Name: time-lapse live imaging and quantification of fast dendritic branch dynamics in developing drosophila neurons
Uploaded: 2019-09-25T15:00:00
Description: Watch this Scientific Journal Video about Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons at JoVE.com

This work is supported by the Intramural Research Program of National Institute of Neurological Disorders and Stroke, National Institutes of Health. Project number 1ZIANS003137.

CAUTION: This protocol involves the use of class IV lasers and will require proper training and safety guidelines to be followed. Avoid eye or skin exposure to direct or scattered laser light. 
NOTE: The protocol includes six steps. The workflow is shown in Figure 1A.
1. Labeling Individual Neurons Using the Flip-out Technique
NOTE: The single labeling of LNvs is ach...

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Here, we describe a protocol we developed to record and quantify the dynamic behavior of dendritic branches in individually labeled neurons in Drosophila larval brains. Notably, our live imaging protocol contains specific parameters that enable us to capture the whole dendritic arbor of a larval LNv and provide a global view of the dynamic state of its dendrite branches. However, because our quantification methods heavily rely on the annotation of branch terminals, neurons with complex dendritic structures and c...

Dendrites are specialized neuronal compartments that receive and process sensory and synaptic input. The complex and stereotyped structure of dendritic arbors has been under intense investigation since their discovery. A number of model systems, including Xenopus optic tectal neurons, chick retinal ganglion cells, and dendritic arborization (da) neurons in the Drosophila system, have been established to study the development, remodeling and plasticity of neuronal dendrites1,2,3,4. Drosophila ventral lateral neurons (LNvs) are a group of visual projection neurons initially identified for their important functions in circadian regulation of fly behaviors5. Studies also revealed the role of larval LNvs as the direct postsynaptic target of the larval photoreceptors (PRs)6,7. Importantly, culturing developing larvae in different light regimes strongly affects the size of LNvs&#39; dendritic arbors, demonstrating the suitability of LNvs as a new model for studying dendritic plasticity7. Recent work from our group further indicates that both the size of the LNv dendrite and the dynamic behavior of the dendritic branches display experience-dependent plasticity8,9. As part of this work, we developed a new live imaging and quantification protocol to perform analysis on the dendrite dynamics of LNvs from 2nd or 3rd instar larvae.
The transparent nature of the Drosophila larval brain makes it ideal for live imaging. However, the dendritic arbors of LNvs are situated in the densely innervated larval optic neuropil (LON) in the center of the larval brain lobe6. To capture images of fine dendrite branches and filopodia in the intact brain tissue, we utilize two-photon microscopy, which increases the depth of light penetration and reduces the phototoxicity during live imaging experiments10. Using this setup, we successfully performed live imaging experiments on LNvs for over 30 min without observing obvious morphological deterioration of the neuron. In addition, genetic manipulations using the Flip-out technique enabled us to label the individual LNvs with a membrane tagged GFP, which is also critical for monitoring the movements of individual branches11,12,13.
To capture the dynamic behavior of all branches on the LNv dendritic arbor with optimal optic resolution, we performed time-lapse 3D imaging on freshly dissected larval brain explants with a high spatial resolution at 1 min per frame for 10 to 30 min. Developing LNv dendrites are highly dynamic, with a large percentage of the branches displaying observable changes within the 10 min window. This leads to one of the main technical challenges in studying dendrite dynamics, quantifying branch behavior based on the 4D image data sets. Previously established methods have various limitations, including lack of accuracy and excessive time requirement. Therefore, we developed a semi-automatic method that combines image post-processing, manual marking of the branch terminals, and automatic 4D spot tracing using an image annotation software. We calculate the movements of branch terminals at different time points based on the 3D coordinates of the spots. The data are then exported and analyzed to produce quantitative measurements of the branch dynamics. This method accurately assesses the duration and extent of extension and retraction events of existing branches, as well as the formation of new branches, allowing us to monitor dendrite dynamics in a large number of neurons.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chameleon Vision II multiphoton laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>high vacuum grease</td>
			<td>Dow Corning</td>
			<td>79751-30</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 780 two-photon laser scanning confocal microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>upright configuration</td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12-544-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td>for processing .csv files</td>
		</tr>
		<tr>
			<td>Huygens Professional</td>
			<td colspan="2">Scientific Volume Imaging</td>
			<td>for drift correction and deconvolution</td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Oxford Instruments</td>
			<td></td>
			<td>for 3D visualization and image annotation</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TES</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

time-lapse live imaging and quantification of fast dendritic branch dynamics in developing drosophila neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Using the live imaging protocol described above, we capture high resolution image stacks for the subsequent analyses and quantification. Supplementary Video 1 shows the maximum intensity projected (MIP) image series collected from a representative individually labeled LNv. Figure 2B shows the corresponding montage of eight frames of the image series. In both panels, arrowheads mark retraction events and arrows mark extension ...

Watch this Scientific Journal Video about Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons at JoVE.com

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

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

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

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

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