We thank the Northwestern Center for Advanced Microscopy for the use of the Nikon A1 Confocal microscope and their assistance in planning and analyzing the experiments. This research was supported by NIGMS (T32GM008061) (A. M. C.), and NIA (R00AG041225) and a NARSAD Young Investigator Grant from the Brain &#38; Behavior Research Foundation (#24133) (A. S. -C.).

Work pertaining to hippocampal primary culture preparation was reviewed and approved by the Northwestern University Animal Care and Use Committee (protocol #IS00001151).
1. Preparation Before Labeling
<ol>
	<li>Preparation and maintenance of primary hippocampal cultures
	<ol>
		<li>Prepare primary hippocampal cultures at a density of 150,000 cells plated on poly-D-lysine-coated (0.1 mg/mL) 18 mm cover glasses. Excellent guides for dissociated neuronal culture preparation are available7,8. 
		NOTE: If required, the cultures may be treated with cytosine arabinoside (Ara-C, 10 &#956;M from DIV1) to avoid glial proliferation in the preparation. 
		NOTE: Alternative coating reagents such as fibronectin (1 mg/mL) or laminin (5 &#956;g/mL) may be used instead of poly-D-lysine.</li>
		<li>Maintain cultures in a cell incubator at 37 &#176;C and 5% CO2 in 2 mL/well of neurobasal media supplemented with B27 and 2 mM L-glutamine. 
		NOTE: Substitutes for L-glutamine (e.g., Glutamax) can be used, if desired.</li>
		<li>On weekly-basis, remove half the volume of media and replace with the same volume of supplemented neurobasal media.</li>
	</ol>
	</li>
	<li>OPTIONAL: Transfection of mutated and/or epitope-tagged receptors 
	NOTE: Neurons should be transfected at least 3&#8211;4 days prior to the analysis time point to allow for receptor expression. The use of young neurons [Days in vitro 6&#8211;9 (DIV6&#8211;9)] results in better transfection efficiency than older (DIV15&#8211;20) neurons, but a sufficient number of transfected cells (&#62;20) can be achieved regardless of the DIV employed.
	<ol>
		<li>For each well of a 12-well plate, dilute 1.5 &#956;g of plasmid containing the construct of interest in 100 &#956;L of fresh neurobasal media without B27 or glutamine supplementation in a microcentrifuge tube and mix by vortexing quickly. 
		NOTE: For successful transfection, it is critical that the neurobasal media used is as fresh as possible, ideally less than 1 week after bottle opening.</li>
		<li>In a second microcentrifuge tube, mix 1 &#956;L of an appropriate lipofection reagent in 100 &#956;L of fresh neurobasal media and mix gently. 
		NOTE: Do not vortex the lipofection reagent mixture. Use of fresh lipofection reagents can improve transfection efficiency.</li>
		<li>Incubate the tubes for 5 min at room temperature (RT).</li>
		<li>Add the lipofection reagent mixture dropwise to the DNA mixture, mix gently, and incubate for 20 min at RT.</li>
		<li>Adjust the volume of media in each well to 1 mL of conditioned media.</li>
		<li>Add the lipofection reagent -DNA mixture dropwise to the well.</li>
		<li>Return cells to the incubator and allow at least 3&#8211;4 days for protein expression. 
		NOTE: For the purposes of the internalization and recycling protocols outlined below, hippocampal neurons were transfected at DIV11&#8211;12 with constructs expressing GluN2B tagged with GFP in the extracellular domain (GFP-GluN2B) and imaged at DIV15&#8211;16.</li>
	</ol>
	</li>
	<li>OPTIONAL: Incubation of cells with drugs (chronically or acutely) in the conditioned media until fixation. 
	NOTE: For acute treatment, begin treating cells before labeling. Depending on the drug treatment protocol used, cells can be maintained in drug-containing media during section 2. In our example, DIV21 cells were subject to a cLTP protocol to increase surface-expressed AMPAR9.
	<ol>
		<li>Exchange conditioned media for extracellular solution (ECS).</li>
		<li>Treat cells with 300 &#956;M glycine in ECS for 3 min at RT. As a control, treat a sister coverslip with ECS (without glycine).</li>
		<li>Wash cells 3x with 37 &#176;C ECS and return the cells in ECS (without glycine) to the cell incubator for 20 min prior to continuing with section 2. 
