Giuliana Pellegrino

Claire Trubert

Jérémy Terrien

Fabien Pifferi

Danièle Leroy

Anne Loyens

Martine Migaud

Marc Baroncini

Claude-Alain Maurage

Christian Fontaine

Vincent Prévot

Ariane Sharif

Resumé

Malgré sa complexité, le cerveau intègre en permanence de nouvelles cellules -à la fois neuronales et gliales -au-delà du développement embryonnaire et ce, tout le long de la vie.

La période postnatale est caractérisée par une gliogenèse intense. A l'âge adulte, de nouveaux neurones et cellules gliales sont produits dans des régions restreintes à partir de cellules souches/progénitrices (CSP) localisées dans des niches. Les deux niches de CSP adultes les mieux décrites sont la zone sous-ventriculaire des ventricules latéraux, qui produit de nouveaux interneurones olfactifs, et la zone sous-granulaire du gyrus denté de l'hippocampe, où de nouveaux neurones en grain sont produits localement. Des travaux menés ces dernières années ont montré qu'une neuro-et une gliogenèse avaient aussi lieu dans l'hypothalamus postnatal, une petite région du diencéphale ventral qui régule des processus physiologiques vitaux tels que le métabolisme, la reproduction, le sommeil et la thermorégulation. Si l'identité des CSP hypothalamiques reste débattue, de nombreux travaux s'accordent sur l'importance de la neurogenèse hypothalamique postnatale dans le contrôle du métabolisme. Cependant, la possibilité que la genèse postnatale de cellules contribue aussi au contrôle de la fonction de reproduction, une autre fonction clé de l'hypothalamus, restait à explorer.

L'objectif premier de mon travail de thèse était de rechercher si la genèse de cellules dans l'hypothalamus postnatal est impliquée dans le contrôle de la reproduction, une fonction physiologique qui requière un haut degré de plasticité. La fonction de reproduction est orchestrée par une petite population de neurones produisant la neurohormone Gonadotrophin-Releasing Hormone (GnRH). Ces neurones, qui naissent en dehors du cerveau, sont en place dans la région préoptique (RPO) de l'hypothalamus à la naissance.

Cependant, ils doivent subir une maturation postnatale pour acquérir le profil de sécrétion qui leur permettra d'initier la puberté et d'assurer la fertilité de l'individu.

Dans une première étude, grâce à une combinaison d'approches in vitro et in vivo, nous avons mis en évidence une vague d'astrogenèse dans l'environnement des neurones à GnRH au sein de la RPO au cours des deux premières semaines de vie postnatale chez la ratte. Nos résultats suggèrent que les neurones à GnRH utilisent la prostaglandine D2 pour attirer les progéniteurs environnants et que ce recrutement est important pour la maturation sexuelle.

Dans une deuxième étude, nous avons recherché si de nouvelles cellules naissent à l'âge adulte dans des régions hypothalamiques qui contrôlent la fonction de reproduction. Nous montrons que des cellules sont produites dans la RPO chez la ratte adulte et que leur taux varie au cours du cycle oestral, suggérant une régulation par les stéroïdes sexuels. De plus, nous montrons que la survenue d'une gestation stimule la néogenèse cellulaire dans une zone de la RPO qui contrôle le comportement maternel.

Si la néogenèse hypothalamique adulte a surtout été étudiée chez les rongeurs de laboratoire, il reste à déterminer si ce phénomène existe aussi chez l'homme. Pour aborder cette question, nous avons évalué dans une troisième étude l'expression de marqueurs de CSP dans l'hypothalamus humain adulte, comparativement au rongeur (souris, rat) et à un primate lémurien, le microcèbe. Nous montrons que l'hypothalamus humain adulte contient des populations de cellules au profil antigénique de CSP, dont certaines semblent propres à l'homme.

Au total, ces travaux montrent que de nouvelles cellules naissent dans des régions hypothalamiques qui contrôlent la fonction de reproduction au cours de la vie postnatale et à l'âge adulte chez la ratte, et que ce phénomène est important pour la maturation sexuelle.

L'observation de CSP putatives dans l'hypothalamus humain adulte suggère que la capacité de l'hypothalamus à produire de nouvelles cellules à l'âge adulte existe aussi dans notre espèce.

Abstract

Despite its complexity, the brain keeps adding new cells -both neuronal and glial -beyond embryonic development and throughout life. The postnatal period is characterized by intense and widespread gliogenesis. During adulthood, both glio-and neurogenesis occur in restricted locations from stem/progenitor cells (NPC) residing in niches. The two bestdescribed niches of adult NPC are the subventricular zone of the lateral ventricles, which provides new interneurons to the olfactory bulb, and the subgranular zone of the hippocampal dentate gyrus that locally produces new granule cells. The last decade has seen an accumulation of studies showing that neuro-and gliogenesis also occur in the postnatal hypothalamus, a small portion of the ventral forebrain surrounding the third ventricle that regulates essential physiological processes such as metabolism, reproduction, sleep and thermoregulation. Even though the identity of hypothalamic NPC remains a matter of debate, a growing body of evidence points to postnatal hypothalamic neurogenesis relevance for the control of metabolism. However, a possible contribution of postnatal hypothalamic cell generation to the central control of reproduction, another key function of the hypothalamus, remained to be explored.

The main aim of my doctoral researches was to evaluate whether the generation of new cells in the postnatal hypothalamus contributes to the central control of reproduction, a physiological function known to require a high degree of plasticity. The reproductive function is controlled by a small population of neurons producing the neurohormone Gonadotrophin-Releasing Hormone (GnRH). These neurons, which are born in the nasal placodes, are in place at birth in the preoptic area (POA) of the hypothalamus. However, they need a postnatal maturation to reach a mature secretory pattern that will trigger puberty and subsequent fertility.

In a first study, using a combination of in vitro and in vivo experiments, we showed that a wave of astrogenesis occurs in the POA from local progenitors in the environment of GnRH neurons during the first weeks of postnatal life in the female rat. We identified prostaglandin D2 as a factor used by GnRH neurons to attract progenitors in their vicinity and showed that impaired progenitor recruitment alters sexual maturation.

In a second study, we evaluated whether cell neogenesis still occurs during adulthood in hypothalamic regions relevant for the reproductive function. Our results showed that new cells are born in the POA of adult female rats. The rate of cell neogenesis varies across the estrus cycle, suggesting a regulatory influence of gonadal steroids. Moreover, we showed that gestation impacts the rate of cell neogenesis in a POA region implicated in the control of maternal behavior.

While cell neogenesis in the adult hypothalamus has been mainly studied in laboratory rodents, it remains to be known whether this phenomenon also occurs in humans. To start addressing this question, we evaluated in a third study the expression of a panel of NPC markers in the adult human hypothalamus and compared it to that found in rodents (mouse, rat) and a lemur primate, the grey mouse lemur. Our results showed that the adult human hypothalamus contains populations of cells with an antigenic profile of NPC, some of which appear specific to humans.

Altogether, this work shows that new cells are born in hypothalamic regions controlling reproduction throughout postnatal and adult life in female rats, and that this process is required for sexual maturation. The identification of NPC marker-expressing cells in the adult human hypothalamus suggests that the capacity for cell neogenesis also exists in the hypothalamus of our species.

Tous les jours, nous interagissons avec un environnement en constante évolution à partir duquel nous recevons une multitude de stimuli différents qui, avec un certain nombre de signaux internes, convergent vers notre cerveau. La délicate coordination entre ces deux acteurs est fondamentale pour toutes les réponses physiologiques.

Nous sommes confrontés à un système où la stabilité, doit coexister avec la plasticité, car le cerveau est un tissu complexe possédant des millions de connexions. La plasticité est la capacité à modifier et adapter les structures et les fonctions pendant la maturation, les pathologies ou les défis environnementaux, au niveau neuronal ou non neuronal, améliorant ainsi les résultats fonctionnels pour mieux s'adapter aux conditions environnementales.

Aujourd'hui nous savons que la plasticité neurale est effectivement maintenue à l'âge adulte, cependant une telle déclaration n'aurait pas été possible il y a 50 ans, quand les premières expériences montrant la capacité du cerveau adulte à générer de nouveaux neurones dans des conditions physiologiques ont commencé à secouer le dogme central de la neuroscience: « dans le système nerveux tout est immuable ».

La région du cerveau principalement impliquée dans la discussion entre l'environnement et l'organisme est l'hypothalamus. Cette petite portion du diencéphale, présente dans le système nerveux de tous les vertébrés, est un régulateur homéostatique des processus comportementaux et physiologiques comme le métabolisme, la reproduction, la thermorégulation, les rythmes circadiens, la peur, les comportements sexuels, toutes les fonctions complexes dépendantes du statut interne de l'animal ( par exemple les niveaux de nutriments) mais aussi sur les informations reçues de l'environnement (par exemple les heures de lumière et d'obscurité, le voisinage d'un danger ou d'un partenaire).

Compte tenu de son rôle essentiel dans le maintien de l'homéostasie physiologique, il ne faut pas s'étonner du niveau élevé de plasticité observé dans la population neuronale et gliale hypothalamique. Néanmoins, l'existence d'une population de cellules souches neurales hypothalamiques n'a été abordée que superficiellement jusqu'à présent. Les deux niches de cellules souches neurales généralement acceptées chez les mammifères adultes se trouvent au niveau de la zone subventriculaire (SVZ) des ventricules latéraux et de la zone subgranulaire (SGZ) de l'hippocampe. En outre, quelques études pionnières ont été consacrées à explorer la possibilité que la production de nouveaux neurones et/ou cellules gliales dans l'hypothalamus adulte puisse contribuer à la flexibilité nécessaire pour intégrer continuellement des signaux provenant d'autres noyaux cérébraux ainsi que de la périphérie et de l'environnement afin de maintenir l'homéostasie énergétique dans le corps. L'hypothalamus est le centre de contrôle de toutes les fonctions végétatives du cerveau des vertébrés. En fonction des signaux concernant l'état physiologique de l'organisme, ainsi que des informations sur l'environnement, cette région doit adapter sa réponse pour coordonner l'action des différents organes par interaction avec l'hypophyse. Les changements dans les entrées synaptiques, ainsi que les interactions neurones-glie ont déjà été décrits comme des caractéristiques de plasticité importantes pour que l'hypothalamus exerce son rôle. De plus, des études initiales chez des rongeurs sur le rôle de la néogenèse cellulaire dans le contrôle du métabolisme, permettent de penser que l'hypothalamus puisse représenter une niche de cellules souches neurales adultes. Cela suggère que l'hypothalamus conserve un niveau de plasticité encore plus élevé que supposé précédemment et ouvre de nombreuses questions sur le fait de savoir si la prolifération cellulaire intervient seulement à l'âge adulte ou pendant le développement postnatal, et de savoir quelles fonctions hypothalamiques peuvent être touchées par ce processus.

La reproduction est la fonction physiologique la plus importante pour une espèce, même si un seul animal peut survivre s'il n'est pas fertile, la perpétuation de la vie dépend de la reproduction. Cette fonction complexe, qui se développe sur une période post-natale, est fortement influencée par l'environnement et l'état métabolique de l'animal, en particulier chez les femmes qui doivent adapter fortement leur corps et leur cerveau pour être en mesure de s'assurer un processus aussi exigeant que la grossesse et les soins à la progéniture, les éléments essentiels pour assurer la survie de l'espèce.

Les régulateurs principaux du système reproducteur sont les neurones GnRH, une petite population de neurones parvocellulaires localisés dans l'aire preoptique de l'hypothalamus, qui ne sont fonctionnels qu'au stade de la puberté, et qui nécessitent donc une période de développement postnatal, dépendant de l'établissement des signaux neuronaux et non neuronaux ainsi que des signaux hormonaux qui agissent pour moduler la synthèse et la libération de la GnRH.

Les objectifs de mon travail ont été:

(i) étudier le rôle de la néogenèse cellulaire dans l'aire preoptique dans la régulation du processus de maturation des neurones GnRH au cours du développement postnatal, un moment où la plupart des cellules gliales sont produites dans le cerveau;

(ii) caractériser la prolifération cellulaire au cours du cycle oestral dans l'aire preoptique et l'éminence médiane à l'âge adulte, les régions où les corps et les fibres des neurones GnRH sont respectivement trouvés, et d'évaluer son implication possible avec la fonction de reproduction;

(iii) évaluer le phénotype progéniteurs/cellules souches des cellules trouvées le long du 3ème ventricule dans l'hypothalamus humain adulte en comparant l'expression d'un panel de progéniteurs neuronaux et de marqueurs de cellules souches entre le cerveau humain et celui d'autres espèces.

Néogenèse cellulaire dans l'hypothalamus postnatal chez la rate: implication dans la maturation sexuelle

Les neurones GnRH sont une petite population de neurones retrouvés au niveau de la région préoptique. Ils contrôlent la reproduction grâce à leur libération de l'hormone gonadotrophine qui induit la libération des gonadotrophines, LH et FSH, qui agiront au niveau des gonades pour induire la gonadogenèse et la libération des hormones gonadiques.

Après la migration embryonnaire des neurones GnRH de la placode olfactive, où ils sont nés, ils s'installent dans l'hypothalamus où ils se trouvent à la naissance. Cependant, une maturation postnatale est nécessaire pour permettre le début de la puberté et la cyclicité oestrale qui en découle.

Le développement postnatal des neurones à GnRH chez les rats femelles peut être divisé en 4 stades, une période néonatale, de la naissance à P7; une période infantile, de P8 au sevrage, une période débutant par la première activation centrée de l'axe HPG, marqué par un pic de FSH à P12; une période juvénile, de P21 à P30, et une période péripubertaire. Au cours de cette maturation postnatale, la libération de GnRH augmente progressivement et culmine au moment de la première poussée de LH, provoquant la première ovulation, qui chez le rat correspond au jour de l'ouverture vaginale.

Pendant ce temps, les neurones GnRH reçoivent des inputs neuronaux et non neuronaux provenant de la même région ainsi que d'autres noyaux de l'hypothalamus, car d'autres fonctions hypothalamiques, telles que l'état métabolique ou l'horloge circadienne, interviennent dans le contrôle de la reproduction et contribue donc à régler le moment où le système acquiert sa maturation complète.

On sait maintenant qu'un rôle important dans la régulation de l'activité de neurones à GnRH est exercé également par les astrocytes de la zone préoptique qui interviennent dans la signalisation des facteurs de croissance de la famille EGF puisque les neurones à GnRH n'expriment pas leurs récepteurs. En réponse à cette voie de signalisation, les astrocytes agissent sur les neurones à GnRH par l'action de la Prostaglandine (PG) E2 qui peut induire la libération de GnRH par la stimulation du déclenchement de ces neurones. Il a également été montré que cette diaphonie est nécessaire au début de la puberté et à l'établissement d'une cyclicité oestrale.

Cependant, alors que les neurones sont nés au cours du développement embryonnaire du cerveau, on sait que les cellules gliales apparaissent au cours du développement postnatal, dans un processus qui n'est pas encore entièrement compris.

Le premier objectif de ma thèse était donc d'étudier si la néogenèse cellulaire dans la zone préoptique pouvait être impliquée dans le processus de maturation des neurones GnRH.

Nous avons utilisé la bromodésoxyuridine (BrdU) pour analyser la prolifération dans la région préoptique de rats femelles à différents âges postnataux. Une intense activité proliférative caractérise la période infantile et ces cellules se différencient principalement en astrocytes. Les astrocytes nouveau-nés sont en relation morphologique avec les neurones à GnRH, par rapport à l'ensemble de la population neuronale de la zone préoptique et leur nombre augmente au fil du temps. Nous avons montré que les neurones à GnRH sont capables de recruter des cellules néonatales à proximité de leur corps cellulaires par la libération de prostaglandine D2 et la signalisation sur son récepteur (DP1). De plus, cette activité proliférative est nécessaire à la maturation des neurones GnRH et au début de la puberté, et le recrutement spécifique des cellules néonatales est impliqué dans la maturation du système GnRH.

Néogenèse cellulaire dans l'hypothalamus adulte des rates

La reproduction dépend de l'ovulation féminine, qui se produit de manière cyclique. Les rats (ainsi que les souris) ont des cycles très courts qui peuvent être divisés en 4 étapes: 2 jours de diestrus, un jour de proestrus quand la montée de GnRH et de LH survient, induisant l'ovulation, et un jour d'estrus. Les hormones gonadiques augmentent graduellement alors que les follicules mûrissent au niveau des ovaires, et exercent à la fois une rétroaction négative et une rétroaction positive, ces dernières se produisant lorsque leurs niveaux sont suffisamment élevés pour causer la poussée de la GnRH et de la LH.

La neurogenèse et la gliogenèse ont été décrites dans les régions hypothalamiques contrôlant la fonction de GnRH, telles que le noyau antéro-ventral périventriculaire (AVPV) de l'hypothalamus, où se trouvent les neurones kisspeptin responsables de la rétroaction positive des oestrogènes sur les neurones GnRH; au niveau de l'organum vasculeux de la lamina terminalis (OVLT), où se trouvent les corps cellulaires des neurones GnRH, et au niveau de l'éminence médiane, où les neurones GnRH projettent leurs terminaisons; ce qui soulève la possibilité que ce processus puisse également participer aux mécanismes de plasticité impliqués dans la régulation neuroendocrinienne de la reproduction.

Le second objectif de mon doctorat était d'explorer la possibilité que la génération de nouvelles cellules dans l'hypothalamus adulte représente un mécanisme de plasticité impliqué dans le contrôle du système à GnRH.

Nous avons observé que la génération de nouvelles cellules se poursuit au-delà de la puberté dans les régions hypothalamiques critiques pour le contrôle des neurones à GnRH: la région préoptique et l'éminence médiane, en outre et que les niveaux de prolifération cellulaire varient selon le cycle oestral. De plus, les cellules nées dans la région préoptique des rats femelles, juste avant l'ovulation, survivent préférentiellement si la gestation survient. Compte tenu du rôle critique de la zone préoptique dans l'apparition du comportement maternel, nous proposons l'hypothèse que l'ajout de nouvelles cellules dans cette région pendant la grossesse participe aux changements adaptatifs qui permettent à ce comportement de se développer.

La niche des cellules souches/progénitrices dans l'hypothalamus humain adulte

Le cerveau adulte contient des niches de cellules souches neurales qui ajoutent continuellement de nouveaux neurones à des circuits sélectionnés tout au long de la vie.

Chez l'homme également, les deux principales niches neurogéniques, la SVZ et la SGZ, ont fait l'objet d'études approfondies, montrant un certain niveau de variabilité par rapport aux autres espèces. Cependant, malgré les données récentes identifiant l'hypothalamus adulte comme une troisième niche neurogénique chez les rongeurs, cette région n'a pas encore été étudiée chez les humains.

Afin d'évaluer si une niche de cellules souches neurales existe également dans l'hypothalamus adulte humain, nous avons effectué plusieurs marqueurs d'immunofluorescence pour évaluer l'expression d'un panel de marqueurs de cellules souches/progénitrices neurales (Sox2, nestine, vimentine, GLAST, GFAP) dans l'hypothalamus humain et nous les avons comparés à la souris, au rat et à une espèce de primate non humain, le lémur gris (Microcebus murinus).

Nos résultats montrent que l'hypothalamus humain adulte contient trois populations distinctes de cellules qui expriment les cinq marqueurs des cellules progénitrices neurales: i) un ruban de petites cellules étoilées qui tapisse la paroi du troisième ventricule derrière un espace hypocellulaire, similaire à celui précédemment décrit le long des ventricules latéraux humains;

ii) des tanycytes qui tapissent le plancher du troisième ventricule dans la région tubéreuse;

iii) une population de petites cellules étoilées dans le noyau suprachiasmatique.

Dans l'hypothalamus de la souris, du rat et du Microcebus murinus, les marqueurs des cellules progénitrices neurales sont co-exprimés dans les tanycytes mais pas dans le noyau suprachiasmatique, et ces espèces n'ont pas de ruban ventriculaire. Notre travail identifie ainsi trois populations cellulaires avec le profil antigénique des cellules progénitrices neurales dans l'hypothalamus humain adulte, dont deux semblent spécifiques aux humains.

Ce travail montre que de nouvelles cellules naissent dans les régions hypothalamiques contrôlant la reproduction tout au long de la vie postnatale et adulte chez les rats femelles, et que ce processus est nécessaire pour la maturation sexuelle. L'identification des cellules exprimant les marqueurs des cellules progénitrices neurales dans l'hypothalamus humain adulte suggère que la capacité de néogenèse cellulaire existe également dans l'hypothalamus de notre espèce.

Poster and oral communications Every day we interact with a constantly changing environment from which we receive a multitude of different stimuli that, together with a number of internal signals, converge into our brain. Coordination between these two players is fundamental to any physiological response as well as extremely delicate: the number, position, subtype and connectivity web of neural cells need to be tightly controlled and can be easily perturbed.

Therefore we are in front of a system where stability, being the brain a complex tissue with millions of intricate connections, has to coexist with plasticity. This is the ability to modify and adapt structures and functions during maturation, pathologies or environmental challenges, at many levels of organization of both neural and non-neural elements, improving functional outcome to better suit the prevailing environmental conditions.

Although early life experiences are those which mainly shape our brain circuits, as they occur at a moment when the brain itself is still developing, neural plasticity is indeed maintained also during adulthood. The discovery of neural stem cells in mammalian brains which could generate new neurons and glial cells in adult animals was a real paradigm shift; however, realising that this process could be influenced by external stimuli highlighted the role of brain plasticity in integrating external information with internal cues to elaborate adapted behavioral and physiological responses; it gave this process a purpose.

The brain region mainly involved in the cross talk between the environment and the organism itself is the hypothalamus. This small portion of the diencephalon, found in all vertebrate nervous systems, is a homeostatic regulator of behavioral and physiological processes as metabolism, reproduction, thermoregulation, circadian rhythms, fear, sexual behaviors, all complex functions dependent on the internal status of the animal (e.g. the nutrient levels) but also on information received from the environment (e.g. the hours of light and dark, the vicinity of a danger or of a mate).

Given its critical role in maintaining physiological homeostasis, it should come with no surprise that high levels of plasticity are observed in the hypothalamic neuronal and glial populations. Nonetheless the existence of a population of hypothalamic neural stem cells has only started to be addressed until now. A few pioneering studies explored the possibility that the production of new neurons and/or glial cells in the adult hypothalamus may contribute to the flexibility necessary to continuously integrate signals coming from other brain nuclei as well as from the periphery and the environment in order to maintain the correct energy homeostasis in the body.

Nevertheless, little is known about the implication of this process in any of the other hypothalamic functions. Reproduction is the most important physiological function for a species. Even though a single animal can survive if not fertile, perpetuation of life depends upon reproduction. This complex function, which matures over the postnatal period, is strongly influenced by the environment and the metabolic state of the animal, especially in females that have to strongly adapt their body and brain to be able to sustain a process as demanding as pregnancy and care of the offspring, all essential elements to ensure survival of the species.

After a general introduction on how neurons and glial cells are produced in the brain, I will first introduce our current knowledge on adult neurogenesis in the two canonical niches, the SVZ and the SGZ. I will then present the current data on the emerging field of cell neogenesis in the post-natal hypothalamus and finally give an overview of how the hypothalamus controls the reproductive function.

Chapter 1

Cell neogenesis in the brain throughout life [START_REF] Daley | Stem cells and the evolving notion of cellular identity[END_REF][START_REF] Xu | Building a central nervous system: The neural stem cell lineage revealed[END_REF]. Indeed, despite the great level of diversity found among the different cells of an organism, they all derive from the unicellular zygote, result of fertilisation between two gametes [START_REF] Ohnuki | Present and future challenges of induced pluripotent stem cells[END_REF]. During embryogenesis, the zygote transforms into a morula and then a blastocyst through mitotic cell division before implantation. The blastocyst is composed by an inner cell mass (ICM), that will form the embryo, and an outer layer called trophoblast, which will give rise to the placenta and yolk sac [START_REF] Ohnuki | Present and future challenges of induced pluripotent stem cells[END_REF][START_REF] Xu | Building a central nervous system: The neural stem cell lineage revealed[END_REF].

Stem cells have not been easy to characterise due to their lack of specific markers [START_REF] Reynolds | Neural stem cells and neurospheres-re-evaluating the relationship[END_REF]. However, they have to fulfil two main characteristics: ability to self-renew and to differentiate. According to their differentiation potency, cells can be distinguished as:

Totipotent: only the zygote and early blastomeres, which can form all the tissues of the embryo itself as well as the supportive extraembryonic tissues.

Pluripotent: cells with robust self-renewal properties and unbiased potential to form all somatic cell lineages and germ cells; these are the embryonic stem cells (ESCs) isolated from the ICM of the blastocyst.

Multipotent: cells that can give rise to all cell types within the same lineage; these are progenitor cells, for example the hematopoietic cells, which can differentiate into the different types of blood cells.

Bi/Unipotent: these cells can also be referred to as progenitor cells as they still retain the ability to proliferate but are committed to differentiate into one or two specific cell types [START_REF] Daley | Stem cells and the evolving notion of cellular identity[END_REF][START_REF] Dulak | Adult stem cells: Hopes and hypes of regenerative medicine[END_REF][START_REF] Martello | The Nature of Embryonic Stem Cells[END_REF].

For many years, embryonic development has been depicted as a one-way process and compared to a ball rolling downhill, with the range of a cell's fate becoming progressively restricted as if cells would roll towards a stable differentiated state from which it was impossible to "escape" [START_REF] Daley | Stem cells and the evolving notion of cellular identity[END_REF][START_REF] Ohnuki | Present and future challenges of induced pluripotent stem cells[END_REF]. However, in the late 1980's, ground breaking experiments led to the discovery that pluripotency is a transcription factor-determined state and can therefore be experimentally induced even in fully differentiated somatic cells, opening the era of the induced pluripotent stem cells [START_REF] Martello | The Nature of Embryonic Stem Cells[END_REF][START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF].

The classical function of somatic stem cells, found in adults within specific germinal niches, is that to sustain homeostatic tissue turnover and enable repair throughout life [START_REF] Martello | The Nature of Embryonic Stem Cells[END_REF]. Multipotent stem cells are able to divide symmetrically, to expand the stem cell pool or asymmetrically, to maintain the stem cell pool while giving rise to the cell types of the tissue in which they reside [START_REF] Xu | Building a central nervous system: The neural stem cell lineage revealed[END_REF]. The direct descendants of stem cells are progenitor cells: cells with a more restricted lineage potential but higher proliferative capacity [START_REF] Xu | Building a central nervous system: The neural stem cell lineage revealed[END_REF].

Adult stem cells can be found in either a quiescent or an active state. The latter are responsible for replenishment of the tissue, while the former are a backup to replace active stem cells. The reasons behind this strategy may be to prevent exhaustion of the stem cell pool and the accumulation of potentially tumorigenic mutations. The quiescent pool can be activated by stochastic mechanisms, feedback signals derived from the loss of the active stem cells, or by extensive tissue damage. The coexistence of these two different pools in the same regions highlights the importance of the presence of a niche where specific factors are produced to maintain the specific proliferative state of the cells [START_REF] Li | Coexistence of Quiescent and Active Adult Stem Cells in Mammals[END_REF].

EMBRYONIC AND POSTNATAL DEVELOPMENT

Neurogenesis occurs during embryonic life

ESCs can be isolated from the developing mouse blastocyst around embryonic day (E) 3.5; by E6.5 the ICM comprises three germ layers: the endoderm, the mesoderm and the ectoderm [START_REF] Ohnuki | Present and future challenges of induced pluripotent stem cells[END_REF][START_REF] Xu | Building a central nervous system: The neural stem cell lineage revealed[END_REF]. The endoderm will form the digestive tube with

the glands that open into it, the urinary bladder, the epithelial parts of trachea, the lungs, the thyroid, the parathyroids [START_REF] Kiecker | Molecular specification of germ layers in vertebrate embryos[END_REF][START_REF] Solnica-Krezel | Gastrulation: Making and Shaping Germ Layers[END_REF]; the mesoderm will form muscles, bones, cartilage, connective tissue, adipose tissue, the circulatory system, the lymphatic system, dermis, the genitourinary system, serous membranes, and the notochord [START_REF] Kiecker | Molecular specification of germ layers in vertebrate embryos[END_REF][START_REF] Solnica-Krezel | Gastrulation: Making and Shaping Germ Layers[END_REF][START_REF] Takemoto | Mechanism of cell fate choice between neural and mesodermal development during early embryogenesis[END_REF]; finally the ectoderm will develop in the surface ectoderm, from which epidermis, hair, nails will derive; the neural crests that will give peripheral nervous system, melanocytes [START_REF] Nitzan | Neural crest and somitic mesoderm as paradigms to investigate cell fate decisions during development[END_REF]; and the neural tube that gives rise to the brain, spinal cord, posterior pituitary and the retina [START_REF] Kiecker | Molecular specification of germ layers in vertebrate embryos[END_REF][START_REF] Solnica-Krezel | Gastrulation: Making and Shaping Germ Layers[END_REF][START_REF] Takemoto | Mechanism of cell fate choice between neural and mesodermal development during early embryogenesis[END_REF].

The process of invagination and closure of the ectoderm, through which the neural tube is formed, is called neurulation; this is a finely regulated process divided into different steps, each of them dependent on the establishment of precise morphogen gradients [START_REF] Takemoto | Mechanism of cell fate choice between neural and mesodermal development during early embryogenesis[END_REF]. The neural tube will give rise to the central nervous system (CNS) therefore being subjected to a rostro-caudal as well as dorso-ventral differentiation process. In its rostral portion, from which the brain will derive, the neural tube balloons into three primary vesicles: the forebrain (cerebral hemispheres, thalamus, hypothalamus, and retina), the midbrain (tectum and motor pathways of the basal ganglia), and the hindbrain (cerebellum and medulla oblongata) [START_REF] Darnell | Neuroembryology[END_REF]. The inner cavity of these vesicles will later be filled with cerebrospinal fluid (CSF) and will give the four ventricles. Meanwhile the dorsoventral axis of the more caudal portion, which will give rise to the spinal cord, is modified by signals coming from the immediate environment: the dorsal side receives sensory inputs, whereas motor signals emanate from the ventral side [START_REF] Darnell | Neuroembryology[END_REF].

Neurons and macroglia of the brain derive from neuroepithelial cells that can be called embryonic neural stem cells (eNSCs), which line the cerebral ventricles, and undergo different amplification steps giving rise to intermediate progenitor cells (IPCs) (Fig. 1).

Cortical neurogenesis begins around E9-10 in mice. By this time, neuroepithelial cells begin to acquire features of glial cells, such as expression of astroglial markers like GLAST (glutamate aspartate transporter), BLBP (brain lipid binding protein), nestin, vimentin and are referred to as radial glia (RG) (Kriegstein & Alvarez-Buylla, 2009;[START_REF] Rowitch | Developmental genetics of vertebrate glial-cell specification[END_REF]. These cells also express GFAP (glial fibrillary acidic protein) in some species, such as in primates, and in mice only from E16 [START_REF] Choi | Prenatal gliogenesis in the developing cerebrum of the mouse[END_REF]. RGs stay in contact with the surface of the ventricle and the pia, with their cell bodies found at the level of the ventricular zone (VZ) and connected by adherens junctions (Kriegstein & Alvarez-Buylla, 2009).

RG cells proliferate and divide asymmetrically to maintain the eNSC pool and to generate either a neuron or an IPC. These IPCs can be committed to the neuronal lineage (nIPC) or to the oligodendrocyte one (oIPC) (Fig. 1).

During cell division, the pial endfeet of the RGs do not retract and the daughter neurons often migrate along these processes. IPCs in turn divide symmetrically to generate either two IPCs or two neurons. RGs but not IPCs are characterised by interkinetic nuclear migration during their cell cycle progression, with nuclei in M-phase being closer to the ventricular surface, nuclei in S-phase migrating towards the apical side and nuclei in G1 or G2-phase found in an intermediary position between the two (Kriegstein & Alvarez-Buylla, 2009). RG cells are able to sense the gradients of the different morphogens that characterise each region of the developing CNS and in turn respond by producing different subtypes of neurons according to their position and stage of development, completing the process of neurogenesis before birth (Kriegstein & Alvarez-Buylla, 2009;[START_REF] Rowitch | Developmental genetics of vertebrate glial-cell specification[END_REF] (Fig. 1).

Gliogenesis occurs during postnatal life

1.2.2.1 Astrogenesis At the end of the embryological development, RG cells disappear in most brain regions (Kriegstein & Alvarez-Buylla, 2009). They will either self-consume by symmetric neurogenic divisions or generate glial and ependymal cells; however, in many mammalian species, RGs can still be found in the adult brain acting as NSCs (Götz et al., 2015;Kriegstein & Alvarez-Buylla, 2009) (see chapter 1.3). The translocation of RG and transformation to astrocytes, mostly studied in the cortex, has been directly visualised by using retroviral labelling and time-lapse imaging [START_REF] Noctor | Distinct behaviors of neural stem and progenitor cells underlie cortical neurogenesis[END_REF].

Since neurons and astrocytes derive from the same progenitor cells, a tight regulation of the switch from a neuronal to an astrocytic potential, which relies upon exogenously secreted cues and intrinsic chromatin changes, is critical for normal brain formation. The main mechanism by which astrogenesis is repressed is by inactivation of the JAK/STAT pathway which plays an important role in astrogenesis. Once JAKs are activated, by autophosphorylation, they phosphorylate STAT3 which forms homodimers that are translocated into the nucleus to induce the expression of astrocytic genes, such as GFAP, therefore promoting astrocytic differentiation [START_REF] Mallamaci | Developmental control of cortico-cerebral astrogenesis[END_REF][START_REF] Qian | Timing of CNS Cell Generation[END_REF].

Moreover, early in forebrain development, the promoters of GFAP and S100B are methylated, therefore silenced, and neuroepithelial cells are insensitive to cytokines. At the onset of astrogenesis, RG cells change their differentiation competence through demethylation of the promoters of astrocytic genes, possibly through epigenetic regulation as for instance the chromatin state of the GFAP promoter changes significantly to allow active gene transcription [START_REF] Mallamaci | Developmental control of cortico-cerebral astrogenesis[END_REF]. Even though the precise timing of this process remains unclear, due to a lack of definitive markers to distinguish astrocyte precursors from radial glia, it is clear that astrocyte number increases abruptly after neurogenesis completion. Accordingly, GFAP + cells, still rare at birth (< 2.0%), double in number every 3-4 days in the first postnatal week, peaking around P3 [START_REF] Qian | Timing of CNS Cell Generation[END_REF] (Fig. 1) and reaching their absolute plateau around P21 [START_REF] Bandeira | Changing numbers of neuronal and nonneuronal cells underlie postnatal brain growth in the rat[END_REF]. The injection of enhanced green fluorescence protein (GFP)-encoding retroviruses into the cortex of P0-P2 mice showed that almost 50% of astrocytes were labelled 10 days after the injection, suggesting that most astrocytes are generated by local proliferating progenitors [START_REF] Mallamaci | Developmental control of cortico-cerebral astrogenesis[END_REF]. Moreover at this stage, neurons secrete gliogenic cytokines promoting gliogenesis themselves. Once new astrocytes are generated glial identity needs to be retained and in order to ensure that, neuronal genes must be repressed. Disruption in any of these mechanisms can potentially affect the timing or efficiency of neurogenesis and astrogenesis, altering the relative ratio between these two cell types, which may contribute to the progression of a variety of neurodevelopmental disorders [START_REF] Sloan | Mechanisms of astrocyte development and their contributions to neurodevelopmental disorders[END_REF].

Oligodendrogenesis

Oligodendrocytes originate in multiple locations and at different moments during development, starting during embryogenesis and continuing during postnatal development.

Oligodendrocyte progenitor cells are nerve/glial antigen 2 (NG2)-expressing proliferating cells distributed throughout the brain; the origin of these cells remains unclear, but they are likely to be derived from RG cells [START_REF] Nishiyama | Lineage, fate, and fate potential of NG2-glia[END_REF].

NG2 cells can be considered as a type of oligodendrocyte precursors that have not yet become terminally differentiated into oligodendrocytes, and which remain capable of symmetrically divide to replenish their pool, throughout all stages of development. A property of NG2 cells that distinguishes them from astrocytes is their robust migratory ability. NG2 cells reach their peak density during the first postnatal week, after which it declines even though they will occupy defined domains through the entire parenchyma.

Genetic fate mapping studies have shown that NG2 cells undergo proliferation in response to a stab wound and some differentiate into oligodendrocytes but not astrocytes.

Nevertheless, it has been shown that NG2 cells could be manipulated to differentiate into neurons in the injured CNS [START_REF] Nishiyama | Lineage, fate, and fate potential of NG2-glia[END_REF].

NSCs IN THE ADULT BRAIN: THE TWO CANONICAL REGIONS

NCSs can still be found in the adult brain within restricted regions where they can give rise to glial as well as neuronal cells (Alvarez-Buylla & Lim, 2004;[START_REF] Gage | Mammalian Neural Stem Cells[END_REF]Ming & Song, 2005;[START_REF] Nottebohm | The road we travelled: Discovery, choreography, and significance of brain replaceable neurons[END_REF]. The two most studied and characterised regions, where the presence of adult NSCs (aNSCs) has been widely accepted, are the subventricular zone of the lateral ventricles (SVZ), adjacent to the ependymal ciliated single cell layer that lines the wall of the lateral ventricles, and the subgranular zone of the dentate gyrus of the hippocampus (SGZ) (Alvarez-Buylla & Lim, 2004;[START_REF] Gage | Mammalian Neural Stem Cells[END_REF]Ming & Song, 2005). However, increasing evidences are pointing towards the hypothesis that more regions retain the ability to produce new neural cells, such as the striatum [START_REF] Bédard | Chemical characterization of newly generated neurons in the striatum of adult primates[END_REF][START_REF] Luzzati | Adult neurogenesis and local neuronal progenitors in the striatum[END_REF][START_REF] Nato | Striatal astrocytes produce neuroblasts in an excitotoxic model of Huntington's disease[END_REF] and the hypothalamus (Djogo et al., 2016;Kokoeva et al., 2005;Mcnay et al., 2012;Migaud et al., 2010;Pencea et al., 2001;Sharif et al., 2014;[START_REF] Xu | Neurogenesis in the ependymal layer of the adult rat 3rd ventricle[END_REF] (see chapter 1.7.3).

The origin of aNSCs in the SVZ has been identified only recently in a subpopulation of quiescent eNSCs [START_REF] Fuentealba | Embryonic Origin of Postnatal Neural Stem Cells[END_REF][START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF]. It appears that around E13.5 and 15.5, a subset of eNSCs strongly slow down their cell cycle, maintaining their undifferentiated state and in this way persisting until postnatal stages when they become reactivated. The rest of them continue dividing to ensure the development of the CNS and are lost during the process [START_REF] Furutachi | Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells[END_REF]. Moreover, it has been shown that as early as E11.5, these cells have acquired a regional specification, due probably to the expression of distinct combinations of transcription factors (TFs), which they will retain throughout postnatal life and that will determine their progeny [START_REF] Fuentealba | Embryonic Origin of Postnatal Neural Stem Cells[END_REF].

NSCs of the DG derive from stem cells located in the subpallium region during embryonic development. However, their exact embryonic origin has not been fully elucidated since the DG is generated by a mosaic of stem cells with different embryonic origins. Embryonic formation of the DG begins around E13.5 in mice. Hippocampal RG cells, which express typical markers such as nestin and GLAST at E15.5, proliferate in a domain of the ventricular zone called the dentate neuroepithelium, which is adjacent to the cortical hem [START_REF] Li | The Ventral Hippocampus Is the Embryonic Origin for Adult Neural Stem Cells in the Dentate Gyrus[END_REF][START_REF] Rolando | Neural Stem Cell of the Hippocampus. Development, Physiology Regulation, and Dysfunction in Disease[END_REF]. From here, these cells will migrate through the dentate migratory stream and spread, covering the DG. At the end of the first postnatal week, following a process of reorganisation, NSCs are found in the SGZ where they will persist throughout life [START_REF] Rolando | Neural Stem Cell of the Hippocampus. Development, Physiology Regulation, and Dysfunction in Disease[END_REF].

Identity of aNSCs -to be or not to be

Adult NSCs are not a homogeneous population. It is true that the classical definition of stem cells implies multipotency and self-renewal. However, these characteristics often emerge only at a population level. This means that most aNSCs are committed to a specific cell type or even to a neuronal subtype; besides, most of them remain quiescent most of their life making them difficult to identify and isolate as there is no single specific marker for aNSCs or progenitors (Kriegstein & Alvarez-Buylla, 2009;[START_REF] Magnusson | Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions[END_REF]) (Fig. 2). These cells retain some features typical of the RG cells and were phenotypically identified as classical astrocytes. Like RGs, they present a primary cilium, localised on their apical surface, which expresses the glycoprotein prominin 1 (CD133 in human), and a long basal process which contacts surrounding blood vessels in mice (Mirzadeh et al., 2008) (Fig. 2). At the same time, they express astrocytic genes such as GFAP or GLAST and they share their ultrastructural and electrophysiological characteristics (Götz et al., 2015;Kriegstein & Alvarez-Buylla, 2009). This brought up the question of whether all astrocytes retain a latent capacity to behave as stem cells. Only astrocytes of the SVZ and SGZ are clearly specialised and behave as NSCs, but the intrinsic potential of parenchymal astrocytes is not well known yet.

For instance, debates are still ongoing on whether new neurons are produced in the striatum and what their physiological function might be. Low levels of striatal neurogenesis have been reported under physiological conditions in rabbits, rats and squirrel monkeys [START_REF] Bédard | Proliferating cells can differentiate into neurons in the striatum of normal adult monkey[END_REF][START_REF] Dayer | New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors[END_REF][START_REF] Magnusson | A latent neurogenic program in astrocytes regulated by Notch signaling in the mouse[END_REF][START_REF] Luzzati | Neurogenesis in the Caudate Nucleus of the Adult Rabbit[END_REF], although these data have not always been replicated [START_REF] Magnusson | Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions[END_REF]. Moreover, strokes triggeres bursts of proliferation in striatal astrocytes in rodents [START_REF] Magnusson | A latent neurogenic program in astrocytes regulated by Notch signaling in the mouse[END_REF], increasing neurogenesis in this region; this capacity to remain quiescent and being activated following a stimulus is reminiscent of aNSCs behaviour [START_REF] Magnusson | Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions[END_REF].

The situation changes in other areas. For instance, cortical astrocytes, can reactivate their proliferation within a week after a stab wound injury [START_REF] Buffo | Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain[END_REF][START_REF] Sirko | Reactive glia in the injured brain acquire stem cell properties in response to sonic hedgehog glia[END_REF]; these subsets of proliferating astrocytes are enriched in the vicinity of the vasculature [START_REF] Bardehle | Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation[END_REF] and, even though they express some more immature markers such as nestin, they remain within their lineage, not showing neurogenic capabilities in vivo [START_REF] Bardehle | Live imaging of astrocyte responses to acute injury reveals selective juxtavascular proliferation[END_REF][START_REF] Buffo | Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain[END_REF]. These differences might be explained in part by the changing microenvironment. For example, experiments where adult progenitor cells of the SVZ or SGZ, were transplanted into the cortex showed that they would lose the ability to generate neurons and would only give rise to glial cells, proving that the cortex is a nonpermissive environment for neuronal differentiation [START_REF] Herrera | Selective suppression of the primary surge of follicle-stimulating hormone in the rat: further evidence for folliculostatin in porcine follicular fluid[END_REF]. Nevertheless, astrocytes are extremely heterogeneous in their form and function throughout the brain, as well as in their mRNA expression profile patterns to an extent that has only started to be unfolded.

Cell differentiation is a complex process involving many different transcription factors which interact to build a fixed network that will determine a cell's properties and identity; such a network will also be self-stabilised by interactions among its different components [START_REF] Magnusson | Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions[END_REF]. However, these transcriptional networks can present variable degrees of stability, giving in turn to the cell different grades of plasticity; in such a case two or more transcriptional networks are expressed and compete against each other keeping the cell on the edge between them two, a situation where even small perturbations would trigger a switch in the cell's identity [START_REF] Graf | Forcing cells to change lineages[END_REF]. The hypothesis is that astrocytes may represent an example of such a state, with no distinct line between "classical" astrocytes and aNSCs but more like a continuum from parenchymal astrocytes that would be able to dedifferentiate to a multipotent intermediate state [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF][START_REF] Magnusson | Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions[END_REF]. In this case, astrocytes from non-neurogenic niches, such as the cortex, may not lack neurogenic capacity but rather possess a less plastic astrocytic identity [START_REF] Magnusson | Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions[END_REF]. For instance, it has been shown that disruption of Notch signalling or overexpression of Sex-determining region Y (SRY)-related HMGbox 2 (SOX2) activates a neurogenic state in mouse striatal astrocytes [START_REF] Magnusson | A latent neurogenic program in astrocytes regulated by Notch signaling in the mouse[END_REF][START_REF] Niu | In vivo reprogramming of astrocytes to neuroblasts in the adult brain[END_REF]. Thus, it appears that Notch promotes astrocyte differentiation while Sox2 stabilises a neurogenic network including activation of Ascl1, which regulates many neurogenic genes [START_REF] Magnusson | Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions[END_REF].

The same approach applies to the switch between a quiescent and an active state within aNSCs; quiescence is a reversible state where cell cycle is arrested but can be rapidly reinitiated in response to environmental cues. The use of single-cell analysis techniques has allowed the characterisation of what are now considered as two different populations within the aNSC pool. In different studies, aNSCs have been purified from both the SVZ and the SGZ, taking advantage of their coexpression of GFAP, or GLAST, and prominin 1 (Fig. 2). In the case of the SVZ, this purified population has proved to divide, to give rise to olfactory bulb (OB) interneurons with no preference towards a specific subtype and these cells were not found in the parenchyma [START_REF] Beckervordersandforth | In vivo fate mapping and expression analysis reveals molecular hallmarks of prospectively isolated adult neural stem cells[END_REF][START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF][START_REF] Shin | Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis[END_REF]. The first feature that allowed the authors to distinguish between active and quiescent NSCs was the lack of EGF receptor (EGFR) expression in the latter [START_REF] Codega | Prospective Identification and Purification of Quiescent Adult Neural Stem Cells from Their In Vivo Niche[END_REF][START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF] (Fig. 2). Single-cell transcriptomic analysis revealed a decrease in the expression of glial genes going through the intermediate states that bring from a quiescent to an active state. Quiescent NSCs are characterised by a glycolytic metabolism, low expression levels of genes involved in protein synthesis and high levels of membrane receptors, cell-cell adhesion molecules and ion channels, probably to promptly sense changes in the microenvironment and respond to them [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF]; they are also characterised by strong expression of Notch receptors and Notch's target genes and surprisingly, at the level of the SVZ, they did not show expression of Nestin [START_REF] Codega | Prospective Identification and Purification of Quiescent Adult Neural Stem Cells from Their In Vivo Niche[END_REF][START_REF] Shin | Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis[END_REF] (Fig. 2).

