Silvia Viana Da Silva

Bernat Gonzalez

Adam Gorlewicz

Vincent Maingret

Jimmy George

Sabine Fièvre

Stefano Zucca

Mario Carta

Marilena Griguoli

Gael Barthet

Thierry Amédée

Christophe Blanchet

Sandrine Pouvreau

Severine Deforges

Audrey Lacquemant

Julie Rumi-Masante

Ashley Kees

Mariela Escande

Nan Jiang

Dario Cupolillo

Ania Ramalho-Goncalves

Tomas Jorda-Siquier

Ruth Betterton

Eva Rodrigues

Susanne Burkhardt

Daniel Riester

Farahnaz Sananbenesi

Sadman Sakib

Rezaul Islam

Eva Benito

Rashi Halder

Tea Berulava

Hendrik Urbanke

Magdalena Navarro

Christian Schiffmann

Lalit Kaurani

Gaurav Jain

Ashish Rajput

Dr Andreas Frick
email: andreas.frick@inserm.fr

Dr Elisabetta Aloisi

Dr. Yoon Cho

Keywords: Alzheimer's, hippocampus, synaptic modifications

Memory encoding is thought to proceed from durable changes in the activity of synaptic circuits, leading to the storage of patterns of electrical events in a sparsely distributed ensemble of neurons. Located at the entry level of hippocampal circuitry, the CA3 region is thought to be important for episodic memory encoding, especially at the initial stage of acquisition, by presumably developing an instant representation of a context. CA3 pyramidal cells receive a variety of inputs, among which the mossy fiber (Mf) inputs draw special attention for their peculiar structure and unique synaptic properties. However, the links between the plasticity of CA3 circuits and memory encoding are not well understood.

This thesis project aimed to address the synaptic mechanisms of episodic memory encoding in physiological conditions as well as in a mouse model of Alzheimer's disease (AD).

AD is characterized at an early stage by impaired episodic memory, which may involve dysregulation of the plasticity of CA3 circuits.

First of all, we searched for synaptic deficits in CA3 local circuit in the early stage of AD pathology in acute slices, taking advantage of a familial AD mouse model: 6month male APP/PS1 mice. We report that there is a reduction in spontaneous IPSC frequency in CA3 pyramidal cells (PCs) together with decreased inhibitory charges of evoked events at Mf-CA3 synapses, whereas the short-term plasticity of these synapses and intrinsic properties of CA3 PCs remain unaffected. Furthermore, there is a robust reduction in Kainate receptor (KAR) mediated currents at Mf-CA3 synapses. The same results were obtained from PSKO mice, suggesting that disturbed function of γsecretase and N-Cadherin processing pathways may underlie the dysfunction of KARs at Mf-CA3 synapses.

In the next step, we explored the changes in CA3 circuits shortly after one-trial contextual fear conditioning in adult C57Bl6j mice. We show that despite hardly any changes in filopodia number of Mf terminals, an increase in spontaneous IPSC frequency can be registered, while the EPSCs and short-term plasticities of these synapses are unaltered. However, this increase cannot be seen anymore 24 hours after the contextual learning. We also tried to do simplified computational modeling of the DG-CA3 neuronal networks, to investigate if and to what extent the local interneurons in CA3 region contribute to memory encoding precision.

Finally, to screen for changes at a transcriptome level, we performed RNA-seq with dissected CA3 tissue from APP/PS1 mice and identified up-and down-regulated genes at this early stage of AD. Moreover, we carried out ChIP-seq for a histone modification marker: H3K4me3, which has been shown to be directly related to onetrial contextual memory, and we report that there is a significant decrease in H3K4me3 levels at the promoter areas of various genes in CA3 PCs. However, these genes are hardly overlapping with the down-regulated genes from RNA-seq result, suggesting that other epigenetic mechanisms may play more important roles in expressing early deficits in this AD mouse model.

Taken together, we show that inhibitory connections of hippocampal CA3 circuits may be important for episodic memory encoding, and in early AD mouse model with memory deficits, there is reduced GABAergic transmission and reduced KARmediated currents in CA3 PCs, together with many active transcriptional regulations across the genome. Our study may contribute to the understanding of early AD pathologies at a synaptic level as well as a transcriptional level, and provide novel insights into the mechanisms underlying rapid encoding of contextual memory.

experience of my doctoral study. It made me more mature, open-minded, and more resilient in the face of frustrations and challenges. To continue in academic or not, I will always cherish this experience at the bottom of my heart. With the advancement of new research tools and numerous devoted scientists, we might be standing in the timepoint where the scope of knowledge of mankind is being broadened at speed never seen, and I feel very lucky to be able to participate in or at least spectate this great cause.

Titre : Modifications synaptiques dans les cellules pyramidales de CA3 de l'hippocampe dans un modèle de souris de la maladie d'Alzheimer

Résumé :

L'encodage de la mémoire dépend de changements durables dans l'activité des circuits synaptiques dans un ensemble de neurones interconnectés. La région CA3 de l'hippocampe reçoit des informations directement ou indirectement (à travers le gyrus denté -GD) en provenance des structures corticales. Des données théoriques et comportementales ont montré que la région CA3 est importante pour l'encodage de la mémoire épisodique, en particulier au stade initial de l'acquisition, en développant vraisemblablement une représentation instantanée d'un contexte. Les neurones pyramidaux CA3 reçoivent une variété de connections afférentes, parmi lesquelles les fibres moussues (FM), les axones des cellules du gyrus denté. Ces connections synaptiques ont attiré une attention par leurs propriétés morphologiques et fonctionnelles uniques. Malgré les nombreuses études, les liens entre plasticité des circuits CA3 et encodage de la mémoire ne sont pas bien compris.

Le cadre général de ce projet de thèse se situe dans l'étude des mécanismes synaptiques de l'encodage de la mémoire épisodique dans des conditions physiologiques ainsi que dans un modèle de souris de la maladie d'Alzheimer (MA).

En effet, la MA se caractérise à un stade précoce par une mémoire épisodique altérée, qui peut être associée à une dysrégulation de la plasticité des circuits CA3. À l'aide de techniques d'enregistrement électrophysiologique, nous avons d'abord exploré les modifications dans les circuits CA3 peu de temps (quelques heures) après conditionnement de la peur contextuelle chez les souris adultes C57Bl6j. Nous avons observé une augmentation de la fréquence des IPSC spontanés accompagnée de changements mineurs dans le nombre de filopodia issus des boutons synaptiques des FM, tandis que les EPSCs et les plasticités à court terme de ces synapses ne sont pas modifiés.

Nous avons ensuite comparé les propriétés synaptiques des circuits CA3 au stade précoce de la pathologie de la MA, en profitant d'un modèle de souris de MA familliale : les souris APP / PS1 males à 6 mois. Nous avons observé une réduction de la fréquence des IPSC spontanés dans les neurones CA3 avec une diminution de la charge des courants synaptiques évoqués des synapses FM-CA3, alors que la plasticité à court terme de ces synapses et les propriétés intrinsèques des neurones CA3 restent inchangées. En outre, il existe une réduction marquée des courants médiés par les récepteurs kainate (KAR) aux synapses FM-CA3 dans ce modèle de MA. Cette réduction est aussi observée en invalidant la préséniline sélectivement dans les cellules pyramidales de CA3. Ceci suggère que perturber des voies de signalisation de la γsecretase, en particulier son action protéolytique sur la N-Cadhérine pourrait soustendre la perte de KAR aux synapses FM-CA3.

Enfin, j'ai comparé le transcriptome des souris contrôle et APP/PS1, et j'ai pu établir une liste de gènes dont l'expression est modifiée à ce stade précoce de la MA.

De plus, nous avons effectué une analyse en ChIP-seq et rapportons ici qu'il existe une diminution des niveaux de H3K4me3, qui s'est révélé être directement lié à la mémoire contextuelle à un coup.

Dans l'ensemble ce travail a révélé que la transmission inhibitrice des circuits locaux CA3 de l'hippocampe pourrait être importante dans l'encodage de la mémoire épisodique. Dans le modèle murin de la MA avec déficit de mémoire, il y a une réduction de la transmission GABAergique et des courants médiés par les KAR réduits cellules pyramidales de CA3. Finalement, avons observé une modification transcriptionnelle d'un certain nombre de gènes dans CA3, à des stades précoces de développement de la pathologie dans notre modèle de MA. Notre étude pourrait contribuer à la compréhension des mécanismes pathologiques précoces de la MA, au niveau synaptique ainsi qu'au niveau transcriptionnel, et fournir des idées nouvelles sur les mécanismes sous-jacents au codage rapide de la mémoire contextuelle.

Morphology of synapses

The connection and communication between neurons (or neurons with other faculties) are realized through synapses. A typical synapse consists of two components: a presynaptic compartment, and a postsynaptic compartment, with a synaptic cleft in between. Depending on the nature of pre-and postsynaptic compartments, the synapses can be called axodendritic, axosomatic, axoaxonic, axosecretory, dendrodendritic synapses, etc., with the vast majority in the mammalian nervous system being axodendritic. In addition, astrocytes also exchange information with neurons, responding to synaptic activity, and regulating neurotransmission in a "tripartite synapse" configuration [START_REF] Perea | Tripartite synapses: astrocytes process and control synaptic information[END_REF]. Further down this path, a concept of "quadripartite synapse" has also been proposed, arguing that the role extracellular matrix (ECM) in regulating synaptic functions and plasticity [START_REF] Sykova | Diffusion in brain extracellular space[END_REF] Synapses can be categorized as chemical synapses and electrical synapses, and in some cases, both at the same synapse [START_REF] Pereda | Electrical synapses and their functional interactions with chemical synapses[END_REF] (Figure 1.1).

Electrical synapses are also known as gap junctions, propagating action potentials (APs) very rapidly. Gap junctions can conduct electrical signal in both directions, and are usually found at systems requiring fast synchronization of neurons. Due to the nature of transmission here is the passive flow of ionic currents, theses synapses lack gain, and the possibility to modify the synaptic strength is very limited.

The much more common form of synapses is chemical synapse. Although more complex, the chemical synapses are more primitive in an evolutionary point of view [START_REF] Bennett | Electrical synapses, a personal perspective (or history)[END_REF], because the sequences of gap-junction-forming proteins are markedly different between vertebrates and invertebrates, while the genes underlying chemical transmission are almost homologs among species [START_REF] Bennett | Electrical synapses, a personal perspective (or history)[END_REF][START_REF] Bruzzone | Pannexins, a family of gap junction proteins expressed in brain[END_REF]. Schematic representation of an electrical synapse. Electrical transmission is mediated by clusters of gap junctions that linking two adjacent cells, directly allowing the bidirectional traffic of electrical currents as well other small molecules. Adapted from [START_REF] Pereda | Electrical synapses and their functional interactions with chemical synapses[END_REF].

The synaptic cleft of a chemical synapse (20~40nm) is much wider than that of an electrical synapse (2~4nm) [START_REF] Goyal | Structure activity relationship of synaptic and junctional neurotransmission[END_REF]. When an AP arrives at presynaptic terminals, vesicles containing neurotransmitters are released into the synaptic cleft. The neurotransmitters can activate receptors on the postsynaptic side, resulting in the opening of the channels and ion flows across the postsynaptic membrane.

These alterations in membrane potential on the postsynaptic side can summate in the soma and generate the next AP once over the threshold. Therefore, the signal is conveyed in an electrical-chemical-electrical manner [START_REF] Pereda | Electrical synapses and their functional interactions with chemical synapses[END_REF]. Chemical synapses are considered to be unidirectional. However, they are subject to certain retrograde modulations, via conventional neurotransmitters, gasses, peptides, growth factors, or membrane-derived lipids [START_REF] Carta | Membrane lipids tune synaptic transmission by direct modulation of presynaptic potassium channels[END_REF][START_REF] Regehr | Activity-dependent regulation of synapses by retrograde messengers[END_REF].

Neuronal transmitters

A chemical synapse is usually described according to the type of neurotransmitters it releases (Table 1.1). These neurotransmitters cause different responses on the postsynaptic side depending on the context, which can be developmental, homeostatic, pathological states, etc. Glutamatergic synapses are excitatory in the vertebrate central nervous system (CNS), while GABAergic synapses are mainly inhibitory, albeit a major excitatory role during development [START_REF] Ben-Ari | Excitatory actions of gaba during development: the nature of the nurture[END_REF]. Most of the other neurotransmitters in the CNS are considered to be modulatory, the effect of which can vary depending mainly on the type of receptors they interact with. Furthermore, the corelease of different neurotransmitters at the same synapse is not rare [START_REF] Hnasko | Neurotransmitter corelease: mechanism and physiological role[END_REF][START_REF] Trudeau | On cotransmission & neurotransmitter phenotype plasticity[END_REF].

Table 1.1 An incomplete list of neurotransmitters

Glutamate receptors

Glutamatergic synapses are the most abundant type of synapses in vertebrate CNS (>50%), comparing to GABAergic synapses (30~40%) and other modulatory synapses (~10%) [START_REF] Hendry | Numbers and proportions of GABA-immunoreactive neurons in different areas of monkey cerebral cortex[END_REF][START_REF] Nadim | Neuromodulation of neurons and synapses[END_REF][START_REF] Roberts | What do GABA neurons really do? They make possible variability generation in relation to demand[END_REF].

The actions of glutamate are mediated via a broad range of ionotropic glutamate receptors (iGluRs, including AMPA, NMDA, Kainate and Delta receptors), and metabotropic glutamate receptors (mGluRs, including group I, II, III). These receptors can locate both pre-and post-synaptically. iGluRs are ligand-gated ion channels. Ligand binding leads to rapid changes in membrane potential and cell excitability [START_REF] Bleakman | Neuropharmacology of AMPA and kainate receptors[END_REF][START_REF] Yamakura | Subunit-and site-specific pharmacology of the NMDA receptor channel[END_REF]. mGluRs are G-protein coupled modulatory receptors, and their activation will trigger a series of reactions in certain signaling pathways and may indirectly open or close other channels [START_REF] Conn | Pharmacology and functions of metabotropic glutamate receptors[END_REF][START_REF] Schoepp | Pharmacological agents acting at subtypes of metabotropic glutamate receptors[END_REF]) (Figure 1.2, 1.3). Ionotropic glutamate receptors are tetramers, including AMPARs (α-amino-3hydroxy-5methyl-4-isoxazolepropionic acid receptors), NMDARs (N-methyl-Daspartic acid receptors), KARs (Kainic acid receptors) and Delta receptors. Their properties are defined by the subunit composition and their modifications [START_REF] Dingledine | The glutamate receptor ion channels[END_REF][START_REF] Dingledine | Peripheral glutamate receptors: molecular biology and role in taste sensation[END_REF].

AMPARs are hetero-oligomers of four subunits: GluA1 to GluA4 [START_REF] Hollmann | Cloned glutamate receptors[END_REF], and are mediating the fast component of excitatory postsynaptic currents (EPSCs), permeable to cations: Na + and K + . The majority of AMPARs in adult CNS are GluA1/2 or GluA2/3 heterodimers [START_REF] Lu | Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach[END_REF][START_REF] Mansour | Heteromeric AMPA receptors assemble with a preferred subunit stoichiometry and spatial arrangement[END_REF][START_REF] Wenthold | Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons[END_REF], whereas GluA4-containing AMPARs can be found in hippocampus during development [START_REF] Zhu | Postnatal synaptic potentiation: delivery of GluR4-containing AMPA receptors by spontaneous activity[END_REF]. However, there's a small portion of AMPARs lacking the GluA2 subunit (e.g. GluA1/3 heterodimers or homomeric GluA1), and thus permeable to Ca 2+ [START_REF] Seeburg | RNA editing of brain glutamate receptor channels: mechanism and physiology[END_REF][START_REF] Wisden | Mammalian ionotropic glutamate receptors[END_REF].

NMDARs are heterotetramers composed of subunits GluN1, GluN2A, GluN2B, GluN2C, and GluN2D (in some rare cases also GluN3A and GluN3B), and mediate the slow component of EPSCs [START_REF] Lieberman | Properties of single NMDA receptor channels in human dentate gyrus granule cells[END_REF][START_REF] Stern | Single-channel conductances of NMDA receptors expressed from cloned cDNAs: comparison with native receptors[END_REF]. As cation channels, NMDARs are highly Ca 2+ permeable [START_REF] Mayer | Permeation and block of N-methyl-Daspartic acid receptor channels by divalent cations in mouse cultured central neurones[END_REF]. Ca 2+ entry triggers several downstream events which are important for synaptic plasticity [START_REF] Berridge | Neuronal calcium signaling[END_REF]. Apart from glutamate binding, the activation of NMDARs also requires the co-agonist glycine and the removal of the blockage by extracellular Mg 2+ . The blockage is voltage-sensitive and can be unblocked upon depolarization [START_REF] Kleckner | Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes[END_REF][START_REF] Schoepfer | Molecular biology of glutamate receptors[END_REF]. Adapted from [START_REF] Verkhratsky | NMDA Receptors in glia[END_REF].

KARs are homo-and heteromeric tetramers composed of 5 subunits: GluK1 -5.

The KAR-mediated synaptic current is much smaller and slower compared to AMPAR current [START_REF] Lerma | Roles and rules of kainate receptors in synaptic transmission[END_REF][START_REF] Traynelis | Glutamate receptor ion channels: structure, regulation, and function[END_REF]. Unlike AMPARs and NMDARs which are normally enriched at excitatory postsynaptic densities (PSDs), KARs can be located at both pre-and postsynaptic sites. In hippocampus, the postsynaptic KARs are predominantly present at Mf-CA3 synapses, where it has been extensively studied [START_REF] Castillo | Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons[END_REF][START_REF] Darstein | Distribution of kainate receptor subunits at hippocampal mossy fiber synapses[END_REF].

The delta receptors (subunit: GluD1 and GluD2) are regarded as ionotropic glutamate receptors solely because of sequence homology. To date, the wild type delta receptors stay closed in the presence of molecules that have been proved to bind its ligand-binding domain, although the ion channel function has been observed in some mutant or chimera delta receptors [START_REF] Orth | The delta subfamily of glutamate receptors: characterization of receptor chimeras and mutants[END_REF]. Knock-out studies suggested they play important roles in cerebellar function and high-frequency hearing [START_REF] Schmid | To gate or not to gate: are the delta subunits in the glutamate receptor family functional ion channels?[END_REF].

Synaptic plasticity

Synaptic plasticity is thought to be one of the mechanisms underlying learning and memory. It exists in many forms, ranging from changes of property in a single synapse to large-scale modifications of synaptic strengths, also including formation or pruning of synaptic connections (Figure 1.4). Synaptic plasticities are normally categorized as short-term and long-term. While short-term synaptic plasticity leads to transient changes in synaptic functions (lasting from milliseconds to minutes) that regulate the moment-to-moment information flow through the circuits, long-term synaptic plasticity leads to persistent changes in synaptic strengths (lasting from hours to the lifetime of the synapse) that adaptively alter synaptic function in response to activities and computational demands [START_REF] Collingridge | Long-term depression in the CNS[END_REF][START_REF] Granger | Expression mechanisms underlying long-term potentiation: a postsynaptic view, 10 years on[END_REF][START_REF] Zucker | Short-term synaptic plasticity[END_REF]. Short-term plasticity can be further divided into presynaptic and postsynaptic plasticities. In the presynaptic forms of short-term plasticity, there can be depression (hundreds of milliseconds to seconds), facilitation (hundreds of milliseconds to seconds), as well as augmentation and post-tetanic potentiation (PTP) (tens of seconds to minutes) [START_REF] Fioravante | Short-term forms of presynaptic plasticity[END_REF]) (Figure 1.5).

Presynaptic depression can be observed at many synapses when stimulated repetitively at short time intervals. Several factors can account for the reduced synaptic strength, including depletion of the readily releasable pool, inactivation of release sites, and decreased presynaptic calcium influx.

Presynaptic facilitation is often found at synapses with a low initial release probability (e.g. mossy fiber synapses), and repeated stimulation at short time intervals can lead to a transient increase in release probability of these synapses. Increased presynaptic residual calcium level, saturation of endogenous calcium buffers, and facilitation of calcium currents are considered to be contributing to this phenomenon [START_REF] Fioravante | Short-term forms of presynaptic plasticity[END_REF]. At Mf-CA3 synapses, this type of plasticity are often registered with two protocols: paired-pulse (PP) or even longer train protocol with repetitive stimulation at short intervals, and frequency facilitation (FF) protocol with increasing stimulation frequency from basal (0.1Hz) to moderate (1-3 Hz) [START_REF] Gundlfinger | Natural spike trains trigger short-and long-lasting dynamics at hippocampal mossy fiber synapses in rodents[END_REF]Nicoll & Schmitz, 2005;[START_REF] Salin | Distinct short-term plasticity at two excitatory synapses in the hippocampus[END_REF]. Post-tetanic potentiation (PTP) and augmentation is another form of short-term plasticity noticed at Mf synapses, which means enhancement in RRP: readily releasable pool of vesicles; Cares: residual calcium. Adapted from [START_REF] Fioravante | Short-term forms of presynaptic plasticity[END_REF] synaptic strength following sustained, high-frequency synaptic activation [START_REF] Vyleta | Plasticity-dependent, full detonation at hippocampal mossy fiber-CA3 pyramidal neuron synapses[END_REF].

Other forms of short-term plasticity involving both pre-and postsynaptic elements and retrograde messengers have been reported, such as endocannabinoids mediated depolarization-induced suppression of inhibition (DSI) [START_REF] Regehr | Activity-dependent regulation of synapses by retrograde messengers[END_REF][START_REF] Wilson | Endocannabinoid signaling in the brain[END_REF], and depolarization-induced potentiation of excitation (DPE) at the Mf synapses [START_REF] Carta | Membrane lipids tune synaptic transmission by direct modulation of presynaptic potassium channels[END_REF].

On the other hand, long-term plasticity also exists in various forms, including but not limited to: NMDA-dependent LTP, presynaptic LTP, NMDA-dependent LTD, mGluR-dependent LTD, eCB-LTD, which were nicely reviewed by [START_REF] Kauer | Synaptic plasticity and addiction[END_REF] (Figure 1.6). Interestingly, albeit the presence of functional NMDA receptors [START_REF] Weisskopf | Presynaptic changes during mossy fibre LTP revealed by NMDA receptor-mediated synaptic responses[END_REF], a form of presynaptic NMDAindependent LTP is characterized at Mf-CA3 synapses, which is quite different from the canonical NMDA-dependent LTP found in other regions of the brain [START_REF] Harris | Long-term potentiation of guinea pig mossy fiber responses is not blocked by N-methyl D-aspartate antagonists[END_REF].

In addition, other forms of plasticity such as homeostatic plasticity (up-or downscaling of excitability), metaplasticity (plasticity of synaptic plasticity), structural plasticity like formation or deletion of synapses are also actively regulating neuronal network activities [START_REF] Fernandes | Mechanisms of homeostatic plasticity in the excitatory synapse[END_REF][START_REF] Holtmaat | Optical imaging of structural and functional synaptic plasticity in vivo[END_REF][START_REF] Hulme | Emerging roles of metaplasticity in behaviour and disease[END_REF]. (PLC) or an increase of intracellular Ca2+ (or both), in the postsynaptic neuron initiates the synthesis of an endocannabinoid (eCB). The eCB is subsequently released from the postsynaptic neuron, travels retrogradely to bind to presynaptic cannabinoid 1 receptors (CB1R), and this prolonged activation of CB1Rs depresses neurotransmitter release via unknown mechanisms. Adapted from [START_REF] Kauer | Synaptic plasticity and addiction[END_REF].

Hippocampus and its circuitry 1.2.1 Learning and memory

Learning and memory are closely related concepts. Learning is the acquisition of skill and knowledge, while memory is the faculty by which the mind stores and remembers information, and is the retention of information over time for the purpose of influencing future action [START_REF] Kazdin | Encyclopedia of Psychology[END_REF]. Learning and memory are so immediately relevant to everyone's daily life, yet remain poorly understood in terms of mechanisms.

Questions such as what is the nature of memory, where the memories are stored, how they are engraved and retrieved have intrigued generations of people till today.

In 400 B.C., Hippocrates was the first one to declare brain as the major controlling center for the body despite Egyptian, biblical, and earlier Greek views, which were heart-centered theory [START_REF] Finger | Minds Behind the Brain: A history of the pioneers and their discoveries[END_REF]. And the first sign that certain parts of the brain might be involved in memory storage came from the very famous case of patient Henry

Molaison. He was an American epileptic patient who had a bilateral medial temporal lobectomy to surgically resect the anterior two thirds of his hippocampi, parahippocampal cortices, entorhinal cortices, piriform cortices, and amygdalae in an attempt to cure his epilepsy. After the surgery, he exhibited severe anterograde amnesia characterized by the inability to form new memories, while his remote memories together with his ability of reasoning and acquiring new motor skills seemed intact (Scoville & Milner, 1957). Numerous clinical case-studies have demonstrated in the last century that the anterior hippocampus and hippocampal gyrus, either separately or together, are critically concerned in the retention of current experience [START_REF] Scoville | Loss of recent memory after bilateral hippocampal lesions[END_REF]. Altogether, it is now widely-accepted that the ability of learning and memory exist in many different forms involving various regions in the brain.

In an effort to categorize forms of memory, Richard Atkinson and Richard Shiffrin proposed in 1968 the Atkinson-Shiffrin model (also known as the multi-store model or modal model) dividing memory as sequential events of: sensory register, short-term memory (also known as working memory, <1min), and long-term memory [START_REF] Spence | The psychology of learning and motivation[END_REF]. Long-term memory involves many processes such as encoding, consolidation, storage and retrieval of memories, and is normally divided into declarative and nondeclarative memories, also known as explicit and implicit memories. Declarative memory is the conscious storage and recollection of data, and can be further divided into semantic and episodic memory [START_REF] Clayton | Looking for episodic memory in animals and young children: prospects for a new minimalism[END_REF][START_REF] Eichenbaum | Declarative memory: insights from cognitive neurobiology[END_REF].

Semantic memory refers to memory that encodes notion, concepts, or specific meaning [START_REF] Grilli | Personal semantic memory: insights from neuropsychological research on amnesia[END_REF], while episodic memory refers to the ability to re-experience a time-and-place-specific event in its original context [START_REF] Schacter | Remembering the past to imagine the future: the prospective brain[END_REF][START_REF] Szpunar | Episodic Future Thought: An Emerging Concept[END_REF]. Declarative memory is usually the primary process thought of when referencing memory (Eysenck, 2012). Declarative memory is a very complicated process involving many different brain regions, among which hippocampus mostly lies in the center of attention [START_REF] Huijgen | The hippocampus: A central node in a large-scale brain network for memory[END_REF] (Figure 1.8). (BIC) model [START_REF] Diana | Imaging recollection and familiarity in the medial temporal lobe: a three-component model[END_REF].

Normally when people refer to memory, declarative memory is usually the one they are thinking of. However, non-declarative memory also plays a significant role in daily life. Non-declarative memory is the storage and recollection of information without conscious attention to learning [START_REF] Fitts | The information capacity of the human motor system in controlling the amplitude of movement[END_REF][START_REF] Ullman | A neurocognitive perspective on language: the declarative/procedural model[END_REF]. An example of a non-declarative learning could be for example acquiring the skills of riding a bicycle or playing the violin. When needed, these memories are unconsciously activated [START_REF] Tulving | Priming and human memory systems[END_REF].

Memory is not a perfect system, and it can be affected by many factors or even manipulated. However, learning and memory is an essential activity in daily life not only for higher organisms but also for invertebrates such as honeybees [START_REF] Menzel | The honeybee as a model for understanding the basis of cognition[END_REF] and Drosophila [START_REF] Ostrowski | Place memory retention in Drosophila[END_REF].

Hippocampus in learning and memory

The earliest description of hippocampal formation comes from the Venetian anatomist Julius Caesar Aranzi ( 1587), who likened it first to a silkworm and then to a seahorse, and that is why it got its name from the Greek word "seahorse". Humans and other mammals have two hippocampi, one in each hemisphere, located in the limbic system of cerebral cortex. The anatomy and neural circuitry of the hippocampal formation are very similar across different species of mammals [START_REF] Anderson | The hippocampus book[END_REF] [START_REF] Strange | Functional organization of the hippocampal longitudinal axis[END_REF]) (Figure 1.9).

Over the years, many theories have been proposed trying to explain the exact role of hippocampus in learning and memory, which are summarized by [START_REF] Bird | The hippocampus and memory: insights from spatial processing[END_REF]) (Table 1.2).

Table 1.2 Theories on the role of the hippocampus in learning and memory.

Declarative Theory [START_REF] Squire | Mechanisms of memory[END_REF][START_REF] Squire | The medial temporal lobe[END_REF] The hippocampus, acting in concert with other medial temporal lobe regions, is crucial for all forms of consciously accessible memory processes (episodic and semantic, recollection and familiarity) for a timelimited period. Ultimately all memories are consolidated to neocortical sites and are thus unaffected by subsequent medial temporal lobe damage.

Multiple-Trace Theory [START_REF] Nadel | Memory consolidation, retrograde amnesia and the hippocampal complex[END_REF] The hippocampus, together with other medial temporal lobe regions, is crucial for the acquisition of episodic and semantic memories. The recollection of episodic memories remains dependent on the hippocampus for the duration of one's life and becomes more resistant to partial damage with repetition and/or rehearsal, whereas semantic memories become independent of the hippocampus and are stored in other brain regions over time.

Dual-Process Theory [START_REF] Aggleton | Episodic memory, amnesia, and the hippocampal-anterior thalamic axis[END_REF][START_REF] Eichenbaum | The medial temporal lobe and recognition memory[END_REF][START_REF] Norman | Modeling hippocampal and neocortical contributions to recognition memory: a complementary-learning-systems approach[END_REF][START_REF] Rugg | Human recognition memory: a cognitive neuroscience perspective[END_REF] The hippocampus is crucial for episodic recollection of the contextual details of an event. Familiarity-based recognition processes are subserved by other medial temporal lobe regions. Recollection is required for the associative recognition of non-unitized items (for example, voiceface pairs).

Relational Theory [START_REF] Cohen | Memory for items and memory for relations in the procedural/declarative memory framework[END_REF] The hippocampus allows the flexible association of information in neocortical modules that could not otherwise communicate. This enables the relations between elements of a scene or event to be retrieved or used for inference in novel situations, in addition to retrieval of the elements themselves. The Cognitive-Map Theory can be subsumed as a special case of spatial relational processing.

Cognitive-Map Theory [START_REF] O'keefe | The Hippocampus as a Cognitive Map[END_REF] A primary role of the mammalian hippocampus is to construct and store allocentric (world-centered) representations of locations in the environment to aid flexible navigation, for example, from a new starting position. In humans, these predominantly spatial processes have evolved to support the spatial-temporal context of episodic memories.

All of these theories acknowledged the importance of hippocampus in learning and memory, especially in consolidation stage of declarative memory and spatial navigation, remaining differences and disputes in whether hippocampus has a timelimited role in episodic memory and hippocampus's function in the acquisition of noncontextual information.

Hippocampal anatomy and circuitry

The hippocampus is made up of four regions or subfields: CA1, CA2, CA3, and CA4. The CA4 region is also known as hilus, or hilar region if considered as a part of the dentate gyrus [START_REF] Amaral | A Golgi study of cell types in the hilar region of the hippocampus in the rat[END_REF][START_REF] Blackstad | Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination[END_REF]. The abbreviation CA comes from "Cornu Ammonis", which is an earlier name of the hippocampus.

The dentate gyrus is composed of three layers: molecular layer (relatives cell-free, with the exception of some interneurons), granular layer (densely packed with granule cells), and polymorphic layer (contains a number of cell types the most prominent being mossy cells) [START_REF] Amaral | The dentate gyrus: fundamental neuroanatomical organization (dentate gyrus for dummies)[END_REF]. The CA regions are also Moreover, in rodents the two hippocampi are highly connected at the stems by the commissure of fornix (also called the hippocampal commissure), while in primates this commissural connection is much sparser [START_REF] Gloor | The human dorsal hippocampal commissure. An anatomically identifiable and functional pathway[END_REF].

Hippocampal CA3 circuits

As is described in the last chapter, the DG has relatively simple connection, receiving inputs from EC and projecting to CA3. The DG is only sparsely activated for any input [START_REF] Chawla | Sparse, environmentally selective expression of Arc RNA in the upper blade of the rodent fascia dentata by brief spatial experience[END_REF], probably because DG has significantly more granule cells (~1,000,000) than cells of EC (~200,000) projecting to DG [START_REF] Amaral | Neurons, numbers and the hippocampal network[END_REF]Rebola, Carta, & Mulle, 2017) (Figure 1.11). This type of connection makes it easier for DG to have distinct, non-overlapping activation patterns during each input from EC [START_REF] O'reilly | Hippocampal conjunctive encoding, storage, and recall: avoiding a trade-off[END_REF]. Besides, DG granular cells are interconnected by excitatory mossy cells and inhibitory interneurons located in the hilus [START_REF] Scharfman | Hilar mossy cells of the dentate gyrus: a historical perspective[END_REF]. However, the DG-CA3 connection is found not to be strictly unidirectional, with CA3 collateral fibers reciprocally influencing DG granule cells through mossy cells and hilar interneurons [START_REF] Myers | Pattern separation in the dentate gyrus: a role for the CA3 backprojection[END_REF]. In contrast to DG, CA3 receives more complicated excitatory inputs: from DG granule cell through mossy fibers [START_REF] Blackstad | Distribution of hippocampal mossy fibers in the rat. An experimental study with silver impregnation methods[END_REF][START_REF] Swanson | An autoradiographic study of the organization of intrahippocampal association pathways in the rat[END_REF], directly from layer II of the EC via the perforant path [START_REF] Witter | Organization of the entorhinal-hippocampal system: a review of current anatomical data[END_REF], and through recurrent collaterals from CA3 itself [START_REF] Myers | Pattern separation in the dentate gyrus: a role for the CA3 backprojection[END_REF]. The Mf-CA3 connection is especially interesting for its peculiar structure. The mossy fibers (Mfs) were named by Ramon y Cajal for the varicosities all along their axons, giving them a "mossy" appearance. They form three morphologically different synaptic terminals, include the large mossy terminals (LMTs), filopodial extensions of the terminals, and smaller "en passant" synapses [START_REF] Acsady | GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus[END_REF]. The mossy fiber boutons are large complex terminals with multiple releasing sites and packed with synaptic vesicles [START_REF] Blackstad | Special axo-dendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of mossy fibers[END_REF][START_REF] Laatsch | Electron microscopic studies of the dentate gyrus of the rat. I. Normal structure with special reference to synaptic organization[END_REF]. They form synapses with the proximal dendritic spines of CA3 PCs in stratum lucidum of CA3 region, consisting the feedforward excitation of Mf-CA3 pathways. These spines on CA3 pyramidal cell dendrites are called "thorny excrescences" (ThEs) [START_REF] Gonzales | Distribution of thorny excrescences on CA3 pyramidal neurons in the rat hippocampus[END_REF].

Meanwhile, the filopodia of LMT form synapses with local interneurons for feedforward inhibition onto CA3 PCs (Ruediger et al., 2011) (Figure 1.12). blue) in P14 and adult mice. Adapted from [START_REF] Wilke | Deconstructing complexity: serial block-face electron microscopic analysis of the hippocampal mossy fiber synapse[END_REF].

A single mossy fiber axon may make as many as 37 contacts with a single CA3

PC, but innervates only about 14 different CA3 PCs [START_REF] Claiborne | A light and electron microscopic analysis of the mossy fibers of the rat dentate gyrus[END_REF]. In contrast, each DG granule cell is innervating as many as 40 to 50 interneurons in CA3 area [START_REF] Acsady | GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus[END_REF]. Intriguingly, these numbers can be dynamically changed by, for example, contextual or spatial learning [START_REF] Crusio | Learning spatial orientation tasks in the radialmaze and structural variation in the hippocampus in inbred mice[END_REF]Ruediger et al., 2011).

On the other hand, each CA3 pyramidal cell receives input from about 50 different DG granule cells [START_REF] Crusio | A quantitative-genetic analysis of hippocampal variation in the mouse[END_REF], as well as ~3600 excitatory inputs from EC layer II via perforant path (PP), and ~12000 contacts from recurrent collaterals of other CA3 PCs [START_REF] Rolls | A quantitative theory of the functions of the hippocampal CA3 network in memory[END_REF]. However, the number of contacts doesn't necessarily mean stronger innervation, considering the synaptic-distancedependent scaling in the brain (de Jong, Schmitz, Toonen, & Verhage, 2012; K. J. [START_REF] Lee | Mossy fiber-CA3 synapses mediate homeostatic plasticity in mature hippocampal neurons[END_REF]. Although sparse, Mf-CA3 synapses at the proximal dendrites of CA3 pyramidal cells are a powerful connection on the main path of information flow, and are thought to enhance the signal-to-noise ratio and optimize the selection of populations of CA3 PCs firing in a correlated manner.

In CA3 area, inhibitory neurons play a pivotal role in information transfer as well.

The CA3 interneurons receive inputs from various sources, including the associational/commissural fibers (A/C), the mossy fibers (MF), and the perforant path (PP). These interneurons serve both the feed-forward and feed-back circuits and can contact hundreds of CA3 pyramidal cells [START_REF] Lawrence | Interneuron diversity series: containing the detonation--feedforward inhibition in the CA3 hippocampus[END_REF]. It has been shown in the last century that MF synapses on interneurons (may be via filopodial extensions or small en passant boutons and occasional large boutons) are significantly more than MF synapses on CA3 pyramidal cells, the ratio being approximately 10 to 1 [START_REF] Acsady | GABAergic cells are the major postsynaptic targets of mossy fibers in the rat hippocampus[END_REF]. This MF preferential innervation of interneurons may underlie the overall inhibitory effect of DG innervation of the CA3 network [START_REF] Bragin | Epileptic afterdischarge in the hippocampal-entorhinal system: current source density and unit studies[END_REF], Penttonen et al., 1997).

