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Glutamatergic N-methyl-d-aspartate receptors (NMDARs) are heterotetrameric proteins whose subunits are derived from three gene families, GRIN1 (codes for GluN1), GRIN2 (GluN2) and GRIN3 (GluN3). In addition to providing binding sites for glutamate and the co-agonist glycine, these subunits in their di (d-) and tri (t-) heteromeric configurations regulate various aspects of receptor function in the brain. For example, the decay kinetics of NMDAR-mediated synaptic currents depend on the type of GluN2 subunit (GluN2A-GluN2D) in the receptor subunit composition. While much is known about the contributions of GluN1 and GluN2 to d-NMDAR function, we know comparatively little about how GluN3 influences the function of t-NMDARs composed of one or more subunits from each of the three gene families. We report here that in addition to altering kinetics and voltage-dependent properties, the GluN3 subunit endows these receptors with ion selectivity wherein influx of Ca2+ is preferred over Na+. This became apparent in the process of assessing Ca2+ permeability through these receptors and is of significance given that NMDARs are generally believed to be nonselective to cations and increased selectivity can lead to enhanced permeability. This was true of two independent brain regions where t-NMDARs are expressed, the somatosensory cortex, where both receptor subtypes are expressed at separate inputs onto single neurons, and the entorhinal cortex, where they are co-expressed at individual synaptic inputs. Based on this data and the sequence of amino acids lining selectivity filters within these subunits, we propose GluN3 to be a regulatory subunit for ion selectivity in t-NMDARs.

Cue-induced cocaine craving progressively intensifies (incubates) after withdrawal from cocaine self-administration in rats and humans. In rats, the expression of incubation ultimately depends on Ca2+-permeable AMPARs that accumulate in synapses onto medium spiny neurons (MSNs) in the NAc core. However, the delay in their accumulation (~1month after drug self-administration ceases) suggests earlier waves of plasticity. This prompted us to conduct the first study of NMDAR transmission in NAc core during incubation, focusing on the GluN3 subunit, which confers atypical properties when incorporated into NMDARs, including insensitivity to Mg2+ block and Ca2+ impermeability. Whole-cell patch-clamp recordings were conducted in MSNs of adult male rats 1-68 d after discontinuing extended-access saline or cocaine self-administration. NMDAR transmission was enhanced after 5 d of cocaine withdrawal, and this persisted for at least 68 d of withdrawal. The earliest functional alterations were mediated through increased contributions of GluN2B-containing NMDARs, followed by increased contributions of GluN3-containing NMDARs. As predicted by GluN3-NMDAR incorporation, fewer MSN spines exhibited NMDAR-mediated Ca2+ entry. GluN3A knockdown in NAc core was sufficient to prevent incubation of craving, consistent with biotinylation studies showing increased GluN3A surface expression, although array tomography studies suggested that adaptations involving GluN3B also occur. Collectively, our data show that a complex cascade of NMDAR and AMPAR plasticity occurs in NAc core, potentially through a homeostatic mechanism, leading to persistent increases in cocaine cue reactivity and relapse vulnerability. This is a remarkable example of experience-dependent glutamatergic plasticity evolving over a protracted window in the adult brain. [ABSTRACT FROM AUTHOR]

