非人类灵长类动物遗传学的大体积、行为相关光照
Summary
建立了一个组织穿透照明灯的协议, 以提供光超过大体积的最小直径。
Abstract
该协议描述了一个大体积的照明灯, 这是为非人类灵长类大脑中的 optogenetic 操纵而开发的。照明灯是一种经蚀刻后的改性塑料光纤, 其发光表面积 #62; 100x。除了描述大容量照明灯的构造外, 本协议还详细介绍了用于确保均匀光分布的质量控制校准。此外, 该协议还描述了插入和移除大容量照明灯的技术。表面和深层结构都可以被照亮。这个大体积的照明灯不需要物理耦合到一个电极, 因为照明灯是由塑料, 而不是玻璃, 它会简单地弯曲的情况下, 传统的光纤将粉碎。因为这个照明灯提供光超过行为相关的组织体积 (≈ 10 mm3) 没有比传统的光纤更大的穿透损伤, 它促进行为研究使用遗传学在非人灵长类动物。
Introduction
Optogenetic 工具, 允许毫秒精确, light-driven 神经元控制被广泛用于研究啮齿动物和无脊椎动物的功能生理学和行为。然而, 技术上的挑战限制了非人类灵长类大脑中遗传学的使用, 它的体积 ~ 100 x 大于啮齿动物的大脑1。
为了促进非人类灵长类动物的遗传学研究, 一个照明灯被设计来解决两个相互竞争的目标: 大体积光照和最小穿透损伤。以前试图解决其中一个问题的做法是代价高昂的。束纤维照亮更大的容量, 但以增加的直径, 并且, 因而, 损坏2,3。锥形玻璃纤维可减少穿透损伤, 但将光线聚焦到发光表面区域和 #60; 100 µm2 4,5。外部的大脑照明通过一个窗口的硬脑膜绕过的挑战, 穿透伤害和可能允许大体积照明, 但它只能用于一些肤浅的大脑区域6。
为了创建一个大体积, 小直径的照明灯 (图 1a), 塑料光纤的尖端是热锥和核心和熔覆蚀刻 (图 1b, c)。不同于将光聚焦到一个窄点的锥形光纤, 蚀刻允许光线在尖端的两侧均匀地逸出, 从而在大面积上分布光源 (图 1d, e)。由于穿透损伤与穿透直径成正比, 因此该照明灯没有比传统纤维更大的穿透损伤, 但它有和 #62; 100x. 光发射表面积和光功率的 1/第100更广泛地提供光密度在脑幻影 (1.75% 琼脂糖) (图 1e)。蒙特卡罗模型 (图 1f) 说明了传统光纤和大体积照明灯在光功率密度相等时与发光表面之间的差异。每个照明灯使用集成球体 (图 2a, b) 进行单独校准, 以确保沿尖端均匀分布 (图 2c)。
这种大体积的照明灯已经验证了 optogenetic 操作的行为和神经元射击在非人灵长类动物。纤维针尖长度可以定制的任何大脑区域和每一个动物的个人接受领域的地图。该照明灯可以配对与穿透电极的神经录音, 跨越的长度照明。此外, 由于纤维可以携带任何颜色的可见光, 它可以与任何可用的 optogenetic 分子配对。
Protocol
Representative Results
Discussion
虽然 optogenetic 工具被广泛用于研究啮齿类动物的疾病和生理学, 但对大脑大容量的照明技术的挑战限制了非人类灵长类动物的遗传学的使用。在猴子的开创性研究中使用了大的光功率密度 (~ 100 兆瓦/毫米2到20瓦特/毫米2) 来照亮小容量, 也许和 #60; 1 mm3, 并报告了轻微的行为影响与兴奋 opsins 在皮层4, 9、10…
Disclosures
The authors have nothing to disclose.
