Name: a high-content assay for monitoring ampa receptor trafficking
Uploaded: 2019-01-28T14:00:00.000+00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about A High-content Assay for Monitoring AMPA Receptor Trafficking at JoVE.com

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/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/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/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

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

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Acyl-PEGyl Exchange Gel Shift Assay for Quantitative Determination of Palmitoylation of Brain Membrane Proteins

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular mechanism for learning and memory. Palmitoylation regulates localization and function of many synaptic proteins including AMPA-Rs, auxiliary factors and synaptic scaffolds in an activity-dependent manner. We identified the synapse differentiation induced gene (SynDIG) family of four genes (SynDIG1-4) encoding brain-specific transmembrane proteins that associate with AMPARs and regulate synapse strength. SynDIG1 is palmitoylated at two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region important for activity-dependent excitatory synapse development. Here, we describe an innovative biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest and demonstrate its utility with the SynDIG family of proteins in mouse brain lysates.

Palmitoylation entails the incorporation of a 16-carbon palmitate moiety to cysteine residues of target proteins in a reversible manner. Here, we describe a biochemical approach, the acyl-PEGyl exchange gel shift (APEGS) assay, to investigate the palmitoylation state of any protein of interest in mouse brain lysates.

Activity-dependent alterations in the levels of synaptic AMPA receptors (AMPARs) within the postsynaptic density (PSD) is thought to represent a cellular ...

Biochemistry

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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 ...

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 ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

A Plate-Based Assay for the Measurement of Endogenous Monoamine Release in Acute Brain Slices

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in monoamine neurotransmitter release and uptake dynamics. Technically complex methods such as electrophysiology, Fast Scan Cyclic Voltammetry (FSCV), imaging, in vivo&#160;microdialysis, optogenetics, or use of radioactivity are required to study monoamine function. The method presented here is an optimized two-step approach for detecting monoamine release in acute brain slices using a 48-well plate containing tissue holders for examining monoamine release, and high-performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) for monoamine release measurement. Briefly, rat brain sections containing regions of interest, including prefrontal cortex, hippocampus, and dorsal striatum were obtained using a tissue slicer or vibratome. These regions of interest were dissected from the whole brain and incubated in an oxygenated physiological buffer. Viability was examined throughout the experimental time course, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The acutely dissected brain regions were incubated in varying drug conditions that are known to induce monoamine release through the transporter (amphetamine) or through the activation of exocytotic vesicular release (KCl). After incubation, the released products in the supernatant were collected and analyzed through an HPLC-ECD system. Here, basal monoamine release is detected by HPLC from acute brain slices. This data supports previous in vivo and in vitro results showing that AMPH and KCl induce monoamine release. This method is particularly useful for studying mechanisms associated with monoamine transporter-dependent release and provides an opportunity to screen compounds affecting monoamine release in a rapid and low-cost manner.

This method introduces a simple technique for the detection of endogenous monoamine release using acute brain slices. The setup uses a 48-well plate containing a tissue holder for monoamine release. Released monoamine is analyzed by HPLC coupled with electrochemical detection. Additionally, this technique provides a screening method for drug discovery.

Monoamine neurotransmitters are associated with numerous neurologic and psychiatric ailments. Animal models of such conditions have shown alterations in ...

An In Vitro Technique to Study Glutamate Receptor Trafficking in Hippocampal Neurons

Source: Chiu, A. M., et al. <a href="https://app.jove.com/v/59982">An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures.</a> J. Vis. Exp. (2019)
This video demonstrates an antibody-feeding approach to study the trafficking of glutamate receptors in primary hippocampal neuronal cultures. This approach allows the observation of receptor populations at both the plasma and internal membranes. Discrimination between these subsets is achieved by labeling the receptors before and after membrane permeabilization, using the same primary antibody but secondary antibodies conjugated to different fluorophores.

1. Preparation Before Labeling

	Preparation and maintenance of primary hippocampal cultures&#8203;
	
		Prepare primary hippocampal cultures at a density ...

A High-content Assay for Monitoring AMPA Receptor Trafficking

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