Synaptic and Extrasynaptic NMDA Receptors Are Gated by Different Endogenous Coagonists

N-methyl-d-aspartate receptors (NMDARs) are located in neuronal cell membranes at synaptic and extrasynaptic locations, where they are believed to mediate distinct physiological and pathological processes. Activation of NMDARs requires glutamate and a coagonist whose nature and impact on NMDAR physiology remain elusive. We report that synaptic and extrasynaptic NMDARs are gated by different endogenous coagonists, d-serine and glycine, respectively. The regionalized availability of the coagonists matches the preferential affinity of synaptic NMDARs for d-serine and extrasynaptic NMDARs for glycine. Furthermore, glycine and d-serine inhibit NMDAR surface trafficking in?a subunit-dependent manner, which is likely to influence NMDARs subcellular location. Taking advantage of this coagonist segregation, we demonstrate that long-term potentiation and NMDA-induced neurotoxicity rely on synaptic NMDARs only. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. Our observations provide key insights into the operating mode of NMDARs, emphasizing functional distinctions between synaptic and extrasynaptic NMDARs in brain physiology.     

D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period

N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC) at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP) at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.     

Organization,control and function of extrasynaptic NMDA receptors

N-methyl d-aspartate receptors (NMDARs) exist in different forms owing to multiple combinations of subunits that can assemble into a functional receptor. In addition, they are located not only at synapses but also at extrasynaptic sites. There has been intense speculation over the past decade about whether specific NMDAR subtypes and/or locations are responsible for inducing synaptic plasticity and excitotoxicity. Here, we review the latest findings on the organization, subunit composition and endogenous control of NMDARs at extrasynaptic sites and consider their putative functions. Because astrocytes are capable of controlling NMDARs through the release of gliotransmitters, we also discuss the role of the glial environment in regulating the activity of these receptors.     

Glutamate-mediated extrasynaptic inhibition: direct coupling of NMDA receptors to Ca(2+)-activated K+ channels

NMDA receptors (NMDARs) typically contribute to excitatory synaptic transmission in the CNS. While Ca(2+) influx through NMDARs plays a critical role in synaptic plasticity, direct actions of NMDAR-mediated Ca(2+) influx on neuronal excitability have not been well established. Here we show that Ca(2+) influx through NMDARs is directly coupled to activation of BK-type Ca(2+)-activated K+ channels in outside-out membrane patches from rat olfactory bulb granule cells. Repetitive stimulation of glutamatergic synapses in olfactory bulb slices evokes a slow inhibitory postsynaptic current (IPSC) in granule cells that requires both NMDARs and BK channels. The slow IPSC is enhanced by glutamate uptake blockers, suggesting that extrasynaptic NMDARs underlie the response. These findings reveal a novel inhibitory action of extrasynaptic NMDARs in the brain.     

Calpain-mediated degradation of myocyte enhancer factor 2D contributes to excitotoxicity by activation of extrasynaptic N-methyl-D-aspartate receptors

Synaptic and extrasynaptic NMDA receptors (NMDARs) appear to play opposite roles in neuronal survival and death. Here we report the new findings on the dysregulation of survival factor, myocyte enhancer factor 2D (MEF2D), by extrasynaptic NMDARs. Excitotoxicity led to the NMDAR-dependent degradation of MEF2D protein and inhibition of its transactivation activity in mature cortical neurons. The activation of extrasynaptic NMDARs alone was sufficient for degradation of MEF2D. Calpain directly cleaved MEF2D in vitro and blocking this protease activity greatly attenuated NMDAR signaled degradation of MEF2D in neurons. Consistently, inhibition of calpain protected cortical neurons from NMDA-induced excitotoxicity. Furthermore, knockdown of MEF2D sensitized neurons to NMDA-induced excitotoxicity, which was not protected by calpain inhibition. Collectively, these findings suggest that dysregulation of MEF2D by calpain may mediate excitotoxicity via an extrasynaptic NMDAR-dependent manner.     

