Waad ku mahadsan tahay booqashada Nature.com. Waxaad isticmaalaysa nooc browser ah oo leh taageero CSS xaddidan. Waayo-aragnimada ugu fiican, waxaan kugula talineynaa inaad isticmaasho browser-ka la cusboonaysiiyay (ama aad damiso Habka Laqabsiga ee Internet Explorer). Intaa waxaa dheer, si loo hubiyo taageerada joogtada ah, waxaan ku tusineynaa goobta aan lahayn qaabab iyo JavaScript.
Waxa uu soo bandhigayaa carousel ka kooban saddex slide hal mar. Isticmaal badhamada hore iyo kan ku xiga si aad ugu gudubto saddex sawir markiiba, ama isticmaal badhamada slider-ka ee dhamaadka si aad ugu gudubto saddex sawir markiiba.
Unugyada neerfayaasha ee iskood isu-urursada waxay ka dhigan yihiin madal fayoqab ah oo rajo leh si ay u qaabeeyaan horumarka iyo cudurrada aadanaha. Si kastaba ha ahaatee, organoids ayaa ka maqan isku xirnaanta ka jirta vivo, taas oo xaddidaysa korriinka oo ka hortagaya la-dhexgalka wareegyada kale ee xakameynaya dabeecadda. Halkan waxaan ku tusinaynaa in unugyada jireed ee bini'aadamka ee ka soo jeeda cortical organoids oo lagu beeray kiliyaha somatosensory ee jiirka qaawan ee dhallaanka ah ay horumariyaan noocyada unugyada qaan-gaarka ah ee ku dhex milma dareenka iyo wareegyada dhiirigelinta la xiriira. MRI waxay daaha ka qaaday kobaca organoid-ka-tallaalidda ka dib ee dhowr xariiqyo unugyada asliga ah iyo xayawaanka, halka falanqaynta hal-core ay daaha ka qaaday horumarka corticogenesis iyo soo bixitaanka barnaamijka ku-tiirsanaanta dhaqdhaqaaqa. Runtii, neerfayaasha kortikal ee la tallaalay waxay soo bandhigaan qaab-dhismeedyo kakan, synaptik, iyo xuubka gudaha marka loo eego dhiggooda vitro, taasoo u oggolaanaysa in la ogaado cilladaha neerfaha ee bukaannada qaba xanuunka Timoteyos. Baafinta anatomical iyo functional waxay muujisay in xubnaha la tallaalay ay helaan agabka thalamocortical iyo corticocortical, iyo in vivo duubista dhaqdhaqaaqa neerfaha waxay soo jeedinayaan in agabyadani ay dhalin karaan jawaabaha dareenka unugyada aadanaha. Ugu dambeyntii, Organoids-ka kortikalku waxay ku fidiyaan axon dhammaan maskaxda jiirka, iyo firfircoonidooda optogenetic waxay horseeddaa dabeecad abaal-marineed. Sidaa darteed, neerfayaasha kortex ee bini'aadamka ee la tallaalay ayaa bislaaday oo ka qaybqaata wareegyada martida loo yahay ee xakameynaya dabeecadda. Waxaan filaynaa in habkan uu fududeeyo ogaanshaha astaamaha heerka-strand ee unugyada bukaan-socodka ee aan lagu ogaan karin siyaabo kale.
Maskaxda bini'aadamka ee soo koraysa waa hab is-abaabulan oo cajiib ah kaas oo unugyadu ay ku bataan, kala soocaan, u haajiraan, oo ay isku xidhaan si ay u sameeyaan wareegyada neerfaha ee shaqeynaya kuwaas oo lagu sifeeyo khibradda dareenka. Dhibaatada ugu muhiimsan ee fahamka horumarka maskaxda bini'aadamka, gaar ahaan marka la eego xaaladda cudurka, waa la'aanta helitaanka unugyada maskaxda. Unugyada is-abaabula, oo ay ku jiraan organoids-ka kortex bini'aadamka (hCO; sidoo kale loo yaqaanno kiliyaha bini'aadamka), waxay dhalin karaan 2,3,4,5,6. Si kastaba ha noqotee, xaddidaadyo dhowr ah ayaa xaddidaya codsigooda ballaaran si ay u fahmaan horumarka iyo shaqada wareegyada neerfaha. Gaar ahaan, ma cadda in korriinka hCO uu xaddiday maqnaanshaha qaar ka mid ah agabyada deegaanka yar yar iyo dareenka ee ku jira vivo. Intaa waxaa dheer, sababtoo ah hCO-yadu kuma jiraan wareegyada kuwaas oo dhalin kara natiijooyinka habdhaqanka, u adeegsigooda qaabaynta hidde ahaan isku dhafan iyo xanuunada maskaxda ee maskaxda ayaa hadda xaddidan.
Ku-tallaalidda hCO ee maskaxda nool ee nool waxay ka gudbi kartaa xaddidaadyadan. Daraasado hore ayaa muujiyay in neerfayaasha bini'aadamka ee lagu tallaalay kiliyaha jiirka ay awoodaan inay noolaadaan, mashruuc, iyo la xiriiraan unugyada jiirka7,8,9,10,11,12. Si kastaba ha ahaatee, tijaabooyinkan waxaa badanaa lagu sameeyaa xayawaanka qaangaarka ah, taas oo xaddidi karta isdhexgalka synaptik iyo axonal. Halkan, waxaan ku sharaxnay jaantuska tallaalka kaas oo aan ku tallaalnay 3D hCO oo ka soo jeeda unugyada hiPS una galnay kiliyaha somatosensory ee aasaasiga ah (S1) ee jiirka difaaca jirka ee marxaladda hore ee horumarinta balaastikada. HCO (t-hCO) neerfayaasha la beddelay waxay maraan korriin la taaban karo, waxay helaan thalamocortical iyo cortical-cortical-ka soo gelida jawaabaha dareenka, waxayna ku kordhiyaan saadaasha axonal maskaxda jiirka si ay u dhaqaajiyaan dabeecad-doonista. Koritaanka korriinka t-hCO ayaa shaaca ka qaaday cilladaha neerfaha ee bukaannada qaba Timoteyos syndrome (TS), cillad hidde-side aad u daran oo ay keento isbeddellada nooca L-xasaasi ah ee CaV1.2 kanaalka calcium (oo ay ku qoran tahay CACNA1C).
Si loo barto neurons-ka bini'aadamka ee wareegyada gudaha vivo, waxaan si stereotactically ah ugu tallaalnay 3D hCO ee saxda ah ee S1 ee jiirka athymic hore ee dhalmada kadib (maalmaha 3-7 dhalmada kadib) (Jaantus. 1a iyo xogta la ballaariyay ee Fig. 1a-c). Halkaa marka ay marayso, saadaasha thalamocortical iyo corticocortical axonal ma aysan dhamaystirin S1 innervation (tixraac. 13). Haddaba, habkan waxaa loogu talagalay in lagu kordhiyo is dhexgalka t-hCO iyadoo la yareynayo saameynta wareegyada endogenous. Si loo sawiro goobta t-hCO ee xayawaanka nool, waxaanu samaynay T2-miisaanka MRI dib-u-dhiska maskaxda ee jiirka 2-3 bilood ka dib tallaalka (Jaantus. 1b iyo xogta la dheereeyey, Fig. 1d). t-hCO si sahal ah ayaa loo arkay iyo cabbirada mugga t-hCO waxay la mid yihiin kuwa laga soo xisaabiyay xaleef go'an (Jaantuska Xogta Dheeraadka ah. 1d,e; P> 0.05). t-hCO si sahal ah ayaa loo arkay iyo cabbirada mugga t-hCO waxay la mid yihiin kuwa laga soo xisaabiyay xaleef go'an (Jaantuska Xogta Dheeraadka ah. 1d,e; P> 0.05). t-hCO легко наблюдались, а объемные измерения t-hCO 1d, P> 0,05). t-hCO si fudud ayaa loo arkay, iyo cabbirada t-hCO ee mugga leh waxay la mid yihiin kuwa lagu xisaabiyo qaybaha go'an (xogta la ballaariyay, Jaantus. 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. 1d, P> 0,05). t-hCO si fudud ayaa loo arkay, iyo cabbirada t-hCO ee mugga leh waxay la mid yihiin kuwa lagu xisaabiyo qaybaha go'an (xogta la ballaariyay, Jaantus. 1d, e; P> 0.05).Waxaan ku go'aaminnay t-hCO 81% ee xoolaha la tallaalay qiyaastii 2 bilood ka dib tallaalka (n = 72 xayawaan; hCO oo ka yimid 10 khadadka unugyada hiPS; khadadka unugyada hiPS ee shaxda Dheeraadka ah 1). Kuwaas, 87% waxay ku yaalaan kortex maskaxda (Jaantus. 1c). Iyadoo la samaynayo baaritaanno MRI oo taxane ah waqtiyo badan oo isku mid ah jiirka la tallaalay, waxaan helnay koror sagaal laab ah oo t-hCO ah gudaha bilaha 3 (Jaantus. 1d iyo xogta la ballaariyay, Fig. 1f). Xayawaanka la tallaalay waxay lahaayeen heer badbaado oo sarreeya (74%) 12 bilood ka dib tallaalka (xog la ballaariyay, Jaantus. 1g iyo Jadwalka Dheeraadka 2), mana jiraan wax mootada muuqda ama xusuusta, gliosis, ama electroencephalogram (EEG) ayaa la helay. Xogta Jaantuska 1g iyo shaxda dheeriga ah 2). 1h-m iyo 3e).
a, Qaabka naqshadaynta tijaabada ah. hCO oo laga soo qaatay unugyada hiPS ayaa lagu beeray S1 ee jiirka qaawan ee dhashay maalmaha 30-60 ee kala duwanaanshaha. b, Sawirada Miisaanka Miisaanka ah ee T2-Miisaanka ah ee Miisaanka ah ee muujinaya t-hCO ee S1 2 bilood ka dib tallaalka. Bar miisaan, 2 mm. c, Qiyaasta heerarka guusha hal-abuurka ee lagu muujiyay xariiq kasta oo hiPS unug ah (n = 108, tirooyinka ku dhex jira baararka waxay muujinayaan tirada t-hCO ee xariiqda unugga hIPS) iyo kortikal ama meel hoose (n = 88). d, Sawirka MRI ee halbowlaha wadnaha (bidix; bar miisaanka, 3 mm) iyo dib u dhiska mugga 3D ee u dhigma (barka miisaanka, 3 mm) oo muujinaya kororka t-hCO ee bilaha 3. e, Dib u eegida qaababka t-hCO ee kiliyaha maskaxda ee jiirka. Bar miisaanka, 1 mm. f, Sawirada difaaca jirka ee matalayaasha t-hCO oo ka muuqda bidix ilaa midig (inta lagu jiro kala duwanaanshaha): PPP1R17 (4 bilood jir), NeuN (8 bilood jir), SOX9 iyo GFAP (8 bilood jir), PDGFRα; (8 bilood), MAP2 (8 bilood) iyo IBA1 (8 bilood). Bar miisaan, 20 µm. Isku-duwidda HNA waxay muujinaysaa unugyada asalka ah ee bini'aadamka. g, snRNA-seq: Midaysan midaysan iyo odoroska (UMAP) sawirka dhimista cabbirka ee dhammaan tayada sare ee t-hCO nuclei ka dib is dhexgalka Seurat (n = 3 t-hCO muunado, n = 2 khadadka unugyada hiPS). Astrocytes, unugyada khadka astrocyte; prog cyc, abtirsiinyada wareega; GluN DL, neerfaha glutamatergic qoto dheer; GluN DL/SP, neerfayaasha glutamatergic qoto dheer iyo sublamellar; GluN UL, lakabka sare ee glutamatergic neurons; oligodendrocytes, oligodendrocytes; OPC, unugyada taranka oligodendrocyte; RELN, relin neurons. h, Gene Ontology (GO) falanqaynta kobcinta gen ee gen si aad ah ayaa loo hagaajiyay (lagu hagaajiyay P <0.05, isbeddelka laablaab> 2, oo lagu muujiyay ugu yaraan 10% nuklei) t-hCO glutamatergic neurons marka la barbar dhigo hCO glutamatergic neurons. h, Gene Ontology (GO) falanqaynta kobcinta gen ee gen si aad ah ayaa loo hagaajiyay (lagu hagaajiyay P <0.05, isbeddelka laablaab> 2, oo lagu muujiyay ugu yaraan 10% nuklei) t-hCO glutamatergic neurons marka la barbar dhigo hCO glutamatergic neurons. h, Анализ обогащения терминов Gene Ontology (GO) для генов. 2. hCO h, Gene Ontology (GO) falanqaynta kobcinta ereyga ee hiddo-wadaha leh firfircooni la taaban karo (lagu hagaajiyay P<0.05, isbeddelka laablaab>2, muujinta ugu yaraan 10% nuclei) ee t-hCO glutamatergic neurons marka la barbar dhigo 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显神经元相比。 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 ， 至少 10%术语富集分析。 h. ядер) в глутаматергических нейронах t-hCO посравнению обогащения. h, hiddo-wadaha si weyn ayaa loo hagaajiyay (lagu hagaajiyay P <0.05, isbeddelka laabashada> 2, oo lagu muujiyay ugu yaraan 10% nuklei) ee t-hCO glutamatergic neurons marka la barbar dhigo hCO glutamatergic neurons Ontological (GO) ee falanqaynta ereyga kobcinta.Xariiqda dhibicda leh waxay muujineysaa qiimaha aq ee 0.05. i, Sawirka UMAP ee noocyada unugyada GluN ee t-hCO iyadoo la adeegsanayo wareejinta summada ee tixraaca 22 snRNA-seq xogta kortexka qaangaarka ee qaangaarka. CT - unugyada corticothalamic, ET - unugyada extracerebral, IT - unugyada telencephalic gudaha, NP - saadaasha u dhow.
