This work was supported by National Institutes of Health NINDS R01 NS105005 (KRG) and NS105005-03S1 (KRG), Department of Defense USAMRMC W81XWH-14-1-0061 (KRG), NIAAA R01AA016852, NIAAA T32AA007565 (CFH), and&#160;a grant from FRAXA Research (CFH) and the Alzheimer&#39;s Association, AARG-NTF-21-852843 (KRG), AARF-19-614794-RAPID (KRG).&#160;

Isolation of cells and tissue from animals was in accordance with the National Institutes of Health&#39;s Guide for the Care and Use of Laboratory Animals and approved by the Wake Forest Institutional Animal Care and Use Committee
NOTE: This protocol is used on samples already treated and fixed per specific experimental paradigms and requirements. For demonstration purposes, synapse formation due to rapid antidepressant treatment is used to highlight this synapse detection technique6. Neurons previously cultured on coverslips, treated, fixed in 4% paraformaldehyde (PFA), and stored in 1x phosphate-buffered saline (PBS) will be used to highlight the in vitro procedures. Previously sliced hippocampal tissue (25 &#181;m thick) from mice treated, transcardially perfused with ice-cold PBS and 4% PFA, and then stored in cryoprotectant will be used to highlight the slice procedures. Please see11,12 for more information about how to culture neurons or transcardially perfuse rodents. See Figure 1 for a graphical representation of this procedure.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63139/63139fig01.jpg" /> 
Figure 1: Graphical representation of DetectSyn assay. After permeabilizing cell membranes, primary antibodies for Synapsin1 and PSD95 bind to these synaptic proteins. Secondaries with oligonucleotide tags then bind to the primary antibodies. If Synapsin1 and PSD95 are within 40 nm, as at a synapse, then the oligonucleotides interact, and a fluorescent tag is amplified. This fluorescent signal can then be imaged via standard microscopy and analyzed. <a href="https://www.jove.com/files/ftp_upload/63139/63139fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Rinse samples
<ol>
	<li>Rinse the samples with 500 &#181;L of 1x PBS + 0.75% glycine for 5 min 3 times with gentle agitation on an orbital shaker to remove residual PFA or cryoprotectant.</li>
</ol>
2. Block and permeabilize samples
<ol>
	<li>Prepare blocking and permeabilization solution (10% normal donkey serum, 0.25% Tween 20) in 1x PBS. Prepare enough to use for blocking, primary, and secondary incubations.</li>
	<li>To samples (e.g., coverslips or free-floating slices) in 24 well plates, add 500 &#181;L of blocking and permeabilization solution. Ensure each well contains a different sample and is appropriately labeled to prevent samples from being switched.</li>
	<li>Incubate the samples at room temperature (RT) for 60 min for cultured cells or 2 h for sliced tissue. Use an orbital shaker for gentle agitation.</li>
</ol>
3. Incubate samples in primary antibodies
<ol>
	<li>Prepare primary antibodies in blocking buffer:
	<ol>
		<li>Prepare Postsynaptic density 95 (PSD95; 1:500, rabbit polyclonal), Synapsin1 (1:500, mouse monoclonal), MAP2 (1:400, chicken polyclonal)</li>
		<li>Prepare a negative control aliquot that omits one of the synaptic pairs (e.g., without PSD95)</li>
	</ol>
	</li>
	<li>Carefully remove the blocking solution with a plastic Pasteur pipette. Try to remove as much as possible without disturbing the cells or tearing tissue.</li>
	<li>For cultured cells:
	<ol>
		<li>Line a large plastic petri dish with parafilm. Carefully transfer coverslips to the parafilm using forceps.</li>
		<li>Carefully add 60 &#181;L of the primary antibody solution to the top of the coverslips. Make sure not to spill the primary antibody solution over the side of the coverslip.</li>
		<li>To provide humidity and prevent samples from drying out during the incubation period, add ultrapure water to a smaller Petri dish and carefully arrange the small Petri dish around the coverslips.</li>
		<li>Cover the large Petri dish and incubate cultured cells for 1 h at RT.</li>
	</ol>
	</li>
	<li>For sliced tissue:
	<ol>
		<li>Carefully add 250 &#181;L of the primary antibody solution to free-floating slices in a 24 well plate.</li>
