Lauriane Harrington

MSci Xavier Viñals

PhD Andrea Herrera-Solís

PhD Africa Flores

BSc Carole Morel

PhD Stefania Tolu

PhD Philippe Faure

PhD Rafael Maldonado

PhD Uwe Maskos

Patricia Dphil

Robledo
email: probledo@imim.es

PhD Patricia Robledo

Nicotinic Acetylcholine Receptors in the Medial Habenula and Interpeduncular Nucleus Modulate Nicotine Reinforcement in Mice

Tobacco is consumed by an estimated 1 billion people world-wide. According to the World Health Organization, tobacco's detrimental health effects cause 6 million deaths per year, naming tobacco consumption as the primary cause of preventable morbidity and mortality. Nicotine is the principal neuroactive compound in tobacco, and exerts neurological effects by binding to nicotinic acetylcholine receptors (nAChRs). These transmembrane receptors are composed of alpha (⍺1-10) or alpha plus beta (β2-4) subunits, forming a diverse variety of homo-and hetero-pentameric ligand-gated ion channels endogenously activated by acetylcholine. Human genetic studies have highlighted the polymorphic nature of the CHRNA5-CHRNA3-CHRNB4 genomic cluster, coding for subunits α5, α3 and β4, and its implication in smoking behaviours. Notably, variants at CHRNB4 alter the risk of nicotine dependence and onset of habitual smoking. The present thesis therefore investigated the role of β4-containing (β4*) nAChRs in nicotine addiction. This was conducted using a combination of transgenic mouse lines, lentivirus technology, and assessment of nicotine-mediated behavioural and physiological outcomes β4* nAChRs in the Medial Habenula and Interpeduncular Nucleus Modulate Nicotine Reinforcement in

Mice

The appetitive nature of nicotine is reduced in the absence of β4 nAChR subunits (β4 knockout, KO) in an intravenous self-administration test. This may be due to the observed aberrant nicotine-mediated responses in the mesolimbic system of β4 KO mice. Viral-mediated restoration of β4 subunits into medial habenula neurons partially rescued the self-administration deficit in β4 KO mice. Re-expression of β4 in interpeduncular nucleus neurons, however, showed a more potent rescue of nicotine self-administration.

β4* nAChRs in the Medial Habenula and Interpeduncular Nucleus Modulate Nicotine Aversion in Mice

The ventral tegmental area (VTA) is an established hub of nicotine reward. Mice will volitionally selfadminister nicotine to this brain structure in an intra-VTA self-administration paradigm. The present thesis demonstrates that the VTA also arbitrates nicotine aversion in this test, an effect that is absent in β4 KO mice. The observed reinforcement of an otherwise aversive concentration of nicotine in the β4 KO mouse suggests that β4* nAChRs are important in regulating the aversive properties of nicotine. Re-expressing β4 in the VTA perpetuates aberrant self-administration of high, aversive doses nicotine, whereas reexpressing β4 in the medial habenula or interpeduncular nucleus attenuates consumption and restores WT-like aversion.

These data demonstrate the importance of habenulo-interpeduncular β4* nAChRs in regulating the positive and negative reinforcing properties of nicotine.

Table of Contents

The tobacco plant, Nicotiana tabacum, is native to South America and Australia. Initially consumed during ritual practices, it was popularised for recreational use in Europe following Columbus' exploration of South America in the late 15th century (see Charlton 2004 for review), and is now consumed by over 1 billion of today's global population [START_REF] Giovino | Tobacco use in 3 billion individuals from 16 countries: an analysis of nationally representative cross-sectional household surveys[END_REF].

French Ambassador Dr. Nicot recognised the medical benefits of tobacco during his post in Lisbon, Portugal, and went on to prescribe tobacco (then called nicotina) for the treatment of dermal ulcers, wounds and lesions, and even prescribed tobacco treatments to the French court [START_REF] Charlton | Medicinal uses of tobacco in history[END_REF]). Tobacco became increasingly popular for its more apparent hedonic and relaxing properties that underlie its popularity ever since its introduction to Europe 500 years ago.

Since its first documented use, tobacco is consumed in a manner that allows rapid uptake of nicotine, tobacco's principal neuro-active substance, into the bloodstream.

Nicot popularised snuff amongst the French bourgeoisie, which continued to be the principal mode of consumption until the 18th century. The following century saw Europeans smoking cigars, and then in the 19th to 20th century, owing to the industrial revolution, manufactured cigarettes became the popular choice of tobacco product [START_REF] Who | WHO report on the global tobacco epidemic[END_REF]. Now, in the 21st century, we see the introduction and increasing popularity of the nicotine-containing electronic cigarette.

ii. Medical diagnosis of dependence

Tobacco is a recognised substance of abuse that, in some smokers, leads to dependence. The psychiatrist's definition of dependence is outlined in the Diagnostic and Statistical Manual of Mental Disorders (DSM), fifth addition (APA 2013). Four overarching clinical criteria describe substance dependence (table 1), allowing psychiatrists to diagnose dependence in the patient, as well as its severity. Dependence is measured on a continuous spectrum along eleven substance-specific criteria, from mild (two-three clinical criteria), moderate (four-five) to severe (six or more), depending on the number of measurable clinical symptoms. The eleven criteria used for tobacco dependence are described in table 2.

Over-arching Features of Substance Dependence

Criterion A Apparition of a reversible, substance-specific syndrome due to recent consumption of said substance

Criterion B

Clinical behavioural or psychological adaptations associated with intoxication, attributable to the pharmacology of the substance in the CNS, developing when the substance is in the system.

Criterion C

The syndrome caused by the substance results in clinically significant distress, and impaired social and occupational functioning.

Criterion D Symptoms cannot be attributed to another medical condition

Table (1) of clinical criteria for diagnosis of substance dependence according to the current edition of the Diagnostic Statistical Manual of Mental Disorders (APA 2013)

Diagnostic Criteria of Tobacco Use Disorder Tobacco is often taken in larger amounts or over a longer period than was intended There is a persistent desire or unsuccessful efforts to cut down or control tobacco use A great deal of time is spent in activities necessary to obtain or use tobacco Craving, or a strong desire or urge to use tobacco Recurrent tobacco use resulting in a failure to fulfil major role obligations at work, school, or home Continued tobacco use despite having persistent or recurrent social or interpersonal problems caused or exacerbated by the effects of tobacco Important social, occupational, or recreational activities are given up or reduced because of tobacco use Recurrent tobacco use in situations in which it is physically hazardous (e.g. smoking in bed) Tobacco use is continued despite knowledge of having a persistent or recurrent physical or psychological problem that is likely to have been caused or exacerbated by tobacco 10 Tolerance, as defined by either of the following: a) A need for markedly increased amounts of tobacco to achieve the desired effect. b) A markedly diminished effect with continued use of the same amount of tobacco 11 Withdrawal, as manifested by either of the following: a) The characteristic withdrawal syndrome for tobacco. b) Tobacco (or a closely related substance, such as nicotine) is taken to relieve or avoid withdrawal symptoms Table (2) of the eleven diagnostic criteria used to define tobacco use disorder and to classify its severity.

According to the current edition of the Diagnostic Statistical Manual of Mental Disorders.

A major driving force for continued tobacco consumption is nicotine dependence.

Regular tobacco smokers usually start during adolescence. Males generally experiment with tobacco at an earlier age than females, a discrepancy that is regressing progressively [START_REF] Giovino | Tobacco use in 3 billion individuals from 16 countries: an analysis of nationally representative cross-sectional household surveys[END_REF]). 50% of daily smokers in the USA attain the DSM criteria of nicotine dependence for over 12 months [START_REF] Apa | Diagnostic and statistical manual of mental disorders: DSM-5[END_REF]. Dependence is also a major obstacle to long-term cessation, with only 3-5% of smokers achieving sustained remission without any nicotine cessation therapy, whilst the remaining 95-97% relapse within 12 months [START_REF] Stead | Nicotine replacement therapy for smoking cessation[END_REF]. This statistic varies with context, with a higher success rate of 15% observed amongst smokers who voluntarily attend smoking clinics, which exposes the patient to medical services (e.g. counselling interventions) that may facilitate cessation [START_REF] Stead | Nicotine replacement therapy for smoking cessation[END_REF]. Such pastoral care is known to increase quit rate successes amongst pregnant women (Hartmann-Boyce et al. 2013).

Whilst addiction can be put into an all-encompassing definition (compulsive use of the drug, consumption despite negative social or occupational effects, tolerance, withdrawal), not all drugs induce the same profile or severity of clinically relevant features. For example, nicotine, unlike alcohol, is not typified by erratic binge intoxications. The half-life of a drug also has implications in withdrawal, with a positive correlation between length of half-life in the system and duration of withdrawal symptoms, and inversely correlated with severity of withdrawal symptoms [START_REF] Apa | Diagnostic and statistical manual of mental disorders: DSM-5[END_REF].

Withdrawal symptoms include bradycardia and digestive discomfort, however the most common symptoms are affective, namely irritability, anxiety and difficulty concentrating, which occur amongst 50% of tobacco users after two days of abstinence [START_REF] Apa | Diagnostic and statistical manual of mental disorders: DSM-5[END_REF].

The DSM-5 clinical diagnosis of tobacco withdrawal is described in tables 3 and 4.

iii. Reducing tobacco consumption

The current estimate of the number of tobacco consumers worldwide stands at 1 billion (WHO 2013; [START_REF] Giovino | Tobacco use in 3 billion individuals from 16 countries: an analysis of nationally representative cross-sectional household surveys[END_REF]). Tobacco consumption is a risk factor for six out of eight leading causes of deaths in the world, resulting in six million preventable deaths per year [START_REF] Who | WHO report on the global tobacco epidemic[END_REF]. The health risks associated with tobacco consumption are multitudinal -smokers are more likely to have cardiovascular disease (including stroke), pulmonary disease and cancer [START_REF] Who | WHO report on the global tobacco epidemic[END_REF]. Manufactured cigarettes are the most common means of tobacco consumption. They are designed to optimise taste sensation, reduce harshness and increase nicotine delivery [START_REF] Cummings | Marketing to America's youth: evidence from corporate documents[END_REF][START_REF] Giovino | Tobacco use in 3 billion individuals from 16 countries: an analysis of nationally representative cross-sectional household surveys[END_REF]. A typical cigarette contains 10-14mg of nicotine, of which 1-1.5mg is absorbed and reaches the brain in 10-20 seconds [START_REF] Benowitz | Nicotine Chemistry, Metabolism, Kinetics and Biomarkers[END_REF]. The half-life of nicotine is approximately 8 minutes, although this varies with age [START_REF] Benowitz | Nicotine Chemistry, Metabolism, Kinetics and Biomarkers[END_REF].

With much resistance from the tobacco industry, policies have been placed in an attempt to reduce tobacco consumption. Strategies such as warning labels on cigarette packaging, increased excise taxes, public smoking bans and squeezing out tobacco advertising all hope to promote tobacco cessation. The number of smokers is decreasing in high-income countries, and the most vulnerable populations are from lowand middle-income countries, which today make up 80% of the world's smokers [START_REF] Giovino | Tobacco use in 3 billion individuals from 16 countries: an analysis of nationally representative cross-sectional household surveys[END_REF]. This is for a multitude of reasons, one of which may be the difference in successful quit rates, with citizens of the UK and US more likely to quit smoking than, for example the Chinese and Russians [START_REF] Giovino | Tobacco use in 3 billion individuals from 16 countries: an analysis of nationally representative cross-sectional household surveys[END_REF]. The discrepancy may also be attributed to eschewed tobacco control policies, and the World Health Organization (WHO)-admonished presence of tobacco industry representatives on government tobacco control boards, as is the case in China [START_REF] Yang | The road to effective tobacco control in China[END_REF].

In 2003, member states adopted the WHO's Framework Convention on Tobacco Control (FCTC), which officially took force in 2005. Thanks to this initiative, and spontaneous governmental policies, 2.3 billion people are now covered by at least one of the antitobacco measures set out by the WHO [START_REF] Who | WHO report on the global tobacco epidemic[END_REF]. The WHO FCTC strategies come into three main categories: prevention (education, advertising bans), reduction (tax excises, warning labels), and cessation (toll-free help-lines for specialist advice). The most adopted of the WHO's FCTC measures are the monitoring of tobacco use and prevention policies (54 countries providing thorough, up-to-date and total demographic examinations), however the insertion of tobacco cessation strategies has only been adopted in 21 countries. Since the implementation of governmental and nongovernmental tobacco control policies, we have seen a ban on smoking in public spaces in several countries worldwide (figure 1). Self-motivated nicotine cessation rates are extremely low, with sustained remission (greater than 12 months) being observed in only 3-5% of smokers [START_REF] Stead | Nicotine replacement therapy for smoking cessation[END_REF].

The risk of relapse is extremely high among dependent smokers, but can be ameliorated by nicotine cessation therapies. The most common of these is nicotine replacement therapy (NRT), which is used to curb cravings, reduce withdrawal symptoms, and wean the smoker off the dependent component of tobacco, nicotine. NRT comes in several forms, including dermal patches, lozenges, nasal sprays and chewing gum. NRTs increase the success rate of quitting by 50 to 70%, with no overall difference in effectiveness between different forms of NRT [START_REF] Stead | Nicotine replacement therapy for smoking cessation[END_REF]. This remains an extremely low rate of success, and other pharmacological treatments have been explored. Two treatments are currently approved for nicotine cessation: bupropion and varenicline (National Health Service UK 2014). Bupropion is an atypical antidepressant and a non-competitive antagonist of nicotinic acetylcholine receptors (nAChRs).

Varenicline is a partial agonist at ⍺4β2 nAChRs and a full agonist at ⍺7, serving to reduce cravings. These medications have been reported to double, and even triple the success rate of long-term tobacco abstinence compared to placebo [START_REF] Knight | The costeffectiveness of an extended course (12 + 12 Weeks) of varenicline compared with other available smoking cessation strategies in the United States: An extension and update to the BENESCO model[END_REF],

and NRT [START_REF] Kotz | Effectiveness of varenicline versus nicotine replacement therapy for smoking cessation with minimal professional support: Evidence from an English population study[END_REF], with varenicline treatment providing the greatest success [START_REF] Knight | The costeffectiveness of an extended course (12 + 12 Weeks) of varenicline compared with other available smoking cessation strategies in the United States: An extension and update to the BENESCO model[END_REF]Hartmann-Boyce et al. 2013). Naltroxone, an opioid antagonist used to treat alcohol dependence, does not improve tobacco cessation (Hartmann-Boyce et al. 2013), highlighting the nAChR as a suitable target for future tobacco cessation therapies.

The present thesis aims to elucidate the neurological mechanisms underlying nicotine addiction, with the goal of both basic understanding of these processes, but also of identification of a novel therapeutic target for nicotine cessation therapies. I shall outline the current knowledge regarding nicotine addiction from the molecular to behavioural aspects including research in nAChRs, the brain circuits influenced by nicotine to reinforce its consumption, and the animal models and experimental techniques that have brought our knowledge to where it is today.

Premise of the thesis

The present thesis was conducted with the aim of elucidating the role of β4-containing (β4*) nicotinic acetylcholine receptors (nAChRs) in nicotine addiction processes. With a continuing lack of efficient nicotine cessation therapies, who principally target the betterstudied β2* nAChRs, it was deemed important to investigate the therapeutic potential of the β4* nAChRs. This idea was further reinforced by human genetic studies showing an important contribution of the β4 subunit in tobacco smoking behaviours. Previously associated with the long-term effects of nicotine exposure, namely nicotine withdrawal, this subtype of nAChRs had yet to be fully explored in the rewarding effects of nicotine.

This was a mere starting point for the thesis, and led to significant findings in β4* nAChR-cholinergic modulation of nicotine aversion.

The cerebro-regional localisation of the nAChR subtypes is of significant importance, serving to integrate cholinergic and nicotinic information, as previously demonstrated in the hosting laboratory. This information, whether relayed as dopamine release in the striatum or glutamate release in the interpeduncular nucleus, has important behavioural outcomes. In order to stratify the region-specific role of β4* nAChRs, I aimed to develop virus constructs for delivery of β4 cDNA to brain regions of interest in the mouse, and analyse the physiological and behavioural impact of this tailored genetic manipulation.

Before describing the results of the thesis, I shall review the molecular characteristics of the receptor as well as its physiological and behavioural function. The distinct pharmacological properties of β4* nAChRs, and their changes in pharmacology with nicotine exposure, shall also be reviewed. In the context of therapeutic exploration, non-β4* nAChRs shall also be reviewed in order to define the function, whether overlapping or distinct, of various nAChR subtypes. Ad hoc genetic manipulation using nonpathogenic viruses shall also be described. Additionally, the introduction sets the scene for the discussion section, which explores important concepts in nicotine addiction and nicotinic pharmacology that may explain the experimental results.

Introduction 1.a Nicotine, acetylcholine, and the nicotinic acetylcholine receptor

Acetylcholine (ACh) is a neurotransmitter and a paracrine-autocrine signalling molecule.

