Learning from NMDA Receptor Trafficking - Semantic Scholar

Review Neurosignals 2004;13:175–189 DOI: 10.1159/000077524

Received: August 12, 2003 Accepted after revision: October 3, 2003

Learning from NMDA Receptor Trafficking: Clues to the Development and Maturation of Glutamatergic Synapses Isabel Pérez-Otaño a Michael D. Ehlers a–c Departments of a Neurobiology, b Cell Biology and c Pharmacology and Cancer Biology, Duke University Medical Center, Durham, N.C., USA

Key Words NMDA receptor W Glutamatergic synapse W Development, brain W Trafficking, receptor W Endocytic cycling

view, focusing on the role that activity plays in altering NMDAR trafficking and how such dynamic regulation of NMDARs may impact on the plasticity of neural circuits. Copyright © 2004 S. Karger AG, Basel

Abstract Activity-dependent changes in excitatory transmission allow the brain to develop, mature, learn and retain memories, and underlie many pathological states of the central nervous system. A principal mechanism by which neurons regulate excitatory transmission is by altering the number and composition of glutamate receptors at the postsynaptic plasma membrane. The dynamic trafficking of glutamate receptors to and from synaptic sites involves a complex series of events including receptor assembly, trafficking through secretory compartments, membrane insertion and endocytic cycling. While these events have become widely appreciated as critical processes regulating AMPA-type glutamate receptors during synaptic plasticity, the mechanisms that control the trafficking of NMDA-type glutamate receptors (NMDARs) are only now beginning to be understood. Until recently, NMDARs were considered immobile receptors, tightly anchored to the postsynaptic membrane. Here, we review recent evidence that challenges this

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Synapses that use glutamate as their transmitter mediate most excitatory neurotransmission in the central nervous system (CNS). Both ligand-gated ion channels (ionotropic receptors) and G protein-coupled receptors (metabotropic receptors) sense the glutamate released from presynaptic terminals and transduce it into electrical or biochemical responses. A key property of the glutamatergic synapse is its plasticity, which enables the developing and mature brain to modify the properties of neural circuits in a long-term fashion and respond adequately to changing needs in the environment. This plasticity allows neuronal connectivity to be regulated at the level of individual synapses as well as in entire synaptic networks over time courses that range from seconds to months [1–6]. During neural development, glutamatergic synapses initially form, and then either stabilize and mature or are eliminated in order to shape neural networks. Once established, most glutamatergic synapses retain the potential for considerable plasticity during later stages of develop-

Isabel Pérez-Otaño Department of Neurobiology, Duke University Medical Center Box 3209, Durham, NC 27710 (USA) Tel. +1 919 681 6140, Fax +1 919 684 4431, E-Mail [email protected] Michael D. Ehlers, E-Mail [email protected]

ment and into adulthood [6–8]. In principle, the strength of a synapse can be modified presynaptically by altering transmitter release or postsynaptically by modifying the number, efficacy or stability of postsynaptic receptors. A number of recent developments have uncovered the importance of postsynaptic mechanisms for plasticity at many CNS synapses, and demonstrated that dynamic changes in the receptor complement at the postsynaptic membrane constitute a fundamental means to generate and remodel a plastic neural network, both by ‘making’ functional synapses and by strengthening or weakening those already formed [for reviews, see ref. 9–11]. While glutamate receptors of the AMPA subtype (AMPARs) mediate most of the rapid excitatory transmission in the mature brain, NMDA-type glutamate receptors (NMDARs) initiate many forms of synaptic plasticity and participate in long-term homeostatic and adaptive brain processes. For instance, NMDAR activity is required for the establishment and refinement of neural circuits during development by contributing to the formation and maturation of dendritic processes, dendritic spines and synaptic connections themselves [7, 10, 12–14]. In mature networks, the activation of NMDARs mediates Hebbian forms of plasticity such as long-term potentiation (LTP) and long-term depression (LTD) that are considered the cellular basis for memory formation and storage [5, 15–17]. Activation of the highly calcium-permeable NMDAR causes the insertion or removal of AMPARs, resulting in changes in synaptic strength, most notably at CA1 hippocampal synapses [for a review, see ref. 11]. While Hebbian plasticity triggers long-lasting, synapsespecific modification of network properties, it tends to destabilize postsynaptic firing rates [2, 18, 19]. Therefore, additional mechanisms are needed to stabilize activity and keep it within an optimal working range (commonly referred to as synaptic homeostasis) and to modify the thresholds at which synaptic stimulation induces LTP and LTD (metaplasticity). Regulation of the synaptic abundance of NMDARs provides a cell biological mechanism that may account for these additional forms of synaptic plasticity [1, 3]. A common denominator of plasticity at glutamatergic synapses is the need for prior synaptic or cellular activity. Diverse forms of plasticity are generated by different patterns of synaptic activation, many of them involving NMDARs [3, 20–23], which requires a tight control over the quality and magnitude of NMDAR-dependent signals – most notably Ca2+ influx. This task can be accomplished both by regulating the numbers and subtypes of NMDARs present at a synapse, and by modifying

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NMDAR composition – and thereby properties – over time periods ranging from minutes to months [24–26]. Given this context, understanding the cell biology and trafficking of the receptor is essential for understanding how different types of NMDARs are placed at or removed from synapses according to the developmental needs of the neuron and in response to changes in the activity of neural networks. Receptor composition can be controlled at two principal levels – subunit expression (e.g. gene transcription and mRNA translation) and intracellular trafficking. While regional or regulated NMDAR expression has received considerable attention over the last 10 years (ever since the first glutamate receptors were cloned), a picture of the cell biological processes that control receptor trafficking is only beginning to emerge [reviewed in ref. 25, 26]. Subunit assembly, forward trafficking along the secretory pathway, local control of endo-/exocytic cycling and lateral diffusion in the plane of the plasma membrane are some of the diverse and complex trafficking events which determine the complement of synaptic receptors. In this review, we will summarize recently identified mechanisms that neurons use to set and change their NMDARs, and describe how such mechanisms help determine the construction, maturation and plasticity of glutamatergic synapses. As key elements of these processes are activity dependent, we will discuss the implications of regulated NMDAR cycling for the developmental and experiencedriven reshaping of neural circuits.

NMDAR Subunits and Receptor Diversity

NMDARs are tetrameric complexes assembled from the ubiquitous NR1 subunit, which is an essential component of all NMDAR complexes, along with various combinations of NR2 or NR3 subunits [24, 27–29]. NR1 is encoded by a single gene, whereas NR2 and NR3 subunits are encoded by four (NR2A–D) and two (NR3A–B) distinct genes, respectively [30–34] (fig. 1). Further molecular diversity arises from alternative splicing of NR1 mRNA, giving rise to eight different splice variants [35]. The functional properties of the receptor complex depend on the specific subunit composition and the stoichiometry in which subunits combine to form the channel [24]. The rules governing NMDAR assembly and thereby controlling receptor stoichiometry have not yet been established, but are likely analogous to those proposed for AMPARs [36–38]. Pharmacological, electrophysiological and biochemical studies of recombinant and native

Pérez-Otaño/Ehlers

Fig. 1. Interaction domains and properties of NMDARs. a Each subunit is composed of an extracellular N-terminal

domain that contains sequence determinants for assembly, four membrane domains and an intracellular carboxyterminal domain. The secondary structure of a typical subunit and the proposed tertiary and quaternary arrangements of the assembled complex are shown. NMDAR subunits have long C-terminal domains, which mediate interactions with many intracellular proteins, including signaling molecules and proteins involved in receptor trafficking. b The final properties of the receptor complex are determined by the specific subunit composition.

NMDARs have demonstrated the existence of both heterodimeric and heterotrimeric assemblies (e.g. NR1/ NR2A/NR2B, NR1/NR2B/NR2D or NR1/NR2A/NR3A [39–43]), a phenomenon that further increases the potential for combinatorial assembly and functional heterogeneity of this receptor family.

