Un grand merci à mon jury d'avoir accepté d'examiner ma thèse, à mes rapporteurs, Pr. K Ki im m D Do o et Pr. T Th ho om ma as s L La am mo on ne er ri ie e, ainsi qu'au Dr. M Ma at tt th hi ia as s G Gr ro os sz ze er r, pour leur regard d'exception. Je voudrais remercier tous les membres du labo d'Alain et du labo de Thérèse pour ces quatre belles années passées avec vous. J'ai beaucoup appris et grandi à vos côtés et je remercie particulièrement les incontournables qui m'ont vraiment touché, non seulement par leur conseils avisés mais surtout par leur générosité : R Ra aj ju u, O Ol li iv vi ia a, A Ar ri ie el l, F Fr ra an nc ci in ne e et C Cl lé ém me en nc ce e. Mais surtout, A Al la ai in n, merci de m'avoir donné cette chance de travailler avec toi, de m'avoir soutenu et poussé à persévérer. Merci de m'avoir donné la responsabilité de faire ce lien si intéressant avec le labo de T Th hé ér rè ès se e et M Ma ar ri ie e--O Od di il le e. G Gw we en n, tu as été d'une chaleureuse aide dans cette collaboration pour coordonner et m'accompagner dans mon travail. Merci à tous les deux pour vos conseils si précieux et tout ce que vous m'avez enseigné. Bien sûr cette thèse a été embellie par la fine équipe des thésards et je garde un super souvenir de nos craquages collectifs en C1.7, au bar, au babyfoot ou, qui l'eut cru, dans le couloir du 1 er étage (et je devrais même inclure Alain avec ses petites blagues). Les acteurs de cette équipe de choc sont F FX X, , K Ka ar re en n, A An na ab be el ll le e, H Ho oc ci in ne e, J Je es ss s, D Da am mi ie en n, E Eu ug gé én ni ie e et C Cl lé ém me en nc ce e avant qu'elle ne nous quitte pour de nouvelles aventures, sniff. Je suis très émue en repensant à chacun d'entre vous qui m'avez apporté beaucoup. Cette thèse n'aurait sans doute jamais eu lieu sans la malignité de B Be er rn na ar rd d Gilly. Merci énormément de m'avoir présenté à Alain et d'avoir enclenché cette belle expérience. Merci aussi pour tous tes conseils d'orientation. Comme dans chaque aventure, il y a tous ceux qui font partie de l'envers du décor. Je ne peux pas imaginer avoir réussi ces 4 années sans l'amour de mes parents. M Ma am ma an n et P Pa ap pa a, mes meilleurs coachs et supporters de ma vie. Je vous aime tellement. Merci de ne m'avoir jamais lâché, même quand j'étais des plus pénibles. Il faut dire que j'ai une super famille. Mes frères, P Pa au ul l e et t H Hu ug gu ue es s que j'aime tant. Avec votre grand coeur et vos grands bras forts, je vous remercie de m'avoir réconforté et de m'avoir offert des moments d'amour si précieux mes pohotes. Je suis tellement fière de vous et de ce que vous accomplissez, et heureuse qu'on soit ensemble pour avancer dans nos vies. Mes cousins C Ch ha ac ch ha a et C Ch ho ou ul l, généreux comme notre grand-père, ont également été à mes côtés avec leur conseils et encouragements. Comme tout le reste de mes cousins et D Da an ni ie el l, I Is sa a (avec nos détentes parisiennes), M Ma am mi il ly y, M Ma ar ri ie e--J Je ea an nn ne e qui ont aussi contribué, mine de rien, à faire qui je suis aujourd'hui. Merci à B Br ri ia an n pour nos discussions passionnantes et pour la passion que tu mets dans nos discussions. Je pense fort à mes grands-pères R Ra ay ym mo on nd d et P Pa ap pi il ly y, à qui j'aurai été très fière de présenter ma thèse. Merci à ma bande de pépettes d'amour, A Al li ic ce e, L Lo ou ul lo ou u, T Ti if ff f, P Pa au ul le et tt te e, N Ni in no ou uc ch he e, C Cl la ai ir ro on n, C Cl la ar ri in ne et tt te e et ma taupe V Vi io o, qui sont aussi belles que gentilles avec leur râleuse de Clem. Les meilleures pour toujours. Dans le genre soutien je crois que vous avez la palme. Bien sûr je remercie l'équipe de choc du Chat Ivre et de la Rivoluzione qui ont adouci mes week-ends et pas que ;) M Ma an no on n, M Ma aë ë, M Mo om mo o, G Ga ab bi in n, I Iq qb ba al l, S Sa ar ra ah h, E Et ti ie en nn ne e, B Ba ab ba a, A Al le ex xi is s, A Ag ga at th he e, , D Do ou ud do ou u, , L La au ur ra a C C et tous les autres fêtards dignes de ce nom, sans oublier T To ou um m. J'ai aussi partagé cette expérience avec les Banyuls-girls L La au ur ri ie e et A An nn ne e--M Ma ar ri ie e. . Toujours là quand il faut c'est bien sûr cette sacrée bande de Lyon : L La au ur ra a S S, T Th ho on no on n, G Ga as s, E Ed do ou ua ar rd d, P Pa au ul li in ne e, L Lo ou ui is s O O, L Lo ou ui is s V V, M Mo o, C Co on nc co on n, P Ph hi il lo ou u, B Br re ee e, P Pr ri is s, C Ch ha ar rl le es s, A Al li ic ce e, L La au ur rè èn ne e, T Th hé éo o, L La au ur ra a B B et C Cl la ar ra a. Toujours à mes cotés, même loin. Merci pour vos fêtes endiablées et vos gentils mots (et de prendre soin de Thomas en mon absence). Je vous aime.

Un autre de mes piliers et non des moindres fut T Th ho om ma as s, Mr. Loucoumi, mon amour de kiki, qui m'a tant apporté toutes ces années. Même si tu n'es pas le meilleur des prodigueurs de soutien, je te remercie de croire en moi, de m'avoir remis les pendules à l'heure quand il fallait et de m'avoir distrait quand il fallait. Il est temps maintenant qu'on mette les voiles vers de nouvelles aventures. Je t'aime.

I. ANXIETY-RELATED BEHAVIORS

According to various clinical surveys, about one third of the population is affected by an anxiety disorder during a lifetime. This includes phobias, panic disorders, generalized anxiety disorders, separation anxiety disorder, post-traumatic stress disorder and obsessivecompulsive disorders [START_REF] Bandelow | Epidemiology of anxiety disorders in the 21st century[END_REF]. But these numbers do not include psychiatric pathologies taking their origin at different levels of the anxiety-regulating system of the brain such as autism, schizophrenia, bipolar disorder or depression.

Fear and anxiety are a set of adaptive defensive reactions to a potential threat to ensure individual survival, and from an evolutionary point of view, promote reproductive success. Indeed, these behaviors are observed in many animal species and testify of an adaptation of the organism to its environment. In humans, when the expression of such behavior is exacerbated it leads to excessive chronic anxiety (such as phobias or obsessional compulsive disorders) or acute fear (such as post traumatic stress disorder, PTSD) and, on the contrary, when the expression is impeded the behavior turns to anhedonic-like (seen in certain phases of schizophrenia or bipolar disorder). Brain states of fear and anxiety can be measured by behavioral, physiological, hormonal and autonomic reactions. Even though fear and anxiety have similar manifestations, they differ in many points and, since the 1960's with the introduction of the classical conditioning, or Pavlovian conditioning, fear has been largely explored whereas anxiety mechanisms are less well understood. Fear is evoked by a specific acute stimulus (an imminent threat) and engages active defensive responses that dissipate when the stimulus is removed. Anxiety is evoked by vague, less predictable stimuli, potentially threatening in the future, and is associated with long-lasting states of arousal, vigilance and apprehension. Anxiety is sometimes referred as unconditioned fear.

Thalamus(

Cortex(

Basolateral) amygdala)

Cortex ( Hippocampus( Dorsal(and(Ventral( Striatum( Central(nucleus(of( the(amygdala( Lateral(bed( nucleus(of(the( stria(terminalis( Memory(of(( emo>onal(events( Approach(or(avoidance( Autonomic,(soma>c(( signs(of(fear( ABen>on(to(significant(s>muli( Autonomic,(soma>c(( signs(of(anxiety( Figure 1. Amygdalar projections and outputs.

The BLA receives sensory information from the thalamus and cortices and channels this information to co-working areas to translate it into an appropriate behaviour. (adapted from [START_REF] Davis | The amygdala[END_REF] Figure 2. Subnuclei of the mouse amygdala

The basolateral amygdala comprises cortex-like subnuclei laterally : the lateral (LA in green), the basolateral (BL in blue) and the basomedial (BM in blue) amygdala ; and striatum-like subnuclei medially : the centrolateral (CEl) and the centromedial (CEm) in yellow.

Intercalated cell masses (ITCs in grey) form clusters at the edges of the BLA. (adapted from Lee, 2013) Fanselow in 1986 described three patterns of threat: the predator has been previously encountered but is no longer present, the predator is seen at a long distance or the predator is present with physical contact. In response to these different situations, anxiety and fear are experienced differently: anxiety is expressed by the potential or distal presence of the threat whereas fear is evoked by its actual presence.

I.1. The amygdala in fear and anxiety

The brain areas and the regulatory pathways as well as the behavioural output circuits of fear and anxiety do not fully overlap. As we will see further, many regions interconnect to adapt behavior to the environment, but a key structure stands at the centre of this network: the amygdala (figure 1). Therefore, for a better understanding of anxiety-related behaviors, I chose to describe the organization of the amygdala and some of its relative connections with other areas of the brain.

A. Anatomy

The amygdala comprises two main areas (figure 2): the basolateral amygdala (BLA) and the central amygdala (CEA). The BLA can be sub-divided into the lateral (LA), the basal (BA) and the basomedial (BMA) amygdala; the CEA into the medial (CEm) and the lateral (CEl) central amygdala. The structural organization and the cellular composition of these nuclei defines the BLA as cortex-like, with approximately 80% of glutamatergic excitatory projection neurons and 20% of GABAergic inhibitory interneurons, whereas the CEA has a majority of striatum-like GABAergic neurons. Adding to its inhibitory functions, the amygdala contains three intercalated cell masses (ITC) along the BLA, composed of GABAergic neurons: the lateral, dorsal and ventral paracapsular ITCs [START_REF] Ehrlich | Amygdala inhibitory circuits and the control of fear memory[END_REF][START_REF] Lee | Inhibitory networks of the amygdala for emotional memory[END_REF].

Figure 3. Difference of fear (up) and anxiety (down) connectivity

The lateral (LA), basal (BA), lateral central and medial central (CEl, CEm) amygdala and the intercalated cell masses (ITC) connect sensory and associative cortices, the prelimbic (PL) and infralimbic (IL) medial prefrontal cortices (mPFC), the thalamus, the periaqueductal grey matter (PAG), the lateral septum (LS), the hypothalamus (HYP), the ventral hippocampus (vHC), the anterodorsal, ventral and oval bed nucleus of stria terminalis (ad, v, ovBNST), the parabrachial nucleus (PB) the ventral tegmental area (VTA), and the locus ceoruleus (LC) and the raphe nuclei (RN).

(adapted from [START_REF] Tovote | Neuronal circuits for fear and anxiety[END_REF] B. Fear Classical Pavlovian fear conditioning is a strong paradigm that is widely used to investigate associative fear acquisition and storage. During fear conditioning, a conditioned neutral stimulus (CS) such as a context or a distinct cue (tone, light, odour) is presented in conjunction with an unconditioned aversive stimulus (US) such as a foot-shock.

Subsequently the CS acquires aversive properties and initiates fear responses when presented in the absence of the US. Sensory signals relevant to the CS and the US converge from the sensory cortices where they are processed, or directly from the thalamus, which convey unprocessed and rapid signals, to the projection neurons of the LA [START_REF] Kim | Mechanisms contributing to the induction and storage of Pavlovian fear memories in the lateral amygdala[END_REF]. The newly integrated information is next transferred to the BA and then to the CEm, activating the expression of motor and autonomic responses via the hypothalamus, the midbrain and brainstem structures. During this process, the BLA dialogues with other regions (figure 3), through feedback circuits, such as the prelimbic (PL) medial prefrontal cortex (mPFC) or the dorsal hippocampus (for context memories related to conditioned fear; [START_REF] Tovote | Neuronal circuits for fear and anxiety[END_REF].

Repeated presentations of the CS alone progressively attenuate fear responses, a neural process termed extinction. This new type of learning does not erase conditioned fear but rather inhibits the fear networks, through the recruitment of local inhibitory interneurons (especially the parvalbumin and cholecystokinin-expressing interneurons) in the BLA and in the ITCs. In contrast with fear conditioning, the BA-mPFC projections fire on the infralimbic part of the mPFC (IL). BA and IL both target the inhibitory neurons of ITCs which, in turn, inhibit CEm, thus dampening fear responses. Extinction being context-dependent, the hippocampus (ventral especially) is also heavily involved in this mechanism by projecting strongly to BA and mPFC [START_REF] Engin | Modulation of anxiety and fear via distinct intrahippocampal circuits[END_REF]. Recent studies increasingly show that BLA and CEA can process information in parallel depending on the type of learning and memory.

A general scheme is starting to be revealed but further investigation on the microcircuitry of the amygdala is needed to shed light on the modulatory events that process fear memories.

C. Anxiety

Anxiety is a more sustained state of vigilance and apprehension. It can be measured in rodents with different paradigms that present aversive contexts with an escape route. The most common are: the openfield, the light-dark box and the elevated-plus maze. The behaviour is assessed by the avoidance or preference of the animal towards the different compartments. In the openfield test, rodents are placed in a relatively large and bright arena and anxiety-like behaviour will be observed in rodents staying close to the walls and avoiding the centre of the arena. In the light-dark box rodents can freely move from a brightly lit stressful compartment to a dark, closed and secured compartment. And in the elevated-plus maze test rodents are placed in cross-shaped maze with two open arms and two arms enclosed by walls, elevated at a height forbidding to jump, and anxiety-like behaviour is observed in rodents avoiding the open arms.

The circuits underlying anxiety have been less investigated than those for fear but the amygdala remains at the centre of this mechanism (figure 3; [START_REF] Davis | Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety[END_REF][START_REF] Tovote | Neuronal circuits for fear and anxiety[END_REF]. Based on anatomical observations [START_REF] Alheid | The neuronal organization of the supracapsular part of the stria terminalis in the rat: the dorsal component of the extended amygdala[END_REF] the concept of extended amygdala was developed because of the columns of cells that connect the CEA and MA to a nucleus in the basal forebrain called the bed nucleus of the stria terminalis (BNST; [START_REF] Davis | Role of bed nucleus of the stria terminalis and amygdala AMPA receptors in the development and expression of context conditioning and sensitization of startle by prior shock[END_REF]. The BNST is divided in three subregions: the oval (oBNST), the anterodorsal (adBNST) and the ventral (vBNST) BNST. The three subregions can have different, and possibly contrasting, effects in relation with their connectivity. For example, the activation of the BLA-CEl-adBNST-hypothalamus circuit induces anxiolysis whereas activation of the adBNST-parabrachial nucleus mediates autonomic anxiety responses. A second example is provided by the projections of vBNST to the non-dopaminergic cells of the ventral tegmental area (VTA) that induce anxiety or anxiolysis, depending on the activation of glutamatergic or GABAergic projections, respectively. The projections between the amygdala and other regions are starting to be known but their functions and the chronology of events inducing anxiety remain unclear. Because, in anxiety, cells respond to conditioned stimuli that were not paired with an aversive one, the afferents triggering the amygdala remain unclear. However it has been observed in rodent models that CEl is strongly activated in anxiety responses, probably through the same circuits involved in fear expression. In addition, BLA activity is rhythmically synchronized to the hippocampus during anxiety behaviours [START_REF] Seidenbecher | Amygdalar and hippocampal theta rhythm synchronization during fear memory retrieval[END_REF]. This synchrony is also observed between the hippocampus and the mPFC, which is not surprising considering the influence of mPFC in fear conditioning [START_REF] Engin | Modulation of anxiety and fear via distinct intrahippocampal circuits[END_REF]. Finally, recent findings show an important role of the lateral septum of the hippocampus in promoting arousal and behavioural inhibition during conflict detection and in anxiogenic uncertain contexts [START_REF] Zarrindast | Histaminergic system of the lateral septum in the modulation of anxiety-like behaviour in rats[END_REF]. The VTA dopaminergic neurons, which encodes positive and negative valence, project to the amygdala and regulate anxiety [START_REF] Lammel | Projection-Specific Modulation of Dopamine Neuron Synapses by Aversive and Rewarding Stimuli[END_REF]). Yet all these interconnections remain to be investigated further. Both for fear and anxiety expression, the amygdala is part of an interplay including the prefrontal cortex, the hippocampus, the BNST, the midbrain (VTA in particular) and the hypothalamus and brainstem regions. Less is known for anxiety mechanisms compared to fear but knowledge on fear conditioning and extinction is of interest when we keep in mind that they share common features. In this perspective, in the following chapters, I will report actual knowledge on anxiety-related behaviors that is mainly based on fear conditional signaling. (Adapted from (Lewis et al., 2005). I.2. The GABAergic system, regulator of excitability A. The inhibitory system Synaptic plasticity at glutamatergic connections in the amygdala is essential for conditioned fear learning and memorization. However, local inhibitory synapses contribute and sometimes directly mediate different aspects of fear conditioning behaviour [START_REF] Spampanato | Interneurons in the basolateral amygdala[END_REF]; [START_REF] Duvarci | Amygdala microcircuits controlling learned fear[END_REF][START_REF] Ehrlich | Amygdala inhibitory circuits and the control of fear memory[END_REF][START_REF] Lee | Inhibitory networks of the amygdala for emotional memory[END_REF]. Following fear conditioning, the mRNA levels of GABA-A receptor subunits and GAD65 and GAD67 (GABAsynthesizing) enzymes decrease, and consequently the level of extracellular GABA is reduced in the BLA. On the contrary, fear extinction is associated with an increase in mRNA levels of GABA-A receptors subunits, GAD67 enzyme and GAT-1, a pre-synaptic GABA reuptake transporter. Indeed, a number of animal studies and clinical observations have shown the importance of the inhibitory system in fear and anxiety behaviours. To name a few: GABA-B receptor subunit 1a, or GAD65 knock-out mice show fear generalization to unconditioned stimuli accompanied by fear extinction deficits (Müller et al., 2015;[START_REF] Shaban | Generalization of amygdala LTP and conditioned fear in the absence of presynaptic inhibition[END_REF]; injection of benzodiazepine, an agonist of GABA-A receptors, in the BLA reduces the avoidance response by enhancing local GABAergic activity [START_REF] Harris | Effects of benzodiazepine microinjection into the amygdala or periaqueductal gray on the expression of conditioned fear and hypoalgesia in rats[END_REF]; administration of a GABA partial inverse agonist GF7142 to rats, alleviates their fear conditioning retrieval thus inducing anxiogenic-like effects [START_REF] Tang | The effects of FG7142 on two types of forgetting in 18-day-old rats[END_REF]; mild traumatic brain injury causing anxiety disorders, is associated with a significant loss of GABAergic neurons [START_REF] Almeida-Suhett | Reduced GABAergic inhibition in the basolateral amygdala and the development of anxiety-like behaviors after mild traumatic brain injury[END_REF]. In all these observations, there is a direct influence of interfering with the enhancement of GABAergic neuron activity on behaviour. In the BLA, inhibitory interneurons represent 20% of the total neuronal population and express calciumbinding proteins characteristic of local cortical GABAergic cells (figure 4): calbindin, calreticulin and parvalbumin, and with the same connecting points on glutamatergic cells.

