọgụgụ sel NeuN + na HNA + site na ọnụ ọgụgụ sel NeuN + n'otu mpaghara ahụ. Iji hụ na a na-agụta naanị mkpụrụ ndụ nwere oghere n'ime ụgbọ elu onyonyo, naanị sel ndị bụkwa Hoechst + ka etinyere na mgbako. Ihe onyonyo abụọ kewapụrụ ma ọ dịkarịa ala 1 mm bụ nkezi iji belata njehie ọnụ ọgụgụ.
Otu izu tupu ịnakọta ihe nlele, tinye anụ ndị na-atụgharị hCO (ihe dị ka ọnwa 8 nke ọdịiche) n'ime ụlọ gbara ọchịchịrị nke a na-akpụ afụ ọnụ iji belata mkpali mmetụta. Emere ikewapụ oghere dị ka akọwara na mbụ, yana mgbanwe ụfọdụ. Na nkenke, e bibiri t-hCO na hCO site na iji ncha cell-mechanical cell lysis na 2 ml glass tissue grinder (D8938, Sigma-Aldrich/KIMBLE). A na-ekpochapụ oghere ndị ahụ site na iji nzacha 40µm wee kechie ya na 320 g maka nkeji 10 na 4 Celsius C tupu emee gradient sucrose. Mgbe nzọụkwụ centrifugation (320 g maka 20 min na 4 ° C), a na-emegharị ihe nlele ahụ na 0.04% BSA / PBS na mgbakwunye nke 0.2 nkeji nke µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) wee gafere site na ntinye 40.µ A kwụsịrị nuclei ndị ahụ kewapụrụ na PBS nwere 0.02% BSA wee tinye ya na mgbawa Chromium Single Cell 3′ (mgbake nke sel 8,000 n'otu ụzọ). Eji Chromium Single cell 3′ GEM, Library & Gel Bead Kit kwadebere ọba akwụkwọ snRNA-seq v3 (10x Genomics). Eji Chromium Single cell 3′ GEM, Library & Gel Bead Kit kwadebere ọba akwụkwọ snRNA-seq v3 (10x Genomics). Библиотеки snRNA-seq были приготовлены с помощью Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). A kwadebere ọba akwụkwọ snRNA-seq site na iji 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). A kwadebere ọba akwụkwọ snRNA-seq site na iji Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics).Achịkọtara ọba akwụkwọ sitere na nlele dị iche iche site na Admera Health na NovaSeq S4 (Illumina).
A na-agbakọ ọkwa mkpụrụ ndụ ihe nketa maka koodu nzuzo nuklia ọ bụla site na iji ngwugwu ngwanrọ nyocha 10x Genomics CellRanger (ụdị 6.1.2). Kpọmkwem, a na-agụ akwụkwọ ndị ahụ kwekọrọ na nchikota mmadụ (GRCh38, Mgbakọ, ụdị 98) na oke (Rnor_6.0, Ensemble, ụdị 100) genomes nke ejiri iwu mkref mepụta yana iji gụta ya na –include-introns=TRUE iwu ka ọnụọgụgụ gụnyere agụmagụ edebere na mpaghara intron. Maka ihe atụ t-hCO, a chọpụtara nuclei mmadụ dabere na ihe nchekwa chọrọ na opekata mpe 95% nke ihe niile a na-agụ maapụ dabara na genome mmadụ. Emere nyocha niile sochirinụ na nzacha nzacha koodu barcode sitere na CellRanger site na iji ngwugwu R (ụdị 4.1.2) Seurat (ụdị 4.1.1)32.
Iji hụ na ọ bụ naanị nuclei dị elu ka etinyere na nyocha nke na-esote, a na-emejuputa usoro nzacha ihe maka nlele ọ bụla. Nke mbụ, nuclei ndị dị ala nwere ihe na-erughị 1000 mkpụrụ ndụ ihe nketa pụrụ iche achọtara na ihe karịrị 20% nke mkpokọta mitochondria na-achọpụta ma wepụ ya. N'ikpeazụ, mkpụrụ ndụ mkpụrụ ndụ mkpụrụ ndụ mkpụrụ ndụ ihe nketa bụ normalized site n'iji ọrụ sctransform (vst.flavor="v2″) arụ ọrụ, nke chọpụtakwara mkpụrụ ndụ ihe nketa 3000 kachasị agbanwe site na iji paramita ndabara. A na-eme mbelata n'elu mkpụrụ ndụ ihe nketa elu site na iji Analysis Component Component (PCA) na ndabara ndabara site na iji data setịpụ n'ikpere33. Saịtị ma jiri ya mee ihe maka nlele niile na nyocha mkpokọta). DoubletFinder33 ngwugwu (pụtara DoubletFinder akara n'elu 95th percentile) sample t-hCO (n=3) na hCO samples (n=3) jikọtara iche iche site na iji IntegrateData ọrụ na n'elu paramita.
