Dynamic modulation of inflammatory pain-related affective and sensory symptoms by optical control of amygdala metabotropic glutamate receptor 4.

Contrary to acute pain, chronic pain does not serve as a warning signal and must be considered as a disease per se. This pathology presents a sensory and psychological dimension at the origin of affective and cognitive disorders. Being largely refractory to current pharmacotherapies, identification of endogenous systems involved in persistent and chronic pain is crucial. The amygdala is a key brain region linking pain sensation with negative emotions. Here, we show that activation of a specific intrinsic neuromodulatory system within the amygdala associated with type 4 metabotropic glutamate receptors (mGlu4) abolishes sensory and affective symptoms of persistent pain such as hypersensitivity to pain, anxiety- and depression-related behaviors, and fear extinction impairment. Interestingly, neuroanatomical and synaptic analysis of the amygdala circuitry suggests that the effects of mGlu4 activation occur outside the central nucleus via modulation of multisensory thalamic inputs to lateral amygdala principal neurons and dorso-medial intercalated cells. Furthermore, we developed optogluram, a small diffusible photoswitchable positive allosteric modulator of mGlu4. This ligand allows the control of endogenous mGlu4 activity with light. Using this photopharmacological approach, we rapidly and reversibly inhibited behavioral symptoms associated with persistent pain through optical control of optogluram in the amygdala of freely behaving animals. Altogether, our data identify amygdala mGlu4 signaling as a mechanism that bypasses central sensitization processes to dynamically modulate persistent pain symptoms. Our findings help to define novel and more precise therapeutic interventions for chronic pain, and exemplify the potential of optopharmacology to study the dynamic activity of endogenous neuromodulatory mechanisms in vivo.

Vision-Based Traffic Sign Detection and Recognition Systems: Current Trends and Challenges.

The automatic traffic sign detection and recognition (TSDR) system is very important research in the development of advanced driver assistance systems (ADAS). Investigations on vision-based TSDR have received substantial interest in the research community, which is mainly motivated by three factors, which are detection, tracking and classification. During the last decade, a substantial number of techniques have been reported for TSDR. This paper provides a comprehensive survey on traffic sign detection, tracking and classification. The details of algorithms, methods and their specifications on detection, tracking and classification are investigated and summarized in the tables along with the corresponding key references. A comparative study on each section has been provided to evaluate the TSDR data, performance metrics and their availability. Current issues and challenges of the existing technologies are illustrated with brief suggestions and a discussion on the progress of driver assistance system research in the future. This review will hopefully lead to increasing efforts towards the development of future vision-based TSDR system.

Improving the performance of thermophilic anaerobic digester through recirculation of low hydrogen biogas.

Biogas recirculation was conducted to improve the performance of two thermophilic anaerobic sequenced batch reactors (ASBRs), in which high concentrations of volatile fatty acids (VFAs) were accumulated. To accelerate degradation of VFAs, facilitating acetate consumption via syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (SAO-HM) was expected to be effective. Hence, to promote the SAO-HM pathway, hydrogen was removed to create low hydrogen partial pressure (pH2) in reactor RH, yet in reactor RB, hydrogen was not treated. The performance of RB and RH on VFAs degradation and methane production processes was compared at steady stage; the VFAs and soluble microbial products (SMP) in the effluents were monitored. The results showed that low pH2 intensified the SAO reaction, thereby accelerating conversion of acetate to methane, as well as acetate production from glucose and VFAs. Glucose fermentation type was also influenced. VFAs and SMP in the effluents were reduced after the introduction of biogas mixing, which proceeded much faster in RH with low pH2. Recirculation of low hydrogen biogas with SAO-HM pathway being promoted should be more effective to alleviate high acid level stress and to improve the reactor performance.

Cinnabarinic acid, an endogenous metabolite of the kynurenine pathway, activates type 4 metabotropic glutamate receptors.

