In the Telencephalon, GluN2C NMDA Receptor Subunit mRNA is Predominately Expressed in Glial Cells and GluN2D mRNA in Interneurons.

N-methyl-D-aspartate receptors (NMDARs) are widely distributed in the brain with high concentrations in the telencephalon where they modulate synaptic plasticity, working memory, and other functions. While the actions of the predominate GluN2 NMDAR subunits, GluN2A and GluN2B are relatively well understood, the function of GluN2C and GluN2D subunits in the telencephalon is largely unknown. To better understand the possible role of GluN2C subunits, we used fluorescence in situ hybridization (FISH) together with multiple cell markers to define the distribution and type of cells expressing GluN2C mRNA. Using a GluN2C-KO mouse as a negative control, GluN2C mRNA expression was only found in non-neuronal cells (NeuN-negative cells) in the hippocampus, striatum, amygdala, and cerebral cortex. For these regions, a significant fraction of GFAP-positive cells also expressed GluN2C mRNA. Overall, for the telencephalon, the globus pallidus and olfactory bulb were the only regions where GluN2C was expressed in neurons. In contrast to GluN2C, GluN2D subunit mRNA colocalized with neuronal and not astrocyte markers or GluN2C mRNA in the telencephalon (except for the globus pallidus). GluN2C mRNA did, however, colocalize with GluN2D in the thalamus where neuronal GluN2C expression is found. These findings strongly suggest that GluN2C has a very distinct function in the telencephalon compared to its role in other brain regions and compared to other GluN2-containing NMDARs. NMDARs containing GluN2C may have a specific role in regulating L-glutamate or D-serine release from astrocytes in response to L-glutamate spillover from synaptic activity.

GluN2D N-Methyl-d-Aspartate Receptor Subunit Contribution to the Stimulation of Brain Activity and Gamma Oscillations by Ketamine: Implications for Schizophrenia.

The dissociative anesthetic ketamine elicits symptoms of schizophrenia at subanesthetic doses by blocking N-methyl-d-aspartate receptors (NMDARs). This property led to a variety of studies resulting in the now well-supported theory that hypofunction of NMDARs is responsible for many of the symptoms of schizophrenia. However, the roles played by specific NMDAR subunits in different symptom components are unknown. To evaluate the potential contribution of GluN2D NMDAR subunits to antagonist-induced cortical activation and schizophrenia symptoms, we determined the ability of ketamine to alter regional brain activity and gamma frequency band neuronal oscillations in wild-type (WT) and GluN2D-knockout (GluN2D-KO) mice. In WT mice, ketamine (30 mg/kg, i.p.) significantly increased [(14)C]-2-deoxyglucose ([(14)C]-2DG) uptake in the medial prefrontal cortex (mPFC), entorhinal cortex and other brain regions, and decreased activity in the somatosensory cortex and inferior colliculus. In GluN2D-KO mice, however, ketamine did not significantly increase [(14)C]-2DG uptake in any brain region examined, yet still decreased [(14)C]-2DG uptake in the somatosensory cortex and inferior colliculus. Ketamine also increased locomotor activity in WT mice but not in GluN2D-KO mice. In electrocorticographic analysis, ketamine induced a 111% ± 16% increase in cortical gamma-band oscillatory power in WT mice, but only a 15% ± 12% increase in GluN2D-KO mice. Consistent with GluN2D involvement in schizophrenia-related neurologic changes, GluN2D-KO mice displayed impaired spatial memory acquisition and reduced parvalbumin (PV)-immunopositive staining compared with control mice. These results suggest a critical role of GluN2D-containing NMDARs in neuronal oscillations and ketamine's psychotomimetic, dissociative effects and hence suggests a critical role for GluN2D subunits in cognition and perception.

Bidirectional Effect of Pregnenolone Sulfate on GluN1/GluN2A N-Methyl-D-Aspartate Receptor Gating Depending on Extracellular Calcium and Intracellular Milieu.

