This study investigated the effects of the pharmacological manipulation of noradrenergic activities on dopaminergic phenotypes in aged rats. Results showed that the administration of L-threo-3,4-dihydroxyphenylserine (L-DOPS) for 21 days significantly increased the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in the striatum and substantia nigra (SN) of 23-month-old rats. Furthermore, this treatment significantly increased norepinephrine/DA concentrations in the striatum and caused a deficit of sensorimotor gating as measured by prepulse inhibition (PPI). Next, old rats were injected with the α2-adrenoceptor antagonist 2-methoxy idazoxan or β2-adrenoceptor agonist salmeterol for 21 days. Both drugs produced similar changes of TH and DAT in the striatum and SN. Moreover, treatments with L-DOPS, 2-methoxy idazoxan, or salmeterol significantly increased the protein levels of phosphorylated Akt in rat striatum and SN. However, although a combination of 2-methoxy idazoxan and salmeterol resulted in a deficit of PPI in these rats, the administration of 2-methoxy idazoxan alone showed an opposite behavioral change. The in vitro experiments revealed that treatments with norepinephrine markedly increased mRNAs and proteins of ATF2 and CBP/p300 and reduced mRNA and proteins of HDAC2 and HDAC5 in MN9D cells. A ChIP assay showed that norepinephrine significantly increased CBP/p300 binding or reduced HDAC2 and HDAC5 binding on the TH promoter. The present results indicate that facilitating noradrenergic activity in the brain can improve the functions of dopaminergic neurons in aged animals. While this improvement may have biochemically therapeutic indication for the status involving the degeneration of dopaminergic neurons, it may not definitely include behavioral improvements, as indicated by using 2-methoxy idazoxan only.

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Computational psychiatry describes psychological phenomena as abnormalities in biological computations. Current available technologies span multiple organizational and temporal domains, but there remains a knowledge gap with respect to neuromodulator dynamics in humans. Recent efforts by members of the Montague Laboratory and collaborators adapted fast scan cyclic voltammetry (FSCV) from rodent experiments for use in human patients already receiving brain surgery. The process of modifying established FSCV methods for clinical application has led improved model building strategies, and a new "random burst" sensing protocol. The advent of random burst sensing raises questions about the capabilities of in-vivo electrochemistry techniques, while opening introducing possibilities for novel approaches. Through a series of in-vitro experiments, this study aims to explore and validate novel electrochemical sensing approaches. Initial expository experiments tested assumptions about waveform design to detect dopamine concentrations by reducing amplitude and duration of forcing functions, as well as distinguishing norepinephrine concentrations. Next, large data sets collected on mixtures of dopamine, serotonin and pH validated a newly proposed "low amplitude random burst sensing" protocol, for both within-probe and out-of-probe modeling. Data collected on the same set of solutions also attempted to establish an order-millisecond random burst sensing approach. Preliminary endeavors into using convolutional neural networks also provided an example of an alternative modeling strategy. The results of this work challenge existing assumptions of neurochemistry, while demonstrating the capabilities of new neurochemical sensing approaches. This study will also act as a springboard for emerging technological developments in human neurochemistry. / Doctor of Philosophy / Neuroscience characterizes nervous system functions from the cellular to the systems level. A gap in available technologies has prevented neuroscientist from studying how changes in the molecular dynamics in the brain relate to psychiatric conditions. Recent efforts by the Montague Laboratory have adapted neurochemistry techniques for use in human patients. Consequently, a new "random burst sensing" approach was developed that challenged existing assumptions about electrochemistry. In this study, in-vivo experiments were conducted to push the limits of electrochemical sensing by reducing the voltage amplitude range and increasing sensing temporal resolution of electrochemical sensing beyond previously established limits. The results of this study offer novel neurochemistry approaches and act as a jumping off point for future technological developments.

