Glutamatergic receptors are targets for many L-glutamate structure analogues, which cause neurotoxicity. This study investigated the actions of two dicarboxylic compounds, the first had cyclic framework and rigid structure, and the other had an acyclic framework and flexible structure, on the glutamatergic neurotransmission, oxidative damage and behavior in mice. The first compound evaluated was D,L-cis-2,3-pyrrolidine dicarboxylate (D,L-cis-2,3-PDC), a new glutamate analogue. D,L-cis-2,3-PDC reduced sodium-independent [3H]-L-glutamate binding by 50% in lysed membrane preparations and had no effect on sodium-dependent glutamate binding. Intracerebroventricular administration (ICV) of D,L-cis-2,3-PDC (7.5 - 25 nmol/ 5μl) induced dose-dependent tonic-clonic convulsions. The co-administration of MK-801 (7 nmol/ 2.5 μl; ICV), a noncompetitive NMDA receptor antagonist, with D,L-cis-2,3-PDC (16.5 nmol/ 2.5 μl; ICV) fully protected the animals against D,L-cis-2,3-PDC-induced convulsions, while the co-administration of DNQX (10 nmol/ 2.5 μl; ICV), a AMPA and KA receptors antagonist, increased the latency to convulsion and did not alter the percentage of animals that had convulsions. These results suggest that D,L-cis-2,3-PDC-induced effects are mediated predominantly by NMDA receptors activation. The second compound studied was succinate, the accumulating substrate in succinate dehydrogenase (SDH) deficiencies and SDH inhibitor intoxication. Adult male mice received an ICV injection of succinate (0.7, 1.0 and 1.7 μmol/ 5 μl) or 0.9% NaCl (5 μl) and had their exploratory behavior assessed in an open field for 10 min. Succinate (0.7 and 1.0 μmol/ 5 μl) decreased locomotor activity behavior and increased thiobarbituric acid reactive substances (TBARS) and protein carbonylation in the forebrain. Conversely, 1.7 μmol of succinate did not alter locomotor activity or oxidative damage parameters. The involvement of NMDA receptors in the succinate-induced increase of total protein carbonylation content and exploratory behavior inhibition was assessed by co-administrating MK-801 (7 nmol/ 2.5 μl, ICV) with succinate (1 μmol/ 2.5 μl, ICV). The co-administration of MK-801 protected against succinate-induced increase of total protein carbonylation and decrease of locomotor activity. These results suggest the involvement of NMDA receptors in these effects of succinate, which may of particular relevance for succinate-accumulating conditions, such as SDH inhibitors intoxication and inherited SDH deficiencies. / Os receptores glutamatérgicos são alvos da ação de muitas neurotoxinas análogas ao L-glutamato. Neste estudo foram investigadas as ações de dois compostos dicarboxílicos, um de cadeia cíclica e estrutura rígida e o outro de cadeia acíclica e estrutura flexível, sobre a neurotransmissão glutamatérgica, dano oxidativo e comportamento em roedores. No primeiro trabalho foi investigado se o D,L-cis-2,3-dicarboxilato de pirrolidina (D,L-cis-2,3-PDC) altera a ligação de [3H]-L-glutamato em membranas plasmáticas de córtex de ratos adultos e se os receptores N-metil-D-aspartato (NMDA) estão envolvidos nas convulsões induzidas por este composto. O D,L-cis-2,3-PDC reduziu a ligação de [3H]-L-glutamato Na+-independente em 50% nas preparações de membranas rompidas e não apresentou efeito sobre a ligação de [3H]-L-glutamato Na+-dependente. A administração intracerebroventricular (ICV) de D,L-cis-2,3-PDC (7,5; 25 nmol/ 5 μl) induziu convulsões generalizadas do tipo tônico-clônica nos camundongos, de uma maneira dose-dependente. A co-administração de MK-801 (7 nmol/ 2,5 μl; ICV), um antagonista não-competitivo dos receptores NMDA, com D,L-cis-2,3-PDC (16,5 nmol/ 2,5 μl; ICV), protegeu totalmente os animais das convulsões induzidas por D,L-cis-2,3-PDC, enquanto que a co-administração de DNQX (10 nmol/ 2,5 μl; ICV), um antagonista dos receptores AMPA e KA, aumentou a latência das convulsões, mas não alterou a percentagem de animais que tiveram