The nematode Caenorhabditis elegans possesses a relatively simple nervous system of only 302 neurons, but is able to perform an impressive range of complex behaviors. This dissertation aims to understand the neurobiology of behavior by quantifying, at the systems-level, the sensorimotor response to carefully controlled stimuli. Through neuronal or genetic perturbations to the system, we can begin to uncouple the behavior from the underlying circuitry. The behavior studied here is thermal nociception, an escape response designed to protect an organism from potential tissue damage or harm from noxious heat. Vertebrates and invertebrates alike possess sensory neurons called nociceptors that detect noxious stimuli and relay the stimulus information to elicit an appropriate escape response. C. elegans is known to perform a reversal or forward response when presented with noxious stimuli at the head or tail, respectively. In this work, we develop a novel thermal stimulus assay with precise spatiotemporal control of an infrared pulse that targets small regions along the worm to spatially dissect the noxious response. We comprehensively quantify the nociceptive behavior, and identify key metrics that scale with intensity, such as speed in the escape state and the probability of certain behavioral states after the stimulus. Furthermore, we have mapped the behavioral receptive field of the worm along its body, and show a previously unreported probabilistic midbody behavior distinct from the head and tail responses. Surprisingly, the worm is able to differentiate localized stimuli at the midbody that are as close as 80 microns. We identified PVD as the thermal nociceptor for the midbody response using calcium imaging, genetic ablation and laser ablation. This suggests PVD could be used as a model to study spatial discrimination at the level of a single nociceptor. This spatial specificity further extends to pharmacological perturbations of the system. In particular, the application of clinically used painkillers to the worm results in a knockdown of this nociceptive response, but does so in a spatially specific manner. These results are promising for future studies building upon the techniques developed here, as they evidentiate the use of C. elegans as a model organism to study pain.

<p><p>Nociceptors are specialized primary sensory neurons that represent the first line of defense against potentially tissue damaging environmental stimuli, and are involved in pathological pain states caused by nerve damage, inflammation and many chronic diseases. In nociception, these neurons detect harmful stimuli and contribute to the reactions to avoid them. Nociceptors transduce noxious stimuli into membrane depolarization, which in turn, triggers action potentials. These action potentials are conducted to synapses in the central nervous system (CNS), resulting in release of neurotransmitters at the presynaptic terminal. The unifying factor in the progression of nociceptive signaling i.e. transduction, action potential propagation, and neurotransmitter release, is the contribution of ion channels. </p><p><p>In this study, I use <italic>Drosophila melanogaster</italic> larvae as a model system to study the contribution of ion channels to nociception. Larvae stimulated with a noxious thermal or mechanical stimulus perform a stereotyped and quantifiable escape behavior. Larvae exhibiting this nocifensive behavior rotate around their long body axis in a corkscrew-like manner thus escaping the damage of the noxious stimulus. This behavior is triggered by the Class IV multidendritic (md) neurons, which are the main larval nociceptors. I describe here, the results of my systematic screen for ion channels required for larval thermal nociception. To perform this screen, I utilized RNAi to knock down the expression of 98% of the predicted ion channels in the <italic>Drosophila</italic> genome. I observed the effects of ion channel knockdown in the thermal nociception behavioral assay. </p><p><p>In