Fani Koukouli
email: fani.koukouli@pasteur.fr

C Ganneau

Uwe Maskos
email: umaskos@pasteur.fr

C Czech

F Grueninger

C Duyckaerts

B Delatour

S Bay

M Dhenain

P Lafaye

T Sklaviadis

Marie Rooy

Jean-Pierre Changeux
email: changeux@noos.fr

Nicotinic receptors modulate ongoing synchronous spontaneous activity of prefrontal cortex: consequences for conscious processing

Keywords: Animal experimentation ethics and welfare level Alzheimer's Disease, AAV, human mutated APP, chronic two-photon imaging Glutamate a7 nicotinic receptor Neurotransmission Synaptic plasticity Nicotine

Koukouli F

Summary

The prefrontal cortex (PFC) underlies higher cognitive processes that are modulated by cholinergic inputs largely via nicotinic acetylcholine receptors (nAChRs). This brain region exhibits spontaneous "default" activity, which is altered in neuropsychiatric disorders, such as schizophrenia (SCZ), and neurodegenerative diseases such as Alzheimer's disease (AD). Both of these disorders have a strong impact and burden on society. Human genetic studies have highlighted the polymorphic nature of specific nAChRs genes that increase risk for smoking and SCZ. Several laboratories, including our own, have shown that mice with altered nAChR gene function exhibit PFC-dependent behavioral deficits, but how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying the behaviors is unknown. Here, mouse models related to SCZ, nicotine dependence and AD were developed and studied. Using in vivo two-photon imaging in the PFC of both awake and anesthetized mice, different nAChR subunits were shown to control spontaneous PFC activity through a hierarchical inhibitory circuit. Genetic manipulations using transgenic mouse lines, lentiviral and adeno-associated viral transduction, CRISPR/Cas9 technology and pharmacological intervention were used to decipher the significant role of distinct nAChR subunits in the modulation of neurotransmission and on neuronal hyperactivity, a dysfunction characteristic of AD pathology. Furthermore, the effect of chronic nicotine administration on brain activity, by delivering concentrations analogous to that observed in smokers, was studied. The impact of nicotine on PFC layer II/III microcircuits altered in pathology can be extended to therapeutic strategies with strong candidates being positive allosteric modulators (PAMs) for defined nAChR subunits. We hope that this work sheds light on the role of cholinergic neurotransmission in the orchestration of cognitive functions and will inspire further research in this direction.

Résumé

Le cortex préfrontal (CPF) est à la base des processus cognitifs supérieurs qui sont modulés majoritairement par des entrées cholinergiques via les récepteurs nicotiniques de l'acétylcholine (nAChRs). Cette région du cerveau présente « par défaut » une activité spontanée, qui est modifiée dans le cas de troubles psychiatriques, tels que la schizophrénie, et de maladies neurodégénératives comme la maladie d'Alzheimer. Ces deux maladies ont un impact et un poids important sur la société. Des études de génétique humaine ont mis en évidence la nature polymorphique de gènes spécifiques codant pour les nAChR qui augmentent les risques de tabagisme et de schizophrénie. De nombreux laboratoires, dont le notre, ont montré que les souris ayant une altération de la fonction du gène nAChR présentent des déficits comportementaux CPF-dépendants. Cependant, la manière dont les polymorphismes humains correspondants altèrent les mécanismes cellulaires et circuits sousjacents aux comportements reste inconnue. De ce fait, nous avons donc développé et étudié des modèles de souris liés à la schizophrénie, à la dépendance à la nicotine et à la maladie d'Alzheimer. L'utilisation in vivo de l'imagerie bi-photon au niveau du CPF de souris éveillées et de souris anesthésiées a montré que différentes sous-unités nAChR sont impliquées dans le contrôle de l'activité spontanée du cortex préfrontal, via un circuit d'inhibition hiérarchique. Des manipulations génétiques utilisant des lignées de souris transgéniques, des lentivirus et adénovirus, la technologie CRISPR/Cas9 et des applications pharmacologiques ont été utilisées pour mettre en lumière le rôle significatif de sous-unités nAChR spécifiques sur la modulation de la neurotransmission et sur l'hyperactivité neuronale, un dysfonctionnement caractéristique de la pathologie MA. De plus, l'effet de l'administration chronique de nicotine sur l'activité cérébrale a été étudié, en fournissant des concentrations analogues à celles observées chez des fumeurs. L'impact de la nicotine sur les micro-réseaux des couches II / III du CPF altérés par des troubles neurologiques, peut être étendu à des stratégies thérapeutiques avec de bons candidats étant des modulateurs allostériques positifs pour les sous-unités nAChR définies. Ce travail met en lumière le rôle de la neurotransmission cholinergique dans l'orchestration des fonctions cognitives et inspirera d'autres recherches dans ce sens.

Aim of the thesis

Disruption in the PFC activity is thought to be a main feature of psychiatric and neurodegenerative diseases. Neurons in the PFC receive significant cholinergic innervation and nicotinic receptors are of particular importance for cognition. The present thesis was conducted with the aim of elucidating the role of nAChRs on the spontaneous activity of the normal brain and in the brain affected by two major pathologies, schizophrenia and AD.

The main objectives of my thesis are summarized below:

1. Functional role of the nAChRs on the modulation of PFC spontaneous neuronal activity of the normal mouse brain.

2. Role of human polymorphisms associated with smoking and schizophrenia on spontaneous PFC activity.

3. Impact of chronic nicotine exposure on cortical activity.

4. Role of nAChRs on the generation of ultra-slow fluctuations and neuronal synchronicity during quiet wakefulness and light general anesthesia.

5. Development of a new AD mouse model based on the delivery of AAVs.

6. Role of the nAChRs on the AD-mediated neuronal hyperactivity.

Before describing the results of the thesis, I shall review current knowledge regarding the nAChRs and their involvement in schizophrenia and AD.

1. Introduction

The cholinergic system

The cholinergic system is composed of an organized network of neurons that use the neurotransmitter acetylcholine (ACh) for signal transduction [START_REF] Picciotto | Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior[END_REF]). These neurons are activated by, or contain and release ACh during the propagation of a nerve impulse. ACh in the brain has a major role on neuronal excitability, synaptic transmission and synaptic plasticity where it is capable of modifying the response of neuronal networks to internal and external inputs (Arroyo et al. 2012[START_REF] Arroyo | Nicotinic modulation of cortical circuits[END_REF]. Cholinergic tone was shown to modulate a large number of brain functions such as cognition, memory, selective attention, and emotional processing (Poorthuis et al. 2013). In the context of my thesis, the action of cholinergic signaling on the cellular properties of neurons is of crucial importance and the ability of ACh to coordinate the response of neuronal networks in the brain makes cholinergic modulation an essential mechanism underlying distinct behaviors.

Acetylcholine, muscarinic and nicotinic acetylcholine receptors

ACh is a fast acting neurotransmitter at the neuromuscular junction and in the autonomic ganglia, with a demonstration of similar fast action in the brain at selected synapses (Arroyo et al. 2012[START_REF] Arroyo | Nicotinic modulation of cortical circuits[END_REF]. Several models have tried to explain the actions of ACh in the central nervous system (CNS) but all these models are consistent with a primary role of ACh as a neuromodulator for changing the state of neurons in response to changing environmental conditions [START_REF] Picciotto | Acetylcholine as a neuromodulator: cholinergic signaling shapes nervous system function and behavior[END_REF].

ACh was the first neurotransmitter identified, for which Herny Dale and Otto Loewi obtained the Nobel Prize in 1936. ACh is synthesized in the cytoplasm by an enzymatic reaction between the enzyme choline acetyltransferase (ChAT) and the substrates choline and acetyl-CoA. ACh is concentrated into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), ready for release at the synaptic cleft. ACh signals through two classes of receptors: metabotropic muscarinic receptors (mAChRs) and ionotropic nicotinic receptors (nAChRs). ACh is then rapidly degraded by acetylcholinesterase (AChE), an enzyme present in the synaptic cleft that regulates the ACh levels, thus modulating the duration of its action on the targeted receptors.

mAChRs comprise a family of five related G protein-coupled receptors (GPCRs)

belonging to the α-branch of class A GPCRs. The mAChRs consist of five distinct subtypes, M1 to M5 that are encoded by the genes CHRM1 to CHRM5. Three of these receptors, the M1, M3 and M5 have been shown to couple to G proteins of the G q/11 family, whereas the M2 and M4 subtypes signal through the G i/0 family of G proteins. mAChRs display an important role in human physiology, regulating heart rate, smooth muscle contraction, glandular secretion and many functions of the CNS [START_REF] Kruse | Muscarinic acetylcholine receptors: novel opportunities for drug development[END_REF]. mAChRs are located both preand postsynaptically in the brain, displaying a major role on brain activity. For example, presynaptic M2/M4 mAChRs can act as inhibitory autoreceptors on cholinergic terminals and reduce glutamate release from corticocortical and corticostriatal synapses, whereas M1/M5 receptors can stimulate dopamine (DA) release from striatal synaptosomes and postsynaptic M1/M5 receptors can increase excitability of cortical pyramidal neurons [START_REF] Thiele | Muscarinic Signaling in the Brain[END_REF].

The nAChRs are ligand-gated ion channels that are present in the peripheral nervous system (PNS), at the skeletal neuromuscular junction and in the autonomic nervous system, and the CNS (Figure 1). The abundant expression of muscle nAChRs in torpedo rays led to a significant number of electrophysiology studies, mainly on Torpedo californica and Torpedo marmorata, with a major achievement in the isolation of the receptor and the suggestion that it is pentameric [START_REF] Changeux | Use of a snake venom toxin to characterize the cholinergic receptor protein[END_REF][START_REF] Hucho | Molecular weight and quaternary structure of the cholinergic receptor protein extracted by detergents from Electrophorus electricus electric tissue[END_REF]. Indeed, nAChRs are transmembrane pentameric receptors that belong to the "cys-loop" superfamily of ligandgated ion channels together with GABA A , GABA C , glycine and 5-hydroxytryptamine (5-HT3) receptors (Le [START_REF] Novère | Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells[END_REF][START_REF] Changeux | Allosteric receptors after 30 years[END_REF]. Common to the cys-loop receptors are a disulfide bridge in the extracellular domain formed between two cysteine residues. They occur as homomeric or heteromeric assemblies of a large family of α and β subunits, which determine the pharmacological and kinetic properties of the receptor [START_REF] Gotti | Neuronal nicotinic receptors: from structure to pathology[END_REF][START_REF] Taly | Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system[END_REF]. They are widely expressed in the brain, with the neuroanatomical localization depending on the receptor subtype [START_REF] Gotti | Heterogeneity and complexity of native brain nicotinic receptors[END_REF]). In general, 17 nAChRs subunits have been identified. In the brain, there are eight α subunits (α2-α7, α9-α10) and three β subunits (β2-β4) (Le [START_REF] Novère | Molecular evolution of the nicotinic acetylcholine receptor: an example of multigene family in excitable cells[END_REF]. ACh mediated innervation acting through nAChRs regulates processes, which are crucial for network operations and influence physiological functions. These functions are involved in pathologies such as AD, SCZ and nicotine addiction (Koukouli and Maskos 2015;Lombardo and Maskos 2015). The structure of nAChRs, their function and involvement in cholinergic neurotransmission will now be discussed in order to provide a better understanding of their implication in these pathologies.

Structure of nAChRs and allosteric modulation

The first structural characterization of nAChRs used Torpedo nAChRs and in subsequent years, these structural definitions were shown to be well conserved in all identified nAChR subunits. All functional members of the Cys-loop family of ligand-gated channels are formed from a pentameric arrangement of subunits that create a central pore.

nAChRs subunits have a molecular mass of approximately 290 kDa. Since nAChR subunits are highly conserved in evolution, high-resolution X-ray crystallography and electron microscopy has been used to show commonalities and contrast in nAChR function based on their differences in structure. These studies mapped out the 4.6-Å electron microscopic structure of the Torpedo nAChR, along with the strutures of ACh-binding proteins (AChBP) in the snail species Aplysia californica, Lymnaea stagnalis and Bulinustruncatus [START_REF] Albuquerque | Mammalian nicotinic acetylcholine receptors: from structure to function[END_REF]). These AChBP are secreted as homopentamers that resemble nAChR-like complexes but lack the transmembrane and cytoplasmic domains. Recently, bacterial homologues of nAChRs have been identified. The candidates with the highest percentage of sequence homology are the Gloebacter violaceus pentameric ligand-gated ion channel (GLIC) and the Erwinia chrysantemi pentameric ligand-gated ion channel (ELIC) [START_REF] Tasneem | Identification of the prokaryotic ligandgated ion channels and their implications for the mechanisms and origins of animal Cysloop ion channels[END_REF][START_REF] Corringer | Structure and pharmacology of pentameric receptor channels: from bacteria to brain[END_REF][START_REF] Prevost | A locally closed conformation of a bacterial pentameric protongated ion channel[END_REF].

Each subunit of the nAChR consists of a large amino-terminal extracellular domain (ECD), a transmembrane domain (TMD) comprising four segments (TM1-TM4) and a variable cytoplasmic domain (Figure 1). There are two to five ACh binding sites within the ECD, which are topographically distinct from the functionally linked cationic ion channel, located on the axis of symmetry of the TMD. Each AChBP molecule has five identical binding sites for ACh and nicotine, located at the boundary between subunits (Taly et al.

2009)

The five subunits of the nAChRs are assembled around a central hydrophilic pore that mediates the migration of the cations K + , Na + and Ca ++ via a partial dehydration mechanism.

The channel pore is located within the 20 α-helical bundle of the TMD, at the level of the αhelically folded TM2, in the axis of symmetry of the nAChR pentamers (Changeux 2012a).

The binding sites for the various channel blockers are distributed throughout the transmembrane channel, in particular within its gate in the closed conformation, and at the entrance of the ion selectivity filter, which consists of rings of hydrophilic residues at the cytoplasmic border [START_REF] Taly | Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system[END_REF].

The nicotinic receptors are allosteric proteins and undergo conformational changes that can modulate the protein's function. Monod, Wyman and Changeux introduced the allosteric model in 1963, known as the MWC model [START_REF] Monod | Allosteric proteins and cellular control systems[END_REF][START_REF] Monod | On the nature of allosteric transitions: A plausible model[END_REF]. The MWC model was first introduced for the conformational changes observed in hemoglobin, but later was applied to other types of proteins, including receptors. Based on this model, the protein exists in three states: active-open, resting-closed and desensitized-closed. Upon binding of an agonist, the active conformation stabilizes causing the pore to open and the subsequent influx of ions. The receptor will desensitize with sustained ligand binding, ranging from seconds to minutes, causing a closing of the channel (conformation change to closed state), stopping the influx of ions. During this closed state, the affinity is very high for the ligand to the binding site. nAChR agonists consist of nicotine and acetylcholine that in the open state of the receptor, have higher affinity whereas antagonists stabilize the desensitized or resting state.

Similar to other receptors types, nAChRs are also sensitive to phosphorylation for modulating their activity [START_REF] Changeux | Allosteric receptors after 30 years[END_REF].

Allosteric modulators are ligands that are able to modulate nAChR function by binding to sites that are distinct from the binding site for ACh, including sites that are located in the transmembrane domain and will be discussed below.

α7-containing receptors have been found in many brain regions and they can presynaptically facilitate the release of neurotransmitters such as glutamate or GABA or exert a direct postsynaptic action by mediating fast synaptic transmission [START_REF] Alkondon | Diversity of nicotinic acetylcholine receptors in rat brain. V. alpha-Bungarotoxin-sensitive nicotinic receptors in olfactory bulb neurons and presynaptic modulation of glutamate release[END_REF][START_REF] Albuquerque | Mammalian nicotinic acetylcholine receptors: from structure to function[END_REF]). Interestingly, co-expression and assembly of α7 nAChR subunits with other nAChR subunits influences the ion permeability of the receptor. nAChRs made up of α7 and β2 nAChR subunits have pharmacological properties distinct from those of homomeric α7 nAChRs [START_REF] Khiroug | Rat nicotinic ACh receptor alpha7 and beta2 subunits co-assemble to form functional heteromeric nicotinic receptor channels[END_REF]. Co-assembly of α7 with α5 nAChR subunits results in receptors with distinct desensitization properties and ion permeability relative to the homomeric α7 nAChR [START_REF] Girod | Heteromeric complexes of alpha 5 and/or alpha 7 subunits. Effects of calcium and potential role in nicotine-induced presynaptic facilitation[END_REF]. The α8-containing receptors are only present in the chick nervous system, where they can also form heteromeric α7-α8 receptors [START_REF] Gotti | Expression of alphabungarotoxin receptor subtypes in chick central nervous system during development[END_REF]). The α9-containing receptors are expressed extra-neuronally and have an unusual mixed nicotinic-muscarinic pharmacological profile [START_REF] Elgoyhen | Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells[END_REF]. When expressed alone in Xenopus laevis oocytes, the α9, but not the α10, subunit is capable of generating functional homomeric nAChRs [START_REF] Elgoyhen | Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells[END_REF][START_REF] Elgoyhen | alpha10: a determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells[END_REF]. Significantly, when α9 and α10

are co-expressed in oocytes, larger whole-cell currents are observed, suggesting that these subunits may normally co-assemble to form a heteromeric receptor.

Heteromeric nAChRs have different combinations of α and β subunits. The receptor stoichiometry determines the receptor's function and upregulation. In general, the distributions of nAChRs in rodent and primate brains are quite similar [START_REF] Gotti | Brain nicotinic acetylcholine receptors: native subtypes and their relevance[END_REF]) (Figure 2). nAChRs are ubiquitously expressed in the rodent brain. α4β2* is the most frequent receptor subtype and can be supplemented by the accessory subunit α5, among others [START_REF] Yakel | Functional and molecular characterization of neuronal nicotinic ACh receptors in rat hippocampal interneurons[END_REF]. Similarly, the most widely expressed nAChR type in humans seems to be the α4β2 [START_REF] Gotti | Brain nicotinic acetylcholine receptors: native subtypes and their relevance[END_REF]). α3β4* is also present, but with regions of strong expression that are very different from the α4β2* types. This receptor is particularly present in the locus coeruleus, medial habenula, interpeduncular nucleus, and hippocampus.

In rat cerebellar tissue about 35% of the nAChRs are α4β2, about 20% α3β4 and about 10% α3α4β2, α3α4β2, α3β2β4 or α3α4β4, with more than 50% of the nAChRs containing the relatively uncommon β4 subunit (for a review; Zoli et al. 2015a). Interestingly, homology exists between rodents, monkeys and humans in the striatum where α6β3β2* and α4β2* prevail. The greatest significant difference between rodents and primates exists in the α2* subunit containing nAChRs. This subunit is highly express in the IPN and sparsely expressed in the hippocampus, amygdala and basal forebrain of mice and rats, whereas α2* expression in the thalamus of monkeys is comparable to that of the α4 subunit. Human expression is less clear, but α2 in brain regions that have been examined, such as the temporal cortex, appear to have higher expression than in rodents (Zoli et al. 2015). Importantly, the α7β2 subtype seems to be expressed in the basal forebrain of mice and humans, but not in many other brain regions [START_REF] Moretti | The novel α7β2-nicotinic acetylcholine receptor subtype is expressed in mouse and human basal forebrain: biochemical and pharmacological characterization[END_REF], suggesting that α7β2 nAChRs may play a specific and important functional role in this circuit. Finally, the α7 homopentameric receptor is also ubiquitously represented, with some regional foci in the hippocampus, hypothalamus and cortex [START_REF] Yakel | Functional and molecular characterization of neuronal nicotinic ACh receptors in rat hippocampal interneurons[END_REF].

Cholinergic signaling through nAChRs

Almost all regions of the brain are innervated by cholinergic neurons and many neurons and glial cells express nAChRs. Projection neurons and interneurons coordinate central cholinergic transmission with the projections neurons having distinct projection patterns segregated into separate cell groups (Ch1-Ch6) (Figure 3). The basal forebrain cholinergic projection neurons (Ch1-Ch4) in the medial septum, diagonal band of Broca, and the nucleus basalis of Meynert are the major source of ACh to cortical and hippocampal regions. More specifically, Ch4 neurons provide the main source of cholinergic projections to the neocortex, including primary and secondary, visual, auditory, somatosensory and higher association cortex and the amygdala. The cholinergic projection neurons with the cell bodies in the pedunculopontine tegmental nucleus (Ch5) and the laterodorsal tegmental nucleus (Ch6) project to the thalamus, pontine reticular formation, and the DA neurons in the ventral tegmental area (VTA) and the substantia nigra (SN). In the caudate-putamen and nucleus accumbens, large cholinergic interneurons are the only source of ACh (Mesulam, Mufson, Levey, et al. 1983;Mesulam, Mufson, Wainer, et al. 1983;[START_REF] Mesulam | Behavioral neuroanatomy of cholinergic innervation in the primate cerebral cortex[END_REF]). Ch7 corresponds to the cholinergic cells in the medial habenula, a densely aggregated structure that projects to the interpeduncular nucleus [START_REF] Oh | Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA[END_REF]. Moreover, the Ch8 nucleus is located in a structure known as parabigeminal. The PFC receives a dense cholinergic innervation that is thought to play an important role in the regulation of PFC related behaviors and shall be further discussed. the neocortex, the hippocampus and the amygdala, while the projections from the mesopontine cholinergic system are directed to the thalamus, habenula and basal forebrain. Adapted from (Woolf and Butcher 2011).

nAChRs in the Prefrontal cortex 1.2.1 The Prefrontal cortex

The PFC is a region of the cerebral cortex that covers the anterior part of the frontal lobe. There is a lot of debate about whether rodents have a PFC and how it functionally contributes to an animal's behavior. Anatomical evidence supports that the rat medial PFC (mPFC) is related to both the primate anterior cingulate cortex (ACC) and the dorsolateral PFC [START_REF] Uylings | Do rats have a prefrontal cortex?[END_REF]. Primate and human ACC are responsible for monitoring actions and outcomes for guiding decisions and the dorsolateral PFC has a more problem solving and prediction role for the maintenance and manipulation of goal-related memories for planning [START_REF] Seamans | Comparing the prefrontal cortex of rats and primates: insights from electrophysiology[END_REF]. Many current theories of consciousness consider the PFC the key player in the emergence of conscious perception (Dehaene and Changeux 2011;Lau and Rosenthal 2011). According to the Global Neuronal Workspace, the PFC serves to distribute information that is processed in unconscious modules to the entire brain (Dehaene and Changeux 2011). Multiple studies have shown that the PFC plays an important role in cognitive processes such as attention (Miller 2000;Dalley et al. 2004), memory and decision making (Euston et al. 2012), working memory (Bartus et al. 1982;Khan and Muly 2011;[START_REF] Funahashi | Space representation in the prefrontal cortex[END_REF]), social behavior [START_REF] Avale | Prefrontal nicotinic receptors control novel social interaction between mice[END_REF], emotions (Miller 2000) and personality [START_REF] Kennis | Neural correlates of personality: an integrative review[END_REF]. In rodents, the mPFC and its sub-areas, the anterior cingulate, prelimbic and infralimbic cortex are strongly involved in attention and regulation of neuronal activity (Muir et al. 1996). Attention and PFC-related behaviors are affected in many neuropsychiatric disorders, including schizophrenia, attention deficit/hyperactivity syndrome (ADHD), addiction, depression and autism, which are characterized by reduced activity of prefrontal areas (Poorthuis and Mansvelder 2013). The PFC receives significant cholinergic innervation, and ACh signaling through nAChRs is crucial for the regulation of these types of behaviors (Bloem et al. 2014). We will now discuss the role of nAChRs in the innervation of the PFC and how it is associated with the control of different behaviors.

Cholinergic modulation of prefrontal cortex by nAChRs in a layer specific manner

The PFC is connected in a topographically specific manner with most other cortices, including sensory and higher-order association areas, and subcortical structures associated with cognition, memory, and emotions (Dehaene and Changeux 2011). Both rodent and primate PFC share a number of connections with other cortical areas, such as motor and somatosensory cortices, sensory cortices and limbic areas including the amygdala, enthorhinal and perirhinal cortex. As mentioned before, in rats, the prelimbic (PrL) area of the PFC is crucially involved in executive functions, making this region functionally homologous to the dorsolateral PFC in humans and other primates [START_REF] Uylings | Do rats have a prefrontal cortex?[END_REF]. It has been shown that most cholinergic axons originate from the basal forebrain, although cholinergic neurons are also present in the cortex (Eckenstein and Baughman 1984). Furthermore, the PFC receives fibers from the pedunculopontine nucleus and the laterodorsal tegmental area (Mesulam, Mufson, Wainer, et al. 1983;[START_REF] Eckenstein | An anatomical study of cholinergic innervation in rat cerebral cortex[END_REF]. Like cortical primary sensory areas, the PFC is organized into a sheet of layers that are characterized by distinct connectivity [START_REF] Eckenstein | An anatomical study of cholinergic innervation in rat cerebral cortex[END_REF].

Recently it has been shown that there is both tonic and phasic cholinergic signaling in the mPFC (Parikh and Sarter 2008). Moreover, it has been investigated whether ACh functions through volume or synaptic transmission (Sarter et al. 2009). Both aspects of transmission are crucial for determining the effects of ACh on the mPFC. A recent study of nicotinic currents evoked by optogenetic stimulation of basal forebrain fibers in layer I (L1) interneurons indicated that α7 receptors mediate synaptic transmission, but proposed that activation of non-α7 receptors is triggered by volume transmission [START_REF] Bennett | Mechanisms generating dual-component nicotinic EPSCs in cortical interneurons[END_REF]. Most likely both are present and there is a complex interplay of tonic and phasic release, and of volume and synaptic transmission, making the precise release parameters crucial for determining the effects on the mPFC (Parikh et al. 2007). Recently, it has been shown that both point-to-point and tonic nicotinic transmission occur in the neocortex depending on the regime of basal forebrain neurons. Low frequency optogenetic stimulation resulted in point-to-point nicotinic transmission occurring synaptically via heteromeric receptors composed of α4, α5, and β2 subunits, whereas high-frequency stimulation resulted in a switch to tonic responses and in the recruitment of extrasynaptic receptors (Hay et al. 2016).

nAChRs are highly expressed across all regions in the cortex (Zoli et al. 2015). In addition to α4, β2 and α7 subunits, the accessory α5 subunit is also expressed. In general, α5 subunits are preferentially expressed in deep cortical layers and its expression is lower, but (Poorthuis et al. 2013). Firstly, the authors distinguished between two types of inhibitory neurons: fast-spiking (FS) interneurons and non-fast-spiking (NFS) interneurons (Kawaguchi and Kubota 1997), which can be distinguished based on morphology and action potential firing characteristics and secondly, using the GIN transgenic mouse line, that expresses eGFP in somatostatin positive cells (SOM). With ACh application, they were able to show a layerspecific modulation of PFC by nAChRs. More specifically, layer II/III pyramidal neurons do not contain nAChRs, layer V pyramidal neurons contain α7 nAChRs, whereas in layer VI, β2* and α5* nAChRs are found. In layer II/III only interneurons express nAChRs, with the SOM interneurons expressing α7 and β2* containing nAChRs and FS interneurons expressing exclusively α7 nAChRs [START_REF] Poorthuis | Layerspecific modulation of the prefrontal cortex by nicotinic acetylcholine receptors[END_REF][START_REF] Poorthuis | Layer-specific interference with cholinergic signaling in the prefrontal cortex by smoking concentrations of nicotine[END_REF]) (Figure 5).

Concerning the expression of the α5 nAChRs in the PFC, electrophysiological recordings showed that nicotinic receptor stimulation in the PFC selectively excites VIP interneurons through the activation of nicotinic receptors containing the α5 subunit [START_REF] Porter | Selective excitation of subtypes of neocortical interneurons by nicotinic receptors[END_REF]. Single cell RT-PCR experiments revealed high levels of α5 nAChRs exclusively in VIP interneurons in the superficial layers of the neocortex [START_REF] Porter | Selective excitation of subtypes of neocortical interneurons by nicotinic receptors[END_REF][START_REF] Férézou | 5-HT3 receptors mediate serotonergic fast synaptic excitation of neocortical vasoactive intestinal peptide/cholecystokinin interneurons[END_REF].

Figure 5: Layer specific modulation of the prefrontal cortex by nAChRs. PFC pyramidal neurons in layer II/III do not express nAChRs and only the interneurons exhibit expression, in comparison to layers V and VI where nAChRs are also expressed by pyramidal neurons. Adapted from (Bloem et al. 2014). MDT: thalamic input.

Contribution of nAChRs to prefrontal cortex associated behaviors

Dysfunction of attention is a main feature of many neuropsychiatric disorders, including SCZ, which are characterized by reduced activity of prefrontal areas including the prelimbic (PrL) cortex. For instance, removing cholinergic innervation of the PFC reduces attention performance, whereas stimulation of cholinergic projections causes enhancement [START_REF] Gill | Sustained visual attention performance-associated prefrontal neuronal activity: evidence for cholinergic modulation[END_REF][START_REF] Peters | Enhanced control of attention by stimulating mesolimbic-corticopetal cholinergic circuitry[END_REF]. Many studies in both animal models and humans have shown that nicotinic receptors are of particular importance for cognitive functions, reward and aging, as KO mice of the α5, α7 and β2 subunits display impaired behavioral performance [START_REF] Picciotto | Abnormal avoidance learning in mice lacking functional high-affinity nicotine receptor in the brain[END_REF][START_REF] Maskos | Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors[END_REF] and attention (Hoyle et al. 2006;Bailey et al. 2010;[START_REF] Guillem | Nicotinic acetylcholine receptor β2 subunits in the medial prefrontal cortex control attention[END_REF].

It has been shown that β2 KO mice show biased behavior when confronted with a choice between conflicting motivations. These mice display increased time in social contact and exaggerated approach movements toward the novel conspecific. However, in the absence of conflicting motivations, β2 KO mice exhibit unaffected cognitive behavior [START_REF] Granon | Executive and social behaviors under nicotinic receptor regulation[END_REF]. Avale and colleagues found that c-Fos expression was specifically activated in the PrL area of PFC of mice exposed to a novel conspecific [START_REF] Avale | Prefrontal nicotinic receptors control novel social interaction between mice[END_REF]). In addition, lesions of the PrL area in wild-type (WT) mice produced the same social pattern as in β2 KO mice and virally mediated re-expression of the β2 subunit in the PrL area of β2 KO mice rescued behavioral components in the social interaction task to normal levels. These data revealed that the presence of functional PrL β2*nAChRs in PrL is necessary for social interactions.

In rodents the 5-choice serial reaction time task is used to test attentional performance since it is analogous to the human continuous performance task [START_REF] Robbins | The 5-choice serial reaction time task: behavioural pharmacology and functional neurochemistry[END_REF].

A brief light stimulus is applied and the rodents are trained to respond in this visuospatial attention task. A cue signal is illuminated in one of five holes and the rodent is rewarded if it responds to the cue hole. Strong impairments in the task occurred in rodents that had lesions in the cholinergic basal forebrain. Genetic deletion of the β2 subunit of the nAChR also resulted in an impairment of attentional performance. β2 KO mice showed a higher level of omissions compared to their wild-type littermates, while their accuracy level was not affected [START_REF] Guillem | Nicotinic acetylcholine receptor β2 subunits in the medial prefrontal cortex control attention[END_REF]). Thus, the absence of the β2 subunit resulted in impaired attention, without effects on spatial choice discrimination. Re-expressing β2 subunits and restoration of functional β2 subunit-containing nAChRs in neurons of the PrL area of the mPFC fully restored attentional performance in the 5-CSRTT, indicating that β2 containing nAChRs in prelimbic mPFC are sufficient for proper attentional performance in this task. In contrast, re-expression of β2 subunits in the adjacent anterior cingulate mPFC did not restore attentional performance, suggesting that cholinergic modulation of anterior cingulate neurons through β2 containing nAChRs does not contribute to attentional behavior. This agrees with previous 5-CSRTT studies that showed no role of cholinergic projections to the anterior cingulate cortex in this task. Also, α7 KO mice in the same task conditions were not affected in their performance contrasting with previous studies that showed reduced omission levels and delayed task acquisition [START_REF] Young | Nicotine improves sustained attention in mice: evidence for involvement of the alpha7 nicotinic acetylcholine receptor[END_REF]). Since both α7 and β2 receptors are expressed in the mPFC, they could both contribute to attentional behavior. Another study investigated the effects of nicotine on the 5-CSRTT using the specific antagonists DHβE and methyllycaconitine (MLA). Co-application of these antagonists with nicotine, suggested that the effects of nicotine are mediated by α7 nAChRs and not by β2* [START_REF] Hahn | Selective nicotinic receptor antagonists: effects on attention and nicotine-induced attentional enhancement[END_REF].

Nonetheless, another study showed that the attention enhancing effects of nicotine are also seen with specific β2* nAChR agonists, but not with α7 nAChRs [START_REF] Young | Nicotinic agonist-induced improvement of vigilance in mice in the 5-choice continuous performance test[END_REF]).

Interestingly, it has also been demonstrated that the α5 subunit is necessary for normal attention behavior (Bailey et al. 2010). It has been shown that in the absence of the α5 subunit, mice show a reduction in accuracy while performing the 5-CSRTT under high attentional demands. The authors suggested that the endogenous ACh responsivity of layer VI neurons in mice expressing the α5 subunit is required for normal performance in the 5-CSRTT under challenging conditions.

Pharmacology of neuronal nAChRs

Nicotinic receptors undergo conformational changes between functional states and the five subunits move relative to each other. The pharmacological properties of each specific subunit depend on the structural feature of the ligand-binding site and on the specific amino acid interactions that determine conformational transitions. Therefore, different receptor compositions show specific pharmacological profiles to agonists, antagonists and modulators [START_REF] Dani | Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system[END_REF].

Agonists and antagonists

Nicotinic agonists are drugs that mimic the action of ACh at nAChRs by interacting with the nicotinic receptor agonist-binding site in order to initiate an allosteric change within the pentameric complex. This interaction leads to the opening of the cation-conducting pore.

The neurotransmitter ACh is the endogenous agonist for nAChRs which binds with high affinity at α4β2 nAChRs but is characterized by lack of selectivity for nAChRs versus mAChRs, thus a mAChR antagonist, for example atropine, and an acetylcholinesterase (AChE) inhibitor is added with ACh in experimental preparations. Other ligands include the agonists nicotine, cytosine, anatoxin A,epibatidine, anabaseine and anabasine. Synthetic agonists range from the classical ganglionic agonist dimethylphenylpiperazine (DMPP), to novel agonists that were created in order to be nicotinic subunit specific (Gotti et al. 2006).

Generally, agonists bind with highest affinity to the α4β2 nAChRs and with 2-3 orders of magnitude lower affinity to α7 nAChRs, whereas they bind to α3* nAChRs with intermediate affinity. Few agonists have sufficient nAChR subtype selectivity for exclusive activation of a particular subtype. Concerning the functional potency, it is similar for the different agonists, however the EC50 values are more similar between α4β2 and α3* nAChRs (Sharples and Wonnacott 2001, for review).

The antagonists of nAChRs are divided into two main categories: competitive antagonists and non-competitive antagonists. The competitive antagonists interact reversibly with the nAChR at, or close to the agonist binding site, stabilizing the receptor in a conformation with the channel closed, preventing access for agonists. Like agonists, the majority of competitive antagonists originate from natural sources. The best established subtype specific nicotinic antagonist is α-Bgt that was essential in the isolation and purification of muscle and Torpedo nAChRs, but is also a highly potent and selective antagonist at α7 nAChRs. Other competitive antagonists include d-tubocurarine (d-TC), dihydro-beta-erythroidine (DHβE), methylcacotinine (MLA) and strychnine. In addition, αconotoxins are small peptide toxins with a highly conserved disulphide-bonding pattern, with specificity for nAChR subtypes. Three well characterized α-conotoxins specific for neuronal nAChRs are α-conotoxins MII, ImI and AuIB (Sharples and Wonnacott 2001, for review).

The non-competitive antagonists (NCAs) interact with sites distinct from the agonistbinding site, thus they do not compete with agonists for binding. Usually they inhibit the nAChR activity by blocking the intrinsic channel pore, by binding to sites within or near the entrance to the channel. One of the most classic NCAs for neuronal nAChRs is mecamylamine that blocks nAChRs with IC 50 values in the low micromolar range. Other

NCAs include hexamethonium, chlorisondamine, ibogaine, dizocilpine, bupoprion and a range of steroids including corticosterone, progesterone, oestradiol, hydrocortisone and aldosterone. It is worth mentioning that several blockers of voltage-operated calcium channels have been reported to inhibit rat α3β4 nAChRs expressed in Xenopus oocytes, whereas α7 nAChRs were unaffected (Sharples and Wonnacott 2001, for review). Interestingly, there are several reports showing that β-amyloid peptide inhibits the α7 nAChRs, but this aspect will be discussed later in this thesis.

Allosteric modulators of nAChRs

Recent research has identified ligands that are able to modulate nAChR function by binding to sites that are distinct from the binding site for ACh, including sites that are located in the transmembrane domain. Allosteric modulators can either potentiate the effects of agonist-activation (positive allosteric modulators; PAMs), or inhibit agonist-activation (negative allosteric modulators; NAMs) [START_REF] Chatzidaki | Allosteric modulation of nicotinic acetylcholine receptors[END_REF]. As mentioned before, the term non-competitive antagonist is also used to describe ligands that are capable to inhibit agonist-evoked responses through a distinct binding site. In addition to PAMs and NAMs, silent allosteric modulators (SAMs) have been reported for nAChRs. These modulators do not potentiate or inhibit responses to orthosteric agonists, but they can block the effect of other allosteric modulators (PAMs, NAMs or allosteric agonists) by binding competitively at an allosteric binding site.

α7 selective PAMs are divided into two categories: type I and type II [START_REF] Bertrand | Allosteric modulation of nicotinic acetylcholine receptors[END_REF][START_REF] Grønlien | Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes[END_REF]. Type I PAMs increase the peak current in the presence of an orthosteric agonist and they do not have an effect on the receptor's desensitization. Type II PAMs have the ability to reduce the fast desensitization of the α7 nicotinic receptors. Also, type II PAMs allow the receptor's reactivation from the desensitized state [START_REF] Grønlien | Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes[END_REF]. Some examples of α7 PAMs include galantamine and the compound PNU-120596 [START_REF] Hurst | A novel positive allosteric modulator of the alpha7 neuronal nicotinic acetylcholine receptor: in vitro and in vivo characterization[END_REF][START_REF] Lopes | Galantamine potentiates the neuroprotective effect of memantine against NMDA-induced excitotoxicity[END_REF]. Furthermore, there are a number of heteromeric PAMs such as desformylflustrabromine (dBr), which is a selective α4β2 PAM that increases the size of the maximum response and has very modest effect on the potency of the agonist [START_REF] Sala | Potentiation of human alpha4beta2 neuronal nicotinic receptors by a Flustra foliacea metabolite[END_REF][START_REF] Kim | Synthesis of desformylflustrabromine and its evaluation as an alpha4beta2 and alpha7 nACh receptor modulator[END_REF]. Recently, PAMs of α3containing nAChRs have also been reported [START_REF] Wu | Morantel allosterically enhances channel gating of neuronal nicotinic acetylcholine alpha 3 beta 2 receptors[END_REF][START_REF] Bürgi | Discovery of potent positive allosteric modulators of the α3β2 nicotinic acetylcholine receptor by a chemical space walk in ChEMBL[END_REF]).

nAChRs and anesthesia

The mechanisms through which general anesthetics, an extremely diverse group of drugs, cause reversible loss of consciousness have been a mystery. Every year, tens of millions of patients are exposed to general anesthetics that fulfill three major criteria:

analgesia, unconsciousness and decreased muscle tone. Although the principal aim of anesthesia today is to insure safety and protection of patients, the mechanisms of action of anesthetic agents on the CNS remain poorly understood. In neuroscience research, anesthetics have been used to identify neuronal factors that are involved in conscious perception, to explain neural mechanisms of sleep and arousal and to elucidate the brain circuits that produce synchronous activity patterns [START_REF] Rudolph | Molecular and neuronal substrates for general anaesthetics[END_REF]Changeux 2012b).

General anesthetics belong to various molecular classes, including alcohol (e.g. ethanol), ethers (e.g. isoflurane, sevoflurane and desflurane), alkanes (e.g. halothane), simple gases (e.g. N 2 O, Xe), barbiturates, alkyl-phenols (propofol) and others like ketamine (Alkire et al. 2008;[START_REF] Forman | Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors[END_REF]. Most general anesthetics are small amphiphilic molecules that can occupy multiple protein cavities [START_REF] Eckenhoff | Promiscuous ligands and attractive cavities: how do the inhaled anesthetics work?[END_REF]. The idea that general anesthetics act by disrupting lipid bilayers or by other nonspecific mechanisms is no longer accepted and general anesthetics are thought to exert their effects on particular neuronal pathways by selective binding to well defined protein targets [START_REF] Forman | Anesthetic sites and allosteric mechanisms of action on Cysloop ligand-gated ion channels[END_REF]. Direct binding to ligand gated ion channels (LGICs) are considered today a main mechanism of action of anesthetics [START_REF] Franks | Molecular and cellular mechanisms of general anaesthesia[END_REF]. The superfamily of LGICs include the inhibitory gammaanimobutiric acid (GABA) A , glycine, the excitatory 5-hydroxytryptamine (5-HT 3 ) and the nAChRs. Structural models place many of the anesthesia interaction sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (e.g. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface (for a review see [START_REF] Forman | Anesthetics target interfacial transmembrane sites in nicotinic acetylcholine receptors[END_REF].

As already mentioned, in many cases, general anesthetics (both intravenous and volatile) do not act by direct steric competition at neurotransmitter binding sites but instead bind to topographically distinct allosteric sites in the TMD. For example, in the case of GLIC (Gloeobacter), propofol and desflurane separately bind within an intrasubunit cavity located in the upper part of the TMD, at the center of the bundle of α-helices composing each subunit and mutations within the binding pocket alter the inhibition of GLIC response to general anesthetics at clinical concentrations [START_REF] Nury | X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel[END_REF]. It is noteworthy that the pharmacology of GLIC inhibition by general anesthetics resembles that of eukaryotic nAChRs (Figure 6). As allosteric modulators, the general anesthetics selectively stabilize either an active (positive modulators) or a resting conformational state (negative modulators), without competing with neurotransmitter binding, thus offering a less disruptive way to control signal processing than orthosteric ligands and explaining the relatively rare occurrence of general anesthetic side-effects (Changeux 2012b).

Most general anesthetics inhibit nAChRs, including Torpedo, mammalian muscle and neuronal nAChRs [START_REF] Forman | Anesthetic sites and allosteric mechanisms of action on Cysloop ligand-gated ion channels[END_REF]. Possible mechanisms for inhibition include, channel blockade, allosteric stabilization of closed channels and desensitization. Different subtypes of nAChRs show distinct sensitivities to general anesthetics, even though they share high sequence homology [START_REF] Mowrey | Insights into distinct modulation of α7 and α7β2 nicotinic acetylcholine receptors by the volatile anesthetic isoflurane[END_REF]. Of particular importance for this thesis, neuronal nAChRs expressed in Xenopus laevis oocytes have been shown to be potently blocked by inhalational anesthetics [START_REF] Flood | Alpha 4 beta 2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but alpha 7-type nicotinic acetylcholine receptors are unaffected[END_REF]Violet et al. 1997). Volatile anesthetics and ketamine are the most potent inhibitors both at α4β2 and α3β4 receptors with clinically relevant IC 50 values (Tassonyi et al. 2002). Co-administration of isoflurane with ACh results in potent, dose-dependent inhibition of the α4β2 receptor current. The inhibition of the α4β2

receptor by isoflurane appears to be competitive with respect to ACh [START_REF] Flood | Alpha 4 beta 2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but alpha 7-type nicotinic acetylcholine receptors are unaffected[END_REF]. In contrast to the potent inhibition of the α4β2 nAChR by volatile anesthetics, and a lesser degree by propofol, the α7 receptors are not affected by either molecule [START_REF] Zhang | Volatile general anaesthetic actions on recombinant nACh alpha 7, 5-HT3 and chimeric nACh alpha 7-5-HT3 receptors expressed in Xenopus oocytes[END_REF].

With the exception of homomeric α7 nAChRs, most neuronal receptors have higher sensitivity than muscle receptors. For example, even at sub-anesthetic levels of volatile agents, both the α3β4 ganglionic and brain α4β2 receptors are inhibited and ketamine is also a potent inhibitor of α3β4 at very low concentrations. In contrast, the α4β2 receptor is only inhibited at clinical concentrations (for a review Tassonyi et al. 2002). Since nAChRs are inhibited at higher than clinical doses by barbiturates, etomidate and propofol, it appears these receptors are not the primary target of these drugs. Both muscle and neuronal nAChRs are effectively inhibited by curares. In contrast, receptor activation and enhanced synaptic activity occur with low concentrations of atracurium and laudosine in the CNS (for a review Tassonyi et al. 2002). Since, ganglionic and brain functions appear to be altered by the interaction between anesthetics and neuronal nAChRs, it is of great interest to decipher these mechanisms of action.

5 Chronic nicotine exposure

Although it is established that neuronal nAChRs are the primary target for nicotine, the mechanisms that lead to nicotine dependence and its behavioral consequences remain to be fully understood. It is well known by now that nicotine can both activate and desensitize nAChRs. There is continuous controversy in the field to what extent the behavioral effects of nicotine result from activation of nAChRs and to what extent receptor desensitization is implicated in behavioral processes. Activation of nAChRs takes place when ACh or nicotine binds at the interface between an alpha and a neighboring alpha or beta subunit, causing a conformational change and opening of the central pore [START_REF] Galzi | Neuronal nicotinic receptors: molecular organization and regulations[END_REF] to allow influx of sodium and calcium [START_REF] Léna | Role of Ca2+ ions in nicotinic facilitation of GABA release in mouse thalamus[END_REF]. Changeux and colleagues originally described the balance between channel opening, desensitization and inactivation proposing an allosteric model of nAChR states [START_REF] Changeux | Acetylcholine receptor: an allosteric protein[END_REF]. This section will explore the dynamics of nAChR expression during chronic nicotine exposure.

Chronic nicotine exposure: upregulation of nAChRs

In humans, in post-mortem brains, early radioligand binding measurements of smokers revealed up to 400% increase in nAChRs in the regions such as cortex and hippocampus [START_REF] Benwell | Desensitization of the nicotine-induced mesolimbic dopamine responses during constant infusion with nicotine[END_REF][START_REF] Perry | Increased nicotinic receptors in brains from smokers: membrane binding and autoradiography studies[END_REF]. It has been found that this effect is dose-dependent and reversible after at least two months of abstinence. In addition, single-photon emission computed tomography (SPECT) imaging of human smokers revealed up-regulation of β2* nAChRs in the cortex and striatum even after seven days of smoking abstinence, required to clear nicotine from the brain [START_REF] Staley | Human tobacco smokers in early abstinence have higher levels of beta2* nicotinic acetylcholine receptors than nonsmokers[END_REF]. Chronic nicotine exposure to animals has also been shown to lead to an increase in the density of nicotine binding sites in the brain.

Both α7 and β2* nAChR up-regulation have been found in several brain regions after chronic nicotine administration, although β2* nAChRs appear to be more consistently up regulated, with β2 knock-out mice not exhibiting up-regulation of nicotine binding sites [START_REF] Picciotto | It is not "either/or": activation and desensitization of nicotinic acetylcholine receptors both contribute to behaviors related to nicotine addiction and mood[END_REF]). While it is clear that α4β2 nAChRs are up-regulated after chronic nicotine exposure, studies of α6/β2* nAChRs have found up-regulation [START_REF] Parker | Upregulation of brain nicotinic acetylcholine receptors in the rat during long-term selfadministration of nicotine: disproportionate increase of the alpha6 subunit[END_REF]), down-regulation [START_REF] Lai | Long-term nicotine treatment decreases striatal alpha 6* nicotinic acetylcholine receptor sites and function in mice[END_REF] or no change [START_REF] Mccallum | Differential regulation of mesolimbic alpha 3/alpha 6 beta 2 and alpha 4 beta 2 nicotinic acetylcholine receptor sites and function after long-term oral nicotine to monkeys[END_REF]).

The cellular mechanisms driving nAChR up-regulation following chronic nicotine exposure have been explored and a number of different mechanisms been reported. For example, it has been shown that the number of nAChRs increases following chronic nicotine exposure, but the mRNA levels do not, however the rate of protein synthesis or degradation may change [START_REF] Marks | Nicotine binding and nicotinic receptor subunit RNA after chronic nicotine treatment[END_REF]). Also, it has been proposed that nicotine acts intracellularly to elicit up-regulation; nicotine can potentiate nAChR assembly in the endoplasmic reticulum (ER) followed by transport of the receptor to the cell surface [START_REF] Kuryatov | Nicotine acts as a pharmacological chaperone to up-regulate human alpha4beta2 acetylcholine receptors[END_REF][START_REF] Sallette | Nicotine upregulates its own receptors through enhanced intracellular maturation[END_REF]. Concomitantly, chronic nicotine reduces proteasome degradation of nAChR in the ER [START_REF] Govind | Nicotine-induced upregulation of native neuronal nicotinic receptors is caused by multiple mechanisms[END_REF].

One important question is whether the increase in nicotinic binding sites after chronic nicotine exposure results in any increase in functional nAChRs, or whether increased binding reflects an increase in desensitized nAChRs [START_REF] Picciotto | It is not "either/or": activation and desensitization of nicotinic acetylcholine receptors both contribute to behaviors related to nicotine addiction and mood[END_REF]. In vitro studies in rat striatal synaptosomes confirm that up-regulated nicotine binding sites are functional receptors and this up-regulation has led many researchers to believe that it is related to the addictive effects of nicotine. Nicotinic activation of nAChRs in the ventral tegmentum enhances glutamatergic inputs to dopaminergic neurons and has been demonstrated to produce long-term potentiation of excitatory inputs in the dopaminergic reward centers [START_REF] Mansvelder | Long-term potentiation of excitatory inputs to brain reward areas by nicotine[END_REF].

There are a few studies showing nicotinic antagonists are also able to elicit nAChR upregulation [START_REF] Wang | Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells[END_REF]). The antagonist DHβE, but not the channel blocker mecamylamine, increases nAChR expression, however antagonist evoked nAChR upregulation has been refuted by other studies [START_REF] Peng | Chronic nicotine treatment upregulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y[END_REF]).

Chronic nicotine exposure: desensitization of nAChRs

nAChRs can undergo desensitization, a reversible reduction in response during a sustained agonist application [START_REF] Giniatullin | Desensitization of nicotinic ACh receptors: shaping cholinergic signaling[END_REF]. Although the mechanism of desensitization remains incompletely understood, several recent studies have elucidated new properties underlying desensitization following chronic nicotine exposure. When medium to high (µM to mM) concentrations of agonist are applied, nAChRs are first activated and can then desensitize with subsequent recovery after the removal of the agonist. This process develops usually in the range of milliseconds, although different subtypes of nAChRs have differential susceptibility to desensitization. Moreover, ACh and nicotine have differential activity to desensitize nAChRs [START_REF] Giniatullin | Desensitization of nicotinic ACh receptors: shaping cholinergic signaling[END_REF]. Desensitization occurs in vitro at nicotine concentrations comparable to plasma nicotine levels in smokers (100 nM) [START_REF] Henningfield | Nicotine delivery kinetics and abuse liability[END_REF]. It has been proposed that desensitization may be necessary for up-regulation and this up-regulation following chronic nicotine exposure may compensate for a loss of receptor function [START_REF] Fenster | Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C[END_REF].

Studies of nAChRs expressed in Xenopus oocytes originally suggested that α7* nicotinic receptors are particularly susceptible to inactivation, making α7 dependent currents difficult to measure unless agonists were delivered rapidly, whereas β2* and β4* exhibited desensitization in response to nicotine exposure and recovered after several minutes of washout [START_REF] Couturier | A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX[END_REF][START_REF] Pidoplichko | Nicotine activates and desensitizes midbrain dopamine neurons[END_REF][START_REF] Picciotto | It is not "either/or": activation and desensitization of nicotinic acetylcholine receptors both contribute to behaviors related to nicotine addiction and mood[END_REF]). In addition,

Mansvelder and colleagues showed that the presynaptic effects of lower concentrations of nicotine, blocked by nicotinic agents that are thought to be selective for α7 nAChRs, persist in the continued presence of nicotine in rats brain slices, whereas presynaptic effects that are blocked with agents thought to be selective for β2* nAChRs desensitize during continued nicotine application [START_REF] Mansvelder | Synaptic mechanisms underlie nicotineinduced excitability of brain reward areas[END_REF].

With one cigarette the β2* nAChR is nearly completely saturated in humans [START_REF] Brody | Cigarette smoking saturates brain alpha 4 beta 2 nicotinic acetylcholine receptors[END_REF]. The responsiveness of β2* nAChRs decreases in the mPFC in smokers while the responsiveness of α7 nAChRs is unaffected. This effect on β2* nAChRs lasts for over 45 minutes after nicotine withdrawal as an indication that nAChRs are desensitized.

Desensitization of β2* nAChRs in the ventral tegmental area also occurs, but specifically on GABAergic interneurons and not on α7 nAChRs on glutamatergic terminals and dopamine neurons [START_REF] Mansvelder | Synaptic mechanisms underlie nicotineinduced excitability of brain reward areas[END_REF]. Prolonged presence of smoking does not desensitize α7

nAChRs in contrast to their displaying rapid desensitization kinetics with robust increases in nicotine. These forms of desensitization (classical and high affinity) are also present in the mPFC [START_REF] Giniatullin | Desensitization of nicotinic ACh receptors: shaping cholinergic signaling[END_REF]. Thus, in the PFC, α7 nAChRs likely are available for activation by fast cholinergic transients during smoking. The desensitizing properties of β2* nAChRs are heterogeneous and depend on the presence of accessory α5 subunits (Bailey et al. 2010;[START_REF] Poorthuis | Layer-specific interference with cholinergic signaling in the prefrontal cortex by smoking concentrations of nicotine[END_REF]). α5 and β2 subunits co-assemble in layer VI pyramidal neurons and layer II/II interneurons of the PFC and therefore these receptors are protected against desensitization by low levels of nicotine [START_REF] Porter | Selective excitation of subtypes of neocortical interneurons by nicotinic receptors[END_REF][START_REF] Poorthuis | Layer-specific interference with cholinergic signaling in the prefrontal cortex by smoking concentrations of nicotine[END_REF].

Since there is evidence that nicotine influences cognitive functions and attentional performance and that, at least some of these effects are mediated by prefrontal nAChRs in rats, it is possible that the effects of nicotine in these types of functions are mediated by desensitization of nAChRs. A number of studies in β2 KO mice have shown that both functional deletion in KO mice and nicotine administration in wild-type mice can lead to similar cellular and behavioral effects, suggesting that nicotine administration and subsequent desensitization of β2* nAChRs contribute these behavioral responses (Bloem et al. 2014).

