Elsevier

Neuroscience

Volume 158, Issue 4, 18 February 2009, Pages 1446-1459
Neuroscience

Molecular Neuroscience
Phospho-regulation of synaptic and extrasynaptic N-methyl-d-aspartate receptors in adult hippocampal slices

https://doi.org/10.1016/j.neuroscience.2008.11.006Get rights and content

Abstract

Recent evidence demonstrates that N-methyl-d-aspartate receptor (NMDAR) trafficking contributes to synaptic plasticity in the hippocampus. Phosphorylation of tyrosine residues, especially NR2B tyrosine 1472, appears to be a mechanism by which NMDAR endocytosis is prevented, suggesting that the tyrosine phosphorylation and surface expression of NMDARs are positively correlated. Previous work from our laboratory and others has confirmed that modulation of tyrosine phosphatase and kinase activity alters the surface expression of NMDARs. However, the changes in NMDAR surface expression described in those studies were in terms of total surface membrane versus intracellular receptors. Within the plasma membrane of glutamatergic synapses, distinct populations of NMDARs exist. Namely, receptors at the surface can be differentiated into synaptic and extrasynaptic pools based on their association with the post-synaptic density (PSD) and availability to glutamate. In the present study, we utilized a subcellular fractionation approach coupled with detergent extraction to prepare synaptic and extrasynaptic NMDARs from adult rat hippocampal slices. Using this method, we examined how tyrosine phosphatase and Src-family tyrosine kinase (SFK) inhibitors modulate the phosphorylation and localization of these different pools of NMDARs. We found that both synaptic and extrasynaptic NMDARs were modulated by tyrosine phosphatase and SFK inhibitors; however subunit- and residue-specific effects were observed. Specifically, phosphorylation of NR2B tyrosine 1472 was associated with enrichment of synaptic NMDARs, whereas phosphorylation of NR2B tyrosine 1336 was associated with enrichment of extrasynaptic NMDARs. Using electrophysiological methods, we also reveal that the biochemical modifications produced by these inhibitors were associated with corresponding changes in NMDAR function.

Section snippets

Antibodies and reagents

Rabbit polyclonal antibodies against NR2A and NR2B were generated previously in our laboratory and described elsewhere (Snell et al 1996, Grosshans and Browning 2001). The phospho-site specific antibodies against NR2B Tyr1252, Tyr1336, and Tyr1472 were obtained from PhosphoSolutions, Inc. (Aurora, CO, USA). Mouse monoclonal anti-phospho-tyrosine clone 4G10, anti–post-synaptic density (PSD) 95, anti-Src, and anti-Yes antibodies were obtained from Upstate Biotechnology (Lake Placid, NY, USA). The

Characterization of methods used to enrich synaptic and extrasynaptic membranes

A modified classical subcellular fractionation approach was utilized in order to separate plasma membrane fractions into synaptic and extrasynaptic membranes (Fig. 1a and see Carlin et al 1980, Cho et al 1992, Davies et al 2007). This method takes advantage of the fact that synaptic junctions and PSDs are insoluble in Triton-X 100 (Cotman and Taylor 1972, Cotman et al 1974, Matus and Taff-Jones 1978, Cohen 1997) and other membranes not tightly bound to the PSD, including membranes from the

Discussion

Accumulating evidence demonstrates that the tyrosine phosphorylation of NMDARs is positively associated with their surface expression (Dunah and Standaert 2001, Goebel et al 2005, Snyder et al 2005, Hallett et al 2006, Braithwaite et al 2006). Given that surface-bound NMDARs can be differentiated into synaptic and extrasynaptic receptors, it has remained unknown how tyrosine phosphatase and kinase inhibitors modulate NMDARs in these different compartments. Another unanswered question concerns

Acknowledgments

We thank Dr. Paul Lombroso for kindly providing the anti-STEP antibody and for helpful discussions. This research was supported by National Institutes of Health grants AA09675-10 and AG04418-19 and by an institutional National Institutes of Health training grant (HD41697-02). M.D.B. has a financial interest in PhosphoSolutions, Inc., a company which provided several of the antibodies used in this study.

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