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Pharmacodynamics of the Glutamate Receptor Antagonists in the Rat Barrel Cortex

Pharmacodynamics of the Glutamate Receptor Antagonists in the Rat Barrel Cortex

to suppress thalamocortical excitatory synaptic responses in slices in vitro (10 µM CNQX and 50 µM dAPV) ( Feldman et al., 1998 ; Laurent et al., 2002 ), in intact preparations of superfused hippocampus of P15-25 rats in vivo (50 µM CNQX) ( Khazipov and Holmes, 2003 ) and in superfusion of the neonatal rat barrel cortex in vivo (20 µM CNQX and 80 µM dAPV) ( Minlebaev et al., 2007, 2009 ). The time course of inhibition produced by epipially applied glutamate receptor antagonists was comparable with bath application in vitro only in superficial L2/3, in deeper layers the inhibitory effects developed much slower following a surface-to-depth gradient. This likely reflects passive drug diffusion across the cortical depth counterbalanced by their removal from the extracellular space through blood microcirculation. Both of these factors are age-dependent with larger extracellular space volume ( Bondareff and Pysh, 1968 ; Bondareff and Narotzky, 1972 ; Lehmenkuhler et al., 1993 ; Sykova et al., 1998 , Sykova et al., 2002 ; Kilb et al., 2006 ) and less developed vascularization ( Keep and Jones, 1990 ; Yu et al., 1994 ; Risser et al., 2009 ) in the immature animals. Together with a thinner cortex in the immature animals, this likely explains the much higher effective antagonist concentrations of the antagonists during their epipial application in older animals compared to neonates.
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Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like receptors.

Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like receptors.

Abstract Whereas agonists bind directly in the heptahelical domain (HD) of most class-I rhodopsin-like G-protein coupled receptors (GPCRs), class-III agonists bind in the extracellular domain of their receptors. Indeed, the latter possess a large extracellular domain composed of a cysteine-rich domain and a venus flytrap module (VFTM). Both the low sequence homology and the structural organization of class-III GPCRs raised the question of whether or not the HD of these receptors functions the same way as rhodopsin-like GPCRs. Here we show that the HD of metabotropic glutamate receptor 5 (mGlu 5 ) displays the same agonist-independent constitutive activity as the
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Activation of a Dimeric Metabotropic Glutamate Receptor by Inter-Subunit Rearrangement

Activation of a Dimeric Metabotropic Glutamate Receptor by Inter-Subunit Rearrangement

opens a crevice allowing interaction with the C- terminus of the G protein α subunit, triggering its activation (5). Many GPCRs have been shown to form dimers, but the functional role of this remains elusive (6- 8). Although a GPCR monomer is sufficient to activate a G protein (8-12), it has been proposed that GPCR dimerization may facilitate their activation, i. e. that allosteric interactions between the protomers, through changes at the dimerization interface or a larger-scale reorientation of the two subunits, may contribute to stabilize the active conformation (13-15). To further elucidate a possible role of an inter- subunit rearrangement in the activation of a dimeric GPCR, we chose a metabotropic glutamate receptor (mGluR) as a model. These GPCRs are clearly established as constitutive dimers, the two subunits being linked by a disulfide bridge (16). Moreover, glutamate and its analogues do not bind to the TMD of an mGluR, but to a distinct extracellular domain called Venus Flytrap (VFT) (17). This separation may help to dissect the relationship between agonist binding and activation within these GPCRs. Agonist binding stabilizes a closed conformation of the VFT (Fig. 1) (18,19), which is both necessary (20) and sufficient (21) for the activation of a class C GPCR. How the VFT closure is in turn transduced into TMD activation, however, is yet unknown. Two mechanisms, not necessarily exclusive, have been proposed.
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Metabotropic glutamate receptor 5 in bulimia nervosa

