Estradiol‐induced increase in novel object recognition requires hippocampal NR2B‐containing NMDA receptors

17β‐estradiol (E2), at high circulating levels, enhances learning and memory in many women, making it a clinical treatment for hormone‐related cognitive decline in aging. However, the mechanisms stimulated by E2, which are responsible for its cognitive enhancing effects, remain incompletely defined. Using an ovariectomized rat model, we previously reported that increasing plasma E2 enhances the magnitude of long‐term potentiation (LTP) at hippocampal CA3‐CA1 synapses, which is caused by a selective increase in current mediated by NR2B‐containing NMDARs, leading to an increase in the NMDAR/AMPAR ratio. Whether the increase in NR2B current is causally related to the ability of E2 to enhance hippocampal dependent learning and memory has yet to be tested. Here, we find that E2 enhances performance in the novel object recognition (NOR) task with the same time course we previously showed E2 enhances the LTP magnitude, temporally linking the increase in LTP to enhanced learning and memory. Furthermore, using the selective NR2B subunit antagonist Ro25‐6981, we find that the E2‐enhanced NOR, like the enhanced LTP, requires hippocampal NR2B‐containing NMDARs, specifically in area CA1. Finally, using whole‐cell recordings and the phosphatase inhibitor orthovanadate, we investigated whether the E2‐induced increase in NMDAR current is caused by an increase in the density of synaptic NMDARs and/or an increase in NMDAR subunit phosphorylation. We find that both mechanisms are responsible for the enhanced NMDAR current in E2‐treated rats. Our results show that the E2‐enhanced NOR requires a functional increase in NR2B‐containing NMDARs, a requirement shared with the E2‐enhanced LTP magnitude at CA3‐CA1 synapses, supporting the hypothesis that the increase in LTP likely contributes to the enhanced learning and memory following an increase in plasma E2 levels. © 2012 Wiley Periodicals, Inc.     

The glutamate receptor GluN2 subunit regulates synaptic trafficking of AMPA receptors in the neonatal mouse brain

The N‐methyl‐d ‐aspartate receptor (NMDAR) plays various physiological and pathological roles in neural development, synaptic plasticity and neuronal cell death. It is composed of two GluN1 and two GluN2 subunits and, in the neonatal hippocampus, most synaptic NMDARs are GluN2B‐containing receptors, which are gradually replaced with GluN2A‐containing receptors during development. Here, we examined whether GluN2A could be substituted for GluN2B in neural development and functions by analysing knock‐in (KI) mice in which GluN2B is replaced with GluN2A. The KI mutation was neonatally lethal, although GluN2A‐containing receptors were transported to the postsynaptic membrane even without GluN2B and functional at synapses of acute hippocampal slices of postnatal day 0, indicating that GluN2A‐containing NMDARs could not be substituted for GluN2B‐containing NMDARs. Importantly, the synaptic α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor (AMPAR) subunit GluA1 was increased, and the transmembrane AMPAR regulatory protein, which is involved in AMPAR synaptic trafficking, was increased in KI mice. Although the regulation of AMPARs by GluN2B has been reported in cultured neurons, we showed here that AMPAR‐mediated synaptic responses were increased in acute KI slices, suggesting differential roles of GluN2A and GluN2B in AMPAR expression and trafficking in vivo. Taken together, our results suggest that GluN2B is essential for the survival of animals, and that the GluN2B–GluN2A switching plays a critical role in synaptic integration of AMPARs through regulation of GluA1 in the whole animal.     

