在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激
Summary
该协议描述了一组方法，利用外体脑切片中的光遗传学刺激，确定来自遥远大脑区域的远程输入的细胞类型特定功能连接。
Abstract
了解细胞型特定突触连接是了解大脑范围神经元回路的关键先决条件。远距离连接的功能调查需要对单个神经元进行有针对性的记录，并结合对已识别的远输入的特定刺激。这通常很难用传统和电刺激技术来实现，因为来自上游大脑区域的正克斯可能混合在目标区域。对特定大脑区域进行立体定向，以表达光敏电通道的病毒介导性，允许选择性地刺激来自该区域的光的斧头。除了整个大脑的其他皮下或皮质区域外，脑内立体性注射可用于很好的限定结构，如前脑核。
这里描述的是一套技术，用于精确立体地注射在小鼠大脑中表达通道性多普辛的病毒载体，随后在脑切片制备中对斧子终端进行光刺激。这些协议简单且广泛适用。结合来自后同步连接神经元的全细胞贴片夹记录，对斧头的光刺激可以检测功能性突触连接、药理表征和评价其强度。此外，记录神经元的生物细胞素填充可用于午睡后神经元的时态形态识别。
Introduction
定义大脑区域之间的连接是了解神经回路所必需的。经典的解剖追踪方法允许建立区域间连接，病变研究有助于理解信息流的分层组织。例如，空间方向和头部方向信号的大脑电路涉及信息从丘脑到前子的定向流动。这已经通过病变研究证明，在下游背水前，以及副海马网格细胞信号1，2降低头部方向信号。
大脑区域之间的功能连接在细胞和亚细胞水平上更难建立。在海马区，高度有组织的解剖允许使用切片制备中的电模拟来研究路径特定的突触连接。放置在CA1地层辐射层的刺激电极可用于专门刺激CA33的舍弗附带输入。置于CA1层乳糖分子的刺激电极将激活对CA14，5的穿孔路径输入。电刺激激活来自斧子端子的神经递质释放;然而，它激活神经元与索马塔附近的刺激位点以及通过的斧子。因此，当不同原产区的纤维在目标结构中混合时，研究来自定义大脑区域的亲子关系是有限用途的，就像新皮质中的典型情况一样。
神经元也可能受到光的刺激。光学方法包括笼中谷氨酸的光活化，可与单光或双光子激光扫描相结合。多个紧密间隔的位点可以按顺序刺激，对组织没有机械损伤6。这已经成功地用于映射突触受体以及激活单个神经元7。虽然谷氨酸未凝固可用于局部电路分析，但它不允许特定激活远程输入。
研究神经元回路中远距离连通性的一种选择方法是使用病毒介导的通道性多普辛表达。使用体内立体注射，如这里所述，光门电通道的表达可以定向，并在空间上限制到所需的大脑区域。通过这种方式，通道性多普辛能够有效地映射从一个区域到其目标的兴奋性或抑制性连接。转染的斧头端子可以在脑切片制备中受到光的刺激，而作为读出的贴片夹记录可以检查大脑中特定电路组件的功能和强度8。光遗传学方法结合病毒的立体注射提供了前所未有的特异性和基因控制9。用光刺激另外允许高时空精度10，11。
前体是海马和准海马形成12、13过渡的六层皮质结构。它接收来自ADN11的重要突触输入，但也从其他几个皮质和皮下区域14接收。因此，在目前切片内选择性刺激的血性偶联体电心端子，在电刺激和谷氨酸未凝固时是不可能的。该协议中所述的方法，使用表达光门通道的病毒载体的精确立体注射来确定大脑区域（ADN 和前子体）之间的功能连接。还描述了在目标区域中投射神经元的斧头终端的光刺激，以及脑切片制备中突触后神经元的全细胞贴片-夹子记录。
Protocol
所有程序均按照欧洲共同体理事会指令（2010/63/EU）执行，并经巴黎笛卡尔大学道德委员会批准。实验者必须获得程序授权，才能遵守当地法规。 1. 实验规划 定义要瞄准的大脑区域。在小鼠大脑图集15的帮助下，确定注射部位的立体坐标。对于右背状的鼻腔核（ADN），坐标为：-0.82后，0.75横向，-3.2深度（毫米）相对于胸膜。坐标可能需要根据不同年?…
Representative Results
这里介绍的程序用于表达一种蓝色光敏通道，通过立体注射异位腺相关病毒，在丘拉他（ADN）的前背核中融合到GFP。立体坐标根据小鼠大脑图图确定，并通过注射200 nL荧光示踪荧光荧光荧光荧光红宝石进行测试。注射后10分钟，动物被牺牲，大脑被提取并固定过夜。冠状脑部分准备检查注射部位，该位置被正确放置在ADN中，并限制在ADN（图1A，B）。 <p class="jov…
Discussion
体内病毒注射以表达光敏蛋白酶在定义的脑区是一种选择方法，用于光遗传学分析远距离功能连接10，11，17，18。立体注射提供了精确定位大脑特定区域的可能性。与荧光报告器的蛋白酶的配合，方便地允许评估精确注射部位的成功表达和确认。使用AAV血清型2/5通常限制表达到目标的大脑区域。通过?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢贝特朗·马顿、梅里·纳萨尔、黄丽文和让·西蒙内特帮助开发早期版本的立体注射方案，并感谢马林·曼努埃尔和帕特里斯·杰古佐的技术支持。这项工作得到了法国教育和研究部（L.R.，L.S.）、国家空间研究中心（M.B.）和国家教育补助协会（ANR-18-CE92-0051-01）的支持。
Materials
| 0.5 mm bur | Harvard Apparatus | 724962 | |
| 10 µL Hamilton syringe | Hamilton | 1701 RN – 7653-01 | |
| 10X PBS solution | Thermofisher Scientific | AM9624 | text |
| 36% PFA | Sigma-Aldrich | F8775 | |
