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Hōʻike ʻia nā ʻāpana neural hui ponoʻī i kahi kahua in vitro hoʻohiki no ka hoʻohālikelike ʻana i ka hoʻomohala kanaka a me nā maʻi. Eia nō naʻe, nele nā ​​organoids i ka pilina i loko o ka vivo, kahi e kaupalena ʻia ai ka makua a pale i ka hoʻohui ʻana me nā ʻāpana ʻē aʻe e hoʻomalu i ka ʻano. Ma ʻaneʻi, hōʻike mākou ua hoʻololi ʻia nā cortical organoids i loaʻa i ke kino kanaka i loko o ka somatosensory cortex o nā ʻiole neonatal nude i hoʻomohala i nā ʻano cell makua i hoʻohui ʻia i loko o nā kaapuni pili i ka sensory a me ka hoʻoikaika. Ua hōʻike ʻo MRI i ka ulu ʻana o ka organoid post-transplant i kekahi mau laina stem cell a me nā holoholona, ​​ʻoiai ʻo ka loiloi hoʻokahi-core i hōʻike i ka holomua o ka corticogenesis a me ka puka ʻana o kahi papahana transcription pili i ka hana. ʻOiaʻiʻo, ʻo nā neurons cortical transplanted e hōʻike i nā waiwai morphological, synaptic, a me nā membrane kūloko ma mua o kā lākou in vitro counterparts, e ʻae ana i ka ʻike ʻana i nā hemahema neuronal i nā maʻi me Timothy's Syndrome. Ua hōʻike ʻia ka ʻimi anatomical a me ka hana ʻana e loaʻa i nā organelles transplanted ka thalamocortical a me ka corticocortical inputs, a ʻo nā hoʻopaʻa vivo o ka hana neural e hōʻike ana e hiki i kēia mau mea hoʻokomo ke hoʻoulu i nā pane sensory i loko o nā pūnaewele kanaka. ʻO ka mea hope loa, hoʻolōʻihi nā cortical organoids i nā axons ma ka lolo o ka ʻiole, a ke alakaʻi nei kā lākou optogenetic activation i ka ʻimi ʻimi uku. No laila, ʻoʻo nā neuron cortex kanaka i hoʻololi ʻia a komo i nā kaapuni o ka mea hoʻokipa e hoʻomalu i ka ʻano. Manaʻo mākou i kēia ala e hoʻomaʻamaʻa i ka ʻike ʻana i nā phenotypes strand-level i nā cell i loaʻa i ka maʻi i hiki ʻole ke ʻike ʻia e nā ala ʻē aʻe.
ʻO ka ulu ʻana o ka lolo kanaka he kaʻina hana hoʻonohonoho ponoʻī kahi e hoʻonui ai, hoʻokaʻawale, neʻe, a hoʻohui ʻia e hana i nā kaʻa neuronal hana i hoʻomaʻemaʻe hou ʻia ma o ka ʻike naʻau. ʻO kahi pilikia nui i ka hoʻomaopopo ʻana i ka ulu ʻana o ka lolo o ke kanaka, ʻoi aku hoʻi i ka pōʻaiapili o ka maʻi, ʻo ia ka loaʻa ʻole o ka lolo. Hiki i nā organelles hoʻonohonoho ponoʻī, me nā organoids cortex kanaka (hCO; ʻike ʻia hoʻi ʻo ke poʻo cortex kanaka), hiki ke hoʻohua i 2,3,4,5,6. Eia nō naʻe, ua kaupalena ʻia kekahi mau palena i kā lākou noi ākea e hoʻomaopopo i ka hoʻomohala ʻana a me ka hana ʻana o nā neural circuits. ʻO ka mea nui, ʻaʻole maopopo i ka palena o ka hCO maturation ma muli o ka loaʻa ʻole o kekahi mau microenvironmental a sensory inputs i loaʻa i loko o vivo. Eia kekahi, no ka mea, ʻaʻole i hoʻohui ʻia nā hCO i loko o nā kaʻa kaʻa e hiki ai ke hoʻopuka i nā hopena kūlohelohe, ua kaupalena ʻia kā lākou pono i ka hoʻohālikelike ʻana i nā genetically complex a me nā maʻi neuropsychiatric behavioral.
ʻO ka hoʻololi ʻana o ka hCO i loko o kahi lolo ola paʻa hiki ke lanakila i kēia mau palena. Ua hōʻike mua nā haʻawina i nā neurons kanaka i hoʻololi ʻia i loko o ka cortex rodent hiki ke ola, hana, a kamaʻilio me nā cell rodent7,8,9,10,11,12. Eia nō naʻe, hana maʻamau kēia mau hoʻokolohua ma luna o nā holoholona makua, hiki ke kaupalena i ka synaptic a me ka hoʻohui axonal. Maʻaneʻi, wehewehe mākou i kahi paradigm transplantation kahi i hoʻololi ai mākou i ka 3D hCO i loaʻa mai nā cell hiPS i loko o ka cortex somatosensory mua (S1) o nā ʻiole immunodeficient i ka wā mua o ka ulu ʻana o ka plastic. Hoʻokomo ʻia nā neurons hCO (t-hCO) i hoʻololi ʻia, loaʻa nā thalamocortical a me nā cortical-cortical inputs e hoʻopuka i nā pane sensory, a hoʻonui i nā kuhi axonal i loko o ka lolo iole e hoʻokau i ka ʻimi ʻimi uku. Ua hōʻike ʻia ka hoʻomāhuahua ʻana o ka t-hCO i nā hemahema neuronal i nā mea maʻi me Timothy's Syndrome (TS), kahi maʻi genetic koʻikoʻi i hoʻokumu ʻia e ka hoʻololi ʻana i ke kahawai calcium L-type CaV1.2 (i hoʻopaʻa ʻia e CACNA1C).
No ke aʻo ʻana i nā neuron cortical kanaka i nā kaapuni i loko o vivo, ua hoʻololi mākou i ka stereotactically intact 3D hCO i loko o S1 o nā ʻiole athymic postnatal mua (lā 3-7 postnatally) (Fig. 1a a hoʻonui i ka ʻikepili o Fig. 1a-c). I kēia manawa, ʻaʻole i pau ka thalamocortical a me ka corticocortical axonal projections i kā lākou S1 innervation (ref. 13). No laila, ua hoʻolālā ʻia kēia ala e hoʻonui i ka hoʻohui ʻana o t-hCO oiai e hoʻemi ana i ka hopena ma nā kaʻapuni endogenous. No kaʻikeʻana i kahi o ka t-hCO i nā holoholona ola, ua hana mākou i nā hana hou o ka lolo MRI T2-kaumaha o nāʻiole 2-3 mahina ma hope o ka hoʻololiʻana (Fig. 1b a me kaʻikepili hoʻonui, Fig. 1d). Ua ʻike koke ʻia ka t-hCO a ua like nā ana nui o ka t-hCO me nā mea i helu ʻia mai nā ʻāpana paʻa (Extended Data Fig. 1d,e; P > 0.05). Ua ʻike koke ʻia ka t-hCO a ua like nā ana nui o ka t-hCO me nā mea i helu ʻia mai nā ʻāpana paʻa (Extended Data Fig. 1d,e; P > 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO были аналогичны рассчитанным для фиксированных срезнов (расных срезнов 1d, e; P> 0,05). Ua maʻalahi ka nānā ʻana i ka t-hCO, a ua like nā ana volumetric t-hCO me nā mea i helu ʻia no nā ʻāpana paʻa (ʻikepili hoʻonui, 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 были аналогичны рассчитанным для фиксированных срезнов (расных срезов) 1d, e; P> 0,05). Ua maʻalahi ka nānā ʻana i ka t-hCO, a ua like nā ana volumetric t-hCO me nā mea i helu ʻia no nā ʻāpana paʻa (ʻikepili hoʻonui, Fig. 1d, e; P > 0.05).Ua hoʻoholo mākou i ka t-hCO ma 81% o nā holoholona i hoʻololi ʻia ma kahi o 2 mau mahina ma hope o ka hoʻololi ʻana (n = 72 holoholona; hCO mai nā laina kelepona 10 hiPS; nā laina kelepona hiPS i ka Papa Hoʻohui 1). ʻO kēia mau mea, aia ka 87% i loko o ka cerebral cortex (Fig. 1c). Ma o ka hana ana i ka serial MRI scans ma na manawa he nui i ka iole transplanted hookahi, ua loaa ia makou he eiwa-palua o ka nui o ka t-hCO iloko o 3 mahina (Fig. 1d a me ka mea i hoonuiia, Fig. 1f). He kiʻekiʻe ke ola o nā holoholona i hoʻololi ʻia (74%) ma 12 mau mahina ma hope o ka hoʻololi ʻana (nā ʻikepili i hoʻonui ʻia, Fig. 1g a me ka Papa Hoʻohui 2), a ʻaʻohe mea i ʻike ʻia i ka mīkini a i ʻole ka hoʻomanaʻo ʻana, gliosis, a i ʻole electroencephalogram (EEG). ʻIkepili Fig. 1g a me ka papa hoʻohui 2). 1h–m a me 3e).
a, Hoʻolālā o ka hoʻolālā hoʻokolohua. Ua hoʻololi ʻia ka hCO i loaʻa mai nā cell hiPS i S1 o nā ʻiole olohelohe hānau hou i nā lā 30-60 o ka hoʻokaʻawale ʻana. b, T2-kaumaha coronal a me nā kiʻi MRI ākea e hōʻike ana i ka t-hCO ma S1 2 mau mahina ma hope o ka hoʻololi ʻana. ʻO ka pā unahi, 2 mm. c, ʻO ka helu ʻana o nā helu kūleʻa engraftment i hōʻike ʻia no kēlā me kēia laina kelepona hiPS (n = 108, nā helu i loko o nā pahu e hōʻike ana i ka nui o t-hCO no ka laina kelepona hIPS) a me kahi cortical a subcortical paha (n = 88). d, ke kiʻi MRI o ke aʻa coronary (hema; pālākiō, 3 mm) a me 3D volumetric reconstruction (scale bar, 3 mm) e hōʻike ana i ka piʻi ʻana o ka t-hCO ma luna o 3 mahina. e, Nānā i nā ʻano t-hCO i loko o ka ʻiole cerebral cortex. Paʻa unahi, 1 mm. f, Nā kiʻi immunocytochemical Lunamakaʻāinana o t-hCO i hōʻike ʻia mai luna hema a i ka ʻākau (i ka wā o ka hoʻokaʻawale ʻana): PPP1R17 (4 mau mahina), NeuN (8 mau mahina), SOX9 a me GFAP (8 mau mahina), PDGFRα; (8 mahina), MAP2 (8 mahina) a me IBA1 (8 mahina). ʻO ka pā unahi, 20 µm. Hōʻike ka ʻōlelo like ʻana o ka HNA i nā cell o ke kumu kanaka. g, snRNA-seq: Unified manifold and projection (UMAP) dimensionality imaging imaging of high quality t-hCO nuclei after Seurat integration (n=3 t-hCO samples, n=2 hiPS cell lines). ʻO nā Astrocytes, nā pūnaewele o ka laina astrocyte; cyc prog, circulating progenitors; ʻO GluN DL, nā neurons glutamatergic hohonu; ʻO GluN DL / SP, nā neurons glutamatergic hohonu a me ka sublamellar; ʻO GluN UL, nā neurons glutamatergic papa luna; oligodendrocytes, oligodendrocytes; OPC, oligodendrocyte progenitor cell; RELN, nā neurons reelin. h, Gene Ontology (GO) hua'ōlelo enrichment anamanaʻo o genes nui upregulated (hoʻololi P <0.05, fold loli> 2, hōʻike 'ia ma ka liʻiliʻi loa 10% o ka nuclei) ma t-hCO glutamatergic neurons hoohalikeia me hCO glutamatergic neurons. h, Gene Ontology (GO) hua'ōlelo enrichment anamanaʻo o genes nui upregulated (hoʻololi P <0.05, fold loli> 2, hōʻike 'ia ma ka liʻiliʻi loa 10% o ka nuclei) ma t-hCO glutamatergic neurons hoohalikeia me hCO glutamatergic neurons. h, Анализ обогащения терминов Gene Ontology (GO) для генов со значительной активацией (скорректированный P <0,05, кратмость иять > экспрессия по крайней мере в 10% ядер) в глутаматергических нейронах t-hCO по сравнению с глутаматергическиромим. h, Gene Ontology (GO) hua'ōlelo enrichment anamanaʻo no nā genes me ka nui ho'ā 'ana (hoʻololi P<0.05, fold loli> 2, hōʻike ma ka liʻiliʻi loa 10% nuclei) ma t-hCO glutamatergic neurons hoʻohālikelike i ka 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 ， 0.5 P <. 2，在至少10% 的细胞核中表达）的基因本体论(GO) 术语富集分析。 h ， 与 hco 谷氨酸 能 元 相比 ， t-hco 谷氨酸 能 神经 元 基因 显着 上调 （后 后 .变化> 2 ， 至少 10% 的 核中 表达） 基因 基因 基因 的 的 的 的 的 的 的 的 的 的 的 的 的 的 )术语富集分析。 h, гены значительно активизировались (скорректированный P <0,05, кратность изменения> 2, экспрессируется по крайне в 1% глутаматергических нейронах t-hCO по сравнению с глутаматергическими нейронами hCO Онтологический (GO) анаглиз тебяониа. h, ua hoʻonui nui ʻia nā genes (hoʻololi ʻia ʻo P <0.05, hoʻololi ʻia> 2, i hōʻike ʻia ma ka liʻiliʻi o 10% o ka nuclei) i nā neurons t-hCO glutamatergic i hoʻohālikelike ʻia me ka hCO glutamatergic neurons Ontological (GO) ka nānā ʻana o ka huaʻōlelo enrichment.Hōʻike ka laina kiko i ka waiwai aq o 0.05. i, UMAP kiʻi o nā ʻano cell GluN i t-hCO me ka hoʻohana ʻana i ka hoʻoili lepili mai kahi hōʻike 22 snRNA-seq kanaka makua kaʻa cortex dataset. CT - corticothalamic cell, ET - extracerebral cell, IT - kūloko telencephalic cell, NP - kokoke i ka projection.