Estradiol potentiation of NR2B‐dependent EPSCs is not due to changes in NR2B protein expression or phosphorylation

The hormone, 17β‐estradiol (E2), influences the structure and function of synapses in the CA1 region of the hippocampus. E2 increases the density of dendritic spines and excitatory synapses on CA1 pyramidal cells, increases CA1 cells' sensitivity to excitatory synaptic input mediated by the NMDA receptor (NMDAR), enhances NMDAR‐dependent long‐term potentiation, and improves hippocampus‐dependent working memory. Smith and McMahon ( 2006 J Neurosci 26:8517–8522) reported that the larger NMDAR‐mediated excitatory postsynaptic currents (EPSCs) recorded after E2 treatment are due primarily to an increased contribution of NR2B‐containing NMDARs. We used a combination of electrophysiology, Western blot, and immunofluorescence to investigate two potential mechanisms by which E2 could enhance NR2B‐dependent EPSCs: An increase in NMDAR subunit protein levels and/or a change(s) in NR2B phosphorylation. Our studies confirmed the E2‐induced increase in NR2B‐dependent EPSC amplitude, but we found no evidence that E2 affects protein levels for the NR1, NR2A, or NR2B subunit of the NMDAR, nor that E2 affects phosphorylation of NR2B. Our findings suggest that the effects of E2 on NMDAR‐dependent synaptic physiology in the hippocampus likely result from recruitment of NR2B‐containing NMDARs to synapses rather than from increased expression of NMDARs or changes in their phosphorylation state. © 2010 Wiley‐Liss, Inc.     

Immunogold electron microscopic evidence of differential regulation of GluN1, GluN2A,and GluN2B,NMDA‐type glutamate receptor subunits in rat hippocampal CA1 synapses during benzodiazepine withdrawal

Benzodiazepine withdrawal‐anxiety is associated with enhanced α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor (AMPAR)‐mediated glutamatergic transmission in rat hippocampal CA1 synapses due to enhanced synaptic insertion and phosphorylation of GluA1 homomers. Interestingly, attenuation of withdrawal‐anxiety is associated with a reduction in N‐methyl‐D‐aspartate receptor (NMDAR)‐mediated currents and subunit expression, secondary to AMPA receptor potentiation. Therefore, in this study ultrastructural evidence for possible reductions in NMDAR GluN1, GluN2A, and GluN2B subunits was sought at CA1 stratum radiatum synapses in proximal dendrites using postembedding immunogold labeling of tissues from rats withdrawn for 2 days from 1‐week daily oral administration of the benzodiazepine, flurazepam (FZP). GluN1‐immunogold density and the percentage of immunopositive synapses were significantly decreased in tissues from FZP‐withdrawn rats. Similar decreases were observed for GluN2B subunits; however, the relative lateral distribution of GluN2B‐immunolabeling within the postsynaptic density did not change after BZ withdrawal. In contrast to the GluN2B subunit, the percentage of synapses labeled with the GluN2A subunit antibody and the density of immunogold labeling for this subunit was unchanged. The spatial localization of immunogold particles associated with each NMDAR subunit was consistent with a predominantly postsynaptic localization. The data therefore provide direct evidence for reduced synaptic GluN1/GluN2B receptors and preservation of GluN1/GluN2A receptors in the CA1 stratum radiatum region during BZ withdrawal. Based on collective findings in this benzodiazepine withdrawal‐anxiety model, we propose a functional model illustrating the changes in glutamate receptor populations at excitatory synapses during benzodiazepine withdrawal. J. Comp. Neurol. 518:4311–4328, 2010. © 2010 Wiley‐Liss, Inc.     

Turning off of GluN2B subunits and turning on of CICR in hippocampal LTD induction after developmental GluN2 subunit switch