Pregnenolone sulfate (PS), one of the most commonly occurring neurosteroids in the central nervous system, influences the function of several receptors. PS modulates N-methyl-D-aspartate receptors (NMDARs) and has been shown to have both positive and negative modulatory effects on NMDAR currents generally in a subtype-selective manner. We assessed the gating mechanism of PS modulation of GluN1/GluN2A receptors transiently expressed in human embryonic kidney 293 cells using whole-cell and single-channel electrophysiology. Only a modest effect on the whole-cell responses was observed by PS in dialyzed (nonperforated) whole-cell recordings. Interestingly, in perforated conditions, PS was found to increase the whole-cell currents in the absence of nominal extracellular Ca(2+), whereas PS produced an inhibition of the current responses in the presence of 0.5 mM extracellular Ca(2+). The Ca(2+)-binding DRPEER motif and GluN1 exon-5 were found to be critical for the Ca(2+)-dependent bidirectional effect of PS. Single-channel cell-attached analysis demonstrated that PS primarily affected the mean open time to produce its effects: positive modulation mediated by an increase in duration of open time constants, and negative modulation mediated by a reduction in the time spent in a long-lived open state of the receptor. Further kinetic modeling of the single-channel data suggested that the positive and negative modulatory effects are mediated by different gating steps which may represent GluN2 and GluN1 subunit-selective conformational changes, respectively. Our studies provide a unique mechanism of modulation of NMDARs by an endogenous neurosteroid, which has implications for identifying state-dependent molecules.

Different NMDA receptor subtypes mediate induction of long-term potentiation and two forms of short-term potentiation at CA1 synapses in rat hippocampus in vitro.

Potentiation at synapses between CA3 and the CA1 pyramidal neurons comprises both transient and sustained phases, commonly referred to as short-term potentiation (STP or transient LTP) and long-term potentiation (LTP), respectively. Here, we utilized four subtype-selective N-methyl-d-aspartate receptor (NMDAR) antagonists to investigate whether the induction of STP and LTP is dependent on the activation of different NMDAR subtypes. We find that the induction of LTP involves the activation of NMDARs containing both the GluN2A and the GluN2B subunits. Surprisingly, however, we find that STP can be separated into two components, the major form of which involves activation of NMDARs containing both GluN2B and GluN2D subunits. These data demonstrate that synaptic potentiation at CA1 synapses is more complex than is commonly thought, an observation that has major implications for understanding the role of NMDARs in cognition.

The continually evolving role of NMDA receptors in neurobiology and disease.

40 years ago, Jeff Watkins and Richard (Dick) Evans (Watkins and Evans, 1981) published their review on excitatory amino acids. The review, combined with the tools that they and their colleagues developed, significantly changed the field of neurobiology. This Special Issue focused on NMDA receptors is one of six that commemorate this anniversary. The broadest impact of the review, and the work of this group (Collingridge and Abraham, 2022; Evans and Watkins, 2021; Watkins, 2000), was to establish the three receptor scheme for the excitatory, l-glutamate-gated ion channels named for their selective agonists: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) (Monaghan et al., 1989; Watkins et al., 1990; Watkins, 2000; Hansen et al., 2021; Lodge, 2009; Watkins and Jane, 2006). The contribution of the Evans and Watkins team was perhaps greatest to the study of NMDA receptors - it was essential. We describe here this fundamental contribution and provide an update on NMDARs as the understanding of their function continues to grow more complex.

Pharmacological characterization of a novel negative allosteric modulator of NMDA receptors, UBP792.

N-methyl-d-aspartate (NMDA) receptors (NMDARs) are a subtype of ionotropic glutamate receptor with important roles in CNS function. Since excessive NMDAR activity can lead to neuronal cell death and epilepsy, there is interest in developing NMDAR negative allosteric modulators (NAMs) as neuroprotective agents. In this study, we characterize the inhibitory properties of a novel NMDAR antagonist, UBP792. This compound displays partial subtype-selectivity by having a varied maximal inhibition of GluN2A-, GluN2B-, GluN2C-, and GluN2D-containing receptors (52%, 70%, 87%, 89%, respectively) with IC50s 4-10 µM. UBP792 inhibited NMDAR responses by reducing l-glutamate and glycine potencies and efficacies. Consistent with non-competitive inhibition, increasing agonist concentrations 30-fold did not reduce UBP792 potency. UBP792 inhibition was also not competitive with the structurally-related positive allosteric modulator (PAM) UBP684. UBP792 activity was voltage-independent, unaffected by GluN1's exon-5, and reduced at low pH (except for GluN1/GluN2A receptors which were more sensitive at acidic pH). UBP792 binding appeared independent of agonist binding and may be entering the plasma membrane to gain access to its binding site. Inhibition by UBP792 is reduced when the ligand-binding domain (LBD) of the GluN2 subunit, but not that of the GluN1 subunit, is cross-linked in the closed-cleft, activated conformation. Thus, UBP792 may be inhibiting by stabilizing an open GluN2-LBD cleft associated with channel inactivation or by stabilizing downstream closed channel conformations allosterically-coupled to the GluN2-LBD. These findings further expand the repertoire displayed by NMDAR NAMs thus expanding the opportunities for developing NMDAR modulators with the most appropriate selectivity and physiological actions for specific therapeutic indications.