A laila loiloi mākou i ka cytoarchitecture a me ka hoʻohui pū ʻana o ka t-hCO. Ua hōʻike ʻia ka hoʻopaʻa ʻana o ka antibody o nā cell endothelial rat i ka vascularization me ka t-hCO, ʻoiai ʻo ka IBA1 staining i hōʻike ʻia i ka hele ʻana o ka microglia ʻiole i loko o ka graft (Fig. 1f a me ka ʻikepili hoʻonui, Fig. 3c, d). Ua hōʻike ʻo Immunostaining i ke kanaka nuklea antigen (HNA) mau pūnana maikaʻi e hōʻike ana i ka PPP1R17 (cortical progenitors), NeuN (neurons), SOX9 a me GFAP (glial-derived cell) a i ʻole PDGFRα (oligodendrocyte progenitors) (Figure 1f). No ke aʻo ʻana i ka haku mele o ka t-hCO ma ka hoʻonā ʻana o ke kelepona hoʻokahi, ua hana mākou i ka sequencing RNA hoʻokahi-core (snRNA-seq) ma hope o 8 mau mahina o ka hoʻokaʻawale ʻana. ʻO ka kānana nui a me ka wehe ʻana i ka nuclei ʻiole i loaʻa i 21,500 mau palapala ʻāina mononuclear kanaka kiʻekiʻe (Fig. 1g a me ka ʻikepili hoʻonui, Fig. 4a,b). ʻO nā hiʻohiʻona hōʻike o nā hōʻailona cell-type maʻamau i ʻike ʻia i nā pūʻulu o nā papa cortical cell nui, me nā neurons glutamatergic hohonu a me ka papa, nā progenitors e holo nei, oligodendrocytes, a me ka laina astrocyte (Fig. 1g, ka ʻikepili i hoʻonui ʻia, Fig. 4c, a me ka Papa Hoʻohui 3). ʻO ka Immunostaining no SATB2 a me CTIP2 i hōʻike ʻia ʻoiai ʻo ka loaʻa ʻana o nā subtypes cortical, ʻaʻole hōʻike ʻo t-hCO i ka stratification anatomical maopopo (ʻike i hoʻonui ʻia, Fig. 3a). ʻO ka snRNA-seq hCO i hoʻohālikelike ʻia i ka pae i hoʻopuka ʻia i nā papa cell like like, me kekahi mau ʻokoʻa, me ka ʻole o nā oligodendrocytes a me ka hele ʻana o nā neurons GABAergic, e hōʻike ana i nā kūlana in vitro maikaʻi i hōʻike mua ʻia no nā cell progenitor lateral15 (ʻike i hoʻonui ʻia, Fig. 4f - i a me ka Papa Hoʻohui 4). Hōʻike ʻia ka hōʻike hōʻike ʻano ʻano like ʻole i nā ʻokoʻa nui o nā neurons glutamatergic ma waena o t-hCO a me hCO (Supplementary Table 5), me ka hoʻoulu ʻana o nā pūʻulu o nā genes e pili ana i ka maturation neuronal e like me ka synaptic signaling, dendritic localization, a me ka hana kaila-gated channel (Figure 1h a me ka Papa Hoʻohui 5). papa 6). No laila, ua hōʻike ʻia nā neurons cortical glutamatergic t-hCO i ka wikiwiki transcriptional maturation.
No ka wehewehe ʻana inā pili kēia mau hoʻololi transcriptional i t-hCO i nā ʻokoʻa morphological ma waena o ka hCO in vitro a me ka t-hCO in vivo, ua kūkulu hou mākou i ka hCO a me ka hCO piha i ka biocytin i ka pae ma nā ʻāpana koʻikoʻi ma hope o 7-8 mau mahina o ka hoʻokaʻawale ʻana. hCO neurons (Fig. 2a). ʻOi aku ka nui o nā neurons t-hCO, he 1.5 ka nui o ke anawaena o ka soma, ʻelua ka nui o nā dendrites, a me ka piʻi ʻana o ka lōʻihi dendritic holoʻokoʻa ʻeono i hoʻohālikelike ʻia me ka in vitro hCO (Fig. 2b). Eia kekahi, ua ʻike mākou i kahi kiʻekiʻe kiʻekiʻe o nā spines dendritic ma nā neurons t-hCO ma mua o nā neurons hCO (Fig. 2c). Hōʻike kēia i nā neurons t-hCO e loaʻa i ka elongation dendritic nui a me ka lālā, i hui pū ʻia me ka hoʻomau ʻana o ka cell proliferation, hiki ke kōkua i ka ulu ikaika o t-hCO ma hope o ka hoʻololi ʻana (Fig. 1d and Extended Data Fig. 1f). Ua koi kēia iā mākou e noiʻi i nā waiwai electrophysiological. ʻEwalu ke kiʻekiʻe o ka capacitance membrane (nā ʻikepili i hoʻonui ʻia, Fig. 8d), ʻoi aku ka hyperpolarized o ka hiki ke hoʻomaha-state (e pili ana i 20 mV), a ua hoʻokomo ʻia ka injection o kēia manawa i kahi kiʻekiʻe kiʻekiʻe o nā neurons t-hCO ma mua o nā neurons hCO. in vitro (Fig. 2d), e), i kūlike me nā hiʻohiʻona morphological nui a paʻakikī o t-hCO. Eia kekahi, ʻoi aku ka kiʻekiʻe o ka manawa o nā hanana postsynaptic excitatory spontaneous (EPSC) i nā neurons t-hCO (Fig 2f), e hōʻike ana i ka hoʻonui ʻia o ka nui o nā spines dendritic i ʻike ʻia i nā neurons t-hCO i pili me ka excitability hana. synapse moe kolohe. Ua hōʻoia mākou i ke ʻano ʻokoʻa o nā neurons hCO in vitro ma o ka hoʻopaʻa ʻana i nā neurons glutamatergic i kapa ʻia (ka ʻikepili hoʻonui, Fig. 6a-c).
a, 3D hana hou o biocytin-piha hCO a me t-hCO neurons ma hope o 8 mahina o ka hoʻokaʻawale. b, Ka helu o nā hiʻohiʻona morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 a me *** P <0.0001). b, Ka helu o nā hiʻohiʻona morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 a me *** P <0.0001). б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,001). b, ka helu o nā hiʻohiʻona morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, a me *** 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 P = 0.0179 。 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 P = 0.0179 。 б, количественная оценка морфологических признаков (n = 8 нейронов hCO, n = 6 нейронов t-hCO; ** P = 0,0084, * P = 0,001). b, ka helu o nā hiʻohiʻona morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179, a me *** P <0.0001).c, hana hou 3D o hCO a me t-hCO dendritic lala ma hope o 8 mahina o ka hoʻokaʻawale. Hōʻike nā asterisk ʻulaʻula i nā spines dendritic putative. Dendritic spine density quantification (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0092). d, Ka helu o ka hikiwawe membrane hoʻomaha (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). d, Ka helu o ka hikiwawe membrane hoʻomaha (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, hoʻomaha hoʻomaha membrane hiki ke helu (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, hoʻomaha hoʻomaha membrane hiki ke helu (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Hoʻoulu hou i ka hana hou i ka hCO a me ka t-hCO i hoʻoulu ʻia e ka hoʻonui ʻana i nā injections o kēia manawa, a me ka helu ʻana o ka nui o ka puhi ahi (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e, Hoʻoulu hou i ka hana hou i ka hCO a me ka t-hCO i hoʻoulu ʻia e ka hoʻonui ʻana i nā injections o kēia manawa, a me ka helu ʻana o ka nui o ka puhi ahi (n = 25 hCO neurons, n = 16 t-hCO neurons; ***P <0.0001). e. возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, hiki ke hana hou i ka hCO a me t-hCO i hoʻoulu ʻia e ka piʻi ʻana o kēia manawa a me ka helu ʻana o ka nui o ka puhi ahi (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e，通过增加电流注入诱导的hCO 和t-hCO 重复动作电位放电，以及最大放电率的量化 = CO2神经元，n = 16 个t-hCO 神经元；***P < 0.0001）。 E ， 通过 增加 电流 注入 的 的 hco 和 t-hco 重复 电位 放电 ， 以及 最 大 的 量化 （n2（n ， n = 16 个 t-hco 神经 ； *** p <0.0001） 。 e. скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, ke puhi hou 'ana o ka hCO a me ka t-hCO mau mana hana i ho'oulu 'ia e ka ho'onui 'ana i ka lako o kēia manawa a me ka helu 'ana i ka nui o ka puhi ahi (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) i hCO a me t-hCO neurons ma 8 mahina o ka hoʻokaʻawale ʻana, a me ka helu ʻana o ka pinepine o nā hanana synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) ma hCO a me t-hCO neurons ma 8 mahina o ka hoʻokaʻawale ʻana, a me ka helu ʻana o ka pinepine o nā hanana 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 частотыч синтич 5 нейронов hCO, n = 17 нейронов t-hCO; *** P <0,0001). f, Spontaneous EPSCs (sEPSCs) i hCO a me t-hCO neurons ma 8 mahina o ka hoʻokaʻawale ʻana a me ka helu ʻana o nā hanana hanana synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P<0.0001). f，分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs (sEPSCs)，以及突触事件频率的量化（n CO = 烈n 的量化（n 17 t-hCO 神经元；***P < 0.0001） . f，分化8 个月时hCO 和t-hCO 神经元中的自发性EPSCs (sEPSCs)，以及突触事件频率的量匼2（n CO =2（n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 месяцев дифференцировки и количественная оценка частотыч синтич 5 нейронов hCO, n = 17 нейронов t-hCO; f, Spontaneous EPSCs (sEPSCs) i hCO a me t-hCO neurons ma 8 mahina o ka hoʻokaʻawale a me ka helu ʻana o nā hanana hanana synaptic (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P<0.0001).No ka bf, hCO a me t-hCO ma ka laina 1208-2 i lawe ʻia mai ka pūʻulu hoʻokaʻawale like i mālama ʻia i ka like. g, Gene set enrichment analysis (hoʻokahi ʻaoʻao ʻo Fisher's exact test) o nā genes i hoʻonui nui ʻia (hoʻololi ʻia P <0.05, hoʻololi ʻia> 2, i hōʻike ʻia ma ka liʻiliʻi o 10% o ka nuclei) i nā neurons t-hCO glutamatergic i hoʻohālikelike ʻia me nā hCO glutamatergic me nā pūʻulu gene o ka pane mua-pane (ERG) a me ka genes pane mua (ERG) noiʻi ʻiole16 a me nā LRG kikoʻī kanaka mai nā neurons in vitro17. g, Gene set enrichment analysis (hoʻokahi ʻaoʻao ʻo Fisher's exact test) o nā genes i hoʻonui nui ʻia (hoʻololi ʻia P <0.05, hoʻololi ʻia> 2, i hōʻike ʻia ma ka liʻiliʻi o 10% o ka nuclei) i nā neurons t-hCO glutamatergic i hoʻohālikelike ʻia me nā hCO glutamatergic me nā pūʻulu gene o ka pane mua-pane (ERG) a me ka genes pane mua (ERG) noiʻi ʻiole16 a me nā LRG kikoʻī kanaka mai nā neurons in vitro17. g. изменения > 2, экспрессия по меньшей мере в 10% ядер) в глутаматергических нейронах t-hCO по сравнению по сравнению с глимичам hCO наборы генов как раннего (ERG), так и позднего (LRG) генов, зависящих от активности, идентифицированных в иванных в иснисла и специфических для человека LRG из нейронов in vitro17. g, ka nānā ʻana o ka hoʻonui ʻana i ka gene set (hoʻokahi-tailed Fisher's exact test) o nā genes me ka hoʻoulu nui ʻana (hoʻololi ʻia P<0.05, hoʻololi ʻia> 2, hōʻike i ka liʻiliʻi o 10% o ka nuclei) i nā neurons t-hCO glutamatergic i hoʻohālikelike ʻia me nā hCO glutamatergic neurons sets o ka hana mua (ERG) a me ka hope (LRG) i ʻike ʻia i loko o ka hana ʻana o ka genesce-midependen (LRG) Nā LRG mai nā neurons in vitro17. g，t-hCO谷氨酸能神经元与hCO谷氨酸能神经元相比，t -hCO谷氨酸能神经元显着上调（调整后P<0.05，倍数变化>2，在至少10%的细胞核中表达）的基因集富集分析（单侧F isher精确检验）从体内小鼠研究中鉴定的早期反应(ERG)和晚期反应(LRG) 活性依赖性基因的基因组16 和体外神经元17 中的人类特异性LRG。 g ， t-hco 谷氨酸 神经 元 与 hco 谷氨酸 神经 元 相比 ， t-hco 谷氨酸 神经 元 上调 元 上调 . ， 倍数> 2 ， 至少 至少 10%的 细胞 核中 表达） 的 集富集 分析 （单侧 fisher 精确）研究 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 的 基因 璌 16神经元 17 中 中 17 中 17的人类特异性LRG。 g. P<0,05, кратность изменения> 2, не менее 10% Анализ обогащения набора генов (односторонний точный тест Фишетра) позднего гены, зависящие от активности ответа (LRG), идентифицированные в исследованиях на мышах in vivo16 и in vivo16 и in l для человека. g, t-hCO glutamatergic neurons ua nui upregulated hoʻohālikelike i ka hCO glutamatergic neurons (hoʻololi P<0.05, fold change>2, ma ka liʻiliʻi loa 10% Early pane (ERG) a me ka hope pane hoʻonui waiwai ana (one-tailed Fisher's exact test) pane hana dependent genes (LRGs) i ikeia i loko o vivo in vitro7 mice16 neurons.Hōʻike ka laina kiko i kahi waiwai P i hoʻoponopono ʻia e Bonferroni o 0.05. h, GluN gene expression (pseudo-package and scaling of each gene) ua hoʻonui nui ʻia i loko o snRNA-seq replicas o LRG genes i t-hCO glutamatergic neurons. i, immunostaining e hōʻike ana i ka SCG2 hōʻike ma t-hCO (luna) a me hCO (lalo) neurons. Hōʻike nā pua keʻokeʻo i nā pūnaewele SCG2+. ʻO ka pā unahi, 25 µm. Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± kaʻe maʻamau.
Ma muli o ka hoʻonui ʻana i ka hana o t-hCO i ʻike ʻia i nā ʻāpana ex vivo, ua hōʻike ʻo snRNA-seq i ka hoʻoponopono ʻana i ka hana o nā transcript gene i t-hCO i hoʻohālikelike ʻia me ka hCO in vitro. ʻO nā neurons Glutamatergic t-hCO i hōʻike i nā pae kiʻekiʻe o nā genes e hoʻoponopono ana i ka hana pane hope (Fig. 2g, h), i loaʻa i nā haʻawina mua i ka ʻiole a me nā neurons16,17. No ka laʻana, BDNF18, SCG2, a me OSTN, he primate-specific activity-regulating gene, hōʻike i ka hoʻonui ʻana i ka ʻōlelo ma nā neurons t-hCO i hoʻohālikelike ʻia me nā neurons hCO (Fig. 2g-i). No laila, ua hōʻike ʻia nā neurons t-hCO i nā hiʻohiʻona maturation i hoʻohālikelike ʻia i nā neurons hCO e ka transcriptional, morphological, a me nā loiloi hana.