Mgbe ewepụchara mkpụrụ ndụ dị ala, achịkọtara dataset agbakwunyere (mkpebi = 0.5) wee tinye ya maka ebumnuche UMAP34. Achọpụtara mkpụrụ ndụ ihe nrịbama maka ụyọkọ ọ bụla site na iji ọrụ FindMarkers nwere paramita ndabara gbakọrọ site na data ngosipụta mkpụrụ ndụ ahaziri ahazi. Anyị na-achọpụta ma na-ekewa klaasị sel ndị bụ isi site na ijikọta datasets ntụaka nke nwa ebu n'afọ na cortical na mkpụrụ ndụ ihe nrịbama 19,20,21,35 na nkọwapụta. Karịsịa, achọpụtara ndị na-ekesa ụzọ site na ngosipụta nke MKI67 na TOP2A. Akọwapụtara ụyọkọ progenitor site na enweghị transcript nke mitotic, nnukwu ndakọrịta yana ụyọkọ progenitor glial multipotent akọwara na ngwụcha metaphase nwa ebu n'afọ, yana okwu EGFR na OLIG1. Anyị na-eji okwu astrocyte gbakọta ọtụtụ steeti dị iche iche nke astrocyte, site na mbubreyo radial glia ruo maturation nke astrocytes. Ụyọkọ Astrocyte na-egosipụta ọkwa dị elu nke SLC1A3 na AQP4 na egosiri na ha na-eji ụdị subtypes nke nwa ebu n'afọ radial glia na/ma ọ bụ astrocytes okenye. OPC na-egosipụta PDGFRA na SOX10 ebe oligodendrocytes na-egosipụta akara myelination (MOG na MYRF). Achọpụtara neurons Glutamatergic site na ọnụnọ nke transcript neuronal (SYT1 na SNAP25), enweghị akara GABAergic (GAD2), na okwu NEUROD6, SLC17A7, BCL11B, ma ọ bụ SATB2. E kewara neurons GluN gaa n'elu (SATB2 okwu na mfu nke BCL11B) na omimi (BCL11B okwu) subclass. Neurons putative subplate (SP) na-egosipụta akara SP18 ama ama dị ka ST18 na SORCS1 na mgbakwunye na akara GluN miri emi. Achọpụtara mkpụrụ ndụ plexus dị ka Choroid site na okwu TTR, na mkpụrụ ndụ meningeal na-egosipụta mkpụrụ ndụ ihe jikọrọ fibroblast na sel pial / vaskụla mapped nke data ntinye aka.
A na-eme nyocha dị iche iche nke mkpụrụ ndụ ihe nketa n'etiti t-hCO na hCO site na iji usoro pseudo-batch emepụtara ọhụrụ nke ewepụtara n'omume ejiri ngwungwu Libra R (ụdị 1.0.0). N'ụzọ doro anya, a na-eme ule EdgeR log-likelihood (ụdị 3.36.0, ngwugwu R) maka otu dị iche iche site n'ịchịkọta ọnụ ọgụgụ mkpụrụ ndụ ihe nketa dị na sel maka klaasị sel enyere maka mmegharị nlele ọ bụla. Maka nleba anya okpomọkụ, a na-agbakọ ụkpụrụ kwa nde (CPM) site na iji EdgeR (cpm () arụ ọrụ) na nha (iji nweta pụtara = 0, ọkọlọtọ ọkọlọtọ = 1). Gene Ontology (GO) nyocha nke ịba ụba nke mkpụrụ ndụ t-hCO GluN mebere nke ukwuu (Benjamini-Hochberg mezie P uru nke na-erughị 0.05 gosipụtara na opekata mpe 10% nke sel t-hCO GluN yana mmụba nke okpukpu abụọ ma ọ dịkarịa ala ugboro abụọ). Emere ya site na iji ToppGene Suite (https://toppgene.cchmc.org/)37. Anyị na-eji ngwa ToppFun nwere parampat ndabara ma na-akọpụta ụkpụrụ P-ụkpụrụ Benjamini-Hochberg agbaziri site na nnwale hypergeometric GO.