Waxaan markaa qiimeynay cytoarchitecture iyo guud ahaan isku dhafka gacanta ee t-hCO. Midabaynta difaaca jirka ee unugyada endothelial ee jiirka ayaa shaaca ka qaaday vascularization with t-hCO, halka IBA1 midabaynta ay muujisay joogitaanka microglia jiirka inta lagu jiro tallaalka (Jaantus. 1f iyo xogta la ballaariyay, Fig. 3c, d). Immunostaining ayaa shaaca ka qaaday antigen nukliyeerka bini'aadamka (HNA) unugyo togan oo wada-shaqeynaya PPP1R17 (progenitors cortical), NeuN (neurons), SOX9 iyo GFAP (unugyada glial-derived) ama PDGFRα (oligodendrocyte progenitors) (Jaantuska 1f). Si loo barto hal-abuurka gacanta ee t-hCO ee xallinta hal unug, waxaanu samaynay taxane hal-core RNA (snRNA-seq) ka dib qiyaastii 8 bilood oo kala duwanaansho ah. Sifeynta badan iyo ka saarida xudunta jiirka waxay keentay 21,500 khariidado mononuclear oo tayo sare leh (Jaantus. 1g iyo xogta la balaariyay, Jaantus. 4a,b). Qaababka muujinta calaamadaha caadiga ah ee nooca unugyada ah ayaa aqoonsaday kooxo ka mid ah fasalada unugyada kortikal ee waaweyn, oo ay ku jiraan neerfayaasha glutamatergic qoto dheer iyo sare, wareegtada wareega, oligodendrocytes, iyo astrocyte lineage (Jaantus. 1g, xogta la ballaariyay, Fig. 4c, iyo Shaxda Dheeraadka ah 3). Immunostaining for SATB2 iyo CTIP2 ayaa muujisay in inkasta oo ay jiraan jiritaanka kortikal-hoosaadyada, t-hCO ma muujin shax cad oo anatomical ah (xog la ballaariyay, Jaantus. 3a). snRNA-seq hCO heerka-ku-habboon waxay soo saartay fasallo unug oo ballaadhan oo isku mid ah, oo ay ku jiraan maqnaanshaha oligodendrocytes iyo joogitaanka GABAergic neurons, taas oo laga yaabo inay ka tarjumto xaaladaha hore ee vitro ee loogu talagalay unugyada taranka dambe15 (xog la ballaariyay, Fig. 4f - i iyo Shaxda Dheeraadka ah 4). Falanqaynta muujinta hidda-wadaha kala duwan ayaa muujisay farqi weyn oo u dhexeeya neerfayaasha glutamatergic ee u dhexeeya t-hCO iyo hCO (Jadwalka Dheeraadka ah 5), oo ay ku jiraan firfircoonida unugyada hiddo-wadaha ee la xidhiidha korriinka neuronal sida calaamadaynta synaptic, deegaan dendritic, iyo dhaqdhaqaaqa kanaalka korantada-gated (Jaantuska 1h iyo Shaxda Dheeraadka ah 5). miiska 6). Sidaa darteed, qanjidhada glutamatergic t-hCO neurons waxay soo bandhigeen korriin qoraal ah oo degdeg ah.
Si loo caddeeyo in isbeddelladan qoraalka ah ee t-hCO ay la xiriiraan kala duwanaanshaha morphological ee u dhexeeya hCO in vitro iyo t-hCO ee vivo, waxaan dib-u-dhisnay heerka-ku-habboon ee biocytin-buuxiyey hCO iyo hCO ee qaybaha ba'an ka dib 7-8 bilood ee kala duwanaanshaha. hCO neurons (Jaantus. 2a). t-hCO neurons aad ayey u weyn yihiin, waxay lahaayeen 1.5 jeer dhexroorka soma, laba jeer ka badan dendrites, iyo guud ahaan lix laab kor u kaca dhererka dendritic dhererka marka la barbar dhigo in vitro hCO (Jaantus. 2b). Intaa waxaa dheer, waxaan aragnay cufnaanta sare ee lafdhabarta dendritic ee t-hCO neurons marka loo eego hCO neurons (Jaantus 2c). Tani waxay soo jeedinaysaa in t-hCO neurons ay maraan ballaarinta dendritic ballaaran iyo laanta, taas oo, marka lagu daro kororka unugyada joogtada ah, laga yaabo inay gacan ka geystaan ​​koritaanka xooggan ee t-hCO ka dib tallaalka (Sawir 1d iyo Xogta Dheeraadka ah Fig. 1f). Tani waxay nagu kalliftay inaan baarno sifooyinka korantada. Awoodda membrane waxay ahayd siddeed jeer ka sarreysa (xogta la ballaariyay, Jaantuska. 8d), awoodda xuubka-nasinta ayaa ahaa mid aad u sarreeya (qiyaastii 20 mV), iyo cirbadeynta hadda waxay keentay heerka sare ee dareenka t-hCO neurons marka loo eego hCO neurons. in vitro (Jaantus. 2d), e), kaas oo la jaan qaadaya sifooyinka morphological ee weyn iyo kakan ee t-hCO. Intaa waxaa dheer, inta jeer ee dhacdooyinka postsynaptic ee degdega ah ee degdega ah (EPSC) ayaa si aad ah uga sarreeya t-hCO neurons (Jaantus. 2f), taas oo soo jeedinaysa in korodhka cufnaanta lafdhabarta dendritic ee lagu arkay t-hCO neurons ay la xiriirto firfircoonida shaqeynta. isu galmoodka. Waxaan xaqiijinay dabeecadda aan qaan-gaarin ee hCO neurons in vitro anagoo diiwaan gelinayna glutamatergic neurons (xog la ballaariyay, Jaantus. 6a-c).
a, dib u dhiska 3D ee biocytin-buuxsan hCO iyo t-hCO neurons ka dib 8 bilood oo kala duwanaansho. b, Qiyaasta astaamaha morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 iyo *** P <0.0001). b, Qiyaasta astaamaha morphological (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0084, * P = 0.0179 iyo *** P <0.0001). б. b, qiyaasida astaamaha morphological (n=8 hCO neurons, n=6 t-hCO neurons; ** P=0.0084, *P=0.0179, iyo ***P <0.0001). b，形态学特征的量化（n = 8 个hCO 神经元。 b，形态学特征的量化（n = 8 个hCO 神经元。 б. b, qiyaasida astaamaha morphological (n=8 hCO neurons, n=6 t-hCO neurons; ** P=0.0084, *P=0.0179, iyo ***P <0.0001).c, dib u dhiska 3D ee hCO iyo t-hCO laamaha dendritic ka dib 8 bilood oo kala duwanaansho. Xiddigo cas ayaa tilmaamaya laf dhabarta dendritic-ka ee jeexan. Tirada cufnaanta laf dhabarta dendritic (n = 8 hCO neurons, n = 6 t-hCO neurons; ** P = 0.0092). d, Qiyaasta awoodda xuubka nasashada (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). d, Qiyaasta awoodda xuubka nasashada (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). d. d, xuubka nasashada ee suurtogalka ah (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, xuubka nasashada ee suurtogalka ah (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, Ficilka soo noqnoqda ee suurtagalka ah ee toogashada hCO iyo t-hCO oo ay keentay kororka irbado hadda jira, iyo qiyaasida heerka ugu sarreeya ee toogashada (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, Ficilka soo noqnoqda ee suurtagalka ah ee toogashada hCO iyo t-hCO oo ay keentay kororka irbado hadda jira, iyo qiyaasida heerka ugu sarreeya ee toogashada (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). e, повторное возбуждение потенциала действия в hCO iyo t-hCO. скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, ficil dib-u-soo-celinta hCO iyo t-hCO oo ay keentay kororka hadda jira iyo qiyaasidda heerka ugu sarreeya ee toogashada (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, повторяюющеся максимальной скорости возбуждения (n = 25 нейронов hCO, n = 16 нейронов t-hCO; *** P <0,0001). e, toogashada soo noqnoqda ee hCO iyo t-hCO suurtagalnimada ficilka ay keentay kororka sahayda hadda jirta iyo qiyaasta heerka ugu sarreeya ee toogashada (n = 25 hCO neurons, n = 16 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) ee hCO iyo t-hCO neurons ee bilaha 8 ee kala duwanaanta, iyo qiyaasta inta jeer ee dhacdooyinka synaptik (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001). f, Spontaneous EPSCs (sEPSCs) ee hCO iyo t-hCO neurons ee bilaha 8 ee kala duwanaanta, iyo qiyaasta inta jeer ee dhacdooyinka synaptik (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, Spontaneous EPSCs (sEPSCs) ee hCO iyo t-hCO neurons ee 8 bilood ee kala duwanaanshaha iyo qiyaasidda heerarka dhacdooyinka synaptik (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） 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, Spontaneous EPSCs (sEPSCs) ee hCO iyo t-hCO neurons ee 8 bilood ee kala duwanaanta iyo qiyaasidda heerarka dhacdooyinka synaptik (n = 25 hCO neurons, n = 17 t-hCO neurons; *** P <0.0001).Wixii ah bf, hCO iyo t-hCO ee xariiqda 1208-2 ayaa laga soo qaatay isla dufcaddii kala duwanaanshiyaha ee lagu hayo is barbar socda. g, Gene dhigay falanqaynta kobcinta kobcinta (tijaabada saxda ah ee Fisher hal dhinac ah) ee hiddo-wadaha si weyn kor loogu qaaday (lagu hagaajiyay P <0.05, isbeddelka laablaab> 2, oo lagu muujiyey ugu yaraan 10% nuklei) t-hCO glutamatergic neurons marka la barbar dhigo hCO glutamatergic neurons oo leh unugyada hidde-side ee labadaba jawaab-celin-celin (RG) laga aqoonsaday daraasadda jiirka in vivo ee 16 iyo bini'aadamka gaarka ah LRGs oo ka yimid in vitro neurons17. g, Gene dhigay falanqaynta kobcinta kobcinta (tijaabada saxda ah ee Fisher hal dhinac ah) ee hiddo-wadaha si weyn kor loogu qaaday (lagu hagaajiyay P <0.05, isbeddelka laablaab> 2, oo lagu muujiyey ugu yaraan 10% nuklei) t-hCO glutamatergic neurons marka la barbar dhigo hCO glutamatergic neurons oo leh unugyada hidde-side ee labadaba jawaab-celin-celin (RG) laga aqoonsaday daraasadda jiirka in vivo ee 16 iyo bini'aadamka gaarka ah LRGs oo ka yimid in vitro neurons17. g, анализ обогащениAYA P <0,05, kratnnosti изmennya > 2, Ekspressiya po mennschey merre 10% yard) глутаматергическими нейронами hCO наborы генов как ранего (ERG), так и позднего (LRG) идентифицированных в исследовании на мыshах in vivo16, и специфических для человека LRG из нейронов in vitro17. g, falanqaynta kobcinta hiddo-wadaha (tijaabada saxda ah ee Fisher hal-tailed) ee hiddo-wadaha leh firfircooni la taaban karo (lagu hagaajiyay P<0.05, isbeddelka laablaab>2, muujinta ugu yaraan 10% nuklei) ee t-hCO glutamatergic neurons marka la barbar dhigo hCO glutamatergic neurons sets ee labadaba hore (ERG) iyo firfircoonida 6-aad ee hore (ERG) LRGs bini'aadamka-gaar ah oo ka yimid neurons gudaha 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 和体外神经们17 g ， t-hco 谷氨酸 神经 元 与 hco 谷氨酸 神经 元 相比, 倍数> 2反究 中 的 早期 反应 反应 反应 和神经元 17 中 中 17 中 17的人类特异性LRG。 g, глутаматергические нейроны t-hCO. (скоррректированный P<0,05, кратность изменения> 2, не менее 10% Анализ обогащения набора генов Фишера) раннего ответа (ERG) iyo позднего гены. мышах in vivo16 iyo нейронах in vitro17. g, t-hCO glutamatergic neurons ayaa si weyn loo hagaajiyay marka la barbar dhigo hCO glutamatergic neurons (lagu hagaajiyay P<0.05, isbeddelka laablaab>2, ugu yaraan 10% Jawaabta hore (ERG) iyo falanqaynta kobcinta jawaabta dambe ee falanqaynta (baaritaanka saxda ah ee Fisher ee saxda ah) waxqabadka waxqabadka ku tiirsan hiddaha (LRGs) ayaa lagu aqoonsaday in 6. LRGsXariiqda dhibicda leh waxay muujinaysaa Bonferroni-lagu saxay qiimaha P ee 0.05. h, muujinta hidda-wadaha GluN (xirmo-xun iyo qolof hiddo-side kasta) ayaa si weyn loogu habeeyey snRNA-seq nuqullada hiddo-wadaha LRG ee t-hCO glutamatergic neurons. i, immunostaining muujinaya muujinta SCG2 ee t-hCO (sare) iyo hCO (hoos) neerfaha. Fallaadhaha cadcad waxay tilmaamayaan unugyada SCG2+. Bar miisaan, 25 µm. Xogta waxaa lagu muujiyay celcelis ahaan ± weecasho caadi ah.
Iyada oo ku saleysan kororka dhaqdhaqaaqa t-hCO ee lagu arkay jeexjeexyada ex vivo, snRNA-seq ayaa shaaca ka qaaday kor u qaadida dhaqdhaqaaqa ku tiirsan ee qoraalada hidda-wadaha ee t-hCO marka la barbar dhigo hCO in vitro. Glutamatergic t-hCO neurons ayaa muujiyay heerar sare oo hiddo-wadayaal ah oo xakameynaya waxqabadka jawaab celinta dambe (Jaantus. 2g, h), kuwaas oo laga helay daraasadihii hore ee jiirka iyo neerfayaasha bini'aadamka16,17. Tusaale ahaan, BDNF18, SCG2, iyo OSTN, oo ah hiddo-wade-hagaajinta dhaqdhaqaaqa asaasiga ah ee gaarka ah, ayaa muujiyay muujinta korodhka t-hCO neurons marka loo eego hCO neurons (Jaantus. 2g-i). Markaa, t-hCO neurons waxay soo bandhigeen sifooyin korriin oo la xoojiyey marka la barbar dhigo hCO neurons iyadoo la adeegsanayo falanqaynta qoraalka, qaab-dhismeedka, iyo shaqada.