However, it has also been suggested that the behavioral consequences of nicotine exposure may result from a simultaneous activation and desensitization of specific nAChRs [START_REF] Picciotto | It is not "either/or": activation and desensitization of nicotinic acetylcholine receptors both contribute to behaviors related to nicotine addiction and mood[END_REF]).

1.6

Human genetic studies of nicotinic acetylcholine receptor genes, smoking and schizophrenia

The CHRNA5/CHRNA3/CHRNB4 gene cluster

Several human genetic studies have elucidated common genetic polymorphisms and rare mutations that contribute to nicotine dependence. Genome-wide association studies (GWAS) allowed the identification of susceptible loci in psychiatric diseases due to the screening of millions of genetic factors in cohorts of thousands of patients and controls. The capture of a substantial proportion of genetic risk facilitates new study designs to investigate the combined effects of genes and the environment [START_REF] Gratten | Large-scale genomics unveils the genetic architecture of psychiatric disorders[END_REF]. A multitude of GWAS showed that variations in nAChR subunits are strongly associated with risk for smoking behavior and nicotine dependence (Tobacco and Genetics Consortium. 2010). The 15q25 gene cluster, which contains the CHRNA5/CHRNA3/CHRNB4 genes, coding for the α5, α3 and β4 nAChR subunits, has shown the most robust evidence as a candidate region contributing to risk of heavy smoking and nicotine addiction [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF][START_REF] Saccone | The CHRNA5-CHRNA3-CHRNB4 nicotinic receptor subunit gene cluster affects risk for nicotine dependence in African-Americans and in European-Americans[END_REF](Tobacco and Genetics Consortium. 2010;[START_REF] Lips | Association between a 15q25 gene variant, smoking quantity and tobacco-related cancers among 17 000 individuals[END_REF]Liu et al. 2010). There are three independently associated groups of highly correlated single nucleotide polymorphisms (SNPs), each of which contributes to the associative signals at the CHRNA5/CHRNA3/CHRNB4 gene cluster [START_REF] Saccone | Multiple independent loci at chromosome 15q25.1 affect smoking quantity: a meta-analysis and comparison with lung cancer and COPD[END_REF]).

The results from three GWAS meta-analyses did not identify SNPs in the CHRNB4 gene as risk factor to the genetic association for nicotine addiction (Tobacco and Genetics Consortium. 2010;[START_REF] Lips | Association between a 15q25 gene variant, smoking quantity and tobacco-related cancers among 17 000 individuals[END_REF][START_REF] Liu | Meta-analysis and imputation refines the association of 15q25 with smoking quantity[END_REF][START_REF] Liu | Meta-analysis and imputation refines the association of 15q25 with smoking quantity[END_REF][START_REF] Liu | Meta-analysis and imputation refines the association of 15q25 with smoking quantity[END_REF][START_REF] Thorgeirsson | Sequence variants at CHRNB3-CHRNA6 and CYP2A6 affect smoking behavior[END_REF]. Interestingly, a study of 2,700 people revealed that the rs12914008 (threonine to isoleucine, T91I) SNP, with a frequency of 4.5% in European-Americans and 0.6% in African-Americans, reduces the risk for nicotine dependence [START_REF] Saccone | The CHRNA5-CHRNA3-CHRNB4 nicotinic receptor subunit gene cluster affects risk for nicotine dependence in African-Americans and in European-Americans[END_REF]). This was replicated by Haller and colleagues, who additionally showed that rs61737499 (threonine to isoleucine T375I) is associated with a reduced risk of nicotine dependence [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF]). In addition, the CHRNA3 variant rs8192475 (R37H), which is in strong disequilibrium with CHRNB4's T91I, exacerbates increased nicotine-evoked current [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF].

Whereas β4 subunit expression in the medial habenula may contribute to murine nicotine aversion, genetic variation within CHRNB4 in European-American populations of smokers does not seem to explain any detectable variance in the nicotine addiction phenotype [START_REF] Berrettini | The CHRNA5-A3-B4 gene cluster in nicotine addiction[END_REF]. The majority of evidence centers on CHRNA5 association to nicotine addiction.

The most notable polymorphism implicated in nicotine addiction risk is the CHRNA5 reference SNP (rs) number 16969968, present at 37% in European populations [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF]. This non-synonymous variant rs16969968 results in an amino acid change in the α5 nicotinic receptor subunit, aspartate to arginine (D398N), and alters the nicotinic receptor's conductance as found in in vitro studies [START_REF] Kuryatov | Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)₂α5 AChR function[END_REF]. This SNP demonstrates an important role in nicotine addiction processes as studied in rodent models [START_REF] Fowler | Habenular α5 nicotinic receptor subunit signalling controls nicotine intake[END_REF][START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. It is worth emphasizing that many of the CHRNA3 and CHRNA5 SNPs are consistently associated with lung cancer and chronic obstructive pulmonary disease (COPD) [START_REF] Amos | Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1[END_REF][START_REF] Thorgeirsson | A variant associated with nicotine dependence, lung cancer and peripheral arterial disease[END_REF][START_REF] Pillai | A genome-wide association study in chronic obstructive pulmonary disease (COPD): identification of two major susceptibility loci[END_REF]. A recent meta-analysis study showed that the rs16969968 SNP predicts delayed smoking cessation and earlier lung cancer diagnosis [START_REF] Chen | CHRNA5 risk variant predicts delayed smoking cessation and earlier lung cancer diagnosis--a meta-analysis[END_REF].

Recently, the same genetic locus was associated with schizophrenia in a major GWAS where 36,989 cases and 113,075 controls were used (Ripke et al. 2014) (Figure 7).

Smoking is extremely common (more than 80% prevalence) in people with schizophrenia and the consortium's identification of an associated variant in the CHRNA5/CHRNA3/CHRNB4 might reflect a dose response relationship between heaviness of smoking and schizophrenia risk, or as well as, a direct relationship [START_REF] Flint | Schizophrenia: genesis of a complex disease[END_REF]. Furthermore, the SNP rs16969968 in the α5 nicotinic subunit, which has been extensively studied in this thesis was reported to be associated with schizophrenia [START_REF] Hong | A CHRNA5 allele related to nicotine addiction and schizophrenia[END_REF]. Hong and colleagues showed that this SNP is associated with schizophrenia in both Caucasian and African-American nonsmoker schizophrenia patients compared with control nonsmokers [START_REF] Hong | A CHRNA5 allele related to nicotine addiction and schizophrenia[END_REF].

Intriguingly, in this study the authors did not report a significant association with smoking status (smokers vs. nonsmokers) in either schizophrenia patients or controls. It has also been

shown that the rs16969968 is associated with decreased intrinsic resting functional connectivity (rsFC) in humans [START_REF] Hong | A genetically modulated, intrinsic cingulate circuit supports human nicotine addiction[END_REF]. rsFC is a functional MRI method that measures the synchronization of intrinsic low-frequency fluctuations between brain regions in the absence of any specific task performance. These circuits correspond closely to networks engaged during task performance, predict brain activation associated with behavioral performance, and are significantly heritable [START_REF] Glahn | Genetic control over the resting brain[END_REF]). An additional study using a larger sample size tested five variants in the 15q25 gene cluster, including the SNP rs16969968 and found an association with risk for schizophrenia and bipolar disorder, and the and GWAS on smoking has not implicated CHRNA7 [START_REF] Thorgeirsson | A variant associated with nicotine dependence, lung cancer and peripheral arterial disease[END_REF]. CHRNA7

variants may be associated with schizophrenia and not with smoking in the disorder [START_REF] Stephens | Association of the 5'-upstream regulatory region of the alpha7 nicotinic acetylcholine receptor subunit gene (CHRNA7) with schizophrenia[END_REF]. More specifically, a SNP, rs3087454, in the 5-UT (untranslated area) of CHRNA7 was associated with schizophrenia, with a positive fMRI improvement in the default network with DMXB-A treatment in schizophrenia [START_REF] Tregellas | Effects of an alpha 7-nicotinic agonist on default network activity in schizophrenia[END_REF].

The α7 nicotinic receptor gene has a partial duplication of exons 5-10 on chromosome 15 that were inserted 1.6 Mb upstream of CHRNA7, interrupting an earlier partial duplication of two other genes [START_REF] Gault | Genomic organization and partial duplication of the human alpha7 neuronal nicotinic acetylcholine receptor gene (CHRNA7)[END_REF]. The chimeric CHRFAM7A gene product, dupα7, assembles with α7 subunits, resulting in dominant negative regulation regulation. The duplication is human specific, occurring neither in primates nor in rodents, but a closely related sequence has been identified in gorillas [START_REF] Shorey-Kendrick | Nicotinic receptors in non-human primates: Analysis of genetic and functional conservation with humans[END_REF]. It is important to notice that the duplicated α7 sequence in exons 5-10 of CHRFAM7A is almost identical to CHRNA7, thus is not completely queried in GWAS (Sinkus et al. 2015). Several studies support the association of the 2 bp deletion in exon 6 of the CHRFAM7A gene with schizophrenia as reviewed by Sinkus and colleagues. Although the 2 bp deletion is more common in Caucasians, association to schizophrenia was found in both African-Americans and Caucasians [START_REF] Sinkus | A 2-base pair deletion polymorphism in the partial duplication of the alpha7 nicotinic acetylcholine gene (CHRFAM7A) on chromosome 15q14 is associated with schizophrenia[END_REF].

Finally, CHRNA4 and CHRNB2 have been associated with smoking [START_REF] Li | Ethnic-and gender-specific association of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) with nicotine dependence[END_REF][START_REF] Hutchison | CHRNA4 and tobacco dependence: from gene regulation to treatment outcome[END_REF][START_REF] Conti | Nicotinic acetylcholine receptor beta2 subunit gene implicated in a systems-based candidate gene study of smoking cessation[END_REF], although no association with schizophrenia or smoking status in schizophrenia has been identified with an exception of an association with the quantity of smoking [START_REF] De Luca | Genetic interaction between alpha4 and beta2 subunits of high affinity nicotinic receptor: analysis in schizophrenia[END_REF][START_REF] Voineskos | Association of alpha4beta2 nicotinic receptor and heavy smoking in schizophrenia[END_REF]).

Schizophrenia

Schizophrenia (SCZ) is a psychotic mental disorder with a lifetime prevalence of approximately 0.3-0.7% (American Psychiatric Association 2013). A new generation of medications and recent developments in neuropathology, brain imaging and molecular genetics have led to a greater understanding of the pathophysiology of SCZ and to improved treatment (Freedman 2003). However, it remains an enigmatic illness that places a substantial burden on society.

A brief introduction to Schizophrenia's history

In Greek mythology, and in the Homerian epics, madness was thought of as a punishment from the Gods and it was not until the time of the Greek physician Hippocrates (460-377 BC) that mental illness first became an object of scientific speculation. Hippocrates thought that madness resulted from an imbalance in the four bodily humors and that it could be cured by rebalancing these humors with such treatments as special diets, purgatives, and blood-letting. The Greek philosopher Aristotle (384-322 BC), and later the Roman physician Galen (129-216), expanded on Hippocrates' humoral theories and both men played an important role in establishing them as Europe's dominant medical model.

The term "schizophrenia" was coined on April 24, 1908, when Professor Bleuler gave a lecture at a meeting of the German Psychiatric Association in Berlin [START_REF] Fusar-Poli | Paul Eugen Bleuler and the birth of schizophrenia[END_REF]. The word schizophrenia, which translates as "splitting of the mind" comes from the Greek verb schizein (σχίζειν, "to split") and phrēn, phren-(φρήν, φρεν-, "mind") and was intended to describe the separation of function between personality, thinking, memory and perception. At that meeting, Professor Bleuler argued that dementia praecox was associated with neither dementia nor precociousness, and emphasized that splitting of psychic functioning is an essential feature of SCZ [START_REF] Ashok | Paul Eugen Bleuler and the origin of the term schizophrenia (SCHIZOPRENIEGRUPPE)[END_REF]. The German alienist Heinrich Schule first used the term dementia praecox in 1886 and then in 1891 Arnold Pick used the same term in a case report of a psychotic disorder.

One of the first to classify the mental disorders into different categories was the German physician Emil Kraepelin in 1899 [START_REF] Decker | The psychiatric works of Emil Kraepelin: a many-faceted story of modern medicine[END_REF]). One of his most important achievements was the connection of pathogenesis and manifestation of psychiatric disorders.

In opposition to the leading theories of his time, Kraepelin did not believe that certain symptoms were characteristic for specific illnesses [START_REF] Ebert | Emil Kraepelin: A pioneer of scientific understanding of psychiatry and psychopharmacology[END_REF]. Clinical observation led him to the hypothesis that specific combinations of symptoms in relation to the course of psychiatric illnesses allow one to identify a particular mental disorder. He differentiated between "dementia praecox" and "manic depression" as the two forms of psychosis. Kraepelin considered "dementia praecox" (which is nowadays known as SCZ) as a biological illness caused by anatomical or toxic processes. For Kraepelin, schizophrenia was a progressive neurodegenerative disease, which automatically resulted in irreversible loss of cognitive functions. In contrast, he described manic depression as an episodic disorder, which does not lead to permanently impaired brain function [START_REF] Kraepelin | Psychiatric observations on contemporary issues[END_REF]. One of the most striking differences between Emil Kraepelin and Bleuler is that Kraepelin collected information about his patients from their records while Bleuler obtained the information by clinical observations. However, both Bleuler and Kraepelin subdivided schizophrenia into categories, based on prominent symptoms and prognoses. Over the years, those working in this field have continued to attempt to classify types of schizophrenia. Five types were delineated: disorganized, catatonic, paranoid, residual, and undifferentiated. The first three categories were originally proposed by Kraepelin [START_REF] Rosenhan | On being sane in insane places[END_REF]. Many researchers are using other systems to classify types of the disorder, based on the preponderance of "positive" vs "negative" symptoms, the progression of the disorder in terms of type and severity of symptoms over time, and the co-occurrence of other mental disorders and syndromes. In the DSM-5 the SCZ subtypes have been eliminated with the reason that these subtypes are not stable conditions and have not afforded significant clinical utility nor scientific validity and reliability (American Psychiatric Association 2013).

Clinical characteristics

SCZ has varied and ominous symptoms that generally begin in late adolescence or early adulthood and usually continue throughout life (Freedman 2003). The characteristic symptoms of SCZ involve a range of cognitive, behavioral and emotional dysfunctions, but no single symptom is pathognomonic of the disorder. The diagnosis involves the recognition of a constellation of signs and symptoms associated with impaired occupational or social functioning. Individuals with the disorder will exhibit most features, as SCZ is a heterogeneous clinical syndrome. It is characterized by positive and negative symptoms that can influence a patient's behavior (American Psychiatric Association 2013). Positive symptoms include hallucinations, voices that converse with or about the patient and delusions, the paranoid belief that external forces are conspiring against the patient. Patients may have some insight that the voices are internal thoughts and that the delusions are not real and this situation provokes confusion and trouble. Negative symptoms include flattened affect, inability to pay attention, loss of sense of pleasure, disorganization and social difficulties.

Cognitive dysfunction, including loss of attention and confused speech, is also an important feature of the illness [START_REF] Green | What are the functional consequences of neurocognitive deficits in schizophrenia?[END_REF].

Criminal behavior is not a concomitant with SCZ, but patients may commit violent acts in response to hallucinations or delusions. Approximately 5%-6% of individuals with SCZ die by suicide, about 20% attempt suicide on one or more occasions, and many have suicidal ideation. Suicidal behavior is sometimes in response to command hallucinations to harm oneself or others (American Psychiatric Association 2013).

Neuropathology

The neuropathology of schizophrenia remains unclear despite the fact that many neurobiologists have investigated this area for over 100 years. It has been for a long time one of the most controversial fields of biomedical research. The morphological or functional abnormalities in the brains of schizophrenia patients have been reported by means of CT or MRI and with advanced functional brain imaging technology such as positron emission tomography or single photon emission computed tomography. These neuroimaging studies have influenced the neuropathological investigation of the schizophrenic brain.

Neuropathological abnormalities were found in almost all areas of the brain in the postmortem schizophrenic brain, but there are more reports describing the temporal lobe and frontal lobe compared to those describing other areas of the brain. These observed neuropathological abnormalities are explained rationally by the hypothesis of a neurodevelopmental disorder in this disease. In recent molecular biology studies, several putative candidate genes were reported, and some of these genes might have a functional effect on neuronal networks.

1.7.3a Macroscopic and histological findings

Advances in brain imaging technology have established that there are significant anatomical changes in brains of patients with schizophrenia. Brain imaging of chronic schizophrenia patients have detected ventricular enlargement accompanied by reductions in brain volume and weight [START_REF] Wright | Meta-analysis of regional brain volumes in schizophrenia[END_REF]. Enlarged lateral and third ventricles and decrease in whole brain, hippocampal, basal ganglia, and thalamic volumes are present in first-episode patients with schizophrenia. There are also abnormalities reported in a diverse range of other parameters, including cortical gyrification, cortical thickness, hippocampal shape and cerebral asymmetry [START_REF] Harrison | The neuropathology of schizophrenia. A critical review of the data and their interpretation[END_REF][START_REF] Harrison | Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence[END_REF].

Of interest for this manuscript, in the PFC there is increased density of pyramidal cells in schizophrenia brains but without a difference in the total cell number. Decreased soma size and decreased neuropil may provoke the more dense packing of neurons [START_REF] Rajkowska | Neuronal and glial somal size in the prefrontal cortex: a postmortem morphometric study of schizophrenia and Huntington disease[END_REF][START_REF] Black | Pathology of layer V pyramidal neurons in the prefrontal cortex of patients with schizophrenia[END_REF]). It has been showed that the soma of pyramidal neurons in the PFC is smaller in schizophrenia brains compared with that in normal controls and that the dendrites are shorter and less branched [START_REF] Bakhshi | The neuropathology of schizophrenia: A selective review of past studies and emerging themes in brain structure and cytoarchitecture[END_REF]. Also, the spine density is lower in schizophrenia [START_REF] Garey | Reduced dendritic spine density on cerebral cortical pyramidal neurons in schizophrenia[END_REF]). Furthermore, there are reductions in several presynaptic markers of pyramidal and other neurons, with decreased expression of genes such as synaptophysin, SNAP-25 and complexin II [START_REF] Honer | Presynaptic proteins and schizophrenia[END_REF]. Concerning the interneurons, several groups have reported molecular changes in GABAergic neurons in the PFC [START_REF] Mirnics | Molecular characterization of schizophrenia viewed by microarray analysis of gene expression in prefrontal cortex[END_REF][START_REF] Lewis | Cortical inhibitory neurons and schizophrenia[END_REF]. More specifically, decrease in a GABAsynthesizing enzyme, glutamic acid decarboxylase-67 (GAD67), has been observed [START_REF] Volk | Decreased glutamic acid decarboxylase67 messenger RNA expression in a subset of prefrontal cortical gammaaminobutyric acid neurons in subjects with schizophrenia[END_REF]. The density of some interneurons, especially parvalbumin-immunoreactive cells and their synaptic projections are reduced in schizophrenia brains [START_REF] Zhang | A selective decrease in the relative density of parvalbuminimmunoreactive neurons in the hippocampus in schizophrenia[END_REF]. This finding is of crucial importance since fast spiking interneurons synchronize pyramidal cell firing and give rise to the gamma oscillations, which are impaired in schizophrenia [START_REF] Gonzalez-Burgos | Alterations of cortical GABA neurons and network oscillations in schizophrenia[END_REF]. Moreover, decreased expression of somatostatin and cholecystokinin in the PFC suggests that GABA neurotransmission is also impaired in the Martinotti and non-fast-spikig basket GABAergic neurons [START_REF] Hashimoto | Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortex of subjects with schizophrenia[END_REF].

Interestingly, a reduction in the number and function of oligodendrocytes is becoming evident from ultrastuctural, microarray and morphometric studies. In the PFC, downregulation of key oligodendrocyte and myelination genes has been found, including transcription factors that regulate these genes [START_REF] Vostrikov | Deficit of perineuronal oligodendrocytes in the prefrontal cortex in schizophrenia and mood disorders[END_REF]. Since oligodendrocytes are known to express excitatory glutamate receptors, the putative deficits in glutamatergic neurotransmission in schizophrenia may reflect oligodendroglial changes [START_REF] Harrison | Schizophrenia genes, gene expression, and neuropathology: on the matter of their convergence[END_REF]. These neuropathological changes may underlie a disturbance in neuronal connectivity in schizophrenia.

A specific interest has arisen in microglia due to a link to the neuroinflammatory response in schizophrenia [START_REF] Najjar | Neuroinflammation and white matter pathology in schizophrenia: systematic review[END_REF]. Increased microglial cell density has been found in the PFC and in the auditory association cortex [START_REF] Radewicz | Increase in HLA-DR immunoreactive microglia in frontal and temporal cortex of chronic schizophrenics[END_REF][START_REF] Fillman | Increased inflammatory markers identified in the dorsolateral prefrontal cortex of individuals with schizophrenia[END_REF]. Increased microglial cells have also been found in the anterior cingulate cortex and mediodorsal thalamus of patients with schizophrenia who had committed suicide during acute psychosis [START_REF] Steiner | Distribution of HLA-DR-positive microglia in schizophrenia reflects impaired cerebral lateralization[END_REF]). In addition, increased levels of pro-inflammatory cytokines have been detected in schizophrenia but it is not clear if they contribute to the pathology or they consist a healthy response [START_REF] Miller | Meta-Analysis of Cytokine Alterations in Schizophrenia: Clinical Status and Antipsychotic Effects[END_REF]).

1.7.3b Brain connectivity in schizophrenia

The application of complex network analysis methods has contributed to the description of altered connectivity in the brains of SCZ patients. However, a significant variability of results exists in structural connectivity studies in all types of SCZ patients and high-risk subjects. These inconsistencies in studies are from differences in methodology, differences in the clinical presentation of schizophrenia, causal mechanism variability and dissimilarity in moderator variables.

Structural connectomic studies have identified altered connectivity within subnetworks in chronic patients, as well as first episode medication naïve patients and siblings of patients. Graph theoretical analyses at the whole network level have predominantly identified network inefficiency and reduced integration between regions, such as the frontal cortex and the limbic areas [START_REF] Wheeler | A review of structural neuroimaging in schizophrenia: from connectivity to connectomics[END_REF]. More specifically, disconnectivity has been demonstrated in fronto-temporal regions, cortico-thalamo-cerebellar loops and interhemispheric fibers crossing in the corpus callosum [START_REF] Cassoli | Disturbed macro-connectivity in schizophrenia linked to oligodendrocyte dysfunction: from structural findings to molecules[END_REF]. A recent metaanalysis study of first episode patients with only marginal treatment showed a reduction in fractional anisotropy in the fronto-limbic circuitry involving the left inferior longitudinal fasciculus, left inferior fronto-occipital fasciculus, and inter-hemispheric fibers of the corpus callosum [START_REF] Yao | White matter deficits in first episode schizophrenia: an activation likelihood estimation meta-analysis[END_REF]). In addition, based on another study, the myelin-associated fraction was decreased in the genu of the corpus callosum of first episode patients, whereas chronic patients showed reductions in the same region along with additional changes in the frontal cortex [START_REF] Flynn | Abnormalities of myelination in schizophrenia detected in vivo with MRI, and post-mortem with analysis of oligodendrocyte proteins[END_REF]. These findings have supported the hypothesis that brain disconnectivity and consequently cognitive disruption in schizophrenia are associated to problematic axon myelination by oligodendrocytes. The identification of affected circuits could guide the application of therapeutic interventions for schizophrenia.

Epidemiology and risk factors

SCZ affects around 0.3 -0.7 % of people at some point in their life (American Psychiatric Association 2013). Diagnostic categories that are biased towards negative symptoms and long duration of illness produce higher incidence rates for men than for women, whereas those including more affective symptoms and brief presentations show similar rates in men and women. These data suggest that the symptomatic expression of schizophrenia and related diagnoses is more severe in men than in women. The finding of an earlier onset in men than in women supports this notion (van Os and Kapur 2009).

There is a peak of morbidity for SCZ in young adulthood. The age of onset varies between men and women, where males tend to have a younger onset. The peak incidence for males and females is in the ages of 15-24. The peak for young adults is more marked for males, and the females have a second peak in the years 55-64. Evidence suggests that males have 30%-40% higher lifetime risk of developing schizophrenia [START_REF] Messias | Epidemiology of schizophrenia: review of findings and myths[END_REF].

A family history of schizophrenia is the most significant risk factor. Other risk factors include perinatal and early childhood factors, such as hypoxia, maternal infection, maternal stress and maternal malnutrition [START_REF] Brown | Prenatal infection as a risk factor for schizophrenia[END_REF]). In addition, systematic reviews of epidemiological studies have indicated that the rate of schizophrenia and related disorders is affected by some environmental factors. For example, some immigrant ethnic groups or children that grow up in urbanized environment have a higher risk of developing psychotic disorders (Cantor-Graae and Selten 2005; Krabbendam and van Os 2005). In addition, studies showed that cannabis use is associated with increased risk for psychosis [START_REF] Moore | Cannabis use and risk of psychotic or affective mental health outcomes: a systematic review[END_REF]).

Smoking and schizophrenia

The prevalence of tobacco smoking among people with SCZ is three to five times higher than in the general population, higher even when compared with many other psychiatric illnesses (de Leon and Diaz 2005). Schizophrenia patients tend to smoke more heavily and have a bigger difficulty quitting. Such patients favor stronger cigarettes and may also extract more nicotine from their cigarettes than other smokers. The etiology for the widespread smoking behavior seen in SCZ is not understood but several possible mechanisms have been suggested. Most of these propose that nicotine serves as a form of self-medication to alleviate the negative symptoms of the disease, ameliorate cognitive deficits, reduce the side effects of antipsychotic treatments or enhance their action. Of particular interest to my thesis, I will present several theories that support or contradict the self-medication concept in schizophrenia.

The self-medication hypothesis: mechanisms mediating schizophrenia-cigarette smoking associations

The self-medication hypothesis has first proposed by Khantzian who noticed that drug-dependent individuals choose a drug related to the symptoms accompanying their mental disorder [START_REF] Khantzian | The self-medication hypothesis of addictive disorders: focus on heroin and cocaine dependence[END_REF]. There are several principles that have been described to compose the self-medication concept: 1) Mental disturbances happen before the initial substance use,

2) Individuals with mental disorders are more susceptible to start the use of psychoactive substances than healthy individuals 3) Drug use leads to symptomatic relief and consequently the patient uses more and becomes an addict, 4) The symptoms of the mental disorder determine the class of drugs that the patient will use [START_REF] Kumari | Nicotine use in schizophrenia: the self medication hypotheses[END_REF][START_REF] Manzella | Smoking in schizophrenic patients: A critique of the self-medication hypothesis[END_REF]. Although Khantzian's theories on the self-medication hypothesis was mainly focused on the abuse of illicit drugs, the concept has been expanded to the use of licit drugs, like alcohol and nicotine.

The most common form of self-medication of nicotine is smoking cigarettes. Nicotine is the main psychotropic agent in tobacco. Schizophrenia patients consume more cigarettes each day and with greater preference for unfiltered cigarettes than the smokers in the general population. It is worth mentioning that schizophrenia patients are more likely to smoke than patients of other psychiatric disorders. However, concerning alcohol and illicit drug use, the rates of schizophrenia patients compared to other psychiatric conditions, like major depression or post-traumatic stress disorder, are similar [START_REF] Brady | Co-occurring mental and substance use disorders: the neurobiological effects of chronic stress[END_REF].

Schizophrenia patients report that smoking reliefs them from schizophrenia symptoms and medication side effects, in addition to the reasons for smoking [START_REF] Barr | Self-reported motivation to smoke in schizophrenia is related to antipsychotic drug treatment[END_REF]. Smoking high-nicotine cigarettes, compared to smoking de-nicotinized cigarettes, was found to reduce negative symptoms without affecting positive symptoms of schizophrenia patients [START_REF] Smith | Effects of cigarette smoking and nicotine nasal spray on psychiatric symptoms and cognition in schizophrenia[END_REF]. Also, there is evidence that smoking contributes to the reduction of antipsychotic side effects. For example, nicotine has been found to restore some of the haloperidol induced disruptions in schizophrenia patients [START_REF] Levin | Nicotine-haloperidol interactions and cognitive performance in schizophrenics[END_REF]). Moreover, it has been reported that a relationship between nicotine use and response to clozapine exists, where patients who smoke seem to gain more therapeutic benefits from clozapine than non-smokers [START_REF] Mcevoy | Smoking and therapeutic response to clozapine in patients with schizophrenia[END_REF].

SCZ patients suffer from multiple sensory processing deficits including auditory sensory processing (P50 gating), pre-pulse inhibition (PPI) abnormalities, eye-tracking deficits, and cognitive deficiencies. According to several studies, smoking has been found to improve all of these deficits in patients [START_REF] Leonard | Smoking, Genetics and Schizophrenia: Evidence for Self Medication[END_REF].

P50 refers to an evoked potential wave, which is elicited approximately 50 msec following exposure to an auditory stimulus. P50 suppression refers to the reduction in P50 amplitude to a second identical stimulus, when that stimulus is presented between msec after the presentation of the first stimulus. When the second stimulus is given, the response is inhibited or gated [START_REF] Adler | Neurophysiological evidence for a defect in neuronal mechanisms involved in sensory gating in schizophrenia[END_REF]. The P50 response is measured by electroencephalography. P50 has been found to be abnormal in patients with schizophrenia, where patients fail to gate out the second response, suggesting that an inhibitory response is taking place [START_REF] Freedman | Neurophysiological evidence for a defect in inhibitory pathways in schizophrenia: comparison of medicated and drug-free patients[END_REF]. The P50 deficit is inherited since 50% of first-degree relatives of schizophrenia patients also have the deficit [START_REF] Waldo | Familial transmission of risk factors in the first-degree relatives of schizophrenic people[END_REF]. It has been shown several times that smoking normalizes the P50 gating deficits, in both schizophrenia patients and their relatives [START_REF] Leonard | Smoking, Genetics and Schizophrenia: Evidence for Self Medication[END_REF]). P50 gating deficits have been linked to the locus of the α7 nicotinic receptor subunit, 15q14 [START_REF] Freedman | Linkage disequilibrium for schizophrenia at the chromosome 15q13-14 locus of the alpha7nicotinic acetylcholine receptor subunit gene (CHRNA7)[END_REF]. The expression of the CHRNA7 gene is low in postmortem hippocampus [START_REF] Freedman | Evidence in postmortem brain tissue for decreased numbers of hippocampal nicotinic receptors in schizophrenia[END_REF], cortex [START_REF] Guan | Decreased protein level of nicotinic receptor alpha7 subunit in the frontal cortex from schizophrenic brain[END_REF]) and in the reticular nucleus of the thalamus of schizophrenia patients [START_REF] Court | Neuronal nicotinic receptors in dementia with Lewy bodies and schizophrenia: alpha-bungarotoxin and nicotine binding in the thalamus[END_REF]. It has been found that treatment of mice with α7 receptor agonists, such as 3-2,4 dimethoxybenzylidene anabaseine (DMXB-A), normalized the sensory gating deficits [START_REF] Stevens | Selective alpha7-nicotinic agonists normalize inhibition of auditory response in DBA mice[END_REF]. The modulatory effect of nicotine on P50 has been hypothesized to result from the nicotine-induced increase in glutamate release following activation of α7 nicotinic receptors [START_REF] Leonard | Nicotinic receptors, smoking and schizophrenia[END_REF]. It has been proposed that a reduction of αbungarotoxin receptor subtypes can result in a reduction of cholinergic activation of inhibitory interneurons, which can lead to decreased inhibition [START_REF] Martin | Alpha-7 nicotinic receptor agonists: potential new candidates for the treatment of schizophrenia[END_REF]). Furthermore, there is evidence that the expression of α4β2* receptors is reduced in the hippocampus of schizophrenia patients, as determined by 3 H-nicotine binding [START_REF] Breese | Abnormal regulation of high affinity nicotinic receptors in subjects with schizophrenia[END_REF], in addition to a relatively recent study that showed a genetic interaction between the α4 and β2 subunits and schizophrenia [START_REF] De Luca | Genetic interaction between alpha4 and beta2 subunits of high affinity nicotinic receptor: analysis in schizophrenia[END_REF]. Also, Radek et al. showed that α4β2* receptors may contribute to the effects of nicotine on sensory gating [START_REF] Radek | Alpha4beta2 nicotinic receptor stimulation contributes to the effects of nicotine in the DBA/2 mouse model of sensory gating[END_REF].

PPI refers to a reliable reduction in the amplitude of the response to a strong sensory stimulus, the pulse, if this is preceded by 30-500 msec with a weak stimulus, the prepulse [START_REF] Graham | Presidential Address, 1974. The more or less startling effects of weak prestimulation[END_REF]. A normal performance is a reduction in the amplitude of the second response, involving an inhibitory mechanism as in the case of P50 gating. Patients with schizophrenia show reduced PPI, the first stimulus does not inhibit the second response (Braff et al. 2001).

The neuronal substrates of PPI involve the hippocampus, amygdala, thalamus, basal ganglia and frontal cortex [START_REF] Kumari | Neural correlates of tactile prepulse inhibition: a functional MRI study in normal and schizophrenic subjects[END_REF]. All these regions are known to be involved in the mechanisms of action of nicotine. It is worth mentioning that PPI deficits appear to be inherited, as for the P50. It is widely accepted that smoking has a positive effect on PPI deficits in schizophrenia. Several studies on animals demonstrated that nicotine enhances PPI, although in some studies the effect was strain dependent. In humans, nicotine intake increases PPI in healthy smokers and has a positive effect in people with schizophrenia (Braff et al. 2001). It has been shown that PPI is impaired by smoking abstinence in schizophrenia and improved by acute smoking reinstatement, mediated by stimulation of nicotinic receptors [START_REF] George | A preliminary study of the effects of cigarette smoking on prepulse inhibition in schizophrenia: involvement of nicotinic receptor mechanisms[END_REF].

Eye-tracking deficits are also inherited and improved by smoking in schizophrenia [START_REF] Holzman | Eye-tracking patterns in schizophrenia[END_REF][START_REF] Holzman | Pursuit eye movement dysfunctions in schizophrenia. Family evidence for specificity[END_REF]. The two eye movement tasks that have received most attention and helped to differentiate healthy individuals from schizophrenia patients are the smooth pursuit (SPEM) and antisaccade tasks. The first measures accuracy of eye movement following a moving target, such as a dot on a computer screen, and the antisaccade paradigm measures an inhibitory response in which the individual is asked to generate an eye movement in the opposite orientation to the target. Several studies have shown that schizophrenia patients show impaired SPEM and generate a high percentage of errors on the antisaccade task. Both types of performance are improved when schizophrenia patients receive nicotine [START_REF] Olincy | Improvement in smooth pursuit eye movements after cigarette smoking in schizophrenic patients[END_REF][START_REF] Olincy | Differential effects of cigarette smoking on performance of a smooth pursuit and a saccadic eye movement task in schizophrenia[END_REF][START_REF] Avila | Effects of nicotine on leading saccades during smooth pursuit eye movements in smokers and nonsmokers with schizophrenia[END_REF]. Also, Tanabe et al. showed that the eye-tracking performance is improved through cholinergic stimulation of the hippocampus and cingulate gyrus [START_REF] Tanabe | Effects of nicotine on hippocampal and cingulate activity during smooth pursuit eye movement in schizophrenia[END_REF].

Studies in experimental animals, but also in humans, have shown that nicotine improves cognitive functions such as working memory and attention, which are impaired in schizophrenia patients [START_REF] Leonard | Smoking, Genetics and Schizophrenia: Evidence for Self Medication[END_REF]). Nicotine, administered via cigarette smoking, skin patches or subcutaneous injections also improves cognitive performance in healthy individuals [START_REF] Kumari | Nicotine use in schizophrenia: the self medication hypotheses[END_REF]. However, overall, existing evidence shows that the beneficial effect of smoking on sustained attention and working memory functions in schizophrenia is greater than seen in healthy populations. Selective nicotinic agonists, such as dimethylaminoethanol, epibatidine, isonicotone, (E)-metanicotine and lobeline have also been shown to improve performance in animal models [START_REF] Levin | Nicotine-haloperidol interactions and cognitive performance in schizophrenics[END_REF](Levin et al. , 2006;;[START_REF] Kumari | Nicotine use in schizophrenia: the self medication hypotheses[END_REF]. Clinical trials relating to the α7 nAChR have advanced enormously, and one compound is in phase 3 for schizophrenia and AD (Preskorn 2014).

Does smoking cause psychosis?

The coincidence of the onset of the smoking with the onset of schizophrenia or psychosis has caused of a lot of debate in research. Recently, a systematic review by Gurillo and colleagues brought together evidence that cigarette smoking might be a cause of psychosis [START_REF] Gurillo | Does tobacco use cause psychosis? Systematic review and meta-analysis[END_REF]. The authors intended to test four hypotheses: 1) an excess of tobacco use is already present in individuals presenting with their first episode of psychosis, 2) the daily tobacco use is associated with an increased risk for psychosis, 3) daily tobacco use is associated with an earlier age at onset of psychotic illness and 4) an earlier age at initiation of smoking is associated with an increased risk of psychotic disorder [START_REF] Gurillo | Does tobacco use cause psychosis? Systematic review and meta-analysis[END_REF]). The authors selected observational studies in which rates of smoking were reported in individuals with psychotic disorders, compared to controls and they calculated the weighed mean difference for age at onset of psychosis and age at initiation of smoking. Interestingly, the prevalence of smoking in patients with their first episode of psychosis was 57% (95% CI [52][53][54][55][56][57][58][START_REF] Lin | Axonal a7 nicotinic ACh receptors modulate presynaptic NMDA receptor expression and structural plasticity of glutamatergic presynaptic boutons[END_REF][START_REF] Zappettini | Nicotinic a7 receptor activation selectively potentiates the function of NMDA receptors in glutamatergic terminals of the nucleus accumbens[END_REF][START_REF] Cheng | Presynaptic a7 nicotinic acetylcholine receptors enhance hippocampal mossy fiber glutamatergic transmission via PKA activation[END_REF][START_REF] Welsby | Nicotinic receptor-mediated enhancement of long-term potentiation involves activation of metabotropic glutamate receptors and ryanodine-sensitive calcium stores in the dentate gyrus[END_REF]. From their analysis of case-control studies, the overall risk of smoking in individuals having their first episode of psychosis was three times higher than that for non-smokers. However, when they restricted their analysis to three studies in which daily smoking was specified, the association disappeared. Also, daily use of tobacco was associated with an earlier onset of psychosis compared with non-smokers. These findings differ from those of a previous meta-analysis that took place in 2012 and the risk of smoking in first-episode psychosis was much higher, but a different set of studies were included in that meta-analysis [START_REF] Myles | Tobacco use before, at, and after first-episode psychosis: a systematic meta-analysis[END_REF]). This study estimated a pooled interval of 5-8 years between age at initiation of daily tobacco use and onset of psychosis. According to Gurillo et al., people with psychosis began smoking 0.44 years earlier than controls, although this difference was not significant. The authors supported their conclusions based on the effect that nicotine has on the DAergic system, since the hypothesis of excess striatal DA is a leading pathogenic theory of schizophrenia [START_REF] Brody | Smoking-induced ventral striatum dopamine release[END_REF][START_REF] Howes | The dopamine hypothesis of schizophrenia: version III--the final common pathway[END_REF].

It is true that the data on the age of onset of smoking do not really help establishing causality, since smoking could advance age at onset without necessarily causing psychosis [START_REF] Alderson | Does cigarette smoking cause psychosis? The lancet Psychiatry[END_REF]. Large, longitudinal, prospective studies are needed to investigate the relationship between smoking and psychosis in order to be able to state that cigarette smoking has a causal role in the development of psychosis. It is of crucial importance to take into consideration that the genes for psychosis overlap with those for cigarette smoking. As mentioned before in this thesis, the Schizophrenia Working Group of the Psychiatric Genomics Consortium described 108 separate genetic loci associated with an increased risk for schizophrenia (Ripke et al. 2014). And of particular interest to my thesis, one of these loci is located in the cluster of genes, CHRNA5/A3/B4, on chromosome 15, that is associated with both early onset of smoking and heavy smoking [START_REF] Hartz | Increased genetic vulnerability to smoking at CHRNA5 in early-onset smokers[END_REF][START_REF] Alderson | Does cigarette smoking cause psychosis? The lancet Psychiatry[END_REF][START_REF] Gage | Rethinking the association between smoking and schizophrenia[END_REF].

1.9 Alzheimer's Disease 8).

In the beginning Alzheimer's observations excited little interest despite an enthusiastic response from Kraepelin, who promptly included "Alzheimer's disease" in his text "Psychiatrie" in 1910 (Weber 1997). Alzheimer published three further cases in 1909 and behavioral issues [START_REF] Burns | Alzheimer's disease[END_REF]. As a person's condition declines, they often withdraw from family and society. The daily activities of AD patients are impaired. Affected patients and their family members may not distinguish AD from the generic dementia clinical syndrome. To AD specialists, able to identify and categorize different patterns of cognitive decline, AD is less a diagnosis of exclusion and more a recognizable procedure [START_REF] Swerdlow | Pathogenesis of Alzheimer's disease[END_REF]. Although no cure for AD is yet available, symptomatic and behavioral treatments may ease the symptoms. In the late stages of the disease the patients are not able to interact with their environment and continuous care is necessary. Physical, emotional and economic pressures can cause great stress to families and caregivers, and support is required from the health, social, financial and legal systems.

Neuropathology

Brain specimens obtained from most cases of AD show a modest degree of cerebral cortical atrophy but not exclusively involving the frontotemporal association cortex (Perl 2010). In particular, this atrophy tends to spare primary motor, sensory, and visual areas.

However, among elderly subjects, there is considerable overlap between brain weight as well as measures of cerebral cortical thickness between age-matched individuals with normal cognitive function and those with AD. In addition, there is an associated loss of brain tissue that generally leads to a symmetrical dilation of the lateral ventricles. By examining the lateral ventricles of advanced elderly individuals, it is not easy to distinguish with certainty age-matched normal brains from patients with AD [START_REF] Terry | Interrelations among the lesions of normal and abnormal aging of the brain[END_REF]). However, significant atrophy of the hippocampus, with an associated selective dilation of the adjacent temporal horn of the lateral ventricle, represents a reasonably reliable element upon dissection of the brain, suggesting that there will be microscopic evidence of AD.

The two primary cardinal lesions associated with AD are the senile plaques and the neurofibrillary lesions (Figure 9). Senile plaques are complex structures that are defined by the presence of a central core accumulation of a 4-kD protein with a beta-pleated sheet configuration called βA4 (Kang et al. 1987).The predominant beta-pleated sheet configuration of this protein confers its ability to bind the planar dye Congo red and produce birefringence when illuminated by polarized light. Thus it corresponds to the physical definition of an amyloid. Plaques are spherical extracellular lesions of 10-200 µm in diameter. Several subtypes have been identified, among those the best known for neuropathological observations being the diffuse and neuritic plaques. Diffuse plaques have been found in a large number of AD brains and represent the earliest identifiable lesion [START_REF] Mann | Saccharides of senile plaques and neurofibrillary tangles in Alzheimer's disease[END_REF].

The typical size of this lesion spans from 10 to 50 µm in diameter. A central core of 6-10 nanometre (nm) fibrillar Aß (Amyloid Beta) composes neuritic plaques [START_REF] Kidd | Alzheimer's disease-An electron Microscopical study[END_REF]. Several studies have found positive tau immunoreactivity, degenerating mitochondria, synaptic vesicles, Aß, and its precursor amyloid precursor protein (APP), to co-localize at neuritic plaques [START_REF] Luse | The ultrastructure of senile plaques[END_REF][START_REF] Perry | Amyloid precursor protein in senile plaques of Alzheimer disease[END_REF][START_REF] Schmidt | Comparative epitope analysis of neuronal cytoskeletal proteins in Alzheimer's disease senile plaque neurites and neuropil threads[END_REF]. The second histopathological feature of AD are the neurofibrillary lesions that are found in cell bodies and apical dendrites as neurofibrillary tangles (NFT), in distal dendrites as neuropil threads, and in the abnormal neurites that are associated with some β-amyloid plaques (neuritic plaques) [START_REF] Götz | Amyloid-induced neurofibrillary tangle formation in Alzheimer's disease: insight from transgenic mouse and tissue-culture models[END_REF]). These structures occur in the regions of the brain responsible for the various cognitive domains that are compromised during the course of AD.

Their major proteinaceous component is abnormal filaments, which are termed either straight or paired helical filaments. The core protein of these filaments is tau, a microtubuleassociated protein [START_REF] Goedert | Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau[END_REF]). In the course of the disease, tau becomes abnormally phosphorylated and it adopts an altered conformation. NFTs are diffusely found in AD, however these lesions are also found in other neurological pathologies such as Parkinson's disease and Down's syndrome, but are also observed as part of the normal aging process.

Braak and Braak in 1991 established a histopathological staging system that is commonly used to determine the severity of AD pathology. This scale describes the progression of ADtype cytoskeletal changes (NFTs and dystrophic neurites), in unrelated humans of increasing age (Braak and Braak 1991). At stage I-II, the neurofibrillary degeneration, found to correlate more with the cognitive deficit compared to plaque deposition, proceeds step-by-step, starting from the transentorhinal area, the entorhinal cortex and the temporal neocortex. A characteristic of stage III-IV is a severe involvement of the trans-entorhinal region and the limbic area. At this stage, even though NFT degeneration is quite prominent, only half of the patients display cognitive impairment. At stage V-VI, there is clearly a cognitive deficit, and at the anatomical level isocortical regions are engaged by the pathology [START_REF] Braak | Vulnerability of cortical neurons to Alzheimer's and Parkinson's diseases[END_REF].

Based [START_REF] Blennow | Alzheimer's disease[END_REF]. The most common form of AD is the sporadic, representing approximately 95% of AD cases. Little is known about the mechanisms involved in the pathogenesis of sporadic AD. First-degree relatives of AD patients have a higher lifetime risk of developing AD than the general population [START_REF] Qiu | Epidemiology of Alzheimer's disease: occurrence, determinants, and strategies toward intervention[END_REF].

Several mutations leading to fAD were identified in different chromosomal loci. There are more than 20 missense mutations in APP known to underlie AD, all of them being clustered in the Aß sequence (ß or γ secretase cleavage sites, or in the middle), with currently no mutations being documented in other regions of the precursor. These mutations are responsible for only 0.01% of all cases. Other genes implicated in fAD are presenilin 1 (PS1) and presenilin 2 (PS2), which are located on chromosome 14 and 1, respectively, and are responsible for aggressive forms of the disease, characterized by an early onset [START_REF] Rogaev | Familial Alzheimer's disease in kindreds with missense mutations in a gene on chromosome 1 related to the Alzheimer's disease type 3 gene[END_REF]. Missense mutations in PS1 and PS2 act on the cleavage pathway of APP, resulting in an increased production of the more toxic Aβ42 over the less amyloidogenic Aβ40. Patients carrying fAD linked PS1 and PS2 mutations have elevated Aβ42 plasma concentrations. It was suggested that the increased extracellular Aβ concentration in these patients could promote Aβ extracellular aggregation into plaques [START_REF] Scheuner | Secreted amyloid betaprotein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease[END_REF].

A major risk factor for the development of AD is the apolipoprotein E (APOE) ε4

allele. The APOE gene comprises in three variants that encode proteins (designated APOE2, APOE3, and APOE4) that differ at two amino acids. The APOE E4 allele, which is present in 10%-20% of various populations increases the risk for AD threefold in individuals carrying one copy and 15-fold for homozygous individuals (Singh et al. 2006). The APOE ε4 allele is the only established genetic factor for both early and late onset AD. With increasing number of the APOE ε4 allele, the risk of AD increases and the age of AD onset decreases, in a dosedependent manner. Many other candidate genes such as angiotensin-I converting enzyme, cholesterol 24-hydroxylase and insulin degrading enzyme genes have been studied but the findings were inconsistent [START_REF] Qiu | Epidemiology of Alzheimer's disease: occurrence, determinants, and strategies toward intervention[END_REF].

Other risk factors for the development of AD include vascular risk factors such as tobacco use, alcohol consumption, overweight and obesity, high total cholesterol, high blood pressure and diabetes. Interestingly, psychosocial factors like high educational attainments and high levels of social engagement may reduce the risk of AD and dementia [START_REF] Qiu | Epidemiology of Alzheimer's disease: occurrence, determinants, and strategies toward intervention[END_REF]). In addition, a recent meta-analysis of 74046 individuals identified eleven new susceptibility loci for AD (Lambert et al. 2013).

1.9.5 Amyloid Precursor Protein

1.9.5a Structure and function

The amyloid precursor protein (APP) is one member of a family of related proteins that includes the amyloid precursor-like proteins (APLP1 and APLP2) in mammals and the amyloid precursor protein-like (APPL) in Drosophila. All are single-pass transmembrane proteins with large extracellular domains, and all are processed in a manner similar to APP.

Only APP generates an amyloidogenic fragment owing to sequence divergence at the internal Aβ site. Alternate splicing of the APP transcript generates 8 isoforms, of which 3 are most common: the 695 amino acid form, which is expressed predominantly in the CNS, and the 751 and 770 amino acid forms, which are more ubiquitously expressed [START_REF] Bayer | It all sticks together--the APP-related family of proteins and Alzheimer's disease[END_REF].

The actual physiological role of APP remains unknown. APP is highly expressed during synaptogenesis [START_REF] Moya | The amyloid precursor protein is developmentally regulated and correlated with synaptogenesis[END_REF]) and undergoes rapid anterograde axonal transport to synaptic terminals [START_REF] Lyckman | Post-translational processing and turnover kinetics of presynaptically targeted amyloid precursor superfamily proteins in the central nervous system[END_REF]. APP overexpression has shown a positive effect on cell health and growth. In transiently transfected cell lines, APP modulates cell growth, motility, neurite outgrowth, and cell survival, functions that can be reproduced by the soluble ectodomain, which is released by cleavage of APP (O'Brien and Wong 2011). Furthermore,

Young-Pearse and colleagues found neuronal migration abnormalities in embryonic rodents injected with APP RNAi [START_REF] Bai | A critical function for beta-amyloid precursor protein in neuronal migration revealed by in utero RNA interference[END_REF]). In adult animals, intracerebral injections of the APP ectodomain improved cognitive function and synaptic density [START_REF] Meziane | Memoryenhancing effects of secreted forms of the beta-amyloid precursor protein in normal and amnestic mice[END_REF]. In addition to a physiological role for APP, the Aβ peptide itself plays an important role in synaptic physiology, regulating synaptic scaling and synaptic vesicle release [START_REF] Abramov | Amyloid-beta as a positive endogenous regulator of release probability at hippocampal synapses[END_REF] Deletion of APP in mice caused only a minor phenotype, suggesting that its loss does not cause any significant deleterious effect in adults. Another study went further and generated triple KO mice for APP, APLP1 and APLP2 and these mice had a scattered cortical migration abnormality [START_REF] Herms | Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members[END_REF]). In addition, presynaptic and postsynaptic markers at the neuromuscular junction and excessive nerve terminal sprouting wasobserved in double KO mice lacking the APP and APLP2 genes [START_REF] Wang | Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2[END_REF]. It is clear that APP is important for neuronal and synaptic function, but further studies are required to dissect the role of these proteins and the pathological mechanism, linking APP to AD.

1.9.5b Amyloid Precursor Protein Processing

APP is synthesized in the endoplasmic reticulum (ER) and then transported through the Golgi apparatus to the trans-Golgi-network (TGN) where the highest concentration of APP is found in neurons at steady state. Aβ is generated in the ER and Golgi/TGN. The pathway that leads to the generation of the Aβ peptide is called amyloidogenic, whereas the pathway that does not lead to its production is called non-amyloidogenic. From the TGN, APP can be transported in TGN-derived secretory vesicles to the cell surface where it is either cleaved by α-secretase to produce a soluble molecule, sAPPα (Figure 10), or re-internalized via an endosomal/lysosomal degradation pathway [START_REF] Zhang | APP processing in Alzheimer's disease[END_REF]. It has been suggested

that Aβ can also be generated in the endosomal/lysosomal system [START_REF] Haass | beta-Amyloid peptide and a 3-kDa fragment are derived by distinct cellular mechanisms[END_REF]). While

Aβ is neurotoxic, studies suggest that sAPPα is neuroprotective [START_REF] Mattson | Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives[END_REF]. It has been found that α-secretase is a zinc metalloproteinase [START_REF] Roberts | Nonamyloidogenic cleavage of the beta-amyloid precursor protein by an integral membrane metalloendopeptidase[END_REF]. Several members of the ADAM (A disintegrin and metalloproteinase) family possess α-secretase-like activity and three of these have been suggested as the α-secretase: ADAM9, ADAM10, and ADAM17.

Like APP, they are also type-I transmembrane proteins. In contrast to Aβ, sAPPα has an important role in neuronal plasticity, survival and is protective against excitotoxicity. sAPPα also regulates neural stem cell proliferation and is important for early CNS development [START_REF] Zhang | APP processing in Alzheimer's disease[END_REF].

The first step in Aβ generation is cleavage of APP by the β-secretase. The amyloidogenic pathway takes place primarily in the endosomal compartment after internalization of APP from the cell surface. β-secretase serves to release the sAPPβ residue.

The remaining β-CTF residue of 99 amino acids is cleaved by γ-secretase to generate Aβ and AICD. When the endosome joins the cell surface the peptides are released into the extracellular space. β-secretase activity was identified in BACE1 (Beta-site APP Cleaving Enzyme) protease [START_REF] Cai | BACE1 is the major beta-secretase for generation of Abeta peptides by neurons[END_REF]. It is important to underline that a pathogenic mutation in APP called "Swedish" enhances the affinity of BACE1 for the APP, resulting in an increase in Aβ levels in brains from patients carrying the mutation. In the amyloidogenic pathway the APP can be cleaved at different sites by both βand γsecretase. This generates a vast variety of Aβ peptides, from N-truncated forms to the most common Aβ 1-40 and Aβ 1-42 , where the latter has two hydrophobic amino acids more than the first at its C-terminal. Both forms are normally synthesized. The Aβ 1-42 is believed to be more toxic than the Aβ 1-40 because it is more hydrophobic and more prone to aggregate. Interestingly, in AD subjects higher levels of the more toxic Aβ 1-42 were found (Benilova et al. 2012). The Aβ peptide, once produced and released into the extracellular space, eventually starts to aggregate in small oligomeric structures, fibrils and plaques. Over the last years, the "toxic Aβ oligomer" hypothesis has attracted attention as a way of resolving the lack of correlation between deposited Aβ in amyloid plaques, in terms of amount, location and cognitive impairment, and neurodegeneration. Aβ oligomers are associated with AD hallmarks, like inducing abnormal tau phosphorylation and synapse loss. Soluble and highly toxic forms of Aβ such as oligomers and protofibrils may be more directly linked to cellular pathology, however the balance between monomeric Aβ, oligomers and insoluble Aβ fibrils is poorly understood (Benilova et al. 2012). 1.9.6 AD hypotheses

1.9.6a The amyloid cascade hypothesis and related treatments

The amyloid cascade hypothesis postulates that the development of AD is caused by the abnormal accumulation of Aβ peptide in various areas of the brain. The amyloid cascade hypothesis is probably the most prevalent theory on the pathogenesis of AD. The idea that amyloid deposition is the driving force in both familial and sporadic AD was proposed in the early 1990s [START_REF] Hardy | Alzheimer's disease: the amyloid cascade hypothesis[END_REF]. Based on this hypothesis, an imbalance of Aβ production and clearance leads to the Aβ accumulation in the brain. The accumulation of Aβ triggers microglia activation, cytokine release, reactive astrocytosis, a multi-protein inflammatory response, synaptic changes, tau phosphorylation and impairment of synaptic transmission.

