NMDA receptors: Biological properties and their roles in neuropsychiatric disorders

Proper signal transmission is the fundamental process of the brain activity. Changes and adaption of neuroplasticity based on the strength of synaptic transmission are essential for the information propagation in the central nervous system, which contribute to cognition, learning, and memory. Being the major excitatory neurotransmitter in the central nervous system, glutamate acts primarily through binding to the glutamate receptors, the glutamate-gated ion channels localized on post-synaptic membrane. The ionotropic glutamate receptors, pharmacologically grouped into α -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, N-methyl-D-aspartic acid (NMDA) receptors, and kainate receptors, have been shown to play distinct roles in excitatory neurotransmission and synaptic plasticity. Due to their high permeability to Ca 2+ , the NMDA receptors have very unique function in neurotransmission and particular importance in the induction of long-term synaptic plasticity. Dysfunction of NMDA receptors causes impairment in synaptic plasticity and learning and memory. In recent years, with the development of genome-wide association studies and next-generation sequencing technology, mutations of NMDA receptor subunits have been in a variety of neuropsychiatric disorders, such as cognitive impairment, schizophrenia, autism or epilepsy. In clinical practice, NMDA receptors are known as the targets for the treatment of many neuropsychiatric disorders. In current review, we summarize current knowledge of NMDA receptors with different subunit compositions in the context of expression pattern, channel properties, protein trafficking, and synaptic plasticity as well as their roles in neuropsychiatric disorders.


Introduction
The ionotropic glutamate receptors play essential roles in excitatory neurotransmission. When action potential propagates through the axons, glutamate released from the vesicles at the presynaptic terminals enters the synaptic cleft, the tiny, and highly organized extracellular space, where the neurotransmission occurs. The glutamate binds to ionotropic glutamate receptors on the post-synaptic membrane and triggers the ion channel function, which gives rise to neuronal signal transfer between neurons. https://doi.org/10.36922/an.v1i2. 148 Advanced Neurology
Neuropsychiatric disorders, including neurodegenerative and neurodevelopmental disorders, such as Alzheimer's disease (AD) and Autism spectrum disorders (ASD), are severely impair the self-cognition and social communication of individuals and may also genetically affect their descendants [2][3][4][5] . As the growing and aging population, researching on the neuropsychiatric disorders is of particular interest to scientists and clinicians. Due to the complexity of these diseases, it is still very challenging for researchers to understand the underlying mechanisms of these diseases [6][7][8][9] . The rapid development of genetic and molecular biotechnology provides an avenue for us to explore the pathogenesis of neuropsychiatric disorders [10,11] . With the support from genome-wide association studies and next-generation sequencing technology, the potential genetic architecture of human psychiatric diseases could be directly investigated [12][13][14] . In combination with genetic modified mouse, the disease-associated risk genes could be further confirmed in different psychiatric disorders, such as SHANK gene that is associated with autisticlike behavior [15] and GRIA3 gene that are associated with aggressive behavior [16] . As numerous genetic variants have been identified in psychiatric patients, potential molecular mechanism underlying these diseases could be gradually elucidated.
Many gene variants, which may act as the causal factors for these neuropsychiatric disorders, have been uncovered to affect the neuroplasticity [17,18] . In the central nervous system (CNS), precise signal transmission is the cornerstone of human physiological processes, and the proper protein structure and function are required to maintain the fine-tune neuronal communication [19,20] . Therefore, numerous efforts have focused on identifying new gene variants in neuropsychiatric disorders and investigating the related mechanism. Understanding of the physiological and pathogenic function of these genes is essential to guide the treatments of the neuropsychiatric disorders.
In this review, we discuss the molecular properties of the NMDA receptors and their roles in neuropsychiatric disorders. We also extend our discussion to the potential therapeutic effects of NMDAR; specifically, it is possible to reverse the improper synaptic transmission and to further mitigate the clinical symptoms of neuropsychiatric disorders by targeting NMDARs.
The GluN1 subunit is encoded by a single gene, but due to alternative splicing, it has eight different isoforms (GluN1-1a-4a and GluN1-1b-4b). Compared to GluN1-a isoforms, GluN1-b isoforms contain exon 5, leading to additional extracellular 21 amino acid extensions (called N1 cassette) that influence the NMDA affinity and pharmacological properties [23,24] . GluN1-1 to -4 were derived from the difference of alternative splicing in exon 21 and exon 22, which alter the C-terminal domain (CTD) length and trafficking capacity [25] .
All ionotropic glutamate receptor subunits consist of four domains [1,26,27] . A long N-terminal domain (NTD) in the extracellular domain is mainly involved in subunit assembly and allosteric regulation. A ligandbinding domain (LBD) consisting of two discontinuous fragments (S1 and S2) is the domain for binding of glycine (or D-serine) in GluN1 or GluN3 and of glutamate in the GluN2 subunit. The transmembrane domain (TMD) of NMDAR is composed of three transmembrane regions (M1, M3, and M4) and a reentrant loop (M2), which form the channel pore for the influx of ions. The final structure is the intracellular CTD, the length of which varies a lot and determines the length difference among NMDAR subunits [1] . CTD is involved in receptor trafficking, post-synaptic protein anchoring and protein-protein interactions, as well as many signaling pathways [22,28,29] .

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NMDA receptors in neuropsychiatric diseases acids that associate as back-to-side heterodimers between GluN1 and GluN2 subunits, adopts a bilobed structure. Upper R2 lobes of GluN1 and GluN2 subunits interact to form a protein-protein interface, while lower R1 lobes connect to the LBD, thus forming a unique dimer-ofdimer arrangement [30][31][32][33][34][35] . Moreover, there are binding sites for allosteric modulators in NTD, including the sites for extracellular Zn 2+ and ifenprodil, the GluN2B-selective antagonist [34,36] ; therefore, the NTD also plays a role in regulating NMDAR gating and function.
The LBD is formed by the S1 and S2 segments, which forms kidney-shaped bilobed structures consisting of an upper lobe and a lower lobe with the agonist binding sites in the gap located between the two lobes [37] . Besides, there are three independent contact regions in the LBD heterodimer crystal structures of GluN1 and GluN2A (referred to as sites I, II, and III). Hydrophobic residues of GluN1 and GluN2 form sites I and III, and non-polar interactions between these residues mediate agonist binding domain (ABD) heterodimerization [37] . The site II of the ABD contains the binding sites of positive and negative allosteric modulators, which are highly selective for GluN2A [38][39][40] .
The TMD is formed by M1, M3, and M4 and a reentrant loop (M2). The M2 is in the intracellular of the ion channel pore, and the M3 forms the extracellular region of the channel pore. The residues of pore region are highly conserved, which indicates the importance of the region. Normally, M3 forms the helical bundle and blocks the pore of channel so that ions cannot pass through the channel when the M3 helical changes its position [41][42][43] . The agonist binding to the LBD is the first step leading to M3 rearrangement [30,31,34,35,44] , followed by multiple shortlived, intermediate conformations, and eventually channel opening [45] . NMDARs are widely distributed throughout the CNS, though the expression of NMDAR subunits varies in different brain regions and developmental stages. Consistent with a broad CNS distribution, the expression of GluN1 subunits generally begins from embryonic E14 and continues into adulthood [46][47][48] . Among the GluN1 splicing isoforms, GluN1-2 is widely distributed. The GluN1-1 and GluN1-4 expression distribution is complementary; the former is distributed in more rostral regions (including cerebral cortex and hippocampus). GluN1-a and GluN1-b subtypes have largely overlapped expression patterns, but their relative abundance varies from region to region. It is noteworthy that GluN1-a is expressed in all principal neurons in the hippocampus, while GluN1-b is mainly confined to the CA3 layer [49] .
Expression of the GluN2 subunit varies in different brain regions during development. In rodents, the GluN2B and GluN2D subunits are highly expressed in the embryonic brain [47,48,50] . GluN2B expression remains high in the postnatal period, but only in forebrain regions. GluN2D expression is significantly reduced in adults; remaining GluN2D is mainly expressed in midbrain structures, including diencephalon and midbrain. The expression of GluN2A starts from birth and gradually increases over time, eventually becoming abundant throughout the CNS. Thus, NMDAR composition of GluN2B changes to predominantly GluN2A during development in the cerebral cortex and hippocampus [51] . The expression of GluN2C begins in the 2 nd week after birth, but is limited to the cerebellum and olfactory bulb. The shift from GluN2B to GluN2C occurs in cerebellar granulosa cells during development, resulting in a sharp decrease in the GluN2B expression in adulthood [48] .
GluN3A and GluN3B subunits also show different expression patterns [52,53] . GluN3A expression is the highest in the early postnatal period and then begins to decline gradually. In contrast, GluN3B expression is increased during development, with high levels of expression in motor neurons in adulthood. GluN2B, GluN2D, and GluN3A subunits are highly expressed in the early development, suggesting that these subunits play important roles in synaptic maturation and synaptogenesis [52,53] . GluN2A and GluN2B are major subunits in the CNS of adult, especially in hippocampus and cortex, suggesting that they play a role in synaptic function and plasticity [46][47][48] .