Iji dakọọ ụyọkọ snRNA-seq anyị nwere ụyọkọ mkpụrụ ndụ nkọwapụta site na nyocha ntụaka nke otu cell RNA-seq ma ọ bụ okenye snRNA-seq19,20,21,22, anyị tinyere usoro njikọta dataset jikọtara ọnụ. Anyị na-eji SCTransform (v2) normalization workflow na Seurat iji jikọta ma tụnyere ụyọkọ ụyọkọ n'etiti datasets (iji otu paramita dị n'elu). Edebere ntọala nke onye ọ bụla na-akpaghị aka ruo sel 500 ma ọ bụ cores kwa ụyọkọ izizi maka ịrụ ọrụ mgbako. N'iji usoro yiri nke ahụ dị ka akọwara na mbụ, a kọwapụtara ụyọkọ ụyọkọ dị ka oke nke mkpụrụ ndụ ma ọ bụ nuclei na ụyọkọ ọ bụla agbakọtara nke jikọtara ya na akara nke ụyọkọ ntụaka. Iji nwetakwuo nkewa GluNs, anyị jiri Seurat's TransferData workflow maka GluN subset data ekenye akara nrụbama n'ime sel GluN anyị.
Iji nyochaa ogo ntozu nke transcriptome zuru ụwa ọnụ nke t-hCO na hCO, anyị jiri ihe atụ pseudo-oke tụnyere BrainSpan/psychENCODE23, nke nwere nnukwu usoro RNA na-emetụta ụbụrụ mmadụ. Anyị rụrụ PCA na usoro okwu mkpụrụ ndụ ihe nketa jikọtara ọnụ site na nlele cortical izu 10 ka a tụchara ime na mgbe e mesịrị, na mkpụrụ ndụ ihe nketa 5567 (yana data anyị) nke achọpụtara na mbụ dị ka ndị na-arụ ọrụ n'ụdị cortical BrainSpan (akọwapụtara dị ka ihe karịrị 50% na mgbanwe mmepe kọwara site na afọ site na iji ụdị cubic)38. Na mgbakwunye, anyị nwetara mkpụrụ ndụ ihe nketa jikọtara ya na nnukwu transcriptome mbinye aka nke mmepe neurodevelopment site na iji ihe na-abụghị nke matrix factorization dị ka akọwara na mbụ. A na-atụba ihe nlere anya atụnyere site na iji usoro nrụpụta matriks na-adịghị njọ na fig. 5b nwere data gbasaara maka nke ọ bụla n'ime mbinye aka ise nke Zhu et al.38 kọwara. Ọzọ, ihe nrịbama ederede dabere na ọrụ sitere na ọmụmụ ndị ebipụtara na mbụ. Karịsịa, ERG na LRG na-emeziwanye nke ọma na neurons glutamatergic chọpụtara site na mkpokọta visual cortex snRNA-seq mgbe mkpali anya sitere na tebụl mgbakwunye 3 Hrvatin et al.16. Enwetara LRG nke mmadụ bara ụba site na omenala ụbụrụ nwa ebu n'afọ na-arụ ọrụ nke KCl wee wepụta 6 awa mgbe mkpali gachara, na mkpụrụ ndụ ihe nketa ndị a gbazere na-edozi nke ọma n'ime mmadụ mana ọ bụghị na òké (Nkwupụta 4). A na-enyocha mmụba nke mkpụrụ ndụ ihe nketa site na iji usoro mkpụrụ ndụ ihe nketa ndị a site na iji nyocha otu ụzọ Fisher.
