Differential association of postsynaptic signaling protein complexes in striatum and hippocampus

Distinct physiological stimuli are required for bidirectional synaptic plasticity in striatum and hippocampus, but differences in the underlying signaling mechanisms are poorly understood. We have begun to compare levels and interactions of key excitatory synaptic proteins in whole extracts and subcellular fractions isolated from micro‐dissected striatum and hippocampus. Levels of multiple glutamate receptor subunits, calcium/calmodulin‐dependent protein kinase II (CaMKII), a highly abundant serine/threonine kinase, and spinophilin, a F‐actin and protein phosphatase 1 (PP1) binding protein, were significantly lower in striatal extracts, as well as in synaptic and/or extrasynaptic fractions, compared with similar hippocampal extracts/fractions. However, CaMKII interactions with spinophilin were more robust in striatum compared with hippocampus, and this enhanced association was restricted to the extrasynaptic fraction. NMDAR GluN2B subunits associate with both spinophilin and CaMKII, but spinophilin‐GluN2B complexes were enriched in extrasynaptic fractions whereas CaMKII‐GluN2B complexes were enriched in synaptic fractions. Notably, the association of GluN2B with both CaMKII and spinophilin was more robust in striatal extrasynaptic fractions compared with hippocampal extrasynaptic fractions. Selective differences in the assembly of synaptic and extrasynaptic signaling complexes may contribute to differential physiological regulation of excitatory transmission in striatum and hippocampus.     

Glutamate Binding to the GluN2B Subunit Controls Surface Trafficking of N-Methyl-D-aspartate (NMDA) Receptors

Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B(-/-) mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.     

Protein kinase C promotes N-methyl-D-aspartate (NMDA) receptor trafficking by indirectly triggering calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation

Regulation of neuronal NMDA receptor (NMDAR) is critical in synaptic transmission and plasticity. Protein kinase C (PKC) promotes NMDAR trafficking to the cell surface via interaction with NMDAR-associated proteins (NAPs). Little is known, however, about the NAPs that are critical to PKC-induced NMDAR trafficking. Here, we showed that calcium/calmodulin-dependent protein kinase II (CaMKII) could be a NAP that mediates the potentiation of NMDAR trafficking by PKC. PKC activation promoted the level of autophosphorylated CaMKII and increased association with NMDARs, accompanied by functional NMDAR insertion, at postsynaptic sites. This potentiation, along with PKC-induced long term potentiation of the AMPA receptor-mediated response, was abolished by CaMKII antagonist or by disturbing the interaction between CaMKII and NR2A or NR2B. Further mutual occlusion experiments demonstrated that PKC and CaMKII share a common signaling pathway in the potentiation of NMDAR trafficking and long-term potentiation (LTP) induction. Our results revealed that PKC promotes NMDA receptor trafficking and induces synaptic plasticity through indirectly triggering CaMKII autophosphorylation and subsequent increased association with NMDARs.     

CaMKII binding to GluN2B is critical during memory consolidation

Memory is essential for our normal daily lives and our sense of self. Ca(2+) influx through the NMDA-type glutamate receptor (NMDAR) and the ensuing activation of the Ca(2+) and calmodulin-dependent protein kinase (CaMKII) are required for memory formation and its physiological correlate, long-term potentiation (LTP). The Ca(2+) influx induces CaMKII binding to the NMDAR to strategically recruit CaMKII to synapses that are undergoing potentiation. We generated mice with two point mutations that impair CaMKII binding to the NMDAR GluN2B subunit. Ca(2+)-triggered postsynaptic accumulation is largely abrogated for CaMKII and destabilized for TARPs, which anchor AMPA-type glutamate receptors (AMPAR). LTP is reduced by 50% and phosphorylation of the AMPAR GluA1 subunit by CaMKII, which enhances AMPAR conductance, impaired. The mutant mice learn the Morris water maze (MWM) as well as WT but show deficiency in recall during the period of early memory consolidation. Accordingly, the activity-driven interaction of CaMKII with the NMDAR is important for recall of MWM memory as early as 24 h, but not 1-2 h, after training potentially due to impaired consolidation.     