Activation of NMDAR
Glycine and glutamate are required for activation of NMDA receptors consisting of GluN1/GluN2 subunits [54][55][56][57][58] . The activation of NMDAR containing of GluN1/GluN3 requires only glycine [53,59] . In the nervous system, glycine is naturally present in the extracellular environment (4.2 ± 1.6 μM of glycine in cerebrospinal fluid) [60] . Other molecules can also activate GluN1/GluN2 receptors as coagonists, such as D-Serine, L-Serine, D-alanine, and L-alanine. In recent years, D-Serine has been proposed as the main coagonist of synaptic NMDARs, while glycine is the main coagonist of NMDARs at extrasynapse [61] . Glutamate, the excitatory neurotransmitter in the CNS, is the native agonist of GluN1/GluN2 NMDARs. Glutamate (L-glutamic acid or D-glutamic acid) can activate NMDARs by binding to the LBD of GluN2 subunit. NMDA, N-methyl-L-aspartic acid, D-aspartic acid, and L-aspartic acid are also the agonists of NMDARs [62] .

Gating function of NMDAR
NMDARs composed of different subunits have different channel characteristics. The GluN2A and GluN2Bcontaining NMDARs have higher conductance, higher Ca 2+ permeability, and higher Mg 2+ sensitivity compared to GluN2C and GluN2D-containing NMDARs, which are regulated by the Ser632 in GluN2A and S633 in GluN2B site in M3 region [68] . Besides, the channel gating properties are different among NMDARs, including the open probability, deactivation kinetics, and agonist potency. The open probability of GluN2A-containing NMDARs is higher than that of other GluN2-containing NMDARs, and the deactivation of GluN2A-NMDAR is faster too [69,70] . Therefore, the channel of GluN2A closes earlier after activation by glutamate, leading to a fast decay time. Interestingly, GluN1 splicing isoforms also affect NMDAR gating kinetics, that is, the NMDARs with GluN1-a deactivates slower than those with GluN1-b [23,24] . For the major NMDAR components in the cortex of adult, the GluN1/GluN2A receptors have the faster decay time, while the GluN1/GluN2B receptors have higher Ca 2+ permeability and charge transfer [71] .

NMDARs trafficking
NMDAR trafficking is mainly mediated by intracellular CTD. The difference in CTD sequences of NMDAR subunits leads to subunit-specific regulations on receptor transport, localization and signal transduction [72][73][74] . The synaptic transmission and escape from the endoplasmic reticulum (ER) of NMDARs are regulated by the C-terminal splicing of GluN1 [25] , a process that appears to be driven by neuronal activity [75] . The different motives in the CTDs of GluN2 and GluN3 diversify the trafficking procedures of NMDARs [76] .

Receptor biogenesis
Typically, NMDARs are first assembled in ER and matured by glycosylation in the Golgi apparatus before being transported to the plasma membrane through vesicles. Cells have strict mechanisms to prevent unassembled or misfolded NMDARs from being transported to the cell surface [77] . The previous studies indicated that only in the form of GluN1/GluN2, NMDAR could escape from the ER [78] . Grin1 gene deletion causes the retention of GluN2 subunit in the ER of hippocampus [79] . Both GluN1 and GluN2 subunits contain ER retention signals, which can be masked by the co-assembly of GluN1 and GluN2 subunit [80] . For example, the CTD of the GluN1 subunit contains positively charged ER retention signals, including lysine-lysine-lysine (KKK), and argininearginine-arginine (RRR) [25,81,82] . The overexpression of GluN2A and GluN2B in cerebellar granular neurons leads to a significant increase in the number of NMDARs and synaptic targeting, probably through the co-assembly with extra GluN1 [83] . An ER retention sequence (HLFY) has been proposed in the CTD of GluN2B subunit [84] . However, subsequent studies showed that HLFY motif was required in the CTD-oriented structure of GluN2B, but its might not serve as an ER retention signal [85] . In a recent study, the KKK879-881 of GluN2A was proven to be an ER retention signal [17] , regulating the surface expression of GluN2A-NMDAR.
GluN2 subunits interact with the proteins of membraneassociated guanylate kinases (MAGUK) family, such as synaptic associated proteins-102 kDa (SAP102) and synaptic associated proteins-97 kDa (SAP97), which is necessary for NMDAR secretion [86,87] . SAP102 is highly expressed in the hippocampus on the 2 nd day after birth, and its PDZ region interaction with GluN2A and GluN2B subunits of NMDARs makes a difference [88,89] . Moreover, SAP102 is also widely present in the cytoplasm and ER [88] . In addition, SAP102 interacts with mPins through its SrChomology 3 (SH3)/guanylate kinase domain to stabilize the SAP102-exocyst-NMDAR complex in ER. This process plays an important role in promoting NMDAR trafficking and membrane targeting [86,87] .
NMDARs also have a trafficking pattern that bypasses the traditional somatic Golgi network. In this pattern, these receptors mix directly within the dendrite Golgi [90] . This strategy can promote more efficient insertion of NMDARs at the post-synaptic density (PSD). Because they contain large protein complexes including scaffold molecules, vesicles produced by this pathway are highly mobile (0.76 μm/s). Mlin7 binds GluN2B with the motor protein KIF17, which promote the long-distance transport of NMDARcontaining vesicles on microtubules along dendrites [90][91][92] . Studies have shown that KIF17-mediated NMDARs trafficking is critical in long-term potentiation (LTP), long-term depression (LTD), learning, and memory [93] . Deletion of kif17 leads to NMDAR degradation due to enhanced ubiquitination, resulting in partial synaptic GluN2A and GluN2B receptors loss. It is interesting that the interaction of CASK leads SAP97 to preferentially bind NMDARs [94] . Meanwhile, SAP97 is phosphorylated by Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) at two key sites, Ser-39 (in the L27 domain) and Ser-232 (in the PDZ1 domain) [95,96] . Phosphorylation of SAP97 at Ser-39 leads to translocation of SAP97 from the ER to the post-synaptic compartment, while phosphorylation at Ser-232 disrupts binding of SAP97 to GluN2A. In https://doi.org/10.36922/an.v1i2.148

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NMDA receptors in neuropsychiatric diseases addition, CaMKII-dependent phosphorylation of KIF17 also facilitates unloading of cargo from microtubules [97] . Therefore, successive phosphorylation of SAP97 and KIF17 may result in the NMDAR escape from the ER and promote the insertion of NMDAR at synapses.
Recent studies have shown that palmitoylation has an important regulatory role in NMDARs post-Golgi trafficking [98,99] . Activity-dependent palmitoylation occurs in two distinct clusters in the CTD of the GluN2A and GluN2B subunits [99,100] . Palmitoylation of GluN2A and GluN2B at the second cluster of cysteines leads to retention of NMDARs in the Golgi apparatus, resulting in decreased surface expression of receptors [100] . Thus, mutation of the cysteines promotes surface expression of NMDARs, although the levels of synaptic NMDARs are not altered [101] .

