Morgane Besson
email: morgane.besson@pasteur.fr

Stefania Guiducci

Sylvie Granon

Jean-Philippe Guilloux

Bruno Guiard

Christelle Repérant

Philippe Faure

Stéphanie Pons

Giuseppe Cannazza

Michele Zoli

Alain M Gardier

Uwe Maskos

Alterations in alpha5* nicotinic acetylcholine receptors result in midbrain-and hippocampus-dependent behavioural and neural impairments

Keywords: Addiction, Nicotine, Vulnerability, Nicotinic receptors, Polymorphism, Mice, Monoamines, Lentiviral vector, Novelty, Anxiety

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Smoking is the most important cause of preventable mortality and morbidity worldwide (World Health Organization, http://www.who.int/tobacco/statistics/tobacco_atlas/en/). Nicotine, the main reinforcing component in tobacco, acts on the brain via nicotinic acetylcholine receptors (nAChRs) [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF]. nAChRs are pentameric ion channels composed of different α and β subunit combinations with different functional properties. nAChRs containing α4 and β2 subunits are predominant in the brain and account for most of the rewarding properties of nicotine [START_REF] Picciotto | Molecular mechanisms underlying behaviours related to nicotine addiction[END_REF].

Recently, the α5 subunit was crucially implicated in nicotine addiction. Genome wide association studies reported a strong association between the CHRNA3/A5/B4 gene cluster, encoding the α3, α5 and ß4 nicotinic subunits, and predisposition to tobacco smoking [START_REF] Berrettini | The CHRNA5-A3-B4 gene cluster in nicotine addiction[END_REF][START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF][START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF]. Notably, a non-synonymous single nucleotide polymorphism, changing an aspartic acid into asparagine, was identified in the α5 gene (α5SNP), and associated with higher scores of nicotine dependence, heavy smoking, and early onset of smoking [START_REF] Berrettini | The CHRNA5-A3-B4 gene cluster in nicotine addiction[END_REF][START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF][START_REF] Grucza | A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence[END_REF][START_REF] Stevens | Nicotinic receptor gene variants influence susceptibility to heavy smoking. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Weiss | A candidate gene approach identifies the CHRNA5-A3-B4 region as a risk factor for age-dependent nicotine addiction[END_REF]. Strikingly, α5SNP homozygous carriers have a two-fold higher risk for tobacco addiction than non-carriers [START_REF] Greenbaum | Differential contribution of genetic variation in multiple brain nicotinic cholinergic receptors to nicotine dependence: recent progress and emerging open questions[END_REF][START_REF] Improgo | The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: dual role in nicotine addiction and lung cancer[END_REF][START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF]).

Although the α5 subunit does not contribute to ligand binding sites, it combines with other α and β subunits and influences the receptor properties [START_REF] Exley | Striatal alpha5 nicotinic receptor subunit regulates dopamine transmission in dorsal striatum[END_REF][START_REF] Kuryatov | Roles of accessory subunits in alpha4beta2(*) nicotinic receptors[END_REF][START_REF] Ramirez-Latorre | Functional contributions of alpha5 subunit to neuronal acetylcholine receptor channels[END_REF][START_REF] Tapia | Ca2+ permeability of the (alpha4)3(beta2)2 stoichiometry greatly exceeds that of (alpha4)2(beta2)3 human acetylcholine receptors[END_REF], probably through allosteric mechanisms [START_REF] Taly | Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system[END_REF].

Mice knockout for the α5 gene (α5 -/-mice) are less sensitive to some nicotine effects including seizures and antinociception [START_REF] Jackson | Role of alpha5 nicotinic acetylcholine receptors in pharmacological and behavioural effects of nicotine in mice[END_REF][START_REF] Salas | The nicotinic acetylcholine receptor subunit alpha 5 mediates short-term effects of nicotine in vivo[END_REF][START_REF] Salas | Nicotinic receptors in the habenulointerpeduncular system are necessary for nicotine withdrawal in mice[END_REF]. They display decreased somatic nicotine withdrawal syndrome [START_REF] Salas | Nicotinic receptors in the habenulointerpeduncular system are necessary for nicotine withdrawal in mice[END_REF]) and decreased sensitivity to nicotine-induced reward as well as aversion and reward-inhibition at high doses [START_REF] Fowler | Habenular alpha5 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]. In vitro studies showed that the α5SNP causes a partial loss of function of nAChRs containing α5 (α5*-nAChRs), with decreased Ca 2+ permeability and current amplitude in response to nicotinic agonists [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF][START_REF] Frahm | Aversion to nicotine is regulated by the balanced activity of beta4 and alpha5 nicotinic receptor subunits in the medial habenula[END_REF][START_REF] Kuryatov | Acetylcholine receptor (AChR) alpha5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (alpha4beta2)(2)alpha5 AChR function[END_REF]. Furthermore, we showed that expressing the α5SNP instead of wild-type α5 (α5WT) in the ventral tegmental area (VTA) causes a rightward shift in the dose response curve to nicotine in both self-administration behaviour and VTA dopamine (DA) neuron activity [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]).

Altogether, these data show that alterations in α5*-nAChRs result in decreased sensitivity to nicotine. Yet, additional work is needed to understand the role of native and polymorphic α5*-nAChRs in the vulnerability to addiction. Only a percentage of individuals exposed to drugs eventually develop addiction. Some personality traits and psychiatric conditions, especially high impulsivity, high novelty/sensation seeking and high anxiety, are strikingly associated with addiction [START_REF] Belin | Responses to novelty and vulnerability to cocaine addiction: contribution of a multi-symptomatic animal model[END_REF][START_REF] Gehricke | Smoking to self-medicate attentional and emotional dysfunctions[END_REF][START_REF] Kreek | Genetic influences on impulsivity, risk taking, stress responsivity and vulnerability to drug abuse and addiction[END_REF]). These traits have been identified as predisposing factors conferring vulnerability to drug, including nicotine, abuse in clinical and preclinical studies [START_REF] Batra | Multidimensional smoker profiles and their prediction of smoking following a pharmacobehavioural intervention[END_REF][START_REF] Diergaarde | Impulsive choice and impulsive action predict vulnerability to distinct stages of nicotine seeking in rats[END_REF][START_REF] Falco | Anxiety status affects nicotine-and baclofen-induced locomotor activity, anxiety, and single-trial conditioned place preference in male adolescent rats[END_REF][START_REF] Kushner | Greater elevation in risk for nicotine dependence per pack of cigarettes smoked among those with an anxiety disorder[END_REF][START_REF] Redolat | Individual differences in noveltyseeking and behavioural responses to nicotine: a review of animal studies[END_REF][START_REF] Suto | Locomotor response to novelty predicts a rat's propensity to self-administer nicotine[END_REF]). α5 -/-mice did not show a high impulsive phenotype in a 5-choice serial reaction time task [START_REF] Bailey | The nicotinic acetylcholine receptor alpha5 subunit plays a key role in attention circuitry and accuracy[END_REF]. However, the role of α5*-nAChRs in, and the impact of the α5SNP on, other neurobehavioural endophenotypes for addiction vulnerability remain unclear. Here we assessed novelty and sensation seeking, modeled as novelty-induced conditioned place preference and locomotor reactivity in novel inescapable environment, respectively [START_REF] Belin | Responses to novelty and vulnerability to cocaine addiction: contribution of a multi-symptomatic animal model[END_REF], and anxiety in α5 -/-mice. We next investigated the consequences of nicotine exposure on the behavioural phenotypes identified. We also assessed, in these mice, spontaneous and nicotine-elicited activities of the DA system, as well the serotonin (5-HT) system for the first time. Finally, we re-expressed, using lentiviral vectors (LVs) [START_REF] Maskos | Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors[END_REF], either the α5WT or α5SNP gene in specific brain areas, and compared their impact on α5 -/-mice behavioural phenotypes.

Materials and Methods

Animals

Experiments were conducted in male C57BL/6J wild-type (WT) mice and constitutive knockout mice lacking the α5 nAChR subunit (α5 -/-mice) [START_REF] Salas | The nicotinic acetylcholine receptor subunit alpha 5 mediates short-term effects of nicotine in vivo[END_REF]. WT and α5 -/-mice were not littermates, however α5 -/-mice have been back-crossed to the C57BL/6J strain for more than 20 generations, WT and α5 -/-mice were both from Charles River, France, bred in the same life conditions, arrived at the animal facility at the same time and were housed in the same room, and were matched for age and sex. They were used in strict accordance with the Guide for the Care and Use of Laboratory Animals (NIH) and the European Commission regulations for animal use in research (CEE n° 86/609), and the IP Animal Care Committee and French Ministry (authorization number 01601.02). The animals were 10 weeks of age at the start of the experiments, housed six per cage with food and water available ad libitum. They were kept under standard conditions (temperature 21±1°C, 60-65% relative humidity) on a reversed 12h light/dark cycle (light on 7.00 p.m.) All experiments were performed between 9.00 a.m. and 6.30 p.m.

