Behavioral Analysis of Locomotor Dysfunction in <em>Drosophila melanogaster</em> as a Readout for Neurotoxicity

Zuzanna Tomkielska; Jorge Frias; Nelson Sim&#245;es; Ana Casas; Duarte Toubarro

doi:10.3791/68517

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Behavioral Analysis of Locomotor Dysfunction in Drosophila melanogaster as a Readout for Neurotoxicity

黑腹果蝇运动功能障碍的行为分析作为神经毒性读数

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07:03 min

July 18, 2025

DOI: 10.3791/68517-v

Zuzanna Tomkielska^1,2, Jorge Frias¹, Nelson Simões¹, Ana Casas², Duarte Toubarro¹

¹Center of Biotechnology of Azores (CBA),University of the Azores, ²Mesosystem Investigação & Investimentos by Spinpark

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Overview

This study presents a high-throughput and cost-effective method for screening neurotoxic compounds using Drosophila melanogaster as an alternative to traditional mammalian models. The protocol assesses neurotoxicity by quantifying locomotor dysfunction, which aids in evaluating the effects of various agents.

Key Study Components

Area of Science

Neurotoxicology
Behavioral Analysis
Model Organisms

Background

Traditional toxicology relies on mammalian models, which are often costly and time-consuming.
Ethical concerns exist regarding the use of these models for neurotoxicity testing.
Drosophila melanogaster provides a sensitive and scalable alternative.
The protocol combines a climbing assay with real-time monitoring for improved sensitivity.

Purpose of Study

To develop a method for detecting early signs of neurotoxicity.
To evaluate the effects of pharmaceutical compounds, environmental agents, and genetic modifications.
To explore locomotor behaviors and quantify motor impairments.

Methods Used

The study utilizes Drosophila melanogaster as a biological model for screening neurotoxic compounds.
Flies are anesthetized and tested for locomotor activity using a climbing assay and video analysis.
The experimental setup includes feeding microcapillaries for controlled dosing and real-time monitoring technology.
Key timelines involve assessing behavior at intervals of 24 and 48 hours post-exposure.
Behavioral phenotypes are quantified by measuring the time taken to reach a specified climbing target.

Main Results

Treated flies showed delayed climbing times and significant motor impairment compared to controls after 48 hours.
Initial hyperactivity was followed by reduced movement, indicating complex behavioral responses to neurotoxic exposure.
Continuous activity tracking revealed progressive loss of rhythmicity in treated flies, related to locomotor deficits.
The study suggests that early signs of neurotoxicity can be detected in this model.

Conclusions

This study demonstrates an effective method for uncovering neurotoxic effects in a cost-efficient and ethical manner.
The approach enables detailed behavioral analysis and early detection of neurotoxic compounds.
Findings contribute to understanding locomotor dysfunctions and neurotoxicity in simplified models.

Frequently Asked Questions

What are the advantages of using Drosophila melanogaster in neurotoxicology?

Drosophila melanogaster offers a cost-effective and ethical alternative to mammalian models. It allows researchers to detect subtle behavioral changes and assess neurotoxic effects in a highly controlled environment.

How is neurotoxicity assessed in the study?

Neurotoxicity is assessed through a climbing assay combined with video analysis to monitor locomotor activity and quantify any impairments over time.

What types of outcomes can be obtained from this method?

The method provides outcomes related to locomotor dysfunction, including changes in climbing behavior, activity levels, and behavioral phenotypes indicative of neurotoxicity.

What limitations should be considered with this model?

While Drosophila offers many advantages, results may not fully extrapolate to mammalian systems, and behavioral assays might miss complex neurotoxic effects observed in higher organisms.

Can this method be adapted for other studies?

Yes, the protocol can be adapted to assess various compounds and genetic modifications by altering exposure conditions and monitoring different behavioral outcomes.