It is the endogenous ligand of muscarinic and nicotinic acetylcholine receptors (nAChRs), which are also activated by the exogenous ligand, nicotine (figure 1.a.i). The effects of nicotine on the central nervous system were first demonstrated in the 1920s (see [START_REF] Silvette | The actions of nicotine on central nervous system functions[END_REF] for review). ACh, however, was not deemed a neurotransmitter until the 1930s, when Sir Henry Dale showed that ACh was present at the vagus nervestomach junction, earning him the Nobel Prize in 1936. Dale also distinguished the two cholinergic receptors subtypes -the muscarinic AChRs, a metabotropic G-proteincoupled class of receptors, which respond uniquely to muscarine; and the nAChRs, which selectively respond to nicotine and belong to the ligand-gated ion channel family, (see Changeux 2012 for review). An abundant expression of muscle nAChRs in torpedo rays resulted in a multitude of nicotine-based physiological studies, primarily involving Torpedo californica and Torpedo marmorata. This eventually led to the isolation of the receptor [START_REF] Changeux | Use of a snake venom toxin to characterize the cholinergic receptor protein[END_REF][START_REF] Miledi | Isolation of the cholinergic receptor protein of Torpedo electric tissue[END_REF][START_REF] Meunier | Separation of the receptor protein of acetylcholine and acetylcholinesterase[END_REF], and a suggestion that this receptor is pentameric [START_REF] Hucho | Molecular weight and quaternary structure of the cholinergic receptor protein extracted by detergents from Electrophorus electricus electric tissue[END_REF]). The nAChR is, indeed, a pentameric ligand-gated ion channel, permeable to monovalent cations and calcium. 17 nAChR subunits and genes have been identified, classed into two main groups: the primary alpha group (possessing two adjacent cysteines for ACh binding), and the complementary non-alpha group, incorporating the beta, gamma, epsilon and delta subunits [START_REF] Corringer | Nicotinic Receptors at the Amino Acid Level[END_REF]. It was found that the muscle nAChR forms obligatory heteromers with an alpha1-gamma-alpha1-delta-beta1 stoichiometry, with the gamma subunit replacing a fetal epsilon subunit [START_REF] Bibliography Albuquerque | Mammalian nicotinic acetylcholine receptors: from structure to function[END_REF]. Eleven subunits, namely alpha2-7, alpha9-10 and beta2-4, are expressed in mammalian neurons and co-assemble as either homomeric pentamers, in the case of alpha7, or as heteromeric pentamers to form functional receptors of varying subunit stoichiometry. A plethora of naturally occurring nAChR antagonists exist, produced by snails (e.g. alpha-conotoxin MII), snakes (e.g. alpha-bungarotoxin), and plants (e.g. ibogaine). Due to the method of administration (intramuscular bite, cutaneous application), these venoms' and toxins' overarching physiological outcome is to paralyse the cholinergic neuromuscular junction. Oral consumption of plant-based nicotinic compounds such as ibogaine has pronounced neurological effects, including hallucinogenesis [START_REF] Baldwin | The Role of the Habenula in Nicotine Addiction[END_REF]. We have been able to exploit these naturally occurring compounds for the study of nAChR subtypes in the central nervous system.

ACh is synthesised in the cytoplasm by an enzymatic reaction between choline, acetyl-CoA and choline-acetyltransferase. ACh is concentrated into synaptic vesicles by vesicular-acetylcholine-transporter-mediated uptake, ready for release at the synaptic cleft ensuing depolarisation of the neuron. ACh can now exert its effects at pre-, postand extra-synaptic nAChRs, before being hydrolysed by acetylcholine-esterase into choline and acetate. Choline is transported back into the cell by the choline transporter, to be recycled (figure 1.a.ii). Nicotine, on the other hand, is metabolised by liver enzymes, principally forming cotinine, a useful biomarker of nicotine consumption.

Figure 1.a.ii. Schema of acetylcholine's life cycle in a neuron, from it's synthesis in the represented presynaptic cell (1), to its vesicle-mediated release to the synaptic cleft, ( 2) its action on the postsynaptic cell's AChR (3), synaptic degradation (4), and reuptake of cleavage products (5).

These functions are impeded in pathologies of cholinergic systems such as Alzheimer's disease (see [START_REF] Lombardo | Role of the nicotinic acetylcholine receptor in Alzheimer's disease pathology and treatment[END_REF] for review), schizophrenia (see [START_REF] Ross | Research review: Cholinergic mechanisms, early brain development, and risk for schizophrenia[END_REF] for review), autosomal dominant nocturnal frontal lobe epilepsy (see [START_REF] Becchetti | The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy[END_REF] for review), and nicotine addiction (see Changeux 2010 for review). Elucidating the structure and function of nAChRs, in addition to their localisation and physiological function in the central nervous system, will allow us to understand the aetiology of these pathologies, and provide useful insight for treatment strategies. The structure and function of the nAChR shall now be elaborated.

1.b Structure and function of the nicotinic acetylcholine receptor: a cys-loop receptor

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC) and a member of the cys-loop receptor family, along with the neurotransmitter receptors glycine, GABA A , GABA C and 5HT 3 . Common to the cys-loop receptors are a disulphide bridge in the extracellular domain formed between two cysteine residues. The neuronal nAChRs are composed of a combination of alpha (⍺2-7, 9-10) and/or beta (β2-4) subunits, whilst ⍺1, β1, gamma, delta and epsilon subunits are found at the neuromuscular junction. The receptor is endogenously activated by ACh, eliciting passive entry of cations across the channel. Each subunit has an extracellular domain with a characteristic cys loop, four ⍺-helical transmembrane domains, and an intracellular domain (figure 1.b.i). Ultrastructural analysis illustrates symmetrical arrangement of the five subunits forming a central ion pore perpendicular to the plasma membrane into which they are integrated [START_REF] Miyazawa | Structure and gating mechanism of the acetylcholine receptor pore[END_REF]. The first ultrastructural analysis of nAChR came from Mitra and colleagues in 1989 (resolution: 22 Å electron microscopy, 12.5 Å x-ray diffraction). Since then, in silico assessment, x-ray diffraction analysis, and electron microscopy observations of nAChR and other pLGICs have developed our understanding of receptor conformation. Over the past few decades, accumulating evidence has demonstrated the structural and functional similarities between eukaryotic and prokaryotic pLGIC, despite low amino acid sequence homology [START_REF] Corringer | Structure and pharmacology of pentameric receptor channels: from bacteria to brain[END_REF]. The 3D structure of neuronal nAChRs is lacking, but parallels can be drawn between the structure and function of different pLGIC, as shall be described. Much of the information below is extracted from analyses of the helminthic glutamate-gated chloride ion channel (GluCl), the serotonin receptor (5HT3A), and bacterial proteins ELIC and GLIC. This is summarised in figure 1.b.ii. The transmembrane domain (TMD). nAChR subunits, along with all cys-loop receptor subunits, have four ⍺-helical TMDs (M1-M4). An M2 helix from each subunit forms the central ion channel [START_REF] Giraudat | Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta subunit is labeled by [3H]chlorpromazine[END_REF], which contains residues that regulate ion permeability [START_REF] Galzi | Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic[END_REF]). In the case of nAChRs, a ring of anionic amino acids are located near the intracellular portion of the receptor at sites -1' or -2' of M2, where prime digits denote amino acid distance from M2's N terminus [START_REF] Konno | Rings of anionic amino acids as structural determinants of ion selectivity in the acetylcholine receptor channel[END_REF][START_REF] Corringer | Molecular basis of the charge selectivity of nicotinic acetylcholine receptor and related ligand-gated ion channels[END_REF] permeability. M1 and M3 associate with the pore-forming M2, and M4 externally encases this bundle, interacting with the lipid bilayer of the plasma membrane. Deletion of M4 in the pLGIC Glycine receptor prevents subunit assembly suggesting that it also has a role in pentamer assembly [START_REF] Haeger | An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors[END_REF]. The formation of the quaternary structure of the transmembrane domain is independent of the extracellular and intracellular domains of the receptor [START_REF] Bondarenko | NMR Structure of the Transmembrane Domain of the n-Acetylcholine Receptor beta2 Subunit[END_REF].

The extracellular domain (ECD). The ECD is composed of β sheets, folded into an immunoglobulin-like β sandwich [START_REF] Corringer | Nicotinic Receptors at the Amino Acid Level[END_REF]. As mentioned above, TMD structural formation is independent of that of the ECD: conversely, we can deduce that the ECD conformation is defined by the ECD itself, since the TMD-less acetylcholine binding protein (AChBP), demonstrates nAChR-like ECD topography. Of prominent importance, this portion of the receptor forms the ligandbinding site. Corringer and colleagues elucidated the fine structural determinants of the formation of the ACh binding site [START_REF] Corringer | Nicotinic Receptors at the Amino Acid Level[END_REF]. This work demonstrated that the binding site lies at the interface between the principal subunit and complementary subunit extracellular domains. So-called loops A, B and C from the principal subunit (typically nAChR ⍺ subunits) plus loops D, E, F and G from the complementary subunit (typically nAChR β subunits) form the ACh binding domain, figure 1.b.iii [START_REF] Corringer | Nicotinic Receptors at the Amino Acid Level[END_REF]. This was later confirmed in an

x-ray crystallography study of the Aplysia californica AChBP, homolog of the nAChR ECD [START_REF] Brejc | Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors[END_REF]). In the case of the nAChR, the two inter-subunit ligand binding sites are created by topographical assembly of ⍺ and β subunits. The loops are additionally responsible for transmission of the agonist binding event to the TMD for channel opening. This is demonstrated by mutations in loops A, D and E resulting in the channel being constitutively open even in the absence of an agonist [START_REF] Torres | Identification of a tyrosine in the agonist binding site of the homomeric rho1 gamma-aminobutyric acid (GABA) receptor that, when mutated, produces spontaneous opening[END_REF]. Note that the ⍺7 homomeric nAChR presents five ACh binding sites, since the ⍺7 subunit has the prerequisites for both the principal and the complementary binding faces [START_REF] Corringer | Nicotinic Receptors at the Amino Acid Level[END_REF]. A second exception is the subunits ⍺5 and β3, deemed accessory subunits since they do not contribute to ligand binding sites [START_REF] Kuryatov | Roles of accessory subunits in α4β2* nicotinic receptors[END_REF]. Some allosteric modulators bind to the ECD and TMD to potentiate or attenuate full agonist effects [START_REF] Taly | Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system[END_REF]. Considering the context of this thesis, modulatory sites shall not be discussed further.

Intracellular domain (ICD). The nAChR ICD regulates pentamer assembly, trafficking to the plasma membrane, and electrophysiological properties of the receptor [START_REF] Kracun | Influence of the M3-M4 intracellular domain upon nicotinic acetylcholine receptor assembly, targeting and function[END_REF]). Variation is observed in the ICD of different nAChR subunits, owing to the variety in the length of its sequence. This may contribute to the equally divergent electrophysiological properties of nAChR subtypes, and the extent to which they are expressed in different brain regions. Particularly noteworthy is the ICD location of the common single nucleotide polymorphism at amino acid residue 398 in the human α5 nAChR subunit, which increases the risk of nicotine dependence [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF]Frahm et al. 2011). Tyrosine residues in the ICD are targeted for phosphorylationmediated regulation of ACh-induced currents, although the downstream mechanism of action underlying this regulation remains unknown [START_REF] Charpantier | Alpha7 neuronal nicotinic acetylcholine receptors are negatively regulated by tyrosine phosphorylation and Src-family kinases[END_REF]. Yeast-twohybrid screening reveals an interaction of cellular proteins with α4β2 nAChRs. By interaction with the receptor's ICD, Cysteine-Rich with EGF-Like Domains (CRELD2), found in the endoplasmic reticulum, negatively regulates the trafficking of nAChRs to the plasma membrane [START_REF] Ortiz | The cysteine-rich with EGF-like domains 2 (CRELD2) protein interacts with the large cytoplasmic domain of human neuronal nicotinic acetylcholine receptor alpha4 and beta2 subunits[END_REF]. ICD sequence substitutions of ⍺4 and β2 nAChR subunits demonstrated that ⍺4's cytoplasmic loop is necessary for functional expression of the receptor, but not the equivalent structure in the β2 subunit [START_REF] Kuo | Roles for Nicotinic Acetylcholine Receptor Subunit Large Cytoplasmic Loop Sequences in Receptor Expression and Function[END_REF]. This study also demonstrates that the cytoplasmic loops regulate the receptor response to antagonists as well as desensitisation to agonists, highlighting an electrophysiological role of the ICD.

The structure of the nAChR ICD is unresolved, and little information can be drawn from its orthologs since the most studied pLGIC structures come from non-metazoan receptors, which do not bear this cytoplasmic domain. First insight into the structure of a vertebrate pLGIC ICD came recently in a study of a cleaved 5HT 3A receptor, with its ECD, TMD and first 20 amino acids of the ICD intact. Ultrastructural information of this receptor suggests that the initial segment of the ICD forms a tight association at the base of the TM pore in the absence of an agonist, which is hypothesised to be impermeable to ions [START_REF] Hassaine | X-ray structure of the mouse serotonin 5-HT3 receptor[END_REF]. However, since this receptor was studied in the absence of the remaining ICD residues, it is possible that the structure, and resulting In the resting state (R) the receptor is twisted, with an enlarged ECD and a closed channel (see Taly et al. 2009 for review). Agonist binding stabilises the intermediate activation state (I) where the ECD has contracted but the receptor has yet to untwist and the channel remains closed [START_REF] Calimet | A gating mechanism of pentameric ligand-gated ion channels[END_REF]. This state favours thermodynamic transition to the active state (A), unlike the less favourable direct R to A. In the active state, the ECD has untwisted and the channel is open due to displacement of M2 towards its associated M1 and M3, enlarging the pore for ion influx [START_REF] Hibbs | Principles of activation and permeation in an anion-selective Cys-loop receptor[END_REF][START_REF] Miyazawa | Structure and gating mechanism of the acetylcholine receptor pore[END_REF].

A R I D

A desensitised state (D) precedes regression to the resting state. In this desensitised state, the receptor is twisted, the agonist is still bound, but the channel is closed [START_REF] Hilf | X-ray structure of a prokaryotic pentameric ligand-gated ion channel[END_REF][START_REF] Cecchini | The nicotinic acetylcholine receptor and its prokaryotic homologues: Structure, conformational transitions & allosteric modulation[END_REF]. The D to R transition is favoured when the agonist unbinds [START_REF] Taly | Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system[END_REF]. Desensitisation is an important step in regulating the delay for receptor re-activation. A second, distinct rapid-onset desensitisation state is proposed for pLGIC [START_REF] Prevost | A locally closed conformation of a bacterial pentameric proton-gated ion channel[END_REF], which also provides an allosteric path from the active to resting state. Mutagenesis and functional analysis of the β2 subunit of nAChR highlight the extracellular domain as regulating desensitisation properties [START_REF] Bohler | Desensitization of neuronal nicotinic acetylcholine receptors conferred by N-terminal segments of the beta 2 subunit[END_REF], and onset of desensitisation in the ⍺7 homopentamer [START_REF] Bouzat | The interface between extracellular and transmembrane domains of homomeric Cys-loop receptors governs open-channel lifetime and rate of desensitization[END_REF].

The conformational transitions that occur during agonist and antagonist binding are distinct. According to AChBP studies, the direction of loop C movement is dependent on whether an agonist or antagonist binds (Hansen et al. 2005), perhaps being the first mechanical signal to determine the net mechanical outcome (channel opening, or not) of the receptor. The molecular mechanisms shall not be discussed further.

Chronic nicotine exposure

This section will explore the dynamics of nAChR expression during chronic nicotine exposure, and its effect on nicotine-related behaviours that contribute to the addiction process. Three main concepts shall be discussed: upregulation, sensitisation and tolerance. These phenomena are an important component of nicotine's plastic effects on smoking behaviours. Chronic nicotine exposure alters nAChR receptor expression and state, with a profound impact on processes such as striatal dopamine release [START_REF] Exley | Striatal dopamine transmission is reduced after chronic nicotine with a decrease in α6-nicotinic receptor control in nucleus accumbens[END_REF]. The cellular response to chronic nicotine shall therefore be discussed, as well as its physiological and behavioural implications.

1.2.a Chronic nicotine exposure: upregulation

Early radioligand binding measurements of smokers' brains revealed up to a 400% increase in nAChRs in the regions such as the cortex and hippocampus [START_REF] Benwell | Evidence that tobacco smoking increases the density of (-)-[3H]nicotine binding sites in human brain[END_REF][START_REF] Perry | Increased Nicotinic Receptors in Brains from Smokers : Membrane Binding and Autoradiography Studies[END_REF], and that this tobacco-mediated effect is dose-dependent, and reversible after at least two months of abstinence [START_REF] Breese | Effect of Smoking History on [ H ] Nicotine Binding in Human Postmortem Brain 1[END_REF]. These data are reinforced by numerous rodent studies revealing nicotine as the component underlying the described cellular deviation [START_REF] Schwartz | In vivo regulation of [3H]acetylcholine recognition sites in brain by nicotinic cholinergic drugs[END_REF][START_REF] Marks | Effects of Chronic Nicotine Infusion on Tolerance and Nicotinic Receptors1[END_REF][START_REF] Marks | Nicotine Binding and Nicotinic Receptor Subunit RNA after Chronic Nicotine Treatment[END_REF]). The term upregulation was coined to describe this increase in nAChR expression.

Chronic nicotine exposure increases functional ⍺4β2 expression [START_REF] Sallette | Nicotine upregulates its own receptors through enhanced intracellular maturation[END_REF][START_REF] Renda | Chronic nicotine pretreatment is sufficient to upregulate α4* nicotinic receptors and increase oral nicotine self-administration in mice[END_REF][START_REF] Kuryatov | Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human alpha4 beta2 Acetylcholine Receptors[END_REF]Nashmi and Lester 2007;Nashmi et al. 2007;[START_REF] Xiao | Chronic nicotine selevtively enhances α4β2* nicotinic acetylcholine receptors in the nigrostriatal dopamine pathway[END_REF][START_REF] Baker | Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug[END_REF]. Stoichiometry is also affected, with the plasma membrane (PM) insertion of high sensitivity, slowly desensitising ⍺4 2 β2 3 increasing at the compromise of low sensitivity ⍺4 3 β2 2 [START_REF] Kuryatov | Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human alpha4 beta2 Acetylcholine Receptors[END_REF][START_REF] Nelson | Alternate stoichiometries of alpha4beta2 nicotinic acetylcholine receptors[END_REF][START_REF] Srinivasan | Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning[END_REF]. Some report that ⍺6β2 is downregulated [START_REF] Marks | Α6Β2*-Subtype Nicotinic Acetylcholine Receptors Are More Sensitive Than Α4Β2*-Subtype Receptors To Regulation By Chronic Nicotine Administration[END_REF] whilst others describe the contrary [START_REF] Henderson | Nicotine exploits a COPI-mediated process for chaperonemediated up-regulation of its receptors[END_REF]). This may be explained by discriminatory stabilisation of non⍺4-⍺6β2 receptors, but not ⍺4⍺6β2 receptors by nicotine [START_REF] Perez | Long-term nicotine treatment differentially regulates striatal alpha6alpha4beta2* and alpha6(nonalpha4)beta2* nAChR expression and function[END_REF]. With the exception of one study, conducted using nicotine concentrations that exceed those pertinent to the smoker [START_REF] Mazzo | Nicotine-Modulated Subunit Stoichiometry Affects Stability and Trafficking of α3β4 Nicotinic Receptor[END_REF], it is widely accepted that β4* nAChRs are resistant to pharmacological upregulation [START_REF] Nguyen | Subtype-Selective Up-Regulation by Chronic Nicotine of High-Affinity Nicotinic Receptors in Rat Brain Demonstrated by Receptor Autoradiography[END_REF][START_REF] Henderson | Nicotine exploits a COPI-mediated process for chaperonemediated up-regulation of its receptors[END_REF][START_REF] Srinivasan | Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning[END_REF][START_REF] Govind | Nicotine-induced upregulation of native neuronal nicotinic receptors is caused by multiple mechanisms[END_REF][START_REF] Wang | Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells[END_REF]. Mutational analysis demonstrated that resistance to pharmacological upregulation is due to the endoplasmic reticulum (ER) export motif of β4 and its lack of ER retention sequence [START_REF] Srinivasan | Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning[END_REF]. Introducing this sequence profile to the β2 subunit results in a β4-like expression phenotype, that is to say that basal levels of trafficking to the PM are high, and are not increased by chronic nicotine. We anticipate in vivo investigations to confirm, or refute, β4* nAChR's resistance to nicotine-induced upregulation.

