Role of Glycine Receptors in Glycine-Induced LTD in Hippocampal CA1 Pyramidal Neurons

Glycine in the hippocampus can exert its effect on both synaptic NMDA receptors (NMDARs) and extrasynaptic functional glycine receptors (GlyRs) via distinct binding sites. Previous studies have reported that glycine induces long-term potentiation (LTP) through the activation of synaptic NMDARs. However, little is known about the potential role of the activated GlyRs that are largely located in extrasynaptic regions. We report here that relatively high levels of glycine achieved either by exogenous glycine application or by the elevation of endogenous glycine accumulation with an antagonist of the glycine transporter induced long-term depression (LTD) of excitatory postsynaptic currents (EPSCs) in hippocampal CA1 pyramidal neurons. The co-application of glycine with the selective GlyR antagonist strychnine changed glycine-induced LTD (Gly-LTD) to LTP. Blocking the postsynaptic GlyR-gated net chloride flux by manipulating intracellular chloride concentrations failed to elicit any changes in EPSCs. These results suggest that GlyRs are involved in Gly-LTD. Furthermore, this new form of chemical LTD was accompanied by the internalization of postsynaptic AMPA receptors and required the activation of NMDARs. Therefore, our present findings reveal an important function of GlyR activation and modulation in gating the direction of synaptic plasticity.     

GLYX-13, a NMDA Receptor Glycine-Site Functional Partial Agonist,Induces Antidepressant-Like Effects Without Ketamine-Like Side Effects

Recent human clinical studies with the NMDA receptor (NMDAR) antagonist ketamine have revealed profound and long-lasting antidepressant effects with rapid onset in several clinical trials, but antidepressant effects were preceded by dissociative side effects. Here we show that GLYX-13, a novel NMDAR glycine-site functional partial agonist, produces an antidepressant-like effect in the Porsolt, novelty induced hypophagia, and learned helplessness tests in rats without exhibiting substance abuse-related, gating, and sedative side effects of ketamine in the drug discrimination, conditioned place preference, pre-pulse inhibition and open-field tests. Like ketamine, the GLYX-13-induced antidepressant-like effects required AMPA/kainate receptor activation, as evidenced by the ability of NBQX to abolish the antidepressant-like effect. Both GLYX-13 and ketamine persistently (24 h) enhanced the induction of long-term potentiation of synaptic transmission and the magnitude of NMDAR-NR2B conductance at rat Schaffer collateral-CA1 synapses in vitro. Cell surface biotinylation studies showed that both GLYX-13 and ketamine led to increases in both NR2B and GluR1 protein levels, as measured by Western analysis, whereas no changes were seen in mRNA expression (microarray and qRT-PCR). GLYX-13, unlike ketamine, produced its antidepressant-like effect when injected directly into the medial prefrontal cortex (MPFC). These results suggest that GLYX-13 produces an antidepressant-like effect without the side effects seen with ketamine at least in part by directly modulating NR2B-containing NMDARs in the MPFC. Furthermore, the enhancement of ‘metaplasticity'' by both GLYX-13 and ketamine may help explain the long-lasting antidepressant effects of these NMDAR modulators. GLYX-13 is currently in a Phase II clinical development program for treatment-resistant depression.     

Developmental switch in requirement for PKA RIIbeta in NMDA-receptor-dependent synaptic plasticity at Schaffer collateral to CA1 pyramidal cell synapses

