Introdução. O transplante de células é possibilidade terapêutica promissora para muitas doenças neurológicas. Nos últimos anos, a possibilidade do isolamento de células-tronco dos tecidos adultos, por exemplo da medula-óssea, atrai a atenção da comunidade científica, estratégia que minimiza os problemas éticos relativos ao uso de tecido fetal para implantes visando ao tratamento de doenças neurológicas. Entretanto, a eficiência da transdiferenciação de células-tronco mesenquimais em neurônios, bem como os mecanismos envolvidos nesse processo, permanecem desconhecidos. A obtenção de neurônios maduros ocorreu somente em sistemas de co-cultura, o que induz a questão se a diferenciação representa um potencial das células per si, ou se é possível somente devido à fusão com neurônios maduros. Objetivos. No presente trabalho, pretendeu-se verificar o potencial de as células-tronco mesenquimais tornarem-se neurônios e esclarecer os possíveis mecanismos envolvidos nesse processo. Material e métodos. Células-tronco mesenquimais foram isoladas de 20 doadores voluntários normais e caracterizadas por análise de separação celular ativada por fluorescência. A multipotencialidade foi investigada ao se diferenciar as células em condrócitos e osteócitos. A capacidade de auto-renovação foi confirmada pelo ensaio de incorporação de BrdU. Ulteriormente, as células foram diferenciadas por uma semana em meio contendo AMPc, IBMX, ou combinação de ambos, e os resultados foram comparados com o cultivo em meio básico. Diferentes bloqueadores de Ca2+ ou inibidores de PKA foram usados como tentativa de se impedir a diferenciação, ocorrência que foi mensurada com imunocitoquímica para NF-200 (marcador de neurônios maduros). O registro eletrofisiológico por meio de patch clamp foi usado para se confirmar o fenótipo neuronal. As figuras foram configuradas em microscopia confocal. Para análise estatística foi utilizada ANOVA com teste post-hoc. Resultados. As células isoladas expressaram CD90, 105, 44 e 13 mas foram negativas para CD34 e 45. Isto significa que não são de origem hematopoiética; 98,74 ± 0,43% das células incorporaram BrdU em 24 horas. Após o isolamento, foi possível diferenciá-las em condrócitos ou osteócitos. Em situação controle, não foram evidenciadas células positivas para NF200. Por outro lado, ocorreu positividade em 10,75% ± 1,35 (p<0,0001) das células sob IBMX e, em 15,18% ± 1,12, sob a combinação cAMP e IBMX (p<0,0001). Foram registradas correntes de Na+ e K+ dependentes de voltagem, mas não potenciais de ação. A diferenciação foi inibida com PKAi (5,73% ± 0,42, p<0,0001), nifedipina (5,79% ± 0,98, p<0,0001), Ni2+ (7,06% ± 1,68, p<0,0001) e Cd2+ (0 ± 0, p<0,0001). Discussão. Isolou-se uma população de células-tronco estromais da medula-óssea de seres humanos que se mostrou multipotencial e auto-renovável. O aumento da concentração de AMPc no meio elevou a concentração de neurônios para 15%. A diferenciação parece depender da via PKA mas também envolve a concentração intracelular de Ca2+. Conclusão. O correto entendimento de como as células-tronco mesenquimais diferenciam-se pode contribuir para aumentar a eficácia do método e, talvez um dia, tornar possível o uso dessa ferramenta no campo clínico. / Introduction. Cell transplantation has been considered a promising therapeutic approach for many neurological diseases. The possibility of isolation of stem cells from adult tissues, i.e. bone marrow, has attracted the attention of the scientific community in the recent years. This strategy is interesting on avoiding the ethical issues regarding the use of fetal tissue for neural implants. Moreover, the efficiency of the transdifferentiation of mesenchymal stem cells (MSCs) into neurons, and the mechanisms involved in this process remain largely unknown. The obtention of mature neurons was described only in coculture systems, what raised the question if the differentiation is a potential of the cells itself, or if it is possible only due to fusion with mature neurons. Objectives. In the present investigation, we aimed to verify the potential of MSCs to differentiate into neurons, and also to clarify the possible mechanisms involved on it. Material and methods. MSCs were isolated from 20 healthy human subjects and characterized by FACS-analysis. Multipotentiality was addressed by differentiating them into chondrocytes and osteocytes. The self-renewal capacity was confirmed with BrdU-incorporation assay. Afterwards, cells were differentiated for 1 week in a medium containing cAMP, IBMX, or a combination of both, and the results were compared with cells treated in basal-medium condition. Different Ca2+-blockers and PKA-inhibitor peptide were used on an attempt to impair differentiation, which was quantified with NF-200 immunostaining (a marker of mature neurons). Patch-clamp recording was used to confirm neuronal phenotype. Pictures were taken in confocal microscope. For statistical analysis ANOVA with a post-hoc test was used. Results. The isolated cells expressed CD90, 105, 44, and 13, but were negative for CD34 and 45, meaning that they were non-hematopoiethic; 98.74 ± 0.43 % of them incorporated BrdU in 6hs. After isolation, they differentiated into chondrocytes and osteocytes. In a control situation, no NF200 positive cell was seen. On the other hand, 10.75% ± 1.35 (p<.0001) of positivity was seen under IBMX and 15.18% ± 1.12 in the combination of cAMP with IBMX (p<.0001). Na+ and K+-voltage gated currents were recorded. Differentiation was impaired with PKAi (5.73% ± 0.42, p<.0001), nifedipin (5.79% ± 0.98, p<.0001), Ni2+ (7.06% ± 1.68, p<.0001), and Cd2+ (0 ± 0, p<.0001). Discussion. We were able to isolate a population of stromal stem cells from the bone marrow of human subjects, since they were multipotential and self-renewable. Increasing the concentration of cAMP raised the percentage of neurons up to 15%. The differentiation seems to be dependent on the PKA pathway, but also involved the intracellular concentration of Ca2+. Conclusions. The complete understanding of how MSC differentiate can contribute to increase the efficiency of the method and thus make possible to use this powerful tool in the clinical practice.