addition, I present detailed characterization of an ion channel that I found to be critical for inhibition of nociceptor excitability, the small conductance calcium-activated potassium channel, SK. This channel inhibits both thermal and mechanical nociception. Results of calcium imaging studies show enhanced excitability of larval nociceptors in <italic>SK</italic> mutant animals. My findings support a role for SK function at the sensory afferents, cell body, and axon. </p><p><p>Another candidate ion channel gene, <italic>shadrach</italic>, encodes a Degenerin/Epithelial Na+ channel (DEG/ENaC) that I found to be required for thermal nociception. DEG/ENaCs are conserved in flies, nematodes, and several vertebrates including humans. These channels are expressed in a variety of tissues including kidney epithelia, muscle, and neurons. Members of this superfamily play a role in a host of biological processes including salt homeostasis, neurodegeneration, proprioception, touch transduction, and nociception. RNAi knockdown of <italic>shadrach</italic> results in increased thermal nociceptive threshold. Optogenetic experiments suggest that shadrach functions downstream of transduction. </p><p><p>Furthermore, I identified seven ion channel genes in the thermal nociception screen, which affect nociceptor dendrite morphology. It is possible that thermal nociception behavioral phenotypes in these RNAi mutants are a consequence of the altered dendritic field. Reduction in segmental coverage by the nociceptors may influence the ability to detect noxious stimuli. Future research in our laboratory will establish the relationship between these ion channels, nociceptor development, and nociceptive behavioral output. </p><p><p> <italic>Drosophila melanogaster</italic> is emerging as a powerful model for the study of pain signaling. I have uncovered several candidate ion channel genes that contribute to thermal nociception; of these, <italic>SK</italic> and <italic>shadrach</italic> are required for the response to noxious heat. I have shown that dendritic field coverage is important for the detection of noxious stimuli, and I have identified many candidate genes that are required for normal dendrite morphology.</p> / Dissertation

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A doenÃa de Parkinson (DP) à uma desordem neurodegenerativa caracterizada pela destruiÃÃo dos neurÃnios nigroestriatais dopaminÃrgicos. O tratamento atual à apenas sintomÃtico e, atà o presente momento, nÃo existem drogas capazes de inibir a degeneraÃÃo neuronal. Assim à grande a procura por agentes neuroprotetores que possam impedir ou retardar a progressÃo desta doenÃa. A Camellia sinensis à uma espÃcie da famÃlia Theaceae, popularmente conhecida como chÃ-verde (CV). VÃrios estudos tÃm demonstrado os efeitos benÃficos desta planta e de seus princÃpios bioativos contra doenÃas neurodegenerativas. O objetivo deste trabalho foi avaliar o efeito neuroprotetor do extrato padronizado do chÃ-verde e de suas catequinas, epicatequina (EC) e epigalocatequina 3-galato (EGCG), no modelo de DP induzida por 6-OHDA em ratos, bem como estudar o efeito do CV em modelos de inflamaÃÃo e nocicepÃÃo. Os animais (ratos Wistar machos, 180-250 g) foram tratados por via oral com CV (25 ou 50 mg/kg), catequinas (EC ou EGCG, na dose de 10 mg/kg), a associaÃÃo de CV (25 mg/kg) + L-DOPA (25 mg/kg) e com L-DOPA sozinha (nas doses de 25 ou 50 mg/kg) durante 14 dias consecutivos; os tratamentos foram iniciados 1h antes da lesÃo estriatal unilateral por 6-OHDA (24 Âg/2ÂL). Nos testes comportamentais, os resultados mostraram que houve um aumento significativo do nÃmero de rotaÃÃes induzidas por apomorfina, diminuiÃÃo da atividade locomotora no teste do campo aberto, aumento do tempo de imobilidade no teste no nado forÃado, aumento do tempo de encontro da plataforma no teste do labirinto aquÃtico e aumento do nÃmero de quedas no teste do rota rod nos animais lesionados com 6-OHDA, em comparaÃÃo com os animais falso-operados. Essas