No ka loiloi hou ʻana i ka hui ʻana o ka maturation t-hCO me ka ulu ʻana o ka lolo o ke kanaka, ua hana mākou i nā hoʻohālikelike transcriptomic o ka fetal a me ka makua cortical cell type19,20 a me ka adult21,22 a me ka ʻikepili nui e pili ana i ka cortical gene expression23 i ka wā o ka hoʻomohala ʻana (ka ʻikepili hoʻonui, Fig. 5). ). me ka hana mua 24, ka honua hCO a me ka t-hCO transcriptome maturation kūlana ma 7-8 mahina o ka hoʻokaʻawale 'ana i laulaha like me ka in vivo ulu manawa a ua like loa me ka hope fetal ola (Extended Data Fig. 5a). ʻO ka mea nui, ua ʻike mākou i ka hoʻonui ʻana o ka transcriptome maturity i ka t-hCO i hoʻohālikelike ʻia me ka hCO i hoʻohālikelike ʻia i ka makahiki, a me ka hoʻoulu ʻana o ka transcriptome e pili ana me synaptogenesis, astrogenesis, a me ka myelination (expanded data, Fig. 5b-d). Ma ka pae cellular, ua ʻike mākou i nā hōʻike o kahi subtype cortex thinner i t-hCO, me nā pūʻulu o nā neurons glutamatergic e uhi ana me nā subtypes neuron L2 / 3, L5, a me L6 (Figure 1i). I ka hoʻohālikelike ʻana, ʻoi aku ka palena o ka puʻupuʻu ma waena o nā neurons glutamatergic t-hCO a me nā neuron cortical fetal i ka waena o ka hāpai ʻana (ka ʻikepili hoʻonui, Kiʻi 5e-j). No ka hoʻoholo ʻana inā he hana like nā neurons t-hCO me nā neocortical neocortical postnatal kanaka, ua hana mākou i nā hoʻopaʻa electrophysiological a me nā anatomical reconstructions o nā neurons pyramidal L2 / 3 kanaka i nā ʻāpana ʻoi o ka cortex postnatal kanaka (ʻike i hoʻonui ʻia, Fig. 7a). ʻO nā mea electrophysiological o L2 / 3 pyramidal neurons ua like ia me nā t-hCO pyramidal neurons (ʻike i hoʻonuiʻia, Fig. 7e). Morphologically, L2 / 3 neurons mai postnatal kanaka laʻana ua oi like me ka t-hCO ma mua o ka hCO, oiai L2/3 pūnaewele ua lōʻihi, i loko o nā lālā oi aku a pau, a he kiʻekiʻe kiʻekiʻe iwi kuamoʻo mānoanoa (Fig. 3g a hoʻonuiʻikepili, Fig. 7b-). G).
a, hoʻololi i ka hCO i hana ʻia e ka mana a me nā laina kelepona TS hiPS i nā ʻiole neonatal. b, hana hou 3D o nā neurons t-hCO piha biocytin ma hope o 8 mau mahina o ka hoʻokaʻawale ʻana. c, ka helu o ka mean dendritic lōʻihi (n = 19 control neurons, n = 21 TS neurons; ** P = 0.0041). d, 3D-reconstructed dendritic lala mai ka mana a me TS t-hCO ma 8 mahina o ka hoʻokaʻawaleʻana, a me ka quantification o dendritic spine density (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001). d, 3D-reconstructed dendritic lala mai ka mana a me TS t-hCO ma 8 mahina o ka hoʻokaʻawaleʻana, a me ka quantification o 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 контроля и количественности дендритных шипов (n = 16 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, hana hou 3D o nā lālā dendritic mai ka mana a me t-hCO TS ma 8 mahina o ka hoʻokaʻawale a me ka 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 煪化（n = 16 煪化21 个TS 神经元，***P < 0.0001）。 d ， 分化 8 个 时 对照 和 ts t-hco 的 3d 重建 分支 分支 以及 树突棘 密度 量化 （n = 窮元 ， n = 21 个 ts 神经 ， *** p <0.0001 ）。 d, 3D-pulepulepule hoʻonaʻauao ветвей контроля и TS t-hCO через 8 месяцев дендритных ветвей контроля и TS t-hCO колести шипов (n = 16 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, hana hou 3D o ka mana dendritic lala a me TS t-hCO ma 8 mahina o ka hoʻokaʻawale a me ka dendritic spine density quantification (n = 16 control neurons, n = 21 TS neurons, *** P <0.0001).Hōʻike nā asterisk ʻulaʻula i nā spines dendritic putative. e, nā EPSC kuʻuna i ka mana a me nā neurons TS t-hCO ma hope o 8 mau mahina o ka hoʻokaʻawale ʻana. f, kumulative frequency plot and quantification of frequency and amplitude of synaptic events (n=32 control neurons, n=26 TS neurons; **P=0.0076 a me P=0.8102). g, Ke kālailai Scholl o TS a me ka hoʻomalu i nā neurons i hCO a me t-hCO. Hōʻike nā laina kuʻi i nā neurons pyramidal L2/3 postnatal kanaka no ka hoʻohālikelike (n = 24 mana t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 mana hCO neurons, a n = 7 TS hCO neurons). Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± maʻamau deviation
ʻO ka hiki i ka t-hCO ke hoʻopili i nā hiʻohiʻona morphological a me ka hana o nā neuron cortex kanaka ma kahi kiʻekiʻe i koi iā mākou e ʻimi inā hiki ke hoʻohana ʻia ka t-hCO e ʻike i nā phenotypes maʻi. Ua kālele mākou iā TS, kahi maʻi neurodevelopmental koʻikoʻi i hoʻokumu ʻia e ka loaʻa ʻana o ka hana hoʻololi i ka gene encoding CaV1.2, ka mea e hoʻomaka ai i ka transcription gene pili i ka hana ma nā neurons. Loaʻa iā mākou ka hCO mai ʻekolu mau maʻi TS e lawe ana i ka hoʻololi maʻamau (p.G406R) a me ʻekolu mau mana (Fig. 3a). Ma hope o ka hoʻololiʻana, uaʻike mākou ua hoʻololiʻia ka dendritic morphology i nā TS neurons i hoʻohālikelikeʻia me nā mana (Fig. 3b a me ka hoʻonuiʻana i kaʻikepili, Fig. 8a, b), me ka piʻiʻelua o ka nui o nā dendrites mua a me ka piʻi nui o ka mean a me ka emiʻana o ka lōʻihi dendritic (Fig. 3c a me kaʻikepili hoʻonui, Fig. 8c). Ua pili kēia me ka hoʻonui nui ʻana o nā spines a me ka hoʻonui pinepine ʻana o nā EPSC spontaneous i TS i hoʻohālikelike ʻia me ka mana o nā neurons (Fig. 3d-f a me ka ʻikepili hoʻonui, Fig. 8g). Ua hōʻike hou ʻia ka nānā ʻana i nā hiʻohiʻona o ka lālā dendritic maʻamau i ka t-hCO TS i hoʻohālikelike ʻia me nā mana, akā ʻaʻole i loko o ka in vitro TS hCO ma kahi pae like o ka ʻokoʻa (Fig. 3g). Kūlike kēia me kā mākou hōʻike mua o ka hoʻohaʻahaʻa dendritic e pili ana i ka hana ma TS a hōʻike i ka hiki o kēia kahua transplant ke ʻike i nā phenotypes maʻi ma vivo.
A laila nīnau mākou i ka nui o ka hoʻohui ʻia ʻana o nā cell t-hCO i loko o ka ʻiole S1. Loaʻa iā S1 i nā rodents nā mea hoʻokomo synaptic ikaika mai ka ipsilateral ventral basal a me posterior thalamic nuclei, a me ka ipsilateral motor a me ka lua somatosensory cortices, a me ka contralateral S1 (Fig. 4a). No ka hoʻihoʻi ʻana i ke ʻano innervation, ua hoʻopili mākou i ka hCO me ka maʻi rabies-dG-GFP/AAV-G a hoʻololi i ka hCO i loko o ka ʻiole S1 3 mau lā ma hope. Uaʻike mākou i ka'ōlelo GFP nui ma nā neurons o ka ipsilateral S1 a me ka ventral basal ganglia 7-14 mau lā ma hope o ka hoʻololiʻana (Fig. 4b, c). Eia kekahi, ua hōʻike ʻia ka hoʻopaʻa ʻana o ka antibody o ka thalamic marker netrin G1 i ka loaʻa ʻana o nā hopena thalamic i t-hCO (Fig. 4d, e). No ka loiloi inā hiki i kēia mau kuhi afferent ke kiʻi i nā pane synaptic i nā cell t-hCO, ua hana mākou i nā hoʻopaʻa kelepona holoʻokoʻa mai nā pūnaewele kanaka ma nā ʻāpana koi o ka papa thalamocortical. ʻO ka hoʻoulu ʻana o ka ʻiole S1, capsule kūloko, mea keʻokeʻo, nā fiber kokoke i ka t-hCO a i ʻole optogenetic activation o opsin-expressing thalamic endings i t-hCO i hoʻokomo ʻia i ka pōkole-latency EPSC i nā t-hCO neurons i hōʻike ʻia i ka AMPA antagonist antagonist NBQX. (Fig. 4f, g a me ka ʻikepili hoʻonui, Fig. 9a-g). Hōʻike kēia mau ʻikepili ua hoʻohui ʻia ka t-hCO i loko o ka lolo ʻiole a hiki ke hoʻāla ʻia e ka ʻiʻo host rat.
a, Kiʻi kiʻi kiʻi o kahi hoʻokolohua ʻimi rabies. b, GFP a me ke kanaka-specific STEM121 hōʻike ma waena o t-hCO a me ka rat cerebral cortex (ka papa luna). Hōʻike pū ʻia ka hōʻike GFP ma ka ʻiole ipsilateral ventral basal nucleus (VB) (hema hema) a me ka ipsilateral S1 (lalo ʻākau). ʻO ka pā unahi, 50 µm. Hōʻike nā ʻāpana ʻulaʻula i nā wahi o ka lolo kahi i lawe ʻia ai nā kiʻi. c, ka helu ʻana o nā cell e hōʻike ana i ka GFP (n = 4 mau ʻiole). d, e - Netrin G1+ mau pahu thalamic i t-hCO. d hōʻike i kahi ʻāpana coronal i loaʻa i ka nuclei t-hCO a me VB. ʻO ka pā unahi, 2 mm. e hōʻike ana i ka ʻōlelo Netrin G1 a me STEM121 i nā neurons t-hCO (hema) a me VB (ʻākau). ʻO ka pā unahi, 50 µm. Hōʻike ka laina kiko ʻalani i ka palena t-hCO. f, g, Nā meheu o kēia manawa o nā neurons t-hCO ma hope o ka hoʻoulu ʻana i ka uila i ka ʻiole S1 (f) a i ʻole ka capsule kūloko (g), me (ʻulaʻula) a i ʻole (ʻeleʻele) NBQX (hema). EPSC amplitudes me NBQX a me ka ʻole (n = 6 S1 neurons, * P = 0.0119; a me n = 6 kūloko capsule neurons, ** P = 0.0022) (waena). ʻO ka pākēneka o nā neurons t-hCO e hōʻike ana i ka EPSC i ka pane ʻana i ka hoʻoulu uila o ka ʻiole S1 (f) a i ʻole ka capsule kūloko (g) (ʻākau). aCSF, ka wai cerebrospinal artificial. h, kiʻi kiʻi kiʻi o ka hoʻokolohua kiʻi 2P (hema). Hōʻike o GCaMP6s ma t-hCO (waena). ʻO ka pā unahi, 100 µm. ʻO ka hala manawa fluorescence o GCaMP6s (ʻākau). i, Z-helu o ka hana kuʻuna fluorescence. j, kii hoohiwahiwa o ka hooulu mui. k, z-scored 2P fluorescence trajectories i loko o hoʻokahi ho'āʻo, i kulike me ka whisker deviation i ka manawa ʻaʻohe (laina kaha) ma ka laʻana cell. l, nā pane z-helu helu o ka heluna kanaka o nā pūnaewele a pau i hoʻohālikelike ʻia me ka hoʻokaʻawale ʻana o ka whisker i ka manawa ʻaʻohe (laina kahakiʻi) (ʻulaʻula) a i ʻole nā ​​kaha manawa i hana ʻia (hina). m. Kiʻikuhi schematic o ka hoʻokolohua ma ka mākaʻikaʻi optical. n, Nā pihi uila makaʻala mai kahi laʻana t-hCO cell i ka wā o ka hoʻoulu ʻana i ka laser polū. Hōʻike nā pua ʻulaʻula i nā wili mua i hoʻokumu ʻia e ka māmā (luna) a i ʻole ma muli o ka haʻalele ʻana o ka whisker (lalo). Hōʻike ka pale hina hina i nā wā o ka haʻalele ʻana o ka ʻumiʻumi. o, Nā ʻano nalu māmā kiʻekiʻe a me nā pane hoʻoheheʻe ʻana. p, nā piko o ka ho'āʻo hoʻokahi, i kūlike me ka ʻae ʻana o nā ʻūhā i loko o nā keena o ka laʻana. Hōʻike ka 0 i ka hoʻokaʻawale ʻana o ka ʻumiʻumi (laina kaha). q, ka helu kiʻi kiʻi z-helu helu helu kanaka no nā keena kiʻi paʻi kiʻi a pau, i hoʻohālikelike ʻia me ka hoʻokaʻawale ʻana i ka whisker i ka manawa ʻaʻohe (laina kaha ʻia) (ʻulaʻula) a i ʻole nā ​​kaha manawa i hana ʻia (hina). r, Hoʻololi nui ʻia ka ʻāpana o nā ʻāpana kiʻi kiʻi kiʻi e ka haʻalele ʻana o ka ʻūhā (n = 3 mau ʻiole) (hema). Peak z-score latency (n = 3 mau ʻiole; n = 5 (māmā ʻōmaʻomaʻo), n = 4 (ʻeleʻele ʻōmaʻomaʻo), a me n = 4 (cyan) mau ʻāpana hoʻololi hoʻololi i ka ʻiole) ('ākau). Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± kaʻe maʻamau
A laila nīnau mākou inā hiki ke hoʻāla ʻia ka t-hCO e nā mea hoʻoulu ʻia i loko o vivo. Hoʻololi mākou i ka hCO e hōʻike ana i nā hōʻailona calcium i hoʻopili ʻia i ka genetically GCaMP6 i nā ʻiole S1. Ma hope o 150 mau lā, hana mākou i ka fiber photometry a i ʻole ʻelua-photon calcium imaging (Fig. 4h a hoʻonui i ka ʻikepili, Fig. 10a). Ua ʻike mākou ua hōʻike ʻia nā cell t-hCO i ka hana rhythmic synchronized (Figure 4i, Expanded Data, Figure 10b and Supplementary Video 1). No ka hoʻohālikelike ʻana i ka hana kiʻekiʻe t-hCO, ua hana mākou i nā hoʻopaʻa hoʻopaʻa electrophysiological extracellular i nā ʻiole transplant anesthetized (ʻikepili hoʻonui, Fig. 10c-f). Ua hana mākou i nā hoʻonohonoho stereotaxic mai nā kiʻi MRI; No laila, ʻo kēia mau ʻāpana i hoʻopaʻa ʻia e hōʻike ana i nā neurons kanaka, ʻoiai ʻo ka electrophysiology wale nō ʻaʻole e ʻae i kahi ʻano kumu e hoʻoholo ai. Ua ʻike mākou i nā puʻupuʻu hana i hoʻonohonoho ʻia (nā ʻikepili i hoʻonui ʻia, Fig. 10d). Ua lōʻihi ka pahū ʻana ma kahi o 460 ms a ua hoʻokaʻawale ʻia e nā manawa hāmau ma kahi o 2 s (ka ʻikepili hoʻonui, Fig. 10d, e). Hoʻokuʻu ʻia nā ʻāpana pākahi i ka awelika o ʻekolu pōʻai i kēlā me kēia pahū, ʻo ia hoʻi ma kahi o 73% o nā ʻāpana i hoʻopaʻa ʻia i kēlā me kēia pahu. Hoʻopili nui ʻia nā hana o kēlā me kēia ʻāpana, a ʻoi aku ka kiʻekiʻe o kēia mau hoʻopili ʻana ma mua o nā ʻāpana i ʻike ʻia i nā holoholona i hoʻopaʻa ʻole ʻia i hoʻopaʻa ʻia ma lalo o nā kūlana like (ʻike i hoʻonui ʻia, Fig. 10f). No ka ʻike hou ʻana i nā pane spike o nā neurons i loaʻa i ke kanaka, ua hana mākou i nā hoʻokolohua hoʻomaʻamaʻa māmā i nā ʻiole anesthetized i hoʻololi ʻia me ka hCO e hōʻike ana i ke kahawai cation māmā rhodopsin 2 (hChR2), ma o ka t-hCO neurons short-latency recognition (emi iho ma mua o 10 ms) i ka pane ʻana i ka stimuli kukui polū (Fiog. Ua hōʻike ʻia nā neurons t-hCO i nā puʻupuʻu o ka hana kuʻuna i nā alapine e like me nā mea i ʻike ʻia ma ke kiʻi calcium, a me nā hoʻopaʻa electrophysiological i hana ʻia ma t-hCO me ka loaʻa ʻole o ka māka kukui (ʻike i hoʻonui ʻia, Fig. 10c-g). ʻAʻole i ʻike ʻia kahi hana kuʻuna i nā pae kūpono o ka hCO i hoʻopaʻa ʻia i loko o ka vitro. No ka hoʻomaopopo ʻana inā hiki ke hoʻāla ʻia ka t-hCO e nā mea hoʻoulu manaʻo, ua hoʻokaʻawale pōkole mākou i nā ʻumi ʻiole mai ka t-hCO (Fig. 4j,m a me ka ʻikepili hoʻonui, Fig. 10h,k). Wahi a nā haʻawina mua 8,10, ua hōʻike ʻia kahi ʻāpana o nā pūnaewele t-hCO i ka hoʻonui ʻana i ka hana ma ka pane ʻana i ka deflection whisker, ʻaʻole i ʻike ʻia i ka wā i hoʻohālikelike ʻia ka ʻikepili me nā peʻa manawa maʻamau (Fig. 4k-q a me ka ʻikepili hoʻonui, Fig. 10h-q). ʻOiaʻiʻo, ma kahi o 54% o nā ʻāpana hoʻokahi opto-labeled i hōʻike i ka piʻi nui ʻana o ka hoʻoulu ʻana ma hope o ka hoʻoulu ʻana i ka whisker, e piʻi ana ma kahi o 650 ms (Fig. 4r). Hoʻopili pū ʻia, hōʻike kēia mau ʻikepili e loaʻa i ka t-hCO nā hoʻokomo hana kūpono a hiki ke hoʻāla ʻia e nā mea hoʻoulu kaiapuni.
