Benoît Forget

Romain Icick

Jonathan Robert

Caroline Correia

Marie S Prevost

Marc Gielen

Pierre- Jean Corringer

Frank Bellivier

Florence Vorspan

Morgane Besson
email: morgane.besson@pasteur.fr

Uwe Maskos
email: uwe.maskos@pasteur.fr

Benoit Forget
email: benoit.forget@pasteur.fr

stages of cocaine addiction: a translational study in transgenic rats and patients

Keywords: cocaine use disorder, transgenic rats, nicotinic receptors, human polymorphisms, relapse, nucleus accumbens

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Alterations in nicotinic receptor alpha5 subunit gene differentially impact early and later stages of cocaine addiction: a translational study in transgenic rats and patients

Benoit C. Forget, Romain Icick, Jonathan Robert, Caroline Correia, Marie S.

Prevost, Marc Gielen, Pierre-Jean Corringer, Frank Bellivier, Florence

Vorspan, Morgane Besson, et al.

To cite this version:

Benoit C. Forget, Romain Icick, Jonathan Robert, Caroline Correia, Marie S. Prevost, et al.. Alterations in nicotinic receptor alpha5 subunit gene differentially impact early and later stages of cocaine addiction: a translational study in transgenic rats and patients. Progress in Neurobiology, 2020, pp.101898. 10.1016/j.pneurobio.2020.101898. pasteur-02928473

Introduction

Cocaine use disorder (CUD) is a clinically devastating neuropsychiatric disease without any approved pharmacotherapy [START_REF] Degenhardt | Mortality among cocaine users: a systematic review of cohort studies[END_REF]. Relapse to cocaine seeking after a phase of abstinence is the main impediment in the treatment of CUD, with 78% of patients relapsing within three months after discharge from hospital detoxification [START_REF] Lima | The role of neurocognitive functioning, substance use variables and the DSM-5 severity scale in cocaine relapse: A prospective study[END_REF]. In such pathological cocaine users, relapse can be precipitated by exposure to cocaine-associated cues, stress, or to cocaine itself [START_REF] Childress | Cue reactivity and cue reactivity interventions in drug dependence[END_REF][START_REF] Jaffe | Cocaine-induced cocaine craving[END_REF][START_REF] O'brien | Stroke in young women who use cocaine or amphetamines[END_REF][START_REF] Sinha | How does stress increase risk of drug abuse and relapse?[END_REF].

Relapse prevention is thus a key target for clinicians in the treatment of substance use disorders in general since it has been associated with the chronicization and the burden of these disorders [START_REF] Hendershot | Relapse prevention for addictive behaviors[END_REF]. Similarly, reinstatement of cocaine-seeking can be elicited in laboratory animals after extinction of cocaine self-administration (SA), by exposure to a conditioned stimulus (CS) previously associated with cocaine intake, stress or a cocaine-priming injection [START_REF] Crombag | Renewal of drug seeking by contextual cues after prolonged extinction in rats[END_REF][START_REF] De Wit | Reinstatement of cocaine-reinforced responding in the rat[END_REF][START_REF] Epstein | Toward a model of drug relapse: an assessment of the validity of the reinstatement procedure[END_REF][START_REF] Fuchs | Predictive validity of the extinction/reinstatement model of drug craving[END_REF].

Cocaine acts primarily by inhibiting monoamine transporters, such as the dopamine transporter (DAT), leading to increased extracellular dopamine concentration in the nucleus accumbens (NAcc), an effect considered essential for its reinforcing property. However, cocaine is also an inhibitor of nicotinic acetylcholine receptors (nAChRs) [START_REF] Damaj | Pharmacological characterization of nicotine's interaction with cocaine and cocaine analogs[END_REF][START_REF] Francis | Subtype-selective inhibition of neuronal nicotinic acetylcholine receptors by cocaine is determined by the alpha4 and beta4 subunits[END_REF], an action that can alter dopamine release in the NAcc [START_REF] Acevedo-Rodriguez | Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release[END_REF]. nAChRs are pentameric ligand-gated ion channels composed of α (α2-α7, α9, α10) and β (β2-β4) subunits that co-assemble according to various combinations with distinct brain localizations and functional properties [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF]. nAChRs have been investigated in preclinical models of cocaine addiction. For example, the nAChR antagonist mecamylamine reduces cocaine-SA and prevents cocaine-SA escalation in rodents [START_REF] Blokhina | Effects of nicotinic and NMDA receptor channel blockers on intravenous cocaine and nicotine self-administration in mice[END_REF][START_REF] Hansen | The nicotinic acetylcholine receptor antagonist mecamylamine prevents escalation of cocaine self-administration in rats with extended daily access[END_REF][START_REF] Levin | The nicotinic antagonist mecamylamine preferentially inhibits cocaine vs. food self-administration in rats[END_REF]. Additionally, 18-MC, an antagonist of the α3β4 containing (*) nAChRs, decreases cocaine-SA in rats [START_REF] Glick | 18-Methoxycoronaridine, a non-toxic iboga alkaloid congener: effects on morphine and cocaine selfadministration and on mesolimbic dopamine release in rats[END_REF][START_REF] Pace | Novel iboga alkaloid congeners block nicotinic receptors and reduce drug self-administration[END_REF], and mice knockout (KO) for the β2 nAChR subunit exhibit decreased cocaine-induced conditioned place preference [START_REF] Zachariou | Nicotine receptor inactivation decreases sensitivity to cocaine[END_REF]. Interestingly, mecamylamine also reduces craving for cocaine in CUD subjects [START_REF] Reid | A nicotine antagonist, mecamylamine, reduces cue-induced cocaine craving in cocaine-dependent subjects[END_REF].

Several independent large-scale human genome-wide association studies (GWAS) identified variants in a region on human chromosome 15 encoding the 3, 5 and 4 nAChR subunits that increase the risk for nicotine dependence [START_REF] Bierut | Novel genes identified in a high-density genome wide association study for nicotine dependence[END_REF][START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF][START_REF] Sherva | Association of a single nucleotide polymorphism in neuronal acetylcholine receptor subunit alpha 5 (CHRNA5) with smoking status and with 'pleasurable buzz' during early experimentation with smoking[END_REF]. One of these variants, the non-synonymous coding SNP rs16969968 found in exon 5 of the 5 gene (5SNP) that causes an amino acid change D398N in the second intracellular loop of the 5 subunit, has been recently introduced into the genome of the rat, resulting in increased nicotine SA at high doses and relapse [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]. This 5SNP is very frequent in the general population and may induce a partial loss of function of the corresponding pentamer [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] without altering the overall expression of the subunit, at least in rats [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]. It does not modify the electrophysiological function of the receptor when expressed in concatemer in Xenopus oocytes [START_REF] Prevost | Concatemers to re-investigate the role of alpha5 in alpha4beta2 nicotinic receptors[END_REF], and it lies away from agonists' binding site, suggesting that it affects other features of the receptor, but its precise mechanism of action is currently unknown [START_REF] Maskos | The nicotinic receptor alpha5 coding polymorphism rs16969968 as a major target in disease: Functional dissection and remaining challenges[END_REF]. Interestingly, in candidate gene association studies, the 5SNP has also been associated with a lower prevalence of CUD in humans [START_REF] Grucza | A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence[END_REF][START_REF] Sherva | Variation in nicotinic acetylcholine receptor genes is associated with multiple substance dependence phenotypes[END_REF]. In addition, three other SNPs in the 5 nAChR subunit, linked to altered levels of 5 mRNA in humans, have also been associated with CUD [START_REF] Sherva | Variation in nicotinic acetylcholine receptor genes is associated with multiple substance dependence phenotypes[END_REF][START_REF] Wang | Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5[END_REF]. These genetic studies suggest an involvement of the 5 nAChR subunit in cocaine dependence, although the precise mechanisms and stages of CUD implicated have never been further assessed so far.

In fact, multiple behaviors related to CUD may be impacted by alterations in 5*nAChR density or mutations in the 5 nAChR subunit gene, that now need to be identified through longitudinal studies to understand how they contribute to the risk for this pathology. For this, the first goal of the present study was to clarify the role of 5*nAChRs and the impact of the 5SNP, found to confer protection for CUD in humans, on the different stages of an intravenous (IV) cocaine-SA procedure, from first cocaine intake to relapse to cocaine seeking after SA extinction, using transgenic rat lines previously generated in the laboratory [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]. At the molecular level, since cocaine has been shown to inhibit nAChR-mediated currents in midbrain DA neurons [START_REF] Acevedo-Rodriguez | Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release[END_REF], we hypothesized that the association between 5*nAChRs and CUD may be linked to an implication of the 5 subunit in nAChR response to cocaine. Thus, the second objective of the study was to characterize the influence of the 5 subunit on nAChR functional response to cocaine in vitro. Finally, for translational purposes, we took advantage of the information generated by the SA longitudinal study implemented in our preclinical models to i) clarify, in humans, the association of the 5SNP with specific CUD phenotypes, and ii) seek for novel polymorphisms in the CHRNA5/A3/B4 gene cluster, linked to 5 expression levels, in association with these CUD phenotypes.

Material and Methods

Cocaine-SA in transgenic rats

Experimental Model and Subject Details

Male rats experimentally naive at the start of the study and initially weighing 250 to 275 g were used for all behavioral experiments. All rats were individually housed in a temperature-controlled environment on a 12-h reverse light/dark cycle (lights off from 08:00 hours to 20:00 hours) at the start of the experiments and were mildly food restricted (20g/day/rat).