The cellular mechanisms driving nicotine-elicited nAChR upregulation have been heavily explored over the past decade. In vitro studies have elucidated the molecular underpinnings of nicotine-mediated nAChR upregulation. There are two major discoveries that formed the upregulation dogma. Firstly, that it is a posttranscriptional incident: whilst the number of nAChRs increases following chronic nicotine exposure, mRNA levels do not [START_REF] Marks | Nicotine Binding and Nicotinic Receptor Subunit RNA after Chronic Nicotine Treatment[END_REF]. Secondly, nicotine acts intracellularly to elicit upregulation [START_REF] Sallette | Nicotine upregulates its own receptors through enhanced intracellular maturation[END_REF]). In the case of heteromeric nAChRs, activation of cell surface nAChRs is not necessary for signalling upregulation. Mutational inhibition of channel opening does not impair upregulation [START_REF] Kuryatov | Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human alpha4 beta2 Acetylcholine Receptors[END_REF]. However, nicotine does indeed act at the agonist binding site, since mutations at binding pocket prevent upregulation [START_REF] Kishi | Role of the agonist binding site in up-regulation of neuronal nicotinic alpha4beta2 receptors[END_REF].

The nicotine concentration of smoker's brain varies from tens of nanomolars after one puff, to mid-hundreds of nanomolars after a full cigarette [START_REF] Rose | Kinetics of brain nicotine accumulation in dependent and nondependent smokers assessed with PET and cigarettes containing 11C-nicotine[END_REF]. This has been considered in the investigations presented below, which allow a synthesis of the cellular events leading to receptor upregulation in the CNS.

1. Nicotine enters the endoplasmic reticulum and stabilises the formation of pentameric nAChRs with particular stoichiometric preferences [START_REF] Kuryatov | Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human alpha4 beta2 Acetylcholine Receptors[END_REF][START_REF] Srinivasan | Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning[END_REF]. Concomitantly, chronic nicotine reduces proteasomal degradation of nAChR subunits in the ER [START_REF] Govind | Nicotine-induced upregulation of native neuronal nicotinic receptors is caused by multiple mechanisms[END_REF].

2. COPI-mediated transport of the nAChR from the ER to the Golgi [START_REF] Henderson | Nicotine exploits a COPI-mediated process for chaperonemediated up-regulation of its receptors[END_REF]).

3. Maturation -glycosylation and trimming of the receptor in the ER is more efficient in presence of nicotine [START_REF] Sallette | Nicotine upregulates its own receptors through enhanced intracellular maturation[END_REF].

4. Mature nAChRs are trafficked to the PM by endocytic vesicles [START_REF] Henderson | Nicotine exploits a COPI-mediated process for chaperonemediated up-regulation of its receptors[END_REF]) Nicotine itself also increases nAChR half-life at the PM [START_REF] Peng | Nicotine-induced increase in neuronal nicotinic receptors results from a decrease in the rate of receptor turnover[END_REF][START_REF] Harkness | Changes in conformation and subcellular distribution of alpha4beta2 nicotinic acetylcholine receptors revealed by chronic nicotine treatment and expression of subunit chimeras[END_REF]. However, evidence to the contrary has also been proposed [START_REF] Vallejo | Chronic nicotine exposure upregulates nicotinic receptors by a novel mechanism[END_REF][START_REF] Govind | Nicotine-induced upregulation of native neuronal nicotinic receptors is caused by multiple mechanisms[END_REF]. The entire process occurs within a three-hour delay [START_REF] Kuryatov | Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human alpha4 beta2 Acetylcholine Receptors[END_REF][START_REF] Sallette | Nicotine upregulates its own receptors through enhanced intracellular maturation[END_REF]. Some studies have shown that nicotinic antagonists are also able to elicit nAChR upregulation. The antagonist DHβE, but not the channel blocker mecamylamine, increases nAChR expression [START_REF] Wang | Chronic nicotine treatment up-regulates human alpha3 beta2 but not alpha3 beta4 acetylcholine receptors stably transfected in human embryonic kidney cells[END_REF]. However antagonist-evoked nAChR upregulation has been refuted by other studies [START_REF] Peng | Chronic nicotine treatment up-regulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y[END_REF][START_REF] Whiteaker | Agonist-Induced Up-Regulation of alpha4 beta2 Nicotinic Acetylcholine Receptors in M10 Cells : Pharmacological and Spatial Definition[END_REF].

ACh is a quaternary amine, with less membrane permeability than the tertiary amine, nicotine. H 3 -epibatidine displacement studies show that nicotine reaches intracellular receptors within 20 minutes [START_REF] Kuryatov | Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human alpha4 beta2 Acetylcholine Receptors[END_REF], whereas ACh may take hours [START_REF] Henderson | Inside-out neuropharmacology of nicotinic drugs[END_REF]. Physiologically, it is not considered that ACh could remain at the synapse for such a time due to the presence of ACh-esterase (AChE) in the synaptic cleft and the rapid uptake of cleaved products back into the presynaptic terminal by choline acetyltransferase. Indeed, Kuryatov and colleagues demonstrate that 300µM ACh can only induce upregulation if AChE is inhibited [START_REF] Kuryatov | Nicotine Acts as a Pharmacological Chaperone to Up-Regulate Human alpha4 beta2 Acetylcholine Receptors[END_REF].

There is therefore perhaps no opportunity for ACh to access intracellular nAChRs under normal physiological circumstances, but this must be confirmed experimentally.

1.2.b Chronic nicotine exposure: sensitisation and tolerance

Chronic nicotine exposure has protracted cellular-level effects in the central nervous system. Nicotine pharmacology is therefore dynamic throughout the smoker's lifetime and may underlie the behavioural changes that occur during nicotine consumption, from occasional use, to abuse. Shifting gears from cellular pharmacology, we shall now explore the physiological and behavioural effects of short-term and chronic nicotine exposure. Two concepts shall be discussed: sensitisation and tolerance. Nicotine sensitisation occurs after short-term pre-exposure to nicotine, causing neuroadaptive changes that increase the organism's response to a given concentration of the drug.

Incentive sensitisation (increased "wanting" of the drug) may represent the physiological underpinnings of the initial steps of drug addiction [START_REF] Robinson | The neural basis of drug craving: an incentivesensitization theory of addiction[END_REF][START_REF] Vanderschuren | Sensitization Processes in Drug Addiction[END_REF]. Chronic nicotine exposure eventually leads to tolerance to nicotine's effects, reducing the physiological or behavioural outcome of a proceeding exposure to nicotine. This is a proposed critical factor in the progression and maintenance of nicotine addiction. Both concepts shall be discussed by assessing experimental data that link conditions known to cause cellular-level upregulation of nAChR expression, or not, and the corresponding physiological and behavioural outcomes.

Common models of chronic nicotine exposure include the use of osmotic minipumps to deliver up to 6mg/kg/h of nicotine for up to several weeks, which models the consumption of a habitual smoker, albeit passive [START_REF] Marks | Nicotine Binding and Nicotinic Receptor Subunit RNA after Chronic Nicotine Treatment[END_REF][START_REF] Renda | Chronic nicotine pretreatment is sufficient to upregulate α4* nicotinic receptors and increase oral nicotine self-administration in mice[END_REF]Nashmi et al. 2007;[START_REF] Henderson | Nicotine exploits a COPI-mediated process for chaperonemediated up-regulation of its receptors[END_REF][START_REF] Nguyen | Subtype-Selective Up-Regulation by Chronic Nicotine of High-Affinity Nicotinic Receptors in Rat Brain Demonstrated by Receptor Autoradiography[END_REF][START_REF] Xiao | Chronic nicotine selevtively enhances α4β2* nicotinic acetylcholine receptors in the nigrostriatal dopamine pathway[END_REF]. A second experimental model presented in the literature is noncontinuous exposure by subcutaneous (s.c) and intra-peritoneal (i.p) injections over the course of several days [START_REF] Baker | Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug[END_REF][START_REF] Ivanová | Nicotine-induced decreases in VTA electrical selfstimulation thresholds: blockade by haloperidol and mecamylamine but not scopolamine or ondansetron[END_REF], which models the initial stages of infrequent nicotine consumption. Intermittent nicotine exposure increases nAChR expression in dopaminergic neurons of the VTA, and increases nicotine-evoked striatal dopamine release [START_REF] Baker | Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug[END_REF]. VTA dopaminergic neurons show an increased response to ACh up to two hours after the last nicotine treatment, yet alterations in glutamatergic input are more persistent, showing an increase in AMPAR-mediated excitatory transmission to the VTA compared to salinetreated controls [START_REF] Baker | Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug[END_REF]. Seven weeks after nicotine pre-treatment, nicotineevoked hyperlocomotion in these rats is increased, despite nAChR expression having returned to baseline [START_REF] Baker | Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug[END_REF]. Similarly, intermittent subcutaneous (s.c.) treatment with nicotine sensitises nicotine-evoked hyperlocomotion in the rat over the course of 5 days, and persists after five days of abstinence from pre-treatment [START_REF] Goutier | The effect of nicotine induced behavioral sensitization on dopamine D1 receptor pharmacology: An in vivo and ex vivo study in the rat[END_REF]. Whilst nAChR upregulation was not determined in this study, the authors observed an increase in efficacy of the accumbal dopamine D1 receptor to dopamine, which may provide an additional mechanism for the nicotine-induced locomotor sensitisation observed by Baker and colleagues [START_REF] Baker | Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug[END_REF]. Three weeks of nicotine exposure increased nicotine-induced hyperlocomotion in rats even seven months after nicotine pre-treatment, which is reflected in the nucleus accumben's increased electrophysiological response to the dopamine D2 receptor agonist, quinpirole, compared to saline pre-treated animals [START_REF] Morud | Nicotine produces chronic behavioral sensitization with changes in accumbal neurotransmission and increased sensitivity to re-exposure[END_REF]. As mentioned, nAChR upregulation of expression in the VTA, at least, can return to baseline within three days [START_REF] Baker | Intermittent nicotine exposure upregulates nAChRs in VTA dopamine neurons and sensitises locomotor responding to the drug[END_REF], and postmortem observations in abstinent human smokers reveals that nicotine-evoked nAChR upregulation is reversed within two months of abstinence in the hippocampus and thalamus [START_REF] Breese | Effect of Smoking History on [ H ] Nicotine Binding in Human Postmortem Brain 1[END_REF]. Whilst nAChR expression was not analysed in Morud's study, the significant abstinence period (seven months) would suggest that other nicotine-evoked neurological adaptations might contribute to the observed sensitisation. With this evidence suggesting that nicotine is capable of sensitising the mesolimbic system, one would therefore question whether intrinsic reward is also altered. Indeed, voluntary [START_REF] Kenny | Nicotine self-administration acutely activates brain reward systems and induces a long-lasting increase in reward sensitivity[END_REF] or passive [START_REF] Ivanová | Nicotine-induced decreases in VTA electrical selfstimulation thresholds: blockade by haloperidol and mecamylamine but not scopolamine or ondansetron[END_REF] daily nicotine exposure decrease the reward threshold for intra-VTA self-stimulation, an effect that persists for over one month after nicotine cessation. Another study, however, shows that volitional, and not experimentercontingent, nicotine self-administration alters the spontaneous activity of VTA dopaminergic neurons due to a nicotine-evoked increase in glutamatergic drive to the VTA from the bed nucleus of stria terminals (BNST)-infralimbic cortex circuit [START_REF] Caillé | Voluntary nicotine consumption triggers in vivo potentiation of cortical excitatory drives to midbrain dopaminergic neurons[END_REF]. In further testament to the malleable nature of the reward pathway in response to nicotine, it was demonstrated that nicotine pretreatment (three s.c. injections at 12h intervals) in adolescent rats is sufficient in increasing nicotine preference and consumption in a two-bottle drinking test (Lee et al. 2015). Saline-injected adolescent rats prefer the water bottle over nicotine, whose bitter taste was not masked by sweeteners, therefore demonstrating that nicotine pre-exposure can motivate nicotine consumption despite adverse effects (taste). This may be an important process in human adolescents during the initiation of tobacco consumption.

Benwell and colleagues report the fine line between prior nicotine exposure and sensitisation or tolerance to further nicotine challenges [START_REF] Benwell | Desensitization of the nicotine-induced mesolimbic dopamine responses during constant infusion with nicotine[END_REF]. Assessing nicotine-induced hyperlocomotion and nicotine-induced striatal dopamine outlfow, the authors show that intermittent pre-treatment with single injections of nicotine for five days sensitises nicotine-mediated hyperlocomotion [START_REF] Benwell | Desensitization of the nicotine-induced mesolimbic dopamine responses during constant infusion with nicotine[END_REF][START_REF] Goutier | The effect of nicotine induced behavioral sensitization on dopamine D1 receptor pharmacology: An in vivo and ex vivo study in the rat[END_REF] and accumbal dopamine release compared to saline-treated controls [START_REF] Benwell | Desensitization of the nicotine-induced mesolimbic dopamine responses during constant infusion with nicotine[END_REF]. 14-day chronic exposure of a low nicotine concentration via minipump sensitises the system (0.25mg/kg/day), but higher doses attenuated the response of the nicotine challenge, arguably a sign of tolerance (1.0 and 4.0mg/kg/day, [START_REF] Benwell | Desensitization of the nicotine-induced mesolimbic dopamine responses during constant infusion with nicotine[END_REF].

Chronic nicotine exposure has been shown to elicit nAChR upregulation in vivo (osmotic minipump, 10 days), however in this regimen, increased receptor expression is specific to midbrain GABAergic inhibitory neurons, resulting in decreased midbrain dopaminergic activity (Nashmi et al. 2007;[START_REF] Xiao | Chronic nicotine selevtively enhances α4β2* nicotinic acetylcholine receptors in the nigrostriatal dopamine pathway[END_REF]. This corresponds to an increase in voluntary nicotine consumption [START_REF] Renda | Chronic nicotine pretreatment is sufficient to upregulate α4* nicotinic receptors and increase oral nicotine self-administration in mice[END_REF], which may be a compensatory response to the decrease in nicotine-evoked accumbal dopamine release [START_REF] Rahman | Neuroadaptive changes in the mesoaccumbens dopamine system after chronic nicotine self-administration: a microdialysis study[END_REF]). β4 knockout (KO) mice show an increase in tolerance to acute nicotine-mediated hypolocomotion and hypothermia following 10 days of chronic nicotine exposure [START_REF] Meyers | Differential expression of the beta4 neuronal nicotinic receptor subunit affects tolerance development and nicotinic binding sites following chronic nicotine treatment[END_REF], whereas β2 KO mice show a decrease in sensitivity to nicotine tolerance in the same test [START_REF] Mccallum | Deletion of the beta 2 nicotinic acetylcholine receptor subunit alters development of tolerance to nicotine and eliminates receptor upregulation[END_REF].

Chronically nicotine-exposed β2 KO mice show no nAChR upregulation, unlike amongst β4 KO mice following chronic nicotine exposure [START_REF] Mccallum | Deletion of the beta 2 nicotinic acetylcholine receptor subunit alters development of tolerance to nicotine and eliminates receptor upregulation[END_REF][START_REF] Meyers | Differential expression of the beta4 neuronal nicotinic receptor subunit affects tolerance development and nicotinic binding sites following chronic nicotine treatment[END_REF]. This supports the idea that β2* and β4* nAChRs have divergent roles in regulating nicotine-mediated neurological and behavioural adaptations. This also highlights the fact that nAChR upregulation is not the sole cellular adaptation that occurs following chronic nicotine exposure. This is supported by a study showing that chronic nicotine exposure in vivo, causing β2* nAChR upregulation, has no effects on β2* receptor-dependent exploratory behaviour, nor on VTA dopaminergic neuronal responses to acute nicotine [START_REF] Besson | Long-term effects of chronic nicotine exposure on brain nicotinic receptors[END_REF]. Since experiments were conducted prior to cessation of chronic nicotine exposure, the authors propose that the alterations in physiological and behavioural events observed may be attributed to the role of upregulated nAChR expression in the absence of nicotine. These neuroadaptive changes following nicotine cessation may underlie the high rate of relapse following attempted abstinence in smokers. The idea that upregulation of nAChRs adapts the neurological homeostatic state only after cessation of nicotine exposure is corroborated by Zhao-Shea and colleagues who show that chronic nicotine increases ⍺3β4 receptor expression on inhibitory IPN interneurons, and in the absence of nicotine permits an increase in glutamatergic input from the MHb to elicit somatic withdrawal symptoms (Zhao-Shea et al. 2013).

