PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (499453)

Clipboard (0)
None

Related Articles

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.  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
4.  Mg2+ block properties of triheteromeric GluN1–GluN2B–GluN2D NMDA receptors on neonatal rat substantia nigra pars compacta dopaminergic neurones 
The Journal of Physiology  2014;592(10):2059-2078.
Native NMDA receptors (NMDARs) are tetrameric channels formed by two GluN1 and two GluN2 subunits. So far, seven NMDARs subunits have been identified and they can form diheteromeric or triheteromeric NMDARs (more than one type of GluN2 subunit). Extracellular Mg2+ is an important regulator of NMDARs, and particularly the voltage dependence of Mg2+ block is crucial to the roles of NMDARs in synaptic plasticity and the integration of synaptic activity with neuronal activity. Although the Mg2+ block properties of diheteromeric NMDARs are fully investigated, properties of triheteromeric NMDARs are still not clear. Our previous data suggested that dopaminergic neurones expressed triheteromeric GluN1–GluN2B–GluN2D NMDARs. Here, using NMDARs in dopaminergic neurones from postnatal day 7 (P7) rats as a model system, we characterize the voltage-dependent Mg2+ block properties of triheteromeric NMDARs. In control conditions, external Mg2+ significantly inhibits the whole cell NMDA-evoked current in a voltage-dependent manner with IC50 values of 20.9 μm, 53.3 μm and 173 μm at −90 mV, −70 mV and −50 mV, respectively. When the GluN2B-selective antagonist ifenprodil was applied, the Mg2+ sensitivity of the residual NMDA-mediated currents (which is mainly carried by GluN1–GluN2B–GluN2D NMDARs) is reduced to IC50 values of 45.9 μm (−90 mV), 104 μm (−70 mV) and 276 μm (−50 mV), suggesting that triheteromeric GluN1–GluN2B–GluN2D NMDARs have less affinity for external Mg2+ than GluN1–GluN2B receptors. In addition, fitting INMDA–V curves with a trapping Mg2+ block model shows the triheteromeric GluN1–GluN2B–GluN2D NMDARs have weaker voltage-dependent Mg2+ block (δ = 0.56) than GluN1–GluN2B NMDARs. Finally, our concentration jump and single channel recordings suggest that GluN1–GluN2B–GluN2D rather than GluN1–GluN2D NMDARs are present. These data provide information relevant to Mg2+ block characteristics of triheteromeric NMDARs and may help to better understand synaptic plasticity, which is dependent on these triheteromeric NMDARs.
doi:10.1113/jphysiol.2013.267864
PMCID: PMC4027860  PMID: 24614743
5.  Activity-dependent regulation of retinogeniculate signaling by metabotropic glutamate receptors 
Thalamocortical neurons in dorsal lateral geniculate nucleus (dLGN) dynamically convey visual information from retina to the neocortex. Activation of metabotropic glutamate receptors (mGluRs) exerts multiple effects on neural integration in dLGN; however, their direct influence on the primary sensory input, namely retinogeniculate afferents, is unknown. In the present study, we found that pharmacological or synaptic activation of type 1 mGluRs (mGluR1) significantly depresses glutamatergic retinogeniculate excitation in rat thalamocortical neurons. Pharmacological activation of mGluR1 attenuates excitatory synaptic responses in thalamocortical neurons at a magnitude sufficient to decrease suprathreshold output of these neurons. The reduction in both N-methyl-D-aspartate (NMDA) and (RS)-α-Amino-3-hydroxy-5-methyl-4-isαoxazolepropionic acid (AMPA) receptor-dependent synaptic responses results from a presynaptic reduction in glutamate release from retinogeniculate terminals. The suppression of retinogeniculate synaptic transmission and dampening of thalamocortical output was mimicked by tetanic activation of retinogeniculate afferent in a frequency dependent manner that activated mGluR1. Retinogeniculate excitatory synaptic transmission was also suppressed by the glutamate transport blocker DL-threo-beta-benzyloxyaspartic acid (TBOA), suggesting that mGluR1 were activated by glutamate spillover. The data indicate that presynaptic mGluR1 contributes to an activity dependent mechanism that regulates retinogeniculate excitation and therefore plays a significant role in the thalamic gating of visual information.
doi:10.1523/JNEUROSCI.0687-12.2012
PMCID: PMC3462222  PMID: 22973005
6.  Clustered burst firing in FMR1 premutation hippocampal neurons: amelioration with allopregnanolone 
Human Molecular Genetics  2012;21(13):2923-2935.
