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JoVE Journal
Neuroscience
斑马鱼癫痫模型中的行为和生理分析
斑马鱼癫痫模型中的行为和生理分析
JoVE Journal
Neuroscience
This content is Free Access.
JoVE Journal Neuroscience
Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

斑马鱼癫痫模型中的行为和生理分析

Full Text
6,290 Views
08:26 min
October 19, 2021

DOI: 10.3791/58837-v

Hortense de Calbiac*1,2, Adriana Dabacan*3, Raul Muresan3, Edor Kabashi1,2, Sorana Ciura1,2

1University Paris Descartes Hospital Necker-Enfants Malades,Institut Imagine, 2Institut du Cerveau et de la Moelle épinière - ICM,Sorbonne Universités Paris, 3Transylvanian Institute of Neuroscience (TINS)

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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 developing and characterizing a zebrafish model of epilepsy through the transient inhibition of the DEPDC5 gene. Utilizing both behavioral and physiological evaluations, the model allows for the examination of epilepsy phenotypes at various developmental stages.

Key Study Components

Area of Science

  • Neuroscience
  • Developmental Biology
  • Genetics

Background

  • Zebrafish are a valuable model for studying neurological disorders due to their transparent embryos and rapid development.
  • Knockdown of the DEPDC5 gene is associated with focal epilepsy, making it a suitable target for this investigation.
  • Behavioral assays and electrophysiological recordings provide comprehensive insights into the epilepsy phenotype.

Purpose of Study

  • To evaluate the effects of DEPDC5 knockdown on seizure-like behaviors in zebrafish.
  • To characterize physiological changes associated with epilepsy using innovative techniques.
  • To establish a foundation for future genetic and chemical modifier studies in epilepsy research.

Methods Used

  • The main platform involves the use of zebrafish embryos for the evaluation of the epilepsy phenotype through behavioral and electrophysiological methods.
  • The zebrafish model includes a transient knockdown of the DEPDC5 gene, allowing the assessment of changes in movement and neuronal activity.
  • Key experimental procedures include microinjections and recording of spontaneous movements and neuronal responses post fertilization.
  • Specific timelines include arranging mating tanks for egg collection one day prior to injection and various assessments at 28 hours and 46 hours post fertilization.
  • Electrophysiological analysis is performed using a patch clamp setup to measure neuronal activity changes.

Main Results

  • The study finds that DEPDC5 knockdown leads to increased spontaneous movements and altered neuronal activity in zebrafish.
  • Notably, experimental models demonstrate a higher occurrence of depolarization events post pentylenetetrazole application, indicating changes in excitability.
  • These results support the notion that DEPDC5 plays a crucial role in regulating seizure-like activity in a developmental context.

Conclusions

  • The study establishes a zebrafish model for understanding the mechanisms underlying epilepsy associated with DEPDC5 inhibition.
  • This model enables further investigations of genetic and pharmacological targets for epilepsy therapies.
  • The findings contribute to a broader understanding of neuronal mechanisms related to seizure disorders.

Frequently Asked Questions

What are the advantages of using zebrafish for epilepsy research?
Zebrafish provide a transparent model system that allows for real-time observation of developmental processes and neurobehavioral assessments, facilitating comprehension of epilepsy mechanisms.
How is the DEPDC5 gene targeted in the zebrafish model?
The DEPDC5 gene is transiently knocked down using microinjections of specific morpholinos, which suppress gene expression and facilitate the study of resulting phenotypic changes.
What types of data are obtained from this protocol?
The protocol yields behavioral data on movement and electrophysiological data through patch clamp recordings, providing insights into neuronal activity and excitability.
Can this method be adapted for other genetic studies?
Yes, this zebrafish model protocol can be adapted for various genetic manipulations, allowing researchers to explore other genes implicated in neurological disorders.
What limitations should be considered with this method?
Limitations include potential non-specific effects of morpholino treatments and the need for careful dose-response evaluations to mitigate toxicity.
How do the recorded changes in neuronal activity contribute to epilepsy understanding?
The changes in neuronal activity recorded during the experiments provide critical insights into the excitatory and inhibitory balance that may disrupt in epilepsy, enhancing understanding of seizure mechanisms.

