Kainate receptor auxiliary subunits NETO1 and NETO2 in anxiety and fear-related behaviors

Anxiety disorders are the most prevalent mental illnesses in Europe, yet, their molecular basis is poorly understood. Unraveling the molecular mechanisms underlying the occurrence and maintenance of anxiety is crucial for effective drug development to treat anxiety disorders. In this thesis work, I focused on the NETO1 and NETO2 auxiliary proteins for kainate receptors (KARs) that tightly modulate the functional properties of the receptor. Because variants in KAR genes have been associated with psychiatric diseases in humans, and with anxiety-like behavior in mice, we hypothesized that NETO1 and NETO2 regulate anxiety through their modulation of KARs. Therefore, the aim of this thesis was to investigate the role of NETO1 and NETO2 in the regulation of anxiety and fear, and to evaluate their potential as novel treatment targets for anxiety disorders. To test our hypothesis, I first carried out a comprehensive behavioral screen of Neto1+/+, Neto1-/-, Neto2+/+ and Neto2-/- mouse anxiety-like and fear-related behaviors. We showed that neither NETO1 nor NETO2 regulated anxiety-like behavior in mice. However, Neto2-/- mice had reduced activity in novel environments without effect on locomotor activity in familiar environments, stress physiology or depression-like behaviors. In cued fear conditioning, Neto2-/- but not Neto1-/- mice had increased fear expression and delayed extinction. To establish the molecular and cellular mechanisms modulating the fear phenotype of the Neto2-/- mice, I investigated its expression pattern by in situ hybridization in the core fear network, composed of the medial prefrontal cortex, the amygdala and the hippocampus. Neto2 was widely expressed in all of these regions and in both excitatory and inhibitory neurons. Accordingly, the NETO2 protein was detectable in the same regions. We next established that in the synapses of these brain regions, the abundance of GLUK2/3 and GLUK5 KAR subunits was reduced 20–40% in the absence of NETO2. By focusing on the amygdala, the central brain region for the processing of fear-inducing stimuli and fear learning, we observed immature features of parvalbumin-expressing inhibitory neurons in Neto2-/- mice. Furthermore, we found a higher amplitude and frequency of miniature excitatory post-synaptic currents specifically in the basolateral amygdala, which is a critical brain region for fear memory consolidation. Concurrent with these results, dendritic spine density in thin dendrites was higher in Neto2-/- compared to Neto2+/+ mice. Taken together, these findings imply stronger glutamatergic synapses within the amygdala in the absence of NETO2. Finally, using the c-Fos immediate early gene as a marker for neuronal activation, we found increased activation of amygdala neurons in Neto2-/- compared to Neto2+/+ mice after fear acquisition. Higher activation of the amygdala may be related to stronger associative learning and be represented behaviorally by higher levels of fear expression during fear conditioning. To summarize, we showed that in the absence of NETO2, mice demonstrate higher conditioned fear expression and extinction delay suggestive of a higher overall conditionability, which is a symptom of posttraumatic stress disorder (PTSD). Furthermore, we established that neither Neto1 nor Neto2 is required for innate anxiety-like behaviors. We propose that the reduced KAR abundance at the synapses of Neto2-/- mice, together with the immaturity and increased excitability of the amygdala, and with the stronger activation of local circuits within the amygdala during fear acquisition underlie the higher conditionability and delayed fear extinction phenotype. Our findings suggest directions for future mechanistic studies on the role of NETO2 in fear conditionability. Taken together, this work showed for the first time that Neto2 is required for normal fear expression and conditioning, and that it modulates amygdala function during associative fear learning, findings with putative therapeutic significance for PTSD.Ahdistuneisuushäiriöt ovat yleisimpiä mielenterveyden häiriöitä, mutta niiden molekyylitason syntymekanismeista tiedetään vasta vähän. Näiden mekanismien selvittäminen on tärkeää henkilökohtaistettujen hoitomuotojen kehittämiseksi. Tässä väitöskirjatyössä tutkittiin NETO1- ja NETO2-proteiineja, jotka sitoutuvat kainaattireseptoreihin (KAR) ja säätelevät niiden toiminnallisia ominaisuuksia. Koska KAR:ien tietyt geenimuodot ovat ihmisellä yhdistetty psykiatrisiin sairauksiin ja hiirillä ahdistuneisuustyyppiseen käyttäytymiseen, hypoteesimme oli, että NETO1 ja NETO2 säätelevät ahdistuneisuutta KAR:ien välityksellä. Väitöskirjatyössä tutkimme NETO1:n ja NETO2:n roolia ahdistuneisuuden ja pelon säätelyssä, sekä mahdollisina uusina hoitokohteina ahdistuneisuushäiriöissä. Selvitimme ensin käyttäytymiskokein Neto1+/+-, Neto1-/--, Neto2+/+- sekä Neto2-/--hiirten ahdistustyyppistä- ja pelkokäyttäytymistä. Osoitimme, ettei Neto1 tai Neto2 säätele ahdistustyyppistä käyttäytymistä hiirellä. Neto2-/--hiirten aktiivisuus oli kuitenkin vähentynyt uudessa ympäristössä. Näiden hiirten aktiivisuus kotihäkissä oli normaalia, eikä niillä ollut muutoksia stressifysiologiassa tai masennustyyppisessä käyttäytymisessä. Testasimme sekä Neto1-/-- että Neto2-/--hiirten pelkoehdollistumista ehdolliseen ärsykkeeseen. Neto1-/--hiiret eivät eronneet verrokeista, mutta Neto2-/--hiirten pelkokäyttäytyminen oli voimakkaampaa ja pelkoehdollistumisen purkautuminen hitaampaa kuin verrokkihiirillä. Tutkimme seuraavaksi in situ hybridisaatiolla missä soluissa Neto2 ilmentyy aivojen pelkoverkostoon kuuluvilla aivoalueilla (mediaalinen etuotsalohko, mantelitumake ja aivoturso). Neto2 ilmentyi laajasti kaikilla näillä alueilla, sekä eksitoivissa että inhiboivissa hermosoluissa. Vastaavasti, NETO2-proteiini ilmentyi samoilla aivoalueilla. Seuraavaksi osoitimme, että NETO2:n puuttuessa näiden alueiden synapseissa GLUK2/3 ja GLUK5 KAR-alayksiköiden määrä väheni 20-40 %. Väitöskirjan toinen osatyö keskittyi tutkimaan mantelitumaketta, joka prosessoi pelkoärsykkeitä ja on tärkeä aivoalue pelko-oppimisessa. Tutkimme amygdalan parvalbumiinia ilmentävien hermosolujen ja perineuronaalisten verkostojen lukumäärää ja osoitimme, että näiden amygdalan erilaistumiseen yhdistettyjen markkereiden määrä oli muuttunut Neto2-/--hiirillä, viitaten siihen, että Neto2 säätelee amygdalan kypsymistä. Lisäksi havaitsimme, että näiden hiirten miniatyyrisilla eksitoivilla postsynaptisilla virroilla oli suurempi amplitudi sekä taajuus basolateraalisessa amygdalassa, joka säätelee pelkoon liittyvien muistijälkien konsolidaatiota. Näihin tuloksiin sopien, Neto2-/--hiirillä oli Neto2+/+-hiiriin verrattuna enemmän haarakkeita ohuissa tuojahaarakkeissa. Tuloksemme viittaavat siihen, että NETO2:n puute mantelitumakkeessa vahvistaa glutamatergisia synapseja. Käyttäen c-FOS-proteiinia hermosolujen aktiivisuuden markkerina, osoitimme, että pelkoon liittyvän muistijäljen muodostuminen liittyi voimakkaampaan mantelitumakkeen hermosolujen aktivoitumiseen Neto2-/-- kuin Neto2+/+-hiirillä. Mantelitumakkeen voimakkaampi aktivaatio voi liittyä vahvempaan assosiatiiviseen oppimiseen, ja se voi esiintyä käyttäytymistasolla voimakkaampana pelon ilmentymisenä pelkoehdollistumisen aikana. Osoitimme siis, että NETO2 tarvitaan normaaliin pelkoehdollistumiseen ja sen purkautumiseen. Molemmat ilmiöt liittyvät korkeampaan ehdollistettavuuteen, mikä on yksi traumaperäisen stressihäiriön oire. Lisäksi osoitimme, etteivät Neto1 tai Neto2 ole välttämättömiä luontaiselle ahdistustyyppiselle käyttäytymiselle. Esitämme, että Neto2-/--hiirten pelkoehdollistumisfenotyyppi johtuu sekä kainaattireseptorien alhaisemmasta määrästä pelkoa säätelevillä aivoalueilla, että erityisesti amygdalan kehityksen ja toiminnan häiriöistä. Tämä väitöskirjatutkimus osoitti ensimmäisenä, että Neto2 tarvitaan normaaliin pelkokäyttäytymiseen ja –ehdollistumiseen, ja että se säätelee mantelitumakkeen toimintaa assosiatiivisessa pelko-oppimisessa. Nämä tulokset auttavat ymmärtämään traumaperäisen stressihäiriön neurobiologisia mekanismeja ja voivat olla avuksi uusien hoitomuotojen kehittämisessä

