Made available in DSpace on 2016-06-02T19:22:01Z (GMT). No. of bitstreams: 1 1385.pdf: 3584070 bytes, checksum: d51ff2677c4163046fa6920e114a1234 (MD5) Previous issue date: 2007-03-15 / Universidade Federal de Minas Gerais / The lateral parabrachial nucleus (LPBN) is a pontine structure located in the dorsal portion of the cerebelar superior peduncle. LPBN receives afferent projections from the area postrema (AP) and the medial portion of the nucleus of tractus solitarius (mNTS) and connects to prosencephalic areas related to hidroelectrolytic control, such as specific nuclei of the hypothalamus and amygdala. So, an important role of the LPBN could be integrating ascending information from AP and mNTS that could affect the activity of prosencephalic areas involved in the hidroelectrolytic control. Previous studies showed the presence of serotonergic, cholecystokininergic and α2-adrenergic mechanisms in the LPBN to control water and sodium intake. The γ-aminobutiric acid (GABA) is an inhibitory neurotransmitter present in the whole central nervous system and binding to two subtypes of receptors: GABAA and GABAB receptors. Previously, it was shown the participation of gabaergic mechanisms to control water, sodium and food intake. Gabaergic activation into the LPBN using bilateral injections of muscimol (GABAA receptor agonist) and baclofen (GABAB receptor agonist) induced a strong ingestion of sodium chloride (NaCl 0.3 M) in a short period of time (3 h) in satiated and normovolemic rats. Besides the presence of gabaergic mechanisms into the LPBN, it was already shown the presence of opioid receptors into the LPBN and the involvement of opioid mechanisms in the control of ingestive behavior. Therefore, the goals of this thesis were: a) to investigate the participation of GABAA and GABAB receptors on NaCl and water intake induced by gabaergic activation in the LPBN in satiated and sodium depleted rats; b) to study the effects of gabaergic activation into the LPBN on c-fos protein expression in satiated and sodium depleted rats, with or without access to NaCl 0.3 M; c) to test if acute lesion of the commissural NTS (cNTS area with the greatest c-fos protein expression in satiated rats not allowed to drink sodium or water and treated with muscimol and baclofen into the LPBN) and anteroventral 3o ventricle region (AV3V an important area involved in the hidroelectrolytic control) could affect the natriorexigenic and water intake induced by gabaergic activation into the LPBN in satiated rats; Abstract d) to study the effects of inhibition of α2-adrenergic and opioid mechanisms and activation of serotonergic mechanisms into the LPBN on NaCl and water intakeinduced by gabaergic activation in the same area in satiated rats; e) to investigate the participation of opioid mechanisms in the LPBN on sodium and water intake in satiated and sodium depleted rats; f) to verify the effects of gabaergic activation in the LPBN on arterial pressure and urinary excretion. Male Holtzman or Sprague Dawley rats with bilateral stainless steel guidecannulas implanted into the LPBN (volume of injection: 0.2 µl) were used. Other groups of rats, besides the cannulas implanted into the LPBN, they were also submitted to electrolytic lesion in the cNTS or AV3V region. In satiated rats, bilateral injections of muscimol (0.5 nmol) and baclofen (0.5 nmol) into the LPBN induced 0.3 M NaCl intake followed by an increase in water intake. The effects on sodium and water intake produced by muscimol injected into the LPBN were reduced by previous treatment with bicuculline (GABAA receptor antagonist - 1.6 nmol), but not CGP 35348 (GABAB receptor antagonist - 50 nmol). Pre treatment with bicuculline or CGP 35348 into the LPBN abolished the effects of baclofen on sodium and water intake. In 24 h sodium depleted rats (treatment with sc furosemide + sodium deficient diet and water for 24 h), muscimol and baclofen bilaterally injected into the LPBN, produced an early inhibition and a late facilitation of 0.3 M NaCl. Bicuculline, but not CGP 35348, abolished the inhibitory and facilitatory effects of muscimol injected into LPBN on sodium intake. In relation to sodium depleted rats treated with baclofen into the LPBN, CGP 35348 abolished the inhibitory effect, while bicuculline abolished the facilitatory effect of baclofen on sodium intake. These results suggest that the natriorexigenic effect in satiated rats and the dual effects on sodium intake in sodium depleted rats produced by muscimol injected into the LPBN depend on GABAA receptors activation. In relation to baclofen, in satiated rats the natriorexigenic effect of baclofen depends on activation of GABAA and GABAB receptors, but in sodium depleted rats, the early inhibition of sodium intake depends on GABAB receptors activation while the late facilitation depends on activation of GABAA receptors. Interestingly, although gabaergic activation promotes a facilitation of sodium intake, maybe there is not tonic gabaergic participation on sodium depletion-induced sodium intake, since inhibition of GABAA (bicuculline) and GABAB (CGP 35348) Abstract receptors into the LPBN did not affect 0.3 M NaCl and water intake in sodium depleted