I would like to thanks my two co-mentors, I couldn't have done it without you. A thesis is done in collaboration and I had probably the best one. Bernard, je ne pourrais assez vous remercier de m'avoir accepté dans votre laboratoire malgré les conditions exceptionnelles et inhabituelles de ma thèse. Merci pour votre présence, votre soutien, votre aide et votre gentillesse à travers l'atlantique. Hannes, I could not thank you enough for taking me into your lab and guiding me during those 4 years. Your mentorship made me grow not only scientifically but also personally. Thank you for being there whenever I needed, for challenging my scientific mind every day, for giving me this project which made me learn so much. I could not have wish for a better lab.

I would like to acknowledge the great scientific environment where I have been lucky to work. This amazing place that is Einstein is due to the people working there. The people of the kitchen: Charlie, Johnny and Ernie. Thank you for your help. Thank you for putting up with the last minute order of plates and other requests. It is also the people of the office: Donna, Karen and Vivian. Thank you so much for helping me along those years with the paperwork but also to listen to my complains and being there to answer to all my questions with patience and kindness even when I was coming up to 10 times a day! Finally, thank you to the C. elegans community of Einstein, especially to Scott and his lab but also Dave, for your help during our weekly meeting. I want to give a special thank to Scott and Steve for collaborating with us on the iBLINC project. Thanks Steve for your help and for answering all of my questions about statistics (among others).

Of course this journey would have not been the same without my awesome labmates, past and present: Matthew, Eillen, Rob, Nathali, John, Yehuda, Nelson, Kevin, Kristian, Dayse, Carlos, and our newest recruit Alu. Thank you to my summer student, Elizabeth, for her help.

I especially want to thanks Matthew and Eillen who taught me most of what I know about worm, worm genetics and molecular biology. In general I want to thanks everyone to always be happy to answer to any of my questions, to share a moment to discuss Science, and life in general, and to compare our experience in living in such different place of the world. The members of this lab are not only colleagues but great friends.

My life in New York would not have been the same without all of my friends: the lunch peeps which includes the lab, the honorary lab members, Raquel and Maria, but also Manon, Anindita, and Abhijit; and the other Einstein friends, especially Laina and Stephania. I want to give a special thanks to my most awesome friends Eillen, Raquel, Dayse and Manon. Eillen, thank you so much to always be ready to put a box on your head to cheer me up, and to always say yes to a complaining dinner. Raquel and Dayse thanks you for being the best comadres ! Raquel I cannot thank you enough for being my personal dictionary and for teaching me all those tremendously fantabulous new words. Dayse thank you for sharing with me the non-Spanish part of the lab and for being as flabbergasted as me about the American life. Manon, merci pour toujours avoir le mot et les conseils qu'il faut afin de me montrer les priorités et les choses les plus importantes. Merci également aux français de Paris pour votre soutien et votre aide durant cette thèse : Georgia, Géraldine, Marie et Nicolas. Merci à mes précieuses amies magistériennes pour votre soutien et pour les conversations « potins » : Amandine, Coline et Emilie. Cette immigration temporaire aurait été bien triste sans vous ! Je veux aussi remercier mes visiteurs qui ont fait le déplacement jusqu'à New York : Les Julie et Floriane (les pionnières), Laure et Cindy, Elodie et Matthieu, et plus récemment Guillaume et sa soeur. En particulier merci Julie pour toujours être partante pour les weekends escapades pour se changer les idées (même si ca implique traverser le Delaware sous la neige!). Merci également de m'écouter raconter ma vie et mes aventures au labo (ce qui inclut les explications de mon apprentissage des injections…). Merci à mes amis de lycée et de l'IUT d'avoir pris le temps de me voir quand je rentrais en France. Surtout merci Quentin de toujours être là pour me remonter le moral même à 6000 kms et pour m'accueillir pendant mes séjours parisiens. Et finalement merci à ma jumelle Elodie de m'avoir suivi tout au long de ses années et de m'avoir encouragé, d'être toujours là pour moi.

I want to give a special thanks to my friends/proofreaders who accepted to read this thesis, to correct the English and give their opinions: Raquel (who is better than google :P), Laina, and Kevin but also Maeva who was my French and naïve eyes. Merci également Maeva de répondre à mes messages de panique et à mes questions parfois ridicules sur le déroulement de l'écriture et de la soutenance de la thèse.

Ces remerciements ne seraient pas complets sans essayer de décrire toute la reconnaissance que j'ai envers ma famille, spécialement envers mes parents pour leur soutien.

Papa et maman merci de m'avoir encouragé de poursuivre mon rêve de petite fille, d'avoir cru en moi tout ce temps et d'avoir mis tous les moyens pour que je réussisse, même si cela à

List of figures and tables

C. elegans nomenclature

Name of strains: two capital letters (related to the lab of the strain's origin) and a number.

Strains created in the Bülow lab are named EB (example: EB2217).

Name of genes: designed with three italicized letters, a hyphen and an Arabic number.

They may be followed by a Roman numeral corresponding to the chromosome to which the gene maps (example: unc-104II). Name abbreviations can refer to the mutant phenotype (example: unc for uncoordinated) or to the predicted protein product or RNA product (example: hst for Heparan SulphoTransferase).

Name of alleles: two letters related to the lab of allele's origin (dz is from the Bülow lab) and a number (example: dz18).

Name of proteins: abbreviations of the gene, in all capital and non-italicized letters (example: UNC for uncoordinated).

Name of phenotypes: non-italicized three-letter abbreviation, often corresponding to the gene name, with the first letter capitalized (example: Unc for uncoordinated).

Transgene's name: italicized name starting with the two-letter laboratory allele prefix, follow by two letters indicating the state of the transgene in the transgenic line (Ex for extrachromosomal array, Is for integrated array) and a number (example: dzEx1240 for an extrachromosomal line or dzIs68 for an integrated line). The name can be followed by genotypic or molecular information describing the transgene in brackets (example: dzIs68:

Abstract

The nervous system is a complex network that senses and processes information and is essential for the survival of both vertebrates and invertebrates as it is involved in behavior responses. Information within the network is transmitted through specialized cell-cell contacts, including synaptic connections. Importantly, the network is not static and is believed to change during development and learning, as well as during pathological or normal agerelated decline. Studying the nervous system in vivo requires the use of animal models such as Caenorhabditis elegans. Understanding of behavior and development requires visualization and analysis of synaptic connectivity. However, existing methods are laborious and may not depend on trans-synaptic interactions, or otherwise 'trap' the synapses by fixing the connections, thus precluding dynamic studies.

In order to study synaptic modifications, we developed a new transgenic approach for in vivo labeling of specific connections in C. elegans, called iBLINC (in vivo Biotin Labeling of

INtercellular Contacts). iBLINC involves the biotinylation of an acceptor peptide (AP) by the

Escherichia coli biotin ligase BirA. Both are fused to two interacting post-and pre-synaptic proteins, respectively. The biotinylated acceptor peptide fusion is detected by a monomer streptavidin fused to a fluorescent protein that is transgenically expressed and secreted into the extracellular space. The method is directional, bright and dynamic. Moreover it correlates well with electron micrograph reconstruction.

Using this new technique to observe synapses, which are part of the thermosensory circuitry of C. elegans, during aging, we could conclude that the connection pattern varies with age and within a population. Changes of the number and size of the synapses were observed during aging. Some segments of the synaptic region seem to be more affected than others by the aging process. Those results were corroborated by using a GABAergic presynaptic marker which allowed us to visualize a decline of the vesicle number with aging.

In summary, in this thesis I explained a new in vivo trans-synaptic method to visualize synapses in C. elegans. Then I demonstrated that a natural decline in the number of synapses as well as the number of vesicles occurs during aging.

Résumé

Le système nerveux est un réseau complexe qui perçoit et analyse les informations

Chapter I: Introduction

I.1 The Nervous system I.1.1 Building the nervous system and its connection

The nervous system is a major organ of invertebrate and vertebrate animals with different degrees of complexity across species. The mammalian nervous system can be divided into two parts, the central nervous system (CNS) and the peripheral nervous system (PNS). While they both derive from neuroectodermal cells, the peripheral and enteric nervous system is derived from neural crest cells ( Neurons communicate through synapses, essential structures for the proper functioning of the nervous system. Two types of synapses exist: chemical and electrical [START_REF] Pereda | Neurobiology: All Synapses Are Created Equal[END_REF]. Chemical synapses are based on the release of neurotransmitters that are recognized by specific specialized receptors which ensure the downstream signaling. These synapses can be formed between two neurons as well as between neuron and muscle, in which case they are called a neuromuscular junction (NMJ). The electrical synapses, also called gap junctions, allow electric currents to flow through, causing voltage changes. This type of synapse will not be further discussed in this study, but is reviewed in [START_REF] Bennett | Electrical synapses, a personal perspective (or history)[END_REF] and [START_REF] Pereda | Neurobiology: All Synapses Are Created Equal[END_REF].

Synapses can form in two ways; en passant -the synaptic boutons (terminals) are distributed along the axon shaft, or terminaux -boutons are formed at the end of the axon branches [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF]). The formation of chemical synapses occurs in several steps: initial contact, differentiation, and maturation [START_REF] Giagtzoglou | Cell Adhesion , the Backbone of the Synapse[END_REF]. After the axon is guided through the cellular environment, the pre-and post-synaptic partners recognize each other. This process is believed to involve cell adhesion molecules. The adhesive interaction between the growth cone and the target cell are the starting point of synaptogenesis [START_REF] Giagtzoglou | Cell Adhesion , the Backbone of the Synapse[END_REF]Hortsch and Umemori 2009). Pre-and post-synaptic components are recruited at the synapse, it has been shown in cell culture that the pre-synaptic molecules are recruited before post-synaptic ones [START_REF] Friedman | Assembly of New Individual Excitatory SynapsesTime Course and Temporal Order of Synaptic Molecule Recruitment[END_REF]. This recruitment occurs rapidly as the neurons produce the synaptic proteins before the initial recognition [START_REF] Ahmari | Assembly of presynaptic active zones from cytoplasmic transport packets[END_REF].

The last step involves the stabilization of the synapse by increasing the number of vesicles in the pre-synaptic partner as well as in some cases by the modification of the shape of the synapse as the contact between the cells increases in surface area. Because the molecular composition does not change drastically, it has been hypothesized that the maturation involves reorganization of cell adhesion molecules. For example, the excitatory synapses change their shape from stubby spine to 'mushroom' spine by stabilizing cytoskeletal proteins [START_REF] Yuste | Genesis of dendritic spines: insights from ultrastructural and imaging studies[END_REF]). This process is proven by the fact that unlike mature synapses, immature synapses are sensitive to actin depolymerization drugs indicating that the cytoskeleton molecules are stabilized during maturation [START_REF] Zhang | Stages of synapse development defined by dependence on F-actin[END_REF]. Moreover, changes in receptor expression are seen in those synapses. In fact, a young mammalian brain expresses NMDA (N-Methyl-D-aspartic acid or N-Methyl-D-aspartate) receptors while in an older brain AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors can be found at the membrane. This change is dependent on the stimulation of the pre-synaptic neuron,

showing that synaptic activity is important in shaping the connections [START_REF] Isaac | Evidence for silent synapses: Implications for the expression of LTP[END_REF][START_REF] Isaac | Silent Synapses during Development of Thalamocortical Inputs[END_REF][START_REF] Durand | Long-term potentiation and functional synapse induction in developing hippocampus[END_REF][START_REF] Wormatlas | Maturation of a central glutamatergic synapse[END_REF][START_REF] Feldman | Experience-Dependent Plasticity and the Maturation of Glutamatergic Synapses[END_REF][START_REF] Liao | Regulation of morphological postsynaptic silent synapses in developing hippocampal neurons[END_REF].

Even though molecules involved in synaptogenesis have been identified, this process is still not fully understood and more proteins implicated in synaptogenesis remain unknown.

Most of the molecules described are part of the cellular adhesion molecule family (CAM), specifically the cadherin or immunoglobulin superfamilly. Several possible roles have been attributed to the CAM's: mechanical stabilization, target recognition molecule, synaptic differentiation, and synaptic structure regulation [START_REF] Yamagata | Synaptic adhesion molecules[END_REF]. Mechanical stabilization of the synapse occurs by tying the membrane of each partner using the "sticky" characteristic of the CAMs which interact across the synaptic cleft. Several proteins are involved in vesicle clustering including FGF22 (fibroblast growth factor 22), Netrin G ligands, signal regulatory proteins (SRP), leucine rich repeat transmembrane proteins (LRRTM) as well as WNT7a, a secreted polypeptide part of the WNT (int/Wingless) family (Hortsch and Umemori 2009). WNT7a protein has also been shown to be able to cluster synapsin I, a protein associated with the vesicles, and has also been associated with growth cone differentiation [START_REF] Hall | Axonal remodeling and synaptic differentiation in the cerebellum is regulated by WNT-7a signaling[END_REF]. FGF22 acts by interacting with FGFR2b (fibroblast growth factor receptor) in vesicle clustering as well as pre-synaptic differentiation [START_REF] Umemori | FGF22 and its close relatives are presynaptic organizing molecules in the mammalian brain[END_REF]. Eph and Ephrins are involved in axon guidance and clustering of NMDA receptor at excitatory synapses only [START_REF] Dalva | EphB Receptors Interact with NMDA Receptors and Regulate Excitatory Synapse Formation[END_REF][START_REF] Henkemeyer | Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus[END_REF], while NARP, a secreted protein, induces the clustering of AMPA receptor in inhibitory neurons only, by binding their extracellular domains [START_REF] Mi | Differing mechanisms for glutamate receptor aggregation on dendritic spines and shafts in cultured hippocampal neurons[END_REF][START_REF] O'brien | Synaptically targeted narp plays an essential role in the aggregation of AMPA receptors at excitatory synapses in cultured spinal neurons[END_REF]. Therefore it is possible that excitatory and inhibitory neurons do not depend on the same molecules to form synapses. SynCAM and thrombospondins have been shown to have synaptogenic effects as well. In the case of thrombospondins, two mechanisms have been proposed: differentiation of the pre-and post-synaptic terminal, or enhanced adhesion of the pre-and post-synaptic membranes [START_REF] Christopherson | Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis[END_REF]. Neurexin and Neuroligin have been shown to be important for synapse maturation and maintenance. Neurexin is a pre-synaptic protein that forms a complex across the synaptic cleft with the post-synaptic molecule

Neuroligin. In cell culture, Neurexin is involved in aggregation of PSD95 (post-synaptic density protein 95) and NMDA receptors at the excitatory synapse, and aggregation of Gephyrin and GABAA (γ-Aminobutyric acid A) receptors at the inhibitory synapse [START_REF] Graf | Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins[END_REF][START_REF] Nam | Postsynaptic assembly induced by neurexin-neuroligin interaction and neurotransmitter[END_REF]. Neuroligin seems to be important for the clustering of vesicles at the site of contact in the pre-synaptic partner. In C. elegans, nlg-1/Neuroligin has also been recently involved in the clustering of GABAergic receptor in synergy with unc-40/DCC and madd-4/Punctin [START_REF] Tu | elegans Punctin Clusters GABAA Receptors via Neuroligin Binding and UNC-40/DCC Recruitment[END_REF]. A working hypothesis is that Neuroligins control the balance between excitatory and inhibitory synapses [START_REF] Graf | Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins[END_REF][START_REF] Chih | Control of excitatory and inhibitory synapse formation by neuroligins[END_REF][START_REF] Chubykin | Activitydependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2[END_REF]). The last step of circuitry formation is synaptic elimination; in this process, already formed synapses are selectively removed in order to shape the nervous system and select the proper connections [START_REF] Giagtzoglou | Cell Adhesion , the Backbone of the Synapse[END_REF]Hortsch and Umemori 2009).

Environmental stimuli have profound effects on the nervous system. These effects lead to the elimination of synapses but also to more subtle changes such as synaptic plasticity, as described later in this introduction. These changes are particularly important during the critical period of embryonic and early life development, in addition to playing an important role in adulthood. Synapse elimination has been studied in NMJ. In rodents, just after birth several axons reach and form synapses with the same muscle cell while in adults only one axon will innervate one muscle cell. At birth, acetylcholine aggregates receive several axons, and later in life, due to active mechanisms, only one remains after the others have retracted.

These observations prove that there is elimination of synapses. Inhibition of muscle cells prevents the elimination of synapses while stimulation of neurons induces it. It has been shown that synapses that are maintained are the ones that are stimulated in a synchronous manner with the post-synaptic cell [START_REF] Walsh | In Vivo Time-Lapse Imaging of Synaptic Takeover Associated with Naturally Occurring Synapse Elimination[END_REF]. Retrograde signaling is also involved in the process. In summary, activity of the synapses and the response from the postsynaptic partner strengthen the synapses and prevent their elimination. The pre-synaptic nerve that does not properly stimulate the post-synaptic cell is eliminated [START_REF] Hebb | The organization of behavior: A Neuropsychological Approach[END_REF]Hortsch and Umemori 2009). [START_REF] Hall | Kinesin-Related Gene unc-104 Is Required for Axonal Transport of Synaptic Vesicles in C . elegans[END_REF]Lodish et al. 2000a).

Once at the terminal, the vesicles can be found in two pools: docked at the plasma membrane or reserved in the active zone (Lodish et al. 2000b). When the neuron is stimulated, an action potential is induced which is translated by the fusion of vesicles with the membrane and release of the neurotransmitters (Figure I-3). The vesicles are then recycled by endocytosis. The action potential provokes an increase in the concentration of the cytosolic calcium (Ca 2+ ). This increase is followed by depolarization of the membrane during which open (Lodish et al. 2000b). The Ca 2+ ions bind synaptotagmin, a protein that is considered to be the "sensor" of the action potential. Synaptotagmin has been shown to bind the vesicles and a plasma-membrane complex composed of neurexin and syntaxin, thereby blocking fusion of the vesicle to the membrane [START_REF] Geppert | Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse[END_REF]. When the ions bind the protein, this complex is displaced, allowing the protein involved in the fusion to bind which provokes the fusion of the vesicle and the membrane. Extra ions are rapidly removed from the cell by Ca 2+ ATPases to allow the synapse to respond to the next action potential (Lodish et al. 2000b;[START_REF] Cheng | Synaptotagmin 1 directs repetitive release by coupling vesicle exocytosis to the Rab3 cycle[END_REF]. membrane and induce a depolarization or a hyperpolarization of the post-synaptic cell [START_REF] Unwin | Neurotransmitter action: Opening of ligand-gated ion channels[END_REF]). The G protein-coupled receptors (in green in Figure I-3) induce a slower response.

Binding of the neurotransmitter to the receptor activates the G protein which can, in most cases, directly bind to the ion-channel protein to trigger a response [START_REF] Hille | G protein-coupled mechanisms and nervous signaling[END_REF]). The consequence of the neurotransmitter release can be an excitation or an inhibition of the postsynaptic cell. The excitatory receptor opens a channel that leads to the entry of sodium (Na + )

or Na + and potassium (K + ) together, while the inhibitory receptors induce K + or chlorine (Cl -) channels. Excitatory activation depolarizes the plasma membrane whereas hyperpolarization is the consequence of inhibition. In order to prevent a constant stimulation, the neurotransmitters are removed or destroyed. They can either diffuse away, be recycled by the pre-synaptic neuron, or be degraded by an enzyme such as choline esterase in the case of acetylcholine (Lodish et al. 2000b).