On the contrary, active NSCs prefer a lipidic metabolism, they express high levels of genes involved in protein synthesis and cell cycle, as they enter mitosis, they also highly express Notch ligands, suggesting the possibility they may act on the quiescent pool to maintain them in a dormant state through Notch signalling [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF]. At the same time however, it appears that quiescent NSCs may regulate their dormancy by an autocrine control since they express high levels of the bone morphogenetic proteins (BMP) signalling pathway, known to promote quiescence [START_REF] Mira | Signaling through BMPR-IA regulates quiescence and long-term activity of neural stem cells in the adult hippocampus[END_REF], i.e. BMP receptor, BMP ligands and targets. Finally, quiescent NSCs are able to enter a primed-quiescent state upon brain injury, switching towards the more active state [START_REF] Llorens-Bobadilla | Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury[END_REF][START_REF] Shin | Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis[END_REF]. Codega et al. 2014, Neuron;Llorens-Bobadilla et al. 2015, Cell Stem Cell;Shin et al. 2015, Cell Stem Cell).

A NICHE OF ONE'S OWN

In contrast to embryonic development, when stem cells are surrounded by a continuously changing environment, aNSCs are stable and housed in a well-defined niche that supports their lifelong maintenance and that includes ependymal cells (in the case of the SVZ), organised vasculature, extracellular matrix, immune cells and axonal terminals. [START_REF] Lim | Adult neural stem cells stake their ground[END_REF][START_REF] Magnusson | Stars from the darkest night: unlocking the neurogenic potential of astrocytes in different brain regions[END_REF]. Moreover, aNSCs are anchored to surrounding endothelial and ependymal cells, a state seen to promote quiescence [START_REF] Ottone | Direct cell-cell contact with the vascular niche maintains quiescent neural stem cells[END_REF].

Stem cell niches express a plethora of different factors and signals deriving from their different constitutive elements: the local vasculature is a source of factors coming from the blood as well as from the endothelial cells themselves; neurotransmitters, such as glutamate, GABA, serotonin, dopamine, are derived from both local neurons and external innervations (Chaker et al., 2016;[START_REF] Fuentealba | Adult neural stem cells bridge their niche[END_REF]. Also the immature neuroblasts, found in intimate contact with NSCs, are able to regulate their own production through different pathways. For instance, they express high levels of Ascl1, known to promote the expression of Notch ligands; as previously said, Notch signalling induces a more quiescent state, suggesting a possible feedback mechanism by direct cell-cell contact (Chaker et al., 2016;[START_REF] Fuentealba | Adult neural stem cells bridge their niche[END_REF]. Furthermore, immature neuroblasts can release GABA, thus activating GABA A receptors on precursor cells inducing their depolarisation with consequent increased Ca 2+ levels and enhanced NeuroD expression [START_REF] Liu | Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAP-expressing progenitors[END_REF][START_REF] Tozuka | GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells[END_REF].

This, in turn, inhibits progenitor proliferation and promotes neuronal differentiation [START_REF] Liu | Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAP-expressing progenitors[END_REF][START_REF] Tozuka | GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells[END_REF], acting again as a negative feedback apt to downregulate their own production (Chaker et al., 2016;[START_REF] Fuentealba | Adult neural stem cells bridge their niche[END_REF]. High levels of BMP signalling promote glial over neuronal differentiation. This signalling is at least partially controlled by the niche as ependymal cells in the SVZ, secrete the BMP antagonist Noggin, and express LRP2, a receptor that sequesters BMP, both of which are required for neurogenesis in vivo.

In the SGZ, radial astrocytes express Noggin, whose levels increase with physical exercise and which promotes neurogenesis [START_REF] Fuentealba | Adult neural stem cells bridge their niche[END_REF][START_REF] Gajera | LRP2 in ependymal cells regulates BMP signaling in the adult neurogenic niche[END_REF].

The SVZ niche

The SVZ is a thin layer of dividing cells located along the walls of the lateral ventricles between the multiciliated ependymal layer and a planar vascular plexus. 2 and 3). These neuroblasts then migrate along the rostral migratory stream (RMS) to reach the OBs where they mature into different subpopulations of inhibitory interneurons of both the granular and periglomerular layers where they will contribute to olfactory learning and discrimination (Alvarez-Buylla & Lim, 2004;[START_REF] Lim | Adult neural stem cells stake their ground[END_REF][START_REF] Tong | SnapShot: Adult Neurogenesis in the V-SVZ[END_REF].

The population of type B cells is not homogeneous. A viral lineage tracing experiment targeting different areas of the adult SVZ showed a strong regional heterogeneity: each region is characterised by the expression of specific TFs, reminiscent of the pattern seen during embryonic development, and cells of each TF domain give rise to specific subtypes of interneurons integrated in different regions of the OB; more importantly, this regionality is already acquired during embryonic development [START_REF] Fuentealba | Embryonic Origin of Postnatal Neural Stem Cells[END_REF]. Type B cells, mainly of the dorsal SVZ, are also able to produce astrocytes of both the corpus callosum and RMS [START_REF] Sohn | The Subventricular Zone Continues to Generate Corpus Callosum and Rostral Migratory Stream Astroglia in Normal Adult Mice[END_REF] and oligodendrocytes of the corpus callosum [START_REF] Ortega | Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signalling[END_REF], in a Sonic hedgehog (Shh)-and Wnt-dependent manner [START_REF] Ortega | Oligodendrogliogenic and neurogenic adult subependymal zone neural stem cells constitute distinct lineages and exhibit differential responsiveness to Wnt signalling[END_REF].

Type B cells are found in a very specific and well characterised niche with a peculiar pinwheel organisation at the ventricular surface. The pinwheel structure is given by the large apical surfaces of the ependymal cells (E1), always containing at least 50 motile cilia, packed around B cells clustered together in the centre with a small apical surface where a short single primary cilium, characterised by the expression of CD133, is found (Mirzadeh et al., 2008) (Fig. 3). This cilium allows B cells to remain in contact with the lumen of the ventricles, filled with the CSF, whose production and composition are dynamically regulated by the choroid plexus, therefore exposing B cells to a constantly changing flux of factors [START_REF] Silva-Vargas | Age-Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells[END_REF]. Intermingled between E1 and B cells, rare biciliated ependymal cells (E2)

were also described, however their function has not been understood yet. Both E1 and E2 cells could be recognised by their expression of CD24, never found in B cells (Fig. 2).

Moreover, E1 and E2 were never found to divide as they were never seen to express Ki67, a marker of cell division expressed throughout the cell cycle, or to incorporate bromodeoxyuridine (BrdU), an analog of thymidine incorporated into the DNA during its synthesis, that is extensively used to label dividing cells (Mirzadeh et al., 2008;[START_REF] Taupin | BrdU immunohistochemistry for studying adult neurogenesis: Paradigms, pitfalls, limitations, and validation[END_REF].

On the other side, B cells present long GFAP + basal processes, running tangential to the wall of the lateral ventricle, almost always terminating in specialised endfeet on a blood vessel (Fig. 3). Moreover, these processes contribute to form the gliotubes along which the immature neuroblasts migrate (Mirzadeh et al., 2008). While the cilium at the apical surface lets B cells sense factors of the CSF, their endfeet give them access to blood-derived factors.

As a matter of fact, clusters of dividing cells are associated with blood vessels where the blood brain barrier (BBB) appears to be leaky, due to the lack of astrocytic or pericytic coverage, therefore allowing entrance of small molecules from the circulation [START_REF] Tavazoie | A Specialized Vascular Niche for Adult Neural Stem Cells[END_REF].

The choroid plexus, a vascularized epithelial structure floating within the brain ventricles, has been recently identified as an active part of the SVZ niche (silva-vargas 2016). This complex structure, able to sense signals coming from the blood and the niche itself and to modulate its secretome accordingly, is essential for physiological brain homeostasis, immune surveillance and inflammatory and repair processes. It has been now shown that it can also affect quiescent and active NSCs as well as intermediate progenitors and neuroblasts within the SVZ niche [START_REF] Silva-Vargas | Age-Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells[END_REF]. BMPs, Wnts (which support oligodendrogenesis), Shh are just some of the factors that can be found in the CSF and that modulate the behaviour of SVZ aNSCs. With aging, the secretome of the choroid plexus is subjected to important modifications, to which only active aNSCs appear to be sensitive, suggesting how it may contribute to the decline in neurogenesis that occurs in older animals [START_REF] Silva-Vargas | Age-Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells[END_REF]. surrounded by multi-ciliated ependymal cells (E) forming pinwheel-like structures. B1 cells give rise to transit-amplifying cells, C cells (green), which divide to generate neuroblasts (type A cells, red). B1 present a thin apical process containing a primary cilium that contacts the lateral ventricle (V), being in direct contact with the CSF, and a long basal process ending on blood vessels (BV, purple). (From Fuentealba et al. 2012, Cell stem Cell).

The SGZ niche

The SGZ is located at the interface between the granule cell layer and the hilus of the DG, therefore the niche is not in contact with the ventricles (Fig. 4). Here the local aNSCs, also referred to as type I progenitors, exhibit a radial process spanning the granule cell layer and arborizing profusely in the molecular layer and express nestin, GFAP, blbp and Sox2 (Fig 2 and4). They give rise to intermediate progenitors, type II progenitors or D cells, characterised by a high proliferative activity, short processes and expression of early neuronal markers (DCX, PSA-NCAM) while they stop expressing GFAP, as they are already committed to a neuronal fate (Fig 2 and4). D cells produce type III cells, which do not show proliferation capacity and will mature into glutamatergic granule cells of the DG (G cells) (Kempermann et al., 2015) (Fig. 4). Moreover, astrocytes, but not oligodendrocytes, were found to be generated in the SGZ, from which they migrate into the hilus, the granule cell layer and the molecular layer [START_REF] Bonaguidi | In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics[END_REF].

Just like B cells, type I cells are highly polarised (Fig. 4); the side facing the hilus presents a primary cilium that has been shown to be necessary for the expansion and establishment of postnatal hippocampal progenitors through activation of Shh signalling [START_REF] Breunig | Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling[END_REF] Y.-G. [START_REF] Han | Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells[END_REF]. Dividing type I progenitors in the SGZ are in close proximity to an extensive network of interconnected blood vessels (Fig. 4). Unlike the SVZ, where endothelial cells are very rarely seen to divide, in the SGZ active angiogenesis is associated with neurogenesis [START_REF] Palmer | Vascular niche for adult hippocampal neurogenesis[END_REF]. Increased angiogenesis results from increased expression of the vascular endothelial growth factor (VEGF), which is also associated with increased progenitor proliferation and neurogenesis, even though its action has not been fully understood [START_REF] Fuentealba | Adult neural stem cells bridge their niche[END_REF]. Furthermore, type I progenitors present an elaborate and extensive set of thin branches in the inner molecular layer of the DG where GABAergic and glutamatergic inputs arrive, as well as dopaminergic, serotonergic or acetylcholinergic projections from other brain regions [START_REF] Zhao | Distinct Morphological Stages of Dentate Granule Neuron Maturation in the Adult Mouse Hippocampus[END_REF]. Disruption of these different neurotransmitter and input systems perturbs adult neurogenesis; however, the specific contribution of each individual system has not been yet identified (Kempermann et al., 2015). Mature granule cells (GCs, brown) send an axon parallel to the SGZ into the hilus, whereas their dendrites branch into the molecular layer (ML). Radial astrocytes are polarized cells with their cell body in the SGZ, where they contact blood vessels (BV, purple) and neighboring radial astrocytes, a long main process that traverses through the granule cell layer (GCL) and then branches diffusely in the inner molecular layer (IML). (From Fuentealba et al. 2012, Cell stem Cell).

PHYSIOLOGICAL RELEVANCE OF ADULT NEUROGENESIS

Neurogenesis increases plasticity on multiple levels; while adding new cells to existing circuits it causes structural remodelling, synaptogenesis, and changes in synaptic strength.

Neurogenesis encompasses different stages: from cell birth through fate determination, survival, until integration and acquisition of functional properties. Of particular relevance is the selection occurring through modulation of the survival rate of the newborn neuroblasts both in the SVZ and the DG that ensures at any moment a readily available pool of cells. All these processes are sensitive to environmental signals and are subject to reduction with aging, possibly through the activation of the hypothalamic-pituitary-adrenal axis [START_REF] Danzer | Depression, stress, epilepsy and adult neurogenesis[END_REF][START_REF] Fuchs | Adult neuroplasticity: More than 40 years of research[END_REF]Gonçalves et al., 2016;[START_REF] Lledo | Adult neurogenesis and functional plasticity in neuronal circuits[END_REF][START_REF] Van Praag | Functional neurogenesis in the adult hippocampus[END_REF].

A role for adult neurogenesis in the SVZ

As previously mentioned, neuroblasts deriving from the SVZ migrate along the RMS, closely associated with each other along a tube-like structure formed by glial cells [START_REF] Peretto | Glial tubes in the rostral migratory stream of the adult rat[END_REF], to reach the OBs where, as early as two weeks later, granule and periglomerular cells can be found in their respective OB layers. These cells make only local contacts in the bulb, directly or indirectly modulating the processing of sensory information [START_REF] Lledo | Adult neurogenesis and functional plasticity in neuronal circuits[END_REF]Lledo & Valley, 2016).

An enriched odour exposure increases the SVZ but not SGZ neurogenesis. The adult-born neurons indeed are more inclined to respond to novel odours and are preferentially activated by the recollection of an odour-reward memory. Therefore, the main role of neurogenesis in the adult SVZ seems to be that to mediate odour-associated learning, allowing an adaptable consolidation of odour-associated memories. This plasticity appears to be achieved by a highly dynamic formation and elimination of dendrodendritic synapses with principal neurons of the OB (mitral and tufted cells) [START_REF] Sakamoto | Continuous Postnatal Neurogenesis Contributes to Formation of the Olfactory Bulb Neural Circuits and Flexible Olfactory Associative Learning[END_REF].

The situation changes in pathological conditions. For instance, ischemic brain insults in the cortex stimulate progenitor proliferation in the SVZ of adult rodents [START_REF] Buffo | Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain[END_REF]. In an experimental stroke model, immature neurons also migrated from the SVZ to the damaged striatal area where they expressed markers for striatal spiny neurons, as to replace the damaged ones. None of these neurons however survive in the environment of the striatum [START_REF] Arvidsson | Neuronal replacement from endogenous precursors in the adult brain after stroke[END_REF][START_REF] Magnusson | A latent neurogenic program in astrocytes regulated by Notch signaling in the mouse[END_REF]. Seizure is also known to stimulate proliferation in the SVZ and to alter neuronal precursor migration in the rat brain by increasing the number of neuroblasts found in the RMS and by inducing a portion of them to exit the RMS prematurely or to migrate ectopically even though the majority of them will fail to survive [START_REF] Parent | Prolonged seizures increase proliferating neuroblasts in the adult rat subventricular zone-olfactory bulb pathway[END_REF].

A role for adult neurogenesis in the SGZ

Newborn neurons of the DG migrate only a short distance to reach the inner granule cell layer from where they rapidly extend long axonal projections to the CA3 pyramidal cell layer within 4 to 10 days after division [START_REF] Markakis | Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles[END_REF], while their dendrites grow in the opposite direction, reaching the molecular layer within two weeks (Ming & Song, 2005;[START_REF] Whitman | Synaptic Integration of Adult-Generated Olfactory Bulb Granule Cells: Basal Axodendritic Centrifugal Input Precedes Apical Dendrodendritic Local Circuits[END_REF]. Nevertheless, as early as 7 days after division, they begin to show GABAergic inputs, followed by glutamatergic ones around two weeks after cell birth (Ming & Song, 2005;[START_REF] Whitman | Synaptic Integration of Adult-Generated Olfactory Bulb Granule Cells: Basal Axodendritic Centrifugal Input Precedes Apical Dendrodendritic Local Circuits[END_REF].

Neurogenesis in the DG is influenced by many signals including adrenal steroids, seizure, exercise, inflammation and antidepressants (Kriegstein & Alvarez-Buylla, 2009). The role of these newborn neurons once integrated in the hippocampal circuits has been deeply studied and mainly linked to spatial learning, memory and possibly mood. Neurogenic activity and survival rate of newborn neurons in the SGZ are increased by physical activity and hippocampus-dependent learning tasks respectively [START_REF] Aimone | Regulation and Function of Adult Neurogenesis: From Genes to Cognition[END_REF]. Moreover, the ablation of adult neurogenesis impairs the ability of the mice to discriminate stimuli with little spatial separation [START_REF] Clelland | A Functional Role for Adult Hippocampal Neurogenesis in Spatial Pattern Separation[END_REF]. Hippocampal neurogenesis also exerts a role in the stress response: when this process is inhibited, a greater stress response has been observed in different behavioural tests in response to non-stressful stimuli [START_REF] Schloesser | Suppression of adult neurogenesis leads to an increased hypothalamo-pituitary-adrenal axis response[END_REF]. Furthermore, blockade of adult neurogenesis affects the response to antidepressants; in fact, antidepressant drugs recruit newborn neurons to re-establish hippocampal regulation of the HPA axis after chronic stress [START_REF] Surget | Antidepressants recruit new neurons to improve stress response regulation[END_REF].

Adult neurogenesis in the hippocampus is altered in various pathological conditions including epilepsy, stroke, degenerative neurological disorders, neuropsychiatric disorders, as well as in the case of alcohol intoxication [START_REF] Danzer | Depression, stress, epilepsy and adult neurogenesis[END_REF]. Ischemic injury increases neurogenesis in the SGZ. Most of the newborn cells differentiate into granule neurons, some of which mislocate in the hilus region for example. Also during epilepsy an increased however disrupted neurogenesis activity characterises the hippocampus with large numbers of adult-generated granule cells integrating abnormally into the DG participating in aberrant network reorganizations [START_REF] Walter | Pilocarpine-Induced Seizures Cause Selective Time-Dependent Changes to Adult-Generated Hippocampal Dentate Granule Cells[END_REF]. On the opposite side, stressful stimuli such as sleep deprivation, social isolation, exposure to predator odour, as well as a condition of depression, have all been found to reduce granule cell neurogenesis, most probably through mediation of corticosterone since adrenalectomy blocks the anti-neurogenic effects of each of these stimuli [START_REF] Danzer | Depression, stress, epilepsy and adult neurogenesis[END_REF].

The interplay with the reproductive function

1.5.3.1 The SVZ The olfactory system is implicated in the control of reproductive behaviour as it plays crucial roles in mating behaviour or interaction with the offspring.

Oestradiol influences SVZ cell proliferation in a dose-and time-dependent manner. If administered in an acute dose, it decreases cell proliferation, leading to a lower number of adult-born neurons in the OB of ovariectomised female mice [START_REF] Brock | Short term treatment with estradiol decreases the rate of newly generated cells in the subventricular zone and main olfactory bulb of adult female mice[END_REF]; chronic administration has no effect on the proliferative activity of the SVZ while it reduces the survival of newborn neurons in the main OB [START_REF] Veyrac | Postnatal and adult exposure to estradiol differentially influences adult neurogenesis in the main and accessory olfactory bulb of female mice[END_REF]) (Fig. 5). Pregnancy also affects adult neurogenesis in the SVZ; it has been shown that during this period, an increase in proliferation occurs in the SVZ, with newborn neurons found to be integrated in the circuits of the OB [START_REF] Shingo | Pregnancy-Stimulated Neurogenesis in the Adult Female Forebrain Mediated by Prolactin[END_REF]. In studies where pseudopregnancy was induced, it was shown that embryo implantation was not necessary for these effects, therefore pointing to a role for circulating maternal hormones in stimulating forebrain neurogenesis. In particular, these effects depend upon the action of prolactin, and appear specific to the SVZ/OB system [START_REF] Shingo | Pregnancy-Stimulated Neurogenesis in the Adult Female Forebrain Mediated by Prolactin[END_REF] (Fig. 5). Prolactin has been shown to mediate increased neurogenesis in both the SVZ and SGZ of male mice that physically interacted with their pups. Indeed, prolactin receptor null mice, albeit showing normal olfaction, do not recognise their own pups toward which they display aggression, as with cage intruders, behaviour that is associated to impaired neurogenesis in the two niches [START_REF] Mak | Paternal recognition of adult offspring mediated by newly generated CNS neurons[END_REF].

1.5.3.2 The SGZ Fluctuations in gonadal hormones can also modulate hippocampal neurogenesis in mice and rats. However, the effects of oestrogens are difficult to summarise as they are extremely variable depending on the species, age, sex, timing of BrdU injections, timing after ovariectomy, dose and type of exogenous oestrogens [START_REF] Galea | Sex, hormones and neurogenesis in the hippocampus: Hormonal modulation of neurogenesis and potential functional implications[END_REF]. Anyhow, effects in males are less robust than in females.

Pregnancy and lactation are characterised by neural and behavioural changes which require a high degree of plasticity for the maternal brain. Besides, the hippocampus controls different components of maternal behaviour, including pup retrieval and nest building [START_REF] Galea | Sex, hormones and neurogenesis in the hippocampus: Hormonal modulation of neurogenesis and potential functional implications[END_REF]. As a general effect, we can say that acutely administered oestradiol enhances proliferation within the SGZ, in agreement with the findings from Tanapat et al.

who showed an increased proliferation in the SGZ of female rats during proestrus, when the levels of oestrogens are higher, compared to diestrus or male animals [START_REF] Tanapat | Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat[END_REF].

Progenitor cells of the hippocampus express ERs and respond to agonists of both Erα and β, with an increase in proliferation, while ER antagonists partially block oestrogen effects [START_REF] Galea | Sex, hormones and neurogenesis in the hippocampus: Hormonal modulation of neurogenesis and potential functional implications[END_REF] (Fig. 5).

Progesterone receptors are found in the hippocampus and treatment with exogenous progesterone increases cell proliferation in ovariectomised female and male rats, and cell survival in male mice. However, when combined with oestrogens, progesterone blocks oestrogen-dependent increase in cell proliferation [START_REF] Liu | Clinically relevant progestins regulate neurogenic and neuroprotective responses in vitro and in vivo[END_REF].

Finally, androgen receptors (ARs) are also expressed in the hippocampus [START_REF] Xiao | Sex Differences, Laterality, and Hormonal Regulation of Androgen Receptor Immunoreactivity in Rat Hippocampus[END_REF]. An upregulation in cell survival with no significant effects on cell proliferation has been shown in male rodents in response to testosterone or dihydrotestosterone, a result that can be blocked by an AR antagonist [START_REF] Hamson | Androgens increase survival of adult-born neurons in the dentate gyrus by an androgen receptor-dependent mechanism in male rats[END_REF].

aNSC AND ADULT NEUROGENESIS IN HUMANS

Data regarding adult neurogenesis in human are more controversial than those obtained from rodents due for instance to difficulties in obtaining human specimens and limits in the experimental strategies that can be applied (Bergmann et al., 2015).

The human SVZ

The human SVZ niche has been detailed and astrocytes with proliferating features have been described (Sanai et al., 2004). During the first 6 months of postnatal life, robust streams of immature DCX/PSA-NCAM expressing neurons tangentially migrate just underneath the ependymal layer of the wall of the lateral ventricle and along the RMS. Between 6 and 18 months, the cytoarchitecture of the SVZ deeply changes until resembling that observed in adults [START_REF] Sanai | Corridors of migrating neurons in the human brain and their decline during infancy[END_REF]. After the sixth month of life, this dense network of putative migratory neurons is completely depleted leaving a hypocellular gap, also observed in macaques and marmosets [START_REF] Gil-Perotín | Adult neural stem cells from the subventricular zone: A review of the neurosphere assay[END_REF], between the ependymal layer and a ribbon of GFAP + and vimentin + astrocytes (Fig. 6a,c). Some of these astrocytes were seen to coexpress the proliferation marker Ki67 and they were able to incorporate BrdU in organotypic slices. Moreover, when these astrocytes were dissected and cultured in vitro, they were able to form neurospheres with the ability to self-renew and differentiate into the three neural cell types. All these data suggest the presence of a subpopulation of mitotically active NSCs (Sanai et al., 2004). In adults, no RMS can be observed, maybe due to the long distance that separates the SVZ from the OBs in humans as well as our microsmatic capabilities.

In 2005, the group of Frisén developed an unusual technique to address the issue of studying adult neurogenesis in humans. They took advantage of the dramatic increase in atmospheric 14 C levels resulting from the tests of nuclear weapons in the 1960's followed by its exponential decline; since 14 C is integrated in the DNA during its synthesis, they showed that the level of 14 C in genomic DNA closely parallels atmospheric levels so it could be used to establish the time point when the DNA was synthesized and cells were born during the life of a person [START_REF] Spalding | Retrospective birth dating of cells in humans[END_REF]. Following this rationale, they showed that in the human OB no significant postnatal neuronal turnover was observed (Bergmann et al., 2012;Spalding et al., 2013) (Fig. 6e).

Nevertheless, the presence of proliferating astrocytes in the SVZ and the absence of migrating neuroblasts in the RMS posed the question of the fate of the putative newborn cells. A study showed the generation of new interneurons in the human striatum both through the detection of neurons positive for iododeoxyuridine (which incorporates into the DNA of dividing cells similarly to BrdU) in post-mortem tissues from cancer patients who had received IdU as an anticancer drug (Ernst et al., 2014), and through 14 C dating approaches. This suggested that a subpopulation of striatal interneurons was subjected to turnover (Ernst et al. 2014). Thus, it may be speculated that neuroblasts born in the adult human SVZ migrate to the striatum and contribute to the neuronal turnover observed in this region (Bergmann et al., 2015) (Fig. 6e).

The human DG

The first experiments able to prove that adult neurogenesis persists within the human hippocampus took advantage of the use of BrdU in cancer patients who had received one intravenous injection for diagnostic purposes. In this study, BrdU-positive cells were found to express a neuronal phenotype in the DG but not in the SVZ (Eriksson et al., 1998). Moreover, DCX and PSA-NCAM-expressing putative neuroblasts were found in the DG (Knoth et al., 2010). Finally, the group of Frisén showed that a subpopulation corresponding to one third of hippocampal neurons was subjected to turnover during human's adult life, with a less pronounced age-dependent decline than rodents, since hippocampal neurons had 14 C levels that corresponded to times after the individual's birth year, unlike neurons from the cortex or the cerebellum (Bergmann et al., 2012;Spalding et al., 2013).

Structural features of the hypothalamus

Due to its role in regulating essential physiological processes for the maintenance of life, the hypothalamus is a conserved structure among vertebrates [START_REF] Xie | Development of the hypothalamus: conservation, modification and innovation[END_REF]. This small region, located in the most ventral part of the forebrain, can be divided both rostro-caudally and medio-laterally. From rostral to caudal, we can find four zones: preoptic (Fig. 7b,f), anterior (Fig. 7c,g), tuberal (Fig. 7d,h) and mammillary (Fig. 7e,i); each of them can be then divided in three levels: periventricular, medial and lateral. However, the embryonic origin of the preoptic region has been subject of debate as it is not clear if it origins from the telencephalon or diencephalon [START_REF] Markakis | Development of the neuroendocrine hypothalamus[END_REF]. These four zones are nevertheless composed by many nuclei containing specific neuronal cell types. Each of these nuclei is involved in controlling a different aspect of physiological homeostasis and behaviour [START_REF] Markakis | Development of the neuroendocrine hypothalamus[END_REF]. For instance, the preoptic area (POA) (Fig. 7b,f) controls reproduction, sleep, thermoregulation and electrolyte balance. The anterior hypothalamus, which includes the supraoptic (SON), suprachiasmatic (SCN), paraventricular (PVN), anteroventral periventricular (AVPV) and anteroventral (AVP) nuclei (Fig. 7b,c,f,g), regulates circadian rhythms, feeding and other homeostatic functions. The tuberal hypothalamus, where we find the arcuate (ARC), ventromedial (VMH) and dorsomedial (DMH) nuclei (Fig. 7d,h), is involved in the control of energy balance, stress and aggression [START_REF] Markakis | Development of the neuroendocrine hypothalamus[END_REF]. Finally, the mammillary hypothalamus, which includes the mammillary bodies (Fig. 7e,i), plays a role in arousal, stress response together with spatial and episodic memory [START_REF] Vann | The mammillary bodies and memory: More than a hippocampal relay[END_REF].

Each of the roles here listed never depends on the action of a single nucleus. The different hypothalamic regions are in fact strongly interconnected between each other and with different areas of the brain, in order to be able to adjust their function according to the physiological status of the organism as well as inputs coming from the surrounding environment.

In order to play its many roles, the hypothalamus links the nervous system to the endocrine system, working in concert with the pituitary gland to which it is morphologically linked. The pituitary gland, or hypophysis, is an endocrine gland composed by two regions with distinct embryological origins. The anterior region, or adenohypophysis, is a neuroepithelial structure composed by different types of endocrine cells defined by the hormones they produce; the posterior region, or neurohypophysis, has a neural nature and is composed by multiple axonal projections arriving from the hypothalamus via the infundibular stalk [START_REF] Cattaneo | Compendio di anatomia umana, 1-3[END_REF].

The hypothalamus contains two different types of neuroendocrine neurons: the magnocellular neurons and the parvocellular neurons. The first ones project into the posterior lobe of the pituitary where they directly release oxytocin and vasopressin; the second ones release their releasing or inhibiting hormones into a small portal blood system directly connected with the anterior pituitary (the hypophyseal portal system) where they act on their specific target to stimulate or inhibit the release of hypophyseal hormones into the systemic circulation [START_REF] Maggi | Neurodevelopmental origin and adult neurogenesis of the neuroendocrine hypothalamus[END_REF]. We can distinguish three main pathways with which the hypothalamus and pituitary exert their neuroendocrine function and whose functions are in many ways entangled: the hypothalamic-pituitary-gonadal (HPG) axis, which regulates the reproductive function; the hypothalamic-pituitary-adrenal (HPA) axis, which regulates the response to stress and the hypothalamic-pituitary-thyroid (HPT) axis, which regulates metabolism.

Building the hypothalamus

Embryonic life

The hypothalamus derives from the forebrain, which further subdivides into the telencephalon and the diencephalon. While the telencephalon gives rise to the cerebral cortex and basal ganglia, the diencephalon is at the origin of the thalamus, the hypothalamus and the epithalamus [START_REF] Xie | Development of the hypothalamus: conservation, modification and innovation[END_REF][START_REF] Ware | Notch signaling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus[END_REF]. Specific patterning of the hypothalamus begins around E8 in mice and is strictly dependent on Shh, which is secreted by the axial mesoderm underlying the neural plate to ventralise the CNS [START_REF] Lupo | Mechanisms of ventral patterning in the vertebrate nervous system[END_REF]. Sonic hedgehog acts in concert with other morphogens such as Wnt, secreted by the posterior neurectoderm and somites, and BMP, secreted by the prechordal mesoderm [START_REF] Barth | Bmp activity establishes a gradient of positional information throughout the entire neural plate[END_REF][START_REF] Braun | Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain[END_REF][START_REF] Kapsimali | Inhibition of Wnt/Axin/β-catenin pathway activity promotes ventral CNS midline tissue to adopt hypothalamic rather than floorplate identity[END_REF]. At E9.5, the hypothalamic primordium is morphologically evident and at this point, Shh is downregulated by local production of BMPs [START_REF] Patten | Opponent activities of Shh and BMP signaling during floor plate induction in vivo[END_REF]. This leads to the subdivision of the primordial hypothalamus into three regions, the anterior hypothalamus, the tuberal hypothalamus and the mammillary hypothalamus [START_REF] Ware | Notch signaling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus[END_REF]. The preoptic neuroepithelial patch gives rise to the medial POA between E12 and E16. The medial preoptic nucleus is generated in two waves, the lateral part first and the central one later, between E14 and E18 while the nuclei of the tuberal region (DMH, VMH and ARC), are generated between E12 and E17 [START_REF] Markakis | Development of the neuroendocrine hypothalamus[END_REF].

Hypothalamic neurogenesis in the mouse begins at E10.5 [START_REF] Shimada | Time of neuron origin in mouse hypothalamic nuclei[END_REF]. As for other brain regions, dividing progenitors reside in the ventricular zone. After a rapid proliferative expansion, they undergo asymmetric divisions to produce both neuronal and glial precursors that migrate to the adjacent parenchyma where they differentiate [START_REF] Xie | Development of the hypothalamus: conservation, modification and innovation[END_REF][START_REF] Ware | Notch signaling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus[END_REF]. Gliogenesis begins just after neurogenesis ends, around E15.5. Production of the correct number of neurons and maintenance of the progenitor population depend upon induction of a Notch/proneural loop [START_REF] Ware | Notch signaling and proneural genes work together to control the neural building blocks for the initial scaffold in the hypothalamus[END_REF]. The axons of the newborn hypothalamic neurons project through the medial forebrain bundle; from here, these axons progress medially to reach the median eminence (ME), depending upon mechanisms that remain unknown [START_REF] Markakis | Development of the neuroendocrine hypothalamus[END_REF]. 1.7.2.2 Post-natal life Not a lot is known regarding cell neogenesis in the postnatal hypothalamus. As for the rest of the brain, this is definitely the moment when most of the astrocytes are produced. However, the exact origin of these cells is not clear. The first weeks of postnatal life are also the moment when tanycytes, glioependymal specialised cells that line the floor of the third ventricle, differentiate (Mirzadeh et al., 2017;[START_REF] Rodríguez | Hypothalamic tanycytes: A key component of brain-endocrine interaction[END_REF].

Neurogenesis has also been reported in the hypothalamus at this stage. For instance, new neurons are added postnatally in the AVPV and in the sexually dimorphic nucleus (SDN) of the POA, two sexually dimorphic nuclei, implicated in female ovulation and male sexual behaviour respectively [START_REF] Ahmed | Pubertal hormones modulate the addition of new cells to sexually dimorphic brain regions[END_REF][START_REF] Mohr | Neurons and glial cells are added to the female rat anteroventral periventricular nucleus during puberty[END_REF]. The addition of new neurons during postnatal development is sexually dimorphic and reflects the differences that will be seen during adulthood: more neurons are added in the female AVPV compared to males, while the opposite is seen for the SDN. These processes depend upon gonadal hormones, as differences are lost in gonadectomised animals. This suggests that gonadal steroids modulate the addition of new cells during puberty to maintain and accentuate sexual dimorphisms in the adult brain [START_REF] Ahmed | Pubertal hormones modulate the addition of new cells to sexually dimorphic brain regions[END_REF][START_REF] Mohr | Neurons and glial cells are added to the female rat anteroventral periventricular nucleus during puberty[END_REF].

Cell neogenesis in the adult hypothalamus

In order to properly fulfil their role of integration of signals from other brain nuclei, the periphery and the environment, the different hypothalamic nuclei need to display a remarkable degree of structural plasticity, hence structural alterations, correlated with changes in the animal's physiological state to deliver an adequate response.

During the past years, different studies have identified several mechanisms through which this adaptation process is accomplished, such as changes in the relationships between neurons and their associated astrocytes, as well as synaptic remodelling, which modifies the type and the efficacy of synaptic inputs and the extent of tonic coupling among different neurons [START_REF] Hatton | A restructuring off hypothalamic synapses is associated with motherhood[END_REF][START_REF] Theodosis | Activity-dependent neuronal-glial and synaptic plasticity in the adult mammalian hypothalamus[END_REF]). These mechanisms have been observed in different contexts. For instance, a modification in the number of dendritic spines in the DMH and VMH neurons is observed in response to variations of gonadal hormones throughout pubertal maturation or across the oestrus cycle [START_REF] Frankfurt | Gonadal Steroids and Neuronal Plasticity[END_REF].

Plasticity is necessary for the function of the circadian clock, which adapts to variations in the light and dark cycles during the day and throughout the year [START_REF] Bosler | Structural plasticity of the circadian timing system. An overview from flies to mammals[END_REF][START_REF] Coomans | Plasticity of circadian clocks and consequences for metabolism[END_REF]; this is examplified by the increase in synaptic innervation of the SCN by glutamatergic fibers at daytime compared to night-time, or by the transitions of chromatin between condensed and decondensed states over the 24-hour cycle. All the mechanisms described so far are rapid and reversible. In addition, an increasing number of studies have described neurogenesis and gliogenesis in the adult hypothalamus across different species suggesting that longer-term plastic remodelling involving the integration of newborn cells into existing circuits may also be at work in the hypothalamus. Newborn neurons and glial cells have been reported postnatally and during adulthood in the hypothalamus of rat, mouse, hamsters, sheep, meadow vole and pig. Even though these reports present many differences, probably due to their use of different experimental strategies, species, sex, and genetic background of the animals as well as quantification methods, they all suggest a role for this process in the physiopathology of the hypothalamus (Djogo et al., 2016;Fowler et al., 2002;Haan et al., 2013;[START_REF] Huang | Photoperiod regulates neuronal bromodeoxyuridine labeling in the brain of a seasonally breeding mammal[END_REF]Kokoeva et al., 2005;Lee et al., 2012;Li et al., 2012;Mcnay et al., 2012;[START_REF] Migaud | Seasonal regulation of structural plasticity and neurogenesis in the adult mammalian brain: Focus on the sheep hypothalamus[END_REF]Pencea et al., 2001;Robins et al., 2013a;Sharif et al., 2014).

Identity of hypothalamic NSCs

Adult hypothalamic neuro and gliogenesis have been mainly studied in the tuberal region of the hypothalamus, i.e. in the ARC, DMH and VMH. Despite the number of works reporting the presence of neural stem/progenitor cells in this region, their precise identity and location remain controversial. Three main and nonexclusive hypotheses have being proposed: (i) the population of stem cells is located in the hypothalamic parenchyma, (ii) they correspond to subependymal astrocytes, or (iii) they correspond to tanycytes (Sharif et al., 2014).

The parenchymal hypothesis

The data pushing towards this hypothesis are based on the observation that shortly after BrdU administration, dividing cells can be found at considerable distance from the third ventricle, with sometimes closely apposed pairs of BrdU-labeled cells that are reminiscent of a recent mitotic event (Pencea et al., 2001;Pérez-Martín et al., 2010;[START_REF] Kokoeva | Evidence for constitutive neural cell proliferation in the adult murine hypothalamus[END_REF]Haan et al., 2013;Robins et al., 2013b). Moreover, lineage tracing studies showed that NG2 glial cells, found in the hypothalamic parenchyma, are able to proliferate both symmetrically to self-renew and asymmetrically to give rise to oligodendrocytes and a smaller population of neurons (Robins et al., 2013b); and that Sox2 + /Nestin + cells, found in the mediobasal hypothalamus, can give rise to new neurons and, to a lesser extent, glial cells, in adult mice (Li et al., 2012). However, when hypothalamic NSCs have been successfully isolated in vitro, the dissected tissue included the third ventricle wall. Whenever cultures were prepared from regions distant from the third ventricle, NSCs were not obtained (Robins et al., 2013a). These results may implicate that, while real stem cells reside within the ventricular wall, multipotent neural progenitor cells, which retain dividing ability, are found in the parenchyma (Sharif et al., 2014). Nonetheless, considering the impossibility to isolate the ventricular lining from the adjacent parenchyma in microdissection procedures, one cannot exclude the possibility that parenchymal cells located in the proximity of the third ventricle could be stem cells.

The subependymal astrocytes hypothesis

Evindences for this hypothesis come from a BrdU-based study. Perez-Martin and collegues (2010) described a population of subependymal GFAP-positive astrocytes located in the middle part of the rat third ventricle that proliferated in response to insulin-like growth factor I (IGF-I). Structural features of this population resemble that of the SVZ neurogenic niche with the GFAP-positive cells contacting the ventricle through an apical process bearing a single cilium and showing many labyrinthine extensions of their basal membrane (Pérez-Martín et al., 2010). However, lineage-tracing experiments will be needed to determine the fate potentiality of these cells.

The tanycyte hypothesis

The ependymal layer of the third ventricle is lined by a single cell layer of multiciliated cubic ependymal cells found in the more dorsal part, an overlapping zone with both ependymal cells and tanycytes, and a ventral zone with non-ciliated tanycytes which retain characteristics of RG cells (Pérez-Martín et al., 2010;[START_REF] Rodríguez | Hypothalamic tanycytes: A key component of brain-endocrine interaction[END_REF] (Fig. 8).

Tanycytes are considered as RG-like cells as they are in contact with the CSF on their apical face while their basal long extensions project into different nuclei of the hypothalamus or into the ME [START_REF] Rodríguez | Hypothalamic tanycytes: A key component of brain-endocrine interaction[END_REF]. They express many different neural stem/progenitor cell markers such as nestin, Sox2, GFAP, GLAST, vimentin, BLBP and components of the Notch or fibroblast growth factor (FGF) signalling pathways (Sharif et al., 2014).

Tanycytes are not a homogeneous cell population; they have been classically subdivided, with respect to their location, spatial relationships, morphology, cytochemistry and ultrastructure, into four groups: α1, α2, β1 and β2 tanycytes [START_REF] Rodríguez | Hypothalamic tanycytes: A key component of brain-endocrine interaction[END_REF]. Alpha tanycytes are located along the lateral walls of the third ventricle: α1 tanycytes send their processes into the VMH and part of the DMH, while α2 tanycytes project to the ARC; β1 tanycytes line the lateral evaginations of the third ventricle in the infundibular recess and project to the latero-external region of the ME; β2 tanycytes line the floor of the third ventricle and extend their projections into the ME to reach the perivascular space of the capillaries of the hypothalamo-hypophysial portal system [START_REF] Recabal | Hypothalamic neurogenesis as an adaptive metabolic mechanism[END_REF][START_REF] Rizzoti | Pivotal role of median eminence tanycytes for hypothalamic function and neurogenesis[END_REF]Robins et al., 2013a;[START_REF] Rodríguez | Hypothalamic tanycytes: A key component of brain-endocrine interaction[END_REF]Sharif et al., 2014) (Fig. 8).

While they all express nestin, Sox2 and vimentin, only α tanycytes express GFAP, and only β tanycytes express BLBP and Fgf10 (Haan et al., 2013;Robins et al., 2013a). BrdU-based studies have always found dividing cells within the tanycytic pool, however, with a much lower frequency than in the parenchyma (Pencea et al., 2001;[START_REF] Kokoeva | Evidence for constitutive neural cell proliferation in the adult murine hypothalamus[END_REF]Haan et al., 2013), a behaviour that may be compared to the quiescent state of SVZ and SGZ NSCs (see chapter 1.3.1). This rather quiescent state is also evidenced by the observation that tanycytes labelled with BrdU during early postnatal development retain BrdU expression into adulthood but can also show co-expression of the cell cycle marker Ki67 (Sharif et al., 2014).

Lineage-tracing studies have been used to explore tanycyte stem cell properties by following their progeny. Even though all studies showed that tanycytes are neurogenic and gliogenic in vivo, results were not homogeneous when different tanycytic subtypes were traced. For instance, a few hours after intracerebroventricular (i.c.v.) injection of a GFP-expressing adenovirus in the third ventricle, only tanycytes were labelled while, 4 weeks later, GFPexpressing neurons were found in the parenchyma of the rat hypothalamus [START_REF] Xu | Neurogenesis in the ependymal layer of the adult rat 3rd ventricle[END_REF].

In young postnatal mice (P4), fate mapping using Nestin:CreER T2 driver animals induces reporter gene expression in tanycytes within a few hours, while reporter-expressing neurons are observed after one month, mainly in the ME (Lee & Blackshaw, 2012).

In the study from Robins et al (2013), a GLAST:: CreER T2 model was used to specifically trace adult dorsal α tanycytes. These cells were able to give rise to other tanycytes, including those of the more ventral region of the third ventricle, as well as astrocytes and sparse numbers of neurons (Robins et al., 2013a). Moreover, when microdissecting the region containing β tanycytes only, they were not able to isolate NSCs in in vitro neurosphere experiments. When Haan and collegues (2013) fate-mapped β tanycytes using a FGF10promoter-driven reporter gene, they identified only neuronal progeny in the parenchyma (Haan et al., 2013). Thus, from these results, it may be speculated that GLAST + dorsal α tanycytes are neural stem cells, able to give rise to other tanycytes, as well as both parenchymal astrocytes and neurons, while β tanycytes are intermediate progenitors with a commetted neuronal fate (Sharif et al., 2014) (Fig. 9).

Modulators of hypothalamic neurogenesis

Most of the studies on hypothalamic neurogenesis have been performed in nonphysiological conditions where proliferation was either stimulated or inhibited. Many different factors have been tested for their ability to influence hypothalamic adult neurogenesis.

Growth factors

An initial work performed in rats showed that infusion of brain derived neurotrophic factor (BDNF) in the lateral ventricles stimulated proliferation and neurogenesis in the hypothalamus (Fig. 9). Interestingly, the BrdU-positive cells did not express the BDNF receptor, TrkB, which was nevertheless found at the level of their neighbouring cells (Pencea et al., 2001). Intracerebroventricular administration of ciliary neurotrophic factor (CNTF) increases proliferation in the hypothalamus at the level of the DMH, VMH, ARC and ME, all regions known to be involved in the control of energy homeostasis (Fig. 9). CNTF induces weight loss in obese animals and humans, and its effect is retained even after termination of the treatment. It appears that this long-term effect depends on the stimulation of hypothalamic neurogenesis, which increases the number of leptin-sensitive neurons in the region. Unlike for the case of BDNF, the newborn cells are directly responsive to CNTF since they express CNTF receptor (Kokoeva et al., 2005). Moreover tanycytes of different subpopulations have been shown to differently respond to stimulatory cues. For instance, FGF2 stimulates proliferation of rat α tanycytes and mouse α2 tanycytes [START_REF] Xu | Neurogenesis in the ependymal layer of the adult rat 3rd ventricle[END_REF], Robins et al., 2013), and IGF-I, whose receptor is expressed by both proliferating and nonproliferating cells, stimulates proliferation of GFAP + α tanycytes in rats (Perez-Martin et al., 2010) (Fig. 9). 1.7.5.2 Diet Adult hypothalamic neurogenesis can also be affected by diet (Fig. 9). In the study from McNay et al. (2012), they showed a decrease in adult hypothalamic neurogenesis due to high fat diet (HFD), as well as an increase in apoptosis of the newborn cells, an effect that was amplified in ob/ob leptin-deficient mice (Mcnay et al., 2012). Li et al. (2012) reported impairment in hypothalamic cell proliferation and differentiation, both in vivo and in vitro, together with a decrease in Sox2 expression in responce to chronic HFD. It is known that the environment of the hypothalamus in HFD or obese conditions is strongly inflammatory and the proinflammatory pathway of nuclear factor-κB (NF-κB) and its upstream IκB kinase-β (IKKβ) is a key mediator of hypothalamic inflammation. Accordingly, NSCs derived from HFDtreated animals were found to display an overactivation of this signalling pathway, which caused both the increased cell death, via the stimulation of pro-apoptotic cytokine secretion, and decreased neuronal differentiation, via activation of the Notch signalling (Li et al., 2012).