A laila ua noiʻi mākou inā hiki i ka t-hCO ke hoʻāla i nā kaapuni i nā ʻiole e kāohi i ka ʻano. Ua noiʻi mua mākou inā pili nā axons o t-hCO neurons i nā ʻiʻo a puni o ka ʻiole. Ua hoʻopili mākou i ka hCO me kahi lentivirus e hoʻopili ana i ka hChR2 i hui pū ʻia me EYFP (hChR2-EYFP). Ma hope o nā lā 110, ua nānā mākou i ka hōʻike EYFP ma nā ʻāpana cortical ipsilateral, me ka auditory, motor, a somatosensory cortices, a me nā ʻāpana subcortical, me ka striatum, hippocampus, a me thalamus (Fig. 5a). No ka nānā ʻana inā hiki i kēia mau manaʻo efferent ke kiʻi i nā pane synaptic i nā cell rat, ua hoʻāla ʻia mākou i nā cell t-hCO e hōʻike ana i ka hChR2-EYFP ma ka hoʻopaʻa ʻana i nā cell cerebral cortex rat i nā ʻāpana o ka lolo. ʻO ka hoʻoulu ʻana o nā axons t-hCO me nā kukui polū i hoʻoulu ʻia i nā EPSC pōkole pōkole i loko o nā neurons cortex pyramidal rat, i kāohi ʻia e NBQX (Fig. 5b-g). Eia kekahi, hiki ke pale ʻia kēia mau pane e ka tetrodotoxin (TTX) a hoʻihoʻi ʻia e 4-aminopyridine (4-AP), e hōʻike ana ua hoʻokumu ʻia lākou e nā pilina monosynaptic (Fig. 5e).
a, Hoʻolālā kiʻi o ka ʻimi axon (hema). t-hCO EYFP hōʻike (akau). ʻO ka pā unahi, 100 µm. A1, apo pepeiao, ACC, cingulate cortex mua, d. striatum, dorsal striatum, HPC, hippocampus; Diaphragm, lateral septum, mPFC, medial prefrontal cortex, piri, piriform cortex, v. striatum, ventral striatum, VPM, ventropostomedial nucleus o ka thalamus, VTA, ventral tegmental region. Hōʻike nā ʻāpana ʻulaʻula i nā wahi o ka lolo kahi i lawe ʻia ai nā kiʻi. b, Kiʻi kiʻi kiʻi o ka hoʻokolohua hoʻoulu. c, d, Nā hiʻohiʻona o ka pane ʻana o ka photocurrent kukui polū (luna) a me ka uila (lalo) i loko o ke kanaka (c) EYFP + t-hCO a i ʻole ʻiole (d) EYFP- pūnaewele. e, f, ʻO nā hiʻohiʻona o kēia manawa o nā neurons iole ma hope o ka hoʻoulu ʻana i ka kukui polū o nā axons t-hCO me TTX a me 4-AR ('ōmaʻomaʻo), TTX (hina) a i ʻole aCSF (ʻeleʻele) (e), me (violet) a i ʻole (ʻeleʻele) ) ) NBQX (e). g, ka lōʻihi o nā pane i hoʻoulu ʻia e ke kukui polū i loko o nā ʻiole (n = 16 cell); Hōʻike nā kaola hoʻopaʻa i ka latency maʻamau (7.13 ms) (hema). ʻO ka nui o nā EPSC i hoʻomālamalama ʻia i hoʻopaʻa ʻia me ka NBQX a ʻole ʻole (n = 7 cell; ***P <0.0001) (waena). ʻO ka nui o nā EPSC i hoʻomālamalama ʻia i hoʻopaʻa ʻia me ka NBQX a ʻole ʻole (n = 7 cell; ***P <0.0001) (waena). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (ma центре). Hoʻopaʻa ʻia ka nui o nā EPSC i hoʻopili ʻia me ka NBQX (n = 7; ***P <0.0001) (waena).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) (ma центре). Hoʻopaʻa ʻia ka nui o nā EPSC i hoʻopili ʻia me ka NBQX (n = 7; ***P <0.0001) (waena).Ka pākēneka o nā ʻiole e hōʻike ana i nā EPSC e pane ana i ke kukui polū (ʻākau). h, kiʻi kiʻi kiʻi o kahi hana kuʻuna. d0, la 0. i. Hana ʻia nā holoholona hoʻohālike i ka lā 1 (hema) a i ʻole lā 15 (ʻākau) o ke aʻo ʻana. ʻO ka helu maʻamau o nā licks i hana ʻia ma ka lā 1 (hema) a i ʻole lā 15 (waena ʻākau) (n = 150 mau hoʻāʻo kukui polū, n = 150 mau hoʻāʻo ʻulaʻula; ***P <0.0001). ʻO ka helu maʻamau o nā licks i hana ʻia ma ka lā 1 (hema) a i ʻole lā 15 (waena ʻākau) (n = 150 mau hoʻāʻo kukui polū, n = 150 mau hoʻāʻo ʻulaʻula; ***P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) a i день 15 (ma центре справа) (n = 150 испытаний с =ситомим с =ситомим испытаний с красным светом; ***P <0,0001). Ka helu maʻamau o nā licks i hana ʻia ma ka lā 1 (hema) a i ʻole lā 15 (waena ʻākau) (n = 150 mau hoʻāʻo kukui polū, n = 150 mau hoʻāʻo ʻulaʻula; ***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) a i день 15 (ma центре справа) (n = 150 испытаний с =ситомим с =ситомим испытаний с красным светом; ***P <0,0001). Ka helu maʻamau o nā licks i hana ʻia ma ka lā 1 (hema) a i ʻole lā 15 (waena ʻākau) (n = 150 mau hoʻāʻo kukui polū, n = 150 mau hoʻāʻo ʻulaʻula; ***P <0.0001).ʻO nā licks kumulative no nā hoʻāʻo ʻulaʻula a me ka uliuli ma ka lā 1 (waena hema) a i ʻole lā 15 (akau). NS, ʻaʻole nui. j, k, Nā hiʻohiʻona o nā holoholona āpau i hoʻololi ʻia me ka t-hCO e hōʻike ana i ka hChR2-EYFP (j) a i ʻole ka mana fluorophore (k) ma ka lā 1 a i ʻole 15 (hChR2-EYFP: n = 9 mau ʻiole, ** P = 0.0049; mana: n = 9, P = 0.1497). l, Evolution o ka helu koho (n = 9 hChR2, n = 9 mana; **P <0.001, ***P <0.0001). l, Evolution o ka helu koho (n = 9 hChR2, n = 9 mana; **P <0.001, ***P <0.0001). l, Эволюция показателя предпочтения (n = 9 hChR2, n = 9 контрольных; **P <0,001, ***P <0,0001). l, Evolution o ka helu koho (n = 9 hChR2, n = 9 mana; **P <0.001, ***P <0.0001). l，偏好评分的演变（n = 9 hChR2，n = 9 对照；**P < 0.001，***P < 0.0001）。 l，偏好评分的演变（n = 9 hChR2，n = 9 对照；**P < 0.001，***P < 0.0001）。 l, Эволюция показателей предпочтения (n = 9 hChR2, n = 9 контролей; **P <0,001, ***P <0,0001). l, Evolution o nā helu koho (n = 9 hChR2, n = 9 mana; **P <0.001, ***P <0.0001).m, ka ʻōlelo FOS i ka pane ʻana i ka optogenetic activation o t-hCO ma S1. Hōʻike ʻia nā kiʻi o ka huaʻōlelo FOS (hema), a me ka helu (n = 3 no kēlā me kēia hui; *P <0.05, **P <0.01 a me ***P <0.001) (ʻākau). Hōʻike ʻia nā kiʻi o ka huaʻōlelo FOS (hema), a me ka helu (n = 3 no kēlā me kēia hui; *P <0.05, **P <0.01 a me ***P <0.001) (ʻākau). Показаны изображения экспрессии FOS (слева) a me количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 и *** P <0,01 и *** P). Hōʻike ʻia nā kiʻi o ka ʻōlelo FOS (hema) a me ka helu (n = 3 no kēlā me kēia hui; *P<0.05, **P<0.01, a me ***P<0.001) hōʻike ʻia (akau).显示了FOS 表达（左）和量化（每组n = 3；*P < 0.05、**P < 0.01 和***P < 0.001）（叾僳）显示了FOS 表达（左）和量化（每组n = 3；*P < 0.05、**P < 0.01 和***P < 0.001）（叾僳） Показаны изображения экспрессии FOS (слева) a me количественного определения (n = 3 на группу; * P <0,05, ** P <0,01 и *** P <0,01 и *** P). Hōʻike ʻia nā kiʻi o ka ʻōlelo FOS (hema) a me ka helu (n = 3 no kēlā me kēia hui; *P<0.05, **P<0.01, a me ***P<0.001) hōʻike ʻia (akau).ʻO ka pā unahi, 100 µm. Hōʻike ʻia ka ʻikepili ma ke ʻano he mean ± hewa maʻamau o BLA, basolateral tonsil, MDT, dorsomedial thalamic nucleus, PAG, periaqueductal hina.
ʻO ka hope, nīnau mākou inā hiki i ka t-hCO ke hoʻololi i ka ʻano ʻiole. No ka hoʻāʻo ʻana i kēia, ua hoʻololi mākou i ka hChR2-EYFP-hōʻike i ka hCO i S1, a me 90 mau lā ma hope, ua hoʻokomo mākou i nā fiber optical i t-hCO no ka lawe māmā ʻana. A laila, hoʻomaʻamaʻa mākou i nā ʻiole me ka hoʻololi ʻana o ka operant conditioning paradigm (Fig. 5h). Hoʻonoho mākou i nā holoholona i loko o kahi keʻena hoʻāʻo ʻano a hoʻohana i ka 5 kekona blue (473 nm) a me ka ʻulaʻula (635 nm) laser stimuli. Ua loaʻa i nā holoholona ka uku wai inā lākou e palu i ka wā o ka hoʻoulu ʻana i ke kukui polū akā ʻaʻole i palu i ka wā o ka hoʻoulu ʻana i ke kukui ʻulaʻula. I ka lā mua o ka hoʻomaʻamaʻa ʻana, ʻaʻohe ʻokoʻa o nā holoholona i ka licking ke hoʻoulu ʻia me ke kukui uliuli a ʻulaʻula paha. Eia naʻe, ma ka lā 15, ua hoʻololi ʻia nā holoholona me ka hCO e hōʻike ana i ka hChR2-EYFP i hōʻike i ka licking ikaika i ka wā i hoʻoulu ʻia me ke kukui polū i hoʻohālikelike ʻia me ka hoʻoulu ʻana i ke kukui ʻulaʻula. ʻAʻole i ʻike ʻia kēia mau hoʻololi ʻana i ka hana licking i nā holoholona hoʻomalu i hoʻololi ʻia me ka hCO e hōʻike ana i ka fluorophore control (ka helu kūleʻa o ke aʻo ʻana: hChR2 89%, EYFP 0%, Kiʻi 5i-1 a me ke wikiō hou 2). Hōʻike kēia mau ʻikepili e hiki i nā cell t-hCO ke hoʻāla i nā neurons rat e hoʻoulu i ka ʻimi ʻimi uku. No ka ʻike ʻana i ka ʻiole t-hCO neural kaapuni i komo i kēia mau ʻano hoʻololi, ua hoʻoikaika mākou i ka t-hCO i loko o nā holoholona i hoʻomaʻamaʻa ʻia a ʻohi ʻia nā ʻiʻo he 90 mau minuke ma hope. Ua hōʻike ʻo Immunohistochemistry i ka hōʻike ʻana o ka protein FOS e pili ana i ka hana ma kekahi mau ʻāpana lolo e pili ana i ka ʻano hoʻoikaika, me ka medial prefrontal cortex, medial thalamus, a me ka periaqueductal hina mea, i hōʻike ʻia i loko o nā holoholona mālama ʻole a i ʻole nā ​​holoholona. laiki. 5m). Hoʻohui pū ʻia, hōʻike kēia mau ʻikepili e hiki i ka t-hCO ke hoʻololi i ka hana neuronal rat e hoʻokele i ka ʻano.
Hōʻike nā neural organoids i kahi ʻōnaehana hoʻohiki no ke aʻo ʻana i ka hoʻomohala ʻana o ke kanaka a me ka maʻi in vitro, akā ua kaupalena ʻia lākou e ka nele o nā pilina ma waena o nā kaapuni i loko o vivo. Ua hoʻomohala mākou i kahi kahua hou kahi i hoʻololi ai mākou i ka hCO i S1 o nā ʻiole postnatal immunocompromised mua e aʻo i ka hoʻomohala ʻana o ke kanaka a me ka hana ma vivo. Ua hōʻike mākou ua hoʻomohala ʻo t-hCO i nā ʻano cell makua ʻaʻole i ʻike ʻia ma vitro28 a ua hoʻohui ʻia ka t-hCO i loko o ka lolo rodent. ʻO ka hoʻohui ʻana o ka t-hCO i loko o nā kaʻa neural rodent ua ʻae iā mākou e hoʻokumu i kahi loulou ma waena o ka hana kelepona kanaka a aʻo i ka ʻano holoholona, ​​​​e hōʻike ana e hiki i nā neurons t-hCO ke hoʻololi i ka hana neuronal iole e hoʻokau i nā pane ʻano.
He ʻoi aku ka maikaʻi o ka paepae a mākou e wehewehe nei ma mua o ka noiʻi mua e pili ana i ka hoʻololi ʻana i nā pūnaewele kanaka i loko o ka lolo rodent. ʻO ka mea mua, ua hoʻololi mākou i ka hCO i loko o ka cortex e ulu ana o nā ʻiole postnatal mua, hiki ke maʻalahi i ka hoʻohui anatomical a me ka hana. ʻO ka lua, ua ʻae ka nānā ʻana i ka t-hCO MRI iā mākou e aʻo i ke kūlana graft a me ka ulu ʻana o nā holoholona ola, e ʻae iā mākou e hana i nā haʻawina multi-holoholona lōʻihi a hoʻokumu i ka hilinaʻi o nā laina kelepona hiPS. ʻO ka mea hope loa, ua hoʻololi mākou i nā organoids intact, ma mua o ka hoʻokaʻawale ʻana i ka hoʻokuʻu ʻia ʻana o ke kelepona hoʻokahi, ʻoi aku ka liʻiliʻi o ka luku ʻana i nā cell kanaka a hiki ke hoʻoikaika i ka hoʻohui ʻana a me ka hana ʻana o nā neuron cortex kanaka i loko o ka lolo ʻiole.