NMDA receptors (NMDARs) are essential for the induction of synaptic plasticity that mediates activity‐dependent refinement of neural circuits during development. GluN2B subunits of NMDARs are abundant at synapses in the immature hippocampus and begin to be replaced by GluN2A subunits with the help of casein kinase 2 activity in the second postnatal week, the critical period for the GluN2 subunit switch (Sanz‐Clemente et al. (2000) Neuron 67:984–996). However, the physiological role of GluN2B subunits in the hippocampus during this critical period has not been elucidated. Here, we report that GluN2B subunits mediate the induction of long‐term depression (LTD) in the CA1 region of the hippocampus only until this period. Ifenprodil and Ro25‐6981, selective inhibitors of NMDARs containing GluN2B subunits, blocked LTD in postnatal Day 11–14 (P11–14) rat hippocampal slices but not in P18–22 hippocampus. Just a few days after P14, synaptic NMDAR currents became narrower than those at P11–14, and calcium influx through NMDARs must be reduced. We found that calcium‐induced calcium release (CICR) through ryanodine receptors starts to support the induction of NMDAR‐dependent LTD at P18–22. Intracellular application of thapsigargin and ryanodine, inhibitors of Ca²⁺‐ATP pumps on internal stores and ryanodine receptors, respectively, did not at all affect LTD in the hippocampus at P11–14 but completely blocked LTD in the P18–22 hippocampus. Therefore, calcium influx through NMDAR with GluN2B subunits is sufficient to induce LTD at P11–14, after which CICR compensates for the decrease in calcium influx during LTD induction. © 2015 Wiley Periodicals, Inc.     

GluN2B‐containing N‐methyl‐D‐aspartate receptors compensate for the inhibitory control of synaptic plasticity during the early critical period in the rat visual cortex

In the visual cortex, synaptic plasticity is very high during the early developmental stage known as the critical period and declines with development after the critical period. Changes in the properties of N‐methyl‐D‐aspartate receptor (NMDAR) and γ‐aminobutyric acid type A receptor (GABA_AR) have been suggested to underlie the changes in the characteristics of plasticity. However, it is largely unknown how the changes in the two receptors interact to regulate synaptic plasticity. The present study investigates the changes in the properties of NMDAR and GABA_AR from 3 to 5 weeks of age in layer 2/3 pyramidal neurons of the rat visual cortex. The impact of these changes on the characteristics of long‐term potentiation (LTP) is also investigated. The amplitude and decay time constant of GABA_AR‐mediated currents increased during this period. However, the decay time constant of NMDAR‐mediated currents decreased as a result of the decrease in the proportion of the GluN2B subunit‐mediated component. Induction of NMDAR‐dependent LTP at 3 weeks depended on the GluN2B subunit, but LTP at 5 weeks did not. Enhancement of GABA_AR‐mediated inhibition suppressed the induction of LTP only at 5 weeks. However, partial inhibition of the GluN2B subunit with a low concentration of ifenprodil allowed the GABA_AR‐mediated suppression of LTP at 3 weeks. These results suggest that changes in the properties of NMDAR‐ and GABA_AR‐mediated synaptic transmission interact to determine the characteristics of synaptic plasticity during the critical period in the visual cortex. © 2015 Wiley Periodicals, Inc.     

GluN3-Containing NMDA Receptors in the Rat Nucleus Accumbens Core Contribute to Incubation of Cocaine Craving

Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca²⁺-permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (∼1 month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg²⁺ block and Ca²⁺ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca²⁺ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain.SIGNIFICANCE STATEMENT “Incubation of craving” is an animal model for the persistence of vulnerability to cue-induced relapse after prolonged drug abstinence. Incubation also occurs in human drug users. AMPAR plasticity in medium spiny neurons (MSNs) of the NAc core is critical for incubation of cocaine craving but occurs only after a delay. Here we found that AMPAR plasticity is preceded by NMDAR plasticity that is essential for incubation and involves GluN3, an atypical NMDAR subunit that markedly alters NMDAR transmission. Together with AMPAR plasticity, this represents profound remodeling of excitatory synaptic transmission onto MSNs. Given the importance of MSNs for translating motivation into action, this plasticity may explain, at least in part, the profound shifts in motivated behavior that characterize addiction.     