O estudo dos efeitos de neurotransmissores e neuromoduladores em circuitos nervosos tradicionalmente consiste em observar o comportamento de um sistema, ou parte dele, quando exposto a concentrações constantes da droga, ignorando flutuações ou padrões temporais de liberação. Neste trabalho implementamos um protocolo dinâmico e dependente da atividade do próprio sistema nervoso, que permitiu estudar experimentalmente a influência da dinâmica de liberação nos efeitos que glutamato (Glu, 1 mM) e serotonina (5-HT, 10 mM) produzem no padrão emergente de um centro gerador de padrões (CPG). Os experimentos foram realizados no sistema nervoso estomatogástrico de Callinectes sapidus e Callinectes danae (CPG pilórico) durante sua operação normal in vitro, que pode ser caracterizada por sua frequência de burst, número de spikes/burst dos neurônios, duty cycle dos neurônios, etc. A partir da atividade de um dos neurônios do CPG, medida extracelularmente, um algoritmo baseado no protocolo dynamic clamp (interação em tempo real entre tecido nervoso vivo e simulação computacional) identifica em tempo real um padrão de atividade pré-escolhido e dispara um injetor de picolitros semelhante a um picospritzer&reg;, aspergindo uma pequena quantidade de solução com a droga nas vizinhanças dos neurônios e sinapses que compõe o CPG, de onde é retirada por perfusão contínua de solução fisiológica. Assim, podemos comparar o comportamento emergente do CPG quando este é estimulado de maneira síncrona com sua atividade ou de maneira assíncrona, mas de forma que a mesma quantidade média de droga seja aspergida nos dois casos. Nossos resultados demonstram que um mesmo estímulo apresentado de duas maneiras diferentes, para um mesmo circuito nervoso, pode produzir respostas diferentes na mesma célula. O protocolo se mostrou mais eficiente em mostrar diferenças quando a atividade da droga ocorre em uma escala de tempo menor que aquela do comportamento do CPG. Novos estudos devem ser realizados com outros neutrotransmissores e moduladores tanto de ação ionotrópica quanto metabotrópica, para se adequar o protocolo aos diferentes tipos de substâncias. / Studying the effect of neurotransmitters and neuromodulators usually consists in observing the behavior of a system, or part of it, when exposed to stationary concentrations of the drugs that completely lack any time dependency. Here we describe a protocol that we developed to study the changes of the emerging rhythmic pattern of a Central Pattern Generator (CPG) due to the effects of the aplications dynamics of tiny puffs of glutamate (Glu, 1mM) or serotonin (5-HT, 10 mM) to the neurons and their neuropil. We experimented on the stomatogastric nervous system of Callinectes sapidus and Callinectes danae (pyloric CPG) during normal operation in vitro, that was characterized using bursting frequency, neurons number of spikes/burst, their duty cycle, etc. From the extracelular activity of one of the CPG neurons, a dynamic clamp based protocol (real time interaction between living nervous tissue and computer simulation) detected in real time a given pattern of activity present in a burst and triggered a picospritzer to puff a small amount of solution with the drug in the neighborhood of the neurons and synapses of the CPG, from where it was washed through constant perfusion of normal saline. Such arrangement allowed us to address what are the differences on the emerging CPG behavior when the stimuli was activity synchronous or asynchronous, but with the same mean amount of drug delivered in both cases. In general, our results showed that the same stimulus presented in different ways to the same nervous circuit may generate different responses even in a cell level. Finally, the protocol proved to be more efficient when the drug activity time scale is smaller than the time scale of the CPG bursting behavior. In the future we should work with other neuromodulators and neurotransmiters to improve the protocol.