Wild-type (WT) rats, homozygous α5KO rats and homozygous rats constitutively carrying the rs16969968 SNP (α5SNP rats), on Long Evans background, all males, were generated, bred and housed at the Institut Pasteur, Paris, France [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]. Homozygous rats were used considering the stronger phenotype on cocaine addiction in patients homozygous for the rs16969968 polymorphism [START_REF] Grucza | A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence[END_REF]. The rats' colonies were regularly back-crossed with WT Long-Evans rats.

Experimental procedures were approved by the institutional Animal Care Committee (agreement N°0355.02). All efforts were made to minimize animal suffering and reduce the number of animals.

Cocaine-SA procedure

Self-administration (SA) sessions were carried out in experimental chambers equipped with two retractable levers (Med Associates, St. Albans, Vt., USA), as previously described [START_REF] Forget | Inhibition of fatty acid amide hydrolase reduces reinstatement of nicotine seeking but not break point for nicotine self-administration--comparison with CB(1) receptor blockade[END_REF]Forget et al., 2010a;[START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]Forget et al., 2010b). Cocaine hydrochloride (Sigma-Aldrich, St Louis, Mo., USA, ref: C5776) was dissolved in saline (0.9%) and administered intravenously (IV) in a volume of 100 μl/kg/injection or subcutaneously (SC) in a volume of 1 ml/kg.

Rats were first trained to press an active lever (AL) under a schedule in which each AL press resulted in the delivery of a 45 mg food pellet (continuous reinforcement, no associated cues, 1h).

Once trained, each animal was surgically prepared with a chronic intravenous catheter implanted in the jugular vein; the catheters exited between the scapulae. Surgery was performed under anesthesia induced by xylazine (10 mg/kg, intraperitoneal (IP)) and ketamine hydrochloride (75 mg/kg, IP). Incision sites were infiltrated with the local anesthetic bupivacaine (0.125%).

Buprenorphine was given for post-operative analgesia (0.01 mg/kg, SC), and a single dose of penicillin (30,000 units, SC) was administered before surgical procedures. Rats were allowed to recover for a 1-week period before drug SA sessions were begun. Every SA session lasted 1h.

Acquisition of cocaine self-administration

WT (n=11), α5SNP (n=10), and α5KO rats (n=12) acquired cocaine SA under a fixed ratio (FR) schedule of reinforcement and the unit dose was 0.2 mg/kg per infusion. Each infusion was associated with the presentation of a visual cue above the AL during a time-out period of 20 sec (the levers were not retracted during the time-out period). During three consecutive sessions, each AL press during the time-in period resulted in the delivery of an infusion (FR-1). The next ten sessions, response requirements were increased to the final value of FR-5 (i.e., rats were required to make five lever presses for each drug infusion). Rats were considered to have acquired stable cocaine-SA when they pressed the AL more than twice the number of times they pressed the inactive lever (IL), and received a minimum of 5 infusions per 1-h session with less than 20% variation in the number of infusions earned per session during two consecutive sessions.

Dose-response curve

The rats that had successfully acquired cocaine-SA behavior at the training dose (9 WT, 8 α5SNP and 11 α5KO rats) were then submitted to SA sessions with different doses of cocaine (0.1; 0.2; 0.4 and 0.8 mg/kg/infusion) according to a Latin square design; with at least three sessions for each dose and a stable responding required, with intermediate sessions at the 0.2 mg/kg/infusion training dose between each testing with another dose. After this testing, rats were submitted to five consecutive sessions with saline substituted to cocaine and the cocaine-associated cue still present. Cocaine-SA at the training dose was then re-established before the progressive ratio (PR) phase. Two α5KO rats were excluded from the study after this phase due to loss of catheter patency.

Progressive Ratio responding

After the establishment of the dose-response curve for cocaine-SA under a FR of reinforcement, rats were switched to a PR schedule wherein the response requirement increased with each successive injection. The response requirement progression was based on the formula 5XEXP(0.2Xinfusion number)-5 [START_REF] Donny | Nicotine self-administration in rats on a progressive ratio schedule of reinforcement[END_REF]. The break point (BP) was defined as the highest ratio completed prior to the first 30 min period without a response on the AL. PR sessions lasted a maximum of 3 h. Under this schedule of reinforcement, the rats were allowed to selfadminister cocaine at 0.2 mg/kg/infusion until stabilization of the behavior (less than 20% variation in the number of infusions earned per session during two consecutive sessions), and then the dose was switched to 0.4 mg/kg/infusion for five consecutive sessions (9 WT, 8 α5SNP and 9 α5KO rats).

Reinstatement of cocaine seeking after extinction

After the PR testing, the rats (9 WT, 8 α5SNP and 9 α5KO rats) were switched to an FR-5 schedule of reinforcement (0.2 mg/kg/injection) for four consecutive sessions. Then, they were submitted to extinction sessions and reinstatement of cocaine seeking sessions induced by reexposure to the visual cue previously associated with cocaine delivery, pharmacological stress or cocaine (10 mg/kg, SC) under a Latin square design.

-Extinction

The extinction phase was conducted by withholding cocaine and its associated cue. Responses on the AL or IL were recorded, but had no consequences. The criterion for extinction was less than 20 AL presses per 1h session with less than 20 % variation over two consecutive days.

-Reinstatement of cocaine seeking Cue-induced reinstatement test was conducted under conditions identical to that of SA, except that 1) a single presentation of the visual cue (light above the active lever for 10s) was delivered response-independently immediately at the start of the session and 2) responses on the AL resulted in contingent presentation of the cue (light above the active lever on) and house-light off for 20 s without cocaine availability (no infusions). The testing sessions lasted 1 h.

Pharmacological stress-induced reinstatement of cocaine seeking consisted of an IP injection of the α2-adrenergic antagonist yohimbine at 1.25 mg/kg, 30 min before the test-session. Yohimbine induces anxiety-and stress-like responses in multiple species (Bremner et al., 1996a, b) and is known to reinstate cocaine seeking [START_REF] Brown | Interaction between noradrenaline and corticotrophinreleasing factor in the reinstatement of cocaine seeking in the rat[END_REF][START_REF] Lee | Pharmacological blockade of alpha2-adrenoceptors induces reinstatement of cocaine-seeking behavior in squirrel monkeys[END_REF].

Cocaine-induced reinstatement of cocaine seeking consisted of an IP injection of cocaine at 10 mg/kg, 10 min before the test-session. No cues were presented.

Rats were submitted again to an extinction phase between each reinstatement session.

The extinction sessions performed before the reinstatement sessions induced by pharmacological stress or cocaine were done with saline priming injection, 30 or10 min before the extinction session, respectively; with the same extinction criterion than during the main extinction phase.

The reinstatement sessions were randomized in a Latin square design.

Statistics:

Cocaine-SA data and in vitro and ex-vivo data were subjected to repeated measures ANOVAs, followed when appropriate by post hoc Bonferroni for multiple comparisons. Differences were considered significant when p<0.05.

For the data on cocaine-SA under the PR schedule, the statistical analysis was made on the total number of injections received during the session before 30 min without any AL press (BP). The timeline of the cocaine-SA study is presented in diagram 1.

Diagram 1: Timeline of the cocaine-SA experiment. The multiples phases of cocaine-SA, doses of cocaine and number of rats per group are presented.

Immunofluorescence and c-Fos counting

At the end of cocaine-induced reinstatement of cocaine seeking sessions or saline priming extinction sessions, the rats were returned to their home cage for 60 min to allow for the synthesis and transport of c-Fos proteins to the nuclei of activated neurons [START_REF] Barros | The pattern of c-Fos expression and its refractory period in the brain of rats and monkeys[END_REF]. The rats were then perfused transcardially with 150 mL of phosphate-buffered saline (PBS), followed by 150 mL 4% paraformaldehyde depolymerized in saline phosphate buffer. The brains were extracted and immersed overnight in the same fixative at 4°c. Sections (50 μm) were cut throughout the entire brain using a vibratome (Leica) and 1 slice over 3 was processed for immunofluorescence.

Free-floating brain sections were incubated 1 h at room temperature in a blocking solution of PBS containing 10% NGS and 0.2% Triton X-100 and then incubated 72h at 4°C with a rabbit anti-c-Fos antibody (Calbiochem #PC38) at 1:15000 dilution in PBS containing 2% NGS and 0.2% Triton X-100. The sections were incubated 3 h at room temperature with Alexa fluor 594-conjugated anti-rabbit secondary antibody (Jackson Immunoresearch) at 1:400 dilution in a solution of 2%NGS and 0.2% Triton in PBS and mounted using Prolong® Gold (Thermofisher scientific).

All the images were acquired using a nanozoomer (Hamamatsu Nanozoomer Digital Pathology, 2.0 HT, its fluorescence unit option, L11600-05, and the NanoZoomer's 3-CCD TDI camera, Hamamatsu Photonics; Cellular Imaging Facility of the Institut de la Vision, Paris) and analyzed with a 20× objective.

The number of c-Fos positive nuclei was automatically counted with the spot detector module of ICY software (Institut Pasteur, Paris, France) as previously described [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]. This program makes possible to select and count cells automatically without experimenter bias (counts were conducted without knowledge of the group assignments). In each coronal section, we sampled both ipsi-and contralateral regions separately and then averaged the density of the spots for each structure per animal. The results were expressed as number of c-Fos positive cells per square millimeter of cerebral tissue.

Data were analyzed using Prism and statview softwares. Data from each brain area were subjected to two-way repeated measures ANOVAs, followed when appropriate by post hoc Bonferroni for multiple comparisons. Changes were considered significant when p<0.05.

Two-electrodes voltage-clamp electrophysiology

Xenopus laevis oocytes were obtained from Centre de Ressources Biologiques-Rennes, France and maintained in modified Barth's medium (88mM NaCl, 1mM KCl, 1mM MgSO4, 2.5 mM NaHCO3, 0.7mM CaCl2, 5mM HEPES pH 7.3). Defolliculated oocytes were submitted to intranucleus injection of a 1:1:0 or 1:1:5 mixture of 3:4:5 or 4:2:5 rat cDNAs and kept at 18°C for 1-2 days before recording.

