Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Anton Dvorzhak; Rosemarie Grantyn

doi:10.3791/60113

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

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

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08:27 min

March 11, 2020

DOI: 10.3791/60113-v

Anton Dvorzhak¹, Rosemarie Grantyn¹

¹Synaptic Dysfunction Group, Neuroscience Research Center,Charité - University Medicine

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a protocol for evaluating the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses using acute slices from adult mice. By employing the fluorescent sensor iGlu u for glutamate detection, researchers can identify local mismatches in transmitter dynamics, aiding in the investigation of dysfunctional synapses in disease contexts.

Key Study Components

Area of Science

Neuroscience
Neuropharmacology
Synaptic physiology

Background

Release and clearance of glutamate are crucial for neurotransmission.
Imaging techniques can identify disruptions in neurotransmitter dynamics.
Pathological conditions may alter synaptic function.
Assessing these changes can provide insights into diseases such as Huntington's disease.

Purpose of Study

To develop a method for assessing synaptic glutamate dynamics.
To investigate how synapses respond to stimulation under different conditions.
To identify functional impairments in synapses associated with neurological disorders.

Methods Used

Ex vivo brain slices from adult mice were used for imaging studies.
Single corticostriatal glutamatergic synapses were the primary focus of investigation.
The fluorescence sensor iGlu u was employed for glutamate detection.
A two-photon microscope setup enabled high-resolution imaging and stimulation.
Custom protocols were followed for precise experimental conditions including electrical stimulation and fluorescence measurement.

Main Results

The protocol allowed for detection of glutamate release and clearance at the level of single synapses.
Differences in glutamate dynamics were observed between wild-type and mutant mice.
Specific decay parameters provided insights into synaptic function and potential dysfunction in Huntington's disease models.
Characterization of synaptic responses revealed differential behaviors in glutamate release.

Conclusions

This study enables detailed assessment of neurotransmitter dynamics at individual synapses, contributing to our understanding of synaptic dysfunction in neurological diseases.
The findings may aid in the identification of targets for therapeutic interventions.
Overall, the method enhances our understanding of glutamatergic transmission mechanisms and their plasticity in health and disease.

Frequently Asked Questions

What are the advantages of using acute brain slices?

Acute brain slices preserve the native environment of synapses, allowing for more accurate assessment of synaptic function and dynamics.

How is the glutamate sensor utilized in this method?

The fluorescent sensor iGlu u specifically detects glutamate release, enabling researchers to visualize and quantify neurotransmitter levels at synapses.

What types of outcomes can be measured with this protocol?

Outcomes include real-time measurements of glutamate release and clearance, as well as insights into synaptic dynamics and potential dysfunction in pathological conditions.

How can this method be adapted for other neurotransmitters?

This imaging protocol could potentially be modified to incorporate sensors specific to other neurotransmitters, allowing for broader applications in synaptic research.

What are the limitations of this imaging technique?

One limitation is the requirement for precise experimental conditions, which can be challenging to maintain, particularly in live slice preparations.

How can findings from this study be applied to neurological disorders?

By identifying dysfunction in glutamate dynamics, this research could lead to the development of targeted treatments for disorders such as Huntington's disease.

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iGlu_u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。

单突触的高分辨率成像表示快速谷氨酸传感器，可检测发射机释放和吸收之间的局部不匹配。在疾病的情况下，此方法可用于识别功能失调的突触。对于自动荧光校正，首先，将感兴趣的小鼠的脑片放入单光子显微镜的录音室。

将切片浸入氧化的人工脑脊液中，并使用 20 倍水浸目标定位后体线状体。在铂琴上用尼龙网格固定切片，以最大限度地减少组织运动，并切换到 63 倍水浸目标。使用 510 纳米的高通滤波器，一起获取自动荧光和谷氨酸传感器正结构的图像。

使用 600 纳米的高通滤波器，单独获取自动荧光结构的第二个图像。要定义范围，请使用 10 个最亮像素和 10 个最暗像素的均值强度缩放红色和黄色图像。然后，对黄色减去红色图像进行减法，重新缩放减去的图像，以生成标准 8 位 TIFF 文件，以方便地可视化感兴趣的回合。

