Excitatory synaptic transmission and plasticity mediated by glutamate receptors are important for many brain functions, including learning and memory. Various molecular and cellular models have been established to study multiple forms of synaptic plasticity that coexist in the hippocampal CA1 region. Metabotropic glutamate receptor-dependent long-term depression (mGluR-dependent LTD) is a form of long lasting synaptic plasticity thought to play critical roles in diverse physiological and pathological processes. The GluR2 subunit of AMPA receptors has been a focus of neuroscience research over the last decade due to its important roles in endocytic trafficking and Ca2+ permeation in many forms of activity-dependent synaptic plasticity and homeostatic plasticity. However, the underlying mechanisms of mGluR-dependent LTD and the possible involvement of GluR2 in this form of plasticity remain unknown. In this project, I utilized GluR2 knockout (KO) mice and tested the requirement of GluR2 in multiple forms of hippocampal synaptic plasticity at different developmental stages. The results showed that although GluR2 is dispensable for long lasting synaptic plasticity in juvenile mice, it is essential for the expression of mGluR-dependent LTD in adult animals. Next, I examined the involvement of a number of GluR2-specific functions in mGluR-dependent LTD and found that GluR2 N-terminal interaction with the cell adhesion molecule N-cadherin is a key process required for GluR2 to regulate the expression of mGluR-dependent LTD. Furthermore, using a combination of approaches including electrophysiology, biochemical assays, and virus-mediated expression of several mutant GluR2 constructs, I identified a signaling cascade involving N-cadherin/β-catenin complex, Rac1 Rho GTPase, LIM-kinase 1 and cofilin, through which GluR2 exerts its effect on actin regulation and mGluR-dependent LTD. Importantly, the impaired LTD in GluR2 KO mice can be fully rescued by manipulating GluR2-N-cadherin N-terminus interaction or cofilin-mediated actin reorganization. Lastly, I showed that this signaling cascade also plays a critical role in the regulation of dendritic spine plasticity during mGluR-dependent LTD. Together, these results reveal a novel signaling process by which GluR2 regulates long lasting synaptic plasticity and provide insights into how functional and structural plasticity are coordinated in the mammalian central nervous system.

Excitatory synaptic transmission and plasticity mediated by glutamate receptors are important for many brain functions, including learning and memory. Various molecular and cellular models have been established to study multiple forms of synaptic plasticity that coexist in the hippocampal CA1 region. Metabotropic glutamate receptor-dependent long-term depression (mGluR-dependent LTD) is a form of long lasting synaptic plasticity thought to play critical roles in diverse physiological and pathological processes. The GluR2 subunit of AMPA receptors has been a focus of neuroscience research over the last decade due to its important roles in endocytic trafficking and Ca2+ permeation in many forms of activity-dependent synaptic plasticity and homeostatic plasticity. However, the underlying mechanisms of mGluR-dependent LTD and the possible involvement of GluR2 in this form of plasticity remain unknown. In this project, I utilized GluR2 knockout (KO) mice and tested the requirement of GluR2 in multiple forms of hippocampal synaptic plasticity at different developmental stages. The results showed that although GluR2 is dispensable for long lasting synaptic plasticity in juvenile mice, it is essential for the expression of mGluR-dependent LTD in adult animals. Next, I examined the involvement of a number of GluR2-specific functions in mGluR-dependent LTD and found that GluR2 N-terminal interaction with the cell adhesion molecule N-cadherin is a key process required for GluR2 to regulate the expression of mGluR-dependent LTD. Furthermore, using a combination of approaches including electrophysiology, biochemical assays, and virus-mediated expression of several mutant GluR2 constructs, I identified a signaling cascade involving N-cadherin/β-catenin complex, Rac1 Rho GTPase, LIM-kinase 1 and cofilin, through which GluR2 exerts its effect on actin regulation and mGluR-dependent LTD. Importantly, the impaired LTD in GluR2 KO mice can be fully rescued by manipulating GluR2-N-cadherin N-terminus interaction or cofilin-mediated actin reorganization. Lastly, I showed that this signaling cascade also plays a critical role in the regulation of dendritic spine plasticity during mGluR-dependent LTD. Together, these results reveal a novel signaling process by which GluR2 regulates long lasting synaptic plasticity and provide insights into how functional and structural plasticity are coordinated in the mammalian central nervous system.

The control of extracellular adenosine is crucial to the regulation of synaptic transmission and neuroprotection. Equilibrative nucleoside transporters (ENTs) are highly expressed in the hippocampus and widely accepted as critical regulators of adenosine tone. However, the mechanisms regulating the surface distribution and transport function of ENTs are largely unknown. Since ENT1 and ENT2 contain consensus sequences for phosphorylation by protein kinase CK2, and because this protein has been reported to regulate synaptic plasticity and ENT function in non-neuronal systems, the present thesis outlines the hypothesis that CK2-induced phosphorylation of ENTs is important for their cellular localization and thus the regulation of adenosine tone and synaptic transmission. Here, a functional interaction between adenosine CK2, ENTs and AMPA receptors in the hippocampus is reported. Western blot analysis shows that a variety of CK2 inhibitors (DMAT, TBB and DRB) significantly reduced the density of ENT1 and ENT2 proteins in hippocampal membrane fractions, suggesting that CK2-mediated phosphorylation of ENTs promotes their surface localization. In contrast, it was found that the ENT1 inhibitor NBTI significantly increased in the membrane localization of ENT1, relative to the control. Moreover, ENTs were found to immunoprecipitate with GluR1 and GluR2-containing AMPA receptors; and CK2 inhibitors caused a decrease in the membrane localization of GluR2 and GluR1 AMPA receptors. These results suggest a novel signaling complex linking CK2-regulated adenosine transport to AMPA receptor trafficking in the rat hippocampus. Although the physiological significance of these findings requires further investigation, this thesis provides insight into an adenosine regulation pathway that may be important for the regulation of synaptic transmission and neuroprotection in the rat hippocampus.

The control of extracellular adenosine is crucial to the regulation of synaptic transmission and neuroprotection. Equilibrative nucleoside transporters (ENTs) are highly expressed in the hippocampus and widely accepted as critical regulators of adenosine tone. However, the mechanisms regulating the surface distribution and transport function of ENTs are largely unknown. Since ENT1 and ENT2 contain consensus sequences for phosphorylation by protein kinase CK2, and because this protein has been reported to regulate synaptic plasticity and ENT function in non-neuronal systems, the present thesis outlines the hypothesis that CK2-induced phosphorylation of ENTs is important for their cellular localization and thus the regulation of adenosine tone and synaptic transmission. Here, a functional interaction between adenosine CK2, ENTs and AMPA receptors in the hippocampus is reported. Western blot analysis shows that a variety of CK2 inhibitors (DMAT, TBB and DRB) significantly reduced the density of ENT1 and ENT2 proteins in hippocampal membrane fractions, suggesting that CK2-mediated phosphorylation of ENTs promotes their surface localization. In contrast, it was found that the ENT1 inhibitor NBTI significantly increased in the membrane localization of ENT1, relative to the control. Moreover, ENTs were found to immunoprecipitate with GluR1 and GluR2-containing AMPA receptors; and CK2 inhibitors caused a decrease in the membrane localization of GluR2 and GluR1 AMPA receptors. These results suggest a novel signaling complex linking CK2-regulated adenosine transport to AMPA receptor trafficking in the rat hippocampus. Although the physiological significance of these findings requires further investigation, this thesis provides insight into an adenosine regulation pathway that may be important for the regulation of synaptic transmission and neuroprotection in the rat hippocampus.

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.

The mechanisms underlying cannabinoid impairment of fear memory is not clear. This study investigated the effects of the synthetic cannabinoid HU210 and the endocannabinoid hydrolysis inhibitor JZL 195 on fear memory following contextual fear conditioning (CFC; an animal model of fear). The long-term depression (LTD)-blocking peptide Tat-GluR2 was utilized to investigate whether the expression of cannabinoid-induced LTD (CB-LTD) is required for the cannabinoid impairment of acquisition and consolidation of contextual fear memory. HU210 reduced freezing throughout the test phase of the acquisition protocol, which was not affected by pre-administration of Tat-GluR2. High and moderate doses of HU210 reduced freezing during the first and last half, respectively, of the test phase of the consolidation protocol, which was prevented by pre-treatment with Tat-GluR2. HU210 did not affect freezing during the test phase of the retrieval protocol. Thus, these results suggest that HU210 impairs acquisition and consolidation, but not retrieval of contextual fear memory, and that in vivo CB-LTD expression is required for HU210 impairment of the consolidation, but not acquisition, of contextual fear memory. We also observed that HU210 and JZL 195 do not facilitate the acquisition of contextual fear memory extinction.

The mechanisms underlying cannabinoid impairment of fear memory is not clear. This study investigated the effects of the synthetic cannabinoid HU210 and the endocannabinoid hydrolysis inhibitor JZL 195 on fear memory following contextual fear conditioning (CFC; an animal model of fear). The long-term depression (LTD)-blocking peptide Tat-GluR2 was utilized to investigate whether the expression of cannabinoid-induced LTD (CB-LTD) is required for the cannabinoid impairment of acquisition and consolidation of contextual fear memory. HU210 reduced freezing throughout the test phase of the acquisition protocol, which was not affected by pre-administration of Tat-GluR2. High and moderate doses of HU210 reduced freezing during the first and last half, respectively, of the test phase of the consolidation protocol, which was prevented by pre-treatment with Tat-GluR2. HU210 did not affect freezing during the test phase of the retrieval protocol. Thus, these results suggest that HU210 impairs acquisition and consolidation, but not retrieval of contextual fear memory, and that in vivo CB-LTD expression is required for HU210 impairment of the consolidation, but not acquisition, of contextual fear memory. We also observed that HU210 and JZL 195 do not facilitate the acquisition of contextual fear memory extinction.

The mechanisms underlying cannabinoid impairment of fear memory is not clear. This study investigated the effects of the synthetic cannabinoid HU210 and the endocannabinoid hydrolysis inhibitor JZL 195 on fear memory following contextual fear conditioning (CFC; an animal model of fear). The long-term depression (LTD)-blocking peptide Tat-GluR2 was utilized to investigate whether the expression of cannabinoid-induced LTD (CB-LTD) is required for the cannabinoid impairment of acquisition and consolidation of contextual fear memory. HU210 reduced freezing throughout the test phase of the acquisition protocol, which was not affected by pre-administration of Tat-GluR2. High and moderate doses of HU210 reduced freezing during the first and last half, respectively, of the test phase of the consolidation protocol, which was prevented by pre-treatment with Tat-GluR2. HU210 did not affect freezing during the test phase of the retrieval protocol. Thus, these results suggest that HU210 impairs acquisition and consolidation, but not retrieval of contextual fear memory, and that in vivo CB-LTD expression is required for HU210 impairment of the consolidation, but not acquisition, of contextual fear memory. We also observed that HU210 and JZL 195 do not facilitate the acquisition of contextual fear memory extinction.