Brain fluid transport is mediated by cerebrospinal fluid (CSF) circulation. This process plays important roles in the function, therefore the health of brain network. For instance, it regulates the intracerebral pressure equilibrium, dynamic distribution of energy fuels such as glucose and lactate, and notably for the removal of waste molecules like amyloid beta. Disturbance in brain flow transport is being recognized as a factor exacerbating neurological pathologies like Alzheimer's disease.

As a major type of glial cells, astrocytes are distributed throughout the brain and strategically located between cerebral vasculature and brain parenchyma. The discovery of the critical involvement of astrocytes in brain fluid transport has led to the emergence of the concept of the glymphatic system. It has been suggested that astrocyte volume dynamics, via regulating the extracellular space, shapes the brain flow transport. Water flux underlies astrocyte volume regulation. Astrocytes express the bidirectional water channel Aquaporin 4 (AQP4) that has been suggested to modulate astrocyte volume change. However, the exact role of AQP4 in astrocyte volume regulation has been debated, causing unresolved controversy on the mechanistic understanding of brain fluid transport and the glymphatic system.

My PhD study acutely discerns the role of AQP4 in astrocyte volume regulation in basal conditions and in response to osmotic changes in brain parenchyma in mouse models.

Interdisciplinary approaches have been adopted during the PhD study, including in vivo chemical astrocyte labeling, ex vivo cellular fluorescence imaging in acute brain slices, in vivo fiberphotometry recording in freely moving animals and by diffusion weighted magnetic resonance imaging (DW-MRI). The obtained results (Chapter I) lead to the finding that AQP4 tonically sculpts astrocyte volume homeostasis, perturbing of which interferes with brain water diffusion. My following study (Chapter II) probed the potential mechanisms implicated in the dysregulated astrocyte signals and volume regulations in pathological β amyloid conditions. A hyper-regulation in astrocyte calcium signaling was observed that could potentially disturb the activity-dependent astrocyte volume dynamics. The possibility that targeting astrocytes AQP4 in vivo to facilitate amyloid clearance is being tested in an Alzheimer's disease mouse model.

These results together shed light on the fundamental role of AQP4 in tuning astrocyte volume and therefore the extracellular route for molecular diffusion, helping to understand brain fluid transport and its implication in health and neurological disorders.

The scientific knowledge from my PhD study has also fostered collaborative works for understanding I) brain astrocyte activity remodeling in obesity-associated metabolic syndromes.

The results of this part contribute to the discovery that the calcium signaling of hypothalamic astrocytes undergoes heterogeneous remodeling in anatomically defined nuclei implicated in systemic metabolism regulation. This work helps to delineate the important participation of astrocytes in energy balance control; and II) the molecular mechanisms underlying the trafficking of calcium-permeable transient potential receptor ion channels. This work suggests testable molecular targets for interrogating the calcium signaling mechanisms in nervous cells including astrocytes.

Résumé (Français)

Le transport du flux cérébral est médié par la circulation du liquide céphalo-rachidien (LCR).

Ce processus joue un rôle important dans la fonction, et donc dans l'homéostasie du cérébrale.

Il régule l'équilibre de la pression intracérébrale, la distribution dynamique des carburants énergétiques tels que le glucose et le lactate, et notamment l'élimination des déchets comme la bêta-amyloïde. La perturbation du transport du flux cérébral est reconnue comme un facteur aggravant des pathologies neurologiques comme la maladie d'Alzheimer.

Etant un type majoritaire de cellules gliales, les astrocytes sont répartis dans tout le cerveau et stratégiquement situés entre le système vasculaire et le parenchyme cérébral. La découverte de l'implication critique des astrocytes dans le transport des fluides cérébraux a conduit à l'émergence du concept de système glymphatique. Il a été suggéré que la dynamique du volume astrocytaire, via la régulation du volume de l'espace extracellulaire, façonne le transport du flux cérébral. Le flux d'eau cellulaire sous-tend la régulation du volume des astrocytes. Les astrocytes expriment le canal d'eau bidirectionnel Aquaporine 4 (AQP4) qui a été suggéré comme modulateur du changement de volume astrocytaire. Cependant, le rôle exact de l'AQP4 dans la régulation du volume des astrocytes a été débattu, provoquant une controverse non résolue sur la compréhension mécanistique du transport des fluides cérébraux et du système glymphatique.

Durant mon doctorat, j'ai étudié le rôle de l'AQP4 dans la régulation du volume astrocytaire dans des conditions basales et en réponse à des manipulations environnementales dans le parenchyme cérébral de modèles murins. Des approches interdisciplinaires ont été développées ou adoptées au cours de cette étude, notamment via ciblage chimiogénique in vivo des astrocytes, d'imagerie par fluorescence cellulaire ex vivo dans des tranches de cerveau ex-vivo, d'enregistrement in vivo par fibrophotométrie chez des animaux en mouvement libre et par imagerie à résonance magnétique de diffusion (DW-MRI). Les résultats obtenus (chapitre I) conduisent à la découverte que l'AQP4 sculpte de manière tonique l'homéostasie du volume des astrocytes dans le parenchyme cérébral, dont la perturbation interfère de manière significative avec le transport du liquide cérébral. J'ai ensuite étudié (chapitre II) a sondé les mécanismes potentiels impliqués dans les dérégulations du volume astrocytaire dans des conditions de pathologies à bêta-amyloïdes. Dans ces conditions, une hyper-régulation de la signalisation calcique astrocytaire a été observée, ce qui pourrait perturber la dynamique du volume astrocytaire dépendant de l'activité. Afin de moduler et faciliter la clairance amyloide dans un contexte murin de la maladie d'Alzheimer, l'activité de l'AQP4 a été impactée in vivo afin de moduler l'homéostasie volumique astrocytaire. Ensemble, ces résultats mettent en lumière le rôle fondamental de l'AQP4 dans le réglage du volume des astrocytes, et donc de la voie extracellulaire du flux cérébral, aidant ainsi à concilier les aspects jusqu'ici débattus du système glymphatique et son implication dans la santé et les troubles neurologiques.

Les connaissances scientifiques issues de mon doctorat ont également favorisé des travaux collaboratifs pour comprendre I) le remodelage de l'activité des astrocytes cérébraux dans les syndromes métaboliques associés à l'obésité. Les résultats de cette partie contribuent à la découverte que la signalisation calcique des astrocytes hypothalamiques subit un remodelage hétérogène dans des noyaux anatomiquement définis impliqués dans la régulation du métabolisme systémique. Ces travaux ont permis de mettre en évidence l'importante participation des astrocytes au contrôle de la balance énergétique; et II) les mécanismes moléculaires sous-jacents au trafic des canaux ioniques récepteurs potentiels transitoires perméables au calcium. Ce travail suggère des cibles moléculaires testables pour interroger les mécanismes de signalisation du calcium dans les cellules nerveuses, y compris les astrocytes. Chapter I: Investigating the role of Aquaporin 4 in astrocyte volume regulation.

List of abbreviations

List of figures

Water is the most prevalent molecule in living creatures, representing the major body component from the vegetal organisms to mammalian animals. For instance ~60% of the body mass of the adult human is made out of water [START_REF] Popkin | Water, hydration, and health[END_REF]. Water is essential for living organism vitality as just a few days without it will prove to be lethal. Water plays crucial roles in physiology to maintain body internal environment. It is indispensable for thermoregulation to sustain a narrow range of temperature highly necessary for routine metabolism and enzyme activities. With very high heat capacity (~4184 J⋅kg -1 ⋅K -1 at 20 °C), water excellently stores heat, and its oscillation from one body compartment to another helps to maintain the temperature constancy. Water is also required as a medium for chemical reactions between, enzymes, ions and proteins due to its heat storage capacity and its stable pH neutrality of 7.0.

So water is a key resource for all metabolic features in living organisms.

Another key feature for water is to protect and lubricate tissues, from the amniotic fluid for a pregnant woman's baby, the pleural fluid lubricating lungs for breath, to the cerebrospinal fluid surrounding the brain and the spinal cord.

Importantly, water serves as a transportation vehicle. Ions, amino acids, vitamins, minerals, and sugars all need be transported by water. Water circulation also appears indispensable for the clearance of waste molecules in the body thereby preventing the build-up of potential toxicities.

I.1 -The cerebrospinal fluid is important for brain homeostasis

Water is a rudimental factor sustaining body homeostasis. Particularly, the central nervous system (CNS) is globally surrounded with the cerebral spinal fluid (CSF). The CSF is a liquid that is found within the tissues of the CNS of all vertebrates, filling all the ventricular spaces in the brain, and the central canal of the brainstem and the spinal cord. Fluid presence in the brain was known since ancient Greece times. Hippocrates described it as "water surrounding the brain" when studying congenital hydrocephalus, while Galen, the first anatomist, was referring to it as an "excremental liquid" [START_REF] Hajdu | A note from history: Discovery of the cerebrospinal fluid[END_REF]. But discovery of the CSF is attributed to Emmanuel Swedenborg. In his manuscript: «The brain: considered anatomically, physiologically and philosophically" he referred to the CSF as a "spirituous lymph" and a "highly gifted juice" that is distributed from the roof of the fourth ventricle to the medulla oblongata (reference). We have to wait until 1912 for a clear description of the chemical properties of the CSF by William Metrezegat [START_REF] Hajdu | A note from history: Discovery of the cerebrospinal fluid[END_REF]

I.1.1 -Cerebrospinal fluid synthesis

The CSF is synthesized by ependymal cells located in the choroid plexus from filtrated blood plasma. The filtrated fluid pass through fenestrated capillaries in the choroid plexus to enter the interstitial space. And then from to the epithelial cell aligned in the choroid plexus in the ventricles. Additional ions and molecules join the CSF from the plasma by being filtered by ionic transporters or co-transporters in the epithelial cells. Ionic transporters will filtered blood plasma and by exchanging different ions with different co transporters and the complete CSF will be then transported into the ventricular systems (Fig. 1) [START_REF] Mitchell | Novel advances in monitoring and therapeutic approaches in idiopathic intracranial hypertension[END_REF][START_REF] Rasmussen | Fluid transport in the brain[END_REF]. The CSF is composed of all necessary physiological ions, essential proteins and amino acids (Table 1). The CSF will then circulates within the ventricles of the brain and to the subarachnoid space and will enter the brain parenchyma via the periarterial space and leave it from the periveinous space, and be drained away by lymph nodes located in the subarachnoid space, as well as the spine. [START_REF] Mitchell | Novel advances in monitoring and therapeutic approaches in idiopathic intracranial hypertension[END_REF].

I.1.2 -Cerebrospinal fluid functions

The CSF serves fundamental functions underpinning the function, integrity and waste clearance of the central nervous system [START_REF] Cousins | The blood-CSF-brain route of neurological disease: The indirect pathway into the brain[END_REF]. As a delicately filtrated fluid, CSF sets the total ionic environment of the neural circuits, being extremely crucial for the operation of information processing in the nerve system [START_REF] Sakka | Anatomy and physiology of cerebrospinal fluid[END_REF]. Equally essential for the CNS function is the efficient diffusion of metabolic molecules throughout the neuron-glia networks [START_REF] Magistretti | Lactate in the brain: from metabolic end-product to signalling molecule[END_REF]. For instance, as a highly energy-consuming organ, the brain relies exclusively on circulating glucose as an energy fuel, where the normal functionality of CSF tightly mediates the extracellular diffusion of glucose and its metabolic derivatives including the lactate [START_REF] Magistretti | Lactate in the brain: from metabolic end-product to signalling molecule[END_REF]. Furthermore, as a wide spread liquid buffer, CSF critically protects the very complex yet super fragile nerve tissues [START_REF] Spector | A balanced view of the cerebrospinal fluid composition and functions: Focus on adult humans[END_REF]. By being submerged by CSF, the brain exists in a neutral buoyancy enabling the brain to maintain its density and therefore its function during daily behaviors. or into the subarachnoid space at the cisterna magna (4). CSF flows within the subarachnoid space and is cleared via the arachnoid villi into the dural venous sinuses (5). (Figure and legend from (Cousins et al., 2022).

I.2 -Astrocytes, a key component in the Central nervous system

In the CNS, two type of cell are prevalent: the neurons and the glial cells. Neurons are electrically excitable cells, while glial cells are electrically non-excitable [START_REF] Verkhratsky | Physiology of Astroglia[END_REF]. The glial cells include oligodendrocytes, ependymal cells, microglia and astrocytes. They represent ~50 % of the cell population in the mammalian CNS, with an approximate of ~100 billion glial cells in the human brain [START_REF] Von Bartheld | The search for true numbers of neurons and glial cells in the human brain: A review of 150 years of cell counting[END_REF]. Among glial cells, astrocytes play important functions in regulating the development, function and homeostasis of the brain. My current study has been focused on brain astrocytes in mouse models.