Behavioural testing

Mice were handled for two days before starting behavioural testing.

For basal identification of behavioural phenotypes in WT and α5 -/-mice, half of the animals were submitted first to the open-field test and 3 days later to the elevated-plus-maze (EPM), while the opposite order was followed for the other half (with equal repartition for both genotypes). The other tests were then conducted in the following order, and with 1 week apart from each other: light-dark box (LDB), novelty preference, novel box exploration for rearing measurement.

For studying the effects of nicotine injections on behavioural phenotypes in WT and α5 -/-mice, each mouse received the same dose of nicotine 10 minutes before each test, with 1 week between each test. The order of running of the tests was pseudo-randomized between novelty-induced rearing behaviour, EPM and LDB tasks, so that each testing sequence was represented in an equal number of mice (for both genotypes). Locomotor activity in the open-field was examined last.

For studying the effects of α5 gene re-expression on behavioural phenotypes in α5 -/-mice, tests were conducted in the following order, and with 1 week apart from each other: novel box exploration for rearing measurement, EPM, LDB. Total distance moved; % of time spent in the periphery; % time spent in inactivity; %PI or %CI; %PA or %CA were measured for 30 min. Rearing behaviour (vertical activity) was also measured, as a distinct measure of novelty-induced exploratory behaviour [START_REF] Lever | Rearing on hind legs, environmental novelty, and the hippocampal formation[END_REF], in an unfamiliar rectangular transparent box (50 x 25 x 50 cm) for 15 min. The light in the testing rooms was set up at 100 lux.

Novelty

Novelty seeking. Novelty preference was measured using a novelty-induced conditioned place preference procedure. The procedure was performed in boxes made of grey Plexiglas of two distinct compartments of equal size (16 × 16 × cm) connected by a central zone (16 × 5 cm), with distinct spatial configurations (one rectangular or triangular object at one of the angles) and two distinct floor textures (grooves or peaks), and that were totally closed at the time of the experiments to be isolated from visual cues in the testing room. Mice were exposed 25 min to one compartment ('familiar'). At the end of this habituation phase, the door isolating the familiar compartment from the other zones was opened to let the animal explore the whole box (central zone, familiar, and new compartment) for 15 min. The index of novelty preference was calculated as the time spent in the new compartment/(time spent in the new compartment + time spent in the familiar compartment) × 100. Light intensity into the boxes was about 0.20 lux. Before starting the experiments, we verified the absence of spontaneous preference for one of the compartments in a separate group of mice (data not shown).

Anxiety. Anxiety was evaluated in the EPM and LDB tests. % of time spent in the EPM open arms and in their extremities (as complementary anxiety detection factor [START_REF] Cruz | Ethopharmacological analysis of rat behaviour on the elevated plus-maze[END_REF])) and total distance moved were measured for 10 min. The light in the testing room was set up at 20 lux. For LDB test, % of time spent in the light side was measured for 5 min. The EPM was elevated 50 cm from the floor, and consisted of two 'open arms', i.e., with no wall (30 x 7 cm), and two 'closed arms', i.e. with 'high walls (30 x 9 x 7 x 18 cm), extending from a central platform (7 x 7 cm). The entire structure was made of grey Plexiglas. The LDB measurements were 44 × 21 × 21 cm. Mice were placed into a box with two chambers, one dark with black floor and walls, and one brightly lit (500 lux) with white floor and walls, connected by an opening.

Drugs

(-)-nicotine bitartrate (Sigma-Aldrich, St. Louis, Missouri) was dissolved in 0.9% saline, and the pH adjusted to 7.2 ± 0.1 with NaOH (0.1N). Doses refer to free base. The different doses of nicotine were tested in independent groups of mice and injected intraperitoneally.

Lentiviral expression vectors

Mice were injected bilaterally into the VTA or dorsal hippocampus with LVs expressing enhanced green fluorescent protein (eGFP) (WT GFP and α5 -/-GFP mice), bicistronic α5WT-IRES2-eGFP-(α5 -/- α5WT mice) or α5D397N-IRES2-eGFP-(α5 -/-α5SNP mice) cDNAs as described in detail in [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. Lentiviruses were derived from pHR' expression vectors [START_REF] Naldini | In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[END_REF] with subsequent modifications [START_REF] Maskos | Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors[END_REF]. The IRES element (Internal Ribosome Entry Site) enables the coordinated expression of two genes within the same vector, allowing the monitoring of the delivery of the gene of interest using eGFP visualization, as we previously validated for the β2-IRES2-eGFP construct [START_REF] Avale | Interplay of beta2* nicotinic receptors and dopamine pathways in the control of spontaneous locomotion[END_REF][START_REF] Maskos | Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors[END_REF]) that we have modified to create the α5WT-IRES2-eGFP and α5D397N-IRES2-eGFP vectors [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. Bicistronic expression of mouse WT or SNP 5 cDNAs and the eGFP cDNA were under the control of the mouse phosphoglycerate kinase (PGK) promoter. Viral particles were generated by co-transfection of HEK-293T cells by the vector plasmid, a packaging plasmid and an envelope plasmid using Lipofectamine Plus (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

At 2 days after transfection, viral particles were harvested in the supernatant, treated with DNaseI and MgCl 2 , filtered through 0.45 mm pores, concentrated by ultracentrifugation and re-suspended 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 HIV-1 p24 antigen immunoassay (ZeptoMetrix Corporation, Buffalo, NY, USA) according to the manufacturer's instructions.

Stereotaxic procedure

Mice were anesthetized using ketamine/xylazine in PBS. Each mouse was introduced into a stereotaxic frame adapted for use with mice. Lentivectors were injected bilaterally at: VTA: antero-posterior 3.4 mm, lateral ± 0.5 mm from Bregma and 4.4 mm from the surface; dorsal hippocampus: antero-posterior -2.0, lateral ± 1.5 mm from Bregma and 1.5 mm from the surface.

Immunostaining

To analyze eGFP transduction in brain cells, sections from GFP, α5WT and α5SNP vectorized mice were stained for GFP (Life Technologies SAS, France) and GFAP antibody (Sigma-Aldrich, St. Louis, Missouri) for astrocyte visualization. Fluorescence immunohistochemistry was performed as follows: free-floating VTA and hippocampus brain sections were incubated 1 h at 4 °C in a fixative solution of PBS containing 10% normal goat serum (Sigma, Lyon, France) and 0.2% Triton X-100, and then overnight at 4 °C in PBS containing a rabbit anti-GFP at 1:2000 dilution or mouse anti-GFAP at 1:200 dilution, 2% normal goat serum and 0.2% Triton X-100.

The next day, sections were rinsed with PBS and then incubated 3 h at room temperature with secondary antibody (AlexaFluor 488-conjugated anti-rabbit, Dylight 594-conjugated anti-mouse, Jackson Immunoresearch, London, UK) at 1:200 dilution in a solution of 2% normal goat serum in PBS. After three rinses in PBS, slices were wet-mounted using Mowiol 4-88 (Calbiochem Corporation, La Jolla, CA, USA). NeuN (Millipore) staining was also tested but not clear-cut enough for reliable utilization in VTA and hippocampus (data not shown).

