激光制导神经跟踪在脑外植体
Summary
我们描述了一种技术，通过顺行或逆行示踪剂注射标记的神经元和它们的过程变成使用体外制备脑核。我们修改的I N通过利用荧光标记的小鼠突变体和基本的光学设备，以便增加贴标精度体外示踪剂电穿孔的现有方法。
Abstract
我们提出结合的体外示踪剂注射方案，它使用了一系列的电气和压力脉冲的过程中，通过电穿孔，增加脑外植体与靶向激光照明和匹配滤波器护目镜染料吸收的技术。 在体外的电本身所描述的技术产生了比较良好的视觉控制目标定位大脑的某些区域。通过将其与荧光遗传标记的激光激发和它们的读出通过带传递滤波器护目镜，其中可以拿起遗传标记的细胞/细胞核和荧光示踪染料的排放量，研究者可以大幅提高注射剂的精度相结合通过找到感兴趣的区域，并更有效地控制了该染料扩散/吸收在注射区域。此外，激光照射技术允许研究给定neurocircuit通过省的功能iding约神经元投射到一特定区域的情况下的GFP表达被链接到发射机的由神经元的亚群表达的类型的类型的信息。
Introduction
为了定义一个特定的神经元（微）电路，必须通过找出所述电路，和它们的连接图形的各个参与者启动。自从沃勒的出版约neurofiber通过损毁1种类繁多的神经解剖跟踪技术跟踪已经建立。这些技术的一些可以在固定的组织验尸2-4被应用，更为依赖于染料的活的神经元的活性的运输，如发现于1971年5-6。后者可进一步细分为两组利用活性逆行方法区分（从注入区域到给定突起的来源 ， 即所突出的所述区的神经元的胞 体）和活性顺行（从注入面积给定突起的目标 ， 即轴突突起和标记的神经元）传输的轴突终端。此外，在一些情况下，TRA减量材料注入活的动物，然后注入由数天或数周（体内示踪剂注射）存活，而在其他情况下取出的大脑注射和注入人工脑脊液后孵育几个小时（体外示踪剂注射） 。
在这个协议中，我们修改了现有的体外电击技术7-8在使用霍乱毒素亚基B和四甲右旋糖酐作为跟踪物质顺行和逆行追踪实验标记神经元胞体和进程。此协议的总体目标是提供神经科学家与一个有效的工具来跟踪不同的大脑核团神经元之间的连接方式，同时采取可转基因小鼠线和基本的光学设备的优势，以便在示踪剂注射，以增加定位精度。虽然顺行和逆行追踪的方法，用霍乱毒素和葡聚糖胺和它们各自的荧光标记的偶联物是不是新的9-13，（由于是电穿孔的方法 ，例如：Haas等14），示踪注射涉及脑组织的块的组合与电穿孔在体外制备是一个比较新的发展7。使用相同类型的示踪染料的活体动物过度神经元的跟踪技术的主要优点是由于较高的效率与该电穿孔的染料被拉紧的神经元的增加的标记强度。一个附加的优点是示踪剂注射期间缩短的潜伏期（用于染料运输）和其增加的目标准确度，因为实验者具有视觉地控制喷射的目标区域。后者也意味着不需要昂贵的立体定向的设备，需要找到感兴趣的核或大脑区域。
为了阿迪tionally增加靶向准确性，我们采取了转基因小鼠系，它表达的GFP在其甘氨酸神经元15和基本的光学设备组成的405nm的波长和匹配带通滤波护目镜的手持式激光指针（450的亚群的优势- 700纳米）。因此，我们在通过其荧光信号识别注射区靶向精度和通过提供一种更细的方式，通过观测土著GFP信号和所述示踪剂的相互作用的时，控制该注射区域内的染料扩散取得显著进一步增加荧光。我们的技术还允许揭示的电路的功能以及通过识别GFP阳性抑制性神经元及其连通（或兴奋性在其它小鼠品系）填充的示踪剂。
总之，我们进一步增强了强大的神经科学工具，研究脊椎动物脑的连接组和评估吨他给定neurocircuit不同的神经解剖学特征。通过使用转基因小鼠以及便宜且广泛可用的光学设备，我们能够显著增加我们注射剂的靶向精度。此外，转基因小鼠使我们能够确定被跟踪的连接，这有助于揭示在听觉脑干的抑制微电路的功能的类型。
Protocol
1.光学基因分型乳鼠1.光学基因分型检查使用适当的激发波长的激光指针的各荧光标记物的表达（在这里描述的实验中为405nm）和相应的过滤器护目镜阻断激发波长不过将发射波长（450 – 700在此处描述的实验纳米） 。点激光指示器在头部或鼠标小狗脊髓的背面（ 参见图1）。避免闪耀的激光进入眼睛和皮肤的激光的冗长曝光。 较早的?…
Representative Results
图1和2示出了如何将激光指针和激光护目镜可用于快速，低成本从垫料基因型GFP阳性的动物。在年轻小鼠幼仔情况下，该技术可以用于通过头骨和覆盖皮肤（图1A – D）的非侵入性地确定在动物的大脑的GFP标签。发射的荧光可以通过皮肤和小鼠幼仔头骨至少高达3日龄（图1C）可以看出。该过程示于图1，为绿色荧光蛋白（GFP…
Discussion
体外示踪剂电穿孔的一般强度，相对于在体内跟踪研究中，是可以让研究人员的利益，因此大脑区域更好的访问，不涉及昂贵立体定向（通常电）设备。此外，所需的脑外植体存活期跨越只有几个小时（1 – 4）中的体内示踪注射的情况下，而不是数天或甚至数周（见关于使用葡聚糖胺和在其他示踪剂的详细审查体内注射由Lanciego和Wouterlood，2011 22，但也罗德里格?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
由美国国立卫生研究院/ NCRR科罗拉多CTSI授权号UL1 RR025780和落基山脉Neurlogical障碍核心中心授予美国国立卫生研究院P30NS048154部分支持NIH / NIDCD R01 DC 011582.成像实验在美国科罗拉多州安舒茨医学校区先进的光学显微镜核心的大学进行支撑。萨沙迪拉克博士索尔克研究所为我们提供了GlyT2-GFP小鼠。
