在活体钙成像小鼠致病性甘里昂神经元对味觉刺激的反应
Summary
在这里，我们介绍如何暴露活的麻醉实验鼠的基因结石，以及如何使用钙成像来测量这些神经元的合奏对味道刺激的反应，允许使用不同的兴奋剂进行多次试验。这允许对哪些神经元对哪些塔斯特反应进行深入比较。
Abstract
在过去十年中，基因编码钙指标（GECIs）的进步促进了活体功能成像的革命。这些技术使用钙作为神经元活动的代理，提供了一种实时监测大型神经元组合中单个细胞对各种刺激的反应的方法。我们和其他人已经应用了这些技术来成像单个基因结核神经元对活麻醉小鼠舌头的刺激的反应。原发性结肠由前舌和味觉内侧阵阵神经元的细胞体以及一些内侧的耳皮皮质的躯体感性神经元组成。通过GCaMP成像单个基因结石神经元的味觉反应，为野生型小鼠中这些神经元的调谐特征提供了重要信息，以及检测基因操纵小鼠周围味觉错误布线表型的方法。在这里，我们演示了暴露基因结石、GCaMP 荧光图像采集、数据分析的初始步骤和故障排除的外科手术。此技术可用于跨基因编码的 GCaMP 或 AAV 介质的 GCAMP 表达，并可修改为映像特定的遗传子集（即 Cre 介质 GCAMP 表达）。总的来说，在活体钙成像的原生结石神经元是一个强大的技术，用于监测外周阵风神经元的活动，并提供补充信息，更传统的全神经和弦丁帕尼录音或味觉行为测定。
Introduction
哺乳动物外周味系统的一个关键组成部分是原生结石。除了一些内侧的耳塞神经元外，基因还包括前舌和味觉内侧阵阵神经元的细胞体。与其他外周感觉神经元类似，原生结核神经元是伪单极神经元，长轴向外周投射到味蕾，并集中投射到孤独道1的脑干核。这些神经元主要通过味觉受体细胞释放ATP来激活，这些受体细胞对口腔中的味道刺激有反应。。。ATP是味觉信号的基本神经递质，阵风结石神经元表达的P2rx受体是激活4的必要条件。鉴于味觉受体细胞表达特定味觉受体的特定味觉模式（甜，苦，咸，乌玛米，或酸），它已被假设，阵风结石神经元的反应，味道刺激也将勉强调整5。
整个神经记录显示，和弦丁帕尼和更大的上级石油神经进行阵风信号代表所有五种口味模式的原生结石6，7。然而，这仍然留下了关于神经元对给定味道的反应的特异性的问题：如果有味道模式特定的神经元，多模式神经元，或两者的混合物。单纤维记录提供了有关单个纤维的活动及其化学敏感性的更多信息 8，9，10，但这种方法仅限于从少量纤维收集数据。同样，在个体大鼠的活体电生理记录中，生成结石神经元提供了有关单个神经元11、12、13的反应的信息，但仍丧失了种群的活性，每只动物的神经元记录相对较少。为了分析神经元合奏的反应模式，而不忽视单个神经元的活性，需要采用新技术。
钙成像，特别是使用基因编码的钙指标，如GCaMP，提供了这一技术突破14，15，16，17，18。GCaMP使用钙作为神经元活性的代名词，随着细胞内钙含量的升高，绿色荧光增加。继续开发新的GCaMP形式，以提高信号与噪声比，调整绑定动力学，并适应专业实验19。GCaMP 提供单个神经元分辨率，与整个神经记录不同，可以同时测量神经元组合的反应，不像单个纤维或单个细胞记录。基因结胶的钙成像已经提供了关于野生型小鼠16、20中这些神经元的调谐特征的重要信息，并已识别出基因操纵小鼠18中的外周味错配表型。
在活体钙成像技术中应用到基因结石中的一个主要困难是，它被封装在骨质的暴躁牛皮中。为了获得光学访问基因，需要精密的手术，以去除骨层，同时保持结节完好无损。为此，我们创建了此指南，以帮助其他研究人员访问基因结石，并成像这些神经元的 GCaMP 介导荧光反应，以品尝体内的刺激。
Protocol
Representative Results
Discussion
这项工作描述了一个分步协议，以手术暴露基因结节和视觉记录其神经元与GCaMP6的活动。这个程序非常类似于之前描述的17，有几个明显的例外。首先，使用头部柱子可以方便地调整手术过程中的头部定位。其次，在刺激性输送方面，吴和德沃里亚奇科夫的方法通过食管17流式刺激，而这个协议则用分配针将液体直接输送到嘴里。这两种方法都可以通过刺激真菌…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
作者感谢胡马云对老鼠的饲养。这项工作的资金部分由UTSA的脑健康联盟毕业生和博士后种子赠款（B.E.F.）和NIH-SC2-GM130411提供给L.J.M。
Materials
| 1 x #5 Inox Forceps | Fine Science Tools | NC9792102 | |
| 1ml Syringe with luer lock | Fisher Scientific | 14-823-30 | |
| 2 x #3 Inox Forceps | Fine Science Tools | M3S 11200-10 | |
| 27 Gauge Blunt Dispensing Needle | Fisher Scientific | NC1372532 | |
| 3M Vetbond | Fisher Scientific | NC0398332 | |
| 4-40 Machine Screw Hex Nuts | Fastenere | 3SNMS004C | |
| 4-40 Socket Head Cap Screw | Fastenere | 3SSCS04C004 | |
| Absorbent Points | Fisher Scientific | 50-930-668 | |
