Name: Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light (Video) | JoVE Journal
Uploaded: 2017-02-10T14:00:00.000+00:00
Duration: 11 min 36 s
Description: Learn to quantify synaptic active zones in Drosophila melanogaster photoreceptors using semi-automated analysis. This method reveals how prolonged light exposure modulates synaptic plasticity in vivo.

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5 minutes

We are grateful to T. St&#252;rner for helpful corrections, discussions and comments on the manuscript; S.L. Zipursky for providing fly stocks; M Sch&#246;lling for performing image processing. Part of the image analysis was performed at A. Kakita&#39;s lab. This work was supported by Alexander von Humboldt Foundation and JSPS Fellowships for Research Abroad (A.S.), JSPS Fellows (S.H.-S.), Grant-In- Aid for Start-up (24800024), on Innovative Areas (25110713), Mochida, Takeda, Inamori, Daiichi-Sankyo, Toray Foundations (T.S.), DZNE core funding (G.T.) and DZNE Light Microscopy Facility (C.M.).

The experimental procedures described in this protocol involve exclusively work with Drosophila and are not subjected to animal welfare laws in Germany and Japan.
1. Light Exposure Conditions
<ol>
	<li>Prepare flies that carry sensless-flippase (sens-flp) and bruchpilot-FRT-STOP-FRT-gfp (brp-FSF-gfp)15 for visualizing Brp-GFP in photoreceptor R8 neurons (R8s). The genomic locus encoding brp within a Bacterial Artificial Chromosome (BAC) was modified to generate this brp-FSF-gfp construct. In the flies carrying this modified BAC the expression of Brp-GFP is under the control of the endogenous regulatory sequences, but limited to R8 photoreceptors after sensless-flippase-mediated removal of the FRT-STOP-FRT cassette15. Note that sens-flp rarely expresses flippase in R7s.</li>
	<li>Keep the flies in a 12 h light/12 h dark cycle (LD) at 25 &#176;C from the larva stage until eclosion (Figure 1A).</li>
	<li>Collect the flies 0 - 6 h after eclosion and place them into a new vial containing fly food.</li>
	<li>Set the vials into a transparent rack made of acrylic resin with three steps (a height of 41 cm, a base of 21 cm, a thickness of 4 cm and a height of 13 cm for each step) (Figure 1B).</li>
	<li>Adjust the distance between the rack and a LED panel to provide the illumination with an average intensity of 1,000 lux using a digital light meter (Figure 1C).</li>
	<li>Keep the flies for 1 - 3 d under one of the following conditions: constant darkness (DD), 12 h light/12 h dark cycle (LD), or constant light (LL) at 25 &#176;C in a small incubator (Figures 1A and 1C).</li>
</ol>
2. Dissection, Immunohistochemistry and Imaging
<ol>
	<li>Dissect fly brains with forceps, fix them with 4% formaldehyde and immunostain with the primary antibody against Chaoptin (1:50) and a second antibody for visualization of photoreceptors based on previous experimental procedures25. Briefly:
	<ol>
		<li>Prepare 0.1% Triton X-100 in PBS (0.1% PBT). This solution is used to improve antibody tissue penetration.</li>
		<li>Anesthetize flies on a CO2 pad, sort out the correct genotype and transfer them to a petri dish filled with 0.1% PBT.</li>
		<li>Stick the forceps underneath the proboscis and pull off the right and left eyes respectively with the other forceps.</li>
		<li>Remove the proboscis and the trachea attached to the brain.</li>
		<li>Transfer the brain with the forceps into a 1.5 mL tube containing 150 &#181;L of 0.1% PBT at RT.</li>
		<li>Within 10 min repeat from 2.1.3 - 2.1.5 to obtain as many brains as possible.</li>
		<li>Add 50 &#181;L of 16% formaldehyde into the tube and mix by pipetting.</li>
		<li>Incubate the brains in fixative at room temperature for 50 min.</li>
		<li>Wash three times with 200 &#181;L of 0.1% PBT with a micropipette, paying attention to leave the brains in the tube.</li>
		<li>After the last wash, remove the 0.1% PBT and add the primary antibody solution, prepared as follows: add to 196 &#181;L of 0.3% PBT (0.3% Triton X-100 in PBS) 4 &#181;L of anti-Chaoptin antibody (final dilution 1:50) for visualization of photoreceptors.</li>
		<li>Incubate the brains in the primary antibody solution at 4 &#176;C O/N.</li>
		<li>Wash 3x with 200 &#181;L of 0.1% PBT with a micropipette.</li>
		<li>After the last wash, remove the 0.1% PBT and add 200 &#181;L of the 0.3% PBT containing a secondary antibody.</li>