Premutation CGG repeat expansions (55–200 CGG repeats; preCGG) within the fragile X mental retardation 1 (FMR1) gene cause fragile X-associated tremor/ataxia syndrome (FXTAS). Defects in neuronal morphology and migration have been described in a preCGG mouse model. Mouse preCGG hippocampal neurons (170 CGG repeats) grown in vitro develop abnormal networks of clustered burst (CB) firing, as assessed by multielectrode array recordings and clustered patterns of spontaneous Ca2+ oscillations, neither typical of wild-type (WT) neurons. PreCGG neurons have reduced expression of vesicular GABA and glutamate (Glu) transporters (VGAT and VGLUT1, respectively), and preCGG hippocampal astrocytes display a rightward shift on Glu uptake kinetics, compared with WT. These alterations in preCGG astrocytes and neurons are associated with 4- to 8-fold elevated Fmr1 mRNA and occur despite consistent expression of fragile X mental retardation protein levels at ∼50% of WT levels. Abnormal patterns of activity observed in preCGG neurons are pharmacologically mimicked in WT neurons by addition of Glu or the mGluR1/5 agonist, dihydroxyphenylglycine, to the medium, or by inhibition of astrocytic Glu uptake with dl-threo-β-benzyloxyaspartic acid, but not by the ionotropic Glu receptor agonists, α-2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl) propanoic acid or N-methyl-d-aspartic acid. The mGluR1 (7-(hydroxyimino)cyclopropa [b]chromen-1a-carboxylate ethyl ester) or mGluR5 (2-methyl-6-(phenylethynyl)pyridine hydrochloride) antagonists reversed CB firing. Importantly, the acute addition of the neurosteroid allopregnanolone mitigated functional impairments observed in preCGG neurons in a reversible manner. These results demonstrate abnormal mGluR1/5 signaling in preCGG neurons, which is ameliorated by mGluR1/5 antagonists or augmentation of GABAA receptor signaling, and identify allopregnanolone as a candidate therapeutic lead.
doi:10.1093/hmg/dds118
PMCID: PMC3373240  PMID: 22466801
7.  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
8.  Structure-based discovery of antagonists for GluN3-containing N-methyl-D-aspartate receptors 
Neuropharmacology  2013;0:10.1016/j.neuropharm.2013.08.003.
NMDA receptors are ligand-gated ion channels that assemble into tetrameric receptor complexes composed of glycine-binding GluN1 and GluN3 subunits (GluN3A-B) and glutamate-binding GluN2 subunits (GluN2A-D). NMDA receptors can assemble as GluN1/N2 receptors and as GluN3-containing NMDA receptors, which are either glutamate/glycine-activated triheteromeric GluN1/N2/N3 receptors or glycine-activated diheteromeric GluN1/N3 receptors. The glycine-binding GluN1 and GluN3 subunits display strikingly different pharmacological selectivity profiles. However, the pharmacological characterization of GluN3-containing receptors has been hampered by the lack of methods and pharmacological tools to study GluN3 subunit pharmacology in isolation. Here, we have developed a method to study the pharmacology of GluN3 subunits in recombinant diheteromeric GluN1/N3 receptors by mutating the orthosteric ligand-binding pocket in GluN1. This method is suitable for performing compound screening and characterization of structure-activity relationship studies on GluN3 ligands. We have performed a virtual screen of the orthosteric binding site of GluN3A in the search for antagonists with selectivity for GluN3 subunits. In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands.
doi:10.1016/j.neuropharm.2013.08.003
PMCID: PMC3865070  PMID: 23973313
NMDA receptor; GluN3 subunit; antagonist; selectivity; virtual screening; Xenopus oocyte electrophysiology
9.  Glutamate transporter-dependent mTOR phosphorylation in Müller glia cells 
ASN NEURO  2012;4(5):e00095.
Glu (glutamate), the excitatory transmitter at the main signalling pathway in the retina, is critically involved in changes in the protein repertoire through the activation of signalling cascades, which regulate protein synthesis at transcriptional and translational levels. Activity-dependent differential gene expression by Glu is related to the activation of ionotropic and metabotropic Glu receptors; however, recent findings suggest the involvement of Na+-dependent Glu transporters in this process. Within the retina, Glu uptake is aimed at the replenishment of the releasable pool, and for the prevention of excitotoxicity and is carried mainly by the GLAST/EAAT-1 (Na+-dependent glutamate/aspartate transporter/excitatory amino acids transporter-1) located in Müller radial glia. Based on the previous work showing the alteration of GLAST expression induced by Glu, the present work investigates the involvement of GLAST signalling in the regulation of protein synthesis in Müller cells. To this end, we explored the effect of D-Asp (D-aspartate) on Ser-2448 mTOR (mammalian target of rapamycin) phosphorylation in primary cultures of chick Müller glia. The results showed that D-Asp transport induces the time- and dose-dependent phosphorylation of mTOR, mimicked by the transportable GLAST inhibitor THA (threo-β-hydroxyaspartate). Signalling leading to mTOR phosphorylation includes Ca2+ influx, the activation of p60src, phosphatidylinositol 3-kinase, protein kinase B, mTOR and p70S6K. Interestingly, GLAST activity promoted AP-1 (activator protein-1) binding to DNA, supporting a function for transporter signalling in retinal long-term responses. These results add a novel receptor-independent pathway for Glu signalling in Müller glia, and further strengthen the critical involvement of these cells in the regulation of glutamatergic transmission in the retina.
doi:10.1042/AN20120022
PMCID: PMC3420017  PMID: 22817638
excitatory amino acid; gene expression regulation; signalling; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; AP-1, activator protein-1; EAAT1-5, excitatory amino acids transporters 1-5; 4E-BP, 4E-binding protein; GLAST, Na+-dependent glutamate/aspartate transporter; iGluR, ionotropic receptor; KA, kainite; MGC, Müller glia cells; mGluRs; mGluRs, G-protein-coupled metabotropic receptors; mTOR, mammalian target of rapamycin; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffer saline; PDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; PKB/Akt, protein kinase B; p70S6K, 70 kDa S6 ribosomal kinase; RTK, receptor tyrosine kinase; Src, non-receptor tyrosine kinase p60src; T3MG, (±)-threo-3-methylglutamic acid; THA, threo-β-hydroxyaspartate
10.  D-aspartate acts as a signaling molecule in nervous and neuroendocrine systems 
Amino acids  2012;43(5):1873-1886.
