Contribution of estrogen and GluN2B subunit to the HtraKO mouse phenotype

Htr1a is one of the most widespread serotonin receptor across the brain, strongly expressed in CAI region of hippocampus. Our laboratory studies the phenotypic alteration in 5HTla- deficient mice (Htr1aK0), characterized an abnormal anxious-like behavior. Our aim is to evaluate the regulation of this cognitive process by understanding the circuitry involved. This phenotype sets up early during development and has durable effect in adulthood. Our laboratory showed that adult Htr1aK0 male mice displaying exuberant dendritic growth of oblique dendrites in a specific layer of a CAI pyramidal neurons, the stratum radiatum. Application of drugs in organotypic cultures and by in vivo injections revealed that GluN2B, a subunit of NMDA receptor highly expressed during development, is responsible for this dendritic exuberance. Immunohistochemistry highlighted in particular a synaptic enrichment of GluN2B in stratum radiatum of Htr1aK0 CAI pyramidal neurons at puberty. Finally, original analysis of Htr1aK0 mouse behavior showed a different response to anxiety between male and female. Htr1a activation down-regulates the CaMKII activity in the CAI pyramidal neurons. CaMKII directly favors the membrane conductance and stability of GluN2B at the synapse. In the context of the Htr1aK0 mouse, GluN2B is the final common pathway of our phenotype. This subunit is well known to regulate the threshold of LTD/LTP and the dendritogenesis during development. In my thesis, I establish a link between the gender differences in the morphology and the physiology in the Htr1aK0 mice during development to understand how these characteristics shape the circuit with prominent cognitive impacts in adulthood. My study highlighted that during development, Htr1aK0 male mice show a constant increase of the dendritic growth of oblique dendrites from early ages until adulthood associated with an increased physiological impact of altered GluN2A/GluN2B ratio. Whereas during puberty, synaptic contribution of GluN2B to NMDA response is higher in Htr1aK0 compared to WT male mice, this ratio comes back to normal values towards adulthood. However, this recovery of the ratio of GluN2A/GluN2B located at the synaptic level is concomitant with the lateral diffusion of excess GluN2B subunits, leading to extrasynaptic enrichment. The main impact was a lowering of the LTP threshold characterized by strong increased potentiation of synaptic strength after 5 Hz low frequency stimulation. Moreover, the extrasynaptic GluN2B overexpression leads to a shift of the maturation phase switch explaining the exuberant morphology. However, Htr1aK0 females characterized during the 3 first weeks of development by an increase of the dendritic growth of oblique dendrites showed starting at puberty that the dendrite arborization returns progressively to WT values. The physiological impact of GluN2B was investigated and directly linked to this morphology, since Htr1aK0 female mice does not show alteration of the synaptic strength during development. These observations show a compensation occurring in Htr1aK0 female, responsible for a rescue of the phenotype morphologically, physiologically and to be tested behaviorally. We highlighted then the biological processes underlying this compensation. During development, sexual hormones such as testosterone and estrogen are responsible to induce sexual differentiation of specific brain regions. I demonstrated that estrogen, but not testosterone, was able to reduce both in vitro and in vivo the dendritic arborization early during development, through activation of GPER-1, a G-coupled protein estrogen receptor, which phenocopy the activation of Htr1a by reducing GluN2B conductance and stability. I then identified a pathway, parallel to Htr1a, able to regulate GluN2B and responsible for the morphological and physiological phenotype in Htr1aK0 female mice. The specific rise of estrogen occurring at puberty in female is responsible for the compensation observed and induces a late rescue of the Htr1aK0 phenotype by activation GPER-1. -- Htr1a est un des récepteurs à la sérotonine les plus répandus dans le cerveau, fortement exprimé dans la région CAI de l'hippocampe. Notre laboratoire étudie les altérations phénotypiques de souris déficientes pour ce récepteur (Htr1aK0), caractérisées par un comportement avec des traits anxieux. Notre objectif est d'évaluer la régulation de ces processus cognitifs en