		<li>Incubate it in the dark at 4 &#176;C O/N.</li>
		<li>Wash three times with 200 &#181;L of 0.1% PBT with a micropipette.</li>
	</ol>
	</li>
	<li>For mounting, put two small drops (2 &#181;L each) of a mounting medium with a micropipette at the center of a microscope slide at approximately 2 cm distance from each other.</li>
	<li>Place two coverslips, one onto each drop and leave a narrow gap between the coverslips of approximately 0.2 mm.</li>
	<li>Place 15 &#181;L of a mounting medium on top of the coverslip and add the brains with a micropipette into the mounting medium.</li>
	<li>Under a dissecting microscope, position the brains with the ventral side up into the narrow gap between the two coverslips (Figure 2).</li>
	<li>Place a coverslip on top and seal the edges of the coverslip using clear nail polish to fix the specimen (Figure 2).</li>
	<li>Obtain images of the brains with a confocal microscope. Use a 63X oil immersion objective lens (1.4 N. A.) and a 2.6X digital zoom. Generate more than 20 optical sections of the second optic ganglion medulla with a step size of 0.5 &#181;m.</li>
</ol>
3. Generation of the Spots,&#160;Surface Objects, Start-points and&#160;End-points
<ol>
	<li>To quantify the number, the distribution and the delocalization level of Brp-GFP puncta, open the stack obtained by confocal microscopy in image analysis software and reconstitute 3D images (Figure 3A). Note that the step in this study was performed with Imaris.</li>
	<li>Identify the Brp-GFP puncta by the spot detection module for each R8 axon terminal.
	<ol>
		<li>Select &#34;Add new Spots&#34;.</li>
		<li>Select &#34;Segment only a Region of Interest&#34; in the algorithm settings. Select an R8 axon terminal in the medulla manually.</li>
		<li>Select the source channel of the Brp-GFP signal. 
		NOTE: The R7 and R8 photoreceptor axons were immunolabeled with anti-Chaoptin (Figure 3A). Only R8, though, contains Brp-GFP puncta and could thus be easily distinguished (Figure 3A). Further, the endpoint of R8 could be identified by the thinning of the anti-Chaoptin-positive axons at the entry point into the M3 medulla layer.</li>
		<li>Set the estimated XY diameter to 0.35 &#181;m and check off &#34;Background subtraction&#34; in the spot detection.</li>
		<li>Select &#34;Quality&#34; in filter type and filter automatically. Then click finish.</li>
		<li>Repeat from 3.2.1 t- 3.2.5 for other R axons (Figure 3B).</li>
	</ol>
	</li>
	<li>To measure the delocalization level of Brp-GFP in R8 axons, generate surface objects with the &#34;Surface&#34; function for each axon (Figure 3C).
	<ol>
		<li>Select &#34;Add new Surfaces&#34;.</li>
		<li>Select &#34;Segment only a Region of Interest&#34; in the algorithm settings. Then select an R8 axon terminals in the medulla manually.</li>
		<li>Select the source channel of the photoreceptors.</li>
		<li>Check off &#34;Smooth&#34;.</li>
		<li>Select &#34;Absolute Intensity&#34; in the threshold.</li>
		<li>Check &#34;Enable&#34;. &#34;Seed Points Diameter&#34; is 0.5 &#181;m.</li>
		<li>Select filter Type &#34;Quality&#34; and &#34;Number of voxels&#34;, and then filter automatically.</li>
		<li>Go to &#34;Edit&#34; and delete the pieces of surfaces generated on other non-interesting axons.</li>
		<li>Repeat from 3.3.1 - 3.3.8 for other R axons (Figure 3C). Note some surface objects were interrupted because of the thinness and the weak signal in photoreceptors at M2 layer, but they were still considered as a single object from start to end points.</li>
	</ol>
	</li>
	<li>To identify the distribution of Brp-GFP puncta along each neuron in the medulla neuropil, define the start-point and end-point with the measurement point function (Figure 3D).
	<ol>
		<li>Select &#34;Add new Measurement Points&#34;. Then select &#34;Edit&#34; and select &#34;Surface of Object&#34; to intersect with the top of the surface objects.</li>
		<li>Put measurement points on top of surface objects of R axons in M1 layer in order. These are now defined as start-points (Figure 3D).</li>
		<li>Select &#34;Add new Measurement Points&#34;. Then select &#34;Edit&#34; and select &#34;Surface of Object&#34; to intersect with the bottom of the surface objects.</li>
		<li>Put measurement points on the bottom of the surface objects of R axons in M3 layer in order. These are now defined as end-points (Figure 3D).</li>
	</ol>
	</li>