Investigamos o papel de condutâncias iônicas lentas na transmissão/codificação de informação entre neurônios que disparam em rajadas ou bursts. Para isso, desenvolvemos um protocolo experimental no qual a interação em tempo real entre computador e neurônio biológico permite isolar o efeito da dinâmica de um determinado tipo de canal iônico e estudar sua inuência nos mecanismos de codificação de informação. Os experimentos foram realizados com neurônios do gânglio estomatogástrico do siri azul, Callinectes sapidus, que não possuem condutâncias lentas capazes de fazê-los apresentar rajadas de disparos quando in vitro, condição na qual apresentam comportamento quiescente ou disparam tonicamente. Durante os experimentos, alteramos artificialmente o comportamento de um destes neurônios, conectando-o a um computador que introduz uma corrente capaz de fazê-lo apresentar rajadas. Essa corrente possui uma componente senoidal (vinda de um gerador de funções) e uma componente devido a uma condutância iônica lenta modelada matematicamente. A condutância lenta pode ser escolhida entre duas versões: uma em que a condutância é calculada em tempo real, a partir do valor instantâneo do potencial de membrana do neurônio biológico, e outra em que o valor da condutância é oriundo de uma série temporal previamente gravada. A fonte de informação utilizada nos experimentos é um neurônio artificial pré-sináptico, que possui uma distribuição de potenciais de ação (spikes) escolhida pelo experimentador, e é conectado ao neurônio biológico modificado através de um modelo de sinapse química inibidora. A quantidade de informação do neurônio artificial (variabilidade dos padrões de disparo) codificada pelo neurônio biológico é inferida calculando-se a informação mútua média entre eles para as duas versões da condutância lenta (dinâmica ou previamente gravada). Nossos experimentos reproduziram qualitativamente as observações feitas por nosso grupo no circuito pilórico intacto do siri, que possui neurônios conectados por mutua inibição que naturalmente apresentam bursts. Além disso, observamos que vários picos de informação mútua média, presentes quando a condutância é dinâmica, desaparecem quando esta é substituída pela série temporal previamente gravada da condutância. Assim, pudemos confirmar os resultados previamente obtidos com simulações computacionais em que foram utilizados apenas modelos matemáticos e na ausência de ruído e demonstramos que as condutâncias iônicas lentas constituem um mecanismo biofísico que permite a codificação de estímulos sinápticos em neurônios que apresentam rajadas. / We investigated the role of slow ionic conductances on information processing by bursting neurons. A real time experimental protocol was developed to allow interacting computer models and biological neurons to address the effect of dynamical details of a single type of ion channel in information coding mechanisms. We experimented on Callinectes sapidus (blue crab) stomatogastric ganglion neurons. Such neurons were chosen because they do not present the slow conductances that can led to bursting activity in vitro (in such conditions they can be found either in a quiescent or in a tonic firing state). The experiments consisted in artificially changing the behavior of one of these neurons by injecting a computer generated current to achieve bursting. Such current has a sinusoidal component (from a function generator) and a component due to mathematical model of a slow ionic conductance. The slow conductance was implemented in two versions: in one of them the instantaneous value of the conductance is computed in real time and according to the membrane potential of the biological neuron, in another version the value of the conductance simply comes from a time series previously stored in the computer. A pre-synaptic artificial neuron, with a spike distribution chosen by the experimenter, provided input for the biological neuron through an artificial chemical inhibitory synapse. The amount of information (variability of spike patterns from the artificial neuron) coded by the biological neuron was inferred by calculating the average mutual information along stimulus and response bursts for the two conditions of the slow conductance (dynamically calculated or from file previously stored). Our experiments reproduced the results found in intact pyloric central pattern generator bursting neurons connected by mutual inhibition. Moreover, we show that the average mutual information peaks, found when the conductance is dynamically calculated, disappear when we use the previously recorded time series of the conductance. Such results validate those only found previously in numerical simulations in the absence of noise and point the role of the slow ionic conductances in a biophysical mechanism that allow bursting motor neurons to encode in a nontrivial fashion the information they receive through a single synapse.

Le récepteur à la glycine est un récepteur neuronal qui appartient à la famille des canaux ligand-dépendants «cys-loop». Avec le récepteur ionotrope GABA ils fournissent la neurotransmission inhibitrice rapide dans le SNC des vertébrés grâce à leur perméabilité sélective au Cl-. Les récepteurs à la glycine participent à différents processus physiologiques comprenant le contrôle de l'activité motrice, la respiration, la sensation de douleur inflammatoire, la perception des stimuli visuels et auditifs. Le développement de modulateurs efficaces des récepteurs à la glycine permettra un contrôle précis de leur activité, ce qui est particulièrement important dans le cas des pathologies des récepteurs à la glycine, comme l'hyperekplexie. En utilisant l'analyse électrophysiologique, la mutagenèse dirigée et l'expression de protéines spécifiques dans un système hétérologue, nous avons identifié les acides ginkgoliques et niflumiques comme nouveaux modulateurs de récepteurs de la glycine, caractérisé leur action sur différentes sous-unités du récepteur et déterminé les sites importants pour la potentialisation ou l'inhibition des récepteurs à la glycine par ces composés. Cette approche est très prometteuse et ouvre de nouvelles voies vers des futures actions thérapeutiques. / Glycine receptor is a ligand-gated neuronal receptor that possesses an ion pore permeable for Cl- and represents an important component of inhibitory neurotransmission in CNS of vertebrates. Glycine receptors participate in the control of motor activity, respiration, inflammatory pain sensation, perception of visual and auditory stimuli. Development of efficient modulators of glycine receptors will allow a precise control of their activity, which is especially important in the case of glycine receptor pathologies, such as hyperekplexia. In the present work we have identified ginkgolic and niflumic acids as novel modulators of glycine receptors, characterized their action on different subunits of the receptor and determined the most probable sites of interaction of the compounds with glycine receptors.

