Name: in vivo calcium imaging of mouse geniculate ganglion neuron responses to taste stimuli
Uploaded: 2021-02-11T15:00:00
Description: Watch this Scientific Journal Video about In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli at JoVE.com

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。

动物协议由德克萨斯大学圣安东尼奥分校机构动物护理和使用委员会审查和批准。
1. 术前设置
注：请注意，此处没有处理设备的初始设置，因为根据泵系统、显微镜、相机和使用的成像软件，设备设置会有所不同。有关设置说明，请参阅设备供应商提供的教学材料。对于作者使用的设备，请参阅 材料表。
<ol><li>确保液体流经所有车辆（水?...

<ol>
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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 x #5 Inox Forceps</td>
			<td>Fine Science Tools</td>
			<td>NC9792102</td>
			<td></td>
		</tr>
		<tr>
			<td>1ml Syringe with luer lock</td>
			<td>Fisher Scientific</td>
			<td>14-823-30</td>
			<td></td>
		</tr>
		<tr>
			<td>2 x #3 Inox Forceps</td>
			<td>Fine Science Tools</td>
			<td>M3S 11200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>27 Gauge Blunt Dispensing Needle</td>
			<td>Fisher Scientific</td>
			<td>NC1372532</td>
			<td></td>
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			<td>3M Vetbond</td>
			<td>Fisher Scientific</td>
			<td>NC0398332</td>
			<td></td>
		</tr>
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			<td>4-40 Machine Screw Hex Nuts</td>
			<td>Fastenere</td>
			<td>3SNMS004C</td>
			<td></td>
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			<td>4-40 Socket Head Cap Screw</td>
			<td>Fastenere</td>
			<td>3SSCS04C004</td>
			<td></td>
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			<td>Absorbent Points</td>
			<td>Fisher Scientific</td>
			<td>50-930-668</td>
			<td></td>
		</tr>
		<tr>
			<td>Acesulfame K</td>
			<td>Fisher Scientific</td>
			<td>A149025G</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Tears</td>
			<td>Akorn</td>
			<td>59399-162-35</td>
			<td></td>
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		<tr>
			<td>BD Allergist Trays with Permanently Attached Needle</td>
			<td>Fisher Scientific</td>
			<td>14-829-6D</td>
			<td></td>
		</tr>
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			<td>Blunt Retractors</td>
			<td>FST</td>
			<td>18200-09</td>
			<td></td>
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			<td>Breadboard</td>
			<td>Thor Labs</td>
			<td>MB8</td>
			<td></td>
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			<td>Citric Acid</td>
			<td>Fisher Scientific</td>
			<td>A95-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cohan-Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-02</td>
			<td></td>
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			<td>Contemporary Ortho-Jet Liquid</td>
			<td>Lang</td>
			<td>1504</td>
			<td></td>
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			<td>Contemporary Ortho-Jet Powder</td>
			<td>Lang</td>
			<td>1520</td>
			<td></td>
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			<td>Cotton Tipped Applicators</td>
			<td>Fisher</td>
			<td>19-062-616</td>
			<td></td>
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			<td>Custom Head Post Holder</td>
			<td>eMachineShop</td>
			<td></td>
			<td>See attached file 202410.ems</td>
		</tr>
		<tr>
			<td>Custom Metal Head Post</td>
			<td>eMachineShop</td>
			<td></td>
			<td>See attached file 202406.ems</td>
		</tr>
		<tr>
			<td>DC Temperature Controller</td>
			<td>FHC</td>
			<td>40-90-8D</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera, sCMOS OrcaFlash4 Microscope Mounted</td>
			<td>Hamamatsu</td>
			<td>C13440</td>
			<td></td>
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			<td>Disection Scope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
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			<td>Hair Clippers</td>
			<td>Kent Scientific</td>
			<td>CL7300-Kit</td>
			<td></td>
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			<td>IMP</td>
			<td>Fisher Scientific</td>
			<td>AAJ6195906</td>
			<td></td>
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			<td>Ketamine</td>
			<td>Ketaved</td>
			<td>NDC 50989-996-06</td>
			<td></td>
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			<td>LED Cold Light Source</td>
			<td>Leica Mcrosystems</td>
			<td>KL300LED</td>
			<td></td>
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			<td>Luer Lock 1/16&#34; Tubing Adapters</td>
			<td>Fisher</td>
			<td>01-000-116</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
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			<td>Mini-series Optical Posts</td>
			<td>Thorlabs</td>
			<td>MS2R</td>
			<td></td>
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			<td>MPG</td>
			<td>Fisher Scientific</td>
			<td>AAA1723230</td>
			<td></td>
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			<td>MXC-2.5 Rotatable probe Clamp</td>
			<td>Siskiyou</td>
			<td>14030000E</td>
			<td></td>
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			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>50-947-346</td>
			<td></td>
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			<td>petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713A</td>
			<td></td>
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			<td>Pressurized air</td>
			<td>Airgas</td>
			<td>AI Z300</td>
			<td></td>
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		<tr>
			<td>Quinine</td>
			<td>Fisher Scientific</td>
			<td>AC163720050</td>
			<td></td>
		</tr>
		<tr>
			<td>Self Sticking Labeling Tape</td>
			<td>Fisher Scientific</td>
			<td>159015R</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Pinch Valve Tubing 1/32&#34; x 1/16&#34; o.d. (per foot)</td>
			<td>Automate Scientific</td>
			<td>05-14</td>
			<td></td>
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			<td>Sola SM Light Engine</td>
			<td>Lumencor</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Snap25-2A-GCaMP6s-D</td>
			<td>JAX</td>
			<td>025111</td>
			<td></td>
		</tr>
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			<td>Student Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
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			<td>Surgical Probe</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6067</td>
			<td></td>
		</tr>
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			<td>Surgical Probe Holder</td>
			<td>Roboz Surgical Store</td>
			<td>RS-6061</td>
			<td></td>
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			<td>Thread</td>
			<td>G&#252;termann</td>
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			<td>BD Intramedic Tubing</td>
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</table>

in vivo calcium imaging of mouse geniculate ganglion neuron responses to taste stimuli

