Elsevier

Neuroscience

Volume 167, Issue 1, 28 April 2010, Pages 68-87
Neuroscience

Cellular Neuroscience
Research Paper
Organization of NMDA receptors at extrasynaptic locations

https://doi.org/10.1016/j.neuroscience.2010.01.022Get rights and content

Abstract

NMDA receptors are found in neurons both at synapses and in extrasynaptic locations. Extrasynaptic locations are poorly characterized. Here we used preembedding immunoperoxidase and postembedding immunogold electron microscopy and fluorescence light microscopy to characterize extrasynaptic NMDA receptor locations in dissociated hippocampal neurons in vitro and in the adult and postnatal hippocampus in vivo. We found that extrasynaptic NMDA receptors on neurons in vivo and in vitro were usually concentrated at points of contact with adjacent processes, which were mainly axons, axon terminals, or glia. Many of these contacts were shown to contain adhesion factors such as cadherin and catenin. We also found associations of extrasynaptic NMDA receptors with the membrane associated guanylate kinase (MAGUKs), postsynaptic density (PSD)-95 and SAP102. Developmental differences were also observed. At postnatal day 2 in vivo, extrasynaptic NMDA receptors could often be found at sites with distinct densities whereas dense material was seen only rarely at sites of extrasynaptic NMDA receptors in the adult hippocampus in vivo. This difference probably indicates that many sites of extrasynaptic NMDA receptors in early postnatal ages represent synapse formation or possibly sites for synapse elimination. At all ages, as suggested in both in vivo and in vitro studies, extrasynaptic NMDA receptors on dendrites or the sides of spines may form complexes with other proteins, in many cases, at stable associations with adjacent cell processes. These associations may facilitate unique functions for extrasynaptic NMDA receptors.

Section snippets

Antibodies

Most antibodies used in this study have been previously described (Sans et al., 2000, Petralia et al., 2002, Petralia et al., 2005, Bezzi et al., 2004, and Yi et al., 2007). These included (1) rabbit polyclonal antibodies: SAP102 (gift of Johannes Hell), flag (Sigma, St. Louis, MO, USA), synapsin (Chemicon, Temecula, CA, USA), NR2A and NR2B (Groc et al., 2006); (2) mouse monoclonal antibodies: PSD-95/93 (MA1-046; Affinity BioReagents, Golden, CO, USA), β-catenin, N-cadherin, and PSD-95 (BD

Immunofluorescence studies of extrasynaptic NMDARs in vitro

We first studied the organization of extrasynaptic NMDARs in cultures of hippocampal neurons at the LM level. We compared live surface labeling of native receptors using anti-NR2A and NR2B rabbit antibodies in cultures of hippocampal neurons at three WIV, with live surface labeling of transfected neurons at two to three WIV using anti-FLAG antibodies to detect flag-NR2A and flag-NR2B. In each case, parallel immunolabeling was carried out with an antibody raised in guinea pigs against the

Discussion

In this study, we combined LM and EM studies in brain and in vitro to show that extrasynaptic NMDARs are localized to discrete locations along dendrites and postsynaptic spines. In most cases, extrasynaptic NMDARs were found accumulated at points in close contact with adjacent cell processes including mainly axons/axon terminals or glia. This is consistent with previous studies showing extrasynaptic membrane localizations of immunogold for NMDARs (Valtschanoff et al., 1999), which includes

Acknowledgments

We thank Nicole Thompson for help formatting the references, and Dr. Inna A. Belyantseva for helpful comments on the text. Research was supported by the NIDCD Intramural Research Program. A.Z. was funded (in part) by ORWH-FAES NIH.

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