Submitted by Ramon Santana (ramon.souza@ufpe.br) on 2015-03-10T19:07:06Z No. of bitstreams: 2 DISSERTAÇÃO Evandro Medeiros Costa.pdf: 971018 bytes, checksum: e4bb7912dc872933b713f9a139303ded (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-10T19:07:06Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Evandro Medeiros Costa.pdf: 971018 bytes, checksum: e4bb7912dc872933b713f9a139303ded (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014-02-21 / INTRODUÇÃO: O consumo excessivo de medicamentos pela população é considerado um problema de saúde pública. Entre esses medicamentos destacam-se os benzodiazepínicos que são comumente utilizados para o tratamento da insônia. Diante dos inúmeros problemas trazidos pelo uso indiscriminado desses insumos a Secretaria Municipal de Saúde do Recife buscou uma alternativa ao seu uso, introduzindo assim o medicamento fitoterápico à base de Valeriana Officinalis L. Esse estudo tem como OBJETIVO avaliar a implantação e utilização do medicamento fitoterápico em ex-usuários de benzodiazepínicos das unidades de Saúde da Família do Distrito Sanitário II. MATERIAIS E MÉTODOS: Avaliou-se através de questionários a qualidade do sono dos usuários do novo medicamento, seu conhecimento sobre a fitoterapia fazendo um paralelo comparativo ao uso dos benzodiazepínicos e entrevistou-se também os prescritotes, através de questionários padronizados, sobre questões relacionadas à fitoterapia com destaque para Valeriana Officinalis L. RESULTADOS E DISCUSSÃO: Participaram do estudo 101 pessoas (93 usuários se 8 médicos). Os usuários relataram conhecer as atividades de vinte e seis espécies diferentes de plantas, utilizadas isoladamente para diversos fins medicinais, destacando-se o maracujá (Passiflora incanata) com 8/63 citações, a camomila (Chamomilla recutita (L.)) com 8/63 citações e a laranja (Inga sp.) com 5/63 citações. O Grupo Valeriana (GV) teve um escore global de 12 pontos e o Grupo Controle (GC) apresentou escore global de 4 pontos no Questionário de Qualidade de Sono de Pittsburg, sendo assim o GV apresentou qualidade do sono muito ruim o GC qualidade do sono boa. Vários fatores podem estar relacionados a esse resultado entre eles o desconhecimento dos prescritores sobre o tratamento o que ficou evidente através dos resultados das entrevistas com os médicos. Quando a pergunta foi relacionada à retirada de benzodiazepínicos utilizando a valeriana, metade dos entrevistados relatou que não se sentem seguros e revelaram desconhecimento em relação ao “desmame”. Em relação ao nível de conhecimento sobre a valeriana 5/8 dos prescritores julgaram como sendo regular, 2/8 muito ruim e 1/8 bom. CONCLUSÃO: É de interesse para este estudo que novas pesquisas sobre o tema sejam realizadas e que nelas sejam envolvidas atividades de acompanhamento farmacoterapêutico e de educação em saúde para os usuários e profissionais de saúde envolvidos no processo. Portanto cabe aos profissionais de saúde a apropriação sobre temas tão importantes como a fitoterapia e a retirada gradual de benzodiazepínicos, uma vez que isso poderá afetar diretamente não somente a qualidade do sono, mas a qualidade de vida dos usuários.

Angelman syndrome (AS) is a rare genetic and neurological disorder presenting with severe developmental delay, ataxia, epilepsy, and lack of speech. AS is associated with a neuron-specific loss of function of the maternal UBE3A allele, a gene encoding an E3 ubiquitin ligase. Currently, no cure exists for this disorder; however, recent research using an AS mouse model suggests that pharmacological intervention is plausible, and can alleviate some of the detrimental phenotypes reported in AS patients. Although there is no curative treatment for AS, seizure medication and behavioral therapies are most commonly prescribed in order to minimize symptoms. However, these options only moderately improve quality of life and can cause adverse side effects, such as alterations in mood and cognition following seizure treatment. Unfortunately, epilepsy is a common cause of death in AS and affects greater than 80% of AS patients, with 77% of those patients remaining refractory. The severity of seizures and lack of consistently effective anti-epileptic medications for AS patients demonstrates a considerable need for other therapeutic options. The goal of this work was to evaluate the effects of seizure therapies that have proven beneficial for treating refractory epilepsy in seizure-related disorders. These studies focused specifically on advances in both a pharmacological and dietary therapy evaluated in the AS mouse model. Previous work in our lab has demonstrated the importance of interneurons and GABAergic tone in hippocampal network regulation and cognition. GABA is an important modulator of synaptic plasticity, and learning increases both inhibitory synaptogenesis and GABA release from hippocampal inhibitory neurons. A neuronal excitatory/inhibitory imbalance, coupled with decreased GABAergic tone, altered synaptic plasticity, and impaired cognition have been reported in the AS mouse model. Therefore, we proposed to examine two therapeutic strategies used in seizure treatment – a ketone ester (KE) supplement, which is thought to increase the [GABA]/[glutamate] ratio via alterations in brain metabolism, and ganaxolone, a positive allosteric modulator of GABAA receptors. We evaluated the effects of each therapeutic on learning and cognitive enhancement, alterations in synaptic function, and anticonvulsant activity. We hypothesized that both the KE and ganaxolone would demonstrate anticonvulsant efficacy in both behavioral and chemiconvulsant seizure models. Additionally, as chronic epilepsy has been linked to progressive cognitive and memory impairment which may be related to GABA deficiencies, we hypothesized that both therapeutics would improve cognition and modulate synaptic plasticity (i.e., synaptic function). KE administration produced sustained ketosis and improved motor coordination, learning and memory, and synaptic plasticity in AS mice. The KE was also anticonvulsant and altered brain amino acid metabolism in AS treated animals. Ganaxolone was anxiolytic, anticonvulsant, and improved motor deficits in AS mice. Four weeks of treatment also led to recovery of spatial working memory and hippocampal synaptic plasticity deficits. This study demonstrates that the KE and ganaxolone ameliorate many of the behavioral abnormalities in the adult AS mouse, possibly through modulations of GABAergic tone. These results support clinical investigation of both the KE and ganaxolone in AS, which may lead to the development of a novel treatment for AS patients.