A novel family of negative and positive allosteric modulators of NMDA receptors.

The N-methyl-D-aspartate (NMDA) receptor family regulates various central nervous system functions, such as synaptic plasticity. However, hypo- or hyperactivation of NMDA receptors is critically involved in many neurological and psychiatric conditions, such as pain, stroke, epilepsy, neurodegeneration, schizophrenia, and depression. Consequently, subtype-selective positive and negative modulators of NMDA receptor function have many potential therapeutic applications not addressed by currently available compounds. We have identified allosteric modulators with several novel patterns of NMDA receptor subtype selectivity that have a novel mechanism of action. In a series of carboxylated naphthalene and phenanthrene derivatives, compounds were identified that selectively potentiate responses at GluN1/GluN2A [e.g., 9-iodophenanthrene-3-carboxylic acid (UBP512)]; GluN1/GluN2A and GluN1/GluN2B [9-cyclopropylphenanthrene-3-carboxylic acid (UBP710)]; GluN1/GluN2D [3,5-dihydroxynaphthalene-2-carboxylic acid (UBP551)]; or GluN1/GluN2C and GluN1/GluN2D receptors [6-, 7-, 8-, and 9-nitro isomers of naphth[1,2-c][1,2,5]oxadiazole-5-sulfonic acid (NSC339614)] and have no effect or inhibit responses at the other NMDA receptors. Selective inhibition was also observed; UBP512 inhibits only GluN1/GluN2C and GluN1/GluN2D receptors, whereas 6-bromo-2-oxo-2H-chromene-3-carboxylic acid (UBP608) inhibits GluN1/GluN2A receptors with a 23-fold selectivity compared with GluN1/GluN2D receptors. The actions of these compounds were not competitive with the agonists L-glutamate or glycine and were not voltage-dependent. Whereas the N-terminal regulatory domain was not necessary for activity of either potentiators or inhibitors, segment 2 of the agonist ligand-binding domain was important for potentiating activity, whereas subtype-specific inhibitory activity was dependent upon segment 1. In terms of chemical structure, activity profile, and mechanism of action, these modulators represent a new class of pharmacological agents for the study of NMDA receptor subtype function and provide novel lead compounds for a variety of neurological disorders.

NMDA receptors containing GluN2C and GluN2D subunits have opposing roles in modulating neuronal oscillations; potential mechanism for bidirectional feedback.

NMDA receptor (NMDAR) antagonists such as ketamine, can reproduce many of the symptoms of schizophrenia. A reliable indicator of NMDAR channel blocker action in vivo is the augmentation of neuronal oscillation power. Since the coordinated and rhythmic activation of neuronal assemblies (oscillations) is necessary for perception, cognition and working memory, their disruption (inappropriate augmentation or inhibition of oscillatory power or inter-regional coherence) both in psychiatric conditions and with NMDAR antagonists may reflect the underlying defects causing schizophrenia symptoms. NMDAR antagonists and knockout (KO) mice were used to evaluate the role of GluN2C and GluN2D NMDAR subunits in generating NMDAR antagonist-induced oscillations. We find that basal oscillatory power was elevated in GluN2C-KO mice, especially in the low gamma frequencies while there was no statistically significant difference in basal oscillations between WT and GluN2D-KO mice. Compared to wildtype (WT) mice, NMDAR channel blockers caused a greater increase in oscillatory power in GluN2C-KO mice and were relatively ineffective in inducing oscillations in GluN2D-KO mice. In contrast, preferential blockade of GluN2A- and GluN2B-containing receptors induced oscillations that did not appear to be changed in either KO animal. We propose a model wherein NMDARs containing GluN2C in astrocytes and GluN2D in interneurons serve to detect local cortical excitatory synaptic activity and provide excitatory and inhibitory feedback, respectively, to local populations of postsynaptic excitatory neurons and thereby bidirectionally modulate oscillatory power.