Another study showed different outcomes of HFD on hypothalamic neurogenesis according to the region and sex of the animals. Indeed, HFD inhibited neurogenesis in the ARC of males and females. However, it increased the proliferative activity in the ME of female mice only (Lee et al., 2014). In the study of Gouazé et al. (2013), HFD was shown to acutely increase cell proliferation in the hypothalamus, without affecting cell survival, and to induce a higher percentage of newborn neurons to adopt a proopiomelanocortin (POMC) phenotype in the ARC. Blocking cell proliferation by chronic i.c.v. infusion of the antimitotic drug AraC accelerated the onset of diet-induced obesity in mice, suggesting that cell renewal in the adult hypothalamus participates in the long-term regulation of food intake (Gouazé et al., 2013). 1.7.5.3 Other modulators Adult neurogenesis in the hypothalamic neurogenic niche is not only modulated by diet but also by the hormonal status in female mice. Shortly after HFD initiation in ovariectomised animals, an increase in cell proliferation and in the number of newborn oestrogen receptor (ER)-expressing neurons in the ARC, VMH and DMH was seen. Oestradiol administration however, prevented the increase proliferation and the weight gain induced by HFD. On the contrary, oestradiol increased cell proliferation in a status of normal diet. This shows that hypothalamic cell proliferation, as previously seen in the SVZ and SGZ, can be influenced by the action of gonadal hormones [START_REF] Bless | Adult Neurogenesis in the Female Mouse Hypothalamus: Estradiol and High-Fat Diet Alter the Generation of Newborn Neurons Expressing Estrogen Receptor[END_REF][START_REF] Bless | Oestradiol and Diet Modulate Energy Homeostasis and Hypothalamic Neurogenesis in the Adult Female Mouse[END_REF] Voluntary exercise stimulates hypothalamic neurogenesis in the adult rat brain after stroke, improving recovery of metabolic homeostatic functions in these animals [START_REF] Niwa | Voluntary exercise induces neurogenesis in the hypothalamus and ependymal lining of the third ventricle[END_REF].

Finally, the proliferative capacity of the hypothalamus is seasonally regulated [START_REF] Migaud | Seasonal regulation of structural plasticity and neurogenesis in the adult mammalian brain: Focus on the sheep hypothalamus[END_REF]. As shown in sheep and hamsters, two seasonal mammalian models, proliferation in the hypothalamus is stimulated during the short photoperiod, when these animals are sexually active [START_REF] Migaud | Seasonal regulation of structural plasticity and neurogenesis in the adult mammalian brain: Focus on the sheep hypothalamus[END_REF].

Functional implications of hypothalamic neurogenesis

After successive divisions, newborn cells are found to express proteins characteristic of migrating, immature neurons, including PSA-NCAM and DCX, which have also been found in the human hypothalamus (Batailler et al., 2014). Later on, newly generated cells differentiate into neurons that integrate within the hypothalamic circuits involved in energy homeostasis as shown by their expression of markers of terminally differentiated hypothalamic neuronal subtypes, such as POMC or neuropeptide Y/agouti-related protein (NPY/AgRP) neurons (Lee & Blackshaw, 2012) and their responsiveness to leptin (Kokoeva et al., 2005). For this reason, virtually all the studies on this subject have focused on the involvement of adut hypothalamic neurogenesis in metabolism.

A BrdU-based study suggested a high degree of neuronal turnover in the ARC. 63% of POMC and 34% of NPY neurons born during embryogenesis are lost between 4 and 12 weeks of age, with no alterations in the total number of ARC neurons, suggesting that this neuronal loss must be balanced by a process of adult neurogenesis, which contributes to remodelling of the network (Mcnay et al., 2012). Adult neurogenesis seems to be able to reshape the feeding hypothalamic circuits to maintain homeostasis in deleterious conditions. Specific deletion of the mitochondrial transcription factor A gene in AgRP neurons, results in progressive neurodegeneration of these neurons due to depletion of mitochondria over a period of several months, which in turn results in increased cell proliferation and generation of new AgRP leptin-sensitive neurons. This compensatory neurogenesis counteracts the deleterious effects of AgRP neuron loss on body weight and food intake (Pierce & Xu, 2010).

To understand the role of adult hypothalamic neurogenesis experiments have been performed to evaluate the effects of its inhibition on metabolic homeostasis. Inhibition of cell proliferation in the hypothalamus has been linked to increased body weight and fat mass. For instance, a study performed specific ablation of NSCs in the mediobasal hypothalamus of adult mice via injection of lentiviruses expressing the Herpes simplex virus thymidine kinase under the Sox2 promoter. This enzyme converts ganciclovir, a non-toxic nucleoside analog into a toxic product that kills cells during DNA replication, hence dividing Sox2-positive cells. This procedure caused increased food intake, weight gain and glucose intolerance (Li et al., 2012). When hypothalamic NSCs were implanted in obese animals, with the purpose of understanding whether they could produce a beneficial effect to these animals, they failed to survive due to the inflammatory state of the hypothalamic parenchyma in obese animals. However, when hypothalamic inflammation was prevented by NF-κB inhibition, grafted hypothalamic NSCs survived, proliferated and counteracted obesity and glucose intolerance [START_REF] Li | Control of obesity and glucose intolerance via building neural stem cells in the hypothalamus[END_REF]. In another study, proliferation blockade through i.c.v. infusion of AraC into the third ventricle, led to increased food intake and body weight. Moreover, this weight gain was preceded by a condition of leptin resistance due to lack of responsiveness of ARC leptin receptor-expressing neurons without affecting downstream effector cells (Djogo et al., 2016). From these data it seems that cell renewal in the adult hypothalamus exerts a protective role against excessive body weight gain under detrimental nutritional conditions. Interestingly, when cell proliferation was inhibited in peripubertal mice at the level of the ME using focal X-ray radiation, HFD-fed animals gained significantly less weight and fat mass compared to non-irradiated animals (Lee et al., 2012). These results may reflect a role for overfeeding-induced neurogenesis, specifically at the level of the ME before establishment of puberty, in reducing baseline energy consumption and promoting energy storage in the form of fat; a response that would optimise adaptation of animals in the wild, where sources of food are rare (Lee & Blackshaw, 2012). Nonetheless, a recent study showed that Gonadotropin releasing hormone (GnRH), the master regulator of the reproductive function (see chapter 2), can stimulate cell proliferation in the hypothalamus of old mice [START_REF] Zhang | Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH[END_REF]. Besides, GnRH treatment decelerates ageing, as attested by improvement of different age-related markers, such as cognitive functions, muscle fibre size and skin thickness [START_REF] Zhang | Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH[END_REF]. Another interesting study regarding the role of hypothalamic cell proliferation in aging came earlier this year from the same group. They showed that hypothalamic NSCs decrease in number with age and that they exert a positive effect on the process of aging. They implanted hypothalamic NSCs derived from newborn mice, in the mediobasal hypothalamus of old animals. The implanted NSCs had been previously modified to survive in the proinflammatory environment of the aged hypothalamus. This procedure caused ageing retardation and lifespan extension [START_REF] Zhang | Hypothalamic stem cells control ageing speed partly through exosomal miRNAs[END_REF], suggesting that hypothalamic NSCs actively regulate systemic aging.

The role of adult hypothalamic neurogenesis has also started to be evaluated in another region of the hypothalamus, the POA, in relation to thermogenesis: the ability of the organism to adapt to changes of the external temperature. It has been shown that in rats, heat exposure stimulates proliferation of neuronal progenitor cells in the hypothalamus, a process that is strongly impaired with aging, and it promotes differentiation of these cells into GABAergic neurons. Moreover, some of the newborn neurons were activated by heat exposure. Inhibiting cell proliferation through infusion of the animitotic drug AraC within the lateral ventricle, the ability of heat tolerance was impaired, therefore suggesting that heat exposure-induced hypothalamic cell proliferation plays a role in the process of thermoregulation [START_REF] Matsuzaki | Proliferation of neuronal progenitor cells and neuronal differentiation in the hypothalamus are enhanced in heat-acclimated rats[END_REF][START_REF] Matsuzaki | Aging attenuates acquired heat tolerance and hypothalamic neurogenesis in rats[END_REF][START_REF] Matsuzaki | Neural progenitor cell proliferation in the hypothalamus is involved in acquired heat tolerance in long-term heatacclimated rats[END_REF].

At the level of the POA, another group explored the neogenic potential of the organum vasculosum of the lamina terminalis (OVLT), one of the circumventricular organs of the CNS, meaning those regions lacking a BBB, therefore able to sense signals found in the circulatory system through fenestrated capillaries. This region was shown to contain proliferative cells able to differentiate into both glial cells and neurons in vitro whereas only gave rise to astrocytes in vivo (Bennett et al., 2009). Interestingly, when nestin-positive cells were isolated from the OVLT and ectopically transplanted into the SVZ of recipient animals, these cells migrated along the RMS and integrated into the OB where they expressed neuronal markers, showing that the OVLT contains multipotent cells whose fate is determined by the environment [START_REF] Bennett | Heterotopically transplanted CVO neural stem cells generate neurons and migrate with SVZ cells in the adult mouse brain[END_REF].

The POA, and expecially the OVLT region, is critical for the regulation of the reproductive function (see next chapter). The identification of a putative niche of neural stem/progenitor cells in this region during adulthood raises the possibility that newborn cells may be implicated in the central control of reproduction.

THE HYPOTHALAMIC-PITUITARY-GONADAL AXIS

The HPG axis is an organized set of neural and endocrine tissues dedicated to the control and regulation of the reproductive function; this means to allow correct development of the gametes, release of gonadal hormones, fertilization and gestation, together with reproductive and parental behaviours [START_REF] Herbison | Control of puberty onset and fertility by gonadotropin-releasing hormone neurons[END_REF].

The axis is composed by: the GnRH neurons of the hypothalamus, the gonadotrope cells of the anterior pituitary and the gonads, testes in males and ovaries in females [START_REF] Herbison | Control of puberty onset and fertility by gonadotropin-releasing hormone neurons[END_REF] (Fig. 10).

Figure 10: The hypothalamic-pituitarygonadal axis. Schematic of the three components of the HPG axis. The GnRH neurons project their terminals to the median eminence portal system; the GnRH decapeptide then acts on anterior pituitary gonadotrophs to control the release of gonadotropins that regulate gonadal function. Gonadal steroid hormones will then feedback on the brain and pituitary gland. GnRH: Gonadotropin-releasing hormone;

FSH: follicle-stimulating hormone; LH: luteinizing hormone (From Herbison A, Nature reviews Endocrinology 2016).

The GnRH neurons are a small population of neurons dispersed between the OB and the mediobasal hypothalamus [START_REF] Cariboni | From nose to fertility: the long migratory journey of gonadotropin-releasing hormone neurons[END_REF] with the majority of the cell bodies being found in the medial septum, POA and anterior hypothalamus [START_REF] Herbison | Chapter 11 -Physiology of the Adult Gonadotropin-Releasing Hormone Neuronal Network[END_REF]. These neurons have a peculiar bipolar morphology as they present two processes originating from the opposite sides of their cell body [START_REF] Jennes | Ultrastructural Characterization of Gonadotropin-Releasing Hormone ( GnRH ) -Producing Neurons[END_REF]. Independently from their location, most of the GnRH neurons project their terminals and release the decapeptide GnRH in a synchronized manner at the level of the ME [START_REF] Herbison | Control of puberty onset and fertility by gonadotropin-releasing hormone neurons[END_REF]. This interesting structure at the base of the hypothalamus is one of eight circumventricular organs of the central nervous system [START_REF] Johnson | Sensory circumventricular organs and brain homeostatic pathways[END_REF]. Furthermore the ME is part of the hypophyseal portal system, one of the three portal systems in the human body, from which blood is drained directly into another capillary bed through veins instead of passing through the heart [START_REF] Green | University of Cambridge[END_REF]. These characteristics make the ME a strategic interface between the hypothalamus and the peripheral endocrine system [START_REF] Yin | The hypothalamic median eminence and its role in reproductive aging[END_REF].

From the ME, GnRH reaches the anterior pituitary and acts on the gonadotrope cells to induce the synthesis and release of the gonadotropins: the luteinizing hormone (LH) and the follicle-stimulating hormone (FSH), which act at the level of the gonads via specific transmembrane receptors. In order to be effective, the GnRH needs to be secreted in a pulsatile manner since continuous administration down regulates the activity of the gonadotropes, leading to infertility [START_REF] Belchetz | Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone[END_REF][START_REF] Christian | The neurobiology of preovulatory and estradiol-induced gonadotropin-releasing hormone surges[END_REF].

The gonads, testes and ovaries, are the primary reproductive organs, responsible for the gametogenesis -spermatogenesis and oogenesis respectively -and for the release of gonadal hormones, which regulate development and maintenance of sexual characteristics and function. The cells directly responsible for the gametogenesis are the germ cells, from which the gametes are obtained following different mitotic and meiotic divisions. However, these are not the only players involved as during this process, specialized cells provide structural and metabolic support to the developing gametes.

In the testes, the Leydig cells are responsible for the release of androgens: testosterone, dihydrotestosterone and androstenedione while the Sertoli cells support and nourish the developing sperm cells [START_REF] Jin | Molecular regulation of hypothalamus-pituitary-gonads axis in males[END_REF][START_REF] Tremblay | Molecular regulation of steroidogenesis in endocrine Leydig cells[END_REF]. In males, LH stimulates the secretion of testosterone from the Leydig cells while FSH regulates the maturation of sperm cells [START_REF] Bliss | GnRH signaling, the gonadotrope and endocrine control of fertility[END_REF][START_REF] Jin | Molecular regulation of hypothalamus-pituitary-gonads axis in males[END_REF].

In the ovaries, each single germ cell, or oocyte, is contained within an ovarian follicle, the functional unit of the ovary [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF]. In this structure, we can distinguish granulosa and outer theca cells. The theca cells provide structure and support to the follicle as it matures and are responsible for the production of androstenedione. The granulosa cells are mainly responsible for the production of oestrogens (oestrone, E1 and oestradiol, E2), which are produced via aromatization of the androgens secreted by the theca cells and, after ovulation occurs, progesterone, which is involved in pregnancy maintenance. Maturation of the follicles goes through primordial, primary, secondary and antral stages. From this stage, only a single follicle in humans, and a few in rodents, will reach the preovulatory stage (Graafian follicles), under cyclic gonadotropin stimulation, while most of them will undergo degeneration [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF]. These Graafian follicles, during each reproductive cycle, ovulate to release the mature oocyte for fertilization, whereas the remaining theca and granulosa cells undergo transformation to become the corpus luteum [START_REF] Mcgee | Initial and cyclic recruitment of ovarian follicles[END_REF]. FSH stimulates follicular growth, with a consequent increase in estradiol secretion, while LH induces ovulation of the mature follicles and their consequent transformation into corpora lutea [START_REF] Bliss | GnRH signaling, the gonadotrope and endocrine control of fertility[END_REF][START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF][START_REF] Simonneaux | A multi-oscillatory circadian system times female reproduction[END_REF].

The gonadal hormones promote the growth of the secondary sex organs and also form important positive and negative feedback loops that act centrally on the GnRH system and at the level of the pituitary [START_REF] Christian | The neurobiology of preovulatory and estradiol-induced gonadotropin-releasing hormone surges[END_REF] to modulate gonadotropin output (see chapter 3.3).

THE RAT OVARIAN CYCLE

The role of the HPG axis is that of ensuring that reproduction occurs; this means optimising the process based on the animal's characteristics and ecosystem; being ovulation and pregnancy very costly in terms of energy demand for females, these processes are finely regulated while spermatogenesis occurs as a continuum throughout adult life in males.

Ovulation is regulated in a cyclic manner with a follicular and a luteal phase: during follicular phase, a follicle develops and produces increasing amounts of oestrogens until ovulation occurs. Then, the follicle turns into the corpus luteum (luteal phase). If the ovum is not fertilised menstruation is induced and a new follicular phase begins [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF]. Animals differ for the duration of their cycles. For instance primate follicular phase lasts at least 2 weeks and they also present a long luteal phase. Domestic animals such as cows, sheep and pigs have a very short follicular phase but a quite long luteal phase with a 17-21-day long cycle. Rats, mice and hamsters have a very short cycle and ovulate every 4-5 days. Finally, a group of mammals that includes rabbits, domestic cats and ferrets, does not ovulate unless coitus occurs (reflex ovulators). It is also possible to distinguish between seasonal and nonseasonal breeders with the former ovulating only during a specific season, like sheep or horses, and the latter cycling throughout the year, as in the case of primates, mice or rats [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF].

Female rats have been extensively used as a model to study reproductive physiology due to their regular and short cycle. As mentioned before, rats ovulate every 4-5 days and it is possible to distinguish four phases that can easily be determined by analysis of vaginal smear: Proestrus, characterised by the presence of nucleated epithelial cells, is the phase that precedes ovulation; in Estrus, when ovulation occurs, cornified squamous epithelial cells are mainly found; Metestrus (or diestrus-1) and Diestrus (or diestrus-2) occur in absence of fertilisation -they correspond to the lutheal phase in primates -and are characterised by predominant presence of leukocytes together with nucleated epithelial cells [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF][START_REF] Westwood | The Female Rat Reproductive Cycle: A Practical Histological Guide to Staging[END_REF] (Fig. 11b).

Patterns of hormonal secretion

2.2.1.1 GnRH While GnRH basal secretion is pulsatile and induces a pulsatile release of LH, a surge of GnRH and therefore LH occurs on the afternoon of proestrus to induce ovulation [START_REF] Herbison | Control of puberty onset and fertility by gonadotropin-releasing hormone neurons[END_REF].

After the surge occurs, GnRH activity declines and basal pulsatility is restored. A peculiar characteristic of the GnRH neurons is their range of different spontaneous firing patterns: in several electrophysiological experiments, neurons were found silent, continuously active, or irregularly active, with bursting activity [START_REF] Constantin | In Vivo Recordings of GnRH Neuron Firing Reveal Heterogeneity and Dependence upon GABAA Receptor Signaling[END_REF][START_REF] Herbison | Chapter 11 -Physiology of the Adult Gonadotropin-Releasing Hormone Neuronal Network[END_REF][START_REF] Sabatier | Phasic spike patterning in rat supraoptic neurones in vivo and in vitro[END_REF]. This heterogeneity of firing activity has been found both in vitro and in vivo, therefore raising the question of whether different firing patterns represent different GnRH neuronal populations with different roles in the generation of a GnRH pulse. For instance, studies performed on animals presenting a mutation resulting in the absence of GnRH expression have shown that after ventricular transplantation of GnRH neurons, as little as 20 of these cells were sufficient to generate pulsatile LH secretion (Kokoris et al., 1988). What appears to be constant is that in order to obtain GnRH pulses, at least a subpopulation of GnRH neurons must be synchronized, but how this synchronization occurs remains still unknown. Two models have been proposed to explain synchronization at the level of GnRH neuron cell bodies: an intrinsic pulse generation, which involves only GnRH neurons, and an extrinsic pulse generation, which involves a separate group of neurons within the GnRH neuronal network. A third hypothesis however, would be that the process of synchronisation only occurs at the level of GnRH neuron terminals in the ME, thus regulating the release of the decapeptide rather than its synthesis [START_REF] Herbison | Chapter 11 -Physiology of the Adult Gonadotropin-Releasing Hormone Neuronal Network[END_REF].

Gonadotropins

The LH surge on the afternoon of proestrus induces ovulation by rupture of the follicle, and formation of the corpora lutea; LH then decreases to basal levels on the morning of estrus (Gallo, 1981;[START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF] (Fig. 11a). FSH levels remain low during late estrus until afternoon of proestrus when there is a rapid increase, in parallel with the increase in LH release, which peaks on the evening of proestrus after which it begins to decline. However, on the morning of estrus, a second peak of FSH occurs, independently from the hypothalamic control, after which basal levels are reached again (Butcher et al., 1974;Gay et al., 1970;[START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF] (Fig. 11a). Unlike LH, FSH in not normally released in a pulsatile manner. The release of LH and FSH is regulated by the frequency of GnRH pulses: a higher pulse frequency increases LH secretion over FSH, whereas low GnRH pulse frequency favours FSH secretion [START_REF] Kaiser | Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic[END_REF][START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF]. This regulation appears to occur at the level of gene transcription in the gonadotropes as the expression of the GnRH receptor is increased by high frequency pulses of GnRH itself, probably increasing the responsiveness of the pituitary to GnRH to switch towards LH release [START_REF] Kaiser | Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic[END_REF].

Prolactin

Prolactin is a hormone produced by the lactotrophs of the anterior pituitary whose release is mainly regulated by the action of three populations of hypothalamic dopaminergic neurons that inhibit its release: the tuberoinfundibular (TIDA), the tuberohypophyseal (THDA) and the periventricular hypophyseal (PHDA) dopaminergic neurons [START_REF] Grattan | The hypothalamo-prolactin axis[END_REF]. While TIDA neurons project to the ME from where dopamine is transported to the anterior pituitary, THDA and PHDA neurons project to the neurohypophysis from where dopamine reaches the anterior pituitary through short portal vessels [START_REF] Grattan | The hypothalamo-prolactin axis[END_REF]. The main role of prolactin, as suggested by its name, is to regulate lactation although many other adaptive roles have been found to be regulated by this hormone in a non-irreplaceable way (see chapter 3.6) [START_REF] Grattan | The hypothalamo-prolactin axis[END_REF]. For instance, prolactin acts on nutrient uptake and metabolism to increase body weight in pregnant females. Prolactin also reduces anxiety and favours establishment of maternal behaviour.

During the oestrous cycle, prolactin secretion is regulated with a peak of release overlapping with the LH surge during afternoon of proestrus (Fig. 11a); however, its role in the events of the oestrus cycle have not been clearly established yet (Butcher et al., 1974;[START_REF] Smith | The control of progesterone secretion during the estrus cycle and early pseudopregnancy in the rat: gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy[END_REF].

Ovarian steroids

During the development of the ovarian follicle increasing amounts of oestrogens are released by granulosa cells until reaching a peak at the beginning of the afternoon of proestrus (Fig. 11a). Two peaks of release have been described for progesterone, one from the preovulatory follicle, on the afternoon of proestrus, and the second from the corpora lutea, in the afternoon of metestrus, independent from the pituitary (Butcher et al., 1974;[START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF]Yoshinaga et al., 1969) (Fig 11a).

REGULATION OF THE GNRH SYSTEM

GnRH neurons are the master regulators of reproduction and therefore their activity is finely modulated by the action of different neuronal populations, which convey different internal and external information to which GnRH activity adapts.

For instance, in order for GnRH surge to occur at the right moment of the day and of the stage of follicle maturation, GnRH neurons receive signals from both the SCN, which conveys signals from the circadian clock [START_REF] Palm | Vasopressin induces a luteinizing hormone surge in ovariectomized, estradiol-treated rats with lesions of the suprachiasmatic nucleus[END_REF], and the AVPV, which integrates oestrogens signals coming from the growing follicle (Wiegand et al., 1978).

Most importantly, GnRH neurons receive positive and negative feedbacks from the gonads through the action of the gonadal hormones that act via multiple neuronal and glial mediators.

Tonic levels of LH and FSH are required for the release of oestrogens; increasing oestrogens and progesterone levels, secreted from metestrus through diestrus, exert a negative feedback, restraining the hypothalamo-pituitary axis and leading to basal secretion of LH during this time. When critical levels are reached, a switch to a positive feedback occurs, therefore stimulating the LH and FSH surge on the afternoon of proestrus (Levine, 2014).

Generally, the action of progesterone on the secretion of LH and FSH seems to be that of potentiating oestrogen's effects. The release of FSH does not only depend on ovarian steroids: the gonads in both males and females also produce inhibins and activins, proteic complex that respectively inhibit and stimulate FSH release at the level of the anterior pituitary. Moreover, a third protein produced by the ovaries, follistatin, has the ability to bind and block the effect of activins, therefore contributing to the inhibition of FSH release [START_REF] Herrera | Selective suppression of the primary surge of follicle-stimulating hormone in the rat: further evidence for folliculostatin in porcine follicular fluid[END_REF][START_REF] Shander | Follicle-Stimulating Hormone and Luteinizing Hormone Affect the Endogenous Release of Pituitary Follicle-Stimulating Hormone and the Ovarian Secretion of Inhibin in Rats[END_REF]. While there seems to be no effect of progesterone on prolactin release [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF], oestrogen is able to induce the proestrus peak of prolactin by acting both at the level of the lactotrophs, where it increases prolactin gene expression [START_REF] Avtanski | Both Estrogen Receptor α and β Stimulate Pituitary GH Gene Expression[END_REF], and at the level of the hypothalamus, where it decreases the inhibitory effect of dopamine on prolactin release (Labrie et al., 1989).

ER and progesterone receptor (PR) are both classic nuclear receptors, able to modulate transcription of different genes by binding at the level of specific sequences found in the promoter of target genes. ER also modulates transcription indirectly as the receptor can also be found at the cell membrane and in this case, it does not translocate into the nucleus as a result of its stimulation [START_REF] Glidewell-Kenney | Nonclassical estrogen receptor alpha signaling mediates negative feedback in the female mouse reproductive axis[END_REF]. Moreover, it appears that during induction of the LH surge, oestrogens can also act through PR in a ligand-independent manner [START_REF] Levine | Progesterone receptors as neuroendocrine integrators[END_REF]. Mice lacking ERα or PR, but not ERβ isoform, exhibit hypergonadotropism and an absence of estrous cyclicity, thus suggesting that only the former is involved in conveying the negative and positive feedback actions of oestradiol during the estrous cycle [START_REF] Chappell | Endocrine Defects in Mice Carrying a Null Mutation for the Progesterone Receptor Gene 1[END_REF][START_REF] Couse | Characterization of the Hypothalamic-Pituitary-Gonadal Axis in Estrogen Receptor (ER) Null Mice Reveals Hypergonadism and Endocrine Sex Reversal in Females Lacking ERα But Not ERβ[END_REF]. Both positive and negative feedbacks act at many different levels: by modulating GnRH neuron pulsatility indirectly, through action on different neuronal populations, by increasing the sensitivity of the pituitary gland to GnRH via modulation of GnRH receptor expression, and by regulating gonadotropin gene expression [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF].

Despite the importance of gonadal hormones in regulating GnRH neuron activity, GnRH neurons are not able to directly sense changing levels of oestrogens as they lack ERα.

Therefore, both positive and negative feedbacks on GnRH release must be exerted through the action of intermediaries [START_REF] Cheong | Effects of neuronspecific Estrogen Receptor (ER) α and ERβ deletion on the acute estrogen negative feedback mechanism in adult female mice[END_REF][START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF].

Neurotransmitters and neuropeptides

GnRH neurons receive synaptic contacts from both GABAergic and glutamatergic neurons.

Moreover, the vast majority of GnRH neurons express postsynaptic ionotropic GABA A receptors and pre-or postsynaptic metabotropic GABA B receptors as well as N-methyl-Daspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) and kainate receptors. The sources of GABA and glutamate inputs to GnRH neurons, which concern not only the GnRH neuron cell bodies but also their terminals at the level of the ME, have not all been clearly identified [START_REF] Herbison | Depolarising and Hyperpolarising Actions of GABAA Receptor Activation on Gonadotrophin-Releasing Hormone Neurones: Towards an Emerging Consensus[END_REF][START_REF] Spergel | GABA-and glutamateactivated channels in green fluorescent protein-tagged gonadotropin-releasing hormone neurons in transgenic mice[END_REF].

2.3.1.1 GABA It appears that GABA neurons located in the AVPV, lateral POA and SCN directly project to GnRH neurons. The general action of GABA seems to be inhibitory over GnRH tone. GABA levels in the POA are elevated at times of negative feedback and then fall just before the GnRH/LH surge (Herbison & Deyer, 1991;[START_REF] Robinson | Changes in the release of gammaaminobutyric-acid and catecholamines in the preoptic septal area prior to and during the preovulatory surge of luteinizing-hormone in the ewe[END_REF]Rotsztejn et al., 1982).

Besides, during proestrus, a time when positive feedbacks are conveyed towards GnRH neurons, a strong reduction in presynaptic GABA B receptor signalling by AVPV afferents occurs compared with other stages of the cycle. This reduction may result from a lower expression of presynaptic GABA B receptors or a change in their efficacy [START_REF] Liu | Estrous cycle-and sex-dependent changes in pre-and postsynaptic GABA B control of GnRH neuron excitability[END_REF]. Moreover, GABA neurons of the AVPV and ARC express ERs [START_REF] Cheong | Expression of ESR1 in Glutamatergic and GABAergic Neurons Is Essential for Normal Puberty Onset, Estrogen Feedback, and Fertility in Female Mice[END_REF][START_REF] Van Vugt | Centrally applied somatostatin inhibits the estrogen-induced luteinizing hormone surge via hypothalamic gonadotropin-releasing hormone cell activation in female rats[END_REF]. The role of these neurons in regulating oestrogen positive and negative feedback has been investigated in mouse lines with selective KO of ERα in GABAergic neurons. The results showed that while functional ERα in GABA neurons was not necessary for the negative feedback, it was critical for the positive feedback, through mechanisms that remain to be elucidated [START_REF] Cheong | Expression of ESR1 in Glutamatergic and GABAergic Neurons Is Essential for Normal Puberty Onset, Estrogen Feedback, and Fertility in Female Mice[END_REF].

Glutamate

Even though we can speculate that glutamatergic inputs arrive from many different brain regions, the only documented input is that of the AVPV. The role of glutamate has not been fully characterised yet. Glutamate levels in the POA increase during the surge of LH (Jarry et al., 1992) and it has stimulatory effects over GnRH tone. This increase is in association with the estradiol-induced LH surge, wich can be blocked with the use of NMDA or AMPA antagonists [START_REF] Ping | Loss-of-function mutations in SOX10 cause Kallmann syndrome with deafness[END_REF]. In accordance, glutamatergic neurons have been shown to express ERα, and this signalling appears to be essential for the establishment of the positive feedback [START_REF] Cheong | Expression of ESR1 in Glutamatergic and GABAergic Neurons Is Essential for Normal Puberty Onset, Estrogen Feedback, and Fertility in Female Mice[END_REF]. Furthermore, it has been shown that oestradiol can increase the GnRH response to NMDA receptors stimulation [START_REF] Arias | Estradiol modulates the LH release response to NMDA in adult female rats[END_REF].

Kisspeptin

It is now well known that the strongest activators of GnRH activity are the kisspeptin neurons, which are found in the ARC and in the AVPV.

The first suggestions for a role of kisspeptin neurons in regulation of the reproductive function came from the discovery that inactivating mutations of the kisspeptin receptor, Kiss1r (or Gpr54), are associated with hypogonadotropic hypogonadism in humans [START_REF] De Roux | Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54[END_REF][START_REF] Seminara | The GPR54 Gene as a Regulator of Puberty[END_REF]. From there, the role of kisspeptin has been described in a variety of other species and transgenic lines have been developed such as mice lacking kisspeptin or Kiss1r, which show total infertility associated with hypogonadotropism and absent ovarian cyclicity [START_REF] Colledge | Transgenic mouse models to study Gpr54/kisspeptin physiology[END_REF]. Moreover, restoring Kiss1r expression specifically in the GnRH neurons has been proven sufficient to totally recover fertility, therefore placing kisspeptin neurons as the master afferents directly controlling GnRH neuron activity [START_REF] Kirilov | Dependence of fertility on kisspeptin-Gpr54 signaling at the GnRH neuron[END_REF].

More than 90% of kisspeptin neurons express ERα [START_REF] Lehman | Anatomy of the kisspeptin neural network in mammals[END_REF]. For this reason, their role in mediating oestrogen feedback has been extensively studied. Firstly, mice with specific ablation of ERα in kisspeptin neurons showed total acyclicity. It has been shown that oestrogens increase kisspeptin mRNA expression in the AVPV and decrease it in the ARC [START_REF] Gottsch | Regulation of Kiss1 and Dynorphin Gene Expression in the Murine Brain by Classical and Nonclassical Estrogen Receptor Pathways[END_REF], and that levels in the AVPV vary at time of LH surge. Besides, infusion of kisspeptin in the ARC affects GnRH pulse generation [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF]. It is now well established that neurons of the AVPV mediate the oestrogen positive feedback and are therefore necessary for the surge of GnRH and LH hence for ovulation to occur [START_REF] Kirilov | Dependence of fertility on kisspeptin-Gpr54 signaling at the GnRH neuron[END_REF]. Moreover, the population of kisspeptin neurons of the AVPV is sexually dimorphic, with female animals displaying a higher number of neurons compared to males (Ahmed et al., 1990). Kisspeptin neurons of the ARC have been suggested to be involved in mediating oestrogen negative feedback, modulating basal pulsatility of GnRH and LH even though this has not been fully demonstrated yet [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF].

It has also been shown that kisspeptin is able to modulate GABA and glutamate transmission to GnRH neurons according to oestrogens levels. For instance, kisspeptin is able to decrease the hyperpolarising effects of GABA B receptor activation, therefore attenuating their inhibitory tone during the stage of oestrogen's positive feedback [START_REF] Zhang | γ-aminobutyric acid B receptor mediated inhibition of gonadotropin-releasing hormone neurons is suppressed by kisspeptin-G protein-coupled receptor 54 signaling[END_REF]. This suggests that GABAergic and glutamatergic tones are also involved in mediating, at least in part, kisspeptin action on GnRH neurons [START_REF] Levine | Neuroendocrine Control of the Ovarian Cycle of the Rat In: Plant TM[END_REF].

Orexigenic and anorexigenic modulators

The metabolic status of the organism is critical for maintenance of ovarian cycles as a status of nutrient deficit could impair pregnancy or lactation, hence decrease the probability of offspring's survival. At the same time, excessive fat accumulation, as in the case of obesity, negatively affects the general health condition of the organism.

Therefore, important modulators of GnRH activity are factors that inform on the energy status of the animal such as leptin, insulin and ghrelin. Leptin is a hormone secreted by the adipocytes, whose levels directly relate to the amount of body fat mass [START_REF] Casanueva | Neuroendocrine Regulation and Actions of Leptin[END_REF], being an indicator of energy sufficiency. Animals which are congenitally deficient for leptin (ob/ob) or leptin receptor (LepR) (db/db) are obese and sterile (Swerdloff et al., 1978). Insulin is a pancreatic hormone released according to glucose levels in the blood, which can act as a modulator of leptin synthesis itself and accordingly stimulate the HPG axis [START_REF] Sonksen | Insulin: understanding its action in health and disease[END_REF]; however, the mechanisms involved are not as well characterised as those for leptin [START_REF] Tena-Sempere | Chapter 35 -Physiological Mechanisms for the Metabolic Control of Reproduction[END_REF]. Ghrelin is an orexigenic circulating factor produced by the stomach [START_REF] Dickson | The role of the central ghrelin system in reward from food and chemical drugs[END_REF]. In accordance to its role as a signal for energy insufficiency, ghrelin has an inhibitory effect over the HPG axis both during puberty and adulthood [START_REF] Tena-Sempere | Chapter 35 -Physiological Mechanisms for the Metabolic Control of Reproduction[END_REF]. Nevertheless, as for oestrogens, these hormones do not act directly on GnRH neurons, even though they are the final output of their actions to regulate reproduction. Different neuronal populations are therefore required in this crosstalk between reproduction and energy homeostasis. Anorexigenic POMC and orexigenic NPY/AgRP neurons of the ARC regulate appetite according to the energy status of the organism, the former by suppressing and the latter by promoting food intake. Because of their ability to sense levels of metabolic hormones and to respond to gonadal hormones, these cells play a crucial role in regulating reproductive function according to the energy status of the animal (Manfredi-Lonzano, 2017; Tena-Sempere, 2015).

POMC neurons

POMC neurons are able to sense leptin, insulin, kisspeptin and NPY. POMC is the precursor for different neuropeptides such as β-endorphin and α-melanocyte stimulating hormone (α-MSH) but these neurons also co-express other neurotransmitters including GABA and glutamate (Manfredi-Lonzano et al., 2017). Leptin acts directly on POMC neurons to regulate energy homeostasis: α-MSH is a signal of energy sufficiency that appears to mediate leptin effects on adult fertility (Maria Manfredi-Lozano et al., 2016) since restoring melanocortin signalling in animals lacking leptin receptor rescues fertility [START_REF] Israel | Effects of leptin and melanocortin signaling interactions on pubertal development and reproduction[END_REF]. Β-endorphin is an endogenous opioid indicating a condition of energy insufficiency that both inhibits basal LH release and blocks LH surge [START_REF] Manfredi-Lozano | Connecting metabolism and gonadal function: Novel central neuropeptide pathways involved in the metabolic control of puberty and fertility[END_REF][START_REF] Nestor | Cross-talk between reproduction and energy homeostasis: Central impact of estrogens, leptin and kisspeptin signaling[END_REF].

Moreover, a subset of POMC neurons express ERα and are therefore capable of responding to oestrogens, which increase POMC activity [START_REF] Gao | Anorectic estrogen mimics leptin's effect on the rewiring of melanocortin cells and Stat3 signaling in obese animals[END_REF]. Loss of ERα from these neurons causes increased weight together with abnormal cyclicity pointing towards a possible role of these cells in mediating oestrogen's negative feedback [START_REF] Xu | Distinct hypothalamic neurons mediate estrogenic effects on energy homeostasis and reproduction[END_REF].

Finally, a possible crosstalk between POMC and kisspeptin neurons is suggested by the findings that inhibition of kisspeptin neurons or ablation of Kiss1r weaken LH response to α-MSH (Manfredi-Lonzano et al., 2017).

2.3.2.2 NPY/AgRP neurons NPY/AgRP neurons act by sensing decrease in leptin levels and therefore stimulating food intake by antagonising the anorexigenic action of α-MSH since AgRP acts as an endogenous antagonist of α-MSH receptors. Food restriction increases NPY expression in the hypothalamus and as a signal of adverse metabolic condition, this has been linked to decreased GnRH and LH secretion. A subset of NPY/AgRP neurons express ERα [START_REF] Sar | Localization of neuropeptide-y immunoreactivity in estradiol-concentrating cells in the hypothalamus[END_REF] and oestrogens have been shown to decrease NPY/AgRP expression [START_REF] Pelletier | Oestrogenic regulation of pro-opiomelanocortin, neuropeptide Y and corticotrophin-releasing hormone mRNAs in mouse hypothalamus[END_REF] and to hyperpolarise these neurons [START_REF] Roepke | Fasting and 17 -Estradiol Differentially Modulate the M-Current in Neuropeptide Y Neurons[END_REF].

Nitric oxide

Nitric oxide (NO) is a gaseous messenger discovered as a signalling molecule in the brain in the 1980's [START_REF] Garthwaite | Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain[END_REF]. NO is produced by NO synthase (NOS); it is not stored in vesicles but it rapidly diffuses and induces formation of cyclic GMP (cGMP) by direct stimulation of soluble guanylate cyclase. Three isoforms of NOS have been identified: an endothelial form (eNOS), an inducible form (iNOS) and a neuronal one (nNOS). This last isoform is considered the main source of NO in the brain and it is indeed widely distributed in many regions with a particularly dense distribution at the level of the OVLT, where most GnRH neuron cell bodies are found, and of the ARC (Chachlaki et al., 2017a). Recent studies from our laboratory have shown the phenotype of these nNOS expressing neurons to be mainly glutamatergic at the level of the OVLT and GABAergic in the ARC (Chachlaki et al., 2017a). Early studies with total KO animals for nNOS showed as their most striking phenotype impairment in the reproductive activity and an increasing number of studies are investigating the role of nNOS neurons within GnRH neuron network. NNOS neurons have been shown to express ERα (Chachlaki et al., 2017a), kisspeptin receptor [START_REF] Hanchate | Kisspeptin-GPR54 Signaling in Mouse NO-Synthesizing Neurons Participates in the Hypothalamic Control of Ovulation[END_REF] and leptin receptor [START_REF] Bellefontaine | Leptin-dependent neuronal no signaling in the preoptic hypothalamus facilitates reproduction[END_REF], putting them at the centre of these entangled circuits and giving them the ability to contribute to the action of these messengers on GnRH release.

For instance, kisspeptin stimulates NO release in the OVLT [START_REF] Hanchate | Kisspeptin-GPR54 Signaling in Mouse NO-Synthesizing Neurons Participates in the Hypothalamic Control of Ovulation[END_REF]. It has also been shown that inhibition of nNOS activity at the level of the POA ablates the stimulatory action of leptin on LH surge [START_REF] Bellefontaine | Leptin-dependent neuronal no signaling in the preoptic hypothalamus facilitates reproduction[END_REF]. Besides, nNOS activity is strictly dependant on its coupling with the NMDA receptor, as the Ca 2+ influx caused by the binding of glutamate favours nNOS phosphorylation, hence activation and therefore linking NO levels to glutamate, another well-known GnRH activator. Moreover, this process appears to be depended on ERα activation, as this can promote the association between nNOS and the NMDA receptor, and nNOS phosphorylation is highest on the day of proestrus, when oestrogen levels peak (Chachlaki et al., 2017b). Even though the absence of nNOS in the brain impairs GnRH function, the direct effect of NO on GnRH neuron electrical activity has been shown to be inhibitory [START_REF] Clasadonte | Activation of neuronal nitric oxide release inhibits spontaneous firing in adult gonadotropin-releasing hormone neurons: A possible local synchronizing signal[END_REF]. Due to its half-life and diffusion constant, NO works as a volume transmitter capable of coordinating neuronal activity in a restricted brain volume. Hence, the main hypothesis on a role for NO is that of being involved in synchronizing the activity of different GnRH neurons to finally promote the release of GnRH in the ME (Chachlaki et al., 2017b).

Autocrine control of the GnRH neurons

Over the years, many neurotransmitters and neuropeptides have been reported to be expressed in GnRH neurons. However their nature and mode of action have not been entirely characterised yet. Among them, we can mention galanin [START_REF] Rossmanith | Galanin Gene Expression in Hypothalamic GnRH-Containing Neurons of the Rat: A Model for Autocrine Regulation[END_REF] and cholecystokinin (CCK) [START_REF] Ciofi | Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin[END_REF][START_REF] Giacobini | Cholecystokinin directly inhibits neuronal activity of primary gonadotropin-releasing hormone cells through cholecystokinin-1 receptor[END_REF], which work as autocrine factors acting on the GnRH neurons themselves; and neurotensin (NT) [START_REF] Ciofi | Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin[END_REF], GABA [START_REF] Zhu | A subpopulation of gonadotropin-releasing hormone neurons in the adult mouse forebrain is γ-Aminobutyric acidergic[END_REF] and glutamate (Chachlaki et al., 2017a;[START_REF] Hrabovszky | Expression of vesicular glutamate transporter-2 in gonadotropin-releasing hormone neurons of the adult male rat[END_REF], whose possible autocrine role has not been demonstrated yet.

Galanin: Galanin is a neuropeptide widely expressed in the CNS, released by both GnRH neurons, that also express galanin receptor [START_REF] Rossmanith | Galanin Gene Expression in Hypothalamic GnRH-Containing Neurons of the Rat: A Model for Autocrine Regulation[END_REF][START_REF] Todman | Profiling neurotransmitter receptor expression in mouse gonadotropin-releasing hormone neurons using green fluorescent protein-promoter transgenics and microarrays[END_REF], and the anterior pituitary, under the control of oestrogens [START_REF] Kaplan | Galanin is an estrogen-inducible, secretory product of the rat anterior pituitary[END_REF]. During the estrous cycle of the rat, galanin and GnRH contents in the ME show an identical profile [START_REF] López | Galanin: a hypothalamic-hypophysiotropic hormone modulating reproductive functions[END_REF]. Galanin's expression by GnRH neurons increases gradually during pubertal maturation and presents a sexually dimorphic pattern, being higher in females (Rossmanith et al., 1994). Galanin is able to increase LH levels [START_REF] Sahu | Effects of neuropeptide Y, NPY analog (norleucine4-NPY), galanin and neuropeptide K on LH release in ovariectomized (ovx) and ovx estrogen, progesterone-treated rats[END_REF], and its levels are higher during the LH surge in response to gonadal steroids [START_REF] Rossmanith | Galanin Gene Expression in Hypothalamic GnRH-Containing Neurons of the Rat: A Model for Autocrine Regulation[END_REF]. It also enhances the ability of GnRH to stimulate LH release [START_REF] López | Galanin: a hypothalamic-hypophysiotropic hormone modulating reproductive functions[END_REF]. It has been hypothesised that galanin works as an autocrine modulator of GnRH neurons: galanin, co-secreted with GnRH at the level of the ME, would inhibit GnRH "leakage" between consecutive pulses, therefore increasing the efficacy of each pulse [START_REF] Rossmanith | Galanin Gene Expression in Hypothalamic GnRH-Containing Neurons of the Rat: A Model for Autocrine Regulation[END_REF].

Cholecystokinin: CCK is a neuropeptide extensively expressed throughout the CNS, with high concentrations in the limbic system. Its levels in several brain nuclei, at the level of the MPO for instance, have been shown to change according to circulating gonadal steroids (Oro et al., 1988). CKK is expressed by at least a subpopulation of GnRH neurons in adult female but not male rats [START_REF] Ciofi | Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin[END_REF]. A strong sexual dimorphism has also been reported in mice, with adult males showing a much lower level of GnRH/CCK coexpression than females. Moreover, CCK inhibits GnRH neuronal activity by acting directly through its receptor [START_REF] Giacobini | Cholecystokinin directly inhibits neuronal activity of primary gonadotropin-releasing hormone cells through cholecystokinin-1 receptor[END_REF].