modificaÃÃes foram, em parte ou totalmente, revertidas apÃs os tratamentos com CV, CV + L-DOPA, EC, EGCG e L-DOPA sozinha. A 6-OHDA provocou morte neuronal, evidenciada pela reduÃÃo dos nÃveis de dopamina e metabolitos (DOPAC e HVA) no estriado direito lesionado quando comparado com o estriado esquerdo nÃo-lesionado (contralateral). Essa depleÃÃo foi significativamente atenuada nos animais lesionados e tratados com CV, L-DOPA, EC, EGCG e associaÃÃo CV 25 + L-DOPA 25; estas mesmas drogas diminuÃram os nÃveis de TBARS (indicador de peroxidaÃÃo lipÃdica) e nitrito/nitrato (indicador de estresse oxidativo) no estriado de ratos submetidos a lesÃo estriatal por 6-OHDA. Experimentos in vitro demonstraram que o CV (0,1-100 Âg/Âl) apresentou um forte efeito antioxidante, ao reduzir a produÃÃo de substÃncias oxidantes em neutrÃfilos humanos estimulados por PMA. Na coloraÃÃo de Nissl, observou-se que nos animais lesionados e tratados com CV, EGCG e CV + L-DOPA houve uma preservaÃÃo dos neurÃnios hipocampais. Os tratamentos com CV e CV+L-DOPA aumentaram a imunorreatividade para TH e diminuÃram a imunomarcaÃÃo para COX-2 no estriado, bem como o CV e EGCG atenuaram a marcaÃÃo para iNOS no hipocampo dos animais tratados, em relaÃÃo ao grupo controle. Observou-se que o CV potencializou os efeitos da L-DOPA, evidenciando um possÃvel efeito sinÃrgico entre essas drogas. Em outra etapa do estudo, avaliou-se a atividade anti-inflamatÃria/antinociceptiva do CV. Assim, no modelo de edema de pata, induzido por carragenina ou dextrano, verificou-se uma diminuiÃÃo do volume do edema da pata dos ratos, apÃs tratamento com CV, em relaÃÃo aos do grupo controle (tratado apenas com Ãgua destilada). O CV produziu efeito antinociceptivo, nos testes da formalina e das contorÃÃes abdominais, induzidas por Ãcido acÃtico, como tambÃm nos testes da placa quente e de Hargreaves, possivelmente atravÃs da ativaÃÃo de receptores opioides e da reduÃÃo do processo inflamatÃrio. Nestes testes de nocicepÃÃo e inflamaÃÃo, o CV potencializou os efeitos da morfina, indometacina e dexametasona. Portanto, nossos resultados evidenciaram os efeitos neuroprotetores do CV e de seus princÃpios bioativos, EC e EGCG, possivelmente decorrentes de suas propriedades anti-inflamatÃrias e antioxidantes, tornando-as opÃÃes terapÃuticas futuras para a prevenÃÃo ou tratamento de doenÃas neurodegenerativas, como a doenÃa de Parkinson.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Carvacrol (5-Isopropil-2-metilfenol) Ã um monoterpeno fenÃlico presente nos Ãleos essenciais de diversas plantas. Ã o principal constituinte dos Ãleos essenciais de orÃgano e thyme. Conhecendo previamente a aÃÃo antidepressiva e ansiolÃtica do carvacrol, este trabalho apresenta uma anÃlise das concentraÃÃes de monoaminas e aminoÃcidos em corpo estriado de camundongos apÃs o tratamento oral com carvacrol nas doses de 25 e 50 mg/Kg. Os resultados mostraram um aumento dos nÃveis de dopamina em ambas as doses e de GABA, aspartato, taurina e glicina na dose de 25 mg/Kg. Sabendo do importante papel do estresse oxidativo na fisiopatologia da depressÃo, foi estudado o efeito do carvacrol nas doses de 25 e 50 mg/Kg em animais previamente submetidos a injeÃÃes de pilocarpina. As Ãreas cerebrais do hipocampo, corpo estriado e cÃrtex prÃ-frontal foram retiradas para anÃlise das concentraÃÃes de MDA, nitrito-nitrato, catalase, GSH e SOD. Os resultados mostraram que houve diminuiÃÃo dos nÃveis de MDA em ambas as doses em todas as Ãreas cerebrais. Os nÃveis de nitrito-nitrato diminuÃram em corpo estriado e hipocampo de animais tratados com a dose de 50 mg/Kg e nos animais tratados com a dose de 25 mg/Kg, houve diminuiÃÃo dos nÃveis de nitrito-nitrato no hipocampo. A concentraÃÃo de catalase diminuiu em ambas as doses na Ãrea cerebral do hipocampo e os nÃveis de GSH aumentaram na dose de 50 mg/Kg nas Ãreas do corpo estriado e hipocampo. Os nÃveis de SOD nÃo foram alterados. Visto que muitas substÃncias antidepressivas apresentam tambÃm aÃÃo