D-Aspartate (D-Asp) is an endogenous amino acid in the central nervous and reproductive systems of vertebrates and invertebrates. High concentrations of D-Asp are found in distinct anatomical locations, suggesting that it has specific physiological roles in animals. Many of the characteristics of D-Asp have been documented, including its tissue and cellular distribution, formation and degradation, as well as the responses elicited by D-Asp application. D-Asp performs important roles related to nervous system development and hormone regulation; in addition, it appears to act as a cell-to-cell signaling molecule. Recent studies have shown that D-Asp fulfills many, if not all, of the definitions of a classical neurotransmitter—that the molecule’s biosynthesis, degradation, uptake, and release take place within the presynaptic neuron, and that it triggers a response in the postsynaptic neuron after its release. Accumulating evidence suggests that these criteria are met by a heterogeneous distribution of enzymes for D-Asp’s biosynthesis and degradation, an appropriate uptake mechanism, localization within synaptic vesicles, and a postsynaptic response via an ionotropic receptor. Although D-Asp receptors remain to be characterized, the postsynaptic response of D-Asp has been studied and several L-glutamate receptors are known to respond to D-Asp. In this review we discuss the current status of research on D-Asp in neuronal and neuroendocrine systems, and highlight results that support D-Asp’s role as a signaling molecule.
doi:10.1007/s00726-012-1364-1
PMCID: PMC3555687  PMID: 22872108
D-aspartate; D-amino acids; nervous system; neurotransmitter; endocrine gland
11.  Molecular Determinants of Agonist Selectivity in Glutamate-Gated Chloride Channels Which Likely Explain the Agonist Selectivity of the Vertebrate Glycine and GABAA-ρ Receptors 
PLoS ONE  2014;9(9):e108458.
Orthologous Cys-loop glutamate-gated chloride channels (GluClR’s) have been cloned and described electrophysiologically and pharmacologically in arthropods and nematodes (both members of the invertebrate ecdysozoan superphylum). Recently, GluClR’s from Aplysia californica (a mollusc from the lophotrochozoan superphylum) have been cloned and similarly studied. In spite of sharing a common function, the ecdysozoan and lophotrochozoan receptors have been shown by phylogenetic analyses to have evolved independently. The recent crystallization of the GluClR from C. elegans revealed the binding pocket of the nematode receptor. An alignment of the protein sequences of the nematode and molluscan GluClRs showed that the Aplysia receptor does not contain all of the residues defining the binding mode of the ecdysozoan receptor. That the two receptors have slightly different binding modes is not surprising since earlier electrophysiological and pharmacological experiments had suggested that they were differentially responsive to certain agonists. Knowledge of the structure of the C. elegans GluClR has permitted us to generate a homology model of the binding pocket of the Aplysia receptor. We have analyzed the differences between the two binding modes and evaluated the relative significance of their non-common residues. We have compared the GluClRs electrophysiologically and pharmacologically and we have used site-directed mutagenesis on both receptor types to test predictions made from the model. Finally, we propose an explanation derived from the model for why the nematode receptors are gated only by glutamate, whereas the molluscan receptors can also be activated by β-alanine, GABA and taurine. Like the Aplysia receptor, the vertebrate glycine and GABAA-ρ receptors also respond to these other agonists. An alignment of the sequences of the molluscan and vertebrate receptors shows that the reasons we have given for the ability of the other agonists to activate the Aplysia receptor also explain the agonist profile seen in the glycine and GABAA-ρ receptors.
doi:10.1371/journal.pone.0108458
PMCID: PMC4178172  PMID: 25259865
12.  Nicotinic α7 receptor activation selectively potentiates the function of NMDA receptors in glutamatergic terminals of the nucleus accumbens 
We here provide functional and immunocytochemical evidence supporting the co-localization and functional interaction between nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartic acid receptors (NMDARs) in glutamatergic terminals of the nucleus accumbens (NAc). Immunocytochemical studies showed that a significant percentage of NAc terminals were glutamatergic and possessed GluN1 and α7-containing nAChR. A short-term pre-exposure of synaptosomes to nicotine (30 µM) or choline (1 mM) caused a significant potentiation of the 100 µM NMDA-evoked [3H]D-aspartate ([3H]D-Asp) outflow, which was prevented by α-bungarotoxin (100 nM). The pre-exposure to nicotine (100 µM) or choline (1 mM) also enhanced the NMDA-induced cytosolic free calcium levels, as measured by FURA-2 fluorescence imaging in individual NAc terminals, an effect also prevented by α-bungarotoxin. Pre-exposure to the α4-nAChR agonists 5IA85380 (10 nM) or RJR2429 (1 µM) did not modify NMDA-evoked ([3H]D-Asp) outflow and calcium transients. The NMDA-evoked ([3H]D-Asp) overflow was partially antagonized by the NMDAR antagonists MK801, D-AP5, 5,7-DCKA and R(-)CPP and unaffected by the GluN2B-NMDAR antagonists Ro256981 and ifenprodil. Notably, pre-treatment with choline increased GluN2A biotin-tagged proteins. In conclusion, our results show that the GluN2A-NMDA receptor function can be positively regulated in NAc terminals in response to a brief incubation with α7 but not α4 nAChRs agonists. This might be a general feature in different brain areas since a similar nAChR-mediated bolstering of NMDA-induced ([3H]D-Asp) overflow was also observed in hippocampal synaptosomes.
