Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Les astrocytes joueraient un rôle central dans la physiopathologie des troubles anxio-dépressifs et dans l’activité thérapeutique des antidépresseurs. En effet, différentes études in vitro suggèrent que les inhibiteurs sélectifs de recapture de la sérotonine (ISRS) stimulent la synthèse de substances neuroactives par ces cellules gliales nécessaires à la prolifération, la maturation et la survie neuronale mais également au maintien de la plasticité synaptique. Le Brain-derived neurotrophic factor (BDNF) fait partie de ces substances mais son origine, notamment astrocytaire, doit encore être démontrée dans les systèmes intégrés tel que l’animal vivant. A partir de ce constat, dans une première partie de ce travail de thèse, nous avons donc voulu préciser si les astrocytes constituent une source cellulaire participant à la synthèse et/ou à la libération de BDNF en réponse à l’administration prolongée d’un ISRS, la fluoxétine d’une part chez des souris naïves et d’autre part chez des souris exposées à la corticostérone pendant plusieurs semaines (modèle « CORT »). Pour cela, nous avons utilisé une stratégie de transfection virale induisant la surexpression de BDNF spécifiquement dans les astrocytes de l'hippocampe. Nos résultats mettent en lumière que cette surexpression provoque des effets de types anxiolytiques-antidépresseurs dépendant de la neurogenèse hippocampique chez des souris naïves soumises au test d’hypophagie induite par la nouveauté mais pas dans le modèle CORT. Nous avons également mis en évidence que le BDNF pouvait agir en retour sur les cellules qui l’ont libéré pour renforcer le réseau astrocytaire (mécanisme autocrine) mais également sur les neurones sérotoninergiques pré-synaptiques (mécanisme paracrine) pour exercer un frein sur la libération de sérotonine dans l’hippocampe. Différents arguments de la littérature suggèrent d’ailleurs que ce dernier mécanisme pourrait être favorable à l’activité anxiolytique de la fluoxétine tel que nous l’avons observé dans ce travail. Sachant que les astrocytes expriment à leur surface, une grande variété de récepteurs sérotoninergiques, nous nous sommes ensuite intéressé à la possibilité que le sous-type de récepteur 5-HT2A pourrait être un élément clé dans la synthèse et/ou la libération de BDNF et de ce fait moduler la réponse au stress et celle à la fluoxétine. Lors de cette seconde étude nous avons appliqué une approche génétique utilisant des souris mutées, privées de manière constitutive du récepteur 5-HT2A (5-HT2A-/-). A l’opposé de la surexpression de BDNF dans les astrocytes, nous avons montré que les souris 5-HT2A-/- sont plus sensibles au stress, modélisé par l’exposition chronique à la corticostérone, et semblent résistantes à la fluoxétine comparées aux souris 5-HT2A+/+. Afin de préciser le mécanisme pouvant rendre compte de ces observations, nous avons mis en évidence que l’inactivation du récepteur 5-HT2A s’accompagne d’une hypersensibilité du frein inhibiteur exercé par les autorécepteurs 5-HT1A somatodendritiques sur le tonus sérotoninergique. / Growing evidence demonstrates that astrocytes could play a crucial role in the pathophysiologies of anxiety and depression as well as in the therapeutic activity of antidepressant drugs. Indeed, in vitro studies suggest that the selective serotonin reuptake inhibitors (SSRIs) stimulate the synthesis of neuroactive substances by these glial cells which are necessary for the proliferation and maturation of neuronal progenitors but also for the maintenance of the neuronal survival. Brain-derived neurotrophic factor (BDNF) is one of these substances, but its cellular origin has yet to be demonstrated in embedded systems such as living animals. In this context, the first part of this thesis was aimed at clarifying whether astrocytes constitute a source of BDNF in response to the chronic administration of the SSRI, fluoxetine, in both naive and anxio-depressive mice exposed to corticosterone (" CORT" model). In this prospect, we used a novel and efficient gene transfer strategy inducing BDNF overexpression specifically in the astrocytes of the hippocampus. Our results indicated that BDNF overexpression produced anxiolytic-/antidepressant-like activity in the novelty suppressed feeding paradigm in relation with the stimulation of hippocampal neurogenesis in naive mice but not in the CORT model. We also showed that BDNF could act on astrocytes themselves (autocrine mechanism) to improve the hippocampal astrocytic network but also on the pre-synaptic serotonergic nerve terminals (paracrine mechanism) to limit the local serotonin release. Different arguments from the literature suggest that the latter mechanism may be favorable to an anxiolytic-like activity of fluoxetine. Given that astrocytes express at the surface a variety of serotonin receptors, we then raised the possibility that the 5-HT2A receptor subtype may be a key element in the synthesis and/or release of BDNF and thereby modulating the vulnerability to depression and/or the response of fluoxetine. In the second part of this thesis, a genetic approach with mutant mice constitutively lacking the 5-HT2A receptor (5-HT2A-/-) was applied. In contrast to Lenti-BDNF mice, we showed that 5-HT2A-/- mice were more prone to develop anxio-depressive-like symptoms in response to the chronic exposure to corticosterone. Moreover, these mutants were resistant to the chronic administration of fluoxetine compared to 5-HT2A+/+ wild type mice. In order to clarify the mechanism underpinning these observations, we demonstrated that the inactivation of the 5-HT2A receptor was associated with a hypersensitivity of a negative feedback exerted by the somatodendritic 5-HT1A autoreceptors on serotonergic tone.Together these data suggest that astrocytes act in concert with neurons to regulate mood and antidepressant drug response, notably through the synthesis and/or release of BDNF following the activation of the 5-HT2A receptor. More generally, our results illustrate the concept of the tripartite synapse in which the bidirectional communication between astrocytes and monoaminergic neurons would be essential in the regulation of higher brain functions.