Hoʻomaopopo mākou ʻoiai ʻo ka holomua ʻana o kēia kahua, nā kaohi ʻana o ke kino, spatial, a me nā ʻano like ʻole e pale i ka hoʻokumu ʻia ʻana o nā kaʻa neural kanaka me ka hilinaʻi kiʻekiʻe, ʻoiai ma hope o ka hoʻololi ʻana i ka wā mua o ka ulu ʻana. No ka laʻana, ʻaʻole maopopo inā ʻo ka hana kuʻuna i ʻike ʻia ma ka t-hCO e hōʻike ana i kahi phenotype hoʻomohala e like me ka hana rhythmic i ʻike ʻia i ka wā o ka ulu ʻana o ka cortical, a i ʻole ma muli o ka nele o nā ʻano cell suppressive i loko o t-hCO. Pēlā nō, ʻaʻole maopopo i ka nui o ka loaʻa ʻole o ka lamination ma t-hCO e pili ana i ka hoʻopili kaulahao30. E nānā ka hana i ka wā e hiki mai ana i ka hoʻohui ʻana i nā ʻano cell ʻē aʻe e like me microglia kanaka, nā cell endothelial kanaka, a me nā ʻano like ʻole o GABAergic interneurons e like me ka hōʻike ʻana me ka hoʻohana ʻana i ka hui 6 in vitro, a me ka hoʻomaopopo ʻana i ke ʻano o ka hoʻohui ʻana a me ka hana ʻana i hiki ke hoʻololi i ka t-hCO. transcriptional, synaptic a me nā pae behavial i nā cell i loaʻa mai nā maʻi.
ʻO ka holoʻokoʻa, hōʻike kēia kahua in vivo i kahi kumuwaiwai ikaika e hiki ke hoʻokō i ka ulu ʻana o ka lolo kanaka a me ka noiʻi maʻi in vitro. Manaʻo mākou e ʻae kēia kahua iā mākou e ʻike i nā phenotypes strand-level hou i nā cell i loaʻa i nā mea maʻi a hoʻāʻo i nā hoʻolālā therapeutic hou.
Hana mākou i ka hCO2.5 mai nā pūnaewele HiPS e like me ka mea i wehewehe mua ʻia. No ka hoʻomaka ʻana i ka hana hCO mai nā cell hiPS i moʻomeheu ʻia ma nā papa feeder, ua wehe ʻia nā kolone paʻa o nā cell hiPS mai nā kīʻaha moʻomeheu me ka hoʻohana ʻana i ka dispase (0.35 mg/mL) a hoʻololi ʻia i nā moʻomeheu plastic ultra-low attachment i loaʻa nā kīʻaha me ka moʻomeheu cell hiPS. (Corning) i hoʻohui ʻia me ʻelua SMAD inhibitors dorsomorphine (5 μM; P5499, Sigma-Aldrich) a me SB-431542 (10 μM; 1614, Tocris) a me ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). I nā lā 5 mua, ua hoʻololi ʻia ka hiPS cell medium i kēlā me kēia lā a ua hoʻohui ʻia ka dorsomorphine a me SB-431542. I ke ono o ka lā o ka hoʻokuʻu ʻana, ua hoʻoneʻe ʻia nā neural spheroids i ka neural medium e loaʻa ana ka neurobasal-A (10888, Life Technologies), B-27 supplement me ka ʻole o ka vitamin A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin a me streptomycin (1:100, Life Technologies) a hoʻohui ʻia me ka e (1 ml − −pidermal growth factor); Nā Pūnaehana R&D) a me ka fibroblast growth factor 2 (FGF2; 20 ng ml−1; R&D Systems) a hiki i ka lā 24. Mai ka lā 25 a hiki i ka lā 42, ua hoʻonui ʻia ka medium me ka brain-derived neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) a me ka neurotrophin 3 (NT3; 20) nā hoʻololi ʻē aʻe i kēlā me kēia lā. I ke ono o ka lā o ka hoʻokuʻu ʻana, ua hoʻoneʻe ʻia nā neural spheroids i ka neural medium e loaʻa ana ka neurobasal-A (10888, Life Technologies), B-27 supplement me ka ʻole o ka vitamin A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin a me streptomycin (1:100, Life Technologies) a hoʻohui ʻia me ka e (1 ml − −pidermal growth factor); Nā Pūnaehana R&D) a me ka fibroblast growth factor 2 (FGF2; 20 ng ml−1; R&D Systems) a hiki i ka lā 24. Mai ka lā 25 a hiki i ka lā 42, ua hoʻonui ʻia ka medium me ka brain-derived neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) a me ka neurotrophin 3 (NT3; 20) nā hoʻololi ʻē aʻe i kēlā me kēia lā.I ke ono o ka lā i hoʻokuʻu ʻia, ua hoʻoneʻe ʻia nā neural spheroids i kahi neural medium i loaʻa ʻo Neurobasal-A (10888, Life Technologies), B-27 supplement me ka ʻole o ka vitamina 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) a me фактором роста фиброб (0 GF2в; 2мбром Nā Pūnaehana R&D) mai 24-го дня. a me streptomycin (1:100, Life Technologies) a hoʻohui ʻia me ka epidermal growth factor (EGF; 20 ng/ml; R&D Systems) a me ka fibroblast growth factor 2 (FGF2; 20 ng/ml; R&D Systems) a hiki i ka lā 24.Mai nā lā 25 a hiki i 42, ua hoʻohui ʻia ka mea neurotrophic i loaʻa i ka lolo (BDNF; 20 ng ml-1, Peprotech) a me ka neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) i ka waena, e hoʻololi ana i ka waena i kēlā me kēia lā.在悬浮的第6 天，将神经球体转移到含有neurobosal-A（10888，Life Technologies）、不含维生素A 的B-27补充剂（12587，Life Technologies）、GlutaMax（1:100，Life Technologies）、青霉素的神经培养基中和链霉素（1:10（1:10 Technologies）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（F ngR&D；20 Systems）直至第24 天。在 悬浮 的 第 第 6 天 将 神经 球体 转移 含有 含有 neurobasal-a （10888 ， Life Technologies） 不 含 的紀2补充剂 （12587 ， Life Technologies） Glutamax （1: 100 ， Life TechNOGIS青霉素 的 神经 培养 基 中 链霉 （1（素 （1（素 （1（素生长 因子 （（（20 ng ml-1 ； r & d Pūnaehana） 成 纤维 细胞 生长 2 （fgf2 ； 20 ng ml- 1；笛紬备＇ No 6-й день суспензии нейросферы были переведены на добавку, содержащую нейробазал-А (10888, Life Technologies), доб-вав7 А (12587, Life Technologies), GlutaMax (1:100, Life Technologies), пенициллин- нейтрализованный стрептомицин (1:100, Life Technologies) с добавлением эпидермальакото (GF22котор; нг мл-1; Ma ka lā 6, ua hoʻololi ʻia nā hoʻokuʻu neurosphere i kahi mea hoʻohui i loaʻa i ka neurobasal-A (10888, Life Technologies), B-27 supplement me ka ʻole o ka vitamina A (12587, Life Technologies), GlutaMax (1:100, Life Technologies), penicillin-neutralized streptomycin (1:100, Life Technologies) i hoʻohui ʻia me ka epidermal 1 & 2 growth factor. fibroblast ulu kumu 2 (FGF2; 20 ng ml-1) 1; Nā Pūnaehana R&D) mai 24-го дня. R&D Systems) a hiki i ka lā 24.Mai nā lā 25 a hiki i ka 42, ua hoʻohui ʻia ka neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) a me ka neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) i ka moʻomeheu i kēlā me kēia lā. Hoʻololi liʻiliʻi i hoʻokahi manawa.E hoʻomaka ana mai ka lā 43, mālama ʻia ka hCO i ke ʻano neurobasal-A i hoʻohui ʻole ʻia (NM; 1088022, Thermo Fisher) me ka loli waena i kēlā me kēia 4-6 lā. No ka loaʻa ʻana o ka hCO mai nā cell hiPS i moʻomeheu ʻia ma lalo o nā kūlana feederless, ua hoʻopili ʻia nā cell hiPS me Accutase (AT-104, Innovate Cell Technologies) ma 37 ° C no 7 mau minuke, hoʻokaʻawale ʻia i loko o nā cell hoʻokahi, a hoʻopaʻa ʻia ma nā pā AggreWell 800 (34815, STEMCELL Technologies) ma kahi kiko o 3 × 106 pūnāwai hoʻohui i ka pūnāwai hoʻokahi ROCK. Y-27632 (10 μM; S1049, Selleckchem). Ma hope o 24 mau hola, ua hoʻopili ʻia ka media i loko o nā pūnāwai i luna a i lalo i loko o ka media e loaʻa ana ka media Essential 6 (A1516401, Life Technologies) i hoʻohui ʻia me dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) a me SB-431542 (10 μM; 1614). , Tocrida). Mai nā lā 2 a hiki i 6, ua hoʻololi ʻia ʻo Essential 6 medium i kēlā me kēia lā me ka dorsomorphine a me ka hoʻohui SB-431542. Mai ke ono o ka lā, ua hoʻoneʻe ʻia nā hoʻokuʻu neurosphere i ke ʻano neurobasal a mālama ʻia e like me ka mea i hōʻike ʻia ma luna.
Ua hana ʻia nā kaʻina hana holoholona a pau e like me nā alakaʻi mālama holoholona i ʻae ʻia e ka Stanford University Laboratory Animal Care Administrative Committee (APLAC). Ua kūʻai ʻia nā ʻiole euthymic RNU (rnu/+) hāpai ʻia (Charles River Laboratories) a i ʻole hale. Mālama ʻia nā holoholona ma kahi pōʻaiapuni māmā-ʻeleʻele he 12 mau hola me ka meaʻai a me ka wai ad libitum. Ua ʻike ʻia nā keiki ʻiole olohelohe (FOXN1–/–) i ʻekolu a ʻehiku mau lā ma muli o ka ulu ʻana o nā ʻumi ʻumi ʻōʻō ma mua o ka ʻoki ʻia ʻana. Hoʻopili ʻia nā puppies (kāne a wahine) me ka 2-3% isoflurane a kau ʻia ma kahi kiʻi stereotaxic. Ua hana ʻia kahi trepanation o ke poʻo me ke anawaena o kahi o 2-3 mm ma luna o S1 i ka wā e mālama ana i ka pono o ka dura mater. A laila e hoʻohana i kahi nila 30-G (ma kahi o 0.3 mm) ma waho pono o ka craniotomy e hou i ka dura. A laila e hoʻopili i ka HCO i kahi parafilm 3x3 knm lahilahi a hoʻoneʻe i ka mea ʻoi aku. Ke hoʻohana nei i kahi pūkini Hamilton i hoʻopili ʻia i kahi nila 23 G, 45°, e huki mālie i ka hCO i ka hope loa o ka nila. A laila e hoʻokomo i ka syringe ma ka pauma syringe i hoʻopili ʻia i ka mea stereotaxic. A laila e kau i ka piko o ka nila ma luna o kahi puka puncture ākea 0.3 mm i hana mua ʻia i loko o ka dura (z = 0 mm) a hoʻokaʻawale i ka syringe 1-2 mm (z = ma kahi o –1.5 mm) a hiki i ka hiki ʻana o ka nila ma waena o ka dura mater A. ua hoʻokumu ʻia kahi sila paʻa. A laila e hoʻokiʻekiʻe i ka syringe i ke kikowaena o ka ʻili cortical ma z = -0.5 mm a hoʻokomo i ka hCO ma ka wikiwiki o 1-2 µl i kēlā me kēia minuke. Ma hope o ka pau ʻana o ka hCO injection, hoʻihoʻi ʻia ka nila ma ka wikiwiki o 0.2-0.5 mm i kēlā me kēia minuke, hoʻopaʻa ʻia ka ʻili, a hoʻokomo koke ʻia ka ʻīlio ma luna o kahi pā hoʻomehana wela a hiki i ka hoʻihoʻi ʻana. Ua hoʻololi ʻia kekahi mau holoholona ma ka lua.
Ua hana ʻia nā kaʻina hana holoholona a pau e like me nā alakaʻi mālama holoholona i ʻae ʻia e ke Kulanui ʻo Stanford APLAC. Hoʻokomo ʻia nā ʻiole (ʻoi aku ma mua o 60 mau lā ma hope o ka hoʻololi ʻana) me ka 5% isoflurane anesthesia a me ka anesthetized me 1-3% isoflurane i ka wā kiʻi. No ka hiʻohiʻona, ua hoʻohana ʻia kahi 7 Tesla i pale ikaika ʻia ʻo Bruker (Bruker Corp.) me ka International Electric Company (IECO) gradient drive, kahi hoʻokomo gradient pale me ka anawaena o loko o 120 mm (600 mT/m, 1000 T/m/s) i hoʻohana ʻia me ka AVANCE. III, 8-channel multi-coil RF a me nā mana multi-core, a me ka papahana Paravision 6.0.1. Hoʻopaʻa ʻia ka hoʻopaʻa ʻana me ka hoʻohana ʻana i ka coil volumetric RF coil me ke anawaena o loko o 86 mm a me kahi coil RF cryo-cooled ʻehā no ka loaʻa wale ʻana. Axial 2D Turbo-RARE (manawa hana hou = 2500 ms, manawa echo = 33 ms, 2 mau awelika) me 16 mau paʻi paʻi, ʻāpana mānoanoa 0.6–0.8 mm, i loaʻa nā laʻana 256 × 256. Ua loaʻa nā hōʻailona me ka hoʻohana ʻana i ka quadrature transceiver volumetric RF coil me ke anawaena o loko o 2 cm (Rapid MR International, LLC). ʻO ka hope loa, e hoʻohana i ka Imaris (BitPlane) i kūkulu ʻia i loko o nā hana hoʻohālikelike ili no ka hana 3D a me ka nānā ʻana i ka leo. Ua wehewehe ʻia kahi hoʻololi kūleʻa e like me kahi i hoʻokumu ʻia ai nā wahi o ka hōʻailona MRI paona T2 mau i loko o ka hemisphere transplanted. Ua wehewehe ʻia ka hōʻole ʻana i ka graft ma ke ʻano he kāpili i hana ʻole i nā wahi o ka hōʻailona MRI hoʻomau T2-kaumaha i ka hemisphere transplanted. Ua kāpae ʻia ka subcortical t-hCO mai ka nānā ʻana ma hope.
