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.
