NMDA-evoked Currents Research Articles

Inhibition of the NMDA Currents by Probenecid in Amygdaloid Kindling Epilepsy Model.

Epilepsy is characterized by a sustained depolarization and repeated discharge of neurons, attributed to overstimulation of N-methyl-D-aspartate receptors (NMDAr). Herein, we propose that probenecid (PROB), an inhibitor of the activity of some ATP binding-cassette transporters (ABC-transporters) can modify NMDAr activity and expression in amygdaloid kindled model. Some studies have suggested that NMDAr expression could be regulated by inhibiting the activity of P-glycoprotein (MDR1) and drug resistance protein-1 (MRP1). Besides, PROB was found to interact with other proteins with proven activity in the kindling model, such as TRPV2 channels, OAT1, and Panx1. Administering PROB at two doses (100 and 300mg/kg/d) for 5 d decreased after-discharge duration and Racine behavioral scores. It also reduced the expression of NR2B and the activity of total NOS and the expression of nNOS with respect to the kindling group. In a second protocol, voltage-clamp measurements of NMDA-evoked currents were performed in CA1 hippocampal cells dissociated from control and kindled rats. PROB produced a dose-dependent reduction in NMDA-evoked currents. In neurons from kindled rats, a residual NMDA-evoked current was registered with respect to control animals, while a reduction in NMDA-evoked currents was observed in the presence of 20mM PROB. Finally, we evaluated the expression of MRP1 and MDR1 in order to establish a relationship between the reduction of kindling parameters, the inhibition of NMDA-type currents, and the expression of these transporters. Based on our results, we conclude that at the concentrations used, PROB inhibits currents evoked by NMDA in dissociated neurons of control and kindled rats. In the kindling model, at the tested doses, PROB decreases the after-discharge duration and Racine behavioral score in the kindling model. We propose a mechanism that could be dependent on the expression of ABC-type transporters.

Changes in NMDA Receptor Function in Rapid Ischemic Tolerance: A Potential Role for Tri-Heteromeric NMDA Receptors.

In this study, we characterize biophysical changes in NMDA receptor function in response to brief non-injurious ischemic stress (ischemic preconditioning). Electrophysiological studies show NMDA receptor function is reduced following preconditioning in cultured rat cortical neurons. This functional change is not due to changes in the reversal potential of the receptor, but an increase in desensitization. We performed concentration–response analysis of NMDA-evoked currents, and demonstrate that preconditioned neurons show a reduced potency of NMDA to evoke currents, an increase in Mg2+ sensitivity, but no change in glycine sensitivity. Antagonists studies show a reduced inhibition of GluN2B antagonists that have an allosteric mode of action (ifenprodil and R-25-6981), but competitive antagonists at the GluR2A and 2B receptor (NVP-AMM077 and conantokin-G) appear to have similar potency to block currents. Biochemical studies show a reduction in membrane surface GluN2B subunits, and an increased co-immunoprecipitation of GluN2A with GluN2B subunits, suggestive of tri-heteromeric receptor formation. Finally, we show that blocking actin remodeling with jasplakinolide, a mechanism of rapid ischemic tolerance, prevents NMDA receptor functional changes and co-immunoprecipitation of GluN2A and 2B subunits. Together, this study shows that alterations in NMDA receptor function following preconditioning ischemia are associated with neuroprotection in rapid ischemic tolerance.

Purinergic P2 and glutamate NMDA receptor coupling contributes to osmotically driven excitability in hypothalamic magnocellular neurosecretory neurons.