doi:10.3389/fncel.2014.00332
PMCID: PMC4199379  PMID: 25360085
nicotinic receptors; NMDA receptors; nicotine treatment; neurotransmitters release; synaptosomes; nucleus accumbens
13.  Rapid Microelectrode Measurements and the Origin and Regulation of Extracellular Glutamate in Rat Prefrontal Cortex 
Journal of neurochemistry  2010;115(6):1608-1620.
Glutamate in the prefrontal cortex (PFC) plays a significant role in several mental illnesses, including schizophrenia, addiction and anxiety. Previous studies on PFC glutamate-mediated function have used techniques that raise questions on the neuronal vs. astrocytic origin of glutamate. The present studies used enzyme-based microelectrode arrays (MEAs) to monitor second-by-second resting glutamate levels in the PFC of awake rats. Locally-applied drugs were employed in an attempt to discriminate between the neuronal or glial components of the resting glutamate signal. Local application of tetrodotoxin (TTX; sodium channel blocker), produced a significant (~40%) decline in resting glutamate levels. In addition significant reductions in extracellular glutamate were seen with locally-applied ω-conotoxin (MVIIC; ~50%; calcium channel blocker), and the mGluR⅔ agonist, LY379268 (~20%), and a significant increase with the mGluR⅔ antagonist LY341495 (~40%), effects all consistent with a large neuronal contribution to the resting glutamate levels. Local administration of D,L-threo-β-benzyloxyaspartate (TBOA; glutamate transporter inhibitor) produced an ~120% increase in extracellular glutamate levels, supporting that excitatory amino acid transporters, which are largely located on glia, modulate clearance of extracellular glutamate. Interestingly, local application of (S)-4-carboxyphenylglycine (CPG; cystine/glutamate antiporter inhibitor), produced small, non-significant bi-phasic changes in extracellular glutamate versus vehicle control. Finally, pre-administration of TTX completely blocked the glutamate response to tail pinch stress. Taken together, these results support that PFC resting glutamate levels in rats as measured by the MEA technology are at least 40-50% derived from neurons. Furthermore, these data support that the impulse flow-dependent glutamate release from a physiologically-evoked event is entirely neuronally derived.
doi:10.1111/j.1471-4159.2010.07066.x
PMCID: PMC2996468  PMID: 20969570
basal; chronic; freely moving; TTX; TBOA
14.  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
15.  Different Mechanisms of Ca2+ Transport in NMDA and Ca2+-permeable AMPA Glutamate Receptor Channels  
The Journal of General Physiology  1998;112(5):623-636.
The channel of the glutamate N-methyl-d-aspartate receptor (NMDAR) transports Ca2+ approximately four times more efficiently than that of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPAR). To investigate the basis of this difference in these glutamate receptors (GluRs), we measured the ratio of Cs+ efflux and Ca2+ influx in recombinant NMDAR and Ca2+-permeable AMPAR channels expressed in human embryonic kidney 293 (HEK 293) cells over a wide voltage range. At any one potential, this biionic flux ratio was measured by quantifying the total charge and the charge carried by Ca2+ using whole-cell currents and fluorometric techniques (dye overload) with Cs+ internally and Ca2+ externally (1.8 or 10 mM) as the only permeant ions. In AMPAR channels, composed of either GluR-A(Q) or GluR-B(Q) subunits, the biionic flux ratio had a biionic flux-ratio exponent of 1, consistent with the prediction of the Goldman-Hodgkin-Katz current equation. In contrast, for NMDAR channels composed of NR1 and NR2A subunits, the biionic flux-ratio exponent was ∼2, indicating a deviation from Goldman-Hodgkin-Katz. Consistent with these results, in NMDAR channels under biionic conditions with high external Ca2+ and Cs+ as the reference ions, Ca2+ permeability (PCa/PCs) was concentration dependent, being highest around physiological concentrations (1–1.8 mM; PCa/PCs ≈ 6.1) and reduced at both higher (110 mM; PCa/PCs ≈ 2.6) and lower (0.18 mM; PCa/PCs ≈ 2.2) concentrations. PCa/PCs in AMPAR channels was not concentration dependent, being around 1.65 in 0.3–110 mM Ca2+. In AMPAR and NMDAR channels, the Q/R/N site is a critical determinant of Ca2+ permeability. However, mutant AMPAR channels, which had an asparagine substituted at the Q/R site, also showed a biionic flux-ratio exponent of 1 and concentration-independent permeability ratios, indicating that the difference in Ca2+ transport is not due to the amino acid residue located at the Q/R/N site. We suggest that the difference in Ca2+ transport properties between the glutamate receptor subtypes reflects that the pore of NMDAR channels has multiple sites for Ca2+, whereas that of AMPAR channels only a single site.