Recordings were performed with a Digidata 1550A (Molecular Devices) digitizer, an Oocyte Clamp OC-725C (Warner Instruments) amplifier and using the pClamp 10.5 software. Oocytes were perfused with recording buffer (100mM NaCl, 2.5mM KCl, 2mM CaCl2, 1mM MgCl2, 10mM HEPES pH 7.3) using a gravity-driven system. Cocaine and acetylcholine were applied after dilution in recording buffer immediately before recording. All currents were measured at -60 mV.

The protocol consisted of a co-application of the given dose of cocaine and 30µM of acetylcholine in between two applications of 30µM acetylcholine. Data are from n ≥ 3 oocytes from at least two injections. Normalized peak current are presented (Ipeak(ACh+cocaine)/Ipeak(ACh)). Recordings were analyzed using ClampFit, AxoGraph X and GraphPad Prism.

Clinical genetic association study

This was both a within-cases and case/control, candidate SNP, preclinically-driven genetic association study.

Study protocol was approved by INSERM institutional review board (IRB00003888) in July 2015.

Participants were genotyped using the Infinium PsychChip array (Illumina, San Diego, CA, USA). Samples were processed by Integragen SA® (Evry, France).

PLINK software [START_REF] Chang | Second-generation PLINK: rising to the challenge of larger and richer datasets[END_REF] was used for quality control, based on a consensus procedure [START_REF] Marees | A tutorial on conducting genome-wide association studies: Quality control and statistical analysis[END_REF] (Diagram 2, Fig. S2), which left 21 SNPs from the chromosome 15 CHRNA5-A3-B4 cluster for analysis (Fig. S2). The candidate SNPs for each study were selected based on previous literature (case/control analysis) and the preclinical results (α5SNP for CUD development, modifiers of CHRNA5 expression for relapse). We evaluated:

-The association of eight SNPs from the CHRNA5-A3-B4 cluster, that could be analyzed with high confidence (quality >90%) after imputation of chromosome 15 SNPs using the best practice pipeline (pre-phasing with SHAPEIT v.2 then IMPUTE2), with the presence of lifetime CUD (N=397 patients with CUD compared to N=2461 controls genotyped at the Wellcome Trust Institute, case-control study).

-The association between CHRNA5 rs16969968 and SNPs from CHRNA5-A3-B4 cluster in high LD, with the time to transition from 1 st to disordered cocaine use (N=350 with CUD, survival model).

-The association of CHRNA5 expression-modifying SNPs from CHRNA5-A3-B4 cluster associated with the delay to relapse to cocaine use after cessation (N =306 after outlier removal, survival model).

Diagram 2: Flowchart of patients and single nucleotide polymorphism (SNP) selection according to ancestry, quality control, and expression data for CHRNA5. SNP, single nucleotide polymorphism; CUD, cocaine use disorder.

Sample selection and clinical assessment

Patients > 18 years seeking treatment for any substance use disorder (SUD) in any of the participating Centre, and fulfilling DSM IV-TR criteria for either lifetime cocaine or opiate use disorder, were consecutively recruited through two multicentric protocols [START_REF] Mouly | Methadone dose in heroindependent patients: role of clinical factors, comedications, genetic polymorphisms and enzyme activity[END_REF][START_REF] Vorspan | Self-reported cue-induced physical symptoms of craving as an indicator of cocaine dependence[END_REF] aimed at deciphering the phenotypic and genotypic architecture of severe SUDs [START_REF] Icick | Serious suicide attempts in outpatients with multiple substance use disorders[END_REF] [START_REF] First | User's guide for the structured clinical interview for DSM-IV axis II personality disorders : SCID-II[END_REF] to ascertain DSM-IV lifetime diagnoses of substance abuse and dependence, along with enhanced information regarding the participants' modalities and trajectory of drug use and sociodemographic data. Investigators used standard survey tips to determine these events with the best possible accuracy [START_REF] Kendig | Adapting data collection methods in the Australian Life Histories and Health Survey: a retrospective life course study[END_REF].

The main phenotype of interest was the delay to relapse to cocaine use after cessation. It was obtained by 1/ asking cocaine use disorder (CUD) participants whether they had ever stopped using cocaine for more than one day, 2/ by checking whether these 'ever-stoppers' were still not using cocaine in the past 30 days and 3/ among those who ever relapsed, estimating the delayin monthsbetween cocaine cessation and relapse. The measure was right-censored by the interview (i.e. non-relapsers at that time could still relapse after the study ended) and was thus analyzed using survival models (see Statistical Analyses subsection). We also considered the presence of CUD and the time to transition from 1 st to disordered cocaine use as our secondary phenotypes of interest (Fig. 3).

Biological sampling

Participants' DNA was extracted from whole blood using a Maxwell 16 PROMEGA® extractor (Promega France, Charbonnières-les-Bains, France). Purity assessment followed the procedures described by the Centre National de Génomique, estimated on a NanoDrop® spectrophotometer by using Picogreen® assay, confirmed by Polymerase Chain Reaction before application on gel. Equilibrium at p < 10 -6 was removed. Thirty individuals were excluded for having a heterozygosity rate deviating more than 3 standard deviations from the mean. Then, cryptic relatedness by Identity-by-Descent was searched for, using a pi-hat threshold of 0.2 (2nd degree relatedness), and Caucasian ancestry was assessed according to their genetic proximity with known Caucasian populations described in the 1000 genomes project (1000G) panel, based on visual inspection of principal component analysis (N=2504 from five superpopulations). Of note, the first two components of this "MDS" analysis were kept as covariates for the case/control analysis, as recommended. These procedures led to exclude four individuals with non-Caucasian ancestry and six due to relatedness. After these procedures, 2461 people and 488479 variants remained.

Imputation

We followed the procedure described on the Oxford University dedicated website (https://mathgen.stats.ox.ac.uk/impute/impute_v2.html). The clinical cases with CUD and the control samples were merged on common, bi-allelic variants with a minor allele frequency >5% using PLINK ad hoc function, leaving 239,578 shared SNPs. After resolving strand issues using the 1000 genomes, phase 3 reference panel for chromosome 15 (and the corresponding genetic map, version b37) [START_REF] Sudmant | An integrated map of structural variation in 2,504 human genomes[END_REF], pre-phasing was performed using SHAPEIT (v2) [START_REF] Delaneau | Integrating sequence and array data to create an improved 1000 Genomes Project haplotype reference panel[END_REF] using default parameters. Imputation for the 21 SNPs identified in the clinical samples was performed using IMPUTE2 [START_REF] Howie | A flexible and accurate genotype imputation method for the next generation of genome-wide association studies[END_REF] on chromosome 15 interval between 78.5 and 79.1 M base pairs, using default parameters except for a buffer of 500 K base pairs, setting the number of template haplotypes to 100 and the addition of the same reference panel (1000 genomes phase 3) to the pre-phased genotypes. Minimal concordance was 88%, and certainty was above 0.995 regarding the SNPs, which were further analyzed.

Case-control analysis

Analysis was performed with PLINK on 397 cases and 2461 controls using sex and the first two components of the MDS (ancestry) analysis as covariates. Bonferroni correction was applied.

Considering 3% lifetime prevalence of CUD in the general population and the odds ratio of rs16969968 risk allele for this disorder, power calculation [START_REF] Sham | Statistical power and significance testing in large-scale genetic studies[END_REF] considering unselected controls yielded  =80% for this analysis (p-value after Bonferroni correction =0.05/16 =0.003125).

Impact of CHRNA5 rs16969968 on the transition from 1 st to disordered cocaine use

In the preclinical experiment, rats carrying the α5SNP showed altered acquisition of cocaine SA.

To investigate this phenomenon in the clinical sample, we tested the association between CHRNA5 rs16969968 and SNPs from CHRNA5-A3-B4 cluster in high linkage disequilibrium (LD) (r 2 ≥0.8, CHRNA3 rs12914385 and CHRNB4 rs950776) with the time to transition from 1 st to disordered cocaine use, using a survival model among CUD individuals only (since rats had all acquired the self-administration behavior in the preclinical study). Tests were performed under both the genotypic (log-rank test for trend) and the recessive models (binary log-rank test). Five patients were excluded from analysis due to extreme values of the transition duration (≥300 months, three of whom were major allele homozygotes), leaving 351 CUD individuals with valid data regarding their age at onset of cocaine experimentation and of CUD. P-values were Bonferroni-corrected for three tests (cut-off =0.05/3 =0.016). Significant associations were further confirmed after multiple adjustment on clinical and sociodemographic variables also associated with the time to transition from 1 st to disordered cocaine use under the recessive model using cox model, after verification of the proportionality of hazard ratios.

Genetic study of relapse to CUD

SNPs from CHRNA5-A3-B4 cluster were further filtered and selected according to their effect on CHRNA5 expression, to perform a candidate association study with relapse to cocaine use in treatment-seeking outpatients. This selection was based on data from an extensive post-mortem study [START_REF] Wang | Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5[END_REF] reporting CHRNA5 mRNA levels according to 44 SNPs from CHRNA5-A3-B4 cluster in their Table 1. We applied Bonferroni correction to these 44 SNPs, of which 28 remained significantly associated with CHRNA5 mRNA levels. Eventually, six of these SNPs were available on our DNA array and could be tested for associations with the delay to relapse to cocaine use in our sample (Diagram 2).