Purinergic and glutamatergic signalling pathways play a key role in regulating the activity of hypothalamic magnocellular neurosecretory neurons (MNNs). However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Here, we report that ATP acts on purinergic P2 receptors in MNNs to potentiate in a Ca2+ -dependent manner extrasynaptic NMDAR function. The P2-NMDAR coupling is engaged in response to an acute hyperosmotic stimulation, contributing to osmotically driven firing activity in MNNs. These results help us to better understand the precise mechanisms contributing to the osmotic regulation of firing activity and hormone release from MNNs. The firing activity of hypothalamic magnocellular neurosecretory neurons (MNNs) located in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) is coordinated by the combined, fine-tuned action of intrinsic membrane properties, synaptic and extrasynaptic signalling. Among these, purinergic and glutamatergic signalling pathways have been shown to play a key role regulating the activity of MNNs. However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Whole-cell patch-clamp recordings obtained from MNNs showed that ATP (100μM) induced an increase in firing rate, an effect that was blocked by either 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]2-pyridinyl]azo]1,3-benzenedisulfonic acid tetrasodium salt (PPADS) (10μM) or kynurenic acid (1mm). While ATP did not affect the frequency or magnitude of glutamatergic excitatory postsynaptic currents (EPSCs), it induced an inward shift in the holding current that was prevented by PPADS or kynurenic acid treatment, suggesting that ATP enhances a tonic extrasynaptic glutamatergic excitatory current. We observed that ATP-potentiated glutamatergic receptor-mediated currents were evoked by focal application of L-glu (1mm) and NMDA (50μM), but not AMPA (50μM). ATP potentiation of NMDA-evoked currents was blocked by PPADS (10μM) and by chelation of intracellular Ca2+ with BAPTA (10mm). Finally, we report that a hyperosmotic stimulus (mannitol 1%, +55mOsm/kgH2 O) potentiated NMDA-evoked currents and increased MNN firing activity, effects that were blocked by PPADS. Taken together, our data support a functional excitatory coupling between P2 and extrasynaptic NMDA receptors in MNNs, which is engaged in response to an acute hyperosmotic stimulus.

RAGE signaling is required for AMPA receptor dysfunction in the hippocampus of hyperglycemic mice

Diabetes in humans has been associated for a long time with cognitive dysfunction. In rodent animal models, cognitive dysfunction can manifest as impaired hippocampal synaptic plasticity. Particular attention has been concentrated on the receptor for advanced glycation end products (RAGE), which is implicated in multiple diabetic complications involving the development of vascular and peripheral nerve abnormalities. In this study, we hypothesize that RAGE signaling alters glutamate receptor function and expression, impairing synaptic transmission in the hippocampus. Using preparations of hippocampal slices from male mice, we show a RAGE-dependent decrease in long-term potentiation (LTP) and an increase in paired-pulse facilitation (PPF) following streptozotocin (STZ)-induced diabetes. Consistently, in hippocampal cultures from male and female neonatal mice, high glucose caused a RAGE-dependent reduction of AMPA- but not NMDA-evoked currents, and an increase in cytosolic reactive oxygen species (ROS). Consistently, when cultures were co-treated with high glucose and the RAGE antagonist FPS-ZM1, AMPA-evoked currents were unchanged. Hippocampi from STZ-induced hyperglycemic wild type (WT) mice showed increased RAGE expression concomitant with a decrease of both expression and phosphorylation (Ser 831 and 845) of the AMPA GluA1 subunit. We found these changes correlated to activation of the MAPK pathway, consistent with decreased pJNK/JNK ratio and the JNK kinase, pMEK7. As no changes in expression or phosphorylation of regulatory proteins were observed in hippocampi from STZ-induced hyperglycemic RAGE-KO mice, we report a RAGE-dependent impairment in the hippocampi of hyperglycemic WT mice, with reduced AMPA receptor expression/function and LTP deficits.

Novel Dithiolane-Based Ligands Combining Sigma and NMDA Receptor Interactions as Potential Neuroprotective Agents.

Sigma receptors (SRs) are recognized as valuable targets for the treatment of neurodegenerative disorders. A series of novel SRs ligands were designed by combining key pharmacophoric amines (i.e., benzylpiperidine or benzylpiperazine) with new 1,3-dithiolane-based heterocycles and their bioisosters. The new compounds exhibited a low nanomolar affinity for sigma-1 and sigma-2 receptors. Five selected compounds were evaluated for their neuroprotective capacity on SH-SY5Y neuroblastoma cell line. They were able to counteract the neurotoxicity induced by rotenone, oligomycin and NMDA. Competition studies with PB212, a S1R antagonist, confirmed the involvement of S1R in neuroprotection from the oxidative stress induced by rotenone. Electrophysiological experiments performed on cortical neurons in culture highlighted the compounds ability to reduce NMDA-evoked currents, suggesting a negative allosteric modulator activity toward the NMDA receptor. Altogether these results qualify our novel dithiolane derivatives as potential agents for fighting neurodegeneration.

