Name: detectsyn: a rapid, unbiased fluorescent method to detect changes in synapse density
Uploaded: 2022-07-22T12:45:00
Description: Watch this Scientific Journal Video about DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density at JoVE.com

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 technique<sup class=...

<ol>
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(65), e3564 (2012).</li><li>Workman, E. R., Niere, F., Raab-Graham, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1-dependent+protein+synthesis+underlying+rapid+antidepressant+effect+requires+GABABR+signaling.">mTORC1-dependent protein synthesis underlying rapid antidepressant effect requires GABABR signaling.</a> Neuropharmacology. 73, 192-203 (2013).</li><li>Li, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTOR-dependent+synapse+formation+underlies+the+rapid+antidepressant+effects+of+NMDA+antagonists.">mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists.</a> Science. 329 (5994), 959-964 (2010).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 diH20 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&#176;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 &#181;L to 500 &#181;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 ...

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