Si loo sii qiimeeyo xidhiidhka t-hCO ee korriinka maskaxda ee horumarinta maskaxda bini'aadamka, waxaanu samaynay isbarbardhigga isbarbardhigga ee uurjiifka iyo noocyada unugyada kortikaalka ee qaangaarka19,20 iyo qaangaarka21,22 iyo sidoo kale xog ballaaran oo ku saabsan muujinta hidda-socodka cortical23 inta lagu jiro horumarinta (xogta la ballaariyay, Fig. 5). ). oo leh shaqadii hore ee 24, heerka korriinka hCO caalamiga ah iyo t-hCO transcriptome ee bilaha 7-8 ee kala duwanaanshiyaha ayaa si ballaaran u waafaqsan wakhtiga horumarinta vivo waxayna u dhigantaa nolosha dambe ee uurjiifka (Xogta Dheeraadka ah 5a). Si gaar ah, waxaan ku aragnay kororka qaan-gaarnimada t-hCO marka la barbar dhigo da'da hCO, iyo sidoo kale firfircoonida transcriptome ee la xiriirta synaptogenesis, astrogenesis, iyo myelination (xog la ballaariyay, Fig. 5b-d). Heerka gacanta, waxaan ka helnay caddayn hoosaad yar oo kiliyaha khafiifka ah ee t-hCO, oo leh kooxo neurons glutamatergic ah oo isku dhafan L2/3, L5, iyo L6 neuron subtypes (Jaantus 1i). Taas bedelkeeda, isku dhafka kooxda ee u dhexeeya glutamatergic t-hCO neurons iyo neerfayaasha kortikal ee uurjiifka ayaa aad u xaddidnaa bartamaha uurka (xog la ballaariyay, Jaantus 5e-j). Si loo go'aamiyo in t-hCO neurons ay si firfircoon ula mid yihiin neerfayaasha neocortical neocortical ee bini'aadamka, waxaan sameynay duubista elektaroonigga ah iyo dib-u-dhiska dib-u-dhiska ee bini'aadamka L2 / 3 pyramidal neurons ee qaybo fiiqan oo ka mid ah kiliyaha dambe ee bini'aadamka (xog ballaaran, Fig. 7a). Qalabka elektaroonigga ah ee L2 / 3 neurons pyramidal waxay la mid yihiin kuwa t-hCO pyramidal neurons (xog ballaaran, Fig. 7e). Morphologically, L2 / 3 neurons ee ka soo baxa shaybaarada bini'aadamka ee dhalmada ka dib waxay la mid yihiin t-hCO marka loo eego hCO, inkastoo unugyada L2 / 3 ay dheer yihiin, waxay ku jiraan laamo badan oo guud ahaan, waxayna lahaayeen cufnaanta laf dhabarta sare (Sawir 3g iyo xogta la ballaariyay, Fig. 7b-). G)
a, ku-tallaalidda hCO ee ay soo saartay xakamaynta iyo TS hiPS khadadka unugga ee jiirka dhallaanka ah. b, Dib u dhiska 3D ee biocytin-buuxsan t-hCO neurons ka dib 8 bilood oo kala duwanaansho ah. c, qiyaasta dhererka dendritic celceliska (n = 19 neurons control, n = 21 TS neurons; ** P = 0.0041). d, Laamaha dendritic-ka ee 3D-dib-u-dhiska ah ee laga soo bilaabo xakamaynta iyo TS t-hCO ee bilaha 8 ee kala duwanaanta, iyo qiyaasta cufnaanta laf-dhabarka dendritic (n = 16 neurons control, n = 21 TS neurons, *** P <0.0001). d, Laamaha dendritic-ka ee 3D-dib-u-dhiska ah ee laga soo bilaabo xakamaynta iyo TS t-hCO ee bilaha 8 ee kala duwanaanta, iyo qiyaasta cufnaanta laf-dhabarka dendritic (n = 16 neurons control, n = 21 TS neurons, *** P <0.0001). d, 3D-реконструкция дендритных ветвей из контроля iyo TS t-hCO плотности дендритных шипов (n = 16 контрольных нейронов, n = 21 TS нейронов, *** P <0,0001). d, Dib-u-dhiska 3D ee laamaha dendritic ee xakamaynta iyo t-hCO TS ee bilaha 8 ee kala duwanaanta iyo qiyaasta cufnaanta laf-dhabarka dendritic (n = 16 kantaroolka 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）。 分化 8 个 时 对照 和 ts t-hco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, Dib-u-dhiska 3D ee laamaha dendritic-ka iyo TS t-hCO ee bilaha 8 ee kala duwanaanta iyo qiyaasta cufnaanta laf-dhabarka dendritic (n = 16 kantaroolka neurons, n = 21 TS neurons, *** P <0.0001).Xiddigo cas ayaa tilmaamaya laf dhabarta dendritic-ka ee jeexan. e, EPSCs iskiis ah oo xakameynaya iyo TS t-hCO neurons ka dib 8 bilood oo kala duwanaansho ah. f, goob joogta ah oo isugeyn iyo qiyaasid inta jeer iyo baaxadda dhacdooyinka synaptika (n=32 neurons control, n=26 TS neurons; ** P=0.0076 iyo P=0.8102). g, Falanqaynta Scholl ee TS iyo xakamaynta neerfayaasha ee hCO iyo t-hCO. Khadadka la jajabiyey waxay muujinayaan bini'aadamka L2 / 3 neurons pyramidal ka dib marka la barbardhigo (n = 24 kantaroolka t-hCO neurons, n = 21 TS t-hCO neurons, n = 8 kantaroolka hCO neurons, iyo n = 7 TS hCO neurons). Xogta waxaa lagu muujiyey sida celceliska ± weecanaanta caadiga ah
Awoodda t-hCO si ay u soo celiso qaababka qaab-dhismeedka iyo shaqada ee neerfayaasha kiliyaha bini'aadamka ee heer sare ah ayaa nagu kallifay inaan sahamno in t-hCO loo isticmaali karo in lagu ogaado phenotypes cudurada. Waxaan diirada saarnay TS, xanuunka korriinka neerfaha ee daran oo ay sababto faa'iido-shaqo-isbedelka hiddasidaha codeeya CaV1.2, kaas oo bilaabaya qoritaanka hidda-socodka ku-tiirsan ee neerfayaasha. Waxaan ka helnay hCO seddex bukaan oo TS ah oo sita bedelka ugu caansan (p.G406R) iyo seddex kontaroolo (Jaantus. 3a). Ka dib markii la beero, waxaan ogaanay in qaabka dendritic morphology lagu bedelay TS neurons marka la barbardhigo kontaroolada (Sawir 3b iyo xogta la ballaariyay, Fig. 8a,b), oo leh laba laab kor u kaca tirada dendrites aasaasiga ah iyo kororka guud ee celceliska iyo guud ahaan hoos u dhaca dhererka dendritic (Jaantus. 3c iyo xogta dheer, sawirka 8c). Tani waxay la xiriirtaa kororka cufnaanta laf dhabarta iyo kororka soo noqnoqda ee EPSC-yada iskood ah ee TS marka la barbardhigo kantaroolka neurons (Jaantus. 3d-f iyo xogta la ballaariyay, Jaantus. 8g). Falanqaynta dheeraadka ah ayaa shaaca ka qaaday qaababka aan caadiga ahayn ee dendritic branching ee t-hCO TS marka la barbardhigo kantaroolka, laakiin aan ku jirin vitro TS hCO marxalad la mid ah oo kala duwanaansho ah (Jaantus. 3g). Tani waxay la socotaa warbixinadeenii hore ee dhaqdhaqaaqa ku-tiirsanaanta dendritic ee TS waxayna muujineysaa awoodda goobtan ku-tallaalidda si loo ogaado phenotypes cudurada ee vivo.
Waxaan markaa waydiinay ilaa intee in le'eg ayay unugyada t-hCO si firfircoon ugu dhex jiraan jiirka S1. S1 ee jiirka ayaa helaya qalabyo synaptik oo xoog leh oo ka yimaada basal ventral ventral basal iyo thalamic nuclei dambe, iyo sidoo kale matoorka ipsilateral iyo cortices somatosensory sare, iyo S1 (Jaantus. 4a). Si loo soo celiyo hannaankii innervation, waxaanu ku qaadnay hCO fayraska raabiyada-dG-GFP/AAV-G waxaana ku tallaalnay hCO gudaha S1 3 maalmood ka dib. Waxaan ku aragnay muujinta cufan ee GFP ee neerfayaasha ipsilateral S1 iyo ventral basal ganglia 7-14 maalmood ka dib tallaalka (Jaantus. 4b, c). Intaa waxaa dheer, midabaynta antibody-ka ee calaamadeeyaha thalamic netrin G1 ayaa shaaca ka qaaday joogitaanka dhamaadka thalamic ee t-hCO (Jaantus. 4d, e). Si loo qiimeeyo in saadaasha afferent-ka ahi ay keeni karto jawaabaha synaptik ee unugyada t-hCO, waxaanu samaynay cajalado unug oo dhan oo laga soo duubay unugyada bini'aadamka qaybo fiiqan oo ka mid ah lakabka thalamocortical. Kicinta korantada ee jiirka S1, kaabsal gudaha ah, walxaha cad, fiilooyinka u dhow t-hCO ama firfircoonida optogenetic ee opsin-muujinaysa dhamaadka thalamic ee t-hCO waxay keentay EPSCs-gaaban ee t-hCO neurons ee soo gaadhay AMPA receptor antagonist NBQX. (Jaantus. 4f, g iyo xogta la dheereeyey, Jaantus. 9a-g). Xogtaasi waxay muujinaysaa in t-hCO ay si anatomically ah ugu dhex milmay maskaxda jiirka oo ay awood u leedahay in ay dhaqaajiso unugga martida loo yahay.
a, Jaantuska jaantuska ee tijaabada raad-raacida rabies. b, GFP iyo muujinta STEM121 ee bini'aadamka u gaarka ah ee u dhexeeya t-hCO iyo kiliyaha maskaxda ee jiirka (guriga sare). Sidoo kale waxaa lagu muujiyay muujinta GFP ee jiirka ipsilateral ventral basal nucleus (VB) (bidix hoose) iyo ipsilateral S1 (midig hoose). Bar miisaan, 50 µm. Labada geesood ee cas ayaa u taagan meelaha maskaxda ee sawirada laga qaaday. c, tirada unugyada muujinaya GFP (n = 4 jiir). d, e - Netrin G1+ terminaalka thalamic ee t-hCO. d waxay tusinaysaa qaybta wadnaha oo ka kooban t-hCO iyo nuklei VB. Bar miisaan, 2 mm. e waxay muujinaysaa Netrin G1 iyo STEM121 muujinta t-hCO (bidix) iyo VB (midig) neerfayaasha. Bar miisaan, 50 µm. Xariiqda liimiga ah ee dhibco leh ayaa tilmaamaysa xadka t-hCO. f,g Baaxadda EPSC oo leh iyo la'aan NBQX (n = 6 S1 neurons, * P = 0.0119; iyo n = 6 neurons capsule gudaha, ** P = 0.0022) (dhex). Boqolkiiba t-hCO neurons muujinaya EPSC iyada oo laga jawaabayo kicinta korantada ee jiirka S1 (f) ama kaabsal gudaha ah (g) (midig). aCSF, dareeraha cerebrospinal macmal ah. h, jaantuska jaantuska tijaabada sawirka 2P (bidix). Muujinta GCaMP6s ee t-hCO (dhexe). Bar miisaan, 100 µm. Wakhtiga luorescence ee GCaMP6s (midig). i, Z-dhibcaha ee fluorescence dhaqdhaqaaqa kediska ah. j, sawir-shaxeedka kicinta shaarubaha. k, z-dhibcaha 2P fluorescence trajectories hal tijaabo, oo la jaanqaadaysa weecasho whisker wakhtiga eber (khadka jajaban) tusaale ahaan unugyada. l, jawaabaha celceliska-z-dhibcaha dadweynaha ee dhammaan unugyada la socda leexashada whiskerka waqtiga eber (xariiq jajaban) (casaan) ama jaantusyada waqtiyada si aan kala sooc lahayn loo soo saaray (cawlan). m. Shaxda jaantuska tijaabada ee calaamadaynta indhaha. n, Qalloocyada korantada ceeriin ee tusaale ahaan unugga t-hCO inta lagu jiro kicinta laysarka buluugga ah ama leexinta whisker-ka. Fallaadhaha gaduudan waxay muujinayaan cirifka ugu horreeya ee uu keeno iftiinka (sare) ama uu keeno leexashada shafka (hoose). Hooska cawlku waxa uu tilmaamayaa xilliyada leexashada shafka. o, Mowjadaha hirarka iftiinka ee ugu sarreeya iyo jawaabaha leexinta whisker p, bislooyin hal isku day ah, oo la jaanqaadaysa leexinta shabagga ee unugyada tusaalaha. 0 waxay tusinaysaa weecasho whisker (xariiq jajaban). q, celceliska dadka-dhibcaha toogashada z-dhibcaha ee dhammaan unugyada sawir-qaadista, oo la jaan-qaadaya leexashada whiskerka wakhtiga eber (khad la jaray) (cas) ama jaantusyada wakhtiyada si aan kala sooc lahayn loo soo saaray (cawlan). r, Saamiga unugyada sawir-qaadista oo si weyn loogu habeeyey leexsanaanta whisker (n = 3 jiir) (bidix). Daahida ugu sarreysa z-dhibcaha (n = 3 jiir; n = 5 (cagaar khafiif ah), n = 4 (cagaar madow), iyo n = 4 (cyan) whisker weecinta unugyada qaabaynta jiirkiiba) (midig). Xogta waxaa lagu muujiyay celcelis ahaan ± weecasho caadi ah
Waxaan markaa waydiinay haddii t-hCO lagu kicin karo kicinta dareenka ee vivo. Waxaan ku tallaalnay hCO inagoo muujinayna tilmaamayaasha kaalshiyamka hidde ahaan lagu calaamadeeyay GCaMP6 una galnay jiirka S1. Ka dib 150 maalmood, waxaanu samaynay sawir-qaadista fiber-ka ama sawir-qaadista kalsiyum ee laba-sawir ah (Jaantus. 4h iyo xogta la ballaariyay, Jaantus 10a). Waxaan ogaanay in unugyada t-hCO ay muujiyeen dhaqdhaqaaqa laxanka isku midka ah (Jaantuska 4i, Xogta La Balaaray, Jaantuska 10b iyo Fiidiyowga Dheeraadka ah 1). Si loo qeexo dhaqdhaqaaqa ugu sarreeya t-hCO, waxaanu samaynay cajalado elektrofisiolojiyeedka ka baxsan unugyada jiirka la suuxiyay (xogta la fidiyay, Jaantuska 10c-f). Waxaan ka soo saarnay isku-duwayaasha stereotaxic sawirada MRI; sidaas awgeed, cutubyadan la diiwaan galiyay waxay u taagan yihiin neerfayaasha bini'aadamka, in kasta oo electrophysiology kaliya aysan ogolayn in la go'aamiyo noocyada asalka ah. Waxaan aragnay dhaqdhaqaaqyo dildilaac ah (xog la fidiyay, Jaantuska 10d). Qaraxyadu waxay socdeen ilaa 460 ms waxaana kala saaray muddo aamusnaan ah oo ku dhow 2 s (xog la fidiyay, Jaantus. 10d, e). Unugyada gaarka ah waxay rideen celcelis ahaan ilaa saddex wareeg qarax kasta, taas oo ah ku dhawaad ​​73% unugyada diwaangashan qarax kasta. Dhaqdhaqaaqyada cutubyada gaarka ah ayaa si aad ah isugu xiran, isku xirnaantaasina waxay ka sarreeyeen kuwa lagu aqoonsaday xoolaha aan la tallaalin ee lagu diiwaan geliyay xaalado isku mid ah (xog la ballaariyay, Jaantus. 10f). Si loo sii tilmaamo jawaabaha sare ee neerfayaasha ka soo jeeda bini'aadamka ee la aqoonsaday, waxaanu samaynay tijaabooyin iftiin leh oo ku saabsan jiirarka la suuxiyay ee lagu beeray hCO oo muujinaya kanaalka iftiinka-xasaasi ah ee rhodopsin 2 (hChR2), kaas oo t-hCO neurons-gaaban aqoonsiga gaaban (in ka yar 10 ms) oo ka jawaabaya iftiinka buluuga ah. t-hCO neurons waxay soo bandhigeen dilaacyo dhaqdhaqaaqyo iskood ah oo isdaba-joog ah oo la mid ah kuwa lagu arkay sawir-qaadista kaalshiyamka, iyo sidoo kale duubista elektaroonigga ah ee lagu sameeyay t-hCO maqnaanshaha calaamadaynta iftiinka (xog ballaaran, Fig. 10c-g). Wax dhaqdhaqaaq ah oo iskiis ah laguma arag marxaladaha u dhigma ee hCO ee lagu duubay vitro. Si loo qiimeeyo in t-hCO lagu hawlgelin karo kicinta dareenka, waxaanu si kooban uga leexinay shafka jiirka ee t-hCO (Jaantus. 4j, m iyo xogta la dheereeyey, Jaantus. 10h,k). Marka loo eego daraasadihii hore ee 8,10, qayb ka mid ah unugyada t-hCO waxay muujiyeen waxqabadyo kordhay oo ka jawaabaya leexinta whisker, taas oo aan la arkin marka xogta la barbar dhigo stamps wakhtiga random (Jaantus. 4k-q iyo xogta la ballaariyey, Fig. 10h-q). Runtii, qiyaastii 54% ee halbeegyada lagu calaamadiyay opto-ku waxay muujiyeen korodhka kacsiga oo aad u kordhay ka dib kicinta whisker-ka, oo gaadhay ugu sarreysa 650 ms (Jaantus. 4r). Isku soo wada duuboo, xogtani waxay soo jeedinaysaa in t-hCO ay hesho agabka shaqayneed ee ku haboon oo ay dhaqaajin karto kicinta deegaanka.