Embryonic Ketamine Produces a Downregulation of Prefrontal Cortex NMDA Receptors and Anxiety-Like Behavior in Adult Offspring

Previous studies have focused on the effects of N-methyl-D-aspartate receptor (NMDAR) blockade on neonates, but little is known about the effect of the embryonic NMDAR blockade on offspring, especially the long-lasting effect, on behavior in adulthood. Here, pregnant rats at E14 were treated with ketamine for 5 successive days and undergone multiple behavior tests, electrophysiology experiment, and Western blotting analysis to detect the alterations in their offspring. We found that embryonic ketamine treatment induced anxiety-like behavior in adulthood (8-week old) offspring. At the same period, we observed an attenuation of NMDA-evoked current as well as decreased NR2A and NR2B membrane expression in the prefrontal cortex (PFC), but not in the hippocampus or amygdala. Selective inhibition experiments with NR2A or NR2B specific antagonists suggested that embryonic ketamine treatment induced NMDAR current attenuation was likely mediated by changes in NR2A subunit. Moreover, at the 4-week time point, NMDA-evoked current was unchanged in PFC, but enhanced in hippocampal CA1 area, which may be caused by the over expression of NR2B in the hippocampus at 4-week time. Furthermore, NR2B knockdown, by using NR2B-shRNA lentivirus, in the hippocampal CA1 area at 3–4-week of age significantly rescued the decrease in NR2A expression in the PFC and anxiety-like behavior observed at 8-week adult offspring rats. In conclusion, our results suggested that embryonic ketamine treatment induced anxiety-like behavior and the downregulation of NMDAR function in PFC in the adulthood period of offspring, which might result from the enhanced function of NMDARs in the hippocampus at the 4-week juvenile time point.

Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine's antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.

Antinociceptive effect of PnTx4(5-5), a peptide from Phoneutria nigriventer spider venom, in rat models and the involvement of glutamatergic system.

ABSTRACTBackground: The venom of Phoneutria nigriventer spider is a source of numerous bioactive substances, including some toxins active in insects. An example is PnTx4(5-5) that shows a high insecticidal activity and no apparent toxicity to mice, although it inhibited NMDA-evoked currents in rat hippocampal neurons. In this work the analgesic activity of PnTx4(5-5) (renamed Γ-ctenitoxin-Pn1a) was investigated. Methods: The antinociceptive activity was evaluated using the paw pressure test in rats, after hyperalgesia induction with intraplantar injection of carrageenan or prostaglandin E2 (PGE2). Results: PnTx4(5-5), subcutaneously injected, was able to reduce the hyperalgesia induced by PGE2 in rat paw, demonstrating a systemic effect. PnTx4(5-5) administered in the plantar surface of the paw caused a peripheral and dose-dependent antinociceptive effect on hyperalgesia induced by carrageenan or PGE2. The hyperalgesic effect observed in these two pain models was completely reversed with 5 µg of PnTx4(5-5). Intraplantar administration of L-glutamate induced hyperalgesic effect that was significantly reverted by 5 μg of PnTx4(5-5) injection in rat paw. Conclusion: The antinociceptive effect for PnTx4(5-5) was demonstrated against different rat pain models, i.e. induced by PGE2, carrageenan or glutamate. We suggest that the antinociceptive effect of PnTx4(5-5) may be related to an inhibitory activity on the glutamatergic system.

Ras inhibitor S-trans, trans-farnesylthiosalicylic acid enhances spatial memory and hippocampal long-term potentiation via up-regulation of NMDA receptor