Sensitisation and tolerance are important contributing factors to nicotine addiction.

Sensitisation increases the physiological and behavioural response to a particular dose of the drug, whilst tolerance is typified by a decrease in drug-evoked response. Koob and Le Moal posit that sensitisation and opposing processes balance homeostatic functions, serving to maintain drug reward and motivation [START_REF] Koob | Drug Addiction , Dysregulation of Reward , and Allostasis[END_REF]. The competing processes are dis-equilibrated by drug taking, which causes dysregulation of reward/motivation networks [START_REF] Koob | Drug Addiction , Dysregulation of Reward , and Allostasis[END_REF] by, for example, changes in nAChR expression and profile and drug-induced facilitation of synaptic long-term potentiation (Nashmi et al. 2007). Whilst such neuroadaptations are perhaps of benefit in nature (e.g. for foraging), nicotine hijacks this system, perpetuating its own consumption as well as that of other drugs of abuse [START_REF] Olausson | Repeated nicotine exposure enhances responding with conditioned reinforcement[END_REF].

Nicotinic acetylcholine receptor localisation in the rodent brain

The nAChR expression pattern in the CNS forms a complicated map, with certain brain regions richly embellished in diverse receptor subtypes (for example the habenulointerpeduncular pathway), and others exposing a more conservative medley of nAChRs.

It is important to elucidate the nAChR subtypes that mediate the cholinergic signalling contributing to important cognitive, affective and motivational functions including attention, arousal and reward.

Due to the lack of specific antibodies against the various types of nAChRs [START_REF] Moser | Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures[END_REF]), we have relied on low-resolution techniques for the localisation of nAChR subtypes, for example radioligand binding and immunoprecipitation. Subunit gene expression, however, can be mapped at a greater resolution thanks to in situ hybridisation, single-cell PCR and fluorphore-tagged subunits, allowing us to characterise the neuronal subtypes that express different nAChR subunits.

The most promiscuously expressed of the nAChRs, are the ⍺4β2* and ⍺7 nAChRs.

Particularly strong ⍺7-specific [I 125 ]⍺-bungarotoxin staining is present at the superior colliculus and hippocampus, and is abolished in ⍺7 KO mice (Baddick and Marks 2011). β2* nAChR localisation has been revealed by [I 125 ]-A85380 binding, illustrating the widespread expression of this receptor subtype in the CNS. Whilst β2 genetic deletion abolished [I 125 ]-A85380 binding, the staining is incompletely reduced in the ⍺4 KO mouse, with residual binding being found in the superior colliculus, interpeduncular nucleus and substantia nigra. [I 125 ]-A85380 binding is reduced in these regions in ⍺6 and β3 KO mice (Baddick and Marks 2011), suggesting a (non-⍺4)β2* conformation of nAChR. This is corroborated in heterologous expression systems, demonstrating a plethora of different subunits that can assemble with β2 to form functional nAChRs, including ⍺3(⍺5)β2 (Fenster et al. 1997;[START_REF] Nelson | Single channel properties of human alpha3 AChRs: impact of beta2, beta4 and alpha5 subunits[END_REF], ⍺6(⍺3)β2 (McIntosh et al. 2004a;[START_REF] Luo | A novel alpha4/7-conotoxin LvIA from Conus lividus that selectively blocks alpha3beta2 vs. alpha6/alpha3beta2beta3 nicotinic acetylcholine receptors[END_REF] and even ⍺7β2, which defies previous perception that ⍺7 is only found as a homopentameric receptor [START_REF] Moretti | The Novel α7β2-nicotinic Acetylcholine Receptor Subtype is Expressed in Mouse and Human Basal Forebrain: Biochemical and Pharmacological Characterisation[END_REF]).

[ 125 I]⍺-conotoxinMII labeling is absent in the mouse brain of ⍺6 KO mice, with marked reductions in the striatum, superior colliculus and VTA [START_REF] Champtiaux | Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice[END_REF]. This is also observed in ⍺4β2 double KO mice [START_REF] Marubio | Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors[END_REF], suggesting that ⍺6 associates with ⍺4 and β2 subunits in the mouse brain. The principal ⍺6-containing conformation, however, is the (⍺4)⍺6β2β3 nAChR, which is particularly represented in the striatum and controls evoked increases in dopamine release by local application of nicotine [START_REF] Quik | Role of alpha6 nicotinic receptors in CNS dopaminergic function: Relevance to addiction and neurological disorders[END_REF]. β4* nAChRs are most infamously expressed in the autonomic ganglia. Not to be neglected, however, is the function and localisation of β4 in the brain. The subunit demonstrates a discreet expression profile, restricted to the medial habenula (MHb), interpeduncular nucleus (IPN), mitral cells of the olfactory bulb, the inferior colliculus, and to a lesser extent the VTA (Scholze et al. 2012;Beiranvand et al. 2014;Salas et al. 2003;Klink et al. 2001). ⍺3 subunit expression grossly overlaps with that of β4 (Frahm et al. 2011;Salas et al. 2004;Shih et al. 2014), which is somewhat expected given that their genes are neighbouring and share regulatory domains [START_REF] Boulter | Alpha 3, alpha 5, and beta 4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster[END_REF]). ⍺3 is the main, if not obligatory, ⍺ subunit partner of β4 in the mouse (Grady et al. 2009), although functional ⍺4β4 conformations have been described in heterologous expression systems using human nAChR subunits (Fenster et al. 1997). β3 can coassemble with a3β4 to form a functional receptor [START_REF] Boorman | Stoichiometry of human recombinant neuronal nicotinic receptors containing the b3 subunit expressed in Xenopus oocytes[END_REF]Grady et al. 2009), as can ⍺5 [START_REF] George | Function of Human α3β4α5 Nicotinic Acetylcholine Receptors is Reduced by the α5(Asp398Asn) Variant[END_REF][START_REF] Nelson | Single channel properties of human alpha3 AChRs: impact of beta2, beta4 and alpha5 subunits[END_REF] and β2 [START_REF] Quick | Alpha3beta4 subunit-containing nicotinic receptors dominate function in rat medial habenula neurons[END_REF]). ⍺5 and β3, deemed accessory subunits since they do not contribute to ligand binding, display an elaborate profile in the CNS [START_REF] Deneris | Beta3: A new member of nicotinic acetylcholine receptor gene family is expressed in brain[END_REF]Hsu et al. 2013). ⍺5 subunits are expressed in the cortex, hippocampus, olfactory bulb, midbrain, raphe nucleus, amygdala, laterodorsal tegmentum, posterodorsal tegmentum, (hypo)thalamus, and IPN [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]Shih et al. 2014;Hsu et al. 2013;[START_REF] Gensat | Gene Expression Nervous System Atlas. 'cholinergic receptor, nicotinic, alpha polypeptide 5[END_REF]. There is debate as to whether ⍺5 is found in the MHb, with transgenic mice arguing against (Hsu et al. 2013) and immunoprecipitation arguing for (Scholze et al. 2012). ⍺5 co-assembles with ⍺4β2 as well as ⍺3β4 (Grady et al. 2009;Scholze et al. 2012). β3, on the other hand, is highly enriched in the MHb and plays an important role at MHb terminals (Shih et al. 2014;Grady et al. 2009). High levels of β3 mRNA are also found in the substantia nigra and VTA, and are important in mediating nigrostriatal and mesostriatal dopamine release [START_REF] Cui | The beta3 nicotinic receptor subunit: a component of alpha-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors[END_REF]. ⍺6 in situ hybridisation reveals rich expression in the VTA [START_REF] Champtiaux | Distribution and pharmacology of alpha 6-containing nicotinic acetylcholine receptors analyzed with mutant mice[END_REF]. The localisation of nAChR subtypes is summarised in figure 1.3. It is important to note that interspecies differences in nAChR brain localisation exist. The localisation of the various nAChR subunits is very similar between the rodent and the macaque. However, whereas ⍺2 subunit expression in the rodent is limited to the hippocampus and IPN, a greatly diffuse expression pattern is observed in the macaque [START_REF] Han | Localization of nAChR subunit mRNAs in the brain of Macaca mulatta[END_REF]Changeux 2010). In this species, ⍺2 expression localisation resembles that of ⍺4 and β2, and may therefore be of wider functional significance in the primate brain than in rodents.

Considering the pharmacological and kinetic differences between the different nAChR subtypes, it is important to identify the localisation of each receptor in its respective neuronal circuit in order to understand the mechanisms of the cognitive functions that they underlie. As with many behaviours, the neurological functions mediating nicotine consumption are far from a simple mono-circuit mechanism, with many neurophysiological processes intertwined in the motivation to consume, or not, nicotine.

The potential complexity increases when considering the neuronal subtypes within a brain region that might express nAChRs, and to which neuronal populations they then project. Nature's complexity may, on the other hand, be advantageous to the clinician in treating nicotine addiction and minimising unwanted side effects.

Neural circuits of nicotine reward

Addiction is a composition of drug-induced physiological and psychological progressions, leading from infrequent use to compulsive abuse [START_REF] Koob | Neurobiological mechanisms in the transition from drug use to drug dependence[END_REF]. As difficult as it is to delineate its unitary stages, is the difficulty in attributing the behavioural components to specific brain regions. Despite this, one main brain region comes to the forefront when discussing addiction: the ventral tegmental area (VTA). Functional magnetic resonance imaging (fMRI) of the anaesthetised mouse demonstrates that acute nicotine activates several cortical regions, the substantia nigra, and the VTA [START_REF] Suarez | Brain activation by short-term nicotine exposure in anesthetized wild-type and beta2-nicotinic receptors knockout mice: A BOLD fMRI study[END_REF]. Furthermore, fMRI of humans shows that nicotine elicits activity in the nucleus accumbens, target of VTA neurons [START_REF] Stein | Nicotine-induced limbic cortical activation in the human brain: a functional MRI study[END_REF]. In fact, optogenetic stimulation of the VTA supports self-administration behaviour and behavioural conditioning (Ilango et al. 2014;[START_REF] Tsai | Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning[END_REF]).

The VTA is a dopaminergic nucleus and predominantly participates in two neuronal pathways: the mesolimbic pathway (VTA to nucleus accumbens) and the mesocortical pathway (VTA to prefrontal cortex). The VTA has been associated with reward prediction error, attributing reward value, motivation, and Pavlovian conditioning of otherwise nonsalient cues. The VTA is activated by natural rewards, such as food and sex, and also by noxious stimuli, allowing implicit shaping of future behaviours when confronted again with such contexts. Dopamine-mediated association of cue, stimulus and reward (whether positive or negative) is rapidly acquired, which is important in the assessment of one's environments (see Di Chiara 2002 for review). The VTA therefore provides a key survival aid in the world's dynamic environment; however, drugs of abuse hijack this system.

To more recent renown, the habenulo-interpeduncular pathway is also involved in nicotine reward, amongst other emotional and cognitive roles, such as anxiety, nicotine withdrawal, nicotine aversion, and primary reinforcement [START_REF] Haller | Functional Characterization Improves Associations between Rare Non-Synonymous Variants in CHRNB4 and Smoking Behavior[END_REF]Kobayashi et al. 2013;Zhao-Shea et al. 2013;Frahm et al. 2011). The cholinergic-glutamatergic medial habenula (MHb) and its target, the GABAergic interpeduncular nucleus (IPN), are richly embellished with nAChRs, and are therefore particularly subject to activation by nicotine. Inhibiting the MHb or the IPN affects nicotine self-administration with a corresponding modulation of striatal dopamine release (Glick et al. 2011;McCallum et al. 2012). The habenulo-peduncular pathway and mesolimbic pathway are therefore intimately linked, and of particular importance in this thesis.

The role of these structures in the rewarding, and positively reinforcing properties of nicotine shall first be discussed. A proceeding section shall broach the roles of these structures in aversion to nicotine.

The ventral tegmental area

The VTA is a midbrain structure found medial to the substantia nigra. It is deemed the central hub of reward prediction error, value coding, motivation and (drug) reinforcement learning, since VTA responding correlates with these behaviours [START_REF] Phillips | Subsecond dopamine release promotes cocaine seeking[END_REF][START_REF] Roitman | Dopamine operates as a subsecond modulator of food seeking[END_REF][START_REF] Schultz | Responses of monkey dopamine neurons to reward and conditioned stimuli during successive steps of learning a delayed response task[END_REF][START_REF] Pan | Dopamine cells respond to predicted events during classical conditioning: evidence for eligibility traces in the reward-learning network[END_REF][START_REF] Berridge | From prediction error to incentive salience: mesolimbic computation of reward motivation[END_REF][START_REF] Tsai | Phasic firing in dopaminergic neurons is sufficient for behavioral conditioning[END_REF]. It integrates information from appetitive and noxious stimuli either by direct activation of VTA neurons or by stimulation of upstream regulatory networks. The magnitude of VTA dopaminergic neuron response correlates with value of the stimulus or the associated sensory cue [START_REF] Schultz | Predictive reward signal of dopamine neurons[END_REF][START_REF] Tobler | Adaptive coding of reward value by dopamine neurons[END_REF][START_REF] Satoh | Correlated coding of motivation and outcome of decision by dopamine neurons[END_REF], although positive responding to noxious stimuli also occurs and shall be discussed in the proceeding section. Dopamine-ascribed reward valence is important in modulating the probability of future approach of the stimulus (Fields et al. 2007). Increasing this behaviour's probability is termed positive reinforcement (e.g. Article One, voluntary self-administration of low doses of nicotine), whereas decreasing the probability of approach is termed negative reinforcement (e.g. Article Two, avoidance of high doses of nicotine). This dogma gains complexity, however, when the subject is presented with a choice of two rewards, and we see that VTA-attributed value is not always representative of explicit preference [START_REF] Hollon | Dopamine-associated cached values are not sufficient as the basis for action selection[END_REF]).

1.4.1.a VTA anatomy and function

The VTA consists of approximately 60% dopaminergic neurons, 35% GABA-ergic neurons and 2-3% glutamatergic (Zhao-Shea et al. 2013;[START_REF] Nair-Roberts | Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat[END_REF], sending predominantly dopaminergic efferents to the nucleus accumbens (NuAcc) core and shell, and the prefrontal cortex (PFC) to form the so-called mesolimbic and mesocortical network, respectively (figure 1.4.1.a.i). Dopaminergic activity is modulated by the VTA's GABAergic neurons [START_REF] Van Zessen | Activation of VTA GABA neurons disrupts reward consumption[END_REF]Tolu et al. 2013) as well as from efferents from the dorsal raphe nucleus, laterodorsal tegmentum, pedunculopontine tegmentum, lateral habenula, and reciprocal regulation from the NuAcc and prefrontal cortex [START_REF] Grace | Regulation of firing of dopaminergic neurons and control of goal-directed behaviors[END_REF]Geisler and Zahm 2005;Fields et al. 2007). The GABAergic interneurons of the VTA, along with afferent input, provide flexibility in the mesolimbic and mesocortical systems, which shall now be discussed. Midbrain dopaminergic activity is typified by two types of firing modes: tonic activity (slow, regular single spike firing, 2-10 Hz) or phasic, burst firing [START_REF] Grace | Regulation of firing of dopaminergic neurons and control of goal-directed behaviors[END_REF]Faure et al. 2014). Tonic firing patterns arise from the cell's intrinsic oscillations in membrane potential, whereas phasic burst firing depends on extrinsic input from the neuron's glutamatergic afferents and cholinergic input from the LDTg [START_REF] Grace | Regulation of firing of dopaminergic neurons and control of goal-directed behaviors[END_REF][START_REF] Gonon | Nonlinear relationship between impulse flow and dopamine released by rat midbrain dopaminergic neurons as studied by in vivo electrochemistry[END_REF]Faure et al. 2014;[START_REF] Blaha | Modulation of dopamine efflux in the nucleus accumbens after cholinergic stimulation of the ventral tegmental area in intact, pedunculopontine tegmental nucleus-lesioned, and laterodorsal tegmental nucleus-lesioned rats[END_REF]. Furthermore, optogenetically activating VTA GABAergic neurons inhibits dopaminergic spontaneous firing via inhibitory postsynaptic currents [START_REF] Tan | GABA neurons of the VTA drive conditioned place aversion[END_REF], confirming the integral role of this subpopulation of neurons on dopaminergic activity. The spontaneous firing mode of these neurons generates a baseline level of dopamine efflux from the neurons' terminals, allowing both positive and negative modulation. Midbrain dopaminergic neurons transition to a phasic firing state when, for example, a cue predicting reward is presented [START_REF] Schultz | Predictive reward signal of dopamine neurons[END_REF][START_REF] Grace | Regulation of firing of dopaminergic neurons and control of goal-directed behaviors[END_REF]. In vivo electrophysiological recordings of VTA dopaminergic neurons in the anaesthetised mouse have further characterised the neurons' electrical profile into four classes: low firing, low bursting (LFLB); low firing, high bursting (LFHB); high firing, low bursting (HFLB); high firing, high bursting (HFHB) (figure 1.4.1.a.ii;[START_REF] Mameli-Engvall | Hierarchical control of dopamine neuron-firing patterns by nicotinic receptors[END_REF]Faure et al. 2014;Changeux 2010). Increased VTA dopaminergic electrophysiological activity is reflected at the terminals, showing increased dopamine release [START_REF] Garris | Different kinetics govern dopaminergic transmission in the amygdala, prefrontal cortex, and striatum: an in vivo voltammetric study[END_REF]. The stimulus-evoked dopamine release profiles in the NuAcc and medial PFC are very different. In vivo electrically evoked dopamine release in the NuAcc can reach an external concentration of 4µM, and is rapidly recuperated by dopamine transporters. The mPFC however, owing to a smaller quantity of dopamine supply, shows a maximal evoked dopamine release concentration of 1µM, which is slowly removed from the extracellular space [START_REF] Garris | Different kinetics govern dopaminergic transmission in the amygdala, prefrontal cortex, and striatum: an in vivo voltammetric study[END_REF].