The interneurons located in the CA3 area are not a homogenous group and can be from [START_REF] Szabadics | Functional specificity of mossy fiber innervation of GABAergic cells in the hippocampus[END_REF].

Although there have been many attempts to explain populational activities through cell-type-specific interactions, we still lack the concrete evidence of how different subtypes of CA3 interneurons control information flow in the local circuits.

DG-CA3 connections have been implicated in pattern separation and assistance in memory encoding and the recall of contextual and spatial memory (Rebola et al., 2017). For example, it has been shown that transgenic inactivation of output of adult versus developmentally born DG neurons has differential impacts on pattern separation and completion [START_REF] Nakashiba | Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion[END_REF]. Some other study has demonstrated that pharmacological impairment of DG-CA3 transmission alters novel contextual representation [START_REF] Daumas | Activation of metabotropic glutamate receptor type 2/3 supports the involvement of the hippocampal mossy fiber pathway on contextual fear memory consolidation[END_REF][START_REF] Lassalle | Reversible inactivation of the hippocampal mossy fiber synapses in mice impairs spatial learning, but neither consolidation nor memory retrieval, in the Morris navigation task[END_REF]. Further evidence includes that mice with impaired Mf LTP are deficient for incremental learning [START_REF] Otto | Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptordeficient mice[END_REF], exposure to novel context regulates electrically induced plasticity [START_REF] Hagena | Learning-facilitated synaptic plasticity at CA3 mossy fiber and commissural-associational synapses reveals different roles in information processing[END_REF], and that there is structural remodeling of Mf boutons upon learning [START_REF] Holahan | Spatial learning induces presynaptic structural remodeling in the hippocampal mossy fiber system of two rat strains[END_REF][START_REF] Routtenberg | Adult learning and remodeling of hippocampal mossy fibers: unheralded participant in circuitry for long-lasting spatial memory[END_REF]Ruediger et al., 2011).

However, there are still open questions and missing evidence about the specific role of Mf-CA3 connections in memory encoding. For instance, whether there is a causal link between Mf LTP and memory encoding is still under debate [START_REF] Kaifosh | Mnemonic Functions for Nonlinear Dendritic Integration in Hippocampal Pyramidal Circuits[END_REF]. Moreover, other questions remain, such as the impact of NMDAR plasticity and its role in metaplasticity and synaptic integration (Hunt, Puente, Grandes, & Castillo, 2013;Rebola, Carta, Lanore, Blanchet, & Mulle, 2011), and the role of Mfdependent heterosynaptic plasticity (input-unspecific synaptic plasticity) in memory encoding.

Computational modeling of CA3 circuits

The relative simplicity of the hippocampal networks, when compared with e.g. neocortex, strongly appealed to computational neuroscientists to build models and test theories about memory mechanisms as early as since the 1970s. The original idea was built on Hebbian theory, stating "when an axon of cell A is near enough to excite cell B and repeatedly or persistently takes part in firing it, some growth process or metabolic change takes place in one or both cells such that A's efficiency, as one of the cells firing B, is increased" [START_REF] Hebb | The Organization of Behavior: A Neuropsychological Theory[END_REF]. In the most influential early model of Marr, he constructed an "autoassociator" that learns to associate all components of an input pattern with all other components of the same pattern [START_REF] Marr | Simple memory: a theory for archicortex[END_REF].

With the computational power and accessibility change in the 1990s, the amount of publication surged in this field. Computational models of the hippocampus, especially of DG-CA3 circuits have been numerously proposed, because this network shares many of the basic connectivity requirements for an autoassociator [START_REF] Hasselmo | Disruption of neurogenesis by amyloid beta-peptide, and perturbed neural progenitor cell homeostasis, in models of Alzheimer's disease[END_REF][START_REF] Korol | Hippocampal synaptic enhancement and spatial learning in the Morris swim task[END_REF].

These models focused mainly on the ability of the hippocampal regions to perform sequential learning, spatial navigation, and the consolidation of episodic memories.

For example, Levy presented a model of hippocampal CA3 region as a sequence predictor, and the model was able to learn and use context by solving sequence and configural learning problems, providing a computational unification of a variety of putative hippocampal-dependent functions [START_REF] Levy | A sequence predicting CA3 is a flexible associator that learns and uses context to solve hippocampal-like tasks[END_REF]. Liaw & Berger described a simple hippocampal network consisting of a small number of neurons connected with dynamic synapses could perform speech recognition, suggesting that the dynamic interplay between synapses (facilitative and inhibitory) results in an emergent "temporal chunking" mechanism for sequential pattern recognition [START_REF] Liaw | Dynamic synapse: a new concept of neural representation and computation[END_REF].

In some more recent works, Myers & Scharfman presented a simple computational model of the dentate gyrus to demonstrate that hilar cells may mediate dynamic regulation of pattern separation, by mediating the backprojection from CA3 to DG [START_REF] Myers | A role for hilar cells in pattern separation in the dentate gyrus: a computational approach[END_REF]. Hiratani et al. constructed an associative memory network model of spiking neurons that stores multiple memory patterns in a connection matrix with a lognormal weight distribution, demonstrating that heavy-tailed distributions of connection weights can generate noise which is useful for associative memory recall [START_REF] Hiratani | Associative memory model with longtail-distributed Hebbian synaptic connections[END_REF].

Computational models provide us a chance to connect data at multiple levels including molecular, cellular, anatomical and behavioral levels, no matter they are developed in a top-down (computational models predicting biological details) or bottom-up (computational models simulating biological data) fashion [START_REF] Gluck | Computational models of the neural bases of learning and memory[END_REF]. The computational and biological fields of neuroscience can inform each other and jointly lead to better understanding of our brain.

Brain plasticity on a molecular level 1.3.1 Epigenetics and chromatin plasticity

Now diving to an even deeper and more basic level under learning and memory, right to the eukaryotic cell nuclei, scientists have been trying to explain plasticities happening in the brain on an epigenetic level.

The notion "epigenetics" was first proposed by C. H. Waddington in 1942 to describe that the genes might interact with each other and with their environment to produce a phenotype [START_REF] Waddington | The Epigenetics of Birds[END_REF].

A gene is a locus or region of DNA and the molecular unit of heredity (Alberts, Johnson, Lewis, Raff, Roberts, & Walter, 2002). Genes are compactly stored on chromosomes (the condensed form) or chromatins (the unraveled form) (Figure 1.14).

The more condensed form of chromatin is called heterochromatin, and the genes located in heterochromatic region usually are less active or not active, while euchromatin is dispersed and loose, making the genes located at euchromatic regions relatively active [START_REF] Grewal | Heterochromatin and epigenetic control of gene expression[END_REF].

Approximately 146 base pair (bp) of DNA strand is wrapped around a histone octamer core protein, forming a structure called nucleosome. The "linker DNA" between two nucleosomes can be up to 80 bp long. The histone octamer consists of 4 types of histones: H2A, H2B, H3, and H4, with two copies of each [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF]. The histone proteins are overall positively charged, making them attracted to negatively charged DNAs. Importantly, each histone protein has an N-terminal tail sticking out, which is subject to many forms of modifications, such as acetylation, methylation, phosphorylation, ubiquitylation and ADP-ribosylation [START_REF] Strahl | The language of covalent histone modifications[END_REF][START_REF] Vaquero | The constantly changing face of chromatin[END_REF]. These post-translational modifications on histone tails actively regulate the interaction between DNA and histones. If the positive charge is reduced by the modification, the attractive force between histone and DNA decreases, thus making DNA more accessible for translational machinery; conversely, when the positive charge is increased, DNAs are less likely to be translated.

Histone Methylation as a transcription regulator

Unlike histone acetylation, which almost always facilitates local gene expression, histone methylation can either promotes or represses gene expression, depending on which residue is modified [START_REF] Justin | Reading, writing and editing methylated lysines on histone tails: new insights from recent structural studies[END_REF][START_REF] Scharf | Every methyl counts--epigenetic calculus[END_REF][START_REF] Shi | Dynamic regulation of histone lysine methylation by demethylases[END_REF][START_REF] Shilatifard | Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation[END_REF]. It occurs on lysine (K) or arginine (R) residues of histone tails, and it can happen more than once on the same residue, resulting in monomethylation, dimethylation or trimethylation [START_REF] Mosammaparast | Reversal of histone methylation: biochemical and molecular mechanisms of histone demethylases[END_REF][START_REF] Santos-Rosa | Active genes are tri-methylated at K4 of histone H3[END_REF][START_REF] Schneider | Molecular regulation of histone H3 trimethylation by COMPASS and the regulation of gene expression[END_REF]. A summary of known histone methylation types is listed in Table 1. 3 (Di Lorenzo & Bedford, 2011).

H4K20me

Transcriptional silencing [START_REF] Nishioka | PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin[END_REF] H4K20me3 Heterochromatin [START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF] H3K4 is the one of the most extensively targeted positions among all possible histone methylation sites [START_REF] Vallianatos | Disrupted intricacy of histone H3K4 methylation in neurodevelopmental disorders[END_REF], and is always facilitating translation. With the advances of technological nowadays, especially chromatin immunoprecipitation (ChIP) together with quantification of associated DNA by nextgeneration sequencing (ChIP-seq), it has been shown that H3K4 methylation has wideranging roles on a genome-wide scale, and is closely related to transcriptional regulation and epigenetic tagging of promoter and enhancer sequences in both neurodevelopment and neuropsychiatric diseases [START_REF] Barski | High-resolution profiling of histone methylations in the human genome[END_REF].

ChIP-seq studies have shown that the distribution of the trimethylated form of histone 3 (H3K4me3) in the human brain shows approximately 30 000 sharp "peaks" genome-wide [START_REF] Cheung | Developmental regulation and individual differences of neuronal H3K4me3 epigenomes in the prefrontal cortex[END_REF][START_REF] Shulha | Human-specific histone methylation signatures at transcription start sites in prefrontal neurons[END_REF]. These "peaks" (1-2 kb in length) can differ among different cell types, and are mostly located around transcription start sites (TSSs) of proximal gene promoters and other regulatory sequences (e.g. CpG islands) [START_REF] Barrera | Genome-wide mapping and analysis of active promoters in mouse embryonic stem cells and adult organs[END_REF][START_REF] Guenther | A chromatin landmark and transcription initiation at most promoters in human cells[END_REF]. In contrast, the di-and mono-methylated forms of histone 3 (H3K4me2 and H3K4me1) are organized as much broader peaks and appear in a much larger proportion of open chromatin, including promoters and enhancers [START_REF] Barski | High-resolution profiling of histone methylations in the human genome[END_REF][START_REF] Maston | Characterization of enhancer function from genome-wide analyses[END_REF].

H3K4me3 in learning and memory

As is mentioned in the last chapter, H3K4me3 markers appear in promoter areas across the genome in a more concentrated and cell-type specific manner compared to H3K4me1 and H3K4me2 [START_REF] Guenther | A chromatin landmark and transcription initiation at most promoters in human cells[END_REF], and is thought to be an important additional layer of transcriptional regulation [START_REF] Shilatifard | Molecular implementation and physiological roles for histone H3 lysine 4 (H3K4) methylation[END_REF][START_REF] Shilatifard | The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis[END_REF].

One study showed that H3K4me3 level is upregulated in hippocampus 1 h following contextual fear conditioning and returned to baseline levels at 24 h [START_REF] Gupta | Histone methylation regulates memory formation[END_REF]. In a more recent study, people have found that retrieval of a recent contextual fear conditioned memory increased global levels H3K4me3 in hippocampal CA1 area, and in vivo knockdown of the H3K4me3 methyltransferase Mll1 in CA1 impaired fear memory [START_REF] Webb | Dynamic association of epigenetic H3K4me3 and DNA 5hmC marks in the dorsal hippocampus and anterior cingulate cortex following reactivation of a fear memory[END_REF]. Another report stated that baseline resting levels of H3K4me3 were altered in the aged rat hippocampus as compared to young adults, and object learning can increase hippocampal H3K4me3 levels in young adult rats but not aged ones with memory deficits, which can be rescued by increasing H3K4me3 levels in aged rats with a histone demethylase inhibitor treatment [START_REF] Morse | Environmental enrichment reverses histone methylation changes in the aged hippocampus and restores age-related memory deficits[END_REF]. In line with these results, increased levels of H3K4me3 were found in the hippocampus of mice with enhanced hippocampal-dependent learning and memory (offspring of high licking/grooming mothers), and this results in higher expression of mGluR1 in hippocampus together with increased mGluR1-induced LTD and pairedpulse depression (PPD) [START_REF] Bagot | Variations in postnatal maternal care and the epigenetic regulation of metabotropic glutamate receptor 1 expression and hippocampal function in the rat[END_REF] Moreover, altered levels of H3K4me3 were also found in a few mouse models with intellectual disabilities. For example, a ketogenic diet modulated H3K4me3 levels in the granule cell layer, rescuing both the neurogenesis defect and hippocampal memory abnormalities in a mouse model of Kabuki syndrome [START_REF] Benjamin | A ketogenic diet rescues hippocampal memory defects in a mouse model of Kabuki syndrome[END_REF].

Disruption of KDM5C in mice, which predominantly represses genes with high levels of H3K4me3 at the promoters, recapitulates adaptive and cognitive abnormalities seen in human patients with X-linked intellectual disability (XLID) [START_REF] Iwase | A Mouse Model of X-linked Intellectual Disability Associated with Impaired Removal of Histone Methylation[END_REF].

Interestingly, abnormal levels of H3K4me3 were also observed in brains of patients with other neuropsychiatric disorders, such as Huntington disease (Vashishtha et al., 2013), autism (Shulha, Cheung, et al., 2012), schizophrenia [START_REF] Kano | Genome-wide profiling of multiple histone methylations in olfactory cells: further implications for cellular susceptibility to oxidative stress in schizophrenia[END_REF], and addiction to cocaine or alcohol [START_REF] Zhou | Substance-specific and shared transcription and epigenetic changes in the human hippocampus chronically exposed to cocaine and alcohol[END_REF].

Collectively, these findings suggest that the levels of H3K4me3, especially in the hippocampus, are actively regulated and closely correlated with learning and memory functions.

Alzheimer's disease (AD) 1.4.1 Historical background and clinical aspects of AD

First reported in 1907 by the German psychiatrist and pathologist Alois Alzheimer [START_REF] Berchtold | Evolution in the conceptualization of dementia and Alzheimer's disease: Greco-Roman period to the 1960s[END_REF]), Alzheimer's disease (AD) has been identified as the most common form of dementia, accounting for 60 -70% of all dementia cases nowadays [START_REF] Burns | Alzheimer's disease[END_REF]. Its global prevalence is considerably high, affecting about 6% of the population over 65 years old, and the incidence increases with age [START_REF] Ferri | Global prevalence of dementia: a Delphi consensus study[END_REF]. According to estimation, over 30 million people worldwide are suffering from AD [START_REF] Querfurth | Alzheimer's disease[END_REF], which makes AD a major public health concern and a research priority in the academic world.

To be more specific, Alzheimer's disease is a chronic and progressive neurodegenerative disorder characterized by various cognitive and non-cognitive symptoms. The cognitive symptoms comprise memory loss, which is normally the first and universal symptom in the vast majority of cases, together with language difficulties, and executive dysfunctions and intellectual impairments [START_REF] Tarawneh | The clinical problem of symptomatic Alzheimer disease and mild cognitive impairment[END_REF].

The non-cognitive or neuropsychiatric symptoms include depression, hallucinations, delusions, and agitation [START_REF] Burns | Psychiatric phenomena in dementia of the Alzheimer type[END_REF]. In addition, difficulties with performing daily life activities and coexistence of vascular diseases are also quite common in AD cases [START_REF] Snowdon | Brain infarction and the clinical expression of Alzheimer disease. The Nun Study[END_REF]. Although the progression of AD can be very slow, the average life expectancy after diagnosis is usually 3 -9 years [START_REF] Todd | Survival in dementia and predictors of mortality: a review[END_REF]. Suggested diagnostic criteria are shown in Figure 1.15 [START_REF] Ballard | Alzheimer's disease[END_REF]. The cause of Alzheimer's disease is still mostly unknown, but many risk factors have been linked with the disease in case-control studies [START_REF] Burns | Alzheimer's disease[END_REF]) (Table 1.4).

Most of the AD cases are sporadic, and only 1% to 5% of cases are familial, where genetic mutations can be identified as single risk factors [START_REF] Reitz | Alzheimer disease: epidemiology, diagnostic criteria, risk factors and biomarkers[END_REF]. The familial AD (FAD) typically has an early onset before the age of 65 [START_REF] Blennow | Alzheimer's disease[END_REF]. In most of the early onset familial AD cases, autosomal dominant inheritance mutations in one of three genes can be found: the amyloid precursor protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2), on chromosomes 21, 14, and 1 respectively [START_REF] Waring | Genome-wide association studies in Alzheimer disease[END_REF]. However, a summary of all possible genetic factors is shown in Table 1.5 [START_REF] Ballard | Alzheimer's disease[END_REF].

Familial AD (FAD) on genetic and molecular level

There are many current hypotheses trying to explain the pathogenic mechanisms of FAD. One of the most popular hypothesis on FAD is "amyloid cascade hypothesis", which postulates that abnormal accumulation of amyloid-β (Aβ) plaques in different areas of the brain is the initiating event in AD pathogenesis, and could trigger various downstream neuropathologies leading to neurodegeneration and full onset of AD [START_REF] Barage | Amyloid cascade hypothesis: Pathogenesis and therapeutic strategies in Alzheimer's disease[END_REF].

The Aβ peptide is derived from amyloid precursor protein (APP), which belongs to an evolutionarily conserved family of type I transmembrane glycoproteins consisted of 695-770 amino acids. APP can be cleaved by α-, β-, or γ-secretase [START_REF] Ling | Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer's disease[END_REF]: α-secretase function can be carried out by 3 enzymes in the ADAM family (a disintegrin and metalloproteinase family enzyme): ADAM9, ADAM10 and ADAM17 [START_REF] Allinson | ADAMs family members as amyloid precursor protein alpha-secretases[END_REF]; β-secretase activity has been found in β-site APP-cleaving enzyme 1 (BACE1) [START_REF] Sinha | Purification and cloning of amyloid precursor protein beta-secretase from human brain[END_REF][START_REF] Vassar | Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE[END_REF]. The γ-secretase has been identified as a complex of enzymes composed of presenilin 1 or 2, (PS1, PS2), nicastrin, anterior pharynx defective (APH-1) and presenilin enhancer 2 (PEN2) [START_REF] Francis | aph-1 and pen-2 are required for Notch pathway signaling, gammasecretase cleavage of betaAPP, and presenilin protein accumulation[END_REF][START_REF] Levitan | PS1 N-and C-terminal fragments form a complex that functions in APP processing and Notch signaling[END_REF][START_REF] Steiner | PEN-2 is an integral component of the gamma-secretase complex required for coordinated expression of presenilin and nicastrin[END_REF][START_REF] Wolfe | Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity[END_REF][START_REF] Yu | Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing[END_REF].

The cleavage and processing of APP can be categorized into a non-amyloidogenic pathway and an amyloidogenic pathway as is shown in Figure 1.16 [START_REF] Nicolas | Amyloid precursor protein and neural development[END_REF]. In the prevalent non-amyloidogenic pathway, APP is cleaved by α-secretase within the Aβ domain, thereby preventing the production of the Aβ peptide. Two fragments are released consequently, the smaller membrane-tethered C-terminal fragment (C83) and the larger N-terminal ectodomain (sAPPα) [START_REF] Kojro | The non-amyloidogenic pathway: structure and function of alpha-secretases[END_REF]. Furthermore, C83 is subsequently cleaved by γ-secretase, producing an even shorter fragment called P3 [START_REF] Haass | beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms[END_REF]. Alternatively, there's also the amyloidogenic pathway for APP processing, leading to the generation of Aβ and later oligomers and plaques. In this pathway, APP is cleaved by β-secretase (β-site APP-cleaving enzyme 1; BACE1), resulting in an ectodomain (sAPPβ) in the extracellular medium, and a membrane-bound C-terminal fragment of 99 amino acids (known as C99). C99 is subsequently cleaved by the γ-secretase complex (consists of presenilin 1 or 2, nicastrin, anterior pharynx defective and presenilin enhancer 2) to release Aβ peptide. Aβ generated in this step are mainly Aβ40 (~90%), but the rarer product Aβ42 (~10%) is more hydrophobic and present as the predominant isoform in cerebral plaques in AD [START_REF] Jarrett | The carboxy terminus of the beta amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease[END_REF][START_REF] Younkin | The role of A beta 42 in Alzheimer's disease[END_REF].

As we have mentioned, FAD-associated mutations are identified as APP, presenilin-1 (PS1) and presenilin-2 (PS2) [START_REF] Waring | Genome-wide association studies in Alzheimer disease[END_REF]. Mutated APP might result in abnormal processing of the protein, for example, favoring the amyloidogenic pathway (the case of APPSwe) [START_REF] Haass | The Swedish mutation causes early-onset Alzheimer's disease by betasecretase cleavage within the secretory pathway[END_REF], or increasing the aggregation of Aβ and fibrils (the case of APPARC) [START_REF] Nilsberth | The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation[END_REF]. Moreover, increased copies of APP gene also result in AD [START_REF] Cabrejo | Phenotype associated with APP duplication in five families[END_REF]Rovelet-Lecrux et al., 2006). Presenilin 1 (PS1) and PS2 are key components of the γ-secretase complex, so their mutations can alter γ-secretase cleavage and promote the production of Aβ42 [START_REF] Guo | Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice[END_REF][START_REF] Jankowsky | Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42specific gamma secretase[END_REF].

Therefore, mutations in these three genes will lead to disturbed metabolism of Aβ and eventually AD pathology [START_REF] George-Hyslop | Molecular biology and genetics of Alzheimer's disease[END_REF].

Mouse models of FAD

Mice are commonly used as models for neuroscience research, because they have comparable brain networks and similar neurobiological processes to humans. Besides, mice are also easy to be manipulated genetically, and can be easily bred in a rapid and inexpensive manner. Therefore, numerous transgenic mouse models of FAD have been created for research [START_REF] Kitazawa | Transgenic mouse models of Alzheimer disease: developing a better model as a tool for therapeutic interventions[END_REF].

Unlike humans, wild type mice do not develop Aβ plaques during normal aging, probably because of small differences in APP gene within the Aβ sequence compared to human [START_REF] Dyrks | Amyloidogenicity of rodent and human beta A4 sequences[END_REF]. When wild type human APP is expressed or even overexpressed in mice, these mice still show no deposition of Aβ plaques and only very mild neuropathological changes [START_REF] Buxbaum | Expression of APP in brains of transgenic mice containing the entire human APP gene[END_REF][START_REF] Lamb | Introduction and expression of the 400 kilobase amyloid precursor protein gene in transgenic mice [corrected[END_REF][START_REF] Mucke | High-level neuronal expression of abeta 1-42 in wildtype human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation[END_REF]. Therefore, transgenic mice with mutant APP genes were necessary for the creation of FAD models.

The age of Aβ pathologies onset varied among these transgenic mice because of different mutations, different transgene promoters, and different expression levels in each mouse line. In general, mice overexpressing mutant APP will exhibit agedependent Aβ neuropathologies in the brain, including cognitive impairment, dystrophic neurites, reactive astrocytes and activated microglia, elevated innate immune and inflammatory responses, and synaptic loss [START_REF] Games | Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein[END_REF][START_REF] Hsiao | Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice[END_REF][START_REF] Sturchler-Pierrat | Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology[END_REF]. However, these mice did not show robust neurodegeneration or tau pathology in the brain.

Again, unlike in humans, PS1 mutations alone do not cause Aβ plaques in mouse brain probably because of different sequence in mouse APP (in Aβ domain). As a result, mutant human APP is often introduced into PS1 transgenic mouse lines. Usually, the double transgenic mice exhibit accelerated production of Aβ42 and earlier formation of Aβ plaques than APP transgenic mice [START_REF] Borchelt | Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins[END_REF]. In addition, knocking out PS1 is lethal for mice in the embryonic stage [START_REF] Shen | Skeletal and CNS defects in Presenilin-1-deficient mice[END_REF][START_REF] Wong | Presenilin 1 is required for Notch1 and DII1 expression in the paraxial mesoderm[END_REF], but postnatal conditional knockout of PS1 is not lethal anymore, suggesting that it plays a vital role during embryonic development.

On the other hand, PS2 deletion in embryos only causes a very mild phenotype with mild pulmonary fibrosis and hemorrhage in senility (Herreman et al., 1999), and the effect of postnatal conditional PS2 KO was also minor. In these PS2 KO models APP cleavage and Notch signaling seem not affected, suggesting the role of PS2 is possibly redundant [START_REF] Franberg | Minor contribution of presenilin 2 for gamma-secretase activity in mouse embryonic fibroblasts and adult mouse brain[END_REF].

Apart from the histochemical pathologies, the AD mouse models need to be validated by behavioral tests for evidence of deficits in either short-term or long-term memory. It is needed to note that whether non-human species display episodic memory is still controversial. As a result, we tend to use terms such as reference memory, or associative learning, for episodic-like memory phenomena in rodents [START_REF] Morris | Episodic-like memory in animals: psychological criteria, neural mechanisms and the value of episodic-like tasks to investigate animal models of neurodegenerative disease[END_REF].

Various behavioral tasks are available for evaluating the cognitive functions in rodent AD models, which have been nicely reviewed by Webster et al. [START_REF] Webster | Using mice to model Alzheimer's dementia: an overview of the clinical disease and the preclinical behavioral changes in 10 mouse models[END_REF]) (Table 1.7).

Table 1.7. Commonly used mouse behavioral tasks for cognitive deficits.

However, it is important to keep in mind that none of the current FAD mouse models fully recapitulate the whole spectrum of pathologies in FAD, including Aβ plaques accumulation, tangle formation, cognitive deficits, synaptic loss, and progressive neurodegeneration. However, transgenic mouse models of FAD remain invaluable tools in AD research to study specific aspects of the disease process, and meanwhile contributing to the evaluation of potential therapeutic strategies before clinical trials.

APPswe/PSEN1dE9 (APP/PS1) mice as an FAD model

Various FAD mouse lines are currently being used for Alzheimer's research worldwide, among which some popular ones are single/multiple APP mutations, single presenilin (PS) mutations, APPxPS1 mutations, APPxPS1xtau mutations (3xTg) and anti-NGF expressing (AD11) mice. Their electrophysiological features have been neatly reviewed by Marchetti and Marie (Marchetti and Marie, 2011).

The FAD model we used for this study, i.e. APPswe/PSEN1dE9 (APP/PS1) mice, were created by crossing mice that express mutant APP (APPswe) with mice that express mutant PS1 (PSEN1dE9). APPswe is a chimeric mouse/human APP (isoform 695) with a Swedish mutation under the mouse prion promoter, resulting in increased total Aβ production by modulating β-secretase cleavage [START_REF] Borchelt | A vector for expressing foreign genes in the brains and hearts of transgenic mice[END_REF][START_REF] Mullan | A pathogenic mutation for probable Alzheimer's disease in the APP gene at the N-terminus of beta-amyloid[END_REF][START_REF] Savonenko | Normal cognitive behavior in two distinct congenic lines of transgenic mice hyperexpressing mutant APP SWE[END_REF]. PSEN1dE9 is the human PS1 lacking exon 9 (M. K. [START_REF] Lee | Hyperaccumulation of FAD-linked presenilin 1 variants in vivo[END_REF], which can disturb γ-secretase cleavage and change it towards the more amyloidogenic direction.

The APP/PS1 mice start to have visible Aβ plaques in cerebral cortex and hippocampus at 6.5-month-old, aggravating with age [START_REF] Jankowsky | Co-expression of multiple transgenes in mouse CNS: a comparison of strategies[END_REF][START_REF] Kim | Analysis of differential plaque depositions in the brains of Tg2576 and Tg-APPswe/PS1dE9 transgenic mouse models of Alzheimer disease[END_REF][START_REF] Seo | Behavioral stress causes mitochondrial dysfunction via ABAD up-regulation and aggravates plaque pathology in the brain of a mouse model of Alzheimer disease[END_REF]. Specifically, in the hippocampus, plaques start to be noticeable at 6-month in the molecular layer (ML) of the DG, later (at 10-month) in both ML of DG and stratum radiatum (SR) of CA3. However, Aβ plaques were not observed in the stratum lucidum (SL) of CA3 (Viana da Silva et al., 2016). In addition, APP/PS1 mice also exhibit tau hyperphosphorylation, but still lack neuronal loss and neurofibrillary tangles (NFTs) [START_REF] Morrissette | Relevance of transgenic mouse models to human Alzheimer disease[END_REF].

Many cognitive impairments in this model have been documented. By the age of 6 months, APPSwe/PSEN1dE9 mice are reported to show working memory deficits in tests of Radial Arm Water Maze (RAWM) [START_REF] Volianskis | Episodic memory deficits are not related to altered glutamatergic synaptic transmission and plasticity in the CA1 hippocampus of the APPswe/PS1deltaE9-deleted transgenic mice model of ss-amyloidosis[END_REF], and Morris Water Maze (MWM) [START_REF] Cao | Intake of sucrose-sweetened water induces insulin resistance and exacerbates memory deficits and amyloidosis in a transgenic mouse model of Alzheimer disease[END_REF][START_REF] Ding | Retinoic acid attenuates beta-amyloid deposition and rescues memory deficits in an Alzheimer's disease transgenic mouse model[END_REF]. Deficits in associative learning have also been described in fear conditioning tasks starting at 6-8 months of age [START_REF] Cramer | ApoE-directed therapeutics rapidly clear beta-amyloid and reverse deficits in AD mouse models[END_REF][START_REF] Knafo | Morphological alterations to neurons of the amygdala and impaired fear conditioning in a transgenic mouse model of Alzheimer's disease[END_REF].

Impairments in reference memory can be detected by 6 months (Bernardo et al., 2009;[START_REF] O'leary | Visuo-spatial learning and memory deficits on the Barnes maze in the 16-month-old APPswe/PS1dE9 mouse model of Alzheimer's disease[END_REF][START_REF] Reiserer | Impaired spatial learning in the APPSwe + PSEN1DeltaE9 bigenic mouse model of Alzheimer's disease[END_REF]. Passive avoidance deficits are developed at 12 months of age (W. [START_REF] Zhang | Soluble Abeta levels correlate with cognitive deficits in the 12-month-old APPswe/PS1dE9 mouse model of Alzheimer's disease[END_REF]. There are also deficits in alternation tasks such as Y-maze of working memory at 6-month (Viana da Silva et al., 2016).

As a link between the histochemical and behavioral findings, electrophysiological experiments have also been done to investigate the pathologies of APP/PS1 mice.

Especially at the beginning stage of the disease, electrophysiological impairments can prelude visible Aβ plaques and behavioral deficits [START_REF] Hsia | Plaque-independent disruption of neural circuits in Alzheimer's disease mouse models[END_REF]. There have been reports about decreased DG LTP in 9-15 months APP/PS1 mice, and this might be due to upregulated GABAA receptor-mediated inhibition [START_REF] Yoshiike | GABA(A) receptor-mediated acceleration of agingassociated memory decline in APP/PS1 mice and its pharmacological treatment by picrotoxin[END_REF]. Besides, some evidence showed impaired SC-CA1 LTP in 3-12 months APP/PS1 mice [START_REF] Volianskis | Episodic memory deficits are not related to altered glutamatergic synaptic transmission and plasticity in the CA1 hippocampus of the APPswe/PS1deltaE9-deleted transgenic mice model of ss-amyloidosis[END_REF]. Furthermore, recently by our team, it has been shown that LTP within the associative network is abolished in CA3 PCs at 6 months of age in the APP/PS1 mice, and this loss of LTP was caused by activation of up-regulated neuronal adenosine A2A receptors (Viana da Silva et al., 2016).

To sum up, although tau protein tangles cannot be observed in this APP/PS1 mouse model, it still captures a lot of other AD pathologies, including synaptic and neuronal loss, Aβ plaques, cognitive impairments, and gliosis, therefore it serves as a good model of AD research, at least for our purpose of amyloid pathology investigation. Our goal was to investigate the AD pathologies at early stage of onset, so we chose to use 6month old mice, where visible plaques and cognitive declines are just about to appear and there's no neuronal loss yet. Therefore, 6-month was considered to be early stage in our case. Moreover, we used male mice only to reduce variability.

Objectives of the work

At the entry level of hippocampus, Mf-CA3 circuitry is thought have a key role in episodic memory encoding, especially at the early stage of acquisition.

Many changes are actively taking place both in healthy learning models and in Alzheimer's disease models. To combine the strengths of my home team of Dr.

Mulle in Bordeaux and my hosting team of Dr. Fischer in Goettingen, and also taking advantage of the opportunity of a one-month workshop in OIST Okinawa, we aimed at investigating the following issues:

1) Possible electrophysiological deficits in Mf-CA3 circuit of early FAD mouse model (APPswe/PS1dE9) at the age of 6-month, with a focus on short-term plasticity of glutamatergic synapses, GABAergic innervations onto CA3 PCs, and NMDAR and KAR-mediated events in CA3 PCs.

2) Electrophysiological changes in 2-month adult C57/Bl6j male mice shortly after contextual fear conditioning (CFC), at 3-hour and 24-hour time point respectively.

3) Screening of gene expression changes in 6-month APP/PS1 mice, and check for contextual memory-related epigenetic marker (histone modification: H3K4me3) changes in the hippocampal CA3 region.

4) Computational modeling of DG-CA3 neuronal networks in healthy condition, and test the fidelity of the output of the system with different spike patterns as inputs (shown in Annexes).

Chapter 2

Materials and Methods

2 Materials and Methods

Animal Use

Ethical Considerations

All procedures (including anesthesia and euthanasia) were carried out in accordance with the European guidelines for the care and use of laboratory animals.

The experiments performed in Bordeaux were approved by the animal care and use committee of Bordeaux Universities (CE50), and the experiments performed in Goettingen were approved by the animal ethics committee of the European Neuroscience Institute, Göttingen, Germany.

Mice

Adult C57/Bl6j male mice (8 weeks) were purchased from Janvier Labs (Daumas et al.). The cages were maintained on a 12-h dark/light cycle and kept in a temperatureregulated room, with free access to food and water. All the experiments were done in the light phase of their circadian cycles.

APP/PS1 mice and their WT littermates were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Throughout their life, all the mice were group-housed, ranging from 4 to 10 animals per cage. Food and water were provided ad libitum. The transparent plexiglass cages (38.1 x 19.1 x 12.7 cm) were maintained on a 12-h dark/light cycle, kept in a temperature-regulated room, and protected from exterior pathogens by a filter. All experiments were performed in the light phase of the circadian cycle in 6 months (26-32 weeks) male APP/PS1 and age-matched male WT littermates.

All mice were genotyped by the local animal facility and re-genotyped after each experiment with tail tissue taken by the experimenter.

APP/PS1 Mice Genotyping

--DNA Isolation from mouse tail tissue.

The tip of the tail was cut in a postmortem way after sacrificing each mouse, and put into a 1.5 ml tube (Eppendorf, Germany). 200 µl of DirectPCR® Lysis Reagent (Peqlab, Germany) were added into each tube together with 0.3 mg/ml Proteinase K (Peqlab, Germany). The tubes were then incubated at 55°C on a thermomixer (Eppendorf, Germany) for 16 h at 550 rpm. Afterwards, the tubes were incubated at 85°C for 45 min without shaking. 1.5 µl of the supernatant containing DNA was taken from the tube and kept at 4°C for later Polymerase Chain Reaction (PCR).

--Polymerase Chain Reaction (PCR):

PCRs were performed targeting APP and PSEN genes to confirm the genotypes of mice used in our experiment. The primers are listed as follows: The PCR mix contained the following to make a total volume of 25 µl each sample:

Dream Tag Green Buffer ----2.5 µl

dNTP mix (2.5 mM) -----------2 µl Primer P255 --------------------0.25 µl Primer P256 --------------------0.25 µl Primer P257 --------------------0.25 µl Primer P258 --------------------0.375 µl Primer P259 --------------------0.25 µl DNA -------------------------------1.5 µl Dream Tag -----------------------0.2 µl milliQ H2O -----------------------17.

Electrophysiology

Anesthesia drug (ketamine 75 mg/kg and xylazine 10 mg/kg) was diluted in saline and injected intraperitoneally to the mouse 5 min before decapitation or transcardial perfusion (for mice older than 2 months) followed by decapitation. The head was immediately placed into a petri dish filled with ice-cold cutting solution (Table 2.2) oxygenated with carbogen (95% O2, 5% CO2). The brain was rapidly removed from the skull, and parasagittal slices (350 mm) were cut with a Leica vibratome (Leica VT 1200S) in the cutting solution. The slices were then kept at 33°C in oxygenated resting solution (Table 2.2) for 20 min, before transferred to aCSF (Table 2.2). The slices were then left at room temperature for a maximum of 6h after cutting. -Igor Pro 6.37 with custom-made macros (WaveMetrics, USA) for off-line analysis

The recording chamber of the electrophysiology set-up was perfused with oxygenated aCSF (Table 2.2). CA3 pyramidal cells were identified by differential interference contrast microscopy at room temperature, and whole-cell patch-clamp configuration was achieved with borosilicate glass capillaries (Harvard Instruments, GC150F-10) pulled by a P-97 puller (Sutter Instrument). The pipettes were filled with variant intracellular solutions (Table 2.3) depending on experiments, and had resistance value ranging from 3 to 5 MΩ.