I.2.1 -Historic

The initial description on glial cells neuroglia was from the pathologist Rudolf Virshow . In 1856, he introduced the term "Nervenkitt" (neuroglia) as a connective substance which forms in the brain, in the spinal cord and in the higher sensory nerve, in which the component of the nervous system are embedded. (Fig. 3). They were first thought of being mere structural cells, hence got their name from the word "glue", being referred as neuroglia. They were then considered as a mere glue holding neurons together. Astrocytes were found to be a major type of glial cells and initially observed in the 19th century along neurons (Verkhasky and Butt, 2013). The name astrocyte was introduced by Michaël von Lenhossék in 1895, coming from the greek astron and kytos, astron meaning star and kytos hollow vessel, later cell, so all together a star like cell.

I.2.2 -Morphology, localisation and organization

They Astrocytes represent from ~20 to ~40% of the cell population of the brain and the most abundant type of glial cells in the CNS [START_REF] Verkhratsky | Glial Physiology and Pathophysiology[END_REF]. Astrocyte are organized as individual domains and are interconnected via gap junctions between them as forming a syncytia network that help for involved in information propagation. Astrocytes are very heterogeneous in terms of following their functions, or their localization and molecular profiles.

By general localization, they can be divided in two main subtypes: protoplasmic astrocytes, or fibrous astrocytes (Fig. 4). Protoplasmic astrocytes are found in the grey matter. They exhibit stem branches that give rise to many fine branch process. Fibrous astrocytes are found in the white matter and show a long fiber like processes morphology [START_REF] Sofroniew | Astrocytes: Biology and pathology[END_REF]. [START_REF] Verkhratsky | Glial Physiology and Pathophysiology[END_REF] Astrocytes are widely distributed in the CNS, present in both grey and white matter. They are organized in non-overlapping domains (Fig. 5). Protoplasmic astrocytes tend to have non overlapping domain in gray matter, so that only the most distal tips of their process are in contact to each other. Astrocytes communicate between each other by gap junctions expressed at the extremity of each domain [START_REF] Wilhelmsson | Redefining the concept of reactive astrocytes as cells that remain within their unique domains upon reaction to injury[END_REF]. Similar domains seem to exist in white matter but have not been reported extensively. Astrocyte are also aligned along blood vessels and part of the brain blood barrier (BBB) [START_REF] Daneman | The blood-brain barrier[END_REF]. Interdigitation of fine cellular processes between adjacent astrocytes is shown here in a threedimensional reconstruction of astrocytes in the dentate gyrus of the hippocampus of a mouse. Note the relatively limited overlap zone between adjacent astrocytes (shown in yellow) [START_REF] Wilhelmsson | Redefining the concept of reactive astrocytes as cells that remain within their unique domains upon reaction to injury[END_REF].

I.2.3 -Functions

As one of the major glial cells, astrocytes have very important functions crucial for the homeostasis and the well-being of the CNS. Astrocytes are at close proximity with neuronal synapses and help for neurotransmission and ions and transmitter homeostasis at the synaptic level [START_REF] Dallérac | Versatile control of synaptic circuits by astrocytes: where, when and how?[END_REF]. Astrocytes also provide metabolic support to neurons. Indeed they provide to neurons the necessary nutrients (e.g., glucose, lactate) for the survival and function of neurons [START_REF] Magistretti | Lactate in the brain: from metabolic end-product to signalling molecule[END_REF]. They also act as repairing cells to safeguard the microenvironment of neuropil and respond to pathological injuries. Indeed upon neuronal injury astrocytes are recruited and contribute to the neuronal repair by secreting neurotropic factors (O' Shea et al., 2017).

Hence, by occupying the strategic position in brain circuits, at the interface between blood vessels and neurons, astrocyte can regulate synaptic activity, blood flow as well as energy metabolism. Unlike neurons, astrocytes do not exhibit action potentials, but they display intracellular calcium (Ca 2+ ) signaling, thought to represent their dynamic activity [START_REF] Khakh | Astrocyte calcium signaling: From observations to functions and the challenges therein[END_REF] . They are part of the tripartite synapse, where their fine perisynaptic processes are adjoin the pre-and post-synaptic elements. This strategic position enables them to regulate synaptic activity; the local concentration of synaptic neurotransmitters is dynamically controlled by astrocytes, via either or both the transmitter uptake/recycling and the intracellular Ca 2+ signaling by ion channels, neurotransmitter receptors and transporters. In turn astrocyte modulation of neuronal activity occurs via multiple mechanisms. For instance, neuroactive molecules (ATP, glutamate, lactate, peptides...) have been suggested to be released by astrocytic Ca 2+ signaling to regulate neuronal excitability and synaptic transmission.

Astrocytes also display changes in their morphology and synapse coverage, which set their proximity to synapses, and thus determine astroglial uptake capacity, released molecules availability and contact-mediated signaling [START_REF] Pannasch | Astroglial networks scale synaptic activity and plasticity[END_REF][START_REF] Kim | Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding[END_REF].

Intercellular astroglial networks are mediated by gap junction channels (GJ) composed of connexin (Cx) subunits, Cx30 and Cx43, which enable long-range exchange of ions, neuromodulators and metabolites mediating network-wide signaling and synchronization [START_REF] Pannasch | Emerging role for astroglial networks in information processing: from synapse to behavior[END_REF][START_REF] Chever | Astrocyte control of neuronal synchrony: mind the GAP![END_REF]. Cx also form channels, such as Cx43 hemichannels (HC), pannexin1 (Px1) or P2X7 [START_REF] Chever | Astroglial connexin43 hemichannels tune basal excitatory synaptic transmission[END_REF][START_REF] Abudara | Synaptic Functions of Hemichannels and Pannexons: A Double-Edged Sword[END_REF] channels, instrumental for the release of neuroactive molecules (Fig. 6). These molecules may also be released via vesicular exocytosis, although this remains controversial [START_REF] Fiacco | Multiple Lines of Evidence Indicate That Gliotransmission Does Not Occur under Physiological Conditions[END_REF][START_REF] Savtchouk | Gliotransmission: Beyond Black-and-White[END_REF].. It is noteworthy that these astroglial molecules can be locally produced in the direct vicinity of neighboring synapses or alternatively they can originate from distal site of the astroglial network and be trafficked via GJ towards neuronal activity hotspots to fuel synapses, as shown for glucose and lactate delivery 20

I.3.1 -A brief historic on aquaporin research

Water is the most prevalent molecule in a cell, around 70% of the intracellular composition being water. Cells membrane delimits the interior cytoplasm from the outside of the cells. Cell membrane is made of lipid bilayer that is crucial for the cell integrity, and a strict control of how water can diffuse through a cell membrane is necessary to maintain this cell integrity.

Water can diffuse through plasma membrane by 2 different mechanisms. First by passive diffusion, and secondly via transporters or channels.

For long researchers hypothesized that passive diffusion was not the only mechanism enabling water molecule to cross cell membrane. As some type of cells were exhibiting higher water permeability than others. But for a long time these mechanisms remained unknown.

In 1957 Sidel and Solomon studied the hypothesis that maybe specialized proteins may exit in order to facilitate water diffusion though biological membrane [START_REF] Sidel | Entrance of water into human red cells under an osmotic pressure gradient[END_REF]. In 1984 Macey et al were able to block water selectivity of red blood cell, and revert it by applying a reducing agent [START_REF] Macey | Transport of water and urea in red blood cells[END_REF]. But it was until 1992 that the first water permeable channel could be isolated by Peter Agre's lab, which awarded him the Nobel Prize in 2003. The isolated protein was then first named CHIP 28 for channel forming integral protein, and later renamed Aquaporin 1 [START_REF] Agre | The aquaporin water channels[END_REF].

I.3.2 -Aquaporin classification

The AQP are divided in 13 families from aquaporin 0 to aquaporin 12. They are all expressed ubiquitously in mammalian tissues (Table 2). They can be subdivided in three different subgroup, first the Aquaporins "purely water channel family" (AQP0, 1, 2, 4, 5, 6 and 8); second, the Aquaglyceroporins (AQP3, 7, 9 and 10); and last and relatively new group of aquaporins the Super-aqua-porins (AQP11, 12) [START_REF] Badaut | Aquaporin and brain diseases[END_REF].

AQP Subtypes

Transport molecules Tissue distribution

I.3.3 -The Aquaporin 4 is the major aquaporin in the CNS

The AQP4 is one of the main AQP present in the CNS, expressed mainly on astrocytes.

The AQP-4 is enriched in astrocyte processes in close contact with blood vessels, being a main component of the glymphatic system. AQP4 was first discovered in 1994 in the rat brain [START_REF] Jung | Molecular characterization of an aquaporin cDNA from brain: candidate osmoreceptor and regulator of water balance[END_REF]). Verkman's and Agre's group independently reported that rat brain expresses a specific AQP cDNA. First named mercury insensitive water channel (MIWC) by Verkman's group but later named AQP4 (Nagelhus and Ottersen, 2013) [START_REF] Morton | Alpha(1A)-adrenoceptors mediate contractions to phenylephrine in rabbit penile arteries[END_REF] The polarized localization of AQP4 pool in astrocytes is maintained by anchoring proteins such as syntrophin (Fig. 7). neuroinflammation, osmosensation, cell migration, and Ca 2+ signaling. AQP4 is also required for normal function of the retina, inner ear, and olfactory system. From [START_REF] Nagelhus | Physiological roles of aquaporin-4 in brain[END_REF] I.4 -Glymphatic system, a recent concept for brain fluid circulation

The glymphatic system (GS) concept has emerged over the last decade. This concept is part of the theory of the intracerebral drainage system, and was first proposed by Iliff in 2012 [START_REF] Iliff | A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β[END_REF]. Glymphatic system leans on the principle of fast pathway for inflow of CSF from the periarteriole space, and the outflow of interstitial fluid to the perivenous space (Fig. 8). This system is featured by three dynamically interlinked aspects: (1) there exists a convective flow wide-spread in brain parenchyma (interstitial flow); (2) this flow is driven by the pulsation of infiltrated arterioles; (3) astrocyte AQP4 modulates the kinetics of glymphatic system. Traditionally, it is known that the pulsation of arteriole creates a local convective flow in perivascular space (i.e., the VirchowRobin space). Glymphatic concept extends this scenario to the parenchyma, which is however still in debate [START_REF] Smith | CrossTalk opposing view: Going against the flow: interstitial solute transport in brain is diffusive and aquaporin-4 independent[END_REF]Mestre et al., 2020b). , copyright [2015]). Figure and legend from [START_REF] Nedergaard | Neuroscience. Garbage truck of the brain[END_REF].

I.4.1 -Brain fluid study to glymphatic system

Study of brain fluid transport go along with the study of the brain itself. And the study of brain fluid transport is as old as the later. The earliest notions for brain fluid transport were found in ancient Greece. But those works were descriptive and often contained arbitrary postulates. It was until the renaissance, which is a period that saw the revival of science that led to many discoveries in many key component of the nervous system. Since then, the understanding for the brain fluid transport has been boosted by the all along scientific discoveries (Fig. 9). Retzius)) [START_REF] Rasmussen | Fluid transport in the brain[END_REF] The dynamics of glymphatic flow was first described in 2012 using bi-photon microscopy in mice by injecting fluorescent tracers into the cisterna magna in order to visualize CSF diffusion in real time [START_REF] Iliff | A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β[END_REF]. Following injection, the tracers quickly flowed along the perivascular space of the cortical surface arteries and the penetrating arterioles, and from there entered the brain parenchyma. Brain fluid flow pathway in the parenchyma was defined by fluorescence imaging of fixed brain slices. A longer-time observation showed that the tracers have accumulated along capillaries and parenchymal venules. This imples that the interstitial fluid (ISF) were drained along the perivenous space surrounding large caliber draining veins.

AQP-4 being localized near periarterial and periveinous space, it was then also hypothesized that AQP4 may play a role in CSF and ISF exchange flux.