PV+$ CCK$ α2$ α1$ α2$
The parvalbumin (PV)-expressing interneurons represent 40% of the GABAergic neurons and are the main sources of perisomatic innervation of glutamatergic principal neurons. PV interneurons which have similar electrophysiological properties preferentially act in concert (connected by gap-junctions) and form the same synapses, thus synchronizing the firing activity of the principal cells in the BA [START_REF] Woodruff | Inhibition and synchronization of basal amygdala principal neuron spiking by parvalbumin-positive interneurons[END_REF]. Moreover, Gogolla et al. reported that the perineuronal nets (PNNs) specific to PV neurons are necessary for protecting fear memories from erasure [START_REF] Gogolla | Perineuronal nets protect fear memories from erasure[END_REF]. Similarly to their function in cortices, PV neurons seem to be of major importance in mediating the inhibitory regulation of signalling in the amygdala. mPFC excitatory/inhibitory balance has also an important role in modulating fear responses [START_REF] Giustino | The Role of the Medial Prefrontal Cortex in the Conditioning and Extinction of Fear[END_REF][START_REF] Pattwell | Dynamic changes in neural circuitry during adolescence are associated with persistent attenuation of fear memories[END_REF]. It is organized in a laminar manner with 80% of glutamatergic cells and 20% of inhibitory interneurons, such as the rest of the cortex (figure 4). As we have seen before, inhibitory signals also emerge from the BNST to the VTA to modulate fear behaviors [START_REF] Davis | Role of bed nucleus of the stria terminalis and amygdala AMPA receptors in the development and expression of context conditioning and sensitization of startle by prior shock[END_REF].

B. Tuning or signaling

On the one hand, the inhibitory interneurons refine the animal behavior by shaping and adjusting the excitatory signal. The LA receives connections from a number of brain regions which are basally suppressed by inhibitory circuits, until the inhibition is released when the organism is exposed to fearful situations. The stimuli activate LA local inhibitory neurons and ITCs interneurons in parallel with the LA glutamatergic projection neurons and by a mechanism of disinhibition that permits the acquisition of fear memory. Tonic suppression helps to avoid unimportant signals and to diminish the noise, thus increasing the signal-tonoise ratio to generate a specific response toward a specific salient event.

On the other hand, the inhibitory interneurons actively play a causal role in signalling. During fear conditioning, Tthe central nucleus and the intercalated cell masses are entirely composed of inhibitory GABAergic interneurons. During fear conditioning, CEl neurons called ON, activated by the CS, suppress CEl-OFF tonic inhibition of CEm [START_REF] Ciocchi | Encoding of conditioned fear in central amygdala inhibitory circuits[END_REF]. This increase of CEm activity by disinhibition leads to an acquisition of fear behaviours. ITCs actively inhibit CEL and CEm when they are activated by LA. During fear extinction, new memories that decouple the CS from the US suppress the expression of fear. Therefore new inhibitory circuits in BA are recruited by the contextual signals [START_REF] Herry | Switching on and off fear by distinct neuronal circuits[END_REF]. In parallel, IL mPFC activate ITCs to inhibit the CEl ON or directly inhibit CEm, suppressing fear expression. In behaviourally naïve mice, mPFC IL and PL projects broadly to the same regions (ie. amygdala, periaqueductal gray) to exert a top-down control over behavioral responses, recruiting PV-cells [START_REF] Arruda-Carvalho | Prefrontal-amygdala fear networks come into focus[END_REF][START_REF] Courtin | Prefrontal parvalbumin interneurons shape neuronal activity to drive fear expression[END_REF]. BLA and ventral hippocampus CA1 project to PL and IL, with a proportion of axons projecting onto inhibitory interneurons, inhibiting mPFC output to downstream targets [START_REF] Jay | Distribution of hippocampal CA1 and subicular efferents in the prefrontal cortex of the rat studied by means of anterograde transport of Phaseolus vulgaris-leucoagglutinin[END_REF].

In anxiogenic environments, there is an increased synchronous activity (theta-frequence) between the hippocampus and the mPFC [START_REF] Adhikari | Synchronized activity between the ventral hippocampus and the medial prefrontal cortex during anxiety[END_REF] mediated by GABAergic interneurons in the hippocampus [START_REF] Cobb | Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons[END_REF]. Very recently, ventral hippocampal PVcells importance have been described in the persistence of fear memory şkan et al.,

2016

). In a model of rodent PTSD, rescue of PV-cells in the hippocampus and mPFC by inhibiting oxidative stress reversed behavioral impairments [START_REF] Sun | Amelioration of oxidative stress-induced phenotype loss of parvalbumin interneurons might contribute to the beneficial effects of environmental enrichment in a rat model of posttraumatic stress disorder[END_REF].

C. Neuromodulators

Inhibitory neurons are major targets of numerous neuromodulators allowing them to adjust their behaviour to the environment. Among these, dopamine (DA) is particularly well-known for its role in fear responses after stressful situations [START_REF] Pape | GABAergic Neurons: Gate Masters of the Amygdala, Mastered by Dopamine[END_REF]. Following a high environmental threat an increased DA turnover is specifically observed in the ventral tegmental area (VTA) permitting a release of dopamine in the BLA. The VTA encodes for positive and negative valence. A release of DA in the amygdala suppresses feedforward inhibition of local LA and ITCs interneurons onto glutamatergic neurons and relieves the cortical brake exerted by the mPFC on the BLA [START_REF] Bissière | Dopamine gates LTP induction in lateral amygdala by suppressing feedforward inhibition[END_REF][START_REF] Marowsky | A specialized subclass of interneurons mediates dopaminergic facilitation of amygdala function[END_REF]. The lateral habenula, thought to be activated by aversive stimuli or when an expected reward does not occur, projects onto mPFC-targeting DA cells of the VTA. In parallel, the lateral habenula activates the tail of the rostromedial tegmental nucleus (RMTg/tVTA) which will then inhibit DA cells of the VTA projecting to the NuAc, usually mediating reward responses. These two pathways contribute to the expression of place avoiding behaviour.

Correlations have been made between defects observed in animal models of PTSD and psychiatric diseases such as schizophrenia or bipolar disorder and deficits in dopamine signalling [START_REF] Seo | Dopamine D3 Receptor Mediates Preadolescent Stress-Induced Adult Psychiatric Disorders[END_REF].

Serotonin (5-HT) is another biogenic amine controlling fear memories and anxiety. BLA receives multiple projections from the dorsal raphe nucleus that inhibit, via serotonin receptors on GABAergic local or ITCs neurons, the excitation of LA projection neurons [START_REF] Prager | The basolateral amygdala γ-aminobutyric acidergic system in health and disease[END_REF]. The mPFC influences the dorsal raphe nucleus activity in situations where the stressor is confined in a controllable situation [START_REF] Amat | Medial prefrontal cortex determines how stressor controllability affects behavior and dorsal raphe nucleus[END_REF][START_REF] Amat | Control over a stressor involves the posterior dorsal striatum and the act/outcome circuit[END_REF]. 5-HT receptor 1A knockout mice display increased anxiety and avoidance in anxiogenic environment with an increase in mPFC theta power involved in the ventral hippocampus-mPFC synchronization [START_REF] Adhikari | Synchronized activity between the ventral hippocampus and the medial prefrontal cortex during anxiety[END_REF].

The inhibitory system is crucial to counterbalance excitation signalling in the brain, especially when the individual needs to adapt to the environment. This circuitry has a delayed development compared to the excitatory, as it shapes from birth to early adulthood during permissive and regulated windows of high sensitivity to internal and external stimuli, called the critical periods.

Figure 5. Age of onset of common psychiatric disorders

The beginning of most of mental disorders occur during childhood, such as phobias, separation anxiety or impulse-control disorders. Other anxiety disorders such as panic, generalized anxiety and PTSD, as well as substance disorders and mood disorders begin later during adolescence. Schizophrenia or bipolar disorders begin in late adolescence.

These illnesses are frequently undetected early and accrue co-morbid disorders. [START_REF] Paus | Why do many psychiatric disorders emerge during adolescence? Nature Reviews[END_REF])

II. CRITICAL PERIODS OF PLASTICITY

II.1. Definition

Psychiatric diseases such as schizophrenia, autism, bipolar disorder or anxiety disorders have their load of family studies where a number of mutations, single nucleotide polymorphisms and structural genomic variants are highlighted. The diversity of these mutations and the absence of specificity toward a disease in particular, not mentioning that some of them were only observed in single families, precludes a simple genetic cause. It is becoming evident that the overlap of genes involved, and phenotypes observed, in these conditions suggests shared common deficits. The genes pointed at are primarily implicated in brain development suggesting that these psychiatric diseases have neurodevelopmental cause(s). Prodromes of psychiatric diseases emerge during childhood and early adolescence when the inhibitory circuits is maturing and the excitation/inhibition (E/I) balance is building (figure 5). Numerous clinical studies have demonstrated PV-cells and GABAergic signalling defects in the hippocampus, prefrontal cortex or amygdala of psychiatric patients [START_REF] Lewis | Cortical circuit dysfunction and cognitive deficits in schizophrenia-implications for preemptive interventions[END_REF][START_REF] Marín | Interneuron dysfunction in psychiatric disorders[END_REF]. GABAergic markers (such as GABA-A receptor, GABAsynthesizing enzymes GAD65 and GAD67, PV and somatostatin) are decreased in major depressive disorders patients (Luscher and Fuchs, 2015;[START_REF] Guilloux | Molecular evidence for BDNF-and GABA-related dysfunctions in the amygdala of female subjects with major depression[END_REF]. In schizophrenia (SZ) patients, PV-cell activity is weakened in the dorsolateral frontral cortex due to a decreased expression of GAD67 and abnormal gamma-frequency oscillations. SZ patients also present decreased PNN density in the amygdala, entorhinal and prefrontal cortices [START_REF] Mauney | Developmental pattern of perineuronal nets in the human prefrontal cortex and their deficit in schizophrenia[END_REF][START_REF] Pantazopoulos | Extracellular matrix-glial abnormalities in the amygdala and entorhinal cortex of subjects diagnosed with schizophrenia[END_REF]. And Autism Spectrum Disorder mouse models show decreased PV-cell numbers along with compromised insular cortex function [START_REF] Gogolla | Sensory integration in mouse insular cortex reflects GABA circuit maturation[END_REF]. Additionally, synaptic plasticity markers such as synaptophysine, NCAM, MAP 1B or GAP 43, are strongly modified in psychiatric disorders pathways emerge earlier than more complex functions such as language and higher cognitive abilities, which require cumulative critical periods rather than a single one.

Critical period plasticity in local cortical circuits. (adapted from Hensch, 2005a) [START_REF] Knable | Molecular abnormalities of the hippocampus in severe psychiatric illness: postmortem findings from the Stanley Neuropathology Consortium[END_REF][START_REF] Vawter | Elevated concentration of N-CAM VASE isoforms in schizophrenia[END_REF]. It is becoming evident that these diseases suffer from neurodevelopmental impairment during postnatal development, affecting the maturation of the inhibitory system and its relative setting towards the excitatory system. In the mouse, excitatory glutamatergic pyramidal cells are generated between embryonic days 11 and 17

(E11-E17) in the dorsal telencephalon and migrate to the neocortex to undergo synaptogenesis, myelination and apoptosis and evolve into a mature neural network [START_REF] Mitsuhashi | Genetic regulation of proliferation/differentiation characteristics of neural progenitor cells in the developing neocortex[END_REF]. Inhibitory interneurons emerge from the ganglionic eminences in the ventral telencephalon from E9.5 and migrate progressively to the cortex to invade the different layers (figure 6). When the interneurons reach their final laminar localization, they overexpress the KCC2 potassium/calcium exchanger, triggering a hyperpolarization that alters their response to ambient extracellular neurotransmitters.

As a result, at birth, the excitatory system is set but the inhibitory circuitry still need to mature to provide inhibitory feedback and shape synchronized oscillations. This E/I network is refining in function of the environment "to establish a neural representation of the surrounding environment" [START_REF] Takesian | Balancing plasticity/stability accross brain development[END_REF].

This heightened plasticity, called critical period (CP), is specific to postnatal development which explains the better performance of young learners or the long-term consequences of adverse early childhood experience. The developing inhibitory circuits are sensitive to earlylife experience as attested by the partial or total loss of inhibitory signals in sensory deprivation conditions such as dark-rearing [START_REF] Morales | Dark rearing alters the development of GABAergic transmission in visual cortex[END_REF], whisker trimming [START_REF] Jiao | Major effects of sensory experiences on the neocortical inhibitory circuits[END_REF] or early hearing loss [START_REF] Takesian | Presynaptic GABA(B) receptors regulate experience-dependent development of inhibitory short-term plasticity[END_REF]. CPs are highly regulated in time and duration. They appear for each sensory areas and more complex functions in a cascade manner (figure 7).

During these windows of permissive high response capacity of neural circuits, spine-pruning and axonal rewiring are actively induced, until molecular brakes accumulate to dampen plasticity: the system becomes consolidated and CP closes. After closure the system cannot be remodelled. To induce plasticity in the adult, one would need to unmask the circuit from ( Hensch, 2005a) these physical brakes. It is becoming more and more clear that disrupted E/I balance, crucial to cortical functioning, is related to neurodevelopmental diseases such as schizophrenia or autism, and the idea of promoting a novel event of rewiring in the adult to correct neurodevelopmental disorder is of potential therapeutic interest.

II.2. Mechanisms of critical periods of plasticity

To understand the mechanisms of CP plasticity, binocular vision was extensively studied, following the demonstration by Hubel and Wiesel in 1963, of the loss of responsiveness of the primary visual cortex to an eye deprived of vision in kittens. Cortical activity results from the competition of the right and left eye inputs, when -after a thalamic relay -they first converge onto individual neurons of the primary visual cortex (V1) (figure 8). In a wild-type mouse, this ocular dominance has a natural bias towards the contralateral eye over the ipsilateral eye. Monocular deprivation (MD) shifts neuronal spiking responses in favour of the open eye, only during the critical period (between post-natal day 20 (P20) and P40 in the mouse). Consequently, V1 adult neurons are unable to respond to the previously closed eye:

this is called amblyopia.

The maturation of a specific subtype of GABAergic interneurons triggers this plasticity. Just after eye opening (P14), GABA release is weak and no loss of visual responsiveness is observed after MD. If diazepam (DZ; a GABAergic agonist) is infused locally in V1 at that time, poor GABA signalling is compensated and the effect of MD is prematurely induced.

Moreover, GAD65 knock-out mice (upon brief MD), similarly to dark-reared mice, fail to shift their visual response, but infusion of benzodiazepines mimicks GABAergic transmission, permitting the onset of ocular dominance plasticity (Hensch and Fagiolini, 2005b;Iwai et al , J Neurosci, 2003). This suggests that a favorable E/I balance, ie a mature inhibition, is during CP (« OFF ») which close the CP. [START_REF] Takesian | Balancing plasticity/stability accross brain development[END_REF] required for plasticity onset. It also shows that CP timing itself is plastic, and can be controlled by the maturation state of the inhibitory system (Figure 9). Of all GABAergic neuron subtypes, only one seems to be implicated in CP regulation: the parvalbuminexpressing interneurons (PV; figure 8), due to their intrinsic properties and the experiencedependent increase of the strength of their input onto pyramidal glutamatergic neurons during CP. PV cells are fast-spiking large basket-shaped neurons which synapse on the soma of pyramidal cells to ensure long-range inhibition and visual cortical synchrony.