In vivo electrophysiology

Extracellular recordings of 5-HT neurons in the Dorsal Raphe Nucleus (DRN) of anesthetized mice were performed in vivo under basal conditions and after i.p. administration of nicotine (0.5 mg/kg). Mice were anaesthetized with chloral hydrate (400 mg/kg i.p.) and placed into a stereotaxic frame. Additional anesthesia (50-100 mg/kg; i.p.) was given as necessary to maintain a full anesthetic state, characterized by the absence of response to a nociceptive tail pinch. Body temperature was maintained at 37°C throughout the experiments using a thermistor-controlled heating pad (Astro-Med, Elancourt, France). The recordings were performed as described in [START_REF] Guiard | Functional interactions between dopamine, serotonin and norepinephrine neurons: an in-vivo electrophysiological study in rats with monoaminergic lesions[END_REF]) using single glass micropipettes (R&D Scientific glass, Spencerville, MD, USA) that were preloaded with 2M NaCl solution. These pipettes were pulled on a pipette puller (Narishige, Japan) with impedances ranging from 2.5 to 5 mV. They were positioned 0.2-0.5 mm posterior to the interaural line on the midline and lowered using a hydraulic micropositioner (Kopf Instruments) into the DRN, usually attained at a depth of 2.5-3.5 mm from the brain surface. To increase the signal-to-noise ratio, we used a current amplifier (BAK Electronics, Mount Airy, MD, USA) connected to the active filter Humbug (Quest scientific, DIPSI, Châtillon, France). The presumed DRN 5-HT neurons were then identified according to the criteria of [START_REF] Aghajanian | Intracellular identification of central noradrenergic and serotonergic neurons by a new double labeling procedure[END_REF], [START_REF] Aghajanian | Intracellular identification of central noradrenergic and serotonergic neurons by a new double labeling procedure[END_REF]) that is, a slow (0.5-2.5 Hz) and regular firing rate and long-duration, positive action potential (2-5 ms) bi-or triphasic extracellular waveform. Neuronal activity was recorded in real time using Spike2 software (Cambridge Electronic Design, Cambridge, UK), which was also used to analyze neurons offline.

In each mouse, all presumed 5-HT neurons were recorded for 2 min to determine the means ± SEM of 1) firing rate and 2) number of DRN 5-HT neurons per track in WT and α5 -/-mice. The last neuron was tested to determine the change in firing rate in response to the intraperitoneal administration of nicotine on DRN 5-HT neuronal activity for 1 min after approximately 10 min of stabilization.

Microdialysis 5-HT microdialysis. Concentric dialysis probes were made of cuprophan fibers (active length of 1.0 mm, outer diameter 0.30 mm). Mice were anaesthetized with chloral hydrate (40mg/kg, i.p.), placed on a stereotaxic frame and implanted with a probe bilaterally into the dorsal hippocampus (Coordinates from Bregma (in mm): anterior = +1.6, lateral = ±1.3, ventral = -1.6). Mice were allowed to recover from surgery overnight. On the next day, 20 hr after the surgery, the probe was continuously perfused with an artificial CSF (composition in mmol/L: NaCl 147, KCl 3.5, CaCl2 1.26, MgCl2 1.2, NaH2PO4 1.0, pH 7.4 ± 0.2) and citalopram 1µm (Sigma) to increase and make detectable the basal extracellular levels of 5-HT as described in [START_REF] Guilloux | Blockade of 5-HT1A receptors by (+/-)-pindolol potentiates cortical 5-HT outflow, but not antidepressant-like activity of paroxetine: microdialysis and behavioural approaches in 5-HT1A receptor knockout mice[END_REF], at a flow rate of 1.5 μL/min using CMA/100 pump (Carnegie Medicine, Stockholm, Sweden). Dialysates were collected at 15 min intervals. Measurements of serotonin content were made using a High Performance Liquid Chromatography (HPLC) system (Ultremex 3u C18, 75 X 4.60 mm, particle size 3 μm, Phenomenex, Torrance, CA) coupled to an amperometric detector (VT03; Antec Leyden, The Netherlands). After 2 hours of balancing perfusion, 4 samples were collected to determine basal values over 60 min. Then, animals received an i.p. systemic administration of 0.9% NaCl and 4 samples were collected before i.p. systemic administration of 0.5 mg/kg of nicotine. Responses to nicotine administration were determined over a 120 -min period (8 samples). At the end of the day, mice were killed by an overdose of chloral hydrate. Brains were removed and stored at +4°C in 2% formalin for 1 week. Brains were then cut using a Kryomat apparatus that maintained them at -25°C to provide 40-μm sections. Each section was photographed using a digital camera, and the implantation site was identified according to the mouse brain atlas. Only mice with probe confined in the dorsal hippocampus were used for subsequent data analysis DA microdialysis. Mice were anesthetized with isofluorane and placed in a flat skull position in a Kopf stereotaxic apparatus fitted with a mouse adaptor. A guide cannula (CMA 7, CMA Microdialysis, Stockholm, Sweden) was inserted into the right nucleus accumbens (NAcb) (Coordinates from Bregma (in mm): anterior = +1.5, lateral = -0.5, ventral = -5.2). The guide cannulae were permanently secured with epoxy glue. After surgery, the mice were placed in a Plexiglas cage and allowed to recover overnight. Twenty-four hours after surgery, the awake animal was gently restrained by the experimenter and a microdialysis probe (CMA 7/2, 2 mm dialysis membrane, CMA Microdialysis, Stockholm, Sweden) was inserted into the guide cannula.

The probe was connected to a microinjection pump (CMA/100, CMA Microdialysis, Stockholm, Sweden) and perfused with artificial cerebrospinal fluid (aCSF: KCl 2.5 mM, NaCl 125 mM, CaCl 2 1.26 mM, MgCl 2 1.18 mM, Na 2 HPO 4 2 mM, pH 7.4) at a flow rate of 1.1 μl/min. Starting 1 h after implantation, dialysate samples were collected every 20 min into glass vials containing 5µl of antioxidant solution (EDTA 0.27mM, l-cysteine 3.3mM, ascorbic acid 0.5mM, acetic acid 0.1M) (27 μl/20 min sample). Three hours after probe insertion animals received an injection of nicotine (0.1-1 mg/kg, i.p., free base) or saline. 6 samples were collected for basal values over 120 min. Responses to i.p. injection of nicotine (0.1-1 mg/kg) or saline were monitored for 120 min (6 samples). For dopamine detection, a 20 μl volume from each sample was injected into an Agilent 6410 triple quadrupole-mass spectrometer with an electrospray ion source operated in positive mode for detection of DA concentrations as previously described [START_REF] Cannazza | Simultaneous measurement of adenosine, dopamine, acetylcholine and 5hydroxytryptamine in cerebral mice microdialysis samples by LC-ESI-MS/MS[END_REF].

Discovery HS-F5 column (150 mm × 2.1 mm, 3 µm) (Sigma-Aldrich, Milan, Italy) was used for chromatographic separations. The HPLC analyses were carried out using an elution profile composed of a first isocratic step water (formic acid 0.1%): acetonitrile (ACN) 95:5 for 1.5 min and then to 30% of ACN over 6 min to separate DA from other analytes at 25 • C. The column was washed with 90% of ACN for 2.5 min followed by the equilibration of the column for 5 min with 5% ACN. The flow rate was 0.3 ml/min. The chromatograms were integrated using Agilent Mass Hunter software. DA detection limit was 0.05 nM. At the end of each experiment, the animals were killed by rapid decapitation, brains were removed from the skull and the positions of the microdialysis probes were verified by visual inspection of the fresh tissue or the frozen brain during cut at the cryostat. The probe track was visible as a small hemorrhagic line. Data from brains with large hemorrhages (more than 1 mm in diameter) were discarded.

The implantation site was identified according to the mouse brain atlas [START_REF] Paxinos | The Mouse Brain in Stereotaxic Coordinates[END_REF].

Statistical analysis

Data were analyzed with Statistica (StatSoft, Inc., France).

Differences in behavioural and electrophysiological phenotypes between WT and α5 -/-mice were analyzed with unpaired Student's t-tests. Two-way ANOVAs were used to analyze the data for novelty preference (genotype as between-and environment as within-subject factors).

Microdialysis data were analyzed with one or two-way repeated measures ANOVAs, (genotype and nicotine dose as between-and time as within-subject factors); paired and unpaired Student's ttests were used for genotype or drug treatment comparisons.

For comparison between GFP, α5WT and α5SNP vectorized groups, data were analyzed using one-way ANOVAs (group as between-subject factor).

Significant main effects (p<0.05) were further analyzed using LSD Fisher post-hoc tests.

Results

(Post-hoc multiple comparisons are reported in Figure legends).