Structural basis of subtype-selective competitive antagonism for GluN2C/2D-containing NMDA receptors.

N-Methyl-D-aspartate receptors (NMDARs) play critical roles in the central nervous system. Their heterotetrameric composition generates subtypes with distinct functional properties and spatio-temporal distribution in the brain, raising the possibility for subtype-specific targeting by pharmacological means for treatment of neurological diseases. While specific compounds for GluN2A and GluN2B-containing NMDARs are well established, those that target GluN2C and GluN2D are currently underdeveloped with low potency and uncharacterized binding modes. Here, using electrophysiology and X-ray crystallography, we show that UBP791 ((2S*,3R*)-1-(7-(2-carboxyethyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) inhibits GluN2C/2D with 40-fold selectivity over GluN2A-containing receptors, and that a methionine and a lysine residue in the ligand binding pocket (GluN2D-Met763/Lys766, GluN2C-Met736/Lys739) are the critical molecular elements for the subtype-specific binding. These findings led to development of UBP1700 ((2S*,3R*)-1-(7-(2-carboxyvinyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) which shows over 50-fold GluN2C/2D-selectivity over GluN2A with potencies in the low nanomolar range. Our study shows that the L-glutamate binding site can be targeted for GluN2C/2D-specific inhibition.

N-methyl-D-aspartate (NMDA) receptor NR2 subunit selectivity of a series of novel piperazine-2,3-dicarboxylate derivatives: preferential blockade of extrasynaptic NMDA receptors in the rat hippocampal CA3-CA1 synapse.

N-Methyl-d-aspartate (NMDA) receptor antagonists that are highly selective for specific NMDA receptor 2 (NR2) subunits have several potential therapeutic applications; however, to date, only NR2B-selective antagonists have been described. Whereas most glutamate binding site antagonists display a common pattern of NR2 selectivity, NR2A > NR2B > NR2C > NR2D (high to low affinity), (2S*,3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid (PPDA) has a low selectivity for NR2C- and NR2D-containing NMDA receptors. A series of PPDA derivatives were synthesized and then tested at recombinant NMDA receptors expressed in Xenopus laevis oocytes. In addition, the optical isomers of PPDA were resolved; the (-) isomer displayed a 50- to 80-fold greater potency than the (+) isomer. Replacement of the phenanthrene moiety of PPDA with naphthalene or anthracene did not improve selectivity. However, phenylazobenzoyl (UBP125) or phenylethynylbenzoyl (UBP128) substitution significantly improved selectivity for NR2B-, NR2C-, and NR2D-containing receptors over NR2A-containing NMDA receptors. Phenanthrene attachment at the 3 position [(2R*,3S*)-1-(phenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP141); (2R*,3S*)-1-(9-bromophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP145); (2R*,3S*)-1-(9-chlorophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP160); and (2R*,3S*)-1-(9-iodophenanthrene-3-carbonyl)piperazine-2,3-dicarboxylic acid (UBP161)] displayed improved NR2D selectivity. UBP141 and its 9-brominated homolog (UBP145) both display a 7- to 10- fold selectivity for NR2D-containing receptors over NR2B- or NR2A-containing receptors. Schild analysis indicates that these two compounds are competitive glutamate binding site antagonists. Consistent with a physiological role for NR2D-containing receptors in the hippocampus, UBP141 (5 muM) displayed greater selectivity than PPDA for inhibiting the slow-decaying component of the NMDA receptor-mediated CA3-CA1 synaptic response in rat hippocampal slices. UBP125, UBP128, UBP141, and UBP145 may be useful tools for determining the function of NMDA receptor subtypes.

Calcium-permeable AMPA receptors containing Q/R-unedited GluR2 direct human neural progenitor cell differentiation to neurons.

