Developmental modification of synaptic NMDAR composition and maturation of glutamatergic synapses: matching postsynaptic slots with receptor pegs.
N-methyl-D-aspartate receptors (NMDARs) are neurotransmitter receptors that are ubiquitous to glutamatergic synapses in the central nervous system. NMDARs are involved in basic synaptic transmission and serve as a triggering point for the induction of long-lasting synaptic plasticity (Malenka and Bear, 2004). NMDAR-dependent synaptic plasticity is strongly implicated in normal forebrain development (Haberny et at., 2002; Dumas, 2005) and is necessary for learning and memory (Wang and Morris, 2010; Rolls, 2010). As well, aberrant NMDAR signaling is observed after brain injury and during chronic stress or disease (Lau and Zuldn, 2007; Hardingham and Baling, 2010; Gladding and Raymond, 2011). A switch in NMDAR subunits occurs throughout the vertebrate forebrain during postnatal development and is the subject of intense investigation because it is associated with dramatic changes in excitatory synaptic transmission and cognitive ability (Dumas, 2005). Combined with its role in neuronal and cognitive development, the ubiquity of this developmental phenomenon (seen in humans, non-human primates, rodents, birds, and amphibians) makes it a highly pertinent model system.
As is the case with most neurotransmitter receptors, NMDARs show developmental and regional heterogeneity (Monyer et al., 1992, 1994; Watanabe et al., 1994). All NMDARs contain two obligatory GluN1 subunits. GluN1 subunits arise from a single gene but exist in multiple isoforms due to alternative splicing. Additional NMDAR subunits fall into two families, G1uN2 and G1uN3, with all isoforms arising from separate genes. This paper focuses on G1uN2A and GluN2B because these subunits are expressed in forebrain and are intimate participants in excitatory synapse maturation and, in turn, cognitive development. The developmental modification in NMDAR composition is described; issues on the molecular level that limit the investigation of NMDAR function across development are explained; and one approach to a more complete understanding of NMDAR function and synapse development is offered, namely the generation of transgenic mice expressing chimeric NMDAR subunits.
GluN2 structure and forebrain synapse development
Like all NMDAR subunits, G1uN2A and GluN2B subunits consist of three basic parts: an extracellular amino region, a series of four transmembrane (TM) segments with connecting extracellular and intracellular loops, and an intracellular carboxy terminus (Fig. i). Briefly, the extracellular amino region contains an amino terminal domain (ATD) that is sensitive to small-molecule modulation and regulates channel open probability and deactivation (Gielen et at., 2009; Yuan et at., 2009), as well as an S1S2 "Venus flytrap" ligand-binding domain (LBD) for glutamate (Paoletti and Neyton, 2007). In addition to forming the ion pore, the TM series has signaling sequences that participate in subunit assembly (Cao et at., 2011; Salussolia et at., 2011), and N-glycosylation sites in the extracellular loop domain that confer either constitutive or activity-dependent synaptic delivery (Storey et at., 2011). The intracellular carboxy terminus contains various sequences that act as sites for interaction with anchoring and signaling proteins in the postsynaptic density (PSD). Well-described signaling domains in the carboxy terminus include binding and phosphorylation sites for alpha calcium-calmodulin kinase type-2 (aCaMKII), a mediator of activity-dependent longterm potentiation (LTP) and long-term depression (LTD) of synaptic efficacy Merrill et at., 2005); phosphorylation sites for the tyrosine kinases, Src and Fyn, that regulate channel function (Sala and Sheng, 1999); an adaptor protein 2 (AP-2) binding site that regulates receptor internalization (YEKL, Lavezzari et at., 2004; Prybylowski et at., 2005); and a proximal ESDV sequence that permits anchoring at PDZ domains of scaffolding proteins (e.g., membrane-associated guanylate kinase proteins, MAGUKs) in the PSD (Sheng, 2001) (Fig. 2). The most proximal ESDV signal has been described as a peg that fits into a PSD anchoring "slot" (Newpher and Ehlers, 2009). Due to postnatal modifications in the types of anchoring proteins that occupy the PSD, this slot and peg phenomenon has important ramifications for developmental regulation of synaptic NMDAR composition as well as proteomic analysis of NMDAR molecular complexes (Husi et at., 2000) and the design of genetically modified mice with altered GluN2 subunit expression.
During postnatal development of forebrain synapses, NMDARs transform from containing GluN2B to GluN2A subunits (Fig. 3, "Wildtype"). This switch occurs in different forebrain regions at different ages (Crair and Malenka, 1995; Nase et al., 1999; Quinlan et al., 1999; Lu et al., 2001; Hsieh et al., 2002), is sensitive to changes in neuronal activity (Bellone and Nicoll, 2007), and is likely regulated at numerous steps of the NMDAR lifecycle (Yashiro and Philpot, 2008). Increasing postnatal age and neuronal activity are associated with increased G1uN2A expression (Monyer et al., 1994; Cull-Candy et al., 2001) and synaptic delivery relative to GluN2B (Barria and Malinow, 2002; Storey et ul., 2011), reduced GiuN2B translation (Chen and Bear, 2007), and increased susceptibility of G1uN2B to internalization (Sanz-Clemente et al., 2010) and ubiquitin-dependent degradation (Turd et al., 2008). All of these factors favor G1uN2A availability at the synapse. Expression appears to be a key regulatory step because the relative levels of GluN2A and GluN2B at synapses can be partially offset by transgenic overexpression of GluN2 subunits, as seen in cultured cerebellar granule cells (Prybylowski et al., 2005), organotypic hippocampal slices (Gambrill and Barria, 2011), and in the hippocampus in vivo (Tang et al., 1999; Wang et al., 2009). However, these factors do not dictate the specific synaptic localization of newly arriving NMDARs either to be near neurotransmitter release sites or to reside perisynaptically.
Importantly, specific placement of NMDARs--that is, synaptic or perisynaptic--can alter receptor function due to distance from transmitter release sites (KulImam) and Asztely, 1998) and interaction with different intracellular signaling streams (Hardingham et at., 2002; Ehlers, 2003; Ivanov et at., 2006). While NMDARs have been reported to be synaptic or perisynaptic, developmental replacement of G1uN2B by GluN2A at hippocampal synapses (Barria and Malinow, 2002) depends on the match between the cytoplasmic carboxy terminal domain of GluN2 and the predominant MAGUK expressed at a given postnatal age (Sheng, 2001; Kiihr, 2006). For instance, during early postnatal development, immature hippocampal synapses contain SAP102 and NMDARs with GluN2B (Fig. 3). During maturation, as SAP102 is replaced by PSD95, NMDARs with GluN2A replace NMDARs with G1uN2B (Sans el al., 2000; Elias et al., 2008). The GluN2B-to-GluN2A shift does not occur in knockout mice that do not express PSD95 (Belque et at., 2006). Functional analyses further suggest that SAP102 associates similarly with G1uN2A or G1uN2B and that PSD95 selectively associates with G1uN2A (Elias et at., 2008), in part through divalent interaction (Bard et at., 2010). Moreover, spontaneous exocytosis of single vesicles primarily activates NMDARs with GluN2A directly beneath transmitter release sites (Townsend et at., 2003; Zhao and Constantine-Paton, 2007); while NIVIDARs with GluN2B appear more concentrated perisynaptically, as shown by G1uN2B pharmacology experiments (Stocca and Vicini, 1998; Dalby and Mody, 2003) and high-frequency synaptic activation (Brickley et at., 2003; Lozovaya et at., 2004). Since biochemical assays show that SAP102 and PSD95 can both interact with GluN2A and G1uN2B (Al-Hallaq et at., 2007), it appears likely that pre-assembled G1uN2A-PSD95 is preferentially inserted at transmitter release sites of more mature animals (Sans et al., 2003; Bessoh et al., 2007). Note that segregated placement is not absolute in that some G1uN2B can be found synaptically (Fujisawa and Aoki, 2003; KOhr et al., 2003; Janssen et al., 2005) and some G1uN2A can be found perisyn-apticaLly (Li et al., 1998; Mohrmann et al., 2002; Thomas et al., 2006), probably due to a tri-heteromeric composition--that is, containing G1uN1, G1uN2A, and G1uN2B subunits (Al-Hallaq et al., 2007). In fact, a substantial number of NMDARs at cortical (Sheng et al., 1994; Luo et at., 1997; Kew et al., 1998), cerebellar (Chazot et al., 1994), and hippocampal synapses (Tovar and Westbrook, 1999; Al-Hallaq et al., 2007) appear to contain both GiuN2A and GluN2B subunits, and display some functional properties that are intermediate to GluN2A and GluN2B di-heteromeric receptors. Triheteromeric NMDARs add further complexity to the study of synaptic NMDAR composition.