Cinnabarinic acid is an endogenous metabolite of the kynurenine pathway that meets the structural requirements to interact with glutamate receptors. We found that cinnabarinic acid acts as a partial agonist of type 4 metabotropic glutamate (mGlu4) receptors, with no activity at other mGlu receptor subtypes. We also tested the activity of cinnabarinic acid on native mGlu4 receptors by examining 1) the inhibition of cAMP formation in cultured cerebellar granule cells; 2) protection against excitotoxic neuronal death in mixed cultures of cortical cells; and 3) protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice after local infusion into the external globus pallidus. In all these models, cinnabarinic acid behaved similarly to conventional mGlu4 receptor agonists, and, at least in cultured neurons, the action of low concentrations of cinnabarinic acid was largely attenuated by genetic deletion of mGlu4 receptors. However, high concentrations of cinnabarinic acid were still active in the absence of mGlu4 receptors, suggesting that the compound may have off-target effects. Mutagenesis and molecular modeling experiments showed that cinnabarinic acid acts as an orthosteric agonist interacting with residues of the glutamate binding pocket of mGlu4. Accordingly, cinnabarinic acid did not activate truncated mGlu4 receptors lacking the N-terminal Venus-flytrap domain, as opposed to the mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). Finally, we could detect endogenous cinnabarinic acid in brain tissue and peripheral organs by high-performance liquid chromatography-tandem mass spectrometry analysis. Levels increased substantially during inflammation induced by lipopolysaccharide. We conclude that cinnabarinic acid is a novel endogenous orthosteric agonist of mGlu4 receptors endowed with neuroprotective activity.

Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors.

Dendritic cells (DCs) form a network comprising different populations that initiate and differentially regulate immune responses. Langerhans cells (LCs) represent a unique population of DCs colonizing epithelium, and we present here observations suggesting that macrophage inflammatory protein (MIP)-3alpha plays a central role in LC precursor recruitment into the epithelium during inflammation. (a) Among DC populations, MIP-3alpha was the most potent chemokine inducing the selective migration of in vitro-generated CD34(+) hematopoietic progenitor cell-derived LC precursors and skin LCs in accordance with the restricted MIP-3alpha receptor (CC chemokine receptor 6) expression to these cells. (b) MIP-3alpha was mainly produced by epithelial cells, and the migration of LC precursors induced by the supernatant of activated skin keratinocytes was completely blocked with an antibody against MIP-3alpha. (c) In vivo, MIP-3alpha was selectively produced at sites of inflammation as illustrated in tonsils and lesional psoriatic skin where MIP-3alpha upregulation appeared associated with an increase in LC turnover. (d) Finally, the secretion of MIP-3alpha was strongly upregulated by cells of epithelial origin after inflammatory stimuli (interleukin 1beta plus tumor necrosis factor alpha) or T cell signals. Results of this study suggest a major role of MIP-3alpha in epithelial colonization by LCs under inflammatory conditions and immune disorders, and might open new ways to control epithelial immunity.

T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines.

Analysis of the cDNA encoding murine interleukin (IL) 17 (cytotoxic T lymphocyte associated antigen 8) predicted a secreted protein sharing 57% amino acid identity with the protein predicted from ORF13, an open reading frame of Herpesvirus saimiri. Here we report on the cloning of human IL-17 (hIL-17), the human counterpart of murine IL-17. hIL-17 is a glycoprotein of 155 amino acids secreted as an homodimer by activated memory CD4+ T cells. Although devoid of direct effects on cells of hematopoietic origin, hIL-17 and the product of its viral counterpart, ORF13, stimulate epithelial, endothelial, and fibroblastic cells to secrete cytokines such as IL-6, IL-8, and granulocyte-colony-stimulating factor, as well as prostaglandin E2. Furthermore, when cultured in the presence of hIL-17, fibroblasts could sustain the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These observations suggest that hIL-17 may constitute (a) an early initiator of the T cell-dependent inflammmatory reaction; and (b) an element of the cytokine network that bridges the immune system to hematopoiesis.

G-protein-coupled receptor oligomers: two or more for what? Lessons from mGlu and GABAB receptors.

G-protein-coupled receptors (GPCRs) are key players in the precise tuning of intercellullar communication. In the brain, both major neurotransmitters, glutamate and GABA, act on specific GPCRs [the metabotropic glutamate (mGlu) and GABA(B) receptors] to modulate synaptic transmission. These receptors are encoded by the largest gene family, and have been found to associate into both homo- and hetero-oligomers, which increases the complexity of this cell communication system. Here we show that dimerization is required for mGlu and GABA(B) receptors to function, since the activation process requires a relative movement between the subunits to occur. We will also show that, in contrast to the mGlu receptors, which form strict dimers, the GABA(B) receptors assemble into larger complexes, both in transfected cells and in the brain, resulting in a decreased G-protein coupling efficacy. We propose that GABA(B) receptor oligomerization offers a way to increase the possibility of modulating receptor signalling and activity, allowing the same receptor protein to have specific properties in neurons at different locations.