Modulation of synaptic plasticity by stress hormone associates with plastic alteration of synaptic NMDA receptor in the adult hippocampus

Stress exerts a profound impact on learning and memory, in part, through the actions of adrenal corticosterone (CORT) on synaptic plasticity, a cellular model of learning and memory. Increasing findings suggest that CORT exerts its impact on synaptic plasticity by altering the functional properties of glutamate receptors, which include changes in the motility and function of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype of glutamate receptor (AMPAR) that are responsible for the expression of synaptic plasticity. Here we provide evidence that CORT could also regulate synaptic plasticity by modulating the function of synaptic N-methyl-D-aspartate receptors (NMDARs), which mediate the induction of synaptic plasticity. We found that stress level CORT applied to adult rat hippocampal slices potentiated evoked NMDAR-mediated synaptic responses within 30 min. Surprisingly, following this fast-onset change, we observed a slow-onset (>1 hour after termination of CORT exposure) increase in synaptic expression of GluN2A-containing NMDARs. To investigate the consequences of the distinct fast- and slow-onset modulation of NMDARs for synaptic plasticity, we examined the formation of long-term potentiation (LTP) and long-term depression (LTD) within relevant time windows. Paralleling the increased NMDAR function, both LTP and LTD were facilitated during CORT treatment. However, 1-2 hours after CORT treatment when synaptic expression of GluN2A-containing NMDARs is increased, bidirectional plasticity was no longer facilitated. Our findings reveal the remarkable plasticity of NMDARs in the adult hippocampus in response to CORT. CORT-mediated slow-onset increase in GluN2A in hippocampal synapses could be a homeostatic mechanism to normalize synaptic plasticity following fast-onset stress-induced facilitation.     

Status epilepticus impairs synaptic plasticity in rat hippocampus and is followed by changes in expression of NMDA receptors

Cognitive deficits and memory loss are frequent in patients with temporal lobe epilepsy. Persistent changes in synaptic efficacy are considered as a cellular substrate underlying memory processes. Electrophysiological studies have shown that the properties of short-term and long-term synaptic plasticity in the cortex and hippocampus may undergo substantial changes after seizures. However, the neural mechanisms responsible for these changes are not clear. In this study, we investigated the properties of short-term and long-term synaptic plasticity in rat hippocampal slices 24 h after pentylenetetrazole (PTZ)-induced status epilepticus. We found that the induction of long-term potentiation (LTP) in CA1 pyramidal cells is reduced compared to the control, while short-term facilitation is increased. The experimental results do not support the hypothesis that status epilepticus leads to background potentiation of hippocampal synapses and further LTP induction becomes weaker due to occlusion, as the dependence of synaptic responses on the strength of input stimulation was not different in the control and experimental animals. The decrease in LTP can be caused by impairment of molecular mechanisms of neuronal plasticity, including those associated with NMDA receptors and/or changes in their subunit composition. Realtime PCR demonstrated significant increases in the expression of GluN1 and GluN2A subunits 3 h after PTZ-induced status epilepticus. The overexpression of obligate GluN1 subunit suggests an increase in the total number of NMDA receptors in the hippocampus. A 3-fold increase in the expression of the GluN2B subunit observed 24 h after PTZ-induced status epilepticus might be indicative of an increase in the proportion of GluN2B-containing NMDA receptors. Increased expression of the GluN2B subunit may be a cause for reducing the magnitude of LTP at hippocampal synapses after status epilepticus.     

