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Introduction
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신경과학

正常小鼠和亨廷顿小鼠急性脑切片中谷氨酸释放和服用的单突触指标

Published: March 11, 2020
doi:
10.3791/60113
,
1Synaptic Dysfunction Group, Neuroscience Research Center,Charité – University Medicine

Summary

我们提出了一个协议，以评估成年小鼠急性切片中单皮质谷胱甘肽突触的谷氨酸释放和间隙之间的平衡。该协议使用荧光传感器iGluu进行谷氨酸检测，sCMOS摄像机用于信号采集和用于聚焦激光照明的设备。

Abstract

突触是高度分节的功能单元，相互独立地运行。在亨廷顿舞蹈症 （HD） 和其他神经退行性疾病中，由于谷氨酸清除不足以及由此产生的溢出和溢出效应，这种独立性可能会受到损害。前突起性终端和/或树突脊柱的天体细胞覆盖率改变，谷氨酸释放部位谷氨酸转运器簇的缩小，都与导致头晕/超激症症状的疾病发病机制有关。然而，导致HD谷胱甘肽突触功能障碍的机制并不十分了解。改进和应用突触成像，我们获得了数据，为阻碍运动启动的机制提供了新的曙光。在这里，我们描述了一种相对便宜的方法，通过使用新的基因编码的超快谷氨酸传感器iGluu，宽场光学，科学的CMOS（sCMOS）相机，473纳米激光和激光定位系统，以评估从年龄适当的健康或患病小鼠急性切片皮质分泌突触的状态，实现单突触分辨率的原理元素。谷氨酸瞬变由单像素或多个像素构造，根据活性区旁边的谷氨酸浓度[Glu]的最大海拔获得谷氨酸释放估计值，ii） 谷氨酸上升，反映在阴囊 [Glu] 的衰减时间常数 （TauD） 中。休息的回合大小和短期可塑性对比模式的差异是确定属于端脑内 （IT） 或金字塔道 （PT） 路径的皮质质间端子的标准。使用这些方法，我们发现，在有症状的HD小鼠+40%的PT型皮质突触显示谷氨酸间隙不足，这表明这些突触可能面临兴奋毒性损伤的风险。研究结果强调了TauD作为亨廷顿小鼠功能失调突触的生物标志物的效用，这种生物标志物具有低动力学表型。

Introduction

属于”单一连接”（即2个神经细胞之间的连接）的每个突触终端的相对影响通常由其对脑后神经元11、22的初始部分的影响来评估。体素和/或从后鼻神经神经元的树突状记录代表最常见的，直到现在，也是澄清自上而下或垂直透视33，4，54,5下信息处理的最有效手段。然而，星形细胞的存在及其离散和（在啮齿动物）非重叠区域可能有助于一个水平视角，基于在突触站点66，7，8，9，107,8,9,10的局部信号交换、集成和同步机制。

因为,众所周知，天体胶原体在神经退行性疾病11、12,12的发病机制中起着重要作用，特别是在谷胱甘肽突触13、14、15、1614的维持和可塑性方面起着重要作用13，可以想象，突触性能的变化会随着星形细胞在不同来源的共享靶区状态而演变。15,16为了进一步探索在健康和疾病方面目标/天体胶衍生的局部监管机制，有必要评估单个突触。制定本办法是为了估计功能性谷氨酸释放和清除指标的范围，并确定可用于识别与运动启动最密切相关的大脑区域（即首先在运动皮层和背纹体）中功能失调（或恢复）突触的标准。

纹状体缺乏内在谷胱甘肽神经元。因此，相对容易识别外分源的谷氨酸。后者主要起源于丘脑中和大脑皮层（见17，18，19，2018,19,20更多）。17皮质科斯特里亚特突触是由在皮质层2/3和5中局部的金字塔神经元的斧子形成的。各自的斧头通过更具有组织性地构成金字塔地带（PT）的光纤系统形成双端脑内 （IT） 连接或 ipsi边连接。另据介绍，IT型和PT型终端的发布特性和尺寸21、22,22不同。鉴于这些数据，人们也可以预期在谷氨酸的处理上会有一些差异。

纹状体是亨廷顿舞蹈症（HD）5中受影响最大的脑区。人类HD是一种严重的遗传性神经退行性疾病。Q175鼠标模型提供了一个机会来研究低动力学刚性形式的HD的细胞基础，这种状态与帕金森主义有很多共同之处。从大约1岁开始，同源Q175小鼠（HOM）表现出低血症的迹象，通过测量在开放场23中不运动所花的时间来揭示出来。目前对异血Q175小鼠（HET）的实验证实了在HOM观察到的先前运动缺陷，此外，观察到的马达缺陷伴随着在皮质分泌突触终端24附近出现星形外皮氨基酸输送器2蛋白（EAAT2）水平的下降。因此，人们推测，天体性谷氨酸的缺乏可能导致功能障碍，甚至失去各自的突触25，26。25,

