A Protein Cross‐Linking Assay for Measuring Cell Surface Expression of Glutamate Receptor Subunits in the Rodent Brain After In Vivo Treatments

Amy C. Boudreau1, Mike Milovanovic1, Kelly L. Conrad1, Christopher Nelson1, Carrie R. Ferrario1, Marina E. Wolf1

1 Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.30
DOI:  10.1002/0471142301.ns0530s59
Online Posting Date:  April, 2012
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Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then, cell surface–expressed proteins are covalently cross‐linked using the membrane‐impermeable, bifunctional cross‐linker bis(sulfosuccinimidyl)suberate (BS3). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS‐PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency, and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after cross‐linking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants. Curr. Protoc. Neurosci. 59:5.30.1‐5.30.19. © 2012 by John Wiley & Sons, Inc.

Keywords: BS3; cell surface expression; glutamate receptors; protein cross‐linking; receptor trafficking

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cross‐Linking Surface‐Expressed Proteins in Freshly Dissected Brain Slices
  • Alternate Protocol 1: BS3 Cross‐Linking of Surface‐Expressed Proteins in Primary Neuronal Cultures
  • Support Protocol 1: Preparing the BS3 Stock Solution
  • Basic Protocol 2: SDS‐Page and Immunoblotting Analysis of Cross‐Linked Glutamate Receptor Subunits
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Cross‐Linking Surface‐Expressed Proteins in Freshly Dissected Brain Slices

  • Lysis buffer (see recipe)
  • 1 M (10×) glycine stock solution in distilled H 2O
  • 52 mM (26×) BS3 stock solution in 5 mM sodium citrate, pH 5.0 (see protocol 3)
  • Artificial cerebrospinal fluid (aCSF), pH 7.4 (see recipe)
  • Live rats or mice
  • Brain matrix (we recommend a metal brain matrix, so that it can be chilled on ice, from ASI Instruments, https://asi‐instruments.com/)
  • Dissecting instruments including chilled tissue punch or scalpel
  • Glass petri dish
  • Whatman no. 1 filter paper (Whatman, cat. no. 1001‐070)
  • Guillotine
  • Double‐edged razor blades for use with brain matrix and tissue chopper
  • McIlwain‐type tissue chopper (Vibratome), set at 400 µm/slice
  • Metal spatula, chilled
  • Flat plate inverter (Thermo Scientific LABQUAKE Shaker, cat. no. C400110; used in rocking mode)
  • Laboratory timer
  • Refrigerated microcentrifuge capable of 20,000 × g
  • Sonicator (Sonic Dismembrator Model 100, Fisher Scientific)

Alternate Protocol 1: BS3 Cross‐Linking of Surface‐Expressed Proteins in Primary Neuronal Cultures

  • Primary rodent brain cell culture (see Chapter 3)
  • Hanks' balanced salt solution (HBSS; appendix 2A)
  • Cell scrapers

Support Protocol 1: Preparing the BS3 Stock Solution

  • 5 mM sodium citrate buffer (prepared from 20× sodium citrate and citric acid stocks; see recipe in Reagents and Solutions
  • Bis(sulfosuccinimidyl)suberate (BS3; Pierce, cat. no. 21580; 50‐mg tubes; store at 4° to 8°C in a desiccator prior to use)

Basic Protocol 2: SDS‐Page and Immunoblotting Analysis of Cross‐Linked Glutamate Receptor Subunits