Glymphatic system was proposed for the removal of the solute and protein wastes in the brain, sustained by the convective exchange between the CSF and ISF (Fig. 10) [START_REF] Iliff | A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β[END_REF][START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF]. As aforementioned, the CSF penetrates the astrocytes end-feet to reach the brain interstitial space through a water channel membrane composed with AQP-4, [START_REF] Iliff | A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β[END_REF]. In the interstitial space, this CSF bulk flow can move brain metabolites and clear them through other perivascular spaces adjacent the cerebral veins. Then, the flow could travel through the cranial nerves, and in the recently described cerebral lymphatic vessels in the dura mater alongside the sinus veins [START_REF] Aspelund | A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules[END_REF][START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF]. Interestingly, the glymphatic activity is dramatically increased during sleep, which may have a role for brain waste clearance, a mechanism seemingly underlying the refreshing effect of sleep [START_REF] Xie | Sleep drives metabolite clearance from the adult brain[END_REF]. [START_REF] Rasmussen | Fluid transport in the brain[END_REF] In this context, dynamic flowing of CSF plays critical roles in regulating brain function and in controlling diffusion of neurotransmitters and metabolic substrates, such as glucose and lactate, thereby shaping synaptic transmission and plasticity [START_REF] Mestre | The Brain ' s Glymphatic System : Current Controversies[END_REF][START_REF] Kessler | Control of cleft glutamate concentration and glutamate spill-out by perisynaptic glia: uptake and diffusion barriers[END_REF][START_REF] Thrane | Paravascular microcirculation facilitates rapid lipid transport and astrocyte signaling in the brain[END_REF][START_REF] Xie | Sleep drives metabolite clearance from the adult brain[END_REF][START_REF] Lundgaard | Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism[END_REF][START_REF] Lundgaard | Glymphatic clearance controls state-dependent changes in brain lactate concentration[END_REF]

I.5 -Scientific question and technical challenges

Brain flow transport plays an important role in regulating extracellular molecular diffusion, therefore the health of brain network [START_REF] Brinker | A new look at cerebrospinal fluid circulation[END_REF][START_REF] Mesquita S Da | Perspective The Meningeal Lymphatic System : A New Player in Neurophysiology[END_REF]. As a recently proposed concept, the glymphatic system extends an active brain flow transport across the parenchyma for physiological homeostasis maintenance as afore summarized. It controls the intracerebral pressure equilibrium, extracellular neurotransmitter diffusion and the dynamic distribution of energy fuels such as glucose and lactate in the brain. Disturbance in brain flow/fluid transport is being recognized as a severe factor exacerbating neurological pathologies like edema development and Alzheimer's disease [START_REF] Tait | Water movements in the brain: role of aquaporins[END_REF][START_REF] Louveau | Structural and functional features of central nervous system lymphatic vessels[END_REF]. As a major type of glial cells, astrocytes are distributed throughout the brain and strategically located between cerebral vasculature and brain parenchyma [START_REF] Barres | The Mystery and Magic of Glia: A Perspective on Their Roles in Health and Disease[END_REF]. Astrocyte volume dynamics is implicated in regulating the extracellular architecture thereby brain flow transport [START_REF] Plog | The Glymphatic System in Central Nervous System Health and Disease: Past, Present, and Future[END_REF]. Water flux underlies astrocyte volume regulation.

Aquaporins (AQP) represent a family of bidirectional water transport channels, with AQP4 being the major subtype expressed in brain astrocytes [START_REF] Nagelhus | Physiological roles of aquaporin-4 in brain[END_REF][START_REF] Papadopoulos | Aquaporin water channels in the nervous system[END_REF].

Figure 12: AQP4 is bidirectionally implicated in astrocyte volume regulation [START_REF] Simard | The neurobiology of glia in the context of water and ion homeostasis[END_REF][START_REF] Lafrenaye | Bursting at the Seams: Molecular Mechanisms Mediating Astrocyte Swelling[END_REF] and modulation of extracellular space thereby brain fluid transport.

AQP4 has been implicated in brain fluid homeostasis, however its role in astrocyte volume regulation remains controversial. At the cellular level, early reports show controversial effects of AQP4 genetic inhibition on astrocyte swelling activity at cellular level; for instance, knocking out of AQP4 reduces astrocyte swelling as shown by [START_REF] Benfenati | An aquaporin-4/transient receptor potential vanilloid 4 (AQP4/TRPV4) complex is essential for cell-volume control in astrocytes[END_REF],while in contrast Murphy et al. showed that AQP4 knocking out facilitated astrocyte swelling [START_REF] Murphy | Hippocampal and Cortical Pyramidal Neurons Swell in Parallel with Astrocytes during Acute Hypoosmolar Stress[END_REF]. Also, reduced extracellular space was observed in AQP4-deficient mouse model [START_REF] Binder | In vivo measurement of brain extracellular space diffusion by cortical surface photobleaching[END_REF][START_REF] Zhang | Microfiberoptic measurement of extracellular space volume in brain and tumor slices based on fluorescent dye partitioning[END_REF], and in mouse model delocating AQP4 by knocking out its anchor protein syntrophin, implying that AQP4 deficiency may swell astrocytic endfeet [START_REF] Amiry-Moghaddam | Anchoring of aquaporin-4 in brain: molecular mechanisms and implications for the physiology and pathophysiology of water transport[END_REF]. Not only being involved in astrocyte volume regulation, astrocyte AQP4 is implicated in brain pathologies such as the edema development.

Nevertheless, while AQP4 knock out ameliorates cytosolic brain edema that reflects the water accumulation in intact cells, deficiency of AQP4 exacerbates the brain swelling in vasogenic edema and hydrocephalus mouse model, which are observed during brain blood barrier disruption and the elevation of intraventricular pressures, respectively. [START_REF] Bloch | Accelerated progression of kaolininduced hydrocephalus in aquaporin-4-deficient mice[END_REF][START_REF] Tait | Water movements in the brain: role of aquaporins[END_REF][START_REF] Verkman | The aquaporin-4 water channel as a potential drug target in neurological disorders[END_REF]. The presence of AQP4 has also been reported to alleviate perihematomal edema in intracerebral hemorrhage [START_REF] Jeon | Upregulation of AQP4 Improves Blood-Brain Barrier Integrity and Perihematomal Edema Following Intracerebral Hemorrhage[END_REF] Recently, dynamic exchange of brain fluid between CSF and parenchyma ISF is suggested to be important for clearing brain environment, leading to the emergence of the concept of glymphatic system (Mestre et al., 2020a). The importance of glymphatic system has been manifested by the removal of waste metabolites and harmful proteins (e.g., β amyloid) from the brain parenchyma. However, controversies exit about the operating mechanisms of glymphatic system [START_REF] Smith | CrossTalk opposing view: Going against the flow: interstitial solute transport in brain is diffusive and aquaporin-4 independent[END_REF], with a major debate raised on the role of AQP4 water channel. Several lines of experiments utilizing different types of AQP4 KO mice and diffusible tracer imaging suggest an indispensable involvement of AQP4 in brain fluid transport [START_REF] Mestre | Aquaporin-4-dependent glymphatic solute transport in the rodent brain[END_REF], However, it was also observed in AQP4 null mice that brain fluid transport is unafected (Smith et al., 2017a). In addition, selectively perturbing astrocyte AQP4 has led to distinct effects on water diffusion in the brain parenchyma as determined by the apparent diffusion coefficient using diffusion-weighted magnetic resonance imaging (DW-MRI) [START_REF] Badaut | Brain water mobility decreases after astrocytic aquaporin-4 inhibition using RNA interference[END_REF][START_REF] Debacker | Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor[END_REF].

One The synthetic compound 2-(nicotinamido)-1,3,4-thiadiazole (TGN-020) is derived from the condensation of nicotinamide and thiadiazole derivatives [START_REF] Burnett | Structural characterization of the aquaporin inhibitor 2-nicotinamido-1,3,4-thiadiazole[END_REF]. TGN-020 has been identified as a selective and potent inhibitor of AQP4 water channel as testified using either the heterologous expression system (Toft-bertelsen and Larsen, 2020) or the AQP4 knockout mouse model [START_REF] Igarashi | Inhibition of aquaporin-4 significantly increases regional cerebral blood flow[END_REF][START_REF] Huber | Identification of Aquaporin 4 inhibitors using in vitro and in silico methods[END_REF]. Application of TGN-020 in vivo reduced ischemia-associated edema in mouse models as revealed by MRI [START_REF] Igarashi | Pretreatment with a novel aquaporin 4 inhibitor, TGN-020, significantly reduces ischemic cerebral edema[END_REF]. Using AQP4 knockout mice, TGN-020 has been engineered as an AQP4 selective ligand for preclinical positron emission tomography imaging to map brain AQP4 distribution [START_REF] Nakamura | Development of a Novel Ligand, [C]TGN-020, for Aquaporin 4 Positron Emission Tomography Imaging[END_REF]. Blocking of AQP4 by TGN application augments the local cerebral blood flow, with effect of TGN-020 was absent in AQP4 knockout mice, indicating the specific targeting of AQP4 [START_REF] Igarashi | Inhibition of aquaporin-4 significantly increases regional cerebral blood flow[END_REF].

Using and results from GCaMP6f mice recording were analyzed using pMAT [START_REF] Bruno | pMAT: An open-source software suite for the analysis of fiber photometry data[END_REF].

Mice preparation and genotyping

The genetically encoded Ca 2+ indicator GCaMP6 was expressed in astrocytes in vivo by crossing a Cre-dependent GCaMP6f mouse line (Ai95, The Jackson Laboratory) with an astrocyte-specific Glast-CreERT2 mouse line [START_REF] Slezak | Transgenic Mice for Conditional Gene Manipulation in Astroglial Cells[END_REF]. Tamoxifen (T5648, Sigma)

was dissolved in ethanol 100 % (at 1 mg/250µL) and vortexed until tamoxifen is fully dissolved.

Corn oil (sigma) were then added to have a final solution at 10% ethanol and 90% oil. Solution is then heated at 37°c for 15 min and vortexed, and put in sonicator for 15 min and then aliquoted. All procedures were made under light-proof protocols. Aliquots were stored at -20 °C until use. GCaMP6f/Glast-CreERT2 mice were injected at ~3-4 weeks of age, once a day (1 mg) for two consecutive days. Genotyping of the Cre-dependent GCaMP6f mouse line (Ai95,

The Jackson Lab) used the standard primers and polymerase chain reaction protocols provided by the supplier. Genotyping of GLAST-CreERT2 mouse line was performed using the primers for Cre recombinase (TK139/141) as reported [START_REF] Slezak | Transgenic Mice for Conditional Gene Manipulation in Astroglial Cells[END_REF].

Imaging set up and procedures

Epifluorescence illumination was provided by a monochromator light source (Polychrome II, TILL Photonics, Germany) directly coupled to the imaging objective via an optical fiber. We used three water immersion imaging objectives: ×10 NA0.3, ×20 NA0.5 and ×40 NA 0.8 (ZEISS). The green fluorescence of GCaMP6 was separated from the red fluorescence of SRB, by spectrally exclusive double-band filters (Di03-R488/561-t3 and FF01-523/610, Semrock).

Fluorescence signal was collected using a digital electron-multiplying charge-coupled device (EMCCD Cascade 512B, Photometrics). The imaging chamber (20 × 30 × 46 mm width, height and length) was custom-designed using 3D modeling software (Trimble SketchUP), produced by a Mojo 3D printer (Stratasys). The chamber was mounted on a motorized PI stage (Physik Instrumente GmbH, Germany) for axial micro-manipulation. Laser excitation and image acquisition were controlled by MetaMorph (Molecular Devices).

In vivo diffusion MRI

Supported by an international exchange program JSPS-CNRS, I got the opportunity to go to the lab of Dr. Tomokazu Tsurugizawa in National Institute of Advanced Industrial Science and Technologies in Tsukuba, to carry out in vivo diffusion MRI experiments to test the effect of astrocyte AQP4 acute inhibition on the rate of brain fluid transport, where apparent diffusion coefficient (ADC) of cerebral water molecule was used.

Ten C57BL6 mice were used for the experiment. MRI experiment was performed with medetomidine anesthesia (0.6 mg/kg/h, i.v.). The mice were injected with TGN020 and saline at 4 weeks interval. For crossover test, the order of injection of TGN020 or saline was randomly determined. The respiration was monitored, and the body temperature was maintained at 36 °C by circulating hot water during the measurement.

The MRI experiments were conducted on a Bruker 7T scanner using a cryo-cooled mouse brain coil. After 10 min from the start of the scanning, TGN-020 (250 mg/kg body weight) or Saline was injected. Then, scanning was continued for 60 min. The body temperature was maintained at 36 °C via the circulating hot water. The respiration rate was monitored and was confirmed within the range of 130-140 /min throughout the experiment. Mice were injected with 250mg/kg of TGN-020. We used a standard diffusion-weighted spin echo EPI sequence, with the following parameters: spatial resolution = 175 x 175 x 500 μm 3 , 3 b-values (b = 0, 250 and 1800 s/mm 2 ), 6 directions, 1 segment, TE = 37.1 ms / TR = 5769 ms, bandwidth = 300 kHz, δ = 3 ms, Δ = 24 ms, total scan time = 5 min. The DWI scan was continued for 70 min (total 14 scans).

The ADC at each time point was calculated from DWI data using DSIstudio (https://dsistudio.labsolver.org/). The percent change of ADC was then calculated following equation, ΔADCi = (ADCi / ADC2 -1) x 100 (%). The ADCi is the ADC at the i-th timepoint. We define the ADC at 2 nd time-point as the baseline because saline injection was performed after 2 nd DWI scan. The averaged ADC changes within the region of interests (ROIs) were calculated using a homemade program. The ROIs of the cortex, the hippocampus, and the striatum were created with reference to Allen mouse brain atlas.

Statistics

Experimental data are expressed as mean ± standard error unless otherwise mentioned. The ttest was performed for two-group comparisons, and significant difference was determined by p values less than 0.05. Mann-Whitney U test was also used to ascertain the significance level, in considering the deviation of the experimental data from the theoretical symmetrical distribution.

In the present study, these two test methods yielded consistent results on statistical significance.

Statistical tests were carried out in Matlab (The MathWorks). The significance levels are shown in figures by *p < 0.05, **p < 0.01, ***p < 0.001.

