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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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Regulation+of+N-Methyl-d-aspartate+(NMDA)+and+alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic+Acid+(AMPA)+Receptors+by+Posttranslational+Modifications.">Dynamic Regulation of N-Methyl-d-aspartate (NMDA) and alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors by Posttranslational Modifications.</a> The Journal of Biological Chemistry. 290 (48), 28596-28603 (2015).</li><li>Traynelis, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+receptor+ion+channels:+structure,+regulation,+and+function.">Glutamate receptor ion channels: structure, regulation, and function.</a> Pharmacological Reviews. 62 (3), 405-496 (2010).</li><li>Molnar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+potentiation+in+cultured+hippocampal+neurons.">Long-term potentiation in cultured hippocampal neurons.</a> Seminars in Cell &amp; Developmental Biology. 22 (5), 506-513 (2011).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+hippocampal+neurons+from+prenatal+mice.">Isolation and culture of hippocampal neurons from prenatal mice.</a> Journal of Visualized Experiments. (65), (2012).</li><li>Nunez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Culture+of+Hippocampal+Neurons+from+P0+Newborn+Rats.">Primary Culture of Hippocampal Neurons from P0 Newborn Rats.</a> Journal of Visualized Experiments. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>CaCl2</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...

Watch this Scientific Journal Video about An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures at JoVE.com

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 ...

an-antibody-feeding-approach-to-study-glutamate-receptor-trafficking

Research

JoVE Journal

Neuroscience

5.6K Views.  Northwestern University. 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.

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

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

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.

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

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 ...

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...