Synapse plasticity is considered to be the mechanism in learning and memory. It is defined by the change in strength or efficacy of synaptic transmission at mature synapses due to their activity [START_REF] Caroni | Structural plasticity upon learning: regulation and functions[END_REF]). The activity of the synapses seems to induce connectivity changes, and in this way it allows the nervous system to adapt to the environment during development as well as adulthood. During development, plasticity can be attributed to the selected elimination of synapses as explained above. Two other types of changes can be observed: long-term and short-term plasticity. The long-term can be split into two effects:

long-term potentiation (LTP) and long-term depression (LTD). LTP is an increase in synaptic strength due to a brief series of stimuli [START_REF] Alger | Long-term and short-term plasticity in the CA1, CA3, and dentate regions of the rat hippocampal slice[END_REF]. LTD shows an opposite response, a reduction of the synaptic strength [START_REF] Ito | Cerebellar Long-Term Depression : Characterization[END_REF]). Short-term plasticity can occur in three forms: facilitation, depression, and post-tetanic potentiation. Facilitation lasts less than a few seconds, depression lasts for tens of seconds, while post-tetanic potentiation can persist for several minutes [START_REF] Abbott | Synaptic computation[END_REF]. Short-term plasticity can be an early stage of LTP or LTD. However short and long-term modification can be differentiated by the molecular pathways that they activate [START_REF] Raymond | NMDA receptor regulation by amyloid-β does not account for its inhibition of LTP in rat hippocampus[END_REF][START_REF] Colbran | Calcium/calmodulin-dependent protein kinase II and synaptic plasticity[END_REF]).

Long-term plasticity seem to be able to induce activation of protein kinases that influence gene expression and protein synthesis. Short-term plasticity seem to induce calcium influx, which will promote phosphorylation of the post-synaptic receptors and retrograde signaling by the endocannabinoid pathway, which influences pre-synaptic transmitter release [START_REF] Basavarajappa | Neuropharmacology of the endocannabinoid signaling systemmolecular mechanisms, biological actions and synaptic plasticity[END_REF]. Presynaptic terminal structural changes affect the probability of release, but the amount of neurotransmitter released is the same. Phosphorylation of the receptors can enhance or reduce their function; this is mediated by modulating the number of functional receptors by modifying either their sensibility or their subunit composition. For example, an augmentation of AMPA receptor function have been show to enhance the excitability of excitatory synapses [START_REF] Sacchetti | Long-term synaptic changes induced in the cerebellar cortex by fear conditioning[END_REF]. In contrast, the excitability of the inhibitory GABAergic neurons is enhanced by increasing the probability of neurotransmitter release [START_REF] Scelfo | Learning-related long-term potentiation of inhibitory synapses in the cerebellar cortex[END_REF]. Another form of synaptic plasticity has been described as homeostatic plasticity. This type of plasticity has been hypothesized to have for objective to maintain a stable function even during development and activity-dependent synaptic changes [START_REF] Turrigiano | Homeostatic plasticity in the developing nervous system[END_REF]. Several mechanisms have been involved in neuronal plasticity such as retrograde signaling through the pre-synaptic Ca 2+ channels and post-synaptic BDNF (Brain-derived neurotrophic factor), TNF-α (Tumor necrosis factor α), and ARC (activityregulated cytoskeleton-associated protein) signaling. In addition, CAM molecules have been hypothesized to play a role [START_REF] Leslie | Postsynaptic depolarization scales quantal amplitude in cortical pyramidal neurons[END_REF][START_REF] Beattie | Control of synaptic strength by glial TNFalpha[END_REF][START_REF] Swanwick | Activity-dependent scaling of GABAergic synapse strength is regulated by brain-derived neurotrophic factor[END_REF][START_REF] Shepherd | Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors[END_REF]).

It has been described than the synaptic spine in mice brain can be maintain for several months and sometimes for their complete life span [START_REF] Grutzendler | Long-term dendritic spine stability in the adult cortex[END_REF][START_REF] Zuo | Development of long-term dendritic spine stability in diverse regions of cerebral cortex[END_REF]).

This observation shows that possible dedicated mechanisms are involved in the maintenance of those synapses. In consequence, it is possible that proper aging of the nervous system is important to avoid the elimination of those connections.

I.1.2. Aging of the nervous system

With age, sensory and motor impairments are observed as for example, delayed reaction time, hearing loss, and reduced eyesight and muscle strength (Riddle 2007). This could be explained by a decline in nervous system capacity. Indeed, anatomical changes as well as synaptic modifications are observed in the aging brains of different species, ranging from mouse to monkeys and humans.

It has been shown that with age, there is a loss of nerve fibers in some areas of the human brain that exhibit a decrease in white matter volume. Moreover, degeneration of the myelin sheath has also been detected in the brains of aging monkeys and mice. It has been suggested

that degeneration is mainly due to the loss of thinner fibers, and that the tracts that are myelinated last are the most affected [START_REF] Marner | Marked loss of myelinated nerve fibers in the human brain with age[END_REF]. Degeneration of the myelin results in shorter internodes and more paranodal structures in the aging brain as new myelination takes place to compensate for the loss (Figure I-4) [START_REF] Lasiene | Age-related myelin dynamics revealed by increased oligodendrogenesis and short internodes[END_REF][START_REF] Peters | A review of the structural alterations in the cerebral hemispheres of the aging rhesus monkey[END_REF]. The cognitive decline observed with age could be partially due to these changes in the structure of the myelin, as the presence of more internodes would result in temporal changes of sequential events, as well as loss of connections between groups of neurons as some nerve fibers are gone [START_REF] Lu | Age-related slowing in cognitive processing speed is associated with myelin integrity in a very healthy elderly sample[END_REF][START_REF] Peters | A review of the structural alterations in the cerebral hemispheres of the aging rhesus monkey[END_REF].

In the aging brain, neurons are not the only population of cells being affected as we can also observe defects in the glia population. Glia can be classified in three groups:

oligodendrocytes, astrocytes, and microglial cells . Oligodendrocytes are responsible for formation of the myelin sheaths around the axons. The number of oligodendrocytes increases (around 50% more) as well as the occurrence of pairs or groups of them in old monkeys brains (after 20 years of age) (Figure I-4) [START_REF] Peters | The neuroglial population in the primary visual cortex of the aging rhesus monkey[END_REF]. This increase could be explained by their need to make more myelin [START_REF] Peters | The neuroglial population in the primary visual cortex of the aging rhesus monkey[END_REF][START_REF] Lasiene | Age-related myelin dynamics revealed by increased oligodendrogenesis and short internodes[END_REF]). The overall number of astrocytes and microglia does not seem to change with age, but some morphological transformations can be observed (Figure I-4) [START_REF] Peters | The neuroglial population in the primary visual cortex of the aging rhesus monkey[END_REF]). In the case of astrocytes, a hypertrophy can be seen with age as they increase in both size and the number of filaments. Therefore they can fill up the space liberated by the loss of nerve fibers.

They also start phagocytosis as they seem to be partially responsible for the degradation of the degenerating myelin. Microglial cells are considered to be the phagocytes of the nervous system. It has been shown that they undergo morphological modification from ramified cells to unbranched cells. Markers of their activation have also been detected in the aging brain.

These two observations indicate that the microglia could play a role in degradation of myelin and other brain components (Figure I-4) (Riddle 2007;[START_REF] Peters | The neuroglial population in the primary visual cortex of the aging rhesus monkey[END_REF].

More subtle changes in the nervous system have also been observed in the aging brain, for example, synaptic function modifications. It has been shown in different organisms that aging is associated with modification of the hyperpolarizing potential (AHP), Ca 2+ homeostasis, LTP and LTD responses, and with a decrease in pre-synaptic markers such as synaptophysin and GAP-43 [START_REF] Kumar | Neurophysiology of Old Neurons and Synapses[END_REF].

Several hypotheses have been formulated to explain the decline of synaptic strength; the loss of synaptic contact, the decrease of neurotransmitter release and/or a decrease in the response to neurotransmitter by the post-synaptic cell. In rhesus monkeys, lost of axodendritic synapses have been observed in some of the brain layers while axosomatic synapses seem to stay unchanged. However, increases in the size of the terminals have been observed in both types of synapses [START_REF] Soghomonian | Effects of age on axon terminals forming axosomatic and axodendritic inhibitory synapses in prefrontal cortex[END_REF]. Moreover, the 'synaptic response:fiber potential' ratio increases in older animals leading to the argument that the surviving synaptic contact is increased, or in other words, the remaining synapses have an increased strength. In some regions of the brain, the functional impairment seems to be due to a decrease in postsynaptic response more so than pre-synaptic cell defects. Indeed, in the hippocampus of rodents, the AMPA and NMDA receptors show a decrease of responsiveness to glutamate [START_REF] Barnes | Region-specific age effects on AMPA sensitivity: electrophysiological evidence for loss of synaptic contacts in hippocampal field CA1[END_REF][START_REF] Jouvenceau | Alteration of NMDA receptor-mediated synaptic responses in CA1 area of the aged rat hippocampus: Contribution of GABAergic and cholinergic deficits[END_REF][START_REF] Gocel | Evidence for loss of synaptic AMPA receptors in anterior piriform cortex of aged mice[END_REF]. Alzheimer's disease and over 500,00 patients have Parkinson's disease, demonstrating the importance of studying these debilitating diseases due to the wide impact that they have (www.alz.org; http://www.ninds.nih.gov). In these diseases, synaptic loss correlates with cognitive impairment (Pienaar et al. 2012;Terry et al. 1991). These results implicate that the maintenance of synapses is disrupted in those diseases and that mechanisms involved in the maintenance of the connections are important for cognition. The increase of the population life expectancy raises the percentage of patients at risk for cognition impairment and neurodegenerative diseases. In consequence studying the nervous system and the consequences of aging on its maintenance is important to find drug targets that can potentially slow down the progression of disease and/or delay its outbreak. The study of animal models is important to understand the functioning of the nervous system. Caenorhabditis elegans is a well suited model for nervous system study and aging study as it has a short life span and a small and defined number of neurons [START_REF] Wolozin | Watching worms whither: Modeling neurodegeneration in C. elegans[END_REF][START_REF] Alexander | Genetics of Aging Use of C . elegans as a Model to Study Alzheimer ' s Disease and Other Neurodegenerative Diseases[END_REF]. England. The Bristol strain has the characteristic of being temperature sensitive and the males are sterile making this strain not suitable for genetic analyses [START_REF] Dougherty | Possible significance of free-living nematodes in genetic research[END_REF].

I.2. C. elegans:

When Dr. Sydney Brenner was looking for a simple animal model to study animal development and behavior, he opted to use the small roundworm. He therefore chose the Bristol strain of C. elegans, which allowed mating with males and growth at different temperatures. Dr Sydney Brenner extensively studied the Bristol strain (N2) and used it to isolate mutants. The so-called N2 strain is now currently used as the reference strain for laboratory studies [START_REF] Brenner | The Genetics of Caenorhabditis elegans[END_REF].

I.2.1.2. General information: life cycle and genetics

C. elegans has two sexes, hermaphrodites and males. The hermaphrodites produce oocytes and sperm and can self-fertilize as the oocyte passes through the spermatica. The hermaphrodites carry 5 pairs of autosomal chromosomes and two X chromosomes, while the male has only one X chromosome for the same number of autosomes. The males are generated by non-disjunction of the X chromosomes during meioses. They can also be obtained by crossing males and hermaphrodites. Once mated, the hermaphrodite will favor the male sperm instead of its own sperm. Self fertilization can give rise to around 300 progenies in a hermaphrodite life while mating can increase its progeny to 1200-1400 [START_REF] Brenner | The Genetics of Caenorhabditis elegans[END_REF]). In the laboratory, C. elegans can be grown on agar plates seeded with the bacteria Escherichia coli (E. coli). It has a short life cycle compared to other animal models as it needs 3.5 days at 20°C to develop from embryo to a fertile adult. Before reaching adulthood it goes through 4 different larval stages (L1 to L4), separated by molts between each stage (Figure I-6) [START_REF] Brenner | The Genetics of Caenorhabditis elegans[END_REF]). An adult worm can live between 2 to 3 weeks. The life span of C. elegans varies with the temperature. The animals live longer at colder temperature and shorter at warmer temperature. Traditionally 15°C is use as colder temperature and 25°C as warmer temperature [START_REF] Brenner | The Genetics of Caenorhabditis elegans[END_REF]). After 7 days of adulthood, age-related decline can be observed including a decline of egg laying, defecation, pharyngeal pumping, motility, but also protein aggregation [START_REF] Bolanowski | Quantitative measures of aging in the nematode Caenorhabditis elegans. I. Population and longitudinal studies of two behavioral parameters[END_REF][START_REF] David | Widespread protein aggregation as an inherent part of aging in C. elegans[END_REF][START_REF] Liu | Functional aging in the nervous system contributes to age-dependent motor activity decline in C. elegans[END_REF]. Under limiting environmental conditions (e.g crowded plates or starvation), the worm larvae can pause its cycle cannot be started [START_REF] Riddle | The Dauer Larva[END_REF]).

The studies done by Brenner and Sulston, show that the cell lineage of C. elegans is invariant. It contains about a thousand somatic cells [START_REF] Brenner | The Genetics of Caenorhabditis elegans[END_REF][START_REF] Sulston | Post-embryonic cell lineages of the nematode, Caenorhabditis elegans[END_REF]. Similar to most round worms, C. elegans body is non-segmented and comprises two cylindrical tubes, the inner and the outer, separated by a cavity called the pseudocoelom. The outer tube is composed of the body wall (cuticle, hypodermis, excretory system, neurons and muscles) while the inner tube is composed of the digestive and reproductive organs. mammalian orthologs [START_REF] Shaye | OrthoList: a compendium of C. elegans genes with human orthologs[END_REF].

I.2.1.3. Tools available to utilize C. elegans as a model

The C. elegans community has grown and collaborated together over the decades to generate numerous tools to facilitate studies of the nematode. One of the most important advances was the sequencing of the full genome which was finished in 1998 and fully available in 2002 [START_REF] The | Genome sequence of the nematode C. elegans: a platform for investigating biology[END_REF]. In addition to sequencing, the genome has been cloned in YAC, cosmid and fosmid libraries while the cDNA has been cloned into lambda phages. All of information about these reagents have been combined with genetic mapping and are freely available online (ACeDB, WormBase). A second useful tool is the possibility of generating transgenic lines carrying transgenes via micro-injections, thus allowing the possibility of expressing foreign DNA [START_REF] Mello | Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences[END_REF]. When foreign DNA is injected into the mitotic zone of the hermaphrodite gonads (Figure I-9) it can be incorporated in a nucleus and as a consequence, forms an extrachromosomal array that can be transmitted during meiosis and mitoses. Those special chromosomes do not contain telomeres and have been shown to have a non-Mendelian transmission [START_REF] Mello | Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences[END_REF]. Indeed, this extrachromosome can be lost during meiotic and mitotic divisions. As a consequence, a hermaphrodite carrying the array will not transmit it to all of its progeny. In addition, all of the cells of the transgenic worm do not necessarily have the extrachromosome. This particularity can allow mosaic analysis and thereby help uncover which cell must express a wild-type version of a gene to rescue a mutant phenotype.

Micro-injection technique took a step forward with the development of the use of fluorescent proteins in the worm [START_REF] Chalfie | Green fluorescent protein as a marker for gene expression[END_REF]. This allowed scientists to uncover temporal-spatial gene expression and also to label cells in vivo by the use of transcriptional and translation fusion between the gene of interest or its promoter, respectively, to a Another advantage of the nematode is the easy generation of mutants by the use of Ethyl methanesulfonate (EMS) to do forward genetic screening. EMS modified guanine pairs with thymine during replication [START_REF] Coulondre | Genetic studies of the lac repressor[END_REF] which induces a G/C to A/T bias, often resulting in the creation of premature stop codons as a consequence [START_REF] Flibotte | Whole-genome profiling of mutagenesis in Caenorhabditis elegans[END_REF]. For this purpose, a simple 'F1 cloning' protocol can be used to obtain mutants. This protocol consists of suspending hermaphrodites at the last larval stage (or young adults) in a solution of EMS for few hours, and after several washes they are placed onto plates containing bacteria. A few days later, F1 worms are single picked, and their progeny are analyzed for the phenotype of interest [START_REF] Brenner | The Genetics of Caenorhabditis elegans[END_REF]).

In addition to forward genetic screening, reverse genetics can be carried out. RNAmediated interference (RNAi) has been largely developed and used to knock-down genes, which allows for the study of essential genes. Clones containing the double stranded DNA (dsDNA) sequence corresponding to the messenger RNA (mRNA) of the gene of interest can be fed to the worm. The dsDNA will then be cleaved into short interfering RNA (siRNA) by the Dicer complex. The siRNA incorporated into the RISC (RNAi induced silencing complex)

will then recognize the corresponding mRNA and lead to its degradation. Subsequently, the amount of protein is also expected to be knocked-down. The effects can be seen in the same worm or in their progeny. The C. elegans genome, almost in its entirety, is available as RNAi clones [START_REF] Fire | Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[END_REF][START_REF] Kamath | Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans[END_REF][START_REF] Kamath | Systematic functional analysis of the Caenorhabditis elegans genome using RNAi[END_REF][START_REF] Joyce | Manipulating and enhancing the RNAi response[END_REF].

More recently, a Drosophila class II transposon, Mos1, has been used to generate mutants and map them directly, as well as for genomic engineering. The transposon is used for a "cut and paste" mechanism. The excision can be provoked experimentally by inducing a double strand break (DSB). The DSB can be repaired using a target sequence that can be designed for knockout or knock in modification of the gene of interest (Figure I-10) [START_REF] Bessereau | Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line[END_REF][START_REF] Boulin | Mos1-mediated insertional mutagenesis in Caenorhabditis elegans[END_REF][START_REF] Robert | Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks[END_REF]).

In the last couple of years, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) has also become available for generating mutants and for genomic engineering in C.

elegans [START_REF] Dickinson | Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination[END_REF][START_REF] Katic | Targeted heritable mutation and gene conversion by Cas9-CRISPR in Caenorhabditis elegans[END_REF]. CRISPR is a method based on Cas9 nuclease, a chimeric fusion of two RNAs called a single guide RNA (sgRNA), and a repair sequence. The design of highly specific sgRNAs allows for recruitment of the Cas9 nuclease to the desired genomic position where it triggers double strand cleavage. The DSB is then repaired through classical Homology Directed Repair (HDR) or Non-Homologous End Joining (NHEJ) repair pathways. Therefore, by providing an exogenous DNA template, the genome can be precisely edited as desired.

Mapping mutations took a step forward by the use of whole genome sequencing which is based on the polymorphisms between the N2 Bristol strain and the Hawaiian strain [START_REF] Doitsidou | elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy[END_REF]). The Hawaiian strain or CB4856 is a natural genetic variation of C. elegans and as its name indicates has been isolated in Hawaii. Forward genetic screening is performed using the N2 strain such that mutation is linked to the N2 genome. Crossing the mutant with the Hawaiian strain and selecting for homozygous F2 mutants creates linkage disequilibrium in which the percentage of N2 polymorphisms will be greater than the Hawaiian polymorphisms at the position of the mutation . Therefore the mutated gene can be map.

