We thank Zachary Allen for technical assistance. This work was supported by the Whitehall Foundation (Grant 2017-05-35) and Cleon C. Arrington Research Initiation Grant Program (RIG-93) to A.M.M. M.A.G. and D.W.Y. were both supported by a Georgia State University Neurogenomics 2CI Fellowship.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) at Georgia State University.
1. Preparation of Primary Hippocampal Neurons (in Laminar Flow Hood)
<ol>
	<li>Isolate primary hippocampal neurons from mixed sex postnatal day (P) 0-1 mice as previously described36.</li>
	<li>Plate neurons in 96-well poly-D-lysine coated microplates at a density of 2 x 104 cells per well.</li>
	<li>Prepare media for feeding the neurons by adding 10 mL of 50x B-27, 5 mL of 100x Glutamine, 242 &#181;L of 10 mg/mL 5-Fluoro-2&#8242;-deoxyuridine (FUDR) and 10 &#181;L of 10 mg/mL Gentamycin to 485 mL of neuronal media (see Table of Materials).</li>
	<li>Feed neurons as previously described36 by removing half (100 &#181;L) of the pre-existing media (referred to as conditioned media, which contains constituents that support neuronal survival) from each well and replacing with 100 &#181;L of 37 &#176;C prewarmed media prepared in step 1.3 every 3-4 days. Store the conditioned media from each feeding at 4&#730;C for step 2.3.</li>
</ol>
2. Measuring AMPA Receptor Trafficking in Response to DHPG (in Laminar Flow Hood)
<ol>
	<li>At Day In Vitro (DIV) 14, prepare a 500 &#181;L stock solution of Tetrodotoxin (TTX) at a concentration of 2 mM using cell culture grade water. 
	CAUTION: TTX is a neurotoxin. Handle carefully and avoid contact with skin.</li>
	<li>Using a multi-channel pipette, remove 100 &#181;L from each well of the 96-well microplate of neurons and pool the media.</li>
	<li>Add 2 &#181;L of the TTX stock solution to 1 mL of the pooled conditioned media from step 2.2 to create a 4 &#181;M solution of TTX. If there is not enough pooled conditioned media, then use some of the stored conditioned media from Step 1.4.</li>
	<li>Treat neurons with 100 &#181;L/well of the 4 &#181;M solution of TTX from step 2.3. Place the microplate in a 5% CO2 incubator at 37 &#176;C for 4 h.</li>
	<li>Attach a sterile glass Pasteur pipette to a suction line and attach a sterile 200 &#181;L pipette end to the tip of the pipette. Remove the media from each well carefully. Avoid touching the bottom of the microplate where the neurons are located.</li>
	<li>Add 200 &#181;L of room temperature neuronal media to each well.</li>
	<li>Incubate the microplate at room temperature for 15 min. Incubation in 5% CO2 is preferred but not necessary.</li>
</ol>
3. Immunolabelling&#160;(in Laminar Flow Hood)
<ol>
	<li>Prepare a 1:150 dilution of the anti-GluA1 or anti-GluA2 antibody in neuronal media.</li>
	<li>Remove the neuronal media added in Step 2.6. Add 50 &#181;L of the anti-GluA1 or anti-GluA2 antibody solutions to corresponding wells for 20 min at room temperature to allow antibody binding. Add 50 &#181;L of neuronal media to the secondary antibody only control wells.</li>
	<li>Remove the antibody solutions added in step 3.2. Wash the microplate 3 times with 100 &#181;L/well room temperature neuronal media to remove any unbound antibodies.</li>
	<li>Make a 50 mM stock solution of DHPG in neuronal media or cell culture grade water. Vortex the solution briefly to fully dissolve the DHPG. Prepare this solution fresh on the same day of the experiment. Avoid using old or thawed solutions.</li>
	<li>Dilute the stock solution in neuronal media to 1:500 to make a 100 &#181;M solution.</li>
	<li>Remove the existing media from each well. Add 100 &#181;L/well of the 100 &#181;M DHPG stock solution from Step 3.5. Incubate the microplate in the incubator at 37 &#176;C in 5% CO2 for 10 min.</li>
	<li>Remove the DHPG solution added in step 3.6 and add 100 &#181;L/well neuronal media. Repeat this step one more time. Place the microplate in the incubator at 37 &#176;C in 5% CO2 for 5 min.</li>
	<li>Add the sucrose to make the 4% paraformaldehyde/4% sucrose solution in PBS on the same day of the experiment. Avoid using old or thawed solutions. Remove the media added in step 3.7. Add 100 &#181;L/well of 4% paraformaldehyde/4% sucrose solution. Repeat steps 3.6 &#8211; 3.8 for each additional time-point. Once complete, incubate the microplate at 4 &#176;C for 20 min. 