comprenant les connexions nerveuses impliquées. Ce phénotype se met en place tôt au cours du développement et présente un effet durable à l'âge adulte. Notre laboratoire a montré que les souris Htr1aK0 mâles adultes se caractérisent par une croissance exubérante des dendrites obliques dans une couche spécifique des neurones pyramidaux du CAI, le stratum radiatum. L'application de drogues sur cultures organotypiques et par injections in vivo ont révélé que GluN2B, une sous-unité du récepteur NMDA fortement exprimée au cours du développement, est responsable de cette exubérance dendritique. Des expériences d'immunohistochimie ont notamment mis en évidence un enrichissement synaptique de GluN2B durant la puberté dans le stratum radiatum des neurones de la région CAI des souris Htr1aK0. Finalement, l'analyse originale du comportement des souris Htr1aK0 a montré une différence de réponse à l'anxiété entre mâles et femelles. L'activation de Htr1a diminue l'activité de la CaMKII dans les neurones pyramidaux du CAI. La CaMKII favorise directement la conductance et la stabilité de la sous-unité GluN2B à la synapse. Dans le contexte de la souris Htr1aK0, GluN2B est le « médiateur » de notre phénotype. Cette sous-unité est particulièrement connue pour réguler le seuil de LTD-LTP ainsi que la dendritogénèse durant le développement. Dans ma thèse, j'ai établi le lien entre les différences dépendant du genre dans la morphologie et physiologie des souris Htr1aK0 au cours du développement pour comprendre comment ces caractéristiques modulent le circuit accompagnés d'impacts cognitifs visibles à l'âge adulte. Mon étude a mis en évidence que durant le développement, les souris mâles Htr1aK0 montrent une constante augmentation de la croissance des dendrites obliques entre les premières semaines et l'âge adulte associée à une augmentation de l'impact physiologique du ratio GluN2A/GluN2B altéré. Alors que durant la puberté, la contribution synaptique de GluN2B à la réponse NMDA est plus haute chez la souris mâle Htr1aK0 que le WT, ce ratio revient à des valeurs normales à l'âge adulte. Cependant, cette récupération de l'expression du récepteur au niveau synaptique est concomitante avec la diffusion des sous-unités GluN2B excédantes, amenant alors à un enrichissement extrasynaptique. Le principal impact est une diminution du seuil de la LTP caractérisée par une forte potentiation de la plasticité après une stimulation basse fréquence à 5 Hz. De plus, la surexpression des GluN2B extrasynaptiques conduit à un décalage de la bascule à la phase de maturation, expliquant la morphologie dendritique exubérante. Cependant, les femelles Htr1aK0 initialement caractérisées pendant les 3 premières semaines du développement par une augmentation de la croissance des dendrites obliques montrent à partir de la puberté que cette arborisation dendritique retourne à des valeurs WT. L'impact physiologique de GLuN2B a été investigué et mis en lien avec cette morphologie, étant donné que les femelles Htr1aK0 ne montrent pas d'altération de la plasticité durant le développement. Ces observations montrent une compensation se produisant chez la femelle Htr1aK0, responsable d'une récupération du phénotype morphologique, physiologique et peut-être comportemental. Nous avons souligné les processus biologiques sous-jacent à cette compensation. Au cours du développement, les hormones sexuelles telles que la testostérone et l'estrogène sont responsables de la différentiation sexuelle de régions du cerveau spécifiques. J'ai démontré que l'estrogène, mais pas la testostérone, était capable de réduire in vitro et in vivo l'arborisation dendritique tôt dans le développement au travers de l'activation du récepteur GPER-1, un récepteur aux estrogènes couplés à un protéine G, qui phénocopie l'activation de Htr1a en réduisant la conductance et la stabilité de GluN2B à la membrane. J'ai identifié une voie de signalisation parallèle à celle de Htr1a, capable de réguler GluN2B et responsable du phénotype morphologique et physiologique de la souris femelle Htr1aK0. La montée spécifique d'estrogène se déroulant à la puberté chez la femelle est responsable de cette compensation et implique une récupération tardive du phénotype Htr1aK0 par l'activation de GPER-1.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats` brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Caracterização da variabilidade de genes relacionados à fertilidade de machos e ao temperamento em eqüinos da raça brasileira Mangalarga