rats. Otherwise, in rats trained to drink 0.3 M NaCl only 2 h per day, CGP 35348, but not bicuculline, injected into the LPBN reduced 0.3 M NaCl, suggesting a tonic participation of GABAB, but not GABAA, receptors on sodium intake control in the protocol studied. (...) / O núcleo parabraquial lateral (NPBL) é uma estrutura pontina, localizada dorsalmente ao pedúnculo cerebelar superior, que recebe projeções aferentes da área postrema (AP) e da porção medial do núcleo do trato solitário (NTSm) e que faz conexões com áreas prosencefálicas envolvidas no controle do balanço hidroeletrolítico, como núcleos específicos do hipotálamo e amígdala. Assim, um papel importante do NPBL seria integrar as informações ascendentes do NTSm e AP que por sua vez poderiam influenciar a atividade das áreas prosencefálicas envolvidas no controle do equilíbrio hidroeletrolítico. Ademais, estudos anteriores mostraram a presença de mecanismos serotoninérgicos, colecistocinérgicos e adrenérgicos α2 para o controle da ingestão de água e de sódio no NPBL. O aminoácido inibitório GABA (ácido γ-aminobutírico) é um neurotransmissor inibitório distribuído por todo o sistema nervoso central, se ligando basicamente em dois subtipos de receptores: os receptores GABAA e os receptores GABAB e estudos anteriores mostraram uma participação de mecanismos gabaérgicos no controle da ingestão de água, sódio e alimento, sendo que estudos do nosso laboratório mostraram que a ativação de receptores do aminoácido inibitório GABA no NPBL através de injeções bilaterais de muscimol ou baclofen (agonistas de receptores GABAA e GABAB, respectivamente) induz intensa ingestão de solução hipertônica de cloreto de sódio (NaCl 0,3 M) num período relativamente curto de tempo (3 h) em ratos saciados e normovolêmicos. Além disso, já foi também verificada a presença de receptores opióides no NPBL e a participação de mecanismos opióides no controle do comportamento ingestivo. Assim sendo, foram objetivos da presente tese: a) investigar a participação dos receptores GABAA e GABAB na ingestão de NaCl 0,3 M e água decorrente da ativação gabaérgica no NPBL em ratos saciados e depletados de sódio; b) estudar os efeitos da ativação gabaérgica no NPBL na expressão da proteína c-fos em ratos saciados ou depletados de sódio, com e sem acesso à solução de NaCl 0,3 M; c) verificar os efeitos da lesão aguda do núcleo do trato solitário comissural (NTSc - área que apresentou maior expressão da proteína c-fos em ratos saciados sem acesso ao sódio e água e tratados com muscimol e baclofen no Resumo NPBL) e da região AV3V (importante área envolvida no controle do equilíbrio hidroeletrolítico) sobre o efeito natriorexigênico e a ingestão de água induzidos pela ativação gabaérgica no NPBL em ratos saciados; d) estudar, em ratos saciados, os efeitos da inibição de receptores adrenérgicos α2 e opióides, bem como a ativação de mecanismos serotoninérgicos, no NPBL sobre os efeitos da ativação gabaérgica na mesma área sobre ingestão de NaCl e água em ratos saciados; e) investigar a participação de mecanismos opióides do NPBL sobre a ingestão de sódio e água em ratos saciados ou depletados de sódio; f) verificar os efeitos da ativação gabaérgica do NPBL sobre a pressão arterial e excreção urinária. Para tanto, foram utilizados ratos Holtzman ou Sprague Dawley com cânulas de aço inoxidável implantadas bilateralmente no NPBL (volume de injeção de 0,2 µl em cada lado do NPBL). Outros grupos de ratos, além do implante de cânulas no NPBL também foram submetidos à lesão eletrolítica do NTSc e região AV3V. Em ratos saciados, injeções bilaterais de muscimol (0,5 nmol) e baclofen (0,5 nmol) no NPBL induziram ingestão de NaCl 0,3 M acompanhada de um aumento na ingestão de água, sendo que os efeitos do muscimol foram reduzidos pelo tratamento prévio com bicuculina (1,6 nmol antagonista de receptores GABAA), mas não pelo tratamento com CGP 35348 (50 nmol antagonista de receptores GABAB). Já os efeitos do baclofen foram abolidos pelas injeções prévias de CGP 35348 e também pela bicuculina. Em ratos depletados de sódio por 24 h (tratamento sc com o diurético furosemide + dieta deficiente de sódio e água por 24 h), muscimol e baclofen injetados bilateralmente no NPBL, promoveram uma redução inicial e uma facilitação tardia da ingestão de NaCl 0,3 M. Bicuculina, mas não CGP 35348, aboliu tanto o efeito inibitório quanto facilitatório do muscimol injetado no NPBL sobre a ingestão de sódio. Em relação aos animais depletados tratados com baclofen, o CGP 35348 aboliu o efeito inibitório, enquanto que a bicuculina aboliu o efeito facilitatório do baclofen sobre a ingestão de sódio. Estes resultados sugerem que tanto o efeito natriorexigênico em ratos saciados quanto o efeito dual sobre a ingestão de sódio em ratos depletados de sódio, do muscimol injetado no NPBL dependem da ativação de receptores GABAA. Em relação ao baclofen, em ratos saciados o efeito natriorexigênico do baclofen depende da ativação tanto de receptores GABAA quanto receptores GABAB, já em animais depletados, a inibição Resumo inicial da ingestão de sódio depende da ativação de receptores GABAB, enquanto que a facilitação tardia depende dos receptores GABAA. (...)