Depression and other mood disorders, as well as cognitive and motor dysfunction have been linked with changes in monoamine levels in the brain. Environmental acetylcholinesterase (AChE) inhibitors, such as organophosphate insecticides (OPs), have also been shown to induce these problems. This study investigated whether insecticide-induced AChE inhibition, induced by chlorpyrifos (CPS), may contribute to the types of forebrain monoaminergic alterations associated with psychiatric, cognitive and motor dysfunction. Increased synaptic ACh, resulting from CPS-induced AChE inhibition, may alter the synthesis or release of monoamines through prolonged action of ACh on monoaminergic neurons that contain ACh receptors. Adult, male Sprague-Dawley rats were subjected to a single subcutaneous dose of CPS or corn oil vehicle. Brains were rapidly removed and the frontal cortex, hippocampus and striatum were bilaterally dissected on ice. These three regions from one side were assayed for AChE activity, while those from the opposite side were processed for high performance liquid chromatography with electrochemical detection (HPLC-ED) analysis of monoamine neurotransmitters and their metabolites. In the initial, exploratory experiment, inhibition of AChE activity was 66.8% in the frontal cortex, 43.8% in the hippocampus and 46.9% in the striatum, 7 days after a 60mg/kg dose of CPS. No significant differences in concentration of monoamine neurochemicals were observed between vehicle control and CPS-treated groups in either the hippocampus or striatum. However, in the frontal cortex of the CPS-treated rats there was a significant increase in median dihydroxyphenylacetic acid (DOPAC) concentration (P=0.019) and a very strong statistical trend toward increased dopamine (DA) concentration (P=0.0506). The second experiment examined the time course of AChE inhibition produced by a higher dose (200mg/kg) of CPS and how monoamine levels changed in conjunction with this pattern of AChE inhibition. Percent inhibition of AChE activity in CPS-treated animals, at 4, 14 and 21 days post-exposure was 77.0%, 86.6% and 81.9% in the frontal cortex, 86.1%, 85.9% and 83.2% in the hippocampus and 90.1%, 89.8% and 85.5% in the striatum. No significant differences in monoamine neurochemicals were observed between vehicle control and CPS-treated groups in either the hippocampus or striatum. A statistical trend toward a decrease in serotonin (5-HT) was seen in the frontal cortex at 14 days (P=0.0753) following CPS exposure. A very consistent, yet non-significant pattern of an increase in monoamines at 4 days post-CPS was observed in all instances, except for 5-hydroxyindoleacetic acid (5-HIAA) in the striatum. Therefore, the final experiment employed a more powerful design to focus on monoamine levels during, or shortly after, the change in AChE activity that rapidly follows exposure to 200mg/kg CPS. This experiment also employed a behavioral analysis on the day of sacrifice to assess the presence or absence of clinical signs of toxicity associated with this dose. Of the 30 CPS-treated rats, only 1 animal displayed a single behavioral sign of cholinergic poisoning. Percent inhibition of AChE activity at 2 and 4 days after treatment was 81.4% and 79.4% in the frontal cortex, 53.4% and 83.5% in the hippocampus, and 80.5% and 87.8% in the striatum. No significant changes in monoamine neurochemicals were observed between vehicle control and CPS-treated groups in either the frontal cortex or hippocampus. However, a significant increase in DOPAC (P=0.0285) in the striatum, 2 days after CPS treatment, was observed. In addition, a strong statistical trend toward decreased striatal 5-HT (P=0.0645) was reported 4 days after CPS treatment. The only significant correlation between AChE activity and monoamine concentration was observed for 5-HIAA in the striatum of CPS-treated, 2 day survivors (P=0.0445). However, it was of low magnitude (r=0.525, r2=0.276). CPS has a limited capacity to produce changes in monoamine neurotransmitters and/or their metabolites in the frontal cortex and striatum of the mammalian brain. These changes are primarily seen in the dopaminergic system. Alterations of monoamines do not appear to be strongly associated with incident levels of AChE inhibition. The biological implication of the limited OP induced changes in central monoamines remains significant, as changes in monoamines in the CNS nervous system have been linked to psychiatric, cognitive and motor dysfunction. / Master of Science