Acknowledgements
LCA 承认来自 NDSEG 奖学金, NSF GRFP, 和麦戈文研究所的朋友的资助。EP 承认资助哈利和尤尼斯 Nohara UROP 基金, 麻省理工学院的 1995年 UROP 基金, 和麻省理工学院 UROP 基金。ESB 承认来自 NIH 2R44NS070453-03A1、”让哈维奖” 和 “纽约干细胞基金会-罗伯逊奖” 的资助。RD 承认来自 NIH EY017292 的资金。迈克尔. 威廉姆斯在拍摄前帮助团队组织和收集补给品。
Materials
| Plastic optical fiber | Industrial fiber optics | SK-10 | 250 micron diameter, Super Eska line |
| Wire stripper | Klein Tools | 11047 | 22 gauge |
| Vise Clamp | Wilton | 11104 | Generic table mount vice clamp |
| Dual temperature heat gun | Milwaukee | 8975-6 | 570 / 1000°F |
| Lab marker | VWR | 52877 | |
| Dissection microscope | VistaVision | 82027-156 | Stereo microscope w/ dual incandescent light, 2x/4x magnification, available from VWR |
| Lab tape | VWR | 89097-972 | 4 pack of violet color; however, tape color does not matter |
| Silicon carbide lapping sheet | ThorLabs | LF5P | 5 micron grit, 10 pack |
| Aluminum oxide lapping sheet | ThorLabs | LF3P | 3 micron grit, 10 pack |
| Aluminum oxide lapping sheet | ThorLabs | LF1P | 1 micron grit, 10 pack |
| Calcined alumina lapping sheet | ThorLabs | LF03P | 0.3 micron grit, 10 pack |
| Hot knife | Industrial fiber optics | IF370012 | 60 Watt, heavy duty |
| Fiber inspection scope | ThorLabs | FS201 | optional |
| Stainless Steel Ferrule | Precision fiber optics | MM-FER2003SS-265 | 265 micron inner diameter |
| 1 mL syringe | BD | 14-823-30 | Luer-lok tip is preferable to reduce risk of leakage, but not strictly needed |
| Plastic epoxy | Industrial fiber optics | 40 0005 | |
| 18 gauge blunt needle | BD | 305180 | 1.5 inch length |
| Lint-free wipe (KimWipe) | ThorLabs | KW32 | available from many vendors |
| Light absorbing foil | ThorLabs | BKF12 | |
| Electrical tape | 3M | Temflex 1700 | Optional, may substitute other brands / models |
| 26 gauge sharp needle | BD | 305111 | 0.5 inch length |
| Micromanipulator | Siskiyou | 70750000E | may substitute other brands/models |
| Steretactic arm | Kopf | 1460 | may substitute other brands/models |
| Laser safety goggles | KenTeK | KCM-6012 | must be selected based on the color of laser used, example given here |
| Laser or other light source | vortran | Stradus 473-50 | example of blue laser |
| Integrating sphere | ThorLabs | S142C | Attached power meter, also available from ThorLabs, item #PM100D |
| Ultem recording chamber | Crist instrument company | 6-ICO-J0 | Customized with alignment notch |
| Tower microdrive with clamps | NAN | DRTBL-CMS | |
| Guide tube | Custom | N/A | Made from 25 gauge spinal needle (BD) or blunt tubing |
| NAN driver system | NAN | NANDrive | |
| Custom grid design | custom | custom | plans available upon request |
| Blunt forceps | FischerScientific | 08-875-8A | generic stainless steel blunt forceps |
| Digital calipers | Neiko | 01407A | available on amazon.com. May select a finer resolution caliper for more precise measurements. |
| Patch cable | ThorLabs | FG200LCC-custom | This is one example of many possible patch cables. As long as the fiber diameter is less than or equal to the fiber diameter of the large volume illuminator and as long as the connectors interface, any patch cable (glass or plastic, vendor purchased or made in the lab) is fine for this application. |
| Clear plastic dust caps | ThorLabs | CAPF | Package of 25 |
| ceramic split mating sleeve | Precision Fiber Products, Inc. | SM-CS1140S |
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