Anesthetize oke na isoflurane, wepụ ụbụrụ na ebe oyi (ihe dị ka 4 Celsius C) oxygenated (95% O2 na 5% CO2) sucrose ngwọta maka ngalaba nwere: 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM. NaH2PO4, 10 mM MgSO4 na 0.5 mM CaCl2 (ihe dịka 310 mOsm). A na-eme akụkụ ụbụrụ ụbụrụ ụbụrụ oke (300-400 µm) nwere t-hCO site na iji Leica VT1200 vibratome dị ka akọwara na mbụ39. A na-ebufe akụkụ n'ime ụlọ nkebi nke nwere ikuku oxygenation na-aga n'ihu n'ime ụlọ nwere aCSF kwadebere site na: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 na 126 mM NaCl (298 mO). opekata mpe nkeji iri anọ na ise tupu edekọ. Edere ngalaba n'ime ụlọ imikpu ebe a na-esi na aCSF (95% O2 na 5% CO2 vial) na-eju ya mgbe niile. Edere data niile na okpomọkụ ụlọ. t-hCO neurons na-ejedebe na borosilicate glass pipette jupụtara na ngwọta nwere 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, na 0.6 mM EGTA, pH 7.2, n'ime ime edozi na KOHs2.9. Iji gbakee, agbakwunyere biocytin (0.2%) na ngwọta ndekọ.
Enwetara data site na iji MultiClamp 700B amplifier (Molecular Devices) na Digidata 1550B digitizer (Molecular Devices), obere ngafe na 2 kHz, digitized na 20 kHz, wee nyochaa site na iji Clampfit (Molecular Devices), Origin (OriginPro). 2021b, OriginLab). na ọrụ MATLAB omenala (Mathworks). Ejiri JPCalc gbakọọ ikike nkwụsịtụ ma gbanwee ndenye ya na uru gbakọrọ nke -14 mV. Ọrụ IV nwere usoro usoro dị ugbu a na usoro 10-25 pA, site na -250 ruo 750 pA.
A na-akpali thalamus, ihe ọcha na ihe S1 na ọkụ eletrik na mpekere thalamocortical n'oge ndekọ mpịachi nke neurons hCO, dị ka akọwara na mbụ. Na nkenke, a na-etinye ụbụrụ n'elu tebụl obibi akwụkwọ 3D gbadaa n'akụkụ 10º, ma na-egbutu ihu ụbụrụ n'akụkụ 35°. A na-ekechikwa ụbụrụ ahụ n'elu ebe a gbuturu ma kewaa ya, na-echekwa axon na-apụta thalamocortical. A na-etinye electrodes tungsten bipolar (0.5 MΩ) na micromanipulator nke abụọ ma debe ya n'ụzọ dabara adaba iji kpalie mpaghara anọ n'otu sel (capsule dị n'ime, ihe ọcha, S1 na hCO). Dekọọ nzaghachi synapti ka 300 µA mkpali phasic na 0.03-0.1 Hz.
hChR2 na-egosipụta hCO neurons na-arụ ọrụ na 480 nm na ọkụ ọkụ nke LED (Prizmatix) mepụtara site na ebumnuche × 40 (0.9 NA; Olympus) iji dekọọ okwu hChR2 n'akụkụ sel. Dayameta nke ọkụ na-enwu bụ ihe dịka 0.5 mm na ike ya bụ 10-20mW. Edobere obosara usu ka ọ bụrụ 10 ms, nke dabara na usu e nyere n'oge nnwale mmụta omume. A na-eji usoro mkpali dị iche iche mee ihe, site na 1 ruo 20 Hz, mana ọ bụ naanị pulse nke mbụ nke usoro ka ejiri mee ihe maka nhazi. Oghere dị n'etiti ụgbọ oloko na-adịkarị ogologo karịa 30s iji belata mmetụta na mgbochi synapti ma ọ bụ ụzọ na-enyere aka. Iji nwalee ma ọ bụrụ na nzaghachi hChR2 bụ monosynaptic, anyị na-etinye TTX (1 μM) na bat ahụ ruo mgbe mmeghachi omume EPSC kwụsịrị, wee tinye 4-aminopyridine (4-AP; 100 μM). Dịka, a na-eweghachi nzaghachi n'ime nkeji ole na ole, na-egbu oge dị ogologo n'etiti ọkụ ọkụ LED na ọgbọ EPSC. Ejiri NBQX (10 μM) nwalee ma ọ bụ ndị nabatara AMPA na-eduzi ya.