Classical NMDARs Classically studied NMDARs are heteromeric NR1/ NR2 complexes and are unique among glutamate receptors in many ways [reviewed in ref. 29]. First, they require dual agonist binding for activation – glycine that binds to the NR1 subunit, and glutamate that binds to NR2. Second, the opening of the ligand-gated cation channel exhib-

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its a profound voltage dependence because the channel is blocked by physiological concentrations of Mg2+ at resting membrane potentials. A partial depolarization of the plasma membrane is required to relieve the Mg2+ block, which allows NMDARs to sense simultaneous inputs of several presynaptic cells and behave as coincidence detectors. The third distinctive property is that the activated channel is highly permeable to Ca2+ ions. This ability to flux Ca2+ couples the NMDAR to intracellular signal transduction pathways and is key to the expression of many forms of synaptic plasticity. Subunit composition determines the channel properties and trafficking of NMDARs. Incorporation of different NR1 splice variants into NMDAR complexes influences such properties as modulation by zinc, polyamines and protein kinase C (PKC), and controls early receptor trafficking between the endoplasmic reticulum (ER) and Golgi apparatus [35, 44–49]. The NR2 subunit determines biophysical characteristics of the channel such as conductance, mean open time and sensitivity to Mg2+ block [31, 50, 51]. In addition, NR2 is required for the clustering and synaptic targeting of NMDARs through interactions with proteins of the PSD-95/SAP90 family [52–54]. The signal determinants for binding to PSD-95 and other proteins containing PDZ domains reside at the very distal portion of the carboxy-termini of NR2 subunits. Gene-targeted mice expressing truncated NR2 subunits that lack the carboxy-terminal domain display impaired synaptic localization of NMDARs [55–57]. Indeed, the phenotypes of the C-terminal-truncated mutants are essentially identical to mice deficient in the respective NR2 subunit, emphasizing the importance of synaptic targeting for NMDAR function [58]. By virtue of their interaction with anchoring proteins such as PSD-95, NMDARs cluster at synaptic sites where they are embedded within the postsynaptic density (PSD), a specialized proteinaceous matrix attached to the postsynaptic membrane. Within the PSD, NMDARs form an extended complex that connects with scaffolding and signaling proteins [59, 60]. Although NMDARs typically concentrate at synapses, exceptions do exist. For example, NMDARs containing NR2D are localized at sites outside the synapse [61], and NR2B-containing receptors display a preferential extrasynaptic distribution [39, 62, 63]. Interestingly, the NR2B and NR2D subunits predominate early in development, but their expression gradually decreases as they are replaced by the ‘mature’ subunits NR2A and NR2C, which are more preferentially targeted to synaptic sites [51, 64]. Thus, in addition to determining intrinsic receptor properties, subunit compo-

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sition confers selective targeting during requisite phases of synapse formation. The NR3A Subunit Forms Nonconventional NMDARs Unlike any of the other known NMDAR subunits, inclusion of NR3 subunits into the NMDAR complex plays an inhibitory role by forming a ‘nonconventional’ NMDAR channel. Receptors that contain NR3 have a smaller conductance, display much lower Ca2+ permeability (approximately 5-fold less) and have a substantially lower sensitivity to Mg2+ blockade [43, 65, 66]. The physiological role of NR3 remains enigmatic, but may involve selective reduction of NMDAR activity (and thus the potential for synaptic plasticity) due to its inability to flux calcium in an ‘NMDAR-like fashion’. For instance, synapses that contain NR3 receptors may be less plastic and unable to undergo LTP or LTD. On the other hand, the relative magnesium insensitivity of NR3-containing receptors may allow them to function during synapse formation when NMDARs are thought to be inactive in the absence of AMPAR activation. In this context, it is interesting to note that the functionally inhibitory NR3A subunit is highly expressed during brain development but undergoes subsequent downregulation in the adult [32, 33, 67]. Consistent with a role in the development of neural circuitry, mice lacking the NR3A subunit show an abnormal proliferation of dendritic spines in neurons of the cerebral cortex [65]. In addition, recent studies have shown that NR1/NR3 assemblies, which lack the glutamate-binding site provided by NR2 subunits, can form functional channels that are inserted at the plasma membrane and may act as excitatory glycine receptors [34, 43]. Although clearly modifying the channel properties of NMDARs, little is known about the contribution of NR3 subunits to NMDAR targeting.

Subunit Assembly and ER Export: Early Trafficking Checkpoints for NMDAR Surface Expression

As discussed above, coassembly between NR1, NR2 and NR3 subunits generates many distinct types of NMDARs. This diversity confers enormous regulatory potential, but presents neurons with the complicated task of coordinating the assembly, trafficking and membrane delivery of specific receptor subtypes to selected synapse populations at different times during development. Regulation of subunit synthesis (i.e. transcription and translation) exerts a first level of control over the NMDAR phe-


notype of individual neurons. Recent studies have shown that an additional checkpoint operates at the level of the ER to recognize and retain unassembled NMDAR subunits and ensure the forward trafficking of only certain subunit combinations. Multimeric membrane proteins assemble early in the secretory pathway as individual subunits are synthesized and fold in the ER. During this process, only some subunit combinations achieve a correctly folded state and are then exported from the ER, through the Golgi apparatus and on to the plasma membrane. Partially or incorrectly assembled complexes are retained in the ER and degraded [68–72]. The first evidence that such ER quality control mechanisms could contribute to NMDAR assembly and transport came from studies on the surface expression of NMDARs. When expressed alone in recombinant systems, NR1 and NR2 subunits were not present at the cell surface, with the exception of NR1 splice variants, which contain the shortest C-terminal tails [73–76]. Subsequent work demonstrated that unassembled NR1 and NR2 subunits were retained intracellularly in the ER, and identified an ER retention motif (RXR) in the alternatively spliced C1 domain in the carboxy-terminus of the NR1 subunit [47–49]. Assembly with NR2 subunits is thought to mask this ER retention signal and facilitate surface expression [74]. Heteromeric assembly is required for NMDAR surface expression, but may not be sufficient. For instance, heteromeric NR2/NR3A complexes are unable to form properly assembled receptors that insert at the plasma membrane [43], indicating an absolute requirement for NR1 in NMDAR surface expression. Indeed, export of nascent NMDARs from the ER is enhanced by the alternatively spliced C2) domain of NR1 [47, 48] and by phosphorylation of serine residues adjacent to the RXR ER retention motif in the C1 domain of NR1 [47, 49]. Since ER export is the rate-limiting step for surface delivery of most integral membrane proteins [77], these findings emphasize the importance of early trafficking checkpoints in controlling the supply of NMDARs to the synapse. Still to be determined are the molecular mechanisms by which ER trafficking signals regulate NMDAR export and synaptic targeting, and the regulation of such trafficking by synaptic activity. A recent study has revealed a first potential molecular link for the sorting of NMDARs from the ER and Golgi apparatus to the synapse, by identifying an interaction between NMDARs, the synaptic scaffold SAP102 and Sec8, one of the components of the exocyst [78]. The exocyst has been best characterized in yeast, where it is known to be involved in targeting vesicles to

specific plasma membrane domains. The interaction between SAP102 and Sec8 might provide a bridge for the exocyst to recognize specific cargo (NMDAR-containing vesicles) and then convey NMDARs, perhaps in a preassembled status with the scaffold protein, to synaptic sites.