No ka hōʻike paʻa ʻana i nā GCaMP6s i ka hCO no ke kiʻi kiʻi calcium ʻelua-photon, ua loaʻa nā maʻi hiPS i ka pLV[Exp]-EF1a ::GcaMP6s-WPRE-Puro a ukali ʻia e ke koho ʻana i nā lāʻau antibiotic. ʻO ka pōkole, ua hoʻokaʻawale ʻia nā cell me EDTA a ua hoʻokuʻu ʻia i ka 1 ml o ka Essential 8 medium ma kahi kikoʻī o kahi 300,000 mau cell i mua o ka polybrene (5 μg / ml) a me 15 μl o ka maʻi. A laila hoʻokomo ʻia nā cell i ka hoʻokuʻu ʻia no 60 mau minuke a kanu ʻia i ka nui o 50,000 mau pūnāwai no ka luawai. Ma hope o ka hui ʻana, mālama ʻia nā cell me 5-10 μg ml-1 puromycin no 5-10 mau lā a i ʻole a hiki i ka ʻike ʻana o nā kolone paʻa. Ua hana ʻia ka maʻi hCO nui e like me ka mea i wehewehe mua ʻia 5 me kekahi mau hoʻololi. No ka pōkole, e hoʻololi i ka lā 30-45 hCO i loko o 1.5 ml Eppendorf microcentrifuge paipu me 100 µl o ke aʻalolo. A laila ma kahi o 90 µl o ka mea waena e wehe ʻia, 3-6 µl o ka high titer lentivirus (mai 0.5 x 108 a i 1.2 x 109) e hoʻohui ʻia i ka paipu, a hoʻoneʻe ʻia ka hCO i ka incubator no 30 mau minuke. A laila e hoʻohui i 90-100 µl o ka medium i kēlā me kēia paipu a hoʻihoʻi i nā paipu i ka incubator i ka pō. I ka lā aʻe, e hoʻoneʻe i ka hCO i ke ʻano nerve hou ma nā pā haʻahaʻa. Ma hope o 7 mau lā, ua hoʻoneʻe ʻia ka hCO i nā pā aniani 24-well no ka nānā ʻana a me ka loiloi ʻana i ka maikaʻi o ka maʻi. pLV[Exp]-SYN1::EYFP-WPRE a me pLV[Exp]-SYN1::hChR2-EYFP-WPRE i haku ʻia e VectorBuilder. Hoʻohana ʻia ʻo Lentivirus i ka hapa nui o nā hoʻokolohua no ka mea ua hoʻohui ʻia i loko o ka genome host, e ʻae ana i ka hōʻike ʻana o ka mea kākau moʻolelo i nā laina kelepona maʻi. No ka rabies follow-up, lā 30-45 hCO i hui pū ʻia me ka rabies-ΔG-eGFP a me AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), holoi maikaʻi ʻia no nā lā 3, a hoʻololi ʻia i loko o nā lā 7-14 i nā lā vivo.
No ka immunocytochemistry, ua hoʻomaʻamaʻa ʻia nā holoholona a hoʻohui ʻia me ka PBS a ukali ʻia e 4% paraformaldehyde (PFA ma PBS; Electron Microscopy Sciences). Hoʻopaʻa ʻia nā lolo i 4% PFA no 2 mau hola a i ʻole pō ma 4 ° C, cryopreserved i 30% sucrose i PBS no 48-72 mau hola, a hoʻokomo ʻia i 1: 1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583, Sakura Finetek) a me ka coronal section 30s. No ka immunohistochemistry o nā ʻāpana mānoanoa, ua hoʻoinu ʻia nā holoholona me ka PBS, a ua ʻoki ʻia ka lolo a ua ʻoki ʻia ka coronally ma 300-400 µm me ka hoʻohana ʻana i kahi vibratome (Leica) a ua hoʻopaʻa ʻia nā ʻāpana me 4% PFA no 30 mau minuke. A laila holoi ʻia nā cryosections a i ʻole nā ​​ʻāpana mānoanoa me ka PBS, paʻa ʻia no 1 hola ma ke ana wela (10% normal donkey serum (NDS) a me 0.3% Triton X-100 i hoʻoheheʻe ʻia i PBS) a hoʻopaʻa ʻia me ka hopena blocking ma 4 ° C. - Hoʻokomo ʻia nā Cryosections incubation i ka pō a hoʻomoʻa ʻia nā ʻāpana mānoanoa no 5 mau lā. ʻO nā antibodies mua i hoʻohana ʻia ʻo: anti-NeuN (iole, 1:500; ab104224, abcam) anti-CTIP2 (iole, 1:300; ab18465, abcam), anti-GFAP (rabbit, 1:1,000; Z0334, Dako), anti-GFP (1:109 Generation, GTX 1000; anti-HNA (iole, 1:200; ab191181, abcam), anti-NeuN (rabbit, 1:500; ABN78, Millipore), anti-PDGFRA (rabbit, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (rabbit, HPA0, Atlasbo1) anti-RECA-1 (iole, 1:50; ab9774, abcam), anti-SCG2 (rabbit, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (kao, 1:500; AF3075, R&D Systems), Netrin G1AFa: 106; 10, R&D Systems anti-STEM121 (iole, 1:200; Y40410, Takara Bio), anti-SATB2 (iole, 1:50; ab51502, abcam), anti-GAD65/67 (rabbit, 1:400; ABN904, Millipore) a me anti-IBA1 (kao, ab500, ab57). ʻO nā antibodies mua i hoʻohana ʻia: anti-NeuN (iole, 1:500; ab104224, abcam) anti-CTIP2 (iole, 1:300; ab18465, abcam), anti-GFAP (rabbit, 1:1,000; Z0334, Dako), anti -GFP (1:1000 Generation, GTX 1000, 18465, abcam) anti-HNA (iole, 1:200; ab191181, abcam), anti-NeuN (rabbit, 1:500; ABN78, Millipore), anti-PDGFRA (rabbit, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (rabbit, HPA0, Atlasbo1) anti-RECA-1 (iole, 1:50; ab9774, abcam), anti-SCG2 (rabbit , 1:100; 20357-1-AP, Proteintech), anti-SOX9 (kao, 1:500; AF3075, R&D Systems), Netrin G1a: 10,6; anti-STEM121 (iole, 1:200; Y40410, Takara Bio), anti-SATB2 (iole, 1:50; ab51502, abcam), anti-GAD65/67 (rabbit, 1:400; ABN904, Millipore) a me ka anti-IBA1 :107; a107 (kao, 607). Использовались следующие первичные антитела: анти-NeuN (мышиные, 1:500; ab104224, abcam), анти-CTIP2 (крысиные, 51b: 304e, 5, abcam; анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA (мышь, 1:200; ab191181, ab1клик), анти-HNA 1:500; ABN78, Millipore), анти-PDGFRA (кролик, 1:200; sc-338, Санта-Круз), анти-PPP1R17 (кролик, 1:200; HPA047819, 1нти-RECA-CA-1; ab9774, abcam), анти-SCG2 (кролик, 1:100; 20357-1-AP, Proteintech), анти-SOX9 (козий, 1:500; AF3075, R&D Systems), нетрин G1a, нетрин G1a (козий 1:500; AF3075, R&D Systems), нетрин G1a (коз10, AF10), R&D Systems анти-STEM121 (мышиный , 1:200; Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:4040, анти-SATB2; (koza, 1 :100; аб5076, абкам). ʻO nā antibodies mua i hoʻohana ʻia ʻo: anti-NeuN (iole, 1:500; ab104224, abcam), anti-CTIP2 (iole, 1:300; ab18465, abcam), anti-GFAP (rabbit, 1:1000; Z0334, Dako), anti-GFP100, 001, GTX1000 (moa, 300) anti-HNA (iole, 1:200; ab191181, abcam), anti-NeuN (rabbit, 1:500; ABN78, Millipore), anti-PDGFRA ( rabbit, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (rabbit, 1:2078; HPA, Atlasbo1, Atlasbo178; HPA, Atlasbo1:2078; anti-RECA-1 (iole, 1:50; ab9774, abcam), anti- SCG2 (rabbit, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (kao, 1:500; AF3075, R&D Systems), netrin G1AFa: 106, R&D10 (kao, 106) STEM121 (iole, 1:200; Y40410, Takara Bio), anti-SATB2 (iole, 1:50; ab51502, abcam), anti-GAD65/67 (rabbit, 1:400; ABN904, Millipore) a me anti-IBA1 (kao, 1:507).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（山羊，1:100；AF1166，R&D Pūnaehana），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），抗HNA（小鼠，1:200；ab191181，abcam），抗NeuN（兔，1:508；小鼠，1:200；ab191181，abcam），抗NeuN（兔，1:508；公re，7 200；sc-338，Santa Cruz），抗PPP1R17（兔，1:200；HPA047819，Atlas 抗体），抗RECA-1（小鼠，1:50；ab9774，ab9774，2 100；20357-1-AP，Proteintech），抗SOX9（山羊，1:500；AF3075，R&D Systems），Netrin G1a（山羊，1:100；AF1166，R&D Systems）;Y40410, Takara Bio), anti-SATB2 (iole, 1:50; ab51502, abcam), anti-GAD65/67 (rabbit, 1:400; ABN904, Millipore) a me anti-IBA1 (kao, 1:100; ab5076, abcam).ʻO nā antibodies mua i hoʻohana ʻia: anti-NeuN (mouse, 1:500; ab104224, abcam), anti-CTIP2 (rat, 1:300; ab18465, abcam), anti-GFAP (rabbit, 1:1000; Z0334, Dako). , anti-GFP (moa, 1:1000; GTX13970, GeneTex), anti-HNA (iole, 1:200; ab191181, abcam), anti-NeuN (rabbit, 1:500; ABN78, Millipore), anti-PDGFRA (rabbit, 1:2308) (rabbit, 1:200; HPA047819, Atlas antibody), anti-RECA-1 (iole, 1:50; ab9774, abcam), anti- SCG2 (rabbit), 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; анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) a me анти-IBA1 (коза, 1:100; аб5076, аб5076). 20357-1-AP, Proteintech), anti-SOX9 (kao, 1:500; AF3075, R&D Systems), Netrin G1a (kao, 1:100; AF1166, R&D Systems), anti-STEM121 (mouse, 1:200; TaBkara Bio, 500; TaBkara Bio, 500; ab51502, abcam), anti-GAD65/67 (rabbit, 1:400; ABN904, Millipore), a me anti-IBA1 (kao, 1:100; ab5076, abkam).Holoi ʻia nā ʻāpana me ka PBS a hoʻomoʻi ʻia me ka antibody lua no 1 hola ma ke ana wela (nā ʻāpana hau) a i ʻole i ka pō ma 4 ° C (mau ʻāpana mānoanoa). Ua hoʻohana ʻia ʻo Alexa Fluor secondary antibody (Life Technologies) i hoʻoheheʻe ʻia i ka 1:1000 i ka hoʻonā pale. Ma hope o ka holoi ʻana me PBS, ʻike ʻia ka nuclei me kahi Hoechst 33258 (Life Technologies). ʻO ka hope, ua kau ʻia nā paheʻe ma kahi microscope me nā uhi uhi (Fisher Scientific) me ka hoʻohana ʻana i kahi Aquamount (Polysciences) a nānā ʻia ma kahi microscope fluorescent Keyence (BZ-X analyzer) a i ʻole Leica TCS SP8 confocal microscope (Las-X) ma ke kiʻi. Ua hana ʻia nā kiʻi me ka polokalamu ImageJ (Fiji). No ka helu ʻana i ka nui o nā neurons kanaka i ka t-hCO a me ka cortex ʻiole, ua lawe ʻia nā kiʻi ʻehā ākea ākea 387.5 μm ma ke kikowaena o ka t-hCO, ma a kokoke paha i ka lihi o ka cortex ʻiole. Ua hoʻoholo ʻia nā palena ʻo Graft ma o ka loiloi ʻana i nā loli i ka ʻike kikoʻī, HNA+ nuclei, a / a i ʻole ke ʻano o ka autofluorescence kiko. Ma kēlā me kēia kiʻi, ua māhele ʻia ka huina o NeuN+ a me HNA+ e ka huina o nā pūnaewele NeuN+ ma ka wahi like. No ka hōʻoia ʻana e helu wale ʻia nā cell me nā nuclei i ka mokulele kiʻi, ʻo nā pūnaewele wale nō ʻo Hoechst+ i hoʻokomo ʻia i ka helu. ʻElua mau kiʻi i hoʻokaʻawale ʻia e ka liʻiliʻi he 1 mm i ʻawelika e hōʻemi i ka hewa helu helu.
Hoʻokahi pule ma mua o ka hōʻiliʻili laʻana, e waiho i nā holoholona hoʻololi hCO (ma kahi o 8 mahina o ka hoʻokaʻawale ʻana) i loko o kahi lumi ʻeleʻele me nā ʻumi ʻumi i ʻoki ʻia e hōʻemi i ka hoʻoulu ʻana i ka naʻau. Ua hana ʻia ka hoʻokaʻawale ʻana o ka nuclei e like me ka mea i wehewehe ʻia ma mua, me kekahi mau hoʻololi. ʻO ka pōkole, ua luku ʻia ka t-hCO a me ka hCO me ka hoʻohana ʻana i ka detergent-mechanical cell lysis a me ka 2 ml glass tissue grinder (D8938, Sigma-Aldrich/KIMBLE). A laila ua kānana ʻia ka nuclei ma ka hoʻohana ʻana i kahi kānana 40 µm a centrifuged ma 320 g no 10 mau minuke ma 4 ° C ma mua o ka hana ʻana i kahi gradient density sucrose. Ma hope o ke kaʻina centrifugation (320 g no 20 min ma 4 ° C), ua hoʻokuʻu ʻia nā laʻana ma 0.04% BSA / PBS me ka hoʻohui ʻana o 0.2 mau ʻāpana o µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) a hele i kahi kānana kahe 40 µm. Ua hoʻokuʻu hou ʻia ka nuclei i hoʻokaʻawale ʻia i PBS i loaʻa ka 0.02% BSA a hoʻouka ʻia ma kahi pahu Chromium Single Cell 3′ (i manaʻo ʻia ka hoʻihoʻi ʻana o nā cell 8,000 i kēlā me kēia ala). Ua hoʻomākaukau ʻia nā hale waihona puke snRNA-seq me ka Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Ua hoʻomākaukau ʻia nā hale waihona puke snRNA-seq me ka Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Библиотеки snRNA-seq были приготовлены с помощью Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). Ua hoʻomākaukau ʻia nā hale waihona puke snRNA-seq me ka hoʻohana ʻana i ka 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). Ua hoʻomākaukau ʻia ka waihona snRNA-seq me ka Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics).Ua hui ʻia nā hale waihona puke mai nā laʻana like ʻole e Admera Health ma NovaSeq S4 (Illumina).
Ua helu ʻia nā pae hōʻike Gene no kēlā me kēia pahu pahu nuklea putative me ka hoʻohana ʻana i ka 10x Genomics CellRanger analysis software package (version 6.1.2). Ma keʻano kikoʻī, ua hoʻohālikelike ʻia nā heluhelu me ka hui pū ʻana o ke kanaka (GRCh38, Ensemble, version 98) a me ka ʻiole (Rnor_6.0, Ensemble, version 100) reference genomes i hana ʻia me ke kauoha mkref a me ka hoʻohana ʻana i ka helu me ka -include-introns=TRUE kauoha i ka quantitation e komo pū nā heluhelu i palapala ʻia i nā ʻāpana intron. No nā laʻana t-hCO, ua ʻike ʻia nā nuclei kanaka ma muli o ke koi conservative ʻo ka liʻiliʻi o 95% o nā heluhelu palapala ʻāina āpau e kūlike me ka genome kanaka. Ua hoʻokō ʻia nā loiloi hope aʻe ma kahi hoʻopuka helu barcode array kānana mai ka CellRanger me ka hoʻohana ʻana i ka pūʻolo R (version 4.1.2) Seurat (version 4.1.1)32.