Mouse hippocampal glutamatergic nerve endings express presynaptic release-regulating NMDA autoreceptors (NMDARs). The presence of GluN1, GluN2A, GluN2B, and GluN3A subunits in hippocampal vesicular glutamate transporter type 1-positive synaptosomes was confirmed with confocal microscopy. GluN2C, GluN2D, and GluN3B immunopositivity was scarcely present. Incubation of synaptosomes with the anti-GluN1, the anti-GluN2A, the anti-GluN2B, or the anti-GluN3A antibody prevented the 30 μM NMDA/1 μM glycine-evoked [3H]d-aspartate ([3H]d-ASP) release. The NMDA/glycine-evoked [3H]d-ASP release was reduced by increasing the external protons, consistent with the participation of GluN1 subunits lacking the N1 cassette to the receptor assembly. The result also excludes the involvement of GluN1/GluN3A dimers into the NMDA-evoked overflow. Complement (1:300) released [3H]d-ASP in a dizocilpine-sensitive manner, suggesting the participation of a NMDAR-mediated component in the releasing activity. Accordingly, the complement-evoked glutamate overflow was reduced in anti-GluN-treated synaptosomes when compared to the control. We speculated that incubation with antibodies had favored the internalization of NMDA receptors. Indeed, a significant reduction of the GluN1 and GluN2B proteins in the plasma membranes of anti-GluN1 or anti-GluN2B antibody-treated synaptosomes emerged in biotinylation studies. Altogether, our findings confirm the existence of presynaptic GluN3A-containing release-regulating NMDARs in mouse hippocampal glutamatergic nerve endings. Furthermore, they unveil presynaptic alteration of the GluN subunit insertion in synaptosomal plasma membranes elicited by anti-GluN antibodies that might be relevant to the central alterations occurring in patients suffering from autoimmune anti-NMDA diseases. [ABSTRACT FROM AUTHOR]

The N-methyl- d -aspartate receptor (NMDAR) is an enigmatic macromolecule that has garnered a good deal of attention on account of its involvement in the cellular processes that underlie learning and memory, following its discovery in the mid twentieth century (Baudry and Davis, 1991). Yet, despite advances in knowledge about its function, there remains much more to be uncovered regarding the receptor's biophysical properties, subunit composition, and role in CNS physiology and pathophysiology. The motivation for this review stems from the need for synthesizing new information gathered about these receptors that sheds light on their role in synaptic plasticity and their dichotomous relationship with the amino acid d -serine through which they influence the pathogenesis of neurodegenerative diseases like temporal lobe epilepsy (TLE), the most common type of adult epilepsies (Beesley et al., 2020a). This review will outline pertinent ideas relating structure and function of t -NMDARs (GluN3 subunit-containing triheteromeric NMDARs) for which d -serine might serve as an inverse co-agonist. We will explore how tracing d -serine's origins blends glutamate-receptor biology with glial biology to help provide fresh perspectives on how neurodegeneration might interlink with neuroinflammation to initiate and perpetuate the disease state. Taken together, we envisage the review to deepen our understanding of endogenous d -serine's new role in the brain while also recognizing its therapeutic potential in the treatment of TLE that is oftentimes refractory to medications. • t -NMDARs are GluN3-containing triheteromeric NMDARs that are highly Ca2+ permeable. • t -NMDAR-mediated responses can be antagonized by D-AP5 and D-serine. • D-serine, an agonist of canonical NMDARs, has the opposite effect on t -NMDARs. • D-serine may be better suited for a role as an inverse co-agonist of t -NMDARs. • t -NMDARs and D-serine are implicated in the pathogenesis of Temporal Lobe Epilepsy. [ABSTRACT FROM AUTHOR]

N-methyl-D-aspartate receptors (NMDARs) are one of three ligand-gated ionotropic channels that transduce the effects of neurotransmitter glutamate at excitatory synapses within the central nervous system. Their ability to influx Ca 2+ into cells, unlike mature AMPA or kainate receptors, implicates them in a variety of processes ranging from synaptic plasticity to cell death. Many of the receptor's capabilities, including binding glutamate and regulating Ca 2+ influx, have been attributed to their subunit composition, determined putatively using cell biology, electrophysiology and/or pharmacology. Here, we show that subunit composition of synaptic NMDARs can also be readily visualized in acute brain slices (rat) using highly specific antibodies directed against extracellular epitopes of the subunit proteins and high-resolution confocal microscopy. This has helped confirm the expression of triheteromeric t -NMDARs (containing GluN1, GluN2, and GluN3 subunits) at synapses for the first time and reconcile functional differences with diheteromeric d -NMDARs (containing GluN1 and GluN2 subunits) described previously. Even though structural information about individual receptors is still diffraction limited, fluorescently tagged receptor subunit puncta coalesce with precision at various magnifications and/or with the postsynaptic density (PSD-95) but not the presynaptic active zone marker Bassoon. These data are particularly relevant for identifying GluN3A-containing t -NMDARs that are highly Ca 2+ permeable and whose expression at excitatory synapses renders neurons vulnerable to excitotoxicity and cell death. Imaging NMDAR subunit proteins at synapses not only offers firsthand insights into subunit composition to correlate function but may also help identify zones of vulnerability within brain structures underlying neurodegenerative diseases like Temporal Lobe Epilepsy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Beesley, Gunjan and Kumar.)