Target‐ and input‐dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus

We examined the synaptic structure, quantity, and distribution of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐ and N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptors (AMPARs and NMDARs, respectively) in rat cochlear nuclei by a highly sensitive freeze‐fracture replica labeling technique. Four excitatory synapses formed by two distinct inputs, auditory nerve (AN) and parallel fibers (PF), on different cell types were analyzed. These excitatory synapse types included AN synapses on bushy cells (AN‐BC synapses) and fusiform cells (AN‐FC synapses) and PF synapses on FC (PF‐FC synapses) and cartwheel cell spines (PF‐CwC synapses). Immunogold labeling revealed differences in synaptic structure as well as AMPAR and NMDAR number and/or density in both AN and PF synapses, indicating a target‐dependent organization. The immunogold receptor labeling also identified differences in the synaptic organization of FCs based on AN or PF connections, indicating an input‐dependent organization in FCs. Among the four excitatory synapse types, the AN‐BC synapses were the smallest and had the most densely packed intramembrane particles (IMPs), whereas the PF‐CwC synapses were the largest and had sparsely packed IMPs. All four synapse types showed positive correlations between the IMP‐cluster area and the AMPAR number, indicating a common intrasynapse‐type relationship for glutamatergic synapses. Immunogold particles for AMPARs were distributed over the entire area of individual AN synapses; PF synapses often showed synaptic areas devoid of labeling. The gold‐labeling for NMDARs occurred in a mosaic fashion, with less positive correlations between the IMP‐cluster area and the NMDAR number. Our observations reveal target‐ and input‐dependent features in the structure, number, and organization of AMPARs and NMDARs in AN and PF synapses. J. Comp. Neurol. 522:4023–4042, 2014. © 2014 Wiley Periodicals, Inc.     

Novel nootropic drug sunifiram enhances hippocampal synaptic efficacy via glycine‐binding site of N‐methyl‐D‐aspartate receptor

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N‐methyl‐D ‐aspartate receptor (NMDAR)‐dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR‐dependent long‐term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10–100 nM significantly enhanced LTP in a bell‐shaped dose‐response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7‐chloro‐kynurenic acid (7‐ClKN), an antagonist for glycine‐binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α‐amino‐3‐hydroxy‐5‐methylisozazole‐4‐propionate receptor (AMPAR) through activation of calcium/calmodulin‐dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1–1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose‐dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser‐657) and Src family (Tyr‐416) activities with the same bell‐shaped dose‐response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser‐657) and Src (Tyr‐416) induced by sunifiram was inhibited by 7‐ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine‐binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation. © 2013 Wiley Periodicals, Inc.     

Synaptic non-GluN2B-containing NMDA receptors regulate tyrosine phosphorylation of GluN2B 1472 tyrosine site in rat brain slices

Activation of N-methyl-D-aspartate receptors（NMDARs）mediates changes in the phosphorylation status of the glutamate receptors themselves.Previous studies have indicated that during synaptic activity,tyrosine kinases（Src and Fyn）or phosphatases（PTPαand STEP）are involved in regulating the phosphorylation of NMDARs.In this study,we used immunoblotting to investigate the role of an NMDAR subpopulation on the phosphorylation level of the GluN2B subunit at the Y1336 and Y1472sites in rat brain slices after NMDA treatment.We found that NMDA stimulation dramatically decreased the phosphorylation level of GluN2B at Y1472 in a dose-and time-dependent manner,but not at Y1336.Extrasynaptic NMDAR activation did not reduce the phosphorylation of GluN2B at Y1472.In addition,ifenprodil,a selective antagonist of GluN2Bcontaining NMDARs,did not abolish the decreased phosphorylation of GluN2B at Y1472 triggered by NMDA.These results suggest that the activation of synaptic GluN2A-containing NMDARs is required for the decreased phosphorylation of GluN2B at Y1472that is induced by NMDA treatment in rat brain slices.     