Há estudos que mostraram que o ácido N-metil-D-aspártico (NMDA), microinjetado nas estruturas do teto mesencefálico (corpos quadrigêmeos) de ratos evoca comportamentos defensivos do tipo pânico que podem ser seguidos por uma resposta antinociceptiva. Tem sido sugerido que respostas defensivas relacionadas ao medo organizadas por neurônios do tronco cerebral podem ser moduladas por projeções ascendentes mediadas pelo neurotransmissor 5-hidroxitriptamina (5-HT) do núcleo dorsal da rafe (NDR), e fenômenos antinociceptivos induzidos pelo medo inato podem ser organizados por vias serotoninérgicas descendentes também originadas no NDR. Os neurônios do NDR que originam tais conexões, por sua vez, podem ser moduladas por monoaminas que recrutam receptores 5-HT2A localizados no NDR. Não obstante, háuma escassez de estudos mostrando o papel dos receptores 5-HT2A do NDR na modulação do comportamento do tipo pânico e da antinocicepção induzida pelo medo inato organizados nos colículos superiores e inferiores. O objetivo deste estudo foi investigar a participação dos receptores 5-HT2A do NDR na modulação do comportamento de defesa organizado pelos corpos quadrigêmeos e da antinocicepção induzida pelo medo evocados por microinjeções de NMDA nos corpos quadrigêmeos. No experimento I, os animais receberam microinjeção de veículo (NaCl 0,9% / 0,2?L) ou 6, 9 e 12 nmol NMDA no CI. No experimento II, foi realizado o pré-tratamento do NDR com microinjeções de veículo ou o antagonista seletivo do receptor 5HT2A (R-96544) nas concentrações de 5, 10 e 15 nM. Dez minutos depois, o NMDA na dose mais efetiva (12 nmol) foi injetado no CI. Em ambos os experimentos, as respostas defensivas foram analisadas quantitativamente durante 10 min e, em seguida, as latências de retirada de cauda foram medidas a intervalos de 10 min durante 70 min. No experimento III, os animais receberam microinjeção de salina fisiológica ou NMDA (6, 9 e 12 nmol) nas cpSC. No experimento IV, a dose mais efetiva de NMDA (12 nmol) ou veículo foi precedida por microinjeções de veículo ou antagonista seletivo do receptor 5HT2A (R- 96544) em diferentes concentrações, 0.5, 5 e 10 nM. Ambos os efeitos pró-eversivos e antinociceptivos provocados pelas injecções intra-cpCS de NMDA foram atenuados pelo pré-tratamento do NDR com R-96544. No experimento V, a análise morfológica mostrou que os receptores 5-HT2A estão presentes nos interneurônios GABAérgicos do NDR. Em conjunto, esses achados sugerem que o bloqueio dos receptores 5-HT2A no NDR é capaz de atenuar tanto o comportamento defensivo do tipo pânico quanto a antinocicepção induzida pelo medo organizada pelos corpos quadrigêmeos. / There are studies that suggest that N-methyl-D-aspartic acid (NMDA) microinjected into the midbrain tectum structures, such corpora quadrigemina, of rats evokes panic-like defensive behaviours that can be followed by an antinociceptive response. It has been suggested that fear-related defensive responses organised by brainstem neurons can be modulated by ascending projections mediated by the neurotransmitter 5-hydroxytryptamine (5-HT) of the dorsal raphe nucleus (DRN), and phenomena of innate fear-induced antinociception can be organised by descending serotonergic pathways also originating from the DRN. The DRN neurons that give rise to such connections, in turn, can be moduled by monoamines that recruit 5-HT2A receptors located in the DRN. Nevertheless, there is a shortage of studies showing the role of DRN 5-HT2A receptors in the modulation of panic-like behaviour and innate fearinduced antinociception organised by superior and inferior colliculi. The purpose of this study was to investigate the participation of DRN 5-HT2A receptors in the modulation of panic-like behaviour and antinociception evoked by corpora quadrigemina injections of NMDA. In experiment I, the animals received microinjection of vehicle (0.9%NaCl/0.2?L) or 6, 9 and 12 nmol NMDA into the IC. In experiment II, it was performed the pretreatment of