The cAMP/protein kinase A (PKA) signaling cascade is crucial for synaptic plasticity in a wide variety of species. PKA regulates Ca²⁺ permeation through NMDA receptors (NMDARs) and induction of NMDAR-dependent synaptic plasticity at the Schaffer collateral to CA1 pyramidal cell synapse. Whereas the role of PKA in induction of NMDAR-dependent LTP at CA1 synapses is established, the identity of PKA isoforms involved in this phenomenon is less clear. Here we report that protein synthesis-independent NMDAR-dependent LTP at the Schaffer collateral-CA1 synapse in the hippocampus is deficient, but NMDAR-dependent LTD is normal, in young (postnatal day 10 (P10)-P14) mice lacking PKA RIIβ, the PKA regulatory protein that links PKA to NMDARs at synaptic sites. In contrast, in young adult (P21-P28) mice lacking PKA RIIβ, LTP is normal and LTD is abolished. These findings indicate that distinct PKA isoforms may subserve distinct forms of synaptic plasticity and are consistent with a developmental switch in the signaling cascades required for LTP induction.     

NMDA Receptors,mGluR5, and Endocannabinoids are Involved in a Cascade Leading to Hippocampal Long-Term Depression

In the CA1 region of the rat hippocampus, metabotropic glutamate receptor-5 (mGluR5) and cannabinoid-1 receptors (CB1Rs) are believed to participate in long-term synaptic depression (LTD). How mGluRs and CB1Rs interact to promote LTD remains uncertain. In this study, we examined LTD induced by CB1R agonists, mGluR5 agonists, and low-frequency electrical stimulation (LFS) of the Schaffer collateral pathway. Synthetic CB1R agonists induced robust LTD that was mimicked by the endocannabinoid (EC), noladin ether (NLDE), but not by anandamide. 2-Arachidonylglycerol (2AG) produced only a small degree of LTD. The selective mGluR5 agonist, namely (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), also induced robust LTD, and CHPG and NLDE occluded each other''s effects. Similarly, CHPG and NLDE occluded LFS-induced LTD, and LTD resulting from all three treatments was blocked by a CB1R antagonist. CHPG-LTD and NLDE-LTD were insensitive to N-methyl--aspartate receptor (NMDAR) block, even though LFS-LTD requires NMDARs. LTD induced by LFS or CHPG, but not NLDE-LTD, was blocked by a selective mGluR5 antagonist. (RS)-3,5-dihydroxyphenylglycine (DHPG), a less selective group I mGluR agonist, also induced LTD, but its effects were not blocked by mGluR5 or CB1R antagonists. Furthermore, DHPG-LTD was additive with LFS-LTD and CHGP-LTD. These results suggest that NMDARs, mGluR5, and CB1Rs participate in a cascade that underlies LFS-LTD and that release of an EC and CB1R activation occur downstream of NMDARs and mGluR5. Furthermore, DHPG induces a form of LTD that differs mechanistically from LFS-induced depression.     

The role of NMDA receptors in regulating group II metabotropic glutamate receptor-mediated long-term depression in rat medial prefrontal cortex

Previous work has shown that brief application of group II metabotropic glutamate receptor (mGluR) agonist (2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl) glycine (DCG-IV) can induce long-term depression (LTD) of excitatory transmission on layer V pyramidal neurons of rat medial prefrontal cortex (mPFC). An unusual feature of this LTD is that it relies on activation of both group II mGluRs and N-methyl-d-aspartate receptors (NMDARs). However, it is not known whether other specific group II mGluR agonists also induce LTD and whether they depend on the conjoint activation of group II mGluRs and NMDARs. We show here that the ability of DCG-IV to induce LTD was mimicked by a more selective group II mGluR agonist, LY379268. The induction of LTD by a lower concentration of DCG-IV (0.2 μM) or LY379268 (0.03 μM) was blocked by the NMDAR antagonist APV or the interruption of synaptic stimulation during drug application. In contrast, application of a higher concentration of DCG-IV (1 μM) or LY379268 (0.1 μM) can induce LTD that was independent of synaptic NMDAR activation. These results suggest that although molecular cooperation between group II mGluRs and synaptic NMDARs may facilitate the induction of group II mGluR-mediated LTD at excitatory synapses onto mPFC layer V pyramidal neurons, enhancing group II mGluR activation may remove NMDAR involvement in this form of synaptic plasticity.     