	<li>To subtract the background signal from the GFP channel, generate surface objects, spots, start-points and end-points for two R axons in M6 layer (Figure 3E).
	<ol>
		<li>Select &#34;Add new Surfaces&#34;.</li>
		<li>Select &#34;Segment only a Region of Interest&#34; in the algorithm settings. Then select a R7 axon terminal between M5 and M6 layer manually.</li>
		<li>Select the source channel of the photoreceptor.</li>
		<li>Check off &#34;Smooth&#34;.</li>
		<li>Select &#34;Absolute Intensity&#34; in the threshold.</li>
		<li>Select filter Type &#34;Quality&#34; and &#34;Number of voxels&#34;, then filter automatically.</li>
		<li>Select &#34;Add new Spots&#34;.</li>
		<li>Select &#34;Skip automatic creation, edit manually&#34;.</li>
		<li>Select &#34;Center of Object&#34; and add a spot in the inner area of the surface object generated from 3.5.1 - 3.5.6.</li>
		<li>Repeat from 3.5.1 -&#160;3.5.9 for another R7 axon terminal.</li>
		<li>Select &#34;Add new Measurement Points&#34;. Then select &#34;Edit&#34; and put measurement points on top of the two surface objects of the tip of the R7 axon terminal in M6 layer. These are now defined as start-points.</li>
		<li>Select &#34;Add new Measurement Points&#34;. Then select &#34;Edit&#34; and put measurement points on the bottom of the two surface objects of the tip of the R7 axon terminal in M6 layer. These are now defined as end-points (Figure 3E).</li>
	</ol>
	</li>
</ol>
4. Calculation of the Number of Spots, Distribution of the Spot Frequency and Level of Enrichment of Brp-GFP with a Customized Image Data Analysis Software
<ol>
	<li>Copy the file &#34;XtquantifySynapseSNR.m&#34; and the &#34;functions&#34; folder into the Imaris XT matlab folder. Note that the calculation in this study was performed with a customized plugin implemented in Matlab. Download the plugin from the website (<a href="https://github.com/cmohl2013/synapse_trafo" target="_blank">https://github.com/cmohl2013/synapse_trafo</a>).</li>
	<li>Open a dataset with n spots, n surfaces and 2 measurement point objects, each containing n measurement points (prepared from 3.1 - 3.4). The first measurement point object defines the start positions for all axons. The second measurement point object defines the end position of all axons. Both measurement point objects need to have the same number of measurement points (that is the number of axons).</li>
	<li>Execute image processing&#62;&#62;CUSTOM&#62;atsushis synapse detection Vs 6.</li>
	<li>Check metadata and change pixel sizes in image analysis software in case it is not correct.</li>
	<li>Select channel for the performance of the intensity analysis of spots regions and cytoplasmic regions (the channel with Brp-GFP).</li>
	<li>Define the filename for exporting the results.</li>
	<li>Define the number of bins and axon length in percent. Enter the number of bins as &#34;10&#34; and range of bins as &#34;100%&#34; (Figures 4A and 4B).</li>
	<li>Define the regions of the spots and the other cytoplasmic regions for the Brp-GFP delocalization analysis. Enter spots radius as &#34;0.35 &#181;m&#34; and width of surrounding area as &#34;50 &#181;m&#34;. The spots radius defines the region for the puncta GFP signal (0.35 &#181;m diameter, centered on one punctum). The width of surrounding area defines the region for the GFP signal in the cytoplasmic area (50 &#181;m diameter, centered on the punctum, but including only R8 and excluding all spot areas in the cytoplasm) (Figures 4A and 4C).</li>
	<li>Wait until all neuron masks are processed and exported as PDF files (Figures 4B and 4C) and a csv table for further analysis in a spreadsheet. The csv table includes the number of the spots, the average signal intensity in the &#34;cytoplasmic area&#34; and the maximum signal intensity in the &#34;spot&#34; in each bin for all R8 axons.</li>
	<li>Open a dataset prepared in 3.5 for subtraction of the background signal.</li>
	<li>Perform the image processing as described from 4.3 - 4.9.</li>
	<li>Calculate the average of the total number and the distribution of the Brp-GFP puncta in a spreadsheet (Figures 4D and 4E).</li>
	<li>Calculate the average of the delocalization of Brp-GFP signal in a spreadsheet by the ratio of the average intensity in the &#34;cytoplasmic area&#34; divided by the maximum intensity in the &#34;spot&#34; subtracted by the average intensity in the background signal (Figure 4F).</li>
</ol>

