成人希马神经前体细胞的乳化电路特定调节
Summary
该协议的目的是描述一种分析成人神经干细胞/祖细胞行为的方法,以响应特定局部神经回路的化学遗传学操作。
Abstract
成人神经发生是一个动态过程,新激活的神经干细胞 (NSC) 在分粒区 (SGZ) 的登牙陀螺(DG) 生成新的神经元,这些神经元集成到现有的神经回路中,并有助于特定的海马功能.重要的是,成人神经发生是高度容易受到环境刺激,这允许各种认知功能的活动依赖调节。来自各个大脑区域的大量神经回路协调这些复杂的认知功能。因此,了解特定神经回路如何调节成人神经发生非常重要。在这里,我们描述了一个协议,以操纵神经回路活动使用设计师受体完全激活由设计师药物(DREADDs)技术,调节NSC和新生后代在啮齿动物。此综合方案包括病毒颗粒的立体注入、特定神经回路的化学遗传学刺激、胸腺素模拟给治、组织处理、免疫荧光标记、共聚焦成像和成像神经前体细胞各个阶段的分析。该协议提供有关用于可视化 NSC 及其后代的抗原检索技术的详细说明,并描述了使用氯扎平 N 氧化物 (CNO) 或含 CNO 的饮用水调节脑回路的一种简单而有效的方法,表达病毒的病毒。该协议的优势在于其适应性,以研究影响从NSC衍生的成人神经发生的各种神经回路。
Introduction
成人神经发生是一个生物过程,新的神经元诞生在成人中,并集成到现有的神经网络1。在人类中,这个过程发生在海马的登当陀螺(DG),每天大约有1,400个新细胞诞生2。这些细胞驻留在DG的内部部分,它蕴藏着一个神经性利基,称为亚粒度区域(SGZ)。在这里,海马成人神经干细胞(NSCs)经历一个复杂的发育过程,成为功能齐全的神经元,有助于调节特定的大脑功能,包括学习和记忆,情绪调节和应激反应3 ,4,5,6.为了影响行为,成人 NSC 通过响应一系列局部和远端化学线索,以活动相关方式受到各种外部刺激的高度监管。这些化学线索包括神经递质和神经调节器,并且以来自各种大脑区域的特定电路方式作用。重要的是,这些化学线索在NSC上的电路广泛收敛,可以独特而精确地调节干细胞的活化、分化和命运决定。
将免疫荧光分析与电路宽操作配对,是询问体内成人NSC电路调节的最有效方法之一。成人NSC的免疫荧光分析是一种常用的技术,其中针对特定分子标记的抗体用于指示成人NSC的发育阶段。这些标记包括:内窝作为径向胶质细胞和早期神经祖传标记,Tbr2作为中间祖传标记,dcx作为神经细胞和未成熟的神经元标记7。此外,通过管理胸腺素类比,如BrdU,CidU,Idu和Edu,经历S相的细胞群可以单独标记和可视化8,9,10。通过结合这两种方法,可以研究广泛的问题,从在特定发育阶段如何调节增殖,到各种线索如何影响NSC分化和神经发生。
有几个选项可以有效地操纵神经回路,包括电刺激、光遗传学和化学遗传学,每个选项都有各自的优缺点。电刺激涉及一个广泛的手术,电极被植入一个特定的大脑区域,后来用于传输电信号来调节目标的大脑区域。然而,这种方法缺乏细胞和电路特异性。光遗传学涉及病毒粒子的传递,这种粒子编码光激活受体,由激光通过植入的光纤激发,但需要大量的操作、巨大的成本和复杂的手术11。化学遗传学涉及病毒粒子的传递,这些粒子编码由设计师药物或DREADD专门激活的设计师受体,随后由称为氯扎平N-氧化物(CNO)的特定和生物惰性配体激活.利用DREADD来操纵控制成人NSC的局部神经回路的好处在于CNO管理的简便性和各种途径。这允许减少动物处理,从而简化耗时的方法,这很容易适应长期研究,以调节神经回路。
该协议中描述的方法是综合收集成功询问成年海马神经发生电路调节所需的各种协议,该控制结合了免疫荧光技术和电路操作使用化学遗传学。以下协议中描述的方法适用于在体内同时刺激或抑制一个或多个回路,以确定其对成人神经发生中的调节功能。如果问题不需要高度的时间解析,则最好使用此方法。需要精确时间控制在一定频率的刺激/抑制的问题,可以更好地使用光遗传学13,14。这里描述的方法很容易适应长期研究,动物处理最少,特别是在压力是主要问题的地方。
Protocol
Representative Results
Discussion
该协议的目的是评估操纵特定神经回路如何利用一系列免疫组化学技术调节体内的成年海马神经发生。由特定神经回路介导的成人神经发生过程分析活性依赖调节是一项有价值的技术,具有很大的修改潜力,可用于研究各种神经回路。这些类型的实验的成功取决于多种因素,包括准确的病毒传递,正确的病毒选择所需的操作,正确交付胸腺素模拟,动物年龄,免疫染色效率,成功跨心灌注,无偏量定量图像。?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
L.J.Q. 得到国立卫生研究院国家精神卫生研究所在多样性补充R01MH111773以及T32培训补助金T32NS007431-20的支持下。该项目得到了NIH(MH1111773、AG058160和NS104530)授予J.S.的赠款的支持。
Materials
| 24 Well Plate | Thermo Fisher Scientific | 07-200-84 | |
| 48 Well Plate | Denville Scientific | T1049 | |
| 5-Ethynyl-2'-deoxyuridine (Edu) | Carbosynth | NE08701 | |
| Alcohol 70% Isopropyl | Thermo Fisher Scientific | 64-17-5 | |
