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1.  Structure and Composition of the Postsynaptic Density during Development 
The Journal of comparative neurology  2010;518(20):4243-4260.
In this study, we used electron tomography as well as immuno-gold labeling to analyze the morphology and distribution of proteins within PSDs isolated from rats before birth (embryonic day 19) and at post-natal days 2, 21 and 60. Our data provides direct evidence of distinct morphological and compositional differences in PSDs throughout development. Not all PSD components are present at the early stages of development, with a near lack of the scaffolding molecule PSD-95 at E19 and P2. The presence of NR1 and NR2b suggests that PSD-95 is not directly required for clustering of N-methyl-D-aspartic acid (NMDA) receptors in PSDs early in development. α-Actinin is abundant by E19 suggesting it is a core structural component of the PSD. Both α and β isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) are present early on, but then rise in labeling density approximately four-fold by P21. Of all the molecules studied, only calmodulin (CaM) was found in higher abundance early in PSD development and then fell in amount over time. Spatial analysis of the immuno-gold label shows a non-random distribution for all the proteins studied, lending support to the idea that the PSD is systematically assembled in an organized fashion. Morphological data from electron tomography shows that the PSD undergoes major structural changes through out development.
doi:10.1002/cne.22451
PMCID: PMC2948241  PMID: 20878786
synapse; electron tomography; synaptogenesis; CaMKII; dendritic spine; immuno-gold labeling
2.  Reduced levels of NR2A and NR2B subunits of NMDA receptor and PSD-95 in the prefrontal cortex in major depression 
Recent neuroimaging and postmortem studies have demonstrated abnormalities in glutamatergic transmission in major depression. Glutamate NMDA (N-methyl-D-aspartate) receptors are one of the major mediators of excitatory neurotransmission in the central nervous system. At synaptic sites, NMDA receptors are linked with postsynaptic density protein-95 (PSD-95) that plays a key role in mediating trafficking, clustering, and downstream signaling events, following receptor activation. In this study, we examined the expression of NMDA receptor subunits NR1, NR2A, and NR2B as well as PSD-95 in the anterior prefrontal cortex (PFC) using Western blot method. Cortical samples were obtained from age, gender and postmortem interval matched depressed and psychiatrically healthy controls. The results revealed that there was a reduced expression of the NMDA receptor subunits NR2A (−54%) and NR2B (−48%), and PSD-95 protein level (−40%) in the PFC of depressed subjects relative to controls, with no change in the NR1 subunit. The alterations in NMDA receptor subunits, especially the NR2A and NR2B, as well as PSD-95 suggest an abnormality in the NMDA receptor signaling in the PFC in major depression. Our findings in conjunction with recent clinical, cellular, and neuroimaging studies further implicate the involvement of glutamate neurotransmission in the pathophysiology of depression. This study provides additional evidence that NMDA receptor complex is a target for discovery of novel antidepressants.
doi:10.1016/j.pnpbp.2008.10.005
PMCID: PMC2655629  PMID: 18992785
major depressive disorder; glutamate receptors; postmortem; postsynaptic density protein; anterior prefrontal cortex
3.  Acute Inactivation of PSD-95 Destabilizes AMPA Receptors at Hippocampal Synapses 
PLoS ONE  2013;8(1):e53965.
Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.
doi:10.1371/journal.pone.0053965
PMCID: PMC3546964  PMID: 23342049
4.  Decreased expression of NMDA receptor-associated proteins in frontal cortex of elderly patients with schizophrenia 
Neuroreport  2009;20(11):1019-1022.
Converging evidence suggests too few activation-ready N-methyl-d-aspartic acid (NMDA) receptor complexes in the postsynaptic density in schizophrenia. Postsynaptic density protein 95 (PSD95), Synaptic GTPase-activating protein (SynGAP), and Multiple PDZ domain protein (MUPP1) are integral components of the NMDA receptor signaling complex, and help facilitate signaling, trafficking, and stabilization. We hypothesized that deficits involving these molecules may contribute to the pathophysiology of schizophrenia. To test our hypothesis, we measured protein expression of PSD95, SynGAP, and MUPP1 in the anterior cingulate cortex and dorsolateral prefrontal cortex. We found decreased PSD95 expression in the anterior cingulate cortex. Antipsychotic medication analyses showed decreased SynGAP expression in the anterior cingulate cortex in patients off medication when analyzed against our comparison group. These data suggest that NMDA receptor complex formation, localization, and downstream signaling may be abnormal in schizophrenia.