		<li>Cover the plate and incubate the tissue overnight at 4 &#176;C with gentle agitation on an orbital shaker.</li>
	</ol>
	</li>
</ol>
4. Wash samples, then incubate in secondary antibodies
<ol>
	<li>Prepare secondary antibodies in the blocking buffer:
	<ol>
		<li>Prepare Donkey anti-mouse (1:5), donkey anti-rabbit (1:5), donkey anti-chicken (1:400).</li>
		<li>At this step, additional technical controls can be obtained by preparing a secondary aliquot that omits either the anti-mouse or anti-rabbit secondary.</li>
	</ol>
	</li>
	<li>For cultured cells:
	<ol>
		<li>Using forceps, carefully tap off the primary solution from coverslips onto a paper towel</li>
		<li>Using forceps, carefully transfer the coverslips back to their original 24 well plate filled with 500 &#181;L of 1x PBS.</li>
	</ol>
	</li>
	<li>For sliced tissue:
	<ol>
		<li>Carefully remove the primary antibody solution with a plastic Pasteur pipette. Try to remove as much as possible without tearing tissue.</li>
		<li>Add 500 &#181;L of 1x PBS</li>
	</ol>
	</li>
	<li>Wash the samples for 10 min 3 times in 1x PBS with gentle agitation on an orbital shaker. During this time, bring all wash buffers to RT.
	<ol>
		<li>During this time, change the parafilm in the large Petri dish</li>
	</ol>
	</li>
	<li>For cultured cells:
	<ol>
		<li>Using forceps, carefully transfer the coverslips back to the parafilmed large Petri dish</li>
		<li>Carefully add 40 &#181;L of the secondary antibody solution to the top of the coverslips. Make sure not to spill the secondary antibody solution over the side of the coverslip.</li>
		<li>If needed, add more ultrapure water to a smaller petri dish and carefully arrange the small petri dish around coverslips.</li>
		<li>Cover the large Petri dish</li>
	</ol>
	</li>
	<li>For sliced tissue:
	<ol>
		<li>Carefully add 250 &#181;L of the secondary antibody solution to free-floating slices in a 24 well plate.</li>
		<li>Cover the plate 
		NOTE: From here on, protect samples from light by wrapping the tops of the plates with foil.</li>
	</ol>
	</li>
	<li>Incubate the samples at 37 &#176;C for 1 h</li>
</ol>
5. Ligation
<ol>
	<li>Mix the ligation stock 1:5 in molecular-grade water.</li>
	<li>As in section 4, carefully transfer the coverslips and remove the secondary mix from the sliced tissue.</li>
	<li>Wash the samples in 500 &#181;L of wash buffer A
	<ol>
		<li>For cultured cells, wash 2 times for 5 min. For sliced tissue, wash 2 times for 10 min. Use gentle agitation on an orbital shaker for both.</li>
		<li>During this time, change the parafilm in the large Petri dish</li>
	</ol>
	</li>
	<li>While keeping the ligase on a cold block, dilute the ligase 1:40 in the ligation stock from step 5.1. Perform this dilution immediately before adding the ligase to the samples.</li>
	<li>As in section 4, remove as much of the wash buffer A as possible from samples before adding the ligase.</li>
	<li>For cultured cells: Transfer coverslips back to the parafilmed Petri dish. Add 40 &#181;L of the ligation mix to coverslips, arrange small water-filled Petri dishes around the coverslips, and cover the large Petri dish.</li>
	<li>For sliced tissue: Add 250 &#181;L of the ligation mix from Step 5.4 to each well and cover the plate.</li>
	<li>Incubate the samples for 30 min at 37 &#176;C.</li>
</ol>
6. Amplification
<ol>
	<li>Mix the amplification stock 1:5 in molecular-grade water.</li>
	<li>As in section 4, carefully transfer the coverslips and remove the ligation mix from the sliced tissue.</li>
	<li>Wash samples in 500 &#181;L of wash buffer A
	<ol>
		<li>For cultured cells, wash 2 times for 2 min. For sliced tissue, wash 2 times for 10 min. Use gentle agitation on an orbital shaker for both.</li>
		<li>During this time, change the parafilm in the large Petri dish</li>
	</ol>
	</li>
	<li>Perform this dilution immediately before adding the polymerase to samples. While keeping the polymerase on a cold block, dilute polymerase
	<ol>
		<li>For cultured cells, dilute polymerase 1:80 in the amplification stock from step 6.1.</li>
		<li>For sliced tissue, dilute polymerase 1:40 in the amplification stock from step 6.1.</li>
	</ol>
	</li>
	<li>As in step 4, remove as much of the wash buffer A as possible from samples before adding the polymerase.</li>
