Seba, Estefani, Graciela Irma Diego

Natalia Karen

Pedro Patricio

Soham, Irini, Yash, Olena, Bodamyr, Katjia Jimmy Mingyue

Nina Lidiia

Oriane Valerie Goguel

Pierre-Yves Musso

Eloise Ghislain

Gonzalo

Johann Lisa

Pierre-Yves Plaçais

Raquel Alexandre

Jun, Khanh Yasmine Olivier

Bmm Brummer

Chk Chaski

-hydroxybutyrate

to be part of this lab. Particularly to Thomas, for your constant motivation, innate curiosity and always have the eager to go for a further step beyond knowledge. I appreciate a lot your passion and genuine interest in neuroscience, it just simply inspires to anyone who see it. Also, for your scientific rigor, expertise and for always aspire to generate high-level science. On the other hand, I would like to thanks Alice for your rigor, order, criticism and scientific support. Thank you both for enriching my scientific culture and critical thinking throughout these years.

I would also like to thank my family. They have always been a constant support throughout these years. To my mother for her wisdom, perseverance and for instilling in me her passion for every type of knowledge, always aspiring to be the best. Thanks to my father for his love, patience and intuition. For instilling in me his mental strength to bravely defy every obstacle that arises in life. And to my brother for always

People I met and made my short stay in Seville more enjoyable and an incredible experience: Mati, Undu and Karina. To Mati and Undu, for the trips around Andalusia and Portugal. Also for sharing good times around the Guadalquivir, talking about science, life, family, among others. And in general for sharing good times around the atmosphere of joy that surrounds the Sevillians. Also to the people of the IBiS laboratory who were a great support during my stay, particularly Alberto Pascual for teaching me the world of proteomics and mass spectrometry, and for allowing me to work in his laboratory. Also, to the people at the mass spectrometry facility for trying a thousand times to help me with the protein identifications I carried out at the beginning of my PhD.

I would also like to thanks to Jorge Campusano, my first mentor, for introduce me into the world of science, neurobiology and Drosophila, when I was an "unmotivated 'biology-allergic' Mechanical Engineer". For have given to me the opportunity to work in your laboratory (despite knowing absolutely nothing about biology), and share your knowledge, experience and values. I appreciate your honesty, words of enthusiasm and encourage for making me pursuing a scientific career and for being always attentive across all this years of my well being.

To Jimena Sierralta, for allowing me work at BNI, for her scientific advises, motivation and constant support until nowadays. Moreover, to Felipe Barros and Bredford Kerr, both who gave me the opportunity for coming to Paris for a short internship, which later on become into a PhD.

Also to the people I met during my stay at Jorge's and Jimena's teams and who we still keep in close contact nowadays: Vicente, Beatriz, Elsa, Sergio, Claudia, Daniela, Nico Fuencha, Pancha, Aquiles, Mauricio. Also, thank you for encouraging me following the science path career and also for sharing unforgettable moments both in the laboratory, as well as outside of it (en "las uruguayas", "Danoi" and "Bar de René").

Thanks to everyone. Thanks for the ones who motivated and supported me during these years, without any of you I could have never reached it so far.

Gracias totales.

1.1 Upon starvation, ketone bodies oxidation in MB neurons is required for sLT-ARM 1.2 Drosophila MB express MCTs that are necessary for sLT-ARM formation 1.3 Silnoon is a lactate transporter that can trans-accelerate the acetoacetate ketone body

Ketone bodies synthesized in cortex glial cells and exported to the mushroom bodies provide essential energy for sLT-ARM……………………

2.1 Ketone bodies export from cortex glial cells requires Chaski MCT for sLT-ARM 2.2 Ketone bodies synthesis from fatty acids in cortex glia is required to support sLT-ARM 3. Lipid droplets are the local intracellular store of fatty acids in cortex glia, which are used to synthesize and export ketone bodies to mushroom body neurons upon starvation………………………………………. Humans have evolved in environments where they have been intermittently

subjected to extended periods of little or no food. This strong selective pressure allowed that higher cognitive abilities evolved as adaptations enabling the invention of tools and methods of hunting, animal domestication, agriculture, food storage and processing [START_REF] Anton | Flipping the Metabolic Switch: Understanding and Applying the Health Benefits of Fasting[END_REF]. In fact, primitive humans encountered a series of metabolic challenges. The need for food searching in a challenging environment established a need for an efficient utilization of the different metabolic fuels under variable internal states. In the need for survival, humans needed to hunt animals, traverse long lands and face intermittently periods of food scarcity. During evolution, to withstand these metabolic challenges, brain function must have acquired the capacity to work costeffectively to adapt to these environmental stress factors in an efficient manner.

Thus, cognitive functions are needed for food-seeking behaviors and processing but at a high-energy cost.

To survive in challenging environments, individuals must have the ability to use different fuels. Individuals need to manage their energy when food is available and perform efficiently in prolonged periods of energy restriction, when food is not available [START_REF] Cahill | Fuel metabolism in starvation[END_REF]. When food is temporally not available, therefore, a rapid shift must occur between the metabolites used in the fed versus food deprived animals, to allow an individual and its brain to keep on working efficiently. Consequently, animals whose brain works optimally in periods of energy deprivation have a greater survival advantage [START_REF] Cahill | Fuel metabolism in starvation[END_REF].

Different brain cognitive skills such as space navigation, decision-making, sociability and creativity have been strongly linked to the success in acquisition of food [START_REF] Mattson | An Evolutionary Perspective on Why Food Overconsumption Impairs Cognition[END_REF]. In fact, food-gathering behaviors require a wide range of cognitive skills, such as remembering the past, planning for the future, and interpreting the behavior of other individuals. For this, humans have had to socialize, acquire information from their environment or from other individuals, and create hunting methods and tools that allow them to obtain food efficiently [START_REF] Berbesque | Hunter-gatherers have less famine than agriculturalists[END_REF]. These features are not exclusives of humans but also of other mammals. For example, wolf packs kill their prey only once every two weeks, and thus are constantly facing long food shortage periods (Mcnulty et al., 2014). Their success depends mainly on a number of factors, such as how brain perform efficiently during hunting in absence of food, how they learn and memorize a strategy to capture and kill their prey in this period and the optimal utilization of energy reservoirs prior and during hunting (McNulty et al., 2014).

Thus, an effective use of energy and an efficient functioning of the brain during food deprivation are crucial to favor the survival of the individuals. Nonetheless, it remains poorly known how brain functions are maintained during long periods of food deprivation.

In fact, despite the brain represents only the 2% of the total body mass, the human brain consumes 20% of the total energy (Mink et al., 1981). Cerebral energy homeostasis has the highest priority over the animal energy metabolism, this is known as the theory of the "selfish brain" (Peters et al., 2004). In fact, human brain mass remains constant during periods of food deprivation, while that of other organs such as the heart, liver, kidney and pancreas lose about 40% of their weight [START_REF] Krieger | Über die Atrophie der menschlichen Organe bei Inanition [On the atrophy of human organs in inanition[END_REF]. This provides evidence that the brain egoistically demands energy from the body to maintain its own energy homeostasis.

Therefore, we can expect that food scarcity imposes a metabolic challenge for the brain and the urgent need for metabolic fuels has created an adaptive response to meet its metabolic demands. But, at which cost?

The brain metabolic cost and adaptations that occur during the transition between the fed and food-deprived states remains still poorly investigated. In my thesis, we studied Drosophila aversive olfactory memory in order to decipher the interplay between metabolism and long-lasting memory formation upon periods of food restriction. In absence of food, how does the brain regulate memory formation to withstands periods of food scarcity? Moreover, what are the metabolic fuels used by the brain to sustain the formation of memory upon starvation? And eventually which are the brain cell types involved in this process?

In Drosophila, the neural networks involved in long-lasting memory formation have been precisely described [START_REF] Bouzaiane | Two independent mushroom body output circuits retrieve the six discrete components of Drosophila aversive memory[END_REF]. Therefore, we focused our study on the main structure involved in memory formation, the mushroom body (Aso et al., 2014). Our aim was to understand which metabolic pathways were involved in mushroom body neurons during memory formation upon food restriction. We further aimed to assess if neuronal energy metabolism is supported by specific substrates derived from glial cells during memory formation upon food deprivation.

In the following sections, since the brain essential needs are the same at the cellular level in fed and food deprived conditions, I will first introduce the energy needs of the brain that enable it to function efficiently under fed physiological conditions. Then, I will present relevant information on how the brain adapts its functioning to challenging conditions such as starvation and many unresolved questions regarding the cellular and molecular mechanisms underlying to brain functioning during this condition. Next, I will provide key information on how Drosophila can be used as a learning and memory model to investigate relevant questions about brain mechanisms during food-deprived states. Finally, I will introduce glial cells and their function as metabolic supporters of neurons, which could have relevant implications for memory formation during starvation states.

Part I: Energy metabolism in the central nervous system

Brain cognitive functions have been shaped throughout evolution by various environmental factors, including food availability (Camandola and Mattson, 2009). In fact, human brain consumes 20% of the total energy of the body (Mink et al., 1981). Among brain cells, neurons consume >90% of this energy for their own demands, while the remaining energy is used by glial cells [START_REF] Harris | Synaptic energy use and supply[END_REF]. More specifically, neurons use energy primarily for synaptic signaling, including pre-and post-synaptic mechanisms: action potentials, resting membrane potentials, vesicular transport, ion gradient restoration and synaptic vesicle cycle [START_REF] Harris | Synaptic energy use and supply[END_REF]. Nevertheless, to obtain this energy, neurons need a local metabolic support. The main metabolic supporters of neurons are glial cells, and they play a key role in providing energy substrates, such as glucose or lactate, to maintain brain function [START_REF] Jha | Glia-neuron energy metabolism in health and diseases: New insights into the role of nervous system metabolic transporters[END_REF]. Understanding energy metabolism of brain cells and their metabolic interactions will increase our understanding of how the brain works and adapts to challenging physiological conditions. Moreover, it will help to elucidate essential mechanisms related to cognitive functions and also in the understanding of certain neurodegenerative diseases, which have a metabolic impairment component [START_REF] Mattson | Impact of intermittent fasting on health and disease processes[END_REF].

Thus, understanding how this energy is distributed in glia and neurons and even within the same cells will be relevant for understanding brain functioning at the metabolic level. But what are the processes that use and regulate energy flux in the brain?

In this section I will present what are the brain energy needs, more specifically, the local energy needs at the cellular level both in neurons and glial cells. Furthermore, I will describe how the brain take up the nutrients necessary for brain functioning and the different metabolic substrates used to maintain its own metabolic demands under fed physiological conditions.

Brain metabolism in the fed state

To allow an optimal brain function, neurons and glial cells require energy to sustain essential cellular processes [START_REF] Harris | Synaptic energy use and supply[END_REF]. In the fed state, this energy is obtained from the catabolism of energy-rich compounds such as carbohydrates and lipids derived from circulation [START_REF] Ebert | Energy contribution of octanoate to intact rat brain metabolism measured by 13C nuclear magnetic resonance spectroscopy[END_REF]Belanger et al., 2011), which provide energy in the form of ATP. Then, this energy must be adequately distributed among the processes that demand energy in these cell types. However, what are these processes that demand energy?

The brain energy needs

What are the processes that use energy in the brain? Based on anatomical and physiological data of the gray matter of rodents, where excitatory synapses are present in high number, it has been estimated that demanding processes such as action potentials, consumes nearly 22% of the energy. On the other hand, synaptic transmission mechanisms accounts for approximately 64% of the energy budget [START_REF] Attwell | An energy budget for signaling in the grey matter of the brain[END_REF][START_REF] Harris | Synaptic energy use and supply[END_REF][START_REF] Sengupta | Balanced excitatory and inhibitory synaptic currents promote efficient coding and metabolic efficiency[END_REF]. Synaptic transmission budget includes postsynaptic receptors and the following ion fluxes (50%), neurotransmitter recycling (4%), presynaptic calcium entry (5%) and vesicle cycling (5%). In addition, membrane potentials for neurons and glia represent 14% of the energy consumption (Figure 1A). This energy budget represents the 75% of the total energy needs of the brain, whereas the other 25% is devoted to housekeeping mechanisms. Housekeeping mechanisms corresponds to basal processes needed in cells for proper functioning, including the turnover of macromolecules such as lipids and proteins, either in neurons or glial cells [START_REF] Attwell | An energy budget for signaling in the grey matter of the brain[END_REF]. Interestingly, there is a strong correlation between the compartment where energy is needed and the density of mitochondria. The mitochondrial fraction observed predicted the energy consumption in these neuronal compartments to be nearly 60% for dendrites and soma whereas for axons terminals is approximately 35% (Figure 1B), which is consistent with the evidence exposed above. Moreover, the energy budget for glial cells is mainly used for membrane potentials (3%) and is considered to be unexpectedly low due to the small numbers of mitochondria (Figure 1B).

Thus, the energy budget of the cell is divided into different costly energy processes. However, how does the brain get this energy and where does it come from specifically? In the following section I will detail how the brain obtains energy precursors from the circulation, and how they are taken up by crossing the so-called BBB.

Brain specificity: the blood brain barrier

Energy production in the brain is based on the carbohydrates catabolism (Belanger et al., 2011), with glucose being the main substrate obtained from the blood. The breakdown of glucose generates reducing agents such as NADH and FADH 2 which can be used as electron donors for OXPHOS and produce energy in the form of ATP (Nelson and Cox, 2003). Glucose is taken up from circulation and needs to be taken up through the so called blood-brain barrier (BBB). The BBB is necessary to preserve a proper microenvironment for neuronal survival and function (Daneman and Prat, 2015). The BBB protects against circulating toxins or pathogens that could cause infections and lead to brain dysfunctions, while at the same time allows nutrients to reach the brain. The vertebrate BBB is formed by endothelial cells that surrounds cerebral microvessels (Figure 3). Brain capillaries are surrounded and closely associated with perivascular endfeet of astrocytic glial cells, microglia, pericytes and neuronal processes. The BBB is a dynamic structure that has specific selective transport systems that regulate the paracellular and transcellular traffics of metabolites. In addition, small molecules such as O 2 and CO 2 can freely diffuse through the cellular membranes.

This selective barrier is conserved across many different species (Brown et al., 2018). For instance, the vertebrate and insect BBB equivalent of Drosophila share common features at the cellular level. The CNS of Drosophila is surrounded by two sheet-like glial cells, which forms the equivalent of the mammalian BBB (Part III; Figure 28,29): perineural glia and subperineural glia [START_REF] Stork | Organization and function of the blood-brain barrier in Drosophila[END_REF].

Subperineural glial cells are linked by septate junctions (similar to the vertebrate tight junctions), creating a tight barrier to prevent paracellular diffusion of polar or charged molecules. On the other hand, perineural cells contribute to the barrier selectivity of larger molecules [START_REF] Stork | Organization and function of the blood-brain barrier in Drosophila[END_REF]. In addition, as in mammals, insect BBB offers a chemical protection against toxic compounds, such as

The brain consumes glucose as their main energy metabolite

Initial studies measured diverse metabolites taken up by the mammalian brain under fed physiological conditions. Measurement of the difference between arterial and venous metabolites concentrations at the brain level led to the conclusion that the main metabolite taken up by the brain was glucose [START_REF] Siesjö | Brain energy metabolism and catecholaminergic activity in hypoxia, hypercapnia and ischemia[END_REF][START_REF] Sibson | Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity[END_REF]. Therefore, it was postulated that under fed conditions, the brain primarily consumes glucose to meet its energy needs. One of the first method developed to identify glucose uptake in the brain was the 2deoxyglucose tracer [START_REF] Sokoloff | The [14C]deoxyglucose method for the measurement of local cerebral glucose utilization: theory, procedure, and normal values in the conscious and anesthetized albino rat[END_REF]. Through this method, it was observed that the glucose uptake occurs preferentially in the gray matter of rodents under resting conditions. In fact, in resting conditions, most of the glucose is completely oxidized to CO 2 and H 2 O in the gray matter, following a stoichiometry of nearly ≈6 molecules of O 2 per glucose [START_REF] Mergenthaler | Sugar for the brain: the role of glucose in physiological and pathological brain function[END_REF]. Interestingly, in humans, in tasks demanding high neuronal activity, the cerebral blood flow and glucose consumption increase much more than the use of O 2 [START_REF] Raichle | Brain work and brain imaging[END_REF][START_REF] Dienel | Brain lactate metabolism: the discoveries and the controversies[END_REF], suggesting that metabolic needs during high neuronal activity is met partly by a complete oxidation of glucose and also but more prominently by non-oxidative glucose metabolism (anaerobic glycolysis). Thus, under fed conditions brain metabolic needs in resting state and during neuronal activity are met by blood circulating glucose. However, how glucose enters the brain passing the BBB will be the focus of the following section.

Glucose transport into the brain

Brain activity increase cerebral glucose utilization in humans [START_REF] Raichle | Brain work and brain imaging[END_REF]. In vitro and in vivo studies have shown that glucose transporters expression parallels changes in cerebral glucose utilization [START_REF] Choeiri | Glucose transporter plasticity during memory processing[END_REF][START_REF] Simpson | Supply and demand in cerebral energy metabolism: the role of nutrient transporters[END_REF]. Thus, an efficient glucose transporting mechanism is needed to meet brain glucose metabolic demands. Glucose is transported specifically through the BBB by the family of glucose transporters (GLUTs) into brain circuits (Figure 4). In the mammalian brain, GLUTs are abundant in endothelial cells, astrocytes endfeets and neurons. Specifically, two types of glucose transporters are present strongly in the mammalian brain: GLUT1 and 5). Authors from this study suggested that rather than oxidizing glucose through glycolysis, neuronal glucose is preferentially metabolized through the pentose phosphate pathway (PPP) [START_REF] Herrero-Mendez | The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1[END_REF][START_REF] Bolaños | Glycolysis: a bioenergetic or a survival pathway?[END_REF]. In the PPP, once glucose is taken up by GLUTs and phosphorylated by hexokinase, it generates glucose-6-phosphate, and then, through a series of ezymatic reactions, ribose-5-phosphate (Figure 5) (Nelson and Cox, 2003). Ribose-5-phosphate can be used as precursor for the synthesis of DNA and RNA, which can be subsequently used in transcriptional processes [START_REF] Yin | Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila[END_REF]. In fact the PPP pathway might have important relevance for protein synthesis events in mammals [START_REF] Beaconsfield | Glucose metabolism via the pentose phosphate pathway relative to nucleic acid and protein synthesis in the human[END_REF] (Figure 6). In addition, the PPP can generate intermediate precursors that branches into glycolysis at the level of fructose-6-phosphate and glyceraldehyde 3-phosphate, contributing to the glycolytic intermediates pool (Figure 5). The PPP pathway generates reducing power in form of NADPH, which is involved in many reactions, including lipid synthesis [START_REF] Wasylenko | Metabolomic and (13)C-metabolic flux analysis of a xylose-consuming Saccharomyces cerevisiae strain expressing xylose isomerase[END_REF] and in the antioxidant cellular defense system in mammals [START_REF] Riganti | The pentose phosphate pathway: an antioxidant defense and a crossroad in tumor cell fate[END_REF] (Figure 6). Thus, the PPP take place in neurons to keep the antioxidative status and synthesize essential metabolic precursors. Nevertheless, whether the PPP mechanism might Metabolic intermediates formed from glucose, such as lactate derived from glial cells, can be oxidized primarily in neurons to obtain energy in the rodent brain [START_REF] Zielke | Direct measurement of oxidative metabolism in the living brain by microdialysis: a review[END_REF] (Figure 5). In fact, lactate is used to feed the Krebs cycle in neurons of the mouse brain to generate ATP [START_REF] Hui | Glucose feeds the TCA cycle via circulating lactate[END_REF]. Astrocytes possess the glycolytic machinery to metabolize glucose efficiently (Belanger et al., 2011;[START_REF] Yellen | Fueling thought: Management of glycolysis and oxidative phosphorylation in neuronal metabolism[END_REF]. In fed conditions, glial cells take up circulating glucose, generate lactate through glycolysis and export them to neurons to provide energy (Figure 5). In fact, numerous evidences support the view that glial cells could use glucose predominantly during neuronal activity [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization[END_REF][START_REF] Chuquet | Predominant enhancement of glucose uptake in astrocytes versus neurons during activation of the somatosensory cortex[END_REF] and generate lactate which can be used as a neuronal oxidative fuel to support energy metabolism, in the astrocyteneuron lactate shuttle (ANLS) model.

Therefore, glucose utilization is compartmentalized in glia and neurons (Figure 5,6).

Yet, there is still a great controversy about the use of glucose in neurons and how this molecule is metabolized during different physiological conditions [START_REF] Barros | Rebuttal from L. F. Barros and B[END_REF]. For instance, it has been shown that glucose can be used primarily by neurons during neuronal activity [START_REF] Lundgaard | Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism[END_REF], in glycolytic-dependent axonal transport events for ATP production [START_REF] Zala | Vesicular glycolysis provides on-board energy for fast axonal transport[END_REF] or to maintain glycolytic-dependent presynaptic function and ATP levels [START_REF] Jang | Glycolytic Enzymes Localize to Synapses under Energy Stress to Support Synaptic Function[END_REF], suggesting that glycolysis can take place in neurons for energy generation, oppositely to what Herrero-Mendez and colleagues have suggested [START_REF] Herrero-Mendez | The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1[END_REF]. Thus, it is still under debate whether neurons can use glucose for energy generation.

In the following section I will introduce evidence supporting the ANLS model.

Astrocyte-neuron lactate shuttle model (ANLS)

Mammalian glial cells are highly glycolytic as compared to neurons [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization[END_REF][START_REF] Herrero-Mendez | The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1[END_REF]Belanger et al., 2011).

According to the astrocyte-neuron lactate shuttle (ANLS) hypothesis, in glutamatergic excitatory synapses, neuronal activity increases extracellular glutamate, which is taken up via a Na + -dependent mechanism by specific Thus, in fed state, either directly or indirectly, glucose is an essential metabolite to fuel the brain.

Can fatty acids be used as an energy fuel in the fed brain?

Despite the fact that glucose is an essential substrate for brain metabolism, evidence suggests that glial cells also use fatty acids as an energy source [START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF][START_REF] Auestad | Fatty acid oxidation and ketogenesis by astrocytes in primary culture[END_REF]Blasquez et al., 1998;Guzman and Blasquez, 2001;[START_REF] Ebert | Energy contribution of octanoate to intact rat brain metabolism measured by 13C nuclear magnetic resonance spectroscopy[END_REF][START_REF] Escartin | Activation of astrocytes by CNTF induces metabolic plasticity and increases resistance to metabolic insults[END_REF], which can account for ≈20% of the brain energy needs [START_REF] Ebert | Energy contribution of octanoate to intact rat brain metabolism measured by 13C nuclear magnetic resonance spectroscopy[END_REF]. Fatty acids are essential components of cell membranes and have crucial functions in different tissues, including the supply of energy through mitochondrial β-oxidation [START_REF] Jensen | Fate of fatty acids at rest and during exercise: regulatory mechanisms[END_REF]. Interestingly, the full oxidation of a molecule of glucose (6 carbons)

generates ≈30 molecules of ATP, whereas a molecule of fatty acid, such as palmitate (16 carbons), generates ≈106 molecules of ATP (Nelson and Cox, 2003). Therefore, in terms of energy production, fatty acids are molecules with a rich energy content. Here, I will present evidence suggesting that in the brain glial fatty acids might also be used as energy source.

In mammals, fatty acids that enter the cell are esterified and stored as triglycerides in lipid droplets (LDs) [START_REF] Olzmann | Dynamics and functions of lipid droplets[END_REF], or oxidized and used as energy by mitochondrial β-oxidation [START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF]. The transport of fatty acids from the blood to the brain is diverse and given the complexity of the BBB additional transport mechanisms are required that I will describe now.

A large body of evidence shows that fatty acids entering the mammalian brain derive from different pools from circulation [START_REF] Bazinet | Polyunsaturated fatty acids and their metabolites in brain function and disease[END_REF], including lipoproteins, lisophospholipids, or unesterified fatty acids associated to albumin (Figure 8). For instance, lipoproteins can bind to their respective lipoprotein receptor and be endocyted [START_REF] Xiao | Receptor-mediated endocytosis and brain delivery of therapeutic biologics[END_REF]. On the other hand, they can also enter through specific transporters such as the major facilitator superfamily domain-containing protein 2A (MFSD2A) [START_REF] Nguyen | Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid[END_REF]. Furthermore, some unesterified fatty acids, associated with albumin, can be taken up by another receptor known as CD36 [START_REF] Mitchell | Fatty acid transport protein expression in human brain and potential role in fatty acid transport across human brain microvessel endothelial cells[END_REF], or alternatively passively diffused into the endothelial membrane [START_REF] Ouellet | Diffusion of docosahexaenoic and eicosapentaenoic acids through the bloodbrain barrier: An in situ cerebral perfusion study[END_REF]. Once they pass 2015; [START_REF] Palikaras | Ectopic fat deposition contributes to age-associated pathology in Caenorhabditis elegans[END_REF][START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF], possess an efficient antioxidant system [START_REF] Herrero-Mendez | The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1[END_REF] and are fully equipped with the enzymes to carry out fatty acid β-oxidation [START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF][START_REF] Auestad | Fatty acid oxidation and ketogenesis by astrocytes in primary culture[END_REF]Blasquez et al., 1998;[START_REF] Escartin | Activation of astrocytes by CNTF induces metabolic plasticity and increases resistance to metabolic insults[END_REF]. The first evidence regarding fatty acid utilization in the brain came from experiments in which the production of CO 2 in the presence of radiolabeled fatty acids was quantified in vitro in different cell types of the developing rat brain, including oligodendrocytes, astrocytes and neurons [START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF]. From these results, it was observed that glial cells, but only astrocytes, produce large amounts of CO 2 , suggesting that fatty acids were metabolized optimally in astrocytes. Later studies using in vitro cultures of the developing rat brain, confirmed that astrocytes can carry out the oxidation of fatty acids [START_REF] Auestad | Fatty acid oxidation and ketogenesis by astrocytes in primary culture[END_REF]Blasquez et al., 1998;Guzman and Blasquez, 2001). In addition, in vivo studies in the rat brain demonstrated that infusions of labelled 13 C-octanoate contribute approximately to the 20% of the total energy consumption in the adult brain [START_REF] Ebert | Energy contribution of octanoate to intact rat brain metabolism measured by 13C nuclear magnetic resonance spectroscopy[END_REF], suggesting that β-oxidation might contribute to brain energy needs. Thus, brain fatty acid β-oxidation might contribute to almost the 20% of the brain energy metabolism under fed conditions, but it remains to be investigated what would occur under starved conditions.

Contrarily to mammalian glial cells, it has been shown that neurons are devoid of LDs structures, which is in agreement with their low β-oxidation capacity (Schonfeld et al., 2013). This low capacity of β-oxidation has been linked

to the low activity of the a rate-limiting step enzyme in the β-oxidation, the 3ketoacyl CoA thiolase in the rat brain [START_REF] Yang | Fatty acid oxidation in rat brain is limited by the low activity of 3-ketoacyl-coenzyme A thiolase[END_REF] (Figure 13). Moreover, the utilization of β-oxidation generate superoxide radicals and consequently increase oxidative stress (Schonfeld et al., 2013) to which neurons are specifically sensitive because of its low antioxidant capacity (Herreo-Mendez et al., 2009;[START_REF] Bolaños | Glycolysis: a bioenergetic or a survival pathway?[END_REF]. In fact, neuronal PPP is active, but lower as compared to glial PPP [START_REF] Herrero-Mendez | The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1[END_REF]. Another hypothesis proposed to support the idea that fatty acids are not used as energy precursors by neurons is that generation of ATP throughout β-oxidation demands much more O 2 than glucose (the full oxidation of a molecule of palmitate requires 23 molecules of O 2 , compared to only 6 molecules of O 2 for glucose) and therefore it increases the risk of neuronal hypoxia. Eventually, ATP generation via fatty acid β-oxidation is estimated to be much slower as compared to glucose oxidation (Schonfeld et al., 2013), since the release of fats reservoirs from adipose tissue is a long and complex process, and in addition, the passage of fatty acids through the BBB and the use of different transporting mechanism might slow this process. In contrast to the evidence exposed above, it has been recently shown that hypothalamic neurons can carry out β-oxidation for ATP generation [START_REF] Timper | Mild Impairment of Mitochondrial OXPHOS Promotes Fatty Acid Utilization in POMC Neurons and Improves Glucose Homeostasis in Obesity[END_REF], and they can use this pathway to mitigate stress and inflamation [START_REF] Mcfadden | Increasing fatty acid oxidation remodels the hypothalamic neurometabolome to mitigate stress and inflammation[END_REF]. Thus, contrary to the findings suggesting that β-oxidation could not take place in neurons, these recent results suggest that β-oxidation would indeed take place in hypothalamic neurons.