PMCID: PMC2229440  PMID: 9806970
Ussing flux-ratio test; Goldman-Hodgkin-Katz current equation; Ca2+ permeation; fractional Ca2+ currents
16.  Endothelial nitric oxide synthase gene polymorphism (Glu298Asp) and development of pre-eclampsia: a case-control study and a meta-analysis 
Background
Pre-eclampsia is thought to have an important genetic component. Recently, pre-eclampsia has been associated in some studies with carriage of a common eNOS gene Glu298Asp polymorphism, a variant that leads to the replacement of glutamic acid by aspartic acid at codon 298.
Method
Healthy women with singleton pregnancies were recruited from 7 district general hospitals in London, UK. Women at high risk of pre-eclampsia were screened by uterine artery Doppler velocimetry at 22–24 weeks of gestation and maternal blood was obtained to genotype the eNOS Glu298Asp polymorphism. Odds ratios (OR) and 95%CI, using logistic regression methods, were obtained to evaluate the association between the Glu298Asp polymorphism and pre-eclampsia. A meta-analysis was then undertaken of all published studies up to November 2005 examining the association of eNOS Glu298Asp genotype and pre-eclampsia.
Results
89 women with pre-eclampsia and 349 controls were included in the new study. The Glu298Asp polymorphism in a recessive model was not significantly associated with pre-eclampsia (adjusted-OR: 0.83 [95%CI: 0.30–2.25]; p = 0.7). In the meta-analysis, under a recessive genetic model (1129 cases & 2384 controls) women homozygous for the Asp298 allele were not at significantly increased risk of pre-eclampsia (OR: 1.28 [95%CI: 0.76–2.16]; p = 0.34). A dominant model (1334 cases & 2894 controls) was associated with no increase of risk of pre-eclampsia for women carriers of the Asp298 allele (OR: 1.12 [95%CI: 0.84–1.49]; p = 0.42).
Conclusion
From the data currently available, the eNOS Glu298Asp polymorphism is not associated with a significant increased risk of pre-eclampsia. However, published studies have been underpowered, much larger studies are needed to confirm or refute a realistic genotypic risk of disease, but which might contribute to many cases of pre-eclampsia in the population.
doi:10.1186/1471-2393-6-7
PMCID: PMC1431561  PMID: 16542455
17.  Identification of Pore Residues Engaged in Determining Divalent Cationic Permeation in Transient Receptor Potential Melastatin Subtype Channel 2*S⃞ 
The Journal of Biological Chemistry  2008;283(41):27426-27432.
The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca2+ and Mg2+ permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg2+ permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca2+ permeability along with concentration-dependent inhibition of Na+-carrying currents by Ca2+. Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca2+-permeable TRPM channels, substantially increased Ca2+ permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca2+ permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.
doi:10.1074/jbc.M801049200
PMCID: PMC2562080  PMID: 18687688
18.  Distinct Functional Roles of Subunits within the Heteromeric Kainate Receptor 
Kainate receptors (KARs) have been implicated in a number of neurological disorders, including epilepsy. KARs are tetrameric, composed of a combination of GluK1–5 subunits. We examined the contribution of GluK2 and GluK5 subunits to activation and desensitization of the heteromeric receptor. Heteromeric GluK2/K5 receptors expressed in HEK 293T cells showed markedly higher glutamate sensitivity than GluK2 homomers and did not desensitize at low glutamate concentrations. Mutation of residue E738 in GluK2 substantially lowered its glutamate sensitivity. However, heteromeric KARs containing this mutant GluK2 [GluK2(E738D)] assembled with wild-type GluK5 showed no change in glutamate EC50 compared to wild-type heteromeric KARs. Instead, higher concentrations of glutamate were required to produce desensitization. This suggested that within the heteromeric receptor, glutamate binding to the high affinity GluK5 subunit alone was sufficient for channel activation but not desensitization, while agonist binding to the low affinity GluK2 subunit was not necessary to open the channel, but instead caused the channel to enter a closed, desensitized state. To test this hypothesis in wild-type receptors, we used the competitive antagonist kynurenate, which has higher affinity for the GluK2 than the GluK5 subunit. Co-application of kynurenate with glutamate to heteromeric receptors reduced the onset of desensitization without affecting the peak current response, consistent with our hypothesis. Our results suggest that GluK2 and GluK5 subunits can be individually activated within the heteromeric receptor and that these subunits serve dramatically different functional roles.
doi:10.1523/JNEUROSCI.3685-11.2011
PMCID: PMC3237056  PMID: 22114280
kainate receptor; GluK5; GluK2; desensitization; glutamate; hippocampus
19.  Metabotropic Glutamate Receptors in the Main Olfactory Bulb Drive Granule Cell-Mediated Inhibition 
Journal of neurophysiology  2006;97(1):858-870.
