Vários estudos mostram que o núcleo mediano da rafe (NMR) e o hipocampo dorsal (HD) estão envolvidos no medo condicionado Pavloviano. Além disso, mecanismos serotoninérgicos do NMR parecem participar da expressão da resposta de medo condicionado contextual. Entretanto, ainda não existe uma abordagem experimental que integre os mecanismos do circuito NMR-HD. Neste trabalho, o paradigma do medo condicionado foi utilizado para testar a influência dos mecanismos serotoninérgicos do circuito NMR-HD no medo condicionado contextual. As respostas de sobressalto e congelamento foram avaliadas após a administração de drogas serotoninérgicas intra-NMR e no HD, 6 h depois a sessões treino, nas quais os ratos eram condicionados com choques nas patas. A redução da transmissão serotoninérgica no NMR é devido a microinjeção do 8-hidroxi-2(di-n-propilamino)-tetralin (8-OH-DPAT), um agonista de receptores 5-HT1A, no NMR promoveu redução das respostas de congelamento, mas não alterou a resposta de sobressalto. Estes resultados são consistentes com a ideia de que mecanismos serotoninérgicos no NMR regulam as respostas de congelamento a um contexto aversivo. A diminuição pós-sináptica da serotonina nas áreas de projeção do NMR ocorre devido a ativação de autoreceptores 5-HT1A nesta estrutura. Com relação ao hipocampo, a microinjeção de cetanserina, um antagonista de receptores 5-HT2, não promoveu alteração nas respostas de congelamento e sobressalto potencializado pelo medo, porém a ativação de receptores 5-HT1A pela injeção de 8-OH-DPAT 6 h após o treino inibiu essas respostas. De acordo com esses resultados, um mecanismo inibitório deva se interpor entre os processos associados à chegada de informação aversiva e os associados à saída delas no HD. As projeções HD-amígdala e córtex pré-frontal medial podem constituir a porta de saída dos processos neurais subjacentes a expressão do medo condicionado contextual, conforme foi observado no experimento em que estudou a imunorreatividade destas estruturas á proteína Fos em ratos submetidos ao mesmo procedimento experimental de medo condicionado contextual / Several studies have shown that the median raphe nucleus (MRN) and dorsal hippocampus (DH) are involved in Pavlovian conditioned fear. Moreover, previous findings have also implicated serotonergic mechanisms of the MRN in the retrieval of contextual conditioned fear. However, studies that examine the integrated involvement of serotonergic mechanisms of the MRN-DH are lacking. This study, a fear conditioning paradigm was used to test whether the serotonergic projections from the MRN to DH can influence contextual fear conditioning. Startle and freezing responses were avaliated after administration of serotoninergics drugs into the MRN or DH, 6 h previously rats received footshocks in the training session. A reduction of 5-HT transmission in the MRN by local infusions of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) decreased freezing in response to the CS but did not reduce fear-potentiated startle. This pattern of results is consistent with the hypothesis that MRN serotonergic mechanisms selectively modulate the freezing response to the aversive context. As for the DH, a decrease in postsynaptic 5-HT receptor activity at projection areas has been proposed to be the main consequence of 5-HT1A receptor activation in the MRN. Infusions of the 5-HT2 receptor antagonist ketanserin into the DH had no effect, but activation of 5-HT1A receptors through intra-DH injections of 8-OH-DPAT inhibited both the freezing and fear-potentiated startle response to the CS. To reconcile these findings, an inhibitory mechanism may exist between the incoming DH 5-HT pathway from the MRN and the presynaptic 5-HT neurons that are part of the DH output to other structures. The DH-amygdala and medial prefrontal cortex projections could well be this output circuit modulating the expression of contextual fear conditioning as revealed by measurements of Fos immunoreactivity in these areas.