A epilepsia do lobo temporal (ELT) é a um dos casos mais frequente epilepsia em humanos e de maior refratariedade nos pacientes. A maioria dos fármacos antiepilépticos são moduladores da atividade neuronal e atuam sobre canais iônicos do receptor GABAA. Estudos vêm demonstrando o papel das células gliais e da neuroinflamação na epileptogênese e a modulação desta resposta pode ser um alvo potencial para drogas adjuvantes aos fármacos anti-epilépticos. Astrócitos são células gliais participantes da sinapse tripartite, moduladores da atividade neuronal. Os astrócitos são capazes de promover a homeostase de íons e de neurotransmissores, são responsáveis pelo metabolismo energético e da produção de fatores neurotróficos, glutationa, glutamina, S100B e citocinas. Neste trabalho, induzimos status epilepticus (SE) em ratos jovens (PN28) através do modelo lítio-pilocarpina que mimetiza alterações neuronais, bioquímicas e morfológicas similares à ELT em humanos. Os animais foram divididos nos tempos 1, 14 e 56 dias após a indução de status epilepticus (SE). Estes períodos são caracterizados respectivamente como a fase aguda, latente e crônica da epilepsia. Inicialmente, analisamos as mudanças neuroquímicas e astrocitárias ao longo do tempo. Foi observada neuroinflamação inicial e transitória que promove morte neuronal e mudanças ao longo do tempo de astrogliose e disfunção astrocitária. Também foi observado que a proteína S100B, proteína ligante de cálcio, predominantemente astrocitária, pode ser considerado um marcador da disfunção neuronal e astrocitária promovida neste modelo de epilepsia. Em seguida, demonstramos que a modulação da secreção de S100B pelo anti-inflamatório dexametasona um dia após indução de SE reverte a neuroinflamação, astrogliose e disfunção astrocitária à curto e à longo prazo. Por conseguinte, observamos que a modulação do receptor GABAA através de agonistas e antagonistas GABAérgicos altera a secreção de S100B em fatias hipocampais agudas e em cultura de astrócitos. Portanto, pode-se sugerir que as alterações astrogliais e a neuroinflamação dependentes do tempo podem estar ligadas à excitabilidade neuronal e/ou à morte neuronal em ratos jovens em modelo de epilepsia; que a proteína S100B pode ser considerada um marcador deste modelo de epilepsia e que a modulação da sua secreção pode ser um possível alvo farmacológico no tratamento da epilepsia. / Temporal lobe epilepsy (TLE) is the most frequent type of epilepsy in humans and is more associated to refractory to anti-epileptic drugs (AED) in patients. The most AEDs are modulators of neuronal activity and act on ion channels, such as GABAA receptor. Studies have been demonstrating the role of glial cells and neuroinflammation in epileptogenesis. The modulation of this response may be a potential target for adjunctive drugs to anti-epileptic drugs. Astrocytes are glial cells that participated in the tripartite synapse and modulated neuronal activity. Astrocytes are able to promote homeostasis of ions and neurotransmitters, are responsible for energy metabolism and the production of neurotrophic factors, glutathione, glutamine, S100B and cytokines. In this work, we induced status epilepticus (SE) in young rats (PN28) through the lithiumpilocarpine model that mimics neuronal, biochemical and morphological alterations similar to ELT in humans. The animals were divided at times 1, 14 and 56 days after the induction of SE. These periods are characterized respectively as the acute, latent and chronic phase of epilepsy. Initially, we analyzed neurochemical and astrocytic changes over time. Initial and transient neuroinflammation was observed and promoted over time neuronal death, astrogliosis and astrocytic dysfunction. It has also been observed that the protein S100B, a calcium-binding protein, predominantly astrocytic, can be considered a marker of neuronal and astrocytic dysfunction promoted by this model of epilepsy. Next, we demonstrate that the modulation of S100B secretion by the antiinflammatory dexamethasone one day after SE induction reverses neuroinflammation, astrogliosis and astrocytic dysfunction in the acute and chronic time. Therefore, we analyzed that modulation of the GABAA receptor through GABAergics agonists and antagonists alters the secretion of S100B in acute hippocampal slices and in astrocyte culture. Therefore, it may be suggested that astroglial changes and time dependent neuroinflammation may be related to neuronal excitability and/or neuronal death in young rats in this epilepsy model; that S100B protein can be considered a marker of this epilepsy model and that the modulation of its secretion may be a possible pharmacological target in the treatment of epilepsy.