The molecular actors of CP for ocular dominance plasticity were categorized in three types (figure 10; see review [START_REF] Takesian | Balancing plasticity/stability accross brain development[END_REF] [START_REF] Ye | Experience-dependent development of perineuronal nets and chondroitin sulfate proteoglycan receptors in mouse visual cortex[END_REF]. A second example is provided by the nogo receptor (NgR), that limit axonal sprouting and consolidate the synapses. Mutants lacking NgR gene exhibit levels of spine turnover similar to that of juvenile mice [START_REF] Yang | Critical period for acoustic preference in mice[END_REF] and dark-reared mice have low levels of NgR in V1 PNNs.

Epigenetic modifications have also been associated with the closure of CP for ocular dominance [START_REF] Baroncelli | Experience Affects Critical Period Plasticity in the Visual Cortex through an Epigenetic Regulation of Histone Post-Translational Modifications[END_REF][START_REF] Putignano | Developmental downregulation of histone posttranslational modifications regulates visual cortical plasticity[END_REF][START_REF] Huang | GAD1 mRNA expression and DNA methylation in prefrontal cortex of subjects with schizophrenia[END_REF][START_REF] Martinowich | DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation[END_REF]. These include DNA methylation and histone deacetylation and occur in response to environmental signals allowing for the regulation of gene expression.

Evidence exist that the same molecular actors are active in other CPs. For example BDNF induces tonotopic map plasticity, with GABA receptors expression induction during auditory cortex CP [START_REF] Anomal | Manipulation of BDNF signaling modifies the experience-dependent plasticity induced by pure tone exposure during the critical period in the primary auditory cortex[END_REF]. PNN formation around PV-cells seems to be a "universal" marker of plasticity as it takes also place during the CP of the mouse barrel sensory cortex (McRae 2007), during zebra finch song learning [START_REF] Balmer | Modulation of perineuronal nets and parvalbumin with developmental song learning[END_REF]) and during CP for fear extinction [START_REF] Gogolla | Perineuronal nets protect fear memories from erasure[END_REF]. It was also observed in the medial prefrontal cortex mediating acoustic preference requiring functional NgR to limit plasticity into adulthood [START_REF] Yang | Critical period for acoustic preference in mice[END_REF].All this suggests that CPs share common mechanisms during the plasticity period that precedes the formation of mature systems in the adult.

II.3. Otx2 in critical period regulation

In the scope of CP regulation, homeoprotein (HP) Otx2 has been demonstrated to be (Adapted from Beurdeley, 2012). (Scale bar: 100µm) two regions, cortical Otx2 is not expressed locally but transferred in an experiencedependent manner to the cortex. Dark-rearing does not affect retinal Otx2 levels but reduces cortical appearance and delays CP onset (Sugiyama, 2008). As for many HPs, Otx2 contains internalization and secretion sequences allowing HP transfer and non-cell autonomous functions (Joliot, Prochiantz 2004). Eye opening may trigger a first step in PNN assembly at the surface of PV-cells (Figure 11) thus forcing the maturation of Otx2 binding sites and triggering Otx2 internalization. As will be described below, one of the extra-cortical Otx2 sources is the choroid plexus (Spattaza, 2013a). Otx2 internalization is mediated by the third helix of the DNA-binding domain (homeodomain, HD) and the specific recognition of the PVcells is mediated by an arginin (R), lysin (K)-rich domain (RK-peptide), overlapping the first helix of the HD. This RK-peptide specifically binds to glycosaminoglycans present in the PNNs that surround the PV-cells (Beurdeley, 2012). Mutating this motif (replacing the RK doublet by two alanines (AA)), thus generating the AA-peptide, does not prevent Otx2 from entering the cells but diminishes cell internalization specificity. As a consequence it takes more time for Otx2 to reach the thresholds for opening and closing plasicity. PV-cell maturation upon Otx2 accumulation can be followed by the increase in PNN assembly. When

Otx2 transfer is blocked, by an antibody capturing Otx2 in the extracellular milieu (Sugiyama, 2008) or by competition for its binding sites within the PNNs following RK-peptide infusion (Beurdeley, 2012 ; figure 12), PV expression is down-regulated, in parallel with a decrease in PNN assembly. Otx2 in PV-cells comes from extracellular sources and one of them was identified as the choroid plexus (ChP) a central structure that secretes the cerebrospinal fluid (CSF) into the ventricles (Spatazza, 2013). Interestingly, Otx2 transfer takes place throughout life and is necessary to maintain a non-plastic adult state. Otx2 is present in PVcells throughout several cerebral areas such as the auditory, somatosensory, prefrontal cortices and limbic structures including the amygdala and the hippocampus CA1. This suggests that non-cell autonomous Otx2 may play in the latter structures a role similar to that discovered in the visual system. In the latter context it is of note that a local knock-down of Otx2 expression in the lateral ChP decreases the levels of Otx2 in V1, reduces the number of mature PV-cells and partially blocks PNN assembly (figure 13).

A model arising from these studies describes Otx2 implication as follows (figure 14 Otx2 internalization with the RK-peptide or reducing its synthesis by the ChP (by recombining the Otx2 locus in this structure) allows one to induce amblyopia in the adult or, and conversely, to restore visual acuity in the lazy eye of an amblyopic mouse (Beurdeley, 2012;Spatazza, 2013). Restoration of plasticity in the adult has also been achieved by the injection of a GAD inhibitor or a GABA-A receptor antagonist [START_REF] Harauzov | Reducing intracortical inhibition in the adult visual cortex promotes ocular dominance plasticity[END_REF], or the digestion of PNN complex sugars by chondroitinase ABC in V1 [START_REF] Pizzorusso | Reactivation of ocular dominance plasticity in the adult visual cortex[END_REF][START_REF] Pizzorusso | Structural and functional recovery from early monocular deprivation in adult rats[END_REF].

Figure 14. Otx2 triggers onset and closure of CP for binocular vision

Early-sensory experience initiates Otx2 accumulation in PV-cells and induces their maturation that triggers CP onset (threshold 1). Otx2 continuously accumulates and the local circuits become stabilized in a mature state: the CP closes (threshold 2). Constant accumulation during adulthood is necessary to maintain this mature state. When Otx2 levels are decreased, a new plasticity event is permitted in the cortex.

III. THE HOMEOPROTEIN SIGNALLING MOLECULE OTX2

III.1. Otx2 multiple functions

Otx2 stands for Orthodenticle Homeobox 2. It is a transcription factor, part a the homeoprotein family characterized by its highly conserved 60 amino-acid long DNA-binding domain, the homeodomain (figure 15A). Among various functions during embryogenesis,

Otx2 is a gap gene that regulates the anterior neuroepithelium specification fated to give rise to the forebrain, midbrain and rostral hindbrain. Consequently, Otx2-/-mice lack the anterior head and die early during development. At later stages, Otx2 interacts with Gbx2 in the formation and positioning of the isthmic boundary separating the midbrain from the hindbrain.

Additionally, in the ventral mesencephalon, Otx2 regulates the proliferation and differentiation of mesencephalic dopaminergic (mesDA) progenitors with a low antero-high posterior gradient response (Omodei, Development, 2008). After E12.5, Otx2 expression is restricted to mesencephalic neurons composing the Ventra Tegmental Area (VTA) and absent in the substantia nigra pars compacta (SNpc). This expression by VTA neurons persists throughout life (Di Salvio et al., 2010a) and is responsible for their low DA reuptake receptor glycoDAT expression which generates a high resistance to the MPTP neurodegenerative toxin (Di Salvio et al., 2010b). Whereas the Otx2-/-genotype is lethal, Otx2+/-mice are viable and fertile but show visual defects. Otx2 is expressed in the optic vesicle starting at E9, and is required to specify the retinal pigment epithelium, photoreceptors and bipolar cells (Martinez- [START_REF] Martinez-Morales | Otx genes are required for tissue specification in the developing eye[END_REF]Nishida et al., 2003). Otx2 expression is then maintained during postnatal life in photoreceptors and bipolar cells (Sugiyama et al., 2008;[START_REF] Fossat | A new GFP-tagged line reveals unexpected Otx2 protein localization in retinal photoreceptors[END_REF][START_REF] Housset | Loss of Otx2 in the adult retina disrupts retinal pigment epithelium function, causing photoreceptor degeneration[END_REF]. Interestingly in the present context, Otx2 protein injected in the eye is taken up by retinal ganglion cells (RGCs) and is transported to the thalamus and cortex. Physiologically, at the time of eye opening blocking Otx2 in the eye in extracellular space prevents its transport from bipolar cells to RGCs and blocks CP opening. This suggests that Otx2 passage into RGCs and possibly to the thalamus and cortex is the first trigger for PV maturation, but this remains to be demonstrated. An unrelated issue is the protective role of Otx2 uptake by RGCs in a model of excitotoxicity [START_REF] Torero Ibad | Otx2 promotes the survival of damaged adult retinal ganglion cells and protects against excitotoxic loss of visual acuity in vivo[END_REF] In the adult, Otx2 expression is maintained in the choroid plexuses and in areas involved in visual information processing: the retina, the thalamus and the superior colliculus (figure 15B). Intriguingly, Otx2 is also imported from extra-cortical sources in many cortical regions. Indeed, during development and maturation to adulthood, Otx2, similarly to many developmental transcription factors, has been recruited for physiological functions, including (in the case of homeoproteins at least and in addition to transcription regulation) the regulation of mRNA translation and that of the epigenetic status of the cells.

III.2. Studying Otx2 non-cell autonomous role

Homeoprotein secretion and internalization sequences are shared by almost all HPs and this capacity to enter cells has been shown for 6 HPs (out of 6 tried) in vivo: Otx2, Pax6, Engrailed 1 and 2, Vax1 and Hoxd1 (Joliot, Prochiantz 2004) and 13 (out of 13) in vitro. This suggests that most, if not all, of them do signal in vivo. These sequences are distinct from classical secretion and endocytosis sequences and the mechanisms are not yet fully understood but the diffusion of HPs is not following the conventional secretion/internalization pathways [START_REF] Joliot | Neurotrophic activity of a homeobox peptide[END_REF][START_REF] Joliot | Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein[END_REF]). The two sequences are part of the homeodomain and, in fact the homeodomain alone can be directly captured by neurons in vitro [START_REF] Joliot | Antennapedia homeobox peptide regulates neural morphogenesis[END_REF]. A difficulty in studies aimed at understanding HP non-cell autonomous functions is that mutating one of these two sequences also disrupt cell-autonomous activity.

An alternative strategy was first tried in the visual paradigm consisting in infusing an anti-Otx2 antibody for 7 days in V1 to block Otx2 internalization by PV cells. A similar strategy was also adopted in the zebrafish where mRNAs encoding a secreted single-chain antibody (scFv) against Pax6 was injected in an embryo (Lesaffre 2007). Pax6 is crucial to the formation of the eye and its extracellular neutralization by the scFv considerably interfered with normal development leading to eye malformation or absence. The scFv strategy was also used with success in transgenic flies allowing the investigators to study the non-cell autonomous role of Engrailed in the formation of the anterior cross vein (Layalle, 2013).

Considering Otx2 ubiquitous distribution in the cerebral cortex, my goal was to investigate if this HP controls physiological functions other than those established for the visual cortex, in particular higher functions related to complex behaviours such as anxiety or fear. Another We provide the first mammalian genetic evidence for a signaling mechanism involving intercellular transfer of a homeoprotein transcription factor. Our single-chain antibody mouse model is a valid strategy for extracellular neutralization that could be applied to other homeoproteins and signaling molecules within and beyond the nervous system.

Introduction

During postnatal life, the cerebral cortex passes through critical periods (CPs) of plasticity allowing the neuronal circuitry to shape in response to environmental stimuli. CPs are driven by the maturation of a subset of inhibitory interneurons, the fast-spiking parvalbumin-expressing GABAergic neurons (PV cells), present in layers III and IV of the cerebral cortex [START_REF] Del Rio | The development of parvalbumin-immunoreactivity in the neocortex of the mouse[END_REF]. Plasticity terminates with the full maturation of PV cells and the consolidation of the Excitation/ Inhibition (E/I) cortical balance [START_REF] Hensch | Critical period plasticity in local cortical circuits[END_REF]. CPs for different sensory, motor or cognitive behaviors are spread out during postnatal development and thus open and close at different times [START_REF] Bavelier | Removing brakes on adult brain plasticity: from molecular to behavioral interventions[END_REF]. In the mouse, plasticity for the establishment of binocular vision opens at post-natal day 20 (P20) and closes at P40 [START_REF] Hensch | Critical period mechanisms in developing visual cortex[END_REF]. Closing one eye during this period (but not before P20 or after P40) leads to an irreversible loss of visual acuity for the temporarily closed eye, a state known as amblyopia, which affects 3% of the human population.

The transfer of the homeoprotein (HP) transcription factor Otx2 from extra-cortical sources into PV cells of the primary visual cortex (V1) regulates CP timing [START_REF] Sugiyama | Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity[END_REF]. Cortical infusion of recombinant Otx2 accelerates both CP onset and closure. Recently, the choroid plexus was identified as one of these sources, as Otx2 knock-down specifically in the choroid plexus reopens a window of plasticity in the adult V1 [START_REF] Spatazza | Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity[END_REF]. Reopening plasticity in the adult was also achieved by blocking Otx2 binding to complex sugars embedded in perineuronal nets (PNNs) that surround PV cells, thus reducing its internalization [START_REF] Beurdeley | Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex[END_REF][START_REF] Miyata | Persistent cortical plasticity by upregulation of chondroitin 6-sulfation[END_REF]. Together, these results led to the important concept that Otx2 accumulation by PV cells leads to a first concentration threshold that opens plasticity at P20 and to a second threshold that closes plasticity at P40; the maintenance of a non-plastic adult state requires the continuous internalization of this HP [START_REF] Spatazza | Homeoprotein signaling in development, health, and disease: a shaking of dogmas offers challenges and promises from bench to bed[END_REF][START_REF] Prochiantz | Homeoprotein Signaling in the Developing and Adult Nervous System[END_REF].

In this report we aimed at developing genetic evidence for direct non-cell autonomous regulation of plasticity by Otx2 in the mouse. An obvious approach would be the production of a "cell autonomous-only" mouse through the mutation of Otx2 import and/or export sequences. This strategy is precluded by the fact that the latter sequences are part of the DNA-binding domain and cannot be modified without modifying the cell autonomous transcription of Otx2 targets [START_REF] Joliot | Transduction peptides: from technology to physiology[END_REF][START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF]. Based on previous studies on non-mammalian embryonic development [START_REF] Lesaffre | Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish[END_REF][START_REF] Wizenmann | Extracellular Engrailed participates in the topographic guidance of retinal axons in vivo[END_REF][START_REF] Layalle | Engrailed homeoprotein acts as a signaling molecule in the developing fly[END_REF][START_REF] Lullo | Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube[END_REF], we turned to a strategy that employs the extracellular expression of neutralizing single-chain variable fragment (scFv) antibodies. We generated conditional knock-in mouse lines (scFvOtx2 tg/o and scFvPax6 tg/o ) expressing secreted scFv antibodies directed against Otx2 or Pax6 (as a control). Targeted expression of anti-Otx2 scFv in juvenile or adult mice resulted in the delay or reactivation of plasticity genes, respectively. This strategy provides the first genetic evidence for HP transfer in mammals and will allow for precise genetic manipulation of noncell autonomous homeoprotein activities to study their functions in embryonic and postnatal development.

Results

We generated knock-in mouse lines expressing inducible secreted Myc-tagged anti-Otx2 or anti-Pax6 single-chain antibodies (scFvOtx2 tg/o and scFvPax6 tg/o ) designed to neutralize HPs in the extracellular space without affecting cell autonomous functions. Tagged anti-Otx2 and anti-Pax6 scFv constructs were generated from hybridoma, cloned behind a floxed STOP cassette, and knocked into the ROSA26a locus (Fig 1A).

To validate scFv expression, fibroblasts cultured from adult scFvOtx2 tg/o ear biopsies were treated with the cell-permeable Cre bacterial recombinase Cre-TAT [START_REF] Joliot | Transduction peptides: from technology to physiology[END_REF]. Cells and media were collected 48h later and analyzed by Western blot (Fig 1B). Anti-Otx2 scFv was only present in cells treated with Cre-TAT and in the medium collected from these cells. Intracellular control RhoA confirms cell integrity. The anti-Myc antibody revealed two bands in the cell extract and in the medium. The lower band corresponds to the scFv recombinant protein and the upper band to the scFv-GFP fusion protein, establishing that the P2A skipping peptide was not 100% efficient. The slightly lower mobility of the antibodies retrieved from the extracellular milieu is due to differences in glycosylation [START_REF] Logan | Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions[END_REF].

The anti-Pax6 scFv functional activity has been demonstrated earlier [START_REF] Lesaffre | Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish[END_REF][START_REF] Lullo | Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube[END_REF]. To examine whether secreted anti-Otx2 scFv binds Otx2 specifically, the media of scFvOtx2 tg/o fibroblasts treated or not with Cre-TAT were used as a source of primary antibodies. Otx2 is recognized by the medium of scFvOtx2 tg/o fibroblasts treated with Cre-TAT (Fig 1C). To confirm scFv in vivo activity, we crossed scFv mice with PV::Cre mice, where the Cre recombinase is driven by the PV promoter [START_REF] Runyan | Response features of parvalbumin-expressing interneurons suggest precise roles for subtypes of inhibition in visual cortex[END_REF], and extracted adult cerebellum, which expresses both HPs [START_REF] Frantz | Otx1 and Otx2 define layers and regions in developing cerebral cortex and cerebellum[END_REF] as well as PV. The anti-Otx2 scFv co-immunoprecipitated with Otx2 and, conversely, Otx2 co-immunoprecipitated with the scFv (Fig 1D). Association with Otx2 protein was not found in PV::Cre or PV::Cre;scFvPax6 tg/o cerebellar extracts.