Responses to novelty and anxiety-related behaviours in α5 -/-mice

We first assessed whether the lack of the α5 subunit would influence the manifestation of behavioural phenotypes strongly associated with drug-, notably nicotine-addiction in humans [START_REF] Batra | Multidimensional smoker profiles and their prediction of smoking following a pharmacobehavioural intervention[END_REF][START_REF] Kreek | Genetic influences on impulsivity, risk taking, stress responsivity and vulnerability to drug abuse and addiction[END_REF][START_REF] Kushner | Greater elevation in risk for nicotine dependence per pack of cigarettes smoked among those with an anxiety disorder[END_REF][START_REF] Salin-Pascual | Nicotine dependence and psychiatric disorders[END_REF], and identified as predisposing factors for drug abuse in clinical [START_REF] Batra | Multidimensional smoker profiles and their prediction of smoking following a pharmacobehavioural intervention[END_REF][START_REF] Kushner | Greater elevation in risk for nicotine dependence per pack of cigarettes smoked among those with an anxiety disorder[END_REF][START_REF] Martin | Sensation seeking and symptoms of disruptive disorder: association with nicotine, alcohol, and marijuana use in early and mid-adolescence[END_REF][START_REF] Martin | Sensation seeking, puberty, and nicotine, alcohol, and marijuana use in adolescence[END_REF][START_REF] Salin-Pascual | Nicotine dependence and psychiatric disorders[END_REF][START_REF] Sargent | Using sensation seeking to target adolescents for substance use interventions[END_REF]) and preclinical studies [START_REF] Abreu-Villaca | Individual differences in novelty-seeking behaviour but not in anxiety response to a new environment can predict nicotine consumption in adolescent C57BL/6 mice[END_REF][START_REF] Falco | Individual differences in the behavioural effects of nicotine: A review of the preclinical animal literature[END_REF][START_REF] Falco | Anxiety status affects nicotine-and baclofen-induced locomotor activity, anxiety, and single-trial conditioned place preference in male adolescent rats[END_REF][START_REF] Redolat | Individual differences in noveltyseeking and behavioural responses to nicotine: a review of animal studies[END_REF][START_REF] Suto | Locomotor response to novelty predicts a rat's propensity to self-administer nicotine[END_REF][START_REF] Wang | Propensity for social interaction predicts nicotine-reinforced behaviours in outbred rats[END_REF]. We addressed, in WT and α5 -/-mice (n=10-12/group), sensation seeking modeled as high novelty-induced exploratory behaviour / locomotor reactivity in novel inescapable environments [START_REF] Belin | In search of predictive endophenotypes in addiction: insights from preclinical research[END_REF][START_REF] Belin | Responses to novelty and vulnerability to cocaine addiction: contribution of a multi-symptomatic animal model[END_REF], shown to predispose to higher nicotine self-administration [START_REF] Redolat | Individual differences in noveltyseeking and behavioural responses to nicotine: a review of animal studies[END_REF][START_REF] Suto | Locomotor response to novelty predicts a rat's propensity to self-administer nicotine[END_REF]; novelty seeking modeled as high novelty preference in a free-choice procedure, that is proposed to reflect propensity for risky activity and predicts drug-, including nicotine-abuse [START_REF] Abreu-Villaca | Individual differences in novelty-seeking behaviour but not in anxiety response to a new environment can predict nicotine consumption in adolescent C57BL/6 mice[END_REF][START_REF] Batra | Multidimensional smoker profiles and their prediction of smoking following a pharmacobehavioural intervention[END_REF][START_REF] Belin | Responses to novelty and vulnerability to cocaine addiction: contribution of a multi-symptomatic animal model[END_REF][START_REF] Redolat | Individual differences in noveltyseeking and behavioural responses to nicotine: a review of animal studies[END_REF]; and high anxiety that predicts transition to tobacco addiction and responsiveness to nicotine reward [START_REF] Falco | Anxiety status affects nicotine-and baclofen-induced locomotor activity, anxiety, and single-trial conditioned place preference in male adolescent rats[END_REF][START_REF] Kushner | Greater elevation in risk for nicotine dependence per pack of cigarettes smoked among those with an anxiety disorder[END_REF][START_REF] Wang | Propensity for social interaction predicts nicotine-reinforced behaviours in outbred rats[END_REF][START_REF] Wong | Anxiety sensitivity as a predictor of acute subjective effects of smoking[END_REF].

We found that α5 -/-mice did not show increased locomotor reactivity to novelty compared to WT mice, with no differences observed between groups in the total distance travelled in a new inescapable open-field, or in other open-field parameters (Figure 1a). α5 -/-mice did not show increased novelty preference in a free choice situation, both WT and α5 -/-mice spending more time within the novel environment than the familiar one [F(1,22)=13.21, p=0.001], with no differences in their % of novelty preference (Figure 1b). α5 -/-mice displayed less rearing behaviour in a novel environment compared to WT mice (p=0.002) (Figure 1c). Moreover, α5 -/-mice showed high levels of anxiety in the EPM, with less time spent in the open arms (p=0.016) and their extremities (p=0.024) compared to WT mice, with no differences in total distance travelled (Figure 1d). High anxiety-like phenotype was also observed in the LDB, with less time spent in the light compartment for α5 -/-mice compared to WT (p=0.0001) (Figure 1e). Thus, α5 -/-mice did not show enhanced responses to novelty. However, they displayed deficits in rearing behaviour and high anxiety-like behaviour.

Effects of nicotine on α5 -/-mice behavioural phenotypes

Several factors have been proposed to explain nicotine abuse, with emphasis on a self-medication hypothesis for cognitive and mood disorders [START_REF] Evans | Nicotine self-medication of cognitive-attentional processing[END_REF][START_REF] Gehricke | Smoking to self-medicate attentional and emotional dysfunctions[END_REF][START_REF] Picciotto | Molecules and circuits involved in nicotine addiction: The many faces of smoking[END_REF]. We and others previously demonstrated that α5 -/-mice exhibit decreased sensitivity to nicotine, including decreased nicotine-elicited DAergic activation, reward, aversion and reward inhibition at high doses [START_REF] Fowler | Habenular alpha5 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]. We next tested whether α5 -/-behavioural impairments may be alleviated by exposure to high doses of nicotine. We found that nicotine injections caused opposite effects in WT and α5 -/-mice (n=8-16/group) on rearing [drug: F(4,75)=7.617, p<0.0001, drug x group: F(4,75)=8.730,p<0.001] (Figure 2). This was also the case on anxiety levels, in the EPM [% time spent in open arms : group: F(1,90)=25,97, p=0.0009; drug: F(4,90)=7.064, p=0.002, drug x group: F(4,90)=9.635,p=0.0002] [[% time spent in extremities: group: F(1,90)=28,26, p=0.0005; drug: F(4,90)=4.809, p=0.023, drug x group: F(4,90)=7.742,p=0.001] and in the LDB [% time spent in light side: group: F(1,96)=4,72, p=0.032; drug x group: F(4,96)=6.186,p=0.0002]. Nicotine significantly decreased rearing in WT mice at the doses of 0.5 mg/kg (p=0.0007) and 1 mg/kg (p<0.0001), while restoring WT-like rearing levels in α5 -/-at the dose of 0.1 mg/kg (p=0.036). The numbers of rearing was significantly different between WT and α5 -/-mice after saline (p=0.014) and nicotine injection at the doses of 0.01 (p=0.008), 0.5 (p=0.010) and 1 (p=0004) mg/kg but similar at the dose of 0.1 mg/kg (Figure 2a). In the EPM, nicotine significantly decreased the % of time spent in the open arms in WT mice, at the doses of 0.5 mg/kg (p<0.0001) and 1 mg/kg (p<0.0001), an effect also seen for open arm extremities, at the doses of 0.5 mg/kg (p<0.0001) and 1 mg/kg (p<0.0001). The % of time spent in the open arms and in their extremities was significantly different between WT and α5 -/-mice after saline (p<0.0001) and nicotine injection at the doses of 0.01 (p=0.002 and p=0.003, respectively) and 0.1 (p=0.008 and p=0.0004, respectively) mg/kg but similar at the doses of 0.5 and 1 mg/kg (Figure 2b). In contrast, nicotine did not significantly affect anxiety in the EPM in α5 -/-mice (Figure 2b). In the LDB, nicotine decreased the % of time spent in the light side for WT mice, at the doses of 0.5 mg/kg (p=0.014) and 1 mg/kg (p=0.001), while it significantly increased this parameter in α5 -/-mice at the dose of 0.5 mg/kg (p=0.03). The % of time spent in the light side was significantly different between WT and α5 -/-mice after saline (p=0.001) and nicotine injection at the dose of 0.01 mg/kg (p=0.0002) but similar at the doses of 0.1, 0.5 and 1 mg/kg (Figure 2c). The doses of nicotine effective in rescuing rearing and LDB anxiety in α5 -/-mice did not affect motor features such as total distance moved in the EPM (Figure 3a) or open-field locomotor activity (Figure 3b).

