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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 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.
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 ne....
Isolation of cells and tissue from animals was in accordance with the National Institutes of Health'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
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.......
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.......
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 a grant from FRAXA Research (CFH) and the Alzheimer's Association, AARG-NTF-21-852843 (KRG), AARF-19-614794-RAPID (KRG).Â
....Name | Company | Catalog Number | Comments |
10x PBS | Fisher Scientific | BP39920 | PBS made in house works, as well. |
24 well plates | Fisher Scientific | FB012929 | For tissue slices, pre-sterilized plates may be unnecessary. |
50 mL conical tubes | Fisher Scientific | 14-432-22 | |
Aluminium foil | Fisher Scientific | 15-078-290 | |
Chicken anti-MAP2 antibody | Abcam | ab5392 | |
Clear nail polish | Fisher Scientific | NC1849418 | Other clear nail polish works, as well. |
Cold block | Fisher Scientific | 13131012 | |
Computer workstation | HP | ||
Confocal or fluorescent microscope | Nikon | A1R HD25 | |
Donkey anti-chicken FITC | Fisher Scientific | SA1-72000 | |
Duolink donkey anti-Mouse PLUS | Sigma | DUO92001 | |
Duolink donkey anti-Rabbit MINUS | Sigma | DUO92005 | |
Duolink In Situ Detection Reagents Far Red | Sigma | DUO92013 | Contains ligation stock, amplification stock, ligase, and polymerase. |
Duolink In Situ Mounting Medium with DAPI | Sigma | DUO82040 | |
Duolink In Situ Wash Buffers, Fluorescence | Sigma | DUO82049 | Contains Wash Buffer A and Wash Buffer B; dilute Wash Buffer B to 1% in diH20 for 1% Wash Buffer B. |
Fine-tipped paintbrush | Fisher Scientific | NC9691026 | Sable hair, size 00 or 000, can also find at craft stores |
Fisherbrand Cover Glasses: Rectangles | Fisher Scientific | 12545MP | Cover glass is unnecessary for cultured neurons already on glass coverslips. |
Fisherbrand Superfrost Plus Microscope Slides | Fisher Scientific | 1255015 | For cultured neurons already on glass coverslips, Superfrost slides may be unnecessary. |
Freezer, -20°C | VWR | 76449-108 | |
Glass coverslips | Fisher Scientific | 125480 | |
Glycine | Fisher Scientific | BP381-1 | |
Image processing software | e.g. NIS Elements, ImageJ | ||
Incubator | Fisher Scientific | 15-015-2633 | |
Large petri dish, 100mm | Fisher Scientific | FB0875712 | |
Molecular grade water | Fisher Scientific | BP24701 | |
Mouse anti-Synapsin1 antibody | Synaptic Systems | 106-011 | |
Normal donkey serum | Jackson ImmunoResearch | 017-000-121 | |
Orbital shaker | Fisher Scientific | 02-106-1013 | |
Parafilm | Fisher Scientific | 13-374-10 | |
Pipette tips | Fisher Scientific | 02-707-025 | |
Pipettes | Fisher Scientific | 14-388-100 | Working volumes range from 3 µL to 500 µL |
Plastic pasteur pipette | Fisher Scientific | 02-708-006 | |
Precision tweezers/foreceps | Fisher Scientific | 12-000-122 | |
Rabbit anti-PSD95 antibody | Abcam | ab18258 | Other antibody pairs may work, as well, with optimization. |
Refrigerator | VWR | 76470-402 | |
Small petri dish, 60 mm | Fisher Scientific | FB0875713A | |
Timer | Fisher Scientific | 14-649-17 | |
Tween 20 | Fisher Scientific | BP337-100 |
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