The inhibitory circuitry of the prefrontal cortex (PFC) is involved in working memory and modulation of brain oscillations. Alterations in this network and especially GABAergic cells that express cholecystokinin (CCK), parvalbumin (PV), or somatostatin (SST) may underlie some of the cognitive deficits observed in schizophrenia. To assess the involvement of CCK+, PV+, and SST+ interneurons in PFC-dependent behaviours, we selectively inactivated these in prelimbic and infralimbic PFC via virus-mediated expression of tetanus toxin light chain (TeLC). We found that functional removal of CCK+ or PV+, but not SST+ neurons leads to specific impairments in working memory, and these represent the main cognitive domains affected in schizophrenia. PV-TeLC and CCK-TeLC mice displayed significant Y-maze alternation index reduction (p < 0.05). Targeting of PV+ prefrontal cells causes anxiety-like phenotype. Moreover, PV+ and SST+, but not CCK+ interneurons, appear to play a role in latent inhibition. Functional removal of CCK+, PV+ and CCK+ cells from PFC does not affect circadian activity and does not cause anhedonia. The involvement of PV-network in generation of neuronal activity and acetylcholine homeostasis was assessed. For neurophysiological recordings, each arm of the Y-maze divided into two equal-sized zones – proximal (close to the central decision point) and distal (far end). Zone entry was event-mapped onto continues local field potential recordings from medial PFC and CA1 region of the hippocampus. PV-TeLC animals displayed significantly lower prefrontal power in the decision zone. This suggests that the PV-TeLC animals are unable to modulate neuronal activity depending on the cognitive demand. Functional removal of prefrontal PV+ interneurons also leads to disturbed acetylcholine homeostasis. These results show that prefrontal GABAergic cells drive different behaviours and control task-relevant neuronal activity in different brain regions engaged with working memory such as hippocampus. Similar signalling anomalies may thus underlie cognitive deficits found in schizophrenia.

The basal ganglia (BG) are a group of nuclei in the basal forebrain critical in movement, goal directed behaviour and action selection. Cortical projections to the largest BG nucleus, the striatum, are highly important in theories of BG function. Therefore, we have investigated the role of striatal neurons in modulating the activity of corticostriatal synapses. In an in-vitro preparation of rodent brain slices, we conducted whole-cell patch clamp recordings of single and pairs of striatal neurons and recorded responses of medium spiny neurons (MSNs) to stimulation of corticostriatal fibres. In the presence of opioid, GABAA, NK1 and cholinergic receptor antagonists, antidromic stimulation of a population of MSNs (5 stims, 50 Hz) caused suppression of subsequently evoked EPSPs in MSNs. This suppression was dependent upon the interval between antidromic MSN stimulation and the stimulation of evoked EPSPs; suppression was larger at 500 ms intervals than at 1 or 2 s intervals. These effects were completely blocked by the GABAB antagonist CGP 52432. Bursts of evoked action potentials (5 APs, 50 Hz) in a single MSN were insufficient to cause these effects in a nearby MSN. Similar spikes in single fast spiking interneurons and low threshold spiking interneurons (LTSIs) were also insufficient. Conversely, single neurogliaform interneurons (NGFIs) could suppress evoked EPSPs in nearby MSNs in a GABAB-dependent manner. This suppression was more likely in NGFI-MSN pairs that exhibited direct GABAergic interactions. We also tested long depolarisations in LTSIs, a protocol that preferentially releases NO, which was shown to suppress evoked EPSPs through a non-GABAergic mechanism. Finally, we tested the application of exogenous NPY to slices, which also inhibited corticostriatal transmission. These results provide the first demonstration of how GABAB receptors at corticostriatal synapses are activated by endogenous GABA released by striatal neurons. They also reveal novel mechanisms through which striatal factors influence these synapses.

