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1.  Unique ionotropic receptors for D-Aspartate are a target for serotonin-induced synaptic plasticity in Aplysia californica✰ 
The non-L-glutamate (L-Glu) receptor component of D-aspartate (D-Asp) currents in Aplysia californica buccal S cluster (BSC) neurons was studied with whole cell voltage clamp to differentiate it from receptors activated by other well-known agonists of the Aplysia nervous system and investigate modulatory mechanisms of D-Asp currents associated with synaptic plasticity. Acetylcholine (ACh) and serotonin (5-HT) activated whole cell excitatory currents with similar current voltage relationships to D-Asp. These currents, however, were pharmacologically distinct from D-Asp. ACh currents were blocked by hexamethonium (C6) and tubocurarine (d-TC), while D-Asp currents were unaffected. 5-HT currents were blocked by granisetron and methysergide (MES), while D-Asp currents were unaffected. Conversely, while (2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid(PPDA) blocked D-Asp currents, it had no effect on ACh or 5-HT currents. Comparison of the charge area described by currents induced by ACh or 5-HT separately from, or with, D-Asp suggests activation of distinct receptors by all 3 agonists. Charge area comparisons with L-Glu, however, suggested some overlap between L-Gluand D-Asp receptors. Ten minute exposure to 5-HT induced facilitation of D-Asp-evoked responses in BSC neurons. This effect was mimicked by phorbol ester, suggesting that protein kinase C (PKC) was involved.
doi:10.1016/j.cbpc.2011.04.001
PMCID: PMC3155736  PMID: 21497673
patch clamping; electrophysiology; NMDA; plasticity; 5-HT; protein kinase C
2.  Changes in D-Aspartate ion currents in the Aplysia nervous system with aging 
Brain research  2010;1343:28-36.
D-Aspartate (D-Asp) can substitute for L-Glutamate (L-Glu) at excitatory Glu receptors, and occurs as free D-Asp in the mammalian brain. D-Asp electrophysiological responses were studied as a potential correlate of aging in the California sea hare, Aplysia californica. Whole cell voltage- and current clamp measurements were made from primary neuron cultures of the pleural ganglion (PVC) and buccal ganglion S cluster (BSC) in 3 egg cohorts at sexual maturity and senescence. D-Asp activated an inward current at the hyperpolarized voltage of −70 mV, where molluscan NMDA receptors open free of constitutive block by Mg2+. Half of the cells responded to both D-Asp and L-Glu while the remainder responded only to D-Asp or L-Glu, suggesting that D-Asp activated non-Glu channels in a subpopulation of these cells. The frequency of D-Asp-induced currents and their density were significantly decreased in senescent PVC cells but not in senescent BSC cells. These changes in sensory neurons of the tail predict functional deficits that may contribute to an overall decline in reflexive movement in aged Aplysia.
doi:10.1016/j.brainres.2010.05.001
PMCID: PMC3062251  PMID: 20452331
A. californica; voltage clamp; D-Asp; glutamate; agonist; NMDA
3.  Synthesis, accumulation, and release of D-aspartate in the Aplysia californica central nervous system 
Journal of neurochemistry  2010;115(5):1234-1244.
D-aspartate (D-Asp) is an endogenous molecule that is often detected in central nervous system and endocrine tissues. Using capillary electrophoresis and a variety of radionuclide detection techniques, we examine the synthesis, release, and uptake/accumulation of D-Asp in the central nervous system of the marine mollusk Aplysia californica. We observe the preferential synthesis and accumulation of D-Asp over L-aspartate (L-Asp) in neuron-containing ganglia compared to surrounding sheath tissues. Little conversion of D-Asp to L-Asp is detected. The Ca2+ ionophore ionomycin and elevated extracellular potassium stimulates release of D-Asp from the cerebral ganglia. Lastly, radioactive D-Asp in the extracellular media is efficiently taken up and accumulated by individual F-cluster neurons. These observations point to a role for D-Asp in cell-to-cell signaling with many characteristics similar to classical transmitters.
doi:10.1111/j.1471-4159.2010.07020.x
PMCID: PMC2972370  PMID: 20874765
D-amino acids; D-aspartate; Aplysia californica; neurotransmitter; hormone
4.  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
5.  Residues at the tip of the pore loop of NR3B-containing NMDA receptors determine Ca2+ permeability and Mg2+ block 
BMC Neuroscience  2010;11:133.
Background
Members of the complex N-methyl-D-aspartate receptor (NMDAR) subfamily of ionotropic glutamate receptors (iGluRs) conventionally assemble from NR1 and NR2 subunits, the composition of which determines receptor properties. Hallmark features of conventional NMDARs include the requirement for a coagonist, voltage-dependent block by Mg2+, and high permeability for Ca2+. Both Mg2+ sensitivity and Ca2+ permeability are critically dependent on the amino acids at the N and N+1 positions of NR1 and NR2. The recently discovered NR3 subunits feature an unprecedented glycine-arginine combination at those critical sites within the pore. Diheteromers assembled from NR1 and NR3 are not blocked by Mg2+ and are not permeable for Ca2+.