Glutamate and GABA:

Even though some evidences show that the vast majority of GnRH neurons are glutamatergic (Chachlaki et al., 2017a;[START_REF] Hrabovszky | Expression of vesicular glutamate transporter-2 in gonadotropin-releasing hormone neurons of the adult male rat[END_REF], there is still no consensus over the actual release of glutamate by these neurons and a possible autoexcitatory action of glutamate on GnRH neuron activity [START_REF] Herbison | Chapter 11 -Physiology of the Adult Gonadotropin-Releasing Hormone Neuronal Network[END_REF].

A subpopulation of GnRH neurons in the mouse forebrain has been found to be GABAergic [START_REF] Zhu | A subpopulation of gonadotropin-releasing hormone neurons in the adult mouse forebrain is γ-Aminobutyric acidergic[END_REF]. The number of these neurons increases during estrus in female mice compared to the diestrus phase or male mice, a sexual dimorphism that becomes evident only after sexual maturation is attained [START_REF] Zhu | A subpopulation of gonadotropin-releasing hormone neurons in the adult mouse forebrain is γ-Aminobutyric acidergic[END_REF].

Neurotensin: NT is a neuropeptide distributed throughout the CNS, with highest levels in the hypothalamus, amygdala and nucleus accumbens. It induces a variety of effects, including analgesia or hypothermia. NT-expressing cells in the AVPV and MPO express ERα and their number increases in the presence of oestrogens, reaching a peak at the moment of the preovulatory LH surge in female mice [START_REF] Dungan Lemko | Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse[END_REF]. NT has also been detected in adult female but not male rats [START_REF] Ciofi | Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin[END_REF]. However, the number of GnRH neurons co-expressing only NT was quite low with most of them found to also co-express CKK [START_REF] Ciofi | Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin[END_REF]. GnRH neurons express NT receptor [START_REF] Dungan Lemko | Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse[END_REF], but a direct role for this peptide on the LH surge has not been clearly demonstrated yet [START_REF] Dungan Lemko | Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse[END_REF].

Glial cells

After many years being neglected as the "glue" of the CNS, there is now a rising number of studies acknowledging the actual importance of glial cells in regulating neuronal activity.

For instance, glial cells of the hypothalamus are critical modulators of the GnRH neurons through different mechanisms: release of paracrine factors, juxtacrine interactions and structural plasticity [START_REF] Sharif | Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion[END_REF]. These cells are mainly astrocytes, both at the level of GnRH cell bodies and terminals, and tanycytes, which interact with GnRH fibers at the level of the ME.

Paracrine factors

The paracrine factors that have been most extensively studied in relation to this matter are prostaglandin E2 (PGE2) and transforming growth factor (TGF) β.

PGE2 is released by both astrocytes and tanycytes in response to erbB receptor signalling activation. It derives from the arachidonic acid through the sequential activity of cyclooxygenases (COX) and a specific synthase (Fig. 12) [START_REF] Crofford | Prostaglandin biology[END_REF]. The erbB receptor family is a group of four tyrosine kinase receptors named erbB1 (or EGF receptor, EGFR), erbB2 (or Neu), erbB3 and erbB4. While erbB1 and 4 are fully functional receptors, with an extracellular ligand binding domain and a cytoplasmic tyrosine kinase domain, erbB2 and 3 lack the extracellular or cytoplasmic domain respectively, and act in the hypothalamus as coreceptor, in heterodimers always including erbB1 or erbB4 [START_REF] Hynes | ERBB Receptors and Cancer: The Complexity of Targeted Inhibitors[END_REF]. These receptors can differentially bind different growth factors of the EGF family such as EGF, TGFα and neuregulins (NRGs). Hypothalamic astrocytes express erbB1, 2 and 4 [START_REF] Sharif | Differential erbB signaling in astrocytes from the cerebral cortex and the hypothalamus of the human brain[END_REF] and tanycytes express erbB1, 2 and 3, while none of these receptors is expressed in GnRH neurons [START_REF] Voigt | Acting Via Tyrosine Kinase Receptors on Luteinizing Neurons[END_REF].

Activation of erbB heterodimers induces production and release of PGE2 which binds to specific E-prostanoid receptors (EPs) (Fig. 12) [START_REF] Woodward | International Union of Basic and Clinical Pharmacology. LXXXIII: Classification of Prostanoid Receptors, Updating 15 Years of Progress[END_REF]. Different subclasses of EPs exist (EP1-4) [START_REF] Woodward | International Union of Basic and Clinical Pharmacology. LXXXIII: Classification of Prostanoid Receptors, Updating 15 Years of Progress[END_REF] (Fig. 12) and GnRH neurons express both EP1 and 2, both at the level of nerve terminals and cell bodies. While activation of EP2 stimulates GnRH firing by acting at the level of the cell body, activation of EP1 induces mobilisation of intracellular calcium stores (Fig. 12) that is involved in the release of GnRH at the level of the ME [START_REF] Clasadonte | Prostaglandin E 2 release from astrocytes triggers gonadotropin-releasing hormone ( GnRH ) neuron fi ring via EP2 receptor activation[END_REF].

Figure 12: The arachidonic acid cascade and PGs signalling. Arachidonic acid is metabolized by cyclooxygenases (COX-1 and 2) to prostaglandin (PG)H2 via the short-lived intermediate PGG2. PGH2 then spontaneously rearranges or is enzymatically isomerized, oxidized, or reduced to yield bioactive prostaglandin isomers. Each PG binds to its specific receptor, which are all transmembrane receptors coupled to different G-proteins. Gq/p signalling results in an increase in the level of intracellular calcium. Gs proteins induce the expression of cAMP. Gi proteins are most often inhibitory to cAMP. AC, adenylate cyclase; DAG, diacylglycerol; IP3, inositol triphosphate; PIP2, phosphatidylinositol diphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C (Modified from Simmons D. L. 2004, Pharmacological reviews andHarris S. G. et al. 2002, Trends in Immunology).

Release of PGE2 in astrocytes depends upon activation of ionotropic and metabotropic glutamate receptors on glial cells (Fig. 13). This results in a cascade of events including recruitment of erbB1 and erbB4 receptors and their pro-ligands, pro-TGFα and pro-NRG, to the cell membrane, and transactivation of these receptors through the action of metalloproteinases such as TGF-α-converting-enzyme (TACE), which cleaves the pro-ligands into their mature forms that will activate erbB receptors [START_REF] Dziedzic | Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates ErbB receptor function in astroglial cells of the neuroendocrine brain[END_REF] (Fig. 13).

Oxytocin, as mentioned before, is produced by magnocellular neurosecretory cells of the supraoptic and paraventricular nuclei. It is able to accelerate GnRH release pulsatility, however, oxytocin receptors are not expressed by GnRH neurons themselves but by adjacent astrocytes. Moreover, its action is abolished if PGE2 synthesis is inhibited.

Therefore, it seems that oxytocin exerts its role on GnRH neurons, just like EGF ligands, through the mediation of astrocytes [START_REF] Parent | Oxytocin facilitates female sexual maturation through a glia-to-neuron signaling pathway[END_REF].

In tanycytes, the activity of COX is stimulated by the TGFα-induced activation of erbB1/erbB2 receptors but also by NO released by adjacent endothelial cells [START_REF] De Seranno | Role of Estradiol in the Dynamic Control of Tanycyte Plasticity Mediated by Vascular Endothelial Cells in the Median Eminence[END_REF].

All these mechanisms may be regulated by the action of gonadal steroids as oestrogens are known to stimulate TGFα release and COX activity [START_REF] Ma | Transforming growth factor-α gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation[END_REF].

The importance of this pathway is shown in animals with disrupted erbB signalling such as those expressing a dominant negative erbB4 in astrocytes which show decreased GnRH release and impaired reproductive function (Prevot, et al., 2003). Moreover, blocking either astrocyte metabolism or COX activity suppresses the spontaneous firing activity of GnRH neurons in brain slices, an effect that can be rescued by administration of exogenous PGE2 [START_REF] Clasadonte | Prostaglandin E 2 release from astrocytes triggers gonadotropin-releasing hormone ( GnRH ) neuron fi ring via EP2 receptor activation[END_REF].

TGFβ1, produced by astrocytes in accordance to oestrogen levels throughout the oestrus cycle [START_REF] Galbiati | Hypothalamic Transforming Growth Factor b 1 and Basic Fibroblast Growth Factor mRNA Expression is Modi ® ed During the Rat Oestrous Cycle[END_REF], is able to regulate GnRH expression and release in vitro in the immortalized hypothalamic cell line GT1. Besides, the expression of its receptors has been demonstrated in vivo at the level of GnRH neuron cell bodies, while it is absent in the ME.

Whether TGFβ1 modulates GnRH neuron activity in the POA remains to be explored [START_REF] Sharif | Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion[END_REF]. 2.3.5.2 Juxtacrine interactions Three major protein complexes have been involved in GnRH neuron-glial cell juxtacrine interactions: the PSA-NCAM, the receptor protein tyrosine phosphatase-β/ contactin/contactin-associated protein-1 (RPTPβ/contactin/CASPR1) complex and synaptic cell adhesion molecule 1 (SynCAM1) [START_REF] Sharif | Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion[END_REF]. Although their specific roles have not been fully clarified yet, they have all been found to be expressed by astrocytes and/or GnRH neurons with expression profiles changing according to reproductive maturation status or oestrus cycle phase. Furthermore, SynCAM1 appears to be necessary for activation of erbB4-dependent PGE2 release [START_REF] Sandau | SynCAM1, a synaptic adhesion molecule, is expressed in astrocytes and contributes to erbB4 receptor-mediated control of female sexual development[END_REF]. The expression of SynCAM1 in the POA decreases in mice with impaired erbB4 signalling; NRG treatment, hence activation of erbB4, results in rapid association of erbB4 with SynCAM1 and activation of SynCAM1 transcription. The importance of this interaction in the astrocytic modulation of GnRH activity was shown in animals with impaired SynCAM1 activity in GFAP-expressing astrocytes, which presented disrupted reproductive capacities as a result of defective astrocytic PGE2 response to NRGs [START_REF] Sandau | SynCAM1, a synaptic adhesion molecule, is expressed in astrocytes and contributes to erbB4 receptor-mediated control of female sexual development[END_REF].

Structural plasticity

As already noticed by Ramon y Cajal in his first description of the CNS, astrocytes enwrap neuronal cell bodies and terminals and these contacts are subjected to profound remodelling in response to different stimuli [START_REF] Theodosis | Activity-dependent neuronal-glial and synaptic plasticity in the adult mammalian hypothalamus[END_REF]. Astrocytes are found to contact GnRH neuron cell bodies and blood vessels at the same time in a tripartite complex sensitive to changing levels of gonadal hormones.

A very specific and peculiar example of how these cytoskeletal remodellings can affect GnRH release is represented by the role of tanycytes in the ME. More specifically, it appears that during periods of low oestrogen levels, such as during diestrus, tanycytic end-feet engulf GnRH terminals separating them from the perivascular space of the ME (Fig. 14). However, during proestrus, when oestrogen levels are high, tanycytes retract their end-feet therefore allowing GnRH terminals to reach the basal lamina of the perivascular space hence favouring the release of GnRH into the portal blood [START_REF] Prevot | Semi-quantitative ultrastructural analysis of the localization and neuropeptide content of gonadotropin releasing hormone nerve terminals in the median eminence throughout the estrous cycle of the rat[END_REF] (Fig. 14). The factors involved in this process have not been all identified. Nevertheless, the same factors involved in the paracrine crosstalk between tanycytes and GnRH neurons appear to have a role in their structural remodelling, as in the case of EGF, TGFβ1, PGE2 or endothelial NO [START_REF] Sharif | Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion[END_REF]. For instance, TGFα, produced by both tanycytes and astrocytes, binds and activates erbB1/B2 heterodimers in both cell types, inducing the release of PGE2. PGE2 then stimulates the release of TGFβ1 from tanycytes, which acts as an autocrine factor to induce endfeet retraction through the activity of metalloproteinases (Prevot et al., 2003) (Fig. 14).

Moreover, as previously said, NO produced from endothelial cells stimulates COX activity and therefore PGE2 production [START_REF] De Seranno | Role of Estradiol in the Dynamic Control of Tanycyte Plasticity Mediated by Vascular Endothelial Cells in the Median Eminence[END_REF]. Recent works from our laboratory have also identified semaphorin7A (sema7A) as a tanycytic factor released according to progesterone levels, being maximal on the afternoon of diestrus-2 and able to stimulate GnRH terminal retraction from the capillary space as well as tanycytes' end-feet expansion [START_REF] Parkash | Semaphorin7A regulates neuroglial plasticity in the adult hypothalamic median eminence[END_REF]. Together with these main modes of action, it is important to mention that hypothalamic astrocytes are not only able to sense gonadal hormones, as they express androgen, oestrogen and progesterone receptors [START_REF] Melcangi | G lial cells : a target for steroid hormones[END_REF], but they are also able to produce neurosteroid hormones, which can act in a paracrine way to modulate different functions such as anxiety, cognition, sleep and aggression [START_REF] Garcia-Segura | Minireview: Role of Glia in Neuroendocrine Function[END_REF].

An interesting example is the ability of astrocytes to respond to oestrogen signalling by producing neuroprogesterone, which is involved in the pre-ovulatory LH surge, as demonstrated by the inability of oestrogens to induce an LH surge in ovariectomised rats treated with a blocker of progesterone synthesis [START_REF] Micevych | The luteinizing hormone surge is preceded by an estrogen-induced increase of hypothalamic progesterone in ovariectomized and adrenalectomized rats[END_REF][START_REF] Micevych | Estradiol stimulates progesterone synthesis in hypothalamic astrocyte cultures[END_REF][START_REF] Terasawa | Neuroestrogen, rapid action of estradiol, and GnRH neurons[END_REF]. Intriguingly, astrocytes from neonatal rats, even though able to sense oestrogen levels, do not respond by releasing progesterone, in agreement with hypothalamic non responsiveness to oestradiol-positive feedback in neonatal animals [START_REF] Micevych | Estradiol stimulates progesterone synthesis in hypothalamic astrocyte cultures[END_REF].

Human findings

Due to the difficulties in performing human studies, it has not been easy to prove the role of glial cells in the regulation of GnRH function in our species. Nonetheless, a tight interaction between GnRH neurons, astrocytes and tanycytes has been described both at the level of their cell body and terminals (Baroncini et al., 2007), as found in other species (Fig. 15). Moreover, magnetic resonance imaging (MRI) studies have been performed in young women to study differences in the hypothalamus throughout the oestrous cycle. Diffusion MRI experiments showed that when the early follicular phase LH pulsatile release occurs, hence the hypothalamus is more active, a higher rate of water molecule diffusion is observed compared to the pill-supplemented period, when the hypothalamus is fully inhibited, or compared to males. Moreover, these changes were not observed in the thalamus, a non-neuroendocrine region [START_REF] Baroncini | Sex steroid hormones-related structural plasticity in the human hypothalamus[END_REF]. Modifications of water diffusion can be linked to shifting of water from the intracellular to the extracellular space, changes in membrane permeability or a decrease in the tortuosity of the extracellular space.

Interestingly, changes in the astrocytic coverage of neurons modify the extracellular space geometry and diffusion parameters during physiological activation [START_REF] Piet | Physiological contribution of the astrocytic environment of neurons to intersynaptic crosstalk[END_REF]. Hence suggesting that the glial coverage of hypothalamic neurons changes according to the activity of the hypothalamus.

Proton MR spectroscopy experiments, which measure freely mobile molecules, performed in the same groups, showed reduced choline levels in the hypothalamus, but not the thalamus, during the pill-free period when compared to the period of oral contraceptive use or to males. Choline is especially concentrated in astrocytes and alterations in choline levels have been linked to disturbances in the formation and degradation of cell membranes [START_REF] Manganas | 1 Grigori Enikolopov, 3* Mirjana Maletic ´-Savatic ´ 1[END_REF].

Altogether these data lead to the speculation that activation of the gonadal axis in women is associated to microstructural changes that cause a reduction in the astroglial coverage of GnRH neurons [START_REF] Baroncini | Sex steroid hormones-related structural plasticity in the human hypothalamus[END_REF].

Finally, insights on the glial control of GnRH neurons in humans come from studies performed on hypothalamic hamartomas (HH), which are rare congenital tumour-like malformations sometimes associated with precocious puberty. It was therefore hypothesised that this effect was due to the ectopic expression of GnRH in the HHs.

Nevertheless, some HHs associated with precocious puberty were shown not to express GnRH, while they were enriched in TGFα-and erbB1 receptor-expressing astrocytes [START_REF] Ma | Transforming growth factor-α gene expression in the hypothalamus is developmentally regulated and linked to sexual maturation[END_REF]. These findings suggest that the glial erbB signalling system may also exist in humans and stimulate the GnRH neurons in a similar way to what has been observed in rodents [START_REF] Sharif | Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion[END_REF].

EMBRYONIC DEVELOPMENT OF THE GNRH SYSTEM

The correct functioning of the HPG axis is tightly dependent on the proper development and establishment of GnRH circuits and correct signalling pattern.

The story of GnRH neurons begins during embryogenesis, more precisely around embryonic day 10.5 (E10.5) in mice and rats (S. [START_REF] Wray | From nose to brain: Development of gonadotrophin-releasing hormone -1 neurones[END_REF] and gestational week 6.5 (GW 6.5) in human [START_REF] Casoni | Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains[END_REF]. Unlike the other parvocellular neuroendocrine neurons, which derive from the proliferative neuroepithelium of the third ventricle [START_REF] Forni | Neural Crest and Ectodermal Cells Intermix in the Nasal Placode to Give Rise to GnRH-1 Neurons, Sensory Neurons, and Olfactory Ensheathing Cells[END_REF] 3V f i h [START_REF] Markakis | Development of the neuroendocrine hypothalamus[END_REF], the GnRH neurons arise from the nasal placode with a single peak of proliferation. However, the identity of their progenitors still remains to be elucidated [START_REF] Markakis | Development of the neuroendocrine hypothalamus[END_REF][START_REF] Wray | From nose to brain: Development of gonadotrophin-releasing hormone -1 neurones[END_REF] as their molecular profile within the placode is not homogeneous as previously thought [START_REF] Forni | Neural Crest and Ectodermal Cells Intermix in the Nasal Placode to Give Rise to GnRH-1 Neurons, Sensory Neurons, and Olfactory Ensheathing Cells[END_REF]. In fact, together with a placode origin a putative neural crest origin has also been proposed to contribute to the GnRH neuron cell lineage [START_REF] Forni | Neural Crest and Ectodermal Cells Intermix in the Nasal Placode to Give Rise to GnRH-1 Neurons, Sensory Neurons, and Olfactory Ensheathing Cells[END_REF].

Being born outside from the brain, these neurons migrate through the nasal mesenchyme, cribriform plate and the OB to finally reach the basal forebrain. This journey ends around E18.5 in mice and rats [START_REF] Cariboni | Defective gonadotropin-releasing hormone neuron migration in mice lacking SEMA3A signalling through NRP1 and NRP2: Implications for the aetiology of hypogonadotropic hypogonadism[END_REF][START_REF] Wray | From nose to brain: Development of gonadotrophin-releasing hormone -1 neurones[END_REF] and GW20 in humans [START_REF] Kim | Gonadotropin-releasing hormone immunoreactivity in the adult and fetal human olfactory system[END_REF] when all the cells have left the nasal compartment and have reached their final destination.

The GnRH neurons migrate along a migratory route formed by the terminal and vomeronasal nerves emerging from the vomeronasal epithelium before migration begins [START_REF] Casoni | Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains[END_REF][START_REF] Wray | Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode (prenatal development/in situ hybridization/histochenustry/immunocytochemistry/[3H]thymidine autoradiography)[END_REF]. Interaction with these axons appears to be fundamental for facilitating correct migration, as proved by the observation that disruption of axonal growth and/or of direct interactions between the fibers and migrating GnRH neurons results into migration defects and consequent deficits in reproduction [START_REF] Cariboni | From nose to fertility: the long migratory journey of gonadotropin-releasing hormone neurons[END_REF][START_REF] Wray | From nose to brain: Development of gonadotrophin-releasing hormone -1 neurones[END_REF]. Nevertheless, vomeronasal sensory axons and terminal nerve axons are not the only components of these migratory scaffold as non GnRH neuronal cells, glial cells and blood vessels have also been shown to bundle together in a structure whose exact nature remains poorly understood [START_REF] Forni | Neural crest and olfactory system: New prospective[END_REF]. One of the components of this path are the olfactory ensheathing cells (OECs), which are glial cells that normally ensheath the olfactory, vomeronasal and terminal nerves and that are involved in their growth and guidance.

Accordingly, mice lacking OECs as a result of a mutation in the Sox10 gene, which is involved in the development of the neural crests, from which OECs derive, present misrouted and defasciculated vomeronasal and terminal nerves and show an abnormal migration of GnRH neurons since these cells fail to enter the brain at E14.5. However, due to the mortality of the mutants, data following this age were impossible to obtain [START_REF] Ping | Loss-of-function mutations in SOX10 cause Kallmann syndrome with deafness[END_REF].

The transition between the nasal mesenchyme and the developing CNS tissue represents a dramatic change in the environment surrounding the migrating GnRH neurons. This transition occurs at the level of the nasal-forebrain junction where the cells appear to pause before entering the brain. The reasons for this delay are not clear but it is hypothesized that it could allow maturation of GnRH neurons or axons before they reach the final appropriate brain regions [START_REF] Wray | From nose to brain: Development of gonadotrophin-releasing hormone -1 neurones[END_REF]. This quite peculiar and long migratory journey traversing different and still developing tissues means that GnRH neurons face a spatio-temporally changing combination of molecules that act as soluble or membrane-bound guidance cues as well as repellent or attractant molecules forming chemoattractive gradients. Several classes of factors have been studied in relation to this process and different mutations have been associated with disrupted migration and therefore reproductive capacity [START_REF] Wray | From nose to brain: Development of gonadotrophin-releasing hormone -1 neurones[END_REF]. These factors include, but are not limited to: guidance molecules such as slits and semaphorins, growth factors, cytokines, chemokines, a variety of neurotransmitters and neuromodulators such as GABA, glutamate and CCK, and even transcription factors. It is important to point out that among the plethora of factors that have been shown to have an impact on the migration of GnRH neurons, a notable absent in rodents is GnRH itself [START_REF] Wray | From nose to brain: Development of gonadotrophin-releasing hormone -1 neurones[END_REF] as demonstrated by studies on two hypogonadal mouse strains, the Gnrh mutant (hpg) mice and the Gnrh receptor mutant mice, that have a normal GnRH neuron population in terms of size, distribution and projection to the ME [START_REF] Gill | The gonadotropin-releasing hormone (GnRH) neuronal population is normal in size and distribution in GnRH-deficient and GnRH receptor-mutant hypogonadal mice[END_REF].

Once their final destination is reached, GnRH neurons detach from their guiding fibers and spread to subsequently extend their terminals to the ME. In adults, GnRH neurons are found in different regions along their main migratory pathway from the OB to the mediobasal hypothalamus. However, this is not the only path they undertake. The existence of a dorsal migratory route has been shown both in mice and humans, from where GnRH neurons reach extrahypothalamic areas such as the hippocampus, piriform cortex and amygdala. The function of these neurons has not been clarified yet [START_REF] Casoni | Development of the neurons controlling fertility in humans: new insights from 3D imaging and transparent fetal brains[END_REF][START_REF] Schwanzel-Fukuda | Distribution of luteinizing hormone-releasing hormone in the nervus terminalis and brain of the mouse detected by immunocytochemistry[END_REF].

Regardless of their location, most GnRH neurons project their terminals to the ME where they are found in rodents from E15-E16 (Eurenius & Jarskar, 1979). However, the first release of the neurohormone coincides with the last two or three days of embryonic development [START_REF] Aubert | Ontogeny of Hypothalamic Luteinizing Hormone-Releasing Hormone (GnRH) and Pituitary GnRH Receptors in Fetal and Neonatal Rats[END_REF]. This precocious release of GnRH seems to be important to prime the HPG axis and more specifically to favour the formation of cell networks in the anterior pituitary, being this the moment when the gonadotroph cells begin to organise (Le [START_REF] Tissier | Anterior pituitary cell networks[END_REF][START_REF] Prevot | Chapter 30 -Puberty in Mice and Rats[END_REF].

POSTNATAL DEVELOPMENT OF THE GNRH SYSTEM

The capacity for sexual reproduction is not a characteristic of newborn mammals even though at birth, both GnRH cell bodies and fibers are in place. This means that at this point the system still needs to undergo a postnatal period of maturation in order to achieve sexual competence corresponding to initiation of puberty.

The onset of puberty implies a switch from a low prepubertal GnRH release pattern to an adult phasic and synchronous activation, characterized by GnRH pulses. Puberty is the result of complex interactions between different regions of the hypothalamus as well as genetic and environmental inputs. Considering that GnRH neurons are able to release GnRH way before the onset of puberty, it is clear that the gatekeepers of the pubertal process' initiation are transsynaptic and glial inputs able to modulate GnRH neurons' activity.

Importantly, initiation of the processes that will lead to puberty does not need an influence from the gonads but depends upon events that occur within the brain. However, puberty itself results from the action of gonadal hormones on GnRH neurons [START_REF] Ojeda | Recent advances in the endocrinology of puberty[END_REF][START_REF] Prevot | Chapter 30 -Puberty in Mice and Rats[END_REF].

When trying to understand a process as long and complex as the establishment of puberty onset (which takes weeks for rodents and years for primates), it is important to take into account not only the process of maturation of the GnRH neurons themselves but also the concomitant development of the surrounding CNS as well as that of the gonads. The postnatal period is in fact a critical moment for the shaping of neural circuits and it is at this moment that the GnRH neural network is established with excitatory and inhibitory transsynaptic inputs, converging from different nuclei of the hypothalamus, as well as nonsynaptic inputs.

Postnatal development in female mice and rats can be divided in four periods: neonatal, infantile, juvenile and peripubertal [START_REF] Prevot | Chapter 30 -Puberty in Mice and Rats[END_REF].

The neonatal period goes from P0 to P7. During the first week of life, the ovarian development occurs independently from the hypothalamic-pituitary axis and by day 7, developed secondary follicles with functional FSH and LH receptors are found in the ovaries [START_REF] Mcgee | Initial and cyclic recruitment of ovarian follicles[END_REF] (Fig. 16).

The infantile period goes from P8 to P21, when pups are weaned from their mothers. The most striking event during this period is a peak in the FSH levels seen at P12 in both mice and rats, which corresponds to the first centrally-driven activation of the HPG axis and may correspond to minipuberty, a phase in the human pubertal maturation when an activation of the HPG axis occurs in the early postnatal life to return quiescent again until time of puberty; in girls, FSH levels increase during the first 3 months and remain high even until 3-4 years of age [START_REF] Kuiri-Hänninen | Activation of the hypothalamic-pituitarygonadal axis in infancy: Minipuberty[END_REF]. This event is gonadal independent but it is critical for the development of the ovarian follicles being FSH a survival factor for early antral follicles that would otherwise undergo apoptosis [START_REF] Mcgee | Initial and cyclic recruitment of ovarian follicles[END_REF]. On the contrary, LH levels remain very low during this period, probably due to the fact that GnRH pulsatile release is at very low frequencies [START_REF] Kaiser | Differential Effects of Gonadotropin-Releasing Hormone (GnRH) Pulse Frequency on Gonadotropin Subunit and GnRH Receptor Messenger Ribonucleic[END_REF]) (Fig. 16).

The juvenile period lasts until P30 in rats and between P25 and P40 in mice. During the juvenile period, both levels of FSH and LH remain very low, while GnRH pulse frequency increases. Meanwhile, oestrogens begin to exert their negative feedback on the GnRH neurons through ARC kisspeptin neuron inputs; however, oestrogen levels are still too low to achieve a positive feedback effect [START_REF] White | Changes in Ovarian Luteinizing Hormone and Follicle-Stimulating Hormone Receptor Content and in Gonadotropin-Induced Ornithine Decarboxylase Activity during Prepubertal and Pubertal Development of the[END_REF]) (Fig. 16).

The peripubertal period culminates with the first ovulation, which occurs around P34-P38 in rats and 6-12 months after the first menstruation in humans. Approaching puberty, GnRH pulses keep increasing in frequency and LH levels acquire a nocturnal pattern with afternoon pulses being higher in amplitude but not in frequency compared to morning pulses [START_REF] Urbanski | The juvenile peripubertal transition period in the female rat: Establishment of a diurnal pattern of pulsatile LH secretion[END_REF]. While the events described so far are gonad-independent, puberty relies on the ability of rising oestrogen levels to trigger a GnRH/LH/FSH surge which will result in the first ovulation [START_REF] Ojeda | The onset of puberty in the female rat: Changes in plasma prolactin, gonadotropins, luteinizing hormone-releasing hormone (LHRH), and hypothalamic LHRH content[END_REF]) (Fig. 16).

Establishment of the GnRH neural network

These first weeks of postnatal life are important for maturation of hypothalamic axonal projections and synapses. GnRH neurons gradually receive synaptic inputs during development seen as an increase in synaptophysin punctae found in close apposition with GnRH neurons' proximal dendrites [START_REF] Xue | Morphological changes of gonadotropin-releasing hormone neurons in the rat preoptic area across puberty[END_REF]. These axonal inputs arrive from different hypothalamic nuclei such as the ARC, VMH and DMH [START_REF] Bouret | Formation of Projection Pathways from the Arcuate Nucleus of the Hypothalamus to Hypothalamic Regions Implicated in the Neural Control of Feeding Behavior in Mice[END_REF], as well as the AVPV [START_REF] Polston | Ontogeny of the projections from the anteroventral periventricular nucleus of the hypothalamus in the female rat[END_REF]) (Fig. 16).

Inhibitory inputs

The progressive changes in the secretion of GnRH seen throughout postnatal development are believed to be due to a gradual decrease in inhibitory and increase in excitatory inputs to the GnRH neurons.

An initial "restraint" is necessary to avoid precocious activation of GnRH neurons and precocious puberty. The main neurotransmitters involved are GABA and opioid peptides [START_REF] Ojeda | The neurobiology of female puberty[END_REF].

Even though the general effect of GABA on GnRH neuron activity is inhibitory both before and after puberty, it is important to point that its role on the progression of puberty onset does not imply only a direct action on GnRH neurons themselves. While, intuitively, blockade of GABA A receptors advances puberty [START_REF] Donoso | Glutamate receptors of the non-N-methyl-Daspartic acid type mediate the increase in luteinizing hormone-releasing hormone release by exicitatory amino acids in vitro[END_REF], the direct activation of these receptors in GnRH neurons results in an excitatory effect due to the high intracellular concentration of chloride present in these cells before puberty [START_REF] Gore | Gonadotropin-releasing hormone neurons, NMDA receptors, and their regulation by steroid hormones across the reproductive life cycle[END_REF]. Therefore, its role in preventing precocious puberty must be exerted indirectly by inhibiting different GnRH excitatory systems. On the other hand, a direct activation of GABA B receptors induces inhibition of GnRH neuron activity [START_REF] Lagrange | Estradiol-17 β and μ-opioid peptides rapidly hyperpolarize GnRH neurons: A cellular mechanism of negative feedback[END_REF] maybe contrasting the excitatory tone mediated by GABA A receptors. It appears that the GABAergic innervation of GnRH neurons does not change throughout postnatal development [START_REF] Cottrell | Postnatal remodeling of dendritic structure and spine density in gonadotropin-releasing hormone neurons[END_REF].

However, the effects of GABAergic signalling do not remain constant across development:

GnRH neurons' intracellular content of chloride decreases while the expression profile of the different GABA A receptor subunits, known to affect sensitivity to GABA itself, is subject to variation [START_REF] Temple | Developmental changes in GABAA receptor subunit composition within the gonadotrophin-releasing hormone-1 neuronal system[END_REF].

The role of endogenous opioids in this picture has not been fully examined yet; however, it has been shown that opioid peptides can inhibit gonadotropin secretion during postnatal development and this effect decreases around the time of puberty [START_REF] Bezzi | Prostaglandins stimulate calcium-dependent glutamate release in astrocytes[END_REF][START_REF] Nakahara | Chronic peripheral administration of kappa-opioid receptor antagonist advances puberty onset associated with acceleration of pulsatile luteinizing hormone secretion in female rats[END_REF][START_REF] Parpura | Glutamatemediated astrocyte-neuron signalling[END_REF].

2.5.1.2 Excitatory inputs While inhibitory signals are necessary to prevent an early puberty, increasing excitatory signals are required to induce an adult pulsatile GnRH secretion pattern [START_REF] Ojeda | The neurobiology of female puberty[END_REF].

The main excitatory inputs come from glutamatergic neurons that stimulate GnRH neurons and other components of the GnRH neuronal network by activating both NMDA and AMPA receptors, two classes of ionotropic glutamate receptors (Eyigor & Jennes, 1997). During postnatal development, the hypothalamic glutamate content rises while glutamatergic projections to the POA increase [START_REF] Goroll | Ontogenic changes in the hypothalamic levels of amino acid neurotransmitters in the female rat[END_REF]Prevot, 2014). It is important to point that hypothalamic glutamate does not only derive from neurons but also from astrocytes, which have been shown to be able to release glutamate in response to calcium waves [START_REF] Charles | Intercellular signaling in glial cells: Calcium waves and oscillations in response to mechanical stimulation and glutamate[END_REF][START_REF] Haydon | GLIA: listening and talking to the synapse[END_REF]. Hypothalamic glutamatergic projections do not only reach GnRH neurons but also the neighbouring GABAergic neurons. It was shown that, during postnatal development, glutamate acts on GABAergic synapses both at a postsynaptic level, by inhibiting the calcium increase induced by GABA-mediated depolarization, and at a presynaptic level by reducing GABA release (van den [START_REF] Van Den Pol | Glutamate inhibits GABA excitatory activity in developing neurons[END_REF]. Therefore, it has been hypothesised that during the postnatal development an increase in glutamatergic inputs precedes and causes the decline of the inhibitory GABAergic tone, leading to the final activation of GnRH neurons (Ojeda et al., 2003).

A further point in favour of the gradual increase in glutamatergic inputs is the evidence that during postnatal development, GnRH neurons undergo a remodelling of their dendritic tree structure and spine density [START_REF] Cottrell | Postnatal remodeling of dendritic structure and spine density in gonadotropin-releasing hormone neurons[END_REF][START_REF] Xue | Morphological changes of gonadotropin-releasing hormone neurons in the rat preoptic area across puberty[END_REF]. Dendritic spines can be used as an index for excitatory inputs and their number dramatically increases between the pre-and the post-pubertal period in male mice, corroborating the idea that GnRH neurons receive increasing excitatory inputs [START_REF] Cottrell | Postnatal remodeling of dendritic structure and spine density in gonadotropin-releasing hormone neurons[END_REF]. A peculiarity of these neurons is also the fact that many spines are found at the level of their cell body and not only on their dendrites.

Neuropeptidergic inputs

AVPV neurons extend and develop their projections from embryonic stage until well after birth. Projections contacting the GnRH neurons have been found as early as E19, while caudal projections to the PVH develop during the first postnatal week [START_REF] Polston | Ontogeny of the projections from the anteroventral periventricular nucleus of the hypothalamus in the female rat[END_REF]. Projections from the ARC are immature at birth and only develop during the second week of life in mice [START_REF] Bouret | Formation of Projection Pathways from the Arcuate Nucleus of the Hypothalamus to Hypothalamic Regions Implicated in the Neural Control of Feeding Behavior in Mice[END_REF]. A mature innervation pattern is reached around P12 with fibers reaching the DMH, VMH, lateral hypothalamus (LHA) and PVN [START_REF] Bouret | Formation of Projection Pathways from the Arcuate Nucleus of the Hypothalamus to Hypothalamic Regions Implicated in the Neural Control of Feeding Behavior in Mice[END_REF]. Kisspeptin neurons are found in both the AVPV and the ARC and, as already mentioned, are responsible for mediating oestrogen's feedbacks to the GnRH neurons. It has been shown that in mice lacking Kiss1r, puberty is totally absent [START_REF] Seminara | The GPR54 Gene as a Regulator of Puberty[END_REF] while mice with an activating mutation of Kiss1r show an early puberty onset [START_REF] Teles | A GPR54-activating mutation in a patient with central precocious puberty[END_REF]. The sensitivity of GnRH neurons to kisspeptin increases during postnatal development with only 25% of them being responsive in juveniles, 50% in prepubertal and up to 90% in adult mice [START_REF] Han | Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty[END_REF]. However, these changes do not depend on differences in Kiss1r expression in GnRH neurons [START_REF] Han | Activation of Gonadotropin-Releasing Hormone Neurons by Kisspeptin as a Neuroendocrine Switch for the Onset of Puberty[END_REF]. At the same time, during late infantile period, kisspeptin fibers from the ARC reach the POA and mediate oestrogen's negative feedback, avoiding a premature activation of GnRH neurons [START_REF] Caron | Alteration in Neonatal Nutrition Causes Perturbations in Hypothalamic Neural Circuits Controlling Reproductive Function[END_REF][START_REF] Mayer | Timing and completion of puberty in female mice depend on estrogen receptorsignaling in kisspeptin neurons[END_REF]. Kisspeptin neurons in the AVPV on the other hand are critical for ovulation since they mediate oestrogen's positive feedback; therefore when maturation is completed, their signalling results in the GnRH and therefore LH surge necessary for the release of the oocyte from the follicle [START_REF] Kirilov | Dependence of fertility on kisspeptin-Gpr54 signaling at the GnRH neuron[END_REF].

In the ARC, anorexigenic POMC and orexigenic NPY/AgRP neurons inhibit or favour the onset of puberty depending on the energy status of the animal. POMC and NPY/AgRP neurons are able to, respectively, inhibit or stimulate GnRH release both directly and indirectly, probably in concert with kisspeptin neurons [START_REF] Manfredi-Lozano | Connecting metabolism and gonadal function: Novel central neuropeptide pathways involved in the metabolic control of puberty and fertility[END_REF][START_REF] Nestor | Cross-talk between reproduction and energy homeostasis: Central impact of estrogens, leptin and kisspeptin signaling[END_REF]. For instance leptin levels, which can be sensed by both these neuronal populations as a signal of energy adequacy, have a permissive role over puberty with leptin deficiency resulting in delayed or absent puberty [START_REF] Vázquez | Roles of leptin in reproduction, pregnancy and polycystic ovary syndrome: Consensus knowledge and recent developments[END_REF]. Moreover, blocking α-MSH signalling delays puberty in female rats and abolishes the permissive effect of leptin [START_REF] Manfredi-Lozano | Defining a novel leptin-melanocortin-kisspeptin pathway involved in the metabolic control of puberty[END_REF] while deletion of NPY signalling in leptin-deficient animals restores the onset of puberty [START_REF] Sainsbury | Y4 receptor knockout rescues fertility in ob / ob mice Y4 receptor knockout rescues fertility in ob / ob mice[END_REF].

nNOS neurons

Unlike other neuronal populations, nNOS neurons do not send projections to GnRH neurons, as NO is a gas that freely diffuses through biological membranes. Nonetheless, unpublished data from our laboratory show that activation of nNOS neurons in the OVLT occurs concomitantly with the peak of FSH observed at P12 in female mice. Moreover, inhibition of nNOS signalling specifically during the infantile period delays puberty onset and perturbs fertility in adult animals (Konstantina Chachlaki and Vincent Prevot, unpublished data). As previously mentioned, nNOS neurons are also able to convey metabolic signals to GnRH neurons. A study performed in leptin-deficient obese mice showed that nNOS inhibition prevented exogenous leptin administration to induce normal sexual maturation even though a normal body weight was rescued [START_REF] Bellefontaine | Leptin-dependent neuronal no signaling in the preoptic hypothalamus facilitates reproduction[END_REF].

Glial cells

Alongside the events involved in the establishment of the neuronal connectivity, postnatal development is characterised by the birth and maturation of most glial cells. In addition to their role in regulating GnRH neurons during adulthood, glial cells are involved in the maturation of the GnRH system.

As seen in a previous chapter (see chapter 3.3.5), glial cells use different strategies to modulate GnRH neuron activity during adulthood. The main factors involved are TGFβ1, EGF, TGFα, NRGs growth factors and the erbB tyrosine kinase receptors [START_REF] Sharif | Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion[END_REF]. The expression of TGFα and erbB receptors increases in concomitance with puberty onset [START_REF] Ma | Expression of epidermal growth factor receptor changes in the hypothalamus during the onset of female puberty[END_REF]. Moreover, animals with disrupted erbB signalling, as a result of a mutation in erbB1 receptor or the expression of a dominant-negative form of erbB4 receptor specifically in astrocytes, have delayed puberty, and this delay is exacerbated when both signalling are disrupted (Prevot et al., 2003). On the contrary, transplantation of TGF-overexpressing cells in the vicinity of GnRH neuron cell bodies and terminals induces precocious puberty [START_REF] Rage | Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway[END_REF].

Adhesion molecules, which are involved in the crosstalk between glial cells and GnRH neurons (see chapter 3.3.5.2), also participate in the maturation of the system. In particular, SynCAM1, whose levels in the hypothalamus increase during sexual maturation, is required for sexual maturation since transgenic mice with an astrocyte-specific disruption of SynCAM1 function show delayed puberty [START_REF] Sandau | SynCAM1, a synaptic adhesion molecule, is expressed in astrocytes and contributes to erbB4 receptor-mediated control of female sexual development[END_REF].

These are only some examples of the molecules involved in the tight interactions between

GnRH neurons and glial cells. However, many others still need to be characterised for their possible contribution to the initiation of puberty.

PREGNANCY AND BEYOND

Pregnancy

The ultimate purpose of the finely regulated process of ovulation is to ensure fertilisation of the ovum following insemination. As previously said, if fertilisation does not occur the corpus luteum formed from residual follicular granulosal and thecal cells degenerates and a new cycle begins.

However, if fertilisation does occur the corpus luteum keeps producing progesterone, which is essential for maintenance of normal pregnancy in mammals, until the placenta begins to take over [START_REF] Pate | Life or death decisions in the corpus luteum[END_REF]. Circulating levels of progesterone are maintained very high throughout almost the entire course of pregnancy until they drop in concomitance with parturition. The corpus luteum, however, does not only produce progesterone but is also important for the production of oestrogens, either by a direct release or by aromatisation of placenta-derived androgens. Oestrogen levels increase gradually during pregnancy until reaching a peak at parturition that drops soon afterwards until oestrus cycle is resumed. At the beginning of pregnancy, prolactin is synthesised by the anterior pituitary in two daily surges, one diurnal and one nocturnal [START_REF] Butcher | Semicircadian rhythm in plasma levels of prolactin during early gestation in the rat[END_REF]; these two surges are gonadal independent as they are initiated even in the absence of gonadal hormones but the latter seem to be required for termination of the surges. The two daily surges of prolactin are responsible for the increased life span of the corpus luteum and its secretion of progesterone early in pregnancy and are thus critical for the maintenance of pregnancy [START_REF] Smith | The control of progesterone secretion during the estrus cycle and early pseudopregnancy in the rat: gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy[END_REF]. Maintenance of the corpus luteum is achieved through two actions of prolactin: increased expression of the LH receptor, leading to increased progesterone synthesis, and repression of the enzymes responsible for progesterone metabolism in the ovary. In the second half of pregnancy, pituitary secretion of prolactin is inhibited by the high release of placental lactogens, which stimulate the activity of the dopaminergic hypothalamic neurons that in turn inhibit the release of prolactin from the anterior pituitary [START_REF] Tonkowicz | Termination of prolactin surges with development of placental lactogen secretion in the pregnant rat[END_REF][START_REF] Grattan | Hypothalamic Control of Prolactin Secretion, and the Multiple Reproductive Functions of Prolactin[END_REF]. During pregnancy, prolactin also prepares the mammary glands in view of lactation.

Lactation

After parturition, the process of suckling mediates important features, necessary for successful nourishment of the offspring themselves: it increases prolactin secretion, which controls milk production; it inhibits GnRH and consequently LH pulsatility; it increases orexigenic and decreases anorexigenic neuronal activity in order to increase maternal food intake [START_REF] Grattan | The hypothalamo-prolactin axis[END_REF].

Prolactin is released in response to a neuroendocrine reflex set in motion by suckling of the pups [START_REF] Selmanoff | Suckling decreases dopamine turnover in both medial and lateral aspects of the median eminence in the rat[END_REF]. This secretion, unlike the surge release, is tightly linked to the stimulus itself as introduction of pups immediately increases prolactin levels and each episode of secretion requires reapplication of the stimulus. During lactation, in many species, there is a suppression of ovarian function meant to avoid overlapping of this process with a new pregnancy due to the high level of energy expenditure that accompanies both these events. However, while some species undergo a period of anovulation (e.g. ewes), others still ovulate but they either have a much longer gestation than lactation so that the end of pregnancy will not overlap with lactation of the previous offspring (as in horses), or lactation can induce a late implantation (as in the case of rats) [START_REF] Goodman | Chapter 33. Neuroendocrine Control of Gonadotropin Secretion: Comparative Aspects[END_REF].

Arcuate NPY and kisspeptin neurons express prolactin receptors and prolactin can be at least partially responsible for NPY neuron activation [START_REF] Li | Neuropeptide Y and tuberoinfundibular dopamine activities are altered during lactation: Role of prolactin[END_REF], which increases food intake, and kisspeptin neuron inhibition, which decrease GnRH neuron activity. This increase in food intake, or hyperphagia, is necessary to avoid exhaustion of maternal energy stores. During gestation, leptin levels are significantly increased due to a hyper production of leptin by the placenta and adipose tissue; therefore, to explain the increase in food intake, a physiological state of leptin resistance seems to be necessary [START_REF] Grattan | Hormonal induction of leptin resistance during pregnancy[END_REF]. Two mechanisms have been proposed to underlie this process: a reduction in the transport of leptin through the BBB, assessed by measuring leptin levels in the CSF of pregnant rats, and an alteration in the hypothalamic response to leptin [START_REF] Trujillo | Hyperphagia and central mechanisms for leptin resistance during pregnancy[END_REF]. In particular, a decrease in leptin receptor levels in the VMH together with a decrease in the number of leptin-responsive cells occurs during pregnancy [START_REF] Ladyman | Suppression of leptin receptor mRNA and leptin responsiveness in the ventromedial nucleus of the hypothalamus during pregnancy in the rat[END_REF], allowing hyperphagia even in a situation of hyperleptinemia.