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The authors have nothing to disclose.

In this study, we showed how to prepare the light conditions to expose flies to an equal light intensity. We quantified not only the number of a synaptic marker puncta but could also spatially resolve the density of synapses along axons and measure the delocalization level of the marker protein in cytoplasmic areas. These three assessments allow us to evaluate the details of synaptic dynamics at single neuron level in different environmental conditions. Our protocol can be adapted to different synaptic proteins but also ...

The modulation of synaptic function contributes to the remarkable ability of the nervous system to precisely respond or adapt to changing environmental stimuli. Adjusting the presynaptic vesicle release probability is one way of controlling synaptic strength1. Synaptic vesicle release takes place at the Active Zone (AZ), a specialized region of the presynaptic membrane2. The AZ is characterized by a cassette of specific proteins3,4. Most proteins contributing to AZ assembly are highly conserved in nematodes, insects and mammals5. Recent studies suggest that the level of neuronal activity regulates the molecular composition of the AZ, which in turn contributes to the regulation of the functional output both in vitro and in vivo6,7,8. We previously found that photoreceptor AZs undergo molecular remodeling in Drosophila after prolonged exposure to natural ambient light9. In this condition, we observed that the number of Bruchpilot (Brp)-positive AZs was reduced in the photoreceptor axons.
The Brp/CAST/ELKS family proteins are fundamental building blocks of AZs in vertebrate and invertebrate synapses10. In Drosophila brp mutants, evoked vesicle release is suppressed11,12. The 17 C-terminal amino acid residues of Brp are essential for synaptic vesicle clustering at the Drosophila Neuromuscular Junction (NMJ)13,14. These studies demonstrated the central role of this molecule in AZ organization and function. With a recently developed genetic tool, Synaptic Tagging with Recombination (STaR), Brp can be observed in vivo in specific cell types, at endogenous expression levels and at a single synapse resolution15. This tool makes it feasible to evaluate the endogenous dynamics of synapses quantitatively in the complex central nervous system.
There have been several studies including synapse quantifications based on data obtained from confocal microscopy. Synaptic alterations have been evaluated by measuring the length, the area, the volume, the density and counting the number based on sophisticated software applications. For instance, the freeware ImageJ provides a quantification method for total synaptic area and synaptic density measures at the Drosophila NMJ16. The number of colocalization sites of pre and postsynaptic markers have been quantified using the plugin &#34;Puncta Analyzer&#34; available on the ImageJ software platform17. Alternatively, a multi-paradigm numerical computing environment based program, Synapse Detector (SynD), can automatically trace dendrites of neurons labeled with a fluorescent marker, and then quantifies the synaptic protein levels as a function of distance from the cell body18. The software Synaptic Puncta Analysis (SynPAnal), has been designed for the rapid analysis of 2D images of neurons acquired from confocal or fluorescent microscopy. The primary function of this software is the automatic and rapid quantification of density and intensity of protein puncta19. Recently, an automatic learning-based synapse detection algorithm has been generated for quantification of synaptic number in 3D20, taking advantage of the 3D Visualization-Assisted Analysis (Vaa3D) software21.
Commercial image analysis software are also powerful tools for synaptic quantifications. For instance, the fluorescently labeled neurotransmitter receptors or a presynaptic AZ component have been quantified in three dimensions with single-synapse resolution in C. elegans22 or the Drosophila olfactory system23,24, allowing hundreds of synapses to be rapidly characterized within a single sample.
Here, we present a method by a customized image analysis software plug-in implemented in a multi-paradigm numerical computing environment that allows to analyze semi-automatically multiple aspects of AZs, including their number, distribution and the level of enrichment of molecular components to the AZ. Thus, this complex analysis allowed us to evaluate the dynamics of synaptic components in axon terminals under different environmental conditions. We investigated the effect of light exposure on the output synapses of adult fly photoreceptors. The procedure is performed in three steps: 1) preparation for light exposure, 2) dissection, immunohistochemistry and confocal imaging, and 3) image analysis.