		NOTE: ECS (in mM): 150 NaCl, 2 CaCl2, 5 KCl, 10 HEPES, 30 Glucose, 0.001 TTX, 0.01 strychnine, and 0.03 picrotoxin at pH 7.4.</li>
	</ol>
	</li>
</ol>
2. Live Labeling of Surface-expressed Receptors
<ol>
	<li>Prepare coverslips for labeling
	<ol>
		<li>To save reagents and facilitate manipulation, transfer coverslips cell side up to a paraffin film-covered tray. 
		NOTE: It is critical to never let the samples dry out.</li>
		<li>Save and maintain conditioned media at 37 &#176;C for incubation and washing steps. 
		NOTE: For an 18 mm coverslip, incubation with 75&#8211;100 &#956;L of media for antibody labeling and 120&#8211;150 &#956;L for internalization/recycling are recommended.</li>
	</ol>
	</li>
	<li>Labeling of surface receptors with primary antibody
	<ol>
		<li>Incubate cells with primary antibody diluted in conditioned media for 15 min at RT. 
		NOTE: For GFP-tagged receptors, rabbit anti-GFP antibody at a dilution of 1:1000 was used. For endogenous GluA1, mouse anti-GluA1 at a 1:200 dilution was used.</li>
		<li>Carefully aspirate off the antibody-containing media using a vacuum pipette and wash cells three times with conditioned media. 
		NOTE: If conditioned media is unavailable, all washing steps may be performed using PBS+ [phosphate buffered saline (PBS) containing 1 mM MgCl2 and 0.1 mM CaCl2]. Manual aspiration using a micropipette may be performed if gentle vacuum aspiration is not available.</li>
	</ol>
	</li>
</ol>
3. Surface Expression (Figure 2)
<ol>
	<li>Secondary antibody labeling of surface-expressed receptors
	<ol>
		<li>Wash once with PBS+.</li>
		<li>Fix cells by incubating with 4% paraformaldehyde (PFA) and 4% sucrose in PBS for 7&#8211;8 min. 
		NOTE: Unlike other fixation methods such as methanol incubation, PFA does not permeabilize the plasma membrane and is therefore suitable for surface-expression analysis. For optimal results, use freshly prepared PFA. Short-term storage of PFA at 4 &#176;C or long-term (up 30 days) storage at -20 &#176;C is permissive for adequate fixation. 
		CAUTION: PFA is a known carcinogen. Use proper personal protective equipment and a safety hood when handling.</li>
		<li>Wash cells three times with regular PBS. 
		NOTE: Alternatively, 0.1 M glycine can be used for washing PFA instead of PBS, as glycine will quench any remaining fixative that may increase the background in the preparation.</li>
		<li>Block nonspecific binding sites by incubating with 10% normal goat serum (NGS) in PBS for 30 min at RT. 
		NOTE: Blocking time can be extended without adverse effects on labeling.</li>
		<li>Incubate with fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT to label primary antibody-labeled receptors (i.e., surface-expressed). 
		NOTE: In these examples, a 1:500 dilution of Alexa 555-conjugated secondary antibodies:goat anti-rabbit for GFP-labeled receptors and goat anti-mouse for GluA1 was used.</li>
		<li>Wash cells with PBS 3x.</li>
	</ol>
	</li>
	<li>Labeling of intracellular receptors
	<ol>
		<li>Permeabilize cells with 0.25% Triton X-100 in PBS for 5&#8211;10 min at RT. 
		NOTE: To check that the initial round of antibody labeling occupies all surface epitopes, this permeabilization step can be skipped in a sister culture. In this case, no signal for intracellular receptors should be obtained. Additionally, to check that no internal receptors have been labeled in the previous section 2 (i.e., showing the integrity of the plasma membranes in culture), the permeabilization step can be skipped in a sister culture, and a primary antibody against an intracellular protein (e.g., PSD-95 or MAP2) can be utilized in step 2.2.1. No signal should be obtained from this primary under these conditions. In this case, a rabbit anti-PSD-95 antibody (1:500) was used.</li>
		<li>Block with 10% NGS in PBS for 30 min at RT.</li>
		<li>Label intracellular receptors by incubating permeabilized cells with the same primary antibody used in section 2.2 diluted in 3% NGS in PBS for 1 h at RT. 
