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 achieved by expressing mCD8::GFP in single LNvs using flippase -mediated stochastic labeling. The genotype of the fly line is: hs-flp; Pdf-Gal4; UAS-FRT-CD2-stop-FRT-mCD8::GFP11,12,13. The frequency of obtaining a single labeled LNv is around 10%. Fly stocks are maintained in standard medium in circadian- and humidity-controlled 25 &#176;C incubators.
<ol>
	<li>Collect 100-200 eggs within a 2 h window post fertilization on a grape juice plate with yeast supplement. Incubate the embryos at 25 &#176;C for 24 h and collect newly hatched first instar larvae for the next step.</li>
	<li>Heat shock the newly hatched first instar larvae at 37.5 &#176;C for 40 min two times, with a 40 min recovery period in between.</li>
	<li>Culture the larvae at 25 &#176;C with circadian and humidity controls to the desired developmental stage(s).</li>
</ol>
2. Dissecting and Mounting Larval Brain Explants
<ol>
	<li>Dissect larval brains in the physiological external saline solution (120 mM NaCl, 4 mM MgCl2, 3 mM KCl, 10 mM NaHCO3, 10 mM Glucose, 10 mM Sucrose, 5 mM TES, 10 mM HEPES, 2 mM Ca2+, PH 7.2) under a dissection microscope (4.5x magnification power) with two pairs of #5 standard tip dissection forceps (11 cm). Use one pair of forceps to hold the larval body in place and the other to carefully dissect out the brain. Preserve the eye disks, brain lobes and the ventral nerve cord. Remove attached muscles to minimize sample movements during imaging.</li>
	<li>Prepare a glass slide (25 x 75 x 1.0 mm3) and use a syringe to draw a square chamber with vacuum grease.</li>
	<li>Add 20 &#181;L of external saline solution to the square chamber with grease barriers.</li>
	<li>Transfer dissected larval brains into the chamber on the glass slide using forceps. Adjust the position of the brains under the dissection scope to ensure the dorsal side faces up.</li>
	<li>Cover the chamber with a glass cover slip (22 x 22 x 0.15 mm3). The larval brain is now mounted on the slide within a chamber filled with the external saline solution (Figure 1B). 
	NOTE: Pressing gently on the coverslip confines the brains and reduces sample drifting in the subsequent imaging session.</li>
</ol>
3. Time-lapse Live Imaging
NOTE: We perform time-lapse imaging experiments using a confocal microscope equipped with a multiphoton laser. The acquisition parameters need to be adjusted for other imaging setups.
<ol>
	<li>Identify brain explants containing individually labeled neurons using a 40X water immersion objective (NA 1.3) and an epifluorescent light source. For image collection, use a two-photon laser tuned to 920 nm and a non-descanned (NDD) detector.</li>
	<li>Collect images at 512 x 512 pixels per frame and 1 min per Z-stack for 10 min. Adjust the optical and digital zoom to achieve a sufficient x-y-z resolution while ensuring the coverage of the whole dendritic arbor within 1 min (Figure 2, Supplementary Video 1). The settings generate images with a typical x-y-z resolution of 0.11 x 0.11 x 0.25 &#181;m3.</li>
	<li>Collect the time lapse image series within 30 min of the brain dissection. Data with excessive drift or obvious morphological deterioration should be excluded from post- processing and quantification.</li>
</ol>
4. Drift Correction and Deconvolution
NOTE: Depending on the image quality, both steps are optional but strongly recommended.
<ol>
	<li>Open an acquired image file with a drift correction software. Edit microscopic parameters to match those used for the experiment. For the software (see Table of Materials), click Edit | Edit microscopic parameters. Set microscope type as widefield for two-photon images if there is no two-photon option and follow the workflow defined by the software. For example, open the tab Deconvolution | Object Stabilizer, and choose Stabilize time frames.</li>
	<li>Open the drift-corrected image in the deconvolution software and follow its workflow. For the software (see Table of Materials), select the stabilized image and then click Deconvolution | Deconvolution Express. To get a better result, fine-tune the parameters using Deconvolution Wizard.</li>
	<li>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 in the image (see Table of Materials).</li>
</ol>
5. Image Annotation
<ol>
	<li>Open the deconvolved image in the image annotation software. Examine and mark branch tips at all time points in 3D. The image annotation software reports and stores the spatial and temporal coordinates of the marked branch tips. Export the coordinate information as a .csv file for subsequent calculations. 
	NOTE: The following two steps are specific to the annotation software we used (see Table of Materials). The workflow may be different for other software.</li>
	<li>Within the Spots module, click &#34;Skip automatic creation, edit manually&#34; and check the bottom &#34;Auto-connect to selected Spot&#34; checkbox (Figure 3A). Go over the frames in the time series and select a branch for annotation. Hold the Shift key and click the branch terminal tip to add a spot. Click through all time points. 
	NOTE: The image annotation software connects the spots between frames and generates a trajectory automatically. The spatial and temporal information of the branch tips is now associated with marked spots. Repeat these steps until all branch tips are annotated (Figure 3B).</li>
	<li>Within the Spots module, click the Statistics tab, choose Detailed and select Specific Values | Position. The recorded spatial and temporal coordinate information will be on display. Click the Save bottom to export that information as a .csv file (Figure 3C).</li>
</ol>
6. Calculating Dendritic Branch Dynamics
NOTE: Using the coordinate information, displacement of the branch tips in 3D can be easily calculated. In our study, all dendritic branch movements are categorized as Extension or Retraction. The steps below describe how to process the .csv files using custom-written R scripts (Supplementary File) and by adding the directional information through manual editing.
<ol>
	<li>Open the .csv file as a spreadsheet. Select the Track ID column, click the Sort Smallest to Largest option and accept Expand the selection. After sorting, Track ID identifies each unique dendritic branch, and spots from the same branch share the same Track ID. The Time column stores the information of different time frames.</li>
	<li>In the spreadsheet, add a Distance column. Calculate the distance of every two temporally adjacent spots using their coordinates and put the values in the Distance column (Figure 4A).</li>
	<li>Minor movements at the single voxel level. Based on the imaging settings, 0.3 &#181;m are usually artifacts from uncorrected drifting or imperfect image annotation. Filter these movements out by resetting all Distance values smaller than 0.3 &#181;m to 0. Process multiple .csv files manually or use our R script batch column filtering.R (Supplementary File).</li>
	<li>Manually generate a new column named Displacement in the spreadsheet. Copy the values from Distance column to Displacement column. Manually assign the extension and retraction events for each branch tip. If it is an extension, leave the Displacement value unchanged, which is a positive value. If it is a retraction, change the corresponding Displacement value to a negative value (e.g., 0.35 to -0.35) (Figure 4B).</li>
	<li>Generate a new column named Event. In this column, manually sum the Displacement values for individual extension and retraction events (Figure 4B).</li>
	<li>Process the modified spreadsheet and quantify the extension and retraction events based on their displacement values using R script batch column sum.R (Supplementary File). The output parameters include Number of extension events, Number of retraction events, Cumulative length extended, Cumulative length retracted, Net length changed, and Total length traveled.</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Branching+out:+mechanisms+of+dendritic+arborization.">Branching out: mechanisms of dendritic arborization.</a> Nature Reviews Neuroscience. 11, 316 (2010).</li><li>Cline, H., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+dendritic+arbor+development+and+plasticity+by+glutamatergic+synaptic+input:+a+review+of+the+synaptotrophic+hypothesis.">The regulation of dendritic arbor development and plasticity by glutamatergic synaptic input: a review of the synaptotrophic hypothesis.</a> The Journal of Physiology. 586 (6), 1509-1517 (2008).</li><li>Helfrich-Forster, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+morphology+of+the+clock-gene-expressing+lateral+neurons+of+Drosophila+melanogaster.">Development and morphology of the clock-gene-expressing lateral neurons of Drosophila melanogaster.</a> Journal of Comparative Neurology. 500 (1), 47-70 (2007).</li><li>Sprecher, S. 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The authors have nothing to disclose.

