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 ...

This protocol provides a new approach to quantifying branch dynamics of the dendritic arbor and developing neurons using live time lapse imaging and a 3D image annotation software. This technique images and tracks the branch terminals providing a fast and efficient method for measuring the dynamic behaviors of dendritic filopodia. Research using this method provides insight into understanding how development and activity regulate dendrite morphogenesis.Our methods are applicable to sparsely labeled neurons in both in vitro and in vivo settings. When performing this procedure, remember that the imaging parameters must be carefully adjusted to achieve a sufficient temporal and spatial resolution. To harvest brains from larval drosophila, under a dissecting microscope use one pair of number five standard tip dissection forceps to hold a larval specimen in place while using the other to carefully dissect out the brain, preserving the eye disks, brain lobes, and ventral nerve cord.Then remove the attached muscles to minimize any movement of the sample during imaging. When all of the brains have been harvested, use vacuum grease to draw a square chamber onto a glass slide and add 20 microliters of external saline solution to the chamber. Use the forceps to transfer the brains to the chamber and adjust the positions of the brains under the microscope with the dorsal sides facing up.Cover the chamber with a glass cover slip and press gently on the cover slip to reduce sample drifting during the imaging procedure. To identify brain explants containing individually labeled neurons, within 30 minutes of the dissection select a 40X water immersion objective and an epifluorescence light source. Select a two photon laser tuned to 920 nanometers and a non Dscan detector and set the imaging parameters to collect the images at 512 by 512 pixels per frame and one minute per Z-stack for 10 minutes.Then, adjust the optical and digital zoom to achieve a sufficient X, Y, Z resolution while ensuring the coverage of the whole dendritic arbor within one minute. The settings will generate images with a typical X, Y, Z resolution of 0.11 by 0.11 by 0.25 micrometers. For drift correction, open the file of interest in a suitable drift correction software program and click Edit and Edit Microscopic Parameters.Set the microscope type to Wide Field for the two photon images if there is a two photon option and follow the workflow defined by the software. For deconvolution, open the drift corrected image in the deconvolution software and follow the workflow. Then, save the deconvolved image in a file type that is supported by the subsequent image annotation software capable of analyzing 4D data and reporting the spatial coordinates of defined spots within the image.For image annotation, open the deconvolved image in the image annotation software and examine and mark the branch tips at all time points in 3D. The image annotation software will report and store the spatial and temporal coordinates of the marked branch tips. Export the coordinate information as a CSV file for subsequent calculations.In the Spots module, click Skip Automatic Creation, Edit Manually, and check the Autoconnect to Selected Spot check box. Review the frames in the time series and select a branch for annotation. Holding the Shift key, click the branch terminal tip to add a spot.Then, click the Statistics tab, select Detailed, Specific Values, and Position. The recorded spatial and temporal coordinate information will be displayed. Click Save to export the data as a CSV file.To calculate the branch tip placement in 3D, open the CSV file as a spreadsheet and select the Track ID column. Click Sort Smallest to Largest and accept Expand the Selection. After sorting, Track ID will identify each unique dendritic branch and the spots from the same branch share the same Track ID.The Time column will store the information from different time frames.In the spreadsheet, add a Distance column, and use the coordinates to calculate the distance of every two temporally adjacent spots. To measure minor movements at the single voxel level, reset all of the distance values smaller than 0.3 micrometers to filter any uncorrected drifting or imperfect image annotation. Next, create a Displacement column and copy the values from the Distance column to the Displacement column.Manually assign the extension and retraction events for each branch tip. If it is an extension, leave the positive displacement value unchanged. If it is a retraction, change the corresponding displacement value to a negative value.Then, in a new Event column, sum the displacement values for individual extension and retraction events. In this video, a maximum intensity projected image series collected from a representative individually labeled ventral lateral neuron can be observed. Single frame images allow evaluation of the retraction and extension events.Semiautomated 4D tracking of the branch terminals allows annotation of the dynamic branch terminals of individually labeled ventral lateral neurons as demonstrated. Calculation of the direction and distance of the branch movements at each time point allows a comparison of the dendritic branch dynamics for individually labeled ventral lateral neurons at different developmental stages. The most important step is preserving the integrity of the brain tissue and dendrite morpholoy during dissection and mounting.The raw image series quality is also critical for successful tracking and quantification. Using this technique we gained the ability to perform quantitative analysis of dendrite filopodia in developing drosophila central neurons and to explore the molecular machinery relating dendrite maturation and synaptogenesis.

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JoVE Journal

Neuroscience

6.1K visualizações.  National Institutes of Health. Este protocolo fornece uma nova abordagem para quantificar a dinâmica do ramo da árvore dendrítica e desenvolver neurônios usando imagens de lapso de tempo ao vivo e um software de anotação de imagem 3D. Esta técnica imagens e rastreia os terminais do ramo fornecendo um método rápido e eficiente para medir os comportamentos dinâmicos da filopodia dendrítica. A pesquisa usando este método fornece uma visão sobre como o desenvolvimento e a atividade regulam a morfogênese dendrite....