Les récepteurs du glutamate de type AMPA (rAMPA) sont les acteurs principaux de la transmission synaptique excitatrice rapide. Leur abondance au niveau de la densité postsynaptique est essentielle pour l'établissement et le maintien de la fonction synaptique, et est le résultat d'un trafic hautement dynamique. De nombreuses études ont permis de caractériser les mécanismes de diffusion membranaire impliqués dans l’adressage des rAMPA jusqu’à la synapse. Le rôle majeur des protéines auxiliaires des rAMPA dans la modulation de cette étape de trafic a été démontré. Par ailleurs, il est suggéré que la localisation synaptique des rAMPA est aussi régulée lors des phases plus précoces du trafic intracellulaire, c’est-à-dire de l'appareil de Golgi vers la membrane plasmique via les vésicules post-Golgiennes. Cependant le trafic vésiculaire post-Golgien des rAMPA n'a jamais été visualisé et reste donc encore très mal compris. En collaboration avec l'équipe de Guus Smit (Amsterdam), j’ai participé à la caractérisation d’une nouvelle protéine auxiliaire des rAMPA, appelée Shisa6. Dans le cadre de ce projet, j’ai pu étudier le rôle de cette protéine sur la diffusion membranaire des rAMPA en utilisant une technique de suivi de particule unique (Quantum dot) développée au laboratoire. Mon projet de thèse principal a consisté à étudier le trafic vésiculaire post-Golgien des rAMPA par le développement d’une nouvelle méthode d’étude. En effet, l'échec dans la visualisation dynamique du trafic vésiculaire des récepteurs pourrait être expliqué par un faible rapport signal/bruit, conséquence d'une faible concentration vésiculaire en rAMPA combinée à un bruit de fond important dû aux marquages provenant du réticulum endoplasmique (RE) et de la membrane plasmique. Dans le but de surpasser cette difficulté, nous avons mis au point un outil ingénieux (système ARIAD) afin de bloquer les rAMPA dans le RE et contrôler, par l'ajout d'un ligand, leur sécrétion du RE jusqu'à la membrane plasmique. Grâce à cet outil, nous avons non seulement augmenté considérablement la concentration des rAMPA dans les vésicules post-Golgiennes, mais aussi éliminé le bruit de fond membranaire. Par la technique de FRAP nous avons pu éliminer le bruit de fond provenant du RE. Une telle approche, combinée à des techniques d'imagerie sur neurones vivants, nous a permis de visualiser pour la première fois le trafic vésiculaire post-Golgien des rAMPA et de l’étudier. / AMPA-type glutamate receptors (AMPAR) are the main actors of the fast excitatory synaptic transmission. Their abundance at the postsynaptic density is essential for the establishment and maintenance of synaptic function, and is the result of a highly dynamic trafficking. Many studies have characterized the membrane diffusion mechanisms involved in the AMPAR synaptic localization, and revealed the critical role of the AMPAR auxiliary proteins in the modulation of this trafficking. Furthermore, it is suggested that AMPAR synaptic localization is also regulated during the early steps of the intracellular trafficking, from the Golgi apparatus to the plasma membrane via the post-Golgi vesicles. However, the post-Golgi vesicular trafficking of AMPAR has never been visualized and therefore remains poorly understood. In collaboration with the Guus Smit team (Amsterdam), I participated in the caracterization of a novel AMPAR auxiliary protein called Shisa6. As part of this project, I studied the role of this protein on the AMPAR membrane diffusion, using a method of single particle tracking (Quantum dot) developed in the laboratory. My main thesis project was to study the post-Golgi vesicular trafficking of AMPAR through the development of a new experimental protocol. Indeed, the failure in the dynamic visualization of the receptor vesicular trafficking could be explained by a low signal/noise ratio resulting of a poor AMPAR vesicular concentration, combined with a high background noise due to receptors localized both in the endoplasmic reticulum (ER) and at the plasma membrane. In order to overcome this difficulty, we have used an ingenious tool (ARIAD system) so as to block AMPAR into the ER and, by adding a ligand, control their trafficking from the ER to the plasma membrane. Thanks to this tool we have not only significantly increased the AMPAR concentration in the post-Golgi vesicles, but also eliminated the plasma membrane background noise. The FRAP imaging technique was used in order to remove the ER background noise. Such methodological approach combined with imaging techniques in living neurons, allowed us to clearly visualize for the first time the post-Golgi vesicular trafficking of AMPAR, and to study the mechanisms involved in this trafficking.