To monitor the access resistance during the whole recording time, a hyperpolarizing voltage step (-5mV, 10ms) was applied at the beginning of each trace.

Series access resistance was <20MΩ, and when it changed by >20%, the recording was discarded. When held at -70mV, neurons with a holding current >300pA were also rejected. Liquid junction potential correction was not used for measurements of membrane potentials.

Mf-CA3 excitatory postsynaptic currents (EPSCs) were evoked using a patch pipette (~5MΩ) filled with aCSF positioned in the hilus of the dentate gyrus. Minimal intensity stimulation was reached as previously described in literature [START_REF] Marchal | Postnatal maturation of mossy fibre excitatory transmission in mouse CA3 pyramidal cells: a potential role for kainate receptors[END_REF][START_REF] Sachidhanandam | Kainate receptors act as conditional amplifiers of spike transmission at hippocampal mossy fiber synapses[END_REF].

Mf synaptic currents were identified according to the following criteria:

--robust frequency facilitation when comparing responses at 0.1Hz and 1Hz, --obvious paired-pulse facilitation --EPSCs had a steep rising phase (∼1ms)

--EPSCs decays free of secondary peaks that might indicate the presence of polysynaptic contamination.

The group II metabotropic glutamate receptor (mGluR) agonist L-CCG-I (10 µM) was applied at the end of each recording. If the EPSCs were reduced by > 80%, we could confirm that the Mf synaptic currents were recorded. All drugs for electrophysiological experiments were obtained from Tocris Biosciences or Sigma-Aldrich, unless otherwise stated.

) CsMSO3 100 -- -- KMSO3 -- 120 -- CsCl -- -- 120 MgCl2 -- 2 2 MgSO4 3 -- -- CaCl2 3.5 1 2 KCl -- 20

Genomic profiling and ChIP-seq 2.3.1 Tissue Preparation and Neuron Sorting

The removal of hippocampal CA3 regions was performed under a stereomicroscope (Motic) as described previously (Sultan FA, 2013). The dissected CA3 tissues were snap frozen in liquid nitrogen immediately for later RNA isolation or further FACS preparation.

FACS were done on the dissected CA3 tissues to separate neurons and nonneuronal cells and debris.

--Nuclei Isolation:

The isolated tissues were homogenized with a mechanical homogenizer in 500 µl of low sucrose buffer (Table 2.4). For crosslinking, 13.5 µl of 37% formaldehyde were added, and samples were rotated 12rpm for 5 min at RT. Crosslinking was quenched by adding 51.4µl 1.25M glycine, and then the samples were kept on a rotator for 5 min before centrifuged at 2000 rcf for 3 min. The supernatant was discarded, and the cell pellet was resuspended in 1 ml low sucrose buffer and homogenize again. Then the samples were carefully pipetted onto 6 ml high sucrose buffer (Table 2.4), followed by centrifugation at 3200 rcf for 10 min at 4ºC. In each tube, the supernatant was removed, and the nuclei pellet was resuspended in 600µl PBS.

--Nuclei Staining:

0.6µl of NeuN-AF488 primary antibody (Millipore MAB 377X) was added to the resuspended solution containing nuclei. After 20 min incubation at 4ºC on a rotator, the samples were centrifuged at 2000 rcf for 3 min. Then the pellets were washed twice with 1 ml PBST (Table 2.4).

--Collection of sorted nuclei:

Nuclei were sorted with a BD FACSAria™ III cell sorter (BD Biosciences, USA) into falcon tubes pre-coated with PBTB (Table 2.4), then centrifuged at 3200 rcf for 15 min at 4ºC. The supernatant was discarded, and the pellet was resuspended in leftover buffer and transferred to a new 1.5 ml low-binding Eppendorf to be centrifuged again at 10000 rcf for 2 min. The nuclei pellets were snap frozen in liquid nitrogen and ready for genomic DNA isolation.

RNA Isolation and qPCR

-RNA isolation:

The dissected hippocampal CA3 tissue was homogenized in 400µl TRI Reagent (Invitrogen, Cat No: 15596-026), and incubated at RT for 5min. Then 80µl chloroform was added. After thorough shake by hand, the samples were incubated at RT for 5min, and centrifuged at 12000 rcf for 15min at 4ºC. The upper aqueous phase was carefully collected to mix with 200μl Isopropanol and 1.5μl Glycoblue (Ambion, 15 mg/mL, AM9515, L/N 1405030) and incubated at -20 ºC for 30min, before centrifugation at 12000 rcf for 15min at 4ºC.

The supernatant was discarded, and 5 μl 10X Incubation Buffer (Ambion), 1 μl DNase I (Ambion, AM2222, 2000U, 2U/μl), 0.5 μl RNase OUT (Invitrogen, P/N 100000840, 5000U, 40U/μl) and 43.5 μl RNase-free water were added to the pellet, and the samples were incubated on thermomixer at 550 rpm for 20min at 37 ºC. After adding 150 μl RNase-free water and 200 μl Phenol/Chloroform/Isoamylalcohol, the samples were vortexed and centrifuged at 13000 rpm at RT for 2 min.

The upper phase containing the RNA was carefully collected to mix with 20 μl 3M Na-acetate pH 4.8, 200 μl isopropanol, and 1 μl Glycoblue, before incubation for 30 min at -20 ºC. Afterwards, the samples were centrifuged at 13000 rcf at 4 ºC for 30min, and the pellets were washed and twice with 75% ethanol. The pellets were dried at RT and dissolved in 50µl of RNase-free water, snap-frozen at -80 ºC, and ready for RNAsequencing.

--cDNA Synthesis:

cDNA synthesis was carried out using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Germany). A mixture of 13µl in volume was made with 1 µg of RNA from the sample, 2 µl random hexamer primers (600 pM/µl), and PCR-grade H2O, and incubated at 65 ºC for 10min to denature the RNA. Then the following were added to each sample: 4 µl Transcriptor Reverse Transcriptase Reaction Buffer, 0.5µl Protector RNase Inhibitor (40U/µl), 2µl Deoxynucleotide Mix (10mM), and 0.5µl Transcriptor Reverse Transcriptase (20U/µl). The samples were subsequently incubated at 25 ºC for 10min, at 55 ºC for 30min, at 85 ºC for 5min. The synthesized cDNA was kept on ice or stored at 4 ºC.

--qRT-PCR (qPCR)

qPCR was performed to check the accuracy of CA3 dissection and the efficiency of ChIP in Light Cycler 480 (Roche Applied Science, Manheim, Germany) and its reaction kit (Roche, Germany). qPCRs were done in duplicate for each sample, and a mixture was pooled from each cDNA sample and diluted serially to the following standard dilutions: 1:1, 1:2, 1:4, 1:8, 1:16, to generate a standard curve for determining cDNA concentration of the samples. PCR-grade H2O was used as a negative control, and the 1:16 cDNA mixture dilution was used as a positive control. In the dissection accuracy check, Tdo2 was used as a marker for DG, Lphn2 for CA3, and Lyd for CA1 region. In the ChIP efficiency check, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was used as the reference gene, and InterG was used as a negative control for the intergenic area. Primers used are listed in Table 2.5. The qRT-PCR mix contained the following to make a total volume of 15 µl in each sample:

- -Shearing check:

Before proceeding, 5 µl from each sample were taken to check the DNA concentration and shearing efficiency. 20µl of EB and 0.1µg/µl RNase A were added to the 5µl chromatin sample and incubated on a thermocycler at 450 rpm at 37 ºC for 30min. Then 1 µl of Proteinase K (20mg/ml) and 20µl of Wiemann Buffer 2X (Table 2.6) were added, and incubated on thermocycler at 13000 rpm at 65 ºC for 2 hours.

Afterwards, 3 µl of LPA and 46µl of SureClean (Bioline, UK) were added, and incubated for 10min at RT, before centrifugation at 20000 rcf for 20min at RT. The supernatant was discarded, and the pellet was washed with 80% ethanol once, before being dried and resuspended in 20µl of EB. Now the DNA was ready for bioanalysis for concentration check and NanoDrop (Thermo Scientific, USA) for fragment size distribution check. EDTA, 500 mM LiCl -Immunoprecipitation:

1.8ml of IP Buffer without SDS were added to the samples in order to dilute the SDS to 0.1%. H3K4Me3 antibody (1µg/500ng of chromatin, Abcam, #ab8580) were added and incubated on a rotator at 4ºC overnight. 15µl of blocked protein A coated magnetic beads were added and incubated on a rotator at 4ºC for 1.5 h. The samples were then put on a magnetic rack, and the supernatant was discarded. The beads were washed twice with 1ml of IP Buffer, then three times with 1ml wash buffer, and 1ml of IP Buffer once again.

-DNA Isolation:

20µl of EB and 0.1µg/µl RNase A were added to the beads and the inputs, and incubated on a thermocycler at 450 rpm at 37 ºC for 30min. Then 1 µl of Proteinase K (20mg/ml) and 20µl of Wiemann Buffer 2X (Table 2.6) were added, and incubated on thermocycler overnight at 800 rpm at 65 ºC. The supernatant was collected on a magnetic rack, and 3 µl of LPA and 60µl of SureClean (Bioline, UK) were added, and incubated for 10min at RT, before centrifugation at 20000 rcf for 20min at RT. The supernatant was discarded and the pellet was washed twice with 80% ethanol, before being dried and resuspended in 30µl of EB. ChIP efficiency was confirmed with qRT-PCR as described in chapter 2.3.2. After concentration check and library preparation, ChIP-seq was performed.

Immunohistochemistry and Imaging

Immunofluorescent Staining for c-Fos

The post-fixation brain was kept in 4% PFA for 24h, before being transferred to PBS 1x (Euromedex, Ref ET330-A), and cut into 50µm slices on a vibratome. The slices were saturated in PBS 1x with 0.3% Triton (Sigma, T9284-100ml) and 2%

Gelatin (Merck Chemicals, 1040721000) for 1h at RT. The primary antibody (rabbit anti-c-Fos, Merck, PC38-100µl) was added at a concentration of 1/10000 and incubated overnight at 4 ºC. The slices were rinsed for 10min with PBS 1x -Triton 0.3%-Gelatin 2% for 3 times and kept in it. Then the biotinylated secondary antibody (donkey antirabbit, Fischer, AP182BMI) was added at a concentration of 1/200 and incubated for 3h at RT. The slices were rinsed for 10 min in PBS 1x for 3 times and transferred into PBS 1x -Triton 0.3%-Gelatin 2% with 5µg/ml Streptavidin-Alexa 568 (Fischer,W3319L) for incubation overnight at 4 ºC. The slices were dried for 5min and mounted on glass slides with coverslips for epifluorescence microscopy.

Confocal Imaging of Mf Terminals

Taking advantage of the genetically encoded fluorescence in Thy1-mGFP-DG mice, we were able to visualize Mf terminals and their filopodia (Ruediger et al., 2011).

Thy1-mGFP-DG mice bred in the local animal facility were anesthetized with intraperitoneal injection of pentobarbital (30 mg/kg), then transcardially perfused with physiological saline (9% NaCl) followed by 4% paraformaldehyde (4% PFA). Brains were removed and post-fixed overnight in 4% PFA. After fixation, coronal sections (70mm) were made on a vibratome (Leica, Germany), then mounted on a glass slide and covered with a thin glass coverslip. The brain sections were imaged on Leica DM6000 TCS SP5 confocal microscope (Leica Microsystems, Germany) for visualization of large mossy fiber terminals and filopodia.

Behavioral tests

Contextual fear conditioning, as one of the most relevant behavioral tests for hippocampal circuitry, was chosen in our experiment to activate Mf-CA3 networks.

Adult male mice were single-caged for at least one week before the start of fear conditioning. Contextual fear conditioning was performed using ANY-maze fear conditioning system and analyzed with its automatic freezing detection software (ANYmaze, Ireland). The mice received foot shocks in context A: the walls of the box were decorated with black-and-white striped papers. Mice were allowed to explore the box for 3min before receiving the first electrical shock. In total 5 mild electrical foot shocks (0.6 mA, 1s duration) were delivered through the metal grid at the bottom of the box, with 30s intervals in between. After the shocks, the mice were allowed to stay in the box for 2 more minutes before being taken out and transferred back to home cage. The box was cleaned with 30% ethanol and air-dried between each session to remove the residual smell. During the fear text, context B was used as the control: the walls were white decorated with orange triangles, and the floor was paved with normal padding.

After the conditioning, the mice were tested at the following time points: 1 hour, 1 day, 2 days, 5 days, 7 days, 14 days. During each test, the mice were first tested in Context B and then Context A, with half an hour in between. Each mouse was tested on maximum three testing days to minimize extinction.

Computational modeling

An open-source Python-based software NEST simulator (NEST initiative, Switzerland) was used to generate artificial neural networks of Mf-CA3 based on simplified anatomical data. Details of the construction of the artificial networks will be described in the first chapter of Annexes.

Statistical Analysis

Statistical analyses were performed with Prism6 (GraphPad Software, USA). First, the normality of data set was tested using the (the GO enrichment analysis) for genomic data was done using the topGO package (Bioconductor, USA) under the "weighted" option. Results were presented as mean ± SEM. Statistical differences were considered significant at P<0.05.

Results

3.1 Input-specific synaptic alterations in CA3 pyramidal cells at an early stage in a mouse model of Alzheimer's disease.

Located at the entry level of hippocampus, the CA3 region is thought to be important for the storage of episodic memory. Meanwhile, loss of hippocampaldependent episodic memory is one of the earliest clinical signs of AD (Sperling, 2007).

Thus, it is intriguing to study the physiological status of CA3 circuits in models of early AD.

CA3 pyramidal cells (CA3 PCs) receive a variety of synaptic inputs which display diverse anatomical and functional properties. Three types of glutamatergic inputs are innervating CA3 pyramidal cells: recurrent collaterals (associational/commissural, A/C fibers), perforant path from the entorhinal cortex, and inputs from the dentate gyrus (DG) through mossy fibers (Mf). These inputs are precisely positioned along apical and basal dendrites, and greatly vary in their structural and functional properties (Rebola et al., 2017). Mfs also make synaptic contacts with GABAergic CA3 interneurons, creating the anatomical basis for robust feedforward inhibition of CA3 PCs (Torborg et al., 2010), which is amenable to structural plasticity upon memory encoding [START_REF] Ruediger | Goal-oriented searching mediated by ventral hippocampus early in trial-and-error learning[END_REF]Ruediger et al., 2011). In AD mouse models, the inhibitory innervation in hippocampal circuits could be impaired (Hazra et al., 2013;Ramos et al., 2006).

We have previously shown that long-term synaptic potentiation (LTP) is abolished at A/C fiber synapses in CA3 pyramidal cells at an early stage (6-month of age) in a mouse model of AD, and this loss of LTP was caused by activation of up-regulated neuronal adenosine A2A receptors and correlated with one-trial memory deficits (Viana da Silva et al., 2016). In this work, we focused on the Mf synapses in the same region of the same mouse model, to investigated early changes in the hippocampal Mf-CA3 circuits during AD pathology. We hypothesized that, in line with early signs of learning and memory impairments and formation of plaques, male APP/PS1 mice might have deficits in glutamatergic synaptic plasticities as well as GABAergic transmissions at

Mf-CA3 synapses at the age of 6 months.

We first characterized the intrinsic properties of CA3 pyramidal cells and looked at the pre-and postsynaptic morphologies of Mf-CA3 synapses in 6-month APP/PS1 mice. Both being unaltered in this early stage of AD, we then explored the presynaptic short-and long-term plasticity of Mf-CA3 synapses in APP/PS1 mice, which are not significantly different from WT littermates either. However, we observed an impaired LTP of NMDA-EPSCs, and some subtle changes in GABAergic transmission and in feedforward inhibition in 6-month APP/PS1 mice.

Taken together, we report here in the form of a manuscript that early synaptic dysfunction in a mouse model of AD is differentially expressed at specific inputs onto a single CA3 neuron.

Input-specific synaptic alterations in CA3 pyramidal cells at an early stage in a mouse model of Alzheimer's disease.

In preparation

Pei Zhang 1* , Silvia Viana da Silva

*equal contribution

Corresponding author: Christophe Mulle, email: christophe.mulle@u-bordeaux.fr Introduction AD is characterized by two neuropathological hallmarks, senile plaques and intracellular neurofibrillary tangles, which result from the accumulation and deposition of the β-amyloid peptide (Aβ) and the aggregation of hyperphosphorylated tau protein, respectively. It is now well accepted that soluble oligomeric forms of Aβ, may play a prominent role in triggering the cognitive deficits by specifically targeting synapses. Synaptic loss and dysfunction better correlates with cognitive impairment in early AD, than amyloid-beta (Aβ) plaques, tangle formation or neuronal loss (Selkoe et al., 2002).

Loss of episodic hippocampal-dependent memory is the earliest clinical sign of AD and hippocampal regions show reduced activation during memory encoding tasks in patients with mild cognitive impairment (Sperling et al., 2007). The encoding of episodic memories is thought to initially require rapid synaptic plasticity within the hippocampus. The CA3 circuits of the hippocampus appear particularly well suited to encode memories, particularly at the earliest stage of acquisition, presumably by developing instant representations of a context [START_REF] Kesner | A computational theory of hippocampal function, and tests of the theory: new developments[END_REF]. It is thus important to study the physiological status of CA3 circuits in models of AD. CA3 pyramidal cells (PCs) form an autoassociative network of recurrent connections which is thought to enable the storage of information through synaptic plasticity (Rebola et al., 2017). Synaptic dysfunction in hippocampal circuits has been observed in a variety of mouse models of AD, particularly in the CA1 region or dentate gyrus (DG), and proposed to correlate with impairment in memory tasks (Marchetti and Marie, 2011;[START_REF] Pozueta | Synaptic changes in Alzheimer's disease and its models[END_REF]. We recently showed that long-term synaptic potentiation (LTP) within the associative network is abolished in CA3 PCs at an early stage in the APP/PS1 mouse model of AD (Viana da Silva et al., 2016). This loss of LTP was caused by activation of up-regulated neuronal adenosine A2A receptors and correlated with onetrial memory deficits (Viana da Silva et al., 2016).

Different synaptic connections onto a single neuron display a variety of anatomical and functional properties, which may be differentially affected in models of AD (Marchetti and Marie, 2011). This segregation of synaptic properties can be particularly well studied in laminated structures such as the hippocampus. CA3 PCs receive three types of glutamatergic inputs, from recurrent CA3 collaterals (associational/commissural, A/C fibers), from the entorhinal cortex (perforant path) and from the dentate gyrus (DG) through mossy fibers (Mf). These inputs are precisely positioned along apical and basal dendrites, and greatly vary in their structural and functional properties (Rebola et al., 2017). In contrast to A/C and perforant path synapses which are single site/single spine type, Mf inputs make synaptic contacts on proximal dendrites of CA3 PCs (in the stratum lucidum) via "giant" Mf boutons with multiple glutamate release sites facing large postsynaptic structures called thorny excrescences [START_REF] Henze | The multifarious hippocampal mossy fiber pathway: a review[END_REF]. At variance with A/C synapses, Mf-CA3 synapses display a wide dynamic range of shortterm plasticity, and express presynaptic forms of LTD and LTP which are independent of NMDA receptor (NMDAR) activation (Nicoll and Schmitz, 2005;Rebola et al., 2017). In addition, at Mf-CA3 synapses, NMDARs are themselves subject to LTP (Kwon and Castillo, 2008;Rebola et al., 2008). Mfs also make synaptic contacts with GABAergic CA3 interneurons creating the anatomical basis for robust feedforward inhibition of CA3 PCs (Torborg et al., 2010), which is amenable to structural plasticity upon memory encoding (Ruediger et al., 2011).

Here we have directly explored whether different synaptic inputs to CA3 PCs were differentially impaired at an early stage in the APP/PS1 mouse model of AD. We have mainly focused on Mf-CA3 synaptic inputs and compared our results with previous observations made at A/C synaptic inputs on the same mouse model (Viana da Silva et al., 2016).

Results

Analysis of intrinsic properties of CA3 PCs in APP/PS1 mice

In order to extend our previous work on A/C synapses, we chose to compare morphological and synaptic properties in CA3 PCs of APP/PS1 and WT mice at 6 months of age, prior to obvious plaque deposits (Viana da Silva et al., 2016). We first performed current-clamp recordings from CA3b PCs in hippocampal slices to analyze their intrinsic properties (Figure 1). The input resistance of CA3 PCs were not altered in APP/PS1 mice (Wt 356.7 ± 41.74 MΩ, n=13, and APP/PS1 406.8 ± 43.21 MΩ, n=12, t-test p value=0.4132). We then examined the firing threshold and firing frequency of CA3 PCs by injecting a ramp of current from 0 to 200 pA current. The firing threshold of CA3b PCs was not altered in APP/PS1 mice as compared to Wt littermates (Wt -43.04 ± 1.13 mV, n=13 and APP/PS1 -40.02 ± 1.37 mV, n=12, t-test p value=0.1005).

Although the firing frequencies during the ramp seemed lower in APP/PS1 mice, twoway ANOVA test of the two I/O curves did not reveal any significant difference (Wt 4.56 ± 0.83 Hz, APP/PS1 3.44 ± 0.65 Hz; Two-way ANOVA test p=0.5894, Wt n=13 and APP/PS1 n=12). No major differences were found between the two genotypes when the aforementioned recordings were made in the presence of NBQX (20μM), Bicuculline (10μM), CGP (3μM), D-AP5 (50μM) (Figure 1 E-H). Therefore, we conclude that the intrinsic properties of CA3b PCs in 6-month APP/PS1 mice are not altered at this early stage of AD pathology, excluding the possibility that this could account for potential differences observed.

Morphometric analysis of Mf boutons and thorny excrescences in 6-month male

APP/PS1 mice. We started our phenotypic comparison of diverse synaptic inputs by comparing the morphological properties of Mf-CA3 synapses in control and 6-months old APP/PS1 mice. For this study two types of recombinant rabies viruses (RV) were stereotaxically injected into the mouse brain to label DG granule cells and CA3 PCs. Local infection was achieved by injecting an anterograde RV expressing a membrane-targeting td-Tomato fluorescent protein in the DG, allowing us to trace DG cell projections as well as presynaptic nerve terminals, i.e. Mf boutons (MfBs). MfBs from Wt and APP/PS1 mice were reconstructed from confocal z-stacks obtained from the CA3b region (the same area in which the electrophysiological experiments were performed). The mean surface area of MfBs did not differ between Wt and APP/PS1 mice (Wt 67.1µm 2 ± 1.8µm 2 , n=898, 5 mice; APP/PS1 67.9µm 2 ± 1.6µm 2 , n=609, 5 mice; Mann-Whitney test p=0.6624), as also indicated by the cumulative distribution of areas (Figure 2 B; KS test p=0.144). In line with this result, the mean volume of MfBs was not different in APP/PS1 and Wt mice (Wt 15.2µm 3 ± 0.4µm 3 n=609, 5 mice; APP/PS1 14.6µm 3 ± 0.4µm 3 , n=898, 5 mice; Mann-Whitney test p=0.1630, Figure 2 C). Importantly, Mf also contact inhibitory interneurons in the hilus and stratum lucidum, through filopodial protrusions emerging from the MfBs. To get an estimate of the number of filopodia, we compared the complexity index of the reconstructed MfBs, since these structures represent a very small volume, but a considerable area of membrane. Our initial analyses show no significant difference in the average complexity index between two genotypes (Wt 3.0 ± 1.2, n=609, 5 mice; APP/PS1 3.5 ± 1.4, n=898, 5 mice; Mann-Whitney test p=0.3116). However, a difference was observed when analyzing the cumulative distribution of MfBs complexity indexes between Wt and APP/PS1 mice (KS test p<0.0001; Figure 2 D). To further analyze the difference in complexity we divided MfBs in three categories based on their volume: small (< 10µm 3 ), medium (10 -20µm 3 ) and large (> 20µm 3 ). We observed a tendency for an increased complexity for the large MfBs in the APP/PS1mice (Wt, 4.3 ± 0.2, n=5 mice; APP/PS1 mice 4.8 ± 0.2 for APP/PS1 mice, n=5 mice) (Figure 2 E). An increase in the complexity index may indicate an increase in the number and/or length of MfB filopodia.

To study the post-synaptic thorny excrescences (ThEs), a RV with the native envelope was used to infect CA3 PCs from their axon terminals. The injection performed in the hippocampal CA1 region resulted in a sparse labeling of CA3 PCs ideal for 3D reconstructions (Figure 2 F). Reconstructions were performed for ThEs located in the stratum lucidum, ThEs located on basal dendrites of CA3 PCs were not included in the study. Only cells with a complete ThE arborization within the 70µm slices were reconstructed and analyzed. The number of ThEs per cell was not different between the two genotypes (Wt 58.2 ± 4.0, n=9 complete cells; APP/PS1 51.6 ± 5.8, n=8 complete cells; t-test p=0.3539, Figure 2 G). We found no change of the mean surface values or distribution of reconstructed CA3 ThEs in APP/PS1 mice (Wt 16.6µm 2 ± 0.6µm 2 , n=1259, 6 mice; APP/PS1 14.2µm 2 ± 0.5µm 2 , n=945, 6 mice; Mann-Whitney test p=0.1074; distribution, KS test p=0.99; Figure 2 H). Similarly, we found no alteration of mean volume of ThEs in APP/PS1 mice (Wt 2.4µm3 ± 0.1µm3, n=1259, 6 mice; APP/PS1 2.0µm3 ± 0.08µm3, n=945, 6 mice; Mann-Whitney test p=0.1221) but a significant alteration in the distribution of volumes of ThEs (KS test p<0.0001; Figure 2 I). Summary plot of the area and volume of each ThE reconstructed (Figure 2 J) shows that the relationship between these two parameters is not altered by chronic overexpression of A during 6 months (Wt linear regression 5.7 ± 0.04; APP/PS1 linear regression 5.6 ± 0.06). Overall, careful morphometric examination of several parameters of Mf synapses showed not major alteration of pre-and post-synaptic compartments in APP/PS1 mice.

Analysis of basal properties and presynaptic plasticity at Mf-CA3 synapses in APP/PS1 mice.

To explore the electrophysiological features of APP/PS1 mice at the early stage of pathology, spontaneous miniature excitatory postsynaptic potentials (mEPSCs) were recorded from the soma of visually identified CA3b PCs in sagittal acute slices in the presence of 1µM TTX to block action potentials. Cells were held at -70 mV and left to stabilize for 8 minutes after whole-cell configuration was established. Synaptic activity was registered for 15 minutes in the presence of the GABAA receptors blocker (bicuculline, 10µM) in order to isolate the glutamatergic inputs.

The mean amplitude of all events occurring during 15 minutes was significantly smaller in APP/PS1 mice compared to Wt (Wt: 24.9pA ± 1.1pA, n=15, 8 mice; APP/PS1: 20.2pA ± 0.6pA, n=15, 9 mice, t-test p=0.0009) (Figure 3). Observing the cumulative probability distributions, we can conclude that the relative difference between the amplitudes is greater for smaller amplitudes, suggesting that APP/PS1 mice have an increased number of smaller events compared to Wt littermates at 6 months. The mean values of mEPSCs inter event intervals (IEI), indicative of the frequency of mEPSCs, was not statistically different between Wt and APP/PS1 mice (Wt: 0.8ms ± 0.1ms, n=15; APP/PS1: 0.7ms ± 0.09ms, n=15; p=0.5863; Figure 3 C), although the large variability in mEPSCs frequency between recordings may preclude drawing a conclusion.

Elimination of DG cells by -irradiation showed that 95% of the mEPSCs recorded from CA3 neurons, with an amplitude above 45pA derive from Mf synapses [START_REF] Henze | Large amplitude miniature excitatory postsynaptic currents in hippocampal CA3 pyramidal neurons are of mossy fiber origin[END_REF], whereas events smaller than 45pA have an equal probability of being from any of the three inputs contacting CA3 PCs. The percentage of mEPSCs larger than 45pA was not significantly different in the APP/PS1 CA3 PCs as compared to Wt (Wt 11.9pA ± 1.5pA, n=15; APP/PS1 8.5pA ± 0.8pA, n=15; Mann-Whitney test p=0.0718) although a tendency for a decrease can be observed (Figure 3 D-F). Since the extent to which different inputs to CA3 PCs are altered cannot be directly assessed with mEPSC recordings, we performed further experiments by recordings evoked Mf-CA3 EPSCs using minimal stimulation conditions [START_REF] Marchal | Postnatal maturation of mossy fibre excitatory transmission in mouse CA3 pyramidal cells: a potential role for kainate receptors[END_REF]. Firstly, we looked at the basal properties of Mf-CA3 synapses. Throughout the following experiments the group II mGluR agonist LCCG-I applied at the end of the recording session resulted in a decrease by more than 80% of the amplitude of EPSCs, confirming that they were Mf-EPSCs. The failure rates registered in the APP/PS1 mice was not significantly different as compared to Wt littermates (data not shown), suggesting comparable stimulation strength was used. Paired pulse facilitation (PPF) is the ability of synapses to increase neurotransmitter release in response to the second of two temporally close afferent stimulations. We first observed that in 6 months-old Wt mice, PPF ratio was smaller when compared with PPF ratio values obtained from 3-4 weeks-old Wt mice (Wt 6 months: 2.11 ± 0.15, n=16) (Pinheiro et al., 2007) [START_REF] Lanore | Impaired development of hippocampal mossy fibre synapses in mouse mutants for the presynaptic scaffold protein Bassoon[END_REF]. No alteration of this presynaptic feature was seen when comparing Wt with APP/PS1 mice of the same age (Figure 4 C, D) (WT 2.11 ± 0.15, n=16, and APP/PS1 mice 2.15 ± 0.16, n=16; t-test p=0.8556). Altogether we observed no indication for a change in basal presynaptic release properties. We also looked at low frequency facilitation, a characteristic presynaptic feature of Mf-CA3 PC synapses (Rebola et al., 2017) which can be observed by switching stimulation frequency from 0.1Hz to 1Hz. The mean value of facilitation was comparable between genotypes as illustrated in the representative traces in Figure 4 (WT 4.67 ± 0.36, n=16; APP/PS1 4.26 ± 0.37, n=16; t-test p=0.4298, Figure 4 A,B).

We also found no different between the genotypes in the facilitation of Mf-EPSCs in response to a train of 10 stimuli of 20Hz (average of 5 traces at the interval of 2min, the final facilitation rate for Wt 7.19 ± 0.81, n=14 and for APP/PS1 6.98 ± 1.34, n=12, Two-way ANOVA test p=0.7745, Figure XX).

We further analyzed presynaptic mechanisms of plasticity at Mf-CA3 synapses in response to high frequency trains which trigger a short lasting post-tetanic potentiation followed by an NMDA-independent form of LTP (Nicoll and Schmitz, 2005). We used a classical high frequency induction protocol consisting of 100 pulses at a frequency of 100Hz repeated 3 times with a 10 s inter train interval, in the presence of bicuculline (10 µM), CGP-55845 (3 µM) and D-AP5 (50 µM) to block GABAA, GABAB and NMDARs, respectively. We compared both the PTP (5 minutes after the train) and LTP levels (30 minutes after the train) in Wt and APP/PS1 mice (Figure 4 G,H). Both genotypes displayed similar levels of PTP, measured as the percentage of increase in Mf-EPSC amplitudes (Wt 702.5% ± 75.1%, n=12; APP/PS1 747.7% ± 112.5%, n=11; t-test p=0.737; Figure 4 I), as well as similar levels of LTP (Wt 182.9% ± 23.7%, n=12; APP/PS1 199.8%±48.7%, n=11; Mann-Whitney p= 0.723; Figure 4 J). Overall, we can conclude that basal properties and presynaptic plasticity, a characteristic feature of Mf-CA3 PC synapses, are mostly unaltered in the APP/PS1 model of AD at the age of 6 months.

Impaired LTP of NMDAR-EPSCs at Mf-CA3 synapses.

Dysregulation of NMDARs has been proposed as a link between A accumulation and disruption of synaptic function and plasticity [START_REF] Malinow | New developments on the role of NMDA receptors in Alzheimer's disease[END_REF]. NMDARs are present at Mf-CA3 PC synapses where they have recently been shown to contribute to synaptic integration and plasticity (Kwon and Castillo, 2008;Rebola et al., 2008;2011;Hunt et al., 2013;Rebola et al, 2011). At normal resting membrane potentials NMDARs are mostly blocked by extracellular Mg 2+ , but the large amplitude of unitary EPSPs at Mf-CA3 PC synapses may lead to the release of the Mg 2+ block, either directly or indirectly by triggering action potentials in CA3 PCs [START_REF] Henze | Single granule cells reliably discharge targets in the hippocampal CA3 network in vivo[END_REF].

After applying a frequency facilitation protocol at -70mV to record AMPAR-EPSCs, we imposed a holding potential of +40mV, and we isolated pharmacologically NMDAR-EPSCs with 20 µM NBQX. With this experimental procedure, we measured the ratio between Mf-EPSC peak amplitude at +40mV and -70mV (NMDAR/AMPAR ratio) in Wt and APP/PS1 mice, at both 0.1Hz and 1Hz (Figure 5). We observed that the NMDAR/AMPAR ratio was similar in both genotypes (Wt 28.3% ± 3.3%, n=9; APP/PS1 29.9% ± 3.4%, n=9; t-test p=0.7449; Figure 5 A-C). Although frequency facilitation of Mf-NMDA-EPSCs (~5 fold) appeared smaller compared to frequency facilitation of AMPAR-EPSCs (~7 fold) in adult Wt mice, the frequency facilitation of Mf-NMDAR-EPSCs was not different between genotypes (Wt 4.78 ± 0.4, n=29; APP/PS1 4.0 ± 0.2, n=31; Mann-Whitney test p=0.1932; Figure 5 D,E). The subunit composition of NMDARs determines its receptor properties [START_REF] Paoletti | NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease[END_REF]. Particularly, the identity of the GluN2 subunit impacts on the affinity for glutamate, modulation by glycine, sensitivity to voltage-dependent block by Mg2+, fractional Ca 2+ current and, consequently, channel kinetics [START_REF] Paoletti | NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease[END_REF][START_REF] Sg | Role of distinct NMDA receptor subtypes at central synapses[END_REF]. Several studies have ascribed a role for the GluN2B subunit in mediating most of the toxic effects of Aβ oligomers applied in culture neurons and acute slices [START_REF] Rönicke | Early neuronal dysfunction by amyloid β oligomers depends on activation of NR2B-containing NMDA receptors[END_REF]Hu et al., 2009). We tested if the chronic accumulation of Aβ peptides in APP/PS1 mice would change the composition of NMDARs at Mf-CA3 PC synapses.

The NMDAR-EPSC decay time is governed by the channel deactivation kinetics and it can be used as an indicator of the identity of the subunits assembling synaptic NMDARs.

Similar decay times of Mf NMDAR-EPSCs were measured in Wt and APP/PS1 mice at 6 months (WT: 103.2ms ± 12.4ms, n=27; APP/PS1: 115.6ms ± 12.0ms, n=30, p=0.4782, t-test, Figure 5 F). The decay of NMDAR-EPSCs at 1Hz stimulation were slightly faster (WT: 97.5ms ± 3.8ms, n=27; APP/PS1: 99.0ms ± 3.6 ms, n=30, p=0.7707, t-test; Figure 5 G). To further examine putative changes in subunit composition, we used a highly selective, activity dependent blocker of GluN2B-containing NMDARs (Ro 25-6981) [START_REF] Fischer | Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro[END_REF]. NMDAR-EPSCs evoked by Mf stimulation at 0.1Hz were recorded at +40mV for eight minutes before applying Ro 25-6981 (1µM) and during 40 minutes following the application. We observed a progressive reduction of both the amplitude and decay time constants of NMDAR-EPSCs after pharmacological application of the GluN2B antagonist in both genotypes (Figure 5 H-K). However, there was no difference in the level of inhibition between the two genotypes, indicating a similar content in GluN2B (Wt 71.8% ± 9.6%, n=11; APP/PS1 66.9% ± 9.2%, n=11; unpaired t-test p=0.8315). In line with this, we found no difference between Wt and APP/PS1 mice in the decay time of NMDARs EPSCs before (Wt control 87.0ms ± 3.2ms, n=11; APP/PS1 control 90.8ms ± 5.0ms, n=11; unpaired t-test p=0.5200) and after the application of Ro 25-6981 (Wt Ro25-6981 71.2ms ± 1.9ms, n=11; APP/PS1 Ro25-6981 77.3ms ± 4.2ms, n=11; unpaired t-test p=0.1964; Figure 5 K). At Mf-CA3 PC synapses, repeated short bursts of Mf stimulation induce robust LTP of NMDAR-EPSCs which spares AMPAR-EPSCs (Rebola et al., 2008;Kwon and Castillo, 2008). Considering the importance of NMDARs for synaptic plasticity and memory formation [START_REF] Nakazawa | Requirement for hippocampal CA3 NMDA receptors in associative memory recall[END_REF][START_REF] Kishimoto | Hippocampal CA3 NMDA receptors are crucial for adaptive timing of trace eyeblink conditioned response[END_REF][START_REF] Rajji | The role of CA3 hippocampal NMDA receptors in paired associate learning[END_REF], it is relevant to study this form of plasticity in our model of AD. NMDAR-EPSCs evoked by Mf stimulation in CA3 PC were recorded at +30mV in the presence of 10µM bicuculline, 3µM CGP-55845, 20µM NBQX to block GABA, AMPA and kainate receptors. After establishment of a stable baseline (10min) LTP was induced by 6 bursts of 6 stimuli at a frequency of 50 Hz with a 150s inter burst interval and recorded over a period of 40 min (Figure 5 L,M). We found that Mf LTP of NMDAR-EPSCs was absent in APP/PS1 mice (Wt 135.3% ± 9.2%, n=10; 98.3% ± 9.3%, n=10; t-test p=0.0109; Figure 5 N,O). Hence, we report that the subunit composition of NMDARs at Mf-CA3 PC synapses is not impacted by chronic accumulation of A in the APP/PS1 mouse model. However, we observe a striking impairment in LTP of NMDARs EPSCs at Mf-CA3 PC synapses.