doi:10.1097/WNR.0b013e32832d30d9
PMCID: PMC3731946  PMID: 19483657
MUPP1; N-methyl-d-aspartic acid receptor; PSD95; schizophrenia; SynGAP
5.  Loose ligation of the rat sciatic nerve elicits early accumulation of Shank1 protein in the postsynaptic density of spinal dorsal horn neurons 
Pain  2010;149(1):152-159.
Plasticity in the spinal dorsal horn may contribute to the development of pain following peripheral nerve injury. Shank proteins are a constituent family of the post-synaptic density (PSD), and they may play a role in synaptic plasticity through activity-dependent synaptic remodeling and growth. In this study we examined the early consequences of the loose ligation of the sciatic nerve on Shank1 protein and message levels in the PSD of spinal dorsal horn neurons.
Four hours after sciatic ligation, the protein levels of Shank1 increased in the ipsilateral PSD of ligated animals. In contrast, no changes were detected in the contralateral PSD of these ligated animals, or either the ipsilateral or contralateral PSD of sham-operated animals. Shank1 was linked to the PSD marker protein PSD-95 and the NR2B subunit of NMDA receptors. The ligated animals also exhibited two early signs of pain behavior, a shift in weight bearing and thermal hyperalgesia. There was no overall change in Shank1 message in either ligated or sham-operated animals. The accumulation of Shank1 in the PSD was abolished by intrathecal pre-treatment with anisomycin or Shank1 siRNA but not non-target siRNA. The same pre-treatment prevented both of the early signs of pain behavior. Intrathecal pre-treatment with either MK-801 or U0126 similarly prevented the Shank1 accumulation and alleviated both of the behavioral signs of pain.
The early accumulation of Shank1 in the PSD of dorsal horn neurons may be a necessary step in the injury-associated plasticity that in time leads to the development of persistent pain.
doi:10.1016/j.pain.2010.02.001
PMCID: PMC2836861  PMID: 20171009
Chronic constriction injury; Glutamate receptors; Neuropathic pain; Nociception; Structural proteins; Synaptic plasticity
6.  Kalirin-7 Is an Essential Component of both Shaft and Spine Excitatory Synapses in Hippocampal Interneurons 
Kalirin, a multifunctional Rho GDP/GTP exchange factor, plays a vital role in cytoskeletal organization, affecting process initiation and outgrowth in neurons. Through alternative splicing, the Kalirin gene generates multiple functionally distinct proteins. Kalirin-7 (Kal7) is the most prevalent isoform in the adult rat hippocampus; it terminates with a PDZ binding motif, is localized to the post-synaptic density, interacts with PSD95 and causes the formation of dendritic spines when over-expressed in pyramidal neurons. Levels of Kal7 are low in the dendrites of hippocampal aspiny interneurons. In these interneurons, Kal7 is localized to the postsynaptic side of excitatory synapses onto dendritic shafts, overlapping clusters of PSD95 and NMDA receptor subunit NR1. Selectively decreasing levels of Kal7 decreases the density of PSD95 positive, bassoon positive clusters along the dendritic shaft of hippocampal interneurons. Over-expression of Kal7 increases dendritic branching, inducing formation of spine-like structures along the dendrites and on the soma of normally aspiny hippocampal interneurons. Essentially all of the spine-like structures formed in response to Kal7 are apposed to VGLUT1 positive, bassoon positive presynaptic endings; GAD positive, VGAT positive inhibitory endings are unaffected. Almost every Kal7 positive dendritic cluster contains PSD95 along with NMDA (NR1) and AMPA (GluR1 and GluR2) receptor subunits. Kal7-induced formation of spine-like structures requires its PDZ binding motif, and interruption of interactions between the PDZ binding motif and its interactors decreases Kal7-induced formation of spine-like structures. Kal7 thus joins Shank3 and GluR2 as molecules whose level of expression at excitatory synapses titrates the number of dendritic spines.
