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Neuroscience
在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生
在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Full Text
9,655 Views
06:35 min
October 8, 2019

DOI: 10.3791/60288-v

Clare E. Hancock1, Florian Bilz1, André Fiala1

1Department of Molecular Neurobiology of Behavior,University of Göttingen

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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 visualizing pre- and postsynaptic calcium in Drosophila to investigate learning and memory. Employing in vivo calcium imaging with synaptically localized sensors, the research combines this with classical olfactory conditioning to explore synaptic plasticity associated with associative learning.

Key Study Components

Area of Science

  • Neuroscience
  • Behavioral Biology
  • Calcium Imaging

Background

  • Drosophila melanogaster serves as a model for studying learning and memory.
  • Understanding synaptic calcium activity can illuminate the physiological processes behind memory formation.
  • Associative learning is fundamental to how memories are encoded and retrieved.
  • The mushroom body in Drosophila is crucial for learning and memory functionalities.

Purpose of Study

  • To visualize calcium activity at synapses during learning processes.
  • To correlate synaptic activity with memory trace formation.
  • To utilize a classical conditioning paradigm to examine synaptic changes.

Methods Used

  • Calcium imaging is performed using genetically encoded calcium indicators in a custom imaging chamber.
  • Drosophila are prepared for imaging following surgical procedures to expose the brain region of interest.
  • The experiment involves a pre-conditioning baseline measurement followed by odor stimulus delivery and post-conditioning measurements.
  • The imaging system includes a multi-photon microscope tuned for specific excitation wavelengths and scanning protocols.
  • Data collection involves measuring calcium transients linked with odor stimuli before and after associative training.

Main Results

  • The study provides insights into synaptic changes associated with olfactory learning.
  • Differences in calcium responses were observed between various genetically altered flies, demonstrating the impact of specific indicators on synaptic activity.
  • Visualizations reveal how olfactory memory is stored and modulated at the cellular level.
  • Results underscore the utility of this method in understanding complex brain structures.

Conclusions

  • This protocol enables real-time visualization of calcium dynamics, contributing to our comprehension of neuronal mechanisms in memory formation.
  • Insights gained through this method can illuminate synaptic plasticity principles and enhance our understanding of learning processes.
  • The results have broad implications for neuroscience, particularly in the context of associative memory storage.

Frequently Asked Questions

What advantages does this imaging method offer?
This method provides real-time observation of calcium activity, allowing for direct correlational studies between synaptic responses and learning.
How are Drosophila prepared for imaging?
Drosophila undergo surgical procedures to expose brain areas and are fixed in an imaging chamber for optimal visualization.
What outcomes can be expected from this protocol?
Outcomes include detailed imaging of calcium transients and insights into synaptic plasticity during learning events.
Can this method be adapted for other models?
While designed for Drosophila, the principles of this imaging technique may be applicable to other model organisms with adaptations.
What are the limitations of this study?
Limitations may include the specificity of the calcium indicators used and potential variations in individual animal responses.

在这里,我们提出了一个协议,其中前和/或午睡钙可以在果蝇学习和记忆的上下文中可视化。体内钙成像使用显着局部钙传感器与经典的嗅觉调节范式相结合,因此可以确定这种类型的关联学习背后的突触可塑性。

该技术有助于实时观察突触钙活性,作为细胞过程的生理参数,在果蝇黑色素素中基础学习和记忆形成。通过将神经元反应与关联训练之前和之后的气味进行比较,我们可以得出突触活动与单个果蝇记忆痕迹的形成之间的直接相关性。生产转基因果蝇,其中感兴趣的特定神经元表示一个基因编码的钙指标交叉雌性处女和雄性苍蝇携带所需的GAL4和UAS构造,并年龄雌性后代,直到三到六天后,围闭。

要准备苍蝇成像,请使用细钳将冰麻醉苍蝇放入定制准备的成像室。使用解剖显微镜将胸部和腿部与腔室底部的电线接触,头部平躺。用透明胶带固定苍蝇,并使用手术刀刀片切割头部周围的胶带中的窗口,使天线被覆盖,只有胸部前部裸露。

使用凹凸钳口夹住的昆虫针,用蓝光固化胶小心地环绕头部的两侧和背面。并使用蓝色发光 LED 灯来设置胶水。当胶水完全固定时,清除飞头后面的任何残留未硬胶,用一滴林格溶液盖住头部的外露角质层。

使用非常细刀片的刺刀,切开穿过头部后部的角质层,从奥佩利开始,将两侧中切到眼睛,形成角质层皮瓣,使用钳子很容易撕掉。去除任何可能阻塞大脑区域的多余的角质层,并使用细钳小心地清除任何气管的后表面,注意避免脑组织本身的中断。必要时拆下并刷新林格溶液,以清除组织碎屑区域。

放置一个皮下气味输送针,距离苍蝇头部约一厘米,注意没有任何东西可以阻碍气味传递到天线。然后通过皮下气味输送针将成像室连接到气味输送系统。让苍蝇10分钟从麻醉和手术中恢复过来。

要可视化基于 GFP 的钙指标,请将装有红外激光和安装在振动隔离台上的多光子显微镜的激光调谐为 920 纳米的激发波长,并安装 GFP 带通滤波器。使用课程 Z 调整旋钮,扫描大脑的 Z 轴以定位感兴趣的大脑区域。使用裁剪功能将扫描重点放在感兴趣的区域上,以最大限度地减少扫描时间。

旋转扫描视图,使头部前部朝下。然后将帧大小调整为 512 x 512 像素,然后选择要扫描的区域。考虑每个帧的计算扫描时间,实现至少 4 赫兹的帧速率。

对于气味唤起钙瞬态可视化,启动能够连接图像采集软件和气味传递程序的预编程宏封装,并在显微镜软件中开始测量 6.25 秒,以建立 F0 基线值。在气味输送系统中,通过特定气味杯阀的打开和关闭触发的 LED 照明,提供此处指示的 2.5 秒气味刺激。接着是气味偏移结束时的 12.5 秒记录。

然后以相同方式重复第二和第三气味的交付。要在此设置中执行关联调节,请使用计算机控制的气味输送系统,在 12 90 伏电击旁边显示 60 秒的调节刺激加气味。休息60秒后,仅出现60秒的调节刺激-减气味,无电击。

在完成训练阶段后三分钟重复训练前气味刺激方案,测量训练后气味再次引起钙的转性。然后以适当的格式保存映像文件,以进行以后的图像分析。这里具有代表性的蘑菇体输出神经元图像,获得的证明是可以观察到的。

dHomer-GCaMP3 传感器的特定分节表达(在神经元的斧弓室中未表达)显示了树突隔间中的点分信号。与表达细胞酸的苍蝇GCaMP6f相比,在表达dHomer-GCaMp3的苍蝇中可以观察到低振幅气味反应。在这里,单个苍蝇的代表性钙痕迹显示了单个制剂之间可获得的噪声水平和振幅的方差。

这种技术开辟了在亚细胞水平上可视化的可能性,嗅觉表示如何调制,记忆相如何通过调节形成。这些实验对于扩大我们对蘑菇体等复杂大脑结构的理解,以及描述关联记忆如何储存在分散的神经元群体中至关重要。

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神经科学 问题 152 果蝇黑色素 蘑菇体 突触可塑性 学习和记忆 嗅觉关联学习 经典调理 光学钙成像 双光子显微镜 记忆

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