I.2.2.1. Anatomy of C. elegans nervous system

The nervous system is the most complex organ of the worm. The adult hermaphrodites' nervous system is composed of 302 neurons and 56 glia. Contacts between neurons but also between neurons and glia, as well as with the muscles, are formed by synapses. The neurons are classified into 118 types based on their morphology and position [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF][START_REF] Altun | WormAtlas and WormImage: The Websites and the Book[END_REF]). Those neurons are then further defined as sensory neurons, interneurons and motor-neurons. [START_REF] Tsalik | Functional mapping of neurons that control locomotory behavior in Caenorhabditis elegans[END_REF]. The gene ttx-3 encodes a LIM homeodomain protein and is important for the functioning of the AIY neurons [START_REF] Hobert | Regulation of Interneuron Function in the C. elegans Thermoregulatory Pathway by the ttx-3 LIM Homeobox Gene[END_REF]). Among the defects observed, the number of body bends is reduced at 20°C, the number of reversal in 3 minutes is increased at 25°C but not at 15 and 20°C, while the number of thrashes in 1 minute is decreased at 25°C. They also travel less on a plate compared to wild type [START_REF] Tsalik | Functional mapping of neurons that control locomotory behavior in Caenorhabditis elegans[END_REF]. Using electron-microscopy, they can be recognized by the presence of a small electron-dense pre-synaptic density where vesicles are also present. The size of the synapses is variable and independent of the neuron type. In fact the same neurons can have synapses of different sizes.

I
However, no post-synaptic specializations are observed by electron microscopy, but labeling of post-synaptic receptors shows that they are clustered close to the pre-synaptic release zone.

C. elegans synapses, as with many organisms, release neurotransmitters including GABA (γ-Aminobutyric acid), acetylcholine, glutamate and monoamines [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF]).

Neuromuscular junctions are chemical synapses that form specifically between a motorneuron and a muscle cell, and show a specific organization. Indeed the muscle sends a process called a muscle arm. Usually, several muscle arms meet at a specialized zone on the motor neuron, next to a pre-synaptic bundle, to form a muscle plate. In this manner, multiple muscle cells can be stimulated simultaneously. c).

Electrical synapses, also called gap junctions, are established by cells in close contact.

They can be made between neurons but also between neuron and muscle cells or between muscle cells. They may be formed between axons but also between an axon and a soma, however they are more rare between two somata [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF][START_REF] Altun | WormAtlas and WormImage: The Websites and the Book[END_REF].

C. elegans nervous system is well suited to study the consequence of aging on its maintenance. Few studies already showed that the nervous system can change with aging [START_REF] Pan | Genetic analysis of age-dependent defects of the Caenorhabditis elegans touch receptor neurons[END_REF][START_REF] Toth | Neurite sprouting and synapse deterioration in the aging Caenorhabditis elegans nervous system[END_REF][START_REF] Chen | Neuronal aging: learning from C. elegans[END_REF][START_REF] Liu | Functional aging in the nervous system contributes to age-dependent motor activity decline in C. elegans[END_REF].

I.2.2.3. Aging of C. elegans nervous system

Aging of the C. elegans nervous system has mainly been studied in touch neurons, but Lesions, such as bubble-like, beading and blebbing, are thought to be different stages in the progression of aging phenotypes. Bubble-like lesions could be an early stage of axon splitting while beading and blebbing could be the consequence of axon transport decline.

Hypotheses have been made that neuronal branching could be the consequence of a decline in the nerve maintenance more than the promotion of sprouting, as regeneration after axotomy is impaired in aging animals [START_REF] Pan | Genetic analysis of age-dependent defects of the Caenorhabditis elegans touch receptor neurons[END_REF]. Moreover, the nerve attachment plays a role in the protection of the axon against the aging phenotypes. The axons of those neurons are normally embedded in the hypodermis. But in some mutants, attachment to the hypodermis is disrupted and this loss of embedding has been linked to an early degeneration of the nerve [START_REF] Pan | Genetic analysis of age-dependent defects of the Caenorhabditis elegans touch receptor neurons[END_REF]). It has also been shown that evoked activity is important for maintenance of the nerves, based on evidence that when the activity in the neurons is genetically reduced or abolished, nerve cells degenerate faster than in wild type worms [START_REF] Pan | Genetic analysis of age-dependent defects of the Caenorhabditis elegans touch receptor neurons[END_REF]. Evidence

shows that locomotion decline is due first, to reduction in neurotransmitter which starts to decrease in middle-aged worms, then second, to the deterioration of the muscles which occurs later in life. Indeed, the post-synaptic current (PSC), which is an indicator of neurotransmitter release, decreases as early as 7 days old in adults, while the contractibility of the muscle starts to decrease at 11 days of age [START_REF] Liu | Functional aging in the nervous system contributes to age-dependent motor activity decline in C. elegans[END_REF]. Other studies have shown that the NMJ has a decreased number of synaptic vesicles and synaptic vesicle fusion followed by defects in neurotransmitter content, synaptic vesicle priming, and docking [START_REF] Toth | Neurite sprouting and synapse deterioration in the aging Caenorhabditis elegans nervous system[END_REF].

Those different studies demonstrated that longevity genes have a role in neuronal decline as Visualizing the nervous system and its synapses has been a challenge for many years.

Several techniques have been developed to accomplish this in C. elegans. Historically, electron micrographs have been used to reconstruct the nervous system of C. elegans [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF]. Those electron micrographs allow us to visualize the pre-synaptic densities and to reconstruct the connectome, in other words determine precisely which neurons form synapses with other ones. Transmission electron microscopy (TEM) was used to generate the micrographs of C. elegans anatomy. TEM transmits an electron beam through the sample which has been sliced. Different tissues absorb the electrons in a different way allowing to visualize them, and to enhance those differences the sample is stained with heavy metals such as lead and uranium. The differences in absorbance of electrons by the sample are detected using a photographic film or a CCD camera [START_REF] Hall | Modern electron microscopy methods for C. elegans[END_REF]. Using these techniques, several samples have been reconstructed from overlapping images, with each sample corresponding to a part of a worm (hermaphrodite adult or L4, or male). Thus, the connectome of the worm was fully reconstructed and has been shared in the resource "Mind of the Worm" [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF]). The connectome is currently undergoing reconstruction using the same original micrographs, but this time with novel software to enhance the analysis by taking into consideration connection weight as well as other factors [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF][START_REF] Jarrell | The Connectome of a Decision-Making Neural Network[END_REF]). Fluorescent markers have now been developed to observe the synapses in vivo. The

I.3.1.2. Methods developed in other organisms

Studying the activity of the neuron historically has been carried out using electrophysiological approaches. However, it is now also possible to observe neuronal activity by calcium imaging using the Cameleon protein [START_REF] Miyawaki | Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin[END_REF]. This protein is sensitive to the entry of Ca Microfluidic devices can also be used to observe the formation and maintenance of synapses in cell culture by choosing the pre-and the post-synaptic partners [START_REF] Deleglise | Synaptoprotective drugs evaluation in reconstructed neuronal network[END_REF]). The device is made of three compartment chips, including two cell culture chambers connected by narrowing microchannels. Cells cultured in the chamber with the widest end of the microchannel can send their axon toward the cells cultured in the second chamber but the opposite is not possible, allowing for a controlled network to be created in vitro. The neuron and the synapses are visualized by fluorescent markers as described above.

In Drosophila a new technique has been developed to label synapses at an endogenous level of the protein. Indeed, in the techniques previously described, the markers are expressed from heterologous regulatory elements and sometimes from multi-copy transgenes. Therefore, the proteins may be overexpressed. In this new technique, called Synaptic Tagging with Recombination (STaR), genomic DNA was modified to tag the synaptic protein with a fluorophore, but in a cell specific manner. To do so, a stop codon flanked by FRT sites was inserted before the tag which is located at the end of the gene coding sequence. The FLP recombinase is expressed specifically in the cell of interest to excise the stop codon and generate the tagged protein. This technique can be used for pre-or post-synaptic specific proteins [START_REF] Chen | Cell-type-specific labeling of synapses in vivo through synaptic tagging with recombination[END_REF]). The observations of the full partnership are based on correlation between pre-and post-synaptic proteins.

I.3.2 Limitation of existing methods

As described above, several methods have been developed to visualize synapses but these techniques have some limitations. Electron microscopy gives static results; we observe the nervous system at one time point -when the worm has been fixed. Moreover, only a limited number of worms at different developmental stages have been reconstructed in this manner since the long process of reconstruction is prohibitive. Therefore, it is not feasible to compare the connectome within a population of worms. Fluorescent pre-and post-synaptic markers can

show specific connections between neurons, but they do not depend on the neuronal partner to be functional. A pre-and a post-synaptic marker can be expressed in the same strain to visualize both synaptic partners, but the conclusion relies on colocalization [START_REF] Nonet | Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions[END_REF]). The trans-synaptic method GRASP circumvents this problem [START_REF] Feinberg | GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems[END_REF]. While this technique was revolutionary, it may not be suitable for aging studies. This is because the interaction between the two fragments that reconstitute GFP is strong and may in fact be irreversible under some conditions [START_REF] Chen | Cell-type-specific labeling of synapses in vivo through synaptic tagging with recombination[END_REF]. Thus, GRASP might stabilize synapses and prevent them from dissociating. Moreover GRASP has been adapted with only a green fluorescent protein and does not for the moment existing in another color of fluorophore. It also has been reported that GRASP do not work for every neuronal connections. Knowledge of these limitations of the current visualization methods highlighted the need for a new visualization technique that would identify two neuronal partners (trans-synaptic) without stabilizing the synapses in order to observe the dynamics of the connections. To answer those criteria we decided to develop a new strategy by applying the method called BLINC (Biotin Labeling of INtercellular Contact) to studies of the nervous system in vivo in C. elegans.

I.4. BLINC in cell culture

The

I.5.1. Biotinylation reaction

Biotinylation is an enzymatic reaction where biotin, also known as vitamin H, is transferred onto a protein. BirA is an E. coli gene which encodes an acetyl-CoA carboxylase biotin holoenzyme synthetase [START_REF] Barker | The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase[END_REF]. Biotin is used as a coenzyme of acetyl-CoA carboxylase in E. coli. The reaction involved several steps which will form 1-Ncarboxybiotinyl carboxylase. The 1-N-carboxybiotinyl carboxylase will then incorporate carboxylate into the acceptor [START_REF] Samols | Evolutionary conservation among biotin enzymes[END_REF]). The acceptor can differ depending on the organism, but a 13 residue consensus peptide has been isolated as the target sequence for biotinylation [START_REF] Schatz | Use of peptide libraries to map the substrate specificity of a peptidemodifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli[END_REF]).

The biotinylation reaction has been used for several years to detect proteins in vitro thanks to the streptavidin affinity for biotin which can be used for detection [START_REF] Weber | Structural origins of high-affinity biotin binding to streptavidin[END_REF]. The tetramer structure of the streptavidin allows it to bind several biotin molecules [START_REF] Weber | Structural origins of high-affinity biotin binding to streptavidin[END_REF]. However it has been shown that this tetramer has limited application in vivo.

Indeed, when used to label receptors (VGLUT1) in neurons in culture, the tetramer has the capacity of aggregating the receptors as it binds the biotin present on each one of them.

Moreover those clusters of receptors did not colocalize with a post-synaptic marker, Neuroligin [START_REF] Howarth | Monovalent, reduced-size quantum dots for imaging receptors on living cells[END_REF]). Hence, great efforts have been made to generate streptavidin that does not form aggregates. One approach is to generate a tetrameric monovalent streptavidin. Engineered monovalent streptavidin tetramer consists of 3 "dead"

(non-functioning) subunits and 1 "alive" (functioning) subunit (Howarth et al. 2006) (Figure I-19a). In order to generate the "dead" subunit (D), 3 amino-acids have been mutated (Table 2) and a 6-his tag was added to the wild-type subunit, giving rise to the "alive" version (A) A second approach has been to generate a monomeric streptavidin using systematic mutagenesis. While initially unsatisfying because of drastic reduction in the affinity for biotin and the stability of the protein, recent work identified new mutations (Table 2) that give a 2-3 fold increased affinity of the streptavidin for biotin compared to previous examples. Moreover the stability of the protein is retained making the mutated streptavidin a good candidate for biotin detection [START_REF] Lim | Engineered streptavidin monomer and dimer with improved stability and function[END_REF].

I.6. Objectives

The development of BLINC in vivo in C. elegans will be carried out using transgenic animals with the same methodology used in cell culture. The use of a monomeric streptavidin is a good option for a detector with high affinity to biotin, but which does not aggregate and is easy to encode genetically. By adapting this technique, we hope to introduce a new tool to the C. elegans community for the functional study of synapses. More specifically, as this technique is dependent on two neuronal partners but should not disrupt the normal plasticity and elimination process of the synapse, it will allow aging and dynamic studies.

The aging process is still not fully understood, but it has been shown that synapses undergo modifications with age. The C. elegans nervous system is well suited to study the consequence of aging on its maintenance. Few studies already showed that the nervous system can change with age [START_REF] Pan | Genetic analysis of age-dependent defects of the Caenorhabditis elegans touch receptor neurons[END_REF][START_REF] Toth | Neurite sprouting and synapse deterioration in the aging Caenorhabditis elegans nervous system[END_REF][START_REF] Liu | Functional aging in the nervous system contributes to age-dependent motor activity decline in C. elegans[END_REF][START_REF] Chen | Neuronal aging: learning from C. elegans[END_REF].

These modifications though have not been well characterized. We hope that by using C.

elegans in vivo BLINC in addition to synaptic markers, we will be able to provide more insights into this process. Enlightenment of synaptic maintenance mechanisms and the nonpathological changes of the connections due to age will help understand the possible mechanisms which are impaired in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

Tab le 2 : M utatio ns car r ied b y strep ta vid in. Amino acid changes at different positions of the streptavidin sequence create the dead subunits for the monovalent streptavidin and for the monomeric streptavidin.

SphI and KpnI enzymes. The final plasmid contains the Pgcy-8 promoter fused with the signal peptide of pat-3 in frame with BirA fused with the nrx-1 cDNA. SphI and XmaI sites were used to exchange the Pgcy-8 promoter to the Pgcy-32 promoter [START_REF] Yu | Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors[END_REF]. All PCR amplified sequences were verified (Table 5 and7).

Tab le 5 : Deta ils o f the SPpat-3:: BirA::n rx -1 cons tr uc t The AFD-AIY GRASP specific plasmids were constructed by switching the Pflp-18 promoter of Pflp-18::nlg-1::spGFP 1-10 for the Pttx-3 promoter, and the Pshr-128 promoter in Pshr-128::nlg-1::spGFP 11 for the Pgcy-8 promoter using SphI and XmaI sites.

a line where the DNA injected follows the Mendelian's law, the transgene can be integrated.

In that goal, worms in the last larva stage which carry the extrachromome were irradiated

(5,000 rads, gamma rays) using a Shepherd Mark I Irradiator (Cesium 137). The irradiation induces breaks in the genomic DNA, which will be repaired. During that repair, the extrachromosome can be inserted in the genomic DNA. The selected lines are then backcrossed at least 4 times to eliminate the background mutations.

Analyses of iBLINC strains

Fluorometric and microscopic analyses

For epifluorescence microscopy, young adult animals were anaesthetized with 5mM levamisole or 0.3mM 2,3-butanedione monoxime (BDM) + 4mM levamisole and analyzed on an AxioImager Z1 compound microscope (Zeiss). In all images, the iBLINC signal was visualized using an exposure time of 1,000 ms at 630x magnification. The GABAergic neuromuscular junction iBLINC signal was imaged at 1,200 ms of exposure time. The cytoplasmic markers were visualized with 10 to 20 ms exposure time at 630x magnification.

Animals were optically sectioned using the Zeiss Apotome and the sections processed to produce maximum intensity projections in some cases. All images were processed subsequently with the Zeiss proprietary Axiovision software and assembled into figures using Adobe Photoshop® and Adobe Illustrator®.

Time-course analysis

To observe the signal at larval stage 1, an egg prep was performed and animals were allowed to develop without food over night. To image the signal during larva stages 2 to 4, an egg prep was performed. The animals were left developing on a plate containing OP50 and staged by gonad morphology. To observe the signal during adulthood, L4 worms were picked and incubated at 20°C for 1 to 15 days. Every two days the worms were transferred onto a fresh plate to avoid starvation and to eliminate the progeny. At relevant time points, animals were imaged as described above.

Analyses of synapse patterns and statistics

To compare the pattern of AFD-AIY synapses, the images of different genotypes were shuffled and blinded for analysis using ImageJ [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]). The staining patterns were isolated from background and worms were placed in the same orientation. Individual punctae were circled and then position, fluorescence intensity, and surface area of each puncta were extracted. Statistical analysis and plots were generated using R (R Development Core Team 2013). The average size of punctae was analyzed using an ANOVA test with a Bonferroni correction for pairwise comparisons.

'Pearl chain' graphs were created by arbitrarily placing punctae into four differently sized bins (0 to 13; 14 to 18; 19 to 25 and larger than 25 square pixels). Total synaptic length was defined by measuring the distance between punctae using a triangulation formula and summing the distances. Punctae were then aligned, and binned depending on quartiles of synaptic length, the first quartile being the most distal part of the process (in relation to the cell body) and the last one being the proximal part of the synaptic length. The number of punctae in each quartile was compared between ages and genotypes by employing a two-way ANOVA test using GraphPad® Prism (version 5.04 for Windows, GraphPad® Software, La Jolla California USA, www.graphpad.com). The analysis of one, three, six, or nine day old wild-type animals was performed using an integrated transgenic line (dzIs68: Is[Pgcy-8::BirA::nrx-1; Pttx-3::AP::nlg-1; Punc-122::streptavidin::tagRFP; Pelt-2::GFP]). The analysis of unc-104 mutant animals was performed using the parental extrachromosomal line dzEx1240.

Behavioral assays

Worms were grown as described at 20°C and assayed at 25°C [START_REF] Tsalik | Functional mapping of neurons that control locomotory behavior in Caenorhabditis elegans[END_REF].

All plates and solutions were equilibrated at 25°C before being used and all experiments were performed blindly. Worms of each genotype were picked the day before the assay at the L4 larval stage and placed on a plate with OP50 bacteria and incubated over night at 20°C. The day of the assay, worms were picked and placed on a plate without food to 'clean' worms from adhering food and then used as follows:

Radial locomotion: Ten worms were placed on the assay plate (lacking food) and their positions were marked on the plate at 5, 10 and 15 minutes. The distances were then measured and Multiple t-tests were performed using GraphPad® software package.

Thrashing assays: Worms were picked individually and placed in a drop of M9 buffer.

The number of thrashes in 1 minute were counted and analyzed with a Mann-Whitney test using GraphPad® software package.

and incubated at 20°C or temperature as indicated for 1 to 15 days. Every two days, the worms were transferred onto a fresh plate to avoid starvation and to eliminate the progeny. At relevant time points, animals were imaged as described below.

Fluorometric and microscopic analyses

For epifluorescence microscopy, young adult animals were anaesthetized with 5mM levamisole and analyzed on an AxioImager Z1 compound microscope (Zeiss). In all images, the juIs1 signal was visualized using an exposure time of 500 ms at 630x magnification.

Animals were optically sectioned using the Zeiss Apotome and the sections processed to produce maximum intensity projections. All images were processed subsequently with the Zeiss proprietary Axiovision software and assembled into figures using Adobe Photoshop® and Adobe Illustrator®.