Results
Employing site-directed mutagenesis of receptor subunits, electrophysiological characterization of mutants in a heterologous expression system, and molecular modeling of the NMDAR pore region, we have investigated the contribution of the unusual NR3 N and N+1 site residues to the unique functional characteristics of receptors containing these subunits. Contrary to previous studies, we provide evidence that both the NR3 N and N+1 site amino acids are critically involved in mediating the unique pore properties. Ca2+ permeability could be rescued by mutating the NR3 N site glycine to the NR1-like asparagine. Voltage-dependent Mg2+ block could be established by providing an Mg2+ coordination site at either the NR3 N or N+1 positions. Conversely, "conventional" receptors assembled from NR1 and NR2 could be made Mg2+ insensitive and Ca2+ impermeable by equipping either subunit with the NR3-like glycine at their N positions, with a stronger contribution of the NR1 subunit.
Conclusions
This study sheds light on the structure-function relationship of the least characterized member of the NMDAR subfamily. Contrary to previous reports, we provide evidence for a critical functional involvement of the NR3 N and N+1 site amino acids, and propose them to be the essential determinants for the unique pore properties mediated by this subunit.
doi:10.1186/1471-2202-11-133
PMCID: PMC2974739  PMID: 20958962
6.  Cortical Kynurenine Pathway Metabolism: A Novel Target for Cognitive Enhancement in Schizophrenia 
Schizophrenia Bulletin  2010;36(2):211-218.
The brain concentration of kynurenic acid (KYNA), a metabolite of the kynurenine pathway of tryptophan degradation and antagonist at both the glycine coagonist site of the N-methyl-D-aspartic acid receptor (NMDAR) and the α7 nicotinic acetylcholine receptor (α7nAChR), is elevated in the prefrontal cortex (PFC) of individuals with schizophrenia. This increase may be clinically relevant because hypofunction of both the NMDAR and the α7nAChR are implicated in the pathophysiology, and especially in the cognitive deficits associated with the disease. In rat PFC, fluctuations in endogenous KYNA levels bidirectionally modulate extracellular levels of 3 neurotransmitters closely related to cognitive function (glutamate, dopamine, and acetylcholine). Moreover, behavioral studies in rats have demonstrated a causal link between increased cortical KYNA levels and neurocognitive deficits, including impairment in spatial working memory, contextual learning, sensory gating, and prepulse inhibition of the startle reflex. In recent human postmortem studies, impairments in gene expression and activity of kynurenine pathway enzymes were found in cortical areas of individuals with schizophrenia. Additional studies have revealed an interesting association between a sequence variant in the gene of one of these enzymes, kynurenine 3-monooxygenase, and neurocognitive deficits seen in patients. The emerging, remarkable confluence of data from humans and animals suggests an opportunity for developing a rational pharmacology by targeting cortical kynurenine pathway metabolism for cognition enhancement in schizophrenia and beyond.
doi:10.1093/schbul/sbq002
PMCID: PMC2833131  PMID: 20147364
kynurenic acid; endophenotype; cognition; schizophrenia; kynurenine 3-monooxygenase; prefrontal cortex; genetic association
7.  Group II metabotropic glutamate receptors modify N-methyl-D-aspartate receptors via Src kinase 
Scientific Reports  2013;3:926.
Group II metabotropic glutamate receptors (mGluR2/3) have emerged as important targets for the treatment of schizophrenia. Since hypofunction of N-methyl-D-aspartate receptors (NMDARs) has also been implicated in the etiology of schizophrenia, we examined whether postsynaptic mGluR2/3 regulate NMDAR function. Activation of mGluR2/3 significantly decreased the ratio of AMPA-to-NMDA excitatory postsynaptic currents at Schaffer Collateral-CA1 synapses and enhanced the peak of NMDA-evoked currents in acutely isolated CA1 neurons. The mGluR2/3-mediated potentiation of NMDAR currents was selective for GluN2A-containing NMDARs and was mediated by the Src family kinase Src. Activation of mGluR2/3 inhibited the adenylyl cyclase-cAMP-PKA pathway and thereby activated Src by inhibiting its regulatory C-terminal Src kinase (Csk). We suggest a novel model of regulation of NMDARs by Gi/o-coupled receptors whereby inhibition of the cAMP-PKA pathway via mGluR2/3 activates Src kinase and potentiates GluN2A-containing NMDAR currents. This represents a potentially novel mechanism to correct the hypoglutamatergic state found in schizophrenia.
doi:10.1038/srep00926
PMCID: PMC3558700  PMID: 23378895
8.  Piperazine-2,3-dicarboxylic acid Derivatives as Dual Antagonists of NMDA and GluK1-Containing Kainate Receptors 
Journal of Medicinal Chemistry  2011;55(1):327-341.