1.4.1.b VTA nAChRs and nicotine reward

By no exception to other drugs of abuse, nicotine reinforcement properties have been attributed to this midbrain structure. Nicotine expedites reward, showing nicotine-evoked facilitation of intracranial self-stimulation [START_REF] Fowler | Role of α5* nicotinic acetylcholine receptors in the effects of acute and chronic nicotine treatment on brain reward function in mice[END_REF][START_REF] Kenny | Nicotine self-administration acutely activates brain reward systems and induces a long-lasting increase in reward sensitivity[END_REF][START_REF] Ivanová | Nicotine-induced decreases in VTA electrical selfstimulation thresholds: blockade by haloperidol and mecamylamine but not scopolamine or ondansetron[END_REF]. Conversely, the reward threshold is increased by the nAChR antagonist mecamylamine [START_REF] Fowler | Role of α5* nicotinic acetylcholine receptors in the effects of acute and chronic nicotine treatment on brain reward function in mice[END_REF].

Glutamatergic input is essential in nicotine's activation of the reward circuit, and an NMDA antagonist can even cause an elevation in the reward threshold in the presence of nicotine [START_REF] Kenny | NMDA receptors regulate nicotine-enhanced brain reward function and intravenous nicotine self-administration: role of the ventral tegmental area and central nucleus of the amygdala[END_REF]. It has been shown that nicotine is sufficient in shifting VTA dopaminergic neuron activity from tonic to high bursting responses, as well as increasing accumbal dopamine release. Nicotine exerts this effect by direct activation of nAChRs in the striatum [START_REF] Rice | Nicotine amplifies reward-related dopamine signals in striatum[END_REF], VTA dopaminergic neuron soma (Tolu et al. 2013), and by recruiting upstream modulatory circuits such as the bed nucleus of stria terminalis-infralimbic cortex [START_REF] Caillé | Voluntary nicotine consumption triggers in vivo potentiation of cortical excitatory drives to midbrain dopaminergic neurons[END_REF]. In further testament to the VTA's orchestration of nicotine reward, mice voluntarily self-administer nicotine directly into this structure (David et al. 2006;Exley et al. 2011). Furthermore, systemically applied nicotine elicits an increase in accumbal dopamine release (Maskos et al. 2005;[START_REF] Besson | Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice[END_REF][START_REF] Reperant | Effect of the alpha4beta2* nicotinic acetylcholine receptor partial agonist varenicline on dopamine release in beta2 knock-out mice with selective re-expression of the beta2 subunit in the ventral tegmental area[END_REF]Picciotto M. R. 1998;[START_REF] Forget | Noradrenergic alpha1 receptors as a novel target for the treatment of nicotine addiction[END_REF]. It has also been demonstrated in acute slices of the NuAcc, that nicotine acting at dopaminergic terminals, and therefore isolated from VTA-evoked alterations in membrane potential, is sufficient in eliciting dopamine release, therefore illustrating the important component of presynaptic modulation in the VTA's targeted brain regions [START_REF] Cragg | Meaningful silences: how dopamine listens to the ACh pause[END_REF][START_REF] Exley | Presynaptic nicotinic receptors: a dynamic and diverse cholinergic filter of striatal dopamine neurotransmission[END_REF]. Conversely, nicotine self-administration is attenuated by pharmacological inhibition of dopamine receptors [START_REF] Corrigall | Selective dopamine antagonists reduce nicotine selfadministration[END_REF].

The VTA displays a rich diversity of nAChR subtypes, which have been heavily investigated in the context of nicotine reinforcement. Thanks to the expression of nAChRs on dopaminergic and GABAergic neuronal populations, as well as presynaptically on the terminals that innervate this structure, VTA neurons can integrate cholinergic and nicotinic stimulation and translate it into increases in dopamine release at its terminals [START_REF] Gonon | Nonlinear relationship between impulse flow and dopamine released by rat midbrain dopaminergic neurons as studied by in vivo electrochemistry[END_REF][START_REF] Blaha | Modulation of dopamine efflux in the nucleus accumbens after cholinergic stimulation of the ventral tegmental area in intact, pedunculopontine tegmental nucleus-lesioned, and laterodorsal tegmental nucleus-lesioned rats[END_REF]. This change in dopamine release is one of the neurological foundations of reward processing, and is a prerequisite to addiction [START_REF] Wise | The development and maintenance of drug addiction[END_REF][START_REF] Wise | Forebrain substrates of reward and motivation[END_REF]) and depends on ⍺4(⍺6)(⍺5)β2* nAChRs (Picciotto M. R. 1998;Maskos et al. 2005;Orejarena et al. 2012;Tolu et al. 2013;Morel et al. 2014;Pons et al. 2008) and ⍺7 nAChRs [START_REF] Besson | Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice[END_REF]. This is reviewed in detail in section 1.6. A single-cell RT-PCR study by Klink and colleagues illustrates the diversity of nAChR subunit expression amongst midbrain dopaminergic and GABAergic neurons (Klink et al. 2001). ⍺4 and ⍺7 are distributed equally amongst neuronal subpopulations, whereas other subunit mRNAs were heterogenous amongst the neuronal populations.

⍺5, ⍺6 and β3 subunits segregate to the dopaminergic neurons, whereas β4, albeit sparse, is concentrated in GABAergic neurons. According to this study, VTA GABA neurons exclusively express ⍺4, β2 and ⍺3 subunits at high levels. Electrophysiological analysis of these neurons highlights ⍺4β2* as the predominant nAChR type expressed in the VTA, with ⍺7 homomeric nAChRs contributing to half of the cells' responses to nicotine. For an overview of nAChR subtype expression according to cell-type and sub- VTA activation and resultant accumbal dopamine release appears to be important neural prerequisite for nicotine reinforcement and self-administration, but other brain regions upstream of the VTA are also modulated by nicotine. Notably, nAChR inhibition in the MHb and IPN affect dopamine release and nicotine self-administration (Glick et al. 2011;McCallum et al. 2012). The habenulo-interpeduncular pathway shall therefore be discussed next.

The medial habenula

The habenulo-interpeduncular network displays the richest diversity of nAChRs in the central nervous system. It is also the almost exclusive site of β4* nAChR expression, the protagonist of this thesis. It is therefore not surprising that this network is implicated in a variety of nicotine-mediated processes, including modulation of nicotine selfadministration, nicotine aversion, and nicotine withdrawal (Antolin-Fontes et al. 2014).

The function of the medial habenula (MHb) and the interpeduncular nucleus (IPN), and their contributions to nicotine dependence processes, shall now be discussed.

1.4.2.a MHb anatomy and function

The habenula flanks the dorsal third ventricle. In mammals, the habenula can be functionally and cytologically subdivided into the lateral and medial portions. The lateral habenula (LHb) projects glutamatergic neurons to the rostromedial tegmental nucleus, which then relays GABAergic signalling onto the dopaminergic neurons of the VTA and substantia nigra [START_REF] Hikosaka | The habenula: from stress evasion to value-based decision-making[END_REF]. The LHb is a source of reward-circuit modulation [START_REF] Velasquez | The role of the habenula in drug addiction[END_REF], but shall not be discussed further in the present thesis.

The MHb demonstrates a simple delineation of its neuronal populations. Substance P (SP)-positive neurons are segregated in the dorsal third of the MHb, and innervate the lateral IPN, its almost sole target (Herkenham and Nauta 1979;Antolin-Fontes et al. 2014). The ventral two thirds of the MHb are densely occupied with cholinergic neurons that co-release glutamate to the central, rostral and caudal IPN (Yamaguchi et al. 2013;Shih et al. 2014;[START_REF] Ren | Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes[END_REF]Qin and Luo 2009;[START_REF] Girod | Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors[END_REF]. The MHb sparsely innervates the LHb [START_REF] Kim | Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus[END_REF] and the median raphe nucleus (Herkenham and Nauta 1979). The MHb receives input primarily from the septum via the stria medullaris and projects its afferents via the fasiculus retroflexus, which traverses the VTA to reach the IPN [START_REF] Andres | Subnuclear organization of the rat habenular complexes[END_REF][START_REF] Hikosaka | The habenula: from stress evasion to value-based decision-making[END_REF]Klemm 2004). The afferent and efferent connections of the MHb are summarised in figure 1.4.2.a. Specific lesions of the triangular septum, which projects to the ventral MHb, attenuates anxiety, whereas ablation of the bed nucleus of the anterior commissure, which projects to the dorsal MHb, increases fear responses in the mouse (Yamaguchi et al. 2013).

These inputs are glutamatergic and GABAergic, respectively, with cholinergic input provided by the posterior septum (Contestabile and Fonnum 1983;Qin and Luo 2009).

Considering the work of Semenova and colleauges showing that the β4 KO mice demonstrate reduced anxiety, one might postulate that this is due to impaired activity of the otherwise β4-rich ventral MHb in response to the triangular septum [START_REF] Semenova | Mice Lacking the β4 Subunit of the Nicotinic Acetylcholine Receptor Show Memory Deficits, Altered Anxiety-and Depression-Like Behavior, and Diminished Nicotine-Induced Analgesia[END_REF]. Genetic depletion of the predominantly SPergic dorsal portion of the MHb results in reduced exercise motivation as well as reduced sucrose preference, a sign of anhedonia (Hsu et al. 2014).

The cholinergic, but not peptidergic, neurons of the MHb display spontaneous tonic firing at a frequency of 1-10Hz ex vivo (Shih et al. 2014;[START_REF] Kim | Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus[END_REF], which is controlled by intrinsic hyperpolarization-activated cyclic nucleotide-gated channels (Görlich et al. 2013). The MHb spontaneous firing frequency in adolescent rats is at 0.1-16Hz (Lee et al. 2015). Importantly, the cholinergic and non-cholinergic neuronal subpopulations are both receptive to nicotine, showing ⍺3β4* and ⍺5* nAChRdependent nicotine-evoked excitatory postsynaptic currents and increases in firing frequency (Görlich et al. 2013;Hsu et al. 2013;Shih et al. 2014;Dao et al. 2014;Lee et al. 2015;[START_REF] Girod | Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors[END_REF]. The subtypes, and expression pattern of nAChR expression shall now be discussed.

1.4.2.b MHb nAChRs and function

Immunoprecipitation studies, autoradiography, in situ hybridisation, electrophysiology and fluorescently tagged nAChR subunits have given insight into the types of nAChRs that are expressed in the MHb. With exquisite precision, Shih and colleagues demonstrated that β4 subunits area found in the ventral, cholinergic portion of the MHb, subdivisions of which project to distinct regions of the IPN (Shih et al. 2014). The same analysis was conducted using ⍺3-, ⍺4-, ⍺6-, β2-and β3-GFP mouse lines, demonstrating the overlap of expression of these nAChR subunits in subdivisions of the ventral MHb. A study by Hsu and colleagues using a GFP-tagged ⍺5 transgenic mouse raises a caveat regarding the interpretation of these mouse lines (Hsu et al. 2013).

Notably, no ⍺5-associated GFP fluorescence was observed in the VTA, contrary to RT-PCR proof of the ⍺5 subunit's expression in this structure (Klink et al. 2001), nor was any ⍺5-GFP detected in the MHb despite immunoprecipitation studies arguing the contrary (Scholze et al. 2012). The authors attribute the discrepancy in ⍺5-GFP localisation to the fact that the "required Chrna5 gene regulatory sequences lie outside the BAC used to generate the transgene". Many studies corroborate the dominance of ⍺3β4* nAChRs in the MHb (Frahm et al. 2011;Scholze et al. 2012;Shih et al. 2014;Baddick and Marks 2011). Functional analysis demonstrates that MHb expresses ⍺3β4* in majority, and that β2* contributes little to the expression profile [START_REF] Quick | Alpha3beta4 subunit-containing nicotinic receptors dominate function in rat medial habenula neurons[END_REF]. (Scholze et al. 2012). This was determined by immunoprecipitation studies.

nAChRs are not only important in mediating ACh-or nicotine-evoked activity in the MHb, but also play an integral role at the MHb terminals in the IPN. The β4* nAChR antagonist, hexamethonium, is most potent at the MHb soma, intermediate at IPN soma, and lowest at MHb terminals in the IPN [START_REF] Mulle | Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system[END_REF], illustrating the differential distribution of β4* nAChRs along the MHb-IPN axis. However, one must not underestimate the contribution of β4* nAChRs to presynaptic pharmacology. Indeed, β4* (namely ⍺3(β3)β4) nAChRs are necessary for ACh release from IPN synaptosomes (Grady et al. 2009;Beiranvand et al. 2014) as well as for noradrenaline release (Beiranvand et al. 2014). In 2000, Girod and colleagues demonstrated that pre-synaptic nAChRs also modulate glutamate release in MHb-IPN co-cultures, naming ⍺7 and ⍺5* receptors as the facilitators of nicotine-mediated glutamate release [START_REF] Girod | Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors[END_REF]. Unfortunately ⍺5's partner in this process was not investigated. Whilst presynaptic nAChRs undoubtedly facilitate neurotransmitter release, we know that action potentials in the MHb axon are sufficient in eliciting glutamate release from their terminals [START_REF] Ren | Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes[END_REF]. A stimulation frequency of up to 20Hz results in glutamate release, and a greater stimulation will result in ACh release [START_REF] Ren | Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes[END_REF]. Note that spontaneous, tonic firing of MHb neurons ex vivo reaches 10Hz (Shih et al. 2014;[START_REF] Kim | Dendritic morphology, local circuitry, and intrinsic electrophysiology of neurons in the rat medial and lateral habenular nuclei of the epithalamus[END_REF], and can achieve 20Hz during nicotine withdrawal (Görlich et al. 2013). One would need to investigate the MHb's electrophysiological responses in vivo in order to determine the conditions in which ACh release is elicited.

1.4.2.c The MHb and nicotine reward

The MHb has been implicated in several components of nicotine addiction, namely nicotine aversion, nicotine withdrawal, and, albeit less explored, nicotine reward.

The stria medullaris, principal input to the LHb and MHb, supports electrical selfstimulation [START_REF] Blander | Anatomical mapping of brain stimulation reward sites in the anterior hypothalamic area: special attention to the stria medullaris[END_REF][START_REF] Vachon | Dorsal diencephalic self-stimulation: a movable electrode mapping study[END_REF], as does the MHb and the fasiculus retroflexus [START_REF] Sutherland | Self-Stimulation of the Habenular Complex in the Rat[END_REF], implicating MHbcontaining circuitry in mediating behavioural reinforcement. It is interesting to note that this self-stimulation is abolished by a lesion of the dorsal raphe nucleus, an important relay to the VTA [START_REF] Sutherland | Self-Stimulation of the Habenular Complex in the Rat[END_REF]. Furthermore, optogenetic selfstimulation of the dorsal portion of the MHb is rewarding, and allows rapid discrimination learning of rewarding and non-rewarding actions (Hsu et al. 2014). Conversely, optogenetic inhibition of the dorsal MHb conditions place aversion (Hsu et al. 2014), highlighting the intrinsic reinforcing (positive and negative) properties of this structure.

Animal studies have also provided evidence that the MHb modulates self-administration of rewarding concentrations of nicotine. Lidocaine-mediated inhibition of MHb increased intra venous self-administration of an otherwise non-salient, high dose of nicotine, as did NMDA receptor antagonism in these structures (Fowler et al. 2011). This finding is surprising, since previous literature suggests that AMPA, and not NMDA receptors, are expressed in the MHb [START_REF] Robertson | Ca2+ permeability and kinetics of glutamate receptors in rat medial habenula neurones: implications for purinergic transmission in this nucleus[END_REF]. Further in vivo pharmacological assessment has elucidated the role of MHb β4* nAChRs on nicotine self-administration. MHb application of the β4* nAChR antagonist, 18MC, reduces nicotine, morphine and amphetamine self-administration in the rat, with no effect on sucrose consumption (Glick et al. 2011;[START_REF] Glick | Brain regions mediating alpha3beta4 nicotinic antagonist effects of 18-MC on methamphetamine and sucrose selfadministration[END_REF][START_REF] Glick | 18-Methoxycoronaridine acts in the medial habenula and/or interpeduncular nucleus to decrease morphine selfadministration in rats[END_REF]). The 18MC-evoked deficit in nicotine self-administration is reflected in the NuAcc, showing impaired nicotine-evoked increases in dopamine release (McCallum et al. 2012). The accumbal dopamine outflow is unaffected, however, by MHb 18MC-induced impairment of morphine self-administration (McCallum et al. 2012).

Other nicotinic ACh receptors in the MHb are known to contribute to the modulation of nicotine self-administration. The ⍺5 KO mouse demonstrates a rightward shift in nicotine self-administration dose-response curve, continuing to consume nicotine at high doses of the drug (Fowler et al. 2011;Morel et al. 2014). This deficit in nicotine titration is restored by viral-mediated re-expression of ⍺5 in the MHb (Fowler et al. 2011), highlighting the contribution of the MHb in modulating nicotine consumption.

The interpeduncular nucleus

We cannot address the role of the MHb in nicotine-mediated behaviours, without assessing its almost sole downstream target, the IPN. Furthermore, the IPN is equally rich in nAChR expression, so its role in the aforementioned nicotine-related behaviours shall therefore be discussed.

