用于同步光遗传学调制和电神经记录的光驱阵列
1Department of Electronic and Electrical Engineering,Ewha Womans University, 2Graduate Program in Smart Factory,Ewha Womans University, 3Department of Electrical and Computer Engineering,Seoul National University, 4Graduate School of Engineering Practice,Seoul National University, 5Department of Brain and Cognitive Sciences,Ewha Womans University
Summary
在这里，我们提出了一种光学系统的制备方法，该系统具有用于光传输的光纤和用于神经记录的电极阵列。表达通道视紫红质-2的转基因小鼠的 体内 实验表明，该系统在同时进行光遗传学刺激和电生理记录方面具有可行性。
Abstract
在过去十年中，光遗传学由于其独特的选择性神经调节或监测能力，已成为研究神经信号的重要工具。由于特定类型的神经元细胞可以被基因修饰以表达视蛋白，因此光遗传学能够对选定的神经元进行光学刺激或抑制。光遗传学的光学系统已经取得了一些技术进步。最近，有人提出将用于光传递的光波导与电生理记录相结合，以同时监测对光遗传学刺激或抑制的神经反应。在这项研究中，开发了一种植入式光驱阵列（2×2光纤），并嵌入了多通道电极。
使用发光二极管（LED）作为光源，并集成了微细结构微透镜阵列，以在光纤尖端提供足够的光功率。光驱阵列系统包括一次性部分和可重复使用部分。一次性部件具有光纤和电极，而可重复使用部分具有用于光控制和神经信号处理的LED和电子电路。除了光驱植入手术、光遗传学刺激和电生理神经记录的程序外，在随附的视频中还介绍了植入式光驱阵列系统的新颖设计。 体内 实验的结果成功地显示了由小鼠海马兴奋神经元的光刺激引起的时间锁定神经尖峰。
Introduction
记录和控制神经活动对于理解大脑在神经网络中和细胞水平上的功能至关重要。传统的电生理记录方法包括使用微量移液管的膜片钳1、2、3、4和使用微肺电极5、6、7、8的细胞外记录。作为一种神经调节方法，电刺激经常被用来通过神经元细胞的直接或间接去极化来直接刺激局灶性大脑区域。然而，电学方法无法区分用于记录或刺激的神经元细胞类型，因为电流向各个方向扩散。
作为一项新兴技术，光遗传学开创了一个新的时代，了解神经系统是如何工作的9，10，11，12，13，14，15，16。光遗传学技术的本质是利用光来控制转基因细胞表达的光敏视蛋白的活性。因此，光遗传学能够在复杂的神经回路14，17中对遗传选择的细胞进行复杂的调节或监测。光遗传学方法的广泛使用需要同时进行神经记录以直接确认光神经调节。因此，具有光控制和记录功能的集成设备将非常有价值16，18，19，20，21，22，23，24，25。
传统的、基于激光的光遗传学刺激存在局限性，这需要笨重且昂贵的光传输系统26、27、28、29、30。因此，一些研究小组采用基于μLED的硅探针来最小化光传输系统的尺寸31，32，33，34。然而，由于LED的能量转换效率低，与μLED直接接触存在热脑损伤的风险。光波导，如光纤，SU-8和氮氧化硅（SiON），已被应用以避免热损伤30，35，36，37，38，39。然而，这种策略也存在一个缺点，因为它在光源和波导之间的耦合效率低。
微透镜阵列先前被引入以增强LED和光纤40之间的光耦合效率。基于微机电系统（MEMS）技术开发了一种光驱系统，用于微米级40的光学刺激和电记录。LED和光纤之间的微透镜阵列将光效率提高了3.13 dB。如图 1所示，2×2光纤阵列在4×4微透镜阵列上对齐，LED位于微透镜阵列下方。安装2×2光纤而不是4×4以减少脑损伤。钨电极阵列位于光棒阵列附近，使用硅通过孔进行电生理记录（图1B）。
该系统由顶部一次性部件和可拆卸底部部件组成。顶部一次性部分，包括光纤阵列，微透镜阵列和钨电极阵列，旨在永久植入大脑 进行体内 实验。底部包括一个LED光源和一个外部电源线，该电源线易于拆卸和重复用于另一个动物实验。可拆卸的塑料盖可在拆卸部件时保护一次性部件。
通过在Ca 2 +/钙调蛋白依赖性蛋白激酶II阳性神经元（CaMKIIα：：ChR2小鼠）中植入表达通道视紫红质-2（ChR2）的转基因小鼠的大脑中来验证该系统的可行性。记录电极用于记录神经元光学刺激期间单个神经元的神经活动。
Protocol
动物护理和外科手术由梨花女子大学机构动物护理和使用委员会（IACUC）批准（编号：20-029）。 1. 光驱阵列的制备（图1 和 图2） 将光纤与微透镜阵列连接。 去除光纤的钝化涂层，并使用精密光纤切割器将其切割成5毫米长的碎片。 将光纤浸入透明UV树脂中，并将光纤放在…
Representative Results
光驱系统被成功制造，以提供足够的光功率来激活靶神经元。钨电极的精细对准是通过微加工硅通过孔实现的。当施加50 mA电流时，在光纤尖端测得的光强度为3.6 mW/mm2 。微透镜将光效提高了3.13 dB。由于微透镜阵列增强了光耦合，因此施加的电流大约是在没有微透镜阵列系统的情况下实现相同光强度所需的电流的一半。由于LED在电流更大的情况下产生更多的热量，因此采用微透镜阵列来降…
Discussion