Competitive N-methyl-D-aspartate receptor (NMDAR) antagonists bind to the GluN2 subunit, of which there are four types (GluN2A-D). We report that some N1-substituted derivatives of cis-piperazine-2,3-dicarboxylic acid display improved relative affinity for GluN2C and GluN2D versus GluN2A and GluN2B. These derivatives also display subtype-selectivity among the more distantly related kainate receptor family. Compounds 18i and (−)-4 were the most potent kainate receptor antagonists and 18i was selective for GluK1 versus GluK2, GluK3 and AMPA receptors. Modeling studies revealed structural features required for activity at GluK1 subunits and suggested that S674 was vital for antagonist activity. Consistent with this hypothesis, replacing the equivalent residue in GluK3 (alanine) with a serine imparts 18i antagonist activity. Antagonists with dual GluN2D and GluK1 antagonist activity may have beneficial effects in various neurological disorders. Consistent with this idea, antagonist 18i (30 mg/Kg i.p.) showed antinociceptive effects in an animal model of mild nerve injury.
doi:10.1021/jm201230z
PMCID: PMC3269097  PMID: 22111545
9.  TCN 201 selectively blocks GluN2A-containing NMDARs in a GluN1 co-agonist dependent but non-competitive manner 
Neuropharmacology  2012;63-540(3-7):441-449.
Antagonists that are sufficiently selective to preferentially block GluN2A-containing N-methyl-d-aspartate receptors (NMDARs) over GluN2B-containing NMDARs are few in number. In this study we describe a pharmacological characterization of 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino]carbonyl]benzyl]benzenesulphonamide (TCN 201), a sulphonamide derivative, that was recently identified from a high-throughput screen as a potential GluN2A-selective antagonist. Using two-electrode voltage-clamp (TEVC) recordings of NMDAR currents from Xenopus laevis oocytes expressing either GluN1/GluN2A or GluN1/GluN2B NMDARs we demonstrate the selective antagonism by TCN 201 of GluN2A-containing NMDARs. The degree of inhibition produced by TCN 201 is dependent on the concentration of the GluN1-site co-agonist, glycine (or d-serine), and is independent of the glutamate concentration. This GluN1 agonist-dependency is similar to that observed for a related GluN2A-selective antagonist, N-(cyclohexylmethyl)-2-[{5-[(phenylmethyl)amino]-1,3,4-thiadiazol-2-yl}thio]acetamide (TCN 213). Schild analysis of TCN 201 antagonism indicates that it acts in a non-competitive manner but its equilibrium constant at GluN1/GluN2A NMDARs indicates TCN 201 is around 30-times more potent than TCN 213. In cortical neurones TCN 201 shows only modest antagonism of NMDAR-mediated currents recorded from young (DIV 9–10) neurones where GluN2B expression predominates. In older cultures (DIV 15–18) or in cultures where GluN2A subunits have been over-expressed TCN 201 gives a strong block that is negatively correlated with the degree of block produced by the GluN2B-selective antagonist, ifenprodil. Nevertheless, while TCN 201 is a potent antagonist it must be borne in mind that its ability to block GluN2A-containing NMDARs is dependent on the GluN1-agonist concentration and is limited by its low solubility.
Highlights
► TCN 201 is a potent and selective GluN1/GluN2A NMDAR antagonist. ► TCN 201 antagonism is dependent on the GluN1-agonist concentration. ► TCN 201 antagonism is independent on the GluN2-agonist concentration. ► TCN 201 blocks GluN2A-containing NMDARs in a non-competitive manner. ► TCN 201 allows pharmacological identification of native GluN2 A-containing NMDAR populations.
doi:10.1016/j.neuropharm.2012.04.027
PMCID: PMC3384000  PMID: 22579927
TCN 201; TCN 213; NMDA receptor; GluN2A-selective; Glycine; d-serine; Schild analysis
10.  Influence of GluN2 subunit identity on NMDA receptor function 
Neuropharmacology  2013;74(100):4-17.
N-methyl-d-aspartate receptors (NMDARs) are ligand-gated ion channels (‘ionotropic’ receptors) activated by the major excitatory neurotransmitter, l-glutamate. While the term ‘the NMDAR’ is often used it obscures the fact that this class of receptor contains within it members whose properties are as different as they are similar. This heterogeneity was evident from early electrophysiological, pharmacological and biochemical assessments of the functional properties of NMDARs and while the molecular basis of this heterogeneity has taken many years to elucidate, it indicated from the outset that the diversity of NMDAR phenotypes could allow this receptor family to subserve a variety of functions in the mammalian central nervous system. In this review we highlight some recent studies that have identified structural elements within GluN2 subunits that contribute to the heterogeneous biophysical properties of NMDARs, consider why some recently described novel pharmacological tools may permit better identification of native NMDAR subtypes, examine the evidence that NMDAR subtypes differentially contribute to the induction of long-term potentiation and long-term depression and discuss how through the use of chimeric proteins additional insights have been obtained that account for NMDAR subtype-dependency of physiological and pathophysiological signalling.
This article is part of the Special Issue entitled ‘Glutamate Receptor-Dependent Synaptic Plasticity’.
Highlights
► Reviews the control of NMDAR functional properties by GluN2 subunits. ► Assesses structural elements of GluN2 subunits that determine NMDAR heterogeneity. ► Considers the utility of orthosteric and allosteric ligands acting at NMDARs. ► Potential of the NMDAR C-terminal domain as a novel therapeutic target is discussed.
doi:10.1016/j.neuropharm.2013.01.016
PMCID: PMC3778433  PMID: 23376022
NMDA receptor; Glutamate receptor; Structure; Biophysics; Pharmacology; Plasticity; Excitoxicity
11.  Prepuberal Stimulation of 5-HT7-R by LP-211 in a Rat Model of Hyper-Activity and Attention-Deficit: Permanent Effects on Attention, Brain Amino Acids and Synaptic Markers in the Fronto-Striatal Interface 
PLoS ONE  2014;9(4):e83003.