1.4.3.a IPN anatomy and function

The interpeduncular nucleus (IPN) is composed of GABAergic interneurons, GABAergic projection neurons, and serotonergic neurons [START_REF] Groenewegen | Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat[END_REF]Zhao-Shea et al. 2013). The principal targets of the IPN are the dorsal and median raphe nucleus, the (latero)dorsal tegmentum and the pedunculopontine nucleus [START_REF] Groenewegen | Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat[END_REF]Hsu et al. 2013;[START_REF] Cornwall | Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat[END_REF][START_REF] Satoh | Cholinergic neurons of the laterodorsal tegmental nucleus: efferent and afferent connections[END_REF][START_REF] Marchand | Interpeduncular nucleus afferents in the rat[END_REF]. The IPN also sparsely innervates the septum, hippocampus, entorhinal cortex, (ventral)thalamus, locus coeruleus and hypothalamus [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] Shibata | Efferent projections of the interpeduncular complex in the rat, with special reference to its subnuclei: a retrograde horseradish peroxidase study[END_REF][START_REF] Jennes | Sites of origin of gonadotropin releasing hormone containing projections to the amygdala and the interpeduncular nucleus[END_REF]Klemm 2004;Antolin-Fontes et al. 2014;[START_REF] Marchand | Interpeduncular nucleus afferents in the rat[END_REF]. Septal and thalamic innervation is reciprocated, as is communication from the raphe nuclei, dorsal tegmentum and locus coeruleus, with the addition of major cholinergic-glutamatergic input from the MHb [START_REF] Jennes | Sites of origin of gonadotropin releasing hormone containing projections to the amygdala and the interpeduncular nucleus[END_REF][START_REF] Cornwall | Afferent and efferent connections of the laterodorsal tegmental nucleus in the rat[END_REF][START_REF] Contestabile | Afferent connections of the interpeduncular nucleus and the topographic organization of the habenulo-interpeduncular pathway: an HRP study in the rat[END_REF]Antolin-Fontes et al. 2014). The IPN therefore receives cholinergic, glutamatergic, SP-positive, serotonergic, and noradrenergic input. The IPN neural network is summarised in figure 1.4.3.a. The IPN's principal input, the MHb, was established as a key cholinergic nucleus, and only recently has its glutamatergic action been established [START_REF] Ren | Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes[END_REF]. Brief optogenetic photostimulation of the fasiculus retroflexus elicits glutamate release in the IPN, whilst tetanic stimulation results in ACh release [START_REF] Ren | Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes[END_REF]. Two types of glutamate-sensitive IPN neurons have been identified based on electrophysiological responses to AMPA (Shih et al. 2014), which provide important relay of glutamatergic input for processes such as nicotine withdrawal (Zhao-Shea et al. 2013).

1.4.3.b IPN nAChRs and function

The IPN is as equally adorned with nAChRs as the MHb, allowing cholinergic activation of these neurons. Type I neurons exhibit lower nicotine-evoked currents compared to type II neurons (Shih et al. 2014). IPN GABAergic neurons show spontaneous firing at 5Hz, and show increases in excitatory currents elicited by 10µM of nicotine (Zhao-Shea et al. 2013). Autoradiography (Baddick and Marks 2011), q-PCR (Zhao-Shea et al. (Beiranvand et al. 2014), and electrophysiological techniques [START_REF] Mulle | Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system[END_REF] have assessed the expression profile of nAChR subtypes, and their function in the IPN. ⍺3β4* are highly expressed in this nucleus, along with ⍺5 subunits (Hsu et al. 2013), ⍺4, β2, and to a lesser extent ⍺2, ⍺6 (Beiranvand et al. 2014;Grady et al. 2009) and β3 (Shih et al. 2014). Electrophysiological assessment of rat IPN cultures shows that they are sensitive to a diversity of nAChR antagonists, reflecting the variety of nAChR subtypes expressed in this nucleus [START_REF] Mulle | Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system[END_REF]. The contribution of ⍺3β4* nAChRs postsynaptically in the IPN has not been extensively explored, and the little information that we have on these receptors raises disagreement.

2013), immunoprecipitation

Mulle and colleagues show that IPN neurons' response to nicotine is sensitive to the ⍺3β4* antagonist hexamethonium [START_REF] Mulle | Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system[END_REF], whereas a more recent study suggests that postynaptic ⍺3β4* nAChRs only contribute to nicotine-evoked excitatory currents following chronic nicotine exposure (Zhao-Shea et al. 2013). Another point of discord is the contribution of postsynaptic ⍺7 nAChRs, with Mulle arguing their absence, and others illustrating the neurons' sensitivity to the ⍺7 antagonist ⍺-bungarotoxin [START_REF] Girod | Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors[END_REF].

Cholinergic agonists also facilitate presynaptic neurotransmitter release in the IPN, with ⍺3(β3)β4 nAChRs responsible for ACh and noradrenaline release, and ⍺7 nAChRs allowing glutamate release [START_REF] Girod | Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors[END_REF]Grady et al. 2009;Beiranvand et al. 2014).

High resolution mapping of nAChR expression in the IPN is lacking due to the absence of specific antibodies for immunostaining. Transgenic mice expressing GFP-tagged subunits have somewhat helped to resolve this, however the masking effect of GFPpositive terminals from the MHb (a nucleus with similar nAChR subunit expression), compromises analysis of the IPN. The only published case where the visualisation of IPN GFP-tagged subunits is clear is in the case of the ⍺5-GFP mouse (Hsu et al. 2013).

This study demonstrates that ⍺5 is localised in the dorsal portion of the IPN along with ⍺4β2* and ⍺3β4* receptors, and that these neurons innervate posterior serotonergic brain regions (Hsu et al. 2013).

1.4.3.c The IPN and nicotine reward

18MC administration to the IPN increases nicotine self-administration and increases accumbal dopamine outflow in response to acute nicotine (Glick et al. 2011;McCallum et al. 2012). This in vivo pharmacological strategy has also highlighted the role of IPN β4* nAChRs in contributing to the neural mechanisms underlying the self-administration of other drugs of abuse. Whilst 18MC infusions into the IPN increase nicotine selfadministration, this pharmacological manipulation causes a decrease in morphine and methamphetamine self-administration in the rat [START_REF] Glick | 18-Methoxycoronaridine acts in the medial habenula and/or interpeduncular nucleus to decrease morphine selfadministration in rats[END_REF][START_REF] Glick | Brain regions mediating alpha3beta4 nicotinic antagonist effects of 18-MC on methamphetamine and sucrose selfadministration[END_REF]. These studies, however, do not have the resolution to attribute this effect to the pre-or post-synaptic β4* receptors in the IPN, a point that shall be addressed again in this thesis (Articles One and Two). Lidocaine or NMDA receptor antagonist infusion into the IPN increases nicotine consumption at otherwise high, nonsalient concentrations of the drug (Fowler et al. 2011), again highlighting the IPN as an important contributor to nicotine self-administration.

Neural circuits of nicotine aversion and withdrawal

Aversion learning is as important in facilitating survival as reward-based learning, showing rapid Pavlovian association of non-salient cues with aversive stimuli. Nicotine is well known as a rewarding stimulus, but at high doses, or in particular physiological contexts, it can also be a punisher. Secondly, withdrawal from chronic nicotine is also a negative reinforcer, and an important contributor to nicotine relapse. The neurological systems involved in these processes shall now be reviewed.

The ventral tegmental area and aversion

The VTA is emerging as an extremely heterogeneous dopaminergic nucleus. VTA neurons, established processors of drug reinforcing properties, also provide the necessary dopaminergic signalling for aversion learning, such as fear conditioning.

Transient activation and inhibition of VTA dopamine neuronal subpopulations have been observed following aversive stimulation [START_REF] Mantz | Effect of noxious tail pinch on the discharge rate of mesocortical and mesolimbic dopamine neurons: selective activation of the mesocortical system[END_REF][START_REF] Zweifel | Activation of Dopamine Neurons is Critical for Aversive Conditioning and Prevention of Generalized Anxiety[END_REF][START_REF] Ungless | Uniform inhibition of dopamine neurons in the ventral tegmental area by aversive stimuli[END_REF]. Other studies observe activation and inhibition of distinct VTA dopamine subpopulations to noxious stimuli (Eddine et al. 2015;[START_REF] Brischoux | Phasic excitation of dopamine neurons in ventral VTA by noxious stimuli[END_REF]. Nicotine increases VTA dopaminergic activity, but can also inhibit a subset of dopaminergic neurons segregated to the medial portion of the VTA (Eddine et al. 2015). This pattern of segregation does not correspond to those of VTA neurons that are activated or inhibited by a noxious stimulus (Eddine et al. 2015).

Inhibition of dopaminergic neurons via GABAergic neuron activation is sufficient to drive conditioned place aversion [START_REF] Tan | GABA neurons of the VTA drive conditioned place aversion[END_REF]. Duration of dopamine neuron inhibition correlates to the attributed negative value of the cues predicting a noxious event [START_REF] Mileykovskiy | Duration of inhibition of ventral tegmental area dopamine neurons encodes a level of conditioned fear[END_REF]. Optogenetic silencing of VTA dopamine neurons elicits place aversion, a process that paradoxically results in activation of NuAcc medium spiny neurons, as measured by cFos expression [START_REF] Danjo | Aversive behavior induced by optogenetic inactivation of ventral tegmental area dopamine neurons is mediated by dopamine D2 receptors in the nucleus accumbens[END_REF]. cFos expression, indicative of neural activation, was also increased in the basolateral amygdala, but not in the lateral habenula nor the medial prefrontal cortex. Lammel and colleagues observed that conditioned place aversion can be elicited by activation of the LHb that innervates VTA dopaminergic projections to the mPFC (Lammel et al. 2012), therefore proposing a distinct neural mechanism for aversion to that of Danjo [START_REF] Danjo | Aversive behavior induced by optogenetic inactivation of ventral tegmental area dopamine neurons is mediated by dopamine D2 receptors in the nucleus accumbens[END_REF]). However, Lammel also observed an activation of RMTg ("VTA tail") GABAergic neurons, and an inhibition of the dopamine neuron subpopulation projecting to the NuAcc shell. Indeed, LHb activation leads to inhibitory postsynaptic currents in the NuAcc lateral shell, but has no effect in the NuAcc medial shell. Together, this suggests that the inhibition of the meso-accumbal network may contribute to aversion. VTA dopaminergic neurons activated by aversive stimuli require glutamatergic signalling to transition from tonic to burst firing [START_REF] Zweifel | Activation of Dopamine Neurons is Critical for Aversive Conditioning and Prevention of Generalized Anxiety[END_REF], perhaps from the LHb.

The MHb and IPN have received great attention in nicotine aversion, and nicotine withdrawal-conditioned aversion. Their role in aversion is important to this thesis, so shall be discussed in detail.

1.5.2.a The medial habenula and aversion

The role of the LHb in aversion processing and its action at the VTA is mentioned above.

In the context of nicotine aversion, however, there is a strong case for the involvement of the neighbouring MHb. The MHb serves as a brake for voluntary nicotine consumption of high, aversive doses. ⍺5 KO mice continue to self-administer high concentrations of nicotine, an effect that is reversed by re-expression of ⍺5 subuints in the MHb (Fowler et al. 2011). Transgenic over-expression of β4 nAChR subunits at endogenous sites results in aberrant avoidance of consumption of low concentrations of nicotine (Frahm et al. 2011). Furthermore, the pairing of a dose of 0.5mg/kg systemic nicotine to an environment was sufficient to condition place aversion in these transgenic mice, but not in WT mice (Frahm et al. 2011). Viral-mediated expression of a loss-of-function ⍺5 mutant (D397N, see section 1.7) in the MHb is able to alleviate nicotine aversion. Overexpression of WT or mutant β4 nAChRs solely in the MHb is sufficient to elicit nicotine aversion, further proof of MHb's role in nicotine aversion (Slimak et al. 2014).

1.5.2.b The medial habenula and withdrawal

Following chronic exposure to nicotine, rats demonstrate a degeneration of the fasiculus retroflexus [START_REF] Carlson | Nicotine produces selective degeneration in the medial habenula and fasciculus retroflexus[END_REF][START_REF] Ellison | Neural degeneration following chronic stimulant abuse reveals a weak link in brain, fasciculus retroflexus, implying the loss of forebrain control circuitry[END_REF]. In humans, the frontoaccumbal and fronto-habenular tracts are disrupted in smokers, as assessed by diffusion tensor imaging of white matter [START_REF] Savjani | Characterizing white matter changes in cigarette smokers via diffusion tensor imaging[END_REF]. Unpublished data from the same study suggests that fasiculus retroflexus degeneration occurs in smokers, but tracing of this nerve bundle in functional imaging is currently insufficient to analyse beyond anecdotal commentary (Salas, Pasteur Institute Neuroscience seminar, 2013).

Animal studies have, however, alluded to the MHb's implication in chronic nicotinemediated physiological and behavioural adaptations. The subject of withdrawal following chronic nicotine exposure, which has received a lot of attention in the MHb field, shall now be broached.

Several studies suggest that MHb neuronal activity adapts to chronic nicotine exposure, or during withdrawal. Chronic (Dao et al. 2014) or sub-chronic (Lee et al. 2015) nicotine pre-treatment attenuates nicotine-evoked increases in firing of MHb neurons, which is also the case during nicotine withdrawal (Shih et al. 2014). This is in contrast to Görlich's observation that chronic nicotine itself does not alter spontaneous electrophysiological activity of MHb cholinergic neurons. Instead, the author demonstrates that the electrophysiological profile of these neurons changes during withdrawal, showing an increase in baseline firing frequency and an increase in the response to nicotine (Görlich et al. 2013). This discrepancy may be due to differences in nicotine exposure in these animals, with Görlich exposing the mice to nicotine in their drinking water for 28 days, Shih using minipumps over 14 days, and Dao's mice being exposed via the dam's milk.

Pharmacological manipulation of the MHb is sufficient in precipitating withdrawal.

Microinjection of the nAChR antagonist mecamylamine directly into the MHb (Salas et al. 2009) or the IPN (Salas et al. 2009;Zhao-Shea et al. 2013) reveals somatic withdrawal symptoms in chronically nicotine-exposed mice. MHb lidocaine infusion was shown to alleviate nicotine withdrawal symptoms (Zhao-Shea et al. 2013). Furthermore, pharmacological inhibition of hyperpolarization-activated cyclic nucleotide-gated channels specifically in the MHb elicits somatic and affective withdrawal symptoms (Görlich et al. 2013) as does inhibition of neurokinin receptors 1 and 3, which are shown to facilitate nicotine's enhancing effect on spontaneous firing in MHb neurons (Dao et al. 2014). These data indicate that non-cholinergic systems are also implicated in the MHb's modulation of nicotine withdrawal.

The interpeduncular nucleus and withdrawal

The IPN has been repeatedly demonstrated to be a central component in nicotine withdrawal. IPN GABAergic neurons are activated during nicotine withdrawal, as shown by increased cFos expression (Zhao-Shea et al. 2013). Furthermore, electrophysiological readings demonstrate these neurons' increased spontaneous excitatory activity and increased current amplitude in response to nicotine compared to saline-treated mice (Zhao-Shea et al. 2013). Whilst systemic administration of the nAChR antagonist mecamylamine precipitates withdrawal in nicotine-exposed mice, manipulation of the habenulo-interpeduncular pathway is sufficient in manifesting somatic and affective withdrawal symptoms. Intra-IPN infusion of mecamylamine in nicotine-exposed mice is sufficient to induce affective and physical withdrawal symptoms, but not intra-VTA mecamylamine infusions (Salas et al. 2009). This may be via inactivation of β4-rich somatostatin interneurons in the IPN, proposed to disinhibit glutamate release from MHb terminals, and subsequently activate other GABAergic neuronal populations (Zhao-Shea et al. 2013). In line with this hypothesis, optogenetic activation of IPN GABAergic neurons mimics mecamylamine-precipitated somatic, but not affective, withdrawal symptoms (Zhao-Shea et al. 2013). Furthermore, NMDA receptor pharmacological inhibition in the IPN alleviates somatic withdrawal symptoms (Zhao-Shea et al. 2013), reinforcing the proposal that the MHb is implicated in nicotine withdrawal. The aversive potential of the optogenetic-precipitated withdrawal was not assessed.

Commentary

An important point must be addressed when considering nicotine aversion. Nicotine can elicit aversive responses in two ways: by nicotine withdrawal-conditioned aversion following chronic nicotine exposure, and nicotine consumption aversion. The former may have implications in relapse, whereas the latter may determine initiation of habitual nicotine consumption. The role of nAChRs in nicotine withdrawal-conditioned place aversion has received much attention. Nicotine withdrawal causes a host of somatic and affective symptoms, including increased anxiety, which themselves may drive conditioned place aversion. Indeed, the models used to induce nicotine withdrawal and condition place aversion, concomitantly increase anxiety [START_REF] Jackson | The role of various nicotinic receptor subunits and factors influencing nicotine conditioned place aversion[END_REF][START_REF] Jackson | Nicotine Reward and Affective Nicotine Withdrawal Signs Are Attenuated in Calcium/Calmodulin-Dependent Protein Kinase IV Knockout Mice[END_REF]). However, optogenetic stimulation of the LHb, which modulates the meso-cortical pathway, conditions place aversion but does not alter anxiety states (Lammel et al. 2012). Further investigations are warranted in order to determine whether the VTA is implicated in nicotine withdrawal-conditioned place aversion.

Knock-out mice: tools for studying the role of nAChRs in vivo

Null mutant mice are useful tools for studying the role of nAChRs in a plethora of physiological and behavioural tests. They have been of particular service to the elucidation of nAChR function in several components of nicotine addiction.

1.6.a The β2 KO mouse

As mentioned in the localisation section, the dominant nAChR expressed in the brain are the high-affinity β2* heteromeric nAChRs. Their expression in the CNS is vast, and their implication in nicotine-related behaviours is just as predominant. Null mutant mice do not self-administer nicotine (Picciotto M. R. 1998), a phenotype that is restored by lentivirusmediated re-expression of β2 in the VTA (Orejarena et al. 2012;Maskos et al. 2005;Tolu et al. 2013). These deficits may be due to a lack of nicotine-elicited increase in burst firing in the VTA (Picciotto M. R. 1998;Maskos et al. 2005;Tolu et al. 2013;[START_REF] Mameli-Engvall | Hierarchical control of dopamine neuron-firing patterns by nicotinic receptors[END_REF]) and lack of nicotine-elicited increases in accumbal dopamine release in the β2 KO (Maskos et al. 2005;[START_REF] Reperant | Effect of the alpha4beta2* nicotinic acetylcholine receptor partial agonist varenicline on dopamine release in beta2 knock-out mice with selective re-expression of the beta2 subunit in the ventral tegmental area[END_REF][START_REF] Salminen | Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice[END_REF]). This is coherent with pharmacological studies in the mouse and rat, where β2* nAChR antagonism impedes nicotine reinforcement, observed as abolished nicotineconditioned place preference (Walters et al. 2006) and a dose-dependent reduction in nicotine self-administration [START_REF] Watkins | Blockade of nicotine self-administration with nicotinic antagonists in rats[END_REF].