Orientador: Alexandre Leite Rodrigues de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T12:05:48Z (GMT). No. of bitstreams: 1 Benitez_SuzanaUlian_M.pdf: 7544701 bytes, checksum: 2e552c7219f0c635a4b0afecffd3204d (MD5) Previous issue date: 2013 / Resumo: Lesões nas raízes dorsais da medula espinal são frequentes e muitas vezes decorrentes de acidentes automobilísticos. Devido à possibilidade de geração de dor neuropática, os procedimentos cirúrgicos não priorizam o reparo do componente aferente, sendo reparado apenas o componente motor. Adicionalmente, a perda das informações sensoriais gera parestesia ou anestesia do membro lesado, bem como descoordenação motora. Nesse contexto, novas terapias precisam ser desenvolvidas para o reparo das raízes dorsais. Uma substância capaz de conectar tecidos por adesão e que promova a hemostase e estabilidade do tecido, como o selante de fibrina (SF), pode ser uma alternativa a ser empregada no reparo de raízes lesadas. Além disso, o emprego conjunto do SF com células-tronco mononuclares de medula óssea (CTMMO) pode potencializar uma eventual regeneração tecidual. Assim, o presente estudo avaliou a resposta glial, a reorganização sináptica, a morfologia das fibras sensoriais e a coordenação motora após reparo com SF e terapia celular. Para isso, foram empregados ratos Lewis fêmeas (6-8 semanas), sendo divididos em três grupos: rizotomia (RZ, n=20), rizotomia reparada com SF (RZ+SF; n=22) e rizotomia reparada com SF e CTMMO (RZ+SF+CT; n=20). O tempo de sobrevida pós-cirúrgico foi de até 8 semanas. Para imunoistoquímica, foram utilizados anticorpos anti-VGLUT1 (terminais pré-sinápticos glutamatérgicos), GAD65 (terminais pré-sinápticos gabaérgicos), sinaptofisina (terminais sinápticos), GFAP (astrócitos), Iba1 (microglia) e BDNF (fator neurotrófico). Além disso, foram realizados citoquímica com Sudan black (coloração para lipídeos) e os testes comportamentais von-Frey eletrônico e walking track test (sistema CatWalk). Os resultados demonstraram regeneração das aferências nos grupos RZ+SF e RZ+SF+CT. Porém, apenas no segundo grupo, houve crescimento axonal até lâminas mais profundas da medula espinal, o que resultou em melhor desempenho nos testes comportamentais. Concluímos que o reimplante de raízes sensitivas com SF e CTMMO pode ser uma alternativa terapêutica para o reparo de lesões dorsais na interface do SNC e SNP / Abstract: Dorsal root lesions are common and often occur in automobile accidents. Due to the possibility of generating neuropathic pain, surgical procedures do not prioritize the repair of the afferent component, focusing on the motor output instead. Moreover, the loss of sensory inputs triggers paresthesis or anesthesia of the injured limb, and motor impairments. In this context, new therapies have to be developed for dorsal root repair. A substance that can promote tissue adhesion and stability and tissue haemostasis, such as fibrin sealant (FS), could be an alternative for the repair of damaged roots. Furthermore, the combined use of FS plus bone marrow mononuclear stem cells (BMSC) may enhance tissue regeneration. Thus, the present study evaluated the glial response, synaptic changes, the cytoarchitecture of the sensory fibers and motor coordination with FS with or without cell therapy for root replantation. Female Lewis rats (6-8 weeks old) were divided into three groups: rhizotomy (RZ, n = 20), rhizotomy repaired with FS (RZ+FS, n = 22) and rhizotomy repaired with SF and BMSC (RZ+FS+SC, n = 20). The survival time after surgery was up to 8 weeks. For immunohistochemistry VGLUT 1 (presynaptic glutamatergic terminals), GAD65 (GABAergic presynaptic terminals), synaptophysin (synaptic terminals), GFAP (astrocytes), Iba1 (microglia) and BDNF (neurotrophic factor) antibodies were used. Also, cytochemistry