In summary, we have discussed in this section that neurons use energy for different cellular processes such as neurotransmission, generation of action potentials, ion gradient restoration, vesicular transport and synaptic vesicle cycle [START_REF] Harris | Synaptic energy use and supply[END_REF]. In fed conditions, glucose is the main metabolite consumed by the mammalian brain [START_REF] Siesjö | Brain energy metabolism and catecholaminergic activity in hypoxia, hypercapnia and ischemia[END_REF][START_REF] Sibson | Stoichiometric coupling of brain glucose metabolism and glutamatergic neuronal activity[END_REF]. The main view is that circulating glucose can cross the BBB, through specific GLUT transporters, and is used by glial cells, to generate lactate and support neuronal energy metabolism [START_REF] Simpson | Supply and demand in cerebral energy metabolism: the role of nutrient transporters[END_REF][START_REF] Chuquet | Predominant enhancement of glucose uptake in astrocytes versus neurons during activation of the somatosensory cortex[END_REF][START_REF] Zala | Vesicular glycolysis provides on-board energy for fast axonal transport[END_REF][START_REF] Lundgaard | Direct neuronal glucose uptake heralds activity-dependent increases in cerebral metabolism[END_REF][START_REF] Jang | Glycolytic Enzymes Localize to Synapses under Energy Stress to Support Synaptic Function[END_REF]. The ANLS hypothesis also suggests that neuronal activity increases glycolysis in astrocytes, producing lactate, which can be oxidized by neurons to generate energy [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization[END_REF]Belanger et al., 2011) Additionnaly, glucose can be taken up from circulation into the brain and contribute directly to neuronal metabolism by entering the PPP [START_REF] Herrero-Mendez | The bioenergetic and antioxidant status of neurons is controlled by continuous degradation of a key glycolytic enzyme by APC/C-Cdh1[END_REF]. In addition to glucose and its intermediates, it has also been suggested that under fed physiological conditions fatty acids could be used by the brain as an energy source in mammals [START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF][START_REF] Auestad | Fatty acid oxidation and ketogenesis by astrocytes in primary culture[END_REF], Blasquez et al., 1998;[START_REF] Escartin | Activation of astrocytes by CNTF induces metabolic plasticity and increases resistance to metabolic insults[END_REF]. Therefore, the oxidation of glucose and its intermediates, as well as the oxidation of fatty acids, are essential to satisfy the energy demands of the brain under fed physiological conditions.

During starvation, the brain still requires an adequate energy supply for its functioning [START_REF] Owen | Brain metabolism during fasting[END_REF]. Thus, if glucose is scarce or not available, what other metabolites could the brain use to maintain its function? Interestingly, astrocytes challenged to glucose deprivation induce an increase of endothelial GLUT1 expression, through the release of unidentified factors, as a compensatory mechanism (Regina et al., 2001), suggesting that in low glucose conditions GLUT1 is upregulated to permit an increased uptake capacity of glucose through the BBB. Additionally, it means that glucose deprivation produces transcriptional changes that might adapt the brain to specific physiological conditions such as during food deprivation. Yet, what would be the overall basis adaptation of the brain to these changes? And moreover, what would be the consequences of glucose unavailability? In the following sections I will bring relevant information with the intention to address these questions based on evidence published in the literature.

in optimal concentrations [START_REF] Cahill | Fuel metabolism in starvation[END_REF] and the need for an alternative fuel become critical for organism bioenergetics and survival. During starvation, the oxidation of fatty acids and the production of KBs turn into the main metabolic fuel of the human body, including the brain [START_REF] Owen | Brain metabolism during fasting[END_REF].

Metabolic switch at the organism level

Studies in different human tissues such as brain [START_REF] Owen | Brain metabolism during fasting[END_REF], muscle [START_REF] Pozefsky | Insulin blockade of amino acid release by human forearm tissues[END_REF], liver and kidney [START_REF] Owen | Liver and kidney metabolism during prolonged starvation[END_REF] revealed how different organs were participating in the distribution of different energy sources under conditions of food deprivation. In the first study, human patients were exposed to 40 days of starvation, supplemented daily with 1.5L water and vitamins [START_REF] Owen | Brain metabolism during fasting[END_REF]. By using brain blood vessels catheterization, blood flow measurements and metabolites concentration of the arteriovenous difference (i.e the metabolite concentration difference between the blood that enters the brain through the arteries and that leave out through the veins), it allowed the determination of the brain circulating KBs concentrations between the fasting and starvation state. Under starvation, both AcAc and βHB, were used as the primary metabolic fuel in the brain, accounting for almost the 60% of brain metabolic needs, whereas glucose only accounted for ≈28%. Even at shorter periods of food deprivation (3 days), KBs can already account for 25% of the brain energy needs (Hasselbach et al., 1994). Thus, as a consequence of starvation, brain glucose consumption decreases progressively with food deprivation, whereas KBs utilization increases (Figure 10) suggesting that the brain can adapt to the usage of KBs as their major oxidative fuel [START_REF] Cahill | Fuel metabolism in starvation[END_REF]. As soon as glycogen stores are progressively depleted, the liver begins to increase gluconeogenesis to counteract the drop in circulating glucose levels (Figure 10). In fact, during starvation, the brain still has a 28% of energy requirement derived from glucose.

Approximately 50% of the glucose synthesized from hepatic and renal gluconeogenesis reaches the brain to fuel its own metabolic needs, whereas some other is used to fuel the glycolytic-dependent red blood cells metabolism [START_REF] Rose | Control of glycolysis in the human red blood cell[END_REF] (Figure 10). Therefore, during starvation, brain glucose needs are filled by hepatic and renal gluconeogenesis [START_REF] Owen | Liver and kidney metabolism during prolonged starvation[END_REF], contributing Interestingly, this switch from glucose to KBs during starvation is not specific of humans and can be found in others species ranging from other mammals to insects. In rats, glycogen stores are lowered after 24 h of food deprivation, being only 7% of the original levels [START_REF] Jensen | Fasting of mice: a review[END_REF], suggesting that a metabolic switch to KBs already started to occur after ≈1 day of food deprivation. In fact, in adult rats subjected to 3 days of food deprivation, circulating concentrations of βHB can reach from ≈0.3 in the fed state up to ≈1.2 mM [START_REF] Hawkins | Regional ketone body utilization by rat brain in starvation and diabetes[END_REF], and they account for 15% of the total cerebral uptake of substrates (Ruderman et al., 1971). Similarly, in Drosophila, food deprivation causes metabolic changes in the hemolymph, the main circulatory system of insects [START_REF] Yamada | Correction: Fat body glycogen serves as a metabolic safeguard for the maintenance of sugar levels in Drosophila[END_REF]; leading to a depletion of trehalose and glucose (the main circulating sugars). Food deprivation deplete trehalose and glycogen stores after ≈18 h, being just 10% of their original levels [START_REF] Yamada | Correction: Fat body glycogen serves as a metabolic safeguard for the maintenance of sugar levels in Drosophila[END_REF]; and similarly to humans, glucose levels decrease on average 40% after ≈1 day of food deprivation, suggesting that a metabolic switch to KBs could take place after ≈1 day of food deprivation. Despite the fact that the metabolism of KBs has not been investigated in flies, in other insect models, such as cockroaches, it has been observed that after 3 days of food deprivation, the concentration of AcAc, the most abundant KB of insects, increases significantly from ≈0.5 mM to ≈3 mM [START_REF] Shah | Enzymes of ketogenesis in the fat body and the thoracic muscle of the adult cockroach[END_REF]. Therefore, these results suggest that in terms of temporal dynamics this metabolic switch from glucose to KBs has been evolutionary conserved from mammals to insects.

Remarkably, two additional conditions can lead to a switch into KBs utilization: high-fat diet (HFD) and the suckling period. Interestingly, rats subjected to 6-weeks of HFD feeding, showed an increase in circulating βHB levels from ≈0.5 mM to ≈1 mM [START_REF] Pierre | Enhanced expression of three monocarboxylate transporter isoforms in the brain of obese mice[END_REF]. On the other hand, during the suckling period of rats, the basal circulating KBs are ≈1.25 mM, which are above the concentration levels found in fed adult rats (≈0.1 mM) [START_REF] Hawkins | Ketone-body utilization by adult and suckling rat brain in vivo[END_REF]).

The explanation for that increase in basal levels is because maternal milk contains an abundant amount of fat [START_REF] Morris | Cerebral ketone body metabolism[END_REF]. Interestingly, in the suckling rats, circulating KBs concentrations can vary from 1.25 mM in the fed state to 2.3 mM after 24h of food deprivation [START_REF] Hawkins | Ketone-body utilization by adult and suckling rat brain in vivo[END_REF]. Those studies demonstrate [START_REF] Rangaraju | Activity-driven local ATP synthesis is required for synaptic function[END_REF]. When hippocampal presynaptic terminals are subjected to glucose deprivation, βHB can partly support the synaptic vesicle cycle, but a reduction in exocytosis and endocytosis is observed (Hyrnevich et al., 2016).

Moreover, higher concentrations of βHB (25 mM) impact specifically endocytosis and plasma membrane depolarization of rat brain synaptosomes [START_REF] Voronina | High Concentration of Ketone Body β-Hydroxybutyrate Modifies Synaptic Vesicle Cycle and Depolarizes Plasma Membrane of Rat Brain Synaptosomes[END_REF]. These high concentrations of KBs, however, might be more representative of what occurs during ketoacidosis, a pathological condition in which KBs concentrations are abnormally high (>20 mM), than during starvation. Thus, KBs are capable of sustaining postsynaptic potentials but just partly preserve presynaptic function. Condition of glucose scarcity, such as starvation, is a stressful stimulus that leads to an upregulation of the antioxidant system in the brain [START_REF] Bhardwaj | Effect of starvation and insulin-induced hypoglycemia on oxidative stress scavenger system and electron transport chain complexes from rat brain, liver, and kidney[END_REF][START_REF] Mattson | Impact of intermittent fasting on health and disease processes[END_REF]. Interestingly, a recent study proposed that KBs could preserve hippocampal synaptic function in presence of hydrogen peroxide (H 2 O 2 ), a ROS produced upon starvation [START_REF] Bhardwaj | Effect of starvation and insulin-induced hypoglycemia on oxidative stress scavenger system and electron transport chain complexes from rat brain, liver, and kidney[END_REF], by increasing the open probability of K ATP channels [START_REF] Kim | Ketones prevent oxidative impairment of hippocampal synaptic integrity through KATP channels[END_REF].

Authors from this study suggest that activation of K ATP channels might lead to reduced synaptic excitability and enhanced mitochondrial redox status, all of them contributing to resistance against stress [START_REF] Kim | Ketones prevent oxidative impairment of hippocampal synaptic integrity through KATP channels[END_REF]. This stress state (high H 2 O 2 levels) might resemble the physiological conditions of the brain during glucose deprivation, such as starvation, and the effect of KBs on preserving synaptic neurotransmission [START_REF] Arakawa | Effect of ketone body (D-3-hydroxybutyrate) on neural activity and energy metabolism in hippocampal slices of the adult guinea pig[END_REF][START_REF] Izumi | beta-Hydroxybutyrate fuels synaptic function during development. Histological and physiological evidence in rat hippocampal slices[END_REF][START_REF] Kim | Ketones prevent oxidative impairment of hippocampal synaptic integrity through KATP channels[END_REF]. In addition, it suggests that during fed conditions the role of the KBs could completely differ from the role that they exert during conditions of glucose scarcity. The clearest example can be observed in the effect of KBs over K ATP channels on neuronal excitability [START_REF] Ma | Ketogenic diet metabolites reduce firing in central neurons by opening K(ATP) channels[END_REF][START_REF] Kim | Ketones prevent oxidative impairment of hippocampal synaptic integrity through KATP channels[END_REF]: on the one hand, KBs can reduce the firing of central neurons in fed conditions [START_REF] Ma | Ketogenic diet metabolites reduce firing in central neurons by opening K(ATP) channels[END_REF], whereas in conditions of stress (i.e H 2 O 2 ), they could maintain synaptic transmission (Kim et al., 2010;[START_REF] Kim | Ketones prevent oxidative impairment of hippocampal synaptic integrity through KATP channels[END_REF].

In food-deprived mammals, KBs inhibit protein synthesis and protein degradation in skeletal muscle, which may be an adaptive mechanism for survival under catabolic states, such as starvation (Thompson and Wu, 1991).

Remarkably, neuronal plasticity mechanisms such as transcription and de novo protein synthesis are essential for long-term memory [START_REF] Yin | Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila[END_REF]. In fact, evidence from mammals and Drosophila suggest that the costly machinery dedicated for protein synthesis is largely confined to neuronal cell bodies [START_REF] Craig | Neuronal polarity[END_REF][START_REF] Rolls | Polarity and intracellular compartmentalization of Drosophila neurons[END_REF][START_REF] Rolls | Neuronal polarity: an evolutionary perspective[END_REF]. Interestingly, histochemical studies of βHB dehydrogenase, a major enzyme participating in KBs degradation, is found strongly stained in the neuronal somata of Purkinje cells, suggesting a potential effect of the KBs at the level of the somas [START_REF] Gesink | A histochemical study of the distribution of betahydroxybutyrate dehydrogenase in developing rat cerebellum[END_REF]. In the starved brain, this mechanism could shut down the costly process of protein synthesis at the level of the somas, in order to keep energy supply to preserve synaptic function.

KBs can also modulate other metabolic pathways, for example: βHB is able to inhibit glycolysis at the phosphofructokinase step, likely by increasing citrate concentration, an allosteric inhibitor of the phosphofructokinase, produced by the oxidation of KBs via the Krebs cycle in rodents [START_REF] Ide | Metabolic interactions of glucose, lactate, and beta-hydroxybutyrate in rat brain slices[END_REF][START_REF] Ruderman | Regulation of glucose and ketone-body metabolism in brain of anaesthetized rats[END_REF][START_REF] Appleton | An Animal Model for the Ketogenic Diet[END_REF]Stanley et al., 1981;[START_REF] Hue | The Randle cycle revisited: a new head for an old hat[END_REF]. This inhibition leads to a decrease in the activity of hexokinase via increasing concentrations of its inhibitor glucose-6-phosphate; thus, decreasing glucose utilization. Therefore, an inhibition of glycolysis is expected to occur during starvation, in which an increased KBs concentration is observed.

The effect of KBs has been studied at the neuronal level but still we lack a full understanding of the mechanisms through which they exert their effect, and under which physiological conditions might optimally act in the brain. Moreover, until now, it is unknown whether their energy budget prevails at the axonal, dendritic or somatic level, and to which extent they could contribute to the different subcellular compartments in terms of synaptic transmission and energy metabolism. These questions are still open and remain a relevant investigation area in the future.

Biochemical pathway of ketone bodies synthesis: ketogenesis

Ketogenesis is a process that occurs in peripheral fat tissues [START_REF] Shah | Enzymes of ketogenesis in the fat body and the thoracic muscle of the adult cockroach[END_REF][START_REF] Mcgarry | Regulation of hepatic fatty acid oxidation and ketone body production[END_REF] and glial cells [START_REF] Auestad | Fatty acid oxidation and ketogenesis by astrocytes in primary culture[END_REF] of different species. The production of KBs, through β-oxidation and ketogenesis requires various enzymatic reactions. Here, I will describe some of the enzymes and the main metabolic pathways underlying the production of KBs.

Generation of the main ketone body precursor: Acetyl-CoA

Fatty acids are oxidized in the mitochondria and can be used as an energy substrate in the mammalian heart, skeletal muscle and liver (McGarry and Ford, 1980). Acetyl-CoA is a central and highly energetic metabolite for fueling the Krebs cycle [START_REF] Krebs | Metabolism of ketonic acids in animal tissues[END_REF] 12). In the carnitine shuttle, first, a free fatty acid molecule needs to be activated in order to enter the mitochondria, this activation is performed by acyl-CoA synthetases in the outer mitochondrial membrane, which generates the activated fatty-acyl-CoA molecule [START_REF] Fujino | Acetyl-CoA synthetase 2, a mitochondrial matrix enzyme involved in the oxidation of acetate[END_REF]. Second, activated fatty acids are transiently attached to a carnitine molecule, thereby forming a fatty acyl-carnitine, a reaction catalyzed by the carnitine palmitoyltransferase 1 (CPT1) on the mitochondrial outer membrane, which is a common enzyme in different species, ranging from mammals to insects [START_REF] Schulz | Glial β-oxidation regulates Drosophila energy metabolism[END_REF]. Then, the fatty-acyl-carnitine molecule is transported through the porin voltage-dependent anion channel (VDAC) (Lee et al., 2011) into the intermembrane space and then it enters the mitochondrial matrix by facilitated diffusion (not shown in Figure 12), with the help of an acylcarnitine/carnitine transporter. Third, once in the matrix, the carnitine group of the fatty-acyl-carnitine molecule is replaced by coenzyme A (CoA), in a reaction catalyzed by the carnitine palmitoyltransferase II (CPT2), yielding a fatty-acyl CoA molecule which then can be catabolized in the β-oxidation pathway and generate ATP (Nelson and Cox, 2003). CPT1 is the rate-limiting step in mitochondrial βoxidation and is considered a key regulatory point [START_REF] Mcgarry | Regulation of hepatic fatty acid oxidation and ketone body production[END_REF].

other tissues (McGarry and Ford, 1980). These enzymes are present in fat tissues and astrocytes of different species, ranging from mammals to insects (McGarry and Ford, 1980;[START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]. Specifically, vertebrate rate-limiting step enzyme HMGS, is expressed in astrocytes [START_REF] Cullingford | Regulation of the ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in astrocytes and meningeal fibroblasts. Implications in normal brain development and seizure neuropathologies[END_REF] and also exhibit a strong expression in satellite glial cells, a type of glial cell surrounding the cell bodies of sensory neurons [START_REF] Wang | HMG-CoA synthase isoenzymes 1 and 2 localize to satellite glial cells in dorsal root ganglia and are differentially regulated by peripheral nerve injury[END_REF]. Moreover, in fruit flies, HMGS is also expressed in glial cells [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]. Thus, these enzymes seem to be present in similar cellular types from different species.

We have seen that there is a metabolic switch from glucose to KBs, and that they might be used to provide energy to the brain. However, what is the origin of these KBs? And more specifically how the brain could obtain KBs? I will now summarize and present the origin of KBs.

Origin of ketone bodies

In mammals, KBs are produced mainly in the mitochondria in response to starvation. Evidence suggests that KBs can be synthesized in the liver and glial cells, and be essential for energy metabolism in mammals [START_REF] Auestad | Fatty acid oxidation and ketogenesis by astrocytes in primary culture[END_REF]Nelson and Cox, 2003). But, how exactly does the body determine which reservoir is used for brain energy purposes?

Ketogenic tissues: liver

In humans and rodents, AcAc and βHB are the two major KBs [START_REF] Morris | Cerebral ketone body metabolism[END_REF]. Upon starvation, they are synthesized from β-oxidation (Figure 13,14) in the liver and transported via circulation to extrahepatic tissues, where they can be reconverted into acetyl-CoA and subsequently oxidized in the Krebs cycle (McGarry and Ford, 1980). KBs can provide an essential fuel for extrahepatic tissues such as skeletal muscle, heart, kidney and most importantly the brain [START_REF] Morris | Cerebral ketone body metabolism[END_REF]. Similarly to vertebrate liver, during starvation insect fat body initiates lipolysis, β-oxidation [START_REF] Chino | Diglyceride release from insect fat body: a possible means of lipid transport[END_REF][START_REF] Arrese | Adipokinetic hormone-induced lipolysis in the fat body of an insect, Manduca sexta: synthesis of sn-1,2-diacylglycerols[END_REF]Gronke et al., 2005Gronke et al., , 2007) ) and production of KBs [START_REF] Shah | Enzymes of ketogenesis in the fat body and the thoracic muscle of the adult cockroach[END_REF], AcAc being the major compound found in the hemolymph. Similarly, in Drosophila, lipids are stored and catabolized in the fat body, a specialized structure that is considered as a functional equivalent of the vertebrate liver and adipocytes [START_REF] Palanker | Drosophila HNF4 regulates lipid mobilization and beta-oxidation[END_REF]. Fat body reservoirs are in close contact with the hemolymph and present in the head, thorax and abdomen; their storage is not only devoted to fat but also to glycogen. The fly fat body contains the enzymatic machinery to oxidize fats through β-oxidation (Arresse et al., 2010) but it remains unknown whether it could carry out ketogenesis. However, other insect species have the enzymatic machinery to carry out ketogenesis in the fat body [START_REF] Shah | Enzymes of ketogenesis in the fat body and the thoracic muscle of the adult cockroach[END_REF], and therefore it seems plausible that flies could carry out this process as well.

Ketogenic tissues: glial cells

The vertebrate liver is generally believed to be the major site of β-oxidation and KBs production to meet brain metabolic needs under starvation. However, several investigations have suggested that the brain can also synthesize, take up and store fatty acids in glial cells as LDs [START_REF] Bazinet | Polyunsaturated fatty acids and their metabolites in brain function and disease[END_REF][START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF]. Remarkably, as compared to glucose, astrocytes have a higher capacity to oxidize fatty acids as their main metabolic fuel substrate in rodents [START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF], similarly to hepatic cells [START_REF] Mcgarry | Regulation of hepatic fatty acid oxidation and ketone body production[END_REF]. Besides, studies in rat primary cultured astrocytes in vitro show that they can synthesize KBs derived from β-oxidation [START_REF] Auestad | Fatty acid oxidation and ketogenesis by astrocytes in primary culture[END_REF]Blasquez et al., 1998). In fact, it has been hypothesized that KBs released from glial cells could contribute to neuronal oxidative metabolism (Guzman and Blasquez, 2001). Therefore, glial cells have the capacity to synthesize KBs, but whether this mechanism would be relevant in vivo during starvation remains unknown. Similarly to mammals, fly glial cells contain an abundant amount of LDs [START_REF] Kis | Specialized Cortex Glial Cells Accumulate Lipid Droplets in Drosophila melanogaster[END_REF]. Could the fly brain take advantage of LDs storing structures in glial cells and use them as an energetic source as it has been suggested for mammalian glial cells? The answer to this question is unclear. Evidence suggests that the fly brain is capable of oxidizing fatty acids [START_REF] Schulz | Glial β-oxidation regulates Drosophila energy metabolism[END_REF] but it remains unknown whether it could carry out ketogenesis. Null mutants for CPT2, a protein involved in fatty acids mitochondria import (Figure 12), accumulate LDs in glial cells. These CPT2 null mutants have a reduced viability, but the genetic rescue of CPT2 in glia restored it to normal levels, suggesting that glial cells carry out β-oxidation in insects. In this context, single-cell transcriptome analyses made in fed flies suggest that the fly brain contain all the enzymes necessary to carry out β-oxidation in glial cells [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF], which suggest a potential involvement of glial cells in βoxidation. However, there is no specific evidence that β-oxidation and ketogenesis could be used in the fly brain for energy purposes.

Thus, fatty acid and KBs metabolism in mammals and insects seems to share many properties. In vertebrates, β-oxidation and ketogenesis could take place in the liver and in astrocytes. Moreover, in insects, β-oxidation and ketogenesis could take place in the fat body. Yet, it remains to be investigated whether under conditions of food deprivation, glial cells could synthesize KBs to supply energy locally to neurons. Nevertheless, a precise regulatory mechanism is necessary for their synthesis. Next, I will give details of the main regulatory mechanisms involved in the regulation of ketogenesis.

Hormonal, enzymatic and transcriptional regulatory mechanisms of ketogenesis

To gain a better understanding of how the metabolism of KBs exerts its action on the brain, we need to gain a deep understanding of the regulatory mechanisms at the hormonal, enzymatic, and transcriptional level.

Hormonal regulation of ketogenesis

In mammals, ketogenesis is not only stimulated by starvation, but also by low levels of insulin and HFDs [START_REF] Mcgarry | The effects of starvation and refeeding on carbohydrate and lipid metabolism in vivo and in the perfused rat liver. The relationship between fatty acid oxidation and esterification in the regulation of ketogenesis[END_REF][START_REF] Morris | Cerebral ketone body metabolism[END_REF][START_REF] Soeters | Effects of insulin on ketogenesis following fasting in lean and obese men[END_REF]. Different hormones can stimulate ketogenesis in hepatocytes, including glucagon, cortisol and catecholamines. These hormones stimulate lipolysis and consequently the production of ketogenic precursors for KBs synthesis [START_REF] Laffel | Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes[END_REF]. Insulin is a core regulator of ketogenesis and whenever insulin levels are low, ketogenesis is triggered [START_REF] Soeters | Effects of insulin on ketogenesis following fasting in lean and obese men[END_REF]. The hormonal regulation of ketogenesis seems to be conserved not only in mammalian tissues but also in insect fat tissues. For instance, at the hormonal level, the insect glucagon homolog, adipokinetic hormone (Akh), released from the Akh-producing cells, acts by stimulating β-oxidation in the fat body (Mayer et al., 1968;[START_REF] Robinson | Adipokinetic hormone and the regulation of carbohydrate and lipid metabolism in a working flight muscle preparation[END_REF]. Likewise, Akh is antagonized by insulin [START_REF] Palanker | Drosophila HNF4 regulates lipid mobilization and beta-oxidation[END_REF]. In fact, increasing the insulin signaling via insulin-like peptides and sugar Likewise, ACC inhibition, releases CPT1 inhibition and thus stimulates βoxidation providing precursors for ketogenesis. In addition, low levels of insulin and high levels of glucagon increase HMGS activity, which eventually stimulates KBs synthesis. Therefore, a low glucagon/insulin ratio inhibits ketogenesis whereas a high glucagon/insulin ratio stimulates ketogenesis. Thus, ketogenesis is promoted by lipolysis and β-oxidation (Table 1). This regulatory mechanism involving ACC/CPT1 axis is conserved in many species (Guzman and Blasquez, 2001), however, whether this mechanism is also conserved in insect is unknown. et al., 2011) and therefore its regulation might have important consequences for brain metabolism. But, might this enzymatic regulation be present in glial cells, as it occurs in hepatic cells? In both human hepatocytes and astrocytes, CPT1 and HMGS are the crucial rate-limiting steps for β-oxidation and ketogenesis, respectively [START_REF] Drynan | Flux control exerted by mitochondrial outer membrane carnitine palmitoyltransferase over beta-oxidation, ketogenesis and tricarboxylic acid cycle activity in hepatocytes isolated from rats in different metabolic states[END_REF]Blasquez et al., 1998;Hegard et al., 1999;Figure 16. AMPK is the master regulator of β-oxidation and ketogenesis. AMPK is activated by AMP when there is a high demand of energy needs (i.e increased AMP:ATP ratio). Phosphorylation of ACC by AMPK, inhibits its activity, and as a consequence diminishes malonyl-CoA levels, releasing the inhibition of CPT1. Thus, activation of AMPK increases shuttling of fatty acids into the mitochondria by CPT1. Mitochondrial acetyl-CoA can be used as a precursor of AcAc and βHB.