We identify calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors on human neural progenitor cells (NPCs) and present a physiological role in neurogenesis. RNA editing of the GluR2 subunit at the Q/R site is responsible for making most AMPA receptors impermeable to calcium. Because a single-point mutation could eliminate the need for editing at the Q/R site and Q/R-unedited GluR2 exists during embryogenesis, the Q/R-unedited GluR2 subunit presumably has some important actions early in development. Using calcium imaging, we found that NPCs contain calcium-permeable AMPA receptors, whereas NPCs differentiated to neurons and astrocytes express calcium-impermeable AMPA receptors. We utilized reverse-transcription polymerase chain reaction and BbvI digestion to demonstrate that NPCs contain Q/R-unedited GluR2, and differentiated cells contain Q/R-edited GluR2 subunits. This is consistent with the observation that the nuclear enzyme responsible for Q/R-editing, adenosine deaminase (ADAR2), is increased during differentiation. Activation of calcium-permeable AMPA receptors induces NPCs to differentiate to the neuronal lineage and increases dendritic arbor formation in NPCs differentiated to neurons. AMPA-induced differentiation of NPCs to neurons is abrogated by overexpression of ADAR2 in NPCs. This elucidates the role of AMPA receptors as inductors of neurogenesis and provides a possible explanation for why the Q/R editing process exists.

Positive and Negative Allosteric Modulators of N-Methyl-d-aspartate (NMDA) Receptors: Structure-Activity Relationships and Mechanisms of Action.

Excitatory activity in the CNS is predominately mediated by l-glutamate through several families of l-glutamate neurotransmitter receptors. Of these, the N-methyl-d-aspartate receptor (NMDAR) family has many critical roles in CNS function and in various neuropathological and psychiatric conditions. Until recently, the types of compounds available to regulate NMDAR function have been quite limited in terms of mechanism of action, subtype selectivity, and biological effect. However, several new classes of NMDAR agents have now been identified that are positive or negative allosteric modulators (PAMs and NAMs, respectively) with various patterns of NMDAR subtype selectivity. These new agents act at several newly recognized binding sites on the NMDAR complex and offer significantly greater pharmacological control over NMDAR activity than previously available agents. The purpose of this review is to summarize the structure-activity relationships for these new NMDAR modulator drug classes and to describe the current understanding of their mechanisms of action.

Investigation of the structural requirements for N-methyl-D-aspartate receptor positive and negative allosteric modulators based on 2-naphthoic acid.

The N-methyl-D-aspartate receptor (NMDAR), a ligand-gated ion channel activated by L-glutamate and glycine, plays a major role in the synaptic plasticity underlying learning and memory. NMDARs are involved in neurodegenerative disorders such as Alzheimer's and Parkinson's disease and NMDAR hypofunction is implicated in schizophrenia. Herein we describe structure-activity relationship (SAR) studies on 2-naphthoic acid derivatives to investigate structural requirements for positive and negative allosteric modulation of NMDARs. These studies identified compounds such as UBP684 (14b), which act as pan potentiators by enhancing NMDAR currents in diheteromeric NMDAR tetramers containing GluN1 and GluN2A-D subunits. 14b and derivatives thereof are useful tools to study synaptic function and have potential as leads for the development of drugs to treat schizophrenia and disorders that lead to a loss of cognitive function. In addition, SAR studies have identified a series of styryl substituted compounds with partial NAM activity and a preference for inhibition of GluN2D versus the other GluN2 subunits. In particular, the 3-and 2-nitrostyryl derivatives UBP783 (79i) and UBP792 (79h) had IC50s of 1.4 µM and 2.9 µM, respectively, for inhibition of GluN2D but showed only 70-80% maximal inhibition. GluN2D has been shown to play a role in excessive pain transmission due to nerve injury and potentially in neurodegenerative disorders. Partial GluN2D inhibitors may be leads for the development of drugs to treat these disorders without the adverse effects observed with full NMDAR antagonists.

The NMDA receptor intracellular C-terminal domains reciprocally interact with allosteric modulators.

