Previous studies have suggested that phosphodiesterase (PDE) hydrolysis of cyclic adenosine monophosphate (cAMP) is important in the generation of specific and segregated cAMP signals within cells. The purpose of this study was to determine if PDE compartmentation was important in cardiac ventricular myocytes. Therefore, we investigated the effects of β-adrenergic (β-AD) stimulation with isoproterenol in the presence of cilostamide, a PDE3 inhibitor, or Ro 20-1724, a PDE4 inhibitor, on unloaded cell shortening, L-type calcium currents and intracellular calcium levels in freshly dissociated adult rat ventricular myocytes. PDE3 inhibition resulted in a 216 ± 17 % (n=8) increase in unloaded cell shortening after ten minutes of isoproterenol exposure, whereas isoproterenol produced a statistically smaller increase of 155 ± 12 % (n=8) in the presence of PDE4 inhibition. There was a non-significant trend for PDE4 inhibition to produce larger increases in calcium currents (179 ± 17 % (n=4) of controls) than PDE3 inhibition (155 ± 10 % (n=6) of controls). Both PDE3 and PDE4 inhibitors had similar effects on isoproterenol-stimulated increases of calcium transient amplitude with values of 209 ± 14 % (n=8) and 185 ± 12 % (n=8), respectively. Determination of sarcoplasmic reticulum (SR) calcium load using caffeine pulse experiments demonstrated that PDE4 inhibition and isoproterenol superfusion produced a statistically larger increase in SR-calcium loading (139 ± 9 % (n=6)) than PDE3 inhibition and isoproterenol superfusion (113 ± 9 % (n=6)). These results suggest that PDE3 may be active in proximity to the contractile apparatus of cardiac myocytes, whereas PDE4 may be localized in a domain consisting of the L-type calcium channel and junctional SR. Consequently, our study provides functional evidence for differential localization of PDE isoforms in cardiac myocytes. / Thesis (Master, Physiology) -- Queen's University, 2008-01-18 10:14:29.671 / CIHR OGS OGSST

Little is known regarding the neurophysiological mechanisms by which the neuropeptide prokineticin 2 (PK2) regulates circadian rhythms. Using whole-cell electrophysiology, we have investigated a potential role for regulation of neuronal excitability by PK2 on neurons of the area postrema (AP), a medullary structure known to influence autonomic processes in the central nervous system. In current-clamp recordings, focal application of 1µM PK2 reversibly influenced the excitability of the majority of dissociated AP cells tested, producing both depolarizations (38%) and hyperpolarizations (28%) in a concentration-dependent manner. Slow voltage ramps and ion substitution experiments revealed a PK2-induced Cl- current was responsible for membrane depolarization, while hyperpolarizations were the result of inhibition of an inwardly rectifying non-selective cation current. In contrast to these differential effects on membrane potential, nearly all neurons that displayed spontaneous activity responded to PK2 with a decrease in spike frequency. These observations are in accordance with voltage-clamp experiments showing that PK2 caused a leftward shift in Na+ channel activation and inactivation gating. Lastly, using post hoc single cell RT-PCR technology, we have shown that 7 out of 10 AP neurons depolarized by PK2 were enkephalin-expressing cells. The observed actions on enkephalin neurons indicate PK2 may have specific inhibitory actions on this population of neurons in the AP acting to reduce their sensitivity to incoming signals. These data suggest that PK2 regulates the level of AP neuronal excitability and may impart a circadian influence on AP autonomic control. / Thesis (Master, Physiology) -- Queen's University, 2009-08-18 11:18:05.977

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

This series of experiments investigated dystrophin localization in the normal cerebellum and examined Purkinje neuron function in normal and dystrophin-deficient mice to better understand the physiological basis for cognitive deficits associated with Duchenne muscular dystrophy (DMD), a common genetic disorder among children. Cognitive impairments are consistently reported in DMD, yet precise mechanisms for their occurrence are unknown. Dystrophin protein, which is absent in DMD, is normally localized to muscles and specific neurons in the brain. Purkinje neurons are rich in dystrophin, specifically in somatic and dendritic membranes. Studies demonstrate perturbed cerebellar function in the absence of dystrophin, suggesting that DMD should be regarded as a cerebellar disorder in addition to being considered a neuromuscular disorder. However, theory and evidence are not generated from overlapping information: research investigating cerebellar involvement in DMD has focused on the vermal region, associated with motor function. The lateral region, implicated in cognition, has not been explicitly examined in DMD. The first experiment revisited the issue of dystrophin distribution in the mouse cerebellum using immunohistochemistry to investigate qualitative and quantitative differences between cerebellar regions. Both regions showed dystrophin localized to Purkinje neuron somatic and dendritic membranes, but dystrophin density was 30% greater in the lateral than the vermal region. The second experiment examined intrinsic electrophysiological properties of vermal and lateral Purkinje neurons from wild-type (WT) mice and from the mdx mouse model of DMD which lack dystrophin. Significant differences in action potential firing frequency, regularity, and shape were found between cerebellar regions. Purkinje neurons from mdx mouse cerebellum exhibited membrane hyperpolarization and irregular action potential firing, regardless of region. Spontaneous action potential firing frequency was reduced in Purkinje neurons from lateral cerebellum in mdx mice relative to controls, demonstrating that a loss of dystrophin causes a potent dysregulation of Purkinje neuron function in the region associated with cognition. This research extends our understanding of cerebellar pathology in DMD and its potential relevance to cognitive deficits in the disorder. Moreover, this research further supports the role of the cerebellum as a structure important for cognition and contributes to our understanding of dystrophin’s role in the brain.