  • Samples: lysates generated according to protocol 1 and removed from −80°C
  • BioRad DC Protein Assay kit
  • 2× sample treatment buffer [STB; see recipe, or purchase from BioRad (cat. no. 161‐0737)]
  • Tris⋅Cl polyacrylamide gradient (4%‐15%) gels (BioRad)
  • 1× electrophoresis running buffer (see recipe)
  • Precision Plus Prestained SDS‐PAGE standards (BioRad) or comparable visible molecular weight markers
  • MagicMark XP Western Standard (Invitrogen, cat. no. LC5602)
  • 1× transblotting buffer (see recipe)
  • 100% methanol
  • Tris buffered saline/Tween‐20 (TBS‐T; see recipe)
  • Blocking buffer (see recipe)
  • Primary antibodies (these are the antibodies that are currently able to recognize AMPA receptor subunits after they have been cross‐linked with BS3; however, each new lot of antibody should be tested with control cross‐linked tissue prior to use in experiments.):
    • Rabbit anti‐GluR1 (Thermo Scientific Pierce, cat. no. PA1‐37776)
    • Rabbit anti‐GluR2/3 (Chemicon, cat. no. AB1506)
    • Mouse anti‐GluR2 (Neuromab; Antibodies Inc., cat. no. 75‐002; http://www.antibodiesinc.com)
    • Rabbit anti‐GluR3 (Cell Signaling, cat. no. 3437)
  • Secondary antibodies:
    • Goat anti‐rabbit IgG‐HRP conjugate (Invitrogen, cat. no. G21234)
    • Goat anti‐Mouse IgG‐HRP conjugate (Invitrogen, cat. no. G21040)
  • ECL substrate (Amersham/GE Healthcare)
  • 0.1% Ponceau S (Sigma, cat. no. P7170‐1L; 1× stock)
  • Heat block
  • Mini‐PROTEAN 3 electrophoresis cell (BioRad, cat. no. 165‐3301)
  • BioRad power supply Power Pac HC (cat. no. 164‐5052)
  • Polyvinylidene fluoride (PVDF) membrane
  • Whatman 3MM chromatography paper
  • Plastic covered containers for incubations (PerfectWestern, http://genhunter.com)
  • Transblot sponges
  • Trans‐blot cell (BioRad, cat. no. 170‐3939)
  • Plate rocker (low to medium rocking)
  • Clear vinyl page protector sheets
  • Film (Amersham Hyperfilm ECL, cat. no. 28906839)
  • Autoradiography cassette (Fisher Scientific, cat. no. FBCA 810)
  • VersaDoc gel documentation system (BioRad; optional), or equivalent system
  • Total Lab (http://www.totallab.com/) or other comparable data analysis program
  • Gel scanner
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Literature Cited