	<li>For cultured cells: Transfer the coverslips back to the parafilmed Petri dish. Add 40 &#181;L of the amplification mix from Step 6.4.1 to coverslips, arrange small water-filled Petri dishes around the coverslips, and cover the large Petri dish. Incubate the samples for 100 min at 37 &#176;C.</li>
	<li>For sliced tissue: Add 250 &#181;L of the amplification mix from Step 6.4.2 to each well and cover the plate. Incubate the samples for 2 h at 37 &#176;C. 
	NOTE: During this time, prepare and label slides.</li>
</ol>
7. Mounting
<ol>
	<li>As in Step 4, carefully transfer the coverslips and remove the amplification mix from the sliced tissue.</li>
	<li>Wash the samples in 500 &#181;L of wash buffer B 2 times for 10 min with gentle agitation on an orbital shaker.</li>
	<li>Wash the samples in 500 &#181;L of 1% wash buffer B for 1 min with gentle agitation on an orbital shaker.</li>
	<li>For cultured cells:
	<ol>
		<li>Drop 3 &#181;L of mounting media onto a slide</li>
		<li>Tap off excess wash buffer from the coverslip and then place the coverslip (with cells facing down) into the mounting media. Seal the sides with a small amount of clear nail polish to seal the coverslip in place.</li>
	</ol>
	</li>
	<li>For sliced tissue:
	<ol>
		<li>Carefully transfer a tissue slice to the prepared slide and arrange it, so the slice is lying flat. Drop between 5-10 &#181;L of mounting media (amount will depend on the size of the slice) onto tissue slice</li>
		<li>Carefully place a glass coverslip over tissue slice and seal with a small amount of clear nail polish along the edge to seal the coverslip in place.</li>
	</ol>
	</li>
	<li>Wait at least 15 min before analyzing under the microscope, or store at -20 &#176;C.</li>
</ol>
8. Obtain digital images with a confocal microscope
<ol>
	<li>Optimize the acquisition settings (e.g., laser power, gain, offset) across samples from all treatments. Ensure that the optimization includes decreasing background noise and enhancing the signal without oversaturating the intensity of the fluorescent signals. Once settings are determined, apply the same acquisition settings across all images obtained. 
	NOTE: The following acquisition details can be used with a Nikon A1 confocal microscope and the Nikon NIS AR Elements software.</li>
	<li>Set the slide with sample on the stage and find the focal plane for the sample using DAPI through the eyepiece.</li>
	<li>Turn off the eye port by clicking on Eye port and choose an optical configuration button to adjust the settings.</li>
	<li>Adjust the gain, offset, and laser power for each fluorescent channel to decrease background noise and enhance the fluorescent signal. Make sure the fluorescent signal does not become oversaturated as mentioned in steps 8.5-8.6.</li>
	<li>Monitor oversaturation using a pseudocolor for the fluorescent signal. At the bottom of the live image, right-click the tab labeled with the fluorescent channel currently being used.</li>
	<li>Next, choose Channel Coloring and pick a pseudocolor like Rainbow Dark to visualize the fluorescence intensity in a heat-map-like pseudocolor. In Rainbow Dark, cooler colors indicate less fluorescent intensity, and hotter colors indicate more fluorescent intensity.</li>
	<li>Once all fluorescent channels are optimized, right-click on the optical configuration button previously chosen and choose Assign Current Camera Setting for this button.</li>
	<li>Verify that the chosen settings are sufficient for a random sample from each treatment group. If the chosen settings oversaturate any of these samples, repeat step 8.4 to eliminate the oversaturation.</li>
	<li>For cultured neurons, follow steps 8.10-8.16.</li>
	<li>Using the eye-port, search for a neuron with dendrites that have minimal overlap with other dendrites.</li>
	<li>Turn off the eye port and use the DAPI channel to visualize the cell body of the chosen neuron. Double-click on the center of the soma to center the neuron in the middle of the field of view.</li>
	<li>Using the MAP2 channel, find the best plane of focus for the MAP2 signal with live scanning.</li>
	<li>Under the ND Acquisition tab, click on Save to File and choose a file to save the image into under Browse. Then, input the filename.</li>