Serotonin (5-HT)-containing neuroepithelial cells (NECs) of the gill filament are believed to be the primary O2 chemosensors in fish. In the mammalian carotid body (CB), 5-HT is one of many neurotransmitters believed to play a role in transduction of hypoxic stimuli, with acetylcholine (ACh) being the primary fast-acting excitatory neurotransmitter. Immunohistochemistry and confocal microscopy was used to observe the presence of the vesicular acetylcholine transporter (VAChT), a marker for the presence of ACh, and its associated innervation in the gills of zebrafish. VAChT-positive cells were observed primarily along the afferent side of the filament, with some cells receiving extrabranchial innervation. No VAChT-positive cells were observed in the gills of goldfish; however, certain key morphological differences in the innervation of goldfish gills was observed, as compared to zebrafish. In addition, in zebrafish NECs, whole-cell current is dominated by an O2-sensitive background K+ current; however, this is just one of several currents observed in the mammalian CB. In zebrafish NECs and the CB, membrane depolarization in response to hypoxia, mediated by inhibition of the background K+ (KB) channels, is believed to lead to activation of voltage-gated Ca2+ (CaV) channels and Ca2+-dependent neurosecretion. Using patch-clamp electrophysiology, I discovered several ion channel types not previously observed in the gill chemosensors, including Ca2+-activated K+ (KCa), voltage-dependent K+ (KV), and voltage-activated Ca2+ (CaV) channels. Under whole-cell patch-clamp conditions, the goldfish NECs did not respond to hypoxia (PO2 ~ 11 mmHg). Employing ratiometric calcium imaging and an activity-dependent fluorescent vital dye, I observed that intact goldfish NECs respond to hypoxia (PO2 ~ 11 mmHg) with an increase in intracellular Ca2+ ([Ca2+]i) and increased synaptic vesicle activity. The results of these experiments demonstrate that (1) ACh appears to play a role in the zebrafish, but not goldfish gill, (2) goldfish NECs likely signal hypoxic stimuli primarily via the central nervous system (CNS), (3) goldfish NECs express a broad range of ion channels as compared to the NECs of zebrafish, and (4) goldfish NECs rely on some cytosolic factor(s) when responding to hypoxia (PO2 ~ 11 mmHg). This thesis represents a further step in the study of neurochemical and physiological adaptations to tolerance of extreme hypoxia.

This series of experiments investigated dystrophin localization in the normal cerebellum and examined Purkinje neuron function in normal and dystrophin-deficient mice to better understand the physiological basis for cognitive deficits associated with Duchenne muscular dystrophy (DMD), a common genetic disorder among children. Cognitive impairments are consistently reported in DMD, yet precise mechanisms for their occurrence are unknown. Dystrophin protein, which is absent in DMD, is normally localized to muscles and specific neurons in the brain. Purkinje neurons are rich in dystrophin, specifically in somatic and dendritic membranes. Studies demonstrate perturbed cerebellar function in the absence of dystrophin, suggesting that DMD should be regarded as a cerebellar disorder in addition to being considered a neuromuscular disorder. However, theory and evidence are not generated from overlapping information: research investigating cerebellar involvement in DMD has focused on the vermal region, associated with motor function. The lateral region, implicated in cognition, has not been explicitly examined in DMD. The first experiment revisited the issue of dystrophin distribution in the mouse cerebellum using immunohistochemistry to investigate qualitative and quantitative differences between cerebellar regions. Both regions showed dystrophin localized to Purkinje neuron somatic and dendritic membranes, but dystrophin density was 30% greater in the lateral than the vermal region. The second experiment examined intrinsic electrophysiological properties of vermal and lateral Purkinje neurons from wild-type (WT) mice and from the mdx mouse model of DMD which lack dystrophin. Significant differences in action potential firing frequency, regularity, and shape were found between cerebellar regions. Purkinje neurons from mdx mouse cerebellum exhibited membrane hyperpolarization and irregular action potential firing, regardless of region. Spontaneous action potential firing frequency was reduced in Purkinje neurons from lateral cerebellum in mdx mice relative to controls, demonstrating that a loss of dystrophin causes a potent dysregulation of Purkinje neuron function in the region associated with cognition. This research extends our understanding of cerebellar pathology in DMD and its potential relevance to cognitive deficits in the disorder. Moreover, this research further supports the role of the cerebellum as a structure important for cognition and contributes to our understanding of dystrophin’s role in the brain.