Metabotropic glutamate receptor 5 in bulimia nervosa

Bulimia nervosa (BN) shares central features with substance-related and addictive disorders. The metabotropic glutamate receptor subtype 5 (mGlu5) plays an important role in addiction. Based on similarities between binge eating and substance-related and addictive disorders, we investigated mGlu5 in vivo in 15 female subjects with BN and 15 matched controls. We measured mGlu5 distribution volume ratio (DVR) with positron emission tomography (PET) using [11 C]ABP688. In BN mGlu5 DVR was higher in the anterior cingulate cortex (ACC), subgenual prefrontal cortex, and straight gyrus (p < 0.05). In BN, higher mGlu5 DVR in various brain regions, including ACC, pallidum, putamen, and caudate, positively correlated with “maturity fears” as assessed using the Eating Disorder Inventory-2 (p < 0.05). In BN and controls, smokers had globally decreased mGlu5 DVR. We present the first evidence for increased mGlu5 DVR in BN. Our findings suggest that pharmacological agents inhibiting mGlu5 might have a therapeutic potential in BN.
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Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells

Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells

A recent study revealed that mGlu2-4 heterodimers are likely present in cortico striatal terminals ( Yin et al., 2014 ). This conclusion is based on co-immunoprecipitation data, and on the lack of effect of PHCCC, an mGlu4 PAM devoid of effects in cells co-expressing mGlu2 and mGlu4, while VU0155041 that is active on both mGlu4 and mGlu2-4 heterodimers, potentiated the response ( Yin et al., 2014 ). Our data also revealed that mGlu2-4 can form in transfected neurons, indicating there are no specific mechanisms in neurons that would prevent the formation of such heterodimers. Most importantly, we found that in a neuronal cell line, responses with the pharmacological charac- teristics of the mGlu2-4 heterodimers can be recorded. Indeed, the synergistic effects of mGlu2 and mGlu4 ligands (both agonists and PAMs) typical of the mGlu2-4 heterodimer were observed in these cells, bringing strong evidence that endogenous mGlu2-4 heterodimers exist in these neuronal cells despite the low expression of both mGlu2 and mGlu4. The synergistic activity of the agonists LY354740 and LSP4-2022 was also observed in the terminals of the medial perforant path in the den- tate gyrus where both mGlu2 and mGlu4 subunits are present ( Shigemoto et al., 1997 ). Such a syn- ergistic activity is no longer observed in slices prepared from mGlu4 KO mice, demonstrating the involvement of mGlu4. However, we cannot rule out that such a synergy may come from the signal- ing cascades activated by both mGlu2 and mGlu4 homodimers. We still think this is unlikely because such a strong synergistic effect has not been observed between Gi-coupled receptors and indeed could not be observed between mGlu2 and the delta opioid receptor co-expressed in the same cells.
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Chemo-enzymatic synthesis of a series of 2,4-syn-functionalized (S)-glutamate analogues: new insight into the structure-activity relation of ionotropic glutamate receptor subtypes 5, 6, and 7.

Chemo-enzymatic synthesis of a series of 2,4-syn-functionalized (S)-glutamate analogues: new insight into the structure-activity relation of ionotropic glutamate receptor subtypes 5, 6, and 7.

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Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells.

Surface expression of metabotropic glutamate receptor variants mGluR1a and mGluR1b in transfected HEK293 cells.

2.1.Mutagenesis Constructs HA-mGluR1 and HA-GB1a and HA-GB2 were used and described in our previous studies (Ango et al., 2000; Galvez et al., 2001; Havlickova et al., 2003; Hlavackova et al., 2005; Pagano et al., 2001). MGluR1b variant was tagged using the same strategy. Shortly, tags HA (hemagglutinin) or c-Myc were introduced in the extreme N-terminal part of the receptor preceded by a signal peptide sequence derived from mGluR5. Mature mGluR1a or mGluR1b (coding sequence starting at Serine 34) were introduced by unique MluI restriction side located after the tag coding sequence. The expressed recombinant receptors thus consist of the signal peptide sequence of mGluR5, HA or c-Myc epitopes followed by mGluR1a or mGluR1b sequence. Mutation F781P was introduced in the i3 loop of HA-mGluR1a as described (Hlavackova et al., 2005) and the YADA (Y226A and D318A) mutations were done in mGluR1 in same manner as described previously for mGluR5 (Kniazeff et al., 2004) using QuickChange site-directed mutagenesis kit (Stratagene, Chemos, Czech Republic). The chimerical proteins GB/C1 consisting of the N-terminal and heptahelical domains of the GABAb receptor GB1a or GB2 subunits followed by the carboxyl-termini of mGluR1a or mGluR1b variants were done using unique Sph1 site on mGluR1 receptor DNA positioned within the portion identical between all the mGluR1 variants. Sph1 site was engineered in the GB1 (after Asp 873) and GB2 (after Arg 759) sequences. Substitution of the carboxyl terminal part were done using Sph1-Xba1 sites for mGluR1a (His 859-Stop 1200) or mGluR1b (His 859-Stop 907) coding sequences. To allow detection of the recombinant receptors on cell surface the N-terminal 16 residues of rat GB1a and N-terminal 41 residues of rat GB2 were replaced by 36 residues encoding the mGluR5 signal peptide, and HA epitope. The adaptor Gqi9 protein, based on sequence of Gq with last 9 residues replaced by extreme C terminal 9 residues from Gi sequence was described (Conklin et al., 1993) and used previously in our studies (Franek et al., 1999; Galvez et al., 2001; Havlickova et al., 2003). All constructs were sequenced using Big Dye Terminater v. 3,1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).
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The glutamate receptor GluK2 contributes to the regulation of glucose homeostasis and its deterioration during aging