Materials
| Sodium chloride | Sigma-Aldrich | S7653 | All chemicals are from Sigma-Aldrich, unless noted otherwise. |
| Potassium chloride | P9333 | ||
| Potassium phosphate monobasic | P5655 | ||
| Sodium phosphate di-basic | S907 | ||
| Magnesium chloride | M2670 | ||
| Calcium chloride | C5080 | ||
| Glucose | G7528 | ||
| Sodium bicarbonate | S6297 | ||
| Ascorbic acid | A4544 | ||
| Myo-inositol | I5125 | ||
| Sodium pyruvate | P2256 | ||
| Bovine serum albumine | A2153 | optional, for additional (immuno)histochemistry | |
| Triton-X-100 | X100 | optional, for additional (immuno)histochemistry | |
| Poly(ethylene glycol), 8000 MW | P2139 | optional, for brain clearing | |
| Formamide | Fisher Scientific | F84 | optional, for brain clearing |
| Choleratoxin subunit-b | Molecular Probes | C-34776 (Alexa 555) | |
| Dextrane tetramethyl-rhodamine | Molecular Probes | D-7162 (Alexa 555) | |
| Fluorescent Nissl | Invitrogen | N-21479 (blue) | optional |
| Paraformaldehyde | Fisher Scientific | SF93 | |
| Agarose | Invitrogen | 16520 | |
| Fluoromount-G | Southern Biotech | 0100-01 | |
| Pentobarbi-tal | Vortech Pharmaceuticals | Fatal-Plus | |
| Borosilicate glass filaments | Harvard Apparatus | G150F-10 | |
| Pipette puller | Zeitz Instruments, Germany | DMZ Universal Puller | |
| Perfusion setup | Custom-made | ||
| Laser pointer | laserpointerpro.com, Hong Kong | HK-88007294 (405 nm) | |
| Filter/safety goggles | Dragon Lasers, China | LSG09 (band-pass 450-700 nm) | |
| Bionocular microscope | Wild Heerbrugg, Switzerland | Wild M3 | Equipped with high-intensity illuminator (MI-150; Dolan-Jenner Inc.) |
| Picospritzer | Parker Instruments | Picospritzer III | |
| PC with installed MC Stimulus software | Multi Channel Sys-tems, Germany (software) | ||
| 2-channel stimulator | Multi Channel Sys-tems, Germany | STG-1002 | |
| Stimulation isolation unit | A.M.P.I., Israel | Iso-Flex | |
| Micromanipulator | Narishige, Japan | YOU-1 | |
| Vibratome | Leica, Germany | VT1000S |
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Citazione di questo articolo
Albrecht, O., Klug, A. Laser-guided Neuronal Tracing In Brain Explants. J. Vis. Exp. (105), e53333, doi:10.3791/53333 (2015).