The cross-talk at the prefronto-striatal interface involves excitatory amino acids, different receptors, transducers and modulators. We investigated long-term effects of a prepuberal, subchronic 5-HT7-R agonist (LP-211) on adult behaviour, amino acids and synaptic markers in a model for Attention-Deficit/Hyperactivity Disorder (ADHD). Naples High Excitability rats (NHE) and their Random Bred controls (NRB) were daily treated with LP-211 in the 5th and 6th postnatal week. One month after treatment, these rats were tested for indices of activity, non selective (NSA), selective spatial attention (SSA) and emotionality. The quantity of L-Glutamate (L-Glu), L-Aspartate (L-Asp) and L-Leucine (L-Leu), dopamine transporter (DAT), NMDAR1 subunit and CAMKIIα, were assessed in prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS), for their role in synaptic transmission, neural plasticity and information processing. Prepuberal LP-211 (at lower dose) reduced horizontal activity and (at higher dose) increased SSA, only for NHE but not in NRB rats. Prepuberal LP-211 increased, in NHE rats, L-Glu in the PFC and L-Asp in the VS (at 0.250 mg/kg dose), whereas (at 0.125 mg/kg dose) it decreased L-Glu and L-Asp in the DS. The L-Glu was decreased, at 0.125 mg/kg, only in the VS of NRB rats. The DAT levels were decreased with the 0.125 mg/kg dose (in the PFC), and increased with the 0.250 mg/kg dose (in the VS), significantly for NHE rats. The basal NMDAR1 level was higher in the PFC of NHE than NRB rats; LP-211 treatment (at 0.125 mg/kg dose) decreased NMDAR1 in the VS of NRB rats. This study represents a starting point about the impact of developmental 5-HT7-R activation on neuro-physiology of attentive processes, executive functions and their neural substrates.
doi:10.1371/journal.pone.0083003
PMCID: PMC3977819  PMID: 24709857
12.  D-Amino acids in rat brain measured by liquid chromatography / tandem mass spectrometry 
Neuroscience letters  2008;445(1):53-57.
Previous work has established that D-amino acids including D-serine (D-Ser) and D-aspartic acid (D-Asp) fulfill specific biological functions in the brain. In this work, the levels and anatomical distribution of D-amino acids in rat brain were determined by using an advantageous liquid chromatography-tandem mass spectrometric analytical method. The study was focused on D-Ser, D-Asp, and D-glutamic acid (D-Glu) because of the significance of L-Asp, L-Glu, and D-Ser in the nervous system. Prenatal, postnatal pups, and 90-day old rats were studied. Results indicated that D-Asp and D-Ser occurred in rat brain at the µg /g tissue level. However, D-Glu was not detected (< 110 ng/ g tissue). Through out the developmental stages D-Asp content in rat brain decreased rapidly from 9.42% of total Asp in 5-day prenatal rats to an undetectable level (< 150 ng /g tissue) in 90-day old rats. In contrast, D-Ser level increased gradually through out the developmental stages. D-Ser percentage (D-Ser / (D-Ser + L-Ser)) changed from 4.94 % in 5-day prenatal rats to 13.7 % in 90-day old rats. Regional levels of D-Ser were found to be significantly higher in cortex, striatum, and hippocampus than in thalamus. D-Ser was not detected in cerebellum (<172 ng /g tissue).
doi:10.1016/j.neulet.2008.08.058
PMCID: PMC2585614  PMID: 18775473
D-Amino acids; Rat brain; Anatomical distribution; Liquid chromatography - tandem mass spectrometry
13.  Effects of NR1 splicing on NR1/NR3B-type excitatory glycine receptors 
BMC Neuroscience  2009;10:32.
Background
N-methyl-D-aspartate receptors (NMDARs) are the most complex of ionotropic glutamate receptors (iGluRs). Subunits of this subfamily assemble into heteromers, which – depending on the subunit combination – may display very different pharmacological and electrophysiological properties. The least studied members of the NMDAR family, the NR3 subunits, have been reported to assemble with NR1 to form excitatory glycine receptors in heterologous expression systems. The heterogeneity of NMDARs in vivo is in part conferred to the receptors by splicing of the NR1 subunit, especially with regard to proton sensitivity.
Results
Here, we have investigated whether the NR3B subunit is capable of assembly with each of the eight functional NR1 splice variants, and whether the resulting receptors share the unique functional properties described for NR1-1a/NR3. We provide evidence that functional excitatory glycine receptors formed regardless of the NR1 isoform, and their pharmacological profile matched the one reported for NR1-1a/NR3: glycine alone fully activated the receptors, which were insensitive to glutamate and block by Mg2+. Surprisingly, amplitudes of agonist-induced currents showed little dependency on the C-terminally spliced NR1 variants in NR1/NR3B diheteromers. Even more strikingly, NR3B conferred proton sensitivity also to receptors containing NR1b variants – possibly via disturbing the "proton shield" of NR1b splice variants.