The developmental GluN2B-to-GluN2A subunit switch alters a number of NMDAR properties including affinity for glutamate, small molecules (phenylethanolamines, PEAs), and ions (protons, zinc); channel open probability and deactivation rates; the presence or absence of activity-dependent synaptic integration; and subunit-selective interactions with intracellular signaling and anchoring proteins (Fig. 2). One or more of these functional properties that are unique to G1uN2A or GluN2B are likely responsible for changes in activity-dependent synaptic plasticity that occur late in postnatal development during the GluN2B-to-GluN2A switch. For instance, prior to the subunit switch, when GluN2B synaptic content is high, NMDAR.-dependent LTP at hippocampal synapses is more difficult to induce but larger in magnitude (Buchanan and Mellor, 2007; Dumas, 2012), and LTD induction is facilitated (Dudek and Bear, 1993). After the GluN2 subunit switch, it is possible to elicit LTP at lower induction frequencies (Dumas, 2012), and LTD is more difficult to induce (Dudek and Bear, 1993). The GluN2 subunit switch also removes an inhibitory signal that prevents the insertion of a-amino-3-hydroxy-5-methy1-4-isoxazolepropionic acid receptors (AMPARs) into the PSD (Hall et al., 2007; Elias et al., 2008; Adesnik et al., 2008). Removal of this AMPAR inhibitory signal induces silent synapses to become active (Gray et al., 2011) and also alters the threshold for induction of synaptic plasticity (Stoneham et al., 2010). One or more of these developmental modifications in synaptic transmission are likely to be involved in concurrent behavioral changes that are observed in learning and memory tests (Dumas, 2005). Thus, molecular dissection of the functional differences between GluN2A and G1uN2B is likely to help explain maturation of glutamate synapses in the forebrain and clarify the involvement of NMDARs in cognitive processes.
GluN2A Domain/Motif AA Number Source Function ATD (1-389) 1 Assembly ATD (25-391) 2, 3, 4 Channel Kinetics (29-404) ATD (1-389) 5 6 Trafficking (44-58) LIVBP + S1 (1-550) 7, 8 LIVBP (1-415) (44, 7 Zinc Binding 102, 105, 107, 128, 233, 266) LIVBP 1-282 10 PEA Binding LIVBP (1-350) 13 Glutamate Binding LAOBP (S1) (473-529) 7 LAOBP (S1) 14, 15 (450, 478, 17, 18 480, 485, 511, 513, 517,518, 465, 466, 671) T671 (671) 18 Synaptic Incorporation 584 (584) 20 Calcium Permeability M1-M4 (539-817) 8, 21 Assembly ER Retention SYTANLAAF (645-653) 23, 24, Channel Activity (M3-M4) (642, 785) 25 S2 (674) 26 Synaptic Delivery LAOBP (S2) (682-695, 14 15, Allosteric 724-738) (654. 17 Modulation of 655, 705, 665, Glutamate Binding 666. 669, 671, 689, 690, 692, 732) Y842, 674-1464 (842, 27,28, Internalization 874-1464) 29 HLFY (839-842) 30 ER Export Y838-875. Y642, (838-875, 842, 27 32, Src Y1267. Y1105, 1267, 1105. 33 Y1109. Y1387 1109, 1387) 934-1203 (934-1203) 34 PKA Spectrin L1244-V1389 (1244-1389 36 CaMKII 1255-1298. 1255-1298, 39 1303, 1303 S1398-V1464, 1398-1464, 37, 32, 1349-1464 1349-1464) 36, 38, 1413-1419 (1413-1419) 39 Y1267 (1267) 27 Zinc Binding 1304-1464 (1304-1464) 41 Degradation o-Actinin Binding Recycling 1159-1464 (1159-1464) 44 Fyn 1408-1429 (1408-1429) 34 PKA 1444-1464 (1444-1464) S1416 (1416) 36 PKC 1444-1464 (1444-1464) 34 R1451. R1452. 1459-1464 18 Synaptic Y1454, K1455, (1451,1452, 19 Incorporation M1456.S1462 1454, 1455, 1457, 1462) LL YEKL ESDV (1319-1320 46 Internalization 53 1454-1457 (AP-2 Binding) 1461-1464) SH3 (1382-1420) 49 PSD95 Anchoring ESDV (1461-1464) 50, 51, 46 L1244-V1309 (1459-1464) 18 S1389-V1464, (1244-1309, 36 43 6 1349-1464, 1389-1464 ) (1349-1464) ESDV (1461-1464) 51 SAP102 1454-1464 (1454-1464) 6 Anchoring GluN2B Domain/Motif Domain/ AA Number Source Motif ATD ATD ATD (25-392) 2, 3 4 (29-404) ATD ATD (1-388) 5 6 (55-60) LIVBP + S1 LIVBP + S1 (1-550) 7, 8 LIVBP LIVBP (1-400) 7 LIVBP LIVBP (1-400), 7, 9, 10, (1-282) (82, 11, 12 101, 114, 150, 176, 236) LIVBP LIVBP (1-350) 13 LAOBP (S1) (400-550) 7, 8 16 15 14, 15 (387, 390, 459, 460, 486, 493, 450, 478, 480, 485) T671 Y531 (531) 19 584 M1-M4 M1-M4 (536-811) 8, 21 M3 (629-655) 22 SYTANLAAF STYTANLAAF (645-653) 23, 24, (M3-M4) (M3-M4) (651) 23 S2 S2 (675) 26 LAOBP (S2) LAOBP (S2) (550-700) 7,8 15 (654, 655, 16 705, 660, 664) Y842, 674-1464 Y843 (843) 27,28, HLFY HLFY T1070. (840-843) 30 31 H1119 (1070, 1119) Y838-875. Y642, Y843, Y1109, (843, 1109, 28. 32, Y1267. Y1105, Y1281, Y1472 1281, 1472) 33 Y1109. Y1387 934-1203 953-1258 (953-1258) 34 SH3. PXXP 1114-1117 35 KKNXN 1292-1296 L1244-V1389 839-1120 (839-1120 28. 37, 1255-1298. 1260-1309 1260-1309 40, 32, 1303, S1398-V1464, S1303 1303) 29, 39 1349-1464 1413-1419 Y1267 1304-1464 839-1482 (839-1482) 1361-1453 (1361-1453) 43 1315-1482 (1315-1482) 43 1159-1464 Y1472 (1472) 28, 32 19,44 1408-1429 1462-1482 (1462-1482) 34 1444-1464 S1416 S1303, S1323 (1303, 1323) 45 1444-1464 1462-1482 (1462-1482) 34 R1451. R1452. S1472, 1477-1482 18 Y1454, K1455, S1480 (1472, 1480) 19 M1456.S1462 LL YEKL ESDV LL YEKL ESDV (1395-1396) 47 19 53 S1480 (1472-1475 1479-1482) (1480) SH3 SH3 (1086-1157) 49 ESDV ESDV (1479-1482) 50, 52 L1244-V1309 1453-1482 (1477-1482) 18 S1389-V1464, 1454-1464 (1453-1482) 43 6 1349-1464, (1454-1464) ESDV 1454-1464 (1454-1464) 6 1454-1464 Figure 2. Chart of known signaling sites for GluN2A and GluN2B. Sites are separated by major segment (amino in white, transmembrane in light gray, or carboxy in dark gray). Motif labels are taken from the primary literature and listed with known amino acids. Bold text indicates a subunit selective interaction. Text boxes are used with citation numbers to indicate gain (no box) or loss (box) of function of the related property with the presence of the motif. Citations numbers are defined as follows: (1) Meddows et al., 2001. (2) Mullasseril et al., 2010, (3) Gielen et al., 2009. (4) Yuan et al., 2009. (5) Qiu et al., 2009. (6) Cousins et al., 2008. (7) Paoletti et al., 2000. (8) Cao et of., 2011. (9) Ng et al., 2008. (10) Rosahl et al., 2006, (11) Karakas et al., 2011, (12) Perin-Dureau et al., 2002, (13) Paoletti and Neyton, 2007, (14) Kalbaugh et al., 2004. (15) Chen et al., 2005, (16) Laube et al., 1997, (17) Anson et al., 1998, (18) Barria and Malinow. 2002. (19) Prybylowski et al., 2005, (20) Meguro et al., 1992. (21) Kittlel. and Schoepler, 1996, (22) Florak et al., 2008. (23) Chang and KUO. 2008, (24) Xu et al., 2012. (25) Talukder and Wollmuth, 2011, (26) Storcy er al., 2011, (27) Vissel ci al., 2001, (28) Wenthold ci al., 2003. (29) Woodward. 2002. (30) Yang ci al., 2007, (31) Hawkins ci al., 2004. (32) Kennedy and Manzerra, 2001, (33) Bessoh ci al., 2007, (34) Leonard and Hell. 1997, (35) Wechsler and Teichherg. 1998. (36) Gardoni ci al., 2001, (37) Strack and Coibran, 1998. (38) Gardoni cial., 1999, (39) Merrill ci at., 2005. (40) Strack cial., 2000. (41) Tang ci al., 2010, (42) Jurd ci al., 2008. (43) Wyszynski ci at., 1997. (44) Rong ci al., 2001, (45) Liao ci cli., 2001, (46) Lavezzari ef t1.. 2004. (47) Roche ci al., 2001, (48) Sanz-Clernente ci al., 2010. (49) Cousins ci al., 2009. (50) Kornau et al., 1995. (51) Sheng and Kim. 19%. (52) Kurshner ci al., 1998. LIV HP, leucine/ isoIeuciie./vahne-bindmg protein domain: LAOB P. lysine/arginine/ornithine-binding protein domain; SYTANLAAF, conserved GIuN moiil: 1 ILFY. ER export signal; LL. dileucine motif: S1-13. Src homology 3 domain.