Kainate receptor auxiliary subunit NETO2 is required for normal fear expression and extinction

NETO1 and NETO2 are auxiliary subunits of kainate receptors (KARs). They interact with native KAR subunits to modulate multiple aspects of receptor function. Variation in KAR genes has been associated with psychiatric disorders in humans, and in mice, knockouts of the Grik1 gene have increased, while Grik2 and Grik4 knockouts have reduced anxiety-like behavior. To determine whether the NETO proteins regulate anxiety and fear through modulation of KARs, we undertook a comprehensive behavioral analysis of adult Neto1(-/-) and Neto2(-/-) mice. We observed no differences in anxiety-like behavior. However, in cued fear conditioning, Neto2(-/-), but not Neto1(-/-) mice, showed higher fear expression and delayed extinction compared to wild type mice. We established, by in situ hybridization, that Neto2 was expressed in both excitatory and inhibitory neurons throughout the fear circuit including the medial prefrontal cortex, amygdala, and hippocampus. Finally, we demonstrated that the relative amount of synaptosomal KAR GLUK2/3 subunit was 20.8% lower in the ventral hippocampus and 36.5% lower in the medial prefrontal cortex in Neto2(-/-) compared to the Neto2(+/+) mice. The GLUK5 subunit abundance was reduced 23.8% in the ventral hippocampus and 16.9% in the amygdala. We conclude that Neto2 regulates fear expression and extinction in mice, and that its absence increases conditionability, a phenotype related to post-traumatic stress disorder and propose that this phenotype is mediated by reduced KAR subunit abundance at synapses of fear-associated brain regions.Peer reviewe