The glutamate receptor GluK2 contributes to the regulation of glucose homeostasis and its deterioration during aging

In contrast, there is considerable evidence for a stimulatory autocrine feedback loop of glutamate in a -cells [ 4 , 18 , 20 ]. The obvious question is whether GluK2 may be speci fically implicated. As a -cells express AMPA as well as kainate receptors, and both are dif ficult to distinguish in pharmacological terms, the molecular identity of the relevant re- ceptors has hitherto only been addressed indirectly. Although previous reports favor a functional role of AMPA rather than of kainate receptors [ 19 ], caution should be exerted. Isolated a -cells may behave differently from cells within islets, as actually observed by the authors [ 19 ], and these findings do not preclude a role of kainate receptors specifically in the autocrine stimulation of glucagon secretion. The situation is even more complex in view of the reported metabotropic action of kainate
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The glutamate receptor GluK2 contributes to the regulation of glucose homeostasis and its deterioration during aging

The glutamate receptor GluK2 contributes to the regulation of glucose homeostasis and its deterioration during aging

In contrast, there is considerable evidence for a stimulatory autocrine feedback loop of glutamate in a -cells [ 4 , 18 , 20 ]. The obvious question is whether GluK2 may be speci fically implicated. As a -cells express AMPA as well as kainate receptors, and both are dif ficult to distinguish in pharmacological terms, the molecular identity of the relevant re- ceptors has hitherto only been addressed indirectly. Although previous reports favor a functional role of AMPA rather than of kainate receptors [ 19 ], caution should be exerted. Isolated a -cells may behave differently from cells within islets, as actually observed by the authors [ 19 ], and these findings do not preclude a role of kainate receptors specifically in the autocrine stimulation of glucagon secretion. The situation is even more complex in view of the reported metabotropic action of kainate
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Alleviating Pain Hypersensitivity through Activation of Type 4 Metabotropic Glutamate Receptor

Alleviating Pain Hypersensitivity through Activation of Type 4 Metabotropic Glutamate Receptor

suggesting that their expression level in these neuronal cell bodies is under the detection limit. We observed that mGlu4 receptors are expressed mostly in the inner lamina II of the dorsal horn of the spinal cord in mice ( Fig. 2 ). In this lamina, we detected mGlu4 immunoreactivity in C-tactile low threshold mechanical receptive (C-LTMR) affer- ents expressing the vesicular glutamate transporter 3 (VGLUT3). Of note, these C-LTMR neurons convey non-noxious touch sen- sations in healthy conditions ( Lo¨ken et al., 2009 ; Li et al., 2011 ) and are involved in mechanical hypersensitivity associated with chronic pain ( Seal et al., 2009 ). A small proportion of mGlu4 receptor staining (⬍10%) merged with peptidergic and nonpep- tidergic C fibers markers, such as substance P (SP), calcitonin gene-related peptide (CGRP), or isolectin B4 (IB4), respectively. The labeling of mGlu4 receptors also partially overlaps with staining of protein kinase C␥ (PKC␥) interneurons in inner lam- ina II ( Fig. 2 ). PKC ␥-expressing interneurons are excitatory neu- rons, which play an important role in the processing of tactile inputs both in physiological and pathological conditions and are notably thought to mediate injury-induced hypersensitivity ( Malmberg et al., 1997 ; Polga´r et al., 1999 ; Miraucourt et al., 2007 ; Neumann et al., 2008 ; Lu et al., 2013 ). By contrast, mGlu4 receptor labeling does not significantly overlap with NF200 ( ⬍1%), a marker of myelinated A␤ or A␦ fibers. In accordance with previous electronic microscopy data indicating that mGlu4 is commonly observed in presynaptic terminals in the dorsal spinal cord ( Azkue et al., 2001 ), most mGlu4 receptor immuno- reactivity merges with that of synaptophysin, a membrane glyco- protein characteristic of presynaptic vesicles ( Wiedenmann and Franke, 1985 ), and bassoon, a protein expressed at the presynap- tic nerve terminals ( tom Dieck et al., 1998 ).
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Metabotropic glutamate receptor 5 in bulimia nervosa

