α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) and N-Methyl-d-aspartate (NMDA) Receptors Adopt Different Subunit Arrangements

Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type, and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here, we use atomic force microscopy imaging of receptor-antibody complexes to show that whereas the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.     

Structural rearrangements of NR1/NR2A NMDA receptors during allosteric inhibition

Ionotropic glutamate receptor (iGluR) subunits contain a large N-terminal domain (NTD) that precedes the agonist-binding domain (ABD) and participates in subunit oligomerization. In NMDA receptors (NMDARs), the NTDs of NR2A and NR2B subunits also form binding sites for the endogenous inhibitor Zn(2+) ion. Although these allosteric sites have been characterized in detail, the molecular mechanisms by which the NTDs communicate with the rest of the receptor to promote its inhibition remain unknown. Here, we identify the ABD dimer interface as a major structural determinant that permits coupling between the NTDs and the channel gate. The strength of this interface also controls proton inhibition, another form of allosteric modulation of NMDARs. Conformational rearrangements at the ABD dimer interface thus appear to be a key mechanism conserved in all iGluR subfamilies, but have evolved to fulfill different functions: fast desensitization at AMPA and kainate receptors, allosteric inhibition at NMDARs.     

Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening

The defining functional feature of N-methyl-d-aspartate (NMDA) receptors is activation gating, the energetic coupling of ligand binding into opening of the associated ion channel pore. NMDA receptors are obligate heterotetramers typically composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits that gate in a concerted fashion, requiring all four ligands to bind for subsequent opening of the channel pore. In an individual subunit, the extracellular ligand-binding domain, composed of discontinuous polypeptide segments S1 and S2, and the transmembrane channel-forming domain, composed of M1-M4 segments, are connected by three linkers: S1-M1, M3-S2, and S2-M4. To study subunit-specific events during pore opening in NMDA receptors, we impaired activation gating via intrasubunit disulfide bonds connecting the M3-S2 and S2-M4 in either the GluN1 or GluN2A subunit, thereby interfering with the movement of the M3 segment, the major pore-lining and channel-gating element. NMDA receptors with gating impairments in either the GluN1 or GluN2A subunit were dramatically resistant to channel opening, but when they did open, they showed only a single-conductance level indistinguishable from wild type. Importantly, the late gating steps comprising pore opening to its main long-duration open state were equivalently affected regardless of which subunit was constrained. Thus, the NMDA receptor ion channel undergoes a pore-opening mechanism in which the intrasubunit conformational dynamics at the level of the ligand-binding/transmembrane domain (TMD) linkers are tightly coupled across the four subunits. Our results further indicate that conformational freedom of the linkers between the ligand-binding and TMDs is critical to the activation gating process.     

Amyloid beta peptide 1-42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that leads to debilitating cognitive deficits. Recent evidence demonstrates that glutamate receptors are dysregulated by amyloid beta peptide (Aβ) oligomers, resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits. Although it is well accepted that neuronal death in AD is related to disturbed intracellular Ca(2+) (Ca(2+)(i)) homeostasis, little is known about the contribution of NMDARs containing GluN2A or GluN2B subunits on Aβ-induced Ca(2+)(i) rise and neuronal dysfunction. Thus, the main goal of this work was to evaluate the role of NMDAR subunits in dysregulation of Ca(2+)(i) homeostasis induced by Aβ 1-42 preparation containing both oligomers (in higher percentage) and monomers in rat cerebral cortical neurons. The involvement of NMDARs was evaluated by pharmacological inhibition with MK-801 or the selective GluN2A and GLUN2B subunit antagonists NVP-AAM077 and ifenprodil, respectively. We show that Aβ, like NMDA, increase Ca(2+)(i) levels mainly through activation of NMDARs containing GluN2B subunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca(2+)(i) rise induced by a high concentration of Aβ (1μM), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca(2+)(i) homeostasis. Moreover, Aβ modulate NMDA-induced responses and vice versa. Indeed, pre-exposure to Aβ (1μM) decrease NMDA-evoked Ca(2+)(I) rise and pre-exposure to NMDA decrease Aβ response. Interestingly, simultaneous addition of Aβ and NMDA potentiate Ca(2+)(I) levels, this effect being regulated by GluN2A and GluN2B subunits in opposite manners. This study contributes to the understanding of the molecular basis of early AD pathogenesis, by exploring the role of GluN2A and GluN2B subunits in the mechanism of Aβ toxicity in AD.     