Volumeninduzierte Chloridströme wurden bereits in einer Reihe von Zelltypen charakterisiert. In dieser Arbeit wurde die Modulation volumenaktivierte Chloridströme ICl,Vol und der Signalweg zu ihrer Aktivierung untersucht. In Endothelzellen aus der Pulmonalarterie des Rindes (CPAE) wurden der Membranstrom unter Anwendung der 'patch clamp' - Technik und simultan dazu die Konzentration des freien intrazellulären Kalziums [Ca2+]i gemessen. Bisher wurde davon ausgegangen, daß die Aktivierung von ICl,Vol kalziumunabhängig erfolgt. In dieser Arbeit wurde ICl,Vol unter Pufferung des [Ca2+]i mit BAPTA und EGTA gemessen. Es konnte gezeigt werden, daß freies intrazelluläres Kalzium in Konzentrationen niedriger als 50 nM zur Stromaktivierung notwendig ist. Bei einer höheren Konzentration verliert der Strom jedoch seine Kalziumabhängigkeit. Chromoglycinsäure (CL) blockiert ICl,Vol in Endothelzellen. Dieser Effekt ist jedoch im Vergleich zu dem klassischen Chloridkanalblocker NPPB geringer (Ki=15mM) und sehr langsam (im Mittel 100 s). Damit sind die blockierenden Eigenschaften auch geringer ausgeprägt als z.B. auf ICl,Vol und Hemmung der Serotoninsekretion in Mastzellen. Weiterhin war die Rolle von durch Proteinphosphorylierung modulierten intrazellulären Signalwegen bei der Aktivierung von ICl,Vol Gegenstand von Experimenten. Weder die Aktivierung der Proteinkinase C (PKC) durch direkte Applikation des Phorbolesters PMA noch deren Ab - Regulation durch 24-stündige Inkubation mit PMA hatten Einfluß auf die Aktivierung von ICl,Vol. Auch Wortmannin, ein Inhibitor sowohl der MAP - Kinasen (und damit des Tyrosinkinase - assoziierten Rezeptor - Signalweges) als auch der PI3 - Kinase, zeigte keinen Effekt auf ICl,Vol. Der Aktivator der MAP - Kinasen und der fokalen Adhaesionskinase p125FAK, die Substanz Lysophosphatsäure (LPA), zeigte weder einen Effekt auf ICl,Vol, noch konnte damit ein Chloridstrom induziert werden. Die Induktion von HSP durch Applizierung von thermischem und chemischem Streß unter Inkubation bei bis zu 45 °C über 60 min und Einwirkung von 0,3 mM Natriumarsenit zeigte keinerlei Einfluß auf ICl,Vol. Es konnte auch keine Aktivierung eines Chloridstromes beobachtet werden. Zur Untersuchung des Aktivierungsmechanismus von ICl,Vol dienten Experimente unter gleichzeitiger Messung von ICl,Vol, Membrankapazität (CM) und (jedoch zeitlich unabhängig voneinander) der Zellhöhe. Die Ergebnisse wurden an Hand dreier Modellvorstellungen zur Aktivierung von ICl,Vol diskutiert. Es bestand eine enge Korrelation zwischen Veränderungen der Zellhöhe und des aktivierten Stromes. Beide korrelieren jedoch nicht mit den Änderungen von CM. Dabei änderte sich CM bei einem Teil der Zellen praktisch nicht, d.h. geringer als 2% des Ausgangswertes vor Applikation hypotoner Lösung (n=17), bei den verbliebenen Teil zeigten sich deutliche Veränderungen von durchschnittlich 10,6 ± 0,9 % ( ± SEM, n=5). In letzterem Fall änderte sich jedoch immer zuerst ICl,Vol und dann CM. Nach Inhibierung des intrazellulären Vesikeltransports durch Brefeldin A war ICl,Vol ohne signifikante Änderungen auslösbar. Unter Cycloheximid, einem Blocker der Proteinsynthese auf Transkribtionssebene, wurden keine spezifischen Veränderungen von ICl,Vol beobachtet. Auf jeden Fall ist CM nicht der primäre Trigger für ICl,Vol. Die Aktivierung von volumeninduzierten Chloridströmen erfolgt nicht maßgeblich durch die Inkorporierung von Ionenkanal - Proteinen enthaltenden Membranvesikeln. / Volume-induced chlorid currents are characterised for many types of cells. I have investigated the modulation and activation of these currents. In cultered pulmonary artery endothelial cells (CPAE) patch clamp maesurements of membrane currents and simultaneous maesurements of free intracellular calcium ([Ca2+]i) were performed. Until now, activation of ICl,Vol was characterised as Calcium-independently. I maesured the current buffering [Ca2+]i with the Calcium chelators BAPTA and EGTA. It could be observed, that [Ca2+]i at concentrations less than 50 nM is required to activate ICl,Vol. At higher concentrations, there is no modulation by [Ca2+]i. Sodiumchromoglycate (CL) blocks ICl,Vol in endothelium cells. This effect is small (Ki=15mM) and slow (100 s) as compared of those of the classical chlorid channel blocker NPPB. Inhibitory effects of CL to ICl,Vol in endothelial cells are weaker than to ICl,Vol and to secretion of serotonin in mast cells. The role of intracellular phosphorylation signal pathways on activation of ICl,Vol was investigated. Neither activation of protein kinase C (PKC) throught direct application of the phorbol ester PMA nor down-regulation through preincubation with PMA had any effect on activation of ICl,Vol. Also Wortmannin, an inhibitor of MAP-kinases, failed to have significant effects on I

Durch Ausübung physiologischer Grundfunktionen spielen Ionenkanäle eine entscheidende Rolle für die reguläre Funktion von Zellen. Zum besseren Verständnis ihrer Struktur und Funktion sind Untersuchungen natürlicher und künstlicher Ionenkanäle wichtige Werkzeuge. Großes analytisches und therapeutisches Potential ist in der Untersuchung künstlicher Kanäle in lebenden Zellen vorhanden, was bisher wenig Beachtung fand. In der vorliegenden Arbeit wurde die Wirkung der künstlichen Ionenkanäle THF-gram, THF-gram-TBDPS sowie linked-gram-TBDPS auf elektrophysiologische Eigenschaften boviner Trabekelwerkszellen des Auges anhand von Patch-Clamp-Untersuchungen im Whole-Cell-Modus analysiert. Die Untersuchung brachte folgende Erkenntnisse: 1. Die Inkorporation aller drei Verbindungen war erfolgreich, was sich durch Anstieg der Stromdichte und Verschiebung des Umkehrpotentials zeigte. 2. Einbau von THF-gram und THF-gram-TBDPS war mit dem Überleben der Zellen vereinbar, während linked-gram-TBDPS aufgrund einer sehr potenten Antwort bereits bei sehr geringen Konzentrationen zum raschen Zelltod führte. 3. Eine Asymmetrie der Stromantwort zugunsten stärkerer Auswärtsströme wurde bei THF-gram und in schwächerer Ausprägung bei THF-gram-TBDPS festgestellt. Linked-gram-TBDPS zeigte keine derartige Asymmetrie. 4. Unter Verwendung von Cs+ als Ladungsträger war der beobachtete Anstieg der Stromdichte bei allen drei Verbindungen eindeutig stärker als unter physiologischen Bedingungen (Na+/K+). 5. Die dargestellten Erkenntnisse sind ein erster Schritt zur therapeutischen Anwendung von künstlichen Ionenkanälen. Eine Weiterentwicklung in Richtung höherer Selektivität und besserer Kontrolle ist jedoch genauso erforderlich wie die Klärung der klinischen Umsetzbarkeit. / Ion channels play a pivotal role for regular cell function. To better understand their structure and function, investigation of both natural and artificial ion channels is being performed to date. Investigation of artificial channels in living cells hides a big potential, however, little attention has been paid to this field so far. In this work, the effect of the artificial ion channels THF-gram, THF-gram-TBDPS and linked-gram-TBDPS on electrophysiological properties of bovine trabecular meshwork cells was investigated with the patch-clamp-technique. Following results were obtained: 1. Incorporation of all three compounds was successful, which was proven by increase of current density and cell depolarisation. 2. The cells survived after incorporation of THF-gram and THF-gram-TBDPS but not after linked-gram-TBDPS, which resulted in cell death at very low concentrations. 3. Larger outward currents were observed with THF-gram and, at a lower extent, with THF-gram-TBDPS. Linked-gram-TBDPS did not show such an asymmetry. 4. With Cs+ as charge carrier all three compunds showed a stronger increase of current density than under physiological conditions (Na+/K+). 5. The decribed results are a first step towards therapeutic application of artificial ion channels, however, further development towards higher selectivity and better control is as necessary as clarification of clinical feasibility.