I.7 -Technical development establishing light sheet microscopy for high quality imaging of mouse brain astrocytes

To achieve high-quality dynamic imaging of astrocyte in situ, I first established light-sheet fluorescence microscope to image astrocytes in mouse acute brain slices. As a basis for astrocyte volume dynamics imaging, I took astrocyte morphology as an example to demonstrate the superior imaging performance of light-sheet microscope. In addition, as astrocytes Ca 2+

represents a highly dynamic signaling underlying their physiology, I also showed that lightsheet microscopy is suitable to imaging astrocyte Ca 2+ signals, which sets the basis for the following physiological experiments of activity-associated astrocyte volume adaptation and the physiopathological alterations. This part of the study has been published [START_REF] Pham | Mapping astrocyte activity domains by light sheet imaging and spatio-temporal correlation screening[END_REF]. To this end, I adopted a rapid in vivo astrocyte chemical labeling strategy where brain astrocytes were specifically labeled by the highly diffusible red fluorescent dye Sulforhodamine B (SRB)

I.8 -Results investigating the role of AQP4 in

following an intraperitoneal injection in awake mice. In acute cortical slices, individual SRBlabeled astrocytes were resolved by a high numerical aperture objective (Fig. 16). This in vivo chemical labeling method labeled indeed widedly distributed astrocyte-like cells in mouse cortex (Fig. 16). The SRB labeling (red fluorescence) was validated to be astrocyte-specific using the fluorescent astrocyte reporter line GFAP-GFP mice (Fig. 17), thus corroborating the early report where an intravenous injection of another dye sulforhodamine 101 also results in the labeling of mouse brain astrocytes [START_REF] Appaix | Specific in vivo staining of astrocytes in the whole brain after intravenous injection of sulforhodamine dyes[END_REF]. In order to follow the global volume dynamics of astrocytes, we then used the wide-file fluorescence microscopy configuration was used to perform single-section time lapse imaging of astrocytes in acute brain slices. This which

AQP4 plays a tonic role in restricting astrocyte swelling

To examine a potential role of AQP4 in maintaining the volume homeostasis in astrocytes, I then acutely inactivated AQP4 in basal conditions using this selective blocker TGN-020 [START_REF] Huber | Identification of Aquaporin 4 inhibitors using in vitro and in silico methods[END_REF].The specificity of TGN-020 in targeting AQP4 water channel has been demonstrated by in vitro using heterologous expression system and in vivo researches using AQP4 knockout mice [START_REF] Nakamura | Development of a Novel Ligand, [C]TGN-020, for Aquaporin 4 Positron Emission Tomography Imaging[END_REF][START_REF] Igarashi | Inhibition of aquaporin-4 significantly increases regional cerebral blood flow[END_REF] (Fig. 19 and20). AQP4 is a bidirectional water channel (Fig. 21A). So, if AQP4 sustains a symmetric water influx and efflux across the astrocyte plasma membrane, acute pharmacological inhibition, in short time window, would not disturb astrocyte volume dynamics. Surprisingly, upon the application of TGN-020 in acute brain slices (Fig. 21B), I observed a moderate yet significant swelling reflected by the drop in the intracellular SRB fluorescence in astrocytes as compared with the application of the control vehicle (Fig. 20C). This result indicates that AQP4 tonically inhibits astrocyte swelling to maintain the volume homeostasis. Intriguingly, this observation suggests that AQP4 mediates a tonic water efflux from astrocytes (Fig. 22), therefore exerting a net shrinking effect in basal conditions to sustain astrocyte volume stability.

Hence, this line of data implies an imbalanced involvement of AQP4 in astrocyte water handling that facilitates the water efflux in the basal states. To validate the basal inhibition effect of AQP4 in astrocyte swelling, I checked the effect of TGN-020 on evoked astrocyte swelling and shrinking. First, I induced astrocyte swelling in acute mouse cortical slices by applying hypo-osmotic solution in the presence or absence of TGN-020 (Fig. 23A). Acute inhibition of AQP4 was observed to facilitate the rate of swelling reflected by a faster initiation time (namely, the shortened starting time) and the more rapid reaching of SRB fluorescence to the peak value (Fig. 23B andC). These results validates the basal contribution of AQP4 for water efflux, whereby (its inhibition enhances the intracellular water accumulation thus the swelling). Meanwhile, I noted that the maximal swelling extent was reduced (Fig. 23B andC). This observation confirms that AQP4 also I also imaged the effect of blocking AQP4 on the shrinking activity of astrocytes induced by hypertonic solution in situ in brain slices (Fig. 24A). I observed shriking kinetics in astrocytes was altered by the presence of AQP4 blocker TGN-020 (Fig. 24B). Quantifying the shrinking kinetics parameters shows that AQP4 inhibition impairs astrocytes shrinking, reflected by a slower reaching to shrinking peak, and a reduced shrinking amplitude as compared with control conditions (Fig. 24C). Although TGN-020 tends to slow down the initiation reflected by the prolonged starting time, the effect is not yet significant. Overall inhibition of AQP4 limits the shrinking activities of astrocytes corroborating the preferential role of AQP4 mediating water efflux in astrocytes. Together, these data confirm that AQP4 plays a tonic role in regulating astrocyte volume responses. Corroboratively, these results suggest that AQP4 in basal states mediates a net water efflux from cortical astrocytes, thereby constraining the basal swelling to maintain brain astrocyte volume stability.

Inhibition of astrocyte AQP4 causes swelling-associated Ca 2+ transients

It is known that astrocyte swelling induces intracellular Ca 2+ increases, likely due to the Ca 2+ influx via the Ca 2+ permeable transient receptor potential (TRPV) ion channels [START_REF] Mola | The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins[END_REF]. Thus to furthermore validate that acute inhibition of basal AQP4 activity causes astrocyte swelling, I performed functional recording of astrocyte Ca 2+ signaling using the GLAST-GCaMP6 mouse line where the genetically encoded Ca 2+ sensor GCaMP6 is selectively expressed in astrocytes [START_REF] Pham | Mapping astrocyte activity domains by light sheet imaging and spatio-temporal correlation screening[END_REF].

Using light-sheet imaging, I first validated that hypo-osmotically induced swelling indeed induced astrocyte Ca 2+ signal increase (Fig. 25A), consistent with previous reports [START_REF] Jarand | Astroglial endfeet exhibit distinct Ca 2 + signals during hypoosmotic conditions[END_REF]. Next I checked the effect of AQP4 inhibition by TGN-020 effect on intracellular Ca 2+ signals in astrocytes. I observed that application of TGN-020 induces significant Ca 2+ increase in astrocytes, suggesting that acute blocking of AQP4 causes astrocyte swelling (Fig. 25B). This observation thus further supports the tonic inhibition role of AQP4 in astrocytes swelling (Fig. 26).

In vivo validation of the tonic inhibition role of AQP4 in astrocyte volume control

Following the observation in acute mouse brain slices, I have started to validate the observation in vivo using fiberphotometry. This technique allows the temporal recording of astrocyte population fluorescence in freely moving animals (Fig. 27A). First I monitored SRB temporal course into S1 cortex following its injection in vivo. An initial sharp entry of SRB into the cortex was noted, which was followed by a gradual fluorescence decrease over time in about 1 hour. Afterward, a long sustained SRB fluorescence plateau was observed lasting for a long time with much reduced decrease rate, indicating the accumulation of SRB in brain astrocytes (Fig. 27B). This time courses is consistent with SRB accumulation in astrocyte as validated by my slice imaging experiment, which is also comparable with the kinetics of the accumulation of sulforhodamine 101 following intravenous injection as previously previous reported [START_REF] Appaix | Specific in vivo staining of astrocytes in the whole brain after intravenous injection of sulforhodamine dyes[END_REF].

Then I tested the effect of TGN-020 on astrocyte swelling in this in vivo setting. TGN-020 was injected at the time when SRB is trapped in astrocytes. In parallel mice I injected saline control solution at the same time as TGN-020 (Fig. 28B).Given the tonic inhibitory effect of AQP4

blocking on astrocyte swelling it was expected that in vivo application of TGN-020 would induce a transient astrocyte swelling thereby a decrease in the SRB fluorescence. Along this line I observed that while the saline control showed no apparent (Fig. 28A), TGN-020 tended to cause a fluorescence decrease in astrocytes (Fig. 28B). Thus far the number of the experimental mice needs to be increased to establish statistical significance. I currently have experiments on going. regions in which water molecules diffuse more freely appear darker (Fig. 29) [START_REF] Bihan D Le | Diffusion Magnetic Resonance Imaging : What Water Tells Us about Biological Tissues[END_REF]. In biological tissues, the effective diffusion coefficient of water depends on several parameters, including the local diffusivity in intracellular and extracellular compartments, the relative volume fraction occupied by cells and the transport of water across the plasma membrane. To disentangle diffusion and relaxation effects on image contrast, one may obtain quantitative images of the "apparent" diffusion coefficient (ADC). The ADC concept is introduced to account for the fact that in biological tissues the diffusion process is complex and reflects several different mechanisms. The ADC incorporates the effects on water diffusion imparted by the cellular membrane and water permeability between intra-cellular and extracellular spaces at the voxel level. diffusion (e.g. within ventricules), the signal is deeply reduced, while in areas of slow water diffusion (e.g. white matter bundles), the signal is only slightly reduced. This differential effect results in a contrast in the diffusion-weighted MRI images, which is not visible in standard MRI images.

I.9 -Discussion

Brain flow transport is mediated by cerebrospinal fluid (CSF) circulation. This process plays important roles in the function, therefore the health of brain network. Disturbance in brain flow transport is being recognized as a severe factor exacerbating neurological pathologies like Alzheimer's disease.

As a major type of glial cells, astrocytes are distributed throughout the brain and strategically located between cerebral vasculature and brain parenchyma. The discovery of the critical involvement of astrocytes in brain fluid transport has led to the emergence of the concept of the glymphatic system [START_REF] Rasmussen | Fluid transport in the brain[END_REF]. It has been suggested that astrocyte volume dynamics, via regulating the volume of the extracellular space, shapes the brain flow transport.

Water flux underlies astrocyte volume regulation. Astrocytes express the bidirectional water channel Aquaporin 4 (AQP4) that has been suggested to modulate astrocyte volume change.

However, the exact role of AQP4 in astrocyte volume regulation has been debated, causing an extended controversy on the mechanistic understanding of brain fluid transport and the glymphatic system.

The Chapter I study acutely discerns the role of AQP4 in astrocyte volume regulation in basal conditions and in response to environmental manipulations in brain parenchyma in mouse models. Interdisciplinary approaches have been developed or adopted, including in vivo chemical astrocyte targeting, ex vivo cellular fluorescence imaging in acute brain slices, in vivo fiberphotometry recording in freely moving animals and by diffusion-weighted magnetic resonance imaging (DW-MRI). The obtained results lead to the finding that AQP4 tonically sculpts astrocyte volume homeostasis in the brain parenchyma, perturbing of which significantly interferes with brain fluid transport. These results together shed light on the fundamental role of AQP4 in tuning astrocyte volume and therefore the extracellular route for brain flow, hence helping to reconcile the thus far debated aspects of glymphatic system and its implication in health and neurological disorders.

Regarding the physiological implications of the finding my data suggest that there is a constant net water efflux from astrocytes under the basal state of the brain. It implies that there is a constitutive water accumulation process in astrocytes. For instance, water is transported from perivascular space to brain parenchyma, which has been suggested to be a complementary mechanism for the production of the CSF in addition of the conventional sources from the choroid plexus [START_REF] Drew | Neurovascular coupling: motive unknown[END_REF]. My finding also recalls the scenario in kidney where AQP4 abundantly expressed, directly underlining the constant efflux of water from local cells [START_REF] Rasmussen | Fluid transport in the brain[END_REF]. The observation of the AQP4 mediating basal water efflux in astrocyte might contribute to the maintenance of the convective flow in the parenchyma as claimed by the glymphatic concept [START_REF] Plog | The Glymphatic System in Central Nervous System Health and Disease: Past, Present, and Future[END_REF]. This extends the early proposed mechanism supported by the arterial pulsation controlled with cardiovascular rhythms (Mestre et al., n.d.).

Taking into account of the polarized distribution of AQP4 in astrocytes, where it is enriched in the perivascular space and pial limitant [START_REF] Nielsen | Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain[END_REF]. AQP4 at basal condition, tonically inhibits astrocyte swelling, suggesting it plays a more prominent role in mediating water efflux from astrocytes. Hence, AQP4 would be important for extruding water from astrocytes, into vascular circulation. It is plausible that a water efflux is critical for astrocytes to maintain their volume homeostasis (thereby the functional stability of the brain), by counterbalancing the constitutive water buildup in astrocytes. The constitutive water buildup in astrocytes could be induced by several mechanisms. In fact, a variety of co-transporters such as Na/K + co transporter (NKCC), bicarbonate cotransporter (NBC) and glutamate or GABA transporters (Fig. 32), together playing a role in bringing water into astrocytes accompanying the ion/transmitter influx (MacAulay and Zeuthen, 2010). In addition the internal water accumulation can be induced by the metabolic processes (references). Thus, it appears necessary for an efficient water efflux pathway to maintain astrocytes water homeostasis. The current observation of AQP4-mediated net water efflux would fulfill this demand. My observation shows that AQP4 is also involved in astrocyte water influx, which though, is inferior to its contribution to water efflux under basal state.