N-Methyl- d-aspartate receptors are localized to synaptic and extrasynaptic sites of dendritic spines and shafts. Here, the ontogenic profiles of GluN3A and GluN3B subunits in the rat brain were determined. A developmental switch from GluN3A to GluN3B proteins was detected within the first two postnatal weeks of crude synaptosomes prepared from forebrain and midbrain. Further fractionation of crude synaptosomes revealed the preferential localization of GluN3B to synaptic regions from P7 onwards. Immunolabeling and biochemical fractionation of rat P7 cultured hippocampal neurons showed that GluN3B was predominantly at synaptic sites. Unlike GluN2A and GluN2B, both GluN3 subunits were mostly associated with peripheral components of the postsynaptic density (PSD) rather than its core. When considering the non-PSD fraction, the overall extrasynaptic/synaptic spatial profile of GluN3B differed from GluN3A. Heterologous expression of GluN3B with GluN1 in HEK293FT cells could not be co-immunoprecipitated with PSD-95 unless co-expressed with a PSD-95-interacting GluN2 subunit, suggesting that anchoring of GluN3B at synaptic sites may require co-assembly with another scaffold-interacting NMDAR subunit. [ABSTRACT FROM AUTHOR]

Hebbian, or associative, forms of synaptic plasticity are considered the molecular basis of learning and memory. However, associative synaptic modifications, including long-term potentiation (LTP) and depression (LTD), can form positive feedback loops which must be constrained for neural networks to remain stable. One proposed constraint mechanism is metaplasticity, a process whereby synaptic changes shift the threshold for subsequent plasticity. Metaplasticity has been functionally observed but the molecular basis is not well understood. Here, we report that stimulation which induces LTP recruits GluN2B-lacking GluN1/GluN3 NMDA receptors (NMDARs) to excitatory synapses of hippocampal pyramidal neurons. These unconventional receptors may compete against conventional GluN1/GluN2 NMDARs to favor synaptic depotentiation in response to subsequent “LTP-inducing” stimulation. These results implicate glycinergic GluN1/GluN3 NMDAR as molecular brakes on excessive synaptic strengthening, suggesting a role for these receptors in the brain that has previously been elusive. [ABSTRACT FROM AUTHOR]

Abstract: N-methyl-d-aspartate receptors (NMDARs) are glutamatergic by virtue of glutamate-binding GluN2 subunits and glycinergic by virtue of glycine-binding GluN1 and GluN3 subunits. The existence, location, and functional-significance of NMDARs containing both GluN2 and GluN3 subunits have as yet remained unelucidated. Here we report on the discovery and characterization of a novel type of NMDARs, found at layer (L)1/primary whisker-motor-cortex inputs onto L5 pyramidal neurons in somatosensory cortex, that are distinct in structure and function from conventional GluN2A-containing NMDARs at thalamic/striatal (Str) inputs onto the same neurons. These receptors had a threshold-like activation at hyperpolarized holding-potentials with strong outward rectification of their current–voltage relationships unlike any known GluN1/GluN2-containing NMDARs. Pharmacology revealed a triheteromeric-receptor with features common to glutamate-activated GluN1/GluN2-containing and glycine-activated GluN1/GluN3-containing diheteromeric NMDARs. However, unlike GluN1/GluN3 receptors, NMDARs at L1 inputs were activated by glutamate and blocked by d-AP5, Ca2+-permeable, and more efficient at integrating and potentiating EPSPs selectively over Str inputs during high-frequency stimulation while obviating the need for AMPAR-mediated depolarization. [Copyright &y& Elsevier]