Placing NMDARs at Synapses: Generating a Substrate for Synaptic Plasticity

Developmental Plasticity and Changes in Synaptic NMDARs NMDARs are initially transported to dendrites along microtubules, transport that relies on adaptor molecules such as mLin10 (Mint1/X11) that couple the NMDARcontaining vesicles to kinesin motors [79–82]. Recent studies in developing neurons in culture have shown that mobile transport packets containing NMDARs are recruited to nascent synapses shortly (from minutes to 1 h) after axodendritic contact [79, 83]. Thus, synapses are ‘born’ with NMDARs that are drawn from an extrasynaptic or intracellular pool of existing subunits. The precise nature of the NMDAR subtypes recruited is unknown, although most clusters seem to contain the NR2B subunit [79]. Critical to understanding how NMDARs become part of nascent synapses is determining the sequence of molecular events that accompany synapse assembly [for a review, see ref. 84]. NMDARs may be recruited to an existing postsynaptic superstructure – the scaffold-first model. Alternatively, the receptors themselves may serve as a focal point for the assembly of associated protein complexes [60] via a receptor-first model. Evidence exists for both paradigms. Washbourne et al. [79] assessed the functional state of nascent synapses to which NMDARs had been recruited by immunocytochemical detection of presynaptic vesicles and postsynaptic markers. Many of the new synapses did not oppose postsynaptic densities as defined by the presence of PSD-95, supporting at least a partial receptor-first model of synapse assembly [79]. Previous studies had instead suggested that synapse development proceeds by first assembling a postsynaptic scaffold, followed by the sequential attachment of glutamate receptors [83–85]. Due to similarities with the well-studied neuromuscular junction [86], the scaffold-first model of glutamatergic synapse assembly is particularly attractive. Simply put, once inserted into the plasma membrane, receptors can diffuse within the plane of the lipid bilayer until they are clustered and stabilized through interac-

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Fig. 2. Developmental changes in synaptic NMDARs. Synaptic activity, during development and in the adult, drives changes in the numbers and composition of NMDARs and their synaptic targeting. a In young neurons, ‘immature’ NMDARs (containing NR2B and perhaps other developmental subunits, such as NR3A) predominate in extrasynaptic sites and are recruited to nascent functional but unspecialized synapses. b As neurons develop and synaptogenesis proceeds, increased levels of synaptic activity trigger the removal of immature NMDAR forms from synapses and their replacement by

NMDAR complexes containing NR2A. Extrasynaptic receptors are also gradually endocytosed. c NR2A-containing receptors are mostly synaptic, and attach to synaptic sites through binding to the postsynaptic protein PSD-95. Synaptic NMDAR restriction may contribute to postsynaptic stabilization, and leads to a specialized, inputspecific localization of NMDARs. At this stage, input-dependent alterations in NMDAR number/composition (or incorporation of AMPARs) may occur.

tions with postsynaptic proteins. For example, binding of ephrin-B promotes an association of the EphB receptor with NMDARs, and promotes NMDAR clustering during synaptogenesis [87]. Further interactions with multivalent scaffolds such as PSD-95 may then increase the stability of postsynaptic NMDARs, perhaps by reducing the rates of endocytosis [88] (reviewed below). Once NMDARs and their associated scaffolds are placed at the postsynaptic membrane, the receptor complexes undergo age-dependent maturation along with the synapse itself (fig. 2). Indeed, early in development, there is an overabundance of synapses formed, which are then selectively stabilized or eliminated to remodel synaptic circuits [7, 86]. The decision about the fate of a synapse – stabilization or elimination – depends on its maturation state. In the case of glutamatergic synapses, maturation is driven by activity and may involve the selective stabiliza-

tion of coactive inputs [14, 89]. A key step in this process is the transition between ‘developmental’ and ‘adult’ forms of the NMDAR, and results in the stabilization of ‘mature’ NMDAR subtypes at synapses during critical periods of development [6, 90]. NMDARs containing ‘immature’ subunits such as NR2B, NR2D and perhaps NR3A are gradually replaced by receptor complexes comprising NR1/NR2A subunits. The first evidence for such a developmental switch in NMDARs came from the observation that the amount of current and duration of NMDAR responses declines rapidly with age and synaptic activity [90–93]. It was later found that this decline reflects a replacement of NR2B by NR2A subunits in the NMDAR complex that requires ongoing synaptic activity and depends on integrin-mediated signaling [39, 94, 95]. Analogous mechanisms transform nascent receptor clusters at neuromuscular junctions into the adult postsy-

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naptic apparatus. The accumulation of neurotransmitter receptors at synaptic sites involves a shift from a uniform extrasynaptic distribution of acetylcholine receptors (AChRs) before nerve contact to highly concentrated clusters of AChRs at synaptic sites. Such accumulation requires the concentration of AChRs in high-density clusters and their disappearance from extrasynaptic sites after muscle innervation. The removal of extrasynaptic receptors seems to result from mechanisms distinct from those responsible for receptor clustering. In addition, the maturation from embryonic to mature endplate coincides with a switch in AChR subunit composition (from Á- to Â-containing AChRs) that changes the receptor Ca2+ permeability and decreases the channel open time [86]. Several parallels emerge from such a comparison, indicating that similar processes may operate at glutamatergic CNS synapses. In particular, there is considerable evidence for the existence of a pool of extrasynaptic NMDARs during neuronal development. Immunofluorescent labeling has demonstrated the presence of numerous nonsynaptic NR1 clusters in young hippocampal and cortical neurons in culture [85, 96]. Electrophysiological approaches confirm that at early stages, extrasynaptic NMDARs – probably NR1/NR2B heteromers – greatly outnumber synaptic receptors (3:1 after 7 days in vitro, DIV) [39]. The functional significance of this pool of extrasynaptic NMDARs is still obscure, but several hypotheses (not mutually exclusive) can be put forth for future testing. Extrasynaptic NMDARs may (1) have a specific function to sense low levels of glutamate from spillover or before synapse formation, (2) act as placeholder elements, which label sites amenable to further activity-dependent stabilization through replacement by mature NMDAR forms, (3) reflect synaptic receptors translocated to endocytic zones prior to internalization, or to other membrane trafficking domains [97], or (4) have specific specialized signaling functions [98]. As synapses develop, the fraction of extrasynaptic receptors decreases, such that in cultured neurons, the population of synaptic NMDARs accounts for 80% of the total surface pool by DIV9-14. The increase in synaptic NMDARs is accompanied by a decline in NR2B expression and an increase in receptors containing NR2A [39, 99, 100]. Notably, this developmental switch is thought to be a major factor in the synaptic restriction of NMDARs that occurs as circuits mature, with NR2A subunits contributing key synaptic targeting information [39, 54, 55, 57]. The incorporation of NR2A subunits coincides with a lowered susceptibility to LTP that closes a critical period for the refinement of connections in the cortex – per-

haps by making synapses ‘less plastic’ [92, 95, 101]. However, critical periods of developmental plasticity do not always correspond with a switch from NR2B to NR2A subunits [102–104], suggesting potential roles for other developmental or ‘immature’ subunits (fig. 2b, c). For instance, it has been recently shown that different sources of presynaptic innervation drive the expression of different NMDAR subytpes in dendrites of CA1 hippocampal neurons [105]. Some of these receptors may not include NR2B, even at early developmental stages, and their actual subunit composition is not known. One potential candidate for a developmentally regulated NMDAR subunit is NR3A, which is highly expressed in the developing hippocampus, cortex and other brain regions prior to downregulation in the adult [32, 33, 67]. As NR3A confers reduced Ca2+ permeability, removal of NR3A-containing receptors during synapse maturation could allow for replacement by Ca2+-permeable NMDA channels carrying synaptic targeting signals and therefore more able to trigger LTP and LTD. The elimination of developmental NMDAR forms may thus serve as a general mechanism to generate inputspecific, temporally restricted transmission at synapses previously subject to more primitive and prolonged levels of transmission. Such a mechanism would ensure that synaptic transmission works efficiently at all stages of maturation of the synapse. Soon after initial contact, the amount of transmitter is low and the postsynaptic membrane is relatively unspecialized, with no zones of high receptor density. The presence of NMDAR channels with long open times, such as those containing NR2B, or relatively insensitive to Mg2+ blockade, such as those containing NR3A, would result in a large and sustained current flow. At mature synapses, the presence of channels with brief open times and high sensitivity to Mg2+ blockade (i.e. NR2A-containing, NR3A-lacking channels) would ensure input-specific coding with a requirement for coincident activity. In this way, the expression of mature forms of the NMDAR confers more tightly determined spatial and temporal NMDAR signaling – spatial by virtue of their concentration at the synapse, temporal because of their more rapid decay kinetics and their susceptibility to Mg2+ blockade. As a result, the developmental switch in the properties of NMDARs shapes neural circuits and dictates their propensity for plasticity. The cellbiological processes that mediate this replacement and the triggers that set them in motion are still unknown, but presumably involve regulated changes in the internalization or insertion of different NMDAR subtypes.