A glândula pineal de mamíferos sintetiza o hormônio melatonina exclusivamente durante o período noturno. A síntese é regulada primordialmente pela via retino-hipotalâmico-pineal e modulada por vários fatores, incluindo o sistema peptidérgico. Assim, o papel da insulina na regulação da síntese de melatonina foi estudado a partir da realização de culturas de glândulas pineais estimuladas com noradrenalina, insulina e noradrenalina associada à insulina, em culturas temporizadas ou não pela noradrenalina, avaliando: a produção de melatonina por HPLC com detecção eletroquímica; as atividades das enzimas envolvidas na síntese da melatonina, por radiometria; assim como, a expressão gênica das enzimas quantificada por Real-Time PCR. Os resultados sugerem uma interação entre as vias de sinalização da noradrenalina e da insulina, com a respectiva potencialização da síntese da melatonina, induzida por noradrenalina, observada pela adição da insulina, efeito esse, que se dá, provavelmente através de mecanismos pós-transcricionais. / The mammalian pineal gland synthesizes the neurohormone melatonin exclusively during the dark phase. Its synthesis is primarily regulated via a retino-hypothalamic-pineal pathway and modulated by many factors, including the peptidergic system. Thus, the role of insulin on the regulation of melatonin synthesis was studied using cultured gland treated with norepinephrine, insulin and norepinephrine associated to insulin. The cultures were also synchronized or not by norepinephrine. Melatonin content was assayed by HPLC (High Performance Liquid Chromatography) with electrochemical detection, melatonin synthesis enzymes activities by radiometry and enzymes gene expressions by Real-Time PCR. The results suggest an interaction between norepinephrine and insulin signaling pathway, with insulinic potentialization on melatonin synthesis norepinephrine-mediated, and this effect, seems to accurs potentially through post-transcriptional events.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-12T18:43:01Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) LIANE DA SILVA DE VARGAS.pdf: 6540879 bytes, checksum: 22a70d7a5b558ca0d495a2273ba31f92 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2017-06-12T18:43:38Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) LIANE DA SILVA DE VARGAS.pdf: 6540879 bytes, checksum: 22a70d7a5b558ca0d495a2273ba31f92 (MD5) / Made available in DSpace on 2017-06-12T18:43:38Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) LIANE DA SILVA DE VARGAS.pdf: 6540879 bytes, checksum: 22a70d7a5b558ca0d495a2273ba31f92 (MD5) Previous issue date: 2016 / A persistência é a principal característica da memória de longa duração (MLD). Uma vez consolidada, a MLD pode persistir por horas, dias ou anos, sendo que a sua persistência irá depender de diferentes fatores. Considerando a importância da memória no cotidiano de cada indivíduo, sendo ela responsável pela construção da personalidade e também pela manutenção das nossas ações, torna-se necessário e indispensável que haja a persistência de algumas memórias. Nesse sentido, é importante investigar os mecanismos envolvidos nesse processo, visando não só o entendimento das suas bases neurobiológicas, as quais ainda não são totalmente claras, mas também, buscar por estratégias que mantenham ou melhorem a memória ao longo do tempo. Diante disso, este trabalho teve como objetivo investigar diferentes estratégias para promoção da persistência das MLD. A tese é composta de dois estudos principais que buscaram investigar: (i) o efeito de uma sessão única de exercício físico, uma estratégia não farmacológica, na persistência da memória de reconhecimento de objetos (RO) em roedores; e, (ii) o efeito do tratamento com a Metilprednisolona (MP), um fármaco glicocorticoide, na persistência da memória aversiva em roedores. Na primeira etapa, demonstramos que a ativação noradrenérgica é necessária para que haja a persistência da memória de RO e que uma sessão única de exercício físico após a aprendizagem é capaz de promover a persistência da memória de RO por meio da ativação do sistema noradrenérgico hipocampal. Na segunda etapa, demonstramos que o tratamento crônico por 10 dias com baixa dose de MP promove a persistência da memória aversiva, além de promover o aumento 10 do influxo de Ca2+ em cultura de células de hipocampo e facilitar a indução da LTP (Potenciação de longa duração) nessa mesma estrutura. Com base nos resultados obtidos, podemos concluir que o exercício