| Alcohol Prep Pads | Thermo Fisher Scientific | 13-680-63 | |
| Alexa-488 Azide | Thermo Fisher Scientific | A10266 | |
| Anti-Chicken Nestin | Aves | NES; RRID: AB_2314882 | |
| Anti-Goat DCX | Santa Cruz | Cat# SC_8066; RRID: AB_2088494 | |
| Anti-Mouse Tbr2 | Thermo Fisher Scientific | 14-4875-82; RRID: AB_11042577 | |
| Betadine Solution (povidone-iodine) | Amazon | ||
| Citiric Acid Stock [.1M] Citric Acid (21g/L citric acid) | Sigma-Aldrich | 251275 | |
| Clozapine N- Oxide | Sigma-Aldrich | C08352-5MG | |
| Confocal Software (Zen Black) | Zeiss Microscopy | Zen 2.3 SP1 FP1 (black) | |
| Copper (II) Sulfate Pentahydrate | Thermo Fisher Scientific | AC197722500 | |
| Cotton Swabs | Amazon | ||
| Coverslip | Denville Scientific | M1100-02 | |
| Delicate Task Wipe Kimwipes | Kimtech Science | 7557 | |
| Drill Bit .5mm | Fine Science Tools | 19007-05 | |
| Ethylene Glycol | Thermo Fisher Scientific | E178-1 | |
| Hamilton Needle 2 inch | Hmailton Company | 7803-05 | |
| Hamilton Syringe 5uL Model 75 RN | Hmailton Company | Ref: 87931 | |
| High Speed Drill | Foredom | 1474 | |
| Infusion Pump | Harvard Apparatus | 70-4511 | |
| Injectable Saline Solution | Mountainside Health Care | NDC 0409-4888-20 | |
| Insulin Syringe | BD Ultra-Fine Insulin Syringes | ||
| Isoflurane | Henry Schein | 29405 | |
| Stereotax For Small Animal | KOPF Instruments | Model 942 | |
| Leica M80 | Leica | ||
| Leica Microtome | Leica | SM2010 R | |
| LSM 780 | Zeiss Microscopy | ||
| Nair (Hair Removal Product) | Nair | ||
| Paraformaldahyde 4% | Sigma-Aldrich | 158127 | |
| Plus Charged Slide | Denville Scientific | M1021 | |
| Phosphate Buffered Solution (PBS) | Thermo Fisher Scientific | 10010031 | |
| Puralube Vet Ointment | Puralube | ||
| Slide Rack 20 slide unit | Electron Microscopy Science | 70312-24 | |
| Slide Rack holder | Electron Microscopy Science | 70312-25 | |
| Small Animal Heating Pad | K&H | ||
| Sucrose | Sigma-Aldrich | S0389 | |
| Super PAP Pen 4 mm tip | PolySciences | 24230 | |
| Surgical Scalpel | MedPride | 47121 | |
| Tris Buffered Solution (TBS) | Sigma-Aldrich | T5912 | |
| Tri-sodium citrate Stock [.1M] Tri-sodium Citrate (29.4g/L tri-sodium citrate) | Sigma-Aldrich | C8532 | |
| Triton X-100 | Sigma-Aldrich | 93443 | |
| Tweezers | Amazon | ||
| Vet Bond Tissue Adhesive | 3M | 1469SB |
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