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Experience- and Activity-Dependent Regulation of NMDARs As described above, developmental plasticity involves a rearrangement of NMDAR transmission that refines originally diffuse synaptic connections and alters their propensity for Hebbian forms of plasticity such as LTP and LTD [14]. Once synapses are established, local alterations in the NMDAR ‘signature’ (i.e. the magnitude, duration and spatial extent of Ca2+ influx) reshape the plastic properties of neuronal networks, and help determine the experience-driven remodeling of neuronal circuits that takes place during adulthood. A principal means by which the magnitude and duration of NMDAR signaling can be altered is by regulating the abundance of receptors at the synapse. Although the placement of Ca2+-permeable NMDARs at synapses is required for plasticity, most evidence to date indicates that both LTP and LTD are expressed as changes in the number of synaptic AMPARs [11]. In mature networks, longer-term manipulations of activity seem to be necessary to modify the number of NMDARs. For example, changes in overall levels of activity cause long-lasting changes in both AMPAR and NMDAR postsynaptic currents, a phenomenon called synaptic scaling or homeostatic plasticity [106–108]. The changes in NMDAR currents parallel a redistribution of receptors to or from synaptic sites; activity blockade with tetrodotoxin or NMDAR antagonists induces an increase in NMDAR clustering at synapses and a concomitant increase in NMDA-mediated currents, while spontaneous activity decreases synaptic clustering [106–109]. This activitydriven synaptic NMDAR scaling operates on a time scale of days, responds to global synaptic blockade and is interpreted as a mechanism to maintain synaptic homeostasis by preserving optimal firing rates in the face of changing synaptic input [2, 18, 19]. Of course, Ca2+ entry through the NMDAR mediates both LTP and LTD – the classical view being that higher levels of NMDAR activation trigger large amounts of Ca2+ flux and induce LTP, whereas smaller amounts result in LTD. Therefore, in addition to stabilizing firing rates, scaling NMDAR currents up or down may constitute an NMDAR-dependent form of metaplasticity, by setting a new threshold of synaptic modification and ensuring that synapses with a history of inactivity do not lose their ability to undergo subsequent synaptic modification. Surprisingly, the molecular mechanisms underlying chronic NMDAR scaling remain elusive, but may involve cAMP-dependent protein kinase signaling [109] or protein degradation via the ubiquitinproteasome system [110].

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In contrast to the idea that changes in NMDAR composition in the adult are driven by long-term changes in neural networks, several recent studies have demonstrated that synapses can acquire or lose NMDARs on a much faster time scale during some forms of synaptic plasticity. For example, LTP may enhance the insertion of NMDARs at mature synapses, as suggested by increases in both the surface expression and synaptosomal localization of NR1 and NR2A subunits 30 min after LTP induction in adult hippocampal CA1 minislices [111]. This increase in NMDARs is both PKC and Src dependent. Conversely, paradigms of LTD induction, such as low-frequency stimulation and metabotropic glutamate receptor activation, decrease the amplitude of NMDAR excitatory postsynaptic currents, coinciding with decreased NR1 surface expression and NR1 protein levels in synaptoneurosomes [112–114]. In addition, experience-dependent changes that mimic the developmental switch in NMDAR subunit composition during development occur relatively rapidly in the visual cortex (within 1 h of light exposure in dark-reared animals) [115, 116], suggesting rapid receptor trafficking. Furthermore, visual sensory deprivation produces a horizontal shift in the frequencyresponse relationship at synapses in superficial layers of the primary visual cortex, consistent with enhanced NMDAR transmission [117] and perhaps due to increased NMDAR abundance at these synapses. Although not discussed in detail here, the formation or rearrangement of synaptic elements (synapses and dendritic spines) generates additional substrate for plasticity and regulates synaptic efficacy not only during development but also in response to experience [10, 12, 118]. The mature brain may be more plastic than traditionally thought, as suggested by live imaging experiments in the barrel cortex in vivo showing that sensory experience can significantly alter spine turnover and thus reshape synaptic connections [119]. Changes in dendritic spine shape, filopodia growth and extensive remodeling of postsynaptic membranes occur in response to synaptic activation, and are both Ca2+ and NMDAR dependent [110, 120, 121]. Spine maintenance may also rely on NMDAR activation, as supported by the finding that knockout mice lacking the inhibitory subunit NR3A display increased spine density [65]. A critical question is whether morphological changes are linked to or triggered by changes in the NMDAR complement at the postsynaptic membrane. The high degree of structural and molecular plasticity exhibited by PSDs [110, 122] could reflect a significant capacity of synaptic membranes to undergo protein recycling, perhaps including NMDARs or other glutamate


receptors. Interestingly, the dynamic remodeling of PSDs differentially affects the abundance of specific NMDAR subunits and is regulated by synaptic activity [110]. In this context, it has been postulated that certain paradigms of LTD induction may initiate synapse elimination by inducing the removal of NMDARs from postsynaptic sites [114], thus connecting synaptic rearrangement to changes in NMDAR composition.

the adult? How are these processes influenced by synaptic activity or coordinated in a synapse-specific manner?

Until quite recently, NMDARs, unlike their highly mobile AMPAR cousins, were viewed as relatively static and confined to the PSD by tight interactions with scaffolding proteins once synapse assembly and maturation had occurred. In support of this idea, the half-life of NMDARs measured in cerebellar granule cells and cortical neurons in culture by pulse-chase receptor labeling and surface biotinylation has been estimated as approximately 20 h for NR1, NR2A and NR2B subunits [110, 123]. This slow constitutive receptor turnover appears to conflict with cases in which rapid changes in the number of surface or synaptic NMDARs have been observed [111–113, 115] and suggests that mechanisms other than receptor synthesis/degradation must exist to allow a rapid control of NMDAR composition. Research over the last 2–3 years has provided evidence that this is, in fact, the case. First, NMDARs redistribute in the plane of the membrane, moving from extrasynaptic to synaptic pools, indicating a dynamic reorganization of NMDARs within the PSD [124]. Second, regulated exocytosis and stabilization occur, and require synaptic activity and the activity of different kinases [47, 125–127]. Third, NMDARs can be rapidly removed from the neuronal surface and/or synapses by endocytosis [63, 88, 114, 128]. The new findings have awakened interest in the mechanisms responsible for fluctuations in NMDAR composition (both slow and rapid), beginning with the different steps in the sorting, insertion and internalization of NMDARs that are subject to regulation (fig. 3). Recent studies have started to unveil the basic underlying mechanisms of local NMDAR trafficking and have given partial answers to key questions such as: What are the receptorintrinsic molecular determinants that control NMDAR trafficking? What are the cell biological mechanisms and relevant cellular machinery? What intracellular signals regulate NMDAR trafficking, and how do they work to mediate receptor replacement during development and in

Lateral Diffusion of Receptors between Synaptic and Extrasynaptic Sites Some of the first evidence that NMDARs were more mobile than generally assumed came from the lab of Gary Westbrook. Using ingenious electrophysiological and pharmacological tools, Tovar and Westbrook [124] demonstrated that NMDARs undergo lateral diffusion within the plasma membrane. The stability of synaptic NMDARs was examined in cultured hippocampal neurons using the irreversible open-channel blocker MK-801 to tag synaptic receptors. An anomalous recovery of NMDAR-mediated excitatory postsynaptic currents was observed after the synaptic block, which could not be accounted for by insertion of new receptors into synapses or a faster rate of MK-801 dissociation, and instead indicated that there was lateral movement of extrasynaptic NMDARs into synapses. The movement reported was bidirectional, in that synaptic receptors could also diffuse into the extrasynaptic pool, and the rate of exchange measured was surprisingly high (65% receptor exchange in !7 min). Lateral movement was not affected by the NMDAR antagonist AP5 or the actin-depolymerizing agent latrunculin. The latter finding was unexpected given that synaptic NMDARs are anchored at synapses through their interaction with scaffolding proteins which link the receptor-scaffold complex to the actin cytoskeleton [129]. Such interactions are believed to stabilize the receptor complex, and one might predict that diffusion would be facilitated if the actin cytoskeleton were transiently dissociated [130, 131]. The results from Tovar and Westbrook [124] force a rethinking of this model, but it is important to point out that these diffusion measurements were conducted on young neurons, in which actin-PSD interactions may not yet be fully developed. In addition, NR1/NR2A complexes (preferentially synaptic) and NR1/NR2B (mostly extrasynaptic) appeared to be equally mobile, in contrast to the prediction that synaptic NR1/ NR2A receptors would be trapped at synapses, and consequently more stable, than their extrasynaptic NR1/NR2B counterparts. Notably, this latter result also argues against a role for lateral diffusion in the developmental NR2BNR2A switch that is believed to underlie progressive synaptic restriction, since both NR1/NR2A and NR1/NR2B complexes diffuse in and out of synapses at similar rates. Given the high degree of lateral mobility exhibited by NMDARs in young neurons, it will be important to determine if lateral movement between synaptic and extra-