doi:10.1523/JNEUROSCI.5283-07.2008
PMCID: PMC2570025  PMID: 18199770
Rho GEF; PSD95; GABA; dendritic growth; synaptogenesis
7.  Size and Receptor Density of Glutamatergic Synapses: A Viewpoint from Left–Right Asymmetry of CA3–CA1 Connections 
Synaptic plasticity is considered to be the main mechanism for learning and memory. Excitatory synapses in the cerebral cortex and hippocampus undergo plastic changes during development and in response to electric stimulation. It is widely accepted that this process is mediated by insertion and elimination of various glutamate receptors. In a series of recent investigations on left–right asymmetry of hippocampal CA3–CA1 synapses, glutamate receptor subunits have been found to have distinctive expression patterns that depend on the postsynaptic density (PSD) area. Particularly notable are the GluR1 AMPA receptor subunit and NR2B NMDA receptor subunit, where receptor density has either a supralinear (GluR1 AMPA) or inverse (NR2B NMDAR) relationship to the PSD area. We review current understanding of structural and physiological synaptic plasticity and propose a scheme to classify receptor subtypes by their expression pattern with respect to PSD area.
doi:10.3389/neuro.05.010.2009
PMCID: PMC2706655  PMID: 19587849
spines; glutamate; AMPAR; NMDAR; mGluR5; PSD
8.  Glutamate signaling proteins and tyrosine hydroxylase in the locus coeruleus of alcoholics 
Journal of psychiatric research  2007;42(5):348-355.
It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and 8 psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (−30%) and middle (−41%)portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.
doi:10.1016/j.jpsychires.2007.02.010
PMCID: PMC2292639  PMID: 17481661
glutamate; NMDA receptor; postsynaptic density protein 95; neuronal nitric oxide synthase; tyrosine hydroxylase; locus coeruleus
9.  Trafficking of the NMDAR2B Receptor Subunit Distal Cytoplasmic Tail from Endoplasmic Reticulum to the Synapse 
PLoS ONE  2012;7(6):e39585.
NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.
doi:10.1371/journal.pone.0039585
PMCID: PMC3384676  PMID: 22761831
10.  Neuronal LRP1 Functionally Associates with Postsynaptic Proteins and Is Required for Normal Motor Function in Mice 
Molecular and Cellular Biology  2004;24(20):8872-8883.
The LDL receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor that is highly expressed on neurons. Neuronal LRP1 in vitro can mediate ligand endocytosis, as well as modulate signal transduction processes. However, little is known about its role in the intact nervous system. Here, we report that mice that lack LRP1 selectively in differentiated neurons develop severe behavioral and motor abnormalities, including hyperactivity, tremor, and dystonia. Since their central nervous systems appear histoanatomically normal, we suggest that this phenotype is likely attributable to abnormal neurotransmission. This conclusion is supported by studies of primary cultured neurons that show that LRP1 is present in close proximity to the N-methyl-d-aspartate (NMDA) receptor in dendritic synapses and can be coprecipitated with NMDA receptor subunits and the postsynaptic density protein PSD-95 from neuronal cell lysates. Moreover, treatment with NMDA, but not dopamine, reduces the interaction of LRP1 with PSD-95, indicating that LRP1 participates in transmitter-dependent postsynaptic responses. Together, these findings suggest that LRP1, like other ApoE receptors, can modulate synaptic transmission in the brain.
doi:10.1128/MCB.24.20.8872-8883.2004
PMCID: PMC517900  PMID: 15456862
11.  Chronic ethanol up-regulates the synaptic expression of the nuclear translational regulatory protein AIDA-1 in primary hippocampal neurons 
Alcohol (Fayetteville, N.Y.)  2012;46(6):569-576.