在这里,我们提出了一个协议,开发和定性斑马鱼模型的癫痫,由于 DEPDC5 基因的暂时抑制。

该协议为评估WC5基因的撞击效果提供了一种快速方法,WC5基因是焦点癫痫的最常见病因。这项技术的主要优点是,我们可以用它来评估癫痫,就像表型在不同的发展阶段使用行为和生理特征一样。在微注射的前一天,设置斑马鱼交配池。

在注射的早晨,取出分隔器,使产卵。使用细筛将卵子转移到100毫米的培养皿中,里面装满了胚胎水。使用塑料巴斯德管道,挑选60至80个鸡蛋,并将鸡蛋排列在硅胶涂层的培养皿中进行注射。

我们移动了大部分的水, 留下足够的钱把鸡蛋盖到一半。将玻璃针垂直放入注射液管中。允许彩色溶液在几分钟内通过毛细管作用填充管子。

当针尖可见注射液时,将针头安装到微型喷油器的注射手柄上,然后打开空气压缩机。调整压力设置以生成两纳米注射体积。并将鸡蛋置于解剖双目显微镜下,放大四倍。

将针尖插入单阶段胚胎的胆汁和蛋黄中,将溶液直接注射到每个细胞内。然后将注射的胚胎转移到标有100毫米的培养皿胚胎水中,并将盘子放在28摄氏度的孵化器中。施肥后28小时,在试盘底部放置一个塑料1.2乘1.2毫米网格。

使用塑料巴斯德移液器将10至12个胚胎放在塑料网状物上。在盘子中装满足够的胚胎水,使胚胎保持浸入水中,但不会漂浮,在必要时用塑料尖端小心地将胚胎移入网格上的位置。然后,使用连接到解剖显微镜的摄像机,记录自发的盘绕活动 10 到 20 分钟。

要分析整个自发运动,请使用 Zebra 实验室系统中的活动定量模块上传录制的视频,并酌情设计每个胚胎周围的跟踪竞技场。将冻结和爆裂阈值分别设置为 10 和 50。当自动视频分析量化每个定义的竞技场内的总活动时。

然后使用适当的数据分析软件将数据集恢复为电子表格进行分析。受精后46小时,使用细钳将胚胎脱钩,并用胚胎水填充130毫米的试验盘。休息前至少15分钟,在28摄氏度的孵化器中加热试盘。

要执行触摸唤起逃生响应测试,请使用塑料巴斯德移液器将胚胎放在测试盘的中心,放在摄像机下。使用每秒 30 帧的采集速率开始录制。使用精细的塑料尖端,用轻弹运动稍微触摸胚胎的尾部。

停止录音,当幼虫已经终止其运动。然后,将胚胎转移到充满新鲜胚胎水的新保鲜盘中,并重复每个实验条件所需的尽可能多的胚胎测试。要准备斑马鱼进行电生理分析,将受精后的一、四至六天放入玻璃底培养皿中。

去除任何多余的,额外的水手介质,以确保鱼将尽可能接近菜的底部。使用塑料巴氏杆移液器添加足够的温暖液体农业覆盖幼虫。当农用硬化物时,使用细钳将鱼腹侧向下定向在菜的中心。

然后加入两毫升的录音溶液,其中含有10微摩尔溴化物到盘子中,以阻止神经肌肉传播。接下来,用记录溶液填充微吐机,并在电压夹配置中使用贴片夹放大器,测量浴缸中的电极电阻,以确认其正确值。使用 20 倍目标,将幼虫的头部放置在中央视野中,并降低微管以达到光学结肠内的记录位置。

将贴片夹放大器切换到当前夹具配置,并将保持电流固定为零毫安。使用一千赫的低通滤波器和一千赫的采集率以及 10 倍的数字增益,记录鱼的自发活动 60 分钟,以确定基线活动水平。在基线记录结束时,在143微升每升五角星溶液的微升下洗澡,最终浓度为每升20毫摩尔。

用五角形溶液记录动物的神经元活动,再记录120分钟。在记录的基线期间,4至6天后受精癫痫模型斑马鱼表现出较高的自发事件发生,而不匹配控制鱼显示很少的波动。在五角形拉佐尔应用后,不匹配的控制和癫痫模型斑马鱼表现出越来越多的深极化事件。

在五角星应用后的第一个期间,在不匹配控制和击倒动物时,观察到每分钟 0.8 个事件的速率,而大多数事件的振幅都很高。在后一个响应期间。去极化事件的速度增加到每分钟一个事件左右。

并且大多数事件的振幅都很低。在执行击倒时,使用剂量响应曲线确定正确的莫福利诺斯剂量非常重要,并执行控制以避免非特定毒性。这一推动使一些下游研究,包括测试遗传或化学修饰剂的影响。

和斑马鱼的行为和神经活动,以了解DC五突变的发展影响。

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