Reduced activity of the N-methyl-D-aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine site on the NR1 subunit that may be a promising therapeutic target for psychiatric illness. Recently, D-serine has been discovered to be a high-affinity endogenous activator of the NMDAR glycine site. Levels of D-serine in the brain are controlled by its synthesis enzyme serine racemase (Srr) and its catabolic enzyme D-amino acid oxidase (DAO). This work investigates the NMDAR glycine site, D-serine, and D-serine-regulatory enzymes Srr and DAO in the pathophysiology and treatment of symptomatology relevant to schizophrenia and other psychiatric disorders. Pharmacological and genetic mouse models were used to alter glycine site function and D-serine availability. Behavioral responses in these models were assessed. Administration of exogenous D-serine and the glycine transporter 1 (GlyT-1) inhibitor ALX-5407 improved performance of C57BL/6J mice in behavioral tests examining prepulse inhibition (PPI) or latent inhibition (LI). These compounds also reversed impairments induced by the NMDAR antagonist MK-801, and produced similar beneficial effects to the classical atypical antipsychotic clozapine. Mice carrying a point mutation that leads to diminished NMDAR glycine site function demonstrated abnormally persistent LI and deficits in social approach and spatial recognition that were reversible by D-serine or clozapine administration. Similarly, mutant mice that lacked Srr function and had a severe reduction in D-serine displayed impairments in sociability, PPI, spatial recognition and memory. Behavioral deficits in mice without Srr were exacerbated by MK-801 and rescued by treatment with D-serine or clozapine. A genetically-induced loss of DAO function in mice resulted in the elevation of brain D-serine levels, and produced improvements in spatial reversal memory and extinction of a learned response in the Morris water maze, consistent with the effects of exogenous D-serine application in wild-type mice. Thus, deficiencies in NMDAR glycine site function and D-serine availability produce behavioral disturbances that are relevant to the negative and cognitive symptoms of schizophrenia. Activation of the NMDAR glycine site by D-serine, GlyT-1 inhibition, or diminished DAO activity may be beneficial for the treatment of schizophrenia and other psychopathologies involving cognitive dysfunction and persistent repetitive behaviors.

This thesis tests the hypothesis that increased neuronal cell death in SIDS infants is related to the ability of risk factors, such as prone sleeping, to expose infants to intermittent hypercapnic hypoxia (IHH). Based on the hypothesis that the NMDA system is linked to neuronal death, by way of excitotoxicity, correlations were also sought between cell death and changes in NMDA receptor (NR1) expression in brainstem nuclei controlling cardiorespiratory function. The first aim of this study was to verify that increased neuronal cell death occurs in SIDS infants. To verify a piglet model of SIDS risk factors, brainstem changes were examined in piglets exposed to IHH, and comparisons were made to changes seen in SIDS infants. The NMDA receptor was characterised in controls for both the human infant and the piglet groups. Comparisons of neuronal changes were made with SIDS infants, and piglets exposed to IHH. Non-radioactive in-situ hybridisation and immunohistochemistry were performed on formalin fixed and paraffin embedded brainstem tissue to identify markers of cell death (caspase-3, active caspase-3, and TUNEL), and to examine NR1 mRNA and protein expressions. Staining was quantified using computerised image analysis software. Eight nuclei from the brainstem medulla (caudal in piglets, and mid in infants), and two nuclei from the rostral pons (infants) were studied. The first dataset included human infants aged 1-6 months with a diagnosis of SIDS (n=15) or non-SIDS (n=10). The second dataset comprised developing piglets aged 13-14 days, with controls (n=6), against those exposed to IHH for 2 (n=6) or 4 (n=5) days. Increased neuronal cell death was not verified in the SIDS infants, but abnormalities in NR1 expression were present in selected nuclei of the medulla. Piglets exposed to IHH had increased neuronal cell death and changes in NR1 in selected nuclei of the medulla. There was also a positive correlation between increased cell death and high NR1 levels. Preliminary data showed that SIDS infants who usually slept prone had some differences in NR1 compared to those who did not usually sleep prone. From these findings, it was concluded that IHH may underlie the abnormalities in NMDA receptor expression that are present in the brainstem of SIDS infants. Although IHH can induce an increase in neuronal cell death, its significance in the aetiology of SIDS is not known. In piglets, IHH induced cell death correlated with high NMDA expression in some brainstem nuclei, supporting the hypothesis that excitotoxicity may be involved in the mechanism for cell death. Moreover, this thesis presents for the first time, �preliminary pathological proof� of an association between prone sleeping and abnormal NMDA receptor expression in SIDS infants.