La Sclérose Tubéreuse de Bourneville est une pathologie génétique rare qui se caractérise par la survenue de crises épileptiques précoces à l’origine du développement de nombreux troubles neurologiques tels que des symptômes autistiques ou des retards mentaux. Les épilepsies retrouvées dans la Sclérose Tubéreuse de Bourneville sont souvent résistantes aux traitements pharmacologiques disponibles soulevant la nécessité de trouver de nouvelles approches médicamenteuses plus efficaces pour traiter les patients. Dans cette étude nous avons mis en évidence que l’AppCH2ppA est une molécule efficace pour bloquer la survenue des crises épileptiques dans un modèle de souris pour la Sclérose Tubéreuse de Bourneville ainsi que sur des résections chirurgicales de tissu provenant de patients humains atteints par la Sclérose Tubéreuse de Bourneville. Nous avons montré que les propriétés antiépileptiques de l’AppCH2ppA s’appuient sur une libération autocrine d’adénosine par les neurones de la couche IV du cortex somatosensoriel et d’une activation consécutive des récepteurs à l’adénosine de type A1. Cette activation a lieu spécifiquement au niveau du compartiment postsynaptique et est responsable d’une activation de conductances potassiques et d’une diminution de l’excitabilité des neurones. L’administration d’AppCH2ppA n’est associé à aucun effet secondaire notables sur la santé des souris. Ainsi l’AppCH2ppA semble être un outil thérapeutique prometteur et peu risqué qui stimule des mécanismes antiépileptiques endogènes naturellement sollicités par le cerveau et efficaces pour stopper et limiter la survenue des crises épileptiques. / Tuberous Sclerosis Complex (TSC) is a rare genetic disease characterized by the presence of epilepsies that appear early and in the life of patients and are responsible for the development of several neurological disorders such as autistic symptoms or mental retardations.In TSC, epileptic seizures often resist to pharmacological approaches raising the importance to find new molecules to treat more efficiently the patients.In this study we showed that AppCH2ppA is an effective molecule to block the onset of epileptic seizures in a mouse model for Tuberous Sclerosis as well as on human patients tissues.We have shown that AppCH2ppA nduce an autocrine release of adenosine by the spiny stellate cells present in the layer IV of the somatosensory cortex. This release is responsible for a subsequent activation of adenosine A1 receptors that occur specifically in the postsynaptic compartment of neurons and is responsible for an activation of potassium channels and a decrease of the excitability of neurons. The administration of AppCH2ppA is not associated with any significant side effects on mouse health. Thus, AppCH2ppA appears to be a promising and low-risk therapeutic tool that stimulates an endogenous antiepileptic pathway that is naturally used in the brain and that is efficient to stop and limit the appearance of epileptic seizures.

Les bourgeons du goût contiennent 50 à 100 cellules gustatives. Parmi elles, on distingue les cellules de type 2 et 3, capables d’émettre des potentiels d’action. Les cellules réceptrices (type 2), qui détectent les stimulus sapides dans la salive, ne possèdent pas de synapse. Les cellules présynaptiques (type 3), équipées d’une synapse, transmettent l’information aux neurones gustatifs. Nous avons réalisé des enregistrements électrophysiologiques (patch-clamp) de cellules de bourgeons du goût de papilles fongiformes in situ, dans un épithélium lingual isolé de souris.L’analyse des courants d’action en configuration cellule-attachée, permet de distinguer des impulsions courtes et longues. Dans ce dernier cas, la phase initiale rapide de dépolarisation est suivie d’une dépolarisation maintenue et d’une repolarisation lente. Ceci dénote l’activation de canaux dépolarisants autres que ceux réglant habituellement le décours temporel du potentiel d’action.Les enregistrements en patch-perforé gramicidine, qui n’altèrent pas le milieu intracellulaire, ont permis de perfuser l’intérieur de la cellule avec du césium, bloqueur des canaux potassiques. Le césium permet de distinguer les cellules de type 2 des cellules de type 3 par la présence d’un courant césium-résistant qui correspondrait, selon la littérature, à un courant résultant de l’ouverture d’hémicanaux permettant la sortie d’ATP. Nos observations sont globalement en accord avec les données de la littérature, mais ne supportent pas totalement la classification proposée actuellement en trois types cellulaires bien définis.Les hémicanaux joueraient donc un rôle central dans la communication intercellulaire au sein du bourgeon du goût. Nous avons, à notre connaissance pour la première fois, enregistré des canaux à grande conductance dans les cellules gustatives, en absence d’ion divalents dans la pipette d’enregistrement. Ces canaux, non spécifiques, présentent un fonctionnement complexe avec, outre l’état fermé et l’état de plus haute conductance (700pS), de nombreux états d’ouvertures intermédiaires et un état de conductance résiduel proche du niveau fermé.Au bilan, nos résultats confortent l’hypothèse de l’ouverture de canaux dont la grande conductance laisserait passer l’ATP dans les cellules réceptrices. / Taste buds contain 50 to 100 taste cells. Among those cells, type 2 and type 3 cells are able to produce action potentials. Receptor cells (type 2) detect sapid stimulus in the saliva, but do not possess synapses. Presynaptic cells (type 3) with synapse deliver information to the taste neurons. We performed electrophysiological (patch-clamp) recordings of fungiform taste bud cells in situ, from a mouse isolated lingual epithelium.Analysis of action currents in cell-attached configuration allows discrimination of short and long impulses. In the long ones, the rapid initial depolarizing phase is followed by a slow and a maintained repolarization. This suggests the activation of depolarizing channels other than the ones implied in usual action potential time decay. Gramicidin perforated-patch recordings, which do not alter the inside of the cell, allowed cells perfusion with cesium, a potassic channel blocker. Cesium allows to distinguish type 2 from type 3 cells by the presence of a cesium-resistant current, that may correspond, according to the literature, to the current flowing through hemichannels responsible for ATP release. Our observations mostly agree with data from the literature, but do not fully support the actual classification in only three defined cell types.Hemichannels have a key role in intercellular communication within the taste bud. Thus, and to our knowledge for the very first time, we recorded large conductance channels from taste cells, using a divalent-free pipet. Those non-specific channels have a complex opening mechanism, which, besides closed and upper conductance states (700pS), present many intermediate opening levels and residual states close to the closed state. Our results support the hypothesis that large conductance channels responsible for ATP release open in receptor cells.