Statins by reducing farnesyl-pyrophosphate or farnesyl transferase inhibitors have been demonstrated to enhance spatial memory and long-term potentiation (LTP). The objective of this study was to investigate effects of the synthetic Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS) on spatial cognitive function in adult mice, synaptic plasticity in hippocampal CA1 regions, and NMDA receptor (NMDAr) activity of pyramidal cells. Here, we show that administering FTS (5 mg/kg, i.p.) enhanced spatial cognitive performance, as assessed via Morris water maze and Y-maze tests. Treating hippocampal slices with FTS (5 μM) for 2 h enhanced selectively NMDAr-dependent LTP without changing the synaptic properties. In comparison with the controls, the FTS-treated slices showed increases in the amplitude of NMDA-evoked currents (INMDA) and the phosphorylation of NMDAr GluN2A/GluN2B subunits and Src. The Src inhibitor PP2 blocked the enhancing effects of FTS on the activity and phosphorylation of NMDAr. In FTS-treated slices, basal levels of CaMKII, ERK2 and CREB phosphorylation did not differ significantly from those of controls; however, high-frequency stimulation-induced increases in CaMKII, ERK2 and CREB phosphorylation were more significant than in the controls, which were sensitive to PP2 and NMDAr antagonist MK801. Furthermore, the phosphorylation of AMPA receptor GluR1 during LTP was higher in FTS-treated slices compared with the control, which depended on Src and ERK1/2 signaling. The results indicate that the Ras inhibition by FTS can enhance NMDAr-dependent LTP by increasing Src activity to promote NMDAr GluN2A/GluN2B phosphorylation, which then leads to spatial memory potentiation.

Oscillatory Synchronous Inhibition in the Basolateral Amygdala and its Primary Dependence on NR2A-containing NMDA Receptors

Synchronous, rhythmic firing of GABAergic interneurons is a fundamental mechanism underlying the generation of brain oscillations, and evidence suggests that NMDA receptors (NMDARs) play a key role in oscillatory activity by regulating the activity of interneurons. Consistent with this, derangement of brain rhythms in certain neuropsychiatric disorders, notably schizophrenia and autism, is associated with NMDAR hypofunction and loss of inhibitory interneurons. In the basolateral amygdala (BLA)—dysfunction of which is involved in a host of neuropsychiatric diseases—, principal neurons display spontaneous, rhythmic “bursts” of inhibitory activity, which could potentially be involved in the orchestration of oscillations in the BLA network; here, we investigated the role of NMDARs in these inhibitory oscillations. Rhythmic bursts of spontaneous IPSCs (0.5 Hz average burst frequency) recorded from rat BLA principal cells were blocked or significantly suppressed by D-AP5, and could be driven by NMDAR activation alone. BLA interneurons generated spontaneous bursts of suprathreshold EPSCs at a similar frequency, which were also blocked or reduced by D-AP5. PEAQX (GluN2A-NMDAR antagonist; 0.4 μM) or Ro-25-6981 (GluN2B-NMDAR antagonist; 5 μM) suppressed the IPSC and EPSC bursts; suppression by PEAQX was significantly greater than that by Ro-25-6981. Immunohistochemical labeling revealed the presence of both GluN2A- and GluN2B-NMDARs on GABAergic BLA interneurons, while, functionally, GluN2A-NMDARs have the dominant role, as suggested by a greater reduction of NMDA-evoked currents by PEAQX versus Ro-25-6981. Entrainment of BLA principal neurons in an oscillatory generation of inhibitory activity depends primarily on activation of GluN2A-NMDARs, and interneuronal GluN2A-NMDARs may play a significant role.

Membrane cholesterol depletion in cortical neurons highlights altered NMDA receptor functionality in a mouse model of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-β-cyclodextrin (MβCD). Interestingly, MβCD chronic treatment has provoked a significant reduction of NMDA-evoked currents in both cellular populations which was dose- and time-dependent but significantly higher in ALS neurons compared to Control. The different MβCD effect on NMDA-evoked currents was not due to a different membrane receptor subunit composition but seemed to cause in both neuronal populations a NMDA receptor membrane redistribution. MβCD treatment effect was receptor-specific since no alterations in the two neuronal populations were detected on AMPA receptors.These results lead us to speculate for an altered proteomic composition of lipid rafts in cortical mutated neurons and suggest the need for further studies on the lipid rafts composition and on their interaction with membrane receptors in ALS cortices.

