Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing <em>Drosophila</em> Neurons

Chengyu Sheng; Uzma Javed; Justin Rosenthal; Jun Yin; Bo Qin; Quan Yuan

doi:10.3791/60287

초파리 뉴런 개발시 빠른 수지상 분지 역학의 시간 경과 라이브 이미징 및 정량화

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

초파리 뉴런 개발시 빠른 수지상 분지 역학의 시간 경과 라이브 이미징 및 정량화

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08:23 min

September 25, 2019

DOI: 10.3791/60287-v

Chengyu Sheng¹, Uzma Javed¹, Justin Rosenthal¹, Jun Yin¹, Bo Qin¹, Quan Yuan¹

¹National Institute of Neurological Disorders and Stroke,National Institutes of Health

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Overview

This study presents a method for imaging highly motile dendritic filopodia in live Drosophila larval brains. Utilizing time-lapse 3D imaging, the protocol quantifies branch dynamics of dendritic arbors in developing neurons, providing insights into dendrite morphogenesis regulated by development and activity.

Key Study Components

Area of Science

Neuroscience
Developmental Biology
Imaging Techniques

Background

Dendritic filopodia play a critical role in synaptogenesis and neural circuit development.
Understanding dendrite dynamics is essential for insights into neuronal plasticity and function.
Live imaging techniques enable real-time observation of neuronal changes during development.
Accurate quantification of dendritic dynamics can provide a better understanding of regulatory mechanisms.

Purpose of Study

To develop a robust method for tracking dendritic filopodia in vivo.
To assess the dynamics of branching in developing neurons.
To facilitate insights into how neuronal activity and development influence dendritic morphology.

Methods Used

Live time-lapse imaging of Drosophila larval brains.
3D image annotation software was utilized to track branch terminals.
Imaging parameters were optimized for temporal and spatial resolution during dissection.
Post-imaging analysis included drift correction, deconvolution, and manual annotation of branch tips.
Quantitative analysis involved calculating distances and movements of dendritic branch tips.

Main Results

The method successfully tracked the dynamic behaviors of dendritic filopodia in developing neurons.
Each dendritic branch's extension and retraction events were quantitatively assessed.
Image quality and tissue integrity were critical for accurate tracking and quantification.
This approach enables comparative analyses across different developmental stages.

Conclusions

This study provides a novel protocol for the quantitative analysis of dendritic filopodia dynamics.
The method enhances understanding of how development and neuronal activity regulate dendritic morphology.
Insights gained could inform research on synaptic formation and neuronal connectivity.

Frequently Asked Questions

What are the advantages of using live imaging in this study?

Live imaging allows for real-time observation of dynamic dendritic changes, offering unique insights that static methods cannot provide.

How are the Drosophila larval brains prepared for imaging?

Brains are dissected under a microscope, ensuring minimal movement and preserving the integrity of the tissue during transfer to a glass slide chamber.

What types of data are obtained using this imaging method?

This method produces quantitative data on dendritic branch movements, including extension and retraction events, at various developmental stages.

Can this technique be adapted for other models?

Yes, the methods described can be applied to various models of sparsely labeled neurons in both in vitro and in vivo experiments.

What limitations should be considered when using this protocol?

Careful optimization of imaging settings is crucial; any deviation can lead to poor data quality and inaccurate assessment of dendritic dynamics.

What is the significance of deconvolution in the analysis?

Deconvolution enhances image clarity, which is essential for accurately identifying and tracking dendritic branches in the dataset.

How does this study contribute to understanding neuronal development?

By quantifying dendritic dynamics, it provides insights into the molecular mechanisms underlying dendrite morphogenesis and synaptic development.

여기에서는 Drosophila 애벌레 뇌의 실시간 준비에서 매우 운동성 수지상 필로포디아를 이미지화하기 위해 사용한 방법과 수상돌기의 정량적 평가를 위해 시간 경과 3D 이미징 데이터 세트를 정량화하기 위해 개발한 프로토콜을 설명합니다. 신경 세포 개발에 역학.

이 프로토콜은 수지상 식목의 분기 역학을 정량화하고 라이브 타임 랩스 이미징 및 3D 이미지 기여 소프트웨어를 사용하여 뉴런을 개발하는 새로운 접근 방식을 제공합니다. 이 기술은 수지상 filopodia의 동적 동작을 측정하기 위한 빠르고 효율적인 방법을 제공하는 분기 단자 이미지를 추적하고 추적합니다. 이 방법을 이용한 연구는 개발 및 활동이 어떻게 말단 형태 발생을 조절하는지 이해하는 통찰력을 제공합니다.

우리의 방법은 시험관 내 및 생체 내 설정 모두에서 드물게 표지 된 뉴런에 적용 할 수 있습니다. 이 절차를 수행할 때 충분한 시간적 및 공간 해상도를 달성하기 위해 이미징 매개 변수를 신중하게 조정해야 합니다. 애벌레 drosophila에서 두뇌를 수확하기 위해, 해부 현미경에서 1 쌍의 표준 팁 해부 집게를 사용하여 다른 한 쌍을 조심스럽게 뇌를 해부하는 동안 애벌레 표본을 제자리에 고정하고 눈 디스크, 뇌 엽 및 복부 신경 코드를 보존하십시오.

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