Conclusion
While functional assembly could be demonstrated for all combinations, not all of the specific interactions seen for NR1 isoforms with coexpressed NR2 subunits could be corroborated for NR1 assembly with NR3. Rather, NR3 abates trafficking effects mediated by the NR1 C terminus as well as the N-terminally mediated proton insensitivity. Thus, this study establishes that NR3B overrides important NR1 splice variant-specific receptor properties in NR1/NR3B excitatory glycine receptors.
doi:10.1186/1471-2202-10-32
PMCID: PMC2669480  PMID: 19348678
14.  Role of the NMDA-receptor in Prepulse Inhibition in the Rat 
Kynurenic acid (KYNA) is an endogenous metabolite of tryptophan. Studies have revealed increased brain KYNA levels in patients with schizophrenia. Prepulse inhibition (PPI) is a behavioral model for sensorimotor gating and found to be reduced in schizophrenia. Previous studies have shown that pharmacologically elevated brain KYNA levels disrupt PPI in the rat. The aim of the present study was to investigate the receptor(s) involved in this effect. Rats were treated with different drugs selectively blocking each of the sites that KYNA antagonizes, namely the glutamate recognition site of the N-methyl-D-aspartate receptor (NMDAR), the α7* nicotinic acetylcholine receptor (α7nAChR) and the glycine site of the NMDAR. Kynurenine (200 mg/kg) was given to replicate the effects of increased levels of KYNA on PPI. In order to block the glutamate recognition site of the NMDAR, CGS 19755 (10 mg/kg) or SDZ 220–581 (2.5 mg/kg) were administered and to antagonize the α7nAChR methyllycaconitine (MLA; 6 mg/kg) was given. L-701,324 (1 and 4 mg/kg) or 4-Chloro-kynurenine (4-Cl-KYN; 25, 50 and 100 mg/kg), a drug in situ converted to 7-Chloro-kynurenic acid, were used to block the glycine-site of the NMDAR. Administration of SDZ 220-581 or CGS 19755 was associated with a robust reduction in PPI, whereas L-701,324, 4-Cl-KYN or MLA failed to alter PPI. Kynurenine increased brain KYNA levels 5-fold and tended to decrease PPI. The present study suggests that neither antagonism of the glycine-site of the NMDA receptor nor antagonism of the α7nAChR disrupts PPI, rather with regard to the effects of KYNA, blockade of the glutamate recognition-site is necessary to reduce PPI.
PMCID: PMC3195246  PMID: 22084584
kynurenic acid; kynurenine; sensorimotor gating; α7* nicotinic acetylcholine receptor; NMDA/glycine-site
15.  Endogenous Activation of Metabotropic Glutamate Receptors Modulates GABAergic Transmission to Gonadotropin-Releasing Hormone Neurons and Alters Their Firing Rate: A Possible Local Feedback Circuit 
Gonadotropin-releasing hormone (GnRH) neurons are the primary central regulators of fertility, receiving input from GABAergic afferents via GABAA receptors. We tested whether metabotropic glutamate receptors (mGluRs) regulate GABA transmission to GnRH neurons and GnRH neuronal firing pattern. Whole-cell recordings were performed under conditions isolating ionotropic GABA postsynaptic currents (PSCs) in brain slices. The broad-spectrum mGluR agonist 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) decreased the frequency of GABAA-mediated spontaneous PSCs in a reversible manner. Amplitude and kinetics were not altered, suggesting that afferent GABA neurons are the primary targets. TTX eliminated the effects of ACPD in most tested neurons. Group II [2-(2,3-dicarboxycyclopropyl) glycine] and III (l-AP-4) mGluR agonists mediated this response; a group I agonist (3,5-dihydroxyphenylglycine) was not effective. The broad-spectrum antagonist α-methyl-4-carboxyphenylglycine (MCPG) and/or (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG) (group III antagonist) enhanced spontaneous PSC frequency, particularly when initial frequency was low, suggesting that endogenous activation of mGluRs regulates GABA transmission to GnRH neurons. Extracellular recordings were used to evaluate GnRH neuron firing rate within the network. ACPD reduced firing rate, and MCPG plus CPPG had an opposite effect, indicating that mGluRs help control excitability of the GnRH network. GnRH neurons express vesicular glutamate transporters, suggesting they may corelease this transmitter. Simulation of firing activity in a GnRH neuron decreased PSC frequency in that cell, an effect blocked by antagonism of mGluRs but not GnRH receptors. These results demonstrate an inhibition of GABAergic inputs to GnRH neurons by mGluRs via a presynaptic mechanism. Through this mechanism, local glutamate milieu, possibly contributed by GnRH neurons themselves, plays an important role in modulating GnRH release and the central regulation of fertility.
