Alterações no receptor N-metil-D-aspartato(NMDAR) marcam o envelhecimento. Um dos efeitos desta via é a ativação da óxido nítrico sintase (NOS) e a produção GMP cíclico (GMPc), promovendo modulação da Na+,K+-ATPase. A proteína αKlotho tem função anti-envelhecimento, e os animais hipomórficos para klotho (kl-/-) são caracterizados por danos periféricos e no Sistema Nervoso Central (SNC). O objetivo deste trabalho é verificar possíveis alterações na via NMDAR-NOS-GMPc e na Na+,K+-ATPase no SNC de camundongos kl-/-. Os dados obtidos apontam para um aumento da fosforilação do NMDAR , atividade da NOS, e redução de GMPc em hipocampo. Em cerebelo ocorre uma redução na fosforilação do NMDAR, atividade da NOS, sem alterações nos níveis de GMPc. Diferenças na expressão das subunidades GluN2 do NMDAR ocorrem em ambas estruturas. Também, observamos uma redução na atividade da α2/α3-Na+,K+-ATPase em cerebelo, e alterações na expressão de α2 e α3 em hipocampo e de α2 em cerebelo. Os dados reforçam a participação destas vias nas alterações em SNC dos animais kl-/-. / Alterations in N-Methyl-D-Aspartate receptor (NMDAR) are typical features of aging. Nitric oxide synthase (NOS) activation and cyclic GMP (cGMP) production, promoting Na+,K+-ATPase modulation are key events in NMDAR signaling. αKlotho protein has anti-aging function, and mice carrying hypomorph allele for klotho gene (kl-/-) are characterized by systemic and central nervous system (CNS) damage. The aim of this work is to verify whether alterations in NMDAR-NOS-cGMP pathway and in Na+,K+-ATPase occur in the CNS of kl-/- mice. Present data point for an increase in NMDAR phosphorylation, NOS activity and reduction in cGMP levels. In cerebellum, a decrease in NMDAR phosphorylation and NOS activity occur, with no changes in cGMP levels. Both situations are followed by changes in GluN2 subunity expression. Furthermore, we saw a reduction in α2/α3-Na+,K+-ATPase activity in cerebellum, and alterations in α2 and α3 in hippocampus and in cerebellar α2. Data presented support these pathways participation in age related conditions, using kl-/- mice as a model to study CNS damage.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Lesões sistêmicas peri e pré-natais alteram o desenvolvimento do SNC, levando a problemas cognitivos e motores em crianças que podem perdurar por toda a vida. Um tipo particular de lesão é a hipóxia-isquemia (HI), caracterizada pela interrupção momentânea ou permanente do fluxo sanguíneo. Um dos mecanismos propostos para as lesões decorrentes da HI é a excitotoxicidade glutamatérgica. O uso de inibidores da neurotransmissão glutamatérgica tem sido estudados em diversos modelos de HI. Neste trabalho, avaliamos os efeitos morfofuncionais da administração de um antagonista não-competitivo do receptor de glutamato NMDA sobre o desenvolvimento do cerebelo. Ratas no 18 dia de gestação foram anestesiadas, os cornos uterinos expostos e as 4 artérias uterinas obstruídas por 45 minutos (Grupo H). Animais controle tiveram os úteros expostos, sem a obstrução (Grupo S). Após a cirurgia a gestação prosseguiu. Somente animais nascidos a termo foram utilizados. Um dia após o nascimento, metade de cada ninhada foi designada para receber MK801, 0,3mg/kg/dia, (grupos SM e HM) e a outra metade recebeu solução salina (grupos SS e HS), por 5 dias. Após anestesia e perfusão-fixação com paraformaldeído 4% aos 9, 23, 30 e 60 dias pós-natais, cortes parassagitais do cerebelo foram obtidos em criótomo e submetidos à imunohistoquímica para calbindina, GFAP, GLAST, PDGFRα e MBP. A partir de 45 dias de vida, os animais foram testados em vários de testes comportamentais: labirinto em cruz elevado (LCE), campo vazado (CV), ROTAROD, teste de caminhada sobre barras (ladder test) e teste do comprimento da passada (stride length). Aos 9 dias, a espessura da árvore dendrítica era menor nos animais SM, HS/HM, demonstrando efeitos deletérios tanto do MK801 quanto da HI. Menor número de células PDGFRα+ foi observado nos animais HS/HM, sem efeitos da administração de MK801. Aos 23 dias, maior número de células PDGFRα+ foi observado nos animais HM comparado aos outros 3 grupos, indicando efeito