	<li>Under the Z tab, select the Symmetric Mode Defined by Range option. Set the focus to the best MAP2 plane and click on the Relative button to set this focal plane as the middle of the z-stack.</li>
	<li>Set the range to 5 &#181;m with 1 &#181;m steps, and make sure to check Close Active Shutter During Z Movement. Under the Wavelength tab, select the name of the optical button with the previously configured acquisition settings under Optical Conf. Then, click on Run Now.</li>
	<li>Repeat steps 8.1.10-8.1.15 for about 10 neurons per coverslip/treatment.</li>
	<li>For sliced tissue, follow steps 8.18-8.22.</li>
	<li>Using the eye-port, search for the region of interest. For example, locate CA1 of the hippocampus.</li>
	<li>Turn off the eye-port and use the MAP2 channel to find the best plane of focus for the MAP2 signal with live scanning.</li>
	<li>Under the Acquire menu, choose Scan Large Image. Next, select the optical button&#39;s name with the previously configured acquisition settings under the Capturing panel from the panel that opens. Also, ensure to select the correct objective in this panel.</li>
	<li>Under the Area panel and the eyepiece, use the arrow keys to set the boundaries of the region of interest. Next, click on Save Large Image to File and create a save path filename for the image.</li>
	<li>Under the Setup panel, make sure Multichannel Capture is checked, and then choose the name of the optical button with the previously configured acquisition settings under Optical Conf. 
	NOTE: A z-stack for a large image is possible but will increase the scan time.</li>
</ol>
9. Analysis
<ol>
	<li>Similar to acquisition settings, use samples from all treatments to optimize threshold settings. Ensure that the threshold optimization focuses on decreasing background noise and enhancing the signal without oversaturating the intensity of the fluorescent signals. Once these settings are determined, apply the same threshold settings across all images used for analysis as described in steps 9.2-9.3.</li>
	<li>In ImageJ, the threshold option is located under the menu Image &#62; Adjust &#62; Threshold. Choose the Dark Background option if the image has a dark background.</li>
	<li>Next, adjust the upper and lower bounds of the threshold per previously determined optimized threshold settings, then click on Apply.</li>
	<li>For cultured cells, use the MAP2 channel and a freehand region of interest (ROI) tool to draw an ROI for each neuron, including dendrites and soma. For sliced tissue, draw a freehand ROI within the slice image that encapsulates the area of interest (e.g., stratum radiatum of the CA1 within the hippocampus).</li>
	<li>Obtain the area of the ROI. In ImageJ, measure the area under the menu Analyze &#62; Measure.</li>
	<li>Detect the number of puncta within each ROI using an automatic detection tool like Particle Analysis in ImageJ following steps 9.7-9.9.</li>
	<li>Find the Particle Analysis option under the menu Analyze &#62; Analyze Particles. First, define the puncta size diameter, typically 0.1-3 &#181;m2.</li>
	<li>Next, choose the Overlay Masks option from the Show drop-down menu and check the Display Results option. Then, click on OK.</li>
	<li>If puncta are not detected with the diameter range chosen, adjust the range until all puncta are detected with this analysis. Make sure to use the same Particle Analysis settings for all images.</li>
	<li>Divide the number of puncta by the area of an individual region of interest by following steps 9.11-9.13.</li>
	<li>Copy and paste the results for each image from the Results pop-up from ImageJ into a spreadsheet.</li>
	<li>First, identify which file and sample the data were obtained from. Then, divide the area of the ROI by the number of puncta.</li>
	<li>Then, clear the data from the&#160;Results&#160;pop-up, and repeat steps 9.2-9.12.</li>
	<li>Normalize results to control samples: Average the results (number of puncta/ROI area) for the control samples. Then, divide the obtained results of all samples by the average of the control to obtain the normalized results. The new average of the control samples should be equal to 1.</li>
</ol>