Cys-loop receptors are ligand-gated ion channels that are involved in fast synaptic neurotransmission in the central and peripheral nervous system. The Cys-loop receptor RDL ('resistant to dieldrin') is a GABA-gated chloride channel from Drosophila melanogaster and is a major target site for insecticides. The aim of this dissertation was to characterise RDL receptors with particular focus on the agonist binding site. To assess the potency of a range of GABA analogues on RDL receptors, I expressed receptors in Xenopus oocytes and used voltage-clamp electrophysiology to detect receptor responses. I carried out computational modelling of these analogues to determine the dipole separation distances and atomic charges. Computational calculations and functional experiments revealed that agonists require a charged ammonium and an anionic centre, with the most potent agonists having a dipole separation distance of ~5 Å. I made a homology model of the extracellular domain of RDL and docked the active analogues into the putative binding site. I then conducted mutagenesis studies to test the accuracy of this model. Functional data from mutagenesis studies broadly support the location of GABA within this model. This model may be useful for further structure-activity studies and rational drug design. Natural compounds from the traditional Chinese medicine 'Ginkgo biloba' (ginkgolide A, ginkgolide B and bilobalide) have potent insecticidal properties and are similar in structure to picrotoxin. I tested the effect of these compounds on RDL receptor function using voltage-clamp electrophysiology. All compounds were found to inhibit RDL receptor function. I probed the binding site of these compounds using site-directed mutagenesis and electrophysiology. Mutations to the 2'A and 6'T channel-lining (M2) residues greatly reduced the potency of these compounds. I then made a homology model of the transmembrane domain of RDL and docked these compounds into the channel. Compounds docked into the channel pore close to the 2' and 6' channel-lining residues and H-bonding interactions were detected at these locations. Ginkgolides are therefore antagonists of RDL receptors, binding in the channel close to the 2' and 6' residues and this may be the mechanism underlying their potent insecticidal properties. The 5-HT3 receptor is a member of the Cys-loop receptor family and shows homology to RDL receptors. To explore different techniques for studying Cys-loop receptor function I assessed the functionality of two brain derived transcripts of the 5-HT3B subunit (Br1 and Br2) using single-channel electrophysiology and a fluorometric assay. Receptors containing Br1 were found to have a conductance identical to the 5-HT3B subunit whilst Br2 receptors were found not to be expressed. This finding has implications for 5-HT3 brain signalling, in which Br1 may play an important role. In conclusion, work here has described how agonists bind to and activate RDL GABA receptors and I have identified a candidate mechanism for the potent insecticidal properties of Ginkgo biloba extracts. I have also confirmed that 5-HT3 receptor brain transcript Br1 forms functional channels with similar properties to the 5-HT3B subunit.

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No description available.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu). / A role has been proposed for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in central cardiovascular control. This proposa 1 was based on the cardiovascular effects of agents which block or mimic the action of GABA on the post-synaptic membrane. This dissertation reported the cardiovascular effects of 3-mercaptopropionic acid (3-MP), an agent which inhibits GABA biosynthesis in the pre-synaptic nerve terminal in anesthetized guinea pigs. 3-MP interrupts GABAergic transmission by decreasing the amount of GABA in the brain. The effect of 3-MP on mean arterial pressure, heart rate and barorflex-induced bradycardia was determined as well as the mechanism(s) involved in observed changes. The content of GABA in four regions of the brain (hypothalamus, medulla, cerebellum and occipital cortex) was determined at the end of each experiment. In anesthetized guinea pigs, 3-MP (195 mg/kg, i.p.) elicited a biphasic response (Type I) in the majority of animals. This response consisted of sympathetically-mediated hypertension and tachycardia superseded by vagally-mediated bradycardia. The other response (Type II) consisted of only the sympathetically-mediated effects. The Type II response was associated with animals in which the vagus nerves were functionally impaired. In all brain regions measured, the GABA levels of both Types I and II were significantly lower than those of control animals. The sympathetically-mediated effects of 3-MP were reversed by chlordiazepoxide, a GABA-facilitory agent. Therefore, the 3-MP-induced cardiovascular effects appeared to reflect GABAergic activity in the bra in which resulted from a reduction of GABA content. The two phases of the Type I response may have resulted from a reduction of GABA content in specific brain regions. A reduction in hypothalamic GABA levels appeared to be related to the sympathetic activation and a reduction in medullary GABA levels appeared to be related to the vagal activation. In unparalyzed animals, 3-MP elicited convulsive movements as well as the described effects on the cardiovascular system. The centrally acting anticonvulsant phenytoin stopped 3-MP-induced motor manifestations of seizure activity without altering either blood pressure or heart rate. Therefore, the cardiovascular effects of 3-MP appeared to occur independent of the convulsive effect of this agent. These results support the hypothesis of GABAergic involvement in the central control of autonomic outflow to the cardiovascular system. The inability of phenytoin to reverse the cardiovascular effects of 3-MP suggests that these effects were independent of 3-MP-induced seizures.

This study investigated the β-subunit of the GABAa receptor and determine if there are changes in the primary sequence of the extracellular N-Terminal domain, which likely regulates GABA binding.

No description available.