Analyses of synapse patterns and statistical analyses

The fluorescence of the dorsal nerve cord was analyzed using ImageJ. A rectangle of 50 pixels wide and 300 pixels long was traced along the distal part of the dorsal nerve cord. A cross-profile along the width was obtained, and the background deducted from the "curve tail" by subtraction from each point along the curve. Normalized values were summed to obtain a total intensity for each genotype and age (Briseño-Roa and Bessereau 2014). The statistical analyses were then performed using a Mann-Whitney test with GraphPad® software package. connections -no or weak signal was observed, whereas worms in which the streptavidin was expressed in the intestine cells localized next to the head showed a stronger signal. This demonstrated that visualization of iBLINC is limited by the ability of secreted streptavidin::tagRFP to travel from its source. This can be overcome by expressing streptavidin::tagRFP in the coelomocytes that will secrete it into the pseudocoelom, which is a fluid-filled space where antibodies have been shown to travel and label the extracellular peripheral membrane [START_REF] Gottschalk | Visualization of integral and peripheral cell surface proteins in live Caenorhabditis elegans[END_REF][START_REF] Attreed | Direct visualization of specifically modified extracellular glycans in living animals[END_REF]. In this manner, the streptavidin should be able to travel throughout the whole body of the worm and recognize the biotinylation anywhere.

Chapter III: Results

III.1. A ne w tool to vis ualize synapses in vivo

To confirm the specificity of iBLINC, we co-expressed iBLINC with a cytoplasmic fluorescent marker for either AIY (Pttx-3::GFP) or AFD (Pgcy-8::GFP) neurons or with a synaptic marker of AIY (Pttx-3::GFP::RAB-3) (Figure III-4). First, we confirmed that iBLINC is juxtaposed with both cytoplasmic markers for AFD and AIY (Figure III-4a,b).

Then we analyzed the juxtaposition of iBLINC signal (post-synaptic site of AIY) and a marker for pre-synaptic specializations in AIY interneurons. They were adjacent to, but largely non-overlapping consistent with the expectation that pre-and post-synaptic sites in AIY neurons are adjacent but non-overlapping (Figure III-4c). This indicated that the iBLINC signal is well localized and probably labels the connections between AFD and AIY. suggest that our labeling approach can capture the directionality of synaptic connections.

Thus, iBLINC seems to work only when the proteins are expressed in the correct synaptic partner. To have a better insight on iBLINC advantages we decided to compare it with GRASP.

We expressed GRASP constructs in the same neurons, AFD and AIY, using the original plasmids of GRASP [START_REF] Feinberg | GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems[END_REF]) and the same promoters as for iBLINC. This last observation could be explained by the fact that part of the GFP present at the membrane is not reconstructed. It has been shown that the C-terminus-fragment can aggregate, in consequence this fragment can not to be part of a fully reconstructed GFP therefore less molecules are reconstituted so the total fluorescence is less strong [START_REF] Cabantous | Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein[END_REF][START_REF] Magliery | Detecting proteinprotein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism[END_REF][START_REF] Blakeley | Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo[END_REF]). Alternatively, one explanation could be that iBLINC is detected with tagRFP which is known to be brighter than GFP [START_REF] Shaner | Advances in fluorescent protein technology[END_REF]. In conclusion, iBLINC appeared more robust and brighter than the analogous GRASP signal . To show that NRX-1 and NGL-1 fusions were localized to synapses and to eliminate the possibility that cellular contact alone was sufficient to establish an iBLINC signal in vivo, we searched for a neuron that is in cellular contact with AIY neurons, but fails to establish synaptic connections. Volumetric reconstruction of an electron micrograph series [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF]

III.1.1.3. Using iBLINC for dynamic studies

To further explore whether iBLINC could lend itself to dynamic studies at the synapse, we fused the monomeric streptavidin detector to the photoconvertible fluorescent protein dendra2. The dendra2 protein changes fluorescent properties upon exposure to blue light, resulting in photo-conversion from green to red, allowing us to perform chase experiments in which 'old' signal (converted/red) can be distinguished from newly formed signal (green). We find that the dendra2 iBLINC signal can be quantitatively converted into red fluorescence, and that within less than one hour new green iBLINC signal appears, which colocalizes with red signal . Since the streptavidin -biotin interaction can be considered irreversible; this suggests that iBLINC could serve to investigate protein turn-over at specific synapses under different experimental conditions.

Moreover, we could observe vesicular signal in the cell body of the post-synaptic partner.

Suggesting that the fusion streptavidin::FP-biotin-AP-NLG-1 (FP for Fluorescent Protein) is internalized, thus implying that the interaction is not stabilized.

III.1.2. Labeling different type of neuronal connections using iBLINC

To test if iBLINC could be used in other types of neurons we expressed it in different classes of neurons: cholinergic and GABAergic motor-neurons, muscle cells and GABAergic motor-neurons which form neuromuscular junction (NMJ), and mechano-sensory and inter neurons.

III.1.2.1. Motor neuron synapses

Two with the muscle [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF]). We expressed the BirA::NRX-1 construct under the kal-1

promoter. kal-1 is known to be expressed in several neurons including cholinergic motorneurons [START_REF] Bülow | Heparan sulfate proteoglycan-dependent induction of axon branching and axon misrouting by the Kallmann syndrome gene kal-1[END_REF]. We specifically expressed the AP::NLG-1 construct in the Dtype motor-neurons using the promoter of one of the gene coding for a transmembrane vesicular GABA transporter, unc-47 (McIntire et al. 1997). Thus, the resulting strain may show a characteristic punctate signal that indicates the connections between the two classes of motor-neurons.

Several independent lines show this pattern. We can observe a punctuated signal at the nerve ring (NR) area, in the ventral (VNC) and dorsal nerve cord (DNC) .

Some commissures are also visible however the signal is not punctuated and faint. Some cell bodies are also observed with the possible internalized fusion AP::NLG-1 tagged by biotin and streptavidin.

Figure II I-11 : iBLINC has a d yna mic sta te. a-c Epifluorescent micrographs of the head region of transgenic animals expressing iBLINC in which the detector is tagged with the photoconvertible protein dendra2. Before photo-conversion, only green fluorescence is observed in the head (a). After 30 seconds exposure to blue light, green is no longer seen in the head while red appeared (b). After only one hour of recovery, green is observed again. The green and red streptavidin colocalization shows that new proteins are at the membrane (c). Adapted from Desbois et al. 2015

III.1.2.2. Neuromuscular junctions

We next tested if we could use iBLINC to visualize neuromuscular junctions. We expressed the BirA::NRX-1 pre-synaptic construct in the D-type motor-neurons using the unc-47 promoter and the AP::NLG-1 construct in the muscle using the muscle-specific myo-3 promoter (Figure . We could observe a punctuated signal in the VNC of the worm . This result being preliminary more experiments need to be done to confirm the pattern and the correct NMJ labeling.

III.1.2.3. Mechanosensory neuron and interneuron synapses

We also used iBLINC to label the mechanosensory neurons and interneurons as they form well known synapses. Those synapses were first used to test pre-synaptic labeling fusion synaptobrevin/SNB-1::GFP [START_REF] Nonet | Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions[END_REF]). The BirA::NRX-1 pre-synaptic construct is expressed in the mechanosensory neurons ALM, AVM, PVM, PLM and FLP using the mec-7

promoter [START_REF] Hamelin | The mec-7 beta-tubulin gene of Caenorhabditis elegans is expressed primarily in the touch receptor neurons[END_REF] One of the possible reasons of this absence of signal is the lack of endogenous neuroligin at the AVA post-synaptic sites [START_REF] Hunter | Neuroligin-deficient mutants of C. elegans have sensory processing deficits and are hypersensitive to oxidative stress and mercury toxicity[END_REF]. To possibly circumvent this problem and knowing that the synapses between PLM and AVA are glutamatergic, we envisioned the use of an accessory protein of the glutamate receptor, GLR-1. The glutamate receptor is dependent on accessory proteins for functionality, including SOL-2, a CUB domain protein [START_REF] Wang | Characterization of Auxiliary Subunits for GLR-1 AMPA Receptors in Caenorhabditis Elegans[END_REF][START_REF] Wang | The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors[END_REF]. Thus, a fusion of the acceptor peptide and sol-2 cDNA was cloned in order to try to make iBLINC specific to glutamate synapses. To this end the BirA::NRX-1 fusion was expressed in PLM among others while an AP::SOL-2 fusion was expressed in AVA among others using the same promoters previously described. But no signal was observed in those lines neither in the head or mid body. This can be explained by the fact that NRX-1 and SOL-2 might not be forming a complex as no experimental data ever described them as partners. In consequence, the enzyme could be too far from the acceptor peptide to be able to transfer biotin to it. f. Epifluorescence micrograph of a head of an animal expressing GRASP. g. Quantification of the transgenic animals showing an iBLINC or a GRASP signal in the head (black bar) or in the head and mid-body (squared pattern bar).

(Figure III-15)

III.1.3. Conclusions

In conclusion, we have developed iBLINC for live imaging of specific synaptic connections in C. elegans. The method is specific, dynamic, directional and brighter than existing methods and the observed signal correlates well with electron microscopy studies of connectivity in C. elegans. This suggests that the labeling method could serve as a useful tool to investigate synaptic connectivity and dynamics under different experimental conditions in nematodes.

III.2. Study of the synapses during aging in C. elegans

The nervous system is adaptive and changes with time. It has been shown that synaptic ultra-structure varies with aging, e.g. the pre-synaptic density is smaller in old animals [START_REF] Toth | Neurite sprouting and synapse deterioration in the aging Caenorhabditis elegans nervous system[END_REF]. Here using two different labeling systems in two different neuronal types, iBLINC in AFD-AIY connections and synaptic vesicle marker in GABAergic neurons, we investigate if an elimination of the synapses can be observed. In addition we will determine if a variation of the number of synaptic vesicles occurs during the aging process.

III.2.1. Progression of AFD-AIY connections with age

The iBLINC signal between AFD and AIY interneurons is visible as early as the first larval stage and continued to be visible throughout life as far as fifteen day old adult worms

(Figure III-16a-c).
To study more thoroughly the synapses made between AFD and AIY we used the same analytical method as described in the first part of Chapter III (III.1.), to quantify the iBLINC signal at different ages. First we determined if the pattern of synapses was different between the left and the right processes. For that, we quantified the number of synapses for each process in one day old (do) adult worms. We found an average of 24.5 ± 1.7 SEM (standard error of the mean) (N=13)

synapses between the left AFD and AIY and 24.0 ± 1.3 (N=14) between the right processes, which is not significantly different . Seeing as we did not observe any obvious bias for the left or right side of the animal we do not need to distinguish between the processes. We can also conclude that AFD-AIY connections do not show any asymmetry. In C. elegans most of the neurons are present as pairs, one being on the left and one on the right.

According to the reconstruction of electron micrographs, most of the neurons do not show obvious asymmetry, and the left and the right neurons make the same amount of contacts at the same strength [START_REF] Durbin | Studies on the development and organisation of the nervous system of Caernorhabditis elegans[END_REF]. However asymmetric gene expression patterns have been observed in bilateral pairs of neurons that correlate with functional differences. For example, the ASE left (ASEL) and right (ASER) neurons show different expression of guanylyl cyclase genes [START_REF] Yu | Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors[END_REF][START_REF] Hobert | The Caenorhabditis elegans lim-6 LIM homeobox gene regulates neurite outgrowth and function of particular GABAergic neurons[END_REF][START_REF] Pierce-Shimomura | The homeobox gene lim-6 is required for distinct chemosensory representations in C. elegans[END_REF]. ASEL expresses the genes gcy-6 and gcy-7 among others, whereas ASER expresses gcy-5 among others [START_REF] Yu | Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors[END_REF]. This asymmetric expression is important for the recognition of different odors. Thus it will be interesting to determine whether these differences are also reflected in asymmetric connectivity of ASE neurons.

We next quantified the number and size of synapses at different ages in order to see if a change could be observed. At the L4 larval stage we found an average of 18.4 ± 0.8 SEM (N=16) synapses per side of the animal which increased to 24.8 ± 1.6 SEM (N=16) punctae in one day old adults . However the size of the synapses is consistent between those two time points. Consistent with a higher number and an unchanged average size of synapses, the total synaptic area increased during early adulthood from 360.2 ± 20.4 at L4 to placed into four different groups: 0 to 13 pixel 2 , 14 to 18 pixel 2 , 19 to 25 pixel 2 , and higher than 25 pixel 2 (px 2 ).

The pearl chains revealed no obvious regularity of puncta between animals at the L4 larval stage, or as one, three, six or nine day old adults . To better visualize the correlation between the positioning and the size of the synapses, we decided to analyze the distribution of the synapses in each percentile depending on their size. We used the same size and percentiles groups as for the pearl chain and calculated the number of synapses of each size depending of their percentile group . We can observe that only one day old adults have significant differences in the number of synapses between size groups in several percentiles. Indeed in the 1 st , 3 rd and last percentile the number of small synapses (0 to 13 px 2 ) is higher than the other sizes . We can observe the same tendency for the third percentile at the 4 th larval stage where there is significantly more small synapses that big ones (19 to 25 px 2 and larger than 25 px 2 ) (Figure . In the case of 6 d-o adult worms we can observe significantly bigger synapses (19 to 25 px 2 and greater than 25 px 2 ) than small synapses (0 to 13 px 2 and 14 to 18 px 2 ) in the first percentile . At the other ages the distribution of the number of synapses regarding their size is equal, no size is more or less represented than the others (Figure f). In conclusion, we did not observe a distinguishable pattern of synapses regarding their size, although they shared a similar number of synaptic connections across the different quartiles.

Then, we wanted to explore whether specific areas of the synaptic connection behaved differently during aging. To this end, we quantified the number of synapses in each quartile We observed dynamic changes in some areas of the connection, whereas others remained relatively unaffected over time . Indeed, the distal part of the process seems to be the most affected as a decline is observed as early as 3 d-o (50 to 75% and 75 to 100% of the synaptic length). These portions are also the only affected ones between the L4 stage and 1 d-o adult where a significant increase of synapse number can be seen. The distal part of the process (0 to 25% of the synaptic length) also shows a decline after 3 d-o adults. However, the region 25 to 50% is constant throughout life.

98

Regarding the difference of number of synapses of each size during aging, we can observe that the number of the small synapses (0 to 13 pixels 2 and 14 to 18 pixels 2 ) varies the most The variation of the number of synapses during aging seems to be due to the variation of the number of the smaller synapses. One possible explanation would be that bigger synapses are stronger so they are less likely to be eliminated.

In conclusion, at the same age, the distribution of the synapses does not seem to follow a pattern but instead it seems that each part of the process manages to balance the number of synapses depending on their sizes. In this manner, the overall synaptic area is maintained even though the pattern of connectivity differs from animal to animal. These findings showing portion-dependent changes are consistent with the notion that dedicated maintenance mechanisms exist to ensure appropriate conservation of synaptic connections and overall connectivity strength [START_REF] Chia | Cell biology in neuroscience: cellular and molecular mechanisms underlying presynapse formation[END_REF]. This leads us to believe that dedicated mechanisms for synapse formation and distribution may exist. As for example PLX-1 which seem to be important for synaptic field restriction of two motor-neurons in the tail [START_REF] Mizumoto | Interaxonal interaction defines tiled presynaptic innervation in C. elegans[END_REF]. Moreover, we can observe a decline of the number of the AFD-AIY synapses with aging. The smaller synapses seem to be eliminated fist.

It has been described in the literature that the number of vesicles decreases with age [START_REF] Toth | Neurite sprouting and synapse deterioration in the aging Caenorhabditis elegans nervous system[END_REF]. So in order to know if we could observe in vivo the same phenomenon and if it was correlated with the decrease of synapses seen with iBLINC, we used a second method and followed a synaptic vesicle marker of the motor-neurons.

III.2.2. Changes of the vesicles number in motor-neurons

Motor-neurons come in two flavors; inhibitory and excitatory. In C. elegans, the inhibitory neurons (D-type) release GABA neurotransmitter while the excitatory neurons (A-and Btype) release acetylcholine (WormAtlas et al.). The cell bodies of the motor-neuron are localized in the ventral nerve cord (VNC) of the worm. They form synapses (neuromuscular junction, NMJ) with the muscle at the VNC but they also send commissures across the animal to form synapses with the muscles at the dorsal nerve cord (DNC). One of the first visible consequences of aging in the worm is the decrease of locomotion. It has been shown that this decline is primarily due to a reduction in the activity of the motor-neurons [START_REF] Liu | Functional aging in the nervous system contributes to age-dependent motor activity decline in C. elegans[END_REF]), so we wanted to investigate if this could be visualized by observing a pre-synaptic At 15°C, the animals have a longer life span than at 20°C [START_REF] Byerly | The life cycle of the nematode Caenorhabditis elegans[END_REF]), so we expected to see a decline later than the one seem at 20°C. Indeed, the differences seen So even though the worms were cultured at the same temperature during adulthood, the temperature at which they were cultured during development appears to affect differently the decline that occurs at older age. Those results suggest a developmental and/or longevity effect on aging.

Those observations lead to the conclusion that the synaptic aging phenotype can be modified by exposure to different temperatures during development, and that this effect on the nervous system decline persists even after several days at a new temperature.

III.2.2.2. Longevity mutants shows developmental defects

To help understand the normal aging of the nervous system, we decided to test two The second longevity model that we tested is the caloric restriction. It has been shown in C. elegans that dietary restriction can increase the life span of the animals [START_REF] Kaeberlein | Lifespan extension in Caenorhabditis elegans by complete removal of food[END_REF]. We observed the fluorescence of one of the mutants of that pathway. The gene eat-2 encodes a pharyngeal nicotinic acetylcholine (nACh) receptor [START_REF] Avery | The genetics of feeding in Caenorhabditis elegans[END_REF][START_REF] Lakowski | The genetics of caloric restriction in Caenorhabditis elegans[END_REF]. Mutants of eat-2 have a decreased rate of pharyngeal pumping which causes a decrease in food intake. The mutants show an increased life span. We tested if the eat-2 These observations support the concept that longevity is not the reason for the differences observed among the wild-type animals grown at different temperatures. Or at least, the decline of the synaptic marker is not dependent on the insulin pathway or caloric intake. However we cannot rule out the possibility that the decline seen in eat-2 mutants is similar to the wild-type one until 12 day old but then is maintain later on raising the possibility than caloric restriction plays a role later in life. In the same way it is possible than daf-2 have a mild effect on synaptic maintenance as it does not show any decline. Nevertheless conditions during development could still have a critical effect on the aging of the nervous system. Moreover, we were surprised that longevity mutants show early defects.

mutant
III.2.2.3. Mutants of genes implicated in the development of the nervous system show mild defects.

We then decided to test if genes known to be involved in the development of the nervous system are important for synapse formation and/or aging of the nervous system.

CAM-1 is a receptor tyrosine kinase (RTK) of the immunoglobulin superfamily [START_REF] Koga | Control of DAF-7 TGF-(alpha) expression and neuronal process development by a receptor tyrosine kinase KIN-8 in Caenorhabditis elegans[END_REF]) that localizes to muscle arms during embryogenesis. It is the ortholog of human PXL-1 and PLX-2 are plexin orthologs that are predicted to encode semaphorin receptors [START_REF] Ikegami | Semaphorin and Eph receptor signaling guide a series of cell movements for ventral enclosure in C. elegans[END_REF]. plx-1 is reported to be expressed in muscles and some of the hypodermal cells while plx-2 seems to be expressed in a subset of neurons. plx-2 has been shown to be important for axon guidance [START_REF] Wang | The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2-mediated axon guidance[END_REF]. plx-1(nc37) mutants do not enf-4 does not seem to be implicated in the maintenance of the GABAergic vesicle pool.