Recent studies have identified synaptic proteins that undergo synapse-to-nucleus translocation in response to neuronal activity that modulate protein synthesis. One such translational regulatory protein of the postsynaptic density (PSD) is AIDA-1d, which binds to PSD-95 via its C-terminus. Activation of synaptic NMDA receptors induces the cleavage of AIDA-1d, and the N-terminus is then shuttled to nuclear Cajal bodies where it plays a role in regulating global protein synthesis. Chronic ethanol exposure has been shown to increase the synaptic clustering of NMDA receptors and PSD-95. Here, we tested the hypotheses that AIDA-1d regulates chronic ethanol-induced increases in synaptic NMDA receptor expression. As reported, we found that AIDA-1 was highly enriched in dendritic spines and co-localized with PSD-95. Acute NMDA treatment increased AIDA-1 colocalization with p80 coilin, a marker of Cajal bodies. Chronic treatment (4 day) of cultures with ethanol (25 – 100 mM) or with the NMDA receptor antagonist AP-V (50 µM) enhanced the clustering of AIDA-1 at synaptic sites. However, chronic ethanol treatment (50 mM) in the presence of the NMDA receptor agonist NMDA (2.5 µM) prevented this increase. Surprisingly, PSD-95 did not seem to play a role in the synaptic distribution of AIDA-1 as this distribution was not affected by declustering PSD-95 from synapses in response to inhibition of palmitoylation. We found that lentiviral knockdown of AIDA-1d did not affect protein expression levels of NMDA receptor subunits GluN1, GluN1 C2’, or GluN2B. The results of this study demonstrate that synaptic AIDA-1 expression is enhanced by chronic ethanol exposure that can be prevented by concurrent stimulation of NMDA receptors. In addition, we found that the association of AIDA-1 with PSD-95 is not required for its localization to the PSD. Moreover, we found that AIDA-1 does not regulate protein expression levels or alternative splicing of the GluN1 subunit of NMDA receptors.
doi:10.1016/j.alcohol.2012.04.004
PMCID: PMC3411901  PMID: 22703994
AIDA-1, chronic ethanol, synapse-to-nucleus signaling; NMDA receptors; Cajal bodies; PSD-95
12.  Distribution of Extrasynaptic NMDA Receptors on Neurons 
The Scientific World Journal  2012;2012:267120.
NMDA receptors are found in both synaptic and extrasynaptic locations on neurons. NMDA receptors also can be found on neurons in early stages prior to synaptogenesis, where they may be involved in migration and differentiation. Extrasynaptic NMDA receptors typically are associated with contacts with adjacent processes such as axons and glia. Extrasynaptic NMDA receptor clusters vary in size and may form associations with scaffolding proteins such as PSD-95 and SAP102. The best-characterized extrasynaptic NMDA receptors contain NR1 and NR2B subunits. Extrasynaptic NMDA receptors may be activated by glutamate spillover from synapses or from ectopic release of glutamate. Consequently, extrasynaptic NMDA receptor activation may occur under different circumstances than that for synaptic NMDA receptors, indicating different functional consequences for the neuron. In some cases, activation of extrasynaptic NMDA receptors may have a negative influence on the neuron, leading to cell damage and death, as may occur in some major diseases of the nervous system.
doi:10.1100/2012/267120
PMCID: PMC3361219  PMID: 22654580
13.  PSD-95 is required to sustain the molecular organization of the postsynaptic density 
PSD-95, a membrane-associated guanylate kinase (MAGUK), is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNAi knockdown of PSD-95 leads to loss of entire patches of PSD material, and EM tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA, but not NMDA receptor type structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.
doi:10.1523/JNEUROSCI.5968-10.2011
PMCID: PMC3099547  PMID: 21525273
PSD-95; molecular conformation; postsynaptic density; hippocampal spines; RNAi; EM tomography; Immuno-EM
14.  Glutamate receptor composition of the post-synaptic density is altered in genetic mouse models of NMDA receptor hypo- and hyperfunction 
Brain research  2011;1392:1-7.