The excitatory, ionotropic glutamatergic AMPA receptor is the most common membrane-bound receptor in the central nervous system. AMPARs and the NMDA receptors are central to synaptic plasticity, memory, and mechanisms of neurodegeneration. The AMPAR is an obligate heterotetramer, composed of subunits GluA1-4. Subunit permutation determines ion conductance, trafficking and other functional characteristics. Few available antibodies are subunit-specific, disabling researchers from accurately visualizing differential AMPAR subunit distribution in the nervous system. This study sought to visualize a novel monoclonal GluA1/2/3 antibody with functional avidity for three of four receptor subunits and to characterize the ultrastructural localization of these receptors using confocal and electron microscopy.

During the critical period of CNS development, dendritic architecture is shaped, in part, by activity-dependent stabilization and elimination of branches. This restructuring is partly dependent on the subunit composition of glutamate receptors in a manner that is both regionally specific and temporally regulated. We used primary cultures of rodent hippocampal neurons to investigate the consequences for hippocampal dendrite development when the glutamate ?-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) subunit composition was altered. Overexpression of the AMPAR subunits GluR1 or GluR2 differentially modified hippocampal dendrite architecture. We investigated signaling pathways known to be involved in activity-dependent circuit development as possible downstream effectors of AMPA-mediated morphogenesis. We identified extracellular signal regulated kinase (ERK) 1/2 as a potential candidate of GluR1-mediated dendrite outgrowth. We found that levels of docosahexaenoic acid (DHA) and a DHA-derived bioactive metabolite, neuroprotectin D1 (NPD1) are differentially regulated by GluR1 and GluR2. DHA, but not NPD1, induced extensive dendritic branching and outgrowth. Overexpression of 15 lipoxygenase 1 (15LOX1), the enzyme responsible for conversion of DHA to NPD1, interrupted outgrowth mediated by GluR1 overexpression. In order to investigate molecular mechanisms that regulated neural circuitry outside of the critical period of CNS development, we examined dendrite morphology across the CNS in response to chronic variable stress (CVS). We found wide-spread changes in circuits implicated in neurocognitive dysfunctions associated with chronic stress, and observed substantial dendritic plasticity in the adult brain. / acase@tulane.edu

Homeostatic synaptic plasticity (HSP) regulates synaptic strength in response to changing neuronal firing patterns. This form of plasticity is defined by neurons’ ability to sense and over time integrate their level of firing activity, and to actively maintain it within a defined range. For instance, a compensatory increase in synaptic strength occurs when neuronal activity is chronically attenuated. However, the underpinning cellular mechanisms of this fundamental neural process remain poorly understood. We previously found that during activity deprivation, HSP leads to an increase in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor function as well as a shift in subunit composition from Ca2+-impermeable GluA2-containing AMPA receptors to Ca2+-permeable GluA2-lacking AMPA receptors not only at synapses, but also at extrasynaptic sites. Neurons therefore appear to be actively enhancing Ca2+ entry, possibly as a compensatory mechanism in response to a prolonged Ca2+ deficit. To test whether the homeostatic response may, at least in part, be mediated by internal Ca2+ stores, we depleted endoplasmic reticulum (ER) Ca2+ stores by using the Sarco/endoplasmic reticulum Ca2+ ATPases (SERCA) pump blocker cyclopiazonic acid (CPA) for a prolonged period. Interestingly, we have found that prolonged Ca2+-store depletion leads not only to an increase in synaptic strength per se, but also a cell-wide increase in synaptic Ca2+-permeable GluA2-lacking AMPARs. This increase in Ca2+ influx following periods of inactivity is conceptually highly reminiscent of a store-operated response, whereby cells re-establish their calcium levels following Ca2+ store depletion using cell surface Ca2+ channels. Our results suggest that neurons use synaptic receptors as means to regulate store Ca2+ levels, thus significantly expanding our understanding of the repertoire used by neurons to modulate cellular excitability.