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 condensed arborizations are not suitable subjects for this analysis.
Sample preparation, image acquisition and post-processing are the critical steps in this protocol. High quality raw images of single LNvs are essential for the accurate quantification of dendrite dynamics. Our data indicate that LNv dendrite morphology and its physiological responses are well-preserved in a carefully prepared larval brain explant within 30 min of dissection. Samples with observable brain tissue deterioration, elongated dendritic arbors and branch breakage should not be used for imaging and quantification. Although optional for datasets with low background and no observable movements, we strongly recommend including the drift correction and deconvolution steps to improve the image quality, which is important for the annotation of branch terminals and automatic spot tracking. Besides the commercial software used in our studies, ImageJ/Fiji&#39;s Correct 3D drift (Plugins | Registration | Correct 3D drift) also works well for drift correction.
One of the major differences between our protocol and existing methods is that we track the movements of individual branch tips but not the whole dendritic arbor. Reconstruction of whole dendritic arbors, although useful for measuring the total dendritic length and architecture, is not ideal for dynamic studies using 3D imaging datasets. For consecutive Z-stack slices, automatic tracing programs often generate different combinations of &#34;branches&#34; and reconstruct the dendritic arbor differently, making the comparison at the single branch level between different time points particularly difficult. In addition, comparing to tracing the whole dendritic arbor, terminal tracking significantly reduces the processing time and the requirements on computation power.
Several modifications can potentially improve the efficiency and analytical features of our method. To extract quantitative positional information from the time-lapse image series, accurate annotation of branch tips at all time points is critically important. This step is currently performed manually, which not only is time consuming, but also introduces variability. New developments in open source 3D visualization software could potentially address current technical limitations and make automation of this critical step possible. Several recently developed software tools provide automatic quantification function for studies on filopodia16,17,18,19,20,21 and can be potentially tested and adopted for studies on dendrite branch dynamics.
Another step that is currently performed manually is the identification of extension vs. retraction events. Automation of this step requires the 3D coordinates of branch bifurcation sites. By comparing distances to the branching site at two time points, a custom-written scrip&#160;can be developed to assign the directionality of the terminal displacements. Following the same logic, additional 3D coordinates along the branch may also be used for analyzing the extension and retraction events occurring within the branch. These add-ons will not only optimize the workflow of our protocol, but also increase the information contents regarding the structural changes of the dendritic arbors at different temporal scales.
In summary, to support our studies on experience-dependent dendrite plasticity, we developed time-lapse live imaging protocols and semi-automated quantification methods to perform assessments of fast branch dynamics in developing neurons. Based on the results generated by the methods described here, our study identified highly motile dendritic filopodia in Drosophila central neurons and revealed a developmental coordination between heightened dendrite dynamics and synaptogenesis. Future improvements at the image acquisition and automation level will greatly enhance the potential of this protocol and facilitate studies on dendrite dynamics and structural plasticity.

