Introdução: Os receptores ionotrópicos do glutamato, como o N-metil-D-Aspartato (NMDA), estão envolvidos em desordens psiquiátricas. NMDARs são complexos heteroméricos que incorporam tres diferentes subunidades: NR1, NR2 e NR3. Objetivos: quantificar os níveis plasmáticos das subunidades NR1 e NR2 NMDAR em pacientes em primeiro episódio psicótico (PEP), em comparação com os irmãos e controles saudáveis. Métodos: Este é um estudo transversal de PEP na região de Ribeirão Preto, Brasil, sendo o grupo controle composto por indivíduos saudáveis, pareados por idade, sexo e mesma área de abrangência dos casos. Foram coletados 5 mL de amostra de sangue próxima a data de diagnóstico de PEP. A quantificação plasmática das subunidades NR1 e NR2 foi realizada por ELISA. Os dados foram analisados por ANOVA (significante se p<0,05) e curva ROC. Resultados: Foram incluídos 166 pacientes em PEP (idade: x = 30,34 ± 12,2 anos; 64% homens), destes 84 com diagnóstico de psicose não afetiva, 51 com transtorno bipolar e 31 com transtorno depressivo. Foram tambem incluídos 76 irmãos e 166 controles saudáveis. Os níveis plasmáticos das subunidades NR1 e NR2 foram significativamente menores em pacientes com transtornos psicóticos (NR1: x = 71,0 ± 100,3 pg/mL, NR2: x = 2,5 ± 2 ng/ml), transtorno bipolar (NR1: x = 185,7 ± 319,5 pg/ml; NR2: x = 2,1 ± 2,2 ng/ml), transtorno depressivo (NR1: x = 83,2 ±185,0 pg/ml; NR2: x = 2,1± 2,1 ng/ml) em comparação com os irmãos (NR1: x = 140,6 ± 193,8 pg/ml; NR2: = 6,2 ± 1,5 ng/ml) e voluntários saudáveis (NR1: x = 146,7 ± 361,1 pg/ml; NR2: x = 4,8 ± 2,2 ng/ml) [NR1 e NR2, p < 0,001]. Indivíduos com valores plasmáticos de NR2 inferiores a 3,648 ng/mL apresentam um risco 14,72 vezes maior de estar doente (PEP) de quem não possui o NR2 abaixo deste valor. Conclusões: Este é o primeiro estudo relatando a quantificação e a redução das concentrações plasmaticas das subunidades NR1 e NR2 em transtornos psiquiátricos graves quando comparados aos irmãos e controles, podendo a subunidade NR2 ser um candidato a biomarcador plasmático em pacientes com PEP. / Background: Ionotropic glutamate receptors, such as N-Methyl-D-Aspartate (NMDA), are involved in pathophysiology of several psychiatric disorders. NMDARs are described as heteromeric complexes incorporating distincts subunits within a repertoire of three types: NR1, NR2 and NR3. Aim: to quantify the NR1 and NR2 subunits NMDAR plasma levels in patients with first episode psychosis (FEP), compared with siblings and healthy controls. Methods: This is a cross-sectional study of FEP conducted in Ribeirão Preto, Brazil. The control group were composed by healthy subjects matched for age, sex and same coverage area of cases. 5 ml of blood sample were collected next to the date of FEP diagnosis. NR1 and NR2 subunits plasmatic quantification was performed by ELISA. Data were analyzed by ANOVA (significant at p < 0.05) and ROC curve. Results: FEP sample comprised 166 patients (age: x = 30.34 ± 12.2 years; 64% men), of these 84 with a diagnosis of psychotic disorder, 51 with bipolar disorder and 31 with depressive disorder. It was also included 76 siblings and 166 healthy controls. NR1 and NR2 subunits plasma levels were significantly lower in patients with psychotic disorders (NR1: x = 71.0 ± 100.3 pg / ml, NR2: x = 2.5 ± 2 ng/ml), bipolar disorder (NR1: x = 185.7 ± 319.5 pg/mL; NR2: x = 2.1 ± 2.2 ng/ml), depressive disorders (NR1: x = 83.2 ± 185.0 pg/mL; NR2: x = 2.1 ± 2.1 ng/ml) compared with siblings (NR1: x = 140.6 ± 193.8 pg/mL; NR2: x = 6.2 ± 1.5 ng/ml) and healthy volunteers (NR1: x = 146.7 ± 361.1 pg / mL; NR2: x = 4.8 ± 2.2 ng/ml) [NR1 and NR2, p < 0.001]. Interestingly, individuals with NR2 plasma values less than 3.648 ng/ml present 14.72 times a higher risk to be in FEP than other patients. Conclusions: This is the first study reporting the measurement and reduction of NR1 and NR2 subunits plasma concentrations in severe psychiatric disorders when compared to siblings and controls. And highlighting that NR2 subunit can be a candidate for plasma biomarker in patients with FEP.