with Sudan black (lipid staining) and the behavioral tests electronic von-Frey and Walking track test (CatWalk system) were carried out. The results showed regeneration of afferent inputs in groups RZ+FS and RZ+FS+SC. However, only in the group with BMSC, the axonal growth was able to reach deeper laminae of the spinal cord, resulting in a better performance in behavioral tests. We conclude that the sensory root replantation with FS and BMSC may be an alternative therapy for the repair of dorsal root injuries in the CNS and PNS interface / Mestrado / Biologia Celular / Mestra em Biologia Celular e Estrutural

Chez les eucaryotes pluricellulaires, la différenciation des cellules repose en partie sur l’activation oula répression des gènes. Les profils d’expression génique mis en place vont perdurer d’une générationcellulaire à l’autre. Ce phénomène met en jeu des mécanismes épigénétiques qui remodèlentlocalement la structure de la chromatine. Chez Drosophila melanogaster, les protéines des groupesPolycomb (PcG) et Trithorax (TrxG) participent au maintien du profil d’expression des gènes au coursdu développement. Les protéines PcG maintiennent les gènes réprimés tandis que les protéines TrxGmaintiennent les gènes activés. Une troisième classe de protéines nommée Enhancers of Trithoraxand Polycomb (ETP) module l’activité des PcG et TrxG. Dorsal Switch Protein 1 (DSP1) est uneprotéine HMGB (High Mobility Group B) classée comme une ETP. Par tamisage moléculaire, nousavions montré que la protéine DSP1 était présente au sein de complexes de poids moléculaire de 100kDa à 1 MDa. Le travail de thèse présenté ici a pour but d’identifier les partenaires de la protéineDSP1 dans l’embryon et de mieux connaître les propriétés biochimiques de DSP1. Premièrement, j’aimis en place puis effectué l’immunopurification des complexes contenant DSP1 dans des extraitsprotéiques embryonnaires. Cette approche nous a permis d’identifier 23 partenaires putatifs de laprotéine DSP1. Parmi ces protéines, nous avons identifié la protéine Rm62 qui est une ARN hélicaseà boîte DEAD. Les relations biologiques entre DSP1 et Rm62 ont été précisées. Deuxièmement, j’aidéterminé, par une approche biochimique, de nouvelles caractéristiques physico-chimiques de laprotéine DSP1. / In multicellular organism, the identity of cell is determined by several factors playing on genesexpression. Once established, the gene expression pattern is transmitted to daughter cells through aprocess involving epigenetic mechanisms that locally reshape the structure of chromatin. In Drosophilamelanogaster, the Polycomb (PcG) and trithorax (trxG) group genes are involved in the maintenanceof gene expression profile during development. Inside multimeric complexes, PcG proteins maintaingenes in repressed state whereas TrxG maintain genes active. A third class of proteins, calledEnhancers of Trithorax and Polycomb, regulate PcG and TrxG activities. Dorsal Switch Protein 1(DSP1) is a High Mobility Group B protein acting as an ETP. But DSP1 has not yet been identified inPcG or TrxG complexes. On the basis of gel filtration analysis of protein complexes in embryo nuclearextracts, it appears that the majority of DSP1 is present in complex(es) from 100 kDa to 1MDa. Aimsof present work are the identification of DSP1 protein partners in drosophila embryo and thecharacterization of biochemical properties of DSP1. Firstly, I used immunopurification from drosophilaembryonic nuclear extracts. The proteins purified with DSP1 were characterized through sequencingof peptides from individual protein bands by mass spectrometry. Among identified proteins, wefocused on the DEAD Box RNA helicase, Rm62. The role of interaction between DSP1 and Rm62 hasbeen characterized. Secondly, I have identified a new physicochemical aspect of DSP1 protein.