KAL-1 is a cell surface protein with a WAP-type protease inhibitor domain and type III fibronectin domains. It is required for axon branching in some neurons [START_REF] Bülow | Heparan sulfate proteoglycan-dependent induction of axon branching and axon misrouting by the Kallmann syndrome gene kal-1[END_REF].

kal-1(gb503) mutants show a significant decrease between 1 and 4 d-o at 25°C, but this seems to be due to a high fluorescent intensity at 1 d-o, during which time they are significantly more fluorescent than the wild-type even though no differences are seen at 4 d-o (Figure d). kal-1 might have an effect on the early regulation of the vesicle pool.

110

In conclusion, it seems that the genes involved in nervous system development have a mild effect on the changes of the vesicle marker that occurs over time. Moreover it seems that there is more variability at 20°C compared to 25°C, as well as at younger ages. The preliminary data of the changes in the vesicle pool imply that some genes known to be involved in neuron development could also have a role in the maintenance of the number of vesicles in the GABAergic motor-neurons. The effect seen in plx-2 mutants might not involve efn-4 since we have shown that efn-4 mutants do not have any phenotype, but more studies need to be done.

III.2.3 Conclusions

These findings demonstrate that not only does the vesicle pool in pre-synaptic terminals decrease with age in vivo as previously described by using electron micrographs [START_REF] Toth | Neurite sprouting and synapse deterioration in the aging Caenorhabditis elegans nervous system[END_REF], but the number of synapses also diminishes. The decline of the number of synapses seems to be compensated by an increase in the size of the remaining synapses. The synaptic aging phenotype can be modified by mutations in neuron development genes. Moreover, no obvious protective effects are observed in longevity models based on insulin pathway or caloric intake mutants.

Chapter IV: Discussion iBLINC a ne w technique to visualize synapses in live ne matode

In the first part of this work, I described a novel technique, iBLINC, developed to visualize synapses in vivo in C. elegans. This method has the advantage of being dependent on two neuronal partners to give a signal (trans-synaptic). It is directional and specific so only the synapses of interest are labeled. Indeed the signal is observed only when the constructs are expressed in the right synaptic partner and is dependent on the formation of the synapses. In fact when expressed in two neurons which are in physical contact but do not form synapses, no signal is observed. This technique is also brighter and more robust than the currently available trans-synaptic markers. Moreover, iBLINC is dynamic, allowing protein turnover studies to be carried out, based on the observance of new signal in less than one hour after photo-conversion. The turnover state also argues in the favor of the non trapping characteristic of the iBLINC. This characteristic is important as it does not stabilize the synapses so their dynamic life can be assessed. This has been reinforced by the observation of vesicular structures in the post-synaptic neuron, which could be the complex streptavidin::FP-Biotin-AP::NLG-1 which has been internalized. This characteristic of iBLINC is important as the stabilization of the synapse by the method used to label them is a great concern. Moreover, the functionality of the synapse does not seem to be disrupted so the signal does not seem to alter the connections.

We used iBLINC to label different connections but we extensively studied the connections between AFD and AIY. We showed that iBLINC specifically labels connections when AFD is pre-synaptic to AIY. The iBLINC signal colocalized with both cytoplasmic marker of AFD and AIY and is also adjacent but does not completely overlap with a pre-synaptic marker of AIY, demonstrating that only post-synaptic site of AIY are labeled when connected to AFD.

Moreover the iBLINC signal corresponds to the electron micrograph reconstruction.

Importantly we showed that iBLINC dotted signal corresponds to synapses. Indeed, as described in the literature we observed less synapses and a smaller total synaptic area in the kinesin/UNC-104 mutant compared to the wild-type [START_REF] Hall | Kinesin-Related Gene unc-104 Is Required for Axonal Transport of Synaptic Vesicles in C . elegans[END_REF]. However those remaining punctae/synapses have similar sizes than the wild-type. Surprisingly we observed a bias in the distribution of the remaining synapses toward the proximal part of the process (closer to the cell body). We can hypothesize than UNC-104 has other function(s) than anterograde transport, or that other mechanisms could be implicated in the distribution of the synapses. It could be possible that dedicated mechanisms ensure that synapses are present all along the process and those pathways guarantee this distribution even when the transport of synaptic components is affected. The possibility of active mechanism is emphasized as we could observe a constant number of synapse in each percentile of the process.

Possible modifications and adaptations for iBLINC

One possible modification of iBLINC would be to create lines with endogenous levels of neurexin and/or neuroligin fusions. By doing so, the technique will be a better representation of the wild-type localization and morphology of the synapses. The development of CRISPR Cas9 associated with Cre and Flip recombinase systems allows us to design that experiment [START_REF] Davis | Gene activation using FLP recombinase in C. elegans[END_REF][START_REF] Dickinson | Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination[END_REF][START_REF] Hubbard | FLP/FRT and Cre/lox recombination technology in C. elegans[END_REF].

Two types of strains could be possible: the first one would be based on the use of Cre and Flip recombinase . In that strain a knock in of the acceptor peptide and the biotin ligase at the locus of neuroligin and neurexin, respectively will be done by using CRISPR. The ligase BirA would be introduced in between the endogenous signal peptide (SP) of neurexin and the first neurexin exon but it would be preceded by a spliced acceptor site (SAS) which would be surrounded by loxP sites . As for the AP, it would be introduced in between the endogenous signal peptide of neuroligin and the first neuroligin exon but it would be preceded by a SAS which would be surrounded by FRT site. A Cre recombinase and Flip recombinase would be expressed specifically in the pre-synaptic neuron of interest and in the post-synaptic partner, respectively using specific promoters (Figure IV-1a). The streptavidin::FP construct would express from the coelomocytes and be integrated in the genome or used as an extrachromosome array.

The second strain would be based on the use of Cre and injection of a plasmid . In that strain, a knock in of the acceptor peptide at the locus of neuroligin would be done by using CRISPR. The AP would be introduced in between the endogenous signal peptide of neuroligin and the first neuroligin exon but it would be present after a SAS which would be surrounded by loxP sites. A Cre recombinase would be expressed specifically in the postsynaptic neuron of interest. As for the ligase it would be injected and present as an extrachromosome with the possibility of integration. This strain would result in an endogenous level of neuroligin but an over expression of neurexin (Figure IV-1b). The streptavidin::FP construct would be integrated in the genome or as extrachromosome and expressed by the coelomocytes as well. The level of the neuroligin is the most crucial one as it is the neuroligin which is labeled by the biotin and the streptavidin. So if neuroligin has an endogenous level of expression it will be representative of the "normal" state of the synapse.

117

In theory, iBLINC could be modified to be used with any molecules that form a complex at the synapse. We could use, for example as in GRASP, the CD4 and PTP-3A protein instead of NRX-1 and NLG-1 for chemical synapses [START_REF] Feinberg | GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems[END_REF]. CD4 is a human T cell protein which is not present in C. elegans [START_REF] Robey | CD4: Collaborator in immune recognition and HIV infection[END_REF] while PTP-3A is a member of the LAR/receptor tyrosine phosphatase family and is comprised of extracellular Ig repeats and fibronectin type III repeats. PTP-3A is localized to pre-synaptic terminals in C. elegans [START_REF] Ackley | The two isoforms of the Caenorhabditis elegans leukocyte-common antigen related receptor tyrosine phosphatase PTP-3 function independently in axon guidance and synapse formation[END_REF]. We can also imagine using any trans-synaptic protein complex which is specific to a subtype of neurons thus making iBLINC highly specific to types of synapse. The homolog of the mammalian pannexin, innexin, could be used to label gap junction. Indeed, those proteins form a channel at both membranes of each electrical partner and form a complex [START_REF] Starich | eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling[END_REF] [START_REF] Liu | Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation[END_REF]). An E. coli lipoic acid ligase (LplA) transfers lipoic acid to a recognition peptide (the Ligase Acceptor Peptide, or LAP). Lipoic acid can be recognized by using antibodies on fixed animals. To avoid the use of lipoic acid which has been shown to modify the worm life span [START_REF] Brown | Beneficial effects of natural antioxidants EGCG and alpha-lipoic acid on life span and age-dependent behavioral declines in Caenorhabditis elegans[END_REF], an engineered LplA with a modified active site [START_REF] Uttamapinant | A fluorophore ligase for site-specific protein labeling inside living cells[END_REF][START_REF] Liu | Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation[END_REF]) can be used. This engineered LplA can transfer a fluorophore on the LAP instead of lipoic acid in consequence the labeling would be possible in vivo. We can envision expressing specifically a LpLAneurexin fusion in the pre-synaptic marker and a fusion LAP-neuroligin in the post-synaptic partner. A fluorophore would be secreted in the extracellular matrix of the worm and be available for transfer to the LAP by the LplA.

Synapses are dynamic and change with age

In this thesis, I also described the changes of two different synaptic markers during development: a pre-synaptic marker of GABAergic neurons and iBLINC in AFD-AIY.

We assessed the effect of wild type aging by studying the connections between AFD and AIY using iBLINC by comparing the number, the size and the distribution of the synapses at different ages. An increase followed by a decrease of the number of synapses is observed before and after 1 day old, respectively. However the decreased number goes with an increase in the size of the synapses. Not surprisingly we observed that the smaller synapses are the ones which are primarily eliminated. Those results show that dynamics mechanisms regulate the synapses during life of the animal with an increase of synapse formation between L4 and adulthood. Those synapses are then regulated and pruned possibly to answer to the needs of the worm dependent on their environment. The connectivity seems to undergo a significant expansion during early adulthood, followed by a consolidation phase and eventual decline. Similar observations of decline have also been made in the aging human brain [START_REF] Bertoni-Freddari | Morphological adaptive response of the synaptic junctional zones in the human dentate gyrus during aging and Alzheimer's disease[END_REF] suggesting that the process is conserved throughout evolution.

The GABAergic pre-synaptic marker allowed us to observe temperature dependent changes of the synaptic vesicle pool. A decline is observed at each temperature but the age at which the decline starts is different. Moreover we showed that the decline is dependent on the temperature at which the animals develop before adulthood. It seems that the decline of the fluorescence of the switch population follows the same pattern than the fluorescence of the population cultured only at the lowest temperature (which corresponds to the developmental one in the switch population). Two criteria seem to be important for the maintenance of the synaptic vesicle pool: (1) the life span of the animals and ( 2) the temperature at which the animals develop.

We also observed important fluorescence variability in the population. Interestingly, the variability is more pronounced at lower temperature (15°C and 20°C) than at higher temperature (25°C). But it is also more variable during adulthood (1 to 8 day old). We could also observe variability of the number and size of synapses in the population with iBLINC.

The variability seen in the population goes with the argument that environment could be important. Indeed even though the worms are treated in the same condition the modulation of their nervous system could be influenced by 'life history" as their movement on the plate, how long they spent their time on and off food, and if they encounter other worms or not in the plate. The absence of a reproducible synaptic pattern between AFD and AIY (at least regarding their size) could fit into this hypothesis. It could also be explained by a lack of need for the thermosensory response of a specific synaptic pattern. In other words, for the circuit to work, the amount of synapse, the overall strength and the establishment of the synaptic field could be more important parameters than the exact location and series of the synapses. As the number and strength change with age this hypothesis seems to be possible, however the possibility of a random distribution of the synapses cannot be ruled out at this point in the study.

To our surprise, the two longevity pathways tested, insulin and caloric restriction do not seem to protect the synaptic vesicle pool. Mutants of those pathways in fact show abnormal development. Even though no decline is observed with age in daf-2 mutants, they actually have less synaptic labeling than the wild-type at 1 and 4 day old at the non permissive temperature. We can hypothesis than DAF-2 has a role in the early increase of vesicle number. eat-2 mutants show a decrease in fluorescence after 4 day old but 1 and 4 day old animals are statistically different from the wild-type.

Some mutants involved in nervous system development show a temperature dependent phenotype. plx-2 has significantly less fluorescence at 1 day old at 25°C but not at 20°C. However an increase is observed at 4 day old at 20°C but not at 25°C compared to the wildtype. In the case of plx-1, no decline is observed between 1 and 4 day old at any temperatures but they have a higher fluorescent value than the wild-type.

Future directions

A forward genetic screen using iBLINC would help uncover molecules implicated in synapse maintenance. The screen would focus on mutants which show a normal developmental pattern but an abnormal aging phenotype. For example, an early or late decrease in synaptic strength (size) or number of synapses or a modification of the synaptic field. iBLINC could also be used to screen for molecules involved in the development of the synapses. In that case mutants showing a defect during early developmental stages would be selected. It could be also an abnormal patterning with synapses localized closer or further away compared to the cell body than in the wild type.

A reverse genetic screen, either candidate based or not, could also be performed to find maintenance mutants using interference RNA (RNAi) mediated gene knock-down. Worm would be fed bacteria carrying the double stranded DNA of the gene of interest at a late stage (L4) of development or early adulthood. Few days later we could then observe if the knock down induces modifications of the synaptic patterns, size, and/or the number of synapses. If a phenotype is observed, we could conclude that the gene plays a potential role in synaptic maintenance.

A candidate screen using known null alleles could also help understand the underlying mechanisms involved in the maintenance of the connectome. One of the genes that could be tested is cima-1. cima-1 is one of the only genes known to be involve in synaptic patterning maintenance in C. elegans [START_REF] Shao | Synapse location during growth depends on glia location[END_REF]. CIMA-1 is homologous to SLC17 family of solute carrier transporters, a lysosomal transporter, it contains 12 transmembrane domains.

The mutant shows ectopic AIY synapses shown by labeling a pre-synaptic protein in the AIY neurons. It has a role in the maintenance of the morphology of a glia cell which is in close proximity to AIY [START_REF] Shao | Synapse location during growth depends on glia location[END_REF]. cima-1 by inhibiting egl-15, a FGF receptor, has a clear role in the distribution of the pre-synaptic terminal of AIY. It would be interesting to see if a mutant of cima-1 affects the synapses made with only one specific partner or all synapses regardless of the post-synaptic partners. To answer the question, iBLINC will be specifically expressed in AIY and its known post-synaptic partners. In that goal, the fusion BirA::nrx-1 would be expressed in AIY and the fusion AP::nlg-1 would be present in the post-synaptic partner (e.g. AIZ, AIB, RIA [START_REF] White | The Structure of the Nervous System of the Nematode Caenorhabditis elegans[END_REF]). The pattern of the synapses would be compared between adult wild type worms and adult cima-1 mutant worms. Moreover it would be interesting to know if it also affects the post-synaptic terminal of AIY. To answer this, iBLINC will be specifically expressed in AIY and its known pre-synaptic partners as for example AFD. In the same way wild-type adult worms and mutant adult worms will be compared. Another set of genes that would be interesting to test are those coding for the core proteins of the heparan sulfate proteoglycans (HSPG) and the heparan sulfate modifying enzymes (HSME). Heparan sulfates (HS) are glycosaminoglycans (GAG). The HS chain is attached to a core protein by specific serine residues to form HSPG and can be modified by the HSME.

The HSPGs are a major components of the extracellular matrix (ECM) and play a role in the diffusion of factors, such as morphogens and growth factors which have been implicated in different development processes [START_REF] Yamaguchi | Heparan sulfate proteoglycans in the nervous system: their diverse roles in neurogenesis, axon guidance, and synaptogenesis[END_REF][START_REF] Bülow | The molecular diversity of glycosaminoglycans shapes animal development[END_REF][START_REF] Bishop | Heparan sulphate proteoglycans fine-tune mammalian physiology[END_REF]). The HSPGs have been shown to be involved in axon guidance and branching formation [START_REF] Bülow | The molecular diversity of glycosaminoglycans shapes animal development[END_REF][START_REF] Tecle | Distinct 3-O-sulfated heparan sulfate modification patterns are required for kal-1-dependent neurite branching in a contextdependent manner in Caenorhabditis elegans[END_REF]. It would be interesting to see if they are also implicated in the formation of synapses. They are good candidates as they are supposed to bind a variety of molecules implicated in different processes of development. The core protein glypican has been shown to be the receptor a leucine-rich repeat transmembrane neuronal protein (LRRTM4) in mammals. Their interaction is HS-dependent. Their transsynaptic interaction is involved in the formation of excitatory synapses [START_REF] De Wit | Unbiased discovery of glypican as a receptor for LRRTM4 in regulating excitatory synapse development[END_REF].

Those results reinforce the idea that HS have a role in synaptic formation and maintenance. To study deeper the changes of the synapses it would be interesting to follow single worms during their life instead of a population. Microfluidic devices can make this possible by restraining the worm mechanically instead of chemically [START_REF] Chokshi | An automated microfluidic platform for calcium imaging of chemosensory neurons in Caenorhabditis elegans[END_REF][START_REF] Chronis | Worm chips: microtools for C. elegans biology[END_REF]. Chemical drugs allow us to immobilize the worms for few hours at most and it usually leads to their death. In the microfluidic device, the worm would be immobilized at L4 until its death by restraining its movements. Time lapse images would be taken to follow the dynamic of the synapses. Environmental cues could also be tightly controlled, such as temperature and amount of food. This comparison of individual worms in addition to population studies could give insight on synapse evolution over time, development and aging.

V.2. Annex II

In this annex, I am presenting different projects in the lab in which I have been involved in and their respective papers.

Direct vis ualization of specifically modified extracellular glycans in living animals

Early in development the cells must make choices such as differentiation or migration [START_REF] Rogers | Morphogen Gradients: From Generation to Interpretation[END_REF]. Those decisions rely on cues like morphogens and growth factors which are diffusible molecules. The diffusion of those factors is regulated by molecules of the extracellular environment. Heparan sulfate (HS) are glycosaminoglycans (GAG) and a major component of the extracellular matrix (ECM). HS are known to be important for the diffusion of some of the factors as morphogens, growth factors and others [START_REF] Yamaguchi | Heparan sulfate proteoglycans in the nervous system: their diverse roles in neurogenesis, axon guidance, and synaptogenesis[END_REF][START_REF] Bülow | The molecular diversity of glycosaminoglycans shapes animal development[END_REF][START_REF] Bishop | Heparan sulphate proteoglycans fine-tune mammalian physiology[END_REF]). The HS GAG is a long and linear polysaccharide chain, it is found in all metazoans and was shown to be important for development [START_REF] Bülow | The molecular diversity of glycosaminoglycans shapes animal development[END_REF]. This HS chain is attached to a core protein by specific serine residues to create a heparan sulfate proteoglycan (HSPG). HSPGs can be found in 3 different forms:

transmembrane, such as syndecan, GPI-anchored, such as glypican, or secreted, such as perlecan (Figure V-1a) [START_REF] Sarrazin | Heparan sulfate proteoglycans[END_REF].