	CAUTION: Handle paraformaldehyde stocks in a fume hood.</li>
	<li>Remove fixative from step 3.8 and add 100 &#181;L/well of cold 1x DPBS.</li>
	<li>Remove DPBS . Add 150 &#181;L/well blocking buffer (a ready-to-use formulation in TBS, refer to Table of Materials) and incubate at room temperature for 90 min. Alternatively, incubate overnight at 4 &#176;C.</li>
</ol>
4. Labelling Surface Receptors
<ol>
	<li>Prepare a 1:1500 dilution of 680RD Goat anti-Mouse IgG secondary antibody in blocking buffer.</li>
	<li>Remove blocking buffer from step 3.10. Add 50 &#181;L/well of the secondary antibody solution and incubate at room temperature for 60 min. Keep the microplate protected from light once the secondary antibodies are added. Make sure to continue to protect the microplate from light during subsequent incubations.</li>
	<li>Remove the solution from step 4.2. Add 100 &#181;L/well TBS (50 mM Tris-HCl, 150 mM NaCl, pH 7.6) and incubate at room temperature for 5 min. Repeat this step 4 additional times.</li>
	<li>Remove TBS from step 4.3. Add 100 &#181;L/well of 4% paraformaldehyde/4% sucrose in PBS and incubate at room temperature for 15 min.</li>
	<li>Remove solution from step 4.4. Add 100 &#181;L/well TBS and incubate at room temperature for 5 min. Repeat this step 2 additional times.</li>
</ol>
5. Labelling the Internalized Receptor Population
<ol>
	<li>Prepare TBS containing 0.2% saponin. Vortex the solution briefly to fully dissolve the saponin powder. Filter the solution using a 0.2 &#181;m filter to remove any particles that could cause auto-fluorescence.</li>
	<li>Add 150 &#181;L TBS containing 0.2% saponin and incubate at room temperature for 15 min.</li>
	<li>Remove saponin solution from step 5.2 and add 150 &#181;L/well blocking buffer. Incubate at room temperature for 90 min.</li>
	<li>Prepare a 1:1500 dilution of 800CW Donkey anti-Mouse IgG secondary antibody in blocking buffer.</li>
	<li>Remove blocking buffer from step 5.3 and add 50 &#181;L/well of the secondary antibody solution. Incubate at room temperature for 60 min.</li>
	<li>Add 100 &#181;L/well TBS and incubate at room temperature for 5 min. Repeat this step 4 additional times.</li>
</ol>
6. Imaging and Analysis
<ol>
	<li>Image the 96-well microplate using an infrared laser imaging system according to manufacturer&#8217;s instructions. Set the scan resolution to 84 &#181;m, the scan quality to medium and the focus offset according to the base height of the 96-well microplate used. Click on the &#34;Image Studio&#34; menu button &#8594; Export &#8594; Image for Digital Media &#8594; Export images at a&#160;resolution of 300 dpi, in TIFF format.</li>
	<li>Download Image J &#34;Fiji&#34; at https://imagej.net/Fiji/Downloads</li>
	<li>Open image in Image J &#34;Fiji&#34;. Split color channels by clicking on the &#34;Image&#34; menu 
	Color &#8594; Split Channels.</li>
	<li>Open the ROI manager from &#34;Analyze&#34; &#8594;&#160;&#34;Tools&#34;. Check the box &#34;labels&#34; in the ROI manager to label the circles with numbers.</li>
	<li>In the red channel (the 680 nm surface receptor pool), select the Region of Interest (ROI) by selecting the circle tool and drawing a circle that accurately fits the first well. Press Ctrl+T.&#160;</li>
	<li>Drag the circle to the next well and press Ctrl+T. Repeat until all wells are circled.</li>
	<li>From the ROI manager, click &#34;Measure&#34;. Select the values that appear and copy them to a spreadsheet.</li>
	<li>Click on the green channel (the 800 nm internal receptor pool) and then transpose the selected ROIs from step 6.6 to the image. From the ROI manager, click &#8220;Measure&#8221;. Select the values that appear and copy them to a spreadsheet.</li>
	<li>Calculate the average integrated density values from the secondary only control wells. Subtract the integrated density values for each experimental well from the average secondary only control integrated density values for the surface receptor pool. Repeat this step for the internal receptor pool.</li>
	<li>Calculate changes in surface receptor expression using Rs/Rt, where Rs represents the integrated density of surface receptors and Rt represents the integrated density of surface receptors + integrated density of internal receptors.</li>
</ol>