Long-term potentiation at hippocampal perforant path-dentate astrocyte synapses

Accumulated evidence indicates that astroglial cells actively participate in neuronal synaptic transmission and plasticity. However, it is still not clear whether astrocytes are able to undergo plasticity in response to synaptic inputs. Here we demonstrate that a long-term potentiation (LTP)-like response could be detected at perforant path-dentate astrocyte synapses following high-frequency stimulation (HFS) in hippocampal slices of GFAP-GFP transgenic mice. The potentiation was not dependent on the glutamate transporters nor the group I metabotropic glutamate receptors. However, the induction of LTP requires activation of the NMDA receptor (NMDAR). The presence of functional NMDAR was supported by isolating the NMDAR-gated current and by identifying mRNAs of NMDAR subunits in astrocytes. Our results suggest that astrocytes in the hippocampal dentate gyrus are able to undergo plasticity in response to presynaptic inputs.     

FRET-FLIM Investigation of PSD95-NMDA Receptor Interaction in Dendritic Spines; Control by Calpain,CaMKII and Src Family Kinase

Little is known about the changes in protein interactions inside synapses during synaptic remodeling, as their live monitoring in spines has been limited. We used a FRET-FLIM approach in developing cultured rat hippocampal neurons expressing fluorescently tagged NMDA receptor (NMDAR) and PSD95, two essential proteins in synaptic plasticity, to examine the regulation of their interaction. NMDAR stimulation caused a transient decrease in FRET between the NMDAR and PSD95 in spines of young and mature neurons. The activity of both CaMKII and calpain were essential for this effect in both developmental stages. Meanwhile, inhibition of Src family kinase (SFK) had opposing impacts on this decrease in FRET in young versus mature neurons. Our data suggest concerted roles for CaMKII, SFK and calpain activity in regulating activity-dependent separation of PSD95 from GluN2A or GluN2B. Finally, we found that calpain inhibition reduced spine growth that was caused by NMDAR activity, supporting the hypothesis that PSD95-NMDAR separation is implicated in synaptic remodeling.     

Differential regulation of CaMKIIα interactions with mGluR5 and NMDA receptors by Ca2+ in neurons

Two glutamate receptors, metabotropic glutamate receptor 5 (mGluR5), and ionotropic NMDA receptors (NMDAR), functionally interact with each other to regulate excitatory synaptic transmission in the mammalian brain. In exploring molecular mechanisms underlying their interactions, we found that Ca²⁺/calmodulin‐dependent protein kinase IIα (CaMKIIα) may play a central role. The synapse‐enriched CaMKIIα directly binds to the proximal region of intracellular C terminal tails of mGluR5 in vitro. This binding is state‐dependent: inactive CaMKIIα binds to mGluR5 at a high level whereas the active form of the kinase (following Ca²⁺/calmodulin binding and activation) loses its affinity for the receptor. Ca²⁺ also promotes calmodulin to bind to mGluR5 at a region overlapping with the CaMKIIα‐binding site, resulting in a competitive inhibition of CaMKIIα binding to mGluR5. In rat striatal neurons, inactive CaMKIIα constitutively binds to mGluR5. Activation of mGluR5 Ca²⁺‐dependently dissociates CaMKIIα from the receptor and simultaneously promotes CaMKIIα to bind to the adjacent NMDAR GluN2B subunit, which enables CaMKIIα to phosphorylate GluN2B at a CaMKIIα‐sensitive site. Together, the long intracellular C‐terminal tail of mGluR5 seems to serve as a scaffolding domain to recruit and store CaMKIIα within synapses. The mGluR5‐dependent Ca²⁺ transients differentially regulate CaMKIIα interactions with mGluR5 and GluN2B in striatal neurons, which may contribute to cross‐talk between the two receptors.