Rapid Movement of NMDARs in and out of the Postsynaptic Membrane

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Fig. 3. A model for local movement of NMDARs in and out of synaptic membranes. NMDARs undergo lateral diffusion within the plane of the plasma membrane and regulated insertion/removal from synaptic sites. Lateral diffusion between synaptic and extrasynaptic sites is constitutive (grey arrows), i.e. not regulated by agonist binding (green arrows) or activity (red arrows). The steady cycling of receptors in and out of synapses provides a chance for altering synaptic strength over a time span of minutes, but would not change the

equilibrium composition of the PSD without the existence of regulated steps. Activity or ligand binding modulate the rate of insertion of NR1/NR2A heteromers and/or internalization of NR1/NR2B complexes. Internalization of synaptic NR1/NR2B complexes may be limited (dashed arrow) by binding to PSD-95; therefore, disengagement from synapses and translocation to extrasynaptic locations may be a prerequisite for receptor internalization (right panel).

synaptic pools is regulated at later stages of development or in response to activity or extracellular signals, as has been found for AMPARs [132]. Lateral movement may mediate receptor insertion into synapses [133, 134] and/ or precede receptor endocytosis by causing the dispersal of receptors from synapses to extrasynaptic sites and thereby facilitating their removal [97]. On the other hand, receptor endo-/exocytic cycling may be the regulated steps, with lateral diffusion constitutively enabling receptors to enter specialized membrane domains dedicated for protein trafficking such as endocytic zones [97].

intra- and extracellular signals. For example, PKC activation stimulates the delivery of new NMDARs to the plasma membrane by two different mechanisms [25]. Coordinated phosphorylation of residues in the C-terminus of the NR1 subunit by PKC and protein kinase A accelerates ER export and follows a slow time course (2–3 h), consistent with the transport kinetics of membrane proteins throughout the secretory pathway [47, 135]. At a later stage of trafficking, PKC triggers the rapid insertion of functional NMDARs into the surface of neuronal dendrites within minutes, as assessed electrophysiologically and by immunofluorescence [127]. Rapid delivery occurs via SNARE-dependent exocytosis, and does not require direct phosphorylation by PKC of NMDAR subunits [127, 136]. On the other hand, experiments by Barria and Malinow [125] in hippocampal slice cultures indicate that

Mechanisms of NMDAR Insertion Supporting this latter view, a number of studies have provided evidence that intracellular NMDARs are mobilized by regulated vesicular transport and exocytosis. The surface delivery of NMDARs can be regulated by both

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delivery of NMDARs to synapses follows different rules depending on the NR2 subunit present in the complex. According to this study, the synaptic insertion of NR2Bcontaining receptors occurs constitutively in an activityindependent manner, whereas the insertion of receptors containing NR2A requires ligand binding to NMDARs and spontaneous synaptic activity without a requirement for ion flux through the channel (fig. 3, left). Such liganddependent incorporation of NR2A-containing NMDARs could be explained by agonist-induced removal of NR2Bcontaining complexes (perhaps via endocytosis, see below), and the subsequent release of ‘synaptic spaces’ which can then by occupied by NR2A subunits. Indeed, the incorporation of NMDARs into synapses appears to be tightly regulated and the number of synaptic slots available for NMDARs limited. Consistent with this notion, overexpression of NR2 subunits in cerebellar granule neurons increases the total surface NMDARs, but does not alter the magnitude of synaptic NMDA excitatory postsynaptic currents [54]. The idea that coordinated, subunit-specific endocytosis and exocytosis drive receptor replacement is a very interesting one, and it emphasizes the need to decipher the constitutive versus the regulated or rate-limiting steps in this process. For instance, new NMDARs could be incorporated into synapses via lateral diffusion (constitutive?) or active insertion (regulated?), whereas receptor internalization (rate limiting if synaptic spaces are limited) would be modulated by activity or protein-protein interactions that trap the receptor at PSDs (fig. 3). Indeed, many of the homeostatic and developmental changes described in the previous sections could be explained by tuning the rates of receptor insertion, removal or degradation [110]. Changes in the rates of ongoing dynamic trafficking events could provide a sensitive and highly adjustable means for neurons to alter synaptic strength and NMDAR signaling. NMDAR Internalization Two recent studies have identified potential internalization signals in the C-terminus of NR2 subunits [88, 137]. Both signals are tyrosine-based motifs – YEKL in the distal portion of the NR2B C-terminus and YWKL in the membrane proximal region of the C-terminus of NR2A – which conform to the consensus YXXØ sequence, where Ø is an amino acid with bulky hydrophobic side chains. YXXØ motifs bind to the endocytic adaptor AP2, which directs internalized cargo for clathrin-mediated endocytosis, and thus causes rapid internalization of plasma membrane proteins [reviewed in ref. 138, 139].

In the case of NR2B, the internalized receptor is transported to recycling endosomes [88], presumably indicating subsequent reinsertion of endocytosed receptors. A very interesting result from this study was that truncation of the PDZ-binding motif in NR2B significantly increased internalization in hippocampal neurons in culture, whereas coexpression of PSD-95 inhibited internalization in heterologous systems. However, the experiments were conducted using fusions of the region of interest (the C-terminus of NR2B) and the surface protein Tac and it is not yet certain whether these findings extend to endogenous receptors in neurons. However, the results do strongly suggest that the rate of NMDAR internalization is controlled by interactions with scaffolding proteins that stabilize the receptor at synaptic sites. Prior to endocytosis, receptors likely need to dissociate from their scaffolds to move away from the PSD and diffuse into the perisynaptic region (fig. 3). Indeed, clathrin coats typically reside in locations adjacent to, but spatially segregated from PSDs [97]. An important question is how receptors are disengaged from the PSD and translocated into specialized endocytic zones in dendrites, and if this process is regulated by neuronal activity, agonist binding or other extracellular modulators and intracellular signals. Yet again, a parallel can be drawn with the neuromuscular junction, where AChR receptor internalization upon denervation or prolonged receptor blockade appears to occur at the lateral margin of the postsynaptic specialization [86, 140]. One predicted consequence of having highly ordered scaffold complexes at synapses is that synaptic and extrasynaptic NMDARs within the same neuron may have different mobility or propensity for downregulation via endocytosis. Consistent with this, Li et al. [63] found that extrasynaptic receptors are preferentially internalized in response to sustained (3- to 5-min) treatment with NMDA and glycine. The differential surface turnover rate of synaptic and extrasynaptic receptors may result from their coupling to distinct signaling pathways and interaction partners [63, 110], and reflect adaptive responses from two distinct receptor populations with different functions. Relative resistance to endocytosis of synaptic receptors would ensure the stability of the network, whereas rapid removal of extrasynaptic receptors would warrant protection under circumstances of prolonged NMDAR stimulation that risk triggering excitotoxic damage [98, 141, 142]. It remains to be definitively established whether different NMDAR subunits distribute into mobile or immobile receptor pools, but one possibility is that different affinities for scaffolding proteins determine



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the degree of synaptic attachment and stability. All NR2 subunits contain a PDZ-binding sequence required for attachment to PSD-95, but such a sequence is not present in the C-terminus of the NR3 subunit, and the degree of enrichment of NR3 in synapses is still unknown [53]. In addition, small deviations of PDZ-binding signals in different NR2 subunits or competitive binding of additional signaling proteins to NMDAR intracellular domains [45, 143] may alter scaffold binding affinity and hence regulate receptor mobility and trafficking. NMDAR internalization is subject to regulation by specific patterns of receptor activation that may work by driving the recruitment of endocytic adaptors and activating intracellular signaling cascades. For instance, usedependent downregulation of NMDARs in heterologous systems requires ligand binding and Src kinase but is independent of ion flux and is prevented by dominant inhibitory Ì2 subunits of the AP-2 complex [137]. In neurons, stimulation of the glycine-binding site initiates a conformational change (or intracellular signal) that primes NMDARs for clathrin-dependent endocytosis by recruiting ß2-adaptin to the receptor complex [128]. However, glycine binding alone does not cause the receptor to be internalized. Rather, activation of both the glutamateand the glycine-binding site is required. Interestingly, NMDARs composed of only NR1 and NR3 subunits lack the glutamate-binding site (conferred by NR2) but have been proposed to act as excitatory glycine receptors [34]. Whether these excitatory glycine receptors are also subject to use-dependent downregulation via endocytosis remains an open question.