The authors have nothing to disclose.

AMPA receptors are an ionotropic glutamate receptor subtype that is integral for neuronal functions which include synapse formation, synapse stability, and synaptic plasticity. AMPA receptor disruption is linked to multiple neurological disorders24 and are considered attractive drug targets40. For example, studies have shown that one of the earliest signs of Alzheimer&#39;s disease (AD) is synapse loss and reduced synaptic AMPA receptor levels41,42. Intriguingly, addition of Amyloid-&#223; oligomers impairs surface GluA1-containing AMPA receptor expression at synapses43. Further, status epilepticus down-regulates GluA2 mRNA and protein in hippocampal neurons preceding their death44. In amyotrophic lateral sclerosis (ALS), TAR DNA-binding protein (TDP-43) pathology, an ALS-specific molecular abnormality, has been linked to inefficient GluA2 Q/R site-RNA editing45.
The high-content AMPA receptor trafficking assay provides an effective means for measuring bulk changes in receptor trafficking profiles within a neuronal network in response to various factors, consuming much less time and materials than alternative methods. A single 96-well microplate provides numerous wells to run multiple technical replicates and controls for different experimental conditions in the same plate. The low plating density of 2 x 104 cells/well relative to the 5 x 105 cells/well density significantly reduces the number of animals and materials required for each experiment (Figure 1). The infrared scanner can image up to six 96-well microplates at a time. The assay can also be modified to a 384-well microplate46, which becomes specifically valuable if the assay is run on precious samples that are difficult to obtain or are expensive to culture (e.g. human samples and induced pluripotent stem cells). The entire assay can be completed and analyzed on the same day, which saves valuable time (Figure 1).
For successful and efficient completion of the assay, some points need to be considered. First, it is important to prepare the DHPG solution on the same day of neuron treatment. Do not reuse or freeze DHPG stocks. The 4% paraformaldehyde/4% sucrose in PBS solution should also be made fresh on the same day of the experiment. Second, control wells treated with TTX only or vehicle are required to ensure that the effects observed are specific to treatment. It is also critical to include wells treated with only the secondary antibodies to control for background fluorescence produced by non-specific binding. Note that the second fixation step (following washout of secondary antibodies) is key to reducing secondary antibody background effects and achieving effective labelling of receptor subunits.
Compromises and limitations, as with any method, exist. This assay does not provide single-cell (or subcellular) resolution and does not allow for real-time tracking of receptor trafficking like other methods32,33,35. Additionally, proper care must be taken during the permeabilization step of neurons, as this method will be sensitive to the leaking of intracellular components in the case of over-permeabilization.