Diss. Stockholm : Karol. inst., 2004.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Submitted by ROBERTO PAULO CORREIA DE ARAÚJO (ppgorgsistem@ufba.br) on 2016-10-18T14:55:15Z No. of bitstreams: 1 Milena Ramos Reis.pdf: 2152638 bytes, checksum: 876fac844a4f4a8b22cb82e9cdeb5f3b (MD5) / Made available in DSpace on 2016-10-18T14:55:15Z (GMT). No. of bitstreams: 1 Milena Ramos Reis.pdf: 2152638 bytes, checksum: 876fac844a4f4a8b22cb82e9cdeb5f3b (MD5) / O carvacrol, um monoterpeno fenólico encontrado nos óleos essenciais de diversas plantas do gênero Origanum, já demonstrou causar hipotensão e vasodilatação em diferentes leitos vasculares de ratos normotensos, porém, seu efeito em ratos hipertensos ainda não foi elucidado. O objetivo deste estudo foi investigar os efeitos cardiovasculares do carvacrol em ratos espontaneamente hipertensos (SHR) e comparar com normotensos Wistar, utilizando ensaios farmacológicos in vitro (estudos funcionais e celulares) e in vivo. Nos ensaios funcionais in vitro, anéis de artéria mesentérica superior isolada de animais hipertensos e normotensos foram précontraídos com FEN (1μM) e o efeito de carvacrol (10-8-10-3M) foi observado. Em SHR, este monoterpeno induziu vasodilatação dependente de concentração (pD2=5,13 ± 0,05; Emáx=115,14 ± 5,46%; N=8) e, após a remoção do endotélio funcional, a potência da droga foi alterada significantemente (pD2=4,91 ± 0,05 N=9; p<0,01), sugerindo que a resposta vasodilatadora induzida por carvacrol, provavelmente, envolve uma via dependente e outra independente do endotélio vascular, porém, esta última parece ser a majoritária e, por isso, os ensaios seguintes foram realizados na ausência do endotélio vascular. Interessantemente, quando comparada com animais normotensos, a potência farmacológica de carvacrol foi reduzida significantemente (pD2=4,91 ± 0,05; N=9; p<0,05). Em anéis de ratos hipertensos, carvacrol reduziu o influxo de Ca2+ por canais Cav tipo-L, SOC e ROC, estes resultados foram semelhantes aos obtidos em ratos normotensos. Em ratos hipertensos, mas não em normotensos, a potência farmacológica do carvacrol em anéis pré-contraídos com FEN e na presença de diferentes inibidores de canais TRP (íon Gd3+, 10-5M; 2-APB, 10-6M ou 10-5M; BCTC, 2μM; 9-fenantrol, 10-5M; ou HC03003-1, 10-5M), foi reduzida em relação ao controle na ausência destes bloqueadores, sugerindo que os canais sensíveis à estes bloqueadores (TRPC1-7, TRPM2, M4 e TRPM8, TRPV1 e TRPA1), provavelmente, estão participando dos efeitos vasculares mediados por carvacrol e podem estar envolvidos no processo hipertensivo. Em estudos de patch-clamp em células de artéria mesentérica dispersas de ratos hipertensos, carvacrol (300μM) reduziu as correntes de entrada de Ba2+ por Cav tipo-L e este efeito foi semelhante em ratos normotensos. Além disso, em células de ratos hipertensos, o Mg2+ (2,5mM), bloqueador do TRPM6 e TRPM7, reduziu as densidades de ITRPM de entrada e saída, assim como carvacrol (100μM e 300μM), na ausência ou presença do 2-APB (100μM), bloqueador de TRPM7. A presença do 2-APB provocou inibição adicional nas densidades de ITRPM pelo carvacrol (100μM, mas não 300μM). Altas concentrações intracelulares de Mg2+ reduziram the magnitude of ITRPM7. Foi evidenciado que a ITRPM no controle é menor em ratos hipertensos que em normotensos. Estes dados obtidos e os relatados na literatura são sugestivos para provável inibição de ITRPM7 por carvacrol em células mesentéricas nativas. O efeito anti-hipertensivo do carvacrol foi avaliado por administração via orogástrica (50mg/kg/dia) durante 20 dias foi capaz de reduzir a pressão arterial média dos animais SHR tratados, no 20º dia do tratamento. O tratamento subcrônico com carvacrol não alterou os pesos cardíaco e corpóreo, nem a reatividade vascular. Em conclusão, esses dados sugerem que carvacrol possui atividade anti-hipertensiva em animais SHR, que pode ser devido ao seu efeito vasodilatador em anéis de artéria mesentérica superior isolada, provavelmente, por inibição do influxo de Ca2+ por Cav tipo-L, ROC, SOC e/ou canais TRPC1, 3 ou 6, além da inibição de correntes tipo-TRPM7 em miócitos mesentéricos.