		NOTE: The antibody dilution for labeling intracellular receptors may be different than that required for labeling surface-expressed receptors. In the example of GluA1, the same antibody dilution (1:200) was used.</li>
		<li>Wash cells 3x with PBS.</li>
		<li>Label with second fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT. 
		NOTE: In these examples, a 1:500 dilution of goat anti-mouse Alexa 647-conjugated secondary antibody (for GluA1) was used.</li>
		<li>Wash cells 3x with PBS.</li>
	</ol>
	</li>
</ol>
4. Internalization (Figure 3)
<ol>
	<li>Internalization of antibody-labeled surface receptors
	<ol>
		<li>After labeling of surface-expressed receptors and antibody washing (section 2.2), maintain cells in conditioned media without antibody and return them to the incubator (37 &#176;C) to allow for internalization. 
		NOTE: For NMDA receptors, 30 min for internalization is recommended. As a control, a sister culture may be maintained with conditioned media at 4 &#176;C during the internalization process. Minimal receptor internalization should occur under these conditions.</li>
	</ol>
	</li>
	<li>Labeling of surface receptors
	<ol>
		<li>Wash cells once with PBS+.</li>
		<li>Fix cells with 4% PFA and 4% sucrose in PBS for 7&#8211;8 min. 
		CAUTION: Use proper personal protective equipment and a safety hood when handling PFA.</li>
		<li>Wash cells 3x with regular PBS.</li>
		<li>Block with 10% NGS in PBS for 30 min at RT to prevent nonspecific binding.</li>
		<li>Incubate samples with fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT to label primary antibody-labeled receptors (i.e., surface-expressed receptors which were not internalized). 
		NOTE: For this example, Alexa 555-conjugated goat anti-rabbit secondary antibody (1:500) was used for labeling.</li>
		<li>Wash cells 3x with PBS.</li>
	</ol>
	</li>
	<li>Labeling of internalized receptors
	<ol>
		<li>Permeabilize cells with 0.25% Triton X-100 in PBS for 5&#8211;10 min.</li>
		<li>Block nonspecific binding by incubation with 10% NGS in PBS for 30 min at RT.</li>
		<li>Incubate samples with fluorescently tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT to label internalized antibody-labeled receptors. 
		NOTE: For this example, Alexa 647-conjugated goat anti-rabbit secondary antibody (1:500) is used for labeling.</li>
		<li>Wash cells 3x with PBS.</li>
	</ol>
	</li>
</ol>
5. Recycling (Figure 4)
<ol>
	<li>Internalization of antibody-labeled surface receptors
	<ol>
		<li>After labeling of surface-expressed receptors and antibody washing (section 2.2), maintain cells in conditioned media without antibody and return them to the incubator (37 &#176;C) to allow for internalization. 
		NOTE: For NMDA receptors, 30 min for internalization is recommended.</li>
	</ol>
	</li>
	<li>Blocking of stable surface expressed receptors
	<ol>
		<li>To block the epitopes on the primary antibody attached to surface-expressed receptors that have not been internalized, incubate cells with unconjugated Fab anti-IgG (H+L) antibody fragments (against the primary used in section 2.2) diluted in conditioned media (20 &#956;g/mL) for 20 min at RT. This treatment prevents future interaction with secondary antibodies. 
		NOTE: For this example, Goat anti-rabbit Fab fragments were used. 
		NOTE: Control experiment: to ensure that complete blocking of surface-expressed receptors has occurred, sister coverslips can be incubated with and without Fab. Cultures should be fixed immediately after Fab treatment, and both cultures are incubated with Alexa 555-conjugated secondary antibody. No Alexa 555 signal in the Fab-incubated cells indicates proper antibody blocking.</li>
		<li>Wash cells 3x with conditioned media.</li>
		<li>Incubate cells with conditioned media containing 80 &#956;M dynasore to prevent further internalization and return cells to the incubator (37 &#176;C) to allow for recycling of internalized receptors. Dynasore is a GTPase inhibitor that inhibits dynamin and therefore prevents internalization. 