GABAergic transmission and feedforward inhibition in CA3 PCs of APP/PS1 mice.

Monosynaptic innervation from Mf onto GABAergic interneurons in CA3 leads to robust feedforward inhibition of CA3 PCs (McBain, 2008), which is subject to structural plasticity upon memory encoding [START_REF] Caroni | Structural plasticity upon learning: regulation and functions[END_REF]. In line with early signs of memory impairments in APP/PS1 mice [START_REF] Kilgore | Inhibitors of Class 1 Histone Deacetylases Reverse Contextual Memory Deficits in a Mouse Model of Alzheimer's Disease[END_REF]Viana da Silva et al., 2016), we hypothesized that GABAergic transmission in CA3 PCs, and more particularly feedforward at Mf-CA3 synapses could be impaired. We first recorded both spontaneous and miniature IPSCs from CA3 PCs, to compare the general properties of GABAergic innervation onto CA3 PCs between the two genotypes. Spontaneous IPSCs were recorded for 10min at -60mV in the presence of 20μM NBQX and 10mM QX-314 added intracellularly to prevent action potentials of the recorded cell. We observed that the frequency of spontaneous IPSCs was significantly reduced in APP/PS1 mice as compared to control mice (Wt, 7.87 ± 1.06 Hz, n=16, 6 mice; APP/PS1 5.01 ± 0.67 Hz n=18, 6 mice; t-test, p=0.0254), whereas the mean amplitudes were unchanged (Wt n=16, 6 mice; APP/PS1 mice n=18, 6 mice; t-test p=0.1147; Figure 6 A-C). This decrease of IPSCs may be caused either by a reduction of the activity of presynaptic interneurons to the recorded CA3 PCs in the slice, or to a change in the number or properties of GABAergic synapses onto CA3 PCs.

To address this question, we recorded miniature IPSCs in the presence of 0.5μMTTX added to the extracellular medium. We observed no difference in the mean frequency (Wt n=15, 6 mice; APP/PS1 n=15, 6 mice; t-test, p=0.9756) and in the mean amplitude (Wt n=15, 6 mice; APP/PS1 n=15, 6 mice; t-test p=0.4292; Figure 6 D-F) of miniature IPSCs in CA3 PCs between APP/PS1 and WT mice. These results suggest that the decrease in the frequency of spontaneous IPSCs observed in APP/PS1 mice can be attributed to decreased spiking activity of presynaptic interneurons rather than to changes in the number of GABAergic synapses. However, this does not preclude changes in the properties of GABAergic synaptic transmission. We thus explored whether these changes may translate into changes in feedforward inhibition at Mf-CA3 PC synapses. For this we measured the excitatory/inhibitory ratio of evoked events at Mf-CA3 synapses using different regimes of presynaptic Mf stimulation (Torborg et al., 2010). We first recorded evoked Mf-EPSCs at -70mV in the presence of D-AP5 (20µM) to block NMDARs. We then switched to +10mV, the reversal potential for cationic ligand-gated channels, to record IPSCs. No significant differences in the ratio were found between APP/PS1 and Wt control mice at 0.1 Hz (Wt n=16, 10 mice; APP/PS1 n=16, 10 mice; t-test p=0.8703; Figure 6 G,H) or at 1 Hz (Wt n=16, 10 mice; APP/PS1 n=16, 10 mice; t-test with Welch's correction for unequal variances p=0.4882; Figure 6 I,J). We further recorded IPSCs evoked by trains of 5 stimuli at 20Hz. Similarly, the ratio between evoked EPSCs and IPSCs was not different during the train (Two-way ANOVA test p=0.9127, p=0.2672 for the normalized plot, Wt n=15 and APP/PS1 n=16, Figure 6 L). But when we normalized the amplitude of IPSCs in the train to the amplitude of the first IPSC to compare short-term plasticity of inhibition in the two genotypes, and found a significant decrease of plasticity in APP/PS1 mice as compared to Wt controls (Two-way ANOVA test p=0.0478, Wt 2.61 ± 0.51, n=14 and APP/PS1 1.62 ± 0.19, n=14; Figure 6 M). Taken together, we observed reduced GABAergic inputs onto CA3 PCs, and reduced inhibitory charges during 20Hz train stimulation at Mf-CA3 PC synapses.

Discussion

In the last decades, growing evidence indicates that synaptic loss and dysfunction, accompanied by neural network alterations, may be the major cause of cognitive impairments in AD, in particular at an early stage of the disease (Canter, Penney, & Tsai, 2016;Selkoe, 2002). It is yet important to precisely understand, for each circuit involved in memory encoding and retrieval, which aspects of synaptic physiology and plasticity is particularly impaired due to the AD pathology. Our work focuses on CA3 circuits which thought to be key for the initial stages of episodic memory encoding and recall (Kesner, Kirk, Yu, Polansky, & Musso, 2016;Rebola et al., 2017). A major question addressed through this work is whether A -induced impairment of synaptic transmission and plasticity is specific for a type of input at the level of a single neuron, here CA3 PCs. This work extends on our previous identification of marked changes in synaptic plasticity at CA3-CA3 synapses before the appearance of amyloid plaques in the APP/PS1 model (Viana da Silva et al., 2016). Our work explored whether pre-and postsynaptic properties of the other major glutamatergic input to CA3 PCs, the Mf-CA3 synapses were equally impaired in 6-month APP/PS1 mice. We also investigated potential alterations GABAergic synaptic transmissions onto CA3 PCs in these mice. After 6 months of overproduction of Aβ peptide and formation of Aβ oligomers, we found no differences in the morphological features of Mf terminals. These results are in agreement with our electrophysiological data, which did not disclose any difference in presynaptic functional parameters, such as in different forms of presynaptic release properties or short-term facilitation. Furthermore, the characteristic presynaptic form of LTP expressed by MfB is normal in 6-month APP/PS1 mice. These results suggest that early behavioral impairment in this AD mouse model at 6 months [START_REF] Kilgore | Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of Alzheimer's disease[END_REF]Viana da Silva et al., 2016) are not due to presynaptic failure at Mf-CA3 synapses. On the postsynaptic side, we found that the morphological features of thorny excrescences on CA3 PCs dendrites in APP/PS1 mice were comparable to those of Wt mice. Accordingly, glutamatergic transmission at Mf-CA3 synapses, in terms of mEPSCs and evoked EPSC amplitudes seemed unaltered. We observed however that postsynaptic LTP of NMDARs, described at Mf synapses (Kwon & Castillo, 2008;Rebola, Lujan, Cunha, & Mulle, 2008), is markedly impaired in the APP/PS1 transgenic mice. This encouraged us to further investigate possible alterations in the number/composition/conductance of NMDARs. In contrast with some publications that describe an alteration of the ratio between NMDAR/AMPAR in models of AD (Cisse et al., 2011;Roberson et al., 2011), we found no change in this ratio in the APP/PS1 mice with 6 months at Mf-CA3 synapses. More importantly, we found also no alterations in the subunit composition of NMDARs present at these synapses after application of Ro 25-6981. This suggests that the levels of synaptic GluN2B containing NMDARs are not affected in this AD mouse model, which is in contradiction with some other in-vitro studies that implicate GluN2B-containing NMDARs as particular mediators of neuronal damage in AD (Cui et al., 2013;Ferreira et al., 2012;Hu, Klyubin, Anwyl, & Rowan, 2009;[START_REF] Ronicke | Early neuronal dysfunction by amyloid beta oligomers depends on activation of NR2B-containing NMDA receptors[END_REF]. Although the composition and conductance of NMDARs is not altered in 6-month APP/PS1 mice, we cannot rule out that the internal cascades activated by NMDAR activation might be altered, or in other words, that the lack of LTP of NMDARs might depend on the postsynaptic function as a whole. Furthermore, LTP of NMDARs is induced and expressed postsynaptically and depends on the co-activation of A2ARs and mGluR5 (Rebola et al., 2008). Previous work of our group has demonstrated that abolition of A/C fiber LTP in CA3 PCs in 6-month APP/PS1 mice was caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signaling or altered dendritic spine morphology (Viana da Silva et al., 2016). Our current results thus may seem at odd with the notion that A2ARs are upregulated in CA3 PCs of 6-months old APP/PS1 mice. We may speculate that overexpression of A2ARs in APP/PS1 mice is accompanied with a change in the signaling mechanism downstream of A2AR activation leading to the loss of LTP of NMDARs at Mf-CA3 synapses.

Last but not least, we found that spontaneous IPSC frequency was decreased, and inhibitory charges were subtly reduced during a 20Hz train stimulation protocol. This result can be indicative of alterations in the synaptic contacts between MfBs and local interneurons, or between local interneurons and CA3 PCs. In line with our results, some studies have reported remodeling of GABAergic neurotransmission in the human AD brain (Fuhrer et al., 2017;Limon, Reyes-Ruiz, & Miledi, 2012) and AD transgenic mouse models (Jo et al., 2014;Wu, Guo, Gearing, & Chen, 2014). Especially, APP/PS1 mice were shown to display spontaneous seizures, which are believed to be associated with an imbalance in the excitatory/inhibitory function in the pathology of AD (Hazra et al., 2013;Ramos et al., 2006). Moreover, in APP/PS1-L166P mice (a transgenic line which includes a different mutant of PS1), it was shown that there are age-dependent concomitant changes in the GABAergic pathway in the APP/PS1 mice (Oyelami et al., 2016). Thus, it would be very interesting to further study the features and activities of GABAergic neurons in hippocampal CA3 region.

Overall, our work explored the pre-and postsynaptic features of hippocampal CA3 synapses in an AD mouse model, providing new insights into synaptic dysfunction at an early stage of AD. As a general principle, this work highlights that early synaptic dysfunction in a mouse model of AD is differentially expressed at specific inputs onto a single neuron. This need to be taken into account when establishing links between synaptic dysfunction and behavioral impairment.

Methods

Mice. APP/PS1 mice were obtained from Jackson Laboratory and used according to regulations of the University of Bordeaux/CNRS Animal Care and Use Committee. Throughout their life, all mice were group-housed, ranging from 4 to 10 animals per cage. Food and water were provided ad libitum. The transparent plexiglass cages (38.1 x 19.1 x 12.7 cm) were maintained on a 12-h dark/light cycle, kept in a temperature-regulated room, and protected from exterior pathogens by a filter. All experiments were performed in the light phase of the circadian cycle in 6 months (26-32 weeks) male APP/PS1 and age-matched male WT littermates.

Electrophysiology.

Anesthesia drug (ketamine 75 mg/kg and xylazine 10 mg/kg) was diluted in saline and injected intraperitoneally to the mouse 5 min before decapitation or transcardial perfusion (for mice older than 2 months) followed by decapitation. The head was immediately placed into a petri dish filled with ice-cold cutting solution (200mM of sucrose, 20mM glucose, 0.4mM CaCl2, 8mM MgCl2, 2mM KCl, 1.3mM NaH2PO4, 26mM NaHCO3, 1.3mM ascorbate, 0.4mM pyruvate and 3mM kynurenic acid, pH 7.3) oxygenated with carbogen (95% O2, 5% CO2). The brain was rapidly removed from the scull and parasagittal slices (350 mm) were cut with a Leica vibratome (Leica VT 1200S) in the cutting solution. The slices were then kept at 33°C in oxygenated resting solution (110mM NaCl, 2.5mM KCl, 0.8mM CaCl2, 8mM MgCl2, 1.25mM NaH2PO4, 26mM NaHCO3, 0.4mM ascorbate, 3mM pyruvate and 14mM glucose, pH 7.3) for 20min, before transferred to aCSF. The slices were then left at room temperature for a maximum of 6h after cutting.

The recording chamber of the electrophysiology set-up was perfused with oxygenated aCSF (125mM NaCl, 2.5mM KCl, 2.3mM CaCl2, 1.3mM MgCl2, 1.25mM NaH2PO4, 26mM NaHCO3 and 14mM glucose, pH 7.4). CA3 pyramidal neurons were identified by differential interference contrast microscopy using an Olympus fixed stage upright microscope (BX51WI) equipped with a ×60 magnification immersion objective at room temperature, and whole-cell patch-clamp configuration was achieved with borosilicate glass capillaries with resistance value ranging from 3 to 5 MΩ. The pipettes were filled with variant intracellular solutions depending on experiments: KMSO3-based solution (120mM KMSO3, 2mM MgCl2, 1mM CaCl2, 20mM KCl, 10mM EGTA, 2mM ATPNa2, 10mM HEPES, pH 7.2) was used for current-clamp recordings, CsCl-based solution (120mM CsCl, 2mM MgCl2, 2mM CaCl2, 5mM EGTA, 5mM phosphocreatine, 2mM ATPNa2, 0.33mM GTP, 10mM HEPES, 10mM QX314, pH 7.2) was used for IPSC recordings, and CsMSO3based solution (100mM CsMSO3, 3mM MgSO4, 3.5mM CaCl2, 20mM EGTA, 5mM phosphocreatine, 3mM ATPNa2, 0.33mM GTP, 10mM HEPES, pH 7.2) was used for all other experiments.

To monitor the access resistance during the whole recording time, a hyperpolarizing voltage step (-5mV, 10ms) was applied at the beginning of each trace. Series access resistance was <20MΩ, and when it changed by >20%, the recording was discarded. When held at -70mV, neurons with a holding current >300pA were also rejected. Mf synaptic currents were identified according to the following criteria: 1) obvious paired-pulse facilitation; 2) EPSCs had a steep rising phase (∼1ms); 3) EPSCs decays free of secondary peaks that might indicate the presence of polysynaptic contamination. Liquid junction potential correction was not used for measurements of membrane potentials.

Recordings were made using an EPC10.0 amplifier (HEKA Elektronik), filtered at 0.5-1kHz and analyzed using IGOR Pro and Neuromatic V2.6 software. All drugs for electrophysiological experiments were obtained from Tocris Biosciences or Sigma-Aldrich, unless otherwise stated. Stereotaxic injections and confocal microscopy. Viral stocks were stored at -80°C until use and were thawed on ice before their in vivo administration. Prior to surgery the mice were anaesthetized by inhalation of isoflurane in an induction chamber connected to tubing that delivered a mix of air and 4% isoflurane. Once the animals were immobile they were placed on a heating pad (36 °C) while they continued to receive anesthesia via a mask (1.5-2% isoflurane) during the surgery. For labeling CA3 pyramidal neurons we injected the glycoprotein deleted rabies virus variant coated with the native glycoprotein (300nL, velocity 40nL/min) [START_REF] Wickersham | Retrograde neuronal tracing with a deletion-mutant rabies virus[END_REF] into the CA1 stratum radiatum (coordinates: anteroposterior: -1.92mm; medio-lateral: ±1.50mm; and ventral: -1.35mm) to achieve a retrograde infection. For labeling mossy fiber terminals we injected an anterograde variant of the glycoprotein deleted rabies virus (500nL, velocity 50nL/min) (Haberl et al., 2014) into the hilus of DG (coordinates: anteroposterior: -1.92mm; medio-lateral: ±1.15mm; and ventral: -2.00mm). The mice were sacrificed 6 days after the injection. Injected mice were anesthetized with intraperitoneal injection of pentobarbital (30 mg/kg), then transcardially perfused with physiological saline (9% NaCl) followed by 4% paraformaldehyde (4% PFA). Brains were removed and post-fixed overnight in 4% PFA. After fixation, coronal sections (70mm) were cut on a vibratome (Leica, Germany), then mounted on a glass slide and covered with a thin glass coverslip. The sections were imaged on Leica DM6000 TCS SP5 confocal microscope (Leica Microsystems, Germany) for visualization of mossy fiber terminals and CA3 thorny excrescences. Statistical analysis. Statistical analyses were performed with Prism6 (GraphPad Software, USA).

First the normality of data set was tested using the D'Agostino-Pearson omnibus normality test. If data was normally distributed, a Student's t-test or two-way ANOVA (Analysis of Variance) was performed; otherwise, nonparametric tests such as Mann-Whitney test (for unpaired data) and Wilcoxon test (for paired data) was used. Data distributions were analyzed using the Kolmogorov-Smirnov test. Results were presented as mean ± SEM (standard error of mean). Statistical differences were considered significant at P<0.05. For Small MfBs the complexity index is 2.6 ± 0.1 for Wt mice and 2.6 ± 0.1 for APP/PS1 mice, displaying no difference between genotypes. Medium sized MfBs are more complex in both genotypes (Wt 3.3 ± 0.2; APP/PS1 3.7 ± 0.17). Finally, for large MfBs with a volume larger than 20µm3, the complexity index was, despite the visible tendency, not significantly altered in the APP/PS1 mice (Wt 4.3 ± 0.2; APP/PS1 4.8 ± 0.2, n=5 mice for each genotype). Data represented as mean ± SEM. F. Example image of the proximal dendrite of a CA3 pyramidal cell infected by RABV∆G-eGFP (RG) acquired with confocal microscopy (left panel) and subsequent reconstruction of ThE in stratum lucidum obtained by IMARIS software. G. Bar graph representing the total number of ThE per pyramidal cell. The number is not different in APP/PS1 and Wt mice. Just ThE on apical dendrites were analyzed and only cells with complete apical dendritic arborization were used (Wt n=9, APP/PS1 n=8, t-test p=0.3539). H. Cumulative distribution of the area from reconstructed ThE shows no difference between Wt (black trace) and APP/PS1 mice (red trace), Ks test p=0.99. There is no difference regarding the mean surface area of reconstructed MfBs in the two genotypes (Wt 16.6µm2 ± 0.6µm2, n=1259, 6 mice; APP/PS1 14.2µm2 ± 0.5µm2, n=945, 6 mice; Mann-Whitney test p=0.1074). I. Cumulative distribution of the volume of reconstructed ThE shows that there is a small difference in the distribution of ThE between Wt (black trace) and APP/PS1 mice (red trace), Ks test p<0.0001. Nevertheless, the mean volume values in the APP/PS1 mice is not different from those in the Wt littermates (Wt 2.4µm3 ± 0.1µm3, n=1259, 6 mice; APP/PS1 2.0µm3 ± 0.08µm3, n=945, 6 mice; Mann-Whitney test p=0.1221). J. Summary plot of the relation between surface area and volume of CA3 thorns in the Wt (black circles) and APP/PS1 mice (green circles). The relation between the two parameters is not altered in the APP/PS1 animal (Wt n=945 and APP/PS1 n=1259). Data represented as mean ± SEM. Black line represents the amplitude distribution of 1500 events from 15 CA3 cells successfully patched in Wt slices (n=8 mice); Mean values: Wt 26.7pA ± 0.7pA; APP/PS1 22.5pA ± 0.6pA; KS test p<0.0001, bin size of 2. E. Bar graphs representing the percentage of the putative Mf events (with an amplitude larger than 45pA) for each of the 15 cells recorded, during a 15-minute period. A tendency was found towards a reduced number of Mf-generated events, which is not statistically significant (Mann-Whitney test p=0.0718). Black bar, Wt cells (n=15, 8 mice). Grey bar, APP/PS1 mice (n=15, 9 mice). Data represented as mean ± SEM. F. Cumulative distribution of the amplitudes of events with high probability of being from Mf synapses is not different between Wt and APP/PS1 mice. Plot used all the events larger than 45pA obtained over a period of 10 minutes per cell (150 minutes in total per genotype). Mean values are 76.8pA for Wt and 78.4pA for APP/PS1; KS test p>0.999, bin size 2. [START_REF] Darstein | Distribution of kainate receptor subunits at hippocampal mossy fiber synapses[END_REF][START_REF] Ruiz | Distinct subunits in heteromeric kainate receptors mediate ionotropic and metabotropic function at hippocampal mossy fiber synapses[END_REF]. Knockout studies have demonstrated that KARs may also play important role in hippocampus-dependent memory [START_REF] Ko | Altered behavioral responses to noxious stimuli and fear in glutamate receptor 5 (GluR5)-or GluR6-deficient mice[END_REF]Rebola et al., 2017). Moreover, there have been many pieces of evidence showing that KARs can modulate slow AHP and neuronal excitability in pyramidal neurons [START_REF] Fisahn | The kainate receptor subunit GluR6 mediates metabotropic regulation of the slow and medium AHP currents in mouse hippocampal neurones[END_REF][START_REF] Melyan | Metabotropic regulation of intrinsic excitability by synaptic activation of kainate receptors[END_REF][START_REF] Melyan | Metabotropic-mediated kainate receptor regulation of IsAHP and excitability in pyramidal cells[END_REF][START_REF] Ruiz | Distinct subunits in heteromeric kainate receptors mediate ionotropic and metabotropic function at hippocampal mossy fiber synapses[END_REF]. Therefore, we hypothesized that there may be dysfunction of KARs at Mf-CA3 synapses in APP/PS1 mice.

Figures and legends

We first recorded evoked Mf-EPSCs from CA3 PCs in the presence of 20µM D-APV, 10µM Bicuculline, 3µM CGP 55845 at 0.1Hz, 1Hz and 3Hz respectively. These Mf-EPSCs corresponded to conductance of AMPA and Kainate receptors (noted as AMPAR+KAR EPSCs). We then added additional 25µM LY303070 to isolate the KAR component of Mf-EPSCs during 0.1Hz, 1Hz, and 3Hz of Mf stimulation. Minimal stimulation was achieved, and the failure rate and AMPAR-EPSC amplitude at 0.1Hz was compared between genotypes to ensure that comparable strength of stimulation was used (For failure rates of AMPAR-EPSCs at 0.1 Hz, WT 13.05 ± 3.54%, n=13; APP/PS1 19.06 ± 6.41%, n=12; Unpaired t-test p=0.4110; For amplitudes of AMPAR-EPSCs at 0.1 Hz, WT 64.46 ± 8.86pA, n=13; APP/PS1 55.91 ± 9.41pA, n=12; Unpaired t-test p=0.5143; Figure 3.1). The AMPAR-EPSCs were comparable between the two groups (At 1Hz, WT 219.0 ± 20.25pA, n=13; APP/PS1 mice 197.8 ± 33.93pA, n=12; t-test p=0.5901; At 3Hz, WT 298.0 ± 21.23pA, n=13; APP/PS1 mice 266.9 ± 41.72pA, n=12; t-test p=0.5032; Figure 3.1).

We observed that the KAR-EPSCs were significantly reduced, and the KAR/AMPAR ratio was significantly decreased in APP/PS1 mice (At 1Hz, WT 0.058 ± 0.005, n=13; APP/PS1 0.032 ± 0.004, n=12; t-test p=0.0003; At 3Hz, WT 0.054 ± 0.004, n=13; APP/PS1 0.030 ± 0.004, n=12; t-test p=0.0005; Figure 3.1). The KAR-EPSCs at 0.1Hz were not shown due to their very small amplitudes and corresponding large errors. These results suggest that there are less functional KARs at the postsynaptic sites of Mf-CA3 synapses. However, whether the number/functional state of extrasynaptic KARs is affected or not is unclear.

KAR-EPSCs of Mf-CA3 synapses is also reduced in PS KO mice

Previous work of our team has demonstrated that N-Cadherin (NCad) can recruit and stabilize KARs by interacting with the GluK2a C-terminal domain, and overexpression of a dominant-negative form of NCad or knockdown of NCad in CA3 PCs could lead to a strong reduction in the amplitude of KAR-EPSCs [START_REF] Fievre | Molecular determinants for the strictly compartmentalized expression of kainate receptors in CA3 pyramidal cells[END_REF]. We suspect that similar mechanism is responsible for the reduction of KAR-EPSCs in APP/PS1 mice. APP/PS1 transgenic mice express a mutated form of Presenilin (i.e. PSEN1 delta E9), which is the catalytic component of the γ-secretase complex. Mutated PS1 could induce loss of function of γ-secretase, potentially leading to accumulation of NCad lacking the extracellular fragment at the cell membrane.

Therefore, we hypothesized that this mutated PS1 gene in APP/PS1 mice could cause accumulation of truncated NCad at the membrane and thus impair the presence or function of KARs at Mf-CA3 synapses. To investigate this question, we took advantage of existing mouse lines at the local institute and generated conditional PS KO mice by injecting 500nl of AAV2.9 Syn Cre-GFP in hippocampal CA3 region of one hemisphere of PS1-floxed/PS2 KO mice. We patched the GFP-positive CA3 PCs as the PS KO cells, and non-fluorescent neurons from the other hemisphere were used as controls.

KAR and AMPAR-mediated EPSC recordings were performed as previously described in Chapter 3.3.5. Again, we observed an significant reduction of the KAR/AMPAR ratio in APP/PS1 mice (At 1Hz, control 0.030 ± 0.005, n=9; PSKO 0.014 ± 0.005, n=10; t-test p=0.0447; At 3Hz, control 0.027 ± 0.005, n=9; PSKO 0.009 ± 0.004, n=10; t-test p=0.0056; Figure 3.2), while the AMPAR-EPSCs were comparable (At 1Hz, control 227.6 ± 24.85pA, n=9;PSKO 178.8 ± 16.27pA, n=10;t-test p=0.1121;At 3Hz, control 317.5 ± 26.06pA, n=9;PSKO 243.8 ± 24.27pA, n=10; t-test p=0.0539; So far, we observed a significant reduction in KAR-EPSCs in CA3 PCs in both APP/PS1 mice and PS KO mice. This suggest that the mutated PS might be the common cause of this phenotype. To hypothesize one step further, the lack of normal NCad resulted from dysfunctional γ-secretase might underlie the dysfunction or inadequate presence of KARs at Mf-CA3 synapses.

CA3 circuits modifications after contextual fear conditioning (CFC)

One-trial CFC can induce changes in CA3 circuits

Located at the entry level of hippocampus, CA3 circuits play a key role in memory encoding. The main purpose of this project was to explore changes in CA3 circuits in regard to learning and memory. We started out by choosing contextual fear conditioning (CFC) as a behavioral paradigm to activate the CA3 circuits during memory encoding and recall. We first validated the one-trial CFC protocol in our lab.

Contextual fear conditioning was performed in adult C57bl6j male mice. Fear conditioning was induced in mice received one-trial foot shocks, and lasted for at least two weeks (Figure 3.3). Immunofluorescent staining of c-Fos was performed 1.5 hours after CFC, to confirm the successful activation of CA3 network. In DG, the number of c-Fos+ cells was not significantly elevated, but the signal strength and distribution pattern seemed different than controls. In CA3, there were significantly more c-Fos+ cells after CFC (Figure 3.4). It was shown previously that there is a robust filopodia growth at Mf terminals after CFC, where the number of filopodia was doubled within 5h and this lasted for at least 10 days (Ruediger et al., 2011). We attempted to reproduce this experiment. We used Thy1-mGFP-DG mice, and sacrificed the mice to count the filopodia numbers at large Mf terminals (LMTs) 5h after CFC. Unexpectedly, we did not see any difference between the two groups (Figure 3.5). Our count of filopodia in control condition is much higher than reported by Reudiger et al. in 2011, but consistent with some other studies in adult mice [START_REF] Wilke | Deconstructing complexity: serial block-face electron microscopic analysis of the hippocampal mossy fiber synapse[END_REF]. Further investigations are needed to interpret this result. Whatsoever, in our hands one-trial CFC can be used as an efficient tool to induce fear memory and cell activity changes in terms of c-Fos expression in the CA3 region in adult mice.

Electrophysiological studies of Mf-CA3 circuit 3h after CFC

As many changes can already happen within hours after CFC at the molecular and structural level in hippocampal neurons [START_REF] Gupta | Histone methylation regulates memory formation[END_REF]Ruediger et al., 2011), we explored the electrophysiological features of Mf-CA3 synapses rapidly after CFC. The mice were sacrificed 3h after CFC, and recordings were performed within the following 5 hours.

We started by investigating the prominent features of short-term plasticity at Mf-CA3 synapses shortly after CFC, by looking at frequency facilitation (FF) and paired-pulse facilitation (PPF). After achieving minimal stimulation (failure rates at 0.1Hz ~20%), we performed FF protocol (0.1Hz to 1Hz) and PPF protocol at 20Hz. With comparable EPSC amplitudes between the two groups (data not shown), no significant differences were found in either type of facilitations (Figure 3.6). We could conclude at this point that the basal presynaptic properties measured from Mf-CA3 synapses were not significantly modified 3h after CFC in adult C57bl6j male mice. We further recorded the spontaneous EPSCs of CA3 pyramidal cells 3h after CFC, and saw no difference in either frequency or amplitude of glutamatergic activities comparing to controls (Figure 3.7). Overall, we did not observe any major change in the plasticities of glutamatergic activities at Mf-CA3 synapses shortly after CFC.

We then explored GABAergic synaptic transmission onto CA3 PCs. We recorded spontaneous IPSCs and miniature IPSCs from CA3 pyramidal cells in mice sacrificed 3h after CFC. We found that the frequency of sIPSCs was significantly increased compared to naïve controls, while the mean amplitude of sIPSCs was not significantly modified. Also, neither the mean frequency nor the mean amplitude of mIPSCs of CFC 3h group was significantly different from controls (Figure 3.8).

These results suggest that the number of inhibitory synapses onto CA3 PCs are largely unchanged, while the spontaneous inhibitory activities are increased rapidly after CFC. This was somehow consistent with our morphological findings in Chapter

Electrophysiological studies of Mf-CA3 circuit 24h after

CFC

We were curious to test whether further changes at Mf-CA3 synapses could be observed at a longer time range after CFC, for example, 24h. We performed again sIPSC recordings in mice sacrificed 24h after CFC, and observed no difference in either frequency or amplitude in CFC group compared to controls (Figure 3.9). We also measured the FF and PPF of evoked synaptic transmission again, to look for changes in presynaptic short-term plasticities in larger time scale after CFC.

Similarly, no significant differences were registered in the CFC group as compared to controls (Figure 3.10). However, it had to be noted that sIPSC frequency at 24h of both groups were at the same level as the CFC 3h group, so that previous conclusions for the 3h experiments had to be tempered.

In a last effort to search for modifications at Mf-CA3 synapses 24h after CFC, we calculated the accumulative charges of the evoked-EPSCs and evoked-IPSCs under the same stimulating intensity, and compared the excitation/inhibition ratio (E/I) between two groups. After recording the evoked Mf-EPSCs in CA3 PCs at -70 mV, we switched the holding potential to +10 mV for evoked-IPSCs recording to explore the excitatory/inhibitory ratio of evoked events at these synapses. The NMDAR antagonist D-APV (20µM) was present throughout the experiments to block possible NMDA activities at positive holding potentials. The E/I ratio was calculated as the accumulative charges of the averaged EPSC event over that of the IPSC event. Still, no significant differences were found in these parameters (Figure 3.11).

Taken together, we observed no electrophysiological alterations at Mf-CA3 synapses 24h after CFC. However, we did register an increased frequency of sIPSCs 3h after CFC. Taken together, these results correspond to the role of hippocampal CA3 region in memory encoding when presented with a novel context, especially at the very initial stage of acquisition (Rebola et al., 2017). However, it will be interesting to further explore the GABAergic innervations onto CA3 PCs rapidly after CFC recall.

Genomic and epigenetic studies of APP/PS1 mice

RNA-sequencing of hippocampal CA3 region in APP/PS1 mice

In pre-symptomatic AD models, many changes may already happen at a transcriptional level, involving signaling cascades, receptor composition and trafficking, and other vital functions in neurons. Identifying these genomic alterations can be very informative on mechanisms of AD onset, and provide us with potential therapeutic targets in treating AD. We were curious to test for genomic changes in our AD mouse model, especially in hippocampal CA3 region. We carried out RNA-seq study with micro-dissected CA3 tissues from 6-month-old male APP/PS1 mice and WT littermates.

We observed that there was a mild change in gene expression pattern in hippocampal CA3 tissue in 6-month-old APP/PS1 mice compared to WT. When the cut-off of p < 0.01 & |fold change| > 1.2 was chosen, there were 24 genes that were upregulated and 16 genes that were down-regulated in APP/PS1 samples (Figure 3.12). Volcano plot displaying the expression levels of various genes in APP/PS1 mice (n=5) compared to WT littermates (n=7). Red dots represent differentially regulated genes compared to WT (p<0.01). To the left side are the down-regulated genes, and to the right side are up-regulated genes.

Among the up-regulated genes, apart from APP and PS1, there are PRNP (encoding for the major prion protein PrP), 2310067B10Rik (an ATPase), and CST7 (encoding a glycosylated cysteine protease inhibitor with a putative role in immune regulation). As for the down-regulated genes, there are Arpp21 (encoding a cAMPregulated phosphoprotein), TRIB2 (an atypical protein kinase), and Dnajb5 (encoding a member of the DNAJ heat shock protein). For a more complete list please refer to Annexes 6.6.

Further pathway analysis revealed the functional group these genes belong in, as is shown in Figure 3.13. These changes indicate that there are impaired functions in many fundamental pathways, and also up-regulation of various genes as response or compensation for the loss of function. Left panel displaying the down-regulated pathways, and right panel the up-regulated pathways. More significant changes [START_REF] Gupta | Histone methylation regulates memory formation[END_REF] are shown in darker color.

H3K4Me3 level is decreased at gene promoters in APP/PS1 mice

As we have noticed the expression levels of many important genes have been up or down-regulated in early AD model of 6-month APP/PS1 mice, we further investigated the epigenetic changes which might contribute to this result. Histone modifications have been proposed as one of the key mechanisms regulating gene expression in eukaryotic organisms [START_REF] Kouzarides | Chromatin modifications and their function[END_REF]B. Li, Carey, & Workman, 2007). Especially trimethylated H3K4 (H3K4Me3) has been reported to be very relevant to contextual memory consolidation, where H3K4Me3 level was elevated after contextual learning, and decreased in mouse models with memory deficits [START_REF] Gupta | Histone methylation regulates memory formation[END_REF][START_REF] Kerimoglu | Histone-methyltransferase MLL2 (KMT2B) is required for memory formation in mice[END_REF].

We hypothesized that there might be a reduction in H3K4Me3 levels at promoters in 6-month-old APP/PS1 mice, leading to the down-regulation of various genes.

Hippocampal CA3 regions were dissected from 6-month-old APP/PS1 mice and WT littermates, and nuclei of only neurons were sorted out using FACS (Figure 3.14). In line with the findings from the RNA-seq, ChIP analysis revealed a downregulation of H3K4me3 level at the gene promoters, while very few genes promoter areas showed increased H3K4Me3 level (Figure 3.15 A). To name just a few of the down-regulated genes detected: Lats1 (large tumor suppressor kinase 1), Dop1R1 (Dopamine 1-like receptor 1), Mlf (Myeloid leukemia factor), Nxn (nucleoredoxin).

For a more complete list please refer to Annexes 6.7.

Again, the pathway analysis was performed, and the down-regulated genes with decreased H3K4Me3 at their promoters are listed in categories (Figure 3.15 B). It is worth noticing that the most affected pathways are mainly involved in neuronal proliferation and function/development of olfactory bulb. This indirectly confirmed that adult neurogenesis could be impaired in 6-month APP/PS1 mice, which have been shown in many AD models. Especially, one study showed that hippocampal neurogenesis is dramatically impaired in 6-month APPswe/PS1dE9 mice. To be more specific, the late survival of newborn cells 4-6 weeks later is significantly diminished [START_REF] Verret | Alzheimer's-type amyloidosis in transgenic mice impairs survival of newborn neurons derived from adult hippocampal neurogenesis[END_REF]. Additional evidence is still needed to confirm whether newborn granule neurons are actively killed by Aβ, or are unable to survive because of changes in the brain associated with Aβ overexpression. However, a closer look at the down-regulated genes with decreased H3K4Me3 levels showed that they were not overlapping much with the down-regulated genes from RNA-seq result. For this we even chose a more tolerant cut-off for the RNA-seq data to include more genes. Still, we detected only one gene which showed both low expression in RNA-seq and low H3K4Me3 levels at the promoter area, which is Synpr (synaptoporin) (Figure 3.16). Synaptoporin is a synaptic vesicle protein that at first was found to be exclusively enriched in the granule cell axons, the mossy fibers in rat hippocampus [START_REF] Grabs | Differential expression of synaptophysin and synaptoporin during pre-and postnatal development of the rat hippocampal network[END_REF]. Later studies showed that synaptoporin also colocalized well with GAD 65 (a marker for GABAergic terminals) in the mouse hippocampus, and further in situ hybridization combined with immunocytochemistry revealed that various subtypes of interneurons expressed synaptoporin, e.g. in 44.2% of PV+ interneurons, 59.9% of VIP+ interneurons and 38.6% of CCK+ interneurons [START_REF] Singec | Synaptic vesicle protein synaptoporin is differently expressed by subpopulations of mouse hippocampal neurons[END_REF]. Therefore, a reduction in synaptoporin expression might implicate that the activities of inhibitory synapses in the hippocampal CA3 area in APP/PS1 mice could be impaired, which coincides with our electrophysiological findings. A fact needs to be taken into consideration is that the RNA-seq was done from whole CA3 tissue whereas the ChIP-seq was performed from sorted CA3 neuronal nuclei. This indicate that the RNA-seq result might include a considerable portion from glia cells in CA3 region, thus can be quite different from the expression profile obtained from ChIP-seq. However, the more plausible explanation is that H3K4Me3 is only one of many epigenetic markers, and other epigenetic mechanisms may play more important role in the down-regulation of genes in early AD mice.