Two models were developed to disrupt non-cell autonomous Otx2 in V1. A first model was based on local scFv secretion by PV cells. A second model consisted in inducing scFv secretion in the cerebrospinal fluid (CSF) from the choroid plexus, which is an endogenous source of extracellular Otx2 [START_REF] Spatazza | Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity[END_REF]. Pax6 is poorly expressed in the postnatal cortex and choroid plexus [START_REF] Duan | Spatiotemporal expression patterns of Pax6 in the brain of embryonic, newborn, and adult mice[END_REF][START_REF] Zeisel | Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq[END_REF], making scFvPax6 tg/o mice an appropriate negative control. To induce scFv expression by PV cells, scFv mice were crossed with PV::Cre mice, where recombination is driven by the PV promoter starting at around P10 in V1 [START_REF] Del Rio | The development of parvalbumin-immunoreactivity in the neocortex of the mouse[END_REF]. At P30, PV cells of PV::Cre;scFvOtx2 tg/o and PV:: Cre;scFvPax6 tg/o mice but not of PV::Cre mice express scFvs in the V1 binocular zone (Fig 2A and S1 Fig). While most PV cells express the scFv, curiously some cells not stained with the PV antibody also express the scFv. This may result from poor PV expression at P30 or by the capture of secreted scFv by PV-negative cells. To induce scFv expression in the choroid plexus and subsequent secretion into the CSF, adult scFvPax6 tg/o and scFvOtx2 tg/o mice were injected with Cre-TAT in the lateral ventricles [START_REF] Spatazza | Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity[END_REF]. Western blot analysis of the CSF confirmed the induction and secretion of both the scFv and scFv-GFP proteins 5 d post-injection (Fig 2B).

The accumulation of Otx2 in V1 PV cells controls CP timing through the maturation of PV cells [START_REF] Prochiantz | Homeoprotein Signaling in the Developing and Adult Nervous System[END_REF]. CP kinetics are paralleled by increasing PV expression and assembly of extracellular matrix perineuronal nets (PNNs) revealed by the WFA lectin [START_REF] Hensch | Critical period plasticity in local cortical circuits[END_REF]. To follow PV cell maturation, P30 V1 sections were stained with antibodies against Otx2 and PV and with fluorescent WFA (Fig 3A). PV::Cre and PV::Cre;scFvPax6 tg/o mice were undistinguishable for all criteria used. In contrast, PV::Cre;scFvOtx2 tg/o mice showed a decrease in the number of PV cells containing detectable amounts of Otx2 (Fig 3B). In addition scFv expression reduced by ~20% the number of cells decorated with Otx2 and PV antibodies or with WFA (Fig 3C). Intensity of Otx2 staining was also reduced by 20%. The number of GABAergic neurons and Calretinin (CR) neurons were not modified by scFv induction (Fig 3C and S2 Fig). This possibly reflects limited antibody diffusion and is in agreement with the fact that Otx2 is primarily captured by PV cells and does not influence the maturation of other interneurons [START_REF] Sugiyama | Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity[END_REF]. Thus, the anti-Otx2 Due to the existence of thresholds, small changes in Otx2 concentrations within PV cells can have dramatic effects on V1 plasticity [START_REF] Spatazza | Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity[END_REF][START_REF] Beurdeley | Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex[END_REF]. Plasticity is paralleled by the expression of plasticity genes in V1 layer IV such as Arc, Fos, Nr4a1 and Egr4 Adult choroid plexus expression of scFv was induced in P90-P120 scFvOtx2 tg/o and scFvPax6 tg/o mice. After 15 d, the number of cells expressing Otx2, PV or stained with WFA (PNN assembly) was analyzed. In binocular V1 of injected scFvOtx2 tg/o compared to injected scFvPax6 tg/o , the number of cells stained with Otx2 as well as the number of cells expressing the two markers of PV cell maturation were reduced by 20 to 30%, with no change in the percentage of CR neurons or of the entire population of GABAergic neurons (Fig 4A and 4B and S2 Fig). In parallel, plasticity transcript expression was dramatically enhanced in V1 layer IV (Fig 4C), suggesting induced brain plasticity similar to the effect obtained by recombination of Otx2 in the adult choroid plexus [START_REF] Spatazza | Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity[END_REF].

To verify if the reactivation of plasticity genes was reflected at a physiological level, we used optical imaging of intrinsic signals to measure visual acuity [START_REF] Lehmann | Age-dependent ocular dominance plasticity in adult mice[END_REF][START_REF] Sato | Distinctive features of adult ocular dominance plasticity[END_REF]

Discussion

The possibility of the anti-Otx2 scFv neutralizing cell autonomous Otx2 cannot be ignored even though the amount of antibody that might escape the secretion pathway is low and the disulfide bond necessary for antibody activity is reduced in the intracellular milieu [START_REF] Der Maur | Antigen-independent selection of intracellular stable antibody frameworks[END_REF][START_REF] Strube | Enhanced intracellular stability of sFv-Fc fusion intrabodies[END_REF]. This risk can be excluded when PV cells express the antibody, given that the Otx2 locus is not active in these cells. In contrast, the issue can be raised for the choroid plexus, which strongly expresses Otx2. However, we have verified that potential Otx2 target genes in the choroid plexus are not quantitatively modified in the recombined scFvOtx2 tg/o plexus. For example an identified target gene is C1qL3 which, compared to GAPDH, is up-regulated from 1.01 ± 0.11 (n = 3) to 5.21 ± 0.62 (n = 3) in the plexus of Otx2 +/-mice and unchanged 1.40 ± 0.07 (n = 3) in the scFv mouse choroid plexus.

The consequences of expressing the anti-Otx2 scFv under the control of the PV promoter are only a modest reduction of Otx2 content, PV expression and PNN assembly at P30. One likely reason is that PV expression starts at around P10 [START_REF] Del Rio | The development of parvalbumin-immunoreactivity in the neocortex of the mouse[END_REF] and that it might take some time before the concentration of extracellular antibody is high enough to completely block Otx2 internalization. Another possibility is that the high affinity (in the nM range) of Otx2 for PNN complex sugars [START_REF] Beurdeley | Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex[END_REF] partially protects Otx2 from being neutralized by the antibody. It is nevertheless clear that the delayed time course in PV cell maturation is enough to delay the expression of plasticity genes.

A similarly modest reduction in cortical Otx2 content occurred after anti-Otx2 scFv expression was induced in the adult choroid plexus. Nonetheless, the level of reduction is comparable to what was previously obtained when Otx2 expression was knocked down in the adult choroid plexus, which resulted in the reopening of V1 binocular plasticity [START_REF] Spatazza | Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity[END_REF]. This suggests that the antibody complexes most of the extracellular transcription factor in the CSF. The remaining Otx2 still present in PV cells after 2 weeks either reflects the stability of protein present before scFv expression or the existence of sources other than the choroid plexus, in particular the eye [START_REF] Sugiyama | Experience-dependent transfer of Otx2 homeoprotein into the visual cortex activates postnatal plasticity[END_REF][START_REF] Samuel | Otx2 ChIP-seq reveals unique and redundant functions in the mature mouse retina[END_REF] and the pineal gland, another established site of Otx2 expression [START_REF] Nishida | Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development[END_REF]. Regardless, this modest reduction is enough to induce a dramatic re-expression of plasticity genes in the adult and an enhancement of visual cortex plasticity. The plasticity observed after 4 d of MD in P105 mice corresponds to an increase in ipsilateral visual acuity with no change to the deprived contralateral eye. Indeed, Sato & Stryker [START_REF] Sato | Distinctive features of adult ocular dominance plasticity[END_REF] previously showed that adult plasticity mainly impacts the response of the non-deprived eye. Furthermore, they showed that 7 d of MD were necessary to induce moderate plasticity in the cortex of adult (P90) mice, while only 4 d were required to induce strong plasticity in juvenile (P30) mice. Thus, Otx2 capture by the scFv antibody in adult (P105) mice accelerates ocular dominance plasticity.

Intercellular transfer has been observed in vivo and/or in vitro for many HPs and the conservation of the transfer sequences in the DNA-binding domain suggests that this is a general property of this transcription factor family. Our single-chain antibody strategy brings clear genetic evidence for Otx2 signaling and will be generally useful to verify HP transport in vivo and to analyze the roles of this novel mode of signal transduction. This same strategy could indeed be applied to the neutralization of any signaling entity within and beyond the nervous system.

This report demonstrates that the extracellular neutralization of Otx2 by the inducible expression of an anti-Otx2 single-chain antibody leads to a reduction in Otx2 capture by PV cells and either delays their maturation or "rejuvenates" them depending on when the antibody is expressed. In both cases, several genes associated with a plastic state are regulated in the expected direction. This provides the genetic evidence for a novel signaling mechanism operating through homeoprotein transfer in the mouse.

Materials and Methods

Ethics statement

All animal procedures, including housing, were carried out in accordance with the recommendations of the European Economic Community (86/609/EEC), the French National Committee (87/848) and French bylaws (AGRG1240332A / AGRG1238724A / AGRG1238767A / AGRG1238729A / AGRG1238753A). For surgical procedures, animals were anesthetized with Xylazine (Rompun 2%, 5mg/kg) and Ketamine (Imalgene 500, 80mg/kg). For biochemical analysis, mice were sacrificed by cervical elongation. This research (project no. 00704.02) was approved by Ethics committee n°59 of the French Ministry for Research and Higher Education.

scFv construction

Hybridoma expressing monoclonal antibodies (CD4 and CD7) against Otx2 were generated, and CD7 hybridoma mRNAs were used to construct scFv minigenes as described in [START_REF] Barbas | Phage Display, a Laboratory Manual[END_REF]. CD7-scFv coding sequence was subcloned as described in [START_REF] Lullo | Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube[END_REF], with an N-terminal signal sequence and C-terminal 6xMyc and His tags followed by a skipping peptide (P2A)-eGFP cassette for expression of either a fusion protein (scFv-myc-his-P2A-GFP) or two separate proteins, depending on P2A peptide efficiency. Anti-Pax6 scFv [START_REF] Lesaffre | Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish[END_REF] was transferred into the same final vector.

Animals

scFvOtx2 and scFvPax6 knock-in mouse lines were generated by the Institut Clinique de la Souris (Strasbourg, France) by transgenesis with targeted insertion in the Rosa26 allele. PV-Cre mice were obtained from The Jackson Laboratory (stock 8069).

Cell culture of ear fibroblasts

Fibroblasts were generated from ear biopsies from adult scFvPax6 tg/o and scFvOtx2 tg/o mice and treated with 10 μM cell-permeable Cre (Cre-TAT, production described in [START_REF] Spatazza | Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity[END_REF]) in DMEM [START_REF] Joliot | Transduction peptides: from technology to physiology[END_REF]. Cells and culture media were collected 48 h later and processed for Western blot analysis. Conditioned media were also used as primary antibodies on Western blots.

Cre-TAT protein injection

Stereotaxic injection (bregma: x = -0.58 mm, y = ±1.28 mm, z = 2 mm) of Cre-TAT in 15% DMSO into the lateral ventricles of anesthetized mice was performed with a Hamilton syringe at a rate of 0.2 μl/min. Five or fifteen days after injection, mice were processed for cerebrospinal fluid sampling (described in [START_REF] Spatazza | Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity[END_REF]), cortical layer dissection or immunohistochemistry.

Layer IV dissection

The dissection of tissue enriched in cortical layer IV was performed in ice cold PBS with micro scalpels. Visual cortex areas were first excised from both brain hemispheres and then cut coronal to produce two thin rectangular strips per hemisphere. These strips were laid on their sides to expose the cortical layers. A lengthwise cut in two equal parts gave the supragranular layer, which was then cut lengthwise in two equal parts to provide enriched layer IV.

Immunoprecipitation

Whole cerebellum was dissected and suspended in immunoprecipitation lysis buffer (20 mM Tris pH8, 120 mM NaCl, 1% NP-40, 1 mM MgCl2, 5% glycerol, Benzonase nuclease and protease inhibitors). Samples were centrifuged (10 min, 20 000 g) at 4°C and the supernatant was incubated overnight at 4°C with rotation with anti-Otx2-coupled Dynabeads (using anti-Otx2 rabbit polyclonal, Abcam ab21990) or with anti-GFP-coupled magnetic beads (Chromotek). The beads were washed with lysis buffer and with 1 M urea before Western blot analysis.

Western blot

Protein extracts were separated on NuPAGE 4-12% Bis-Tris pre-cast gels (Invitrogen) for 1 h at 200 V and transferred onto a methanol-activated PVDF membrane at 400 mA for 1 h. The following primary antibodies were used: anti-Myc (rabbit polyclonal, 1/4000, Sigma-Aldrich C3956), anti-Otx2 (rabbit monoclonal, 1/2000, Abcam ab92326), anti-RhoA (mouse monoclonal, 1/200, Santa Cruz sc418). Membranes were imaged with a LAS-4000 gel imager (Fujifilm) and quantified by densitometry with ImageJ.

Immunohistochemistry

Mice were perfused transcardially with PBS followed by 4% paraformaldehyde prepared in PBS. Brains were post-fixed 1 h at 4°C and immunohistochemistry was performed on cryosections (20 μm) encompassing the entire visual cortex. Primary antibodies included anti-Myc (rabbit, 1/400, Sigma-Aldrich C3956), anti-Otx2 (mouse monoclonal, in house), anti-PV (rabbit, 1/500, Swant PV25), anti-CR (mouse monoclonal, 1/500, Swant), anti-GABA (rabbit, 1/ 300, Sigma), and anti-cFos (rabbit monoclonal, 1/300, Cell Signaling). Secondary antibodies were Alexa Fluor-conjugated (Molecular Probes). Biotinylated WFA (1/100, Sigma-Aldrich L1516) with Alexa Fluor-conjugated streptavidin was used to reveal perineuronal nets. Sections were mounted in Fluoromount (Southern Biotech). Images were acquired with a Leica SP5 confocal microscope and analyzed with ImageJ.

In situ hybridization

In situ hybridization on cryosections (20 μm) were performed as previously described [START_REF] Nardo | Dendritic localization and activity-dependent translation of Engrailed1 transcription factor[END_REF]. Briefly, hybridization was carried out overnight at 70°C with DIG-labeled probes. Sections were washed and incubated with alkaline phosphatase-conjugated anti-DIG (1/2000, Roche) overnight at 4°C. Sections were then washed and the color reaction was carried out at room temperature. Reaction was stopped by PBS washes and sections were mounted with Fluoromount (Southern Biotech). Mosaïc images (10x magnification) were acquired with a Nikon 90i microscope.

Quantitative PCR

Layer IV of visual cortex was manually dissected and RNA was extracted using the RNeasy Lipid Tissue Mini (Qiagen) according to manufacturer's instructions. cDNA was synthesized from 500 ng of total RNA with the QuantiTect Reverse Transcription kit (Qiagen). For realtime PCR, triplicates were analyzed with a LightCycler 480 II (Roche). Gene-to-GAPDH ratios were determined using the 2 -∆∆Ct method.

Monocular deprivation and surgery for optical imaging

For monocular deprivation, mice were anesthetized prior to suturing of the left eye as described in [START_REF] Gordon | Experience-dependent plasticity of binocular responses in the primary visual cortex of the mouse[END_REF]. Animals were checked daily to ensure sutures remained intact. The eye was opened immediately before recording.

For optical imaging, mice were anesthetized with urethane (1.2 g/kg, i.p.) and a sedative (chlorprothixene, 8 mg/kg i.m.). Atropine (0.1 mg/kg) and dexamethasone (2 mg/kg) were injected subcutaneously and body temperature was maintained at 37°C. Mice were placed in a stereotaxic instrument and craniotomy was made over the primary visual cortex in the hemisphere contralateral to the deprived eye. The exposed area was covered by 2.5% agarose and a glass coverslip.

Visual stimulation

Visual stimuli were randomly displayed on a 21" LCD monitor located 20 cm in front of the animal. Coordinates of the monitor were reprogrammed in order to maintain spatial and temporal frequencies constant across the visual field's eccentricities (See examples in Fig 5C, [START_REF] Yokoo | An optimization approach to signal extraction from noisy multivariate data[END_REF]). Each stimulus consisted of oriented sine-wave gratings drifting in one direction perpendicular to the orientation at a temporal frequency of 1.5 Hz. Ten spatial frequencies (SFs) were tested, ranging from 0.0303 to 1 cycle per degree (cpd) in a logarithmic scale. Monocular stimuli were displayed for 5s. A grey screen was presented between trials for 10s. Each SF was displayed 16 times with a different orientation/direction.

Intrinsic signal optical imaging

Optical images of intrinsic signals were recorded with a Dalsa 1M60 CCD camera controlled with an Imager 3001 system (Optical Imaging Ltd). Images were acquired after a 2x2 pixels spatial binning with a resolution of * 10.5 μm/pixel using a 135x50 mm tandem lens (Nikon) configuration. The focal plane was set 400 μm below the cortical surface, and intrinsic signals were acquired with a 700 nm illumination wavelength. The size of the recorded region was * 2.5 x 3.6 mm. Five frames of 1s duration each were stored right after the stimulus onset.

For data analysis, the last three recorded frames, which correspond to the strongest intrinsic signal, were averaged and divided by the first recorded frame. The generalized indicator function method was applied to identify the stimulus-related activity patterns [START_REF] Yokoo | An optimization approach to signal extraction from noisy multivariate data[END_REF]. An additional low-pass filter with a Gaussian kernel of 3 pixels SD was applied to smooth the data. The ipsilateral eye was stimulated, the area of interest was delineated over the primary visual cortex and the SF tuning curve in the binocular zone was calculated by averaging the response for each SF stimulus. This curve was least-square fitted to extract visual acuity. For each eye the response integral (trapezoidal approximation) elicited by SFs was calculated. Ocular dominance values ranged between -1 and 1 (ipsilateral and contralateral eye response, respectively) and calculated for each SF threshold with the contrast formula from the response integral of each eye.