RETRACTED: Rett-like Severe Encephalopathy Caused by a De Novo GRIN2B Mutation Is Attenuated by D-serine Dietary Supplement

N-Methyl-D-aspartate receptors (NMDARs) play pivotal roles in synaptic development, plasticity, neural survival, and cognition. Despite recent reports describing the genetic association between de novo mutations of NMDAR subunits and severe psychiatric diseases, little is known about their pathogenic mechanisms and potential therapeutic interventions. Here we report a case study of a 4-year-old Rett-like patient with severe encephalopathy carrying a missense de novo mutation in GRIN2B(p.P553T) coding for the GluN2B subunit of NMDAR. We generated a dynamic molecular model of mutant GluN2B-containing NMDARs. We expressed the mutation in cell lines and primary cultures, and we evaluated the putative morphological, electrophysiological, and synaptic plasticity alterations. Finally, we evaluated D-serine administration as a therapeutic strategy and translated it to the clinical practice. Structural molecular modeling predicted a reduced pore size of mutant NMDARs. Electrophysiological recordings confirmed this prediction and also showed gating alterations, a reduced glutamate affinity associated with a strong decrease of NMDA-evoked currents. Moreover, GluN2B(P553T)-expressing neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of GluA1 at stimulated synapses. Notably, the naturally occurring coagonist D-serine was able to attenuate hypofunction of GluN2B(p.P553T)-containing NMDARs. Hence, D-serine dietary supplementation was initiated. Importantly, the patient has shown remarkable motor, cognitive, and communication improvements after 17months of D-serine dietary supplementation. Our data suggest that hypofunctional NMDARs containing GluN2B(p.P553T) can contribute to Rett-like encephalopathy and that their potentiation by D-serine treatment may underlie the associated clinical improvement.

Antagonistic action on NMDA/GluN2B mediated currents of two peptides that were conantokin-G structure-based designed

BackgroundThe GluN2B subunit of the N-methyl-d-aspartate receptor (NMDAr) modulates many physiological processes including learning, memory, and pain. Excessive increase in NMDAr/GluN2B activity has been associated with various disorders such neuropathic pain and neuronal death following hypoxia. Thus there is an interest in identifying NMDAr antagonists that interact specifically with the GluN2B subunit. Recently based on structural analysis between the GluN2B subunit and conantokin-G, a toxin that interacts selectively with the GluN2B subunit, we designed various peptides that are predicted to act as NMDAr antagonists by interacting with the GluN2B subunit. In this study we tested this prediction for two of these peptides EAR16 and EAR18.ResultsThe effects of EAR16 and EAR18 in NMDA-evoked currents were measured in cultured rat embryonic hippocampal neurons and in HEK-293 cells expressing recombinant NMDAr comprised of GluN1a–GluN2A or GluN1a–GluN2B subunits. In hippocampal neurons, EAR16 and EAR18 reduced the NMDA-evoked calcium currents in a dose-dependent and reversible manner with comparable IC50 (half maximal inhibitory concentration) values of 241 and 176 µM, respectively. At 500 µM, EAR16 blocked more strongly the NMDA-evoked currents mediated by the GluN1a–GluN2B (84%) than those mediated by the GluN1a–GluN2A (50%) subunits. At 500 µM, EAR18 blocked to a similar extent the NMDA-evoked currents mediated by the GluN1a–GluN2B (62%) and the GluN1a–GluN2A (55%) subunits.ConclusionsThe newly designed EAR16 and EAR18 peptides were shown to block in reversible manner NMDA-evoked currents, and EAR16 showed a stronger selectivity for GluN2B than for GluN2A.

STEP activation by Gαq coupled GPCRs opposes Src regulation of NMDA receptors containing the GluN2A subunit.

N-methyl-D-aspartate receptors (NMDARs) are necessary for the induction of synaptic plasticity and for the consolidation of learning and memory. NMDAR function is tightly regulated by functionally opposed families of kinases and phosphatases. Herein we show that the striatal-enriched protein tyrosine phosphatase (STEP) is recruited by Gαq-coupled receptors, including the M1 muscarinic acetylcholine receptor (M1R), and opposes the Src tyrosine kinase-mediated increase in the function of NMDARs composed of GluN2A. STEP activation by M1R stimulation requires IP3Rs and can depress NMDA-evoked currents with modest intracellular Ca2+ buffering. Src recruitment by M1R stimulation requires coincident NMDAR activation and can augment NMDA-evoked currents with high intracellular Ca2+ buffering. Our findings suggest that Src and STEP recruitment is contingent on differing intracellular Ca2+ dynamics that dictate whether NMDAR function is augmented or depressed following M1R stimulation.