analgÃsica, o presente trabalho apresentou um estudo das aÃÃes comportamentais do carvacrol em modelos animais de nocicepÃÃo, tais como teste da formalina, placa quente e contorÃÃes induzidas por Ãcido acÃtico. Para estes testes, foram utilizadas doses de 50 e 100 mg/Kg. No teste das contorÃÃes induzidas por Ãcido acÃtico, carvacrol em ambas as doses diminuiu o nÃmero de contorÃÃes. No teste da placa quente, carvacrol em ambas as doses apresentou um aumento no tempo de latÃncia de dor no tempo de 60 min. No modelo de nocicepÃÃo induzida pela formalina, os animais tratados com carvacrol na dose de 50 mg/Kg apresentaram uma diminuiÃÃo do tempo de lambedura da pata na primeira fase do teste. JÃ os animais tratados com carvacrol na dose de 100 mg/Kg apresentaram uma diminuiÃÃo do tempo de lambedura tanto na primeira quanto na segunda fase do teste. O prÃ-tratamento dos animais com naloxona ou L-arginina nÃo foi capaz de reverter o efeito antinociceptivo do carvacrol na dose de 100 mg/Kg no teste das contorÃÃes induzidas por Ãcido acÃtico. No modelo da nocicepÃÃo induzida pela formalina, o prÃ-tratamento dos camundongos com naloxona ou L-arginina tambÃm nÃo foi capaz de reverter o efeito antinociceptivo do carvacrol na dose de 100 mg/Kg na primeira e segunda fase do teste. O teste do campo aberto e rota rod nÃo demonstrou alteraÃÃo na dose de 100 mg/Kg, demonstrando que essa substÃncia nÃo alterou a atividade locomotora dos animais. Foi realizada uma avaliaÃÃo toxicolÃgica aguda em camundongos previamente tratados com carvacrol nas doses de 300 e 2000 mg/Kg. A anÃlise histolÃgica do fÃgado, rins e cÃrebro nÃo mostrou nenhuma alteraÃÃo desses ÃrgÃos apÃs o tratamento com carvacrol. / Carvacrol (5-Isopropyl-2-methylphenol) is a monoterpenic phenol present in the essencial oil of many plants. It is the major component of the essential oil fraction of oregano and thyme. Knowing the antidepressant and anxiolytic actions of carvacrol, this work presents an analysis of monoamines and amino acids concentrations in mice striatum after oral treatment with carvacrol at doses of 25 and 50 mg / Kg. The results showed increased expression of dopamine in both doses of carvacrol and increased levels of GABA, aspartate, glycine and taurine at the dose of 25 mg / kg. Knowing the important role of oxidative stress in the pathophysiology of depression, the present work studied the effects of carvacrol at doses of 25 and 50 mg/Kg in animals submitted to previous injection of pilocarpine. The brain areas of hippocampus, striatum and prefrontal cortex were removed for analysis of MDA, nitrite-nitrate, catalase, SOD and GSH concentrations. The results showed that there was a decrease in MDA levels in both doses in all the studied brain areas. Nitrite and nitrate levels decreased in the striatum and hippocampus of animals treated with the dose of 50 mg/Kg and the animals treated with 25 mg/Kg showed a decrease in nitrite and nitrate levels in the hippocampus. The concentration of catalase at both doses decreased in the hippocampus and the levels of GSH increased in the dose of 50 mg/Kg in mice striatum and hippocampus. The SOD levels did not change. Since many antidepressant substances also have analgesic action, this work presented a study of the behavioral actions of carvacrol in animal models of nociception such as the formalin test, hot plate and acetic acid-induced abdominal constriction test. For these tests we used carvacrol at doses of 50 and 100 mg/Kg. In acetic acid induced-abdominal constriction test, carvacrol at both doses reduced the number of writhes. In the hot plate test, carvacrol at both doses showed an increase in pain latency at the time of 60 min. In the formalin test, animals treated with carvacrol at a dose of 50 mg/Kg showed a decrease in paw licking time in the first phase of the test. The animals treated with carvacrol at a dose of 100 mg/Kg showed a reduction in paw licking time in both first and second phases of the test. Pretreatment with naloxone or L-arginine