However, the fact that Aβ peptide accumulation does not correlate well with neuronal loss and cognitive dysfunction raised significant concerns regarding this hypothesis [START_REF] Herrup | The case for rejecting the amyloid cascade hypothesis[END_REF]. Interestingly, humans can present with few, if any, clinical symptoms of dementia and yet carry substantial amyloid burdens in their brains. Having a detectable amyloid burden increases the risk that a healthy individual will progress to AD about fourfold, however, the question still remains of how long amyloid deposits can persist without cognitive dysfunction [START_REF] Cairns | Absence of Pittsburgh compound B detection of cerebral amyloid beta in a patient with clinical, cognitive, and cerebrospinal fluid markers of Alzheimer disease: a case report[END_REF]). Moreover, a big variety of transgenic AD mouse models have been developed and these mice show impaired cognitive symptoms, produce substantial deposits of amyloid in their brains, nonetheless no NFTs appear and there is little or no neurodegeneration. Surprisingly, mice can live three-quarters of their lives with extensive amyloid plaque accumulation. Thus, it has been suggested that there is an intermediate stage between amyloidosis and neurodegeneration [START_REF] Herrup | The case for rejecting the amyloid cascade hypothesis[END_REF]. On the other hand, active and passive immunization against the Aβ peptide has been shown to be effective for the clearing of the plaques from the mouse brain and the mice had improved performance in behavioral tasks, approaching the levels of WT animals [START_REF] Schenk | Immunization with amyloid-beta attenuates Alzheimerdisease-like pathology in the PDAPP mouse[END_REF][START_REF] Cramer | ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models[END_REF].

Analogous studies were initiated in humans with early sporadic AD. The plaque burden of these patients was greatly reduced, however, dementia has not improved and continued to worsen [START_REF] Orgogozo | Subacute meningoencephalitis in a subset of patients with AD after Abeta42 immunization[END_REF][START_REF] Holmes | Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial[END_REF][START_REF] Serrano-Pozo | Beneficial effect of human anti-amyloid-beta active immunization on neurite morphology and tau pathology[END_REF]. Recent reports of human trials using anti-amyloid antibody therapy also failed even after 80 weeks of therapy, but plaque burden was reduced [START_REF] Doody | Phase 3 trials of solanezumab and bapineuzumab for Alzheimer's disease[END_REF][START_REF] Salloway | Phase 3 trials of solanezumab and bapineuzumab for Alzheimer's disease[END_REF]. The success of the human trials in reducing amyloid load forces us to accept that an individual with dementia, but no plaques, might very well have AD. As it has been stated, the implication is that as there can be plaques without AD, there can also be AD without plaques [START_REF] Herrup | The case for rejecting the amyloid cascade hypothesis[END_REF].

With the amyloid cascade hypothesis, Aβ remains as a target for therapies that concentrate on the cleavage pathway and ways to prevent Aβ aggregation. Two drugs have been developed, rosiglitazone and proglitazone, both are for treating type 2 diabetes and are in clinical trials since they stimulate the nuclear peroxisome proliferator-activated receptor γ (PPARγ) thereby inhibiting β-secretase [START_REF] Landreth | PPARγ agonists as therapeutics for the treatment of Alzheimer's disease[END_REF]. Reduction in the amount of soluble Aß occurred with the PPARγ agonist Bexarotene which decreased the plaque burden and restored memory deficits in AD mouse models [START_REF] Cramer | ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models[END_REF]. Unfortunately these results were irreproducible in four independent studies ranging in results from not showing a reduction in soluble Aβ or effect on plaques, to no amelioration of memory loss and also an absence of drug efficacy [START_REF] Fitz | Comment on "ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models[END_REF][START_REF] Price | Comment on "ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models[END_REF][START_REF] Tesseur | Comment on "ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models[END_REF][START_REF] Veeraraghavalu | Comment on "ApoE-directed therapeutics rapidly clear β-amyloid and reverse deficits in AD mouse models[END_REF]).

1.9.6b The cholinergic hypothesis and related treatments

The cholinergic hypothesis in AD is the oldest hypothesis and is based on a mechanism of cholinergic dysfunction. The cholinergic hypothesis for AD was proposed in the early 1980's, and since then many experiments were performed to investigate its validity, and the actual role of cholinergic pathways in the AD pathogenic process.

Systematic biochemical analysis of brains from AD patients showed reduction in activity of choline acetyltranseferase (ChAT) and acetlylcholinesterase (AChE) in cerebral cortex and other areas below the normal levels observed in normal brains, along with loss of cholinergic neurons in the nucleus basalis of Meynert and decreased ACh release. More precisely, ChAT activity was reduced by 0-60% in the neocortex and up to 97% in the hippocampus. The cholinergic nuclei in the basal forebrain are also affected, with a 65% reduction in the diagonal band of Broca and 55% in the medial septal nucleus [START_REF] Henke | Cholinergic enzymes in neocortex, hippocampus and basal forebrain of non-neurological and senile dementia of Alzheimer-type patients[END_REF]. Modifications comparable to those observed for ChAT were also found for AChE. These modifications are common among AD patients, but substantial differences are found when comparing early-onset against late-onset AD. In late-onset AD, the major decrease in ChAT activity is found in hippocampus (Bird et al., 1983).

On the other hand, there are cases where a negative correlation was observed between

ChAT alteration and plaque deposition in frontotemporal regions of the cortex [START_REF] Perry | Neurochemical activities in human temporal lobe related to aging and Alzheimer-type changes[END_REF], and in other studies the correlation was linked to the amount of tau tangles, which in general are more related to the severity of the dementia compared to amyloid deposition [START_REF] Wilcock | Alzheimer's disease. Correlation of cortical choline acetyltransferase activity with the severity of dementia and histological abnormalities[END_REF]. It is not clear whether reduced cholinergic activity is a specific feature of AD or not, since opposing results are found in the literature. Some studies indicate that there is no decrease in ChAT activity in cerebrovascular dementia or in aged subjects [START_REF] Wilcock | Alzheimer's disease. Correlation of cortical choline acetyltransferase activity with the severity of dementia and histological abnormalities[END_REF]. Others show a decrease in ChAT activity related to diseases and ageing [START_REF] Perry | Cholinergic correlates of cognitive impairment in Parkinson's disease: comparisons with Alzheimer's disease[END_REF][START_REF] Murdoch | Cortical cholinergic dysfunction after human head injury[END_REF][START_REF] Smith | Age-associated neuronal atrophy occurs in the primate brain and is reversible by growth factor gene therapy[END_REF].

Over the last two decades, the development of different strategies including cholinesterase inhibitors, choline precursors, presynaptic and postsynaptic cholinergic stimulation with muscarinic and nicotinic agonists have been investigated. Nonetheless, clinical studies showed that the use of precursors for the presynaptic releasing agent and muscarinic agonists are not effective and provoke severe side effects. Tacrine, donepezil, rivastigmine and galantamine are four FDA approved AChE inhibitors for the treatment of mild to moderate AD. Tacrine was widely used, but side effects like hepatotoxicity led to the development of second-generation molecules. Clinical trials based on long-term administration of AChE inhibitors to patients with mild cognitive impairment failed to reduce the risk or delay AD onset. AChE inhibitors, together with memantine, an NMDA receptor antagonist, are the only drugs in use for the treatment of AD symptoms [START_REF] Terry | The cholinergic hypothesis of age and Alzheimer's diseaserelated cognitive deficits: recent challenges and their implications for novel drug development[END_REF]. These drugs are widely used even though they are not always effective and they do not stop the progression of the disease. Moreover, patients develop tolerance for these drugs, and an explanation that has been given is a compensatory upregulation of AChE at the synapse [START_REF] Darreh-Shori | Long-lasting acetylcholinesterase splice variations in anticholinesterase-treated Alzheimer's disease patients[END_REF]).

1.9.6c Other hypotheses

Several other theories have been examined and tried to explain the etiology of AD.

Among them is the hypothesis of oxidative stress and oxidative imbalance. It is well recognized that oxidative imbalance and stress can play a crucial role in the pathogenesis of neuronal degeneration and death. A pathological mechanism called the "two-hit" hypothesis was proposed. It states that although either oxidative stress or abnormalities in mitotic signaling can independently serve as initiators, both processes are necessary to propagate disease pathogenesis [START_REF] Zhu | Alzheimer's disease: the two-hit hypothesis[END_REF]). Other theories include the conception of mild cognitive impairment, the nitric oxide theory, the chronic stress theory, the hypothesis of microtubule instability and the theory of glutamate neurotoxicity [START_REF][END_REF]).

AD mouse models

Numerous animal models have been developed to mimic the human context of AD for understanding the pathogenic mechanisms that lead to the development of the disorder and in order to develop efficient therapeutic strategies. The main purpose of these animal models is to reproduce the neuropathological findings of AD followed by the correlation of cognitive dysfunction. However, a major concern is whether the neuropathological agents have similar biological consequences in humans and in animal models. Both knock-in and knock-out models were created and extensively studied over the past years.

Transgenic technology provided a unique opportunity to reproduce the cause of familial AD by expressing the mutant human APP. Mice have extensively been used as transgenic models and facilitated our understanding of the molecular mechanisms associated with Aβ-production, deposition and clearance and the effects of Aβ on neuronal network and synapses that play important roles in cognitive function [START_REF] Laurijssens | Animal models of Alzheimer's disease and drug development[END_REF]. In general, mice harboring APP pathogenic mutations display extracellular Aß accumulation and neuritic plaques. Many different mutations were used to generate these mice, such as the Indiana mutation (V717F) [START_REF] Games | Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein[END_REF], the Swedish double mutation (K670N/M671L) [START_REF] Sturchler-Pierrat | Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology[END_REF], Dutch mutation (E693Q) [START_REF] Herzig | Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis[END_REF]) and London mutation (V717I) [START_REF] Moechars | Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain[END_REF]). However, these transgenic mouse models failed to develop NFTs, an important histopathological hallmark of AD resulting from intraneuronal aggregation of hyperphosphorylated tau protein. In order to obtain NFTs, a further genetic modification was required. In addition to mutated hAPP, mutated human tau is also required [START_REF] Lewis | Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP[END_REF]. Oddo and colleagues presented for the first time a triple transgenic mouse model where both plaques and NFTs were found in AD-relevant brain regions (Oddo et al. 2003). These mice carried the mutated PS1 (M146V), the APP swe and the tau P301L. They also developed extracellular amyloid beta deposits before the formation of NFTs and exhibited impaired synaptic plasticity including long-term potentiation, the key correlate for cognitive function. Inflammation, oxidative stress and mitochondrial dysfunction have also been shown in AD mouse models [START_REF] Du | Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease[END_REF][START_REF] Lee | Inflammation and Alzheimer's disease[END_REF]. Despite these observation, the hallmark of AD is still lacking in most of these models: progressive neuronal loss in the neocortex and hippocampus [START_REF] Laurijssens | Animal models of Alzheimer's disease and drug development[END_REF]. Also, sporadic AD, the most prominent form, is not represented since these mouse models were derived from genes in the familial form of AD, which constitutes less than 1% of all AD cases. In the field of AD, there is no single mouse model that can faithfully reproduce the pathogenesis and disease progression of sporadic/late-onset AD.

In addition to transgenic mouse models for the insertion of different human mutations, several genetically modified mice, based on gene ablation, have been developed. Although these mouse models do not reproduce the human pathological phenotype, they are useful tools for the elucidation of molecular mechanisms that are involved in AD pathology. PS1 KO mice were produced in the late 1990s and the homozygous KO was lethal with developmental defects in both the CNS and skeletal systems [START_REF] Shen | Skeletal and CNS defects in Presenilin-1-deficient mice[END_REF]). Hence, the production of these mice were the first indications that PS, and the γ-secretase complex, had crucial roles apart from the production of Aβ, and that their inhibition may lead to undesirable effects.

BACE1 is the other key enzyme of Aβ production that has β-secretase activity. The BACE1 KO mouse appears healthy, without any gross anatomical brain modification. In these mice the production of Aβ peptide is abolished, confirming the central role of BACE1 enzyme in the amyloidogenic pathway [START_REF] Luo | Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished betaamyloid generation[END_REF]. Furthers studies showed that BACE1 is important for axon myelination of central and peripheral nerves. Null mutant mice exhibit hypomyelinated axons and at the behavioral level they display increased pain sensitivity and reduced grip strength [START_REF] Willem | Control of peripheral nerve myelination by the betasecretase BACE1[END_REF]).

Moreover, it has been shown that crossing APP transgenic mice onto a tau KO background prevents all cognitive deficits associated with the presence of APP and Aβ [START_REF] Roberson | Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model[END_REF], including reduced spontaneous seizures (Palop and Mucke 2009) and long-term potentiation [START_REF] Shipton | Tau protein is required for amyloid {beta}-induced impairment of hippocampal long-term potentiation[END_REF]. The absence of tau has no effect on the development of Aβ pathologies, including plaque load. Hence, endogenous murine tau is necessary for APP and Aβ to mediate its effects on cognition, LTP and hyperexcitibility of neurons (LaFerla and Green 2012).

Overall, animal models continue to have a critical role in AD research and help to improve our knowledge regarding AD development and progression. However, better models that allow us to investigate the early AD stage are required and this is an important challenge.

Nicotinic receptors and AD

As discussed before in the Introduction, a significant reduction in nicotine and ACh binding sites in cerebral cortex of AD patients has been shown using binding methods of

[3H]-nicotine and [3H]-ACh [START_REF] Perry | Neurochemical activities in human temporal lobe related to aging and Alzheimer-type changes[END_REF][START_REF] Gotti | Selective nicotinic acetylcholine receptor subunit deficits identified in Alzheimer's disease, Parkinson's disease and dementia with Lewy bodies by immunoprecipitation[END_REF]. Moreover, the activity of the ChAT enzyme, and therefore the synthesis of ACh, is decreased in AD brains.

All these led to the formation of the cholinergic hypothesis that has been discussed in this thesis, according to which the reduction in cholinergic innervation is responsible for the cognitive decline observed in AD patients.

In this chapter, we will focus on the role of nAChRs in AD by their interaction with the Aβ peptide, the functional consequences of this interaction and the pharmacological approaches that have been used.

The interaction between nAChRs and the Aβ peptide

The most widely demonstrated interaction of nAChRs and the Aβ peptide is with α7 nAChRs. The first demonstration was by Wang and colleagues who showed co-localization of the α7 subunit with Aβ 1-42 in amyloid plaques of brain slices obtained from patients with sporadic AD (Wang, Lee, D'Andrea, et al. 2000). Immunoprecipitation experiments and Western Blot analysis further showed that this interaction is specific for the α7 nAChRs and the sequence of Aβ that is responsible for this interaction corresponds to the amino acid residues 12-28 of the Aβ sequence (Wang, Lee, Davis, et al. 2000). More specifically, by using [3H]-methyllycaconitine and [3H]-cytisine, two selective nicotinic receptor ligands, it was revealed that α7 nAChRs bind the less toxic petide Aβ 1-40 but with lower affinity compared to the Aβ 1-42 (Wang, Lee, D'Andrea, et al. 2000). Incubation of α7 nAChRs with Aβ and αBgt suggested that α7 and Aβ compete for the same ligand-binding domain because αBgt was able to decrease the amount of Aβ that bound to the α7 nAChRs (Wang, Lee, Davis, et al. 2000). Molecular docking studies performed on the AChBP, a homologue of α7 nAChRs, and Aβ 1-42 showed that monomeric Aβ has a high affinity for the ligand binding site of the receptor and that Aβ interacts with loop C, an important site in the agonist binding site pocket [START_REF] Espinoza-Fonseca | Molecular docking of four beta-amyloid1-42 fragments on the alpha7 nicotinic receptor: delineating the binding site of the Abeta peptides[END_REF]. Moreover, the addition of mutations in the receptor's sequence confirmed that the loop C is important for binding, as well as with key amino acids in loop A. These critical residues are Tyrosine-188 and Tyrosine-195 in loop C, and Tyrosine-93 in loop A [START_REF] Tong | Role of key aromatic residues in the ligand-binding domain of alpha7 nicotinic receptors in the agonist action of betaamyloid[END_REF]. The immunoprecipitation of α7 with the Aβ peptide was further demonstrated in vivo, using the APPswe/PS1ΔE9 AD mouse model [START_REF] Søderman | The nicotinic alpha7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-beta1-42[END_REF].

Even though research has mainly focused on the study of the interaction between α7 nAChR and Aβ and its functional consequences, it has been demonstrated that also non-α7

nAChRs bind Aβ, such as β2* nAChRs. Several electrophysiological studies support this interaction (Sudweeks and Yakel 2000). Blockade of β2* nAChRs has been observed, in hippocampal interneurons and oocytes, and this block could be prevented by dFBr (desformylflustrabromine), which is a PAM specific for the β2 nicotinic subunit (Pandya and Yakel 2011). Concerning the affinity of the interaction, Wang et al. showed that it occurs with a lower affinity compared to α7 homomeric receptor, at least 100 -5000 times lower (Wang, Lee, Davis, et al. 2000). There seems to be a contradiction in the literature as to the role of the interaction between α7 nAChRs and Aβ, with inhibition or activation following Aβ binding, depending on the study (Pettit et al. 2001;[START_REF] Tozaki | The inhibitory and facilitatory actions of amyloid-beta peptides on nicotinic ACh receptors and AMPA receptors[END_REF][START_REF] Puzzo | Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus[END_REF]).

This variability depends on 1) the different approaches used, such as the difference in receptor population (homo-or hetero-pentameric receptors), 2) the system used for recordings (Xenopus oocytes that express transiently the subunits versus organotypic slices), 3) the Aβ peptide preparation used. Recordings in slices demonstrated the first evidence for a functional interaction between nAChRs and Aβ where hippocampal GABAergic neurons had their nAChR currents inhibited by Aβ 1-42 application. The most effective concentration was 500 nM, but inhibition was also found at the lower concentration of 100 nM. This inhibition on nAChR was reversible. With the use of selective antagonists it was possible to determine that inhibition operates on both α7 and non-α7 receptors (Pettit et al. 2001). Several later studies determined the activation and inhibition of nAChRs by Aβ as summarized in Table 2.

Generally, short incubation and lower concentrations of Aβ lead to receptor activation, while longer incubation periods and higher peptide concentrations give rise to an inhibitory effect [START_REF] Dougherty | Beta-amyloid regulation of presynaptic nicotinic receptors in rat hippocampus and neocortex[END_REF][START_REF] Puzzo | Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus[END_REF]Lombardo and Maskos 2015). influx. Studies on organotypical brain slices, where a concentration of 10 pM of Aβ was used, showed that the calcium influx activates ERK and MAPK via the PI3K pathway, and the final result is the activation of CREB [START_REF] Dineley | Beta-amyloid activates the mitogen-activated protein kinase cascade via hippocampal alpha7 nicotinic acetylcholine receptors: In vitro and in vivo mechanisms related to Alzheimer's disease[END_REF][START_REF] Young | Oligomeric aggregates of amyloid beta peptide 1-42 activate ERK/MAPK in SH-SY5Y cells via the alpha7 nicotinic receptor[END_REF]. Incubation with 400 nM Aβ in primary neuronal cultures activated pathways involved in Akt phosphorylation with subsequent activation of CREB thereby causing induction of a survival pathway, as has been shown to be activated with nicotine treatment [START_REF] Abbott | Abeta(1-42) modulation of Akt phosphorylation via alpha7 nAChR and NMDA receptors[END_REF]. Still, the level of Aβ can greatly change the results since the same pathway is downregulated at a higher concentration. Interestingly, in Tg2576 AD model mice these pathways are downregulated, which is correlated with a very high level of Aβ peptide [START_REF] Dineley | Beta-amyloid activates the mitogen-activated protein kinase cascade via hippocampal alpha7 nicotinic acetylcholine receptors: In vitro and in vivo mechanisms related to Alzheimer's disease[END_REF]).

The role of the α7 nAChRs in cognitive functions of AD mouse models

The role of α7 nAChRs in AD was also studied in vivo by generating AD mouse models where the α7 nAChRs were knocked-out. Dziewczapolski and colleagues crossed the PDAPP mouse, a model for AD that expresses APP with the Swedish (KM670/671NL) and the Indiana (V717F) mutations [START_REF] Mucke | High-level neuronal expression of abeta 1-42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation[END_REF], with the α7 KO mouse (Orr-Urtreger et al. 1997) and created the APP-α7 KO mouse line. Then, they studied the behavioral performance of these mice. More specifically, the PDAPP mice displayed behavioral deficits in the Morris water maze at 13-16 months of age, whereas the APP-α7 KO mice exhibited a better performance. Indeed, APP-α7 KO mouse performance was comparable to that obtained with control WT and α7 KO mice, indicating that α7 KO mice do not have memory deficits.

It was suggested that the absence of the α7 subunit of the nicotinic receptor restores the behavioral deficit caused by the expression of the mutated forms of APP in the PDAPP mouse line. It has been shown that PDAPP mice display several pathological modifications, like loss of synaptic terminal markers [START_REF] Games | Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein[END_REF]) such as synaptophysin and reduction in the dendritic marker MAP2. The lack of α7 was sufficient to preserve synaptic terminals and dendrites, rescuing levels of synaptophysin and MAP2 to the control levels. In addition, no neuronal loss was detected in any of the mouse lines analyzed. Western Blot and ELISA experiments confirmed that the level of expression of APP and the consequent synthesis of Aβ were comparable between mouse lines, demonstrating that the difference in cognitive deficit and neuropathology were mediated exclusively by the nicotinic receptor. In addition, APP mice demonstrated a deficit in LTP while APP-α7 KO mice did not. However, this study did not include any quantification of the amount of intracellular Aβ, a very important aspect since the Aβ interaction with that α7 nAChRs leads to the peptide's internalization into the cells [START_REF] Nagele | Intracellular accumulation of beta-amyloid(1-42) in neurons is facilitated by the alpha 7 nicotinic acetylcholine receptor in Alzheimer's disease[END_REF]. The authors suggested that the absence of α7 could prevent Aβ intracellular accumulation ameliorating the cognitive neuropathology and the phenotype associated with it (Dziewczapolski et al. 2009).

Interestingly, opposite outcomes were obtained in a study published one year later when the authors crossed α7 KO mice with a transgenic AD line Tg2576 that expresses the Swedish mutation in the human APP sequence [START_REF] Hsiao | Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice[END_REF] to create a different APP-α7 KO line (Hernandez et al. 2010). The mice performed behavioral tests in order to investigate the role of α7 in early AD (5 month old mice). Cognitive deficits worsened in both the contextual fear conditioning and novel object recognitions tasks with α7 nicotinic receptor being deleted. These were the main findings, although the APP-α7 KO mice exhibited high levels of Aβ, but still less than in APP mice, whereas the levels of APP was equivalent in the two lines. The plaque load was reduced in APP-α7 KO mice as a consequence of the lower Aβ concentration. In addition, there was a decrease in MAP2 immunoreactivity in the hippocampus, suggesting that neuroprotective pathways were activated due to the Aβ-α7 nAChR interaction while also preventing Aβ aggregation as accumulated in AD thereby being toxic to neurons (Parri et al. 2011).

Overall, α7 nAChRs seem to have a dual effect depending on the age of the mice tested and the different AD models used, since distinct mutations cause AD pathology under different mechanisms, making it difficult to perform a direct comparison between the studies.

Nicotine in AD

Over the past two decades numerous studies have focused on the consequences of nicotine and smoking on amyloid production and clearance [START_REF] Dome | Smoking, nicotine and neuropsychiatric disorders[END_REF]. Finding a link between AD onset and cigarette smoking was the focus of numerous epidemiological studies which yielded inconclusive and conflicting results because some studies showed increased risk of developing AD with smoking [START_REF] Almeida | Smoking as a risk factor for Alzheimer's disease: contrasting evidence from a systematic review of case-control and cohort studies[END_REF] and others the opposite [START_REF] Van Duijn | Interaction between genetic and environmental risk factors for Alzheimer's disease: a reanalysis of case-control studies[END_REF]. Even with pooling the data from the studies, the outcome was still inconclusive [START_REF] López-Arrieta | Efficacy and safety of nicotine on Alzheimer's disease patients[END_REF]. In 2010, a NIH report found an increased risk for developing AD in smokers [START_REF] Williams | Preventing Alzheimer's disease and cognitive decline[END_REF].

The first attempt to treat an AD mouse model with nicotine was performed by Nordberg and colleagues. Treatment of the AD mouse model Tg2576 with nicotine in the drinking water for a period of 5.5 months reduced plaque burden by 80% in treated mice compared to non-treated, age matched controls. The treatment started at 9 months of age when this particular model already exhibits an observable plaque deposition. The analysis of the Aβ fraction reduced by nicotine showed that mainly insoluble Aβ 1-40/42 was affected while there was no change in soluble Aβ [START_REF] Nordberg | Chronic nicotine treatment reduces beta-amyloidosis in the brain of a mouse model of Alzheimer's disease (APPsw)[END_REF]. In addition to this finding, the same group used a different approach to deliver nicotine [START_REF] Hellström-Lindahl | Nicotine reduces A beta in the brain and cerebral vessels of APPsw mice[END_REF]). Nine month old Tg2576 mice were injected with a subcutaneous solution of nicotine (0.45 mg/kg/day) for ten consecutive days. This protocol also led to a significant reduction in cortical insoluble Aβ 1-40/42 . In a recent study, APP/PS1 mice were treated for one month with intraperitoneal nicotine injections and the effects of nicotine were studied in both young and old mice [START_REF] Inestrosa | Nicotine prevents synaptic impairment induced by amyloid-β oligomers through α7-nicotinic acetylcholine receptor activation[END_REF]. Six and twelve month old mice showed improvements in working and episodic memory compared to non-treated transgenic mice. A reduction in the Aβ oligomeric form was found after the nicotine injections. It was suggested that AD mouse models display a reduction of α7 binding sites and PSD-95 puncta reflecting a synaptic deficit. Nicotine treatment increased both α7 binding sites and PSD-95 puncta.

Chronic nicotine administration was also performed in order to study the consequences of nAChR activation on tau pathology in 3xTg-AD mice (Oddo et al. 2003).

Nicotine treatment in the drinking water for 5.5 months caused faster tau aggregation in CA1

and it is believed that nicotine enhances the aggregation of tau through the activation of p38-MAP kinase. This result shows that even though nicotine has a positive effect on reducing the amyloid burden, it has negative effects on tau pathology.

Moreover, nicotine treatment in the drinking water of 12 month old AD mice harboring the Swedish mutation improved the memory deficits, as found in the Morris water maze task [START_REF] Shim | Nicotine leads to improvements in behavioral impairment and an increase in the nicotine acetylcholine receptor in transgenic mice[END_REF]. Different doses were used (5 mg/kg, 30 mg/kg and 180 mg/kg) and the treatment lasted 6 months. In the group of mice that received the highest dose of nicotine, the levels of α7 nAChR were restored to the levels found in wild-type mice. This dose dependent effect is in contrast with previous studies [START_REF] Oddo | Chronic nicotine administration exacerbates tau pathology in a transgenic model of Alzheimer's disease[END_REF].

In conclusion, one important issue is that smoking causes a much more complex chemical makeup than just administering pure nicotine, therefore this could cause a bias in the effect of nicotine on AD. Irrespective of this, deciphering the role of nicotine on AD is of great interest since it is the main ingredient in tobacco that directly interacts with the cholinergic system.

The default network activity

Shulman and colleagues first noted that a constellation of areas in the human cerebral cortex consistently reduced its activity while performing various novel, non-self-referential, goal-directed tasks when these tasks were compared with a control state of quiet repose [START_REF] Shulman | Common Blood Flow Changes across Visual Tasks: II. Decreases in Cerebral Cortex[END_REF]. In 2001, Raichle and colleagues used positron emission tomography (PET) measurements of regional blood flow and oxygen consumption to show, by established metabolic criteria for activation, that areas consistently exhibiting activity reductions during task performance were not activated in the resting state. Marcus Raichle was the first to coin the term 'default-mode network' (DMN) in relation to resting state brain function [START_REF] Raichle | A default mode of brain function[END_REF]. In their study, they concluded that the brain areas observed to decrease their activity during attention-demanding, goal-directed tasks were not activated in the resting state but, rather, were indicative of a heretofore-unrecognized organization within the brain's intrinsic or ongoing activity.

The brain's DMN consists of discrete, bilateral and symmetrical cortical areas, in the medial and lateral parietal, medial prefrontal, and medial and lateral temporal cortices.

Resting brain states and the DMN have been principally investigated using techniques such as PET [START_REF] Raichle | A default mode of brain function[END_REF], fMRI [START_REF] Greicius | Default-mode activity during a passive sensory task: uncoupled from deactivation but impacting activation[END_REF] and electrophysiology [START_REF] Helps | Very low frequency EEG oscillations and the resting brain in young adults: a preliminary study of localisation, stability and association with symptoms of inattention[END_REF]. The general features of the default mode network have been identified in the monkey [START_REF] Vincent | Intrinsic functional architecture in the anaesthetized monkey brain[END_REF], cat [START_REF] Popa | Contrasting activity profile of two distributed cortical networks as a function of attentional demands[END_REF], rat [START_REF] Lu | Rat brains also have a default mode network[END_REF], and mouse [START_REF] Stafford | Large-scale topology and the default mode network in the mouse connectome[END_REF]. The DMN in the rat, monkey, and human share similarities but they exhibit clear differences.

The DMN is characterized by very low frequency neuronal oscillations providing temporal synchrony between functionally specific and diverse brain regions (Sonuga-Barke and Castellanos 2007). Biswal and colleagues were the first to observe the coherence between such low frequency oscillations and widely distributed neuro-anatomical networks. Since then, research on the brain's DMN and the brain's intrinsic activity has moved in many directions producing a literature that has become quite extensive. Importantly, altered DMN activity has been identified in mental disorders, such as AD [START_REF] Greicius | Default-mode activity during a passive sensory task: uncoupled from deactivation but impacting activation[END_REF], SCZ [START_REF] Pomarol-Clotet | Failure to deactivate in the prefrontal cortex in schizophrenia: dysfunction of the default mode network?[END_REF], anxiety [START_REF] Zhao | Altered default mode network activity in patient with anxiety disorders: an fMRI study[END_REF], epilepsy (He and Liu 2008), autism spectrum disorder [START_REF] Kennedy | Failing to deactivate: resting functional abnormalities in autism[END_REF]) and attention deficit/hyperactivity disorder (ADHD) [START_REF] Helps | Very low frequency EEG oscillations and the resting brain in young adults: a preliminary study of localisation, stability and association with symptoms of inattention[END_REF]).

Changes of the default mode network in SCZ

Schizophrenic patients have been found to exhibit atypical functional connectivity in resting state networks, especially in the DMN [START_REF] Broyd | Defaultmode brain dysfunction in mental disorders: a systematic review[END_REF]). In the eyes closed resting condition, it was shown that schizophrenic patients have non-specific disconnectivity [START_REF] Liang | Widespread functional disconnectivity in schizophrenia with resting-state functional magnetic resonance imaging[END_REF]). Even though there was a high degree of overlap in samples, there was an increase in the resting state functional connectivity in the DMN and task-positive network in the same group which was consistent with increased anti-correlations between the networks in paranoid schizophrenics that participated in the study [START_REF] Zhou | Functional disintegration in paranoid schizophrenia using resting-state fMRI[END_REF]. Different analytical techniques for measuring functional connectivity and the diversity of the disorder are reasons for such disparity. Increased connectivity in the task-positive network and DMN suggests increased sensitivity to self-referential or introspective thought as well as the external environment is what schizophrenics are experiencing. In this patient group, the strength of the anti-correlation between the two networks shows that these processes are in elevated competition [START_REF] Zhou | Functional disintegration in paranoid schizophrenia using resting-state fMRI[END_REF]).

Pomarol-Clotet and colleagues showed that in an n-back working memory task, schizophrenic patients had lower activation of the dorsolateral PFC which correlated with decreased task performance whereas reduced deactivation of medial frontal area DMN did not correlate with task performance [START_REF] Pomarol-Clotet | Failure to deactivate in the prefrontal cortex in schizophrenia: dysfunction of the default mode network?[END_REF]). In the auditory oddball task, ICA analysis showed difference in both temporal and spatial connectivity of the DMN [START_REF] Garrity | Aberrant "default mode" functional connectivity in schizophrenia[END_REF]. In contrast to Pomarol-Clotet et al. this study revealed that schizophrenic patients had more deactivation of the frontal gyrus DMN with decreased anterior cingulate cortex activation which may be related to attentional deficits as seen in schizophrenia [START_REF] Garrity | Aberrant "default mode" functional connectivity in schizophrenia[END_REF]. In schizophrenic patients the parahippocampal gyrus was included in the DMN and their low frequency oscillations were significantly higher (0.08 -0.24 Hz) than control patients (0.03 Hz). Two other studies confirmed these task-related findings where the medial prefrontal, posterior cingulate cortex, lateral parietal and cerebellar regions had disconnectivity in low-frequency oscillatory (<0.1 Hz) activity during rest [START_REF] Bluhm | Spontaneous low-frequency fluctuations in the BOLD signal in schizophrenic patients: anomalies in the default network[END_REF] and during the auditory oddball task [START_REF] Calhoun | Modulation of temporally coherent brain networks estimated using ICA at rest and during cognitive tasks[END_REF]. In contrast, using spatial ICA, an increased connectivity between the DMN and other resting state networks was discovered and this has been proposed to be a mechanism for schizophrenic patient's distractions and hallucinations [START_REF] Jafri | A method for functional network connectivity among spatially independent resting-state components in schizophrenia[END_REF]. Finding a link between schizophrenia and the selfmonitoring function of regions within the DMN will be the focus for further research.

Differences in methodologies and the results of these studies emphasize the need for consistency in the methods in order to fully understand a causal link between DMN functional connectivity and the mechanisms that cause schizophrenia symptoms [START_REF] Williamson | Are anticorrelated networks in the brain relevant to schizophrenia?[END_REF].

Inter-default functional connectivity, in contrast to intra-default connectivity, also showed abnormalities in schizophrenic patients between DMN and ventral attention and frontoparietal control networks. In a recent study, resting-state fMRI data were acquired between healthy and schizophrenic patients where DMN connectivity was estimated and showed in schizophrenic patients decreased connectivity between lateral DMN and unimodal sensory cortex but increased connectivity with the heteromodal association areas. Negative and anergia factor scores in the schizophrenic patients were correlated with increased connectivity between the lateral DMN and right control network while the anterior and posterior DMN had increased connectivity with control and visual networks, respectively [START_REF] Williamson | Are anticorrelated networks in the brain relevant to schizophrenia?[END_REF].

Therefore, hallucinations, thought disturbances, delusions and negative symptoms may be caused by altered DMN connectivity in schizophrenia.

To summarize, the DMN subsystem has been demonstrated in these studies to have a distinct role in schizophrenia and future treatments can focus on the DMN including being a method to better diagnose schizophrenia.

Neuronal hyperactivity in AD

Initially the impairment of cognition in AD was thought to result from a reduction in neuronal activity and subsequently neuronal death. Recent in vivo evidence mainly from mouse models, but also humans, indicates that neurons are hyperactive instead of hypoactive.

More specifically, in vivo two-photon calcium imaging using the Oregon Green Bapta-1 calcium indicator in mouse models of AD showed hyperactive neurons in the cerebral cortex and hippocampus. In the frontal cortex of lightly anesthetized APP23xPS45 mice, more than 20% of supragranular layer II/III neurons were found to be hyperactive (Busche et al. 2008).

These hyperactive neurons were found close to the amyloid plaques, less than 60 µm from the plaque load, whereas the fractions of the simultaneously imaged silent cells increased with plaque distance. In addition to these observations, the GABA A agonist diazepam reduced activity in hyperactive cells to normal levels, whereas application of the GABA A antagonist gabazine increased their activity. These findings suggested a spatial redistribution of excitatory and inhibitory synaptic activities (Busche and Konnerth 2015). Using the same technical approach, hyperactivity was also found in the primary visual cortex [START_REF] Grienberger | Staged decline of neuronal function in vivo in an animal model of Alzheimer's disease[END_REF]) and in the CA1 area of the hippocampus (Busche et al. 2012) of APP23xPS45 mice where an increased fraction of silent cells was also detected. An additional study that used a different AD mouse model, the APPswe/PS1d9, confirmed the presence of hyperactive neurons close to amyloid plaques [START_REF] Rudinskiy | Orchestrated experience-driven Arc responses are disrupted in a mouse model of Alzheimer's disease[END_REF]). Whole-cell patch clamp recordings in the CA1 of the hippocampus in APPswe/PS1M146V mice showed elevated firing rates, when compared with age-matched wild-type mice (Šišková et al. 2014).

Furthermore, it has been showed that soluble Aβ peptide is able to produce neuronal hyperactivity and that amyloid plaques are not an essential requirement. In vivo two-photon calcium imaging revealed that already in young APP23xPS45 mice without plaques, more than 25% of the neurons were hyperactive and a single oral dose of a gamma-secretase inhibitor, which significantly reduced the concentrations of soluble Aβ, was sufficient to rescue from hyperactivity. In addition, local application of nanomolar concentrations of crosslinked Aβ dimers to CA1 neurons in wild-type mice elevated the rates of calcium transients, indicating increases action potential firing (Busche et al. 2012). These findings support previous in vitro studies where soluble Aβ acutely induced inward currents in hippocampal neurons, leading to increased action potential firing [START_REF] Snyder | Regulation of NMDA receptor trafficking by amyloid-beta[END_REF][START_REF] Sanchez-Mejia | Phospholipase A2 reduction ameliorates cognitive deficits in a mouse model of Alzheimer's disease[END_REF][START_REF] Alberdi | Amyloid beta oligomers induce Ca2+ dysregulation and neuronal death through activation of ionotropic glutamate receptors[END_REF]Busche and Konnerth 2015).

Asymptomatic humans with high amyloid load were found to display abnormally increased activation in the default-mode network using blood-oxygen-level dependent (BOLD) fMRI. This increase was mainly found in the medial prefrontal cortex, the precuneus and the posterior cingulate cortex (Sperling et al. 2009). In the hippocampus, elevated restingstate BOLD activity was observed already during pre-dementia stages of AD [START_REF] Dickerson | Increased hippocampal activation in mild cognitive impairment compared to normal aging and AD[END_REF][START_REF] O'brien | Longitudinal fMRI in elderly reveals loss of hippocampal activation with clinical decline[END_REF]. Increased hippocampal activation has also been reported in asymptomatic carriers of genetic mutations for fAD [START_REF] Quiroz | Hippocampal hyperactivation in presymptomatic familial Alzheimer's disease[END_REF]) and carriers of the APOE ε4 allele [START_REF] Filippini | Distinct patterns of brain activity in young carriers of the APOE-epsilon4 allele[END_REF]). In addition, hyper-synchronous activity across large populations of neurons was also detected.

Overall, these findings from mice and human studies, suggest an amyloid-mediated elevation in the balance between synaptic excitation and inhibition. However, the exact mechanisms that lead to this disruption of neuronal activity in AD is indeed poorly understood and further studies must be performed in order to elucidate the complexity of AD mediated hyperactivity.

Anesthesia differentially modulates spontaneous network dynamics

It is widely acceptable that anesthesia suppresses brain activity [START_REF] Friedman | A conserved behavioral state barrier impedes transitions between anesthetic-induced unconsciousness and wakefulness: evidence for neural inertia[END_REF].

Brain imaging studies have shown that most general anesthetics cause a global reduction in cerebral blood flow when loss of consciousness occurs (for a review see Changeux 2012b). In propofol infusion, it was demonstrated that cortical EEG values decreased robustly at the exact moment that consciousness was lost [START_REF] Velly | Differential dynamic of action on cortical and subcortical structures of anesthetic agents during induction of anesthesia[END_REF]). Functional magnetic resonance imaging (fMRI) in awake and anesthetized primates have revealed altered stimulus-evoked responses and functional connectivity induced by anesthesia [START_REF] Liu | fMRI in the awake marmoset: somatosensory-evoked responses, functional connectivity, and comparison with propofol anesthesia[END_REF]). Furthermore, resting-state fMRI (rs-fMRI) in humans has demonstrated reduced functional connectivity during anesthesia compared with the awake state, which scaled with depth of anesthesia [START_REF] Peltier | Functional connectivity changes with concentration of sevoflurane anesthesia[END_REF]. There is also evidence that anesthesia does not affect all cortical areas similarly. In particular, rs-fMRI studies indicate that propofol-induced loss of consciousness correlates with decreased corticocortical and thalamocortical connectivity in frontoparietal networks, whereas connectivity is preserved in sensory cortices [START_REF] Boveroux | Breakdown of within-and betweennetwork resting state functional magnetic resonance imaging connectivity during propofol-induced loss of consciousness[END_REF].

Positron emission tomography data in humans has also shown that anesthesia induced loss of consciousness by isoflurane or halothane is accompanied by decreased corticocortical and thalamocortical connectivity [START_REF] White | Impaired thalamocortical connectivity in humans during general-anesthetic-induced unconsciousness[END_REF].

In a more recent study, Sellers and colleagues examined how anesthesia modulates spontaneous network activity in the primary visual cortex and in PFC by electrophysiological recordings of local field potential and multiunit spiking activity in awake and anesthetized ferrets (Sellers et al. 2013). They used isoflurane at three concentrations (0.5%, 0.75%, and 1.0%, each with continuous and equal xylazine administration for maintaining adequate sedation) and found that the effects of anesthesia on these two cortical areas were vastly different. In V1, modulation induced by anesthesia in input layer IV (granular layer) differed from modulation of activity in supragranular and infragranular layers. In contrast, in PFC, anesthesia altered network dynamics and induced highly rhythmic activity patterns with fewer differences across cortical layers (Sellers et al. 2013).

Overall, taking into consideration all these studies, anesthetics produce changes in the brain activity via at least two mechanisms: the dose-dependent global and regionally specific suppression of neuronal activity and the disruption of functional interactivity within distributed neural networks (Heinke and Koelsch 2005).

Methods

Using viral vectors as a research tool

The Throughout this thesis, adapted lentiviruses and adeno-associated viruses have both been developed and exploited, allowing us to compare the functional contributions of certain nAChR subunits in the PFC and shall be discussed.

HIV-1 lentivirus, its genome and life cycle

Lentiviruses are a subgroup of the Retriviridae family, which replicate in a host cell through the process of reverse transcription. Lentiviruses can deliver a significant amount of viral RNA into the DNA of the host cell and have the unique ability among retroviruses of being able to infect non-dividing cells, so they are one of the most efficient methods of a gene delivery vector. Lentiviruses can infect non-dividing mature cells, as was first demonstrated by Naldini and colleagues, who injected the adult rat brain with a non-replicative human immunodeficency virus type 1 (HIV-1) based vector (Naldini et al. 1996). They obtained high expression of the reporter gene, which was stable even several months after a single injection.

Many other vectors have been developed from a variety of lentiviruses such as feline immunodeficiency virus (FIV), human immunodeficiency virus type 2 (HIV-2) and bovine immunodeficiency virus (BIV).

The HIV-1 virus consists of a diploid single-stranded RNA genome surrounded by a protein coat and outer phospholipid envelope. Its genome contains three essential retroviral genes as well as regulatory genes that modulate host interactions. The gag gene is responsible for glycoprotein structure, the env gene transcribes envelope proteins, and the pol gene transcribes the reverse transcriptase, responsible for producing a DNA copy of the HIV RNA genome. Viral genes vif, vpr, vpu, rev, nef and tat transcribe virulence proteins. All proviral genes, those that become integrated into the host (gag, pol, pro, vif, vpr, vpu, tat, and env), are bordered by two long terminal repeats (LTRs) essential for gene regulation. Viral promoters and enhancers are located in the LTR U3 region. The packaging signal, Ψ, is located in the 5' end of the genome, and is responsible for packaging genes downstream of itself into the viral capsid during replication (Trono 1994).

The HIV-1 life cycle can be split into an early and a late phase of replication. The early steps begin with the attachment of the virion at the cell surface, and finish with the integration of the proviral DNA into the host genome. The succeeding late part of the viral replication extends until virion release. Upon infection of an organism, the HIV-1 particle travels mainly in the blood or lymphatic system until it attaches to the surface of a target cell with a low-specificity interaction between the virus and cellular proteins. The specificity of this interaction defines the tropism of the virus, in other words, the host spectrum. The fusion of the cell and virus membranes allows the release of the viral core into the cell. The viral reverse transcriptase generates a double stranded DNA molecule using its viral RNA genome as a template. The structural end product of the reverse transcription process, called the "preintegration complex" (PIC), is imported into the nucleus. The precise mechanisms that permit this active transport are still a matter of debate. Replication of the viral genome next occurs and copies of RNA from the viral gene are made. Viral structural proteins then migrate to the cell surface, assemble and their presence causes the membrane to weaken, making a membrane-coated viral particle that is released (Trono 1994). As described is what occurs in nature whereas engineered viruses can be modified to be replication-deficient therefore they cause stable genomic integration into the host cell genome, but without the proliferation of virus particles (Naldini et al. 1996).

2.1.1a Genetically modified HIV-1 as a research tool

HIV-1 is genetically modified in order to serve as a research tool and several modifications have been made for a production of lentiviruses that exhibit efficient transgene expression and safety. Lentiviral vectors are usually created in a transient transfection system in which a cell line is transfected with three separate plasmid expression systems. These include the transfer vector plasmid, the packaging plasmid or construct, and a plasmid with the heterologous envelop gene (env) of a different virus (Naldini et al. 1996). The three plasmids components of the vector are put into a packaging cell, which is then inserted into the HIV shell. The virus portions of the vector contain insert sequences so that the virus cannot replicate inside the cell system.

In greater detail, the packaging plasmid is the backbone of the virus system and contains the genes gag, pol, rev and tat which are necessary for the production of structural The vectors are then transfected into HEK-293T cells in vitro to produce lentiviruses.

Ultracentrifugication separates the virus produced from HEK cell debris and a titer of viral particles is measured.

Despite the important advantages that lentiviruses display such as their application to dividing and non-dividing cells, the large cloning capacity and the stable transgene expression, there are some disadvantages. For example, the integration into the cell genome may cause deleterious mutations. Another disadvantage is the limited amount of viral particles that can be injected in a tissue. In response to these limitations adeno-associated viruses (AAV) are commonly used in research.

Adeno-associated virus as a research tool

AAV belongs to the group of Parvoviruses, which is not linked to any known symptoms or pathology in humans. AAV is commonly used in research due to its wide tropism, robust, long-term expression, and the low immunogenicity. Similar to the lentiviral vector, the AAV can transduce non-dividing cells. In contrast to the wild-type AAV that is capable of integrating into the host cell genome, the recombinant AAV depleted of the rep and cap genes is integration-defective (Daya and Berns 2008). However, the integration of the and VA RNAs, should be provided by either adenovirus infection or by transfecting into production cells as a third plasmid that provides these adenovirus helper factors. Given that HEK293 cells, a commonly used AAV production cells, already contain the E1A/E1b genes, the only helper factors that need to be delivered are E2A, E4ORF6 and VA RNAs.

Eleven serotypes of AAV have been identified, with the most commonly used being AAV2. These serotypes differ in their tropism, or the types of cells they infect, making AAV a very useful system for preferentially transducing specific cell types. With lysis induced by the helper virus, the virions are release. This lytic cycle can also occur without helper virus when metabolic inhibitors DNA damaging agents or genotoxic.

AD models that use viral delivery of the mutated APP

A first effort to develop an AD model using viral delivery technology used a vector that was derived from the murine leukemia virus to drive the expression of APP695 and APP995-Swedish mutations in embryonic rat brain cells which were then grafted into adult rat brains (Bayer et al. 1996). A long lasting expression of the transgene in transplanted rats was found with some signs of neural degeneration. Interestingly, six months after the transplantation, APP positive plaques were detected, but no Aβ positivity (Bayer et al. 1996).

In another study, the expression of human APP695 was driven in the ventral hippocampus of adult rats with a replication-defective adenovirus (Nishimura et al. 1998). The authors observed degeneration of neurons but no Aβ was detected. It was suggested that the degeneration was caused by APP accumulation inside the neurons.

Jaworski and colleagues developed an AAV where they inserted the double Swedish (KM670/671NL), Austrian (T714I) and London (V717I) mutations in the APP and performed injections into the hippocampus of adult mice. Twelve weeks after the viral injection the expression of the transgene was detected with intracellular accumulation of Aβ but no corresponding neuronal death. The transgenic human APP was expressed two fold higher than the endogenous murine APP, as identified by Western Blot. The expression of the virus was followed up to six months after the injection, however no plaque deposition was observed in APP-SLA expressing mice [START_REF] Jaworski | AAV-tau mediates pyramidal neurodegeneration by cell-cycle re-entry without neurofibrillary tangle formation in wild-type mice[END_REF]. Moreover, in another study, virally Taken together these studies show that the use of viral vectors is a useful tool to generate AD like pathology. There are, however, several limitations in the system that prevent the development of Aβ accumulation such as the choice of the promoter that drives the expression of the transgene.

Two-photon microscopy

The

Principles of two-photon excitation microscopy

Two-photon excitation microscopy is based on the principle that two low-energy photons cooperate to cause a higher energy electronic transition in a fluorescent molecule (Denk et al. 1990) (Figure 11). Each photon carries approximately half the energy necessary to excite the molecule. Similarly, three or more photons can combine to cause excitation. The efficiency of multiphoton absorption depends on the physical properties of the molecule and on the spatial and temporal distribution of the excitation light [START_REF] Helmchen | Deep tissue two-photon microscopy[END_REF]. In particular, since two-photon excitation depends on the simultaneous absorption of two photons, the probability of two-photon absorption by a fluorescent molecule is a quadratic function of the excitation intensity. This quadratic relationship between excitation and emission gives rise to many of the significant advantages associated with two-photon excitation microscopy. Under sufficiently intense excitation, three-photon and higher photon excitation is also possible and deep UV microscopy has been developed.

As reviewed by Helmchen and Denk, to generate sufficient signal, excitation has to be concentrated in space and time. High spatial densities are generated by focusing a laser beam through a high numerical aperture (NA) objective. For example, the typical two-photon excitation point spread function has a full width at half-maximum of 0.3 µm in the radial direction and 0.9 µm in the axial direction when 960 nm excitation light is focused by a 1.25

NA objective. Concentration in the time domain requires the use of lasers that emit pulses (less than a picosecond long) with corresponding high peak intensities.

The two-photon set up

Figure 11D shows the typical components in a two-photon microscope. The excitation light is reflected by a dichroic mirror to the microscope objective and is focused in the specimen. Two-photon induced fluorescence is generated at the diffraction-limited volume.

The images are constructed by raster scanning the fluorescent volume in two dimensions using a galvanometric scanner. The emission signal is collected by the same objective and transmitted through the dichroic mirror along the emission path. A barrier filter in front of the detector is also used to block scattered excitation light.

Concerning the lasers used, mode-locked Ti:sapphire lasers have nearly ideal properties for two-photon excitation microscopy. They produce a stream of pulses with repetition rates of approximately 100 MHz. The pulse duration produced by turn-key systems is on the order of 100 fs, shorter pulses would be severely distorted by dispersion in the microscope optics (Guild et al. 1997). The excitation wavelength is tunable from 670 nm to above 1000 nm, allowing excitation of most useful fluorophores. Ti:sapphire lasers perform relatively poorly at wavelengths that are optimal for the excitation of red fluorescent proteins (>1000 nm). In this wavelength regime, other powerful light sources are available, for example mode-locked Ytterbium-doped lasers (fixed wavelength: ranging from 1030 nm to 1060 nm) or Cr:forsterite lasers (tunable, 1200-1300 nm) (Svoboda and Yasuda 2006).

As mentioned before, the majority of laser scanning microscopes use galvanometer mirrors, which provide excellent optical properties and allow image rotation. As far as the detectors used for two-photon microscopy, photomultiplier tubes (PMTs) are best for most applications. Finally, an important element for two-photon microscopy is the objective.

Important factors that need to be taken into consideration include 1) the NA, which determines the resolution (combined with wavelength used) and the collection angle for fluorescence, 2) the magnification, which determines the field of view, 3) the working distance and 4) the efficiency of transmission of the excitation light (Svoboda and Yasuda 2006).

Two-photon versus single-photon microscopy

Compared to single-photon techniques, two-photon provides several advantages for microscopy in scattering specimens. First, the excitation wavelengths used in two-photon microscopy penetrate tissue better than the visible wavelengths used in single-photon microscopy. This is due to reduced scattering and reduced absorption by endogenous chromophores. Second, scattered excitation photons are too diluted to cause appreciable fluorescence (Figure 12). Even deep in tissue, under conditions where most of the incidence photons are scattered, excitation is therefore still mainly limited to a small focal volume.

Third, because of localization of excitation, all fluorescence photons, ballistic and scattered, constitute useful signal if they are detected. Confocal microscopy uses the emission pinhole aperture to reject out of focus light but inside thick specimens, scattering of the fluorescent photon is inevitable. Two-photon microscopy requires no pinhole aperture and minimizes signal loss (Denk et al. 1990;[START_REF] Helmchen | Deep tissue two-photon microscopy[END_REF].

absorption occurs, mainly in pigmented samples. Finally, the intense electromagnetic field produced by femtosecond laser pulses can cause photodamage.

The depth limit

Depending on the properties of the tissue, the two-photon microscope can image up to a depth of 1 mm in tissue. The maximum achievable imaging is proportional to the scattering and depends on the laser power used. In addition, imaging depth is limited by fluorescence generated at the surface of the sample by out of focus light, which degrades localization of excitation and contrast. Increased laser power or pulse energy can enhance background and signal. If deeper structures need to be reached, mechanical penetration or removal of the overlying tissue is necessary. Efforts in this direction have been made using very narrow objective lenses made from gradient-index (GRIN) material or microprisms (Jung et al. 2004;Levene et al. 2004;Low et al. 2014).

Labeling techniques

The two-photon excitation spectra of several useful fluorescent molecules have been measured. Two-photon excitation cross-sections are expressed in units of Goppert-Mayer (GM h 10 -50 cm 4 s). Compared to one-photon spectra, two-photon spectra tend to be broader and shifted toward the blue. EGFP, YFP, CFP, and red proteins all have good two-photon cross-sections (approximately 100 GM) (Xu et al. 1996). Even some endogenous chromophores can produce usable fluorescence signals under two-photon. In addition, developments of photo-activatable and switchable fluorescence proteins allow measurements of protein transport and recycling. Two-photon cross-sections of photo-activatable GFP are sufficiently high to visualize proteins in individual spines.

