Simulation of Postsynaptic Glutamate Receptors Reveals Critical Features of Glutamatergic Transmission

Activation of several subtypes of glutamate receptors contributes to changes in postsynaptic calcium concentration at hippocampal synapses, resulting in various types of changes in synaptic strength. Thus, while activation of NMDA receptors has been shown to be critical for long-term potentiation (LTP) and long term depression (LTD) of synaptic transmission, activation of metabotropic glutamate receptors (mGluRs) has been linked to either LTP or LTD. While it is generally admitted that dynamic changes in postsynaptic calcium concentration represent the critical elements to determine the direction and amplitude of the changes in synaptic strength, it has been difficult to quantitatively estimate the relative contribution of the different types of glutamate receptors to these changes under different experimental conditions. Here we present a detailed model of a postsynaptic glutamatergic synapse that incorporates ionotropic and mGluR type I receptors, and we use this model to determine the role of the different receptors to the dynamics of postsynaptic calcium with different patterns of presynaptic activation. Our modeling framework includes glutamate vesicular release and diffusion in the cleft and a glutamate transporter that modulates extracellular glutamate concentration. Our results indicate that the contribution of mGluRs to changes in postsynaptic calcium concentration is minimal under basal stimulation conditions and becomes apparent only at high frequency of stimulation. Furthermore, the location of mGluRs in the postsynaptic membrane is also a critical factor, as activation of distant receptors contributes significantly less to calcium dynamics than more centrally located ones. These results confirm the important role of glutamate transporters and of the localization of mGluRs in postsynaptic sites in their signaling properties, and further strengthen the notion that mGluR activation significantly contributes to postsynaptic calcium dynamics only following high-frequency stimulation. They also provide a new tool to analyze the interactions between metabotropic and ionotropic glutamate receptors

Événements moléculaires et cellulaires associés à l'épileptogenèse dans deux modèles murins d'injection intrahippocampique de toxiques. Implication des mécanismes neuro-inflammatoires

