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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
Chapters
Summary February 11th, 2021
Please note that all translations are automatically generated.
Click here for the English version.
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。
Transcript
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。
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