| 470 nm LED | Cairn Research | P1105/470/LED DC/59022m | use with matched excitation filter 470/40x and emission filter for GFP |
| AAV5.Syn.Chronos-GFP.WPRE.bGH | Penn Vector Core | AV-5-PV3446 | lot V6026R, qTiter GC/ml 4.912e12, ddTiter GC/ml 2.456e13 |
| All chemicals | Sigma | ||
| Bath temperature controler | Luigs & Neumann | SM7 | Set at 34°C |
| beveled metal needle | Hamilton | 7803-05 | 33 gauge, 13mm, point style 4-20° |
| Big scissors | Dahle Allround | 50038 | |
| Biocytin | Sigma | B4261 | final 1-3 mg/ml |
| Borosilicate Capillaries | Havard Apparatus | GC150-10 | 1.5 mm outer, 0.86 inner diameter |
| Brown Flaming electrode puller | Sutter Instruments | P-87 | |
| BupH Phosphate Buffered Saline pack | Thermofisher Scientific | 28372 | |
| butterfly needle for perfusion | Braun | Venofix A | 24G |
| CCD Camera | Andor | DL-604M | |
| Confocal Microscope | Zeiss | LSM710 | 20X |
| curved forceps | FST | 11011-17 | |
| CY5 configuration (confocal) | Helium-Neon 633nm (5,0 mW) laser; Mirror: MBS 488/561/633 | ||
| CY5 configuration (epifluo) | Nikon/Chroma | Fluorescent light (Intensilight); Excitation filter: BP645/30; Dichroic mirror: 89100 BS ; Emission filter: BP705/72 | |
| DAPI | Sigma | D9542 | |
| DAPI configuration (epifluo) | Nikon/Chroma | Fluorescent light (Intensilight); Cube: Semrock Set DAPI-5060C-000-ZERO (Excitation: BP 377/50; Mirror: BS 409; Emission: BP 447/60) | |
| Digidata 1440A | Axon Instruments | ||
| Digital handheld optical meter | ThorLabs | PM100D | Parametered on 475 nm |
| Double egde stainless steel razor blades | Electron Microscopy Sciences | 72000 | Use half of the blade in the slicer |
| Dual Fluorescent Protein Flashlight | Nightsea | DFP-1 | excitation, 440-460 nm; emission filter on glasses, 500 nm longpass. |
| EGTA | Sigma | E4368 | final 0,2 mM |
| Epifluorescence Microscope | Nikon | Eclipse TE-2000E | 10 or 20X |
| Filter paper | Whatman | ||
| Fluoro-Ruby 10% | Millipore | AG335 | disolve 10 mg in 100 µl of distilled water ; inject 150 to 300 nl |
| GFP configuration (epifluo) | Nikon/Chroma | Fluorescent light (Intensilight); Cube: Filter Set Nikon B-2E/C FITC (Excitation: BP 465-495; Mirror: BS 505; Emission: BP 515-555) | |
| Heatingplate | Physitemp | HP4M | |
| Heparin choay 5000 U.I./ml | Sanofi | 5 ml vial | |
| HEPES | Sigma | H3375 | final 10 mM |
| High speed rotary micromotor kit | Foredom | K.1070 | maximum drill speed 38,000 rpm |