convulsões. Estes resultados sugerem que os efeitos induzidos por D,L-cis-2,3-PDC são mediados principalmente pela ativação dos receptores NMDA. No segundo estudo, foi investigado se o sucinato, substrato que se acumula nas deficiências da enzima sucinato desidrogenase (SDH) e nas intoxicações por inibidores da SDH, causa lipoperoxidação e carbonilação protéica, e se os receptores NMDA estão envolvidos no dano oxidativo induzido por sucinato. Camundongos machos adultos receberam uma injeção ICV de sucinato (0,7; 1,0; 1,7 μmol/ 5 μl) ou 0,9 % de NaCl (5 μl) e seu comportamento foi analisado em um campo aberto por 10 minutos. Sucinato (0,7; 1,0 μmol/ 5 μl) diminuiu a atividade locomotora e aumentou as substâncias que reagem ao ácido tiobarbitúrico (TBARS) e carbonilação protéica no cérebro. Por outro lado, 1,7 μmol de sucinato não alterou a atividade locomotora ou os parâmetros de dano oxidativo. O envolvimento dos receptores NMDA no aumento induzido por sucinato do conteúdo de carbonilação protéica e da inibição do comportamento exploratório foi avaliado pela co-administração de MK-801 (7nmol/ 2,5 μl, ICV) com sucinato (1 μmol/ 2,5 μl, ICV). A co-administração de MK801 protegeu contra o aumento induzido por sucinato da carbonilação protéica e na diminuição da atividade locomotora. Esses resultados sugerem o envolvimento dos receptores NMDA nesses efeitos do sucinato, os quais são de grande relevância nas condições em que acumula sucinato, tais como as intoxicações com inibidores da SDH e deficiências dessa enzima causadas por erros inatos do metabolismo.

The contamination of seafood with methylmercury (MeHg) is a global health issue, as MeHg is a well known neurotoxin. Since dietary nutrients may interact with MeHg toxicity, and oxidative stress is one of the primary mechanisms underlying MeHg neurotoxicity, we characterized dietary antioxidant-MeHg interactions. Firstly, we used an ethnobotanical study to confirm the antioxidant activity of Northern Labrador Tea, Rhododendron tomentosum ssp. subarcticum (Tea), for the Canadian Inuit, a population with elevated MeHg exposure. Secondly, we determined the ability of Tea to ameliorate MeHg-induced toxicity in a rat perinatal exposure study. MeHg exposure (2 mg/KgBW/d) was associated with perturbed development and behaviour, elevated brain N-methyl-D-aspartate receptors, and serum lipid peroxidation. Surprisingly, Tea co-exposure (100 mg/KgBW/d) modulated MeHg’s effects on brain NMDA-R levels and lipid peroxidation, but also increased mercury serum concentrations. Thirdly, using a toxicogenomics approach we determined that MeHg exposure caused the down-regulation of Nr4a2 and its protein product Nurr1. These novel MeHg targets are implicated in developmental learning functions and were corrected with MeHg + Tea co-exposure. Lastly, we conducted a risk assessment survey and cross-sectional dietary epidemiology study in Costa Rica to further investigate dietary nutrient-MeHg interactions. Costa Rica is a Central American country with multiple sources of Hg and a high per capital fish consumption. Here, 5 of the 14 populations we studied exceeded the recommended MeHg provisional tolerable daily intake (pTDI) of 0.2 µg/KgBW/d. In Heredia the pTDI was exceeded by 34% of woman participants, primarily associated with canned tuna consumption. Interestingly, we detected that Hg body burden was significantly reduced by the consumption of antioxidant-rich dietary items. Considering our collective results, we hypothesized that MeHg toxicokinetics may be altered by dietary nutrients at the site of intestinal absorption from the disruption of gut flora, or at the site of cellular demethylation in tissues from the improvement of cellular redox state. The interaction of dietary nutrients on MeHg outcomes has a large impact on risk assessment and may provide a public health approach for managing the risk associated with MeHg exposure without reducing local fish consumption.

La synapse glutamatergique est formée par une présynapse axonale et une postsynapse dont le support est l'épine dendritique. L'épine présente des récepteurs membranaires du glutamate liés à des protéines d'échafaudage sous-membranaires. Ces protéines de la densité postsynaptique (PSD) permettent de relier les récepteurs à leurs voies de signalisation. Les récepteurs NMDA sont reliés aux récepteurs métabotropiques du glutamate (mGluR1/5) via le complexe PSD95/GKAP/Shank/Homer. Au cours de ma thèse, j'ai caractérisé la dynamique d'interactions protéiques au sein de ce complexe et étudié les conséquences fonctionnelles sur l'activité des récepteurs.Homer est une protéine multimérique reliant mGluR5 au complexe PSD95/GKAP/Shank. La forme monomérique Homer1a est incapable de relier mGluR5 à Shank. Nous avons montré que la rupture du complexe par l'expression de Homer1a permet une interaction directe entre les récepteurs NMDA et mGluR5 et une inhibition des courants NMDA. Nous avons validé que ce processus intervient lors de la potentialisation synaptique. J'ai également étudié le rôle de l'interaction entre GKAP et DLC2, une chaîne légère de transporteurs moléculaires. Après avoir caractérisé l'occurrence et la dynamique de l'interaction GKAP-DLC2, j'ai montré que l'activité neuronale entraîne une augmentation de cette interaction et une accumulation synaptique de GKAP. De plus, cette interaction permet d'acheminer PSD95 dans les épines et d'augmenter les courants NMDA. L'ensemble de ces résultats montre que les protéines d'échafaudage participent à la signalisation des récepteurs, modulent la transmission synaptique et sous-tendent les mécanismes de plasticité à long terme. / The glutamatergic synapse is composed by an axonal presynapse and a postsynapse which is supported by a dendritic spine. The spine contains membrane glutamatergic receptors connected to sub-membrane scaffolding proteins. These postsynaptic density (PSD) proteins allow to link receptors to their signaling pathways. NMDA receptors are associated to metabotropic glutamate receptors (mGluR1/5) through the PSD95/GKAP/Shank/Homer protein complex. During my PhD, I have characterized protein-protein interactions dynamic in this complex and studied functional consequences on receptor activity.Homer is a multimeric protein linking mGluR5 to the PSD95/GKAP/Shank complex. The monomeric form Homer1a is unable to connect mGluR5 to Shank. We have shown that complex disruption by Homer1a expression induces a direct interaction between NMDA and mGluR5 and subsequent inhibition of NMDA currents. We have shown that this process occurs during synaptic potentiation.I have also studied the interaction between GKAP and DLC2, a light chain shared by molecular transporters. I have characterized the occurrence and dynamic of GKAP-DLC2 interaction and shown that neuronal activity increases this interaction leading to synaptic accumulation of GKAP. Moreover, this interaction allows PSD95 targeting into dendritic spines and NMDA currents increase. Together, these results show that scaffolding proteins participate to receptor signaling, modulate synaptic transmission and underlie long-term synaptic plasticity mechanisms.

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.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / In Alzheimer s disease (AD), β-amyloid peptide (Aβ) has been linked with synaptic loss and cognitive dysfunction, albeit the precise mechanism remains unknown. An involvement of N-Methyl-D-Aspartate receptors (NMDAR) in AD is proposed, since its inhibition attenuates some aspects of AD s neuropathology. In this regard, polyamines, like spermidine and spermine, positive modulators of NMDARs, have been shown to have both concentration and synthesis increased by Aβ. Using the novel object recognition task we showed that negative modulation of polyamine system, been trough blockade of its binding site at NMDAR by arcaine (0.02 nmol/site), traxoprodil (0.002 nmol/site), or inhibition of polyamine synthesis by DFMO (2.7 nmol/site), reverses Aβ25-35-induced memory impairment in mice. The activation of polyamine binding site at NMDAR located at extrasynaptic sites might underlie the cognitive deficits of Aβ25-35-treated mice, since incubation of hippocampal neuron cultures with spermidine (400 μM) or Aβ25-35 (10 μM) significantly increased nuclear accumulation of jacob protein, a marker of extrasynaptic NMDAR activation. Moreover, traxoprodil (4nM), arcaine (4 μM) or DFMO (5 μM) blocked the Aβ-induced jacob nuclear translocation. Activation of extrasynaptic NMDAR in neurons leads to striping of synaptic contacts and simplification of neuronal cytoarchitecture. Incubation of hippocampal neuron cultures with traxoprodil (4 Nm), arcaine (4 μM) or DFMO (5 μM) reversed the deleterious effects of Aβ25-35 on dendritic spine number and spine morphology. We also evaluated the involvement of A-type K+ currents on the Aβ25-35-induced memory impairment. Administration of Tx3-1 (3 100 pmol/site), a selective IA blocker, restored memory of mice injected with Aβ25-35 and tested on the novel object recognition task The reversal of memory impairment and the protective effect on dendritic spine alterations exerted by the modulators of the polyamine system suggest the polyamine binding site at extrasynaptic NMDAR a potential player in Aβ-induced cognitive deficit. / O peptídeo β-amiloide (Aβ), reconhecido como agente tóxico na Doença de Alzheimer (DA) é implicado como causador de danos cognitivos e sinápticos, apesar de os mecanismos não serem completamente compreendidos. O envolvimento do receptor N-metil-D-aspartato (NMDA) na DA é sugerido, visto que o seu bloqueio atenua alguns aspectos neuropatológicos da DA. Nesse contexto, tem sido demonstrado que as poliaminas, como espermidina e espermina, moduladores positivos do receptor NMDA, possuem níveis e síntese elevada tanto no cérebro de pacientes com DA como em preparações in vitro utilizando o peptídeo Aβ. Neste estudo demonstrou-se que a modulação do sistema das poliaminas, através do bloqueio do seu sítio de ligação no receptor NMDA por arcaína (0,02 nmol/sítio), traxoprodil (0,002 nmol/sítio) ou da inibição de sua síntese por DFMO (2,7 nmol/sítio), reverte o déficit cognitivo induzido pela injeção de Aβ25-35 em camundongos testados na tarefa de reconhecimento de objetos. A ativação do sítio de ligação das poliaminas em receptores NMDA extrassinápticos pode subjazer o déficit cognitivo de camundongos injetados com Aβ25-35, visto que a incubação de culturas primárias de neurônios hipocampais com espermidina (400 μM), NMDA (200 μM) ou Aβ25-35 (10 μM) aumenta o acúmulo nuclear de jacob, um marcador de ativação de receptores NMDA extrassinápticos, de maneira significante. Ademais, traxoprodil (4 nM), arcaína (4 μM) ou DFMO (5 μM) bloquearam o acúmulo nuclear de jacob induzido por Aβ. A ativação de receptores NMDA extrassinápticos em neurônios leva a simplificação da citoarquitetura neuronal e a diminuição de contatos sinápticos. Aqui demonstrou-se que a incubação de culturas de neurônios hipocampais com traxoprodil (4 nM), arcaína (4 μM) ou DFMO (5 μM) reverte as alterações na a densidade e morfologia de espinhas dendríticas induzido pela incubação com Aβ25-35. Ainda, também avaliou-se o envolvimento de correntes de K+ do tipo A no déficit cognitivo induzido pela injeção i.c.v. de Aβ25-35. A administração de Tx3-1 (3 100 pmol/sítio), um bloqueador seletivo de correntes IA, reverteu o prejuízo de memória de camundongos injetados com Aβ25-35 e testados na tarefa de reconhecimento de objetos. A reversão dos danos cognitivos e sinápticos induzidos por Aβ25-35 através da modulação do sistema das poliaminas sugere a estimulação do sítio de ligação das poliaminas no receptor NMDA, possivelmente extrassínaptico, como um dos mecanimos por trás do déficit cognitivo induzido pelo peptídeo Aβ.

Networks of interneurons within the spinal cord coordinate the rhythmic activation of muscles during locomotion. These networks are subject to extensive neuromodulation, ensuring appropriate behavioural output. Astrocytes are proposed to detect neuronal activity via Gαq-linked G-protein coupled receptors and to secrete neuromodulators in response. However, there is currently a paucity of evidence that astrocytic information processing of this kind is important in behaviour. Here, it is shown that protease-activated receptor-1 (PAR1), a Gαq-linked receptor, is preferentially expressed by glia in the spinal cords of postnatal mice. During ongoing locomotor-related network activity in isolated spinal cords, PAR1 activation stimulates release of adenosine triphosphate (ATP), which is hydrolysed to adenosine extracellularly. Adenosine then activates A1 receptors to reduce the frequency of locomotor-related bursting recorded from ventral roots. This entails inhibition of D1 dopamine receptors, activation of which enhances burst frequency. The effect of A1 blockade scales with network activity, consistent with activity-dependent production of adenosine by glia. Astrocytes also regulate activity by controlling the availability of D-serine or glycine, both of which act as co-agonists of glutamate at N-methyl-D-aspartate receptors (NMDARs). The importance of NMDAR regulation for locomotor-related activity is demonstrated by blockade of NMDARs, which reduces burst frequency and amplitude. Bath-applied D-serine increases the frequency of locomotor-related bursting but not intense synchronous bursting produced by blockade of inhibitory transmission, implying activity-dependent regulation of co-agonist availability. Depletion of endogenous D-serine increases the frequency of locomotor-related but not synchronous bursting, indicating that D-serine is required at a subset of NMDARs expressed by inhibitory interneurons. Blockade of the astrocytic glycine transporter GlyT1 increases the frequency of locomotor-related activity, but application of glycine has no effect, indicating that GlyT1 regulates glycine at excitatory synapses. These results indicate that glia play an important role in regulating the output of spinal locomotor networks.