Introdução: Os Benzodiazepínicos estão entre as drogas mais prescritas no mundo. Possuem características ansiolíticas, hipnóticas, miorelaxantes e anticonvulsivantes. Estudos prévios evidenciam inadequações nas prescrições de benzodiazepínicos, como uso equivocado para quadros inespecíficos, tratamento prolongado e abuso por idosos. Benzodiazepínicos podem trazer sérios efeitos adversos, sobretudo em idosos, como sonolência diurna, deterioro da memória e funções cognitivas, desequilíbrio e quedas. Objetivos: Analisar a prevalência do uso de benzodiazepínicos nos pacientes do Centro de Atenção Psicossocial (CAPS) de Campo Bom-RS em um período de 24 meses (Junho de 2013 a Maio de 2015), faixa etária, frequência de dispensações, dosagens, CIDs e especialidades dos médicos prescritores; bem como possíveis relações com a função cognitiva, afetiva, e quedas em idosos. Métodos: Estudo transversal, com informações coletadas e tabuladas a partir dos prontuários e prescrições médicas do CAPS Campo Bom, bem como do seu sistema informatizado de gerenciamento (Software Multi 24 Horas). Foram obtidos dados como idade, gênero, identificação dos CIDs; bem como informações sobre prescrições de Benzodiazepínicos, como frequência, quantidade, tipo de medicamento, posologia, profissional prescritor e demais medicamentos utilizados. Critério de inclusão: utilização de qualquer benzodiazepínico disponível na rede SUS: Clonazepam 0,5mg, Clonazepam 2,5 mg/ml, Clonazepam 2mg e Diazepam 5mg. Foram aplicados os testes MEEM e GDS nos pacientes idosos, em entrevistas individuais, nas quais também se avaliou a escolaridade, ocorrência e frequência de quedas, além das comorbidades auto relatadas. Para construção do banco de dados foi utilizado o Software Microsoft® Office Excel® 2007, e para análise estatística o Software PASW V18 (SPSS®). Resultados: O número total de pacientes ativos identificados no CAPS foi de 855 indivíduos, sendo 543 (63,5%) mulheres e 84 idosos (9,8%). A prevalência de utilização de BZD nesta população representou 47,2% (n=404 indivíduos). Foram identificadas 12.680 prescrições médicas dispensadas e, deste total, a prevalência de prescrições de benzodiazepínicos foi de 21,7% (n=2.748). Dentre os pacientes que receberam BZD, 297 (73,5%) eram mulheres e 59 idosos (14,6%), dos quais 56 foram entrevistados. Dentre os idosos entrevistados, 42 (75,0%) possuíram significativa sintomatologia depressiva (GDS≥5) e 32 (57,1%) apresentaram duas ou mais quedas no período de 1 ano. Dezoito pacientes idosos demonstraram fazer uso de dois ou mais BZDs. Houve correlação linear negativa entre os escores do MEEM e do GDS (r = - ,416; p=,002). Houve também uma correlação linear negativa entre o escore do MEEM e número de quedas em idosos (r = -,327 p=,016). Conclusões: Os benzodiazepínicos corresponderam a 21,7 % do total de prescrições do CAPS e do total dos indivíduos 47,2% receberam benzodiazepínicos. Proporcionalmente aos homens, as mulheres tenderam a receber mais prescrições de benzodiazepínicos (p<.001). A prevalência de significativa sintomatologia depressiva nos idosos usuários do CAPS foi alta (75,0%), assim como a incidência de quedas, sendo que mais da metade dos idosos caiu 2 ou mais vezes no período. Identificou-se uma correlação linear negativa entre a função cognitiva como avaliada pelo MEEM e a sintomatologia depressiva avaliada pelo GDS; bem como houve uma