A Few Answers and Many New Questions

In a remarkably short time, our view of the NMDAR has changed from a stable and stolid component of the PSD reliably fluxing Ca2+ to effect synaptic change, to a wandering wayfarer moving in and out of synapses. Perhaps it is not so surprising that NMDARs are dynamic, and that their transport and trafficking are subject to regulation at numerous cellular levels. After all, NMDARs are among the most central signaling molecules in the CNS. The emergence of neuronal cell biology, and membrane trafficking in particular, as key loci for synaptic plasticity has revealed a realm of previously underappreciated mechanisms for regulating the abundance of NMDARs at synapses. From the mechanistic to the multicellular, many questions remain. How do the various signals and motifs for

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endocytosis or ER export coordinate and contribute to trafficking of different heteromeric NMDAR complexes? What are the relevant adaptors and what is the relationship of these adaptors to the PSD or the cytoskeleton? Where precisely do the endocytic and exocytic events occur? What is the identity and location of intracellular compartments or organelles that process and deliver NMDARs? How does trafficking of NMDARs differ from that of AMPARs? What are the mechanisms that regulate NMDAR delivery at early stages of secretory trafficking (e.g. ER export) and at later stages of terminal exocytosis? How are these trafficking events regulated by activity? Finally, the overarching question is the physiological relevance of NMDAR trafficking. How is the insertion or removal of specific subtypes of NMDARs linked to synapse formation/elimination, to rearrangement of dendritic spines or to global or synapse-specific changes in the plasticity of neural circuits? Likewise, to what extent does abnormal NMDAR trafficking contribute to pathophysiological changes in the numerous brain disorders linked to NMDAR function, e.g. stroke, epilepsy, schizophrenia, addiction, alcoholism, neurodegeneration and traumatic injury, among others. Addressing these questions will require an understanding of the fundamental cell biological mechanisms, assisted by recent advances in live-cell imaging and fluorescence microscopy, together with electrophysiological approaches for studying synaptic plasticity and in vivo genetic manipulations of receptor trafficking. Clearly, the future holds considerable promise for uncovering the tracks and trails of the surprisingly mobile NMDAR.

Acknowledgments I.P.O. was supported by grants from the NIH (NS32742 to Donald C. Lo) and NARSAD Foundation. Research in the laboratory of M.D.E. is supported by grants from the NIH (NS39402 and MH64748) and a grant from the Ruth K. Broad Foundation. We thank Donald C. Lo and John F. Wesseling for graciously providing intellectual support and stimulating discussion, and Thomas Blanpied, Juliet Hernandez, Kathryn Hawk, April Horton, Derek Scott and Yuanyue Mu for critical readings of the manuscript.


References 1 Abbott LF, Nelson SB: Synaptic plasticity: Taming the beast. Nat Neurosci 2000;3(suppl): 1178–1183. 2 Turrigiano GG, Nelson SB: Hebb and homeostasis in neuronal plasticity. Curr Opin Neurobiol 2000;10:358–364. 3 Abraham WC, Bear MF: Metaplasticity: The plasticity of synaptic plasticity. Trends Neurosci 1996;19:126–130. 4 Zito K, Svoboda K: Activity-dependent synaptogenesis in the adult Mammalian cortex. Neuron 2002;35:1015–1017. 5 Bliss TV, Collingridge GL: A synaptic model of memory: Long-term potentiation in the hippocampus. Nature 1993;361:31–39. 6 Feldman DE, Knudsen EI: Experience-dependent plasticity and the maturation of glutamatergic synapses. Neuron 1998;20:1067–1071. 7 Katz LC, Shatz CJ: Synaptic activity and the construction of cortical circuits. Science 1996; 274:1133–1138. 8 Cohen-Cory S: The developing synapse: Construction and modulation of synaptic structures and circuits. Science 2002;298:770–776. 9 Barry MF, Ziff EB: Receptor trafficking and the plasticity of excitatory synapses. Curr Opin Neurobiol 2002;12:279–286. 10 Luscher C, et al: Synaptic plasticity and dynamic modulation of the postsynaptic membrane. Nat Neurosci 2000;3:545–550. 11 Malinow R, Malenka RC: AMPA receptor trafficking and synaptic plasticity. Annu Rev Neurosci 2002;25:103–126. 12 Nikonenko I, et al: Activity-induced changes of spine morphology. Hippocampus 2002;12: 585–591. 13 Cline HT: Dendritic arbor development and synaptogenesis. Curr Opin Neurobiol 2001;11: 118–126. 14 Constantine-Paton M, Cline HT, Debski E: Patterned activity, synaptic convergence, and the NMDA receptor in developing visual pathways. Annu Rev Neurosci 1990;13:129–154. 15 Hebb DO: The Organization of Behavior; A Neuropsychological Theory. New York, Wiley, 1949. 16 Dudek SM, Bear MF: Homosynaptic long-term depression in area CA1 of hippocampus and effects of N-methyl-D-aspartate receptor blockade. Proc Natl Acad Sci USA 1992;89:4363– 4367. 17 Martin SJ, Grimwood PD, Morris RG: Synaptic plasticity and memory: An evaluation of the hypothesis. Annu Rev Neurosci 2000;23:649– 711. 18 Renart A, Song P, Wang XJ: Robust spatial working memory through homeostatic synaptic scaling in heterogeneous cortical networks. Neuron 2003;38:473–485. 19 Royer S, Pare D: Conservation of total synaptic weight through balanced synaptic depression and potentiation. Nature 2003;422:518–522. 20 Malenka RC, Nicoll RA: NMDA-receptor-dependent synaptic plasticity: Multiple forms and mechanisms. Trends Neurosci 1993;16: 521–527.


21 Kirkwood A, Rioult MC, Bear MF: Experience-dependent modification of synaptic plasticity in visual cortex. Nature 1996;381:526– 528. 22 Craig AM: Activity and synaptic receptor targeting: The long view. Neuron 1998;21:459– 462. 23 Shouval HZ, Bear MF, Cooper LN: A unified model of NMDA receptor-dependent bidirectional synaptic plasticity. Proc Natl Acad Sci USA 2002;99:10831–10836. 24 Cull-Candy S, Brickley S, Farrant M: NMDA receptor subunits: Diversity, development and disease. Curr Opin Neurobiol 2001;11:327– 335. 25 Carroll RC, Zukin RS: NMDA-receptor trafficking and targeting: Implications for synaptic transmission and plasticity. Trends Neurosci 2002;25:571–577. 26 Wenthold RJ, et al: Trafficking of NMDA receptors. Annu Rev Pharmacol Toxicol 2003; 43:335–358. 27 Schorge S, Colquhoun D: Studies of NMDA receptor function and stoichiometry with truncated and tandem subunits. J Neurosci 2003; 23:1151–1158. 28 Laube B, Kuhse J, Betz H: Evidence for a tetrameric structure of recombinant NMDA receptors. J Neurosci 1998;18:2954–2961. 29 McBain CJ, Mayer ML: N-methyl-D-aspartic acid receptor structure and function. Physiol Rev 1994;74:723–760. 30 Hollmann M, Heinemann S: Cloned glutamate receptors. Annu Rev Neurosci 1994;17:31– 108. 31 Monyer H, et al: Heteromeric NMDA receptors: Molecular and functional distinction of subtypes. Science 1992;256:1217–1221. 32 Sucher NJ, et al: Developmental and regional expression pattern of a novel NMDA receptorlike subunit (NMDAR-L) in the rodent brain. J Neurosci 1995;15:6509–6520. 33 Ciabarra AM, et al: Cloning and characterization of chi-1:A developmentally regulated member of a novel class of the ionotropic glutamate receptor family. J Neurosci 1995;15: 6498–6508. 34 Chatterton JE, et al: Excitatory glycine receptors containing the NR3 family of NMDA receptor subunits. Nature 2002;415:793–798. 35 Zukin RS, Bennett MV: Alternatively spliced isoforms of the NMDARI receptor subunit. Trends Neurosci 1995;18:306–313. 36 Rosenmund C, Stern-Bach Y, Stevens CF: The tetrameric structure of a glutamate receptor channel. Science 1998;280:1596–1599. 37 Leuschner WD, Hoch W: Subtype-specific assembly of alpha-amino-3-hydroxy-5-methyl-4isoxazole propionic acid receptor subunits is mediated by their n-terminal domains. J Biol Chem 1999;274:16907–16916. 38 Ayalon G, Stern-Bach J: Functional assembly of AMPA and kainate receptors is mediated by several discrete protein-protein interactions. Neuron 2001;31:103–113.