Opposing action of conantokin-G on synaptically and extrasynaptically-activated NMDA receptors

Synaptic and extrasynaptic activation of the N-methyl-D-aspartate receptor (NMDAR) has distinct consequences on cell signaling and neuronal survival. Since conantokin (con)-G antagonism is NR2B-selective, which is the key subunit involved in extrasynaptic activation of the receptor, its ability to specifically elicit distinct signaling outcomes in neurons with synaptically or extrasynaptically-activated NMDARs was evaluated. Inhibition of Ca(2+) influx through extrasynaptic NMDAR ion channels was neuroprotective, as it effectively enhanced levels of activated extracellular signal-regulated kinase 1/2 (ERK1/2), activated cAMP response element binding protein (CREB), enhanced mitochondrial viability, and attenuated the actin disorganization observed by extrasynaptic activation of NMDARs. Conversely, the pro-signaling pathways stimulated by synaptically-induced Ca(2+) influx were abolished by con-G. Furthermore, subunit non-selective con-T was unable to successfully redress the impairments in neurons caused by extrasynaptically-activated NMDARs, thus indicating that NR2B-specific antagonists are beneficial for neuron survival. Neurons ablated for the NR2B subunit showed weak synaptic Ca(2+) influx, reduced sensitivity to MK-801 blockage, and diminished extrasynaptic current compared to WT and NR2A(-/-) neurons. This indicates that the NR2B subunit is an integral component of both synaptic and extrasynaptic NMDAR channels. Altogether, these data suggest that con-G specifically targets the NR2B subunit in the synaptic and extrasynaptic locations, resulting in the opposing action of con-G on differentially activated pools of NMDARs.     

Activation of NR2B-containing NMDA receptors is not required for NMDA receptor-dependent long-term depression

The triggering of both NMDA receptor-dependent long-term potentiation (LTP) and long-term depression (LTD) in the CA1 region of the hippocampus requires a rise in postsynaptic calcium. A prominent hypothesis has been that the detailed properties of this postsynaptic calcium signal dictate whether LTP or LTD is generated by a given pattern of synaptic activity. Recently, however, evidence has been presented that the subunit composition of the NMDA receptor (NMDAR) determines whether a synapse undergoes LTP or LTD with NR2A-containing NMDARs triggering LTP and NR2B-containing NMDARs triggering LTD. In the present study, the role of NR2B-containing synaptic NMDARs in the induction of LTD in CA1 pyramidal cells has been studied using the selective NR2B antagonists, ifenprodil and Ro25-6981. While both antagonists reduced NMDAR-mediated synaptic currents, neither prevented induction of LTD. These results demonstrate that activation of NR2B-containing NMDARs is not an absolute requirement for the induction of LTD in the hippocampus.     

The role of NMDAR subtypes and charge transfer during hippocampal LTP induction

Activation of NMDA receptors (NMDARs) is a requirement for persistent synaptic alterations, such as long-term potentiation of synaptic transmission (LTP). NMDARs are composed of NR1 and NR2 subunits, and NR2 subunit-dependent gating properties of NMDAR subtypes cause dramatic differences in the timing of charge transfer. These postsynaptic temporal profiles are further influenced by the frequency of synaptic activation. Here, we investigated in the CA1 region of hippocampal slices from P28 mice, whether particular NMDAR subtypes are recruited based on NR2 subunit-specific gating following different induction protocols. For high frequency afferent stimulation (HFS), we found that genetic impairment of NR2A or pharmacological block of NR2A- or NR2B-type NMDARs can reduce field LTP. In contrast, when pairing low frequency synaptic stimulation with postsynaptic depolarization (LFS pairing) in single CA1 neurons, pharmacological antagonism of either subtype modestly reduced the charge transfer during LFS pairing without reducing the LTP magnitude. These results indicate that HFS-triggered LTP is induced by more than one NMDAR subtype, whereas a single subtype is sufficient during LFS pairing. Analysis of charge transfer during LFS pairing in 13 different conditions revealed a threshold for LTP induction, which was independent of the NR2 antagonist tested. Thus, at least for LFS pairing, the amount of charge transfer, and thus Ca2+ influx, during LTP induction is a factor more critical than the participation of a particular NMDAR subtype.     