NAcb DA release in response to nicotine in α5 -/-mice nAChRs are key regulators of mesolimbic DA neurons [START_REF] Faure | Role of nicotinic acetylcholine receptors in regulating dopamine neuron activity[END_REF]. We previously showed that anesthetized α5 -/-mice display decreased sensitivity to nicotine-induced VTA DA neuron response [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]). To complete these data, we measured nicotine-elicited changes in DA perfusate levels in the NAcb, the main target of DA input from the VTA, from freely moving α5 -/-and WT mice (n=4-6/group) (Figure 4a, left). No differences were found in mean baseline DA concentrations between WT (mean±SEM: 0.57±0.09 nM) and α5 -/-mice (0.73±0.16 nM). Two-way repeated measures ANOVAs on DA outflow over time showed significant genotype effect [F(1,22)=6.791, p=0.002] and nicotine dose effect [F(3,22)=5.434, p=0.006] (Figure 4a, middle). Furthermore, DA perfusate levels during the first hour post-injection were significantly higher in response to nicotine at the doses of 0.2 mg/kg (p=0.025), 0.4 mg/kg (p=0.04) and 1 mg/kg (p=0.011) compared to saline in WT mice (Figure 4a, right). In α5 -/-mice, DA perfusate levels during the first hour post-injection were significantly higher in response to nicotine compared to saline only at the dose of 1 mg/kg (p=0.021).

DRN 5-HT neuron activity in response to nicotine in α5 -/-mice

Besides DA, 5-HT plays an important role in drug abuse, including in nicotine dependence [START_REF] Fletcher | Serotonin receptors as potential targets for modulation of nicotine use and dependence[END_REF][START_REF] Kirby | Contributions of serotonin in addiction vulnerability[END_REF][START_REF] Muller | The role of serotonin in drug use and addiction[END_REF]. The involvement of 5-HT neurotransmission in nicotine action on the brain is firmly established, although the underlying mechanisms are much less elucidated [START_REF] Hernandez-Lopez | Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus[END_REF]. Nicotine influences 5-HT activity, notably in the DRN that contains 5-HT neurons, and in the dorsal hippocampus, that contains 5-HT terminals [START_REF] File | The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety[END_REF][START_REF] Hernandez-Lopez | Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus[END_REF]). Yet, the contribution of α5*-nAChRs to 5-HT neurotransmission has not been assessed so far to our knowledge. We examined whether α5 -/-mice may show alterations in 5-HT neurotransmission, notably in response to nicotine, and performed extracellular recordings of DRN 5-HT neurons (Figure 4b). The mean firing frequency of 5-HT neurons was similar in WT and α5 -/-mice (n=5/group) (Figure 4b, left).

However, the mean number of spontaneously active 5-HT neurons recorded per tract in α5 -/-mice was twice as high as in WT mice (p=0.001) (n=11-17 cells/group) (Figure 4b, middle). Strikingly, nicotine at the dose of 0.5 mg/kg caused differential effects on DRN 5-HT neuronal activity. In WT mice, nicotine inhibited 5-HT neuron firing rate, while in α5 -/-mice, nicotine increased 5-HT neuron firing rate (% change from baseline, WT vs. α5 -/-mice: p=0.0016) (Figure 5b, right).

Hippocampus 5-HT extracellular levels in response to nicotine in α5 -/-mice

We next measured nicotine-induced changes in extracellular 5-HT levels in the dorsal hippocampus, one of the discrete brain regions where 5-HT levels are affected in response to nicotine [START_REF] Muller | The role of serotonin in drug use and addiction[END_REF] and where α5*-nAChRs are significantly expressed (Gensat, http://www.gensat.org/GeneProgressTracker.jsp?gensatGeneID=675) [START_REF] Broide | Increased sensitivity to nicotine-induced seizures in mice expressing the L250T alpha 7 nicotinic acetylcholine receptor mutation[END_REF][START_REF] Hsu | Medial habenula output circuit mediated by alpha5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus[END_REF][START_REF] Salas | The nicotinic acetylcholine receptor subunit alpha 5 mediates short-term effects of nicotine in vivo[END_REF][START_REF] Sudweeks | Functional and molecular characterization of neuronal nicotinic ACh receptors in rat CA1 hippocampal neurons[END_REF][START_REF] Wada | The distribution of mRNA encoded by a new member of the neuronal nicotinic acetylcholine receptor gene family (alpha 5) in the rat central nervous system[END_REF], in freely moving α5 -/- and WT mice under citalopram perfusion (n=9-10/group) (Figure 4c). No differences were found in mean baseline 5-HT concentrations between WT (mean±SEM: 0.26±0.02 nM) and α5 -/-mice (0.29±0.03 nM). Repeated measures ANOVAs on 5-HT outflow over time showed significant time effect [F(11,264)=6.380, p˂0.0001] and time x genotype interaction [F(11,264)=4.780, p˂0.0001].

Post-hoc analysis showed that nicotine increased hippocampal 5-HT outflow during the first time sample post-injection in α5 -/-mice (p˂0.0001) with no effect on WT mice (Figure 4c, middle).

Analyzing the AUC values during the first hour post-injection further showed that 5-HT outflow was significantly higher after nicotine injection compared to saline in α5 -/-mice (p=0.005) but not in WT mice (Figure 4c, right).

Effects of α5WT or α5SNP re-expression in the VTA on α5 -/-mice behavioural phenotypes

Since we have highlighted altered mesolimbic DA activity in response to nicotine in α5 -/-mice [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]) (and present data, see above), and we also previously showed that nAChRs crucially contribute to VTA-dependent exploratory behaviour [START_REF] Avale | Interplay of beta2* nicotinic receptors and dopamine pathways in the control of spontaneous locomotion[END_REF][START_REF] Besson | Long-term effects of chronic nicotine exposure on brain nicotinic receptors[END_REF][START_REF] Faure | Role of nicotinic acetylcholine receptors in regulating dopamine neuron activity[END_REF][START_REF] Maskos | Nicotine reinforcement and cognition restored by targeted expression of nicotinic receptors[END_REF], we next investigated the roles of VTA α5WT and α5SNP in α5 -/-behavioural phenotypes. For this, we used LV to transduce eGFP alone (WT GFP and α5 -/-GFP mice), bi-cistronic α5WT and eGFP (WT α5WT and α5 -/-α5WT mice), or bi-cistronic α5SNP and eGFP (WT α5SNP and α5 -/-α5SNP mice) selectively in the VTA as previously described in [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]) (n=12-20/group) (Figure 5a). We detected eGFP expression in the whole VTA including in astrocytes (Figure 5a). VTA re-expression of α5WT, but not of α5SNP, restored WTlike rearing levels in α5 -/-mice [group: F(3,59)=7.88, p=0.0002] (Figure 5b). In contrast, anxiety

Effects of α5WT or α5SNP re-expression in the hippocampus on α5 -/-mice behavioural phenotypes

We have observed altered 5-HT activity in the dorsal hippocampus in α5 -/-mice (see above). The hippocampus, notably its dorsal part, has been particularly implicated in the action of nicotine on anxiety [START_REF] Cheeta | Hippocampal and septal injections of nicotine and 8-OH-DPAT distinguish among different animal tests of anxiety[END_REF][START_REF] Engin | The effects of intra-cerebral drug infusions on animals' unconditioned fear reactions: a systematic review[END_REF][START_REF] File | The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety[END_REF][START_REF] File | Bimodal modulation by nicotine of anxiety in the social interaction test: role of the dorsal hippocampus[END_REF][START_REF] Piri | The effects of nicotine on nitric oxide induced anxiogenic-like behaviours in the dorsal hippocampus[END_REF][START_REF] Seth | Nicotinic--serotonergic interactions in brain and behaviour[END_REF]. Also, this brain region expresses significant levels α5*-nAChRs, presumably more than the other areas that have mainly been involved in the action of nicotine on anxiety such as the amygdala or the septum, according to the few studies reporting α5*-nAChR brain distribution in mice (Gensat, http://www.gensat.org/index.html) [START_REF] Broide | Increased sensitivity to nicotine-induced seizures in mice expressing the L250T alpha 7 nicotinic acetylcholine receptor mutation[END_REF][START_REF] Hsu | Medial habenula output circuit mediated by alpha5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus[END_REF][START_REF] Salas | The nicotinic acetylcholine receptor subunit alpha 5 mediates short-term effects of nicotine in vivo[END_REF][START_REF] Sudweeks | Functional and molecular characterization of neuronal nicotinic ACh receptors in rat CA1 hippocampal neurons[END_REF][START_REF] Wada | The distribution of mRNA encoded by a new member of the neuronal nicotinic acetylcholine receptor gene family (alpha 5) in the rat central nervous system[END_REF]). We next investigated the roles of hippocampal α5WT and α5SNP in the anxiety-like phenotype observed in α5 -/-mice. In mice transduced with bi-cistronic LV in the hippocampus, we detected eGFP mostly in the CA1 area, dentate gyrus, including in astrocytes (n=11-20/group) (Figure 6a). Hippocampal α5*-nAChR re-expression, contrary to VTA re-expression, did not rescue rearing behaviour in α5 -/- mice [group: F(3,46)=5.94, p=0.002] (Figures 6b). 6d). By contrast, expressing the α5SNP did not alleviate anxiety in the EPM (Figure 6c) or the LDB (Figure 6d).