In vivo labeling techniques for staining cells in living tissues have greatly advanced.

Several classes of fluorescent labels can be distinguished. A first group comprises synthetic organic dyes, often referred to as "traditional" dyes, which need to be introduced into cells by physical or chemical means. Fluorescent proteins establish a second class; they are genetically encoded, and their expression can be induced in specific cell types. A third "mixed" is to attach a fluorophore to a protein of interest by tagging the protein with a specific short peptide sequence (Helmchen 2009). Traditional dyes can be loaded into cells using a variety of methods, including uptake from the extracellular space, intracellular recordings, or electroporation. Often these methods lack cell type specificity. An exception is the red fluorescent dye sulforhodamine-101which specifically labels astroglia in the rodent neocortex

In vivo calcium imaging

The first synthetic fluorescent dyes developed by Roger Tsien around 1980 were based on calcium-chelating molecules such as BAPTA (Tsien 1989). Since then several synthetic calcium indicators have been created. Since there is such a large range of calcium concentrations within cells from nanomolar to micromolar levels, it is desirable to have a range of affinities. The most suitable dyes for in vivo two-photon imaging are non-ratiometric indicators that have a single-photon excitation near 488 nm (Oregon Green BAPTA) (Figure 13). This translates into two-photon excitations between 800-900 nm, well within the range of a two-photon laser, along with traditional UV-excitable dyes like Fura-2 (Helmchen 2009).

Red fluorescent calcium indicators (Rhod-2) are more rare due to problems in loading cells

where Rhod-2 AM, for example, preferentially loads glial cells so is not well suited for neuronal applications. Ideally, labeling functional neurons that make up the entire local population would be performed in order to dissect network dynamics in vivo.

A major breakthrough was achieved in 2003 in Arthur Konnerth's group, when they successfully loaded cells in vivo using the membrane-permeable, acetoxymethyl (AM)-ester form of calcium indicator dye (Stosiek et al. 2003). Since then, multicell bolus loading has become a widespread method for in vivo investigations of the functional organization of local circuits.

Article 1 Overview

One of the strongest predictors of failing to quit smoking is nicotine dependence, a heritable trait with an estimated 50% of its variability attributable to genetics. Smoking is the leading cause of morbidity and mortality worldwide and it is therefore very important to establish and dissect genetic mechanisms leading to smoking. In this part of my thesis, I focused on the human genetic polymorphism rs16969968, that predisposes one to smoking, found in the gene coding for the α5 nAChR, and verified by extensive meta-analysis studies.

As this same polymorphism (α5 SNP) has also recently been linked to schizophrenia, there is a developing hypothesis that heavy smoking could have a "causal" effect on schizophrenia.

Smoking is highly prevalent in schizophrenic patients, and is seen as a form of selfmedication. fMRI studies showed that humans with this polymorphism had hypo-function in the prefrontal cortex (hypofrontality) in the absence of performing a task. Here using twophoton calcium imaging and transgenic mice, I show that expression of the human polymorphism linked to smoking and schizophrenia causes a substantial deficit in prefrontal function, and this function is restored with chronic nicotine administration.

Briefly, the main findings of this part of my thesis can be summarized as follows:

1. α5 SNP mice exhibit impairments in social interaction and deficiencies in the prepulse inhibition task, characteristic markers of psychiatric disorders.

2. nAChRs modulate cortical activity through a hierarchical circuit of inhibitory neurons.

3. α5 SNP mice are characterized by decreased PFC neuronal activity.

4. Chronic nicotine administration reverses the hypofrontality to control levels.

These new findings lead us to conclude that there exists a genetic core dysfunction in the brain due to the altered nicotinic gene function, and that smoking is secondary to this primary lesion. Chronic nicotine administration restores the "hypofrontality", paving the way to new therapeutic strategies targeting nicotinic receptors in mental disorders.

My personal contribution for this article was to design and perform all the experiments except the two behavioral tests. I performed the cranial windows, stereotaxic injections, in vivo calcium imaging recordings, targeted lentiviral expression, CRISPR/Cas9 and nicotine restoration experiments. I significantly contributed to the design of the calcium imaging code and participated in the data analysis. Finally, I wrote the manuscript.

Nicotine reverses hypofrontality in animal models of addiction and schizophrenia

Fani Koukouli The prefrontal cortex (PFC) underlies higher cognitive processes 1 that are modulated by cholinergic inputs via nicotinic acetylcholine receptors (nAChRs) 2 . It exhibits spontaneous "default" activity 3 , which is altered in neuropsychiatric disorders 4 , including schizophrenia 5 , that in turn is accompanied by heavy smoking 6 Impairment of cognitive processes that are critically reliant on the PFC is a signature of schizophrenia 5 and is associated with a low default network activity 3 . However, the cellular and circuit deficits underlying this "hypofrontality" remain unexplored. Because acetylcholine (ACh) release exerts a strong modulation of cortical activity via nAChRs 14-16 , it is not surprising that human nAChR gene variants are strongly linked to mental disorders with cognitive deficits 17 . Genetic variants within the CHRNA3/A5/B4 nAChR gene cluster contribute to risk for habitual smoking 7 . Among the series of polymorphisms composing this human haplotype, the rs16969968 SNP leads to a substitution of aspartic acid 398 by asparagine (D398N) in the human α5 subunit 18 . In humans, this SNP is associated with reduced resting-state functional connectivity (rsFC) and circuits involving the PFC are altered 12 .

Recently, the same genetic locus was associated with schizophrenia in a major GWAS 8 , but whether the SNP is responsible for the "hypofrontality" observed in these patients, and thus implicates altered nicotinic signaling directly in the pathophysiology of schizophrenia, is not yet clear. One obstacle for further mechanistic dissection is the absence of animal models. Because genetic deletion of the α5 nicotinic subunit in the mouse leads to PFC-linked behavioral deficits, altered cholinergic excitation and aberrant neuronal morphology in the PFC 19 , we developed a mouse line to express the human α5 variant to investigate its impact on behaviors associated with schizophrenia and to determine the neuronal and circuit mechanisms underlying cognitive deficits.

Research on social cognition suggests that social interaction deficits, similar to emotion dysfunctions, represent characteristic markers of psychiatric disorders 20 . Because an impaired social ability is a common feature of schizophrenia-spectrum disorders 20 , we used the three-chamber social test to determine whether the α5SNP mouse exhibits impairments in social interaction 21 . The results indicated that the α5SNP mice exhibit deficits in sociability, with wild-type (WT) littermates having a clear preference for the social object, spending 69.6 ± 2.9% of their time interacting with the social object, while the α5 SNP mice showed no preference between the social and inanimate object (44.1 ± 5.3%) (Fig. 1a). The differences in social interaction cannot be attributed to genotype effects on activity or general exploratory behavior since the WT and α5SNP mice did not differ in locomotor activity or combined time in the social and inanimate object interaction areas (Fig. 1a).

Deficiencies in the prepulse inhibition (PPI) task are a measure of impaired sensorimotor gating and are observed in several neuropsychiatric disorders 22 . PPI has been particularly important for studying the neurobiology of schizophrenia because cognitive information gating deficits are a clinically important feature of the disorder 22 . The PPI task measures the ability of an animal to inhibit its startle in response to an acoustic tone (pulse) when it is preceded by a lower-intensity stimulus (prepulse). There is a high homology between measures of PPI in rodents and humans, consistent with the use of PPI as a crossspecies measure of sensorimotor gating 22 . We found that α5SNP mice exhibit decreased PPI across a range of prepulse intensities (4, 8, 12 and 20 db above background noise) relative to WT littermates (Fig. 1b), suggesting that sensorimotor gating is disrupted by the α5 variant.

To determine the circuit mechanisms underlying the cognitive deficits attributed to dysfunction of α5 containing nAChRs and the human SNP, we assessed resting state network activity using in vivo two-photon imaging in the prelimbic cortex (PrLC) of the PFC in awake mice lacking the α5 (α5KO) and with the α5SNP, compared to WT. Neurons were transduced with an adeno-associated viral vector (AAV) expressing the genetically encoded calcium indicator GCaMP6f in order to monitor Ca 2+ transients as a proxy for action potential firing (Fig. 1c and Supplementary Fig. 1). In layer II/III we estimate that more than 80% of the recorded cells are pyramidal neurons (see Supplementary Fig. 2). Interestingly, we found the median frequency and duration of Ca 2+ transients reduced in the transgenic mice compared to WT (Supplementary Fig. 3). Spontaneous Ca 2+ transients were deconvolved with a putative unitary (action potential-evoked) fluorescence response in order to estimate neuronal firing rates (Supplementary Figs. 456and Supplementary Table 1). We found layer II/III neuronal activity significantly decreased in PrLC of α5KO (8.2 ± 0.5 spikes/min, 7 mice) and α5SNP (20.3 ± 0.6 spikes/min, 11 mice) compared to WT mice (27.5 ± 0.9 spikes/min, 11 mice, P < 0.001 for all comparisons) (Fig. 1d-f). Thus, loss of function of α5 containing nAChRs causes a decrease in layer II/III microcircuit activity, which parallels the decrease in default activity ("hypofrontality"), a defining feature of schizophrenia in humans 5 .

The α5 subunit is present only in vasoactive intestinal polypeptide (VIP) interneurons in layer II/III 23 , which exert inhibitory control of somatostatin (SOM) and parvalbumin (PV) interneurons in the PFC 24 . Since layer II/III pyramidal neurons do not express nAChRs, we hypothesized that loss of function of the α5 subunit would result in disinhibition of PV and SOM interneurons and thus reducing pyramidal cell activity (Fig. 1g). Because dendritic targeting SOM interneurons express different nicotinic receptor subtypes, α7 and β2 containing nicotinic receptors 15,25 , while the perisomatic targeting PV cells 15,26 exclusively express α7 nAChRs, we used transgenic animals with deletions of the nAChR subunits expressed in PV and SOM interneurons. We observed a strong increase in the median spike frequency in both KO lines, with the β2KO exhibiting the highest frequencies (85 ± 1.1 spikes/min, 7 mice) compared to α7KO (45.9 ± 1.8 spikes/min, 4 mice) and WT mice (P < 0.001 for all comparisons) (Fig. 1h-j). Thus, the absence of either α7 or β2 subunits in the GABAergic interneurons in layer II/III decreases pyramidal neuron-targeted interneuron inhibition, and thus increases pyramidal neuron activity.

Because α5 subunits are expressed in other cortical layers 15 , we selectively reexpressed the α5 WT subunit only in the interneurons of layer II/III in the PFC of α5KO-GAD Cre mice by using a conditional lentivirus (Fig. 2a,b). We observed full restoration of pyramidal neuron activity to control levels, whereas when the α5SNP was re-expressed in the α5KO-GAD Cre mice, the pyramidal cell activity was only partially recovered between the level of control mice and α5KO-GAD Cre (Fig. 2c). Because PV interneurons do not express the requisite β2 subunit to form a functional α5 nAChR, it is only possible that VIP and SOM interneurons express the α5 subunit in these experiments. Because the activity in pyramidal neurons is restored, re-expression of α5 in VIP interneurons is likely the dominant effect on the circuit behavior. These data are consistent with the hypothesis that the hypofrontality in the α5KO and α5SNP mice is attributed exclusively to an effect on the hierarchy of layer II/III GABAergic interneurons.

In addition, because β2 subunits are also expressed in other cortical layers 15 , we monitored pyramidal cell activity in β2KO mice where we rescued expression of the β2 subunit specifically in layer II/III of PrLC. β2 was re-expressed with tdTomato as a reporter, by targeted injection of a β2-IRES2-tdTomato bi-cistronic lentiviral vector into layer II/III of the PrLC of β2KO mice (β2KO VEC ) (Fig. 2d and Supplementary Fig. 8). In the PrLC, only the interneurons in layer II/III express nAChR α subunits, and therefore the lentivirusmediated expression results in functional nAChRs containing β2 subunits exclusively in interneurons 27 . We found that the neuronal activity of β2KO VEC mice was rescued to the levels of WT mice (Fig. 2e,f). These results are consistent with a layer II/III re-expression of β2 in SOM and VIP interneurons. Together these manipulations demonstrate the critical role of layer II/III interneurons in controlling pyramidal neuron activity within the same layer.

Thus far we have used the specific expression of nAChR subtypes to confirm that decreased disinhibition in α5KO and α5SNP mice accounts for hypofrontality. To further decipher the mechanism, we examined the spiking activity in VIP, PV, and SOM interneurons under α5 knock-down. We used the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 technology 28 for the deletion of the α5 subunits in vivo. We delivered the Cas9 and guide RNA using a lentiviral vector (α5-Cas9 lentiviral vector) to target the CHRNA5 gene in layer II/III of PFC in VIP-Cre/tdTomato mice (VIP CRISPR ) (Fig. 3a,b). The absence of α5 nAChR expression resulted in strong reduction in the median spike frequency of VIP interneurons in VIP CRISPR mice (3.49 ± 0.66 spikes/min, 4 mice) compared to control mice (21.5 ± 2.17 spikes/min, 4 mice, control: VIP-Cre mice injected with a CRISPR non target control lentiviral vector, see Materials and Methods for details) (Fig. 3c-f and Supplementary Fig. 9). We then examined how the deletion of the α5 subunit in VIP interneurons could affect the spontaneous activity of PV and SOM interneurons. We injected again the α5-Cas9 lentiviral vector into layer II/III of PFC in PV-Cre mice (PV CRISPR ) and did not observe any significant difference in PV CRISPR interneuronal activity (6.07 ± 0.60 spikes/min, 4 mice) compared to control mice (5.62 ± 0.45 spikes/min, 4 mice) (Fig. 3g-j). These results are consistent with weak innervation of PV interneurons by VIP interneurons 24,29 . In contrast, targeted α5-Cas9 lentiviral injection into layer II/III of SOM-Cre mice (SOM CRISPR ) revealed a robust increase in SOM interneuron spontaneous activity (39.12 ± 3.14 spikes/min, 4 mice) compared to control mice (5.57 ± 1.31 spikes/min, 4 mice) (Fig. 3k-n), whereas the pyramidal cell activity was reduced (Fig. 3o-r). Our data reveal a central role for the α5 subunit in controlling VIP-mediated disinhibition via SOM interneuron inhibition of pyramidal neurons in layer II/III (Fig. 3s).

With the evidence that nicotine levels obtained from smoking can improve cognitive abilities 30 , and can mitigate certain schizophrenic symptoms 6,31 , we administered nicotine to WT and α5SNP mice by mini-pump infusion and examined alterations in layer II/III neuronal firing before and after infusion (Fig. 4a). The dose of nicotine was selected to maintain a plasma concentration analogous to that observed in smokers 32 . The pyramidal neuron activity significantly increased after two days of nicotine administration in WT mice and remained elevated after two weeks (Fig. 4b-e and Supplementary Fig. 10). We also found that nicotine treatment from one to two weeks increased the pyramidal neuron activity in α5SNP mice to the levels of WT baseline activity (Fig. 4b-e). An explanation for these data is that chronic nicotine exposure desensitizes the β2 nAChRs expressed by interneurons. β2-dependent nAChR currents, but not α7, in interneurons of PFC layer II/III completely desensitize after exposure to smoking concentrations of nicotine in slice preparations 15 . To test this in vivo, we administered the same dose of nicotine in SOM-Cre mice and observed a significant reduction in SOM activity compared to control mice (Fig. 4g,h). These results suggest that the normalization of neuronal firing in α5 deficient mice by nicotine administration is mediated by a dominant nicotine-evoked desensitization of the β2-containing receptors on SOM interneurons. As a result, SOM inhibition of pyramidal cells is reduced. Together these data support the conclusion that layer II/III PFC activity is significantly controlled by different nAChR subunits expressed specifically in different classes of interneurons. These experimental results argue strongly that nicotine, or nicotinic agonists, could normalize PFC activity and thus reduce some of the cognitive deficits found in humans.

In summary, we show that endogenous ACh distinctly recruits specific interneuron types within an interneuron hierarchy that controls pyramidal neurons in layer II/III of PrLC, by differential expression of nAChR subunits. We also provide the first experimental evidence that the α5 human polymorphism, which predisposes to nicotine addiction, provokes a decrease in neuronal activity that mirrors the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction 5 . We further demonstrate that chronic nicotine treatment of mice carrying the α5 SNP restores their neuronal activity deficits, which together provide the basis for examining new pharmacotherapeutic strategies.

Indeed, positive allosteric modulators, for defined nAChR subunits, like the polymorphic α5, are strong candidates 33 . Finally, our results support the hypothesis that subjects with polymorphic CHRNA5 smoke to self-medicate in order to ameliorate at least some symptoms of cortical dysfunction. The GAD67-Cre mice were published previously 4,5 . For the generation of GAD67-Cre/tdTomato, GAD67-Cre mice were crossed with a ChAT-Cre/tdTomato line 6 . GAD67-Cre mice were bred onto a homozygous α5KO background to obtain α5KO-GAD Cre .

For the generation of the VIPcre/tdTomato mice, homozygous Vip-ires-Cre (VIP tm1(cre)Zjh , The Jackson Laboratory, Bar Harbor, USA) mice were crossed with homozygous Ai9 mice (B6.Cg-Gt(ROSA) 26Sortm9(CAG-tdTomato)Hze /J, The Jackson Laboratory) as previously described 7 .

PV-Cre (no: 008069, The Jackson Laboratory) and SOM-Cre (no: 013044, The Jackson Laboratory) mice were bred onto a C57BL/6J background.

All experiments were performed using male mice.

Social interaction test. 8 to12-week-old male WT and α5SNP littermates were tested for social behavior using a three-chamber social approach paradigm 8 . Experimental mice were habituated for 1 h in separate clean holding cages and then introduced into a three-chamber arena with only empty object-containment cages (circular metallic cages, Stoelting

Neuroscience (Wood Dale, IL)) for a 10-min acclimation phase in two 5-min sessions in a 3-4 h period (Habituation Periods I and II). The following day the mice were placed in the center chamber (without access to the left and right social test areas) and allowed to explore the center area for 5 min (Open Field Period). After this exploration period, barriers to adjacent chambers were removed, allowing mice to explore the left and right arenas, which contained a social object (i.e. unfamiliar C57BL/6J male mouse) in one chamber and an inanimate object (plastic toy) in the other chamber. Experimental mice were given 10 min to explore both chambers and measured for approach behavior as interaction time (i.e. sniffing, approach)

with targets in each chamber (within 2 cm, excluding non-nose contact or exploration).

Sessions were video-recorded and object exploration time and total distance moved were analyzed using the Noldus tracking system (Leesburg, VA). A social preference index was calculated as the percentage of time spent investigating the social target out of the total exploration time of both objects. The analysis was conducted with investigators blind to the genotypes of subjects. Arenas and contents were thoroughly cleaned between testing sessions. Multiple social targets from different home cages were used for testing to prevent potential odorant confounds from target home cages.

Prepulse inhibition of the startle reflex. Male WT and α5SNP littermates ranging in age from 60-75 days of age were used for the study. The prepulse inhibition (PPI) apparatus (SR-Lab, San Diego Instruments, San Diego, CA, USA) consisted of a sound-attenuating chamber and a vented Plexiglas animal enclosure (4 cm diameter) that rests on a 20x13 cm Plexiglas frame. Background (70 dB), prepulse (74, 78, 82 and 90 dB) and pulse (120 dB) bursts were generated through a computer using SR-Lab software (San Diego Instruments, San Diego, CA, USA) and presented through a speaker mounted (13 cm) above the animal enclosure.

Magnitude of the startle response was measured by a piezoelectric accelerometer mounted below the Plexiglas animal enclosure.

The session was initiated with a 5 minute acclimation period followed by initiation of the test session with 5 pulse alone trials (white noise, 120 dB/40 ms); the session also concluded with 5 identical pulse only trials. Both the pre and post pulse trials were excluded from analysis.

The remaining trials consisted of pseudorandomized presentation within 10 trial blocks of a blank (no stimulus) trial, pulse only trial, and four different types of prepulse+pulse trials Stereotaxic injections and chronic cranial windows. Mice were anesthetized with ketamine (Imalgen 1000, 10% in PBS; Rhone Mérieux) and xylazine (Rompun, 2% in PBS; Bayer AG), 10 ml/kg i.p. and placed into a stereotaxic frame. Mice were kept on a thermal blanket and their eyes were protected with artificial tear ointment. The scalp was washed with three alternating swabs of 70% ethanol and betadine. Surgical tools were sterilized using a hot glass-bead sterilizer. The skull was exposed and 1% xylocaine applied to the periosteum. A chronic cranial window was prepared as previously described 9 (Supplementary Fig. 1).

Briefly, the skull was gently thinned using a dental drill around the region of interest and the thinned bone was removed using forceps leaving the dura intact. GC/ml, was injected bilaterally at the same coordinates as described above. The glass pipette was left in situ for an additional 5 min, before being slowly withdrawn from the brain. The optical window was covered with a circular coverglass (5mm diameter) and was sealed to the skull with dental cement (Coffret SUPERBOND complet, Phymep). A sterile, small stainless steel bar with a screw hole was embedded into the acrylic over the cerebellum to head-fix the mouse for the imaging sessions and all exposed skull was covered with dental cement.

Lentiviral expression vector and stereotaxic procedure. The lentiviral expression vectors are derived from the pHR' expression vectors first described by Naldini et al 10 with several subsequent modifications. In the lentivirus used in this study, expression of the mCherry cDNA is under the control of the mouse phosphoglycerate kinase (PGK) promoter. Further details can be found in 11 . Briefly, to create the pLV-PGKmCherry-WPRE, a two-step strategy was used: First, the mCherry cDNA was amplified from pRSET plasmid (a kind gift of Dr. Shahragim Tajbakhsh's laboratory), using the following primers, forward: 5'-GTACTCGAGCCACCATGGTGAGCAAGGGC-3', and reverse: 5'-GCTGACGCGGCCGCTTACTTGTACAGCTCGTCCATG-3', and the T/A cloning strategy was used to introduce mCherry cDNA into a pGMET Easy vector® (Promega Corporation Madison, USA). Next, we took advantage of the presence of the XhoI site in the multiple cloning region of the vector to replace in the pre-existing pTRIP-PGK-eGFP lentiviral vector 11 , the fragment coding for eGFP by the cDNA of mCherry between the XhoI and BsrGI sites. This viral vector was used as a control injection for the β2 rescue experiments.

To create the pLV pmPGK-β2-IRES-tdTomato-WPRE we used a PTP-β2-IRES-GFP-WPRE previously described 11 and replaced the full IRES-GFP sequence with IRES-tdTomato available from Clotech (pLVX-IRES-tdTomato) by using the gateway system. Lentivectors were diluted in PBS before stereotaxic injection to achieve a dose corresponding to 250 ng of p24 protein in 2 µl.

The production of the conditional lentivectors was performed as previously described 12 .

Briefly, we used the lentivector pTrip PDGF-floxed-mCherry-eGFP-WPRE 5 CRISPR/Cas9 Nuclease RNA-guided genome editing. We used the CRISPR-associated endonuclease (Cas)9 technology for the deletion of the α5 subunit (Sigma-Aldrich). After screening of several target sites on the CHRNA5 gene, the following target site was chosen:

TGTCACTCAGGTGTTCCACAGG. The lentiviral vector uses a one-plasmid system consisting of an EF1-alpha-driven Cas9, a U6-driven guide RNA (gRNA) with both puromycin and GFP co-expressed with Cas9. 2.5µg U6-gRNA were nucleofected with 5µg

Cas9 plasmid. In vitro validation was performed in mouse N2a (Neuro2a) neuroblastoma cell line (Sigma-Aldrich). Insertions and deletions were detected at the gRNA target via mismatch assay (CEL-1 endonuclease) using the following primers, forward: 5' -GATTATTGATAACATTGAGTTTC -3' and reverse: 5' -CTCTAGTGGCTAACTCACAC-3'. As control we used a lentiviral plasmid vector that includes a gRNA sequence that does not target known human, mouse, or rat genes (CRISPR12-1EA Sigma-Aldrich). Viral particles were generated by co-transfection of HEK-293T cells with the vector plasmid, as described above. For the α5-Cas9 lentiviral vector 49 ng of p24 protein in 1 µl were locally injected in layer II/III of PFC, whereas the control virus was diluted in PBS before stereotaxic injection to achieve a dose corresponding to 50 ng of p24 protein in 1 µl. In addition, 100 nl of AAV1.CAG.tdTomato.WPRE.SV40 1.52e13 GC/ml (University of Pennsylvania Vector Core) were added in the solution to help us localize the cells transduced with the CRISPR.

Mouse handling for awake imaging. Mice were habituated to the imaging environment by handling and training for 4 days, as described previously 13,14 . Briefly, the animals were habituated to the following conditions, sequentially: 1) gentle handling, 2) free exploration of a 50 ml open ended support tube (for the mouse to rest in during imaging), 3) incremental periods of mild head fixation by briefly securing the head post (5s to 15s) and 4) extended periods of head fixation on the mouse stage while resting in the support tube (5 min to 60 min).

Immunofluorescence. At the end of the final two-photon imaging session, immunofluorescence was performed in order to verify the injection site. Fixation was carried out by trans-cardiac perfusion of anesthetized mice using 4% PFA, the brains were removed ALZET DURECT) were implanted s.c. at the nape of the neck as previously described 15 .

Nicotine or saline were delivered at a constant rate (2.4 mg/kg per day for nicotine).

In vivo two-photon imaging. In vivo imaging was performed with an Ultima IV two-photon laser-scanning microscope system (Bruker), using a 16x 0. Regions of interest (ROIs) were manually selected in FIJI and detection of Ca 2+ transients of individual neurons was performed automatically by using a custom-written toolbox in MATLAB (Mathworks). For details on the analysis method see Supplementary Information. Transients larger than 4x the standard deviation of the baseline fluctuations were selected for further analysis.

We next set out to identify putative unitary Ca 2+ transients that result from single action potentials, for use as a kernel for a deconvolution algorithm to estimate action potential (spike) times. Ca 2 transients were first selected according to their shape. It is a reasonable assumption that unitary calcium transients detected with GCaMP6f should have a rapid rise period, followed by a single peak value and a longer decay period 2 . For kernel estimation, we first selected transients exhibiting a single peak above 4σ (Supplementary Fig. 4a). Second, we selected a subset of these traces in which the decay was well fit by an exponential function for the entire decay and were not contaminated by a second event. We fit the first 750 ms of the decay with an exponential decay function, then examined the ratio between the duration when the signal was above the fitted curve and the total decay time (Supplementary Fig. 4b andc). Ratios above 0.9 were rejected. This criterion essentially eliminates transient second events and/or large fluorescence fluctuations not associated with spikes. Third, we rejected transients with rise times slower than the decay times (Supplementary Fig. 4c). Transients that passed this "shape control" were used for further analysis of their amplitude, rise time and decay time distributions selecting putative unitary events (Supplementary Fig. 5). In order to determine a kernel, we selected transients with amplitudes and fastest rise time and decay time distributions centered on the mode of the distributions. The kernel is then obtained from the fitting of an alpha function to the average of all selected Ca 2+ transients. This procedure was performed on all recorded transients separately for each experimental condition (i.e., WT or KO, etc).

Validation of spike detection algorithm. In order to test the efficacy of our spike selection algorithm, we simulated calcium signals through the convolution of the estimated kernel for WT mice and a simulated spike train, generated through a Poisson process (Supplementary Fig. 6a), with the addition of Gaussian noise. After the use of the transient detection algorithm on this simulated signal, we used the deconvolution algorithm on the selected transients. First, selected calcium transients were smoothed using a Gaussian filter. The filtered calcium transients were then deconvolved by computing the inverse Fast Fourier

Transform of the signal's Fast Fourier Transform divided by the kernel's Fast Fourier

Transform (Supplementary Fig. 6b). Next, we computed the firing rate error between the reconstructed spike trains and the original spike trains, for different Gaussian noise levels and different mean firing rates. See Supplementary Fig. 6c1, c2 for the noise level distribution for each recorded cell in WT mice. The noise level was estimated in each signal as the standard deviation in a segment of ~7.5 seconds duration with the lowest variability. We considered noise levels between µ + σ (µ, mean, σ, standard deviation of the distribution) and µσ for the computation of reconstruction error. Most reconstruction error values corresponded to an underestimation (with a maximum of 4 spikes/min for the highest noise levels) of the original spike train activity. They were not dependent on the firing rate. For the mean noise level computed in the experimental signals, the mean reconstruction error corresponded to an underestimation of 0.1 spikes/min of the original spike train activity, which is very low compared to the order of magnitude of the spiking activities computed in the signal (tens of spikes/min). Furthermore, the kernel estimation algorithm permits a good reconstruction of the original WT kernel (Supplementary Fig. 6d).

Supplementary Discussion

Social interaction deficit of α5 SNP mice. The social preference index was calculated as the amount of time exploring in the social interaction zone (within 2 cm of the enclosed mouse)

divided by the total time spend exploring both interaction zones. The amount of time the mice spent in each of the chambers containing objects (social or inanimate) was also determined.

Similar to the results from the social preference index, α5 SNP mice showed no preference for the chamber with the social object (46.8 ± 8.4% of total time in either chamber) while the WT mice exhibited a preference for the chamber with the social object (62.4 ± 6.5%). The percentage of time spent in the social chamber relative to the chamber with the inanimate object differed significantly by genotype (P < 0.05).

PPI deficit of

Article 2 Overview

The human brain exhibits spontaneous activity that is characterized by ultra-slow fluctuations (USFs). This phenomenon is considered to play an important role in conscious processing in humans. The aim of my work was to identify USFs in mice and characterize the role of specific nAChR subunits in the generation of USFs and the synchronous firing of neurons during quiet wakefulness or light general anesthesia. Using transgenic mice and twophoton calcium imaging in the PFC, a brain region involved in higher cognitive processes, I showed for the first time that:

1. Mice exhibit USFs that contribute to PFC spontaneous neuronal activity.

2. General anesthesia disrupts neuronal synchronicity.

3. Both the α7 and β2 nAChR subunits play a key role in the generation of USFs.

4. The β2 subunit is specifically required for synchronized activity patterns.

5. Chronic application of mecamylamine, an antagonist of nAChRs, disrupts the generation of USFs and mimics the β2 KO phenotype.

Overall, these findings allow further dissection of the mechanisms involved in the generation of USFs in the awake and anesthetized state and provide new insights into the role of cholinergic neurotransmission in cognitive functions.

My personal contribution for this article was to design and perform all the experiments. I also participated in the data analysis and I wrote the manuscript.

Introduction

The PFC plays an important role in cognitive processes such as attention (1), working memory ( 2), decision making (3), social behavior (4) and emotions (5). Current theories consider the PFC as a key player in conscious processing (6)(7)(8). Deficits in prefrontal function, including attention, are noted in several neuropsychiatric disorders, including schizophrenia, attention deficit/hyperactivity syndrome (ADHD), addiction, depression and autism (9).

In humans, as in rodent models, cholinergic innervation of the PFC regulates cognitive processes and neuronal activity. For instance, in rodents, removing PFC cholinergic innervation reduces attention performance, whereas stimulation of cholinergic projections causes enhancement (10). Many studies in both animal models and humans have shown that nAChRs are of particular importance for cognitive functions, reward, aging and for pathologies like Alzheimer's Disease (AD) (11,12). For instance, deletion of the α7 and β2 nAChR subunits in mice impairs behaviors, such as exploration, and attention (13)(14)(15)(16)(17).

Importantly, lesions of the prelimbic cortex (PrLC) in wild-type (WT) mice cause deficits in social behavior, similar to those observed in β2 KO mice, whereas re-expression of the β2 subunit in the PrL of β2 KO mice rescues their social interaction (4). Similarly, re-expression of β2 subunits in the PrL cortex of β2 KO mice fully restored their attentional performance in the 5-choice serial reaction time task (5-CSRTT) (17).

Ongoing spontaneous activity is known to occur in developing and adult brain and its physiological importance on the "default" network of the resting brain has been emphasized (18). Recordings in humans indicate that ongoing activity constantly fluctuates in a tightly correlated manner across distant brain regions forming reproducible patterns with rich temporal dynamics and spatial organization (19). The potential contribution of spontaneous activity to conscious processing has been suggested and simulations helped characterize two main modes of activity: an active mode characterized by rapid and sustained activity (''ignition'') (20) which contributes to the signature of "conscious access", and a resting mode with spontaneous ultra-slow (<0.1 Hz) fluctuations (USFs) of low amplitude (19). It has been suggested that ignition and ultra-slow spontaneous fluctuations (19), share similar mechanisms. Independently, electrophysiological recordings in animal models have distinguished "up" and "down" states (21), and a possibility is considered that "up" states coincide with USFs in humans. Furthermore, in vivo recordings in animal models (22) reveal that, similar to humans, USFs persist under general anesthesia, yet with a lower amplitude (23)(24)(25). Under anesthesia, the dominating functional configurations have low information capacity and lack negative correlations (26). Conversely, in the awake state a dynamic exploration of a rich, flexible repertoire of functional configurations takes place, including ignition, in the course of conscious access. Yet, to our knowledge, the pharmacology and biochemistry of these processes remain unexplored.

To address this gap in our knowledge, using in vivo Ca 2+ imaging, we recorded and compared the spontaneous activity patterns in PrLC of both awake mice and mice under general anesthesia, a condition where conscious processing is known to be altered (27). These recordings point to spontaneous and infrequent coherent states with high firing rates, analogous to USFs. Our findings reveal a distinct role of nAChRs in the ongoing neuronal activity of PFC in awake and anesthetized animals, shedding light on a possible involvement of nAChRs in conscious processing.

Results

Ultra-slow fluctuations (USFs) contribute to mouse PFC spontaneous activity

The discovery of the "default mode" network ( 28) started a long-lasting interest in the significance of the human brain's ongoing or intrinsic activity, which is characterized by synchronous ultra-slow fluctuations of activity, often taking seconds to develop (19). USFs have been recorded in the cortex of awake humans and primates, and also under anesthesia (29)(30)(31). The neuronal mechanisms that generate spontaneous resting-state fluctuations, together with the ignition dynamics, are unknown and remain a matter of debate.

To explore these issues in vivo, we used the mouse as a model together with twophoton imaging of spontaneous neuronal activity recorded through a chronic cranial window in layer II/III of PrLC (Figs. S1A andB). Neurons of PrLC were transduced with an adenoassociated viral vector (AAV) expressing the fluorescent calcium indicator GCaMP6f (see

Materials and Methods

). Four weeks after AAV injection, the majority of layer II/III neurons exhibited green fluorescence (Figs. S1C to E). We first studied the activity patterns of three month old awake head-fixed WT mice, where we simultaneously monitored the spontaneously occurring somatic GCaMP6f Ca 2+ transients in multiple individual cells, and estimated the neural spiking rates through deconvolution of calcium transients (see Materials and Methods). The mice were awake and reactive, as monitored by an infrared camera. A typical example of neural population activity is shown in Fig. 1A. In order to identify patterns of activity and USFs in populations of simultaneously recorded neurons, we studied the distribution of their time varying mean activity (see Materials and Methods for details). In Fig. 1, the USFs correspond to population activities in red, and basal activities correspond to population activities in blue (Figs. 1B andC). Interestingly, awake WT mice exhibited USFs with an average interval of 14.43 ± 1.54 s. This period corresponds to a frequency of 0.069 ± 0.0074 Hz and is similar to that observed in humans (below 0.1 Hz) (32,33). These frequencies are slower than those observed in the cardiac (0.6 to 1.2 Hz) and respiratory cycles (0.1 to 0.5 Hz) (32). We then analyzed the activity patterns to detect synchronous activity in populations of simultaneously imaged neurons as previously described (21) (Materials and Methods). The number of simultaneously imaged cells ranged from 4 to 71 neurons, with a mean value of 36 neurons. In the awake state, we detected robust synchronicity in the neuronal populations (Fig. 1D1). Our recordings indicate that, similar to awake humans, the ongoing activity in a mouse brain constantly fluctuates and exhibits synchronously firing neuronal activity, making the mouse a reliable model for studying some of the elementary physiological processes associated with conscious processing in humans.

USFs persist under general anesthesia, but synchronicity is disrupted

General anesthesia differentially alters states of consciousness and access to conscious content (8). The ability of general anesthesia to induce safe and reversible loss of consciousness poses the most complex question of how a simple chemical can affect conscious experience (34). General anesthetics may directly or indirectly affect conscious processing by the cerebral cortex, however, the neurobiological mechanisms involved in this interaction are largely unknown. We thus performed Ca 2+ imaging of pyramidal cells in the PrLC of lightly anesthetized WT mice (0.8% isoflurane), and compared their activity patterns with the ones recorded in awake mice (Fig. 1A2-D2). Interestingly, the USFs persist under anesthesia, with no significant difference between the populations that exhibit USFs in the awake state (90.30 ± 5.03 %), when compared to the anesthetized state (88.38 ± 5.82 %) (ANOVA test) (Fig. 1E). Anesthetized WT mice exhibited USFs with an average interval of 12.97 ± 2.16 s, corresponding to a frequency of 0.077 ± 0.013 Hz. However, we observed a robust reduction of the synchronously firing populations under anesthesia (55.87 ± 11.38 %), compared to awake mice (95.35 ± 3.67) (P<0.001, ANOVA) (Fig. 1F). Therefore, USFs share similar mechanisms between awake and anesthetized conditions but anesthesia shows strong inhibitory effects on the generation of neuronal synchronicity. KO mice (n = 3459 cells in 11 mice) with a significant difference between WT and β2 KO groups (P = 0.0130, ANOVA, Fig. S2A1). In each population of simultaneously recorded neurons with USFs/basal activity transitions, we determined the percentage of cells that exhibit an activity pattern in accordance with the population activity. In awake WT animals, 96.6 ± 1.8 % of cells fire in accordance with their population activity, in α7 KO, 88.23 ± 4.12 % and in β2 KO 84.37 ± 2.27 % (Fig. S2B1). In order to compute the percentage of cells with USFs/basal activity, for each mouse group, we multiplied the percentage of populations with USFs/basal activity with the mean percentage of cells in accordance with the population patterns of activity, for each mouse. Interestingly, we found that 82.41 ± 5.28 % of simultaneously recorded cells exhibited USFs in WT, 69.31 ± 7.4 % in α7 KO mice and 54.85 ± 5.9 % in β2 KO mice, with a significant difference between WT and β2 KO (P = 0.007, ANOVA, Fig. 3A). Next, we computed the USFs/basal activity properties in all populations.

Differential role of nAChRs on the generation of

The basal activity duration was significantly lower for the β2 KO (7.36 ± 1.19 s), when compared to α7 KO (11.69 ± 2.43 seconds) (P = 0.0021) and WT mice (14.43 ± 1.54 s) (P < 0.001, Kruskal-Wallis) (Fig. 3B), whereas the USFs duration was significantly higher for the the β2 KO (5.11 ± 0.64 s), when compared to α7 KO (3.89 ± 0.65 seconds) (P = 0.0022) and WT mice (3.03 ± 0.38 s) (P < 0.001, Kruskal-Wallis) (Fig. 3C).

We then analyzed and compared the synchronicity in the different animal groups. In the WT and α7 KO mice, neurons displayed strong synchronous activity in 95.35 ± 3.67 % (40 populations) and 88.33 ± 11.66 % (38 populations) of the recorded populations, respectively, with no significant difference between groups (P = 0.70, ANOVA) (Fig. 3D). In the β2 KO mice, the patterns of activity were remarkably different from that in WT (P < 0.001) and α7 KO mice (P = 0.046), with synchronous activity detected in only 63.91 ± 5.38 % of the recorded populations (99 populations) (Fig. 3D). The number of synchrony peaks detected was similar between WT mice (30.59 ± 3.73 peaks/min) and α7 KO (31 ± 6.77 peaks/min) (P = 0.99, ANOVA) whereas a robust decrease was observed in the case of β2 KO mice (4.52 ± 0.72 peaks/min) (P < 0.001, ANOVA) (Fig. 3E). In addition, the percentage of coactive cells in the peaks of synchrony was significantly higher for α7 KO (53.34 ± 0.51 %) and β2 KO mice (53.30 ± 1.05 %) compared to the WT mice (47.71 ± 0.47 %) (P < 0.001, ANOVA, Fig. 3F).

Overall, USFs were still detected in mice with deleted nAChR subunits. Yet, β2 KO mice showed reductions in the percentage of cells that exhibit USFs and a strong decrease in the percentage of synchronously firing neuronal populations. These data indicate a potentially major role for β2 subunits in the generation of physiological phenomena associated with conscious processing in the awake state.

Role of nAChRs in ongoing activity under general anesthesia

The nAChRs are possible direct/indirect targets of general anesthetics and as a consequence may interfere with cholinergic transmission. For instance, the volatile anesthetic isoflurane shows high affinity for nAChRs (35,36). Yet, isoflurane inhibition has been found

to vary with nAChR subunit composition (37). We aimed to study in vivo the contribution of defined nAChR subunits to spontaneous activity under conditions of light general anesthesia.

Typical examples of the effect on the neuronal population activity in α7 and β2 KO anesthetized mice are shown in Figure 4. We found that 88.4 ± 5.8 % of simultaneously recorded populations exhibited USFs/basal activity in WT, 76.6 ± 14.5 % in α7 KO mice and 85.7 ± 4.9 % in β2 KO mice, with no significant difference between the groups (Fig. S2A2).

Furthermore, in WT mice 96.42 ± 4.28 % of cells fire in accordance with their population activity, 91.66 ± 6.55 % in α7 KO, and 83.82 ± 2.2 % in β2 KO (Fig. S2B2).

We also found that 76.68 ± 4.35 % of simultaneously recorded cells exhibited USFs in WT, 65.89 ± 14.16 % in α7 KO and 68.35 ± 4.3 % in β2 KO mice, with no significant difference (Fig. 5A). Next, we computed the USFs/basal activity properties in all populations.

Under anesthesia, there were no significant changes of basal activity state duration between animal types (P > 0.14, Kruskal-Wallis), nor any significant changes in variability (P > 0.49, Ansari-Bradley) (Fig. 5B). Conversely, the USFs duration was significantly higher for the α7 (129 populations), with no significant differences (P > 0.9, ANOVA) between groups (Fig. 5D). Interestingly, in the anesthetized state, the number of synchrony peaks detected was similar between WT mice (0.79 ± 0.16 peaks/min) and α7 KO (1.41 ± 0.52 peaks/min), whereas a robust increase was observed in the case of β2 KO mice (4.58 ± 0.62 peaks/min) (P < 0.001, ANOVA) (Fig. 5E). Finally, the percentage of coactive cells in the peaks of synchrony was significantly higher for the WT (41.03 ± 1.03 %) and α7 KO (46.30 ± 1.25 %) compared to β2 KO mice (21.74 ± 0.19 %) (P < 0.001, ANOVA) (Fig. 5F).

In conclusion, the deletion of nAChR α7 and β2 subunits has differential effects in awake mice on USFs duration, while, in the anesthetized state, β2 deletion alone increases synchronicity and decreases the percentage of coactive cells in the peaks of synchrony, compared to WT and α7 KO.

Brain exposed to chronic mecamylamine resembles nAChR KO phenotype

Finally, we aimed to compare the neuronal activity patterns in the PFC of WT mice after chronic application of mecamylamine, a non-selective, non-competitive antagonist of nAChRs. Osmotic minipumps for the infusion of either saline or mecamylamine (1mg/kg per day for mecamylamine) were implanted s.c. at the nape of the neck of WT mice. We recorded the neuronal activity patterns 7 days after the inmpantation of the minipumps. Typical examples of neuronal population activities in awake mice under saline or mecamylamine are shown in Figure 6. We found that 94.44 ± 5.55 % of simultaneously recorded populations exhibited USFs/basal activity in WT under saline condition (n = 743 cells in 3 mice) and 69.44 ± 2.77 % in WT under mecamylamine (n = 446 cells in 3 mice) with a significant difference between the two groups (P = 0.015, ANOVA, Fig. S3A). In awake WT animals under saline conditions, 98.21 ± 2.4 % of cells fire in accordance with their population activity, whereas in WT under mecamylamine 90.32 ± 2.4% (P = 0.041, ANOVA, Fig. S3B).

Discussion

In humans, two distinct modes of electrical activation of the cerebral cortex have been recorded: a rapid and sustained activation termed "ignition", which is viewed as a signature of conscious access, whereas synchronous ultra-slow fluctuations with low amplitude characterize the spontaneous activity mode termed "resting state". In the resting state, in the absence of explicit task performance or external stimulus perception, the cortex exhibits a highly informative mode of spontaneous activity. Studies in anesthetized animals (22) and in humans with BOLD fMRI data ( 29), together with animal recordings of firing rate modulations (23), and EEG (25) and ECoG in humans (23,38) reveal ultra-slow (<0.1 Hz) dynamic activations with low amplitude, that we termed USFs, which may be viewed as characterizing the resting state. Here, we aimed to elucidate the mechanisms engaged in these cortical activity dynamics at single cell resolution using in vivo two-photon imaging in the mouse. We recorded ongoing spontaneous activity in the PFC of WT and mice deleted for specific nAChRs subunits, both in awake and anesthetized states. Interestingly, we recorded USFs in the mouse cortex and found that anesthesia shows inhibitory effects by disrupting the synchronous USFs firing of neurons. We further showed that, first, nAChRs have an important role in the generation of USFs, yet to a different extent during quiet wakefulness and anesthesia, and that the β2 subunit is specifically required for synchronized activity patterns. Our recordings indicate that, similar to humans, the ongoing activity in mouse brain constantly fluctuates and exhibits synchronously firing neuronal activity, making the mouse a reliable, though simplified model, for studying mechanisms of conscious processing in humans.

Current theories consider the PFC as a key player in conscious processing (6,39).

According to the Global Neuronal Workspace (GNW) theory, a subset of cortical pyramidal cells with long-range excitatory axons, that are particularly dense in prefrontal, cingulate, and parietal regions, together with the relevant thalamo-cortical loops, form a horizontal "neuronal workspace" interconnecting the multiple specialized, automatic, and nonconscious processors. A conscious content is assumed to be encoded by the "all or none" sustained and synchronous ignition of a fraction of GNW neurons (7,8). In these circumstances, the nonamplified neurons would be inhibited and the GNW sustained activity would represent the conscious content. In the PFC, pyramidal neurons in layer II/III do not express nAChRs, only interneurons do, in contrast to layers V and VI, where nAChRs are also expressed by pyramidal neurons (40). Also, pyramidal neurons with long axons, which have been postulated to play a critical role in the GNW theory (41), are more abundant in these particular layers of the cerebral cortex and especially in the PFC (42). By recording neuronal activity patterns in layer II/III of PFC, we found that α7 and β2 nAChRs play an important role in the generation of USFs that occur yet to a different extent during quiet wakefulness and anesthesia. We showed that the balance of recurrent excitation and inhibition is disrupted in the case of awake β2 KO mice, and more specifically, the neuronal synchronicity. It has been reported that the β2 subunit is involved in the dendritic morphogenesis of pyramidal neurons, and in particular, in the circuits that contribute to the high-order functional connectivity of the cerebral cortex (43). These defects in the maturation of the cerebral cortex that have been reported in the β2 KO mice could contribute to the observed behavioral deficits (4,14,17). However, our data reveal that pharmacological intervention with nicotinic antagonists is enough for the disruption of USFs mechanisms. Our findings suggest an important contribution of nAChRs in the processing of neural information during quiet wakefulness.

Moreover, an important role of the cholinergic innervation of the cerebral cortex was postulated (44) to be the mediation of arousal state. This hypothesis was based on the initial observation that the cholinergic inputs are diffusely distributed in the cerebral cortex and, most interestingly for us, exhibits a slow release of ACh on the same time scale as the USFs.

Several studies have further shown that cholinergic projections to the cortex are involved in sustained attention and cue detection, with the level of ACh efflux in the PFC correlating with the demand upon attention during attentional tasks (45). The slow fluctuations in ACh levels correlate with a shift in behavior, indicating that these fluctuations could correlate with decision-making (45,46). Indeed, experimental evidence from both rodents and humans revealed that ACh release contributes to mechanisms that mediate the integration of external cues with internal representations to initiate and guide behavior (47). At the scale of seconds, ACh undergoes phasic release, which may exert a top down control over defined cognitive operations, while muscarinic ACh receptors also play an important role in attentional behavior and cue detection (48). Recently, it has been shown in vitro that endogenously released ACh can modulate up and down states through the activation of nAChRs (49). The alterations of neuronal activity patterns that our results revealed in the nAChR KO mice could further our understanding of the cholinergic system in higher brain functions and, also, when disrupted, the contribution of nAChRs to cognitive disorders.

The focus of the present study was to elucidate the role of nAChRs in PFC activity when comparing resting state dynamics in awake and anesthetized animals. Since ongoing spontaneous activity has been postulated to have a significant functional role in cortical functions (50), we focused on how it differs between awake and anesthetized states. The production of ultra-slow fluctuations, or USFs, was identified and compared among the different conditions investigated. Our work demonstrates, in vivo, that nAChRs play a crucial role in the modulation of PFC synchronous spontaneous neuronal activity. Furthermore, with recent studies probing the mechanisms of general anesthesia as causing loss of consciousness, our work exploring the effects of anesthesia on nAChRs in the PFC network in vivo, at cellular resolution, reveals an elaborate mechanism of modulation of cortical activity. Our results provide a starting point for understanding the relationship between nAChRs and loss of consciousness under light anesthesia in mice, that might potentially be a reliable, though highly simplified, model for studying mechanisms of conscious processing in humans. As a future goal, it is important to consider additional studies in order to evaluate the presence of USFs during behavioral assays. Since several studies have shown that deletion in mice of the α7 and β2 subunits impairs behaviors, such as exploratory behavior and attention, it will be important to explore the behavioral deficits in parallel with neuronal recordings to establish a causal relationship.

Materials and Methods

Mice.

All experiments were performed on male mice at 3 months of age. Male α7 KO ( 51 and the mouse was secured in a stereotaxic frame. The eyes were protected with artificial tear ointment and body temperature was maintained with a feedback heating pad. The scalp was washed thoroughly with betadine and 70% ethanol and all surgical tools were sterilized.

Xylocaine (1%) was used for local anesthesia and the skull was exposed. As previously described ( 52), a chronic cranial window as prepared after injection of 200 nl of (serotype 1 (rep/cap: 2/1) AAV virus (AAV.syn.GCaMP6f.WPRE.SV40, 2.2e13 GC/ml, University of Pennsylvania Vector Core) was injected bilaterally at the following coordinates PrLC: AP, +2.8 mm from bregma; L, ±0.5 mm; and DV, -0.5 to -0.1 mm from the skull using a Nanoject II TM (Drummond Scientific) at the slow infusion setting. The glass pipette was left for 5 min in the brain before slowly being removed. A 5 mm diameter circular coverglass was placed on the brain surface and the edge was sealed to the skull with dental cement (Coffret SUPERBOND complete, Phymep). A sterile small stainless steel head bar with a screw hole was glued to the skull and the surrounding exposed skull was covered with dental cement.

Surgical implantation of minipumps. Mice were anesthetized with a xylazine and ketamine combination (15% xylazine/2.5%ketamine/82.5% PBS). Osmotic minipumps (model 2004, ALZET DURECT) were implanted s.c. at the nape of the neck as previously described 15 .

Mecamylamine or saline were delivered at a constant rate (1 mg/kg per day for mecamylamine).

Mouse handling for awake imaging As previously described (53,54), for 3 to 5 days, mice were habituated to the imaging environment by handling and training.

Immunofluorescence. Immunofluorescence was performed in order to identify the area of injection at the end of the two-photon imaging experiments. Mice were transcardiac-perfused with 4% PFA, the brains were removed and post-fixed with immersion in PFA for 2 days at 4°C, followed by immersed in 30 % sucrose in PBS overnight for cryoprotection. Using a sliding microtome (Leica Microsystems), serial 40 µm coronal sections were cut, mounted on slides and cover-slipped with ProLong Gold antifade reagent containing DAPI (Invitrogen).

Microscopy was carried out using a Zeiss fluorescence microscope.

In vivo two-photon imaging. In vivo imaging was performed with an Ultima IV two-photon laser-scanning microscope system (Bruker), using a 16x 0.8 NA water immersion objective (Nikon) with a femtosecond laser (MaiTai DeepSee, Spectra Physics, Mountain View, CA, USA) tuned to 950 nm for imaging of GCaMP6f expressing cells. Time-series movies of neuronal populations expressing GCaMP6f were acquired at 7 Hz (182 x 182 µm FOV; 0.71 µm/pixel). Each focal plane movie duration was 3.6 minutes (1500 frames) to track the spontaneous neuronal activity. For anesthetized mice, recordings were conducted under light isoflurane anesthesia (0.8% isoflurane in O 2 ).

Recordings in anesthetized mice. The mouse was placed into a transparent induction chamber (Harvard Apparatus) that allows observing the mouse and anesthesia was induced at a concentration of 4% vaporized isoflurane/O 2 (Forene, Abbott France; vaporizer, Harvard Apparatus) leading to a rapid induction of anesthesia within 30 seconds as tested by loss of righting reflex and tail pinch. The mouse was then removed from the induction chamber and immediately secured to the mouse frame on the two-photon microscope stage. The mice were then continuously supplied with 0.8% isoflurane/O 2 through a facemask. Body temperature was maintained at 37° using a thermometer feedback thermal blanket. Artificial tear ointment was also applied to the eyes to prevent dryness. The recordings started approximately 15 minutes after placing the animal on the stage and anesthesia was maintained at 0.8% until the end of the experiments. Under these conditions the mice were completely immobile and had no reaction to tail pinch.

Data analysis.

Image analysis was performed off-line with ImageJ software. The time series were corrected using the FIJI plugin 'Image Stabilizer' (K. Li, "The image stabilizer plugin for ImageJ," http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html,). Regions of interest (ROIs) were manually selected in FIJI and detection of Ca 2+ transients of individual neurons was performed automatically using a custom-written toolbox in MATLAB. A baseline correction algorithm was utilized in order to remove the slow time scale (< 0.05 Hz) changes in the fluorescence as described previously (55). We assumed the smallest and fasted Ca 2+ transients were a result of a single action potential. We used the mean shape and amplitude of this unitary event as a kernel for deconvolution to best estimate action potential or spike frequency. The custom-written toolbox in MATLAB (Mathworks, 2014b) is available upon request.

