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

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::GFP^11,12,13. The frequency of obtaining a single labeled LNv is around 10%. Fly stocks are maintained in standard medium in circadian- and humidity-controlled 25 °C incubators.

1. Collect 100-200 eggs within a 2 h window post fertilization on a grape juice plate with yeast supplement. Incubate the embryos at 25 °C for 24 h and collect newly hatched first instar larvae for the next step.
2. Heat shock the newly hatched first instar larvae at 37.5 °C for 40 min two times, with a 40 min recovery period in between.
3. Culture the larvae at 25 °C with circadian and humidity controls to the desired developmental stage(s).

2. Dissecting and Mounting Larval Brain Explants

1. 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.
2. Prepare a glass slide (25 x 75 x 1.0 mm³) and use a syringe to draw a square chamber with vacuum grease.
3. Add 20 µL of external saline solution to the square chamber with grease barriers.
4. 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.
5. 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.

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.

1. 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.
2. 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 µm³.
3. 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.

4. Drift Correction and Deconvolution

NOTE: Depending on the image quality, both steps are optional but strongly recommended.

1. 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.
2. 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.
3. 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).

5. Image Annotation

1. 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.
2. Within the Spots module, click "Skip automatic creation, edit manually" and check the bottom "Auto-connect to selected Spot" 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).
3. Within the Spots module, click the Statistica tab, choose Detailed and select Specific Values | Posizione. 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).

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.

1. 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.
2. 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).
3. Minor movements at the single voxel level. Based on the imaging settings, 0.3 µm are usually artifacts from uncorrected drifting or imperfect image annotation. Filter these movements out by resetting all Distance values smaller than 0.3 µm to 0. Process multiple .csv files manually or use our R script batch column filtering.R (Supplementary File).
4. 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).
5. Generate a new column named Event. In this column, manually sum the Displacement values for individual extension and retraction events (Figure 4B).
6. 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.