The overall goal of this protocol is to analyze dendric routing of adult drosophila medulla neurons in columns and layers to characterize dendric patterns, determine cell types based on morphology, and identify mutants. This method can help answer key questions in the field of neuroscience, including mapping the brain circuitry, and understanding the wiring mechanism of the dendrite. The main advantage of this technique is that it passes down the dendritic properties in three dimensional space, thus improving the understanding of dendritic wiring logic and functions.
The goal of this procedure is to acquire two image stacks of the neurons of interest in two orthogonal, horizontal, and frontal orientations. Using the standard techniques, stain dissected fly brains with rabbit anti-GFP and an antibody that labels photoreceptor axons primary antibodies. Then label the primary antibodies with fluorescent secondary antibodies and clear the brain in 70%glycerol and 1X PBS.
To mount the brain in the horizontal orientation, transfer a glycerol cleared brain into a 20 microliter drop of antifade mounting medium deposited centrally on the microscope slide. Then on a coverslip, attach small patches of clay at the four corners to prevent the coverslip from crushing the brain. Now under a dissecting microscope, position the brains in the ventral up position.
Use the convex dorsal surface of the brain as a landmark to identify the orientation of the brain sample. This will provide a horizontal view of the neuron. Then attach the coverslip.
Using a confocal microscope, ideally equipped with GaAsP detectors, obtain the horizontal view image stack with a high numerical aperture objective and digital zoom of 2.5. Acquire at least 180 optical sections, have 512 square pixels, and a step size of 2 microns. After taking the first image stack, remove the coverslip from the slide and reposition the brain in the anterior up position which provides a frontal view.
Then using the same technique, make a new image stack of the frontal view of the same neurons imaged in the horizontal view. Be sure to also record the location of the neuron of interest with respect to the medulla neuropal. Identifying the same neurons is feasible under lower magnification.
However, if signal is lost because the tissue is deep, just image the ventral half of your brain. Also check if the sample moved while acquiring images by examining the image stacks. Image de convolution and image combining are detailed in the text protocol.
The goal of this procedure is to trace neurites and to assign reference points for registration. First open the recombined image file. Then go to edit.
Then show display adjustment and turn off the photoreceptor channel, which is red. Next visualize the image in the surpass mode. If the computer is equipped with a stereograph system, turn on stereo and use the quad buffer mode to visualize 3D images.
Add new filaments, go to surpass, then filaments, and click on the tab labeled skip automatic creation, edit manually. Then click on the draw tab and select AutoDepth. Next select settings.
Toggle the line option and input the appropriate pixel number for better visualization. Then toggle show dendrites, beginning point, and branching point, and set the render quality to 100%Now select the draw tab, and start tracing neurites. Start with the axon, then move to the dendrites.
The axon and dendrites of the trans medulla neurons should be easy to differentiate. After tracing the neurites, go back to settings, and de toggle both beginning point and branching point. Then go to surpass, then export.
Select object, and save the filament as an inventor file. The next step is to assign reference points. To begin, select show display adjustment and turn on both imaging channels.
Then go to surpass, then measurement. And under the edit tab, toggle specific channel, and select the photoreceptor channel in this case. Now assign reference points for the top layer.
Select surpass, then measurement points, and mark the beginning of the m1 layer as a top layer. Use the clipping plane function to facilitate the point assignment. The order of the points is equatorial, anterior equatorial, anterior, anterior ventral, ventral, posterior ventral, posterior, posterior equatorial, and center.
To find the center photoreceptor, it's the one associated with the most dendritic processes. Next assign the reference points for the R8 and R7 layers using the same technique. After defining each set of reference points, export the coordinates.
Select statistics, then detailed, specific values, and position. Then select export statistics on tab display to file, and save the coordinates as a CSV file. Next open the three CSV files in a spreadsheet editor.
Copy and paste the coordinates of the 27 reference points into a new file, following the order of top, R8, and then R7.Proceed by following text protocol for rigid body and TPS non-linear registration standardization to right ventral configuration in calculation of dendritic branching and terminating frequencies. Ultimately, the data is used to make various informative plots. Using the dual view imaging procedure presented here, a fly brain containing sparsely labeled tm 20 neurons was imaged in two orthogonal directions.
Image recombination improves axial resolution. The recombined image has a better resolution than those of the horizontal view and frontal view images. After consulting 3D models, dendritic traces were collected from 15 tm 20 neurons and compared with dendric traces from 15 tm 2 neurons.
The branching and terminating probabilities of these neurons were analyzed. Both types were similar for segments of less than 4 microns. Layer-specific analysis of dendritic termination showed the two neuron types distribute in distinct layer specific manners.
Tm 2 dendrites receive inputs in m2 and in m5. While tm 20 dendrites terminate in the m1 to m3 layer. Planar analysis show that tm 2 dendrites project anteriorly, while tm 20 dendrites project posteriorly.
After watching this video, you should have a good understanding of how to acquire two of your confocal images. Read your study marks on the imaging files and extract the dendritic properties from these images. While attempting this procedure, it's important to mark the sample properly before confocal imaging.
As the imaging combination will fail if the sample moves during imaging.