Molecular basis of positive allosteric modulation of GluN2B NMDA receptors by polyamines

NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.     

Neurosteroid binding to the amino terminal and glutamate binding domains of ionotropic glutamate receptors

The endogenous neurosteroids, pregnenolone sulfate (PS) and 3α-hydroxy-5β-pregnan-20-one sulfate (PREGAS), have been shown to differentially regulate the ionotropic glutamate receptor (iGluR) family of ligand-gated ion channels. Upon binding to these receptors, PREGAS decreases current flow through the channels. Upon binding to non-NMDA or NMDA receptors containing an GluN2C or GluN2D subunit, PS also decreases current flow through the channels, however, upon binding to NMDA receptors containing an GluN2A or GluN2B subunit, flow through the channels increases. To begin to understand this differential regulation, we have cloned the S1S2 and amino terminal domains (ATD) of the NMDA GluN2B and GluN2D and AMPA GluA2 subunits. Here we present results that show that PS and PREGAS bind to different sites in the ATD of the GluA2 subunit, which when combined with previous results from our lab, now identifies two binding domains for each neurosteroid. We also show both neurosteroids bind only to the ATD of the GluN2D subunit, suggesting that this binding is distinct from that of the AMPA GluA2 subunit, with both leading to iGluR inhibition. Finally, we provide evidence that both PS and PREGAS bind to the S1S2 domain of the NMDA GluN2B subunit. Neurosteroid binding to the S1S2 domain of NMDA subunits responsible for potentiation of iGluRs and to the ATD of NMDA subunits responsible for inhibition of iGluRs, provides an interesting option for therapeutic design.     

A critical role for GluN2B-containing NMDA receptors in cortical development and function

The subunit composition of N-methyl D-aspartate receptors (NMDARs) is tightly regulated during cortical development. NMDARs are initially dominated by GluN2B (NR2B), whereas GluN2A (NR2A) incorporation increases after birth. The function of GluN2B-containing NMDARs during development, however, is incompletely understood. We generated a mouse in which we genetically replaced GluN2B with GluN2A (2B→2A). Although this manipulation restored NMDAR-mediated currents at glutamatergic synapses, it did not rescue GluN2B loss of function. Protein translation-dependent homeostatic synaptic plasticity is occluded in the absence of GluN2B, and AMPA receptor contribution is enriched at excitatory cortical synapses. Our experiments indicate that specificity of GluN2B-mediated signaling is due to its unique interaction with the protein effector alpha calcium-calmodulin kinase II and the regulation of the mTOR pathway. Homozygous 2B→2A mice exhibited high rates of lethality, suppressed feeding, and depressed social exploratory behavior. These experiments indicate that GluN2B-containing NMDARs activate unique cellular processes that cannot be rescued by replacement with GluN2A.     

Palmitoylation at Two Cysteine Clusters on the C-Terminus of GluN2A and GluN2B Differentially Control Synaptic Targeting of NMDA Receptors

Palmitoylation of NMDARs occurs at two distinct cysteine clusters in the carboxyl-terminus of GluN2A and GluN2B subunits that differentially regulates retention in the Golgi apparatus and surface expression of NMDARs. Mutations of palmitoylatable cysteine residues in the membrane-proximal cluster to non-palmitoylatable serines leads to a reduction in the surface expression of recombinant NMDARs via enhanced internalization of the receptors. Mutations in a cluster of cysteines in the middle of the carboxyl-terminus of GluN2A and GluN2B, leads to an increase in the surface expression of NMDARs via an increase in post-Golgi trafficking. Using a quantitative electrophysiological assay, we investigated whether palmitoylation of GluN2 subunits and the differential regulation of surface expression affect functional synaptic incorporation of NMDARs. We show that a reduction in surface expression due to mutations in the membrane-proximal cluster translates to a reduction in synaptic expression of NMDARs. However, increased surface expression induced by mutations in the cluster of cysteines that regulates post-Golgi trafficking of NMDARs does not increase the synaptic pool of NMDA receptors, indicating that the number of synaptic receptors is tightly regulated.     