Epileptiform curents in rat cortical neurons increase over time of the in vitro culture period

Here we show that in primary culture of rat cortical neurons the number of episodes of epileptiform curents (EC) provoked by extracellular magnesium removal increases over time. We demonstrate that NMDA receptor agonists in low concentrations induce an elevation of frequency of miniature postsynaptic currents followed by their synchronization resulting in EC. Ifenprodil did not block EC but strongly inhibited NMDA-evoked whole-cell currents, which say for a key role of the ifenprodil-resistant synaptic GluN2A-containing NMDA receptors in the generation of EC. We suppose that in cultured neurons the onset of EC and a gradual increase of the EC amplitude over the time of culture period is directed by an increase of synaptic connections density and displacement of the GluN2B subunit by GluN2A in synapses.

Altered NMDA receptor function in primary cultures of hippocampal neurons from mice lacking the Homer2 gene.

N-Methyl-D-Aspartate (NMDA) receptors are inhibited during acute exposure to ethanol and are involved in changes in neuronal plasticity following repeated ethanol exposure. The postsynaptic scaffolding protein Homer2 can regulate the cell surface expression of NMDA receptors in vivo, and mice with a null mutation of the Homer2 gene exhibit an alcohol-avoiding and -intolerant phenotype that is accompanied by a lack of ethanol-induced glutamate sensitization. Thus, Homer2 deletion may perturb the function or acute ethanol sensitivity of the NMDA receptor. In this study, the function and ethanol sensitivity of glutamate receptors in cultured hippocampal neurons from wild-type (WT) and Homer2 knock-out (KO) mice were examined at 7 and 14 days in vitro (DIV) using standard whole-cell voltage-clamp electrophysiology. As compared with wild-type controls, NMDA receptor current density was reduced in cultured hippocampal neurons from Homer2 KO mice at 14 DIV, but not at 7 DIV. There were no genotype-dependent changes in whole-cell capacitance or in currents evoked by kainic acid. The GluN2B-selective antagonist ifenprodil inhibited NMDA-evoked currents to a similar extent in both wild-type and Homer2 KO neurons and inhibition was greater at 7 versus 14 DIV. NMDA receptor currents from both WT and KO mice were inhibited by ethanol (10-100 mM) and the degree of inhibition did not differ as a function of genotype. In conclusion, NMDA receptor function, but not ethanol sensitivity, is reduced in hippocampal neurons lacking the Homer2 gene.

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.

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.

Acute 5-HT7 receptor activation increases NMDA-evoked currents and differentially alters NMDA receptor subunit phosphorylation and trafficking in hippocampal neurons

BackgroundN-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2–24 h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) β receptors which can protect neurons against NMDA-induced neurotoxicity.ResultsIn contrast to long-term activation of 5-HT7 receptors, acute (5 min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered.ConclusionsIn contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor.

Group II metabotropic glutamate receptors modify N-methyl-D-aspartate receptors via Src kinase

Group II metabotropic glutamate receptors (mGluR2/3) have emerged as important targets for the treatment of schizophrenia. Since hypofunction of N-methyl-D-aspartate receptors (NMDARs) has also been implicated in the etiology of schizophrenia, we examined whether postsynaptic mGluR2/3 regulate NMDAR function. Activation of mGluR2/3 significantly decreased the ratio of AMPA-to-NMDA excitatory postsynaptic currents at Schaffer Collateral-CA1 synapses and enhanced the peak of NMDA-evoked currents in acutely isolated CA1 neurons. The mGluR2/3-mediated potentiation of NMDAR currents was selective for GluN2A-containing NMDARs and was mediated by the Src family kinase Src. Activation of mGluR2/3 inhibited the adenylyl cyclase-cAMP-PKA pathway and thereby activated Src by inhibiting its regulatory C-terminal Src kinase (Csk). We suggest a novel model of regulation of NMDARs by Gi/o-coupled receptors whereby inhibition of the cAMP-PKA pathway via mGluR2/3 activates Src kinase and potentiates GluN2A-containing NMDAR currents. This represents a potentially novel mechanism to correct the hypoglutamatergic state found in schizophrenia.