Organization of NMDA receptors at extrasynaptic locations

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 MAGUKs, 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. KeywordsPSD-95; SAP102; cadherin; catenin; NR2A; NR2B Extrasynaptic NMDA receptors (NMDARs) are common on neurons but are little understood compared to synaptic NMDARs. In hippocampal neuronal cultures, physiological studies showed that ~ 75% of NMDARs are extrasynaptic at about 1 WIV (= week or weeks in vitro; Rosenmund et al., 1995; Westbrook, 1999, 2002) but this decreases with the published values ranging from ~ 20% to 50% at 2 WIV (Ivanov et al., 2006).Correspondence: Ronald S. Petralia 50 South Drive, Rm. 4142 NIDCD/NIH Bethesda, MD 20892-8027 301-496-3804 petralia@nidcd.nih.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2011 April 28. Published in final edited form as:Neuroscience. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptImmunocytochemical studies indicate that about 80-90% of NMDARs are extrasynaptic at 1 WIV. These comprise both NR2A-and NR2B-containing receptors. At 1 WIV, the majority of N...

Organization, control and function of extrasynaptic NMDA receptors

“…These extrasynaptic sites spread to an average width of less than 100 nm, which means they are substantially narrower than synaptic sites that have an estimated width of 195 nm in the CA1 region [37]. Interestingly, extrasynaptic NMDAR clusters seem to colocalize with intercellular adhesion molecules such as b-catenins and some scaffolding proteins such as SAP102 and PSD95 in culture [22], although it is not clear in this work whether any specific MAGUKs are preferentially interacting with extrasynaptic NMDARs. These observations strongly suggest that extrasynaptic NMDARs are clustered and organized at the plasma membrane in a similar manner to their synaptic homologues.…”

“…Because they tend to interact with SAP102 rather than with PSD95, GluN2B-NMDARs are thought to be more mobile, widespread and not confined to synapses in adults [45,47]. In fact, the expression of GluN2A and GluN2B subunits closely follows the expression of PSD95 and SAP102, respectively, during development [47], and evidence suggests that (i) neonatal GluN2B-NMDARs are held at synapses through their interaction with SAP102 during early stages of development; (ii) the expression of PSD95 increases throughout postnatal development and competes with SAP102 for insertion into the PSD; and (iii) this displaces SAP102 and GluN2B subunits outside of synapses, whereas PSD95 and GluN2A become predominant at synaptic sites [22,45,48,49]. Along with the fact that neurons of the visual cortex and superior colliculus still show significant expression of SAP102 and GluN2B subunits in adulthood [45,47], these observations together strongly fuel the idea that GluN2B-NMDARs exist at extrasynaptic sites more abundantly than GluN2A-NMDARs, which are preferentially found at synapses.…”

“…This difficulty arises from the fact that what one considers as extrasynaptic based on morphological clues can still be part of the synaptic space as far as signal transmission is concerned. From the morphological point of view, receptors are often considered extrasynaptic if they lie more than 100 nm away from the post-synaptic density (PSD) [22,23]. However, this morphological definition does not take into account glutamate spillover during synaptic transmission, which probably activates receptors located on the neck of dendritic spines (i.e.…”

“…Interestingly, it seems that extrasynaptic NMDARs are organized as clusters, with a discrete distribution along the perisynaptic zone (45%) and dendritic shaft (55%) [22]. In particular, these clusters often correspond to contact area between a dendrite and adjacent cell processes, including glial (approx.…”

Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

“…Immunogold electron microscopy (EM) Post-embedding immunogold labeling was based on established methods (Petralia et al, 2005(Petralia et al, , 2010. In brief, rats were perfused with 4% to express shRNA in neurons under the control of a U6 promoter while expressing GFP under the control of an independent CMV promoter as an indication of infection efficiency.…”