correlação linear negativa entre o MEEM e o número de quedas em idosos. / Introduction: Benzodiazepines are among the most prescribed drugs in the world, they have characteristics such as anxiolytic, hypnotic, muscle relaxants and anticonvulsants. Studies have pointed out distortions in benzodiazepines’ prescriptions, such as misuse for unspecific cases, prolonged treatment and use by elderly. Such medications may cause serious damage, particularly in the elderly, and its continued use causes side effects such as daytime somnolence, imbalance, memory and cognitive function loss, increased incidence of falls. Objectives: To assess the prevalence of benzodiazepine use among patients of the Center for Psychosocial Care in Campo Bom-RS within the period of June 2013 to May 2015. The variables analyzed were: age, frequency of dispensations, dosages, ICDs and specialties of prescribing doctors; as well as possible correlations between cognitive function, emotional function and falls in the elderly. Methods: Cross-sectional study, with data collected and tabulated from medical records and prescriptions, as well as from the management system from CAPS (Software Multi 24 hours). Data obtained was age, gender, ICDs identification; as well as frequency of prescriptions, quantity and type of medication, dosage, prescribing professional, others used drugs. Inclusion criteria: Use of any benzodiazepine available in the Health Unic System: Clonazepam 0,5 mg, Clonazepam 2,5 mg/ml, Clonazepam 2 mg and Diazepam 5mg. MMSE and GDS tests have been applied in the elderly, through individual interviews, in which we found about educational level, occurrence and frequency of falls and other self-reported comorbidities. The software Microsoft® Excel® 2007 was used to build database, and for statistical analysis the software PASW V18 (SPSS) was used. Results: The total number of active patients identified at CAPS was 855 individuals, of these 543 were women (63.5%) and 84 elderly (9.8%). The prevalence of benzodiazepines’ use in this population was 47.2 % (n = 404). We have found 12.680 prescriptions dispensed and the prevalence of benzodiazepines’ prescriptions was 21.7 % (n = 2.748). Among patients who received benzodiazepines, 297 (73.5%) were women and 59 elderly (14.6%), of these 56 were interviewed. From the interviewed patients, 42 (75.0%) had significant depressive symptomatology (GDS≥5) and 32 (57.1%) have suffered two or more falls. Eighteen elderly patients demonstrated to use two or more benzodiazepines. There was a negative linear correlation between MMSE and GDS scores (r = -.416, p =.002). There was also a negative linear correlation between MMSE scores and number of falls in elderly (r = -.327 p =. 016). Conclusions: Benzodiazepines accounted for 21.7% of the total CAPS’ prescriptions, and 47.2% individuals treated at CAPS received benzodiazepines. Women tended to receive more prescriptions of benzodiazepines (p <.001) than men. The prevalence of significant depressive symptomatology in the elderly was very high (75.0%). As well as the incidence of falls, since more than half of the elderly patients presented two or more falls in the period. We identified a negative linear correlation between cognitive function as assessed by MMSE and depressive symptoms assessed by the GDS; and there was a negative linear correlation between MMSE and the number of falls in the elderly.