neuroprotetor do MK801. Nessa idade, menor número de fibras mielinizadas (MBP+) foi observada nos animais HS, e a administração de MK801 parece reverter estes efeitos. Aos 9 dias a distribuição de GLAST estava alterada nos animais HS, com os efeitos da HI parcialmente revertidos pelo MK801. Não foram observados efeitos da HI ou do MK801 sobre comportamentos relacionados a ansiedade pelo LCE, assim como na latência de queda no ROTAROD. HI piora a performance motora no ladder test. No teste do CV, não observamos efeitos da HI sobre a busca por novidade assim como sobre a atividade locomotora espontânea. No entanto, MK801 diminui comportamentos de autolimpeza e a atividade locomotora espontânea. Menor variação das passadas foi observada em decorrência da administração de MK801 no stride length, com nenhum efeito da HI. Nossos resultados demonstram que a inibição do receptor NMDA tem um efeito neuroprotetor sobre os progenitores de oligodendrócitos e mielinização, provavelmente pela manutenção da capacidade proliferativa por um período maior. A atividade do receptor NMDA exerce importante papel na diferenciação das células de Purkinje, assim como na distribuição do transportador GLAST, corroborando a importância deste receptor na gênese das lesões causadas pela HI. / Peri and prenatal systemic lesions alter CNS development leading to motor and cognitive problems in children that might persist throughout life. A particular kind of injury, the hypoxic ischemic (HI), is characterized by a permanent or temporary blockage of blood flow. One of the proposed mechanisms downstream from a HI event is called glutamatergic excitotoxicity. The administration of glutamate inhibitors has been studied in HI models for several years. In this work, we evaluated the effects of administration of a non-competitive antagonist of glutamate receptor, NMDA, on cerebellar development and behavioral tests of HI animals. Pregnant rats in the 18th gestational day were anesthetized, the uterine horns were exposed and the four uterine arteries were clamped for 45 minutes (group H). Sham controls had the uterine horns exposed, but no arteries were clamped (group S). Gestation proceeded after surgery. Only full term animals were used. One day after birth half the animals was assigned to receive either SALINE (groups SS and HS) or MK801 (groups SM and HM). Animals were anesthetized and perfused with 4% paraformaldehyde at 9, 23, 30 and 60 days of age. Parasagittal cerebellar sections were submitted to Calbindin, GFAP, GLAST, PDGFRα and MBP immunohistochemistry. Beginning at P45 animals were subjected to a battery of behavioral tests: elevated plus maze (EPM), hole board (HB), ROTAROD, ladder test and stride length. At P9 the dendritic tree of Purkinje cells were thinner in SM, HS/HM animals, indicating that both HI and MK801 are deleterious regarding this Purkinje cell differentiation. A lower number of PDGFRα+ cells was observed in HS/HM animals, with no effects of MK801 administration. At P23 a greater number of PDGFRα+ cells was found in HM animals when compared to the other 3 groups, demonstrating a neuroprotector effect of MK801. A lower number of myelinated fibers (MBP+) was observed in HS animals at P9, and MK801 administration reverse this effect. At P9, GLAST distribution was altered in HS animals, and MK801 partially reverse this altered distribution. No effects of HI and MK801 were observed in the EPM and ROTAROD tests. HI decreased motor performance of hind limbs in the ladder test, though no effect of MK801 was noted. In the HB test, we do not observe HI effects regarding the novelty seeking behavior and locomotor activity, otherwise the administration of MK801 decreased the number of grooming and locomotor activity. In the stride length test, we do not observed effects of HI although MK801 augmented the length variation of the fore limbs. Our results show that inhibition of NMDA receptors exerts a neuroprotector effect on oligodendrocyte progenitor cells and myelination, probably by temporarily inhibiting differentiation of those, providing more time to proliferate. NMDA activity exerts a crucial role in Purkinje cell differentiation as well as in GLAST distribution. Taken together our results lead us to conclude that NMDA receptor activity has an important role in the genesis of lesions caused by HI events.