Η πλαστικότητα - η ικανότητα του εγκεφάλου να αναδιοργανώνει τις συνδέσεις του δομικά και λειτουργικά σε απάντηση μεταβολών της αισθητικής εμπειρίας - είναι απαραίτητη για την ανάπτυξη των νευρικών κυκλωμάτων και την δυνατότητα προσαρμογής του νευρικού συστήματος στο περιβάλλον. Το οπτικό σύστημα έχει για χρόνια αποτελέσει το έδαφος των περισσότερων μελετών της εξαρτώμενης από την εμπειρία πλαστικότητας και αυτό χάριν του εύκολου χειρισμού της οπτικής εμπειρίας καθώς και των συνεπειών αυτής, που δύνανται να μελετηθούν σε ανατομικό και μοριακό επίπεδο αλλά και σε επίπεδο φυσιολογίας. Σε ζώα που μεγάλωσαν στο σκοτάδι από την γέννηση, οι φλοιϊκοί νευρώνες εμφανίζουν ιδιότητες ανώριμου οπτικού συστήματος όπως μεγαλύτερα υποδεκτικά πεδία, μειωμένη ικανότητα προσανατολισμού και κατεύθυνσης και μειωμένη οπτική οξύτητα, όπως αυτών που παρατηρείται κατά την στιγμή του ανοίγματος των οφθαλμών. Η πιο κλασική μορφή πλαστικότητας που χρησιμοποιήθηκε σαν μοντέλο για την κατανόηση του τρόπου με τον οποίο η δραστηριότητα συμμετέχει στην δημιουργία των νευρικών κυκλωμάτων είναι αυτή της οφθαλμικής επικράτησης (Hubel and Wiesel, 1963). Παρά το γεγονός ότι το οπτικό σύστημα αποτελεί για πολλά χρόνια ένα από τα καλύτερα μοντέλα για την μελέτη του φαινομένου της πλαστικότητας, εντούτοις οι περισσότερες εργασίες αναφέρονται κυρίως στον οπτικό φλοιό, στο έξω γονατώδες σώμα και στο άνω διδύμιο. Λιγότερες μελέτες αναφέρονται στον αμφιβληστροειδή όπου από τα πειράματα των Chalupa et al (1993) αλλά και Tian et al (2003) φαίνεται ότι η πλαστικότητα ίσως δεν είναι προνόμιο μόνο του οπτικού φλοιού. Οι μελέτες αυτές έδειξαν ότι τόσο φαρμακολογικοί παράγοντες όσο και η οπτική αποστέρηση δύνανται να οδηγήσουν σε αναδιοργάνωση της φυσιολογικής δομής του ιστού του αμφιβληστροειδούς. Το γλουταμινικό οξύ είναι ο κύριος διεγερτικός νευροδιαβιβαστής στο κεντρικό νευρικό σύστημα. Ένας από τους υποδοχείς μέσω των οποίων ασκεί τη δράση του είναι οι υποδοχείς του Ν-μέθυλο-D-ασπαρτικού οξέος (NMDA) οι οποίοι έχουν σημαντικό ρόλο στην πλαστικότητα λόγω της διττής ιδιότητας του να είναι ιοντο-εξαρτώμενοι και τασο-εξαρτώμενοι υποδοχείς. Πληθώρα ερευνών έχει δείξει ότι οι υποδοχείς αυτοί συμμετέχουν στους κυτταρικούς μηχανισμούς για την πλαστικότητα των νευρωνικών κυκλωμάτων όπως LTP (μακρόχρονη ενδυνάμωση) και LTD (μακρόχρονη καταστολή). Μελέτες είτε με φαρμακολογικό αποκλεισμό είτε με γενετική τροποποίηση των υποδοχέων NMDA έχουν δείξει την εξάρτηση της πλαστικότητας των κυκλωμάτων του οπτικού συστήματος από τους υποδοχείς NMDA. Γνωρίζουμε ότι μεταφραστικές και μετα-μεταφραστικές τροποποιήσεις, καθώς και ρύθμιση της διακίνησης των υποδοχέων NMDA παίζουν ενεργό ρόλο στον μηχανισμό πλαστικότητας της γλουταμινεργικής σύναψης. Ένας από τους βασικούς