Introduktion: Extrapyramidala systemet är ett nätverk i det centrala nervsystemet(CNS) som har en viktig roll i att samordna kroppsrörelser. De komponenter somtillhör det extrapyramidala systemet är basala ganglier, cerebellum och flera olikaledningsbanor och bland dessa har basala ganglier störst betydelse. Extrapyramidalasymtom, även kallade EPS, är ett samlingsbegrepp för motoriska biverkningar,exempelvis muskelkramper, muskelstelhet och skakningar, som uppkommer omextrapyramidala systemet i hjärnan utsätts för störningar. Vissa läkemedelsgrupper,såsom äldre antipsykotiska och antidepressiva läkemedel, är kända för att orsaka EPSgenom sin påverkan på det extrapyramidala systemet. Det finns även andra läkemedelsom också kan orsaka EPS, vilket inte är lika välkänt. Ett exempel på detta ärciprofloxacin, ett ofta förskrivet antibiotikum. Trots att extrapyramidala symtom kanupplevas besvärliga för patienter och trots att ciprofloxacin är ett mycket välanväntläkemedel, finns det inte mycket publicerad/beskriven information om risken attdrabbas av EPS vid användning av ciprofloxacin. Därför är det värdefullt att ta reda påom ciprofloxacin kan ge EPS och hur detta läkemedel kan påverka nervsystemet. Syfte: Syftet med denna studie är att ta reda på eventuellt samband mellan intag avciprofloxacin och EPS. Syftet är även att undersöka möjliga mekanismer bakom dessasymtom. Metod: Detta är en litteraturstudie som baseras på fallstudier inhämtade fråndatabasen PubMed samt inkomna biverkningsrapporter i Läkemedelsverkets databas,Biverkningsrapportering och Signaldetektion (BiSi), och den amerikanskaläkemedelsmyndighetens (The Food and Drug Administration) databas, FDA AdverseEvent Reporting System (FAERS). Resultat/Diskussion/Slutsats: Denna studie visar att det, trots att frekvensen avEPS är låg, är troligt att användning av ciprofloxacin kan utlösa EPS, framförallttremor, muskelkramper och muskuloskeletal stelhet. Detta resultat kan dock, på grundav det begränsade materialet, inte generaliseras till att omfatta alla patienter. Det äroklart om ciprofloxacin, även hos en frisk person, kan orsaka EPS och om andrafysiologiska faktorer såsom lever- eller njurfunktion hos patienten har betydelse förframkallande av EPS eftersom nedsatt lever- eller njurfunktion ökar toxiciteten avläkemedlet. Det går heller inte att utesluta påverkan av läkemedelsinteraktion medsamtidigt intagna läkemedel. För att kunna ge ett svar med högre tillförlitlighetangående frågeställningen: eventuellt samband mellan intag av ciprofloxacin ochextrapyramidala symtom, krävs det vidare forskning, exempelvis genom att studerasamtliga biverkningsrapporter som innehåller information om patientens profil,dosering av ciprofloxacin och samtidig medicinering. Möjliga mekanismer bakomuppkomsten av EPS vid användning av ciprofloxacin anses vara att ciprofloxacinfungerar som en GABA-A-antagonist i basala ganglier och den förändrade GABA-ergahämningen, på grund av antagonismen, stör den normala rörelsereglerande funktionenhos de basala ganglierna. Avslutningsvis, baserat på resultatet av denna studie kan detvara av värde att iaktta försiktighet vid förskrivning av ciprofloxacin till patienter somhar nedsatt njur- eller leverfunktion och till patienter som har samtidig medicinering,särskilt med läkemedel som är kända för benägenhet att orsaka EPS såsomantipsykotiska och antidepressiva läkemedel.

Comprendre les mécanismes mis en place au sein du système nerveux pour générer des répertoires locomoteurs complexes reste l'un des grands défis des neurosciences systémiques. Le travail présenté dans ce manuscrit vise à comprendre comment les neurones de la moelle épinière contribuent à la production et à la modulation de l'activité locomotrice. Pour répondre à ce problème, nous utilisons le poisson-zèbre comme organisme modèle et avons développé de nouvelles approches génétiques et optiques afin de disséquer l'architecture du circuit formé par une classe de neurones sensoriels de la moelle et qui est conservée chez tous les vertébrés. Ces neurones sont appelés les neurones au contact du liquide céphalo-rachidien (Nc-LCR) et nous proposons de sonder leur(s) fonction(s) in vivo. Ces neurones sensoriels forment une interface unique entre le liquide céphalo-rachidien et le réseau de neurones impliqué dans le contrôle du mouvement dans la moelle épinière. Cependant, leur diagramme de connectivité demeure complètement inconnu. Afin de comprendre comment ces " Nc-LCR ou CSF-cNs " modulent la locomotion chez les vertébrés, nous avons développé un projet combinant des approches génétiques, électrophysiologiques, d'imagerie, et d'analyse du comportement, afin de cartographier le circuit qu'elles forment avec les neurones de la moelle épinière. Nos résultats montrent que les CSF-cNs projettent sur de nombreux éléments du centre générateur de rythme de la moelle. Notre approche révèle également la capacité des CSF-cNs à moduler la locomotion selon l'état dans lequel se trouve l'animal, une propriété caractéristique des circuits proprioceptifs dans la moelle épinière. / Understanding how the central nervous system generates motor sequences, coordinates limbs and body orientation in an ever-changing environment, while adapting to sensory cues remains a central question in the field of systems neuroscience. The work presented here aims to understand how local sensory neurons in the spinal cord contribute to the production and/ or the modulation of locomotor activity. We focused our work on a conserved class of spinal sensory neurons termed cerebrospinal fluid contacting neurons (CSF-cNs). These neurons lie at the interface between the CSF and spinal interneurons controlling motor output and represent an interesting yet poorly understood sensorimotor loop in the vertebrate spinal cord. However, the connectivity of CSF-cNs remains completely uncharacterized. To understand how CSF-cNs modulate locomotion in vertebrates, we combined genetics, imaging, optogenetics, electrophysiology, and behavior analysis to map the functional connectivity of these sensory neurons and test their function in the zebrafish larva. Our results demonstrate that CSF-cNs target several elements thought to be part of the locomotor central pattern generator in zebrafish, including glutamatergic spinal neurons involved in slow and fast swimming. We show that CSF-cNs can modulate the duration and occurrence of spontaneous locomotor events in a state dependent manner and tune the frequency of evoked fast escape responses. Altogether our work dissecting sensorimotor integration in the spinal cord bridged single cell function in vivo to behavior in zebrafish and should contribute to a better understanding of the role of sensory feedback during locomotion in vertebrates.