Metabotropic glutamate receptor 5 in bulimia nervosa

Positive t-values indicate higher mean [11C]ABP688 DVR in subjects receiving antidepressant treatment than in subjects not receiving antidepressant medication.. All three subjects with[r]

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Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3

Chloride ions stabilize the glutamate-induced active state of the metabotropic glutamate receptor 3

Supplemental figure 6: Molecular determinants of mGlu3 chloride lock A. Sequence alignment of the 8 mGluRs performed using Align123 on Discovery Studio suite and then optimized. Sequences were retrieved from Uniprot database (identifiers reported on the figure, middle column) and only the ECD parts were used (residue interval in right column). Numbers correspond to mGlu3R residue numbering. Blue color intensity is correlated by residue identity (dark), high similarity (medium) or low similarity (light). Key position in Cl‐ binding are indicated in red (site 1) and yellow (site 2). mGlu2/3R cation‐pi interlobe connection amino‐acids are in orange. Loop intervals indicated with black double arrows. B. Bar plot of mGlu3 mutants pEC50 values determined by TR‐FRET mGlu sensor assay in high‐Cl (open) and low‐Cl (hatched) media. C. Calcium mobilisation assay of mGlu3 receptor mutants in high‐Cl medium. Data are normalized by TR‐FRET largest window defined by top and bottom of the mGlu3 WT dose‐response curve in low‐Cl. D. Expression of HA SNAP and HA mGlu3 constructs as determined by ELISA. Open bar: cell surface expression; hatched bar: total expression. All constructs dose‐response parameters are listed in supplemental table 2. Glu: glutamate. VEH: vehicle.
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Optocontrol of glutamate receptor activity by single side-chain photoisomerization

Optocontrol of glutamate receptor activity by single side-chain photoisomerization

Photosensitive ABD domains to control receptor activity with light The activation of NMDARs requires binding of two different agonists, glutamate and glycine (or D-serine), a unique feature among ligand-gated ion channels. These co-agonists bind at appropriate clamshell-like domains that close thereupon to transduce the signal to the ion channel pore. Within a tetrameric GluN1/GluN2 receptor, the ABDs assemble as two heterodimers with the glycine-binding GluN1 ABD and the glutamate-binding GluN2 ABD, pairing through back-to-back interactions within each dimer ( Furukawa et al., 2005 ; Karakas and Furukawa, 2014 ; Lee et al., 2014 ) ( Figure 2a ). To endow NMDARs with optical sensitivity, we initially targeted the GluN1/GluN2A ABD heterodimer interface for PSAA incorporation since this region critically controls receptor functionality ( Furukawa et al., 2005 ; Gielen et al., 2008 ; Borschel et al., 2011 ). Replacing the highly conserved proline residue GluN1-P532 by the PSAA resulted in functional receptors, as represented by large and robust co-agonist activated currents ( Figure 2—figure supplement 1 ) as well as a pronounced modulation of receptor activity induced by application of light ( Figure 2 ). Specifically, following receptor activation with saturating co-agonist concentrations, illumination with UV light (365 nm) to switch the PSAA from the trans- to the cis-isomer, produced a remarkable current inhibition of 48.9 ± 1% (n = 23; Figure 2b and g ). Crucially, this effect could be fully reversed by blue light (460 nm) that regenerates the trans-version of the PSAA. The degree of photoinactivation was similar when UV illumination occurred prior to agonist application (48.4 ± 1.6% [n = 7]; Figure 2c and d ) or during application of competitive antagonists ( Figure 2—figure supplement 2 ), indicating that the effect is not dependent on the functional state of the receptor. Application of blue light in the resting state was ineffective to alter the current
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Alleviating Pain Hypersensitivity through Activation of Type 4 Metabotropic Glutamate Receptor

