混合微驱系统,带可回收的光电探头和Tetrode,用于自由移动小鼠的双位点高密度记录
Summary
该协议描述了混合微驱阵列的构建,该阵列允许在自由移动的小鼠的两个大脑区域中植入9个独立可调节的四分线体和一个可调光硅探头。还演示了一种用于多种用途的安全回收和重复使用光硅探头的方法。
Abstract
多区域神经记录可以为理解多个大脑区域之间的精细时间级交互提供重要信息。然而,传统的微驱设计通常只允许使用一种类型的电极从单个或多个区域进行记录,从而限制了单单元或深度轮廓记录的产量。它还经常限制将电极记录与光遗传学工具相结合以靶向通路和/或细胞类型特定活动的能力。这里介绍的是一个混合微驱阵列,用于自由移动的小鼠,以优化产量,并描述其制造和重用微驱阵列。目前的设计采用九个四分线和一个光硅探头,同时植入两个不同的大脑区域,在自由移动的小鼠中。四分线和光硅探头可沿大脑中的多索文轴独立调节,以最大化单位和振荡活动的收率。此微驱阵列还集成了光、中介光遗传学操作的设置,以研究远程神经回路的区域或细胞类型特定响应和功能。此外,光硅探头可以在每次实验后安全回收和重复使用。由于微驱阵列由 3D 打印部件组成,因此可轻松修改微驱的设计以适应各种设置。首先介绍了微驱阵列的设计以及如何将光纤连接到硅探头进行光遗传学实验,然后制作 ttrode 捆绑和将阵列植入小鼠大脑。记录局部场势和单位尖峰,结合光遗传学刺激,也证明了微驱阵列系统在自由移动小鼠的可行性。
Introduction
通过调查不同大脑区域如何动态地相互作用,了解神经元活动如何支持认知过程(如学习和记忆)至关重要。为了阐明认知任务背后的神经活动动力学,在微驱阵列1、2、3的帮助下,在自由移动的动物身上进行了大规模的细胞外电生理学研究。 4.在过去的二十年里,已经开发出几种微驱阵列,为大鼠5、6、7、8和小鼠9植入多个大脑区域。10,11,12.尽管如此,目前的微驱设计一般不允许使用多种探头类型,迫使研究人员选择具有特定优点和限制的单电极类型。例如,ttrode 阵列适用于人口稠密的大脑区域,如背海马 CA11,13,而硅探针为研究解剖连接提供了更好的几何轮廓14,15.
特龙德和硅探头常用于体内慢性记录,各有优缺点。除了成本效益和机械刚度外,Tetrodes在单单元隔离方面比单电极16、17具有显著优势。当与微驱动器8、18、19、20相结合时,它们也能提高单单元活动的收益率。增加同时记录的神经元的数量对于理解神经回路21的功能至关重要。例如,需要大量细胞来调查功能异质细胞类型的小群体,如与时间相关的22或奖励编码23个细胞。需要更高的细胞数来提高尖峰序列13、24、25的解码质量。
然而,特龙在记录空间分布的细胞(如皮层或丘拉他)方面处于不利地位。与特罗伊德相比,硅探头可以提供局部场势(LLFP)的空间分布和相互作用,并在局部结构14、26中进行探针活动。多柄硅探头进一步增加了记录站点的数量,并允许跨单个或相邻结构录制 27。然而,与特龙相比,这种阵列在电极位点的定位上不太灵活。此外,高密度探测器需要复杂的尖峰排序算法,以提取有关相邻通道作用电位的信息,以镜像Tetrodes28、29、30获取的数据。因此,单单位的总体产量往往小于四元组。此外,硅探头由于其脆弱性和高成本而处于不利地位。因此,对硅探头的选择取决于记录的目的,即是否优先考虑在记录点获得高产量的单单元或空间分析。
除了记录神经活动,光遗传学操作已成为神经科学中更强大的工具之一,以检查特定细胞类型和/或通路如何促进神经回路功能13,31, 32,33.然而,光遗传学实验在微驱阵列设计中需要额外的考虑,以将光纤连接器连接到刺激光源34,35,36。通常,连接光纤需要相对较大的力,这可能导致探头在大脑中发生机械移动。因此,将植入式光纤与传统的微驱阵列相结合并非易事。
出于上述原因,研究人员需要根据记录的目的优化电极类型的选择或植入光纤。例如,在海马1,13中,四叶酸用于实现更高的单位产量,而硅探针用于研究皮质区域的层状深度轮廓,如中端皮质皮层(MEC)37。目前,据报道,为大鼠5、11同时植入三分线体和硅探头的微驱。然而,由于微驱的重量、鼠标头上空间有限以及设计采用不同探头的微驱的空间要求,在小鼠中植入多个四头和硅探头是极具挑战性的。虽然可以在没有微驱的情况下植入硅探头,但此过程不允许调整探头,降低了硅探针回收12、38的成功率。此外,光遗传学实验在微驱阵列设计中需要额外的考虑。该协议演示如何构建和植入用于自由移动小鼠的慢性记录的微驱阵列,这允许植入 9 个独立可调节的四分线体和一个可调节的光电探头。这种微驱阵列还有助于光遗传学实验和硅探头的回收。
Protocol
这里描述的所有方法都已获得得克萨斯大学西南医学中心机构动物护理和使用委员会(IACUC)的批准。 1. 微驱阵列部件的准备 使用牙科模型树脂使用 3D 打印机打印微驱阵列部件(图 1A,B)。确保单个 3D 打印层的厚度小于 50 μm,以保持打印零件上的小孔清晰可行。注:微驱阵列由五个部分组成(图1C):(1)微驱阵列的主体,包括9个用于四驱的微驱螺钉和一个用于硅探?…
Representative Results
微驱阵列在5天内建成。表2描述了微驱准备的时间表。使用这种微驱,将9个四分线和一个硅探头分别植入小鼠的海马CA1和MEC[21周大/29克体重男性pOxr1-Cre(C57BL/6背景)]中。这种转基因小鼠在MEC层III金字塔神经元中表达Cre。在电极植入前10周,将200 nL的AAV5-DIO-ChR2-YFP(奶口:7.7 x 1012 gc/mL)注射到MEC中。使用低通滤波器(1-500 Hz)记录LLFP,并使用高通滤波器(0.8-5 kHz)检测到尖峰单元。光刺激(+ = 450…
Discussion
该协议演示如何构建和植入混合微驱阵列,允许使用独立可调的四轮驱动和硅探针记录来自两个大脑区域的神经活动。并演示了光遗传学实验和硅探头在实验后的恢复。虽然可调硅探针33或光硅探头36植入先前在小鼠中演示,但该协议在同时Ttrode阵列和光硅探头植入中具有明显优势,可提供灵活的选择植入式探头类型。植入式探头的类型可根据实验目的进行切换,如?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作部分得到了日本科学海外研究促进协会(HO)、授予学者计划(TK)、人类前沿科学计划(TK)、脑研究基金会(TK)、学院科学和技术获取和保留计划 (TK)、大脑和行为研究基金会 (TK) 和住友基金会研究资助 (JY)、NARSAD 青年调查员研究资助 (JY)。我们感谢W.Marks在编写稿件期间提出的宝贵意见和建议。
Materials
| #00-90 screw | J.I. Morris | #00-90-1/8 | EIB screws |
| #0-80 nut | Small Parts | B00DGB7CT2 | brass nut for holding fiber ferrule holder |
| #0-80 screw | Small Parts | B000FMZ57G | brass machine screw for probe connector mount, fiber ferrule holder, and shielding cone |