MTOR est une sérine/thréonine kinase appartenant au complexe mTORC1 (mTOR Complexe 1), un régulateur clé de la traduction. Ce complexe joue un rôle au sein de la LTP (Potentialisation à Long Terme), une forme de plasticité synaptique qui requiert la synthèse de nouvelles protéines pour renforcer la transmission synaptique. La première partie de ma thèse porte sur les mécanismes de régulation de la voie mTORC1 dans les neurones. Dans les cellules non neuronales, cette voie de signalisation est classiquement régulée par deux voies distinctes. D’une part, les acides aminés induisent le recrutement du complexe mTORC1 à la membrane des endo-lysosomes où la protéine Rheb est enrichie et favorisent ainsi l’activation de mTORC1. D’autre part, les facteurs de croissance activent mTORC1 en stimulant la voie PI3K/Akt/TSC/Rheb. Nos résultats indiquent que les neurones sont capables d’ "utiliser" le mécanisme responsable de la translocation de mTORC1 en réponse à la supplémentation en acides aminés pour coupler l’induction de la plasticité synaptique à l’activation de mTORC1. En effet, les récepteurs NMDA et le BDNF, deux acteurs centraux de la LTP, augmentent le recrutement de mTORC1 à la membrane des endo-lysosomes même en absence d’acides aminés, et activent mTORC1. Par des stratégies induisant la translocation de mTORC1 à la membrane des endo-lysosomes, nous avons montré que ce mécanisme est important pour l’activation de mTORC1 mais n’est pas suffisant : il faut également une activation de la protéine Rheb. Le second aspect de mon projet porte sur la régulation de mTORC1 dans le cadre de la maladie d’Alzheimer, une maladie neurodégénérative caractérisée par une perte progressive de la mémoire. Les déficits cognitifs s’accompagnent d’un dysfonctionnement progressif des synapses suivi par la perte neuronale, tous deux causés par une accumulation anormale du peptide amyloïde β (Aβ). Les données de la littérature montrent que les oligomères toxiques du peptide Aβ (AβO) inhibent la plasticité synaptique dans les stades précoces de la maladie. Cependant, les mécanismes restent obscurs. Plusieurs études mettent en évidence une altération de la voie mTORC1. Nos résultats montrent que les AβO inhibent le recrutement de mTORC1 à la membrane des endo-lysosomes. Ce mécanisme est rétabli par une inhibition pharmacologique de l’AMPK. Ainsi, ces données indiquent que les AβO inhibent l’adressage de mTORC1 aux compartiments endo-lysosomaux via l’AMPK. Cela aurait pour conséquence une inhibition de la synthèse protéique décrite dans la littérature et contribuerait ainsi au dysfonctionnement synaptique. / MTOR is a serine/threonine kinase that belongs to mTORC1 (mTOR complex 1), a key regulator of translation. This complex is involved in LTP (Long Term Potentiation), a form of synaptic plasticity requiring new protein synthesis to reinforce synaptic transmission. The first part of my thesis investigates the mechanism of mTORC1’s regulation in neurons. In non-neuronal cells, mTORC1 pathway is commonly activated by two distinct pathways. On the one hand, amino acids induce mTORC1 recruitment to the membrane of endo-lysosomes where Rheb is enriched and can thus promote mTORC1 activation. On the other hand, growth factors activate mTORC1 via the PI3K/Akt/TSC/Rheb pathway. Our results indicate that neurons are capable of “using” amino acid-induced translocation of mTORC1 to connect synaptic plasticity induction to mTORC1 activation. Indeed, NMDA receptors and BDNF, two main actors of synaptic plasticity, increase mTORC1 recruitment to the membrane of endo-lysosomes even in the absence of amino acids, and activate mTORC1. Using strategies targeting mTORC1 to endo-lysosomes, we show that this mechanism promotes activation of mTORC1 but is not sufficient: Rheb activation is also required. The second part of my project is focused on the regulation of mTORC1 in Alzheimer’s disease, a neurodegenerative pathology characterized by a progressive memory loss. Cognitive deficits are widely believed to result from a progressive dysfunction of synapses, followed by a loss of neurons, both caused by an abnormal accumulation of the amyloid β peptide (Aβ). Data from others show that toxic Aβ oligomers (AβOs) inhibit synaptic plasticity at early stages of the disease. However, the mechanisms remain poorly understood. Several studies indicate an alteration of the mTORC1 pathway. Our results show that AβOs inhibit mTORC1 recruitment to the membrane of endo-lysosomes and that this effect can be rescued by a pharmacological inhibition of AMPK. Thus our data indicate that AβOs inhibit mTORC1 translocation to endo-lysosomal compartments via AMPK. This could lead to the impairment of protein synthesis reported in other studies and thus alter synaptic function.