No ka hōʻoia ʻana i nā nuclei kiʻekiʻe wale nō i hoʻokomo ʻia i ka nānā ʻana ma hope, ua hoʻokō ʻia kahi kaʻina kānana iterative no kēlā me kēia hāpana. ʻO ka mua, ʻike ʻia a wehe ʻia nā nuclei haʻahaʻa haʻahaʻa me ka liʻiliʻi ma mua o 1000 mau genes kūikawā a ʻoi aku ma mua o 20% o ka mitochondria a pau. Ma hope iho, ua hoʻohālikelike ʻia ka matrix helu gene raw e ka regression binomial negative regularized me ka hoʻohana ʻana i ka hana sctransform(vst.flavor=”v2″), a ua ʻike pū ʻia ka 3000 o nā genes nui loa me ka hoʻohana ʻana i nā ʻāpana paʻamau. ka nānā ʻana i nā kahua kuli a hoʻohana ʻia no nā hoʻohālike āpau a me nā analy ensemble (ʻo ia ka helu DoubletFinder ma luna o ka 95th percentile (n=3) a me nā hCO samples (n=3) i hoʻohui like ʻia me ka hana IntegrateData me nā ʻāpana i luna.
Ma hope o ka wehe ʻana i nā kernels haʻahaʻa haʻahaʻa, ua hui pū ʻia ka ʻikepili i hoʻohui ʻia (hoʻonā = 0.5) a hoʻokomo ʻia no nā kumu ʻike maka UMAP34. Hoʻoholo ʻia nā genes marker no kēlā me kēia puʻupuʻu me ka hoʻohana ʻana i ka hana FindMarkers me nā ʻāpana paʻamau i helu ʻia mai ka ʻikepili hōʻike gene maʻamau. Hoʻomaopopo a hoʻokaʻawale mākou i nā papa kelepona nui ma o ka hoʻohui ʻana i ka fetal a me ka makua cortical reference datasets me ka marker gene expression 19,20,21,35 a me ka annotation. ʻO ka mea kūikawā, ua ʻike ʻia nā precursors e hoʻolaha ana e ka ʻōlelo o MKI67 a me TOP2A. Ua wehewehe ʻia nā pūʻulu progenitor e ka nele o nā transcript mitotic, overlap kiʻekiʻe me nā pūʻulu progenitor glial multipotent i wehewehe ʻia i ka hope metaphase fetal cortex, a me ka EGFR a me ka OLIG1 hōʻike. Hoʻohana mākou i ka huaʻōlelo astrocyte e hoʻopuni i kekahi mau mokuʻāina o ka ʻokoʻa astrocyte, mai ka radial glia hope a hiki i ka oʻo ʻana o nā astrocytes. Hōʻike nā pūʻulu Astrocyte i nā pae kiʻekiʻe o SLC1A3 a me AQP4 a ua hōʻike ʻia i ka palapala ʻāina me nā subtypes o ka fetal radial glia a / a i ʻole nā ​​​​astrocytes makua. Hōʻike nā OPC i ka PDGFRA a me SOX10 aʻo nā oligodendrocytes e hōʻike i nā hōʻailona myelination (MOG a me MYRF). Ua ʻike ʻia nā neurons Glutamatergic e ka hele ʻana o nā transcripts neuronal (SYT1 a me SNAP25), ka nele o nā māka GABAergic (GAD2), a me ka ʻōlelo o NEUROD6, SLC17A7, BCL11B, a i ʻole SATB2. Ua hoʻokaʻawale hou ʻia nā neurons GluN i luna ('ōlelo SAB2 a me ka nalowale o BCL11B) a me nā subclasses hohonu (BCL11B expression). Hōʻike nā neurons Putative subplate (SP) i nā māka SP18 i ʻike ʻia e like me ST18 a me SORCS1 i hoʻohui ʻia i nā māka hohonu GluN. Ua ʻike ʻia nā cell plexus-like Choroid e ka hōʻike TTR, a ua hōʻike ʻia nā ʻano meningeal-like i nā genes pili i ka fibroblast a me nā pūnaewele pial/vascular o ka hoʻonohonoho ʻikepili kuhikuhi.
Hana ʻia ka loiloi ʻokoʻa o ka hōʻike gene ma waena o nā subclasses t-hCO a me hCO me ka hoʻohana ʻana i kahi ʻano hana pseudo-batch hou i hana hou ʻia i nā laʻana i hoʻohana ʻia me ka pūʻolo Libra R (version 1.0.0). Ma ke ʻano kikoʻī, ua hana ʻia nā hoʻokolohua edgeR log-likelihood (version 3.36.0, package R) no nā pūʻulu ma ka hōʻuluʻulu ʻana i ka helu o nā genes i loko o nā cell no kahi papa cell no kēlā me kēia hōʻano hou. No ka ʻike wela wela, helu ʻia nā waiwai maʻamau i kēlā me kēia miliona (CPM) me ka hoʻohana ʻana i ka hana edgeR (cpm()) a hoʻonui ʻia (no ka loaʻa ʻana o ka mean = 0, ʻokoʻa maʻamau = 1). ʻO Gene Ontology (GO) hoʻonui i ka loiloi o nā genes t-hCO GluN i hoʻonui nui ʻia (Benjamini-Hochberg i hoʻoponopono i ka waiwai P ma lalo o 0.05 i hōʻike ʻia ma ka liʻiliʻi o 10% o nā pūnaewele t-hCO GluN a me ka piʻi ʻana o ka piʻi ʻana o ka loli o ka liʻiliʻi o 2 mau manawa). hana ʻia me ka ToppGene Suite (https://toppgene.cchmc.org/)37. Hoʻohana mākou i ka app ToppFun me nā ʻāpana paʻamau a hōʻike i nā waiwai P-hoʻoponopono ʻia ʻo Benjamini-Hochberg i helu ʻia mai nā hoʻokolohua hypergeometric GO-annotated.
No ka hoʻohālikelike ʻana i kā mākou mau pūʻulu snRNA-seq me nā puʻupuʻu cell annotated mai nā haʻawina kuhikuhi o ka RNA-seq pākahi-nui a i ʻole snRNA-seq19,20,21,22 makua, ua noi mākou i kahi ala hoʻohui hoʻohui data. Ua hoʻohana mākou i ke kahe hana maʻamau o SCTransform (v2) ma Seurat e hoʻohui a hoʻohālikelike i nā puʻupuʻu overlaps ma waena o nā waihona (e hoʻohana ana i nā ʻāpana like e like me luna). Ua hoʻokaʻawale ʻia nā ʻikepili pākahi a hiki i 500 mau pūnaewele a i ʻole nā ​​cores no kēlā me kēia pūʻulu kumu no ka pono o ka helu ʻana. Me ka hoʻohana ʻana i kahi ala like e like me ka mea i wehewehe ʻia ma mua, ua wehewehe ʻia ka hui ʻana ma ke ʻano o ka nui o nā cell a i ʻole nuclei i loko o kēlā me kēia puʻupuʻu i hoʻopili ʻia me ka lepili o ka hui kuhikuhi. No ka hoʻokaʻawale hou ʻana i nā GluN, ua hoʻohana mākou i ka holo ʻana o Seurat's TransferData no ka ʻikepili subset GluN e hāʻawi i nā hōʻailona hōʻike hōʻike i kā mākou mau pūnaewele GluN.
No ka loiloi i ke kūlana maturation o ka honua transcriptome o t-hCO a me hCO laʻana, ua hoʻohālikelike mākou i kā mākou pseudo-bulk samples me BrainSpan / psychENCODE23, aia i kahi kaʻina RNA nui e pili ana i ka ulu ʻana o ka lolo kanaka. Ua hana mākou i ka PCA ma ka hui pū ʻana o nā ʻano huaʻōlelo maʻamau mai nā cortical samples 10 mau pule ma hope o ka hāpai ʻana a ma hope, ma 5567 genes (me kā mākou ʻikepili) i ʻike mua ʻia he ikaika ma BrainSpan cortical samples (i wehewehe ʻia he ʻoi aku ka nui ma mua o 50% i ka hoʻomohala hoʻomohala i wehewehe ʻia e ka makahiki me ka hoʻohana ʻana i ka model cubic)38. Eia kekahi, ua loaʻa iā mākou nā genes e pili ana i nā inoa transcriptome nui o ka neurodevelopment me ka hoʻohana ʻana i ka non-negative matrix factorization e like me ka mea i wehewehe mua ʻia. Hoʻopili ʻia nā mea kaupaona hāpana i helu ʻia me ka hoʻohana ʻana i ke kaʻina hana matrix non-negative matrix. 5b me ka ʻikepili i hoʻonui ʻia no kēlā me kēia o nā pūlima ʻelima i wehewehe ʻia e Zhu et al.38. Eia hou, ua loaʻa nā māka transcriptional pili i ka hana mai nā haʻawina i paʻi mua ʻia. ʻO ka mea nui, ua hoʻonui nui ʻia ʻo ERG a me LRG i nā neurons glutamatergic i ʻike ʻia e ka hōʻiliʻili ʻana o ka cortex visual cortex snRNA-seq ma hope o ka hoʻoulu ʻia ʻana mai ka Papa Hoʻohui 3 Hrvatin et al.16. Ua loaʻa nā LRG i hoʻonui ʻia i ke kanaka mai ka KCl-hoʻoulu ʻia i nā moʻomeheu o ka lolo fetal kanaka a hōʻiliʻili i nā hola 6 ma hope o ka hoʻoulu ʻana, a ua hoʻonui nui ʻia nā genes kānana i nā kānaka akā ʻaʻole i nā rodents (Supplementary Table 4). Ua hana ʻia ka ʻike ʻana o ka hoʻonui ʻana i nā gene set me ka hoʻohana ʻana i ka hoʻāʻo pololei a Fisher.
Hoʻomaʻamaʻa i nā ʻiole me ka isoflurane, wehe i ka lolo a waiho i ke anuanu (ma kahi o 4°C) oxygenated (95% O2 a me 5% CO2) solution sucrose no nā ʻāpana i loaʻa: 234 mM sucrose, 11 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM. NaH2PO4, 10 mM MgSO4 a me 0.5 mM CaCl2 (ma kahi o 310 mOsm). Ua hana ʻia nā ʻāpana coronal brain brain (300-400 µm) i loaʻa i ka t-hCO me ka hoʻohana ʻana i kahi vibratome Leica VT1200 e like me ka mea i wehewehe mua ʻia39. A laila ua hoʻoneʻe ʻia nā ʻāpana i kahi keʻena ʻāpana me ka oxygenation wela o ka lumi mau i loaʻa i ka aCSF i hoʻomākaukau ʻia mai: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 a me 126 mM NaCl (298 mO). ma kahi o 45 mau minuke ma mua o ka hoʻopaʻa ʻana. Ua hoʻopaʻa ʻia nā ʻāpana i loko o kahi keʻena i hoʻopaʻa ʻia kahi i hoʻoinu mau ʻia me ka aCSF (95% O2 a me 5% CO2 vial). Hoʻopaʻa ʻia nā ʻikepili a pau ma ka lumi wela. Ua hoʻopau ʻia nā neurons t-hCO me kahi pipette aniani borosilicate i hoʻopiha ʻia me ka hopena i loaʻa ka 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, a me 0.6 mM EGTA, pH 7.2, hoʻoponopono kūloko me KOHs (m290 m290). I mea e ola hou ai, ua hoʻohui ʻia ka biocytin (0.2%) i ka hopena hoʻopaʻa leo.
Loaʻa ka ʻikepili me ka hoʻohana ʻana i kahi mea hoʻonui MultiClamp 700B (Molecular Devices) a me Digidata 1550B digitizer (Molecular Devices), kānana haʻahaʻa ma 2 kHz, helu ʻia ma 20 kHz, a nānā ʻia me ka hoʻohana ʻana iā Clampfit (Molecular Devices), Origin (OriginPro). 2021b, OriginLab). a me nā hana MATLAB maʻamau (Mathworks). Ua helu ʻia ka mana hui me ka hoʻohana ʻana iā JPCalc a ua hoʻoponopono ʻia nā mea komo i ka waiwai i helu ʻia o -14 mV. ʻO ka hana IV he pūʻulu o nā ʻanuʻu i kēia manawa ma nā ʻanuʻu 10-25 pA, mai -250 a 750 pA.
ʻO ka thalamus, ka mea keʻokeʻo, a me ka S1 afferents ua hoʻoulu ʻia i nā ʻāpana thalamocortical i ka wā o ka hoʻopaʻa ʻana i nā neurons hCO, e like me ka mea i wehewehe mua ʻia. ʻO ka pōkole, ua kau ʻia ka lolo ma luna o kahi pākaukau paʻi 3D i hili ʻia ma kahi kihi 10 °, a ʻoki ʻia ka mua o ka lolo ma kahi kihi 35 °. Hoʻopili ʻia ka lolo i ka ʻili i ʻoki ʻia a ʻokiʻoki ʻia, e mālama ana i nā axons protruding thalamocortical. Ua kau ʻia nā electrodes tungsten bipolar (0.5 MΩ) ma kahi micromanipulator lua a ua hoʻonohonoho pono ʻia e hoʻoulu i ʻehā mau ʻāpana o ke kelepona (ka capsule i loko, mea keʻokeʻo, S1 a me hCO). E hoʻopaʻa i nā pane synaptic ma hope o 300 µA phasic stimulation ma 0.03-0.1 Hz.
Ua hoʻohana ʻia nā neurons hChR2-hōʻike i ka hCO ma 480 nm a ua hoʻohana ʻia nā pulse māmā i hana ʻia e kahi LED (Prizmatix) ma o kahi pahuhopu × 40 (0.9 NA; Olympus) e hoʻopaʻa i ka ʻōlelo hChR2 kokoke i nā cell. ʻO ke anawaena kahua hoʻomālamalama ʻia ma kahi o 0.5 mm a ʻo ka nui o ka mana he 10-20 mW. Ua hoʻonohonoho ʻia ka laulā pulse i 10 ms, e like me ka pulse i hāʻawi ʻia i ka wā o ka hoʻokolohua aʻo ʻana. Ua hoʻohana ʻia nā alapine hoʻoulu like ʻole, mai 1 a 20 Hz, akā ʻo ka pulse mua wale nō o ka moʻo i hoʻohana ʻia no ka helu ʻana. ʻOi aku ka lōʻihi ma waena o nā kaʻaahi ma mua o 30 mau kekona e hōʻemi i ka hopena ma nā ala hoʻopono synaptic. No ka hoʻāʻo ʻana inā he monosynaptic ka pane hChR2, hoʻohana mākou i ka TTX (1 μM) i ka ʻauʻau a hiki i ka nalo ʻana o ka hopena EPSC, a laila hoʻohana i ka 4-aminopyridine (4-AP; 100 μM). ʻO ka maʻamau, e hoʻihoʻi ʻia kahi pane i loko o kekahi mau minuke, me kahi lohi iki ma waena o ke kī ʻana o LED a me ka hanauna EPSC. Ua hoʻohana ʻia ʻo NBQX (10 μM) e hoʻāʻo inā e alakaʻi ʻia ka pane e nā mea hoʻokipa AMPA.
Ua hana ʻia nā ʻāpana hCO koʻikoʻi e like me ka mea i wehewehe mua ʻia. ʻO ka pōkole, ua hoʻokomo ʻia nā ʻāpana hCO i 4% agarose a hoʻololi ʻia i nā cell i loaʻa ka 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 a me 10 mM d-(+) -0glucose i ʻoki ʻia i loko o ka pauku (+) -0glucose. µm ma ka lumi wela me ka hoʻohana ʻana i ka vibrator Leica VT1200 a mālama ʻia ma ASF ma ka wela lumi. A laila, hoʻopaʻa ʻia ka hoʻopaʻa ʻana i ka hoʻopaʻa ʻana i nā cell holoʻokoʻa ma nā ʻāpana hCO ma lalo o kahi microscope SliceScope pololei (Scientifica). Hoʻopili ʻia nā ʻāpana me ka aCSF (95% O2 a me 5% CO2) a ua hoʻopaʻa ʻia nā hōʻailona kelepona ma ka lumi wela. Hoʻohana ʻia nā neurons hCO me ka pipette aniani borosilicate i hoʻopiha ʻia me kahi hopena i loaʻa ka 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, a me 0.6 mM EGTA, pH 7, 2 i loko, i hoʻoponopono ʻia me KOH290 (osmolarity). No ka hoʻihoʻi hou ʻana, e hoʻohui i ka 0.2% Biocytin i ka hopena o loko.