     

Computational investigation of the changing patterns of subtype specific NMDA receptor activation during physiological glutamatergic neurotransmission

NMDA receptors (NMDARs) are the major mediator of the postsynaptic response during synaptic neurotransmission. The diversity of roles for NMDARs in influencing synaptic plasticity and neuronal survival is often linked to selective activation of multiple NMDAR subtypes (NR1/NR2A-NMDARs, NR1/NR2B-NMDARs, and triheteromeric NR1/NR2A/NR2B-NMDARs). However, the lack of available pharmacological tools to block specific NMDAR populations leads to debates on the potential role for each NMDAR subtype in physiological signaling, including different models of synaptic plasticity. Here, we developed a computational model of glutamatergic signaling at a prototypical dendritic spine to examine the patterns of NMDAR subtype activation at temporal and spatial resolutions that are difficult to obtain experimentally. We demonstrate that NMDAR subtypes have different dynamic ranges of activation, with NR1/NR2A-NMDAR activation sensitive at univesicular glutamate release conditions, and NR2B containing NMDARs contributing at conditions of multivesicular release. We further show that NR1/NR2A-NMDAR signaling dominates in conditions simulating long-term depression (LTD), while the contribution of NR2B containing NMDAR significantly increases for stimulation frequencies that approximate long-term potentiation (LTP). Finally, we show that NR1/NR2A-NMDAR content significantly enhances response magnitude and fidelity at single synapses during chemical LTP and spike timed dependent plasticity induction, pointing out an important developmental switch in synaptic maturation. Together, our model suggests that NMDAR subtypes are differentially activated during different types of physiological glutamatergic signaling, enhancing the ability for individual spines to produce unique responses to these different inputs.     

Neurogranin enhances synaptic strength through its interaction with calmodulin

Learning‐correlated plasticity at CA1 hippocampal excitatory synapses is dependent on neuronal activity and NMDA receptor (NMDAR) activation. However, the molecular mechanisms that transduce plasticity stimuli to postsynaptic potentiation are poorly understood. Here, we report that neurogranin (Ng), a neuron‐specific and postsynaptic protein, enhances postsynaptic sensitivity and increases synaptic strength in an activity‐ and NMDAR‐dependent manner. In addition, Ng‐mediated potentiation of synaptic transmission mimics and occludes long‐term potentiation (LTP). Expression of Ng mutants that lack the ability to bind to, or dissociate from, calmodulin (CaM) fails to potentiate synaptic transmission, strongly suggesting that regulated Ng–CaM binding is necessary for Ng‐mediated potentiation. Moreover, knocking‐down Ng blocked LTP induction. Thus, Ng–CaM interaction can provide a mechanistic link between induction and expression of postsynaptic potentiation.     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

The Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca²⁺/CaM but outlasts this initial Ca²⁺-stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phospho-mimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.     

Directional gating of synaptic plasticity by GPCRs and their distinct downstream signalling pathways