Therefore, AMPK stimulates fatty acid mobilization and mitochondrial βoxidation in response to high-energy demands and is an essential energy sensor conserved between different species. Here we have discussed the main hormonal and enzymatic mechanisms that regulate β-oxidation and consequently ketogenesis. Under starvation, in order to activate ketogenesis, insulin levels must be low so that glucagon can exert its effect [START_REF] Hirota | Hepatocyte nuclear factor-4 is a novel downstream target of insulin via FKHR as a signal-regulated transcriptional inhibitor[END_REF]. In fact, activation of glucagon receptors leads to activation of transcriptional mechanisms that are essential to trigger ketogenesis. I will now describe briefly some of these transcription factors and their contribution to the production of KBs.

Transcriptional regulation of ketogenesis

Glucagon triggers the activation of nutritional sensors that participate in the response to starvation in mammals [START_REF] Contreras | PPAR-α as a key nutritional and environmental sensor for metabolic adaptation[END_REF]. The most well studied, the peroxisome proliferator-activated receptor (PPARα), hepatocyte nuclear factor (HNF4) and the FoxO transcription factor, regulate the transcription of several enzymes involved in the liver β-oxidation and ketogenesis in mice (Martinez-Jimenez et al., 2009), and fat tissues of other species such as fruit flies (Junger et al., 2003;[START_REF] Palanker | Drosophila HNF4 regulates lipid mobilization and beta-oxidation[END_REF]. In fed state, HNF4 repress several target genes involved in liver β-oxidation, whereas in the starved state, HNF4 is replaced by activated PPARα, inducing the transcription of β-oxidation genes (Martinez-Jimenez et al., 2009). On the other hand, FoxO can interact and suppress the function of PPARs transcription factors in hepatic tissues [START_REF] Wang | SIRT2 suppresses adipocyte differentiation by deacetylating FOXO1 and enhancing FOXO1's repressive interaction with PPARgamma[END_REF]. Interestingly, although HNF4 and FoxO are expressed in glial cells of insects [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF] it remains unknown whether they might be also involved in the regulation of ketogenesis in this cell type. Thus, transcriptional regulations of these pathways are subjected to different mechanisms between different nuclear transcription factors even within the same tissue. This highlights [START_REF] Halestrap | The monocarboxylate transporter family--Structure and functional characterization[END_REF]. In addition, normal plasma concentrations values were obtained from Psychogios et al. (2011). Net rates of transport of any monocarboxylate is determined by the difference between influx and efflux. NA -Not assayed. a D, L racemix mix used in these studies. b uptake too low to measure accurately.

Although MCT1 expression is subjected to transcriptional regulation under starvation in different mouse tissues [START_REF] Schutkowski | Tissue-specific expression of monocarboxylate transporters during fasting in mice[END_REF], it remains unknown whether this adaptive mechanism also occurs in the brain. Earlier studies have shown that a HFD causes an increase in MCT1 expression in the rat BBB (Leino et al., 2001), suggesting that MCT1 expression might be regulated by circulating KBs levels. Moreover, during early development of rodents, which coincides with a high concentration of circulating KBs [START_REF] Hawkins | Ketone-body utilization by adult and suckling rat brain in vivo[END_REF], there is a maximal expression of MCT1 mRNA and protein levels in the BBB and astrocytes endfeets, which decline over the postweaning period [START_REF] Leino | Monocarboxylate transporter (MCT1) abundance in brains of suckling and adult rats: a quantitative electron microscopic immunogold study[END_REF][START_REF] Rafiki | Highly differential expression of the monocarboxylate transporters MCT2 and MCT4 in the developing rat brain[END_REF]Vanucci and Simpson, 2003;[START_REF] Pierre | Monocarboxylate transporters in the central nervous system: distribution, regulation and function[END_REF].

Therefore, MCT1 expression in the BBB may facilitate the uptake and utilization of KBs to sustain brain energy metabolism.

On the other hand, MCT2 expression is largely confined to neuronal somata and dendrites, but not glial cells in rodents [START_REF] Pierre | Monocarboxylate transporters in the central nervous system: distribution, regulation and function[END_REF].

Moreover, MCT2 is also found at neuronal postsynaptic densities [START_REF] Bergersen | A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is co-localized with delta-glutamate receptors in postsynaptic densities of parallel fiber-Purkinje cell synapses[END_REF][START_REF] Pierre | MCT2 is a major neuronal monocarboxylate transporter in the adult mouse brain[END_REF]. In fact, this location may be relevant for the facilitation and uptake of energy rich monocarboxylates to fuel neuronal oxidative metabolism [START_REF] Suzuki | Astrocyte-neuron lactate transport is required for long-term memory formation[END_REF]. Interestingly, rats subjected to 2 days food deprivation exhibit an increase in MCT2 mRNA expression in the brain stem [START_REF] Matsuyama | Food deprivation induces monocarboxylate transporter 2 expression in the brainstem of female rat[END_REF], suggesting its expression is regulated by food deprivation in the brain. Moreover, mice subjected to a HFD, display a strong increase in MCT2 expression in neuronal cell bodies in the cortex and the hippocampus [START_REF] Pierre | Enhanced expression of three monocarboxylate transporter isoforms in the brain of obese mice[END_REF], suggesting that the uptake of KBs might be facilitated at the level of neuronal somata. During early post-natal stage, MCT2 mRNA and protein levels are transiently increased in rodents brains [START_REF] Rafiki | Highly differential expression of the monocarboxylate transporters MCT2 and MCT4 in the developing rat brain[END_REF], which is in agreement with the fact that brain of suckling rats uptake more KBs during this period [START_REF] Hawkins | Ketone-body utilization by adult and suckling rat brain in vivo[END_REF]. Thus, as well as MCT1, MCT2 expression in neuronal somata might facilitate the uptake of KBs under different conditions.

In Drosophila, the MCT family contains 22 homologs expressed in glia and neurons, which might contribute to the transport of monocarboxylates [START_REF] Volkenhoff | Glial Glycolysis Is Essential for Neuronal Survival in Drosophila[END_REF][START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]. Interestingly, the Chaski gene encodes a MCT1 homolog that is necessary for survival during food deprivation [START_REF] Delgado | Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress[END_REF], suggesting that Chaski might be relevant during conditions of glucose scarcity. Nevertheless, whether Chaski could play a relevant role in the transport of KBs in the fly brain remains unknown. Thus, the MCT family of different species might have conserved a common function during food deprivation.

These studies confirm that when KBs increase considerably, either during starvation, a HFD, or during the suckling period, there is a brain adaptation of the to upregulate the expression of MCT carriers. Specifically, this adaptation is reflected by an upregulation of MCT1 in the BBB and glial cells, and a further increase of MCT2 in neuronal somata, which might be essential for the uptake and transport of KBs to maintain brain energy metabolism. I will now discuss some additional properties of the MCTs carriers.

Transacceleration property of monocarboxylate transporters

In physiological conditions, the main known function of MCT1 and MCT2 is the H + -linked transport of L-lactate (either efflux or influx), which depend in the substrate concentration and the pH in cell compartments [START_REF] Poole | Transport of lactate and other monocarboxylates across mammalian plasma membranes[END_REF]. Glia derived L-lactate and export through MCT1, and neuronal import through MCT2, is considered an essential mechanism in the ANLS model (Belanger et al., 2011). Despite MCTs present a high affinity for L-lactate, their affinity properties also permit the transport of several other relevant substrates, including D-βHB and AcAc (Table 2).

In terms of molecular mechanism, the substrate binding of MCTs follows an ordered mechanism (Pool and Halestrap, 1993) (Figure 17). At first, MCT binds a H + , and then a specific monocarboxylate in the extracellular side. Next, the MCT undergoes into a conformational modification that exposes both substrates to the other side of the membrane. Eventually, both substrates are released, being at first the monocarboxylate and then the H + . Nevertheless, under certain conditions Thus, MCTs possess the properties to transport KBs. However, it remains unknown whether their transport might have a physiological relevance in the brain during food restriction periods. Once KBs are taken up, how are they oxidized in the brain? I will now discuss the catabolism of KBs.

Ketone bodies utilization

Since KBs cannot be used for structural purposes upon food deprivation periods, they are used to produce energy [START_REF] Morris | Cerebral ketone body metabolism[END_REF].

Ketone bodies catabolism

Once KBs are taken up by neurons they are imported into mitochondria for their final oxidation [START_REF] Page | Activities of enzymes of ketonebody utilization in brain and other tissues of suckling rats[END_REF][START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF]. D-βHB is converted to AcAc by the enzyme D-βHB dehydrogenase 1 (BDH1). Then, AcAc is converted by successive action of β-ketoacyl CoA transferase (or SCOT) and a mitochondrial acetoacetyl-CoA thiolase (ACAT1) [START_REF] Maurer | Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy[END_REF][START_REF] Grabacka | Regulation of Ketone Body Metabolism and the Role of PPARα[END_REF][START_REF] Abdelkreem | Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency[END_REF], into one molecule of coenzyme A and two acetyl-CoA molecules, which can finally enter the Krebs cycle and thus provide energy to the cell. Enzymes involved in KBs degradation are present in the brain of species ranging from human and rodent to insects [START_REF] Page | Activities of enzymes of ketonebody utilization in brain and other tissues of suckling rats[END_REF][START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]. In the adult rat brain, the enzymatic activity of BDH1 increases 5 times after 72 h of starvation [START_REF] Smith | Induction of brain D(--)-betahydroxybytrate dehydrogenase activity by fasting[END_REF], suggesting a greater utilization of KBs during starvation. Thus, the brain upregulates the activity of KBs degradation enzymes during starvation to fuel its energy needs.

In the rat brain, both BDH1, SCOT and ACAT1 are upregulated during development, followed by a slow fall in adult rats [START_REF] Page | Activities of enzymes of ketonebody utilization in brain and other tissues of suckling rats[END_REF][START_REF] Middleton | The acetoacetyl-coenzyme A thiolases of rat brain and their relative activities during postnatal development[END_REF]. More specifically, histochemical studies of BDH1 in the developing rat brain is found strongly stained in the somatas of Purkinje cells, suggesting a somatic effect of KBs [START_REF] Gesink | A histochemical study of the distribution of betahydroxybutyrate dehydrogenase in developing rat cerebellum[END_REF]. These changes reflects the increased utilization of KBs due to changes in KBs circulation and BBB transporting activity in the suckling rat brain. On the other hand, in flies, ACAT1 and D-βHB dehydrogenase are also found strongly expressed in neurons [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]. However, their role as KBs degradation enzymes remains to be investigated. Thus, KBs degradation enzymes are also present in insects but their role remains unknown.

Therefore, reasonably under periods of food deprivation or during the suckling period, KBs degradation enzymes are active to maintain the energetic requirements of brain metabolism. Nevertheless, similar to what occurs with glucose, which is consumed during neuronal activity in the fed state [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization[END_REF], could it also be possible that KBs are used during neuronal activity upon conditions of food restriction? I will try to give some insights about this idea in the following section.

Are ketone bodies used during neuronal activity?

The favored positions of astrocytes make them the ideal candidates to receive information from neurons in the form of neurotransmitters, since they have a large number of receptors for neurotransmitters (Belanger et al., 2011). Astrocytes express G s -protein receptors, which stimulates cAMP production (Kimelberg et al., 1994). In fact, increasing concentrations of cAMP in astrocytes enhances glycogen breakdown, glycolysis and lactate release [START_REF] Coggan | Norepinephrine stimulates glycogenolysis in astrocytes to fuel neurons with lactate[END_REF][START_REF] Dienel | Does shuttling of glycogen-derived lactate from astrocytes to neurons take place during neurotransmission and memory consolidation[END_REF], suggesting that astrocytic receptors activation is essential to maintain synaptic activity. Remarkably, cAMP activates ketogenesis in rat cultured astrocytes (Blasquez et al., 1998), by a mechanism involving ACC and CPT1 axis, which suggests that G s -protein receptor activation in glial cells could trigger ketogenesis in response to neuronal activity. In fact, it has been shown that endocannabinoids (Blasquez et al., 1999) and glutamate, by an unpublished communication (Guzman and Blasquez, 2001), activate ketogenesis in cultured astrocytes. Thus, similarly to lactate generation, ketogenesis could be activated during neuronal activity.

Interestingly, the impact of synaptic activity on lipid metabolism in astrocytes and neurons has been studied in vitro in mice [START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF].

Using primary cultures, it was demonstrated that neurons transfer toxic peroxidated fatty acids to glial cells that store them in LDs in response to neuronal activity. These stored fatty acids are later consumed via mitochondria βoxidation in glial cells. These in vitro studies suggest that, under neuronal activity, astrocytes are capable of oxidizing fatty acids. Yet, it remains to be investigated whether β-oxidation is accompanied by an increase in ketogenesis to provide energy to neurons, and whether this mechanism might be relevant in vivo.

In summary, we have presented in this section that in absence of glucose, the oxidation of fatty acids and the production of KBs are key metabolic pathways that supply energy during periods of energy restriction (Guzman and Blasquez, 2001. In addition, we have seen that ketogenesis is also regulated at the hormonal [START_REF] Laffel | Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes[END_REF]), enzymatic (Blasquez et al., 1998) and transcriptional level [START_REF] Palanker | Drosophila HNF4 regulates lipid mobilization and beta-oxidation[END_REF], which shows the complex regulation mechanisms underlying this metabolic pathway. We have detailed that in the vertebrate and insect BBB, there are specific MCT mechanisms that would allow the transport of KBs during starvation, either from the circulation, or from glial cells into neurons [START_REF] Morris | Cerebral ketone body metabolism[END_REF][START_REF] Pierre | Enhanced expression of three monocarboxylate transporter isoforms in the brain of obese mice[END_REF][START_REF] Delgado | Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress[END_REF], suggesting a potential role of glial cells as local suppliers of KBs to neurons.

Finally, whether the KBs can be used during neuronal activity to produce energy upon starvation remains unknown yet.

In the following section I will describe the Drosophila as a model organism to study memory function and the neural pathways involved in this process.

Part II: Drosophila and memory

Drosophila melanogaster has been successfully used as a simple model to study brain function and behavior [START_REF] Krashes | Sequential use of mushroom body neuron subsets during drosophila odor memory processing[END_REF]. Remarkably, fruit flies exhibit a variety of well-known behavioral responses, including courtship, aggression, sleep, learning and memory, among others [START_REF] Krashes | Sequential use of mushroom body neuron subsets during drosophila odor memory processing[END_REF].

Drosophila is a small, harmless and ease to breed organism. Its life cycle is relatively short: approximately 10 days at 25ºC until eclosion. Moreover, flies exhibit a reduced number of chromosomes (i.e 4 chromosome pairs), which allows a straightforward identification of gene mutations with high precision [START_REF] Heisenberg | Mushroom body memoir: from maps to models[END_REF]. Moreover, Drosophila is a genetically tractable organism, thanks to the enormous array of genetic tools, to manipulate circuits and gene function [START_REF] Krashes | Sequential use of mushroom body neuron subsets during drosophila odor memory processing[END_REF]. For instance, tools to disrupt gene function, to inhibit and stimulate the function of genetically identifiable neurons, and tools to optically image them and record neuronal activity provide numerous advantages to study brain function in fruit flies [START_REF] Krashes | Sequential use of mushroom body neuron subsets during drosophila odor memory processing[END_REF]Aso et al., 2014;[START_REF] Hige | Heterosynaptic Plasticity Underlies Aversive Olfactory Learning in Drosophila[END_REF][START_REF] Plaçais | Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory[END_REF].

In fact, numerous availability of transgenes along with tools to precisely control brain function make it an ideal model in a wide variety of studies, including electrophysiology, cell biology and behavior [START_REF] Heisenberg | Mushroom body memoir: from maps to models[END_REF]Aso et al., 2014;Fourakis et al., 2015). Indeed, thanks to this rich repertoire of experimental methods has also strengthened the experimental advantages of using Drosophila to dissect the cellular and molecular pathways mediating learning and memory events [START_REF] Mcguire | The role of Drosophila mushroom body signaling in olfactory memory[END_REF]Kitamoto et al., 2001;Aso et al., 2014a).

Memory is defined as a brain function that enables living organisms to acquire, store and retrieve the information under a given context. Associative memory can be defined as the capacity to learn and remember relationships between unrelated elements. Historically, the classical conditioning, also known as Pavlovian conditioning, is a type of associative learning described by Ivan Pavlov (Pavlov, 1906). The idea of this associative learning is based on training dogs to associate a meal -the unconditioned stimulus (US)-, together with an x1) flies form a memory that decay rapidly within the first day. 5 cycles of massed conditioning (without rest intervals between each cycle) or 5 cycles of spaced training (with resting intervals between each cycle) leads to the formation of consolidated memories that last for more than one day. Figure modified from [START_REF] Tully | Genetic dissection of consolidated memory in Drosophila[END_REF] I will present in the following sections the neural networks involved during associative olfactory memory formation in the fly brain.

Neuronal network of olfactory associative memory in Drosophila

Neural networks and their coordinated functioning are essential during memory formation. In order to generate olfactory memories, the fly requires mechanisms to sense the odor chemical components (CS) as well as neuronal pathways to convey the aversive information (US) to the center of olfactory memory. I will present now the details of the neural pathways involved in transducing this information (both the CS and US pathways). Then, I will present the main integrative center of the fly brain, the Mushroom Bodies (MBs), where olfactory memories are encoded and stored [START_REF] Heisenberg | Mushroom body memoir: from maps to models[END_REF]Isabel and Pascual, 2001;[START_REF] Gervasi | PKA dynamics in a Drosophila learning center: coincidence detection by rutabaga adenylyl cyclase and spatial regulation by dunce phosphodiesterase[END_REF] and how at the molecular level, the coincident detection between the CS and US generate new memories in MB neurons.

Conditioned stimulus pathway (CS): olfactory structures conveying the odor information

In the fly olfactory system, odorants are detected by a specific family of seven transmembrane domain receptors, known as Drosophila odorant receptors (OR), which are expressed in the olfactory receptor neurons (ORNs) [START_REF] Gomez-Diaz | The Two Main Olfactory Receptor Families in Drosophila, ORs and IRs: A Comparative Approach[END_REF]. ORNs extend their axons towards the antennal lobe (Figure 21), which has a high anatomical and functional homology to the vertebrate olfactory bulb [START_REF] Hildebrand | Mechanisms of olfactory discrimination: converging evidence for common principles across phyla[END_REF]. The antennal lobe is constituted of 43 glomeruli, and it is suggested that each one has a different functional activity for odor processing [START_REF] Laissue | Three-dimensional reconstruction of the antennal lobe in Drosophila melanogaster[END_REF]). Interestingly, it has been shown that the ORNs that express the same olfactory receptor project towards the same common glomerulus [START_REF] Jefferis | Target neuron prespecification in the olfactory map of Drosophila[END_REF][START_REF] Wong | Spatial representation of the glomerular map in the Drosophila protocerebrum[END_REF][START_REF] Marin | Representation of the glomerular olfactory map in the Drosophila brain[END_REF]. In Drosophila, the brain maintains a spatial map of OR activation, which can be chemical treatment with hydroxyurea, which affects the normal development of MBs, also caused memory deficits (Belle & Heisenberg, 1994). Thus, it has been hypothesized very early that the MB was the main olfactory memory center of fruit flies.

We have described in detail the neurons forming the lobes of the MB.

However, what other cell types innervate the MB? In fact, two types of neurons contact the MB lobes, dopaminergic neurons (DANs) and mushroom body output neurons (MBONs) (Aso et al., 2014a;[START_REF] Aso | Mushroom body output neurons encode valence and guide memory-based action selection in Drosophila[END_REF].

In the fly brain there are 3 dopaminergic neurons clusters that make synaptic contact with the MB: PPL2a, PPL1 and PAM cluster (Figure 23). PPL2a cluster innervate the calyx region, while PPL1 and PAM neurons innervate the MB lobes (Aso et al., 2014a). PPL1 projects neurites on the peduncle and vertical lobe, while the PAM cluster extends its projections on the medial lobes [START_REF] Claridge-Chang | Writing memories with light-addressable reinforcement circuitry[END_REF]. We have seen that olfactory inputs coming from projections neurons make synaptic contact at the level of the calyx of the MB, whereas DANs inputs contact the MB at the level of the lobes. In addition, Kenyon cells contact with another type known as mushroom body output neurons (MBONs).

MBONs dendritic arborizations make synaptic contact at the level of the MB lobes (Figure 26). Specifically, the dendritic tree of each MBON covers a spatially defined region on MB that can be defined as subcompartments along the medial and horizontal lobes [START_REF] Aso | Mushroom body output neurons encode valence and guide memory-based action selection in Drosophila[END_REF].

Thus, the MB is the main center of olfactory memory and is surrounded by different neuronal types. However, in order to form memories the odor information but also the electric shock should converge on this structure. But how?

Unconditioned stimulus pathway (US): US stimulus processing

We have previously seen how olfactory information is conveyed to the MB, and now I will present how the valence of the US stimulus is encoded in the fly brain. Dopaminergic systems projecting on the MB have been shown to encode the valence of the aversive unconditioned stimulus (Shroll et al. 2006, Honjo et al. 2012;[START_REF] Burke | Layered reward signalling through octopamine and dopamine in Drosophila[END_REF][START_REF] Liu | A subset of dopamine neurons signals reward for odour memory in Drosophila[END_REF]. Interestingly, it was possible to induce aversive memory formation by replacing the electroshock by activating the PPL1 dopaminergic cluster [START_REF] Claridge-Chang | Writing memories with light-addressable reinforcement circuitry[END_REF]. In addition, the activation of some dopaminergic neurons of the PAM and of the PPL1 clusters are required to induce aversive memory formation [START_REF] Aso | Specific dopaminergic neurons for the formation of labile aversive memory[END_REF][START_REF] Aso | Three dopamine pathways induce aversive odor memories with different stability[END_REF], whereas blocking these neurons impaired the electroshock-dependent aversive memory. Interestingly, by using the optogenetics technique [START_REF] Schroll | Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae[END_REF]Clardige-Chang, 2009), it was shown that dopaminergic neuronal activation is capable of replace the use of a US (electric shock) for aversive memory formation. Thus, these data show that dopaminergic neurons convey the electroshock information.

Therefore, dopaminergic neurons encode the valence of the aversive stimulus in the fly. However, how the MB is capable to integrate both the CS and US signal to form memories? Memory acquisition occurs when the fly learns an association in a given context (i.e when flies associate an odor with electroshocks). Then, during consolidation, which can last from a few minutes to several days, this memory is converted from a labile into a more stable form. Besides, during the retrieval, the animal is returned to the conditioning context, where the memory association is assessed [START_REF] Abel | Molecular mechanisms of memory acquisition, consolidation and retrieval[END_REF]. MB neurons are involved in the acquisition, consolidation and retrieval of olfactory memories [START_REF] Mcguire | Thirty years of olfactory learning and memory research in Drosophila melanogaster[END_REF][START_REF] Krashes | Sequential use of mushroom body neuron subsets during drosophila odor memory processing[END_REF]. In fact, blocking α'/β' neurons during conditioning or 1 hour after training impaired the acquisition and consolidation of aversive memories, respectively. However, the neurotransmission blockade of α'/β' neurons during the testing phase have no influence on memory formation, showing that memory retrieval is not affected. On the other hand, the blockade of the synaptic transmission in α/β neurons only has an effect on memory recall [START_REF] Krashes | Sequential use of mushroom body neuron subsets during drosophila odor memory processing[END_REF]). Thus, the MB and its subpopulations are involved in memory acquisition, consolidation and retrieval of new memories.

Memories are encoded in the MB neurons [START_REF] Bouzaiane | Two independent mushroom body output circuits retrieve the six discrete components of Drosophila aversive memory[END_REF]. In fact, immediately after conditioning a short-term memory (STM) and a short-term anesthesia-resistant memory (ST-ARM) can be formed. STM is encoded in the γ neurons, whereas ST-ARM in α/β neurons [START_REF] Bouzaiane | Two independent mushroom body output circuits retrieve the six discrete components of Drosophila aversive memory[END_REF]. Moreover, 3 hours after conditioning, a middle-term memory (MTM) and a middle-term anesthesia-resistant memory (MT-ARM) can be observed after one conditioning cycle, being encoded in the α/β and γ neurons, respectively (Figure 25). As we presented in previous sections, one training cycle is not sufficient to induce a perdurable memory. In fact, specific protocols are necessary to induce a stable and long-lasting memory (Figure 20). Both long-lasting memories, LTM and LT-ARM are differentially encoded in α/β and α'/β' neurons, respectively (Bouzaiane Thus, numerous evidences suggest that there are critical changes in neural networks and in the synapses that store information, specifically in KC-MBONs, to shape synaptic plasticity and memory formation.

I will present now a more detailed description of the cellular and molecular mechanisms underlying the two key consolidated memories in the fly brain: LTM and LT-ARM.

Long-lasting consolidated memories

LTM and LT-ARM, two mutual exclusive consolidated memories

We have previously seen that the formation of LT-ARM and LTM depends on different conditioning protocols. Moreover, these two memories require different signaling pathways. Indeed, LT-ARM relies on the radish gene [START_REF] Tempel | Reward learning in normal and mutant Drosophila[END_REF], serotonin synthesis (Lee et al., 2011;[START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF] and is independent of protein synthesis [START_REF] Tully | Genetic dissection of consolidated memory in Drosophila[END_REF]. In contrast, LTM is dependent on protein synthesis and requires CREB transcription factor (Tully et al., 1994;[START_REF] Yin | CREB and the formation of long-term memory[END_REF], a hallmark of long-lasting consolidated memories across different species [START_REF] Kogan | Long-term memory underlying hippocampus-dependent social recognition in mice[END_REF].

Evidence from our lab show that LTM and LT-ARM are mutually exclusive consolidated memories [START_REF] Isabel | Exclusive consolidated memory phases in Drosophila[END_REF][START_REF] Lagasse | Functional and evolutionary tradeoffs co-occur between two consolidated memory phases in Drosophila melanogaster[END_REF], Plaçais et al. 2012). Our lab was strongly involved over the past years in defining the molecular and circuit mechanisms involved in the regulation of the LTM/LT-ARM balance. At the circuit level, the oscillatory activity of dopaminergic neurons of the PPL1 cluster were involved this process [START_REF] Plaçais | Slow oscillations in two pairs of dopaminergic neurons gate longterm memory formation in Drosophila[END_REF]. More specifically, MV1 and MP1 (Figure 23) neurons activity is increased after spaced training and their activity is required for the formation of LTM. Additionally, blocking the activity of MV1 and MP1 during the rest intervals inhibited LTM formation. In contrast, synaptic transmission blockade of MV1 and MP1 increased the formation of LT-ARM, while its activation decreased LT-ARM [START_REF] Plaçais | Slow oscillations in two pairs of dopaminergic neurons gate longterm memory formation in Drosophila[END_REF]. Thus, this support that LT-ARM and LTM are mutually exclusive consolidated memories and that a gating mechanism allows the consolidation of one or the other of these memories.

Food deprivation shapes consolidated memories

Unlike LT-ARM, LTM requires protein synthesis and therefore it is likely to be energetically costly. The cost of forming a long-lasting memory was investigated several years ago in Drosophila by [START_REF] Mery | A cost of long-term memory in Drosophila[END_REF]. Flies were subjected to four different protocols: associative conditioning (spaced or massed), non-associative conditioning, shock only and naive (untreated). Then, flies were food deprived until reaching death. Interestingly, flies subjected to spaced training died earlier than controls and massed trained flies. This result suggested that LTM has an energetic cost and might be unfavored in conditions of food deprivation, while LT-ARM remains unaltered and might be resistant to conditions of energy restriction. Thus, it seems that LTM and LT-ARM are sustained by different energy-costly mechanisms [START_REF] Mery | A cost of long-term memory in Drosophila[END_REF].