This thesis investigates the phytochemistry and pharmacology of two neotropical plants used traditionally to treat anxiety and stress, Souroubea sympetala (Marcgraviaceae) and Piper amalago (Piperaceae). A method of phytochemical analysis was developed to characterize S. sympetala extracts, identifying and quantifying four triterpenes, which were present in higher amounts in bark as compared to leaf. Subsequently, a standardized supercritical CO2 extraction procedure for S. sympetala was developed and compared favourably with conventional extraction methods in terms of its anxiety-reducing effects in a behavioural assay of anxiety and content of the active principle, betulinic acid (BA). All of these materials demonstrated anxiolytic properties. The pharmacological mode of action of S. sympetala raw plant, extracts and isolated active principle were examined in rodent behavioural models of anxiety. The extracts were shown to have affinity for the γ-amino butyric acid (GABA)a benzodiazepine (GABAa- BZD) receptor of the central nervous system in vitro, in a competitive binding assay. Pre-treatment of animals with the GABAa-BZD antagonist flumazenil, followed by plant extract and pure compound extinguished the anxiety-reducing effect, demonstrating that S. sympetala and BA act at the GABAa- BZD receptor in vivo. The effect of S. sympetala in stressed animals, specifically its cortisol-lowering ability was investigated in vitro and in vivo in rainbow trout. Both leaf extract and BA significantly lowered cortisol in response to an adrenocorticotropic hormone (ACTH) challenge in vitro and a standardized net restraint assay in vivo. The anxiety-reducing effect of P. amalago was examined and the bioactive principle identified by bioassay-guided fractionation. P. amalago extract significantly reduced anxiety-like behaviour in rats and demonstrated affinity for the GABAa-BZD receptor in vitro. The bioactive molecule was determined to be a furofuran lignan. Together these results provide a pharmacological basis for the traditional use of S. sympetala and P. amalago to treat anxiety and elucidate their mode of action and active principles. S. sympetala is now thoroughly characterized and represents an excellent candidate plant for development as a natural health product.

This PhD thesis focuses on improving the limit of detection (LOD) of Raman biosensors by using surface enhanced Raman scattering (SERS) and/or hollow core photonic crystal fibers (HC-PCF), in conjunction with statistical methods. Raman spectroscopy is a multivariate phenomenon that requires statistical analysis to identify the relationship between recorded spectra and the property of interest. The objective of this research is to improve the performance of Raman biosensors using SERS techniques and/or HC-PCF, by applying partial least squares (PLS) regression and principal component analysis (PCA). I began my research using Raman spectroscopy, PLS analysis and two different validation methods to monitor heparin, an important blood anti-coagulant, in serum at clinical levels. I achieved lower LOD of heparin in serum using the Test Set Validation (TSV) method. The PLS analysis allowed me to distinguish between weak Raman signals of heparin in serum and background noise. I then focused on using SERS to further improve the LOD of analytes, and accomplished simultaneous detection of GLU-GABA in serum at clinical levels using the SERS and PLS models. This work demonstrated the applicability of using SERS in conjunction with PLS to measure properties of samples in blood serum. I also used SERS with HC-PCF configuration to detect leukemia cells, one of the most recurrent types of pediatric cancers. This was achieved by applying PLS regression and PCA techniques. Improving LOD was the next objective, and I was able to achieve this by improving the PLS model to decrease errors and remove outliers or unnecessary variables. The results of the final optimized models were evaluated by comparing them with the results of previous models of Heparin and Leukemia cell detection in previous sections. Finally, as a clinical application of Raman biosensors, I applied the enhanced Raman technique to detect polycystic ovary syndrome (PCOS) disease, and to determine the role of chemerin in this disease. I used SERS in conjunction with PCA to differentiate between PCOS and non-PCOS patients. I also confirmed the role of chemerin in PCOS disease, measured the level of chemerin, a chemoattractant protein, in PCOS and non-PCOS patients using PLS, and further improved LOD with the PLS regression model, as proposed in previous section.