Statistical analysis

Statistical analysis was performed with Prism software (GraphPad). Pairwise comparison was done using Student t-test.

II. Otx2 in anxiety-related behaviors INTRODUCTION

During postnatal development, the homeoprotein Otx2 is exported from the choroid plexus, one of the major source of Otx2 in the brain, to the primary visual cortex to trigger the maturation of the fast-spiking GABAergic parvalbumin-expressing (PV) interneurons (Sugiyama et al, 2008;Spatazza et al. 2013). Thanks to a glycosaminoglycan-recognition motif, rich in arginin (R) and lysine (K) aminoacids, Otx2 binds the perineuronal nets (PNNs) surrounding PV cells (PV-cells) and is internalized specifically by these cells (Beurdeley at al, 2012). PV-cell maturation is required for the onset of the critical period (CP) for binocular vision between postnatal day 20 (P20) and P40 in the mouse. Infusing Otx2 before P20 in the primary visual cortex, or blocking its transfer during postnatal development, induces a premature or a delayed CP, respectively. Beyond this window, shift of ocular dominance is not possible, unless Otx2 is brought back at a "juvenile level" in the cortex, restoring a plasticity event in the adult (Spatazza et al., 2013).

PV-cell and E/I balance defects have been associated to the physiopathology of neurodevelopmental disorders (see review Marin, 2012). Indeed, a decrease in many factors participating in GABA signaling has been observed in PV-and somatostatin (SOM)expressing cells from the prefrontal and cingulate cortices, as well as in the amygdala, of major-depressive patients (Luscher and Fuchs, 2015). In schizophrenia (SZ) patients, PVcell activity is reduced in the dorsolateral frontal cortex due to decreased expression of GAD67, in parallel with abnormal gamma-frequency oscillations. SZ patients also present a decrease in PNN density, with a deregulation of CSPG expression, in the entorhinal and prefrontal cortices and amygdala [START_REF] Pantazopoulos | Extracellular matrix-glial abnormalities in the amygdala and entorhinal cortex of subjects diagnosed with schizophrenia[END_REF]. It is also of note that the number of PV-cell is decreased in the neocortex of many mouse models based on the identification of genes, loci for autism susceptibility or prenatal perturbation found in syndromes associated with autistic-like behavior [START_REF] Gogolla | Common circuit defect of excitatory-inhibitory balance in mouse models of autism[END_REF]. Similarly, several studies have demonstrated the implication of PV-cells and SOM-cells in the physiopathology of Rett Syndrome (Tomassy, J Neurochem 2014;Ito-Ishida Neuron 2014).

The symptoms of psychiatric illnesses often emerge between peri-adolescence and adulthood when the GABAergic system matures in the prefrontal cortex (Paus et al., 2010), leading to the hypothesis that several late appearing psychiatric diseases may be of neurodevelopmental origin and related to an abnormal CP regulation in specific regions of the cortex. Otx2 is present in PV-cells of most if not all cortical and subcortical regions of the brain (Spatazza et al., 2013). Its established role in CP for binocular vision, raises the possibility that it acts as a general CP regulator, including in regions of the cortex associated with complex behaviors.. In this study, it is shown that Otx2 heterozygous mice (Otx2 +/GFP ) display hypoanxious behaviors in the Elevated-Plus maze and the Light-Dark box tests. This behavior appears during postnatal development and persist in the adult. Otx2 +/GFP mice display no change in dopamine neurons number in the Ventral Tegmental Area (VTA) where Otx2 is expressed and show normal dopamine and serotonin cortical content and metabolism. This strongly suggests a role of non-cell autonomous Otx2 activity in the regulation of complex behaviors in the mouse.

RESULTS

1.Otx2 heterozygote mice present low anxiety-related behaviors

Three month old Otx2 +/GFP mice with one allele of Otx2 replaced by the fluorescent protein GFP (A. Simeone) and their wild-type sibling were analyzed for several behaviors (figure 16A). No difference was found between the two genotypes in locomotor activity (openfield), locomotor coordination (accelerated rotarod) and short-term memory (Y-maze). The tailsuspension and the forced swimming tests did not show any difference in depressive-like behaviors. In contrast, a strong hypoanxiety phenotype is associated with the loss of one Otx2 allele as demonstrated in the light-dark box (LDB) and the elevated-plus maze (EPM) tests (figure 16B). Otx2 +/GFP mice spent more time in the lighted compartment of the LDB (132.02 ± 17.23 vs 60.54 ± 9.81; F(1.59)=12.79 p<0.001) with no significant sex x genotype effect (F=1.59 p>0.05) and on the open arms of the EPM (56.02 ± 13.63 vs 10.79 ± 4.02; F(1.41)=7.18 p=0.0105) with no sex x genotype effect. No deficient behavior in Otx2 +/GFP mice capacity to gate sensory motor information was found with the Prepulse Inhibition test, but males displayed an elevated amplitude of startle compared to Otx2 +/GFP females and males (data not shown).

In the Otx2AA knock in mutant mouse, an arginine (R) and a lysin (K) have been replaced by the doublet of Alanine (A) within a domain allowing the recognition of PV-cells in layer III/IV of the cortex (Beurdeley et al., 2012). In Otx2 +/AA mice 75% of Otx2 transcriptional activity is preserved [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF] but the homeoprotein is not specifically targeted to PV-cells.

As a result Otx2AA accumulation in these cells is reduced and CP opening for binocular vision takes place at P100 instead of P20 (Bernard et al., in revision). Otx2 +/AA mice also displayed hypoanxiety behaviors in the LDB and the EPM tests (figure 1A). In the LDB tests Otx2 +/AA mice do not spend more time in the lighted box but they transit more often between the two compartments than their WT littermates (27.47 ± 1.21 vs 21.52 ± 1.85 ; F(1.49)=9.3 p<0.01). In the EPM, Otx2 +/AA mice spend more time on the open arms than the control group (2.,96 ± 3.33 vs 15.01 ± 3.43 ; F(1.49)=4.1 p<0,05). In the openfield, Otx2 +/AA mice have a higher locomotor activity (F(1.49)=16.8 p<0.001); females are more active than males (F(1.49)=9.8 p<0.01) but the heterozygous hyperactivity is similar within a gender as there is no gender x genotype interaction. Also, Otx2 +/AA mice spend less time doing thigmotaxis as they cross more frequently the center zone, as a sign of low anxiety (F(1.49)=4.8 p<0.05).

Here again, females spend more time in the central zone than males (F(1.49)=4.7 p<0.05) even though the difference between Otx2 +/AA and WT does not differ between genders (no interaction gender X genotype). In the other tests Otx2 +/AA mice behave similarly to Otx2 +/GFP and WT mice. It is of note that an interaction period x genotype in the Forced Swimming test is observed revealing that Otx2 +/AA mice are freezing for a longer time than WT siblings during the first half-time of the test (205.05 ± 19.03 vs 148.19 ± 13.36 F(1.47)=5 p<0.05).

Since the phenotypes of the Otx2 +/GFP and Otx2 +/AA converged to demonstrate similar hypoanxious phenotypes, we decided to focus our study on the Otx2 +/GFP genotype and completed this first behavior screening by analyzing Otx2 +/GFP behavior on the EPM at different ages. As shown in figure 16C, the increased time spent in the open arms is already present in Otx2 +/GFP in 1 month-old animals with no evolution at P60, P90 and P120 (no age x genotype interaction).

Otx2GFP/+ mice do not present defects in fear conditioning learning and extinction

Anxiety-related behaviors include the basal anxiety, a state of high arousal and vigilance towards a potential threat, and fear, an active defensive response evoked by a specific acute stimulus. Fear is the result of a process of associative learning followed thanks to a wellestablished fear conditioning paradigm. In this test a conditioned stimulus (CS) is paired with an aversive unconditioned stimulus (here an electric foot-shock) such that the presentation of the CS activates fear responses (measured by the duration of freezing events over the total time period). Given the low anxiety phenotype observed in Otx2 +/GFP mice, we wondered if they presented a decrease in fear memory storage. The mice were assessed in two types of fear conditioning differing by the CS, either the context (figure 17A), or a tone (figure 17B). In both protocols Otx2 +/GFP and WT mice learned similarly to associate the CS to the US as shown by the increased freezing following the CS-US, over time (figure 17). Fear extinction recalls the activation of inhibitory interneurons in the amygdala and synaptic plasticity in the medial prefrontal cortex (mPFC;Myers, Davis Mol.Psy. 2007;Pattwell et al, PNAS 2016).

Thus deficits in GABAergic development in Otx2 +/GFP mice could interfere with the acquisition of fear extinction. To measure fear extinction, following learning in both contextual and tone fear conditioning, mice are repeatedly presented with the initial CS in the absence of the US.

Figures 17 C andD illustrate that Otx2 +/GFP and WT mice present the same extinction rate, strongly suggesting that fear conditioning memory formation and extinction are not modified in Otx2 +/GFP mice.

Otx2GFP/+ hypoanxiety is not a dopaminergic issue

During embryogenesis, Otx2 regulates de proliferation and differentiation of mesencephalic dopaminergic progenitors. After birth, Otx2 expression is maintained in the dopaminergic cells of the ventral tegmental area (VTA;Di Salvio et al., 2010a) and controls the identity of the ventro-medial VTA neuron sub-types [START_REF] Simeone | Otx genes in neurogenesis of mesencephalic dopaminergic neurons[END_REF]. Considering the importance of the VTA dopaminergic control of several limbic areas such as the mPFC, the amygdala, the bed nucleus of the stria terminalis and the nucleus accumbens, it was of interest to investigate its implication in the Otx2 +/GFP phenotype. A first result is that, at 3 months, the number of tyrosine hydroxylase-positive dopaminergic neurons in the VTA is identical in Otx2 +/GFP and WT mice (figure 18B). Secondly, dopaminergic activity was evaluated by measuring the amount of DA and of its main metabolites in four large areas, namely, the anterior cortex comprising the mPFC and the amygdala, the hippocampus, the posterior cortex (which does not receive VTA projections) and the striatum (innervated by dopaminergic axons from the substantia nigra). In addition to DA and its metabolite DOPAC and HVA, we also measured -for control -the amount of serotonin (5HT) and its metabolite 5HIAA. Figure 18A demonstrates the absence of difference between the two phenotypes in DA, 5HT and their metabolite content. These results rule out the idea that VTA dopaminergic activity in Otx2-expressing cells controls the anxiety behaviors in the mouse and support the hypothesis of a non-cell autonomous role of Otx2 in anxiety regulation.

Otx2 is exported from the choroid plexus to regulate anxiety

Otx2 expression in the postnatal brain is restricted to the retina, the superior colliculus, the choroid plexuses (ChP), and the VTA. In addition, non-cell autonomous Otx2 is present in hippocampus CA1, prefrontal cortex and basolateral amygdala (Spatazza et al., 2013), three regions implicated in the regulation of anxiety-related behaviors. Interestingly, both the VTA and the ChP have direct access to these regions and could thus provide them with Otx2.

Therefore we developed gain-and loss-of-function experiments to evaluate the respective contributions of the VTA and ChP, to limbic non-cell autonomous Otx2. AAV5-Otx2 and AAV2-Otx2 were injected in the lateral ventricles or the VTA respectively, of 2 month-old Otx2 +/GFP mice to increase he amount of Otx2 in these structures (AAV5 injected in the ventricles specifically targets the ChPs; Spatazza et al., 2013). In parallel, AAV5-shRNAOtx2 was injected in the lateral ventricles or VTA of WT of 2 month-old mice to decrease the amount of Otx2. Five weeks later, the mice were tested on the EPM in comparison with noninjected mice. Although these experiments need to be completed by increasing the number of mice, the preliminary data shown in figure 19 suggest that hypoanxiety in Otx2 +/GFP may be rescued following AAV5-Otx2 injection in the lateral ventricles but not that of AAV2-Otx2 in the VTA. In contrast, and much to our surprise, the acute Otx2 loss-of-function in the ChP of WT mice (AAV5-shRNAOtx2) considerably reduced (in fact abolished) the time spent on open arms, compared to non-injected mice, suggesting a hyperanxious phenotype. Again, no effect was seen following the injection of the same virus in he VTA. Although we have no explanation for the opposite results observed between the Otx2 +/GFP mice and an acute Otx2 loss of function, these results show that Otx2 levels of expression in the ChP (and not the VTA) control the anxiety-related behaviors.

The discussion of this paper, presented here as a manuscript in preparation, will be developed within the global discussion of the thesis (page 83).

EXPERIMENTAL PROCEDURES

Mice

Otx2 +/GFP and Otx2-AA mouse lines were generated in the laboratory of Antonio Simeone (CEINGE, Naples). In the Otx2 +/GFP mouse, the Otx2 coding sequence and introns are replaced by GFP (Acampora 2009). Otx2 +/GFP males were crossed with B6D2F1 females to obtain Otx2 +/GFP heterozygous mice. In Otx2-AA mouse line, the R( 36)K(37) doublet in Otx2 was mutated into an alanine doublet (Beurdeley, 2012). Otx2 AA/+ males were crossed with B6D2F1 females to obtain Otx2 AA/+ heterozygous mice. All animals have a B6D2 genetic background.

Mice were raised from birth in a 12 hour light/dark cycle with 2 to 5 (males) or 6 (females) animals per cages. Temperature is controlled (21+-3°C), and food and water are provided ad libitum. All surgery and manipulations were carried out in accordance to the recommendations of the European Economic Community (86/609/EEC), the French National Committee (87/848) and French bylaws (AGRG1240332A / AGRG1238724A / AGRG1238767A /AGRG1238729A / AGRG1238753A). Before surgery, mice were anesthetized by intraperitoneal injection of Xylazine (Rompun 2%, 5mg/kg) and Ketamine (Imalgene 500, 80mg/kg).

AAV injection

Three Adeno-Associated Viruses (AAV) were generated by Vector Biolabs: AAV5(CMV)-HAOtx2-2A-mCherry, AAV2(CMV)-Otx2HA-mCherry, AAV5(U6)-shRNAOtx2-GFP.

Stereotaxic injection was performed with a Hamilton serynge at a rate of 0.3µl/min: 2µl/lateral ventricule (bregma: x = ±1.28 mm, y = -0.58 mm, z = 2 mm) and 1µl/side in the ventral tegmental area (bregma: x = ±0.4 mm, y = -3.0 mm, z = -3.3 mm). 3 weeks post-injection mice are tested in the light-dark box, 5 weeks post-injection they were tested on the elevated-plus maze.

Behavior

During the initial experiment, tests were performed on a group of female and male WT mice and Otx2 +/GFP mice in the following order: locomotor activity, Y-maze, elevated-plus maze, light-dark box, rotarod, tail-suspension, prepulse inhibition, forced swimming test. Mice were taken at P90 for the first test and achieved P120 at the last test. Except for rotarod, tailsuspension and prepulse inhibition tests, behavior is registered using a semi-automated infrared system (Viewpoint, Lyon). Experiments with Otx2 +/AA mice followed the same procedures.

Locomotor activity

Spontaneous locomotor activity is measured during 60 min in an openfield of 40 x 40 x 30 cm enlighted at 50 lux. A virtual squared area is designed at 8cm from the borders. Time spent in this central zone is taken as a measure of reduced anxiety.

Y maze

Short-term spatial memory is evaluated by the spontaneous alternative exploration of the Ymaze during 10 min. The maze consist of 3 allies diverging at 120° from one another, 40 cm long, 15 cm width, with black 30 cm high Perspex borders decorated differently in each arm, enlighted at 50 lux. The mouse is put down at the tip of an arm and the time it visits each arm sequentially is counted as a memory performance.

dB) with interval of 70 ms. The mouse is settled in a see-through Plexiglas cylinder fixed on a platform sensitive to movements. Tones are emitted randomly, every 15-20 s with a speaker at 26 cm above the animal. During the protocol a background sound is maintained at 68 dB.

The prepulse inhibition percentage is measured by the amplitude of startle following, or not, a prepulse.

Fear conditioning

Fear conditioning and extinction tests were performed in a chamber equipped with an infrared camera, a speaker and a grid floor connected to a shock generator (Med Associates). For contextual fear conditioning: mice (WT=8/ Otx2 +/GFP = 11) were placed in an enlighted chamber, previously cleaned with acetic acid 1% (day 1). After 180s of habituation, they were exposed to 5 shocks (0.6 mA, 1 s) at 120 s intervals. During contextual extinction (day 2) mice were placed in the same context during 30 min and freezing measured every 120 s after a first period of habituation of 180s.

For tone conditioning: mice (WT=6/ Otx2 +/GFP = 4) were placed in an enlighted chamber for 5 min in the morning for acclimatization (day 1). In the afternoon, after 180s of habituation in the same chamber they were exposed to 30s tones (82 dB) co-terminating with a foot shock mice in the Y-maze proves their ability to correctly localize themselves in space. It is thus safe to conclude that hypoanxiety is a characteristic of Otx2 GFP/+ and Otx2 +/AA mice.

The latter comparison between genotypes allows us to propose a dose-dependent role of Otx2 in anxiety-related behaviors. Indeed, as demonstrated earlier [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF],

Otx2AA transcriptional activity is 75% that of Otx2. If we add the fact that Otx2AA is not internalized normally by PV-cells, it is easy to calculate that the level of Otx2 activity in Otx2 +/AA ranks between those of WT and Otx2GFP/+ mice, paralleling the anxiety deficit. This observation is in line with the pre-cited work where retinal structure and visual behaviour analyses of Otx2 +/+ , Otx2 GFP/+ , Otx2 +/AA and Otx2 AA/AA demonstrated that dose changes in the range of 10-20% have major phenotypic consequences [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF].