N-methyl-d-aspartate receptors (NMDARs) have multiple prominent roles in CNS function but their excessive or insufficient activity contributes to neuropathological/psychiatric disorders. Consequently, a variety of positive and negative allosteric modulators (PAMs and NAMs, respectively) have recently been developed. Although these modulators bind to extracellular domains, in the present report we find that the NMDAR's intracellular C-terminal domains (CTDs) significantly influence PAM/NAM activity. GluN2 CTD deletion robustly affected NAM and PAM activity with both enhancing and inhibiting effects that were compound-specific and NMDAR subunit-specific. In three cases, individual PAMs became NAMs at specific GluN2-truncated receptors. In contrast to GluN2, GluN1 CTD removal only reduced PAM activity of UBP684 and CIQ, and did not affect NAM activity. Consistent with these findings, agents altering phosphorylation state or intracellular calcium levels displayed receptor-specific and compound-specific effects on PAM activity. It is possible that the GluN2's M4 domain transmits intracellular modulatory signals from the CTD to the M1/M4 channel gating machinery and that this site is a point of convergence in the direct or indirect actions of several PAMs/NAMs thus rendering them sensitive to CTD status. Thus, allosteric modulators are likely to have a marked and varied sensitivity to post-translational modifications, protein-protein associations, and intracellular ions. The interaction between PAM activity and NMDAR CTDs appears reciprocal. GluN1 CTD-deletion eliminated UBP684, but not pregnenolone sulfate (PS), PAM activity. And, in the absence of agonists, UBP684, but not PS, was able to promote movement of fluorescently-tagged GluN1-CTDs. Thus, it may be possible to pharmacologically target NMDAR metabotropic activity in the absence of channel activation.

Multiple roles of GluN2B-containing NMDA receptors in synaptic plasticity in juvenile hippocampus.

In the CA1 area of the hippocampus N-methyl-d-aspartate receptors (NMDARs) mediate the induction of long-term depression (LTD), short-term potentiation (STP) and long-term potentiation (LTP). All of these forms of synaptic plasticity can be readily studied in juvenile hippocampal slices but the involvement of particular NMDAR subunits in the induction of these different forms of synaptic plasticity is currently unclear. Here, using NVP-AAM077, Ro 25-6981 and UBP145 to target GluN2A-, 2B- and 2D-containing NMDARs respectively, we show that GluN2B-containing NMDARs (GluN2B) are involved in the induction of LTD, STP and LTP in slices prepared from P14 rat hippocampus. A concentration of Ro (1 µM) that selectively blocks GluN2B-containing diheteromers is able to block LTD. It also inhibits a component of STP without affecting LTP. A higher concentration of Ro (10 µM), that also inhibits GluN2A/B triheteromers, blocks LTP. UBP145 selectively inhibits the Ro-sensitive component of STP whereas NVP inhibits LTP. These data are consistent with a role of GluN2B diheretomers in LTD, a role of both GluN2B- and GluN2D- containing NMDARs in STP and a role of GluN2A/B triheteromers in LTP. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

A single-channel mechanism for pharmacological potentiation of GluN1/GluN2A NMDA receptors.

NMDA receptors (NMDARs) contribute to several neuropathological processes. Novel positive allosteric modulators (PAMs) of NMDARs have recently been identified but their effects on NMDAR gating remain largely unknown. To this end, we tested the effect of a newly developed molecule UBP684 on GluN1/GluN2A receptors. We found that UBP684 potentiated the whole-cell currents observed under perforated-patch conditions and slowed receptor deactivation. At the single channel level, UBP684 produced a dramatic reduction in long shut times and a robust increase in mean open time. These changes were similar to those produced by NMDAR mutants in which the ligand-binding domains (LBDs) are locked in the closed clamshell conformation by incorporating a disulfide bridge. Since the locked glutamate-binding clefts primarily contributes to receptor efficacy these results suggests that UBP684 binding may induce switch in conformation similar to glutamate LBD locked state. Consistent with this prediction UBP684 displayed greater potentiation of NMDARs with only the GluN1 LBD locked compared to NMDARs with only the GluN2 LBD locked. Docking studies suggest that UBP684 binds to the GluN1 and GluN2 LBD interface supporting its potential ability in stabilizing the LBD closed conformation. Together these studies identify a novel pharmacological mechanism of facilitating the function of NMDARs.

Mechanism and properties of positive allosteric modulation of N-methyl-d-aspartate receptors by 6-alkyl 2-naphthoic acid derivatives.

