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 (50 X), 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&#8242;-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 &#34;Blocking Buffer&#34; 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 &#34;neuronal Media&#34; in the protocol</td>
		</tr>
		<tr>
			<td>28 mm Diameter Syringe Filters, 0.2 &#181;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 colspan="2">https://imagej.nih.gov/ij/ ; RRID: SCR_003070</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>

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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 ...

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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

Eye Movement Monitoring of Memory

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank yesterday."), however such reports can often be unreliable or sensitive to response bias 1, and may be unobtainable in some participant populations. Furthermore, explicit reports only reveal when information has reached consciousness and cannot comment on when memories were accessed during processing, regardless of whether the information is subsequently accessed in a conscious manner. Eye movement monitoring (eye tracking) provides a tool by which memory can be probed without asking participants to comment on the contents of their memories, and access of such memories can be revealed on-line 2,3. Video-based eye trackers (either head-mounted or remote) use a system of cameras and infrared markers to examine the pupil and corneal reflection in each eye as the participant views a display monitor. For head-mounted eye trackers, infrared markers are also used to determine head position to allow for head movement and more precise localization of eye position. Here, we demonstrate the use of a head-mounted eye tracking system to investigate memory performance in neurologically-intact and neurologically-impaired adults. Eye movement monitoring procedures begin with the placement of the eye tracker on the participant, and setup of the head and eye cameras. Calibration and validation procedures are conducted to ensure accuracy of eye position recording. Real-time recordings of X,Y-coordinate positions on the display monitor are then converted and used to describe periods of time in which the eye is static (i.e. fixations) versus in motion (i.e., saccades). Fixations and saccades are time-locked with respect to the onset/offset of a visual display or another external event (e.g. button press). Experimental manipulations are constructed to examine how and when patterns of fixations and saccades are altered through different types of prior experience. The influence of memory is revealed in the extent to which scanning patterns to new images differ from scanning patterns to images that have been previously studied 2, 4-5. Memory can also be interrogated for its specificity; for instance, eye movement patterns that differ between an identical and an altered version of a previously studied image reveal the storage of the altered detail in memory 2-3, 6-8. These indices of memory can be compared across participant populations, thereby providing a powerful tool by which to examine the organization of memory in healthy individuals, and the specific changes that occur to memory with neurological insult or decline 2-3, 8-10.

Eye movement monitoring (or eye tracking) reveals where in space the eyes linger, when and for how long. Here, we demonstrate how eye tracking can be used to investigate the integrity of memory in multiple participant populations, without requiring verbal, or otherwise explicit, reports.

Explicit (often verbal) reports are typically used to investigate memory (e.g. "Tell me what you remember about the person you saw at the bank ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Real-time In Vitro Monitoring of Odorant Receptor Activation by an Odorant in the Vapor Phase

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition follows a combinatorial coding scheme, where one OR can be activated by a set of odorants and one odorant can activate a combination of ORs. Through such combinatorial coding, organisms can detect and discriminate between a myriad of volatile odor molecules. Thus, an odor at a given concentration can be described by an activation pattern of ORs, which is specific to each odor. In that sense, cracking the mechanisms that the brain uses to perceive odor requires the understanding odorant-OR interactions. This is why the olfaction community is committed to &#34;de-orphanize&#34;&#160;these receptors. Conventional in vitro systems used to identify odorant-OR interactions have utilized incubating cell media with odorant, which is distinct from the natural detection of odors via vapor odorants dissolution into nasal mucosa before interacting with ORs. Here, we describe a new method that allows for real-time monitoring of OR activation via vapor-phase odorants. Our method relies on measuring cAMP release by luminescence using the Glosensor assay. It bridges current gaps between in vivo and in vitro approaches and provides a basis for a biomimetic volatile chemical sensor.

Physiologically, odorant receptors are activated by odorant molecules inhaled in the vapor phase. However, most in vitro systems utilize liquid phase odorant stimulation. Here, we present a method that allows real-time in vitro monitoring of odorant receptor activation upon odorant stimulation in vapor phase.

Olfactory perception begins with the interaction of odorants with odorant receptors (OR) expressed by olfactory sensory neurons (OSN). Odor recognition ...