Backpropagating action potentials enable detection of extrasynaptic glutamate by NMDA receptors

Synaptic NMDA receptors (NMDARs) are crucial for neural coding and plasticity. However, little is known about the adaptive function of extrasynaptic NMDARs occurring mainly on dendritic shafts. Here, we find that in CA1 pyramidal neurons, back-propagating action potentials (bAPs) recruit shaft NMDARs exposed to ambient glutamate. In contrast, spine NMDARs are "protected," under baseline conditions, from such glutamate influences by peri-synaptic transporters: we detect bAP-evoked Ca(2+) entry through these receptors upon local synaptic or photolytic glutamate release. During theta-burst firing, NMDAR-dependent Ca(2+) entry either downregulates or upregulates an h-channel conductance (G(h)) of the cell depending on whether synaptic glutamate release is intact or blocked. Thus, the balance between activation of synaptic and extrasynaptic NMDARs can determine the sign of G(h) plasticity. G(h) plasticity in turn regulates dendritic input probed by local glutamate uncaging. These results uncover a metaplasticity mechanism potentially important for neural coding and memory formation.     

Calcium-Dependent Desensitization of NMDA Receptors

Glutamate receptors play the key role in excitatory synaptic transmission in the central nervous system (CNS). N-methyl-D-aspartate-activated glutamate receptors (NMDARs) are ion channels permeable to sodium, potassium, and calcium ions that localize to the pre- and postsynaptic membranes, as well as extrasynaptic neuronal membrane. Calcium entry into dendritic spines is essential for long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission. Both LTP and LTD represent morphological and functional changes occurring in the process of memory formation. NMDAR dysfunction is associated with epilepsy, schizophrenia, migraine, dementia, and neurodegenerative diseases. Prolonged activation of extrasynaptic NMDARs causes calcium overload and apoptosis of neurons. Here, we review recent findings on the molecular mechanisms of calcium-dependent NMDAR desensitization that ensures fast modulation of NMDAR conductance in the CNS and limits calcium entry into the cells under pathological conditions. We present the data on molecular determinants related to calcium-dependent NMDAR desensitization and functional interaction of NMDARs with other ion channels and transporters. We also describe association of NMDARs with lipid membrane microdomains.     

Neuroprotective strategies for NMDAR-mediated excitotoxicity in Huntington’s Disease

BACKGROUND

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disease causing severe neurodegeneration of the striatum as well as marked cognitive and motor disabilities. Excitotoxicity, caused by overstimulation of NMDA receptors (NMDARs) has been shown to have a key role in the neuropathogenesis of HD, suggesting that targeting NMDAR-dependent signaling may be an effective clinical approach for HD. However, broad NMDAR antagonists are generally poor therapeutics in clinical practice. It has been suggested that GluN2A-containing, synaptically located NMDARs activate cell survival signaling pathways, while GluN2B-containing, primarily extrasynaptic NMDARs trigger cell death signaling. A better approach to development of effective therapeutics for HD may be to target, specifically, the cell-death specific pathways associated with extrasynaptic GluN2B NMDAR activation, while maintaining or potentiating the cellsurvival activity of GluN2A-NMDARs.

OBJECTIVE

This review outlines the role of NMDAR-mediated excitotoxicity in HD and overviews current efforts to develop better therapeutics for HD where NMDAR excitotoxicity is the target.

METHODS

A systematic review process was conducted using the PubMed search engine focusing on research conducted in the past 5-10 years. 235 articles were consulted for the review, with key search terms including “Huntington’s Disease,” “excitotoxicity,” “NMDAR” and “therapeutics.”

RESULTS

A wide range of NMDAR excitotoxicity-based targets for HD were identified and reviewed, including targeting NMDARs directly by blocking GluN2B, extrasynaptic NMDARs and/or potentiating GluN2A, synaptic NMDARs, targeting glutamate release or uptake, or targeting specific downstream cell-death signaling of NMDARs.