was unable to reverse the antinociceptive effect of carvacrol at a dose of 100 mg/Kg in acetic acid induced-abdominal constriction test. In the formalin test, pretreatment of mice with naloxone or L-arginine was also unable to reverse carvacrol antinociceptive effect at a dose of 100 mg/Kg in the first and second phases of the test. The open field and rota rod tests were performed at dose of 100 mg/Kg and showed no significant changes, demonstrating that the substance did not changed locomotor activity in mice. Acute toxicological evaluation was performed in mice pretreated with carvacrol at doses of 300 and 2000 mg/Kg. Histological analysis of liver, kidney and brain showed no change in these organs after treatment with carvacro.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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Despite the development of antiretroviral therapy (ART), human immunodeficiency virus (HIV)-associated distal sensory polyneuropathy (HIV-DSP) has remained prevalent. To recapitulate chronic pain during HIV infection, simian immunodeficiency virus (SIV)-infected rhesus macaques were examined to dissect the molecular mechanisms of peripheral and central sensitization. Previous studies identified atrophy in nociceptive neurons during SIV infection, which was associated with monocyte infiltration into the dorsal root ganglia (DRG). However, the sensory signaling mechanism connecting this pathology to symptomology remains unclear, especially because pain persists after resolution of high viremia and inflammation with ART. We hypothesize that residual DRG and dorsal horn neuroinflammation contributes to nociceptive sensitization. Using three cohorts of macaques (uninfected, SIV-infected, and SIV+/ART), we showed an increase in the cellular and cytokine inflammatory profiles in the DRG of SIV+/ART macaques compared to uninfected animals. We observed significant increased expression of nociceptive ion channels, transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) among DRG neurons in SIV+/ART compared to uninfected animals. SIV-infected and SIV+/ART animals showed reduced innervation of the non-peptidergic nociceptors into the dorsal horn compared to uninfected animals. Finally, there were significantly higher numbers of CD68+ macrophages in the dorsal horn of SIV+/ART macaques compared to uninfected animals. As pain persists in people with HIV (PWH) on ART, mechanisms outside of viral load and DRG pathology were investigated. Here, bulk RNAseq of DRGs from the three cohorts of macaques revealed a significant increase in upstream regulation by interferon γ (IFNγ) and lipopolysaccharide (LPS) with SIV infection. The introduction of ART decreased the signaling of IFNγ and LPS, yet in SIV+/ART animals, there was a significant increase in expression of the IFNγ inducible chemokines, CXCL9 and CXCL10. To assess if CXCL9 and/or CXCL10 influence TRPV1/TRPA1 expression, we utilized an induced pluripotent stem cell-derived peripheral sensory neuron model (IPSC-PSN). Treatment of IPSC-PSNs with CXCL9 and/or CXCL10 induced a significant increase in TRPV1, but not TRPA1 expression. Since macrophages persist in the DRGs of SIV+/ART macaques, monocyte-derived macrophages (MDMs) were treated with IFNγ or LPS. MDMs treated with IFNᵧ but not LPS, significantly increased the release of CXCL9/CXCL10. Conditioned media from MDMs treated with IFNγ, but not LPS, significantly increased expression of TRPV1 in IPSC-PSNs. Inhibition of the CXCL9/CXCL10 receptor, CXCR3, in IPSC-PSNs partially blocked TRPV1 upregulation following treatment with conditioned media from IFNᵧ treated MDMs. In summary, these data demonstrate that neuroinflammation, characteristics of nociceptor sensitization, and central terminal atrophy persists in SIV infection with ART. Mechanistically, these data indicate a potential role of IFNᵧ signaling in the sensitization of nociceptors in HIV-DSP despite ART. / Biomedical Sciences

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