Figure 32: AQP4 mediates water efflux in astrocytes

The observation that AQP4 tonically inhibits the swelling of astrocytes lends support to the observation reported by Fiacco's group [START_REF] Murphy | Hippocampal and Cortical Pyramidal Neurons Swell in Parallel with Astrocytes during Acute Hypoosmolar Stress[END_REF] where knock-out of AQP4 facilitates astrocytes swelling induced by hypo-osmotic solution. However, as we here performed acute pharmacological inhibition of AQP4, the controversy between the results obtained with AQP4 KO animals needs be further investigated [START_REF] Amiry-Moghaddam | Anchoring of aquaporin-4 in brain: molecular mechanisms and implications for the physiology and pathophysiology of water transport[END_REF][START_REF] Benfenati | An aquaporin-4/transient receptor potential vanilloid 4 (AQP4/TRPV4) complex is essential for cell-volume control in astrocytes[END_REF][START_REF] Murphy | Hippocampal and Cortical Pyramidal Neurons Swell in Parallel with Astrocytes during Acute Hypoosmolar Stress[END_REF].

The mechanism underlying the imbalanced engagement of AQP4 in astrocyte water influx and efflux could stem from at least two possibilities. One possibility is that the asymmetric role of AQP4 in water flux might be regulated at the channel level, namely AQP4 itself has an intrinsic property that facilitates more the water efflux from the cell while contributes less to the water influx. Another plausible scenario might be that AQP4 engagement in water flux is coordinated with other molecular machineries underlying water flux in astrocytes. As aforementioned, because diverse pathways could contribute to water buildup (Fig. 32), a net water efflux thus occurs via AQP4 at basal conditions to avoid water over accumulation in astrocytes.

Besides the physiological implications, the current results have several tiers of implications for brain pathology. In several pathologies like Alzheimer's disease, Parkinson disease, the polarization of AQP4 in astrocytic endfeet is lost and more accumulated in somatic regions [START_REF] Ikeshima-Kataoka | Neuroimmunological Implications of AQP4 in Astrocytes[END_REF]. Because intracellular metabolism is upregulated in reactive astrocytes in neurodegenerative disease and aging [START_REF] Chen | Brain Energy Metabolism: Astrocytes in Neurodegenerative Diseases[END_REF], one possibility might be that hyper-upregulated astrocytic metabolism occurs in somatic region, enriched with mitochondria [START_REF] Jackson | Regulation of mitochondrial dynamics in astrocytes: Mechanisms, consequences, and unknowns[END_REF], which would cause upregulated water production in the somatic region and thus requires more AQP4 to export water. This chronic adaptation could eventually shift the global distribution of AQP4 in astrocytes to pathological accumulation in the soma as an attempt to prevent the potential development of cytotoxic edema.

AQP4 is highly implicated in the formation and development of brain edema, but its role in the different phases of edema. In the brain, two types of edema have been documented: cytotoxic edema induced by over accumulation of water in astrocytes, and vasogenic edema caused by the massive water influx resulted from ruptured vascular system [START_REF] Tait | Water movements in the brain: role of aquaporins[END_REF]. Reducing AQP4 in astrocytes has been introduced as a putative strategy to slow down the formation of edema [START_REF] Igarashi | Pretreatment with a novel aquaporin 4 inhibitor, TGN-020, significantly reduces ischemic cerebral edema[END_REF], however the specific type of edema concerned has not been discerned. My current data suggest that AQP4 mediates constant water efflux. Thus, in conditions where cytotoxic edema firstly develops, keeping AQP4 in place, rather than simply blocking its function, would facilitate water efflux so slowing down the edema formation.

However, in situations where vasogenic edema dominates, inhibiting AQP4 needs be considered at priority. Therefore, my current data suggest a mechanistic basis for conditiondependent intervention for cerebral edema amelioration.

Furthermore, my data provides a mechanistic hint for understanding the DW-MRI data.

The DW-MRI data of my current study and that of [START_REF] Debacker | Diffusion MRI reveals in vivo and non-invasively changes in astrocyte function induced by an aquaporin-4 inhibitor[END_REF] both show that acutely blocking astrocyte AQP4 with TGN-020 increases water diffusion dynamics in mouse brain. My volume imaging data show that TGN-020 blocks AQP4-mediated net water efflux to cause astrocyte swelling, suggesting TGN-020 would decrease extracellular space while increasing intracellular space in astrocytes. Accordingly, the water diffusion rate would be increased inside astrocytes and reduced in extracellular space. DW-MRI provides a readout on the overall water diffusion in both intracellular and extracellular space (Fig. 29). Hence, a net increase in ADC in my observation reflects that TGN-020 causes an appreciable astrocyte swelling thereby an increase in the intracellular water diffusion, which surpasses the water diffusion reduction in extracellular space imposed by the swelling.

Alternatively but not exclusively, the glymphatic concept has proposed that the brain fluid in the extracellular space of parenchyma is not pure of Brownian motion, rather it represents a convective flow sustained by the arteriole pulsation and reaching the perivenous spaces (Mestre et al., 2020b). In this sense, a diminution in extracellular space as caused by TGN-020-induced astrocytes swelling would transiently increase the water diffusion rate along the glymphatic routes. Nevertheless, the current data MRI lacks sufficient spatial resolution to claim the convective flow occurring throughout the parenchyma. It is also possible that the convective flow mainly exists in perivascular space generated by local vascular pulsation as already acknowledged by conventional views on brain fluid transport [START_REF] Smith | CrossTalk opposing view: Going against the flow: interstitial solute transport in brain is diffusive and aquaporin-4 independent[END_REF].

Extending the volume imaging data, the DW-MRI results testify the important role of astrocyte AQP4 in regulating brain water diffusion. Furthermore to follow CSF/ISF diffusion in the extracellular space of brain parenchyma, specific MRI based neuroimaging techniques need to be applied. For example the diffusion weighted magnetic resonance spectroscopy (DW-MRS) has been used to follow exogenously injected extracellular tracers like sucrose [START_REF] Vincent | Characterizing extracellular diffusion properties using diffusion-weighted MRS of sucrose injected in mouse brain[END_REF].

In conclusion, my current study unveils a tonic role of AQP4 in regulating brain astrocyte volume dynamics, which helps to understand the cellular mechanism underlying astrocyte volume homeostasis, its pathological adaptation to neurological diseases and the interpretation of the MRI neuroimaging data. These data will thus contribute to the understanding of astrocyte water handling in neurophysiology and in brain disorders.

Chapter II: Probing disturbed astrocytes signals and volume regulation in amyloid pathologies

II.1 -Astrocytes and glymphatic system impairment in neuropathology

Astrocytes plays import role in brain physiology and the maintenance of body homeostasis through interactions with neurons and other glial cells. They display extensive adaptation to pathological environment, such as in neurodegenerative diseases like Alzheimer's disease, metabolic disorders, and brain injury [START_REF] Buckman | Regional astrogliosis in the mouse hypothalamus in response to obesity[END_REF][START_REF] Chiarini | Amyloid β-Exposed Human Astrocytes Overproduce Phospho-Tau and Overrelease It within Exosomes, Effects Suppressed by Calcilytic NPS 2143-Further Implications for Alzheimer's Therapy[END_REF]. Astrocytes show morphology remodeling, functional dysregulation, and molecular profile deviation by releasing or sensing of multiple types of cytokines and chemokines [START_REF] Sofroniew | Astrocytes: Biology and pathology[END_REF]) (Fig. 33). Such pathological adaptation of astrocytes impairs their interaction with synaptic transmission and the regulation of neurovascular coupling. In addition, the communication between astrocytes and other glial cells including microglia, oligodendrocytes and NG2 cells can also be impaired. Such pathological changes have been proposed to either protect or to exacerbate disease development [START_REF] Escartin | Reactive astrocyte nomenclature, definitions, and future directions[END_REF]. can modulate synaptic transmission leading to the concept of the tripartite synapse [START_REF] Verkhratsky | Physiology of Astroglia[END_REF]. In addition astrocyte Ca 2+ was propose to trigger to release neuromodulatory signaling molecules, called the gliotransmitter [START_REF] Harada | Gliotransmitter release from astrocytes: Functional, developmental, and pathological implications in the brain[END_REF]. [START_REF] Shigetomi | Probing the Complexities of Astrocyte Calcium Signaling[END_REF]. At the systemic level astrocyte Ca 2+ is implicated in cognitive behavior such as memory consolidation, spatial learning, and memory retrieval [START_REF] Denizot | Simulating calcium signaling in fine astrocytic processes To cite this version : HAL Id : tel-02406699 Simulation de la signalisation calcique dans les prolongements fins astrocytaires[END_REF] Furthermore astrocytes Ca 2+ signals are found to be important for sensory processing, visual stimuli, somatosensory cortex plasticity, motor processing, motor coordination, and sleep [START_REF] Nagai | Behaviorally consequential astrocytic regulation of neural circuits[END_REF].

Figure 35: Functional role of astrocytic Ca 2+ signals [START_REF] Denizot | Simulating calcium signaling in fine astrocytic processes To cite this version : HAL Id : tel-02406699 Simulation de la signalisation calcique dans les prolongements fins astrocytaires[END_REF] Astrocyte Ca 2+ is altered in brain pathologies

Glia was suggested as a contributor in brain pathologies, particularly astrocyte Ca 2+ signals has been shown to be altered in neurological and psychiatric disorders [START_REF] Denizot | Simulating calcium signaling in fine astrocytic processes To cite this version : HAL Id : tel-02406699 Simulation de la signalisation calcique dans les prolongements fins astrocytaires[END_REF].

Studies using Alzheimer's disease mouse models showed that astrocyte Ca 2+ basal levels and transients were increased, near β-amyloid plaques [START_REF] Verkhratsky | Astroglial Calcium Signaling in Aging and Alzheimer's Disease[END_REF]). An increase of astrocyte Ca 2+ signals was also observed in response to amyloid-β plaques [START_REF] Herrera Moro Chao | 2 nd thematic days of the neuroendocrinolgy society (SNE). Imaging Ca 2+ signals in hypothalamic astrocytes in diet induced obesity[END_REF]. In Hungtinton's disease, basal Ca 2+ signals are reduced, however astrocytes still respond to cortical stimulation with Ca 2+ signals [START_REF] Jiang | Dysfunctional Calcium and Glutamate Signaling in Striatal Astrocytes from Huntington's Disease Model Mice[END_REF]. In addition Ca 2+ signaling of astrocyte has been shown to be up regulated in Parkinson's disease and epilepsy [START_REF] Nedergaard | Glial calcium and diseases of the nervous system[END_REF][START_REF] Heuser | Astrocytic Ca(2+) Signaling in Epilepsy[END_REF]. These data raise the possibility that astrocyte Ca 2+ signaling contributes in specific ways to brain disorders.

II.1.2 -Astrocyte Ca 2+ signal modulates volume dynamics

Experimental investigations have indicated that astrocytes display Ca 2+-dependent cell volume dynamics. For instance using calcein based volume measurement in cultured astrocytes, Nedergaard's group has shown that ATP evocked Ca 2+ rises in astrocytes increased their volume (Fig. 36; [START_REF] Takano | Receptor-mediated glutamate release from volume sensitive channels in astrocytes[END_REF]). (Figures and legends from [START_REF] Takano | Receptor-mediated glutamate release from volume sensitive channels in astrocytes[END_REF])

Also, the early study of our team has demonstrated that chelating basal Ca 2+ in astrocytes using BAPTA-AM impaired the spontaneous remodeling of the plasma membrane (Fig. 37A). We also showed that Ca 2+ increase in astrocytes could induce astrocytes volume change, using dual color imaging of calcein-based astrocyte volume change and glutamate-evoked Ca 2+ dynamics in cultured astrocytes (Fig. 37B). These results suggest that astrocyte Ca 2+ plays a role in regulating the volume dynamics. In situ this phenomenon would likely regulate the extracellular space therefore the brain fluid transport in glymphatic system.