EMBO J 31 4, 805–816 (2012); published online December202011Synaptic plasticity, the activity-dependent modification of synaptic strength, plays a fundamental role in learning and memory as well as in developmental maturation of neuronal circuitry. However, how synaptic plasticity is induced and regulated remains poorly understood. In this issue of The EMBO Journal, Yang and colleagues present sets of exciting data, suggesting that G-protein-coupled receptors (GPCRs) selectively execute distinct signalling pathways to differentially regulate induction thresholds of hippocampal long-term potentiation (LTP) and long-term depression (LTD), thereby governing the direction of synaptic plasticity. These results shed significant light on our current understanding of how bidirectional synaptic plasticity is regulated.Synaptic plasticity has been demonstrated at synapses in various brain regions; the most well-characterized forms are LTP and LTD at hippocampal CA1 glutamatergic synapses (Collingridge et al, 2004). In experimental models, LTP and LTD can be, respectively, induced by high-frequency stimulation (HFS) and low-frequency stimulation (LFS) via activation of the N-methyl-D-aspartic acid (NMDA) subtype ionotropic glutamate receptor (NMDAR). However, how HFS and LFS activate NMDARs and thereby lead to synaptic plasticity remains poorly understood and highly controversial. It is even more unclear how the bidirectional synaptic plasticity is produced and regulated in response to physiological or pathological changes.Functional NMDARs consist primarily of two GluN1 subunits and two GluN2 subunits, with GluN2A and GluN2B subunits being the most common NMDAR subunits found in the cortical and hippocampal regions of the adult brain (Cull-Candy et al, 2001). GluN2A and GluN2B subunits may confer distinct gating and pharmacological properties to NMDARs and couple them to distinct intracellular signalling machineries (Cull-Candy et al, 2001). Moreover, the ratio of these two subpopulations of NMDARs at the glutamatergic synapse is dynamically regulated in an activity-dependent manner (Bellone and Nicoll, 2007; Cho et al, 2009; Xu et al, 2009). Although controversial, GluN2A- and GluN2B-containing NMDARs have been suggested to have differential roles in regulating the direction of synaptic plasticity (Collingridge et al, 2004; Morishita et al, 2007). Among the factors shown to regulate NMDAR function, Src family tyrosine kinases may be the best characterized, with both Src and Fyn able to upregulate NMDAR function, and thus LTP induction (Salter and Kalia, 2004). However, if these kinases modulate NMDAR function in a NMDAR subunit-specific manner remains unknown. To explore this concept, Yang et al (2012) investigated the potential subunit-specific regulation of NMDARs by Src and Fyn using whole-cell patch clamp recording of NMDAR-mediated currents from acutely dissociated CA1 hippocampal neurons or from rat hippocampal slices. They found that intracellular perfusion of recombinant Src or Fyn increased the NMDAR-mediated currents. By applying subunit-preferential antagonists of GluN2A- or GluN2B-containing NMDARs, or by using neurons obtained from GluN2A knockout mice, they discovered that Src and Fyn differentially enhanced currents gated through GluN2A- and GluN2B-containing NMDARs, respectively.Can physiological or pathological factors differentially activate Src or Fyn, thereby exerting subunit-specific regulation of NMDAR function? To answer this question, Yang et al focused their investigation on the role of GPCRs, specifically pituitary adenylate cyclase activating peptide receptor (PAC1R) and dopamine D1 receptor (D1R), both of which have recently been shown to potentiate NMDARs through Src family kinases (Macdonald et al, 2005; Hu et al, 2010). Indeed, they found that activation of PAC1R specifically increased GluN2A-NMDAR-mediated currents without affecting currents gated through GluN2B-NMDARs, and this potentiation was prevented by the Src-specific inhibitory peptide Src(40–58) (Salter and Kalia, 2004). To rule out the contribution of Fyn, the authors developed a novel-specific Fyn inhibitory peptide Fyn(39–57), and demonstrated that it had little effect on PAC1R potentiation. In contrast, activation of D1R potentiated GluN2B- (but not GluN2A-) NMDAR-mediated currents, and this potentiation was specifically eliminated by Fyn(39–57), but not by Src(40–58). The authors further demonstrated that stimulation of PAC1Rs resulted in a selective activation of Src kinase and consequent tyrosine phosphorylation of the GluN2A subunit, whereas activation of D1Rs led to a specific increase in Fyn-mediated tyrosine phosphorylation of the GluN2B subunit. To provide convincing evidence that these subunit-differential modulations are indeed the result of tyrosine phosphorylation of the respective NMDAR subunits, the authors then performed electrophysiological experiments using neurons from two knockin mouse lines GluN2A(Y1325F) and GluN2B(Y1472F), in which the tyrosine phosphorylation residues in native GluN2A and GluN2B subunits were, respectively, replaced with non-phosphorylatable phenylalanine residues. As expected, the authors found that PAC1R-mediated potentiation of NMDA currents was lost in neurons from GluN2A(Y1325F) mice (but maintained in neurons from GluN2B(Y1472F) mice), while D1R-mediated enhancement of NMDA currents was only observed in neurons from GluN2A(Y1325F) mice. Together, as illustrated in Figure 1, the authors have made a very convincing case that PAC1R and D1R, respectively, enhance function of GluN2A- and GluN2B-containing NMDARs by differentially activating Src- and Fyn-mediated phosphorylation of respective NMDAR subunits.Open in a separate windowFigure 1GPCRs regulate the direction of synaptic plasticity via activating distinct signalling pathways. Synaptic NMDA receptors, both GluN2A- and GluN2B-containing, play key roles in the induction of