		NOTE: 45 min for NMDAR recycling is recommended. Note that Dynasore exclusively blocks the dynamin-dependent internalization process (e.g., NMDARs internalization). However, internalization of other synaptic protein (dynamin-independent) can still occur in the presence of Dynasore.</li>
	</ol>
	</li>
	<li>Labeling of recycled receptors
	<ol>
		<li>Wash cells once with PBS+.</li>
		<li>Fix cells with 4% PFA and 4% sucrose in PBS for 7&#8211;8 min. 
		CAUTION: Use proper personal protective equipment and a safety hood when handling PFA.</li>
		<li>Wash cells 3x with PBS.</li>
		<li>Block with 10% NGS in PBS for 30 min at RT to prevent nonspecific binding.</li>
		<li>Label cells with first fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT. 
		NOTE: For this example, Alexa 555-conjugated goat anti-rabbit antibody (1:500) was used for labeling.</li>
		<li>Wash cells 3x with PBS. 
		NOTE: Longer washes with PBS (5&#8211;10 min) may help to reduce background in the preparation.</li>
	</ol>
	</li>
	<li>Labeling of internalized receptors
	<ol>
		<li>Permeabilize cells with 0.25% Triton X-100 in PBS for 5&#8211;10 min.</li>
		<li>Block with 10% NGS in PBS for 30 min at RT.</li>
		<li>Label with second fluorescently-tagged secondary antibody diluted in 3% NGS in PBS for 1 h at RT. 
		NOTE: For this example, Alexa 647-conjugated goat anti-rabbit antibody (1:500) was used for labeling.</li>
		<li>Wash cells 3x with PBS.</li>
	</ol>
	</li>
</ol>
6. Mounting and Imaging of Samples
<ol>
	<li>Mount cells by gently placing the coverslips cell side down on 12&#8211;15 &#956;L of the appropriate mounting media. 
	NOTE: Aspiration of excess mounting media will improve the quality of images.</li>
	<li>Image cells on an appropriate confocal microscope. 
	NOTE: It is recommended to image a z-stack at 60x magnification with 0.35 &#956;m steps, encompassing the entire thickness of the neuron.</li>
</ol>
7. Time Considerations
<ol>
	<li>This is a long protocol that can be stopped at several points. If desired, perform blocking and primary antibody incubation steps overnight at 4 &#176;C in a humid chamber.</li>
	<li>Alternatively, if desired, use a microwave tissue processor to vastly speed up post-fixation incubation times. For all steps, use 150 W at 30 &#176;C for &#8220;On&#8221; settings.
	<ol>
		<li>To block, run the processor at 2 min &#8220;On,&#8221; 1 min &#8220;Off,&#8221; and 2 min &#8220;On.&#8221;</li>
		<li>For primary and secondary antibody incubation steps, run the processor at 3 min &#8220;On,&#8221; 2 min &#8220;Off,&#8221; and 3 min &#8220;On.&#8221; 
		NOTE: We observe no difference in quality by making the above alterations to the protocol.</li>
	</ol>
	</li>
</ol>
8. Image Analysis
<ol>
	<li>It is recommended to use FIJI &#60;https://fiji.sc/&#62; to conduct image analysis, as it is compatible with multiple file formats. For our data, images in the Nikon ND2 file format were acquired.</li>
	<li>A macro script is provided for easy batch quantification of different parameters pre-selected by FIJI. The following steps are included in the macro: 
	NOTE: For these examples, &#8220;integrated intensity&#8221; was measured.</li>
	<li>Open the image files in FIJI and separate channels.</li>
	<li>Z-project each channel stack as a maximum intensity projection.</li>
	<li>Set a lower threshold for each channel. 
	NOTE: Thresholds should be empirically determined for each experimental data set. While each channel can have a separate lower threshold value, it crucial that channel threshold values are consistently maintained for all images of the same data set.</li>
	<li>Select three to five secondary or tertiary dendrites and save them as regions of interest (ROIs).</li>
	<li>Measure the integrated density of each ROI in surface and intracellular channels.</li>
	<li>Normalize the signal for each ROI by dividing the integrated density value of the surface channel by the intracellular channel.</li>
	<li>Repeat the measurements for all control and experimental images and normalize experimental values to control values (e.g., GluN2B WT or no-glycine conditions).</li>
</ol>