Copy Number Variants (CNVs)

We followed the pipeline used in a recent study [START_REF] Huguet | Measuring and Estimating the Effect Sizes of Copy Number Variants on General Intelligence in Community-Based Samples[END_REF], starting from Illumina® final report file generated by GenomeStudio v.2, that involves two detection programs, PennCNV [START_REF] Wang | PennCNV: an integrated hidden Markov model designed for high-resolution copy number variation detection in whole-genome SNP genotyping data[END_REF] and QuantiSNP [START_REF] Colella | QuantiSNP: an Objective Bayes Hidden-Markov Model to detect and accurately map copy number variation using SNP genotyping data[END_REF] optimized in a combined algorithm allowing for thorough quality check. Both are based on hidden Markov chains model, but show different sensitivity and specificity. The aim of such analysis was to identify additional variants that would be relevant to fuel further in vitro and in vivo functional analyses. It was thus performed at the genome-wide and sample-wide levels. We only considered individuals and CNV meeting stringent control criteria (Diagram 2).

Statistical analyses

Data is described by means (standard deviation, SD) or median (interquartile range, IQR) when continuous and counts (frequencies) when categorical.

In the clinical sample, non-parametric Chi 2 , Spearman's rho and Mann-Whitney U were used to identify the sociodemographic and clinical variables associated with the three phenotypes of interests. Of note, both the delay to transition and the delay to relapse had a highly skewed distribution with Shapiro-Wilk test indicating lack of normality at p <2.2 x 10 -16 . The variables were thus log-transformed for bivariate analyses. Before fitting survival models, multivariate identification of outliers was also conducted. It was deemed relevant because 1/ the distribution was highly skewed (Fig. S1) and 2/ some respondents reported delays to relapse as low as a few days, whichalthough it was not corrected by the interviewer in order to maintain a fully naturalistic and observational study designshould be considered as continuous use or withdrawal relief rather than relapse. To do so, the cook.distance function was applied to the linear fit of the log-transformed delay variables and the most influential values were removed [START_REF] Cook | Residuals and influence in regression[END_REF].

Across all human genetic studies, ancestry was thoroughly controlled for, since the minor allele frequency of the 5SNP (rs16969968) ranges from in 2.3% among African-Americans to 36.6% in Europeans. Thus, mixing these populations would deeply bias the interpretation of group comparisons.

Cox proportional models and curves were built separately for each SNP associated with the delays of transition and relapse. After verification of the proportional hazard assumption, hazard ratios (HR) were computed to detect significant differences in those delays according to binary genotypes under the recessive model (major allele homozygotes vs. carriers of any number of minor allele copies), considered significant at Bonferroni-corrected p=0.05. In case HR were significantly associated with genotypes, clinical and sociodemographic correlates of the given delay (transition or relapse) identified in bivariate analyses were incorporated in final cox models to test the independence of genetic associations. Analyses were performed by R 3.5.3 (through R studio 1.185) on Mac OS X.12.6.

1.2.1.5.11 "Weighted" NES calculation: in order to test if the genotyped SNPs possibly associated with CUD relapse yielded a reduction of CHRNA5 expression, i.e. if they would mimic the results from the preclinical study, we investigated the effects of both genotyped and non-genotyped SNPs known to be coinherited SNPs with our lead SNP(s). To do so, we searched gene expression data from the GTEx portal (available at https://gtexportal.org), which is based on actual gene expression measurements in multiple tissue samples [START_REF] Baran | The landscape of genomic imprinting across diverse adult human tissues[END_REF], using a LD threshold of r 2 >0.6, considering the signed normalized effect size (NES) of each SNP significantly associated with CHRNA5 expression in eight brain regions. There were a total of 311 variants in high LD in the CHRNA5-CHRNB4-CHRNA3 cluster. LD scores of all SNPs with available and significant association with CHRNA5 expression were used as coefficients to weight each NES, so that final NES =LD 1 *NES 1 +LD 2 *NES 2 ….+LD i *NES i . We also searched for associations between our lead SNP(s) and DNA methylation of CHRNA5 in the mQTLdb database (available at http://www.mqtldb.org), which is also based on actual cellular experimentation [START_REF] Gaunt | Systematic identification of genetic influences on methylation across the human life course[END_REF].

Limitations: Our Human genetic study shows some limitations. In particular, they have been performed on a limited set of SNPs. However, selection was driven by results from our preclinical study and from reliable expression data in the literature. The model used to estimate the effects of rs950776 on CHRNA5 expression was purely empirical and intended for discussion purposes only. Binary genotypes under the recessive model were mostly considered, as previously published [START_REF] Grucza | A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence[END_REF]. Accordingly, direct causality cannot be drawn from the present human association studies results.

Results

Cocaine-SA and relapse in transgenic rats

Acquisition of cocaine-SA, dose-response curve, saline substitution and motivation for cocaine-SA in α5SNP, α5KO and WT rats

α5SNP, α5KO and WT rats acquired the food training similarly (continuous reinforcement, no associated cues; Fig. 1A), suggesting an absence of learning or motor deficit in α5SNP and α5KO rats.

Rats were then submitted to chronic IV cocaine-SA. The three groups increased similarly their lever pressing behavior when the ratio progressively increased (FR-1 then FR-2 then FR-3 then FR-4 and finally FR-5; Fig. 1B). However, there was a significant interaction between sessions and genotypes on the number of cocaine infusions received during cocaine-SA acquisition (Fig. 1C), indicating that the evolution of SA acquisition was different between groups. Indeed, there was a session effect only in α5SNP rats, showing that they were unable to maintain their level of cocaine intake when the ratio increased, contrary to WTs and α5KOs. In addition, the mean number of cocaine infusions received during the fixed-ratio 5 (FR-5) sessions was significantly reduced compared to FR-1 sessions in α5SNP rats but not in WTs or α5KOs (Fig. 1D), confirming the deficit in cocaine-SA acquisition of α5SNP rats. There were no significant differences between groups on inactive lever (IL) presses or on perseverative responding during the time-out period (wasted presses) (Table S1) or on the number of sessions necessary to reach the criterion (Table S2).

α5SNP, α5KO and WT rats were then allowed to self-administer multiple doses of cocaine according to a Latin-square design (0.2-0.8 mg/kg/injection) under FR-5, to establish a doseresponse curve. We did not observe significant differences on the number of cocaine infusions received during these sessions between groups (Fig. 1E). There were also no differences between groups on IL or wasted presses (Table S1). The fact that the deficit in cocaine-SA acquisition of α5SNP rats was no longer present at the same dose (0.2 mg/kg) throughout the dose effect investigation suggest that the protective effect of the α5SNP is temporary and restricted to an early phase of cocaine-SA.

When saline was substituted for cocaine during five consecutive sessions, α5SNP, α5KO and WT rats displayed similar AL pressing extinction (Fig. 1F), indicating that cocaine expectation drives the lever pressing behavior.

We then shifted the schedule of reinforcement to a progressive ratio (PR) [START_REF] Donny | Nicotine self-administration in rats on a progressive ratio schedule of reinforcement[END_REF], allowing the measurement of the incentive motivational and rewarding properties of cocaine [START_REF] Paterson | The metabotropic glutamate receptor 5 antagonist MPEP decreased break points for nicotine, cocaine and food in rats[END_REF]. We found no significant differences between genotypes for 0.2 and 0.4 mg/kg of cocaine, i.e. on the number of reinforcements received during the test (Fig. 1G), nor on IL or wasted presses (Table S1).

An extinction phase was then conducted by withholding cocaine and its associated visual cue, inducing a progressive and similar decrease of AL responding in the three groups (Fig. 1H), without a difference in the number of sessions necessary to reach the extinction criterion (Table S2).

Reinstatement of cocaine seeking after extinction in α5SNP, α5KO and WT rats

Rats were then tested for reinstatement of cocaine seeking induced by cue, pharmacological stress or cocaine in a Latin-square design. WT, α5SNP and α5KO rats exhibited similar cueand stress-induced reinstatement of cocaine seeking (Figs. 2A andB, respectively). However, we observed a strong increase in cocaine-induced reinstatement of cocaine seeking in α5KO rats compared to WTs and α5SNPs (Fig. 2C), without modifying IL pressing behavior (Table S1).

This indicates that the absence of nAChR α5 subunit, and not α5SNP presence, leads to higher level of relapse after re-exposure to cocaine, but not to cocaine-associated cue nor after exposure to stress.

Neuronal activation during cocaine-induced reinstatement of cocaine seeking

To dissect the effect of the absence of the α5 subunit on relapse, we investigated the expression of the c-Fos immediate early gene product, considered as a marker of neuronal activation (Forget et al., 2010a), during cocaine-induced reinstatement of cocaine seeking or extinction session with saline injection, in multiple brain areas of WT and α5KO rats. Cocaine-induced reinstatement of cocaine seeking was associated with an increase of c-Fos expression in WT and α5KO rats compared to saline condition in prelimbic, infralimbic and cingulate cortices, in the shell and core of the NAcc, the ventral tegmental area (VTA), the dorsal striatum and the primary motor cortex (M1) (Fig. 2D). Interestingly, a significantly higher expression of c-Fos was observed in the shell of the NAcc of α5KO rats, which relapsed more intensely compared to WTs, during cocaineinduced relapse but not extinction.

