Aux synapses Fibres Parallèles - Cellules de Purkinje, le récepteur mGluR4 est le seul mGluR du groupe III à moduler la neurotransmission en inhibant les influx calciques qui régulent la libération de glutamate. Dans des systèmes hétérologues, il a été montré que mGluR4 était lié à des protéines G de type Gi/o couplées négativement à l'adénylate cyclase (AC). Afin de rester au plus proche des interactions physiologiques, nous avons débuté notre étude par la définition du réceptosome des récepteurs mGluR4 natifs dans le cervelet de rat. Nous avons identifié 184 partenaires putatifs du récepteur. Afin de confirmer ces interactions, mais aussi de recenser d'autres interacteurs éventuels, nous avons réalisé une approche complémentaire et indépendante de chromatographie d'affinité. Nombre de protéines ont été retrouvées par cette deuxième approche, en particulier des protéines appartenant aux familles de l'exocytose et du trafic cellulaire. Nos résultats suggèrent que le contrôle de la neurotransmission par mGluR4 pourrait s'effectuer, au moins partiellement, par une interaction avec ce type de protéines. D'autre part, nos approches biochimiques n'ont pas mis en évidence de protéines de la voie AC, mais au contraire plusieurs protéines identifiées appartiennent à la voie Phospholipase C/ Protein Kinase C (PLC/PKC). Ces résultats biochimiques corroborent certains résultats fonctionnels du laboratoire et ouvrent de nouvelles pistes quant à la modulation négative de la neurotransmission par les récepteurs mGluR4 natifs dans le cervelet / At Purkinje Cell - Parallel Fiber synapses, mGluR4 receptors are the only glutamatergic metabotropic receptors of group III to modulate glutamatergic transmission by inhibiting calcium presynaptic influx controlling glutamate release. In heterologous systems, mGluR4 has been shown to activate G proteins of type Gi/o that would be negatively linked to adenylate cyclase (AC). In order to conserve most of physiological interactions, we first studied the receptosome of native mGluR4 in rat cerebellum. We identified 184 putative partners of the receptor. Moreover, in order to confirm these interactions, but also to find other partners, we decided to perform an independent and complementary approach of chromatography affinity. Numerous proteins have been found by this method, particularly proteins belonging to exocytosis and cellular trafficking families. Our results suggest that a partial control of neurotransmission could be due to interaction of mGluR4 with these kinds of proteins. On the other hand, biochemical approaches did not reveal interactions of mGluR4 with some proteins belonging to AC pathway, but with proteins of PLC/PKC pathway. These results are consistent with some functional studies of our lab and gave the way for elucidating the native molecular mechanisms of the cerebellar neurotransmission modulation by mGluR4.

The central complex is a prominent neuropil found in the middle of the insect brain. It is considered as a higher center for motor control and information processing. Multiple neuropeptides and neurotransmitters are produced in neurons of the central complex, however, distribution patterns and functional roles of signaling substances in this brain region are poorly known. Thus, this thesis focuses on the distribution of signaling substances and on modulatory roles of neuropeptides in the central complex of Drosophila. Immunocytochemistry in combination with GAL4/UAS technique was used to visualize various signaling substances in the central complex. We revealed different central-complex neurons expressing the neuropeptides; Drosophila tachykinin (DTK), short neuropeptide F (sNPF), myoinhibitory peptide (MIP), allatostatin A, proctolin, SIFamide, neuropeptide F and FMRFamide. Subpopulations of DTK, sNPF and MIP-expressing neurons were found to co-localize a marker for acetylcholine. In addition, five metabotropic neurotransmitter receptors were found to be expressed in distinct patterns. Comparison of receptor/ligand distributions revealed a close match in most of the structures studied. By using a video-tracking assay, peptidergic modulation of locomotor behavior was studied. Different DTK and sNPF-expressing neurons innervating the central complex were revealed to modulate spatial distribution, number of activity-rest phases and activity levels, suggesting circuit dependent modulation. Furthermore, neurosecretory cells in the Drosophila brain that co-express three types of neuropeptides were shown to modulate stress responses to desiccation and starvation. In summary, we have studied two different neuropeptides (DTK and sNPF) expressed in interneuronal circuits and neurosecretory cells of the Drosophila brain in more detail. We found that these neuropeptides display multiple actions as neuromodulators and circulating hormones, and that their actions depend on where they are released. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Manuscript.