39 Tovar KR, Westbrook GL: The incorporation of NMDA receptors with a distinct subunit composition at nascent hippocampal synapses in vitro. J Neurosci 1999;19:4180–4188. 40 Vicini S, et al: Functional and pharmacological differences between recombinant N-methyl-Daspartate receptors. J Neurophysiol 1998;79: 555–566. 41 Luo J, et al: The majority of N-methyl-D-aspartate receptor complexes in adult rat cerebral cortex contain at least three different subunits (NR1/NR2A/NR2B). Mol Pharmacol 1997;51: 79–86. 42 Brickley SG, et al: NR2B and NR2D subunits coassemble in cerebellar Golgi cells to form a distinct NMDA receptor subtype restricted to extrasynaptic sites. J Neurosci 2003;23:4958– 4966. 43 Perez-Otano I, et al: Assembly with the NR1 subunit is required for surface expression of NR3A-containing NMDA receptors. J Neurosci 2001;21:1228–1237. 44 Durand GM, Bennett MV, Zukin RS: Splice variants of the N-methyl-D-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C. Proc Natl Acad Sci USA 1993;90:6731–6735. 45 Wyszynski M, et al: Competitive binding of alpha-actinin and calmodulin to the NMDA receptor. Nature 1997;385:439–442. 46 Hollmann M, et al: Zinc potentiates agonistinduced currents at certain splice variants of the NMDA receptor. Neuron 1993;10:943– 954. 47 Scott DB, et al: An NMDA receptor ER retention signal regulated by phosphorylation and alternative splicing. J Neurosci 2001;21:3063– 3072. 48 Standley S, et al: PDZ domain suppression of an ER retention signal in NMDA receptor NR1 splice variants. Neuron 2000;28:887–898. 49 Xia H, Hornby ZD, Malenka RC: An ER retention signal explains differences in surface expression of NMDA and AMPA receptor subunits. Neuropharmacology 2001;41:714–723. 50 Stern P, et al: Single-channel conductances of NMDA receptors expressed from cloned cDNAs: Comparison with native receptors. Proc R Soc Lond B Biol Sci 1992;250:271– 277. 51 Monyer H, et al: Developmental and regional expression in the rat brain and functional properties of four NMDA receptors. Neuron 1994; 12:529–540. 52 Niethammer M, Kim E, Sheng M: Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J Neurosci 1996;16:2157–2163. 53 Kornau HC, et al: Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 1995;269: 1737–1740. 54 Prybylowski K, et al: Relationship between availability of NMDA receptor subunits and their expression at the synapse. J Neurosci 2002;22:8902–8910.


187

55 Mori H, et al: Role of the carboxy-terminal region of the GluR epsilon2 subunit in synaptic localization of the NMDA receptor channel. Neuron 1998;21:571–580. 56 Mohrmann R, et al: Deletion of the C-terminal domain of the NR2B subunit alters channel properties and synaptic targeting of N-methylD-aspartate receptors in nascent neocortical synapses. J Neurosci Res 2002;68:265–275. 57 Steigerwald F, et al: C-Terminal truncation of NR2A subunits impairs synaptic but not extrasynaptic localization of NMDA receptors. J Neurosci 2000;20:4573–4581. 58 Sprengel R, et al: Importance of the intracellular domain of NR2 subunits for NMDA receptor function in vivo. Cell 1998;92:279–289. 59 Sheng M, Sala C: PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 2001;24:1–29. 60 Husi H, et al: Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nat Neurosci 2000;3:661–669. 61 Vicini S, Rumbaugh G: A slow NMDA channel: In search of a role. J Physiol 2000;525:283. 62 Rumbaugh G, Vicini S: Distinct synaptic and extrasynaptic NMDA receptors in developing cerebellar granule neurons. J Neurosci 1999; 19:10603–10610. 63 Li B, et al: Differential regulation of synaptic and extra-synaptic NMDA receptors. Nat Neurosci 2002;5:833–834. 64 Sheng M, et al: Changing subunit composition of heteromeric NMDA receptors during development of rat cortex. Nature 1994;368:144– 147. 65 Das S, et al: Increased NMDA current and spine density in mice lacking the NMDA receptor subunit NR3A. Nature 1998;393:377–381. 66 Sasaki YF, et al: Characterization and comparison of the NR3A subunit of the NMDA receptor in recombinant systems and primary cortical neurons. J Neurophysiol 2002;87:2052– 2063. 67 Wong HK, et al: Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain. J Comp Neurol 2002;450: 303–317. 68 Hampton RY: ER-associated degradation in protein quality control and cellular regulation. Curr Opin Cell Biol 2002;14:476–482. 69 Wickner S, Maurizi MR, Gottesman S: Posttranslational quality control: Folding, refolding, and degrading proteins. Science 1999;286: 1888–1893. 70 Zerangue N, et al: A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 1999;22: 537–548. 71 Margeta-Mitrovic M, Jan YN, Jan LY: A trafficking checkpoint controls GABA(B) receptor heterodimerization. Neuron 2000;27:97–106. 72 Ellgaard L, Helenius A: Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 2003;4:181–191. 73 McIlhinney RA, et al: Assembly intracellular targeting and cell surface expression of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A in transfected cells. Neuropharmacology 1998;37:1355–1367.

188

74 McIlhinney RA, et al: Cell surface expression of the human N-methyl-D-aspartate receptor subunit 1a requires the co-expression of the NR2A subunit in transfected cells. Neuroscience 1996;70:989–997. 75 Okabe S, Miwa A, Okado H: Alternative splicing of the C-terminal domain regulates cell surface expression of the NMDA receptor NR1 subunit. J Neurosci 1999;19:7781–7792. 76 Ehlers MD, Tingley WG, Huganir RL: Regulated subcellular distribution of the NR1 subunit of the NMDA receptor. Science 1995;269: 1734–1737. 77 Lodish HF, et al: Hepatoma secretory proteins migrate from rough endoplasmic reticulum to Golgi at characteristic rates. Nature 1983;304: 80–83. 78 Sans N, et al: NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat Cell Biol 2003;5: 520–530. 79 Washbourne P, Bennett JE, McAllister AK: Rapid recruitment of NMDA receptor transport packets to nascent synapses. Nat Neurosci 2002;5:751–759. 80 Setou M, et al: Kinesin superfamily motor protein KIF17 and mLin-10 in NMDA receptorcontaining vesicle transport. Science 2000;288: 1796–1802. 81 Wong RW, et al: Overexpression of motor protein KIF17 enhances spatial and working memory in transgenic mice. Proc Natl Acad Sci USA 2002;99:14500–14505. 82 Guillaud L, Setou M, Hirokawa N: KIF17 dynamics and regulation of NR2B trafficking in hippocampal neurons. J Neurosci 2003;23: 131–140. 83 Friedman HV, et al: Assembly of new individual excitatory synapses: Time course and temporal order of synaptic molecule recruitment. Neuron 2000;27:57–69. 84 Ziv NE, Garner CC: Principles of glutamatergic synapse formation: Seeing the forest for the trees. Curr Opin Neurobiol 2001;11:536–543. 85 Rao A, et al: Heterogeneity in the molecular composition of excitatory postsynaptic sites during development of hippocampal neurons in culture. J Neurosci 1998;18:1217–1229. 86 Sanes JR, Lichtman JW: Induction, assembly, maturation and maintenance of a postsynaptic apparatus. Nat Rev Neurosci 2001;2:791–805. 87 Dalva MB, et al: EphB receptors interact with NMDA receptors and regulate excitatory synapse formation. Cell 2000;103:945–956. 88 Roche KW, et al: Molecular determinants of NMDA receptor internalization. Nat Neurosci 2001;4:794–802. 89 Cline HT, Wu GY, Malinow R: In vivo development of neuronal structure and function. Cold Spring Harb Symp Quant Biol 1996;61: 95–104. 90 Stocca G, Vicini S: Increased contribution of NR2A subunit to synaptic NMDA receptors in developing rat cortical neurons. J Physiol 1998; 507:13–24. 91 Hestrin S: Developmental regulation of NMDA receptor-mediated synaptic currents at a central synapse. Nature 1992;357:686–689.


