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Neuroscience
前体钙成像用于可视化大脑对果蝇内分泌信号的反应
前体钙成像用于可视化大脑对果蝇内分泌信号的反应
JoVE Journal
Neuroscience
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JoVE Journal Neuroscience
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

Full Text
9,773 Views
06:49 min
June 2, 2018

DOI: 10.3791/57701-v

Hiroshi Ishimoto1, Hiroko Sano2

1Division of Biological Science, Graduate School of Science,Nagoya University, 2Department of Molecular Genetics, Institute of Life Science,Kurume University

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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 ex vivo calcium imaging of the Drosophila brain, aiming to explore neuronal responses to endocrine signals. The method allows for the testing of natural or synthetic compounds to activate specific neurons, providing insights into the intersections of endocrinology and neuroscience.

Key Study Components

Area of Science

  • Neuroscience
  • Endocrinology
  • Neurobiology of Drosophila

Background

  • The study investigates the direct effects of endocrine signals on the brain.
  • Utilizes Drosophila to provide a model for understanding hormonal networks.
  • Focuses on how specific peptides can influence neuronal activation.
  • Established the groundwork for further research on receptor specificity.

Purpose of Study

  • To develop a protocol that allows for the assessment of brain responses to endocrine signals.
  • To enable researchers to examine neuronal activity separate from other tissues.
  • To facilitate screening of compounds that may influence neuronal function.

Methods Used

  • The method involves dissection and imaging of the Drosophila larval brain using calcium indicators.
  • Larvae are collected and prepared for imaging approximately 90-120 hours after egg laying.
  • Calcium fluorescence imaging parameters are established, allowing for detailed observation of neuronal activity.
  • Application of test peptides enables measurement of the brain's responsive signals over time.

Main Results

  • Successful imaging of GCaMP6 signals reveals neuronal activation in response to various peptides.
  • Statistical analysis incorporates baseline signal intensity to evaluate neuronal responses post-peptide application.
  • The findings confirm the usefulness of this technique for exploring hormonal control of brain function.

Conclusions

  • This study demonstrates a valuable protocol for investigating endocrine influence on neuronal activity in Drosophila.
  • It enables exploration of receptor specificity and hormonal networks in neuroscience research.
  • The implications extend to understanding plasticity and mechanisms underlying brain function and responses.

Frequently Asked Questions

What are the advantages of using Drosophila for calcium imaging?
Drosophila offers a simplified model system where genetic modifications can easily be made, allowing for targeted studies on neuronal functions and responses to hormones.
How is the larval brain prepared for imaging?
The larval brain is dissected using fine forceps to separate it from other tissues, then mounted in an imaging chamber for calcium fluorescence assessment.
What outcomes can be obtained from this imaging method?
The method provides real-time data on neuronal calcium signals, enabling insights into neuronal activation and response to hormonal signals.
How long does it take to perform the entire procedure?
With proper preparation, the technique can be completed in about one hour, making it efficient for screening experiments.
What considerations should be taken into account when preparing the brain sample?
It is crucial to prepare the brain sample quickly and handle it gently to avoid any damage during dissection, which can affect imaging results.
Can this protocol be adapted for other types of experiments?
Yes, the protocol can be combined with genetic manipulations to explore different receptor types and their specific roles in neuronal signaling.

本文介绍了 果蝇脑的体外钙成像协议。在这种方法中, 自然或合成化合物可以应用到缓冲, 以测试他们的能力, 激活特定神经元的大脑。

这种方法可以帮助回答内分泌学领域以及神经科学领域的关键问题,例如我们审查大脑对内分泌信号反应的研究。这种技术的主要优点是您可以检查内分泌信号对与其他组织分离的大脑的直接影响。要开始此过程,请使用镊子刮擦塑料培养皿的底部中心,以创建一个凹痕,以便在解剖后更容易安装幼虫脑的腹侧神经节。

用牙签在凹痕的两侧滴一小滴强力胶,然后将钨棒连接到胶水上。然后,使用一小块类似油灰的可重复使用粘合剂,在凹痕周围制作一堵圆形墙。为了解剖幼虫的大脑,在产卵后 90 到 120 小时收集幼虫并用蒸馏水清洗至少 3 次,以去除粘附的食物残渣。

接下来,将幼虫放在装满冰冷 PBS 的一英寸半方形手表玻璃杯上。用一把镊子轻轻抓住幼虫的中间部分。用另一对镊子轻轻抓住并拉动口钩,将包含大脑的幼虫前部与身体其他部分分开。

然后,用镊子握住前尖端,将幼虫翻过来。去除附着在大脑上的外来组织,例如假想盘、脂肪体和环腺。轻轻地将大脑与口器分开。

之后,将 200 微升 PBS 涂抹在成像室中较早制备的粘合环内侧。用 PBS 轻轻地将解剖的大脑吸入巴斯德移液器中,然后将其转移到成像室中。在成像室中,抓住从腹侧神经节伸出的肌肉纤维,将大脑轻轻插入钨丝下方的凹痕中。

然后,将钨丝稍微向上拉,将大脑置于正确的成像位置。要获取钙荧光图像,请将包含脑外植体的成像室置于显微镜下。降低物镜直到它接触到 PBS,然后在明场照明下定位大脑并使其聚焦。

切换到荧光灯并调整 GCaMP 成功标记细胞的焦点。在水冷模式下,以 250 毫秒/帧的分辨率和 512 x 512 像素的分辨率开始采集。然后,调整曝光时间以获得 CCD 相机动态范围内的荧光值,但不要低于 1000 任意单位,16 位图像。

确定成像参数后,在肽给药前拍摄图像 1 分钟以检测基线信号强度。随后,将 100 微升制备的肽溶液移液到幼虫浴中,直接施用测试肽。记录 GCaMP 成功发出几分钟。

要分析数据,请打开分析软件并使用肽应用之前的第一帧作为参考图像。然后,依次选择 Plugins(插件)、TurboReg(TurboReg)。选择串行映像文件作为 Source (源),选择参考映像作为 Target (目标)。

接下来,检查 刚体 和 精确 分别了解加工方法和质量。随后,单击 Batch 开始图像处理。使用图像托盘中的 Analyze、Tools、ROI Manager 选择多个感兴趣的区域。

然后,通过在 ROI Manager 窗口中单击 More (更多)、Multi Measure (多重测量)、OK (确定) 来测量像素强度。在本实验中,野生型脑暴露于 CCHa2 、 Ghrelin 和 Nociceptin ,而 CCHa2 受体突变型脑暴露于 CCHa2。通过共聚焦显微镜以 250 ms/帧的速度检测胰岛素产生细胞中的 GCaMP6 信号,以下是选定时间点的静止图像。

绘制每个时间点 ROI 强度与基线信号强度变化的比率。ROI 设置在同一焦平面中检测到的细胞体上。实线表示 5 到 10 个样本的平均值,虚线标记平均值标准误差的上限和下限。

阴影区域表示实验中信号的变化。一旦掌握,如果作得当,这项技术可以在一小时内完成。在尝试此程序时,请务必记住快速准备脑样本,以免对其造成损害。

该程序可以与遗传学相结合,以回答其他问题,例如受体特异性。该协议中使用的系统易于准备和重复使用。因此,该协议将很有用,例如在筛选中。

开发后,这项技术为内分泌学和神经科学领域的研究人员探索控制果蝇大脑功能的激素网络铺平了道路。

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