This conclusion is further supported by the RNA sequencing data (A. Prochiantz lab, unpublished) on layer IV of V1 dissected from Otx2 GFP/+ and Otx2 +/AA mice at P30 and P100.

In WT mice, plasticity genes such as Arc, Egr4 or Gadd45b are up-regulated at the peak of CP for binocular vision at P30 and down-regulated in the adult at P100. In contrast the same genes are poorly expressed at P30 and up-regulated at P100 in Otx2 P45 in the rat basolateral amygdala coincident with a switch in fear memory erasure (Gogolla et al., 2009a). Experiments of fear extinction on juvenile P16 and P23 rats revealed a critical window when fear memories can be erased before PNN assembly (at P23) prevents such erasure and forces the system to contextualize the memory by a second learning episode, as in adult rats. In line with this study, GABA-A receptor alpha1 subunit expression is initiated between P14 and P21 and gradually increases to a maturation state of inhibitory synaptic transmission by P30 in the rat BLA (Ehrlich, J Neurophysiol 2013).

Converging evidence from human and rodent studies show that a correct E/I balance in the mPFC is required for the regulation of fear expression and extinction. They show that a potentiation of prelimbic (PL) mPFC excitatory synapses occurs in the amygdala after contextual fear acquisition. Moreover, after extinction learning, infralimbic (IL) mPFC suppresses amygdala activity in parallel with the inhibition by hippocampal projections of PL sustained activity, dampening fear expression. This hippocampus-mPFC-amygdala circuit is common to contextual fear conditioning and cued fear extinction as both require spatial information in the environment. The dynamic regulation of fear expression by the mPFC is absent in adolescent mice at P29 [START_REF] Pattwell | Altered fear learning across development in both mouse and human[END_REF]. This coincides with the slow development of cortical GABAergic transmission during postnatla development: at P30 there is an increase of pruning on pyramidal neurons in PL happens at P30, along with a peak of amygdalo-prefrontal and ventral hippocampal-prefrontal connectivities formation [START_REF] Pattwell | Dynamic changes in neural circuitry during adolescence are associated with persistent attenuation of fear memories[END_REF], supporting the idea that mPFC critical maturation is crucial to fear expression.

Very recently Guirado et al demonstrated that the BLA and ventral hippocampus compete onto mPFC neurons to shape its connectivity. This competition is similar to that shaping the cortical connections coming from the two eyes during the critical period for binocular vision [START_REF] Guirado | Evidence for Competition for Target Innervation in the Medial Prefrontal Cortex[END_REF].

All in all, the existence of a critical period for fear conditioning during post-natal development is a very likely possibility. Interestingly, Otx2 regulates the maturation of PV-cells in mPFC during the CP for acoustic preference between P15 and P25 (Bernard et al., in revision at Mol. Psy.). It is unclear if different mPFC cell populations are involved in the several complex functions controlled at the level of the mPFC, such as acoustic preference, fear expression or decision-making. However, but the role of Otx2 in the maturation of this region lends weight to the idea that Otx2 may regulate the CP for anxiety-related behaviours. As anxiety expression is the product of a number of interconnected regions, it is possible that CP for anxiety integrates multiple information and is more complex than those that have served a experimental models, including CP for binocular vision.

For the Otx2 GFP/+ mouse, we hypothesize that GABAergic interneurons start maturing late but never reach full maturation (at least not before 4 months) and that mice remain plastic, thus incapable of deciphering between negative and positive valences. This situation is in strong contrast with many studies on the role of inhibitory interneurons in the amygdala where knockouts of GABAergic mediators, such as GAD65 or GABA-A receptor alpha1 subunit foster a hyperanxious behaviour in response to amygdala hyperexcitability. Finding the regions implicated in Otx2-regulated anxiety by quantifying PV expression and PNN assembly in the FPC and amygdala of Otx2 GFP/+ mice would support the role of Otx2 in the maturation of PV-cells responsible for anxiety. In addition, it may shed light on the precise structures involved in anxiety control.

In the past decade, fear mechanisms have been thoroughly investigated, much more than those controlling anxiety. Strikingly, Otx2 GFP/+ mice do not present any defect in fear learning or extinction. It would be interesting to complete this analysis by observing fear extinction retention more than 7 days post-conditioning in order to explore Otx2GFP/+ ability to store fear memories on a long-term. It must be kept in mind that fear conditioning is associated with pain (at least in these experiments) and on active learning. This makes it is conceptually very different from basal anxiety evaluated in the EPM or light-dark box. Rodent studies have demonstrated a dissociation between cued fear learning and contextual fear learning, the first happening earlier (<P24) than the second. This suggests that multiple plasticity windows occur for this complex behaviour. Whatever the explanation, the fact that our mutant

Otx2 GFP/+ mice are hypoanxious but do not differ from WT sibling when it comes to fear conditioning/extinction strongly suggest that fear and anxiety circuits do not totally overlap and that the mechanisms underlying the maturation of anxiety behaviours differs from those regulating fear.

II. Non-cell autonomous function of Otx2

The fact that Otx2 +/AA mice are also hypoanxious suggests that Otx2 regulates anxiety in a non-cell autonomous manner. We cannot totally preclude however that anxiety regulation might in part be due to cell-autonomous transcriptional defects as Otx2AA activity is reduced by 30%, compared to that of Otx2 [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF]. A better tool to study non-cell autonomous functions only is provided by the single-chain antibody (scFv) mouse. Designed PV::Cre and PV::Cre x scFvPax6 tg/0 mice. This modest reduction t can be explained two nonexclusive arguments. First the scFv-Otx2 interaction in the extracellular space is weaker than the PNN-Otx2 interaction or a proportion of scFv is not directly accessible to Otx2 thus leading to a incomplete blockade of the HP. Secondly, PV expression starting at P10 the expression of the scFv (Cre is under the PV promoter) is still too limited at P14 to fully block Otx2 capture by many PV-cells. However, a 20% reduction in the maturation state is enough to delay visual cortical plasticity as indicated by the low expression of four major plasticity genes.

When the antibody is expressed in the adult ChP, we observe the same modest decrease in the number of cells expressing PV and Otx2 or stained with WFA. This again may have several explanation, including possible other sources of Otx2 (for example the pineal gland)

or, and very likely so given previous experiments (Spatazza et al., 2013), the stability of Otx2 within PV-cells. Whatsoever, a 20% reduction of Otx2 cortical content is sufficient to activate plasticity in the adult (Beurdeley et al., 2012;Spatazza et al., 2013) and forcing scFvOtx2 secretion by the adult ChP reduced Otx2 cortical concentration in PV-cells of V1, induced the re-expression of plasticity genes and reopened physiological plasticity in the adult. As described previously, adult plasticity enhances visual acuity of the non-deprived eye and has little effect, at least within a four-day period, on the deprived eye. This scFv model provides the first genetic evidence for Otx2 paracrine signalling. It will be of great interest to recombine scFvOtx2 tg/0 mice in different regions of the limbic system to identify Otx2 target regions regulating anxiety-related behaviors.

One of the major sources of Otx2 in the cerebral cortex is the choroid plexus suggesting that this structure regulates anxiety through Otx2 secretion and transport to PV-cells in anxietycoding cortical region. As the VTA influences directly the BLA, the hippocampus and the mPFC, among other structures, and also expresses Otx2, it is possible that it provides Otx2 to these regions through axonal transport and synaptic release. The number of mice is still insufficient to conclude with certainty but the preliminary results obtained after addressing AAV5-Otx2HA to the choroid plexus or VTA of Otx2 GFP/+ mice (figure 4) suggests that Otx2 comes from the choroid plexus. As for the loss of function experiments using shOtx2expressing AAVs, they suggest that modulating Otx2 content in the VTA or the choroid plexus does not modify anxiety in WT mice.

In addition to a possible direct non-cell autonomous activity of Otx2 made in the VTA, just discussed and unlikely as it seems, Otx2 may have an indirect effect since VTA dopaminergic neurons project onto limbic structures, including the prefrontal cortex and amygdala, and that any change in the VTA physiology may impact this pathway. This possible effect through the regulation of DA metabolism at the level of the cortex can probably be ignored since our data comparing Otx2 GFP/+ and WT mice do not indicate any significant difference in the content of DA or of its metabolites in any of the tested regions, including the cortex, the hippocampus, the amygdala and the striatum (figure 3).

Similarly to what was discussed for the visual cortex, which receives Otx2 from the ChP (Spatazza et al., 2013) but could also import the HP from the eye (Sugiyama et al., 2008) or the pineal gland, one could hypothesize that anxiety circuits import Otx2 from multiple sources, including areas sensing changes in the external and internal environment. An interesting possibility is represented by the hypothalamus that participates in the control of sexual hormones levels, a key role indeed during adolescence (Holder and Blaustein, 2013).

This structure is a possible source for Otx2 as it expresses this HP at postnatal stages and is in close relation with the amygdala. The hypothalamus is also highly responsive to stress and sensitive to circadian rhythms such as the BNST, one of its projecting target. In this context, it can be recalled that clock genes are involved in CP timing [START_REF] Kobayashi | Clock genes control cortical critical period timing[END_REF] and that the pineal gland, which is highly sensitive to the circadian rhythm synthesizes high amounts of Otx2. A last interesting possible source is the superior colliculus where it seem that a group of cells expressing parvalbumin can trigger fear expression in the amygdala (Shang et al., 2015).

This work proposes that Otx2 plays a role in the regulation of anxiety, a complex social behaviour. Discovering the regions involved in this regulation would be of great interest to understand the mechanisms of action of the HP but also to better identify the many pathways involved in this behaviour and which poorly defined. This extension of Otx2 functions to complex behaviours may be of some therapeutic interest for several psychiatric diseases such as anxiety-disorders, autism and schizophrenia. Indeed, the work done in the laboratory has demonstrated that, in the case of experimentally-induced amblyopia in the mouse, binocular vision can be restored in the adult. It remains to be seen if this can be generalized to other, and more complex, neurodevelopmental diseases.

I. GENETIC EVIDENCE FOR A NON-CELL AUTONOMOUS HOMEOPROTEIN SIGNALLING

I.1 The single chain antibody approach

As already developed in the general introduction, direct non-cell autonomous signalling by homeoproteins (HPs) has been highly debated. Since the first proposal of the existence of such an unsuspected signalling pathway in animals, the evidence has accumulated in support of the hypothesis, which has been demonstrated in many systems, in vitro and in vivo. However, it must be understood that only the genetic evidence is final. This evidence was a real challenge to provide, due to the fact that, as recalled in figure 20, the two transfer sequences are in the homeodomain. This meant that any mutation affecting the transfer sequences could potentially modify HP cell autonomous properties precluding the generation of knock-in mouse models with mutations in the transfer sequences. This has led us to develop the single chain antibody (scFv) approach, illustrated in figure 21 and at the core of the first article (result section).

Indeed in such an experimental paradigm the protein is blocked in the extracellular space but its cell-autonomous activities are not modified. The model had already been tested by injection of a scFv in the fish embryo for Pax6 (Lesaffre et al., 2007), by electroporation in the frog and chick for Engrailed and Pax6 (Wizenmann et al., 2009;Di Lullo et al., 2011) and by transgenesis in the fly for Engrailed (Layalle et al., 2013), but never in a mamal and never for Otx2. Although the conservation of the transfer sequences is such that it is quite likely that most HPs can do it, it must be clear that we have presently shown it with certainty in vivo for only 3

HPs out of 250, or so. Indeed, one could produce as many scFv mouse lines as wished, based on the same principle. A more parsimonious approach would be to produce an anti-tag mouse (anti-myc scFv, for example) and to genetically tag any desired HP. Indeed this approach could be generalized to any type of signalling entity, including, classical growth factor or growth factor receptor. Whatsoever, we think that the demonstration presented in this thesis [START_REF] Bernard | A Mouse Model for Conditional Secretion of Specific Single-Chain Antibodies Provides Genetic Evidence for Regulation of Cortical Plasticity by a Non-cell Autonomous Homeoprotein Transcription Factor[END_REF] represents a turning point in the still young domain of HP signalling.

One possible pitfall in this approach is when the antibody is expressed by cells that also produce the targeted HP. Indead, it is always possible that part of the antibody escapes the secretion pathway, thus neutralizing the cell autonomous HP. This danger is minimized by the fact that scFvs are very sensitive to the reductive milieu of the cytoplasm that reduces the S-S bounds indispensible for epitope recognition (Lesaffre et al., 2007). But minimized as it What is known and what remains to be analysed concerning signalling is summarized in figure 23. The hypothetical scheme is as follows. First the HP recognizes a complex sugar that provides the specificity for cell addressing (Beurdeley et al., 2012). Second, the protein internalized goes to the nucleus and, in cooperation with other pathways, regulates transcription and also the epigenetic state of the chromatin, at least for Engrailed [START_REF] Rekaik | Engrailed Homeoprotein Protects Mesencephalic Dopaminergic Neurons from Oxidative Stress[END_REF]. Third, and as shown in the case of Engrailed but not generalized to other HPs, the HP regulates local translation [START_REF] Brunet | The transcription factor Engrailed-2 guides retinal axons[END_REF]; many of the translated messenger RNAs encode mitochondrial proteins leading to an up-regulation of ATP synthesis (Wizenmann et al., 2009[START_REF] Stettler | Engrailed homeoprotein recruits the adenosine A1 receptor to potentiate ephrin A5 function in retinal growth cones[END_REF]. In as much as the present work is concerned, the co-signalling aspect is particularly important when one considers that in the visual cortex, the same homeoprotein Otx2 opens plasticity at P20 and closes it at P40 (figure 24).

Figure 24. A two-threshold model for Otx2 regulation of cortical plasticity

This means that the same protein can have, depending on its concentration within PV-cells, two opposite effects. We speculate that the genes regulated differ at the two concentrations and it is tempting to propose that this is due to the existence of distinct co-signalling pathways. An exciting possibility would be that it co-signals with Brain Derived Neurotrophic Factor (BDNF; [START_REF] Hanover | Brain-derived neurotrophic factor overexpression induces precocious critical period in mouse visual cortex[END_REF]) at P20 and with Sema3A at P40 [START_REF] Vo | The chemorepulsive axon guidance protein semaphorin3A is a constituent of perineuronal nets in the adult rodent brain[END_REF], leading to the expression of different target genes. This obviously needs to be further explored.

The same figure suggests that the protein, which is ubiquitously expressed by ChPs (or the pineal gland) and thus present in the extracellular environment, starts to be internalized only after the sensory stimulation (eyes open at P14 in the mouse). Based on dark-reared experiments (Sugiyama, 2008), it seems that this sensory signal, of an unknown nature, triggers PNN assembly thus initiating Otx2 internalization. Because Otx2 further enhances PNN assembly, this may initiate a virtuous circle whereby Otx2 maintains PNN levels allowing an ongoing internalization of Otx2. Indeed, removing PNNs in the adult also reopens plasticity [START_REF] Pizzorusso | Reactivation of ocular dominance plasticity in the adult visual cortex[END_REF] and a possible mechanism, based on our own data (Beurdeley et al., 2012) is that this reflects a lower internalization of Otx2, allowing re-entry in the plasticity zone. The fact that initial PNN assembly is activity-dependent also explains why, even though Otx2 seems to be a general regulator of plasticity (Bernard in revision at Molecular Psychiatry), all CPs are not synchronous in the post-natal cerebral cortex. Indeed this underscores the importance of PNNs in the regulation of cerebral cortex plasticity but does not limit PNN functions to Otx2 internalization control. Indeed this structure presents many important function related to plasticity, in particular in the context of PV-cell redox status [START_REF] Do | Targeting Oxidative Stress and Aberrant Critical Period Plasticity in the Developmental Trajectory to Schizophrenia[END_REF].

An issue that has certainly not escaped the attention of the reader is that the model developed in our laboratory means that plasticity is the default state of the cerebral cortex and that the permanent capture of Otx2 by PV-cells is necessary to maintain a non-plastic state. From an evolutionary viewpoint, this probably has the advantage to maintain the longterm engrams associated with cortical memory. Indeed the price to pay is the difficulty to regenerate parts of the circuits following a lesion (as is seen in chicks and fishes), for example a stroke. Indeed being able to release the inhibition, either by blocking Otx2 directly or neutralizing the complex sugars may facilitate recovery. In this context, the experiments done in the laboratory consisting in restoring visual acuity in a mouse model of amblyopia, suggests that this strategy is worth exploring.

III. WHAT ABOUT PSYCHIATRIC DESEASES and COGNITION?

III.1. Critical periods for psychiatric diseases, a hypothesis As already developed in the introduction of this document, several psychiatric disease, in particular schizophrenia are seen as possible neurodevelopmental diseases. This is a physiological and developmental statement independent of whether the causes are genetic, environmental or multifactorial. Figure 25 recalls that many psychiatric disorders emerge during adolescence. suggests that the latter period when inhibition strongly increases is a period of plasticity in the PFC, possibly allowing an adaptation to the dramatic changes taking place during puberty.

An interesting hypothesis is that some psychiatric diseases may find their origin in an incomplete adaptation during this period. This paragraphs owes to the work of the school of Wolf Singer (see Uhlhaas et al, 2009;[START_REF] Prochiantz | Signaling with homeoprotein transcription factors in development and throughout adulthood[END_REF]. Figure 27 illustrates in the prefrontal cortex the presence of the gamma-bands created by the high speed oscillations (40 to 80 hertz) resulting from the interaction between PV-cells and pyramidal cells, after the PV-cells have matured and reached their full inhibitory power.

