The pedunculopontine tegmental (PPT) nucleus is a brainstem structure thought to be important in the regulation of sleep/wake states. The PPT is comprised of three distinct types of neurons (cholinergic, GABAergic, and glutamatergic), and each may serve different functions. It remains unknown how PPT neurons affect specific sleep/wake states and which of their axonal projections mediate their effect. Therefore, we used optogenetics to selectively activate glutamatergic PPT (PPTglut) neurons at both the cell soma and axon terminals in a temporally and spatially precise manner. The purpose of these experiments was to determine the role of PPTglut neurons during wake, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep, and to identify the key projections through which PPTglut neurons produce their effects. Using transgenic mice, we transfected PPTglut neurons with an adeno-associated viral vector to induce expression of a light-dependent ion channel, channelrhodopsin-2 (ChR2). While recording electroencephalography (EEG), electromyography (EMG), and video feed, we photostimulated the transfected PPTglut neurons and measured the effects on sleep/wake states of the mice. Stimulation of the PPTglut soma during NREM sleep produced a frequency-dependent wake response. With increasing frequency of stimulation, we observed an increase in the speed of the wake response, as well as the amplitude and duration of the wake response. Stimulating the PPTglut soma increased time spent in wake, decreased NREM sleep, and slightly decreased REM sleep. We also noticed that mice did not exhibit spontaneous body movements during stimulation of the PPTglut soma. Stimulating individual PPTglut axon terminal fields partially recapitulated the phenotype observed with stimulation of the cell soma. Photostimulation of axon terminals in the basal forebrain, lateral hypothalamus, and thalamus elicited a fast wake response, stimulation of both the basal forebrain and lateral hypothalamus terminal fields produced a strong wake response, but long-lasting wakefulness was observed only with high-frequency stimulation of axon terminals in the lateral hypothalamus. In summary, photostimulation of PPTglut neurons promotes wake, and slightly decreases REM sleep. Our experiments strongly support the role of PPTglut neurons in promoting wakefulness from NREM sleep, and this wake response is carried out through several axonal projections which, in sum, recapitulate the wake phenotype observed with stimulation of the cell soma in the PPT itself. Further exploration of the axonal projections of PPTglut neurons is warranted to elucidate the neuronal targets through which this response is carried out. / 2019-07-03T00:00:00Z

O floroglucinol é um composto fenólico precursor de diversas moléculas com atividades biológicas já descritas na literatura, com destaque para a antidepressiva. O modelo experimental com zebrafish tem sido bastante utilizado em várias linhas de pesquisa biológica, como, por exemplo, para avaliação da atividade anticonvulsivante. A partir de estudos que mostram uma correlação entre compostos antidepressivos e anticonvulsivantes, nosso grupo administrou floroglucinol e dois derivados sintéticos (composto 7 e composto 8) em zebrafish para observação da atividade locomotora e exploratória no open tank e, posteriormente, à avaliação através do modelo convulsivo induzido por pentilenotetrazol (PTZ). Além disso, os níveis de captação de glutamato e a toxicidade dos compostos foram avaliados em cérebro total de zebrafish. O comportamento dos animais não sofreu alteração em relação ao controle para nenhum dos compostos testados. O composto 7 aumentou significativamente o tempo para os animais atingirem a primeira convulsão além de reduzir a intenidade da crise convulsiva. Também se observou aumento na captação de glutamato para esse composo, sem sinais de toxicidade envolvidos. Desta forma, nossos resultados contribuem para a busca de compostos potencialmente ativos frente a crises convulsivas induzidas por PTZ. / Phloroglucinol, a phenolic compound, which is precursor of several molecules with biological activities are described in the literature, mainly for antidepressant activity. The zebrafish experimental model has been widely used in many kinds of biological research, for example, to evaluate the anticonvulsant activity. From studies that shows correlation between antidepressants and anticonvulsant compounds, our group managed phloroglucinol and two synthetic derivatives (compound 7 and compound 8) in zebrafish in order to observe the locomotor and exploratory activity on open tank and subsequently, conduct the evaluation through the seizure model induced by pentylenetetrazol (PTZ). Furthermore, glutamate uptake and toxicity levels of the compounds were evaluated in zebrafish’s whole brain. The animals' behavior did not change compared to control for any of the tested compounds. The compound 7 increased significantly the time for the animals reach the first seizure and reduce the seizure intensity. It was also observed an increase in glutamate uptake for this compound without signs of toxicity involved. Thus, our results contribute to the search for potentially active compounds against seizures induced by PTZ.

O floroglucinol é um composto fenólico precursor de diversas moléculas com atividades biológicas já descritas na literatura, com destaque para a antidepressiva. O modelo experimental com zebrafish tem sido bastante utilizado em várias linhas de pesquisa biológica, como, por exemplo, para avaliação da atividade anticonvulsivante. A partir de estudos que mostram uma correlação entre compostos antidepressivos e anticonvulsivantes, nosso grupo administrou floroglucinol e dois derivados sintéticos (composto 7 e composto 8) em zebrafish para observação da atividade locomotora e exploratória no open tank e, posteriormente, à avaliação através do modelo convulsivo induzido por pentilenotetrazol (PTZ). Além disso, os níveis de captação de glutamato e a toxicidade dos compostos foram avaliados em cérebro total de zebrafish. O comportamento dos animais não sofreu alteração em relação ao controle para nenhum dos compostos testados. O composto 7 aumentou significativamente o tempo para os animais atingirem a primeira convulsão além de reduzir a intenidade da crise convulsiva. Também se observou aumento na captação de glutamato para esse composo, sem sinais de toxicidade envolvidos. Desta forma, nossos resultados contribuem para a busca de compostos potencialmente ativos frente a crises convulsivas induzidas por PTZ. / Phloroglucinol, a phenolic compound, which is precursor of several molecules with biological activities are described in the literature, mainly for antidepressant activity. The zebrafish experimental model has been widely used in many kinds of biological research, for example, to evaluate the anticonvulsant activity. From studies that shows correlation between antidepressants and anticonvulsant compounds, our group managed phloroglucinol and two synthetic derivatives (compound 7 and compound 8) in zebrafish in order to observe the locomotor and exploratory activity on open tank and subsequently, conduct the evaluation through the seizure model induced by pentylenetetrazol (PTZ). Furthermore, glutamate uptake and toxicity levels of the compounds were evaluated in zebrafish’s whole brain. The animals' behavior did not change compared to control for any of the tested compounds. The compound 7 increased significantly the time for the animals reach the first seizure and reduce the seizure intensity. It was also observed an increase in glutamate uptake for this compound without signs of toxicity involved. Thus, our results contribute to the search for potentially active compounds against seizures induced by PTZ.

Glutamate dehydrogenase a (GDHa) has been suggested as a potential drug target against the malaria parasite Plasmodium falciparum. GDHa knockout cell line was generated and needed a phenotypic description by means of molecular biology and biochemistry. The knockout cell line was tested for higher oxidative stress sensitivity, levels of relevant proteins and gene transcripts were quantified. Furthermore, concentrations of two key molecules enabling redox homeostasis, glutathione and NADPH, were attempted to quantify. Finally, we attempted to disrupt a gene of another glutamate dehydrogenase, gdhb, which did not result in formation of viable parasites. In conclusion, GDHa is not a suitable drug target and GDHb needs to be further elucidated.

No description available.

Chez les mammifères, le glutamate est un neuromédiateur bien connu au niveau du système nerveux central et plus récemment un rôle immunomodulateur lui a été reconnu. Le glutamate est le ligand de récepteurs ionotopiques (iGluRs) qui sont des récepteurs-canaux perméables à divers cations dont le calcium (non-selective cation channels, NSCC). Chez Arabidopsis thaliana, une famille de 20 gènes de iGluRs homologues des iGluRs de mammifères a été identifiée et leur implication dans divers processus biologiques est suggérée. Dans ce travail où nous utilisons des suspensions de cellules de tabac (Nicotiana tabacum var Xanthi), divers arguments suggèrent que ces iGluR sont fonctionnels dans le tabac : influx de calcium et élévation rapide et transitoire de la concentration en calcium cytosolic libre en réponse à l’addition de glutamate, inhibition de ces effets par 4 antagonistes de iGluRs animaux (compétitifs ou non compétitifs), désensibilisation, et pH dépendance des effets. Pour la première fois chez les plantes nous montrons que le glutamate induit la production de NO très vraisemblablement via l’activation de iGluRs. De plus, nous démontrons que ce(s) iGluRs sont impliqués dans le mode d’action, via les flux de calcium, de la cryptogéine une protéine de 10 kDa de Phytophthora cryptogea, éliciteur des réactions de défense chez le tabac. Néanmoins, à ce niveau, les iGluRs ne sont pas impliqués dans la plupart des événements calcium-dépendants induits par la cryptogéine dont l’activation des MAPKs et de canaux anioniques, la production de H2O2 (activation de la NADPH-oxydase) et la réponse hypersensible. En revanche, ils sont tout ou partiellement responsables de la production de NO décrite pour la première fois par le passé en réponse à la cryptogéine. Ces résultats suggèrent que différents types de canaux calciques activés par divers médiateurs, génèrent, via le calcium, des messages spécifiques décodés par des protéines associées à chacun de ces types de canaux et impliquées dans des réponses biologiques différentes. Dans le mode d’action de la cryptogéine, nous démontrons que l’activation des iGluRs est possible grâce à l’exocytose de glutamate dans l’apoplaste, induite par la cryptogéine. Ainsi, ce travail est la première démonstration du rôle de iGluRs potentiels dans la défense chez les plantes et de leur implication dans la production de NO. Nos résultats sont un argument supplémentaire à la conservation des mécanismes de la défense dans le monde vivant et posent le problème du rôle du glutamate dans la signalisation chez les plantes. / Glutamate is recognized as the primary excitatory neurotransmitter in the mammalian central nervous system (CNS) but recent studies have shown that glutamate has an important additional immunomodulator role. Glutamate is the ligand of ionotropic glutamate receptors (iGluRs), which are non-selective cation channels (NSCC), permeable to calcium. In plants, animal iGluR homologs were found that were involved in many developmental processes. Here we demonstrate the involvement of putative iGluRs in calcium signalling in response to cryptogein which is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea and is an elicitor of defence in tobacco. Using transformed tobacco cell suspensions expressing aequorin in the cytosol or in the nucleus, our results have shown that glutamate induces a strong and transient [Ca2+]cyt elevation without [Ca2+]nuc changes. Glutamate-induced [Ca2+]cyt elevation was a result of calcium influx from the extracellular medium and was inhibited by different GluR inhibitors. This data suggest the presence of functional calcium channels of GluRs-type in tobacco. Nevertheless, glutamate does not induce some of the calcium-dependent characteristic events of the defence pathways, which are H2O2 production, MAPK activation and hypersensitive response, but promoted NO production. Further, Ca2+ influx,[Ca2+]cyt elevation and NO production induced by cryptogein were shown to be partially inhibited by the glutamate receptor inhibitors, suggesting that cryptogein treatment could activate a calcium channel of the GluR-type leading to plant defense signalling through NO production. We have also demonstrated that cryptogein induces an efflux of glutamate in the apoplast by the process of exocytosis thus activating the GluRs in tobacco. This is the first demonstration for a potential GluR(s) involvement in plant defense signalling, furthermore by mechanisms that showed homology with glutamate effect on neuronal cells.

No description available.

Le glutamate est le principal neurotransmetteur excitateur du système nerveux central des vertébrés, et le codage de l’information cérébrale repose en partie sur des modulations de l’amplitude et de la fréquence des transmissions synaptiques glutamatergiques. De ce fait, la résolution spatiale et temporelle de ces transmissions nécessite un contrôle fin de la présence de glutamate dans la fente synaptique. Cette durée de vie du glutamate dans les synapses dépend directement de l’action de transporteurs spécifiques exprimés à la surface des astrocytes, en particulier les transporteurs de type GLT-1, qui retirent le neurotransmetteur et permettent ainsi de « nettoyer » la fente synaptique avant la survenue d’un nouvel épisode de neurotransmission. / A classic understanding of neurotransmitter clearance at glutamatergic synapses is that, in order to ensure sufficient glutamate uptake on a fast timescale, it is necessary to have high numbers of glutamate transporters in the vicinity of release sites to compensate for their slow transport kinetics. Using a combination of single molecule imaging and electrophysiological approaches, we now challenge this view by first demonstrating that GLT-1 transporters are not static but highly mobile at the surface of astrocytes, and that their surface diffusion is dependent upon both neuronal and glial cell activities. In the vicinity of glutamate synapses, GLT-1 dynamics are strongly reduced favoring their retention within this strategic location. Remarkably, glutamate uncaging at synaptic sites instantaneously increases GLT-1 diffusion, displacing the glutamate-bound transporter away from this compartment. Functionally, impairment of the transporter lateral diffusion through an antibody-based surface cross linking, both in vitro and in vivo, significantly slows the kinetics of excitatory postsynaptic currents. Taken together, these data reveal the unexpected and major role of the astrocytic surface GLT-1 fast dynamics in shaping glutamatergic synaptic transmission.Keywords:

No description available.

The symbiotic relationship between cnidarians and their photosynthetic dinoflagellate symbionts underpins the success of coral reef communities in oligotrophic, tropical seas. Despite several decades of study, the cellular and molecular mechanisms that regulate the symbiotic relationship between the dinoflagellate algae and the coral hosts are still not clear. One of the hypotheses on the metabolic interactions between the host and the symbiont suggests that ammonium assimilation by the host can be the underlying mechanism of this endosymbiosis regulation. An essential intermediate of the ammonium assimilation pathway is glutamate, which is also known for its glutamatergic signaling function. Interestingly, recent transcriptomic level and DNA methylation studies on sea anemone Aiptasia showed differences in metabotropic glutamate signaling components when comparing symbiotic and non-symbiotic animals. The changes in this process on transcriptional and epigenetic levels indicate the importance of glutamate signaling in regard to cnidarian symbiosis. In this study, I tested glutamatergic signaling effect on symbiosis in sea anemone Aiptasia using a broad-spectrum glutamate receptor inhibitor 7- CKA and glutamate. Significantly decreased cell density was observed in animals with inhibitor treatment suggesting a possible correlation between glutamate signaling and the establishment or maintenance of symbiosis. Using RNA-Seq, I was able to obtain transcriptional profiles of the animals under inhibitor and glutamate treatment. Differential gene expression and gene ontology analyses indicated changes in amino acid metabolism, lipid metabolism and such signaling pathways as MAPK, NF-kappa B and phospholipase C. Although amino acid and lipid metabolism could be a result of the reduced symbiotic state of inhibitor treated Aiptasia, the signaling pathways which are related to apoptosis and immune response provide an exciting venue for direct regulatory interaction between symbiosis and glutamatergic signaling. However, as these signaling pathways mainly act via signal transduction through protein phosphorylation, further studies looking at changes on a post-translational level might provide further insight into the mechanisms underlying the observed phenotype.