The bradykinin system in stress and anxiety in humans and mice

Pharmacological research in mice and human genetic analyses suggest that the kallikrein-kinin system (KKS) may regulate anxiety. We examined the role of the KKS in anxiety and stress in both species. In human genetic association analysis, variants in genes for the bradykinin precursor (KNG1) and the bradykinin receptors (BDKRB1 and BDKRB2) were associated with anxiety disorders (p <0.05). In mice, however, neither acute nor chronic stress affected B1 receptor gene or protein expression, and B1 receptor antagonists had no effect on anxiety tests measuring approach-avoidance conflict. We thus focused on the B2 receptor and found that mice injected with the B2 antagonist WIN 64338 had lowered levels of a physiological anxiety measure, the stress-induced hyperthermia (SIH), vs controls. In the brown adipose tissue, a major thermoregulator, WIN 64338 increased expression of the mitochondrial regulator Pgc1 alpha and the bradykinin precursor gene Kng2 was upregulated after cold stress. Our data suggests that the bradykinin system modulates a variety of stress responses through B2 receptor-mediated effects, but systemic antagonists of the B2 receptor were not anxiolytic in mice. Genetic variants in the bradykinin receptor genes may predispose to anxiety disorders in humans by affecting their function.Peer reviewe

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Modeling Debris Disk Evolution

Understanding the formation, evolution, and architectures of planetary systems requires detailed knowledge of their components. Debris disks provide a means with which we can study them. The next decade will deliver a wealth of new information on the nearest systems. Parallel advances in modeling will be necessary to interpret these new datasets

Glucocorticoid-Responsive Tissue Plasminogen Activator (tPA) and Its Inhibitor Plasminogen Activator Inhibitor-1 (PAI-1): Relevance in Stress-Related Psychiatric Disorders

Stressful events trigger a set of complex biological responses which follow a bell-shaped pattern. Low-stress conditions have been shown to elicit beneficial effects, notably on synaptic plasticity together with an increase in cognitive processes. In contrast, overly intense stress can have deleterious behavioral effects leading to several stress-related pathologies such as anxiety, depression, substance use, obsessive-compulsive and stressor- and trauma-related disorders (e.g., post-traumatic stress disorder or PTSD in the case of traumatic events). Over a number of years, we have demonstrated that in response to stress, glucocorticoid hormones (GCs) in the hippocampus mediate a molecular shift in the balance between the expression of the tissue plasminogen activator (tPA) and its own inhibitor plasminogen activator inhibitor-1 (PAI-1) proteins. Interestingly, a shift in favor of PAI-1 was responsible for PTSD-like memory induction. In this review, after describing the biological system involving GCs, we highlight the key role of tPA/PAI-1 imbalance observed in preclinical and clinical studies associated with the emergence of stress-related pathological conditions. Thus, tPA/PAI-1 protein levels could be predictive biomarkers of the subsequent onset of stress-related disorders, and pharmacological modulation of their activity could be a potential new therapeutic approach for these debilitating conditions

Socialisations sportives enfantines : méthode et premiers résultats d’enquête

International audienceCette communication présentera les premiers résultats d’une enquête quantitative (Programme Les nouvelles formes de gouvernement des enfants : normes, réseaux, dispositifs, territoires, Labex SMS Toulouse, 2014-2016) réalisée en 2014-2015 dans trois communes françaises d’environ 10 000 habitants chacune diversifiées sur le plan de leur composition sociale. Un questionnaire a été administré à des parents d’enfants (en école élémentaire) inscrits dans un club sportif, association ou structure proposant une activité sportive ou physique. Les données présenteront les résultats recueillis dans une vingtaine de club. Le questionnaire s’attache à différents aspects des socialisations enfantines et parentales et vise à approfondir des travaux récents sur la socialisation corporelle des enfants qui montrent que les cultures somatiques différencient toujours les familles selon leurs caractéristiques sociales, même si elles se sont partiellement modifiées depuis l’enquête pionnière de Boltanski (1971). Dans un premier temps nous expliciterons le dispositif d’enquête, les choix méthodologiques opérés, le protocole retenu en montrant son intérêt et ses limites. Dans un second temps, nous présenterons les premiers résultats obtenus par le croisement d’indicateurs des modes de socialisations sportives des enfants d’une part et des propriétés socio-sexuées des enfants d’autre part