For the analysis of USFs, the time varying population activity was computed as the mean activity across neurons for each time bin, and was smoothed through Gaussian filtering. The distribution (Probability density function estimate) of the activity values was plotted at each time point that exhibits multi-modality or uni-modality. In order to identify the type of modality of the distribution, we used Gaussian filtering (black lines in Fig. 1C). The number of peaks determined the types of activity: multiple peaks are the signatures for USFs vs basal activity states. The threshold between USFs vs basal activity states was computed by taking the minimum of the multimodal distributions between the highest amplitude peak and its neighboring peak (red dotted lines in Fig. 1C). In some cases, where one of the activity states was very short (in the case of WT), a Gaussian or logistic function could be fitted around the highest amplitude peak (according to the RMSE output), and the activity state with low durations could be identified by taking values > 99% or < 1% of the cumulative sums. The USFs correspond to population activities in red, and basal activities correspond to population activities in blue (Fig. 3C). Our analysis method is based on a previously described technique (57).

We identified synchronously firing neurons based on a method that was previously described (21). For each population, small time bins (~0.144 seconds) were used to sum the spiking activity of all the cells. To identify peaks of synchronous activity that included more cells than expected by chance, interval reshuffling (randomly reordering of intervals between events for each cell) was used to create sets of event sequences. Reshuffling was carried out 1,000 times for each population and a histogram was constructed for each reshuffling. The threshold corresponding to a significant level of P < 0.05 (green dotted line) was estimated as the number of coactive cells exceeded in a time bin in only 1% of these histograms.

Statistical analysis.

Kruskal-Wallis one-way analysis of variance combined with multiple comparison testing was applied to the activities (spikes/min) of the neurons in all mouse groups in order to study the statistical similarities. For each mouse category, we used interpolation in order to account for the different number of neurons recorded in the different animals. In this way, our statistical analysis assigned the same weight to each neuronal population recorded in the different mice of a given category. One-way analysis of variance (ANOVA test) was used to compare the populations with USFs, the cells that exhibit USFs, the mean synchronously firing populations, the mean coactive cells in synchrony peaks and the number of synchrony peaks.

Article 3 Overview

Alzheimer's Disease is a major social and financial burden throughout the world. No treatment exists for the disease and there still is an enormous lack in our understanding of the disease mechanisms. The available animal models consisting of mostly transgenic and KO mice strategies have constitutive, ubiquitous over-expression of human genes throughout the life of the animal.

The aim of this article was to characterize a novel non-transgenic mouse model that I developed during my thesis. This model allows inducing Aβ pathology in a defined cortical area of adult mice. This is achieved with the stereotaxic injection of an adeno-associated viral vector that expresses the mutated human amyloid precursor protein, hAPP, in PFC. I then tracked the gradual accumulation of soluble Aβ species and their effect on cortical physiology using longitudinal two-photon imaging.

The main findings of this article can be summarized as follows:

1. The presence of soluble Aβ species changes the activity of cortical pyramidal cells in layer II/III of prelimbic cortex already one month after the injection of the vector.

2. The increase in pyramidal cell activity continues at six months of hAPP expression, contrasting with a decrease in the control as part of "normal" mouse brain aging. This new approach can also be used in species where transgenic approaches have not been established, like in non-human primates. In addition, my work sheds a different light on previously published work that implied a role of plaques in cortical hyperactivity by demonstrating that the soluble form of Aβ is sufficient in the very early stages of the disease to induce cortical hyperactivity.

My personal contribution for this article was to design and perform all the experiments. I also participated in the data analysis and I wrote the manuscript.

Introduction

Alzheimer's Disease (AD) is a devastating neurodegenerative condition that greatly impacts society, primarily affecting the elderly population, and will become an enormous burden as the population ages [START_REF] Castellani | Alzheimer disease[END_REF]. In the broad category of dementia, AD amounts to 70% of all cases and thereby is the most common form of dementia [START_REF] Fratiglioni | Worldwide prevalence and incidence of dementia[END_REF]. Although there are familial genetic mutations linked to AD, the sporadic form is the most prevelant. There is currently no cure available for AD, only symptomatic treatments are administered in order to slow down the progression of the clinical manifestations [START_REF] Small | Current and Future Treatments for Alzheimer Disease[END_REF].

Several genes have been implicated in AD in humans, most notably, those encoding the mutated APP, presenilin 1 and presenilin 2 (McGowan et al., 2006). Consequently, various transgenic mouse models of AD harboring mutations in these genes have been established to decipher the disease mechanism. Moreover, a significant advance in the field came from the development of transgenic rodent models that exhibit tau pathology (Oddo et al., 2003a;[START_REF] Cohen | A transgenic Alzheimer rat with plaques, tau pathology, behavioral impairment, oligomeric aβ, and frank neuronal loss[END_REF]. Although the results from these models have unraveled specific components of the disease pathology and given indications for developing potential therapies, there is no transgenic model that replicates the broad spectrum of AD pathology. In some cases, the mutations are associated with other pathologies, like frontotemporal dementia that is not part of AD pathology [START_REF] Götz | Animal models of Alzheimer's disease and frontotemporal dementia[END_REF]. Moreover, in transgenic mice that express human familial AD mutations, the gene is expressed throughout the brain, making it impossible to study disease induced changes in specific brain regions.

The two primary features associated with AD pathology are the senile plaques and the neurofibrillary lesions. However, over the last years, the 'toxic Aβ oligomer' hypothesis has attracted attention as a way of resolving the lack of correlation between deposited Aβ in amyloid plaques, in terms of amount, location and cognitive impairment, and neurodegeneration (Benilova et al., 2012). Aβ oligomers are associated with AD hallmarks, like inducing abnormal tau phosphorylation, oxidative stress and synapse loss [START_REF] De Felice | Alzheimer's disease-type neuronal tau hyperphosphorylation induced by A beta oligomers[END_REF]. Soluble and highly toxic forms of Aβ such as oligomers and protofibrils may be more directly linked to cellular pathology, however the balance between monomeric Aβ, oligomers and insoluble Aβ fibrils is poorly understood and the lack of an experimental description of the toxic Aβ oligomer makes conclusions difficult (Benilova et al., 2012). In addition, the acute short-term injection of Aβ oligomers, sometimes of defined composition, into the brain of rodents and non-human primates [START_REF] Forny-Germano | Alzheimer's disease-like pathology induced by amyloid-β oligomers in nonhuman primates[END_REF] does not allow studies on the progression of the disease over time. There still is a need for a model that replicates the regionality and timecourse of the disorder.

Here, we developed an adeno-associated virus (AAV) based model for replicating AD-like pathology. The AAV vector expresses the mutated form of human APP harboring three pathogenic mutations: Swedish, London and Austrian (hAPP-SLA) [START_REF] Goate | Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease[END_REF][START_REF] Mullan | A pathogenic mutation for probable Alzheimer's disease in the APP gene at the Nterminus of β-amyloid[END_REF][START_REF] Kumar-Singh | Nonfibrillar diffuse amyloid deposition due to a gamma(42)-secretase site mutation points to an essential role for N-truncated A beta(42) in Alzheimer's disease[END_REF]. We chose these mutatons because they are associated with early onset of the disease. We targeted the expression of hAPP-SLA to the PFC, due to this region's key role in cognitive processes and its crucial implication in AD pathology (Grady et al., 2001). Stereotaxic injections and chronic cranial windows. Twelve week old mice were anesthetized with ketamine (Imalgen 1000, 10% in PBS; Rhone Mérieux) and xylazine (Rompun, 2% in PBS; Bayer AG), 10 ml/kg i.p. The mice were placed in a stereotaxic frame, they were kept on a thermal blanket and their eyes were protected with artificial tear ointment. The scalp was washed with three alternating swabs of 70% ethanol and betadine. Surgical tools were sterilized using a hot glass-bead sterilizer. The skull was exposed and 1% xylocaine applied to the periosteum. A chronic cranial window was prepared as previously describe (Holtmaat et al., 2009). Briefly, the skull was carefully thinned using a dental drill over the region of interest and the thinned bone was removed using forceps, leaving the dura intact. Quantitative analysis of immunofluorescent images. The analysis of the Aβ diffusion, in vivo microglia images and the immunolabeling of astrocytes was performed using Fiji (ImageJ, NIH). For the microglia, 1024x1024 resolution images (163.225 x 163.225 um) were analyzed for both sham operated and AAV-hAPP injected mice. The same settings (laser power and gain) were used during the acquisition of the images. First, we set the scale in µm by adding the distance in pixels. For the quantification of cell area (somas and processes) the perimeter of the cells' process tips was manually marked using the polygon selection tool, whereas for the soma area, we manually traced the somas of the cells within the whole field.

Materials and Methods

Adeno

In the set measurements menu we selected "area and then measure". For the Aβ diffusion 5 slices were analyzed from 3 control mice and 5 slices from 3 AAV-hAPP-SLA injected mice.

For the microglia analysis, 10 in vivo two-photon images from 3 control mice and 8 in vivo In vivo two-photon imaging. In vivo imaging was performed with an Ultima IV two-photon laser-scanning microscope system (Bruker), using a 16x 0.8 NA water immersion objective (Nikon) with a femtosecond laser (MaiTai DeepSee, Spectra Physics, Mountain View, CA, USA) tuned to 950 nm for imaging of GCaMP6f expressing cells. Time-series movies of neuronal populations expressing GCaMP6f were acquired at 7 Hz (182 x 182 microns). Each focal plane movie duration was 3.6 minutes (1500 frames) to track spontaneous neuronal activity. Care was taken to use less than 10 mW of laser power at the surface of the tissue. For in vivo two-photon imaging of microglia cells of the CX3CR1 mice (also injected with the AAV1.CAG.tdTomato vector), the femtosecond laser tuned to 960 nm and laser power was kept below 5 mW to avoid phototoxic effects. Time series were acquired (1024x1024 pixels) at a 10-second interval for a total of 10 min (60 iterations).

Two-photon data analysis. Image analysis (also for immunostained images) was performed off-line with ImageJ software. The time series were registered using the "Image Stabilizer" plugin (K. Li, http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html). Regions of interest (ROIs) were manually selected in FIJI and processing of Ca 2+ transients of individual neurons was performed automatically by using a custom-written toolbox in MATLAB (Mathworks) based on a previously published method (Dombeck and Tank, 2014). A baseline correction algorithm was used in order to remove the slow time scale (< 0.05 Hz) changes in the fluorescence as previously described (Dombeck and Tank, 2014).

Code availability.

Results

Generation of an AAV vector to express human mutated hAPP

The mutant hAPP-SLA contained the Swedish, London and Austrian mutations. The Swedish mutation was chosen because it leads to familial AD, as seen in patients harboring just one allele of this dominant mutation [START_REF] Mullan | A pathogenic mutation for probable Alzheimer's disease in the APP gene at the Nterminus of β-amyloid[END_REF]. It has been widely used to establish transgenic mouse models of AD, for example the Tg2576 line [START_REF] Hsiao | Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice[END_REF], and in combination with other fAD associated mutations, such as PS1 (M146V) and tau (P301L) in the 3x-Tg mouse model (Oddo et al., 2003b). We also added the Austrian (T714I) (Kumar- Singh et al., 2000) and London (V717I) [START_REF] Goate | Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease[END_REF] mutations that when present in hAPP are known to drive a higher production of Aβ. We also added a FLAG tag at the Cterminus of the hAPP sequence to be able to detect the protein with anti-FLAG tag antibodies.

We thus produced a hAPP-SLA-FLAG construct (Fig. 1A).

Protein expression was confirmed in vitro in the HEK cell line. HEK cells were transduced with the vector expressing hAPP-SLA. The anti-FLAG antibody confirmed the synthesis of the hAPP protein (Fig. 1B)

In vivo detection of hAPP in the PFC

The main cellular pathology of AD consists of damage and loss of neurons in widespread areas of the cortex and hippocampus (Serrano-Pozo et al., 2011a). The cognitive impairments characteristic of dementia in humans, such as attentional deficits and short-term memory loss, indicate PFC pathology [START_REF] Larson | Cognitive impairment: dementia and Alzheimer's disease[END_REF][START_REF] Storandt | Rates of progression in mild cognitive impairment and early Alzheimer's disease[END_REF]. For this reason, we targeted the prelimbic area of the PFC, PrLC. A series of stereotaxic injections were performed in vivo to verify the efficiency of the AAV-hAPP-SLA in the PrLC of 3 month old WT mice. The vector showed high diffusion in the brain and sufficient expression of the transgene, as visualized one month post-injection using an anti-FLAG antibody (Fig. 2 panel 1) with the hAPP diffusing throughout the PFC. As shown in the mosaics, there is no labeling in other parts of the brain outside PFC (Fig. 2 panel 1).

AAV-hAPP-SLA drives Aß oligomer synthesis and intracellular accumulation

Most AD transgenic models exhibit memory impairments, with the cognitive deficits occuring earlier than the appearance of extracellular plaques. Research shifted to identify the precursors to plaque formation and to determine whether, and how, aggregation of Aβ was crucial to its toxicity. This led to the focus on soluble oligomeric Aβ species. As in AD transgenic mouse models, cognitive decline in humans is not proportional to Aβ plaque load [START_REF] Terry | Physical basis of cognitive alterations in Alzheimer's disease: synapse loss is the major correlate of cognitive impairment[END_REF], but does correlate with soluble Aβ species [START_REF] Wang | The levels of soluble versus insoluble brain Abeta distinguish Alzheimer's disease from normal and pathologic aging[END_REF].

Intraneuronal Aβ has gained experimental support in recent years, as, similar to humans, many hAPP AD transgenic mice exhibit intraneuronal amyloid accumulation [START_REF] Laferla | Intracellular amyloid-beta in Alzheimer's disease[END_REF]. The accumulation of intracellular Aβ has been shown to precede deposition.

Interestingly, it was found that intraneuronal Aβ strongly correlates with initial deficits on a hippocampal-based memory task [START_REF] Billings | Intraneuronal Abeta causes the onset of early Alzheimer's disease-related cognitive deficits in transgenic mice[END_REF] and that intraneuronal Aβ is more neurotoxic than extracellular Aβ [START_REF] Casas | Massive CA1/2 neuronal loss with intraneuronal and N-terminal truncated Abeta42 accumulation in a novel Alzheimer transgenic model[END_REF].

We investigated whether the expression of the hAPP-SLA protein was able to drive Aβ oligomer accumulation in our model. The presence of oligomeric Aβ was confirmed with anti-VHH 31-1 antibody, specific for oligomeric forms of Aβ (Lafaye et al., 2009).

Immunofluorescent images with this antibody in WT mice injected with AAV-hAPP-SLA showed abundant intracellular Aβ oligomer expression in the PFC at one month post-injection (1 mpi) of viral vector (Fig. 2 panels 2 to 3). Aβ synthesis was strictly related to hAPP-SLA expression, since Aβ oligomers were not observed in other brain areas, without viral transduction. There was no detection of Aβ oligomers in sham mice injected with the control vector AAV-CAG-tdTomato (Fig. 2E,F). We followed the Aβ oligomer accumulation over time and performed immunostaining analysis in mouse brains at various time points.

Significant Aβ production was also detected 4 and 6 mpi post injection of AAV-hAPP-SLA.

The diffusion of Aβ was significantly higher 4 and 6 mpi in comparison to 1 mpi of the AAV-hAPP-SLA (Fig. 2G).

Activation of migroglia by AAV-hAPP-SLA

Neurological disorders trigger local inflammation and consequently activation of the immune response. Specifically, AD is characterized by an inflammatory response to Aβ, including the activation of microglia and the recruitment of astrocytes around Aβ deposits (Sastre et al., 2006). Cause or consequence of this activation in the disease progression is still not clear.

Several studies in animal models suggest that microglia activation precedes amyloid plaques [START_REF] Itagaki | Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease[END_REF][START_REF] Jung | Fibrillar amyloid plaque formation precedes microglial activation[END_REF] and the formation of NFTs [START_REF] Bellucci | Induction of inflammatory mediators and microglial activation in mice transgenic for mutant human P301S tau protein[END_REF][START_REF] Yoshiyama | Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model[END_REF]. Once activated, microglia prominently change their morphology: the ramified processes swell and withdraw, while their cell bodies enlarge [START_REF] Stence | Dynamics of microglial activation: a confocal time-lapse analysis in hippocampal slices[END_REF][START_REF] Jonas | The spider effect: morphological and orienting classification of microglia in response to stimuli in vivo[END_REF]. Microglia activation occurs in the AD brain with microglia clusters forming around amyloid deposits as an early indicator of pathology and little is known about how this interaction is initiated. Here, we specifically focused on the early stages of the pathology and we validated our AD model by characterizing in vivo the process by which the microglia become activated in the presence of Aβ peptide.

CX3CR1-GFP +/-mice [START_REF] Virgone-Carlotta | Mapping and kinetics of microglia/neuron cell-to-cell contacts in the 6-OHDA murine model of Parkinson's disease[END_REF] were used to visualize microglia in order to characterize the morphological dynamics of microglia activation. The knockin of GFP at the Cx3cr1 locus in CX3CR1-GFP mice results in GFP-labeling of microglia.

CX3CR1-GFP +/-mice were injected with a mixture of AAV-hAPP-SLA and the AAV1.CAG.tdTomato, whereas control mice were injected only with the AAV1.CAG.tdTomato vector specifically in the PrLC. A cranial window was implanted (see Materials and Methods for details) and four weeks after the injection, the microglia were imaged by two-photon microscopy with the mouse lightly anesthetized with isoflurane. Tensecond interval imaging of control mice showed microglia that were characterized by a small cell body and highly elaborated thin processes, with multiple branches extending radially, a feature of the resting state (Fig. 3A1 and Supplementary Movies 1 and 2). Interestingly, in the mice injected with the AAV-hAPP-SLA vector, microglia were characterized by an amoeboid form, a feature of microglial activation (Fig. 3A2 and Supplementary Movie 3).

Their processes were retracted and the soma enlarged. Most of the activated microglia had a large round morphology with one short or complete lack of processes. We estimated the level of microglial extension by measuring the cell area, including the soma and the processes and we found a significant difference between the two groups. The mean cell area of microglial cells in the sham mice was 1335 ± 50.21 µm 2 , and 172.9 ± 13.66 µm 2 for the AAV-hAPP-SLA mice (Student's test, p < 0.0001, n = 150 cells, 3 mice) (Fig. 3D). The mean soma area of microglial cells in the sham mice was 30.66 ± 1.269 µm 2 , and 87.35 ± 3.623 µm 2 for the AAV-hAPP-SLA mice (Student's test, p < 0.0001, n = 150 cells, 3 mice) (Fig. 3 E). Thus, the AAV-hAPP-SLA induces robust microglial activation.

AAV-hAPP-SLA induces astrocyte activation

Reactive gliosis, including astrocyte activation, detected by increased glial fibrillary acidic protein (GFAP) expression, is another important characteristic of AD neuropathology (Serrano-Pozo et al. 2011). Therefore, we aimed to investigate whether our model induces astrocyte activation. Sections from the PFC of control or AAV-hAPP-SLA injected mice were immunostained for the presence of astrocytes using an anti-GFAP antibody. The AAV-hAPP-SLA injected mice showed markedly increased immunoreactivity for GFAP, compared to the control (Fig. 3F to I). This suggests that the presence of astrocyte-mediated inflammatory processes is associated with the Aβ oligomers.

Amyloid plaque and neurofibrillary tangle formation in AAV-hAPP-SLA injected mice

As the disease advances, amyloid peptides accumulate and aggregate, eventually forming amyloid plaques. We were able to detect typical amyloid plaques in the PFC of AAV-hAPP-SLA mice at 12 mpi, but not in control mice (Fig. 4). Another hallmark pathology of human AD is the intra-neuronal aggregation of hyperphosphorylated tau forming NFTs. We aimed to evaluate abnormal tau phosphorylation in our model. We investigated the levels of paired helical filaments (PHFs) reactive to the anti-AT100 antibody, which recognizes tau phosphorylated at serine 212 and threonine 214 residues [START_REF] Forny-Germano | Alzheimer's disease-like pathology induced by amyloid-β oligomers in nonhuman primates[END_REF]. AT100-positive neurons represent early stage markers of tau pathology. Immunostaining with AT100 revealed tau pathology in the PFC of mice injected with the AAV-hAPP-SLA, but no immunoreactivity was observed to the PFC of control mice (Fig. 4). No amyloid plaques or tau pathology were detected before 12 mpi.

The amyloid cascade hypothesis predicts that tau hyperphosphorylation occurs as a downstream consequence of Aβ accumulation (LaFerla and Green, 2012). APPoverexpressing transgenic mice have provided evidence both for and against this. Unlike humans with AD, many mouse models do not develop NFTs, yet many do show increased tau hyperphosphorylation.

Higher rates of neuronal activity in layer II/III of PrLC in AAV-hAPP-SLA injected mice

There is accumulating experimental evidence that neuronal hyperactivity as a result of amyloid pathology is a major indicator of AD associated dysfunction (Busche and Konnerth, 2015).

Experimental analysis using various approaches, from single neurons to neuronal populations to large-scale networks, with a variety of electrophysiological and imaging techniques, have revealed two forms of AD-related hyperactivity and provided first insights into the synaptic mechanisms. A striking early observation from in vivo two-photon calcium imaging in mouse models of AD was the unexpected abundance of hyperactive neurons in networks of the cerebral cortex and the hippocampus. For instance, in the frontal cortex of amyloid plaquebearing hAPP and PS1 double transgenic mice (the APP23 PS45 model), more than 20% of supragranular layer II/III neurons were found to be hyperactive (Busche et al., 2008). These hyperactive neurons were located mostly in the direct vicinity of amyloid plaques (that is, less than 60 µm from the plaque border), whereas the fractions of the simultaneously present functionally silent neurons increased with plaque distance. This observation indicated a spatial redistribution of excitatory and inhibitory synaptic activities.

In order to assess neuron function in the PFC of our mouse model, we used dynamic twophoton microscopy to image the activity of neurons in vivo through a chronic imaging window (Fig. 5A). Neurons of PrLC were transduced with an AAV expressing the genetically encoded calcium indicator GCaMP6f driven by the synapsin promoter [START_REF] Chen | Ultrasensitive fluorescent proteins for imaging neuronal activity[END_REF].

Four weeks after AAV injection, the majority of layer II/III neurons exhibited green fluorescence (Fig. 5B). The activity patterns of lightly anesthetized mice (isoflurane anesthesia) were recorded. Three-month-old WT mice injected with the AAV-hAPP-SLA (hAPP) and sham mice, injected with the control vector, were used to monitor the spontaneously occurring somatic Ca 2+ transients in individual cells (Fig. 5C). We analyzed the spontaneous neuronal activity at two time points: 1 and 6 mpi (Fig. 6A).

The distribution of spontaneous Ca 2+ transients for the different time points is shown in Figure 6B. We found that the median frequency distribution of Ca 2+ transients shifted towards higher values in hAPP mice at 6 mpi (1.75 ± 0.08 transients/min, 160 cells, n = 4 mice), compared with the sham (1.458 ± 0.04 transients/min, 829 cells, n = 4 mice, Kruskall Wallis test; P < 0.001), although at 1 mpi no significant difference was found (sham; 1.29 ± 0.05 transients/min in 451 cells, hAPP; 1.47 ± 0.049 transients/min in 729 cells, n= 4 mice in each group) (Fig. 6C). We also analyzed the duration of Ca 2+ transients between the different mouse groups. For the sham mice the median transient duration was 4.74 ± 0.17 seconds (2542 transients) and 4.04 ± 0.15 for the hAPP mice (3960 transients analyzed, Kruskall

Wallis test; P < 0.001), at 1 mpi of our vector. Interestingly, the sham mice at 6 mpi exhibited a much lower calcium transient duration when compared to the hAPP mice (sham; 2.32 ± 0.04 seconds of 5524 transients analyzed, hAPP; 4.63 ± 0.29 seconds of 887 transients analyzed n = 4 mice, Kruskall Wallis test; P < 0.001) (Fig. 5D). Based on the difference of the calcium transient durations between the different groups, and the fact that action potential firing causes calcium influx into the cytoplasm via the opening of voltage-gated calcium channels and therefore one calcium transient in not necessarily translated to one action potential, we deconvolved spontaneous Ca 2+ transients with a putative unitary (spike-evoked) event in order to estimate neuronal firing rates.

Briefly, Ca 2+ transients were selected according to their shape. Unitary calcium transients detected with GCaMP6f should have a rapid rise period, followed by a single peak value and a longer decay period [START_REF] Chen | Ultrasensitive fluorescent proteins for imaging neuronal activity[END_REF]. For the kernel estimation, we first selected transients exhibiting a single peak above 4σ and then selected a subset of these traces in which the decay was well fitted by an exponential function for the entire decay and was not contaminated by a second event. We fitted the first 750 ms of the decay with an exponential decay function, then examined the ratio between the duration when the signal was above the fitted curve and the total decay time. Ratios above 0.9 were rejected. This criterion essentially eliminated transient second events and/or large fluorescence fluctuations not associated with spikes. Finally, we rejected transients with rise times slower than the decay time. The selected transients were used for further analysis of their amplitude, rise time and decay time distributions for selection of putative unitary events. We selected transients with the 80 % lowest amplitudes and fastest rise time and decay time distributions, centered on the mode of the distributions. The kernel was then obtained from the fitting of an alpha function to the average of all selected Ca 2+ transients. This procedure was performed on all recorded transients separately for each experimental condition (i.e., Sham and hAPP, 1 and 6 mpi) (Fig. 6 E to H and Table 1).

We found layer II/III neuronal activity significantly increased in PrLC of hAPP mice (27.14 ± 3.33 spikes/min) compared to WT-control mice (4.46 ± 0.71 spikes/min, Kruskall Wallis test; P < 0.001), 6 mpi of vector. There was no significant difference between the mice imaged at 1 mpi (WT control; 12.70 ± 2.35 spikes/min, hAPP; 12.65 ± 1.38 spikes/min, p = 0.397 Kruskall Wallis test; Fig. 6I,J). Interestingly, sham mice showed a decrease in neuronal activity over time.

Discussion

AD is a complex disease affecting discrete brain areas with defined specificity for certain regions and pathways (Braak and Braak, 1991). Degeneration, at the cellular level in dendrites and synapses, causes changes in connectivity, disrupting brain function at the circuit level.

Therefore, to more effectively study AD pathology, it is necessary to dissect the pathological mechanisms using in vivo models and recording methods identifying the functional changes.

Here, we generated an AAV model that created AD-like pathology. The AAV was made to express mutant human APP harboring the Swedish, London and Austrian mutations that are associated with early onset of AD. Production of different AAVs for the expression of the tau protein or the mutant APP have previously been described without including any functional analysis on the role of soluble Aβ [START_REF] Jaworski | AAV-tau mediates pyramidal neurodegeneration by cell-cycle re-entry without neurofibrillary tangle formation in wild-type mice[END_REF]. With injection of our viral vector into the PFC of WT mice, we successfully induced Aβ production as early as 1 mpi. This production of Aβ oligomers in the initial phase of the pathology has the potential to greatly advance the understanding of the mechanisms centrally implicated in the early stages of AD pathogenesis, with applications for therapeutic development. It is thought that in AD the balance between Aβ production and clearance is disrupted therefore causing an increase in cellular Aβ. Over time, this disrupted balance leads to large amyloid deposits. The deposition of Aβ is a slowly progressive process that starts in the neocortex and then expands hierarchically into other brain regions, representing different phases of Aβ deposition [START_REF] Thal | Parenchymal and vascular Abeta-deposition and its effects on the degeneration of neurons and cognition in Alzheimer's disease[END_REF]. In accordance with the sequential involvement of brain regions in AD, we targeted the prefrontal area of the brain, a region associated with cognitive functions that could provide a basis for the understanding of the early phases of the disease. We were able to identify microglia activation, a feature of AD pathology, at 1 mpi of the vector. We followed the disease progression and identified hallmarks of AD pathology, such as the presence of amyloid plaques and tau hyper-phosphorylation that appeared one year after vector injection.

Our method could be very useful to investigate region specific vulnerability in AD linked to Aβ deposition and to follow the accumulation of amyloid deposition from one targeted brain structure to connected areas. In contrast, transgenic mouse models that express mutated hAPP globally in the brain throughout pre-and post-natal development into adulthood complicate the ability to measure the disease progression. Since all brain regions express the mutated APP, it is impossible to determine how the disease pathology spreads between an affected brain region to other initially unaffected areas. Our model allows targeted induction of ADlike pathology and therefore future studies exploring the spread to other brain regions will be possible.

Another advantage of our model is that it is compatible with chronic long-term in vivo imaging and recording techniques. The importance of this is illustrated by previous slice and primary neuronal culture studies that concluded that high levels of Aβ cause a reduction in excitatory neuronal transmission resulting in hypoactivity and synaptic failure [START_REF] Selkoe | Alzheimer's disease is a synaptic failure[END_REF].

However, clinical observations suggested that patients with AD, and in particular early-onset familial AD, have a higher incidence of epileptic seizures, indicating an increase rather than decrease in neuronal excitability (Palop and Mucke, 2009). In vivo studies in the last few years have further confirmed these human results in mouse models. It was shown that hyperactive neurons were found in close proximity to amyloid plaques, suggesting a synaptotoxic microenvironment around the AD lesions (Busche et al., 2008). Importantly, one study, in the visual cortex, demonstrated that a progressive deterioration of neuronal tuning for the orientation of visual stimuli occurs in parallel with the age-dependent increase of the Aβ load and this deterioration was found only in neurons that are hyperactive during spontaneous activity (Grienberger et al., 2012). The mechanisms underlying the changed neuronal activity in the diseased visual cortex are likely to involve a redistribution of synaptic inhibition and excitation, as it has been suggested for the impaired spontaneous activity in the frontal cortex (Busche et al., 2008). In addition, local application of soluble Aβ oligomers in the form of synthetic dimers provoked hyperactivity of CA1 neurons in WT mice [START_REF] Busche | Critical role of soluble amyloid-β for early hippocampal hyperactivity in a mouse model of Alzheimer's disease[END_REF].

Here, we examined the alterations in PFC neuronal activity at two different timepoints. By using in vivo two photon imaging, chronic cranial windows and monitoring neuronal activity with a genetically encoded calcium indicator, we were able to track neuronal activity in the same mice, at 1 and 6 mpi of AAV-hAPP-SLA. Interestingly, we identified a robust increase in the median frequency of PFC neurons injected with the AAV-hAPP-SLA as compared to control mice. At 6 mpi of our vector, we did not detect amyloid plaques or NFT formation, however, the amyloid oligomer enriched environment can explain the occurrence of this neuronal activity increase. Our results are in accordance with human studies where asymptomatic humans with high amyloid load were found to display abnormally increased activation in the default-mode network using blood-oxygen-level dependent (BOLD) fMRI.

This increase was mainly found in the medial prefrontal cortex, the precuneus and the posterior cingulate cortex (Sperling et al., 2009). In addition, we observed reduced neuronal activity in the PFC of control mice at 6 mpi. This finding is in accordance with neuroimaging studies in humans where older adults have shown reduced activity in PFC and other brain regions critical for cognitive functions (Morcom and Friston, 2012). Our data show an evolution in the functional properties of the neurons and network since the brain activity of the same mouse was followed over time. A crucial role of soluble Aβ on the spontaneous brain activity was identified, since increased pyramidal cell activity was detected before the formation of amyloid plaques. These findings can help us to elucidate the mechanisms that lead to the increase in PFC neuronal activity.

Another advantage of this approach is that it is applicable to every animal model, strain or species. This includes knock-out animals for specific factors involved in the pathways that lead to AD pathology. Notably, the cholinergic pathway, and cholinergic neurons located in the basal forebrain in particular, are subject to degeneration in AD (Auld et al., 2002).

Nicotinic acetylcholine receptors are crucially implicated in cognitive processing and have been proposed to be involved in the cognitive decline observed in AD patients [START_REF] Dineley | Nicotinic ACh receptors as therapeutic targets in CNS disorders[END_REF]. In human tissue, Aβ oligomers have been identified in cholinergic neurons, suggesting a role in cholinergic deficiency [START_REF] Baker-Nigh | Neuronal amyloid-β accumulation within cholinergic basal forebrain in ageing and Alzheimer's disease[END_REF]. The AAV vector we developped and characterized in vivo can be used to rapidly study the implications of different nicotinic receptor knock-out animals by using a single injection and therefore avoids the time and resource consuming crossbreeding between transgenic lines.

In conclusion, our model induces Aβ accumulation, astrocyte and microglia activation, amyloid plaque formation, and abnormal tau phosphorylation. Functional in vivo two-photon imaging of AAV-hAPP injected mice revealed an important role of soluble Aβ on spontaneous brain activity, indicating that the default network activity is disrupted in an ADlike brain before the formation of amyloid plaques. These findings can further our understanding of AD mechanisms of pathogenesis since it replicates important features of AD and can therefore be applied to improve our efforts to develop future therapies, already targeting early AD stages. However, the exact mechanisms that lead to the disruption of neuronal activity in AD is indeed poorly understood and further studies must be performed in order to elucidate the complexity of AD mediated hyperactivity.

Article 4 Overview

Several hypotheses have tried to explain the etiology of AD. Among them the "cholinergic hypothesis" postulates that a reduction in cholinergic innervation is responsible for the cognitive decline observed in AD patients. It has been demonstrated that nAChRs interact with Aβ, however how this interaction contributes to the pathophysiology of AD remains unknown. At the same time, it has been shown that AD brains are characterized by neuronal hyperactivity in the cerebral cortex and hippocampus. My work aimed to shed light to the possible involvement of nAChRs in AD mediated neuronal hyperactivity.

The main findings of article 4 are summarized below:

1. Soluble Aβ species induce pyramidal cell hyperactivity in the PFC of awake WT mice.

2. α7 KO mice are protected from Aβ -induced hyperactivity in the early stages of the disease.

3. β2 KO mice are protected from Aβ -induced hyperactivity early in the disease.

This part of my thesis reveals that nAChR-Aβ can influence the neuronal activity in PFC and suggests that the absence of α7 and/or β2 subunits may have a protective role towards the pyramidal cell hyperactive observed in AD mouse brain. This study provides further support to the continued effort to decipher the mechanism of nAChR neuroprotection for the development of effective treatments.

Role of the nicotinic acetylcholine receptors in Alzheimer's disease hyperactivity in prefrontal cortex

Fani Koukouli 1, 2 , Marie Rooy 3 , Geoffroy Goujon 1, 2 , Christiane Bils 1, 2 , Jean-Pierre

Introduction

Alzheimer's Disease is the most common neurodegenerative disease that is characterized by slowly progressing impairment of memory, ultimately leading to dementia and death. Several hypotheses have been examined and tested to explain the etiology of AD.

Among them emerged the "cholinergic hypothesis" (1), postulating that a reduction in cholinergic innervation is responsible for the cognitive decline observed in AD patients.

Anatomical studies in AD patients showed specific loss of cholinergic neurons in the basal forebrain (2). Basal forebrain neurons provide the primary cholinergic innervation to the neocortex, hippocampus, and other limbic regions (3). In addition, the activity of the enzyme choline acetyltransferase, the enzyme involved in ACh production, and acetylcholinesterase, the enzyme that metabolizes ACh after its release in the synaptic cleft (2), have reduced expression in AD brains. Interestingly, currently approved human therapies are restricted to enhancing cholinergic signaling for the symptomatic treatment of AD patients (4).

It was demonstrated that amyloid beta (Aβ) interacts with nAChRs. The most widely demonstrated interaction of nAChRs and the Aβ peptide is with the α7 nAChR subunit.

Studies showed co-localization of the α7 subunit with Aβ 1-42 in amyloid plaques of brain slices obtained from patients with sporadic AD (5). It was also revealed that α7 nAChRs bind the less toxic peptide Aβ 1-40 , but with lower affinity compared to the Aβ 1-42 peptide (6).

Immunoprecipitation of the α7 subunit with the Aβ peptide was further demonstrated in vivo, using the APPswe/PS1ΔE9 AD mouse model (7). In vivo behavioral paradigms attempted to dissect the role of α7 nAChRs in AD (8,9). In these studies, AD mouse models were crossed with α7 knock-out (KO) mice. However, these studies came to opposite conclusions, improvement and worsening, respectively, of AD-associated phenotypes, such as memory and synaptic deficits. Even though research has mainly focused on the study of the interaction between the α7 nAChR subunit and Aβ and its functional consequences, it has been also demonstrated that non-α7 nAChRs bind Aβ, such as β2* nAChRs, with electrophysiological approaches also supporting this interaction (10)(11)(12).

Overall, it is unknown whether Aβ activates or inhibits nAChRs, and how this interaction contributes to AD pathology (12)(13)(14). Importantly, nicotine treatment of different AD mouse models reduced plaque burden 80% in treated mice compared to non-treated, age matched controls (15) or led to a significant reduction in cortical insoluble Aβ 1-40/42 (16). In a recent study, APP/PS1 mice, after treatment with intraperitoneal nicotine injections, showed improvements in working and episodic memory compared to non-treated transgenic mice (17). Additionally, a reduction in the Aβ oligomeric form was found after the nicotine injections.

Initially the impairment of cognition in AD was thought to result from a reduction in neuronal activity and subsequently neuronal death. Recent in vivo evidence, mainly from mouse models, but also in humans, indicates that in AD neurons are hyperactive instead of hypoactive (18). More specifically, in vivo two-photon calcium imaging in mouse models of AD showed hyperactive neurons in the cerebral cortex and hippocampus. In the frontal cortex of lightly anesthetized APP23xPS45 mice, more than 20% of supragranular layer II/III neurons were found to be hyperactive (19). Hyperactivity was also found in the primary visual cortex (20) and in the CA1 area of the hippocampus ( 21) of APP23xPS45 mice where an increased fraction of silent cells were also detected. An additional study that used a different AD mouse model (APPswe/PS1d9) confirmed the presence of hyperactive neurons close to amyloid plaques (22). However, the exact mechanisms that lead to this disruption of neuronal activity in AD are poorly understood and further studies are required to elucidate the complexity of AD mediated hyperactivity.

Here, we hypothesized that nAChRs are involved in AD mediated neuronal hyperactivity and this was tested using two-photon imaging of awake mice. AAV-hAPP injected and wild-type (WT) mice were compared as to their PFC pyramidal cell hyperactivity while the activity was also recorded in genetically modified mice for the deletion of the two major nicotinic subunits involved in AD pathology, the α7 and β2 subunits. Our data indicate an important role of nAChRs subunits in cortical activity of AD brains, with the β2 subunit being a key player. and placed in a stereotaxic frame. Temperature was maintained with a thermometer feedback thermal blanket and the eyes were protected with artificial tear ointment. The scalp was washed with three alternating swabs of 70% ethanol and betadine and surgical tools were sterilized using a hot glass-bead sterilizer. The skull was exposed and 1% xylocaine was applied to the periosteum for local anesthesia. A chronic cranial window was prepared as previously described (27) and 200 nl of (serotype 1 (rep/cap: 2/1) AAV virus (AAV.syn.GCaMP6f.WPRE.SV40, 2.2e13 GC/ml, University of Pennsylvania Vector Core)

Materials and Methods

Adeno

was injected bilaterally at the following coordinates PrLC: AP, +2.8 mm from bregma; L, ±0.5 mm; and DV, -0.5 to -0.1 mm from the skull using a Nanoject II TM (Drummond Scientific) at the slow infusion setting. For WT control mice, 2 µl of AAV1.CAG.tdTomato.WPRE.SV40 1.52e13 GC/ml (University of Pennsylvania Vector Core, catalog number; AV-1-PV2126) diluted in PBS 1X, was injected bilaterally at the same coordinates as described above. For the WT-hAPP mice, 2 µl of AAV-EF1a-hAPP-SLA-FLAG (2,2e12 GC/ml.) was also injected bilaterally at the same coordinates. The glass pipette was left in place for an additional 5 min before being slowly withdrawn from the brain. The craniotomy was covered with a circular coverglass (5 mm diameter) and the glass edge was sealed to the skull with dental cement (Coffret SUPERBOND complete, Phymep). A sterile, small stainless steel bar with a screw hole was embedded and secured to the skull over the cerebellum to head-fix the mouse for the imaging sessions and the exposed skull was covered with dental cement.

Immunofluorescent staining. Mice were deeply anesthetized with a lethal dose of ketamine/xylazine before intracardiac perfusion with ice cold PBS, followed by 4% paraformaldehyde (PFA) (Sigma-Aldrich, Saint Louis, MO, USA). The brains were removed Mouse handling for awake imaging. Mice were habituated to the imaging environment by handling and training for 4 days, as previously described (29,30).

In vivo two-photon imaging. In vivo imaging was performed with an Ultima IV two-photon laser-scanning microscope system (Bruker), using a 16x 0.8 NA water immersion objective (Nikon) with the femtosecond laser (MaiTai DeepSee, Spectra Physics, Mountain View) tuned to 950 nm for imaging of cells expressing GCaMP6f and tdTomato. Time-series movies of neuronal populations expressing GCaMP6f were acquired at the frame rate of 33 HZ ( 405x 405 µm, FOV; 0.79 µm/pixel). Each focal plane movie duration was 165 seconds (5000 frames). Care was taken to use less than 10 mW of laser power at the brain surface.

Data analysis. Image analysis was performed off-line with ImageJ software (NIH). The time series were registered using the FIJI plugin 'Image Stabilizer' (K. Li, "The image stabilizer plugin for ImageJ," http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html). Regions of interest (ROIs) were manually selected in FIJI and detection of Ca 2+ transients of individual neurons was performed automatically using a custom-written toolbox in MATLAB.

Statistical analysis.

Kruskal-Wallis one-way analysis of variance combined with multiple comparison testing was applied to the activities (spikes/min) of the neurons in all mouse groups in order to study statistical similarities. For each mouse category, we used interpolation in order to account for the different number of neurons recorded between animals. In this way, our statistical analysis assigned the same weight to each neuronal population recorded in the different mice of a given category.

Results

AAV-hAPP induces pyramidal cell hyperactivity in WT mice.

We developed an adeno-associated virus (AAV) based model for replicating AD-like pathology (For details see article 3). The AAV vector expresses the mutated form of human APP harboring three pathogenic mutations: Swedish, London and Austrian (hAPP-SLA) (31)(32)(33). These mutations are associated with early onset of the disease. We targeted the expression of hAPP-SLA to the PFC, due to this region's key role in cognitive processes and its crucial implication in AD pathology (34). Wild-type (WT) AAV-injected mice developed AD-like pathological features such as Aβ oligomer deposition. We then aimed to track pyramidal cell activity in PFC as the disease progressed. We used in vivo two-photon imaging in awake head fixed mice (Figure 2A) and spontaneous pyramidal neuron activity was simultaneously monitored the in PrLC of control WT and WT-APP mice. The control WT mice were also injected with a control AAV (see Materials and Methods for details). Neurons of PrLC of all mouse groups were transduced with an AAV expressing the genetically encoded calcium indicator GCaMP6f driven by the synapsin promoter (35). Four weeks after AAV injection, the majority of layer II/III neurons exhibited green fluorescence (Figure 2B).

We then monitored the spontaneously occurring somatic Ca 2+ transients in individual cells (Figure 2C) and found layer II/III pyramidal neuron activity significantly increased in PrLC of WT -APP mice (25 ± 0.92 spikes/min, n = 720 cells), compared to WT-control mice (17.78 ± 0.60 spikes/min, n = 1510 cells) one-month post-injection of virus (***P < 0.001).

These data indicate a key role of soluble Aβ in the properties of cortical neurons, early in the disease.

α7 KO-APP mice exhibit less activity than α7 KO mice one-month post-injection.

Because a direct interaction of α7 nAChRs and Aβ peptide has been previously reported (5,6), we aimed to investigate the role of α7 nicotinic subunits in AD neuronal hyperactivity. We recorded the spontaneous pyramidal cell activity in the PrLC of control α7 KO and α7 KO mice injected with the AAV-hAPP vector (α7 KO -APP). We observed a strong increase in the median spike frequency in α7 KO (71.57 ± 3.56 spikes/min, n = 304 cells) and α7 KO -APP (42.79 ± 3.65 spikes/min, n = 257 spikes/min) compared to WT and WT -APP mice (Figure 2D andE).

α7 KO mice are protected from Aβ -induced hyperactivity 3 months post-injection of the AAV-hAPP.

We aimed to track the disease progression and record the pyramidal neuron activity in the same mice over time. Interestingly, the median spike frequency of WT-APP mice (63.19 ± 4.17 spikes/min, n = 348 cells) was significantly increased compared to WT control mice (27.62 ± 1.41 spikes/min, n = 473 cells) (Figure 3A andB). Conversely, we did not observe significant difference between the spontaneous neuronal activity of α7 KO (63.57 ± 3.68 spikes/min, n = 600 cells) and α7 KO -APP (71.47 ± 3.89 spikes/min, n = 196 cells) mice (P = 0.60). To further study the possible involvement of α7 nAChRs in AD hyperactivity, we locally re-expressed the α7 nicotinic subunits in layer II/III of α7 KO -APP mice using the lentiviral vector LV-PGK-α7-IRES-eGFP-WPRE (α7 KO -APPV EC mice, see Materials and Methods for details). We found that the pyramidal cell activity of α7 KO -APP VEC mice in PrLC was restored to the levels of WT control mice (24.89 ± 1.48 spikes/min, n = 346 cells) (P = 0.99) (Figure 3A and B). We then followed the spontaneous activity of the same mice 6 months post-injection. WT-APP exhibited increased activity (55.09 ± 2.7 spikes/min, n = 640 cells) compared to WT (29.81 ± 3.67 spikes/min, n = 334 cells) (***P < 0.001, Kruskal-production of Aβ. We targeted the expression of hAPP to the PFC, due to this region's key role in cognitive processes and its crucial implication in AD pathology (34). With two-photon imaging in awake mice, we were able to observe robust pyramidal cell hyperactivity in layer II/III of awake WT mice at the site of Aβ expression, already at one month post-injection of the virus.

The literature presents conflicting results about functional consequences of Aβ binding to nAChRs, eliciting in some studies receptor activation, and in others inhibition (12)(13)(14).

These conflicting findings can partly be explained by the variability in the experimental approaches that were utilized including the Aβ preparation that was selected including the aggregation status and concentration variability, the recording method for neuronal activity and differences in receptor subtype (homo-or hetero-pentameric receptors). When comparing between oligomeric and fibrillary Aβ, there is high variability between the characteristics of toxicity (34). We used our AAV-hAPP that we developed and focused our work on the α7 nicotinic subunits since these were shown to interact with Aβ (6,43). In addition, we investigated a potential role of the β2 subunits in AD pathology. The β2 subunit and its interaction with Aβ was mainly investigated in electrophysiological studies (44,45) and less is known about the downstream pathways that are activated by this interaction. In addition, Liu et al. postulated the existence of an α7β2 nAChR heteropentamer in basal forebrain cholinergic neurons (46). This novel nAChR subtype is characterized by a high sensitivity to

Aβ inhibition (47).

The aim of our study was to investigate if nAChRs play a role in the Aβ peptide mediated hyperactivity that was observed in AD brains. Interestingly, the pyramidal neurons of layer II/III in PFC of α7 and β2 KO mice are hyperactive compared to WT mice. nAChRs in layer II/III of the PFC are expressed only by GABAergic interneurons (48). When the α7 and/or β2 subunits are deleted, the interneurons can no longer inhibit the pyramidal cells that become hyperactive. When the AAV-hAPP was injected to the α7 KO mice, we observed a decrease in pyramidal cell activity compared to age-matched α7 KO mice injected with the control virus at one month post-injection, whereas there was no significant difference between the two groups three months later. Surprisingly, in the case of β2 KO mice, the levels of pyramidal cell activity were the same between β2 KO control and β2 KO-APP injected mice.

This could lead to the supposition that Aβ peptide interacts with β2 nicotinic subunits in the GABAergic interneurons of layer II/III of the PFC, and this interaction leads to pyramidal cell hyperactivity and, with the absence of β2 subunits, this interaction cannot occur, leading to similar levels of neuronal activity. To further investigate this hypothesis, we locally reexpressed the β2 subunit in the β2 KO-APP injected mice, where we observed a strong reduction to neuronal activity. However, the activity was not fully restored to WT mice levels.

Recently, we showed that the absence of the β2 subunit is protective against the memory loss induced by Aβ expression in the dentate gyrus (Lombardo 2016). The lack of the β2 subunit prevents Aβ-induced memory loss, since β2 KO animals were protected from the recognition memory impairment induced by hAPP expression in control mice.

In conclusion, our data reveal that the nAChR-Aβ interaction can alter the functional activity of interneurons and Aβ inhibition of nAChRs could interfere with their regulatory function, leading to pyramidal cell hyperactivity in the PFC. In β2 KO mice, the absence of the subunit can be protective by preventing the nAChR-Aβ toxic interaction. This study provides further support to the continued effort to decipher the mechanism of nAChR neuroprotection for the development of effective drugs for AD based on disrupting or enhancing nAChR function.

Discussion

Measuring spontaneous default brain activity in the PFC

The brain's default mode network consists of discrete, bilateral and symmetrical cortical areas, in the medial and lateral parietal, medial prefrontal, and medial and lateral temporal cortices of the human, nonhuman primate, cat, and rodent brains (Raichle 2015).

This network was a surprising finding that was revealed in positron emission tomography studies where quiet eye-closed or simple visual fixation tasks were compared to novel, attention-demanding and non-self-referential tasks. The discovery of the default mode network started a longstanding interest in the significance of the brain's ongoing or intrinsic activity since attention, sensory expectation and other cognitive functions to interpret the environment of an animal, are thought to be encoded in the cerebral cortex through spontaneous neuronal activity (Arieli et al. 1996;Dehaene and Changeux 2005). This activity is dependent on top-down modulation and attention, varying with behavioral states, to be essential for normal brain function.

This thesis focused on studying spontaneous activity of the PFC, a brain area that has been considered a key player in the emergence of conscious perception (Dehaene and Changeux 2011;Lau and Rosenthal 2011), since it serves to distribute information that is processed in unconscious modules to the entire brain. In vivo recordings in awake WT and genetically modified mice KO and KI for several nAChR subunits revealed that endogenous ACh distinctly recruits specific interneurons within a hierarchical circuit that controls pyramidal cell activity in layer II/III of PFC. Furthermore, recordings in awake mice were compared to recordings from anesthetized mice and showed that nAChRs have an important role in the modulation of neurotransmission and generation of ultra-slow fluctuations that occur both during quiet wakefulness and anesthesia.

The mechanisms through which general anesthetics, an extremely diverse group of drugs, cause reversible loss of consciousness has been a mystery. Every year, tens of millions of patients are exposed to general anesthetics that fulfill three major criteria: analgesia, unconsciousness and decreased muscle tone. Although the principal aim of anesthesia today is to insure the safety and protection of patients, the mechanisms of action of anesthetic agents on the CNS remains poorly understood. Now, it is widely accepted that anesthetics act selectively at the molecular level by binding directly to specific protein targets [START_REF] Franks | General anaesthesia: from molecular targets to neuronal pathways of sleep and arousal[END_REF].

In neuroscience research, anesthetics have been used to identify neuronal factors that are involved in conscious perception, to explain neural mechanisms of sleep and arousal and to elucidate the brain circuits that produce synchronous activity patterns [START_REF] Rudolph | Molecular and neuronal substrates for general anaesthetics[END_REF]Changeux 2012). Anesthesia has significant effects on large-scale neuronal networks by specifically modulating activity, and while also causing suppression in brain activity [START_REF] Mccarthy | Dynamical changes in neurological diseases and anesthesia[END_REF]. Anesthesia also has multiple effects including changing electroencephalogram oscillatory activity from high frequency, low-amplitude activity to low frequency, high-amplitude activity [START_REF] Voss | Monitoring consciousness: the current status of EEG-based depth of anaesthesia monitors[END_REF]. Therefore, long range coherence and integration between brain regions are disrupted, causing a reduction in the cortical information capacity (Alkire et al. 2008).

The first two articles presented in this thesis allowed for the examination of neuronal activity levels in the PFC of awake and anesthetized animals and the findings provided the basis for 1) elucidating the role of nicotinic receptors in default brain activity in the normal and diseased brain, 2) understanding the role of cholinergic neurotransmission in the orchestration of cognitive functions, such as consciousness, 3) re-evaluating the large number of neuroscience studies performed in anesthetized animals and 4) provided fundamental insight into cortical network dynamics.

In the context of human genetic studies

In order to fully understand the genetic contribution to many human diseases, human genetic variation studies have and are actively being performed. These studies provide a powerful tool to expand our understanding of the role that genes have in the development and progression of disease. Significant amounts of money have been directed towards these large genetic studies, with the permission of society, based on an assumption that it will provide enormous benefits to improving human health. This has lead to a significant increase in the number of organizations and people involved in human genetics (NIH 2007). Thanks to unbiased screening of millions of genetic factors in cohorts of thousands of patients and controls, we are beginning to elucidate the genetic etiology of diseases.

Schizophrenia and nicotine addiction are highly heritable. The heritability is 60-75%

for nicotine dependence and 60-80% for schizophrenia [START_REF] Hong | A CHRNA5 allele related to nicotine addiction and schizophrenia[END_REF]. A genetic attribute to this smoking-schizophrenia comorbidity has often been proposed. Finding a crosscorrelation with schizophrenics having a higher level of smoking is more complicated than a simple-case control association test. It is hard to separate between whether a candidate variant was having an effect on both smoking and schizophrenia as an additive effect, or instead, to be specific to smoking in schizophrenia, being a distinct variant separate from other genetic variants exclusively linked to smoking.

My thesis studied a functional smoking-related nicotinic receptor α5 subunit gene nonsynonymous SNP (Asp398Asn) [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF], that has been also associated to schizophrenia (Ripke et al. 2014), aiming to identify the cellular and circuit mechanisms underlying a possible direct or indirect relationship between nicotine and schizophrenia. My thesis showed that endogenous ACh distinctly recruits specific interneurons within a hierarchical circuit that controls pyramidal cell activity in a specific layer of the PFC and provided the first experimental evidence that the α5 human polymorphism produces a decrease in neuronal activity, mirroring the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction. Chronic nicotine treatment of mice carrying the α5 SNP restored their neuronal activity deficits, which together provide the basis for examining new pharmacotherapeutic strategies and support the hypothesis that subjects with polymorphic CHRNA5 smoke to self-medicate in order to ameliorate an underlying cortical dysfunction. But is smoking the proper therapy for medicating hypofrontality?

Alternatives to smoking for schizophrenia

The current estimate of the number of tobacco consumers worldwide stands at about 1 billion people. Six out of eight leading causes of death have tobacco as a risk factor, causing six million preventable deaths per year. Manufactured cigarettes are the most common means of tobacco consumption which are designed to optimize taste sensation, reduce harshness and increase nicotine delivery [START_REF] Giovino | Tobacco use in 3 billion individuals from 16 countries: an analysis of nationally representative cross-sectional household surveys[END_REF]. A typical cigarette contains 10-14 mg of nicotine, of which 1-1.5 mg is absorbed, reaching the brain in 10-20 seconds [START_REF] Benowitz | Nicotine Psychopharmacology, Handbook of experimental pharmacology, Handbook of Experimental Pharmacology[END_REF]). The half-life of nicotine is approximately 8 minutes, but this depends on the age of the smoker.