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The authors have nothing to disclose.

The interaction between a cell and its environment (e.g., communication with other cells, response to different stimuli, etc.), heavily relies on the correct expression of receptors at the cell surface. The rapid and fine-tuned regulation in surface-expressed receptor content enables proper cellular response to a constantly changing environment. In the particular case of neurons, alterations in the number, localization, and subunit composition of synaptically expressed receptors heavily influences synaptic communication,...

Cells utilize the active process of trafficking to mobilize proteins to specific subcellular localizations and exert strict spatiotemporal regulation over their function1. This process is especially important for transmembrane receptors, as cellular responses to different environmental stimuli rely on intracellular cascades triggered by receptor activation. Cells are able to modify these responses by altering the density, localization, and subunit composition of receptors expressed at the cell surface via receptor subcellular trafficking regulation2. Insertion of newly synthetized receptors into the plasma membrane, along with endocytosis and recycling of existing receptors are examples of trafficking processes that determine the net pool of surface-expressed receptors2. Many molecular mechanisms cooperate to regulate protein trafficking, including protein-protein interactions and posttranslational modifications such as phosphorylation, ubiquitination, or palmitoylation2.
Regulation of receptor trafficking is particularly required in strongly polarized cells with highly specialized structures. The paradigmatic example is the control of neuronal function by regulated trafficking of glutamate receptors (GluRs)3,4. Glutamate, the main excitatory neurotransmitter, binds and activates surface-expressed GluRs to control fundamental physiological neuronal functions such as synaptic neurotransmission and synaptic plasticity. The fact that altered GluR trafficking has been observed in a broad spectrum of neuropathies, ranging from neurodevelopmental disorders to neurodegenerative diseases, highlights the importance of this process5. Thus, understanding the molecular events that control GluR trafficking is of interest in many areas of research.
In this protocol, an antibody-feeding based method is used to quantify the level of surface-expressed GluRs in primary hippocampal neurons as well as evaluate how changes in internalization and recycling result in the observed net surface expression. The use of pharmacology and/or overexpression of exogenous receptors harboring specific mutations makes this protocol a particularly powerful approach for studying molecular mechanisms underlying neuronal adaptation to different environmental stimuli. A final example of the utility of this protocol is studying how multifactorial changes in the environment (such as in a disease models) affects GluR trafficking through the examination of surface expression in such models.
Using specific examples, it is initially demonstrated how a pharmacologic manipulation mimicking physiological synaptic stimulation [chemical LTP (cLTP)] increases the surface expression of the endogenous GluA1 subunit of the AMPA-type of GluRs (AMPARs)6. The trafficking of an overexpressed phospho-mimetic form of the GluN2B subunit of NMDA-type of GluRs (NMDARs) is also analyzed to exemplify how this protocol can be used to study the regulation of GluR trafficking by specific posttranslational modifications. Though these specific examples are used, this protocol can easily be applied to other GluRs and other receptors and proteins that possess antigenic extracellular domains. In the case that there are no antibodies available for extracellular domains, overexpression of extracellular epitope-tagged (e.g., Flag-, Myc-, GFP-tagged, etc.) proteins can assist in protein labeling.
The current protocol provides instructions for quantifying specific GluR subtype density and trafficking using specific antibodies. This protocol can be utilized to study 1) total GluR surface expression, 2) GluR internalization, and 3) GluR recycling. To study each process individually, it is advised to begin with sections 1 and 2 and continue with either section 3, 4, or 5. In all cases, finish with sections 6 and 8 (Figure 1).