Impact of the α5 subunit on cocaine-induced inhibition of cholinergic currents on α3β4-or α4β2-containing nicotinic receptors in oocytes

Since biologically relevant concentrations of cocaine can inhibit nAChR-mediated currents in midbrain dopaminergic neurons, leading to an alteration of dopamine release in the dorsal and ventral striatum [START_REF] Acevedo-Rodriguez | Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release[END_REF]; we next investigated whether the increased cocaine-induced relapse observed in α5KO rats may be due to an alteration of cocaine-induced inhibition of nAChRs in the absence of α5. To do so, we measured cholinergic currents in Xenopus oocytes expressing α3β4-or α4β2-nAChRs in the presence or absence of α5. As expected, we observed a dose-dependent inhibition of cholinergic currents in oocytes expressing α3β4-or α4β2-nAChRs by cocaine (0-100 µM) (Fig. S3A andB, left panels). However, the addition of the α5 subunit did not modify the effect of cocaine on either nAChR subtypes (Fig. S3A andB, right panels), suggesting that α5 does not contribute directly to cocaine-induced inhibition of nAChR currents, at least in this heterologous expression system.

Genetic association study in patients

We then sought to establish the role of α5SNP and CHRNA5 expression levels in CUD patients.

The flowchart of the study is described in Diagram 2 and the sample is described in Table S3.

Patients were 39 +/-9 years old, 76% men, with a median delay to CUD relapse of 13 months, the distribution of which was highly skewed (Fig. S1). There were no significant associations among the eight SNPs from the CHRNA5-A3-B4 cluster and the presence of lifetime CUD (Table 1). In particular, we did not replicate the association between CHRNA5 rs16996698 SNP and CUD [START_REF] Grucza | A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence[END_REF][START_REF] Sherva | Variation in nicotinic acetylcholine receptor genes is associated with multiple substance dependence phenotypes[END_REF]. However, CHRNA5 rs16969968 was significantly associated with a slower transition from 1 st to disordered cocaine use. Under the recessive model, hazard ratio (HR) was 0.73 (95% CI =0.58-0.91, corrected p =0.014, Fig. 3A) for carriers of at least one copy of the minor allele. Considering the increased cocaine-induced relapse intensity observed in α5KO rats during the SA experiment, genotyped SNPs from the CHRNA5-A3-B4 cluster were further filtered by their effect on CHRNA5 expression in human brain [START_REF] Wang | Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5[END_REF] and selected to perform a candidate association study with relapse to cocaine use in the CUD patients. Among the six final SNPs that were selected based on their previously reported effect on CHRNA5 mRNA expression, five were intron variants and one (rs3743074) was a splice region variant.

One SNP, rs950776 in CHRNB4, was significantly associated with a shorter delay to relapse to cocaine use (HR = 1.58, 95% CI=1.41-1.75, Bonferroni-corrected p =0.04) (Fig. 3C). Shorter delay to relapse was also associated with a higher number of DSM-IV criteria for cocaine abuse/dependence (HR=1.41, 95% CI =1.27-1.58, p <0.001) while both alcohol (HR=0.58, 95% CI=0.4-0.86, p=0.006) and sedative medication (HR =0.69, 95% CI=0.48-0.98, p=0.037) use disorders were independently associated with a longer delay to relapse to cocaine (Fig. 3D). No CNVs were identified in the CHRNA5-A3-B4 cluster.

Discussion

Phenotypes of α5SNP and α5KO rats in cocaine-SA and relapse

We first observed that the presence the α5SNP did not prevent rats from acquiring cocaine-SA, and did not alter the dose-response curve for cocaine-SA. Yet, contrary to WTs, α5SNP rats did not adapt their AL pressing to maintain constant amounts of cocaine intake when the ratio schedule of reinforcement was increased (Fig. 1C andD). This seems in line with previous indications of a protective effect of this polymorphism on cocaine dependence in humans [START_REF] Grucza | A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence[END_REF][START_REF] Sherva | Variation in nicotinic acetylcholine receptor genes is associated with multiple substance dependence phenotypes[END_REF]. However, the deficit in cocaine-SA acquisition of α5SNP rats was no longer present at the same dose after extensive SA sessions, and we further found that the α5SNP did not alter cocaine-SA extinction and relapse to cocaine seeking induced by cocainecue, pharmacological stress or cocaine priming. These results suggest that this polymorphism protects against the risk to develop cocaine abuse through altered cocaine effects during early stages of drug experimentation, but has no longer an impact on later stages of the disease when CUD is already installed.

The absence of the α5 nAChR subunit had no effect on cocaine-SA acquisition and dose-response curve, nor on the extinction or reinstatement of cocaine seeking induced by cocaine-cue or pharmacological stress. However, KO rats showed a strong increase in relapse intensity induced by cocaine priming. This constitutes an important finding considering the high frequency of relapse in cocaine addiction [START_REF] Sinha | New findings on biological factors predicting addiction relapse vulnerability[END_REF] and is in line with the fact that three SNPs in the CHRNA5 gene previously associated with cocaine dependence (rs684513, rs621849 and rs615470 [START_REF] Sherva | Variation in nicotinic acetylcholine receptor genes is associated with multiple substance dependence phenotypes[END_REF]) are also associated with modifications in CHRNA5 mRNA expression in the human brain [START_REF] Wang | Risk for nicotine dependence and lung cancer is conferred by mRNA expression levels and amino acid change in CHRNA5[END_REF]. It would now be interesting to evaluate whether these SNPs specifically affect the risk to relapse to cocaine seeking in humans.

Additional work will also now be needed to further understand how the α5SNP, which has been proposed to confer a partial loss of function of the receptor although the underlying mechanisms are still unclear [START_REF] Maskos | The nicotinic receptor alpha5 coding polymorphism rs16969968 as a major target in disease: Functional dissection and remaining challenges[END_REF][START_REF] Prevost | Concatemers to re-investigate the role of alpha5 in alpha4beta2 nicotinic receptors[END_REF], may confer protection against CUD development by modifying the effects of cocaine at early stages of exposure, while a full absence of α5nAChRs rather seems to increase the risk to relapse after prior long-term exposure.

Neuronal activation during cocaine-induced relapse

We also found that cocaine-induced reinstatement of cocaine seeking was associated with increased neuronal activation in WT and α5KO rats, in most of the brain areas investigated: the prelimbic, infralimbic and cingulate cortices, the shell and core of the NAcc, the posterior VTA, the dorsal striatum and the primary motor cortex (M1). This is in line with a previous study showing an increase in c-Fos expression in the NAcc and prefrontal cortex during cocaineinduced reinstatement of cocaine seeking in the SA paradigm [START_REF] Wright | Methyl supplementation attenuates cocaine-seeking behaviors and cocaine-induced c-Fos activation in a DNA methylation-dependent manner[END_REF], and adds the cingulate cortex, the posterior VTA, the dorsal striatum and M1 to the list of brain areas activated during this behavior.

Interestingly, only the shell of the NAcc was associated with a significant increase in neuronal activation in α5KO rats compared to WTs. However, we observed similar tendencies in the prelimbic, infralimbic and cingulate cortices and in the NAcc core, all part of the neuronal ensemble involved in relapse to drug seeking [START_REF] Cruz | Using c-fos to study neuronal ensembles in corticostriatal circuitry of addiction[END_REF]. It is thus possible that the activation of these brain areas is also affected by the absence of the α5 subunit but that the present analysis is not powerful enough to detect it. Interestingly, we did not observe any difference or tendency between α5KO and WT rats on c-Fos expression in M1, a structure mainly involved in motor control and procedural skill learning [START_REF] Papale | Circuit changes in motor cortex during motor skill learning[END_REF]; suggesting that the increase in neuronal activation in α5KO rats is not due to a non-specific motor effect. The NAcc shell is crucial for cocaine-induced relapse since infusion of cocaine into this structure is sufficient to reinstate cocaine seeking after extinction in rats [START_REF] Reed | Assessing contributions of nucleus accumbens shell subregions to reward-seeking behavior[END_REF], while inhibition of D1-like dopamine receptor in the NAcc shell attenuates cocaine priming-induced reinstatement of cocaine seeking [START_REF] Anderson | Administration of the D1-like dopamine receptor antagonist SCH-23390 into the medial nucleus accumbens shell attenuates cocaine priming-induced reinstatement of drug-seeking behavior in rats[END_REF]. In addition, stimulation of dopamine receptors in the shell, but not the core, of the NAcc reinstates cocaine seeking [START_REF] Schmidt | Stimulation of D1-like or D2 dopamine receptors in the shell, but not the core, of the nucleus accumbens reinstates cocaine-seeking behaviour in the rat[END_REF]. Finally, MRI studies found that NAcc activity was positively correlated with craving in subjects with CUD exposed to cocaine [START_REF] Breiter | Acute effects of cocaine on human brain activity and emotion[END_REF][START_REF] Risinger | Neural correlates of high and craving during cocaine self-administration using BOLD fMRI[END_REF].

α5*nAChRs are present in dopaminergic cell bodies in the VTA [START_REF] Azam | Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons[END_REF] and in dopaminergic terminals in the striatum [START_REF] Zoli | Identification of the nicotinic receptor subtypes expressed on dopaminergic terminals in the rat striatum[END_REF]. Since the absence of α5 can modify the pharmacological properties, calcium permeability and acetylcholine sensitivity of nAChRs [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] Sciaccaluga | Crucial role of nicotinic alpha5 subunit variants for Ca2+ fluxes in ventral midbrain neurons[END_REF], it may also alter the inhibition of nAChRs caused by cocaine, thereby contributing to enhanced relapse to cocaine seeking in α5KO rats. However, we did not observe any difference in the presence or absence of α5 on cocaine-induced inhibition of cholinergic currents in oocytes expressing either α3β4or α4β2-containing nAChRs, at cocaine concentrations comparable to plasma level in rats after IV SA [START_REF] Booze | Dose-response cocaine pharmacokinetics and metabolite profile following intravenous administration and arterial sampling in unanesthetized, freely moving male rats[END_REF]. This suggests that the increased cocaine-induced relapse in α5KO rats is not mediated by a direct effect on cocaine-induced nAChR inhibition. Nevertheless, this may differ in vivo since the absence of α5 may modify the interaction of nAChRs with intracellular partners [START_REF] Kabbani | Intracellular complexes of the beta2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics[END_REF], which are not present in our heterologous expression system.