Maternal behaviour

Parental care is indispensable for survival of the offspring and is therefore finely regulated.

Parenting behaviour is usually sexually dimorphic and most of mammalian species display maternal but not paternal behaviour. Maternal behaviour includes many different components such as: nest building, care of the young, recognition and bonding, nursing and defensive behaviour as well as aggression against intruders.

For maternal behaviour to be initiated, adaptation of different neural circuitries of the maternal brain needs to occur prior to parturition. The pregnancy hormones prolactin, oestrogens and progesterone are the main responsible for this process of reorganisation.

The main brain circuits involved are the OB, the medial POA (MPO) and the mesolimbic dopaminergic motivation system. The OB circuitry is critical for recognition of the offspring as olfaction is the primary sensory mediator of social information in rodents (Lonstein et al., 2014). In relation to this, it has been shown that during pregnancy, prolactin stimulates the birth of new olfactory interneurons with important functional consequences for the associated maternal behaviour [START_REF] Shingo | Pregnancy-Stimulated Neurogenesis in the Adult Female Forebrain Mediated by Prolactin[END_REF].

The MPO is the key hypothalamic area for the establishment of maternal behaviour (Fisher, 1956). A number of studies have shown that functional impairment of the MPO through different approaches, such as wounds, pharmacological or electrolytic injuries, lesion of the axonal projections emanating from this area, all result in disrupted maternal behaviour (Lonstein et al., 2014). This region is able to sense ovarian and pituitary hormones, thanks to a widespread expression of different receptors whose levels are increased at the end of pregnancy, and then send the information to the motivation system [START_REF] Brunton | The expectant brain: adapting for motherhood[END_REF]. This crosstalk is necessary for the newly mothers to become attracted by newborns, through activation of the reward dopaminergic circuitry [START_REF] Carlson | Elevated prolactin levels immediately precede decisions to babysit by male meerkat helpers[END_REF], and to suppress their fear response to pups, through active GABAergic inhibition of the periaqueductal gray [START_REF] Brooks | Dopamine agonist treatment before and after the birth reduces prolactin concentration but does not impair paternal responsiveness in Djungarian hamsters, Phodopus campbelli[END_REF]. Nonetheless, the role of the MPO in regulating maternal behaviour does not rely on activation of endocrine pathways but rather on its electrical activity. Brain adaptation to motherhood depends on the structural plasticity displayed by this region. Even though most of these events, i.e. changes in the size of neuronal cell bodies or in the number of dendritic branches, appear to be temporary, the remodelling of MPO circuits is in part permanent since after the first pregnancy, female rats are more prone to act maternally in the future [START_REF] Bridges | Long-term alterations in neural and endocrine processes induced by motherhood in mammals[END_REF][START_REF] Brunton | The expectant brain: adapting for motherhood[END_REF][START_REF] Champagne | Plasticity of the Maternal Brain Across the Lifespan[END_REF]. One example is an increase in GFAP-immunoreactive astrocytes found in the MPO after the first pregnancy, which is dependent on the direct interaction with the offspring [START_REF] Keyser-Marcus | Alterations of medial preoptic area neurons following pregnancy and pregnancy-like steroidal treatment in the rat[END_REF].

Chapter 3

Aims

The hypothalamus is the centre of control of all vegetative functions in the vertebrate brain.

According to signals informing on the physiological state of the organism, as well as information from the environment, this small region of the ventral forebrain adapts its response to coordinate the action of different organs via the intermediary of the pituitary gland. Changes in synaptic inputs, as well as neuron-glia interactions have been identified as important plasticity mechanisms used by the hypothalamus to exert its roles. Moreover, accumulating data suggest that the adult hypothalamus hosts a niche of neural stem cells that give birth to new neurons and glial cells involved in the control of energy metabolism. This suggests that the hypothalamus retains an even higher level of plasticity than once thought and opens many questions on whether the post-natal generation of new cells contributes to the regulation of hypothalamic functions others than the control of metabolism.

During my Ph.D., I have explored the possible contribution of hypothalamic cell neogenesis to the control of reproduction, a function governed by the hypothalamus and known to require a high degree of plasticity. I have addressed this question in two contexts: the postnatal maturation of the GnRH system and the adaptation of the maternal brain to the occurrence of a pregnancy. Moreover, while the existence of a neural stem cell niche in the adult hypothalamus has been postulated from studies performed in different mammalian species, mostly rodents, it remained to be determined whether a niche also exists in the human hypothalamus.

The specific aims of my work were:

• to explore the interactions between GnRH neurons and cells born in their environment during the post-natal period, and the possible contribution of this dialogue to the maturation of the GnRH system.

• to map cell proliferation in hypothalamic regions involved in the control of reproduction during adulthood to explore whether such a process may be relevant for this function.

• to evaluate whether the adult human hypothalamus contains cells with molecular properties of neural stem/progenitor cells and address this question in the frame of a comparative approach with rodents and a non-human primate. Dunedin, New Zealand), in which EGFP expression is driven by the rat GnRH promoter [START_REF] Fujioka | Generation of transgenic rats expressing enhanced green fluorescent protein in gonadotropin-releasing hormone neurons[END_REF]. All animals were maintained at 23-25°C on a 12-hour (h) light/12-h dark cycle (lights on at 07:00 a.m.) with food and water available ad libitum.

BRDU INJECTIONS

Postnatal animals received a single intraperitoneal (i.p.) injection of BrdU at a dose of 300 mg/kg body weight (Sigma) at different postnatal ages and were allowed to survive for 2 hr, 24 hr, 7 days or ≥ 60 days.

Adult animals received either a single i.p. injection of BrdU at a dose of 300 mg/kg body weight or 3 injections of 100 mg/kg body weight each at 9 am, 1 pm and 5 pm on one day of the estrus cycle and were sacrificed 2 hr later. Animals receiving 3 injections in diestrus 2 were mated or not on the following day and then sacrificed 7, 14 or 21 days later.

PUBERTY ONSET AND ESTROUS CYCLICITY

Animals were inspected daily for imperforation of the vaginal membrane (i.e. vaginal opening). Thereafter, oestrous cycle was monitored by daily inspection of vaginal smears under an inverted microscope: diestrus 1 and 2 were defined by the presence of a predominance of leukocytes in the vaginal lavage, proestrus was characterized by a predominance of nucleated rounded epithelial cells, and oestrous was distinctively characterized by large numbers of cornified squamous epithelial cells forming clusters.

TISSUE PREPARATION

Animals were anesthetized by i.p. injection of pentobarbital (70 mg/kg). They were perfused transcardially with a rinse of saline solution (0.9% NaCl), followed by 100 ml (postnatal animals) or 400 ml (adult animals) of 4% paraformaldehyde (PFA) in 0.1 M PB (pH 7.4). The brains were removed and immersed in the same fixative for 2-3 hours at 4°C. They were then transferred to PB containing 20% sucrose until they had sunk. After embedding in OCT embedding matrix (CellPath, Newtown, UK), brains were frozen in liquid nitrogen and stored at -80°C. Frontal coronal sections (14 µm thick) containing the preoptic region only (0.20 mm to -0.30 mm relative to bregma) or the full extent of the hypothalamus, were cut on a cryostat (CM3050S, Leica, Nussloch, Germany) and mounted on chrom-alum-gelatin-coated glass slides. Sections were collected in five series with 6 sections per slide and stored at -80°C until use. Anatomical landmarks were determined using the rat brain atlas of Swanson [START_REF] Swanson | Brain Maps: Structure of the Rat Brain[END_REF].

IMMUNOFLUORESCENCE

Antibodies

Immunofluorescent stainings were performed using the following primary antibodies and working concentrations: rat monoclonal anti-BrdU (1:100, AbD Serotec, Oxford, UK), rabbit polyclonal anti-GnRH (1:3,000), mouse monoclonal anti-PCNA (1:10,000, Santa Cruz Biotechnology, Dallas, TX), rabbit polyclonal anti-GFAP (1:1,000, DakoCytomation, Glostrup, Denmark), mouse monoclonal anti-GFAP (clone G-A-5, 1:500; Sigma, St. Louis, MO), mouse monoclonal anti-HuC/D (1:500, Molecular Probes, Eugene, OR), goat polyclonal anti-Olig2

(1:100, RnD Systems, Minneapolis, MN), mouse monoclonal anti-O4 (1:300, RnD Systems), mouse monoclonal anti-APC (clone CC-1, 1:100, Merck Millipore, Darmstadt, Germany), mouse monoclonal anti-MAP2 (1:500, Sigma), goat polyclonal anti-Sox2 (1:500, Santa Cruz Biotechnology), rabbit polyclonal anti-ki67 (1:2,000, Abcam), mouse monoclonal anti-Nestin (clone rat-401, 1:300, Merck Millipore, Darmstadt, Germany), rabbit polyclonal anti-Blbp

(1:500, Merck Millipore, Darmstadt, Germany). The secondary antibodies used were Alexa Fluor 488-, Alexa Fluor 568-, Alexa Fluor 647-, Alexa Fluor 350-conjugated anti-IgG (1:400, Molecular Probes) and fluorescein-conjugated anti-mouse IgM (1:400, Vector Laboratories, Burlingame, CA).

Immunohistochemistry

Sections were washed with 0.1 M phosphate-buffered saline (PB). For BrdU immunodetection, the sections were submitted to microwave pretreatment (4 minutes at 800 W followed by 2 cycles 5 minutes each at 400 W in 0.1 mol/L sodium citrate buffer, pH 6) before the immunolabeling procedure. Sections were then rinsed and incubated with antibodies diluted in PB containing 0.3% Triton X-100 and 10% normal goat or donkey serum (Sigma).

The primary antibodies were applied for 1 or 2 nights at 4°C and the secondary antibodies

for 1 hour at room temperature. Cell nuclei were stained with Hoechst 33258 bis-Benzimide

(1:10,000; Molecular Probes). After washing, slides were coverslipped with Mowiol mounting medium. Control sections were incubated in the absence of primary antibodies.

Immunocytochemistry

Coverslips were quickly rinsed in phosphate-buffered saline (PBS) and fixed in 4% PFA for 20 minutes before being processed for immunolabelling.

Nonspecific sites were blocked with PBS 0.1 M containing 0.1% Triton X-100 and 0.3% normal goat or donkey serum (Sigma) for 30 minutes at room temperature. Sections were then incubated with primary antibodies diluted in the same solution overnight at 4°C.

Secondary antibodies were applied for 1 hour at room temperature. Cell nuclei were stained with Hoechst 33258 bis-Benzimide (1:10,000; Molecular Probes). After washing, coverslips were mounted on slides using Mowiol mounting medium. For O4 immunodetection, the anti-O4 antibody was applied to living cells for 2 hr at 37°C before fixation.

MICROSCOPIC ANALYSIS

Postnatal animals

Immunofluorescence was examined on a fluorescent microscope (Imager.Z1, Zeiss or DMRB, Leica) with appropriate filters. Images were acquired using an Axio Imager.Z1 ApoTome microscope (AxioCam MRm camera, AxioVision 4.6 software system; Zeiss). Illustrations were prepared with the help of Photoshop CS5 software (Adobe).

All analyses were made in the preoptic region, as defined in the Swanson stereotaxic brain atlas, between plate 17 (i.e. beginning of the OVLT) and plate 20 (i.e., crossing of the anterior commissure), a region where the GnRH neurons that stimulate the LH surge are located. This region extended over 840 µm anteroposteriorally, which corresponded to 60 coronal sections. Analyses were made on one section every 70 µm (i.e. one out of every 5 sections) with a mean of 8.8 ± 0.3 sections analyzed per animal. Estimation of total cell populations in the preoptic region was obtained by multiplying the number of cells per section by the total number of sections per preoptic region, and then correcting for split nuclei. BrdU+ cells were counted at the x40 objective with the help of an optical grid; 2,654.9 ± 725.1 BrdU+ cells were counted per group (n = 3 to 4 animals per group).

Adult animals

Analysis of sections and acquisition of images were done using an Axio Imager Z2 Apo-Tome microscope equipped with a motorized stage (Zeiss, Germany) and an ORCAFlash 4.0 V2 camera (Hamamatsu, Japan) driven by the Zen imaging software (Zeiss). The number of BrdU+ cells within the ME was evaluated together with the interaction of these cells with the GnRH fibers as described above.

4.6.2.3 Analysis at the level of the DG In the DG of the hippocampus, 27 to 40 sections were counted per animal in a one-in-five series of sections (14 μm thickness) using a 20x objective, throughout the rostrocaudal extent of the granule cell layer. The number of BrdU-positive cells was calculated as described above.

STATISTICAL ANALYSIS

The data are presented as mean ± s.e.m. Statistical differences were evaluated using Student t-test for comparisons of two groups or one-way analysis of variance (ANOVA) with Tukey post-hoc test for comparisons of more than two groups. When the criterion for normality or equal variances was not met, the Mann-Whitney Rank Sum Test or the Kruskal-Wallis One Way ANOVA on Ranks Test was used to compare two or more groups, respectively. P < 0.05 was considered significant. All statistics were performed using SigmaStat software.

HYPOTHALAMIC PROGENITOR CULTURES

The preoptic region of rats aged 1 or 2 days was dissected and minced on 20 µm nylon mesh (Buisine, Clermont de l'Oise, France). The dissection excluded the subventricular zone of the lateral ventricle. The cell suspension was transferred to a culture flask (BD Biosciences, San Jose, CA) coated with Poly-D-lysine (10 µg/ml, Sigma) and laminin (10 µg/ml, Sigma) and cultured in "neurosphere medium" composed of Dulbecco's Modified Eagle's Medium (DMEM)/F-12 with L-glutamine, 15 mM HEPES supplemented with 2% (v/v) B27, 20 ng/ml EGF (human recombinant), 20 ng/ml FGF2 (human recombinant), 2 mM L-glutamine, and 100 units of penicillin and 100 µg of streptomycin/ml (all from Gibco, Carlsbad, CA) under a humid atmosphere of 5% CO2-95% air at 37°C. Cell culture medium was changed every 3 to 4 days. Cells were passaged once before performing the experiments, when 70-80% confluence was reached. Cells were used for migration or proliferation assays (see below) or were snap frozen in dry ice for gene expression analysis (see chapter 4.13). The phenotype of the cells was characterized by immunocytochemistry, as described above.

IN VITRO MIGRATION ASSAY

Boyden microchemotaxis chambers were used according to manufacturer's instructions (Neuro Probe, Gaithersburg MD, USA). In brief, the cells grown in complete neurosphere medium were harvested and the suspension (500 cells/μl in control medium: neurobasal medium (Gibco, Carlsbad, CA) with L-glutamine, 15 mM HEPES supplemented with 2% (v/v) B27, 2 mM L-glutamine, and 100 units of penicillin and 100 µg of streptomycin/ml) plated on the open-bottom wells of the upper compartment, previously coated with poly-D-lysine and laminin. The two compartments of each well were separated by a polyvinylpyrrolidone-free polycarbonate porous membrane (8 μm pore size). The lower chamber was filled with control medium containing one of the following treatment: GnRH 100 ng/ml (Genecust), prostaglandin D2 (PGD2) 1 μM (Sigma), galanin 2 μM (Tocris), glutamate 10 μM (Sigma), GABA 2 μM (Sigma), EGF 100 ng/ml (Gibco) (positive control). Four different experiments were run, with 3 to 6 wells per condition in each of them.

For the experiments with BWA868C (Cayman Chemical), this compound was added to the cell suspension in the upper compartment to block DP1 receptor, at either 10 nM or 1 μM in the absence or presence of PGD2 in the lower chamber. Three to six wells per condition were analysed in three different experiments.

After 18 hours of incubation, cells attached to the upper side of the filter were mechanically removed with a cotton swab. Cells that had migrated to the lower side were fixed with 4% PFA (30 minutes at room temperature) and stained with Hoechst (1:10,000). The stained cells were photographed at 20X magnification and counted on ImageJ.

IN VITRO PROLIFERATION ASSAY

For proliferation experiments, cells were transferred to 24-well plates on poly-Dlysine/laminin-coated glass coverslips and grown in DMEM/F-12 with L-glutamine, 15 mM HEPES supplemented with 2% (v/v) B27, 20 ng/ml EGF (human recombinant), 20 ng/ml FGF2 (human recombinant), 2 mM L-glutamine, and 100 units of penicillin and 100 µg of streptomycin/ml. Once cells were 70-80% confluent, they were starved in growth medium devoid of FGF2 (starvation medium) for 24 hours prior to the treatment with GnRH, PGD2, Galanin, glutamate or GABA (same concentration as in chapter 4.10) in starvation medium for 24 hours. FGF2 was added to starvation medium at a concentration of 100 ng/ml as a positive control. Cells were then labelled for Ki67, as described above, to mark cycling cells.

Six coverslips per condition were analyzed in 3 different experiments. Results are presented as the percentage of Ki67 + cells over the total number of Hoechst-stained nuclei; 663 ± 36.7 nuclei were counted in each coverslip.

IMMUNOBLOTTING

Three different hypothalamic progenitor cultures were plated in 12-well-plates coated with poly-D-lysine and laminin. Cells were starved for 24 hours in DMEM/F-12 with L-glutamine, 15 mM HEPES supplemented with 2 mM L-glutamine, 100 units of penicillin and 100 µg of streptomycin/ml, 5 μg/ml insulin (Sigma) and 16 μg/ml putrescine (Sigma). On the day after, new starvation medium was added to all wells and the cells were treated 1 hour later for 7 minutes at room temperature with PGD2 1 μM and/or BWA868C (10 nM or 1 μM). Cells were then snap frozen in dry ice and stored at -80°C until use.

Protein extracts were prepared in 60 μl of lysis buffer per well (pH 7.4, 25 mM Tris, 50 mM bglycerophosphate, 1.5 mM EGTA, 0.5 mM EDTA, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 mg/ml leupeptin and pepstatin, 10 mg/ml aprotinin, 100 mg/ml phenylmethyl sulfonyl fluoride and 1% Triton X-100) by trituration through a 26-G needle.

The cell lysates were cleared by centrifugation at 12,000 g for 15 min and protein content was determined using the Bradford method (BioRad, Hercules, CA). The samples were stored at -80°C until use. After thawing, 4X sample buffer (Invitrogen) and 10X reducing agent (Invitrogen) were added and the samples were boiled for 5 min before electrophoresis at 150 V for 60 min in precast 3%-8% SDS-polyacrylamide Tris-acetate gels according to the protocol supplied with the NuPAGE system (Invitrogen, Carlsbald, CA). After sizefractionation, the proteins were transferred onto Nitrocellulose membranes (0.2 mm poresize membranes; Invitrogen) in the blot module of the NuPAGE system (Invitrogen) for 90 min at room temperature. Membranes were blocked for 1 h in TBS containing 0.05% Tween 20 (TBST) and 5% non-fat milk at room temperature, incubated overnight at 4°C with primary antibodies and washed four times with TBST before being exposed to horseradish peroxidase-conjugated secondary antibodies diluted in 5% non-fat milk-TBST for 1 h at room temperature. The immunoreactions were detected with enhanced chemiluminescence (PerkinElmer, Boston, MA). The rabbit polyclonal anti-phospho-p44/42 MAPK (p-Erk1/2) (Thr202/Tyr204; 1:1,000) and the rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (1:1,000)

were purchased from Cell Signaling Technology (Beverly, MA, USA). The mouse monoclonal anti-actin (1:1,000) was purchased from Sigma. The secondary antibodies used were antimouse (1:2,000) and anti-rabbit (1:2,000) horseradish peroxidase-conjugated antibodies purchased from Sigma (Saint-Quentin Fallavier, France).

ISOLATION OF HYPOTHALAMIC GNRH NEURONS USING FLUORESCENCE-ACTIVATED CELL SORTING (FACS)

The preoptic region of Gnrh::EGFP rats was microdissected and enzymatically dissociated

QUANTITATIVE REAL TIME-PCR ANALYSES

Gene expression analyses were performed on FACS-sorted GnRH neurons and hypothalamic progenitor cultures. For the latter, RNA was extracted with phenol-chloroform and precipitated with ethanol. Briefly, 100 µl of Trizol (Life Scientific, Carlsbad CA, USA) was used to dissociate cells before adding 100 µl of chloroform (Merck, Darmstadt, Germany) and centrifuging at 12,000 g for 15 minutes at 4°C. The aqueous phase was carefully collected and added to isopropanol at 1:1 before centrifuging at 12,000 g for 10 minutes at 4°C. The aqueous phase was then discarded and the pellet washed in 70% ethanol.

After centrifugation at 12,000 g for 5 minutes at 4°C, the pellet was left to air-dry and then diluted in 10 µl DEPC water. The purity and quantity of RNA was determined by UV spectroscopy (Nanodrop 1000. Thermo Scientific, Waltham MA, USA).

Messenger-RNAs obtained from FACS-sorted GnRH neurons or hypothalamic progenitor cell cultures were reverse transcribed using SuperScript III Reverse Transcriptase (Life Technologies). In the case of sorted cells, a linear preamplification step was performed using the TaqMan PreAmp Master Mix Kit protocol (Applied Biosystems). Real time-qPCR was carried out on Applied Biosystems 7900HT Fast Real-Time PCR System using exon-boundaryspecific TaqMan Gene Expression Assays (Applied Biosystems): Gnrh1 (Rn00562754_m1); level of the bregma, according to a rat brain atlas (Paxinos and Franklin, 1987). A stainless steel 5 mm cannula filled with BWA868C mixed with melted cocoa butter (10 nM) or cocoa butter alone was stereotaxically implanted at the level of the bregma (as described in Ojeda and Ramirez, 1969). After implantation, the cannulas were fixed to the skull with superglue, also used to suture the skin wound. Blood was collected from animals at P12 for plasma FSH measurements (see chapter 4.18). Four controls and 5 BWA-injected animals were sacrificed 7 days after the surgery and analysed for morphological association between GnRH neurons and BrdU + cells (see chapter 4.6.1.1). Other animals were followed for puberty onset and oestrous cyclicity (described in chapter 4.3), and blood was collected on the day prior to vaginal opening as well as on the day of proestrus.

HORMONE LEVELS MEASUREMENT

Plasma LH was measured using a high sensitivity Enzyme-Linked Immunosorbent Assay (ELISA) as described elsewhere [START_REF] Steyn | Development of a Methodology for and Assessment of Pulsatile Luteinizing Hormone Secretion in Juvenile and Adult Male Mice[END_REF]. Serum FSH levels were measured using radioimmunoassay as previously described [START_REF] Martini | Comparative analysis of the effects of ghrelin and unacylated ghrelin on luteinizing hormone secretion in male rats[END_REF]. The accuracy of hormone measurements was confirmed by the assessment of rodent serum samples of known concentration (external controls).

RESULTS

INTRODUCTION

The most important hypothalamic function for a species is that of controlling the reproductive physiology and behaviour. The master regulators of reproduction are the GnRH neurons, a small population of neurons found at the level of the POA. Following embryonic migration from the olfactory placode, where these neurons are born, they settle down in the hypothalamus where they are found at birth. However, they require postnatal maturation to determine the timely onset of puberty and subsequent oestrous cyclicity. The postnatal development of GnRH neurons in female rats can be divided into 4 stages, a neonatal period, from birth to P7; an infantile period, from P8 to weaning, during which a peak of FSH occurs at P12, corresponding to the first centrally-driven activation of the HPG axis; a juvenile period, from P21 to P30; and a peripubertal period. During this postnatal period, GnRH release gradually increases and culminates in the first LH surge, causing the first ovulation, which in rats corresponds to the day of vaginal opening [START_REF] Prevot | Chapter 30 -Puberty in Mice and Rats[END_REF]. Meanwhile, GnRH neurons receive neuronal inputs from their close environment as well as from other hypothalamic nuclei, such as those controlling the metabolic state or the circadian clock, which contribute to the fine tuning of the maturation of the GnRH system. In addition to neurons, astrocytes play an important role in regulating GnRH neuron activity, as well as ensuring correct onset of puberty and establishment of oestrous cyclicity.

Considering that the postnatal period is the moment when most glial cells are born in the brain, a project has been initiated in the laboratory with the aim to explore whether the birth of new cells in the environment of GnRH neurons was involved in their postnatal maturation. Previous results obtained by a former Ph.D. student (Cécile Allet), which I will briefly summarise, showed that the birth of new astrocytes morphologically associated with GnRH neurons during the infantile period was important for sexual maturation of female rats.

Starting from here, the question I addressed concerned the mechanisms involved in the establishment of the crosstalk between maturating GnRH neurons and newborn astrocytes.

Our findings identify a signalling pathway involving PGD2 and its receptor DP1, responsible for the morphological interactions between GnRH neurons and newborn cells during the infantile period in female rats and suggest a role for this interaction in the correct onset of sexual maturation.

PREVIOUS RESULTS

Proliferation was analysed in the POA of female rats at different postnatal ages through i.p.

injections of BrdU (Fig. 1a,b). The proliferative activity was highest in the infantile period (P8, P12) compared to the juvenile period (P20, P24) (Fig. 1c). The BrdU-positive cells were multipotent progenitors, as attested by their ability to differentiate into the three neural lineages in vivo (Fig. 1d-g). However, most newborn cells differentiated into astrocytes at all ages studied (Fig. 1d,g). In vitro experiments confirmed the presence of neural progenitors in the postnatal POA since neurospheres that proliferated, self-renewed and differentiated in all neural lineages could be obtained from microdissected postnatal POA (not shown). When co-immunolabelling for BrdU and GnRH was performed at different postnatal ages, close morphological interactions were seen between some GnRH neuronal cell bodies and newborn cells (Fig. 2a). The association was most prominent during the infantile period (Fig. 2b) and was preferential towards GnRH neurons compared to the whole neuronal population of the POA (not shown). When animals were injected with BrdU at P8 and sacrificed 7 days later, the association between GnRH neurons and newborn cells was found to increase (Fig. 2b), while the total number of GnRH neurons remained unchanged (not shown), suggesting that GnRH neurons were able to recruit newborn cells in their vicinity.

Moreover, this partnership was still observed in adult animals (P8+60d,Fig 2b). This suggests that during a specific window of the infantile period, newborn cells preferentially form close morphological associations with GnRH neuronal cell bodies, and that GnRH neurons retain their newborn companion cells into adulthood (Fig 2c). To study the fate of the population of cells born on P8 and found in close association with GnRH neurons, triple immunolabelling were performed for GnRH, BrdU and markers of the astroglial (GFAP) or neuronal (HuC/D) lineages (Fig. 3). Seven days after the BrdU injection, 69.3 ± 7.1% of these cells expressed GFAP (Fig. 3a,d), a proportion that was similar to that observed in the whole preoptic region (compare with Fig. 1g; Student t-test, p = 0.681). Only rare neurons were observed (Fig. 3c). I completed this characterisation by evaluating the oligodendroglial differentiation, and showed that 26.7 ± 14.5% of BrdU + cells expressed the oligodendroglial marker APC (Fig. 3b). Moreover, I analysed the phenotype of BrdU+ cells associated with GnRH neurons at the moment of their birth, i.e. 2 hours after the injection of BrdU, by performing immunolabelings for Sox2, a marker of immature undifferentiated cells, or GFAP. Notably, most cells born on P8 in the vicinity of GnRH neurons did not express GFAP at the moment of their birth (7.14 ± 7.14%; 2 hr vs. 7d, P < 0.001) (Fig. 3d), while most of them expressed Sox2 (66.33 ± 5.71%) (Fig. 3d), suggesting they were progenitors that subsequently differentiated into astrocytes.

RESULTS

The previous results suggest that astrogenesis in the environment of GnRH neurons during the infantile period is important for the onset of puberty. GnRH neurons appear to establish preferential and long-lasting partnerships with glial progenitors. Moreover, the increase in the proportion of GnRH neurons associated with newborn cells seen between P8 and P15 (Fig. 2b) suggests that GnRH neurons are able to attract local progenitors in their vicinity.

However, the molecular determinants involved in these interactions remained to be explored.

Identification of candidate factors

In order to identify factors produced by GnRH neurons and able to affect their neighbouring glial progenitors, we used a candidate approach. In addition to GnRH itself, we selected from the literature a series of factors reported to be expressed in GnRH neurons: NT, CCK, galanin, glutamate and GABA [START_REF] Herbison | Chapter 11 -Physiology of the Adult Gonadotropin-Releasing Hormone Neuronal Network[END_REF][START_REF] Ciofi | Phenotypical segregation among female rat hypothalamic gonadotropin-releasing hormone neurons as revealed by the sexually dimorphic coexpression of cholecystokinin and neurotensin[END_REF]. However, since these factors were identified in different contexts (mouse or rat, embryonic or adult life), we first evaluated their expression in GnRH neurons of infantile rats. Double immunostainings showed that GnRH neuron cell bodies express galanin but lack CCK and NT during the infantile period (Fig. 5). To determine whether GnRH neurons produce glutamate or GABA, we evaluated the expression of v-Glut and v-GAT, the vesicular transporters of glutamate and GABA respectively, found in nerve terminals. We observed the co-localisation of GnRH with v-Glut or v-GAT at the level of their terminals in the OVLT and most prominently in the ME (Fig. 6). Moreover, in collaboration with the laboratory of Dr. Francois Pralong in Switzerland, a transcriptomic analysis was performed on GnV3 cells, an in vitro model of mature rat GnRH neurons [START_REF] Mansuy | Phenotypic and molecular characterization of proliferating and differentiated GnRHexpressing GnV-3 cells[END_REF], exposed or not to hypothalamic astrocyte-conditioned medium. A series of genes were found to be over-or underexpressed in GnV3 cells treated with astrocyte factors compared to untreated GnV3 cells. The most upregulated gene was Ptgds, the gene coding for the brain-type prostaglandin D2 (PGD2) synthase, which showed a 40.6-fold increase in the presence of astrocyte factors, suggesting that GnRH neurons increase their capacity of PGD2 synthesis in a context of gliogenesis. We therefore added PGD2 to the panel of candidate factors.

Development and characterisation of hypothalamic progenitor cultures

In order to evaluate whether the selected factors are able to influence the motility and/or the proliferation of progenitor cells, we developed primary cultures of hypothalamic progenitor cells from microdissections of the POA of P1-P2 rats (Fig. 7a).

The cultures obtained were characterised for the expression of different markers expressed by immature cells (Nestin, Sox2, Blbp), glial (Olig2, GFAP) or neuronal lineages (HuC/D, Tuj), as well as the proliferation marker Ki67 (Fig. 7b-e). Almost all the cells expressed Blbp (98.53 ± 0.32%), Nestin (95.47 ± 0.79%) and Sox2 (88.83 ± 1.75%), as expected for immature progenitors. A smaller percentage of cells expressed Olig2 (42.44 ± 2.88%) and GFAP (25.77 ± 2.86%). While very few cells expressed HuC/D (7.25 ± 3.1%), none were positive for Tuj. Ki67

was expressed by 7.74 ± 2.06% of cells (Fig. 7e). This suggested that our primary cultures have an antigenic profile of neural progenitors and proliferate at a low level.

Figure 7. Characterization of the primary cultures of rat POA progenitors. The preoptic region of P1-P2 rats was dissected (a), mechanically dissociated and grown in DMEM/F12 + EGF + FGF2. Adherent cells were labelled for Nestin (b, c, d green), Sox2 (b, red), Blbp (c, red), Olig2 (d, red), GFAP, HuC/D, Tuj and Ki67 (not shown). The percentage of cells expressing each marker was calculated over the total number of Hoechst-positive cells (e). Nuclei were counterstained with Hoechst (blue). Mean ± s.e.m.

PGD2 attracts POA progenitors in vitro via DP1 signalling

To explore whether GnRH neurons produce factors able to recruit POA progenitors, we used the transwell assay (Fig. 8a) to evaluate the ability of GnRH, PGD2, galanin, glutamate and GABA to attract hypothalamic progenitors. EGF was used as a positive control, as it has been previously shown to stimulate the migration of neural stem and progenitor cells [START_REF] Sun | Asymmetric distribution of EGFR receptor during mitosis generates diverse CNS progenitor cells[END_REF][START_REF] Zhang | BDNF promotes EGF-induced proliferation and migration of human fetal neural stem/progenitor cells via the PI3K/Akt pathway[END_REF]. Out of all the factors tested, only PGD2 showed the ability to attract progenitor cells (Fig. 8b, n = 4 experiments).

In order to show that PGD2 was acting through a direct action on progenitor cells, we used RT-qPCR to evaluate the expression of the 2 receptors known to bind PGD2, DP1 and DP2, in hypothalamic progenitor cultures. Both receptors were found to be expressed by these cells in vitro (Fig. 8c). Since DP1 has been reported to be the most abundant PGD2 receptor in the brain (Mizoguchi et al., 2001), we subsequently focused on this receptor. In order to evaluate whether the migratory effect of PGD2 involves DP1, we used BWA868C, a specific antagonist of the DP1 receptor extensively used in the literature [START_REF] Kumar | Role of Lipocalin-type prostaglandin D<inf>2</inf> synthase (L-PGDS) and its metabolite, prostaglandin D<inf>2</inf>, in preterm birth[END_REF][START_REF] Ohinata | Central prostaglandin D2 stimulates food intake via the neuropeptide Y system in mice[END_REF][START_REF] Schratl | The Role of the Prostaglandin D2 Receptor, DP, in Eosinophil Trafficking[END_REF]. To test the efficacy of BWA868C in blunting PGD2 signalling, hypothalamic progenitor cultures were treated with PGD2 alone or in the presence of BWA868C and activation of the mitogen activated protein kinase (MAPK) pathway was analysed by western blot analysis (Fig. 8d). Treatment with BWA868C inhibited the PGD2induced phosphorylation of ERK (Fig. 8d).

BWA868C was then tested in a transwell assay for its ability to block PGD2-mediated attraction. While the presence of BWA868C alone did not affect the motility of progenitor cells, it totally blocked the stimulatory effect of PGD2 on cell migration (Fig. 8e), suggesting that PGD2 attracts progenitor cells in vitro via the activation of the DP1 receptor.

The candidate factors do not affect hypothalamic progenitor proliferation in vitro

To explore whether factors produced by GnRH neurons create a micro-environment that could also regulate the proliferation of progenitor cells, the same factors were tested in vitro for their ability to affect proliferation of POA progenitor cultures. In this experiment, we used FGF2 as a positive control since it has been shown to be a more powerful stimulator of proliferation of hypothalamic progenitor cells than EGF [START_REF] Xu | Neurogenesis in the ependymal layer of the adult rat 3rd ventricle[END_REF].

Cultures were starved for 24 hours, treated for 24 hours with the different factors and proliferative cells were identified by their expression of the cell cycle marker Ki67 (Fig 9a).

The percentage of cells expressing Ki67 was not affected by any of the treatments (Fig. 9b), suggesting that none of the candidate factors regulates the proliferation of POA progenitors.

In vivo expression of PGDS and DP1

To address whether the PGD2/DP1 signalling pathway also regulates the migration of POA progenitors in vivo, the expression of PGDS and DP1 in the postnatal POA was first analysed using two approaches.

a. In situ hybridization

The RNAscope technique was used to perform FISH on the POA of female rats at P4, P8 and P12. Triple in situ hybridisation was performed with three different probes directed against GnRH, the progenitor marker Sox2 and either PGDS (Fig. 10a) or DP1 (Fig. 10b).

The distribution of each staining was coherent with what is expected from the literature (Allen brain atlas, http://mouse.brain-map.org/experiment/show/77280331; Herbison, 2015; Urade et al.,1987). The signals obtained with the GnRH and the Sox2 probes were comparable to immunohistochemistry results; Sox2 was, for example, enriched at the level of the SVZ. PGDS and DP1 were, as expected, more abundant at the level of the meninges.

PGDS was also strongly expressed at the level of the choroid plexus. Together with the observation that no signal was observed when a negative control probe was used, these data support the specificity of the hybridisation probes.

In the POA, the PGDS signal was enriched at the level of the OVLT and could be observed in GnRH neurons as well as non-GnRH cells (Fig. 10a). The signal for DP1 was detected in Sox2expressing cells, as well as in GnRH neurons and other cells in the parenchyma (Fig. 10b).

b. Cell sorting

In a second approach, GnRH neurons were isolated from the POA of GnRH::EGFP female rats at P8, P12 and P20 (Fig. 11a). EGFP-positive cells were compared to negative ones, showing at each age a strong enrichment in the expression of GnRH (Fig. 11b-d). The levels of GnRH mRNA in the EGFP-positive cells were highest at P8 and P12, and then decreased at P20 (Fig. 11e). The expression of PGDS was then compared among the different ages both within the sorted GnRH neurons (Fig. 11f) and within the negative cells (Fig. 11g) of the POA. An increase in the expression of PGDS in GnRH neurons was observed between P8 and P20 (Fig. 11f), while the levels of PGDS mRNA in the negative cells remained unchanged during the same period (Fig. 11g).

Impairment of DP1 signalling in the POA during the infantile period perturbs the onset of oestrous cyclicity

Finally, whether activation of the DP1 signalling during the infantile period was functionally important for sexual maturation was evaluated.

BWA868C was delivered to the POA of P8 female rats who had just received a single injection of BrdU. A modified protocol was used to allow longer term delivery of the compound (as described in chapter 4.15.2). Animals were monitored for FSH levels at P12, when a peak is known to occur, puberty onset and oestrous cyclicity (Fig. 13a).

Animals that had received BWA868C showed a decrease in the levels of FSH compared to control animals (Fig. 13b).

In a group of animals sacrificed 7 days after the injection of BrdU (i.e. P15), we evaluated the association between GnRH neurons and BrdU + cells and observed a decreased proportion of GnRH neurons associated with BrdU + cells in BWA868C-treated animals compared to controls (Fig. 13c), while the total number of BrdU + cells in the POA was not affected (Fig. 13d).

When animals were analysed for puberty onset, no difference was seen in the day of vaginal opening or first estrus (Fig. 13e), as well as in the weight on the day of vaginal opening (Fig. 13f).

Monitoring of the oestrus cycle showed that BWA868C-treated animals spent more time in estrus at the expense of the time spent in proestrus during the first 15 days after the onset of puberty compared to control animals (Fig. 13g). After this time window, the cycle became regular. Animals were sacrificed on the day of proestrus (between P62-65) and LH levels were measured (Fig. 13a), confirming no differences between the two groups (Fig. 13h).

Altogether, these results suggest that integrity of the DP1 signalling during the infantile period is important for the recruitment of glial progenitors in the environment of GnRH neurons and for the correct initiation of estrous cyclicity. Chapter 6

Cell neogenesis in the hypothalamus of the adult female rat

INTRODUCTION

A growing number of studies conducted in various mammalian species have documented both neurogenesis and gliogenesis in the postnatal hypothalamus and have linked them to the regulation of energy homeostasis (Lee et al., 2012;Li et al., 2012;Kokoeva et al., 2005).

However, this is only one of the many critical physiological functions regulated by the hypothalamus.

The orchestration of the reproductive axis is another key hypothalamic function, which requires a high degree of structural plasticity between GnRH neurons, the master regulators of reproduction, and their neuronal and non-neuronal partners (Prevot et al., 2010;Ojeda et al., 2010).

Reproduction depends upon female ovulation, which occurs in a cyclic manner. Rats (as well as mice) present a very short cycle that can be divided into 4 stages: 2 days of diestrus, one day of proestrus and a day of estrus. During these stages, ovarian follicles gradually mature and secrete increasing levels of gonadal hormones, which feedback onto the hypothalamus.

Between proestrus and estrus, ovulation is induced by a surge of GnRH and LH, which depends on the positive feedback exerted by high levels of ovarian oestrogens.

Neurogenesis and gliogenesis have been previously described in hypothalamic regions controlling reproduction, such as the OVLT, which is found in the POA, where most GnRH neuron cell bodies are found in rodents, or the ME, where GnRH nerve terminals release the GnRH neurohormone into the pituitary portal blood vessels for delivery to the anterior pituitary (Lee et al., 2012;Bennett et al., 2009). These observations raised the possibility that cell neogenesis might participate in mechanisms of plasticity involved in the neuroendocrine regulation of reproduction.

The second aim of my Ph.D. was to map cell proliferation in the adult hypothalamus of female rats at the level of the two main regions involved in the control of GnRH neuron activity, the POA and the ME, throughout the different stages of the estrous cycle, and to explore whether the reproductive status influences cell proliferation.

6.2 RESULTS

Cell proliferation throughout the estrous cycle

In order to evaluate whether cell proliferation is physiologically regulated according to the reproductive status, cell proliferation was studied across the estrous cycle in adult female rats in different regions. Animals (n = 5 per group) received a single injection of BrdU (300 mg/kg body weight) at each stage of the cycle (i.e. diestrus 1, diestrus 2, proestrus and estrus) and were sacrificed 2 hours later to analyse cell proliferation (Fig. 1a).

6.2.1.1 Cell proliferation at the level of the POA Cell proliferation occurs in the POA (Fig. 1b) of adult female rats. It reaches its peak on the day of diestrus 2, and its lowest levels on the day of estrus (Fig. 1c).

Due to the observation that newborn cells are found in association with GnRH neurons during the infantile period in the POA, this relationship was also evaluated during adulthood.

The percentage of BrdU + cells associated with GnRH neurons was evaluated at each stage of the cycle; however, the values were extremely low and did not show variation throughout the cycle (Fig. 1d).

Cell proliferation at the level of the ME

The GnRH neurons project their terminals to the ME, a region where adult neurogenesis has been previously described in relation to energy homeostasis (Lee et al., 2012).

Analysis of cell proliferation in the ME (Fig. 2a) across the estrous cycle revealed a higher level of proliferation during the day of diestrus 2 when compared to estrus (Fig. 2b).

The association of newborn cells with GnRH neuron terminals in the ME was then evaluated (Fig. 2c). The percentage of BrdU + cells found associated with GnRH fibers was higher in the 17 20 18

two days of diestrus (27.26 ± 2.49% in diestrus 1 and 29.96 ± 2.76% in diestrus 2) compared to estrus (16.87 ± 1.96%) (Fig. 2d). 6.2.1.3 Cell proliferation at the level of the DG As previously said, the two regions where adult neurogenesis has been widely accepted are the SVZ of the lateral ventricles and the DG of the hippocampus. A previous study reported variations in cell proliferation in the DG of adult female rats across the estrous cycle [START_REF] Tanapat | Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat[END_REF], therefore this region (Fig. 3a) was also included to the analysis.

As in the ME, proliferation was higher on the day of diestrus 2 compared to the day of estrus (Fig. 3b). These results are discordant with those of [START_REF] Tanapat | Estrogen stimulates a transient increase in the number of new neurons in the dentate gyrus of the adult female rat[END_REF] who found a peak of proliferation on the day of proestrus. However, this difference could be explained by differences in the quantification method used. In particular, we restricted our quantification to the subgranular zone and quantified the full rostro-caudal extent of the dentate gyrus while Tanapat and colleagues quantified proliferation in the subgranular, granular and hilus regions and they did not specify the extent of the region quantified.

Pregnancy increases survival of cells born before mating

To analyse the evolution over time of the population of cells born in diestrus 2, animals were injected with BrdU and sacrificed 2 hours, 7, 14 and 21 days later (Fig. 4a). In order to label the entire population of cells born in diestrus 2, the animals received 3 injections of BrdU across the day (100 mg/kg body weight each, at 9 am, 1 pm and 5 pm).

The POA is a complex region composed by different nuclei where different neuronal populations can be found. For instance, postnatal neurogenesis and gliogenesis have been described in hypothalamic regions controlling GnRH function, such as the organum vasculosum of the lamina terminalis (OVLT), where GnRH neurons' cell bodies are found, together with nNOS neurons, known to regulate their activity; as well as at the level of the anteroventral periventricular nucleus (AVPV), which contains kisspeptin neurons responsible for mediating the oestrogen positive feedback onto GnRH neurons (Bennett et al., 2009;[START_REF] Ahmed | Pubertal hormones modulate the addition of new cells to sexually dimorphic brain regions[END_REF].

Therefore, cell proliferation was evaluated in the different nuclei of the POA, i.e. the OVLT, the median preoptic nucleus (MEPO), the medial preoptic nucleus (MPO), the AVPV and the anteroventral nucleus (AVP), using the Swanson atlas for anatomical landmarks (Fig. 4b).

At the level of the MPO, the number of newborn cells increased 7 days after the BrdU injections, to drastically decrease during the following two weeks, suggesting that after a period of proliferation, only a fraction of newborn cells survives in the region (Fig. 4c). In the other POA nuclei, the population of BrdU + cells did not show significant variations over time.

Noteworthy, a decrease in the number of BrdU + cells can result from a dilution of the BrdU content over successive divisions rather than cell death. However, the BrdU staining in cell nuclei was as intense and homogeneous in animals analysed at 14 and 21 days compared to 7 days. Moreover, mitotic figures were not observed in animals analysed at 14 and 21 days, suggesting that the disappearance of BrdU + cells in these animals is a consequence of cell death. Among its functions, the MPO is the region involved in the control of maternal behaviour, a process that is established during pregnancy and is indispensable for survival of the offspring. The possibility that cell survival may be influenced by the occurrence of a pregnancy was then addressed.

Animals received 3 injections of BrdU on the day of diestrus 2 and were put in the presence of a male rat on the next day (proestrus). Animals were separated on the day after to make sure that mating would occur directly following the BrdU injections. Animals were then sacrificed 14 or 21 days later and the number of BrdU + cells within the different nuclei of the POA were compared to non-pregnant animals at the same time points (Fig. 5a).

No differences were observed at the level of the OVLT, MEPO, AVPV and AVP. However, a higher number of cells born on the day of diestrus 2 survived in the MPO when pregnancy occurred, as observed both 14 (Fig. 5b) and 21 days (Fig. 5c) after the BrdU injections. The observation that pregnancy enhances survival of newborn cells selectively in the MPO raises the possibility that these cells may be involved in the plasticity processes necessary for the establishment of maternal behaviour, which are known to develop during pregnancy until birth of the offspring.