Epileptogenesis, which leads to the occurrence of sponteanous electroencephalographic fits that are a key feature of epilepsy, arises from mostly unknown and probably diverse mechanisms. Recent studies stress that cerebral inflammation may be involved in the early events of epileptogenesis. The mesiotemporal lobe epilepsy (MTLE) syndrome is often associated to a unilateral hippocampal neuronal loss stressing that hippocampus is a particularly sensitive region of the brain. Our work focused on epileptogenesis that follows the intrahippocampal injection of chemical toxicants that are able to induce local tissular and cellular modifications in C57BL/6 mice. The potential role of neuro-inflammation was of particular interest. In our first model, a cholinergic imbalance was created by the injection of soman, a potent cholinesterase inhibitor. It was able to induce epileptogenesis without an initial status epilepticus and a deficit in the contextual fear conditioning test. Interestingly no major tissular changes (neuronal damage, edema and neuro-inflammation) could be observed. Conversely, in the murine MLTE model obtained with the injection of kainate, a strong microglial reaction could quickly be observed in the areas showing neuronal damage. Astrogliosis was also present. Thanks to reverse transcription quantitative polymerase chain reaction (RT-qPCR), we could show that changes in mRNA levels of some neuroinflammatory mediators (especially d'IL-1β) were related to the time course of neuronal degenerescence in areas showing damage. For the RT-qPCR data, from samples obtained from these regions, to be reliably considered, appropriate sets of reference genese have to be determined. We showed that a group of five genes (Hprt1, Ppia, Tbp, Actb et Arbp) were highly stable in the hippocampus of mice experiencing MTLE and could be used to normalize raw data from RT-qPCR in this model. All in all, ours results stress the fact that, in mice, neurochemical modifications are able to induce epileptogenesis even when no tissular lesions could be noticed and that massive neuro-inflammation is not a sine qua non condition. To determine if neuro-inflammation is beneficial or deleterious is still a major research thrust as this may lead to the discovery of new antiepileptogenic drugs.Le processus d'épileptogenèse, qui conduit à la production de décharges électro-encéphalographiques spontanées caractéristiques de la maladie épileptique, implique des mécanismes inconnus pour la plupart et probablement divers. Des études récentes soulignent le rôle potentiel de l'inflammation cérébrale dans les mécanismes précoces de l'épileptogenèse. Le syndrome d'épilepsie de la face mésiale du lobe temporal (EMLT) est souvent associé à une perte neuronale unilatérale et sélective dans l'hippocampe, suggérant que cette structure est particulièrement sensible. Notre travail a donc été consacré à l'étude de l'épileptogenèse faisant suite à l'injection intrahippocampique de toxiques chimiques capables d'entraîner des modifications tissulaires et cellulaires locales chez la souris C57BL/6. Le rôle potentiel de la neuro-inflammation a été plus particulièrement recherché. Dans notre premier modèle, un déséquilibre cholinergique est induit par l'injection de soman, un puissant inhibiteur des cholinestérases. Il est capable d'induire un processus d'épileptogenèse sans état de mal initial, associé à un déficit de la réponse émotionnelle conditionnée contextuelle mais en l'absence de remaniements tissulaires majeurs (neurodégénérescence, œdème et neuro-inflammation). En revanche, dans le modèle d'EMLT obtenu par l'injection de kaïnate, une forte activation microgliale est détectée précocement dans les zones de neurodégénérescence. Une astrogliose est également observée. Grâce à la technique quantitative de transcription inverse suivie de polymérisation en chaîne (RT-qPCR), nous avons également pu mettre en évidence que certains médiateurs moléculaires de l'inflammation sont également liés dans le temps et dans l'espace (particulièrement la transcription d'IL-1β) aux événements neurodégénératifs. Pour s'assurer de la fiabilité des données analytiques de RT-qPCR dans les régions cérébrales touchées par ces modifications, le choix des gènes de référence doit faire l'objet d'études spécifiques. Nous avons pu montrer qu'un ensemble de cinq gènes (Hprt1, Ppia, Tbp, Actb et Arbp) avait une forte stabilité dans la structure hippocampique dans ce modèle murin d'EMLT et pouvait être utilisé pour normaliser les données brutes de RT-qPCR dans ce modèle. Nos résultats indiquent que, chez la souris, des altérations neurochimiques sont capables d'initier l'épileptogenèse même en l'absence de lésions tissulaires notables et qu'une réponse neuro-inflammatoire massive n'est pas une condition sine qua non. Déterminer le caractère bénéfique ou délétère de la neuro-inflammation reste un enjeu majeur pour la découverte de nouvelles thérapeutiques anti-épileptogènes

Evénements moléculaires et cellulaires associés à l'épileptogenèse dans deux modèles murins d'injection intrahippocampique de toxiques (implication des mécanismes neuro-inflammatoires)

Le processus d'épileptogenèse, qui conduit à la production de décharges électro-encéphalographiques spontanées caractéristiques de la maladie épileptique, implique des mécanismes inconnus pour la plupart et probablement divers. Des études récentes soulignent le rôle potentiel de l'inflammation cérébrale dans les mécanismes précoces de l'épileptogenèse. Le syndrome d'épilepsie de la face mésiale du lobe temporal (EMLT) est souvent associé à une perte neuronale unilatérale et sélective dans l'hippocampe, suggérant que cette structure est particulièrement sensible. Notre travail a donc été consacré à l'étude de l'épileptogenèse faisant suite à l'injection intrahippocampique de toxiques chimiques capables d'entraîner des modifications tissulaires et cellulaires locales chez la souris C57BL/6. Le rôle potentiel de la neuro-inflammation a été plus particulièrement recherché. Dans notre premier modèle, un déséquilibre cholinergique est induit par l'injection de soman, un puissant inhibiteur des cholinestérases. Il est capable d'induire un processus d'épileptogenèse sans état de mal initial, associé à un déficit de la réponse émotionnelle conditionnée contextuelle mais en l'absence de remaniements tissulaires majeurs (neurodégénérescence, œdème et neuro-inflammation). En revanche, dans le modèle d'EMLT obtenu par l'injection de kaïnate, une forte activation microgliale est détectée précocement dans les zones de neurodégénérescence. Une astrogliose est également observée. Grâce à la technique quantitative de transcription inverse suivie de polymérisation en chaîne (RT-qPCR), nous avons également pu mettre en évidence que certains médiateurs moléculaires de l'inflammation sont également liés dans le temps et dans l'espace (particulièrement la transcription d'IL-1b) aux événements neurodégénératifs. Pour s'assurer de la fiabilité des données analytiques de RT-qPCR dans les régions cérébrales touchées par ces modifications, le choix des gènes de référence doit faire l'objet d'études spécifiques. Nous avons pu montrer qu'un ensemble de cinq gènes (Hprt1, Ppia, Tbp, Actb et Arbp) avait une forte stabilité dans la structure hippocampique dans ce modèle murin d'EMLT et pouvait être utilisé pour normaliser les données brutes de RT-qPCR dans ce modèle. Nos résultats indiquent que, chez la souris, des altérations neurochimiques sont capables d'initier l'épileptogenèse même en l'absence de lésions tissulaires notables et qu'une réponse neuro-inflammatoire massive n'est pas une condition sine qua non. Déterminer le caractère bénéfique ou délétère de la neuro-inflammation reste un enjeu majeur pour la découverte de nouvelles thérapeutiques anti-épileptogènes.Epileptogenesis, which leads to the occurrence of sponteanous electroencephalographic fits that are a key feature of epilepsy, arises from mostly unknown and probably diverse mechanisms. Recent studies stress that cerebral inflammation may be involved in the early events of epileptogenesis. The mesiotemporal lobe epilepsy (MTLE) syndrome is often associated to a unilateral hippocampal neuronal loss stressing that hippocampus is a particularly sensitive region of the brain. Our work focused on epileptogenesis that follows the intrahippocampal injection of chemical toxicants that are able to induce local tissular and cellular modifications in C57BL/6 mice. The potential role of neuro-inflammation was of particular interest. In our first model, a cholinergic imbalance was created by the injection of soman, a potent cholinesterase inhibitor. It was able to induce epileptogenesis without an initial status epilepticus and a deficit in the contextual fear conditioning test. Interestingly no major tissular changes (neuronal damage, edema and neuro-inflammation) could be observed. Conversely, in the murine MLTE model obtained with the injection of kainate, a strong microglial reaction could quickly be observed in the areas showing neuronal damage. Astrogliosis was also present. Thanks to reverse transcription quantitative polymerase chain reaction (RT-qPCR), we could show that changes in mRNA levels of some neuroinflammatory mediators (especially d'IL-1b) were related to the time course of neuronal degenerescence in areas showing damage. For the RT-qPCR data, from samples obtained from these regions, to be reliably considered, appropriate sets of reference genese have to be determined. We showed that a group of five genes (Hprt1, Ppia, Tbp, Actb et Arbp) were highly stable in the hippocampus of mice experiencing MTLE and could be used to normalize raw data from RT-qPCR in this model. All in all, ours results stress the fact that, in mice, neurochemical modifications are able to induce epileptogenesis even when no tissular lesions could be noticed and that massive neuro-inflammation is not a sine qua non condition. To determine if neuro-inflammation is beneficial or deleterious is still a major research thrust as this may lead to the discovery of new antiepileptogenic drugs.GRENOBLE1-BU Sciences (384212103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude de la réaction inflammatoire cérébrale au cours de l'épileptogenèse dans un modèle murin d'épilepsie mésio-temporale

DIJON-BU Médecine Pharmacie (212312103) / SudocSudocFranceF

Selection of reference genes for real-time quantitative reverse transcription-polymerase chain reaction in hippocampal structure in a murine model of temporal lobe epilepsy with focal seizures.

International audienceReference genes are often used to normalize expression of data from real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and only a validation of their stability during a given experimental paradigm leads to reliable interpretations. The present study was thus designed to validate potential reference genes in a mouse model of mesiotemporal lobe epilepsy (MTLE) with focal seizures after unilateral intrahippocampal injection of kainate (KA). Ipsilateral and contralateral hippocampi were removed during nonconvulsive status epilepticus (5 hr), epileptogenesis (7 days), and the chronic period of recurrent focal seizures (21 days). Naive animals were equally studied. The stability of eight potential reference genes (hypoxanthine phosphoribosyltransferase, Hprt1; peptidylprolyl isomerase A, Ppia; TATA box binding protein, Tbp; beta-actin, Actb; acidic ribosomal phosphoprotein P0, Arbp; glyceraldehyde-3-phosphate dehydrogenase, Gapdh; ribosomal RNA 18S, 18S rRNA; and glucuronidase beta, Gusb) were determined using geNorm and NormFinder software. The first five (Hprt1, Ppia, Tbp, Actb, and Arbp) were found to be stable across the different phases of the disease and appeared adequate for normalizing RT-qPCR data in this model. This was in contrast to the other three (18S rRNA, Gapdh, and Gusb), which showed unstable expressions and should be avoided. The analysis of KA-induced changes in the expression of glial fibrillary acidic protein (Gfap) gene resulted in various relative expressions or even a completely different pattern when unstable reference genes were used. These results highlight the absolute need to validate the reference genes for a correct interpretation of mRNA quantification

Null Variance Altitudes for the photolysis rate constants of species with barometric distribution: Illustration on Titan upper atmosphere modeling

International audienceThe precision of the rates of the photolysis processes initiating the complex chemistry of Titan's upper atmosphere conditions strongly the predictivity of photochemical models. Recent studies in sensitivity analysis of such models point out photolysis rate constants as key parameters. However, they have been treated approximately so far. We deal here directly with uncertainty in the absorption cross-sections to derive the uncertain altitude-dependent photolysis rate constants. We observe that the uncertainty on the photolysis rate constants of the major species, N2 and CH4, varies strongly with altitude and rather surprisingly vanishes at specific altitudes. We propose a simple model to interpret these features and we demonstrate that they are transferable to any major absorber distributed barometrically in an atmosphere

Energetics of Photoinduced Charge Migration within the Tryptophan Tetrad of an Animal (6–4) Photolyase

International audienceCryptochromes and photolyases are flavoproteins that undergo cascades of electron/hole transfers after excitation of the flavin cofactor. It was recently discovered that animal (6–4) photolyases, as well as animal cryptochromes, feature a chain of four tryptophan residues, while other members of the family contain merely a tryptophan triad. Transient absorption spectroscopy measurements on Xenopus laevis (6–4) photolyase have shown that the fourth residue is effectively involved in photoreduction but at the same time could not unequivocally ascertain the final redox state of this residue. In this article, polarizable molecular dynamics simulations and constrained density functional theory calculations are carried out to reveal the energetics of charge migration along the tryptophan tetrad. Migration toward the fourth tryptophan is found to be thermodynamically favorable. Electron transfer mechanisms are sought either through an incoherent hopping mechanism or through a multiple sites tunneling process. The Jortner–Bixon formulation of electron transfer (ET) theory is employed to characterize the hopping mechanism. The interplay between electron transfer and relaxation of protein and solvent is analyzed in detail. Our simulations confirm that ET in (6–4) photolyase proceeds out of equilibrium. Multiple site tunneling is modeled with the recently proposed flickering resonance mechanism. Given the position of energy levels and the distribution of electronic coupling values, tunneling over three tryptophan residues may become competitive in some cases, although a hopping mechanism is likely to be the dominant channel. For both reactive channels, computed rates are very sensitive to the starting protein configuration, suggesting that both can take place and eventually be mixed, depending on the state of the system when photoexcitation takes place

Statistical Analysis of Ion Mobility Spectrometry. II. Adaptively Biased Methods and Shape Correlations

Following a recent effort [J. Am. Soc. Mass Spectrom. 23, 386-396 (2012)], we continue to explore computational methodologies for generating molecular conformations to support collisional cross sections suggested by ion mobility measurements. Here, adaptively biased molecular dynamics (ABMD) simulations are used to sample the configuration space and to achieve flat-histogram sampling along the reaction coordinates of the first two moments of the gyration tensor. The method is tested and compared with replica-exchange simulations on triply-protonated bradykinin and on a larger 25-residue peptide. It is found to have a much higher efficiency for producing large sets of conformations in a broad range of diffusion cross-sections, whereas it does not compete with conventional replica-exchange molecular dynamics in locating the lowest-energy structure. Nevertheless, the broad sampling obtained from the ABMD method allows to quantitatively correlate the diffusion cross-section Omega with other geometric order parameters that have simpler interpretation. The strong correlations found between the diffusion cross-section and the radius of gyration, the surface area and the volume of the convex hull suggest an optimal template for accurately mimicking the variations of Omega in a broad range of conformations, using only geometrical information and doing so at a very moderate computational cost. The existence of such a correlation is confirmed on the much larger protein alpha-lactalbumin