Στόχος: Ο υποδοχέας του γλουταμινικού οξέος ΝMDA (N-methyl-D-aspartate), αποτελεί μόριο-κλειδί που διαμεσολαβεί πολλούς τύπους συναπτικής πλαστικότητας στο κεντρικό νευρικό σύστημα. Στο οπτικό σύστημα η πλαστικότητα ξεκινά στο επίπεδο του αμφιβληστοειδούς χιτώνα. Κατά την ανάπτυξη του αμφιβληστροειδούς οι υποδοχείς NMDA συμμετέχουν σε φαινόμενα πλαστικότητας εξαρτώμενα από την εμπειρία όπως ο λειτουργικός διαχωρισμός των ON και OFF μονοπατιών. Η ανάπτυξη και η οπτική αποστέρηση επηρεάζουν επίσης τις ηλεκτροφυσιολογικές ιδιότητες των υποδοχέων NMDA στον αμφιβληστροειδή του επίμυος καθώς και την έκφραση των υπομονάδων του NR1 και NR2. Η βασική υπομονάδα NR1 υφίσταται εναλλακτικό μάτισμα με αποτέλεσμα να εμφανίζεται σε οκτώ διαφορετικές ισομορφές που προσδίδουν μοριακή ποικιλότητα στον υποδοχέα. Το ερώτημα που ετέθη ήταν εάν η έκφραση των ισομορφών της υπομονάδας NR1 ρυθμίζεται κατά την ανάπτυξη του αμφιβληστροειδούς χιτώνα του επίμυος και εάν μεταβάλλεται από την οπτική εμπειρία. Μέθοδος: Χρησιμοποιήθηκαν αμφιβληστροειδείς επίμυων (Wistar) που μεγάλωσαν είτε σε φυσιολογικό ημερήσιο κύκλο 12 ώρες φως/12 ώρες σκοτάδι [normal-reared (NR)], είτε σε διαρκές σκοτάδι [dark-reared (DR)] από την 9η έως την 60η ημέρα μετά τη γέννηση. Η μελέτη της έκφρασης των ισομορφών του αμινοτελικού (NR1a, NR1b) και του καρβοξυτελικού (NR1-1, NR1-2, NR1-3, NR1-4) άκρου της υπομονάδας NR1 έγινε με τη μέθοδο της real-time PCR. Αποτελέσματα: Το αναπτυξιακό προφίλ όλων των ισομορφών εμφάνισε διαφορετική αύξηση κατά τη διάρκεια της δεύτερης και τρίτης εβδομάδας, με μέγιστη έκφραση στο τέλος της τρίτης εβδομάδας. Μεταξύ των ισομορφών του αμινοτελικού άκρου, η NR1b εκφραζόταν σταθερά σε υψηλότερα επίπεδα σε σχέση με την NR1a, ενώ μεταξύ των ισομορφών του καρβοξυτελικού άκρου, η NR1-2 εκφραζόταν σε υψηλότερα επίπεδα από την NR1-4, ενώ τόσο η NR1-1 όσο και η NR1-3 εκφράζονταν σε χαμηλά επίπεδα. Η ανάπτυξη στο σκοτάδι μείωσε την έκφραση όλων των ισομορφών σε πολλά αναπτυξιακά στάδια και στο ενήλικο ζώο. Σημαντική αλληλεπίδραση μεταξύ ηλικίας και οπτικής εμπειρίας προέκυψε για τις ισομορφές NR1a, NR1-2 και NR1-4. Συμπεράσματα: Η έκφραση όλων των ισομορφών της υπομονάδας NR1 μεταβάλλεται κατά την ανάπτυξη του αμφιβληστροειδούς του επίμυος, ενώ ορισμένες (NR1a, NR1-2 and NR1-4) ρυθμίζονται τόσο από την ηλικία όσο και από την οπτική εμπειρία. Τέτοιες μεταβολές μπορεί να παίζουν σημαντικό ρόλο σε φαινόμενα πλαστικότητας που λαμβάνουν χώρα στον αμφιβληστροειδή χιτώνα. / Purpose: The N-methyl-D-aspartate (NMDA) type of glutamate ionotropic receptor is a key molecule mediating plasticity related processes in the central nervous system. Visual system plasticity begins in the retina. During postnatal retinal development NMDA receptor has been shown to be involved in experience dependent plasticity such as the functional segregation of ON and OFF pathways. Development and visual deprivation have been found to affect the kinetics of NMDA receptor in rat retina and the expression of its main subunits NR1 and NR2. The NR1 fundamental subunit of NMDA receptor exists in eight distinct splice isoforms. Knowing that alternative splicing of the NR1 subunit offers a further molecular diversity to the receptor, we have addressed the question of whether the alternative splicing of NR1 subunit of the NMDA receptor is regulated during postnatal retinal development and whether this regulation is altered by visual experience. Methods: Retinas were dissected from eyes of Wistar rats raised either in normal 12-hour light/12-hour dark cycle [normal-reared (NR)], or in complete darkness [dark-reared (DR)] at postnatal days 9 to 60. Real-time PCR was performed in order to assess the mRNA expression of NR1 isoforms using oligonucleotide primers specific for N- terminal (NR1a, NR1b) and C-terminal splice variants (NR1-1, NR1-2, NR1-3, NR1-4). Results: The developmental profiles of mRNA expression levels of both N- and C-terminal NR1 isoforms showed differential increases during the second and third postnatal weeks, while their expression peaked at the end of the third week. Among N-terminal isoforms NR1b was constantly expressed at higher levels compared to NR1a and among the C-terminal isoforms, NR1-2 was expressed at higher levels than NR1-4, while both NR1-1 and NR1-3 were expressed at low levels. Dark-rearing led to reductions in both N- and C-terminal NR1 variants in several developmental ages and in adult retina. A significant age and experience interaction was observed at NR1a N-terminal isoform, and at the most abundant C-terminal isoforms NR1-2 and NR1-4. Conclusions: Our results have demonstrated that all NR1 splice isoforms are developmentally regulated in rat retina and some of them (NR1a, NR1-2 and NR1-4) are also bidirectionally regulated by age and visual experience. Such changes may play an important role in the plastic and activity-dependent events taking place in retina.