Receptor exocytosis
Exocytosis of NMDARs occurs primarily at extrasynaptic sites [102] , where receptors spread to synapses through lateral diffusion [103] . In general, a family of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) proteins mediate the fusion of vesicles to the plasma membrane, including target SNARE syntaxin, synaptosome-associated protein (SNAP), and vesicular SNARE vesicle-associated membrane protein [104] . Although the role of the SNARE complex in NMDAR exocytosis is critical, the precise role of specific members of this protein family remains controversial.
SNP25 plays an important role in mediating NMDAR exocytosis in CA3 pyramidal neurons and Xenopus oocytes [105,106] . In these studies, botulinum toxin A, which inactivates SNAP25, could block mGluR1-and PKCdependent NMDAR exocytosis. SNAP25 is a substrate of PKC; mutation of the Ser-187 phosphate site inhibits PKC-mediated NMDAR insertion [107] . Meanwhile, knockdown of SNAP25 with shRNA also reduces NMDAR transmission [108] . Studies have also shown that another member of the SNAP family, SNAP23, is highly expressed at excitatory synapses and regulates NMDAR exocytosis [109,110] . Reducing SNAP23 by shRNA knockdown or gene deletion impairs surface expression of NMDAR [110] . Taken together, both SNAP23 and SNAP25 are important in regulating the exocytosis of NMDARs.

Receptor lateral diffusion
Lateral diffusion of NMDAR on the membrane is along extrasynapse and synapse [111] . GluN2A-NMDAR and GluN2B-NMDAR appear to be differentially located on the neuronal surface. GluN2A-NMDAR is preferentially expressed in the synapse, while the expression of GluN2B-NMDAR tends to be higher in extrasynapse during development phase [112][113][114][115] . The underlying mechanism seems to be that the interaction between CTD of GluN2A with PDZ domain of post-synaptic density protein-95 (PSD-95) is relatively stabler [116,117] . Furthermore, the peptides mimicking GluN2A or GluN2B PDZ binding motif only causes a decrease in synaptic GluN2A-NMDAR but not in synaptic GluN2B-NMDAR, indicating that the PDZ-domain dependent regulation is subunit-related.

Receptor endocytosis
The surface receptors are also regulated through receptor internalization. NMDAR endocytosis is dependent on development and neuronal activity; the rate of internalization decreases gradually as neurons mature [124] . The CTD of GluN2A and GluN2B contains endocytosis motifs, but GluN2B subunits have relatively higher endocytosis rates in mature neurons [125] .
The phosphorylation of Y1472 by Fyn kinase induces GluN2B binding with MAGUKs, which stabilizes GluN2B in the synaptic membranes. Reduction in Y1472 phosphorylation induces the interaction between YEKL1472-1475 endocytic motif and adaptor protein-2 (AP-2), resulting in increased internalization of GluN2B [124,126,127] . On the other hand, Ser-1480 phosphorylation by CK2 contributes to increased GluN2B internalization [83,128] . Therefore, the phosphorylation/ dephosphorylation plays an important role in receptor internalization. In GluN2A subunit, the endocytosis is mediated by the di-leucine LL1319-1320 [125] .
GluN3 endocytosis has been relatively poorly studied. In the GluN3A subunit, PACSIN1 binds to the CTD of GluN3A through its asparagine-proline-phenylalanine (NPF) domain, thereby regulating the internalization of the GluN3A receptor. Moreover, this regulation mode is activity-dependent, and destruction of the function of PACSIN1 leads to the accumulation of GluN3A receptors at the synapse [76] . https://doi.org/10.36922/an.v1i2.148

NMDA receptors in neuropsychiatric diseases
Thus, the NMDARs undergo various regulation of protein synthesis, trafficking, and internalization. These regulations also ensure the dynamic abundance and composition of NMDARs on the synaptic membrane, thereby allowing physiological functions regulated by neuronal activity and plasticity.

NMDARs in neuroplasticity
Synaptic plasticity refers to the long-lasting change in morphology and function of synapses caused by the neural activity induced by experience [130] . Two classic types of synaptic plasticity, LTP and LTD, have been studied at hippocampal excitatory synapses; both requires the activation of NMDARs [131] . LTP strengthens synapse function and is induced by high-frequency presynaptic stimulation, while LTD weakens synapse function and requires low presynaptic stimulation to induce. NMDARdependent synaptic plasticity is the basis of learning and memory in hippocampus. Blocking hippocampal NMDARs before training impairs rodent learning ability [132,133] , but memory is enhanced after hippocampaldependent task training [134,135] .
In hippocampal synapses, AMPARs and NMDARs involve the forms of LTP and LTD. AMPARs and NMDARs are glutamate receptors that are permeable to Na + and K + , while all NMDARs and part of AMPARs are also permeable to Ca 2+ . When the post-synaptic membrane is at resting potential, NMDARs cannot conduct currents because Mg 2+ blocks the channel pore. When AMPAR current causes local membrane depolarization, Mg 2+ is removed from the NMDAR channel, allowing Ca 2+ flow into the cell. LTP and LTD require NMDARs-mediated influx of Ca 2+ ; LTP requires a large elevation of Ca 2+ in spine while that it is much less for LTD. When NMDARs mediate a large influx of Ca 2+ , protein kinases, especially CaMKII acting on receptor proteins, accessory receptors, or transcriptional regulators increase glutamate receptor activity and/or levels in the synapses, thus inducing the induction and maintenance of LTP [136,137] . When NMDARmediated Ca 2+ elevation is modest, Ca 2+ /calmodulindependent protein phosphatase, protein phosphatase 1, and calcineurin are phosphorylated and activated, thereby inducing the dephosphorylation of AMPAR that results in the internalization of AMPAR from the synapses and LTD [138][139][140] .
Synaptic plasticity requires Ca 2+ , and the composition of NMDARs has different permeability to Ca 2+ , which could couple with the phosphatase pathway and downstream signaling to regulate the plasticity [140] . However, how specific subunit composition determines synaptic plasticity remains unclear and controversial.