E kere akụkụ hCO dị nkọ dị ka akọwara na mbụ. Na nkenke, a na-etinye akụkụ hCO na 4% agarose wee bufee ya na sel nwere 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 na 10 mM NaHCO3 na 10 mM dse (+ 0) - glut n'ime ngalaba 3 (+ 0) µm na ụlọ okpomọkụ na-eji Leica VT1200 vibrator ma chekwaa na ASF n'ime ụlọ okpomọkụ. Mgbe ahụ, a na-eme ndekọ patch-camps nke mkpụrụ ndụ niile na ngalaba hCO n'okpuru microscope SliceScope (Scientifica). Ejiri akụkụ aCSF (95% O2 na 5% CO2) mejupụta akụkụ yana akara ekwentị ka edekọtara na okpomọkụ ụlọ. A na-etinye ihe ntanetị hCO site na iji ọkpọkọ iko borosilicate jupụtara na ngwọta nwere 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, na 0.6 mM EGTA, n'ime pH 7, 2, gbanwee na KOH (osmolarity). Maka ebumnuche mgbake, tinye 0.2% Biocytin na ngwọta dị n'ime.
Enwetara data Clampex (Clampex 11.1, Molecular Devices) site na iji MultiClamp 700B amplifier (Molecular Devices) na Digidata 1550B digitizer (Molecular Devices), obere ngafe na 2 kHz, digitized na 20 kHz, wee nyochaa site na iji Clampfit.6 ngwaọrụ na ụdị MAT. Ọrụ (MATLAB 2019b, Mathworks). Ejiri JPCalc gbakọọ ikike nkwụsịtụ ma gbanwee ndenye ndị ahụ ka enwere ike gbakọọ ọnụ nke -14 mV. Ọrụ IV nwere usoro usoro dị ugbu a na usoro 5-10 pA site na -50 ruo 250 pA.
Maka nrụgharị morphological nke neurons pinched, 0.2% biocytin (Sigma-Aldrich) agbakwunyere na ngwọta dị n'ime. A na-emezi mkpụrụ ndụ ndị ahụ opekata mpe nkeji iri na ise ka ha wachara. A na-eji nwayọọ nwayọọ na-adọta pipette maka 1-2 min ruo mgbe akpụkpọ ahụ edebanyere aha na-emechi kpamkpam. N'ịgbaso usoro physiology nke ngalaba, a na-edozi akụkụ n'otu abalị na 4 ° C. na 4% PFA, na-asa ya na PBS X3, ma gbasaa 1: 1000 na streptavidin-conjugated DyLight 549 ma ọ bụ DyLight 405 (Vector Labs). Edebere mkpụrụ ndụ jupụtara na biocytin (2%; Sigma-Aldrich) n'oge a na-edekọ mkpọchi n'ime ụlọ maka awa 2. A na-etinye akụkụ ndị ahụ na slide microscopy site na iji Aquamount (Thermo Scientific) wee hụ ya n'echi ya na Leica TCS SP8 confocal microscope site na iji ebumnobi imikpu mmanụ nwere oghere ọnụọgụ ×40 1.3, mmụba ×0.9-1.0, xy. Ọnụego nlele bụ ihe dịka 7 pikselụ kwa micron. E nwetara usoro Z-stacks na 1 µm n'usoro, na z-stack mosaics na akpaaka nke dabeere na Leica ka emere iji kpuchie osisi dendritic dum nke neuron ọ bụla. A na-eji aka neuTube 40 nyochaa neurons wee mepụta faịlụ SWC. Ebugoro faịlụ ndị ahụ na ngwa mgbakwunye SimpleNeuriteTracer41 Fiji (ImageJ, ụdị 2.1.0; NIH).
Enwetara anụ ahụ cortical mmadụ site na nkwenye amata dịka ụkpụrụ nke Institutional Review Board nke Mahadum Stanford kwadoro. A na-enweta ihe nlele abụọ nke anụ ahụ ụmụ mmadụ (3 na 18 afọ) site na resection nke frontal cortex (etiti frontal gyrus) dị ka akụkụ nke ịwa ahụ maka refractory epilepsy. Mgbe nhazichara, anụ ahụ na-ewe ihe ubi na ice-oyi NMDG-aCSF nwere: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 2 mM thiourea, 5 mMM thiourea, 5 mMpy5 mMM, sodium ascorvate. CaCl2 4H2O na 10 mM MgSO4 7H2O. Titrate na pH 7.3-7.4 nwere hydrochloric acid gbadoro anya. A na-ebuga anụ ahụ na ụlọ nyocha n'ime nkeji 30 ma were akụkụ coronal dịka usoro akọwara n'elu.