Based on these pioneer studies [START_REF] Mery | A cost of long-term memory in Drosophila[END_REF], it was hypothesized that the feeding status might have consequences on the formation of LTM and LT-ARM. In fact, it was shown that starved flies do not form LTM since the memory formed is insensitive to a protein synthesis inhibitor (Figure 27A) [START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF]. However, this 24-memory formed in starved condition is sensitive to an ARM inhibitor, showing that under starvation flies only form LT-ARM (Figure 27B). Moreover, starved flies subjected to massed conditioning showed a significant increase in memory performance (Figure 27C). Thus, even in conditions of food deprivation, the brain maintains and upregulates the formation of LT-ARM, but it remains unknown what are the cellular and molecular mechanisms underlying its maintenance.

Part III: Glial cells and memory in Drosophila

As we have seen in the previous section long-term memory is an energetic costly process [START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF], and the metabolic pathways that support it have recently begun to be elucidated [START_REF] Plaçais | Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory[END_REF]. In mammals, glial cells are the main metabolic supporters of neurons (Belanger et al., 2011). However, in fruit flies, the functional roles of glial cells on memory formation and brain energy metabolism have been poorly investigated.

As we have seen previously the so-called "blood-brain barrier" of Drosophila shares many physical and functional properties with their mammalian counterpart [START_REF] Stork | Organization and function of the blood-brain barrier in Drosophila[END_REF]. It acts as a physical selective barrier, prevents the paracellular diffusion of charged molecules, offers protection against infection, toxic compounds and plays an essential role in the transfer of energy-rich compounds into the CNS [START_REF] Stork | Organization and function of the blood-brain barrier in Drosophila[END_REF][START_REF] Volkenhoff | Glial Glycolysis Is Essential for Neuronal Survival in Drosophila[END_REF]. In the CNS, glial cells and neurons can obtain energy-rich substrates from the general circulation, which must cross the BBB. Alternatively, neurons can also obtain energetic molecules from glial cells to support neuronal oxidative metabolism [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization[END_REF][START_REF] Volkenhoff | Glial Glycolysis Is Essential for Neuronal Survival in Drosophila[END_REF]. Therefore, investigating how these energy-rich substrates are obtained and delivered, either from circulation or directly from glial cells into neurons, will be essential for understanding brain energy metabolism and its adaptation to environmental constrains.

Here I will first present the Drosophila glial cells morphology and function.

In a second section, I will present the available evidence regarding their involvement in metabolism and memory formation.

1.2 Glia cell around the soma: cortex glia.

Cortex glia surrounds neuronal cell bodies of the cortex region (Figure 28).

A single glial cell can surround up to ≈100 cell bodies [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF], forming a honeycomb-like structure (Figure 28,29). Given the proximity of the cortex glia to the cells of the BBB, it resides in a privileged location to efficiently receive and transport nutrients from circulation towards the CNS [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF]). Yet, there is little evidence showing the involvement of this glial type in metabolic support of neurons. The first studies attempted to elucidate the function of cortex glia during the development. It was found that neurotrophic factors released from neurons are important for the development of cortex glia [START_REF] Avet-Rochex | Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila postembryonic brain[END_REF]. Moreover, it has also been suggested that both the cortex glia, together with the SPG, regulate the proliferation of neuroblasts in response to nutritional status. [START_REF] Chell | Nutrition-responsive glia control exit of neural stem cells from quiescence[END_REF]Sousa-Nunes et al. 2011;[START_REF] Coutinho-Budd | Probing the enigma: unraveling glial cell biology in invertebrates[END_REF]. However, the mechanism behind this regulation remains poorly understood.

In adult flies, cortex glia has been shown to be involved in maintaining neuronal firing, acting as a K + buffering system at the neuronal somata. Zydeco, a Na + /Ca 2+ ,K + exchanger was identified in Drosophila and mutants exhibit increased susceptibility to seizures and display higher intracellular Ca 2+ levels in cortex glia [START_REF] Melom | Mutation of a NCKX eliminates glial microdomain calcium oscillations and enhances seizure susceptibility[END_REF]) (Figure 30). Ca 2+ oscillations occur in cortex glia, which were blocked in zydeco mutants. Acute induction of Ca 2+ influx in cortex glia mimics seizure susceptibility of zydeco mutants. Furthermore, zydeco function depends on the activation of Ca 2+ /calmodulin and calcineurin signaling [START_REF] Weiss | Glial Ca(2+)signaling links endocytosis to K(+) buffering around neuronal somas to regulate excitability[END_REF]. In addition, mutants for Sandman, a K + channel, reproduces seizures in cortex glia [START_REF] Weiss | Glial Ca(2+)signaling links endocytosis to K(+) buffering around neuronal somas to regulate excitability[END_REF], and plasma membrane levels in cortex glia are reduced in zydeco mutants, suggesting a link between glial Ca 2+ signaling and K + buffering. Thus, these studies suggest that cortex glia influence neuronal excitability at the somatic level acting as a K + buffering system. In addition to this K + buffering function, could cortex glia play a functional role as a metabolic support?

Glia in the neuropil

Two other glial types, ENG and ALG extend their processes along axons and dendrites of the neuropil, respectively [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF]) (Figure 28). Glial cell bodies are located between the cortex and neuropil regions, and often between neuropil regions [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF].

ENG has a morphology that resembles mammalian oligodendrocytes [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF]. Transcriptomic data suggests an evolutionary similarity between ENG of flies and mammalian oligodendrocytes [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]. However, myelin, which is produced by oligodendrocytes, is not formed in the CNS of insects (Yildrim et al., 2018). In the adult fly brain, ENG responds to injury and clean the axonal debris in the neuropil [START_REF] Macdonald | The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons[END_REF]. Similarly, in vertebrates, Schwann cells surrounding axons clear cellular debris and coordinate repair processes after peripheral nerve injury [START_REF] Zuchero | Glia in mammalian development and disease[END_REF]. Thus, it seems that mammalian and insect glial cells around axons shares common functions. In vertebrates, oligodendrocytes glutamate homeostasis, via glutamine synthetase, is crucial to support neuronal neurotransmission [START_REF] Xin | Oligodendrocytes Support Neuronal Glutamatergic Transmission via Expression of Glutamine Synthetase[END_REF]. Given the spatial position of ENG around axonal tracts, one could ask if similarly to vertebrate oligodendrocyte, ENG might be essential for homeostasis of neurotransmitters and neuronal function. Evidence supporting this hypothesis comes from a recent study that shows the gene shopper, which encodes a mitochondrial sulfite oxidase, is involved in the regulation of glutamate-glutamine cycle in ENG and neuronal activity [START_REF] Otto | The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia[END_REF]. Thus, glial glutamate homeostasis mechanisms are conserved between mammals and insects.

Eventually, the last glia cell-type, the ALG has a similar morphology than mammalian astrocytes [START_REF] Freeman | Drosophila Central Nervous System Glia[END_REF]. Most of astrocyte cell bodies are located at the boundaries of the cortex and the neuropil (Figure 28,29), and they extend fine ramifications to different parts of the neuropil, including synapses [START_REF] Stork | Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes[END_REF]. This direct association faithfully reflects the "tripartite synapse" [START_REF] Strauss | A Distinct Perisynaptic Glial Cell Type Forms Tripartite Neuromuscular Synapses in the Drosophila Adult[END_REF], which is believed to be essential for neurotransmitter recycling and modulation of synaptic activity in mammals (Perea et al., 2009). But whether ALG in the Drosophila CNS might play a similar role remains poorly investigated. In Drosophila, astrocytes play a critical role in neurotransmitter recycling at the synapse and altering its function impacts animal behavior and survival [START_REF] Rival | Decreasing glutamate buffering capacity triggers oxidative stress and neuropil degeneration in the Drosophila brain[END_REF]. Astrocytes express various transporters involved in neurotransmitter clearance, including excitatory amino acid transporter (EAAT1) and GABA transporter. EAAT1 is involved in the glutamate buffering capacity of astrocytes, whose disruption leads to neurodegeneration and reduced survival [START_REF] Rival | Decreasing glutamate buffering capacity triggers oxidative stress and neuropil degeneration in the Drosophila brain[END_REF]. Therefore, ALG plays an essential role in neurotransmitter recycling at the synapse and behavior. But, would they also play a role in synaptic activity?

In adult flies, astrocytes have spontaneous Ca 2+ activity in different brain regions [START_REF] Liu | Astrocyte-like glial cells physiologically regulate olfactory processing through the modification of ORN-PN synaptic strength in Drosophila[END_REF][START_REF] Zhang | Astrocyte Ca(2+) Influx Negatively Regulates Neuronal Activity[END_REF], including the antennal lobe [START_REF] Liu | Astrocyte-like glial cells physiologically regulate olfactory processing through the modification of ORN-PN synaptic strength in Drosophila[END_REF]. When astrocytes are stimulated using optogenetics, they inhibit the odorevoked activity of the projection neurons [START_REF] Liu | Astrocyte-like glial cells physiologically regulate olfactory processing through the modification of ORN-PN synaptic strength in Drosophila[END_REF]. Moreover, astrocytic processes that infiltrate the neuropil exhibit a robust Ca 2+ oscillatory activity [START_REF] Zhang | Astrocyte Ca(2+) Influx Negatively Regulates Neuronal Activity[END_REF]. Induction of astrocytic Ca 2+ entry leads to paralysis and blockade of neuronal activity by increasing GABA levels at the synapse.

Therefore, these two studies suggest that astrocytes can regulate neuronal activity. Thus, Ca 2+ signaling is essential in astrocytes to maintain synaptic function and neuronal plasticity. In summary, these studies show the importance of each glial subtype in behavior, development, plasticity, energy metabolism and function of neural circuits. However, could some of these mechanisms, including the ones related to energy metabolism, have a relevant role during memory formation?

Role of glial cells during memory formation in Drosophila

The role of glial cells during memory formation has been studied in mammalian models [START_REF] Suzuki | Astrocyte-neuron lactate transport is required for long-term memory formation[END_REF]Kol et al., 2019). In the adult Drosophila, first studies on glial function and memory were carried out more than a decade ago. Crammer gene, which encodes an inhibitor of a cysteine proteinase, is found predominantly in glial cells and MB neurons [START_REF] Comas | Drosophila long-term memory formation involves regulation of cathepsin activity[END_REF]. The overexpression of crammer in glial cells induces a specific defect for LTM, but not LT-ARM, suggesting that glial cysteine proteinases are required for LTM.

Further studies in adult flies attempted to decipher the role of adhesion molecules during memory formation. Cell-cell adhesion molecules are important for brain morphology and function [START_REF] Sakisaka | Cell adhesion molecules in the CNS[END_REF]. An adhesion molecule called Klingoon, found at the junction between neurons and glial cells, is required independently in each compartment for the formation of LTM [START_REF] Matsuno | Long-term memory formation in Drosophila requires training-dependent glial transcription[END_REF]. Klingoon mutants did not affect LT-ARM formation. In this study, the specificity and requirement of each glial subtype was not investigated. Therefore, Klingoon adhesion molecule is critical for neuron-glia interactions during LTM formation.

Recently, it has been investigated the role of glial cells in memory upon aging [START_REF] Yamazaki | Glial dysfunction causes age-related memory impairment in Drosophila[END_REF]. When aged flies are subjected to one conditioning cycle and tested after 1 h they exhibit memory defects defined as age-related memory impairment (AMI). In an effort to identify new mutations involved in AMI, a proteomic screening in mutants for PKA was carried out, leading to the identification of the enzyme pyruvate carboxylase (PC), which showed altered expression upon aging. The PC participates in the conversion of pyruvate to oxaloacetate in the gluconeogenesis pathway. Reduction of PC activity in glial cells significantly suppresses AMI. In contrast, overexpression of PC in glia causes memory defects, which can be restored by administration of D-serine, an agonist of glutamatergic activity. In this study, the requirement of each glial subtype was not investigated. Therefore, these results suggest that during aging, PC activity and glutamatergic signaling in glial cells may be underlying age related memory defects.

Repo, a transcription factor required during glial development, is also involved in the formation of LTM [START_REF] Matsuno | Long-term memory formation in Drosophila requires training-dependent glial transcription[END_REF]. Interestingly, Repo increases the expression of EAAT1 [START_REF] Matsuno | Inhibiting Glutamate Activity during Consolidation Suppresses Age-Related Long-Term Memory Impairment in Drosophila[END_REF]. The involvement of EAAT1 during memory formation was studied in aged flies. Remarkably, aged flies display LTM defects, but normal LT-ARM formation [START_REF] Tonoki | Aging impairs protein-synthesis-dependent long-term memory in Drosophila[END_REF].

Matsuno and colleagues showed that EAAT1 levels decreased upon aging, but their overexpression in glial cells rescued LTM deficits observed during aging.

Specifically, the overexpression of EAAT1 in astrocytes, but not ENG, rescued age-related impairments in LTM. Interestingly, using an anti-excitotoxic agent in aged flies also restored LTM. These results suggest that inhibition of astrocytic glutamate activity in aged flies could suppress LTM defects related to aging.

The involvement of cortex glia in energy metabolism and memory formation was recently investigated in our lab (de Tredern et al., in revision).

Indeed, a metabolic coupling between neurons and cortex glia is essential for LTM formation (Figure 32). During LTM formation, a cholinergic signal originated from MB neurons activates cortex glia. This activation leads to an intracellular Ca 2+ increase, which triggers insulin-like peptide 4 (Ilp4) releases that acts in an autocrine manner on cortex glia by activating insulin receptor (InR). Then, insulin activates trehalase, triggering increased glucose synthesis. Next, glucose is shuttle from cortex glia to Kenyon cells somatas, suggesting that glucose substrates derived from cortex glial cells are essential for LTM formation. These results provide the first insights about the role of glial cells in memory formation with a potential link to energy metabolism. As this mechanism is specific to LTM, which, as we previously described, is inhibited upon starvation [START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF], it remains an open question to know how neuron-glia coupling are modified in this state, and whether they are also important in the formation of LT-ARM.

Aims

We have seen that energy metabolism in various physiological situations, such as in fed and starved state needs to be tightly regulated for proper brain functioning [START_REF] Owen | Brain metabolism during fasting[END_REF][START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF].

We have also seen that memory formation requires glia-neuron interactions (de Tredern et al., in revision) and metabolic regulations [START_REF] Plaçais | Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory[END_REF]. The role of energy metabolism and its impact on memory formation is extremely challenging to investigate in the mammalian brain, due to cell diversity and the complexity of its networks. Drosophila has been successfully used as model to study the cellular and molecular mechanisms underlying memory. Given its powerful array of genetic tools, it allows precise spatiotemporal manipulation of brain circuits and their study through in vivo imaging techniques. Therefore, we used Drosophila to study metabolism during memory formation.

During my thesis, we wanted to address the question of how the brain withstands to periods of food deprivation while preserving brain plasticity processes and memory formation. To address this question, we first questioned what are the metabolic fuels used by the brain underlying long-lasting memory upon starvation. For this, we have dissected the involvement of KBs pathway during long-lasting memory upon starvation. Eventually, we aimed to address the question of which brain cell types are involved in memory formation upon starvation. To address this question, we have investigated the role of neurons and glial cells, by combining genetics, behavior, immunohistochemistry and in vivo imaging techniques.

Materials and methods

Genetics tools

Drosophila rearing

Flies were raised on standard food medium containing yeast, cornmeal and agar in a 12:12 light-dark cycle at 18ºC with 60% of humidity. All lines were outcrossed for at least five generations to flies carrying a Canton-S wild-type background.

1.2 Gal4/UAS and TARGET systems One of the greatest advantages of the fruit fly as a biological model has been its wide range of genetic intervention tools. Among these tools, the most widely used corresponds to the GAL4/UAS binary expression system, which allows the selective expression of a gene in a tissue-specific and even cellspecific manner (Brand & Perrimon, 1993). Gal4 encodes a protein that activates galactose-induced transcription in the Saccharomyces cerevisiae yeast [START_REF] Matsumoto | Function of positive regulatory gene gal4 in the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae: evidence that the GAL81 region codes for part of the gal4 protein[END_REF][START_REF] Laughon | Primary structure of the Saccharomyces cerevisiae GAL4 gene[END_REF]. When the GAL4 protein is expressed, it can bind to the UAS response element (Upstream Activating Sequence), allowing the activation of the transcription of genes that are downstream of the UAS sequence (Figure 33). Thus, when Gal4 (driver) binds to the UAS response element (effector), it can initiate transcription of a gene of interest in a tissue or cell-specific manner (Phelps & Brand, 1998) (Figure 33). A, The structure of the CRIMIC cassette resembles to that of Trojan Gal4. It consists of a T2A-Gal4 followed by a polyA signal followed by a 3xP3 promoter and GFP reporter tag, to verify the insertion of the construct. The T2A is a viral ribosomal skipping site that arrests translation, which becomes reinitiated after the site, producing untagged Gal4 protein. B, Scheme of the CRIMIC integration by CRISPR/Cas9 in a coding intron. It consists of the CRIMIC vector and two homology arms by homology directed repair (HDR). The integration of the CRIMIC vector allows the expression of a Gal4 in the intronic region of a gene and to assess its expression they can be crossed to transgenic flies expressing membrane red fluorescent protein (UAS-mCD8::RFP). Figure from [START_REF] Lee | A gene-specific T2A-GAL4 library for Drosophila[END_REF]. Briefly, groups of 40-60 were subjected to conditioning. Conditioning was performed by exposing the flies to one odor paired with electric shocks (CS + ), with a subsequent exposure to a second odor in the absence of electroshock (CS -) [START_REF] Tully | Genetic dissection of consolidated memory in Drosophila[END_REF]. Custom-built barrels were used for conditioning (Figure 37A). Specifically, the conditioning started with the presentation of an air flux without odor during 90 s, followed by 60 s of the conditioned odorant in parallel with 12-pulses of 60V electric shocks (Figure 37B). Then, after 45 s of air flux presentation, the second odor was presented for 60 s without electroshock, and next 45 s of neutral air flux.

Behavioral assays

Memories were tested 24 h after conditioning. For this, flies were exposed simultaneously to both odorants in a T-maze for 1 min under darkness (to avoid environmental light cues). The performance index (PI) was calculated as the number of flies attracted to the unconditioned stimulus (CS -) minus the number of flies attracted to the (CS + ), divided by the total number of flies in the experiment. This is calculated as (N CS(-) -N CS(+) )/(N CS(-) +N CS(+) ) (where N is the number of flies). A single memory PI value is the average of the scores from two groups of flies of the same genotype trained with either 3-octanol or 4-methylcyclohexanol as the conditioning stimulus.

Olfactory acuity

To test for shock avoidance response, flies were kept at 30ºC for 2 days in the fed condition and were food-deprived for 16 h prior to test in the starved condition. Since the delivery of electroshocks can modify olfactory acuity (Preat, 1998), flies were first exposed to an odor -octanol or methylcyclohexanol-paired with electroshocks. Then, flies were exposed simultaneously to both the nontested odor and the paraffin oil alone for 1 min under darkness in a T-maze. The position of the odor and paraffin oil bottles was switched in every single experiment. Olfactory avoidance scores were calculated as follow: number of flies choosing paraffin oil minus the number of flies choosing the non-tested odor divided by the total number of flies.

Shock avoidance

To test for shock avoidance response, flies were kept at 30ºC for 2 days in the fed condition. Flies were induced 2 days at 30ºC and then were then fooddeprived for 16 h in the starved condition. The shock response was tested for 1 min at 25ºC under darkness, by placing flies in two connected barrels: electroshocks were delivered in only one of the barrels. Shock avoidance scores were calculated as follow: number of flies choosing the non-shocked barrel minus the number of flies choosing the shocked barrel divided by the total number of flies.

Immunohistochemistry and lipid staining

Fly brain immunostaining

Female flies carrying CRIMIC.TG4.0]Silnoon CR00519-TG4.0 were crossed to male flies carrying the UAS-mCD8::RFP construct to obtain the F1 (CRIMIC.TG4.0]Silnoon CR00519-TG4.0 >UAS-mCD8::RFP). Prior to dissection female flies of 2-4 days from the F1 were fixed in 4% paraformaldehyde in PBST (PBS containing 1% of Triton X-100, (Sigma-Aldrich)) at 4ºC overnight. Fly brains were dissected on ice in PBS solution and rinsed three times for 20 min in PBST. Then, brains were blocked with 2% BSA in PBST for 2 h. Next, they were incubated with primary antibodies 1:250 rabbit anti-RFP (catalogue number: 632496;

Clontech) and 1:100 mouse anti-nc82 (catalogue reference: nc82, DSHB). nc82 is a neuropile marker that labels active zones (Wagh et al., 2006). The incubation of primary antibodies was performed in the blocking solution (2% BSA in PBST) at 4ºC overnight. The following day, brains were rinsed 3 times for 20 min with PBST and then incubated for 3 h at room temperature with secondary antibodies diluted in blocking solution at 1:400 anti-mouse conjugated to Alexa Fluor 488 (catalogue number: A11029, Invitrogen Molecular Probes) and 1:400 anti-mouse conjugated to Alexa Fluor 594 (catalogue number: A11005, Invitrogen Molecular Probes).

Brains were then rinsed one time in PBST for 20 min, and two times in PBS for 20 min. After rinsing, brains were mounted using Prolong Mounting Medium (Lifetechnology). Acquisition was done with a Nikon A1R confocal microscope and confocal z-stacks acquired in 1 μm slices were imported into NIH ImageJ software for analyses.

Lipid droplet staining

Neutral LDs were detected with a non-polar fluorescent probe BODIPY 493/503 (Cabirol-Pol et al., 2017;[START_REF] Liu | The Glia-Neuron Lactate Shuttle and Elevated ROS Promote Lipid Synthesis in Neurons and Lipid Droplet Accumulation in Glia via APOE/D[END_REF] or BODIPY Red C12 [START_REF] Rambold | Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics[END_REF]. BODIPY staining was carried out according to a previous published protocol (Cabirol-Pol et al., 2017). Flies carrying the tub-Gal80 ts ;R54H02 cortex glia specific driver were crossed with a RNAi for CPT1, HMGS, Chaski, Brummer or Hormone-sensitive lipase. Moreover, flies carrying the tub-Gal80 ts ;VT30559 MB specific driver were crossed with a Silnoon RNAi or ACAT1 RNAi.

Briefly, flies were raised at 18ºC. Then, female flies of 2-4d old from the F1 were induced for 2 days at 30.5ºC and placed in starvation bottles during 16 h prior dissection. Group of flies from the same bottle were separated into fed and starved conditions. Prior to dissection, flies were anesthetized on ice. Brains were dissected in PBS. Then, brains were fixed in 4% paraformaldehyde in PBS. Next, brains were rinsed three times with PBS and incubated for 30 min with 1 μM BODIPY 493/503 under darkness. Brains were rinsed three times with PBS. After rinsing, brains were mounted using Prolong Mounting Medium (Lifetechnology).

Mounting and image acquisition was carried out the same day. Image acquisition was obtained around the cortex region, in the proximity of the MB calyx. Confocal z-stacks of LDs were imported into CellProfiler Analyst Software for analyses [START_REF] Jones | CellProfiler Analyst: data exploration and analysis software for complex image-based screens[END_REF].

A single plane in the cortex region, in the proximity of the MB calyx, was selected and converted into a 8-bit grayscale image (0-255 range). A rectangular specific area (84x80 μm) in close proximity to the calyx was selected and analyzed (Figure 38 A1). The threshold value was set up manually for each grayscale image using the following procedure. Indeed, since BODIPY label also the plasma membrane we have to apply a threshold to remove them from the quantification (Figure 38 threshold is not applied many non-specific particles linked to plasma membrane staining are identified by CellProfiler. After thresholding and applying size-limit object filter, LDs are positively identified (in green) and objects outside of the LDsize range are excluded from the analysis (in magenta) (Figure 38 B3). For each ROI, the area of each LD is calculated. Plotted area is expressed in μm 2 units.

In vivo two-photon microscopy imaging

In images were acquired at a rate of one image every two seconds.

For the lactate trans-acceleration experiments, Acetoacetate (Sigma A8509) was diluted into physiological solution or free-glucose solution at a final concentration of 100 mM. After 2 minutes of baseline acquisition, 10 μL of the solution was added to the 90 μL recording solution drop on the top of the brain, reaching a final concentration of 10 mM. Images were acquired during 10 min.

Image analysis was performed using a custom-written Matlab script [START_REF] Plaçais | Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory[END_REF]. Regions of interest (ROI) were delimited by hand around each visible Kenyon cells somas region. The average intensity of mTFP and Venus channels over each ROI were calculated over time after background substraction. FRET Laconic ratio was calculated by dividing mTFP intensity by Venus intensity.

Traces of the Kenyon cell somatas from the two hemispheres of a single fly were pooled. The indicated 'n' is the number of flies that were assayed for each condition.

Statistical analysis

Memory scores are displayed as mean ± SEM.. Statistical analyses were performed by one-way ANOVA followed by the Newman-Keuls pairwise comparisons test (significance was set at p<0.05). ANOVA results are given as the value of the Fisher distribution F (x,y) , where x is a number of degrees of freedom numerator and y is the total number of degrees of freedom denominator.

For imaging experiments, one replicate corresponds to the mean of the Laconic ratio of one fly brain and displayed as mean ± SEM. For LDs experiments, one replicate corresponds to the mean LDs area of one fly brain, and results are displayed as mean ± SEM. Two-tailed unpaired t-test was used to compare two conditions. Results from the unpaired t-test are given as the value t x of the t distribution, where x is a number of degrees of freedom. Asterisks in each figure refer to the least significant post hoc comparison between the genotype of interest and the genotypic controls. The nomenclature used corresponds to *p<0.05; **p<0.01; ***p< 0.001; ns: not significant, p>0.05. All statistical studies were performed using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA).

Results

Upon starvation mushroom body neurons import and oxidize ketone bodies to sustain sLT-ARM

Food deprivation periods lead to changes in long-lasting memory formation [START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF]. More specifically, when flies are starved for 24 hours LTM formation is shut down, while the formation of LT-ARM is upregulated, showing that the brain is still able to form memory under starvation.

From now I will name this memory starved LT-ARM (sLT-ARM), to differentiate it with LT-ARM formed in fed conditions. Remarkably, evidence from our lab showed that in normal conditions flies double their sugar intake after LTM conditioning, which is linked to upregulated glucose and pyruvate fluxes in the MB [START_REF] Plaçais | Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory[END_REF]de Tredern et al., in revision)

Upon starvation, ketone bodies oxidation in MB neurons is required for sLT-ARM

We have seen in the introduction that the brain is able to use KBs upon starvation in mammals [START_REF] Owen | Brain metabolism during fasting[END_REF]. However, do neurons have the machinery to use them to sustain memory process? This is the first question that I will address here. To investigate if MB neurons are able to use directly KBs as energy substrates, I tested the requirement of mitochondrial ACAT1, a key rate-limiting step enzyme of KBs oxidation (Figure 14), in adult MB for sLT-ARM formation. Consequently, I drove the expression of a specific RNAi that targets ACAT1 under the control of the MB VT30559-Gal4 driver, in combination with the ubiquitously expressed thermosensitive GAL4 inhibitor Gal80 (tubulin-Gal80 ts ), to restrict the RNAi expression to the adulthood. To test the hypothesis about the importance of KBs in memory formation upon starvation, I have used an aversive associative conditioning, where flies are upon starvation during 16 hours prior conditioning and during the following 24 hours, as it was done previously in [START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF]. Downregulation of ACAT1 expression in the adult MB induced a strong sLT-ARM impairment (Figure 39 A1), whereas LT-ARM (Figure 39 A2) and LTM (Figure 39 A3) were normal in fed flies. In absence of RNAi induction, sLT-ARM is normal (Figure 39 A4). Finally, the shock (Figure 39 A5) and olfactory acuity controls (Figure 39 show that ACAT1 is required specifically in adult MB neurons for sLT-ARM, suggesting that KBs are used by neurons as an energy substrate during food deprivation periods to form consolidated memories (Figure 40).