<table><tbody><tr><td>Vial</td><td>Hightech, Japan</td><td>MKC-20</td><td></td></tr><tr><td>Plug</td><td>Thermo Fisher Sciehtific, USA</td><td>AS-275</td><td></td></tr><tr><td>Customized transparent rack made of acrylic resin&amp;nbsp;</td><td>Shin-Shin Corporation, Japan</td><td></td><td>a height of 41 cm, a base of 21 cm, a thickness of 4 cm and a height of 13 cm for each step</td></tr><tr><td>Cool incubator</td><td>MITSUBISHI ELECTRIC, Japan</td><td>CN-40A</td><td></td></tr><tr><td>LED panel</td><td>MISUMI, Japan</td><td>LEDXC170-W</td><td></td></tr><tr><td>Digital light meter</td><td>CEM</td><td>DT-1301</td><td></td></tr><tr><td>Fly pad</td><td>Tokken, Japan</td><td>TK-HA03-S</td><td></td></tr><tr><td>Petri dish (35 x 10 mm)</td><td>Greiner Bio-One International, Germany</td><td>627102</td><td></td></tr><tr><td>PBS tablet</td><td>Takara, Japan</td><td>T900</td><td></td></tr><tr><td>Triton X-100</td><td>Wako, Japan</td><td>160-24751</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>1.5 mL tube</td><td>Sarstedt, Germany</td><td>A. 152X</td><td></td></tr><tr><td>Formaldehyde 16%</td><td>NEM, Japan</td><td>3152</td><td></td></tr><tr><td>Pipetman P-200</td><td>Gilson</td><td>F123601&amp;nbsp;</td><td></td></tr><tr><td>Pipetman P-20</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>Pipetman P-2</td><td>Gilson</td><td>F144801</td><td></td></tr><tr><td>anti-chaoptin antibody</td><td>DSHB</td><td>24B10</td><td></td></tr><tr><td>Alexa568-conjugated anti-mouse antibody</td><td>Life Technologies</td><td>A-11031</td><td></td></tr><tr><td>VECTASHIELD Mounting Medium</td><td>Vector Laboratories, Inc.</td><td>H-1000</td><td></td></tr><tr><td>Microscope slide (76 x 26 mm)</td><td>Thermo Fisher Scientific Gerhard Menzel B.V. &amp;amp; Co. KG, Germany</td><td></td><td></td></tr><tr><td>Coverslip (18 x 18 mm, 0.17 mm)</td><td>Zeiss, Germany</td><td>474030-9000-000</td><td></td></tr><tr><td>Industrial Microscopes</td><td>Olympus, Japan</td><td>SZ61-C-SET</td><td></td></tr><tr><td>Stereo Microscope Lighting</td><td>Olympus, Japan</td><td>KL 1600 LED</td><td></td></tr><tr><td>confocal microscopy</td><td>Zeiss, Germany</td><td>LSM780</td><td></td></tr><tr><td>Imaris</td><td>Bitplane, Switzerland</td><td></td><td>Version 7.6.4 or above</td></tr><tr><td>Matlab</td><td>The MathWorks, Inc., USA</td><td></td><td></td></tr><tr><td>Excel for Mac&amp;nbsp;</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

analyzing synaptic modulation of drosophila melanogaster photoreceptors after exposure to prolonged light

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

The compound eye of Drosophila comprises ~780 ommatidia, each containing eight types of photoreceptors (R1-8). R7 and R8 project their axons to the second optic ganglion, the medulla, where they form synapses in layers M6 and M3, respectively26. To investigate the effect of prolonged exposure to light on the molecular composition of photoreceptor R8 active zones, we took advantage of the STaR method15. Endogenous expression levels o...

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

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.

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JoVE Journal

Neuroscience

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Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

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