Discussion

We demonstrated that α5 -/-mice display deficits in rearing behaviour and enhanced anxiety. These two phenotypes could be rescued by exposure to nicotine while nicotine caused α5 -/--like impairments in WT mice. This was not related to nicotine-elicited general locomotor modifications. We also observed that higher doses of nicotine are required to increase DA release in the NAcb in α5 -/-mice. Furthermore, we identified for the first time altered 5-HT activity in α5 -/- mice, with an increased number of spontaneously active 5-HT neurons in the DRN and nicotineelicited increase in 5-HT release in the dorsal hippocampus at a dose ineffective on this parameter in WT mice. Finally, re-expressing the α5WT gene in the VTA and the hippocampus of α5 -/-mice restored WT levels of rearing behaviour and anxiety, respectively, while re-expressing the α5SNP resulted in knockout-like phenotypes on both parameters.

Alterations in α5*-nAChRs and addiction vulnerability behavioural phenotypes

Only a proportion of individuals exposed to drugs develop addiction, as this pathology requires the interaction between polygenic and external factors [START_REF] Belin | In search of predictive endophenotypes in addiction: insights from preclinical research[END_REF][START_REF] Degenhardt | Toward a global view of alcohol, tobacco, cannabis, and cocaine use: findings from the WHO World Mental Health Surveys[END_REF]). The α5 gene constitutes a very interesting candidate for studying vulnerability to tobacco addiction, and potentially for novel treatment strategies, since the α5SNP [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF][START_REF] Frahm | Aversion to nicotine is regulated by the balanced activity of beta4 and alpha5 nicotinic receptor subunits in the medial habenula[END_REF][START_REF] Kuryatov | Acetylcholine receptor (AChR) alpha5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (alpha4beta2)(2)alpha5 AChR function[END_REF]) is frequent in Caucasians [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF]) and consistently associated with heavy smoking [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF][START_REF] Improgo | The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: dual role in nicotine addiction and lung cancer[END_REF][START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF][START_REF] Ware | From men to mice: CHRNA5/CHRNA3, smoking behaviour and disease[END_REF]. We previously showed that deletion of the α5 gene and expression of the α5SNP in the VTA of α5 -/-mice results in decreased sensitivity to nicotine reward in a self-administration procedure [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. It may appear counterintuitive that subjects with impaired α5*-nAChR function would be less sensitive to nicotine reward and yet show higher risk for nicotine addiction. We postulated that there may be additional mechanisms underlying the impact of α5*-nAChR dysfunction on the risk for heavy smoking. Specific behavioural endophenotypes of presumed genetic origin (e.g. trait anxiety, high response to novelty and impulsivity) were shown to contribute to drug addiction according to different modalities [START_REF] Belin | In search of predictive endophenotypes in addiction: insights from preclinical research[END_REF].

High responses to novelty were shown to predispose to greater propensity to acquire drug, including nicotine, self-administration and to loss of control over drug use [START_REF] Abreu-Villaca | Individual differences in novelty-seeking behaviour but not in anxiety response to a new environment can predict nicotine consumption in adolescent C57BL/6 mice[END_REF][START_REF] Batra | Multidimensional smoker profiles and their prediction of smoking following a pharmacobehavioural intervention[END_REF][START_REF] Belin | In search of predictive endophenotypes in addiction: insights from preclinical research[END_REF][START_REF] Belin | Responses to novelty and vulnerability to cocaine addiction: contribution of a multi-symptomatic animal model[END_REF][START_REF] Redolat | Individual differences in noveltyseeking and behavioural responses to nicotine: a review of animal studies[END_REF][START_REF] Suto | Locomotor response to novelty predicts a rat's propensity to self-administer nicotine[END_REF]. Also high impulsivity impacts both processes [START_REF] Belin | In search of predictive endophenotypes in addiction: insights from preclinical research[END_REF][START_REF] Diergaarde | Impulsive choice and impulsive action predict vulnerability to distinct stages of nicotine seeking in rats[END_REF]). Here, we did not observe enhanced response to novelty in α5 -/-mice and a previous study showed no impulsive phenotype in these mice in a 5-choice serial reaction time task [START_REF] Bailey | The nicotinic acetylcholine receptor alpha5 subunit plays a key role in attention circuitry and accuracy[END_REF], suggesting that α5*-nAChRs do not impact these processes. But mood and cognitive disorders appear to confer vulnerability to nicotine abuse according to dissociable processes more related to "coping" or self-medication strategy, to regulate affective or cognitive symptoms [START_REF] Gehricke | Smoking to self-medicate attentional and emotional dysfunctions[END_REF]. This is particularly well documented for high anxiety [START_REF] Baker | Motivational influences on cigarette smoking[END_REF][START_REF] Kushner | Greater elevation in risk for nicotine dependence per pack of cigarettes smoked among those with an anxiety disorder[END_REF][START_REF] Picciotto | Effect of nicotine and nicotinic receptors on anxiety and depression[END_REF][START_REF] Salin-Pascual | Nicotine dependence and psychiatric disorders[END_REF][START_REF] Sonntag | Are social fears and DSM-IV social anxiety disorder associated with smoking and nicotine dependence in adolescents and young adults?[END_REF][START_REF] Wong | Anxiety sensitivity as a predictor of acute subjective effects of smoking[END_REF]). Here we demonstrate that both α5 gene deletion and targeted α5SNP expression lead to decreased rearing and increased anxiety-like behaviour. Rearing has been proposed to have a major exploratory information-gathering function [START_REF] Lever | Rearing on hind legs, environmental novelty, and the hippocampal formation[END_REF]. It can also reflect other factors such as fear and anxiety, suggesting that the decreased rearing and increased anxiety we observed in α5 -/-mice may be associated phenotypes, although our α5*-nAChR targeted reexpression experiments showed that rescuing one of these phenotypes did not affect the other.

Exploratory behaviour reflects many neuropsychological and genetic correlates. Fine exploratory activity such as rearing can be dissociated from general ambulatory activity, with nAChRs independently modulating these behaviours [START_REF] Avale | Interplay of beta2* nicotinic receptors and dopamine pathways in the control of spontaneous locomotion[END_REF][START_REF] Somm | Concomitant alpha7 and beta2 nicotinic AChR subunit deficiency leads to impaired energy homeostasis and increased physical activity in mice[END_REF]. α5 impairmentrelated deficits in rearing, which we found distinct from general locomotor dysfunction, indicate abnormal response to novel environment features that might be associated with attentional or motivational impairments. Interestingly, α5 -/-mice also display deficits in attention under highly demanding conditions [START_REF] Bailey | The nicotinic acetylcholine receptor alpha5 subunit plays a key role in attention circuitry and accuracy[END_REF].