Recent literature has shown that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors lacking the GluR2 subunit (thus calcium permeable) are widely expressed in the CNS, especially in interneurons and glia, where they contribute to synaptic transmission and plasticity. Studies have also indicated that calcium permeable AMPARs (CP-AMPARs) are expressed and participate in synaptic regulation in principal neurons, including hippocampal pyramidal neurons. Furthermore, CP-AMPARs and their resultant calcium influx are implicated in various pathophysiological conditions such as ischemia and seizures. However, the synaptic events activated by calcium influx through CP-AMPARs remain unknown. I took advantage of genetically altered mice without (GluR2-/-) or with reduced GluR2 (GluR2+/-), thus allowing the expression and detailed analysis of synaptic CP-AMPARs in hippocampal pyramidal neurons. Utilizing electrophysiological techniques, I demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR-dependent LTP) through a number of different induction protocols, including high-frequency stimulation (HFS) and theta-burst stimulation (TBS). This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP) at CA1 synapses that is NMDA-receptor (NMDAR) independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460. Surprisingly, calcium/calmodulin-dependent kinase II (CaMKII), the key protein kinase that is indispensable for NMDAR-dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR-dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine) on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDAR dependent LTP in control studies. In contrast, inhibitors for the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade (PD98059 and U0126) significantly attenuated this CP-AMPAR-dependent LTP. Additional studies with knockout mice revealed that the ERK/MAPK signalling cascade is likely acting through p-21 activated kinase 1 (or PAK1, a Rho-GTPase associated kinase) dependent mechanisms. These results suggest that distinct synaptic signalling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces the recent notions that CP-AMPARs are important facilitators of synaptic plasticity in the brain.

Recent literature has shown that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors lacking the GluR2 subunit (thus calcium permeable) are widely expressed in the CNS, especially in interneurons and glia, where they contribute to synaptic transmission and plasticity. Studies have also indicated that calcium permeable AMPARs (CP-AMPARs) are expressed and participate in synaptic regulation in principal neurons, including hippocampal pyramidal neurons. Furthermore, CP-AMPARs and their resultant calcium influx are implicated in various pathophysiological conditions such as ischemia and seizures. However, the synaptic events activated by calcium influx through CP-AMPARs remain unknown. I took advantage of genetically altered mice without (GluR2-/-) or with reduced GluR2 (GluR2+/-), thus allowing the expression and detailed analysis of synaptic CP-AMPARs in hippocampal pyramidal neurons. Utilizing electrophysiological techniques, I demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR-dependent LTP) through a number of different induction protocols, including high-frequency stimulation (HFS) and theta-burst stimulation (TBS). This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP) at CA1 synapses that is NMDA-receptor (NMDAR) independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460. Surprisingly, calcium/calmodulin-dependent kinase II (CaMKII), the key protein kinase that is indispensable for NMDAR-dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR-dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine) on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDAR dependent LTP in control studies. In contrast, inhibitors for the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade (PD98059 and U0126) significantly attenuated this CP-AMPAR-dependent LTP. Additional studies with knockout mice revealed that the ERK/MAPK signalling cascade is likely acting through p-21 activated kinase 1 (or PAK1, a Rho-GTPase associated kinase) dependent mechanisms. These results suggest that distinct synaptic signalling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces the recent notions that CP-AMPARs are important facilitators of synaptic plasticity in the brain.

The IEM 1460 is a potential age-specific anticonvulsant and an indicator of the distribution of AMPA receptor subtypes among rat brain cells. It is a derivative of adamantane, that was tested in previous studies on models of human myoclonic and generalized tonic-clonic seizures with promising results. In this thesis we evaluated its effect on the motor activity of rat offspring in the age of 12, 18 and 25 days, we used 90 animals in total . The effect was evaluated 30 minutes after intraperitoneal administration of IEM 1460 in two doses, 10 mg/kg or 20 mg/kg, and was compared to the control animals with physiological solution applied intraperitoneally in amount of 20 mg/kg. To test the animals we used Open field test, righting reflex, negative geotaxis, horizontal bar test, rope climbing test, regular and irregular horizontal ladder test. The tests were applied to animals in mentioned order. There were found significant changes influencing motor behaviour, primarily in the 12 days old animals with the dose of 20 mg/kg IEM 1460 and in the 25 days old animals with both doses of IEM 1460, 10 and 20 mg/kg. In the 18 days old animals the results were less significant. Powered by TCPDF (www.tcpdf.org)

Tem sido demonstrado o envolvimento do glutamato, através de diferentes receptores, nos mecanismos excitotóxicos que levam à morte neuronal na maioria das doenças neurodegenerativas do Sistema Nervoso Central (SNC). Adicionalmente, a Parawixina 10 (Pwx 10) tem sido demonstrada possuir efeito neuroprotetor em modelos de lesão atuando sobre o transporte de glutamato. Os objetivos gerais deste trabalho foram: i) estudar, em um curso temporal (24h, 1, 2 e 4 semanas), as alterações na expressão dos receptores AMPA no hipocampo de ratos induzidas pela injeção local de NMDA e ii) estudar o efeito neuroprotetor da Pwx 10 neste modelo. Foram utilizados ratos Wistar machos, submetidos à cirurgia estereotáxica para a microinjeção de salina ou NMDA no hipocampo dorsal. Alguns grupos de animais foram tratados com Pwx 10 a partir de 1h ou 24h após NMDA. O teste comportamental no labirinto aquático de Morris (LAM) e a coloração de Nissl foram realizados para verificar a extensão e eficácia da lesão por NMDA e o efeito neuroprotetor da Pwx 10. A expressão dos receptores foi estudada através do método de imunoistoquímica. Foram também realizados experimentos de imunofluorescência para GFAP e NeuN para avaliação da gliose e presença de neurônios na área lesada. Foi observado comprometimento das funções de aprendizado e memória no LAM, além de intensa perda de células neuronais e proliferação glial na região do CA1 que recebeu o NMDA, comprovando a eficiência da lesão pelo agonista. Observamos um curso temporal de diferentes alterações na expressão das subunidades GluR1 e GluR2 dos receptors AMPA no hipocampo, que podem ser relacionadas ao complexo mecanismo que ocorre em resposta à microinjeção de NMDA resultando em uma lesão local e na ativação da plasticidade neuronal. O tratamento com Pwx 10 apresentou efeito neuroprotetor, sendo este mais pronunciado quando a toxina foi administrada a partir de 1h após o agonista. / It has been shown the involvement of glutamate, through different receptors, on the excitotoxic mechanisms which result on the neuronal death reported in most neurodegenerative disorders of the CNS. In addition, Parawixina 10 (Pwx 10) has been demonstrated to act as neuroprotective in models of injury regulating the glutamatergic neurotransmission through glutamate transporters. The aims of this work were: i) to study, in a time course (24h, 1, 2 and 4 weeks), the changes on the expression of AMPA receptors in rat hippocampus induced by NMDA intrahippocampal injection, and ii) to study the neuroprotective effect of Pwx 10 in this moldel. Male Wistar rats has been used, submitted to stereotaxic surgery for saline or NMDA microinjection into dorsal hippocampus. Some groups of animals were treated with Pwx 10 from 1h or 24h after NMDA. The behavioral test on Morris water maze (MWM) and the Nissl staining were performed for evaluating the extension and efficacy of the NMDA injury and the neuroprotective effect of the Pwx 10 . The expression of the receptors was analyzed by immunohistochemistry. The expression of GFAP and NeuN on the lesioned area has also been investigated by immunofluorescency. It was observed the impaiment of learning and memory functions in the MWM, and intense loss of neuronal cells and glial proliferation in CA1 that received the NMDA, confirming the efficiency of the injury by the agonist. We observed a time course of distinct changes on the expression of GluR1 and GluR2 subunits of AMPA receptors in hippocampus, which may be related to the complex mechanism triggered in response to NMDA injection resulting in a local injury and on the activation of neuronal plasticity. The treatment with Pwx 10 showed neuroprotective effect, being most pronounced when the toxin was administrated from 1h after NMDA.

Tem sido demonstrado o envolvimento do glutamato, através de diferentes receptores, nos mecanismos excitotóxicos que levam à morte neuronal na maioria das doenças neurodegenerativas do Sistema Nervoso Central (SNC). Adicionalmente, a Parawixina 10 (Pwx 10) tem sido demonstrada possuir efeito neuroprotetor em modelos de lesão atuando sobre o transporte de glutamato. Os objetivos gerais deste trabalho foram: i) estudar, em um curso temporal (24h, 1, 2 e 4 semanas), as alterações na expressão dos receptores AMPA no hipocampo de ratos induzidas pela injeção local de NMDA e ii) estudar o efeito neuroprotetor da Pwx 10 neste modelo. Foram utilizados ratos Wistar machos, submetidos à cirurgia estereotáxica para a microinjeção de salina ou NMDA no hipocampo dorsal. Alguns grupos de animais foram tratados com Pwx 10 a partir de 1h ou 24h após NMDA. O teste comportamental no labirinto aquático de Morris (LAM) e a coloração de Nissl foram realizados para verificar a extensão e eficácia da lesão por NMDA e o efeito neuroprotetor da Pwx 10. A expressão dos receptores foi estudada através do método de imunoistoquímica. Foram também realizados experimentos de imunofluorescência para GFAP e NeuN para avaliação da gliose e presença de neurônios na área lesada. Foi observado comprometimento das funções de aprendizado e memória no LAM, além de intensa perda de células neuronais e proliferação glial na região do CA1 que recebeu o NMDA, comprovando a eficiência da lesão pelo agonista. Observamos um curso temporal de diferentes alterações na expressão das subunidades GluR1 e GluR2 dos receptors AMPA no hipocampo, que podem ser relacionadas ao complexo mecanismo que ocorre em resposta à microinjeção de NMDA resultando em uma lesão local e na ativação da plasticidade neuronal. O tratamento com Pwx 10 apresentou efeito neuroprotetor, sendo este mais pronunciado quando a toxina foi administrada a partir de 1h após o agonista. / It has been shown the involvement of glutamate, through different receptors, on the excitotoxic mechanisms which result on the neuronal death reported in most neurodegenerative disorders of the CNS. In addition, Parawixina 10 (Pwx 10) has been demonstrated to act as neuroprotective in models of injury regulating the glutamatergic neurotransmission through glutamate transporters. The aims of this work were: i) to study, in a time course (24h, 1, 2 and 4 weeks), the changes on the expression of AMPA receptors in rat hippocampus induced by NMDA intrahippocampal injection, and ii) to study the neuroprotective effect of Pwx 10 in this moldel. Male Wistar rats has been used, submitted to stereotaxic surgery for saline or NMDA microinjection into dorsal hippocampus. Some groups of animals were treated with Pwx 10 from 1h or 24h after NMDA. The behavioral test on Morris water maze (MWM) and the Nissl staining were performed for evaluating the extension and efficacy of the NMDA injury and the neuroprotective effect of the Pwx 10 . The expression of the receptors was analyzed by immunohistochemistry. The expression of GFAP and NeuN on the lesioned area has also been investigated by immunofluorescency. It was observed the impaiment of learning and memory functions in the MWM, and intense loss of neuronal cells and glial proliferation in CA1 that received the NMDA, confirming the efficiency of the injury by the agonist. We observed a time course of distinct changes on the expression of GluR1 and GluR2 subunits of AMPA receptors in hippocampus, which may be related to the complex mechanism triggered in response to NMDA injection resulting in a local injury and on the activation of neuronal plasticity. The treatment with Pwx 10 showed neuroprotective effect, being most pronounced when the toxin was administrated from 1h after NMDA.

Fragile X Syndrome (FXS) is the leading cause of inherited intellectual disability. The disorder is caused by a trinucleotide expansion that silences the Fragile X Mental Retardation 1 (Fmr1) gene resulting in the loss of its protein product, the Fragile X Mental Retardation Protein (FMRP). FXS patients show broad clinical phenotypes including intellectual disability, as well as a number of cognitive and behavioral problems. The lack of FMRP is believed to be the direct cause of the deficits seen in FXS patients. FMRP is an RNA-binding protein that is expressed in the brain and testes. This protein is believed to form a messenger ribonucleoprotein complex with mRNAs in the nucleus and subsequently export them to polyribosomes in the cytoplasm, therefore influencing translation of its bound mRNAs. Importantly, FMRP has long been suspected to be involved in synaptic plasticity due to its ability to bind several mRNAs that encode for proteins important in synaptic plasticity. Such proteins include the GluN1, GluN2A and GluN2B subunits of the N-methyl-D- aspartate receptor (NMDAR). FMRP is expressed in the hippocampus, a region of the brain involved in learning and memory processes. Recently, impaired NMDAR functioning in the dentate gyrus (DG) subregion of the hippocampus has been observed in Fmr1 knockout (-/y) mice. This impairment also resulted in reduction in long-term potentiation (LTP) and long-term depression (LTD) of synaptic efficacy, two biological models of learning and memory. In the present study, I focused on the levels of the NMDAR GluN1, GluN2B and Glu2B subunits in order to determine the synaptic plasticity alterations seen in the DG of Fmr1-/y mice. Using Western blotting, I found that there is a decrease in the GluN1, GluN2A and GluN2B subunits in the DG of young adult Fmr1-/y mice, indicating that these mice have significantly lower amounts of total NMDARs. These results could explain the altered LTP and LTD seen in Fmr1-/y mice at the molecular level and might contribute to the intellectual impairments seen in these KO mice. NMDARs appear to be important in the development and maturation of synapses. The GluN2A and GluN2B subunits are developmentally regulated, where GluN2B is predominantly expressed early in development and GluN2A in the adult brain. A dysregulation of GluN2A and GluN2B subunits has been proposed to affect the maturation and formation of synapses. Intriguingly, FMRP is also believed to play a functional role in early brain development. Thus, this study also focused on the developmental expression of the GluN1, GluN2A and GluN2B subunits in the DG, Cornu Ammonis, prefrontal cortex and cerebellum of Fmr1-/y mice, all of which are brain regions implicated in FXS. We found that the developmental expression of these subunits is altered in Fmr1-/y mice in specific brain regions. Together, these results demonstrate that the loss of FMRP differentially affects GluN1, GluN2A and GluN2B subunit expression both developmentally and spatially, further implicating NMDARs in the pathophysiology of FXS. / Graduate