KBs cannot be synthesized in neuronal compartments due to the lack of LDs and activity of enzymes involved in β-oxidation in mammals [START_REF] Morris | Cerebral ketone body metabolism[END_REF]Schonfeld et al., 2013), and therefore is mandatory that they are taken up into neurons upon energy demand. In the following section I will investigate how MB neurons uptake KBs to form memories upon starvation.

1.2 Drosophila MB expresses MCTs that are necessary for sLT-ARM formation Since KBs are small polar molecules that cannot freely diffuse across the plasma membrane, the question that I will now address is how they enter neurons. In vertebrates, KBs are transported into the cell by MCTs [START_REF] Halestrap | The monocarboxylate transporter family--Structure and functional characterization[END_REF][START_REF] Hugo | A monocarboxylate transporter required for hepatocyte secretion of ketone bodies during fasting[END_REF]. The Drosophila genome harbors 22 MCTs homologs that share functional properties with vertebrates MCTs [START_REF] Volkenhoff | Glial Glycolysis Is Essential for Neuronal Survival in Drosophila[END_REF].

However, in Drosophila, the role of MCTs homologs on ketone body transport remains unknown. 41A). To further verify that the transcriptomic data faithfully represents the MB pattern of expression I have used a published CRIMIC reporter line for Silnoon [START_REF] Lee | A gene-specific T2A-GAL4 library for Drosophila[END_REF]. CRIMIC lines contain a T2A-Gal4 insertion in the intronic region of a gene of interest [START_REF] Lee | A gene-specific T2A-GAL4 library for Drosophila[END_REF], which allows, in combination with a UAS-reporter line, determine the expression pattern of a given gene (see material and methods for details). We crossed the CRISPR-derived CRIMIC Silnoon line with UAS-mCD8::RFP flies. We then carried out immunohistochemistry on the F1 flies (CRIMIC-Silnoon T2A-Gal4 >UAS-mCD8 ::RFP), and confirmed that Silnoon is expressed broadly in the brain, including MB neurons (Figure 41B-D). Thus, this evidence support that Silnoon transporter is expressed in MB neurons.

Unfortunately I did not find any gene reporter line for CG8034. I will now investigate if these transporters have structural features that may allow them to transport KBs within the MBs.

The human neuronal MCT2 and liver-specific Zebrafish Slc16a6a transporters have been described as well-known KBs carriers [START_REF] Halestrap | The monocarboxylate transporter family--Structure and functional characterization[END_REF][START_REF] Hugo | A monocarboxylate transporter required for hepatocyte secretion of ketone bodies during fasting[END_REF]. In Drosophila, Silnoon has been described as a butyrate and lactate transporter [START_REF] Jang | LKB1 induces apical trafficking of Silnoon, a monocarboxylate transporter, in Drosophila melanogaster[END_REF][START_REF] Delgado | Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress[END_REF] Human MCT1-4 transport monocarboxylates through a H + -coupled mechanism [START_REF] Halestrap | Monocarboxylic acid transport[END_REF], contrarily to MCT8/MCT10 that are H +independent. Two regions in the proximity to TM1 and TM8 domains have been previously suggested to play key roles in mediating monocarboxylates transport in MCT1-4 [START_REF] Manoharan | The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity[END_REF], with three charged residues: K38 close to TM1, and D309 and R313 in TM8 that are necessary for the H + -coupled mechanism of these transporters. We found that these three residues are present in the sequence of Silnoon and CG8034 (Figure 42). These analyses suggest that Silnoon and CG8034 may transport monocarboxylates via H + -coupled mechanism. Thus, bioinformatics analyses show that both Silnoon and CG8034 share sequence identity with the human MCT2 and contain key residues for the H + -coupled transport of monocarboxylates. Thus these analyses point CG8034

and Silnoon as potential candidates for transporting KBs in MB neurons. 44). Although Silnoon shares sequence with described KBs transporter (Figure 42), the capacity of Silnoon for transport KBs remains unknown. I hypothesized that Silnoon may mediate the uptake of KBs at the Kenyon cells somata, because vertebrate MCT2 transporter are preferentially expressed in neuronal cell bodies [START_REF] Pierre | Enhanced expression of three monocarboxylate transporter isoforms in the brain of obese mice[END_REF]. Because KBs in vivo imaging is not technically possible in the absence of proper reporter, to address this question I used the trans-acceleration property of MCTs [START_REF] Halestrap | Monocarboxylic acid transport[END_REF] (Figure 17). Trans-acceleration is a property of MCTs by which the transport in the manner of a symporter-type carrier can be activated by the in trans occupancy of a monocarboxylate [START_REF] Halestrap | The monocarboxylate transporter family--Structure and functional characterization[END_REF], which in this experiment can be observed as extrusion of lactate by the import of acetoacetate ketone body (Figure 45A). The extracellular application of acetoacetate and consequent extrusion of lactate can allow us to decipher in an indirect manner that Silnoon may transport KBs. Thus to assess the ability of Silnoon to uptake KBs at Kenyon cells somata, I drove the expression of a FRET Laconic sensor (San Martin et al., 2013) in MB neurons to observe, using in vivo 2-photon imaging, lactate trans-acceleration changes in Kenyon cells somata upon KBs application (Figure 45A-C). I chose to use acetoacetate for trans-acceleration experiments, as it is considered the main circulatory ketone body present in insects upon starvation [START_REF] Shah | Enzymes of ketogenesis in the fat body and the thoracic muscle of the adult cockroach[END_REF].

I have first bath-applied acetoacetate on the whole fly brain in order to assess if we could observe an efflux of lactate from the Kenyon cells somas due to an increased uptake of acetoacetate. Application of acetoacetate 10 mM on the whole fly brain led to a decrease lactate ratio signal in the Kenyon cell somata (Figure 45D), suggesting that lactate is extruded from Kenyon cells somata and therefore being trans-accelerated in presence of acetoacetate in normal fed conditions. Thus, acetoacetate application in the fly brain allows the extrusion of lactate from Kenyon cells somata.

Next, I compared trans-acceleration experiments in fed and starved flies in presence of acetoacetate (Figure 45E). As we previously observed, acetoacetate application in fed flies extruded lactate from Kenyon cells somatas. However, in starved flies, lactate extrusion is significantly reduced as compared to fed flies (Figure 45E). These results combined with the behavioral data suggest that Silnoon is likely to be the MCT required for the transporting of KBs into MB neurons to sustain memory formation upon starvation. Our results can be further supported by performing biochemical in vitro assays [START_REF] Jang | LKB1 induces apical trafficking of Silnoon, a monocarboxylate transporter, in Drosophila melanogaster[END_REF][START_REF] Halestrap | The monocarboxylate transporter family--Structure and functional characterization[END_REF] to determine whether Silnoon can transport KBs. ), suggesting that transacceleration is not taking place in starved flies. F, we mirrored the starved condition and artificially pushed the system by adding acetoacetate 10 mM in fed flies. Acetoacetate 10 mM application in fed flies expressing Laconic in MB along with Silnoon RNAi did not induce lactate extrusion from Kenyon cells somas, whereas as previously it did in control fed flies (n=10-11, t 40 =4,260, P=0.0001). Each curve is the mean of several recordings in different single flies. The bar graph is the quantification of 100 s at the plateau as indicated in the figures. Quantification has been done during the last 100 s of a 10 min recording. One replicate corresponds to one fly brain and displayed as mean ± SEM.

Therefore, KBs oxidation and uptake in the MB neurons is necessary for memory formation upon starvation (Figure 46). However, what is the cellular origin of KBs? In the following section I will address the cellular origin of these molecules.

cortex glial cells, that are therefore more than a transient transfer site. Eventually, I will demonstrate that KBs are synthetized in cortex glia from LDs.

2.1 Ketone bodies export from cortex glial cells requires Chaski MCT for sLT-

ARM

In Drosophila, six putative MCTs carriers are expressed in glial cells [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]: CG8389, Silnoon [START_REF] Jang | LKB1 induces apical trafficking of Silnoon, a monocarboxylate transporter, in Drosophila melanogaster[END_REF], CG13907, Hermes [START_REF] Velentzas | The Proton-Coupled Monocarboxylate Transporter Hermes Is Necessary for Autophagy during Cell Death[END_REF], dMCT1 [START_REF] Volkenhoff | Glial Glycolysis Is Essential for Neuronal Survival in Drosophila[END_REF] and Chaski [START_REF] Delgado | Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress[END_REF]. The constitutive knockdown of Silnoon, CG13907, dMCT1 or Chaski in glial cells causes lethality in adult fed flies [START_REF] Volkenhoff | Glial Glycolysis Is Essential for Neuronal Survival in Drosophila[END_REF][START_REF] Delgado | Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress[END_REF], pointing out the relevance of these MCTs in glial cells for organism survival. We thus hypothesized that any of these glial expressed MCTs might be required for KBs export during sLT-ARM formation and carried out a behavioral screen. We drove the expression of a specific RNAi that targets each of the six MCTs using the tub-Gal80ts;R54H02 cortex glia driver (de Tredern et al., in revision). Knocking down the expression of CG8389 (Figure 47A), Silnoon (Figure 47B), CG13907

(Figure 47C), Hermes (Figure 47D) or dMCT1 (Figure 47E) in cortex glial cells did not affect sLT-ARM or LT-ARM. Thus, many of the glial MCTs are not involved in sLT-ARM formation. However, the knockdown of Chaski in the adult cortex glia resulted in a strong sLT-ARM defect (Figure 48A), whereas LT-ARM (Figure 48B) as well as LTM were unaffected in control fed flies (Figure 48C). In absence of RNAi induction, sLT-ARM formation is normal (Figure 48D). Finally, the shock (Figure 48E) and olfactory acuity controls (Figure 48F-G) were normal in flies in which Chaski is downregulated in adult cortex glia. These results show that Chaski expression in the adult cortex glia is required specifically for sLT-ARM.

These results support the hypothesis that Chaski is the MCT involved in KBs export to neurons to sustain memory formation upon starvation. In the near future, I will investigate if Chaski is necessary for trans-acceleration of KBs and lactate in cortex glia, as I have shown for Silnoon in MB neurons (Figure 45).

However, such trans-acceleration mechanisms in cortex glia have never been reported yet in Drosophila. To conclude, our data suggest that a KBs transfer from cortex glia to MB neurons, via Chaski, is necessary to sustain memory upon starvation (Figure 49). Nevertheless, these data let open question about the origin of these molecules. In the following section I will address the question whether they could be locally synthesized in cortex glial cells or other glial subtypes.

the brain via surface glia and cortex glia, being cortex glia just an intermediate Nevertheless, I asked if memory formation upon starvation might rely on KBs synthesis from other glial cell types. I decided to address this question with adult ensheathing glia, because it surrounds another compartment than cortex glia, i.e the MB axons, and it shows a strong expression of CPT1 [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]. I did not investigate astrocyte-like glia due to the low astrocytic density processes around the MB [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF]. Knocking down CPT1 in the adult ensheathing glia did not affect sLT-ARM (Figure 51 A1) or LT-ARM in fed condition (Figure 51 A2). Similarly, downregulation of HMGS expression in adult ensheathing glia did not impair sLT-ARM (Figure 51 B1) or LT-ARM in fed flies (Figure 51 B2). Altogether, these results show that mitochondrial fatty acid uptake through CPT1, as well as mitochondrial KBs synthesis via HMGS, are required specifically in cortex glia for sLT-ARM. Thus, it strongly suggests that cortex glia is the main provider of KBs to MB neurons to sustain long-lasting memory formation upon starvation (Figure 52). As we have seen, KBs are synthesized out of fatty acids, thus I will show in the next section whether these fatty acids molecules are locally stored in cortex glia.

Lipid droplets are the local intracellular store of fatty acids in cortex glia, which are used to synthesize and export ketone bodies to mushroom body neurons upon starvation

In mammals, KBs derive from fatty acid β-oxidation [START_REF] Mcgarry | Regulation of hepatic fatty acid oxidation and ketone body production[END_REF]. Moreover, these fatty acids molecules might derive from internal LDs stores (Olzmann and Carvalho, 2018). In Drosophila, fatty acids are stored as LDs [START_REF] Palm | Lipoproteins in Drosophila melanogaster--assembly, function, and influence on tissue lipid composition[END_REF][START_REF] Kis | Specialized Cortex Glial Cells Accumulate Lipid Droplets in Drosophila melanogaster[END_REF]Olzmann and Carvalho, 2018). These LDs are mainly constituted of triglycerides, phospholipids and sterols (Olzmann and Carvalho, 2018), and fatty acids derived from LDs can be mobilized through the action of several lipases (Gronke et al., 2005). Here I will first address whether cortex glia present such internal stores of fatty acids in the form of LDs.

Adult cortex glia contains cytoplasmic LDs

It has been shown that Drosophila larvae cortex glial cells contain an abundant number of LDs [START_REF] Kis | Specialized Cortex Glial Cells Accumulate Lipid Droplets in Drosophila melanogaster[END_REF][START_REF] Zheng | A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster[END_REF]. However, whether this reservoir of lipid is still present at the adult stage in cortex glia was unclear.

To investigate the presence of LDs in the cortex glia at adult stage, I have used non-polar fluorescent dyes BODIPY 493/503 and BODIPY Red C12 both of them are molecules that identify neutral LDs [START_REF] Rambold | Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics[END_REF]. We drove the expression of a specific fluorescent reporter UAS-mCD8::RFP, under the control of the cortex glia specific R54H02-Gal4 driver (Coutinho-Budd et al., 2017) and then we stained these fly brains with BODIPY 493/503 (Figure 53A). BODIPY 493/503 staining revealed that LDs are present in the adult cortex glia (Figure 53B). Interestingly, BODIPY 493/503 staining exhibits a homogenous expression pattern that resembles cortex glia staining (Figure 53B). In fact, BODIPY reaches regions inside the plasma membrane of the cortex glia (Figure 53B), which is also observed by others [START_REF] Rambold | Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics[END_REF][START_REF] Cabirol-Pol | Glial lipid droplets and neurodegeneration in a Drosophila model of complex I deficiency[END_REF]. This explains why, in addition to the high intensity LD staining observed (Figure 53B), a diffuse staining that colocalize with the plasma membrane staining of cortex glia is also detected. To confirm that the high intensity objects are indeed LDs, I have used a

LDs coating protein LSD-2::GFP reporter line [START_REF] Teixeira | Drosophila Perilipin/ADRP homologue Lsd2 regulates lipid metabolism[END_REF] and BODIPY Red C12 (because of fluorophore compatibility), which also labels neutral LDs [START_REF] Rambold | Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics[END_REF]. Coating proteins are present in LDs, but not all of them contain LSD-2 (Olzmann and Carvalho, 2018). Using the LDs specific protein reporter (UAS-LSD-2::GFP) expressed in cortex glia I found that LSD-2 reporter overlap with most of the LDs stained with BODIPY Red C12 (Figure 53C 48,50). We carried out LDs area analysis in starved and fed flies using CellProfiler Software Analysis as it has been validated in the previous section (Figure 53D, and see material and methods section for more details). We first assessed the role of CPT1, the entry point of fatty acids into mitochondria for KBs synthesis, on LDs mobilization in starved and fed flies.

Knocking down CPT1 in cortex glia of starved flies showed a significant increase in LDs area as compared to control starved flies (Figure 54 A,B1). On the contrary, downregulation of CPT1 expression in cortex glia of fed flies did not show any significant difference with the control group (Figure 54 B2). These results suggest that under starvation, and not in fed conditions, cortex glia LDs are mobilized and provide fatty acids to mitochondria through CPT1. In fed condition, the flux of fatty acids into mitochondria is either, not dependent on CPT1, or more likely, not large enough that blocking it does not significantly affect the size of LD.

As we have seen previously, fatty acids taken up into mitochondria are then used to generate acetyl-CoA (Blasquez et al., 1998). If the acetyl-CoA produced is then used for KBs synthesis, by affecting the ketogenesis pathway in cortex glia we should observe a similar effect on LD, as the one observed by knocking-down CPT1 (Figure 54). Thus I tested this hypothesis by knocking down HMGS in cortex glia, the rate-limiting step in the ketogenesis pathway, and characterizing LDs upon starvation. Downregulation of HMGS in cortex glia of starved flies showed a significant increase in LDs area than control flies (Figure 54 C,D1). In fed flies, the knockdown of HMGS in cortex glia did not alter LDs area as compared to the control group (Figure 54 D2). These results show that in cortex glia the two key steps of ketogenesis that are critical to sustain sLT-ARM formation in MB neurons (Figure 50), are downstream of LD mobilization upon starvation. Altogether, these data suggest that upon starvation the main source of fatty acids for ketogenesis in cortex glia is their own internal store of fatty acids in the form of LDs.

Once they have been produced in cortex glia, we have shown that KBs can be exported to sustain sLT-ARM formation in MB neurons via the Chaski MCT (Figure 48). We thus investigate if, as for ketogenesis (Figure 54 In Drosophila, several lipases are able to release fatty acids from LDs that are then available for a variety of cellular process, including ketogenesis. Among them, the 4 lipases Brummer, Hormone-sensitive Lipase (dHSL), Lipase 4 (Lip4)

and CG5966 are expressed in glial cells [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF] (Figure 55A). To further identify the molecular pathway involved in LDs mobilization to provide fatty acids for KBs synthesis upon starvation, I first started a screen on LDs area using the BODIPY dye. Preliminary data suggest that when Brummer expression is reduced in adult cortex glia, LD area is increased in starved condition (Figure 55B). These results seem specific of Brummer since the knockdown of HSL in adult cortex glia has no effect on LD area upon starvation (Figure 55B). These results need to be completed with experiments in fed conditions and the other glial lipases, but it suggests that at least Brummer is required for LDs mobilization in cortex glia in starved condition. This result will allow us to test whether Brummer lipase expression in cortex glia is required for sLT-ARM. This later result will be crucial to bring direct evidence that LDs mobilization in cortex glia is required to sustain memory formation upon starvation. However, another way to address this question in a more indirect way is to investigate if MB neuronal KBs import and oxidation can be linked to LD mobilization in cortex glia. This approach will be detailed in the next section.

significant increase in LDs area in starved flies as compared to controls (n=3, t 4 =3,717, P=0.0205). B, HSL RNAi expression in adult cortex glia did not change LDs area in starved flies (n=3, t 4 =1,300, P=0.2636). Each dot represents the mean of LDs area of one single fly brain (in μm). Data are shown as mean ± SEM. Statistical analyses were performed using unpaired t-test (*p<0.05, ns: not significant). LDs analyses were carried out with CellProfiler Software Analyst.

3.3 Influence of MB neuronal ketone bodies oxidation and uptake on cortex glial

LDs

In the first part of the results section, I demonstrated that MB neurons use KBs to sustain sLT-ARM formation and that these KBs are mainly provided by cortex glia. I have just shown in the previous section that cortex glia mobilizes its own fatty acids internal store upon starvation to produce KBs and export them.

Here we want to investigate if a direct link between the use of KBs by MB Altogether these data suggest that blocking the KBs use in MB neurons upon starvation does not impact significantly LDs mobilization in cortex glia. This can be explained either by the fact that cortex glia LDs are not the source of KBs precursor used by cortex glia to provide these molecules to neurons, or alternatively, that affecting KBs only in MBs that represent a small subset of total neurons in the brain is not sufficient to globally affect cortex glial LDs. Thus, impairing KBs utilization in only a fraction of neurons might be masked by the use of KBs by other neurons in the brain that are also depending on cortex glia for providing them. This hypothesis will be further tested using a pan-neuronal driver to knockdown Silnoon or ACAT1, and investigate cortex glia LDs mobilization.

can cortex glia switch to KBs production depending on brain energy status and how does it tune its production of KBs to the MB neurons needs? Indeed, I have seen that in fed condition, KBs linked pathways (i.e synthesis, transport and catabolic use) are not involved to sustain LT-ARM or LTM as well as in LDs use, but they are needed upon starvation to feed neuronal MB needs. In the last results section, I will start addressing these questions.

The activity of AMPK energy sensor in cortex glia is required for sLT-ARM formation

We have seen previously that the uptake of fatty acids into the mitochondria is a key step in cortex glia KBs production. Interestingly, CPT1

activity is regulated by AMPK via ACC phosphorylation in rodents (Blasquez et al., 1999;[START_REF] Kim | The effect of clozapine on the AMPK-ACC-CPT1 pathway in the rat frontal cortex[END_REF] (Figure 16). Thus, fatty acid entry into mitochondria can be regulated by AMPK. AMPK is a heterotrimeric complex composed of three different subunits (α, β and γ) (Figure 57A). The α subunit is the catalytic unit, whereas the γ sense the AMP:ATP levels [START_REF] Kim | AMPK activators: mechanisms of action and physiological activities[END_REF]. Moreover, the β subunit has been shown to be required for glycogen metabolism (Polekhina et al., 2003). AMPK regulates several core pathways, including mTOR signaling (by inhibiting protein synthesis), activation of autophagy pathway and the activation of the ATGL lipase (Figure 57B). Moreover, AMPK activation inhibits the synthesis of glycogen, fatty acids and cholesterol [START_REF] Kim | AMPK activators: mechanisms of action and physiological activities[END_REF] (Figure 57B). AMPK is a major energy sensor of the cell, and whenever the AMP:ATP ratio is high, such as upon starvation, AMPK is activated [START_REF] Miyamoto | Compartmentalized AMPK signaling illuminated by genetically encoded molecular sensors and actuators[END_REF].

To investigate how cortex glia tunes their KBs synthesis to the energy status of the brain and if AMPK would be the sensor of this regulation, I tested the effect of knocking down either the α-or γ-subunit of AMPK in adult cortex glia on sLT-ARM. I decided to focus on the α-and γ-subunit because the α-subunit is the catalytic unit, and for the γ subunit because it is the unit sensing the AMP/ATP ratio in the cell (Figure 57A). Downregulation of AMPK-α expression in adult cortex glia induced a strong sLT-ARM impairment (Figure 58 A1), whereas LT-ARM was unaffected in fed flies (Figure 58 A2). In addition, the knockdown of AMPK-α in the adult cortex glia did not impair LTM in fed flies (Figure 58 A3).

Non-induced controls and sensory controls will be done in the future. On the other hand, downregulation of AMPK-γ expression in adult cortex glia did not alter sLT-ARM (Figure 58 B1), LT-ARM in fed flies or LTM (Figure 58 B2,B3). Thus, these results show that AMPK α subunit is critical in cortex glia for sLT-ARM.

Although further experiments will be needed to confirm the negative results obtained with the γ-subunit, such as using another RNAi line and verifying the efficiency of the RNAi construct. Therefore, AMPK activity is required for sLT-ARM, likely because it regulates the activity of downstream critical effectors, such as CPT1 or Brummer lipase involved in LDs mobilization. Eventually, these results

suggest that in cortex glia upon starvation AMPK regulates KBs synthesis via CPT1 regulation. Therefore, the brain could adapt its metabolism to provide KBs to neurons to sustain memory formation upon starvation.

In conclusion, I have found that upon starvation cortex glial cells synthetize KBs from their fatty acids internal stores and export these KBs to MB neurons to sustain memory formation. In addition, I have shown that KBs uptake and oxidation in the MB Kenyon cells is critical for memory upon starvation.

Eventually, I found that the master energy sensor AMPK is required in cortex glia for memory formation upon starvation likely via regulation of fatty acid flux into mitochondria to generate KBs. Thus, I have proposed a model in which the starved brain initiates a glia-to-neuron KBs shuttling to provide neurons with energy rich substrates during associative long-lasting memory formation. This model is depicted in Figure 59 and could be named, in reference to the ANLS model [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization[END_REF], the glia-to-neuron KBs shuttle model.

Discussion

Even though extensive evidence reveals the importance of the neural circuits surrounding the MB in memory formation, the metabolic mechanisms that affect aversive long-lasting memory formation have recently started to being revealed [START_REF] Plaçais | Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory[END_REF]de Tredern et al., in revision).

What is the cost of forming a long-lasting memory? We have introduced that LTM and LT-ARM have a different cost [START_REF] Mery | A cost of long-term memory in Drosophila[END_REF]. When flies form LTM or LT-ARM in fed conditions and then are subjected to a food and water deprivation challenge, flies that form LTM died earlier than those forming LT-ARM [START_REF] Mery | A cost of long-term memory in Drosophila[END_REF]. This prompted to suggest that LTM and LT-ARM might be sustained by mechanisms involving different metabolic costs. But, how do flies form a long-lasting memory when glucose is unavailable?

Glucose unavailability during starvation periods has consequences on LTM and LT-ARM formation [START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF]. Indeed, starved flies subjected to spaced conditioning do not form LTM, but rather form a memory that is resistant to a protein synthesis inhibitor and sensitive to a LT-ARM inhibitor (Figure 27), suggesting that in absence of food flies only form LT-ARM. Indeed, flies subjected to massed conditioning upon starvation showed increased memory performance compared to fed flies. This indicates that flies upon starvation only form sLT-ARM while LTM formation is precluded (Figure 27). Thus, in conditions of food deprivation flies still form a long-lasting memory.

Interestingly, evidence from mammals show that mice subjected to alternate days of food deprivation (i.e alternate day fasting) showed an improved memory formation in the Barnes maze and contextual fear conditioning assays [START_REF] Li | Chronic intermittent fasting improves cognitive functions and brain structures in mice[END_REF], suggesting that the maintenance of memory upon food deprivation periods is a mechanism conserved from mammals to insects.

During food deprivation periods the human brain switch into a major utilization of KBs as their main oxidative fuel [START_REF] Owen | Brain metabolism during fasting[END_REF]. KBs can be oxidized in vitro by mammalian neurons, and to a lesser extent, by astrocytes [START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF][START_REF] Pan | 2,4-13 C2 ]-beta-Hydroxybutyrate metabolism in human brain[END_REF]. Pioneer studies demonstrated that astrocytes synthesize KBs in vitro in rodents (Blasquez et al., 1998), which prompted to hypothesize the ongoing idea of the existence of a KBs shuttling mechanism between neurons and glial cells (Guzman and Blasquez, 2001).

However, until now the existence in vivo of this shuttling mechanism upon periods of food restriction remained unknown.

The work done here provides the first evidence in vivo that a glia-to-neuron KBs transport mechanism support memory formation under periods of food deprivation in the fruit fly brain (Figure 59). More specifically, we have shown that during memory formation upon starvation cortex glial cells activate lipolysis from LDs stores to provide substrates for ketogenesis (Figure 50,54,55). Then, KBs synthesized in cortex glia are transported into MB neurons by specific MCT carrier systems (Figure 44,48). Eventually, KBs are oxidized in MB neurons for energy production (Figure 39). In addition, we showed that in the starved brain AMPK expression in cortex glial cells is required during memory formation as a regulatory enzyme of this KBs transport mechanism (Figure 58).

These findings highlight the relevance of glial lipid metabolism in the production of energy during memory formation and open new questions regarding the neuronal utilization of KBs under different brain processes upon starvation.

The proposed glia-to-neuron ketone body transport

Although we show that a glia-to-neuron ketone body transport mechanism is required during memory formation upon starvation (Figure 59), this hypothesis has not been yet explored in the mammalian brain. In fact, in the fed mammalian brain the ANLS has been proposed as the model to provide energy to the brain under neuronal activity (Figure 7). However, could it be possible that the Drosophila KBs transport proposed here might also be relevant under starvation in response to neuronal activity in the mammalian brain?

