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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.  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.  Metabotropic glutamate receptor subtypes mediating slow inward tail current (IADP) induction and inhibition of synaptic transmission in olfactory cortical neurones 
British Journal of Pharmacology  1997;120(6):1083-1095.
The pharmacological features of the pre- and postsynaptic metabotropic glutamate receptors (mGluRs) present in the guinea-pig olfactory cortex, were examined in brain slices in vitro by use of a conventional intracellular current clamp/voltage clamp recording technique.Bath-application of trans-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (50 μM) produced a sustained membrane depolarization, increase in cell excitability and induction of a post-stimulus inward (afterdepolarizing) tail current (IADP) (measured under ‘hybrid' voltage clamp) similar to those evoked by the muscarinic receptor agonist oxotremorine-M (OXO-M, 2 μM).L-Glutamate (0.25–1 mM, in the presence of 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 μM DL-amino-5-phosphono valeric acid (DL-APV)) or the broad spectrum mGluR agonists 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 10 μM), 1S,3S-ACPD (50 μM), ibotenate (Ibo; 25 μM, in the presence of 100 μM DL-APV), the selective mGluR I agonists (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG, 10 μM), (S)-3-hydroxyphenylglycine ((S)-3HPG, 50 μM), or quisqualate (10 μM, in the presence of 20 μM CNQX), but not the mGluR II agonist 2S,1′S,2′S-2-(2′-carboxycyclopropyl)-glycine (L-CCGI, 1 μM) or mGluR III agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 1 mM), were all effective in producing membrane depolarization and inducing a post-stimulus IADP. Unexpectedly, the proposed mGluR II-selective agonist (2S,1′R,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)-glycine (DCG-IV, 10 μM, in the presence of 100 μM DL-APV) was also active.The excitatory effects induced by 10 μM 1S,3R-ACPD were reversibly antagonized by the mGluR I/II antagonist (+)-α-methyl-4-carboxyphenylglycine ((+)-MCPG, 0.5–1 mM), as well as the selective mGluR I antagonists (S)-4-carboxyphenylglycine ((S)-4CPG) and (S)-4-carboxy-3-hydroxyphenyl glycine ((S)-4C3HPG) (both at 1 mM), but not the nonselective mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3, 1 mM) or the selective mGluR III antagonist (S)-α-methyl-L-AP4 (MAP4, 1 mM).The excitatory postsynaptic potentials (e.p.s.ps), induced by single focal stimulation of cortical excitatory fibre tracts, were markedly reduced by 1S,3R-ACPD or L-AP4 (both at 10 μM), and by the selective mGluR II agonists (mGluR I antagonists) (S)-4CPG or (S)-4C3HPG (both at 1 mM) but not (S)-3,5-DHPG or (S)-3HPG (both at 100 μM).The inhibitory effects of 1S-3R-ACPD, but not L-AP4, were reversibly blocked by (+)-MCPG (1 mM), whereas those produced by L-AP4, but not 1S,3R-ACPD, were blocked by the selective mGluR III antagonist MAP4 (1 mM).It is concluded that a group I mGluR is most likely involved in mediating excitatory postsynaptic effects, whereas two distinct mGluRs (e.g. group II and III) might serve as presynaptic inhibitory autoreceptors in the guinea-pig olfactory cortex.
doi:10.1038/sj.bjp.0701021
PMCID: PMC1564578  PMID: 9134221
Metabotropic glutamate receptors (mGluRs); 1S,3R-ACPD; L-AP4; depolarization; slow inward tail current (IADP); synaptic transmission; olfactory cortex; intracellular recording
5.  A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes 
PLoS ONE  2015;10(5):e0126314.
Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase.
doi:10.1371/journal.pone.0126314
PMCID: PMC4425671  PMID: 25954808
6.  NMDA receptors control vagal afferent excitability in the nucleus of the solitary tract 
Brain research  2014;1595:84-91.
