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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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

Representative Results

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.......

Discussion

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.......

Acknowledgements

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). 

....

Materials

NameCompanyCatalog NumberComments
10x PBSFisher ScientificBP39920PBS made in house works, as well.
24 well platesFisher ScientificFB012929For tissue slices, pre-sterilized plates may be unnecessary.
50 mL conical tubesFisher Scientific14-432-22
Aluminium foilFisher Scientific15-078-290
Chicken anti-MAP2 antibodyAbcamab5392
Clear nail polishFisher ScientificNC1849418Other clear nail polish works, as well.
Cold blockFisher Scientific13131012
Computer workstationHP
Confocal or fluorescent microscopeNikonA1R HD25
Donkey anti-chicken FITCFisher ScientificSA1-72000
Duolink donkey anti-Mouse PLUSSigmaDUO92001
Duolink donkey anti-Rabbit MINUSSigmaDUO92005
Duolink In Situ Detection Reagents Far RedSigmaDUO92013Contains ligation stock, amplification stock, ligase, and polymerase.
Duolink In Situ Mounting Medium with DAPISigmaDUO82040
Duolink In Situ Wash Buffers, FluorescenceSigmaDUO82049Contains Wash Buffer A and Wash Buffer B; dilute Wash Buffer B to 1% in diH20 for 1% Wash Buffer B.
Fine-tipped paintbrushFisher ScientificNC9691026Sable hair, size 00 or 000, can also find at craft stores
Fisherbrand Cover Glasses: RectanglesFisher Scientific12545MPCover glass is unnecessary for cultured neurons already on glass coverslips.
Fisherbrand Superfrost Plus Microscope SlidesFisher Scientific1255015For cultured neurons already on glass coverslips, Superfrost slides may be unnecessary.
Freezer, -20°CVWR76449-108
Glass coverslipsFisher Scientific125480
GlycineFisher ScientificBP381-1
Image processing softwaree.g. NIS Elements, ImageJ
IncubatorFisher Scientific15-015-2633
Large petri dish, 100mmFisher ScientificFB0875712
Molecular grade waterFisher ScientificBP24701
Mouse anti-Synapsin1 antibodySynaptic Systems106-011
Normal donkey serumJackson ImmunoResearch017-000-121
Orbital shakerFisher Scientific02-106-1013
ParafilmFisher Scientific13-374-10
Pipette tipsFisher Scientific02-707-025
PipettesFisher Scientific14-388-100Working volumes range from 3 µL to 500 µL
Plastic pasteur pipetteFisher Scientific02-708-006
Precision tweezers/forecepsFisher Scientific12-000-122
Rabbit anti-PSD95 antibodyAbcamab18258Other antibody pairs may work, as well, with optimization.
RefrigeratorVWR76470-402
Small petri dish, 60 mmFisher ScientificFB0875713A
TimerFisher Scientific14-649-17
Tween 20Fisher ScientificBP337-100

References

  1. Südhof, T. C. Towards an understanding of synapse formation. Neuron. 100 (2), 276-293 (2018).
  2. Bliss, T. V., Collingridge, G. L. A synaptic model of memory: long-term potentiation in the hippocampus.

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