CONCLUSION

The current review identifies NMDAR-mediated excitotoxicity as a key player in HD pathogenesis and points to various excitotoxicity-focused targets as potential future preventative therapeutics for HD.

     

Distribution of extracellular glutamate in the neuropil of hippocampus

Reported values of extracellular glutamate concentrations in the resting state depend on the method of measurement and vary ～1000-fold. As glutamate levels in the micromolar range can cause receptor desensitization and excitotoxicity, and thus affect neuronal excitability, an accurate determination of ambient glutamate is important. Part of the variability of previous measurements may have resulted from the sampling of glutamate in different extracellular compartments, e.g., synaptic versus extrasynaptic volumes. A steep concentration gradient of glutamate between these two compartments could be maintained, for example, by high densities of glutamate transporters arrayed at the edges of synapses. We have used two photon laser scanning microscopy and electrophysiology to investigate whether extracellular glutamate is compartmentalized in acute hippocampal slices. Pharmacological blockade of NMDARs had no effect on Ca(2+) transients generated in dendritic shafts or spines of CA1 pyramidal neurons by depolarization, suggesting that ambient glutamate is too low to activate a significant number of NMDARs. Furthermore, blockade of transporters did not flood the synapse with glutamate, indicating that synaptic NMDARs are not protected from high concentrations of extrasynaptic glutamate. We suggest that, in the CA1 region of hippocampus, glutamate transporters do not create a privileged space within the synapse but rather keep ambient glutamate at very low levels throughout the neuropil.     

Sustained saturating level of glycine induces changes in NR2B-containing-NMDA receptor localization in the CA1 region of the hippocampus

Post-synaptic actions of glycine are terminated by specialized transporters. There are two genes encoding glycine transporters, GlyT1 and GlyT2. Glycine acts as a co-agonist at N -methyl- d -aspartate glutamatergic receptors (NMDARs). Blockage of GlyT1 enhances NMDAR function by controlling ambient glycine concentrations. Using whole-cell patch-clamp recordings of acute hippocampal slices, we investigated NMDAR kinetics of CA1 pyramidal neurons of mice expressing 50% of GlyT1 (GlyT1+/−). In this study, we report that the glycine modulatory site of the NMDAR at CA1 synapses is saturated in GlyT1+/− but not in wild-type (WT) mice. We also found that the effect of ifenprodil, a highly selective NR2B-containing-NMDAR antagonist, is significantly reduced at CA1 synapses in GlyT1+/− compared to WT mice while immunoblotting experiments do not show significant differences for NR1, NR2A-B-C-D subunits in both types of mice, suggesting alteration in NR2B-containing-NMDAR localization under a state of chronic saturating level of endogenous glycine. Using a pharmacological approach with MK-801 and DL-TBOA, we discriminated synaptic vis-à-vis extra-synaptic NMDARs. We found that NR2B-containing-NMDARs are expressed at a higher level in the extra-synaptic area of CA1 pyramidal neurons from GlyT1+/− compared to WT mice. Our results demonstrate that chronic saturating level of glycine induces significant changes in NMDAR localization and kinetic. Therefore, results from our study should help to gain a better understanding of the role of glycine in pathological conditions.     

Glycine Induces Bidirectional Modifications in N-Methyl-d-aspartate Receptor-mediated Synaptic Responses in Hippocampal CA1 Neurons