In our glia-to-neuron ketone body transport mechanism we were unable to distinguish if i) KBs production and utilization is the baseline fueling mechanism during starved states and ii) whether there is a specific signaling from MB neurons to cortex glia to trigger a KBs transfer during memory formation. In this entire section we will bring evidence to discuss around these two potential hypotheses. More specifically, I will first discuss the current view of transport mechanisms between neurons and glial cells and how the proposed model can be integrated in this general scheme. Then I will discuss the regulatory mechanisms of this glia-to-neuron ketone body transport and how it responds to brain changing needs.

1.1 ANLS model and a glia-to-neuron ketone body transport model Nowadays, the main view on how the mammalian brain supports neuronal oxidative metabolism in the fed state is based on the ANLS model [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization[END_REF], where glucose-derived lactate in astrocytes fuels neuronal oxidative metabolism in response to neuronal activity (Figure 60). In addition, it has been proposed an alternative to this model, in which glucose derived from glial cells fuel neuronal energy metabolism in fed conditions (Diaz-Garcia et al.,

2018).

To date, there is no evidence supporting that the ANLS model would occur during starved states. In fact, during starvation glycogen stores are depleted after a few hours [START_REF] Mattson | Intermittent metabolic switching, neuroplasticity and brain health[END_REF] and glucose-derived lactate is expected to be low in this condition. Therefore, the ANLS model cannot provide energetic fueling under starvation at the synapse due to a lack of carbohydrates stores.

On the other hand, in mammals, it has been mentioned, as an unpublished observation, that glutamate application enhances ketogenesis in astrocytes (Guzman and Blasquez, 2001). Authors from that study suggested that glutamate release from neurons might activate astrocytic shuttling of KBs. Nevertheless, as it occurs in the ANLS model with glucose, it is unclear if neuronal activity would also trigger circulating fatty acid uptake in glial cells (Figure 60). Interestingly, glutamate receptor activation in astrocytes leads to LDs mobilization and increased β-oxidation in vitro [START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF]. Thus, in mammals, glutamate release upon neuronal activity might favor lipolysis [START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF], ketogenesis and KBs shuttling from astrocytes (Guzman and Blasquez, 2001).

Our glia-to-neuron ketone body transport model proposes an additional transport mechanism, which provide energy to the brain in food-deprived conditions (Figure 60). It is interesting to note that both astrocytes and satellite glial cells (Figure 61), a perisomatic glial cell that surrounds the cell bodies of sensory neurons, and that is morphologically similar to Drosophila cortex glial cells, express HMG-CoA synthase, suggesting that the synthesis of KBs may be occurring in these cells in vivo (Hegardt et al., 2000[START_REF] Wang | HMG-CoA synthase isoenzymes 1 and 2 localize to satellite glial cells in dorsal root ganglia and are differentially regulated by peripheral nerve injury[END_REF]. Thus, a KBs transporting mechanism may take place between neurons and glial cells from different species. released from glial cells is greater [START_REF] Cahill | Fuel metabolism in starvation[END_REF], suggesting that in terms of net ATP production KBs might replace glucose-derived substrates in a more efficient manner. Thus, in the starved state, KBs metabolism can replace lactate to sustain neuronal oxidative metabolism. Intriguingly in the Drosophila starved brain, only sLT-ARM is formed whereas the long-lasting form of memory dependent of protein synthesis (LTM) and relying on pyruvate metabolism is not formed [START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF][START_REF] Plaçais | Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory[END_REF], the reason for this is thus not based only on the ATP generation but likely rely on regulatory mechanisms sensing the level of energy at the entire brain level.

Thus, in the mammalian brain glial-derived lactate might fuel neuronal oxidative metabolism upon glutamatergic activity in the fed state. However, it is unclear whether a KBs shuttling might occur in vivo. It remains unknown if activating Drosophila cortex glia would lead to a greater production of KBs in response to neuronal activity during starvation. In the following section I will discuss more in deep the question about the utilization of KBs as a baseline fueling mechanism versus a KBs transfer triggered by an increased neuronal activity.

1.2 Are ketone bodies synthesized as a baseline fueling mechanism or upon demand?

The glia-to-neuron KBs transport model opens the question regarding whether KBs fueling from glia to neurons is the baseline state in the starved condition or if there is an activation of KBs synthesis and transport upon memory formation. Could it be possible that KBs are produced during the first hours of food deprivation or are they synthesized and used upon demand?

A hallmark of the glia-to-neuron KBs transport model we propose is that it depends on a local source of energy in cortex glial cells (Figure 59). It should be noted that vertebrate and insect glial cells store a rich amount of energy in the form of glycogen and fatty acids (Belanger et al., 2011;Kiss et al., 2015), which can be mobilized for instance during events of glucose scarcity [START_REF] Wigglesworth | The Nutrition of the Central Nervous System in the Cockroach <em>Periplaneta Americana&lt[END_REF][START_REF] Wender | Astrocytic glycogen influences axon function and survival during glucose deprivation in central white matter[END_REF]. Thus, when glial carbohydrates stores are depleted during the first hours of food deprivation [START_REF] Mattson | Impact of intermittent fasting on health and disease processes[END_REF], the cortex glial LDs stores would become the main substrate to fuel neuronal demands for a prolonged period of time [START_REF] Wigglesworth | The Nutrition of the Central Nervous System in the Cockroach <em>Periplaneta Americana&lt[END_REF]. Thus, a KBs transport would provide local energy to fuel neurons upon food restriction as a baseline fueling mechanism.

But, more specifically, when do KBs are synthesized in glial cells? We subjected flies to starvation during 16 hours prior and then 24 hours after conditioning. In fact, in fruit flies, food deprivation depletes carbohydrates stores after approximately 18 h (at 25ºC), being just 10% of their original levels [START_REF] Yamada | Correction: Fat body glycogen serves as a metabolic safeguard for the maintenance of sugar levels in Drosophila[END_REF]. However, in our experimental condition, we starved the flies during 16 h at 30.5ºC (see material and methods), which may deplete glycogen stores even earlier than the 18 h showed by Yamada and colleagues. This evidence suggests that a metabolic switch to KBs synthesis could take place more likely before conditioning. In fact, our LDs analyses suggest that after 16 h of food deprivation, the synthesis of KBs is already taking place in glial cells in naive flies (Figure 54). Thus, synthesis of KBs might be produced as a baseline mechanism to support neuronal energy metabolism. However, it remains unknown whether glia could increase KBs synthesis from LDs fatty acids or transport upon demand during memory formation.

If the synthesis of KBs takes place even before conditioning, then we can ask whether KBs can be stored. Evidence from humans, archeas and bacteria

show that KBs can be stored as polymers in the form of polyhydroxybutyrate (PHB) in specialized organelles known as carbonosomes [START_REF] Elustondo | Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes[END_REF].

Interestingly, PHB polymers can be used in conditions of nutrient deprivation [START_REF] Elustondo | Identification of the polyhydroxybutyrate granules in mammalian cultured cells[END_REF][START_REF] Kaewbai-Ngam | Increased accumulation of polyhydroxybutyrate in divergent cyanobacteria under nutrientdeprived photoautotrophy: An efficient conversion of solar energy and carbon dioxide to polyhydroxybutyrate by Calothrix scytonemicola TISTR 8095[END_REF]. But, are these PHB polymers present in the fly brain and could they be used in conditions of nutrient restriction? In Drosophila, it is unknown if such PHB polymers exist in cortex glia.

However, if PHB would be present in this structure, we can hypothesize PHB polymers could be used to provide KBs, bypassing the LDs mobilization and fatty acid oxidation steps, during neuronal activity. Currently, the only detection method available to measure and quantify PHB polymers is through gas chromatography [START_REF] Kansiz | Quantitative determination of the biodegradable polymer Poly(beta-hydroxybutyrate) in a recombinant Escherichia coli strain by use of mid-infrared spectroscopy and multivariative statistics[END_REF] and might be used to detect PHB polymers in Drosophila. PHB synthase and depolymerase are the main enzyme responsible for the conversion of molecules of acetyl-CoA into PHB polymers and degradation into β-hydroxybutyrate in bacterial cells, respectively [START_REF] Kansiz | Quantitative determination of the biodegradable polymer Poly(beta-hydroxybutyrate) in a recombinant Escherichia coli strain by use of mid-infrared spectroscopy and multivariative statistics[END_REF]. However, there are no identified orthologs of these enzymes in Drosophila yet. Therefore, PHB could be synthesized as an early storing mechanism of KBs.

Thus, the storage of KBs as PHB polymers might ensure a faster action of KBs in providing energy to neurons upon demand.

I have shown that KBs transfer from cortex glia to MB neurons occurs during memory formation upon starvation (Figure 59). Indeed, since cortex glial cells cover the majority of cell bodies in the cortex region [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF], we can ask if this transport occurs also for others neurons than MB, such as the central complex or insulin-producing cells [START_REF] Zheng | A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster[END_REF]. When we knocked down genes involved in ketogenesis we observed a global accumulation of LDs in cortex glia (Figure 54). Oppositely, when we downregulated ACAT1 or Silnoon in the MB neurons we did not observe changes in cortex glial LDs (Figure 56), suggesting that the effect of this transport might be spread around all the cortex glia. This will be confirmed by the experiment in which we will knockdown either ACAT1 or Silnoon pan-neuronally and observe LDs changes in cortex glia. Therefore, our data may support the hypothesis that KBs might serve as a basic fueling mechanism, but it remains to be determined whether a KBs transfer might be regulated in response to neuronal activity and more specifically upon memory formation.

In the following section I will discuss the cellular localization of the KBs transport within glia and neurons.

1.3 Cellular localization of the glia-to-neuron ketone body transport: main components Our data brings in vivo evidence to support the glia-to-neuron ketone body transport model with a transfer of KBs from cortex glia to neurons (Figure 59).

However, where is occurring this transporting mechanism within glia and neurons? I will now discuss evidence to address the question about the cellular localization within these cellular types.

Cellular localization of the ketone body transport within cortex glial cells

Approximately ≈2600 cortex glial cells are present in the fly brain [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF] and extend their ramifications around neuronal cell bodies. In the cortex glia region we have shown by immunohistochemistry and confocal imaging that there are an abundant number of LDs (Figure 53), but we could not determine the intracellular localization of LDs more than being cytoplasmic (Figure 53). A 3D-reconstruction of the whole Drosophila brain in the fed state was recently carried out using electron microscopy [START_REF] Zheng | A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster[END_REF]. Similarly to what we observed with BODPY staining (Figure 53), LDs are found in the cytoplasmic region of cortex glial cells in close proximity to the neuronal somas of the Kenyon cells (Figure 62). But the high-resolution of electronic microscopy provide evidence using a GFP tag for Brummer (UAS-Bmm::GFP) (Gronke et al., 2005) expressed in cortex glia to confirm its expression in close contact with LDs.

To test this hypothesis we can use this Brummer tag with BODIPY Red C12 in starved flies to observe if they are in close contact with LDs. On the other hand, similarly as we hypothesized with Brummer, are CPT1 or HMGS in close contact to LDs during starvation? KBs synthesis enzymes, such as CPT1 and HMGS are present in cortex glia and expected to be preferentially located in mitochondria [START_REF] Schulz | Glial β-oxidation regulates Drosophila energy metabolism[END_REF]. To test if CPT1 or HMGS are in close contact with LDs we can use antibodies available for these two enzymes [START_REF] Escartin | Activation of astrocytes by CNTF induces metabolic plasticity and increases resistance to metabolic insults[END_REF][START_REF] Patel | Effects of 4-hydroxynonenal on mitochondrial 3-hydroxy-3-methylglutaryl (HMG-CoA) synthase[END_REF] combined with BODIPY, to observe if they are in close contact upon starvation as compared to the fed state. Thus, it is yet unclear if under starvation the KBs transporting components might be in close contact to favor KBs synthesis.

Eventually, we can wonder if the KBs transport system in glia (i.e Chaski) is specifically localized at the cell membrane in the place of close proximity with Kenyon cells soma or if it is homogenously distributed across cell membrane of cortex glia. Starvation or demand from specific subset of neurons could induce a re-localization of Chaski in a specific cell membrane region. Those questions would need further investigation using a homemade antibody or alternatively an endogenous tag for Chaski in order to be addressed.

Thus, glial cells LDs are likely to be in close proximity to mitochondria upon starvation [START_REF] Rambold | Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics[END_REF], which would facilitate KBs synthesis. Moreover, a re-localization of MCT transporters to specific cell membrane regions would facilitate KBs flux towards neurons. I will now discuss the localization of the KBs transport mechanism within neurons.

Localization of the ketone body transport within MB neurons

KBs are presumably delivered at the Kenyon cells somatas because of cortex glial cells localization. However for now we did not show that KBs uptake (Figure 44) and oxidation (Figure 39) are localized at the Kenyon cells soma. The laconic sensor and trans-acceleration experiments provide evidence that uptake of KBs occurs at Kenyon cells somatas (Figure 45). However, it does not rule out the possibility that uptake of KBs also occurs in the MB lobes: which can be addressed by similar experiments carrying out in vivo imaging at the MB lobes. In fact, KBs can be imported at the level of the somas and then diffuse to the calyx or lobes where they can be oxidized. Although there are no antibodies available for ACAT1, it might be interesting to generate an antibody and determine its subcellular localization in neurons during starved conditions. This will help us to determine the subcellular localization of ACAT1 enzyme and the potential site of KBs oxidation, either at the somas, axons or synaptic compartments.

Interestingly, immunohistochemical evidence in mammals show that neuronal cell bodies of Purkinje cells strongly express KBs oxidative enzymes [START_REF] Gesink | A histochemical study of the distribution of betahydroxybutyrate dehydrogenase in developing rat cerebellum[END_REF], suggesting that neuronal somatas are well equipped for oxidizing these molecules. Therefore, it will be crucial to determine the subcellular localization of the enzymes participating in KBs oxidation in Drosophila.

I will now discuss some of the mechanisms involved in the regulation of this KBs transporting mechanism, with emphasis in AMPK master cell sensor.

Regulation of the ketone body transport

The transport of KBs needs to be tightly regulated at different levels within glia and neurons. This may allow an efficient production and release of KBs in glia and consequent oxidation in neurons as a baseline mechanism or upon demand, which might be essential during long-lasting memory formation. However, what are the mechanisms involved in the regulation of this KBs transport mechanism? I will now discuss the mechanisms involved in the regulation of this transport mechanism with emphasis in AMPK master cell sensor.

1.4.1 Sensitivity to energy level via the master energy sensor: AMPK

We have identified AMPK as a one key regulatory step enzyme involved in sLT-ARM (Figure 58). In vertebrates, AMPK activation can lead to inhibition of mTOR (which in turn inhibit protein synthesis), activation of autophagy and inhibition of fatty acid synthesis [START_REF] Mihaylova | The AMPK signalling pathway coordinates cell growth, autophagy and metabolism[END_REF] (Figure 57). On the other hand, vertebrate AMPK participate in the upregulation of catabolic processes, including lipolysis [START_REF] Daval | Functions of AMP-activated protein kinase in adipose tissue[END_REF], mitochondrial β-oxidation and ketogenesis in glial cells (Blasquez et al., 1998) (Figure 57). We can hypothesize that the status of the animal and after conditioning, using the FRET AMPK sensor developed by [START_REF] Miyamoto | Compartmentalized AMPK signaling illuminated by genetically encoded molecular sensors and actuators[END_REF]. Another approach to investigate the activation of the AMPK/CPT1/ACC axis would be to determine the level of phosphorylation of ACC, using a specific Drosophila antibody [START_REF] Hardie | Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase[END_REF], in the starved brain compared to the fed brain, with the expectation that AMPK activation would lead to phosphorylation of ACC. Thus, these experiments will help us to show a direct link between AMPK activation, ACC inhibition and further CPT1 activation. Moreover, the AMPK imaging experiments will allow us to determine if the activation of a KBs transporting mechanism might be taking place upon demand, after conditioning. However, how can AMPK activation be linked to the mobilization of LDs stores in cortex glia?

1.4.2 Regulation of LD mobilization in cortex glia by lipase regulation LDs mobilization mediated by glial lipase is a critical event to provide fatty acids to mitochondria for KBs production. In vertebrates, upon starvation, AMPK activation can enhance lipolysis by activating the ATGL lipase (Figure 57), which can enhance LDs mobilization and further mitochondrial fatty acid oxidation in brown adipose tissue [START_REF] Rambold | Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics[END_REF][START_REF] Kim | AMPK activators: mechanisms of action and physiological activities[END_REF]. It is interesting to note that in Drosophila, Brummer gene encodes the homolog of the human ATGL lipase (Gronke et al., 2005). Brummer is expressed in cortex glial cells [START_REF] Davie | A Single-Cell Transcriptome Atlas of the Aging Drosophila Brain[END_REF]) and as we have shown is required for LDs mobilization (Figure 55). If

Brummer is a downstream target of AMPK we should also observe an increase in LDs area in presence of a AMPK RNAi expressed in cortex glia, although this increase might be partial or complete because might be not the only signal to activate lipolysis. Eventually, in order to have a direct link between LDs mobilization and memory formation we expect to use a Brummer RNAi expressed in cortex glia and test sLT-ARM formation. Thus, a Brummer activity in cortex glial cells may also be necessary during memory upon starvation. (Heier and Kuhnlein, 2018). However, the presence of Akh receptor in cortex glia and downstream pathways remain to be proved. Moreover, we did not observe changes in LDs in the knockdown of dHSL in cortex glia (Figure 55), suggesting that this pathway might not be necessary for LDs mobilization in cortex glia. Starvation might increase the number of MCT present at the cell membrane, either by increasing gene expression or by regulation of trafficking and anchoring processes. We will discuss now if such mechanisms can occur in cortex glia to increase KBs transport between glia and neurons. For instance, Chaski expression in cortex glial cells is required for the survival of the fly during starvation, and not in fed conditions [START_REF] Delgado | Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress[END_REF]. This specific requirement of Chaski can be considered a regulation of monocarboxylates transport at the level of the brain in food-deprived states. In order to prove that hypothesis, we can determine Chaski expression in the brain and compare those changes between the fed and starved brain by carrying out qPCR experiments. A second potential mechanism of regulation is to increase the number of MCTs at the plasma membrane. Such regulation of MCT trafficking has already been described in flies [START_REF] Buyse | Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE[END_REF][START_REF] Jang | LKB1 induces apical trafficking of Silnoon, a monocarboxylate transporter, in Drosophila melanogaster[END_REF], although it has not been shown yet in cortex glia or MB neurons. For instance, in cell lines, leptin upregulates butyrate uptake by increasing the intracellular pool of MCT1 transporters, translocating a complex of MCT1 and the anchoring protein CD147

to the apical plasma membrane of these cells [START_REF] Buyse | Luminal leptin enhances CD147/MCT-1-mediated uptake of butyrate in the human intestinal cell line Caco2-BBE[END_REF]. In the wing discs of Drosophila, Silnoon trafficking from the apical to the basolateral membrane has been described and is dependent on LKB1 activation [START_REF] Jang | LKB1 induces apical trafficking of Silnoon, a monocarboxylate transporter, in Drosophila melanogaster[END_REF], a kinase that is upstream of AMPK and controls its activation, as a mechanism to provide butyrate and lactate monocarboxylates during apoptosis.

Thus, membrane trafficking of MCT may be a potential mechanism to adjust KBs flux between cortex glia and MB neurons.

Thus, starvation might increase the expression of MCT in the cell membrane and regulate the trafficking of these transporters to control the flux of monocarboxylates upon demand in cortex glia and neurons.

Therefore, several regulatory mechanisms are required to allow the control of the transport of KBs from glial cells to neurons. However, it is still unclear whether these regulatory mechanisms can be activated upon demand. In the following section I will discuss if the KBs flux is sensitive to changes in neuronal activity.

Ketone bodies flux sensitivity to neuronal activity

Can the KBs flux be regulated by neuronal activity? Although we did not address this question several evidences from the literature suggest that, similarly to ANLS model [START_REF] Pellerin | Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization[END_REF]

Cortex glia morphology and function in ketone body transport

Despite the remarkably morphology, cortex glial cells are the most understudied of Drosophila CNS glial subtypes [START_REF] Melom | Mutation of a NCKX eliminates glial microdomain calcium oscillations and enhances seizure susceptibility[END_REF][START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF]de Tredern et al., in revision). Given the privileged position of cortex glial cells, interacting directly with neuronal cell bodies, its study might be relevant to understand glia-neuron communication at the level of the somas in the mammalian CNS [START_REF] Freeman | Drosophila Central Nervous System Glia[END_REF]. Nevertheless, what could be the equivalent of cortex glia in mammals? Do they also have LDs internal stores? More specifically, what is the general role of cortex glia in Drosophila and its impact in the KBs transport? And how cortex glia can regulate when and how transfer energetic nutrients to neurons?

I will now discuss the morphology of cortex glial and its equivalent in mammals. Then, I will discuss the general role of cortex glia in Drosophila and how it regulates the transfer of energetic rich nutrients to neurons upon demand.

Eventually I will discuss the origin of cortex glia fatty acids.

Cortex glial cells resemble morphologically and energetically to mammalian glial cells

A single cortex glia cell densely infiltrate from 10 up to 100 neuronal cell bodies in Drosophila [START_REF] Kremer | The glia of the adult Drosophila nervous system[END_REF] (Figure 62). In mammals, a similar cellular ensheathment has been found in protoplasmic astrocytes and satellite glial cells [START_REF] Halassa | Synaptic islands defined by the territory of a single astrocyte[END_REF]Sun and Jacobs, 2012;[START_REF] Hanani | Role of satellite glial cells in gastrointestinal pain[END_REF]. In fact, protoplasmic astrocytes are the major glial cell of CNS gray matter [START_REF] Halassa | Synaptic islands defined by the territory of a single astrocyte[END_REF]. Indeed, in the rodent cortex, one protoplasmic astrocyte domain can contact from 4 to 8 neuronal somatas (Figure 65A). On the other hand, satellite glial cell is a type of glia present in the PNS and can envelop the soma of a sensory neuron (Figure 65B). Thus, similarly to cortex glial cells, mammalian protoplasmic astrocytes and satellite glial cells can envelop neuronal somatas [START_REF] Freeman | Drosophila Central Nervous System Glia[END_REF].

bodies may be required for neurotransmission, but is unclear whether a similar mechanism might take place in the mammalian brain.

Interestingly, fly cortex glia is required for the maintenance of neuronal firing in Drosophila [START_REF] Melom | Mutation of a NCKX eliminates glial microdomain calcium oscillations and enhances seizure susceptibility[END_REF]. For instance, mutations in the cortex glia specific Na + /Ca 2+ , K + exchanger Zydeco resulted in a rapid and reversible induction of seizures and hyperexcitability (Figure 30), suggesting that cortex glia is required for neuronal excitability. On the other hand, evidence from our lab suggests that acetylcholine release from Kenyon cells somatas activate nAChR-α7 in cortex glia during LTM (de Tredern et al., in revision). These data suggest that, similarly to mammalian perisomatic glial cells [START_REF] Enes | Satellite glial cells modulate cholinergic transmission between sympathetic neurons[END_REF], a release of acetylcholine at the level of neuronal somas might activate glial cells in Drosophila.

In Drosophila, KBs have been shown to reduce the induction of seizures (Li et al., 2017) 59), respectively, and therefore it may ensure that proper metabolic substrates can be delivered to Kenyon cells somatas upon demand. Since cortex glial cells associate with the BBB they are well positioned to conduits the efficient transfer of nutrients from circulation to neuronal cell bodies [START_REF] Freeman | Drosophila Central Nervous System Glia[END_REF]. Remarkably, cortex glia, in concert with the BBB, regulates neuroblasts proliferation in fly larvae in response to nutritional control [START_REF] Chell | Nutrition-responsive glia control exit of neural stem cells from quiescence[END_REF]. For instance, when the neuroblasts division stop between embryonic development and larval stage, a fat body derived signal allows the release of insulin-like peptide 6 from the BBB and cortex glia, which in turn activates insulin receptor in neuroblasts, allowing neuroblast exit from quiescence [START_REF] Chell | Nutrition-responsive glia control exit of neural stem cells from quiescence[END_REF]. Thus, the BBB and cortex glia are sensitive to nutritional signals in larval stages. However, the impact of nutritional signals in the adult cortex glia on the transfer of energy rich metabolites to neuronal somatas has just recently begun to be studied.

Indeed, in adult flies, it has been shown that acetylcholine release can activate nAChR-α7 in cortex glia during LTM (Figure 32). nAChR-α7 activation leads to insulin-like peptide 4 release and autocrine activation of insulin receptor.

Insulin receptor activation increases the activity of trehalase, and therefore glucose synthesis, which is then shuttled to Kenyon cells somatas during LTM formation. This shows that secreted neuronal signals might control energy metabolism in cortex glia and allow the transfer of nutrients to neuronal somatas upon demand.

On the other hand, in our work here I have demonstrated that KBs transfer from cortex glia to neuronal somatas provide energy for sLT-ARM (Figure 59).

However, we did not provide evidence of released signals from Kenyon cells somatas that could regulate energy metabolism in cortex glia upon starvation. To further identify the signal derived from MB neurons to activate cortex glia and LDs mobilization, as a first approach, we can use BODIPY and perform a screening of neurotransmitters involved in lipolysis (Nelson and Cox, 2003) to observe whether glial LDs can be mobilized upon starvation. This will help us to determine whether neurotransmitters can exert a lipolytic effect in glial LDs during starvation.

It is generally believed in mammals and insects that the canonical production of KBs to fuel brain bioenergetics reside in the fat stores of peripheral tissues [START_REF] Shah | Enzymes of ketogenesis in the fat body and the thoracic muscle of the adult cockroach[END_REF][START_REF] Mcgarry | Regulation of hepatic fatty acid oxidation and ketone body production[END_REF]. Since we found a strong impairment of sLT-ARM when we knocked down genes involved in KBs synthesis in cortex glia (Figure 50), a substantial contribution of circulating LDs mobilization in cortex glia is crucial to provide KBs upon starvation.

However, what would be the origin of these fatty acid molecules? In this section I will first discuss the cellular origin of fatty acids from circulation. Then, I will discuss some evidence regarding a neuronal origin of fatty acids and whether they could be locally synthesized in cortex glial cells.

Eventually, I will discuss the advantage of having a local rather than a peripheral source of KBs.

between these cellular compartments. We can test the hypothesis that fatty acids are transferred from circulation into the cortex glia through the BBB by knocking down FATPs and lipoprotein receptors in the BBB or cortex glia during memory upon starvation. This will help us to determine if fatty acids from circulation into the cortex might be an important source of energy during memory upon starvation.

Alternatively, neuron-derived fatty acids might contribute to glial LDs stores. Evidence from mammals and flies suggests that glial LDs fatty acid stores might have a neuronal origin [START_REF] Liu | Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration[END_REF][START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF]. This raises the question whether cortex glial fatty acids may have a neuronal origin as well (Figure 66,b). To limit ROS damaging linked to neuronal activity, a similar mechanism than the one proposed by [START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF] (Figure 64) was earlier described in the adult Drosophila brain between photoreceptors neurons and glial cells [START_REF] Bailey | Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila[END_REF][START_REF] Liu | Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration[END_REF][START_REF] Liu | The Glia-Neuron Lactate Shuttle and Elevated ROS Promote Lipid Synthesis in Neurons and Lipid Droplet Accumulation in Glia via APOE/D[END_REF]. In basal condition as well as in response to high ROS levels, glial lactate can fuel lipogenesis in neurons (Figure 67). Remarkably, under high ROS conditions, fatty acids can suffer peroxidation and then peroxidated fatty acids are transported to glial cells and stored as LDs in flies [START_REF] Bailey | Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila[END_REF][START_REF] Liu | Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration[END_REF][START_REF] Liu | The Glia-Neuron Lactate Shuttle and Elevated ROS Promote Lipid Synthesis in Neurons and Lipid Droplet Accumulation in Glia via APOE/D[END_REF]. But whether LDs are consumed through β-oxidation remains unknown yet.