Genetic studies of GluN2
Much of the evidence for relationships between the GluN2 subunits, synaptic function, and behavior comes from studies that employ molecular and genetic techniques to modify or terminate NMDAR subunit expression. Early studies encountered neonatal lethality with genetic deletion of GluN1 (Li et al., 1994; Forrest et al., 1994), GluN2B (Kutsuwada et al., 1996), or in mice lacking only the carboxy terminus of G1uN2B (Sprengel et al., 1998), but not after deletion of GluN2A. Initial investigation of adult mice with genetic deletion of G1uN2A revealed impaired spatial learning and contextual fear conditioning (Sakimura et al., 1995; Kiyama et al., 1998). LTP was induced at hippocampal synapses only after multiple high-frequency bursts were applied, suggesting that LTP could be obtained by activation of NMDARs with G1uN2B subunits, but with a much higher threshold for induction (Sakimura et al., 1995; Kiyama et al., 1998). Combined, these physiological and behavioral results might suggest that G1uN2A allows for LTP to be induced more easily or more rapidly, which could explain developmental modifications in LTP induction threshold (Dumas, 2010) and learning and memory abilities (Dumas, 2005). A more recent study employing the same mutant mouse lines lacking GluN2A showed intact spatial reference memory but impaired spatial working memory and impaired single-trial learning (Bannerman et al., 2008). A loss of G1uN2A-dependent lowering of LTP threshold might explain the disruption of learning and memory abilities that require more rapid processing, yet maintenance of slower developing reference memory formation in G1uN2A knockout mice. Related experiments examined mice expressing truncated GluN2A subunits having no carboxy terminus. Learning and reference memory as well as motor impairments were observed (Sprengel et al., 1998). Furthermore, in the presence of a specific G1uN2B antagonist, synaptic NMDAR currents were still present but LTP induction was blocked, indicating that calcium entry alone was insufficient for LTP induction and implicating the C-terminus of G1uN2A in LTP induction (Kohr et al., 2003; Berberich et al., 2007). While informative on molecular and physiological levels, these preliminary studies lacked regional anatomical specificity, hindering a greater understanding of brain-to-behavior relationships; and they did not investigate the specific synaptic location of the mutated NMDARs or possible modifications in PSD organization.
Recordings made in hippocampal slices taken from primary G1uN2B knockout mice maintained until postnatal day (P) P2 or P3 by hand-feeding revealed impaired induction of LTD (Kutsuwada et al., 1996). Unfortunately, no assessments of baseline synaptic strength or LTP were made. Using different means to circumvent the lethality produced by G1uN2B deletion and increase the age at which recordings can be made, cortical cultures were prepared from late-stage embryonic mice and maintained alive for weeks after preparation (Hall et al., 2007). Obviously, this manipulation precludes behavior testing, but physiological experiments revealed that deletion of G1uN2B induced greater surface expression of AMPARs. These findings were corroborated in organotypic slices overexpressing GluN2A or GluN2B (Gambrill and Barria, 2011). While these papers provide a robust description of the mechanisms involved in the GluN2B regulation of AMPAR insertion, no plasticity experiments were performed. More recent GluN2B knockout models, limiting deletion to the late postnatal period and restricted brain structures, further implicated this subunit in functional development of glutamatergic synapses. For instance, mosaic G1uN2B deletion from hippocampal neurons enhanced the functional induction of silent synapses during development (Gray et al., 2011). GluN2B deletion limited to cortical and hippocampal pyramidal cells resulted in viable, healthy animals (von Engelhardt et at., 2008; Brig-man et al., 2010). As adults, these animals performed poorly in spatial learning and memory tests and displayed impaired induction of LTP. Interestingly, when GluN2B was deleted from CAI pyramidal cells only, animals performed similar to controls in spatial reference memory tasks, but like G1uN2A knockouts, were impaired in working memory for recently visited places (von Engelhardt et al., 2008). In both studies, increased afferent stimulation produced LTP at hippocampal synapses, indicating that G1uN2B subunits are not absolutely necessary for LTP induction. Although a deficit in LTD at hippocampal synapses was reported, a glutamate transporter blocker, which likely increased activation of perisynaptic NMDARs, was necessary for the induction of LTD, confounding interpretations about the function of synaptic NMDARs (Brigman et al., 2010). G1uN2A may be preferentially activated in adults due to synaptic positioning closer to neurotransmitter release sites. So, under physiological conditions, it appears that GluN2B-containing NMDARs may play a modulatory role, either by providing greater calcium entry when transmitter release is high or by recruiting intracellular plasticity proteins to the site of calcium entry. The differential impact on rapid versus slower spatial learning in GluN2A or GluN2B knockouts may be a function of the specific neurons that undergo the mutation (i.e., CA1 pyramidal cells). However, deletion of G1uN1 from CA3 pyramidal cells (Nakazawa et at., 2003) or dentate gyrus granule cells (McHugh et al., 2007) and deletion of AMPAR subunits in CAI pyramidal cells all produce the same behavioral phenotype (Schmitt et al., 2005). So, similarity between the effects of GluN2A and GluN2B deletion could be an artifact resulting from compensatory changes in synaptic organization. More sophisticated mutations to GluN2 subunits, greater neuroanatomical and temporal control over transgene expression, and finer assessment of synaptic placement of mutated subunits will provide greater clarification.
Next-generation genetic studies of the developmental G1uN2 switch: Anatomically specific chimeric GluN2 subunit expression in transgenic mice
The developmental shift in the type of GluN2 subunits found at synapses is mediated by changes in PSD scaffolding proteins (Sheng, 2001). As SAP102 is replaced by PSD95, G1uN2B is replaced by G1uN2A (Sans et al., 2000; Elias et al., 2008). When GluN2A-containing NMDARs are inserted at synapses, they also displace G1uN2B-containing NMDARs perisynaptically (Groc et al., 2009). This information is important not only for understanding synaptic development and plasticity, but also in the design of transgenic mice to study the functional implications of changes in NMDAR composition. That is, the age of the animal and hence the "context" of the PSD must be considered (Kiihr, 2006). This problem is exemplified in studies investigating the effects of G1uN2B overexpression on glutamatergic synaptic transmission in adult visual cortical slices (Philpot et al., 2001) or hippocampal slice cultures after the SAP102-to-PSD95 shift (Foster et al., 2010). Negative physiological findings were accompanied by immunolabeling evidence that NMDARs built from transgenic GluN2B subunits do not displace NMDARs with GluN2A, most likely because the adult PSD anchoring composition did not match the transgenic G1uN2B carboxy termini of the overexpressed NMDARs.