various forms of synaptic plasticity at the hippocampal CA1 glutamatergic synapse. Under the basal level of GluN2A and GluN2B ratio, stimulation with a train of pulses at frequencies from 1 to 100 Hz produces a frequency and plasticity (LTD–LTP) curve, with maximum LTD and LTP being, respectively, induced at 1 and 100 Hz. Activation of PAC1R with its agonist PACAP38 activates Src and thereby results in tyrosine phosphorylation and consequent functional upregulation of GluN2A-containing NMDARs, resulting in an increase in the ratio of functional GluN2A and GluN2B. The increased ratio in turn causes a left shift of frequency and plasticity curve, favouring LTP induction. In contrast, activation of D1R by the receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF81297","term_id":"1156277425","term_text":"SKF81297"}}SKF81297 triggers Fyn-specific tyrosine phosphorylation and functional upregulation of GluN2B, causing a reduction of GluN2A and GluN2B ratio. This decreased ratio results in a right shift of the curve, favouring LTD induction. The ability of GPCRs to differentially activate distinct downstream signalling pathways involved in synaptic plasticity suggests the potential roles of GPCRs in governing the direction of synaptic plasticity.Given the coupling of NMDARs to the induction of synaptic plasticity, it is then reasonable to ask if activation of the two GPCRs can selectively affect the induction of LTP or LTD at CA1 synapses. Yang and colleagues investigated the effects of pharmacological activation of PAC1R and D1R on the induction of LTP and LTD by recording the field excitatory postsynaptic potentials from hippocampal slices. Consistent with differential roles of NMDAR subunits in governing directions of synaptic plasticity, the authors observed that activation of PAC1Rs reduces the induction threshold of LTP, while stimulation of D1Rs favours LTD induction (Figure 1). Facilitation of LTP by PAC1R and LTD by D1R were, respectively, prevented in the brain slices obtained from GluN2A(Y1325F) and GluN2B(Y1472F) knockin mice, supporting the differential involvements of Src-mediated GluN2A phosphorylation and Fyn-mediated GluN2B phosphorylation.Taken together, the authors'' results have demonstrated that activation of PAC1R and D1R can control the direction of synaptic plasticity at the hippocampal CA1 synapse by differentially regulating NMDAR_EPSCs in a subunit-specific fashion (Figure 1). Specifically, PAC1R enhances the function of GluN2A-containing NMDARs by increasing Src phosphorylation of GluN2A subunit at Y1325, whereas D1R upregulates GluN2B-containing NMDARs through increased Fyn phosphorylation of GluN2B at Y1472. Moreover, by regulating the ratio of functional GluN2A- and GluN2B-containing NMDARs, PAC1R and D1R in turn modulate the direction of synaptic plasticity, favouring the production of LTP and LTD, respectively.While consistent with the recently proposed hypothesis that GluN2A and GluN2B may have preferential roles in the induction of hippocampal CA1 LTP and LTD (Collingridge et al, 2004; but see also Morishita et al, 2007), the current study further emphasizes the importance of GluN2A/GluN2B ratios in regulating LTP and LTD thresholds: increased ratio favours LTP, while reduced ratio promotes LTD. However, this seems to contradict some recent studies where the reduction and increase in the GluN2A/GluN2B ratio appeared to, respectively, favour LTP (Cho et al, 2009; Xu et al, 2009) and LTD (Xu et al, 2009). Therefore, the direction of plasticity change is likely modulated not only by the GluN2A/GluN2B ratio, but also by additional factors such as experimental conditions, developmental stages, and brain regions.Under many experimental conditions, LTP and LTD are usually induced by HFS and LFS stimulating protocols, respectively, but it remains essentially unknown how LTP and LTD are physiologically or pathologically generated in animals. To this end, the identification of different GPCRs as the endogenous upstream regulators of NMDA receptor subpopulations, and hence regulators of synaptic plasticity, is the major novelty of Yang and colleagues'' work. Future studies are needed to investigate if and how PAC1R and/or D1R are critically involved in the production of LTP or LTD in animals under physiological or pathological conditions. Given the fact that Src family kinases may be required for LTP induced by HFS in hippocampal slices (Salter and Kalia, 2004), an equally intriguing question would be whether these GPCRs are actually required for LTP/LTD induced by HFS/LFS experimental paradigms. In line with this conjecture, it would be interesting to determine if ligands for various GPCRs co-exist in the glutamatergic presynaptic terminals and, if so, can be differentially co-released with glutamate in a frequency-dependent manner, thereby contributing to either HFS-induced LTP or LFS-induced LTD.The findings by Yang and colleagues establish an exciting mechanistic model by which GPCRs can govern the direction of synaptic plasticity by determining the contributions of GluN2A- and GluN2B-NMDARs through differential tyrosine phosphorylation of respective NMDA receptor subtypes. Additional studies further validating this model under physiological and pathological conditions will greatly improve our understanding of the molecular mechanisms underlying synaptic plasticity and cognitive brain functions. In addition, NMDARs, depending on their subunit composition and/or subcellular localization, may also have complex roles in mediating neuronal survival and death (Lai et al, 2011). Considering that neurotoxicity produced by over-activation of NMDARs is widely accepted to be a common mechanism for neuronal loss in a number of acute brain injuries and chronic neurodegenerative diseases, Yang and colleagues'' finding of the differential regulation of NMDAR subunits by different GPCRs could have wider implications beyond synaptic plasticity.     