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-01-13T10:59:16Z No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-01-20T14:53:34Z (GMT) No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) / Made available in DSpace on 2017-01-20T14:53:34Z (GMT). No. of bitstreams: 1 AnnaraYveMouraSoares_DISSERT.pdf: 884763 bytes, checksum: df8a008c0603a2358c6331d336c3bd21 (MD5) Previous issue date: 2016-04-05 / Esse estudo objetiva avaliar se a express?o da enzima de reparo de DNA Neil3 ? importante para o desenvolvimento funcional dos neur?nios. Estudos previos tem demonstrado que Neil3 interfere tanto na neurog?nese adulta como na fazer embrionaria. Eu utilizei whole cell patch clamp para estudar propriedades sinapticas e de membrana das c?lulas granulares do giro denteado. O giro denteado ? uma das regi?es com maior express?o de Neil3 no c?rebro e estudos previos tem demonstrado que a neurog?nese reativa em camundongos adultos ? afetada pela ausencia da enzima de reparo Neil3. Eu encontrei que a maioria das propriedades de membrana nas c?lulas granulares de camundongos knockout para Neil3 s?o normais com exce??o ? resposta de membrana ?s correntes de hiperpolariza??o e p?s-hiperpolariza??o. Diferentemente de neur?nios imaturos, as c?lulas granulares do giro denteado de camundongos com aus?ncia de Neil3, na qual as correntes de hiperpolariza??o ativadas s?o geralmente as ultimas a aparecerem durante o desenvolvimento. Al?m disso, correntes sinapticas excitatorias foram similar em amplitude mas apresentaram um decaimento ligeiramente mais rapido em c?lulas de camundongos knockout de Neil3. Esses resultados podem indicar um balan?o diferente entre os receptores AMPA e NMDA em camundongos knockout. Analises morfologicas de neur?nios preenchidos com biotina e reconstru??o post hoc n?o apresentaram grandes diferen?as na morfologia dendritica entre animais controle e knockout. Esse estudo mostra que, em rela??o diferen?as entre animais controle, neur?nios do giro denteado de animais knockout de Neil3 n?o podem ser classificados como imaturos. Eu encontrei diferen?as pontuais na corrente de hiperpolariza??o e pequenas diferen?as em propriedades sinapticas. Ainda devem ser avaliadas se essas diferen?as podem ser respons?veis por altera??es comportamentais encontradas em camundongos Neil3-knockout. Al?m disso, estudos futuros utilizando marcadores de neur?nios rec?m-nascidos s?o necess?rios para analisar o efeito da elimina??o da enzima Neil3 no desenvolvimento de neur?nios. / This study aims to assess whether the expression of the DNA repair enzyme Neil3 is important for the functional development of neurons. Previous studies have demonstrated that Neil3 interferes with both adult and embryonic neurogenesis. I have used whole cell patch clamp to study membrane and synaptic properties of granule cells of the dentate gyrus. The dentate gyrus is one of the regions with the highest expression of Neil3 in the brain, and previous studies have shown that reactive neurogenesis in adult mice is affected by Neil3 deletion. I found that most membrane properties of granule cells in Neil3-knockout mice are normal except from the membrane response to hyperpolarization currents and afterhyperpolarization currents. Different from immature neurons, granule cells of the dentate gyrus from Neil3-knockout mice, in which hyperpolarizing activated currents, are generally the last to appear during development. In addition, excitatory synaptic currents were similar in amplitude but showed a slightly faster decay in cells from Neil3-knockout mice. These results could indicate a different balance between AMPA and NMDA receptors in Neil3-knockout mice cells. Morphological analysis of neurons filled with biocytin and reconstructed post hoc showed no gross difference in dendritic morphology between dentate gyrus neurons of control and Neil3-knockout mice. This study shows that, while different from those of control littermates, dentate gyrus neurons of Neil3-knockout mice cannot be classified as ?immature?. I found specific differences in hyperpolarizing activated currents and small differences in synaptic properties. Whether these differences may account for behavioral changes found in Neil3-knockout mice is yet to be assessed. In addition, future studies using markers of newly born neurons are necessary for analyzing the effect of Neil3 deletion in developing neurons.