Nevertheless, these results confirmed that H3K4Me3 plays a key role in regulating gene expressions in neurons of hippocampal CA3 region, and H3K4Me3 level was significantly decreased at the promoter areas of many genes in 6-month male APP/PS1 mice.

Chapter 4 Discussion

Discussion

CA3 circuits deficits in early AD mouse models

Growing evidence indicates that synaptic loss and dysfunction, accompanied by neural network alterations, may be the major cause of cognitive impairments in AD (Canter et al., 2016;Selkoe, 2002). Even at the early stage of AD and still free of Aβ plaques, the synaptic function can be already altered by abnormal amyloid metabolism [START_REF] Cleary | Natural oligomers of the amyloid-beta protein specifically disrupt cognitive function[END_REF][START_REF] Haass | Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide[END_REF][START_REF] Klyubin | Alzheimer's disease Abeta assemblies mediating rapid disruption of synaptic plasticity and memory[END_REF][START_REF] Lesne | A specific amyloid-beta protein assembly in the brain impairs memory[END_REF][START_REF] Shankar | Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway[END_REF][START_REF] Tanzi | The synaptic Abeta hypothesis of Alzheimer disease[END_REF]. Therefore, it is important to explore which aspects of synaptic physiology and plasticity is particularly impaired in early AD, especially at circuits involved in memory encoding and retrieval.

The project of this thesis focused on CA3 circuits which is thought to be key for the initial stages of episodic memory encoding and recall (Kesner et al., 2016;Rebola et al., 2017). Previous work of our team has identified marked changes in synaptic plasticity at CA3-CA3 synapses in 6-month APP/PS1 mice (Viana da Silva et al., 2016).

We set out to explore whether pre-and postsynaptic properties of the other major glutamatergic input to CA3 PCs, the Mf-CA3 synapses, were equally impaired in 6month APP/PS1 mice. Additionally, we also investigated potential alterations in GABAergic synaptic transmissions onto CA3 PCs.

Unaltered presynaptic features and loss of LTP of NMDARs at Mf-CA3 synapses

In line with our observation that the morphology of Mf terminals is unaltered in 6-month APP/PS1 mice, we registered no difference in presynaptic functional parameters, such as different forms of presynaptic release properties, short-term facilitation, and the characteristic presynaptic form of LTP expressed by MfB. While on the postsynaptic sites, we found no difference in the morphological features of thorny excrescences on CA3 PCs dendrites in APP/PS1 mice; likewise, glutamatergic transmission at Mf-CA3 synapses in terms of miniature and evoked EPSC amplitudes also seemed unaltered. However, we observed that postsynaptic LTP of NMDARs was markedly impaired in 6-month APP/PS1 mice. This encouraged us to further investigate possible alterations in the number/composition/conductance of NMDARs. In contrast with some publications that describe an alteration of the ratio between NMDAR and AMPAR in models of AD (Cisse et al., 2011;Roberson et al., 2011), and some other in-vitro studies that implicate GluN2B-containing NMDARs as particular mediators of neuronal damage in AD (Cui et al., 2013;Ferreira et al., 2012;Hu et al., 2009;[START_REF] Ronicke | Early neuronal dysfunction by amyloid beta oligomers depends on activation of NR2B-containing NMDA receptors[END_REF], we found no change in either NMDAR/AMPAR ratio or NMDAR composition in terms of GluN2B component in the 6 months old APP/PS1 mice at Mf-CA3 synapses.

Furthermore, this form of LTP of NMDARs depends on the co-activation of postsynaptic A2ARs and mGluR5 (Rebola et al., 2008). Previous work of our group has demonstrated that abolition of A/C fiber LTP in CA3 PCs in 6-month APP/PS1 mice was caused by activation of upregulated neuronal A2ARs (Viana da Silva et al., 2016).

Our current results thus may seem at odd with the notion that A2ARs are upregulated in CA3 PCs of 6-months old APP/PS1 mice. Nevertheless, we cannot rule out that changes in the signaling pathways downstream of A2AR activation are responsible for the loss of LTP of NMDARs at Mf-CA3 synapses, or in other words, that the lack of LTP of NMDARs might depend on the postsynaptic function as a whole. Moreover, it will be extremely interesting to examine whether the decreased NMDAR-LTP in the MF pathway is directly responsible for any cognitive impairment of APP/PS1 mice, because this would further extend our understanding of the functional role of this circuit in behavior.

Interestingly, although we observed no impairment in MF LTP (mainly mediated by AMPAR response), some different results have been shown for Tg2576 mice.

Tg2576 mice is a widely used AD model harboring the human APPswe695 gene, exhibits early synaptic abnormalities starting around the age of 4-6 months and displays several neuropathological features of AD later in its life [START_REF] D'amelio | Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer's disease[END_REF][START_REF] Jacobsen | Early-onset behavioral and synaptic deficits in a mouse model of Alzheimer's disease[END_REF]. Jung et al. found that the MF LTP (in terms of fEPSP potentiation) is significantly enhanced in 12-month Tg2576 mice [START_REF] Jung | Pathway-specific alteration of synaptic plasticity in Tg2576 mice[END_REF], while Witton et al. recorded significantly reduced MF LTP in the same model by the age of 24 months (Witton et al., 2010). We cannot explain where the discrepancies come from, but one possibility is that the PS1 transgene in APP/PS1 mice might contribute to the LTP phenotypes at MF synapses.

In addition, we found unexpectedly a marked and selective alteration of the KAR component at Mf-CA3 synapses (see below). This indicates that alteration of glutamatergic function at early stage of AD, even at the level of a single neuron is selective for the type of input and for the type of post-synaptic receptors.

Decreased GABAergic transmissions onto CA3 PCs

CA3 PCs receive diverse GABAergic inputs. The interneurons involved can be categorized as parvalbumin (PV)-positive, somatostatin (SOM)-positive, cholecystokinin (CCK)-positive, vasoactive intestinal polypeptide (VIP)-positive, and other incompletely characterized interneurons, each located in defined layers of CA3 and innervating different segments of CA3 PCs dendrites [START_REF] Freund | Perisomatic inhibition[END_REF][START_REF] Hajos | Spike timing of distinct types of GABAergic interneuron during hippocampal gamma oscillations in vitro[END_REF].

These interneurons actively influence CA3 circuit activities, in that they can control the spiking of CA3 PCs by regulating neuronal excitability, firing and synaptic integration (Hajos et al., 2013;[START_REF] Losonczy | Persistently active cannabinoid receptors mute a subpopulation of hippocampal interneurons[END_REF][START_REF] Schlingloff | Mechanisms of sharp wave initiation and ripple generation[END_REF][START_REF] Szabo | Anatomically heterogeneous populations of CB1 cannabinoid receptor-expressing interneurons in the CA3 region of the hippocampus show homogeneous inputoutput characteristics[END_REF]. For example, it was shown in organotypic slice cultures that a single AP in a DG granule cell leads to net inhibition in CA3 PCs through feedforward inhibition, and this can be shifted to excitation when the stimulation frequency increases to the 20-40 Hz range [START_REF] Mori | A frequency-dependent switch from inhibition to excitation in a hippocampal unitary circuit[END_REF]. In slice preparation, feedforward inhibition potently limits postsynaptic depolarization and burst firing of CA3 PCs (Torborg, Nakashiba, Tonegawa, & McBain, 2010). Recent studies from the lab have demonstrated that CA3 interneurons control spike transfer at hippocampal Mf synapses in vivo through both GABAA and GABAB receptors, with an optimum frequency of presynaptic activity at 10 Hz [START_REF] Zucca | Control of Spike Transfer at Hippocampal Mossy Fiber Synapses In Vivo by GABAA and GABAB Receptor-Mediated Inhibition[END_REF]. Altogether, presynaptic plasticity and feedforward inhibition define the temporal rules for efficient information transfer at DG-CA3 synaptic connections in the intact circuit [START_REF] Zucca | Control of Spike Transfer at Hippocampal Mossy Fiber Synapses In Vivo by GABAA and GABAB Receptor-Mediated Inhibition[END_REF]. In this work, I report that the spontaneous but not miniature IPSC frequency was significantly decreased. Inhibitory accumulative charges were also reduced during a 20Hz train stimulation protocol in 6-month APP/PS1 mice. These results suggest that the number of GABAergic connections of CA3 PCs may not be modified in general, but the presynaptic interneurons are giving less inputs to CA3 PCs. This change may be due to either alterations in the synaptic contacts between MfBs and local interneurons, or changes in the spiking activity of presynaptic CA3 interneuron. In line with our results, some studies have reported remodeling of GABAergic neurotransmission in the human AD brain (Fuhrer et al., 2017;Limon et al., 2012) and AD transgenic mouse models (Jo et al., 2014;Wu et al., 2014). Especially, APP/PS1 mice were shown to display spontaneous seizures, which are believed to be associated with an imbalance in the excitatory/inhibitory function in the pathology of AD (Hazra et al., 2013;Ramos et al., 2006). In APPswePS1dE9 mice, Minkeviciene et al. found that as early as 3.5 month there was persistently decreased resting membrane potential and firing threshold in neocortical layer 2/3 pyramidal cells and dentate granule cells, leading to increased network excitability, and bath-applied Aβ (proto-)fibrils achieved similar effects in juvenile rodent brain [START_REF] Minkeviciene | Amyloid beta-induced neuronal hyperexcitability triggers progressive epilepsy[END_REF]. Moreover, in APP/PS1-L166P mice, it was shown that there are age-dependent concomitant changes in the GABAergic pathway in the APP/PS1 mice (Oyelami et al., 2016). However, none of these studies above has pinpointed the specific subtypes of interneurons involved or other mechanisms underlying the altered GABAergic innervations. Intriguingly, Verret et al. recorded spontaneous epileptiform discharges in hAPP mice primarily during reduced gamma oscillatory activity. Because this oscillatory rhythm is generated by inhibitory parvalbumin (PV) neurons, they suspected that this network dysfunction arised from impaired PV cells. Supporting this hypothesis, hAPP mice and AD patients showed decreased levels of the interneuron-specific and PV cell-predominant voltage-gated sodium channel subunit Nav1.1. Restoring Nav1.1 levels in hAPP mice not only increased inhibitory synaptic activity and gamma oscillations, but also reduced hypersynchrony and memory deficits. This is consistent with the results from Donato et al., demonstrating that PV neurons are highly relevant in memory encoding and memory precision in healthy rodents [START_REF] Donato | Parvalbumin-expressing basket-cell network plasticity induced by experience regulates adult learning[END_REF][START_REF] Donato | Early-and late-born parvalbumin basket cell subpopulations exhibiting distinct regulation and roles in learning[END_REF]. Thus, it would be very interesting to further explore the intrinsic properties and/or synaptic connections of PV+ interneurons in hippocampal CA3 region.

Reduced KAR-mediated EPSCs in CA3 PCs

While AMPARs and NMDARs mediate most of basal excitatory synaptic transmission, KARs also play distinct roles in regulating the activity of CA3 circuits.

KARs are abundantly present in the stratum lucidum of the CA3 region, both pre-and postsynaptically at Mf synapses [START_REF] Darstein | Distribution of kainate receptor subunits at hippocampal mossy fiber synapses[END_REF][START_REF] Ruiz | Distinct subunits in heteromeric kainate receptors mediate ionotropic and metabotropic function at hippocampal mossy fiber synapses[END_REF]. The analysis of KAR knockout mice has provided us information about the potential role of KARs in hippocampus-dependent memory [START_REF] Carta | Kainate receptors in the hippocampus[END_REF]. For instance, GluK4 KO mice exhibit moderate deficits in spatial memory acquisition and recall but not in working memory tasks [START_REF] Catches | Genetic ablation of the GluK4 kainate receptor subunit causes anxiolytic and antidepressant-like behavior in mice[END_REF]; Lowry, Discussion 123 [START_REF] Lowry | The GluK4 kainate receptor subunit regulates memory, mood, and excitotoxic neurodegeneration[END_REF]. GluK2 KO mice were impaired in contextual fear conditioning [START_REF] Ko | Altered behavioral responses to noxious stimuli and fear in glutamate receptor 5 (GluR5)-or GluR6-deficient mice[END_REF], but did not show any deficits in the Morris water maze (Mulle et al., 1998).

At postsynaptic sites, KARs are present at only a subset of glutamatergic synapses in CA3 PCs and in a subpopulation of interneurons, influencing the integrative features of synaptic transmission by mediating the slow component of postsynaptic EPSCs [START_REF] Castillo | Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons[END_REF]Mulle et al., 1998;[START_REF] Pinheiro | Selective block of postsynaptic kainate receptors reveals their function at hippocampal mossy fiber synapses[END_REF]. In this work, we showed for the first time that there is a robust reduction in KAR-mediated currents at MF-CA3 synapses in any model of AD. We propose that there may be dysfunctional KARs or less KARs at postsynaptic sites of MF-CA3 synapses. Whether extrasynaptic KARs are affected or not is still unclear.

The function of KARs at Mf synapses is further complicated by the presence of presynaptic KARs. Presynaptic KARs can be activated by a single AP, resulting in the facilitation of synaptic transmission in a few milliseconds range, which can be observed during pairs or trains of stimuli as well as during spike transmission [START_REF] Sachidhanandam | Kainate receptors act as conditional amplifiers of spike transmission at hippocampal mossy fiber synapses[END_REF]. In addition, presynaptic KARs also facilitate the induction of the presynaptic LTP at Mf-CA3 synapses [START_REF] Contractor | Kainate receptors are involved in short-and long-term plasticity at mossy fiber synapses in the hippocampus[END_REF][START_REF] Lauri | Synaptic activation of a presynaptic kainate receptor facilitates AMPA receptor-mediated synaptic transmission at hippocampal mossy fibre synapses[END_REF]Pinheiro et al., 2007;[START_REF] Schmitz | Presynaptic kainate receptor mediation of frequency facilitation at hippocampal mossy fiber synapses[END_REF]. However, we did not observe any change in the presynaptic short-term and longterm plasticity at the Mf-C3 synapses in APP/PS1 mice. Thus, for now we do not have a reason to suspect deficits of KARs on the presynaptic sites in this AD mouse model.

Intriguingly, a reduction of KAR-mediated EPSCs were also observed in conditional PS KO mice. Presenilin (PS) is the catalytic component of the γ-secretase complex, so its mutation can alter γ-secretase cleavage and promote the production of Aβ42 [START_REF] Guo | Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock-in mice[END_REF][START_REF] Jankowsky | Mutant presenilins specifically elevate the levels of the 42 residue beta-amyloid peptide in vivo: evidence for augmentation of a 42specific gamma secretase[END_REF]. Apart from APP, N-cadherin (NCad) is also an important substrate of γ-secretase. NCads are synaptic adhesion proteins implicated in synapse formation and maturation [START_REF] Basu | The classic cadherins in synaptic specificity[END_REF]. It has been shown previously by our group, that activation of NCad recruits GluK2 subunit to cadherin/catenin complexes, stabilizing KARs at the synaptic membrane [START_REF] Coussen | Recruitment of the kainate receptor subunit glutamate receptor 6 by cadherin/catenin complexes[END_REF]. Both overexpression of a dominant-negative form of NCad and knockdown of NCad in CA3 PCs could lead to a strong reduction in the amplitude of KAR-EPSCs, while the amplitude of AMPAR currents were not significantly reduced [START_REF] Fievre | Molecular determinants for the strictly compartmentalized expression of kainate receptors in CA3 pyramidal cells[END_REF]. Our findings in APP/PS1 mice and PS KO mice suggest that the lack of normal NCad resulting from dysfunctional γ-secretase might underlie impaired recruitment of stabilization of KARs at MF-CA3 synapses.

Moreover, the impaired KAR-EPSCs and reduced GABAergic inputs in CA3 PCs might not be independent events, because there is also considerable expression of KARs in GABAergic interneurons in hippocampus. It has been shown that KAR agonists can activate presynaptic KARs, regulating GABAergic transmission onto CA1 pyramidal cells [START_REF] Clarke | A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission[END_REF][START_REF] Rodriguez-Moreno | Kainate receptor modulation of GABA release involves a metabotropic function[END_REF]. In fact, low concentrations of kainate facilitate GABA release, whereas high concentrations of KAR agonists depress GABAergic synapses in CA1 region [START_REF] Jiang | A kainate receptor increases the efficacy of GABAergic synapses[END_REF]. Kainate also increases GABAergic transmission between CA1 interneurons [START_REF] Cossart | Presynaptic kainate receptors that enhance the release of GABA on CA1 hippocampal interneurons[END_REF][START_REF] Mulle | Subunit composition of kainate receptors in hippocampal interneurons[END_REF]. However, we do not see any solid evidence of KARs activation by endogenous glutamate affecting GABAergic transmission. It will be interesting to study KARs expression and function in CA3 interneurons, or in Mf synapses onto these interneurons.

Finally, trains of high-frequency stimulation (50 stimuli at 25 Hz) can induce both NMDAR LTP and KAR LTD at Mf synapses, the latter depends on the activation of adenosine A2A receptors [START_REF] Chamberlain | Long-term depression of synaptic kainate receptors reduces excitability by relieving inhibition of the slow afterhyperpolarization[END_REF]Rebola et al., 2008). Since we observed a loss of NMDAR LTP in APP/PS1 mice, it might be interesting to check if KAR LTD is affected or not. However, considering the significantly reduced amplitudes of KAR-EPSCs in APP/PS1 mice, it might be difficult to register a difference.

CA3 circuits modifications after contextual learning

Being at the entry level of hippocampal circuitry, the CA3 region of hippocampus is thought to be important for episodic memory encoding, especially at the initial stage of acquisition. However, the links between the plasticity of CA3 circuits and memory encoding are not well understood (Rebola et al., 2017).

Paired recordings revealed that Mf can innervate up to four different types of CA3 interneurons through projections to different districts of the CA3 PC arborization [START_REF] Szabadics | Functional specificity of mossy fiber innervation of GABAergic cells in the hippocampus[END_REF]. These interneurons in the stratum lucidum innervated by filopodial or small en passant Mf synapses are the anatomical basis for feedforward inhibition of DG-CA3 circuits [START_REF] Evstratova | Information processing and synaptic plasticity at hippocampal mossy fiber terminals[END_REF][START_REF] Galvan | Multiple forms of long-term synaptic plasticity at hippocampal mossy fiber synapses on interneurons[END_REF]McBain, 2008;Torborg et al., 2010), which is thought to be closely related to episodic memory. Within hours, one-trial contextual fear conditioning and Morris water maze learning can lead to robust and reversible outgrowth of filopodia at Mf terminals that synapse onto PV-positive fast-spiking interneurons. This plasticity has been proved to be crucial for the precision of contextual memory (Ruediger et al., 2011). The increased number of filopodia at Mf terminals is thought to enhance DG-CA3 feedforward inhibition, possibly by enhancing the signal-to-noise ratio and building a more salient representation of the context from the DG inputs. Further work is needed, since there are still open questions about the actual time point at which the new synaptic contacts become fully functional and are integrated into the CA3 circuits for memory encoding. Plus, the involvement of other types of interneurons than PVexpressing cells is not yet fully investigated.

In line with these results, we showed that the spontaneous IPSC frequency was increased 3 hours after CFC, and this increase disappeared 24 hours after the contextual learning. This imply that the feedforward inhibition onto CA3 PCs undergoes rapid and reversible strengthening after CFC. However, unlike Reudiger et al. [START_REF] Ruediger | Goal-oriented searching mediated by ventral hippocampus early in trial-and-error learning[END_REF]Ruediger et al., 2011) have reported, we did not discover any changes in LMT filopodia numbers after CFC, suggesting the structural plasticity of MF synapses might already be at ceiling level before the contextual learning. This could be due to many reasons, for instance, the housing conditions of our animal facility, or the way we handled the subjects. Moreover, we observed increased sIPSCs but not mIPSCs, suggesting that probably not so many new functional inhibitory synapses were formed onto CA3 PCs rapidly after CFC in our experimental conditions, but the existing GABAergic connections were more active after learning.

However, a non-neglectable factor might be complicating the scenario, i.e. the mice were single-housed for one week before experiment at the age of 7 weeks. Social deprivation/isolation is a type of stress and can lead to reduced brain BDNF levels in adult C57BL/6J mice (Barry et al., 2012), reduced neurogenesis as well as poorer learning and memory performance in rodents [START_REF] Famitafreshi | Social isolation is associated with reduced neurogenesis, impaired spatial working memory performance and altered anxiety levels in male rats[END_REF]. Particularly, some studies have shown that social isolation can alter the GABAergic activities in hippocampus. For example, male adult mice socially isolated for 4 weeks showed reductions in levels of allopregnanolone in hippocampal CA3 pyramidal neurons and DG granular cells (Agís-Balboa, [START_REF] Agís-Balboa | Downregulation of neurosteroid biosynthesis in corticolimbic circuits mediates social isolation-induced behavior in mice[END_REF][START_REF] Nin | Neurosteroids reduce social isolation-induced behavioral deficits: a proposed link with neurosteroidmediated upregulation of BDNF expression[END_REF], while allopregnanolone being a GABAA receptor active neurosteroid that facilitates the inhibitory actions of GABA and up-regulates BDNF in the adult mouse [START_REF] Nin | Neurosteroids reduce social isolation-induced behavioral deficits: a proposed link with neurosteroidmediated upregulation of BDNF expression[END_REF][START_REF] Pinna | In a mouse model relevant for post-traumatic stress disorder, selective brain steroidogenic stimulants (SBSS) improve behavioral deficits by normalizing allopregnanolone biosynthesis[END_REF]. Although in our study, the mice were only single-house for one week, we could not exclude the possibility that social isolation and stress might mask or exaggerate some changes in GABAergic circuits.

In addition, expression of immediate early genes in CA3 PCs can provide a link between behavioral experience and the molecular events that are necessary for memory encoding [START_REF] Kubik | Using immediate-early genes to map hippocampal subregional functions[END_REF]. We showed in this work that the C-Fos positive CA3 PCs were increased rapidly after CFC. However, other immediate early genes may serve as better labels for memory traces. For example, it has been shown that the activity-dependent transcription factor NPAS4 is selectively expressed in CA3 PCs after presentation of a novel context, and selective deletion of the gene encoding NPAS4 in CA3 impairs contextual fear conditioning [START_REF] Ramamoorthi | Npas4 regulates a transcriptional program in CA3 required for contextual memory formation[END_REF]. Moreover, NPAS4 is also shown to be involved in the activity-dependent regulation of inhibitory synapse development [START_REF] Lin | Activity-dependent regulation of inhibitory synapse development by Npas4[END_REF]. Therefore, it will be interesting to express fluorescent proteins or even channelrhodopsins under NPAS4 promoter, so that we can identify memory-related CA3 PCs to record from or manipulate. In this way, we may be able to deepen our understanding of CA3 circuits modifications after contextual learning.

Transcriptome changes in CA3 region in early AD

As we know, epigenetic markers are actively regulating gene expressions on a genomic level, both during learning in healthy subjects and in neuropathological situations. For example, histone-methyltransferase MLL2 (KMT2B) is shown to be essential for memory formation in mice [START_REF] Kerimoglu | Histone-methyltransferase MLL2 (KMT2B) is required for memory formation in mice[END_REF]; histone modification profile can be altered in AD mouse models, and systemic application of histone deacetylase inhibitor (HDACi) can rescue the memory deficits in these mice [START_REF] Kilgore | Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of Alzheimer's disease[END_REF]. In the same direction, we first performed RNA-seq with dissected CA3 tissue from APP/PS1 mice to screen for changes on a transcriptome level, and found a list of up-and down-regulated genes at this early stage of AD. Then we carried out ChIP-seq for a histone modification marker: H3K4me3, which has been shown to be directly related to one-trial contextual memory [START_REF] Gupta | Histone methylation regulates memory formation[END_REF], and we found that there was a concrete decrease in H3K4me3 levels at the promoter areas of various genes in CA3 PCs, especially those involved in neuronal proliferation. Adult neurogenesis is known to be very important for encoding new information [START_REF] Leuner | Is there a link between adult neurogenesis and learning?[END_REF][START_REF] Aimone | Potential role for adult neurogenesis in the encoding of time in new memories[END_REF]. Our finding is yet another piece of evidence among reports of altered neurogenesis in various models of AD (Haughey et al., 2002;[START_REF] Jin | Enhanced neurogenesis in Alzheimer's disease transgenic (PDGF-APPSw, Ind) mice[END_REF][START_REF] Dong | Modulation of hippocampal cell proliferation, memory, and amyloid plaque deposition in APPsw (Tg2576) mutant mice by isolation stress[END_REF][START_REF] Donovan | Decreased adult hippocampal neurogenesis in the PDAPP mouse model of Alzheimer's disease[END_REF][START_REF] Zhang | Long-lasting impairment in hippocampal neurogenesis associated with amyloid deposition in a knock-in mouse model of familial Alzheimer's disease[END_REF]. Especially, one study using the same AD model as our group showed that these mice have diminished new-born neuronal survival in hippocampus but no change in overall cell proliferation, resulting in decreased number of newborn neurons in DG [START_REF] Verret | Alzheimer's-type amyloidosis in transgenic mice impairs survival of newborn neurons derived from adult hippocampal neurogenesis[END_REF].

However, the down-regulated genes with low H3K4me3 marker are not overlapping much with the down-regulated genes from RNA-seq result, suggesting that other epigenetic mechanisms might play more important roles in expressing early deficits in this AD mouse model.

Perspectives

Our work mainly explored the pre-and postsynaptic features of hippocampal CA3 synapses in an AD mouse model. We showed that LTP of NMDARs is abolished at the Mf-CA3 synapses, GABAergic innervations of CA3 PCs is slightly reduced, and postsynaptic KARs in CA3 PCs shows a marked decrease in 6-months old APP/PS1 mice. Moreover, we showed that in CA3, at this early stage of AD, many transcriptional regulations are already taking place across the whole genome. Last but not least, we showed changes of GABAergic synaptic transmission in CA3 circuits rapidly after contextual fear conditioning, confirming its role in memory encoding.

As a next step, we would like to further explore the phenomenon of reduced KARmediated EPSCs at Mf-CA3 synapses. For example, it is important to know whether the reduced conductance of KARs is due to reduced number of functional KARs at the postsynaptic sites, or less KARs are present on cell membrane in general. To this end we can for instance wash in Kainate in the aCSF while record the KAR events from CA3 PCs. This can also be accompanied by immunohistochemistry staining of KARs in CA3 region as a confirmation.

In another direction, it will be also interesting to explore which subtypes of interneurons are mediating modifications in CA3 GABAergic transmission in AD models as well as in healthy subjects after learning. For this we might need genetically encoded fluorescent markers for interneurons, so that we can register their properties and connections with CA3 PCs. Of course, with all these data being collected from in vitro acute hippocampal slices, we would be interested to see some confirmation by in vivo electrophysiology studies from rather intact CA3 circuits.

Overall, CA3 circuits are worth studying in that they have all the anatomical and functional attributes to serve in the rapid encoding of episodic memory, and are also among the first targets to be impaired when early AD pathologies occur. Hopefully these experiments provide novel insights into synaptic dysfunction differentially expressed at specific inputs onto a single CA3 PC at an early stage of AD, and contribute to the understanding of the circuits and mechanisms underlying contextual memory encoding. At the hub of all connection, a multi-compartment conductance-based leaky integrate-and-fire neuron model (iaf_cond_alpha_mc) was used for CA3 pyramidal cells [START_REF] Bernander | Synaptic background activity influences spatiotemporal integration in single pyramidal cells[END_REF][START_REF] Meffin | An analytical model for the "large, fluctuating synaptic conductance state" typical of neocortical neurons in vivo[END_REF] (Figure 6.1), and for DG granular cells and interneurons a simpler leaky integrate-andfire neuron model was used [START_REF] Gewaltig | Propagation of cortical synfire activity: survival probability in single trials and stability in the mean[END_REF][START_REF] Rotter | Exact digital simulation of time-invariant linear systems with applications to neuronal modeling[END_REF]. The model iaf_cond_alpha_mc contains three compartments: soma, proximal dendrite, and distal dendrite. An action potential is generated when somatic membrane potential exceeds the threshold, and there's a refractory period after a spike. However, the dendritic membrane potentials are not manipulated in this case.

We set excitatory or inhibitory conductance-based synapses onto each Moreover, 400 interneurons for feedforward inhibition (FFI interneuron) and 200 interneurons for feedback inhibition (FBI interneuron) was added to the system. Each FFI interneuron received inputs from a circular area (radius: 0.8, kernel: 0.9, weights=5) of cells in DG layer, and projected onto the CA3 layer in a donut manner (inner radius: 0.2, outer radius: 0.8, kernel: 0.8) through proximal inhibitory synapses. While the FBI interneurons received general input from CA3 layer (followed pairwise Bernoulli distribution with a probability of 0.9, weights: 1), and projects back to CA3 layer in a similar manner through proximal inhibitory synapses. Noise was introduced with 2

Poisson spike generators (at 9700Hz and 14300Hz respectively) (Figure 6.3). Six different binary input sequences were designed. When run through the network, an output spike pattern was generated for each input signal (Figure 6.4). Parameters of the network were adjusted to achieve a more physiological activity pattern for the 4 groups of neurons. In the end, the average firing rate of each population was calculated: DG 14. 3 Hz,CA3 15.2 Hz,FFI interneuron 19.2 Hz,and FBI interneuron 25.3 Hz.

Test of the model with input/output correlation

The fidelity of this artificial network was tested using an input/output correlation equation as follows:

Where η μ was the binary input vector, and X Δt (t) was the binarized output spike pattern.

The test was carried out multiple times with different FFI and FBI weights to optimize the fidelity of the network (Figure 6.5). After being fit to a curve, we could see that both too high and too low inhibition weights decreased the I/O correlation.

Another thing to note is that as the I/O correlation was increasing together synaptic weights before reaching the plateau on the curve, the computational burden for each neuron was also increasing, resulting in unnaturally high firing rate in the neuron.

Therefore, a slightly conservative inhibitory synaptic weight should be picked for the interneurons in order to have a more physiological network model. Overall, we built this over-simplified computational model of the DG-CA3 neuronal networks, to investigate if and to what extent the local interneurons in CA3 region contribute to memory encoding precision. This model can be further improved by implementing the spike timing-dependent plasticity (STDP) rule at the synapses [START_REF] Dan | Spike timing-dependent plasticity: from synapse to perception[END_REF][START_REF] Kobayashi | Spike train timing-dependent associative modification of hippocampal CA3 recurrent synapses by mossy fibers[END_REF]. STDP happens when the pre-and postsynaptic sides are excited within a specific time window, e.g. normally LTP is induced when stimulation of presynaptic fibers happens before the postsynaptic cell fires. This protocol is called STDP and LTP is normally induced when stimulation of presynaptic fibers happens before the postsynaptic cell fires [START_REF] Caporale | Spike timing-dependent plasticity: a Hebbian learning rule[END_REF][START_REF] Feldman | The spike-timing dependence of plasticity[END_REF]. For example, in a CA3-CA1 computational model, Jang et al. especially applied STDP rule to two groups of interneurons for feedforward and feedback inhibition, and found that the two pathways differentially modulate synaptic activity, and inhibitory synaptic weight and synaptic location influenced the STDP profile, suggesting a role of inhibitory network of STDP and in memory processing of hippocampal circuits [START_REF] Jang | GABAA receptor-mediated feedforward and feedback inhibition differentially modulate hippocampal spike timing-dependent plasticity[END_REF]. Moreover, Zalay et al. simulated the hippocampal Mf-CA3 network with both di-synaptic inhibition and monosynaptic excitation, and found that feedforward inhibition is crucial for the high-pass filter function of Mf-CA3 circuits [START_REF] Fortune | Short-term synaptic plasticity as a temporal filter[END_REF].

Taken together, this is only a very primitive model of DG-CA3 neuronal networks, and the reality is much more complicated than the constructed model. However, it might serve some purposes in neuroscience research. With more improvement, such models could be potentially used for generating disease models where the behavior of a certain type of neurons was altered. It could also be used for predicting, for example, interneuron subtypes in certain circuits by fine-tuning properties of the neuronal population.

INTRODUCTION

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and best-known cause of autism (reviewed by [START_REF] Hagerman | The physical and behavioural phenotype[END_REF]Abrahams and Geschwind, 2008;Bassel and Warren, 2008). In most cases FXS is caused by transcriptional silencing of the FMR1 gene and the ensuing lack of encoded Fragile X Mental Retardation Protein (FMRP) [START_REF] Penagarikano | The pathophysiology of fragile x syndrome[END_REF], an RNA-binding protein that regulates translation and trafficking of its interacting mRNAs in dendrites and axons [START_REF] Antar | Metabotropic glutamate receptor activation regulates fragile x mental retardation protein and FMR1 mRNA localization differentially in dendrites and at synapses[END_REF][START_REF] Zalfa | A new function for the fragile X mental retardation protein in regulation of PSD-95 mRNA stability[END_REF][START_REF] Bassell | Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function[END_REF][START_REF] De Diego-Otero | Transport of fragile X mental retardation protein via granules in neurites of PC12 cells[END_REF][START_REF] Ferrari | The fragile X mental retardation protein-RNP granules show an mGluR-dependent localization in the post-synaptic spines[END_REF][START_REF] Penagarikano | The pathophysiology of fragile x syndrome[END_REF][START_REF] Santoro | Molecular mechanisms of fragile X syndrome: a twenty-year perspective[END_REF]. During the last decade numerous FMRP target mRNAs have been identified (Chen et al., 2003;Darnell et al., 2001, Brown et al., 2001;[START_REF] Darnell | FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism[END_REF]. In contrast, how these molecules contribute to different features of FXS pathology has been only partially unraveled [START_REF] Gkogkas | Pharmacogenetic inhibition of eIF4E-dependent Mmp9 mRNA translation reverses fragile X syndromelike phenotypes[END_REF][START_REF] Zhang | Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1(-/y) mice[END_REF]. Studies from Fmr1 knockout (KO) mouse model of FXS provide compelling evidence that an increased expression of a subset of synaptic proteins  and subsequent alteration in synaptic plasticity  underlie the cognitive aspects of this disorder (reviewed by [START_REF] Pfeiffer | The state of synapses in fragile X syndrome[END_REF]). In particular, exaggerated group-I metabotropic glutamate receptor subtype 5 (mGluR5)/protein synthesis-dependent hippocampal long-term depression (LTD) of -amino-3-hydroxy-5methyl-4-isoxazole propionic acid receptor (AMPAR) currents is a hallmark feature of FXS [START_REF] Huber | Altered synaptic plasticity in a mouse model of fragile X mental retardation[END_REF][START_REF] Nosyreva | Metabotropic receptor-dependent long-term depression persists in the absence of protein synthesis in the mouse model of fragile X syndrome[END_REF]. These findings are central to the mGluR5 theory of FXS [START_REF] Bear | The mGluR theory of fragile X mental retardation[END_REF]. In agreement with this notion, correction of the aberrant mGluR5 signaling, through either pharmacological or genetic means, leads to the rescue of a number of disease phenotypes [START_REF] Dölen | Correction of fragile X syndrome in mice[END_REF][START_REF] Dölen | Mechanism-based approaches to treating fragile X[END_REF][START_REF] Michalon | Chronic pharmacological mGlu5 inhibition corrects Fragile X in adult mice[END_REF][START_REF] Michalon | Chronic metabotropic glutamate receptor 5 inhibition corrects local alterations of brain activity and improves cognitive performance in fragile X mice[END_REF]. Although much work has focused on the protein-synthesis dependent functional consequences of mGluR5 activation, other findings suggest that the intrinsic properties and signal transduction mechanisms of mGluR5 might also be altered in FXS (reviewed by [START_REF] D'antoni | Dysregulation of group-I metabotropic glutamate (mGlu) receptor mediated signalling in disorders associated with Intellectual Disability and Autism[END_REF]). Indeed, previous work has demonstrated an important link between long Homer proteins and mGluR5 at the synapse. In the absence of FMRP, this interaction is reduced [START_REF] Giuffrida | A reduced number of metabotropic glutamate subtype 5 receptors are associated with constitutive homer proteins in a mouse model of fragile X syndrome[END_REF], likely contributing to an altered mGluR5-mediated signaling, neocortical circuit defects and behavioral dysfunction in Fmr1 KO mice [START_REF] Ronesi | Homer interactions are necessary for metabotropic glutamate receptor-induced long-term depression and translational activation[END_REF][START_REF] Ronesi | Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome[END_REF][START_REF] Tang | Homer protein-metabotropic glutamate receptor binding regulates endocannabinoid signaling and affects hyperexcitability in a mouse model of fragile x syndrome[END_REF]. Homer proteins are a family of post-synaptic density (PSD) scaffolding proteins responsible for the link between mGluR5 and other PSD proteins [START_REF] Tu | Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors[END_REF][START_REF] Tu | Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins[END_REF]. Both long (Homer1b/c, Homer2, and Homer3 -here collectively referred to as Homer) and short (Homer1a) isoforms have been identified; the long Homer isoforms are constitutively expressed, multimerize, and link mGluR5 to signaling pathways within the PSD (reviewed by [START_REF] Shiraishi-Yamaguchi | The Homer family proteins[END_REF]). Homer1a, on the other hand, is an immediate early gene inducible by synaptic activity and functions as a dominant negative regulator of group-I mGluR signaling by disrupting the binding between mGluR5 and Homer [START_REF] Xiao | Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins[END_REF]. Interestingly, mGluR5 and NMDA receptor (NMDAR) are linked together in the PSD by a Homer-containing complex [START_REF] Brakeman | Homer: A protein that selectively binds metabotropic glutamate receptors[END_REF][START_REF] Scannevin | Postsynaptic organization and regulation of excitatory synapses[END_REF][START_REF] Perroy | Direct interaction enables cross-talk between ionotropic and group I metabotropic glutamate receptors[END_REF]. This organization precludes the direct mGluR5/NMDAR association but can be disrupted by Homer1a [START_REF] Bertaso | Homer1a-dependent crosstalk between NMDA and metabotropic glutamate receptors in mouse neurons[END_REF][START_REF] Moutin | Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability[END_REF]. The disassembly of the synaptic multimeric mGluR5/Homer complex by Homer1a promotes physical and functional interactions between NMDAR and mGluR5, resulting in mGluR5-mediated inhibition of NMDA currents [START_REF] Moutin | Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability[END_REF]. The dynamic movement of synaptic components has emerged as a key feature of synaptic transmission and plasticity (reviewed by (Triller andChoquet, 2005, 2008;[START_REF] Anggono | Regulation of AMPA receptor trafficking and synaptic plasticity[END_REF]). Indeed, receptors on the neuronal surface constantly switch between mobile and immobile states, driven by thermal agitation and reversible binding to stable elements such as scaffolding proteins, cytoskeletal anchoring slots or extracellular anchors (reviewed by [START_REF] Choquet | The dynamic synapse[END_REF]). The local enrichment of receptors at the PSD is thought to result from receptor immobilization by stable elements that interact in a biochemical and structural network. Despite the prominent role of mGluR5 in the regulation of synaptic plasticity and cognition, little is known about mGluR5 surface dynamics at the synapse and how these dynamics modulate interactions with synaptic partners  both under physiological and pathological condition. Importantly, interaction of mGluR5 with Homer proteins at the postsynaptic site regulates lateral diffusion of mGluR5 (Sergè et al., 2002). Here, we explored the dynamics of mGluR5 at hippocampal synapses and the consequences of a potential disrupted interaction with Homer proteins for NMDAR function and plasticity, as well as for related cognitive deficits in Fmr1 KO mice. We addressed this question using a powerful combination of high-resolution single molecule tracking, electrophysiological and knockdown approaches in hippocampal neurons from wild type (WT) and Fmr1 KO mice together with behavioral analysis. We found that the lateral mobility of mGluR5 was increased specifically at the synaptic sites in Fmr1 KO hippocampal neurons and correlated with an increased synaptic confinement and co-clustering of mGluR5 and NMDAR. These changes were most likely the result of the mGlu5/Homer disruption. This led us to investigate changes in synaptic NMDA currents and their long-term depression following mGluR5 activation. Importantly, we found that restoring the mGluR5/Homer interaction by reducing the expression of Homer1a in the hippocampus rescued abnormal NMDAR function and plasticity as well as cognitive deficits in Fmr1 KO mice. Our data corroborate the view that the regulation of the interaction of mGluR5 with long Homer isoforms represents a promising therapeutic target for FXS.