Studies supporting the self-medication hypothesis show that nicotine is particularly effective in reducing depression, anxiety and in improving attention in psychiatric sub-populations [START_REF] Gehricke | Smoking to self-medicate attentional and emotional dysfunctions[END_REF][START_REF] Picciotto | Effect of nicotine and nicotinic receptors on anxiety and depression[END_REF][START_REF] Picciotto | Molecules and circuits involved in nicotine addiction: The many faces of smoking[END_REF]. Consistently, here, in α5 -/-mice, nicotine alleviates impairments in both rearing and anxiety, at doses that have opposite effects in control mice. Interestingly, anxiety sensitivity has been shown to predict greater positive affect in response to acute nicotine in smokers [START_REF] Wong | Anxiety sensitivity as a predictor of acute subjective effects of smoking[END_REF]. While anxiogenic nicotine action in control mice was observed in the LDB and the EPM, nicotine rescued WT anxiety levels in α5 -/- mice in the LDB with no significant effects in the EPM. Both tests are based on approachavoidance conflict, yet differences in the external input such as elevation vs. bright illumination may cause different subjective experiences with different sensitivity to pharmacological manipulation. As regards nicotine rescue of rearing behaviour in α5 -/-mice, cholinergic neurotransmission is a key regulator of exploratory behaviour through DAergic modulation [START_REF] Faure | Role of nicotinic acetylcholine receptors in regulating dopamine neuron activity[END_REF]. Accordingly, α5*-nAChRs critically contribute to nicotine-elicited activation of the mesolimbic system, with higher doses of nicotine required to enhance NAcb DA release in α5 -/- mice, similar to VTA neuron activity [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF].

Identification of the VTA and the hippocampus as key areas for α5 -/-mice behavioural phenotypes

Our LV-based targeted α5 re-expression strategy identified that VTA, but not hippocampal, α5*-nAChRs regulate rearing behaviour. Mice knockout for the β2 subunit show high ambulation and low fine exploration [START_REF] Besson | Long-term effects of chronic nicotine exposure on brain nicotinic receptors[END_REF][START_REF] Granon | Executive and social behaviours under nicotinic receptor regulation[END_REF], the latter being rescued by VTA β2 re-expression [START_REF] Avale | Interplay of beta2* nicotinic receptors and dopamine pathways in the control of spontaneous locomotion[END_REF]. Interestingly, this unbalanced exploratory behaviour was associated with impairments in VTA DA activity, and rescued by nicotine exposure [START_REF] Besson | Long-term effects of chronic nicotine exposure on brain nicotinic receptors[END_REF]). In the VTA, α5 is associated with α4 and β2 subunits [START_REF] Millar | Diversity of vertebrate nicotinic acetylcholine receptors[END_REF]. Our present results suggest that α5 may be a key partner of VTA α4β2*-nAChR function in fine exploratory control. Further, α5*-nAChRs expressed in the hippocampus, but not in the VTA, modulate anxiety. The hippocampus has been widely implicated in anxiety and nicotine effects on anxiety [START_REF] Cheeta | Hippocampal and septal injections of nicotine and 8-OH-DPAT distinguish among different animal tests of anxiety[END_REF][START_REF] Engin | The effects of intra-cerebral drug infusions on animals' unconditioned fear reactions: a systematic review[END_REF][START_REF] File | The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety[END_REF][START_REF] File | Bimodal modulation by nicotine of anxiety in the social interaction test: role of the dorsal hippocampus[END_REF][START_REF] Piri | The effects of nicotine on nitric oxide induced anxiogenic-like behaviours in the dorsal hippocampus[END_REF], although the role of the different nAChRs is not well established [START_REF] Picciotto | Effect of nicotine and nicotinic receptors on anxiety and depression[END_REF]). Here we observed partial rescue of the anxiety phenotype after hippocampal α5*-nAChR re-expression.

The contribution of α5*-nAChRs in the lateral septum, DRN or amygdala [START_REF] Picciotto | Effect of nicotine and nicotinic receptors on anxiety and depression[END_REF][START_REF] Seth | Nicotinic--serotonergic interactions in brain and behaviour[END_REF], might be required for fully balanced anxiety processing. With our LV and ubiquitous PGK promoter, transduction is not cell-type specific. The endogenous α5 expression in the hippocampus is not well documented. Two publications suggest that in the hippocampal CA1 region of C57BL/6J mice, nAChR expression is found on astrocytes and GABAergic interneurons (Gahring et al. 2004a;Gahring et al. 2004b). Hippocampal astrocytes were recently implicated in anxiolytic-like responses [START_REF] Quesseveur | BDNF overexpression in mouse hippocampal astrocytes promotes local neurogenesis and elicits anxiolytic-like activities[END_REF]). In the CA1 region, GABAergic interneurons are the main neuronal type expressing high affinity nicotinic receptors [START_REF] Sudweeks | Functional and molecular characterization of neuronal nicotinic ACh receptors in rat CA1 hippocampal neurons[END_REF]. An important population of these interneurons is also receptive to serotonergic modulation from the DRN [START_REF] Chittajallu | Dual origins of functionally distinct O-LM interneurons revealed by differential 5-HT(3A)R expression[END_REF]. Further investigations beyond the scope of the present work are required to identify the role of α5 expression on different cell types.

A key role for α5*-nAChRs in 5-HT neurotransmission

We reveal impairments in spontaneous and nicotine-elicited 5-HT activity in α5 -/-mice. DRN 5-HT neurons are either silent or spontaneously active with slow regular firing pattern [START_REF] Vandermaelen | Electrophysiological and pharmacological characterization of serotonergic dorsal raphe neurons recorded extracellularly and intracellularly in rat brain slices[END_REF]. The number of spontaneously active 5-HT neurons is enhanced in α5 -/-mice, indicating that the absence of α5 changes the excitability of 5-HT neurons.

DRN cholinergic input derives from laterodorsal tegmental and pedunculopontine nuclei and was proposed to modulate DRN 5-HT activity, through nAChRs located on noradrenergic and glutamatergic neurons arising from many areas [START_REF] Hernandez-Lopez | Nicotinic modulation of serotonergic activity in the dorsal raphe nucleus[END_REF][START_REF] Li | Presynaptic nicotinic receptors facilitate monoaminergic transmission[END_REF][START_REF] Soiza-Reilly | Glutamatergic drive of the dorsal raphe nucleus[END_REF]. DRN glutamate neurotransmission influences 5-HT tone through α4β2*-nAChRs [START_REF] Garduno | Presynaptic alpha4beta2 nicotinic acetylcholine receptors increase glutamate release and serotonin neuron excitability in the dorsal raphe nucleus[END_REF]. Our data indicate that α5 contributes to synaptic gating of α4β2*-nAChRs in preventing the transition from silent to active state of DRN 5-HT neurons. We also found that nicotine has differential effects on DRN 5-HT neuron firing and hippocampal 5-HT release between WT and α5 -/-mice. Alterations in the number of spontaneously active 5-HT neurons in α5 -/-mice might also contribute to the differential responses of these neurons in WT and α5 -/-mice [START_REF] File | The role of the dorsal hippocampal serotonergic and cholinergic systems in the modulation of anxiety[END_REF][START_REF] Seth | Nicotinic--serotonergic interactions in brain and behaviour[END_REF]. The highest levels of α5 expression in the brain are found by far in the interpeduncular nucleus (IPN) (Gensat, http://www.gensat.org/GeneProgressTracker.jsp?gensatGeneID=675) [START_REF] Hsu | Medial habenula output circuit mediated by alpha5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus[END_REF], mostly on GABA neurons [START_REF] Hsu | Medial habenula output circuit mediated by alpha5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus[END_REF]. In WT animals, nicotine may thus decrease DRN 5-HT neuron firing by activating, through α5*-nAChRs, IPN GABA inhibitory neurons projecting to the DRN [START_REF] Montone | Serotonergic and nonserotonergic projections from the rat interpeduncular nucleus to the septum, hippocampal formation and raphe: a combined immunocytochemical and fluorescent retrograde labelling study of neurons in the apical subnucleus[END_REF][START_REF] Dorocic | A whole-brain atlas of inputs to serotonergic neurons of the dorsal and median raphe nuclei[END_REF]. The lack of α5*-nAChRs may prevent IPNelicited inhibition of 5-HT DRN neurons, favoring an excitation of these neurons and an increase in 5-HT release in hippocampal 5-HT terminals in response to nicotine, as we observed in α5 -/- mice.

These results demonstrate that alterations in α5*-nAChRs result in vulnerability phenotypes, decreased rearing behaviour and enhanced anxiety, which depend at least partly upon the VTA and the hippocampus, respectively. Exposure to high doses of nicotine rescued these phenotypes in α5 - /-mice, suggesting that self-medication to reduce behavioural or affective disorders may be a critical factor for the initiation and maintenance of smoking in subjects with altered cholinergic signaling through α5*-nAChR dysfunction.