| 22 Ga polyetheretherketone tubes | Small Parts | SLPT-22-24 | for attaching to the shuttle, 0.025 inches inner diameter |
| 23 Ga stainless tubing | Small Parts | HTX-23R | for tetrode |
| 23 Ga stainless wire | Small Parts | HTX-23R-24-10 | for L-shape/support wire |
| 26 Ga stainless wire | Small Parts | GWX-0200 | for guide-posts |
| 30 Ga stainless wire | Small Parts | HTX-30R | for tetrode |
| 3-D CAD software package | Dassault Systèmes | SolidWorks 2003 | |
| 3D printer | FormLab | Form2 | |
| 5.5mil polyimide insulating tubes | HPC Medical | 72113900001-012 | |
| aluminum foil tape | Tyco | Tyco Adhesives 617022 Aluminum Foil Tape | for the alternative shielding cone |
| conductive paste | YSHIELD | HSF54 | for shielding cone |
| customized screws for silicon-probe microdrive | AMT | UNM1.25-HalfMoon | half-moon stainless screw, 1.5 mm diameter, 300 µm thread pitch |
| customized screws for tetrode microdrive | AMT | Yamamoto_0000-160_9mm | slotted stainless screw, 0.5 mm diameter, 160 µm thread pitch, custom-made to order for our design |
| dental acrylic | Stoelting | 51459 | |
| dental model resin | FormLab | RS-F2-DMBE-02 | |
| Dremel rotary tool | Dremel | model 800 | a grinder |
| drill bit | Fine Science Tool | 19007-05 | |
| electric interface board | Neuralynx | EIB-36-Narrow | |
| epoxy | Devcon | GLU-735.90 | 5 minutes epoxy |
| eye ointment | Dechra | Puralube Ophthalmic Ointment | to prevent mice eyes from drying during surgery |
| fiber polishing sheet | Thorlabs | LFG5P | for polishing the optical fiber |
| fine tweezers | Protech International | 15-368 | for loading/recovering the silicon probe |
| gold pins | Neuralynx | EIB Pins Small | |
| ground wire | A-M Systems | 781500 | 0.010 inch bare silver wire |
| headstage preamp | Neuralynx | HS-36 | |
| impedance meter | BAK electronics | Model IMP-2 | 1 kHz testing frequency |
| mineral oil | ZONA | 36-105 | for lubricating screws and wires |
| optical fiber | Doric | MFC_200/260-0.22_50mm_ZF1.25(G)_FLT | |
| Recording system | Neuralynx | Digital Lynx 4SX | |
| ruby fiber scribe | Thorlabs | S90R | for cleaving the optical fiber |
| silicon grease | Fine Science Tool | 29051-45 | |
| silicon probe | Neuronexus | A1x32-Edge-5mm-20-177 | Fig. 3, 4A, 4B, 5 |
| silicon probe | Neuronexus | A1x32-6mm-50-177 | Fig. 4C |
| silicon probe washing solution | Alcon | AL10078844 | contact lens cleaner |
| silicone lubber | Smooth-On | Dragon Skin 10 FAST | for preparation of microdrive mold |
| silver paint | GC electronic | 22-023 | silver print II coating, used for ground wires |
| skull screw | Otto Frei | 2647-10AC | 0.8 mm diameter, 0.200 mm thread pitch |
| standard surgical scissors | ROBOZ | RS-5880 | |
| stereotaxic apparatus | Kopf | Model 942 | |
| super glue | Loctite | LOC230992 | for applying to guide-posts |
| surgical tweezers | ROBOZ | RS-5135 | |
| Tetrode Twister | Jun Yamamoto | TT-01 | |
| tetrode wires | Sandvik | PX000004 |
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Citar este artigo
Osanai, H., Kitamura, T., Yamamoto, J. Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice. J. Vis. Exp. (150), e60028, doi:10.3791/60028 (2019).