Gamma bands are highly activated during a cognitive tasks such as recognizing a contrasted face in an upside down position (Mooney face, figure 27). This recognition takes between 400 and 1000 msec. It is very rapid in early adolescence but deteriorates in late adolescence before going back to normal in the adult (bottom middle part). This period of deterioration is marked by the decrease in gamma-band spectral power suggests that circuit remodelling accompanies this period spanning between early adolescence (blue curve) and late adolescent (grey curve). Indeed everything becomes stabilized in the adult (black curve).

CONCLUSION

Taking the most complicated disease, schizophrenia, as an example, I would like to briefly discuss the idea that there is no animal model for complex disease. Indeed, transgenic mouse lines designed with gene mutations encountered in some forms of the human disease, will tell us a lot about the function of this gene and shed some light on the human disease. However, we cannot call it a mouse model for schizophrenia.

In contrast, if we speculate that schizophrenia is a critical period disease, then understanding, even in the mouse, the mechanisms involved in the opening and closure of a critical period may be very instructive. Of course, the human cortex differs dramatically from that of a mouse, but it is constructed on the same principles and it is tempting to believe that learning a very complex social skill does not differ from learning binocular vision. In this perspective, amblyopia in the mouse might be seen as a model to study schizophrenia. Let us dream that the cure of a neurodevelopmental disease, even in the mouse, may help understanding complex human diseases and developing novel categories of "plasticity drugs", that would happily complement the present pharmacology toolbox.

During postnatal development, neural circuits are refined in response to experience to map neural representations of the environment. This plasticity takes place during time windows called critical periods (CP), which differ between each sensory modalities and more complex behaviors. This cortical plasticity remodeling is driven by the maturation of parvalbuminexpressing (PV-cells) fast-spiking GABAergic interneurons, resulting in a shift in the excitation/inhibition balance. In the primary visual cortex, CP for binocular vision is triggered by PV-cells internalization of Otx2 homeoprotein, expressed and secreted by the choroid plexus. This transfer is necessary and sufficient to open visual plasticity at P20 and close it at P40, when the visual cortex looses plasticity; and its constant accumulation throughout adulthood maintains this consolidated non-plastic state. Accordingly, reducing the levels of Otx2 in the adult reopens a period of plasticity.

Many studies report a correlation between defaults of PV-cell maturation and psychiatric disorders such as major depressive disorders, schizophrenia, autism or anxiety disorders, causing an imbalance between excitation and inhibition. These pathologies often manifest during late childhood and adolescence, suggesting a possible pathological derailment in a late plasticity period, possibly a critical period for complex "social behaviors". Considering Otx2 ubiquitous source (the choroid plexus) and its presence in PV-cells throughout the cortex, we hypothesized that Otx2 may regulate other region's plasticity.

We first addressed this question by investigating different behaviors of mice lacking one allele of Otx2 (Otx2 +/GFP ), for which we had observed a delayed CP opening in the visual cortex. Among many traits that were analyzed, we find Otx2 +/GFP mice are less anxious than their wild-type littermates. This hypoanxious phenotype was observed as early as one month old, suggesting that CP for anxiety takes place earlier. Otx2 is expressed in dopaminergic neurons of the ventral tegmental area and in the amygdala, two connected regions that participate in the regulation of anxiety-related behaviors, fostering the idea that Otx2 could act at two levels to regulate the maturation of complex "social" behaviors. Analysis of the dopaminergic metabolism (dopamine and metabolites cerebral dosage and dopaminergic neurons counting in the VTA) showed that the reduced Otx2 levels in the VTA of Otx2 +/GFP mice does not affect cortical dopaminergic levels, suggesting a non-cell autonomous regulation of anxiety by Otx2. Preliminary results of gain-of-function in the choroid plexus or the VTA of Otx2 +/GFP , show a reduction of hypoanxiety towards a wild-type behavior only in mice that were completed in Otx2 in the choroid plexus, lending weight to the idea that Otx2 coming from the choroid plexus, and not the VTA, regulates anxiety-related behaviors.

To have a better insight into the non cell-autonomous mechanisms of Otx2, we have developed transgenic mice secreting a single-chain antibody (scFv) that neutralizes Otx2 in the extracellular space and blocks its signaling activities. Indeed, this was the only strategy possible since the sequences allowing secretion and internalization are in the DNA-binding domain (homeodomain) and cannot be modified without perturbing Otx2 cell-autonomous activities. We tested this tool in the context of CP for binocular vision. In the developing cortex of PV::Cre;scFvOtx2 tg/0 mice, Otx2 neutralization delays the expression of plasticity genes, including Arc and Fos, thus retarding PV-cell maturation and the opening of plasticity. Conversely, the induced secretion of the anti-Otx2 scFv by the adult choroid plexus leads to the reactivation of plasticity genes in the visual cortex, allowing a new event of plasticity (as confirmed by optical imaging recordings on mice challenged by monocular deprivation). This model, tested in the context of anxiety, should allow us to decipher the role of Otx2 in the regulation of plasticity for anxiety.

  ABBREVIATIONS

Figure 4 .

 4 Figure 4. Cortical interneurons synapse onto glutamatergic pyramidal cells. The cortex is organized in 6 layers with different subtypes of GABA-releasing inhibitory interneurons, different by their morphology, connectivity or expression of calcium-binding proteins or neuropeptide (cholecystokinin-expressing cells (CCK), chandelier cell (Ch), double bouquet cell (DB), Cajal-Retzius cell (CRC), neurogliaform neuron (Ng), Martinotti neuron (M)). PV-cells (PV+) specifically contact soma and axon initial segment (ais) of pyramidal neurons (green cell), through the α1-subunit containing GABA-A receptor.

Figure 6 .Figure 7 .

 67 Figure 6. Corticogenesis in the rodent forebrain Interneurons from the lateral and the medial ganglionic eminences (LGE, MGE) in the ventral forebrain (VF) migrate tangentially toward the neocortex in the dorsal telencephalon and express different markers in function of their origin: calretinin (CALR), neuropeptide Y (NPY), somatostatin (SST) or parvalbumin (PVALB). Projection neurons come from the ventricular zone in the dorsal forebrain (DF). (adapted from[START_REF] Rakic | Evolution of the neocortex: a perspective from developmental biology[END_REF]

Figure 8 .Figure 9 .

 89 Figure 8. GABAergic interneurons underlying critical period in mouse visual cortex.Inputs from from the two eyes converge in the binocular zone of the primary visual cortex, via the lateral geniculate nucleus of the thalamus (LGN). Large basket parvalbumin (PV) cells activate the critical period for binocular vision, via the alpha1 subunit of GABA-A receptors.Other subtypes of interneurons (cholecystokinin CCK, calretinin CR, somatostatin SOM) also contact the pyramidal cells via other subtypes of GABA-A receptors.[START_REF] Sugiyama | From brain formation to plasticity: insights on Otx2 homeoprotein[END_REF]

Figure 10 .

 10 Figure 10. Molecular actors controlling onset and closure of a critical period for binocular vision Early factors prevents precocious onset of CP (« OFF») during the precritical period. Then, factors « ON » promote PV maturation, triggering CP onset. Progressively a balance between excitatory circuits (E) and inhibitory circuits (I) is achieved, resulting from the intervention of neuromodulators (CAMKII, ERK…). Finally molecular brakes accumulate

Figure 11 .

 11 Figure 11. Otx2 specifically enters PNN-surrounded PV-cell and regulates their maturation (A) distribution of Otx2 (red) and PV (green) in the layers II-IV of V1 before (Pre-CP) and during (CP) critical period (adapted from Sugyama 2008). (B) Otx2 (green) is present preferentially in cells surrounded by PNNs (WFA) in the layer IV of V1.

Figure 12 .

 12 Figure 12. Reversible control of Otx2-dependent PV cell maturation by RK peptide Immunostaining of Otx2 (green; B,F), PNNs by WFA lectin (red; C,G) and PV (blue; D,H) in layers II-IV of V1 in untreated (upper line) vs RK-peptide (white) infused mice (lower line ; 7 days of infusion in V1).

Figure 13 .

 13 Figure 13. Choroid plexus Otx2 transfers to primary visual cortical PV cells and maintains ther mature phenotype Immunostaining of Otx2 (red ; A,F), PV (green ; B,G) and PNNs by WFA lectin (blue ; C,H) in the layers II-IV of V1, in vehicle (left column) vs CRE-Tat recombinase (right column) ChPinjected Otx2flox/flox mice. (scale bar: 100µm). Following injection in the CSF, Cre-Tat is specifically captured by the choroid plexus where it recombines the floxed Otx2 locus (Adapted from Spatazza et al., 2013a).

  ): Otx2 progressive accumulation by PV-cells starts with the opening of the eyes and reaches a first threshold that opens CP for binocular vision. The level of cortical Otx2 increases during CP and induces PV-cell maturation until a second threshold is achieved and CP closes. The system is no more plastic and Otx2 accumulation in the adult maintains PV-cells in a mature state precluding plasticity. However manipulations allowing a decrease of PV-cell Otx2 content in the adult can transiently or permanently reopen plasticity. Considering this model, playing with Otx2 juvenile cerebral cortical levels permits to modulate natural CP onset and closure. Early Otx2 infusion in V1 increases PV cell markers such as PV, GAD65, Kv3.1b potassium channel and GABA-A alpha1 receptor subunit. The two latter molecules are responsible for the installation of fast-spiking properties of the PV-cells allowing the rapid 40-80 Hertz oscillations of the excitatory-inhibitory circuit. In the adult, lowering Otx2 levels has also considerable physiological effects on cerebral cortex plasticity. For example, blocking

Figure 15 .

 15 Figure 15. Otx2 homeoprotein signalling molecule (A) Common composition of a homeoprotein and the different domains' possible interactors. (B) Differential expression patterns of the HPs Otx2 and Engrailed 1 and 2 during embryogenesis and postnatal life. (Adapted from[START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF]

  part of my project was to develop the scFv tool to study HP non-cell autonomous functions. It is in the latter context that a transgenic mouse line expressing an inducible anti-Otx2 scFv was developed and tested in the visual paradigm, allowing us to genetically prove the role of Otx2 signalling in the regulation of cortical plasticity.RESULTSI. A new tool to study non-cell autonomous Otx2 roleRESEARCH ARTICLE A Mouse Model for Conditional Secretion of Specific Single-Chain Antibodies Provides Genetic Evidence for Regulation of Cortical Plasticity by a Non-cell Autonomous Homeoprotein Transcription Factor Clémence Bernard ☯ , Clémentine Vincent ☯ , Damien Testa ☯ , Eva Bertini, Jérôme Ribot, Ariel A. Di Nardo, Michel Volovitch, Alain Prochiantz* Centre for Interdisciplinary Research in Biology (CIRB), Collège de France, CNRS UMR 7241/INSERM U1050, PSL Research University, Paris, France ☯ These authors contributed equally to this work. * alain.prochiantz@college-de-france.frAbstractDuring postnatal life the cerebral cortex passes through critical periods of plasticity allowing its physiological adaptation to the environment. In the visual cortex, critical period onset and closure are influenced by the non-cell autonomous activity of the Otx2 homeoprotein transcription factor, which regulates the maturation of parvalbumin-expressing inhibitory interneurons (PV cells). In adult mice, the maintenance of a non-plastic adult state requires continuous Otx2 import by PV cells. An important source of extra-cortical Otx2 is the choroid plexus, which secretes Otx2 into the cerebrospinal fluid. Otx2 secretion and internalization requires two small peptidic domains that are part of the DNA-binding domain. Thus, mutating these "transfer" sequences also modifies cell autonomous transcription, precluding this approach to obtain a cell autonomous-only mouse. Here, we develop a mouse model with inducible secretion of an anti-Otx2 single-chain antibody to trap Otx2 in the extracellular milieu. Postnatal secretion of this single-chain antibody by PV cells delays PV maturation and reduces plasticity gene expression. Induced adult expression of this single-chain antibody in cerebrospinal fluid decreases Otx2 internalization by PV cells, strongly induces plasticity gene expression and reopens physiological plasticity.

Fig 1 .

 1 Fig 1. Inducible scFv knock-in mice. A) Construct for scFv knock-in mouse. The single chain antibody (scFv) is knocked in the ROSA26 locus of C57Bl6 mice. The scFv plasmid contains an IgK signal peptide for secretion, 6 Myc tags for detection and a skipping peptide (P2A) before EGFP. A STOP cassette is inserted before the scFv construct, flanked by two LoxP sites. B) Western blot showing expression (cells) and secretion (media) of anti-Otx2 scFv, upon treatment with Cre-TAT protein of scFvOtx2 tg/o ear fibroblasts. The scFv was revealed with an anti-Myc antibody and RhoA served as an intracellular protein control. C) Western blot with recombinant Otx2 protein, revealed with media of scFvOtx2 tg/o fibroblasts treated or not with Cre-TAT. D) Co-immunoprecipitation of Otx2 protein and anti-Otx2 scFv from P30 PV::Cre, PV::Cre;scFvPax6 tg/o and PV::Cre;scFvOtx2 tg/o cerebellum lysates. Co-immunoprecipitations were performed using Otx2 (left) or GFP (right) antibodies and probed on Western blots using anti-Otx2 or anti-Myc antibodies. doi:10.1371/journal.pgen.1006035.g001

  [START_REF] Andreasson | Role of immediate early gene expression in cortical morphogenesis and plasticity[END_REF][START_REF] Kaczmarek | Sensory regulation of immediate-early gene expression in mammalian visual cortex: implications for functional mapping and neural plasticity[END_REF][START_REF] Shepherd | New views of Arc, a master regulator of synaptic plasticity[END_REF][START_REF] Valles | Genomewide analysis of rat barrel cortex reveals time-and layer-specific mRNA expression changes related to experience-dependent plasticity[END_REF][START_REF] Li | The neuroplasticity-associated arc gene is a direct transcriptional target of early growth response (Egr) transcription factors[END_REF][START_REF] Tognini | Experience-dependent DNA methylation regulates plasticity in the developing visual cortex[END_REF]. Quantitative mRNA analysis of V1 layer IV extracts at P30 demonstrated that the expression of the anti-Otx2 scFv, in addition to delaying PV cell maturation, significantly decreased the up-regulation of Fos, Egr4 and Nr4a1 transcripts (Fig 3D). Arc and c-Fos are expressed throughout V1 layers, and Egr4 and Nr4a1, although showing higher levels of expression in V1 layer IV (Fig 3E and S3 Fig), are not necessarily specifically expressed in PV cells. Thus, changes in layer IV gene expression reflect the global effect on plasticity caused by Otx2 reduction.

Fig 2 .Fig 3 .

 23 Fig 2. Strategies for inducing expression of scFv in PV cells or in CSF. A) Co-staining for Myc and PV in layers II-IV of the binocular zone of the visual cortex (V1b) of P30 PV::Cre, PV::Cre;scFvPax6 tg/o and PV::Cre; scFvOtx2 tg/o mice (scale bar: 100μm). The enlarged view shows the immunofluorescence overlap. B) Western blot of cerebrospinal fluid (CSF) protein extracts from scFvOtx2 tg/o and scFvPax6 tg/o adult mice injected (+Cre) or not (ctrl) with Cre-TAT protein in lateral ventricles. Anti-Otx2 and anti-Pax6 scFvs (revealed with anti-Myc antibody) are secreted in the CSF upon choroid plexus recombination. doi:10.1371/journal.pgen.1006035.g002

  . Two weeks after intra-ventricular injection of Cre-TAT or NaCl, adult (P105) scFvOtx2 tg/o mice underwent 4 d of monocular deprivation (MD) prior to optical imaging in the opposite hemisphere (Fig 5A). The responses to different spatial frequencies were measured and quantified, as illustrated in Fig 5B and 5C. The expression of anti-Otx2 scFv enhances visual acuity of the ipsilateral eye (Fig 5D) but does not modify that of contralateral eye (Fig 5E), leading to a shift in ocular dominance for the highest spatial frequencies (above 0.14 cpd, Fig 5F).

  scFvPax6 tg/o and PV::Cre;scFvOtx2 tg/o mice (in percentage of PV::Cre mice, dashed line indicates PV::Cre levels). Absolute cell numbers are provided in S2 Fig. D) Quantification of Arc, c-Fos, Nr4a1, Egr4 and HPRT mRNA contents (relative to GAPDH) in V1b layer IV extracts from P30 PV::Cre, PV::Cre;scFvPax6 tg/o and PV::Cre;scFvOtx2 tg/o mice. E) Co-staining for c-Fos and WFA in V1b of P30 PV::Cre mice. (t-tests and oneway ANOVAs; 2-9 mice per group; *p<0.05, **p<0.01, ***p<0.001; error bars indicate SEM) doi:10.1371/journal.pgen.1006035.g003

Fig 4 .

 4 Fig 4. Anti-Otx2 scFv in adult CSF activates plasticity genes. A) Immunostaining for Otx2, PV, WFA, CR and GABA in V1b layers II-IV of adult scFvPax6 tg/o and scFvOtx2 tg/o mice, 15 days after intracerebroventricular (icv) injection of Cre-TAT protein (scale bar: 100μm). B) Quantification of number of Otx2-, PV-, WFA, CR-and GABA-positive cells in V1b layers II-IV of adult scFvOtx2 tg/o and scFvPax6 tg/o mice, 15 days after icv injection of Cre-TAT (in percentage of scFvPax6 tg/o mice, dashed line indicates scFvPax6 tg/o levels). Absolute numbers are provided in S2 Fig. C) Quantification of Arc, Fos, Nr4a1, Egr4 and HPRT mRNA content (relative to GAPDH) in V1 layer IV extracts from adult scFvOtx2 tg/o and scFvPax6 tg/o mice, 15 d after icv injection of Cre-TAT. (t-tests; 3-12 mice per group; *p<0.05, **p<0.01, ***p<0.001; error bars indicate SEM) doi:10.1371/journal.pgen.1006035.g004

Fig 5 .