One of the neuronal populations found in the MPO and that have been linked to one of the components of maternal behaviour, i.e. maternal aggression, are the nNOS neurons (Gammie & Nelson, 1999), which are also involved in the regulation of GnRH neuron activity (see chapter 2.3.3). For this reason, the association between BrdU + cells and nNOS neurons was analysed 14 days after BrdU injection. Due to the low number of BrdU + cells and the high number of nNOS neurons in the POA, only 0.92% ± 0.13 (n = 3 animals) of nNOS neurons were found associated with BrdU + cells in non-pregnant animals. This proportion was not affected by pregnancy (1.12% ± 0.14 nNOS neurons associated with BrdU + cells in pregnant animals (n = 3); P = 0.35 vs non-pregnant rats). The percentage of newborn cells associated with nNOS neurons in the MPO was also evaluated. In non-pregnant animals, 12.19% ± 2.57 (n = 3 animals) of BrdU + cells were associated with nNOS neurons. This proportion was not affected by pregnancy (11.23% ± 2.72 BrdU + cells associated with nNOS neurons in pregnant animals (n = 3); P = 0.81 vs non-pregnant rats).

These results suggest that if newborn cells regulate the activity of MPO neuronal circuits, they do so by interacting with neurons other than the nNOS neurons.

INTRODUCTION

Neural stem cells persist in the brain of vertebrates beyond development [START_REF] Doetsch | The glial identity of neural stem cells[END_REF]. In adult mammals, the two best-described neural stem cell niches are the subventricular zone (SVZ) that lines the wall of the lateral ventricles, and the subgranular zone (SGZ) at the base of the hippocampal dentate gyrus (Alvarez-Buylla & Lim, 2004;Ming & Song, 2005). These niches harbor neural stem cells that self-renew throughout life and generate new neurons and glial cells that are functionally integrated into selected circuits and participate in specific brain functions such as learning, memory and odor discrimination (Gonçalves, Schafer, & Gage, 2016;Lledo & Valley, 2016). Almost two decades ago, a study performed in the golden hamster reported for the first time the generation of new neurons in the adult hypothalamus [START_REF] Huang | Photoperiod regulates neuronal bromodeoxyuridine labeling in the brain of a seasonally breeding mammal[END_REF], a small diencephalic region surrounding the third ventricle and involved in the control of vital processes such as energy metabolism, reproduction, biological rhythms and other homeostatic processes. Since then, a number of studies have accumulated suggesting that neurons and glial cells are generated from local neural stem/progenitor cells (NPCs) in the hypothalamus throughout life (Batailler et al., 2014;Chaker et al., 2016;Gouazé et al., 2013;Haan et al., 2013;[START_REF] Jourdon | Prss56, a novel marker of adult neurogenesis in the mouse brain[END_REF]Kokoeva, Yin, & Flier, 2005[START_REF] Taupin | BrdU immunohistochemistry for studying adult neurogenesis: Paradigms, pitfalls, limitations, and validation[END_REF]Lee et al., 2012;Li, Tang, & Cai, 2012;McNay, Briançon, Kokoeva, Maratos-Flier, & Flier, 2012;Pencea, Bingaman, Wiegand, & Luskin, 2001;Pérez-Martín et al., 2010;Pierce & Xu, 2010;S. C. Robins et al., 2013;[START_REF] Robins | Evidence for NG2-glia derived, adult-born functional neurons in the hypothalamus[END_REF].

Hypothalamic NPCs are regulated by various physiological factors and pathophysiological conditions [START_REF] Rojczyk-Gołębiewska | Hypothalamic subependymal niche: a novel site of the adult neurogenesis[END_REF]Sharif, Ojeda, & Prevot, 2014), and hypothalamic neurogenesis, while it occurs at a lower rate than in the two classic niches (Migaud et al., 2010), has functional significance for the control of energy metabolism (Djogo et al., 2016;Gouazé et al., 2013;Kokoeva et al., 2005;Lee et al., 2012Lee et al., , 2014;;Li et al., 2012;[START_REF] Li | Control of obesity and glucose intolerance via building neural stem cells in the hypothalamus[END_REF]Pierce & Xu, 2010). Several different, and nonexclusive, hypotheses have been proposed concerning the identity and location of hypothalamic NPCs: some studies suggest that they reside within the hypothalamic parenchyma [START_REF] Kokoeva | Evidence for constitutive neural cell proliferation in the adult murine hypothalamus[END_REF]Li et al., 2012;[START_REF] Robins | Evidence for NG2-glia derived, adult-born functional neurons in the hypothalamus[END_REF], some that they are subependymal astrocytes located in a region of the mid third ventricle (Pérez-Martín et al., 2010), and others that they are a subpopulation of tanycytes, ependymoglial cells that line the ventral portion of the third ventricle (Chaker et al., 2016;Haan et al., 2013;[START_REF] Jourdon | Prss56, a novel marker of adult neurogenesis in the mouse brain[END_REF]Lee et al., 2012;S. C. Robins et al., 2013).

Most of our knowledge of adult neural stem and progenitor cells comes from rodent models, and few studies have been conducted in humans. Neuroanatomical studies and the recently developed technique of 14 C dating have confirmed that cells exhibiting neural stem cell properties and neurogenesis persist in the adult human brain (Bergmann, Spalding, & Frisén, 2015). While the hippocampal neurogenic system in adult humans exhibits features similar to that of rodents, i.e. the local generation of dentate granule neurons from nearby neural stem cells (Eriksson et al., 1998;Ernst et al., 2014;Knoth et al., 2010;Spalding et al., 2013), the SVZ neurogenic system appears to differ significantly between human and rodents. Not only does the cellular organization of the niche differ [START_REF] Quiñones-Hinojosa | Cellular composition and cytoarchitecture of the adult human subventricular zone: a niche of neural stem cells[END_REF]Sanai et al., 2004), but newborn neurons adopt a different fate, migrating into the adjacent striatum to become medium spiny neurons (Ernst et al., 2014) instead of migrating long distances along the rostral migratory stream to become part of the olfactory bulb circuit (Bergmann et al., 2012) as in rodents (Ming & Song, 2005).

A recent neuroanatomical study has revealed the presence of cells expressing the neuroblast marker doublecortin (DCX) in the adult human hypothalamus (Batailler et al., 2014), raising the possibility that neurogenesis, and a neural stem cell niche, may exist in this region in humans. While the expression of the NPC markers nestin and vimentin has been reported in the adult human hypothalamus (Baroncini et al., 2007;[START_REF] Dahiya | Comparative characterization of the human and mouse third ventricle germinal zones[END_REF][START_REF] Nogueira | Existence of a potential neurogenic system in the adult human brain[END_REF], there has been no detailed mapping of the expression of a repertoire of NPC markers in the adult human hypothalamus as yet.

Here, we carried out a neuroanatomical characterization of the expression of a combination of NPC markers in the adult human hypothalamus, along with the SVZ and SGZ.

To evaluate potential inter-species variability, the expression of NPC markers in the adult hypothalamus was concomitantly analyzed in the mouse, rat and a non-human primate species, the grey mouse lemur (Microcebus murinus).

MATERIALS AND METHODS

Tissue Human

Tissues were made available in accordance with French bylaws (Good Practice Concerning the Conservation, Transformation and Transportation of Human Tissue to be Used Therapeutically, published on December 29, 1998). Permission to use human tissues was obtained from the French Agency for Biomedical Research (Agence de la Biomédecine, Saint-Denis la Plaine, France, protocol no. PFS16-002).

A total of nine brains were obtained from autopsies (average age 81 years, range 60-92 years; average postmortem delay 23 hours, range 8-41.5 hours). The brain samples were taken from patients that donated bodies to Science in compliance with the French laws on bioethics. Structures were identified by reference to an atlas of the human brain [START_REF] Mai | Atlas of the human brain, 3rd Edition[END_REF].

One 11-week old (menstrual age) human fetus was obtained after elective abortion from the Gynaecology Department of the University Hospital of Lille. Written informed consent had been previously obtained from the parent.

One de novo glioblastoma multiforme (GBM) sample was obtained from the Neurosurgery Clinic of the University Hospital of Lille. The GBM specimen was obtained at initial surgery of a 65-year old male. The tumor was classified and graded by a neuropathologist according to the World Health Organization system [START_REF] Louis | The 2007 WHO classification of tumours of the central nervous system[END_REF].

Written informed consent had been previously obtained from the patient and/or his family members. The tissue was processed as described previously [START_REF] Duhem-Tonnelle | Differential distribution of erbB receptors in human glioblastoma multiforme: expression of erbB3 in CD133-positive putative cancer stem cells[END_REF].

Rodents

Grey mouse lemur (Microcebus murinus)

Tissues were obtained from one male (1.9 year-old) and one female (5.9 year-old) grey mouse lemur (Microcebus murinus) bred in the laboratory breeding colony of Brunoy (MNHN, France, license approval N° A91.114.1). The two animals were sacrificed during the long day period (14-hours of light/day), which corresponds to the period of reproductive activity. Mouse lemur studies were approved by the Cuvier Ethics Committee for the Care and Use of Experimental Animals of the Muséum National d'Histoire Naturelle (n°2083-2015090311335786).

Fluorescent immunostaining Human

After whole brain removal, blocks of 20 mm per side encompassing the hypothalamus were harvested with the optic chiasm as the anterior limit and the mammillary bodies as the posterior limit. The hippocampus was collected from a cut posterior to the mammillary bodies. The dorso-lateral and ventral walls of the body of the lateral ventricles were collected on a width of about 5 mm towards the parenchyma. Tissue blocks were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 1 week at 4°C, cryoprotected in PB containing 0.9% sodium chloride (PBS) and sucrose at 10% for 48 hours followed by 20% for 3 days, embedded in Tissue Tek® (Sakura Finetek, Villeneuve d'Ascq, France) and frozen in isopentane cooled with liquid nitrogen. Coronal cryostat sections (14 or 20 m) were mounted on chrome-alum-gelatin coated slides, air-dried, and subjected to immunofluorescent stainings. After rehydration in a solution of sodium citrate 0.01 M (pH 6) (citrate buffer), the sections were subjected to microwave pretreatment in citrate buffer for 4 minutes at 800 W followed by 2 cycles 4 minutes each at 400 W for antigen retrieval. After cooling at room temperature and washing in PB, sections were incubated for 15 minutes in PBS containing 0.3% Triton X-100 and 2% normal donkey serum (PBSTS) at room temperature to block the nonspecific sites. Sections were then incubated with primary antibodies diluted in PBSTS for one to two nights at 4°C in a humid chamber. The characteristics of the primary antibodies used are provided in Table 1. After rinsing in PB, secondary antibodies diluted in PBSTS were deposited on sections and incubated at room temperature for 1 hour. The characteristics of the secondary antibodies used are provided in Table 2. After washing in PB, cell nuclei were stained with Hoechst 33258 bis-Benzimide (Molecular Probes, Eugene, OR, USA) diluted at 1/1000 in PB for 2 minutes and then washed in PB. To avoid the strong autofluorescence caused by lipofuscin granules usually present in adult human brain tissue, sections were immersed in a solution of 0.3% Sudan Black B (Sigma, St Quentin Fallavier, France) in 70% ethanol for 5 minutes [START_REF] Romijn | Double immunolabeling of neuropeptides in the human hypothalamus as analyzed by confocal laser scanning fluorescence microscopy[END_REF]. This treatment completely blocked autofluorescence. After washing in PB, sections were coverslipped in Mowiol mounting medium (20% Mowiol, 2.5% DABCO in Tris 0.2 M pH 8.5).

Control sections were incubated in the absence of primary antibody. Omission of the primary antibody resulted in no staining.

The human fetus was immersion fixed in 4% paraformaldehyde for 4 days at 4°C, cryoprotected in 30% sucrose/PBS overnight at 4°C, and then processed and subjected to immunofluorescent stainings as described above.

Rat

Animals were anesthetized by intraperitoneal injection of pentobarbital (70 mg/kg). They were perfused transcardially with a rinse of saline solution (0.9% NaCl), followed by 500 ml of 4% paraformaldehyde in 0.1 M PB (pH 7.4). The brains were removed and immersed in the same fixative for 2-3 hours at 4°C. They were then transferred to PB containing 20% sucrose until they had sunk, embedded in Tissue Tek® (Sakura Finetek, Villeneuve d'Ascq, France), and frozen in liquid nitrogen. Frozen 14-m coronal sections were prepared using a cryostat (CM3050, Leica) and collected throughout the full extent of the third ventricle. The sections were mounted onto chrome-alum-gelatin coated slides, air-dried and subjected to immunofluorescent stainings as described above. Anatomical landmarks were determined using the rat brain atlas of Swanson [START_REF] Swanson | Brain Maps: Structure of the Rat Brain[END_REF].

Mouse

Animals were anesthetized by intraperitoneal injection of xylazine 10 mg/kg (Rompun 2%, Bayer) and ketamine 200 mg/kg (Ketalar, Parke-Davis), perfused transcardially with a rinse of saline solution, followed by 100 ml of 4% paraformaldehyde in 0.1 M PB (pH 7.4). After brain removal, post-fixation and freezing, 30 m free-floating coronal sections encompassing the full extent of the third ventricle were prepared using a cryostat and collected in ice-cold PB.

Free-floating sections were subjected to immunofluorescent stainings following the abovedescribed procedure, mounted on chrome-alum-gelatin coated slides, air-dried and coverslipped in Mowiol mounting medium. Anatomical landmarks were determined using the mouse brain atlas from [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates[END_REF].

Grey mouse lemur (Microcebus murinus)

Animals were anesthetized by intraperitoneal injection of pentobarbital 5 mg/kg, perfused transcardially with a rinse of saline solution, followed by 120 ml of 4% paraformaldehyde in 0.1 M PB (pH 7.4). After brain removal, post-fixation and freezing, frozen 14-m coronal sections were prepared using a cryostat and collected throughout the full extent of the third ventricle. The sections were mounted onto chrome-alum-gelatin coated slides, air-dried and subjected to immunofluorescent stainings as described above. Anatomical landmarks were determined using the Microcebus murinus brain atlas of Bons [START_REF] Bons | A stereotaxic atlas of the grey lesser mouse lemur brain (Microcebus murinus)[END_REF].

Antibody characterization Human

We used human fetal cerebral cortex and GBM as positive control tissues to verify the specificity of primary antibodies in human tissues.

In the fetal cerebral cortex, the SRY (sex determining region Y)-box 2 (Sox2) antibody (goat polyclonal # sc-17320 from Santa Cruz) yielded a nuclear staining with decreasing density from the ventricular to the pial surface (Fig. 1a), as reported by others [START_REF] Vinci | Immunohistochemical markers of neural progenitor cells in the early embryonic human cerebral cortex[END_REF]. This antibody detects a single band of 34 kDa on Western blots of human neural progenitor and GBM stem-like cell lysates [START_REF] Guichet | Cell death and neuronal differentiation of glioblastoma stem-like cells induced by neurogenic transcription factors[END_REF]. Moreover, immunoreactivity is lost when human embryonic stem cells are treated with a small interfering RNA (siRNA) directed against Sox2 [START_REF] Adachi | Role of SOX2 in maintaining pluripotency of human embryonic stem cells[END_REF]. In the adult hypothalamus, a double labeling with another Sox2 antibody (mouse monoclonal # ab79351 from Abcam, 1/300) showed an overlap of the two stainings (Fig. 1i,j).

In agreement with previous reports, the nestin antibody stained radial glial cells in the fetal cerebral cortex (Fig. 1e) [START_REF] Vinci | Immunohistochemical markers of neural progenitor cells in the early embryonic human cerebral cortex[END_REF], tumoral cells in the GBM specimen (Fig. 1h) [START_REF] Ludwig | Molecular markers in glioma[END_REF] and hypothalamic adult tanycytes (Fig. 6) (Baroncini et al., 2007).

This antibody detects a band at 177 kDa, the expected molecular weight, on Western blots of human GBM stem-like cell lysates [START_REF] Ciechomska | BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells[END_REF]. Moreover, inhibition of nestin expression using short hairpin RNA or siRNA transfection in a human lung adenocarcinoma cell line results in decreased immunolabeling [START_REF] Narita | Nestin regulates proliferation, migration, invasion and stemness of lung adenocarcinoma[END_REF].

Vimentin was detected in radial glial cells in the fetal cerebral cortex (Fig. 1c), as described elsewhere [START_REF] Hofman | Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis[END_REF][START_REF] Vinci | Immunohistochemical markers of neural progenitor cells in the early embryonic human cerebral cortex[END_REF]. It was strongly expressed in adult tanycytes (Fig. 6b) in agreement with other reports (Baroncini et al., 2007;[START_REF] Koopman | Age-Related Changes in the Morphology of Tanycytes in the Human Female Infundibular Nucleus/Median Eminence[END_REF]. The M0725 antibody from Dako detects a band at 54 kDa on Western blots of human mesenchymal stromal cell lysates [START_REF] Talele | Expression of α-Smooth Muscle Actin Determines the Fate of Mesenchymal Stromal Cells[END_REF]. Treatment of a human cholangiocarcinoma cell line with siRNA against vimentin suppressed immunoblot and immunofluorescent signals with this antibody [START_REF] Saentaweesuk | Activation of Vimentin is Critical to Promote a Metastatic Potential of Cholangiocarcinoma Cells[END_REF]. The two anti-vimentin antibodies used yielded a similar expression profile.

We had previously used the Glial Fibrillary Acidic Protein (GFAP) antibody (rabbit polyclonal Z0334 from Dako) to stain cerebro-cortical [START_REF] Duhem-Tonnelle | Differential distribution of erbB receptors in human glioblastoma multiforme: expression of erbB3 in CD133-positive putative cancer stem cells[END_REF] and hypothalamic (Baroncini et al., 2007) astrocytes in the adult human brain, and tumoral cells and reactive astrocytes in human GBM [START_REF] Duhem-Tonnelle | Differential distribution of erbB receptors in human glioblastoma multiforme: expression of erbB3 in CD133-positive putative cancer stem cells[END_REF]. This antibody stained radial glial cells in the fetal cerebral cortex (Fig. 1f, g), as described by others [START_REF] Hofman | Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis[END_REF] and it detects a single band at 50 kDa on Western blots of human GBM cell lysates [START_REF] Tardito | Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of glutamine-restricted glioblastoma[END_REF]. A co-immunolabeling with another anti-GFAP antibody (mouse monoclonal MAB3402 from Chemicon) previously used by others to stain the ribbon of SVZ astrocytes [START_REF] Quiñones-Hinojosa | Cellular composition and cytoarchitecture of the adult human subventricular zone: a niche of neural stem cells[END_REF]Sanai et al., 2004) yielded a strict co-localization (Fig. 1k,l). The expression profile observed with the two antibodies along the lateral ventricles replicates the pattern of mRNA expression determined by in situ hybridization (Allen brain atlas; http://human.brain-map.org/ish/experiment/show/159107343), with a labeling of ependymal, subependymal and small parenchymal cells.

The Glutamate-Aspartate Transporter (GLAST) antibody labeled radial glial cells in the fetal cerebral cortex (Fig. 1d) following an expression profile described elsewhere [START_REF] Hofman | Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis[END_REF]. The expression profile observed along the lateral ventricles replicates the pattern of mRNA expression determined by in situ hybridization, with a strong enrichment in ependymal and ribbon cells, as well as scattered parenchymal cells (Allen brain atlas; http://human.brain-map.org/ish/experiment/show/160133315). This antibody detects a single band at 65 kDa on Western blots of human cerebrocortical homogenates [START_REF] Roberts | Localization of excitatory amino acid transporters EAAT1 and EAAT2 in human postmortem cortex: a light and electron microscopic study[END_REF]. Based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), we determined the GLAST immunogen peptide (ab127026, Abcam) sequence to be from amino acid 522-541 (data not shown).

The Ki67 antibody has been used previously to stain proliferative cells in human GBM [START_REF] Duhem-Tonnelle | Differential distribution of erbB receptors in human glioblastoma multiforme: expression of erbB3 in CD133-positive putative cancer stem cells[END_REF]. It yielded a dense nuclear staining in the fetal cerebral cortex following a gradient with the highest density of positive cells in the ventricular and subventricular zones (Fig. 1b), as described elsewhere [START_REF] Bayatti | A molecular neuroanatomical study of the developing human neocortex from 8 to 17 postconceptional weeks revealing the early differentiation of the subplate and subventricular zone[END_REF][START_REF] Harkin | Distinct expression patterns for type II topoisomerases IIA and IIB in the early foetal human telencephalon[END_REF]. In the adult human brain, the Ki67 antibody produced a patchy staining in the nucleus of cells, some of which exhibited mitotic figures (Fig. 8). This antibody detects a band at 360 kDa on Western blots of primary human dermal fibroblast cell lysates [START_REF] Chierico | The role of the two splice variants and extranuclear pathway on Ki-67 regulation in non-cancer and cancer cells[END_REF].

The immunoblot signal is lost following siRNA-mediated knock-down of Ki67 in various human cell lines [START_REF] Sun | Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells[END_REF].

The Neuronal Nuclei (NeuN) antibody has been used previously to stain mature neurons in the adult human cerebral cortex [START_REF] Duhem-Tonnelle | Differential distribution of erbB receptors in human glioblastoma multiforme: expression of erbB3 in CD133-positive putative cancer stem cells[END_REF] and hippocampus (Knoth et al., 2010). This antibody detects a doublet at 46-50 kDa, the expected apparent molecular weight, in Western blots of human hippocampus autopsy homogenates (Engel et al., 2011).

The Vasoactive intestinal peptide (VIP) antibody has been used previously to immunostain neurons in the monkey and human enteric nervous system [START_REF] Noorian | Neurochemical phenotypes of myenteric neurons in the rhesus monkey[END_REF] and produced a staining pattern in the suprachiasmatic nucleus (Fig. 7a) similar to that described in the literature with other antibodies [START_REF] Hofman | Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis[END_REF][START_REF] Wang | Suprachiasmatic neuron numbers and rest-activity circadian rhythms in older humans[END_REF] or using in situ hybridization [START_REF] Van Wamelen | Suprachiasmatic nucleus neuropeptide expression in patients with Huntington's Disease[END_REF].

The CD68 antibody has been used previously by others to stain microglia in the human brain [START_REF] Monier | Distribution and differentiation of microglia in the human encephalon during the first two trimesters of gestation[END_REF]. As expected for a lysosomal membrane protein, it yielded a cytoplasmic punctuate staining (Fig. 8g). This antibody detects bands at 110, 70, 40 kDa in Western blots of human lung, spleen and U937 monocytic cell line extracts using reducing conditions. An additional band is detected at 220 kDa in spleen extracts using non-reducing conditions [START_REF] Pulford | KP1: a new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections[END_REF].

Microcebus murinus

The specificity of the Sox2 and vimentin antibodies was inferred from the similarity in the expression pattern with rodents. Noteworthy, we tested 2 anti-nestin antibodies, MAB1259

from R&D Systems and MAB353 clone rat-401 from Millipore, recognizing human and rodent nestin, respectively, but none of them yielded any staining in the grey mouse lemur.

Histological staining

For anatomical identification of hypothalamic regions and nuclei, coronal cryostat sections were immersed in a solution of Toluidine Blue dissolved at 0.5% in a solution of 1% sodium borate for 6 minutes at room temperature. After three rinses in water, sections were dehydrated in successive alcoholic solutions: 2 times 5 minutes in ethanol 70°, 7 minutes in ethanol 70° containing 0.2% acetic acid, 2 times 5 minutes in ethanol 100° and 2 times 3 minutes in xylene. Sections were coverslipped in Eukitt mounting medium (Sigma).

Microscopy

Analysis of sections and acquisition of images were done using an Axio Imager Z2 Apo-Tome microscope equipped with a motorized stage (Zeiss, Germany) and an ORCA- (NA = 0.75). All images were captured in a stepwise fashion over a defined z-focus range corresponding to all visible fluorescence within the section and consistent with the optimum step size for the corresponding objective and the wavelength (500 nm). To create photomontages, ApoTome images were captured using the z-stack module coupled to the MosaiX module of the Zen imaging software and a Zeiss 5x (NA = 0.15) or 20x objective for each filter sequentially. Images to be used for figures were pseudocolored, adjusted for brightness and contrast and merged using Photoshop (Adobe Systems, San Jose, CA).

Analysis Human

Co-expression of nestin and Sox2 was systematically analyzed on sections distributed across the antero-posterior extent of seven hypothalami. Then, triple stainings for nestin/Sox2/vimentin, nestin/Sox2/GLAST, nestin/Sox2/GFAP, nestin/Sox2/Ki67 and vimentin/Sox2/NeuN were performed on representative sections. Double and triple immunostainings were performed on a total of 73 ± 6 (mean ± SEM) (range, 47 -94) sections per specimen.

Quantification of stainings in the hypothalamic ependymal layer, ribbon cells and buds was performed on the seven specimens. A total of 2044 ± 226 (mean ± SEM) ependymal cells, 2117 ± 423 (mean ± SEM) ribbon cells and 836 ± 260 (mean ± SEM) bud cells were counted per specimen. Noteworthy, the border of the third ventricle was usually less well preserved at its floor, where tanycyte cell bodies are found, thus precluding the quantification of stainings in this cell type. Quantification of stainings in the suprachiasmatic nucleus was made on three hypothalami. While the rodent suprachiasmatic nucleus appears as a high cell density spherical structure clearly delimited after counterstaining for cell nuclei, it is not the case in the human. The localisation of the human suprachiasmatic nucleus was determined using an atlas [START_REF] Mai | Atlas of the human brain, 3rd Edition[END_REF] and further verified by an immunostaining for VIP, which is produced by a subpopulation of suprachiasmatic neurons [START_REF] Hofman | Suprachiasmatic nucleus of the human brain: an immunocytochemical and morphometric analysis[END_REF]. For quantitative analysis of the stainings, a total of 2931 ± 235 (mean ± SEM) cells were counted on five to seven sections per suprachiasmatic nucleus.

Expression of NPC markers in the dentate gyrus of the hippocampus was analyzed on specimens collected from three brains. Double and triple immunostainings were performed on a total of 20 ± 4 (mean ± SEM) (range, 12 -26) sections per specimen.

The SVZ was analysed on samples collected from six brains. Three samples were collected in the dorsal portion of the body of the lateral ventricle, 4 in its ventral portion and 3 in the temporal horn. Notably, three ventral SVZ samples were present in the tissue block containing the third ventricle, enabling comparison between these regions on the same sections. Double and triple immunostainings were performed on a total of 33 ± 10 (mean ± SEM) (range, 12 -94) sections per specimen.

Rat

Co-immunolabelings of nestin, Sox2 and vimentin were performed and analysed on 42-204 sections per animal regularly spaced between plate 17 (i.e. the anterior-most part of the third ventricle above the optic chiasm) and plate 38 (i.e., posterior-most part of the third ventricle and end of mammillary nuclei) of the rat brain atlas from Swanson [START_REF] Swanson | Brain Maps: Structure of the Rat Brain[END_REF].

Mouse

Triple nestin/Sox2/vimentin immunostainings were performed and analysed on 9-12 sections per animal regularly spaced between plate 34 (i.e. a region containing the optic chiasm and the anterior part of the suprachiasmatic nucleus) and plate 53 (i.e., posteriormost part of the third ventricle and mammillary nuclei) of the mouse brain atlas from [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates[END_REF].

Microcebus murinus

Co-expression of vimentin and Sox2 was analysed on the male and female specimens on a total of 78 and 60 sections, respectively, encompassing the rostro-caudal extent of the third ventricle, i.e. plate 39 to 51 of the Microcebus murinus brain atlas of Bons [START_REF] Bons | A stereotaxic atlas of the grey lesser mouse lemur brain (Microcebus murinus)[END_REF].

Statistics

Differences in the percentage numbers of NPC marker-expressing cells between the different cell populations were analyzed by One Way Analysis of Variance (ANOVA) using the SigmaStat software. The level of significance was set at p<0.05.

RESULT

The human hypothalamus

For the neuroanatomical analysis, the hypothalamus was antero-caudally subdivided into four regions: the anterior-most region surrounding the preoptic recess (Fig. 2a), the anterior hypothalamic area containing the optic chiasm, the anterior commissure, the paraventricular and supraoptic nuclei (Fig. 2b), the tuberal region containing the ventromedial, dorsomedial, infundibular nuclei and the median eminence (Fig. 2c,d), and the mammillary region containing the mammillary bodies and the posterior hypothalamic nucleus (Fig. 2e) [START_REF] Mai | Atlas of the human brain, 3rd Edition[END_REF].

In order to identify putative NPCs, we first mapped the co-expression of the NPC markers nestin and Sox2 in the whole hypothalamus (Fig. 3) and then performed triple stainings for nestin, Sox2 and vimentin, GLAST or GFAP in representative regions (Fig. 4, 5, 6, 7 and Table 3). A similar expression profile was found in the seven hypothalami analyzed.

Expression of nestin and Sox2 was primarily detected in a region bordering the third ventricle (Fig. 3). Deeper in the parenchyma, labeling for Sox2 was absent while labeling for nestin was only seen in capillary vessels (Fig. 3, asterisks). Ependymal cells, which line the ventricle, showed intense Sox2 (Fig. 3, 4), low to high vimentin (Fig. 4a,c) and intense GLAST (Fig. 4h,j) immunoreactivity in their vast majority, while expression of nestin (Fig. 4b,i,p)

and GFAP (Fig. 4o,q) was less ubiquitously detected (Table 3). Co-expression of nestin and Sox2 was detected in 32.1 ± 4.0% of ependymal cells. Among the population of nestin/Sox2 co-expressing ependymal cells, 96.8 ± 1.4% expressed vimentin, 98.9 ± 0.3% expressed GLAST and 81.1 ± 7.1% expressed GFAP, implying that at least 77% of nestin/Sox2-positive cells also expressed vimentin, GLAST and GFAP. Ependymal cells often invaginated into the underlying parenchyma to form crowns (Fig. 3f) or rosettes (Fig. 3b-e). Heterogeneity in the antigenic profile and organization of ependymal cells was not specific to a particular area of the third ventricle.

A ribbon of small process-bearing cells expressing nestin, Sox2, vimentin, GLAST and GFAP consistently lined the border of the ventricle, separated from it by a hypocellular gap (Fig. 3a 1 , a 3 , b, c, d, Fig. 4a, h, o, table 3). The ribbon was variable in thickness, from one (Fig. 3a 1 ) to multiple cell layers (Fig. 3a 3 ), as well as the gap that ranged from ~20 µm (Fig. 3a 1 ) to ~100-150 µm (Fig. 3d). Noteworthy, the hypocellular gap was consistently at its largest extent in the mammillary region (Fig. 3d). Co-expression of nestin and Sox2 was detected in 18.0 ± 3.7% of ribbon cells. Nestin/Sox2 co-expressing ribbon cells frequently sent long processes contacting the ependymal layer (Fig. 3b, inset) and/or capillaries (Fig. 3g). Triple co-immunolabelings showed that nestin/Sox2 co-expressing ribbon cells also expressed vimentin (Fig. 4e-g), GLAST (Fig. 4l-n) and GFAP (Fig. 4s-u) in 83.8 ± 4.7%, 87.7 ± 4.2% and 80.9 ± 9.4% of them, respectively, implying that at least 52% of nestin/Sox2-positive cells also express vimentin, GLAST and GFAP. The hypocellular gap contained nestin-and vimentin-expressing processes (Fig. 3 and4), presumably corresponding to ependymal and/or ribbon cell processes, and was enriched in GLAST (Fig. 4h) and GFAP (Fig. 4o)

immunoreactivity. The gap and ribbon were devoid of NeuN-immunoreactive neurons.

NeuN-immunoreactive cells were primarily found in the parenchyma at distance from the subependymal region (not shown) and occasionally in the proximal parenchyma underlying the subependymal region (Fig. 5j-l).

Notably, some portions of the third ventricular wall were devoid of the gap-and-ribbon organization but exhibited a thick border enriched in intermingled nestin/Sox2-positive cells (Fig. 3a 2 ) sometimes bulging into the ventricle (Fig. 3a 4 , g, h) and showing systematic interruption of the ependymal layer. These buds were highly variable in size and shape, from long and flat (Fig. 3a 2 , a 4 ) to ovoid and protuberant (Fig. 3g,h), with frequent strengthening in nestin staining (Fig. 3h). They contained a rich network of NPC marker-expressing cells (Table 3) but were devoid of NeuN-immunoreactive cells (Fig. 5j-l). Co-expression of nestin and Sox2 was detected in 46.8 ± 5.5% of bud cells. Among the population of nestin/Sox2 coexpressing cells, 72.3 ± 7.8% expressed vimentin (Fig. 5a-c), 98.5 ± 1.3% expressed GLAST (Fig. 5d-f) and 95.5 ± 2.1% expressed GFAP (Fig. 5g-i), implying that at least 66% of nestin/Sox2-positive cells also express vimentin, GLAST and GFAP. These structures were found anywhere along the ventricular wall of all hypothalami examined.

In the tuberal region, the floor of the third ventricle does not exhibit the gap-andribbon organization and is lined by tanycytes whose processes extend into the median eminence (Fig. 6a). Co-immunolabelings showed that Sox2, nestin, vimentin, GLAST and GFAP were expressed by tanycytes (Fig. 6). While nestin and vimentin, GLAST or GFAP were frequently co-expressed in tanycyte processes, long processes expressing each marker separately were also seen in variable proportions between the different specimens.

The rodent and grey mouse lemur hypothalamus

The organization of the third ventricular border significantly differed between human and rodents. The rodent third ventricle wall did not exhibit the hypocellular gap and ribbon of NPC marker co-expressing cells (Fig. 9a-k). Co-expression of nestin, Sox2 and vimentin was essentially restricted to the tanycytes with the exception of very occasional ependymal cells (Fig. 9a-k and Table 4). Noteworthy, significant differences were seen between the mouse and rat third ventricle wall. While the mouse ventricular border was composed of a regular layer of ependymal cells (Fig. 9a,e), the rat one was much more irregular with thickenings and evaginations of the ependymal layer frequently protruding in the dorsal part of the ventricle (Fig. 9i). The mouse (Fig. 10a-d) and rat (Fig. 10e-h) suprachiasmatic nucleus contained a population of Sox2-positive cells that did not co-express nestin nor vimentin.

The organization and expression profile of the grey mouse lemur third ventricle region was reminiscent of that found in mice. The third ventricle wall was composed of a regular layer of ependymal cells devoid of a gap-and-ribbon organization (Fig. 9l,o). Co-expression of Sox2 and vimentin was detected in ependymal cells (Fig. 9l-n) and tanycytes (Fig. 9o-q and Table 4). The suprachiasmatic nucleus contained Sox2-positive cells that did not co-express vimentin (Fig. 10i-k).

The human SVZ and the dentate gyrus of the hippocampus

In parallel with the analysis of the human hypothalamus, we evaluated the expression of NPC markers in the two classical neurogenic niches, the SVZ and the hippocampal dentate gyrus.

The SVZ exhibited a similar pattern of NPC marker expression as the third ventricle, with a ribbon of small process-bearing cells co-expressing nestin, Sox2, vimentin, GLAST and GFAP (Fig. 11 and Table 3), separated from the ependymal layer by a hypocellular gap enriched in GLAST (Fig. 11i,k) and GFAP immunoreactivity (Fig. 11m,o). Ependymal cells expressed Sox2, GLAST, and, at varying levels, vimentin, nestin and GFAP (Fig. 11 and Table 3). This expression profile was observed at the different rostro-caudal and dorso-ventral levels of the lateral ventricle analyzed. However, differences were noted compared to the third ventricle. The SVZ exhibited a more uniform organization, with a more regular ribbon and a thinner gap compared to the third ventricle (e.g. compare Fig. 11a-d to Fig. 3b-d).

While buds similar to those found in the third ventricle were observed (not shown), crowns and rosettes of ependymal cells were not seen. With regard to the expression of NPC markers, detection of nestin in ependymal cells was much rarer than in the third ventricle while strong vimentin immunoreactivity was more consistently observed in these cells (Fig. 11e,g).

In the hippocampus, a population of radial stem cells has been described in the SGZ of the dentate gyrus in several species including rodents and non-human primates [START_REF] Aizawa | Primate-specific alterations in neural stem/progenitor cells in the aged hippocampus[END_REF]Gonçalves et al., 2016). We failed to detect Sox2expressing cells in the dentate gyrus and nestin immunoreactivity was restricted to capillaries (Fig. 12a). The lack of Sox2 staining was not due to a technical detection problem since Sox2 expression was readily observable on the same sections along the border of the temporal horn of the lateral ventricle in ependymal and ribbon cells (Fig. 12a, inset). No GLAST immunoreactivity was observed in the dentate gyrus region (not shown). Moreover, while GFAP and vimentin were detected in small stellate cells close to the dentate gyrus, they were not seen in subgranular cells with a radial morphology (Fig. 12b,c).

DISCUSSION

Studies conducted in rodents have shown that the adult hypothalamus contains a neurogenic niche, with local NPCs that give birth to glial and neuronal cells in the nearby hypothalamic parenchyma (Chaker et al., 2016;Haan et al., 2013;Li et al., 2012;S. C. Robins et al., 2013;[START_REF] Robins | Evidence for NG2-glia derived, adult-born functional neurons in the hypothalamus[END_REF]. In the adult human hypothalamus, the recent identification of cells expressing the neuroblasts marker DCX, some of which exhibit a morphology characteristic of very immature neuronal progenitors (Batailler et al., 2014), raised the possibility that a local niche of NPCs may exist within this structure. Here we identify in the adult human hypothalamus four populations of cells, ependymal cells, ventricular ribbon cells, tanycytes and suprachiasmatic small stellate cells, which exhibit an antigenic profile of NPCs. In rodents and grey mouse lemur, a similar antigenic profile is essentially restricted to tanycytes.

In order to identify NPCs in the human hypothalamus, we used a series of five markers known to be expressed in these cell types during embryogenesis and adulthood (Götz, Sirko, Beckers, & Irmler, 2015;Kriegstein & Alvarez-Buylla, 2009;[START_REF] Kuhn | Detection and Phenotypic Characterization of Adult Neurogenesis[END_REF][START_REF] Vinci | Immunohistochemical markers of neural progenitor cells in the early embryonic human cerebral cortex[END_REF]. At present, a single marker specific for neural stem cells is not available since markers found in neural stem cells are also expressed in progenitors and/or mature cells. For instance, this is the case for GFAP, which is expressed both in neural stem cells and mature astrocytes, or nestin, which is expressed in neural stem cells, neural progenitors and endothelial cells [START_REF] Kuhn | Detection and Phenotypic Characterization of Adult Neurogenesis[END_REF][START_REF] Von Bohlen Und Halbach | Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus[END_REF]. Higher specificity can however be obtained by using a combination rather than a single marker. In the two main neurogenic niches, the SVZ and the SGZ, neural stem cells have been shown to express a repertoire of markers including GFAP, nestin, Sox2, GLAST, vimentin, BLBP and Pax6 (Kriegstein & Alvarez-Buylla, 2009;[START_REF] Von Bohlen Und Halbach | Immunohistological markers for proliferative events, gliogenesis, and neurogenesis within the adult hippocampus[END_REF]. Therefore, we performed triple immunostainings for nestin, Sox2 and vimentin, GLAST or GFAP and we considered that cell populations expressing all markers were the best candidates for being considered as neural stem or progenitor cells [START_REF] Kuhn | Detection and Phenotypic Characterization of Adult Neurogenesis[END_REF]. Noteworthy, technical constraints preclude the concomitant detection of the five markers of interest at the same time. However, our quantitative analysis showed that at least a fraction of nestin/Sox2positive cells also expressed vimentin, GLAST and GFAP. Interestingly, GFAP discriminates neural stem cells from neural progenitors in the two classical niches in rodents since neural stem cells express GFAP but neural progenitors lose its expression [START_REF] Doetsch | The glial identity of neural stem cells[END_REF]. In addition, a recent study in mouse suggested that among tanycytes, a subpopulation of GFAPpositive alpha tanycytes exhibited neural stem cell properties (S. C. Robins et al., 2013).

Therefore, if the repertoire of markers expressed by rodent neural stem cells is conserved in humans, it may be speculated that the nestin/Sox2/vimentin/GLAST/GFAP-immunoreactive ribbon, ependymal, suprachiasmatic cells and tanycytes are neural stem cells.

The ribbon of NPC marker-immunoreactive small stellate cells lining the human third ventricle is highly reminiscent of the ribbon of astrocytes described by Sanai and colleagues along the human lateral ventricles (Sanai et al., 2004). In their study, the authors identified a band of stellate cells separated from the ependyma by a hypocellular gap. SVZ ribbon cells expressed GFAP, vimentin and some of them were immunoreactive for Ki67. Importantly, by preparing astrocyte cultures from intraoperative specimens, they showed that SVZ astrocytes were capable of generating multipotent, self-renewing neurospheres, suggesting that they were bona fide neural stem cells. In the present study, and in agreement with Sanai and colleagues, the SVZ ribbon lining the lateral ventricles was found to express GFAP, vimentin as well as the other NPC markers Sox2, nestin and GLAST. Given the similar morphology and NPC marker expression profile between SVZ ribbon cells and hypothalamic ribbon cells, it may be speculated that hypothalamic ribbon cells exhibit neural stem cell properties as their SVZ counterpart. Noteworthy, the hypothalamic ribbon appears to be a characteristic feature of the human brain since it was not observed in the rodent or grey mouse lemur hypothalamus and has not been previously reported in the literature in other species. A striking difference between the lateral and the third ventricular border was the higher degree of irregularity in the aspect of the third ventricular border that exhibited frequent invaginations of ependymal cells, highly variable width of the gap and thickness of the ribbon. Such heterogeneity may explain why the hypothalamic ribbon was not previously identified from small samples of the human third ventricular wall (Sanai et al., 2004). In both the lateral and the third ventricular wall, we observed occasional bulges of the wall characterized by an interruption of the ependymal layer and enrichment in NPC markers.

Such protrusions are described by neuropathologists as nodular gliosis (or ependymal granulations or nodular ependymitis). Even though they have been associated with central nervous system infection or ventriculomegaly [START_REF] Johnson | Granular ependymitis. Occurrence in myxovirus infected rodents and prevalence in man[END_REF][START_REF] Shook | Ventriculomegaly associated with ependymal gliosis and declines in barrier integrity in the aging human and mouse brain[END_REF], they are also observed in non pathological brains and occur at a similar rate in young (20-40 years) and aged brains (60 and over) [START_REF] Gilmore | Analysis of ependymal abnormalities in subjects with schizophrenia, bipolar disorder, and depression[END_REF][START_REF] Johnson | Granular ependymitis. Occurrence in myxovirus infected rodents and prevalence in man[END_REF].

The suggested origin for these structures is a proliferation of subependymal glial cells [START_REF] Gilmore | Analysis of ependymal abnormalities in subjects with schizophrenia, bipolar disorder, and depression[END_REF] as a consequence of reactive gliosis. Reactive gliosis involves several characteristic features including cellular hypertrophy, up-regulation of GFAP expression [START_REF] Sofroniew | Reactive astrocytes in neural repair and protection[END_REF] and can be accompanied by the re-expression of NPC markers such as nestin and vimentin [START_REF] Goc | A spatiotemporal study of gliosis in relation to depth electrode tracks in drug-resistant epilepsy[END_REF]Götz et al., 2015). While the ventricular protrusions were highly enriched in NPC marker-expressing cells, these cells did not exhibit signs of hypertrophy and the expression of GFAP in protrusions was as intense as in the nearby hypocellular gap lying behind an intact ependyma. Moreover, NPC markerexpressing cells found in protrusions appear to be in continuity with adjacent ribbon cells.

Therefore, our observations raise the possibility that protrusions may originate from the local proliferation of ribbon cells rather than reactive astrocytes. Along the same line, ventricular protrusions are not observed in mice [START_REF] Shook | Ventriculomegaly associated with ependymal gliosis and declines in barrier integrity in the aging human and mouse brain[END_REF], which lack a ventricular ribbon.

Co-expression of NPC markers was also detected in a substantial fraction of human third ventricle ependymal cells. Whether ependymal cells have neural stem cell properties has been a matter of debate since almost two decades but recent rodent studies suggest that they do not (discussed in [START_REF] Pastrana | Eyes wide open: a critical review of sphere-formation as an assay for stem cells[END_REF]. Accordingly, and in agreement with in situ expression profile data (Allen brain atlas), rodent third ventricle ependymal cells showed ubiquitous and robust Sox2 and vimentin immunoreactivity, while most of them exhibited low to undetectable levels of nestin. Notably, studies using different expression profiling strategies and/or different antibodies (Chaker et al., 2016;[START_REF] Chen | Single-Cell RNA-Seq Reveals Hypothalamic Cell Diversity[END_REF]Lin et al., 2015;Mirzadeh et al., 2017;S. C. Robins et al., 2013) have reported variable results as to the presence of nestin in ependymal cells of the rodent third ventricle. Whether these apparently discrepant results reflect the existence of several alternative splicing-derived isoforms and/or post-translational modifications, some of them being more specific to stem cells, remains to be determined. Another population of NPC marker co-expressing cells seen in the human hypothalamus but not in the other species was found in the suprachiasmatic nucleus. In mammals, the suprachiasmatic nucleus is the endogenous masterclock that coordinates endogenous rhythms with the external light-dark cycle thereby controlling circadian physiology and behaviour. We also found a rich population of Sox2-expressing cells in the mouse, rat and grey mouse lemur suprachiasmatic nucleus. However, these cells did not co-express nestin and/or vimentin, suggesting that they are not NPCs. Accordingly, a previous study showed that Sox2-positive cells in the rodent suprachiasmatic nucleus mostly co-expressed neuronal markers [START_REF] Hoefflin | Neuronal expression of SOX2 is enriched in specific hypothalamic cell groups[END_REF]. In the human suprachiasmatic nucleus, we did not find evidence for the expression of GLAST and Sox2 in NeuN-positive mature neurons. It must be noted that NeuN immunostaining is weak and only reveals a small fraction of suprachiasmatic nucleus neurons, as previously shown in the rat [START_REF] Geoghegan | A novel site of adult doublecortin expression: neuropeptide neurons within the suprachiasmatic nucleus circadian clock[END_REF], calling for caution in the interpretation of data. However, the morphological characteristics of NPC-marker expressing cells, ie, small stellate, argue in favour of a non-neuronal identity. Altogether, the observation of cells with the same NPC antigenic profile as ribbon cells in the human ependymal layer and suprachiasmatic nucleus raises the intriguing possibility that these regions may contain neural stem cells.