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The authors report no conflict of interest.

DetectSyn is a rapid assay that uses a proximity ligation assay to detect proteins within 40 nm of each other, which allows for the detection of synapse formation. This technique improves current fluorescent assays, which serve only as proxy measurements for synapse formation. DetectSyn detects quantifiable changes in synaptic proteins localized within 40 nm, i.e., within the synaptic cleft, of each other. Further, DetectSyn is more cost-effective and takes less time than techniques, like electron microscopy and array to...

Synapses are the fundamental unit of communication between neurons1. Many synapses between neurons within the same regions give rise to circuits that mediate behavior2. Synapses consist of a presynaptic terminal from one neuron that releases neurotransmitters or neuropeptides that relay information to postsynaptic receptors of another neuron. The summation of presynaptic signals determines whether the postsynaptic neuron will fire an action potential and propagate the message to other neurons.
Synaptopathology, the break down of synapses, arises in diseases and disorders marked by decreased neural volume, like Alzheimer&#39;s disease and major depressive disorder, resulting in circuits that no longer optimally perform3,4,5. Restoring synapse density likely underlies the efficacy of potential treatments for these disorders. For example, it was recently demonstrated that increasing synapses underlies the behavioral efficacy of rapid antidepressants6. To rapidly screen possible synaptopathology treatments, researchers require techniques that quickly identify changes to synapse numbers.
Current methodologies are either time-consuming and expensive (electron microscopy, array tomography), or they only examine postsynaptic changes without incorporating presynaptic engagement (spine analyses, immunofluorescence/colocalization). Dyes like DiI or fluorescent proteins like GFP help visualize neurons and characterize postsynaptic spines. However, spine analysis uses researcher-defined ratios to determine morphology, which can decrease reproducibility7. Further, how the different spine classes relate to functional synapses is still being uncovered8. Spine formation can be transient and may reflect postsynaptic plasticity, but these spines could be eliminated before stabilizing into a synapse with a presynaptic neuron9.
Colocalization provides a better proxy for synapses than spine analysis because one can immunostain for presynaptic and postsynaptic proteins. However, synaptic proteins may yield low colocalization values because the proteins are juxtaposed and may not consistently overlap. Thus, because the proteins are not wholly superimposed, colocalization techniques may not accurately measure changes to synapse formation due to this missing information. Finally, although both electron microscopy (EM) and array tomography provide high-resolution images of synapses, they are time-consuming. EM further requires specialized equipment, and researchers are limited to small volumes of tissue for any given experiment. While array tomography elegantly provides the ability to screen for many proteins on ultrathin sections and can be combined with EM10, this technique may be too labor-intensive and beyond the scope of experiments that need to scan rapidly for changes to synapse formation.
DetectSyn is a specific application of the Duolink Proximity Ligation Assay. The PLA assay allows for the general detection of protein-protein interactions. DetectSyn bridges proxy postsynaptic measures by amplifying a fluorescent signal emitted by tagged pre- and postsynaptic proteins within 40 nm of each other. If the synaptic proteins are within 40 nm, as within a synaptic cleft, then the secondary antibodies, which contain DNA probes, will hybridize into circular DNA. This hybridized circular DNA expresses a fluorescent probe, which is then amplified and detected with standard fluorescent microscopy techniques (see Figure 1). Crucially, unlike EM and array tomography, this technique does not require specialized equipment and takes about the same amount of time as standard immunohistochemistry. The accessibility of this technique, thus, enables investigators outside of research-intensive institutions to participate in synaptopathology research. Further, this technique can examine changes to synaptic density in multiple brain regions within a single experiment, offering a more holistic representation of synaptic changes due to disease or treatment.