The aims of the present study were to propose a PCR-RFLP genotyping method for the AJ_315378:c.110A>G and AB_264325:c. 771G>C SNPs in the equine CRISP1 and HTR1A genes, respectively, as well as to characterize these and another polymorphism, AB_098561:c.1470G>A of the SLC6A4 gene, in order to provide a basis for future studies investigating the association between DNA markers and traits of interest in this breed. For this, 151 Mangalarga horses of both sexes, representatives of the population of the Sate of São Paulo, Brazil, were used. PCR-RFLP was found to be adequate for the genotyping of the SNPs AJ_315378: c.110A>G of the CRISP1 and AB_264325:c.771G>C of the HTR1A. However, the polymorphism of the CRISP1 probably does not occur in Mangalarga horses, a fact impairing association studies of this marker with traits related to male fertility. The estimative of the population genetic parameters obtained for the polymorphisms AB_264325:c.771G>C of the HTR1A and AB_098561:c.1470G> A of the SLC6A4 in the studied sample discourage the conduct of research addressed the association between markers and traits related to temperament.

Caracterização da variabilidade de genes relacionados ao desenvolvimento muscular, fertilidade e temperamento em equinos da raça mangalarga

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Changes in hippocampal gene expression by 7-nitroindazole in rats submitted to forced swimming stress

Nitric oxide (NO) is an atypical neurotransmitter that has been related to the pathophysiology of major depression disorder. Increased plasma NO levels have been reported in depressed and suicidal patients. Inhibition of neuronial nitric oxide synthase (nNOS), on the other hand, induces antidepressant effects in clinical and pre-clinical trials. The mechanisms responsible for the antidepressant-like effects of nNOS inhibitors, however, are not completely understood. In this study, genomic and proteomic analyses were used to investigate the effects of the preferential nNOS inhibitor 7-nitroindazole (7-NI) on changes in global gene and protein expression in the hippocampus of rats submitted to forced swimming test (FST). Chronic treatment (14 days, i.p.) with imipramine (15 mg/kg daily) or 7-NI (60 mg/kg daily) significantly reduced immobility in the FST. Saturation curves for Serial analysis of gene expression libraries showed that the hippocampus of animals submitted to FST presented a lower number of expressed genes compared to non-FST stressed groups. Imipramine, but not 7-NI, reverted this effect. GeneGo analyses revealed that genes related to oxidative phosphorylation, apoptosis and survival controlled by HTR1A signaling and cytoskeleton remodeling controlled by Rho GTPases were significantly changed by FST. 7-NI prevented this effect. In addition, 7-NI treatment changed the expression of genes related to transcription in the cAMP response element-binding pathway. Therefore, this study suggests that changes in oxidative stress and neuroplastic processes could be involved in the antidepressant-like effects induced by nNOS inhibition.

Distribution of mRNA coding for 5-hydroxytryptamine receptor subtypes in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation

OBJECTIVE: To investigate the distribution of mRNA coding for 7 subtypes of 5-hydroxytryptamine receptors (5-HTRs) in the intestines of healthy dairy cows and dairy cows with cecal dilatation-dislocation (CDD). SAMPLE POPULATION: Full-thickness intestinal wall biopsy specimens were obtained from the ileum, cecum, proximal loop of the ascending colon, and external loop of the spiral colon (ELSC) of 15 cows with CDD (group 1) and 15 healthy dairy cows allocated to 2 control groups (specimens collected during routine laparotomy [group 2] or after cows were slaughtered [group 3]). PROCEDURE: Amounts of mRNA coding for 7 subtypes of 5-HTRs (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, and 5-HT4) were measured by quantitative real-time reverse transcriptase-PCR assay. Results were expressed as the percentage of mRNA expression of a housekeeping gene. RESULTS: Expression of mRNA coding for 5-HTR1B, 5-HTR2B, and 5-HTR4 was significantly lower in cows with CDD than in healthy cows. For 5-HTR2B and 5-HTR4, significant differences between cows with CDD and control cows were most pronounced for the ELSC. Expression of mRNA for 5-HTR1D, 5-HTR1F, and 5-HTR2A was extremely low in all groups, and mRNA for 5-HTR1A was not detected. CONCLUSIONS AND CLINICAL RELEVANCE: Relative concentrations of mRNA coding for 5-HTR1B, 5-HT2B, and 5-HTR4 were significantly lower in the intestines of cows with CDD than in the intestines of healthy dairy cows, especially for 5-HT2B and 5-HTR4 in the ELSC. This supports the hypothesis that serotonergic mechanisms, primarily in the spiral colon, are implicated in the pathogenesis of CDD.