验证了该系统同时进行光遗传学刺激和电生理记录的可行性（图6）。光刺激过程中的大尖峰是与光刺激同时发生的光电伪影（图6A）。这在红色虚线矩形波形的放大视图中很明显（图6A）。如图 6A所示，从记录的波形中可以清楚地分辨出光电伪影，并清楚地识别出海马信号通路的光遗传学刺激所引起的神经?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了韩国国家研究基金会（NRF）的人体增强融合技术研发计划的支持，由科学和信息通信技术部（NRF-2019M3C1B8090805）资助，并由韩国政府（MSIT）资助的韩国国家研究基金会（NRF）资助（编号：2019R1A2C1088909）。我们感谢韩国大田KAIST生物科学系的Lee Seung-Hee实验室为转基因小鼠提供了优质小鼠。
Materials
| 5-pin Connector | NW3 | HD127K | 1.27 mm (.050") pitch |
| Bovie | Fine Science Tools(F.S.T) | 18010-00 | High Temperature Cautery Kit |
| Data Acquisition Software | Intan Technologies, LLC | USB Interface Board software | Work with the RHD USB Interface Board |
| Dental Cement | Lang Dental Manufacturing Company, Inc. | 1223CLR | Use Jet Liquid and powder in jet denture repair package |
| Digital Manipulator Arm | Stoelting Co. | 51904/51906 | Left, Right each Digital Manipulator Arm, 3-Axes, Add-On |
| Gel Foam | Cutanplast | Standard (70*50*10 mm) | Sterile re-absorbable gelatin sponge with a haemostatic effect |
| Headstage Preamplifier | Intan Technologies, LLC | #C3314 | RHD 16-Channel Recording Headstages |
| Heating Pad | Stoelting Co. | 53800R | Stoelting Rodent Warmer X1 with Rat Heating Pad |
| LED | OSLON | GB CS8PM1.13 | λ typ. 470 nm, Viewing angle 80 °, Forward voltage 2.85 V |
| MATLAB | MathWorks, Inc. | R2019a | |
| Micro Clamp | SURGIWAY | 12-1002-04 | Straight type, Serre-fine DIEFFENBACH droite 3.5 cm |
| Optical Fiber | Thorlabs, Inc. | FT200UMT | 0.39 NA, Ø 200 µm Core Multimode Optical Fiber, High OH for 300 – 1200 nm |
| PFA-Coated Tungsten Wire | A-M System | Custom ordered | Rod type, Ø 101.6 μm (.004") |
| Photodiode | Thorlabs | S121C | |
| power meter | Thorlabs Inc. | PM100D | |
| Precision cleaver | FITEL | S326 | Fiber slicer tool |
| Prism | GraphPad | 5.01 version | |
| Scalpel | Feather™ | #20 | Scalpel blade with 100mm long Scalpel Handle |
| screw | Nasa Korea | stainless steel | diameter: 1.2 mm, length: 3 mm |
| Silver Wire | The Nilaco Corporation | AG-401265 | Ø 200 µm |
| Stereotaxic Fxrame | Stoelting Co. | 51500D | Digital new standard stereotaxic, rat and mouse |
| suture | ETHICON | W9106 | suture size: 4-0, length:75 cm, wire diameter: 4-0 |
| Vaseline | Unilever PLC | Original | 100% pure petroleum jelly |
| Wave_Clus | N/A | N/A | https://github.com/csn-le/wave_clus |
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Citer Cet Article
Lee, Y., Ryu, D., Jeon, S., Lee, Y., Cho, Y. K., Ji, C., Kim, Y., Jun, S. B. Optrode Array for Simultaneous Optogenetic Modulation and Electrical Neural Recording. J. Vis. Exp. (187), e63460, doi:10.3791/63460 (2022).