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes.

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.

Get receptive to metabotropic glutamate receptors.

Glutamate activates not only ionotropic glutamate receptors, but also G-protein-coupled receptors, called metabotropic glutamate receptors. Recent studies have revealed that these metabotropic receptors share distinctive structural properties and that they form a subgroup within the heptahelical receptor family. The development of ligands that bind specifically to these receptors has provided a means of characterizing the important roles they play in the tuning of fast synaptic transmission, including the induction of long-term changes in synaptic strength. Their involvement in the control of movement, spatial and olfactory memory and nociception has recently been demonstrated.

Dendritic and axonal targeting of type 5 metabotropic glutamate receptor is regulated by homer1 proteins and neuronal excitation.

The physiological actions of neurotransmitter receptors are intimately linked to their proper neuronal compartment localization. Here we studied the effect of the metabotropic glutamate receptor (mGluR)-interacting proteins, Homer1a, b, and c, in the targeting of mGluR5 in neurons. We found that mGluR5 was exclusively localized in cell bodies when transfected alone in cultured cerebellar granule cells. In contrast, mGluR5 was found also in dendrites when coexpressed with Homer1b or Homer1c, and in both dendrites and axons when cotransfected with Homer1a. In dendrites, cotransfected mGluR5 and Homer1b/c formed clusters that colocalized with the synaptic marker synaptophysin. Interestingly when transfected alone, the Homer proteins were also translocated to neurites but did not form such clusters. Depolarization of the neurons with a mixture of ionotropic glutamate receptor agonists, NMDA and kainate, or potassium channel blockers, tetraethylammonium and 4-aminopyridine, induced transient expression of endogenous Homer1a and persistent neuritic localization of transfected mGluR5 even long after degradation of Homer1a. These results suggest that Homer1a/b/c proteins are involved in the targeting of mGluR5 to dendritic synaptic sites and/or axons and that this effect can be regulated by neuronal activity. Because the activity-dependent effect of endogenous Homer1a was also long-lasting, the axonal targeting of mGluR5 by this protein is likely to play an important role in synaptic plasticity.

C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABA(b) receptors.

Assembly of fully functional GABA(B) receptors requires heteromerization of the GABA(B(1)) and GABA(B(2)) subunits. It is thought that GABA(B(1)) and GABA(B(2)) undergo coiled-coil dimerization in their cytoplasmic C termini and that assembly is necessary to overcome GABA(B(1)) retention in the endoplasmatic reticulum (ER). We investigated the mechanism underlying GABA(B(1)) trafficking to the cell surface. We identified a signal, RSRR, proximal to the coiled-coil domain of GABA(B(1)) that when deleted or mutagenized allows for surface delivery in the absence of GABA(B(2)). A similar motif, RXR, was recently shown to function as an ER retention/retrieval (ERR/R) signal in K(ATP) channels, demonstrating that G-protein-coupled receptors (GPCRs) and ion channels use common mechanisms to control surface trafficking. A C-terminal fragment of GABA(B(2)) is able to mask the RSRR signal and to direct the GABA(B(1)) monomer to the cell surface, where it is functionally inert. This indicates that in the heteromer, GABA(B(2)) participates in coupling to the G-protein. Mutagenesis of the C-terminal coiled-coil domains in GABA(B(1)) and GABA(B(2)) supports the possibility that their interaction is involved in shielding the ERR/R signal. However, assembly of heteromeric GABA(B) receptors is possible in the absence of the C-terminal domains, indicating that coiled-coil interaction is not necessary for function. Rather than guaranteeing heterodimerization, as previously assumed, the coiled-coil structure appears to be important for export of the receptor complex from the secretory apparatus.

Molecular determinants of metabotropic glutamate receptor signaling.

Metabotropic glutamate (mglu) receptors are implicated in the regulation of many physiological and pathological processes in the CNS, including synaptic plasticity, learning and memory, motor coordination, pain transmission and neurodegeneration. Several recent studies have elucidated the molecular determinants of mglu receptor signaling and show that several mechanisms acting at different steps in signal propagation are involved. We attempt to offer an integrated view on how homologous and heterologous mechanisms regulate the initial steps of signal propagation, mainly at the level of mglu-receptor-G-protein coupling. Particular emphasis is placed on the role of phosphorylation mechanisms mediated by protein kinase C and G-protein-coupled receptor kinases, and on the emerging importance of some members of the regulators of G-protein signaling family, such as RGS2 and RGS4, which facilitate the GTPase activity that is intrinsic to the alpha-subunits of G(q) and G(i).

Allosteric functioning of dimeric class C G-protein-coupled receptors.

Whereas most membrane receptors are oligomeric entities, G-protein-coupled receptors have long been thought to function as monomers. Within the last 15 years, accumulating data have indicated that G-protein-coupled receptors can form dimers or even higher ordered oligomers, but the general functional significance of this phenomena is not yet clear. Among the large G-protein-coupled receptor family, class C receptors represent a well-recognized example of constitutive dimers, both subunits being linked, in most cases, by a disulfide bridge. In this review article, we show that class C G-protein-coupled receptors are multidomain proteins and highlight the importance of their dimerization for activation. We illustrate several consequences of this in terms of specific functional properties and drug development.

Three-dimensional model of the extracellular domain of the type 4a metabotropic glutamate receptor: new insights into the activation process.

Metabotropic glutamate receptors (mGluRs) belong to the family 3 of G-protein-coupled receptors. On these proteins, agonist binding on the extracellular domain leads to conformational changes in the 7-transmembrane domains required for G-protein activation. To elucidate the structural features that might be responsible for such an activation mechanism, we have generated models of the amino terminal domain (ATD) of type 4 mGluR (mGlu4R). The fold recognition search allowed the identification of three hits with a low sequence identity, but with high secondary structure conservation: leucine isoleucine valine-binding protein (LIVBP) and leucine-binding protein (LBP) as already known, and acetamide-binding protein (AmiC). These proteins are characterized by a bilobate structure in an open state for LIVBP/LBP and a closed state for AmiC, with ligand binding in the cleft. Models for both open and closed forms of mGlu4R ATD have been generated. ACPT-I (1-aminocyclopentane 1,3,4-tricarboxylic acid), a selective agonist, has been docked in the two models. In the open form, ACPT-I is only bound to lobe I through interactions with Lys74, Arg78, Ser159, and Thr182. In the closed form, ACPT-I is trapped between both lobes with additional binding to Tyr230, Asp312, Ser313, and Lys317 from lobe II. These results support the hypothesis that mGluR agonists bind a closed form of the ATDs, suggesting that such a conformation of the binding domain corresponds to the active conformation.

The Ca2+/C1- dependent L-[3H]glutamate binding: a new receptor or a particular transport process?

Ca2+/C1- increases the L-[3H]glutamate binding to rat brain synaptic membranes. It was suggested that Ca2+/C1- expresses a new class of glutamate receptors. We report several lines of evidence suggesting that Ca2+/C1- in fact favours a glutamate transport into membrane vesicles. This finding may serve to reconcile most of the discrepancies found in the literature on the glutamate binding and its pharmacology.

The metabotropic glutamate receptors: structure and functions.

Glutamate is the main excitatory neurotransmitter in the brain. For many years it has been considered to act only on ligand-gated receptor channels--termed NMDA, AMPA and kainate receptors--involved in the fast excitatory synaptic transmission. Recently, glutamate has been shown to regulate ion channels and enzymes producing second messengers via specific receptors coupled to G-proteins. The existence of these receptors, called metabotropic glutamate receptors, is changing our views on the functioning of fast excitatory synapses.

Phenylglycine derivatives discriminate between mGluR1- and mGluR5-mediated responses.

The effects of the phenylglycine derivatives, alpha-methyl-4-carboxyphenylglycine (MCPG), 4-carboxyphenylglycine (4CPG), 4-carboxy-3-hydroxyphenylglycine (4C3HPG), 3-hydroxyphenylglycine (3HPG) and 3,5-dihydrohyphenylglycine (DHPG) were tested on LLC-PK1 cells transiently expressing the rat mGluR1a or mGluR5a receptors. As previously reported by others, (S)-3HPG and (RS)-DHPG were found to be partial agonists at mGluR1a, whereas(+)-MCPG,(S)-4CPG and (S)-4C3HPG competitively antagonized the effect of Glu. Surprisingly, the 4-carboxy derivatives of phenylglycine antagonized the effect of 1S,3R-ACPD on mGluR1a with lower KB values. On mGluR5a, (S)-3HPG and (RS)-DHPG are also partial agonists. However, in contrast to their effects on mGluR1a,(S)-4CPG did not inhibit the effect of Glu or 1S,3R-ACPD, and (S)-4C3HPG acted as an agonist at high concentration. Whereas no significant antagonism of the Glu effect on mGluR5a was observed with 1 mM (+)-MCPG, this compound was found to potently and competitively antagonize the effect of 1S,3R-ACPD. Finally, the effect of 4CPG was also examined on cultured cortical and cerebellar neurons that express mGluR5 and mGluR1 mRNA, respectively. 4CPG inhibited 1S,3R-ACPD-stimulated IP production in cerebellar neurons only. These results(1) demonstrate that phenylglycine derivatives can be used to discriminate between effects mediated by mGluR1 and mGluR5 and (2) suggest that the apparent potency of phenylglycine antagonists depends on the agonist used to activate these receptors.

Comparative effect of L-CCG-I, DCG-IV and gamma-carboxy-L-glutamate on all cloned metabotropic glutamate receptor subtypes.

In a previous study we reported that the addition of a carboxylic group to the mGlu receptor agonist aminocyclopentane-1,3-dicarboxylate (ACPD) changes its properties from agonist to antagonist at both mGlu1 and mGlu2 receptors, and resulted in an increase in affinity at mGlu4 receptors, with isomers being either agonists or antagonists. In the present study, the effect of gamma-carboxy-L-glutamic acid (Gla) and (2S,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), two carboxylic derivatives of non-selective agonists, were examined on all cloned mGlu receptors. We found that this additional carboxylic group on glutamate prevents its interaction with group-I mGlu receptors and generates a potent group-II antagonist (K(B) = 55 microM on mGlu2). At group-III mGlu receptors, Gla was found to be either an antagonist (mGlu7 and mGlu8 receptors) or a partial agonist (mGlu4 and mGlu6 receptors). We show here that L-CCG-I is a general mGlu receptor agonist activating all cloned receptors. We also confirm that DCG-IV, which corresponds to L-CCG-I with an additional carboxylic group, is a selective group-II agonist. However, this additional COOH group changes the properties of L-CCG-I from an agonist to an antagonist at all group-III receptors, making this compound one of the most potent group-III mGlu receptor antagonist known so far. These observations will be useful for the development of more potent and selective mGlu receptor agonists and antagonists.

Extended glutamate activates metabotropic receptor types 1, 2 and 4: selective features at mGluR4 binding site.

To get an insight into the bioactive conformation of glutamic acid and its topological environment at the mGluR4 binding site, a pharmacophore model was constructed using molecular modeling. Agonists of known activities were used to run the Apex-3D program or to validate the resulting model. An extended glutamate conformer, two selective hydrophilic sites and bulk tolerance regions are disclosed. Selective features of mGluR1, mGluR2 and mGluR4 are discussed.

mGluR7-like receptor and GABA(B) receptor activation enhance neurotoxic effects of N-methyl-D-aspartate in cultured mouse striatal GABAergic neurones.

Presynaptic metabotropic glutamate receptors (mGluRs) of group III constitute possible targets for putative neuroprotective drugs acting against glutamate excitotoxic insults. Indeed, in glutamatergic cerebellar granule neurones in culture, high concentrations of L-2-amino-4-phosphonobutyrate (L-AP4, above 0.3 mM, thus activating mGluR7) inhibit NMDA-induced cell death. In contrast, in striatal cultures which are enriched in GABAergic neurones, we show that high concentrations of L-AP4 increased neuronal death in control as well as in NMDA-stimulated cultures. Moreover, similar results were obtained with the GABA(B)R agonist. baclofen. Both the neuroprotective effects in cerebellar granule cells and the neurotoxic effects in striatal neurones were mediated via Gi-Go-coupled mGluRs, suggesting that these effects were probably mediated by mGluR7a or b and GABA(B)R expressed in these neurones. In striatal neurones, we found that L-AP4 and baclofen inhibited both basal and NMDA-stimulated GABA release. These inhibitions of GABA release may be responsible for the increase in basal and NMDA-stimulated neuronal death. Indeed, blockade of GABA(A) receptors with bicuculline increased neuronal death of control and NMDA-treated striatal cultures. Taken together, these results suggest that L-AP4 and baclofen, via mGluR7 and GABA(B)R, reduced the neuroprotective effect of GABA present in striatal cultures acting via GABA(A) receptors. Although caution must be taken when extrapolating from in vitro to in vivo situations, the present experiments and the recent observations that mGluR7 and GABA(B)R are expressed in heterologous synapses, should be taken into consideration when evaluating the neuroprotective action of future mGluR7 specific agonists or GABA(B)R specific antagonists.