Waxaan markaa baarnay in t-hCO ay dhaqaajin karto wareegyada jiirka si loo xakameeyo dhaqanka. Waxaan marka hore baarnay in axon ee t-hCO neurons ay mashruuca ku galaan unugyada ku hareeraysan jiirka. Waxaan ku qaadnay hCO-ga lentivirus-ka hChR2 oo ku dhegan EYFP (hChR2-EYFP). Ka dib 110 maalmood, waxaan ku aragnay muujinta EYFP ee gobollada kortikaalka ee ipsilateral, oo ay ku jiraan kortiyada maqalka, mootada, iyo somatosensory, iyo sidoo kale gobollada hoose, oo ay ku jiraan striatum, hippocampus, iyo thalamus (Jaantus. 5a). Si loo qiimeeyo in saadaashan firfircooni ay keeni karto jawaabaha synaptik ee unugyada jiirka, waxaanu si muuqaal ah u hawlgelinay unugyada t-hCO ee muujinaya hChR2-EYFP annagoo duubnayna unugyada kortex maskaxda ee qaybaha maskaxda ee fiiqan. Dhaqdhaqaaqa t-hCO axon oo leh iftiin buluug ah oo keenay EPSCs daahitaan gaaban oo ku jira jiirka kortex neurons, kuwaas oo ay xannibeen NBQX (Sawir 5b-g). Intaa waxaa dheer, jawaabahaas waxaa lagu xannibi karaa tetrodotoxin (TTX) waxaana dib loo soo celiyay 4-aminopyridine (4-AP), taas oo soo jeedinaysa in ay sababeen isku-xirnaanta monosynaptic (Jaantus. 5e).
a, Jaantuska jaantuska raadraaca axon (bidix). t-hCO EYFP muujinta (midig). Bar miisaan, 100 µm. A1, kortex maqal, ACC, kortex cingulate hore, d. striatum, dorsal striatum, HPC, hippocampus; Diaphragm, septum lateral, mPFC, medial prefrontal cortex, piri, piriform cortex, v. striatum, ventral striatum, VPM, ventropostomedial nucleus ee thalamus, VTA, gobolka ventral tegmental. Labada geesood ee cas ayaa u taagan meelaha maskaxda ee sawirada laga qaaday. b, Jaantuska jaantuska ee tijaabada kicinta. c, d, Tusaalooyinka jawaabta iftiinka buluuga ah ee iftiinka buluuga ah ee sawir-qaadaha (sare) iyo danab (hoose) ee bini'aadamka (c) EYFP+ t-hCO ama rat (d) unugyada EYFP. e.f g, daahitaanka jawaabaha uu keenay iftiinka buluuga ah ee unugyada jiirka (n = unugyada 16); baararka siman waxay muujinayaan daahitaan celcelis ahaan (7.13 ms) (bidix). Baaxadda EPSC-yada iftiinka-kicinaya ee lagu duubay NBQX ama aan lahayn (n = 7 unug; *** P <0.0001) (dhexe). Baaxadda EPSC-yada iftiinka-kicinaya ee lagu duubay NBQX ama aan lahayn (n = 7 unug; *** P <0.0001) (dhexe). Амплитуда вызванных светом EPSC, зарегистрированных с или без NBQX (n = 7 клеток; ***P <0,0001) (в центре). Balaadhinta EPSC-yada iftiinka leh ee lagu duubay NBQX ama aan lahayn (n = 7 unug; *** P <0.0001) (dhex).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). Balaadhinta EPSC-yada iftiinka leh ee lagu duubay NBQX ama aan lahayn (n = 7 unug; *** P <0.0001) (dhex).Boqolkiiba unugyada jiirka ee muujinaya EPSC-yada ka jawaaba iftiinka buluuga ah (midig). h, Jaantus shaxeed hawsha habdhaqanka. d0, maalin 0. i. Waxqabadka xoolaha ku dayashada mudan maalinta 1 (bidix) ama maalinta 15 (midig) ee tababarka. Celceliska tirada leefooyinka la sameeyay maalinta 1 (bidix) ama maalinta 15 (xarunta midig) (n = 150 tijaabo nalka buluuga ah, n = 150 tijaabo nalka cas; *** P <0.0001). Celceliska tirada leefooyinka la sameeyay maalinta 1 (bidix) ama maalinta 15 (xarunta midig) (n = 150 tijaabo nalka buluuga ah, n = 150 tijaabo nalka cas; *** P <0.0001). Среднее количество облизываний, выполненных в день 1 (слева) n = 150 испытаний с красным светом; *** P <0,0001). Tirada celceliska leefooyinka la sameeyay maalinta 1 (bidix) ama maalinta 15 (midigta dhexe) (n = 150 tijaabo nalka buluuga ah, n = 150 tijaabo nalka cas; ***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) n = 150 испытаний с красным светом; *** P <0,0001). Tirada celceliska leefooyinka la sameeyay maalinta 1 (bidix) ama maalinta 15 (midigta dhexe) (n = 150 tijaabo nalka buluuga ah, n = 150 tijaabo nalka cas; ***P <0.0001).Leefis isugeyn ah oo loogu talagalay tijaabooyinka iftiinka cas iyo buluugga maalinta 1 (bidix dhexe) ama maalinta 15 (midig). NS, ma aha mid muhiim ah. j,k, Astaamaha hab-dhaqanka ee dhammaan xayawaanka lagu beeray t-hCO oo muujinaya hChR2-EYFP (j) ama xakamaynta fluorophore (k) maalinta 1 ama 15 (hChR2-EYFP: n = 9 jiir, ** P = 0.0049; xakamaynta: n = 9, P = 0.1497). l, Kobcinta dhibcaha doorbidida (n = 9 hChR2, n = 9 xakamaynta; ** P <0.001, *** P <0.0001). l, Kobcinta dhibcaha doorbidida (n = 9 hChR2, n = 9 xakamaynta; ** P <0.001, *** P <0.0001). l, Эvolyutsyja pokzatelya предпочтения (n = 9 hChR2, n = 9 kontrolьныh; ** P <0,001, *** P <0,0001). l, Kobcinta dhibcaha doorbidida (n = 9 hChR2, n = 9 kontaroolada; ** P <0.001, *** P <0.0001). l. l. l, Эvolюця показателей предпочтения (n = 9 hChR2, n = 9 kontroley; ** P <0,001, *** P <0,0001). l, Kobcinta buundooyinka doorbidida (n = 9 hChR2, n = 9 kontaroolada; ** P <0.001, *** P <0.0001).m, FOS muujinta ka jawaabista firfircoonida optogenetic ee t-hCO ee S1. Sawirada muujinta FOS (bidix), iyo qiyaas (n = 3 koox kasta; * P <0.05, ** P <0.01 iyo *** P <0.001) (midig) ayaa la muujiyay. Sawirada muujinta FOS (bidix), iyo qiyaas (n = 3 koox kasta; * P <0.05, ** P <0.01 iyo *** P <0.001) (midig) ayaa la muujiyay. Покаzanы изображения эkspressы FOS (sleva) iyo kolychestvenno opredelennya (n = 3 на групу; * P <0,05, ** P <0,01 , p <0,01 , p <0,01 , p <0,01 . Sawirada muujinta FOS (bidix) iyo qiyaas (n = 3 koox kasta; * P <0.05, ** P<0.01, iyo *** P <0.001) ayaa la muujiyay (midig).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） Покаzanы изображения эkspressы FOS (sleva) iyo kolychestvenno opredelennya (n = 3 на групу; * P <0,05, ** P <0,01 , p <0,01 , p <0,01 , p <0,01 . Sawirada muujinta FOS (bidix) iyo qiyaas (n = 3 koox kasta; * P <0.05, ** P<0.01, iyo *** P <0.001) ayaa la muujiyay (midig).Bar miisaan, 100 µm. Xogta waxaa lagu muujiyay celcelis ahaan ± qaladka caadiga ah ee BLA, qumanka basolateral, MDT, dorsomedial thalamic nucleus, PAG, cawl periaqueductal.
Ugu dambeyntii, waxaan weydiinay haddii t-hCO ay wax ka bedeli karto dhaqanka jiirka. Si tan loo tijaabiyo, waxaanu ku tallaalnay hChR2-EYFP-muujinaysa hCO gudaha S1, iyo 90 maalmood ka dib, waxaanu ku tallaalnay fiilooyinka indhaha ee t-hCO si loo iftiimiyo. Ka dib waxaan ku tababarnay jiirarka qaab qaboojiyaha wax ka beddelka ah (Jaantus. 5h). Waxaan ku dhejinay xayawaanka qolka tijaabada dabeecadda waxaana si aan kala sooc lahayn u codsanay 5 buluug labaad (473 nm) iyo casaan (635 nm) kicinta laysarka. Xayawaanku waxay heleen abaal-marin biyaha haddii ay leefleefi jireen xilliga kicinta nalka buluugga ah laakiin ma ay leefin xilliga kicinta iftiinka cas. Maalintii ugu horeysay ee tababarka, xayawaanku ma muujin wax farqi ah oo leef ah marka lagu kiciyo iftiin buluug ama casaan ah. Si kastaba ha ahaatee, maalintii 15, xayawaanka lagu beeray hCO oo muujinaya hChR2-EYFP waxay muujiyeen leefsi firfircoon marka lagu kiciyo iftiin buluug ah marka loo eego kicinta iftiinka cas. Isbeddelladan hab-dhaqanka leefka laguma arag xawayaanka kantaroolka ee lagu tallaalay hCO oo muujinaya kontoroolka fluorophore (hChR2 89%, EYFP 0%, Jaantuska 5i-1 iyo Fiidiyowga Dheeraadka ah 2). Xogtaasi waxay soo jeedinaysaa in unugyada t-hCO ay dhaqaajin karaan neerfaha jiirka si ay u kiciyaan habdhaqanka abaal-marineed. Si loo ogaado wareegyada neerfaha ee t-hCO ee laga yaabo inay ku lug yeeshaan isbeddelladan dabeecadeed, waxaan si optogenetically u hawlgelinnay t-hCO ee xayawaanka tababbaran iyo unugyada la goostay 90 daqiiqo ka dib. Immunohistochemistry ayaa shaaca ka qaaday muujinta borotiinka FOS ku tiirsan ee dhaqdhaqaaqa ku jira dhowr gobol oo maskaxda ah oo ku lug leh dabeecad dhiirigelin leh, oo ay ku jiraan kiliyaha prefrontal cortex, medial thalamus, iyo walxaha cawlan ee periaqueductal, kaas oo lagu muujiyay xayawaanka xakamaynta aan la kicin ama xayawaanka. bariis 5m). Isku soo wada duuboo, xogtani waxay soo jeedinaysaa in t-hCO ay wax ka beddeli karto dhaqdhaqaaqa neerfaha ee jiirka si loo wado dabeecadda.
Neural organoids waxay u taagan yihiin nidaam rajo leh oo lagu baranayo horumarka aadanaha iyo cudurka in vitro, laakiin waxay xaddidan yihiin la'aanta xiriirka ka dhexeeya wareegyada ka jira vivo. Waxaan samaynay madal sheeko cusub kaas oo aan ku tallaalnay hCO una galnay S1 ee tallaalka difaaca ee hore ee jiirka dhalmada ka dib si aan u barano horumarka unugyada bini'aadamka iyo shaqada gudaha vivo. Waxaan tusnay in t-hCO ay horumariso noocyada unugyada qaan-gaarka ah ee aan lagu arkin vitro28 iyo in t-hCO ay tahay mid anatomically ah oo si shaqaynaysa u dhexaysa maskaxda jiirka. Isku dhafka t-hCO ee wareegyada neerfayaasha jiirka ayaa noo ogolaatay inaan sameyno xiriir ka dhexeeya dhaqdhaqaaqa gacanta ee bini'aadamka iyo dabeecadda xayawaanka ee la bartay, taas oo muujinaysa in t-hCO neurons ay bedeli karaan dhaqdhaqaaqa neerfaha jiirka si ay u kiciyaan jawaabaha habdhaqanka.
Madal aan ku sifayneyno waxay leedahay faa'iidooyin dhowr ah marka loo eego cilmi-baaris hore oo ku saabsan in unugyada aadanaha loogu beddelo maskaxda jiirka. Marka hore, waxaanu ku tallaalnay hCO-da kortex ee jiirka hore ee dhalmada ka dib, kaas oo fududayn kara isdhexgalka anatomical iyo functional. Marka labaad, t-hCO MRI kormeerka ayaa noo ogolaatay inaan barano booska tallaalka iyo korriinka xoolaha nool, taasoo noo ogolaaneysa inaan sameyno daraasado xayawaanno badan oo waqti dheer ah oo aan dejino kalsoonida dhowr xariiqo unug oo hiPS ah. Ugu dambayntii, waxaanu ku tallaalnay organoid-ka saxda ah, halkii aan ka ahaan lahayn xannibaadyo unug keliya ah oo go'doonsan, kuwaas oo aan waxyeello u geysan unugyada bini'aadamka oo kor u qaadi kara is-dhexgalka iyo jiilka neurons-ka bini'aadamka ee maskaxda jiirka.