Alleviating Pain Hypersensitivity through Activation of Type 4 Metabotropic Glutamate Receptor

suggesting that their expression level in these neuronal cell bodies is under the detection limit. We observed that mGlu4 receptors are expressed mostly in the inner lamina II of the dorsal horn of the spinal cord in mice ( Fig. 2 ). In this lamina, we detected mGlu4 immunoreactivity in C-tactile low threshold mechanical receptive (C-LTMR) affer- ents expressing the vesicular glutamate transporter 3 (VGLUT3). Of note, these C-LTMR neurons convey non-noxious touch sen- sations in healthy conditions ( Lo¨ken et al., 2009 ; Li et al., 2011 ) and are involved in mechanical hypersensitivity associated with chronic pain ( Seal et al., 2009 ). A small proportion of mGlu4 receptor staining (⬍10%) merged with peptidergic and nonpep- tidergic C fibers markers, such as substance P (SP), calcitonin gene-related peptide (CGRP), or isolectin B4 (IB4), respectively. The labeling of mGlu4 receptors also partially overlaps with staining of protein kinase C␥ (PKC␥) interneurons in inner lam- ina II ( Fig. 2 ). PKC ␥-expressing interneurons are excitatory neu- rons, which play an important role in the processing of tactile inputs both in physiological and pathological conditions and are notably thought to mediate injury-induced hypersensitivity ( Malmberg et al., 1997 ; Polga´r et al., 1999 ; Miraucourt et al., 2007 ; Neumann et al., 2008 ; Lu et al., 2013 ). By contrast, mGlu4 receptor labeling does not significantly overlap with NF200 ( ⬍1%), a marker of myelinated A␤ or A␦ fibers. In accordance with previous electronic microscopy data indicating that mGlu4 is commonly observed in presynaptic terminals in the dorsal spinal cord ( Azkue et al., 2001 ), most mGlu4 receptor immuno- reactivity merges with that of synaptophysin, a membrane glyco- protein characteristic of presynaptic vesicles ( Wiedenmann and Franke, 1985 ), and bassoon, a protein expressed at the presynap- tic nerve terminals ( tom Dieck et al., 1998 ).
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A polymorphism in the glutamate metabotropic receptor 7 is associated with cognitive deficits in the early phases of psychosis

A polymorphism in the glutamate metabotropic receptor 7 is associated with cognitive deficits in the early phases of psychosis

4 the pioneering trial about oral glycine on negative symptoms in schizophrenia (Leiderman et al., 1996), several studies have tested modulators of the metabotropic glutamate receptors (Matosin and Newell, 2013) and modulators of the N-methyl-D-aspartate receptor (NMDAR) including D- serine or glycine in add-on in chronic schizophrenia (Kantrowitz et al., 2010). Despite mixed findings of their attenuation of cognitive deficits in later stages (Iwata et al., 2015), these treatments may have efficacy when used earlier, during UHR stages (Kantrowitz et al., 2015). Modulation of N-methyl-D-aspartate receptor has also been reported to improve psychotic symptoms in patients carrying a rare copy-number variant encompassing glutamatergic genes (Bodkin et al., 2019) suggesting efficacy in patients with specific genetic factors. Indeed, rare variants in NMDAR have been associated with schizophrenia (Tarabeux et al., 2011). Common variants associated with schizophrenia pointed towards genes related to synaptic function and expressed in pyramidal glutamatergic neurons (Skene et al., 2018). In addition, N-methyl-D- aspartate-type glutamate receptor (NMDAR) antagonists, such as ketamine, induce cognitive deficits and symptoms closely resembling those of schizophrenia (Anticevic et al., 2012; Vinckier et al., 2016). Consequently, it has been postulated that dysregulation in glutamate neurotransmission may play a significant role in cognitive impairments observed in schizophrenia (Thomas et al., 2017) and may also play a role in the emergence of psychotic symptoms. A meta- analysis of brain spectroscopy studies in patients with UHR revealed that medial frontal glutamate plus glutamine levels are increased compared to healthy controls (Merritt et al., 2016). Recent reports suggest that glutamate level could be higher in FEP compared to controls in the cingular cortex (Chiu et al., 2017; Kim et al., 2017) and striatum (Plitman et al., 2016).
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AMPA-sst2 somatostatin receptor interaction in rat hypothalamus requires activation of NMDA and/or metabotropic glutamate receptors and depends on intracellular calcium.