LTP
To investigate the possible specific role of GluN2 in synaptic plasticity, both genetic and pharmacological approaches have been applied.
NMDARs containing GluN2B showed a greater current [48] and carried more Ca 2+ [71] . Besides, GluN2B preferentially interacts with CaMKII [141] , and it is speculated that the GluN2B subtype is more likely to induce LTP than the GluN2A subtype. For example, ifenprodil, the GluN2B antagonist, blocks the pairing-induced LTP in hippocampal slices [142] and in the barrel cortex [143] . Moreover, the GluN2B antagonist could block the pairing-and theta burst-induced LTP in the anterior cingulate cortex at 6-8-week-old mice [144] . The tetanus induced-LTP in hippocampal CA3 synapses was abolished in mice with conditional GluN2B knockout [145] . Lacking GluN2B in the Cornu ammonis 1 (CA1), the LTP is impaired [146] . Another study showed that LTP is deficient by pairing protocol of GluN2B knockout in the forebrain [147] . LTP is enhanced when GluN2B is overexpressed in hippocampal neurons of 4-6-month-old mice [148] . Disruption of the interaction between GluN2B and CaMKII by overexpressing the CTD of GluN2B eliminates the LTP of 3-4-month-old mice [149] . Genetic deletion of GluN2A has no effect on the LTP in P28 mice, indicating that GluN2B is essential for the LTP [150][151][152] . Other genetic methods that influence the level of GluN2B also impairs the induction of LTP. For instance, knockout of the KIF17, a protein that transports GluN2B to the synapses, reduces synaptic GluN2B and abolishes LTP [93] . Besides, Cdk5 knockout mice show increased GluN2B and enhanced LTP [153] . Recently, it has been found that enhanced GluN2A surface and synaptic expression causes LTP impairment, likely through compensatory GluN2B decrease [17] . Hence, GluN2B-containing NMDARs play a role in the induction of LTP.
However, some studies have shown that GluN2A is important for LTP [153,154] . In pharmacological experiments, blocking of GluN2A by NVP-AAM077 could prevent tetanus-and pairing-induced LTP in 3-4-week-old rats, while the ifenprodil and Ro 25-6981, the antagonists of GluN2B, have no effect on LTP but could block the induction of LTD [154] . Another study that used the same antagonist concentration and rats of the same age found that NVP-AAM077 completely blocks LTP, and ifenprodil and Ro 25-6981 partially block LTP [155] . Low Zn + which selectively inhibited GluN2A impairs LTP [72] . GluN2A knockout mice [156] and deletion CTD of GluN2A mice [72] show impaired LTP in hippocampal synapses.
For LTP, a possibility is that both GluN2A and GluN2B subunits mediate Ca 2+ influx, so both subunits are involved in the induction of LTP [157,158] . However, the exact extent of involvement of these two subunits in LTP remains unclear. https://doi.org/10.36922/an.v1i2.148

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NMDA receptors in neuropsychiatric diseases

LTD
There are also many contradictory results on the LTD with the composition of NMDAR subunits. One study suggested that ifenprodil blocks LTD in hippocampus [154] , while the results of other research groups indicated that ifenprodil does not affect LTD [159] or even enhances LTD [160] . Disruption the interaction of GluN2B and PSD95 has no effect on LTD, although the level of synaptic GluN2B is reduced [161] . Moreover, the overexpression of GluN2B does not affect the LTD [162] , while the LTD is deficient in GluN2B knockout mice and KIF17 knockout mice, due to the reduction of synaptic GluN2B [93,146] . Thus, these conflicting findings point to the ambiguous function of the GluN2B subunit in LTD, and to the fact that the experimental conditions are important for the induction of LTD.
On the other hand, some studies found that NVP-AAM077 not only impaired LTP but also blocked LTD [155,163] . By contrast, the NVP-AAM077 only affected LTP but did not impair LTD in slices [154] or in vivo [164] . Besides, overexpression GluN2A induced decreased LTD [152] and GluN2A knockout mice displayed no impairment of LTD [165] . However, the LTD could be induced by the 0.5 Hz stimulation in GluN2A knockout mice [165] .
More experiments are warranted to clarify the role of NMDAR subunits in the LTD.

NMDARs pharmacology
NMDARs play a role in the neuropsychiatric disorders. The dysfunction of NMDARs involved in many disease, such as AD, Parkinson's disease (PD), epilepsy, and schizophrenia [166] . Therefore, in the past decades, a great deal of money has been spent to develop NMDAR antagonists and agonists to cure the diseases associated with NMDAR. For example, the ketamine and rapastinel (GLYX-13) are used for the treatment of depression [167][168][169] . Memantine, a NMDAR blocker, has been proven for use in treating AD and increasing cognitive behavior of AD patients [170,171] . Unfortunately, due to the off-target or side effects of excessive inhibition of NMDAR, many drugs have failed in most clinical trials [166,172] . Despite the challenges, there are still opportunities to develop NMDAR drugs.
Many positive and negative allosteric modulators (PAMs and NAMs, respectively) have been found recently. In addition to the antagonists and agonists of NMDAR, the allosteric modulators can positively and negatively regulate the NMDAR activity. Compared with antagonist or channel blocker, the allosteric modulators have many potential advantages in therapeutics development. The binding domain of allosteric modulators with NMDARs is not highly conserved ligand-binding site or channel pore. Therefore, allosteric agents have better subunit selectivity, which reduces side effects and off-target. Besides, the inhibition of NAMs is <100%, so partial function of NMDARs can be preserved, so as to avoid excessive blockade of receptors. This partial inhibition of NAMs has a better safety profile than competitive antagonists and channel blockers. In schizophrenia caused by NMDAR hypofunction, or other cognitive disorders, PAMs only enhanced the activity of weakly activated NMDARmediated signals to restore normal function. This is unlike the NMDAR agonists, which could activate all receptors and lead to side effects and excitotoxicity. Therefore, a better understanding of these allosteric modulators can improve their usage in clinical practice.

The antagonists of NMDARs
The previous studies have shown that most NMDAR antagonists basically interact with NMDARs by glutamate binding site, glycine binding sites, NTD binding sites, or ion channel pores. The glutamate is an activator of NMDARs. Agonists and antagonists that interact with glutamate binding sites were first identified [56] , such as D-α-amino adipic acid and D-AP5. Since glutamate binding affinity is the strongest in GluN2D followed by GluN2B/2C, and the lowest in GluN2A, the competitive antagonists generally have the strongest inhibition on GluN2A, then on GluN2B/2C. For example, CPP is more sensitive to GluN2A subunit than to other subunits [173] . Quinoxalinedione derivative (1RS,1'S)-PEAQX, a GluN2A competitive antagonist, has no effect on GluN2B [174] . At present, the NVP-AAM077 ([1R,1'S]-stereoisomer) purified from (1RS,1'S)-PEAQX is widely used to inhibit GluN2A-NMDARs.
The channel sequence and structure of NMDARs are highly conserved, so the selectivity of channel blocker is little. The NMDAR blockers, such as ketamine and phencyclidine (PCP), act as the anesthetics that bind the ion channel pore [179,180] . However, when the Mg 2+ are present, the memantine appears to be more sensitive to GluN1/GluN2C and GluN1/GluN2D receptors. Besides, the MK-801 is also a NMDAR channel blocker, which binds to channel pore when the NMDARs are activated [181] .

NAMs
The binding sites of NMDAR subunits with NAMs are not a highly conserved ligand binding site or channel https://doi.org/10.36922/an.v1i2.148

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NMDA receptors in neuropsychiatric diseases pore, so the subunit-specificity of NAMs is greater than the antagonists. Due to the noncompetitive glutamate and glycine binding site and independent NTD regulation to activity, the NAMs are safer to be used in the clinical settings. Many NAMs have been found in recent years, including sulfonamide series, quinazoline-4-one series, and phenanthroic and naphthaloic acid NAMs. Ifenprodil, CP101, and RO25 also belong to the category of NAMs.
Ifenprodil was studied in treatment in stroke and then was proven a GluN1/GluN2B receptor antagonist, which binds the NTD of the subunit [182,183] . Subsequently, CP101, 606 [184] and Ro 25-6981 [185] were developed as GluN1/ GluN2B antagonists, which have been studied for clinical use.
The TCN-201, one of sulfonamide series, was first found as a noncompetitive GluN2A NAM [186] , which binds with dimer interface between the LBD [40] . TCN-201 has a higher affinity with GluN2A over other GluN2 subunits [186,187] . Studies indicated that the TCN-201, which reduces the potency of glycine and D-serine, is a noncompetitive antagonist [187,188] . The inhibition of TCN-201 is abolished at high concentrations of glycine [186] .
The quinazoline-4-one series inhibitor has been identified to be more sensitive to GluN2C and GluN2D compared with GluN2A and GluN2B [189] . QNZ46, one of this type of noncompetitive NAMs, exerts differential effect on GluN2A and GluN2D. Point mutations and chimeric molecules were used to identify the binding site of QNZ46, and S2 domain of GluN2 is found to be critical for QNZ46 activity [190] . Unlike TCN-201, QNZ46 increases the potency of glutamate but not glycine, although QNZ46 is a NAM. The feature of QNZ46 inhibition is needed in the binding with glutamate. After QNZ46/ glycine preincubation, glutamate plus QNZ46/glycine application produced a transient peak response followed by homeostasis inhibition. Thus, the glutamate binding is essential for the inhibition of QNZ46. The QNZ46 inhibits NMDAR function by binding in S2, and the binding site of QZN46 with GluN2 is exposed by glutamate binding.
Phenanthroic and naphthaloic acid NAMs exhibit different subunit selectivity and have enhanced or inhibited activity in different subunits. UBP512 not only is a potentiator of GluN2A but also is an inhibitor of GluN2C and GluN2D-containing NMDARs [191] . Meanwhile, the UBP551 showed PAM activity on GluN2D at high concentration of 30 μM, but inhibited GluN2A, GluN2B, and GluN2C subunits at IC50s around 10 μM [191] . Besides, the UBP618 is an inhibitor of all GluN2 subunits, and the CTD is critical for the inhibition [192] . In addition, the UBP608 shows inhibitory effects on GluN2A-NDMA receptor [192] .