Authors from this study proposed that glial cells provide lactate to neurons as a fatty acid precursor, instead of providing directly fatty acids, to ensure that fatty acid synthesis could produce undamaged fatty acids in neurons and avoid peroxidation of fatty acids in previous steps in glial cells. However, this mechanism is unlikely to occur due to the low amount of carbohydrates during starvation in flies [START_REF] Yamada | Correction: Fat body glycogen serves as a metabolic safeguard for the maintenance of sugar levels in Drosophila[END_REF], and particularly because neuronal lactate is expected to be low during starved states (Figure 45). Thus, neuronal originated fatty acids are unlikely to contribute to glial LDs. might originate from circulation, rather than from a neuronal or a local origin.

In the following section I will discuss how neurons and glial cells can sense the energy status of the animal during the starved state.

The glia-to-neuron ketone body transport might rely on signals that sense the starved state

Reasonably, this transport mechanism requires that neurons and glial cells sense the food-deprived state and get adapted to these metabolic changes progressively. In our work we have found that AMPK is a crucial energy sensor in glial cells that sense the starved state and allow the activation of downstream effectors that participates in the synthesis of KBs (Figure 59) (see section 1.4).

However, despite AMPK activation seems sufficient to explain the switch into KBs synthesis, the remaining signals involved in this transition remain still poorly understood. In the following section I will discuss some of these molecular changes and how neurons and glial cells adapt to this transition. Eventually, I will discuss the effect of KBs metabolism in the aged and pathological brain and the relevance of this transport mechanism under these contexts.

3.1 Hormonal signals in cortex glia allow the mobilization of energy stores to form long-lasting memories upon starvation

We have previously introduced that in the fed state insulin hormonal signaling within cortex glia is crucial for glucose synthesis and delivery to neurons to allow LTM formation (Figure 32). In our work we did not investigate a hormonal signal that might be required to allow LDs mobilization and therefore sLT-ARM formation. However, in the transition from the fed to the starved state, insulin the fed to the starved state in the fat body [START_REF] Choi | Feeding and Fasting Signals Converge on the LKB1-SIK3 Pathway to Regulate Lipid Metabolism in Drosophila[END_REF]. For instance, LKB1 expression and LDs mobilization. We can test this hypothesis by measuring LDs changes during the first hours of food deprivation with BODIPY dyes and FOXO RNAi expressed in cortex glia. This will help us to determine the early steps on cortex glia activation and the molecular pathways involved on this process. Thus, cortex glia might sense the change to starvation first, by lowering insulin signaling, and then, activating FOXO and Brummer lipase to trigger LDs mobilization.

We have discussed how different hormonal signals can sense the transition from fed to the starved state in neurons and glia. During aging and certain pathological conditions, the brain becomes inefficient to utilize glucose in mammals [START_REF] Kapogiannis | Brain glucose and ketone utilization in brain aging and neurodegenerative diseases[END_REF] and should necessarily rely on different metabolic fuels, among them, KBs. Whether KBs might have a physiological relevance under these conditions in the brain remains unknown yet. I will now discuss the importance of KBs metabolism in the aged and neuropathological brain.

3.4 A ketone body transport mechanism might be relevant in the aged and neuropathological brain A hallmark of the vertebrate aging brain is a reduction in glucose utilization [START_REF] Kapogiannis | Brain glucose and ketone utilization in brain aging and neurodegenerative diseases[END_REF] and cognitive performance [START_REF] Murman | The Impact of Age on Cognition[END_REF].

Interestingly, the administration of a ketogenic diet, which increases circulating KBs, can reduce mortality and improve memory performance in aged mice [START_REF] Newman | Ketogenic Diet Reduces Midlife Mortality and Improves Memory in Aging Mice[END_REF]. This supports the notion that during aging, KBs might exert beneficial cognitive effects in the brain. Here we provided evidence that upon starvation KBs are synthesized in glial cells and transported to neurons during memory formation (Figure 59). But, could this transport mechanism be also relevant in the aged brain? We have discussed previously that a KBs transport mechanism might take place in the mammalian brain in vitro (Blasquez et al., 1998). However, whether this mechanism might be required during aging remains unknown yet. But, what about under pathological conditions?

Numerous CNS pathologies have a metabolic component affected. For instance, a reduction of the cerebral metabolic rate for glucose is observed in AD [START_REF] Mosconi | Multicenter standardized 18F-FDG PET diagnosis of mild cognitive impairment, Alzheimer's disease, and other dementias[END_REF], which may explain the cognitive deficits observed during this condition. However, could KBs metabolism exert a positive effect in cognition, as well as in aging, during AD? Interestingly, intermittent periods of food deprivation or a ketogenic diet led to an amelioration of cognitive deficits and a reduction of Aβ plaques observed in AD mice models [START_REF] Wang | Caloric restriction attenuates beta-amyloid neuropathology in a mouse model of Alzheimer's disease[END_REF][START_REF] Patel | Caloric restriction attenuates Abeta-deposition in Alzheimer transgenic models[END_REF][START_REF] Halagappa | Intermittent fasting and caloric restriction ameliorate agerelated behavioral deficits in the triple-transgenic mouse model of Alzheimer's disease[END_REF][START_REF] Rusek | Ketogenic Diet in Alzheimer's Disease[END_REF], suggesting that KBs can ameliorate certain aspects of AD. Thus, food deprivation and the synthesis of KBs might exert beneficial effects on cognition in the aged and pathological brain.

General conclusions and perspectives

The results I obtained during my thesis support the view that specific metabolic pathways are required in memory-relevant brain structures during longlasting memory formation in Drosophila, as it has been demonstrated in vertebrates models [START_REF] Suzuki | Astrocyte-neuron lactate transport is required for long-term memory formation[END_REF]. The utilization of KBs by the brain upon periods of starvation was early stated in the 60s [START_REF] Owen | Brain metabolism during fasting[END_REF] and almost two decades ago a KBs shuttle has been hypothesized (Guzman and Blasquez, 2001) based on in vitro data [START_REF] Edmond | Capacity for substrate utilization in oxidative metabolism by neurons, astrocytes, and oligodendrocytes from developing brain in primary culture[END_REF][START_REF] Auestad | Fatty acid oxidation and ketogenesis by astrocytes in primary culture[END_REF]Blasquez et al., 1998). However, these in vitro data did not proof a biological significance of this KBs transport mechanism. In our work, we propose that cortex glial cells transport KBs to Kenyon cells somas to support long-lasting memory upon starvation. We describe a molecular pathway that links KBs synthesis in glial cells and its oxidation in MB neurons to support memory formation upon starvation (Figure 59). The molecular pathway supporting this model is that starvation leads to the activation of Brummer lipase that triggers LDs mobilization (Figure 55) and unknown. This energy might be used not only for energy generation but also for neurotransmitter synthesis at the synapse (i.e acetylcholine out of acetyl-CoA). In addition, KBs might protect against stress and neuronal death by upregulating the antioxidant system in somas, axons or in the synapse [START_REF] Haces | Antioxidant capacity contributes to protection of ketone bodies against oxidative damage induced during hypoglycemic conditions[END_REF]. It will be interesting to clarify if the effects of KBs are globally across all the neuronal compartments or whether there is a specificity of their effects.

We have shown that a glia-to-neuron ketone body transport is required to support olfactory memory formation upon starvation (Figure 59). However, as I discussed previously, it is likely that this transport is also required to support other complex behaviors upon starvation such as foraging, sleep, aggression, and memory involving other sensorial modalities. It is of particular relevance as it is becoming increasingly evident that food deprivation periods such as caloric restriction or intermittent fasting have profound consequences in promoting neuroplasticity, resistance of the brain to injury and neurodegenerative diseases [START_REF] Mattson | Intermittent metabolic switching, neuroplasticity and brain health[END_REF]. Thus, a glia-to-neuron ketone body transport mechanism in the starved state may have profound health benefits for the brain.

Introduction

Le jeûne représente un défi métabolique majeur pour le cerveau et les processus qui assurent son bon fonctionnement sont considérés comme cruciaux pour la survie de l'organisme [START_REF] Mattson | Impact of intermittent fasting on health and disease processes[END_REF] [START_REF] Plaçais | To favor survival under food shortage, the brain disables costly memory[END_REF].

Récemment, nous avons démontré qu'après la formation de la MLT les mouches doublent leur consommation de sucre et augmentent leur flux de pyruvate mitochondrial dans le centre de la mémoire olfactive, les Corps pédonculés (CP) [START_REF] Plaçais | Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory[END_REF]. Néanmoins, en l'absence de glucose, comment le cerveau régule-t-il la formation de la mémoire pour résister aux périodes de jeûne? De plus, quels sont les substrats métaboliques utilisés par le cerveau pour soutenir la formation d'une mémoire persistante en cas de jeûne? Et finalement, quels sont les types de cellules cérébrales impliqués dans ce processus?

Les corps cétoniques (CC) sont des métabolites riches en énergie, dérivés du métabolisme des acides gras et utilisés par le cerveau dans des conditions de stress métabolique tel que le jeûne [START_REF] Owen | Brain metabolism during fasting[END_REF]. Cependant, on ignore encore si les neurones sont capables d'utiliser directement les CCs pour maintenir leur activité physiologique et en particulier la formation de la mémoire.

Dans ma thèse, j'ai abordé le rôle in vivo du métabolisme des CCs dans la formation de la mémoire olfactive aversive lors du jeûne (j-MRA), les voies

3. 1

 1 Adult cortex glia contains cytoplasmic LDs 3.2 In cortex glia, LDs are the main source of fatty acids precursors for ketone bodies synthesis upon starvation 3.3 Influence of MB neuronal ketone bodies oxidation and uptake on cortex glial LDs 4. The activity of AMPK energy sensor in cortex glia is required for sLT-ARM formation………………………………………………………………….. Discussion…………………………………………………………………………….. 1. The proposed glia-to-neuron ketone body transport……………………. 1.1 ANLS model versus a glia-to-neuron ketone body transport model 1.2 Are ketone bodies synthesized as a baseline fueling mechanism or upon demand? 1.3 Cellular localization of the glia-to-neuron ketone body transport : main components 1.3.1 Cellular localization of the ketone body transport within cortex glial cells 1.3.2 Localization of the ketone body transport within MB neurons 1.4 Regulation of ketone body transport 1.4.1 Sensitivity to energy level via the master energy sensor: AMPK 1.4.2 Regulation of LD mobilization in cortex glia by lipase regulation 1.4.3 Regulation of ketone bodies flux by regulation of MCT 1.4.4 Ketone bodies flux sensitivity to neuronal activity 2. Cortex glia morphology and function in ketone bodies shuttling………...162 2.1 Cortex glial cells resemble morphologically and energetically to mammalian glial cells 162 2.2 General role of cortex glia in Drosophila 163 2.3 Cortex glia can regulate the transfer of nutrients to neuronal somatas which may determine LTM and sLT-ARM formation 164 2.4 Cortex glia fatty acids may originate from circulation, neurons or locally synthesized 166 3. The glia-to-neuron ketone body transport might rely on signals that sense the starved state……………………………………………………………... 171 3.1 Hormonal signals in cortex glia allow the mobilization of energy stores to form longketone bodies shuttling mechanism might be relevant in the aged and neuropathological

Figure 20 .

 20 Figure20. Memory retention curves after different conditioning protocols. After one cycle of conditioning (x1) flies form a memory that decay rapidly within the first day. 5 cycles of massed conditioning (without rest intervals between each cycle) or 5 cycles of spaced training (with resting intervals between each cycle) leads to the formation of consolidated memories that last for more than one day. Figure modified from[START_REF] Tully | Genetic dissection of consolidated memory in Drosophila[END_REF]

Figure 23 .

 23 Figure 23. Location of dopaminergic neurons innervating the olfactory memory center. PPL2a contact the calyx of the MB, whereas PAM and PPL1 innervate the MB at the level of the lobes. Figure obtained from Aso et al., 2014a.

2. 4

 4 Coincidence detection of CS and US form memories in the mushroom body In order to identify the molecular mechanisms underlying memory formation, several behavioral screenings were initially carried out to identify new mutations affecting memory formation. The first identified genes involved in memory are mutants involved in the cyclic AMP signaling cascade (cAMP): dunce and rutabaga. Dunce gene encodes a cAMP phosphodiesterase, whereas

Figure 24 .

 24 Figure24. Molecular mechanism driving memory formation in KCs upon arrival of the CS and US. Cholinergic inputs from PNs drives odor information (CS) into the MB, which leads to an increase in intracellular Ca 2+ levels. The valence of the US is encoded by dopaminergic neurons. When both CS and US simultaneously coincide onto KCs, activation of adenylate cyclase (Rut-AC; adenylate cyclase, rutabaga) leads to an increase in cAMP. By increasing the amount of cAMP, protein kinase A (PKA) is activated. Ca 2+ /CAM, calcium/calmodulin; Rut-AC calcium/calmodulin dependent adenylyl cyclase. Illustration from[START_REF] Heisenberg | Mushroom body memoir: from maps to models[END_REF]

Figure 26 .

 26 Figure 26. Triad and location mushroom body output neurons innervating the olfactory memory center. A, Triad between dopaminergic neurons (DANs), Kenyon cells (KC) and MBONs are shown: MP1 DAN and MBON MVP2 innervating the y1 subcompartment of the MB. B, MBONs innervate specific subcompartments of the MB. Two MBONs are shown: MB-V2α and MB-MVP2, which surrounds vertical lobes and the peduncle, respectively. Figure A and B obtained from Hige et al., 2015 and Aso et al., 2014 respectively.

Figure 31 .

 31 Figure 31. Cortex glial cells contain LDs in different insect models.A, cockroach surface and cortex glia are drawn in the picture. In addition, neuronal somata of ganglion cells, glycogen granules and LDs are present in the cortex region. B, Drosophila perineural glia (PG) and superineural glia (SPG) contain some LDs (arrows). Cortex glia (CG) LDs are highly abundant in the cytosolic compartment surrounding neurons (N). Figure A was obtained from[START_REF] Wigglesworth | The Nutrition of the Central Nervous System in the Cockroach <em>Periplaneta Americana&lt[END_REF]. Figure B was obtained from Kis et al., 2015.

  Figure 31. Cortex glial cells contain LDs in different insect models.A, cockroach surface and cortex glia are drawn in the picture. In addition, neuronal somata of ganglion cells, glycogen granules and LDs are present in the cortex region. B, Drosophila perineural glia (PG) and superineural glia (SPG) contain some LDs (arrows). Cortex glia (CG) LDs are highly abundant in the cytosolic compartment surrounding neurons (N). Figure A was obtained from[START_REF] Wigglesworth | The Nutrition of the Central Nervous System in the Cockroach <em>Periplaneta Americana&lt[END_REF]. Figure B was obtained from Kis et al., 2015.

Figure 36 .

 36 Figure36. Structure of CRIMICs. A, The structure of the CRIMIC cassette resembles to that of Trojan Gal4. It consists of a T2A-Gal4 followed by a polyA signal followed by a 3xP3 promoter and GFP reporter tag, to verify the insertion of the construct. The T2A is a viral ribosomal skipping site that arrests translation, which becomes reinitiated after the site, producing untagged Gal4 protein. B, Scheme of the CRIMIC integration by CRISPR/Cas9 in a coding intron. It consists of the CRIMIC vector and two homology arms by homology directed repair (HDR). The integration of the CRIMIC vector allows the expression of a Gal4 in the intronic region of a gene and to assess its expression they can be crossed to transgenic flies expressing membrane red fluorescent protein (UAS-mCD8::RFP). Figure from[START_REF] Lee | A gene-specific T2A-GAL4 library for Drosophila[END_REF]

2. 1

 1 Aversive associative conditioningIn the fed condition, GAL4 activity was released by transferring adult flies to a 30ºC incubator for 2 days (induced flies). The non-induced control flies, in which RNAi expression is inhibited, were kept at 18ºC. After induction of a specific RNAi, the flies were kept at either 25ºC (during the training period and memory test) or 18ºC (24 h storage period) for LTM and LT-ARM experiments. In the starved condition, flies were food-deprived for either 16 h at 30ºC (for 2-days induced flies) or 21 h at 25ºC (for non-induced control flies) before conditioning in bottles containing filter paper soaked with water. Flies were kept in starvation bottles at either 25ºC (during the training period and memory test) or 18ºC (24 h storage period) during sLT-ARM experiments.

  A1). The pixel intensity histogram of the grayscale ROI was exported from ImageJ to Prism software (Figure38 A2). For each image, a Gaussian fit is performed on the intensity histogram (Intensity values of x<3 were excluded from the fitting of the Gaussian curve) from which the mean and SD parameter are extracted (Fig 38A2) and then used to calculate the threshold value which was set to: [mean intensity + 4*(SD/255)] (Figure38 A2). Then, the image is open in CellProfiler and we defined LDs diameter from 0.37-1.5 μm 2 , according to previous LDs data published in the literature(Cabirol-Pol et al., 2017;[START_REF] Van Den | Physiological and pathological roles of FATP-mediated lipid droplets in Drosophila and mice retina[END_REF][START_REF] Velentzas | The Proton-Coupled Monocarboxylate Transporter Hermes Is Necessary for Autophagy during Cell Death[END_REF], and discarding the objects outside this diameter range. The threshold previously calculated above was manually added to CellProfiler Software analyses. As depicted in figure38 B2, if

  photon imaging was performed on a Leica TCS-SP5 upright microscope, equipped with a ×25, 0.95 NA water-immersion objective. Two-photon excitation of mTFP was achieved using a Mai Tai DeepSee laser tuned to 825 nm. 512 × 256

  , demonstrating that carbohydrates are key substrates during LTM formation. Therefore, what would be the energetic substrate used by MB neurons for sLT-ARM formation? Interestingly, after ≈14 hours of food deprivation, carbohydrates stores are depleted(Haller and Bines, 2005;[START_REF] Mattson | Intermittent metabolic switching, neuroplasticity and brain health[END_REF] and the use of fatty acid and KBs turn into the main energetic fuel for the brain of vertebrates[START_REF] Owen | Brain metabolism during fasting[END_REF]. Thus, I first reasoned that under periods of food shortage, flies may switch into KBs oxidation to fuel brain energetic demands during memory formation upon starvation. Given that KBs are key substrates used by the brain during starvation periods, I studied whether KBs were the main metabolic substrate in neurons during memory formation upon starvation, and how KBs enter MB neurons. Then, I investigated the cellular origin of KBs and asked if they were locally synthetized in the brain. Eventually, I looked for a potential regulation of the local synthesis of KBs in the brain.

  A6,7) were normal in induced starved flies. To further confirm the specific role of ACAT1 in sLT-ARM, I used a second nonoverlapping RNAi construct for ACAT1. As we observed for the first RNAi (Figure 39A), flies expressing ACAT1 RNAi only in the adult MB presented a strong sLT-ARM impairment (Figure 39 B1), whereas in fed condition consolidated memories both LT-ARM (Figure 39 B2) and LTM (Figure 39 B3) were normal. In absence of induction sLT-ARM formation remained normal (Figure 39 B4). Neither the shock (Figure 39 B5) nor the olfactory acuity were affected by this second ACAT1 RNAi expression in the adult MB of starved flies (Figure 39 B6,7). Therefore, our data

Figure 41 .

 41 Figure 41. Silnoon expression in the fly brain. A, transcriptomic analyses in MB . Silnoon and CG8034 are expressed in MB neurons. Expression levels goes from red (strong) to black (weak). B, Immunohistochemistry of the whole Drosophila brain of SLN CR00519-T2A-Gal4 >UAS-mCD8::RFP flies. Silnoon is expressed broadly in the brain, including the MB neurons. A single focal plane of the anterior part of the brain with the MB lobes is shown in the picture (dashed lines). Silnoon is also expressed in antennal lobes (AL). Scale bar: 40 μm. C, Anterior part of the brain showing the expression of Silnoon in MB calyx (Ca). Scale bar: 40 μm. D, Magnification of figure B (square), showing the expression of Silnoon in MB neurons (in the calyx region). Scale bar: 20 μm. nc82: bruchpilot, a neuropile marker and RFP (red fluorescent protein) were used.

Figure 42 .

 42 Figure42. Multiple sequence alignment: Silnoon and CG8034 have homology with human MCT2 transporter. Amino acid sequence comparison of Silnoon and CG8034 with MCT2 from human and Slc16a6a (Slc16) of Zebrafish. A black line in top of the alignments are pointing TM domains of MCT transporters. The color code is depicted in the top, from red denoting the conserved amino acids to blue representing the nonconserved amino acids. In the proximity of the TM1 and TM8 region are highlighted key residues for the binding of protons (red rectangle). Close to TM1 charged residues of Lysine (K) are necessary for proton binding. Moreover, in the TM8 region two residues Aspartate (D) and Arginine (R) are important for the binding of a proton. Multiple sequence alignment was carried out with PRALINE (Bawono and Heringa, 2014); for further details see material and section method.To test if Silnoon or CG8034 expression in MB neurons were involved in KBs transport upon starvation and thus required during sLT-ARM, I have downregulated the expression of each MCT in the adult MB using a specific RNAi. Downregulation of CG8034 in MB neurons restricted to the adulthood, led to a normal sLT-ARM (Figure43 A1). In addition, LT-ARM remained normal in fed conditions (Figure43 A2). To confirm this negative result, I have used a second non-overlapping RNAi to downregulate CG8034 expression in MB. As for the first RNAi (Figure43A), expression of CG8034 RNAi in adult MB neurons has no effect on sLT-ARM (Figure43 B1) as well as for LT-ARM in fed flies (Figure43 B2).Thus, these data indicate that CG8034 is not required for sLT-ARM and consequently not involved in KBs transport to sustain memory upon starvation.

  and a local provider. An alternative hypothesis is that glial cells would synthesize KBs from their own lipid energy stores upon starvation to provide energy to neurons: either cortex glia and/or other glial subtypes could be the site of this local synthesis of KBs. It has been shown that vertebrate glial cells can synthesize KBs out of fatty acids as a molecule precursor(Blasquez et al., 1998).Thus, in a first attempt, I wanted to test the hypothesis of a local synthesis of KBs in cortex glia and/or another glial cell type, rather than an import from circulation to MB neurons, during memory formation upon starvation. To test this hypothesis, I focused on the two key steps of KBs synthesis(Figure 13,14). First, in the import of fatty acids into mitochondria, via carnitine palmitoyltransferase 1 (CPT1), which is the rate-limiting step for β-oxidation and acetyl-CoA production (Figure12). And secondly, in the mitochondrial synthesis of KBs out of acetyl-CoA precursor, via the rate-limiting step enzyme of ketogenesis HMG-CoA synthase (HMGS) (Figure14). To investigate if cortex glia is a local provider of KBs to neurons, I have tested memory formation upon starvation in flies in which the flux of KBs precursors, namely fatty acids, into the mitochondria is impaired.CPT1 is the entry point of fatty acid into the mitochondria and then fatty acids are oxidized up to acetyl-CoA molecules via β-oxidation[START_REF] Mcgarry | Regulation of hepatic fatty acid oxidation and ketone body production[END_REF], that will then be used for KBs synthesis in mammals (Figure14). Downregulation of CPT1 expression in cortex glia induced a strong sLT-ARM impairment (Figure50 A1), while LT-ARM memory scores were normal in fed flies (Figure50 A2). Knocking down CPT1 in the adult cortex glia did not affect LTM in fed flies (Figure50 A3). Additionally, in absence of CPT1 RNAi induction sLT-ARM scores are normal (Figure50 A4). Moreover, shock (Figure50 A5) and olfactory controls (Figure50 A6-7) were normal in starved flies. Thus, it suggests that KBs precursors import into mitochondria of cortex glial cells is critical for memory formation upon starvation.Fatty acids entry into mitochondria is followed by their breakdown via βoxidation, which generates acetyl-CoA, the main precursor of KBs. In order to determine the role of ketogenesis during memory, I then assessed the role of HMGS during sLT-ARM. Downregulation of HMGS expression in the adult cortex glia induced a strong sLT-ARM defect (Figure50 B1), whereas LT-ARM was unaffected in fed flies (Figure50 B2). Moreover, reducing HMGS expression in the adult cortex glia did not impair LTM in fed flies (Figure50 B3). sLT-ARM is normal in absence of RNAi induction (Figure50 B4). Shock acuity (Figure50B5) and olfactory controls were normal in starved flies (Figure 50 B6,7). To further confirm our results with HMGS, I used a second non-overlapping RNAi against HMGS. As for the first RNAi against HMGS, expression of a second non-overlapping RNAi against HMGS in the adult cortex glia resulted in a strong sLT-ARM defect (Figure 50 C1), which was not observed in LT-ARM in fed flies (Figure 50 C2). Additionally, LTM was normal in induced fed flies expressing RNAi HMGS in adult cortex glia (Figure 50 C3). Moreover, in absence of thermal induction, flies exhibited normal sLT-ARM formation (Figure 50 C4). Shock (Figure 50 C5) and olfactory acuity (Figure 50 C6,7) were unaffected by expression of HMGS RNAi in adult cortex glia. Therefore, together with the results on CPT1, I have shown that KBs synthesis in cortex glial cells is required to sustain sLT-ARM formation.

  ),showing that high intensity object stained by BODIPY are LDs coated with the LSD-2 protein. However, there are several proteins coat LD in Drosophila, and because LSD-2 is only one of them, I observe here only a partial overlap between LSD-2::GFP and BODIPY Red C12 signals.Eventually, we carried out LDs area analyses with CellProfiler Software Analyst[START_REF] Jones | CellProfiler Analyst: data exploration and analysis software for complex image-based screens[END_REF] (see material and methods for details), with the aim of determine the role of β-oxidation and ketogenesis in LDs dynamics. After background subtraction LDs are positively identified (in green) (Figure53C, middle panel). Objects outside of the size limits are shown in magenta. Each individual LD for which the area is calculated can be visualized in artificial colors (Figure53C, right panel). Thus, we can identify LDs in the cortex glia. We measured the area of the LDs stained with BODIPY 493/503 and our results show that the average area for LDs in cortex glia is ≈0.15-0.25 μm 2 , which is in agreement with the area of published LDs data[START_REF] Cabirol-Pol | Glial lipid droplets and neurodegeneration in a Drosophila model of complex I deficiency[END_REF][START_REF] Zheng | A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster[END_REF]. Altogether, these results show that Drosophila adult cortex glia contains LDs that can be identified with BODIPY staining. Nevertheless, whether this fatty acid reservoir can be mobilized upon starvation to sustain memory formation is an open question that I will address in the next section.

  C,D), the export of KBs is downstream of LDs mobilization in cortex glia upon starvation. Downregulation of Chaski in cortex glia led to a strong increase in LDs area of starved flies compared to control group flies (Figure 54 E,F1), whereas in fed condition LDs area remained unchanged (Figure 54 F2). These results show that the export of KBs through Chaski is downstream of LD mobilization upon starvation.reservoirs in cortex glial cells. Oppositely, in fed flies LDs size remained unchanged, suggesting that they are not used as energy reservoir for KBs synthesis. Although we cannot discard that under fed conditions LDs might be mobilized for other purposes. Here I have shown that the same genes required during memory formation upon starvation (i.e CPT1, HMGS and Chaski) (Figure48,50) were also downstream of LDs mobilization (Figure54), suggesting that there is a strong correlation between the LDs mobilization and memory formation upon starvation. Altogether these results show that upon starvation cortex glial LDs are mobilized to act as a local energy reservoir to fuel mitochondrial fatty acid uptake for KBs synthesis and export.

  neurons and cortex glia LDs can be evidenced. Thus, I have downregulated either Silnoon or ACAT1 expression in adult MB neurons while measuring LDs mobilization in cortex glia. Downregulating the MB neuronal uptake of KBs, through Silnoon, did not cause significant changes in glial LDs area in either starved or fed flies (Figure56 A1,A2). These results suggest that MB neurons uptake of KBs did not cause changes in LDs mobilization in cortex glia upon starvation. Knockdown of ACAT1 in MB neurons did not affect LDs area either in starved (Figure56 B1) or in fed conditions (Figure56 B2), showing that MB neuronal degradation of KBs did not mobilize LDs of cortex glia upon starvation.

