The overall goal of this procedure is to observe the impact of the SOX5 gene on the complexity of the dendritic arborization neurons during the neuronal development in Drosophila. This method can help study morphogenesis of neuro-dendrites, and the gene function in the development of nervous system, to better understand neurodegenerative disease genes. Ultimately this technique provides a quantitative analysis of dendrites complexity, which can be used in many different types of neuro-dendrites.
The implications of this technique extend toward genetic mechanism of neuro-degenerative disease, because mutation holds the key to understanding gene function. Set-up crosses of UAS-GFP;ppk-GAL4 with the UAS-Sox102F-RNAi strain flies or W118 controls. Culture the flies under standard conditions at 25 degrees Celsius.
In about five to six days, use forceps to carefully collect the third instar larvae for dissection. Place a larva in a dissection dish made of silicon elastomer base, and a tissue-culture Petri dish. Then place the dish under the dissection microscope.
Now pin the tail of the larva to expose the midline. Then pin the larva mouth hooks so the larva's dorsal side is up. Now add 200 microliters of PBS to the dish to immerse the larva in solution.
Next, cut the tail and mouth of larva with small incisions. Then cut the larva open along the dorsal midline and between the two tracheas, going from caudal to rostral. Then place a pin at each of the four corners of the body so the body lies flat.
Now fix the larva body wall in four percent PFA for 25 minutes at room temperature. Then wash the larva with PBS three times for five minutes per wash. After the washes, remove the pins and transfer the tissue onto a glass slide.
Then immerse the tissue in antifade mounting medium, and mount it with a cover slip. Allow the mounted tissue to air dry for an hour before sealing on the cover slip with fingernail polish. Store these slides in the dark at four degrees Celsius.
Capture z-series images with a confocal microscope using a 20X objective. First, set the range of the z-steps to include all of the DA neurons in the larva body wall, and set the step size to a half micrometer. Then store the images as TIF or ND2 files for processing.
Next, evaluate the number and morphology of the dendrites on the DA neurons. Begin with assessing their lengths. First, in Fiji ImageJ, split the images into separate channels if necessary.
Only the GFP channel needs to be assessed. Next, make a z-projection. In the new window, select max intensity for projection type, and choose the start and stop slices.
Then click OK to produce a z-projection image with clear dendrite projections. Now trace the neurites using the plug-in tool. First, go to the soma of the neuron of interest and click where one dendrite emerges from the cell body.
Then click at the tip of this dendrite. If the blue line that connects the two points runs the length of the neurite, press Y for yes. Then if this line fits the complete path of the neurite, click complete path.
If the line does not fit the path, then click on points further along this neurite to bend the path into multiple line segments that will fit the length of the neurite. Then, click complete path and proceed with marking the path for the next neurite. After completing all the tracing paths, select the Analysis, Measure Paths option to export the paths to a CSV file.
Use this data to calculate and average value of path lengths for analysis. Next, calculate the surface area of the neuron. First, select the free hand drawing tool from the Fiji ImageJ window.
Then, connect the end points of the neuron of interest. Then, select the Measure option from the Analyze window. The result appears in a new box with the value for the area selected.
Copy this value to the data analysis software. Finally, calculate the total number of branches using the Analyze Skeletonized Paths plugin. The dendrites of DA neurons were labeled by co-overexpressing GFP in their soma and dendrite arbors for GFP florescence imaging analysis.
The morphology of dendrites was imaged using an inverted confocal microscope. The dendrites of DA neurons were traced as described. The file was used to estimate the dendrite length.
Silencing of Sox102F in DA neurons in the third instar larva led to a significant reduction in the total number of dendrites with about half of the branch length and with a simpler structure, showing only half the number of branches. Logically, this led to reduction in arbor surface area. This protocol provides a quick method to assess development of DA sensory neurons.
While attempting this procedure, be sure to take an ample number of images of each group of DA neurons to get a good coverage. Following this procedure, other methods like dendrite analysis can be performed in order to answer additional questions about dendrite development. Since it's development, this technique has helped researchers in the field of neurosciences make inferences into neurodegenerative disease such as Alzheimer's using model systems.