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-14T17:55:12Z No. of bitstreams: 1 RosaHelenaSilva.pdf: 2716670 bytes, checksum: 07dee1e77d704e91281c33c31e8c4938 (MD5) / Made available in DSpace on 2017-06-14T17:55:12Z (GMT). No. of bitstreams: 1 RosaHelenaSilva.pdf: 2716670 bytes, checksum: 07dee1e77d704e91281c33c31e8c4938 (MD5) Previous issue date: 2016-10-19 / Borreria verticillata (L.) G. Mey species known as broom vassourinha has antibacterial, antimalarial, hepatoprotective, antioxidative, analgesic and antiinflammatory activities; however, its antinociceptive action still demands more thorough investigation. The present study was to assess the antinociceptive activity of B. verticillata crude hydroalcoholic extract (EHBv) and the ethyl acetate fraction (FAc) by means of in vivo and in silico studies. In vivo assessment included the paw edema test, the writhing test, the formalin test and the tail flick test. Wistar rats and Swiss mice were divided into 6 groups and given the following treatments oral: 0.9% NaCl control group (CTL), 10 mg/kg memantine (MEM), 10 mg/kg indomethacin (INDO), 500 mg/kg EHBv (EHBv 500), 25 mg/kg FAC (FAc 25), 50 mg/kg and FAc (FAC 50). EHBv, FAc 25 and 50 treatments exhibited anti-edematous and peripheral antinociceptive effects. For in silico assessment, compounds found in FAc were subjected to molecular docking, and the leading compound was selected for molecular dynamics (MD) simulations. Ursolic acid exhibited better affinity parameters with the enzyme COX-2 and the NMDA receptor subunits GluN1a and GluN2B on molecular docking. In MD simulations, AU exhibited highly frequent interactions with residues Arg120 and Glu524 in the COX-2 active site and NMDA, whereby it might prevent COX-2 and NMDA receptor activation. Treatment with ursolic acid 10mg / Kg (AU) showed peripheral and central antinoceceptivo effect. The antinociceptive effect of B. verticillata might be predominantly attributed to peripheral actions, including the participation of anti-inflammatory components. Ursolic acid is the main active component and seems to be a promising source of COX-2 inhibitors and NMDA receptor antagonists / Borreria verticillata (L.) G. Mey espécie conhecida como vassourinha apresenta atividade antibacteriana, antimalárica, hepatoprotetora, antioxidante, analgésica e anti-inflamatória, entretanto sua atividade antinociceptiva é pouco estudada. O objetivo deste trabalho foi avaliar atividade antinociceptiva do extrato hidroalcoólico bruto (EHBv) e fração acetato de etila (FAc) de B. verticillata realizando estudos in vivo e in silico. Para avaliação in vivo, foram utilizados os testes do edema de pata, contorções abdominais, formalina e tail flick. Ratos Wistar e camundongos Swiss foram tratados via oral e divididos em 6 grupos: controle-NaCl 0.9%(CTL), memantina 10 mg/Kg (MEM), indometacina 10 mg/Kg (INDO), EHBv 500 mg/kg (EHBv 500), FAc 25 mg/Kg (FAc 25), FAc 50 mg/Kg (FAc 50). O tratamento com EHBv 500, FAc 25 e 50 apresentou efeito antiedematogênico e antinociceptivo periférico. Para avaliação in silico os compostos identificados na FAc foram submetidos a docagem molecular, o melhor composto foi selecionado para simulações de dinâmica e testado in vivo molecular. O ácido ursólico apresentou melhores parâmetros de afinidade com COX-2, GluN1a e GluN2B durante a docagem molecular. Nas simulações por dinâmica molecular, o ácido ursólico apresentou alta frequência de contatos com Arg120 e Glu524 do local ativo da COX- 2 e com o domínio LBD da Glun1a e GluN2B podendo com isso, impedir a ativação da COX-2 e do receptor NMDA. O tratamento com ácido ursólico 10mg/Kg (AU) apresentou efeito antinoceceptivo periférico e central. Sugere-se que o efeito antinociceptivo periférico de B. verticillata pode ser atribuído predominantemente à ação de compostos com ação anti-inflamatória. O ácido ursólico é o principal composto ativo, sendo um composto promissor para o desenvolvimento de fármacos inibidores da COX-2 e antagonistas dos receptores NMDA.

Acredita-se que diferenças hodológicas e diferente padrão de expressão gênica ao longo do eixo dorso/ventral do hipocampo seriam responsáveis pela distinta função entre a porção dorsal (HD) e ventral do mesmo (HV). HD seria responsável por processos cognitivos, tais como memória e aprendizagem espacial, e o HV pelas respostas neuroendócrinas e emocional-motivacionais ao estresse. No entanto, não há muitos estudos acerca de diferenças entre HD e HV na modulação de comportamentos relacionados à neurobiologia da depressão. Há, contudo, dados indicando que o bloqueio de receptores glutamatérgicos do tipo NMDA ou da síntese de NO no HD induz efeito semelhante ao dos antidepressivos (i.e. do tipo antidepressivo) no teste do nado forçado (TNF). Quanto ao HV, a função da neurotransmissão glutamatérgica/nitrérgica na neurobiologia da depressão permanece não investigado. Dessa forma, o objetivo do presente estudo foi realizar o bloqueio reversível do HD ou do HV, em diferentes momentos, em animais submetidos ao TNF. O próximo passo foi realizar a micro-injeção intra-HD ou intraHV do antagonista NMDA AP-7 ou do inibidor da óxido nítrico sintase neuronal (nNOS), N-PLA, ou do inibidor da guanilato ciclase solúvel (sGC), ODQ. Os resultados mostram que o bloqueio do HD ou do HV com cloreto de cobalto (CoCl2, bloqueador da neurotransmissão sináptica dependente de cálcio) não modificou o comportamento dos animais no TNF. No entanto a administração de AP-7 ou N-PLA ou ODQ no HV antes do teste, reduziu o tempo de imobilidade no TNF. Por outro lado, a injeção dessas drogas no HD foi capaz de reduzir o tempo de imobilidade quando administradas tanto depois do pré-teste quanto antes do teste. Estes resultados sugerem que as vias NMDA-NO do HD e do HV, estariam envolvidas na modulação da resposta comportamental frente ao estresse do nado forçado. Além disso, os dados indicam que a participação dessas estruturas é importante em diferentes momentos após a exposição ao estresse. / It is believed that hodological and genetic pattern of expression differences along dorsal/ventral hippocampal axis would be responsible for distinct functions attributed to its dorsal (DH) and ventral (VH) poles. DH would be responsible for cognitive process, such as spatial memory and learning, whereas the VH would be responsible for neuroendocrine and emotional-motivation responses to stress. However, there is no many studies about possible differences between DH and VH in the modulation of behavioral responses related to the neurobiology of depression. Though, there are data showing that the blockade of glutamatergic NMDA receptors blockers or NO synthesis inhibition within the DH induces similar effect to that of antidepressant drugs (like antidepressant effect) in the forced swimming test (FST). On the VH, The role of the glutamatergic/nitrergic neurotransmission remains to be investigated. Thus, the aim of this work was to perform the reversible blockade of DH or VH, at different times, in animals subjected to FST. Additionally, the next step was to perform a microinjections into the DH or the VH of NMDA antagonist (AP-7), inhibitor of neuronal nitric oxide synthase (nNOS, N-PLA), or the inhibitor of soluble guanylate cyclase (sGC, ODQ). The results show that blocking the VH or the DH (cobalt chloride, calcium dependent neurotransmission inhibitor) did not modify the behavior of animals during the TNF. However administration of AP-7, N-PLA or ODQ intra-HV, before testing, caused antidepressant-like effects. Moreover, injection of such drugs, intra-HD, was able to induce similar results when administered both after the pre-test and before testing. These results suggest that NMDA-NO pathway of both VH and DH is involved in the modulation of emotional responses to the forced swim stress, although there may be an interest differential participation of these structures at different times after exposure to stress.

Acredita-se que diferenças hodológicas e diferente padrão de expressão gênica ao longo do eixo dorso/ventral do hipocampo seriam responsáveis pela distinta função entre a porção dorsal (HD) e ventral do mesmo (HV). HD seria responsável por processos cognitivos, tais como memória e aprendizagem espacial, e o HV pelas respostas neuroendócrinas e emocional-motivacionais ao estresse. No entanto, não há muitos estudos acerca de diferenças entre HD e HV na modulação de comportamentos relacionados à neurobiologia da depressão. Há, contudo, dados indicando que o bloqueio de receptores glutamatérgicos do tipo NMDA ou da síntese de NO no HD induz efeito semelhante ao dos antidepressivos (i.e. do tipo antidepressivo) no teste do nado forçado (TNF). Quanto ao HV, a função da neurotransmissão glutamatérgica/nitrérgica na neurobiologia da depressão permanece não investigado. Dessa forma, o objetivo do presente estudo foi realizar o bloqueio reversível do HD ou do HV, em diferentes momentos, em animais submetidos ao TNF. O próximo passo foi realizar a micro-injeção intra-HD ou intraHV do antagonista NMDA AP-7 ou do inibidor da óxido nítrico sintase neuronal (nNOS), N-PLA, ou do inibidor da guanilato ciclase solúvel (sGC), ODQ. Os resultados mostram que o bloqueio do HD ou do HV com cloreto de cobalto (CoCl2, bloqueador da neurotransmissão sináptica dependente de cálcio) não modificou o comportamento dos animais no TNF. No entanto a administração de AP-7 ou N-PLA ou ODQ no HV antes do teste, reduziu o tempo de imobilidade no TNF. Por outro lado, a injeção dessas drogas no HD foi capaz de reduzir o tempo de imobilidade quando administradas tanto depois do pré-teste quanto antes do teste. Estes resultados sugerem que as vias NMDA-NO do HD e do HV, estariam envolvidas na modulação da resposta comportamental frente ao estresse do nado forçado. Além disso, os dados indicam que a participação dessas estruturas é importante em diferentes momentos após a exposição ao estresse. / It is believed that hodological and genetic pattern of expression differences along dorsal/ventral hippocampal axis would be responsible for distinct functions attributed to its dorsal (DH) and ventral (VH) poles. DH would be responsible for cognitive process, such as spatial memory and learning, whereas the VH would be responsible for neuroendocrine and emotional-motivation responses to stress. However, there is no many studies about possible differences between DH and VH in the modulation of behavioral responses related to the neurobiology of depression. Though, there are data showing that the blockade of glutamatergic NMDA receptors blockers or NO synthesis inhibition within the DH induces similar effect to that of antidepressant drugs (like antidepressant effect) in the forced swimming test (FST). On the VH, The role of the glutamatergic/nitrergic neurotransmission remains to be investigated. Thus, the aim of this work was to perform the reversible blockade of DH or VH, at different times, in animals subjected to FST. Additionally, the next step was to perform a microinjections into the DH or the VH of NMDA antagonist (AP-7), inhibitor of neuronal nitric oxide synthase (nNOS, N-PLA), or the inhibitor of soluble guanylate cyclase (sGC, ODQ). The results show that blocking the VH or the DH (cobalt chloride, calcium dependent neurotransmission inhibitor) did not modify the behavior of animals during the TNF. However administration of AP-7, N-PLA or ODQ intra-HV, before testing, caused antidepressant-like effects. Moreover, injection of such drugs, intra-HD, was able to induce similar results when administered both after the pre-test and before testing. These results suggest that NMDA-NO pathway of both VH and DH is involved in the modulation of emotional responses to the forced swim stress, although there may be an interest differential participation of these structures at different times after exposure to stress.

Les récepteurs NMDA (NMDARs) sont des canaux cationiques activés par le glutamate. Les NMDARs participent au développement cérébral, à la plasticité synaptique, à l'apprentissage, à la mémoire et aux fonctions cognitives supérieures. Des variants pathogènes de GRIN2A, codant pour la sous-unité GluN2A des NMDARs, peuvent causer des épilepsies focales et encéphalopathies épileptiques de l'enfance avec troubles du langage et de la parole, connue sous le nom de spectre des épilepsies-aphasies (EAS). Les caractéristiques communes de l'EAS comprennent une activité épileptiforme âge-dépendante activée pendant le sommeil lent associées à des troubles de la parole, de la cognition et du comportement qui peuvent persister à l'âge adulte. Afin de commencer à identifier les événements précoces possiblement associés aux altérations de GluN2A, nous avons exploré le modèle correspondant de souris knock-out (KO) du gène Grin2a. Nous avons notamment recherché des altérations précoces de la communication vocale, de la (micro)structure cérébrale, et de l'activité électrique néocorticale. Nos données démontrent l'existence de plusieurs altérations à ces différents niveaux, parfois transitoirement à des stades spécifiques. De plus, les enregistrements néocorticaux mettent en évidence des anomalies de divers types liées au sommeil lent. Nos résultats indiquent également un rôle de GluN2A dans la communication vocale, dans l'organisation de la microstructure cérébrale, et dans la maturation des activités d’ondes lentes. Ces données suggèrent que les souris KO Grin2a représentent un modèle fiable pour appréhender les mécanismes physiopathologiques associés à l’EAS et leur séquence temporelle. / NMDA receptors (NMDARs) are cation channels that are gated by glutamate - the major excitatory neurotransmitter of the central nervous system. NMDARs participate in brain development, synaptic plasticity, learning, memory and high cognitive functions. Pathogenic variants in the GRIN2A gene, which encodes the GluN2A subunit of the NMDARs, can cause a group of childhood focal epilepsies and epileptic encephalopathies with speech and language dysfunction, known as the epilepsy-aphasia spectrum (EAS). Features shared in common by EAS disorders include age-dependent epileptiform activity activated in sleep associated with speech, neuropsychological and behavioral deficits that may persist in adulthood. In order to start in deciphering the early events possibly associated with the dysfunctioning of GluN2A-containing NMDARs, we have explored the corresponding Grin2a knock-out (KO) mouse model. That consisted in looking for early alterations of vocal communication, of brain (micro)structure, and of neocortical electrical activity. Our data demonstrated the existence of several alterations at those various levels. Some alterations were transient only, being detected at selective stages; also, neocortical recordings pointed for sleep-related anomalies of various types. Our data also indicated a role for GluN2A-containing NMDA receptors in vocal communication, fine organization of brain microstructure, and proper maturation of slow wave activity in sleep. Altogether, our data suggest that the Grin2a KO mice represent a reliable model to further elucidate the pathophysiological mechanisms associated with the disorders of EAS and their temporal sequences.

Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.

In this study we tested whether the prevalence or synaptic distribution of NMDA receptor subunits would be altered in the brain of the MeCP2-null mouse model of Rett syndrome. Detergent resistant membranes (DRMs) and post-synaptic densities (PSDs) were isolated from the synaptic membranes treated with TritonX-100, and resolved by sucrose density gradient centrifugation. Immunoblot analysis of the resulting density gradient fractions revealed that the relative distribution of the different NMDA receptor subunits between the DRM fractions, soluble fractions, and insoluble postsynaptic density fractions was preserved in the MeCP2-null brain. However, analysis of the overall NMDA receptor subunit prevalence within these fractions revealed a significant decrease in the expression of the NR1 and NR2A subunits, but not the NR2B subunit, in the MeCP2-null brain. The preservation of distribution of NMDAR subunits to the synaptic membranes, together with the decrease in NR1 and NR2A prevalence, suggest an imbalance in equilibrium between the mature and the immature synapses in a mouse model of Rett syndrome.

In this study we tested whether the prevalence or synaptic distribution of NMDA receptor subunits would be altered in the brain of the MeCP2-null mouse model of Rett syndrome. Detergent resistant membranes (DRMs) and post-synaptic densities (PSDs) were isolated from the synaptic membranes treated with TritonX-100, and resolved by sucrose density gradient centrifugation. Immunoblot analysis of the resulting density gradient fractions revealed that the relative distribution of the different NMDA receptor subunits between the DRM fractions, soluble fractions, and insoluble postsynaptic density fractions was preserved in the MeCP2-null brain. However, analysis of the overall NMDA receptor subunit prevalence within these fractions revealed a significant decrease in the expression of the NR1 and NR2A subunits, but not the NR2B subunit, in the MeCP2-null brain. The preservation of distribution of NMDAR subunits to the synaptic membranes, together with the decrease in NR1 and NR2A prevalence, suggest an imbalance in equilibrium between the mature and the immature synapses in a mouse model of Rett syndrome.

Anoxia is an environmental stress that few air-breathing vertebrates can tolerate for more than a few minutes before extensive neurodegeneration occurs. Some facultative anaerobes, including the freshwater western painted turtle Chrysemys picta bellii, are able to coordinately reduce ATP demand to match reduced ATP availability during anoxia, and thus tolerate prolonged insults without apparent detriment. To reduce metabolic rate, turtle neurons undergo channel arrest and spike arrest to decrease membrane ion permeability and neuronal electrical excitability, respectively. However, although these adaptations have been documented in turtle brain, the mechanisms underlying channel and spike arrest are poorly understood. The aim of my research was to elucidate the cellular mechanisms that underlie channel and spike arrest and the neuroprotection they confer on the anoxic turtle brain. Using electrophysiological and fluorescent imaging techniques, I demonstrate for the first time that: 1) the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) undergoes anoxia-mediated channel arrest; 2) delta opioid receptors (DORs), and 3) mild mitochondrial uncoupling via mitochondrial ATP-sensitive K+ channels result in an increase in cytosolic calcium concentration and subsequent channel arrest of the N-methyl-D-aspartate receptor, preventing excitotoxic calcium entry, and 4) reducing nitric oxide (NO) production; 5) the cellular concentration of reactive oxygen species (ROS) decreases with anoxia and ROS bursts do not occur during reoxygenation; and 6) spike arrest occurs in the anoxic turtle cortex, and that this is regulated by increased neuronal conductance to chloride and potassium ions due to activation of γ–amino-butyric acid receptors (GABAA and GABAB respectively), which create an inhibitory electrical shunt to dampen neuronal excitation during anoxia. These mechanisms are individually critical since blockade of DORs or GABA receptors induce excitotoxic cell death in anoxic turtle neurons. Together, spike and channel arrest significantly reduce neuronal excitability and individually provide key contributions to the turtle’s long-term neuronal survival during anoxia. Since the turtle is the most anoxia-tolerant air-breathing vertebrate identified, these results suggest that multiple mechanisms of metabolic suppression acting in concert are essential to maximizing anoxia-tolerance.