要搜索响应灵敏的电管，您需要一个合适的玻璃微管进行电刺激。使用微移管拉拔器从内尖直径约一微米的硅酸盐玻璃毛细管中产生刺激移液器。要从一组候选的电毒中寻找谷氨酸的操作潜在依赖释放，请选择 63 倍放大倍率和 510 纳米发射过滤器。

加载减除的图像，以便将玻璃刺激电极放置在荧光度旁边，避免靠近额外的轴子。在什么变种与最大分叉或分配在切片的更深部分。将刺激电极放在感兴趣的电位附近并关闭灯，因为录音必须在完全黑暗中进行。

然后，打开多通道浴池应用系统，一个通道提供标准沐浴解决方案，其他通道提供离子通道、运输器或膜受体（包括四恶英）的必要阻滞剂，以阻止作用潜在的生成。控制记录现场的流量，打开刺激器，向刺激移液器提供2至10微安的去极化电流脉冲。释放现在通过直接钙通过电压门控通道流入激活。

要在间隙中可视化谷氨酸释放，使用显微镜 X、Y 驱动器，将测试的谷氨酸传感器阳性 bouton 置于视场中心附近。停止采集后，单击鼠标左键的图像以确定休息的回合中心的 X、Y 位置。将显示设置光标的 X、Y 坐标。

使用校准数据，使用指示的公式计算应发送激光束以激发谷氨酸传感器荧光的站点的坐标。若要在激光控制软件中创建一个点序列，请在激光控制软件序列页面上的"添加到序列"框中选择点，并将运行和运行延迟设置为零，并在 TLL 上运行序列。然后，单击"开始"序列。

在摄像机控制软件中，选择适当的成像参数，然后为触发模式选择外部启动。单击相机控制软件中的接收信号。然后，启动为触发装置制定的实验协议，并在适当的时间轴上实现实验协议试验，使摄像机在一次试验中获得400帧，频率为2.48千赫，重复频率为0.1赫兹或更低。

要识别病理突触，请打开高程例程并计算所选感兴趣区域的荧光强度、平均值和标准偏差。确定和装箱由荧光强度大于平均值加上三个标准差的像素占用的区域，并假设以超阈值区域的圆形形式以微米为单位的虚拟直径。根据时间绘制荧光强度，因为实际荧光强度值与休息时荧光强度值之间的差值除以余点荧光强度值。

确定荧光响应的峰值振幅。对荧光响应峰值的衰变执行单一远位拟合，并确定衰减的时间常数 TauD。要估计给定突触的最大振幅，请选择荧光强度变化最大的像素，这是向突触的间隙机械呈现的谷氨酸负载的最佳指标。

单突触成像可用于使用大小和配对脉冲比标准识别两类皮质突触。在20至50毫秒的刺激间隔下，较小的肠内间终端容易成对脉冲凹陷，而较大的金字塔路端子则显示配对脉冲促进。对表达亨廷顿表型的野生型小鼠和小鼠进行的运动行为测试，揭示了在开放领域中运行的总路径结果与延迟步骤之间的显著正相关关系。

此外，单突触谷氨酸成像显示，有症状的亨廷顿舞蹈鼠表现出的朱克斯塔辛帕氏谷氨酸衰变速度的不足，反映在谷氨酸对单突触刺激反应的TauD值中。在野生类动物中，只有在使用选择性的、不可运输的谷氨酸吸收抑制剂后，才观察到这种延长。对野生型小鼠切片中给定 TauD 值的发生概率的评估，以及表达亨廷顿舞蹈症症状的小鼠，发现在野生型动物中，TauD 的发生概率永远不会超过 15 毫秒。

然而，在症状性亨廷顿舞蹈症中，40%的突触在16至58毫秒之间表现出TauD值，尽管释放的谷氨酸的量有减少的趋势。因此，TauD可能被视为亨廷顿舞蹈症中功能失调突触的生物标志物，并可能进一步用于验证针对星体谷氨酸运输的实验中的功能恢复。这种在单个皮质突触中谷氨酸监测的协议可能有助于澄清谷氨酸吸收缺乏在神经退行性疾病发病机制中的作用。

单突触成像对于探索兴奋突触的突触前位点特别有用。

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