Alors que les synapses sont des structures relativement stables, les éléments qui la composent sont, eux, en permanent échange dans le temps et dans l'espace. Les composants des densités postsynaptiques sont renouvelés avec des cinétiques caractéristiques de chaque molécule et de chaque sous compartiment synaptique. La compatibilité entre le comportement dynamique des composants de la synapse et son maintien structural et fonctionnel à long terme implique une conception de ces assemblages multimoléculaires en équilibre dynamique. De nombreux paramètres peuvent influencer la dynamique des récepteurs aux neurotransmetteurs (RNT) dans les synapses, y compris l'activité synaptique et les protéines de la matrice extracellulaire. Cependant, le rôle des cellules gliales dans ce mécanisme est inconnu. Mon travail de thèse a porté sur l'exploration d'une possible contribution de la microglie, les cellules immunitaires du système nerveux central, à la stabilité des RNT et à l'efficacité des synapses inhibitrices de la moelle épinière. Mon travail de thèse démontre pour la première fois comment et en quoi la microglie est un partenaire clé de l'équilibre dynamique qui régit la structure et la fonction de la synapse inhibitrice dans la moelle. Par conséquent, il donne un éclairage nouveau sur la façon de concevoir l'efficacité synaptique et sa régulation de façon non neurone autonome. / Whereas synapses are relatively stable structures, their molecular constituents are continuously recycled and exchanged in time and space. Each of the molecules that contribute to build synaptic structures is renewed with specific kinetics, depending on their organisation in the postsynaptic densities. The compatibility between a dynamic behaviour and a long-term maintenance of synapses implies to think synapses as multi-molecular assemblies in a dynamic equilibrium. Several parameters can influence the dynamics of receptors to neurotransmitters(RNT) at synaptic sites, including neuronal activity and extracellular matrix proteins. However,the role of glial cells in this mechanism is unknown. During my thesis work, I explored the roleof microglia, the resident immune cells of the central nervous system, on the lateral diffusion ofRNT and synaptic efficacy at spinal cord inhibitory postsynaptic densities. My work demonstrates for the first time a partnership between microglia and synapses. It shows that immune cells can take part to the regulation of synaptic strength very rapidly but also at basal state, by regulating RNT dynamics. Furthermore it identifies microglia as a key partner for a heterocellular stabilization of synaptic receptors. This work raises the intriguing possibility that the general regulation of network activity may also be explained by a fine modulation of receptors stability at the synapse controlled by microglia.

Changes in spontaneous spike activities recorded from murine frontal cortex networks grown on substrate-integrated microelectrodes were used to determine the dissociation constant (KB) of three GABAA antagonists. Neuronal networks were treated with fixed concentrations of GABAA antagonists and titrated with muscimol, a GABAA receptor agonist. Muscimol decreased spike activity in a concentration dependent manner with full efficacy (100% spike inhibition) and a 50% inhibitory concentration (IC50) of 0.14 ± 0.05 µM (mean ± SD, n=6). At 10, 20, 40 and 80 µM bicuculline, the muscimol IC50 values were shifted to 4.3 ± 1.8 µM (n=6), 6.8 ± 1.7 µM (n=6), 19.3 ± 3.54 µM (n=10) and 43.5 µM (n=2), respectively (mean ± SD). Muscimol titration in the presence of 10, 20, 40 µM of gabazine resulted in IC50s values of 20.1 (n=2), 37.17 (n=4), and 120.45 (n=2), respectively. In the presence of 20, 80, and 160 µM of TMPP (trimethylolpropane phosphate) the IC50s were 0.86 (n=2), 3.07 (n=3), 6.67 (n=2) µM, respectively. Increasing concentrations of GABAA antagonists shifted agonist log concentration-response curves to the right with identical efficacies, indicating direct competition for the GABAA receptor. A Schild plot analysis with linear regression resulted in slopes of 1.18 ± 0.18, 1.29 ± 0.23 and 1.05 ± 0.03 for bicuculline, gabazine and TMPP, respectively. The potency of antagonists was determined in terms of pA2 values. The pA2 values were 6.63 (gabazine), 6.21 (bicuculline), and 5.4 (TMPP). This suggests that gabazine has a higher binding affinity to the GABAA receptor than bicuculline and TMPP. Hence, using spike rate data obtained from population responses of spontaneously active neuronal networks, it is possible to determine key pharmacological properties of drug-receptor interactions.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第21714号 / 薬科博第105号 / 新制||薬科||11(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 金子 周司, 教授 小野 正博, 教授 土居 雅夫 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM