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

Imágenes en vivo de lapso de tiempo y cuantificación de la dinámica de ramas dendríticas rápidas ...

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JoVE Journal Neuroscience

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

Imágenes en vivo de lapso de tiempo y cuantificación de la dinámica de ramas dendríticas rápidas en el desarrollo de neuronas drosophila

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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.

Aquí, describimos el método que empleamos para crear imágenes de filopodia dendrítica altamente móviles en una preparación en vivo del cerebro larvario Drosophila, y el protocolo que desarrollamos para cuantificar conjuntos de datos de imágenes 3D de lapso de tiempo para evaluaciones cuantitativas de la dendrita dinámica en el desarrollo de neuronas.

Este protocolo proporciona un nuevo enfoque para cuantificar la dinámica de las ramas del árbol dendrítico y desarrollar neuronas utilizando imágenes de lapso de tiempo en vivo y un software de anotación de imágenes 3D. Esta técnica realiza imágenes y seguimientos de los terminales de bifurcación proporcionando un método rápido y eficiente para medir los comportamientos dinámicos de la filopodia dendrítica. La investigación con este método proporciona información sobre cómo el desarrollo y la actividad regulan la morfogénesis de dendrita.

Nuestros métodos son aplicables a las neuronas escasamente etiquetadas en entornos in vitro e in vivo. Al realizar este procedimiento, recuerde que los parámetros de imagen deben ajustarse cuidadosamente para lograr una resolución temporal y espacial suficiente. Para cosechar cerebros de escoria larvaria, bajo un microscopio diseccionado utilice un par de fórceps de disección de punta estándar número cinco para mantener un espécimen larval en su lugar mientras usa el otro para diseccionar cuidadosamente el cerebro, preservando los discos oculares, los lóbulos cerebrales y el cordón nervioso ventral.

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