Les plantes médicinales constituent un réservoir important de substances naturelles pour la découverte de nouvelles molécules thérapeutiques. L’intérêt de ce travail est d'explorer le potentiel thérapeutique des plantes médicinales connues pour leurs vertus neuromodulatrices et potentiellement d’intérêt pour le traitement de maladies neurologiques, y compris l’épilepsie, en étudiant leur capacité à cibler l’activité des canaux calciques de type T qui jouent un rôle important dans l’hyperexcitabilité neuronale et la physiopathologie des épilepsies. Le premier objectif de ma thèse était d’étudier l’effet des extraits de plantes méditerranéennes ; Lavandula stoechas, Rosmarinus officinalis et Peganum harmala, ainsi que leurs principes actifs ; le linalol, l’acide rosmarinique et l’harmaline, respectivement, sur des courants calciques de type T en utilisant la technique patch-clamp en configuration cellule-entière. Les enregistrements électrophysiologiques à partir de cellules HEK-293 exprimant les canaux T montrent que la lavande, le romarin et l’harmal réduisent significativement les courants de type T sur la gamme de potentiel membranaire testée. Les produits naturels arrivent à déplacer l'état stable d’inactivation vers des potentiels de membrane plus négatifs et certains (Peganum harmala) accélèrent significativement la cinétique d'inactivation des canaux T. Le deuxième objectif était d’étudier l’effet anticonvulsivant et/ou antiépileptique de ces plantes et du TTA-A2 ; un bloqueur sélectif des canaux T, sur un modèle animal d’épilepsie. Nos résultats valident le PTZ et la 4-AP comme inducteurs de crises chez le poisson zèbre, ces deux modèles permettant le criblage pour des molécules anticonvulsivantes et/ou antiépileptiques. Nos résultats montrent que le romarin, la lavande ainsi que le TTA-A2 inhibent les crises pseudo-épileptiques chez ces deux modèles. Dans l’ensemble, ce projet suggère que les canaux T seraient impliqués dans les propriétés neuroprotectrices et anticonvulsivantes des plantes médicinales étudiées et valide le rôle des plantes médicinales comme source intéressante de produits thérapeutiques. / Medicinal plants represent an interesting reservoir of natural substances for the discovery of new therapeutic molecules. The interest of this work is to explore the therapeutic potential of medicinal plants, which are known for their neuromodulation effects, by studying their ability to target the activity of T-type calcium channels which play a major role in neuronal hyperexcitability and the pathophysiology of epilepsy and other neurological diseases.The first objective of my thesis was to study the effect of Mediterranean plant extracts; Lavandula stoechas, Rosmarinus officinalis and Peganum harmala and their active ingredients; linalool, rosmarinic acid and harmaline, respectively, on T-type calcium currents using the patch clamp technique in whole-cell configuration. Electrophysiological recordings from HEK-293 cells expressing T-type channels show that lavender, rosemary and Harmal significantly reduce T-type currents over the potential range tested. The natural products shifted steady-state inactivation towards more negative membrane potentials and some plants (Peganum harmala) significantly accelerate the inactivation kinetics of T-type channels. The second objective was to study the anticonvulsant / antiepileptic activity of these plants as well as TTA-A2, a selective T-type channel blocker, in an epilepsy model in zebrafish. Our results validate the PTZ and 4-AP as inducers of convulsions in zebrafish and both models could be used to screen for anticonvulsant and/or antiepileptic molecules. Our results show that rosemary, lavender and TTA-A2 inhibit seizures-like activity in these two models. Overall, this project suggests that T-type channels are involved in the neuroprotective and anticonvulsant properties of the studied medicinal plants and validates the role of medicinal plants as a valuable source of therapeutic products.