Glycine can persistently potentiate or depress AMPA responses through differential actions on two binding sites: NMDA and glycine receptors. Whether glycine can induce long-lasting modifications in NMDA responses, however, remains unknown. Here, we report that glycine induces long-term potentiation (LTP) or long-term depression (LTD) of NMDA responses (Gly-LTP_NMDA or Gly-LTD_NMDA) in a dose-dependent manner in hippocampal CA1 neurons. These modifications of NMDA responses depend on NMDAR activation. In addition, the induction of Gly-LTP_NMDA requires binding of glycine with NMDARs, whereas Gly-LTD_NMDA requires that glycine bind with both sites on NMDARs and GlyRs. Moreover, activity-dependent exocytosis and endocytosis of postsynaptic NMDARs underlie glycine-induced bidirectional modification of NMDA excitatory postsynaptic currents. Thus, we conclude that glycine at different levels induces bidirectional plasticity of NMDA responses through differentially regulating NMDA receptor trafficking. Our present findings reveal important functions of the two glycine binding sites in gating the direction of synaptic plasticity in NMDA responses.     

NMDA receptor function: subunit composition versus spatial distribution

NMDA receptors (NMDARs) play a pivotal role in the regulation of neuronal communication and synaptic function in the central nervous system. The subunit composition and compartmental localization of NMDARs in neurons affect channel activity and downstream signaling. This review discusses the distinct NMDAR subtypes and their function at synaptic, perisynaptic, and extrasynaptic sites of excitatory and inhibitory neurons. Many neurons express more than one of the modulatory NR2 subunits that participate in the formation of di- and/or triheteromeric channel assemblies (e.g., NR1/NR2A, NR1/NR2B, and/or NR1/NR2A/NR2B). Depending on the subunit composition and presence or absence of intracellular binding partners along the postsynaptic membrane, these NMDAR subtypes are allocated to distinct synaptic inputs converging onto a neuron or are distributed differentially among synaptic or extrasynaptic sites. These sites can carry NR2A and NR2B subunits, supporting the hypothesis that the spatial distribution of scaffolding and signaling complexes critically determines the full spectrum of NMDAR signaling.The author thanks the Deutsche Forschungsgemeinschaft for financial support (Ko 1064/5).     

D-Serine inhibits AMPA receptor-mediated current in rat hippocampal neurons

D-Serine, a recently identified gliotransmitter, serves as an endogenous coagonist binding to the glycine site of N-methyl-D-aspartate (NMDA) receptors. However, it is not clear whether this native ligand is able to bind to and modulate alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptors. In the present study, we showed that D-serine was able to concentration-dependently inhibit kainate-induced AMPA receptor-mediated current in acutely isolated hippocampal neurons. The blocking action of D-serine on AMPA receptors was characterized by a shift in concentration-response curve of kainate-induced current to the right with no change in the maximal response and independent of holding potential in the range of -80 to +60 mV. This is consistent with a model that D-serine is a competitive antagonist on AMPA receptors. In contrast, L-serine did not exert such an inhibitory action. Consistent with this observation, we found that several D-isoforms, but not L-isoforms, of endogenous and exogenous amino acids were able to block AMPA receptors. These results indicate that there is a low affinity and stereo-selective site at the agonist binding pocket of AMPA receptors for these D-amino acids. More importantly, vesicular-released endogenous D-serine from astrocytes could potentially modulate AMPA receptors in synaptic transmission in hippocampus.     

Comparison of the pharmacological properties of GK11 and MK801, two NMDA receptor antagonists: towards an explanation for the lack of intrinsic neurotoxicity of GK11

Over-stimulation of NMDA receptors (NMDARs) is involved in many neurodegenerative disorders. Thus, developing safe NMDAR antagonists is of high therapeutic interest. GK11 is a high affinity uncompetitive NMDAR antagonist with low intrinsic neurotoxicity, shown to be promising for treating CNS trauma. In the present study, we investigated the molecular basis of its interaction with NMDARs and compared this with the reference molecule MK801. We show, on primary cultures of hippocampal neurons, that GK11 exhibits neuroprotection properties similar to those of MK801, but in contrast with MK801, GK11 is not toxic to neurons. Using patch-clamp techniques, we also show that on NR1a/NR2B receptors, GK11 totally blocks the NMDA-mediated currents but has a six-fold lower IC₅₀ than MK801. On NR1a/NR2A receptors, it displays similar affinity but fails to totally prevent the currents. As NR2A is preferentially localized at synapses and NR2B at extrasynaptic sites, we investigated, using calcium imaging and patch-clamp approaches, the effects of GK11 on either synaptic or extrasynaptic NMDA-mediated responses. Here we demonstrate that in contrast with MK801, GK11 better preserve the synaptic NMDA-mediated currents. Our study supports that the selectivity of GK11 for NR2B containing receptors accounts contributes, at least partially, for its safer pharmacological profile.     