Waxaan qiraynaa in inkasta oo horumarka laga sameeyay goobtan, ku-meel-gaadhka, ku-meel-gaadhka ah, iyo caqabadaha noocyada kala-gudbinta ah ay ka hortagayaan samaynta wareegyada neerfaha ee bini'aadamka oo leh daacadnimo sare, xitaa ka dib tallaalka marxaladda hore ee horumarinta. Tusaale ahaan, ma cadda in hawsha iskeed ah ee lagu arkay t-hCO ay u taagan tahay phenotype korriin la mid ah dhaqdhaqaaqa laxanka ee lagu arkay inta lagu jiro koritaanka kortikal, ama haddii ay sabab u tahay maqnaanshaha noocyada unugyada xakamaynta ee ku jira t-hCO. Sidoo kale, ma cadda ilaa xadka maqnaanshaha lamination ee t-hCO ay saameynayso isku xirka silsiladda30. Shaqada mustaqbalka waxay diiradda saari doontaa isku-dhafka noocyada kale ee unugyada sida microglia bini'aadamka, unugyada endothelial ee bini'aadamka, iyo qaybaha kala duwan ee GABAergic interneurons sida lagu muujiyay isticmaalka golaha 6 ee vitro, iyo sidoo kale fahamka sida isdhexgalka neerfaha iyo farsamaynta ay u dhici karto t-hCO oo la beddelay. transcriptional, synaptik iyo heerarka dabeecadda ee unugyada laga helay bukaanka.
Guud ahaan, tan in vivo platform waxay ka dhigan tahay kheyraad awood leh oo kaabi kara horumarinta maskaxda bini'aadamka iyo cilmi-baarista cudurrada. Waxaan filaynaa in goobtani ay noo ogolaan doonto in aan ogaano noocyada cusub ee heerka strand ee unugyo bukaan-socod oo kale ah oo aan tijaabinno xeelado daweyn oo cusub.
Waxaan ka soo saarnay hCO2.5 unugyada HiPS sidii hore loogu sharraxay. Si loo bilaabo wax soo saarka hCO ee unugyada hiPS ee lagu beeray lakabyada quudiyaha, kolonyadii aan fiicneyn ee unugyada hiPS ayaa laga saaray suxuunta dhaqanka iyadoo la adeegsanayo daadinta (0.35 mg/mL) waxaana loo wareejiyay dhaqamo caag ah oo aad u hooseeya oo ay ku jiraan suxuun leh dhexdhexaad dhaqanka unugga hiPS. (Corning) oo lagu daray laba SMAD inhibitors dorsomorphine (5 μM; P5499, Sigma-Aldrich) iyo SB-431542 (10 μM; 1614, Tocris) iyo ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). Inta lagu jiro 5-tii maalmood ee ugu horreeyay, dhexdhexaadinta unugga hiPS waa la beddelay maalin kasta waxaana lagu daray dorsomorphine iyo SB-431542. Maalintii lixaad ee joojinta, spheroids neural waxaa loo wareejiyay dhexdhexaad neural ah oo ay ku jiraan neurobasal-A (10888, Life Technologies), B-27 dheeraad ah oo aan vitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin iyo streptomycin (1: 100, Technology Life; 1 ml) iyo kobcinta 0. Nidaamyada R & D) iyo factor koritaanka fibroblast 2 (FGF2; 20 ng ml-1; R & D Systems) ilaa maalinta 24. Laga soo bilaabo maalinta 25 ilaa maalinta 42, dhexdhexaadinta waxaa lagu kabay factor neurotrophic maskaxda ka soo jeeda (BDNF; 20 ng ml-1, Peprotech) iyo neurotrophin 3 (NT3) - 10 ml maalin kasta oo kale neurotrophin. Maalintii lixaad ee joojinta, spheroids neural waxaa loo wareejiyay dhexdhexaad neural ah oo ay ku jiraan neurobasal-A (10888, Life Technologies), B-27 dheeraad ah oo aan vitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin iyo streptomycin (1: 100, Technology Life; 1 ml) iyo kobcinta 0. Nidaamyada R & D) iyo factor koritaanka fibroblast 2 (FGF2; 20 ng ml-1; R & D Systems) ilaa maalinta 24. Laga soo bilaabo maalinta 25 ilaa maalinta 42, dhexdhexaadinta waxaa lagu kabay factor neurotrophic maskaxda ka soo jeeda (BDNF; 20 ng ml-1, Peprotech) iyo neurotrophin 3 (NT3) - 10 ml maalin kasta oo kale neurotrophin.Maalintii lixaad ee joojinta, spheroids neural waxaa loo wareejiyay dhexdhexaad ah neural ka kooban Neurobasal-A (10888, Life Technologies), B-27 dheeraad ah oo aan vitamin A (12587, Life Technologies), GlutaMax (1: 100, Life Technologies), penicillin.iyo streptomycin (1:100, Life Technologies) iyo dhopolnenы эpydermal phaktorom rosta (EGF; 20 ng/ml; R&D Systems) (FGF2; 20 нг/мл; R&D Systems) iyo 24-го дня. iyo streptomycin (1: 100, Teknolojiyada Nolosha) oo lagu kabo factor koritaanka epidermal (EGF; 20 ng / ml; R & D Systems) iyo factor koritaanka fibroblast 2 (FGF2; 20 ng / ml; R & D Systems) ilaa maalinta 24.Laga soo bilaabo maalmaha 25 ilaa 42, maskaxda ka soo jeeda neurotrophic factor (BDNF; 20 ng ml-1, Peprotech) iyo neurotrophin 3 (NT3; 20 ng ml-1, Peprotech) ayaa lagu daray dhexdhexaadinta, beddelidda dhexdhexaadka maalin kasta oo kale.在悬浮的第6 天，将神经球体转移到含有neurobasal-A（10888，Life Technologies Teknolojiyadda, GlutaMax (1:100, Teknolojiyadda Nolosha) Teknolojiyada）并辅以表皮生长因子（EGF；20 ng ml-1；R&D Systems Nidaamyada)直至第24 天.在 浮 的 第第 6 天 将 神经 球体 转移 含有 含有 neurobasal-a补充剂 （12587 ， Technology Life） Glutamax (1: 100生长 因子 (20 ng ml-1 ； r & d Systems) 成 纤维 细胞 生长 2 На 6-й день суспензии нейросфERы были переведены на добавку, содержащую нейробазазал-А (10888), Life Technology витамина А (12587, Technology Life), GlutaMax (1:100, Life Technology), пеницилинный стрептомины (1:100, Technology Life) фактора роста (EGF; 20 нг мл-1; R&D Systems) iyo фактора роста фибробластов 2 (FGF2; 20 нг мл-1) 1; Maalinta 6, joojinta neurosphere ayaa loo beddelay kabitaan ay ku jiraan neurobasal-A (10888, Teknolojiyadda Nolosha), B-27 oo aan lahayn fitamiin A (12587, Teknolojiyadda Nolosha), GlutaMax (1: 100, Teknolojiyadda Nolosha), penicillin-dhexdhexaad ah streptomycin (1: 100, Teknolojiyadda Nolosha) 2 oo lagu kabay korriinka EGF. Nidaamyada) iyo koritaanka fibroblast 2 (FGF2; 20 ng ml-1) 1; R&D Systems) ilaa 24-го дня. Nidaamyada R&D) ilaa maalinta 24.Laga soo bilaabo maalmaha 25 ilaa 42, factor neurotrophic maskaxda ka soo jeeda (BDNF; 20 ng ml-1, Peprotech) iyo neurotrophic factor 3 (NT3; 20 ng ml-1, Peprotech) ayaa lagu daray dhexdhexaadinta dhaqanka maalin kasta oo kale. Isbeddel dhexdhexaad ah hal mar.Laga bilaabo maalinta 43, hCO waxaa lagu hayaa dhexdhexaad neurobasal-A (NM; 1088022, Thermo Fisher) oo leh isbeddel dhexdhexaad ah 4-6 maalmood kasta. Si aad u heshid hCO unugyada hiPS ee lagu dhaqmo xaaladaha feeder la'aanta, unugyada hiPS waxaa lagu darey Accutase (AT-104, Innovate Cell Technologies) 37°C ee daqiiqadaha 7, oo loo kala qaybiyey unugyo keliya, waxaana lagu dhejiyay AggreWell 800 taarikada (34815, STEMCELL Technologies) cufnaanta Eeg10 ee unug kasta 3 × dhexdhexaad ah ROCK inhibitor Y-27632 (10 μM; S1049, Selleckchem). 24 saacadood ka dib, warbaahinta ceelasha ayaa kor iyo hoos loogu shubay warbaahinta ka kooban warbaahinta muhiimka ah ee 6 (A1516401, Technology Life) oo lagu daray dorsomorphine (2.5 μM; P5499, Sigma-Aldrich) iyo SB-431542 (10 μM; 1614). , Tocrida). Laga soo bilaabo maalmaha 2 ilaa 6, Dhexdhexaadinta Muhiimka ah ee 6 waxaa lagu bedelay maalin kasta dorsomorphine waxaana lagu daray SB-431542. Laga soo bilaabo maalintii lixaad, joojinta neurosphere waxaa loo wareejiyay dhexdhexaadiyaha neurobasal waxaana lagu hayaa sida kor lagu sharaxay.
Dhammaan hababka xayawaanka waxaa loo sameeyay si waafaqsan xeerarka daryeelka xayawaanka ee ay ansixiyeen Guddiga Maamulka Daryeelka Xayawaanka ee Jaamacadda Stanford (APAC). Jiirka euthymic RNU (rnu/+) ee uurka leh ayaa la iibsaday (Charles River Laboratories) ama guriyeeyay. Xayawaanka waxaa lagu haayay wareeg iftiin 12-saac ah oo mugdi ah oo leh cunto iyo biyo ad libitum. Qaawan (FOXN1–/-) Dhallaanka jiirka ee da'doodu u dhaxayso saddex ilaa toddobo maalmood ayaa lagu aqoonsaday korriinka shabagga aan qaan-gaarin ka hor inta aan la jarin. Dhallaanka (lab iyo dheddig) ayaa lagu suuxiyay 2-3% isoflurane waxaana la saaray qaab stereotaxic ah. Wareegga madaxa oo leh dhexroor qiyaastii 2-3 mm ka sarreeya S1 ayaa la sameeyay iyadoo la ilaalinayo daacadnimada dura mater. Ka dib isticmaal irbad 30-G ah (qiyaastii 0.3 mm) meel ka baxsan craniotomy si aad u muddo durayga. Dabadeed mari HCO dhuuban 3 × 3 sm oo khafiif ah oo ka saar dhexdhexaadinta xad dhaafka ah. Adigoo isticmaalaya saliingaha Hamilton ee ku dheggan 23G, 45° irbad, si tartiib ah hCO ugu soo jiid cidhifka ugu fog ee irbadda. Kadibna ku dheji saliingaha bamka saliingaha ee ku xidhan aaladda stereotaxic. Kadibna cidhifka cirbadda dul dhig dalool dalool oo ballaciisu yahay 0.3 mm oo ballac ah oo dura ah (z = 0 mm) oo cidhiidhi saliingaha 1–2 mm (z = qiyaastii -1.5 mm) ilaa ay irbaddu u dhexayso dura mater A. shaabad cufan ayaa la sameeyay. Dabadeed kor u qaad saliingaha badhtamaha kortikaalka z = -0.5 mm kuna duri hCO xadiga 1-2 µl daqiiqadii. Ka dib marka la dhammeeyo duritaanka hCO, cirbadda ayaa dib loo soo celiyaa qiyaasta 0.2-0.5 mm daqiiqadii, maqaarka ayaa la tolay, cunugga isla markiiba waxaa la dhigayaa suuf diiran oo diirran ilaa uu ka soo kabsado. Xoolaha qaar ayaa lagu beeray laba dhinac.
Dhammaan hababka xayawaanka waxaa loo sameeyay si waafaqsan Jaamacadda Stanford ee APLAC-ay ansixisay xeerarka daryeelka xayawaanka. Jiirka (in ka badan 60 maalmood ka dib tallaalka) ayaa lagu kiciyay 5% suuxdinta isoflurane waxaana lagu suuxiyay 1-3% isoflurane inta lagu guda jiro sawirka. Aragtida, 7 Tesla ayaa si firfircoon u difaacay iskaanka godka riigga ah ee Bruker (Bruker Corp.) oo wata shirkad koronto oo caalami ah (IECO), galinta gradient gashan leh dhexroor gudaha ah 120 mm (600 mT/m, 1000 T/m/s) ayaa la isticmaalay iyadoo la isticmaalayo AVANCE. III, sideed kanaal multi-coil RF iyo awoodaha badan ee asaasiga ah, iyo Paravision 6.0.1 ee la socota. Duubista waxaa lagu sameeyay iyadoo la adeegsanayo gariiradda RF mugga leh ee si firfircoon loo kala saaray oo leh dhexroor gudaha ah oo ah 86 mm iyo gariiradda RF afar-channel cryo-qabow si loo helo oo keliya. Axial 2D Turbo-RARE (waqtiga ku celcelinta = 2500 ms, echo time = 33 ms, 2 celceliss) oo leh 16 xabbo oo xabbo ah, dhumucdiisuna tahay 0.6-0.8 mm, kana kooban 256 × 256 muunado. Calaamadaha waxaa lagu helay iyadoo la adeegsanayo gariiradda RF mugga quadrature transceiver ah oo leh dhexroor gudaha ah 2 cm (Rapid MR International, LLC). Ugu dambeyntii, isticmaal Imaris (BitPlane) ku dhex-dhisan hawlaha qiyaasidda dusha sare ee samaynta 3D iyo falanqaynta mugga. Ku-tallaalid guul leh ayaa lagu qeexay inay tahay mid ka mid ah meelaha calaamadaha MRI ee Miisaanka ah ee T2 ee joogtada ah lagu sameeyay hemisphere la tallaalay. Diidmada graft-ga waxaa lagu qeexay sida tallaalid aan soo saarin aagagga joogtada ah ee T2-miisaanka MRI ee hemisphere la tallaalay. Subcortical t-hCO ayaa laga saaray falanqaynta xigta.