G protein-coupled receptors control NMDARs and metaplasticity in the hippocampus

Long-term potentiation (LTP) and long-term depression (LTD) are the major forms of functional synaptic plasticity observed at CA1 synapses of the hippocampus. The balance between LTP and LTD or "metaplasticity" is controlled by G-protein coupled receptors (GPCRs) whose signal pathways target the N-methyl-D-asparate (NMDA) subtype of excitatory glutamate receptor. We discuss the protein kinase signal cascades stimulated by Galphaq and Galphas coupled GPCRs and describe how control of NMDAR activity shifts the threshold for the induction of LTP.     

Microtubule regulation of N-methyl-D-aspartate receptor channels in neurons

N-Methyl-D-aspartate (NMDA) receptors (NMDARs), which play a key role in synaptic plasticity, are dynamically regulated by many signaling molecules and scaffolding proteins. Although actin cytoskeleton has been implicated in regulating NMDAR stability in synaptic membrane, the role of microtubules in regulating NMDAR trafficking and function is largely unclear. Here we show that microtubule-depolymerizing agents inhibited NMDA receptor-mediated ionic and synaptic currents in cortical pyramidal neurons. This effect was Ca(2+)-independent, required GTP, and was more prominent in the presence of high NMDA concentrations. The NR2B subunit-containing NMDA receptor was the primary target of microtubules. The effect of microtubule depolymerizers on NMDAR currents was blocked by cellular knockdown of the kinesin motor protein KIF17, which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Neuromodulators that can stabilize microtubules, such as brain-derived neurotrophic factor, significantly attenuated the microtubule depolymerizer-induced reduction of NMDAR currents. Moreover, immunocytochemical studies show that microtubule depolymerizers decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Taken together, these results suggest that interfering with microtubule assembly suppresses NMDAR function through a mechanism dependent on kinesin-based dendritic transport of NMDA receptors.     