Main olfactory bulb (MOB) granule cells (GCs) express high levels of the group I metabotropic glutamate receptor (mGluR), mGluR5. We investigated the role of mGluRs in regulating GC activity in rodent MOB slices using whole cell patch-clamp electrophysiology. The group I/II mGluR agonist (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) or the selective group I agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) depolarized (~20 mV) and increased the firing rate of GCs. In the presence of ionotropic glutamate and GABA receptor antagonists, DHPG evoked a more modest depolarization (~8 mV). In voltage clamp, DHPG, but not group II [(2S,2′R,3)-2-(2′,3′-dicarboxycyclopropyl) glycine, DCG-IV] or group III [L(+)-2-amino-4-phosphonobutyric acid, L-AP4] mGluR agonists, induced an inward current. The inward current reversed polarity near the potassium equilibrium potential, suggesting mediation by closure of potassium channels. The DHPG-evoked inward current was unaffected by the mGluR1 antagonist (S)-(+)-α-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385), was blocked by the group I/II mGluR antagonist (αS)-α-amino-α-[(1S,2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid (LY341495), and was absent in GCs from mGluR5 knockout mice. LY341495 also attenuated mitral cell-evoked voltage-sensitive dye signals in the external plexiform layer and mitral cell-evoked spikes in GCs. These results suggest that activation of mGluR5 increases GC excitability, an effect that should increase GC-mediated GABAergic inhibition of mitral cells. In support of this: DHPG increased the frequency of spontaneous GABAergic inhibitory postsynaptic currents in mitral cells and LY341495 attenuated the feedback GABAergic postsynaptic potential elicited by intracellular depolarization of mitral cells. Our results suggest that activation of mGluR5 participates in feedforward and/or feedback inhibition at mitral cell to GC dendrodendritic synapses, possibly to modulate lateral inhibition and contrast in the MOB.
doi:10.1152/jn.00884.2006
PMCID: PMC2788779  PMID: 17093122
20.  Effect of inhibition of spinal cord glutamate transporters on inflammatory pain induced by formalin and complete Freund’s adjuvant 
Anesthesiology  2011;114(2):412-423.
Background
Spinal cord glutamate transporters clear synaptically released glutamate and maintain normal sensory transmission. However, their ultrastructural localization is unknown. Moreover, whether and how they participate in inflammatory pain has not been carefully studied.
Methods
Immunogold labeling with electron microscopy was carried out to characterize synaptic and non-synaptic localization of glutamate transporters in the superficial dorsal horn. Their expression and uptake activity after formalin- and complete Freund’s adjuvant (CFA)-induced inflammation were evaluated by Western blot and glutamate uptake assays. Effects of intrathecal glutamate transporter activator [(R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline], inhibitors [DL-threo-β-benzyloxyaspartate (TBOA), dihydrokainate, and DL-threo-beta-hydroxyaspartate], or TBOA plus a group III metabotropic glutamate receptor antagonist [(RS)-α-methylserine-O-phosphate] on formalin- and CFA-induced inflammatory pain were examined.
Results
In the superficial dorsal horn, excitatory amino acid carrier 1 is localized in pre-synaptic membrane, postsynaptic membrane, and axonal and dendritic membranes at non-synaptic sites, whereas glutamate transporter-1 and glutamate/aspartate transporter are prominent in glial membranes. Although expression of these three spinal glutamate transporters was not altered at 1 h after formalin injection or 6 h after CFA injection, glutamate uptake activity was decreased at these time points. Intrathecal (R)-(−)-5-methyl-1-nicotinoyl-2-pyrazoline had no effect on formalin-induced pain behaviors. In contrast, intrathecal TBOA, dihydrokainate, and DL-threo-beta-hydroxyaspartate reduced formalin-evoked pain behaviors in the second phase. Intrathecal TBOA also attenuated the CFA-induced thermal hyperalgesia at 6 h after CFA injection. The antinociceptive effects of TBOA were blocked by coadministration of (RS)-α-methylserine-O-phosphate.
Conclusion
Our findings suggest that spinal glutamate transporter inhibition relieves inflammatory pain through activation of inhibitory pre-synaptic group III metabotropic glutamate receptors.
doi:10.1097/ALN.0b013e318205df50
PMCID: PMC3057540  PMID: 21245732
21.  Prolonged post-inhibitory rebound firing in the cerebellar nuclei mediated by group I mGluR potentiation of L-type Ca currents 
Neurons in the cerebellar nuclei fire at accelerated rates for prolonged periods after trains of synaptic inhibition that interrupt spontaneous firing. Both in vitro and in vivo, however, this prolonged rebound firing is favored by strong stimulation of afferents, suggesting that neurotransmitters other than GABA may contribute to the increased firing rates. Here, we tested whether metabotropic glutamate receptors modulate excitability of nuclear cells in cerebellar slices from mouse. In current clamp, the prolonged rebound firing rate after high-frequency synaptic stimulation was reduced by a variety of group I mGluR antagonists, including CPCCOEt (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester), JNJ16259685 ((3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone)+MPEP, or 3-MATIDA (α-amino-5-carboxy-3-methyl-2-thiopheneacetic acid) +MPEP, as long as both mGluR1 and mGluR5 were blocked. This mGluR-dependent acceleration of firing was reduced but still evident when IPSPs were prevented by GABAA receptor antagonists. In voltage clamp, voltage ramps revealed a non-inactivating, low-voltage-activated, nimodipine-sensitive current that was enhanced by the selective group I mGluR agonist s-DHPG ((S)-3,5-dihydroxyphenylglycine). This putative L-type current also increased when mGluRs were activated by trains of evoked synaptic currents instead of direct application of agonist. In current clamp, blocking L-type Ca channels with the specific blocker nifedipine greatly reduced prolonged post-stimulus firing and occluded the effect of adding group I mGluR antagonists. Thus, potentiation of a low-voltage-activated L-type current by synaptically released glutamate accounted nearly fully for the mGluR-dependent acceleration of firing. Together, these data suggest that prolonged rebound firing in the cerebellar nuclei in vivo is most likely to occur when GABAA and mGluRs are simultaneously activated by concurrent excitation and inhibition.