físico pode ser adotado como estratégia comportamental para a promoção da persistência das MLD. Além disso, o uso de glicocorticoides também tem potencial para ser utilizado como estratégia farmacológica que melhora a memória, entretanto seu efeito depende da dose, e estudos futuros são necessários para melhor elucidar os mecanismos de ação envolvidos, bem como seus efeitos colaterais. / Persistence is the main characteristic of long-term memory (LTM). When consolidated the LTM may persist for hours, days or years, and the persistence will depend of different factors. Considering the importance of memory in individual's daily life, being responsible for personality construction and also for the maintenance of our actions, it is necessary and essential that some memories persist along the time. Therefore, it is important to investigate the mechanisms involved in this process, not only to understanding of its neurobiology, which is not entirely clear, but also to find strategies to maintain or improve memory over time. Thus, this study aimed to investigate different strategies for promoting LTM persistence. This thesis is composed of two main studies that pursued to investigate: (i) the effect of one-single physical exercise session, a non-pharmacological strategy, in the persistence of object recognition memory (OR) in rodents; and (ii) the effect of treatment with methylprednisolone (MP), a glucocorticoid drug, on persistence of aversive memory in rodents. In the first stage, we show that noradrenergic activation is required to the persistence of OR memory and that one-single exercise session after learning promotes OR memory persistence through noradrenergic hippocampal system activation. In the second stage, we demonstrated that a chronic treatment for 10 days with low MP dose promotes aversive memory persistence, promotes increased Ca2+ influx in hippocampal cell culture and facilitates LTP induction in the same structure. Based on the results obtained, we can conclude that physical exercise can be adopted as a behavioral strategy for promoting the persistence of LTM. In addition, the use of glucocorticoids 12 also has potential to be used as a pharmacologic strategy that improves memory. However its effect depends on the dose, and future studies are needed to better elucidate the mechanisms involved, as well as its side effects.

<p>Stress can affect several behavioural patterns, such as food intake and the general activity level of an animal. The central monoamine neurotransmitters serotonin, dopamine, and norepinephrine are important in the mediation of both behavioural and neuroendocrine stress effects. This thesis describes studies of two salmonid fish model systems: Fish that become socially dominant or subordinate when reared in pairs, and rainbow trout (<i>Oncorhynchus mykiss</i>) genetically selected for high (HR) and low (LR) stress responsiveness, in terms of stress induced cortisol release. Socially subordinate individuals are often subject to chronic stress, and it was found that plasma cortisol and brain monoaminergic activity rapidly increased in subordinate fish during the initial 24 h period following fights for social dominance in pairs of rainbow trout. In pairs of Arctic char (<i>Salvelinus alpinus</i>), subordinate individuals were characterised by an inhibition of food intake and aggression, and low spontaneous locomotion. Appetite inhibition in subordinate fish was reversed by subsequent rearing in isolation, and this effect was probably related to a concomitant decrease in brain serotonergic activity. Furthermore, differential stress responsiveness in HR and LR rainbow trout was associated with differences in behaviour, as well as changes in brain monoaminergic activity. HR fish displayed higher locomotor activity when challenged by a conspecific intruder. This response was probably related to a larger stress induced activation of brain dopaminergic systems in these fish. Finally it was shown that the steroid 'stress-hormone' cortisol has dose- and context-dependent behavioural effects in fish, as has been described in mammals. Specifically, short- term cortisol treatment elevated the behavioural response to a territorial intruder, while long-term treatment, like chronic stress, had the opposite effect, inhibiting locomotor activity and aggression. It is concluded that the signalling systems involved in behavioural and neuroendocrine control during stress display extensive similarities between teleost fishes and mammals.</p>