. Cessation of chronic nicotine exposure results in somatic and affective withdrawal symptoms in both humans and rodents. Nicotine withdrawal-induced anhedonia is precluded in the β2 KO mouse [START_REF] Stoker | Null mutation of the β2 nicotinic acetylcholine receptor subunit attenuates nicotine withdrawal-induced anhedonia in mice[END_REF], as is nicotine withdrawal-induced place aversion and anxiety [START_REF] Jackson | Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs[END_REF]. The somatic component of nicotine withdrawal symptoms, however, is attributed to the β4* nAChRs as shall be discussed below. It has also been demonstrated, thanks to the β2 KO mouse, that β2* nAChRs are necessary for tolerance to nicotine-mediated hypothermia and hypolocomotion [START_REF] Mccallum | Deletion of the beta 2 nicotinic acetylcholine receptor subunit alters development of tolerance to nicotine and eliminates receptor upregulation[END_REF]). These mice require a higher concentration of nicotine during the 10-day exposure to reduce the hypolocomotion and hypothermia induced by a proceeding acute challenge of nicotine.

The importance of β2* nAChRs in nicotine reinforcement, tolerance and withdrawal has not gone unnoticed, with the partial ⍺4β2 agonist, varenicline, being used as a nicotine cessation therapy.

1.6.b The β3 KO mouse

As mentioned previously, the β3 subunit is highly expressed in the substantia nigra, the VTA and the MHb [START_REF] Cui | The beta3 nicotinic receptor subunit: a component of alpha-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors[END_REF]. Whilst few studies have explored the role of the β3 subunit, there is demonstrated evidence that β3 contributes to behaviours that are, in part, regulated by the nigrostriatal and mesostriatal pathways. β3 KO resulted in an overall increase in nicotine-elicited striatal dopamine release, although ⍺-conotoxin MIIsensitive nicotine-evoked striatal dopamine release was abolished [START_REF] Cui | The beta3 nicotinic receptor subunit: a component of alpha-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors[END_REF][START_REF] Salminen | Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice[END_REF], suggesting that β3 functionally assembles with ⍺6β2* nAChRs. This physiological aberrance may underlie the nigrostriatal and mesostriatal behavioural alterations, with β3 KO mice showing an increase in locomotion in an open field and a decrease in prepulse inhibition of an acoustic startle response [START_REF] Cui | The beta3 nicotinic receptor subunit: a component of alpha-conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors[END_REF]. Further assessment revealed that an altered anxiety state in the β3 KO may also contribute to the increased locomotion previously observed. β3 KO mice indeed demonstrate a baseline anxiolytic state, with greater time spent in the open arms of an elevated plus maze [START_REF] Booker | Decreased anxiety-like behavior in beta3 nicotinic receptor subunit knockout mice[END_REF]. It remains to be determined whether this is due to altered activity in the normally β3-rich MHb, which has been implicated in anxiety (Yamaguchi et al. 2013).

Affective state is an important predictor of smoking relapse after attempted abstinence [START_REF] Leventhal | Anhedonia, depressed mood, and smoking cessation outcome[END_REF], and conversely nicotine can alter affective states such as anxiety [START_REF] Mcgranahan | Α4Β2 Nicotinic Acetylcholine Receptors on Dopaminergic Neurons Mediate Nicotine Reward and Anxiety Relief[END_REF]) and withdrawal-induced anhendonia [START_REF] Stoker | Null mutation of the β2 nicotinic acetylcholine receptor subunit attenuates nicotine withdrawal-induced anhedonia in mice[END_REF]. It would therefore be interesting to assess the role of β3-containing nAChRs in nicotine-evoked anxiolysis as well as nicotine withdrawal-evoked anxiogenesis.

1.6.c The β4 KO mouse

As focus of this thesis, the use of β4 KO mice in the study of nicotine addiction processes shall be described in detail. β4's role has also been ascribed to such processes thanks to in vivo pharmacological assessment, which shall also be recounted.

The β4 KO mouse was first published in 1999, demonstrating the non-viable nature of the β2-β4 double KO (Xu et al. 1999), which seems to have discouraged further use of the β4 KO line. Some years later β4 KO mice underwent behavioural characterisation revealing reduced anxiety in a battery of tests, with the exception of social isolation anxiety (Salas et al. 2003). Behavioural characterisation was further developed by Markou and colleagues, who corroborate the aforementioned reduced anxiety, and also demonstrated a deficit in fear conditioning [START_REF] Semenova | Mice Lacking the β4 Subunit of the Nicotinic Acetylcholine Receptor Show Memory Deficits, Altered Anxiety-and Depression-Like Behavior, and Diminished Nicotine-Induced Analgesia[END_REF]. No learning and memory deficits were observed in multiple tasks, with the exception of a small spatial memory deficit [START_REF] Semenova | Mice Lacking the β4 Subunit of the Nicotinic Acetylcholine Receptor Show Memory Deficits, Altered Anxiety-and Depression-Like Behavior, and Diminished Nicotine-Induced Analgesia[END_REF]. Article One demonstrates that β4 KO are not handicapped in an operant task.

The β4 KO mouse has not been greatly exploited by neuroscientists, with preference for pharmacological inhibition dominating the literature. Somatic signs of nicotine withdrawal are reduced in the β4 KO mouse, and in WT mice intra-cerebrally administered with the ⍺3β4 antagonist ⍺-conotoxin AuIB [START_REF] Salas | Decreased signs of nicotine withdrawal in mice null for the beta4 nicotinic acetylcholine receptor subunit[END_REF]Jackson et al. 2013). This is paradoxical to a recent article demonstrating that the ⍺3β4 antagonist SR16584 administered locally to the IPN is sufficient in eliciting somatic withdrawal signs in nicotine-naive and nicotine-exposed mice (Zhao-Shea et al. 2013). Intracerebroventricular administration of the ⍺3β4 antagonist ⍺-conotoxin AuIB, or systemic administration of hexamethonium, does not alter withdrawal-elicited anxiety in the elevated plus maze, nor does it mimic mecamylamine's (general nAChR antagonist) ability to condition place aversion as a model of withdrawal-elicited negative affective state (Jackson et al. 2013;[START_REF] Jackson | The role of various nicotinic receptor subunits and factors influencing nicotine conditioned place aversion[END_REF]. Overall, these studies show that β4 has a role in the somatic, but not affective, components of nicotine withdrawal.

Interest in the β4 subunit concerning its role in various components of nicotine addiction continues to grow following the discovery of the polymorphic nature of the human CHRNA5-CHRNA3-CHRNB4 gene cluster, and its contribution to smoking risk (see section 1.7). The β4 over-expressing mouse avoids consumption of 5-50µg/ml nicotine in a two-bottle choice test, which elicits mild-to-no preference in WTs (Frahm et al. 2011). A later study showed that over-expression of β4 in the MHb was sufficient in eliciting this aberrant aversion to nicotine (Slimak et al. 2014). The use of ⍺3β4-specific antagonists has also alluded to the fact that these receptors are important in nicotine reward. Intra-cerbroventricular administration of ⍺-conotoxin AuIB precludes nicotineconditioned place preference in the mouse (Jackson et al. 2013). Furthermore, local inhibition of ⍺3β4* nAChRs in the MHb or basolateral amygdala reduces nicotine selfadministration in the rat, whereas inhibition at the IPN increases self-administration, with no effect of antagonism at the VTA (Glick et al. 2011). The effects of ⍺3β4 antagonism at the MHb and IPN is reflected in a decrease and increase in accumbal dopamine release, respectively (McCallum et al. 2012), which we cannot attribute to impaired function at striatal terminals in mediating dopamine release, since the β4 KO mouse shows WT-like agonist-evoked striatal dopamine release ex vivo [START_REF] Salminen | Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice[END_REF].

It is interesting to note that MHb-targeted pharmacological inhibition of ⍺3β4 nAChRs also reduces morphine self-administration [START_REF] Glick | 18-Methoxycoronaridine acts in the medial habenula and/or interpeduncular nucleus to decrease morphine selfadministration in rats[END_REF]) without affecting morphineevoked increases in accumbal dopamine release (McCallum et al. 2012).

The work of the present thesis aimed to investigate the role of β4* nAChRs in nicotine reinforcement (Article One) as well as aversion (Article Two), as modelled in the β4 KO mouse.

1.6.d The ⍺2 KO mouse

In rodents, ⍺2 is highly expressed in the IPN, a region that has heavily been implicated in nicotine withdrawal properties (Beiranvand et al. 2014;Zhao-Shea et al. 2013;Salas et al. 2009). ⍺2 subunits are necessary for somatic signs of nicotine withdrawal (Salas et al. 2009), as are other nAChR subunits that are found to equally high expression levels in the IPN referenced in this section.

⍺2* nAChRs are believed to mediate the non-desensitising nicotine-evoked inward currents at hippocampal interneurons of the stratum oriens-alveus [START_REF] Jia | α2 Nicotine receptors function as a molecular switch to continuously excite a subset of interneurons in rat hippocampal circuits[END_REF]. It is not known whether this is dependent on specific stochiometric assemblies of ⍺2 with other nAChR subunits, and therefore whether the ⍺2β2 found in the rodent IPN (Grady et al. 2009) also exhibits this unique pharmacological profile.

1.6.e The ⍺3 KO mouse

Unfortunately the specific role of ⍺3* nAChRs has yet to be extensively explored owing to the lethal nature of the ⍺3 KO mouse (Xu et al. 1999). To circumvent this obstacle, ⍺3+/mice have been used to assess the in vivo contribution of this subunit to nicotineinduced behaviours. As with the β4 KO line, ⍺3+/mice demonstrate a reduced sensitivity to nicotine-evoked seizures, but show no change in nicotine-stimulated hypolocomotion (0.5mg/kg i.p.) compared to ⍺3+/+ mice (Salas et al. 2004). The similarity in ⍺3+/and β4-/-may be due to the fact that these two subunits are principal partners in the central and peripheral nervous system (see nAChR localisation section).

In fact, immunoprecipitation studies have shown that ⍺3 subunit expression is almost abolished in the MHb (Scholze et al. 2012) and IPN (Beiranvand et al. 2014) of the β4 KO mouse. It is therefore possible that the β4 KO mirrors many ⍺3-dependent functions, but this remains to be demonstrated experimentally.

1.6.f The ⍺4 KO mouse

The ⍺4 subunit of nAChRs contributes to high-sensitivity nAChR subtypes in the brain.

VTA ⍺4 is necessary for nicotine intra-venous (Pons et al. 2008) and intra-VTA (Exley et al. 2011) self-administration as demonstrated by the attenuated self-administration in the KO mouse, and rescue of this deficit by re-expression of the subunit specifically in the VTA. Dopamine neuron-specific genetic depletion of ⍺4 is sufficient to abolish nicotine CPP [START_REF] Mcgranahan | Α4Β2 Nicotinic Acetylcholine Receptors on Dopaminergic Neurons Mediate Nicotine Reward and Anxiety Relief[END_REF], as does non-discriminate genetic depletion [START_REF] Sanjakdar | Differential Roles of α6β2* and α4β2* Neuronal Nicotinic Receptors in Nicotine-and Cocaine-Conditioned Reward in Mice[END_REF]) and pharmacological inhibition of ⍺4β2 nAChRs [START_REF] Tobey | Effects of the specific α4β2 nAChR antagonist, 2-fluoro-3-(4-nitrophenyl) deschloroepibatidine, on nicotine reward-related behaviors in rats and mice[END_REF]. The deficit in nicotine reinforcement can be attributed in part to the incomplete reduction of nicotine-evoked excitatory currents [START_REF] Marubio | Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors[END_REF]) and burst firing (Exley et al. 2011) at VTA dopaminergic neurons of ⍺4 KO mice. However, there is an increase of function at the terminals as demonstrated by baseline hyperdopaminergia in the nucleus accumbens, but no nicotine-evoked increases in dopamine release as measured by in vivo microdialysis [START_REF] Marubio | Effects of nicotine in the dopaminergic system of mice lacking the alpha4 subunit of neuronal nicotinic acetylcholine receptors[END_REF]. Contrary to these findings, acute slice fast-scan cyclic voltammetry recordings demonstrate that ⍺4 KO mice show a deficit in ACh-or nicotine-evoked accumbal dopamine release (Exley et al. 2011). This discrepancy may be due to the isolation of the nucleus accumbens from important input from, for example, the infra-and pre-limbic cortices, medial frontal cortex, hippocampus and amygdala [START_REF] Groenewegen | Cortical afferents of the nucleus accumbens in the cat, studied with anterograde and retrograde transport techniques[END_REF][START_REF] Kirouac | Topographical organization in the nucleus accumbens of afferents from the basolateral amygdala and efferents to the lateral hypothalamus[END_REF][START_REF] Salgado | The Nucleus Accumbens: A Comprehensive Review[END_REF].

1.6.g The ⍺5 KO mouse

The ⍺5 KO mouse has been a useful tool in assessing the role of ⍺5* nAChRs in nicotine addiction processes. ⍺5 can associate with both high-affinity, ⍺4β2* nAChRs, and low affinity ⍺3β4* nAChRs [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]Scholze et al. 2012;Grady et al. 2009). Nicotine-conditioned place preference (0.5mg/kg s.c.) is still present in the ⍺5 KO mouse (Jackson et al. 2013), and ⍺5 KO mice self-administer classically reinforcing doses of nicotine. However, this genetic ablation of ⍺5 results in continued self-administration at higher doses of nicotine (Fowler et al. 2011;Morel et al. 2014). Two hypotheses were drawn from these studies using local ⍺5 re-expression analysis: one implicates the habenulo-interpeduncular pathway in inhibiting consumption of high doses of nicotine (Fowler et al. 2011), and the other suggests that ⍺5 in VTA dopaminergic neurons is required for correct modulation of nicotine consumption (Morel et al. 2014). Undoubtedly both of these structures mediate important ⍺5 nicotine function, and discrepancies may be a reflection of the subunits to which ⍺5 associates in these structures. Further analysis demonstrates that high doses of nicotine continue to reduce intra-cranial self-stimulation reward thresholds in the ⍺5 KO mouse at doses that no longer have a threshold-lowering effect in WTs [START_REF] Fowler | Role of α5* nicotinic acetylcholine receptors in the effects of acute and chronic nicotine treatment on brain reward function in mice[END_REF].

Additionally, high doses of nicotine continue to be reinforcing to ⍺5 KO mice in a conditioned place preference paradigm [START_REF] Jackson | Role of alpha5 nicotinic acetylcholine receptors in pharmacological and behavioral effects of nicotine in mice[END_REF]. Together these data, thanks to the use of the ⍺5 KO mouse, demonstrate that this subunit, albeit an accessory subunit that does not participate in ligand binding, has an important role in dampening brain reward responses to high doses of nicotine.

⍺5 nAChR subunits contribute to ACh-evoked striatal dopamine release. The ⍺3β2* and ⍺6β2 antagonist ⍺-conotoxin MII (McIntosh et al. 2004b) reduces striatal synaptosome dopamine release, however in the absence of ⍺5, dopamine release is increased in this ⍺-conotoxin MII (⍺-ctxMII)-sensitive portion. Non ⍺-ctxMII-sensitive nAChRs (e.g. ⍺4β2), however, depend on ⍺5 for dopamine release, since KO of this subunit reduces the ⍺-ctxMII-resistant ACh-evoked synaptosomal dopamine release [START_REF] Salminen | Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice[END_REF]).

These ⍺5-dependent events may be the physiological substrates that allow the motivation to self-administer high doses of nicotine to persist, which could facilitate the smoker's transition from infrequent smoking to habitual consumption and ultimately addiction.

With regards to nicotine withdrawal, ⍺5 nAChR subunits do not appear to play a significant role, since ⍺5 KO mice show WT-like withdrawal-conditioned place aversion and only a mild reduction in the number of somatic withdrawal symptoms [START_REF] Jackson | Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs[END_REF]). However, ⍺5 KO mice do not display nicotine withdrawal-induced increases in brain reward thresholds, suggesting that ⍺5* nAChRs do indeed participate in certain aspects of the negative affective state that follows nicotine withdrawal [START_REF] Fowler | Role of α5* nicotinic acetylcholine receptors in the effects of acute and chronic nicotine treatment on brain reward function in mice[END_REF].

1.6.h The ⍺6 KO mouse

⍺6 KO mice appear to be less sensitive to the rewarding properties of nicotine, showing impaired nicotine-conditioned place preference [START_REF] Sanjakdar | Differential Roles of α6β2* and α4β2* Neuronal Nicotinic Receptors in Nicotine-and Cocaine-Conditioned Reward in Mice[END_REF]) and absence of nicotine intravenous self-administration (Pons et al. 2008). The latter is rescued by VTA re-expression of the subunit (Pons et al. 2008). This is in contrast, however, to the WTlike intra-VTA nicotine self-administration amongst ⍺6 KO mice, as well as the WT-like response of VTA dopaminergic neurons to nicotine in in vivo electrophysiological recordings (Exley et al. 2011). This may be due to the fact that in this intra-VTA selfadministration test, nicotine does not reach the ⍺6-rich terminals of the dopaminergic neurons in the NuAcc, which are necessary for modulation of dopamine release elicited by local nicotine (Exley et al. 2011;[START_REF] Salminen | Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice[END_REF][START_REF] Exley | Presynaptic nicotinic receptors: a dynamic and diverse cholinergic filter of striatal dopamine neurotransmission[END_REF]. The role of ⍺6β2* nAChRs in vivo, however, is somewhat contested. Champtiaux and colleagues demonstrated that nicotine-evoked accumbal dopamine release was similar in WT and ⍺6 KO mice [START_REF] Champtiaux | Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice[END_REF]. This study highlights the disadvantage of using KO mouse lines for the study of nAChRs: compensatory increases of other nAChR subunits, for example increased ⍺4 in the ⍺6 KO mouse [START_REF] Champtiaux | Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knock-out mice[END_REF]. Pharmacological tools for short-term inhibition of nAChRs can overcome this.