doi:10.1523/JNEUROSCI.1834-11.2011
PMCID: PMC3155995  PMID: 21753005
22.  mGluR5 Upregulation Increases Excitability of Hypothalamic Presympathetic Neurons through NMDA Receptor Trafficking in Spontaneously Hypertensive Rats 
The Journal of Neuroscience  2014;34(12):4309-4317.
The hypothalamic paraventricular nucleus (PVN) is critically involved in elevated sympathetic output and the development of hypertension. However, changes in group I metabotropic glutamate receptors (mGluR1 and mGluR5) and their relevance to the hyperactivity of PVN presympathetic neurons in hypertension remain unclear. Here, we found that selectively blocking mGluR5 significantly reduced the basal firing activity of spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs), but not in normotensive Wistar-Kyoto (WKY) rats. However, blocking mGluR1 had no effect on the firing activity of PVN neurons in either group. The mRNA and protein levels of mGluR5 in the PVN and rostral ventrolateral medulla were significantly higher in SHRs than in WKY rats. The group I mGluR selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) similarly increased the firing activity of PVN neurons in WKY rats and SHRs. In addition, blocking NMDA receptors (NMDARs) through bath application or intracellular dialysis not only decreased the basal firing in SHRs, but also eliminated DHPG-induced excitation of spinally projecting PVN neurons. DHPG significantly increased the amplitude of NMDAR currents without changing their decay kinetics. Interestingly, DHPG still increased the amplitude of NMDAR currents and caused reappearance of functional NMDAR channels after initially blocking NMDARs. In addition, protein kinase C (PKC) inhibition or intracellular dialysis with synaptosomal-associated protein of 25 kDa (SNAP-25)-blocking peptide abolished DHPG-induced increases in NMDAR currents of PVN neurons in SHRs. Our findings suggest that mGluR5 in the PVN is upregulated in hypertension and contributes to the hyperactivity of PVN presympathetic neurons through PKC- and SNAP-25-mediated surface expression of NMDARs.
doi:10.1523/JNEUROSCI.4295-13.2014
PMCID: PMC3960469  PMID: 24647951
23.  Aplysia synapse associated protein (APSAP): identification, characterization, and selective interactions with Shaker-type potassium channels 
Journal of neurochemistry  2007;105(3):1006-1018.
The vertebrate post-synaptic density (PSD) is a region of high molecular complexity in which dynamic protein interactions modulate receptor localization and synaptic function. Members of the membrane-associated guanylate kinase (MAGUK) family of proteins represent a major structural and functional component of the vertebrate PSD. In order to investigate the expression and significance of orthologous PSD components associated with the Aplysia sensory neuron-motor neuron synapse, we have cloned an Aplysia Dlg-MAGUK protein, which we identify as Aplysia synapse associated protein (ApSAP). As revealed by western blot, RT-PCR, and immunocytochemical analyses, ApSAP is predominantly expressed in the CNS and is located in both sensory neuron and motor neurons. The overall amino acid sequence of ApSAP is 55– 61% identical to Drosophila Dlg and mammalian Dlg-MAGUK proteins, but is more highly conserved within L27, PDZ, SH3, and guanylate kinase domains. Because these conserved domains mediate salient interactions with receptors and other PSD components of the vertebrate synapse, we performed a series of GST pull-down assays using recombinant C-terminal tail proteins from various Aplysia receptors and channels containing C-terminal PDZ binding sequences. We have found that ApSAP selectively binds to an Aplysia Shaker-type channel AKv1.1, but not to (i) NMDA receptor subunit AcNR1-1, (ii) potassium channel AKv5.1, (iii) receptor tyrosine kinase ApTrkl, (iv) glutamate receptor ApGluR1/4, (v) glutamate receptor ApGluR2/3, or (vi) glutamate receptor ApGluR7. These findings provide preliminary information regarding the expression and interactions of Dlg-MAGUK proteins of the Aplysia CNS, and will inform questions aimed at a functional analysis of how interactions in a protein network such as the PSD may regulate synaptic strength.
doi:10.1111/j.1471-4159.2007.05202.x
PMCID: PMC3909683  PMID: 18182049
Aplysia; Aplysia synapse associated protein; membrane-associated guanylate kinase; post-synaptic density; potassium channel
24.  Selective inhibition of GluN2D-containing N-methyl-D-aspartate receptors prevents tissue plasminogen activator-promoted neurotoxicity both in vitro and in vivo 
Background
Tissue plasminogen activator (tPA) exerts multiple functions in the central nervous system, depending on the partner with which it interacts. In particular, tPA acts as a positive neuromodulator of N-methyl-D-aspartate glutamatergic receptors (NMDAR). At the molecular level, it has been proposed that the pro-neurotoxicity mediated by tPA might occur through extrasynaptic NMDAR containing the GluN2D subunit. Thus, selective antagonists targeting tPA/GluN2D-containing NMDAR signaling would be of interest to prevent noxious effects of tPA.