More work will now be needed to support this hypothesis. Importantly, the α5SNP was previously identified as predisposing to increased addiction-like behaviors for both nicotine [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF][START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF] and alcohol [START_REF] Besson | Profound alteration in reward processing due to a human polymorphism in CHRNA5: a role in alcohol dependence and feeding behavior[END_REF], and to enhanced appetence for food [START_REF] Besson | Profound alteration in reward processing due to a human polymorphism in CHRNA5: a role in alcohol dependence and feeding behavior[END_REF], suggesting global alterations in reward processing. The apparently opposite influence of the α5SNP on cocaine-related behaviors, as compared to what was observed for other rewards, may rely on the specific inhibitory action of cocaine on nAChRs.

The absence of effect of α5 deletion on cueand stress-induced reinstatement of cocaine seeking may be due to differences in the brain areas involved compared to cocaine-induced reinstatement (see [START_REF] Kalivas | Brain circuitry and the reinstatement of cocaine-seeking behavior[END_REF] and [START_REF] Farrell | Modeling cocaine relapse in rodents: Behavioral considerations and circuit mechanisms[END_REF] for reviews). For example, the basolateral amygdala appears critical for cue-induced but not for cocaine-or stress-induced reinstatements [START_REF] Kalivas | Brain circuitry and the reinstatement of cocaine-seeking behavior[END_REF]. The VTA, dorsal prefrontal cortex and NAcc shell are crucial to cocaine-induced reinstatement while the central nucleus of the amygdala and bed nucleus of the stria terminalis are necessary for stress-induced reinstatement [START_REF] Kalivas | Brain circuitry and the reinstatement of cocaine-seeking behavior[END_REF]. In this line, α5 is expressed at relatively high levels in the VTA and NAcc, but absent in the amygdala [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]. The lack of effect of Chrna5 deletion on cue-and stress-induced relapse seems in accordance with the physiological absence of α5 containing nAChRs in brain areas specifically involved in these modalities of relapse.

Genetic association study in patients with CUD

Attempting to translate these novel preclinical insights in a well-characterized human sample of patients with CUD, we first observed a lack of associations between SNPs from the cluster and CUD compared to controls, in contrast with two previous candidate gene association studies in humans reporting a protective effect of 5-SNP on cocaine dependence [START_REF] Grucza | A risk allele for nicotine dependence in CHRNA5 is a protective allele for cocaine dependence[END_REF][START_REF] Sherva | Variation in nicotinic acetylcholine receptor genes is associated with multiple substance dependence phenotypes[END_REF]. This discrepancy may be explained by the different populations used as CUD cases. Thus, in our clinical sample, other biological and environmental risk factors for CUD and pleiotropy between nAChRs genes and several SUDs may blur possible associations between CHRNA5-B4-A3 genes and CUD. Moreover, if the protective effect of the α5SNP acts at early stages of cocaine use, as discussed above, it is not surprising not to detect it in our patients under a case/control design, since they mainly suffer from highly severe and comorbid "end-stage"

CUDs. Indeed, in the sample of CUD patients, we observed a slower transition from 1 st to disordered cocaine use in carriers of at least one copy of the α5SNP, which is in line with the preclinical experiment, further supporting its action on an early phase of CUD to mediate its putative protective effect. However, before testing 5-altering medication for CUD prevention, an extensive preclinical validation step would be required. Thus, although the prevention of the transition from ultra-high risk (UHR) states to full-syndrome disorders remains a major goal in psychiatry in general in order to reduce the burden of mental illness, it has yet been operationalized for schizophrenia only [START_REF] Mchugh | The Ultra-High-Risk for psychosis groups: Evidence to maintain the status quo[END_REF], with emerging evidence regarding CUD [START_REF] Casey | Reduced dopamine response to amphetamine in subjects at ultra-high risk for addiction[END_REF]. Selecting individuals with frequent cocaine use and homozygous for the 5-SNP genotype to provide them with preventive medication would require additional criteria, especially other biomarkers that remain to be identified. With that regard, a study recently identified addiction-like striatal activation patterns in self-reported cocaine users, representing a putative biomarker for CUD in people who did not fulfill CUD criteria [START_REF] Cox | Cocaine Cue-Induced Dopamine Release in Recreational Cocaine Users[END_REF]. Eventually, combined with 5-SNP genotyping, this could open new avenues toward preventive clinical trials, while also probably raising ethical issues since such individuals would not be clinically ill.

Focusing on SNPs that alter CHRNA5 expression in the brain, we further identified a SNP in CHRNB4, rs950776, associated with a shorter delay to relapse to cocaine use. Our focus was driven by the preclinical results showing increased cocaine relapse strength in 5KO rats, the equivalent mutations of which in humans rather alter gene expression than protein structure (unlike rs16969968). The rs950776 SNP was previously associated with nicotine and alcohol dependence [START_REF] Hancock | A multiancestry study identifies novel genetic associations with CHRNA5 methylation in human brain and risk of nicotine dependence[END_REF][START_REF] Joslyn | Chromosome 15q25.1 genetic markers associated with level of response to alcohol in humans[END_REF], but not with CUD. This SNP is a synonymous and frequent mutation that does not affect CHRNB4 expression. It is associated with increased CHRNA5 expression, with the third largest Normalized Effect Size (NES) among the six SNP that we tested with CUD relapse (see Table 1). SNPs showing a larger NES than rs950776 had negative values but were not statistically associated with the relapse phenotype.

Interestingly, rs950776 shows many associations with DNA enzymes, transcription factors and chromatin states, supporting its role as a regulatory variant. Finally, to verify whether this finding was in line or against our initial hypothesis that a more severe relapse phenotype in 5-KO rats should correspond to genetic associations resulting in decreased CHRNA5 expression in patients, and in line with recent developments in the polygenic nature of human phenotypes and disease [START_REF] Cano-Gamez | From GWAS to Function: Using Functional Genomics to Identify the Mechanisms Underlying Complex Diseases[END_REF], we had to describe the meaning of this association in the broader genetic context of the CHRNA3-CHRNB4-CHRNA5 regulome [START_REF] Barrie | The CHRNA5/CHRNA3/CHRNB4 Nicotinic Receptor Regulome: Genomic Architecture, Regulatory Variants, and Clinical Associations[END_REF].

Using methylation and expression data from available literature and online databases, we first found that rs950776 had been associated with the cis-methylation of CHRNA5 promoter region [START_REF] Hancock | A multiancestry study identifies novel genetic associations with CHRNA5 methylation in human brain and risk of nicotine dependence[END_REF] in the human brain. Second, we took into account the functional data from SNPs in the cluster that are in high LD (i.e. "co-inherited") with rs950776. Although rs950776 alone is associated with increased CHRNA5 expression (NES =0.63), it is thus in strong LD with 61 other SNPs significantly associated with either increased or decreased CHRNA5 expression (not genotyped in the current study, data from the GTEx portal database, Table S4); resulting in a "weighted" NES of -2.46 in the NAcc (Table 2; 1.2.1.5.11 section for the "weighted" NES calculation). This suggests that rs950776 may eventually be associated with decreased CHRNA5 expression through complex haplotypic and methylation effects, without evidence of other functional impact in the cluster. The impact of this SNP on relapse to cocaine use may thus be due to reduced CHRNA5 expression, paralleling the increased relapse observed in α5KO rats.

Among the other five SNPs tested with CUD relapse, CHRNA5 rs680244 & rs667282 and CHRNA3 rs3743074 & rs6495308 are associated with decreased CHRNA5 expression, yet without being associated with CUD relapse in our study (see Table 1). We believe this is explained by the fact that CHRNB4 rs950776 is the only SNP we tested that enhances expression of the CHRNA5 antisense RNA RP11-650L12.2, which is decreased by rs680244, rs3743074 and rs938682. This effect toward reduced CHRNA5 expression at the post-transcriptional level is likely to overcome that of other SNPs associated with decreased CHRNA5 expression.

Conclusion

We showed that deletion of the α5 nAChR subunit had no effect on cocaine-SA or on its extinction, but induced a major increase in cocaine-induced reinstatement of cocaine seeking.

This effect was associated with increased neuronal activation in the NAcc shell. We further highlighted that the α5SNP rs16969968 was associated with impaired cocaine intake pattern at early stages of cocaine exposure but not at later stages, as supported both by our preclinical and clinical data. Finally, we identified a SNP in CHRNB4, rs950776, associated with a shorter delay to relapse to cocaine use in a sample of CUD patients, which may result from a reduction in CHRNA5 expression.

These results suggest that CHRNA5 expression may represent a novel biomarker for an increased risk of relapse to cocaine use and that positive allosteric modulators of α5-containing nAChRs could represent a novel therapeutic strategy against relapse to CUD. Such relapse prevention strategy is at the core of treatment guidelines against SUDs in general in order to limit their chronicization and reduce their consequences. Future clinical trials based on this mechanism are greatly expected since CUD remains a devastating disorder that still lacks effective psychopharmacological treatment [START_REF] Kampman | The treatment of cocaine use disorder[END_REF]. The two-way ANOVA performed on the number of food-pellet self-administered revealed no significant effect of genotype (F 2,30 =0.77, p= 0.47, NS; partial η 2 =0.1), but a significant effect of session (F 3,90 = 90.99, p<0.0001; partial η 2 =0.75); and no interaction (F 6,90 = 0.79, p=0.58, NS; partial η 2 =0.05).

Figure and Table Legends

(B) WT, α5SNP and α5KO rats show no difference in active lever presses during the acquisition of the cocaine-SA (0.2 mg/kg/infusion).