Metabotropic glutamate receptor 1α (mGluR1α) exerts important effects on numerous neurological processes. Although mGluR1α is known to respond to extracellular Ca2+ ([Ca2+]o) and the crystal structures of the extracellular domains (ECDs) of several mGluRs have been determined, the calcium-binding site(s) and structural determinants of Ca2+-modulated signaling in the Glu receptor family remain elusive. Here, we identify a novel Ca2+-binding site (Site 1) in the ECD-mGluR1α using a recently developed computational algorithm. This predicted site (D318, E325, D322 and the bound L-Glu) is situated in the hinge region in the ECD-mGluR1α adjacent to the reported Glu-binding site. Mutagenesis studies indicated that binding of L-Glu and Ca2+ to their distinct but partially overlapping binding sites synergistically modulated mGluR1α activation of intracellular Ca2+ ([Ca2+]i) signaling. Mutating the Glu-binding site completely abolished Glu signaling while leaving its Ca2+-sensing capability largely intact. Mutating the predicted Ca2+-binding residues abolished or significantly reduced the sensitivity of mGluR1α not only to [Ca2+]o and [Gd3+]o but also, in some cases, to Glu. In addition, the Ca2+ effects on drugs targeting mGluR1α were investigated. Ca2+ enhances L-Quis response of the receptor by increasing L-Quis binding to ECD-mGluR1α and promotes the potency of Ro 67-4853, a positive allosteric modulator of mGluR1α. Increasing Ca2+ concentration, the inhibitory effects of a competitive antagonist ((s)-MCPG) and a non-competitive negative allosteric modulator (CPCCOEt), were eliminated. Furthermore, we also identified another potential Ca2+ binding pocket (Site 2) consists of S165, D208, Y236 and D318, which completely overlapped with L-Glu. Thapsigargin (TG) induced ER Ca2+ depletion reduced surface expression of mGluR1α, and D208I and Y236I also decreased the receptor trafficking to plasma membrane suggesting the role of Ca2+ binding in protein folding and trafficking in the ER. Further, to measure ER Ca2+, a series of genetically encoded biosensors were designed by placing a Ca2+ binding pocket at the chromophore sensitive region of red florescent protein mCherry. The designed sensors are able to bind Ca2+ and monitor Ca2+ concentration change both in vitro and in cells. The findings in this dissertation open up new avenues for developing allosteric modulators of mGluR function that target related human diseases.

The retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and thalamic relay neurons, undergoes extensive remodeling and refinement in the first few postnatal weeks. While many studies have focused on this process, little is known about the factors that influence excitatory transmission during this dynamic period. A major goal of my dissertation research was to identify mechanisms that regulate glutamate release and clearance at the developing synapse. First, we investigated the role of glutamate transporters and metabotropic glutamate receptors (mGluRs) in shaping excitatory transmission. Early in development, we found presynaptic group II/III mGluRs are present and are activated by glutamate released from RGCs following optic tract stimulation at natural frequencies. This response was found to diminish with age, but glutamate transporters continued to shape synaptic currents throughout development. The finding that glutamate is able to escape the synaptic cleft and bind extrasynaptic high-affinity mGluRs led us to speculate that glutamate might also diffuse to neighboring synapses and bind ionotropic glutamate receptors opposing quiescent release sites. Excitatory currents recorded from immature, but not mature, retinogeniculate synapses display a prolonged decay timecourse. We found evidence that both asynchronous release of glutamate as well as spillover of glutamate between neighboring synapses contributes to these slowly decaying synaptic currents. Furthermore, we uncovered and characterized a novel, purely spillover-mediated current from immature relay neurons, which strongly supports the presence of glutamate spillover between boutons of different RGCs. The results of my studies indicate that far more RGCs contribute to relay neuron firing than would be predicted by the anatomy alone. Finally, in an ongoing study, we investigated the functional role of the neuronal glutamate transporter GLT-1 at the immature retinogeniculate synapse. While GLT-1 has been found in both neurons and glia, excitatory currents at the retinogeniculate synapse were largely unaffected in mice lacking neuronal GLT-1, suggesting non-neuronal glutamate transporters are responsible for the majority of glutamate removal from the developing synapse. Taken together, these results provide insight into the synaptic environment of the developing retinogeniculate synapse and identify a number of mechanisms that shape excitatory transmission during this period of synaptic maturation and refinement.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Sept. 22, 2008). Includes bibliographical references.