<table><tbody><tr><td>10x PBS</td><td>Fisher Scientific</td><td>BP39920</td><td>PBS made in house works, as well.</td></tr><tr><td>24 well plates</td><td>Fisher Scientific</td><td>FB012929</td><td>For tissue slices, pre-sterilized plates may be unnecessary.</td></tr><tr><td>50 mL conical tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Aluminium foil</td><td>Fisher Scientific</td><td>15-078-290</td><td></td></tr><tr><td>Chicken anti-MAP2 antibody</td><td>Abcam</td><td>ab5392</td><td></td></tr><tr><td>Clear nail polish</td><td>Fisher Scientific</td><td>NC1849418</td><td>Other clear nail polish works, as well.</td></tr><tr><td>Cold block</td><td>Fisher Scientific</td><td>13131012</td><td></td></tr><tr><td>Computer workstation</td><td>HP</td><td></td><td></td></tr><tr><td>Confocal or fluorescent microscope</td><td>Nikon</td><td>A1R HD25</td><td></td></tr><tr><td>Donkey anti-chicken FITC</td><td>Fisher Scientific</td><td>SA1-72000</td><td></td></tr><tr><td>Duolink donkey anti-Mouse PLUS</td><td>Sigma</td><td>DUO92001</td><td></td></tr><tr><td>Duolink donkey anti-Rabbit MINUS</td><td>Sigma</td><td>DUO92005</td><td></td></tr><tr><td>Duolink In Situ Detection Reagents Far Red</td><td>Sigma</td><td>DUO92013</td><td>Contains ligation stock, amplification stock, ligase, and polymerase.</td></tr><tr><td>Duolink In Situ Mounting Medium with DAPI</td><td>Sigma</td><td>DUO82040</td><td></td></tr><tr><td>Duolink In Situ Wash Buffers, Fluorescence</td><td>Sigma</td><td>DUO82049</td><td>Contains Wash Buffer A and Wash Buffer B; dilute Wash Buffer B to 1% in diH&lt;sub&gt;2&lt;/sub&gt;0 for 1% Wash Buffer B.</td></tr><tr><td>Fine-tipped paintbrush</td><td>Fisher Scientific</td><td>NC9691026</td><td>Sable hair, size 00 or 000, can also find at craft stores</td></tr><tr><td>Fisherbrand Cover Glasses: Rectangles</td><td>Fisher Scientific</td><td>12545MP</td><td>Cover glass is unnecessary for cultured neurons already on glass coverslips.</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>1255015</td><td>For cultured neurons already on glass coverslips, Superfrost slides may be unnecessary.</td></tr><tr><td>Freezer, -20&amp;deg;C</td><td>VWR</td><td>76449-108</td><td></td></tr><tr><td>Glass coverslips</td><td>Fisher Scientific</td><td>125480</td><td></td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-1</td><td></td></tr><tr><td>Image processing software</td><td>e.g. NIS Elements, ImageJ</td><td></td><td></td></tr><tr><td>Incubator</td><td>Fisher Scientific</td><td>15-015-2633</td><td></td></tr><tr><td>Large petri dish, 100mm</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Molecular grade water</td><td>Fisher Scientific</td><td>BP24701</td><td></td></tr><tr><td>Mouse anti-Synapsin1 antibody</td><td>Synaptic Systems</td><td>106-011</td><td></td></tr><tr><td>Normal donkey serum</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td></td></tr><tr><td>Orbital shaker</td><td>Fisher Scientific</td><td>02-106-1013</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-10</td><td></td></tr><tr><td>Pipette tips</td><td>Fisher Scientific</td><td>02-707-025</td><td></td></tr><tr><td>Pipettes</td><td>Fisher Scientific</td><td>14-388-100</td><td>Working volumes range from 3 &amp;micro;L to 500 &amp;micro;L</td></tr><tr><td>Plastic pasteur pipette</td><td>Fisher Scientific</td><td>02-708-006</td><td></td></tr><tr><td>Precision tweezers/foreceps</td><td>Fisher Scientific</td><td>12-000-122</td><td></td></tr><tr><td>Rabbit anti-PSD95 antibody</td><td>Abcam</td><td>ab18258</td><td>Other antibody pairs may work, as well, with optimization.</td></tr><tr><td>Refrigerator</td><td>VWR</td><td>76470-402</td><td></td></tr><tr><td>Small petri dish, 60 mm</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Timer</td><td>Fisher Scientific</td><td>14-649-17</td><td></td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>BP337-100</td><td></td></tr></tbody></table>