Distinct trafficking and expression mechanisms underlie LTP and LTD of NMDA receptor‐mediated synaptic responses

Although an increasing number of studies have demonstrated the plasticity of NMDA receptor‐mediated synaptic transmission, little is known about the molecular mechanisms that underlie this neurologically important process. In a study of NMDAR‐mediated synaptic responses in hippocampal Schaffer‐CA1 synapses whose AMPA receptor (AMPAR) activity is totally blocked, we uncovered differences between the trafficking mechanisms that underlie the long‐term potentiation (LTP) and long‐term depression (LTD) that can be induced in these cells under these conditions. The LTP‐producing protocol failed to induce a change in the amplitude of NMDAR‐mediated postsynaptic currents (NMDAR EPSCs) in the first 5–10 min, but induced gradual enhancement of NMDAR EPSCs thereafter that soon reached a stable magnitude. This “slow” LTP of NMDAR EPSCs (LTP_NMDA) was blocked by inhibiting exocytosis or actin polymerization in postsynaptic cells. By contrast, LTD of NMDAR EPSCs (LTD_NMDA) was immediately inducible, and, although it was blocked by the actin stabilizer, it was unaffected by exocytosis or endocytosis inhibitors. Furthermore, concomitant changes in the decay time of NMDAR EPSCs suggested that differential switches in NR2 subunit composition accompanied LTP_NMDA and LTD_NMDA, and these changes were blocked by the calcium buffer BAPTA or an mGluR antagonist. Our results suggest that LTP_NMDA and LTD_NMDA utilize different NMDAR trafficking pathways and express different ratios of NMDAR subunits on the postsynaptic surface. © 2009 Wiley‐Liss, Inc.     

NMDA Receptor GluN2 Subtypes Control Epileptiform Events in the Hippocampus

NMDA receptors (NMDARs) play a key role in synaptic plasticity and excitotoxicity. Subtype-specific role of NMDAR in neural disorders is an emerging area. Recent studies have revealed that mutations in NMDARs are a cause for epilepsy. Hippocampus is a known focal point for epilepsy. In hippocampus, expression of the NMDAR subtypes GluN1/GluN2A and GluN1/GluN2B is temporally regulated. However, the pharmacological significance of these subtypes is not well understood in epileptic context/models. To investigate this, epilepsy was induced in hippocampal slices by the application of artificial cerebrospinal fluid that contained high potassium but no magnesium. Epileptiform events (EFEs) were recorded from the CA1 and DG areas of hippocampus with or without subtype-specific antagonists. Irrespective of the age group, CA1 and DG showed epileptiform activity. The NMDAR antagonist AP5 was found to reduce the number of EFEs significantly. However, the application of subtype-specific antagonists (TCN 201 for GluN1/GluN2A and Ro 25-69811 for GluN1/GluN2B) revealed that EFEs had area-specific and temporal components. In slices from neonates, EFEs in CA1 were effectively reduced by Ro 25-69811, but were largely insensitive to TCN 201. In contrast, EFEs in DG were equally sensitive to both of the subtype-specific antagonists. However, the differential sensitivity for the antagonists observed in neonates was absent in later developmental stages. The study provides a functional insight into the NMDAR subtype-dependent contribution of EFEs in hippocampus of young rats, which may have implications in treating childhood epilepsy and avoiding unnecessary side effects of broad spectrum antagonists.     

Transient appearance of Ca2+‐permeable AMPA receptors is crucial for the production of repetitive LTP‐induced synaptic enhancement (RISE) in cultured hippocampal slices