The two-way ANOVA performed on the number of active lever presses during these 13 sessions indicated a significant effect of session (F 12,360 =22.31, p<0.0001; partial η 2 =0.42), but not of genotype (F 2,30 =0.63, p=0.54, NS; partial η 2 =0.047) nor interaction (F 24,360 =1.43, p=0.089, NS; partial η 2 =0.087).

(C) Alteration in the number of cocaine infusions during the acquisition of the cocaine-SA (0.2 mg/kg/infusion) in α5SNP rats compared to WT and α5KO rats.

The two-way ANOVA performed on the number of cocaine infusions during these 13 sessions indicated a significant effect of session (F 12,360 =2.386, p=0.0057; partial η 2 =0.07) and interaction (F 24,360 =1.69, p=0.024; partial η 2 =0.1), but not of genotype (F 2,30 =2.68, p=0.085, NS; partial η 2 =0.19). The one-way ANOVA performed on each group indicated a significant effect of session for α5SNP rats (F 12,108 =2.79, p=0.0024**), but not for WT or α5KO rats (F 12,132 =1.79,p=0.14,NS;and F 12,108 =2.79,p=0.056,NS).

(D) Reduction in the number of cocaine infusions between the mean of 3 sessions under FR-1 and FR-5 during the acquisition of the cocaine-SA (0.2 mg/kg/infusion) in α5SNP rats but not WT or α5KO rats.

The two-way ANOVA performed on the number of cocaine infusions received between FR-1 and FR-5 sessions indicated an absence of effect of session (F 1,30 =1.24, p=0.27, NS; partial η 2 =0.04) or genotype (F 2,30 =1.88, p=0.175, NS; partial η 2 =0.25), but a significant interaction (F 2,30 =4.6, p=0.018; partial η 2 =0.23). Post-hoc analysis (paired t-test) FR-1 vs FR-5: WT (p=0.35, NS); α5SNP (p=0.0136*) and α5KO (p=0.21, NS) on reinstatement (NS on extinction). (E) WT, α5SNP and α5KO rats show no difference in the number of cocaine infusions during cocaine (0.1-0.8 mg/kg/infusion)-SA under FR-5 schedule of reinforcement.

The two-way ANOVA indicated a significant effect of cocaine concentration (F 3,75 =53.88, p<0.0001; partial η 2 =0.68), but not of genotype (F 2,25 =0.13, p=0.88, NS; partial η 2 =0.018) nor interaction (F 6,75 =0.45, p=0.84, NS; partial η 2 =0.03).

(F) WT, α5SNP and α5KO rats show no difference in the number of active lever presses during five consecutive sessions with saline substituted for cocaine and cocaine associated cue still present.

The two-way ANOVA indicated a significant effect of session (F 4,92 =51.46, p<0.0001; partial η 2 =0.69) but not of genotype (F 2,23 =0.84, p=0.44, NS; partial η 2 =0.05) nor of interaction (F 8,92 =0.82, p=0.58, NS; partial η 2 =0.07).

(G) WT, α5SNP and α5KO rats show no difference in number of cocaine infusions under a progressive ratio of reinforcement schedule at the cocaine doses of 0.2 and 0.4 mg/kg/injection. The two-way ANOVA indicated no significant effect of genotype (F 2,23 =0.57, p=0.57, NS; partial η 2 =0.29), cocaine concentration (F 1,23 =1.13, p=0.3, NS; partial η 2 =0.047), or interaction (F 2,23 =0.29, p=0.75; partial η 2 =0.025). Hazard ratios were all proportional (lowest p-value =0.093).

  , between April 2008 and July 2016. They provided written informed consent for both the clinical and genetic assessments. Study protocol was approved by INSERM institutional review board (IRB00003888) in July 2015. Eligible participants were excluded if they had severe cognitive impairment or insufficient mastery of the French language preventing their proper understanding of the study purposes and assessments, had no social insurance, or were under compulsory treatment. A unique standardized interview conducted by trained investigators (psychologists or M.D) allowed for collecting extensive phenotypic data regarding participants' history of SUD with the Structured Clinical Interview for DSM-IV (SCID-IV)

  CHRNA3 rs12914385 and CHRNB4 rs950776, which are in high LD with rs16969968, were not significantly associated with transition from 1 st to disordered cocaine use [corrected p-values of 0.2 for both SNPs under the genotypic model; and 0.063 (rs12914385) and 0.26 (rs950776) under the recessive model]. The association withheld adjustment on gender, protocol of origin, preferred route of cocaine administration during the self-reported worst period, the number of DSM-IV criteria for CUD, and the presence of opiate use disorder (Fig.3B; only significant associations are shown). The adjusted HR was 0.73 (95% CI =0.59-0.92, p =0.007) for carriers of at least one copy of the CHRNA5 rs16969968 as compared to major allele homozygotes.

  This work was supported by the Institut Pasteur, Centre National de la Recherche Scientifique UMR 3571, Fondation pour la Recherche Médicale (SPF20140129365, DPA20140629803 and EQU201903007822), Agence Nationale de la Recherche (ANR), Neuroscience "SNP-NIC" and BLANC, LABEX BIO-PSY, FP7 ERANET program NICO-GENE grant agreement convention ANR n° 2010-NEUR-004-01, European Commission FP7 RTD Project HEALTH-2009-Neurocyp.08-202088 grant 242167, French National Cancer Institute grant CANCEROPOLE IDF 2016-1-TABAC-01-IP-1 MASKOS, European ERANET programme iPS&BRAIN (all to U.M.). The laboratory of U.M. is part of the Ecole des Neurosciences de Paris Ile-de-France RTRA network. R.I., U.M., P-J.C, F.V. and F.B. are members of the LABEX BIO-PSY. This work was supported by French state funds managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX-0004-02, and by the Programme Hospitalier de Recherche Clinique (PHRC) from the French Ministry of Health (AOM10165). The authors are thankful to the Direction de le Recherche Clinique et de l'Innovation from the Assistance Publique -Hôpitaux de Paris that acted as the study promotor, and to Pr Georges Brousse (Clermont-Ferrand), Dr Maeva Fortias, Ms Emily Karsinti, Dr Vanessa Bloch (CSAPA Murger Paris), Dr Cyrille Orizet (CSAPA Monte Christo, Paris), Dr Philippe Coeuru (CSAPA Aurore-EGO, Paris), Pr Olivier Cottencin (Lille), Dr Philippe Batel (Clichy), Dr Alice Deschesneau (CSAPA Liberté, Ivry), Dr Philipppe Lack (Lyon), Dr Aurélia Gay (St Etienne) and Dr Trabut (Limeil-Brevannes) who helped collecting the data, and to the patients who participated. The genetic study in humans was designed and conducted during a 'Poste d'accueil' research fellowship from Assistance Publique -Hôpitaux de Paris and LABEX BIO-PSY obtained by R.I. 1.5.2 Conflict of interest: RI has received honoraria and support to attend congresses from Indivior and honoraria and consulting fees from Lundbeck. FV has received an honorarium from Lundbeck for a single lecture. FB has received honoraria or research or educational conference grants from Bristol-Myers Squibb, Otsuka, Eli Lilly and Co., Servier, Sanofi Aventis, Lundbeck, AstraZeneca, the European Space Agency and has received peer review research funding from French Ministry of research, Assistance Publique -pitaux de Paris, the National Institute for Research (INSERM) and the NARSAD. None of the support received by RI, FV and FB were related to the theme of the present study. All other authors declare that they have no competing financial interests in relation to the work described.

Fig. 1 .

 1 Fig. 1. Cocaine self-administration in WT, α5SNP and α5KO rats. (A) WT, α5SNP and α5KO rats show no difference in active lever presses during food training.The two-way ANOVA performed on the number of food-pellet self-administered revealed no significant effect of genotype (F 2,30 =0.77, p= 0.47, NS; partial η 2 =0.1), but a significant effect of session (F 3,90 = 90.99, p<0.0001; partial η 2 =0.75); and no interaction (F 6,90 = 0.79, p=0.58, NS; partial η 2 =0.05). (B) WT, α5SNP and α5KO rats show no difference in active lever presses during the acquisition of the cocaine-SA (0.2 mg/kg/infusion). The two-way ANOVA performed on the number of active lever presses during these 13 sessions indicated a significant effect of session (F 12,360 =22.31, p<0.0001; partial η 2 =0.42), but not of genotype (F 2,30 =0.63, p=0.54, NS; partial η 2 =0.047) nor interaction (F 24,360 =1.43, p=0.089, NS; partial η 2 =0.087). (C) Alteration in the number of cocaine infusions during the acquisition of the cocaine-SA (0.2 mg/kg/infusion) in α5SNP rats compared to WT and α5KO rats. The two-way ANOVA performed on the number of cocaine infusions during these 13 sessions indicated a significant effect of session (F 12,360 =2.386, p=0.0057; partial η 2 =0.07) and interaction (F 24,360 =1.69, p=0.024; partial η 2 =0.1), but not of genotype (F 2,30 =2.68, p=0.085, NS; partial η 2 =0.19). The one-way ANOVA performed on each group indicated a significant effect of session for α5SNP rats (F 12,108 =2.79, p=0.0024**), but not for WT or α5KO rats (F 12,132 =1.79, p=0.14, NS; and F 12,108 =2.79, p=0.056, NS). (D) Reduction in the number of cocaine infusions between the mean of 3 sessions under FR-1 and FR-5 during the acquisition of the cocaine-SA (0.2 mg/kg/infusion) in α5SNP rats but not WT or α5KO rats. The two-way ANOVA performed on the number of cocaine infusions received between FR-1 and FR-5 sessions indicated an absence of effect of session (F 1,30 =1.24, p=0.27, NS; partial η 2 =0.04) or genotype (F 2,30 =1.88, p=0.175, NS; partial η 2 =0.25), but a significant interaction (F 2,30 =4.6, p=0.018; partial η 2 =0.23). Post-hoc analysis (paired t-test) FR-1 vs FR-5: WT (p=0.35, NS); α5SNP (p=0.0136*) and α5KO (p=0.21, NS) on reinstatement (NS on extinction). (E) WT, α5SNP and α5KO rats show no difference in the number of cocaine infusions during cocaine (0.1-0.8 mg/kg/infusion)-SA under FR-5 schedule of reinforcement. The two-way ANOVA indicated a significant effect of cocaine concentration (F 3,75 =53.88, p<0.0001; partial η 2 =0.68), but not of genotype (F 2,25 =0.13, p=0.88, NS; partial η 2 =0.018) nor interaction (F 6,75 =0.45, p=0.84, NS; partial η 2 =0.03). (F) WT, α5SNP and α5KO rats show no difference in the number of active lever presses during five consecutive sessions with saline substituted for cocaine and cocaine associated cue still present. The two-way ANOVA indicated a significant effect of session (F 4,92 =51.46, p<0.0001; partial η 2 =0.69) but not of genotype (F 2,23 =0.84, p=0.44, NS; partial η 2 =0.05) nor of interaction (F 8,92 =0.82, p=0.58, NS; partial η 2 =0.07). (G) WT, α5SNP and α5KO rats show no difference in number of cocaine infusions under a progressive ratio of reinforcement schedule at the cocaine doses of 0.2 and 0.4 mg/kg/injection. The two-way ANOVA indicated no significant effect of genotype (F 2,23 =0.57, p=0.57, NS; partial η 2 =0.29), cocaine concentration (F 1,23 =1.13, p=0.3, NS; partial η 2 =0.047), or interaction (F 2,23 =0.29, p=0.75; partial η 2 =0.025).