II.1.3 -Glymphatic system impairment in Alzheimer's disease

Glymphatic system, featured with the dynamic exchange between cerebrospinal fluid (CSF)

and interstitial fluid (ISF), has emerged as a brain-wide clearance pathway for potentially toxic substances [START_REF] Louveau | Understanding the functions and relationships of the glymphatic system and meningeal lymphatics[END_REF] (Fig. 38). Glymphatic pathway is driven by the convective flow of the peri-arteriole CSF, through the parenchyma ISF, to the recirculation of CSF along the draining veins [START_REF] Louveau | Understanding the functions and relationships of the glymphatic system and meningeal lymphatics[END_REF]. A functional link between the glymphatic system and the meningeal lymphatic vessels has been proposed for brain molecular clearance [START_REF] Mesquita S Da | Perspective The Meningeal Lymphatic System : A New Player in Neurophysiology[END_REF]. In the Chapter II study, I (1) contributed to the exploration of pathologically altered Ca 2+ responses of astrocytes to neurotoxic amyloid peptide (Pham et al., 2021); (2) initiated the in vivo exploration of modulating astrocyte volume dynamics in order to examine its ameliorative effect on amyloid deposition in an Alzheimer's mouse model. Here by fluorescence imaging of signaling pathways, we dissected astrocytic responses to Aβ25-35 peptide, a neurotoxic Aβ fragment present in AD patients. In native health astrocytes, Aβ25-35 evoked Ca 2+ elevations via purinergic receptors, being also dependent on the opening of connexin (CX) hemichannels. Aβ25-35, however, induced a Ca 2+ diminution in Aβpreconditioned astrocytes as a result of the potentiation of the plasma membrane Ca 2+ ATPase (PMCA). The PMCA and CX protein expression was observed with immunostaining in the brain tissue of hAPPJ20 AD mouse model. We also observed both Ca 2+ -independent and Ca 2+dependent glutamate release upon astrocytic Aβ exposure, with the former mediated by CX hemichannel and the latter by both anion channels and lysosome exocytosis. Our results suggest that Aβ peptide causes state-dependent responses in astrocytes, in association with a multiphasic release of signaling molecules. This study therefore helps to understand astrocyte engagement in AD-related amyloidopathy. This part of the study has been published (Pham et al., 2021).

II.3 -Results of experimental investigations

II.3.2 -Initial in vivo experiment to modulate glymphatic diffusion in an

Alzheimer's mouse model

In Chapter I study, combining the multimodal imaging approaches and the selective AQP4 blocker TGN-020, I have examined the acute influence of AQP4 on astrocyte volume regulation.

The results suggest that acutely blocking AQP4 activity with TGN-020 induces astrocyte swelling, indicating AQP4 tonically constrains astrocyte swelling, and accordingly, acute inhibition of AQP4 facilitates hypotonically evoked astrocyte swelling and impedes hypertonically induced astrocyte shrinking. These results suggest that pharmacologically targeting AQP4 will shape astrocyte volume dynamics, and thereby the extracellular route, which might be used to modulate glymphatic clearance of amyloid β peptide in AD brain.

To probe this hypothesis, I have started to employ the hAPPJ20 AD mouse model, used in my recent study (Pham et al., 2021), to design experiments to examine the potential effect of modulating astrocytic AQP4 on brain to Aβ1-42 peptide deposition.

As the hAPPJ20 AD mouse model start forming Aβ1-42 fibrils/plaques after 4-5 month of age (Fig. 39). I have decided to manipulate AQP4 function in vivo in pre-plaque phase, thereby enabling the evaluation of astrocyte impact on the formation of Aβ fibrils, and the inference on the clearance of diffusible Aβ peptides. I therefore started using 4-month old mice to pharmacologically manipulate AQP4 functions in vivo for 1 week, so as to determine the effect on the subsequent Aβ1-42 deposition. In addition to the AQP4 blocker TGN-020, I also used the AQP4 facilitator, TGN-073 (N-(3-benzyloxypyridin-2-yl)-benzene-sulfonamide) [START_REF] Huber | Aquaporin-4 facilitator TGN-073 promotes interstitial fluid circulation within the bloodbrain barrier : [ 17 O ] H 2 O JJVCPE MRI study[END_REF], thus allowing the paired comparison between the loss-and gain-of-function interventions.

128 The expected results will hence provide clues on the therapeutic potential of targeting astrocyte AQP4. Pharmacological strategy holds the advantage that it could be readily tested in vivo over flexible time windows and by different doses.

II.4 -Perspectives

The proposed experiments in this chapter possess multiple perspectives. First, the results suggest a mechanism link between astrocyte Ca 2+ dynamics and volume control. Early studies mainly investigated the phenomena using cultured astrocytes [START_REF] Takano | Receptor-mediated glutamate release from volume sensitive channels in astrocytes[END_REF][START_REF] Mola | The speed of swelling kinetics modulates cell volume regulation and calcium signaling in astrocytes: A different point of view on the role of aquaporins[END_REF]. Because astrocytes in culture show different features from their in vivo states [START_REF] Zamanian | Genomic analysis of reactive astrogliosis[END_REF], so it is necessary to explore astrocyte Ca 2+ regulation of volume dynamics in situ.

The experimental strategies I explored so far enabled the study of this important scientific question at multiscale level, from brain slices to all brain scale in vivo in mouse model.

  benzyloxypyridin-2-yl)-benzene-sulfonamide)

Figure 1 :Figure 2 :

 12 Figure 1: The major ion channels responsible for CSF secretion in the choroid plexus Figure 2: The classic model of cerebrospinal fluid (CSF) dynamics.

Figure 3 :Figure 4 :

 34 Figure 3: Virchow's illustrations of neuroglia Figure 4: Two main type of astrocytes.

Figure 5 :

 5 Figure 5: Astrocytes in the adult brain typically do not reach into domains of their astrocyte neighbors.

Figure 6 :

 6 Figure 6: Astrocyte function in the CNS.

Figure 7 :

 7 Figure 7: Distribution of AQP4 in the brain.

Figure 8 :

 8 Figure 8: Outline of the glymphatic system.

Figure 9 .

 9 Figure 9. Timeline of significant events and discoveries pertaining to brain fluid transport.

Figure 10 :

 10 Figure 10: Plausible fluid transport routes and their dependence on the polarized expression of aquaporin 4 (AQP4) at perivascular astrocytic end-feet.

Figure 11 .

 11 Figure 11. Glymphatic system and its role in clearance, nourishment, and volume transmission.

Figure 12 :

 12 Figure 12: AQP4 is bidirectionally implicated in astrocyte volume regulation.

Figure 13 :Figure 14 :Figure 15 :

 131415 Figure 13: Water content in mouse brain in conditional AQP4 KO mice Figure 14: Complex morphology of mouse brain astrocyte Figure 15: The principle of FIT imaging of astrocyte volume change.

Figure 16 :

 16 Figure 16: In vivo chemical labelling of astrocytes in mouse brain.

Figure 17 :Figure 18 :

 1718 Figure 17: Validation of in vivo SRB labelling of mouse cortical astrocytes Figure 18: Experimental validation of The FIT imaging method of astrocyte volume dynamics.

Figure 19 :Figure 20 :Figure 21 :

 192021 Figure 19: Protocol for acute blocking of astrocytic AQP4 by TGN-020 Figure 20: Selectivity of TGN-020 for AQP4 validated using oocytes expressing different type of aquaporin channels Figure 21: Acutely blocking AQP4 in astrocytes in situ in brain slices causes swelling.

Figure 22 :

 22 Figure 22: Potential explanation for the tonic role of AQP4 in astrocyte volume homeostasis.

Figure 23 :

 23 Figure 23: Blocking AQP4 facilitate astrocyte swelling.

Figure 24 :

 24 Figure 24: Blocking AQP4 acutely impaired astrocyte volume shrinking

Figure 25 :

 25 Figure 25: Acutely blocking AQP4 triggered swelling-associated astrocyte Ca 2+ increase.

Figure 26 :

 26 Figure 26: Swelling induced by AQP4 blocking elicits Ca 2+ increase in astrocytes.

Figure 27 :

 27 Figure 27: Fiberphotometry fluorescence recording of astrocyte volume in vivo.

Figure 28 :

 28 Figure 28: In vivo fiberphotometry suggest that AQP4 blocking shows a tendency to swell astrocytes.

Figure 29 :

 29 Figure 29: Diffusion weighted magnetic resonance imaging principles.

Figure 30 :

 30 Figure 30: Diffusion MRI-detected water diffusion rate in mouse brain following the acute inhibition of astrocytic AQP4.

Figure 31

 31 Figure 31 ADC mean values following saline or TGN-020 injection derived from diffusion MRI recordings.

Figure 32 :

 32 Figure 32: AQP4 mediates water efflux in astrocytes.

Figure 33 :

 33 Figure 33: Astrocyte maladaptive changes in pathologic brain.

Figure 34 :

 34 Figure 34: Excitatory and/or inhibitory signals trigger Ca 2+ elevations in astrocytes and lead to gliotransmitter release.

Figure 35 :

 35 Figure 35: Functional role of astrocytic Ca 2+ signals.

Figure 36 .

 36 Figure 36. Astrocytic Ca 2+ increases are associated with a transient increase in cell volume.

Figure 37 :

 37 Figure 37: Astrocytic Ca2+ signal in morphology and volume dynamics.

Figure 38 :

 38 Figure 38: Impaired glymphatic system in Alzheimer's disease.

Figure 39 :

 39 Figure 39: Amyloid β plaque formation time course in hAPP J20 mice.

Figure 40 :

 40 Figure 40: Experiments for exanimating the effect of AQP4 examination on Aβ clearance in AD mouse model.

Figure 1 :

 1 Figure 1: The major ion channels responsible for CSF secretion in the choroid plexus. The major ion channels responsible for CSF secretion in the choroid plexus are shown with sites of action of acetazolamide, AZD4017 and exenatide. Cortisone is converted to the active cortisol by 11ß-HSD1, cortisol binds to the GR and MR receptors, which upregulate Na + /K + ATPase activity; AZD4017 inhibits 11ß-HSD1 reducing local availability of cortisol.Exenatide binds and activates GLP-1R stimulating the conversion of ATP to cAMP by AC. cAMP activates PKA, which inhibits the Na + H + exchanger reducingNa + re-absorption and also inhibits the Na + /K + ATPase reducing Na + excretion. Carbonic anhydrase catalyzes the conversion of H2 O and CO2 to H + and HCO3 -, which is important in the establishment of the osmotic gradient. Both acetazolamide and topiramate inhibit carbonic anhydrase function. AC, adenylate cyclase; AE2, anion exchange protein 2; cAMP, cyclic adenosine monophosphate; CSF,

Figure 2 :

 2 Figure 2: The classic model of cerebrospinal fluid (CSF) dynamics. Ventricular CSF is produced from the blood via the choroid plexuses in the walls of each of the four ventricles (red arrows). CSF flows (black arrows) from the lateral into the third ventricle (1), then flows into the fourth ventricle (2) via the cerebral aqueduct. From the fourth ventricle CSF flow is either into the central canal of the spine (3)

FIGURE 3 :

 3 FIGURE 3: Virchow's illustrations of neuroglia. (A) Ependyma and neuroglia in the floor of the fourth ventricle. Between the ependyma and the nerve fibers is "the free portion of the neuroglia with numerous connective tissue corpuscles and nuclei." Numerous corpora amylacea are also visible, shown enlarged below the main illustration (ca). E, ependymal epithelium; N, nerve fibers; v, blood vessels. (B) Elements of neuroglia from white matter of the human cerebral hemispheres. A, free nuclei with nucleoli; b, nuclei with partially destroyed cell bodies; c, complete cells. Source: Reproduced with permission from Verkhratsky A, Butt AM. Glial physiology and pathophysiology. Oxford, UK: Wiley-Blackwell; 2013 (Figure 1.14). (Figure and legends from astrocyte and epilepsy Hubbard et al Science 2016)

Figure 4 :

 4 Figure 4: Two main type of astrocytes. (A) Images of astrocytes drawn by Michael von Lenhossek (Lenhossek, 1895). (B) rotoplasmic and fibrous astrocytes drawn by William Andriezen (Andriezen, 1893).Both preparations were stained with Golgi technique.Figure and legends from(Verkhratsky and

  Figure and legends from

Figure 5 :

 5 Figure 5: Astrocytes in the adult brain typically do not reach into domains of their astrocyte neighbors.

Figure 6 :

 6 Figure 6: Astrocyte function in the CNS (Sofroniew and Vinters, 2010)

Figure 7 :

 7 Figure 7: Distribution of AQP4 in the brain. There is evidence accumulated to suggest that AQP4 is involved in such diverse functions as regulation of extracellular space volume, potassium buffering (references), cerebrospinal fluid circulation (references), interstitial fluid resorption, waste clearance,

Figure 8 :

 8 Figure 8: Outline of the glymphatic system. This figure illustrates that perivascular clearance comprises perivascular drainage and glymphatic pathways. (1) Cerebrospinal fluid flows into the brain parenchyma via the periarterial space, which is the perivascular space surrounding the parenchymal arteries. From this perivascular space surrounding the artery, cerebrospinal fluid enters the interstitium of the brain tissue via aquaporin 4 (AQP4)-controlled water channels. These are distributed in the end feet of astrocytes that constitute the outer wall of the perivascular space. (2) Cerebrospinal fluid entering the interstitial fluid flows by convection, and the cerebral spinal fluid (CSF)-interstitial fluid (ISF) exchange within the brain parenchyma. (3) After washing the waste proteins from the tissue, it flows into the perivenous space, which is the perivascular space around the deep-draining vein, and is subsequently discharged outside the brain. (Reprinted by permission from Macmillan Publishers Ltd:

Figure 9 .