在这里，我们描述了一种新方法，它允许人们根据释放的神经递质的量评估单个突触谷氨酸间隙。新的谷氨酸传感器iGluu在皮质激素金字塔神经元中表达。它由卡塔林Türük27开发，代表了先前引入的高亲和力，但缓慢的谷氨酸传感器iGluSnFR28的修改。两个传感器都是增强型绿色荧光蛋白（EGFP）的衍生物。有关光谱和动力学特性，请参阅赫拉萨等人27。简而言之，iGluu是一种低亲和力传感器，具有快速去活化动力学，因此特别适合研究谷氨酸释放突触端子的谷氨酸间隙。iGluu的分离时间常数是在停止流装置中确定的，在 20°C 下，Tau的脱位值为 2.1 毫秒，但推断到温度为 34 °C27时为 0.68 毫秒。在2光子显微镜下，在有机细胞海马培养的CA1区域，在34°C下探测的单舍弗辅助终端，平均衰变时间常数为2.7毫秒。

Protocol

所有工作均按照欧盟第2010/63/欧盟动物实验指令进行，并在柏林卫生保护和技术安全办公室（G0233/14和G0218/17）注册。 注：Q175 野生类型 （WT） 和异种 （HETs） 的录音可以在任何年龄和性别进行。在这里，我们研究了51至76周的男性和女性。 1. 注射谷氨酸传感器 iGluu，用于在科蒂科斯塔阿克斯中表达 使用移液器拉拔器（一步模?…

Representative Results

两种皮质质谷类性变异性鉴定IT 和 PT 亲和力分别源自第 2/3 层和第 5 层，在 ipsi边和反向（仅限 IT 终端）纹状体中表现出差分冲压和终止模式。仍然鲜为人知的谷氨酸释放和清除在重复激活条件下的特性，在运动开始期间观察到，但它是有充分记录，相应的谷氨酸释放变数在大小22不同。应用尺寸标准，发现IT和PT终端呈现出对比形式的短期可塑性24?…

Discussion

实验涉及一个普遍感兴趣的问题 ——突触不依赖及其在神经退化过程中可能失去的问题，我们描述了一种识别老年（>1年）小鼠急性脑切片中受影响的突触的新方法。利用最近引入的谷氨酸传感器 iGluu的改进动力学特性，实验以前所未有的方式阐明突触谷氨酸释放和服用之间的关系。

谷氨酸清除对突触功能和维持的影响没有很好地阐明，虽然谷氨酸引起的兴奋毒性可…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作得到了CHDI（A-12467）、德国研究基金会（Exc 257/1和DFG项目ID327654276 + SFB 1315）和Charité校内研究基金的支持。我们感谢伦敦圣乔治大学K.Türék和利物浦大学的N.Helassa，感谢他们的iGluu质粒和许多有益的讨论。D. 贝坦斯和A.舍恩赫尔提供了出色的技术援助。

Materials

Stereo microsope WPI PZMIII Precision Stereo Zoom Binocular Microscope
Stereotaxic frame Stoelting 51500D Digital Lab New Standard stereotaxic frame
High speed drill equipment Stoelting 514439V Foredom K1070 cromoter Kit
Injection system Stoelting 53311 Quintessential Stereotaxic Injector (QSI)
Hamilton syringe 5 µl Hamilton 87930 75RN Syr (26s/51/2)
Laser positioning system Rapp OptoElectronic UGA-40 UGA-40
Blue laser for iGluu excitation Rapp OptoElectronic DL-473-020-S 473 nm laser
Dichroic mirror for 473 nm Rapp OptoElectronic ROE TB-355-405-473 Dichroic
1P upright microscope Carl Zeiss 000000-1066-600 Axioskop 2 FS Plus
Objective 63x/1.0 Carl Zeiss 421480-9900 W Plan-Apochromat
4x objective Carl Zeiss 44-00-20 Achroplan 4x/0,10
Dichroic mirror for iGluu Omega optical XF2030
Emission filter for iGluu Omega optical XF3086
Dichroic mirror Omega optical QMAX_DI580LP
Emission filter for autofluorescence subtr. Omega optical QMAX EM600-650
sCMOS camera Andor ZYLA4.2PCL10 ZYLA 4.2MP Plus
Acqusition software Andor 4.30.30034.0 Solis
AD/DA converter HEKA Elektronik 895035 InstruTECH LIH8+8
Aquisition software HEKA Elektronik 895153 TIDA5.25
Electrode positioning system Sutter Instrument MPC-200 Micromanipulator
Electrical stimulator Charite workshops STIM-26
Slicer Leica VT1200 S Vibrotome
Brown/Flaming-type puller Sutter Instr SU-P1000 P-1000
Glass tubes for injection pipettes WPI 1B100F3
Glass tubes forstimulation pipettes WPI R100-F3
Tetrodotoxin Abcam ab120054 TTX
iGluu plasmid Addgene 106122 pCI-syn-iGluu
Q175 mice Jackson Lab 27410 Z-Q175-KI
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Tags

Single SynapseGlutamate ReleaseGlutamate UptakeAcute Brain SlicesHuntington MiceOne-photon MicroscopeAutofluorescence CorrectionDorsal StriatumStimulation PipetteAction Potential Dependent ReleaseIon Channel BlockersMembrane ReceptorsTetrototoxin
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Dvorzhak, A., Grantyn, R. Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice. J. Vis. Exp. (157), e60113, doi:10.3791/60113 (2020).
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