<table><tbody><tr><td>18 mm dia. #1.5 thick coverglasses</td><td>Neuvitro</td><td>GG181.5</td><td></td></tr><tr><td>Alexa 555-conjugated goat anti-mouse secondary</td><td>Life Technologies</td><td>A21424</td><td></td></tr><tr><td>Alexa 555-conjugated goat anti-rabbit secondary</td><td>Life Technologies</td><td>A21429</td><td></td></tr><tr><td>Alexa 647-conjugated goat anti-mouse secondary</td><td>Life Technologies</td><td>A21236</td><td></td></tr><tr><td>Alexa 647-conjugated goat anti-rabbit secondary</td><td>Life Technologies</td><td>A21245</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C7902</td><td></td></tr><tr><td>Corning Costar Flat Bottom Cell Culture Plates</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>Dynasore</td><td>Tocris</td><td>2897</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Glycine</td><td>Tocris</td><td>0219</td><td></td></tr><tr><td>Goat anti-rabbit Fab fragments</td><td>Sigma</td><td>SAB3700970</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H7006</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen</td><td>11668019</td><td></td></tr><tr><td>Mouse anti-GluA1 antibody</td><td>Millipore</td><td>MAB2263</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S6546</td><td></td></tr><tr><td>Neurobasal Media</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>NGS</td><td>Abcam</td><td>Ab7481</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM999</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Pelco BioWave</td><td>Ted Pella</td><td>36500</td><td></td></tr><tr><td>PFA</td><td>Alfa Aesar</td><td>43368</td><td></td></tr><tr><td>Picrotoxin</td><td>Tocris</td><td>1128</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma</td><td>P7280</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Life Technologies</td><td>P36934</td><td></td></tr><tr><td>Rabbit anti-GFP antibody</td><td>Invitrogen</td><td>A11122</td><td></td></tr><tr><td>Rabbit anti-PSD-95 antibody</td><td>Cell Signaling</td><td>2507</td><td></td></tr><tr><td>Strychnine</td><td>Tocris</td><td>2785</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389</td><td></td></tr><tr><td>Superfrost plus microscope slides</td><td>Fisher</td><td>12-550-15</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>TTX</td><td>Tocris</td><td>1078</td><td></td></tr></tbody></table>

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 protocol to study glutamate receptor trafficking is based on differential labeling of receptors expressed at the cell surface and those expressed in internal membranes. Segregation is achieved by the labeling the receptors before and after membrane permeabilization, using the same primary antibody but a secondary antibody conjugated to a different fluorophore. As outlined by the optional steps included the protocol, this is a very versatile method for interrogating different receptor...

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

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 ...

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Research

JoVE Journal

Neuroscience

5.6K Views.  Northwestern University. Cellulaire reacties op externe stimuli zijn sterk afhankelijk van de set eiwitten die op een bepaald moment op het celoppervlak worden uitgedrukt. Dienovereenkomstig worden het getal, de subcellulaire lokalisatie en subeenhedensamenstelling van deze eiwitten dynamisch gecontroleerd. Hier zullen we een antilichaamvoedingsbenadering gebruiken om het niveau van oppervlakte-uitgedrukte receptoren in celculturen te kwantificeren, zolang de internalisatie en recycling worden hervat.Dit is ee...

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

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

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 ...

Biochemistry

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

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 ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (&#947;-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. 
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. 
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

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

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

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

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

A 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.

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 ...

Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as &#34;autoimmune encephalitis&#34; (AE). There are currently 17 known AE syndromes, and all are associated with antibodies against the neuronal cell surface or synaptic proteins. The clinical syndromes are complex and vary according to the type of associated antibody. The best-known of these diseases is anti-N-methyl D-aspartate receptor (NMDAR) encephalitis, which is a prominent neuropsychiatric disorder associated with severe memory and behavioral impairments. The associated antibodies react with the GluN1 subunit of the NMDAR at the N-terminal domain. The approach most frequently used for the discovery and characterization of AE antibodies includes the culture of dissociated, fetal, rodent hippocampal neurons. During the process of antibody characterization, live neurons in culture are exposed to patients&#39; serum or CSF, and the detection of reactivity indicates that the serum or CSF samples of the patient contain antibodies against neuronal surface antigens. Hippocampal cultures can also be used to determine whether the antibodies in patients are potentially pathogenic by examining if they cause structural or functional alterations of the neurons. The level of success of these studies depends on the quality of the cultures and on the protocols used to obtain and detect the reactivity of patient samples. This article provides an optimized protocol for primary cell culture of fetal rat hippocampal neurons combined with immunostaining to determine the presence of antibodies in the serum or CSF of patients. An example of how to examine the potential pathogenic effects of NMDAR antibodies using cultured neurons and calcium imaging is also presented.

Autoimmune encephalitis is a new category of antibody-mediated diseases of the central nervous system. Hippocampal neurons can be used to discover and characterize these antibodies. This article provides a protocol for primary cell culture and immunostaining to determine autoantibodies in the serum and cerebrospinal fluid of patients.

Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as ...

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 ...

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

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