Ionotropic glutamate receptor subunit distribution on hypoglossal motoneuronal pools in the rat

The expression of ionotropic glutamate receptor subunits in the motoneuronal pools of the hypoglossal nucleus was studied using specific antibodies against subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes. The highest numbers of intensely immunolabelled motoneurons were found in the dorsal tier and caudoventromedial part of the hypoglossal nucleus with all antibodies except that against the GluR1 AMPA subunit. Labelling for the GluR1 subunit was weak except for caudally located groups of motoneurons which innervate tongue muscles related to respiratory activity. By contrast, most motoneurons were intensely immunostained with antibodies against GluR2/3 and GluR4 subunits of the AMPA subtype. The low staining observed using an antibody specific for the GluR2 subunit (which prevents Ca²⁺-entry through AMPA channels) strongly suggests that AMPA receptors in hypoglossal motoneurons are Ca²⁺-permeable. Immunolabelling for the GluR5/6/7 kainate receptor subunits was found in many motoneuronal somata as well as in thin axon-like profiles and puncta that resembled synaptic boutons. Most motoneurons were intensely immunostained for the NMDA receptor subunit NR1. These results show that the hypoglossal nucleus contains five heterogeneous pools of motoneurons which innervate functionally defined groups of tongue muscles. The uneven expression of the different receptor subunits analysed here could reflect diverse phenotypic properties of hypoglossal motoneurons which might be expected to generate different patterns of motor responses under different physiological or pathological conditions.     

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with K_b values of 21–63 nm at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.     

Two N-glycosylation Sites in the GluN1 Subunit Are Essential for Releasing N-methyl-d-aspartate (NMDA) Receptors from the Endoplasmic Reticulum

NMDA receptors (NMDARs) comprise a subclass of neurotransmitter receptors whose surface expression is regulated at multiple levels, including processing in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, internalization, recycling, and degradation. With respect to early processing, NMDARs are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications, including phosphorylation and palmitoylation. However, the role of N-glycosylation, one of the most common posttranslational modifications, in regulating NMDAR processing has not been studied in detail. Using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy, we found that NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (Asn-203 and Asn-368) are N-glycosylated. Although the GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.     

Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo

During development there is an activity-dependent switch in synaptic N-Methyl-D-aspartate (NMDA) receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of this?switch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find that both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2B-containing receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A, which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence for?a large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.     

Apparent homomeric NR1 currents observed in Xenopus oocytes are caused by an endogenous NR2 subunit

Functional N-methyl-d-aspartate receptors NMDARs are thought to be heteromeric receptor complexes consisting of NR1 and NR2 subunits. However, recombinant NR1 subunits expressed in Xenopus oocytes assemble functional ion channels even without exogenous NR2 subunits and with a different pharmacology, suggesting a homomeric subunit stoichiometry. To explain this phenomenon, we screened oocytes for Xenopus NR2 subunits and found all four subunit-encoding mRNAs (XenNR2A-XenNR2D) to be present endogenously, with those encoding the XenNR2B subunit being particularly abundant. We cloned the full-length XenNR2B cDNA and co-expressed it with NR1 in oocytes. A detailed electrophysiological characterization revealed that the pharmacology of NR1/XenNR2B was identical with that of the presumed homomeric NMDARs expressed from NR1 subunits. By contrast, heteromeric receptors containing the rat NR2B subunit showed significant pharmacological differences compared with NR1/XenNR2B receptors. These results demonstrate that recombinant NR1 subunits expressed in Xenopus oocytes interact with an endogenously expressed NR2B subunit and form hybrid heteromeric NMDARs. These findings confirm the current views that NMDARs are obligatory heteromeric complexes and that functional homomeric NMDARs do not exist.     

GluA and GluN receptors regulate the surface density of GluN receptor subunits in cultured neocortical interneurons

J. Neurochem. (2012) 121, 597-606. ABSTRACT: In cultured rat neocortical interneurons, we have studied the effect of long-term application of NMDA or AMPA on the surface density of the NMDA (GluN) receptor subunits GluN1 and GluN2B. Stimulation of Ca(2+) -permeable AMPA (GluA) receptors located on the interneurons decreased the response of GluN receptors. The reduction was caused by a decrease in the surface density of GluN1/GluN2B subunits. In contrast, stimulation of GluN receptors located on the interneurons enhanced the surface density of GluN1/GluN2B subunits. Both effects could be induced by network activation.     

GluN2D‐containing NMDA receptors inhibit neurotransmission in the mouse striatum through a cholinergic mechanism: implication for Parkinson's disease

The GluN2 subunits that compose NMDA receptors (NMDARs) determine functional and pharmacological properties of the receptor. In the striatum, functions and potential dysfunctions of NMDARs attributed to specific GluN2 subunits have not been clearly elucidated, although NMDARs play critical roles in the interactions between glutamate and dopamine. Through the use of amperometry and field potential recordings in mouse brain slices, we found that NMDARs that contain the GluN2D subunit contribute to NMDA‐induced inhibition of evoked dopamine release and of glutamatergic neurotransmission in the striatum of control mice. Inhibition is likely mediated through increased firing in cholinergic interneurons, which were shown to express GluN2D. Indeed, NMDA‐induced inhibition of both dopamine release and glutamatergic neurotransmission is reduced in the presence of muscarinic receptor antagonists and is mimicked by a muscarinic receptor agonist. We have also examined whether this function of GluN2D‐containing NMDARs is altered in a mouse model of Parkinson's disease. We found that the inhibitory role of GluN2D‐containing NMDARs on glutamatergic neurotransmission is impaired in the 6‐hydroxydopamine lesioned striatum. These results identify a role for GluN2D‐containing NMDARs and adaptive changes in experimental Parkinsonism. GluN2D might constitute an attractive target for the development of novel pharmacological tools for therapeutic intervention in Parkinson's disease.

     

Stimulation of GluN receptors decreases the surface density of GluN1/GluN2B subunits in cultured neocortical interneurons

Changes in the density of NMDA (GluN) receptors in the neuronal membrane are critical for plasticity, whereas malfunction of precisely regulated GluN receptor activity may be involved in neurotoxicity. In cultured rat neocortical interneurons, we have studied the regulation of the surface density of GluN1, GluN2A and GluN2B subunits. Application of 5 μMol NMDA for 24 h followed by a washout period of 24 h decreased the response of GluN receptors for at least 2?days. The reduction was caused by a decrease in the surface density of GluN1/GluN2B subunits, whereas GluN2A subunits remained unaffected. Our data indicate that long but reversible low level activation of GluN receptors can cause long-term changes in their subunit composition in cultured interneurons.     

Topiramate reduces AMPA-induced Ca(2+) transients and inhibits GluR1 subunit phosphorylation in astrocytes from primary cultures