doi:10.1523/JNEUROSCI.0913-05.2005
PMCID: PMC1201448  PMID: 15958740
GnRH; mGluR; GABA; presynaptic; IPSC; firing pattern
16.  Diversity in NMDA receptor composition: many regulators, many consequences 
N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptor, which play a central role in learning, memory, and synaptic development. NMDARs are assembled as tetramers composed of two GluN1 subunits and two GluN2 or GluN3 subunits. Although NMDARs are widely expressed throughout the central nervous system, their number, localization, and subunit composition are strictly regulated and differ in a cell- and synapse-specific manner. The brain area, developmental stage and level of synaptic activity are some of the factors that regulate NMDARs. Molecular mechanisms that control subunit-specific NMDAR function include developmental regulation of subunit transcription/translation, differential trafficking through the secretory pathway, post-transcriptional modifications such as phosphorylation, and protein-protein interactions. The GluN2A and GluN2B subunits are highly expressed in cortex and hippocampus and confer many of the distinct properties on endogenous NMDARs. Importantly, the synaptic NMDAR subunit composition changes from predominantly GluN2B-containing to GluN2A-containing NMDARs during synaptic maturation and in response to activity and experience. Some of the molecular mechanisms underlying this GluN2 subunit switch have been recently identified. In addition, the balance between synaptic and extrasynaptic NMDARs is altered in several neuronal disorders. Here, we summarize the recent advances in the identification of NMDAR subunit-specific regulatory mechanisms.
doi:10.1177/1073858411435129
PMCID: PMC3567917  PMID: 22343826
17.  Glutamine deamidation: Differentiation of Glutamic acid and γ-Glutamic acid in peptides by electron capture dissociation 
Analytical chemistry  2010;82(9):3606-3615.
Due to its much slower deamidation rate comparing to that of asparagine (Asn), studies on glutamine (Gln) deamidation have been scarce, especially on the differentiation of its isomeric deamidation products: α- and γ-glutamic acid (Glu). It has been shown previously that electron capture dissociation (ECD) can be used to generate diagnostic ions for the deamidation products of Asn: aspartic acid (Asp) and isoaspartic acid (isoAsp). The current study explores the possibility of an extension of this ECD based method to the differentiation of the α- and γ-Glu residues, using three human crystallin peptides (αA (1-11), βB2 (4-14), and γS (52-71)) and their potentially deamidated forms as model peptides. It was found that the z•-72 ions can be used to both identify the existence and locate the position of the γ-Glu residues. When the peptide contains a charge carrier near its N-terminus, the c+57 and c+59 ions may also be generated at the γ-Glu residue. It was unclear whether formation of these N-terminal diagnostic ions is specific to the Pro-γ-Glu sequence. Unlike the Asp containing peptides, the Glu containing peptides generally do not produce diagnostic side chain loss ions, due to the instability of the resulting radical. The presence of Glu residue(s) may be inferred from the observation of a series of zn•-59 ions, although it was neither site specific, nor without interference from the γ-Glu residues. Finally, several interference peaks exist in the ECD spectra, which highlights the importance of using high performance mass spectrometers for confident identification of γ-Glu residues.
doi:10.1021/ac9028467
PMCID: PMC2872026  PMID: 20373761
18.  Free amino acids in the nervous system of the amphioxus Branchiostoma lanceolatum. A comparative study 
The cephalochordate amphioxus is the closest invertebrate relative to vertebrates. In this study, using HPLC technique, free L-amino acids (L-AAs) and D-aspartic acid (D-Asp) have been detected in the nervous system of the amphioxus Branchiostoma lanceolatum. Among other amino acids glutamate, aspartate, glycine, alanine and serine are the amino acids found at the greatest concentrations. As it occurs in the nervous system of other animal phyla, glutamate (L-Glu) and aspartate (L-Asp) are present at very high concentrations in the amphioxus nervous system compared to other amino acids, whereas the concentration of taurine and γ-aminobutyric acid (GABA) is very low. Interestingly, as it is the case in vertebrates, D-aspartic acid is present as an endogenous compound in amphioxus nervous tissues. The physiological function of excitatory amino acids, and D-aspartate in particular, are discussed in terms of evolution of the nervous system under an Evo-fun (Evolution of function) perspective.
PMCID: PMC1458427  PMID: 16733539
Amphioxus; amino acids; GABA; nervous system; chordates; evolution.
19.  NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina 
Molecular Brain  2013;6:34.
Background
Loss of retinal ganglion cells (RGCs) is a hallmark of various retinal diseases including glaucoma, retinal ischemia, and diabetic retinopathy. N-methyl-D-aspartate (NMDA)-type glutamate receptor (NMDAR)-mediated excitotoxicity is thought to be an important contributor to RGC death in these diseases. Native NMDARs are heterotetramers that consist of GluN1 and GluN2 subunits, and GluN2 subunits (GluN2A–D) are major determinants of the pharmacological and biophysical properties of NMDARs. All NMDAR subunits are expressed in RGCs in the retina. However, the relative contribution of the different GluN2 subunits to RGC death by excitotoxicity remains unclear.
Results
GluN2B- and GluN2D-deficiency protected RGCs from NMDA-induced excitotoxic retinal cell death. Pharmacological inhibition of the GluN2B subunit attenuated RGC loss in glutamate aspartate transporter deficient mice.