HSPGs are essential for most organisms as mutations in one or several of the genes encoding the core proteins or the enzymes responsible for HS GAG chains assembly are lethal. This lethality as well as the other defects observed could be due to disruption of the diffusion of morphogens and other factors [START_REF] Nybakken | Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila[END_REF]. In addition to being important for viability, HSGPs and HS GAGs are important for the development of organs like the nervous systems which also relies on their ability to interact with molecules in the ECM [START_REF] Bülow | The molecular diversity of glycosaminoglycans shapes animal development[END_REF][START_REF] Tecle | Distinct 3-O-sulfated heparan sulfate modification patterns are required for kal-1-dependent neurite branching in a contextdependent manner in Caenorhabditis elegans[END_REF].

HS GAGs can be modified by several HS modifying enzymes (HSME). Those modifications are variable and can give rise to regions of lower or higher amounts of modifications . These regions are differentiated by the state of the glucosamine within the region: N-acetylglucosamine (NA), N-sulfoglucosamine (NS) or NA/NS, for regions composed of both glucosamines [START_REF] Lindahl | Regulated Diversity of Heparan Sulfate[END_REF]). The separation of the HS in specific combinations of modifications of 4 to 12 saccharides which provide bindings sites for proteins can also be used to designate the regions.

Minimal HS-binding domains for few proteins have been discovered as the FGF binding domain . Different FGF proteins require different domains. Indeed FGF-1 requires a fully modified disaccharide unit containing iduronic acid with 2-O-sulfation and Nsulfoglucosamine with 3-O-and 6-O-sulfation [START_REF] Kreuger | Characterization of fibroblast growth factor 1 binding heparan sulfate domain[END_REF] while FGF-2 does not require this fully modified disaccharide, but rather, requires a 2-O-sulfated iduronic acid [START_REF] Maccarana | Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor[END_REF]. In contrast endostatin, a vascular growth inhibitor, recognizes preferentially a HS fragment of 10 to 12 saccharides comprised of two NS domains separated by one or more N-acetylglucosamines [START_REF] Kreuger | Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function[END_REF].

Those domains which allow the interaction between the HS GAG and the proteins in the ECM are clearly important for the development of the organism. However these interactions as well as the synthesis by the cell of the HS GAGs and modification are not well understood. This is partially due to the fact that no method allows their visualization in vivo. In this work

5«

  Fait de ta vie un rêve et d'un rêve, une réalité. » Antoine de Saint-Exupéry « Pour ce qui est de l'avenir, il ne s'agit pas de le prévoir mais de le rendre possible.» Antoine de Saint-Exupéry 6 Acknowledge ments First I would like to thanks the committee members, Dr. Isabelle Dusart, Dr. Chantal Mathis, Pr. Jean-Louis Bessereau and Dr. Thomas Boulin. I am honored that you accepted to participate in my final examination.

  impliquer vivre loin. Merci d'avoir encouragé ma curiosité et d'avoir accepté de subir mes conversations et mon enthousiasme des sciences. Merci à mes soeurs pour être là pour moi et aussi pour avoir eu d'aussi beaux, parfait et adorable garçons, des neveux de rêve . Merci a mes grands-parents d'avoir cru en moi. Sans vous -ma famille-votre soutien, votre présence et votre confiance en moi, cette thèse n'aurait pu être possible. Elle vous est entièrement dédiée. show mild defects. ...................................................................................................... III.2.3 Conclusions ............................................................................................... Chapter IV: Discussion ........................................................................................................ iBLINC a new technique to visualize synapses in live nematode ............................ Possible modifications and adaptations for iBLINC ................................................ Synapses are dynamic and change with age ............................................................. Future directions ....................................................................................................... Chapter V: Annexes ............................................................................................................. V.1. Annex I.............................................................................................................. V.2. Annex II ............................................................................................................ Direct visualization of specifically modified extracellular glycans in living animals ...................................................................................................................... Skin-Derived Cues Control Arborization of Sensory Dendrites in Caenorhabditis elegans ...................................................................................................................... References ..............................................................................................................................

Figure I- 1 :

 1 Figure I-1: Schematic of the nervous system formation. .........................................................

Figure I- 2 :

 2 Figure I-2: Schematic of vesicle transport by kinesin. .............................................................

Figure I- 3 :

 3 Figure I-3: Schematic of a synapse. .........................................................................................

Figure I- 4 :

 4 Figure I-4: Modification of the nervous system during aging..................................................

Figure I- 5 :

 5 Figure I-5: Differential interference contrast (DIC) picture of an adult C. elegans hermaphrodite. ..........................................................................................................................

Figure

  Figure I-6: C. elegans life cycle ...............................................................................................

Figure

  Figure I-7: C. elegans anatomy ................................................................................................

Figure I- 8 :

 8 Figure I-8: Schematic of a chromosome ..................................................................................

Figure I- 9 :

 9 Figure I-9: Schematic of a gonad .............................................................................................

Figure I- 10 :

 10 Figure I-10: Schematic of MosTIC principle. ..........................................................................

Figure I- 11 :

 11 Figure I-11: Schematic of Whole Genome Sequencing principle. ...........................................

Figure I- 12 :

 12 Figure I-12: Schematic of the nervous system of C. elegans ...................................................

Figure I- 13 :

 13 Figure I-13: The sensory circuitry ............................................................................................

Figure I- 14 :

 14 Figure I-14: Schematic of chemical synapses ..........................................................................

Figure I- 15 :

 15 Figure I-15: Neuronal aging defects. ........................................................................................

Figure I- 16 :

 16 Figure I-16: Schematic of two techniques to visualize synapses in vivo. ................................

Figure I- 17 :

 17 Figure I-17: BLINC in cell culture ...........................................................................................

Figure I- 18 :

 18 Figure I-18: Tetrameric structure of streptavidin form by two dimers ....................................

Figure

  Figure I-19: Tetrameric monovalent streptavidin.....................................................................

Figure III- 1 :

 1 Figure III-1: in vivo BLINC in C. elegans ...............................................................................

Figure III- 2 :

 2 Figure III-2: Expression of iBLINC in AFD and AIY .............................................................

Figure

  Figure III-3: Control experiments to demonstrate iBLINC specificity. ...................................

Figure

  Figure III-4: Localization of iBLINC signal ............................................................................

Figure

  Figure III-5: iBLINC is directional. ........................................................................................

Figure

  Figure III-6: iBLINC is brighter and more robust than GRASP .............................................

Figure

  Figure III-7: iBLINC does not obviously compromise AIY functions during locomotory behaviors...................................................................................................................................

Figure

  Figure III-8: iBLINC is dependent of the formation of synapses ............................................

Figure III- 9 :

 9 Figure III-9: Quantification of synapses between AFD sensory neurons and AIY interneurons ..................................................................................................................................................

Figure

  Figure III-10: iBLINC punctae correspond to synapses...........................................................

Figure

  Figure III-11: iBLINC has a dynamic state ..............................................................................

Figure

  Figure III-12: Motor-neuron connections.................................................................................

Figure

  Figure III-13: Motor-neurons connections ...............................................................................

Figure

  Figure III-14: Mechanosensory and interneuron connections ..................................................

Figure

  Figure III-15: The glutamate receptor and its accessory proteins ............................................

Figure

  Figure III-16: Epifluorescence micrograph of iBLINC in the head of animals at different ages: ..................................................................................................................................................

Figure

  Figure III-17: The number of synapses is identical between the right and left processes........ 94

  Abbreviations

  Figure I-1) (Le Douarin and Dupin 2014) while the central nervous system arises from the neural tube (Figure I-1) (Morest and Silver 2003; Preston and Sherman 2011). The neural tube is formed by invagination and fusion of the ends of the neuroectoderm which is part of the neural plate (Figure I-1). The nervous system is composed of neurons and glia (Figure I-4a). The neurons are the cells that allow cell to cell communication. The glia carry different functionalities such as phagocytosis or production of myelin among others, and includes neuroglia (astrocytes and oligodentrocytes) and microglia. Neurons are highly polarized cells composed of axons and dendrites which can propagate action potentials. The dendrites receive input either byenvironmental or synaptic stimulation while the axon is the output structure. In some cases, neurons migrate from the place where they were born to their final destination. Once located at their final destination, they send their axons through the extracellular matrix to find their target cells. To do that, they follow guidance cues which can be attractive or repulsive, and which can work short-or long-range thanks to the growth cone that senses the environment[START_REF] Tessier-Lavigne | The molecular Biology of axon guidance[END_REF][START_REF] Dickson | Molecular mechanisms of axon guidance[END_REF] .

Figure I- 1 :

 1 Figure I-1 : Sc he matic o f the ner vo us s yste m for matio n. The nervous system derives from neuroectoderm. The neural plate invaginates to form the neural tube. The neurons can then migrate and differentiate. Adapted from Preston et al. 2011

  The pre-synaptic terminal is composed of vesicles containing neurotransmitters (Figure I-3). Different types of synapses exist and can be classified based on the neurotransmitters they employ, e.g. serotonergic, GABAergic, acetylcholinergic, and glutamatergic synapses. The vesicles are formed and packed in the cell body where the machinery for protein synthesis is present. Vesicles are then actively transported to the terminal along microtubules with the help of kinesin (Figure I-2). Kinesin is a dimer of two heavy chains, each one binding a light chain. Three domains can be defined: the head domain which binds to the microtubules and ATP (Adenosine Triphosphate), the long central stalk, and the tail domain which binds the vesicle

Figure I- 2 :

 2 Figure I-2 : Sc he matic o f ves ic le tra nsport b y k ines in. The kinesin (in blue) is a dimer protein which interacts with microtubules (in black) to transport the vesicles (purple circle) packed with neurotransmitters (red dots) from the cell body to the synapse.

Fusion

  Figure I-3 : Sc he matic o f a s ynap se. The pre-synaptic bouton contains vesicles which enclose the neurotransmitter. The neurotransmitter is released in the synaptic cleft by exocytosis. It can then bind two types of receptors: ligandgated ion channels (red) and G protein-coupled receptors (green).

Figure I- 4 :

 4 Figure I-4 : Mod ific atio n o f the nervo us s yste m d uring a ging. a. Schematic of the morphology of glia and neurons in a young brain. b. Schematic of their morphology in an aging brain.

  Caenorhabditis elegans is a free-living nematode measuring about 1mm long from the family Rhabditida (Figure I-5). It was first described by Maupas in 1900 and named Rhabditis elegans[START_REF] Maupas | Modes et formes de reproduction des nématodes[END_REF]. Osche categorized it under the subgenus Caenorhabditis in 1952. In the late 1940s, two strains of Caenorhabditis elegans (C. elegans) have been isolated one named Bergerac, which was isolated in the center of France and a strain named Bristol from

Figure I- 5 :

 5 Figure I-5 : Differe ntia l interfe re nce co ntras t ( DIC) picture o f a n ad ult C. elegans her ma p hrod ite. Scale bar indicates 0.1mm. Adapted from WormAtlas.

  Figure I-6 : C. elegan s life c yc le. Embryo development starts in utero before being laid at the gastrula stage. After hatching, the worm goes through 4 larval stages before reaching adulthood. Every stage is separated by a molt. Adapted from WormAtlas.

Figure I- 9 :

 9 Figure I-9 : Sc he matic o f a go nad. The foreign DNA is injected in the mitotic zone of the gonad.

Figure

  Figure I-10 : Sc he matic o f Mos TIC pr inc ip le. Double strand break is induced by excision of a Mos1 element. The break is possibly repaired by the DNA template of interest. From Robert and Bessereau 2007

Figure

  Figure I-11 : Sche matic o f W ho le Ge no me Seq ue nc ing pr inc ip le. Mutant with a N2 genomic background is crossed with the Hawaiian strain. The mutation is genetically link to N2 polymorphisms. Modified from Doitsidou et al. 2010

Figure I- 12 :

 12 Figure I-12 : Sc he matic o f the ner vo us s yste m o f C. elegan s. The nervous system is composed of the nerve ring (NR), dorsal (DNC) and a ventral (VNC) nerve cords and ganglia (G). The ganglia contain cell bodies of the neurons. The NR is the major neuropil of the worm.

Figure I- 13 :

 13 Figure I-13 : The se nso r y c irc uitr y. a. Schematic of the sensory circuitry including the temperature circuit. b. Schematic of three neurons involved in the thermosensory circuit. AFD is the sensory neuron, AIY the primary interneuron, and AVA the command interneuron. Modified from Tsalik et al., Nishida et al. and WormAtlas

  Chemical synapses can be monadic (one pre-and one post-synaptic partner) or polyadic (one pre-synaptic partner but several post-synaptic partners). They occur en passant between neighboring parallel nerve processes, or between nerve processes and muscle (NMJ). A synaptic swelling can be observed at the localization of the synapse.

  Figure I-14 : Sc he matic o f c he mica l s ynapse s. a. Synapses can have one or several post-synaptic partners. b. Synapses made between two neurons en passant, or between a motor neuron and a muscle cell (NMJ). The muscle cell sends a muscle arm to form a NMJ. c. Details of a NMJ showing the different subcellular locations of the pre-and post-synaptic partners. Adapted from WormAtlas.

Figure I- 15 :

 15 Figure I-15 : Ne uro na l a ging de fects. Schematic showing the different defects of the sensory neurons (ALM, PLM) observed in aging worms compared to their shape in young worms.

  most common ones are the use of a vesicle protein or a receptor subunit tagged with a fluorophore[START_REF] Nonet | Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions[END_REF] .Today, fluorescent proteins are available in a wide spectrum of colors. Most of these are derived from the green fluorescent protein (GFP), which is naturally produced by the jellyfish Aequorea victoria[START_REF] Shimomura | Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan[END_REF]). The fluorescent protein can be genetically fused with the gene encoding a synaptic protein or the receptor subunit[START_REF] Chalfie | Green fluorescent protein as a marker for gene expression[END_REF]. Expression of this fusion is driven by either a ubiquitous, tissue or cell specific promoter (e.g. pan-neuronal promoter), allowing for visualization of the entire nervous system or specific neurons. More recently, a trans-synaptic method (Figure I-16b), based on complementary GFP fragments, has been developed and called GRASP (GFP Reconstitution Across Synaptic Partners)[START_REF] Feinberg | GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems[END_REF]. In this technique, a part of the GFP is fused to neuroligin and expressed on the pre-synaptic partner, while the second part (also fused to neuroligin) is specifically expressed on the post-synaptic partner. Only under the condition in which the two partners form a synapse will the GFP be reconstituted and fluoresce. GRASP, originally developed for cell culture and for C. elegans, is now also available for Drosophila and mice[START_REF] Gordon | Motor control in a Drosophila taste circuit[END_REF][START_REF] Yamagata | Transgenic strategy for identifying synaptic connections in mice by fluorescence complementation (GRASP)[END_REF].

Figure I- 16 :

 16 Figure I-16 : Sc he matic o f two tec hniq ues to vis ua lize s ynapse s in v iv o. a. Green Fluorescent Protein (GFP) fused with a vesicle protein allows visualization of the pre-synaptic terminal (green) and Red Fluorescent Protein (RFP) fused with a subunit of a receptor allows visualization of the post-synaptic terminal (red). b. Schematic of GRASP (GFP Reconstitution Across Synaptic Partners)

  2+ , which occurs due to stimulation (see part I.1 of the introduction). Upon entry, the Ca 2+ binds to calmodulin and leads to modification of the conformation of the Cameleon protein. This conformational change induces a FRET (Fluorescence Resonance Energy Transfer) reaction and a subsequent change in the emission wavelength which changes the color of fluorescence, thereby indicating that stimulation has occurred. This calcium imaging technique is now, also available in C. elegans.

  Ting team at Harvard University developed BLINC (Biotin Labeling of INtercellularContact) in cell culture[START_REF] Thyagarajan | Imaging Activity-Dependent Regulation of Neurexin-Neuroligin Interactions Using trans-Synaptic Enzymatic Biotinylation[END_REF][START_REF] Liu | Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation[END_REF]. BLINC is based on the enzymatic transfer of biotin onto a 15 amino acid peptide (the acceptor peptide, AP) by the E. coli biotin ligase BirA. The enzyme was fused to Neurexin while the AP was fused to Neuroligin. Neurexin and Neuroligin are two cell adhesion molecules which formed a complex across the synaptic cleft. Cells transfected with either the enzyme-fusion or the AP-fusion were then cultured together. To induce the biotinylation, ATP and biotin were added to the cell culture. To detect the biotinylation a monovalent streptavidin tag with a fluorophore was added to the culture (Figure I-17). The authors were able to show that a signal was visible only when two HEK cells expressing the two different constructs are in contact (Figure I-17b). I.5. Labeling by biotinylation.

Figure I- 17 :

 17 Figure I-17 : BLINC in ce ll c ulture. a. Schematic of BLINC reaction. b. BLINC expressed in HEK cells in culture. Green cells express the fusion BirA-NRX, blue cells express AP-NLG, and BLINC signal is represented in red. Modified from Liu et al. 2013

Figure I- 18 :

 18 Figure I-18 : Tetra me r ic s tr uc ture o f str epta vid in fo r m b y two d ime rs. a. in silico structure. Dotted line represents the tetramer interface, the arrows indicate the dimer interface. b. Schematic of the four subunits forming two dimers that interact to form the final structure. Adapted from Lim et al. 2011

Figure I- 19 :

 19 Figure I-19 : Tetra me r ic mo no va le nt strepta vid in. a. Schematic of a tetravalent streptavidin that has been modified to a monovalent streptavidin.b. Different steps to engineer the monovalent streptavidin. First, mutated subunits and wild-type subunits tagged with 6His are generated and mixed. After folding, the right composition of subunits (3 deads, 1 alive) is purified using the 6His tag. Modified from[START_REF] Howarth | A monovalent streptavidin with a single femtomolar biotin binding site[END_REF]

  As in mammals, C. elegans NLG-1/neuroligin is a post-synaptic partner of NRX-1/neurexin[START_REF] Haklai-Topper | The neurexin superfamily of Caenorhabditis elegans[END_REF]. NLG-1/neuroligin reporter constructs are expressed in a subset of cells in the nervous system, including VA, DA, and AIY neurons used in this study[START_REF] Hunter | Neuroligin-deficient mutants of C. elegans have sensory processing deficits and are hypersensitive to oxidative stress and mercury toxicity[END_REF] whereas NRX-1/neurexin shows widespread expression in the nervous system[START_REF] Haklai-Topper | The neurexin superfamily of Caenorhabditis elegans[END_REF]. In C. elegans, biotin is naturally present in the extracellular matrix as it is provided by the food. For the development of in vivo BLINC (iBLINC) in C. elegans, we generated transgenic strains in which BirA is fused to the N-terminus of the pre-synaptic cell adhesion protein NRX-1 (Figure III-1a,c) while the 15 amino-acid acceptor peptide (AP) was inserted in tandem at the N-terminus of the post-synaptic protein NLG-1 (Figure III-1a,c). In consequence BirA and AP are present in the synaptic cleft and come in proximity when NRX-1 and NLG-1 form a complex. We predicted that, as in cell culture, pre-and post-synaptic expression of these fusions would result in enzymatic biotinylation of the AP on NLG-1 across the synaptic cleft (Figure III-1a) as biotin is present in the extracellular matrix. The AP and BirA fusions follow an exogenous signal peptide from the PAT-3 protein. To detect the biotinylated AP in vivo we designed a monomeric streptavidin::tagRFP 'detector' (Figure III-1c) that is transgenically secreted into the extracellular space from six scavenger cells in C. elegans termed coelomocytes allowing its diffusion across the animal (Figure III-2b).