As discussed throughout this thesis, heavier tobacco smoking among people with SCZ has been suggested to reflect self-medication of mainly negative symptoms. Nicotine enhances sensory, alerting, attentional and mnemonic processes in SCZ [START_REF] Leonard | Smoking, Genetics and Schizophrenia: Evidence for Self Medication[END_REF]).

However, smoking is obviously not a good treatment for SCZ because of its adverse health risks and since the receptors are quickly desensitized, the effect is short-lived. For this reason, several nicotinic agonists have been introduced. Preclinical trials of α7 nAChR agonists have shown positive effects on various animal models of SCZ [START_REF] Toyohara | α7 Nicotinic Receptor Agonists: Potential Therapeutic Drugs for Treatment of Cognitive Impairments in Schizophrenia and Alzheimer's Disease[END_REF].

For example, DMXBA (3-(2,4-dimethoxybenzylidene)-anabaseine-GTS-21) is a synthetic derivative of anabaseine, an alkaloid present in several species of marine worms. It is a drug that acts as a partial agonist a neural nAChRs [START_REF] Martin | Alpha-7 nicotinic receptor agonists: potential new candidates for the treatment of schizophrenia[END_REF]). It binds to both the α4β2 and α7 subtypes, but activates only the α7 to a significant extent. DMXBA was found to normalize aberrant auditory gating and to enhance cognition. A phase I trial showed improvements in both the sensory processing P50 deficit and in cognition. A phase II trial is underway in which effects in smokers with SCZ will be assessed.

EVP-6124 is a synthetic molecule that was found to bind with high affinity to α7 nAChRs. Animal data indicated that EVP-6124 acts as a potent partial and selective agonist to α7 nAChRs. A phase IIb clinical trial showed that EVP-6124 had a clinically meaningful and statistically significant impact on the patients' overall cognition. This compound is now in phase III for SCZ, and also AD [START_REF] Deardorff | Safety and clinical effects of EVP-6124 in subjects with Alzheimer's disease currently or previously receiving an acetylcholinesterase inhibitor medication[END_REF][START_REF] Van Goethem | Continuous infusion of the α7 nicotinic acetylcholine receptor agonist EVP-6124 produces no signs of tolerance at memory-enhancing doses in rats: a pharmacokinetic and behavioral study[END_REF].

Nicotine replacement products could also be beneficial for people trying to quit smoking who also search for the beneficial effects of nicotine. There are many products available, including: nicotine patch, gum, lozenges, nasal spray and nicotine inhalers. All of these products are used as a substitution for the nicotine that users would consume with a smoked product. Numerous unhealthy substances are found in cigarettes (especially with combustion), chewing tobacco and snuff, therefore nicotine replacements seem to be a preferred method of intake.

It is worth underlining that in the general population, self-motivated nicotine cessation rates are extremely low, with sustained remission being observed in only 3-5% of smokers [START_REF] Stead | Nicotine replacement therapy for smoking cessation[END_REF]. Two treatments are currently approved for nicotine cessation, bupropion and varenicline; the effect of varenicline on neuronal activity was studied during this thesis.

Bupropion, which is an antidepressant and a non-competitive antagonist of nAChRs and varenicline, a partial agonist of α4β2 and a full agonist of α7, have been reported to triple the success rate of long-term tobacco abstinence compared to placebo, with varenicline providing the greatest success [START_REF] Hartmann-Boyce | Efficacy of interventions to combat tobacco addiction: Cochrane update of 2013 reviews[END_REF]. A combination of a smoking cessation therapy with nicotine replacement products could be used in the case of schizophrenia patients to help ease their symptoms.

Therapeutic considerations: are nAChRs an appropriate therapeutic target for genetic diseases?

As outlined in numerous sections of this thesis, the therapeutic potential for nAChR related disorders not only provides an avenue for symptomatic treatments, but also gives more understanding to neuropharmacology and disease mechanisms. Currently, approved nicotinic agonist drugs are for smoking cessation, depression, attention deficit, hyperactivity disorder, schizophrenia, Parkinson's disease and Alzheimer's disease. The hope of molecular therapies for these disorders is within reach due to significant advances in understanding the exact genetic causes for nAChR disorders. With the great need and societal benefic of treating nicotine addiction, there is heightened interest in the pharmacological industry, therefore more rare orphan diseases caused by improper nicotinic signaling could benefit from this interest.

The broad scope of the nAChR system makes for difficult development of pharmacological substances. Lack of efficacy, narrow therapeutic windows or side effects have caused many clinical trials to fail. Optimal combinations of pharmacodynamics and pharmacokinetics are required for drugs in order for them to be specific to defined receptor subtypes, within a proper dose range, while not affecting other receptor subtypes to minimize side effects.

Positive allosteric modulators are receiving greater attention beyond nicotinic agonists since they lack intrinsic activity when not in the presence of ACh, thereby causing a potentiation of its effect. Selectivity of allosteric modulators can be achieved at a greater level than cholinergic drug agonists since allosteric binding sites are less conserved between receptor subtypes and are distinct from the agonist binding site. A particularly attractive strategy is to combine positive allosteric modulators with cholinergic agonists since this could oppose agonist-induced desensitization. Due to their great potential in targeted therapies for neurodegenerative diseases and complex genetic disorders, the fields of neuroscience, psychiatric, behavioral and neuro-clinical fields are increasingly appreciating the importance of nAChRs and future drugs for their manipulation.

Advantages of our new AD model

As described in the third article, we generated an AAV that allowed us to create a We then used this AAV-based model to investigate the role of nicotinic receptors in the pathology. We focused our work on the α7 and β2 subunits since these subunits were found to interact with Aβ (Wang et al. 2000;[START_REF] Yakel | Functional and molecular characterization of neuronal nicotinic ACh receptors in rat hippocampal interneurons[END_REF]. The use of AAV vectors to cause an AD-like phenotype is a potentially powerful tool. The major advantage of this approach is its flexibility and versatility. The model presented in my thesis gives the ability to target a discrete brain region and permits the study of region-specific vulnerability to AD lesions and their spread throughout the brain. In my study, I focused on the PFC brain area, but since the virus is be targeted with stereotaxic delivery, it is possible to affect any desired brain region.

In the future, modifications of the virus, for example adding a different promoter, could facilitate the infection to more diverse brain regions or specific cell types. Another big advantage of this AD model is the possibility to use different mouse lines, for example knockout mice for the remaining nAChR subunits that were not studied here. By using this powerful AAV method, we avoid crossing different mouse lines, which is a time consuming approach that can also generate undesired diversity in the genetic background of the resulting lines. In addition, our model can be a method of choice for non-rodent models.

I should mention that beside its numerous advantages, this approach has some disadvantages that should be taken into consideration. For one, it is not possible to infect the totality of the brain with the AAV, without cytotoxic consequences. We consider, however, that this region-restricted infection by the virus is a major advantage. In addition, because of the nature of the virus, the number of copies of the transgene that integrate into each cell is variable and it is not possible to control the integration site. For this reason, it is important to compare the AAV-hAPP injected mice with a control group injected with a different virus expressing a reporter gene to assure that the observed effect with hAPP is not due to tissue damage caused by the injection, or a possible infection.

The role of nAChRs in AD pathology

It is worth discussing that the two major in vivo studies investigating the role of the α7 nAChRs in AD found opposite results. In both studies, AD mouse models were crossed to α7 nAChR KO mice using the PDAPP and Tg2576 lines, respectively (Dziewczapolski et al. 2009;Hernandez et al. 2010). In the first case, the loss of α7 nAChR improved cognition, and in the second, it exacerbated the disease. A possible explanation would be that nicotinic receptors exert different functions in early and late phases of the disease, depending on the Aβ concentrations present in the brain. Indeed, it was already shown that at low Aβ levels, its interaction with α7 nAChR can activate neuro-protective pathways and eventually Aβ becomes toxic at high concentrations [START_REF] Puzzo | Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus[END_REF]Parri et al. 2011). Taking into account both studies, we could assume that at an early stage the interaction with α7 nAChR is protective, and its absence exacerbates the disease (Hernandez et al. 2010), however, at a more advanced stage, the interaction with α7 mediates Aβ-induced neuronal impairment (Dziewczapolski et al. 2009). Our AAV based AD model gives the opportunity to investigate the role of nAChRs in the same mouse line, avoiding the difficulties that could arise from comparisons between different AD models.

We focused our work on the α7 nicotinic subunits since these were shown to interact with Aβ (Wang et al. 2000;Parri et al. 2011). In addition, we investigated a potential role of the β2 subunits in AD pathology. The β2 subunit and its interaction with Aβ was mainly investigated in electrophysiological studies [START_REF] Wu | beta-Amyloid directly inhibits human alpha4beta2-nicotinic acetylcholine receptors heterologously expressed in human SH-EP1 cells[END_REF][START_REF] Pym | Subtype-specific actions of beta-amyloid peptides on recombinant human neuronal nicotinic acetylcholine receptors (alpha7, alpha4beta2, alpha3beta4) expressed in Xenopus laevis oocytes[END_REF] and less is known about the downstream pathways that are activated by this interaction. The aim of our study was to investigate if nAChRs play a role in the Aβ peptide mediated hyperactivity observed in AD brains. By injecting our AAV-hAPP to layer II/III of the PFC of WT mice, we observed a very early, robust increase in neuronal activity four weeks post injection. We used the same virus to inject α7 and β2 KO mice in the PFC and performed in vivo two photon microscope calcium activity recordings. As described in my thesis, the pyramidal neurons of layer II/III in PFC of α7 and β2 KO mice are hyperactive compared to WT mice.

As explained, nAChRs in layer II/III in PFC are expressed only by GABAergic interneurons.

When the α7 and/or β2 subunits are deleted, the interneurons can no longer inhibit the pyramidal cells that become hyperactive. Interestingly, when the AAV-hAPP was injected to the α7 KO mice, we observed a decrease in the pyramidal cell activity compared to aged matched α7 KO mice injected with the control virus, one month post injection, whereas there was no significant difference between the two groups three months later. In the case of β2 KO mice, it is promising that the levels of pyramidal cell activity were the same between β2 KO control and β2 KO-APP injected mice. This could lead to the assumption that the Aβ peptide interacts with β2 nicotinic subunits in the GABAergic interneurons of layer II/III in the PFC, and this interaction leads to the pyramidal cell hyperactivity and with the absence of β2 subunits, this interaction cannot occur, leading to similar levels of neuronal activity. To further investigate this hypothesis, we locally re-expressed the β2 subunits in the β2 KO-APP injected mice. We observed a strong reduction to neuronal activity but it was not restored to the levels of WT mice. These experiments reveal a potential role of both α7 and β2 nicotinic subunits in AD hyperactivity. It is important to mention that we continue these experiments in order to track the disease progression over time to find at which stage of the disease there is a possible interaction between Aβ peptide and nAChRs. Several Cre lines for specific interneuronal types are now available in the lab, and could be used in order to elucidate not only which nicotinic subunit could be responsible for the AD pyramidal cell hyperactivity by interacting with the Aβ peptide, but also in which specific cell type this interaction takes place. The double KO mice for the α7 and β2 nicotinic subunits could also be used in the future to better dissect our hypothesis.

Future perspectives

Several opportunities have emerged for optical brain imaging studies in behaving mammals: those involving head-fixed animals and those permitting unconstrained behavior.

Both approaches play important roles in the study of cellular and neural circuit dynamics during active animal behavior. Studies in head-fixed animals differ from studies in freely moving animals, with freely moving studies requiring miniaturization of the entire, or part of, the microscope so that the animal can carry it on the cranium during unrestrained behavior [START_REF] Hamel | Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach[END_REF].

As described in my thesis, Ca 2+ imaging studies in awake head-restrained rodents involve relatively simple behaviors such as quiet wakefulness [START_REF] Greenberg | Population imaging of ongoing neuronal activity in the visual cortex of awake rats[END_REF]).

However, behavioral assays have now progressed, making use of the head-fixed preparation's suitability for controlled delivery of sensory stimuli and with training, animals can perform various perceptual discrimination tasks. Some examples are associative fear conditioning [START_REF] Lovett-Barron | Dendritic inhibition in the hippocampus supports fear learning[END_REF], visual and odor discrimination and in very advanced setups, virtual reality systems with a head-restrained mouse controlling its position in a virtual maze for studying motor behavior [START_REF] Harvey | Intracellular dynamics of hippocampal place cells during virtual navigation[END_REF][START_REF] Dombeck | Functional imaging of hippocampal place cells at cellular resolution during virtual navigation[END_REF]. These studies enable sensory manipulations that would be difficult to re-create in the physical realm of freely behaving animals. For instance, one can create artificial mismatches between an animal's motor behavior and the visual feedback signals it receives in response. Virtual reality approaches also allow imaging of neural activity related to spatial navigation behaviors that have usually been studied in freely moving animals. Researchers are still exploring the extent to which neural responses during virtual-navigation tasks in restrained animals mimic those occurring during normal unrestrained navigation.

In compliment to head fixed techniques, miniaturization has allowed for headmounted optical approaches to allow freely moving mammal brain imaging [START_REF] Hamel | Cellular Level Brain Imaging in Behaving Mammals: An Engineering Approach[END_REF]. These techniques are important since some behavioral tasks are impossible to be performed head fixed. Examples include the social behaviors such as fighting, mating, caregiving, and other forms of interaction; behaviors probing stress or anxiety that might be influenced by the stress of head restraint; motor behaviors involving motion of the head and neck; olfactory behaviors involving active exploration of an odor landscape; and vestibulardependent behaviors. Freely moving studies in operant, social, sensory, learning, navigation and motor behaviors have been performed with miniaturized optical devices. Overall, both head-restrained and freely behaving optical studies are becoming more common and allow addressing very deep questions in neuroscience.

During my thesis, we explored the behavioral deficits of KI mice for the α5 subunit human polymorphism (Asp398Asn) associated to nicotine dependence and schizophrenia and we discovered social interaction deficits, as described to article one. As a future goal, it is important to consider additional studies in order to evaluate the neuronal activity, more specifically the hypofrontality of these mice during behavioral assays. In addition, since several studies have shown that deletion in mice of the α5, α7 and β2 subunits impairs behaviors, such as exploratory behavior and attention, it will be important to explore the behavioral deficits in parallel with neuronal recordings to decipher a causal relationship.

Furthermore, the hippocampus is a prime target of AD yet little is known about how the disease affects neuronal activity patterns in the hippocampus in vivo. Pyramidal cell hyperactivity has been demonstrated in CA1 of hippocampus of AD mouse models [START_REF] Busche | Critical role of soluble amyloid-β for early hippocampal hyperactivity in a mouse model of Alzheimer's disease[END_REF], however a potential contribution of nAChRs in hippocampal AD hyperactivity has not been examined. The hippocampus exhibits a rich variety of nAChR subtypes, with different subunit assemblies being expressed on different neuronal populations that exert distinct functions. Sudweeks and Yakel showed a specific expression of α7 exclusively in hippocampal interneurons within the CA1 subfield (Sudweeks and Yakel 2000). The β2 subunit is also expressed in the hippocampus, and it was postulated that it can assemble with α7 subunits in hippocampal interneurons to form a receptor with a distinct electrophysiological kinetic profile compared to α7 homopentamers or β2* heteromers [START_REF] Yakel | Functional and molecular characterization of neuronal nicotinic ACh receptors in rat hippocampal interneurons[END_REF]Liu et al. 2012). A potential role of nAChRs in hippocampal AD hyperactivity has not been examined. This information has been difficult to obtain because of the inaccessibility of the hippocampus for cellular-level functional imaging, due to its depth.

However, technology has advanced and different techniques can be applied, such as removal of a part of the cortex by local aspiration, implantation of glass microprisms, grin lenses or special fiber optics in order to have access deep in the brain [START_REF] Helmchen | Deep tissue two-photon microscopy[END_REF][START_REF] Dombeck | Functional imaging of hippocampal place cells at cellular resolution during virtual navigation[END_REF][START_REF] Attardo | Impermanence of dendritic spines in live adult CA1 hippocampus[END_REF][START_REF] Gonçalves | In vivo imaging of dendritic pruning in dentate granule cells[END_REF]). In the future, I believe it will be of crucial importance to investigate the patterns of neuronal activity in different KO mice for distinct nAChRs that will be injected with the AAV-hAPP and combine in vivo calcium recordings with behavioral assays associated to AD pathology.

Concluding statement

The body of work presented in my thesis demonstrates a crucial role of α5, α7 and β2 nAChRs and associated human polymorphisms in modulation of PFC ongoing spontaneous activity. Our findings provide new insights on the default network dynamics during the awake and anesthetized state, on the role of cholinergic neurotransmission in the orchestration of cognitive functions in the normal and diseased brain and the results provide a starting point for understanding the complex interaction between nAChRs and pathologies such as schizophrenia and Alzheimer's disease. I hope this work will contribute to further decipher the role of nAChRs in these pathologies and have a therapeutic impact in the future.

INTRODUCTION

Brain penetration of targeted compounds, such as high affinity antibodies, is a prerequisite for the treatment and diagnosis of central nervous system pathologies. However, the entry into the central nervous system of most of blood-circulating molecules is seriously limited by the blood-brain barrier (BBB). It has been shown that only 0.02% -0.1% of conventional immunoglobulins present in serum can penetrate into brain parenchyma [START_REF] Bard | Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease[END_REF][START_REF] Coloma | Transport across the primate blood-brain barrier of a genetically engineered chimeric monoclonal antibody to the human insulin receptor[END_REF]. Hijacking the BBB receptor-mediated transcytosis system has been envisioned as a way of promoting passage of agents in the brain. Antibodies directed against the transferrin receptor [START_REF] Niewoehner | Increased brain penetration and potency of a therapeutic antibody using a monovalent molecular shuttle[END_REF], or the insulin receptor [START_REF] Boado | Blood-brain barrier molecular trojan horse enables imaging of brain uptake of radioiodinated recombinant protein in the rhesus monkey[END_REF] have hence been used as Trojan horses to transport therapeutic molecules across the BBB.

Unfortunately, receptor-mediated transcytosis targets are highly and broadly expressed in tissues and are often implicated in critical cellular functions, therefore raising possible safety risks [START_REF] Couch | Addressing Safety Liabilities of TfR Bispecific Antibodies That Cross the Blood-Brain Barrier[END_REF][START_REF] Ohshima-Hosoyama | A monoclonal antibody-GDNF fusion protein is not neuroprotective and is associated with proliferative pancreatic lesions in parkinsonian monkeys[END_REF]. Artificial BBB opening by ultrasound [START_REF] Matharu | Gadolinium-complexed Aβbinding contrast agents for MRI diagnosis of Alzheimer's Disease[END_REF][START_REF] Santin | Fast in vivo imaging of amyloid plaques using µ-MRI Gd-staining combined with ultrasound-induced blood-brain barrier opening[END_REF] or by means of BBB permeability enhancers (e.g. mannitol [START_REF] Sigurdsson | A non-toxic ligand for voxelbased MRI analysis of plaques in AD transgenic mice[END_REF]) is also an option to facilitate the penetration of compounds within the brain but these methods also have safety limitations [START_REF] Rodriguez | Neurosurgical Techniques for Disruption of the Blood-Brain Barrier for Glioblastoma Treatment[END_REF].

Another approach of targeting intracerebral epitopes relies on the use of homodimeric heavy chain-only antibodies naturally produced in camelidae [START_REF] Hamers-Casterman | Naturally occurring antibodies devoid of light chains[END_REF].

These "non-conventional" single-domain antibodies are devoid of light chains and their heavy chain variable domain (VHH) acts as a fully functional binding moiety [START_REF] Muyldermans | Nanobodies: natural single-domain antibodies[END_REF].

VHHs have several unique features such as relatively low molecular weight, high production yield, high stability and solubility with an ability to specifically bind target epitopes at subnanomolar affinity. Due to their small size (15 kDa), VHHs occupy only 1/10 of the volume filled by conventional antibodies [START_REF] Hamers-Casterman | Naturally occurring antibodies devoid of light chains[END_REF]. Consequently,

In vivo labeling of Aβ-positive lesions

Diffusion of R3VQ after stereotaxic brain injection

To test the ability of R3VQ to diffuse in the brain and to label in vivo amyloid plaques, six micrograms of R3VQ was stereotaxically injected into the cortex of 12-month old PS2APP mice (n=2). The mice were sacrificed 2 hours after injection. IHC revealed that R3VQ diffused in a large spherical region (approximatively 3 mm diameter) and amyloid plaques were detected in this volume (Figure S2A). The results demonstrate a good ability of R3VQ

to diffuse through living tissue and to label Ab lesions quite rapidly (< 2 hours). The same experiment was performed in mice sacrificed 24 hours after intracerebral injections of R3VQ:

the diffusion halo narrowed down considerably within a 1 mm diameter sphere and a reduced staining of amyloid plaques was observed, suggesting elimination of R3VQ over time in the brain (data not shown). To summarize, the VHH R3VQ was able to diffuse in the brain and specifically target amyloid plaques in vivo.

BBB transit and diffusion of R3VQ after intravenous injection

To investigate if R3VQ-S-AF488 crossed the BBB, a 50-mg/kg dose was injected iv into two 12-month-old PS2APP mice. In parallel, two age-matched PS2APP were injected with PBS (negative controls) or with AlexaFluor 488 conjugated anti-Ab mAb 4G8 (4G8-S-AF488; 50 mg/kg) as controls for BBB passage of standard immunoglobulins. Four hours after injection, the mice were sacrificed and paraffin brain sections were prepared. In R3VQ-S-AF488 injected mice immunostaining using anti-His mAb showed extensive plaque labeling throughout the brain. This demonstrates that the VHH conjugate was able to cross the BBB, diffuse widely in the living brain and label its target. In contrast, no staining was found in mice injected with PBS (Figure S3) and only few plaques in restricted brain regions were detected following 4G8-S-AF488 injections, underlining very limited BBB permeability for regular immunoglobulins (Figure S3).

Real-time imaging of R3VQ after intravenous injection

A 50-mg/kg dose of R3VQ-S-AF488 was injected in the tail vein of a one-year-old PS2APP mouse. The conjugate extravasation and diffusion in the brain was recorded for 4 hours post injection using two-photon microscopy of a cortical brain window (from surface to 360 µm deep). Figure 3A displays in vivo imaging of the same region of interest at 1 and 30 min postinjection of R3VQ-S-AF488. A few seconds after iv injection, strong labeling of arborescent vessels was observed. After 20 min only few capillary vessels were still labeled. This suggested a short half-life (10-20 min) of conjugated VHH in the blood circulation [START_REF] Nabuurs | In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease[END_REF]. Shortly after injection a green fluorescent "cloud" formed and spread in the parenchymal space, reminiscent of the diffusion observed following stereotaxic injection of R3VQ (Figure S2A). Thirty minutes after injection, both amyloid plaques and vascular Aβ (CAA) began to be visualized. In vivo labeling of Ab deposition in plaques and vessels remained up to four hours after injection (Figure 3B and Supplementary Movie S1), suggesting a half-life of R3VQ-S-AF488 extending over several hours in the brain. Four hours after the intravenous injection of R3VQ-S-AF488, the brain was harvested and paraffin sections were prepared.

Using anti-His mAb, immunodetection of amyloid plaques by R3VQ-S-AF488 was observed throughout the entire brain with an accompanying background signal which could correspond to the diffusion halo of the VHH (Figure 3C). Additional experiments were performed with a lower dose of R3VQ-S-AF488 (10 mg/kg) and in vivo detection of Ab deposition was observed but with decreased intensity. These results demonstrated that R3VQ brain penetration and its ability to label brain Ab lesions were dose-dependent, an observation which was also confirmed by IHC (Figure 3D).

The basic pI of VHHs has been proposed to be an important parameter predicting their ability to transmigrate across the BBB [START_REF] Li | Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging[END_REF]. R3VE-S-AF488 conjugate was prepared with a pI of ca. 7.5 (Figure S4A-B). A 10 mg/kg dose of R3VE-S-AF488 was iv injected in a oneyear-old PS2APP mouse. Forty-five minutes after injection, only cerebral amyloid angiopathy was shown without signs of extravasation or labeling of amyloid plaques (Figure S4C). Four hours after injection, the mouse was sacrificed. IHC was then performed to detect the presence of R3VE-S-AF488 in the brain. Compared to the staining obtained with the R3VQ-S-AF488 injected mouse (10 mg/kg), only a very light labeling of the plaques was observed (data not shown), supporting the hypothesis that the positive charges present on the surface of VHHs play a critical role for brain penetration of these antibodies across the BBB.

Finally, R3VQ-S-AF488 at 50 mg/kg was intravenously injected in a wild-type mouse.

Except for initial blood vessel labeling, no specific in vivo staining in the brain parenchyma was observed using two-photon microscopy imaging. A fluorescent cloud appeared 1 hour after injection (suggesting presence of unbound R3VQ-S-AF488 in the brain) but then rapidly vanished (Figure S5). These data indicate that R3VQ-S-AF488 crosses the BBB in wild-type mice but is not retained in the absence of amyloid plaques.

In summary, using two-photon microscopy after intravenous injection of R3VQ-S-AF488, we demonstrated the ability of the VHH R3VQ to cross the BBB, to reach its extracellular cerebral target and to transport fluorescent probes across the BBB, allowing its real-time visualization.

(Figure 5B). Specific staining of neurofibrillary tau inclusions with A2 was also readily obtained on free floating sections from Tg4510 mice (Figure S6A). Comparable staining was observed using the reference anti-pTau AT8 mAb (Mercken et al., 1992). No labeling was noted with tissues from age-matched wild type mice (Figure S6A). Double labeling performed on mouse sections using the A2 and reference antibody AT8 showed strong colocalization of the two immunostainings (Figure 5C). The load of tau lesions detected by A2 and AT8 antibodies were highly correlated underlining similar sensitivity-specificity profiles (Figure S6B).

Using paraffin-fixed AD brain sections, VHH A2 detected neurofibrillary tau tangles in neuronal soma, tortuous fibers and dystrophic neurites was also obtained in (Figure 5A).

Additional experiments (Figure S7) showed that VHH A2 also recognized tau-positive lesions from other tauopathies e.g. glial inclusions in tissues from patients with fronto-temporal dementia (oligodendroglial coiled bodies) or with progressive supranuclear palsy (astrocytic tufts).

To our knowledge, this is the first VHH that has been shown to detect tau lesions in both tau transgenic mouse and human tauopathy brain tissues.

In vivo labeling of tau-positive lesions:

Diffusion of A2 after stereotaxic brain injection

To test the ability of A2 to immunolabel tau lesions in vivo, A2 was stereotaxically injected into the cortex of 10-month-old Tg4510 mice. The mice were sacrificed 2 hours after injection. IHC revealed that A2 had diffused throughout the entire brain and bound to pTau aggregates in numerous cortical and subcortical regions (Figure S2B).

of the engineered VHHs. The presence of VHHs throughout the brain was subsequently confirmed by immunohistochemistry. Labeling of amyloid plaques and NFTs by R3VQ and A2 respectively was detectable until 4 hours post-injection with high signal intensity. These data suggest a long half-life of VHHs in the brain, at least when bound to target. Indeed, Nabuurs and collaborators indicated that their anti-Aβ VHH remained detectable in the brain for at least 24 hours post-injection [START_REF] Nabuurs | In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease[END_REF].

R3VQ-S-AF488 demonstrated rapid diffusion (0.5-1 h post-injection) throughout the brain of PS2APP mice and showed high specificity for Aβ deposits in both parenchymal (Aβ plaques)

and vascular spaces (CAA). In the Tg4510 mice receiving iv injection of A2-S-AF488, the specific staining of NFTs was observed 2h post-injection, much later than with R3VQ-S-AF488, probably because A2-S-AF488 needed to cross two barriers: the BBB and the cell membrane of neurons containing NFTs.

Traumatic uncontrolled BBB opening during the two-photon imaging procedure related to local brain bleeding due to craniotomy could have explained our results. Such a possibility is implausible. First we observed that both R3VQ and A2 VHHs were distributed throughout the whole brain following iv injection and not only in the vicinity of the cranial window as would be the case if there was local bleeding. Second, four hours after iv injection into transgenic mice without any cranial surgery both fluorescent VHHs labeled their respective targets in the brain.

It remains possible that BBB passage of VHHs results from a "naturally-compromised" barrier in the transgenic mice. Indeed, during normal aging, the permeability of the BBB increases, and disruption of the BBB occurs in a number of neurological disorders, including AD [START_REF] Erickson | Blood-brain barrier dysfunction as a cause and consequence of Alzheimer's disease[END_REF][START_REF] Marques | Blood-brain-barriers in aging and in Alzheimer's disease[END_REF]. A recent study indicates however a lack of BBB permeability in the PS2APP transgenic line we used for R3VQ imaging [START_REF] Bien-Ly | Lack of Widespread BBB Disruption in Alzheimer's Disease Models: Focus on Therapeutic Antibodies[END_REF], even at the oldest ages. Evidence of BBB leakage has been reported in Tg4510 mice but starting at 9 months of age [START_REF] Blair | Tau depletion prevents progressive blood-brain barrier damage in a mouse model of tauopathy[END_REF] while we performed in vivo imaging in younger animals. We confirmed the absence of BBB leakage in the two models we used by demonstrating that conventional immunoglobulin specific for the targeted antigens did not significantly penetrate into the CNS following iv injection.

Finally, it could be argued that high VHH concentrations (from 10 to 50 mg/kg) induce a transient disruption of the BBB and an extravasation of the VHH. However, our previous study using an Evans Blue dye exclusion assay showed that VHHs do not compromise the BBB even using VHH at very high concentration (133 mg/kg, i.e. 4 mg for an adult mouse) [START_REF] Li | Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging[END_REF].

To summarize we showed that both R3VQ and A2 can cross the BBB after peripheral administration and bind to extracellular (plaques) and intracellular (tangles) brain targets.

The factors influencing VHH transit across the BBB are poorly understood. Basic pI appears to be an important parameter since a VHH variant with a near-neutral pI (R3VE) showed only limited brain penetration. In our previous study using an anti-GFAP VHH we showed that BBB crossing was not energy-dependent, suggesting active transport is not involved [START_REF] Li | Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging[END_REF]. The transmigration of basic VHHs through the BBB probably occurs by adsorptivemediated endocytosis initiated by non-specific, charge-based interactions with proteins at the endothelial cell surface [START_REF] Abbott | Blood-brain barrier structure and function and the challenges for CNS drug delivery[END_REF][START_REF] Abulrob | The blood-brain barrier transmigrating single domain antibody: mechanisms of transport and antigenic epitopes in human brain endothelial cells[END_REF][START_REF] Bickel | Delivery of peptides and proteins through the blood-brain barrier[END_REF][START_REF] Vorbrodt | Ultracytochemical characterization of anionic sites in the wall of brain capillaries[END_REF]. Recent studies demonstrated that proteins with high net positive charge have indeed the ability to penetrate mammalian cells and deliver macromolecules into cells in vitro and in vivo [START_REF] Cronican | Potent delivery of functional proteins into Mammalian cells in vitro and in vivo using a supercharged protein[END_REF][START_REF] Cronican | A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo[END_REF]. Understanding the precise mechanism of VHH BBB passage will be important for optimizing efficient targeted probes in the future. Tissues from PS2APP mice injected with R3VQ or PBS were developed with anti-His mAb.

FIGURES

Tissues from Tg4510 mice injected with A2 or PBS were developed with anti-A2 polyclonal antibody. Tissue from transgenic mice injected with standard immunoglobulins were processed with a regular anti-mouse secondary antibody.

Two brain rostro-caudal levels are illustrated, with no noticeable differences in staining. C: In vivo imaging of R3VQ-S-AF488 and R3VE-S-AF488 using two-photon microscopy after iv injection (10mg/kg) in one-year-old PS2APP mice. Forty-five minutes after iv injection,both extracellular amyloid deposits and CAA were detected in the mouse injected iv rabbits with alpaca IgGs isolated on protein A and protein G columns [START_REF] Muyldermans | Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains[END_REF].

Library construction, phage preparation and phage display

The library was constructed as previously described in (Lafaye et al., 2009). E.coli TG1 strains (Lucigen) transformed with the recombinant phagemid (pHEN1) in 100 ml of 2YT (Yeast, Tryptone) + A (Ampicilline 100 µg/ml), + G (Glucose 1%) were cultured for approximately 2h30 at 37°C at 150 rpm from an OD 600 of 0.05 to an OD 600 of 0.5 which indicated that bacterial growth was in the exponential phase and corresponded approximately to 10 10 bacteria. VCS M13 helper phages (Stratagene, 2x10 11 ) in 100 µl were added in the culture, which was incubated for 30 min at 37°C without shaking followed by 30 min at 37°C with shaking. The culture was then centrifuged at 5000 rpm for 10 min and the pelleted cells were resuspended and incubated in 100 ml of 2YT +A +K (kanamycine 50 µl/ml) + 1mM IPTG overnight at 20°C to induce expression of recombinant phage-VHHs. The culture was then centrifuged and phage-VHHs present in the supernatant were precipitated after adding 20 ml of 20% PEG (polyethylene glycol) + 2.5M NaCl for 4 h at 4°C. After centrifugation at 8000 rpm the pellet was resuspended in 40 ml of water and 8 ml of 20% PEG + 2.5M NaCl and incubated for 1h at 4°C. The pellet was then resuspended after another centrifugation in 1ml PBS. To eliminate remaining bacterial debris, an additional centrifugation was performed at 10 000 rpm for 2 min. Finally, the supernatant containing phage-VHHs was stored at 4°C until required for use.

Phage Display technology allows the selection of antigen specific phage-VHHs. A large number of phage-VHHs (10 13 ) were used to performed a round of panning. A different blocking agent was used at each of the three rounds of panning: 2% skimmed milk, Licor blocking buffer (Biosciences) diluted with PBS in the ratio 1:4, and 4% BSA were respectively used. After blocking step, phage-VHHs were incubated with antigen precoated immunotubes or streptavidin-beads for 2h on wheel at room temperature. To remove non specific binders, a 6 x PBS Tween 0.1% and 4 x PBS washing procedure was performed, specific phage-VHHs were then eluted in 100 mM TEA (triethylamine) during 5 min on wheel and the excess TEA was neutralized immediately in 0.1M Tris-HCl, pH 7.6. E.coli TG1 at exponential growth phase was then infected with eluted phage-VHHs and then incubated for 30 min at 37°C without stirring then 30 min at 37°C under stirring. The infected bacteria (100µl) at different dilutions, 10 -2 , 10 -3 ,10 -4 were spread on 2YT+A+G Petri dishes for an overnight incubation at 37°C to calculate the OUTPUT of each round of panning (OUTPUT represents the number of selected phage-VHHs). The remaining bacteria were centrifuged at 4000 rpm for 15 min and the pellet was resuspended in 1 ml of 2YT and spread on 2YT+A+G Bio-assay dish (24 cm x24 cm) for an overnight incubation at 30°C and bacteria were recovered the next day with 4ml of 2YT containing 8% of DMSO (Dimethyl sulfoxide Sigma-Aldrich) and were stored at -80°C in aliquots of 1 ml. A decreased ELISA signal showed an inhibitory effect of corresponding Ab fragment on the phage-VHH, i.e. the Aβ fragment was recognized by the phage-VHH.

Panning of the library of anti-pTau VHH

The library (>6x10 8 clones) was panned against full-length pTau protein (Roche, 441 aminoacids, phosphoylated at multiple sites). Nunc Immunotubes (Maxisorp) tubes were coated overnight at 4°C with the antigen (10 µg/ml) in PBS. Phages (10 13 ) were panned by incubation with the coated tubes for 1h at 37°C with gentle agitation. ELISA experiments were performed on both full-length pTau protein and non phosphorylated tau protein.

5%. The conjugation was performed for 2 hours at room temperature, under protection from light. The non-conjugated fluorophore was then removed by extensive dialysis in PBS containing 300 mM NaCl with Dialysis Cassette (Thermo Scientific Slide-A-Lyzer ® MWCO 3500Da).

Protein quantification

Protein concentration was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific) using measurements of the protein absorbance at 280 nm. For an accurate protein quantification, quantitative amino acid analysis (AAA) was performed. Protein samples were submitted to hydrolysis with 6N HCl + 0.2% phenol in glass tubes for 20 hours at 110 °C, in presence of known amount of NorLeucine as internal standard. After HCl evaporation the samples are analysed on a Beckman 6300 analyser (post column derivatization with ninhydrin after ion-exchange chromatography separation).

Enzyme-linked Immunosorbent Assay (ELISA)

A modified version of a standard ELISA was used to test for the presence of VHH.

Streptavidin-coated microtiter plates (Thermo Scientific) were coated by incubation 15 min at room temperature with 1 µg/ml of biotinylated Aβ40 or 42 (Bachem) diluted in PBS.

Maxisorp Nunc-Immuno plates (Thermo Scientific) were coated with 1 µg/ml of pTau or Ova-pS422 (CSIDMVDS(PO 3 H 2 )PQLATLAD coupled to Ovalbumine) overnight at 4°C.

Plates were washed with buffer 0.1% Tween 20 in PBS and His-tagged VHHs were diluted in buffer 0.5% gelatin 0.1% Tween 20 in PBS. After 2 hours incubation at 37 °C, plates were washed again before adding respectively a rabbit anti-His tag antibody (eBiosciences), followed by peroxidase labeled goat anti-rabbit immunoglobulins (Abcam). OPD (o-Phenylenediamine Dako) was used as substrate. The optical density was measured spectrophotometrically at 490 nm using a Magellan microplate reader (Sunrise Tecan).

99% formic acid for 5 min, washed again under running tap water, quenched for endogenous peroxidase with 3% hydrogen peroxide and 20% methanol, and finally washed in water.

Sections were then blocked by incubating the sections for 30 min in 4% bovine serum albumin (BSA) in 0.05M Tris Buffered Saline, with 0.05% Tween 20, pH8 (TBS-Tween) (Sigma). Appropriate dilutions of primary antibodies (1-5 µg/ml of His tagged VHH) were then applied overnight in a humidified chamber at 4°C. Slides were washed with TBS-Tween and incubated with secondary antibodies rabbit anti-His tag at 1/1000 dilution or anti-His Tag mAb at 1/200 (Sigma) in TBS-Tween at room temperature for 1 h (Rabbit anti-His tag polyclonal was used to detect His-tag at the C terminus, while anti-His tag mAb was used to detect His tag at the N terminus). Slides were then incubated with reagents of Dako REAL TM Detection System, Peroxidase/DAB+ according to manufacturer's instructions. Chromogenic (DAB) revelation was developed until a good signal-to-noise ratio was obtained (about 5 min). After washing with water, slides were counter-stained with hematoxylin (Reactifs RAL) and then dehydrated in graded ethanol solutions (70, 90 and 100%) and cleared in xylene, and finally mounted in Eukitt mounting medium (Chem-Lab).

Immunohistochemistry -fresh tissues

Fresh frozen brain sections (10µm thick) from AD brains were obtained using a Leica CM 1950 Cryostat. Sections were fixed with pre-cooled (-20°C) acetone for 10 min. After washing in water, endogenous peroxidase was quenched, sections were washed again in water and blocked in 2% BSA in TBS-Tween 0.05% for 30 min. The following steps until final development in DAB were performed as described above.

Immunohistochemistry -free floating sections

Free-floating sections (50µm thick) were obtained with a vibratome (VT1000S Leica) or a microtome (Microm HM450 Thermo Scientific) and collected in 0.1 M PBS (pH 7.4). The sections were pretreated to neutralize endogenous peroxidase and non-specific binding sites All sections were first digitized using a slide scanner (Nanozoomer, Hamamatsu). Images were then exported in tif format at maximum resolution for subsequent morphological analysis.

Amyloid loads detected by 4G8 and R3VQ antibodies were calculated using the ICY program (http://icy.bioimageanalysis.org). Images were first converted to 8-bit gray levels images and then binarized using the thresholder plugin. Unified plugin parameters were applied for all sections. For each region of interest the thresholded surface reported to the total surface was calculated to give a regional amyloid load expressed in percent.

NFT loads derived from AT8 and A2 immunostaining were extracted using the ImageJ freeware (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2014). After 8-bits conversion a "maxentropy" thresholding was applied to get a binarized image allowing to estimate the NFT load (percent of tissue stained over total surface of the region of interest).

Double immunofluorescence staining

Paraffin-embedded sections obtained from young PS2APP or Tg4510 transgenic mice were first de-paraffinized and pretreated with formic acid as described above. They were then preincubated with TBS-tween containing 3% Normal Goat serum and 4% BSA for 1h at room temperature. For PS2APP brain tissue, processed sections were incubated with a cocktail of 2µg/ml of the anti Ab VHH R3VQ plus biotinylated 4G8 in PBS-Tween overnight at 4°C. For Tg4510 brain tissue, sections were incubated with 2µg/ml of the anti pTau VHH A2 and biotinylated AT8 (Thermo Scientific, 1/200) and were also incubated overnight at 4°C. All sections were then treated with rabbit anti His polyclonal (eBioscience, 2h), followed by a goat anti rabbit AlexaFluor 555 antibody (Life Technologies, 1/1000) and DyLight 488 streptavidin (KPL, 1/200). Finally all sections were stained with DAPI and mounted with Mowiol medium before image analysis using an Apotome.2 system for fluorescence microscopy (Zeiss).

Craniotomy Surgery for two-photon microscopy

Twelve-month-old PS2APP mice and 8-month-old Tg4510 mice were anesthetized by inhalation of isoflurane (1.5% vol/vol in pure O 2 ) and the body temperature was maintained with a thermocouple feedback warming blanket (37 °C). The head was immobilized in a stereotaxic frame. Fifty microliters of 2% lidocaine was injected subcutaneously under the skin over the skull for local anesthesia before incision and skin retraction. A small craniotomy (2 mm diameter) was performed above the posterior cortex of the right hemisphere. The dura mater was kept intact. To avoid movement artifacts, the skull opening was covered with 2% low melting point agarose and a coverglass. The optical window was secured and sealed to the skull with dental cement, covering the exposed skull and coverglass edges.

Administration of AF488-conjugated VHH and monoclonal antibodies

Intravenous injections: R3VQ-S-AF488 at a dose of 50 mg/kg or 10 mg/kg in 200µl and A2-S-AF488 at a dose of 10mg/kg in 150µl were slowly injected into the caudal vein of PS2APP and Tg4510 mice respectively. Two-photon imaging was then performed during the 2-4 subsequent hours.

For control experiments relying on peripheral injections with no in vivo imaging the same procedure were applied (iv injections of AlexaFluor conjugated 4G8 (50 mg/kg), muRb86 (10 mg/kg), R3VQ (50 mg/kg) and A2 (10 mg/kg) antibodies and of PBS) but no surgery was performed and mouse brains were harvested 4 hours after injections for histological examination.

1. Introduction nAChRs have multiple roles in regulating synaptic transmission of numerous neurotransmitter pathways. Neurotransmission is enhanced by both pre-and postsynaptic nAChRs, where they produce cell autonomous changes in signalling and in activity dependent mechanisms. This modulation translates into synaptic plasticity associated with development, learning, memory and addiction, in addition to contributing to neurodegenerative diseases and psychiatric conditions like Alzheimer's disease (AD) and schizophrenia [1]. Of interest in this review, nAChRs containing the a7 subunit have high Ca 2+ permeability, giving them strong influence for enhancing glutamate release, and in synaptic plasticity. Animal models, novel drugs and advances in electrophysiological techniques have opened up new avenues to further probe the specific role of a7 nAChRs in the normal and pathological mechanisms of the CNS. This review will concentrate on the role of the a7 nAChR on modulating glutamate release, starting with a brief overview of glutamatergic neurotransmission.

Glutamatergic neurotransmission

The role of glutamatergic neurotransmission has been established through decades of research, for more than 75 years [2]. Initially, glutamate was considered to solely have a metabolic function in the CNS due to it being an essential amino acid and an intermediate metabolite in the Krebs cycle [3]. The first excitatory action of glutamate was demonstrated in 1954 by Hayashi [4], where injecting glutamate into the brain or carotid arteries produced body convulsions, and by Curtis and Watkins in 1960 who showed excitation and depression of spinal neurons with administration of amino acids [5]. Since these pioneering studies, a tremendous amount of research has greatly expanded our knowledge of the glutamatergic system.

Glutamate is the principal excitatory neurotransmitter in the mammalian central nervous system and is a major modulator of neuroplasticity, as it participates in normal synaptic transmission, long-term potentiation and long-term depression, neuronal maturation and synaptogenesis [6]. In parallel, glutamate has been indicated as the primary factor responsible for excitotoxicity, a process where excessive glutamate release occurs in association with neurological disorders such as amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, epilepsy, cerebral ischemia, Huntington's disease, autism and schizophrenia [7][8][9][10][11][12].

Glutamate employs its signaling function by binding to and activating specific receptors, either being ionotropic receptors, which are ion channels that flux cations, or metabotropic receptors, which indirectly activate or inhibit second messenger signaling through their interaction with G-proteins [13]. In general, ligand-gated ion channel receptors, including glutamate receptors, are multimeric assemblies of individual subunits and the combinatorial composition of these subunits define the functional properties of the receptor [14].

Ionotropic glutamate receptors

Ionotropic glutamate receptors are divided into three classes:

N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4isoxazole propionic acid (AMPA) and kainate receptors. They are composed of four subunits and their channel properties are determined by their subunit composition and expression, giving them diverse response properties. In the mammalian brain, about 70% of synapses contain NMDARs or AMPARs [15]. Three families of NMDAR subunits have been characterized: GRIN1, GRIN2A through D and GRIN3A and B. NMDARs are permeable to Ca 2+ , which induces signaling cascades linked to neuronal transmission. Calcium influx through the NMDAR is one of the major mechanisms contributing to long-term potentiation (LTP) and long-term depression (LTD) of the synapse. NMDARs require the simultaneous binding of two different agonists in order to activate, with the primary binding site typically being occupied by glutamate, and a binding cofactor in a separate site which can be glycine or a polyamine, such as spermine [16,17]. Of great importance for its role in synaptic plasticity, NMDARs contain an internal binding site for Mg 2+ , thereby creating a voltage-dependent block at resting potential, under physiological conditions [18]. Upon depolarization above a certain threshold, mainly by the activation of AMPARs and kainate receptors, or by back propagating action potentials, the Mg 2+ is removed thereby allowing ion flux through the NMDAR [19].

AMPARs have lower glutamate affinity than NMDARs and also have faster kinetics, thus they are responsible for rapid excitatory neurotransmission [20]. The AMPAR family contains four different subunit types, from GRIA1 to GRIA4, with the subunit composition of an individual tetrameric AMPAR and the post-transcriptional editing determining the specificity of its cation influx [21].

Kainate receptors have been shown to regulate excitatory and inhibitory synaptic transmission [22]. They are also tetrameric assemblies, like AMPARs, but are composed of a combination of GRIK1-3 and GRIK4-5 subunits. The kainate receptors have limited distribution in the brain, in comparison to NMDARs and AMPARs, and are less studied. Kainate receptors share similar properties to AMPARs by being mainly localized in the postsynaptic membrane [23]. Finally, delta receptors (GRID1 and GRID2), another class of ligand-gated excitatory amino acid receptor subunits, have been demonstrated to exist in cerebellar Purkinje cells with the receptor having similar functions to AMPARs [24].

Metabotropic glutamate receptors

The metabotropic glutamate receptors (mGluRs) are a sub-type of glutamate receptor that act primarily through the activation of intracellular G-proteins and downstream signal transduction pathways. Eight mGluRs have been identified and they are subdivided into three groups (groups I-III) based on their protein sequence, pharmacology and signaling cascade mechanism [25]. They are localized both pre-and post-synaptically and are highly expressed in the hippocampus, cerebellum and cerebral cortex, where they modulate neurotransmission, synaptic plasticity and neuronal development. Postsynaptic activation of mGluR can potentiate or inhibit ion channel activity, whereas presynaptic mGluR has been shown to decrease excitatory glutamatergic and inhibitory GABAergic neurotransmission [26]. mGluRs also regulate neurotransmission presynaptically by exerting their control on voltage dependent calcium channels and SNARE-dependent vesicular release [27]. Presynaptic group II and III metabotropic receptor modulators are actively being developed as potential drugs for the treatment of neuropsychiatric diseases [28]. 1.2. Neuronal a7 nicotinic acetylcholine receptor Nicotinic acetylcholine receptors (nAChRs) are expressed throughout the brain, forming ligand-gated ion channels composed of five subunits with the various combinations of nAChR subunits determining the properties, pharmacology and ultimately, function of the mature receptor. The nAChR subunits have a large extracellular N-terminal domain that contributes to ligand binding, three hydrophobic transmembrane regions (M1-M3), a large intracellular loop, a fourth transmembrane region (M4) and a short extracellular C-terminal. The nAChRs shift between three main states: closed, open and desensitized [29]. When the channel is in the closed state, it is capable of binding ligand, but the channel does not conduct. The open state requires agonist binding to the receptor, which allows the flux of cations. Upon desensitization, the channel blocks the flux of cations.

The a7 nAChR represents a major nicotinic subtype highly expressed in mammalian brain regions including the olfactory bulb, cerebral cortex, hippocampus, hypothalamus and amygdala [30]. Initially, the a7 nAChR was considered to be functional exclusively as a homopentameric receptor, but recently the co-expression and assembly of a7 nAChR subunits with other nicotinic subunits has been shown [31]. This co-assembly results in receptors with different ion permeability and desensitization properties when compared to the homomeric a7 nAChR [32,33]. Receptors composed of a7 subunits desensitize rapidly and have a high Ca 2+ to Na + permeability ratio, that exceeds that of NMDARs [34][35][36]. Thus, opening of a7 containing nAChR channels can cause a significant increase in the intracellular Ca 2+ concentration, due to second messenger-mediated release of calcium from intracellular stores or from voltage-gated Ca 2+ channels (VGCCs) [37]. In some cases, this type of signaling may be independent of channel activation as it has shown an effect of a7 nAChRs in non-neuronal cells, particularly cells of the immune system, which is independent of generating ion currents [38]. Such modulation of immune cell function has been reported to be most effectively accomplished by ligands that have little or no ion channel efficacy in cells that express a7 nAChR that are capable of ion channel function, suggesting that the intracellular domain directly mediates signal transduction [39][40][41]. This alternative signaling modality may also occur in neurons.

Electrophysiological recordings from intact brain neurons have increased our knowledge regarding the localization of specific a7 nAChR subunits to defined neuronal compartments. The a7 nAChR is localized in neurons both pre-and post-synaptically, at dendrites, pre-terminal axon regions, axon terminals and the soma [1]. The localization of the a7 nAChR, in combination with the activation of voltage-dependent Ca 2+ channels and NMDARs, improved our understanding of its role in synaptic plasticity, in the formation of synapses and in the modulation of excitatory synaptic transmission [42,43]. These mechanisms will be explored later in this review.

1.3. a7 nAChRs facilitate glutamate release 1.3.1. Presynaptic a7 nAChRs In the stereotypical excitatory neurotransmission mechanism, with the generation of a pre-synaptic neuron action potential, neurotransmitter is released from pre-synaptic terminals, causing depolarization of the post-synaptic neuron. Depolarization of the efferent neuron activates pre-synaptic voltage-gated Ca 2+ channels (VGCCs) causing a rise in intracellular calcium levels and leads to calcium dependent vesicular neurotransmitter release into the synapse [37]. Under specific conditions, opening of a7 containing nAChRs alone can provide sufficient Ca 2+ influx to depolarize the cell and trigger neurotransmitter release [44]. The localization of nAChRs subtypes in the brain is important for understanding how nicotinic receptors affect neurotransmission.

Activation of presynaptic a7 nAChRs can elevate Ca 2+ levels restricted to the axonal bouton by either direct calcium gating or through local depolarization [45]. For example, the frequency of miniature excitatory postsynaptic potentials induced by glutamatergic neurons from the hippocampus or the medial habenula has been demonstrated to increase after the induction of nicotinic currents [46]. Measurements of intracellular Ca 2+ in single mossyfiber presynaptic terminals in the CA3 of the hippocampus revealed that nAChRs containing the a7 subunit can also elevate Ca 2+ influx to sufficient levels for the induction of vesicular neurotransmitter release [44]. Direct electrophysiological experiments showed that nicotine application induced inward currents in presynaptic calyces and the blockade of this effect by a-bungarotoxin suggested the involvement of presynaptic a7 nAChRs [47].

In rat auditory cortex, presynaptic nAChRs of the a7 subtype have been shown to stimulate glutamate release and selectively potentiate NMDA receptor-mediated synaptic transmission [48]. These mechanisms are present only in early postnatal life and might contribute to the activity dependent plasticity of the developing sensory cortex. Studies on synaptosomes, where nicotinic agonists induced the release of preloaded transmitters, confirmed that a7 nAChRs are found presynaptically [49,50].

More specifically, experiments performed in rat striatal slices revealed that activation of a7 nAChRs on glutamatergic axons stimulated glutamate release, which consequently, via presynaptic glutamate receptors on dopamine terminals, stimulated dopamine release. Later, Marchi et al. demonstrated that a7 nAChRs are localized on human and rat brain glutamatergic terminals where they enhance release when the terminals are also depolarized [51].

Numerous other studies showed that the a7 subunit participates in presynaptic nAChRs that facilitate the release of glutamate at several synapses, including in medial habenulainterpeduncular nucleus synapses [52], intra-olfactory bulb synapses [53], synapses between ventral lateral geniculate nucleus neurons [54], olfactory bulb-amygdala synapses [55], corticoamygdala synapses [56] and more extensively, cortico-limbic synapses [57] (Table 1). In developing hippocampal neurons, a7 nAChRs on presynaptic glutamatergic terminals, by facilitating the release of glutamate, contribute to converting silent synapses to functional synapses [58]. Lin et al. [59] demonstrated that axonal a7 nAChRs modulate presynaptic NMDAR expression and the structural plasticity of glutamatergic presynaptic boutons during early synaptic development. Chronic inactivation of a7 nAChRs markedly increased cell surface NMDAR expression as well as the number and size of glutamatergic axonal varicosities in cortical neuronal cultures [START_REF] Lin | Axonal a7 nicotinic ACh receptors modulate presynaptic NMDA receptor expression and structural plasticity of glutamatergic presynaptic boutons[END_REF]. Additionally, immunocytochemical studies showed that a significant percentage of nucleus accumbens (NAc) terminals were glutamatergic and possessed GluN1 and a7 containing nAChRs. Nicotinic a7 receptor activation selectively potentiated the function of NMDA receptors in the glutamatergic terminals of the NAc. The authors postulated that this may be a general feature in various brain areas since a similar nAChR-mediated bolstering of NMDA-induced ([ 3 H]D-Asp) overflow was also observed in hippocampal synaptosomes [START_REF] Zappettini | Nicotinic a7 receptor activation selectively potentiates the function of NMDA receptors in glutamatergic terminals of the nucleus accumbens[END_REF]. Lastly, patch-clamp recordings from hippocampal CA3 pyramidal neurons revealed that presynaptic a7 nAChRs enhance hippocampal mossy fiber glutamatergic transmission via PKA activation [START_REF] Cheng | Presynaptic a7 nicotinic acetylcholine receptors enhance hippocampal mossy fiber glutamatergic transmission via PKA activation[END_REF]. They showed that nicotine, in combination with NS1738, an a7 nAChR positive allosteric modulator (PAM), strongly potentiated the amplitude of evoked EPSCs and reduced the EPSC paired-pulse ratio. The action of nicotine and NS1738 was mimicked by PNU-282987, an a7 nAChR agonist, and was absent in a7 nAChR knock-out mice. 1.3.2. Mechanisms by which presynaptic a7 nAChRs alter glutamate release

The pre-synaptic mechanisms of a7 nAChR enhancement of glutamate release are currently being actively pursued and numerous studies have demonstrated its multiple modes of action. a7 nAChRs can directly control the gating of calcium and also cause local depolarizations via a mechanism of activating voltage gated calcium channels, thereby causing release of calcium from internal stores [37,45]. These stores can either be ryanodine receptor or inositol triphosphate stores, as demonstrated in hippocampal astrocytes and hippocampal axons, respectively [START_REF] Welsby | Nicotinic receptor-mediated enhancement of long-term potentiation involves activation of metabotropic glutamate receptors and ryanodine-sensitive calcium stores in the dentate gyrus[END_REF][START_REF] Zhong | Nicotine elicits prolonged calcium signaling along ventral hippocampal axons[END_REF].