Tanycytes are highly specialized ependymoglial cells that line the floor and the basolateral walls of the third ventricle in the tuberal region of the hypothalamus. Tanycytes have recently emerged as hypothalamic neural stem cells [START_REF] Goodman | Hypothalamic tanycytes-masters and servants of metabolic, neuroendocrine, and neurogenic functions[END_REF]. In agreement with previous studies (Bennett et al., 2009;Haan et al., 2013;Lee et al., 2012;Pérez-Martín et al., 2010;S. C. Robins et al., 2013), we here confirm that rodent tanycytes express NPC markers and expand these results to the grey mouse lemur concerning the two markers analysed, Sox2 and vimentin. In contrast to the ventricular ribbon that is only seen in humans, our study and others show that mice, rats, sheep (Batailler et al., 2014), grey mouse lemur and humans (Baroncini et al., 2007;[START_REF] Koopman | Age-Related Changes in the Morphology of Tanycytes in the Human Female Infundibular Nucleus/Median Eminence[END_REF][START_REF] Sidibe | Expression of the orphan GPR50 protein in rodent and human dorsomedial hypothalamus, tanycytes and median eminence[END_REF] share NPC marker-expressing tanycytes. In adult mice, lineage-tracing studies have shown that tanycytes give birth to neurons that populate nearby hypothalamic regions including the arcuate and ventromedial nuclei (Chaker et al., 2016;Haan et al., 2013;S. C. Robins et al., 2013). Interestingly, DCX-expressing cells are detected in the arcuate and ventromedial nuclei of the sheep and human hypothalamus (Batailler et al., 2014), raising the possibility that tanycytes may also be neurogenic in these species.

At present, the organization of the rodent hypothalamic stem cell niche remains vague. While we find in the human hypothalamus a gap-and-ribbon organization reminiscent of that found in the human SVZ (Sanai et al., 2004), the situation is different in rodents in which the architecture of the SVZ stem cell niche is not recapitulated in the hypothalamus (Mirzadeh, Merkle, Soriano-Navarro, Garcia-Verdugo, & Alvarez-Buylla, 2008). Lineage-tracing studies performed in adult mice have shown that new hypothalamic neurons can be produced not only from tanycytes but also from parenchymal cells that express Sox2 and/or NG2 (Li et al., 2012;[START_REF] Robins | Evidence for NG2-glia derived, adult-born functional neurons in the hypothalamus[END_REF]. Whether tanycytes and parenchymal neurogenic cells correspond to distinct NPC populations or whether they are linked by lineage relationship is currently unknown. However, if the combination of markers expressed in the adult rodent SVZ and SGZ [START_REF] Semerci | Transgenic mouse models for studying adult neurogenesis[END_REF]) is shared by the hypothalamic niche, our observation of NPC marker co-expression in tanycytes but not in parenchymal cells suggests that tanycytes are neural stem cells while parenchymal cells are more committed progenitors.

Neuroanatomical (Eriksson et al., 1998;Knoth et al., 2010) and, more recently, 14 C dating studies (Spalding et al., 2013), have brought evidence that a neurogenic niche exists in the human hippocampus. Here, we failed to detect a population of NPC marker-expressing cells in the dentate gyrus of the human hippocampus. In an extensive immunofluorescent study of neurogenesis-associated markers in the human hippocampus, Knoth and colleagues (2010) did not find radial cells, the putative neural stem cells in the rodent hippocampus (von Bohlen und Halbach, 2011) but detected Sox2/DCX and nestin/DCX co-expressing cells, i.e. early neuronal precursors, in humans up to 79 and 100 years of age, respectively.

However, the expression of these markers declined with age. Since our study was conducted on samples from aged brains ( 77 year-old), we cannot rule out the possibility that we missed these cells if present in very low number.

In the present study, our criteria for identifying NPCs were based on their molecular signature. However, proving that these cells are bona fide neural stem cells will require functional arguments, which is challenging in human studies. Neural stem cells are defined by two canonical properties, the ability to extensively self-renew and to differentiate into all three neural lineages, i.e. neuronal, astroglial and oligodendroglial. Even if it has some limitations, the neurosphere assay is commonly used to probe in vitro whether a tissue contains cells with neural stem cell properties [START_REF] Pastrana | Eyes wide open: a critical review of sphere-formation as an assay for stem cells[END_REF]. A study reported the possibility to grow neurospheres from rapid autopsy samples of SVZ from elderly human subjects [START_REF] Leonard | Subventricular zone neural progenitors from rapid brain autopsies of elderly subjects with and without neurodegenerative disease[END_REF]. A key to success certainly resides in the short post-mortem interval (less than 5 hours in the study by [START_REF] Leonard | Subventricular zone neural progenitors from rapid brain autopsies of elderly subjects with and without neurodegenerative disease[END_REF] and may explain our inability to grow neurospheres from hypothalami with post-mortem intervals over 7 hours, the shortest post-mortem delay reached in our study (unpublished observations). Future neuroimaging developments enabling to detect and measure levels of adult neurogenesis in living human brains [START_REF] Ho | In vivo imaging of adult human hippocampal neurogenesis: progress, pitfalls and promise[END_REF]) are eagerly awaited.

Comparative studies of adult neurogenesis in the two classical niches have revealed a great diversity in this process among species [START_REF] Lindsey | A comparative framework for understanding the biological principles of adult neurogenesis[END_REF]. The general view that adult neurogenesis has undergone a phylogenetic reduction has recently been reevaluated to propose that this process may rather have evolved to optimize the functioning of neuronal networks that have to deal with new situations [START_REF] Kempermann | New neurons for "survival of the fittest[END_REF]. Even though we need comparative data from many more species to start to draw a phylogenetic tree of adult hypothalamic neurogenesis, our results raise the intriguing possibility that the human brain may have expanded its hypothalamic neural stem cell niche compared to rodents and lemur primates. Whether this translates into higher functionally-relevant neurogenesis and a higher degree of plasticity and adaptability in hypothalamic circuits will be challenging future questions to address. Most ependymal cells showed low to undetectable nestin labeling. The rare ependymal cells with marked, unambiguous nestin immunoreactivity were scattered or gathered at varying dorso-ventral and antero-posterior locations along the third ventricular border. ‡ Occasional small vimentin-positive stellate cells were detected in the parenchyma surrounding the dorsal part of the third ventricle. NA, not available. Nestin (green) and Sox2 (magenta) were co-immunodetected with vimentin (a, c, d, f, g, white), GLAST (h, j, k, m, n, white) or GFAP (o, q, r, t, u, white). The ependymal layer (ep) and gap (G) are shown at higher magnification in upper right panels (b-d, i-k, p-r) while the ribbon (rb) is shown at higher magnification in lower right panels (e-g, l-n, s-u). Plain arrowheads show ependymal cells that co-express nestin, Sox2 and vimentin, GLAST or GFAP. While GLAST was strongly detected in most ependymal cells (h), vimentin showed highly variable expression levels (a). Some ependymal cells showed low levels of vimentin in their soma (a-d, empty arrowheads) but prominent expression in their basal processes, some of which co-expressed nestin (crossed arrows in b-d). Ependymal cells were heterogeneous for the expression of GFAP, with GFAP-negative (q, empty arrowhead) next to GFAP-positive cells (q, plain arrowhead). Note the strong expression of GLAST (h, j) and GFAP (o, q) in the hypocellular gap. Arrows point to ribbon cells that co-express nestin, Sox2 and vimentin, GLAST or GFAP. Figure 9. Expression of NPC markers along the third ventricle of the mouse (a-h), rat (i-k) and grey mouse lemur (l-q). Representative photographs were taken at the level of the tuberal region of the hypothalamus. The rat third ventricle is shown as a whole while the third ventricle from the mouse and grey mouse lemur is shown in its most dorsal (a-d, l-n) and ventral (e-h, o-q) portions. Coimmunostainings of Sox2 (magenta, a, e, i, l, o), nestin (green, b, f, j) and vimentin (white, c, g, inset in j, m, p). Merge images of all but hoechst stainings are shown in d, h, k, n, q. In the dorsal part of the third ventricle, ependymal cells co-express Sox2 and vimentin (empty arrowheads, a-d, i-k, l-n) with very occasional nestin co-labeling (a-d, plain arrowhead). In the ventral part of the third ventri -express Sox2, nestin and vimentin. While the third ventricle wall is composed by a regular layer of ependymal cells in the mouse (a, e) and grey mouse lemur (l, o), that of the rat shows thickenings frequently protruding inside the ventricle (crossed arrows in i). The yellow asterisk in p shows a capillary wrapped by tanycyte processes. Note that both tanycyte processes and endothelial cells are vimentin-immunoreactive (see Fig. 10j for examples of vimentin-positive capillaries). Nuclei were counterstained with Hoechst (blue, a, e, i, l, o). 3V, third ventricle; Arc, arcuate nucleus; ME, median eminence; PT, pars tuberalis. Scale bars = 50 µm.

Figure 10. Expression of NPC markers in the suprachiasmatic nucleus of the mouse (a-d), rat (e-h) and grey mouse lemur (i-k). A high density of Sox2-expressing cells (magenta) is detected in the suprachiasmatic nucleus of all three species (a, e, i). In the mouse, the suprachiasmatic nucleus is devoid of vimentin (b) and nestin (d) immunoreactivity. In the rat, it also lacks vimentin expression (h) but contains nestin-immunoreactive capillaries (f, g, arrows with stars). In both species, the third ventricle ependymal layer (empty arrowheads) expresses Sox2, vimentin but shows low to undetectable levels of nestin. In the grey mouse lemur, the suprachiasmatic nucleus contains rare

DISCUSSION

From the moment when adult cell proliferation in the mammalian brain has been considered possible, an increasing number of studies have tried to describe this process and its functions. While for many years the scientific community has focused on 2 brain regions only, the SVZ and the SGZ, different groups have now turned their attention towards the possibility that stem cell niches may reside in the postnatal mammalian brain, as for instance at the level of the striatum or the hypothalamus (Lin & Iacovitti, 2015).

The hypothalamus is a well-conserved structure in all mammalian species, devoted to the control of all vegetative functions, as metabolism, reproduction, circadian rhythms, control of sleep, thirst, fear, indispensable functions for survival of the individual and of the species itself. In order to fulfil its many roles, the hypothalamus relies on a surprisingly high number of different neuronal populations, for such a small region. These neurons, located in discrete nuclei, communicate with each other and with extra-hypothalamic brain regions. Cell proliferation in the hypothalamus is still a poorly studied subject both in relation to the different brain developmental stages and to the different hypothalamic functions (Sharif et al., 2014).

Here, we have explored how the birth of new cells in the postnatal and adult female rat hypothalamus may be relevant for the reproductive function, a function known to require a high level of plasticity. Moreover, we provide the first description of the neural stem cell niche in the adult human hypothalamus.

Cell proliferation in the female rat POA is maintained throughout postnatal development and during adulthood

Through the use of BrdU as a marker of dividing cells, we described cell proliferation in the POA of infantile, juvenile and adult female rats.

The highest levels of proliferation are observed during the infantile stage, when newborn cells mainly differentiate into astrocytes, in accordance with data obtained in other brain regions, such as the cerebral cortex, where most astrocytes are born at this stage. The drop in cell proliferation, which can already be seen during the juvenile period, is even more evident during adulthood, when the levels of cell neogenesis become very low.

The process of astrogenesis is still not entirely understood; the exact origin of hypothalamic astrocytes is still not clear. Recent works suggest that they derive from the same radial glial cells that give rise to neurons during embryonic development, which lately migrate into the parenchyma to give rise to astrocytes. Using the in vitro neurosphere assay, previous experiments performed in the lab showed the presence of neural stem/progenitor cells in the POA of infantile rats as neurospheres could be obtained, passaged up to 31 times (the longest time examined) and differentiated into the three neural lineages. Moreover, enrichment in Sox2-expressing progenitor cells was attested by both in situ hybridisation (Chapter 5.3.5.1) and immunohistochemistry (personal observations) in this region. Sox2 mRNA and protein levels were extremely high at the level of the SVZ, as expected, and were particularly enriched in the POA at the level of the OVLT.

Newborn cells are associated with GnRH neurons and are necessary for their postnatal development

Interestingly, during the infantile period, some of the newborn cells were found in a morphological association with the GnRH neurons that was preferential compared to the entire neuronal population of the POA. During the postnatal period, the GnRH neurons undergo a very specific and peculiar developmental process, as, differently from other neurons, they are not functional until puberty is established.

It is well known that the crosstalk between GnRH neurons and glial cells at the level of their cell body and terminals is important for the modulation of GnRH neuron activity and secretion. In the POA, tight morphological interactions occur between GnRH neurons and astrocytes. For instance, adhesion molecules regulating the adhesiveness between these 2 cell types have been shown to play an important role in the onset of puberty [START_REF] Sharif | Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion[END_REF]. At the same time, astrocytes regulate GnRH neuron activity and secretion through the release of gliotransmitters, such as PGE2, in a crosstalk that is established during postnatal maturation.

When cell proliferation was inhibited specifically within this region during the infantile period, the maturation process of GnRH neurons was altered as attested by delayed puberty and perturbed oestrous cyclicity, proving that the birth of astrocytes in the region is necessary for the correct maturation of the system.

8.1.3

The recruitment of newborn cells by GnRH neurons is necessary for the correct maturation of the system Given the observation that newborn cells were preferentially found in association with GnRH neurons and that the percentage of neurons associated with newborn cells increased over time during a 7-day-window of the infantile period starting at P8, we asked the question of whether GnRH neurons are able to create a microenvironment around them that would facilitate the proliferation and/or the recruitment of the surrounding progenitor cells. The possibility that GnRH neurons would also regulate the differentiation of the newborn cells was not analysed since the fate of the newborn cells found in association with the GnRH neurons was not different compared to that of the whole population of the POA.

We used a candidate approach to identify factors that could be released by GnRH neurons at the level of the POA and recruit progenitors. GnRH, galanin, glutamate, GABA and PGD2, a member of the prostaglandin family, were selected and were tested in vitro. Only PGD2 had an attractive effect on progenitor cultures derived from rat POA through its DP1 receptor.

However, none of the factors tested was able to alter the proliferative activity of these cells.

Prostaglandins are a family of unsaturated fatty acids derived from the arachidonic acid, which are not stored intracellularly, but rather are synthesised and released immediately.

DP1 is a transmembrane receptor coupled to a Gs protein that stimulates cAMP formation and subsequent activation of PKA and MAPK pathways. PGD2 has been already identified as a chemoattractant for eosinophils, through the activation of DP1, in processes involved in the allergic response [START_REF] Schratl | The Role of the Prostaglandin D2 Receptor, DP, in Eosinophil Trafficking[END_REF].

We showed that this pathway is active in vivo, as blocking DP1 receptor activation in the POA of infantile female rats decreases the association between GnRH neurons and newborn cells. Moreover, we saw that blocking this pathway altered FSH levels at P12 and affected the establishment of estrous cyclicity, although the animals did not show altered vaginal opening or disrupted fertility during adulthood. The absence of an effect on the age of puberty and fertility may be due to different reasons; first of all, we do not know for how long the DP1 receptor antagonist remains active within the brain and what percentage of newborn cells is affected. An effect of compensation can be easily imagined since cell proliferation continues over the juvenile period and the proliferation rate is not affected, even though we showed that at this stage the rate of cell proliferation is lower; nonetheless, GnRH neurons may still be able to attract cells born later on and manage to rescue normal activity in adulthood. If this were the case, it would also explain why complete inhibition of cell proliferation in the entire region resulted in a stronger phenotype.

Moreover, it has been shown that the number of functional GnRH neurons necessary to induce ovulation is relatively small [START_REF] Herbison | Gonadotropin-releasing hormone neuron requirements for puberty, ovulation, and fertility[END_REF], indicating that disrupting the process of recruitment of newborn astrocytes only in a percentage of neurons may not be sufficient to affect the onset of puberty.

Proposed model and future perspectives

Results from transcriptomic analyses showed that exposure to astrocyte conditioned medium increases the expression of the PGD2 synthase gene in GnV3 neurons, an in vitro model for mature GnRH neurons. Besides, the expression levels of PGDS within sorted GnRH neurons increase between P8 (early infantile) and P20 (late infantile). Therefore, we propose a model in which GnRH neurons at P8 attract, via PGD2/DP1 signalling, newborn immature cells, which subsequently differentiate into astrocytes. The recruited astrocytes would then be able to induce an increase in the expression levels of PGDS, probably causing the attraction of more newborn cells from the vicinity, establishing a positive loop that would help GnRH neurons establish their glial network before the onset of puberty, when astrocytes are known to regulate the activity of GnRH neurons via multiple mechanisms, including adhesive, juxtacrine and paracrine interactions [START_REF] Sharif | Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion[END_REF] (Fig. 1).

Figure 1: Proposed model. During the infantile period, GnRH neurons release PGD2 to attract DP1expressing newborn cells from their vicinity. This period of intense recruitment is associated to increased expression of PGD2 synthase in GnRH neurons. The associated newborn cells then differentiate into astrocytes. At puberty, the astrocytes associated with GnRH neurons release PGE2 to stimulate their electrical and secretory activity, thus participating in the induction of the first ovulation that marks puberty onset. PGDS, PGD2 synthase.

While the role of glial-to-GnRH neuron interactions is well known, here for the first time we show that this interaction is not unidirectional. Our results show that the GnRH neurons actively shape their glial environment over their postnatal maturation, attracting the newborn astrocytes found in their vicinity. This would allow GnRH neurons to control the establishment of their interactions with glial cells, which are necessary for the regulation of GnRH function during adulthood. Furthermore, during postnatal maturation, GnRH neurons receive an increasing number of neuronal inputs coming from different nuclei of the hypothalamus. The ability to control the association with neighbouring astrocytes would also allow the GnRH neurons to modulate their level of glial coverage, which is known to affect accessibility to neuronal inputs [START_REF] Clarke | Emerging roles of astrocytes in neural circuit development[END_REF]. Among the neurons that establish their projections over this time window, there are ARC neurons, known to regulate gonadal negative feedbacks [START_REF] Bouret | Formation of Projection Pathways from the Arcuate Nucleus of the Hypothalamus to Hypothalamic Regions Implicated in the Neural Control of Feeding Behavior in Mice[END_REF], necessary to restrain the system until proper maturation of the follicles is attained. An adequate balance over postnatal development, between the level of glial coverage and of the inputs driving negative feedbacks onto the GnRH neurons, may therefore be critical for the correct activation of the axis.

To overcome the difficulties encountered in inducing a longer blockage of DP1 signalling in vivo, different approaches could be used. For instance, an option may be that of injecting a virus in the POA of infantile female rats, carrying a siRNA against PGDS, under the control of the GnRH promoter, in the presence of a reporter gene. This would allow us not only to block this signalling in a more controlled manner, since expression of the siRNA could be checked through the presence of the reporter gene, but also to specifically knock it down in the GnRH neurons, avoiding possible interference with different hypothalamic functions exerted by PGD2, such as the control of sleep [START_REF] Urade | Prostaglandin D2 and sleep/wake regulation[END_REF]. Moreover, it would be interesting to evaluate whether interactions with newborn cells over the infantile period affect the electrophysiological properties of the GnRH neurons. Finally, to understand to which extent glial coverage changes over the postnatal maturation and its control by the GnRH neurons themselves, ultrastructural experiments using electron microscopy could be performed at different ages and after disruption of the PGDS/DP1 signalling.

Cell neogenesis in the hypothalamus of adult female rats during the estrous cycle

As previously mentioned, we observed cell proliferation in the POA of female rats during adulthood, even though at a lesser extent than during postnatal development. Moreover, we saw that the rate of cell birth is affected by estrous cyclicity. The highest levels of proliferation are found on the day before ovulation and the lowest ones on the day of estrus. In both the ME and the DG, we observed an increase in proliferation during the second day of diestrus when compared to the day of estrus. A higher percentage of newborn cells were found to be associated with GnRH terminals in the ME during the 2 days of diestrus compared to estrus, while no association was observed between GnRH neuron cell bodies and cells born during adulthood at the level of the POA.

After their birth, cells require a couple of weeks to differentiate and integrate within preexisting circuits [START_REF] Carlén | Functional integration of adult-born neurons[END_REF][START_REF] Ramirez-Amaya | Integration of New Neurons into Functional Neural Networks[END_REF]. Considering that rats ovulate every four days, it is improbable that the variations in cell proliferation over the estrous cycle regulate the cycle itself. We therefore searched for an explanation that would take into account the need for a long-term plasticity process, such as pregnancy.

Pregnancy increases cell survival in the MPO

The POA contains different nuclei with different roles and functions; when we focused on each nucleus separately, we observed an initial increase in the number of BrdU + cells in the MPO, followed by a drop 2 weeks afterwards. The final goal of ovulation is that of ensuring pregnancy; it has already been shown by [START_REF] Shingo | Pregnancy-Stimulated Neurogenesis in the Adult Female Forebrain Mediated by Prolactin[END_REF], that during pregnancy prolactin enhances cell proliferation at the level of the SVZ, and this is required for normal postpartum maternal interactions with pups [START_REF] Shingo | Pregnancy-Stimulated Neurogenesis in the Adult Female Forebrain Mediated by Prolactin[END_REF]Larsen et al., 2008;Larsen and Grattan, 2010). Indeed the success of pregnancy requires profound modifications in brain functioning to trigger the cerebral and physiological adaptations necessary for pregnancy completion, parturition, preparation of lactation and onset of maternal responsible for regulating the peak in proliferation that occurs before ovulation as well as the increase in cell survival in case of pregnancy.

What do these cells become?

Another important point that needs to be elucidated is the phenotypical fate of the newborn cells that survive during pregnancy. Given the gliogenic nature of the POA described during postnatal development, our hypothesis points towards an astrocytic differentiation fate.

Besides, astrocytes could efficiently play a role in supporting the changes in pre-existing neuronal circuits described in the MPO at the time of parturition. Indeed, astrocytes are able to modulate synaptic activity, dendritic spine number, as well as extracellular levels of ions and neurotransmitters [START_REF] Clarke | Emerging roles of astrocytes in neural circuit development[END_REF][START_REF] Oliveira | Do stars govern our actions? Astrocyte involvement in rodent behavior[END_REF]. Moreover, a recent work has shown how hypothalamic astrocytes are involved in the regulation of energy homeostasis through plastic changes in the morphology of their processes that depend on the feeding state and modulate extracellular levels of GABA with functional consequences on the activity of anorexigenic and orexigenic neurons [START_REF] Zhang | Hypothalamic stem cells control ageing speed partly through exosomal miRNAs[END_REF]. It has been estimated that one astrocyte can contact up to 100,000 synapses [START_REF] Halassa | Synaptic Islands Defined by the Territory of a Single Astrocyte[END_REF].

Therefore, even considering that the total number of new cells in the MPO is not as elevated as in regions such as the OB, it is clear that even few astrocytes can affect the activity of thousands of neurons in the region.

For these reasons, our preliminary results brought us to hypothesise that cell neogenesis in the MPO, at the onset of gestation, participates to the adaptive changes that enable development of the maternal behaviour.

Where do these newborn cells come from?

An important question that remains unanswered concerns the origin of newborn cells. The existence of adult neural stem cell niches outside of the SVZ and SGZ is still a matter of debate and new regions are not easily accepted. A growing number of studies conducted in both mice and rats show that cell proliferation persists during adulthood in the tuberal region of the hypothalamus, is influenced by diet and affects metabolic homeostasis. By analogy to what has been proposed in the tuberal region, newborn cells of the POA may derive from progenitor cells located within the parenchyma or migrate from a neighbouring stem cell niche. The proposed stem cell niche for the tuberal region of the hypothalamus resides within the wall of the third ventricle, where stem cells could correspond to tanycytes, specialised ependymoglial cells that exhibit morphological, molecular and functional characteristics of radial glia [START_REF] Rodríguez | Hypothalamic tanycytes: A key component of brain-endocrine interaction[END_REF]. Tanycytes can also be found at the level of the POA along the beginning of the third ventricle. Recent works have shown that also at this level, these cells express stem and progenitor cell markers such as nestin, Sox2, GFAP, Vimentin, as well as the proliferation marker Ki67 (Bennett et al., 2009;[START_REF] Furube | Characterization of neural stem cells and their progeny in the sensory circumventricular organs of adult mouse[END_REF]Langlet et al., 2013). Moreover, neurospheres were obtained from this region, and could proliferate and differentiate into both glial and neuronal cells (Bennet et al., 2009). While these in vitro data show that stem/progenitor cells can indeed be found at the level of the POA, they do not clarify their location. To address the hypothesis that tanycytes may be the candidate stem cells of the POA, it would be interesting to perform fate mapping experiments of tanycytes of the OVLT, as previously done for tanycytes of the ME, to see if they are able to give rise to parenchymal glial and/or neuronal cells.

What is the role of these newborn cells?

To answer this question, cell proliferation could be selectively inhibited at the level of the MPO, before or after mating and the consequences over maternal behaviour evaluated.

The adult human hypothalamus likely contains neural stem/progenitor cells

Different findings suggest that the process of adult neurogenesis has not disappeared throughout evolution but that it has rather adapted to larger structures and different necessities. The third ventricular niche remains a subject of debate among scientists, due to the variability of the data collected at present. The recent identification of cells expressing the neuroblast marker DCX in the human hypothalamus raised the possibility that a local niche may exist in this region (Batailler et al., 2014); however, the hypothalamus had never been characterised in human for the expression of markers used to identify stem/progenitor cells.

Due to the absence of a single marker able to unambiguously label neural stem cells, we decided to use a series of five markers known to be expressed in these cells during embryogenesis and adulthood (Kriegstein & Alvarez-Buylla, 2009;Götz et al., 2015). We identified in the adult human hypothalamus four populations of cells, ependymal cells, ventricular ribbon cells, tanycytes and suprachiasmatic small stellate cells, which exhibit an antigenic profile of neural stem cells (NSCs). Tanycytes was the only population showing a similar antigenic profile between rodents, grey mouse lemur and human.

A very interesting feature was the description of a ribbon of cells expressing NPCs markers, separated from the ependymal layer by a hypocellular gap, which was highly reminiscent of the astrocyte ribbon described along the lateral ventricles by Sanai and colleagues (2004).

The SVZ is the largest germinal zone in the adult brain. From this region, a chain of migrating newborn neurons, identified by their expression of DCX, is seen to travel to the OB in rodents and in the non-human primates rhesus monkey (Whang et al., 2011) and grey mouse lemur (personal observations). However, when adult human brains were analysed for the persistence of neurogenesis in the OB, such a phenomenon could not be observed using either neuroanatomical approaches [START_REF] Sanai | Corridors of migrating neurons in the human brain and their decline during infancy[END_REF] or 14 C dating (Spalding et al., 2013).

Nevertheless, when Sanai and colleagues (2011) described the SVZ in newborn children, they surprisingly saw a very strong staining for DCX and a high number of migrating neurons reaching the OB, as well as a subregion of the prefrontal cortex, along a route never seen before in other species. These chains of migrating cells were found between the ependymal layer and the ribbon of cells, before entering the RMS, and had completely disappeared as early as the second year of life, leaving in their place a void corresponding to the hypocellular gap seen along the adult lateral ventricles [START_REF] Sanai | Corridors of migrating neurons in the human brain and their decline during infancy[END_REF]. The observation of a similar gap along the adult third ventricle raises really interesting questions regarding the possibility that the gap could serve during early postnatal development as a path for migrating neurons reaching different regions of the hypothalamus or adjacent structures, therefore contributing to the establishment of networks involved in the maturation of different systems of the neonatal brain. Due to obvious difficulties in obtaining specimens at these young ages, the role of newborn neurons at the level of the OB and of the prefrontal cortex in neonatal human brain is still not known. In the same way, the characteristics of the postnatal development of the human hypothalamus remain obscure.

The presence of engorged areas, referred to as buds, along the ventricle, where the expression of the stem/progenitor markers was strengthened was quite intriguing. Together with the striking difference between the lateral and the third ventricular border in regards of the much higher degree of irregularity in the aspect of the latter, it may be speculated that this area is indeed quite plastic and actively shaping its niche. Once again, it would be very interesting to study the differences between specimens from young and old persons, to understand if this morphological variability can be observed already during postnatal development or only during adult life, maybe being the result of enhanced or erroneous proliferation.

Finally, we estimated the percentage of cells co-expressing the whole panel of NPC markers in the four populations of cells identified in the human hypothalamus. These values were extremely variable among the same population in different specimens, as well as among different regions of the same hypothalamus. For this reason, another point that could be interesting to develop would be that to understand if this variability reflects differences in the neurogenic potential of these different populations; if this was the case, the next question that naturally arises is whether any of this variability could be explained by the age or the presence of different clinical conditions in the patients, such as obesity or neurodegenerative diseases. For example, high fat diet has been shown to cause a decrease in the levels of cell proliferation within the hypothalamus in rodents (Li et al., 2012;McNay et al., 2012), while aging [START_REF] Zhang | Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH[END_REF] and neurodegenerative diseases are known to affect proliferation within the DG both in rodents and humans.

CONCLUDING REMARKS

When Ramòn y Cajal's most famous proclamation is cited to introduce the topic of neural plasticity ("In adult centers the nerve paths are something fixed, ended, immutable.

Everything may die, nothing may be regenerated"), we tend to forget mentioning the second part of his statement: "It is for the scientists of the future to change, if possible, this harsh decree". Since then, many scientists have accepted his challenge and the view of the brain as a static structure has now been definitely abandoned in favor of that of a system actively adapting to the different inputs that it constantly receives. The new questions now concern to which extent this happens, and which are the regions that have not been found yet.

The work of my thesis suggests that cell neogenesis during postnatal development and adulthood in the female rat preoptic region of the hypothalamus may be involved in the control of the reproductive function.

The process of postnatal maturation that leads to puberty is still a subject that has not been fully understood. Deviations in the age at which the onset of puberty occurs are becoming more and more common in young girls, due in part to environmental factors such as nutrition, stress and exposure to endocrine-disrupting chemicals. The consequences of this disruption tend to be underestimated but have been associated to psychological and behavioural disorders, increased risk of obesity, type 2 diabetes and cardiovascular diseases in adulthood [START_REF] Leka-Emiri | The mystery of puberty initiation: genetics and epigenetics of idiopathic central precocious puberty (ICPP)[END_REF]. This highlights the importance of gaining new insights on the mechanisms involved in pubertal maturation. Our results show that postnatal astrogenesis is involved in the onset of puberty, and the ability of GnRH neurons to attract these newborn cells from their neighbourhood seems to be necessary for their proper maturation, therefore adding postnatal cell proliferation to the processes involved in this complex puzzle.

Moreover, we observe that during adulthood cell proliferation in the POA is influenced by both estrous cyclicity and pregnancy, suggesting that this process may play a role also in the plastic adaptation of the mother brain during and/or after pregnancy.

Finally, we show for the first time the presence of different cell populations expressing a repertoire of stem cell markers in the adult human hypothalamus, suggesting the existence of a hypothalamic neurogenic niche and opening interesting questions on the possible roles played by these putative stem cells in regards to the different hypothalamic functions in humans.

I would like to start my aknowledgements by thanking the members of the jury who have agreed to read and comment this manuscript, and Dr. Vincent Prevot as the head of the lab.

Thank you Vincent, for accepting me in the lab when I came as a master student. Since then, you have been not only the head of the team but also a teacher for me. I have learnt a lot with you and not only regarding how to perform experiments but also about how to become a better scientist. Your constant enthusiasm and kidness make you a great boss and are always inspiring. I am glad I have worked for and with you.

I deeply thank Dr. Ariane Sharif, my supervisor. These three years have not always been easy, a Ph.D. probably never is. I am incredibly grateful for your help and your presence at every step of this path. You made me feel trusted and valued at each time and even if I will never be a perfectionist as you are, maybe you managed to make me a slightly more organised person.

Dr. Paolo Giacobini, cause you are the reason why I came to Lille in the first place. Thank you for mentoring me when I was a student of yours and to believe in me enough to suggest me to stay. Your help and suggestions are always very valuable to me. Céline, Sophie, Michelle and Nathalie from the secretery, who do an amazing job for us and always have a smile or a nice word to give.

The staff of the DHURE, who has been very nice to work with, Nathalie from the sorting facility who is always available for the "VP team" and who adjusts to our many problems and requests, Meryem from the confocal facility. I feel incredibly lucky for the team I have had the pleasure to work with during these 3 years.

Everyone made me feel part of a group, where everyone was always ready to help eachother, a feeling that really helps "surviving" in a lab. Special thanks go to Andrea, Irene and Filippo, the Italians who helped me when I arrived, to Charlottine, who we all really miss. Thank you for your kindness and your support.

To Sam and Nadia, without whom I could not even imagine Lille.

Sam, my companion in this journey. We started this together and here we are, still "shoulder to shoulder". Maybe we did not really know what to expect from it and we have made our lives more complicated in some moments but you have been a good friend and amazing collegue. Thank you for every moment, for your jokes and your silly questions, for your suggestions and interesting conversations. You have helped me open my mind sometimes towards ideas that would otherwise be still too far for me to accept.

Nadia, an amazing collegue and a friend I could always trust to find when in need. Thank you for making me feel like I was not alone, even when it was difficoult to believe otherwise.

Considering how picky we both are when it comes to people we open to, I believe it is an amazing coincidence we liked eachother.

To Sarah, who deals with my messy desk every day without complaining. You are an incredible person, I have never met someone so determined in her life, everything you want to accomplish, you do. Your help in the lab is priceless and we would all be lost without you.

To Maria, Monica, Nour and Valerie, because working with your friends make life easier and happier. You are all incredible people and I am really glad you have joint us. You all make me feel part of a group who would not leave anyone aside.

To Anne and Daniele, the pillars of the lab, whose experience is irreplaceable. Thank you for your help and your suggestions. I would like to thank all my friends from outside the lab and expecially Justine, who was the right person in the right moment, I loved being your roommate.

Finally, my thanks go to India, Martina, Elena, Francesca, Giulio, Cristina, Bianca, Federica e Giorgia, and all my friends from Catania and from Turin who stuck with me, even though I am always far away.

Clearly to my parents and my brother, although I cannot put my gratitude for them in words.

Mamma, Papà, Dami (e anche little Emily), grazie per il vostro amore incondizionato. So che siete orgogliosi di me e che è difficile per voi sapermi sempre lontana, spero un giorno di poter tornare e vedere di nuovo Papà costruire i suoi muretti. Casa rimane il posto più bello di tutti, non importa quanto lontano si viaggi.

Last but not least, to Giulio, Paola and Fabio, because I would have never survived here without you, I will never stop thanking you and the destiny that made me cross your path.

  Pellegrino G, Allet C, Leroy D, Siepmann J, Baroncini M, Prevot V, Sharif A. GnRH neurons shape their glial environment during postnatal maturation through the action of PGD2. The 21 st Annual meeting of the LARC-Neuroscience. Lille, France. 13 November 2017. Selected on abstract. Posters:  2017 : Pellegrino G, Allet C, Leroy D, Siepmann J, Baroncini M, Prevot V, Sharif A. Postnatal astrogenesis is critical for sexual maturation. 13 e EUROGLIA meeting, Edinburgh, 8-11 July 2017.  2017 : Pellegrino G, Allet C, Leroy D, Siepmann J, Baroncini M, Prevot V, Sharif A. GnRH neurons use PGD2 to shape their glial environment during postnatal maturation. Endo 2017, Orlando, Florida, USA, 1-4 April 2017.  2017 : Romero-Ruiz A, Torres-Jiménez E, Pellegrino G, Sharif A, Argente J, Chowen J, Roa J, Pinilla L, Colledge W.H, Tena-Sempere M. Identification of a novel kisspeptin signalling pathway in glial cells. Endo 2017, Orlando, Florida, USA, 1-4 April 2017.  2017 : Sharif A, Pellegrino G, Trubert C, Wyart E, Leroy D, Baroncini M, Maurage CA, Fontaine C, Prévot V. Une niche de cellules souches dans l'hypothalamus humain adulte? 22es Journées du Collège des Histologistes, Embryologistes et Cytogénéticiens, Reims, France.  2015 : Sharif A, Allet C, Pellegrino G, Leroy D, Caillet A, Loyens A, Siepmann J, Corfas G, Ojeda SR & Prevot V. The birth of astrocytes in the environment of GnRH neurons during early post-natal development is required for the timely onset of puberty in rodents. Colloque de la Fondation Ipsen, Paris, France.  2015 : Pellegrino G, Prevot V, Baroncini M, Sharif A. Cell neogenesis in the hypothalamus: a new mechanism of control of the reproductive function? 40 ème colloque de la Société de Neuroendocrinologie (SNE-BSN joint meeting), Lille, France.  : Sharif A, Allet C, Pellegrino G, Leroy D, Caillet A, Loyens A, Siepmann J, Corfas G, Ojeda SR & Prevot V. « The timely onset of puberty in rodents requires postnatal astrogenesis in the environment of GnRH neurons ». 12 ème congrès de la Société des Neurosciences Françaises, Montpellier, France.  : Pellegrino G, Prevot V, Baroncini M, Sharif A. « Cell neogenesis in the hypothalamus: a new mechanism of control of the reproductive function? » COST Third Annual Training School in Neuroendocrinology, Prato, Italy.  : Sharif A, Allet C, Pellegrino G, Leroy D, Caillet A, Loyens A, Siepmann J, Corfas G, Ojeda SR & Prevot V. Postnatal astrogenesis and female sexual maturation in rodents. Reprosciences 2015, Rennes, France.

  Such a statement would have been considered impossible until no more than 50 years ago, when the first experiments showing the ability of the adult brain to generate new neurons in physiological conditions started shaking one of the central dogma of neuroscience: "In adult centers the nerve paths are something fixed, ended, immutable. Everything may die, nothing may be regenerated." (Degeneration and Regeneration of the Nervous System, Ramon y Cajal, 1928).

Figure 1 :

 1 Figure 1: Neural stem cells (NSCs) from embryo to adult. (Upper panel) During brain development neuroepithelial cells elongate and convert into radial glial (RG) cells that contact the ventricle apically and the meninges, basal lamina, and blood vessels basally. RG can give rise to different type of intermediate progenitors, committed towards a neuronal (nIPC) or oligodendrocytic (oIPC) fate. At the end of embryonic development, some RGs convert into ependymal cells, others detach and convert into astrocytes while a subpopulation retains apical contact and continue functioning as NSCs in the neonate and adult animal. Dashed arrows are hypothetical. (Lower panel) Waves of neuro-, astro-and oligodendro-genesis from embryological to postnatal development. MA: mantle; MZ: marginal zone; NE: neuroepithelium; nIPC: neurogenic progenitor cell; oIPC: oligodendrocytic

Figure 2 :

 2 Figure 2: Summary of the expression of markers used to identify different cell types of the adult subventricular zone (a) and subgranular zone (b) neurogenic niches. aNSC, active neural stem cells; IPC, intermediate progenitor cells; qNSC, quiescent neural stem cells; TAC, transit amplifying cells. *data obtained from transcriptomic analyses only; #data obtained with the use of transgenic reporter lines only. (Scheme modified from Semerci & Maletic-Savatic 2016, Frontiers in Biology;Codega et al. 2014, Neuron; Llorens-Bobadilla et al. 2015, Cell Stem Cell; Shin et al. 2015, Cell Stem Cell).

Figure 3 .

 3 Figure 3. Schematic of the Different Domains of the Adult SVZ niche. (Upper left panel) A frontal cross-section of the adult mouse brain showing the location of the SVZ. (Lower panel) Cellular composition of the adult SVZ niche. Neural stem cells (NSCs) correspond to type B1 cells (blue),surrounded by multi-ciliated ependymal cells (E) forming pinwheel-like structures. B1 cells give rise to transit-amplifying cells, C cells (green), which divide to generate neuroblasts (type A cells, red). B1 present a thin apical process containing a primary cilium that contacts the lateral ventricle (V), being in direct contact with the CSF, and a long basal process ending on blood vessels (BV, purple).(From Fuentealba et al. 2012, Cell stem Cell).

Figure 4 .

 4 Figure 4. Schematic of the Different Domains of adult SGZ niche. (Upper panel) A frontal crosssection of the adult mouse brain showing the hippocampal formation (left). The insert shows a higher magnification indicating the location of the dentate gyrus (DG) (right). (Lower panel) Cellular composition of the adult DG domains. Radial astrocytes (RA), also known as type I cells (blue) give rise to intermediate progenitor cells (IPCs, green), or type II cells, which progressively (via IPC1, light green and IPC2, dark green) differentiate into immature granule cells (IGCs, or type III cells, red).Mature granule cells (GCs, brown) send an axon parallel to the SGZ into the hilus, whereas their dendrites branch into the molecular layer (ML). Radial astrocytes are polarized cells with their cell body in the SGZ, where they contact blood vessels (BV, purple) and neighboring radial astrocytes, a long main process that traverses through the granule cell layer (GCL) and then branches diffusely in the inner molecular layer (IML).(From Fuentealba et al. 2012, Cell stem Cell).

Figure

  Figure 5: Effects of reproduction hormones on adult neurogenesis. Gonadal steroids negatively affect cell proliferation in the OB, and enhance it in the DG. Prolactine is known to stimulate neurongenesis in the OB. DG, dentate gyrus; HYP, hypothalamus; OB, olfactory bulbs; RMS, rostral migratory stream; SVZ, subventricular zone.

Figure 6 :

 6 Figure 6: The SVZ niche in the adult human brain. (a) Coronal section revealing a region of high cellularity (Rb) that is separated from the ependymal (Ep) by a hypocellular gap. Nuclei were counterstained with DAPI (blue). (b) Vibratome section showing GFAP expression in SVZ astrocyte ribbon and GFAP-positive fibre bundles filling the subependymal gap. (c) Panoramic electron micrograph of a postmortem adult human SVZ, showing the ependymal lining, gap region and astrocyte ribbon. (d,e) Whereas neuroblasts from the adult SVZ in rodents migrate to the olfactory bulb to become interneurons, and enter the striatum only under pathological conditions such as ischemia (d), in humans hardly any adult neurogenesis can be found in the adult OB, but it occurs to a substantial degree in the striatum (e). Scale bars, 40 µm (a,b); 10 µm (c). LV, lateral ventricle; SVZ, subventricular zone (a-c are modified from Sanai 2004, Nature; d,e are from Kempermann 2014, Cell).

Figure 7 :

 7 Figure 7: The hypothalamic nuclei. (A) Sagittal view of a mouse brain, the red square corresponds to the region shown in B-I. (B-E) Coronal view of the four different levels of the hypothalamus showing from rostral to caudal the preoptic (b), anterior (c), tuberal (d) and mammillary (e) regions. (F-I) The boxed regions in B-E are shown at higher magnification.3V, third ventricle; 3Vm, 3V mammillary recess; ac, anterior commissure; ARC, arcuate nucleus; AVP, anteroventral nucleus; AVPV, anteroventral periventricular nucleus; DMH, dorsomedial hypothalamus; LHA, lateral hypothalamus; LM, lateral mammillary nucleus; LPO, lateral preoptic area; LV, lateral ventricle; ME, median eminence; MEPO, median preoptic nucleus; MM, medial mammillary nucleus; MPO, medial preoptic nucleus; och, optic chiasm; opt, optic tract; PH, posterior hypothalamic nucleus; SCN, suprachiasmatic nucleus; SON, supraoptic nucleus; SUM, supramamillary nucleus; VMH, ventromedial hupothalamus; VTA, ventral tegmental area (Swanson Atlas).

Figure 8 :

 8 Figure 8: The tanycytes of the third ventricle. Left hand panel: coronal section of the adult mouse third ventricle (3V), immunolabelled with vimentin (white). Tanycytes were false-coloured to distinguish them from ependymocytes. Right-hand panels: high-power magnifications of boxed regions in the left panel (Modified from Robins 2013, Nature Communications).

Figure 9 :

 9 Figure 9: Summary of our current knowledge on cell neogenesis in the adult rodent hypothalamus. Left hand panel: Tanycytes of the different regions differentially give rise to new tanycytes, glial cells and neurons. Proliferating cells, indicated by a red nucleus, are also found at the level of the

Figure 11 :

 11 Figure 11: The rat's oestrus cycle. (A) The pattern of GnRH, progesterone, prolactin, oestradiol, LH and FSH release throughout the 4-day oestrous cycle. The concentrations were obtained at 2-h intervals from five to six different rats. The numbers below represent the time-of-day in terms of the 24-h clock. (B) Vaginal smear from rat, stained with crystal violet throughout the stages of the oestrus cycle. The relative enrichment in the different cell types allows to identify the oestrus cycle stage. The two days of diestrus are characterised by a predominance of leukocytes (black arrows); in proestrus, only nucleated epithelial cells are found (white arrows); estrus is characterised by an

Figure 13 :

 13 Figure 13: Schematic representation of GnRH neurons-astrocytes interactions in the POA. Neuronal glutamate (Glu) co-activates metabotropic glutamatergic (mGluR) and AMPA glutamatergic receptors (GluR) in astrocytes, stimulating the activity of metalloproteinases of the ADAM family that subsequently release mature TGFα and NRG, which activate erbB1/erbB2 and erbB4/erbB2 heterodimers, respectively. Activation of erbB receptors promotes the release of PGE2, which binds to EP2 receptors in GnRH neurons and induces a cAMP/PKA pathway that leads to a reversible membrane depolarization and initiation of spike firing through the activation of a nonselective cation current (From Clasadonte J. 2011, Frontiers in Neuroendocrinology).

Figure 14 :

 14 Figure 14: Schematic representation of GnRH neuron-glial cell interactions in the ME. The liganddependent activation of erbB1 receptors in tanycytes results in plastic changes characterized by a phase of retraction of their endfeet, which requires PGE2 synthesis, production of TGFβ1 and metalloproteinase activity. In addition, PGE2 released by tanycytes and astrocytes is able to directly stimulate GnRH release at nerve endings via binding to EP1 receptors (Modified by Clasadonte J. 2011, Frontiers in Endocrinology).

Figure 15 :

 15 Figure 15: Close relationship between GnRH neurons and glial cells in mammals. (a, d, g) Microphotographs representing GFAP-immunoreactive astrocytes (red, arrows) enwrapping the cell bodies and processes of GnRH neurons (green) in the mouse (a), microcebus murinus (a non-human primate) (d), and human (g) hypothalamus. (b, e, h) Microphotographs the median eminence showing interaction between dense GnRH-terminals (green) and vimentin-immunoreactive (b,e) or nestin-immunoreactive (h) tanycytic processes in the mouse (b), microcebus murinus (e) and human (h). c, f and i show higher magnification views of b, e and h respectively. Arrows point to GnRH terminals (green) closely apposed to tanycytic processes (red). 3V, third ventricle; Inf S, infundibular stem. Scale bars: 120 μm in h; 100 μm in b and e, 50 μm in c anf f; 30 μm in i; 20 μm in a, d and g. (a panel from Ojeda et al. 1999; b-c panels from Prevot 2010, Frontiers in Neuroendocrinology; g-I panels from Baroncini 2007, Journal of Neuroendocrinology; d-f panels, personal observation).