RESULTS

Increased lateral diffusion of mGluR5 at synapses in Fmr1 KO neurons

To better dissect the mechanism(s) by which mGluR5 dysfunction causes synaptic abnormalities in Fmr1 KO mice, we first investigated the lateral mobility of mGluR5 within synapses of hippocampal neurons by employing a quantum dot (QD) based single-molecule tracking approach (Figure 1A). Synapses were labeled using an active mitochondria marker (MitoTracker) to distinguish them from extrasynaptic sites, as previously described [START_REF] Groc | Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies[END_REF]. The analysis of the trajectories of single mGluR5 molecules revealed that their diffusion coefficient was significantly enhanced within the synaptic compartment of Fmr1 KO as compared with WT neurons (Figure 1B and C; +38,89%; P < 0.001). This result indicates an increased mobility of mGluR5 within the synaptic membrane of Fmr1 KO neurons. Accordingly, the fraction of mobile mGluR5 (diffusion coefficient > 0.005 μm 2 /s) at the synapse was higher in Fmr1 KO neurons (+15.24%; P < 0.001). In contrast to the synaptic sites, lateral mobility of mGluR5 at extrasynaptic sites was comparable between Fmr1 KO and WT neurons (Figure 1D; diffusion coefficient: P = 0.085; mobile fraction: P = 0.7957). To determine whether the observed increase in membrane mobility was specific for mGluR5, or a more general phenomenon affecting other glutamate receptors as well, we also quantified the mobility of individual AMPA-type (AMPAR) and NMDA-type (NMDAR) glutamate receptors. To this end, we tracked GluA2 and GluN1 receptor subunits comprising the AMPAR and NMDAR tetramer complexes, respectively. The dynamics of these receptor subunits have been extensively characterized previously using QD-based tracking approaches [START_REF] Groc | Differential activitydependent regulation of the lateral mobilities of AMPA and NMDA receptors[END_REF][START_REF] Michaluk | Matrix metalloproteinase-9 controls NMDA receptor surface diffusion through integrin beta1 signaling[END_REF][START_REF] Opazo | CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin[END_REF]Ladépêche et al., 2013;[START_REF] Zhang | Regulation of AMPA receptor surface trafficking and synaptic plasticity by a cognitive enhancer and antidepressant molecule[END_REF][START_REF] Hafner | Lengthening of the Stargazin Cytoplasmic Tail Increases Synaptic Transmission by Promoting Interaction to Deeper Domains of PSD-95[END_REF]. We found no differences in the lateral diffusion and the mobile fraction of AMPAR within the synaptic compartment (diffusion coefficient: P = 0.723; mobile fraction: P = 0.9094), whereas a small but significant reduction was detected in the extrasynaptic compartment (diffusion coefficient: -4.39%, P < 0.001; mobile fraction: -3.44%; P < 0.001) of Fmr1 KO neurons (Figure S1). Conversely, NMDAR showed a small yet statistically significant increase in the lateral diffusion both in the synaptic (diffusion coefficient: +12.73%; P < 0.001) and the extrasynaptic sites (diffusion coefficient: +11.26%; P < 0.001) of Fmr1 KO neurons, which was not sufficient to affect the fraction of mobile receptors in both compartments (synaptic sites: P = 0.1734; extrasynaptic sites: P = 0.5004; Figure S2). These data suggest that the absence of FMRP differentially impacts the mobility of mGluR5, AMPAR and NMDAR, with a major effect for mGluR5 at synaptic sites.

Increased mGluR5 lateral diffusion is caused by the disruption of the mGluR5/Homer scaffold

Since Homer isoforms function as anchoring molecules for mGluR5 at synapses [START_REF] Tu | Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors[END_REF][START_REF] Tu | Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins[END_REF], we hypothesized that the previously described reduction in mGluR5/Homer interaction [START_REF] Giuffrida | A reduced number of metabotropic glutamate subtype 5 receptors are associated with constitutive homer proteins in a mouse model of fragile X syndrome[END_REF] leads to the exaggerated membrane mobility of mGluR5 in Fmr1 KO neurons reported here. If our prediction was correct, then disrupting the specific mGluR5/Homer interaction in WT neurons should mimic the disease phenotype. We tested this hypothesis, using a cell-permeable peptide containing the Homer binding motif of mGluR5 (TAT-mGluR5ct; characterized previously in [START_REF] Mao | The scaffold protein Homer1b/c links metabotropic glutamate receptor 5 to extracellular signal-regulated protein kinase cascades in neurons[END_REF]Ronesi et al., 2008, Figure 2A). Indeed, pre-incubation of WT neurons with TAT-mGluR5ct caused an increase in the lateral diffusion and mobile fraction of mGluR5 in the synaptic compartment (Figure 2B-D; diffusion coefficient; +62.63%, P < 0.001; mobile fraction +20.23%, P < 0.001; WT TAT-mGluR5ct vs WT TAT-mGluR5mu). Importantly, both parameters were now comparable to those of Fmr1 KO neurons without the peptide (Figure 2C and D; diffusion coefficient, P > 0.05; mobile fraction, P = 0.5047). As expected, pre-incubation of WT neurons with a peptide containing a mutated Homer binding motif (TAT-mGluR5mu; [START_REF] Mao | The scaffold protein Homer1b/c links metabotropic glutamate receptor 5 to extracellular signal-regulated protein kinase cascades in neurons[END_REF][START_REF] Ronesi | Homer interactions are necessary for metabotropic glutamate receptor-induced long-term depression and translational activation[END_REF] had no effect on the lateral diffusion of mGluR5 (Figure 2C and D; diffusion coefficient, P > 0.05, mobile fraction P = 0.8943). Moreover, neither TAT-mGluR5ct nor TAT-mGluR5mu treatment had any effect on the mGluR5 mobility in Fmr1 KO neurons (Figure 2C and D; diffusion coefficient, P > 0.05; mobile fraction, P = 0.1038; Fmr1 KO TAT-mGluR5ct vs Fmr1 KO TAT-mGluR5mu). Taken together, these experiments provide strong correlative evidence that changes in the lateral diffusion of mGluR5 detected in Fmr1 KO neurons are indeed due to a disrupted link between the long Homer scaffolding proteins and mGluR5.

Disrupted mGluR5/Homer scaffold leads to an increased surface co-clustering of mGluR5 and NMDAR in Fmr1

KO neurons

In addition to anchoring mGluR5 at synapses, Homer containing complexes also provide a physical link to NMDARs [START_REF] Brakeman | Homer: A protein that selectively binds metabotropic glutamate receptors[END_REF][START_REF] Scannevin | Postsynaptic organization and regulation of excitatory synapses[END_REF]. We thus explored whether the disrupted mGluR5-Homer scaffold might also alter the interaction between mGluR5 and NMDAR in Fmr1 KO neurons. In a first set of experiments, we took advantage of the detection accuracy of single QDs (reviewed in [START_REF] Groc | Surface trafficking of neurotransmitter receptor: comparison between single-molecule/quantum dot strategies[END_REF][START_REF] Triller | New concepts in synaptic biology derived from single-molecule imaging[END_REF]) by measuring the synaptic fraction of mGluR5-QD and GluN1-NMDAR-QD co-localized with MitoTracker (Figure 3A). We found that the synaptic fraction of both mGluR5-QD and GluN1-QD was increased in Fmr1 KO neurons (Figure 3B; mGluR5, P < 0.001; GluN1, P < 0.01). This finding provides direct evidence that mGluR5 and NMDAR are more confined within the synapse in Fmr1 KO neurons, likely increasing the probability of physical interactions between these receptors within a given time window. To examine this further, we performed a triple immunofluorescence labeling experiment for mGluR5, NMDAR and Homer1 (the latter used as a synaptic marker), together with confocal microscopy and posthoc image analysis (Figure 3C-F). Quantitative analysis of the proportion of mGluR5-/Homer1-positive or GluN1-NMDAR-/Homer1-positive intensity (expressed as a function of total mGluR5 or GluN1-NMDAR signal) suggests in increased localization of both mGluR5 and NMDAR at the synapse in Fmr1 KO neurons (Figure 3D andE, respectively; mGluR5 P < 0.001; NMDAR P < 0.001). In addition, combined analysis of all three markers points to a higher degree of co-clustering of mGluR5 and NMDAR at synaptic sites (Figure 3F; P < 0.05) further supporting a tighter association of these receptors in the absence of FMRP. To further examine whether disrupted mGluR5/Homer scaffold might provide a causal mechanism for this increased coclustering of mGluR5 and NMDAR in Fmr1 KO neurons, we again exploited the interfering peptide. As expected, the pre-incubation of WT neurons with TAT-mGluR5ct resulted in a significant increase in the mGluR5/NMDAR colocalization (Figure 3G; P < 0.05), reflecting the increased percentage of synaptic mGluR5 and NMDAR (Figure S3). However, pre-incubation with TAT-mGluR5mu had no effect (Figure 3G; P = 0.3426, Figure S3). Taken together, these data provide strong correlative evidence that the disruption of the mGluR5/Homer crosstalk causes a tighter physical association between mGluR5 and NMDAR at synapses in Fmr1 KO neurons.

Reduced NMDAR function and plasticity in Fmr1 KO neurons

The finding that mGluR5 and NMDAR are more closely associated at synaptic sites in Fmr1 KO neurons prompted us to probe alterations in NMDAR function. Synaptic NMDAR-mediated excitatory postsynaptic currents (EPSCsNMDA) induced by Schaffer collateral stimulation were investigated using whole-cell patch-clamp recordings from CA1 pyramidal neurons in acute hippocampal slices (Figure 4A). EPSCsNMDA displayed lower amplitudes in Fmr1 KO neurons when compared with WT neurons (Figure 4B and C; 46.5 ± 8 nA vs 175 ± 24 nA; P < 0.001). Consistently, the NMDA/AMPA ratio was significantly lower in Fmr1 KO neurons (Figure 4C; Fmr1 KO: 0.86 ± 0.05; WT: (1,60 ± 0.29; P = 0.0094). These defects in NMDAR function were mimicked in WT neurons by application of the interfering peptide TAT-mGluR5ct (Figure 4B and C; P < 0.01; 4B P < 0.05). These data thus strongly support our hypothesis that alterations in the membrane dynamics of mGluR5 and its tighter coupling with NMDAR, as a consequence of mGlu5/Homer disruption, mediate abnormal NMDAR function in Fmr1 KO neurons. It is well established that the activation of group-I mGluR induces an exaggerated LTD of EPSCAMPA in hippocampal Fmr1 KO neurons [START_REF] Huber | Altered synaptic plasticity in a mouse model of fragile X mental retardation[END_REF]. LTD of EPSCsNMDA by group-I mGluR activation [START_REF] Baskys | Agonists at metabotropic glutamate receptors presynaptically inhibit EPSCs in neonatal rat hippocampus[END_REF][START_REF] Snyder | Internalization of ionotropic glutamate receptors in response to mGluR activation[END_REF][START_REF] Watabe | Postsynaptic induction and presynaptic expression of group 1 mGluR-dependent LTD in the hippocampal CA1 region[END_REF][START_REF] Ireland | Mechanisms of group I mGluR-dependent long-term depression of NMDA receptor-mediated transmission at Schaffer collateral-CA1 synapses[END_REF][START_REF] Bhouri | mGlu1 receptor-induced LTD of NMDA receptor transmission selectively at Schaffer collateral-CA1 synapses mediates metaplasticity[END_REF], however, has not been investigated in Fmr1 KO mice. In WT hippocampal neurons, DHPG application (100 µM, 5 min) induced a strong and long-lasting reduction in EPSCsNMDA (Figure 4D). In contrast, this form of synaptic NMDAR plasticity was largely absent in Fmr1 KO neurons (Figure 4D; P < 0.001). As with the enhanced mGluR5/NMDAR co-clustering, this phenotype could also be recapitulated in WT neurons by use of our peptide mimicking approach, suggesting the defective mGluR5/Homer interaction as the underlying cause (Figure 4E-F; P < 0.01). These data suggest that the disruption of the mGluR5/Homer scaffold compromises NMDAR function under basal conditions as well as during synaptic plasticity in Fmr1 KO neurons.

Reduction of Homer1a expression rescues NMDAR function and plasticity

Homer1a is known to antagonize the interaction between mGluR5 and Homer. Thus, we asked whether knocking down Homer1a -an approach that fosters the mGluR5/Homer interaction by increasing the Homer1a/Homer balance [START_REF] Ronesi | Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome[END_REF] -could correct the dysfunction in NMDAR currents in Fmr1 KO neurons. To address this question we exploited an AAV vector expressing a small interfering hairpin RNA (shRNA) targeted against the unique 3'untranslated region of Homer1a mRNA [START_REF] Tappe | Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain[END_REF][START_REF] Klugmann | Targeting Homer genes using adeno-associated viral vector: lessons learned from behavioural and neurochemical studies[END_REF]. A similar vector expressing scrambled shRNA served as control [START_REF] Tappe | Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain[END_REF][START_REF] Klugmann | Targeting Homer genes using adeno-associated viral vector: lessons learned from behavioural and neurochemical studies[END_REF]. AAV vectors were stereotaxically injected into the CA1 area of the hippocampus of Fmr1 KO mice, and EPSCsNMDA from CA1 pyramidal neurons in acute brain slices were measured four weeks later (Figure 5A). EPSCsNMDA displayed higher amplitudes in Fmr1 KO neurons expressing the shRNA for Homer1a when compared to Fmr1 KO neurons expressing the scrambled shRNA (Figure 5B; P < 0.05). In addition, the mGluR-dependent LTD of EPSCsNMDA was significantly increased in Fmr1 KO neurons expressing the shRNA for Homer1a (Figure 5C; P < 0.001). Both findings confirm our hypothesis that mGlu5/Homer disruption can cause abnormal NMDA function and plasticity in Fmr1 KO neurons.

Reduction of Homer1a expression rescues cognitive defects in Fmr1 KO neurons

Can the correction of the disrupted mGluR5/Homer scaffold also rescue cognitive defects linked to NMDAR dysfunction? To evaluate the effects of Homer1a reduction on hippocampus dependent memory formation, we used two behavioral procedures -an object-recognition task and contextual fear-conditioning. Both tasks induce robust hippocampus dependent learning with a single training episode, and have previously been utilized for the investigation of memory defects in Fmr1 KO mice (Paradee et al. 2005;[START_REF] Ventura | Object recognition impairment in Fmr1 knockout mice is reversed by amphetamine: involvement of dopamine in the medial prefrontal cortex[END_REF][START_REF] Busquets-Garcia | Targeting the endocannabinoid system in the treatment of fragile X syndrome[END_REF][START_REF] King | Lithium treatment alleviates impaired cognition in a mouse model of fragile X syndrome[END_REF][START_REF] Oddi | Early social enrichment rescues adult behavioral and brain abnormalities in a mouse model of fragile X syndrome[END_REF]. Consistent with the aforementioned studies employing the first-generation Fmr1 KO mouse line, the second-generation model, used here, exhibited similar decreases in the discrimination index (DI) in the object-recognition task (Figure S5; P < 0.001) and in the percentage of freezing compared with WT mice (Figure S5; P < 0.001). We then tested whether mGluR5/Homer crosstalk modulation could correct these defects in another batch of behaviorally naive Fmr1 KO and WT mice, stereotaxically injected with the aforementioned AAV vectors into the CA1 area of the hippocampus. Importantly, Homer1a knockdown in the hippocampus corrected defects in both the object recognition memory (Figure 5D; P < 0.001) and context-based fear memory (Figure 5E; P < 0.05) in adult Fmr1 KO mice. Taken together with the aforementioned results, these data strongly support the idea that disruption of mGluR5/Homer scaffold contributes to impairment of hippocampal dependant cognitive function in Fmr1 KO mice.

DISCUSSION

In spite of the widespread acceptance of the mGluR theory of FXS, the sub-cellular mechanisms underlying mGluR5dependent alterations in synaptic function and plasticity, as well as the subsequent cognitive phenotypes remain poorly investigated. This is because most studies so far have focused on the altered translational processes and their effect on synaptic plasticity following mGluR5 activation. Recent evidence [START_REF] Ronesi | Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome[END_REF][START_REF] Tang | Homer protein-metabotropic glutamate receptor binding regulates endocannabinoid signaling and affects hyperexcitability in a mouse model of fragile x syndrome[END_REF], however, suggests that certain FXS phenotypes may be mediated by a less well-characterized aspect of mGluR5 dysfunction, namely by a defective mGluR5/Homer interaction leading to a reduced physical association with intracellular signaling pathways. Such defects are suggested to be upstream of protein translation [START_REF] Ronesi | Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome[END_REF] and may represent a novel therapeutic target for the treatment of FXS.

Here we demonstrate a novel role for mGluR5/Homer interaction in the regulation of mGluR5 surface dynamics and in the mGluR5-dependent modulation of NMDA currents under normal physiological conditions, as well as their pathophysiological modification in FXS. Specifically, we found that the disruption of the mGluR5/Homer association increased the lateral diffusion of mGluR5 at synaptic-, but not extrasynaptic sites, mimicking the phenotype observed in Fmr1 KO neurons. This increased motility (specifically at the synapse) is in coherence with previous findings showing that long Homer isoforms are enriched at synaptic sites [START_REF] Xiao | Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins[END_REF]. These alterations in receptor dynamics are specific for mGluR5 because the AMPAR subunit, GluA2, which does not bind Homer proteins, did not exhibit alterations in trafficking. Furthermore, although GluN1 also exhibited a slightly higher diffusion coefficient in Fmr1 KO neurons, this difference was observed both at synaptic and extrasynaptic sites. Other mechanisms, including higher activity of metalloproteinases 9 or CamKII, which are both increased in FXS [START_REF] Janusz | The Fragile X mental retardation protein regulates matrix metalloproteinase 9 mRNA at synapses[END_REF][START_REF] Zalfa | The fragile X syndrome protein FMRP associates with BC1 RNA and regulates the translation of specific mRNAs at synapses[END_REF], may impact on NMDAR lateral diffusion [START_REF] Michaluk | Matrix metalloproteinase-9 controls NMDA receptor surface diffusion through integrin beta1 signaling[END_REF][START_REF] Dupuis | Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses[END_REF]). In addition to the altered receptor dynamics, we observed an increased co-localization of mGluR5 and NMDAR in hippocampal synapses from both Fmr1 KO neurons or WT neurons treated with the interfering peptide. The higher mobility of mGluR5 in Fmr1 KO neurons (or interfering peptide-treated WT neurons) and the associated exaggerated synaptic confinement of both mGluR5 and GluN1, might increase the probability of transient interactions between these two receptor types at synapses. Similar findings have been reported following the induction of the immediate early gene Homer1a, leading to the mGluR5-mediated inhibition of NMDA currents [START_REF] Moutin | Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability[END_REF]. In accordance with these findings, we found that EPSCsNMDA evoked by Schaffer collateral stimulation showed lower amplitudes in Fmr1 KO neurons. Once again, the Fmr1 KO phenotype was mimicked by application of the competing peptide. Moreover, knocking down Homer1a in the hippocampus of Fmr1 KO mice rescued the aforementioned defect in EPSCsNMDA. Taken together, our findings suggest that this novel defect in NMDA function in the CA1 of Fmr1 KO mice is specifically mediated by an altered crosstalk between mGluR5 and NMDA and that this altered crosstalk is influenced by a disruption of the physical interaction between mGluR5 and long Homer isoforms (see model in Figure 6). NMDAR function has been the subject of previous studies in Fmr1 KO mice, yet there is little consensus concerning the type and nature of changes observed. While no change in NMDA currents was previously found in CA1 hippocampal pyramidal cells (Hu et al., 2008) andcortex (Desai et al., 2006), several studies show a reduction of synaptic NMDA currents in several brain regions of Fmr1 KO mouse including the anterior piriform cortex [START_REF] Gocel | Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse[END_REF], dentate gyrus [START_REF] Yun | Fragile X mice: reduced long-term potentiation and N-Methyl-D-Aspartate receptor-mediated neurotransmission in dentate gyrus[END_REF], prefrontal cortex [START_REF] Martin | Age-Dependent Long-Term Potentiation Deficits in the Prefrontal Cortex of the Fmr1 Knockout Mouse Model of Fragile X Syndrome[END_REF], and nucleus accumbens [START_REF] Neuhofer | Functional and structural deficits at accumbens synapses in a mouse model of Fragile X[END_REF]. One study, conversely, reports a transient increase in NMDAR-dependent LTP in the CA1 of 2-week old animals but not in adults (Pilpel et al., 2009). These differences reported in the literature likely reflect the range of brain regions, experimental protocols and ages of mice used. Interestingly, in accordance with our suggestion that a synaptic deficit of NMDAR in FXS might be caused by a mGluR5-mediated inhibition, the mGluR5 inverse agonist MPEP acutely rescued the increased AMPA/NMDA ratio detected in Fmr1 KO mice [START_REF] Martin | Age-Dependent Long-Term Potentiation Deficits in the Prefrontal Cortex of the Fmr1 Knockout Mouse Model of Fragile X Syndrome[END_REF]. In line with our hypothesis that altered mGluR5 dynamics may impinge on different aspects of NMDAR function in FXS, we also found that mGluR5-mediated LTD of EPSCsNMDA was reduced in Fmr1 KO neurons. Activation of group-I mGluRs is involved in the induction of NMDAR-dependent synaptic plasticity and it can induce Long Term Potentiation (LTP) or LTD depending on whether the currents are elicited by exogenous NMDA application or synaptic stimulation. Exogenous NMDAR mediated currents are transiently potentiated by group-I mGluR activation [START_REF] Benquet | Two distinct signaling pathways upregulate NMDA receptor responses via two distinct metabotropic glutamate receptor subtypes[END_REF][START_REF] Grishin | Differential calcium-dependent modulation of NMDA currents in CA1 and CA3 hippocampal pyramidal cells[END_REF][START_REF] Heidinger | Metabotropic glutamate receptor 1-induced upregulation of NMDA receptor current: mediation through the Pyk2/Src-family kinase pathway in cortical neurons[END_REF][START_REF] Mannaioni | Metabotropic glutamate receptors 1 and 5 differentially regulate CA1 pyramidal cell function[END_REF][START_REF] Skeberdis | mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C[END_REF][START_REF] Snyder | Internalization of ionotropic glutamate receptors in response to mGluR activation[END_REF]. Conversely, synaptically elicited EPSCsNMDA undergo depression in response to group-I mGluR activation [START_REF] Baskys | Agonists at metabotropic glutamate receptors presynaptically inhibit EPSCs in neonatal rat hippocampus[END_REF][START_REF] Snyder | Internalization of ionotropic glutamate receptors in response to mGluR activation[END_REF]Watabe et al. 2002 Ireland and[START_REF] Ireland | Mechanisms of group I mGluR-dependent long-term depression of NMDA receptor-mediated transmission at Schaffer collateral-CA1 synapses[END_REF]. The reasons for this differential response and the mechanisms underlying the mGluR-induced depression of the EPSCsNMDA are poorly understood. Here we suggest that the dynamic remodelling of the link between mGluR5 and Homer proteins plays a key role in the in the induction of LTD of synaptic EPSCsNMDA in response to group-I mGluR activation. A possible explanation for the reduced mGluR5-dependent NMDA-LTD observed in Fmr1 KO mice and in the presence of TAT-mGluR5ct peptide is that the increased physical interaction between NMDAR and mGluR5 might prevent NMDAR removal from the synapse, a mechanism that is believed to be responsible for depression of synaptic NMDA currents [START_REF] Ireland | Mechanisms of group I mGluR-dependent long-term depression of NMDA receptor-mediated transmission at Schaffer collateral-CA1 synapses[END_REF]. In line with this interpretation, NMDAR is more confined at synapses in the Fmr1 KO neurons and after incubation with the TAT-mGluR5ct peptide. Alternatively, mGluR5-mediated post-traslational modification of NMDAR facilitated by their physical interaction may influence the ion channel properties of NMDARs. Altered synaptic plasticity of NMDAR might be associated with cognitive impairment in the Fmr1 KO mouse. Mechanisms of LTP and LTD are believed to be the underlying cellular basis of learning and memory formations and altered mGluR-dependent NMDAR-LTD might impact on these functions in FXS. A role for a reduced Homer-mGluR5 interaction in hippocampal-dependent cognitive impairment induced by social defeat stress, has been recently documented in mice [START_REF] Wagner | Homer1 mediates acute stress-induced cognitive deficits in the dorsal hippocampus[END_REF]. Likewise, a selective overexpression of Homer1a in the dorsal hippocampus impairs spatial working memory [START_REF] Celikel | Select overexpression of homer1a in dorsal hippocampus impairs spatial working memory[END_REF]. Our finding that mGluR-dependent LTD of NMDA currents is reduced in Fmr1 KO mice, and that this reduction is strongly correlated with a disruption of the mGluR5/Homer scaffold has important implications, not only for our understanding of the pathophysiology of FXS, but also for future therapeutic approaches. NMDA hypofunction has been considered significant in the context of autism and schizophrenia and its correction by means of mGluR5 positive allosteric modulators has been proposed [START_REF] Gandal | Mice with reduced NMDA receptor expression: more consistent with autism than schizophrenia? Genes[END_REF]. Genetic or pharmacological reduction of mGluR5 activity coverslips and kept at 37°C in 5% CO2. The original plating Neurobasal culture medium (Gibco) supplemented with B27 (Gibco, 2%) and complemented with 5% fetal bovine serum was replaced with a serum free medium on day in vitro (DIV) 2. Cytosine B-D-arabinofuranoside (Sigma, 5 μM) was added on DIV 4. All the experiments were performed at DIV 12/15.

Pharmacological treatments

A cell-permeable (TAT-fused) peptide containing the proline-rich motif (PPXXF) of the mGluR5 C-terminal tail that binds the EVH1 domain of Homer, TAT-mGluR5ct (YGRKKRRQRRR-ALTPPSPFR), and a control peptide with a mutated Homer binding motif, mGluR5mu (YGRKKRRQRRR-ALTPLSPRR), were synthesized at the UT Southwestern Protein Chemistry Technology Center and kindly provided by Prof. K. Huber (Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA). The peptides were dissolved in H20 at a concentration of 5 mM, and aliquots of this stock concentration were stored at -20°C. Frozen aliquots of both TAT-fused peptides were used within 10 days and diluted to the desired final concentration. Hippocampal cultures were treated with TAT-mGluR5ct or TAT-mGluR5mu for 1 hour with a final concentration of 5 µM in serum free culture medium at 37°C. Slices were incubated during 4 hours with either TAT-mGluR5ct or TAT-mGluR5mu (each at 5µM) in oxygenated ACSF at room temperature (21-22° C). (S)-3,5-Dihydroxyphenylglycine (DHPG) (Abcam, # ab120007, 100 µM) was dissolved in ACSF and applied by bath perfusion.

Single-Particle (Quantum Dot) Tracking and Surface Diffusion Calculation

For single-molecule tracking experiments, neurons were first exposed for 10 minutes to either mouse monoclonal anti-NH2 mGluR5 antibody (1:20) [START_REF] Mion | Bidirectional regulation of neurite elaboration by alternatively spliced metabotropic glutamate receptor 5 (mGluR5) isoforms[END_REF], mouse monoclonal anti-GluA2 AMPA receptor (AMPAR) subunit antibody (Millipore, #MAB397, 1:200), or rabbit polyclonal anti-GluN1 NMDA receptor (NMDAR) subunit antibody (Alomone Laboratories, #AGC-001, 1:200) at 37°C. Neurons were then incubated for 10 minutes in a solution containing quantum dots (QD) 655 coupled to goat anti-mouse IgG (Invitrogen, #Q11022MP) or coupled to goat antirabbit IgG (Invitrogen, #Q-11421MP) (final dilution 1:5000/1:10.000) at 37°C. To label synaptic sites, neurons were incubated for 40 seconds at RT (~22°C) in a solution containing the orange mitochondria marker MitoTracker (Invitrogen, #M-7510, 20 nM). A fraction of coverslips was also incubated for 1 hour with TAT-mGluR5ct or TAT-mGluR5mu (5 µM) in culture medium at 37°C before and during the incubation with the primary antibodies. For QD 655 fluorescence imaging we used an EM-CCD camera (Evolve 512, Photometrics) with a 512 x 512 imaging array together with an HXP-120 light source (Zeiss) and the appropriate filters for excitation and emission. Images were acquired at an integration time of 50 milliseconds for up to 500 consecutive frames (24 seconds) as described previously [START_REF] Groc | Differential activitydependent regulation of the lateral mobilities of AMPA and NMDA receptors[END_REF]Ladépêche et al., 2013;[START_REF] Hafner | Lengthening of the Stargazin Cytoplasmic Tail Increases Synaptic Transmission by Promoting Interaction to Deeper Domains of PSD-95[END_REF]. QD movements were followed on randomly selected healthy looking dendritic regions for up to 20 minutes, and analyzed using Metamorph software (Universal Imaging Corporation, PA, USA). Briefly, the instantaneous diffusion coefficient, D, was calculated for each trajectory, from linear fits of the first 4 points of the mean-square-displacement versus time function using the following equation: MSD (t) = < r2 > (t) = 4Dt. To assign synaptic localization, trajectories were sorted into extrasynaptic (i.e. MitoTracker-negative pixels) and synaptic regions (MitoTracker-positive pixels). To determine the distribution and synaptic fraction of single QD complexes, frame stacks were obtained and on each frame the receptor/QD complexes were precisely located in synaptic and extrasynaptic compartments. Then, those locations were projected on a single image, providing a highresolution distribution of the receptor/QD complexes.

Immunocytochemistry and confocal analysis

The surface expression of mGluR5 was studied using an antibody against the NH2 terminal of the mGluR5 in nonpermeabilized neurons (see above QD tracking experiments). After removing the medium, cell cultures were incubated with the antibody (1:10) for 30 minutes at 37°C. Subsequently, cultures were fixed with a solution containing 4% paraformaldeyde (PFA) and 4% sucrose for 10 minutes at RT, permeabilized in PBS containing 0.1% Triton-X for 10 min, incubated with blocking solution containing 4% BSA for 45 minutes at RT, followed by incubation with the rabbit monoclonal anti-GluN1-NH2 antibody (Alomone Labs, #AGC-001, 1:200) and the Guinea pig polyclonal anti-Homer1 antibody (Synaptic Sistems, #160 004, 1:500) for 1 hour at RT. After washing, cultures were incubated for 45 minutes at RT with the appropriate secondary fluorescent antibodies (Alexa Fluor 647 anti-mouse, Invitrogen, #A-21236, 1:750; Alexa Fluor 555 anti-rabbit, Invitrogen, #A-21428, 1:750; Alexa Fluor 488 anti-Guinea pig, Invitrogen, #A-11073, 1:750). A fraction of coverslips was also incubated for 1 hour with TAT-mGluR5ct or TAT-mGluR5mu (final concentration for each: 5 µM in culture medium) at 37°C before the incubation with anti-mGluR5 antibodies. Images were acquired to measure co-localization of mGluR5, GluN1 and Homer1, using a commercial Leica DMI6000 TCS SP5 confocal microscope with identical settings for all conditions. Ten individual confocal images per coverslip were acquired at 12 bit-depth with a pixelsize of 96.2 nm x 96.2 nm (63x objective, 1.4 NA, 2.5 digital zoom, 1024 x 1024 pixel per image, 50 Hz scanning speed, 98.41 µm x 98.41 µm field of view). Images were processed with AutoquantX software (MediaCybernetics) and ImageJ software. A minimum of eight randomly chosen cells per condition was acquired and analyzed. A 2D blind deconvolution algorithm was first applied to each image in order to retrieve better data from our images. Then, analysis of the co-localization of mGluR5, GluN1 and Homer1 was performed using the "Co-localization" module of ImageJ (version 1.49; Scion Image, Frederick, MD). A custom-made macro was used to analyze the dendritic part of each image by measuring the intensity of each label using fixed threshold intensities.

Virus production and titration of recombinant adeno-associated virus (AAV)

Homer1a mRNA was targeted by RNA interference-mediated silencing. The recombinant AAVs were composed of biscistronic expression of short hairpin RNA, driven by the mouse RNA polymerase III U6 promoter (oligonucleotides corresponding to Homer1a-specific shRNA (shH1a; sense strand, 5'-GGAGCAUUGAGCUAAUUAUTT-3'; antisense strand, 5'-AUAAUUAGCUCAAUGCUCCTT-3'; Sigma Genosyses), and of control shRNA (sense strand, 5'-GUACUGCUUACGAUACGGTT-3'; antisense strand, 5'-CCGUAUCGUAAGCAGUACUTT-3'), combined with GFP, expressed under the control of the CBA promoter. The entire cassette was flanked by AAV2 inverted terminal repeats (ITRs). Briefly, rAAV2/1 vectors (shCtrl and shH1a) were produced by polyethylenimine (Collingridge et al.)-mediated triple transfection of HEK293 cells. HEK 293 cells were cotransfected with the AAV cis plasmid (pAAV-U6-shRNA-CBA-GFP-WPRE-bGH, control-shRNA or Homer1a-specific shRNA [START_REF] Tappe | Synaptic scaffolding protein Homer1a protects against chronic inflammatory pain[END_REF][START_REF] Klugmann | Targeting Homer genes using adeno-associated viral vector: lessons learned from behavioural and neurochemical studies[END_REF], the AAV1 (pH21) and AAV2 (pRV1) helper plasmids, and the adenovirus helper plasmid (pFD6). Seventy-two hours after transfection, cells were harvested and lysed by three sequential freeze-thaw cycles (-80°C/37°C). The AAV vector were purified using heparin affinity columns (GE HealthCare), concentrated using Amicon ultra-4 centrifugal filter units with a 100,000 molecular weight cutoff (Millipore) and filtered through a 13 mm diameter 0.2 μm syringe filter. Genomic titers were determined on the basis of an AAV2 ITR sequence-specific qPCR [START_REF] Aurnhammer | Universal real-time PCR for the detection and quantification of adenoassociated virus serotype 2-derived inverted terminal repeat sequences[END_REF], using a LightCycler® 480 Real-Time PCR System (Roche, Meylan, France).

AAV vector administration

Briefly, WT and Fmr1 KO mice (postnatal age 21 days) were anaesthetized with an isofluorane/air mix (3% for initial induction and 1.5%-2% for maintenance), head-fixed in a stereotaxic apparatus (David Kopf Instruments), and placed on a heating pad (HP-1M, Physitemp Instruments Inc) connected to a controller (TCAT-2LV, Physitemp Instruments Inc) set to maintain the body temperature at 37°C. Three hundred nanoliters of either AAV-shScr or AAV-shH1a were injected bilaterally into the dorsal hippocampus (Paxinos and Franklin, 2001; mouse brain atlas; coordinates: -1.95 mm AP, ± 1.25 mm ML, -1.35 mm DV from bregma). The viruses were injected at the rate of 75 nL/min. The injections were performed using a 34-gauge needle (World Precision Instruments) attached to a 10 µL-NanoFil microsyringe (Nanofil, World Precision Instruments). The microsyringe was driven by an electronic micropump system (UltraMicroPump, World Precision Instruments) connected to a microprocessor controller (Micro4, MicroSyringe Pump Controller). Electrophysiological experiments began 4 weeks after virus infusion when transgene protein expression had peaked to remain at stable levels (During et al., 2003). Behavioral training started 9 weeks after virus infusion.