  Alternatively, another pathway might be involved in LDs mobilization: LDs mobilization in fat tissues relies on hormonal signaling via Akh receptor (Heier and Kuhnlein, 2018), which activates the cAMP/PKA pathway. Activation of the cAMP/PKA pathway leads to phosphorylation of the LDs scaffold membrane protein Perilipin-1, which allows the translocation of dHSL to the inner face of LDs

  Thus, upon starvation Brummer lipase might enhance LDs mobilization to provide fatty acids to mitochondria and favor KBs synthesis through a mechanism dependent on AMPK activation(Figure 50,(55)(56)(57). Moreover, it remains to be investigated whether Brummer might be required during memory upon starvation. Once KBs are synthesized in glial cells how the flux can be regulated at the level of MCT transporters to allow an efficient delivery to neurons?1.4.3 Regulation of ketone bodies flux by regulation of MCTWe have discussed that KBs synthesis might be regulated by the master energy sensor AMPK (Figure57,58), likely by controlling ACC/CPT1 axis, and also by a regulation of the Brummer lipase in cortex glia. But, could this regulation be extended, for instance, at the level the MCT transporters as well?

2. 4 .

 4 Cortex glia fatty acids may derive from circulation, neurons or locally synthesized Our data suggest that upon starvation, cortex glial LDs fatty acids are used as metabolic precursors of KBs (Figure54,55), which in turn provide energy to neurons during memory formation (Figure50). Yet an open question is what supplies cortex glia LDs internal stores with fatty acids so they can be continuously delivered into mitochondria for β-oxidation in starved conditions? Are they transferred from circulation to cortex glial cells through the BBB (i.e surface glia)? (Figure66,a) Could it be possible that these molecules may have an alternative source of origin such as from neurons? (Figure66,b) or are these locally synthesized in cortex glial cells? (Figure66,c).

  mitochondrial fatty acids utilization in cortex glia, which are taken up into mitochondria by CPT1 for β-oxidation(Figure 50,54). Then, β-oxidation generates molecules of acetyl-CoA, which are used for KBs synthesis through HMGS enzyme(Figure 50,54). Then, a transfer of KBs from cortex glia to Kenyon cells somatas, through specific MCT transporters, allow their oxidation by ACAT1 enzyme, into acetyl-CoA molecules for energy generation(Figure 39,44,48). This transport is regulated by the energy sensing system of cortex glia, AMPK cell sensor (Figure58), which likely regulates downstream effectors, including CPT1 and Brummer lipase(Figure 57,63). However, two main questions are still pending: if and how this transport is regulated by neuronal activity, to respond neuronal specific demands depending on their activity and how the brain switches from a metabolism based on sugar in the fed stated to a metabolism based on fatty acid breakdown and KBs utilization upon starvation. We have shown that the dependence on KBs metabolism in Kenyon cells is specific for sLT-ARM, but is not present in fed condition for either LT-ARM or LTM (Figure39). Previous evidence in our lab has shown that glucose consumption in the Kenyon cells somata is necessary after LTM formation (de Tredern et al., in revision) (Figure32). Oppositely, it still remains unknown what is the metabolic pathways underlying LT-ARM formation in fed conditions, a future interesting research area that might uncover novel or alternative substrates sustaining memory formation.We have shown that KBs oxidation in Kenyon cells is used for the generation of acetyl-CoA (Figure40), which can enter the Krebs cycle and provide energy to neurons. However, are KBs used mainly at the level of the soma? Or alternatively are transported in other neuronal compartment, such as the calyx or the lobes of MB, to provide energy locally at the sites of synaptic activity? If KBs may be also used in axons or synapses for energy generation remains still

  métaboliques nécessaires et les types cellulaires impliqués dans ce processus en combinant la génétique, les tests comportementaux, des marquages fluorescents des lipides et de l'imagerie in vivo à 2-photons à l'aide de l'organisme modèle Drosophila melanogaster.RésultatsLa première question est de déterminer si, en condition de jeûne, les neurones des CPs utilisent directement les CCs comme substrat énergétique lors de la formation de la mémoire. Pour tester cette hypothèse, en utilisant un promoteur spécifique des CPs inductible à l'âge adulte, nous avons diminué l'expression de l'acétyl-CoA acétyl transférase 1 (ACAT1), l'enzyme de l'étape limitante de l'oxydation mitochondriale des CCs, et testé la j-MRA dans ces mouches. Cette diminution de l'expression d'ACAT1 dans les CPs de mouches adultes, entraine un défaut j-MRA, alors qu'en condition nourrie, les deux mémoires type de mémoire persistantes MLT et MRA sont normales. Ces résultats montrent que la formation de j-MRA est dépendante de l'oxydation mitochondriale des CCs par les neurones de CP. Les CC ne peuvent pas diffuser librement à travers la membrane plasmique, nous avons donc cherché à identifier le transporteur de monocarboxylates (TMC) neuronal permettant l'import des CCs dans les CPS. En combinant analyse de séquence protéique, immunohistochimie et la réalisation d'un crible comportemental des différents TMC, nous avons pu identifier Silnoon comme transporteur probables des CCs dans les neurones de CPs. En effet, la diminution de l'expression de Silnoon dans les CPs adulte entraine un défaut spécifique j-MRA sans affecter les mémoires persistantes en condition nourrie. Il n'y a actuellement aucun outil disponible pour mesurer in vivo les flux de CCs. Ainsi, pour confirmer que Silnoon est responsable de l'import des CCs dans les neurones des CPs lors du jeûne, j'ai utilisé une propriété des TMC, la trans-accélération, qui leur permet de coupler et accélèrer le transport d'un monocarboxylate (ici le lactate) en échange d'un autre monocarboxylate (ici les CCs) suivant leur gradient de concentration. J'ai donc mesuré in vivo le flux de lactate dans les CPs avec la sonde FRET laconique (San Martin et al., 2013) lors de l'application de CCs sur le cerveau de la mouche. L'application de CCs entraine une diminution de la quantité de lactate dans les CPs ce qui suggèrent un export de celui-ci lorsque la concentration en CCs est artificiellement augmentée dans le milieu extracellulaire. Lorsque l'expression de la sonde laconique est couplée à l'expression d'un ARN interférant (ARNi) contre Silnoon dans les neurones MB adultes, l'application de CCs ne modifie pas la quantité de lactate intracellulaire suggérant qu'il n'y a pas de trans-accélération dans ce contexte. Ces résultats, combinés aux données de comportement et d'analyse de séquence suggèrent que Silnoon est responsable de l'import des CCs, substrat énergétique pour la formation de la j-MRA, dans les neurones des CPs en condition de jeûne.Dans la seconde partie de ma thèse, je me suis intéressé à l'origine de ces CCs. Deux hypothèses sont envisageables : une origine périphérique avec un import des CCs par le cerveau depuis circulation générale ou une origine locale avec une synthèse à partir de réserves lipidiques présente dans la glie. Chez la drosophile, un type spécifique de glie, la glie corticale cerne les corps cellulaires des neurones des CP et sont riches en réserves lipidiques (sous forme de gouttelette lipidique notée GL)[START_REF] Zheng | A Complete Electron Microscopy Volume of the Brain of Adult Drosophila melanogaster[END_REF]. Nous avons émis l'hypothèse que ce type de cellule gliale pouvait localement fournir aux neurones les CCs en période de jeune. Pour tester cette hypothèse, nous avons effectué un crible comportemental de j-MRA en testant différents TMC dans la glie corticale chez l'adulte. Ceci m'a permis d'identifier Chaski comme TMC indispensable dans les cellules de la glie corticale pour la formation de j-MRA dans les neurones des CPs. J'ai ensuite testé si, dans la glie corticale chez l'adulte, une diminution de l'expression de enzymes limitantes de la voie cétogénique affectait la formation de j-MRA. Ainsi la diminution de l'expression de CPT1, transporteur-clé permettant l'import des acides gras dans les mitochondries, dans la glie corticale chez l'adulte entraine un défaut de j-MRA sans affecter en condition nourrie la MLT et la MRA. Le même phénotype est observé lorsqu'on diminue l'expression de la HMG-CoA-synthétase (HMG-S), une enzyme mitochondriale clé situé à la fin de la voie cétogénique. Afin de déterminer si ces CCs sont synthétisés à partir des réservoirs locaux d'acide gras contenus dans la glie corticale, les GL, j'ai utilisé une sonde fluorescente non polaire (BODIPY) pour marquer les lipides neutres présents dans ces GL(Cabirol-Pol et al., 2017). Lorsqu'on inhibe génétiquement à l'aide d'ARNi exprimé spécifiquement dans la glie corticale adulte la voie de synthèse des CCs (CPT1, HMG-S) ou leur export (Chaski), on observe une augmentation de la taille des GLs en condition de jeûne et non en condition nourrie. Ces résultats montrent qu'en condition nourrie, les GLs ne sont pas utilisés pour la synthèse de CCs et que lors du jeûne il y a une activation de la lipolyse des GLs pour fournir des précurseurs des CCs sous forme d'acide gras. Des résultats préliminaires avec la sonde BODIPY suggèrent que Brummer pourrait être la lipase impliquée dans la mobilisation des acides gras contenus dans les GLs.Les résultats des expériences de BODIPY montrent que les acides gras des GLs ne sont pas mobilisés pour la synthèse des CCs lorsque la mouche est nourrie. Nous avons donc cherché à identifier quelle voie de signalisation cellulaire pouvait être impliquée dans l'activation de cette voie lors du jeûne. De part son rôle de senseur majeur de l'état énergétique cellulaire, nous avons voulu tester l'implication d'AMPK dans la mise en place de cette voie de synthèse des CCs. La diminution de l'expression d'AMPK dans la glie corticale chez l'adulte entraine une diminution de la jMRA sans affecter les mémoires MLT et MRA en condition nourrie. Il a de plus été montré qu'AMPK régule positivement l'activité de CPT1(Blasquez et al., 1999). Ainsi l'activation d'AMPK en période de jeûne pourrait être le signal d'activation de la mobilisation des réserves lipidiques locales dans la glie corticale afin de synthétiser les CCs nécessaires à la formation de la mémoire jMRA.ConclusionSur la base de ces données, nous pouvons proposer un modèle d'interactions glie corticale-neurones des CP spécifique aux conditions de jeûne dans lequel les CC synthétisés par la glie à partir de leur propre stock de lipides sont exportés vers les neurones pour soutenir la formation d'une mémoire persistante. Nos données suggèrent qu'au moins dans les cellules gliales, l'AMPK pourrait être l'une des clés pour activer ce modèle en cas de jeûne. Le travail présenté ici est particulièrement pertinent dans le cadre d'études récentes montrant que les périodes de privation alimentaire telles que la restriction calorique ou le jeûne intermittent pourraient avoir des effets bénéfiques sur la cognition dans les déficiences cognitives liées à l'âge.The physiological role of the amyloid precursor protein-like during appetitive long-term memory formationDuring my pre-PhD research I have worked on a side project related to the role of the amyloid precursor protein-like and its protein interactants during appetitive long-term memory formation. This study leads to the publication of a scientific article in Journal of Neurogenetics, which is titled as: "Interactions between amyloid precursor protein-like (APPL) and MAGUK scaffolding proteins contribute to appetitive long-term memory in Drosophila melanogaster". The published article is attached below.

Table 1 . Effects of insulin and glucagon on key enzymes controlling ketogenesis (Laffel, 1999). Hormone

 1 -sensitive lipase, ACC and HMG-CoA synthase enzymes are shown. The location, action and their functional role are explained for each of them. The effect that insulin and glucagon exert on each of them is depicted in the table. Tableadaptedfrom[START_REF] Laffel | Ketone bodies: a review of physiology, pathophysiology and application of monitoring to diabetes[END_REF]

	Enzyme	Location Action	Result	Effect of insulin (Ketogenesisê )	Effect of glucagon (ketogenesisé )
			breaks
	Hormone-	peripheral	down	elevated
	sensitive	adipocyte	triglyceride	serum fatty	inhibited	stimulated
	lipase	s	s into fatty	acids
			acids
	Acetyl-CoA carboxylase	hepatocyt es	converts acetyl-CoA CoA to malonyl-	malonyl-CoA blocks fatty mitochondria into acid transport	stimulated	inhibited
	HMG-CoA synthase	hepatic mitochon dria	converts acetoacetyl -CoA into te acetoaceta	rate-limiting step in ketone bodies series of producing the first of the	inhibited	stimulated
	As we have seen previously, glial cells support neuronal function (Belanger

Table 2 . K m values of MCT isoforms for the main monocarboxylates metabolites

 2 . K m values for MCT1 and MCT2 in Xenopus laevis oocytes expressing them. In parenthesis are shown the values obtained from a mouse cancer cell line expressing MCT1 endogenously

Table 4 .

 4 RNAi (in light orange) and reporter lines (in light blue) used in this study. Targeted genes are depicted in the table.

	Targeted gene	CG	Reference
			VDRC GD7132
	acetoacetyl-CoA tholase 1 (ACAT1)	CG10932	TRiP HMC03340,
			Bloomington #51785
			TRiP HMS00331,
	CG8034 (putative MCT)	CG8034	Bloomington #32340
			NIG-R1 Kyoto
	Silnoon	CG8271	VDRC GD1940 VDRC KK104306
	Chaski (Chk)	CG3409	VDRC GD1829
	CG8389 (putative MCT)	CG8389	VDRC KK105290
	CG13907 (putative MCT)	CG13907	VDRC KK110059
	Hermes (Hrm)	CG11665	VDRC GD1807
	Drosophila monocarboxylate transporter 1 (dMCT1)	CG3456	VDRC KK108618
	HMG-CoA Synthase (HMGS)	CG4311

  Figure38. CellProfiler analyses. A1, a rectangular ROI (dashed white rectangle) of 84x80 μm is selected (left panel) and thresholded (middle panel) to remove BODIPY plasma membrane staining from the analyses. A magnification of a zone inside the ROI is shown on the right panel. A2, the histogram of the grayscale ROI is exported to GraphPad Prism Software for analyses. A Gaussian fit curve is performed (red line) by excluding intensity values of x<3. The mean (red dashed line) and standard deviation (SD) (green dashed line) parameters are extracted and used to calculate the threshold value. The threshold value is set to: [mean intensity + 4*(SD/255)]. B1, the ROI with LDs is analyzed in CellProfiler Software. The LDs diameter range was defined according to data published in the literature, discarding objects that are outside this range. B2, when the threshold is not applied non-specific particles are identified by CellProfiler. B3, after threshold LDs are positively identified (in green) and non-specific objects (in magenta). A colored LDs image is shown in the right with positively identified LDs (non-specific particles are not shown).

  To test if KBs are transported into MB neurons by Drosophila MCT homologs, I first examine which MCTs are expressed in the Drosophila MB neurons. In a recent brain single-cell transcriptome analyses, I found that, among the MCTs present in the Drosophila genome, two of them, CG8034 and Silnoon, are strongly expressed in MB neurons (Davie et al., 2018) (Figure

Figure 45. Silnoon can transport KBs in MB Kenyon cells somata

  . A, Model of transacceleration between lactate (Lac) and acetoacetate (AcAc). Presence of extracellular acetoacetate stimulates lactate efflux through the MCT. This process involves a facilitated conformational switch of the MCT substrate binding-site across the cell membrane when acetoacetate is bound. Under normal conditions acetoacetate concentrations are too low to compete with lactate at MCT. Whenever extracellular acetoacetate levels increase, such as during starvation, MCTs bind acetoacetate, which result in a reduced binding site availability for lactate, a reduction of lactate influx and an increase of acetoacetate influx. Then, the binding site translocates from the extra-to the intracellular space. If there is lactate inside the cell, the increased number of intracellular sites subsequently causes an increased lactate efflux. The combination of reduced lactate influx and increased lactate efflux results in a decrease of intracellular lactate concentration. B, Lactate FRET sensor scheme. The donor and acceptor fluorophores, mTFP and Venus, are represented in blue and yellow respectively. When L-lactate binds to its binding domain it induces a conformational change of Laconic sensor, that separates mTFP from Venus, and thus FRET ceases. C, representative image of the Laconic signal in Kenyon cell somatas in one focal plane, with the regions of interest (white dashed lines) in the two hemispheres in which lactate flux is analyzed. Scale bar: 20 μm. D, after 2 minutes of baseline recording, acetoacetate is applied on the whole fly brain. Acetoacetate 10 mM application decreased lactate signal in Kenyon cell somata (n=6, t 5 =5,961, P<0,0001; one sample t-test is significantly different from zero). E, comparison of lactate efflux between fed and starved flies in presence of acetoacetate 10 mM. In fed conditions KBs levels are supposed to be low, whereas in starved conditions KBs levels are expected to be high. Acetoacetate 10 mM application decreased lactate signal in fed flies, but in starved flies acetoacetate application did not decrease lactate signal (n=10 flies, t 33 =4.685, P<0.0001

  induced control flies showed a normal sLT-ARM (n=16, F 2,45 =1.582, P=0.2169). C5-7, shock acuity (n=9, F 2,24 =0.8465, P=0.4413) and olfactory controls for octanol (n=8, F 2,21 =0.5644, P=0.5771) and methylcyclohexanol (n=8, F 2,21 =0.2450, P=0.7926) were normal in starved flies. Data are shown as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by a post hoc Newman-Keuls test (*p<0.05, **p<0.01, ***p<0.001, ns: not significant).

Figure 53. In the adult Drosophila brain, cortex glia contains cytoplasmic LDs

  . A, protocol used for LDs staining. Drosophila brain stained with BODIPY 493/503. BODIPY 493/503 labels cytoplasmic neutral LDs. After brain dissection brains are fixated (without detergent), washed and incubated 30 min with BODIPY 493/503. Next, brains are washed to remove remaining BODIPY dye and images are acquired under a confocal microscope. B, BODIPY staining revealed that LDs are homogenously distributed in the cortex region. The overlap between BODIPY and a cortex glial reporter (R54H02-Gal4>UAS-mCD8::RFP) confirm that LDs are localized in cortex glia. Since BODIPY diffuse across the plasma membrane is it possible to observe a homogenous pattern of expression that resembles cortex glia. Scale bar: 20 μm. Magnifications (rectangle) are shown for each figure. Scale bar 5 μm. C, BODIPY Red C12 and a lipid droplet surface protein reporter (LSD-2) expressed in cortex glia (R54H02>UAS-LSD-2::GFP) show that many LDs identified with BODIPY overlap with the LSD-2 protein reporter. A similar protocol, as for BODIPY 493/503, was used for BODIPY Red C12. BODIPY Red C12 also labels neutral LDs, as well as BODIPY 493/503[START_REF] Rambold | Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics[END_REF]. D, Gray scale image (left) of BODIPY staining in the cortex region. LDs (white dots) are possible to observe in the image. After threshold (to remove plasma membrane staining) LDs were counted with CellProfiler (middle); objects outside of the size limits are shown in magenta. Each individual LD for which the area is calculated can be visualized in artificial colors on the right panel. Scale bar: 10 μm.

3.2 In cortex glia, LDs are the main source of fatty acids precursors for ketone bodies synthesis upon starvation Here I wanted to investigate if cortex glia LD can be used as a main reservoir of KBs precursors upon starvation to feed ketogenesis. To test this hypothesis, I have reasoned that if fatty acids LDs are used as precursors of KBs we should observe dynamic changes in LDs mobilization upon starvation. I have used BODIPY to investigate LDs structure in cortex glia of flies expressing RNAi against genes of which downregulation in cortex glia previously resulted in starvation memory defects (Figure

  Tredern et al., in revision). These data suggested that acetylcholine, the Kenyon cells neurotransmitter, would stimulate the delivery of glucose by cortex glia during LTM. Nevertheless, whether this mechanism would also work in the starved brain by activating cortex glia for promoting KBs release during sLT-ARM remains an open question yet. To test this hypothesis we can use an acetylcholine receptor RNAi in cortex glia during sLT-ARM. Alternatively, we can use BODIPY and bath-applied cholinergic drugs to observe LDs lipolysis in the normal and knocked down condition upon starvation. This will help to prove if the activation of cholinergic receptors in cortex glia might be required for both mobilize LDs and memory upon starvation.Additionally, we can ask if KBs molecules are as efficient substrates, compared to lactate, in terms of energy production to sustain neuronal oxidative metabolism? Theoretically the number of molecules of ATP produced in neurons from a single molecule of β-hydroxybutyrate or acetoacetate (26 and 23 mol of ATP, respectively) as compared to a single lactate molecule (18 mol of ATP)

	Therefore, in mammals, upon glutamatergic activity in the fed state, glial-
	derived lactate might fuel neuronal oxidative metabolism, whereas in the starved
	state upon glutamatergic activity glial-derived KBs may provide energy to
	neurons. Nevertheless, is there a similar mechanism in response to neuronal
	activity in Drosophila? And more specifically how cortex glia metabolism might be
	activated during long-lasting memory formation upon starvation?

  Thus, we cannot discard that cortex glial cells might be supplying KBs to other structures different from MB neurons. Although these experiments would favor the hypothesis about a baseline fueling mechanism, in either case, it remains open the possibility that KBs mobilization from cortex glia into MB neurons might be also regulated upon demand. To test this later hypothesis we could use BODIPY and measure LDs mobilization after memory formation in wild-type and in a context where we knockdown enzymes involved in KBs metabolism.

Figure 64. Neurons transfer peroxidated fatty acids to astrocytes under neuronal activity.

  Hyperactive neurons transfer fatty acids to glia via ApoE-lipoprotein particles. Then, glial cells consume the fatty acids stored in LDs via mitochondrial β-oxidation in response to neural activity. LDs lipolysis is mediated by glial lipases. Figure obtained from[START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF].

	, the delivery of KBs by glia could
	be stimulated by neuronal activity. In mammals, application of glutamate or
	endocannabinoids, a family of endogenous neuromodulatory ligands of
	cannabinoid receptors, can activate CPT1 and ketogenesis in cultured astrocytes
	(Blasquez et al., 1999), a mechanism that involves the CPT1/ACC axis. This
	suggests that the KBs transport can be sensitive to neurotransmitters and
	neuromodulators in mammals. However, this requirement may vary in Drosophila
	and whether these ligands might regulate a KBs transport in flies remains an open
	question yet.
	Interestingly, in vertebrates, a mechanism to protect neurons from toxic

peroxidated fatty acids involve glial NMDA receptors activation by neuronal glutamate to mobilize LDs and activate β-oxidation in vitro

[START_REF] Ioannou | Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity[END_REF]

  KBs tosLT-ARM formation is unlikely. Although we cannot rule out the possibility that circulating KBs might contribute to a different type of memory upon starvation such as short-term memories. Yet, this asks the question of what would be the advantage to have a local provider for KBs directly in the brain (i.e cortex glia) versus being dependent on the general KBs pool from circulation? The most evident advantage to have a local provider in the brain is that it allows a local release of KBs and therefore they can be used faster and efficiently into neurons upon demand. Contrarily, relying on circulating KBs might take longer time to transport these molecules to the site of energy demand. Thus, it is more likely that local KBs, rather than a circulating source, might contribute to the local events in the brain during memory formation upon starvation.Thus, communication between cortex glia and Kenyon cells somata is essential for the transfer of energy rich metabolites during memory formation in the fed condition. However, it remains to be determined if the glia-neuron KBs transport is regulated during memory formation upon starved states. Yet, the impact of secreted signals from neuronal somas in cortex glia during periods of nutritional restriction remains still unknown.

  signaling decrease progressively in the fly body(Heier and Kuhnlein, 2018), allowing that the Akh glucagon homolog activates fatty acid mobilization and oxidation from peripheral LDs stores. We can hypothesize that similarly to the fat body, a mechanism involving LDs mobilization and oxidation might occur in cortex glial cells during sLT-ARM. To test this hypothesis we can use a Akh receptor RNAi expressed in cortex glia and evaluate memory upon starvation.We have discussed briefly some hormonal signals that are involved in the mobilization of energy stores in fed and starved state. But, how sharp or progressive is occurring this transition within neurons and glial cells? And what mechanisms do neurons and glial cells have to sense those changes? I will discuss now evidence about neuronal sensing mechanisms in the starved state. Jimenez and Gimenez-Cassina, 2019). This mechanism could thus prepare the brain for the first hours of starvation, preventing drastic metabolic changes. Nevertheless, it remains unknown if it does exist a mechanism describing a similar property of FGF hormones on KBs utilization in flies.The Drosophila genome encodes FGF ligands and FGF receptors (FGFR)[START_REF] Avet-Rochex | Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila postembryonic brain[END_REF]. Interestingly, expression of FGFR in cortex glia and FGF ligand in neurons are necessary and sufficient for glial proliferation[START_REF] Avet-Rochex | Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila postembryonic brain[END_REF], suggesting that neurons signal directly to adjacent cortex glia by secreting FGF ligands to control gliogenesis. Since FGF signal may exerts its action on AMPK as a downstream effector, it would be interesting to test whether FGF signaling might be also relevant in MB neurons as a mechanism to increase the utilization of KBs via AMPK. This can be tested with a FGFR RNAi or AMPK RNAi in MB neurons during memory formation upon starvation.Alternatively, we can test if secreted neuronal FGF might activate FGFR in cortex glial cells, and consequently increase AMPK signaling as well. Therefore, neurons may sense very early the food deprived states by hormonal signals. Nevertheless, if more hormonal signals are required to sense early or prolonged starvation in neurons remains unknown yet. Thus, neurons need mechanisms to sense the starved state. However, how glial cells sense this transition? I will now discuss some evidence to address this question.3.3 Glial sensing of the starved stateIn Drosophila, feeding and starved signals can trigger the switch between

	3.2 Neuronal energy sensing in the starved state
	The increased ability of neurons to utilize KBs upon starvation might rely
	on signals that allow a sharp and efficient metabolic switch in the brain. In
	primary cortical neurons, the fibroblast growth factor 21 (FGF21) hormone, which
	contributes to modulate systemic adaptation to starvation, increases the ability to
	utilize KBs in vitro during the first hours of food deprivation, by phosphorylating
	AMPK (Katzu-
	This will prove if Akh receptor is necessary during sLT-ARM formation. Thus,
	hormonal signals may allow the mobilization of carbohydrate stores in the fed
	state and lipid stores in the starved state, which may underlie the formation of
	long-lasting memories.

  . Le cerveau est le régulateur central de l'homéostasie énergétique et il donne la priorité à son propre apport par rapport aux organes périphériques. Cependant, les mécanismes par lesquels l'état énergétique régule la plasticité cérébrale restent largement inconnus. Nous avons précédemment montré chez l'organisme modèle Drosophila melanogaster que le cerveau est soumis à une plasticité adaptative et que lors d'un stress métabolique, tel que le jeûne (c'est-à-dire lorsque le niveau du substrat énergétique principal du cerveau, le glucose, est rare), le cerveau désactive la formation de la mémoire à long terme dépendante de la synthèse protéique (MLT) pour favoriser la survie, et parallèlement favorise une autre forme de mémoire à long terme consolidée appelée MRA

Acknowledgements

I would like to start to thanks Thomas and Alice for giving me the professional opportunity

Annex

Résumé détaillé