The ESDV sequence at the tip of the carboxy terminus of the GluN2 subunit interacts directly with PSD anchoring proteins (Gladding and Raymond, 2011). Multiple MAGUKs, including SAP102 and PSD95, interact with GluN2A and GluN2B at this site (Rutter and Stephenson, 2000), suggesting that other direct or indirect subunit-specific interactions exist to confer greater precision placement of GluN2A at synaptic active zones (Cousins et al., 2009), including a divalent structure unique to G1uN2A (Bard et al., 2010). Thus, unless the PSD context is experimentally modified to transgenically express GluN2 subunits that undergo synaptic insertion, the carboxy terminus must match the type of anchoring protein that predominates at the PSD at the intended age of study (Fig. 3). This means that overexpressed GluN2B subunits would be inserted near neurotransmitter release sites at immature synapses having a SAP102, but not at mature synapses having a PSD95 phenotype. In contrast, since G1uN2A binds similarly with SAP102 and PSD95, overexpressed G1uN2A subunits could be inserted near neurotransmitter release sites at immature or mature synapses (Wang et al., 2011). Thus, in contrast to GluN2B overexpression at mature synapses. G1uN2A overexpression at immature synapses would be expected to alter NMDAR composition near release sites. Functional changes induced by G1uN2A or GluN2B overexpression at mature synapses likely reflect alterations in perisynaptic NMDARs or total numbers of synaptic and perisynaptic receptors.
To more fully understand the effects of the developmental GluN2 subunit switch on synapse composition, synaptic function, and behavior, it is necessary to create genetically modified animals in which the mutation is anatomically restricted to pertinent neurons and is compatible with the composition of the PSD at the age of interest. This requires the generation of chimeric G1uN2 subunits in which functional domains and synaptic placement domains are chosen, fused together, and placed under conditional transcriptional control. Some chimeric GluN2 subunits have been generated and used to identify (1) a site in the carboxy tail of G1uN2B that induces NMDAR recycling (Tang etal., 2010) and (2) a N-glycosylation site in the extracellular loop between TM3 and TM4 that confers activity-dependency to G1uN2A synaptic insertion (Storey et al., 2011). Chimeric GluN2 constructs have also been expressed in organotypic hippocampal slice cultures. Under these conditions, it was shown that the G1uN2B carboxy terminus, but not calcium entering through NMDARs with GluN2B subunits, was essential for LTP induction and that the GluN2A carboxy tail served as a dominant negative regulator of LTP induction (Foster et al., 2010). A pre- and postsynaptic pairing protocol was used to slowly induce LTP (in contrast to high-frequency induction). Given these caveats, it is possible that the carboxy tail of G1uN2B slowly recruits molecules to positions near GluN2A calcium channels to facilitate calcium-induced activation of plasticity-related signaling cascades. Shorter bursts of LTP-inducing stimulation are necessary to more closely simulate neuronal activation patterns during sensory stimulation or spatial exploration. Additional chimeras are needed to control synaptic placement of NMDARs having specific GluN2A or GluN2B conductance phenotypes (i.e., open probability, response decay rate) at immature and mature synapses.
To change the calcium conductance phenotype of NMDARs that are inserted into synapses, one must generate G1uN2 chimeras consisting of protein segments with motifs that regulate calcium conductance fused to carboxy terminus domains that allow for synaptic insertion. For instance, fusing the amino and TM regions of GluN2A to the carboxy terminus of GluN2B should produce an NMDAR that conducts as if it contained GluN2A, but can be inserted into juvenile synapses that contain SAP102 (Fig. 3, "ABC). This would produce synapses that retain an immature G1uN2 carboxy complement, but with a more mature NMDAR functional capacity in terms of calcium conductance. Insertion of non-native GluN2A subunits has previously been demonstrated at synapses in developing cortical neurons in GluN2B [right qrrow] GluN2A replacement mice (Wang et al., 2011) and in immature hippocampal organotypic slices (Gambrill and Barria, 2011). In contrast to the ABc chimera, these constructs contain GluN2A carboxy termini (Fig. 3, "GluN2A"). Alternatively, fusing the amino and TM regions of GluN2B to the carboxy terminus of GluN2A (Fig. 3, "BAc") would overcome previous limitations in attempts to reintroduce an immature GluN2B phenotype to mature synapses.
We have generated a chimeric subunit that has the G1uN2A amino and TM segments fused to the G1uN2B carboxy terminus (termed ABc) and, vice versa, a chimera that has the GluN2B amino and TM segments fused to the G1uN2A carboxy terminus (BAc). We placed these constructs downstream from the tetracycline response element (TRE) and generated transgenic mice with genomic integration of either of these constructs. We crossed these animals with a line of mice that express the tetracycline transactivator (tTA) under transcriptional control of the minimal CaMKII promoter (Mayford et at., 1995). Native CaMKII expression is robust in the 2-week-old rat hippocampus (Burgin et al., 1990; Petralia et al., 2005; Liu et at., 2012), and tTA expression driven by the minimal CaMKII promoter can be observed at 2 weeks of age in the mouse hippocampus (Mayford et al., 1995). These results suggest that it is possible to use the minimal CaMICH promoter and tTA expression to drive chimeric GluN2 subunit expression prior to the end of the third postnatal week. Predicted hippocampal synaptic placement profiles for NMDARs with ABc or BAc chimeric subunits are shown in Figure 3. An ABc mutant NMDAR would be expected to mimic overexpression of GluN2A at immature synapses, due to the non-specificity of the predominating PSD anchoring protein, SAP102, for GluN2A or G1uN2B. ABc-containing NMDARs would not be expected to infiltrate the core of the mature synapse, due to binding restrictions between the G1uN2B carboxy terminus and PSD95. However, perisyn-aptic scaffolds might incorporate NMDARs with the chimeric ABc subunit. BAc subunits would not be expected to alter NMDAR conductance phenotype at immature synapse, due to a match with amino and TM regions of endogenous synaptic GluN2B containing NMDARs. However, unlike GluN2B overexpression at mature synapses, NMDARs with the BAc subunits can be inserted into the synaptic core due to the G1uN2A carboxy tail. As stated above, previous experiments examining the impact of transgenic G1uN2B overexpression in mice reported increased LTP magnitude in hippocampal slices and improved spatial learning and memory abilities (Tang et al., 1999; Wang et al., 2009). Subsequent studies showed that these transgenic GluN2B-containing NMDARs were not inserted into synapses and likely resided perisynaptically (Philpot et at., 2001; Foster et al., 2010). With the newly proposed constructs, it is possible to achieve a GluN2B functional phenotype at the synapse core in adult animals and address more detailed questions about GluN2-dependent modifications in calcium conductance. In preliminary experiments, we have observed enhanced hippocampal-dependent spontaneous alternation in immature mice expressing the ABc chimeric subunit (Fig. 4). When allowed to freely explore a symmetrical Y-maze, normal intact adult mice alternate at a rate of 60%-70% (Sarter et al., 1988). At postnatal days Pi 7--P19, when the G1uN2A to GluN2B ratio is lower, wildtype mice alternate at chance levels (near 50%). At P17-19, ABc mice alternated at a rate that was significantly higher than that observed in wildtype littermates of the same age and was similar to the alternation rate seen at P22-24 and in adult wildtype mice. Because different results were observed with overexpression of ABc or BAc subunits, the behavioral effect appears more closely related to the arrangements of the peptide sequences than to the presence of transgenic proteins. Similarly, differential effects of each chimera across age groups are likely due to specific interactions with PSD anchoring proteins that vary as synapses mature. Due to the mismatch between the amino and TM portions of ABc with native G1uN2B subunits at P17-19, these data suggest that a shift from GluN2B-type to G1uN2A-type calcium conductance dynamics, and not a change in carboxy terminus-dependent intracellular signaling, is a limiting factor in the developmental emergence of spatial navigation abilities. Furthermore, observing adult-like hippocampal-dependent behavior at P17-19 after introduction of a chimeric subunit alone strongly supports the idea that the developmental GluN2 shift is a rate-limiting step in the final maturation of the hippocampus. Immunohistochemical labeling of a unique hemagglutinin (HA) tag fused to the ABc and BAc constructs indicates chimeric subunit expression in developing and adult animals (Fig. 5). Additional assays of synapse ultrastructure, synaptic transmission, and learning and memory will help complete our understanding of the importance of developmental changes in NMDAR composition in synaptic and cognitive maturation.
Detailed understanding of how changes in NMDARs relate to alterations in synaptic transmission and behavior require sophisticated molecular techniques to modify specific aspects of NMDAR function in a temporally and anatomically specific manner. Experimental investigation of the developmental G1uN2B-to-G1uN2A shift is complicated by subunit-selective synaptic insertion constraints imposed by age-related changes in PSD anchoring proteins. Generation of transgenic mice that express chimeric GluN2 subunits should allow for specific alterations in synaptic NMDAR calcium conductance while minimally perturbing the molecular organization of the PSD. Such animals will provide substantial new information about parcellation of G1uN2 amino acid motifs as pertains to synaptic transmission and cognition. As shown by preliminary behavioral assays, chimeric G1uN2 subunit expression broadens our understanding of synaptic development and its relationship to cognitive development and will provide new inroads into understanding the mechanisms that support adult learning and memory.