| Internal solution compounds : | |||
| Isolated Pulse Stimulator | A-M Systems | 2100 | |
| KCl | Sigma | P4504 | final 1,2 mM |
| Ketamine 1000 | Virbac | ||
| Ketofen 10% | Merial | 100 mg/ml : dilute 1 µl in 1ml total (0,1%) | |
| Laocaine (lidocaine) | MSD | 16,22 mg/ml : dilute 1 ml in 4 ml total (around 4%) | |
| LED hi power spot for surgery | Photonic (via Phymep) | 10044 | |
| LED Power Supply | Cairn Research | OptoLED Light Source | |
| Manipulators | Luigs & Neumann | SM-7 | |
| Mg-ATP 2H20 | Sigma | A9187 | final 4 mM |
| MgCl2 | Sigma | 63069 | final 2 mM |
| Micro temperature controler | Physitemp | MTC-1 | |
| Milk powder | Carnation | ||
| MultiClamp 700B | Axon Instruments | ||
| Na Phosphocreatine | Sigma | P7936 | final 10 mM |
| Na3-GTP 2H20 | Sigma | G9002 | final 0.4 mM |
| needle holder/hemostat | FST | 13005-14 | |
| pClamp acquisition software | Axon Instruments | ||
| Peristaltic pump | Gilson | Minipuls 3 | 14-16 on the display for 2-3 ml/min |
| Potassium gluconate (K-gluconate) | Sigma | G4500 | Final 135 mM |
| ProLong Gold antifade mounting medium | Thermofisher Scientific | P36390 | |
| Rompun 2% (xylazine) | Bayer | ||
| small scissors | FST | 14060-09 | |
| Sodium chloride 0.9% | Virbac | dilute 8.5 mL in 10 ml total | |
| Stereomicroscope VISISCOPE SZT | VWR | 630-1584 | |
| Stereotaxic frame with digital display | Kopf | Model 940 | Small animal stereotaxic instrument |
| Streptavidin-Cy3 conjugate | Life technologies | 434315 | |
| Streptavidin-Cy5 conjugate | Thermofisher Scientific | S32357 | |
| Superglue3 Loctite | Dutscher | 999227 | 1g tube |
| Suture filament Ethilon II 4-0 polyamid | Ethicon | F3210 | |
| Syringe pump | kdScientific | Legato 130 – 788130 | Use Infuse and Withdraw modes |
| Tissue slicer | Leica | VT1200S | speed 0.07, amplitude 1. |
| tubing | Gilson | F117942, F117946 | Yellow/Black, Purple/Black |
| upright microscope | Olympus | BX51W1 | |
| Versi-dry bench absorbant paper | Nalgene |
References
- Goodridge, J. P., Taube, J. S. Interaction between the postsubiculum and anterior thalamus in the generation of head direction cell activity. The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 17 (23), 9315-9330 (1997).
- Winter, S. S., Clark, B. J., Taube, J. S. Spatial navigation. Disruption of the head direction cell network impairs the parahippocampal grid cell signal. Science. 347 (6224), 870-874 (2015).