Figure 16 :

 16 Figure 16: Centrally driven activation of the female HPG axis during postnatal development. Ovarian maturation: Scheme of follicules and consequent E2 secretion during postnatal development. Hormonal profiles: Schematic diagrams illustrating changes in GnRH, FSH and LH profiles during female postnatal development. Hypothalamic development: Gradual establishment

4. 6

 6 .1.1 Morphological association between GnRH neurons and BrdU + cells Quantification was made under the fluorescent microscope at the x40 objective. A BrdU+ cell was considered morphologically associated with a GnRH neuron when it was located at a distance corresponding to the size of a BrdU + nucleus (i.e., 10 µm) from the GnRH neuron cell body with no interposed cell nucleus in between. To characterize the morphological association during postnatal development in rats (n = 3 to 10 animals per group), 163.6 ± 13.0 GnRH neurons were analyzed per animal. For the group of rats that received multiple injections of BrdU (n = 4), 194.8 ± 8.3 GnRH neurons were analyzed per animal. For microsphere-injected rats (n = 3 animals per group), 125.3 ± 24.1 GnRH neurons were analyzed per animal. 4.6.1.2 Phenotypic identity of BrdU + cells To determine the phenotype of BrdU+ cells in the preoptic region, double immunofluorescent labeling of BrdU and GFAP, APC or HuC/D at the four postnatal ages were analysed on photomontages of the whole preoptic region taken at the x20 objective. 257.8 ± 25.3 BrdU + cells were examined per animal (n = 3 animals per group). Results are displayed as the percentage number of co-labeled cells over the total number of BrdU+ cells analyzed. To determine the phenotype of BrdU+ cells morphologically associated with GnRH neurons, triple immunofluorescent labelings of GnRH, BrdU and GFAP, APC or HuC/D were analyzed under the microscope at the x40 objective. 124.8 ± 12.0 GnRH neurons were analyzed and 23.2 ± 3.9 BrdU + cells have been identified at their neighbourhood per animal (n = 3 to 4 animals per group). Results are displayed as the percentage of co-labeled cells over the total number of BrdU + cells associated with GnRH neurons analyzed.

  figures were pseudocolored, adjusted for brightness and contrast and merged using Photoshop (Adobe Systems, San Jose, CA).

  using a Papain Dissociation System (Worthington, Lakewood, NJ) to obtain single-cell suspensions. FACS was performed using an EPICS ALTRA Cell Sorter Cytometer device (BD Bioscience). The sort decision was based on measurements of EGFP fluorescence (excitation: 488 nm, 50 mW; detection: GFP bandpass 530/30 nm, autofluorescence bandpass 695/40 nm) by comparing cell suspensions from Gnrh::Egfp and wild-type animals. For each animal, 80 to 300 EGFP-positive cells were sorted directly into 10 µl of extraction buffer containing 0.1% Triton X-100 (Sigma) and 0.4 unit/µl RNaseOUT (Life technologies).

Figure 1 .

 1 Figure1. The postnatal POA contains multipotent progenitors that mainly differentiate into astrocytes. (a) Diagram of the experimental protocol. Female rats received a single intraperitoneal injection of BrdU (300 mg/kg) at P8 (early infantile), P12 (infantile), P20 (early juvenile) or P24 (juvenile). Animals were perfused at different survival times: 2 hr and 24 hr to study proliferation, 7 d to evaluate the phenotypic identity of newborn cells and 60 d to monitor the long-term survival of newborn cells. (b) Coronal section of the preoptic region of a P12-injected animal immunolabeled for BrdU (red). (c) Quantification of the total number of BrdU+ cells in the preoptic region of animals injected with BrdU at P8, P12, P20 or P24 and analysed 2 hr later. Note the drop between the infantile and the juvenile period (n = 3-4 animals per group). (d-g) Female rats were injected with BrdU at P8, P12, P20 or P24. Brains were collected 7 d later and processed for immunodetection of BrdU (red) and markers (green) of the astroglial (GFAP, d), oligodendroglial (APC, e) or neuronal (Hu, f) lineages. Arrows point to co-labeled cells. (g) Quantification of the phenotypic identity of newborn cells calculated as the percentage of cells co-labeled for BrdU and GFAP, APC or Hu over the total number of BrdU+ cells in the preoptic region (n = 3 animals per group). Sections were counterstained with the nuclear marker Hoechst (white). Scale bars: 100 µm (in b); 10 µm (d-f). Mean ± s.e.m.; *** P < 0.001 relative to P8 or P12. 3V, third ventricle.

Figure 2 .

 2 Figure 2. Morphological interactions between GnRH neurons and newborn cells in the postnatal POA. (a) Coronal section of the preoptic region immunolabeled for GnRH (green) and BrdU (red). Arrow and arrowheads point to GnRH neuron cell bodies. Inset, higher magnification view of the GnRH neuron pointed by the arrow, showing the close morphological interaction with a BrdU+ cell (crossed arrow). (b) Female rats received a single injection of BrdU at P8, P12, P20 or P24 and the proportion of GnRH neurons associated with BrdU + cells was quantified 2 hr, 7 d or 60 d later (see Fig. 1a) (n = 3-10 animals per group). (c) Schematic of the proposed model. At P8, cells are generated (red) in the vicinity of GnRH neurons (green). Within the following week, additional GnRH neurons

Figure 3 .

 3 Figure 3. Phenotypic identity of newborn cells morphologically associated with GnRH neurons. (a-c) Triple immunolabeling for GnRH (green), BrdU (red) and GFAP (a, white), APC (b, white) or HuC/D (c, white) of sections from the preoptic region of female rats injected with BrdU at P8 and sacrificed 7 d later. Arrows point to BrdU+ cells morphologically associated with GnRH neurons. (d) Quantification of the phenotypic identity of newborn cells associated with GnRH neurons 2hr or 7 d after an injection of BrdU at P8, calculated as the percentage of cells co-labeled for BrdU and Sox2, GFAP, APC or HuC/D over the total number of BrdU+ cells (n = 3-4 animals per group). Scale bars: 20 µm. *** P < 0.001.

Figure 5 .

 5 Figure 5. GnRH neurons express galanin but lack cholecystokinin (CKK) or neurotensin (NT) during the infantile period. Double immunolabelings of GnRH (red, upper panels) and CCK, NT or galanin (GAL) (green, lower panels) in P11 rats. Arrows point to GnRH neuron cell bodies that co-express galanin (Philippe Ciofi, U862, Bordeaux).

Figure 6 .

 6 Figure 6. GnRH neurons express v-Glut and v-GAT during the infantile period. Double immunolabelings showing the co-expression of GnRH (red) and the vesicular glutamate transporter (v-Glut) (green, upper panels) or the vesicular GABA transporter (v-GAT) (green, lower panels) in P8 female rats at the level of the organum vasculosum of the lamina terminalis (OVLT) (left) and the median eminence (right). Nuclei were counterstained with Hoechst (blue). 3V, third ventricle.

Figure 8 .

 8 Figure 8. PGD2 attracts POA progenitor cells in vitro. (a) Schematic of the transwell assay; cells are seeded on a porous membrane placed in a well containing the factor to test. If cells are attracted by this factor, they migrate through the membrane to the bottom surface, where they can be subsequently fixed, stained, and counted. (b) Quantification of transwell assays showing that PGD2 attracts progenitor cells. EGF was used as a positive control (n = 4 experiments). (c) Real-time PCR analysis of DP1 and DP2 mRNA levels in progenitor cultures (n = 4 cultures). (d) Western blot experiment showing that progenitors respond to PGD2 treatment with increased phosphorylation of the MAPK ERK1/2, an effect that is inhibited by treatment with the DP1 receptor antagonist BWA868C (n = 3). (e) Quantification of transwell assays showing that treatment with BWA868C inhibits the PGD2-dependent migration of POA progenitors (n = 3 experiments).

Figure 9 .

 9 Figure 9. The candidate factors do not affect the proliferation of POA progenitors in vitro (a) Immunolabelling of Ki67 (green) in POA progenitor cultures untreated (control) or treated with FGF2 or PGD2. Nuclei were counterstained with Hoechst (blue). Scale bar: 100 μm. Mean ± s.e.m.; * P < 0.05; ** P < 0.01. . (b) Quantification of the percentage of Ki67+ cells in response to GnRH, PGD2, Galanin, Glutamate and GABA. FGF2 was used as a positive control (n = 3 experiments).

FGF2

Figure 10 .

 10 Figure 10. In situ hybridisation experiment showing that GnRH neurons express PGDS and progenitors express DP1 in the postnatal POA in vivo. (a,b) Representative confocal images of fluorescent in situ hybridization experiments using the RNAscope technology on brain sections of P8 female rats, showing the presence of GnRH (a, b, green), Sox2 (a, b, white), PGDS (a, red) and DP1 (b, red) mRNAs. The boxed panels in a and b are shown at higher magnification in the right panels. Arrows point to a GnRH/PGDS co-expressing cell (a) and Sox2/DP1 co-expressing cells next to a GnRH neuron (b). Scale bars: 10 μm in a and b panels; 20 μm in high-magnification views.

Figure 11 .

 11 Figure 11. Cell sorting experiment showing that postnatal GnRH neurons express PGDS. (a) Schematic of the cell sorting procedure used to sort GnRH neurons from the POA of Gnrh::EGFP rats. (b-d) Real-time PCR analysis of the expression of GnRH in EGFP-positive versus negative FACS-sorted

Figure 13 .

 13 Figure 13. Integrity of DP1 signalling during the infantile period is necessary for the correct establishment of the estrus cycle. (a) Schematic of the experimental protocol. (b) Circulating FSH levels at P12 are lower in BWA868C-injected animals compared to controls. (c)The percentage of GnRH neurons associated with newborn cells 7 days after the injection of BrdU is lower in BWA868Cinjected animals, while the total number of BrdU+ cells in the POA is not affected (d) compared to control animals. (e) The day of vaginal opening and (f) the weight on the day of vaginal opening do not change between the two conditions. (g) During the first 15 days after vaginal opening, BWAinjected animals spend more time in estrus and less in proestrus compared to controls. After this time window, the cycle goes back to normal, as also seen by LH levels on the day of proestrus, which do not show any difference between BWA868C-treated and contro animals (h).

Figure 1 .

 1 Figure 1. A peak in cell proliferation occurs in the POA of adult female rats on the day of diestrus 2. (a) Schematic of the protocol. (b) Representative coronal sections of the rat brain, corresponding to levels 17, 18 and 20 of the Swanson brain atlas, to show the area analysed for the quantification. (c) The number of BrdU-expressing cells in the POA of adult female rats is highest on the day of diestrus 2 and lowest on the day of estrus. (d) BrdU + cells born during adulthood are not associated with GnRH neurons (n = 5 animals per group). i.p., intraperitoneal. Mean ± s.e.m.; ** P < 0.01; *** P < 0.001.

Figure 2 .

 2 Figure 2. Cell proliferation in the ME of adult female rats across the estrous cycle. (a) Representative coronal section of the rat brain, corresponding to level 29 of the Swanson brain atlas, to show the area analysed for the quantification. (b) Proliferation in the ME is higher in diestrus 2 compared to estrus (n = 5 animals per group). (c) Coronal section of an animal injected with BrdU and labelled for GnRH (green) and BrdU (red). The lower panels are high-magnification views of the boxed areas in the upper panel. Nuclei were counterstained with Hoechst (blue). (d) The percentage of BrdU+ cells associated with GnRH fibers within the ME is higher during the two days of diestrus compared to estrus. Scale bars: 50 μm in c top panel, 20 μm in c bottom panels. Mean ± s.e.m.; * P < 0.05; ** P < 0.01. ARC, arcuate nucleus; DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus; 3V, third ventricle.

Figure 3 .

 3 Figure 3. Cell proliferation in the hippocampal dentate gyrus of adult female rats across the estrous cycle. (a) Representative coronal sections of the rat brain, corresponding to levels 29, 31, 34 and 36 of the Swanson brain atlas to show the area analysed for the quantification. (b) Proliferation in the DG is higher in diestrus 2 compared to estrus (n = 5 animals per group). Mean ± s.e.m.; * P < 0.05.

Figure 4 .

 4 Figure 4. Follow-up of the population of cells born in diestrus 2 in the different POA nuclei. (a) Schematic of the protocol. (b) Representative coronal section of the rat brain showing the different nuclei of the POA according to the Swanson brain atlas. (c) Quantification of the total number of BrdU + cells in the different POA nuclei 2 hours, 7, 14 and 21 days after a triple injection of BrdU on the day of diestrus 2. Ac, anterior commissure; AVP, anteroventral nucleus; AVPV, anteroventral periventricular nucleus; MEPO: median preoptic nucleus; MPO, medial preoptic nucleus; och, optic chiasm; OVLT, Organum vasculosum of the lamina terminalis; 3V, third ventricle. Mean ± s.e.m.; * P < 0.05.

Figure 5 .

 5 Figure 5. Pregnancy increases survival of newborn cells in the MPO. (a) Schematic of the protocol. 14 (b) and 21 (c) days after the injections, the total number of BrdU + cells is higher in the MPO of pregnant rats compared to non-pregnant animals. Mean ± s.e.m.; * P < 0.05; ** P < 0.01.

  Animal studies were approved by the Institutional Ethics Committee for the Care and Use of Experimental Animals of the University of Lille; all experiments were performed in accordance with the guidelines for animal use specified by the European Union Council Directive of September 22, 2010 (2010/63/EU). Tissues were obtained from 7 adult female Sprague-Dawley rats (2-3 months, Janvier Laboratories, Saint Berthevin, France) and 3 adult female mice (3-4 months, Charles River, L'Arbresle, France). The animals were maintained on a 12-hour light/12-hour dark cycle (lights on at 07.00 h), with food and water available ad libitum.

  Flash 4.0 V2 camera (Hamamatsu, Japan) driven by the Zen imaging software (Zeiss). Specific filter cubes were used for the visualization of green (Alexa 488 fluorochrome) (Excitation wavelength: 475/40 nm, beam splitter: 500 nm, emission wavelength: 530/50 nm), red (Alexa 568 fluorochrome) (excitation wavelength: 550/25 nm, beam splitter: 570 nm, emission wavelength: 605/70 nm), far red (Alexa 647 fluorochrome) (excitation wavelength: 640/30 nm, beam splitter: 660 nm, emission wavelength: 690/50 nm) and blue fluorescence (Hoechst) (excitation wavelength: 365 nm, beam splitter: 395 nm, emission wavelength: 445/50 nm). High magnification photomicrographs represent maximal intensity projections derived from 15-52 triple or quadruple-ApoTome images collected using the z-stack module of the Zen imaging software and a Zeiss 20x (numerical aperture NA = 0.8) or 40x objective

Figure 1 .

 1 Figure1. Characterization of the specificity of antibodies used to detect NPC markers in human tissues. a-g: Expression of Sox2 (a, magenta, stained with the goat polyclonal # sc-17320 from Santa Cruz), Ki67 (b, magenta), vimentin (c, green, stained with the mouse monoclonal M0725 clone V9 from Dako),GLAST (d, green), nestin (e, green) and GFAP (f, magenta, stained with the rabbit polyclonal Z0334 from Dako) in the cerebral cortex of a 11-week-old human fetus. All markers are detected in radial glial cells, which have their cell body lining the ventricle (LV) and extend a long process across the nervous tissue toward the pial surface (top left corner, not visible). Note the colocalisation of nestin and GFAP in radial glial cells (g, merge). h: Expression of nestin in an adult human GBM. i, j: Double immunofluorescent labeling using two different anti-Sox2 antibodies (i, magenta: goat polyclonal # sc-17320 from Santa Cruz; j, green: mouse monoclonal # ab79351 from Abcam) in the adult human hypothalamus yielded the same expression profile. Arrowheads point to examples of double-labeled cells. k, l: Double immunofluorescent labeling using two different anti-GFAP antibodies (k, green: mouse monoclonal MAB3402 from Chemicon; l, magenta: rabbit polyclonal Z0334 from Dako) in the adult human SVZ yielded the same expression profile. Nuclei were counterstained with Hoechst (all panels except g, blue). 3V, third ventricle; CP, cortical plate; IZ, intermediate zone; LV, lateral ventricle; SVZ, subventricular zone; VZ, ventricular zone. Scale bars = 50 µm in a-g, k, l; 20 µm in h, i, j.

Figure 2 .

 2 Figure 2. Representative coronal histological sections stained with Toluidine Blue across the rostrocaudal extent of the human hypothalamus. a: anterior-most region containing the preoptic recess of the third ventricle. b: anterior hypothalamic area. c, d: tuberal region. e: mammillary region. 3V, third ventricle; ac, anterior commissure; DMH, dorsomedial hypothalamic nucleus; FLV, frontal horn of lateral ventricle; fx, fornix; Inf, infundibular nucleus (or arcuate nucleus of the hypothalamus); InfS, infundibular stalk; IthA, interthalamic adhesion; LT, lamina terminalis; ME, median eminence; ML, medial mammillary nucleus, lateral part; MM, medial mammillary nucleus, medial part; MPO, medial preoptic nucleus; oc, optic chiasm; opt, optic tract; P3V, preoptic recess of the third ventricle; Pa, paraventricular nucleus; PaPo, paraventricular hypothalamic nucleus, posterior part; PH, posterior hypothalamic area; PV, paraventricular thalamic nucleus; SCh, suprachiasmatic nucleus; SO, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus. Scale bar = 2 mm.

Figure 3 .

 3 Figure3. Expression of nestin and Sox2 along the wall of the human third ventricle. a: Lowmagnification photomontage of Hoechst counterstaining (grey) showing the third ventricle border at an antero-posterior level corresponding to the end of the anterior hypothalamic area and the beginning of the tuberal region. a 1 -h: Double immunofluorescent labeling of nestin (green) and Sox2 (magenta). Nuclei were counterstained with Hoechst (blue). a 1 -a 4 : High-magnification images of the areas indicated in a. Insets in a 1 , a 2 : High-magnification images of the boxed areas. Most of the third ventricular border is lined by a ribbon of small nestin/Sox2 double-labeled cells with multiple processes (arrows in a 1 , a 2 , b, c, d) separated from the ependymal layer by a hypocellular gap (G, double arrow in a 1 , a 3 , b, c, d). These cells cohabit with Sox2-positive/nestin-negative cells (arrowheads in a 1 , a 2 ). Some nestin/Sox2 double-labeled cells in the ribbon extend long processes (crossed arrow) to contact ependymal cells (inset in b) or capillaries (g). The nestin-positive subependymal area shows occasional thickenings (a 2 , a 3 ) that can protrude into the ventricle (a 4 , g, h). Protrusions are associated with an interruption of the ependymal layer (empty arrowheads). Empty arrows point to invaginations of Sox2-positive/nestin-negative ependymal cells in the underlying parenchyma forming rosettes(b, c, d, e) or crowns (f). Regular invaginations of ependymal cells associated with bulging of the wall gives the border a crenelated aspect (b, c). Asterisks indicate nestin-positive capillaries. 3V, third ventricle; ep, ependymal layer; G, hypocellular gap. Scale bars = 1 mm in a; 50 µm in a 1 , a 2 , a 3 , a 4 , b, c, d, f, h; 20 µm in e, g and insets.

Figure 4 .

 4 Figure 4. Expression of NPC markers in ependymal and ribbon cells of the human third ventricle.Nestin (green) and Sox2 (magenta) were co-immunodetected with vimentin (a, c, d, f, g, white),GLAST (h, j, k, m, n, white) or GFAP (o, q, r, t, u, white). The ependymal layer (ep) and gap (G) are shown at higher magnification in upper right panels (b-d, i-k, p-r) while the ribbon (rb) is shown at higher magnification in lower right panels (e-g, l-n, s-u). Plain arrowheads show ependymal cells that co-express nestin, Sox2 and vimentin, GLAST or GFAP. While GLAST was strongly detected in most ependymal cells (h), vimentin showed highly variable expression levels (a). Some ependymal cells showed low levels of vimentin in their soma (a-d, empty arrowheads) but prominent expression in their basal processes, some of which co-expressed nestin (crossed arrows in b-d). Ependymal cells were heterogeneous for the expression of GFAP, with GFAP-negative (q, empty arrowhead) next to

Figure 5 .

 5 Figure 5. Expression of NPC markers in ventricular protrusions of the human third ventricle. Protrusions of various sizes and forms are found all along the third ventricle. The continuous ependymal layer (plain arrowheads) is interrupted on top of the protrusions (empty arrowheads).These structures contain nestin (a-i, green)/Sox2 (magenta)-immunoreactive cells that co-express vimentin (a-c, j-l), GLAST (d-f) and GFAP (g-i) (arrows, co-expressing cells are shown at higher magnification in insets). The subependymal region and buds are devoid of NeuN-immunoreactive cells, which are found in the deeper parenchyma (j-l, white, crossed arrow). Nuclei were counterstained with Hoechst (blue). 3V, third ventricle; P3V, preoptic recess of the third ventricle. Scale bars = 50 µm in main panels and 20 µm in insets.

Figure 6 .

 6 Figure 6. Expression of NPC markers in the median eminence of the human hypothalamus. a: Nestin (green) and Sox2 (magenta) are expressed in tanycytes, which line the floor of the third ventricle. Crossed arrows point to tanycyte nuclei and arrows show tanycyte processes. b-d: coimmunodetection of nestin (green) and vimentin (b, white), GLAST (c, white) or GFAP (d, white). The three panels below each main panel show a high magnification view of the boxed area. Nestinimmunoreactive tanycyte processes co-express vimentin (b, arrow), GLAST (c, arrow) and GFAP (d, arrow). Plain arrowheads point to nestin-positive processes devoid of vimentin (b), GLAST (c) or GFAP (d) immunoreactivity. Vimentin-positive (b, empty arrowhead) and GFAP-positive (d, empty arrowhead) processes devoid of nestin immunoreactivity were also seen. Nuclei were counterstained with Hoechst (blue, panel a). 3V, third ventricle, ME, median eminence. Scale bars = 50 µm in a, 20 µm in other panels.

Figure 7 .Figure 8 .

 78 Figure 7. Expression of NPC markers in the human suprachiasmatic nucleus. a: Immunofluorescent staining of VIP (white) was performed to ascertain the correct localization of the suprachiasmatic nucleus on human hypothalamus sections. Crossed arrows point to VIP-immunoreactive neurons. b: Double immunofluorescent staining of nestin (green) and Sox2 (magenta). The inset is a highmagnification view of the boxed area in the main panel. Note the co-existence of small processbearing nestin/Sox2 co-expressing cells (arrows) with Sox2-positive/nestin-negative cells (arrowheads). c-k: triple immunofluorescent stainings of nestin (green), Sox2 (magenta) and vimentin (white, d, e), GLAST (white, g, h) or GFAP (white, j, k). Arrows point to triple labelled cells and arrowheads show nestin-negative/Sox2-positive cells. l, m: Co-immunodetection of GLAST (green),

Figure 11 .

 11 Figure 11. Expression of NPC markers in the human SVZ. a-d: Double immunofluorescent staining of nestin (green) and Sox2 (magenta) in the SVZ showing the ribbon (rb) of nestin/Sox2-co-expressing cells (arrows), separated from the ependymal layer (arrowheads) by the hypocellular gap (G). e-h:Vimentin (white) is expressed in ependymal cells (plain arrowheads), ribbon cells (arrows) and parenchymal astrocytes (crossed arrows in e). Note the variable levels of vimentin expression in ependymal cells, some of which express low to undetectable levels of the protein (empty arrowheads). The ribbon contains a population of cells that co-express nestin, Sox2 and vimentin (arrows, f-h). i-l: GLAST (white) is expressed in ependymal cells (arrowheads) and is enriched in the gap and ribbon. The ribbon contains a population of cells that co-express nestin, Sox2 and GLAST (arrows, j-l). m-p: GFAP (white) is enriched in the gap and ribbon. The ribbon contains a population of cells that co-express nestin, Sox2 and GFAP (arrows, n-p). GFAP is also expressed in parenchymal astrocytes (crossed arrows in m) but is absent from most ependymal cells (empty arrowheads). Rare ependymal cells express GFAP (plain arrowhead, n-p). Nuclei were counterstained with Hoechst

Figure 12 .

 12 Figure12. Expression of NPC markers in the dentate gyrus of the human hippocampus. a: Double immunofluorescent staining of nestin (green) and Sox2 (magenta). In the dentate gyrus, Sox2 was not detected and nestin was restricted to capillaries (asterisks). Note that Sox2 and nestin were readily detectable along the ventricular border of the temporal horn of the lateral ventricle on the same section (inset). Arrows point to nestin/Sox2 co-expressing cells; the arrowhead points to Sox2positive/nestin-negative ependymal cells. b: Immunodetection of GFAP (white) reveals the presence of numerous astrocytes (arrows). c: Vimentin immunoreactivity (white) was detected in capillaries (asterisks) and small stellate cells (arrows). Nuclei were counterstained with Hoechst (blue). DG, dentate gyrus. LV, lateral ventricle. Scale bars = 50 µm.

  1.1 STEM CELLS In the 1960s the work from James Till and Ernest McCulloch for the first time introduced the concept of developmental hierarchies of cells, where a single stem cell can give rise to multiple differentiated offspring

5: Effects of reproduction hormones on adult neurogenesis. Gonadal

	steroids negatively affect cell
	proliferation in the OB, and
	enhance it in the DG. Prolactine
	is	known	to	stimulate
	neurongenesis in the OB. DG,
	dentate	gyrus;	HYP,
	hypothalamus; OB, olfactory
	bulbs; RMS, rostral migratory
	stream; SVZ, subventricular
	zone.

  Animal studies were approved by the Institutional Ethics Committee for the Care and Use of Experimental Animals of the University of Lille. All experiments were performed in accordance with the guidelines for animal use specified by the European Union Council

	4.1 ANIMALS
	Chapter 4
	Directive of September 22, 2010 (2010/63/EU). Tissues were obtained from female Sprague-
	Materials and Methods Dawley rats (Janvier Laboratories, Saint Berthevin, France). GnRH neurons were sorted from
	transgenic female Wistar rats, kindly provided by Prof. A. Herbison (University of Otago,

Table 1 .

 1 Primary antibodies used in the study.

			Host &		Reference
	Antigen	Clonality	Dilution	(Company)	Immunogen
						Peptide corresponding to amino acids 277-293 of
	Sox2		goat polyclonal	1/300	sc-17320 (Santa Cruz)	human Sox2 (YLPGAEVPEPAAPSRLH), affinity-purified serum (Poché, Furuta, Chaboissier, Schedl, & Behringer,
						2008).
	Nestin  † mouse monoclonal	1/400	MAB1259 (R&D Systems)	NS0 mouse myeloma cell line transfected with human N estin
					MAB353 clone
	Nestin	‡	mouse		rat-401
			monoclonal	1/500	(Millipore)	Nestin purified from embryonic rat spinal cord
	Vimentin	mouse monoclonal	1/300	M0725 clone V9 (Dako)	Purified vimentin from porcine eye lens
	Vimentin	chicken polyclonal	1/500	AB5733 (Millipore)	Recombinant Golden Syrian hamster vimentin
	GFAP		rabbit polyclonal	1/500	Z0334 (Dako)	GFAP isolated from cow spinal cord
	GFAP		mouse monoclonal	1/500	MAB3402 (Chemicon)	Purified glial filament
	GLAST		rabbit polyclonal	1/300	ab416 (Abcam)	Synthetic peptide corresponding to amino acids 522-541 of rat EAAT1 (PYQLIAQDNEPEKPVADSET) $ .
	Ki67		rabbit polyclonal	1/100	ab15580 (Abcam)	Synthetic peptide conjugated to KLH corresponding to amino acids 1222-1234 of human Ki67 (C-EDLAGFKELFQTP).
			mouse		M0814 clone
	CD68		monoclonal	1/300	KP1 (Dako)	Lysosomal fraction of human lung macrophages
	NeuN		mouse monoclonal	1/300	MAB377 (Chemicon)	Purified cell nuclei from mouse brain
	VIP		goat polyclonal	1/300	sc-21041 (Santa Cruz)	16-Residue synthetic peptide representing the carboxy-terminal domain of human VIP from amino acids 125-140
	 † antibody used for immunostainings on human tissue; ‡ antibody used for immunostainings
	on rodent tissue. CD68, cluster of differentiation 68; EAAT1, Excitatory Amino Acid
	Transporter 1 (synonymous GLAST, Glutamate-Aspartate Transporter); GFAP, Glial Fibrillary
	Acidic Protein; NeuN, Neuronal Nuclei; Sox2, SRY (sex determining region Y)-box 2; VIP,

Table 2 .

 2 Secondary antibodies used in the study.

	Species	Conjugated Dilution	Reference (Company)
	Donkey anti-mouse IgG Alexa 568 1/500	A10037 (Molecular Probes)
	Donkey anti-mouse IgG Alexa 488 1/500	A21202 (Molecular Probes)
	Donkey anti-rabbit IgG	Alexa 568 1/500	A10042 (Molecular Probes)
	Donkey anti-rabbit IgG	Alexa 488 1/500	A21206 (Molecular Probes)
	Donkey anti-goat IgG	Alexa 568 1/500	A11057 (Molecular Probes)
	Donkey anti-goat IgG	Alexa 647 1/500	A21447 (Molecular Probes)
	Donkey anti-chicken IgG Alexa 488 1/500	703-545-155 (Jackson Immunoresearch)

Table 3 .

 3 Summary of the expression of NPC markers in the adult human hypothalamus, SVZ, parenchyma and hippocampal dentate gyrus.

				Hypothalamus				SVZ		Parenchyma	Dentate gyrus
		ependy mal cells	gap	ribbon cells	buds	tany cytes	SCh	ependy mal cells †	gap	ribbon cells	buds	endothe lial cells	astro cytes ‡	endothe lial cells	astro cytes ‡
		+		+	+		+
	Sox2	(84.5 ±	+	(29.0 ±	(58.3 ±	+	(30.6 ±	+	+	+	+	-	-	-	-
		2.3%)		5.4%)	6.1%)		4.0%)
		+		+	+		+
	nestin	(38.1 ±	+	(37.0 ±	(67.7 ±	+	(16.4 ±	+/--	+	+	+	+	-	+	-
		4.1%)		3.7%)	4.9%)		1.3%)
		+		+	+		+
	vimentin	(83.6 ±	+	(49.0 ±	(57.6 ±	+	(38.8 ±	++/-	+	+	+	+	+/-	+	+/-
		3.8%)		2.7%)	5.7%)		3.4%)
		+		+	+		+
	GLAST	(97.1 ±	++	(80.6 ±	(97.6 ±	+	(28.0 ±	+	++	+	+	-	+/-	-	-
		0.7%)		3.6%)	1.1%)		7.5%)
		+		+	+		+
	GFAP	(60.9 ±	++	(78.0 ±	(92.6 ±	+	(42.7 ±	+/-	++	+	+	-	+	-	+
		8.0%)		9.5%)	3.0%)		13.0%)
	† Ependymal cells that line the lateral ventricles are more rarely nestin-immunoreactive (+/--)
	but show higher vimentin immunoreactivity (++/-) compared to ependymal cells of the third
	ventricle. ‡ In the parenchyma lying behind the hypocellular gap of the third and lateral
	ventricles, and in the hippocampal dentate gyrus, vimentin and/or GLAST are detected in
	small stellate cells at a lower frequency than GFAP-positive cells (+/-), suggesting that they
	are expressed by subpopulations of astrocytes. SCh, suprachiasmatic nucleus; SVZ,
	subventricular zone of the lateral ventricles. Quantification of the stainings in the
	hypothalamus is given as mean ± SEM.

Table 4 .

 4 Summary of the expression of NPC markers in the adult hypothalamus of the mouse, rat and grey mouse lemur.

		Mouse hypothalamus		rat hypothalamus		Mouse lemur hypothalamus
		ependy	tany	parenchy	endothe	ependy	tany	parenchy	endothe	ependy	tany	parenchy	endothe
		mal cells	cytes	mal cells	lial cells	mal cells	cytes	mal cells	lial cells	mal cells	cytes	mal cells	lial cells
	Sox2	+	+	+	-	+	+	+	-	+	+	+	-
	nestin	+ rare †	+	-	+/-	+ rare †	+	-	+	NA	NA	NA	NA
	vimentin	+	+	-	+/-	+	+	+ rare ‡	+	+	+	+ rare ‡	+
	†

  The same cells are pointed in main panels (a, h, o) and high-magnification views. Empty arrows in main panels point to vimentin-(a), GLAST-(h) or GFAP-expressing cells (o) in the parenchyma behind the ribbon. Stars indicate capillaries whose endothelial cells express vimentin (a) but lack GLAST (h) and GFAP (o). Nuclei were counterstained with Hoechst (blue). 3V, third ventricle; ep, ependymal layer; G, hypocellular gap; rb, ribbon. Scale bars = 50 µm in a, h, o; 20 µm in all other panels.

Résumé substantiel

This work was supported by a doctoral fellowship from the University of Lille to G.P., Inserm grant U1172, the Agence Nationale de la Recherche (ANR, France) ANR-15-CE14-0025 (to V.P.) and ANR-16-CE37-0006 (to M.M. and V.P.), the Association pour l'Etude des Anomalies Congénitales (to A.S.) and European Regional Development Fund (ERDF) attributed to Normandy region. We thank Elisabeth Wyart for her help with the immunolabeling of rat sections, Maurice De Meulaere (Laboratory of Anatomy, School of Medicine, Lille) for his skillful help during dissection of human brains and Philippe Chan Tchi Song from the PISSARO proteomic platform (Rouen) for mass spectrometry analysis.

Abbreviations Chapter 2

The central control of reproduction Gpr44 (or PtgdrII) (Rn02349421_s1); PtgdrI (Rn00824628_m1); Ptgds (Rn00564605_m1).

Control housekeeping genes: Actb (Rn00667869_m1) and Rn45s;H (Rn03928990_g1).

Gene expression data were analyzed using SDS 2.4.1 and Data Assist 3.0.1 software (Applied Biosystems) with R45S and actin as control housekeeping mRNAs following a standardized procedure.

STEREOTACTIC BRAIN INFUSION OF BWA868C

Acute injections

Female rats were i.p. injected with BrdU at P8. A group (n = 4) was sacrificed 2 hours after the injection. The other animals were placed in a stereotaxic frame (Kopf Instruments, California) under anesthesia (isoflurane), and 2 burr holes were drilled 0.5mm apart from the bregma, according to a rat brain atlas (Paxinos and Franklin, 1987). A 10 µl Hamilton syringe was slowly inserted into the POA (5.2 mm deep relative to the dura), and 1 µl of solution containing either vehicle (saline solution 0.9%) either BWA868C 10 nM was injected bilaterally using an infusion pump (KD Scientific, Holliston, MA) over 5 min. Animals were killed 24 hours later and analysed for morphological association between GnRH neurons and BrdU + cells (see chapeter 4.6.1.1).

Long-term injections

Female rats were i.p. injected with BrdU at P8, placed in a stereotaxic frame (Kopf Instruments, California) under anesthesia (isoflurane), and one burr hole was drilled at the Chapter 5

Cell neogenesis in the postnatal hypothalamus in the female rat: involvement in sexual maturation

To address the functional role of this infantile wave of astrogenesis in the central control of reproduction, proliferation in the POA was selectively inhibited in infantile female rats by stereotaxic injection of microparticles loaded with the antimitotic drug paclitaxel (Fig. 4a-c).

Paclitaxel-treated animals exhibited a marked delay in the onset of puberty, as attested by late vaginal opening and first estrus compared to control animals (Fig. 4d-f). These data suggest that astrogenesis in the POA during the infantile period is required for the timely onset of puberty. To this end, BWA868C was stereotaxically injected in the POA of P7 female rats that had just received an injection of BrdU (Fig. 12a). Three experimental groups were analysed: a group of animals sacrificed 2 hours after the BrdU injection (no stereotaxic injection), a group of animals stereotaxically injected with saline and sacrificed 24 hours later, and a group of animals stereotaxically injected with 10 nM BWA868C and sacrificed 24 hours later. We then quantified the morphological association between GnRH neurons and BrdU + cells in the three conditions.

Two hours after the BrdU injection, 11.79 ± 0.89% (n = 4 animals) GnRH neurons were associated with BrdU + cells (Fig. 12b). This proportion increased to 27.16 ± 3.14% (n = 5 animals) in animals injected with saline and sacrificed 24 hours later. However, this increase was not seen in animals injected with BWA868C (15.79 ± 1.48% GnRH neurons associated with BrdU + cells; n = 5 animals).

Quantification of the total number of BrdU + cells in the POA showed that the population of BrdU + cells increased between 2 hours and 24 hours (Fig. 12c). Treatment with BWA868C did not affect the total number of BrdU + in the POA, suggesting that the decreased association between GnRH neurons and BrdU + cells was not due to a general depletion of the BrdU + cell population but rather to an inhibition of the recruitment of BrdU + cells in the vicinity of GnRH neurons.

Chapter 7

The stem/progenitor cell niche in the adult human hypothalamus

The Journal of Comparative Neurology A comparative study of the neural stem cell niche in the adult hypothalamus of human, mouse, rat and grey mouse lemur (Microcebus murinus)

Abstract

The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Two niches have been extensively studied in various mammalian species including humans, the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Recently, studies conducted mainly in rodents have identified a third neurogenic niche in the adult hypothalamus. In order to evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescence labeling to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus and compared them with the mouse, rat and a non-human primate species, the grey mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four distinct populations of cells that express the five NPC markers: i) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap, similar to that found along the lateral ventricles, ii) ependymal cells, iii) tanycytes, which line the floor of the third ventricle in the tuberal region, and iv) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, coexpression of NPC markers is primarily restricted to tanycytes, and these species lack a ventricular ribbon. Our work thus identifies four cell populations with the antigenic profile of NPCs in the adult human hypothalamus, of which three appear specific to humans.

Rodent

The Sox2 antibody (goat polyclonal # sc-17320 from Santa Cruz) yielded a strong staining in the SVZ (not shown) and in the hypothalamus (Fig. 9a, e, i, Fig. 10a,e) that recapitulated expression patterns previously described with other antibodies in the mouse (Haan et al., 2013;Li et al., 2012) and rat (Bennett, Yang, Enikolopov, & Iacovitti, 2009;[START_REF] Hoefflin | Neuronal expression of SOX2 is enriched in specific hypothalamic cell groups[END_REF]. It also replicates the pattern of mRNA expression determined by in situ hybridization in the adult mouse brain (Allen brain atlas; http://mouse.brainmap.org/experiment/show/77280331). This antibody detects a single band at 34 kDa in Western blots of mouse embryonic stem cell lysates (manufacturer's technical information).

Moreover, it yields an immunostaining in a mouse mesenchymal stem cell line stably overexpressing Sox2 by retroviral infection while no staining is observed in non-infected cells [START_REF] Ding | Over-expression of Sox2 in C3H10T1/2 cells inhibits osteoblast differentiation through Wnt and MAPK signalling pathways[END_REF].

The nestin antibody stained tanycytes and capillaries (Fig. 9b, f, j, Fig. 10f), as previously reported in the mouse [START_REF] Alliot | Pericytes and periendothelial cells of brain parenchyma vessels co-express aminopeptidase N, aminopeptidase A, and nestin[END_REF]Haan et al., 2013;Lee et al., 2012) and rat (Bennett et al., 2009;[START_REF] Takamori | Nestin-positive microglia in adult rat cerebral cortex[END_REF]. This expression profile replicates the pattern of mRNA expression determined by in situ hybridization in the adult mouse brain (Allen brain atlas; http://mouse.brain-map.org/experiment/show/1387). This antibody detects a doublet in the 200-220 kDa range on Western blots of rat brain homogenates [START_REF] Shigemoto-Mogami | Microglia enhance neurogenesis and oligodendrogenesis in the early postnatal subventricular zone[END_REF]. Moreover, rat cardiac ventricular fibroblasts infected with a lentivirus containing a shRNAmir directed against nestin show reduced immunocytofluorescent and immunoblot signals [START_REF] Béguin | Nestin expression is lost in ventricular fibroblasts during postnatal development of the rat heart and reexpressed in scar myofibroblasts[END_REF].

Vimentin was detected in ependymal cells, tanycytes and capillaries (Fig. 9c, g, inset in j, Fig. 10b,h), as previously reported in the mouse (Langlet, Mullier, Bouret, Prevot, & Dehouck, 2013;[START_REF] Mullier | Differential distribution of tight junction proteins suggests a role for tanycytes in blood-hypothalamus barrier regulation in the adult mouse brain[END_REF] and rat (Pérez-Martín et al., 2010). The two anti-vimentin antibodies used yielded a similar expression profile. This expression profile replicates the pattern of mRNA expression determined by in situ hybridization in the adult mouse (Allen brain atlas; http://mouse.brainmap.org/experiment/show/79907904) and rat brain [START_REF] Helfer | A neuroendocrine role for chemerin in hypothalamic remodelling and photoperiodic control of energy balance[END_REF]. The Dako antibody detects a single band at 54 kDa on Western blots of rat hepatic stellate cell lysates [START_REF] Mezaki | Differential increases in the expression of intermediate filament proteins and concomitant morphological changes of transdifferentiating rat hepatic stellate cells observed in vitro[END_REF].

While nestin and Sox2 expression was mainly restricted to the proximal border of the third ventricle, an exception was seen in the suprachiasmatic nucleus (Fig. 2a, b, Fig. 7a), which contained a population of small stellate nestin/Sox2 co-expressing cells at distance from the ventricle (Fig. 7b). The five NPC markers were detected in the suprachiasmatic nucleus (Table 3). Eleven ± 2.0% of suprachiasmatic cells co-expressed nestin and Sox2, and among this population, 74.7 ± 10.4% expressed vimentin (Fig. 7c-e), 80.9 ± 5.5% expressed GLAST (Fig. 7f-h) and 65.6 ± 10.6% expressed GFAP (Fig. 7i-k), implying that at least 21% of nestin/Sox2-positive cells also express vimentin, GLAST and GFAP. Since Sox2 expression was previously reported to co-localise with neuronal markers in the rodent suprachiasmatic nucleus [START_REF] Hoefflin | Neuronal expression of SOX2 is enriched in specific hypothalamic cell groups[END_REF], we performed a triple staining for Sox2, GLAST and NeuN to evaluate whether NPC markers could be found in NeuN-expressing neurons. NeuN was only expressed in a small fraction of suprachiasmatic neurons (Fig. 7m), as already observed in rats [START_REF] Geoghegan | A novel site of adult doublecortin expression: neuropeptide neurons within the suprachiasmatic nucleus circadian clock[END_REF]. We found no evidence of GLAST/Sox2 co-expression in NeuN-positive neurons (Fig. 7l,m).

Proliferative cells, identified by their expression of the cell cycle marker Ki67, were detected near the third ventricle border in variable proportions in 6/7 hypothalami. We identified only two nestin/Sox2-positive cells that co-expressed Ki67 in one specimen in the ribbon and at the level of a bud (Fig. 8a-e). Most Ki67-labeled cells co-expressed the microglia marker CD68 (Fig. 8f-h).

Comparison of the stainings between the different cell populations showed that the fraction of Sox2-positive cells that co-expressed nestin was higher in the ribbon (63.2 ± 3.4%) compared to ependymal (39.3 ± 4.1%) and suprachiasmatic cells (37.3 ± 4.8%) (ANOVA with Tukey post-hoc test, p<0.01). However, when the population of nestin/Sox2-positive cells was considered, the fraction that co-expressed vimentin, GFAP or GLAST was not different between the ribbon, the ependymal layer and the suprachiasmatic nucleus (Kruskal-Wallis

One Way Analysis of Variance on Ranks with Dunn's post-hoc test, p>0.05). In buds, the population of nestin/Sox2-positive cells was enriched (ANOVA with Tukey post-hoc test, p<0.001) and the fraction of nestin/Sox2-positive cells that also expressed GLAST was higher compared to the ribbon and the suprachiasmatic nucleus (Kruskal-Wallis One Way Analysis of Variance on Ranks with Dunn's post-hoc test, p<0.05).

Chapter 8

Discussion and Conclusions behaviour. We therefore explored whether cells born just before ovulation may serve a role in this process. We evaluated how cell survival may be influenced in case of pregnancy. We could indeed observe that more cells born before ovulation in the MPO survive if a pregnancy occurs.

The MPO is the region involved in the control of maternal behaviour; this develops around parturition and involves recognition, emotionality and attachment, necessary to provide nutrition and protection to the pups and hence allow their survival. In this complex picture, the MPO represents the central hub in the neural networks that organises the different components of maternal behaviour in response to pregnancy hormones (gonadal steroids, prolactin…) (Brunton & Russel, 2015). Expression of maternal behaviour is triggered by restructuring of hypothalamic neurons. This process is quite dynamic as mothers need to rapidly adapt to the changing developmental needs of the offspring. To this aim, at the level of the MPO there is an increased neural complexity by the end of pregnancy: MPO neurons are activated in order to signal to dopaminergic neurons of the ventral tegmental area, involved in the reward circuitry; neuronal connectivity and primary dendritic branches increase, as well as neuronal firing activity; at the same time the expression of receptors for prolactin and oestrogens, crucial hormones for regulating the progress and success of pregnancy, is upregulated [START_REF] Bridges | Long-term alterations in neural and endocrine processes induced by motherhood in mammals[END_REF][START_REF] Brunton | The expectant brain: adapting for motherhood[END_REF][START_REF] Champagne | Plasticity of the Maternal Brain Across the Lifespan[END_REF]. In such a complex context, our results raise the possibility that addition of new cells to the MPO may be an additional mechanism of plasticity involved in the maternal brain adaptation to pregnancy.

Open questions

Which factors influence cell proliferation in the POA during the oestrous cycle and pregnancy?

The oestrous cycle is characterised by specific fluctuations in hormonal levels including gonadal steroids and pituitary hormones. These same hormones are then finely modulated to ensure pregnancy. Therefore, one of the next steps to better understand the process of adult cell neogenesis in the rat MPO will be to characterise which of these hormones is Bibliography