Methadone and ketamine have been used in neuropathic pain management, but the benefits of the association of both drugs are uncertain. Aiming to enhance the analgesic efficacy and to reduce the occurrence of adverse-effects, an oral solutionof methadone plus ketamine was tested in patients suffering of neuropathic pain of different etiologies and resistant to other analgesics. All patients were treated in the Clinical Care & Pain Management of Santa Maria University Hospital (HUSM) between October 2006 and March 2008. Pain (primary aim) was analyzed before and several times after starting the treatment, through the visual analogical scale (VAS), allodynia and burning or shooting pain evaluation. The development of adverse-effects (secondary aim), such as somnolence, nausea, vomit, dizziness, hallucination, constipation and headache, was also investigated. The first part of this thesis presented the case report of 18 neuropathic pain patients treated with the oral solution of methadone plus ketamine during 6 months.Except for one patient that discontinued the treatment due to intolerable adverse effect, all patients reported pain relief and lowprevalence of adverse effects throughout the treatment. The second part of this thesis presents arandomized, double-blind study conducted with 37 neuropathic patients. Patients were randomly assigned in three treatment groups: methadone group (n=13), ketamine group (n=11) and methadone plus ketaminegroup (n=13). All patients received the designated treatment by oral route, during 3 months. Methadone, ketamine or methadone plus ketamine treatmentgradually (better effect in 30 than in 7 days) and largely (inhibition of about 70% at the end of treatment) reduced the level of neuropathic pain (VAS) with nodifference between the treatment groups.The proportion of patients presenting burning or shooting pain was similarly reduced in all treatmentgroups, but the prevalence of allodynia was significantly reduced only in ketamine group. Excepted for the somnolence that was more prevalent in methadone and methadone plus ketamine groups, the prevalence of the adverse effects was very similar in all treatment groups. All together, the present data show that the combination of methadone plus ketamine was effective to reliefneuropathic pain resistant to other analgesics, though the combination of the two drugs did not show superior efficacy than those reached by methadone or ketamine alone. This study strengthens the use of methadone or ketamine as alternatives to treat neuropathic pain. The drug of choice, however, should be based on the patient background. / Metadona e cetamina têm sido usados no tratamento da dor neuropática, mas os benefícios da associação das duas drogas são desconhecidos. Com o objetivo de melhorar a eficácia analgésica e reduzir a ocorrência de efeitos adversos, uma solução oral de metadona mais cetamina foi testada em pacientes que sofrem de dor neuropática de diferentes etiologias e resistentes a outros tipos de tratamento. Todos os pacientes foram tratados no ambulatório da dor do Hospital Universitário de Santa Maria (HUSM) entre outubro de 2006 e março de 2008. A dor foi analisada antes e várias vezes depois de iniciar o tratamento, através da escala visual analógica (EVA), bem como alodinia, dor em queimação e/ou em choque. Efeitos adversos, como sonolência, náuseas, vômitos, tontura, alucinação, constipação e dor de cabeça, também foram investigados. A primeira parte desta tese apresenta um relato de caso de 18 pacientes com dor neuropática, submetidos ao tratamento com a solução oral de metadona mais cetamina durante seis meses. Exceto para um paciente que descontinuou o tratamento devido a efeitos adversos intoleráveis, todos os pacientes relataram alívio da dor e de baixa prevalência de efeitos adversos ao longo do tratamento. A segunda parte desta tese apresenta um estudo duplo-cego randomizado, realizado com 37 pacientes apresentando dor neuropática de diferentes etiologias. Os pacientes foram divididos aleatoriamente em três grupos de tratamento: grupo metadona (n = 13), grupo cetamina (n = 11) e grupo metadona mais cetamina (n = 13). Todos os pacientes receberam o tratamento designado por via oral, durante 3 meses. O tratamento com metadona, cetamina ou metadona mais cetamina, gradualmente (melhor efeito em 30 do que em 7 dias) e em grande parte (inibição de cerca de 70% no final do tratamento) reduziu o nível de dor neuropática sem apresentar diferença significativa entre os grupos. A dor em queimação ou em choque apresentada pelos pacientes foi igualmente reduzida em todos os grupos de tratamento, mas a prevalência de alodinia foi significativamente reduzida apenas no grupo cetamina. A sonolência foi mais prevalente nos grupos da metadona e metadona mais cetamina, a manifestação dos demais efeitos adversos foi muito semelhante em todos os grupos de tratamento.

L’évolution du Syndrome Douloureux Régional Complexe (SDRC) peut aboutir à un état de douleur chronique invalidant et difficile à traiter. Parmi les stratégies thérapeutiques utilisables, la kétamine, administrée sur 5 jours à des doses infra-anesthésiques, a une place de choix. Cependant, il s'agit d'un traitement invasif et dont le mécanisme d'action antalgique n'est pas connu. Notre travail a porté sur deux aspects du traitement du SDRC par la kétamine: la prédiction de l’efficacité thérapeutique et la compréhension des mécanismes d'action. Dans un premier temps, chez 105 patients SDRC, nous avons évalué l'intérêt de la scintigraphie osseuse au Technétium 99 réalisée avant le traitement pour prédire l’efficacité thérapeutique de la kétamine. Cette efficacité était corrélée à l’augmentation relative de l'activité inflammatoire et du remodelage osseux objectivée par scintigraphie. Dans une deuxième étude, portant sur l’évaluation de l’excitabilité corticale par stimulation magnétique transcrânienne et incluant 19 patients SDRC, nous avons observé que la kétamine réprimait fortement et de façon bilatérale la facilitation intracorticale, paramètre de transmission glutamatergique, et en revanche restaurait l’inhibition intracorticale correspondant au côté douloureux, paramètre gabaergique qui était très altéré avant le traitement. L'effet antalgique de la kétamine était corrélé à cette restauration d'inhibition ainsi qu'à la réduction de la facilitation correspondant au côté sain. Ainsi, la kétamine semble jouer un rôle dans la balance d'influences gabaergiques et glutamatergiques transcalleuses. Ce travail a permis de caractériser de nouveaux mécanismes physiopathologiques ainsi qu’une nouvelle justification de l’efficacité thérapeutique de la kétamine dans le SDRC. / Regional Complex Pain Syndrome (CRPS) can result in a chronic pain condition that is disabling and difficult to treat. Among the therapeutic strategies that can be used, ketamine, administered over 5 days at infra-anesthetic doses, has a place of choice. However, it is an invasive treatment and the mechanism of analgesic action is not known. Our work focused on two aspects of the treatment of CRPS by ketamine: predicting therapeutic efficacy and understanding mechanisms of action. Initially, in 105 CRPS patients, we assessed the benefit of pre-treatment Technetium 99 bone scintigraphy to predict the therapeutic efficacy of ketamine. This efficacy was correlated with the relative increase in inflammatory activity and bone remodeling detected by scintigraphy. In a second study, evaluating cortical excitability by transcranial magnetic stimulation and including 19 CRPS patients, we observed that ketamine strongly and bilaterally repressed intracortical facilitation, a glutamatergic transmission parameter, and on the other hand restored the intracortical inhibition corresponding to the pain side, a gabaergic parameter which was very altered before the treatment. The analgesic effect of ketamine was correlated with this restoration of inhibition as well as the reduction of the facilitation corresponding to the healthy side. Thus, ketamine seems to play a role in the balance of gabaergic and glutamatergic transcallosal influences. This work allowed new physiopathological mechanisms to be characterized as well as a new justification of the therapeutic efficacy of the ketamine in the CRPS.

Introdução: Os receptores ionotrópicos do glutamato, como o N-metil-D-Aspartato (NMDA), estão envolvidos em desordens psiquiátricas. NMDARs são complexos heteroméricos que incorporam tres diferentes subunidades: NR1, NR2 e NR3. Objetivos: quantificar os níveis plasmáticos das subunidades NR1 e NR2 NMDAR em pacientes em primeiro episódio psicótico (PEP), em comparação com os irmãos e controles saudáveis. Métodos: Este é um estudo transversal de PEP na região de Ribeirão Preto, Brasil, sendo o grupo controle composto por indivíduos saudáveis, pareados por idade, sexo e mesma área de abrangência dos casos. Foram coletados 5 mL de amostra de sangue próxima a data de diagnóstico de PEP. A quantificação plasmática das subunidades NR1 e NR2 foi realizada por ELISA. Os dados foram analisados por ANOVA (significante se p<0,05) e curva ROC. Resultados: Foram incluídos 166 pacientes em PEP (idade: x = 30,34 ± 12,2 anos; 64% homens), destes 84 com diagnóstico de psicose não afetiva, 51 com transtorno bipolar e 31 com transtorno depressivo. Foram tambem incluídos 76 irmãos e 166 controles saudáveis. Os níveis plasmáticos das subunidades NR1 e NR2 foram significativamente menores em pacientes com transtornos psicóticos (NR1: x = 71,0 ± 100,3 pg/mL, NR2: x = 2,5 ± 2 ng/ml), transtorno bipolar (NR1: x = 185,7 ± 319,5 pg/ml; NR2: x = 2,1 ± 2,2 ng/ml), transtorno depressivo (NR1: x = 83,2 ±185,0 pg/ml; NR2: x = 2,1± 2,1 ng/ml) em comparação com os irmãos (NR1: x = 140,6 ± 193,8 pg/ml; NR2: = 6,2 ± 1,5 ng/ml) e voluntários saudáveis (NR1: x = 146,7 ± 361,1 pg/ml; NR2: x = 4,8 ± 2,2 ng/ml) [NR1 e NR2, p < 0,001]. Indivíduos com valores plasmáticos de NR2 inferiores a 3,648 ng/mL apresentam um risco 14,72 vezes maior de estar doente (PEP) de quem não possui o NR2 abaixo deste valor. Conclusões: Este é o primeiro estudo relatando a quantificação e a redução das concentrações plasmaticas das subunidades NR1 e NR2 em transtornos psiquiátricos graves quando comparados aos irmãos e controles, podendo a subunidade NR2 ser um candidato a biomarcador plasmático em pacientes com PEP. / Background: Ionotropic glutamate receptors, such as N-Methyl-D-Aspartate (NMDA), are involved in pathophysiology of several psychiatric disorders. NMDARs are described as heteromeric complexes incorporating distincts subunits within a repertoire of three types: NR1, NR2 and NR3. Aim: to quantify the NR1 and NR2 subunits NMDAR plasma levels in patients with first episode psychosis (FEP), compared with siblings and healthy controls. Methods: This is a cross-sectional study of FEP conducted in Ribeirão Preto, Brazil. The control group were composed by healthy subjects matched for age, sex and same coverage area of cases. 5 ml of blood sample were collected next to the date of FEP diagnosis. NR1 and NR2 subunits plasmatic quantification was performed by ELISA. Data were analyzed by ANOVA (significant at p < 0.05) and ROC curve. Results: FEP sample comprised 166 patients (age: x = 30.34 ± 12.2 years; 64% men), of these 84 with a diagnosis of psychotic disorder, 51 with bipolar disorder and 31 with depressive disorder. It was also included 76 siblings and 166 healthy controls. NR1 and NR2 subunits plasma levels were significantly lower in patients with psychotic disorders (NR1: x = 71.0 ± 100.3 pg / ml, NR2: x = 2.5 ± 2 ng/ml), bipolar disorder (NR1: x = 185.7 ± 319.5 pg/mL; NR2: x = 2.1 ± 2.2 ng/ml), depressive disorders (NR1: x = 83.2 ± 185.0 pg/mL; NR2: x = 2.1 ± 2.1 ng/ml) compared with siblings (NR1: x = 140.6 ± 193.8 pg/mL; NR2: x = 6.2 ± 1.5 ng/ml) and healthy volunteers (NR1: x = 146.7 ± 361.1 pg / mL; NR2: x = 4.8 ± 2.2 ng/ml) [NR1 and NR2, p < 0.001]. Interestingly, individuals with NR2 plasma values less than 3.648 ng/ml present 14.72 times a higher risk to be in FEP than other patients. Conclusions: This is the first study reporting the measurement and reduction of NR1 and NR2 subunits plasma concentrations in severe psychiatric disorders when compared to siblings and controls. And highlighting that NR2 subunit can be a candidate for plasma biomarker in patients with FEP.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-10T08:13:00Z No. of bitstreams: 2 Dissertação - Jaqueline Andrade Ribeiro da Silva - 2015.pdf: 2794453 bytes, checksum: 2542dd17b29db760b73cc18f188703fd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-10T08:14:50Z (GMT) No. of bitstreams: 2 Dissertação - Jaqueline Andrade Ribeiro da Silva - 2015.pdf: 2794453 bytes, checksum: 2542dd17b29db760b73cc18f188703fd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2015-12-10T08:14:50Z (GMT). No. of bitstreams: 2 Dissertação - Jaqueline Andrade Ribeiro da Silva - 2015.pdf: 2794453 bytes, checksum: 2542dd17b29db760b73cc18f188703fd (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-04-29 / This study evaluated the analgesic effects of ketamine administered by three intravenous continuous infusionprotocols (IC) in bitches undergoing mastectomy. Twenty-five bitches were distributedrandomly in three groups: intraoperative continuous infusion group (CONTROL, n=8), receiving the IC only during the intraoperative period; 12-hour continuous infusion group (IC12, n=8), receiving the IC during the intraoperative period and for 12 hours after surgery; and 24-hour continuous infusion group (IC24, n=8), receiving the IC during the intraoperative period and for 24 hours after the surgery. Cardiorespiratory evaluations were performed during the perioperative period, and two researchers evaluated the postoperative analgesia by the interactive visual analog scale (VASi) and by the Short form of Glasgow composite pain scale (GCPS-SC), 10 minutes and 2, 4, 6, 8, 12, 18 and 24 hours after extubation. Owners evaluated their dogs using a questionnaire adapted from the "Helsinki Chronic Pain Index" on the 4th and 10th daysafter the surgical procedure. Pain scores and postoperative opioid requirements did not differ among groups. Rescue analgesia was administered to 7/8, 7/8 e 6/8 bitches in CONTROL, IC12 and IC24 groups, respectively. The main adverse effects observed were tachycardia, tachypnea, hypertension and excitement. According to the owners’ criteria, the animals of IC12 and IC24 groups had lower pain scores on the 4th and 10th days after the surgical procedure. We concluded that, although some mild adverse effects could occur, IC of ketamine is an effective and safe analgesic adjuvant in the early postoperative period. However, the main benefit of the prolonged Ketamine IC is probably related to the best quality of life andrecovery in the last postoperative period. / Neste estudo foi avaliada a eficácia analgésica da cetamina, administrada em três diferentes regimes de infusão intravenosa contínua (IC), em cadelas submetidas a mastectomia unilateral. Foram incluídas no estudo 24 cadelas, distribuídas em três grupos denominados: grupo infusão contínua transoperatória (CONTROLE, n=8), nas quais a IC da cetamina foi administrada somente durante o período transoperatório; grupo infusão contínua de 12 horas (IC12, n=8), as quais receberam a IC durante o período transoperatório e 12 horas após o término da cirurgia; e grupo infusão contínua de 24 horas (IC24, n=8), as quais receberam a IC durante o período transoperatório e 24 horas após o término da cirurgia. Os parâmetros cardiorrespiratórios foram avaliados durante o período perioperatório e aanalgesia pós-operatória foi avaliada de acordo com as escalas análoga visual interativa (VASi) eComposta de Glasgow(GCPS-SC). As avaliações foram realizadaspor dois pesquisadores 10 minutos, 2, 4, 6, 8, 12, 18 e 24 horas pós-extubação.Os proprietários avaliaram seus animais por meiodo―Helsinki Chronic Pain Index‖ no 4º e no 10º diasapós o procedimento cirúrgico. Os escores de dor e o requerimento de morfina no período pós-operatório não diferiram estaticamente entre os grupos. O resgate analgésico foi administrado em 7/8, 7/8 e 6/8 animais nos grupos CONTROLE, IC12 e IC24, respectivamente. Os principais efeitos adversos da IC de cetamina observados foram taquipnéia e hipertensão. Na avaliação dos proprietários os grupos IC12 e IC24 tiveram menores escores de dor no quarto e no décimo dia de pós-operatório. Concluiu-se que, apesar de poder ocasionar efeitos adversos discretos, a IC de cetamina é eficaz como adjuvante no fornecimento de analgesia no período pós-operatório imediato, maso principal benefício de seu uso provavelmente esteja relacionado com melhor recuperação e qualidade de vida no período pós-operatório tardio.

Les encéphalites à anticorps (Ac) anti-récepteur NMDA (NMDAR) sont des pathologies auto-immunes nouvellement décrites ciblant un récepteur majeur du système nerveux central, le NMDAR. Les synapses glutamatergiques assurent la majeure partie de la transmission excitatrice du cerveau et les récepteurs ionotropiques au glutamate de type NMDA ont un rôle clé dans la plasticité synaptique, c'est-à-dire les modifications de la force de transmission synaptique constituant le corrélat cellulaire des processus d'apprentissage et de mémoire. Les études in vitro et in vivo de l'effet des Ac anti-NMDAR tendent à montrer une altération de la dynamique des NDMAR et une internalisation. Les Ac auraient un effet pathogénique pouvant expliquer des symptômes. Au cours de ma thèse, j'ai souhaité approfondir la compréhension de cette pathologie sur le plan clinique et mécanistique. Pour cela j'ai étudié les caractéristiques cliniques et histologiques des patients présentant une tumeur maligne associée, me permettant d'établir des recommandations de prise en charge au vu du taux de mortalité plus élevé chez ces patients et d'une fréquence plus importante de tératomes ovariens immatures que dans la population générale. En parallèle j'ai étudié l'effet des Ac anti-NMDAR sur la transmission et la plasticité synaptique dans un modèle murin d'administration chronique des anticorps. Ces études menées en électrophysiologie sur tranches aigües m'ont permis de mettre en évidence un effet des anticorps du LCR sur la plasticité synaptique significatif mais d'amplitude moindre que les études in vitro l'avaient suggéré. Nous avons également montré l'importance de la durée d'exposition aux anticorps / Anti-NMDAR autoimmune encephalitis is a newly described pathology, characterized by the presence of IgG antibodies (Abs) directed against NMDA receptor (NMDAR). Glutamatergic synapses are the main component of excitatory transmission in the adult brain and the ionotropic glutamate receptor NMDAR has a key role in synaptic plasticity. Synaptic plasticity is defined by the synapses property to modify their transmission strength and seems to be the cellular correlate of learning and memory. In vitro and in vivo studies on anti-NMDAR Abs effects showed an altered dynamic at the membrane followed by an internalization of NMDAR. Thus, Abs seem to have a pathogenic effect, able to explain clinical symptoms. During my thesis, I wanted to deepen the understanding of this pathology on the clinical and mechanistic level. To this end, I studied the clinical and histological features of patients with an associated tumor. Results obtained allow me to establish management recommendations considering the high mortality rate in these patients and a higher frequency of immature ovarian teratomas than in the general population. Simultaneously, I studied the effect of anti-NMDAR Abs on synaptic transmission and plasticity in a murine model allowing chronic Abs infusion. These results obtained by electrophysiology on acute slices allowed me to demonstrate an effect of CSF Abs on synaptic plasticity but of less amplitude that the in vitro studies had suggested. Results also highlighted the importance of the duration to antibodies exposure

The polyamines, putrescine, spermidine and spermine, are present in high concentrations in the central nervous system and, because of their policationic nature, they can interact with diverse cellular anionic targets (nucleic acids and proteins) and modulate the learning and memory by interacting with the polyamine binding site at N-methyl-D-aspartate receptor. In this study we investigated the effects of the systemic administration of spermidine and arcaine on the memory of the inhibitory avoidance task in rats. It was also determined whether the effects of the spermidine and arcaine involve state dependence. The animals were trained in an inhibitory avoidance apparatus (0.4 mA, 3s) and tested in the same apparatus 24 hours later. Immediate post-training administration of spermidine (50 mg/kg, i.p.) improved, while arcaine (10 and 30 mg/kg, i.p.) impaired step-down latencies in the inhibitory avoidance test. Administration of spermidine (50 mg/kg, i.p.) 15 minutes before testing did not alter the performance of rats which were injected with spermidine (50 mg/kg, i.p.) or vehicle immediately after training. However, administration of arcaine (30 mg/kg, i.p.) 15 minutes before testing reverted the impairment of memory caused by the administration of arcaine (30 mg/kg, i.p.) immediately after training. Administration of arcaine (30 mg/kg, i.p.) or MK-801 (0.03 mg/kg, i.p.) before testing partially reverted the impairment of memory caused by the administration of arcaine (30 mg/kg, i.p.) or MK-801 (0.03 mg/kg, i.p.) immediately after training, characterizing a crossed state dependence. The results suggest that memory improvement caused by the administration of spermidine immediately after training is not due to state dependence. In contrast, the impairment of memory induced by arcaine is due to state dependence. The crossed state dependence between arcaine and MK-801 supports that state dependence induced by arcaine is related to NMDA receptor hypofunction. / As poliaminas, putrescina, espermidina e espermina estão presentes em altas concentrações no sistema nervoso central e, por sua natureza policatiônica, podem interagir com sítios aniônicos de macromoléculas (ácidos nucléicos e proteínas) e modulam o aprendizado e a memória interagindo com o sítio de ligação das poliaminas no receptor N-metil-D-apartato. Neste estudo nós investigamos os efeitos da administração sistêmica de espermidina e arcaína sobre a memória da tarefa de esquiva inibitória em ratos. Também foi determinado se os efeitos da espermidina e arcaína envolvem dependência de estado. Os animais foram treinados em um aparelho de esquiva inibitória (0,4 mA, 3 seg) e testados no mesmo aparelho, 24 horas depois. A administração imediatamente após o treino de espermidina (50 mg/kg, i.p.) melhorou, enquanto que arcaína (10 e 30 mg/kg, i.p.) prejudicou a latência de descida da plataforma no teste da esquiva inibitória. A administração de espermidina (50 mg/kg, i.p.) 15 minutos antes do teste não afetou a performance dos ratos que foram injetados com espermidina (50 mg/kg, i.p.) ou veículo imediatamente após o treino. Entretanto, a administração de arcaína (30 mg/kg, i.p.) 15 minutos antes do teste reverteu o prejuízo da memória causado pela administração de arcaína (30 mg/kg, i.p.) imediatamente após o treino. A administração de arcaína (30 mg/kg, i.p.) ou MK-801 (0,03 mg/kg, i.p.) antes do teste reverteu parcialmente o prejuízo da memória causado pela administração de arcaína (30 mg/kg) ou MK-801 (0,03 mg/kg) imediatamente após o treino, caracterizando dependência de estado cruzada. Estes resultados sugerem que a melhora da memória causada pela administração de espermidina imediatamente após o treino não é devido à dependência de estado. Em contraste, o prejuízo da memória induzido pela arcaína é devido a uma dependência de estado. A dependência de estado cruzada entre arcaína e MK-801. sugere que a dependência de estado induzida pela arcaína envolve a hipofunção do receptor NMDA.

La maladie d’Alzheimer (MA) se caractérise par une perte des fonctions cognitives liée à une dégénérescence neuronale induite par l’accumulation de peptides amyloïdes-β (Aβs) dans des régions vulnérables du cerveau comme l’hippocampe. Au niveau moléculaire, les peptides Aβs se lient préférentiellement à la densité post-synaptique des synapses excitatrices, espace au niveau duquel la protéine d’échafaudage PSD-95 organise l’ancrage des récepteurs NMDA (RNMDAs) et régule leur mobilité membranaire. A l’aide d’une stratégie intégrative qui favorise des niveaux d’analyse verticaux (du phénotype aux événements moléculaires) et qui combine un ensemble d’approches corrélatives et invasives chez des souris double transgéniques APPswe/PS1dE9 modèles de la MA, nous avons mis en évidence que les peptides Aβs déstabilisent l’organisation synaptique (altération de l’expression de la PSD-95) et augmentent le pool extrasynaptique de sous-unités GluN2B des RNMDAs dans l’hippocampe. Cette réorganisation se traduit par une perturbation des fonctions mnésiques. Par ailleurs, il a été montré que certaines oscillations de l’activité hippocampique, comme les « sharp-wave ripples » (SWRs) générées pendant les périodes de sommeil, jouent un rôle crucial dans la formation de la mémoire. De façon surprenante, l’accumulation des peptides Aβs semble épargner la dynamique d’expression des SWRs durant les comportements de routine. Afin d’examiner l’effet potentiel des Aβs sur les SWRs chez des animaux confrontés à des challenges cognitifs, nous avons soumis des souris adultes injectées intracérébralement avec une solution d’Aβs à un test de reconnaissance spatiale. Alors qu’elles sont capables de former une mémoire à court terme, les souris Aβs montrent un oubli plus rapide, suggérant qu’elles encodent avec succès, mais qu’elles sont incapables de stabiliser et de rappeler une information acquise antérieurement. En l’absence d’une demande cognitive préalable, les propriétés des SWRs ne sont pas altérées par les Aβs. En revanche, lorsqu’elles doivent résoudre un test cognitif, les pics de SWRs normalement observés après encodage ou reconnaissance chez les souris témoins sont abolis chez les souris Aβs, indiquant une perturbation du traitement hippocampique de l’information spatiale. Pris dans leur ensemble, ces résultats identifient deux nouveaux mécanismes délétères sous-tendant les déficits de mémoire spatiale associés à la MA. / Cognitive impairments in Alzheimer’s disease (AD) are thought to be related to degenerative synaptic changes caused by the accumulation of amyloid-β peptides (Aβs) in vulnerable brain regions such as the hippocampus. At the molecular level, Aβs bind preferentially to the postsynaptic density of neuronal excitatory synapses, where the scaffolding post-synaptic protein-95 (PSD-95) organizes NMDA receptor (NMDAR) location as well as its downstream signaling. By using an integrative strategy which favoured vertical levels of analyses (from phenotype to molecular events) and combined a set of interrelated correlative and invasive approaches in a double transgenic mouse model of AD (APPswe/PS1dE9 mice), we were successful in establishing that Aβs destabilize the synaptic organization (reduction of expression of PSD-95) and increase the extrasynaptic pool of GluN2B-containing NMDAR in the hippocampus, a reorganization which translates into impaired memory functions. It is also well-known that hippocampal sharp wave-ripples (SWRs) generated during sleep periods are crucial for memory formation but accumulation of soluble Aβs, surprisingly seems to spare SWR dynamics during routine behavior. To unravel a potential effect of Aβs on SWRs in cognitively-challenged animals, we submitted vehicle- and Aβ-injected mice to spatial recognition memory testing. While capable of forming short-term memory, Aβ mice exhibited faster forgetting, suggesting successful encoding but an inability to adequately stabilize and/or retrieve previously acquired information. Without prior cognitive requirements, similar properties of SWRs were observed in both groups. In contrast, when cognitively challenged, the post-encoding and -recognition peaks in SWR occurrence observed in controls were abolished in Aβ mice, indicating impaired hippocampal processing of spatial information. Altogether these results identify two new disruptive mechanisms for the spatial memory deficits associated with AD.