The magnitude and temporal dynamics of neuronal excitability is largely dependent on the availability and composition of surface receptor populations which transduce electrochemical signals. Whereas the synthesis of new receptors (or receptor subunits) is generally an energetically costly and relatively protracted process, a host of cellular machinery dedicated to the endo- and exocytosis of existing receptors provide a means for their fast insertion and removal to and from the membrane1. Therefore, in addition to the transcriptional and translational regulation of receptors, posttranslational receptor trafficking is an important modulator of neuronal excitability.
Synaptic plasticity, or the changing strength of connections between neurons with experience, is thought to form the basis of learning and memory2,3. The strengthening and weakening of synapses over time, termed long-term potentiation (LTP) and long-term depression (LTD), respectively, can be modulated through the trafficking of &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors4,5. AMPA receptors are heterotetramers composed of four subunits (GluA1-4), and mediate the majority of fast excitatory synaptic transmission in the brain6. Thus, neuron excitability is in large part a function of the quantity of AMPA receptors at the postsynaptic surface available to be activated by glutamate. LTD is commonly associated with an increase in AMPA receptor endocytosis, whereas LTP is predominantly associated with an increase in AMPA receptor exocytosis. Postsynaptic surface expression of AMPA receptors requires endosomal delivery to exocytotic proteins, where fusion with the plasma membrane then occurs in a calcium-dependent manner7,8,9,10,11,12,13,14. There also exist a host of mechanisms that regulate activity-dependent AMPA receptor endocytosis. One example of this is via the immediate early gene Arc/Arg3.1 (Arc). Among other functions15, Arc is known to mediate metabotropic glutamate receptor (mGluR)-dependent LTD by promoting AMPA receptor endocytosis through its binding partners, which include the endocytic proteins AP-2, endophilin-3, and dynamin-216,17,18,19, at clathrin-coated pits20,21. Internalized AMPA receptors can either be recycled back to the plasma membrane or earmarked for degradation22,23.
Importantly, the subunit composition of AMPA receptors contributes to their trafficking dynamics24. Of major relevance is the intracellular C-terminal domain of the subunits, where the majority of posttranslational modifications and trafficking-related protein interactions occur. GluA1 and GluA4 subunit-containing AMPA receptors are particularly apt to being trafficked to the cell surface during LTP, in part due to the presence of their PDZ ligands, ~90 amino acid sequences that promote membrane anchoring via interactions with various PDZ domain-containing proteins25,26. On the other hand, AMPA receptors containing GluA2 and lacking GluA1 or GluA4 tend to be trafficked constitutively but accumulate intracellularly with synaptic activity27. GluA2 subunits undergo RNA editing which, in addition to promoting retention in the endoplasmic reticulum28, renders the channel pore impermeable to calcium29, further implicating subunit-specific trafficking as a key mediator of neuronal homeostasis and plasticity. Interestingly, disruption of ubiquitin-dependent Arc degradation has been shown to increase GluA1 endocytosis and increase the surface expression of GluA2 subunit-containing AMPA receptors after induction of mGluR-LTD with the selective group I mGluR agonist (S)-3,5-Dihydroxyphenylglycine (DHPG), indicating that much remains to be learned regarding the mechanisms and role of subunit-specific AMPA receptor trafficking30.
Methods for observing changes in surface expression of receptors are often cumbersome, time consuming, or introduce unnecessary confounds. Biotin-based assays are a pervasive and commercially available approach. Affinity purification of biotinylated surface receptors represents one such example, however the necessity of performing electrophoresis requires a large amount of sample material and can render the screening of multiple treatments a prohibitively lengthy process31. Extensions of this assay, where multiple time points of labeling and immunoprecipitations are performed to quantify the gradual degradation of an initial signal, similarly neglect the addition of new &#8212; or recycled &#8212; receptors to the cell surface and only exacerbate the time and material requirements.
Other approaches make use of chimeric constructs or the addition of fluorescent tags to observe receptor trafficking32, sometimes using live cell imaging33,34. While potentially powerful, these designs can affect the normal trafficking patterns of receptors due to the mutations or dramatic changes in molecular weight introduced in these proteins. The use of dyes that label subcellular compartments by targeting low pH34,35 are non-specific and make distinguishing between different intracellular compartments important for receptor trafficking (e.g. lysosomes and proteasomes) difficult. Finally, the use of confocal microscopy to visualize the colocalization of receptors with markers associated with trafficking and degradation, such as endosomal proteins or clathrin, while potentially providing useful insight into specific localization with subcellular resolution, are time, labor-intensive, and costly due to the necessity of individually analyzing each cell and the requirement of confocal or super-resolution microscopy.
Here, we demonstrate a high-content receptor trafficking assay that is compatible with primary neuronal culture preparations30. This method separately labels the surface and intracellular receptor pools of fixed neurons, enabling the presentation of data as a ratio of normalized surface or internalized receptor density to the overall density for that receptor. The high-content nature of this method is ideal for screening multiple treatments and/or genotypes in a short time frame, and requires only standard cell culturing and antibody incubation expertise.
Briefly, primary neurons are grown in standard 96-well microplates and then treated as dictated by experimental design, incubated with primary antibodies, washed, and fixed. Cells are then incubated with a secondary antibody to label surface receptors, followed by another fixation step. Permeabilization then occurs and a second secondary antibody is used to label internal receptor pools. Finally, cells are imaged using an infrared fluorescent microplate scanner to quantify the integrated density of each receptor population. Figure 1 summarizes our high-content assay in comparison to a traditional biotinylation assay.
While the protocol offered here is optimized and specific for AMPA receptor trafficking in primary hippocampal neuron cultures, this procedure could, in theory, be extended and adapted for different receptors in a variety of cell types.