We have previously shown that repetitive induction of long‐term potentiation (LTP) by glutamate (100 μM, 3 min, three times at 24‐hr intervals) provoked long‐lasting synaptic enhancement accompanied by synaptogenesis in rat hippocampal slice cultures, a phenomenon termed RISE (repetitive LTP‐induced synaptic enhancement). Here, we examined the role of Ca²⁺‐permeable (CP) AMPA receptors (AMPARs) in the establishment of RISE. We first found a component sensitive to the Joro‐spider toxin (JSTX), a blocker of CP‐AMPARs, in a field EPSP recorded from CA3‐CA1 synapses at 2–3 days after stimulation, but this component was not found for 9–10 days. We also observed that rectification of AMPAR‐mediated current appeared only 2–3 days after stimulation, using a whole‐cell patch clamp recording from CA1 pyramidal neurons. These findings indicate that CP‐AMPAR is transiently expressed in the developing phase of RISE. The blockade of CP‐AMPARs by JSTX for 24 hr at this developing phase inhibited RISE establishment, accompanied by the loss of small synapses at the ultrastructural level. These results suggest that transiently induced CP‐AMPARs play a critical role in synaptogenesis in the developing phase of long‐lasting hippocampal synaptic plasticity, RISE.     

Synthetic conantokin peptides potently inhibit N‐methyl‐D‐aspartate receptor‐mediated currents of retinal ganglion cells

Retinal ganglion cells (RGCs), which are the sole output neurons of the retina, express N‐methyl‐D‐aspartate receptors (NMDARs), rendering these cells susceptible to glutamate excitotoxicity, with implications for loss of normal RGC excitatory responses in disorders such as glaucoma and diabetic retinopathy. Therefore, antagonists that inhibit NMDAR‐mediated currents specifically by targeting the GluN2B component of the ion channel have the potential to serve as a basis for developing potential therapeutics. The roles of peptidic conantokins, which are potent brain neuronal NMDAR inhibitors, were studied. By using patch‐clamp whole‐cell analyses in dissociated RGCs and retinal whole‐mount RGCs, we evaluated the effects of synthetic conantokin‐G (conG) and conantokin‐T (conT), which are small γ‐carboxyglutamate‐containing peptides, on NMDA‐mediated excitatory responses in mouse RGCs. Both conG and conT inhibited the NMDA‐mediated currents of dark‐adapted dissociated and whole‐mount RGCs in a dose‐dependent, reversible, noncompetitive manner. Inhibition of NMDA‐mediated steady‐state currents by NMDAR nonsubunit‐selective conT was approximately threefold greater than GluN2B‐selective conG or ifenprodil, demonstrating its potential ability to inhibit both GluN2A‐ and GluN2B‐containing ion channels in RGCs. Because the extent of inhibition of NMDA‐evoked currents by conG and the pharmacologic GluN2B‐selective inhibitor ifenprodil were similar (40–45%) to that of the GluN2A‐selective antagonist NVP‐AAM0077, we conclude that the levels of GluN2A and GluN2B subunits are similar in RGCs. These results provide a novel basis for developing effective neuroprotective agents to aid in the prevention of undesired glutamatergic excitotoxicity in neurodegenerative diseases of the retina and demonstrate functional assembly of NMDARs in RGCs. © 2014 Wiley Periodicals, Inc.     

GluN2D Subunit-Containing NMDA Receptors Control Tissue Plasminogen Activator-Mediated Spatial Memory

Tissue plasminogen activator (tPA) is a serine protease with pleiotropic actions in the CNS, such as synaptic plasticity and neuronal death. Some effects of tPA require its interaction with the GluN1 subunit of the NMDA receptor (NMDAR), leading to a potentiation of NMDAR signaling. We have reported previously that the pro-neurotoxic effect of tPA is mediated through GluN2D subunit-containing NMDARs. Thus, the aim of the present study was to determine whether GluN2D subunit-containing NMDARs drive tPA-mediated cognitive functions. To address this issue, a strategy of immunization designed to prevent the in vivo interaction of tPA with NMDARs and GluN2D-deficient mice were used in a set of behavioral tasks. Altogether, our data provide the first evidence that tPA influences spatial memory through its preferential interaction with GluN2D subunit-containing NMDARs.     