AMPA-sst2 somatostatin receptor interaction in rat hypothalamus requires activation of NMDA and/or metabotropic glutamate receptors and depends on intracellular calcium.

represented more than 95% of the total recorded inward current, thus indicating a 5% contribution of NMDA activation, similar to that recorded for synaptic responses in slices. As previously reported, at the agonist concentrations used herein, AMPA-mediated responses displayed no fast peak current (Swandulla et al., 1994). As shown in Figure 5A and 5B, the fast application of octreotide + glutamate decreased the glutamate-induced inward current in 43% of the tested neurones (3/7) with a maximum average decrease of –15.9 + 4.1 % (p<0.001 vs control values before octreotide in responsive cells, n=3, and values under octreotide in non responsive cells, n=4). As opposed to slice experiments, octreotide effect desensitized rapidly before the end of application. Octreotide-induced decrease was also found when all glutamate receptor subtypes were activated by coapplying AMPA, NMDA and trans(+)-ACPD in 38% of the tested cells (3/8) with a comparable maximum average decrease of –12.5 + 0.4 % (p<0.001 vs control values before octreotide in responsive cells, n=3, and values under octreotide in non responsive cells, n=5 ; Fig 5C,D). Fast application of AMPA with octreotide on 9 cells never led to a decreased AMPA current (Fig 5E,F) while octreotide coapplied with AMPA and NMDA decreased the AMPA current by –8.4 + 1.6 % (p<0.001 vs control values before octreotide in responsive cells, n=5, and values under octreotide in non responsive cells, n=8, Fig 5G,H). In a second series of experiments, we tested whether calcium influx through the coactivated NMDA receptors could be responsible for restoring octreotide modulation of the AMPA current. As shown in Figure 5I and 5J, omitting calcium from the extracellular medium totally abolished the decrease of AMPA current during coapplication of AMPA, NMDA and octreotide in all 15 tested neurones. However, octreotide was still able to decrease glutamate response by –14.1 + 1.7 % in 3 out of 7 tested cells even in the absence of extracellular calcium (data not shown) suggesting that mGluRs were involved in sst2/AMPA receptor interactions.
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Higher ambient synaptic glutamate at inhibitory versus excitatory neurons differentially impacts NMDA receptor activity

Higher ambient synaptic glutamate at inhibitory versus excitatory neurons differentially impacts NMDA receptor activity

To isolate GluN2A NMDAR EPSCs, neurons were held at −60 mV in the presence of NBQX (10 μM), picrotoxin (50 μM), Mg 2+ (0.5 mM), and piperindine 18 (1 μM; selective GluN2B-NMDAR antagonist). All drugs were applied in the bath. For experiments testing the effect of CPG on holding current and MK-801 on ambient glutamate responses, neurons were held at +40 mV. After obtaining whole-cell configuration, 5–10 min was allowed for recordings to stabilize. In experiments using γ-DGG, 10 or 100 μM D -APV was used to block NMDAR. TBS ( five pulses at 250 Hz) was delivered every 90 s to avoid inducing synaptic plasticity. For puffing experiments, glass pipettes (1–2 MΩ) filled with D -APV (5 mM) or GNE-8324 (3 mM) were positioned ~50 μm from the recorded neuron, with pressure (10 psi for 50 ms) from a Picospritzer III (Parker). For measuring the ambient NMDAR responses with CPG incubation (50 μM, >20 min), D -APV was fast perfused into the recording chamber by a tube positioned at the edge of the recording chamber. In the experiments where NMDA was bath applied, both 5 and 7 μM NMDA was used either on the same neurons or on different neurons (each neuron was exposed to either 5 or 7 μM NMDA, but not both). Similar changes in the holding current and noise were seen between these two conditions, and hence results were pooled. For testing the effect of bath-applied NMDA on GNE-8324, neurons were exposed to either 5 or 7 μM NMDA, but not both. In the DHK at 32 °C experiment, 150 μM DHK was used to maintain an adequate recording conditions (such as voltage control).
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Rôles physiologiques du transporteur vésiculaire du glutamate VGLUT2 dans les neurones dopaminergiques

Rôles physiologiques du transporteur vésiculaire du glutamate VGLUT2 dans les neurones dopaminergiques

neurons prior to the identification of VGLUTs showed that the vast majority of axon varicosities formed by DA neurons in single neuron microculture were double-labeled for TH and glutamate, suggesting that most terminals have the capacity to release both transmitters 60 (see also 62, 65 for early ultrastructural data suggesting that DA neurons establish two morphologically distinct subsets of axon terminals). Close examination of VGLUT2 immunoreactivity in vitro 28 and in vivo 66 , however, suggests that DA neurons possess different subsets of axon terminals, perhaps as much as three: a first that contains only TH, a second, perhaps smaller, containing both TH and VGLUT2, and a third containing VGLUT2 but not TH (Figure 2). The possibility that TH might be absent from some axonal branches and/or axon terminals of DA neurons may come as a surprise in light of the fact that such biosynthetic enzymes are known to be mostly cytosolic. However, there is some evidence that TH may be membrane bound, even to synaptic vesicles, notably in the striatum 67 , and may therefore be unevenly distributed among axon terminals. Indeed, isolated DA neurons in culture have a large number of VGLUT2 immunopositive, TH immunonegative varicosities, confirmed as axon terminals by the presence of synaptic vesicle 2- related protein (SV2) 28 . A recent study has suggested that VGLUT2 is only found in a small subset of TH- and VMAT2-containing axon terminals in cultured DA neurons 68 . The notion of a phenotypical heterogeneity among axon terminals of DA neurons might also explain why, in adult (as opposed to P15) rats, axon terminals dually labeled for TH and VGLUT2 are no longer observed in ventral or dorsal striatum 34 . This is even the case after a partial 6-hydroxydopamine lesioning of the mesencephalic DA neurons, a condition known to activate expression of Vglut2 and increase the colocalization of TH and VGLUT2 in the DA axon terminals of postnatal rat 34 . These results now need to be re-evaluated by quantifying the colocalization of VGLUT2 with other dopaminergic markers such as DAT as well as VMAT2.
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en fr Paracrine transmitters as informative signals: GABA and glutamate modulate neuronal migration Modulation de la migration neuronale par les neurotransmetteurs GABA et glutamate : aspects fondamentaux et implications pathologiques

Paracrine transmitters as developmental signals An increasing amount of evidences, including those provided in our study, suggest that transmitters act as developmental signals (Nguyen et al., 2001;Owens and Kriegstein, 2002). These “infor- mative” transmitters may establish morphogenetic gradients that guide migrating neuroblasts to their target fields (i.e., positional signaling), or create a permissive substrate for migration, giving to the neuroblasts the conditions required for motility, or a com- bination of both. Transmitters may also establish and maintain a level of activity adequate for migrating neuroblasts to respond to the “classical” guidance cues. In addition, transmitters could stimulate the secretion of these guidance cues from surrounding cells, glial cells (astrocytes or radial glial cells) or more mature neurons, present along the migrating pathway and/or at the level of the target field. A “guiding action” of transmitters is unlikely because the leading process orientation (considered as an index of the direction of migration) is unaffected after treatments with antagonists in our study. However, we measured the leading pro- cess orientation of migrating neuroblasts “freezed” in the posi- tion they had just before fixation, bypassing the fact that the leading process is a highly dynamic structure. Additional studies, more dynamic (i.e., time-lapse high-power microscopy), are re- quired and in process to answer that question. Interestingly, a study by Zheng et al. (1996) reported a turning response of the growth cones of Xenopus spinal neurons submitted to glutamate
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The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene.

The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene.

developmental defects, such as impaired node formation and gastrulation (Pare et al., 2004). During adulthood, LRH-1 plays important roles in cholesterol and lipid homeostasis by controlling the expression of genes involved in these metabolic pathways (for review, see Fayard et al., 2004). Several studies reported elevated expression of LRH-1 in granulosa cells and corpus luteum of the ovary, suggesting potential functions for this nuclear receptor in follicular growth and function (Falender et al., 2003; Hinshelwood et al., 2003). By controlling expression of cyclin E1 and D1 through interaction with ß-catenin, LRH-1 has recently been shown to promote intestinal cell renewal (Botrugno et al., 2004). However, despite its transcriptional control on G 1 phase cyclins, the contribution of LRH-1 to
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