 9 Figure 9. Timeline of significant events and discoveries pertaining to brain fluid transport. The black arrow superimposed over the cerebral artery illustrates the passage of time. The timeline spans over millenniums and includes the first clinical description of cerebral spinal fluid (CSF) in ancient Egypt found in the Edwin Smith papyrus scroll from 1500 BC, the first morphologically correct drawing of the ventricular system by Leonardo da Vinci in 15th century, and the description of CSF flow as the third circulation by Harvey Cushing in the 20th century. Many other substantial discoveries extending beyond those illus-trated in the figure established the foundation to our modern understanding of brain fluid transport but due to space limitations cannot be included in the fig-ure (see FIGURE 3). Images are courtesy of The New York Academy of Medicine Library (Edwin Smith Papyrus scroll) and from Ref. 31 (Da Vinci drawings; with permission from Springer International), (schematic by Harvey Cushing; with

Figure 10 :

 10 Figure 10: Plausible fluid transport routes and their dependence on the polarized expression of aquaporin 4 (AQP4) at perivascular astrocytic end-feet. Directional fluid transport from arterial to venous vasculature would require differential conditions at the site of influx (left) and efflux (right ) of fluid. Possible flow pathways can be largely divided into transcellular or paracellular transport routes. Transcellular routes (top row) would consist of direct entry of fluid through AQP4 channels, and could be subdivided into the following: 1: trans-astrocytic fluid transport, where fluid enters the astrocyte at the endfoot and continues through the soma and the greater glial syncytium following a largely intracellular pathway, or 2: trans-endfoot fluid transport, where fluid enters into the endfoot and rapidly exits into the extracellular space (ECS) without entering the soma, thus following a largely extracellular pathway interspersed with short segments of intracellular transport. Alternatively, fluid may also follow paracellular routes (bottom row), which would consist of fluid transport through the clefts formed by neighboring endfeet. This primarily extracellular transport route would rely indirectly on AQP4 water channel expression. 3: Hydrostatic pressure gradients, between the arterial and venous sides of the extracellular space could drive directional fluid flow toward the region of lower pressure (P artery > P ECS > P vein). 4: osmotic pressure gradients could be generated by astrocytic ionic fluxes (e.g., K1 spatial buffering) that in part depend on AQP4 polarization. Directional transport of extracellular osmoles could potentially drive fluid entry between endfeet toward efflux sites (rpvein > rpECS > rpartery). PVS, perivascular space.Figure and legends from[START_REF] Rasmussen | Fluid transport in the brain[END_REF]

  Figure and legends from[START_REF] Rasmussen | Fluid transport in the brain[END_REF] I.4.2 -Physiological functions of the glymphatic systemThe glymphatic system plays roles in brain fluid circulation, metabolite clearance, delivery of nutrient and in volume neurotransmission. Glymphatic system thus helps the brain to get rid of excess of extracellular fluid, metabolite and protein waste products (Fig.11).

Figure 11 .

 11 Figure 11. Glymphatic system and its role in clearance, nourishment, and volume transmission. The glymphatic system serves various physiological functions. By establishing extracellular fluid efflux, the glymphatic system clears the brain parenchyma of extracellular metabolites and waste products. Furthermore, the influx of cerebrospinal fluid (CSF) to the brain along periarterial spaces also provides nourishment to the brain, whereby the glymphatic system distributes glucose to neurons and astrocytes, and delivers lipids and apolipoprotein E arising in the choroid plexus throughout the brain. Certain vitamins like folate and ascorbate are exclusively delivered to the brain parenchyma by CSF. Finally, the glymphatic sys-tem may play a role in local volume transmission, which refers to the transport of "spill-over" neurotransmitter from a synapse to neighboring non-post-synaptic cells. Glymphatic flow in the extracellular space would theoretically increase local volume transmission, as may occur during nonrapid eye movement (NREM) sleep, when large volumes of CSF wash through the neuropil.

  reason underlying the aforementioned controversial observations (at the cellular, network and MRI level) on the role of astrocyte AQP4 in brain fluid transport, might be that chronically inactivating astroctye AQP4 by genetic interventions have caused variable alterations in cerebral water environment, or induced unexpected cellular adaptation. Indeed, it has been observed that in AQP4 KO mice, the water content in the brain parenchyma is significantly altered compared to intact animals (Haj-yasein et al., 2011); Fig. 13). This, likely have rendered it difficult to clarify the function of astrocyte AQP4 in brain fluid circulation. Hence, acutely targeting AQP4 water channel is necessary to pinpoint its role in astrocyte volume regulation in brain physio-and pathology.

Figure 13 :

 13 Figure 13: Water content in mouse brain in conditional AQP4 KO mice (Haj-Yasein et al., 2011).

  mouse model and combining the selective targeting of AQP4 with TGN0-020, I developed ex vivo and in vivo fluorescent intensity-translated astrocyte volume imaging methodology and resorted to diffusion MRI to assess brain water diffusion. The results of my PhD study show that AQP4 water channel plays a tonic role in inhibiting astrocyte swelling, suggesting AQP4 mediating a constitutive water efflux from astrocytes. Acutely blocking AQP4 by TGN-020 caused astrocyte swelling in mouse somatosensory cortex. Accordingly, AQP4 inhibition facilitated osmotically evoked astrocyte swelling while delayed the shrinking responses. I then observed that manipulating astrocyte AQP4 in vivo altered global water diffusion in mouse brain using diffusion-weighted MRI. Together, my study proposes an asymmetric engagement of AQP4 in homeostatic astrocyte water flux which helps to maintain the brain water diffusion. This finding provides an updated mechanism for understanding the cellular basis of astrocyte AQP4 in brain fluid transport and helps to better understand its involvement in brain glymphatic system.I.6 -Material and methodsBrain slices preparation, and in vivo preparationExperiments were undertaken in accordance with European Community guiding principles on the care and use of animals (86/609/CEE), and the rules of the host institute. Coronal slices comprising somatosensory cortex and hypothalamus were acutely prepared from C57BL/6 mice of both sexes at ages as indicated. Mice were deeply anesthetized by isoflurane (Axcience) evaporation in a closed plexiglass box. For mice less than 4 weeks old, the brain was taken out and placed in ice-cold artificial cerebrospinal fluid (aCSF, mM: 124 NaCl, 4.5 KCl, 1.2 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, and 10 D-Glucose), and cut into 300-μm thick slices by a vibratome (Leica VT1200S). For mice older than 4 weeks, the brain was placed in a modified aCSF for slicing (in mM: 30 NaCl, 4.5 KCl, 1.2 NaH2PO4, 1 MgCl2, 26 NaHCO3, 10 D-Glucose, and 194 sucrose)[START_REF] Jiang | Dysfunctional Calcium and Glutamate Signaling in Striatal Astrocytes from Huntington's Disease Model Mice[END_REF] maintained at 4 °C during sectioning. Brain slices were recovered in normal aCSF at 37 °C for about 1 h. Mice of similar age were used also for in vivo labeling of astrocytes, sulforhodamine B (10 mg/ml, Sigma) was intraperitoneally injected into awake mice at a dose of10 μL/g[START_REF] Appaix | Specific in vivo staining of astrocytes in the whole brain after intravenous injection of sulforhodamine dyes[END_REF].

Fiberphotometry 4 -

 4 photometry system (FP3002). A branched fiber optic patch cord (BFP_200/230/900-0.37_FC-MF1.25, Doric Lenses) connected to the fiber photometry apparatus was attached to the implanted fiber optic cannula using a cubic zirconia sleeve. To record fluorescence signals from SRB, light from 560-nm LED; for GCaMP6f, light from a 470-nm LED was band-pass filtered, collimated, reflected by a dichroic mirror and focused by a ×20 objective. LED light was delivered at a power that resulted in 50 µW of 560-and 470-nm light at the tip of the patch cord.Emitted SRB and GCaMP6f fluorescence was band-pass filtered and focused on the sensor of a CCD camera. To account for auto-fluorescence and possible motion artifacts during testing, a 410-nm light stimulation corresponding to isosbestic GCaMP6f signal was used. This signal was similarly directed into the brain and subsequently measured with the CCD camera.Simultaneous recording of -560 470-and 410-nm channels was achieved through an integrated camera and image splitter (Flir Blackfly S series). Signals were collected at a rate of 1 Hz for SRB and 16 Hz for GCaMP6f and visualized using the open-source software Bonsai 2.4 (http://bonsai-rx.org). Results of SRB recording were analyzed in Igor software (Wavemetrics),

  role of AQP4 in astrocyte volume regulation, we sought to image in situ in a real-time manner the astrocyte volume dynamics. Astrocytes bear extended three-dimensional morphology sitting in nanoscale distances with other cell types in the brain (Bindocci et al., 2017) (Fig. 14). Performing video-rate three-dimensional imaging at nanoscale resolution remains difficult. I therefore developed fluorescent intensity-translated (FIT) imaging to follow astrocyte volume change in high temporal resolution. In this method, selective delivery of a highly water soluble fluorescent dye in astrocytes can enable the imaging of its volume change by following the associated fluorescence oscillation. Fluorescence increases upon transient shrinking because it augments the astrocyte dye density in spatially limited focus depth and the field of view, while fluorescence signal decreases during swelling (Fig. 15).

Figure 14 :Figure 15 :

 1415 Figure 14: Complex morphology of mouse brain astrocyte (From bindocci et al 2017)

  Figure 16: In vivo chemical labelling of astrocytes in mouse brain. (A) Illustration of experimental protocol for in vivo astrocyte labelling by SRB. Coronal slices were prepared acutely for ex vivo imaging of astrocytes in S1 cortex using epifluorescence microscopy, which enables the acquisition of the fluorescence along the z-axis, so suitable for following the global fluorescence signals caused by volume changes. (B) Representative fluorescence images of astrocytes in S1 cortex labelled by SRB under low (×20) and high magnification (×63).

Figure 17 :

 17 Figure 17: Validation of in vivo SRB labelling of mouse cortical astrocytes. To resolve fine astrocytic structure dual-color, light-sheet fluoresce microscopy was used. Experiment was performed in the GFAP-EGFP mice. Colocalization was observed between the red fluorescence dyeSRB and EGFP-positive astrocytes. Note that, in mouse cortex, just a subpopulation of astrocytes express GFAP, while SRB labels more astrocytes, which yields a sub-regional colocalization but meanwhile excluding any potential bleed through from the red SRB imaging channel to the EGFP channel.

Figure 18 :

 18 Figure 18: Experimental validation of The FIT imaging method of astrocyte volume dynamics. (A) Top, time course of SRB fluorescence following hypo-osmotic solution application for different time periods. Bellow quantification of swelling amplitude (maximum fluorescence drop) and speed (temporal initial slope). (B) Top, time course of SRB fluorescence following hyper-osmotic solution application for different time periods. Bellow quantification of shrinking amplitude (maximum fluorescence peak) and speed (temporal initial slope). (n=52 astrocytes, from 8 slice from 4 mice).

Figure 19 :

 19 Figure 19: Protocol for acute blocking of astrocytic AQP4 by TGN-020.

Figure 20 :

 20 Figure 20: Selectivity of TGN-020 for AQP4 validated using oocytes expressing different type of aquaporin channels (Toft bertelsen et al 2020).

Figure 21 :

 21 Figure 21: Acutely blocking AQP4 in astrocytes in situ in brain slices causes swelling. (A) Acute blocking of astrocytic AQP4 should if equally bidirectional should not impact astrocytic volume. (B) Imaging protocol for acute blocking of AQP4 by TGN-020 (20 µM) or control (CTRL). (C) Time course of SRB fluorescence following TGN-020 application or CTRL solution

Figure 22 :

 22 Figure 22: Potential explanation for the tonic role of AQP4 in astrocyte volume homeostasis. The results suggest that AQP4 mediates a net water efflux in basal conditions. This supports the observation that the acute AQP4 blocking by TGN-020 induces a transient swelling of astrocytes (right, from dotted line changing to the solid line).

  astrocyte swelling in presence of TGN-020 or control solution. Astrocyte swelling caused a decreased SRB fluorescence in both conditions. (C) Comparison between different swelling conditions in presence of TGN-020 or control solution. In presence of TGN-020 the swelling starting time (time between the reaching of the hypo-osmotic solution and the first changing pointing fluorescence curve), the time to swelling peak and the maximum swelling amplitude decreased compared to control. On the other hand initial astrocyte swelling speed was increased in presence of TGN-020 compared to control. (n=12 astrocytes, from 3 slices from 2 mice).