The N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) are ionotropic glutamate receptors responsible for excitatory neurotransmission in the brain. These excitatory synapses are found on dendritic spines, with the abundance of receptors concentrated at the postsynaptic density (PSD). We utilized two genetic mouse models, the serine racemase knockout (SR−/−) and the glycine transporter subtype 1 heterozygote mutant (GlyT1+/−), to determine how constitutive NMDAR hypo- and hyperfunction, respectively, affect the glutamate receptor composition of the PSD in the hippocampus and prefrontal cortex (PFC). Using cellular fractionation, we found that SR−/− mice had elevated protein levels of NR1 and NR2A NMDAR subunits specifically in the PSD-enriched fraction from the hippocampus, but not from the PFC. There were no changes in the amounts of AMPAR subunits (GluR1, GluR2), or PSD protein of 95kDa (PSD95) in either brain region. GlyT1+/− mice also had elevated protein expression of NR1 and NR2A subunits in the PSD, as well as an increase in total protein. Moreover, GlyT1+/− mice had elevated amounts of GluR1 and GluR2 in the PSD, and higher total amounts of GluR1. Similar to SR−/− mice, there was no protein changes observed in the PFC. These findings illustrate the complexity of synaptic adaptation to altered NMDAR function.
doi:10.1016/j.brainres.2011.03.051
PMCID: PMC3225000  PMID: 21443867
serine racemase; glycine transporter; postsynaptic density; NMDA receptor; AMPA receptor; schizophrenia
15.  Quantifying the Effects of Elastic Collisions and Non-Covalent Binding on Glutamate Receptor Trafficking in the Post-Synaptic Density 
PLoS Computational Biology  2010;6(5):e1000780.
One mechanism of information storage in neurons is believed to be determined by the strength of synaptic contacts. The strength of an excitatory synapse is partially due to the concentration of a particular type of ionotropic glutamate receptor (AMPAR) in the post-synaptic density (PSD). AMPAR concentration in the PSD has to be plastic, to allow the storage of new memories; but it also has to be stable to preserve important information. Although much is known about the molecular identity of synapses, the biophysical mechanisms by which AMPAR can enter, leave and remain in the synapse are unclear. We used Monte Carlo simulations to determine the influence of PSD structure and activity in maintaining homeostatic concentrations of AMPARs in the synapse. We found that, the high concentration and excluded volume caused by PSD molecules result in molecular crowding. Diffusion of AMPAR in the PSD under such conditions is anomalous. Anomalous diffusion of AMPAR results in retention of these receptors inside the PSD for periods ranging from minutes to several hours in the absence of strong binding of receptors to PSD molecules. Trapping of receptors in the PSD by crowding effects was very sensitive to the concentration of PSD molecules, showing a switch-like behavior for retention of receptors. Non-covalent binding of AMPAR to anchored PSD molecules allowed the synapse to become well-mixed, resulting in normal diffusion of AMPAR. Binding also allowed the exchange of receptors in and out of the PSD. We propose that molecular crowding is an important biophysical mechanism to maintain homeostatic synaptic concentrations of AMPARs in the PSD without the need of energetically expensive biochemical reactions. In this context, binding of AMPAR with PSD molecules could collaborate with crowding to maintain synaptic homeostasis but could also allow synaptic plasticity by increasing the exchange of these receptors with the surrounding extra-synaptic membrane.
Author Summary
One of the most accepted theories of information storage in neurons is that it is partially localized in the strength of synaptic contacts. Evidence suggests that at the cellular level, in combination with other cellular mechanisms, this is implemented by increasing or decreasing the concentration of a particular type of membrane molecules. Two opposing mechanisms have to coexist in synapses to allow them to store information. On one hand, synapses have to be flexible, to allow the storage of new memories. On the other hand, synapses have to be stable to preserve previously learned information. Although much is known about the molecular identity of synapses, the biophysical mechanisms by which molecules can enter, leave and remain in the synapse are unclear. Our modeling work uses fundamental biophysical principles to quantify the effects of molecular collisions and biochemical reactions. Our results show that molecular collisions alone, between the diffusing proteins with anchored molecules in the synapse, can replicate known experimental results. Molecular collision in combination with biochemical binding can be fundamental biophysical principles used by synapses for the formation and preservation of memories.
doi:10.1371/journal.pcbi.1000780
PMCID: PMC2869312  PMID: 20485563
16.  S-Nitrosylation and S-Palmitoylation Reciprocally Regulate Synaptic Targeting of PSD-95 
Neuron  2011;71(1):131-141.
PSD-95, a principal scaffolding component of the post-synaptic density, is targeted to synapses by palmitoylation where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction.
doi:10.1016/j.neuron.2011.05.033
PMCID: PMC3181141  PMID: 21745643
17.  PSD-93 MEDIATES TYROSINE-PHOSPHORYLATION OF THE N-METHYL-D-ASPARTATE RECEPTORS 
Neuroscience  2008;153(3):700-708.
Src family protein kinases (SFKs)-mediated tyrosine-phosphorylation regulates N-methyl-D-aspartate (NMDA) receptor synaptic function. Some members of the membrane-associated guanylate kinase (MAGUK) family of proteins bind to both SFKs and NMDA receptors, but it is unclear whether the MAGUK family of proteins is required for SFKs-mediated tyrosine-phosphorylation of the NMDA receptors. Here, we showed by co-immunoprecipitation that PSD-93, a member of the MAGUK family of proteins, interacts with the NMDA receptor subunits NR2A and NR2B as well as with Fyn, a member of the SFKs, in mouse cerebral cortex. Using a biochemical fractionation approach to isolate subcellular compartments revealed that the expression of Fyn, but not of other members of the SFKs (Lyn, Src, and Yes), was significantly decreased in synaptosomal membrane fractions derived from the cerebral cortex of PSD-93 knockout mice. Interestingly, we found that PSD-93 disruption causes reduction of tyrosine-phosphorylated NR2A and NR2B in the same fraction. Moreover, PSD-93 deletion markedly blocked the SFKs-mediated increase in tyrosine-phosphorylated NR2A and NR2B through the protein kinase C pathway after induction with 4β-PMA in cultured cortical neurons. Our findings indicate that PSD-93 appears to mediate tyrosine-phosphorylation of the NMDA receptors and synaptic localization of Fyn.
doi:10.1016/j.neuroscience.2008.03.006
PMCID: PMC2696054  PMID: 18423999
Src family tyrosine kinases; Fyn; MAGUK; PSD-95; NR2A; NR2B
18.  NMDA NR2A and NR2B Receptors in the Rostral Anterior Cingulate Cortex Contribute to Pain-related Aversion in Male Rats 
Pain  2009;146(1-2):183-193.
NMDA receptors, which are implicated in pain processing, are highly expressed in forebrain areas including the anterior cingulate cortex (ACC). The ACC has been implicated in the affective response to noxious stimuli. Using a combination of immunohistochemical staining, western blot, electrophysiological recording and formalin-induced conditioned place avoidance (F-CPA) rat behavioral model that directly reflects the affective component of pain, the present study examined formalin nociceptive conditioning-induced changes in the expressions of NMDA receptor subunits NR1, NR2A, and NR2B in the rostral ACC (rACC) and its possible functional significance. We found that unilateral intraplantar (i.pl.) injection of dilute formalin with or without contextual conditioning exposure markedly increased the expressions of NMDA receptor subunits NR2A and NR2B but not NR1 in the bilateral rACC. NMDA-evoked currents in rACC neurons were significant greater in formalin-injected rats than that of naïve or normal saline-injected rats. Selectively blocking either NR2A or NR2B subunit in the rACC abolished the acquisition of F-CPA and formalin nociceptive conditioning-induced Fos expression, but did not affect formalin acute nociceptive behaviors and non-nociceptive fear stimulus-induced CPA. These results suggest that both NMDA receptor subunits NR2A and NR2B in the rACC are critically involved in pain-related aversion. Thus, a new strategy targeted at NMDA NR2A or NR2B subunit might be raised for the prevention of pain-related emotional disturbance.
doi:10.1016/j.pain.2009.07.027
PMCID: PMC2764121  PMID: 19695778
NR1; NR2A; NR2B; Anterior cingulate cortex; Formalin-induced conditioned place avoidance (F-CPA); Fos; Pain affect; Rat
19.  Strain differences in the distribution of N-Methyl-D-Aspartate and Gamma (γ)-aminobutyric acid-A receptors in rat brain 
Life sciences  2009;85(23-26):794-799.