Si loo muujiyo GCaMP6s ee hCO ee sawir-qaadista laba-sawir ee calcium, unugyada hiPS waxaa ku dhacay pLV[Exp]-EF1a:: GcaMP6s-WPRE-Puro oo ay ku xigtay xulashada antibiyootiga. Si kooban, unugyo ayaa lagu kala saaray EDTA waxaana lagu hakiyay 1 ml oo ah dhexdhexaad ah 8 dhexdhexaad ah cufnaanta qiyaastii 300,000 unug marka ay joogaan polybrene (5 μg/ml) iyo 15 μl ee fayraska. Unugyada ayaa markaa lagu darey ganaax 60 daqiiqo ah waxaana lagu beeray cufnaanta 50,000 unug kasta. Isku darka ka dib, unugyada waxaa lagu daaweeyay 5-10 μg ml-1 puromycin muddo 5-10 maalmood ah ama ilaa ay ka soo baxeen gumeysi xasilloon. Infekshanka hCO ee ba'an ayaa la sameeyay sidii hore loogu sharraxay5 iyadoo wax laga beddelay. Si kooban, maalinta u wareejinta 30-45 hCO geli 1.5 ml Eppendorf tuubooyinka microcentrifuge oo ka kooban 100 µl dhexdhexaad ah neerfaha. Ka dib qiyaastii 90 µl dhexdhexaad ah ayaa la saaraa, 3-6 µl oo lentivirus titer sare ah (laga bilaabo 0.5 x 108 ilaa 1.2 x 109) ayaa lagu daraa tuubada, hCO waxaa loo wareejiyaa kubaatarka 30 daqiiqo. Dabadeed ku dar 90-100 µl dhexdhexaad ah tuubo kasta oo ku celi tuubooyinka habeenkii oo dhan. Maalinta ku xigta, u wareeji hCO dhexdhexaad dareeme cusub oo taargooyin hoose ah. 7 maalmood ka dib, hCO waxaa loo wareejiyay taarikada hoose ee 24-si fiican si loo arko loona qiimeeyo tayada caabuqa. pLV[Exp]-SYN1::EYFP-WPRE iyo pLV[Exp]-SYN1::hChR2-EYFP-WPRE waxaa soo saaray VectorBuilder. Lentivirus waxaa loo isticmaalaa tijaabooyinka badankood sababtoo ah waxay ku dhex milmeen genome-ka martida loo yahay, taas oo u oggolaanaysa in weriyaha uu muujiyo khadadka unugyada cudurka qaba. Dabagalka rabitaanka, maalinta 30-45 hCO waxaa la wadaagay cudurka raabiyada-ΔG-eGFP iyo AAV-DJ-EF1a-CVS-G-WPRE-pGHpA (plasmid #67528, Addgene), si fiican loo dhaqay 3 maalmood, waxaana lagu beeray jiirka S1 waxaana lagu hayaa vivo maalmo 7-1.
Immunocytochemistry, xayawaanku waa la suuxiyay oo si toos ah loogu shubay PBS oo ay ku xigto 4% paraformaldehyde (PFA ee PBS; Electron Microscope Sciences). Maskaxda waxaa lagu hagaajiyay 4% PFA saacadaha 2 ama habeen dhaxe 4°C, cryopreserved in 30% sucrose in PBS for 48-72 hours, and gund in 1:1, 30% sucrose: OCT (Tissue-Tek OCT Compound 4583 (Leica). Immunohistochemistry ee qaybaha qaro weyn, xayawaanka waxaa lagu shubay PBS, maskaxdana waa la kala gooyay oo loo qaybiyay 300-400 µm iyadoo la adeegsanayo vibratome (Leica) iyo qaybaha waxaa lagu hagaajiyay 4% PFA 30 daqiiqo. Ka dib qaybaha dhogorta leh ama qaybaha qaro weyn ayaa lagu dhaqay PBS, oo lagu xannibay 1 saac heerkulka qolka (10% serum dameerka caadiga ah (NDS) iyo 0.3% Triton X-100 oo lagu qasi jiray PBS) waxaana lagu xannibay xalka xannibaadda 4 ° C. -Cryosections incubation ayaa la shiilay habeenkii oo qaybo qaro weyn ayaa la shiilay 5 maalmood. Unugyada difaaca aasaasiga ah ee la isticmaalay waxay ahaayeen: anti-NeuN (mouse, 1:500; ab104224, abcam) anti-CTIP2 (jiir, 1:300; ab18465, abcam), anti-GFAP (bakayle, 1:1,000; Z0334, Dako), anti-GFP:1, GTXT,00 anti-HNA (jiirka, 1:200; ab191181, abcam), anti-NeuN (bakayle, 1:500; ABN78, Millipore), anti-PDGFRA (bakayle, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (bakayle, 19,2008), HPAdies. anti-RECA-1 (jiirka, 1:50; ab9774, abcam), anti-SCG2 (bakayle, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (ri, 1:500; AF3075, R & D Systems), Netrin G1a (Riyaha, 6, 1:10), anti-STEM121 (jiirka, 1:200; Y40410, Takara Bio), anti-SATB2 (jiirka, 1:50; ab51502, abcam), anti-GAD65/67 (bakayle, 1:400; ABN904, Millipore) iyo anti-IBA1 (ri, 1:006; abcam). Unugyada difaaca aasaasiga ah ee la isticmaalay waxay ahaayeen: anti-NeuN (mouse, 1:500; ab104224, abcam) anti-CTIP2 (jiir, 1:300; ab18465, abcam), anti-GFAP (bakayle, 1:1,000; Z0334, Dako), anti-GFP:1ex, GTX 00 anti-HNA (jiirka, 1:200; ab191181, abcam), anti-NeuN (bakayle, 1:500; ABN78, Millipore), anti-PDGFRA (bakayle, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (bakayle, 19,2008), HPAdies. anti-RECA-1 (jiirka, 1:50; ab9774, abcam), anti-SCG2 (bakayle, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (ri, 1:500; AF3075, R & D Systems), Netrin G1a (Riyaha, AF & D), anti-STEM121 (jiirka, 1:200; Y40410, Takara Bio), anti-SATB2 (jiirka, 1:50; ab51502, abcam), anti-GAD65/67 (bakayle, 1:400; ABN904, Millipore) iyo anti-IBA1: 070, abcam. Использовались ab18465, abcam), анти-GFAP (кроличьи, 1:1000; Z0334, Dako), анти- -GFP (курица, 1:1000; GTX13970, GeneTex), анти-HNA ( ab19cam), 1:9cam, 1; анти-NeuN (кролик, 1:500; ABN78, Millipore), анти-PDGFRA (кролик, 1:200; sc-338, Санта-Круз), анти-PPP1R17 (kр1040, HPA, 1900, 1000, 1990, HPAt: 1900, 1900, 1990, HPAt: 1900, 1900, 1900, 1000, 1000, 1000, 1000, 1000, 1000, 1000, 1900, HPA-At. Unugyada difaaca jirka), анти-RECA-1 (мышь, 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 (мышь, ab51), 50; анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) iyo анти-IBA1 (коза, 1:100; аб5076, абкам). Unugyada difaaca aasaasiga ah ee la isticmaalay waxay ahaayeen: anti-NeuN (jiirka, 1:500; ab104224, abcam), anti-CTIP2 (jiir, 1:300; ab18465, abcam), anti-GFAP (bakayle, 1:1000; Z0334, Dako), anti-GFP (chicken, 1030; GTX 10ex), GFP anti-HNA (jiirka, 1:200; ab191181, abcam), anti-NeuN (bakayle, 1:500; ABN78, Millipore), anti-PDGFRA (bakayle, 1:200; sc-338, Santa Cruz), anti-PPP1R17 (bakayle, 1:0408), HPAdies anti-RECA-1 (jiirka, 1:50; ab9774, abcam), anti-SCG2 (bakayle, 1:100; 20357-1-AP, Proteintech), anti-SOX9 (ri, 1:500; AF3075, R & D Systems), netrin G1a (Riyaha, AF, 106, AF), STEM121 (jiirka, 1:200; Y40410, Takara Bio), anti-SATB2 (mouse, 1:50; ab51502, abcam), anti-GAD65/67 (bakayle, 1:400; ABN904, Milipore) iyo anti-IBA1 (ri, ab51502).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 Nidaamyada), 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 -GFP（鸡，1:1,000；GTX13970，GeneTex），抗HNA（小鼠，1:200；ab191181：abcam））NeuN（兔，1:500；兔，1:500；兔，1:500；兔，1:500；小鼚1；小鼠，1:200 200；sc-338، Santa Cruz））抗PPP1R17（兔，1:200；HPA047819， Atlas 100 20357-1-APY40410, Takara Bio), anti-SATB2 (jiirka, 1:50; ab51502, abcam), anti-GAD65/67 (bakayle, 1:400; ABN904, Millipore) iyo anti-IBA1 (ri, 1:100; ab5076, abcam).Unugyada aasaasiga ah ee la isticmaalay waxay ahaayeen: anti-NeuN (mouse, 1: 500; ab104224, abcam), anti-CTIP2 (rat, 1:300; ab18465, abcam), anti-GFAP (bakayle, 1: 1000; Z0334, Dako) . , anti-GFP (digaag, 1: 1000; GTX13970, GeneTex), anti-HNA (jiirka, 1: 200; ab191181, abcam), anti-NeuN (bakayle, 1: 500; ABN78, Millipore), anti-PDGFRA (bakayle, 1: 3300), anti-Sta-PPZ1 anti-NeuN. (bakayle, 1:200; HPA047819, Atlas antibody), anti-RECA-1 (jiirka, 1:50; ab9774, abcam), anti-SCG2 (bakayle), 1:100;20357-1-AP, Proteintech), анти-SOX9 (коза, 1:500; AF3075, R & D Systems), Нетрин G1a (коза, 1:100; AF1166, R & D Systems), iyo -STEM120; Y40410, Takara Bio), анти-SATB2 (мышь, 1:50; ab51502, abcam), анти-GAD65/67 (кролик, 1:400; ABN904, Millipore) iyo анти-IBA1 (коза0); cabkaam). 20357-1-AP, Proteintech), anti-SOX9 (ri, 1:500; AF3075, R&D Systems), Netrin G1a (ri, 1:100; AF1166, R & D Systems), anti-STEM121 (jiirka, 1:200; Y40410; SAA: TB,50, Takara Bio), ab51502, abcam), anti-GAD65/67 (bakayle, 1:400; ABN904, Millipore), iyo anti-IBA1 ( ri, 1:100; ab5076, abkam).Qaybaha ayaa markaa lagu maydhay PBS waxaana lagu galiyay antibody-ka labaad ilaa 1 saac heerkulka qolka (qaybo barafaysan) ama habeenkii 4°C (qaybo qaro weyn). Alexa Fluor secondary antibody (Life Technologies) oo lagu qasi jiray 1:1000 ee xakamaynta xalka ayaa la isticmaalay. Ka dib markii lagu maydhay PBS, nuclei-yada waxaa lagu sawiray Hoechst 33258 (Technology Life). Ugu dambeyntii, sawirada sawirada waxaa lagu dhejiyay mikroskoob leh daboolo (Fisher Scientific) iyadoo la adeegsanayo Aquamount (Polysciences) waxaana lagu falanqeeyay mikroskoob Keyence fluorescent (BZ-X analyzer) ama Leica TCS SP8 mikroskoob confocal (Las-X) ee sawirka. Sawirada waxaa lagu farsameeyay barnaamijka ImageJ (Fiji). Si loo qiyaaso saamiga neerfayaasha bini'aadamka ee t-hCO iyo kiliyaha jiirka, 387.5 μm sawiro leydi ah oo ballaaran ayaa laga soo qaaday bartamaha t-hCO, meel u dhow ama u dhow cidhifka kiliyaha jiirka. Margins graft waxaa lagu go'aamiyay iyadoo la qiimeynayo isbeddellada daahfurnaanta unugyada, HNA+ nuclei, iyo/ama joogitaanka autofluorescence unugga. Sawir kasta, wadarta tirada guud ee unugyada NeuN+ iyo HNA+ waxaa loo qaybiyay wadarta tirada unugyada NeuN+ ee isku meel. Si loo hubiyo in kaliya unugyada nuclei ee diyaaradda sawirka la tiriyo, kaliya unugyada sidoo kale Hoechst + ayaa lagu daray xisaabinta. Laba sawir oo kala soocay ugu yaraan 1 mm ayaa la isku celceliyay si loo yareeyo khaladka tirakoobka.
Hal usbuuc ka hor inta aan la qaadin muunada, dhig xayawaanka ku-tallaalidda hCO (qiyaastii 8 bilood oo kala duwanaansho) qol mugdi ah oo shabag la jarjaray si loo yareeyo kicinta dareenka. Go'doominta nuclei-yada ayaa la sameeyay sidii hore loogu sharraxay, iyadoo wax laga beddelay. Si kooban, t-hCO iyo hCO waa la burburiyay iyadoo la adeegsanayo saabuunta-mashiinka lysis unugga iyo 2 ml oo Furimaha Dagaalka ah (D8938, Sigma-Aldrich/KIMBLE). Nuclei-yada cayriin ayaa markaa la sifeeyay iyadoo la adeegsanayo shaandheyn 40 µm waxaana lagu xareeyay 320 g 10 daqiiqo at 4 ° C ka hor inta aan la samaynin cufnaanta sukrose. Ka dib tilaabada centrifugation (320 g 20 min at 4 ° C), shaybaarada ayaa dib loogu celiyay 0.04% BSA/PBS iyada oo lagu daray 0.2 cutubyada µl-1 RNase inhibitor (40 u µl-1, AM2682, Ambion) waxayna soo mareen shaandhada qulqulka 40.µ Nukleiyadii kala go'ay ayaa dib loo hakiyay gudaha PBS oo ka kooban 0.02% BSA waxaana lagu raray Chromium Single Cell 3′ chip (qiyaastii soo kabashada 8,000 unug halkii haad). snRNA-seq maktabadaha waxaa lagu diyaariyay Chromium Single cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics). snRNA-seq maktabadaha waxaa lagu diyaariyay 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). Maktabadaha snRNA-seq waxaa lagu diyaariyay 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). Maktabada snRNA-seq waxa lagu diyaariyey iyadoo la isticmaalayo Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics).Maktabadaha laga soo qaaday muunado kala duwan ayaa la isku geeyay oo ay isku xigxigeen Admera Health on NovaSeq S4 (Illumina).
Heerarka muujinta hidda-wadaha ee barcode kasta oo nukliyeer ah ayaa lagu qiyaasey iyadoo la isticmaalayo 10x Genomics CellRanger xirmada falanqaynta software (nooca 6.1.2). Gaar ahaan, akhrintu waxa ay isku beegmeen isku darka aadanaha (GRCh38, Ensemble, nooca 98) iyo jiirka (Rnor_6.0, Ensemble, nooca 100) genomes tixraaceed ee lagu sameeyay amarka mkref iyo adeegsiga tirinta -include-introns=Amarka TRUE si loo qiyaaso waxaa ka mid ah akhrinta loo habeeyey gobollada gudaha. Tusaalooyinka t-hCO, nukleiyada bini'aadamka ayaa la aqoonsaday iyadoo lagu salaynayo shuruudaha muxaafidka ah ee ah in ugu yaraan 95% dhammaan akhrinta khariidad ay la mid tahay genome-ka aadanaha. Dhammaan falanqaynta soo socota waxaa lagu sameeyay soo-saar codeed sifaysan oo ka yimid CellRanger iyadoo la adeegsanayo xirmada R (nooca 4.1.2) Seurat (nooca 4.1.1)32.