| Acesulfame K | Fisher Scientific | A149025G | |
| Artificial Tears | Akorn | 59399-162-35 | |
| BD Allergist Trays with Permanently Attached Needle | Fisher Scientific | 14-829-6D | |
| Blunt Retractors | FST | 18200-09 | |
| Breadboard | Thor Labs | MB8 | |
| Citric Acid | Fisher Scientific | A95-3 | |
| Cohan-Vannas Spring Scissors | Fine Science Tools | 15000-02 | |
| Contemporary Ortho-Jet Liquid | Lang | 1504 | |
| Contemporary Ortho-Jet Powder | Lang | 1520 | |
| Cotton Tipped Applicators | Fisher | 19-062-616 | |
| Custom Head Post Holder | eMachineShop | See attached file 202410.ems | |
| Custom Metal Head Post | eMachineShop | See attached file 202406.ems | |
| DC Temperature Controller | FHC | 40-90-8D | |
| Digital Camera, sCMOS OrcaFlash4 Microscope Mounted | Hamamatsu | C13440 | |
| Disection Scope | Leica | M80 | |
| Hair Clippers | Kent Scientific | CL7300-Kit | |
| IMP | Fisher Scientific | AAJ6195906 | |
| Ketamine | Ketaved | NDC 50989-996-06 | |
| LED Cold Light Source | Leica Mcrosystems | KL300LED | |
| Luer Lock 1/16" Tubing Adapters | Fisher | 01-000-116 | |
| Microscope | Olympus | BX51WI | |
| Mini-series Optical Posts | Thorlabs | MS2R | |
| MPG | Fisher Scientific | AAA1723230 | |
| MXC-2.5 Rotatable probe Clamp | Siskiyou | 14030000E | |
| NaCl | Fisher Scientific | 50-947-346 | |
| petri dishes | Fisher Scientific | FB0875713A | |
| Pressurized air | Airgas | AI Z300 | |
| Quinine | Fisher Scientific | AC163720050 | |
| Self Sticking Labeling Tape | Fisher Scientific | 159015R | |
| Silicone Pinch Valve Tubing 1/32" x 1/16" o.d. (per foot) | Automate Scientific | 05-14 | |
| Sola SM Light Engine | Lumencor | ||
| Snap25-2A-GCaMP6s-D | JAX | 025111 | |
| Student Fine Scissors | Fine Science Tools | 91460-11 | |
| Surgical Probe | Roboz Surgical Store | RS-6067 | |
| Surgical Probe Holder | Roboz Surgical Store | RS-6061 | |
| Thread | Gütermann | 02776 | |
| BD Intramedic Tubing | Fisher Scientific | 22-046941 | |
| Two Stage Gas Regulator | Airgas | Y12FM244B580-AG | |
| Tygon vinyl tubing – 1/16" | Automate Scientific | 05-11 | |
| Valvelink8.2 digital/manual controller | Automate Scientific | 01-18 | |
| Valvelink8.2 Pinch Valve Perfusion System | Automate Scientific | 17-pp-54 | |
| Xylazine | Anased | NADA# 139-236 |
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