Résumé : les benzodiazépines (BZD) et les antidépresseurs sont efficaces pour réduire l’anxiété, mais devant les effets indésirables que les BZD induisent et face au délai d’action des antidépresseurs, le développement de nouvelles stratégies constitue un besoin primordial. Récemment, il a été montré que l’activation du récepteur 5-HT4 (5-HT4R) pouvait représenter une cible d’action prometteuse. Bien qu’un certain nombre d’études ait évalué l’activité anxiolytique des agonistes du 5-HT4R, après une administration chronique, aucune étude n’a examiné leurs effets après une administration unique, ni même le circuit cérébral à la base de cette réponse comportementale. Nous avons donc cherché à savoir si l’activation aiguë du 5-HT4R via le recrutement des terminaisons neuronales glutamatergiques du cortex préfrontal médian (CPFm) se projetant sur le noyau du raphé dorsal (NRD), un circuit impliqué dans les processus émotionnels, induisait des effets anxiolytiques rapides, grâce à des outils pharmacologiques, électrophysiologiques et optogénétiques. L’administration unique par voie systémique ou intra-CPFm d’un agoniste du 5-HT4R a produit des effets anxiolytiques rapides chez la souris, associés à une augmentation de l’activité des neurones sérotoninergiques du NRD. L’activation des projections glutamatergiques du CPFm vers le NRD a permis de réduire l’anxiété des souris, alors que l’inhibition de ces projections a bloqué les effets anxiolytiques induits par l’injection unique intra-CPFm d’une BZD (diazépam) ou d’un agoniste du 5-HT4R (RS 67333). Toutefois, ces effets ne sont que partiellement bloqués après l’administration par voie systémique de ces composés, suggérant que le circuit cortex-raphé est un carrefour nécessaire mais non suffisant à l’activité anxiolytique du diazépam et du RS 67333. Enfin, l’administration prophylactique d’un agoniste du 5-HT4R avant l’induction d’un stress a prévenu le développement d’un phénotype anxio-dépressif chez la souris, laissant penser que cette molécule pourrait renforcer la résilience au stress des populations à risque. / Benzodiazepines (BZD) and antidepressants are effective in reducing anxiety, but adverse effects of BZD and the delayed onset of action of antidepressants emphasize the need to develop fast-acting new drugs. Recent studies indicated that activation of 5-HT4 receptor (5-HT4R) could be a promising target. Although a number of studies have assessed 5-HT4R agonist anxiolytic activity after chronic treatment, few of them have neither evaluated their anxiolytic profile acutely, neither the brain circuits involved in this behavioral activity. Here, we evaluated whether acute 5-HT4R activation in glutamatergic axon terminals arising from the medial prefrontal cortex (mPFC) to the dorsal raphe nucleus (DRN), a circuit involved in emotional processes, induced fast anxiolytic effects, using pharmacologic, electrophysiologic and optogenetic tools. Acute systemic administration and intra-mPFC infusion of 5-HT4R produced fast anxiolytic effects in mice and increased DRN serotonin cell firing. Optogenetically activating mPFC terminals targeting the DRN reduced anxiety in mice whereas silencing this circuit blocked BZD (diazepam) and 5-HT4R agonist (RS 67333) mPFC infusion -induced anxiolytic effects. However, anxiolytic effects induced by an acute systemic administration of both molecules were partially blocked after optogenetically inhibiting cortical glutamatergic terminals in the DRN, suggesting that cortex-brainstem neural circuit is necessary but not sufficient for a rapid activity of diazepam and RS 67333. Finally, the prophylactic administration of a 5-HT4R agonist before stress prevented the development of an anxio-depressive phenotype in mice, suggesting that this molecule could reinforce the resilience of population at risk.

The dorsal dentate gyrus (DDG) of the hippocampus plays a role in the expression of different forms of flexible behaviour mainly due to its ability to sustain neurogenesis throughout life. In the present thesis, we examined the role that the DDG and its adjacent areas, both collectively referred to as dorsal medial hippocampus (DMH), play in flexible, adaptive behaviour and cognitive processing. We used the neurotoxin, colchicine, to induce lesions of the DDG, which were found to affect neighbouring areas. Thus these lesions will be referred to as lesions of the DMH. In the first experiment, rats were tested for (1) perseverative behaviour before and after receiving chronic methamphetamine (METH) treatment, (2) METH-induced locomotor activity and stereotypy in an open field, and (3) working memory in a T-maze. The results showed that rats with lesions of the DMH exhibited perseveration and supersensitivity to the locomotor- and stereotypy-inducing effects of METH (0, 0.1, 0.3, 1 mg/kg i.p.) as well as increased long-term METH sensitization. Rats with DMH lesions also showed significant working memory deficits. Taken together, these results reveal specific forms of behavioural inflexibility in rats with lesions of the DMH that are mainly associated with perseveration, drug-related behaviours, including stimulant motor supersensitivity and drug sensitization, and impaired working memory functions.