Literature Cited
   Archibald, K., Perry, M.J., Molnar, E., and Henley, J.M. 1998. Surface expression and metabolic half‐life of AMPA receptors in cultured rat cerebellar granule cells. Neuropharmacology 37:1345‐1353.
   Boudreau, A.C. and Wolf, M.E. 2005. Behavioral sensitization to cocaine is associated with increased AMPA receptor surface expression in the nucleus accumbens. J. Neurosci. 25:9144‐9151.
   Boudreau, A.C., Reimers, J.M., Milovanovic, M., and Wolf, M.E. 2007. Cell surface AMPA receptors in the rat nucleus accumbens increase during cocaine withdrawal but internalize upon cocaine challenge in association with altered activation of mitogen‐activated protein kinases. J. Neurosci. 27:10621‐10635.
   Boudreau, A.C., Ferrario, C.R., Glucksman, M., and Wolf, M.E. 2009. Signaling pathway adaptations and novel protein kinase A substrates related to behavioral sensitization to cocaine. J. Neurochem. 110:363‐377.
   Broutman, G. and Baudry, M. 2001. Involvement of the secretory pathway for AMPA receptors in NMDA‐induced potentiation in hippocampus. J. Neurosci. 21:27‐34.
   Clayton, D.A., Grosshans, D.R., and Browning, M.D. 2002. Aging and surface expression of hippocampal NMDA receptors. J. Biol. Chem. 277:14367‐14369.
   Collingridge, G.L., Isaac, J.T., and Wang, Y.T. 2004. Receptor trafficking and synaptic plasticity. Nat. Rev. Neurosci. 5:952‐962.
   Conrad, K.L., Tseng, K.Y., Uejima, J.L., Reimers, J.M., Heng, L.J., Shaham, Y., Marinelli, M., and Wolf, M.E. 2008. Formation of accumbens GluR2‐lacking AMPA receptors mediates incubation of cocaine craving. Nature 454:118‐121.
   Conrad, K.L., Ford, K.A., Marinelli, M., and Wolf, M.E. 2010. Dopamine receptor expression and distribution dynamically change in the nucleus accumbens after withdrawal from cocaine self‐administration. Neuroscience 169:182‐194.
   Ferrario, C.R., Li, X., Wang, X., Reimers, J.M., Uejima, J.L., and Wolf, M.E. 2010. The role of glutamate receptor redistribution in locomotor sensitization to cocaine. Neuropsychopharmacology 35:818‐833.
   Ferrario, C.R., Loweth, J.A., Milovanovic, M., Ford, K.A., Galiñanes, G.L., Heng, L.‐J., Tseng, K.Y., and Wolf, M.E. 2011. Alterations in AMPA receptor subunits and TARPs in the rat nucleus accumbens related to the formation of Ca2+‐permeable AMPA receptors during the incubation of cocaine craving. Neuropharmacology 61:1141‐1151.
   Ford, K.A., Wolf, M.E., and Hu, X.‐T. 2009. Plasticity of L‐type Ca2+ channels after cocaine withdrawal. Synapse 63:690‐697.
   Gao, C. and Wolf, M.E. 2008. Dopamine receptors regulate NMDA receptor surface expression in prefrontal cortex neurons. J. Neurochem. 106:2489‐2501.
   Gerges, N.Z., Tran, I.C., Backos, D.S., Harrell, J.M., Chinkers, M., Pratt, W.B., and Esteban, J.A. 2004. Independent functions of hsp90 in neurotransmitter release and in the continuous synaptic cycling of AMPA receptors. J. Neurosci. 24:4758‐4766.
   Greger, I.H. and Esteban, J.A. 2007. AMPA receptor biogenesis and trafficking. Curr. Opin. Neurobiol. 17:289‐297.
   Grosshans, D.R., Clayton, D.A., Coultrap, S.J., and Browning, M.D. 2002a. LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1. Nat. Neurosci. 5:27‐33.
   Grosshans, D.R., Clayton, D.A., Coultrap, S.J., and Browning, M.D. 2002b. Analysis of glutamate receptor surface expression in acute hippocampal slices. Sci. STKE 2002(137):pl8.
   Hall, R.A. and Soderling, T.R. 1997a. Differential surface expression and phosphorylation of the N‐methyl‐D‐aspartate receptor subunits NR1 and NR2 in cultured hippocampal neurons. J. Biol. Chem. 272:4135‐4140.
   Hall, R.A. and Soderling, T.R. 1997b. Quantitation of AMPA receptor surface expression in cultured hippocampal neurons. Neuroscience 78:361‐371.
   Hall, R.A., Hansen, A., Andersen, P.H., and Soderling, T.R. 1997. Surface expression of the AMPA receptor subunits GluR1, GluR2, and GluR4 in stably transfected baby hamster kidney cells. J. Neurochem. 68:625‐630.
   Li, X. and Wolf, M.E. 2011. Brain‐derived neurotrophic factor rapidly increases AMPA receptor surface expression in rat nucleus accumbens. Eur. J. Neurosci. 34:190‐198.
   Mangiavacchi, S. and Wolf, ME. 2004. D1 dopamine receptor stimulation increases the rate of AMPA receptor insertion onto the surface of cultured nucleus accumbens neurons through a pathway dependent on protein kinase A. J. Neurochem. 88:1261‐1271.
   Meredith, G.E. and Totterdell, S. 1999. Microcircuits in nucleus accumbens' shell and core involved in cognition and reward. Psychobiology 27:165‐186.
   Mickiewicz, A.L. and Napier, T.C. 2011. Repeated exposure to morphine alters surface expression of AMPA receptors in the rat medial prefrontal cortex. Eur. J. Neurosci. 33:259‐265.
   Nelson, C.L., Milovanovic, M., Wetter, J.B., Ford, K.A., and Wolf, M.E. 2009. Behavioral sensitization to amphetamine is not accompanied by changes in glutamate receptor surface expression in the rat nucleus accumbens. J. Neurochem. 109:35‐51.
   Safferling, M., Tichelaar, W., Kümmerle, G., Jouppila, A., Kuusinen, A., Keinänen, K., and Madden, D.R. 2001. First images of a glutamate receptor ion channel: Oligomeric state and molecular dimensions of GluRB homomers. Biochemistry 40:13948‐13953.
   Stackpole, C.W., De Milo, L.T., Hammerling, U., Jacobson, J.B., and Lardis, M.P. 1974. Hybrid antibody‐induced topographical redistribution of surface immunoglobulins, alloantigens, and Concanavalin A receptors on mouse lymphoid cells. Proc. Natl. Acad. Sci. U.S.A. 71:932‐936.
   Sun, X., Milovanovic, M., Zhao, Y., and Wolf, M.E. 2008. Acute and chronic dopamine receptor stimulation modulates AMPA receptor trafficking in nucleus accumbens neurons co‐cultured with prefrontal cortex neurons. J. Neurosci. 28:4216‐4230.
   Thomas‐Crusells, J., Vieira, A., Saarma, M., and Rivera, C. 2003. A novel method for monitoring surface membrane trafficking on hippocampal acute slice preparation. J. Neurosci. Meth. 125:159‐166.
   Triller, A. and Choquet, D. 2005. Surface trafficking of receptors between synaptic and extrasynaptic membranes: And yet they do move! Trends Neurosci. 28:133‐139.
   Xie, Z., Srivastava, D.P., Photowala, H., Kai, L., Cahill, M.E., Woolfrey, K.M., Shum, C.Y., Surmeier, D.J., and Penzes, P. 2007. Kalirin‐7 controls activity‐dependent structural and functional plasticity of dendritic spines. Neuron 56:640‐656.
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