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。

按照协议，转基因Snap25-GCaMP6s动物被镇定，基因结节被暴露，在录制视频时将烤面包涂在舌头上。实验的目的是确定哪些塔斯特引起每个细胞的反应。18 种溶解在 DI 水中，5 mM 奎宁、60 mM NaCl、50 mM IMP = 1 mM MPG、50 mM 柠檬酸）18 被溶解在 DI 水中，并应用于舌头 2 s 由 13 s DI 水分离。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62172/62172fi...

Watch this Scientific Journal Video about In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli at JoVE.com

In vivo Calcium Imaging of Mouse Geniculate Ganglion Neuron Responses to Taste Stimuli

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。

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

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

这种技术可以回答重要的功能问题，神经元反应的味道在原生结石，内和弦丁帕尼味通路的关键部分。该技术可用于监测单个实验试验中多个单个神经元的实时反应，因为每只动物记录的细胞明显高于通常通过电生理学方法观察到的细胞。将头柱安装的鼠标放在加热垫上的上部位置，在喉咙上的皮肤上，从胸骨到下巴，在皮肤上切口两厘米。并收回皮肤和亚最大腺体，以充分暴露肌肉。在将接缝定位在副切口肌肉中后，用钝解剖将接缝分离，并缩回组织以打开它。小心地切开气管顶部的开口，大到足以容纳一块聚乙烯管，而不会将气管直径减半，并将管子插入气管朝向肺部。重新定位再造器以释放副切除肌肉并收回亚最大腺体。然后，使用少量的兽医胶水将副切口粘合在管子上。要打破丁巴尼亚牛皮，轻轻地调戏所需的肌肉向上，拉开结缔组织。在肌肉前端切口，避免血管。后拉， 直到清除暴躁的公牛。稍微向后倾斜头部，抬起暴躁的公牛，并将胡萝卜动脉前部的分支定位到大块肌肉的后插入点。苍白只是后，这个血管的凸起结构的暴躁牛皮，并定位在肌肉的接缝。使用两套细钳，钝解剖接缝，直到清澈的牛皮骨可见，并使用缩回器保持清晰的骨骼视图。找到前后在牛皮上运行的接缝，并使用手术探头在接缝中心的骨骼上戳一个洞，然后，使用一套细端剪刀在骨骼中切割圆形区域，注意不要切断前部和后部的血管，以或在牛皮下方。要暴露基因，找到科奇莱亚。前科奇莱亚是紧张廷帕尼肌肉。使用弹簧剪刀切割和去除此肌肉。使用手术探针在耳蜗海角上戳一个洞，并立即使用吸力吸气任何流出孔的液体。放大球菌中的孔，注意不要损坏包围科奇莱亚的血管，损伤到后部和横向边缘。将鼠标的头部向前倾斜，以定位前耳蜗结构下方的时间骨孔。注意直接位于第七神经上的孔的脊前。并在洞中插入一个手术探头，让时间骨被小心地抬起来暴露第七个神经。如果基因不能完全可见，轻轻地将动物的头向后倾斜，并试图将骨骼前部拉到神经上。如果黑帮仍然模糊不清，从下面拉起更多的骨头，小心不要把探针放在骨头的深处，因为这可能会损坏原生体。要运行 tastant 面板，请使用吸气从生源性上去除液体，并将鼠标放在解剖显微镜下的吸水垫上。使用牛皮剩下的孔、时间骨的孔和第七神经来定位原生结石。并使用表皮上 FITCGFP 过滤器检查单个 GCaMP 表达致病性结核神经元。将一根口香管的配药针牢牢地放入动物的嘴里，并在嘴下放置一个培养皿，以捕捉任何液体。将视频录制的开始与 tastant 演示的开始同步，在录制过程中观看实时源以获得响应、漂移和渗漏。如果渗漏发生，吸吸液体，直到基因视图清晰，并重新清除烤面包。如果发生漂移，请检查前哨的所有部分是否都牢固地拧紧。如果没有反应，请检查液体是否流动，显微镜和摄像头是否聚焦在适当的位置，而不会遮挡视野。当所有所需的实验完成后，轻轻地放松缩回器，并重复动物另一侧的暴露和口感分析。观察到，应用于舌头的味觉刺激应导致 GCaMP 荧光的快速瞬时增加，导致响应神经元之间的亮度发生明显变化。对荧光视频片段的分析允许生成与荧光变化对应的痕迹，从而在一段时间内在个别感兴趣的区域内对基线响应进行跟踪。荧光强度超过阈值水平的变化被认为是一个积极的反应。除了破坏黑帮，重要的是要避免出血。如果出血发生，等待血液凝固，然后应用盐水和吸力从视觉场中取出血液。基因结胶的可视化使研究人员能够直接测量神经元对味觉刺激的反应，并使用依赖Cre的GCaMP等工具识别这些神经元。

Research

JoVE Journal

Neuroscience

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

在气味诱发反应的钙成像果蝇触角叶

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

光学相干断层扫描: 成像小鼠视网膜神经节细胞在体内

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments

在神经生理学实验中利用神经元刺激活性触发闭环刺激

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems ...

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses

在行为和刺激反应期间为整个大脑成像准备成人德罗索菲拉褪黑麦片加斯特

We present a method developed specifically to image the whole Drosophila brain during ongoing behavior such as walking. Head fixation and dissection are ...