The mammalian central nervous system maintains a delicate balance between neuronal excitation and inhibition. Conventional synaptic inhibition is mediated through the transient activity of postsynaptic γ-aminobutyric acid (GABA) at type A GABA (GABAA) receptors. A subset of GABAA receptors is also located outside of inhibitory synapses. These extrasynaptic receptors generate a tonic inhibitory conductance in response to low concentrations of extracellular GABA. Tonic inhibition broadly suppresses neuronal activity and regulates many vital processes such as sleep, consciousness and memory formation. This thesis examines the physiological effects of tonic inhibition at the cellular level and in the behaving animal. This thesis also explores whether gabapentin, a commonly used sedative, anxiolytic, and analgesic drug, enhances tonic GABAergic inhibition. I hypothesize that: (1) tonic GABAA receptor activity reduces the intrinsic excitability of neurons; (2) the activity of tonically active GABAA receptors in spinal pain pathways attenuates nociception; and (3) tonic inhibition can be upregulated by gabapentin. The results show that a tonic inhibitory current generated by α5 subunit-containing GABAA (α5GABAA) receptors reduces the excitability of hippocampal pyramidal neurons excitability by increasing the rheobase, but does not change the gain of action potential firing. A similar shunting inhibition is present in spinal cord lamina II neurons that is generated by δ subunit-containing GABAA receptors. The activity of these receptors in spinal nociceptive pathways reduces acute thermal nociception and may constrain central sensitization in a behavioural model of persistent pain. Finally, gabapentin increases a tonic inhibitory current in cultured hippocampal neurons independent from changes in the expression of α5GABAA receptors or in the concentration of GABAA receptor ligands. The results of this thesis demonstrate that tonically active GABAA receptors play an important role in the regulation of neuronal activity and nociception, and that tonic inhibition represents a novel target of therapeutic drugs.

The aims of the thesis were to examine i) whether the endogenous opioid system (EOS) is lateralized in human brain areas involved in processing of emotions and pain; ii) whether EOS responses to unilateral brain injury depend on side of lesion, and iii) whether in human alcoholics, this system is involved in molecular adaptations in brain areas relevant for cognitive control of addictive behavior and habit formation. The main findings were that (1) opioid peptides but not opioid receptors and classic neurotransmitters are markedly lateralized in the anterior cingulate cortex involved in processing of  positive and negative emotions and affective component of pain. The region-specific lateralization of neuronal networks expressing opioid peptides may underlie in part lateralization of higher functions in the human brain including emotions and pain. (2) Analysis of the effects of traumatic brain injury (TBI) demonstrated predominant alteration of dynorphin levels in the hippocampus ipsilateral to the injury, while injury to the right hemisphere affected dynorphin levels in the striatum and frontal cortex to a greater extent than that to the left hemisphere. Thus, trauma reveals a lateralization in the mechanisms mediating the response of dynorphin expressing neuronal networks in the brain. These networks may differentially mediate effects of left or right brain injury on lateralized brain functions. (3) In human alcoholics, the enkephalin and dynorphin systems were found to be downregulated in the caudate nucleus and / or putamen that may underlie in part changes in goal directed behavior and formation of a compulsive habit in alcoholics. In contrast to downregulation in these areas, PDYN mRNA and dynorphins in dorsolateral prefrontal cortex, k-opioid receptor mRNA in orbitofrontal cortex, and dynorphins in hippocampus were upregulated in alcoholics. Activation of the k-opioid receptor by upregulated dynorphins may underlie in part neurocognitive dysfunctions relevant for addiction and disrupted inhibitory control. We conclude that the EOS exhibits region-specific lateralization in human brain and brain-area specific lateralized response after unilateral TBI in mice; and that the EOS is involved in adaptive processes associated with specific aspects of alcohol dependence.