 5 Fig 5. Anti-Otx2 scFv in adult CSF restores ocular dominance plasticity. A) scFvOtx2 tg/o adult (P90) mice were injected with NaCl or Cre-TAT and, after 15 d, the left eye was sutured for 4 d. Intrinsic optical imaging was then performed in the right visual cortex to measure visual acuity for each eye. B) Intrinsic signals recorded for the ipsilateral eye stimulation in scFvOtx2 tg/o adult mice injected with NaCl (top) or Cre-TAT (bottom). Ten spatial frequencies (SFs) ranging from 0.03 to 1 cycle/degree were tested. The binocular zone is delineated in white (A, Anterior; M, Medial; Scale bar, 1 mm). C) Estimation of the ipsilateral eye visual acuity for the two mice shown in (B). Each point corresponds to the average magnitude in the binocular zone. For each animal, data were normalized by the maximal amplitude among SFs and visual acuity was defined as the intersection of a fitted line segment with the X-axis. Representative stimuli are shown below. D) Visual acuity of the ipsilateral eye in adult scFvOtx2 tg/o injected with NaCl or Cre-TAT. E) Same as (D) for the contralateral eye. F) Ocular dominance index at different SF thresholds in adult scFvOtx2 tg/o mice injected with NaCl or Cre-TAT. Positive values indicate a preference for the contralateral eye. (t-tests; *p<0.05; error bars indicate SEM) doi:10.1371/journal.pgen.1006035.g005

  S3 Fig. Localization of Arc, Erg4 and Nr4a1 expression in V1b by in situ hybridization. The left panels show the staining for sense probes and the middle panels the staining for antisense probes. The right panel shows WFA immunostaining in V1b to visualize layer IV. (TIF)

  During CPs, that open and close at different time points, depending on the cortical functional areas involved, a heightened plasticity allows the remodeling of neural circuits in an experience-dependent manner. These periods of plasticity are paralleled by PV-cell maturation and thus correspond to a shift in the excitatory/inhibitory balance. During CP for binocular vision, inputs from the two eyes converge to the primary visual cortex and compete to form synapses on individual neurons. Monocular deprivation during the CP shifts the ocular dominance in favor of the open eye leading to a low visual acuity for the closed eye.

Figure 16 .Figure 17 .

 1617 FIGURES

Figure 18 .Figure 19 .

 1819 Figure 18. Otx2GFP/+ hypoanxiety is independent of VTA dopaminergic pathways (A) dosage of dopamine (DA) and serotonin (5-HT) and metabolites DOPAC, HVA, 5-HIAA, in anterior (Cx ant) and posterior (Cx post) cortices, striatum (Str) and hippocampus (Hipp) of Otx2 +/GFP (n=7) and WT (n=5) mice at 3 months of age. (B) TH+ cells numbers determined by stereology in the VTA of 3 month-old Otx2 +/GFP (n=4) and WT (n=5) mice.

( 0 .

 0 4 mA, 1 s) at 120 s intervals. During tone extinction (days 2, 3 and 4), mice were placed in a different context (white floor, triangular black roof, dark). 10 tones of 30 s (82 dB) were emitted at 60 s intervals.DosageBrains were dissected in ice-cold PBS1X and dry tissue was kept at -80°C. The samples were dosed by HPLC in the lab of Daniel Alvarez Fischer, Germany.CountingAnesthetized mice (n= 5 wt , 4 Otx2 +/GFP ) were intracardially perfused with 20ml of PBS and 30ml of paraformaldehyde 4% at 10ml/min; dissected brains were post-fixed in paraformaldehyde 4% at 4°C for 1h, rinsed in PBS and incubated in sucrose 20% at 4°C for 12-24h and then frozen. Immunohistochemistry was performed on free-floating cryostat sections (40µm). First, endogenous peroxidase was removed with a 5min incubation in H2O2 0.3%, Methanol 0.2%. After permeabilization with Triton 1% for 20 min, paraformaldehyde demasking was performed with Glycine 100mM during 20 min. Sections were then blocked with PBS 1X, Triton 0.2%, Normal Goat Serum (NGS) 10% during 30-45 minutes and incubated during 12-24h with an anti-Tyrosine Hydroxylase antibody (rabbit, Pel Freeze EPM in total darkness. Under 50 lux and darkness, the mutant mice spent, on average 8%, of the total time of the test on open arms. Moreover, the normal performance of Otx2GFP/+

  to block specifically Otx2 (scFvOtx2), Pax6 (scFvPax6) and Engrailed 1 and 2 (scFvEn) homeoprotein transfer, these mouse lines conditionally express and secrete the scFv in the extracellular space, thus preventing HP internalization without interfering with their cell autonomous functions. (A complete analysis of scFvOtx2 tg/0 mice is furnished in the article Bernard, Vincent, Testa et al 2016) At P30, we observed a 20% reduction in the number of PV-positive, Otx2-positive and WFApositive cells accompanied by a 20% reduction of Otx2 staining intensity in the supragranular layers (area V1, cerebral cortex) of PV::Cre x scFvOtx2 tg/0 mice. This is in comparison with

Figure 20 .

 20 Figure 20. Homeoprotein non cell-autonomous signalingHomeoproteins can transfer between cells, thanks to secretion and internalization sequences overlapping the homeodomain. Once in the target cell, it can regulate transcritption, translation and epigenetic events. (Adapted fromProchiantz and Di Nardo, 2015)

Figure 21 .

 21 Figure 21. The single-chain antibody toolA secreted single-chain variable fragment (scFv) of antibody targeting specifically a homeoprotein (HP) is secreted in the extracellular space and binds the HP, preventing its internalization in the target cell. (adapted from[START_REF] Prochiantz | Signaling with homeoprotein transcription factors in development and throughout adulthood[END_REF]

Figure 22 .

 22 Figure 22. HP signalling in synergy with classical signalling factors.In absence or gain of function of calissical signalling, HP is useless. (adapted from[START_REF] Prochiantz | Signaling with homeoprotein transcription factors in development and throughout adulthood[END_REF]

Figure 23 .

 23 Figure 23. General scheme of HP signalling hypothesis.HPs recognize specific target cells through their binding to complex sugars; inside the cells they regulate translation, transcription and epigenetic state in cooperation with other factors. Important up-regulated translational targets are mitochondrial leading to an up-regulation of ATP synthesis. (adapted fromSpatazza et al., 2013)

Figure 25 .

 25 Figure 25. Age of onset of psychiatric disorders (adapted from Paus et al., 2008)

Figure 26 .

 26 Figure 26. Developmental model of schizophrenia Normal cortical development involves decrease in excitatory and increase in inhibitory synaptic strengths during adolescence, the period of emergence of prodromes of psychosis. Reduced myelination and altered excitatory-inhibitory balance would alter connectivity and cause schizophrenia. III.2. Development of neuronal synchrony during late maturation of the cerebral cortex

Figure 27 .

 27 Figure 27. High speed oscillations in prefrontal cortex during a cognitive task and default in schizophrenia patients (left) Interaction of PV-cells (GABA) and pyramidal cells (glutamate) generate oscillations. (middle) Gamma-band oscillations at different developmental stages. (right) Neural synchrony during a perception task (Mooney face upside down) in adult controls and schizophrenia (ScZ) patients. (adapted from Uhlhaas 2009)

Figure 28 .

 28 Figure 28. Otx2 regulation of cortical oscillations hypothesis.Otx2 from the choroid plexus is internalized in GABAegic PV-cells (round). The induced maturation of PV-cells permits high-speed oscillations in the cortices (CX1, 2, 3) serving the cognitive tasks. This mechanism is reglated by aminergic neuromodulators.

  GFP/+ mice, suggesting that these mice, which have a delayed CP, open plasticity at P100. As expected from our behaviour experiments, Otx2 +/AA mice showed an intermediate profile of gene expression.

	Hypoanxiety is similar in one month-old and adult (3 months) Otx2 GFP/+ mice, which supports
	neurodevelopmental rather than neurodegenerative mechansisms. Several studies converge
	to propose that there is a CP for anxiety behaviours during postnatal development. Windows
	of plasticity occur between the maturation of an optimal Excitatory/Inhibitory (E/I) balance
	and an emerging set of molecular brakes consolidating synapses and dampening plasticity
	such as PNNs. Gogolla et al reported in 2009 the progressive formation of PNNs from P16 to

PLOS Genetics | DOI:10.1371/journal.pgen.1006035May 12, 2016

PLOS Genetics | DOI:10.1371/journal.pgen.1006035May 12, 2016 3 / 15

PLOS Genetics | DOI:10.1371/journal.pgen.1006035May 12, 2016 4 / 15

PLOS Genetics | DOI:10.1371/journal.pgen.1006035May 12, 2016 5 / 15

PLOS Genetics | DOI:10.1371/journal.pgen.1006035May 12, 2016 6 / 15

PLOS Genetics | DOI:10.1371/journal.pgen.1006035May 12, 2016 9 / 15

Acknowledgments

We thank Eliane Ipendey and Anthony Perrot for excellent technical work.

Data Availability Statement: All relevant data are within the paper and its Supporting Information files.

Funding:

The study was supported by ERC Advanced Grant HOMEOSIGN n°339379, ANR-11-BLAN-069467 BrainEver, Région Ile de France, Fondation Bettencourt Schueller and GRL program N °2009-00424. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Summary

Classically, cell signaling is based on the secretion of molecules that bind cell surface receptors. Lipophilic agents can do without cell-surface receptors due to their ability to diffuse through the plasma membrane, but this is normally not the case for proteins, which cannot pass the membrane barrier. However, homeoprotein transcription factors represent an exception as they are secreted and internalized by live cells owing to two peptidic domains. An important illustration of this novel signaling mechanism is provided by Otx2, a homeoprotein that travels from the choroid plexus to specific inhibitory neurons in the cerebral cortex, where it regulates physiological plasticity throughout life. Because the two transfer peptides are in the DNA-binding domain of Otx2, it is impossible to mutate them without altering both cell signaling and cell-autonomous functions. We have therefore developed a mouse in which a secreted anti-Otx2 single-chain antibody can be induced to trap extracellular Otx2 while leaving its cell autonomous function untouched. We show that neutralizing extracellular Otx2 modifies the expression of plasticity genes in the visual cortex, thus providing the first genetic demonstration for homeoprotein signaling in a mammal.

Competing Interests: The authors have declared that no competing interests exist.

Supporting Information

Elevated-plus maze

This test is made of a cross-shape maze elevated at 70 cm from the floor, enlighted at 50 lux.

Two "closed" arms facing each other are protected from the height by black Perspex walls (20 cm high), crossed perpendicularly by the two others, "opened". The mouse is put down at the center of the cross and the time spent on the open arms is measured as an index of reduced anxiety, during a 10 min test.

Light-dark box

A double two-compartment box allows the registration of two mice at the same time. In each box, a dark compartment (walls, floor and roof) is linked by a small opening to a white box (walls and floor, no roof) enlighted at 150 lux (equal volume of 20 x 20 x 30 cm). The mouse is put down in the light box facing the opening, and anxiety is measured as the increased time spent in the dark compartment of the box, during a 10 min test.

Rotarod

Equilibrium and motor coordination is measured in an accelerating rotarod (model 47600, Ugo Basile, Italy). A cylinder accelerates progressively from 2 rpm to 40 rpm during 5 min.

Test is performed in a schedule of 3 trials per day separated by 90 min, during 2 days.

Tail-suspension

Mice are suspended by the tail with adhesive to a hook linked to a gauge which detects movement (automated system BIOSEB, France), during 6min. Immobility is interpreted a sign of depression.

Forced swimming

Also named Porsolt test, this test evaluates despair (depressive) behaviors by registering periods of freezing when the mouse is placed in a see-through Perspex cylinder full of water (25°C). The cylinder is 25 cm high, 13 cm of diameter and filled with water up to 20 cm. On the first day the animal is accustomed to the test during 10 min, on the second day the test lasts 6 min (an infra-red panel is positioned vertically behind the cylinder for movement detection).

Prepulse Inhibition

Sensory-motor information gating consist of reducing a startle reflex to a tone stimulus (120 dB; the pulse) when a tone of lower intensity has previously been presented (72, 76, 80 or 84 Biologicals, 1/1000e) in PBS 1X, Triton 0.1%, NGS 3% at 4°C. On the second day, the slides were washed 3 times in PBS 1X, Triton 0.1% and a second biotin-coupled anti-rabbit antibody (1/2000e) was added for 2h at room temperature. After 3 washes in PBS 1X, Triton 0.1%, the biotin was revealed usisng the Vectastain Elite ABC HRP kit and Peroxidase Substrate kit (1h of ABC buffer + revelation with DAB) from Vectorbiolabs until the signal was found appropriate and the slides washed in ddH2O2. Sections were mounted in Dapifluoromount milieu and kept at 4°C until counting. Every two section (containing VTA) was counted by stereology using Stereo Investigator software (MBF Bioscience Inc.) at a 40X magnification.

DISCUSSION

The discussion on the genetic evidence for Otx2 transfer can be found in the published article [START_REF] Bernard | A Mouse Model for Conditional Secretion of Specific Single-Chain Antibodies Provides Genetic Evidence for Regulation of Cortical Plasticity by a Non-cell Autonomous Homeoprotein Transcription Factor[END_REF] and will be enlarged in the next chapter (CONCLUSIONS AND PERSPECTIVES). This section is thus primarily focused on the last part of my work (still in progress) on anxiety regulation.

I. Otx2 in the anxiety pathway

Otx2 transfers from different extra-cortical sources to PV-cells in the primary visual cortex (V1) to induce their maturation and regulate CP for binocular vision. This is now wellestablished even if the molecular mechanisms involved are not really characterized.

Recently, the laboratories of A. Prochiantz and T. Hensch have found that non-cell autonomous Otx2 regulates plasticity not only in the visual cortex but also in the primary auditory cortex (tonotopic map expansion) and in the medial prefrontal cortex for the acoustic preferences acquisition (Bernard et al., in revision at Mol. Psy.).

In the previous chapter we have investigated a possible role for Otx2, in the regulation of anxiety-related behaviors. When one allele of Otx2 has been deleted (Otx2 GFP/+ ) thus probably reducing Otx2 production by 50% (although we did not quantify Otx2 protein levels), some anxiety-related behaviours are modified (figure 1). Compared to their WT littermates, Otx2 GFP/+ mice spend more time on the elevated-plus maze open arms and in the enlighted compartment of the light-dark box, suggesting that they are less anxious than normal mice.

We can observe the same phenotype in the Otx2 +/AA mice, though at a lesser level. On the EPM, Otx2 +/AA average time spent on the open arms is nearly half that measured for Otx2GFP/+ and in the light-dark box, Otx2 +/AA mice do not explore more the enlighted compartment compared to WT but they enter it more frequently. The latter behaviour cannot be attributed to short-term spatial memory as no defects was observed in these mice in the Y-maze. On the contrary it was seen that these mice are more active in the openfield suggesting an increased exploration behaviour (or a pure abnormal motor behaviour).

Nevertheless in the open field, Otx2 +/AA mice are not afraid to cross the central zone, a probable sign of reduced anxiety.

Considering the role of Otx2 in retinal and visual cortex development, one could attribute the Otx2 GFP/+ mice behaviour, in particular on the EPM, to visual defects. This is probably not he case since Otx2 +/AA mice visual acuity is normal acuity P100 [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF] while exhibiting the hypoanxiety phenotype. In addition, Otx2 GFP/+ mice were also tested on the

CONCLUSIONS AND PERSPECTIVES

is, it must be taken into account. In this thesis, the problem can be ignored when the anti-Otx2 antibody is produced and secreted by PV-cells that do not express Otx2, but it must be kept in mind when the production of the antibody is induced in the adult ChP, itself a source of Otx2. Although Otx2 target gene expression in ChP is not modify following scFv induction in this structure, this word of caution remains valid since we do not yet have the entire catalogue of Otx2 target genes in this structure.

I.2 The Otx2AA mouse

In fact, and somehow in contradiction with what was just discussed we have produced an Otx2AA mouse in which a RK doublet necessary for PV-cell recognition was replaced by an alanine doublet. This mouse develops correctly suggesting that the basic Otx2 cell autonomous functions are not affected. However, it is still possible that more subtle functions, in particular in the adult, may be affected in a cell autonomous way. Indeed the Otx2AA protein is not fully active at a transcriptional level as it was demonstrated, using a bona fide promoter that only 50% of Otx2 transcriptional activity is preserved, thus 75% in the Otx2AA/+ mouse [START_REF] Bernard | Graded Otx2 activities demonstrate dose-sensitive eye and retina phenotypes[END_REF]. Thus, it s still possible that the phenotype of the Otx2AA/+ mouse results from a combination of changes in the cell autonomous and non-cell autonomous properties of the transcription factor. For example, some important classical factors, controlled by Otx2 in the ChP, may not be synthesized at a proper level modifying Otx2 indirect non-cell autonomous activity.

II. CO-SIGNALLING BETWEEN HPs AND CLASSICAL FACTORS

As important as it might be both in development and in the adult, it is clear from previous experiments achieved in the laboratory that HP signalling cooperates with more classical modes of signal transduction. Indeed, Engrailed co-signals with DPP and EphrinA5/Adenosine in the fly wing disk and the superior colliculus, respectively (Layalle et al., 2013;Wizenmann et al., 2009;Stetller et al., 2012). This is an important concept as one could see HP signalling as a way to make the cells more sensitive to classical signals operating at low concentration (figure 22).