Conclusions
Our data suggest that GluN2B- and GluN2D-containing NMDARs play a critical role in NMDA-induced excitotoxic retinal cell death and RGC degeneration in glutamate aspartate transporter deficient mice. Inhibition of GluN2B and GluN2D activity is a potential therapeutic strategy for the treatment of several retinal diseases.
doi:10.1186/1756-6606-6-34
PMCID: PMC3733768  PMID: 23902942
NMDA receptor; GluN2B; GluN2D; Excitotoxicity; Retina; Glaucoma; Glutamate transporter
20.  Glycine transport accounts for the differential role of glycine vs. D-serine at NMDA receptor coagonist sites in the salamander retina 
In this study, we demonstrate that D-serine interacts with N-methyl-D-aspartate receptor (NMDAR) coagonist sites of retinal ganglion cells of the tiger salamander retina by showing that exogenous D-serine overcomes the competitive antagonism of 7-chlorokynurenic acid for this site. Additionally, we show that exogenous D-serine was more than 30 times as effective at potentiating NMDAR currents compared with glycine. We thus examined the importance of glycine transport through the application of selective antagonists of the GlyT1 (NFPS) and GlyT2 (ALX-5670) transport systems, while simultaneously evaluating the degree of occupancy of the NMDAR coagonist binding sites. Analysis was carried out with electrophysiological recordings from the inner retina, including whole-cell recordings from retinal ganglion cells and extracellular recordings of the proximal negative field potential. Blocking the GlyT2 transport system had no effect on the light-evoked NMDAR currents or on the sensitivity of these currents to exogenous D-serine. In contrast, when the GlyT1 system was blocked, the coagonist sites of NMDARs showed full occupancy. These findings clearly establish the importance of the GlyT1 transporter as an essential component for maintaining the coagonist sites of NMDARs in a non-saturated state. The normal, unsaturated state of the NMDAR coagonist binding sites allows modulation of the NMDAR currents, by release of either D-serine or glycine. These results are discussed in light of contemporary findings which favor D-serine over glycine as the major coagonist of the NMDARs found in ganglion cells of the tiger salamander retina.
doi:10.1111/j.1460-9568.2010.07135.x
PMCID: PMC2913692  PMID: 20374282
GlyT1; PNFP; retinal ganglion cell; whole-cell recording
21.  Electrophysiological Characterization of AMPA and NMDA Receptors in Rat Dorsal Striatum 
The striatum receives glutamatergic afferents from the cortex and thalamus, and these synaptic transmissions are mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. The purpose of this study was to characterize glutamate receptors by analyzing NMDA/AMPA ratio and rectification of AMPA and NMDA excitatory postsynaptic currents (EPSCs) using a whole-cell voltage-clamp method in the dorsal striatum. Receptor antagonists were used to isolate receptor or subunit specific EPSC, such as (DL)-2-amino-5-phosphonovaleric acid (APV), an NMDA receptor antagonist, ifenprodil, an NR2B antagonist, CNQX, an AMPA receptor antagonist and IEM-1460, a GluR2-lacking AMPA receptor blocker. AMPA and NMDA EPSCs were recorded at -70 and +40 mV, respectively. Rectification index was calculated by current ratio of EPSCs between +50 and -50 mV. NMDA/AMPA ratio was 0.20±0.05, AMPA receptor ratio of GluR2-lacking/GluR2-containing subunit was 0.26±0.05 and NMDA receptor ratio of NR2B/NR2A subunit was 0.32±0.03. The rectification index (control 2.39±0.27) was decreased in the presence of both APV and combination of APV and IEM-1460 (1.02±0.11 and 0.93±0.09, respectively). These results suggest that the major components of the striatal glutamate receptors are GluR2-containing AMPA receptors and NR2A-containing NMDA receptors. Our results may provide useful information for corticostriatal synaptic transmission and plasticity studies.
doi:10.4196/kjpp.2009.13.3.209
PMCID: PMC2766737  PMID: 19885039
Striatum; AMPA; Glutamate receptor; NMDA; Patch clamp
22.  Glutamate Residues In the Second Extracellular Loop of the Human A2a Adenosine Receptor Are Required for Ligand Recognition 
Molecular pharmacology  1996;49(4):683-691.
SUMMARY
The A2a adenosine receptor, a member of the G protein-coupled receptor family, is important in the regulation of dopaminergic pathways of the brain and in platelet and cardiovascular functions. In this study, the role of extracellular loops in ligand binding to the human A2a receptor was explored through site-directed mutagenesis. Four glutamate/aspartate residues (Glu151, Glu161, Glu169, and Asp170) in the second extracellular loop (E2) and a cysteine residue (Cys262) in the third extracellular loop (E3) were individually replaced with alanine and other amino acids. A proline residue (Pro173) in E2 was mutated to arginine, the homologous amino acid in A3 receptors. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. The mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, thus allowing their detection in the plasma membrane with immunological techniques. High affinity specific binding of [3H]2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5′-N-ethylcarboxamidoadenosine (15 nm) and [3H]8-[4-[[[[(2-aminoethyl)-amino]carbonyl]methyl]oxy]phenyl]-1,3- dipropylxanthine (4 nm), an A2a agonist and antagonist, respectively, was not observed with four of the mutant receptors, E151A, E151Q, E151D, and E169A, although they were well expressed at the cell surface. The E151A and E169A mutant receptors showed nearly full stimulation of adenylyl cyclase at ~103-fold higher concentrations of 2-[4-[(2-carboxyethyl)phenyl]ethyl-amino]-5′-N-ethylcarboxamidoadenosine. The E161A mutant receptor showed an increase in affinity for the nonxanthine adenosine antagonist 9-chloro-2-(furyl)[1,2,4]triazolo[1,5-c]quinazolin-5-amine (6-fold) but not for other ligands. An E169Q mutant gained affinity (5–22-fold) for adenosine derivatives (agonists) substituted at N6 but not at C2 or C5′ positions. Mutant receptors D170K and P173R were similar to wild-type receptors in binding of both agonist and antagonist radioligands. A C262G mutant also resembled the wild-type receptor in radioligand binding, indicating that a potential disulfide bridge with another cysteine residue in proximity is not required for the structural integrity of the receptor. Our data suggest that certain amino acids in the second extracellular loop may be directly or indirectly involved in ligand binding.