We thank Robert Gardner for critical evaluation of the manuscript. This research was supported by the Jeffress Memorial Trust Fund, the Kxasnow Institute for Advanced Study at George Mason University, and a Multiple University Research Initiative award from the Department of Defense (ONR# N00014-10-1-0198).
Adesnik, H., G. Li, M. J. During, S. J. Pleasure, and R. A. Nicoll. 2008. NMDA receptors inhibit synapse unsilencing during brain development. Proc. Natl. Acad. Sci. USA 105: 5597-5602.
Al-Hallaq, R. A., T. P. Conrads, T. ft Veenstra, and R. J. Wenthold. 2007. NMDA di-heteromeric receptor populations and associated proteins in rat hippocampus. J. Neurosci. 27: 8334-8343.
Anson, L. C., P. E. Chen, D. J. Wyllie, D. Colquhoun, and R. Schoepfer. 1998. Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR I /NR2A NMDA receptors. J. Neurosci. 18: 581-589.
Bannerman, D. M., B. Niewoehner, L. Lyon, C. Romberg, W. B. Schmitt, A. Taylor, D. J. Sanderson, J. Cottam, R. Sprengel, P. H. Seeburg, G. Kiihr, and J. N. Rawlins. 2008. NMDA receptor subunit NR2A is required for rapidly acquired spatial working memory but not incremental spatial reference memory. J. Neurosci. 28: 36233630.
Bard, L., M. Sainlos, D. Bouchet, S. Cousins, L. Mikasova, C. Breillat, F. A. Stephenson, B. Imperiali, D. Choquet, and L. Groc. 2010. Dynamic and specific interaction between synaptic NR2--NMDA receptor and PDZ proteins. Proc. Natl. Acad. Sci. USA 107: 1956119566.
Barria, A., and R. Malinow. 2002. Subunit-specific NMDA receptor trafficking to synapses. Neuron 35: 345-353.
Beique, J. C., D. T. Lin., M. G. Kang, H. Aizawa, K. Takamiya, and R. L. Huganir. 2006. Synapse-specific regulation of AMPA receptor function by PSD-95. Proc. Natl. Acad. Sci. USA 103: 19535-19540.
Bellone, C., and R. A. Nicoll. 2007. Rapid bidirectional switching of synaptic NMDA receptors. Neuron 55: 779-785.
Berberich, S., V. Jensen, 0. Hvalby, P. H. Seeburg, and G. Kohr. 2007. The role of NMDAR subtypes and charge transfer during hippocampal LTP induction. Neuropharmacology 52: 77-86.
Bessoh, S., S. Chen, I. R. Brown, and J. W. Gurd. 2007. Developmental changes in the association of NMDA receptors with lipid rafts. J. Neurosci. Res. 85: 1876-1883.
Brickley, S. G., C. Misra, M. H. Mok, M. Mishina, and S. G. Cull-Candy. 2003. NR2B and NR2D subunits coassemble in cerebellar Golgi cells to form a distinct NMDA receptor subtype restricted to extrasynaptic sites. J. Neurosci. 23: 4958-4966.
Brigman, J. L., T. Wright, G. Talani, S. Prasad-Mulcare, S. Jinde, G. K. Seabold, P. Mathur, M. I. Davis, R. Bock, R. M. Gustin et al. 2010. Loss of GluN2B-containing NMDA receptors in CAI hippocampus and cortex impairs long-term depression, reduces dendritic spine density, and disrupts learning. J. Neurosci. 30: 4590-4600.
Buchanan, K. A., and J. R. Mellor. 2007. The development of synaptic plasticity induction rules and the requirement for postsynaptic spikes in rat hippocampal CA I pyramidal neurones. J. PhysioL 585: 429-445.
Burgin, K. E., M. N. Waxham, S. Pickling, S. A. Westgate, W. C. Mobley, and P. T. Kelly. 1990. In situ hybridization histochemistry of [Ca.sup.2+]/calmoldulin-dependent protein kinase in developing rat brain. J. Neurosci. 10: 1788-1798.
Cao, J. Y., S. Qiu, J. Zhang, J. J. Wang, S. M. Zhang, and J. H. Luo. 2011. Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly. J. Biol. Chem. 286: 27698-27705.
Chang, H. R., and C. C. Kuo. 2008. The activation gate and gating mechanism of the NMDA receptor. J. Neurosci. 28: 1546-1556.
Chazot, P. L., S. K. Coleman, M. Cik, and F. A. Stephenson. 1994. Molecular characterization of N-methyl-D-aspartate receptors expressed in mammalian cells yields evidence for the coexistence of three subunit types within a discrete receptor molecule. J. Biol. Chem. 269: 24403-24409.
Chen, P. E., M. T. Geballe, P. J. Stansfeld, A. R. Johnston, H. Yuan, A. L. Jacob, J. P. Snyder, S. F. Traynelis, and D. J. Wyllie. 2005. Structural features of the glutamate binding site in recombinant NR1/NR2A N-methyl-D-aspartate receptors determined by site-directed mutagenesis and molecular modeling. Mol. Pharmacol. 67: 1470-1484.
Chen, W. S., and M. F. Bear. 2007. Activity-dependent regulation of NR2B translation contributes to metaplasticity in mouse visual cortex. Neuropharmacology 52: 200 -214.
Cousins, S. L., M. Papadakis, A. R. Rutter, and F. A. Stephenson. 2008. Differential interaction of NMDA receptor subtypes with the postsynaptic density-95 family of membrane associated guanylate kinase proteins. J. Neurochem. 104: 903-913.
Cousins, S. L., A. V. Kenny, and F. A. Stephenson. 2009. Delineation of additional PSD-95 binding domains with NMDA receptor NR2 subunits reveals differences between NR2A/PSD-95 and NR2B/PSD-95 association. Neuroscience 158: 89-95.
Crair, M. C., and R. C. Malenka. 1995. A critical period for long-term potentiation at thalamocortical synapses. Nature 375: 325-328.
Cull-Candy, S., S. Brickley, and M. Farrant. 2001. NMDA receptor subunits: diversity, development and disease. Curr. Opin. Neurobiol. 11: 327-335.
Dalby, N. 0., and I. Mody. 2003. Activation of NMDA receptors in rat dentate gyrus granule cells by spontaneous and evoked transmitter release. J. Neurophysiol. 90: 786-797.
Dudek, S. M., and M. B. Bear. 1993. Bidirectional long-term modification of synaptic effectiveness in the adult and immature hippocampus. J. Neurosci. 13: 2910-2918.
Dumas, T. C. 2005. Developmental regulation of cognitive abilities: modified composition of a molecular switch turns on associative learning. Prog. Neurobiol. 76: 189-21 1 .
Dumas, T. C. 2012. Postnatal alterations in induction threshold and expression magnitude of long-term potentiation and long-term depression at hippocampal synapses. Hippocampus 22: 188-199.
Ehlers, M. D. 2003. Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system. Nat. Neurosci. 6: 231-242.
Elias, G. M., L. A. Elias, P. F. Apostolides, A. R. Kriegstein, and R. A. Nicoll. 2008. Differential trafficking of AMPA and NMDA receptors by SAP102 and PSD-95 underlies synapse development. Proc. Natl. Acad. Sci. USA 105: 20953-20958.
Forrest, D., M. Yuzaki, H. D. Soares, L. Ng, D. C. Luk, M. Sheng, C. L. Stewart, J. I. Morgan, J. A. Connor, and T. Curran. 1994. Targeted disruption of NMDA receptor I gene abolishes NMDA response and results in neonatal death. Neuron 13: 325-338.
Foster, K. A., N. McLaughlin, D. Edbauer, M. Phillips, A. Bolton, M. Constantine-Paton, and M. Sheng. 2010. Distinct roles of NR2A and NR2B cytoplasmic tails in long-terrn potentiation. J. Neurosci. 30: 2676-2685.
Fujisawa, S., and C. Aoki. 2003. In vivo blockade of N-methyl-D-aspartate receptors induces rapid trafficking of NR2B subunits away from synapses and out of spines and terminals in adult cortex. Neuroscience 121: 51-63.
Gambrill, A. C., and A. Barria. 2011. NMDA receptor subunit composition controls synaptogenesis and synapse stabilization. Proc. Natl. Acad. Sci. USA 108: 5855-5860.