Si loo hubiyo in xudunta tayada sare leh oo kaliya lagu daro falanqaynta xigta, hannaan shaandhayn ku celcelin ah ayaa la fuliyay muunad kasta. Marka hore, nuklei- tayadoodu hoosayso oo leh in ka yar 1000 hiddo-sideyaal gaar ah ayaa la helay oo in ka badan 20% wadarta guud ee mitochondria waa la aqoonsaday oo laga saaray. Intaa ka dib, tirada hiddo-wadaha ceyriinka ah waxaa lagu caadaystay dib-u-celin taban oo joogto ah iyadoo la adeegsanayo shaqada sctransform(vst.flavor=”v2″), kaas oo sidoo kale aqoonsaday 3000 ee hiddo-wadaha doorsoomiyaha badan iyadoo la adeegsanayo cabbirada caadiga ah. goobaha oo loo isticmaalo dhammaan shaybaarada iyo falanqaynta ensemble). Xirmada DoubletFinder33 (macnaha DoubletFinder dhibcaha ka sarreeya boqolleyda 95) muunadaha t-hCO (n=3) ayaa si gooni gooni ah la isugu daray iyadoo la adeegsanayo IntegrateData oo leh cabbirrada kore.
Ka dib markii meesha laga saaro kernels tayada hooseeya, xogta isku dhafan ayaa la kooxeeyay (xarka = 0.5) waxaana lagu dhejiyay ujeedooyinka UMAP34. Hidde-sideyaasha koox kasta waxaa lagu go'aamiyay iyadoo la adeegsanayo shaqada FindMarkers oo leh cabbiraadyo aan caadi ahayn oo laga soo xisaabiyay xogta muujinta hidda-wadaha caadiga ah. Waxaan aqoonsanaa oo aan kala saarnaa fasalada unugyada waaweyn anagoo isku darayna xogta tixraaca ee uurjiifka iyo dadka waaweyn ee kortikalka leh muujinta hidde-sidaha 19,20,21,35 iyo sharraxaad. Gaar ahaan, horu-yaasha wareega ayaa lagu aqoonsaday muujinta MKI67 iyo TOP2A. Kooxaha hore waxaa lagu qeexay maqnaanshaha qoraalada mitotic-ga, isku dhejinta sare oo leh kooxo fara badan oo glial ah oo lagu sharaxay kortex uurjiifka metaphase dambe, iyo EGFR iyo OLIG1 muujinta. Waxaan u isticmaalnaa ereyga astrocyte si aan u koobno ​​dhowr gobol oo kala duwanaanshaha astrocyte, laga bilaabo radial glia dambe ilaa korriinka astrocytes. Kooxaha Astrocyte waxay muujinayaan heerar sare oo SLC1A3 iyo AQP4 ah waxaana la tusay inay khariidad ku sameeynayaan noocyada hoose ee radial glia uurjiifka iyo/ama astrocytes dadka waaweyn. OPC-yadu waxay muujinayaan PDGFRA iyo SOX10 halka oligodendrocytes ay muujinayaan calaamadaha mylination (MOG iyo MYRF). Glutamatergic neurons waxaa lagu aqoonsaday joogitaanka qoraallada neuronal (SYT1 iyo SNAP25), maqnaanshaha calaamadaha GABAergic (GAD2), iyo muujinta NEUROD6, SLC17A7, BCL11B, ama SATB2. GluN neurons ayaa loo sii kala qaybiyay sare (SATB2 muujinta iyo luminta BCL11B) iyo qoto dheer (BCL11B muujinta). Subplate (SP) neerfayaasha ayaa muujiya calaamadaha SP18 ee caanka ah sida ST18 iyo SORCS1 marka lagu daro calaamadaha GluN qoto dheer. Unugyada loo yaqaan 'Choroid plexus-like' waxaa lagu aqoonsaday muujinta TTR, iyo unugyada meningeal-sida waxay muujiyeen hiddo-wadaha fibroblast-ku xiran iyo unugyada pial / vascular ee xogta tixraaca.
Falanqaynta kala duwan ee muujinta hidda-wadaha ee u dhexeeya t-hCO iyo hCO waa la sameeyay iyadoo la adeegsanayo hab cusub oo dufcaddii been-abuurka ah oo lagu soo saaray muunado lagu hirgeliyay xirmada Libra R (nooca 1.0.0). Gaar ahaan, imtixaannada u-ur-galnimada EdgeR (nooca 3.36.0, xirmada R) ayaa loo sameeyay kooxaha iyadoo la soo koobay tirada hiddo-wadaha unugyada ee fasalka unugga ee muunad kasta. Aragtida kulaylka, qiimaha caadiga ah ee hal milyan (CPM) waxaa lagu xisaabiyaa iyadoo la adeegsanayo EdgeR (cpm () function) oo la miisaamay (si loo gaaro celceliska = 0, jaangooyada caadiga ah = 1). Gene Ontology (GO) falanqaynta kobcinta ee t-hCO GluN hiddo-wadaha si weyn loo hagaajiyay ayaa la sameeyay (Benjamini-Hochberg ayaa saxay qiimaha P ee ka yar 0.05 oo lagu muujiyay ugu yaraan 10% unugyada t-hCO GluN iyo kororka isbeddelka ugu yaraan 2 jeer). lagu sameeyay iyadoo la isticmaalayo ToppGene Suite (https://toppgene.cchmc.org/)37. Waxaan u isticmaalnaa app-ka ToppFun oo leh cabbirada caadiga ah waxaanan ka warbixineynaa Benjamini-Hochberg-la saxay P-qiimaha laga soo xisaabiyay GO-fasaxeed hypergeometric tests.
Si loo waafajiyo rucubyadayada snRNA-seq ee leh rucubyada unugga la sharraxay ee laga soo xigtay daraasadaha tixraaca ee hal unug ee aasaasiga ah ee RNA-seq ama snRNA-seq19,20,21,22, waxaanu isticmaalnay hab isku dhafan xogta xogta. Waxaan u isticmaalnay SCTransform (v2) socodka shaqada ee caadiga ah ee Seurat si aan u dhexgalno oo aan isbarbar dhigno isku dhafka isku dhafka ah ee u dhexeeya xog-ururinta (iyadoo la adeegsanayo cabbirro la mid ah kuwa kore). Xog-ururinta shaqsiga ah ayaa si aan kala sooc lahayn loo dejiyay ilaa 500 unug ama unugyo koox kasta oo asalka u ah waxtarka xisaabinta. Isticmaalka hab la mid ah sidii hore loogu sharraxay, isku-xidhka kooxdu waxa lagu qeexay saamiga unugyada ama nukleiyada koox kasta oo la ururiyey oo ku dahaaran calaamadda kooxda tixraaca. Si aan u sii kala saarno GluNs, waxaanu u isticmaalnay Seurat's TransferData shaqada socodka xogta GluN si aanu ugu meelayno sumadaha kaydka xogta unugyadeena GluN.
Si loo qiimeeyo heerka korriinka ee qoraalada caalamiga ah ee t-hCO iyo hCO, waxaanu is barbar dhignay shay-baarkayada been abuurka ah ee BrainSpan/psychENCODE23, kaas oo ka kooban taxane RNA oo ballaadhan oo ku saabsan horumarka maskaxda aadanaha. Waxaan ku samaynay PCA-ga isku-darka muujinta hidde-sidaha caadiga ah ee laga soo qaaday muunadaha kortikal 10 toddobaad ka dib markii la raray iyo ka dib, 5567 hiddo-wadaha (oo ay weheliso xogtayada) kuwaas oo hore loogu aqoonsaday inay firfircoon yihiin muunadaha kortikaalka ee BrainSpan (oo lagu qeexay in ka badan 50% kala duwanaanshaha korriinka ee lagu sharaxay da'da iyadoo la adeegsanayo qaab cubic)38. Intaa waxaa dheer, waxaan ka soo saarnay hiddo-wadaha la xidhiidha saxeexyada transcriptome-ga weyn ee horumarinta neurodevelopment iyadoo la adeegsanayo wax-soo-saarka matrix-ka ee aan taban ahayn sidii hore loo sharraxay. Miisaanka muunada ee la xisaabiyay iyadoo la adeegsanayo habka is-saaraynta shaxanka aan taban waxa lagu sawiray berdihii. 5b oo leh xog la ballaadhiyey mid kasta oo ka mid ah shanta saxeex ee ay sifeeyeen Zhu et al.38. Mar labaad, calaamadaha ku-tiirsanaanta dhaqdhaqaaqa ayaa laga soo qaatay daraasado hore loo daabacay. Gaar ahaan, ERG iyo LRG ayaa si weyn loogu hagaajiyay neerfayaasha glutamatergic ee lagu aqoonsaday ururinta kiliyaha muuqaalka ee snRNA-seq ka dib kicinta muuqaalka ee Shaxda Dheeraadka ah 3 Hrvatin et al.16. LRGs-ka bini'aadamka ay ku qanceen ayaa laga helay dhaqamada maskaxda uurjiifka ee bini'aadamka ee KCl waxaana la goostay 6 saacadood ka dib kicinta, iyo hiddo-wadaha la miiray ayaa si weyn loogu habeeyey bini'aadamka laakiin maaha jiirka (Shaxda Dheeraadka ah 4). Falanqaynta kobcinta hidda-sidaha iyada oo la adeegsanayo noocyada hidde-sideyaashan ayaa la sameeyay iyada oo la adeegsanayo tijaabada saxda ah ee Fisher hal-jid ah.
Ku suuxdin jiirka oo leh isoflurane, ka saar maskaxda oo dhig qabow (qiyaastii 4°C) ogsijiin leh (95% O2 iyo 5% CO2) xal sukrose ee qaybaha ka kooban: 234 mM sukrose, 11 mM gulukoos, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaH2PO4, 10 mM MgSO4 iyo 0.5 mM CaCl2 (qiyaastii 310 mOsm). Qaybaha xididdada maskaxda ee jiirka (300-400 µm) oo ay ku jiraan t-hCO ayaa la sameeyay iyadoo la adeegsanayo vibratome Leica VT1200 sida hore loogu sharraxay39. Qaybaha ayaa markaa loo wareejiyay qolka qaybinta oo leh oksijiinta heerkulka qolka joogtada ah oo ay ku jirto aCSF laga diyaariyey: 10 mM glucose, 26 mM NaHCO3, 2.5 mM KCl, 1.25 mM NaHPO4, 1 mM MgSO4, 2 mM CaCl2 iyo 126 mM NaCl (298 mO) ugu yaraan 45 daqiiqo ka hor duubista. Qaybaha waxaa lagu duubay qol la quusiyay halkaasoo si joogto ah loogu shubay aCSF (95% O2 iyo 5% CO2 vial). Dhammaan xogta waxaa lagu duubay heerkulka qolka. t-hCO neurons ayaa lagu joojiyay pipette galaas borosilicate ah oo ay ka buuxaan xal ka kooban 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, iyo 0.6 mM EGTA, pH 7.2, xal gudaha ah oo lagu hagaajiyay m0hs2.9. Si loo soo kabsado, biocytin (0.2%) ayaa lagu daray xalka duubista.
Xogta waxaa lagu helay iyadoo la adeegsanayo MultiClamp 700B amplifier (Aaladaha Molecular) iyo Digidata 1550B digitizer (Aaladaha Molecular), baas hooseeya oo lagu sifeeyay 2 kHz, digitized at 20 kHz, waxaana lagu falanqeeyay iyadoo la adeegsanayo Clampfit (Aaladaha Molecular), Asalka (OriginPro). 2021b, OriginLab). iyo hawlaha caadiga ah ee MATLAB (Mathworks). Awoodda isgoysyada waxaa la xisaabiyay iyadoo la adeegsanayo JPCalc iyo gelinta ayaa lagu hagaajiyay qiimaha la xisaabiyay ee -14 mV. Hawlgalka IV wuxuu ka kooban yahay tillaabooyin taxane ah oo hadda socda 10-25 pA tillaabo, min -250 ilaa 750 pA.
Thalamus-ka, walxaha cad, iyo afferents-ka S1 ayaa si koronto ah loogu kiciyay jeexjeexyada thalamocortical inta lagu guda jiro duubista balastar-ku-qabsiga ee neerfayaasha hCO, sida hore loogu sharraxay. Si kooban, maskaxda waxaa la saaray miiska daabacaadda 3D oo u janjeera xagal 10°, dhanka hore ee maskaxda waxaa la jaray xagal 35° ah. Maskaxda ayaa markaa lagu dhejiyay dusha sare ee la gooyay oo la qaybiyay, iyada oo la ilaalinayo thalamocortical axon ee soo baxa. Laba-cirifoodka tungsten electrodes (0.5 MΩ) ayaa lagu rakibay micromanipulator labaad oo si xeeladaysan loo meeleeyay si loo kiciyo afar gobol halkii unug (kaabsal gudaha, walxaha cad, S1 iyo hCO). Ku duub jawaabaha synaptik ka dib kicinta wajiga 300 µA ee 0.03–0.1 Hz.
hChR2-muujinta hCO neurons ayaa lagu hawlgeliyay 480 nm iyo garaacyada iftiinka ah ee ay soo saartay LED (Prizmatix) ayaa lagu dabaqay ujeedada × 40 (0.9 NA; Olympus) si loo duubo muujinta hChR2 ee u dhow unugyada. Dhexroorka beerta iftiimay waa ku dhawaad ​​0.5 mm wadarta guudna waa 10-20mW. Balaadhka garaaca wadnaha ayaa loo dejiyay 10 ms, kaas oo u dhigma garaaca garaaca wadnaha ee la bixiyay intii lagu jiray tijaabada barashada habdhaqanka. Noocyo kala duwan oo kicin ah ayaa la isticmaalay, laga bilaabo 1 ilaa 20 Hz, laakiin kaliya garaaca garaaca ugu horreeya ee taxanaha ayaa loo isticmaalay qiyaasid. Inta udhaxeysa tareenadu waxay badanaa ka badan yihiin 30s si loo yareeyo saameynta ka hortagta synaptik ama wadooyinka fududeeya. Si loo tijaabiyo haddii jawaabta hChR2 ay tahay monosynaptic, waxaanu isticmaalnay TTX (1 μM) qubayska ilaa falcelinta EPSC ay meesha ka baxayso, ka dibna ka codsanay 4-aminopyridine (4-AP; 100 μM). Caadi ahaan, jawaabta ayaa lagu soo celiyaa dhowr daqiiqo gudahood, iyadoo waxyar ka dheer daahitaan inta u dhaxaysa toogashada LED-ka iyo jiilka EPSC. NBQX (10 μM) ayaa loo isticmaalay in lagu tijaabiyo in jawaabta ay wadaan soo-qabayaasha AMPA.
Qaybaha hCO ee fiiqan ayaa la sameeyay sidii hore loogu sharraxay. Si kooban, qaybaha hCO waxaa lagu dhejiyay 4% agarose waxaana loo wareejiyay unugyo ka kooban 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 1 mM MgSO4, 2 mM CaCl2, 26 mM NaHCO3 iyo 10 mM dse (+) µm heerkulka qolka iyadoo la adeegsanayo gariiriyaha Leica VT1200 waxaana lagu kaydiyaa ASF heerkulka qolka. Kadibna, duubista xerada balastar ee unugyada oo dhan ayaa lagu sameeyay qaybaha hCO ee hoos yimaada mikroskoob toos ah SliceScope (Scientifica). Qaybaha waxaa lagu shubay aCSF (95% O2 iyo 5% CO2) iyo calaamadaha unugyada waxaa lagu duubay heerkulka qolka. hCO neurons ayaa lagu dabaqay iyadoo la adeegsanayo pipette galaas borosilicate ah oo lagu buuxiyey xal ka kooban 127 mM potassium gluconate, 8 mM NaCl, 4 mM magnesium ATP, 0.3 mM sodium GTP, 10 mM HEPES, iyo 0.6 mM EGTA, pH 7, 2, oo lagu hagaajiyay KOH (osmolarity). Ujeedooyinka soo kabashada, ku dar 0.2% Biocytin xalka gudaha.