The Dorsal Raphe Nucleus (DRN) is a complex brain region that has been implicated in disorders such as anxiety and depression. The DRN is divided into subregions through its rostrocaudal and mediolateral axis. It has been reported that after a single restraint session there is differential spatial activation of nitric oxide synthase (NOS) across the DRN. The temporal profile of NOS activity during acute stress is not known but it is important because duration of acute stress is associated with different general responses. In this report rats were restrained for 1, 3, or 6 hours and nicotinamide adenine phosphate diaphorase (NADPH-d) was stained as an index to NOS activity to determine the spatio-temporal profile of NOS throughout a 6 hour restraint. Astrocyte reactivity was also measured to determine whether NOS activation correlated with GFAP expression since astrocytes react to neural activity and store and release l-arginine, the precursor for nitric oxide production. The results showed that the DRN had a dynamic response to acute restraint stress, most notably in the caudal lateral wings where activation increased after 3 hours of restraint (p = > 0.001) but neuron count decreased after 6 hours (p = 0.040). Astrocytes did not correlate with NOS activation but they showed spatio-temporal differences as well whereas they were more active in the rostral half of the DRN. In conclusion, the present study suggests that NOS produced in the DRN may have a role in prolonged exposure to acute stress and that subregions show differential NOS activation.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

<p>Mainstream electrical stimulation therapies, e.g., spinal cord stimulation (SCS) and deep brain stimulation, use pulse trains that are delivered at rates no higher than 200 Hz. In recent years, stimulation of nerve fibers using kilohertz-frequency (KHF) signals has received increased attention due to the potential to penetrate deeper in the tissue and to the ability to block conduction of action potentials. As well, there are a growing number of clinical applications that use KHF waveforms, including transcutaneous electrical stimulation (TES) for overactive bladder and SCS for chronic pain. However, there is a lack of fundamental understanding of the mechanisms of action of KHF stimulation. The goal of this research was to analyze quantitatively KHF neurostimulation. </p><p>We implemented a multilayer volume conductor model of TES including dispersion and capacitive effects, and we validated the model with in vitro measurements in a phantom constructed from dispersive materials. We quantified the effects of frequency on the distribution of potentials and fiber excitation. We also quantified the effects of a novel transdermal amplitude modulated signal (TAMS) consisting of a non-zero offset sinusoidal carrier modulated by a square-pulse train. The model revealed that high-frequency signals generated larger potentials at depth than did low frequencies, but this did not translate into lower stimulation thresholds. Both TAMS and conventional rectangular pulses activated more superficial fibers in addition to the deeper, target fibers, and at no frequency did we observe an inversion of the strength-distance relationship. In addition, we performed in vivo experiments and applied direct stimulation to the sciatic nerve of cats and rats. We measured electromyogram and compound action potential activity evoked by pulses, TAMS and modified versions of TAMS in which we varied the amplitude of the carrier. Nerve fiber activation using TAMS showed no difference with respect to activation with conventional pulse for carrier frequencies of 20 kHz and higher, regardless the size of the carrier. Therefore, TAMS with carrier frequencies >20 kHz does not offer any advantage over conventional pulses, even with larger amplitudes of the carrier, and this has implications for design of waveforms for efficient and effective TES. </p><p>We developed a double cable model of a dorsal column (DC) fiber to quantify the responses of DC fibers to a novel KHF-SCS signal. We validated the model using in vivo recordings of the strength-duration relationship and the recovery cycle of single DC fibers. We coupled the fiber model to a model of SCS in human and applied the KHF-SCS signal to quantify thresholds for activation and conduction block for different fiber diameters at different locations in the DCs. Activation and block thresholds increased sharply as the fibers were placed deeper in the DCs, and decreased for larger diameter fibers. Activation thresholds were > 5 mA in all cases and up to five times higher than for conventional (~ 50 Hz) SCS. For fibers exhibiting persistent activation, the degree of synchronization of the firing activity to the KHF-SCS signal, as quantified using the vector strength, was low for a broad amplitude range, and the dissimilarity between the activities in pairs of fibers, as quantified using the spike time distance, was high and decreased for more closely positioned fibers. Conduction block thresholds were higher than 30 mA for all fiber diameters at any depth and well above the amplitudes used clinically (0.5 – 5 mA). KHF-SCS appears to activate few, large, superficial fibers, and the activated fibers fire asynchronously to the stimulation signal and to other activated fibers. </p><p>The outcomes of this work contribute to the understanding of KHF neurostimulation by establishing the importance of the tissue filtering properties on the distribution of potentials, assessing quantitatively the impact of KHF stimulation on nerve fiber excitation, and developing and validating a detailed model of a DC fiber to characterize the effects of KHF stimulation on DC axons. The results have implications for design of waveforms for efficient and effective nerve fiber stimulation in the peripheral and central nervous system.</p> / Dissertation