PAMs
The function of PAMs is to enhance the NMDAR function. Endogenous PAMs include ATP [193] , histamine [194] , Mg 2+ , polyamines such as spermine [195] , and pregnenolone sulfate (PS) [196] . The enhancement of histamine and ATP is both enhanced at high glutamate concentrations. The low concentrations of ATP can enhance the GluN2A-, GluN2B-, and GluN2C-containing NMDARs [193] . The effects of spermine on NMDARs are different; it can either increase the potency of glycine, or allosterically interact with GluN2B-and GluN1-lacked exon 5, that is, the glycine-independent [194,195,197] . PS potentiates GluN2A-and GluN2B-containing NMDARs at micromolar concentrations, while inhibits the GluN2C and GluN2D subunits [198] . The mechanism of the potentiation of PS is the increased open probability of NMDARs through phosphorylation [199,200] . Similar results have been found by single channels analysis that the frequency of channel openings is enhanced by PS [201,202] . Besides, the studies have proven that the S2 domain of GluN2A may be the interaction site of PS with GluN2A [200] , while the S2 and M4 domain of GluN2B play an important role in the potentiation [203] .
Other PAMs of NMDARs, such as phenanthrene, naphthalene, and coumarin derivatives, have been reported in many studies [204] . UBP512, a phenanthroic acid, enhanced the GluN2A-contaning NMDAR, but not the GluN2B-containing NMDAR, and inhibited the GluN2C and GluN2D subunits. The UBP710 enhanced both GluN2A-and GluN2B-containing NMDARs, while inhibited the GluN2C and GluN2D subunits. The UBP551, a naphthoic acid NMDAR PAM, potentiated the GluN2D subunit, while had no effect on the other three GluN2 subunits [191] . Another naphthoic acid NMDAR PAM is the UBP684, which could enhance the effect on all GluN1/ GluN2 receptors by increasing the open probability and mean open time [205,206] . The UBP714, a coumarin derivative, slightly potentiated the GluN2A, GluN2B and GluN2D [207] . The CIQ is a potentiator of GluN2C-or GluN2D-containing NMDAR [208,209] . Studied indicated that the linker between NTD and LBD and T592 of GluN2D is the important sites of the GluN2D to activate the CIQ [208] .
Recently, the GEN family has been reported as the PAMs of GluN2A. According to the analysis of the GluN2A binding with GEN-6901, we understand that the V783 of GluN2A is a necessary site for binding with GEN-6901 specifically [38] . The potentiation of GEN-8324, instead of GEN-6901, in GluN2A increased the potency of glutamate, but both of them had no effect on the sensitivity to glycine. Meanwhile, they also decreased the NMDAR deactivation [38] . However, there are differences between https://doi.org/10.36922/an.v1i2.148

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NMDA receptors in neuropsychiatric diseases the two potentiators. The GEN-8324 has an impact on the inhibitory neurons only, while the GEN-6901 plays a role in both inhibitory and excitatory neurons [38] ; however, the underlying principle remaining to be explored. The GEN-0723 had been shown to enhance GluN2A not only in vitro but also in vivo [39] . The GEN-9278 potentiated not only the GluN2A-NMDARs but also the GluN2B-, GluN2C-and GluN2D-NMDARs [210] . The GEN-9278 increased the glutamate and glycine potency and reduced the deactivation of glutamate.

NMDAR in neuropsychiatric disorders
NMDARs play an important role in neuronal development. NMDARs dysfunction in subunits expression, localization, and trafficking may cause brain disorders. Therefore, both NMDAR antagonists and enhancers have the potential to be utilized as CNS treatment agents. In this section, we focus on some neuropsychiatric disorders with NMDAR dysfunction, with special emphasis on subunit specificity.

AD
AD is a dementia, which is characterized by increased beta-amyloid (Aβ) peptide and the intracellular tangles composed of tau protein in hyperphosphorylation. Aβ may aggregate to form soluble oligomers composed of multiple polypeptides or insoluble amyloid plaques. Hyperactivation of NMDAR is also associated with AD ( Figure 1). Memantine, which is an NMDAR channel blocker, is currently used to treat patients with AD. Moreover, the antagonists of NMDAR, such as APV and ifenprodil, as well as the channel blocks, ketamine, and MK-801 can abolish the synaptic depression induced by Aβ [211] . Some studies, further, suggested that GluN2B is essential for the harmful effects of Aβ. Synaptic loss [212] , impairment of LTP [212][213][214] , and increased LTD induced by Aβ [215] can all be rescued by GluN2B antagonists. Some of these mechanisms may be the activation of extrasynaptic GluN2B receptors induced by changes in glutamate uptake [215] . Phosphorylation of the GluN2B by the tau and tyrosine protein kinase FYN trafficking to the dendritic spine enhances the interaction with PSD95, which leads to downstream excitatory toxicity [216] . Besides, the induction of Aβ is needed for the activation of extrasynaptic GluN2B-containing NMDARs [217] . A synthetic peptide that interferes with the interaction between GluN2B and PSD95 enhances memory and longevity in AD mouse models [216] . However, this peptide did not impair NMDAR-mediated synaptic currents, but reduced ischemic cell death in stroke model [216,218] . In addition, it should be noted that much of the evidence supporting the role of GluN2B in mediating Aβ-induced excitatory toxicity is obtained through synthetic Aβ preparations in vitro. Therefore, it remains to be clarified whether the GluN2B antagonists can prevent or reduce synapse loss and improve cognition in AD models.

PD
PD is a neurodegenerative disease, which is characterized by the degeneration of nigral dopaminergic neurons and the loss of dopamine in the striatum. Dopaminergic and glutaminergic signaling are involved in controlling motor function. Studies have shown that the NMDAR is impaired in the disorder (Figure 1). For example, in the PD model, the expression of GluN1 and GluN2B, but not GluN2A, was reduced, which can be rescued by L-DOPA [219] . In addition, after L-DOPA treatment in dyskinetic animals, GluN2B was transferred from the synaptic to the extrasynaptic region,

NMDA receptors in neuropsychiatric diseases
and GluN2A was increased in the synaptic region [220] . In addition, the expression of MAGUK protein was decreased, which is probably due to the impaired interaction between MAGUK with GluN2B in the PD models [220] . However, the GluN2B selective antagonists have inconsistent effects on PD models and human PD patients [221,222] . However, in the rat model of PD, specific interference of GluN2A in synaptic localization can reduce motor dysfunction [223] .
Although these studies help us further understand the pathophysiology of PD, the disease involves many changes in organs and brain regions. Therefore, a complete cure for PD remains a major challenge.

Schizophrenia
Schizophrenia is psychiatric disorder with hallucinations and delusions (positive symptoms), aversion and anhedonia (negative symptoms), and cognitive dysfunction [224] . Studies have shown that excessive release of dopamine in striatum is the reason of the positive symptoms [225] , but the mechanism of negative symptoms and cognitive dysfunction remains elusive. Changes in glutamate signal may be a pathological basis for the schizophrenia [226] , especially dysregulated NMDARs may induce the imbalance between excitation and inhibition in PV neuron (Figure 1).
Studies have indicated that reduction in NMDAR expression causes schizophrenia-like behavior in mice [227] . Impaired dopamine release is found in mice with GluN1 deletion in PV neurons [228] . Recently, other studies in clinic proved the NMDAR hypofunction in schizophrenia. The noncompetitive NMDAR antagonists, PCP, and ketamine could induce schizophrenic phenotypes in people [229][230][231] . Furthermore, the MK-801 also induced the PCP-like behavior in many species [232,233] . The autoantibodies against NMDARs, which are found in patients with the anti-NMDAR encephalitis, cause NMDAR internalization. These patients show the symptoms similar to schizophrenia [234] .
These studies suggested that the NMDAR hypofunction may cause schizophrenia, providing new ideas for developing treatment of schizophrenia in the future.

Huntington's disease (HD)
HD in human results from an inherited huntingtin (HTT) gene defect, which induces progressive degeneration of neurons in the brain and impairs movement, cognitive functions, and emotions. HD is defined by repeated CAG mutation in HTT gene [235] . Some studies have indicated the NMDAR function is increased in HD mouse models [236] , and the balance of synaptic and extrasynaptic NMDAR plays an important role in HD (Figure 1). The data found that the balance of synaptic and extrasynaptic NMDAR is disrupted in mice model of HD [237] . The experiments also showed that in the HD model, caspase-6 cleavage, a process of the production of toxic mutation Htt fragments, increased extransynaptic NMDA receptor-induced currents, and signaling pathways [237] . In addition, the HD mice were treated with memantine, which only antagonized extrasynaptic NMDAR at low dose and rescued impaired learning as indicated by a test on rotarod motor [237] . Another study also presented the similar results that the activation of synaptic NMDAR rendered Htt mutationexpressing cortical cells resistant to cell death, while the activation of extrasynaptic NMDAR rendered the cell more vulnerable to cell death [238] . Clinical trials have shown that treating HD mice with low doses rather than high doses of memantine rescued adverse neuropathological and impaired behaviors [238] .
Binding of β-amyloid to synaptic NMDAR increases post-synaptic Ca 2+ level, which impairs synaptic transmission and further affects LTP/LTD. The stimulation of extrasynaptic NMDAR could activate calpain which degrade tau protein to a toxic peptide and also phosphorylate p38-MAPK signaling pathways and leads to cell death. Moreover, tau protein is required to transport kinase Fyn to the post-synaptic localization, and to further promote the interaction with PSD95 and phosphorylation of NMDAR, which lead to the impaired LTP/LTD in AD. Dysregulated NMDAR could induce ER stress and DNA damage, probably through the overloading of Ca 2+ ions and related signaling pathways. The impaired interaction between PSD95 and NMDAR may also affect the synaptic transmission and lead to dopaminergic neurons death in PD. Hypofunction of NMDAR affects the spinogenesis and also reduces the release of dopamine, resulting in an unbalance between excitatory and inhibitory transmission and schizophrenia. Increased level of extrasynaptic NMDAR attracts more Ca 2+ ions, which induce mitochondrial dysfunction and further lead to neuron death. In addition, caspase 6 cleavage of HTT is essential to increase the extrasynaptic NMDAR current and subsequent excitotoxicity in HD.

Major depressive disorder (MDD)
MDD is currently one of the most common mental disorders in the world and frequently occurs as a complication in other diseases [239] . The efficacy of drug treatment for MDD patients usually lasts from weeks to even years. In addition to its severe effect on the social communication and cognitive functioning, the comorbid MDD and anxiety symptoms are also a challenge for pharmaceutical approaches [240,241] .

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NMDA receptors in neuropsychiatric diseases ketamine, which was tested in MDD patients, displayed potential clinical effectiveness [167] . Two enantiomers of ketamine, arketamine and esketamine, show antidepressant activity with different degree of affinity to NMDAR [243] . Recently, the treatment of esketamine for MDD has been approved by the USA and Europe FDA [244] . It has been suggested that the dysfunction of glutamatergic transmission may associated with MDD, and NMDAR attracts considerable attention with respect to its biological function in the CNS [245] . Studies with MDD patients showed significantly reduction of GluN2A and GluN2B subunits of NMDAR but did not change GluN1 protein level compared to the controls, along with reduced level of PSD-95 in prefrontal cortex, indicating an abnormal signaling in the synaptic transmission within MDD [246] . However, in the lateral amygdala and locus coeruleus of depressed subjects, increased levels of GluN2A and GluN2C expression have been detected [247,248] . A recent research showed that α7nAChR-NMDAR complex may play a role in the MDD, and disruption of this complex with a peptide exhibits antidepressant effects [249] . Moreover, another study suggested that the antidepressant mechanism of ketamine is NMDAR-independent but related to AMPAR [250] . Taken together, these studies reveal a crucial role of NMDAR in MDD, underlining the significance of NMDAR in the development of next-generation antidepressants.

Stroke and traumatic brain injury (TBI)
In stroke and TBI, extracellular glutamate is continuously elevated, causing excitotoxicity and acute neuronal death [251] . The NMDAR-mediated excitotoxicity is a major cause of acute neuronal death after ischemia or injury. Studies have suggested that NMDAR antagonists could inhibit ischemic cell death [252,253] . NMDAR antagonists protect neurons against ischemic cell death if they have been applied before [254][255][256] , but not 30 min [254] or 3 h [255] after stroke in animal models. However, it has also been reported that GluN2B antagonists applied 2 h poststroke could reduce brain infarct volume [257] . Intriguingly, NMDAR-mediated excitotoxicity appears to be subunit-dependent: GluN2A antagonist aggravates but GluN2B antagonist blocks the ischemic cell death [255,256] . Probably due to the intolerable side effects of NMDAR antagonists and the level of elevated extracellular glutamate that last less than an hour, the clinical trials concerning the application of NMDAR antagonists in stroke have so far been unsuccessful [218] . In addition to the direct inhibition of NMDAR activity, neuronal protection can be achieved by disrupting the interactions between NMDARs and their scaffold proteins, including PSD-95, phosphatase and tensin homolog, and associated signaling molecules in stroke animal models [258,259] and in humans [260] . This could be a new strategy targeting NMDAR-associated signaling in stroke.
The clinical trials concerning NMDAR antagonists, including selective GluN2B antagonists, for use in the treatment of TBI were also unsuccessful [261] . Although overactivation of NMDARs is toxic, functional recovery after TBI requires physiological activation of NMDARs [262] .

Epilepsy
Epilepsy is a very common neuropsychiatric disorder that causes abnormal brain activity, seizures, unusual behavior sensations, and sometimes, loss of consciousness. Glutamate-mediated excitability changes could be involved in the pathogenesis of epileptic discharge [263] . NMDAR may be involved in the seizure-induced excitotoxic cell death of hippocampal neuronal populations, as NMDAR antagonists provide protection against such damage [264] . Many animal models of epilepsy have been developed, including chemical induction models (such as kainic acid, pilocarpine, picrotoxin, or bicuculline), physical models (such as hyperthermia, or photic or auditory stimuli), genetic models (such as mutations, transgenes, or knockouts), electrical stimulation models, and spontaneous seizure models (such as post-kindling). Due to the differences in animal models, brain regions, and NMDAR subunits examined, the results of these studies vary. A study found that seizure enhances expression of GluN1 mRNA and protein in rat cerebral cortex [265] . Another study indicated that the mRNA of GluN1 is continuously increased in cortex of amygdaloid kindled rat [266] . However, application of picrotoxin (500 μM) caused a decrease in mRNA levels of GluN2A and GluN2B, while the mRNA level of GluN1 remained unchanged [267] . Besides, a study demonstrated that mRNA and protein levels of GluN2A and GluN2B were increased in spontaneous seizure, but not in kindled seizure [268] . Kainic acid-induced seizure reduces the mRNA level of GluN1 in CA1 and CA3 pyramidal cells, but not dentate gyrus [269] .
NMDAR antagonists are proven to be anticonvulsant in several animal models of epilepsy. Felbamate (Felbatol ® ) is used in patients with intractable partial seizures, infantile spasms, or Lennox-Gastaut syndrome [270,271] . Some studies reported that felbamate inhibits the NMDAR by binding to the glycine site [271][272][273] , while others showed that felbamate binds with the site of channel pore [274] . Therefore, the binding site remains unsolved. Ifenprodil, a GluN2Bselective antagonist, has been reported to have effects on many animal models of seizure [275][276][277] , except for seizure induced by imipenem or pefloxacin in DBA/2 mice [278] . Memantine, a blocker of NMDARs, exerts anticonvulsant effects in seizures [279][280][281] by blocking the NMDAR ion channel pore. However, many clinical trials have failed due to the side effects. In experimental models of epilepsy, the combination of conventional antiepileptic drugs with https://doi.org/10.36922/an.v1i2.148

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NMDA receptors in neuropsychiatric diseases low-dose NMDAR antagonists has shown significant efficacy but minimal side effects [282] , although the potential advantage also requires further validation.

Neuropathic pain
Neuropathic pain is a type of chronic pain induced by the damage of neurons or nerves in the nervous system. During injury and inflammation, glutamate is released to activate NMDARs in the peripheral terminals of primary sensory afferents, leading to pain-related behaviors [283] . During sensitization of the pain, GluN2B expression is increased in the nociceptive neurons in the dorsal horn of the spinal cord, a process that NMDARs activity appears to be involved [283,284] . In animal models of peripheral nerve injury, NMDA-activated whole-cell current and calcium influx in spinal lamina II neurons appear to be increased in nerve-ligated rats [285] . The phosphorylation of GluN1 subunit in the dorsal horn is increased in partial ligation of the sciatic nerve [286] . However, the total protein levels of GluN1 and GluN2A-D subunits were unchanged in the spinal cord after nerve injury [286,287] . Therefore, increased NMDAR phosphorylation may increase surface NMDAR function and is critical for central sensitization induced by nerve injury.
Blocking NMDARs could reduce the hypersensitivity of spinal dorsal horn neurons in neuropathic pain models. Memantine, ketamine, and MK-801, which are the NMDAR antagonists, attenuate evoked responses of dorsal horn neuron in spinal nerve-ligated rats [288] . Besides, a study showed that ifenprodil, a GluN2B subunit-specific antagonist, reduces the amplitude of NMDAR currents in nerve-ligated mice [289] . In clinic, intravenous infusion of ketamine can relieve pain in patients with refractory complex regional pain syndrome, but does not prevent chronic neuropathic pain caused by thoracotomy [290][291][292] . Intravenous infusion of amantadine, another NMDAR antagonist, reduces persistent neuropathic pain in cancer patients after surgery, but often causes intolerable side effects [293,294] . Therefore, there could be additional mechanisms underlying the enhanced NMDAR activity after nerve injury. Further studies along this direction may facilitate the development of neuropathic pain therapies.

Post-traumatic stress disorder (PTSD)
PTSD is a complex and chronic neuropsychiatric disorder characterized by recurrent appearance of unpleasant or even painful memories resulted from traumatic events, especially for the military members and also people who have recently been infected with COVID-19 [295][296][297] . The symptoms severely impair physical and mental health and lower the quality of life [298] .
It has been suggested that over-response of fear function in the amygdala and a combination of weakened inhibitory effects of the projection from prefrontal cortex to amygdala may lead to PTSD [299] . The functional brain circuits require the proper synaptic connectivity which can attribute to glutamate with its role in maintenance the neuronal activity [300] . Ketamine plays a crucial role in repairing the synaptic network and also shows positive effects on the PTSD [301,302] . The benefits of ketamine in PTSD may result from its anti-depressant and antiinflammatory effects that improve the brain-derived neurotrophic factor levels and further re-establish the neuronal connectivity [303] . High activity of NMDAR could impair formation of spontaneous intrusive memories that may contribute to the development of PTSD [302] . A study on rat models of PTSD-induced by contextual fear showed that the level of brain-derived neurotrophic factor was strongly reduced, which could be reversed by ketamine treatment, and NMDAR inhibition interrupted the consolidation of fear conditioning related to hippocampus and hypothalamus-pituitary-adrenal axis [303][304][305] . Taken altogether, the hyperactivity of NMDAR is tightly linked to PTSD and may lead to synaptic abnormalities and decreased expression of brain-derived neurotrophic factor especially under traumatic event. Ketamine is currently an effective pharmaceutical intervention for PTSD. In the future, the studies should focus on optimization of ketamine with appropriate therapeutic dose and duration, and on reducing the side effects.

NMDARs mutations in diseases
Mutations in NMDARs are related with neuropsychiatric diseases. Nowadays, the pathogenesis of and therapy development for brain diseases can be explored using genomics approaches following the emergence of wholeexome/genome sequencing and targeted therapy. Human genetics studies indicated that more than 500 variants of NMDARs subunit mutations have been found in patients with brain disorders, such as intellectual disability, hyperactivity disorder, ASD, schizophrenia, or epilepsy. These mutations are more prevalent in the GluN2A and GluN2B subunits of the amino terminal domain (ATD), ABD, TMD, and CTD regions. Discovering and analyzing the function of these mutations can help determine the role they play in these neuropsychiatric disorders. In addition, functional analysis of these mutations can advance the understanding in the etiology of the disease and facilitate the formulation of treatment plans in clinic.

Advanced Neurology
NMDA receptors in neuropsychiatric diseases been found to be related to many diseases, including the ID, autism spectrum, and epilepsy; the function of some of the mutations has been studied, while others remain unclear. The function of mutations, including D227H, R306E, A349S, S549R, P557R, M641I, and N650K, has not been studied by biological techniques [306,307] . The mutations of D552E, Q556*, S560dup, P557R, G620R, Y647S, G815R, F817L, and G827R are loss-of-functions. The S560 is located in pre-TM1 region, and the mutation of S560dup decreases the activity of NMDARs and changes the structure of the pore region [308] . The G815R and F817L mutations are adjacent to each other, thus their functions are similar, that is, they decreased sensitivity to glutamate and glycine [309] . However, the E662K, A645S, and R844C mutations did not change the function of NMDARs [309][310][311][312] . On the contrary, the mutations of Y647C, R659W, and R794Q enhanced the function of NMDARs [306] . The R659W and R794Q enhance the potency of glutamate and glycine, while the mutation of Y647C increases potency of glycine, but not glutamate.

The GluN2A mutations
GluN2A subunit is encoded by GRIN2A in the human chromosome 16p13. More than 240 mutations have been found in GRIN2A. Many patients had normal delivery at birth, with good appearance, facial expression, and vital signs scores. However, the patients at the 1 st year after birth may begin to exhibit neurological abnormalities, such as abnormal electroencephalogram and myoclonic convulsions [313] , and then progress to epilepsy, which is possibly due to a gradual increase in GluN2A expression during development [47] . Some studies indicated that the patients carrying GluN2A mutations were more likely to show epilepsy, such as benign focal epilepsy with centrotemporal spikes and Landau-Kleffner syndrome [314][315][316] . Moreover, the mutations of GluN2A can produce different effects: gain-of-function and loss-offunction.
Some mutations are gain-of-function. The GluN2A_ N447K is found in a male patient with Rolandic epilepsy and is located in the S1 domain of ABD. The GluN2A_ N447K enhanced the current density of NMDAR by electrophysiological recording. The potency of glutamate was increased, while the inhibition of Mg 2+ was decreased by GluN2A_N447K. Lamotrigine and valproate treatment could rescue the epilepsy in patient [317] . The patient is a child with impaired cognitive and epileptic encephalopathy. Another mutation, GluN2A_L812M, is located in the linker between S2 of ABD and M4 of TMD. Studies have shown that the GluN2A_L812M and M817V enhanced the open probability and potency with glutamate and glycine of NMDAR, and decreased the inhibition of Mg 2+ [318,319] . The patient with GluN2A_L812M was treated by memantine, which attenuated epilepsy [313] . The other mutation GluN2A_N615K is in the channel pore of M2 TMD domain. The patient was a 3-year-old female with abnormal electroencephalogram, developmental delay, and earlyonset epileptic encephalopathy [320] . The GluN2A_N615K mutation changed the channel characteristics, including the decreased Ca 2+ permeability and Mg 2+ sensitivity [321][322][323] . The reduction of Mg 2+ sensitivity to NMDARs increased the NMDAR current.
Meanwhile, the mutations of GluN2A are loss-of-function. The GluN2A_D731N was found in a child with developmental delay and epilepsy. The missense mutation is in the ABD of GluN2A. The results indicated that the mutation decreased the NMDAR current, glutamate sensitivity, and enhanced the potency of NAM [324] . The GluN2A_V685G is located in the S2 of ABD region and caused the developmental delay and epilepsy. The mutation caused a low glutamate potency with NMDAR and decreased NMDAR current with reduced surface expression [325] .
Besides, the mutations also lead to impairment in trafficking. The GluN2A_S1459G, which is located in the CTD-related to the protein trafficking of GluN2A, was found in the epilepsy patients [314] . The GluN2A_ S1459G induced the impaired binding with SNX27 and PSD95, which led to deficits in NMDAR trafficking and spine density, and synaptic transmission in excitatory neurons [326] .
From the studies, we understand that the mutations may exist in any domains of GluN2A to cause the disorders. Since each mutation is specific, we need to better understand the pathogenesis and changes in function of the mutation so as to increase the efficacy of targeted therapy in the patients.

The GluN2B mutations
GRIN2B gene encodes the GluN2B subunit and is in the human chromosome 12p13. Over 200 mutations have been found in GRIN2B in the patients not only with epilepsy, epileptic encephalopathy [325,327] , intellectual disability [320,325,327,328] , and ASD [320,325,327] , but also with the AD [329] and schizophrenia [330][331][332][333] . The mutations exist in all regions of GluN2B. Some of the mutations have been functionally analyzed.
Many GluN2B mutations are loss-of-function in patient with intellectual disability or ASD. The GluN2B_E413G is located in the ATD domain, which is adjacent to the glutamate-binding site. The E413G reduced the potency of glutamate and deactivation, resulting in the loss-offunction of NMDAR [334] . Another study reported the function of GluN2B_C456Y mutation. They constructed the heterozygous GluN2B_C456Y mutation mice and https://doi.org/10.36922/an.v1i2.148

Advanced Neurology
NMDA receptors in neuropsychiatric diseases found that the protein levels of GluN2B was decreased. Besides, the NMDAR current was reduced and the LTD was impaired, while the LTP was normal. The behavior data showed that the mice represented the anxious behavior with normal social interaction. In rescue experiments, the D-cycloserine had an effect on the NMDAR currents and LTP and recued the impaired behavior in adult mice [335] . The mutation GluN2B_C461F is located in the S1 domain of ABD in patient with Lennox-Gastaut syndrome and autism. Functional analysis described the C461F mutation decreased the glutamate potency [327] . In the same study, the P553L was found in the patient with intellectual disability. The mutation, which is located in the pre-MI domain, decreased the glutamate potency and NMDAR current in the neurons [327] . Another study found the mutation, which, however, mutated to the threonine (P553T), at the same site. The mutation was found in a 5-year-old patient with Rett-like syndrome. The P553T reduced the potency of glutamate, NMDAR currents and spine density in the neurons. Furthermore, the D-serine could restore the deficits induced by GluN2B_P553T [336] .
The mutations of N615I and V618G are gain-offunction and found in the patients with West syndrome and intellectual disability [337] . The two mutations reduced the inhibition of Mg 2+ , although the glutamate potency was unchanged. Furthermore, the mutation of R540H increased the sensitivity to glutamate and Ca 2+ permeability [338] . The gain-of-function mutations may lead to neuronal hyperexcitability, resulting in the disorders. Therefore, we can treat the diseases by inhibiting NMDAR function in gain-of-function mutations.

The challenge and opportunity in treatment of diseases
In biological experiments, the addition of glycine, D-serine, and D-cycloserine can enhance the function of NMDAR, which can provide a new drug choice for clinical use as NMDAR potentiators [336] . Besides, in some studies, patients carrying GluN2A and GluN2B mutations were treated with drugs, such as memantine and the blocker of NMDARs [319] . Likewise, the other drugs, such as ketamine, magnesium, and TCN-201, have effects on the inhibition of NMDAR current in vitro [318,319,339] . Since different patients respond differently to the drug, personalized treatment is needed for patients with NMDAR mutations.
However, due to the wide distribution and function of NMDARs, drugs targeting NMDARs may cause severe adverse effects limiting their clinical potential. The competitive NMDAR antagonists, which prevent glutamate-mediated neurotoxicity by inhibiting NMDAR function, could cause extensive inhibition of NMDAR function. Inhibition of NMDAR with non-competitive antagonists is the most attractive therapeutic intervention, because the effect of antagonists requires pre-activation of the NMDA receptor [340] . However, NMDAR hypofunction has an influence on the brain function. Even low doses of NMDAR antagonists can cause decreased excitability of NMDAR, leading to memory dysfunction and learning disabilities. Besides, enhancing the function of NMDARs may cause NMDAR hyperexcitability due to the inability to distinguish between extrasynaptic and synaptic NMDARs. Comprehensive weighing of the benefits versus adverse effects need be carried out during the clinical development of drugs targeting NMDARs.

Summary
NMDAR plays an important role in many processes, including learning and memory, development, and neuroplasticity. Many functions of NMDAR depend primarily on the GluN2 subunit. GluN2 subunits are mainly expressed in the cortex and hippocampus, and they differ in expression patterns, protein trafficking, channel dynamics, synaptic plasticity, and neuropsychiatric disorders. This difference also determines the different functions of NMDAR. In addition, the composition of the NMDAR subunit is also regulated by neural activity, which, further, indicates that the composition of NMDARs is consistent with neural function. However, it is still difficult to distinguish the functions and effects of individual subunits, which also leads to the difficulty in clinical treatment of neuropsychiatric disorders. Therefore, in addition to using new drugs and genetic approaches to specifically target different subunits and related signaling pathways in the treatment of neuropsychiatric disorders, it would be also interesting to identify the auxiliary subunits of NMDAR, which is involved in the regulation of the ions accessibility, and how this interaction affects the downstream signaling pathways may contribute to the understanding of the cellular mechanism in different brain diseases. The double-sided function of NMDAR poses great challenges for clinical application, so we still need to make continuous efforts in the treatment of neuropsychiatric disorders targeting NMDARs.