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the vertebrate CNS. Classified as AMPA, kainate, delta and NMDA receptors, iGluRs are central drivers of synaptic plasticity widely considered as a major cellular substrate of learning and memory. Surprisingly however, five out of the eighteen vertebrate iGluR subunits do not bind glutamate but glycine, a neurotransmitter known to mediate inhibitory neurotransmission through its action on pentameric glycine receptors (GlyRs). This is the case of GluN1, GluN3A, GluN3B, GluD1 and GluD2 subunits, all also binding the D amino acid d -serine endogenously present in many brain regions. Glycine and d -serine action and affinities broadly differ between glycinergic iGluR subtypes. On 'conventional' GluN1/GluN2 NMDA receptors, glycine (or d -serine) acts in concert with glutamate as a mandatory co-agonist to set the level of receptor activity. It also regulates the receptor's trafficking and expression independently of glutamate. On 'unconventional' GluN1/GluN3 NMDARs, glycine acts as the sole agonist directly triggering opening of excitatory glycinergic channels recently shown to be physiologically relevant. On GluD receptors, d -serine on its own mediates non-ionotropic signaling involved in excitatory and inhibitory synaptogenesis, further reinforcing the concept of glutamate-insensitive iGluRs. Here we present an overview of our current knowledge on glycine and d -serine agonism in iGluRs emphasizing aspects related to molecular mechanisms, cellular function and pharmacological profile. The growing appreciation of the critical influence of glycine and d -serine on iGluR biology reshapes our understanding of iGluR signaling diversity and complexity, with important implications in neuropharmacology. • 5 out of the 18 iGluR subunits do not bind glutamate but glycine or d -serine. • GluD and GluN1/GluN3 receptors are activated by glycine (or d -serine) alone. • Glycinergic iGluR subunits display a wide range of agonist sensitivities. • Glycine and d -serine can mediate non-ionotropic signaling. • Glycine agonism reshapes our understanding of iGluR signaling and pharmacology. [ABSTRACT FROM AUTHOR]

Glutamatergic N-methyl-d-aspartate receptors (NMDARs) are heterotetrameric proteins whose subunits are derived from three gene families, GRIN1 (codes for GluN1), GRIN2 (GluN2) and GRIN3 (GluN3). In addition to providing binding sites for glutamate and the co-agonist glycine, these subunits in their di ( d -) and tri ( t -) heteromeric configurations regulate various aspects of receptor function in the brain. For example, the decay kinetics of NMDAR-mediated synaptic currents depend on the type of GluN2 subunit (GluN2A-GluN2D) in the receptor subunit composition. While much is known about the contributions of GluN1 and GluN2 to d -NMDAR function, we know comparatively little about how GluN3 influences the function of t -NMDARs composed of one or more subunits from each of the three gene families. We report here that in addition to altering kinetics and voltage-dependent properties, the GluN3 subunit endows these receptors with ion selectivity wherein influx of Ca 2+ is preferred over Na + . This became apparent in the process of assessing Ca 2+ permeability through these receptors and is of significance given that NMDARs are generally believed to be nonselective to cations and increased selectivity can lead to enhanced permeability. This was true of two independent brain regions where t -NMDARs are expressed, the somatosensory cortex, where both receptor subtypes are expressed at separate inputs onto single neurons, and the entorhinal cortex, where they are co-expressed at individual synaptic inputs. Based on this data and the sequence of amino acids lining selectivity filters within these subunits, we propose GluN3 to be a regulatory subunit for ion selectivity in t -NMDARs., (© 2020 The Author(s).)