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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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 "antibody-feeding" 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.

Introduction

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, alon....

Protocol

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

  1. Preparation and maintenance of primary hippocampal cultures
    1. 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.......

Representative Results

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.......

Discussion

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,.......

Acknowledgements

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 & Behavior Research Foundation (#24133) (A. S. -C.).

....

Materials

NameCompanyCatalog NumberComments
18 mm dia. #1.5 thick coverglassesNeuvitroGG181.5
Alexa 555-conjugated goat anti-mouse secondaryLife TechnologiesA21424
Alexa 555-conjugated goat anti-rabbit secondaryLife TechnologiesA21429
Alexa 647-conjugated goat anti-mouse secondaryLife TechnologiesA21236
Alexa 647-conjugated goat anti-rabbit secondaryLife TechnologiesA21245
B27Gibco17504044
CaCl2SigmaC7902
Corning Costar Flat Bottom Cell Culture PlatesCorning3513
DynasoreTocris2897
GlucoseSigmaG8270
GlycineTocris0219
Goat anti-rabbit Fab fragmentsSigmaSAB3700970
HEPESSigmaH7006
KClSigmaP9541
L-GlutamineSigmaG7513
Lipofectamine 2000Invitrogen11668019
Mouse anti-GluA1 antibodyMilliporeMAB2263
NaClSigmaS6546
Neurobasal MediaGibco21103049
NGSAbcamAb7481
ParafilmBemisPM999
PBSGibco10010023
Pelco BioWaveTed Pella36500
PFAAlfa Aesar43368
PicrotoxinTocris1128
Poly-D-lysine hydrobromideSigmaP7280
ProLong Gold Antifade MountantLife TechnologiesP36934
Rabbit anti-GFP antibodyInvitrogenA11122
Rabbit anti-PSD-95 antibodyCell Signaling2507
StrychnineTocris2785
SucroseSigmaS0389
Superfrost plus microscope slidesFisher12-550-15
Triton X-100SigmaX100
TTXTocris1078

References

  1. Enns, C. Overview of protein trafficking in the secretory and endocytic pathways. Current Protocols in Cell Biology. , (2001).
  2. Bedford, F. K., Binder, M. D., Hirokawa, N., Windhorst, U. . Encyclopedia of Neuroscience. , 3385-3389 (2009).
  3. D....

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