Fig. 3 .

 3 Fig. 3. Crude and adjusted associations between the delay to transition from 1 st to disordered cocaine use and CHRNA5 rs16969968 genotype; and between the delay to relapse of cocaine use disorder and CHRNB4 rs950776 genotype. (A) Increased duration of the transition from 1 st cocaine use to cocaine use disorder in patients (n=351) with cocaine use disorder carrying at least one copy of the rs16969968 polymorphism (CHRNA5). Survival curve, time is expressed in months. Corrected p-value results from log-rank test. (B) The time to transition from 1 st to disordered cocaine use is associated with rs16969968 binary genotype and the presence of lifetime opiate use disorder but not with the preferred route for cocaine administration during the self-reported worst period or the number of DSM-IV criteria for cocaine use disorder (Full cox model). Hazard ratios were all proportional (lowest p-value =0.093). Model is also adjusted for gender, and patients' protocol of origin, none of which were significant and thus not displayed (lowest p-value =0.49 for gender). *p <0.05, **p <0.01. (C) Reduction in the delay to relapse of cocaine use disorder in patients (n=306) with cocaine use disorder carrying at least one copy of the rs950776 polymorphism (CHRNB4). Survival curve, time is expressed in months. P-value results from the cox proportional hazards test without additional covariates Survival curves (D) The delay to relapse of cocaine use disorder is associated with rs960776 binary genotype, the presence of lifetime alcohol and sedative medication use disorders, and the number of DSM-IV criteria for cocaine use disorder (full cox model).Hazard ratios were all proportional (lowest p-value =0.093).

  participants were known brothers and two were unknown cousins according to piHat scores) and another 75 individuals due to non-Caucasian ancestry were excluded. After these procedures, 21 SNPs from the CHRNA5-A3-B4 cluster on chromosome 15 were identified on the DNA array for further analysis (Fig.S2).

	1.2.1.5.4 Control sample
	The dataset EGA 000100000290 was downloaded from the Wellcome Trust initiative through a
	registered procedure from a dedicated website. It consists of 2502 UK blood donors genotyped on
	-6 were
	analyzed, leaving a total of 579 individuals and 260,853 markers. Then, cryptic relatedness by
	Identity-by-Descent was searched for, using a pi-hat threshold of 0.2 (2 nd degree relatedness), and
	Caucasian ancestry was assessed according to their genetic proximity with known Caucasian
	populations described in the 1000 genomes project (1000G) panel, based on visual inspection of

Participants were genotyped using the Infinium PsychChip array (Illumina, San Diego, CA, USA). Samples were processed in two stages (2014 and 2017) by Integragen SA® (Evry, France) and genotype files were merged for the present study, keeping only bi-allelic variants common to both extractions.

1.2.1.5.3 Bioinformatic quality control for genetic analyses

PLINK software

[START_REF] Chang | Second-generation PLINK: rising to the challenge of larger and richer datasets[END_REF]

was used for quality control, based on a consensus procedure

[START_REF] Marees | A tutorial on conducting genome-wide association studies: Quality control and statistical analysis[END_REF]

(see flowchart, Diagram 2). At the whole-sample (N=581) and genome-wide (566,932 single nucleotide variants (SNVs)) levels, SNVs and individuals with > 2% genotype missingness were excluded. Sex discrepancy was also verified, and only SNVs with minor allele frequency (MAF) ≥ 5% (thus targeting single nucleotide polymorphisms, hereafter referred to as SNPs), and without deviation from Hardy-Weinberg Equilibrium at p < 10 principal component analysis (N=2502 from five superpopulations). As a result, 31 individuals with the lowest genotyping rates from 62 pairs (58 pairs were duplicate inclusions across original protocols, two the Affymetryx-500k® DNA array. These subjects underwent the same stringent quality control as clinical cases. At the whole-sample (N=2502) and genome-wide (514,967 single nucleotide variants (SNVs)) levels, 1546 SNVs and 0 individual with > 2% genotype missingness were excluded; one due to sex discrepancy. One SNVs with deviation from Hardy-Weinberg

(H) WT, α5SNP and α5KO rats show no difference in Extinction behavior when cocaine and cocaine-associated cues where withheld. The two-way ANOVA indicated a significant effect of session (F 7,161 =26.1, p<0.0001; partial η 2 =0.53); but not of genotype (F 2,23 =0.007, p=0.99, NS; partial η 2 =0.0004) nor interaction (F 14,161 =0.82, p=0.64, NS; partial η 2 =0.066). Mean and SEM are represented. The two-way ANOVA performed on cue-induced reinstatement indicated a significant effect of reinstatement (F 1,23 =67.5, p<0.0001; partial η 2 =0.74); but not of genotype (F 2,23 =0.14, p=0.87, NS; partial η 2 =0.013) nor interaction (F 2,23 =0.14, p=0.87, NS; partial η 2 =0.012). ###: p<0.0001 on extinction vs reinstatement for all genotypes (Bonferroni's multiple comparison test). (B) WT, α5SNP and α5KO rats show no difference in reinstatement of cocaine seeking (active lever presses) after extinction induced by pharmacological stress (Yohimbine 0.125 mg/kg; IP). The two-way ANOVA performed on stress-induced reinstatement indicated a significant effect of reinstatement (F 1,23 =22.3, p<0.0001; partial η 2 =0.49); but not of genotype (F 2,23 =0.08, p=0.92, NS; partial η 2 =0.007) nor interaction (F 2,23 =0.137, p=0.87, NS; partial η 2 =0.012). ###: p<0.0001 on extinction vs reinstatement for all genotypes (Bonferroni's multiple comparison test). (C) Enhanced reinstatement of cocaine seeking (active lever presses) after extinction induced by cocaine administration (10 mg/kg; IP) in α5KO rats but not in α5SNP or WT rats. The two-way ANOVA performed on cocaine-induced reinstatement indicated a significant effect of reinstatement (F 1,23 =24.35, p<0.0001; partial η 2 =0.51); of genotype (F 2,23 =6.47, p=0.006; partial η 2 =0.34) and interaction (F 2,23 =5.97, p=0.0082; partial η 2 =0.34). Post-hoc analysis (Bonferroni's multiple comparison test) α5KO vs α5SNP or WT: p<0.001 (***) on reinstatement (NS on extinction). ###: p<0.0001 on extinction vs reinstatement for α5KO but not α5SNP or WT rats (NS). (D) Enhanced expression of the c-Fos protein (number of c-Fos positive cells/mm 2 ) during cocaine-induced reinstatement of cocaine seeking (coc) in the nucleus accumbens shell of α5KO rats compared to WT rats. The two-way ANOVAs performed on the number of c-Fos positive cells/mm 2 indicated a significant effect of treatment in the prelimbic cortex (PreL, F 1,18 =19.5; p=0.0003), infralimbic cortex (IL, F 1,23 =22.33; p=0.0002), cingulate cortex (Cg, F 1,18 =20.6; p=0.0003), shell and core of the nucleus accumbens (shell, F 1,18 =27.78; p<0.0001 and core, F 1,18 =35.75; p<0.0001), anterior (a) and posterior (p) ventral tegmental area (VTAa, F 1,15 =8.1; p=0.012 and pVTA, F 1,18 =17.2; p=0.0006), anterior (a) and posterior (p) dorsal striatum (DSa, F 1,17 =31.4, p<0.0001; DSp, F 1,17 =48.6, p<0.0001) and primary motor cortex (M1, F 1,16 =29.22, p<0.0001). The genotype effects were only significant in the NAcc shell (F 1,18 =4.68, p=0.044), Post-hoc analysis (Bonferroni's multiple comparison test) WT vs α5KO *: p<0.05 on reinstatement (NS on extinction with saline condition). Saline vs cocaine ###: p<0.0001; ##: p<0.01; #:p<0.05. Mean and SEM are represented.