Gardoni, F., L. H. Schrama, J. J. van Dalen, W. H. Gispen, F. Cat-tabeni, and M. Di Luca. 1999. AlphaCaMKE binding to the C-terminal tail of NMDA receptor subunit NR2A and its modulation by autophosphorylation. FEBS Lett. 456: 394-398.
Gardoni, F., L. H. Schrama, A. Kamal, W. H. Gispen, F. Cattabeni, and M. Di Luca. 2001. Hippocampal synaptic plasticity involves competition between [Ca.sup.2+]/calmodulin-dependent protein kinase 11 and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor. J. Neurosci. 21: 1501-1509.
Gielen, M., R. B. Siegler, L. Mony, J. W. Johnson, and P. Paoletti. 2009. Mechanism of differential control of NMDA receptor activity by NR2 subunits. Nature 459: 703-707.
Gladding, C. M., and L. A. Raymond. 2011. Mechanisms underlying NMDA receptor synaptic/extrasynaptic distribution and function. Moil. Cell. Neurosci. 48: 308-320.
Gray, J. A., Y. Shi, H. Usui, M. J. During, K. Salrimura, and R. A. Nicoll. 2011. Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo. Neuron 71: 1085-1101.
Groc, L., L. Bard, and D. Choquet. 2009. Surface trafficking of N-methyl-D-aspartate receptors: physiological and pathological perspectives. Neuroscience 158: 4-18.
Haberny, K. A., M. G. Paule, A. C. Scallet, F. D. Sistare, D. S. Lester, J. P. Hanig, and W. Slikker, Jr. 2002. Ontogeny of the N-methyl-D-aspartate (NMDA) receptor system and susceptibility to neurotoxicity. Toxicol. Sci. 68: 9-17.
Hall, B. J., B. Ripley. and A. Ghosh. 2007. NR2B signaling regulates the development of synaptic AMPA receptor current. J. Neurosci. 27: 13446-13456.
Hardingham, G. E., and H. Bading. 2010. Synaptic versus extrasyn-aptic NMDA receptor signalling: implications for neurodegenerative disorders. Nat. Rev. Neurosci. 11: 682-696.
Hardingham, G. E., Y. Fukunaga, and H. Bading. 2002. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. Neurosci. 5: 405-414.
Hawkins, L. M., K. Prybylowski, K. Chang, C. Moussan, F. A. Stephenson, and R. J. Wenthold. 2004. Export from the endoplasmic reticulum of assembled N-methyl-D-aspartic acid receptors is controlled by a motif in the c terminus of the NR2 subunit. J. Biol. Chem. 279: 28903-29000.
Horak, M., K. Chang, and R. J. Wenthold. 2008. Masking of the endoplasmic reticulum retention signals during assembly of the NMDA receptor. J. Neurosci. 28: 3500-3509.
Hsieh, C. Y., Y. Chen, F. M. Leslie, and R. Metherate. 2002. Postnatal development of NR2A and NR2B mRNA expression in rat auditory cortex and thalamus. J. Assoc. Res. Otolaryngol. 3: 479487.
Husi, H., M. A. Ward, J. S. Choudhary, W. P. Blackstock, and S. G. Grant. 2000. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nat. Neurosci. 3: 661-669.
Ivanov, A., C. Pellegrino, S. Rama, I. Dumalska, Y. Salyha, Y. Ben-Ari, and I. Medina. 2006. Opposing role of synaptic and extrasynaptic NMDA receptors in regulation of the extracellular signal-regulated kinases (ERK) activity in cultured rat hippocampal neurons. J. PhysioL 572: 789-798.
Janssen, W. G., P. Vissavajjhala, G. Andrews, T. Moran, P. R. Hof, and J. H. Morrison. 2005. Cellular and synaptic distribution of NR2A and NR2B in macaque monkey and rat hippocampus as visualized with subunit-specific monoclonal antibodies. Exp. Neural. 191: S28-S44.
Jurd, R., C. Thornton, J. Wang, K. Luong, K. Phamluong, V. Khara-zia, S. L. Gibb, and D. Ron. 2008. Mind bomb-2 is an E3 ligase that ubiquitinates the N-methyl-D-aspartate receptor NR2B subunit in a phosphorylation-dependent manner. J. Biol. Chem. 283: 301-310.
Kalbaugh, T. L., H. M. VanDongen, and A. M. VanDongen. 2004. Ligand-binding residues integrate affinity and efficacy in the NMDA receptor. Mal. Pharmacol. 66: 209-219.
Karakas, E., N. Simorowski, and H. Furukawa. 2011. Subunit arrangement and phenylethanolamine binding in GluNI/GluN2B NMDA receptors. Nature 475: 249-253.
Kennedy, M. B., and P. Manzerra. 2001. Telling tails. Proc. Natl. Acad. Sci. USA 98: 12323-12324.
Kew, J. N., J. G. Richards, V. Mutel, and J. A. Kemp. 1998. Developmental changes in NMDA receptor glycine affinity and ifenprodil sensitivity reveal three distinct populations of NMDA receptors in individual rat cortical neurons. J. Neurosci. 18: 1935-1943.
Kiyama, Y., T. Manabe, K. Sakimura, F. Kawakami, H. Mori, and M. Mishina. 1998. Increased thresholds for long-term potentiation and contextual learning in mice lacking the NMDA-type glutamate receptor epsilonl subunit. J. Neurosci. 18: 6704-6712.
Kohr, G. 2006. NMDA receptor function: subunit composition versus spatial distribution. Cell Tissue Res. 26: 439-446.
Kohr, G.. V. Jensen, H. J. Koester, A. L. Mihaljevic, J. K. Utvik, A. Kvello, 0. P. Ottersen, P. H. Seeburg, R. Sprengel, and 0. Hvalby. 2003. Intracellular domains of NMDA receptor subtypes are determinants for long-term potentiation induction. J. Neurosci. 23: 1079110799.
Kornan, H. C., L. T. Schenker, M B. Kennedy, and P. H. Seeburg. 1995. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269: 1737-1740.
Kullmann, D. M., and F. Asztely. 1998. Extrasynaptic glutamate spillover in the hippocampus: evidence and implications. Trends Neurosci. 21: 8-14.
Kuner, T., and R. Schoepfer. 1996. Multiple structural elements determine subunit specificity of Mg2- block in NMDA receptor channels. .1. Neurosci. 16: 3549-3558.
Kurschner, C., P. G. Mermelstein, W. T. Holden, and D. J. Surmeier. 1998. CIPP, a novel multivalent PDZ domain protein, selectively interacts with Kir4.0 family members. NMDA receptor subunits, neur-exins, and neuroligins. Mol. Cell. Neurosci. 11: 161-172.
Kutsuwada, T., K. Sakimura, T. Manabe, C. Takayama, N. Katakura, E. Kushiya, R. Natsume, M. Watanabe, Y. Inoue, T. Yagi et aL 1996. Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice. Neuron 16: 333-344.
Lau, C. G., and R. S. Zukin. 2007. NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat. Rev. Neurosci. 8: 413-426.
Laube, B., H. Hirai, M. Sturgess, H. Betz, and J. Kuhse. 1997. Molecular determinants of agonist discrimination by NMDA receptor subunits: analysis of the glutamate binding site on the NR2B subunit. Neuron 18: 493-503.
Lavezzari, G., J. McCallum, C. M. Dewey, and K. W. Roche. 2004. Subunit-specitic regulation of NMDA receptor endocytosis. J. Neuro-sci. 24: 6383-6391.
Leonard, A. S., and J. W. Hell. 1997. Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-D-aspartate receptors at different sites. J. Biol. Chem. 272: 12107-12115.
Li, Y., R. S. Erzurtunlu, C. Chen, S. Jhaveri, and S. Tonegawa. 1994. Whisker-related neuronal patterns fail to develop in the trigeminal brainstem nuclei of NMDAR1 knockout mice. Cell 76: 427-437.
Li, J. H., Y. H. Wang, B. B. Wolfe, K. E. Krueger, L. Corsi, G. Stocca, and S. Vicini. 1998. Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons. Eur. J. Neurosci. 10: 1704-1715.
Liao, G. Y., D. A. Wagner, M H. Hsu, and J. P. Leonard. 2001. Evidence for direct protein kinase-C mediated modulation of N-methyl-D-aspartate receptor current. Mol. Pharmacol. 59: 960-964.