Contribution of NMDA receptors to dorsolateral prefrontal cortical networks in primates

Cognitive disorders such as schizophrenia and Alzheimer’s disease are associated with dysfunction of the highly evolved dorsolateral prefrontal cortex(dl PFC), and with changes in glutamatergic N-methyl-D-aspartate receptors(NMDARs). Recent research on the primate dl PFC discovered that the pyramidal cell circuits that generate the persistent firing underlying spatial working memory communicate through synapses on spines containing NMDARs with NR2 B subunits(Glu N2B) in the post-synaptic density. This contrasts with synapses in the hippocampus and primary visual cortex, where Glu N2 B receptors are both synaptic and extrasynaptic. Blockade of Glu N2 B in the dl PFC markedly reduces the persistent firing of the Delay cells needed for neuronal representations of visual space. Cholinergic stimulation of nicotinic α7 receptors within the glutamate synapse is necessary for NMDAR actions. In contrast, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors have only subtle effects on the persistent firing of Delay cells, but contribute substantially to the firing of Cue and Response cells. Systemic administration of the NMDAR antagonist ketamine reduces the persistent firing of Delay cells, but increases the firing of some Response cells. The reduction in persistent firing produced by ketamine may explain why this drug can mimic or worsen the cognitive symptoms of schizophrenia. Similar actions in the medial PFC circuits representing the emotional aspects of pain may contribute to the rapid analgesic and anti-depressant actions of ketamine.     

AMPA and NMDA Receptor Trafficking at Cocaine-Generated Synapses

Cocaine experience generates AMPA receptor (AMPAR)-silent synapses in the nucleus accumbens (NAc), which are thought to be new synaptic contacts enriched in GluN2B-containing NMDA receptors (NMDARs). After drug withdrawal, some of these synapses mature by recruiting AMPARs, strengthening the newly established synaptic transmission. Silent synapse generation and maturation are two consecutive cellular steps through which NAc circuits are profoundly remodeled to promote cue-induced cocaine seeking after drug withdrawal. However, the basic cellular processes that mediate these two critical steps remains underexplored. Using a combination of electrophysiology, viral-mediated gene transfer, and confocal imaging in male rats as well as knock-in (KI) mice of both sexes, our current study characterized the dynamic roles played by AMPARs and NMDARs in generation and maturation of silent synapses on NAc medium spiny neurons after cocaine self-administration and withdrawal. We report that cocaine-induced generation of silent synapses not only required synaptic insertion of GluN2B-containing NMDARs, but also, counterintuitively, involved insertion of AMPARs, which subsequently internalized, resulting in the AMPAR-silent state on withdrawal day 1. Furthermore, GluN2B NMDARs functioned to maintain these cocaine-generated synapses in the AMPAR-silent state during drug withdrawal, until they were replaced by nonGluN2B NMDARs, a switch that allowed AMPAR recruitment and maturation of silent synapses. These results reveal dynamic interactions between AMPARs and NMDARs during the generation and maturation of silent synapses after cocaine experience and provide a mechanistic basis through which new synaptic contacts and possibly new neural network patterns created by these synapses can be manipulated for therapeutic benefit.SIGNIFICANCE STATEMENT Studies over the past decade reveal a critical role of AMPA receptor-silent, NMDA receptor-containing synapses in forming cocaine-related memories that drive cocaine relapse. However, it remains incompletely understood how AMPA and NMDA receptors traffic at these synapses during their generation and maturation. The current study characterizes a two-step AMPA receptor trafficking cascade that contributes to the generation of silent synapses in response to cocaine experience, and a two-step NMDA receptor trafficking cascade that contributes to the maturation of these synapses after cocaine withdrawal. These results depict a highly regulated cellular procedure through which nascent glutamatergic synapses are generated in the adult brain after drug experience and provide significant insight into the roles of glutamate receptors in synapse formation and maturation.     