Emere usoro anụmanụ niile dabere na ụkpụrụ nlekọta anụmanụ na Mahadum Stanford APLAC kwadoro. Ejiri 5% isoflurane anesthesia butere oke (karịa ụbọchị 140 ka a tụgharịchara ya) wee were 1-3% isoflurane tinye ya n'ime ahụ. A na-etinye anụmanụ n'ime stereotaxic frame (Kopf) na ntọhapụ na-adịgide adịgide buprenorphine (SR) ka a na-agbanye ya n'okpuru akpụkpọ ahụ. A na-ekpuchi okpokoro isi, kpochaa ma tinye skru 3-5. Iji lekwasịrị anya t-hCO, anyị mepụtara nhazi stereotaxic site na onyonyo MRI. A gwuru oghere burr na saịtị mmasị na eriri (400 µm dayameta, NA 0.48, Doric) wedara 100 µm n'okpuru elu hCO wee chekwaa ya na okpokoro isi ya na ciment eze na-agwọta UV (Relyx).
Emere ndekọ fotometric fiber dị ka akọwara na mbụ42. Iji dekọọ ọrụ mberede, etinyere oke n'ime oghere dị ọcha yana eriri 400 µm dayameta fiber optic patch cable (Doric) ejikọrọ na sistemụ nweta data fiber optic photometric na eriri fiber optic etinyere. N'ime ndekọ 10 nkeji nke ọrụ moto, ụmụ anụmanụ nweere onwe ha inyocha oghere ahụ. Iji dekọọ ọrụ ewepụrụ, oke (ihe karịrị ụbọchị 140 ka a tụgharịchara ya) ka ejiri 5% isoflurane kụnwụ ahụ maka ntinye yana 1-3% isoflurane maka mmezi. Debe anụ ahụ n'ime stereotactic frame (Kopf) na afụ ọnụ nke dị n'akụkụ nke ọzọ nke t-hCO na-edozi ihe dịka 2 cm wee gafee ntupu ejikọrọ na piezoelectric actuator (PI). Ejikọrọ eriri 400µm fiber optic patch cable (Doric) na eriri etinyere wee jikọọ na sistemụ nnweta data. A na-atụgharịkwa afụ ọnụ nke dị n'akụkụ nke ọzọ nke t-hCO ugboro 50 (2 mm na 20 Hz, 2 s kwa ngosi) n'oge enweghị usoro site na piezoelectric mbanye n'ime oge ndekọ 20 nkeji. Jiri ngwugwu nkwado Arduino MATLAB iji jikwaa oge ngbanwe site na iji koodu MATLAB omenala. A na-emekọrịta ihe omume na ngwanrọ nnweta data site na iji transistor-transistor logic (TTL).
Ejiri 5% isoflurane anesthesia butere oke (karịa ụbọchị 140 ka a tụgharịchara ya) wee were 1-3% isoflurane tinye ya n'ime ahụ. A na-etinye anụmanụ n'ime stereotaxic frame (Kopf) na buprenorphine SR na dexamethasone ka a na-agbanye n'okpuru akpụkpọ ahụ. A na-ekpuchi okpokoro isi, kpochaa ma tinye skru 3-5. Iji lekwasịrị anya t-hCO, anyị mepụtara nhazi stereotaxic site na onyonyo MRI. A na-eme craniotomy okirikiri (ihe dị ka 1 cm na dayameta) na-agba ọsọ dị elu ozugbo n'elu hCO a tụgharịrị. Ozugbo ọkpụkpụ ahụ dị gịrịgịrị dị ka o kwere mee, ma tupu ịgbanye ọkpụkpụ ahụ dum, jiri forceps wepụ diski pelvic fọdụrụ na-adịghị emebi emebi iji kpughee t-hCO dị n'okpuru. craniotomy juputara na saline na-adịghị mma, na ihe mkpuchi na ntụtụ isi pụrụ iche ka ejiri ciment eze gwọọ UV (Relyx).