DRN with microinjections of vehicle or the 5HT2A receptor selective antagonist (R-96544) in a concentration of 5, 10 and 15 nM. Ten minutes later, NMDA at the most effective dose (12nmol) was injected in the IC. In both experiments, the defensive responses were quantitatively analysed for 10 min and then the tail-flick withdrawal latencies were measured at 10 min-intervals for 70 min. In experiment III, the animals received microinjection of physiological saline or NMDA (6, 9 and 12 nmol) into the deep layers of SC (dlSC). In experiment IV, the most effective dose of NMDA (12 nmol) or vehicle was preceded by microinjections of vehicle or 5HT2A receptor selective antagonist (R-96544) at different concentrations (0.5, 5, and 10 nM). Both proaversive and antinociceptive effects elicited by intra-dlSC injections of NMDA were attenuated by the pretreatment of the DRN with R-96544. In experiment V, the morphological analysis showed that 5-HT2A receptors are present in GABAergic interneurons in the DRN. Taken together, these findings suggest that the blockade of DRN 5-HT2A receptors decreased both panic attack-like defensive behaviour and fear- induced antinociception organised by the corpora quadrigemina neurons.

Classically it was assumed that the compounds with therapeutic effect exert their action interacting with a single receptor. Nowadays it is widely recognized that the pharmacological effect of most drugs is more complex and involves a set of receptors, some associated to their positive effects and some others to the side effects and toxicity. Antipsychotic drugs are an example of effective compounds characterized by a complex pharmacological profile binding to several receptors (mainly G protein-coupled-receptors, GPCR). In this work we will present a detailed study of known antipsychotic drugs and the receptors potentially involved in their binding profile, in order to understand the molecular mechanisms of the antipsychotic pharmacologic effects.The study started with obtaining homology models for all the receptors putatively involved in the antipsychotic drugs receptorome, suitable for building consistent drug-receptor complexes. These complexes were structurally analyzed and compared using multivariate statistical methods, which in turn allowed the identification of the relationship between the pharmacological properties of the antipsychotic drugs and the structural differences in the receptor targets. The results can be exploited for the design of safer and more effective antipsychotic drugs with an optimum binding profile. / Tradicionalmente se asumía que los fármacos terapéuticamente efectivos actuaban interaccionando con un único receptor. Actualmente está ampliamente reconocido que el efecto farmacológico de la mayoría de los fármacos es más complejo y abarca a un conjunto de receptores, algunos asociados a los efectos terapéuticos y otros a los secundarios y toxicidad. Los fármacos antipsicóticos son un ejemplo de compuestos eficaces que se caracterizan por unirse a varios receptores simultáneamente (principalmente a receptores unidos a proteína G, GPCR). El trabajo de la presente tesis se ha centrado en el estudio de los mecanismos moleculares que determinan el perfil de afinidad de unión por múltiples receptores de los fármacos antipsicóticos.En primer lugar se construyeron modelos de homología para todos los receptores potencialmente implicados en la actividad farmacológica de dichos fármacos, usando una metodología adecuada para construir complejos fármaco-receptor consistentes. La estructura de estos complejos fue analizada y se llevó a cabo una comparación mediante métodos estadísticos multivariantes, que permitió la identificación de asociaciones entre la actividad farmacológica de los fármacos antipsicóticos y diferencias estructurales de los receptores diana. Los resultados obtenidos tienen interés para ser explotados en el diseño de fármacos antipsicóticos con un perfil farmacológico óptimo, más seguros y eficaces.