The theory that N-methyl-d-aspartate receptor (NMDAR) hypofunction is responsible for the symptoms of schizophrenia is well supported by many pharmacological and genetic studies. Accordingly, positive allosteric modulators (PAMs) that augment NMDAR signaling may be useful for treating schizophrenia. Previously we have identified several NMDAR PAMs containing a carboxylic acid attached to naphthalene, phenanthrene, or coumarin ring systems. In this study, we describe several functional and mechanistic properties of UBP684, a 2-naphthoic acid derivative, which robustly potentiates agonist responses at each of the four GluN1a/GluN2 receptors and at neuronal NMDARs. UBP684 increases the maximal l-glutamate/glycine response while having minor subunit-specific effects on agonist potency. PAM binding is independent of agonist binding, and PAM activity is independent of membrane voltage, redox state, and the GluN1 exon 5 N-terminal insert. UBP684 activity is, however, markedly pH-dependent, with greater potentiation occurring at lower pHs and inhibitory activity at pH 8.4. UBP684 increases channel open probability (Po) and slows receptor deactivation time upon removal of l-glutamate, but not glycine. The structurally related PAM, UBP753, reproduced most of these findings, but did not prolong agonist removal deactivation time. Studies using cysteine mutants to lock the GluN1 and GluN2 ligand-binding domains (LBDs) in the agonist-bound states indicate that PAM potentiation requires GluN2 LBD conformational flexibility. Together, these findings suggest that UBP684 and UBP753 stabilize the GluN2 LBD in an active conformation and thereby increase Po. Thus, UBP684 and UBP753 may serve as lead compounds for developing agents to enhance NMDAR activity in disorders associated with NMDAR hypofunction.

Regional variations in NMDA receptor downregulation in streptozotocin-diabetic rat brain.

Insulin insufficiency has multiple actions on the CNS. Three weeks after streptozotocin-induced diabetes in rats, we found a preferential downregulation of l-[(3)H]glutamate-labeled NMDA receptors in primary sensory cortical regions. Layers I-III of the parietal cortex and superficial piriform cortex were the most sensitive followed by other cortical regions and, in turn, deeper brain structures. These effects on NMDA receptors can potentially explain some of the known CNS effects of diabetes.

The NMDA receptor GluN2C subunit controls cortical excitatory-inhibitory balance, neuronal oscillations and cognitive function.

Despite strong evidence for NMDA receptor (NMDAR) hypofunction as an underlying factor for cognitive disorders, the precise roles of various NMDAR subtypes remains unknown. The GluN2C-containing NMDARs exhibit unique biophysical properties and expression pattern, and lower expression of GluN2C subunit has been reported in postmortem brains from schizophrenia patients. We found that loss of GluN2C subunit leads to a shift in cortical excitatory-inhibitory balance towards greater inhibition. Specifically, pyramidal neurons in the medial prefrontal cortex (mPFC) of GluN2C knockout mice have reduced mEPSC frequency and dendritic spine density and a contrasting higher frequency of mIPSCs. In addition a greater number of perisomatic GAD67 puncta was observed suggesting a potential increase in parvalbumin interneuron inputs. At a network level the GluN2C knockout mice were found to have a more robust increase in power of oscillations in response to NMDAR blocker MK-801. Furthermore, GluN2C heterozygous and knockout mice exhibited abnormalities in cognition and sensorimotor gating. Our results demonstrate that loss of GluN2C subunit leads to cortical excitatory-inhibitory imbalance and abnormal neuronal oscillations associated with neurodevelopmental disorders.

The effect of competitive antagonist chain length on NMDA receptor subunit selectivity.

The widely-used N-methyl-D-aspartate (NMDA) receptor antagonists (R)-4-(3-phosphonopropyl) piperazine-2-carboxylic acid ((R)-CPP) and (R)-2-amino-7-phosphonoheptanoate ((R)-AP7) are frequently used as general NMDA receptor antagonists and assumed not to display significant selectivity among NMDA receptor NR2 subunits. However, electrophysiological studies have suggested that certain longer chain N-methyl-D-aspartate (NMDA) receptor competitive antagonists, such as (R)-CPP are ineffective at subpopulations of NMDA receptors in the red nucleus, superior colliculus, and hippocampus. Using recombinant receptors expressed in Xenopus oocytes, we have examined the effect of antagonist chain length on NR2 subunit selectivity. All antagonists displayed the potency order (high to low affinity) of NR2A > NR2B > NR2C > NR2D, however the longer chain antagonists (having 7 instead of 5 bond lengths between acidic groups) displayed much greater subunit selectivity than their short-chain homologues. For example (R)-CPP displayed a 50-fold difference in affinity between NR2A-containing and NR2D-containing NMDA receptors, while the shorter chain homologue 4-(phosphonomethyl) piperazine-2-carboxylic acid (PMPA) displayed only a 5-fold variation in affinity. These results can account for the earlier physiological findings and suggest that longer chain antagonists such as (R)-CPP and (R)-AP7 should not be used as general NMDA receptor antagonists.