92 Crair MC, Malenka RC: A critical period for long-term potentiation at thalamocortical synapses. Nature 1995;375:325–328. 93 Carmignoto G, Vicini S: Activity-dependent decrease in NMDA receptor responses during development of the visual cortex. Science 1992;258:1007–1011. 94 Chavis P, Westbrook G: Integrins mediate functional pre- and postsynaptic maturation at a hippocampal synapse. Nature 2001;411: 317–321. 95 Flint AC, et al: NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex. J Neurosci 1997;17: 2469–2476. 96 Li JH, et al: Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons. Eur J Neurosci 1998;10:1704–1715. 97 Blanpied TA, Scott DB, Ehlers MD: Dynamics and regulation of clathrin coats at specialized endocytic zones of dendrites and spines. Neuron 2002;36:435–449. 98 Hardingham GE, Fukunaga Y, Bading H: Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci 2002;5: 405–414. 99 Rosenmund C, Feltz A, Westbrook GL: Synaptic NMDA receptor channels have a low open probability. J Neurosci 1995;15:2788– 2795. 100 Liao D, et al: Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons. Nat Neurosci 1999;2: 37–43. 101 Tang YP, et al: Genetic enhancement of learning and memory in mice. Nature 1999; 401:63–69. 102 Shi J, Townsend M, Constantine-Paton M: Activity-dependent induction of tonic calcineurin activity mediates a rapid developmental downregulation of NMDA receptor currents. Neuron 2000;28:103–114. 103 Barth AL, Malenka RC: NMDAR EPSC kinetics do not regulate the critical period for LTP at thalamocortical synapses. Nat Neurosci 2001;4:235–236. 104 Livingston FS, White SA, Mooney R: Slow NMDA-EPSCs at synapses critical for song development are not required for song learning in zebra finches. Nat Neurosci 2000;3: 482–488. 105 Kawakami R, et al: Asymmetrical allocation of NMDA receptor epsilon2 subunits in hippocampal circuitry. Science 2003;300:990– 994. 106 Watt AJ, et al: Activity coregulates quantal AMPA and NMDA currents at neocortical synapses. Neuron 2000;26:659–670. 107 Rao A, Craig AM: Activity regulates the synaptic localization of the NMDA receptor in hippocampal neurons. Neuron 1997;19:801– 812. 108 O’Brien RJ, et al: Activity-dependent modulation of synaptic AMPA receptor accumulation. Neuron 1998;21:1067–1078.


109 Fong DK, et al: Rapid synaptic remodeling by protein kinase C: Reciprocal translocation of NMDA receptors and calcium/calmodulindependent kinase II. J Neurosci 2002;22: 2153–2164. 110 Ehlers MD: Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system. Nat Neurosci 2003;6: 231–242. 111 Grosshans DR, et al: LTP leads to rapid surface expression of NMDA but not AMPA receptors in adult rat CA1. Nat Neurosci 2002;5:27–33. 112 Montgomery JM, Madison DV: State-dependent heterogeneity in synaptic depression between pyramidal cell pairs. Neuron 2002;33: 765–777. 113 Heynen AJ, et al: Bidirectional, activity-dependent regulation of glutamate receptors in the adult hippocampus in vivo. Neuron 2000; 28:527–536. 114 Snyder EM, et al: Internalization of ionotropic glutamate receptors in response to mGluR activation. Nat Neurosci 2001;4:1079–1085. 115 Quinlan EM, et al: Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo. Nat Neurosci 1999;2: 352–357. 116 Philpot BD, et al: Visual experience and deprivation bidirectionally modify the composition and function of NMDA receptors in visual cortex. Neuron 2001;29:157–169. 117 Philpot BD, Espinosa JS, Bear MF: Evidence for altered NMDA receptor function as a basis for metaplasticity in visual cortex. J Neurosci 2003;23:5583–5588. 118 Harris KM: Structure, development, and plasticity of dendritic spines. Curr Opin Neurobiol 1999;9:343–348. 119 Trachtenberg JT, et al: Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 2002;420: 788–794.


120 Maletic-Savatic M, Malinow R, Svoboda K: Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 1999;283:1923–1927. 121 Engert F, Bonhoeffer T: Dendritic spine changes associated with hippocampal longterm synaptic plasticity. Nature 1999;399: 66–70. 122 Toni N, et al: Remodeling of synaptic membranes after induction of long-term potentiation. J Neurosci 2001;21:6245–6251. 123 Huh KH, Wenthold RJ: Turnover analysis of glutamate receptors identifies a rapidly degraded pool of the N-methyl-D-aspartate receptor subunit, NR1, in cultured cerebellar granule cells. J Biol Chem 1999;274:151– 157. 124 Tovar KR, Westbrook GL: Mobile NMDA receptors at hippocampal synapses. Neuron 2002;34:255–264. 125 Barria A, Malinow R: Subunit-specific NMDA receptor trafficking to synapses. Neuron 2002;35:345–353. 126 Skeberdis VA, et al: Insulin promotes rapid delivery of N-methyl-D-aspartate receptors to the cell surface by exocytosis. Proc Natl Acad Sci USA 2001;98:3561–3566. 127 Lan JY, et al: Protein kinase C modulates NMDA receptor trafficking and gating. Nat Neurosci 2001;4:382–390. 128 Nong Y, et al: Glycine binding primes NMDA receptor internalization. Nature 2003;422:302–307. 129 Sheng M, Kim MJ: Postsynaptic signaling and plasticity mechanisms. Science 2002; 298:776–780. 130 Allison DW, et al: Role of actin in anchoring postsynaptic receptors in cultured hippocampal neurons: Differential attachment of NMDA versus AMPA receptors. J Neurosci 1998;18:2423–2436. 131 Choquet D, Triller A: The role of receptor diffusion in the organization of the postsynaptic membrane. Nat Rev Neurosci 2003;4:251– 265. 132 Borgdorff AJ, Choquet D: Regulation of AMPA receptor lateral movements. Nature 2002;417:649–653.

133 Passafaro M, Piech V, Sheng M: Subunit-specific temporal and spatial patterns of AMPA receptor exocytosis in hippocampal neurons. Nat Neurosci 2001;4:917–926. 134 Chen L, et al: Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms. Nature 2000;408:936–943. 135 Scott DB, Blanpied TA, Ehlers MD: Coordinated PKA and PKC phosphorylation suppresses RXR-mediated ER retention and regulates the surface delivery of NMDA receptors. Neuropharmacology 2003;45:755–767. 136 Zheng X, et al: Protein kinase C potentiation of N-methyl-D-aspartate receptor activity is not mediated by phosphorylation of N-methyl-D-aspartate receptor subunits. Proc Natl Acad Sci USA 1999;96:15262–15267. 137 Vissel B, et al: A use-dependent tyrosine dephosphorylation of NMDA receptors is independent of ion flux. Nat Neurosci 2001;4: 587–596. 138 Bonifacino JS, Traub LM: Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu Rev Biochem 2003;72:395– 447. 139 Slepnev VI, De Camilli P: Accessory factors in clathrin-dependent synaptic vesicle endocytosis. Nat Rev Neurosci 2000;1:161–172. 140 Akaaboune M, et al: Rapid and reversible effects of activity on acetylcholine receptor density at the neuromuscular junction in vivo. Science 1999;286:503–507. 141 Hardingham GE, Bading H: The Yin and Yang of NMDA receptor signalling. Trends Neurosci 2003;26:81–89. 142 Aarts M, et al: Treatment of ischemic brain damage by perturbing NMDA receptor- PSD95 protein interactions. Science 2002;298: 846–850. 143 Gardoni F, et al: Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase II and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor. J Neurosci 2001;21:1501–1509.


189