NMDA receptor activity bidirectionally controls active decay of long‐term spatial memory in the dorsal hippocampus

The time‐dependent forgetting of long‐term spatial memories involves activation of NMDA receptors (NMDARs) in the hippocampus. Here, we tested whether NMDARs regulate memory persistence bidirectionally, decreasing or increasing the rate of forgetting. We found that blocking NMDAR activation with AP5 or the GluN2B‐selective antagonist Ro25‐6981 in the dorsal hippocampus (dHPC) prevented the natural forgetting of long‐term memory for the locations of objects in an open field arena. In contrast, while enhancing NMDAR function with the partial agonist D‐Cycloserine did not affect the speed of forgetting for these types of memories, infusing the NMDAR co‐agonist D‐Serine significantly shortened their persistence. These results suggest that NMDAR activity can modulate the speed of constitutive long‐term memory decay in the dHPC and that regulating NMDAR expression and D‐Serine availability could provide a mechanism to control the duration of long‐term memory.     

Protein Kinase C-Mediated Phosphorylation and α2δ-1 Interdependently Regulate NMDA Receptor Trafficking and Activity

N-methyl-d-aspartate receptors (NMDARs) are important for synaptic plasticity associated with many physiological functions and neurologic disorders. Protein kinase C (PKC) activation increases the phosphorylation and activity of NMDARs, and α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-1 coexpression significantly increased NMDAR activity in HEK293 cells transfected with GluN1/GluN2A or GluN1/GluN2B. PKC activation with phorbol 12-myristate 13-acetate (PMA) increased receptor activity only in cells coexpressing GluN1/GluN2A and α2δ-1. Remarkably, PKC inhibition with Gӧ6983 abolished α2δ-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/GluN2A or GluN1/GluN2B. Treatment with PMA increased the α2δ-1–GluN1 interaction and promoted α2δ-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of α2δ-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in α2δ-1 knockout mice. Furthermore, inhibiting α2δ-1 with pregabalin or disrupting the α2δ-1–NMDAR interaction with the α2δ-1 C-terminus peptide abolished the potentiating effect of PMA on NMDAR activity. Additionally, using quantitative phosphoproteomics and mutagenesis analyses, we identified S929 on GluN2A and S1413 (S1415 in humans) on GluN2B as the phosphorylation sites responsible for NMDAR potentiation by PKC and α2δ-1. Together, our findings demonstrate the interdependence of α2δ-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic α2δ-1–NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity.SIGNIFICANCE STATEMENT A major challenge in studies of protein phosphorylation is to define the functional significance of each phosphorylation event and determine how various signaling pathways are coordinated in response to neuronal activity to shape synaptic plasticity. PKC phosphorylates transporters, ion channels, and G-protein-coupled receptors in signal transduction. In this study, we showed that α2δ-1 is indispensable for PKC-activation-induced surface and synaptic trafficking of NMDARs, whereas the α2δ-1–NMDAR interaction is controlled by PKC-induced phosphorylation. Our findings reveal that α2δ-1 mainly functions as a phospho-binding protein in the control of NMDAR trafficking and activity. This information provides new mechanistic insight into the reciprocal roles of PKC-mediated phosphorylation and α2δ-1 in regulating NMDARs and in the therapeutic actions of gabapentinoids.     

Regulation of AMPA receptor recruitment at developing synapses

Fast synaptic current at most excitatory synapses in the brain is carried by AMPA and NMDA subtypes of ionotropic glutamate receptors (AMPARs and NMDARs). During development there is an increase in the ratio of AMPAR- to NMDAR-mediated current at these synapses. Recent studies indicate that NMDAR signaling early in development negatively regulates AMPAR expression and function at multiple levels, which likely accounts for the small AMPAR current at developing synapses. This contrasts with the positive role of NMDAR signaling in recruiting AMPARs to synapses during long-term potentiation in the adult brain. Thus, NMDARs exert differential effects on the recruitment of AMPA receptors to synapses depending on the developmental state of the neural circuit.