detectsyn: a rapid, unbiased fluorescent method to detect changes in synapse density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

Data modified from Heaney et al.6 are presented to demonstrate an experiment where increased synapse formation is expected (please see6 for more information and a more in-depth discussion of the mechanism). Previously, it was demonstrated that rapid antidepressants require activation of the inhibitory metabotropic receptor, GABAB (gamma-aminobutyric acid subtype B), to be effective13. Further, previous data indicated that rapid antidepressants increa...

Watch this Scientific Journal Video about DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density at JoVE.com

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

detectsyn-rapid-unbiased-fluorescent-method-to-detect-changes-synapse

Research

JoVE Journal

Biochemistry

3.2K Views.  Wake Forest School of Medicine. DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

Video: DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Neuroscience

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

Presynaptically Silent Synapses Studied with Light Microscopy

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise damaging insults from becoming neurotoxic.  We have been studying a form of presynaptic plasticity that is interesting in part because it is expressed as a digital switching on and off of a presynaptic terminal s ability to release vesicles containing the neurotransmitter glutamate.  Here we demonstrate a protocol for visualizing the activity status of presynaptic terminals in dissociated cell cultures prepared from the rodent hippocampus.  The method relies on detecting active synapses using staining with a fixable form of the styryl dye FM1-43, commonly used to label synaptic vesicles.  This staining profile is compared with immunostaining of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status.   We find that depolarizing stimuli induce presynaptic silencing.  The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways.

Glutamatergic synapses can switch from an active mode to a silent mode. We demonstrate that presynaptic activity status in dissociated culture of rodent neurons is visualized using a fixable form of the FM1-43 dye to visualize active synapses and immunostaining with vGluT-1 antibody to visualize all glutamate synapses.

Synaptic plasticity likely underlies the nervous system's ability to learn and remember and may also represent an adaptability that prevents otherwise ...

Biology

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

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.

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

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 ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Summary

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Chapters in this video

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Automated Quantification of Synaptic Fluorescence in C. elegans

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Presynaptically Silent Synapses Studied with Light Microscopy

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

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

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins