Thesis (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Elizabeth Tietz. Includes abstract. Title from title page of PDF document. Bibliography: pages 88-94, 130-136, 178-189, 218-266.

Mammalian neurons rapidly undergo excitotoxic cell death during anoxia, while neurons from the anoxia-tolerant painted turtle can survive without oxygen for hours without apparent damage. An anoxia-mediated decrease in AMPA receptor currents are an important part of the turtle’s natural defence however the mechanism underlying it is unknown. Here I investigate a mechanism that involves activation of a mitochondrial KATP channel that subsequently signals a decrease in AMPAR currents. Whole-cell AMPAR currents were stable during normoxia, but anoxia or pharmacological activation of mKATP channels resulted in a 50% decrease in AMPAR currents. Conversely, mKATP antagonists blocked the anoxia-mediated decrease. Mitochondrial KCa channel modulators responded similarly. Blocking the Ca2+-uniporter also reduced normoxic AMPAR currents by 40%, and including BAPTA in the recording abolished the anoxia or agonist-mediated decrease. Therefore, the mKATP channel is involved in the anoxia-mediated down-regulation of AMPAR activity and is a common mechanism to reduce glutamatergic excitability.

Mammalian neurons rapidly undergo excitotoxic cell death during anoxia, while neurons from the anoxia-tolerant painted turtle can survive without oxygen for hours without apparent damage. An anoxia-mediated decrease in AMPA receptor currents are an important part of the turtle’s natural defence however the mechanism underlying it is unknown. Here I investigate a mechanism that involves activation of a mitochondrial KATP channel that subsequently signals a decrease in AMPAR currents. Whole-cell AMPAR currents were stable during normoxia, but anoxia or pharmacological activation of mKATP channels resulted in a 50% decrease in AMPAR currents. Conversely, mKATP antagonists blocked the anoxia-mediated decrease. Mitochondrial KCa channel modulators responded similarly. Blocking the Ca2+-uniporter also reduced normoxic AMPAR currents by 40%, and including BAPTA in the recording abolished the anoxia or agonist-mediated decrease. Therefore, the mKATP channel is involved in the anoxia-mediated down-regulation of AMPAR activity and is a common mechanism to reduce glutamatergic excitability.

Disruptions in memory are a hallmark feature of several psychiatric diseases. These illnesses are often marred by an inability to recognize that a stimulus or event as been previously experienced, a phenomenon known as recognition memory. Previous study has demonstrated that cognitive disruptions reflect aberrant signaling, including disruptions in synaptic plasticity, in key regions of the brain, such as prefrontal cortex (PFC), hippocampus, and perirhinal cortex (PRh). However, in the case of recognition memory, how these disruptions arise and what specific plasticity mechanisms are involved is less clear. An understanding of the etiological factors underlying disruption and the synaptic processes involved in recognition will greatly advance the treatment and prevention of psychiatric disorders. As a result, the present thesis examined recognition memory in rodents in two experiments. In the first experiment, we blocked the endocytosis of AMPA receptors during the encoding, consolidation, or retrieval phase of object recognition memory using local PRh infusions of the cell membrane permeable Tat-GluA23Y interference peptide. Tat-GluA23Y infusion before the encoding and consolidation phases did not alter memory. In contrast, Tat-GluA23Y infusion prior to the retrieval phase significantly disrupted memory. These results indicate a distinct role for AMPA receptor endocytosis during a specific phase (retrieval) of visual recognition memory. In the second experiment, pregnant dams were treated with PolyI:C (4mg/kg, i.v.) on gestational day (GD) 15, and both the male and female offspring of these rats were tested as young adults in three different recognition memory tests: spontaneous novel object recognition, novel object location recognition, and object-in-place recognition. Male, but not female, rats were impaired in an object-in-place memory test that depends on processing between medial temporal lobe and PFC. However, neither male nor female rats were impaired on tests of simpler discriminations dependent on the medial temporal lobe. These findings support clinical studies demonstrating impaired object location binding in clinical populations and further demonstrate the plausibility of prenatal immune activation as an etiological factor in neurodevelopmental disease. Taken together, these results highlight the importance of a specific form of synaptic plasticity during the recognition of familiar stimuli and demonstrate that early life adversity can disrupt recognition memory processes.

Disruptions in memory are a hallmark feature of several psychiatric diseases. These illnesses are often marred by an inability to recognize that a stimulus or event as been previously experienced, a phenomenon known as recognition memory. Previous study has demonstrated that cognitive disruptions reflect aberrant signaling, including disruptions in synaptic plasticity, in key regions of the brain, such as prefrontal cortex (PFC), hippocampus, and perirhinal cortex (PRh). However, in the case of recognition memory, how these disruptions arise and what specific plasticity mechanisms are involved is less clear. An understanding of the etiological factors underlying disruption and the synaptic processes involved in recognition will greatly advance the treatment and prevention of psychiatric disorders. As a result, the present thesis examined recognition memory in rodents in two experiments. In the first experiment, we blocked the endocytosis of AMPA receptors during the encoding, consolidation, or retrieval phase of object recognition memory using local PRh infusions of the cell membrane permeable Tat-GluA23Y interference peptide. Tat-GluA23Y infusion before the encoding and consolidation phases did not alter memory. In contrast, Tat-GluA23Y infusion prior to the retrieval phase significantly disrupted memory. These results indicate a distinct role for AMPA receptor endocytosis during a specific phase (retrieval) of visual recognition memory. In the second experiment, pregnant dams were treated with PolyI:C (4mg/kg, i.v.) on gestational day (GD) 15, and both the male and female offspring of these rats were tested as young adults in three different recognition memory tests: spontaneous novel object recognition, novel object location recognition, and object-in-place recognition. Male, but not female, rats were impaired in an object-in-place memory test that depends on processing between medial temporal lobe and PFC. However, neither male nor female rats were impaired on tests of simpler discriminations dependent on the medial temporal lobe. These findings support clinical studies demonstrating impaired object location binding in clinical populations and further demonstrate the plausibility of prenatal immune activation as an etiological factor in neurodevelopmental disease. Taken together, these results highlight the importance of a specific form of synaptic plasticity during the recognition of familiar stimuli and demonstrate that early life adversity can disrupt recognition memory processes.

D-Serine is regarded as an obligatory co-agonist required for the activation of NMDA-type glutamate receptors (NMDARs). In the retina D-serine and a second NMDAR coagonist, glycine, are present at similar concentration and the cells that produce and release them are in close apposition. This arrangement allows for an abundant supply of coagonists and under certain conditions the NMDAR coagonist binding site could be saturated. There is also evidence suggesting that D-serine can act in an inhibitory manner at AMPA/kainate-type glutamate receptors (GluRs). Glutamate receptor activation can lead to direct and indirect elevation of intracellular calcium (Ca2+) concentration ([Ca2+]i). Therefore, in this thesis, I predominantly used Ca2+ imaging techniques to study the effect of D-serine on GluR activation in the mammalian retina. I first describe a novel method I developed to load retinal cells with Ca2+ indicator dye using electroporation and show that retinas remain viable and responsive following electroporation. This technique was used to explore the excitatory role of D-serine at NMDARs and its potential inhibition of AMPA/kainate receptors using cultured retinal ganglion cells (RGCs) and isolated retina preparations. Using cultured RGCs I demonstrated that D-serine and glycine enhance NMDAR-mediated Ca2+ responses in a concentration-dependent manner and are equally effective as coagonists. In isolated retinas I showed that D-serine application enhanced NMDA-induced responses consistent with sub-saturating endogenous coagonist concentration. Degradation of endogenous D-serine reduced NMDAR-mediated Ca2+ responses supporting the contribution of this coagonist to NMDAR activation in the retina. Using imaging and two different electrophysiological approaches, I found that D-serine reduced AMPA/kainate receptor-mediated responses in cultured RGCs and isolated retinas at concentrations that are saturating at NMDARs. Antagonist experiments suggest that the majority of inhibition is due to D-serine acting on AMPA receptor activity. Degradation of endogenous D-serine enhanced AMPA/kainate-induced responses of some cells in isolated retina suggesting that, under these conditions, D-serine concentration may be sufficient to inhibit AMPA receptor activity. Overall, the work in this thesis illustrates the utility of electroporation as a method to load Ca2+-sensitive fluorescent dyes into retinal cells and highlights the potential role for D-serine as a modulator of ionotropic GluRs in the CNS.

Impulse control, an executive process that restrains inappropriate actions, is impaired in numerous psychiatric conditions. This thesis reports three experiments that utilized a novel animal model of impulse control, the response inhibition (RI) task, to examine the substrates that underlie learning this task. In the first experiment, rats were trained to withhold responding on the RI task, and then euthanized for electrophysiological testing. Training in the RI task increased the AMPA/NMDA ratio at the synapses of pyramidal neurons in the prelimbic, but not infralimbic, region of the medial prefrontal cortex. This enhancement paralleled performance as subjects underwent acquisition and extinction of the inhibitory response. AMPA/NMDA was elevated only in neurons that project to the ventral striatum. Thus, this experiment identified a synaptic correlate of impulse control. In the second experiment, a separate group of rats were trained in the RI task prior to electrophysiological testing. Training in the RI task produced a decrease in membrane excitability in prelimbic, but not infralimbic, neurons as measured by maximal spiking evoked in response to increasing current injection. Importantly, this decrease was strongly correlated with successful inhibition in the task. Fortuitously, subjects trained in an operant control condition showed elevated infralimbic, but not prelimbic, excitability, which was produced by learning an anticipatory signal that predicted imminent reward availability. These experiments revealed two cellular correlates of performance, corresponding to learning two different associations under distinct task conditions. In the final experiment, rats were trained on the RI task under three conditions: Short (4-s), long (60-s), or unpredictable (1-s to 60-s) premature phases. These conditions produced distinct errors on the RI task. Interestingly, amphetamine increased premature responding in the short and long conditions, but decreased premature responding in the unpredictable condition. This dissociation may arise from interactions between amphetamine and underlying cognitive processes, such as attention, timing, and conditioned avoidance. In summary, this thesis showed that learning to inhibit a response produces distinct synaptic, cellular, and pharmacological changes. It is hoped that these advances will provide a starting point for future therapeutic interventions of disorders of impulse control. / Thesis (Ph.D, Neuroscience Studies) -- Queen's University, 2011-07-11 09:44:54.815

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

Made available in DSpace on 2013-08-07T19:05:10Z (GMT). No. of bitstreams: 1 000409596-Texto+Completo-0.pdf: 723209 bytes, checksum: caf0c117dfa3ea5f2b501cc60cc59052 (MD5) Previous issue date: 2008 / Introduction: Memory is the acquisition, consolidation and evocation of information. It always involves an emotional component, which is added to the information of a cognitive nature. When a memory is the consequence of a particularly stressful and traumatic situation, it involves emotions of that nature and is established through the amygdala and hippocampus and is more resistant to extinction and to be forgotten. Anxiety and stress influence the initial phase of the consolidation of memory through several modulatory paths, the effect of which is incorporated within the rest of the content of each memory. In the case of particularly intense stress, there is tendency for the evocation of the traumatic memory to be reiterated, causing persistent avoidance of any stimulus associated with it. TOP is an efficacious medicine in the treatment of epilepsy, which has among its side effects, which are concentration dependent, a reduction in working memory and verbal fluency, leading to confusion and numbness. Materials, methods and assessed hypotheses: The action of TOP (10mg/kg) in the consolidation and extinction of long-term memory was assessed in 84 Wistar rats divided into 6 experimental groups and one control group. The action of TOP on consolidation was assessed by administering it immediately following, or three hours after the training. In order to assess extinction, TOP was administered for 14 days before, or 5 days during the extinction. In all the experiments, the animals initiated tests 15 days after the training. The training consisted of measuring the latency time taken to descend from the platform, in an inhibitory avoidance paradigm, when they received a 1 mA shock for 2 seconds. The action of TOP in consolidation and extinction was assessed in repeated tests, without shock, in which the latency of the descent from the platform was measured at the times T1 to T5 and in the final test T6, 48 hours after. A counter-test was also carried out in order to assess whether there was a direct effect of TOP in the loss of memory when administered for 5 days without there occurring an extinction procedure. Results: When administered post-training, TOP interfered with the consolidation of memory. The result was more efficient when it was administered 3 h after training. TOP failed to induce memory extinction when administered when administered for 14 days prior to extinction, though it facilitated extinction when administered for 5 days during extinction. When TOP was administered for a week and there was no extinction procedure, it did not provoke memory loss. Suggestion: TOP may be of use in the treatment of patients with post-traumatic stress, as well as those considered borderline that exhibit self-destructive behavior related to childhood trauma. / Introdução: A memória é aquisição, consolidação e evocação de informações. Envolve sempre um componente emocional, que se acrescenta às informações de índole cognitiva. Quando a memória é conseqüência de uma situação estressante e traumática, envolve emoções dessa índole e se estabelece através da amídala e do hipocampo, sendo mais resistentes à extinção e ao esquecimento. A ansiedade e o estresse influenciam a fase inicial da consolidação da memória, através de várias vias modulatórias, cujo efeito se incorpora ao restante do conteúdo de cada memória. No caso de estresse particularmente intenso, há tendência à evocação reiterada da memória traumática, provocando uma esquiva persistente a qualquer estímulo que seja associado à mesma. O TOP é um medicamento eficaz na epilepsia, que tem entre seus efeitos colaterais, que são concentração dependente, a diminuição da memória de trabalho e da fluência verbal, provocando confusão e torpor. Materiais, métodos e hipóteses avaliadas: Foi avaliada a ação do TOP (10mg/kg) na consolidação e extinção da memória de longa duração em 84 ratos Wistar, divididos em 6 grupos caso e um grupo controle. A ação do TOP sobre a consolidação foi avaliada por sua administração imediatamente após, ou 3 horas após o treino. Na avaliação da extinção, o TOP foi administrado por 14 dias antes, ou 5 dias durante a extinção. Em todos os experimentos os animais iniciaram os testes 15 dias após o treino. O treino consistiu em medir o tempo de latência para descer da plataforma no paradigma de esquiva inibitória, momento em que receberam um choque de 1 mA por 2 segundos. A ação do TOP sobre a consolidação e extinção foi avaliada em testes repetidos, sem o choque, em que se mediu a latência de descida da plataforma nos tempos T1 a T5 e no teste final T6, 48 horas após. Foi realizada também uma contra prova para avaliar se havia ação direta do TOP na perda da memória quando administrado por 5 dias sem passar pelo procedimento de extinção. Resultados: O TOP administrado pós-treino interferiu com a consolidação da memória. O resultado foi mais eficaz quando administrado 3 h após o treino. O TOP não induziu a extinção da memória quando administrado antes da extinção por 14 dias, porém a facilitou quando administrado por 5 dias durante a mesma. O TOP administrado por uma semana, sem passar pelo procedimento de extinção, não provocou a perda da memória. Sugestão: O TOP talvez possa ser um medicamento que auxilie pacientes com estresse pós-traumático, assim como aqueles considerados “borderline”, que apresentam um comportamento autodestrutivo relacionado a traumas na infância.

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated glutamatergic ion channels that mediate most excitatory neurotransmission in the brain. Alterations in AMPAR synaptic accumulation mediate synaptic plasticity, including long-term potentiation, long-term depression and homeostatic synaptic plasticity. AMPAR abundance in neurons is determined by balanced processes of protein translation and degradation. Changes in AMPAR function and trafficking have direct impacts on synaptic transmission and cognitive functions. However, the molecular mechanisms regulating AMPAR expression and dynamics in neurons remain largely unknown. In this thesis, two molecular mechanisms that regulate AMPAR translation and protein stability through two different signaling pathways, 5' adenosine monophosphate-activated protein kinase (AMPK) and sirtuin 2 (SIRT2), are described. It is shown that SIRT2, a NAD+-dependent protein deacetylase, directly controls AMPAR stability by regulating AMPAR acetylation. For the first time, we discovered that AMPARs are subject to lysine acetylation, a novel form of post-translational modification for glutamate receptors. Under basal conditions, AMPARs are highly acetylated at their intracellular C termini, which protects against ubiquitination to antagonize AMPAR endocytosis and degradation, leading to prolonged receptor half-life. SIRT2 is also identified as the enzyme responsible for AMPAR deacetylation. Knockdown of SIRT2 led to elevated AMPAR acetylation and reduced ubiquitination, and consequently, increased AMPAR levels and synaptic transmission. SIRT2 knockout mice displayed weakened synaptic plasticity and impaired learning and memory. Resveratrol is a phytoalexin that has been shown to increase AMPAR expression and synaptic accumulation in neurons. The resveratrol effect on AMPAR expression is independent of sirtuin 1, the conventional target of resveratrol, but rather is mediated by AMPK and its downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Application of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), to neurons mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis through the AMPK-PI3K pathway activation. These studies describe novel regulatory mechanisms responsible for the control of AMPAR protein amount and subcellular distribution in neurons, providing insights into our understanding of synaptic plasticity, brain function and neurological disorders. / 2017-12-06T00:00:00Z