Emere onyonyo foto abụọ site na iji microscope multiphoton Bruker nwere ebumnuche Nikon LWD (×16, 0.8 NA). Emere onyonyo GCaMP6 na 920 nm nwere mmụba 1.4x otu z-ụgbọ elu yana nkezi 8x nke 7.5fps. Ejiri 5% anesthesia isoflurane butere oke ma jiri 1-3% isoflurane dokwa ya. A na-etinye oke ndị ahụ n'ụdị edozi isi emebere ma debe ya n'okpuru oghere. Enwetara ndekọ ndabere nke nkeji 3 nke ọrụ moto. N'ime nkeji 20 nke ndekọ, a na-ebunye puffs 50 (ihe ngosi ọ bụla dị 100 ms ogologo) na enweghị usoro na pad whisker chere ihu t-hCO site na iji picospricer. Jiri ngwugwu nkwado Arduino MATLAB iji jikwaa oge mgbawa na koodu MATLAB omenala. Mekọrịta mmemme na ngwanrọ nnweta data (PrairieView 5.5) site na iji pulses TTL. Maka nyocha, a na-edozi ihe onyonyo a maka mmegharị xy site na iji mmezi affine na mmemme MoCo ewepụtara na Fiji. Mwepụta nke akara fluorescent sitere na sel nke ọ bụla na-eji CNMF-E43. E weputara fluorescence maka mpaghara ọ bụla nke mmasị, tụgharịrị na akụkụ dF/F, wee gbanwee gaa na z-scores.
Ejiri 5% isoflurane anesthesia butere oke (karịa ụbọchị 140 ka a tụgharịchara ya) wee were 1-3% isoflurane tinye ya n'ime ahụ. A na-etinye anụmanụ n'ime stereotaxic frame (Kopf) na buprenorphine SR na dexamethasone ka a na-agbanye n'okpuru akpụkpọ ahụ. A na-egbutu ahịhịa ndị dị n'akụkụ nke ọzọ nke t-hCO na ihe dịka 2 cm wee tinye ya na ntupu ejikọrọ na piezoelectric actuator. A na-ekpuchi okpokoro isi ma kpochaa ya. A na-ejikọta eriri igwe anaghị agba nchara na okpokoro isi. Iji lekwasịrị anya t-hCO, anyị mepụtara nhazi stereotaxic site na onyonyo MRI. Mee craniotomy okirikiri (ihe dị ka 1 cm na dayameta) na-agba ọsọ dị elu karịa t-hCO. Ozugbo ọkpụkpụ ahụ dị gịrịgịrị dị ka o kwere mee, ma tupu ịgbanye ọkpụkpụ ahụ dum, jiri forceps wepụ diski pelvic fọdụrụ na-adịghị emebi emebi iji kpughee t-hCO dị n'okpuru. Edere mkpụrụ ndụ nke ọ bụla site na iji 32-channel ma ọ bụ 64-channel high-density silicon probes (Cambridge Neurotech) gbadoro ụkwụ na skru ala ma jiri RHD amplifiers (Intan). Jiri manipulator wedata electrodes na saịtị ebumnuche site na craniotomy, nke juputara na saline na-adịghị ọcha. A na-eme nchịkọta data n'ogo nke 30 kHz site na iji usoro nnweta data Open Ephys. Ndekọ a gara n'ihu naanị mgbe anyị chọpụtara ọrụ rhythmic nke ukwuu jikọtara ya na ihe karịrị ọwa 10, na-atụ aro na electrodes dị na graft (dabere na data foto calcium foto abụọ). Enwetara ndekọ ndabere nke nkeji 10 nke ọrụ moto. A na-atụgharịkwa afụ ọnụ nke dị n'akụkụ nke ọzọ nke t-hCO ugboro 50 (2 mm na 20 Hz, 2 s kwa ngosi) n'oge enweghị usoro site na piezoelectric mbanye n'ime oge ndekọ 20 nkeji. Iji ngwugwu nkwado MATLAB maka Arduino (MATLAB 2019b), jikwaa oge ngbanwe na koodu MATLAB omenala. Jiri TTL pulses mekọrịta mmemme na ngwanrọ nnweta data.
Maka nnwale nrịbama anya, 200 µm optical patch cord (Doric) ejikọrọ na 473 nm laser (Omicron) jikọtara ya na eriri anya 200 µm etinyere n'elu craniotomy. Ozugbo tupu nke a, gbanwee ike jumper na 20 mW. Jiri manipulator wedata electrodes na saịtị ebumnuche site na craniotomy, nke juputara na saline na-adịghị ọcha. Na mmalite nke ndekọ ahụ, ọkụ iri 473 nm (ugboro ugboro 2 Hz, pulse duration 10 ms). A kọwapụtara mkpụrụ ndụ nwere mmetụta dị ka sel ndị gosipụtara nzaghachi dị elu n'ime 10 ms nke ìhè na 70% ma ọ bụ karịa nke ule.

Oge nzipu: Nov-19-2022