- Fan, Y., et al. Activity-dependent decrease of excitability in rat hippocampal neurons through increases in I(h). Nature Neuroscience. 8 (11), 1542-1551 (2005).
- Takahashi, H., Magee, J. C. Pathway Interactions and Synaptic Plasticity in the Dendritic Tuft Regions of CA1 Pyramidal Neurons. Neuron. 62 (1), 102-111 (2009).
- Dolleman-van der Weel, M. J., Lopes da Silva, F. H., Witter, M. P. Interaction of nucleus reuniens and entorhinal cortex projections in hippocampal field CA1 of the rat. Brain Structure & Function. 222 (5), 2421-2438 (2017).
- Callaway, E. M., Yuste, R. Stimulating neurons with light. Current Opinion in Neurobiology. 12 (5), 587-592 (2002).
- Fino, E., et al. RuBi-Glutamate: Two-Photon and Visible-Light Photoactivation of Neurons and Dendritic spines. Frontiers in Neural Circuits. 3, 2 (2009).
- Mao, T., et al. Long-range neuronal circuits underlying the interaction between sensory and motor cortex. Neuron. 72 (1), 111-123 (2011).
- Zhang, F., et al. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nature Protocols. 5 (3), 439-456 (2010).
- Simonnet, J., et al. Activity dependent feedback inhibition may maintain head direction signals in mouse presubiculum. Nature Communications. 8, 16032 (2017).
- Nassar, M., et al. Anterior Thalamic Excitation and Feedforward Inhibition of Presubicular Neurons Projecting to Medial Entorhinal Cortex. Journal of Neuroscience. 38 (28), 6411-6425 (2018).
- Fricker, D., et al. Pyramidal cells of rodent presubiculum express a tetrodotoxin-insensitive Na+ current. The Journal of Physiology. 587, 4249-4264 (2009).
- Simonnet, J., Eugène, E., Cohen, I., Miles, R., Fricker, D. Cellular neuroanatomy of rat presubiculum. The European Journal of Neuroscience. 37 (4), 583-597 (2013).
- Simonnet, J., Fricker, D. Cellular components and circuitry of the presubiculum and its functional role in the head direction system. Cell and Tissue Research. 373 (3), 541-556 (2018).
- Paxinos, G., Franklin, K. B. J. . The Mouse Brain in Stereotaxic Coordinates. , (2013).
- Huang, L. -. W., et al. Laminar Localization and Projection-Specific Properties of Presubicular Neurons Targeting the Lateral Mammillary Nucleus, Thalamus, or Medial Entorhinal Cortex. eNeuro. 4 (2), (2017).
- Cruikshank, S. J., Urabe, H., Nurmikko, A. V., Connors, B. W. Pathway-specific feedforward circuits between thalamus and neocortex revealed by selective optical stimulation of axons. Neuron. 65 (2), 230-245 (2010).
- Gonzalez-Sulser, A., et al. GABAergic Projections from the Medial Septum Selectively Inhibit Interneurons in the Medial Entorhinal Cortex. Journal of Neuroscience. 34 (50), 16739-16743 (2014).
- Mathon, B., et al. Increasing the effectiveness of intracerebral injections in adult and neonatal mice: a neurosurgical point of view. Neuroscience Bulletin. 31 (6), 685-696 (2015).
- Nassar, M., et al. Diversity and overlap of parvalbumin and somatostatin expressing interneurons in mouse presubiculum. Frontiers in Neural Circuits. 9, 20 (2015).
- Klapoetke, N. C., et al. Independent optical excitation of distinct neural populations. Nature Methods. 11 (3), 338-346 (2014).
- Hass, C. A., Glickfeld, L. L. High-fidelity optical excitation of cortico-cortical projections at physiological frequencies. Journal of Neurophysiology. 116 (5), 2056-2066 (2016).
Cite This Article
Richevaux, L., Schenberg, L., Beraneck, M., Fricker, D. In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices. J. Vis. Exp. (151), e59534, doi:10.3791/59534 (2019).