μηχανισμούς ρύθμισης των υποδοχέων NMDA είναι η φωσφορυλίωση που παίζει ρόλο τόσο στην ενδοκυττάρια διακίνηση του υποδοχέα όσο και στις ιδιότητες του διαύλου του υποδοχέα μεταβάλλοντας κατά αυτόν τον τρόπο την συναπτική ισχύ και εν τέλει συμμετέχοντας σε διάφορες μορφές συναπτικής πλαστικότητας. Με βάση τα παραπάνω, σκοπός της παρούσας μελέτης είναι να διερευνήσει τη συμβολή του γλουταμινεργικού υποδοχέα NMDA στην πλαστικότητα του αναπτυσσόμενου οπτικού συστήματος. Η παρούσα διατριβή μελετά την επίδραση της οπτικής εμπειρίας στην ρύθμιση των υπομονάδων του υποδοχέα NMDA στον αμφιβληστροειδή και τον οπτικό φλοιό επίμυων. Συγκεκριμένα, πρώτος στόχος της εργασίας είναι να μελετήσουμε την μεταβολή των επιπέδων έκφρασης των υπομονάδων NR2A και NR2B του υποδοχέα NMDA κατά την ανάπτυξη των επίμυων στο σκοτάδι (οπτική αποστέρηση) στον αμφιβληστροειδή. Δεύτερος στόχος να μελετήσουμε την επίδραση του οπτικής αποστέρησης στην φωσφορυλίωση της υπομονάδας ΝR2B στον αμφιβληστροειδή και στον οπτικό φλοιό. Τρίτος στόχος είναι να μελετήσουμε τα επίπεδα έκφρασης και φωσφορυλίωσης των υπομονάδων του υποδοχέα NMDA μετά από έκθεση στο φως των επίμυων που μεγάλωσαν στο σκοτάδι με σκοπό να εξετάσουμε εάν η ρύθμιση τους από την οπτική εμπειρία είναι αμφίδρομη. / Experimental manipulation of experience during development can have profound effects on the functioning of the resulting circuits. N-methyl-d-aspartate glutamate receptor (NMDAR) activity is required for the establishment and refinement of neural circuits during development. In the present study, the authors addressed the issue of experience-dependent regulation of NMDARs by examining the effects of visual experience and deprivation on subunit composition and subunit phosphorylation of NMDAR in the retina and visual cortex. Methods. Total homogenates were prepared from retinas and visual cortices of 30-day-old (P30) Wistar rats, raised either in a normal 12-hour light/12-hour dark cycle (normal-reared [NR]) or in complete darkness from birth (dark-reared [DR]). Some of the DR animals were exposed to light for 6 hours at P30 (DR+6h). Immunoblotting was performed for the NMDAR subunits, NR2A and NR2B, and for the phosphorylated NR2B subunit protein at serine 1303 (pNR2B-Ser1303). Results. Dark rearing for 1 month decreased the NR2A/NR2B ratio and increased the level of phosphorylation of NR2B subunit at Ser1303 in the retina and visual cortex. Light exposure at P30 reversed the effects of visual deprivation on NMDAR composition and NR2B phosphorylation in both regions. Conclusions. These results indicated that NMDAR subunit composition and NR2B phosphorylation at Ser1303 is regulated bidirectionally by visual experience and deprivation in rat retina and visual cortex.

Pregnenolone (PREG), the precursor of all neurosteroids, is synthesized in the nervous system from cholesterol and recent clinical studies indicate that reduced cognitive symptoms of schizophrenia correlate with elevated serum levels of pregnenolone sulfate (PregS), its immediate sulfated metabolite. PregS fulfills most of the classical criteria for an endogenous modulator of excitatory synaptic transmission, including: presence in nervous tissue at physiologically relevant concentrations, potentiation of N-methyl-D-aspartate receptor (NMDAR) mediated synaptic activity, and a mechanism for its inactivation. As NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, defects in neurosteroid metabolism might play a role in its associated cognitive dysfunction. PregS improves memory performance in rodents and augments long-term potentiation (LTP), an electrophysiological correlate of synaptic plasticity that is stabilized by phosphorylation of the cAMP response element binding protein (CREB). We have previously demonstrated that PregS at low picomolar (pM) concentrations increases intracellular Ca2+ and CREB via synaptic NMDARs. Therefore, we hypothesized that low pM concentrations of PregS might potentiate spontaneous excitatory postsynaptic currents (sEPSCs) and promote molecular events underlying synaptic plasticity. Here, using whole-cell patch clamp recordings, we report that PregS enhances the frequency of sEPSCs of cultured hippocampal neurons by about 2-fold while not altering their amplitude or passive membrane properties. This suggests that PregS acts presynaptically by increasing the frequency of neurotransmitter release or postsynaptically by activating silent synapses. We then investigated the hypothesis that PregS increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and NMDAR subtypes at synapses as a molecular switch for this enhancement. We measured receptor redistribution and phosphorylation using fluorescence imaging and Western blot technology. The results demonstrate that PregS (50pM, 10min): (1) Increases AMPAR (GluA1)/PSD95 colocalization (dependent on L-type voltage-gated Ca+2 channel and synaptic NMDAR activity), and increases phosphorylation of GluA1 at serine-831/845; (2) Increases casein kinase 2 (CK2) dependent surface NMDAR2A (GluN2A) but not GluN1 or GluN2B; and (3) Increases GluN2B serine-1480 phosphorylation. The results show that PregS increases the frequency of excitatory synaptic transmission and increases surface/synaptic AMPARs and surface GluN2A (but not GluN1 or GluN2B) NMDARs, shifting the molecular composition of young glutamatergic synapses toward the adult GluN2A enriched synaptic phenotype.

Dor neuropática (DN) é definida como “dor iniciada ou causada por lesão primária ou disfunção em sistema nervoso”, porém sua prevalência depende do tipo de trauma e da disfunção relacionada. Apesar desta condição dolorosa ser considerada altamente prevalente e debilitante, os tratamentos disponíveis são relacionados a efeitos adversos dificultando a adesão. Devido a isso, buscam-se alternativas não farmacológicas para o tratamento deste tipo de dor, entre elas, as técnicas de neuromodulação periférica, como acupuntura (AC) e eletroacupuntura (EA). Estas técnicas podem ser combinadas com intervenções farmacológicas e não farmacológicas e têm apresentado resultados promissores no tratamento da dor neuropática. No entanto, seus mecanismos de ação não estão totalmente elucidados, desta forma a utilização de modelos animais é de grande valia para o estudo destes mecanismos no tratamento da dor neuropática e da patofisiologia deste tipo de dor crônica. É importante salientar que a aplicação de AC e EA em animais acordados é complexa, visto que gera desconforto e pode alterar a analgesia induzida pelo tratamento. Em muitos estudos a anestesia com isoflurano é utilizada durante a aplicação dos tratamentos, porém sua utilização pode gerar um viés no estudo, considerando a possível interferência do fármaco nos resultados comportamentais e neuroquímicos. Outro importante foco de estudo consiste em comparar as duas técnicas, AC e EA, buscando determinar qual destas é a mais eficaz no tratamento da dor neuropática. Considerando o exposto acima, os objetivos desta tese foram: 1) avaliar os parâmetros comportamentais e neuroquímicos dos efeitos da utilização de anestesia na aplicação de AC e EA em ratos submetidos ao modelo de DN; 2) comparar os efeitos da AC e EA em modelo animal de DN por meio de parâmentros comportamentais, neuroquímicos e histológicos. Considerando os resultados obtidos nesta tese, concluímos que o isoflurano aumenta a analgesia promovida por AC e EA, provavelmente diminuindo o efeito do estresse gerado pela aplicação dos tratamentos em animais acordados, resultado que é corroborado pela diminuição do nível de S100β periférico (marcador de morte neuronal central); Por outro lado, o isoflurano diminuiu os níveis de fator de crescimento neuronal (NGF) no nervo periférico lesado, indicando diminuição do processo de regeneração neural, enquanto a EA aumentou. Ao mesmo tempo, o isoflurano alterou os efeitos dos tratamentos nos comportamentos exploratórios e nos níveis de N-metil D-aspartato em tronco encefáfio e medula espinhal. A AC apresentou-se mais eficaz no tratamento da DN em comparação à EA, porém nenhum dos tratamentos foi capaz de alterar os danos causados pela indução da DN no músculo gastrocnemio esquerdo dos animais demonstrado na histologia. Todavia, este resultado não alterou a analgesia gerada pelos tratamentos. / Neuropathic pain (NP) is defined as "pain initiated or caused by primary injury or dysfunction in the nervous system," but its prevalence depends on the type of trauma and related dysfunction. Although this painful condition is considered to be highly prevalent and debilitating, the available treatments are related to adverse effects, making adherence difficult. Because of this, non-pharmacological alternatives for the treatment of this type of pain are sought, among them, the techniques of peripheral neuromodulation, such as acupuncture (AC) and electroacupuncture (EA). These techniques can be combined with pharmacological and non-pharmacological interventions and have shown promising results in the treatment of neuropathic pain. However, its mechanisms of action are not fully elucidated, so the use of animal models is of great value for the study of these mechanisms in the treatment of neuropathic pain and the pathophysiology of this type of chronic pain. It is important to emphasize that the application of AC and EA in awake animals is complex, since it generates discomfort and can alter the analgesia induced by the treatment. In many studies, anesthesia with isoflurane is used during the application of the treatments, but its use may generate a bias in the study, considering the possible interference of the drug in the behavioral and neurochemical results. Another important focus of the study is to compare the two techniques, AC and EA, seeking to determine which is the most effective in the treatment of neuropathic pain. Considering the above, the objectives of this thesis were: 1) to evaluate the behavioral and neurochemical parameters of the effects of the use of anesthesia in the application of AC and EA in rats submitted to the DN model; 2) to compare the effects of AC and EA on animal model of DN by means of behavioral, neurochemical and morphological parameters. Considering the results obtained in this thesis, we conclude that isoflurane increases the analgesia promoted by AC and EA, probably decreasing the effect of the stress generated by the application of the treatments in agreed animals, a result that is corroborated by the decrease in the level of peripheral S100β (biomarker of central neuronal injury); On the other hand, isoflurane decreased the levels of neural grown factor (NGF) in the injured peripheral nerve, indicating a decrease in the neural regeneration process, while the EA increased. At the same time, isoflurane altered the effects of treatments on exploratory behaviors and N-metil-D-aspartato (NMDA) levels in the brainstem and spinal cord. AC was more effective in the treatment of DN compared to EA, but none of the treatments was able to alter the damage caused by DN induction in the left gastrocnemius muscle of the animals showed in histology. However, this result did not alter the analgesia generated by the treatments.

Master of Science / Department of Psychological Sciences / Mary Cain / Research has demonstrated that an enriched rearing environment improves learning in many tasks. However, growing evidence suggests that an enriched environment may not provide the same benefits during a fear conditioning paradigm. In fact, it appears that an isolated rearing environment may facilitate acquisition of fear to an aversive stimulus. The neural mechanisms responsible for this disparity in fear learning among differentially reared animals are currently unknown. The NR2B subunit of the NMDA receptor has been shown to be involved in the acquisition of fear and influenced by differential rearing, making it a prime candidate to begin investigating these underlying neural mechanisms. Therefore, this study assessed the expression of the NR2B subunit in brain regions important for the acquisition of fear (amygdala and hippocampus) among differentially reared rats. Rats were reared in an enriched, an isolated, or a standard condition for 30 days. They received four tone-footshock pairings, after which their brains were removed and expression of the NR2B subunit was quantified in the basolateral amygdala (BLA), central nucleus of the amygdala (ACe), and the CA3 region of the hippocampus. Analyses found that the isolated rats began to acquire fear to the aversive stimulus faster than the enriched and standard housed rats. However, the isolated rats showed the least amount of NR2B expression in the BLA while there were no rearing differences in expression within the ACe or the CA3. The results from this study provide further insight to the importance of the rearing environment in learning and memory, especially the learning of fear, and its central neural basis.

Dor neuropática (DN) é definida como “dor iniciada ou causada por lesão primária ou disfunção em sistema nervoso”, porém sua prevalência depende do tipo de trauma e da disfunção relacionada. Apesar desta condição dolorosa ser considerada altamente prevalente e debilitante, os tratamentos disponíveis são relacionados a efeitos adversos dificultando a adesão. Devido a isso, buscam-se alternativas não farmacológicas para o tratamento deste tipo de dor, entre elas, as técnicas de neuromodulação periférica, como acupuntura (AC) e eletroacupuntura (EA). Estas técnicas podem ser combinadas com intervenções farmacológicas e não farmacológicas e têm apresentado resultados promissores no tratamento da dor neuropática. No entanto, seus mecanismos de ação não estão totalmente elucidados, desta forma a utilização de modelos animais é de grande valia para o estudo destes mecanismos no tratamento da dor neuropática e da patofisiologia deste tipo de dor crônica. É importante salientar que a aplicação de AC e EA em animais acordados é complexa, visto que gera desconforto e pode alterar a analgesia induzida pelo tratamento. Em muitos estudos a anestesia com isoflurano é utilizada durante a aplicação dos tratamentos, porém sua utilização pode gerar um viés no estudo, considerando a possível interferência do fármaco nos resultados comportamentais e neuroquímicos. Outro importante foco de estudo consiste em comparar as duas técnicas, AC e EA, buscando determinar qual destas é a mais eficaz no tratamento da dor neuropática. Considerando o exposto acima, os objetivos desta tese foram: 1) avaliar os parâmetros comportamentais e neuroquímicos dos efeitos da utilização de anestesia na aplicação de AC e EA em ratos submetidos ao modelo de DN; 2) comparar os efeitos da AC e EA em modelo animal de DN por meio de parâmentros comportamentais, neuroquímicos e histológicos. Considerando os resultados obtidos nesta tese, concluímos que o isoflurano aumenta a analgesia promovida por AC e EA, provavelmente diminuindo o efeito do estresse gerado pela aplicação dos tratamentos em animais acordados, resultado que é corroborado pela diminuição do nível de S100β periférico (marcador de morte neuronal central); Por outro lado, o isoflurano diminuiu os níveis de fator de crescimento neuronal (NGF) no nervo periférico lesado, indicando diminuição do processo de regeneração neural, enquanto a EA aumentou. Ao mesmo tempo, o isoflurano alterou os efeitos dos tratamentos nos comportamentos exploratórios e nos níveis de N-metil D-aspartato em tronco encefáfio e medula espinhal. A AC apresentou-se mais eficaz no tratamento da DN em comparação à EA, porém nenhum dos tratamentos foi capaz de alterar os danos causados pela indução da DN no músculo gastrocnemio esquerdo dos animais demonstrado na histologia. Todavia, este resultado não alterou a analgesia gerada pelos tratamentos. / Neuropathic pain (NP) is defined as "pain initiated or caused by primary injury or dysfunction in the nervous system," but its prevalence depends on the type of trauma and related dysfunction. Although this painful condition is considered to be highly prevalent and debilitating, the available treatments are related to adverse effects, making adherence difficult. Because of this, non-pharmacological alternatives for the treatment of this type of pain are sought, among them, the techniques of peripheral neuromodulation, such as acupuncture (AC) and electroacupuncture (EA). These techniques can be combined with pharmacological and non-pharmacological interventions and have shown promising results in the treatment of neuropathic pain. However, its mechanisms of action are not fully elucidated, so the use of animal models is of great value for the study of these mechanisms in the treatment of neuropathic pain and the pathophysiology of this type of chronic pain. It is important to emphasize that the application of AC and EA in awake animals is complex, since it generates discomfort and can alter the analgesia induced by the treatment. In many studies, anesthesia with isoflurane is used during the application of the treatments, but its use may generate a bias in the study, considering the possible interference of the drug in the behavioral and neurochemical results. Another important focus of the study is to compare the two techniques, AC and EA, seeking to determine which is the most effective in the treatment of neuropathic pain. Considering the above, the objectives of this thesis were: 1) to evaluate the behavioral and neurochemical parameters of the effects of the use of anesthesia in the application of AC and EA in rats submitted to the DN model; 2) to compare the effects of AC and EA on animal model of DN by means of behavioral, neurochemical and morphological parameters. Considering the results obtained in this thesis, we conclude that isoflurane increases the analgesia promoted by AC and EA, probably decreasing the effect of the stress generated by the application of the treatments in agreed animals, a result that is corroborated by the decrease in the level of peripheral S100β (biomarker of central neuronal injury); On the other hand, isoflurane decreased the levels of neural grown factor (NGF) in the injured peripheral nerve, indicating a decrease in the neural regeneration process, while the EA increased. At the same time, isoflurane altered the effects of treatments on exploratory behaviors and N-metil-D-aspartato (NMDA) levels in the brainstem and spinal cord. AC was more effective in the treatment of DN compared to EA, but none of the treatments was able to alter the damage caused by DN induction in the left gastrocnemius muscle of the animals showed in histology. However, this result did not alter the analgesia generated by the treatments.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

The sigma-1 receptor (sig-1R) is a unique endoplasmic reticulum (ER) chaperone protein that interacts with a variety of voltage- and ligand-gated ion channels, which are components of an intricate system that regulates neuronal functioning. While there is an extensive body of knowledge pertaining to the sig-1R, many questions remain. The first question this thesis addresses is how the sig-1R modulates the functioning of the N-methyl-D-aspartate receptor (NMDAR). Using a heterologous expression system, I provide evidence that the mechanism of modulation is likely not a direct interaction between sig-1R and NMDAR and that this is not affected by the presence or absence of the membrane-associated guanylate kinases (MAGUK) protein PSD-95. The next question addressed investigates the impact of sig-1R absence on the synaptic physiology and action potential firing of CA1 pyramidal neurons. It was found that there is not a significant difference in these parameters, suggesting a non-essential role of the sig- 1R under normal physiological conditions. The third topic covered in my studies explores the sig-1R KO mouse in the Aβ25-35 infusion model of Alzheimer’s disease (AD). Preliminary results suggest that there is a dysfunction in the action potential characteristics and after- hyperpolarization characteristics of challenged sig-1R KO mice. Overall my results provide the groundwork for future experiments that will lead to a better understanding of the sig-1R and its role in cellular and synaptic physiology.

Ischemic strokes cause excessive release of glutamate, leading to overactivation of N-methyl-D-aspartate receptors (NMDARs) and excitotoxicity-induced neuronal death. For this reason, inhibition of NMDARs has been a central focus in identifying mechanisms to avert this extensive neuronal damage. N-acetyl-aspartyl-glutamate (NAAG), the most abundant neuropeptide in the brain, is neuroprotective in ischemic conditions in vivo. Despite this evidence, the exact mechanism underlying its neuroprotection, and more specifically its effect on NMDARs, is currently unknown due to conflicting results in the literature. Here, we uncover a pH-dependent and subunit specific action of NAAG on NMDARs. Using whole-cell electrophysiological recordings on acute hippocampal slices from adult mice and on HEK293 cells, we found that NAAG increases synaptic GluN2A-containing NMDAR excitatory postsynaptic currents (EPSCs), while effectively decreasing extrasynaptic GluN2B-containing NMDAR EPSCs in physiological pH. Intriguingly, the results of our study further show that in low pH, which is a physiological occurrence during ischemia, NAAG depresses GluN2A-containing NMDAR EPSCs and amplifies its inhibitory effect on GluN2B-containing NMDAR EPSCs, as well as upregulates the surface expression of the GluN2A subunit. Altogether, our data demonstrate that NAAG has differential effects on NMDAR function based on subunit composition and extracellular pH levels. These findings suggest that the role of NAAG as a neuroprotective agent during an ischemic stroke is likely mediated by its ability to reduce NMDAR excitation. The inhibitory effect of NAAG on NMDARs and its enhanced function in acidic conditions makes NAAG a prime therapeutic agent for the treatment of ischemic events.