Figure

  Figure II I-1 : in v iv o BLINC in C. elegans. a. Schematic of iBLINC: the biotin ligase BirA is fused to the pre-synaptic protein neurexin/NRX-1, while the acceptor peptide, AP, is fused to the post-synaptic protein neuroligin/NLG-1. When the synapse is formed BirA can transfer biotin to the AP. The biotinylation can be detected using a secreted monomer streptavidin tagged with a fluorophore. b. Schematic of a worm with the 6 coelomocytes secreting streptavidin::Fluorophore. c. Schematic of the constructs injected that encode each of the different protein fusions. Adapted from Desbois et al. 2015

Figure

  Figure II I-3 : Contro l e xper ime nts to de mo nstrate iBLINC spec ific ity. a-c. Epifluorescent micrographs of the head region of transgenic animals expressing only the streptavidin 'detector' fusion (a), the post-synaptic AP::NLG-1/neuroligin with the streptavidin 'detector' fusion (b), or the pre-synaptic BirA::NRX-1/neurexin with the streptavidin 'detector' fusion (c). Scale bar indicates 20 μm and anterior is to the left in all micrograph panels. d-e. Epifluorescent micrographs of the head region of transgenic animals expressing the iBLINC constructs using monomeric streptavidin fused to tandem superfolder GFP (2xsfGFP) as a detector instead of tagRFP (d, (dzEx1330)). The corresponding detector alone (e, dzIs66)) shows no signal. f-g. Epifluorescent micrographs of the head region of transgenic animals expressing the iBLINC constructs but secreting the detector from the intestine using the Pelt-2 promoter instead of the Punc-122 coelomocytes specific promoter (f, (dzEx1321)). The corresponding detector alone (g, (dzEx1322)) shows no signal. h. Quantification of transgenic lines as indicated. The percentage of animals showing signal is shown by black bars (N=8-54/transgenic line). Adapted from Desbois et al. 2015

Figure

  Figure II I-4 : Loc a lizatio n o f iBLINC s igna l. a-b. Epifluorescent micrographs of the head region of transgenic animals expressing AFD/AIY iBLINC (dzEx1240) and a cytoplasmic marker for AFD (oyIs17) (a) or AIY (mgIs18) (b) showing juxtaposition of iBLINC signals with cytoplasmic markers. a', b'. Optical sections of the indicated area in a, b, demonstrating juxtaposition of both signals. c. Quantification of animals showing juxtaposition of iBLINC signals with the AIY or AFD cytoplasmic markers (N=24 and 30, respectively). d. Epifluorescent micrograph showing post-synaptic sites of AIY in magenta (iBLINC) and pre-synaptic sites in green (wyIs45 (Is[pAIY::GFP::RAB-3])). e. Colocalization of post-and pre-synaptic sites in AIY was measured by a line scan and showed by and large mutually exclusive staining. 100% of animals showed immediately juxtaposed pre-and post-synaptic sites in AIY neurons (N=14). Adapted from Desbois et al. 2015

  Comparing the signal in animals and in different lines, we could observe a lower percentage of animals showing a signal in each GRASP extrachromosomal line compared to iBLINC lines (Figure III-6a) and the puncta observed were smaller and less bright (Figure III-6b,c).

Figure

  Figure II I-5 : iBLINC is d irectio na l. a. Epifluorescence micrograph of the head of a worm expressing the post-synaptic protein fusion BirA::NLG-1 in the pre-synaptic partner AFD, and the pre-synaptic protein fusion AP::NRX-1 in the post-synaptic partner AIY. b. Epifluorescence micrograph of the head of a worm showing correct activation of iBLINC by expressing the enzyme, BirA, in the post-synaptic neuron, AIY, and the AP in the pre-synaptic neuron, AFD. Adapted from Desbois et al. 2015

  identified the pair of URX sensory neurons. The URX neurites run along the neurites of the AIY interneurons without forming synapses (Figure III-8b). Using green and red fluorescent cytoplasmic markers of AIY and URX, respectively, we confirmed that the neurites of both pairs of neurons appeared immediately adjacent in 89% of the animals (N=28)(Figure III-8g,h). Expression of the BirA::NRX-1/neurexin pre-synaptic fusion in URX with the post-synaptic AP::NLG-1/neuroligin in AIY failed to result in any signal (0/5 transgenic lines (N=15-48), Figure III-8b,c,f). In contrast, expression of the BirA::NRX-1/neurexin pre-synaptic fusion in URX and the AP::NLG-1/neuroligin in RIG interneurons, bona fide post-synaptic partners of URX sensory neurons (White et al. 1986) (Figure III-8d,e), resulted in specific labeling (4/4 transgenic lines (N=22-51), Figure III-8f). These results are consistent with the conclusion that the iBLINC signal requires the presence of synapses and that cellular contact alone is insufficient to bring the NRX-1/neurexin and NLG-1/neuroligin components of the system into sufficient physical proximity for interaction.

Figure

  Figure II I-7 : iBLINC does no t ob vio us ly co mpro mise AIY func tio ns d uring lo co moto ry be ha vio rs. a. Trashing assays: number of trashes per minute at 25°C. ttx-3 mutants show a higher number than the wild-type. iBLINC transgenic worms are not significantly different than N2. b. Reversal assay: number of reversals in 3 minutes at 25°C. ttx-3 mutants make more reversal than the wild-type. iBLINC transgenic worms are not significantly different than N2 wild-types. c. Radial locomotion: distance travelled in 5, 10 and 15 minutes in a plate. ttx-3 mutants travel less distance than the wild-type. iBLINC transgenic and non-transgenic siblings shows a slight defect at 5 minutes only. d. Body bends assay: number of body bends in 20 seconds. Statistical significance compared to N2 and non transgenic siblings is indicated in red or blue, respectively: ns, not significant; *, P<0.05. Adapted from[START_REF] Desbois | Directional Trans-Synaptic Labeling of Specific Neuronal Connections in Live Animals[END_REF]

Figure

  Figure II I-10 : iBLINC punc tae correspo nd to s ynapses. a-b. Epifluorescent micrographs of a one day old wild-type (a) or one day old unc-104(e1265) mutant animals (b).Anterior is to the left in all micrograph panels. c. 'Pearl chain' graphs illustrating the sequential pattern of synapses. Each row is an individual animal. Synapses were quantified and placed into four different sized bins based on fluorescent area as indicated by different sizes and shades. Four different synaptic areas were defined as shown in the schematic. The total synaptic length was defined by the distance between the first and last dots and then split in four equally sized areas. d-g Quantification of the number of synapses per animal (d) and box plots of the average synaptic area per animal (e), the total synaptic area per animal (f), and the number of synapses per animal in the different synaptic sections as defined in c (g) in genotypes as indicated. In e and f, each dot represents the mean of one animal (with the mean of means indicated by a line) and the box shows the 25th and 75th percentile, respectively. Statistical significance is indicated explicitly or as: ns, not significant; *, P<0.05; **, P<0.005, ***, P<0.0005. Size units are square pixels. Adapted from[START_REF] Desbois | Directional Trans-Synaptic Labeling of Specific Neuronal Connections in Live Animals[END_REF]

  types of motor-neurons are present in C. elegans, the D-type which are inhibitory and allow the relaxation of the muscle by releasing GABA and the A and B-type neurons which are excitatory neurons and stimulates the contraction of the muscle by releasing acetylcholine (WormAtlas et al.). Those two types of neurons form a triad (Figure I-14a, Figure III-12a,b)

Figure

  Figure II I-12 : Moto r-ne uro n c o nnectio ns. a. Schematic of a triad where a cholinergic motor-neuron (blue) is pre-synaptic to GABAergic (beige) motorneuron and a muscle cell (green). b. Electron micrograph showing a triad synapse: the neuron VA01 circled in blue (cholinergic) is pre-synaptic to DD01, circle in beige (GABAergic) next to a muscle arm circled in green. c. Epifluorescent micrograph of a worm expressing iBLINC in the motor-neurons.

  Figure II I-13 : Moto r-ne uro ns co nnectio ns. a. Schematic of a triad where a GABAergic (beige) motor neuron is pre-synaptic to a muscle cell (green) next to a cholinergic motor neuron (blue). b. Electron micrograph showing a triad synapse: the neuron DD01, circle in beige (GABAergic) is pre-synaptic to a muscle arm circled in green next to the neuron VA02 circled in blue (cholinergic). c. Epifluorescent micrograph of the DNC of an animal expressing iBLINC (dzEx1436) and a cytoplasmic marker of the GABAergic neurons (juIs76).

Figure

  Figure II I-15 : The gluta mate recep tor and its ac cessor y p rote ins. GLR-1 is the glutamate receptor, its localization and function dependent on SOL-1 and SOL-2, two CUB-domains proteins and STG-1 or STG-2, two TARP proteins. Adapted from Wang, 2010

Figure

  Figure II I-16 : Ep ifluoresce nce micro grap h o f iBLINC in the he ad o f anima ls at d iffe re nt a ges : first larval stage (L1) (a), 1 day old adult (b), 15 day old adult (c). Adapted from Desbois et al. 2015

Figure

  Figure II I-17 : The number o f synap ses is ide ntica l betwee n the r ight a nd le ft proces ses in 1 day old adult worms. n.s.: non-significant. Adapted from Desbois et al. 2015

  independent (Figure III-20) or dependent (Figure III-21) on their size and compared them between ages.

  with age (Figure III-21). The number of the smaller ones (lighter grey) increases between L4 and 1 d-o in 2 out of 4 synaptic percentiles (0 to 25% and 75 to 100%). The number then decreases after one day old in 3 out of 4 percentiles (0 to 25%, 25 to 50% and 75 to 100%). With less statistical certainty their number also decreases between 1 d-o and 6 d-o in the percentile 25 to 50 % of the synaptic length (Figure III-21).

Figure

  Figure II I-20 : Orga niza tio n o f the s ynap tic p atter n depe nd ing o n the r e gio n o f the proces s. Quantification of the number of synapses per animal in the different synaptic sections at the indicated ages. Each dot represents the mean of one animal with the mean of means indicated by a line. Statistical significance as: ns, not significant; *, P<0.05; **, P<0.005; ***, P<0.0005. Size units are square pixels. Adapted from Desbois et al. 2015

Figure

  Figure II I-21 : Orga niza tio n o f the s ynap tic p attern depe nd ing o n the ir s ize a nd the re gio n o f the p rocess. Comparison between ages of the quantification of the number of synapses per animal in the synaptic sections 0 to 25 % (a), 25 to 50% (b), 50 to 75% (c), 75 to 100% (d). Each dot represents the mean of one animal with the mean of means indicated by a line of each size and age as indicated. Statistical significance is indicated explicitly or as: *, P<0.05; **, P<0.005; ***, P<0.0005. Size units are square pixels. Adapted from Desbois et al. 2015

  between 1 and 12 d-o are less significant compared to worms grown at 20°C. However, a strong decline is observed between 1 and 15 d-o (Figure III-23b,c). We can also observe a decrease of fluorescence between 4 and 15 d-o, 8 d-o with 12 and 15 d-o, and 12 and 15 d-o (Figure III-23c). Therefore a decline in fluorescence is observed at lower temperature but this decrease seems to be delayed compared to animals grown at 20°C. At 25°C, a temperature at which the animals have a shorter life span than at 20°C (Byerly et al. 1976), we can observe a progressive decline of the signal (Figure III-23b,d). Four, 8 and 12 d-o adults are statistically different from 1 d-o. Those results correlate with the fact that life span of the animals at 25°C is shorter which imply a "faster" aging.

  different longevity mutants. The first carries a mutation in daf-2, the insulin/IGF receptor ortholog which is part of the insulin pathway. The allele e1370 used is temperature sensitive: the mutants can reach adulthood at 15°C (permissive temperature) but if grown at 25°C (nonpermissive temperature) they enter a dauer stage, the alternative survivor stage. In addition the mutant show an increase of the life span when grow at 15°C only or when grown at 15°C until L4 stage (stage post dauer stage critical period) and then switched to 25°C[START_REF] Kenyon | A C. elegans mutant that lives twice as long as wild type[END_REF][START_REF] Tissenbaum | An insulin-like signaling pathway affects both longevity and reproduction in Caenorhabditis elegans[END_REF]. Surprisingly we did not observe any strong protective effect in this mutant. While no decline of the signal is observed in the mutant over time, a defect is apparent in 1 d-o and 4 d-o worms when transferred to the non-permissive temperature at L4 stage. A lower intensity is observed when compared to wild-type animals at those ages. Therefore it seems that at the non-permissive temperatures, the fluorescence is reduced compared to wild-type animals but is otherwise stable along the lifespan.

Figure

  Figure II I-25 : GABAer gic pre-s ynap tic s igna l de c line depe nd o n the te mperature o f la rva de ve lop me nt. Comparison of the quantification with wild type animals at different ages and temperatures, as indicated. **, P<0.005; ***, P<0.0005

  allele ad465 shows a different progression of the GABAergic synapses at 25°C. We can observe a decline in eat-2 adults older than 4 d-o as compared to 1 d-o, and 4 d-o mutants have significantly higher fluorescent synapses than 8 and 12 d-o (Figure III-28a). Interestingly, we can observe a difference between the mutant and the wild-type at 1 and 4 do, but not at 8 and 12 d-o (Figure III-28b).

Figure

  Figure II I-28 : Age-depe nde nt GABAergic pre-s ynaptic s igna l o f eat-2 muta nts at 25 °C. a. Quantification of the signal in eat-2 mutant animals at different ages and temperatures, as indicated. b. Comparison of wild-type and eat-2 mutant animals. **, P<0.005; ***, P<0.0005

  ROR1 and ROR2. It has been shown to play a role in axon outgrowth and the localization of the nAch receptor subunit, ACR-16 (Francis et al. 2005). It is also part of the Wnt signaling pathway. It is localized among other RTKs to muscle arms during embryogenesis. cam-1(ks52) mutants do not show any fluorescence decrease between 1 and 4 d-o adults, whether the worms are grown at 20 or 25°C (Figure III-29a,c). Moreover, the fluorescent signal is not significantly different from the wild-type at any of those temperatures (Figure III-29b,d). In conclusion cam-1 gene does not seem to be involved in the maintenance of the vesicle pool of GABAergic motor-neurons at least it does not induce an early decrease of the vesicle pool.

  show any changing of fluorescence intensity between 1 and 8 d-o, neither at 20 nor 25°C (Figure III-29a,c). But interestingly, it has significantly higher fluorescence at 20°C than the wild-type in 1 d-o adults. However, at 25°C only 8 d-o worms exhibit a significant difference compared to the wild-type, whereas no changes are observed between 1 and 8 d-o (Figure III-29b,d). In the case of plx-2(ev773) mutants, we can observe a significant increase in fluorescence between 1 and 4 d-o of adulthood at 20°C (Figure III-29a). This increase is also observed at 25°C and is follow by a decrease between 4 and 8 d-o adults (Figure III-29d). While 1 d-o plx-2 mutants are not different from wild-type at 20°C, 4 d-o mutants do exhibit differences (Figure III-29b). The opposite phenotype is seen at 25°C, in which case mutants are significantly different from wild-type at 1 d-o, but not at 4 or 8 d-o (Figure III-29d). The plexin pathway might have a role in the maintenance of the vesicle pool of the GABAergic neurons. PLX-1 has been shown to be important for synaptic tiling in a subset of neurons in C. elegans. In plx-1 mutants the synapses formed between the neurons DA8 or DA9 with muscle cells display overlapping synaptic fields while in the wild-type those synaptic fields tile (Mizumoto and Shen 2013). A modification of the synaptic area of the GABAergicmuscles synaptic field could explain the differences observed. efn-4 encodes a ligand of the ephrin family. It has been shown to be important for neuroblast migrations (Ikegami et al. 2012). It is expressed in neurons as well as other cell types. efn-4(bx80) mutants do not show any phenotype at 20°C (Figure III-29a). There are no significant differences between ages or between the mutant and the wild-type (Figure III-29b).

Figure

  Figure II I-29 : Age-depe nde nt GABAergic pre-s ynaptic s igna l in d iffere nt muta nts. a. Quantification of the signal of the different mutants at 20°C at different ages, as indicated. b. Comparison between wild-type and mutants at 20°C. c. Quantification of the signal of the different mutants at 25°C at different ages, as indicated. d. Comparison between wild-type and mutants at 25°C.*, P<0.05; **, P<0.005; ***, P<0.0005.

  ; Phelan and Starich 2001) (Figure IV-3). Additionally, double labeling experiments could be imagined using alternative transsynaptic techniques developed in cell culture, such as ID-PRIME (Interaction-Dependent PRobe Incorporation Mediated by Enzymes) where lipoic acid is enzymatically transferred across the synaptic cleft

Figure IV- 3 :

 3 Figure IV-3: Inne xins for m a co mp le x a t the gap junctio n. Possible complexes of innexins which bring the membranes of the two partners closer. Adapted from Phelan et al. 2001

Figure

  Figure IV-4: M ice b ra in spa ined us ing CLARITY. Hippocampus of a mouse showing neurons (green), parvalbumin-positive neurons (red) and glial fribrillary acidic protein (blue). Alv, alveus. Scale bar, 200µm. Adapted from Chun et al. 2013

  The semaphorin pathway seems to be important for pre-synaptic GABAergic vesicle pool. Interestingly the two semaphorin receptors have different phenotypes. The other pathways tested, ephrin and adhesion molecules pathway, do not seem to be implicated in the maintenance of the GABAergic synaptic vesicles during early adulthood.Analyses of the different parts of the AFD-AIY synapses area suggest that dedicated mechanisms are involved for the maintenance of the synapses as we can observe a different evolution of their number dependent on the portion of the synaptic area observed. The distal part of the process seems to be less affected by the decline than the proximal part. Different possibilities of mechanisms can be postulated one of them could be the mechanisms involved in synapses plasticity as for example activity of the synapses and/or trans-synaptic proteins could also be implicated for their maintenance along the life. Those molecules remain to be discovered. Few candidates exist as cima-1[START_REF] Shao | Synapse location during growth depends on glia location[END_REF] which is shown to be involved in AIY pre-synaptic terminal maintenance or unc-40/DCC and unc-6/Netrin[START_REF] Colón-Ramos | Glia Promote Local Synaptogenesis Through UNC-6 (Netrin) Signaling in C. elegans[END_REF]) which have been implicated in pre-synaptic terminal formation.

  The genes unc-40/DCC (Deleted in Colorectal Cancer) and unc-6/Netrin would also be interesting to test. UNC-40 encodes a transmembrane protein part of the immunoglobulin superfamily and is a receptor of UNC-6/Netrin, a guidance molecule. They are both implicated in axon guidance during neural development[START_REF] Dickson | Molecular mechanisms of axon guidance[END_REF]. In a screen for abnormal distribution of AIY pre-synaptic terminal, unc-40 has been identified with a 123 phenotype showing an abnormal distribution and a reduction of several pre-synaptic markers but without showing an axon guidance defect. unc-6 mutants phenocopy the Unc-40 phenotype. In wild-type animals, UNC-40 proteins accumulate at the pre-synaptic zones in AIY, this accumulation in dependent onUNC-6 (Colón-ramos et al. 2007). It would be interesting to see if it also affects the post-synaptic terminal of AIY.

