Name: an antibody feeding approach to study glutamate receptor trafficking in dissociated primary hippocampal cultures
Uploaded: 2019-08-02T15:00:00
Description: Watch this Scientific Journal Video about An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures at JoVE.com

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...

<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. (19), (2008).</li><li>Lu, W., 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=Activation+of+synaptic+NMDA+receptors+induces+membrane+insertion+of+new+AMPA+receptors+and+LTP+in+cultured+hippocampal+neurons.">Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons.</a> Neuron. 29 (1), 243-254 (2001).</li><li>Sanz-Clemente, A., Nicoll, R. A., Roche, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+in+NMDA+Receptor+Composition:+Many+Regulators,+Many+Consequences.">Diversity in NMDA Receptor Composition: Many Regulators, Many Consequences.</a> The Neuroscientist: A Review Journal Bringing Neurobiology, Neurology and Psychiatry. 19 (1), 62-75 (2013).</li><li>Tham, D. K. L., Moukhles, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+Cell-surface+Expression+and+Endocytic+Rate+of+Proteins+in+Primary+Astrocyte+Cultures+Using+Biotinylation.">Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.</a> Journal of Visualized Experiments. (125), (2017).</li><li>Bermejo, M. K., Milenkovic, M., Salahpour, A., Ramsey, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Synaptic+Plasma+Membrane+and+Postsynaptic+Density+Proteins+Using+a+Discontinuous+Sucrose+Gradient.">Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient.</a> Journal of Visualized Experiments. (91), e51896 (2014).</li><li>Makino, H., Malinow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+incorporation+into+synapses+during+LTP:+the+role+of+lateral+movement+and+exocytosis.">AMPA receptor incorporation into synapses during LTP: the role of lateral movement and exocytosis.</a> Neuron. 64 (3), 381-390 (2009).</li><li>Bailey, D. M., Kovtun, O., Rosenthal, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-Conjugated+Single+Quantum+Dot+Tracking+of+Membrane+Neurotransmitter+Transporters+in+Primary+Neuronal+Cultures.">Antibody-Conjugated Single Quantum Dot Tracking of Membrane Neurotransmitter Transporters in Primary Neuronal Cultures.</a> Methods in Molecular Biology. 1570, 165-177 (2017).</li><li>Trussell, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+and+analyzing+synaptic+currents+and+synaptic+potentials.">Recording and analyzing synaptic currents and synaptic potentials.</a> Current Protocols in Neuroscience. Chapter 6, Unit. , (2001).</li><li>Barreto-Chang, O. L., Dolmetsch, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Imaging+of+Cortical+Neurons+using+Fura-2+AM.">Calcium Imaging of Cortical Neurons using Fura-2 AM.</a> Journal of Visualized Experiments. (23), e1067 (2009).</li></ol>

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 ...

Cellular responses to external stimuli heavily rely on the set of proteins that are expressed on the cell surface in a given moment. Accordingly, the number, the sub-cellular localization, and subunit composition of these proteins are dynamically controlled. Here, we will use an antibody feeding approach to quantify the level of surface expressed receptors in cell cultures, as long as the internalization and recycling resume.This is a very versatile protocol, that provides mechanistic information of the regulation of surface expressed proteins. In our example, we will study glutamine receptors in primary hippocampal neurons. This protocol can be adapted to any protein possessing an anagenic extracellular epitope, if antibodies aren't available, transvection of tagged constructs can be useful for both labeling and studying specific mutations.In our examples, we use antibodies against endogenous GluA1, and tagged Glu12 B.To begin this procedure, transfer the cover slips cell side up to a paraffin film covered tray, to save reagents, and facilitate manipulation. Save and maintain conditioned media at 37 degrees Celsius for incubation and washing steps. Incubate the cells with primary antibodies diluted in conditioned media, at room temperature, for 15 minutes.After this, use a vacuum pipette to carefully aspirate off the antibody containing media, and wash the cells three times with conditioned media. If studying surface versus intracellular receptor expression fix the cells with paraformaldehyde after this step, as outlined in the text protocol. Maintain the cells in conditioned media without antibodies, and return them to the incubator at 37 degrees Celsius to allow for internalization.If studying internalization process, fix the cells with paraformaldehyde after this step, as outlined in the text protocol. To block the epitopes on the primary antibody attached to the surface expressed receptors that have not been internalized, incubate cells with unconjugated Fab anti-IgG antibody fragments diluted in conditioned media, for 20 minutes at room temperature. After this, wash the cells three times with conditioned media.Incubate the washed wells with conditioned media containing 80 micromolar Dynasore, at 37 degrees Celsius, to allow for recycling of internalized receptors. After finishing the modifications on live cells, wash the cells once with PBS plus. Add a solution of 4%paraformaldehyde and 4%sucrose in PBS to the cells, and incubate in a fume hood at room temperature for seven to eight minutes, to fix the cells.Then wash the cells three times with regular PBS. Add a solution of 10%Normal Goat Serum in PBS to the cells, and incubate at room temperature for 30 minutes, to block non-specific binding sites. Next, incubate the cells with fluorescently tagged secondary antibody diluted in 3%NGS in PBS at room temperature for one hour, to label primary antibody label receptors.After this, wash the cells three times with PBS. To begin labeling the intracellular receptors, add a solution of 0.25%TritonX in PBS, and incubate at room temperature for five to 10 minutes, to permeabilize the cells. Then block with 10%NGS at room temperature for 30 minutes.If studying surface versus total, incubate the cells with the same primary antibody used previously, diluted with 3%NGS in PBS, at room temperature for one hour, to label the intracellular receptors. Next, wash the cells three times with PBS. Label with second fluorescently tagged secondary antibody, diluted in 3%NGS in PBS, at room temperature for one hour.After this wash the cells three times with PBS. First gently place the cover slips, cell side down, on 12 to 15 microliters of the appropriate mounting media to mount the cells. Then image the cells on the appropriate confocal microscope, and analyze the cells as outlined in the text protocol.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. After acquiring confocal images, the fluorescent signal can be easily quantified to show an increase in surface expression, relative to intracellular population, following chemical LTP. No signal for PSD-95 can be obtained in non-permeabilized cells, demonstrating the integrity of the plasma membrane.This indicates that the signal obtained for surface GluA1 indeed corresponds to surface expressed receptors. Importantly, a minimal signal for internalized GluA1 can be observed under non-permeabilization conditions, showing that all surface epitopes are occupied by the initial round of antibody labeling. Receptors that have been internalized are then identified.This exampled highlights that GluN2B phosphorylation at S1480 promotes receptor internalization. As the phosphomimetic mutant S1480E displayed a much higher internalization ratio compared to wild-type receptors. As expected, no signal is obtained for internalized receptors in the control.Next, recycled receptors and internalized receptors are identified. The generated recycling ratio shows that GluN2B S1480E has no effect on NMDAR recycling. In the control, a strong signal can be observed for surface expressed GluN2B in the absence of Fab blocking.This signal disappears in Fab-treated cultures, demonstrating that the blocking protocol is sufficient to completely block the surface expressed epitopes, and that the surface signals observed after recycling indeed correspond to receptors trafficked back to the plasma membrane. The current protocol is just one of the methods available for studying the regulation of surface expressed proteins. Other approaches can be taken to expand or verify the data obtained here.Those include biochemical methods, as biotinylation, life imaging techniques, or functional approaches, like electrophysiology or calcium imaging. We use PFA to freeze the localization of receptors, however PFA is a known carcinogen, so it's crucial to perform steps using PFA under a fume hood, while wearing appropriate PPE.

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