  -induced locomotor and exploratory activity. Locomotor reactivity to novelty (ambulation, i.e. horizontal activity) was measured as the total distance travelled in an unfamiliar 1-m-diameter circular open-field. The open-field was virtually divided into a central (65 cm) and a peripheral (35 cm) zone to further measure the instantaneous velocity divided into two categories: activity (A, > 8.75 cm/sec) and inactivity (I, < 8.75 cm/sec). Different states were thus observed: activity or inactivity in the central zone (CA or CI), activity or inactivity in the peripheral zone (PA or PI).

  All procedures were carried out in accordance with European Commission directives 219/1990 and 220/1990, and approved by Animalerie centrale and Medecine du travail, Institut Pasteur. The authors carrying out surgery hold an Animal Surgery Authorization from the French Ministry of Agriculture.

  levels were not affected by VTA α5*-nAChRs re-expression in α5 -/-mice as measured by the % of time spent in the EPM open arms [group: F(3,62)=3.14, p=0.032] and their extremities [group: F(3,62)=3.40, p=0.023] (Figure 5c) and in the LDB light side [group: F(3,63)=8.95, p<0.0001] (Figure 5d).

  However, hippocampal α5WT re-expression resulted in partial rescue of anxiety levels: α5 -/-α5WT mice displayed anxiety at intermediate levels between WT GFP and α5 -/-GFP , as measured by the % of time spent in the EPM open arms [group: F(3,59)=6.40, p=0.0008] and their extremities [group: F(3,59)=4.02, p=0.0011] (Figure 6c), as well as in the LDB light side [group: F(3,64)=3.85, p=0.013] (Figure

Figure 1 :

 1 Figure 1: Response to novelty and anxiety-like behaviour in α5 -/-mice. a, Novelty-induced locomotor activity expressed as total distance travelled in the openfield (left); % time spent at the periphery of the open-field, in inactivity, in inactivity in the periphery (PI) or center (CI). b, Novelty preference in a free-choice procedure expressed as time spent in familiar vs novel environment (left) and % of novelty preference (right). c, Novelty-induced rearing behaviour expressed as number of rearings. d, EPM anxiety-related behaviour measured as % of time spent in open arms (left) and extremities (right). e, LDB anxiety-related behaviour expressed as % of time spent in the light side. Data plotted as mean±SEM. Genotype effect: *p≤0.05, **p≤0.01, ***p≤0.001. Environment effect for novelty preference: ## p≤0.01. n=10-12/group.

Figure 2 :

 2 Figure 2: Effects of nicotine exposure on α5 -/-mice behavioural phenotypes. a, Noveltyinduced rearing behaviour in response to different doses of nicotine, expressed as number of rearings in WT (left) and α5 -/-mice (right). b, EPM anxiety-related behaviour expressed as % of time spent in open arms (upper) and in their extremities (bottom) in WT (left) and α5 -/-mice (right). c, LDB anxiety-related behaviour expressed as % of time spent in the light side in WT (left) and α5 -/-mice (right). Data plotted as mean±SEM. Nicotine effect: # p≤0.05, ### p≤0.001 compared to saline. n=8-16/group.

Figure 3 :

 3 Figure 3: Effects of nicotine exposure on locomotion in α5 -/-mice. a, Total distance moved in the EPM in WT (left) and α5 -/-mice (right). b, Total distance moved (upper); and % time spent at the periphery, in inactivity, in inactivity in the periphery (PI) or center (CI) in the open-field in WT(left) and α5 -/-mice (right). Data plotted as mean±SEM. Nicotine effect: # p≤0.05, ## p≤0.01, ### p≤0.001 as compared to saline.

Figure 4 :

 4 Figure 4: Effects of nicotine exposure on DA and 5-HT neurotransmission in WT and α5 -/- mice. a, Microdialysis of NAcb DA extracellular levels: Schematic representation of probe location (optimal position and range of accepted positions (dashed lines)) (left); Time course of DA extracellular levels as % of basal levels after nicotine injection (middle); Area under the curve values (AUC) as % of basal values for the amount of DA outflow during the 1st hour postinjection (right), n=4-6/group. b, DRN 5-HT neurons firing: Basal firing frequency (left), n=5/group; Number of spontaneous active cells per tract (middle), n=11-17/group; Changes in firing rate in response to nicotine as % of basal values, n=5/group. c, Microdialysis of dorsal hippocampus 5-HT extracellular levels: Schematic representation of probe location (optimal

This work was supported by the Institut Pasteur, Centre National de la Recherche Scientifique UMR 3571& 7102, Agence Nationale pour la Recherche, Fondation pour Neuropole de Recherche Francilien of Ile de France, National Cancer Institute INCa BIO-SILC programme, Ecole des Neurosciences de Paris, EU FP7 ERANET Neuron NICO-GENE network, LabEx BIOPSY funded by ANR, Italian Ministry of Health RF2009-154961. We thank M. Soudant for lentivirus production, C. Morel for sharing vectorized mice and B. Forget for comments on the work and manuscript.

position and range of accepted positions (dashed lines)) (left); Time course of 5-HT extracellular levels as % of basal levels after saline and nicotine injection (middle); AUC as % of basal values for the amount of 5-HT outflow during the 1st hour post-injection (Right) n=9-10/group. Data plotted as mean±SEM. Genotype effect: **p≤0.01, ***p≤0.001. Nicotine effect: # p≤0.05, ## p≤0.01 compared to saline. zooms from α5 -/-α5SNP mouse (right). Arrowheads point to cells with absence of co-expression. b, Number of rearings (WT GFP /α5 -/-GFP : p=0.001; WT GFP /α5 -/-α5WT : NS; α5 -/-GFP /α5 -/-α5SNP : NS;

WT GFP /α5 -/-α5SNP : p=0.0004; α5 -/-α5WT /α5 -/-α5SNP : p=0.0009). c, EPM anxiety-related behaviour: % of time spent in open arms (WT GFP /α5 -/-GFP : p=0.008; WT GFP /α5 -/-α5WT : p=0.01; WT GFP /α5 -/-α5SNP : p=0.09; α5 -/-GFP /α5 -/-α5WT : NS; α5 -/-α5WT /α5 -/-α5SNP : NS) and their extremities (WT GFP /α5 -/-GFP : p=0.005; WT GFP /α5 -/-α5WT : p=0.02; WT GFP /α5 -/-α5SNP : p=0.04; α5 -/-GFP /α5 -/-α5WT : NS; α5 -/-α5WT /α5 -/- α5SNP : NS). d, LDB anxiety-related behaviour: % of time spent in the light side (WT GFP /α5 -/-GFP : p=0.0006; WT GFP /α5 -/-α5WT : p=0.0007; WT GFP /α5 -/-α5SNP : p=0.00001; α5 -/-GFP /α5 -/-α5WT : NS; α5 -/- α5WT /α5 -/-α5SNP : NS). Data plotted as mean±SEM. Group effect: *p≤0.05, **p≤0.01, ***p≤0.001. n=12-20/group. examples from α5 -/-GFP (upper) and α5 -/-α5WT (bottom) mice; zooms from α5 -/-α5SNP mouse (right and bottom). Arrowheads point to cells with absence of co-expression. b, Number of rearings (WT GFP /α5 -/-GFP : p=0.002; WT GFP /α5 -/-α5WT : p=0.002; WT GFP /α5 -/-α5SNP : p=0.0007; α5 -/-GFP /α5 -/-α5WT : NS; α5 -/-α5WT /α5 -/-α5SNP : NS). c, EPM anxiety-related behaviour: % of time spent in open arms (WT GFP /α5 -/-GFP : p=0.04; WT GFP /α5 -/-α5WT : NS; α5 -/-GFP /α5 -/-α5WT : p=0.07; WT GFP /α5 -/-α5SNP : p=0.0002, α5 -/-α5WT /α5 -/-α5SNP : p=0.02) and extremities (WT GFP /α5 -/-GFP : p=0.02; WT GFP /α5 -/-α5WT : NS; α5 -/-GFP /α5 -/-α5WT : p=0.07; WT GFP /α5 -/-α5SNP : p=0.005, α5 -/-α5WT /α5 -/-α5SNP : p=0.02). d, LDB anxiety-related behaviour: % of time spent in the light side (WT GFP /α5 -/-GFP : p=0.04; WT GFP /α5 -/- α5WT : NS; α5 -/-GFP /α5 -/-α5WT : NS; WT GFP /α5 -/-α5SNP : p=0.002, α5 -/-α5WT /α5 -/-α5SNP : p=0.04). Data plotted as mean±SEM. Group effect: *p≤0.05, **p≤0.01, ***p≤0.001. n=11-20/group.