Previous behavioral studies have demonstrated that presynaptic N-methyl-D-aspartate (NMDA) receptors expressed on vagal afferent terminals are involved in food intake and satiety. Therefore, using in vitro live cell calcium imaging of prelabeled rat hindbrain slices, we characterized which NMDA receptor GluN2 subunits may regulate vagal afferent activity. The nonselective NMDA receptor antagonist D,L-2-amino-5-phosphonopentanoic acid (D,L-AP5) significantly inhibited vagal terminal calcium influx, while the excitatory amino acid reuptake inhibitor D,L-threo-β-benzyloxyaspartic acid (TBOA), significantly increased terminal calcium levels following pharmacological stimulation with ATP. Subunit-specific NMDA receptor antagonists and potentiators were used to identify which GluN2 subunits mediate the NMDA receptor response on the vagal afferent terminals. The GluN2B-selective antagonist, ifenprodil, selectively reduced vagal calcium influx with stimulation compared to the time control. The GluN2A-selective antagonist, 3-chloro-4-fluoro-N-[4-[[2-(phenylcarbonyl)hydrazino] carbonyl] benzyl]benzenesulfonamide (TCN 201)produced smaller but not statistically significant effects. Furthermore, the GluN2A/B-selective potentiator (pregnenolone sulfate) and the GluN2C/D-selective potentiator [(3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroisoquinolin-2(1 H)-yl)methanone; (CIQ)] enhanced vagal afferent calcium influx during stimulation. These data suggest that presynaptic NMDA receptors with GluN2B, GluN2C, and GluN2D subunits may predominantly control vagal afferent excitability in the nucleus of the solitary tract.
doi:10.1016/j.brainres.2014.11.010
PMCID: PMC4330085  PMID: 25446446
NST; calcium imaging; presynaptic; GluN2B; GluN2C; GluN2D
7.  Pre-synaptic nicotinic receptors evoke endogenous glutamate and aspartate release from hippocampal synaptosomes by way of distinct coupling mechanisms 
British Journal of Pharmacology  2010;161(5):1161-1171.
BACKGROUND AND PURPOSE
The present work aimed to investigate whether and through which mechanisms selective α7 and α4β2 nicotinic receptor (nAChR) agonists stimulate endogenous glutamate (GLU) and aspartate (ASP) release in rat hippocampus.
EXPERIMENTAL APPROACH
Rat hippocampal synaptosomes were purified on Percoll gradients and superfused in vitro to study endogenous GLU and ASP release. The synaptosomes were superfused with selective α7 and α4β2 nAChR agonists and antagonists. The excitatory amino acid (EAA) content of the samples of superfusate was determined by HPLC after pre-column derivatization and separation on a chromatographic column coupled with fluorimetric detection.
KEY RESULTS
Choline (Ch), a selective α7 receptor agonist, elicited a significant release of both GLU and ASP which was blocked by the α7 receptor antagonist methyllycaconitine (MLA), but was unaltered by the α4β2 receptor antagonist dihydro-β-erythroidine (DHβE). The stimulant effect of Ch was strongly reduced in a Ca2+-free medium, was not inhibited by Cd2+ and tetrodotoxin (TTX), but was antagonized by dantrolene, xestospongin C and thapsigargin. 5-Iodo-A-85380 dihydrochloride (5IA85380), a selective α4β2 receptor agonist, elicited EAA release in a DHβE-sensitive, MLA-insensitive fashion. The 5IA85380-evoked release was dependent on extracellular Ca2+, blocked by Cd2+ and TTX, but unaffected by dantrolene.
CONCLUSIONS AND IMPLICATIONS
Our study shows for the first time that rat hippocampal synaptosomes possess α7 and α4β2 nAChR subtypes, which can enhance the release of endogenous GLU and ASP via two distinct mechanisms of action. These results extend our knowledge of the nicotinic modulation of excitatory synaptic transmission in the hippocampus.
doi:10.1111/j.1476-5381.2010.00958.x
PMCID: PMC2998695  PMID: 20633015
nicotinic receptor subtypes; endogenous glutamate; endogenous aspartate; synaptosomes; rat hippocampus; neurotransmitter release
8.  PAR1-Activated Astrocytes in the Nucleus of the Solitary Tract Stimulate Adjacent Neurons via NMDA Receptors 
The Journal of Neuroscience  2015;35(2):776-785.
Severe autonomic dysfunction, including the loss of control of the cardiovascular, respiratory, and gastrointestinal systems, is a common comorbidity of stroke and other bleeding head injuries. Previous studies suggest that this collapse of autonomic control may be caused by thrombin acting on astrocytic protease-activated receptors (PAR1) in the hindbrain. Using calcium imaging and electrophysiological techniques, we evaluated the mechanisms by which astrocytic PAR1s modulate the activity of presynaptic vagal afferent terminals and postsynaptic neurons in the rat nucleus of the solitary tract (NST). Our calcium-imaging data show that astrocytic and neuronal calcium levels increase after brain slices are treated with the PAR1 agonist SFLLRN-NH2. This increase in activity is blocked by pretreating the slices with the glial metabolic blocker fluorocitrate. In addition, PAR1-activated astrocytes communicate directly with NST neurons by releasing glutamate. Calcium responses to SFLLRN-NH2 in the astrocytes and neurons significantly increase after bath application of the excitatory amino acid transporter blocker dl-threo-β-benzyloxyaspartic acid (TBOA) and significantly decrease after bath application of the NMDA receptor antagonist dl-2-amino-5-phosphonopentanoic acid (dl-AP5). Furthermore, astrocytic glutamate activates neuronal GluN2B-containing NMDA receptors. Voltage-clamp recordings of miniature EPSCs (mEPSCs) from NST neurons show that astrocytes control presynaptic vagal afferent excitability directly under resting and activated conditions. Fluorocitrate significantly decreases mEPSC frequency and SFLLRN-NH2 significantly increases mEPSC frequency. These data show that astrocytes act within a tripartite synapse in the NST, controlling the excitability of both postsynaptic NST neurons and presynaptic vagal afferent terminals.
doi:10.1523/JNEUROSCI.3105-14.2015
PMCID: PMC4293422  PMID: 25589770
calcium imaging; electrophysiology; gliotransmitter; GluN2B; mEPSC; tripartite synapse
9.  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
10.  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(Pt 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
11.  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
12.  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
13.  Local N-Methyl-D-Aspartate Receptor Antagonism in the Prefrontal Cortex Attenuates Spatial Cognitive Deficits Induced by Gonadectomy in Adult Male Rats 
Neuroscience  2014;288:73-85.
Gonadectomy in adult male rats significantly impairs spatial working memory, behavioral flexibility and other functions associated with the prefrontal cortex (PFC). However, the mechanisms through which this occurs are largely unknown. In this study, intracortical drug challenge with the selective N-methyl-D-aspartate glutamate receptor (NMDAR) antagonist D(-)-2-amino-5-phosphonopentanoic acid (APV) was combined with Barnes maze testing, gonadectomy and hormone replacement (17β estradiol, testosterone propionate) to explore the contributions of NMDAR-mediated activity within the PFC to hormone effects on spatial cognition in adult male rats. Previous studies have shown that Barnes maze testing reveals significant estrogen-dependent, gonadectomy-induced deficits in spatial working memory and androgen-sensitive, gonadectomy-induced deficits in spatial search strategy. Here we found that bilateral infusion of APV into the medial prefrontal cortex prior to testing significantly improved both sets of behaviors in gonadectomized rats and significantly worsened performance measures in gonadally intact controls. In hormone-replaced cohorts, we further found that behaviors that are normally similar to controls were significantly disrupted by APV, and those that are normally similar to gonadectomized rats were rescued by intracortical APV infusion. There were, however, no residual effects of APV on retention testing conducted 24 hours later. Together these findings suggest that hormone regulation of NMDAR-mediated activity specifically within the PFC may be fundamental to the effects of gonadal steroids on spatial cognition in males. Our findings further identify NMDAR antagonists as potentially novel, non-steroidal means of attenuating the cognitive deficits that can accompany gonadal hormone decline in human males in aging, clinical cases of hypogonadalism and in certain neurologic and psychiatric illnesses. Accordingly, it may be important to obtain in males the kind of detailed knowledge concerning hormone effects on, for example, the channel and electrophysiological properties of NMDAR that currently exists for the female brain.
doi:10.1016/j.neuroscience.2014.12.032
PMCID: PMC4323667  PMID: 25545712
working memory; Barnes maze; estrogen; androgen; schizophrenia; executive function
14.  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
15.  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
16.  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
17.  Effect of the umami peptides on the ligand binding and function of rat mGlu4a receptor might implicate this receptor in the monosodium glutamate taste transduction 
British Journal of Pharmacology  1999;128(5):1027-1034.
The effect of several metabotropic ligands and di- or tripeptides were tested on the binding of [3H]-L(+)-2-amino-4-phosphonobutyric acid ([3H]-L-AP4) on rat mGlu4 receptor. For selected compounds, the functional activity was determined on this receptor using the guanosine-5′[γ-35S]-thiotriphosphate [γ-35S]-GTP binding assay.Using the scintillation proximity assay, [3H]-L-AP4 saturation analysis gave binding parameters KD and Bmax values of 150 nM and 9.3 pmoles mg−1 protein, respectively. The specific binding was inhibited concentration-dependently by several mGlu receptor ligands, and their rank order of affinity was established.Several peptides inhibited the [3H]-L-AP4 binding with the following rank order of potency: glutamate-glutamate>glutamate-glutamate-leucine=aspartate - glutamate>>glutamate - glutamate-aspartate>lactoyl-glutamate>>aspartate-aspartate. Aspartate-phenylalanine-methyl ester (aspartame) was inactive up to 1 mM and guanosine-5′-monophosphate and inosine-5′-monophosphate were inactive up to 100 μM.The [γ-35S]-GTP binding functional assay was used to determine the agonist activities of the different compounds. For the rat mGlu4 agonists, L-AP4 and L-glutamate, the correlation between their occupancy and activation of the receptor was close to one. The peptides, Glu-Glu, Asp-Glu and Glu-Glu-Asp failed to stimulate the [γ-35S]-GTP binding at receptor occupancy greater than 80% and Glu-Glu-Leu appeared to be a weak partial agonist. These peptides did not elicit a clear dose-dependent umami perception. However, Glu-lac showed a good correlation between its potency to stimulate the [γ-35S]-GTP binding and its affinity for displacement of [3H]-L-AP4 binding. These data are in agreement with the peptide taste assessment in human subjects, which showed that the acid derivatives of glutamate had characteristics similar to umami.
doi:10.1038/sj.bjp.0702885
PMCID: PMC1571727  PMID: 10556940
Umami peptide; metabotropic glutamate receptor; [3H]-L-AP4; rat mGlu4a receptor; [γ-35S]-GTP binding; radioligand binding; taste transduction
18.  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
19.  Pharmacology of (S)-homoquisqualic acid and (S)-2-amino-5-phosphonopentanoic acid [(S)-AP5] at cloned metabotropic glutamate receptors 
British Journal of Pharmacology  1998;123(2):269-274.
In this study we have determined the pharmacological profile of (S)-quisqualic acid, (S)-2-amino-4-phosphonobutyric acid ((S)-AP4) and their higher homologues (S)-homoquisqualic acid, (S)-2-amino-5-phosphonopentanoic acid ((S)-AP5), respectively, and (R)-AP5 at subtypes of metabotropic (S)-glutamic acid (mGlu) receptors expressed in Chinese hamster ovary cells.(S)-Quisqualic acid was a potent mGlu1/mGlu5 agonist (EC50 values of 1.1 μM and 0.055 μM, respectively) showing no activity at mGlu2 and weak agonism at mGlu4 (EC50∼1000 μM).(S)-Homoquisqualic acid displayed competitive antagonism at mGlu1 (KB=184 μM) and full agonism at mGlu5 (EC50=36 μM) and mGlu2 (EC50=23 μM), but was inactive at mGlu4.(S)-AP4 was a potent and selective mGlu4 agonist (EC50=0.91 μM) being inactive at mGlu1, mGlu2 and mGlu5 both as agonist and antagonist.(S)-AP5 displayed very weak agonist activity at mGlu4. At the mGlu2 receptor subtype (S)-AP5 acted as a competitive antagonist (KB=205 μM), whereas the compound was inactive at mGlu1 and mGlu5. (R)-AP5 was inactive at all mGlu receptor subtypes tested both as agonist and antagonist.These studies demonstrate that incorporation of an additional carbon atom into the backbone of (S)-glutamic acid and its analogues, to give the corresponding homologues, and replacement of the terminal carboxyl groups by isosteric acidic groups have profound effects on the pharmacological profiles at mGlu receptor subtypes. Furthermore, (S)-homoquisqualic acid has been shown to be a potentially useful tool for differentiating mGlu1 and mGlu5.
doi:10.1038/sj.bjp.0701616
PMCID: PMC1565167  PMID: 9489615
Metabotropic glutamate receptors; mGluR; (S)-quisqualic acid; (S)-homoquisqualic acid; (S)-AP4 (L-AP4); (S)-AP5 (L-AP5); (R)-AP5 (D-AP5).
20.  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
21.  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
22.  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
23.  Metabotropic glutamate receptors, transmitter output and fatty acids: studies in rat brain slices. 
British Journal of Pharmacology  1996;117(1):189-195.
1. The effects of (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), a non-selective agonist of the metabotropic glutamate receptors (mGluRs), have been studied in rat cortical and striatal slices by measuring the depolarization-induced output of D-[3H]-aspartate (D-[3H]-Asp) and of [3H]-glutamate ([3H]-Glu), neosynthesized from [3H]-glutamine. 2. In cortical slices, 1S,3R-ACPD potentiated the depolarization-induced (KCl, 30 mM) output of both D-[3H]-Asp and [3H]-Glu. The potentiation, obtained at 300 microM 1S,3R-ACPD was 65 +/- 6% for D-[3H]-Asp and 56 +/- 10% for [3H]-Glu. Conversely, in striatal slices, 1S,3R-ACPD reduced the depolarization-induced transmitter output. The reduction, obtained at 300 microM of the agonist, was 60 +/- 8% for D-[3H]-Asp and 50 +/- 5% for neosynthesized [3H]-Glu. 3. Bovine serum albumin (BSA, 15 microM), which is able to bind locally produced fatty acids, completely eliminated the potentiating effect 1S,3R-ACPD had on D-[3H]-Asp output from cortical slices. Low concentrations of arachidonic acid (1-10 microM) or of oleic acid (1-10 microM) added to BSA-containing perfusion medium, restored this potentiating effect. BSA, however, had no effect on the inhibitory action of 1S,3R-ACPD in striatal slices. 4. Bromophenacyl bromide (100 microM), an inhibitor of phospholipase A2, and RG80267 (100 microM), an inhibitor of diacylglycerol lipase, have been shown to inhibit fatty acid production. These compounds prevented the potentiating effect of 1S,3R-ACPD on D-[3H]-Asp-output in cortical slices. 5. Indomethacin (100 microM), an inhibitor of cyclo-oxygenases, plus nordihydroguaiaretic acid (100 microM), an inhibitor of lipoxygenases, increased D-[3H]-Asp output in cortical slices perfused with BSA-containing medium. 6. These experiments suggest that the mGluR-mediated potentiation of transmitter output requires the availability of unsaturated fatty acids, such as arachidonic or oleic acids, in cortical slices. In contrast, the mGluR-induced inhibition of transmitter output is not dependent upon fatty acid availability in striatal slices. The requirement of both unsaturated fatty acids and 1S,3R-ACPD in the facilitation of transmitter exocytosis may play an important role in the regulation of synaptic plasticity.
PMCID: PMC1909368  PMID: 8825362
24.  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
25.  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

Results 1-25 (622379)