Xogta waxaa helay Clampex (Clampex 11.1, Qalabka Molecular) iyadoo la adeegsanayo MultiClamp 700B amplifier (Aaladaha Molecular) iyo Digidata 1550B digitizer (Aaladaha Molecular), baas hooseeya oo lagu sifeeyay 2 kHz, digitized at 20 kHz, oo lagu falanqeeyay iyadoo la adeegsanayo qalabka Clampfit.6 hawlaha (MATLAB 2019b, Mathworks). Awoodda isgoysyada waxaa la xisaabiyay iyadoo la adeegsanayo JPCalc gelintana waxaa lagu hagaajiyay awoodda isgoysyada la xisaabiyay ee -14 mV. Hawlgalka IV wuxuu ka kooban yahay tillaabooyin taxane ah oo hadda socda oo ah 5-10 pA tilaabo laga bilaabo -50 ilaa 250 pA.
Dib-u-dhiska morphological ee neerfayaasha qanjaruufo, 0.2% biocytin (Sigma-Aldrich) ayaa lagu daray xalka gudaha. Unugyada waxaa la nadiifiyaa ugu yaraan 15 daqiiqo ka dib marka la jabsado. pipette si tartiib ah ayaa loo soo jiidaa 1-2 daqiiqo ilaa xuubka diiwaangashan si buuxda loo xiro. Iyadoo la raacayo nidaamka physiology qaybta, qaybaha waxaa lagu hagaajiyay habeenkii 4 ° C. ee 4% PFA, lagu dhaqay PBS X3, oo lagu qasi jiray 1:1000 oo leh streptavidin-conjugated DyLight 549 ama DyLight 405 (Vector Labs). Unugyada ay ka buuxaan biocytin (2%; Sigma-Aldrich) ayaa la calaamadeeyay inta lagu guda jiro duubista balastar heerkulka qolka 2 saacadood. Qaybaha ayaa markaa lagu rakibay muraayadaha microscopy iyadoo la adeegsanayo Aquamount (Thermo Scientific) waxaana lagu sawiray maalinta xigta Leica TCS SP8 mikroskoob iyadoo la adeegsanayo ujeeddada immersion saliidda leh dalool lambar ×40 1.3, weyneynta ×0.9-1.0, xy. Qiyaasta muunadku waa ku dhawaad ​​7 pixels halkii micron. Xirmooyinka Z ee dhexda 1 µm ayaa la helay si isdaba joog ah, iyo mosaics-ka z-stack iyo auto-tolida ku salaysan Leica ayaa la sameeyay si loo daboolo dhammaan geedka dendritic ee neuron kasta. Neurons-ka ayaa markaa la raadraacay badh-gacmeed iyadoo la adeegsanayo neuTube 40 interface waxaana la sameeyay faylalka SWC. Faylasha ayaa markaa lagu dhejiyay SimpleNeuriteTracer41 Fiji plugin (ImageJ, version 2.1.0; NIH).
Nudaha kortikal ee bini'aadamka waxaa lagu helay ogolaansho la wargaliyay iyadoo la raacayo nidaamka ay ansixiyeen Guddiga Dib-u-eegista Hay'adaha ee Jaamacadda Stanford. Labo muunado oo ah unugyada dhalmada kadib (3 iyo 18 sano jir) ayaa lagu helay dib-u-soo-noqoshada kiliyaha hore (girus hore ee hore) taasoo qayb ka ah qalliinka suuxdinta refractory. Dib-u-soo-celinta ka dib, goosashada nudaha ku jira NMDG-aCSF-qabow oo ka kooban: 92 mM NMDG, 2.5 mM KCl, 1.25 mM NaH2PO4, 30 mM NaHCO3, 20 mM HEPES, 25 mM gulukoos, 2 mM thiourea, 5 mMpy5 mMM, sodium ascorvate. CaCl2 4H2O iyo 10 mM MgSO4 7H2O. Titrate ilaa pH 7.3-7.4 oo leh hydrochloric acid. Unugyada waxaa lagu geeyey shaybaadhka 30 daqiiqo gudahood waxaana qaybaha wadnaha laga soo qaaday habraaca kor lagu sharaxay.
Dhammaan hababka xayawaanka waxaa loo sameeyay si waafaqsan Jaamacadda Stanford ee APLAC-ay ansixisay xeerarka daryeelka xayawaanka. Jiirka (in ka badan 140 maalmood ka dib-tallaalidda) ayaa lagu kiciyay 5% suuxdinta isoflurane waxaana lagu suuxiyay 1-3% isoflurane gudaha qalliinka. Xayawaanka waxaa lagu dhejiyay qaab stereotaxic ah (Kopf) iyo sii-deynta joogtada ah ee buprenorphine (SR) ayaa lagu duray maqaarka hoostiisa. Dhafoorku waa bannaan yahay, waa la nadiifiyey, waxaana la geliyey 3-5 boolal lafo ah. Si loo beegsado t-hCO, waxaan ka soo saarnay iskuduwayaal stereotaxic sawirada MRI. Dalool daloola ayaa laga qoday goobta xiisaha iyo fiilooyinka (dhexroorka 400 µm, NA 0.48, Doric) ayaa hoos loo dhigay 100 µm ka hooseeya dusha hCO waxaana lagu hubiyay dhakada sibidh ilkeed oo lagu daweyn karo UV (Relyx).
Duubista sawir-qaadista fiber-ka ayaa la sameeyay sidii hore loogu sharraxay42. Si loo diiwaan geliyo dhaq-dhaqaaqa kediska ah, jiirarka waxaa la geliyey qafis nadiif ah iyo 400 µm dhexroorka fibre optic patch cable (Doric) oo ku xiran nidaamka xog-helidda xogta fiber optic photometric ayaa lagu xiray fiilada fiber optic-ga ee la geliyey. Inta lagu jiro duubista 10-daqiiqo ee dhaqdhaqaaqa dhaqdhaqaaqa, xayawaanku waxay xor u ahaayeen inay sahamiyaan qafiska. Si loo diiwaan geliyo dhaqdhaqaaqa la kiciyay, jiirarka (in ka badan 140 maalmood ka dib tallaalka) ayaa lagu suuxiyay 5% isoflurane si loo sameeyo iyo 1-3% isoflurane si loo dayactiro. Ku rid neefka qaab muuqaal ah (Kopf) iyo shabaqyada dhinaca ka soo horjeeda ee t-hCO waa la jarjaray ilaa 2 cm waxaana la dhex maraa mesh ku xiran qalabka korontada ku shaqeeya (PI). Fiber optic patch 400 µm (Doric) ayaa lagu xidhay fibre-gaabkii la geliyey oo lagu xidhay nidaamka xogta. Shabaqyada dhinaca ka soo horjeeda ee t-hCO ayaa markaa la leexiyay 50 jeer (2 mm at 20 Hz, 2 s per bandhig) waqtiyo aan kala sooc lahayn iyada oo la adeegsanayo piezoelectric drive in ka badan 20 daqiiqo duubis. Adeegso Xirmada Taageerada Arduino MATLAB si aad u xakamayso wakhtiga leexashada oo leh koodka MATLAB ee caadiga ah. Dhacdooyinka waxa lala jaanqaadayaa software-ka xog-helidda iyadoo la adeegsanayo garaaca wadnaha transistor-transistor logic (TTL).
Jiirka (in ka badan 140 maalmood ka dib-tallaalidda) ayaa lagu kiciyay 5% suuxdinta isoflurane waxaana lagu suuxiyay 1-3% isoflurane gudaha qalliinka. Xayawaanka waxaa lagu dhejiyay qaab stereotaxic ah (Kopf) iyo buprenorphine SR iyo dexamethasone ayaa lagu duray maqaarka hoostiisa. Dhafoorku waa bannaan yahay, waa la nadiifiyey, waxaana la geliyey 3-5 boolal lafo ah. Si loo beegsado t-hCO, waxaan ka soo saarnay iskuduwayaal stereotaxic sawirada MRI. Craniotomy wareeg ah (qiyaastii 1 cm dhexroor) ayaa lagu sameeyay layli xawaare sare leh oo toos ah hCO-da la beeray. Marka laftu ay khafiif noqoto intii suurtogal ah, laakiin ka hor inta aanad qodin lafta oo dhan, isticmaal xoog xoog leh si aad uga saarto saxanka miskaha ee hadhay si aad u muujiso hoosta t-hCO. Craniotomy waxaa ka buuxsamay cusbo nadiif ah oo nadiif ah, iyo dabool daboolan iyo biin madax gaar ah ayaa lagu dhejiyay madaxa oo leh shamiitada ilkaha ee UV-la daweeyay (Relyx).
Sawir-qaadasho laba-sawir ah ayaa la sameeyay iyadoo la adeegsanayo mikroskoob-ka-muuqaalka badan ee Bruker oo leh ujeeddada Nikon LWD (×16, 0.8 NA). Sawirka GCaMP6 waxaa lagu sameeyay 920 nm oo leh 1.4x hal-weyn oo diyaaradeed iyo 8x celcelis ahaan 7.5fps. Jiirka waxaa lagu keenay 5% suuxdinta isoflurane waxaana lagu ilaaliyay 1-3% isoflurane. Jiirarka waxa lagu dhejiyay qalab madaxeed oo caado loo sameeyay waxaana la dhigay muraayada hoosteeda. 3-daqiiqo oo taariikhi ah oo duuban dhaqdhaqaaqa dhaqdhaqaaqa ayaa la helay. Intii lagu guda jiray 20 daqiiqo ee duubista, 50 puffs (bandhig kasta 100 ms dheer) ayaa si aan kala sooc lahayn loogu geeyey suufka suufka ee ka soo horjeeda t-hCO iyadoo la isticmaalayo picospricer. Adeegso Xirmada Taageerada Arduino MATLAB si aad u xakamayso wakhtiga dilaaca koodka MATLAB ee caadada u ah. Isku-dubarid dhacdooyinka software-ka iibsiga xogta (PrairieView 5.5) adoo isticmaalaya garaaca TTL. Falanqaynta, sawirada waxaa lagu saxay dhaqdhaqaaqa xy iyadoo la adeegsanayo sixitaanka affine ee barnaamijka MoCo ee laga bilaabay Fiji. Soo saarista raadadka fluorescent ee unugyada gaarka ah iyadoo la adeegsanayo CNMF-E43. Fluorescence ayaa loo soo saaray gobol kasta oo xiiso leh, waxaa loo beddelay qalloocyada dF/F, ka dibna loo beddelay z-dhibcaha.
Jiirka (in ka badan 140 maalmood ka dib-tallaalidda) ayaa lagu kiciyay 5% suuxdinta isoflurane waxaana lagu suuxiyay 1-3% isoflurane gudaha qalliinka. Xayawaanka waxaa lagu dhejiyay qaab stereotaxic ah (Kopf) iyo buprenorphine SR iyo dexamethasone ayaa lagu duray maqaarka hoostiisa. Shabaqyada dhinaca ka soo horjeeda ee t-hCO ayaa la gooyay ilaa 2 cm waxaana lagu dhejiyay shabag ku xiran qalabka korontada ku shaqeeya. Dhafoorku waa bannaan yahay oo waa la nadiifiyaa. Qalab bir ah oo dhulka ah ayaa ku dheggan madaxa. Si loo beegsado t-hCO, waxaan ka soo saarnay iskuduwayaal stereotaxic sawirada MRI. Samee craniotomy wareeg ah (qiyaastii 1 cm dhexroorka) oo leh layli xawaare sare ka sarreeya t-hCO. Marka laftu ay khafiif noqoto intii suurtogal ah, laakiin ka hor inta aanad qodin lafta oo dhan, isticmaal xoog xoog leh si aad uga saarto saxanka miskaha ee hadhay si aad u muujiso hoosta t-hCO. Unugyada shakhsi ahaaneed ayaa la duubay iyadoo la adeegsanayo 32-channel ama 64-channel cufnaanta sare ee silikoonka (Cambridge Neurotech) oo ku salaysan boolal dhulka oo horay loogu sii kordhiyay RHD amplifiers (Intan). Isticmaal manipulator-ka si aad u hoos ugu dhigto electrodes-ka goobta la beegsanayo iyada oo loo marayo craniotomy, kaas oo ay ka buuxaan salineed nadiif ah. Xog ururinta waxaa lagu sameeyay inta jeer ee 30 kHz iyadoo la adeegsanayo nidaamka xogta Ephys ee furan. Duubista ayaa sii socotay kaliya markii aan ogaanay dhaq-dhaqaaq lama filaan ah oo laxanka ah oo aad isku xira in ka badan 10 kanaal, taasoo soo jeedinaysa in korantadu ay ku yaalliin tallaalka (oo ku salaysan xogta sawir-qaadista kalsiyam ee laba-sawir ah). Duub 10-daqiiqo ah taariikhda dhaqdhaqaaqa dhaqdhaqaaqa ayaa la helay. Shabaqyada dhinaca ka soo horjeeda ee t-hCO ayaa markaa la leexiyay 50 jeer (2 mm at 20 Hz, 2 s per bandhig) waqtiyo aan kala sooc lahayn iyada oo la adeegsanayo piezoelectric drive in ka badan 20 daqiiqo duubis. Adigoo isticmaalaya Xirmada Taageerada MATLAB ee Arduino (MATLAB 2019b), kontorool wakhtiga leexinta ee leh koodka MATLAB ee caadiga ah. Isticmaal garaaca TTL si aad dhacdooyinka ula socodsiiso software-ka xog-helidda.
Tijaabooyinka calaamadaynta indhaha, 200 µm xadhig balastar indhaha ah (Doric) oo ku xidhan 473 nm laser (Omicron) ayaa lagu xidhay 200 µm fiber indhaha oo la dulsaaray craniotomy. Isla markiiba ka hor, ku hagaaji awoodda jumper ilaa 20 mW. Isticmaal manipulator-ka si aad u hoos ugu dhigto electrodes-ka goobta la beegsanayo iyada oo loo marayo craniotomy, kaas oo ay ka buuxaan salineed nadiif ah. Bilowgii duubista, toban garaac oo iftiin ah 473 nm (soo noqnoqda 2 Hz, garaaca garaaca 10 ms) ayaa la sii daayay. Unugyada xasaasiga ah waxaa lagu qeexay unugyo muujiyay jawaab celin sare 10 ms oo iftiin ah 70% ama ka badan tijaabooyinka.