Ua loaʻa ka ʻikepili e Clampex (Clampex 11.1, Molecular Devices) me ka hoʻohana ʻana i kahi mea hoʻonui MultiClamp 700B (Molecular Devices) a me kahi Digidata 1550B digitizer (Molecular Devices), kānana haʻahaʻa ma 2 kHz, helu ʻia ma 20 kHz, a nānā ʻia me ka hoʻohana ʻana i ka Clampfit (version 10, version MAT) hana maʻamau. (MATLAB 2019b, Mathworks). Ua helu ʻia ka mana hui me ka hoʻohana ʻana iā JPCalc a ua hoʻoponopono ʻia nā mea komo i ka hiki ke hoʻohālikelike ʻia o -14 mV. ʻO ka hana IV he pūʻulu o nā ʻanuʻu i kēia manawa ma nā ʻanuʻu 5-10 pA mai -50 a 250 pA.
No ka hana hou morphological o nā neurons pinched, 0.2% biocytin (Sigma-Aldrich) i hoʻohui ʻia i ka hopena kūloko. Hoʻomaka ʻia nā cell no ka liʻiliʻi he 15 mau minuke ma hope o ka hacking. A laila huki mālie ʻia ka pipette no 1-2 min a hiki i ka hoʻopaʻa ʻia ʻana o ka membrane i hoʻopaʻa ʻia. Ma hope o ke kaʻina hana physiology, ua hoʻopaʻa ʻia nā ʻāpana i ka pō ma 4 ° C. ma 4% PFA, holoi ʻia me PBS X3, a hoʻoheheʻe ʻia 1: 1000 me streptavidin-conjugated DyLight 549 a i ʻole DyLight 405 (Vector Labs). Ua hoʻopaʻa inoa ʻia nā cell i hoʻopiha ʻia me ka biocytin (2%; Sigma-Aldrich) i ka wā o ka hoʻopaʻa ʻana i ka hoʻopaʻa paʻi ʻana i ka lumi lumi no 2 mau hola. A laila, kau ʻia nā ʻāpana ma luna o nā paheʻe microscopy me ka hoʻohana ʻana i kahi Aquamount (Thermo Scientific) a ʻike ʻia i ka lā aʻe ma kahi microscope confocal Leica TCS SP8 me ka hoʻohana ʻana i kahi pahuhopu ʻaila me kahi puka helu × 40 1.3, hoʻonui × 0.9–1.0, xy. ʻO ka helu hoʻohālike ma kahi o 7 pixels no ka micron. Ua loaʻa nā Z-stacks ma 1 µm intervals, a ua hana ʻia nā mosaics z-stack a me Leica-based auto-stitching e uhi i ka lāʻau dendritic holoʻokoʻa o kēlā me kēia neuron. A laila nānā ʻia nā neurons me ka lima me ka hoʻohana ʻana i ka neuTube 40 interface a ua hana ʻia nā faila SWC. Ua hoʻouka ʻia nā faila i ka plugin SimpleNeuriteTracer41 Fiji (ImageJ, version 2.1.0; NIH).
Ua loaʻa ka ʻiʻo cortical kanaka me ka ʻae ʻia e like me kahi protocol i ʻae ʻia e ka Institutional Review Board o Stanford University. ʻElua mau laʻana o ka ʻiʻo postpartum kanaka (3 a me 18 mau makahiki) i loaʻa ma ka ʻoki ʻana i ka cortex mua (middle frontal gyrus) ma ke ʻano he ʻoki no ka epilepsy refractory. Ma hope o ka wehe ʻana, e ʻohi i ka ʻiʻo i loko o ka NMDG-aCSF anu anuanu i loaʻa: 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 thiourea. 0.5 mM CaCl2 4H2O a me 10 mM MgSO4 7H2O. Titrate i ka pH 7.3-7.4 me ka waika hydrochloric concentrated. Hāʻawi ʻia nā ʻili i ka hale hana i loko o 30 mau minuke a lawe ʻia nā ʻāpana coronal e like me ke kaʻina hana i hōʻike ʻia ma luna.
Ua hana ʻia nā kaʻina hana holoholona a pau e like me nā alakaʻi mālama holoholona i ʻae ʻia e ke Kulanui ʻo Stanford APLAC. ʻO nā ʻiole (ʻoi aku ma mua o 140 mau lā ma hope o ka hoʻololi ʻana) ua hoʻoulu ʻia me ka 5% isoflurane anesthesia a me ka anesthetized me 1-3% isoflurane intraoperatively. Hoʻokomo ʻia nā holoholona i loko o kahi stereotaxic frame (Kopf) a hoʻokuʻu ʻia ka buprenorphine (SR) i hoʻokuʻu ʻia ma lalo o ka ʻili. Hōʻike ʻia ka iwi poʻo, hoʻomaʻemaʻe ʻia a hoʻokomo ʻia nā iwi iwi 3-5. No ka huli ʻana i ka t-hCO, ua hana mākou i nā hoʻonohonoho stereotaxic mai nā kiʻi MRI. Ua wili ʻia kahi lua burr ma ke kahua o ka hoihoi a me nā fibers (400 µm anawaena, NA 0.48, Doric) i hoʻohaʻahaʻa ʻia ma 100 µm ma lalo o ka ʻili o ka hCO a hoʻopaʻa ʻia i ke poʻo me ka sima niho hiki ke hoʻōla ʻia UV (Relyx).
Hana ʻia nā hoʻopaʻa ʻana o ka fiber photometric e like me ka mea i wehewehe mua ʻia42. No ka hoʻopaʻa ʻana i ka hana maʻamau, ua hoʻokomo ʻia nā ʻiole i loko o ka hale paʻa maʻemaʻe a ua hoʻopili ʻia kahi 400 µm anawaena fiber optic patch cable (Doric) i hoʻopili ʻia i kahi ʻōnaehana kiʻi kiʻi kiʻi kiʻi fiber optic i hoʻopili ʻia i ke kaula fiber optic i hoʻokomo ʻia. I loko o ka hoʻopaʻa ʻana o ka hana kaʻa he 10 mau minuke, ua noa nā holoholona e ʻimi i ka hale. No ka hoʻopaʻa ʻana i ka hana i hoʻāla ʻia, ua hoʻopaʻa ʻia nā ʻiole (ʻoi aku ma mua o 140 mau lā ma hope o ka hoʻololi ʻana) me 5% isoflurane no ka hoʻokomo ʻana a me 1-3% isoflurane no ka mālama. E kau i ka holoholona i loko o kahi kiʻi stereotactic (Kopf) a ʻoki ʻia nā ʻumiʻumi ma ka ʻaoʻao ʻē aʻe o ka t-hCO ma kahi o 2 knm a hele i loko o kahi ʻupena i hoʻopili ʻia me kahi mea hana piezoelectric (PI). Ua hoʻopili ʻia kahi 400 µm fiber optic patch cable (Doric) i ka fiber i hoʻokomo ʻia a hoʻopili ʻia i ka ʻōnaehana ʻikepili. ʻO nā ʻumikū ma ka ʻaoʻao ʻē aʻe o t-hCO ua hoʻohuli ʻia he 50 mau manawa (2 mm ma 20 Hz, 2 s i kēlā me kēia hōʻike) i nā manawa like ʻole e kahi kaʻa piezoelectric ma kahi manawa hoʻopaʻa 20 minuke. E hoʻohana i ka Arduino MATLAB Support Package e hoʻomalu i ka manawa deflection me ka code MATLAB maʻamau. Hoʻonohonoho ʻia nā hanana i ka polokalamu loaʻa ʻikepili me ka hoʻohana ʻana i nā pulses transistor-transistor logic (TTL).
ʻO nā ʻiole (ʻoi aku ma mua o 140 mau lā ma hope o ka hoʻololi ʻana) ua hoʻoulu ʻia me ka 5% isoflurane anesthesia a me ka anesthetized me 1-3% isoflurane intraoperatively. Hoʻokomo ʻia nā holoholona i loko o kahi kiʻi stereotaxic (Kopf) a ua hoʻokomo ʻia ka buprenorphine SR a me ka dexamethasone ma lalo o ka ʻili. Hōʻike ʻia ka iwi poʻo, hoʻomaʻemaʻe ʻia a hoʻokomo ʻia nā iwi iwi 3-5. No ka huli ʻana i ka t-hCO, ua hana mākou i nā hoʻonohonoho stereotaxic mai nā kiʻi MRI. Ua hana ʻia kahi craniotomy pōʻai (ma kahi o 1 knm ke anawaena) me kahi drill wikiwiki kiʻekiʻe ma luna o ka hCO transplanted. I ka wā e lahilahi ai ka iwi, akā ma mua o ka wili ʻana i ka iwi holoʻokoʻa, e hoʻohana i nā pūlima e wehe i ke koena pelvic disc e hōʻike i ke kumu o ka t-hCO. Ua hoʻopiha ʻia ka craniotomy me ka paʻakai sterile, a ua hoʻopili ʻia kahi uhi uhi a me kahi pine poʻo kūikawā i ka iwi poʻo me ka sima niho hoʻōla UV (Relyx).
Ua hana ʻia ʻelua kiʻi kiʻi me ka Bruker multiphoton microscope me kahi pahuhopu Nikon LWD (×16, 0.8 NA). Ua hana ʻia ke kiʻi GCaMP6 ma 920 nm me 1.4x hoʻokahi z-plane magnification a me 8x awelika o 7.5 fps. Hoʻokomo ʻia nā ʻiole me 5% isoflurane anesthesia a mālama ʻia me 1-3% isoflurane. Ua hoʻokomo ʻia nā ʻiole i loko o ke poʻo i hana ʻia a hoʻonoho ʻia ma lalo o ka lens. Ua loaʻa kahi hoʻopaʻa hope 3 mau minuke o ka hana kaʻa. I loko o 20 minuke o ka hoʻopaʻa ʻana, 50 puffs (kekahi hōʻike ʻana he 100 ms ka lōʻihi) i hāʻawi ʻole ʻia i ka ʻūhiwi e kū pono ana i ka t-hCO me ka hoʻohana ʻana i kahi picospricer. E hoʻohana i ka Arduino MATLAB Support Package no ka mālama ʻana i ka manawa pahū me ka code MATLAB maʻamau. Hoʻopili i nā hanana me ka polokalamu loaʻa ʻikepili (PrairieView 5.5) me ka hoʻohana ʻana i nā pulse TTL. No ka nānā ʻana, ua hoʻoponopono ʻia nā kiʻi no xy motion me ka hoʻohana ʻana i ka hoʻoponopono affine ma ka papahana MoCo i hoʻokumu ʻia ma Fiji. ʻO ka unuhi ʻana i nā meheu fluorescent mai nā pūnana pākahi me ka hoʻohana ʻana iā CNMF-E43. Wehe ʻia ka Fluorescence no kēlā me kēia māhele o ka hoihoi, hoʻololi ʻia i nā pihi dF/F, a laila hoʻololi ʻia i nā helu-z.
ʻO nā ʻiole (ʻoi aku ma mua o 140 mau lā ma hope o ka hoʻololi ʻana) ua hoʻoulu ʻia me ka 5% isoflurane anesthesia a me ka anesthetized me 1-3% isoflurane intraoperatively. Hoʻokomo ʻia nā holoholona i loko o kahi kiʻi stereotaxic (Kopf) a ua hoʻokomo ʻia ka buprenorphine SR a me ka dexamethasone ma lalo o ka ʻili. Ua ʻoki ʻia nā ʻumiʻumi ma ka ʻaoʻao ʻē aʻe o ka t-hCO a ma kahi o 2 knm a hoʻopaʻa ʻia i loko o kahi mesh i hoʻopili ʻia i kahi mea hoʻokele piezoelectric. Hōʻike a hoʻomaʻemaʻe ʻia ke poʻo. Hoʻopili ʻia kahi wili lepo kila kila i ka iwi poʻo. No ka huli ʻana i ka t-hCO, ua hana mākou i nā hoʻonohonoho stereotaxic mai nā kiʻi MRI. E hana i ka craniotomy poepoe (ma kahi o 1 knm ke anawaena) me ka puolo kiekie ma luna pono o ka t-hCO. I ka wā e lahilahi ai ka iwi, akā ma mua o ka wili ʻana i ka iwi holoʻokoʻa, e hoʻohana i nā pūlima e wehe i ke koena pelvic disc e hōʻike i ke kumu o ka t-hCO. Ua hoʻopaʻa ʻia nā kelepona pākahi me ka hoʻohana ʻana i ka 32-channel a i ʻole 64-channel high-density silicon probes (Cambridge Neurotech) i hoʻopaʻa ʻia i ka lepo a hoʻonui ʻia me nā mea hoʻonui RHD (Intan). E hoʻohana i ka manipulator e hoʻohaʻahaʻa i nā electrodes i ka pae i hoʻopaʻa ʻia ma o ka craniotomy, i hoʻopiha ʻia me ka saline sterile. Hana ʻia ka hōʻiliʻili ʻikepili ma ke alapine o 30 kHz me ka hoʻohana ʻana i ka ʻōnaehana ʻikepili Open Ephys. Ua hoʻomau ʻia ka hoʻopaʻa ʻana i ka wā i ʻike ai mākou i ka hana kuʻuna rhythmic i hoʻopili pono ʻia ma mua o nā kahawai 10, e hōʻike ana aia nā electrodes i loko o ka graft (ma muli o ʻelua-photon calcium imaging data). Ua loaʻa kahi hoʻopaʻa hope 10 mau minuke o ka hana kaʻa. ʻO nā ʻumikū ma ka ʻaoʻao ʻē aʻe o t-hCO ua hoʻohuli ʻia he 50 mau manawa (2 mm ma 20 Hz, 2 s i kēlā me kēia hōʻike) i nā manawa like ʻole e kahi kaʻa piezoelectric ma kahi manawa hoʻopaʻa 20 minuke. Ke hoʻohana nei i ka Pūʻulu Kākoʻo MATLAB no Arduino (MATLAB 2019b), kaohi i ka manawa deflection me ka code MATLAB maʻamau. E hoʻohana i nā pulu TTL e hoʻonohonoho i nā hanana me ka lako polokalamu ʻikepili.
No nā hoʻokolohua ʻike maka, ua hoʻopili ʻia kahi 200 µm optical patch cord (Doric) i hoʻopili ʻia i kahi laser 473 nm (Omicron) i kahi fiber optical 200 µm i kau ʻia ma luna o ka craniotomy. Ma mua koke o kēia, hoʻoponopono i ka mana jumper i 20 mW. E hoʻohana i ka manipulator e hoʻohaʻahaʻa i nā electrodes i ka pae i hoʻopaʻa ʻia ma o ka craniotomy, i hoʻopiha ʻia me ka saline sterile. I ka hoʻomaka ʻana o ka hoʻopaʻa ʻana, ua hoʻokuʻu ʻia nā ʻumi pulse o ka māmā 473 nm (frequency 2 Hz, pulse lōʻihi 10 ms). Ua wehewehe ʻia nā cell Photosensitive e like me nā cell e hōʻike ana i ka pane spike i loko o 10 ms o ka mālamalama ma 70% a i ʻole o nā hoʻokolohua.