Liu, W., L. Chang, Y. Song, X. Gao, W. Ling, T. Lu, Y. Zhang, and Y. Wu. 2012. Immunolocalization of CaMKII and NR2B in hippocampal subregions of rat during postnatal development. Acta Histochem. In press. doi: 10.1016./j.acthis.20 I 2.07.007
Lozovaya, N. A., S. E. Grebenyuk, T. Sb. Tsintsadze, B. Feng, D. T. Monaghan, and 0. A. Krishtal. 2004. Extrasynaptic NR2B and NR2D subunits of NMDA receptors shape 'superslow' afterburst EPSC in rat hippocampus. J. PhysioL 558: 451-463.
Lu, H. C., E. Gonzalez, and M. C. Crair. 2001. Barrel cortex critical period plasticity is independent of changes in NMDA receptor subunit composition. Neuron 32: 619-634.
Luo, J., Y. Wang, R. P. Yasuda, A. W. Dunah, and B. B. Wolfe. 1997. The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebellar cortex contain at least three different subunits (NRI/NR2A/NR2B). Mol. Pharmacol. 51: 79-86.
Malenka, R. C., and M. F. Bear. 2004. LTP and LTD: an embarrassment of riches. Neuron 44: 5-21.
Mayford, M., J. Wang, E. R. Kandel, and T. J. O'Dell. 1995. CaMKIT regulates the frequency-response function of hippocampal synapses for the production of both LTD and LTP. Cell 81: 891-904.
McHugh, T. J., M. W. Jones, J. J. Quinn, N. Balthasar, R. Coppari, J. K. Elinquist, B. B. Lowell, M. S. Fanselow, M. A. Wilson. and S. Tonegawa. 2007. Dentate gyms NMDA receptors mediate rapid pattern separation in the hippocampal network. Science 317: 94-99.
Meddows, E., B. Le Bourdelles, S. Grimwood, K. Wafford, S. Sandhu, P. Whiting, and R. A. Mcilhinney. 2001. Identification of molecular determinants that are important in the assembly of N-methyl-D-aspartate receptors. J. Biol. Chem. 276: 18795-18803.
Meguro, H., H. Mori, K. Araki, E. Kushiya, T. Kutsuwada, M. Ya. mazaki, T. Kumanishi, M. Arakawa, K. Sakimura, and M. Mishina. 1992. Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs. Nature 357: 70-74.
Merrill, M. A., Y. Chen, S. Strack, and J. W. Hell. 2005. Activity-driven postsynaptic translocation of CaMKII. Trends Pharmacol. SeL 26: 645-653.
Mohrmann, R., G. Kohr, H. Hatt, R. Sprengel, and K. Gottmann. 2002. Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methyl-D-aspartate receptors in nascent neocortical synapses. J. Neurosci. Res. 68: 265-275.
Monyer, H., R. Sprengel, R. Schoepfer, A. Herb, M. Higuchi, H. Lomeli, N. Burnashev, B. Sakmann, and P. H. Seeburg. 1992. Heteromeric NMDA receptors: molecular and functional distinction of subtypes. Science 256: 1217-1221.
Monyer, H., N. Burnashev, D. J. Laurie, B. Salunann, and P. H. Seeburg. 1994. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 12: 529-540.
Mullasseril, P., K. B. Hansen, K. M. Vance, K. K. Ogden, H. Yuan, N. L. Kurtkaya, R. Santangelo, A. G. Orr, P. Le, K. M. Vellano et al. 2010. A subunit-selective potentiator of NR2C- and NR2D-con-taining NMDA receptors. Nat. Commun. 1: 90.
Nakazawa, K., L. D. Sun, M. C. Quirk, L. Rondi-Reig, M. A. Wilson, and S. Tonegawa. 2003. Hippocampal CA3 NMDA receptors are crucial for memory acquisition of one-time experience. Neuron 38: 305-315.
Nase, G., J. Weishaupt, P. Stern, W. Singer, and H. Monyer. 1999. Genetic and epigenetic regulation of NMDA receptor expression in the rat visual cortex. Eur. J. Neurosci. 11: 4320-4326.
Newpher, T. M., and M. D. Ehlers. 2009. Spine microdomains for postsynaptic signaling and plasticity. Trends Cell Biol. 19: 248-227.
Ng, F. M., M. T. Geballe, J. P. Snyder, S. F. Traynelis, and C. M. Low. 2008. Structural insights into phenylethanolamines high-affinity binding site in NR2B from binding and molecular modeling studies Mol. Brain 1: 16.
Paoletti, P., and J. Neyton. 2007. NMDA receptor subunits: function and pharmacology. Curr. Opin. Pharmacol. 7: 39-47.
Paoletti, P., F. Perin-Dureau, A. Fayyazuddin, A. Le Goff, I. Callebaut, and J. Neyton. 2000. Molecular organization of a zinc binding n-terminal modulatory domain in a NMDA receptor subunit. Neuron 28: 911-925.
Perin-Dureau, F., J. Rachline, J. Neyton, and P. Paoletti. 2002. Mapping the binding site of the neuroprotectant ifenprodil on NMDA receptors. J. Neurosci. 22: 5955-5965.
Petralia, R. S., N. Sans, Y. X. Wang, and R. J. Wenthold. 2005. Ontogeny of postsynaptic density proteins at glutamatergic synapses. Mol. Cell. Neurosci. 29: 436-452.
Philpot, B. D., M. P. Weisberg, M. S. Ramos, N. B. Sawtell, Y. P. Tang, J. Z. Tsien, and M. F. Bear. 2001. Effect of transgenic overexpression of NR2B on NMDA receptor function and synaptic plasticity in visual cortex. Neuropharmacology 41: 762-770.
Prybylowski, K., K. Chang, N. Sans, L. Kan, S. Vicini, and R. J. Wenthold. 2005. The synaptic localization of NR2B containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2. Neuron 47: 845-857.
Qiu, S., X.-M. Zhang, J.-Y. Cao, W. Yang, Y.-G. Yan, L. Sham, J. Zheng, and J.-H. Luo. 2009. An endoplasmic reticulum retention signal located in the extracellular amino-terminal domain of the NR2A subunit of N-Methyl-D-aspartate receptors. J. Biol. Chem. 284: 2028520298.
Quinlan, E. M., D. H. OLstein, and M. F. Bear. 1999. Bidirectional, experience-dependent regulation of N-methyl-D-aspartate receptor subunit composition in the rat visual cortex during postnatal development. Proc. Nail. Acad. Sci. USA 96: 12876-12880.
Roche, K. W., S. Sthndley, J. McCallum, L. C. Dune, M. D. Ehlers, and R. J. Wenthold. 2001. Molecular determinants of NMDA receptor internalization. Nat. Neurosci. 4: 794- 802.
Rolls, E. T. 2010. A computational theory of episodic memory formation in the hippocampus. Behav. Brain Res. 215: 180-196.
Rong, Y., X. Lu, A. Bernard, M. Khrestchatisky, and M. Baudry. 2001. Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95. J. Neurochem. 79: 382-390.
Rosahl, T. W., P. B. Wingrove, V. Hunt, R. L. Fradley, J. M. Lawrence, R. P. Heavens, P. Treacey, M. Usala, A. Macaulay, T. P. Bonnert et aL 2006. A genetically modified mouse model probing the selective action of ifenprodil at the N-methyl-D-aspartate type 28 receptor. Mol. Cell. Neurosci. 33: 47-56.
Rutter, A. R., and F. A. Stephenson. 2000. Coexpression of postsynaptic density-95 protein with NMDA receptors results in enhanced receptor expression together with a decreased sensitivity to L-glutamate. J. Neurochem. 75: 2501-10.
Sakimura, K., T. Kutsuwada, I. Ito, T. Manabe, C. Takayama, E. Kushiya, T. Yagi, S. Aizawa, Y. Inoue, H. Sugiyama, and M. Mishina. 1995. Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon I subunit. Nature 373: 151155.
Sala, C., and M. Sheng. 1999. The fyn art of N-methyl-D-aspartate receptor phosphorylation. Proc. Natl. Acad. Sci. USA 96: 335-337.
Salussolia, C. L., M. L. Prodromou, P. Borker, and L. P. Wollmuth. 2011. Arrangement of subunits in functional NMDA receptors. J. Neurosci. 31: 11295-11304.
Sans, N., R. S. Petralia. Y. X. Wang, J. Blahos, 2nd, J. W. Hell, and R. J. Wenthold. 2000. A developmental change in NMDA receptor-associated proteins at hippocampal synapses. J. Neurosci. 20: 12601271.
Sans, N., K. Prybylowski, R. S. Petralia, K. Chang, Y. X. Wang, C. Racca, S. Vicini, and R. J. Wenthold. 2003. NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat. Cell Biol. 5: 520-530.