PMCID: PMC3425639  PMID: 8609897
23.  Selective extracellular stimulation of individual neurons in ganglia 
Journal of neural engineering  2008;5(3):287-309.
Selective control of individual neurons could clarify neural functions and aid disease treatments. To target specific neurons, it may be useful to focus on ganglionic neuron clusters, which are found in the peripheral nervous system in vertebrates. Because neuron cell bodies are found primarily near the surface of invertebrate ganglia, and often found near the surface of vertebrate ganglia, we developed a technique for controlling individual neurons extracellularly using the buccal ganglia of the marine mollusc Aplysia californica as a model system. We experimentally demonstrated that anodic currents can selectively activate an individual neuron and cathodic currents can selectively inhibit an individual neuron using this technique. To define spatial specificity, we studied the minimum currents required for stimulation, and to define temporal specificity, we controlled firing frequencies up to 45 Hz. To understand the mechanisms of spatial and temporal specificity, we created models using the NEURON software package. To broadly predict the spatial specificity of arbitrary neurons in any ganglion sharing similar geometry, we created a steady-state analytical model. A NEURON model based on cat spinal motorneurons showed responses to extracellular stimulation qualitatively similar to those of the Aplysia NEURON model, suggesting that this technique could be widely applicable to vertebrate and human peripheral ganglia having similar geometry.
doi:10.1088/1741-2560/5/3/003
PMCID: PMC2574815  PMID: 18714126
24.  Aspartate-444 Is Essential for Productive Substrate Interactions in a Neuronal Glutamate Transporter 
The Journal of General Physiology  2007;129(6):527-539.
In the central nervous system, electrogenic sodium- and potassium-coupled glutamate transporters terminate the synaptic actions of this neurotransmitter. In contrast to acidic amino acids, dicarboxylic acids are not recognized by glutamate transporters, but the related bacterial DctA transporters are capable of transporting succinate and other dicarboxylic acids. Transmembrane domain 8 contains several residues that differ between these two types of transporters. One of these, aspartate-444 of the neuronal glutamate transporter EAAC1, is conserved in glutamate transporters, but a serine residue occupies this position in DctA transporters. When aspartate-444 is mutated to serine, cysteine, alanine, or even to glutamate, uptake of d-[3H]-aspartate as well as the inwardly rectifying steady-state currents induced by acidic amino acids is impaired. Even though succinate was not capable of inducing any steady-state transport currents, the dicarboxylic acid inhibited the sodium-dependent transient currents by the mutants with a neutral substitution at position 444. In the neutral substitution mutants inhibition of the transients was also observed with acidic amino acids. In the D444E mutant, acidic amino acids were potent inhibitors of the transient currents, whereas the apparent affinity for succinate was lower by at least three orders of magnitude. Even though L-aspartate could bind to D444E with a high apparent affinity, this binding resulted in inhibition rather than stimulation of the uncoupled anion conductance. Thus, a carboxylic acid–containing side chain at position 444 prevents the interaction of glutamate transporters with succinate, and the presence of aspartate itself at this position is crucial for productive substrate binding compatible with substrate translocation.
doi:10.1085/jgp.200609707
PMCID: PMC2151622  PMID: 17535962
25.  Differential roles of GluN2A- and GluN2B-containing NMDA receptors in neuronal survival and death 
Glutamate-induced neurotoxicity is the primary molecular mechanism that induces neuronal death in a variety of pathologies in central nervous system (CNS). Toxicity signals are relayed from extracellular space to the cytoplasm by N-methyl-D-aspartate receptors (NMDARs) and regulate a variety of survival and death signaling. Differential subunit combinations of NMDARs confer neuroprotection or trigger neuronal death pathways depending on the subunit arrangements of NMDARs and its localization on the cell membrane. It is well-known that GluN2B-contaning NMDARs (GluN2BRs) preferentially link to signaling cascades involved in CNS injury promoting neuronal death and neurodegeneration. Conversely, less well-known mechanisms of neuronal survival signaling are associated with GluN2A-comtaining NMDARs (GluN2AR)-dependent signal pathways. This review will discuss the most recent signaling cascades associated with GluN2ARs and GluN2BRs.
PMCID: PMC3544217  PMID: 23320134
NMDA receptor; GluN2A; GluN2B; neuronal survival; neuronal death

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