Topiramate (TPM) is a structurally novel broad spectrum anticonvulsant known to have a negative modulatory effect on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate subtypes of glutamate receptors and some types of voltage-gated Na(+) and Ca(2+) channels, and a positive modulatory effect on some types of gamma-aminobutyric acid(A) (GABA(A)) receptors and at least one type of K(+) channels in neurons. In an earlier work, we showed that the negative modulatory effect of TPM (100 mum) on AMPA/kainate receptors in neurons is dependent on TPM modulation of the phosphorylation state of these receptors. In this work, we investigated the effect of TPM on AMPA-induced intracellular calcium ([Ca(2+)](i)) responses in cultured rat cortical astrocytes, with special interest in intracellular mechanisms. Here, we report that the ability of TPM (1-100 mum) to inhibit AMPA-induced accumulation of Ca(2+) in astrocytes is inversely related to the level of protein kinase A (PKA) -mediated phosphorylation of channels activated by AMPA. The level of receptor phosphorylation was further determined with western blot using phosphorylation specific antibodies that recognize the glutamate receptor 1 (GluR1) subunit phosphorylated on Ser845. These results demonstrated that, even in cultured cortical astrocytes, TPM significantly reduced the phophorylation level of GluR1 subunits. Furthermore, it was shown that TPM binds to AMPA receptors in the dephosphorylated state and thereby exerts an allosteric modulatory effect on the ion channel.     

Modulation of triheteromeric NMDA receptors by N-terminal domain ligands

NMDA receptors (NMDARs) are heteromeric assemblies of NR1 and NR2(A-D) subunits with properties heavily influenced by the type of NR2 subunit incorporated. While NMDARs with only one type of NR2 subunit have been extensively characterized, little is known about receptors containing two different NR2 subunits, despite compelling evidence that such triheteromeric receptors exist in vivo. We used a point-mutation approach that allows isolation of recombinant triheteromeric NMDARs possessing two different NR2 N-terminal domains (NTDs). We show that in receptors associating the NR2A-NTD (sensing nanomolar Zn) and the NR2B-NTD (sensing ifenprodil), each NTD binding site retains selective high affinity for its ligand. However, each ligand produces only partial inhibition, and maximal inhibition requires occupancy of both NR2-NTDs by their respective ligands. Similarly, NR1/2A/2C receptors are inhibited by zinc with high potency but low efficacy. Therefore, interactions between homologous N-terminal domains determine the unique pharmacological properties of triheteromeric NMDARs.     

GluN2B-containing NMDA receptors as possible targets for the neuroprotective and antidepressant effects of fluoxetine

Accumulating evidence has indicated the involvement of glutamatergic neurotransmission in the pathophysiology of excitotoxicity and in the mechanism of action of antidepressants. We have previously shown that tricyclic desipramine and the selective serotonin reuptake inhibitor fluoxetine inhibit NMDA receptors (NMDARs) in the clinically relevant, low micromolar concentration range. As the different subtypes of NMDARs are markedly different in their physiological and pathological functions, our aim was to investigate whether the effect of antidepressants is subtype-specific. Using whole-cell patch-clamp recordings in rat cortical cell cultures, we studied the age-dependence of inhibition of NMDA-induced currents after treatment with desipramine and fluoxetine, as the expression profile of the NMDAR subtypes changes as a function of days in vitro. We also investigated the inhibitory effect of these antidepressants on NMDA-induced currents in HEK 293 cell lines that stably expressed rat recombinant NMDARs with GluN1a/GluN2A or GluN1a/GluN2B subunit compositions. The inhibitory effect of desipramine was not age-dependent, whereas fluoxetine displayed a continuously decreasing inhibitory profile, which was similar to the GluN1/GluN2B subtype-selective antagonist ifenprodil. In HEK 293 cells, desipramine equally inhibited NMDA currents in both cell lines, whereas fluoxetine showed an inhibitory effect only in cells that expressed the GluN1/GluN2B subtype. Our data show that fluoxetine is a selective inhibitor of GluN2B-containing NMDARs, whereas desipramine inhibits both GluN1/GluN2A and GluN1/GluN2B subtypes. As the clinical efficacy of these drugs is very similar, the putative NMDAR-associated therapeutic effect of antidepressants may be mediated only via inhibition of the GluN2B-containing subtype. The manifestation of the GluN1/GluN2B-selectivity of fluoxetine suggests the neuroprotective potential for this drug in both acute and chronic neurodegenerative disorders.