Cellular NeuroscienceResearch PaperOrganization of NMDA receptors at extrasynaptic locations
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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