Nesse estudo investigamos os efeitos da hipóxia tecidual aguda (HA) sobre as propriedades eletrofisiológicas intrínsecas dos neurônios pré-simpáticos bulboespinhais da área rostro-ventrolateral do bulbo (RVLM) de ratos jovens adultos submetidos previamente à hipóxia crônica intermitente (HCI) e os seus respectivos controle. Para marcarmos os neurônios pré-simpáticos bulboespinhais da RVLM, ratos Wistar jovens (P19-P21) anestesiados com ketamina e xilazina, receberam microinjeções bilaterais de rodamina, um traçador fluorescente retrógrado, na coluna intermediolateral da medula espinhal (T3-T6) e 2 dias após a recuperação da cirurgia, os animais foram submetidos ao protocolo de HCI, enquanto que ratos controle foram mantidos em condições de normóxia, durante 10 dias. No décimo primeiro dia, os ratos foram novamente anestesiados para a remoção do cérebro e as fatias do tronco cerebral contendo neurônios pré-simpáticos com marcação positivas foram registrados. Utilizamos a técnica de whole cell patch-clamp para estudo das propriedades eletrofisiológicas desses neurônios. As propriedades eletrofisiológicas intrínsecas foram analisadas antes e após a HA, a qual foi produzida pela perfusão das fatias do tronco cerebral com uma solução hipóxica (95% N2 + 5% CO2) durante 2 minutos na presença de bloqueadores sinápticos excitatórios e inibitórios. Todos os neurônios pré-simpáticos apresentaram característica intrínseca de autodespolarização e a frequência de disparos basal de potenciais de ação (PAs) desses neurônios de ratos do grupo controle e HCI foram similares [Controle= 5,03 ± 0,4 Hz (n=39) vs HCI= 6,31 ± 0,7 Hz (n=31); p > 0,05]. No grupo controle, a HA não alterou a frequência média de disparos de PAs (BS = 5,03 ± 0,4 Hz vs HA = 5,24 ± 0,3 Hz (n=39); p > 0,05], porém revelou diferentes perfis de disparo de PAs após 2 min de exposição à HA: i) 11 neurônios com aumento na frequência de disparos (BS = 5,1 ± 0,7 Hz vs HA = 7 ± 0,7 Hz; p < 0,05]; ii) 21 neurônios sem alteração na frequência de disparos (BS = 4,8 ± 0,5 Hz vs HA = 5,36 ± 0,6 Hz; p > 0,05] e iii) 7 neurônios com diminuição na frequência de disparos (BS = 7,3 ± 1,1 Hz vs HA = 3,6 ± 0,7 Hz; p < 0,05). No grupo HCI, a HA produziu aumento na frequência média de disparos (BS= 6,31 ± 0,7 Hz vs HA= 7,25 ± 0,8 Hz; n=31 - p < 0,05) e na análise do perfil de disparo de PAs, a HA revelou 2 subpopulações: i) 9 neurônios com aumento na frequência de disparos (BS = 4,7 ± 0,8 Hz vs HA = 8,2 ± 1,4 Hz; p < 0,05) e ii) 22 neurônios sem alteração na frequência de disparos (BS = 7,0 ± 1,0 Hz vs HA = 6,8 ± 1,0 Hz; p > 0,05). Esse estudo nos permitiu revelar diferentes subpopulações de neurônios pré-simpáticos que responderam de forma distintas à HA. Os resultados também sugerem que a HCI teria um efeito pré- condicionante na excitabilidade intrínseca dos neurônios pré-simpáticos em resposta à HA / In this study we evaluated the effects of acute hypoxia (AH) on the intrinsic electrophysiological properties of presympathetic neurons from rostro ventrolateral medulla (RVLM) of juvenile rats exposed to chronic intermittent hypoxia (CIH) or normoxic condition (control group). To label the RVLM bulbospinal presympathetic neurons, young Wistar rats (P 19 - 21) anesthetized with ketamine and xylazine, received bilateral microinjections of a fluorescent retrograde tracer (rhodamine retrobeads) were performed into the intermediolateral column of spinal cord (T3-T6) and two days after recovery of the surgery, the animals were submitted to CIH or normoxic protocol, during 10 days. On the 11th day, under anesthesia, brainstem slices were obtained and only the labeled RVLM presympathetic neurons were recorded, using whole-cell patch-clamp approach to study the electrophysiological properties of these neurons. The intrinsic electrophysiological properties were analyzed before and after AH, which was produced by slice perfusion with hypoxic solution (95% N2 and 5% CO2) during 2 min in the presence of excitatory and inhibitory synaptic antagonists. All recorded RVLM presympathetic neurons presented intrinsic pacemaker activity and the baseline firing frequency of these neurons from control and CIH group were similar [Control= 5,03 ± 0,4 Hz (n=39) vs HCI= 6,31 ± 0,7 Hz (n=31); p > 0,05]. In the control group, AH do not change the firing rate (BS = 5,03 ± 0,4 Hz vs HA = 5,24 ± 0,3 Hz (n=39); p > 0,05), but revealed different pattern of firing frequency after 2 min of AH: i) 11 neurons increased the firing frequency (BS = 4,9 ± 0,9 Hz vs HA = 6,9 ± 1,0 Hz; p < 0,05) ; ii) 21 neurons do not change the firing frequency (BS = 4,8 ± 0,5 Hz vs HA = 5,36 ± 0,6 Hz; p > 0,05) and iii) 7 neurons decreased the firing frequency (BS = 7,3 ± 1,1 Hz vs HA = 3,6 ± 0,7 Hz; p < 0,05). In the CIH group, the AH increased the firing rate comparing with basal condition (SB= 6,31 ± 0,7 Hz vs AH= 7,25 ± 0,8 Hz; n=31 - p < 0,05) and analyzing the pattern of action potential, AH revealed 2 subpopulations in this group: i) 9 neurons increased the firing frequency (SB = 4,7 ± 0,8 Hz vs AH = 8,2 ± 1,4 Hz; p < 0,05) and ii) 22 neurons do not change the firing frequency (SB = 7,0 ± 1,0 Hz vs AH = 6,8 ± 1,0 Hz; p > 0,05).. The data shows that AH revealed different subpopulations of presympathetic neurons and suggest that CIH plays a preconditioning in the intrinsic excitability of presympathetic neurons in response to acute hypoxia