Support was provided by startup-funds from the Pharmacology and Chemical Biology Department at the University of Pittsburgh School of Medicine. Acknowledgement of Jacob lab members who contributed to the video submission: Nicholas Graff.

All protocols described below are in accordance with the IACUC and IRB institutional review boards of the University of Pittsburgh School of Medicine.
1. Preparation of Hippocampal Neuronal Cultures in Tissue Culture Hood
Note: Use sterile technique and reagents throughout Protocol&#160;1.
<ol>
	<li>Prepare poly-D-lysine (0.1 mg/ml in H2O) coated glass coverslips as a substrate for the neuronal culture.
	<ol>
		<li>Place 4-5 round glass coverslips inside each 3.5&#160;cm tissue culture dish.</li>
		<li>Spot 70 &#956;l poly-D-lysine onto each 12&#160;mm coverslip. Note: for live imaging, a glass bottomed 3.5-cm tissue culture dish can be used (spot 200 &#956;l poly-D-lysine onto embedded 14&#160;mm glass coverslip).</li>
		<li>Leave the prepared dishes in the tissue culture hood O/N. To minimize poly-D-lysine evaporation and keep surfaces sterile in the tissue culture hood, close down the window sash and turn off the blower O/N. Note: UV lights are not used during the O/N incubation.</li>
		<li>The next morning, wash the dishes 3x with 2 ml of H2O.</li>
		<li>After removing the last H2O wash, add 2 ml of media to each 3.5&#160;cm dish and leave dishes in incubator until ready to plate the neurons in step 1.4.</li>
	</ol>
	</li>
	<li>Hippocampal neurons are prepared from embryonic day 18-19 (E18-19) rats (modified from&#160;Goslin22).</li>
	<li>Transfect freshly dissociated neurons on the day of culturing with 1-4 &#956;g of maxiprepped construct DNA. 
	Note: typically 3 &#956;g of DNA is transfected into 1-2 million neurons, with a viability of 50% and a transfection efficiency of 60% (e.g.&#160;starting with 2 million neurons: ((2 x&#160;106 neurons x&#160;0.5)0.6) provides approx. 1 million neurons; 600,000 of which are transfected1. Different amounts of construct DNA can be transfected when troubleshooting potential issues of over-expression, followed by appropriate characterization of construct localization and function.</li>
	<li>Plate the transfected neurons on the poly-D-lysine coated coverslips at a final density of approximately 200,000 neurons per 3.5&#160;cm dish. Note: for a glass bottomed dish, plate 40,000-50,000 neurons on the 14&#160;mm glass area.</li>
	<li>Replace the media 4-24 hr&#160;after preparation of neuronal cultures, then allow neurons to develop in the incubator until 14-17 days in vitro (DIV) or desired stage.</li>
</ol>
2. Endocytosis Assay
CAUTION: Note on &#945;-bungarotoxin and tubocurarine waste and handling:
<ol>
	<li>Handling of all peptide neurotoxins is recommended within the guidelines of Biosafety Level-2 (BL-2) research protocols. All proper personal protective gear must be worn. Lyophilized toxin powders should be reconstituted according to manufacturer&#39;s instructions. Toxin waste should be disposed of in compliance with the governing institution&#39;s Environmental Health and Safety guidelines.</li>
	<li>To remove potential peptide aggregates that may have formed during storage, &#945;-bungarotoxin aliquots should be centrifuged briefly before use, and the supernatant should be used for the experiment.</li>
	<li>&#945;-bungarotoxin (Bgt) stocks are resuspended to a concentration of 1 mg/ml in sterile H2O&#160;and stored in 10 &#956;l aliquots at -20 &#176;C. Fluorescently coupled Bgt stocks are used at 3 &#956;g/ml. Tubocurarine is used at a final concentration of 150 &#956;M.</li>
</ol>
<ol>
	<li>Set bench top heater block to 16 &#176;C in cold room with thin aluminum or stainless steel plate on top. Alternatively, if available, a bench top cooling/heating device can be used for each step requiring 16 &#176;C.</li>
	<li>Cool extracellular Hepes-buffered saline (HBS containing in mM: 135 NaCl, 4.7 KCl, 10 HEPES, 11 glucose, 1.2 MgCl2, and 2.5 CaCl2, pH 7.4) to 16 &#176;C in a water bath.</li>
	<li>Transfer neuron dishes to aluminum plate at 16 &#176;C and cool for 5 min.</li>
	<li>Remove media (keep conditioned media and reserve in incubator for step 2.8, endocytosis assay time points) and replace with 1 ml of 16 &#176;C HBS + tubocurarine (150 &#956;M) for 2 min.</li>
	<li>Remove HBS + tubocurarine to labeled waste container and replace with 1 ml of 16 &#176;C HBS + tubocurarine (150 &#956;M) + &#945;-bungarotoxin Alexa 594 [3 &#956;g/ml], incubating at 16 &#176;C for 15 min.</li>
	<li>Remove HBS + tubocurarine + &#945;-bungarotoxin Alexa 594 to labeled waste container and wash dishes 3x with 2 ml of 16 &#176;C HBS (the washes can be collected via aspiration in a vacuum flask containing 50 ml of 50 % bleach).</li>
	<li>For live imaging time series experiments following receptor endocytosis using a glass bottomed 3.5&#160;cm culture dish, perform steps 2.1-2.6, then transfer dish to heated stage (peltier device) on microscope, and begin imaging with a confocal or TIRF microscope setup.</li>
	<li>Transfer T = 0 time point coverslips into a dish of RT 4% paraformaldehyde /4% sucrose for 20 min, continuing with other coverslips to step 2.9.</li>
	<li>For other time points, replace HBS with conditioned equilibrated 37 &#176;C media (reserved in step 2.4) and return to incubator for endocytosis at 37 &#176;C. Note: suggested additional time points of T = 15, 30, and 60.</li>
	<li>At time points needed, take dishes from incubator, wash quickly twice with 2 ml of RT DPBS (DPBS no calcium, magnesium) then fix in 4% paraformaldehyde/4% sucrose for 20 min.</li>
	<li>After 20 min&#160;fixation, remove 4% paraformaldehyde /4% sucrose to waste container and wash dishes twice with 2 ml RT&#160;DPBS.</li>
	<li>Incubate coverslips at RT for 10 min in immunofluorescence block solution (block solution = 5% horse serum, 0.5% BSA in DPBS) containing 0.2% Triton X-100 to permeabilize the neurons and enable anti-GFP immunostaining of intracellular receptor pool and/or labeling of other proteins of interest.</li>
	<li>Remove block + 0.2% Triton X-100 and incubate coverslips in immunofluorescence block solution without Triton X-100 for 20-30 min.</li>
	<li>Perform primary antibody incubations of coverslips in block for several hours at RT or O/N&#160;at 4 &#176;C. Note: incubation of coverslips with primaries at 4 &#176;C O/N&#160;routinely produces improved immunofluorescence results.</li>
	<li>Wash coverslips 3x with DPBS (5 min for each wash step).</li>
	<li>Incubate coverslips with secondary antibodies in block solution for 1 hr at RT.</li>
	<li>Wash coverslips 3x with DPBS (5 min for each wash step).</li>
	<li>Mount coverslips, handling each coverslip individually: remove excess liquid from back of coverslip with lab tissue, then invert coverslip onto 4 &#956;l of mounting medium on a glass slide.</li>
	<li>Allow slides to dry at RT covered for 30 min then store at 4 &#176;C until ready to perform microscopy.</li>
	<li>Image acquisition and analysis of experiment with confocal microscopy (blind to experimental condition). 60X oil NA 1.49 immersion objective with the following lasers: Argon gas 488 nm, 561 diode, 640 diode.
	<ol>
		<li>Using the same image acquisition settings, acquire single Z section images with the neuronal cell body and several dendritic processes in focus.</li>
		<li>Quantify &#945;-bungarotoxin Alexa fluorescence signal and GFP immunostaining along 20 &#956;m of 3-4 proximal dendrites per neuron, using the same threshold for analysis of all endocytosis data.</li>
		<li>Analyze data from 10-12 neurons for each time point, repeating the experiment with several independent neuronal cultures.</li>
	</ol>
	</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+mobility+of+functional+GABA(A)+receptors+at+inhibitory+synapses.">Dynamic mobility of functional GABA(A) receptors at inhibitory synapses.</a> Nat. Neurosci. 8, 889-897 (2005).</li><li>Wilkins, M. E., Li, X., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+Cell+Surface+GABAB+Receptors+Using+an+&#945;-Bungarotoxin+Tag.">Tracking Cell Surface GABAB Receptors Using an &#945;-Bungarotoxin Tag.</a> J. Biol. Chem. 283, 34745-34752 (2008).</li><li>Saliba, R. S., Pangalos, M., Moss, 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=The+ubiquitin-like+protein+Plic-1+enhances+the+membrane+insertion+of+GABAA+receptors+by+increasing+their+stability+within+the+endoplasmic+reticulum.">The ubiquitin-like protein Plic-1 enhances the membrane insertion of GABAA receptors by increasing their stability within the endoplasmic reticulum.</a> J. Biol. 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U.S.A. 107, 16679-16684 (2010).</li><li>Katchalski-Katzir, E., 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=Design+and+synthesis+of+peptides+that+bind+alpha-bungarotoxin+with+high+affinity+and+mimic+the+three-dimensional+structure+of+the+binding-site+of+acetylcholine+receptor.">Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor.</a> Biophys. Chem. 100, 293-305 (2003).</li><li>Scherf, T., 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=A+beta+-hairpin+structure+in+a+13-mer+peptide+that+binds+alpha+-bungarotoxin+with+high+affinity+and+neutralizes+its+toxicity.">A beta -hairpin structure in a 13-mer peptide that binds alpha -bungarotoxin with high affinity and neutralizes its toxicity.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 6629-6634 (2001).</li><li>Wilkins, M. E., Li, X., Smart, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+cell+surface+GABAB+receptors+using+an+alpha-bungarotoxin+tag.">Tracking cell surface GABAB receptors using an alpha-bungarotoxin tag.</a> J. Biol. Chem. 283, 34745-34752 (2008).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Jacob, T. C., 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=Benzodiazepine+treatment+induces+subtype-specific+changes+in+GABAA+receptor+trafficking+and+decreases+synaptic+inhibition.">Benzodiazepine treatment induces subtype-specific changes in GABAA receptor trafficking and decreases synaptic inhibition.</a> Proc. Natl. Acad. Sci. U.S.A. 109, 18595-18600 (2012).</li><li>Anderson, M. J., Cohen, 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=Fluorescent+staining+of+acetylcholine+receptors+in+vertebrate+skeletal+muscle.">Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle.</a> J. Physiol. 237, 385-400 (1974).</li><li>Axelrod, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinkage+and+visualization+of+acetylcholine+receptors+on+myotubes+with+biotinylated+alpha-bungarotoxin+and+fluorescent+avidin.">Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin.</a> Proc. Natl. Acad. Sci. U.S.A. 77, 4823-4827 (1980).</li><li>Axelrod, D., 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=Lateral+motion+of+fluorescently+labeled+acetylcholine+receptors+in+membranes+of+developing+muscle+fibers.">Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers.</a> Proc. Natl. Acad. Sci. U.S.A. 73, 4594-4598 (1976).</li><li>Sekine-Aizawa, Y., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+of+receptor+trafficking+by+using+alpha-bungarotoxin-binding-site-tagged+receptors.">Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 17114-17119 (2004).</li><li>Jacob, T. C., 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=GABAA+receptor+membrane+trafficking+regulates+spine+maturity.">GABAA receptor membrane trafficking regulates spine maturity.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 12500-12505 (2009).</li><li>Hannan, S., 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=GABAB+receptor+internalisation+is+regulated+by+the+R2+subunit.">GABAB receptor internalisation is regulated by the R2 subunit.</a> J. Biol. Chem. , (2011).</li><li>Saliba, R. S., Kretschmannova, K., Moss, 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=Activity-dependent+phosphorylation+of+GABAA+receptors+regulates+receptor+insertion+and+tonic+current.">Activity-dependent phosphorylation of GABAA receptors regulates receptor insertion and tonic current.</a> EMBO. 31, 2937-2951 (2012).</li><li>Beqollari, D., Betzenhauser, M. J., Kammermeier, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+G-Protein+Coupling+in+an+mGluR6+Point+Mutant+Associated+with+Congenital+Stationary+Night+Blindness.">Altered G-Protein Coupling in an mGluR6 Point Mutant Associated with Congenital Stationary Night Blindness.</a> Mol. Pharmacol. 76, 992-997 (2009).</li><li>Terunuma, M., 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=Prolonged+activation+of+NMDA+receptors+promotes+dephosphorylation+and+alters+postendocytic+sorting+of+GABAB+receptors.">Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 13918-13923 (2010).</li><li>Goslin, K. G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+Nerve+Cells.">Culturing Nerve Cells.</a> , (1998).</li></ol>

The authors have nothing to disclose.

The BBS based fixed and live techniques described here can be used to track receptor or other plasma membrane protein trafficking in cell lines, neurons, and other primary cells. This method has been successfully used to study membrane insertion and removal of ligand-gated ion channels and GPCR and assess changes in trafficking due to the presence of receptor agonists and modulators. Key aspects include appropriate localization of the tag to an extracellular location and performing controls to ensure that addition of the BBS (as standard for other reporters, such as GFP) is functionally silent. For use in cells that express nicotinic acetylcholine (nAChR) receptors, the nAChR antagonist tubocurarine must be used throughout the protocol as indicated. In the case where the additional GFP tag is not used, total BBS tagged protein level can be determined via staining with an additional &#945;-bungarotoxin coupled to another fluorophore after fixation and permeabilization. Choice of time points for measuring endocytosis rates of the receptor/protein of interest requires assessing the current knowledge of trafficking for the receptor/protein, combined with initial use of more time points. This technique also can be readily employed with conventional immunostaining. We also describe an alternative approach for tracking receptor endocytosis via live confocal imaging experiments. Equipment availability, time requirements for live experiments, and other experimental constraints will guide choice of method, both being viable and published techniques. Future applications of BBS techniques include live or fixed imaging combined with endosomal and lysosomal markers to follow receptor fate, i.e. targeting to recycling endosomes and redelivery to the plasma membrane or degradation via lysosomes.

The use of fluorescently coupled &#945;-bungarotoxin to study receptor localization and dynamics was pioneered in studies of the nicotinic acetylcholine receptor13-15, the toxin&#39;s endogenous target. Subsequently, incorporation of the minimal Bgt binding peptide (BBS) has been used to study trafficking of both excitatory and inhibitory ligand-gated ion channels and G protein coupled receptors2,10,16-21. This BBS-based technique provides advantages to other approaches used for trafficking studies such as surface biotinylation methods, antibody labeling of live cells with antibodies to extracellular epitopes, and fluorescence recovery after photobleaching (FRAP). During cell surface biotinylation free amines are covalently modified, with the potential to affect cellular activity. Antibody based studies have often been hampered by surface antigen clustering or capping, which can alter trafficking events. Due to the bleaching step for FRAP studies, an important concern is damaging the underlying cellular structure. An additional advantage is that BBS tagged constructs can also be used for biochemical methodologies with biotin-coupled bungarotoxin to asses receptor trafficking. This technique is readily applicable to cell lines and primary cells. For use in cells that express nicotinic acetylcholine (nAChR) receptors, the nAChR antagonist tubocurarine must be used throughout the protocol as indicated. Performing a simple surface Bgt labeling (equivalent to the T=0 time point for the endocytosis protocol) on untransfected cells in the absence of tubocurarine will provide evidence of endogenous nAChR.
An important consideration for using this technique is appropriate insertion of the BBS so that it is present at an extracellular location when the protein of interest is delivered to the plasma membrane. For example, the N-terminal domains of GABAAR subunits reside in the vesicular lumen during trafficking and become extracellular after receptor insertion in the plasma membrane, allowing specific labeling of cell surface receptors and assessment of their removal from the cell surface by endocytic events. We have previously shown that the addition of GFP, myc, or BBS epitopes to this domain of GABAAR subunits is functionally silent. Standard controls should be performed to ensure that the tagged protein is expressed at similar levels to an untagged construct, that it appropriately localized, and that it does not affect receptor function. This characterization of transfected constructs&#160;will also aid in troubleshooting overexpression concerns.

<table><tbody><tr><td>P3 Primary Cell 4D kit&amp;nbsp;</td><td>Lonza</td><td>V4XP-3024</td><td></td></tr><tr><td>&amp;alpha;-Bungarotoxin, Alexa Fluor 647 conjugate</td><td>Molecular Probes</td><td>B35450</td><td></td></tr><tr><td>&amp;alpha;-Bungarotoxin, Alexa Fluor 488 conjugate</td><td>Molecular Probes</td><td>B13422</td><td></td></tr><tr><td>&amp;alpha;-Bungarotoxin, Alexa Fluor 594 conjugate</td><td>Molecular Probes</td><td>B13423</td><td></td></tr><tr><td>(+)-Tubocurarine chloride</td><td>Tocris</td><td>2820</td><td></td></tr><tr><td>Mounting medium&amp;nbsp;</td><td>DAKO</td><td>cS703</td><td></td></tr><tr><td>DPBS no calcium, magnesium</td><td>Invitrogen</td><td>14190-136</td><td></td></tr><tr><td>Poly-D-lysine&amp;nbsp;</td><td>Sigma</td><td>P6407</td><td></td></tr><tr><td>Gephyrin antibody</td><td>Synaptic Systems</td><td>147 011</td><td>1:300</td></tr><tr><td>VIAAT/VGAT antibody</td><td>Synaptic Systems</td><td>131 002</td><td>1:1,000</td></tr><tr><td>anti GFP</td><td>Life Technologies</td><td>A6455</td><td>1:2,000</td></tr><tr><td>Glass bottom tissue culture dish</td><td>MatTek Corporation</td><td>P35G-1.5-14-C - Mattek Dishes</td><td></td></tr><tr><td>Coverslips (round cover glass), #1 thickness, 12 mm</td><td>Warner Instruments</td><td>640-0702</td><td></td></tr></tbody></table>

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.

Characterization of a BBS tagged construct includes important controls such as determining if the expressed protein assembles properly (particularly with receptors composed of multiple subunits), traffics to the cell surface and localizes appropriately. Inhibitory synapses are composed of GABA A receptor surface clusters that colocalize with the inhibitory scaffold protein gephyrin and are apposed to presynaptic inhibitory terminals, identified by the vesicular inhibitory amino acid transporter that loads GABA and glycine into synaptic vesicles (VIAAT/VGAT). Shown in Figure 2 are &#945;2pHGFP+BBS expressing neurons with surface GABAAR labeled with Bgt, followed by immunostaining for total receptor population with anti-GFP antibody and either the postsynaptic inhibitory scaffold protein gephyrin (Figure 2A) or VIAAT (Figure 2B).
Figure 3 shows the endocytosis of &#945;2 containing GABAAR in hippocampal neurons at 15 DIV. Bgt fluorescence measurements of individual dendritic processes as shown in the panels (Figure 3A) are normalized to the level of construct expression via the GFP antibody staining. Final comparison of time points is made by normalization to the T=0 measurement, as shown in Figure 3B. Quantification of internalization of receptors over a 60 min period (Figure 3B) from fluorescence signal changes over time (Mean &#177; SEM: T0 = 100 &#177; 7.2, T15 = 74.7 &#177; 12.9, T30 = 44.7 &#177; 5.6, and T60 = 19.1 &#177; 3.4 min) was best described by a single exponential (at 37 &#186;C: t1/2 = 26 &#177; 4.8 min).
An alternative approach, shown in Figure 4, is to follow receptor endocytosis in a single neuron by live imaging time-lapse confocal microscopy. The punctate and overlapping fluorescence of surface pHGFP tagged GABAAR and Bgt labeled receptors is visible at the start of the experiment (T0). Over the time course, Bgt labeled GABA AR signal decreases, as receptors endocytose, while the pHGFP signal remains high due to new receptor insertion. The minimal decrease in phGFP signal is due to cell surface receptor distribution changes and photobleaching.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51365/51365fig1highres.jpg" width="500" /> 
Figure 1. Diagram of a BBS and pHGFP tagged GABAAR subunit and the endocytosis assay. Endocytosis is measured by first applying a fluorescently labeled bungarotoxin to neurons or cells, followed by a brief wash to remove unbound toxin, then fluorescence loss is monitored as receptors are internalized.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51365/51365fig2highres.jpg" width="500" /> 
Figure 2. Confocal imaging of BBS and pHGFP tagged GABAAR showing appropriate localization with inhibitory synapse components. Surface GABAAR in &#945;2pHGFP+BBS expressing neurons were labeled with &#945;-bungarotoxin Alexa 594 (red), followed by immunostaining, enlargements of dendrites are shown to the right of each neuron. Merged panel (&#945;-bungarotoxin Alexa 594 in red, GFP in green, gephyrin or VIAAT in blue). Surface Bgt labeled GABAAR show colocalization with A) the postsynaptic inhibitory scaffold protein gephyrin and B) apposition to the presynaptic marker VIAAT. (Scale bars, 10 &#956;m.)
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51365/51365fig3highres.jpg" width="500" /> 
Figure 3. Confocal imaging of GABAAR endocytosis in 15 DIV hippocampal neurons. A) Surface GABAAR in &#945;2pHGFP+BBS neurons were live-labeled with &#945;-bungarotoxin Alexa 594 (red), and then unbound &#945;-bungarotoxin Alexa 594 was removed by washing, followed by incubation at 37 &#176;C. At T = 0, 15, 30, and 60 min, samples were removed and fixed. Total receptor number was assayed by permeabilization and staining with anti-GFP antibody (green). Panels are enlargements of dendrites (Scale bars, 10 &#956;m). B) Graph represents the surface GABAAR &#945;-bungarotoxin Alexa 594 fluorescence loss over time with endocytosis (normalized to T = 0) (10-12 neurons per time point, 4 processes analyzed per neuron; error bars represent MEAN &#177; SEM).
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51365/51365fig4highres.jpg" width="500" /> 
Figure 4. Live confocal imaging of GABAAR endocytosis in 15 DIV hippocampal neurons. A) B3pHGFP+BBS containing surface GABAAR in neurons were live-labeled with &#945;-bungarotoxin Alexa 594 (red), followed by brief removal of unbound &#945;-bungarotoxin Alexa 594 by washing. Neurons were imaged by confocal microscopy at 37 &#176;C in HBS for 20 min at a 1 min interval (Scale bars, 10 &#956;m). Bgt Labeled GABAARs were localized to the cell surface at T0, as seen by colocalization with the pHGFP signal at T0. Over the time course, Bgt labeled GABA AR signal decreases, as receptors endocytose, new pHGFP expressing receptors are continually being inserted, so the pHGFP signal remains nearly constant.