Synaptic plasticity of NMDA receptors: mechanisms and functional implications

Beyond their well-established role as triggers for LTP and LTD of fast synaptic transmission mediated by AMPA receptors, an expanding body of evidence indicates that NMDA receptors (NMDARs) themselves are also dynamically regulated and subject to activity-dependent long-term plasticity. NMDARs can significantly contribute to information transfer at synapses particularly during periods of repetitive activity. It is also increasingly recognized that NMDARs participate in dendritic synaptic integration and are critical for generating persistent activity of neural assemblies. Here we review recent advances on the mechanisms and functional consequences of NMDAR plasticity. Given the unique biophysical properties of NMDARs, synaptic plasticity of NMDAR-mediated transmission emerges as a particularly powerful mechanism for the fine tuning of information encoding and storage throughout the brain.     

G protein-coupled receptors control NMDARs and metaplasticity in the hippocampus

Long-term potentiation (LTP) and long-term depression (LTD) are the major forms of functional synaptic plasticity observed at CA1 synapses of the hippocampus. The balance between LTP and LTD or “metaplasticity” is controlled by G-protein coupled receptors (GPCRs) whose signal pathways target the N-methyl-D-asparate (NMDA) subtype of excitatory glutamate receptor. We discuss the protein kinase signal cascades stimulated by Gαq and Gαs coupled GPCRs and describe how control of NMDAR activity shifts the threshold for the induction of LTP.     

Long-term potentiation in cultured hippocampal neurons

Studies performed on low-density primary neuronal cultures have enabled dissection of molecular and cellular changes during N-methyl-D-aspartate (NMDA) receptor-dependent long-term potentiation (LTP). Various electrophysiological and chemical induction protocols were developed for the persistent enhancement of excitatory synaptic transmission in hippocampal neuronal cultures. The characterisation of these plasticity models confirmed that they share many key properties with the LTP of CA1 neurons, extensively studied in hippocampal slices using electrophysiological techniques. For example, LTP in dissociated hippocampal neuronal cultures is also dependent on Ca(2+) influx through post-synaptic NMDA receptors, subsequent activation and autophosphorylation of the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and an increase in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor insertion at the post-synaptic membrane. The availability of models of LTP in cultured hippocampal neurons significantly facilitated the monitoring of changes in endogenous postsynaptic receptor proteins and the investigation of the associated signalling mechanisms that underlie LTP. A central feature of LTP of excitatory synapses is the recruitment of AMPA receptors at the postsynaptic site. Results from the use of cell culture-based models started to establish the mechanism by which synaptic input controls a neuron's ability to modify its synapses in LTP. This review focuses on key features of various LTP induction protocols in dissociated hippocampal neuronal cultures and the applications of these plasticity models for the investigation of activity-induced changes in native AMPA receptors.     

CaMKII‐dependent phosphorylation of GluK5 mediates plasticity of kainate receptors

Calmodulin‐dependent kinase II (CaMKII) is key for long‐term potentiation of synaptic AMPA receptors. Whether CaMKII is involved in activity‐dependent plasticity of other ionotropic glutamate receptors is unknown. We show that repeated pairing of pre‐ and postsynaptic stimulation at hippocampal mossy fibre synapses induces long‐term depression of kainate receptor (KAR)‐mediated responses, which depends on Ca²⁺ influx, activation of CaMKII, and on the GluK5 subunit of KARs. CaMKII phosphorylation of three residues in the C‐terminal domain of GluK5 subunit markedly increases lateral mobility of KARs, possibly by decreasing the binding of GluK5 to PSD‐95. CaMKII activation also promotes surface expression of KARs at extrasynaptic sites, but concomitantly decreases its synaptic content. Using a molecular replacement strategy, we demonstrate that the direct phosphorylation of GluK5 by CaMKII is necessary for KAR‐LTD. We propose that CaMKII‐dependent phosphorylation of GluK5 is responsible for synaptic depression by untrapping of KARs from the PSD and increased diffusion away from synaptic sites.