Sanz-Clemente, A., J. A. Matta, J. T. Isaac, and K. W. Roche. 2010. Casein kinase 2 regulates the NR2 subunit composition of synaptic NMDA receptors. Neuron 67: 984-996.
Sarter, M.. G. Bodewitz, and D. N. Stephens. 1988. Attenuation of scopolamine-induced impairment of spontaneous alteration behaviour by antagonist but not inverse agonist and agonist beta-carbolines. Psychopharmacology (Berl.) 94: 491-495.
Schmitt, W. B., R. Sprengel, V. Mack, R. W. Draft, P. H. Seeburg, R. M. J. Deacon, J. N. P. Rawlins, and D. M. Bannerman. 2005. Restoration of spatial working memory by genetic rescue of GluR-A-deficient mice. Science 8: 270-272.
Sheng, M. 2001. The postsynaptic NMDA-receptor-PSD-95 signaling complex in excitatory synapses of the brain. J. Cell. Sci. 114: 1251.
Sheng, M., and E. Kim. 1996. Ion channel associated proteins. Curr. Opin. NettrobioL 6: 602-608.
Sheng, M., J. Cummings, L. A. Roldan, Y. N. Jan, and L. Y. Jan. 1994. Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 368: 144-147.
Sprengel, R., B. Suchanek, C. Amico, R. Brusa, N. Burnashev, A. Rozov, 0. Hvalby, V. Jensen, 0. Paulsen, P. Andersen et aL 1998. Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell 92: 279-289.
Stocca, G., and S. Vicini. 1998. Increased contribution of NR2A subunit to synaptic NMDA receptors in developing rat cortical neurons. J. PhysioL 507: 13-24.
Stoneham, E. T., E. M. Sanders, M. Sanyal, and T. C. Dumas. 2010. Rules of engagement: factors that regulate activity-dependent synaptic plasticity during neural network development. Biol. Bull. 219: 81-99.
Storey, G. P., X. Opitz-Araya, and A. Barria. 2011. Molecular determinants controlling NMDA receptor synaptic incorporation. J. Neuro-sci. 31: 6311-6316.
Strack, S., and R. J. Colbran. 1998. Autophosphorylation-dependent targeting of calcium/calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor. J. Biol. Chem. 273: 20689-20692.
Strack, S., R. B. McNeil, and R. J. Colbran. 2000. Mechanism and regulation of calcium/calmodulin-dependent protein kinase 11 targeting to the NR2B subunit of the N-methyl-D-aspartate receptor. J. Biol. Chem. 275: 23798-23806.
Talukder, I., and L. P. Wollmuth. 2011. Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening. J. Gen. Physiol. 138: 179-194.
Tang, T. T., J. D. Badger, 2nd, P. A. Roche, and K. W. Roche. 2010. Novel approach to probe subunit-specific contributions to N-methyl-D-aspartate (NMDA) receptor trafficking reveals a dominant role for NR2B in receptor recycling. J. Biol. Chem. 285: 20975-20981.
Tang, Y.-P., E. Shimizu, G. R. Dube, C. Rampon, G. A. Kerchner, M. Zhuo, G. Liu, and J. Z. Tsien. 1999. Genetic enhancement of learning and memory in mice. Nature 401: 63-69.
Thomas, C. G., A. J. Miller, and G. L. Westbrook. 2006. Synaptic and extrasynaptic NMDA receptor NR2 subunits in cultured hippocampal neurons. J. Neurophysiol. 95: 1727-1734.
Tovar, K. R., and G. L. Westbrook. 1999. The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J. Neurosci. 19: 4180-4188.
Townsend, M., A. Yoshii, M. Mishina, and M. Constantine-Paton. 2003. Developmental loss of miniature N-methyl-D-aspartate receptor currents in NR2A knockout mice. Proc. Natl. Acad. Sci. USA 100: 1340-1345.
Vissel, B., J. J. Krupp, S. F. Heinemann, and G. L. Westbrook. 2001. A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux. Nat. Neurosci. 4: 587-596.
von Engelhardt, J., B. Doganci, V. Jensen, [PHI]. Hvalby, C. Gingrich, A. Taylor, C. Barkus, D. J. Sanderson, J. N. Rawlins, P. H. Seeburg et al. 2008. Contribution of hippocampal and extra-hippocampal NR2B-containing NMDA receptors to performance on spatial learning tasks. Neuron 60: 846-860.
Wang, C. C., R. G. Held, S. C. Chang, L. Yang, E. Delpire, A. Ghosh, and B. J. Hall. 2011. A critical role for GluN2B-containing NMDA receptors in cortical development and function. Neuron 72: 789-805.
Wang, D., Z. Cui, Q. Zeng, H. Kuang, L. P. Wang, J. Z. Tsien, and X. Cao. 2009. Genetic enhancement of memory and long-term potentiation but not CAI long-term depression in NR2B transgenic rats. PLoS One 4: e7486.
Wang, S. H., and R. G. Morris. 2010. Hippocampal-neocortical interactions in memory formation, consolidation, and reconsolidation. Annu. Rev. Psychol. 61: 49-79, C1-4.
Watanabe, M., M. Mishina, and Y. Inoue. 1994. Distinct spatiotemporal expressions of five NMDA receptor channel subunit mRNAs in the cerebellum. J. Comp. Neurol. 343: 513-551.
Wenthold, R. J., K. Prybylowski, S. Standley, N. Sans, and R. S. Petralia. 2003. Trafficking of NMDA receptors. Anna. Rev. Phar-macol. Toxicol. 43: 335-358.
Wechsler, A., and V. I. Teichberg. 1998. Brain spectrin binding to the NMDA receptor is regulated by phosphorylation, calcium and calmodulin. EMBO J. 17: 3931-3939.
Woodward, J. J. 2002. Prostacyclin-induced rundown of N-methyl-D-aspartate receptor currents in HE1C293 cells is protein kinase A-dependent and NR2 subunit-selective. J. Neurochem. 80: 598-604.
Wyszynski, M., J. Lin, A. Rao, E. Nigh, A. H. Beggs, A. M. Craig, and M. Sheng. 1997. Competitive binding of alpha-actinin and calmodulin to the NMDA receptor. Nature 385: 439-442.
Xu, M., C. T. Smothers, J. Trudell, and J. J. Woodward. 2012. Ethanol inhibition of constitutively open N-methyl-D-aspartate receptors. J. Pharmacol. Exp. Ther. 340: 218-226.
Yang, W., C. Zheng, Q. Song, X. Yang, S. Qiu, C. Liu, Z. Chen, S. Duan, and J. Luo. 2007. A three amino acid tail following the TM4 region of the N-methyl-D-aspartate receptor (NR) 2 subunits is sufficient to overcome endoplasmic reticulum retention of NR1-1 a subunit. J. Biol. Chem. 282: 9269-9278.
Yashiro, K., and B. D. Philpot. 2008. Regulation of NMDA receptor subunit expression and its implications for LTD, LTP, and metaplas-ticity. Neuropharmacology 55: 1081-1094.
Yuan, H., K. B. Hansen, K. M. Vance, K. K. Ogden, and S. F. Traynelis. 2009. Control of NMDA receptor function by the NR2 subunit amino-terminal domain. J. Neurosci. 29: 1204512058.
Zhao, J.-P., and M. Constantine-Paton. 2007. [NR2A.sup.-1-] mice lack longterm potentiation but retain NMDA receptor and L-type [Ca.sup.2+] channel-dependent long-term depression in the juvenile superior colliculus. J. Neurosci. 27: 13649-13654.
ERIN M. SANDERS, MICHAEL A. NGUYEN, KEVIN C. ZHOU, MARY E. HANKS, KAVVTHAR A. YUSUF, DANIEL N. COX, THEODORE C. DUMAS *
Molecular Neuroscience Department, Krasnow Institute for Advanced Study, George Mason University, Fairfax, Virginia 22030
Received 5 January 2012; accepted 13 November 2012.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
Abbreviations: AMPAR, a-amino-3-hydroxy-5-methy1-4-isoxazolepro-pionic acid receptor; LTD, long-term depression; LIP, long-term potentiation; MAGUK, membrane-associated guanylate kinase protein; NMDAR. N-methyl-D-aspartate receptor; PSD, postsynaptic density; TM, transmembrane.
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|Title Annotation:||N-methyl-D-aspartate receptors|
|Author:||Sanders, Erin M.; Nguyen, Michael A.; Zhou, Kevin C.; Hanks, Mary E.; Yusuf, Kawthar A.; Cox, Daniel|
|Publication:||The Biological Bulletin|
|Date:||Feb 1, 2013|
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