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Using an &#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 ...

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Nanyang Technological University

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Neuroscience

15.8K Views.  University of Pittsburgh School of Medicine. Here we demonstrate the use of fluorescent Alexa dye coupled to α-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 α-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

Video: Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

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 ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

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

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

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

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

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 ...

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity &#947;-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

The method of autoradiography is routinely used to study binding of radioligands to tissue sections for determination of qualitative or quantitative pharmacology.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The ...

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 ...

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

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Biology

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the animal kingdom and can be recognized as either adaptive or maladaptive responses to a wide range of stress modalities. Rodents provide a proven experimental model for translational studies addressing the integrative mechanisms of anxiety at the molecular, cellular, and circuit levels. In particular, the chronic psychosocial stress paradigm elicits maladaptive responses mimicking anxiety-/depressive-like behavioral phenotypes that are analogous between humans and rodents. While previous studies show significant effects of chronic stress on neurotransmitter contents in the brain, the effect of stress on neurotransmitter receptor levels is understudied. In this article, we present an experimental method to quantitate the neuronal surface levels of neurotransmitter receptors in mice under chronic stress, especially focusing on gamma-aminobutyric acid (GABA) receptors, which are implicated in the regulation of emotion and cognition. Using the membrane-impermeable irreversible chemical crosslinker, bissulfosuccinimidyl suberate (BS3), we show that chronic stress significantly downregulates the surface availability of GABAA receptors in the prefrontal cortex. The neuronal surface levels of GABAA receptors are the rate-limiting process for GABA neurotransmission and could, therefore, be used as a molecular marker or a proxy of the degree of anxiety-/depressive-like phenotypes in experimental animal models. This crosslinking approach is applicable to a variety of receptor systems for neurotransmitters or neuromodulators expressed in any brain region and is expected to contribute to a deeper understanding of the mechanisms underlying emotion and cognition.

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain

The BS3 chemical crosslinking assay reveals reduced cell surface GABAA receptor expression in mouse brains under chronic psychosocial stress conditions.

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

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