  The genes discovered to affect the development and maintenance of the synapses in vivo in C. elegans could be then tested in microfluidic neuronal culture (see Introduction III-1.2)[START_REF] Deleglise | Synaptoprotective drugs evaluation in reconstructed neuronal network[END_REF]). The microfluidic device would allow us to test if the Human homolog of the worm also has a role in the development and/or maintenance of synapses of human neurons in culture.iBLINC could be a useful tool to understand what roles environmental cues play on the remodeling of synaptic connections. In the case of AFD-AIY which are part of the thermosensory circuitry it would be interesting to see if the number and strength of the synapses are the same when the worms are cultured at different temperatures(15, 20, 25°C) but also to observe the dynamics of the connectome when the worms are switched to another 124 temperature at different time-points in development (first, second, third, or fourth larval stage).

Figure V- 1 :

 1 Figure V-1 : The hepa ra n s ulfate c ha in a nd proteo glyca ns. a. The heparan sulfates are located in the extracellular matrix and are attached to core proteins, the proteoglycans. b. Heparan sulfate chain with different modifications distributed along the chain, and schematic of the set of heparan sulfate modifying enzymes which introduce specific modifications and form domain along the chain. Adapted from Díaz-Balzac et al. 2014

  we developed a new technique to visualize specifically modified HS domains in vivo in C. elegans. This method allows for the visualization of HS domains throughout development. This new technique should help uncovering the biosynthesis of the HS GAG domains. Due to the low immunogenicity of the HS GAGs the technique is based on the use of single chain variable fragment (scFv) antibodies. Using those antibodies, HS domains were found in a subcellular-specific manner and are partially associated with synapses. My contribution to this work was helping with the creation of the single chain variable fragment antibodies, by site directed mutagenesis or fusion PCR, of the different plasmids encoding the scFv.

List of genes mentioned C. elegans genes

	List of C. elegans neurons mentioned
	Name Name		Organism Location*	Protein Type*
	Name Arc AFD acr-16 AIY bpl-1 AMPA AIZ ALM	Human Ortholog* Homo sapiens Lateral ganglia in head nicotinic cholinergic receptor alpha 7 Ventral ganglion in head holocarboxylase synthetase Homo sapiens Ventral ganglion in head Lateral in mid-body	activity-regulated cytoskeleton-associated Thermosensory and CO2-sensory Protein encoded* Interneuron protein subunit of nicotinic acetylcholine receptor biotin ligase/ holocarboxylase synthetase Interneuron Glutamate receptor Mechanosensory
	cam-1 Bir A AVA	ROR1	Escherichia coli Lateral ganglia in head	receptor tyrosine kinase of the immunoglobulin Biotin Ligase Interneuron
	BDNF AVM		Homo sapiens Lateral	superfamily member of the neurotrophin family of Mechanosensory
	cima-1 daf-2 growth factors solute carrier family 17, member 5 IGF receptor Right side of the anterior half of the body memb rane solute transporter receptor tyrosine kinase eat-2 nicotinic cholinergic receptor alpha 7 Cre P1 Bacteriophage AWC Lateral ganglia in head Odorsensory and thermosensory tyrosine recombinase enzyme subunit of nicotinic acetylcholine receptor efn-4 Ephrin -B2 ephrin family of ligands CD4 Homo sapiens FLP Head Poly modal nociceptive fo r mechanosensation and glycoprotein thermosensation
	egl-15 Flip Motor-neurons	FGFR1-4 Saccharomyces cerevisiae recombinase fibroblast growth factor receptor Body (ventral nerve cord) Motorneurons
	glr-1 GAP-43 PLM	isoform Flop of glutamate receptor 1 Homo sapiens Lu mbar ganglia	AMPA-type ionotropic glutamate receptor Neuromodulin Mechanosensory
	innexin kal-1 lipoic acid ligase Pannexin Kal-1 PVD Lu mbar ganglia Escherichia coli myo-3 Myosin-3 Mos1 Drosophila mauritiana nlg-1 Neuroligin Neuroligin Homo sapiens PVM Lateral Left side of the posterior half of the body gap junction proteins Poly modal nociceptive fo r mechanosensation and cell surface protein thermosensation Mariner Mos1 transposase myosin heavy chain synaptic protein Mechanosensory cell surface protein
	nrx-1 Neurexin RIA	Neurexin Homo sapiens Head	synaptic protein cell surface protein Interneuron
	plx-1 NMDA RIG	Plexin	Homo sapiens Head	semaphorin receptor glutamate-gated ion channels Interneuron
	plx-2 ptp-3 ROR1 URX	Plexin Leukocyte Co mmon Antigen Related Homo sapiens Head	semaphorin receptor receptor-like tyrosine phosphatase Tyrosine-protein kinase transmembrane Oxygen and minor CO 2 sensory and ring
	rab-3	Rab-3C		Ras GTPase superfamily receptor
	rec-1 Streptavidin	unknown Streptomyces avidinii	unknown, affects reco mbination frequency
	snb-1 Syp	Vesicle-Associated Membrane Protein 1 Homo sapiens	synaptic vesicle protein (synaptobrevin) Synaptophysin
	sol-2 TNF-α	Cubulin/ Neto Homo sapiens	CUB and LDLa domain-containing type I Tumor necrosis factor α
	ttx-3 SLC17	LIM/homeobox protein Lh x2 Homo sapiens	transmembrane protein LIM ho meodomain p rotein Vesicular glutamate transporter
	unc-6	Netrin		netrin/guidance molecule
	unc-40	Deleted in Co lorectal Carcino ma	netrin receptor
	unc-47	Vesicular inhib itory amino acid transporter GA BA transporter
	unc-104	Kinesin		kinesin-like motor protein

*source http://www.wormbase.org Other organis ms genes interneuron *source www.wormatlas.org

  . Ces informations sont transmises entre cellules grâce à des connections synaptiques et des jonctions communicantes. Ce réseau n'est pas statique et évolue au cours du développement, de l'apprentissage mais aussi durant le processus de vieillissement -naturels ou pathologiques. Comprendre le système nerveux et son fonctionnement requiert une analyse des connections synaptiques in vivo chez un animal model tel que Caenorhabditis elegans.

	Cependant les techniques actuellement disponibles pour C. elegans sont laborieuses, ne
	dépendent pas forcément d'une interaction trans-synaptique ou fixent la synapse. Par

conséquent, ces approches ne permettent pas de réaliser des études de populations et dynamiques des modifications synaptiques. Dans ce manuscrit, je décris tout d'abord une nouvelle technique pour visualiser les synapses in vivo chez le vers C. elegans. Cette technique appelé iBLINC (in vivo Biotin Labeling of INtercellular Contacts) consiste en la biotinylation d'un peptide par une ligase d'Escherichia coli, BirA. Ces deux molécules sont fusionnées à des protéines transmembranaires qui forment un complexe à la synapse. La biotinylation est détectée grâce à une streptavidin monomérique taguée avec un fluorophore qui est secrétée dans l'espace extracellulaire. J'ai démontré que cette technique est directionnelle et dynamique. En utilisant iBLINC pour visualiser des synapses faisant partie du circuit sensoriel de C. elegans, une évolution du nombre et de la taille des synapses a pu être observée avec l'âge. Il semblerait que ce changement soit dépendant du segment de la zone synaptique observée. Ces résultats ont été corroborés par l'observation d'une diminution du nombre de vésicules pendant le vieillissement grâce à un marqueur pré-synaptique des synapses GABAergique de la jonction neuromusculaire Pour conclure, ce manuscrit décrit une nouvelle technique permettant d'observer les synapses chez le vers vivant et démontre une évolution naturelle du nombre de synapses et du nombre de vésicules pré-synaptiques avec l'âge.

Chapter II: Materials and Methods

Strains

Worms were cultured as previously described [START_REF] Brenner | The Genetics of Caenorhabditis elegans[END_REF]. The worms are grown on a lawn of Escherichia coli (strain named OP50) at 20°C unless otherwise indicated. Mutants and integrated lines were maintained either by chunking a piece of agar and placing it into a plate containing OP50, or by picking individual worms and transferring them to a new plate.

Extrachromosome lines were maintained by picking few worms that carried the co-injection marker and by transferring them to a fresh plate. Below are two tables of the different strains used in this thesis, listed in order of appearance: promoter [START_REF] Rogers | Inhibition of Caenorhabditis elegans social feeding by FMRFamide-related peptide activation of NPR-1[END_REF] using NotI/BamHI, and the ttx-3 promoter was switched using with XmaI/ApaI (Table 6 and7). promoter using NotI and BamHI sites and further to the Pmec-7 promoter using SphI/XmaI (Table 7).

The nrx-1 cDNA was amplified using custom primers from N2 cDNA and subcloned into pPD96.41 using XhoI/XmaI sites; then subcloned into the plasmid Pmec-7::BirA::nlg-1 using Bsu36I and SmaI sites. Finally the Pmec-7 promoter was switched to Pgcy-8 promoter using Pgcy-8::AP::nrx-1

The Pgcy-8 from Pgcy-8::BirA::nrx-1 was cloned into Pmec-7::AP::nrx-1 with SphI/XmaI Pgcy-8::BirA::nlg-1

A Pgcy-8 fragment was ligated to a BirA::nlg-1 fragment using SphI/XmaI

Pgcy-8::BirA::nrx-1

The Pgcy-8 from Pgcy-8::GFP was cloned into Pmec-7::BirA::nrx-1 with SphI/KpnI

Pgcy-8::nlg-1::spGFP11

The Pgcy-8 from Pgcy-8::BirA::nrx-1 was cloned into Psrh-128::nlg-1::spGFP 11 with SphI/BamHI

Pkal-1 3/4 ::BirA::nrx-1

The Pkal-1 3/4 from Pkal-1 3/4 ::ceKal-1 was cloned into Pgcy-32::BirA::nrx-1 with SphI/XmaI Pmec-7::AP::nlg-1

The Pmec-7 from Pmec-7::sdn-1 was cloned into Pflp-18::AP::nlg-1 with PaeI/XmaI Pmec-7::AP::nrx-1

The Pmec-7::AP from Pmec-7::AP::nlg-1 was cloned into Pmec-7::BirA::nlg-1 with SpeI/Bsu36I

Pmec-7::BirA::nlg-1

The Pmec-7 from Pmec-7::sdn-

Micro-injection and Integration

To create transgenic worms, iBLINC constructs were injected at 25ng/μl with pRF4 (rol-6(su1006)) as a dominant co-injection marker, a gut fluorescent marker (Pelt-2::GFP or Pelt-2::tagRFP) or AIY neurons cytoplasmic marker (Pttx-3::mcherry) as fluorescent co-injection marker.

The total DNA mix concentration is 100ng/μl, this include the plasmids which are coinjected, the co-injection marker plasmid and Bluescript plasmid (empty vector) if needed to reach the final concentration. The DNA mix was injected in the distal arm of the gonad. This area of the gonad contains germ cell nuclei localized in a central core of cytoplasm. Hence the DNA injected can be distributed to the progeny. The DNA will form an extrachromosome which can be inherited to the F2 and thus generate a line [START_REF] Mello | Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences[END_REF]).

The extrachromosome does not follow the Mendelian's law and can be lost during meiosis, therefore only part of the progeny will carry it [START_REF] Mello | Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences[END_REF]. In order to obtain

Reversal assays: Worms were placed on a fresh plate without food. After one minute of recovery, the number of reversals in 3 minutes was counted. Every change of movement from forward to backward was considered as a reversal. Statistical analyses were performed using a Mann-Whitney test with GraphPad® software package.

Body bends: Worms were placed on a fresh plate without food and the number of body bends per 20 seconds was counted. Statistical analyses were performed using a Mann-Whitney test with GraphPad® software package.

Photo-conve rsion experiments

For photo-conversion experiments, young adult animals were anaesthetized using 0.15mM BDM + 4mM levamisole and analyzed on an AxioImager Z1 compound microscope (Zeiss).

The slide was sealed using Vaseline® petroleum jelly. Images were taken first in the red channel (591-652 nm) at 400 ms exposure time and gain x4, then in the green channel (500-550nm) at 400 ms exposure time and gain x4 to avoid photo-conversion by the blue light.

Then the worm was exposed 30 seconds to blue light using a DAPI filter (365 nm). A postphoto-activation image series was taken using the same exposure time. The worm was left on the slide for an hour at room temperature. Finally the recovery images were taken using the same parameters.

Volumetric Reconstruction of the C. elegans nervous system

Digitized serial-section transmission electron micrographs of an L4 animal (JSH series)

and approximately 3 day old adult hermaphrodite (N2U) were aligned manually, segmented, and rendered using TrakEM2 [START_REF] Cardona | TrakEM2 software for neural circuit reconstruction[END_REF][START_REF] Walhovd | Unraveling the secrets of white matter--bridging the gap between cellular, animal and human imaging studies[END_REF]. Synaptic connectivity between neurons was annotated by counting the number of consecutive serialsections containing a pre-synaptic density using Elegance [START_REF] Xu | Computer Assisted Assembly of Connectomes from Electron Micrographs: Application to Caenorhabditis elegans[END_REF].

Analysis of the GABAergic synapses at the neuromuscular junction

Strains and culture

The GABAergic synapses were visualized using the marker juIs1 [Punc-25::snb-1::GFP + lin-15(+)]. The strains were crossed to the mutants and genotyped by phenotype or PCR.

They were grown at the permissive temperature or at 20°C. For aging studies, the worms were cultured as described above. To observe the signal during adulthood, L4 worms were picked than when the mutant is kept at 15°C the brood size is normal while when they are switched to 25°C it is decreased [START_REF] Tissenbaum | Genetics, life span, health span, and the aging process in Caenorhabditis elegans[END_REF]. This observation in addition to the temperaturedependent dauer stage entrance confirmed that the function of the protein is not completely abolished at 15°C. This partial functioning could be sufficient to induce a wild-type decline. a. BirA sequence will be introduced at the nrx-1 locus after a splice acceptor site (SAS) sequence surrounding by 2 loxP sites. AP sequence will be introduced at the nlg-1 locus after a splice acceptor site sequence surrounding by 2 FRT sites. The excision of the SAS would be cell specific by expressing the Cre or the Flip recombinase under specific promoter of the pre-synaptic partner (neuron A) or postsynaptic partner (neuron B). b. BirA sequence will be fused to nrx-1 sequence and drive under a specific promoter of pre-synaptic neuron (neuron A). AP sequence will be introduced at the nlg-1 locus after a splice acceptor site sequence surrounding by 2 loxP sites. The excision of the SAS would be cell specific by expressing the Cre recombinase under a specific promoter of the post-synaptic partner (neuron B). Key: orange rectangle: nrx-1 gene, purple rectangle: nlg-1 gene, orange triangle: loxP site sequence, red triangle: FRT site sequence, grey rectangle: BirA, blue rectangle: AP, pink rectangle: A neuron specific promoter sequence, SAS: splice acceptor site sequence, pink circle: neuron A, green circle: neuron B, red dots: iBLINC signal.

iBLINC could be adapted to study synaptic connection in others model organisms, such as mice, as well as to study other types of connections. In worm, iBLINC could also be used to observe any transient contact between two cells by using transmembrane proteins which have either the subcellular location of interest or are distributed along the membrane. For example we could observe the contact between two axons, or two dendrites as for instance PVD and FLP axons, these neurons are a pair of two nociceptor neurons. FLP neurons innervate the head and neck of the worm while PVD neurons innervate the body. The dendritic arborization patterns of these neurons are characterized by a 4 level order [START_REF] Hall | How does morphology relate to function in sensory arbors?[END_REF]. These two neurons seem to tile, but the exact mechanism underlying this phenomenon is poorly understood and has not been extensively studied [START_REF] Smith | Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans[END_REF]). The development of FLP and PVD as well as how they determine the dendritic field are still unclear; we do not know if FLP and PVD physically touch or only come into close proximity before retracting. To answer this question we could express the biotin ligase in one of the neurons, e.g. PVD, and the acceptor peptide in the other neuron, e.g. FLP. If the dendrites of the neurons do touch before retracting labeling will be observed. But if they come into close proximity without touching no signal would be observed.

In mice, the fusion BirA::Neurexin and AP::Neuroligin could specifically be expressed by the use of tissue specific promoters. In the absence of coelomocytes-type cells in vertebrates we could imagine that the streptavidin could be delivered by intravenous injection or secreted by another type of tissue. Another possibility would be to stain the tissue after its extraction by using a fluorescently labeled streptavidin in association with another method like CLARITY [START_REF] Chung | Structural and molecular interrogation of intact biological systems[END_REF]. CLARITY is a novel technique to visualize the connectivity in an intact brain by making it optically transparent and permeable to macromolecule (Figure IV-4). For that a hydrogel-tissue fusion and electrophoretic-clearing technology has been developed [START_REF] Chung | Structural and molecular interrogation of intact biological systems[END_REF].

Skin-Derived Cues Control Arborization of Sensory Dendrites in

Caenorhabditis elegans

Neurons can be highly branched, and it exists a vast variety of arborization. The branching of those neurons is tightly regulated and controlled by extracellular and intracellular molecules. Indeed the cytoskeleton is an important intracellular cue for this process whereas in the extracellular compartment branching factors can inhibit or induce branching. Few branching factors have been identified and there are either implicated in axon or dendrite branching.

To study dendrite branching in C. elegans the neurons PVD are used as a model system.

PVDs are two polymodal nociceptor neurons for mechanosensation and thermosensation [START_REF] Hall | How does morphology relate to function in sensory arbors?[END_REF]. Several factors have been identified for its branching using unbiased genetics screens such as the LIM homeodomain transcription factor mec-3 [START_REF] Smith | Time-lapse imaging and cell-specific expression profiling reveal dynamic branching and molecular determinants of a multi-dendritic nociceptor in C. elegans[END_REF][START_REF] Smith | Sensory neuron fates are distinguished by a transcriptional switch that regulates dendrite branch stabilization[END_REF] or hpo-30/claudin which act to stabilize the PVD dentritic arbor [START_REF] Smith | Sensory neuron fates are distinguished by a transcriptional switch that regulates dendrite branch stabilization[END_REF].

Molecules involved in intracellular transport (protein, organelles, mRNA and others molecules transport) have also been implicated in PVD branching. Axon guidance molecules as UNC-6/Netrin, UNC-5 and UNC-40/DCC (receptor deleted in colorectal cancer) have also been involved to mediate self-avoidance mechanism [START_REF] Smith | Netrin (UNC-6) mediates dendritic self-avoidance[END_REF]. EFF-1/fusogen and KPC-1/Furin, a proprotein convertase, have also been shown to be important either for pruning mechanism or for the dauer specific arborization respectively [START_REF] Oren-Suissa | The fusogen EFF-1 controls sculpting of mechanosensory dendrites[END_REF][START_REF] Schroeder | Dauer-specific dendrite arborization in C. elegans is regulated by KPC-1/furin[END_REF] as well as the PVD arborization [START_REF] Salzberg | The Proprotein Convertase KPC-1/Furin Controls Branching and Self-avoidance of Sensory Dendrites in Caenorhabditis elegans[END_REF]. Moreover a leucine-rich repeat ortholog to a human-insulin growth factor binding protein, called DMA-1, which is localized at the dendrite, has been shown to be important for dendritogenesis [START_REF] Liu | The transmembrane LRR protein DMA-1 promotes dendrite branching and growth in C. elegans[END_REF]. However few non-autonomous proteins have been discover to be involved in PVD arborization.

In this study, we showed that two skin-derived molecules, the novel menorin-1 (mnr-1) gene and sax-7/L1CAM gene, are involved in normal branching in PVD neurons. My contribution to this work has been to design and create plasmids as well as injects them and select lines for the revision experiments.

181