Aims
Previous studies have shown that the Wistar-Kyoto (WKY) rat strain exhibits depressive symptoms such as anhedonia, psychomotor retardation, ambivalence and negative memory bias following exposure to stress. Given the involvement of excitatory glutamate and inhibitory gamma (γ)-aminobutyric acid (GABA) signaling pathways in influencing depressive behavior, the present study investigated strain differences in the distribution of central N-methyl-D-aspartate (NMDA) and GABAA receptor sites in WKY compared to their inbred counterpart, Wistar (WIS) rats.
Main methods
Quantitative autoradiographic analysis was used to map the binding and distribution of NMDA and GABAA receptors in various brain regions in WKY and WIS rats.
Key findings
Results indicated a significant difference between the two strains. Lower NMDA receptor binding was found in the anterior cingulate cortex, caudate putmen, nucleus accumbens, CA1 region of the hippocampus and the substantia nigra pars reticulata in WKY compared to WIS rats. Conversely, higher GABAA receptor binding was found in the amygdala, caudate putmen, dentate gyrus, CA2 and CA3 fields of the hippocampus, periaqueductal gray and substantia nigra pars reticulata in WKY compared to WIS rats.
Significance
Given that these two rat strains differ in their behavioural, endocrine and neurochemical profile, the observed strain differences in NMDA and GABAA receptor binding suggests that these two neurotransmitter systems may be involved in the depressive and stress sensitive phenotype of the WKY rat strain.
doi:10.1016/j.lfs.2009.10.010
PMCID: PMC2810679  PMID: 19874829
N-Methyl-D-Aspartate Receptors; Gamma (γ)-aminobutyric acid Receptors; Wistar Kyoto Rat; Quantitative Autoradiography
20.  CXCL12 inhibits expression of the NMDA receptor's NR2B subunit through a histone deacetylase-dependent pathway contributing to neuronal survival 
Cell Death & Disease  2010;1(4):e33-.
Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl--aspartic acid (NMDA) receptor's subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains.
doi:10.1038/cddis.2010.10
PMCID: PMC3032300  PMID: 21364640
chemokine; neuron; CXCR4; cell death; calcium
21.  Molecular pharmacology of human NMDA receptors 
Neurochemistry international  2011;61(4):601-609.
N-methyl-D-aspartate (NMDA) receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. NMDA receptors are also important drug targets that are implicated in a number of pathophysiological conditions. To facilitate the transition from lead compounds in pre-clinical animal models to drug candidates for human use, it is important to establish whether NMDA receptor ligands have similar properties at rodent and human NMDA receptors. Here, we compare amino acid sequences for human and rat NMDA receptor subunits and discuss inter-species variation in the context of our current knowledge of the relationship between NMDA receptor structure and function. We summarize studies on the biophysical properties of human NMDA receptors and compare these properties to those of rat orthologs. Finally, we provide a comprehensive pharmacological characterization that allows side-by-side comparison of agonists, un-competitive antagonists, GluN2B-selective non-competitive antagonists, and GluN2C/D-selective modulators at recombinant human and rat NMDA receptors. The evaluation of biophysical properties and pharmacological probes acting at different sites on the receptor suggest that the binding sites and conformational changes leading to channel gating in response to agonist binding are highly conserved between human and rat NMDA receptors. In summary, the results of this study suggest that no major detectable differences exist in the pharmacological and functional properties of human and rat NMDA receptors.
doi:10.1016/j.neuint.2011.11.016
PMCID: PMC3335988  PMID: 22197913
ionotropic glutamate receptor; NMDA; pharmacology; electrophysiology; structure-function
22.  Genetic Deletion of NR3A Accelerates Glutamatergic Synapse Maturation 
PLoS ONE  2012;7(8):e42327.
Glutamatergic synapse maturation is critically dependent upon activation of NMDA-type glutamate receptors (NMDARs); however, the contributions of NR3A subunit-containing NMDARs to this process have only begun to be considered. Here we characterized the expression of NR3A in the developing mouse forebrain and examined the consequences of NR3A deletion on excitatory synapse maturation. We found that NR3A is expressed in many subcellular compartments, and during early development, NR3A subunits are particularly concentrated in the postsynaptic density (PSD). NR3A levels dramatically decline with age and are no longer enriched at PSDs in juveniles and adults. Genetic deletion of NR3A accelerates glutamatergic synaptic transmission, as measured by AMPAR-mediated postsynaptic currents recorded in hippocampal CA1. Consistent with the functional observations, we observed that the deletion of NR3A accelerated the expression of the glutamate receptor subunits NR1, NR2A, and GluR1 in the PSD in postnatal day (P) 8 mice. These data support the idea that glutamate receptors concentrate at synapses earlier in NR3A-knockout (NR3A-KO) mice. The precocious maturation of both AMPAR function and glutamate receptor expression are transient in NR3A-KO mice, as AMPAR currents and glutamate receptor protein levels are similar in NR3A-KO and wildtype mice by P16, an age when endogenous NR3A levels are normally declining. Taken together, our data support a model whereby NR3A negatively regulates the developmental stabilization of glutamate receptors involved in excitatory neurotransmission, synaptogenesis, and spine growth.
doi:10.1371/journal.pone.0042327
PMCID: PMC3411625  PMID: 22870318
23.  Perturbing PSD-95 Interactions With NR2B-subtype Receptors Attenuates Spinal Nociceptive Plasticity and Neuropathic Pain 
Molecular Therapy  2011;19(10):1780-1792.
Peripheral inflammation or nerve injury induces a primary afferent barrage into the spinal cord, which can cause N-methyl -aspartate (NMDA) receptor-dependent alterations in the responses of dorsal horn sensory neurons to subsequent afferent inputs. This plasticity, such as “wind-up” and central sensitization, contributes to the hyperexcitability of dorsal horn neurons and increased pain-related behavior in animal models, as well as clinical signs of chronic pain in humans, hyperalgesia and allodynia. Binding of NMDA receptor subunits by the scaffolding protein postsynaptic density protein-95 (PSD-95) can facilitate downstream intracellular signaling and modulate receptor stability, contributing to synaptic plasticity. Here, we show that spinal delivery of the mimetic peptide Tat-NR2B9c disrupts the interaction between PSD-95 and NR2B subunits in the dorsal horn and selectively reduces NMDA receptor-dependent events including wind-up of spinal sensory neurons, and both persistent formalin-induced neuronal activity and pain-related behaviors, attributed to central sensitization. Furthermore, a single intrathecal injection of Tat-NR2B9c in rats with established nerve injury-induced pain attenuates behavioral signs of mechanical and cold hypersensitivity, with no effect on locomotor performance. Thus, uncoupling of PSD-95 from spinal NR2B-containing NMDA receptors may prevent the neuronal plasticity involved in chronic pain and may be a successful analgesic therapy, reducing side effects associated with receptor blockade.
doi:10.1038/mt.2011.42
PMCID: PMC3188755  PMID: 21427709
24.  PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and Synaptic Plasticity 
Current opinion in neurobiology  2011;21(2):306-312.
Activity-dependent modification of excitatory synaptic transmission is fundamental for developmental plasticity of the neural circuits and experience-dependent plasticity. Synaptic glutamatergic receptors including AMPA receptors and NMDA receptors (AMPARs and NMDARs) are embedded in the highly organized protein network in the postsynaptic density. Overwhelming data have shown that PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs), as a major family of scaffold proteins at glutamatergic synapses, regulate basal synaptic AMPAR function and trafficking. It is now clear that PSD-MAGUKs have multifaceted functions in terms of regulating synaptic transmission and plasticity. Here we discuss recent advancements in understanding the roles of PSD-95 and other family members of PSD-MAGUKs in synaptic plasticity, both as an anchoring protein for synaptic AMPARs and also as a signaling scaffold for mediating the interaction of the signaling complex and NMDARs.
doi:10.1016/j.conb.2011.03.001
PMCID: PMC3138136  PMID: 21450454
25.  Coactivation of NMDA receptors by glutamate and -serine induces dilation of isolated middle cerebral arteries 
N-methyl--aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, -serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl--aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of -serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, -2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and -serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and -serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.
doi:10.1038/jcbfm.2011.161
PMCID: PMC3293118  PMID: 22068228
-serine; eNOS; glutamate; middle cerebral artery; NMDA receptor; NR2C

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