The overall goal of this experimental procedure is to understand synaptic dynamics in a single neuron under different activation conditions. This method can help answer key questions in the synaptic plasticity field such as revealing changes in the molecular composition of synapses upon maturation of the neuron's activity. The main advantage of this technique is that it allows the semi-automated analysis of multiple aspects of synapses including their number, distribution, and the level of enrichment of molecular components after synapses.
For this experiment, collect the flies into normal vials within six hours of eclosion. Load the collection vials into a transparent acrylic rack. In a small incubator set to 25 degrees celsius, position the rack at a precise distance from an LED panel where the light exposure is at an average of 1000 lux.
Then, rear flies for one to three days using one of the following conditions, either constant darkness, 12 hours of light followed by 12 hours of darkness, or constant light. Later, dissect and stain the brains using standard techniques. To mount the fly brains, load a micropipet with mounting medium and deposit two 2 microliter drops at the center of a microscope slide about 2 cm apart.
Place a cover slip onto each drop and the gape between about 0.2 mm. Then deposit 15 microliters of mounting medium over the cover slips and into the gap. Under a dissecting microscope, deposit the brain into the gap from micropipet.
And then position the brains, ventral side up. Finally, attach a cover slip and seal it along the edges using clear nail polish. The brains can then be imaged using standard techniques to reconstitute 3D images.
In this case, GFP luminescence is documented in cells with active Brp expression using the star method. Also in this example, R7 and R8 photoreceptor axons were immunolabled with anticiaoptin, which was viewed using RFP-tagged secondary antibodies. To quantify the number, distribution, and delocalization level of the Brp GFP puncta first load reconstituted 3D images of the brain made from image stacks.
Brp puncta are displayed in white. Now, find the region of interest. In this case, R8 axon's terminals are located by following the thinning of the anticiaoptin positive axons at the entry point into the M3 medulla layer.
At each R8 axon terminal identify the Brp GFP puncta using the spot detection module. Select add new spots then select segment only region of interest in the algorithm settings. Once the analysis region is defined, set the source channel to Brp GFP then set the estimated XY diameter to 0.35 microns.
And then, check off background subtraction in the spot detection. Next, select quality as the filter type, and the puncta are filtered automatically. Complete this step by clicking finish.
Repeat the spot detection method for all of R8 axons in the dataset. The next region to identify is the axon cytoplasm. First, generate surface objects with the surface function.
Then, select add new surfaces, and under the algorithm settings, toggle segment only region of interest. Now, manually select the region of interest. Next, set the calculation perimeters.
To find the photoreceptor channel as source, select the smooth option. For the threshold, select absolute intensity. Enable the split touching objects option.
Set the seat points diameter to 0.5 microns and use the default filter settings for quality and number of voxels. The filter is then applied automatically. Now, go to edit and delete the pieces of surface generated outside of the region of interest.
In this case, the other axons. After repeating the axon region detection for all R8 axons in the dataset, all the Brp puncta and R8 axons are identified as a set of spot objects. And the corresponding cytoplasmic regions are identified as surface objects.
Now, proceed by manually defining each axon's orientation and space. This is important for later quantification of Brp GFP density along each neuron in the medulla neuropil. To do this, first define their start points and end points.
Select add new measurement points, then select edit followed by select surface of object to work with the top of the surface objects. To define the start points, put measurement points on all the surface objects of the R axons in the M1 layer. Place these points in a systematic reproducible order.
Next, define the end points. Select add new measurement points, edit, and surface of object again. Then, repeating the same systematic order, place measurement points on the bottom of the R axons in M3 layer.
To quantify the Brp background intensity, analyze the cytoplasmic regions of two R7 axons. Select add new surfaces and manually define the region of an R7 axon as was done for the R8 axons. Use identical settings except do not toggle on the enable split touching objects option.
Leave this off. Next, add a dummy spots object inside a defined R7 axon region. Select add new spots, and select skip automatic creation, and edit manually.
Then, select center of object and click on the surface object to place the dummy spots object inside the R7 axon. Now, repeat the surface and spot detection steps for a second R7 axon. And then define the start and end terminal points of the R7 axons as done with the R8 axons.
For this analysis, be certain to have the correct software installed. Open the dataset containing the spot data, surfaces data, and two measurement point objects, each containing the same number of measurement points. Inspect the data.
Both of the measurement point objects must have the same number of measurement points for the calculations to work. Once the spots, axon regions, and start and end points have been defined, start the plugin. First, check the metadata, in particular, the voxel size.
Open image properties from edit, then check the 3 dimensions of the voxel and microns under coordinates in geometry. Next, select a channel for the intensity analysis. In this case, the channel showing Brp GFP is selected.
Then define the file name for the results. Next, define the number of bins as 10, and set the axon length to 100%Then define the regions of the puncta by setting the spot radius to 0.35 microns and the surrounding cytoplasmic region to 50 microns. Finally, execute the synapse detection command, and later, statistically analyze the output.
Following the described protocols, Brp GFP puncta were analyzed in the R8 synapses of flies exposed to constant darkness, constant light, or a normal light/dark cycle. The number of Brp puncta was significantly reduced in R8 photoreceptors of flies kept in constant light. The distribution of the puncta was calculated using a custom plugin.
R8 synapses were distributed all along the axonal shaft from the M1 to the M3 layer, but their density was higher at the M1 and M3 layers. Similarly, delocalization levels of the puncta were calculated. They were unchanged in all conditions, however delocalization levels of Brp GFP differed from other reporters, like Brp-short-cherry which becomes clearly defused under constant light.
It is possible that there is inappropriate processing of the Brp short fragment after disassembling from the AZ.After watching this video, you should have a good understanding of how to analyze synaptic plastic properties in a single neuron. Such plastic properties includes the synapse number, their distribution, and the label of enrichment of a specific molecular component after the synapses.
