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

Characterization of microdot apodizers for imaging exoplanets with next-generation space telescopes

A major science goal of future, large-aperture, optical space telescopes is to directly image and spectroscopically analyze reflected light from potentially habitable exoplanets. To accomplish this, the optical system must suppress diffracted light from the star to reveal point sources approximately ten orders of magnitude fainter than the host star at small angular separation. Coronagraphs with microdot apodizers achieve the theoretical performance needed to image Earth-like planets with a range of possible telescope designs, including those with obscured and segmented pupils. A test microdot apodizer with various bulk patterns (step functions, gradients, and sinusoids) and 4 different dot sizes (3, 5, 7, and 10 $\mu$m) made of small chrome squares on anti-reflective glass was characterized with microscopy, optical laser interferometry, as well as transmission and reflectance measurements at wavelengths of 600 and 800 nm. Microscopy revealed the microdots were fabricated to high precision. Results from laser interferometry showed that the phase shifts observed in reflection vary with the local microdot fill factor. Transmission measurements showed that microdot fill factor and transmission were linearly related for dot sizes >5 $\mu$m. However, anomalously high transmittance was measured when the dot size is <5x the wavelength and the fill factor is approximately 50%, where the microdot pattern becomes periodic. The transmission excess is not as prominent in the case of larger dot sizes suggesting that it is likely to be caused by the interaction between the incident field and electronic resonances in the surface of the metallic microdots. We used our empirical models of the microdot apodizers to optimize a second generation of reflective apodizer designs and confirmed that the amplitude and phase of the reflected beam closely matches the ideal wavefront.Comment: Space Telescopes and Instrumentation 2018: Optical, Infrared, and Millimeter Wav

The Habitable Exoplanet (HabEx) Imaging Mission: preliminary science drivers and technical requirements

HabEx is one of four candidate flagship missions being studied in detail by NASA, to be submitted for consideration to the 2020 Decadal Survey in Astronomy and Astrophysics for possible launch in the 2030s. It will be optimized for direct imaging and spectroscopy of potentially habitable exoplanets, and will also enable a wide range of general astrophysics science. HabEx aims to fully characterize planetary systems around nearby solar-type stars for the first time, including rocky planets, possible water worlds, gas giants, ice giants, and faint circumstellar debris disks. In particular, it will explore our nearest neighbors and search for signs of habitability and biosignatures in the atmospheres of rocky planets in the habitable zones of their parent stars. Such high spatial resolution, high contrast observations require a large (roughly greater than 3.5m), stable, and diffraction-limited optical space telescope. Such a telescope also opens up unique capabilities for studying the formation and evolution of stars and galaxies. We present some preliminary science objectives identified for HabEx by our Science and Technology Definition Team (STDT), together with a first look at the key challenges and design trades ahead

Ring-apodized vortex coronagraphs for obscured telescopes. I. Transmissive ring apodizers

The vortex coronagraph (VC) is a new generation small inner working angle (IWA) coronagraph currently offered on various 8-meter class ground-based telescopes. On these observing platforms, the current level of performance is not limited by the intrinsic properties of actual vortex devices, but by wavefront control residuals and incoherent background (e.g. thermal emission of the sky) or the light diffracted by the imprint of the secondary mirror and support structures on the telescope pupil. In the particular case of unfriendly apertures (mainly large central obscuration) when very high contrast is needed (e.g. direct imaging of older exoplanets with extremely large telescopes or space- based coronagraphs), a simple VC, as most coronagraphs, can not deliver its nominal performance because of the contamination due to the diffraction from the obscured part of the pupil. Here we propose a novel yet simple concept that circumvents this problem. We combine a vortex phase mask in the image plane of a high-contrast instrument with a single pupil-based amplitude ring apodizer, tailor designed to exploit the unique convolution properties of the VC at the Lyot-stop plane. We show that such a ring-apodized vortex coronagraph (RAVC) restores the perfect attenuation property of the VC regardless of the size of the central obscuration, and for any (even) topological charge of the vortex. More importantly the RAVC maintains the IWA and conserves a fairly high throughput, which are signature properties of the VC.Comment: 10 pages, 6 figure

Resolving the delta Andromedae spectroscopic binary with direct imaging

We present a direct image of the innermost companion to the red giant delta Andromedae using the Stellar Double Coronagraph at the Palomar Observatory. We use a Markov chain Monte Carlo based algorithm to simultaneously reduce the data and perform astrometry and photometry of the companion. We determine that the companion is most likely a main-sequence K-type star and is certainly not the previously hypothesized white dwarf.Comment: ApJ, accepted. 10 pages, 3 figure

Reflected spectroscopy of small exoplanets III: probing the UV band to measure biosignature gasses

Direct-imaging observations of terrestrial exoplanets will enable their atmospheric characterization and habitability assessment. Considering the Earth, the key atmospheric signatures for the biosphere is O$_2$ and the photochemical product O$_3$. However, this O$_2$-O$_3$ biosignature is not detectable in the visible wavelengths for most of the time after the emergence of oxygenic photosynthesis life (i.e., the Proterozoic Earth). Here we demonstrate spectroscopic observations in the ultraviolet wavelengths for detecting and characterizing O$_2$ and O$_3$ in Proterozoic Earth-like planets, using ExoReL$^\Re$. For an O$_2$ mixing ratio 2 to 3 orders of magnitude less than the present-day Earth, and an O$_3$ mixing ratio of $10^{-7}-10^{-6}$, we find that O$_3$ can be detected and its mixing ratio can be measured precisely (within $~1$ order of magnitude) in the ultraviolet ($0.25-0.4\ \mu$m) in addition to visible-wavelength spectroscopy. With modest spectral resolution ($R=7$) and S/N ($\sim10$) in the ultraviolet, the O$_3$ detection is robust against other potential gases absorbing in the ultraviolet (e.g., H$_2$S and SO$_2$), as well as the short-wavelength cutoff between 0.2 and 0.25 $\mu$m. While the O$_3$ detection does not rely on the near-infrared spectra, extending the wavelength coverage to the near-infrared ($1-1.8\ \mu$m) would provide essential information to interpret the O$_3$ biosignature, including the mixing ratio of H$_2$O, the cloud pressure, as well as the determination of the dominant gas of the atmosphere. The ultraviolet and near-infrared capabilities should thus be evaluated as critical components for future missions aiming at imaging and characterizing terrestrial exoplanets, such as the Habitable Worlds Observatory.Comment: 13 pages, 8 figures, 3 tables, accepted for publication in A