Aromatase is an enzyme that converts androgens to estrogens. In teleosts, brain aromatase, also known as aromatase B (cy19a1b), is only expressed in radial glial cells (RGCs). This is in contrast to aromatase A, which is expressed in gonads. Estrogens such as estradiol (E2) modulate neurogenesis in the adult teleost brain. Recent studies show that E2 also differentially regulates aromatase B expression in goldfish RGCs. As a result, teleost RGCs are suggested to be involved in regulating neurogenesis. In addition, aromatase B expression in goldfish RGC is under the control of dopamine suggesting that neurons and neurotransmitters can regulate RGC function. Interestingly, goldfish RGC transcriptome data shows the expression of one group of metabotropic glutamate receptors (mGluRs), group III mGluRs, which suggests that glutamate may affect RGC function. In this thesis, I present my findings regarding potential glutamatergic regulation of RGCs. Firstly, I investigated the distribution of glutamatergic synaptic vesicles and RGCs in the female goldfish forebrain. Double-staining immunohistochemistry shows that vesicular glutamate transporter (vGLUT) 1/2-labelled glutamatergic synaptic vesicles are in close anatomical proximity to aromatase B-labelled RGCs, which suggests potential regulation of RGCs by glutamate. Glutamatergic regulation of cyp19a1b, cyclin D1 (ccnd1), cyclin A2 (ccna2), mGluR6b (grm6b), mGluR7 (grm7), and mGluR8b (grm8b) expression in cultured adult female goldfish RGCs was also examined. Results from pharmacological manipulations and qPCR data analysis show that selective activation of group III mGluRs decreased cyp19a1b, ccnd1, and ccna2 mRNA via inhibition of cAMP/PKA signalling. Furthermore, grm7 mRNA is positively regulated by cAMP-dependent signalling. The glutamate analog L-glutamic acid decreased cyp19a1b mRNA and increased ccnd1 and grm6b mRNA in a dose-dependent manner. This suggests that ccnd1 and grm6b expression may be regulated by glutamate receptors other than group III mGluRs, for example, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are expressed in cultured goldfish RGCs. It was found that E2 upregulated cyp19a1b, ccnd1 and grm7 mRNA. However, selective activation of group III mGluRs decreases the stimulatory effect of E2 on ccnd1 expression. My findings show that glutamate finely regulates RGC neurogenic and steroidogenic genes, which may implicate glutamate in the regulation of RGC differentiation, RGC proliferation, and neurogenesis in surrounding cells.

No description available.

Schizophrenia is a chronic psychotic illness affecting ~21 million people globally. Currently available antipsychotic agents act through a dopamine D2 receptor mechanism, and produce extrapyramidal or metabolic side effects. Hence, there is a need for novel targets and agents. The mGlu2/5-HT2A receptor heteromer has been implicated in the action of antipsychotic agents, and represents a novel and attractive therapeutic target for the treatment of schizophrenia. A long-term goal of this project is to synthesize bivalent ligands where a 5-HT2A receptor antagonist is tethered to an mGlu2 PAM via a linker. The goals of the investigation were to study the SAR of risperidone (an atypical antipsychotic agent) at 5-HT2A receptors using a “deconstruction-reconstruction-elaboration” approach to determine the minimal structural features of risperidone that contribute to its 5-HT2A receptor affinity and antagonism, and to determine where on the “minimized risperidone” structure an mGlu2 PAM can be introduced. Additional goals included studying the binding modes of various mGlu2 PAMs and identifying where on an mGlu2 PAM a risperidone “partial” structure could be introduced. Biological studies of deconstructed/elaborated analogs of risperidone suggest that the entire structure of risperidone is not necessary for 5-HT2A receptor affinity and antagonism, and that a fluoro group contributes to 5-HT2A binding. 6-Fluoro-3-(4-piperidinyl)-1,2-benz[d]isoxazole that has only half the structural features of risperidone retains 5-HT2A receptor affinity and antagonist activity, and represents the “minimized risperidone” structure with the piperidine nitrogen atom representing a potential linker site for eventual construction of bivalent ligands. Molecular modeling studies at 5-HT2A receptors suggest that risperidone and its analogs have more than one binding mode. Modeling studies to evaluate binding modes of various PAMs at mGlu2 receptors, coupled with known SAR information, were used to identify a PAM (JNJ-40411813), and the pyridone nitrogen atom of JNJ-40411813 as a potential linker site. Additionally, potential synthetic routes for JNJ-40411813 were explored that might be of value in the synthesis of bivalent ligands. Based on the structural features of 6-fluoro-3-(4-piperidinyl)-1,2-benz[d]isoxazole, a new pharmacophore for 5-HT2A receptor antagonists, consisting of one aromatic region, a basic protonated amine and hydrogen bond acceptors, has been proposed.

of the thesis Glutamate is a main excitatory neurotransmitter in the brain of mammals, which activates both ionotropic and metabotropic glutamate receptors. Ionotropic receptors are responsible for fast synaptic transmission leading to membrane depolarization and Ca2+ influx into the cell. On the other hand mGlu receptors play an important role in regulation of the transmission via heterotrimeric G-proteins and activation of various signaling pathways. Postsynaptically localized group I mGlu receptors (mGluR1, 5) together with ionotropic NMDA and AMPA receptors share common large receptor signaling complexes, or signalosome facilitating glutamate signal transductions. Individual mGluR1 splice variants are differently associated with signalosome including scaffold proteins like PSD-95 which organize postsynaptic density (PSD). Heterodimerization of different mGluR1 splice variants is a focal point of my thesis together with investigation of recently discovered protein IL1RAPL1 (interleukin-1 receptor accessory protein-like 1) and its role in organization of postsynaptic signalosome. Using biochemical, immunocytochemical and functional assays we showed heterodimers of mGluR1a/1b were expressed on the plasma membrane and that heterodimers are fully functional in the recombinant system. Next we showed...

Os receptores glutamatérgicos metabotrópicos (mGluRs) estão envolvidos nos processos de plasticidade, neurodegeneração e neuroproteção. Avaliamos a expressão de mGluRs no sistema visual de ratos e pintos em diferentes tempos após enucleação ocular. Os animais foram avaliados pelo método de imuno-histoquímica, immunoblotting e RT-PCR, para detecção dos receptores mGluR1,2/3,5 e 7. Foi observado aumento da imunorreatividade (IR) de mGluR1, 5 e 7 no colículo superior, porém não foi observada diferença no núcleo geniculado lateral. Houve aumento na expressão protéica para mGluR1, 5 e 7 e aumento da expressão gênica para mGluR1,5 e 7; por outro lado, ocorreu uma diminuição de mGluR3. No TeO, foi observado aumento da IR para mGluR1 e 5 e diminuição para mGluR2/3. As análises de immunoblotting confirmaram o aumento observado de mGluR1 e diminuição de mGluR2/3. Os resultados indicam uma modulação diferencial na expressão gênica e protéica dos mGluRs, sugerindo a participação desses receptores em processos plásticos decorrentes de lesões no sistema visual adulto. / The metabotropic glutamate receptors (mGluRs) are involved in neuronal plasticity and neuroprotection. We analyzed the expression of mGluRs in the visual system of rats and chicks in several periods after ocular enucleation. The localization and expression of mGluR1, 5, 2/3 and 7 receptors were evaluated by standard immunoperoxidase, immunoblotting and real-time PCR protocols. The immunorreativity, protein and gene expression of mGluR1, 5 and 7 receptors in the superior colliculus showed an increase, whereas no changes were seen in the lateral geniculate nucleus. For mGluR3, gene expression was decreased. In the TeO, mGluR1 and 5 increased for all survival periods analyzed. Immunoblotting analyses confirmed the increases for mGluR1 and 5, decreases for mGluR2/3. These results indicate that the expression of mGluRs is regulated by the glutamatergic retinal input, and add data on a possible role of these receptors in neuroplasticity in adult animals.