Results
Here, we compared three putative antagonists of GluN2D-containing NMDAR and we showed that the new compound UBP145 ((2R*,3S*)-1-(9-bromophenan-threne-3-carbonyl)piperazine-2,3-dicarboxylic acid) is far more selective for GluN2D subunits than memantine and PPDA (phenanthrene derivative (2S*, 3R*)-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid). Indeed, in vitro, in contrast to the two other compounds, UBP145 prevented NMDA toxicity only in neurons expressing GluN2D (ie, in cortical but not hippocampal neurons). Furthermore, in cultured cortical neurons, UBP145 fully prevented the pro-excitotoxic effect of tPA. In vivo, we showed that UBP145 potently prevented the noxious action of exogenous tPA on excitotoxic damages. Moreover, in a thrombotic stroke model in mice, administration of UBP145 prevented the deleterious effect of late thrombolysis by tPA.
Conclusions
In conclusion, tPA exerts noxious effects on neurons by acting on GluN2D-containing NMDAR and pharmacological antagonists of GluN2D-containing NMDAR could be used to prevent the ability of tPA to promote neurotoxicity.
doi:10.1186/1750-1326-6-68
PMCID: PMC3204249  PMID: 21975018
Tissue plasminogen activator; GluN2D; UBP145; excitotoxicity; stroke; glutamatergic neurotransmission
25.  GluN2B-containing NMDA receptors regulate depression-like behavior and are critical for the rapid antidepressant actions of ketamine 
eLife  null;3:e03581.
A single, low dose of the NMDA receptor antagonist ketamine produces rapid antidepressant actions in treatment-resistant depressed patients. Understanding the cellular mechanisms underlying this will lead to new therapies for treating major depression. NMDARs are heteromultimeric complexes formed through association of two GluN1 and two GluN2 subunits. We show that in vivo deletion of GluN2B, only from principal cortical neurons, mimics and occludes ketamine's actions on depression-like behavior and excitatory synaptic transmission. Furthermore, ketamine-induced increases in mTOR activation and synaptic protein synthesis were mimicked and occluded in 2BΔCtx mice. We show here that cortical GluN2B-containing NMDARs are uniquely activated by ambient glutamate to regulate levels of excitatory synaptic transmission. Together these data predict a novel cellular mechanism that explains ketamine's rapid antidepressant actions. In this model, basal glutamatergic neurotransmission sensed by cortical GluN2B-containing NMDARs regulates excitatory synaptic strength in PFC determining basal levels of depression-like behavior.
DOI: http://dx.doi.org/10.7554/eLife.03581.001
eLife digest
Depression is the leading cause of disability worldwide, with hundreds of millions of people living with the condition. The ‘gold standard’ for depression treatment involves a combination of psychotherapy and medication. Unfortunately, current antidepressant medications do not help everyone, waiting lists for psychotherapy are often long, and both normally take a number of weeks of regular treatment before they begin to have an effect. As patients are often at a high risk of suicide, it is crucial that treatments that act more quickly, and that are safe and effective, are developed.
One substance that may fulfill these requirements is a drug called ketamine. Studies have shown that depression symptoms can be reduced within hours by a single low dose of ketamine, and this effect on mood can last for more than a week. However, progress has been hindered by a lack of knowledge about what ketamine actually does inside the brain.
Neurons communicate with one another by releasing chemicals known as neurotransmitters, which transfer information by binding to receptor proteins on the surface of other neurons. Drugs such as ketamine also bind to these receptors. Ketamine works by blocking a specific receptor called the n-methyl d-aspartate (NMDA) receptor, but how this produces antidepressant effects is not fully understood.
The NMDA receptor is actually formed from a combination of individual protein subunits, including one called GluN2B. Now Miller, Yang et al. have created mice that lack receptors containing these GluN2B subunits in neurons in their neocortex, including the prefrontal cortex, a brain region involved in complex mental processes such as decision-making. This allowed Miller, Yang et al. to discover that when the neurotransmitter glutamate binds to GluN2B-containing NMDA receptors, it limits the production of certain proteins that make it easier for signals to be transmitted between neurons. Suppressing the synthesis of these proteins too much may cause depressive effects by reducing communication between the neurons in the prefrontal cortex.
Both mice lacking GluN2B-containing receptors in their cortical neurons and normal mice treated with ketamine showed a reduced amount of depressive-like behavior. This evidence supports Miller, Yang et al.'s theory that by blocking these NMDA receptors, ketamine restricts their activation. This restores normal levels of protein synthesis, improves communication between neurons in the cortex, and reduces depression.
Understanding how ketamine works to alleviate depression is an important step towards developing it into a safe and effective treatment. Further research is also required to determine the conditions that cause overactivation of the GluN2B-containing NMDA receptors.
DOI: http://dx.doi.org/10.7554/eLife.03581.002
doi:10.7554/eLife.03581
PMCID: PMC4270067  PMID: 25340958
depression; cortex; synapse; ketamine; electrophysiology; protein synthesis; mouse; rat

Results 1-25 (499453)