Due to the high Ca 2+ permeability of a7 nAChRs, in comparison to other nAChR subunits, the impact of their activation can have large effects on synaptic output. By activating nicotinic receptors on ventral hippocampal inputs to medium spiny neurons with the direct application of nicotine, sustained increases in presynaptic extracellular calcium, and thereby increased glutamate release, occurred for up to 30 min [START_REF] Zhong | Nicotine elicits prolonged calcium signaling along ventral hippocampal axons[END_REF][START_REF] Zhong | Presynaptic type III neuregulin 1 is required for sustained enhancement of hippocampal transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine receptors[END_REF]. This activation was IP3-dependent and was shown to also be reliant on presynaptic calmodulin-dependent kinase and phospholipase C signaling.

Moreover, direct interaction of a7 nAChRs has been reported

with G-protein signaling, further increasing the complexity of the a7 nAChR mode of presynaptic action [START_REF] Nordman | An interaction between a7 nicotinic receptors and a G-protein pathway complex regulates neurite growth in neural cells[END_REF]. 1.3.3. Postsynaptic and extrasynaptic a7 nAChRs In addition to their presynaptic localization, a7 nAChRs are also found postsynaptically in various brain regions including the neocortex and hippocampus. Postsynaptic a7 nAChRs are found on both inhibitory interneurons and excitatory neurons where they cause fast inward cationic currents. They can therefore cause amplification of weak inputs on pyramidal cells directly by activation of their postsynaptic a7 nAChRs or indirectly by inhibition of GABAergic interneurons that directly synapse onto pyramidal cells. The strengthening of Schaffer collateral pathway inputs in the hippocampus by a7 nAChRs also has an impact on synaptic plasticity. [START_REF] Ji | Inhibition and disinhibition of pyramidal neurons by activation of nicotinic receptors on hippocampal interneurons[END_REF][START_REF] Ji | Timing and location of nicotinic activity enhances or depresses hippocampal synaptic plasticity[END_REF]. Of great interest, Li et al. demonstrated the presence of an a7 nAChR-NMDA protein complex in rat hippocampal tissue and by using in vitro binding assays, they provided evidence that the a7 nAChR and GRIN2A subunits interact directly [START_REF] Li | The a7nACh-NMDA receptor complex is involved in cue-induced reinstatement of nicotine seeking[END_REF]. nAChRs, in general, are also found extrasynaptically where they have non-synaptic function, and can modulate the local excitability thereby having an effect on neurotransmitter release. Specifically, activation of extrasynaptic nAChRs caused induction of spike discharge and enhanced GABA release in interpeduncular nucleus interneurons [START_REF] Léna | Evidence for preterminal nicotinic receptors on GABAergic axons in the rat interpeduncular nucleus[END_REF][START_REF] Lucas-Meunier | Cholinergic modulation of the cortical neuronal network[END_REF]. Overall, there are limited studies of these extrasynaptic receptors and moreover, no direct demonstration exclusive to a7 nAChRs, and therefore further studies using knockout models could further decipher the potential extrasynaptic function of a7 nAChRs. 1.4. a7 nAChRs and synaptic plasticity

The cellular processes of synaptic plasticity are critical for cognitive function and in particular, synaptic plasticity has been associated with learning, memory, behavior, attention and working memory [57,[START_REF] Malenka | LTP and LTD: an embarrassment of riches[END_REF]. Synaptic plasticity is the ability of synapses to increase or decrease their strength, thereby changing the information flow between specific synapses. There are multiple forms of synaptic plasticity that have been demonstrated, mainly studied in the hippocampus, including short-term potentiation, long-term potentiation (LTP), and long-term depression (LTD), all of which have been associated with memory [START_REF] Placzek | Nicotinic mechanisms influencing synaptic plasticity in the hippocampus[END_REF].

The timing of action potentials in presynaptic and postsynaptic neurons influences the induction of LTP or LTD. LTP can be induced experimentally using several protocols, but the most common method of in vitro LTP induction combines presynaptic high frequency stimulation with a postsynaptic depolarization. The combination of nAChR mediated presynaptic facilitation of excitatory neurotransmitter release, with a coincident postsynaptic stimulus, can directly produce synaptic plasticity or modulate its direction and duration [START_REF] Dan | Spike timing-dependent plasticity of neural circuits[END_REF]. 1.4.1. a7 nAChRs modulate LTP and LTD Several studies have highlighted the role of a7 nAChRs in modulating LTP and LTD. In vitro hippocampal slice excitatory postsynaptic potential recordings suggested that a7 nAChR desensitization and non-a7 nAChR activation were involved in the induction of LTP by nicotine [START_REF] Fujii | Acute and chronic nicotine exposure reverse agerelated declines in the induction of long-term potentiation in the rat hippocampus[END_REF]. It was also demonstrated that nicotine accelerates depotentiation (reversal of LTP), enhances LTD in the rat hippocampal CA1 region, and the nicotine effect appeared to be at least partly mediated by nicotine-induced desensitization of a7 nAChRs [START_REF] Fujii | Nicotine accelerates reversal of long-term potentiation and enhances long-term depression in the rat hippocampal CA1 region[END_REF]. Field recordings obtained from rat dentate gyrus in acute slices showed that nicotine enhances stimulation-induced LTP in an a7 nAChR and NMDAR-dependent manner [START_REF] Welsby | Nicotinic receptor-mediated enhancement of long-term potentiation involves activation of metabotropic glutamate receptors and ryanodine-sensitive calcium stores in the dentate gyrus[END_REF]. Nevertheless, nAChR enhanced LTP required activation of mGluR5, Ca 2+ influx through L-type VGCCs and Ca 2+ induced Ca 2+ release from ryanodine-sensitive intracellular Ca 2+ stores, and it was demonstrated that chronic application of nicotine in vivo (two weeks of twice-daily nicotine injections) also enhanced LTP induction in slices. This LTP induction was dependent on the activation of mGluRs coupled with Ca 2+ release from ryanodinesensitive intracellular stores, suggesting that acute in vitro, or chronic in vivo nicotine administration, follow similar pathways [START_REF] Welsby | Nicotinic receptor-mediated enhancement of long-term potentiation involves activation of metabotropic glutamate receptors and ryanodine-sensitive calcium stores in the dentate gyrus[END_REF].

The in vivo data on nAChRs and their regulation of synaptic plasticity are sparser. In vivo experiments performed on guinea pig dentate gyrus revealed that nicotine blocks the inhibition of LTP caused by Angiotensin II [START_REF] Wayner | Nicotine blocks angiotensin II inhibition of LTP in the dentate gyrus[END_REF]. In addition, a challenge dose of nicotine (0.4 mg kg À1 s.c.) produced a long-lasting potentiation of field excitatory postsynaptic potentials evoked in the dentate gyrus by stimulation of the medial perforant path of urethaneanaesthetized rats that were primed four weeks previously with seven daily injections of nicotine (0.8 mg kg À1 s.c.) [START_REF] Hamid | Nicotine induces long-lasting potentiation in the dentate gyrus of nicotine-primed rats[END_REF]. The effects of nicotine were blocked by mecamylamine, confirming activation of central nAChRs and these effects were also blocked by propranolol, a b-adrenergic receptor antagonist, implying that the effects were secondary to nicotine stimulation of noradrenaline release. Both hippocampal noradrenalin release, and an increase in tyrosine hydroxylase activity, are associated with nicotine priming, therefore it seems that significant amounts of noradrenaline were released in the primed rats, being sufficient to cause b-adrenoreceptor induced long lasting potentiation [START_REF] Mitchell | Increases in tyrosine hydroxylase messenger RNA in the locus coeruleus after a single dose of nicotine are followed by time-dependent increases in enzyme activity and noradrenaline release[END_REF].

Further experiments showed that systemic application of nicotine or choline, induced the long lasting potentiation in vivo in the intact mouse dentate gyrus, signifying that a7 nAChRs may contribute to the induction of LTP by nicotine [START_REF] Matsuyama | Activation of nicotinic acetylcholine receptors induces long-term potentiation in vivo in the intact mouse dentate gyrus[END_REF]. More specifically, intraperitoneal application of nicotine at a dose of 3.0 mg/kg, but not 0.03 or 0.3 mg/kg, produced a gradually developing, long-lasting increase for 2 h, similar to tetanic LTP. In an additional study, the same authors showed that epibatidine, a b2 selective agonist, induced LTP via a4b2 nAChRs and that both a7 and a4b2 nAChRs were essential for full-scale LTP, implying that both of these nAChRs play an important role in synaptic plasticity [START_REF] Matsuyama | Epibatidine induces long-term potentiation (LTP) via activation of alpha4beta2 nicotinic acetylcholine receptors (nAChRs) in vivo in the intact mouse dentate gyrus: both alpha7 and alpha4beta2 nAChRs essential to nicotinic LTP[END_REF].

There is strong evidence that the nicotinic effects on synaptic plasticity in the hippocampus are highly mediated by the activation of inhibitory GABAergic interneurons, because nAChRs are more densely expressed on these interneurons [START_REF] Mckay | Regulation of synaptic transmission and plasticity by neuronal nicotinic acetylcholine receptors[END_REF]. nAChRs control the excitatory transmission indirectly through GABAergic interneurons and modulate the glutamatergic system by inhibiting or stimulating pyramidal neurons, depending on the timing of their activation [START_REF] Griguoli | Regulation of hippocampal inhibitory circuits by nicotinic acetylcholine receptors[END_REF].

Nicotinic receptors are also known to regulate synaptic plasticity in brain areas that play an important role in reward and addiction, such as the ventral tegmental area (VTA). In the VTA, a7 nAChRs and NMDARs have complementary roles in inducing a long-term enhancement of excitation of the brain reward system. Upon the presence of nicotine in the VTA, activation of presynaptic a7 nAChRs allows entry of Ca 2+ into glutamatergic terminals, enhancing glutamate release. This enhancement of glutamate release results in increased activation of NMDARs, as well as NMDA-insensitive ionotropic glutamate receptors, leading to Ca 2+ influx into the postsynaptic DA neuron, causing an induction of LTP [START_REF] Mansvelder | Long-term potentiation of excitatory inputs to brain reward areas by nicotine[END_REF]. The increased delivery of glutamate caused activation of postsynaptic AMPARs coupled with nAChR-mediated postsynaptic depolarization, thereby facilitating the release of Mg 2+ blockade of the NMDARs, which contributed to LTP induction. Interestingly, this differs from in vitro hippocampus preparations where persistent high frequency stimulation of presynaptic cells combined with depolarization of the postsynaptic cell, with nAChR activation, are all required to produce LTP [START_REF] Mckay | Regulation of synaptic transmission and plasticity by neuronal nicotinic acetylcholine receptors[END_REF]. Within the VTA, as described above, one major mechanism by which nicotine contributes to LTP of glutamatergic synapse onto dopamine neurons is through sustained activation of presynaptic a7 nAChRs to augment glutamate transmission onto dopaminergic neurons. 1.4.2. a7 nAChRs modulate the morphology of glutamatergic synapses

Recent studies suggest that a7 nAChRs, in combination with the facilitation of glutamatergic neurotransmission, influence the shape, morphology and density of glutamatergic synapses in hippocampus and cortex. It has been shown that a7 nAChRs contribute to the organization of dendrites of individual neurons and modulate the tempo of adult-born neuron physiological and morphological development [START_REF] Campbell | Endogenous signaling through alpha7-containing nicotinic receptors promotes maturation and integration of adult-born neurons in the hippocampus[END_REF]. It was demonstrated that functional a7 nAChRs are necessary for normal survival, maturation, and integration of adult-born neurons in the dentate gyrus of mice, and a7 KO mice have less elaborate dendritic structure, a longer maintenance of a depolarizing chloride gradient coupled with immature GABAergic current kinetics.

Additionally, mice lacking the a7 subunit had a reduced dendritic branching pattern in CA1 hippocampal and cortical pyramidal neurons during postnatal development (P24) and a 64% increase in thin (L-type) dendritic spines on the CA1 basilar tree when compared to WT mice [START_REF] Morley | Dendritic spine alterations in the hippocampus and parietal cortex of alpha7 nicotinic acetylcholine receptor knockout mice[END_REF].

Further studies revealed that a7 KO mice have an altered balance in the excitatory/inhibitory input they receive, and significantly reduced numbers of glutamatergic synapses.

Nicotinic activation of a7 nAChRs in WT organotypic culture, as well as in cell culture, increased the number of glutamatergic synapses, thus the reduction of a7 with RNA interference produced a critical loss of glutamatergic synapses [START_REF] Lozada | Glutamatergic synapse formation is promoted by a7-containing nicotinic acetylcholine receptors[END_REF]. On the contrary, as previously described in this review, chronic inactivation of a7 nAChRs increases the numbers of presynaptic glutamatergic boutons by enhancing presynaptic NMDAR expression and implicates presynaptic a7 nAChR/NMDAR interactions in synaptic development, showing that desensitization of a7 nAChRs also plays an important role on synaptic rearrangements [START_REF] Lin | Axonal a7 nicotinic ACh receptors modulate presynaptic NMDA receptor expression and structural plasticity of glutamatergic presynaptic boutons[END_REF].

Finally, a novel mechanism by which nicotinic signaling modulates synaptic function and increases synaptic strength in the hippocampus was demonstrated in mouse organotypic slice and in rat cell culture by inducing the cells to express super-ecliptic pHluorin-tagged GluA1-containing AMPA receptors [START_REF] Halff | A novel mechanism for nicotinic potentiation of glutamatergic synapses[END_REF].

Nicotine, acting through a7-containing nAChRs on the postsynaptic neuron, caused the stabilization and accumulation of GluA1-containing AMPA receptors on dendritic spines. These changes were not dependent on AMPAR or NMDARs, suggesting cell-autonomous regulation, independent of simultaneous rapid synaptic transmission.

1.5. Involvement of the glutamatergic system in nicotine dependence: the role of a7 nAChRs Nicotine is the main psychoactive component of tobacco that leads to tobacco dependence, a major public health problem resulting in significant morbidity and mortality [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF]. Despite the availability of numerous smoking-cessation medications, most individuals who attempt to quit smoking eventually relapse. Nicotine dependence is characterized by three phases: (1) acquisition and maintenance of nicotine seeking behavior, (2) withdrawal symptoms upon abstaining from smoking and (3) vulnerability to relapse [START_REF] Markou | Neuronal mechanisms underlying development of nicotine dependence: implications for novel smoking-cessation treatments[END_REF]. Nicotine is self-administered by humans, non-human primates and rodents, affecting mood, cognition and various body functions [START_REF] Fowler | Subtypes of nicotinic acetylcholine receptors in nicotine reward, dependence, and withdrawal: evidence from genetically modified mice[END_REF]. The pharmacological effects of nicotine on the CNS are due to its binding to the orthosteric site of nAChRs [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF]. Nicotine binding activates nAChRs, but at the concentrations observed in the blood of regular smokers, it desensitizes most heteromeric nAChRs and induces a long-term increase in receptor numbers, called up-regulation [START_REF] Colombo | Biogenesis, trafficking and upregulation of nicotinic ACh receptors[END_REF].

At the circuit level, activation of nicotinic receptors in the mesolimbic reward system is at the center of nicotine's rewarding effects. Dopamine signaling from the VTA to the NAc, hippocampus, amygdala and prefrontal cortex encompass the bulk of the mesolimbic reward system affecting memory and emotion. The dopaminergic neurons of the mesolimbic system have presynaptic nAChRs and therefore nicotine binding causes enhanced release of neurotransmitter [START_REF] Balfour | Nicotine Psychopharmacology[END_REF]. Additionally, GABAergic VTA neurons have nAChRs and nicotine binging causes GABA release, inhibiting dopaminergic neurons. Interestingly, this inhibition can be overridden by excitation due to GABAergic neurons having more rapid desensitization, at lower doses than a7 homomeric nAChRs on the excitatory neurons, thereby causing increased dopamine release.

Both glutamate and GABA neurotransmission play significant roles in nicotine dependence. Experimental evidence suggests that b2-, a4-, a6 and a7-containing nAChRs mediate the reinforcing and behavioral effects of nicotine [START_REF] Review | Neurobiology of nicotine dependence[END_REF], but here we will focus on the a7-containing nAChRs.

As mentioned previously, nicotine increases glutamatergic transmission by acting on excitatory a7 nAChRs located on glutamatergic afferents in the VTA and NAc. In parallel, several studies performed in rats showed that blockade of glutamatergic neurotransmission attenuated the motivational effects of nicotine, with the NMDA, mGlu2/3 and mGlu5 receptors playing an important role in the regulation of the reinforcing effects of nicotine [START_REF] Liechti | Metabotropic glutamate 2/3 receptors in the ventral tegmental area and the nucleus accumbens shell are involved in behaviors relating to nicotine dependence[END_REF][START_REF] Kenny | NMDA receptors regulate nicotine-enhanced brain reward function and intravenous nicotine self-administration: role of the ventral tegmental area and central nucleus of the amygdala[END_REF][START_REF] Markou | Metabotropic glutamate receptor 5 antagonist 2methyl-6-(phenylethynyl) pyridine (MPEP) microinfusions into the nucleus accumbens shell or ventral tegmental area attenuate the reinforcing effects of nicotine in rats[END_REF]. Consistent with these findings, Markou and Paterson showed that a7 nAChR subtype plays a critical role in the reinforcing effects of acute nicotine administered intravenously, but not on nicotine dependence, because of lack of precipitation of the nicotine withdrawal syndrome in nicotine-treated animals [START_REF] Markou | The nicotinic antagonist methyllycaconitine has differential effects on nicotine self-administration and nicotine withdrawal in the rat[END_REF]. Specifically, methyllycaconitine (MLA), a relatively selective antagonist of the a7 nAChR subtype, was administered to rats that were allowed to self-administer nicotine intravenously, and also to rats that had been prepared with nicotine-containing osmotic mini-pumps and trained in a brain stimulation reward procedure. The data demonstrated that pretreatment with the highest doses of MLA reduced self-administration at two doses of self-administered nicotine, although MLA administration, at every dose tested, had no effect on brain reward thresholds and on somatic signs of withdrawal during chronic nicotine administration. To conclude, MLA administration reduced the positive-reinforcing effects of nicotine in non-nicotine-dependent subjects. In contrast, Grottick et al. have reported no effect of a similar dosing regimen on self-administration when rats had a two-day wash-out period between MLA doses [START_REF] Grottick | Evidence that nicotinic alpha(7) receptors are not involved in the hyperlocomotor and rewarding effects of nicotine[END_REF]. Differences in experimental procedures possibly contributed to the contradictory results in these two studies.

Recently, brain slice patch recordings revealed that both a7 and b2 nAChRs contribute to the increase in the AMPAR/NMDAR current ratio induced by in vivo nicotine administration, which leads to an increase of presynaptic and postsynaptic glutamatergic function, and consequently initiates glutamatergic synaptic plasticity, which may be an important early neuronal adaptation to nicotine reward and reinforcement [START_REF] Gao | Mechanisms involved in systemic nicotine-induced glutamatergic synaptic plasticity on dopamine neurons in the ventral tegmental area[END_REF]. On the contrary, brief exposure of nicotine to brain slices from drug-naive adult rats, followed by a period of recovery, resulted in an NMDAR-dependent increase of AMPAR/NMDAR ratio in VTA dopaminergic neurons. This effect, which is consistent with the induction of LTP, required the excitation of dopaminergic neurons mediated by nicotineelicited activation of somatodendritic a4b2 nAChRs, whereas a7 receptors had only a minor effect on dopaminergic neuron excitability.

KO of the a7 nAChR subunit had no effect on conditioned place preference in mice, but in rats, blocking a7 nAChRs, specifically in the NAc, caused increased nicotine self-administration [START_REF] Brunzell | Alpha7 nicotinic acetylcholine receptors modulate motivation to self-administer nicotine: implications for smoking and schizophrenia[END_REF].

These effects in rats were reversed with an a7 nAChR agonist, implying that reinforcement requires enhanced glutamate transmission whereas decreased transmission blocks the primary reinforcing and motivational results with nicotine administration. On the other hand, withdrawal after chronic nicotine exposure is characterized by decreased glutamatergic transmission [START_REF] Mansvelder | Synaptic mechanisms underlie nicotine-induced excitability of brain reward areas[END_REF]. MLA increased somatic withdrawal signs in wild-type and b2 KO mice following chronic nicotine treatment [START_REF] Jackson | Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs[END_REF], although, under similar treatment conditions, somatic withdrawal signs were diminished in a5, a7, and b4 knockout mice [START_REF] Fowler | Subtypes of nicotinic acetylcholine receptors in nicotine reward, dependence, and withdrawal: evidence from genetically modified mice[END_REF]. A recent study of Salas et al. indicated that a7 nAChRs control the severity of the nicotine withdrawal syndrome [START_REF] Salas | Decreased withdrawal symptoms but normal tolerance to nicotine in mice null for the alpha7 nicotinic acetylcholine receptor subunit[END_REF]. Therefore, there is no clear defined role of a7 nAChRs in nicotine reinforcement and further studies probing a7 KO mice, or rats, with self-administration could resolve its function in nicotine dependence. Our in vivo work suggests that a7 nAChR is not crucial for nicotine self administration [START_REF] Pons | Crucial role of alpha4 and alpha6 nicotinic acetylcholine receptor subunits from ventral tegmental area in systemic nicotine self-administration[END_REF] and a7 KO mice exhibit only a slightly altered electrophysiological response to nicotine injection when DAergic neurons are recorded in vivo [START_REF] Mameli-Engvall | Hierarchical control of dopamine neuron-firing patterns by nicotinic receptors[END_REF].

a7 nAChRs as a target in CNS diseases

The exact mechanism through which nicotinic signaling regulates the glutamatergic system remains unclear, but it involves neuronal processes relevant in cognitive functions and major pathological conditions have been linked to decrease expression of nAChRs, with the a7 subunit having a significant contribution. Nicotinic and glutamatergic receptors have been both implicated in numerous important neuropathological disorders, but here we will focus on Alzheimer's disease (AD) and schizophrenia.

Alzheimer's disease

AD is a neurodegenerative disease characterized by memory and cognitive loss, representing the major cause of dementia in the aged population greater than 60 years old. A hallmark for synaptic and neuronal dysfunction in AD is the aberrant accumulation of amyloid-b (Ab) peptide [31]. Ab is formed through proteolytic cleavage of its precursor protein, a type 1 membrane protein, amyloid precursor protein (APP). Ab plaques, and soluble oligomers, can have dramatic effects on glutamatergic systems by inducing excitotoxicity through stimulating glutamate release and inhibiting its uptake, thereby causing effects on glutamatergic signaling and receptor activation [START_REF] Rudy | The role of the tripartite glutamatergic synapse in the pathophysiology of Alzheimer's disease[END_REF].

An a7 nAChR interaction with Ab was first described 15 years ago, however the exact nature of this interaction in not well understood. Wang et al. showed that a7 nAChRs and Ab are co-localized in AD cortical regions, and also in the hippocampus, suggesting that a7 nAChRs and Ab may bind with high-affinity [START_REF] Wang | beta-Amyloid 1À42 binds to alpha7 nicotinic acetylcholine receptor with high affinity. Implications for Alzheimer's disease pathology[END_REF][START_REF] Wang | Amyloid peptide Abeta 1À42 binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors[END_REF]. However, more recent data indicate that the Ab peptide mediates the inactivation of a7 nAChRs [START_REF] Wang | S 24795 limits beta-amyloid-alpha7 nicotinic receptor interaction and reduces Alzheimer's disease-like pathologies[END_REF]. For a more extensive review on the nature of Ab binding to a7 nAChRs, see the review of Parri et al. [110].

AD patient tissue displays significant loss of glutamatergic neurons, abnormalities in NMDA and AMPA receptor expression, dysfunctions in glutamate neurotransmission and inhibition of synaptic plasticity [START_REF] Francis | Rationale for combining glutamatergic and cholinergic approaches in the symptomatic treatment of Alzheimer's disease[END_REF]. The effect of Ab on LTP, specifically at the level of NMDAR density reduction, has been demonstrated to directly involve a7 nAChRs. Applying Ab42 to cultured neurons caused endocytosis of NMDARs and also in Tg2576 mice that overexpress Ab42. Consequently, NMDAR-evoked currents were reduced [START_REF] Snyder | Regulation of NMDA receptor trafficking by amyloid-beta[END_REF]. a7 nAChRs were implicated in the mechanism since Ab42 binds to a7 nAChRs in the synaptic cleft causing the activation of protein phosphatase 2B (PP2B; calcineurin). This activation then causes the dephosphorylation of striatal enriched phosphatase-46 (STEP 46 ), causing its activation, thus striatal enriched phosphatase-46 phosphorylates the NR2B NMDAR subunit. This phosphorylation causes the endocytosis of NMDARs and thus impairment of glutamatergic transmission (Fig. 1).

On the other hand, several studies revealed that Ab can increase glutamate release from neurons and astrocytes, resulting in elevated glutamate extracellular levels, capable of activating pathological extrasynaptic NMDA (eNMDA) receptors. The eNMDA receptors are located on the spine neck, dendritic shaft or soma and are activated only by high glutamate concentrations [START_REF] Rudy | The role of the tripartite glutamatergic synapse in the pathophysiology of Alzheimer's disease[END_REF]. In hippocampal slice it was shown that astrocytic calcium, as gated through astrocytic a7 nAChRs, can be activated by only picomolar concentrations of Ab 42 [START_REF] Pirttimaki | a7 Nicotinic receptor-mediated astrocytic gliotransmitter release: Ab effects in a preclinical Alzheimer's mouse model[END_REF][START_REF] Talantova | Ab induces astrocytic glutamate release, extrasynaptic NMDA receptor activation, and synaptic loss[END_REF]. Ab induced astrocytic glutamate release increases eNMDA receptor currents and decreases synaptic NMDA receptor currents, leading to the loss of synapses.

Overall, the specific role of the a7 nAChR subunit in AD, with its affinity to Ab peptide, has an important role in synaptic dysfunction in glutamatergic transmission, specifically with its role on eNMDA receptors. This, in effect, causes neuronal death through excitotoxicity and therefore the a7 nAChR subunit is a prime target for therapeutic interventions for the amelioration of AD and possibly other diseases and excitotoxic mechanism.

Schizophrenia

Schizophrenia is a chronic, disabling neurodevelopmental disorder characterized by a broad spectrum of severe cognitive impairments [START_REF] Ripke | Biological insights from 108 schizophrenia-associated genetic loci[END_REF]. Several studies have suggested that schizophrenia is related to deficits of neurotransmitter classes including dopaminergic, glutamatergic, GABAergic and cholinergic systems [START_REF] Bencherif | Alpha7 nicotinic cholinergic neuromodulation may reconcile multiple neurotransmitter hypotheses of schizophrenia[END_REF]. Various studies suggest that glutamate plays a prominent role in schizophrenia. Clinical observations propose that schizophrenia is the consequence of deficient glutamate-mediated excitatory neurotransmission through NMDA receptors [START_REF] Moghaddam | Bringing order to the glutamate chaos in schizophrenia[END_REF]. NMDA receptor antagonists have been shown to have strong effects on prefrontal cortex by impairing working memory in rodents and primates [START_REF] Verma | NMDA receptor antagonists impair prefrontal cortex function as assessed via spatial delayed alternation performance in rats: modulation by dopamine[END_REF][START_REF] Dudkin | Neurophysiological correlates of delayed visual differentiation tasks in monkeys: the effects of the site of intracortical blockade of NMDA receptors[END_REF].

Blockade of a7 nAChRs in rodents has been demonstrated to cause schizophrenia-like symptoms including sensory gating deficits and disrupted memory consolidation via mechanisms consistent with altered modulation of glutamate release [START_REF] Mansvelder | Long-term potentiation of excitatory inputs to brain reward areas by nicotine[END_REF][START_REF] Bencherif | Alpha7 nicotinic cholinergic neuromodulation may reconcile multiple neurotransmitter hypotheses of schizophrenia[END_REF].

Preclinical trials of a7 nAChR agonists have shown positive effects on various animal models of schizophrenia [START_REF] Toyohara | a7 nicotinic receptor agonists: potential therapeutic drugs for treatment of cognitive impairments in schizophrenia and Alzheimer's disease[END_REF]. Clinical trials relating to the a7 nAChR have advanced enormously, and one compound is in phase 3 for AD and schizophrenia [START_REF] Preskorn | The role of proof of concept (POC) studies in drug development using the EVP-6124 POC study as an example[END_REF][START_REF] Van Goethem | Continuous infusion of the a7 nicotinic acetylcholine receptor agonist EVP-6124 produces no signs of tolerance at memory-enhancing doses in rats: a pharmacokinetic and behavioral study[END_REF][START_REF] Deardorff | Safety and clinical effects of EVP-6124 in subjects with Alzheimer's disease currently or previously receiving an acetylcholinesterase inhibitor medication[END_REF].

Recent studies suggest that a combination of multi-target directed ligands may be particularly effective in schizophrenia. For example, Koola et al. reviewed that the combination of galantamine and memantine could increase the selective cognition enhancement produced by either medication alone [START_REF] Koola | Potential role of the combination of galantamine and memantine to improve cognition in schizophrenia[END_REF]. This combination of memantine, which decreases the tonic NMDA current and galantamine, which enhances the NMDA induced action potentials, caused increased transmission, which was proposed to increase the signal to noise, thereby having a neuroprotective effect [START_REF] Lopes | Galantamine potentiates the neuroprotective effect of memantine against NMDA-induced excitotoxicity[END_REF].

Finally kynurenic acid (KYNA) has been identified as a potent antagonist of NMDA and a7 nACh receptors [START_REF] Parsons | Novel systemically active antagonists of the glycine site of the Nmethyl-D-aspartate receptor: electrophysiological, biochemical and behavioral characterization[END_REF]. Early developmental elevations of brain KYNA were associated with cognitive impairments in adult rats, which were reversed with galantamine [START_REF] Alexander | Early developmental elevations of brain kynurenic acid impair cognitive flexibility in adults: reversal with galantamine[END_REF]. Interestingly, since KYNA was linked with cognitive deficits in schizophrenia, it is possible that galantamine and memantine may have an effect on a7 and NMDA by reducing KYNA, causing increased mental ability [START_REF] Wonodi | Cortical kynurenine pathway metabolism: a novel target for cognitive enhancement in Schizophrenia[END_REF].

Of potential importance, the partial duplication of CHRNA7 was recently reported, causing the creation of a new gene CHRFAM7A. Deletions in these genes, which occur rarely, have a high correlation with developing schizophrenia, specifically with a 2 bp deletion in exon 6 of CHRFAM7A [129,[START_REF] Sinkus | The human CHRNA7 and CHRFAM7A genes: a review of the genetics, regulation, and function[END_REF]. Since this duplication is restricted to humans and no other known species, animal models do not exist, making testing of drugs that target this mutation to be very difficult to translate to clinical trials.

Perspective

Based on the data reviewed above, many important aspects of the mechanisms by which a7 nicotinic receptors regulate glutamate neurotransmission and participate in the pathology of psychiatric and neurodegenerative diseases remain to be resolved. Considering the progress that has been made to date using animal models, continuing advances in this field will provide answers to important questions and help to develop efficient therapies.

Further studies probing the specific role of a7 using knock-out approaches, modifications of downstream regulators, behavioural assays, in vivo approaches to probe modifications of neuronal networks and novel drug targets, will further our understanding.

Due to the strong modulatory role of a7 nChRs, with its high permeability to calcium, its effects as demonstrated on brain function, neuropathology and neurological and psychiatric disorders make it a promising target for further research.

Figure 3 :

 3 Figure 3: Cholinergic networks in the rodent CNS. The nuclei present in the basal forebrain project to

Figure 9 :

 9 Figure 9: The two main neuropathological features of AD. The extracellular amyloid plaques and the

Figure 10 :

 10 Figure 10: Figure 10: Secretase processing of APP. 1) APP, under nonamyloidogenic mechanisms, is

  addition, several studies have shown that the dynamics of large-scale neuronal networks are modulated by anesthetics[START_REF] Mccarthy | Dynamical changes in neurological diseases and anesthesia[END_REF]. Anesthesia alters the electroencephalogram (EEG) by shifting oscillatory activity from high-frequency, low amplitude patterns to lowfrequency, high amplitude activity[START_REF] Voss | Monitoring consciousness: the current status of EEG-based depth of anaesthesia monitors[END_REF], disrupts integration of information across brain regions through decreasing long-range coherence and reduces cortical information capacity by affecting distinct activity patterns(Alkire et al. 2008). Following cortical EEG and subcortical (ESCoG) by intracranial measurements during sevoflurane and

  proteins and enzymes that are packaged into virions. The transfer vector plasmid contains cisacting genetic sequences necessary for the vector to infect the target cell. It contains a promoter, the transgenes of interest, LTRs for integration of cDNA, RRE and PPT. However, it lacks gag, pol, env, rev and tat in order to prevent viral replication. In addition, new generation lentivirus constructs contain the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE) that increases the level of transgene expression, improving the performance of the vector(Zufferey et al. 1999) and the central polypurine tract of HIV that increases the titre. Finally, the envelope vector produces capsid glycoproteins and defines the virus' infection selectivity. The envelope commonly used in our laboratory is the VSV-G glycoprotein(Cronin et al. 2005).

2. 1 .

 1 2a Adeno-associated virus lifecycle AAV attaches to and infects a cell via membrane-associated heparin sulfate proteoglycan(Summerford and Samulski 1998). The use of ubiquitous heparan sulphate proteoglycans as docking sites explains the broad tropism of this virus. After entry into the host cell nucleus, AAV can follow either one of two distinct and interchangeable pathways of its life cycle: the lytic and the lysogenic. The former develops in cells infected with a helper virus such as adenovirus or herpes simplex virus (HSV), whereas the latter is established in host cells in the absence of a helper virus. When AAV infects a human cell alone, its gene expression program is auto-repressed and latency is ensued by preferential integration of the virus genome into a region of roughly 2-kb. This site-specific integration involves the AAV ITRs and Rep proteins. A helper virus is used to super-infect the cell using adenovirus or herpes simplex virus which causes the AAV Rep-mediated rescue of the provirus DNA that is contained in the host chromosome and final replication and packaging of the viral genome.

  mediated gene transfer was used to selectively increase hippocampal levels of human Aβ 42 and Aβ 40 in adult rats, allowing examination of the contribution of each to the cognitive deficits and pathology seen in AD. The results showed that while expression of Aβ 42 alone is sufficient to initiate Aβ deposition and that both Aβ 42 and Aβ 40 may contribute to cognitive deficits (Lawlor et al. 2007).

  traditional optical microscopy techniques use linear (one-photon) absorption processes for contrast generation and are limited to use near the tissue surface for highresolution imaging. At greater depths multiple scattering causes blurring of the images. A large part of this problem was solved with the invention of new optical microscopy techniques that use non-linear light-matter interactions in order to generate signal contrast and have the ability to create less scattering while acquiring high-resolution imaging in tissue. The development of the two-photon excitation microscopy, in combination with fluorescence labeling techniques, has opened the way of imaging intact tissues and living animals. Maria Goppert Mayer first proposed two-photon excitation in 1936 in her doctoral dissertation, but it wasn't until the invention of the two-photon fluorescence light microscopy by Winfred Denk in 1990 that caused a revolution in three-dimensional in vivo imaging of cells and tissues (Denk et al. 1990).

(

  Nimmerjahn et al. 2004). Fluorescent protein expression can be achieved using methods such as viral transfections and targeted single-cell DNA electroporation or more globally by generating transgenic animals. A major advantage is that cell type specificity can be achieved if a specific promoter is available. Activity dependent probes have been also developed that have the ability to change their fluorescent properties in response to a specific cellular activity. Voltage-sensitive fluorescent dyes and fluorescent ion indicators are examples of traditional activity-dependent probes. The most commonly applied activity-dependent probes are fluorescent calcium indicators, as it will be discussed below.

Figure 13 :

 13 Figure 13: A) Acute cranial window over the PFC. The bright spots indicate two injections of OGB1.

  . Recently, robust genome-wide association studies (GWAS) identified single nucleotide polymorphisms (SNPs) in the human CHRNA5 gene, coding for the α5 nAChR subunit, that increase risk for both smoking and schizophrenia 7,8 . Mice with altered nAChR gene function exhibit PFC dependent behavioral deficits 9-11 , but how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying the behavior is unknown. Here, we show that α5SNP mice, expressing the human α5 D398N coding SNP, rs16969968, exhibit deficits in behaviors thought to represent human cognitive alterations in schizophrenia. Using awake two-photon imaging of mouse models, we demonstrate that nicotine can differentially influence PFC pyramidal cell activity. This occurs through specific nAChR modulation of a hierarchical circuit of inhibitory neurons within layer II/III. Specifically for α5SNP and α5 knock-out (α5KO) mice, decreased activity of vasoactive intestinal polypeptide (VIP) interneurons resulted in an increased somatostatin (SOM) interneuron inhibitory drive of layer II/III pyramidal neurons. The decreased activity observed in α5SNP mice coincidently mirrors the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction 5,12 . Chronic nicotine administration reversed this hypofrontality to control levels, suggesting a therapeutic strategy for the treatment of schizophrenia, and a physiological basis for the tendency of patients with schizophrenia to self-medicate by smoking 13 .

FiguresFigure 1 Figure 2 Figure 3 Figure 4

 1234 Figures

(

  20ms at74,78,82, and 90 dB) followed by pulse (120 dB) 100 ms after prepulse. PPI was calculated using the means of 10 trials for each type of stimulus: percent PPI= (pulse only score-[prepulse+pulse score])/pulse only score *100.

  and we replaced the eGFP cDNA by the fragment α5 WT-IRES2-eGFPP or the polymorphic version of the α5 nicotinic subunit (α5 SNP) using the fragment α5 SNP-IRES2-eGFPP . The sequences of generated by co-transfection of HEK-293T cells with the vector plasmid, a packaging plasmid and an envelope plasmid using the calcium phosphate protocol. At 2 days after transfection, viral particles were harvested in the supernatant, treated with DNaseI and MgCl 2 , passed through a 0.45 µm filter, concentrated by ultracentrifugation and resuspended in a small volume of phosphate-buffered saline (PBS). Viral stocks were stored in small aliquots at -80° C before use. Viral titers were estimated by quantification of the p24 capsid protein using the HIV-1 p24 antigen immunoassay (ZeptoMetrix Corporation, Buffalo, NY, USA) according to the manufacturer's instructions. Lentivectors were diluted in PBS before stereotaxic injection to achieve a dose corresponding to 150 ng of p24 protein in 2 µl for the reexpression of α5 subunits. As controls we used vectors carrying only eGFP, under the same promoter and we injected the same volumes. The lentiviral vectors were injected bilaterally into the PrLC at the following coordinates PrL: AP, +2.8 mm from Bregma; L, ±0.5 mm; and DV, -0.25 to -0.1 mm from the skull using a Nanoject II TM (Drummond Scientific) at the slow infusion setting.

  and post-fixed by immersion in PFA for 2 days at 4° C. The brains were then immersed in 30% sucrose in PBS overnight for cryoprotection at 4°C. Serial 40 µm coronal sections were cut with a sliding microtome (Leica Microsystems), transferred to PBS, mounted on slides and cover-slipped with ProLong Gold antifade reagent with DAPI (Invitrogen). Microscopy was carried out with a Zeiss epifluorescent microscope.To determine the expression pattern of the β2-IRES2-tdTomato bi-cistronic vector and PDGF-floxed-mCherry-α5 WT/SNP-IRES2-eGFPP-WPRE, β2KO and α5 KO-GAD Cre injected mice respectively, were anesthetized and perfused as described above. Serial 40µm coronal sections were cut with a sliding microtome, collected and washed in PBS, treated with 0.2% Tween 20 (PBS-T), immersed in 10% normal horse serum blocking solution, and incubated overnight with primary antibodies against GFP(1:2000, Abcam, ab38689) and RFP (1:2000, Molecular Probes, R10367) followed by incubation with secondary antibodies conjugated to Alexa Fluor 488 (green 1:500, Jackson ImmunoResearch Laboratories, 715-545-150) and Alexa Fluor 568 (red 1:500, Abcam, ab175470). The sections were mounted on slides and cover-slipped with ProLong Gold antifade reagent with DAPI. Microscopy was carried out with a Zeiss LSM-700 confocal microscope, see Supplementary Fig.8. Surgical implantation of minipumps. Mice were anesthetized with a xylazine and ketamine combination (15% xylazine/2.5%ketamine/82.5% PBS). Osmotic minipumps(model 2004,

  8 NA water immersion objective (Nikon) with the femtosecond laser (MaiTai DeepSee, Spectra Physics, Mountain View) tuned to 950 nm for imaging of cells expressing GCaMP6f, and to 1020 nm for imaging of cells expressing tdTomato. Time-series movies of neuronal populations expressing GCaMP6f were acquired at the frame rate of 6.926 Hz (182 x 182 µm, FOV; 0.71 µm/pixel). Each focal plane movie duration was 216.564 seconds (1500 frames) to track spontaneous neuronal activity. For the interneuron recordings, time-series movies of interneuronal populations expressing the floxed.GCaMP6f were acquired at the frame rate of 32.97 Hz (405 x 405 µm, FOV; 0.79 µm/pixel). Each focal plane movie duration was 164.897 seconds (5000 frames).Care was taken to use less than 10mW of laser power at the surface of the tissue.Randomization and blinding. The mice from all experimental groups, baseline (and nicotine treated, were imaged in a random order. Blinded experiments were performed for the β2 reexpression, VIP-Cre, PV-Cre baseline activities and SOM-Cre nicotine treatment recordings but not for the rest of the experiments. Blinded analysis was performed for the social interaction test data and baseline activities of the α5KO, α5SNP, β2 re-expression groups, PV-Cre, SOM-Cre recordings and SOM-Cre nicotine treatment recordings, but not for the rest of the experiments. Two-photon data analysis. Image analysis was performed off-line with ImageJ software. The time series were corrected using the 'Image Stabilizer' (K. Li, "The image stabilizer plugin for ImageJ," http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html, February, 2008).

  Code availability: The custom-written toolbox in MATLAB (Mathworks, 2014b) is available upon request.Statistical analysis. Two-photon data: Kruskal-Wallis one-way analysis of variance combined with multiple comparison testing was applied on the aggregated activity (transients/min and spikes/min) of the neurons in all mouse groups in order to study the statistical similarities between them. For each mouse category, we used interpolation in order to account for the different number of neurons recorded in the different animals. In this way, our statistical analysis assigned the same weight to each neuronal population recorded in the different mice of a given category. In each boxplot, we show the distribution of mean neural activities between the different mice of a given mouse type, such that extreme neural activities are not shown in this representation. Levene's test was used to compare the variances of the neural distributions that were similar between groups that were statistically compared. We used Welch's F Test ANOVA as a complementary test for heteroscedasticity. This test gave similar results as the Kruskal-Wallis test with the same level of significance. Animals were not excluded from the analysis. The number of mice needed for our study was determined by preliminary experiments in anesthetized mice and pilot experiments in awake mice. Boxplots, histograms and empirical cumulative distribution functions (Kaplan-Meier estimate) were used to compare the different distributions of neural activity between the mouse types. On the empirical cumulative distribution plots, 95% lower and upper confidence bounds were computed with Greenwood's formula. Behavioral data: Statistical analysis was performed using Prism (Graphpad) software. Social interaction task differences were assessed using compute the noise level without including the calcium transients. To remove fluorescence fluctuations due to movement along the optical axis, we examined the ratio between positive and negative going transients for fluorescence transients with varying durations and amplitude15 . The selected transients have an amplitude and a duration for which the ratio of negative to positive transients is below a threshold = 0.05. Positive going transients with amplitude and duration defined by this threshold have a <5% chance of being caused by brain motion and a >95% chance of being a calcium transient, according to Dombeck and Tank 1 .

  USFs and synchronicity in the awake state Many studies in both animal models and humans have identified a contribution of nAChRs to cognitive functions, we thus investigated the occurrence of USFs transitions in knockout (KO) mice of α7 and β2 nAChR subunits. Typical examples of neuronal population activities in awake α7 and β2 KO mice are shown in Figure 2. We found that 90.30 ± 5.03 % of simultaneously recorded populations exhibited USFs/basal activity in WT (n = 2900 cells in 11 mice), 82.08 ± 7.2 % in α7 KO mice (n = 1403 cells in 5 mice) and 65.86 ± 5.7 % in β2

FiguresFigure 1 .

 1 Figures

  Figure 1. USFs and synchronously firing neurons of simultaneously recorded neurons in WT

Figure 2 .Figure 3 .

 23 Figure 2. USFs and synchronously firing neurons of simultaneously recorded neurons in α7

Figure 4 .

 4 Figure 4. USFs and synchronously firing neurons of simultaneously recorded neurons in α7

Figure 5 .

 5 Figure 5. Comparison of the properties of USFs and synchronicity in anesthetized mice. (A)

Figure 6 .

 6 Figure 6. Brain exposed to chronic mecamylamine resembles nAChR KO phenotype. (A)

3 . 4 .

 34 These phenotypes are observed before the appearance of amyloid plaques and neurofibrillary tangles, and thus demonstrate a crucial role of soluble Aβ in pathological changes of cortical activity. The early induction of microgliosis and astrocytosis, and the late appearance of plaques and tangles at 12 months further validate the model.

  -associated viral construction. The hAPP cDNA harboring the Swedish mutation (KM670/671NL) was recovered from a pcDNA 3.1 plasmid. The cDNA sequence was modified with directed mutagenesis PCR (Agilent Technologies, Stratagene, Santa Clara, CA, . The same procedure was used to delete the stop codon at APP 3' in order to add a FLAG tag(Einhauer and Jungbauer, 2001). The obtained construct was inserted into a pGEM-T vector (Promega, Madison, WI, USA), resulting in an APP-SLA-FLAG-pGEM-T plasmid. The hAPP-SLA-FLAG cassette was amplified with Expand High Fidelity PCR (Roche, Meylan, France) (Fw primer : The hAPP-SLA-FLAG cassette was recovered from pGEM-T vector with XbaI and EcoRV restriction enzymes and inserted in an AAV-EF1a vector. The AAV-EF1a vector was derived from an AAV-EF1a-DIO-ChetA-EYFP plasmid (http://www.everyvector.com/sequences/show_public/7300) that was digested with the same restriction enzymes. Ligation of the two fragments with T4 DNA ligase (M0202, NEB) resulted in the adeno-associated viral vector AAV-EF1a-hAPP-SLA-FLAG. Virus production was performed by INSERM U649 Vector Core of Nantes University to a final titer of 2.2x10 12 vg/ml. Animals. Experiments were performed with male wild-type mice (C57Bl/6J line) and were bred at Charles River Laboratories (L'Arbresle, France). All mice were transported to our facilities at eight weeks of age, housed under a 12h light-dark cycle with ad libitum access to food and water. CX3CR1-GFP +/-mice were kindly provided by the Unite d'Histopathologie humaine et modeles animaux of the Institut Pasteur in Paris, France. The experiments described in the present work were conducted in accordance with the guidelines on the ethical use of animals from the European Community Council Directive of 24 November 1986 (86/609/EEC) and in accordance with institutional animal welfare guidelines and were approved by the CETEA Ethics committee, Animalerie Centrale and Médecine du Travail, Institut Pasteur.

  200 nl ofGCaMP6f expressing Serotype 2.1 AAV virus under the synapsin-1 promoter (AAV.syn.GCaMP6f.WPRE.SV40, 2.2e13 GC/ml, University of Pennsylvania Vector Core, catalog number; AV-1-PV2822, lot; CS0261WL) was injected bilaterally at the following coordinates into PrLC: AP, +2.8 mm from bregma; L, ±0.5 mm; and DV, -0.3 to -0.1 mm from the skull using a Nanoject II TM (Drummond Scientific) at the slow infusion setting. For WT control mice, 2 µl of AAV1.CAG.tdTomato.WPRE.SV40 1.52e13 GC/ml (University of Pennsylvania Vector Core, catalog number; AV-1-PV2126) diluted in PBS 1X, was injected bilaterally at the same coordinates as described above. For the WT-hAPP mice, 2 µl of AAV-EF1a-hAPP-SLA-FLAG (2,2e12 GC/ml.) was also injected bilaterally at the same coordinates. The glass pipette was left in situ for an additional 5 min before being slowly withdrawn. The cranial window was covered with a circular coverglass (5mm diameter) and was sealed to the skull with dental cement (Coffret SUPERBOND complet, Phymep).Immunofluorescent staining. Mice were deeply anesthetized with a lethal dose of ketamine/xylazine before intracardiac perfusion with ice cold PBS, followed by 4% PFA (Sigma-Aldrich, Saint Louis, MO, USA). The brains were removed and post-fixed by immersion in 4% PFA for 2 days at 4° C. The brains were then immersed in 30% sucrose in PBS overnight at 4° C for cryoprotection. Serial 40 µm coronal sections were cut using a sliding microtome (Leica Microsystems) and transferred to PBS. Slices were incubated in 10% normal goat serum (NGS) and 0.2% Triton X-100 in PBS for one hour, then washed in PBS and incubated with various combinations of primary antibodies: rabbit anti-GFP(1:2000; Life Technologies, Invitrogen, Carlsbad, CA, USA), rabbit anti-GFAP (1:1000; Chemicon, AB5804, Temecula, CA), mouse anti-VHH V31-1 (1:500; kindly provided by Pierre Lafaye(Lafaye et al., 2009)) and mouse anti-FLAG (1:1000; Sigma Life Science, F1804, France). A mouse anti-His antibody (1:1500; Sigma-Aldrich, Saint Louis, MO, USA) was used for amplifying the VHH signal. Fluorophore-conjugated secondary antibodies were used with cy3-anti-mouse and Alexa 488-anti-rabbit (Life technologies, Eugene, OR, USA) at a dilution of 1:500 for 3 hours at RT. After DAPI (Sigma-Aldrich, Saint Louis, MO, USA) incubation, the slices were mounted on slides ProLong Gold Antifade Reagent mounting medium (Life Technologies, Molecular Probes, Carlsbad, CA, USA). Images were aquired with a Zeiss epifluorescent microscope and a confocal microscope (Zeiss LSM 700, Heidelberg, Germany).Immunohistochemistry. 40 µm coronal sections were incubated for 30 min in NH4Cl 50 mM, Lysine 1 mM and Glycine 1 mM in PBS, then one hour in 3% H 2 O 2 in PBS for neutralisation of endogenous aldehydes and peroxidases, respectively. Slices were incubated in 10% NGS and 0.2% Triton X-100 in PBS (Tx PBS) for 1 hour and then with the primary biotinylated antibody, mouse anti-4G8 (1:500; Covance, Dedham, MA, lot: 09EC00860), diluted in 2% NGS and 0.2% Triton X-100 in PBS and incubated overnight at 4°C. Slices were rinsed with Tx-PBS and incubated in ABC mixture for 30 min with gentle shaking (Elite ABC, PK-6100, Vector Laboratories, Burlingane, CA). After 3 washes with Tx-PBS, the sections were developed in DAB solution (Vector Novared substrate kit, SK-4800, Vector Laboratories, Burlingane, CA) for 5-10 minutes. Finally, slices were washed with distilled H 2 O and mounted in aqueous mounting media. Amyloid plaques were visualized with light microscopy.

  two-photon images from 3 AAV-hAPP injected mice were analyzed, n = 150 cells. For the analysis of astrocytic density between sham and AAV-hAPP injected mice, the rectangle tool was used to selecte a region of interest (ROI) of 200 x 200 um. The same size of ROI was selected for both sham and hAPP brain slices in the injection area. A ROI was also drawn in an area without fluorescence to be used for background subtraction. The net average fluorescence intensity in the ROI was calculated for the different groups and the average intensity values of AAV-hAPP injected mice were normalized by values obtained in sham mice. 6 slices were analyzed from 3 control mice and 6 slices from 3 AAV-hAPP-SLA injected mice. For these experiments, all parameters during image acquisition were kept identical.Quantitative analysis of immunohistochemical labeling. The analysis of optical densities for the immunohistochemical labeling of amyloid plaques and neurofibrillary tangles was also performed in Fiji. The images first underwent color deconvolution. The H DAB setting was selected as the labeling method from the vectors and the analysis was performed and measurements were set to the mean grey value. ROIs were selected (the prelimbic cortex area was validated by taking mosaics of each brain slice), always maintaining the same size of box for both sham and hAPP brain slices. Optical density numbers were acquired using the formula OD = log (max intensity/mean intensity). The values were normalized by values obtained in sham mice. For the quantification of amyloid plaques, 8 slices were analyzed from 4 control mice and 6 slices from 3 AAV-hAPP injected mice, whereas for the quantification of NFTs, 6 slices were analyzed from 3 control mice and 6 slices from 3 AAV-hAPP injected mice.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Detection of hAPP and Aβ oligomers in the PFC at one-month post injection of

Figure 3 :

 3 Figure 3: AAV-hAPP-FLAG induces microglial activation and astrocytosis. In vivo twophoton imaging of microglial activation in AAV-hAPP-SLA injected CX3CR1-GFP +/-mice.