HIV-1 Glycoprotein 120 Enhancement of N-Methyl-D-Aspartate NMDA Receptor-Mediated Excitatory Postsynaptic Currents: Implications for HIV-1-Associated Neural Injury

It is widely accepted that human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120) plays an important role in HIV-1-induced neural injury and pathogenesis of HIV-1-associated dementia (HAND). Multiple pathways have been proposed for gp120-induced neurotoxicity, amongst is the activation of N-Methyl-D-Aspartate receptors (NMDARs). It has been shown that gp120 causes neuronal injury or death and gp120 transgenic mice exhibit neurological similarity to that of HAND, all of which can be blocked or attenuated by NMDAR antagonists. Several lines of evidence indicate the subtype and location of activated NMDARs are key determinants of the nature of NMDAR physiology. To examine the subtype and the location of NMDARs affected by gp120, we studied gp120 on subtype NMDAR-mediated EPSCs in the CA1 region of rat hippocampal slices through “blind” whole-cell patch recordings. Our results showed bath application of gp120 increased both NR2A- and NR2B-mediated EPSCs possibly via a presynaptic mechanism, with much stronger effect on NR2B-mediated EPSCs. In contrast, gp120 failed on enhancing AMPA receptor-mediated EPSCs. Ca²⁺ imaging studies revealed that gp120 potentiated glutamate-induced increase of intracellular Ca²⁺ concentration in rat hippocampal neuronal cultures which were blocked by a NMDAR antagonist, but not by an AMPA receptor antagonist, indicating gp120 induces Ca²⁺ influx through NMDARs. Further investigations demonstrated that gp120 increased the EPSCs mediated by extrasynaptic NR2BRs. Taken together, these results demonstrate that gp120 interacts with both NR2A and NR2B subtypes of NMDARs with a predominant action on the extrasynaptic NR2B, implicating a role NR2B may play in HIV-1-associated neuropathology.     

Depression of synaptic transmission and evoked NMDA Ca2+ influx in hippocampal neurons by adenosine and its blockade by LTP or ischemia

Neuronal Ca²⁺ influx in response to repetitive synaptic activation was determined in rat hippocampal slices by measuring the evoked decreases of the extracellular Ca²⁺ concentration with ion-sensitive electrodes in the synaptic layer (ΔCaSpad) and in the CA₁ pyramidal cell layer (ΔCaSpyr). The generation of NMDA receptor-mediated Ca²⁺ fluxes (NMDA Ca²⁺ fluxes) was uncovered by measuring the proportion of ΔCa blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (APV); 1 μmol adenosine increased the critical input frequency required to generate synaptic MNDA Ca²⁺ influx. This effect is apparently exerted by limiting the postsynaptic membrane depolarization and is no longer seen, if the NMDA Ca²⁺ channels have lost their Mg-dependent voltage sensitivity. It has been recently reported by Ben-Ari and co-workers [(1992) Trends Neurosci 15:333–339] that such a loss of voltage sensitivity, leading to an “upregulation” and persistent generation of NMDA Ca²⁺ currents, Can be elicited by an activation of the protein kinase C (PKC) and may be responsible for the initiation of synaptic long-term potentiation (LTP) and for ischemia-induced nerve cell damage. Consistent with this hypothesis, we found that the depressive effect of 1 μmol adenosine on the generation of NMDA Ca²⁺ influx or synaptic transmission can be no longer elicited in hippocampal slices after LTP and after preceding transient brain ischemia in vivo. These findings suggest that the mosaic of different adenosine actions includes some which are related to PKC activation. Accordingly, we observed a synaptic modulation by adenosine which was characterized by a reduced Mg-sensitivity and blocked by phorbol ester treatment. © 1993 Wiley-Liss, Inc.     

Mutant mice with reduced NMDA-NR1 glycine affinity or lack of D-amino acid oxidase function exhibit altered anxiety-like behaviors

Several compounds that promote activation of the N-methyl-d-aspartate receptor (NMDAR) glycine site have been proposed as treatments for schizophrenia, but the impact of these putative antipsychotics on anxiety remains unclear. In this study, we employed genetic and pharmacological mouse models of altered NMDAR glycine site function to examine the effects of these proposed treatments in unconditioned tests of anxiety. In the elevated plus-maze, open field, and novel object test, homozygous Grin1^D481N mutant mice that have a five-fold reduction in NMDAR glycine affinity demonstrated an anxiolytic-like phenotype. In contrast, d-serine, a direct activator of the NMDAR glycine site, and ALX-5407, a glycine transporter-1 (GlyT-1) inhibitor, enhanced anxiety-like behaviors in wild-type and Grin1^D481N mutant animals. Homozygous Dao1^G181R mutant mice that lack function of the d-serine catabolic enzyme, d-amino acid oxidase (DAO), displayed an elevation in anxiety. Deficient DAO activity also reversed the anxiolytic effects of diminished NMDAR function in mice carrying both the homozygous Grin1^D481N and Dao1^G181R mutation. Thus, a direct agonist of the NMDAR glycine site, a GlyT-1 inhibitor, and suppression of DAO function induced anxiogenic-like behaviors. Consequently, application of these treatments for amelioration of schizophrenic symptoms necessitates caution as an enhancement of comorbid anxiety disorders may result.     

Increased expression, but not postsynaptic localisation, of ionotropic glutamate receptors during the late-phase of long-term potentiation in the dentate gyrus in vivo

Long-term potentiation (LTP) is extensively studied as a cellular mechanism of information storage in the brain. The induction and early expression mechanisms of LTP depend on activation and rapid modulation of ionotropic glutamate receptors. However, the mechanisms that underlie maintenance of LTP over the course of days or longer are poorly understood. Here, we have investigated the overall expression of AMPA- and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively), as well as their levels at the synaptic surface membrane and in the postsynaptic density (PSD), in the dentate gyrus at 48 h following the induction of LTP at perforant path synapses in awake rats. We found a high-frequency stimulation-dependent increase in the overall levels of AMPAR subunits GluA1 and GluA2, but not GluA3 in the dentate gyrus. The increases in GluA1 and GluA2 levels were partially NMDAR-dependent, but were not found in biochemically isolated synaptic surface membrane or PSD fractions. In contrast, we found that the core NMDAR subunit, GluN1, increased in the synaptic surface-membrane fraction but it also was not targeted to the PSD. The GluA1 and GluA2 expression and the surface localisation of GluN1 returned to baseline levels by 2 weeks post-LTP induction. These data suggest that the late-phase LTP is not mediated by an overt increase in the AMPAR content of perforant path synapses. The increase in surface expression NMDARs may influence thresholds for future plasticity events.     

Phorbol 12-Myristate 13-Acetate Enhances Long-Term Potentiation in the Hippocampus through Activation of Protein Kinase Cδ and ε

Many intracellular proteins and signaling cascades contribute to the sensitivity of N-methyl-D-aspartate receptors (NMDARs). One such putative contributor is the serine/threonine kinase, protein kinase C (PKC). Activation of PKC by phorbol 12-myristate 13-acetate (PMA) causes activation of extracellular signal-regulated kinase (ERK) and promotes the formation of new spines in cultured hippocampal neurons. The purpose of this study was to examine which PKC isoforms are responsible for the PMA-induced augmentation of long-term potentiation (LTP) in the CA1 stratum radiatum of the hippocampus in vitro and verify that this facilitation requires NMDAR activation. We found that PMA enhanced the induction of LTP by a single episode of theta-burst stimulation in a concentration-dependent manner without affecting to magnitude of baseline field excitatory postsynaptic potentials. Facilitation of LTP by PMA (200 nM) was blocked by the nonspecific PKC inhibitor, Ro 31-8220 (10µM); the selective PKCδ inhibitor, rottlerin (1µM); and the PKCε inhibitor, TAT-εV1-2 peptide (500 nM). Moreover, the NMDAR blocker DL-APV (50µM) prevented enhancement of LTP by PMA. Our results suggest that PMA contributes to synaptic plasticity in the nervous system via activation of PKCδ and/or PKCε, and confirm that NMDAR activity is required for this effect.     

Pharmacological study on Alzheimer's drugs targeting calcium/calmodulin-dependent protein kinase II

In the brain of Alzheimer's disease patients, down-regulation of both cholinergic and glutamatergic systems have been found and is thought to play an important role in impairment of cognition, learning, and memory. Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as a cognitive-enhancing effect. The present study was undertaken to elucidate mechanisms underlying the action of nefiracetam on glutamatergic receptors and intracellular protein kinases. N-Methyl-D-aspartate (NMDA)-evoked currents were recorded from rat cortical neurons in long-term cultured primary neurons using the whole-cell patch-clamp technique. NMDA-evoked currents were greatly and reversibly potentiated by bath application of nefiracetam, resulting in a bell-shaped dose-response curve. The maximum potentiation of 170% relative to the control was produced at 10 nM. Treatment with an inhibitor of the glycine binding site of the NMDA receptor, 7-chlorokynurenic acid, at 1 μM prevented augmentation of NMDA-evoked currents by nefiracetam. In rat hippocampal CA1 slices, field excitatory postsynaptic potentials were recorded by stimulation of Schaffer collateral/commissural pathways. Nefiracetam treatment significantly enhanced long-term potentiation (LTP) with the same bell-shaped dose-response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with calcium/calmodulin-dependent protein kinase II (CaMKII) activation with concomitant increase in phosphorylation of AMPA-type glutamate receptor subunit 1 (GluA1) (Ser-831) as a postsynaptic CaMKII substrate. In conclusion, nefiracetam enhances NMDA-receptor function through stimulation of its glycine binding site and nefiracetam-induced CaMKII activation likely contributes to improvement of cognition, learning, and memory.     

Reduction of Endogenous Kynurenic Acid Formation Enhances Extracellular Glutamate, Hippocampal Plasticity, and Cognitive Behavior

At endogenous brain concentrations, the astrocyte-derived metabolite kynurenic acid (KYNA) antagonizes the α7 nicotinic acetylcholine receptor and, possibly, the glycine co-agonist site of the NMDA receptor. The functions of these two receptors, which are intimately involved in synaptic plasticity and cognitive processes, may, therefore, be enhanced by reductions in brain KYNA levels. This concept was tested in mice with a targeted deletion of kynurenine aminotransferase II (KAT II), a major biosynthetic enzyme of brain KYNA. At 21 days of age, KAT II knock-out mice had reduced hippocampal KYNA levels (−71%) and showed significantly increased performance in three cognitive paradigms that rely in part on the integrity of hippocampal function, namely object exploration and recognition, passive avoidance, and spatial discrimination. Moreover, compared with wild-type controls, hippocampal slices from KAT II-deficient mice showed a significant increase in the amplitude of long-term potentiation in vitro. These functional changes were accompanied by reduced extracellular KYNA (−66%) and increased extracellular glutamate (+51%) concentrations, measured by hippocampal microdialysis in vivo. Taken together, a picture emerges in which a reduction in the astrocytic formation of KYNA increases glutamatergic tone in the hippocampus and enhances cognitive abilities and synaptic plasticity. Our studies raise the prospect that interventions aimed specifically at reducing KYNA formation in the brain may constitute a promising molecular strategy for cognitive improvement in health and disease.     

P2Y1 receptors inhibit long-term depression in the prefrontal cortex

Long-term depression (LTD) is a form of synaptic plasticity that may contribute to information storage in the central nervous system. Here we report that LTD can be elicited in layer 5 pyramidal neurons of the rat prefrontal cortex by pairing low frequency stimulation with a modest postsynaptic depolarization. The induction of LTD required the activation of both metabotropic glutamate receptors of the mGlu1 subtype and voltage-sensitive Ca²⁺ channels (VSCCs) of the T/R, P/Q and N types, leading to the stimulation of intracellular inositol trisphosphate (IP3) receptors by IP3 and Ca²⁺. The subsequent release of Ca²⁺ from intracellular stores activated the protein phosphatase cascade involving calcineurin and protein phosphatase 1. The activation of purinergic P2Y₁ receptors blocked LTD. This effect was prevented by P2Y₁ receptor antagonists and was absent in mice lacking P2Y₁ but not P2Y₂ receptors. We also found that activation of P2Y₁ receptors inhibits Ca²⁺ transients via VSCCs in the apical dendrites and spines of pyramidal neurons. In addition, we show that the release of ATP under hypoxia is able to inhibit LTD by acting on postsynaptic P2Y₁ receptors. In conclusion, these data suggest that the reduction of Ca²⁺ influx via VSCCs caused by the activation of P2Y₁ receptors by ATP is the possible mechanism for the inhibition of LTD in prefrontal cortex.     

Ifenprodil, a NR2B-selective antagonist of NMDA receptor, inhibits reverse Na(+)/Ca(2+) exchanger in neurons

Glutamate-induced delayed calcium dysregulation (DCD) is causally linked to excitotoxic neuronal death. The mechanisms of DCD are not completely understood, but it has been proposed that the excessive influx of external Ca²⁺ is essential for DCD. The NMDA-subtype of glutamate receptor (NMDAR) and the plasmalemmal Na⁺/Ca²⁺ exchanger operating in the reverse mode (NCX_rev) have been implicated in DCD. In experiments with “younger” neurons, 6–8 days in vitro (6–8 DIV), in which the NR2A-containing NMDAR expression is low, ifenprodil, an inhibitor of NR2B-containing NMDAR, completely prevented DCD whereas PEAQX, another NMDAR antagonist that preferentially interacts with NR2A-NMDAR, was without effect. With “older” neurons (13–16 DIV), in which NR2A- and NR2B-NMDARs are expressed to a greater extent, both ifenprodil and PEAQX applied separately failed to prevent DCD. However, combined application of ifenprodil and PEAQX completely averted DCD. Ifenprodil and ifenprodil-like NR2B-NMDAR antagonists Ro 25-6981 and Co 101244 but not PEAQX or AP-5 inhibited gramicidin- and Na⁺/NMDG-replacement-induced increases in cytosolic Ca²⁺ mediated predominantly by NCX_rev. This suggests that ifenprodil, Ro 25-6981, and Co 101244 inhibit NCX_rev. The ability of ifenprodil to inhibit NCX_rev correlates with its efficacy in preventing DCD and emphasizes an important role of NCX_rev in DCD. Overall our data suggest that both NR2A- and NR2B-NMDARs are involved in DCD in “older” neurons, and it is necessary to inhibit both NMDARs and NCX_rev to prevent glutamate-induced DCD.     

L-687,414, a low efficacy NMDA receptor glycine site partial agonist in vitro,does not prevent hippocampal LTP in vivo at plasma levels known to be neuroprotective

1. N-methyl-D-aspartic acid (NMDA) receptors are known to play a key role in the induction phase of long-term potentiation (LTP) at certain hippocampal synapses and to represent some component of spatial learning in animals. The ability of NMDA receptor antagonists (or gene knockout) to impair LTP has led to the suggestion that the therapeutic use of such antagonists may impair cognitive function in humans. The present study compares the effects on LTP of NMDA receptor ion channel block by MK-801 and glycine-site antagonism by 3R(+)cis-4-methyl-pyrrollid-2-one (L-687,414).
2. In vitro experiments using rat cortical slices revealed L-687,414 to be ∼3.6 fold more potent than its parent analogue, R(+)HA-966 at antagonizing NMDA-evoked population depolarizations (apparent K_bs: 15 μM and 55 μM, respectively).
3. Whole-cell voltage-clamp experiments using rat cultured cortical neurones revealed L-687,414 to shift to the right the concentration-response relationship for NMDA-evoked inward current responses (pK_b=6.2±0.12). L-687,414 affinity for the glycine site on the NMDA receptor complex was also determined from concentration-inhibition curves, pKi=6.1±0.09. In the latter experiments, L-687,414 and R(+)HA-966 were unable to completely abolish inward current responses suggesting each compound to be a low efficacy partial agonist (estimated intrinsic activity=∼10 and 20% of glycine, respectively).
4. L-687,414 and MK-801 were compared for their effects on NMDA receptor-dependent LTP in the dentate gyrus of anaethestized rats following high frequency stimulation of the medial perforant path (mPP) afferents. Control rats, administered saline (0.4 ml kg⁻¹ followed by 0.0298 ml min⁻¹), showed a robust augmentation of the population e.p.s.p. risetime (LTP) recorded in the dentate hilus following tetanic stimulation of the mPP. LTP was effectively abolished in a separate group of rats treated with an MK-801 dosing regimen (0.12 mg kg⁻¹ i.v. followed by 1.8 μg kg⁻¹ h⁻¹) known to produce maximal neuroprotection in a rat stroke model but, by contrast, remained largely intact in a third group of animals given a similarly neuroprotective L-687,414 treatment (28 mg kg⁻¹ i.v. followed by 28 mg kg⁻¹ h⁻¹).
5. These experiments suggest that a low level of intrinsic activity at the glycine site may be sufficient to support NMDA receptor-dependent LTP but in circumstances where there is likely to be an excessive NMDA receptor activation the agonism associated with a low efficacy partial agonist, such as L-687,414, is dominated by the antagonist properties. Thus, an NMDA receptor partial agonist profile may offer a therapeutic advantage over neutral antagonists by permitting an acceptable level of `normal'' synaptic transmission whilst curtailing excessive receptor activation.

     

IDRA-21, a positive AMPA receptor modulator, inhibits synaptic and extrasynaptic NMDA receptor mediated events in cultured cerebellar granule cells

IDRA-21 (7-chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide) reduces alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptors desensitisation in vitro and restores learning and cognitive impairment in vivo. In this study, we show that in cerebellar granule cells (CGCs) in culture IDRA-21 reduces N-methyl-d-aspartate receptor (NMDAR) whole-cell currents. The effect is neither competitive nor voltage-dependent. The reduction of NMDA currents is stronger at low glycine concentrations suggesting an interaction with this site. IDRA-21 shortens miniature excitatory postsynaptic currents mediated by NMDARs (NMDA-mEPSCs) in CGCs grown in low potassium with no effect on peak amplitudes. By using fast glutamate application onto CGCs nucleated patches, we found that IDRA-21 decreases both decay time constant and amplitude of the current. Experiments performed on recombinant NMDAR expressed in HEK 293 cells showed that IDRA-21 was more effective on NR1a-NR2B than NR1a-NR2A receptors highlighting a subunit selectivity of the drug. Our findings make light on a novel target for IDRA-21: NMDA receptors function is negatively modulated and the different action at the level of extrasynaptic and synaptic receptors could be ascribed to a partial selectivity for NR2B subunits.