Slice preparation and electrophysiological recordings of NMDA currents

Acute hippocampal slices were prepared from male WT and Fmr1 KO mice (post natal age 11-15 days). The brains were removed and placed in oxygenated ice-cold artificial cerebrospinal fluid (ACSF; in mM: NaCl 124; KCl 3.0; NaH2PO4 1.2; MgSO4 1.2; CaCl2 2.0; NaHCO3 26; D-glucose 10; pH 7.3). The two brain hemispheres were separated and transverse slices of dorsal hippocampus (300 µm thick) were cut using a vibratome (Leica VT1200). Slices were allowed to recover for at least 3 hours at RT while continually perfused with oxygenated ACSF. Some of the slices were incubated for 4 hours with 5-10 µM TAT-mGluR5ct or TAT-mGluR5mu in oxygenated ACSF at RT. For electrophysiological recordings, hippocampal slices were transferred to the recording chamber and visualized using a Leica DMLFS microscope equipped with 20X/0.30 and 40X/0.80 objectives, infrared filter and differential interference contrast (Mulle et al.). A tungsten monopolar electrode (WPI) was placed in the stratum radiatum to stimulate Schaffer collaterals with negative current pulses (duration 0.3 milliseconds), delivered every 30 seconds by a stimulus generator (A310 Accupulser with A360 stimulus isolator unit, WPI, USA). Stimulation intensity was set to induce 80% of maximal EPSC amplitude. Evoked EPSCs were recorded at RT from CA1 pyramidal neurons in the whole-cell patch-clamp configuration at a holding potential of -60 mV using an EPC7-plus amplifier (HEKA, Germany). Current traces were filtered at 3 kHz and digitized at 10 kHz. The NMDA-to-AMPA ratio (NMDA/AMPA) of evoked EPSCs was measured following standard procedures [START_REF] Myme | The NMDA-to-AMPA ratio at synapses onto layer 2/3 pyramidal neurons is conserved across prefrontal and visual cortices[END_REF]. Patch pipettes (open-tip resistance of 1.5-3 MΩ) were filled with intracellular solution containing cesium (130 mM) and tetraethylammonium (TEA, 10 mM). Mixed NMDA/AMPA EPSCs were evoked in ACSF containing bicuculline (5 µM) and glycine (10 µM). EPSCs were evoked every 15 sec and recorded at a holding potential of -90 mV, 0 mV, and +50 mV. For NMDA/AMPA ratio measurement, at least twenty traces were averaged at holding potential (HP) of -90 and +50 mV respectively. At a HP -90 mV, EPSCs were entirely mediated by activation of AMPA receptors (EPSCAMPA). The time to peak of EPSCAMPA at HP of -90 mV was used to establish the time window for measuring the peak EPSCAMPA at +50 mV. A complete decay to baseline of EPSCAMPA at HP of -90 mV generally occurred 40 ms after the stimulus artifact. This delay was used as a time window to measure the amplitude of the current mediated by NMDA receptors (EPSCNMDA) at a HP +50 mV. The amplitude of EPSCNMDA at +50mV divided by the amplitude of EPSCAMPA at +50mV was taken as the NMDA/AMPA ratio. For the LTD experiments, patch pipettes were filled with intracellular solution containing (in mM): K-gluconate 140; HEPES 10; NaCl 10; MgCl2 2; EGTA 0.2; QX-314 1; Mg-ATP 3.5; Na-GTP 1; pH 7.3. Slices were continuously perfused with ACSF at a flow rate of 1.0 ml/min. Following whole-cell access, the slice was perfused with Mg 2+ -free ACSF containing CNQX (10 μM), glycine (10 μM) and bicuculline (5 μM) to isolate EPSCs mediated by NMDA receptors (EPSCNMDA). DHPG (100 µM) was dissolved in the same ACSF and bath-applied for 5 min. Data were acquired and analyzed with Signal software (Cambridge Electronic Design, England). EPSCNMDA amplitude values were measured as the difference between peak current and baseline, averaged over 1 minutes and expressed as percentage of control (calculated from EPSCs recorded during at least 15 minutes prior to DHPG application). For the electrophysiological recordings of NMDA currents from Fmr1 KO mice injected with AAV-sh H1a or AAV-scr, hippocampal slices were prepared four weeks following vial injection (age of mice: 7-8 weeks). The brains were removed and placed in oxygenated ice-cold cutting solution (in mM: Choline-Cl 110; KCl 2.5; NaH2PO4 1.25; MgCl2 7; CaCl2 0.5; NaHCO3 25; D-glucose 20; Na-Ascorbate 5; Piruvate Acid 3,pH 7.3). The two brain hemispheres were separated and sagittal slices of dorsal hippocampus (350 μm thick) were cut using a vibratome (Leica VT1200S). Slices recovered during at least 30 minutes at 34°C in with oxygenated resting solution (in mM: NaCl 110; KCl 2.5; NaH2PO4 1.25; MgCl2 7; CaCl2 0.8; NaHCO3 25; D-glucose 20, Na-Ascorbate 1.3; Piruvate Acid 3,pH 7.3). Then the slices were Group values are expressed as mean ± s.e.m. Comparisons between groups for instantaneous diffusion coefficients were performed using Mann-Whitney test (pair comparison) or Kruskal-Wallis followed by Dunn's Multiple Comparison test (group comparison). Comparisons of cumulative distribution of instantaneous diffusion coefficients were performed using Kolmogorov-Smirnov test. The comparison of the percentage of immobile and mobile receptors was performed using Chi-square test with Yates' correction. All of the other comparisons between groups were performed using parametric statistical tests, Student's t-test (pair comparison), one or two-way Anova (group comparison) followed by Tukey's or Sidak's Multiple Comparison tests when appropriate. Significance levels were defined as *P < 0.05, **P < 0.01, and ***P < 0.001. represents the detection of a single receptor during 1-sec acquisition time), revealing the synaptic site as a trapping zone (green). (B) Relative fractions of synaptic mGluR5-QD (left panel) and GluN1-QD (right panel) particles. These values are increased in Fmr1 KO neurons (mGluR5-QD, WT, 8.053 ± 0.504 %, n= 16 dendritic fields; Fmr1 KO, 15.95 ± 0.685 %, n = 14 dendritic fields; P < 0.001 by unpaired Student's t-test; GluN1-QD, WT, 8.318 ± 0.382 %, n = 63 dendritic fields; Fmr1 KO, 10.02 ± 0.446 %, n = 51 dendritic fields; P < 0.01 by unpaired Student's t-test). (C) Representative images of cultured hippocampal neurons from WT and Fmr1 KO mice that were triple-labeled with the primary antibodies anti-mGlu5-NH2, anti-GluN1-NH2 and anti-Homer1. (D and E). Group data show a significant increase in the expression of mGluR5 and GluN1 in the synaptic area of Fmr1 KO neurons calculated as percentage of mGluR5/Homer1 or GluN1/Homer1 co-labeled area over total mGluR5 or GluN1 labeled area/field (mGluR5, WT, 9.527 ± 0.959 %, n = 27 dendritic fields; Fmr1 KO, 35.62 ± 4.618 %, n = 21 dendritic fields; P < 0.001 by unpaired Student's t-test; GluN1, 15.75 ± 1.841 %, WT n = 29 dendritic fields; Fmr1 KO 37.15 ± 5.324 %, n = 18 dendritic fields; P < 0.001 by unpaired Student's t-test). (F) Group data show a significant increase in the percentage of synaptic GluN1 co-localized with mGluR5 in Fmr1 KO neurons (WT, 63.97 ± 2.414 %, n = 26 dendritic fields; Fmr1 KO, 72.14 ± 2.081 %, n = 23 dendritic fields; P < 0.05 by unpaired Student's t-test). (G) Pretreatment with TAT-mGluR5ct peptide (5 µM, 1h) leads to a significant increase in mGluR5 and GluN1 co-localization in the synaptic area of WT neurons (WT, 63.97 ± 2.414 %, n = 26 dendritic fields; WT TAT-mGluR5mu, 61.19 ±3.489 %, n = 10 dendritic fields; WT TAT-mGluR5ct, 71.79 ± 1.528 %, n = 27 dendritic fields; P < 0.05 by one way Anova test). No differences were observed for co-localization in Fmr1 KO neurons treated with either TAT-mGluR5mu or TAT-mGluR5ct (both 5 µM, 1h) (Fmr1 KO, 72.14 ± 2.081 %, n = 23 dendritic fields; Fmr1 KO TAT-mGluR5mu, 67.32 ± 2.832 %, n = 20 dendritic fields; Fmr1 KO TAT-mGluR5ct, 67. ) was significantly lower than in WT (175.7 ± 21.9 pA, n = 11, P = 0.0004 by unpaired Student's t-test). In a similar manner, in WT slices treated with TAT-mGluR5-ct, the amplitude of EPSCsNMDA was significantly lower than in control conditions (88.1 ± 12.7 pA, n = 13, P = 0.0016 by unpaired Student's t-test). In neurons from WT slices incubated with TAT-mGluR5-mu, the amplitude of EPSCsNMDA was comparable to control (125,9 ± 16.8 pA n = 8, P = 0.11 by unpaired Student's t-test). Right panel: mixed AMPA-and NMDA-EPSCs were recorded at a holding potential of +50 mV to evaluate the NMDA/AMPA ratio. The NMDA/AMPA ratio in Fmr1 KO slices (0.86 ± 0.05, n = 8) was significantly lower than in WT (1,60 ± 0.29, n = 5, P = 0.0094 by unpaired Student's t-test). A reduction in NMDA/AMPA ratio was also observed in slices treated with TAT-mGluR5-ct (1,14 ± 0.03, n = 4). (D, F) EPSCsNMDA were recorded from CA1 pyramidal neurons at a HP -60 mV, in the presence of bicuculline and CNQX. Bath application of the group-I mGluR agonist DHPG (100 µM, 5 min) induced a long-term depression (mGluR-LTD) of EPSCsNMDA recorded in WT slices (EPSCsNMDA amplitude 30 min after LTD induction: 24.9 ± 2 % of control, n = 8). mGluR-LTD of EPSCsNMDA was not observed in Fmr1 KO neurons (EPSCsNMDA amplitude: 107.8 ± 25 % of control, n = 6, a value significantly different from WT, P = 0.004 by unpaired Student's t-test). (E, F) The lack of mGluR-mediated plasticity of EPSCsNMDA could be mimicked in WT slices after pretreatment with TAT-mGluR5-ct peptide (EPSCsNMDA amplitude: 100.6 ± 15 % of baseline, n = 8, a value significantly different from WT, P = 0.0006 by unpaired Student's t-test) but not with its control peptide TAT-mGluR5-mu (EPSCsNMDA amplitude: 56.6 ± 11 % of control, n = 7). Data are shown as mean ± s.e.m. ***P < 0.001, **P < 0.01, *P < 0.05. Figure 6. Model for dysfunction of the NMDAR/mGluR5 crosstalk in Fmr1 KO neurons. In WT neurons, long Homer proteins anchor mGluR5 to a chain of PSD proteins in the synapse and prevent a direct interaction with NMDAR. Under those conditions, a co-clustering of mGluR5 and NMDAR is prevented. In Fmr1 KO neurons, however, mGluR5 is less associated with the long Homer proteins and more associated with the short isoform, Homer1a. This disengagement from the long Homer protein-containing complex increases the lateral diffusion of mGluR5 and promotes the interaction with synaptic NMDAR. This constellation prevents boosting of NMDAR currents under control conditions and their LTD following mGluR5 stimulation.

Publication 3

Figure 1 . 1 .

 11 Figure 1.1. The two main modalities of synaptic transmission. A. Schematic representation of a chemical synapse. Chemical transmission requires sophisticated presynaptic machinery that releases neurotransmitters upon depolarization of the presynaptic terminal (e.g. the arrival of an AP). An equally complex postsynaptic machinery includes inotropic and metabotropic receptors is capable of detecting and translating the presynaptic message (neurotransmitter) into various postsynaptic events (e.g. resting potential changes or signaling cascades). B.

Figure 1 . 2 .

 12 Figure 1.2. Classification of glutamate receptors.

Figure 1 . 3 .

 13 Figure 1.3. Ionotropic and metabotropic glutamate receptors. Schematic representations of the two classes of glutamate receptors. The ionotropic receptors were further subdivided into three distinct subtypes AMPA, NMDA, and Kainate receptors. The iGluRs are assembled from four subunits, which will determine their functional properties. The AMPARs and KARs are predominantly permeable for Na + and K + , although there are certain Ca 2+ -permeable subtypes, whereas NMDA receptors are highly Ca 2+ -permeable. When activated by glutamate, AMPA receptors undergo rapid desensitization (time constant ~100 ms); KA receptors desensitize slower, and NMDA receptors show almost no desensitization. Metabotropic glutamate receptors (mGluRs) control intracellular second messenger signaling cascades as represented. Adapted from[START_REF] Verkhratsky | NMDA Receptors in glia[END_REF].

Figure 1 . 4 .

 14 Figure 1.4. Different forms of synaptic plasticity.

Figure 1 . 5 .

 15 Figure 1.5. Presynaptic mechanisms of use-dependent short-term plasticity. Schematic illustrations of different mechanisms for use-dependent short-term plasticity: A. depression, B. facilitation, C. post-tetanic potentiation (PTP) and augmentation.

Figure 1 .

 1 Figure 1.6. Well-described forms of LTP and LTD. Highly simplified diagrams of the induction and expression of synaptic plasticity observed in the rodent brain. A. NMDAR-dependent LTP is dependent on postsynaptic NMDAR activation and calcium/CaMKII signaling pathway for its initiation. The voltage-dependent relief of the magnesium block of the NMDAR allows the synapse

Figure 1 . 7 .

 17 Figure 1.7. Different forms of memory and the brain regions involved. Scheme of categorization of mammalian memory. Adapted from Squire et al. (Squire[START_REF] Squire | Conscious and unconscious memory systems[END_REF]

Figure 1 . 8 .

 18 Figure 1.8. Proposed roles of different medial temporal lobe regions in declarative memory tasks by fMRI studies. Scheme of the anatomical connections between the medial temporal lobe regions and the proposed roles of the hippocampus, entorhinal cortex, parahippocampal cortex, and perirhinal cortex in recognition memory according to the 'binding of item and context'

  structured in layers: stratum oriens (SO, inhibitory basket cells and horizontal trilaminar cells, recurrent fibers contacting the basal dendrites of pyramidal neurons here), stratum pyramidale (SP, contains the cell bodies of the pyramidal neurons and some interneurons), stratum lucidum (SL, only found in the CA3 where mossy fibers course through), stratum radiatum (SR, contains some interneurons, and Schaffer collateral fibers, part of recurrent fibers contacting the apical dendrites of pyramidal neurons here), stratum lacunosum-moleculare (SLM, perforant path fibers form synapses onto the apical tufts of the apical dendrites of pyramidal cells) (van Strien, Cappaert, & Witter, 2009) (Anderson, Morris, Amaral, Bliss, & O'Keefe, 2007). The input of hippocampus comes from neurons in Entorhinal cortex (EC) via the perforant path. Neurons in layers II of entorhinal cortex (EC) project to DG and CA3(the indirect pathway), whereas layer III neurons project to CA1 and the subiculum (the direct pathway). Subsequently, cells in DG project to CA3 through mossy fibers (Mf), and CA3 PCs project to CA1 through the Schaffer collaterals[START_REF] Witter | Architecture of spatial circuits in the hippocampal region[END_REF]. The hippocampal output is made by CA1 and the subiculum, reciprocally projecting to the deeper layers of EC (layer V and VI). There are also substantial projections of CA3 PCs to other CA3 PCs, through associational/commissural or recurrent fibers (Le[START_REF] Duigou | Recurrent synapses and circuits in the CA3 region of the hippocampus: an associative network[END_REF][START_REF] Li | The hippocampal CA3 network: an in vivo intracellular labeling study[END_REF] (Figure 1.10).

Figure 1 . 10 .

 110 Figure 1.10. Basic circuitry of the hippocampus. Schematic drawing of basic circuitry of the hippocampus. DG: dentate gyrus. Sub: subiculum. EC: entorhinal cortex. Adapted from hand drawings of Santiago Ramón y Cajal (1852 -1934).

Figure 1 .

 1 Figure 1.11. CA3 circuits and their proposed role in memory. This schematic illustration shows the different elements of CA3 circuits and their hypothesized involvement in memory encoding and recall. The CA3 PCs receives three types of glutamatergic inputs, including the input from EC through perforant path, from DG through mossy fibers, and from CA3 layer itself through A/C fibers. The CA3 PCs also receive inhibitory signals from local interneurons, this can be either feedforward inhibition coming from DG, or feedback inhibition coming from CA3 cells. Adapted from Rebola, Carta & Mulle, 2017.

Figure 1 .

 1 Figure 1.12. Mossy fiber terminals. A. Schematic representation of the hippocampus with the region of mossy fiber terminals indicated (dashed box). B. Dil-labeled MfBs. Scale bar, 5μm. C. CA3 neuron with TE spines labeled by intracellular injection of LY dye in fixed tissue. Scale bar, 5μm. D. Reconstructed Mf axons (red) with boutons (regions of vesicle accumulation;

  classified into various subtypes by their morphology, physiology, molecular expression patterns (such as receptors, neuropeptides or calcium-binding proteins), or biophysical features. For the convenience of our MF synapse electrophysiological studies, one functional classification of CA3 interneurons receiving MF inputs by Szabadics and Soltesz is shown below (Figure1.13)[START_REF] Szabadics | Functional specificity of mossy fiber innervation of GABAergic cells in the hippocampus[END_REF]. To be more specific, there are fast spiking basket cells and spiny lucidum cells receiving more MF inputs, which exhibit low release probability and small EPSC amplitudes; there are also regular spiking basket cells and Ivy cells receiving fewer MF inputs, which have high release probability and large EPSCs amplitudes. Long-term plasticities of MFinterneuron synapses in CA3 area is review byGalván et al. (Galván et al., 2011).

Figure 1 .

 1 Figure 1.13. Functional classification of some CA3 interneurons. A. Non-normalized averages from each cell type that received monosynaptic EPSCs from MFs after five presynaptic spikes at 50 Hz (top in gray: average of action

Figure 1 .

 1 Figure 1.14. Schematic representation of chromosome, chromatin, nucleosome, and histone octamers. A. Illustration showing how DNA is packaged into a chromosome. DNA wraps around the histone cores to form nucleosomes. These units then condense into a chromatin fiber, which condenses further to form a chromosome. Image copyright: Genome Research Ltd. B. Nucleosome as a "beads-on-a-string" structure. The nucleosome core is comprised of a histone octamer with histone 2A, 2B, 3 and 4, two copies each. The DNA double helix strand is wrapped around (~1.7 times) the histone octamer. Image copyright: Pearson Education Inc.

Figure 1 .

 1 Figure 1.15. Diagnostic criteria of AD (revised from NINCDS-ADRDA criteria)

Figure 1 .

 1 Figure 1.16. Scheme of the proteolytic processing of APP. APP (shown in the middle in the illustration) can be cleaved by α-, β-and γ-secretases. The cleavage sites of these proteases are α-, β-and γ-sites respectively. When APP undergoes non-amyloidogenic pathway (to the left side): it is cleaved by α-secretase within the Aβ sequence to generate sAPPα and the membrane-tethered αAPP-CT, which is then further cleaved by γ-secretase to release the P3 peptide and AICD; when APP undergoes amyloidogenic pathway (to the right side), it is cleaved by β-secretase resulting in the formation of soluble APPβ (sAPPβ) and βAPP-CTF, which is then further cleaved by γ-secretase releasing Aβ and AICD (amyloid precursor protein intracellular domain). Adapted from Nicolas & Hassan, 2014.

  425 µlDream Tag Green Buffer, Dream Tag Polymerase, and dNTP mix were purchased from Fermentas, Germany. The reaction mix was pipetted into 0.2 ml PCR tubes (Nerbe Plus, Germany) and spun down, and the reaction was performed using a Mastercycler ep gradient S (Eppendorf, Germany). The reaction conditions are described as follows:

  were conducted using the following setup: -Nikon FN-S2N microscope (equipped with 10×/0.30 and 60×/1.0W objectives) -Micromanipulator SM7 (Luigs & Neumann, Germany) -EPC 10 amplifier (HEKA Elektronik) (filtered at 2.9 kHz with a Bessel filter, digitized at 10 kHz) -DS3 constant current isolated stimulator (Digitimer Ltd., England) -LED excitation system at 490nm (CooLED, UK) -Greyscale CCD camera CoolSNAP HQ (Photometrics, USA) -PatchMaster data acquisition software (HEKA, Germany).

  ChIP) was performed to determine the H3K4Me3 level at promoter areas of variant genes. The frozen nuclei pellets of CA3 PCs were resuspended in 200 µl of RIPA buffer (Table2.6), and incubated on a rotator for 10min at 4ºC. The samples were sonicated for 35 cycles in Bioruptor Plus (30s ON/30s OFF, high power), and centrifuged at 18000 rcf for 5min. The supernatant containing sheared chromatin was transferred to a new DNA low-binding tube for further immunoprecipitation.

1 .

 1 Figure 1. The intrinsic properties of CA3 pyramidal neurons are not altered in APP/PS1 mice A. Representative traces of current-clamp recording during steps of increasing current injections for Wt (black traces) and APP/PS1 (red traces). B. Bar graph representing no difference in mean input resistance of CA3 pyramidal neurons between Wt (n=13) and APP/PS1 (n=12), t-test p value=0.4132, Data represented as mean ± SEM. C. Box plot representing no difference in mean firing threshold of CA3 pyramidal neurons between Wt (n=13) and APP/PS1 (n=12), t-test p value=0.1005, Data represented as mean ± SEM. D. Line chart representing the firing frequency during increasing current injections. Wt represented in black and APP/PS1 in red. Two-way ANOVA test p=0.6342 between Wt (n=13) and APP/PS1 (n=12). Data represented as mean ± SEM. E. F. G. H. the same experiments as in A-D but with the presence of NBQX (20μM), Bicuculline (10μM), CGP (3μM), D-AP5 (50μM), n=10 for Wt and n=11 for APP/PS1. F. t-test p=0.9949, G. t-test p=0.7722. H. Two-way ANOVA test p=0.1643 (dots at 40pA and 80pA showed significant difference in t-test).

Figure 2 .

 2 Figure 2. Morphology of the MfBs and postsynaptic thorny excrescences of CA3 cells is unaltered in APP/PS1 mice at 6 months of age. A. Example image of Mf axons along stratum lucidum acquired with confocal microscopy (upper panel). Zoom in on two examples of MfBs (lower left panel) and a representative reconstruction obtained with IMARIS software (lower right panel). This type of 3D reconstruction allows accurate measurements of surface area and volume. B. Cumulative distribution of MfB area from reconstructed MfBs for Wt (black trace) and APP/PS1 mice (red trace), Ks test p=0.144. C. Cumulative distribution of volume from reconstructed MfBs from Wt (black trace) and APP/PS1 mice (red trace), Ks test p>0.999. D. Cumulative distribution of the complexity index calculated for reconstructed MfBs showing a significant difference between Wt (black trace) and APP/PS1 mice (red trace), Ks test p<0.0001. This index uses both area and volume of each MfB analyzed. E. Summary plot representing the complexity index of three classes of MfBs. Smaller MfB have a volume smaller than 10 µm3, medium MfBs have a volume ranging from 10 to 20µm3, and large MfBs have volumes values above 20µm3.For Small MfBs the complexity index is 2.6 ± 0.1 for Wt mice and 2.6 ± 0.1 for APP/PS1 mice, displaying no difference between genotypes. Medium sized MfBs are more complex in both genotypes (Wt 3.3 ± 0.2; APP/PS1 3.7 ± 0.17). Finally, for large MfBs with a volume larger than 20µm3, the complexity index was, despite the visible tendency, not significantly altered in the APP/PS1 mice (Wt 4.3 ± 0.2; APP/PS1 4.8 ± 0.2, n=5 mice for each genotype). Data represented as mean ± SEM. F. Example image of the proximal dendrite of a CA3 pyramidal cell infected by RABV∆G-eGFP (RG) acquired with confocal microscopy (left panel) and subsequent reconstruction of ThE in stratum lucidum obtained by IMARIS software. G. Bar graph representing the total number of ThE per pyramidal cell. The number is not different in APP/PS1 and Wt mice. Just ThE on apical dendrites were analyzed and only cells with complete

Figure 3 .

 3 Figure 3. Mossy fiber input of CA3 neurons in terms of mEPSC is not selectively affected in 6-month APP/PS1 mice. A. Representative recording of mEPSCs (1mM TTX) during one minute, illustrating the difference of amplitude registered between CA3 cells from Wt and APP/PS1 mice. B. Summary plot of the mean mEPSCs amplitude registered during 15 minutes showing a reduction in mEPSCs amplitude from the APP/PS1 cells. Black bar represents Wt cells (n=15, 8 mice), grey bar represents cells from APP/PS1 mice (n=15, 9 mice), t-test, p=0.0009. C. Summary graph showing no difference between genotypes of the mEPSCs mean inter event intervals registered during a 15-minute period. Black bar represents Wt cells (n=15, 8 mice) and grey bar represents cells from APP/PS1 mice (n=15, 9 mice), t-test p=0.5863. Data represented as mean ± SEM, *** represents p<0.001. D. Cumulative probability distribution of the amplitudes from the first 100 mEPSCs registered from each cell.Black line represents the amplitude distribution of 1500 events from 15 CA3 cells successfully patched in Wt slices (n=8 mice); Mean values: Wt 26.7pA ± 0.7pA; APP/PS1 22.5pA ± 0.6pA; KS test p<0.0001, bin size of 2. E. Bar graphs representing the percentage of the putative Mf events (with an amplitude larger than 45pA) for each of the 15 cells recorded, during a 15-minute period. A tendency was found towards a reduced number of Mf-generated events, which is not statistically significant (Mann-Whitney test p=0.0718). Black bar, Wt cells (n=15, 8 mice). Grey bar, APP/PS1 mice (n=15, 9 mice). Data represented as mean ± SEM. F. Cumulative distribution of the amplitudes of events with high probability of being from Mf synapses is not different between Wt and APP/PS1 mice. Plot used all the events larger than 45pA obtained over a period of 10 minutes per cell (150 minutes in total per genotype). Mean values are 76.8pA for Wt and 78.4pA for APP/PS1; KS test p>0.999, bin size 2.

Figure 4 .

 4 Figure 4. Normal presynaptic short-term plasticity and presynaptic LTP of Mf synapses in 6months APP/PS1 mice. A. Representative average traces of the amplitude increase of Mf-EPSCs with FF. EPSCs obtained at 0.1Hz (30 sweeps) and 1Hz (30 sweeps) for Wt and APP/PS1 mice. B. Bar graph displaying comparable mean values of FF observed in Wt and APP/PS1. WT (4.67 ± 0.36) and APP/PS1 mice (4.26 ± 0.37). t-test p=0.4298, n=16 for WT and n=16 for APP/PS1. C. Average of 20 traces illustrating PPF at an inter-stimulus interval of 40ms for Wt (black traces) and APP/PS1 (red traces) Mf synapses. D. Bar graph representing the ratio between the amplitude of the second over the first EPSCs obtained during PP stimulating protocol. WT (2.11 ± 0.15) and APP/PS1 mice (2.15 ± 0.16). t-test p=0.8556, n=16 for WT and n=16 for APP/PS1. E. Average of 5 traces illustrating 10-stimuli 20Hz train at the interval of 2min apart, for Wt (black traces) and APP/PS1 (red traces) mice. F. Normalized amplitudes in the 10-stimuli 20Hz train. Two-way ANOVA test p=0.7745 between Wt (n=14) and APP/PS1 (n=12). Data represented as mean ± SEM. G. Summary plot of the LTP time course for Wt (black circles, n=12) and APP/PS1 (red circles, n=11) cells. The LTP protocol (100 pulses at a frequency of 100Hz repeated 3 times with a 10s inter train interval) was applied at the time indicated by the arrow. Mf-EPSCs were averaged every minute and normalized to the mean amplitude of the 10min period preceding the LTP induction protocol. H. Traces represent Mf-EPSCs obtained at a stimulus frequency of 0.1Hz during Baseline (10min), PTP (5min), LTP (30-40min after the train) and LCCG (5min after drug application). Black and red traces from representative cells in Wt and APP/PS1 mice, respectively. I. Bar graph with average values of PTP recorded 5minutes after the high frequency train was applied shows no difference between the two animal genotypes. Values are expressed as percentage of the baseline amplitude of Mf EPSPs. J. Bar graph representing the mean amplitude of Mf-EPSCs recorded between 30 to 40 minutes after LTP induction. Values are expressed as percentage of the baseline Mf-EPSCs amplitude. Data represented as mean ± SEM.

Figure 5 .

 5 Figure 5. NMDAR-EPSCs and Short-term plasticity of NMDAR is maintained in APP/PS1 mice, whereas a loss of Mf-NMDAR dependent LTP is observed in APP/PS1 mice. A. Representative traces of Mf-EPSCs recorded at negative (-70mV) and positive potentials (+40mV), with and without NBQX (20µM) illustrating no major differences between results obtained in the two genotypes. Bicuculline was present throughout all experiments. B. Bar graph resuming mean values of NMDAR/AMPAR ratios obtained with isolated currents at -70mV (AMPAR) and +40mV (NMDAR with NBQX). Black bar represents results from 9 cells from Wt mice and red bar represents results from 9 APP/PS1 cells. C. Bar graph representing mean values obtained for NMDAR/AMPAR ratio performed at +40mV in the presence of bicuculline. The ratio, calculated as the amplitude 40ms after the peak divided by the peak amplitude, was not different between the Wt (n=9) and APP/PS1 (n=9) cells analyzed. Data represented as mean ± SEM. D. Representative traces of NMDAR-mediated EPSCs recorded at +40mV, illustrating that both genotypes perform equivalent FF at positive potentials. Black traces illustrate average of 40 traces at 0.1Hz and 60 at 1Hz (from 0.1Hz to 1Hz) obtained from Wt cells (n=29). Red traces were obtained from APP/PS1 mice (n=31). E. Bar graph summarizing mean FF values obtained from NMDAR-EPSCs recorded at positive potentials from Wt and APP/PS1 cells, Mann-Whitney test p=0.1932. F. Summary plot of the mean decay time of Mf NMDAR-mediated EPSCs recorded at a frequency of 0.1Hz. Black and Red bars represent respectively Wt and APP/PS1 values of Mf NMDAR-EPSCs decay time (ms). Average decay times are not statistically different t-test, p=0.4782. G. Summary plot of the mean decay time of Mf NMDAR-mediated EPSCs recorded at a 1Hz frequency. Average decay times are not statistically different (t-test, p=0.7707) between Wt (black bars) and APP/PS1 mice (red bar). Data represented as mean ± SEM. H. Summary plot describing the time course of

Figure 6 .

 6 Figure 6. The frequency of spontaneous IPSCs and inhibitory charges in CA3 neurons are reduced, while the E/I ratio is maintained in APP/PS1 mice. A. Representative traces of spontaneous IPSCs (20μM NBQX extracellularly, 10mM QX-314 intracellularly) during 20s, recorded at -60mV, from CA3 cells of Wt and APP/PS1 mice. B. Summary graph showing no difference between genotypes of the sIPSCs mean amplitude registered during a 10-minute period. Black bar represents Wt cells (n=16, 6 mice) and red bar represents cells from APP/PS1 mice (n=18, 6 mice), t-test p=0.1147. C. Summary plot of the mean sIPSCs frequency registered during 10 minutes showing a reduction in sIPSCs frequency from the APP/PS1 cells. Black bar represents Wt cells (n=16, 6 mice), red bar represents cells from APP/PS1 mice (n=18, 6 mice), t-test, p=0.0254. Data represented as mean ± SEM, * represents p<0.05. D. Representative traces of spontaneous IPSCs (20μM NBQX and 0.5μM TTX extracellularly, 10mM QX-314 intracellularly) during 20s, recorded at -60mV, from CA3 cells of Wt and APP/PS1 mice. E. Summary graph showing no difference between genotypes of the mIPSCs mean amplitude registered during a 10-minute period. Black bar represents Wt cells (n=15, 6 mice) and red bar represents cells from APP/PS1 mice (n=15, 6 mice), t-test p=0.4292. F. Summary plot of the mean mIPSCs frequency registered during 10 minutes showing no difference in mIPSCs frequency from the APP/PS1 cells. Black bar represents Wt cells (n=15, 6 mice), red bar represents cells from APP/PS1 mice (n=15, 6 mice), t-test, p=0.9756. Data represented as mean ± SEM. G. Representative traces of Mf-EPSCs recorded at negative (-70mV) and IPSCs recorded at positive potentials (+10mV) at 1Hz stimulation, for Wt (black traces) and APP/PS1 (red traces), D-AP5 (20µM) was present through the experiment. H. Bar graph representing slightly elevated E/I ratio in APP/PS1 mice, but no significant difference was found. Black bar represents Wt cells (n=16, 10 mice), red bar represents cells from APP/PS1 mice (n=16, 10 mice), t-test with Welch's correction for unequal variances p=0.4882. Data represented as mean ± SEM. I. Representative traces of Mf-EPSCs recorded at negative (-70mV) and IPSCs recorded at positive potentials (+10mV) at 0.1Hz stimulation, for Wt (black traces) and APP/PS1 (red traces), D-AP5 (20µM) was present through the experiment. J. Bar graph illustrating no major differences between

Figure 3 . 1 .

 31 Figure 3.1. KAR-mediated EPSCs at Mf-CA3 synapses are significantly reduced in 6-month APP/PS1 mice. A. Representative traces illustrating evoked Mf-EPSCs of AMPAR and KAR during 1Hz stimulation, and was an average of 30 sweeps. 20µM D-APV, 10µM Bicuculline, 3µM CGP 55845 was present through the experiment to isolate AMPAR+KAR currents, and for KAR response recording, additional 25µM LY303070 was present. In the graph, black color was used for WT and red for APP/PS1 mice. B. Dot plot displaying the ratio of KAR-EPSCs amplitudes over AMPAR-EPSCs amplitudes at 1Hz, and there was a significant reduction in mean KAR/AMPAR ratio in the APP/PS1 mice comparing to WT littermates. Black dots represent WT (0.058 ± 0.005, n=13, 9 mice) and red dots represent APP/PS1 (0.032 ± 0.004, n=12, 6 mice), t-test p=0.0003. *** represents p<0.001. C. Box plot displaying the amplitude of AMPAR-EPSCs at 1Hz observed in Mf-CA3 synapses in WT (219.0 ± 20.25pA, n=13, 9 mice) and APP/PS1 mice (197.8

Figure 3 . 2 )

 32 Figure 3.2). The failure rates and AMPAR-EPSC amplitudes at 0.1Hz were also comparable between genotypes (For failure rates of AMPAR-EPSCs at 0.1 Hz, control 13.05 ± 3.54%, n=9; PSKO 19.06 ± 6.41%, n=10; t-test p=0. 6673; For amplitudes of AMPAR-EPSCs at 0.1 Hz, control 65.67 ± 15.18pA, n=9; PSKO 74.07 ± 12.07pA, n=10; t-test p=0.5143; Figure 3.2).

Figure 3 . 2 .

 32 Figure 3.2. KAR-mediated EPSCs at Mf-CA3 synapses are significantly reduced in PSKO mice. A. Representative traces illustrating evoked Mf-EPSCs mediated by AMPARs and KARs at 1Hz stimulation, average of 30 sweeps. 20µM D-APV, 10µM Bicuculline,

Figure 3 . 3 .

 33 Figure 3.3. One-trial contextual fear conditioning induced long-lasting freezing behavior in adult WT mice. Line chart displaying freezing time of mice after one-trial CFC. Mice received 5 foot shocks in Context A on the first day, and Context B was neutral and not fear-related. After CFC, mice were tested at 1 hour, 1 day, 2 days, 5 days, 7 days, and 14 days respectively, n=6 for each data point, and each mouse was tested in for a maximum of 3 timepoints to avoid possible extinction. Data represented as mean ± SEM. *represents p<0.05.

Figure 3 .

 3 Figure 3.4. c-Fos staining and cell count in DG and CA3 region after CFC. A. c-Fos signal in hippocampal sagittal slices of control mice. B. c-Fos signal in hippocampal sagittal slices of mice 1.5h post-CFC. C. Bar graph illustrating mean value of c-Fos+ cell count in each slice. n=12 slices in each group, 2 mice per group. Data represented as mean ± SEM. *represents p<0.05.

Figure 3 . 5 .

 35 Figure 3.5. Filopodia number count after one-trial CFC at large Mf terminals. A. Representative confocal image of LMTs and their filopodia in control mice. B. Representative confocal image of LMTs and their filopodia in mice sacrificed 5h after CFC. C. Bar graph displaying mean filopodia count per LMT in mice having received CFC vs. controls. 50 LMTs were examined for each mouse, and 5 mice were used in each group. Filopodia/LMT was 2.02 ± 0.10 for controls; 2.15 ± 0.10 for CFC 5h group. Unpaired t-test p=0.3109. Data represented as mean ± SEM.

Figure 3 . 6 .