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 events.
Next, we perform semi-automated 4D tracking of the branch terminals using an image annotation software (Table of Materials). Figure 3B shows annotations of the dynamic branch terminals from a representative individually labeled LNv. Using this software, we visualize the image stacks in 3D, manually mark all branch terminals and use the Spots module to track the terminals&#39; movements through time. This software generates time-stamped trajectories for every annotated branch terminal, serving as visual representations of the dynamic events on the dendritic arbor.
We then use the coordinate information from annotated branch tips to calculate the direction and distance of branch movements at each time point. Screenshots in Figure 4A,B show how this is achieved. The processed spreadsheets generate output files that we use to quantify dendrite dynamics with four parameters, including percentage of dynamic branches, number of new branches, cumulative distance traveled, and number of branch movement events. Figure 4C shows comparisons of dendritic branch dynamics for individually labeled LNvs from different developmental stages. Consistent with the findings in mammalian neurons and zebrafish tectal cells, the LNv dendrite dynamics are developmentally regulated14,15. LNv dendrites in younger larvae, 48-72 h after egg laying (AEL), are significantly more dynamic compared to those in older larvae, 96-120 h AEL, as measured by all four parameters, and the developmental transition from the dynamic to stable state occur between 72 to 96 h AEL8.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60287/60287fig1v2.jpg" /> 
Figure 1: Workflow of the dendrite dynamics imaging and quantification protocol. (A) The protocol contains six steps covering sample preparation, image collection, image processing and semi-automated quantification of dendrite dynamics. (B) A schematic diagram illustrating an imaging chamber containing a larval brain explant mounted with the dorsal side up. The brain explant has an individually labeled LNv in each lobe and is immersed in the external saline solution. The vacuum grease barriers support the weight of the coverslip and forms a small chamber that prevents the brain from moving. <a href="https://www.jove.com/files/ftp_upload/60287/60287fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60287/60287fig2v2.jpg" /> 
Figure 2: Time-lapse imaging of dendritic branch dynamics in 3D. (A) The dendritic arbor of an individually labeled LNv from a 3rd instar larva. (B) The corresponding montage image of (A) illustrating the representative branch movements. Arrowheads (Green) mark retraction events; arrows (Red) mark extension events. <a href="https://www.jove.com/files/ftp_upload/60287/60287fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60287/60287fig3v2.jpg" /> 
Figure 3: Manual annotation and automatic tracking of dendritic branch terminals in an image annotation software. (A) A screenshot shows the settings for branch terminal tracking using the Spots module. The red oval outlines the Manual Tracking option. (B) Representative time-stamped trajectories of annotated branch terminals (yellow spots) generated by the image annotation software in an individually labeled LNv. Scale bar = 5 &#181;m. (C) A screenshot shows the interface for viewing and exporting the coordinate information from the Spots module. Figure 3B has been modified from Sheng, C. et al. 20188. <a href="https://www.jove.com/files/ftp_upload/60287/60287fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60287/60287fig4v2.jpg" /> 
Figure 4: Quantification of dendritic branch dynamics using the coordinate information of the branch terminals. (A) A screenshot shows the spreadsheet containing the X, Y and Z coordinates, Track IDs and formula used for calculating displacement of spots in the adjacent frames. (B) A screenshot shows representative results obtained from tracking one branch. The minor movements (&#60;0.3 &#181;m) were filtered out. The retractions are marked by assigning their value to negative (Distance column). By summing up adjacent positive/negative values, the displacement of each biological extension/retraction is computed (Events column). (C) Representative quantification of results showing the developmental regulation of dendrite dynamics. Data are presented as a box plot (box, 25-75%; center line, median) overlaid with a dot plot (individual data points). Figure 4C has been modified from Sheng, C. et al. 20188. <a href="https://www.jove.com/files/ftp_upload/60287/60287fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Video 1" src="/files/ftp_upload/60287/60287vid1.jpg" /> 
Supplementary Video 1: Ten maximum-intensity-projection (MIP) images played at a speed of 2 frames per second. The whole dendritic arbor was imaged at 1 min per Z-stack. <a href="https://www.jove.com/files/ftp_upload/60287/60287vid1.mp4" target="_blank">Please click here to view this video (Right click to download).</a>
Supplementary File 1: Batch Column Filtering.R Script. <a href="https://www.jove.com/files/ftp_upload/60287/batch_column_filtering.R" target="_blank">Please click here to view this file (Right click to download).</a>
Supplementary File 2: Batch Column Sum.R Script. <a href="https://www.jove.com/files/ftp_upload/60287/batch_column_sum.R" target="_blank">Please click here to view this file (Right click to download).</a>