Interactions between glycine transporter type 1 (GlyT-1) and some inhibitor molecules - glycine transporter type 1 and its inhibitors (review)

Glycine is a mandatory positive allosteric modulator of N-methyl-D-aspartate (NMDA)-type ionotropic glutamate receptors in the central nervous system. Elevation of glycine concentrations by inhibition of its reuptake in the vicinity of NMDA receptors may positively influence receptor functions as glycine B binding site on NR1 receptor subunit is not saturated in physiological conditions. Synaptic and extrasynaptic concentrations of glycine are regulated by its type-1 glycine transporter, which is primarily expressed in astroglial and glutamatergic cell membranes. Alteration of synaptic glycine levels may have importance in the treatment of various forms of endogenous psychosis characterized by hypofunctional NMDA receptors. Several lines of evidence indicate that impaired NMDA receptor-mediated glutamatergic neurotransmission is involved in development of the negative (and partly the positive) symptoms and the cognitive deficit in schizophrenia. Inhibitors of glycine transporter type-1 may represent a newly developed therapeutic intervention in treatment of this mental illness. We have synthesized a novel series of N-substituted sarcosines, analogues of the glycine transporter-1 inhibitor NFPS (N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-propyl]sarcosine). Of the pyridazinone-containing compounds, SzV-1997 was found to be a potent glycine transporter-1 inhibitor in rat brain synaptosomes and it markedly increased extracellular glycine concentrations in conscious rat striatum. SzV-1997 did not exhibit toxic symptoms such as hyperlocomotion, restless movements, respiratory depression, and lethality, characteristic for NFPS. Besides pyridazinone-based, sarcosine-containing glycine transporter-1 inhibitors, a series of substrate-type amino acid inhibitors was investigated in order to obtain better insight into the ligand-binding characteristics of the substrate binding cavity of the transporter.     

A Monte Carlo model reveals independent signaling at central glutamatergic synapses

We have developed a biophysically realistic model of receptor activation at an idealized central glutamatergic synapse that uses Monte Carlo techniques to simulate the stochastic nature of transmission following release of a single synaptic vesicle. For the a synapse with 80 AMPA and 20 NMDA receptors, a single quantum, with 3000 glutamate molecules, opened approximately 3 NMDARs and 20 AMPARs. The number of open receptors varied directly with the total number of receptors, and the fraction of open receptors did not depend on the ratio of co-localized AMPARs and NMDARs. Variability decreased with increases in either total receptor number or quantal size, and differences between the variability of AMPAR and NMDAR responses were due solely to unequal numbers of receptors at the synapse. Despite NMDARs having a much higher affinity for glutamate than AMPARs, quantal release resulted in similar occupancy levels in both receptor types. Receptor activation increased with number of transmitter molecules released or total receptor number, whereas occupancy levels were only dependent on quantal size. Tortuous diffusion spaces reduced the extent of spillover and the activation of extrasynaptic receptors. These results support the conclusion that signaling is spatially independent within and between central glutamatergic synapses.     

Effects of Quinolinate-Induced Lesion of the Medial Prefrontal Cortex on Prefrontal and Striatal Concentrations of d-Serine in the Rat

Neurochemical Research - d-Serine has been shown to play an important role in the expression and control of a variety of brain functions by acting as the endogenous coagonist for the...