 36 Figure 3.6. Normal short-term plasticity of Mf-CA3 glutamatergic synapses 3h after CFC in adult mice. A. Representative of superimposed traces illustrating frequency facilitation (FF) at Mf-CA3 synapses. The evoked-EPSCs were obtained at 0.1Hz (60 sweeps) and 1Hz (60 sweeps) respectively. B. Representative of superimposed traces illustrating pairedpulse facilitation (PPF) at Mf-CA3 synapses. Paired-pulse recordings were performed every 30s for 20 sweeps, and the inter-stimuli interval is 50ms (20Hz). C. Box plot

Figure 3 . 7 .

 37 Figure 3.7. The spontaneous EPSCs in CA3 PCs are unaltered 3h after CFC in adult male mice. A. Representative traces of spontaneous EPSCs (10μM Bicuculline in aCSF) during 10s, recorded at -70mV from hippocampal CA3 pyramidal cells of adult C57bl6j mice. B. Dot plot of the mean sEPSC frequency registered during 10 minutes showing no significant difference between groups. Black dots represent controls (n=16) and blue dots represent CFC 3h group (n=10), t-test p=0.9253. C. Dot plot showing no difference in mean amplitude of sEPSCs between groups. Black dots represent controls (n=16) and blue dots represent CFC 3h group (n=10), t-test p=0.8702.

3. 3 .

 3 1 regarding filopodia numbers at LMTs, indicating no significant change in number of synapses in the Mf-CA3 interneuron feedforward inhibition after CFC. However, the increased frequency of sIPSCs could imply that the interneurons in CA3 region might be firing more actively rapidly after CFC, giving more GABAergic signals to CA3 PCs.This might be due to enhanced excitability of CA3 interneurons after CFC, or facilitated synaptic transmission at synapses of feedforward inhibition circuits, or some other unknown mechanisms. It will be interesting to further investigate the feedforward inhibition connections in CA3 region.

Figure 3 .

 3 Figure 3.8. The frequency but not the amplitude of spontaneous IPSCs in CA3 PCs is increased 3h after CFC in adult mice A. Representative traces of sIPSCs (20μM NBQX in aCSF) during 10s, recorded at -60mV, from CA3 pyramidal cells. B. Dot plot of the mean sIPSC frequency registered during 10 minutes showing a significant increase in sIPSC frequency in CFC 3h group comparing to controls. Black dots represent controls (n=15) and blue dots represent CFC 3h group (n=15), t-test p=0.0182. * represents p<0.05. C. Dot plot showing no difference in mean amplitude of sIPSCs between groups. Black dots represent controls (n=15) and blue dots represent CFC 3h group (n=15), t-test with Welch's correction p=0.0563. D. Representative traces of miniature IPSCs (20μM NBQX and 0.5μM TTX in aCSF) during 10s, recorded at -70mV, from CA3 pyramidal cells. E. Dot plot of the mean mIPSC frequency registered during 10 minutes showing no significant alteration between groups. Black dots represent controls (n=13) and blue dots represent CFC 3h group (n=12), Mann-Whitney test p=0.0846. F. Dot plot showing no difference in mean amplitude of mIPSCs between groups. Black dots represent controls (n=13) and blue dots represent CFC 3h group (n=12), t-test with Welch's correction p=0.0578.

Figure 3 . 9 .

 39 Figure 3.9. The sIPSCs were unaltered 24h after CFC in adult male mice. A. Representative traces of spontaneous IPSCs (20μM NBQX in aCSF) during 10s, recorded at -60mV, from CA3 pyramidal cells. B. Dot plot of the mean sIPSC frequency registered during 10 minutes showing no significant difference in sIPSC frequency in CFC 24h group comparing to controls. Black dots represent controls (n=18, 6 mice) and blue dots represent CFC 3h group (n=18, 6 mice), t-test p=0.2429. C. Dot plot showing no difference in mean amplitude of sIPSCs between groups. Black dots represent controls (n=18, 6 mice) and blue dots represent CFC 24h group (n=18, 6 mice), t-test p=0.8922.

Figure 3 . 10 .

 310 Figure 3.10. Normal short-term plasticity at Mf-CA3 glutamatergic synapses 24h after CFC in adult mice. A. Representative of averaged traces illustrating the frequency facilitation (FF) at Mf-CA3 synapses. The evoked-EPSCs were obtained at 0.1Hz (30 sweeps) and 1Hz (30 sweeps) respectively. B. Representative of averaged traces illustrating the paired-pulse facilitation (PPF) at Mf-CA3 synapses. The paired-pulses were performed every 30s for 20 sweeps, and the inter-stimuli interval is 50ms (20Hz). C. Box plot displaying the FF ratio observed in Mf-CA3 synapses in CFC 24h group (5.32 ± 0.49) and control (4.48 ± 0.51). t-test p=0.2468, n=10 for control and n=10 for CFC 24h group. D. Box plot displaying the FF ratio observed in Mf-CA3 synapses in CFC 24h group (2.38 ± 0.23) and controls (2.67 ± 0.45). t-test p=0.5428, n=8 for control and n=10 for CFC 24h group.

Figure 3 .

 3 Figure 3.11. The ratio between evoked EPSCs and IPSCs at Mf-CA3 synapses is largely maintained in adult mice 24h after CFC. A. Representative traces illustrating Mf-EPSCs (recorded at -70mV) and IPSCs (recorded at +10mV) at 0.1Hz stimulation, and was an average of 30 sweeps. 20µM D-APV was present through the experiment. B. Bar graph illustrating no significant differences in the IPSC charges at 0.1Hz between two groups. Black bar represents controls (3.52 ± 1.65pA, n=10), blue bar represents CFC 24h group (3.15 ± 1.00, n=10), t-test p=0.8504. Data represented as mean ± SEM. C. Bar graph displaying no changes in the E/I ratio at 0.1Hz between two groups. Black bar represents controls (0.33 ± 0.14, n=9), blue bar represents CFC 24h group (0.21 ± 0.08, n=9), t-test p=0.4665. Data represented as mean ± SEM. D. Representative averaged traces illustrating Mf-EPSCs and IPSCs at 1Hz stimulation. E. Bar graph illustrating no significant differences in the IPSC charges at 1Hz between groups. Black bar represents controls (8.20 ± 3.04pA, n=10), blue bar represents CFC 24h group (7.20 ± 1.89pA, n=10), t-test p=0.7818. Data represented as mean ± SEM. F. Bar graph displaying no changes in the E/I ratio at 1Hz between groups. Black bar represents controls (0.58 ± 0.22, n=9), blue bar represents CFC 24h group (0.39 ± 0.13, n=9), t-test p=0.4810. Data represented as mean ± SEM.

Figure 3 .

 3 Figure 3.12. Down-regulated and up-regulated genes in 6-month APP/PS1 mice. A. Heatmap of gene expression in hippocampal CA3 region of 6-month-old APP/PS1 mice (n=5) compared to WT littermates (n=7) (cut-offs: p<0.01, fold change > 1.2). B.

Figure 3 .

 3 Figure 3.13. Pathway analysis of genes differentially down-regulated and upregulated in 6-month APP/PS1 mice.

Figure 3 .

 3 Figure 3.14. Example result of sorted neuronal nuclei by FACS. Scatter plot of one sample displaying separated neuronal nuclei from non-neuronal cells and debris using FACS. Area P4 (shown in magenta) represents collected neurons, and area P3 (shown in blue) represents non-neuronal components. Note that the yield of neurons per tissue sample is just around 10%.

Figure 3 .

 3 Figure 3.15. H3K4Me3 level is decreased at the promoter areas of many genes in hippocampal CA3 PCs of 6-month APP/PS1 mice. A. Volcano plot of ChIP-Seq results displaying H3K4Me3 levels at promoters of various genes in APP/PS1 mice (n=4) compared to WT littermates (n=4). Red dots represent differential binding. To the left side are the numerous gene promoters with low H3K4Me3 levels, and to the right side are few gene promoters with high H3K4Me3 levels. B. Pathway analysis of genes with decreased H3K4Me3 at their promoters.

Figure 3 .

 3 Figure 3.16. Down-regulated genes from RNA-seq and ChIP-seq in APP/PS1 mice. Venn diagram depicting down-regulated genes from RNA-seq (shown in yellow) and gene promoters with decreased H3K4me3 from ChIP-seq (shown in purple) and their overlap, in CA3 region of 6-month-old APP/PS1 mice. Note that RNA-seq was performed from whole CA3 tissue and ChIP-seq was performed from sorted neuronal nuclei.

  The modeling of hippocampal DG-CA3 networks was performed with a Pythonbased software NEST, in Oracle VM VirtualBox environment.

Figure 6 . 1 .

 61 Figure 6.1. A simplified illustration of a multi-compartment conductance-based leaky integrate-and-fire neuron.

Figure 6 . 3 .

 63 Figure 6.3. A more complete illustration with two types of interneurons for FFI and FBI respectively in the network.

Figure 6 . 4 .

 64 Figure 6.4. Spike patterns of four distinctive populations of neurons. Scatter plot illustrating spikes of each population of neurons. CA3 layer was represented in blue, DG in red, FFI interneurons in green and FBI interneurons in black. 6 distinctive inputs were delivered sequentially, with interactions of multiple factors in the network, each resulted in a unique spike pattern.

Figure 6 . 5 .

 65 Figure 6.5. Relations between inhibitory synaptic weight and I/O correlation index for FFI and FBI interneurons. A. Line chart of FBI weights plotted against the I/O correlation index, with an exponential curve fit. The firing frequency of the FBI interneurons were: 3.1Hz at weight = 0.5, 35.5Hz at weight = 1.0, 57.2Hz at weight = 1.5, 76.2Hz at weight = 2.0, 91.4Hz at weight = 2.5, 105 Hz at weight = 3.0. B. Line chart of FFI weights plotted against the I/O correlation index, with an exponential curve fit. The firing frequency of the FFI interneurons were: 1.2Hz at weight = 2, 3.5Hz at weight = 4, 9.6Hz at weight = 5, 19.2Hz at weight = 6, 42.8Hz at weight = 8, 67.3 Hz at weight = 10.

Figure 1 .

 1 Figure 1. Membranous mGluR5 displays an increased lateral diffusion rate within the synaptic compartment of hippocampal Fmr1 KO neurons. (A) Experimental setup. Upper panel: schematic drawing of the endogenous mGluR5 in the dendritic membrane labeled with a QD-antibody complex targeting the extracellular domain of the receptor. Lower panel: reconstructed trajectories of surface mGluR5-QD complexes in the dendritic membrane of hippocampal neurons are depicted in red and shown on a phase contrast (left) and epifluorescence image showing MitoTracker labeling of synaptic sites in green (Holcomb et al.). Scale bar = 5 μm. (B) Representative trajectories of single surface mGluR5-QD in WT and Fmr1 KO neurons. The synaptic sites are represented by the green shapes. Scale bar = 1 μm. (C) Mean distribution (left panel) and cumulative distribution (right panel) of the instantaneous diffusion coefficient of mGluR5-QDs in the synaptic compartment. The lateral diffusion is significantly higher in Fmr1 KO neurons (WT, 0.018 ± 0.001, n = 1633 trajectories; Fmr1 KO, 0.025 ± 0.001, n = 1451 trajectories; P < 0.001 by Mann-Whitney test on plot distribution; P < 0.001 by Kolmogorov-Smirnov test on cumulative distribution). (D) Mean distribution (left panel) and cumulative distribution (right panel) of the instantaneous diffusion coefficient of mGluR5-QDs in the extrasynaptic area of WT and Fmr1 KO neurons (WT, 0.045 ± 0.002, n = 1907 trajectories; Fmr1 KO, 0.051 ± 0.003, n = 1350 trajectories; P = 0.085 by Mann-Whitney test on plot distribution; P = 0.057 by Kolmogorov-Smirnov test on cumulative distribution). Data are shown as mean ± s.e.m. ***P < 0.001.

Figure 2 .

 2 Figure 2. Disruption of the link between mGluR5 and Homer in WT neurons mimics the Fmr1 KO phenotype. (A) Schematic illustration of the effect of the cell permeable TAT-mGluR5ct peptide. This peptide disrupts the mGluR5/Homer link, mimicking the situation in Fmr1 KO neurons. (B) Representative trajectories of single surface mGluR5-QD in WT and Fmr1 KO neurons treated with TAT-mGluR5ct and its mutated TAT-mGluR5mu control peptide (both peptides 5 µM, 1h). The synaptic sites are represented by the green shapes. Scale bar = 1 μm. (C) Normalized instantaneous diffusion coefficient of mGluR5-QDs in the synaptic area of WT and Fmr1 KO neurons treated with TAT-mGluR5mu and TAT-mGluR5ct. The lateral diffusion rate of mGluR5-QDs in WT neurons treated with TAT-mGluR5ct peptide is comparable to that in Fmr1 KO neurons under basal conditions (WT, 0.016 ± 0,002, n = 636 trajectories; WT TAT-mGluR5mu, 0.016 ± 0.002, n = 1798 trajectories; WT TAT-mGluR5ct, 0.027 ± 0.002, n = 1444 trajectories; Fmr1 KO, 0.025 ± 0.002, n = 793 trajectories; Fmr1 KO TAT-mGluR5mu, 0.023 ± 0.001, n = 1419 trajectories; Fmr1 KO TAT-mGluR5ct, 0.022 ± 0.003, n = 489 trajectories; P < 0.001 by Kruskal Wallis test). (D) Cumulative distribution of the instantaneous diffusion coefficient of mGluR5-QDs within the synaptic area of WT and Fmr1 KO neurons treated with TAT-mGluR5mu and TAT-mGluR5ct (P < 0.001 by Kolmogorov-Smirnov test). Data are shown as mean ± s.e.m. ***P < 0.001.

Figure 3 .

 3 Figure 3. mGluR5 and GluN1 are more confined within the synaptic compartment in Fmr1 KO neurons. (A) Representative surface distribution of mGluR5-QD (upper panel) and GluN1-QD (lower panel) clusters (each dot

  58 ± 2.69 %, n = 15 dendritic fields; P = 0.343 by one way Anova test). Data are shown as mean ± s.e.m. ***P < 0.001, **P < 0.01, *P < 0.05. Scale bar = 2 μm (except panel C for low magnification scale bar = 10 μm).

Figure 4 .

 4 Figure 4. Disruption of mGluR5/Homer coupling alters synaptic NMDAR function and plasticity in Fmr1 KO neurons. (A) NMDA receptor-mediated excitatory post-synaptic currents (EPSCsNMDA) were recorded under whole-cell patch clamp from CA1 pyramidal neurons in hippocampal slices following stimulation of Schaffer collaterals. (B) Representative traces of EPSCsNMDA recorded from CA1 neurons in slices from WT, Fmr1 KO, WT treated with TAT-mGluR5-ct and TAT-mGluR5-mu (both peptides 5 µM, 4 hours). (C) Left panel: the amplitude of EPSCsNMDA in CA1 neurons from Fmr1 KO slices (46.4 ± 8.4 pA, n = 7) was significantly lower than in WT (175.7 ± 21.9 pA, n = 11, P = 0.0004 by unpaired Student's t-test). In a similar manner, in WT slices treated with TAT-mGluR5-ct, the amplitude of EPSCsNMDA was significantly lower than in control conditions (88.1 ± 12.7 pA, n = 13, P = 0.0016 by unpaired Student's t-test). In neurons from WT slices incubated with TAT-mGluR5-mu, the amplitude of EPSCsNMDA was comparable to control (125,9 ± 16.8 pA n = 8, P = 0.11 by unpaired Student's t-test). Right panel: mixed AMPA-and NMDA-EPSCs were recorded at a holding potential of +50 mV to evaluate the NMDA/AMPA ratio. The NMDA/AMPA ratio in Fmr1 KO slices (0.86 ± 0.05, n = 8) was significantly lower than in WT (1,60 ± 0.29, n = 5, P = 0.0094 by unpaired Student's t-test). A reduction in NMDA/AMPA ratio was also observed in slices treated with TAT-mGluR5-ct(1,14 ± 0.03, n = 4). (D, F) EPSCsNMDA were recorded from CA1 pyramidal neurons at a HP -60 mV, in the presence of bicuculline and CNQX. Bath application of the group-I mGluR agonist DHPG (100 µM, 5 min) induced a long-term depression (mGluR-LTD) of EPSCsNMDA recorded in WT slices (EPSCsNMDA amplitude 30 min after LTD induction: 24.9 ± 2 % of control, n = 8). mGluR-LTD of EPSCsNMDA was not observed in Fmr1 KO neurons (EPSCsNMDA amplitude: 107.8 ± 25 %

Figure 5 .

 5 Figure 5. Reduction of Homer1a expression rescues synaptic NMDAR dysfunction and cognitive defects in Fmr1 KO mice. (A) Schematic representation of the experimental procedure. WT and Fmr1 KO mice (postnatal age 21 days) were bilaterally injected into the dorsal hippocampus with either AAV-shH1a or AAV-Scr. Experiments were performed four weeks (electrophysiology, B and C) or nine weeks (behavioral training, D and E) following virus injection. Objectrecognition memory was assayed in the L-maze. On day 1, mice were habituated for 10 min to the maze in which the task was performed. On the second day, mice were put back in the maze for 10 min, and two identical objects were presented. The mice were again placed in the maze 24 h later for 10 min, one of the familiar objects was replaced with a novel object and the total time spent exploring each of the two objects was computed. During the fear conditioning task, mice received five 2-seconds electric shocks (0.4 mA, 50 Hz, ITI=1 minute) after 2 minutes of habituation. During the test phase (24 hours after conditioning) mice were put back in the same chambers for 6 minutes, without receiving any shock. (B) The EPSCNMDA amplitude of AAV-sh H1a injected Fmr1 KO mice was significantly larger than that of AAV-scr injected Fmr1 KO mice (Fmr1 KO AAV-scr, EPSCNMDA amp. = 160.4 ± 27.8, n = 12; Fmr1 KO AAV-sh H1a, EPSCNMDA amp. = 311.3 ± 58, n = 12; P < 0.05, unpaired Student's t-test). (C) AAV-sh H1a infection of CA1 pyramidal neurons in Fmr1 KO mice rescues normal long-term depression of EPSCNMDA induced by bath application of the group-I mGluR agonist DHPG (100 µM, 10 min) (EPSCNMDA amplitude 40 min after LTD induction: 70.4 ± 6 % of control, n = 6). mGluR dependent LTD of EPSCNMDA was not observed in Fmr1 KO mice injected with AAV-scr (EPSCNMDA amplitude: 106.8 ± 7 % of control, n = 7, P < 0.001, by unpaired Student's t-test). (D) Discrimination index values in the object recognition test of mice after bilateral intra-hippocampal microinjection of AAV-sh H1a (300 nl per side) or AAV-scr (WT AAV-scr, 0.244 ± 0.042, n = 10; Fmr1 KO AAV-scr, -0.030 ± 0.032, n = 9; Fmr1 KO AAV-sh H1a, 0.235

Table 1 .

 1 3 Histone tail methylations.

	Histone Modification	Role	Reference
	H3	H3K4me2	Permissive euchromatin	(Santos-Rosa et al., 2002)
		H3K4me3	Transcriptional elongation; active	(Roguev et al., 2001; Strahl,
			euchromatin	Ohba, Cook, & Allis, 1999)
		H3K9me3	Transcriptional repression; imprinting; DNA	(Rea et al., 2000; K. Zhang
			methylation	et al., 2002)
		H3R17me	Transcriptional activation	(Bauer, Daujat, Nielsen,
				Nightingale, & Kouzarides,
				2002; Chen et al., 1999)
		H3K27me3	Transcriptional silencing; X-inactivation;	(K. Zhang et al., 2002)
			bivalent genes/gene poising
		H3K36me3	Transcriptional elongation	(K. Zhang et al., 2002)
	H4	H4R3me	Transcriptional activation	(Wang et al., 2001)

Table 1 .

 1 4. Risk factors for Alzheimer's disease.

		Alzheimer's disease
		Premorbid cognitive reserves: longer education and higher intelligence are protective
	Medical history	Head injuries: anti-inflammatory drugs are associated with a reduction in risk
		Oestrogen replacement: no consistent evidence
		Vascular risk factors: hypertension, diabetes, homocysteine, and cholesterol are all implicated
		Depression: associated with Alzheimer's disease
		Herpes simplex: a risk factor, possibly mediated by the presence of ApoE e4
	Habits	Alcohol: drinking wine is protective
		Smoking: no consistent evidence
		Diet: no consistent evidence (including for aluminium)
		Occupational and recreation factors: no consistent evidence

Table 1 .

 1 5. Genetic factors of AD.

	In clinical practice of treating AD, apart from psychosocial interventions, a few
	pharmacotherapies are available (Ballard et al., 2011; Burns & Iliffe, 2009) (Table 1.6)
	to ameliorate symptoms, but none of these seem disease-modifying.

Table 1 .

 1 6. Drug treatments for Alzheimer's disease. Due to so many non-modifiable risk factors, interventions by drug treatments are unlikely to result in a large-scale reduction in AD incidence. However, the picture is being changed by lifestyle interventions and perhaps ultimately by progress in basic research.

Table 2 .

 2

	1. Primers of PCR
	Primer	Targeted Gene	Sequence (5'-3')
	P255	PSEN	AATAGAGAACGGCAGGAGCA
	P256	PSEN	GCCATGAGGGCACTAATCAT
	P257	APP	AAGCGGCCAAAGCCTGGAGGGTG
	P258	APP	GTGGATAACCCCTCCCCCAGCCTAGACCA
	P259	APP	GTTGAGCCTGTTGATGCCCG

Table 2 .

 2 2. Composition of extracellular solutions.

	Ingredients	Cutting solution	Resting solution	aCSF
		(mM)	(mM)	(mM)
	Sucrose	200	--	--
	Glucose	20	20	20
	CaCl2	0.5	0.8	2.3
	MgCl2	7	7	1.3
	NaCl	--	110	125
	KCl	2.5	2.5	2.5

Table 2 .

 2 3. Composition of intracellular solutions.

	Ingredients	For voltage-clamp	For current-clamp	For sIPSCs and
		experiments (mM)	experiments (mM)	mIPSCs (mM

Table 2 .

 2

	4. Buffers for FACS
	Low sucrose buffer 0.32M sucrose, 5mM CaCl2, 5mM Mg(Ac)2, 0.1mM EDTA,
		10mM HEPES (pH 8.0), 1mM DTT, 0.1% Triton X-100
	High sucrose buffer 1M sucrose, 3mM Mg(Ac)2, 10mM HEPES (pH 8.0), 1mM
		DTT
	PBTB (pH 7.5)	5% BSA, 0.2% Tween-20 in 1×PBS, prepared freshly and
		filtered
	PBST (pH 7.5)	0.2% Tween-20 in 1×PBS

Table 2 .

 2 5.

	Primer	Forward sequence	Reverse sequence
	GAPDH promoter	TTCACACCGACCTTCAC	GGCCACGCTAATCTCAT
	InterG	AAAACTTCAACTACAGCAAC GAGACGATCGTACTAAGTG
	Tdo2	TCCAGGGAGCACTGATGATA CTGGAAAGGGACCTGGAATC
	Lphn2	GCCGAGTGAGAAGGATTCG	CCCTGCATGTCTTCTCTCGT
	Lyd	AGTGTGTCCATCGCCTGTG	CGTCACTAGCCCTGCATTCT

Table 2

 2

	.6. Buffers for ChIP
	RIPA buffer	140mM NaCl, 1mM EDTA, 0.1% Na-Deoxycholate, 1% Triton
		X-100, 10mM Tris (pH 8.0), 1% SDS
	Wiemann Buffer	20mM EDTA, 100mM Tris (pH 8.0), 2% SDS
	2X
	IP Buffer	140mM NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 50mM Tris
		(pH 8.0), 20mM EDTA, 0.1% SDS
	Wash Buffer	100mM Tris (pH 8.0), 1% NP-40, 1% Na-Deoxycholate, 20mM

  1,2* , Matthias Georg Haberl, Noëlle Grosjean 1 ,

	Virginie Labrousse, Gaël Barthet and Christophe Mulle 1 .
	1 University of Bordeaux, Interdisciplinary Institute for Neuroscience, CNRS UMR
	5297, F-33000 Bordeaux, France.
	2 BEB PhD program CNC Coimbra, Portugal.

Acknowledgements

First of all, I would like to express my heartfelt thanks to Dr. Christophe Mulle, for granting me this opportunity to explore in science, also for his patient instruction and help along my way in the last four years.

ACKNOWLEDGEMENTS

This work was supported by the International PhD Program in Neuropharmacology from the University of Catania (Italy), Labex Bordeaux BRAIN, Telethon (GGP07264), Oasi M SS Troina ("Ricerca Finalizzata"of the Italian Ministry of Health). We thank Silvia Viana da Silva, Arnau Busquets Garcia, Laurent Ladépêche, Jennifer Peterson, and Guillaume Bony for constructive discussions and scientific advice. We thank the team of Daniel Choquet for initial help with the method of quantum dot imaging and its analysis. We also thank Natalie Sans and Mireille Montcouquiol (both Neurocentre Magendie, Bordeaux), and Carmela M. Bonaccorso and Giuseppina Bonaccorso (both Oasi Maria SS, Troina) for technical assistance. Some data were acquired using equipment of the Bordeaux Imaging Center (BIC).

suggestions.

I am very grateful to my funding, Erasmus

Abbreviations

A/C

Associative/commissural AC Adenylate cyclase aCSF Chapter 3

Results

Abstract

Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimer's disease (AD). The CA3 region of the hippocampus is thought to be important for the storage of episodic memory. Like many neuronal populations, CA3 pyramidal cells receive a variety of synaptic inputs which display diverse anatomical and functional properties. Here we explored whether the morphology, function and plasticity of specific synaptic inputs are differentially impaired in CA3 pyramidal cells in a mouse model of AD. We have previously shown that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage in a mouse model of AD. We now show that the basal synaptic properties of a different glutamatergic input to CA3 pyramidal cells, the mossy fiber input, are not impaired at an early stage in a mouse model of AD. Whereas presynaptic short-and long-term

Chapter 6

Annexes compartment with alpha-function time course. Each compartment also received current input from a current generator (e.g. to create noise). We also set synaptic weights for each type of connection.

Network design of DG-CA3 circuits

Four types of neurons were taken into consideration in this model: DG granular cells, CA3 pyramidal cells, interneurons for feedforward inhibition, and interneurons for feedback inhibition.

A matrix of 40×40 neurons was created for DG granular cell layer, and also a matrix of 40×40 was created as the CA3 pyramidal layer. Each DG cell was innervating neurons in the CA3 layer in a circular area (radius: 0.6, kernel: 0.9) with somatic excitatory synapses. Apart from this input, the CA3 layer also received input from EC (created by a Poisson spike generator) and CA3-CA3 recurrent input. The recurrent input was designed as each CA3 neuron was connecting a 10×10 grid of CA3 PCs in the vicinity, and the strength of connection followed Gaussian distribution (Figure 6.2).

Publication 2 (as co-second author)

Reduced interaction between mGluR5 and Homer alters the surface dynamics of mGluR5 leading to synaptic NMDA receptor dysfunction and cognitive defects in the mouse model of Fragile X Syndrome

Elisabetta Aloisi 1,2 , # Katy Le Corf 1,2 , # Pei Zhang 3,4 , # Virginie Labrousse 1,2,3,4 , # Julien Dupuis 3,4 , Matthias Georg Haberl 1,2 , Lara Costa 5 , Melanie Ginger 1,2 , Ryuichi Shigemoto 6 , Rohini Kuner 7 , Anke Tappe Theoder 7 , Filippo Drago 8 , Pier Vincenzo Piazza 1,2 , Christophe Mulle 3,4 , Laurent Groc 3,4 , Lucia Ciranna 8 , § Maria Vincenza Catania 9,10 and § Andreas Frick

SUMMARY

Dysfunction of the metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS), but the underlying sub-cellular mechanisms are incompletely understood. Here, we probed the consequences of the disrupted mGluR5/Homer scaffold for synaptic and behavioral phenotypes in Fmr1 knockout (KO) mice. Using single molecule tracking, we found that the lateral mobility and confinement of mGluR5 were exaggerated at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and N-methyl-D-aspartate receptor (NMDAR). Interestingly, we detected a reduction in the amplitude of synaptic NMDAR currents and a lack of their mGluR5-activated long-term depression in the hippocampus of Fmr1 KO mice. All these phenomena were recapitulated in wild-type neurons by peptide-based disruption of mGluR5/Homer scaffold. Importantly, knockdown of the short Homer1a isoform reversed both NMDAR dysfunction and associated cognitive deficits in Fmr1 KO mice, unveiling novel targets for mGluR5 based therapeutics.

lead to a consistent amelioration of numerous disease phenotypes in the Fmr1 KO mouse [START_REF] Bear | The mGluR theory of fragile X mental retardation[END_REF][START_REF] De Vrij | Rescue of behavioral phenotype and neuronal protrusion morphology in Fmr1 KO mice[END_REF][START_REF] Dölen | Correction of fragile X syndrome in mice[END_REF][START_REF] Dölen | Mechanism-based approaches to treating fragile X[END_REF][START_REF] Michalon | Chronic pharmacological mGlu5 inhibition corrects Fragile X in adult mice[END_REF][START_REF] Michalon | Chronic metabotropic glutamate receptor 5 inhibition corrects local alterations of brain activity and improves cognitive performance in fragile X mice[END_REF]. In spite of this promise, large scale clinical trials (Hessl et al. 2008;Berry Kravis et al. 2009;Jacquemont et al., 2011;Clinicaltrials.gov) employing novel mGluR5 antagonists have recently been cancelled because of lack of efficacy of the treatment over placebo. Here we propose that new therapeutic approaches aimed at restoring the normal mGluR5/Homer interaction might be considered in FXS. In particular, modulation of mGluR5/Homer interaction might correct phenotypes that are related to NMDA dysfunction. How the loss of FMRP leads to an altered mGluR5/Homer crosstalk is still unknown. Phosphorylation of mGluR5 at the C-terminal Homer interaction domain reduces the affinity of mGluR5 for Homer [START_REF] Orlando | Phosphorylation of the homer-binding domain of group I metabotropic glutamate receptors by cyclin-dependent kinase 5[END_REF]. Therefore, post-translational modification of mGluR5 and/or Homer in Fmr1 KO mice may underlie the decreased interactions.

Given the dynamic nature of mGluR5/Homer interaction, it is tempting to speculate that the effect of mGluR5 blockade on FXS phenotype might critically depend on the condition which exacerbates disruption of mGluR5/Homer interaction.

Further studies in human specimens and elucidation of the mechanisms underlying the reduced mGluR5/Homer interaction of FXS might help to clarify this issue.

Our findings demonstrate for the first time an alteration of the mGluR5 dynamics at synapses of Fmr1 KO neurons and provide a new cellular mechanism by which mGluR5 dysfunction disrupts normal NMDAR synaptic plasticity. This study is not only the first to show that the surface dynamics of mGluR5 at synapses are altered in FXS, but also establishes evidence of how this alteration could contribute to the pathophysiology of FXS. The discovery that an abnormal mGluR5/Homer interaction accounts for much of the complex dysfunction of mGluR5 in FXS will help to facilitate the development of alternative, targeted therapies for this disorder and its co-morbidities, and provide mechanistic links to other genetic causes of autism.

EXPERIMENTAL PROCEDURES

Animals

All experiments were conducted in strict compliance with the European Directive (2010/63/EU), and French and Italian law governing the use of laboratory animals and were approved by the Bordeaux Ethics Committee (C2EA50, authorization #5012024-A) and by the Ethics Committee of Catania University (project # 181). Mice were housed in a SPF animal facility prior to experiments, kept on a 12 h-12 h light-dark cycle and had ad libitum access to food and water at all times. Second generation Fmr1 KO mice (Mijenties et al., 2006) were used in our study. These mice are distinct from the original Fmr1 KO mouse genetic line, because they are deficient for both Fmr1 mRNA and FMRP protein. Mice were backcrossed six generations into a C57BL/6J (Charles River, L'Abresle, France) background and maintained in this mixed background for all experiments. For dissociated neuronal cultures WT and Fmr1 KO embryos were generated by crossing homozygous (Fmr1 +/+ X Fmr1 +/y or Fmr1 -/-X Fmr1 -/y ) progenitor mice. For electrophysiology, male WT and Fmr1 KO littermates were generated by crossing a heterozygous (Fmr1 +/-) female mouse with a wild-type (Fmr1 +/y ) male mouse as described previously [START_REF] Zhang | Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1(-/y) mice[END_REF]. Mice were subsequently regenotyped after the experiment by tail-PCR as described by [START_REF] Mientjes | The generation of a conditional Fmr1 knock out mouse model to study Fmrp function in vivo[END_REF]. Some electrophysiology experiments (see Figure 4) were carried out on first-generation Fmr1 KO mice (FVB background; Dutch-Belgian Fragile X Consortium (1994). Genotype was assessed by tail-PCR as described previously [START_REF] Musumeci | Audiogenic seizure susceptibility is reduced in fragile X knockout mice after introduction of FMR1 transgenes[END_REF]. Only Fmr1 -/y mice were used for electrophysiology and behavior.

Primary cell cultures

Cultures of hippocampal neurons and glial cells were prepared from E18 WT and Fmr1 KO embryos. Pregnant mice were sacrificed by decapitation after deep anaesthesia with isoflurane and the uterine horn dissected. Hippocampi were subsequently dissected from the embryos in ice-cold dissection solution and then dissociated in trypsin (Sigma, 0.25%). Briefly, cells were plated at a density of 100 to 200 x 10 3 cells per milliliter on poly-L-lysine (Sigma) precoated removed from the water bath and kept at RT. The slices were transferred to the recording chamber, and were continuously superfused with ACSF (in mM NaCl 125; KCl 2.5; NaH2PO4 1.25; MgCl2 1.3; CaCl2 2.3; NaHCO3 25; D-glucose 20, pH 7.3) at a flow rate of 1.7 ml/min. The ACSF also contained NBQX (20 μM), glycine (10 μM) and bicuculline (10 μM) to isolate EPSCs mediated by NMDA receptors (EPSCNMDA). Cells were visualized using a Nikon FN-S2N microscope equipped with 10X/0.30 and 60X/1.0W objectives and differential interference contrast (Mulle et al.), and fluorescent cells were selected using a LED excitation system at 490nm (CooLED, UK). A stimulating glass pipette (open-tip resistance of 3.5-5 MΩ) was placed in the stratum radiatum to stimulate Schaffer collaterals with positive current pulses (duration 0.2 milliseconds), delivered every 20 seconds by a stimulus generator (DS3 constant curent isolated stimulator, Digitimer Ltd., England). Evoked EPSCNMDA were recorded at RT from CA1 pyramidal neurons with green fluorescence (successfully infected with AAV-sh H1a or AAV-scr) in the whole-cell patch-clamp configuration at a holding potential of +40 mV using an EPC10 amplifier (HEKA, Germany). Current traces were filtered at 3 kHz and digitized at 10 kHz. The patch pipettes (open-tip resistance of 2.5-4 MΩ) were filled with intracellular solution containing (in mM): CsMeSO3 133; HEPES 10; NaCl 4; MgCl2 2; EGTA 0.2; Phosphocreatine 5; Na2-ATP 3; Na-GTP 0.4; pH 7.25. For determining the EPSCNMDA Input/Output curve (I/O curve), at least three traces were averaged for each stimulation intensity (10µA, 20µA, 30µA, 40µA, 50µA, 60µA, 70µA, 80µA respectively). For LTD experiments, DHPG (100 µM) was dissolved in the same ACSF and bath-applied for 10 min. Data were acquired using PatchMaster software (HEKA, Germany) and analyzed with Igor software (WaveMetrics Inc., USA). EPSCNMDA amplitude values were measured as the difference between peak current and baseline, expressed as percentage of control (calculated from EPSCs recorded during at least 10 minutes prior to DHPG application).

Contextual fear conditioning task

Contextual fear conditioning was assayed as previously described (Oddi et al., 2014). The task took place in a plexiglas box (30 × 24 × 22 cm high) giving access to the visuo-spatial cues in the experimental room. The floor of this conditioning chamber consisted of stainless-steel rods (2 mm diameter), spaced 5 mm apart and connected to a shock generator (Imetronic, Talence, France). The four sides of the chamber and the rods of the floor were cleaned with 70% ethanol before each trial. During conditioning, mice received five 2-second-long electric shocks (0.4 mA, 50 Hz, ITI=1 minute) after 2 minutes of habituation. During the test phase (24 hours after conditioning) mice were put back in the same chambers for 6 minutes, without receiving any shock; freezing time was manually scored as the absence of any body movement beside respiration and normalized by the test session length to obtain a percent measure. All tests were conducted by an observer blinded to the treatments.

Novel object recognition task

Novel object recognition memory was assayed as described previously [START_REF] Busquets-Garcia | Targeting the endocannabinoid system in the treatment of fragile X syndrome[END_REF]. Objectrecognition memory was assayed in the L-maze made out of grey Plexiglas with two corridors (30 cm long × 4.5 cm wide, and 15 cm high walls) set at a 90°angle. On day 1, mice were habituated for 10 min to the maze in which the task was performed. On the second day, mice were put back in the maze for 10 min, two identical objects were presented and the time that the mice spent exploring each object was recorded. The mice were again placed in the maze 24 h later for 10 min, one of the familiar objects was replaced with a novel object and the total time spent exploring each of the two objects was computed. Object exploration was defined as the orientation of the nose to the object at a distance of less than 2 cm. In each test session, a discrimination index (DI) was calculated as the difference between the time spent exploring the novel (TN) and the familiar object (TF) divided by the total exploration time (TN+TF): DI=[TN-TF]/[TN+TF]. A higher discrimination index is considered to reflect greater memory retention for the familiar object.

Data representation and