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 ...

time-lapse-live-imaging-quantification-fast-dendritic-branch-dynamics

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

6.1K Views.  National Institutes of Health. 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.

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

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured neurons for extended durations1-3. This is now possible through the use of vital dyes and fluorescent proteins with which cytoskeletal components, organelles, and other structures in living cells can be labeled and then visualized via dynamic fluorescence microscopy. For example, in embryonic chicken sympathetic neurons, mitochondrial movement was characterized using the vital dye rhodamine 1234. In another study, mitochondria were visualized in rat forebrain neurons by transfection of mitochondrially targeted eYFP5. However, imaging of primary neurons over minutes, hours, or even days presents a number of issues. Foremost among these are: 1) maintenance of culture conditions such as temperature, humidity, and pH during long imaging sessions; 2) a strong, stable fluorescent signal to assure both the quality of acquired images and accurate measurement of signal intensity during image analysis; and 3) limiting exposure times during image acquisition to minimize photobleaching and avoid phototoxicity.
Here, we describe a protocol that permits the observation, visualization, and analysis of mitochondrial movement in cultured hippocampal neurons with high temporal resolution and under optimal life support conditions. We have constructed an affordable stage-top incubator that provides good temperature regulation and atmospheric gas flow, and also limits the degree of media evaporation, assuring stable pH and osmolarity. This incubator is connected, via inlet and outlet hoses, to a standard tissue culture incubator, which provides constant humidity levels and an atmosphere of 5-10% CO2/air. This design offers a cost-effective alternative to significantly more expensive microscope incubators that don't necessarily assure the viability of cells over many hours or even days. To visualize mitochondria, we infect cells with a lentivirus encoding a red fluorescent protein that is targeted to the mitochondrion. This assures a strong and persistent signal, which, in conjunction with the use of a stable xenon light source, allows us to limit exposure times during image acquisition and all but precludes photobleaching and phototoxicity. Two injection ports on the top of the stage-top incubator allow the acute administration of neurotransmitters and other reagents intended to modulate mitochondrial movement. In sum, lentivirus-mediated expression of an organelle-targeted red fluorescent protein and the combination of our stage-top incubator, a conventional inverted fluorescence microscope, CCD camera, and xenon light source allow us to acquire time-lapse images of mitochondrial transport in living neurons over longer durations than those possible in studies deploying conventional vital dyes and off-the-shelf life support systems.

We describe a protocol that allows imaging of mitochondria in living neurons via fluorescence microscopy over long durations. Imaging over extended periods is accomplished through lentivirus-mediated expression of a mitochondrially targeted fluorescent protein and use of an inexpensive stage-top incubator that was designed and built in our laboratory.

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.