<table><tbody><tr><td>Mouse monoclonal (RH95) anti-GluA1 N-terminal</td><td>EMD Millipore</td><td>Cat#MAB2263MI, RRID: AB_11212678, LOT#2869732</td><td></td></tr><tr><td>Mouse monoclonal (6C4) anti-GluA2</td><td>ThermoFisher Scientific</td><td>Cat#32-0300, RRID: AB_2533058, LOT#TE268315</td><td></td></tr><tr><td>IRDye 680RD Goat anti-Mouse IgG (H+L)</td><td>Li-COR Biosciences</td><td>Cat# 926-68070, RRID: AB_10956588, LOT#C60107-03</td><td></td></tr><tr><td>IRDye 800CW Donkey anti-Mouse IgG (H+L)</td><td>Li-COR Biosciences</td><td>Cat# 926-32212, RRID: AB_621847, LOT#C60524-15</td><td></td></tr><tr><td>(RS)-3,5-DHPG</td><td>Tocris Bio-Techne</td><td>Cat#0342</td><td></td></tr><tr><td>Tetrodotoxin Citrate (TTX)</td><td>Tocris Bio-Techne</td><td>Cat#1069</td><td></td></tr><tr><td>Poly-D-Lysine Hydrobromide (PDL)</td><td>Sigma-Aldrich</td><td>Cat#P7280-5X5MG</td><td></td></tr><tr><td>Saponin</td><td>ACROS Organics</td><td>Cat# 419231000</td><td></td></tr><tr><td>B-27 Supplement (50x), Serum Free</td><td>Gibco</td><td>Cat#17504044</td><td></td></tr><tr><td>Glutamax Supplement</td><td>Gibco</td><td>Cat#35050061</td><td></td></tr><tr><td>5-Fluoro-2&amp;prime;-deoxyuridine (FUDR)</td><td>Sigma-Aldrich</td><td>Cat#F0503</td><td></td></tr><tr><td>Gentamycin (10 mg/mL)</td><td>Gibco</td><td>Cat#15710064</td><td></td></tr><tr><td>Paraformaldehyde, EM Grade, Purified</td><td>Electron Microscopy Sciences</td><td>Cat#19210</td><td></td></tr><tr><td>Sucrose (EP/BP/NF)</td><td>FisherScientific</td><td>Cat#S2500GM</td><td></td></tr><tr><td>Odyssey Blocking Buffer (TBS)</td><td>Li-COR</td><td>Cat#927-50003</td><td>Refered to as &quot;Blocking Buffer&quot; in the protocol</td></tr><tr><td>Cell Culture Grade Water</td><td>HyClone</td><td>Cat#SH30529.02</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>Cat#14190250</td><td></td></tr><tr><td>Neurobasal Medium, minus phenol red</td><td>Gibco</td><td>Cat#12348017</td><td>Referred to as &quot;neuronal Media&quot; in the protocol</td></tr><tr><td>28 mm Diameter Syringe Filters, 0.2 &amp;micro;m Pore SFCA Membrane, Sterile</td><td>Corning</td><td>Cat#431219</td><td></td></tr><tr><td>96-well Black/Clear and White/Clear Bottom Polystyrene Microplates</td><td>Corning</td><td>Cat#3603</td><td></td></tr><tr><td>ImageJ</td><td>NIH</td><td>https://imagej.nih.gov/ij/ ; RRID: SCR_003070</td><td></td></tr><tr><td>FIJI (Fiji is Just ImageJ)</td><td>NIH</td><td>http://fiji.sc/ ; RRID: SCR_002285</td><td></td></tr><tr><td>Odyssey CLx imaging system</td><td>Li-COR Biosciences</td><td></td><td></td></tr></tbody></table>