Figure 4 :

 4 Figure 4: Detection of amyloid plaques and NFTs in the PFC of AAV-hAPP-SLA injected

Figure 6 :

 6 Figure 6: Higher rates of neuronal activity in layer II/III of PrLC in AAV-hAPP-SLA injected mice. (A), Representative spontaneous Ca 2+ transients of sham-operated mice and

Changeux 2 , 2 1

 22 Uwe Maskos 1, Institut Pasteur, Neurobiologie intégrative des systèmes cholinergiques, Paris, France 2 CNRS UMR 3571, Paris, France 3 Group for Neural Theory, Laboratoire de Neurosciences Cognitives, INSERM Unité 969, Département d'Études Cognitive, École Normale Supérieure, Paris, France

  -associated viral construction. The hAPP cDNA harboring the Swedish mutation (KM670/671NL) was recovered from a pcDNA 3.1 plasmid. The cDNA sequence was modified with directed mutagenesis PCR (Agilent Technologies, Stratagene, Santa Clara, CA, . The same procedure was used to delete the stop codon at APP 3' in order to add a FLAG tag(23). The obtained construct was inserted into a pGEM-T vector (Promega, Madison, WI, USA), resulting in an APP-SLA-FLAG-pGEM-T plasmid. The hAPP-SLA-FLAG cassette was amplified The hAPP-SLA-FLAG cassette was recovered from the pGEM-T vector with XbaI and EcoRV restriction enzymes and inserted in an AAV-EF1a vector. The AAV-EF1a vector was derived from an AAV-EF1a-DIO-ChetA-EYFP plasmid (http://www.everyvector.com/sequences/show_public/7300) that was digested with the same restriction enzymes. Ligation of the two fragments with T4 DNA ligase (M0202, NEB) resulted in the adeno-associated viral vector AAV-EF1a-hAPP-SLA-FLAG. Virus production was performed by INSERM U649 Vector Core of Nantes University to a final titer of 2.2x10 12 vg/ml.Mice. Male α7 KO(24), β2 KO(25) and WT (C57BL/6J) mice used in this study were maintained at Charles River Laboratories (L'Arbresle, France) and experiments were performed on 3 month old male mice. The experiments described were conducted in accordance with the guidelines on the ethical use of animals from the European Community Council Directive of 24 November 1986 (86/609/EEC) and in accordance with institutional animal welfare guidelines that were approved by the Animalerie Centrale and Médecine du Travail, Institut Pasteur.Lentiviral expression vector, stereotaxic injections and chronic cranial windows. The lentiviral expression vectors were derived from the pHR' expression vectors first described byNaldini et al (26) with several subsequent modifications. Mice were anesthetized with ketamine (Imalgen 1000; Rhone Mérieux) and xylazine (Rompun; Bayer AG), 10 ml/kg i.p.

  and post-fixed by immersion in 4% PFA for 2 days at 4° C. The brains were then immersed in 30% sucrose in PBS overnight at 4° C for cryoprotection. Serial 40 µm coronal sections were cut using a sliding microtome (Leica Microsystems) and transferred to PBS. Brain slices were incubated in 10% normal goat serum (NGS) and 0.2% Triton X-100 in PBS for one hour, then washed in PBS and incubated with various combinations of primary antibodies: rabbit anti-GFP (1:2000; Life Technologies, Invitrogen, Carlsbad, CA, USA), rabbit anti-GFAP (1:1000; Chemicon, AB5804, Temecula, CA) and mouse anti-VHH V31-1 (1:500; kindly provided by Pierre Lafaye(28)). A mouse anti-His antibody (1:1500; Sigma-Aldrich, Saint Louis, MO, USA) was used for amplifying the VHH signal. Fluorophore-conjugated secondary antibodies were used with cy3-anti-mouse and Alexa 488-anti-rabbit (Life technologies, Eugene, OR, USA) at a dilution of 1:500 for 3 hours at RT. After DAPI (Sigma-Aldrich, Saint Louis, MO, USA) incubation, the slices were mounted on slides ProLong Gold Antifade Reagent mounting medium (Life Technologies, Molecular Probes, Carlsbad, CA, USA). Images were acquired with a Zeiss epifluorescent microscope and a confocal microscope (Zeiss LSM 700, Heidelberg, Germany).

FiguresFigure 1

 1 Figures

Figure 2 60 Figure 3

 2603 Figure 2 AAV-hAPP induces pyramidal cell hyperactivity in WT mice whereas reduces the

Figure 4 β2

 4 Figure 4 β2 KO mice are protected from hAPP -induced hyperactivity 1-month postinjection. (A) Representative spontaneous Ca 2+ transients of β2 KO and β2 KO -APP

Figure 5 β2

 5 Figure 5 β2 KO mice are protected from hAPP -induced hyperactivity 3 months post-

  novel model of AD by expressing the human mutated APP into the mouse brain. By injecting the hAPP harboring the Swedish, London and Austrian mutations that are associated with early onset of AD, we generated a new model displaying core features of AD allowing the study of early pathology. This model causes the induction of AD-like deficits in adults, thereby more accurately modeling human pathology. AAV-hAPP expression caused rapidly accumulation of Aβ oligomers, microglial activation, astrocytosis and the gradual formation of amyloid plaques and NFTs. Importantly, in vivo two-photon imaging revealed an increase in neuronal activity, a dysfunction characteristic of the pathology. This AD model allowed us to track brain activity changes during disease progression, in the absence of plaques, indicating an important role of the soluble form of Aβ peptide on brain physiology.

Figure 1 :

 1 Figure 1: Biochemical characterization of R3VQ and its variants.

Figure 2 :

 2 Figure 2: Immunohistochemical characterization of R3VQ as a specific probe for labeling

Figure 3 :

 3 Figure 3: In vivo imaging of R3VQ using two-photon microscopy.

Figure 4 :

 4 Figure 4: Biochemical characterization of A2 and its variants.

Figure 5 :

 5 Figure 5: Immunohistochemical characterization of A2 VHH as a specific probe for

Figure 6 :

 6 Figure 6: In vivo imaging of A2-S-AF488 using two-photon microscopy.

Figure S2 :

 S2 Figure S2: Intracerebral diffusion of R3VQ and A2 VHHs after stereotaxic injections.

Figure S3 :

 S3 Figure S3: Brain penetration and diffusion of VHHs vs standard immunoglobulins 4

Figure S4 :

 S4 Figure S4: In vivo imaging of R3VQ and R3VE using two-photon microscopy.

Figure S5 :

 S5 Figure S5: In vivo imaging of R3VQ-S-AF488 in WT (plaques-free) mice.

Figure S6 :

 S6 Figure S6: Immunohistochemical detection of tau lesions with A2.

  37°C without shaking followed by 30 min at 37°C with shaking and then centrifuged at 2500 rpm for 10 min. The pelleted cells in each well were resuspended in 500 µl of 2YT+A+K+IPTG. The cultures were then incubated overnight at 30°C with shaking to allow expression of phage-VHHs by bacteria. Each well contained a single selected phage-VHH. In parallel, plate III (Nunc Thermo Scientific) was coated with antigen overnight at 4°C. In the case of anti Ab phage-VHHs, a streptavidin coated plate (Nunc Immobilizer Thermo Scientific) was used as plate III and different biotinylated Aβ fragments (Aβ40, Aβ42) were coated 15 min before ELISA experiments. Between each ELISA step, plates were washed 6x with PBS-Tween 0.1%. The following day, plates III were first saturated with PGT (PBS-Gelatin 0.5%-Tween 0.1%) for 30 min at 37°C (100 µl/well). Plates II were centrifuged at 2500 rpm for 10 min to precipitate bacteria and to retrieve the supernatant containing phage-VHHs. Phage-VHHs were then diluted with PGT in a ratio of 1/5 and transferred into the plates III and incubated at 37°C for 1h. Anti-phage M13 IgG conjugated to HRP (GE Healthcare) diluted in PGT (100µl) at 1/5000 were added for 1h at 37°C. Subsequently, the reactions were developed by adding 100 µl of OPD (o-Phenylenediamine, Dako) for about 10 min in darkness and stopped by adding 50 µl of 3N HCl. The optical density was measured spectrophotometrically at 490 nm using Magellan microplate reader (Sunrise Tecan). A clone was considered as positive when SNR (signal-to-noise ratio) was greater than or equal to 4. In order to enhance the accuracy of determination of positive clones, preparation of monoclonal (individual) phage-VHH in 100 ml culture was also performed and ELISA was performed using the same antibodies.Panning of the library of anti-Ab VHH and binding epitope determination of monoclonal phage-VHHThe library was panned for reactivity with biotinylated Aβ42 and Aβ40 peptides respectively (Bachem). Phages(10 13 transducing units) were panned by incubating with the biotinylated peptides for 1h at 37 • C under gentle agitation then the mixture was incubated with the streptavidin magnetic beads (Invitrogen) for 15 min at 37°C. The concentration of biotinylated peptides used decreased at each round of panning with respectively 100 nM, 50 nM and 10 nM of protein. R3VQ, one of monoclonal phage-VHH directed against Ab42 was chosen for epitope mapping. A constant amount of phage-VHH in 100 µl PGT was incubated respectively with 10 µM of overnight at 4°C. The mixture was then deposited on 1 µg/ml of biotinylated Aβ42 coated in a streptavidin plate (Thermo Scientific) for 30 min at 4°C. After washes with PBS-Tween 0.1%, binding of the free phage-VHHs to Aβ42 was then analyzed by ELISA using an anti-phage M13 IgG coupled to HRP. The same amount of phage-VHH without incubation with Ab fragments was deposited in parallel to determine a maximum (100%) binding signal.

Fig. 1 .

 1 Fig.1. Glutamatergic transmission in Alzheimer's disease. Ab oligomers enhance the presynaptic glutamate release. Ab forms complexes with the a7 nAChRs causing increased glutamate release. a7 nAChR activation also causes an increase in astrocyte intracellular calcium, thereby inducing astrocytic glutamate release by unknown mechanisms. Synaptic loss, neuronal shrinkage and cell death are results of this excitotoxicity, while also causing downregulation of survival pathways including pCREB, ERK and CAMK II. Additionally, dephosphorylation of the NR2B NMDA receptor subunit is dependent on Ab binding to the a7 nAChR, thereby causing the internalization of NMDA receptors and disrupted glutamatergic transmission[START_REF] Snyder | Regulation of NMDA receptor trafficking by amyloid-beta[END_REF].

1.9.1 A brief introduction to Alzheimer's Disease history

	In 1906, a clinical psychiatrist and neuroanatomist, Alois Alzheimer, reported "A peculiar
	severe disease process of the cerebral cortex" to the 37th Meeting of South-West German
	Psychiatrists in Tubingen. He described a 50-year-old woman, whose name was Auguste D,
	whom he had followed from her admission for paranoia, progressive sleep and memory
	disturbance, aggression, and confusion, until her death 5 years later. Alzheimer applied his
	expertise in histology and performed several tests on Auguste's brain. Anatomical studies
	showed a widespread atrophy and a massive cell loss throughout the brain while histological
	analysis, performed in collaboration with Geatano Perusini and Francesco Bonfiglio, two
	neuropathologists who were visiting Alzheimer's institute in Munich, displayed what they
	defined as 'strange changes in neurofibrils' and 'military foci', today known as tangles and
	plaques, respectively (Hippius and Neundörfer 2003) (Figure

Table 2 .

 2 Aβ activates or inhibits nAChRs.

	Type of Aβ	Aβ concentration nAChR	Functional effect Preparation	Reference
	Aβ	1-42			100 nM-1µM	α7	Inhibition		slices Rat hippocampal	(Pettit et al. 2001)
	Aβ	1-42	-Aβ	1-40	100 nM	α7	Inhibition		neurons Rat hippocampal	(Liu et al. 2001)
	Aβ	1-42	-Aβ	1-40	100 nM	α4β2	inhibition Insensitive	to	neurons Rat hippocampal	(Liu et al. 2001)
	Aβ	1-40			0.1-10µM	α7, α4β2 Inhibition		X. leavis oocytes	(Tozaki et al. 2002)
	Aβ	1-42			1-100 pM	α7	Activation		X. leavis oocytes	(Dineley et al. 2002)
	Aβ	1-42			100 pM	α7	Activation		Rat synaptosomes (Dougherty et al. 2003)
	Aβ	1-42			100 nM	Non-α7	Activation		Hippocampus and cortex	(Dougherty et al. 2003)
	Aβ	1-42			1 nM	α4β2	Inhibition		SHEP1 cell line	(Wu et al. 2004)
	Aβ	1-42	-Aβ	1-40	10 nM	activation α7, α4β2 Inhibition,	after first	X. leavis oocytes	(Pym et al. 2005)
							inhibition
	Aβ	1-40

  development of powerful viral gene transfer techniques has facilitated the study of gene function. Viral vectors have been exploited for gene therapy and basic research by serving as biological mediators of transgene integration into living cells. Viral vectors are used in vitro in order to infect cells that are difficult to transduce and in vivo in order to drive

transgene expression. By genetically modifying the genomes, scientists are able to disrupt the replication and pathogenicity, but at the same time maintain stability, long-term expression and post-mitotic cell infection. An ideal gene delivery would: (1) transfer genes into both developing and mature animals; (2) transduce cells with high efficiency; (3) mediate high level, long-term expression; (4) cause limited cytotoxicity; (5) elicit a small/ negligible immune response in vivo;

(6)

incorporate sufficient lengths of DNA so that transgenes of interest can be accommodated and (

7

) mediate expression that can be regulated

(Howarth et al. 2010)

. All these characteristics are difficult to find in a single vector system and thus, a variety of viral gene delivery methods have been developed.

  1, 2 , Marie Rooy 3 , Dimitrios Tziotis 4 , Kurt A. Sailor2, 5 , Heidi O'Neill 6 , Josien Levenga 6 , Mirko Witte 7 , Michael Nilges 4 , Jean-Pierre Changeux 2 , Charles Hoeffer 6 , Jerry A.Stitzel 6 , Boris Gutkin3, 8 , David A. DiGregorio2, 9 & Uwe Maskos1, 2*

	Affiliations:

1 Institut Pasteur, Integrative Neurobiology of Cholinergic Systems, Paris, France 2 CNRS UMR 3571, Paris, France 3 Group for Neural Theory, Laboratoire de Neurosciences Cognitives, INSERM Unité 969, Département d'Études Cognitive, École Normale Supérieure, Paris, France

  For the recordings of pyramidal neurons, 200 nl of Serotype 2.1 AAV virus (AAV.syn.GCaMP6f.WPRE.SV40, 2.2e13 GC/ml, from University of Pennsylvania Vector Core) was injected bilaterally at the following coordinates PrL: AP, +2.8 mm from Bregma; L, ±0.5 mm; and DV, -0.3 to -0.1 mm from the skull using a Nanoject II TM (Drummond Scientific) at the slow infusion setting.

	For	the	recordings	of	interneurons	in	the	Cre	lines,	200nl	of
	AAV.Syn.Flex.GCaMP6f.WPRE.SV40 (University of Pennsylvania Vector Core), 2.1e13

Table 1 :

 1 , as in b4. Here, b1, b2 and b3 are selected for further analysis. (c) In a final step, the algorithm compares the rise time, t peak , to the decay time, t 1/2 of the selected transients. When the rise time exceeds the decay time, the transient is rejected, as in b4. (d) Between the 5 Kernel estimation of amplitude, tau rise and tau decay of PFC layer II/III neurons for the different mouse groups.

	368 ± 2.951%

α5 SNP mice. 4 dB level over background: WT mice showed 25.between WT and α5 SNP at the 12 dB level over background with P = 0.0023. rejected2%; α5 SNP vehicle, 86 ± 3%; α5 SNP nicotine, 123 ± 4%). (f) Comparison of the normalized transient duration (sec) of WT and α5 SNP with vehicle or nicotine infusion 14 days after mini-pump implantation (WT vehicle, 100 ± 1%; WT nicotine, 111 ± 2%; α5 SNP vehicle, 74 ± 1%; α5 SNP nicotine, 75 ± 1%). (g) Comparison of the normalized mean activity (transients/min) of SOM interneurons with vehicle or nicotine infusion 7 days after minipump implantation (SOM vehicle, 100 ± 5%; SOM nicotine, 44 ± 4%). (h) Comparison of the normalized transient duration (sec) of SOM interneurons with vehicle or nicotine infusion 7 days after mini-pump implantation (SOM vehicle, 100 ± 3%; SOM nicotine, 121 ± 10%).

  The custom-written toolbox in MATLAB (Mathworks, 2014b) is available upon request. We used Welch's Test ANOVA as a complementary test for heteroscedasticity. This test gave similar results as the Kruskal-Wallis test with the same level of significance.

	Statistical analysis. Data are presented as ± SEM. The p values were obtained by a two-tail
	Students's t test comparing control and APP injected groups' images. Kruskal-Wallis one-

way analysis of variance combined with multiple comparison testing was applied on the activities (transients/min and spikes/min) of the neurons in all mouse groups in order to study the statistical similarities.

Table 1 . Kernel estimation of amplitude, tau rise and tau decay of PFC layer II/III neurons in the different mouse groups.

 1

	Mouse group	Amplitute (ΔF/F) (%) tau rise (msec)	tau decay (msec)
	WT control 1 month	21.5202 ± 0.576	200 ± 5	390 ± 10
	WT APP 1 month	21.7686 ± 0.4782	160 ± 1	350 ± 1
	WT control 6 months	21.5057 ± 0.35565	190 ± 1	370 ± 5
	WT APP 6 months	22.2633 ± 0.99118	180 ± 1	370 ± 10
	Data presented as mean ± SEM.

Selection of specific phage-VHHs by ELISA

  Three microliters of each colony were cultured in 200 µl of 2YT A+G in a 96 deepwell plate (Fisher Bioblock) (Plate II). After an incubation of 1h30 min at 37°C with shaking, the bacteria present in each well were infected with 1x10 9 VCS M13 helper phages. The plate II was then incubated for 30 min at

	Ninety six individual colonies from the third round of panning were picked from Petri dishes
	and were cultured in a 96 well plate (Plate I) (Corning Incorporated Costar) containing 200 µl

of 2YT +A+G/well overnight at 37°C with shaking. This plate (Plate I) was used to seed a secondary plate (Plate II) in order to express phage-VHH.

Table 1

 1 Presynaptic a7 nAChRs facilitate glutamate release.

	Brain area	Methodology	Species	Main outcome

Activation of a7 nAChRs at presynaptic sites, via PKA activation, enhances synaptic efficiency of hippocampal mossy fiber transmission

[START_REF] Cheng | Presynaptic a7 nicotinic acetylcholine receptors enhance hippocampal mossy fiber glutamatergic transmission via PKA activation[END_REF]

F.Koukouli, U. Maskos / Biochemical Pharmacology 97 (2015) 378-387

F.Koukouli, U. Maskos / Biochemical Pharmacology 97 (2015) 378-387

20 dB level over background: WT mice showed 36.333 ± 3.858% inhibition whereas α5 SNP mice 21.169 ± 7.093%. Bonferroni's multiple comparisons test showed a significant difference between WT and α5 SNP at the 20 dB level over background with P = 0.0218. Two-way repeated measures ANOVA showed significant difference in % inhibition between WT and α5 SNP mice with P = 0.0028.

Acknowledgements

F.K. is a scholar of the Pasteur Paris University Doctoral Program (PPU) and received a stipend from the Stavros Niarchos Foundation. This work was supported by the CNRS UMR 3571, the Fondation de la recherche m.dicale (FRM), the Agence nationale de la recherche (ANR), the Laboratoire d'Excellence BIO-PSY and the programme PasteurInnov 2012. The laboratory of U.M. is part of the Ecole des Neurosciences de Paris Ile-de-France RTRA network. U.M. is member of the Laboratory of Excellence, LabEx BIO-PSY. We would like to thank Dr. David DiGregorio for maintaining the imaging platform of the Department of Neuroscience at the Pasteur Institute and Dr. Kurt Sailor for advice on awake imaging and editing of the manuscript. We also thank the GENIA Program and the Janelia Research Campus and specifically Vivek Jayaraman, Ph.D., Douglas S. Kim, Ph.D., Loren L. Looger, Ph.D., Karel Svoboda, Ph.D. from the GENIE Project, Janelia Research Campus, Howard Hughes Medical Institute for making the AAV.Syn.GCaMP6 available. Acknowledgements: F.K. is a scholar of the Pasteur Paris University Doctoral Program (PPU) and received a stipend from the Stavros Niarchos Foundation. This work was supported by the CNRS UMR 3571, the Fondation de la recherche médicale (FRM), the Agence nationale de la recherche (ANR), the Laboratoire d'Excellence BIO-PSY and the programme PasteurInnov 2012. The laboratory of U.M. is part of the École des Neurosciences de Paris Ile-de-France RTRA network. U.M. is member of the Laboratory of Excellence, LabEx BIO-PSY. We would like to thank Dr David DiGregorio for maintaining the imaging platform of the Department of Neuroscience at the Pasteur Institute, Dr Franck Verdonck and Dr Fabrice Chrétien for providing the CX3CR1 mice. We also thank the GENIA Program and the Janelia Research Campus and specifically Vivek Jayaraman, Ph.D., Douglas S. Kim, Ph.D., Loren L. Looger, Ph.D., Karel Svoboda, Ph.D. from the GENIE Project, Janelia Research Campus, Howard Hughes Medical Institute for making the AAV.Syn.GCaMP6 and AAV.CAG.tdTom available. Acknowledgements: F.K. is a scholar of the Pasteur Paris University Doctoral Program (PPU) and received a stipend from the Stavros Niarchos Foundation. We would like to thank Dr David DiGregorio for maintaining the imaging platform of the Department of Neuroscience at the Pasteur Institute. We also thank the GENIA Program and the Janelia Research Campus and specifically Vivek Jayaraman, Ph.D., Douglas S. Kim, Ph.D., Loren L. Looger, Ph.D., Karel Svoboda, Ph.D. from the GENIE Project, Janelia Research Campus, Howard Hughes Medical Institute for making the AAV.Syn.GCaMP6 and AAV.CAG.tdTom available.

ACKNOWLEDGEMENTS

We would like to thank the GIE Neuro-CEB of Hôpital de la Pitié-Salpêtrière for providing human AD brain tissue. We gratefully acknowledge financial support from the Institut Roche de Recherche et Médecine Translationnelle. This work was also supported by the "Foundation France Alzheimer" and the "Foundation Georges Pompidou". The present work was also supported by funding from the program "Investissements d'avenir ANR-10-IAIHU-06". The immunohistochemistry of this work was carried out on the HISTOMICS platform of the ICM and we sincerely acknowledge all staff involved. Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 Acknowledgments Fani Koukouli is a scholar of the Pasteur Paris University Doctoral Program (PPU) and received a stipend from the Stavros Niarchos Foundation. We would like to thank Anncharlott Berglar for illustrating the graphical abstract.

To my parents

Maria and Christos

Author Contributions F.K. and U.M. conceived and designed research. Author Information Reprints and permissions information is available at www.nature.com/reprints/index.html. The authors declare no competing financial interests.

Supplementary Information

Supplementary Methods

Two-photon data analysis. After the selection of ROIs, a baseline correction algorithm was utilized in order to remove the slow time scale (< 0.05 Hz) changes in the fluorescence as described previously 1 . The algorithm uses the distribution of fluorescence in a time window (20 seconds) around each sample time point. Each of those points is then normalized by the 8% percentile value. In order to prevent distortions of calcium transients with long durations (∼10 seconds), recorded in cells with high activities, a minimal slope threshold was used for detection. The shape of these long duration calcium transients was preserved by an algorithm based on the computation of the mean drift in the signal.

Baseline fluorescence was computed using an algorithm that searched for the longest suitable time window in which no transients were detected using an iterative linear regression over the entire signal. The algorithm compared the mean squared error of the optimal time window determined for a given size, and for this size divided by two. If the relative difference was lower than 50 %, then the algorithm preserved the higher size time window, otherwise, it preserved the lower size time window. The algorithm repeated this operation iteratively until the minimum time window duration (7.5 seconds). The maximum time window was 30 seconds and the minimum time window was 7.5 seconds. ΔF/F signals were computed as the difference between each fluorescence time point and the mean of the selected baseline, divided by the mean of the selected baseline.

We then used a spike selection algorithm as previously described 1 . Briefly, baseline fluorescence fluctuations were estimated in signal windows in which the standard deviation was less than 0.1 ΔF/F. This threshold was determined by visual inspection in order to

Supplementary Figures

Supplementary Figure 1

Abstract

The prefrontal cortex (PFC) plays an important role in cognitive processes, including access to consciousness. The PFC receives significant cholinergic innervation and nicotinic acetylcholine receptors (nAChRs) contribute significantly to the effects of acetylcholine (ACh) signaling. Using in vivo two-photon imaging of awake and anesthetized mice, we recorded spontaneous, ongoing pyramidal neuron activity in layer II/III in the PFC of wildtype (WT) and mice deleted for different nAChR subunits. Similar to humans, this activity is characterized by synchronous ultra-slow fluctuations of low amplitude with general anesthesia disrupting the generation of neuronal synchronicity. Both the α7 and β2 nAChR subunits have an important role in the generation of ultra-slow fluctuations that occur to a different extent during quiet wakefulness and light general anesthesia. The β2 subunit is specifically required for synchronized activity patterns. Furthermore, chronic application of mecamylamine, a no-competitive antagonist of nAChRs, disrupts the generation of ultra-slow fluctuations. Our findings provide new insights into the default network dynamics in the awake and anesthetized state, and the role of cholinergic neurotransmission in the orchestration of cognitive functions.

Significance statement

The human brain exhibits ongoing spontaneous activity characterized by very slow frequency fluctuations. These synchronously firing populations are considered to play a key role in consciousness. We identified ultra-slow fluctuations (USFs) in awake and anesthetized mice using two-photon imaging in the prefrontal cortex (PFC), a brain region involved in higher cognitive processes. We demonstrate a crucial role for nicotinic acetylcholine receptors (nAChRs) in the modulation of PFC spontaneous activity, the generation of USF and synchronicity, processes that are affected by the interaction of nAChRs with general anesthetics like isoflurane. This work allows dissecting the underlying mechanisms, and predicts that in humans with nAChR polymorphisms or copy number variation (CNV), these processes should be altered, which can be tested experimentally.

Abstract

Alzheimer's Disease (AD) is the most common form of dementia. The condition predominantly affects the cerebral cortex and hippocampus and is characterized by the spread of amyloid plaques and neurofibrillary tangles (NFTs). But soluble amyloid-β (Aβ) oligomers have also been identified to accumulate in the brains of AD patients and correlate with cognitive dysfunction more than the extent of plaque deposition. Here, we developped an adeno-associated viral vector expressing the human mutated amyloid precursor protein (AAV-hAPP). Intracranial injection of the AAV into the prefrontal cortex (PFC) allowed the induction of AD-like deficits in adults, thereby modelling human pathology. The possibility to target a discrete brain region dissects regional vulnerability to AD lesions and potential spreading of the disease. AAV-hAPP expression caused accumulation of Aβ oligomers, microglial activation, astrocytosis and the gradual formation of amyloid plaques and NFTs.

Importantly, in vivo two-photon imaging revealed an increase in neuronal activity, a dysfunction characteristic of the pathology, already during the accumulation of soluble oligomers, not detected in previous work. This AD model allowed us to track brain activity changes during disease progression, in the absence of plaques, and is also a method of choice for models other than mice.

Significance statement

AD is a progressive neurodegenerative disease with a strong impact on society, which becomes a major burden as our population ages. To investigate its underlying neurobiological mechanisms, different mouse models of AD were used in the past. However, these models show important limitations, mainly for the study of the different steps occurring in the course of the disease. Here, we have developed a novel approach to study the early phases of AD in the mouse brain. Our model allows targeted induction of AD-like pathology, reveals a major role of soluble Aβ on the spontaneous activity of cortical neurons, before the formation of amyloid plaques and provides a tool for studying the disease progression, including in species without available transgenic techniques. My personal contribution for this article was to design and perform all the experiments. Geoffroy Goujon and Christiane Bils participated in preparing cranial windows and stereotaxic injections. I also participated in the data analysis and I wrote the manuscript.

Abstract

Alzheimer's Disease (AD) is the most common form of dementia characterized by the spread of amyloid plaques and neurofibrillary tangles (NFTs). Soluble amyloid-β (Aβ) oligomers have also been identified to accumulate in the brains of AD patients and correlate with cognitive dysfunction. AD mainly affects the cerebral cortex and hippocampus and is accompanied by a loss of cholinergic tone and acetylcholine (ACh) levels in the brain, which was hypothesized to be responsible for the observed cognitive decline. Here, we used an adeno-associated viral vector expressing the human mutated amyloid precursor protein (AAV-hAPP) sequence harboring three pathogenic mutations associated with early-onset familial AD. In vivo two-photon Ca 2+ imaging of awake mice showed increased neuronal activity at the site of Aβ peptide expression, a dysfunction characteristic of the pathology.

Because an interaction of nicotinic acetylcholine receptors (nAChRs) and Aβ peptide has been previously reported, we also injected the AAV-hAPP into brains of α7 and β2 nAChR subunit knock-out mice to investigate their role in AD hyperactivity in prefrontal cortex (PFC), a brain region involved in higher cognitive processes. Our data suggest that distinct nAChR subunits enable a diverse regulation of cortical activity in the AD brain and could provide a mechanistic basis for understanding the pathophysiology of the disorder.

Keywords: Alzheimer's Disease, neuronal activity, prefrontal cortex, nicotinic receptors

Discussion

Neuronal circuits become hyperactive instead of hypoactive in the early stages of AD as shown in various in vivo studies in humans and in mouse models (18). This hyperactivity was shown in large-scale networks with imaging techniques and also at the single cell level with electrophysiology (18). What is surprising is that hyperactivity is a sign of AD neuronal dysfunction since it would have been assumed that hypoactivity would instead be a plausible mechanism. Asymptomatic humans with high amyloid load were found to display abnormally increased activation in the default-mode network using blood-oxygen-level dependent (BOLD) fMRI (36). This increase was mainly found in the medial prefrontal cortex, the precuneus and the posterior cingulate cortex. In the hippocampus, elevated resting-state BOLD activity was observed already during the pre-dementia stages of AD (37,38).

Increased hippocampal activation has also been reported in asymptomatic carriers of genetic mutations for familial AD (39).

Early-onset AD is associated with mutations in the APP, presenilin-2 or presenilin-1 genes. All these mutations cause either an increase in the total production of amyloid-β or a relative increase in the fraction of the longer 42 amino-acid isoform of Aβ, which appears to be more closely associated with the development of amyloid plaques than the shorter 40 amino-acid isoform (40)(41)(42). To track neuronal activity in the early stages of AD, before the formation of amyloid plaques, we used an AAV vector that expresses the mutated form of human APP harboring three pathogenic mutations: Swedish, London and Austrian (31)(32)(33).

We chose these mutations because they are associated with early onset of the disease. The Swedish mutation was chosen because it leads to familial AD, as seen in patients harboring just one allele of this dominant mutation (31). We also added the Austrian (T714I) (32) and London (V717I) (33) mutations that when present in hAPP are known to drive a higher

Conflict of interest:

The authors declare no competing financial interests.

Article in collaboration

Camelid single-domain antibodies: A versatile tool for in vivo imaging of extracellular and intracellular brain targets

Tengfei Li, 1,2,3,10 Matthias Vandesquille, 4,5,6,7,10 Fani Koukouli, 8 Clémence Dudeffant, 2,6,7 Ihsen Youssef, 2 Pascal Lenormand, 1 Christelle Ganneau, 4,5 Uwe Maskos, 8 Christian Czech, 9 Fiona Grueninger, 9 Charles Duyckaerts, 2 Marc Dhenain, 6,7 Sylvie Bay, 4,5 Benoît Delatour, 2 and Pierre Lafaye

SUMMARY

Detection of intracerebral targets with imaging probes is challenging due to the nonpermissive nature blood-brain barrier (BBB). The present work describes two novel singledomain antibodies (VHHs or nanobodies) that specifically recognize extracellular amyloid deposits and intracellular tau neurofibrillary tangles, the two core lesions of Alzheimer's disease (AD). Following intravenous administration in transgenic mouse models of AD, in vivo real-time two-photon microscopy showed gradual extravasation of the VHHs across the BBB, diffusion in the parenchyma and labeling of amyloid deposits and neurofibrillary tangles. Our results demonstrate that VHHs can be used as specific BBB-permeable probes for both extracellular and intracellular brain targets and suggest new avenues for therapeutic and diagnostic applications in neurology. they can diffuse more rapidly and deeply in fixed tissues [START_REF] Perruchini | Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies[END_REF]. They have a better biodistribution profile in vivo than conventional antibody fragments [START_REF] Cortez-Retamozo | Efficient tumor targeting by single-domain antibody fragments of camels[END_REF][START_REF] Li | Influence of molecular size on tissue distribution of antibody fragments[END_REF]. Depending on their binding properties, VHHs can be used either to visualize and/or to interact with intracellular targets [START_REF] Van Audenhove | Mapping cytoskeletal protein function in cells by means of nanobodies[END_REF][START_REF] Helma | Direct and dynamic detection of HIV-1 in living cells[END_REF][START_REF] Li | Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging[END_REF][START_REF] Maier | Real-time analysis of epithelialmesenchymal transition using fluorescent single-domain antibodies[END_REF][START_REF] Rocchetti | Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody[END_REF][START_REF] Rothbauer | Targeting and tracing antigens in live cells with fluorescent nanobodies[END_REF][START_REF] Traenkle | Monitoring interactions and dynamics of endogenous beta-catenin with intracellular nanobodies in living cells[END_REF].

We recently demonstrated that some VHHs with a basic isoelectric point (pI) are able to readily transmigrate across the BBB in vivo after peripheral injection, without the need of any invasive or hazardous procedures (such as opening of the barrier using chemical or ultrasound sonication) [START_REF] Li | Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging[END_REF]. These VHHs can be used as specific transporters as demonstrated by in vivo labeling of astrocytes with a VHH-fluorophore conjugate targeting the intracellular protein GFAP [START_REF] Li | Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging[END_REF].

In the present work we engineered two new VHHs that were able to detect extracellular or intracellular pathological brain targets, namely the two main neuropathological lesions in Alzheimer's disease (amyloid plaques and neurofibrillary tangles -NFTs). We designed sensitive and specific anti-Aβ and anti-tau VHHs that allowed detection of plaques and NFTs in brain tissue from patients with Alzheimer's disease and from mouse models of the disease.

We also showed that plaques and tangles could be visualized in vivo in mice following intravenous administration of these probes.

RESULTS

Selection and characterization of a VHH recognizing extracellular targets

Generation and optimization of R3VQ

One alpaca was immunized with a fibrillar synthetic Aβ42 peptide and a specific VHH library was constructed from cDNA encoding VHH domains isolated from lymphocytes. The total size of the library contained 1x 10 8 phage-VHHs. VHHs were selected by Phage Display through 3 panning cycles with biotinylated Aβ42 and Aβ40 peptides at different concentrations of antigens, buffer and washing conditions. Four hundreds individual clones were tested by ELISA using biotinylated Aβ42. Two VHHs were obtained, called R3VQ and R3VE respectively. They differed by only one amino acid: a glutamine (Q) of R3VQ was replaced by a glutamic acid (E) in R3VE. R3VQ recognized an epitope located in the central part of the Aβ peptide between aa 17-28 (Figure S1A). C terminal His tagged VHH R3VQ and R3VE were obtained after subcloning of their genes in pET23d vector. Both VHHs had high production yields about 20 and 9mg/l of culture, respectively. Note that in the present study, R3VQ and R3VE represent R3VQ-His and R3VE-His antibodies unless otherwise mentioned. As a basic pI favors BBB passage [START_REF] Li | Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging[END_REF], we decided to focus our study on R3VQ, which had a higher pI than R3VE (see below). R3VQ showed good purity with only one band observed by SDS-PAGE. It had a basic pI > 8.3 (Figure 1A-B). By ELISA, R3VQ showed strong binding to Ab40 and Ab42 (Figure 1C).

We engineered a fluorescent conjugate to allow in vivo imaging with microscopic resolution.

For this purpose, an additional cysteine residue was inserted in the C terminal part of the sequence of R3VQ (referred as R3VQ-SH), thus allowing a C terminal thio-addition of a maleimido-Alexa fluor dye (AF488) by a site specific approach (Vandesquille et al., in prep).

The coding sequence of R3VQ-SH contained a N-terminal 6-histidine tag for purification and detection.

In this way we obtained a well-defined conjugate with only one AF488 molecule (Figure 1A), as confirmed by mass spectrometry (data not shown). This final compound was referred to as R3VQ-S-AF488. The pI of R3VQ-SH and R3VQ-S-AF488 were analyzed by NEPHGE using IEF 3-10 gel. The pI of R3VQ-SH was between 8.3 and 9.5 (Figure 1B), similar to that of R3VQ. The addition of maleimido-AF488 to R3VQ-SH decreased its pI to ca. 8.3 (Figure 1B). The two bands on IEF gel indicate the presence of two isoforms for both R3VQ-SH and R3VQ-S-AF488.

Immunodetection of Ab brain lesions in mice and in human brain sections

VHH immunohistochemistry (IHC) was performed in plaques-enriched AD tissues (isocortical samples) and in PS2APP mice (Ozmen et al., 2009;Weidensteiner et al., 2009) harboring amyloid deposits. R3VQ showed excellent sensitivity and selectivity in detecting extracellular amyloid plaques and cerebral amyloid angiopathy (CAA), both on human and mouse paraffin sections pre-treated with formic acid (Alafuzoff et al., 2009) (Figure 2A-B).

Immunodetection of amyloid plaques was also possible on fresh brain tissues from AD patients without the use of any antigen retrieval pre-treatement (Figure S1B). Double labeling performed on mouse sections using 4G8 (as a gold-standard anti-Ab monoclonal antibody (mAb)) and R3VQ showed a very good colocalization of the two antibodies (Figure 2C).

Comparison of amyloid loads detected by R3VQ and 4G8 showed excellent correlation between the results obtained with the two antibodies (Figure S1C). No labeling was observed following R3VQ IHC on tissues from age-matched wild type C57Bl/6 mice (Figure S1D).

The R3VE variant showed similar immunodetection to R3VQ on PS2APP mouse tissue (Figure S1E).

Selection and characterization of a VHH recognizing intracellular targets

Generation and engineering of A2

One alpaca was immunized with AD phospho-tau (pTau) enriched brain extracts and a second alpaca was immunized with a mono-phosphorylated peptide derived from the C-terminus of the tau protein (pS422 peptide) coupled to the KLH protein. The sera of immunized alpacas recognized both pTau and non-phosphorylated tau. The immunized animals generated 2 phage-VHH libraries of about 3x10 8 clones each. In order to expand the diversity of VHHs, a pooled library from these two animals was used for panning experiments by Phage Display.

After 3 panning cycles with pTau protein, one clone was found to bind specifically to pTau and was referred to as A2. C-terminally His tagged A2 was produced as described above for R3VQ VHH (yield 5 mg/l of culture). Only one band was observed by SDS-PAGE (Figure 4A). Analysis by IEF-NEPHGE showed that the pI of A2 was higher than 9.5 (Figure 4B).

ELISA was performed on pTau and pS422 peptide coupled to ovalbumin. pTau corresponded to the full-length tau protein phosphorylated at multiple sites including S422. A2 bound to both full-length pTau protein and the pS422-containing peptide. Lack of binding to nonphosphorylated-tau demonstrated that A2 was specific for a phosphorylated epitope (Figure 4C).

For the purpose of in vivo analysis, an A2-SH variant was prepared and conjugated to AF488 as previously described for R3VQ-S-AF488 (see above). The pI of A2-SH and A2-S-AF488 was between 9.5 and 10.7. The addition of maleimido-AF488 to A2-SH slightly decreased the pI of A2-SH as already observed with R3VQ-S-AF488 (Figure 4B).

Immunodetection of tau brain lesions in mice and in human brain sections

VHH A2 allowed detection of intracellular neurofibrillary tangle-like structures in Tg4510 mouse brain tissues using paraffin sections with or without any antigen retrieval pretreatment

BBB transit and diffusion of A2 after intravenous injection

To investigate if A2-S-AF488 crosses the BBB, the same systematic approach as implemented for R3VQ-S-AF488 (see above) was applied. A 10 mg/kg dose of A2-S-AF488 was injected iv in two 8-month-old Tg4510 mice that were sacrificed 4 hours later.

Immunostaining using anti-A2 polyclonal antibody showed binding to a large number of NFT-like structures in the brain, demonstrating that A2-S-AF488 crossed the BBB, diffused extensively throughout the brain in vivo and labeled pTau aggregates. By contrast, no staining was found in the mice injected with PBS or with a conventional antibody specific for pTau (Figure S3).

Real-time imaging A2 after intravenous injection

VHH A2-S-AF488 (10 mg/kg) was injected in the tail vein of Tg4510 mice. A2-S-AF488 extravasation and diffusion in the brain was monitored for 4 hours post injection using twophoton microscopy imaging of the cortical surface up to 350 µm deep. Immediately after iv injection, cerebral blood vessels showed strong fluorescence, as observed for R3VQ.

Identification of NFT-like structures was possible 2 hours after injection (Figure 6A). A persistent and specific labeling of NFTs was observed even 4 hours after injection (Figure 6B) and the 3D reconstruction of the immuno-detected objects revealed the standard flame-shaped morphology of tangles (Supplementary Movie S2). Four hours after injection, the mice were sacrificed, immunohistochemistry was performed using anti-A2 polyclonal antibody to demonstrate the presence of A2-S-AF488 in the brain. Extensive tau immunostaining throughout the brain was also confirmed in postmortem tissue (Figure 6C).

In summary, VHH A2-S-AF488 efficiently crossed the BBB and penetrated into neurons to reach its cytoplasmic target. As for in vivo R3VQ experiments, target labeling was visible four hours after administration demonstrating slow turnover of target-associated VHHs in the brain.

DISCUSSION

VHHs bind to both extracellular and intracellular brain targets

VHHs were generated to Aβ42 and phospho-tau (R3VQ and A2, respectively). Their specificities were biochemically assessed and then confirmed by immunohistochemistry on both human and AD transgenic mouse brain tissues. To the best of our knowledge, the VHH A2 presented here is the first engineered VHH capable of immunodetecting AD-associated intraneuronal NFT pathology.

By means of stereotaxic injection we then confirmed the ability of R3VQ and A2 to label their targets in vivo after rapid diffusion in brain tissue, a result that may be explained by the small size of the constructs [START_REF] Perruchini | Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies[END_REF]. Notably, the antigen binding properties of both VHHs were preserved after conjugation with AlexaFluor 488, therefore opening up opportunities to use labeled VHHs for in vivo imaging.

VHHs cross the BBB and efficiently diffuse in the brain

A few VHHs have been shown to cross the BBB by receptor-mediated transcytosis [START_REF] Muruganandam | Selection of phagedisplayed llama single-domain antibodies that transmigrate across human blood-brain barrier endothelium[END_REF][START_REF] Rutgers | Differential recognition of vascular and parenchymal beta amyloid deposition[END_REF]. We have previously shown that VHH E9 with basic pI was able to passively cross the BBB, diffuse in the brain parenchyma and label astrocytes in vivo [START_REF] Li | Cell-penetrating anti-GFAP VHH and corresponding fluorescent fusion protein VHH-GFP spontaneously cross the blood-brain barrier and specifically recognize astrocytes: application to brain imaging[END_REF]).

An anti-Aβ VHH (pa2H) was previously reported to cross the BBB in vivo [START_REF] Nabuurs | In vivo detection of amyloid-β deposits using heavy chain antibody fragments in a transgenic mouse model for Alzheimer's disease[END_REF]. However pa2H brain uptake was too low to allow in vivo imaging. More recently the same research group designed a glutathione PEGylated liposomal encapsulation system to enhance the delivery of pa2H across the BBB [START_REF] Rotman | Enhanced glutathione PEGylated liposomal brain delivery of an anti-amyloid single domain antibody fragment in a mouse model for Alzheimer's disease[END_REF]. SPECT imaging performed in this study did not however allow the visualization of pa2H brain penetration. In our study plaques and NFTs were detected in vivo after a single iv injection of fluorescent R3VQ and A2 respectively, confirming high BBB crossing potential and exquisite sensitivity

VHH potential for AD diagnosis

Until now the development of imaging markers for AD brain lesions has focused on PET ligands [START_REF] Brendel | microPET Imaging of Tau Pathology with [18F]-THK5117 in two Transgenic Mouse Models[END_REF][START_REF] Clark | Use of florbetapir-PET for imaging beta-amyloid pathology[END_REF][START_REF] Kimura | PET Quantification of Tau Pathology in Human Brain with 11C-PBB3[END_REF][START_REF] Klunk | Imaging brain amyloid in Alzheimer's disease with Pittsburgh Compound-B[END_REF][START_REF] Maruyama | Imaging of tau pathology in a tauopathy mouse model and in Alzheimer patients compared to normal controls[END_REF][START_REF] Okamura | Non-invasive assessment of Alzheimer's disease neurofibrillary pathology using 18F-THK5105 PET[END_REF][START_REF] Sehlin | Antibodybased PET imaging of amyloid beta in mouse models of Alzheimer's disease[END_REF]. However PET imaging suffers from numerous drawbacks, namely, low spatial resolution, exposure to radiation, limited availability and high cost. Conversely, MRI provides information with high spatial resolution, does not rely on ionizing radiations and is widely available for clinical use.

Previous attempts have been made to use MR contrast agents for visualizing amyloid deposits in AD mouse models. However these studies relied on artificial BBB opening by ultrasound [START_REF] Matharu | Gadolinium-complexed Aβbinding contrast agents for MRI diagnosis of Alzheimer's Disease[END_REF][START_REF] Santin | Fast in vivo imaging of amyloid plaques using µ-MRI Gd-staining combined with ultrasound-induced blood-brain barrier opening[END_REF] or BBB permeability enhancers such as mannitol [START_REF] Sigurdsson | A non-toxic ligand for voxelbased MRI analysis of plaques in AD transgenic mice[END_REF]. With the goal of developing a more physiological approach we recently described a method for site-specifically conjugating VHHs to paramagnetic gadolinium (Vandesquille et al., in prep). The VHHs described in the present work could be conjugated with gadolinium to provide a novel MR imaging agents for AD.

Conclusions

We describe novel VHHs that could be potentially used as in vivo imaging agents able to detect brain lesions observed in human diseases, e.g. Alzheimer's disease. VHH R3VQ binds to brain Aβ deposits and VHH A2 recognizes neuronal pathological tau inclusions. VHHs are able to penetrate the brain more efficiently than conventional IgGs and they can be readily site-specifically modified for in vivo imaging of neurodegenerative diseases with extracellular or intracellular misfolded protein aggregates. Aβ immunization was described in (Lafaye et al., 2009). To induce an Aβ-directed immune reaction a two-year-old male alpaca was immunized with 1mg of fibrillar synthetic Aβ42 peptide (Bachem). The aggregated conformation of Aβ42 peptide was previously confirmed by DLS experiment.

EXPERIMENTAL PROCEDURES

All

Tau immunization was partly performed using tangles-enriched extracts (Mercken et al., 1992). Cortex isolated from 4 AD patients (0.2 g) was homogenized in 10 volumes of 10 mM Tris, 1mM EGTA, 0.8 M NaCl pH 7.4 containing 10 % sucrose and was centrifuged at 27,000 g for 20 min at 4°C. The pellet was removed and the supernatant was adjusted to 1% Nlaurylsarcosine and 1% beta-mercaptoethanol and incubated while rotating for 2.5 hours at 37°C. The supernatant mixture was centrifuged at 100,000 g for 35 min at 20°C. The PHF containing pellet was gently washed with PBS and finally resuspend in the same buffer. One alpaca was immunized with the final Tau pellet. Another alpaca was immunized with a synthetic mono-phosphorylated peptide derived from the C-terminus of the tau protein (sequence CSIDMVDS(PO 3 H 2 )PQLATLAD) coupled to the Keyhole Limpet Hemocyanin (KLH) protein (Eurogentec). The same phospho-peptide has been used for the generation of the muRb86 anti-Tau-pS422 mAb [START_REF] Grueninger | Phosphorylation of Tau at S422 is enhanced by Abeta in TauPS2APP triple transgenic mice[END_REF]. Two hundred and fifty microliters (250 µg) of both antigens (human extract and synthetic peptide) was mixed with

Expression of VHHs

The coding sequences of the selected VHHs in vector pHEN1 were sub-cloned into a bacterial expression vector pET23d (Novagen) containing a 6-Histidine tag using NcoI and NotI The coding sequences of the Cys-engineered VHH (R3VQ-SH or A2-SH) were cloned into the expression vector pET23d using NcoI and XhoI restriction sites. The different genes were synthetized by GenArt (Life technologies). Transformed E. coli BL21 (DE3) pLysS cells were used as described before for protein expression. After purification, VHHs were dialyzed in 0.1M PBS buffer containing 300 mM NaCl and stored at 4°C.

Preparation of AlexaFluor 488-conjugated VHH and mAb

The cysteine present in the C-terminal region of VHH and the SH group in the hinge region of mAbs (4G8 and muRb86) were used to realize the conjugation with the maleimido-AF488 fluorophore (Invitrogen). The pH of the solution containing VHH-SH or mAb was adjusted between 6.8 and 7. The solution was then gently stirred with 10-fold molar excess of tris (2carboxyethyl) phosphine (TCEP, Acros Organics) at room temperature for 30 min, to allow complete reduction of any intermolecular disulfide bond. A 10-fold molar excess of maleimido-AF488 dissolved in dimethylformamide (DMF) was added. Notably, for high efficiency of conjugation, the volume percentage of DMF in the final solution was kept below

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Polyacrylamide Gel electrophoresis (PAGE) was performed using NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) according to manufacturer's instructions. PageRuler TM Prestained Protein ladder was used as molecular weight maker and Instant Blue (Expedeon UK) was used to stain SDS-PAGE gel.

Determination of pI by IsoElectric Focusing (IEF)

The pI of VHHs was determined by isoelectric focusing using IEF 3-10 Gel (Invitrogen). NEPGHE (non equilibrium pH gradient gel electrophoresis) with sample application at the anode was also used because it allows optimal protein analysis in the basic range of the gel including pI 8.5 to 10.5. Briefly, for NEPHGE the polarity of the electrophoresis unit (compared to standard IEF) was switched, gels were always cooled during the electrophoresis and the following conditions were used: Anode buffer 40mM glutamic acid and cathode buffer 20mM NaOH. The wells were filled with 10-30 µl of protein solution. NEPHGE was carried out first for 1 hour at 100 volts then 20 min at 200 volts and finally 5-10 min for 500 volts (SERVA Gel IEF 3-10 instruction manual).

Animal models

PS2APP mice overexpressing hAPP Swedish mutation combined with PS2 N141I mutation (Ozmen et al., 2009;Weidensteiner et al., 2009) were used as animal models harboring Aβpositive lesions. PS2APP mice start to develop overt Aβ deposition in the brain at approximately 6 months of age.

Tg4510 mice with the hMAPT P301L gene mutation [START_REF] Santacruz | Tau suppression in a neurodegenerative mouse model improves memory function[END_REF] were used as NFT-bearing mouse model. NFT start to appear in the cortex by 4 months of age and one month later in the hippocampus.

Stereotaxic injections of VHHs

Stereotaxic injections of anti Aβ VHH R3VQ were performed in anesthetized PS2APP mice (one-year-old, n=2). Two microliters (6µg) of R3VQ was injected into the frontal cortex of one mouse at the rate of 0.2µl /min. The stereotaxic coordinate was +0.86 mm from bregma, -1.5mm from the midline, -0.65mm ventral from dura. Two hours after the injection, the mouse was euthanized and given an intracardiac perfusion of 4% paraformaldehyde in PBS (pH 7.6). In parallel, another mouse was stereotaxically injected with the same amount of R3VQ at the same coordinates, but the mouse was euthanized 24 hours later. Brains were removed and postfixed in the same fixative overnight at 4°C. Paraffin embedded sections (4µm thick) were obtained with a Microm HM340E microtome (Thermo Scientific). The presence of the VHH in cerebral tissue was detected using the standard immunohistochemical procedures as described below.

For the stereotaxic injection of anti pTau VHH A2, the same procedures were performed in two 10-month-old Tg4510 mice, except A2 was bilaterally injected into mice brains.

Reference antibodies

Control immunostaining of amyloid plaques made use of biotinylated 4G8 mAb (Covance, 1/10000) as the gold standard anti-Aβ mAb (Alafuzoff et al., 2009;[START_REF] Wisniewski | Amyloid beta 1-42 deposits do not lead to Alzheimer's neuritic plaques in aged dogs[END_REF].

Conversely control immunolabeling of NFTs relied on biotinylated AT8 mAb (Thermo Scientific, 1/200 for paraffin-sections and 1/5000 for free-floating sections) (Mercken et al., 1992). The muRb86 mAb directed specifically against pS422 [START_REF] Grueninger | Phosphorylation of Tau at S422 is enhanced by Abeta in TauPS2APP triple transgenic mice[END_REF] was used as control for BBB passage after iv injection.

Immunohistochemistry -paraffin sections

Immunostaining of brain (human and mouse) tissue was performed on 4µm thick paraffin sections. Sections were de-paraffinized in xylene, rehydrated through ethanol (100%, 90%, and 70%) and finally brought to running tap water for 10 min. They were then incubated in prior to immunolabeling as described previously. Between steps, the sections were rinsed 3 times in PBS-triton-X100 0.2%. The sections were then incubated overnight at 4°C with the primary His tagged VHH antibody, diluted to 1-5 g/ml in PBS-triton 0.2%. Sections exposed to His tagged VHH were then treated successively with rabbit anti-His-tag antibodies (eBioscience) for 1h at room temperature, then with goat biotinylated anti-rabbit IgG(H+L) (Vector) for 1h at room temperature, and finally with ABC complex (Vector) for 30 min.

DAB was used as chromogen. Reacted sections were mounted on superfrost+ glass slides (Thermo Scientifc), air-dried for 1h at 55°C then dehydrated in graded ethanol solutions as described above.

Immunohistochemistry -VHH-exposed tissue

Brain sections obtained from mice that received intracerebral or peripheral (iv) injections of VHHs were treated using the same protocols as described above but starting with secondary antibody incubation after formic acid pre-treatment, endogenous peroxidase and non-specific binding blocking steps. All tissue were processed using the same anti-His antibodies as described above with the sole exception of brain tissue obtained from Tg4510 mice that received A2-S-AF488 and that were incubated with a house-made anti VHH A2 serum providing better signal-to-noise ratio. Direct use of anti-His or anti-A2 antibodies finally allowed the detection of resident VHHs that were able to diffuse after stereotaxic injection or to cross the BBB after peripheral administration.

Estimation of lesion loads by VHHs and reference antibodies

Comparative lesion loads were assessed from PS2APP and Tg4510 paraffin brain sections immunostained using reference (4G8, AT8) or VHH (R3VQ, A2) antibodies. Paired comparisons of lesion loads were performed between 4G8 vs R3VQ and between AT8 and A2 immunostaining, using the same regions of interest from adjacent brain sections.

In vivo two-photon imaging

Two-photon imaging was performed with a two-photon laser-scanning microscope system and PrairieView software (Prairie Technologies, Middelton, WI), using a 16x 0.8 NA water immersion objective (Nikon, Tokyo, Japan) with the two-photon laser tuned to 920 nm (MaiTai DeepSee, Spectra Physics, Mountain View, CA). The images were acquired at 512 x 512 with a pixel size of 0.5 um. Care was taken to use less than 20 mW of laser power in the tissue. The raw data obtained were processed using Image J software for analysis and 3D rendering.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.