该方案提出了一种高通量、经济高效的方法，通过量化运动功能障碍作为传统哺乳动物模型的替代方案来评估果蝇的神经毒性。它旨在使用敏感、可重复且道德上有利的模型评估药物化合物、环境因素或基因改造的神经毒性作用。

我们研究的目标是开发一种具有成本效益且符合道德标准的神经毒性化合物筛选方法。毒理学测试的传统方法通常依赖于虚拟速率模型，正如我们所知，这些模型既昂贵又耗时，否则存在伦理问题。我们想探索是否可以使用既灵敏又可扩展的黑腹果蝇替代模型来检测神经毒性的早期迹象。该协议帮助我们检测行为表型，例如运动模式改变和活动，以及进行性运动衰退或安全书面中断，并且还测试了广泛的偏好。

我们的协议优势在于，它将经典的攀爬测定与实时监测技术相结合，这使我们能够捕获可能被遗漏的细微运动障碍。通过传统方法提供更高的灵敏度和更精确的行为数据。

[旁白]首先，轻轻地将苍蝇转移到空瓶子中。将装有苍蝇的瓶子放入装满冰块的盒子中，确保整个瓶子都被覆盖。一分钟后，轻轻敲击瓶子，确认所有苍蝇都已麻醉。准备一个装满冰块的小容器。将培养皿或平坦干净的表面放在冰上，形成一个冷藏平台。现在将管子倒置在冷冻的培养皿上，轻轻敲击以将苍蝇释放到冰冷的表面上。小心地将苍蝇转移到空小瓶中。让苍蝇在25摄氏度的受控环境中从麻醉中恢复30分钟，12小时的明暗循环和50%的湿度。接下来，对于微毛细管进料制备，使用移液器手动将 10 微升测试溶液加载到每个微毛细管中，确保所有样品的体积一致。在每个微毛细管的开口端加入三到五微升矿物油，以防止实验过程中蒸发和泄漏。将预装微毛细管插入棉瓶塞上的孔中，确保牢固贴合。将装有苍蝇的小瓶放入设置为25摄氏度的培养箱中，并以12小时的明暗循环进行，并在实验期间将小瓶留在培养箱中。每 12 小时将苍蝇转移到干净的空小瓶中，以评估它们的运动活动。使用永久性记号笔测量并标记距小瓶底部七厘米的线。该标记将作为攀登目标。将相机或手机放置在稳定的位置以记录攀爬行为。确保适当的照明和透明背景，以实现清晰的可见性和分析。确认实验设置与相机的视野兼容 view 并开始录制。查看视频记录并测量每只苍蝇到达七厘米标记所需的时间。如前所示，用样品和矿物油封准备进料微毛细管。然后使用切成约六厘米长的透明塑料吸管准备单独的运动室。用透明薄膜密封每根吸管的一端，形成气密封口。在吸管的密封端附近，创建一个小孔以插入进料微毛细管，确保其紧密贴合，并且在测定过程中不会泄漏或移动。如前所述麻醉和分离苍蝇后，使用细刷将一只麻醉的苍蝇轻轻放入每个管中。用透明薄膜或棉塞密封每个管子的开口端，允许气流，同时防止逸出。现在通过指定的孔将预装的微毛细管插入每个准备好的腔室中。将组装好的试管放入测定场，确保微毛细管保持可访问性以供日常更换。将整个装置放置在保持在 25 摄氏度的腔室内，具有 12 小时的明暗循环和 50% 的湿度。将设备连接到本地跟踪系统或网络。访问软件平台并找到分配给实验的设备。确保系统根据视觉标记正确跟踪每只苍蝇。输入所有实验元数据并开始记录测定。在 24 小时和 48 小时使用负趋地测定法评估的攀爬能力显示，对照组和治疗组在 24 小时时表现相似，表明没有直接运动缺陷。到 48 小时时，经过治疗的苍蝇爬升所需的时间几乎是对照组的两倍，这反映了严重的运动障碍。持续的活动跟踪表明，经过治疗的苍蝇最初表现出高度运动，但它们的活动在 12 小时后急剧下降，这表明早期过度兴奋，随后运动功能下降。到 24 到 40 小时时，与对照组相比，经过治疗的苍蝇表现出减少的运动。总体而言，经过治疗的苍蝇在 40 小时内活动减少约 20%。经过治疗的苍蝇最初对光线的反应延迟，然后是短暂的昼夜节律对齐，但在 48 小时内逐渐失去节律性，这可能是由于机车缺陷的结果。经过治疗的苍蝇比对照更喜欢光区。

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