a high-content assay for monitoring ampa receptor trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Arc/Arg3.1 accelerates AMPA receptor endocytosis through interaction with AP-2, endophilin-3 and dynamin-216,18 following mGluR activation37. In the Arc knock-in mouse (ArcKR), lysines 268 and 269 in the Arc protein are mutated to arginine, which interferes with Arc ubiquitination. This impairs proteasome-dependent turnover of Arc and prolongs its half-life in neurons21,30. In this experiment, the persistence of Arc in regulating AMPA receptor trafficking was examined using the high-content AMPA receptor trafficking assay. Neurons were treated with the Na+ channel blocker TTX, which inhibits action potentials and reduces Arc levels38, followed by DHPG, which induces Arc translation and ubiquitination37,39. Both surface and internalized pools of GluA1- (Figure 2A) and GluA2-containing (Figure 3A) AMPA receptor subunits were measured at 5 and 15 min after DHPG washout. This particular experiment used three technical replicates from 3 independent experiments. ArcKR neurons showed increased GluA1 endocytosis when treated with DHPG compared to WT neurons, an effect that was not seen when neurons were treated with TTX only (Figure 2B). Surface expression of GluA2 subunits was significantly increased at short time points compared to WT neurons (Figure 3B), indicating a potential subunit replacement30. Some wells were treated with secondary antibodies only to control for background fluorescence caused by nonspecific binding.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59048/59048fig1.jpg" /> 
Figure 1: Comparison of the AMPA receptor high-content trafficking assay to the receptor biotinylation assay. The high-content trafficking assay consumes less time and resources compared to the standard biotinylation assay. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59048/59048fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59048/59048fig2.jpg" /> 
Figure 2: Surface and internal populations of GluA1 AMPA receptor subunits in WT and ArcKR hippocampal neurons. (A)&#160;Surface (sGluA1) and internalized (iGluA1) GluA1-containing AMPA receptor subunits in WT and ArcKR hippocampal neurons at 5 and 15 min after DHPG washout. Compared to WT, ArcKR neurons have a further decrease in the sGluA1-containing AMPA receptor pool 5 and 15 min after DHPG washout. (B)&#160;Graph represents surface fluorescence normalized to the total fluorescence intensity. Statistical comparisons were carried out using a one-way ANOVA, paired and unpaired Student&#39;s t tests. *p &#8804; 0.05; n = 3 technical replicates from 3 independent experiments. Values represent mean &#177; SEM. This figure has been modified from Wall, M. J. et al. 201830. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59048/59048fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59048/59048fig3.jpg" /> 
Figure 3: Surface and internal populations of GluA2 AMPA receptor subunits in WT and ArcKR hippocampal neurons.&#160;(A) The same experimental condition as Figure 2. Compared to WT, ArcKR neurons have an increase in the sGluA2-containing AMPA receptor pool 5 min after DHPG washout. (B)&#160;Graph represents surface fluorescence normalized to the total fluorescence intensity. Statistical comparisons were carried out using one-way ANOVA, paired and unpaired Student&#39;s t tests. *p &#8804; 0.05; n = 3 technical replicates from 3 independent experiments. Values represent mean &#177; SEM. This figure has been modified from Wall, M. J. et al. 201830. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59048/59048fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A High-content Assay for Monitoring AMPA Receptor Trafficking at JoVE.com

A High-content Assay for Monitoring AMPA Receptor Trafficking

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

a-high-content-assay-for-monitoring-ampa-receptor-trafficking

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

7.6K Views.  Georgia State University. Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

Video: A High-content Assay for Monitoring AMPA Receptor Trafficking

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Bioengineering

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

Biochemistry

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

Chemistry

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Studying Protease-Activated Receptor Ligands in Endothelial Cells

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of G&#945;q signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

Author Spotlight: Developing Parmodulins to Target Protease-Activated Receptors for Inflammation Control

An improved protocol for a calcium mobilization assay with endothelial cells, used to identify ligands of protease-activated receptors (PARs), has been developed. The new protocol reduces total assay time by 90-120 min and yields reproducible concentration-response curves.

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding ...

A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR malfunction, expressed as either over- or under-activity caused by mutations, altered expression, trafficking, or localization, can contribute to numerous diseases, especially in the central nervous system. Therefore, understanding the receptor&#39;s biology as well as facilitating the discovery of compounds and small molecules is crucial in ongoing efforts to combat neurological diseases. Current approaches to studying the receptor have limitations including low throughput, high cost, and the inability to study its functional abilities due to the necessary presence of channel blockers to prevent NMDAR-mediated excitotoxicity. Additionally, the existing assay systems are sensitive to stimulation by glutamate only and lack sensitivity to stimulation by glycine, the other co-ligand of the NMDAR. Here, we present the first plate-based assay with high-throughput power to study an NMDA receptor with sensitivity to both co-ligands, glutamate and D-serine/glycine. This approach allows the study of different NMDAR subunit compositions and allows functional studies of the receptor in glycine- and/or glutamate-sensitive modes. Additionally, the method does not require the presence of inhibitors during measurements. The effects of positive and negative allosteric modulators can be detected with this assay and the known pharmacology of NMDAR has been replicated in our system. This technique overcomes the limitations of existing methods and is cost-effective. We believe that this novel technique will accelerate the discovery of therapies for NMDAR-mediated pathologies.

The goal of this protocol is to facilitate the study of NMDA-receptors (NMDAR) at a larger scale and allow the examination of modulatory effects of small molecules and their therapeutic applications.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Medicine

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

Summary

Explore More Videos

Chapters in this video