Figure 24 :

 24 Figure 24: Blocking AQP4 acutely impaired astrocyte volume shrinking: (A) Imaging protocol for astrocyte shrinking when blocking AQP4 by TGN-020 (20µM) or in control conditions (CTRL). Astrocyte shrinking was triggered by application of a 400 mOsM solution. (B) SRB fluorescence time course following astrocyte shrinking in presence of TGN-020 or CTRL. Astrocyte shrinking caused an

Figure 25 :Figure 26 :

 2526 Figure 25: Acutely blocking AQP4 triggered swelling-associated astrocyte Ca 2+ increase. (A) Astrocyte swelling was triggered by application of a 100 mOsM solution, which triggered Ca 2+ increase. Brain slices were taken from Glast-Cre ERT2 /GCaMP6 floxP mice where GCaMP6f is specifically expressed in astrocytes. Light-sheet fluoresce microscopy was used in order to sensitively track Ca 2+ signals. Left, swelling induced Ca 2+ signals time courses of individual responses regions. Middle to right, bar comparison between the signals integral values (area under the curve) and their histograms of the signals before and after hypo-osmotic solution challenge. (B) Left, TGN-020 induced Ca 2+ signals time courses of individual responses regions. Middle to right, bar comparison between the signals integral values (area under the curve) and their histograms of the signals before and after TGN-020 application.(n=89 astrocytes, from 8 slices from 5 mice)

Figure 27 :

 27 Figure 27: Fiberphotometry fluorescence recording of astrocyte volume in vivo. (A)Fiberphotometry allows the recording of temporal fluorescence signals from populations of cells in freely moving animals.

Figure 29 :

 29 Figure 29: Diffusion weighted magnetic resonance imaging principles. (A) Contrast and signal levels in the diffusion-weighted image (left) reflect water diffusion behavior (random walk) (right). Diffusion behavior is modulated by tissue structure at the cellular level (middle): For instance, diffusion can be restricted within cells, water may escape when cell membranes are permeable and might then experience a tortuous pathway in the extracellular space (hindrance). (B) In the presence of a magnetic field gradient (variation of the magnetic field along one spatial direction), magnetized water molecule hydrogen atoms are dephased. The amount of dephasing is directly related to the diffusion distance (a few micrometers) covered by water molecules during measurement (a few tens of milliseconds). Given the great many water molecules experiencing individual random walk displacements, the overall effect of this dephasing is an interference, which reduces MRI signal amplitudes. In areas with fast water

Figure 31 :

 31 Figure 31: ADC mean values following saline or TGN-020 injection derived from diffusion MRI recordings. Time-course of averaged ADC changes in (A) the cortex, (B) the striatum, and (C) the hippocampus. Arrowhead indicates the injection of saline or TGN020. Averaged ADC changes between 30 and 60 min after injection of saline or TGN020 in (D) the cortex, (E) the striatum, and (F) the hippocampus. *p<0.05 by paired t-test following ANOVA (n= 10 mice for saline injection, n= 9 mice for TGN-020 injection.)

Figure 33 :

 33 Figure 33: Astrocyte adaptations in pathologic brain (from Sofroniev et al 2010)

Figure 34 :

 34 Figure 34: Excitatory and/or inhibitory signals trigger Ca 2+ elevations in astrocytes and lead to gliotransmitter release. Scheme depicting input signals that trigger astrocyte Ca 2+ elevations and respective transmitter release. Both excitatory (+) and inhibitory (-) signals cause global or focal Ca 2+ elevations in astrocytes (Left), and precede gliotransmitter release that might exert excitation or inhibition of neighboring synapses (Right). For each reference, the region studied is indicated in black

Figure 36 :

 36 Figure 36: Astrocytic Ca 2+ increases are associated with a transient increase in cell volume. (A) Exposure to ATP (100µM) induces swelling of cultured astrocytes. Confocal vertical cross-sectional images of confluent astrocyte cultures with 3-5 cells in the field of view loaded with calcein AM (5M for30 min) were constructed from repetitive x-z line scans at 488 nm excitation. Two images of crosssectional area before (red) and 1 min after the exposure to ATP (green) are overlapped to indicate the change in cell volume. Over-lapped areas (no change before and after ATP exposure) are displayed as white. Hypotonicity (214 mOsM) also induced cellular swelling. (B) Quantification of relative changes in cross-sectional areas 1 min after addition of vehicle (control,n12); ATP (100M,n23); ATP to cultures preloaded with BAPTA (20M for 30 min,n11); UTP (100M,n15) and hypotonicity (214mOsM,n6).*,P0.01 compared with control, Tukey-Kramer test. (C)Coulter counter analysis of relative changes in astrocytic cell volume evoked by ATP. ATP exposure of astrocytes in suspension triggered a transient increase in cell volume at 30 and 60 sec. (C Inset) Hypotonicity induced a large

Figure 37 :

 37 Figure 37: Astrocytic Ca 2+ signal modulates morphology and volume dynamics. A, Astrocytic Ca2+ signaling is indispensable in maintaining cellular morphology and plasma membrane dynamics. The plasma membrane of primary cultured mouse cortical astrocytes was labeled by Lck-EGFP. In CTR condition, thin filaments attaching to the plasma membrane could be observed (red line scan), and the kymograph (left bottom) shows the dynamic changes of the local plasma membrane (positioned by the white dotted line) over time. In contrast, in astrocytes where intracellular Ca 2+ signal was chelated by BAPTA-AM, near-membrane thin filaments were retracted and plasma membrane became static over time. B, Ca 2+ rise in astrocytes alters cellular volume. The Ca 2+ red chemical indicator Xrhod-1 and the membrane-impermeable volume marker calcein were coloaded into cultured astrocytes in their AM form. Local application of 100 µM glutamate triggered transient Ca 2+ elevation accompanied by cell volume change reflected by the corresponding decrease in Calcein fluorescence. (Unpublished data of the host team).

Figure 38 :

 38 Figure 38: Impaired glymphatic system in Alzheimer's disease. (A) Vascular endfeet of astrocytes create the perivascular spaces through which CSF enters the brain and pervades its interstitium. CSF enters these perivascular spaces from the subarachnoid space and is propelled by arterial pulsatility deep into the brain, from where CSF enters the neuropil, facilitated by the dense astrocytic expression of the water channel AQP4, which is arrayed in nanoclusters within the endfeet. CSF mixes with fluid in the extracellular space and leaves the brain via the perivenous spaces, as well as along cranial and spinal nerves. Interstitial solutes, including protein waste, are then carried through the glymphatic system and exported from the CNS via meningeal and cervical lymphatic vessels. (B)Amyloid-b plaque

  II.3.1 -Astrocyte Ca 2+ signal dysregulation by neurotoxic amyloid peptideExcessive amounts of amyloid β (Aβ) peptide have been suggested to dysregulate synaptic transmission in Alzheimer's disease (AD). As a major type of glial cell in the mammalian brain, astrocytes regulate neuronal function and undergo activity alterations upon Aβ exposure. Yet the mechanistic steps underlying astrocytic responses to Aβ peptide remain to be elucidated.

Figure 39 :

 39 Figure 39: Aβ plaque formation time course in hAPP J20 mice[START_REF] Mucke | High-Level Neuronal Expression of Aβ1-42 in Wild-Type..Synaptotoxicity without Plaque Formation.pdf[END_REF]

Figure 40 :

 40 Figure 40: Experiments for exanimating the effect of AQP4 on Aβ clearance in AD mouse model (on going).

  Secondly, thus far the effect of regulating astrocyte AQP4 on the clearance of amyloid β or other waste molecules remains to be clarified. My current results so far suggest that AQP4 plays an important role in maintaining the volume homeostasis of astrocytes in situ, making it possible to target astrocyte AQP4 to regulate extracellular space, and the brain fluid transport.Further experiments to establish the efficacy of this treatment on amelioration of cognitive behavior will pave the way for preclinical tests. There is a change in the polarization AQP4 distribution at the astrocytic endfeet in several neuropathologies including Alzheimer's disease (Ikeshima-Kataoka, 2016) So alternatively to understands the mechanism of the disruption in AQP4 localisation under neuropathology will help to rescue the functional distribution thereby preventing disease development. A recent report suggest that the clinical drug trifluoperazine can modulate the trafficking of AQP4 in vivo[START_REF] Kitchen | Article Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema ll ll Article Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema[END_REF] which ameliorates the recovery from brain edema. Therefore this strategy could be also used to control AQP4 dysfunction in neurodegenerative disease. Thirdly, my Ph.D study also shows that diffusion MRI is noninvasive method and sensitive for brain fluid transport detection. This technique could be used for early diagnostics of brain fluid transport disruption linked pathologies. It can further extended to follow the development of other neurological disease, and metabolism disorders in the brain.III.1 -Contribution to the understanding of brain astrocytes adaptation to systemic metabolic disorders. III.2 -Contribution to the mechanistic study on the cellular trafficking of the Ca 2+ ion channel TRPV

Table 1 :

 1 CSF and blood plasma composition.

Table 2 :

 2 Distribution and molecule transport of the different type of aquaporin in the CNS.

Table 1 : CSF and blood plasma composition (Brown et al., 2004)

 1

Table 2 : Distribution and molecule transport of the different type of aquaporin in the CNS. Adapted from (Trillo-contreras et al 2022)

 2

mediates water influx, hence inhibition of AQP4 over extended time windows reduces the total amount of water influx thereby the absolute swelling amplitude. During my Ph.D study I have obtained an international exchange grant between the CNRS and the Japanese Society for Promotion of Science (JSPS). This allowed me to perform in vivo DW-MRI experiment in the National institute of Advanced industrial Science and technologies (AIST) in Tsukuba, Japan in Dr. Tomokazu Tzurugizawa's lab. We used a 7T MRI to image the role of astrocyte AQP4 in brain water diffusion. The effect of AQP4 blocking by the watersoluble TGN-020 on brain water diffusion was examined in mildly anesthetized mice by medetomidine. We followed the water diffusion rate in the mouse brain at different time points following TGN-020 application (Fig. 30). Compared to the control saline injection in vivo application of TGN-020 significantly increased the water apparent diffusion coefficient (ADC) at different cerebral areas including the cortex, hippocampus and the striatum (Fig. 31). This result hence indicates that astrocytic AQP4 plays a critical role in mediating brain water diffusion, a process that can be imaged by the DW-MRI. [START_REF] Nedergaard | Glymphatic failure as a final common pathway to dementia[END_REF] Among the neurodegenerative disorders, Alzheimer's disease (AD) severely impacts the life quality of patients and imposes recurrent burden on the society [START_REF] Scheltens | Seminar Alzheimer ' s disease[END_REF]. As a potential risk, the excessive brain accumulation of the amyloid β (Aβ) peptide is implicated in AD pathogenesis and the cognitive deficits [START_REF] Scheltens | Seminar Alzheimer ' s disease[END_REF]. Therapeutic strategies aiming at relieving cerebral Aβ depositions have provided promising results for further pre-clinical and clinical investigations [START_REF] Lobello | Targeting Beta Amyloid : A Clinical Review of Immunotherapeutic Approaches in Alzheimer ' s Disease[END_REF].

Efflux of Aβ across the brain vasculatures underlies its clearance [START_REF] Roberts | Amyloid-b Efflux from the Central Nervous System into the Plasma[END_REF], hence highlighting the important involvement of glymphatic system. Glymphatic clearance has been suggested to rely on the water channel aquaporin 4 (AQP4), the most abundant water channel in the mammalian brain and predominantly expressed in astrocytes [START_REF] Papadopoulos | Aquaporin water channels in the nervous system[END_REF]. Despite the need for further validations [START_REF] Smith | Test of the ' glymphatic ' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma[END_REF][START_REF] Mestre | Aquaporin-4-dependent glymphatic solute transport in the rodent brain[END_REF], recent data in rodent models show that genetic or pharmacological inactivation of AQP4 impairs the glymphatic clearance of Aβ and tau [START_REF] Harrison | Impaired glymphatic function and clearance of tau in an Alzheimer's disease model[END_REF][START_REF] Rosu | Inhibition of Aquaporin 4 Decreases Amyloid A β 40 Drainage Around Cerebral Vessels[END_REF].

Manipulating astrocyte AQP4, therefore, might regulate the efficacy of glymphatic clearance so to ameliorate AD amyloidopathy. However, it remains elusive how glymphatic clearance is impaired in brain pathology such as AD-associated amyloid deposition.

II.2 -Hypothesis and initial explorations

Chapter III: Collaborative works

The scientific knowledge from my PhD study has also fostered collaborative works for understanding:

I)

Brain astrocyte activity remodeling in obesity-associated metabolic syndromes. The results of this part contribute to the discovery that the calcium signaling of hypothalamic astrocytes undergoes heterogeneous remodeling in anatomically defined nuclei implicated in systemic metabolism regulation. This work has helped to delineate the important participation of astrocytes in energy balance control. This part of the study has been published (Herrera Moro Chao et al, 2022).

II)

The molecular mechanisms underlying the trafficking of calcium-permeable transient potential receptor ion channels. This work suggests testable molecular targets for interrogating the calcium signaling mechanisms in nervous cells including astrocytes (Tian et al, 2019).

List of publications, scientific communications, and rewards

Publications from the PhD study:

Academic laureats from the PhD study:

France Alzheimer Foundation, Individual Fellowship, 2021-2022

Japan Society for the Promotion of Science (JSPS) -CNRS Summer program, 2022