Notably, local pharmacological inhibition of ⍺6β2* receptors in the NuAcc shell reduces the motivation to seek nicotine, as demonstrated by a reduced breaking point in a progressive ratio schedule for acquisition of a reinforcing dose of nicotine [START_REF] Brunzell | Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors in the nucleus accumbens shell regulate progressive ratio responding maintained by nicotine[END_REF]. Furthermore, pharmacological assessment of the role of ⍺6β2* nAChRs in nicotine addiction processes has revealed that these receptors are required for certain affective signs of precipitated nicotine withdrawal, namely reduced anxiety in the elevated plus maze and a decrease in withdrawal-conditioned place aversion [START_REF] Jackson | The Role of a6-Containing Nicotinic Acetylcholine Receptors in Nicotine Reward and Withdrawal[END_REF]).

1.6.i The ⍺7 KO mouse ⍺7 homomeric nAChRs have a low affinity for nicotine and ACh, are rapidly activated and are highly calcium permeable. ⍺7 KO mice show a decreased sensitivity to the reinforcing effects of nicotine in an intra-VTA self-administration paradigm, which is further validated in the WT mouse and rat pre-treated systemically with the ⍺7 antagonist MLA, showing attenuated nicotine self-administration [START_REF] Besson | Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice[END_REF][START_REF] Markou | The nicotinic antagonist methyllycaconitine has differential effects on nicotine self-administration and nicotine withdrawal in the rat[END_REF]. The Pavlovian reinforcing properties of nicotine, however, does not depend on ⍺7 nAChRs, since ⍺7 KO mice show WT-like nicotine-conditioned place preference (Walters et al. 2006). Furthermore, an acute intra-venous nicotine selfadministration paradigm demonstrates that ⍺7 nAChRs do not modulate nicotinereinforced self-administration behaviour (Pons et al. 2008). The ⍺7 KO mouse shows a persistent elevation in accumbal dopamine release in response to an acute administration of nicotine systemically, which is in contrast to the rapid-onset and rapid return to baseline of the dopamine release peak in WT mice [START_REF] Besson | Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice[END_REF]). As mentioned previously, ⍺-ctxMII reduces agonist-evoked dopamine release from striatal synaptosomes in the mouse. This ⍺-ctxMII-sensitive portion of receptors is also sensitive to the antagonising effects of MLA, therefore suggesting that ⍺7 nAChRs play a role in striatal dopamine release [START_REF] Salminen | Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice[END_REF]). However, this article also presents a WT-like dopamine release from striatal synaptosomes in the ⍺7 KO mouse, conflicting with the pharmacological results. It is therefore unclear whether the aberrant accumbal dopamine release observed during in vivo microdialysis may be due to nicotine's action at ⍺7 nAChRs in the NuAcc, the VTA itself, or even at other brain regions regulating the VTA.

The role of ⍺7 nAChRs in the manifestation of nicotine withdrawal is somewhat ambiguous. The ⍺7 KO mouse shows no alterations in affective or somatic symptoms following precipitated or spontaneous nicotine withdrawal, with the exception of withdrawal-induced hyperalgesia [START_REF] Jackson | Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs[END_REF][START_REF] Grabus | Nicotine physical dependence in the mouse: Involvement of the α7 nicotinic receptor subtype[END_REF]. This is in contrast to another study demonstrating reduced somatic symptoms of mecamylamine-precipitated withdrawal amongst ⍺7 KO mice, who also show no alteration in nicotine-induced tolerance to nicotine hypolocomotion compared to WT mice (Salas et al. 2007). Two studies show that MLA is sufficient in precipitating withdrawal following chronic nicotine exposure [START_REF] Damaj | Characterization of spontaneous and precipitated nicotine withdrawal in the mouse[END_REF], including in the ⍺7 KO mouse (Salas et al. 2007), therefore suggesting that MLA can also act at non-⍺7 nAChRs. The role of ⍺7 in affective and somatic nicotine withdrawal is again put into question by a study showing that MLA is insufficient in eliciting withdrawal symptoms in the rat [START_REF] Markou | The nicotinic antagonist methyllycaconitine has differential effects on nicotine self-administration and nicotine withdrawal in the rat[END_REF]. This reinforces the importance of the use of knockout mice in the investigation of nAChR subunits.

An illustrative summary of the roles of nAChR subunits in nicotine addiction processes, elucidated by KO models, is summarised below:

Human genetic studies of nicotinic acetylcholine receptor genes and smoking

There is a large body of human genetic studies that have highlighted common genetic polymorphisms 1 , and rare mutations 2 as contributing to nicotine dependence. Genomewide association studies (GWAS) of human disease have changed the way we view pathology. Thanks to unbiased screening of millions of genetic factors in cohorts of thousands of patients and controls, we are able to stratify the genetic aetiology of diseases. The advent of technologies allowing the screening of entire genomes in large samples has greatly advanced the robust identification of susceptibility loci in psychiatric diseases, the hardest group to tackle [START_REF] Gratten | Large-scale genomics unveils the genetic architecture of psychiatric disorders[END_REF]. A multitude of GWAS, and candidate gene analyses have demonstrated the polymorphic nature of the gene cluster coding for ⍺5, ⍺3 and β4 nAChR subunits (CHRNA5-CHRNA3-CHNRΒ4, figure 1.7), and that a number of these variants contribute to nicotine dependence and phenotypic traits that lead, or protect, from dependence [START_REF] Tobacco | Genome-wide meta-analyses identify multiple loci associated with smoking behavior[END_REF]Saccone et al. 2009;[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] Flora | Functional characterization of SNPs in CHRNBA3/B4 intergenic region associated with drug behaviors[END_REF][START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF]. The most notable polymorphism implicated in nicotine addiction risk is the CHRNA5 single nucleotide polymorphism (SNP), reference SNP (rs) number 16969968, aspartate to arginine (D398N), present in 37% of European populations [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF]. This SNP demonstrates an important role in nicotine addiction processes as analysed in rodent models (Morel et al. 2014;Fowler et al. 2011). Furthermore, it has been 1 Polymorphismcommon. Occurs in more than 1% of the population 2 Mutation -rare. Occurs in less than 1% of the population demonstrated that the rodent ⍺5 D397N SNP affects β4* nAChR-mediated nicotine aversion (Frahm et al. 2011).

Figure 1.7. The CHRNA5-CHRNA3-CHRNB4 genetic cluster, encoding ⍺5, ⍺3 and β4 nAChR subunits.

The cluster is located on chromosome 15 of the human genome (note, the cluster is located on chromosome 9 of the mouse genome). CHRNA3 and CHRNB4 genes are located on the opposite strand to CHRNA5, therefore are oriented oppositely to the CHRNA5 gene. Black squares represent exons (to scale), red squares represent the 5' untranslated region (UTR), and orange squares denote the 3' UTR.

The linked 3 single nucleotide variants rs1948 (in CHRNB4 3' untranslated region, UTR) and rs8023462 (intergenic, in CHRNA3 promoter and CHRNB4 downstream domain) have been associated with early initiation of alcohol and tobacco use (Schlaepfer et al. 2008). Furthermore, the rs1996371 single nucleotide variant upstream of CHRNB4 was identified as decreasing the age of onset of habitual smoking by one year in homozygous risk allele carriers [START_REF] Kapoor | Variants located upstream of CHRNB4 on chromosome 15q25.1 are associated with age at onset of daily smoking and habitual smoking[END_REF]). Risk variants have also been identified in the coding region of CHRNB4. A study of 2700 people revealed that the rs12914008 (threonine to isoleucine, T91I) SNP, with a frequency of 4.5% in European-Americans (EA) and 0.6% in African-Americans, reduces the risk of nicotine dependence, with an odds ratio of 0.73 in EA (Saccone et al. 2009). This was replicated by Haller and colleagues, who additionally showed that rs61737499 (threonine to isoleucine T375I) is associated with a reduced risk of nicotine dependence, exhibiting a higher frequency in non-dependent smokers than dependent smokers [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF]. It is important to note that the rs1948 variant is linked to rs8023462, and rs12914008 to the CHRNA3 SNP rs8192475 (arginine to histidine, R37H), so the interpretation of these CHRNΒ4 variants' contributions to nicotine dependence, is limited. However, the independent and additive nature of several CHRNΒ4 variants have been addressed by in vitro and in vivo studies, who reveal the extent of pharmacological and behavioural alterations caused by mutations in the β4 subunit gene, and its partner subunit ⍺3 [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF]Slimak et al. 2014). The nonsynonymous mutations in the coding region of CHRNΒ4 highlighted above exhibit gains of function, increasing nicotine-mediated inward currents of mutant β4-containing nAChRs in oocytes [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF]) and in rat hippocampal culture (Slimak et al. 2014). Furthermore, the CHRNA3 variant rs8192475 (R37H), which is in strong linkage disequilibrium 3 with CHRNΒ4's T91I, exacerbates the increased nicotine-evoked current [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF]. So far only one study has investigated the effects of CHRNΒ4 mutations in vivo, by inducing the expression of mouse T374I in the MHb, host of β4, in WT mice. Co-expressing mutant and WT β4 induced aversion to nicotine. However, a caveat with this study is that the overexpression of WT β4 in the MHb also induces aversion to nicotine. 3 Linkage disequilibrium is the occurrence of some combinations of alleles or genetic markers in a population more or less often than would be expected from a random association via meiotic DNA recombination. An increase in occurrence of two genetic markers, e.g. SNPs, is common when the loci are located close together on the chromosome, which decreases the chance of chromosomal cross-over between the two markers during meiosis, therefore increasing the probability of concomitant inheritance.

Behavioural models of nicotine addiction in the mouse

Nicotine addiction is led by a constellation of events. The neural mechanisms underlying the transition from infrequent use to loss of control of consumption are complex. Whilst it is the interplay of differing nicotinic actions that contribute to the final addiction pathology, for the sake of research, it is important to stratify these components and model them in animal models. A useful laboratory test must therefore provide an empirical measure of a component of addiction. However, whilst it is to the experimenter's advantage to delineate these neurological components, we must not neglect the importance of assessing the results in the context of the human smoker's dynamic experience with nicotine.

Animal models are useful tools since we can create a homogeneous population of subjects to test. We can control the environment, and employ inbred colonies of subjects to minimise intra-group variation, which cannot be said of humans with genetic, environmental predispositions to drug abuse. Furthermore, we are able to model a variety of the DSM's criteria for nicotine addiction in rodents, as shall be outlined.

1.8.a.i Intra venous nicotine self-administration

Nicotine self-administration, whether this is by intravenous or intracranial route, is a measure of the appetitive value of nicotine. It closely models the human behaviour, since administration is contingent on the animal. Another advantage to this paradigm is that different doses of nicotine can be tested, either by successively increasing the dose after a given number of sessions (e.g. Fowler et al. 2011) or by changing the dose in a latin square design (e.g. [START_REF] Contet | Patterns of responding differentiate intravenous nicotine self-administration from responding for a visual stimulus in C57BL/6J mice[END_REF]). The resultant dose-response curve can therefore be compared between experimental groups, such as genotype, age or pharmacological treatment.

Procedure

The self-administration procedure typically occurs in a box where the animal can selfadminister a reward by nose-poking into a magazine or by pressing a lever. There is oftentimes a light cue situated above the magazine or lever that illuminates during reward delivery. To control for indiscriminate activity, there is a control magazine or lever that is not paired to cue or reward administration (figure 1.8.a.i). Reduced activity at the control magazine/lever is demonstrative of the individual's capacity to discriminate between reward-paired and non reward-paired act-outcome events, and therefore confirms the appetitive nature of the reward received at the "active" magazine/lever. A typical method of intravenous delivery is via a catheter implanted into the jugular vein (e.g. Orejarena et al. 2012). The apparatus is adapted for optimal motility of the mouse during the session, and fitted with an automated system for delivery of the drug. A typical nicotine dose range tested in the mouse is ≤100µg/kg/infusion, over 1-2h sessions, 5-6 consecutive days of daily sessions per dose, for up to 10 weeks, limited by maintenance of patency of the catheter (Gallego et al. 2012;Orejarena et al. 2012;[START_REF] Contet | Patterns of responding differentiate intravenous nicotine self-administration from responding for a visual stimulus in C57BL/6J mice[END_REF][START_REF] Fowler | Intravenous nicotine self-administration and cueinduced reinstatement in mice: Effects of nicotine dose, rate of drug infusion and prior instrumental training[END_REF].

Food-restricted animals can be first trained to self-administer food pellets in this paradigm prior to nicotine self-administration assessment. This allows the experimenter to eliminate animals that physically or cognitively are incapable of performing the task (e.g. due to illness), and also acts to facilitate acquisition of nicotine self-administration, as demonstrated experimentally in the rat [START_REF] Garcia | Effect of Food Training and Training Dose on Nicotine Self-Administration in Rats[END_REF]. A visual or auditory cue is a palpable signal of drug delivery, reinforcing the act-outcome series of events. The cue itself eventually becomes a conditioned stimulus due to associations with the rewarding effect of nicotine infusion as shown in the rat [START_REF] Caggiula | Cue dependency of nicotine self-administration and smoking[END_REF][START_REF] Chaudhri | Self-administered and noncontingent nicotine enhance reinforced operant responding in rats: Impact of nicotine dose and reinforcement schedule[END_REF]), but this reinforcement rapidly dissipates when nicotine is withheld (Orejarena et al. 2012). However, a study conducted in the rat demonstrates that the cue associated with nicotine delivery eventually is sufficient to maintain responding for this stimulus during intermittent sessions, despite nicotine being withheld [START_REF] Cohen | Nicotine-associated cues maintain nicotine-seeking behavior in rats several weeks after nicotine withdrawal: reversal by the cannabinoid (CB1) receptor antagonist, rimonabant (SR141716)[END_REF]. Conversely, removal of a cue reduces responding for nicotine in the rat [START_REF] Caggiula | Cue dependency of nicotine self-administration and smoking[END_REF]. This highlights the influential properties of nicotine in evoking incentive saliency of nicotine cues.

After extinction of nicotine self-administration (by replacing the drug with saline), the cue can be sufficient to elicit relapse of drug-seeking behaviour, as can a single dose of nicotine administered by the experimenter [START_REF] Contet | Patterns of responding differentiate intravenous nicotine self-administration from responding for a visual stimulus in C57BL/6J mice[END_REF][START_REF] Fowler | Intravenous nicotine self-administration and cueinduced reinstatement in mice: Effects of nicotine dose, rate of drug infusion and prior instrumental training[END_REF].

It is not an aberrant inclusion in the modelling of human smoking behaviours since nicotine-associated cues pose a barrier to sustained smoking abstinence in the human.

One can implement a fixed ratio (FR) schedule to determine the number of nose pokes/lever presses required for reward delivery. In the intra venous self-administration paradigm described in Article One, animals are trained on FR1 and FR3 schedules (1 and 3 nose-pokes for delivery of one food pellet, respectively), and then continue on an FR3 schedule for nicotine self-administration assessment. An FR>1 is useful in observing the motivational aspect of a particular dose of nicotine. Furthermore, it improves stability of infusions since only consecutive nose-pokes/lever presses will be considered for the administration of reward. The progressive ratio schedule, where the number of nose pokes/lever presses required for drug delivery increases, allows us to determine the breaking point, which is the point at which the animal no longer responds for reward [START_REF] Garcia | Effect of Food Training and Training Dose on Nicotine Self-Administration in Rats[END_REF]Gallego et al. 2012). This estimates the subject's motivation to receive a drug infusion.

1.8.a.ii Intra-cranial nicotine self-administration

A model of morphine self-administration directly into the ventral tegmental area was first described by Welzl and colleagues [START_REF] Welzl | Self-administration of small amounts of morphine through glass micropipettes into the ventral tegmental area of the rat[END_REF] and has been further developed by Dr. Vincent David (David andCazala 1994b, 1994a) for intra-VTA nicotine self-administration (David et al. 2006;Exley et al. 2011;[START_REF] Besson | Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice[END_REF]Tolu et al. 2013). Similarly to the intra venous self-administration paradigm, this test allows us to measure the appetitive nature of nicotine. There are, however, some fundamental differences.

Procedure

The self-administration is conducted in a three-arm Y-shaped apparatus. The mouse, who is fitted with a permanent intracranial cannula guide, is placed in the start arm and is allowed to move towards the left or right arm, one of which is associated with nicotine delivery (figure 1.8.a.ii). Once it has reached the end of the chosen arm a sliding door is closed to confine the mouse in the compartment for the duration of nicotine delivery (50nl, 4 seconds), or not, plus 25 seconds. The mouse is guided back to the start arm for brief confinement before a new trial begins. There are a total of ten trials per session. Right. WT mice self-administer nicotine to the VTA, but not saline (Ringer, David et al. 2006). The number of nicotine self-administrations increases over the course of seven sessions (A1-A7), whereas saline is insufficient in acquiring self-administration behaviour.

There are ten trials per session, and one session per day for several days (typically seven). Nicotine is automatically delivered in an attributed arm (left or right) throughout the duration of the experiment. Nicotine pairing is evenly distributed amongst the experimental group. Nicotine is delivered directly into the ventral tegmental area via a cannula connected to catheter tubing. Unlike many self-administration paradigms, this procedure does not use any associative cues contingent with drug delivery.

The subject rapidly learns the association between its actions and the self-administration of nicotine into the VTA. One can measure this acquisition of self-administration over the course of the experiment, illustrating the nicotine-reinforced goal-directed behaviour. A typically reinforcing range of nicotine concentration is 10-100ng/infusion, which elicits a gradual increase in the average number of self-administrations over the course of seven sessions (figure 1.8.a.ii), as well as a decrease in latency to reach the chosen arm (David et al. 2006). A great advantage of this 10-trial choice system is that we can assess the aversive effects of nicotine. This is observed by self-administration at lessthan chance level (<5/10 per session), and is addressed in Article Two.

Modelling nicotine addiction processes: overview of 1.8.a.i and 1.8.a.ii Nicotine self-administration is therefore a useful tool in measuring operant, selfcontingent, long-term drug consumption. It reflects the reinforcing and motivational properties of nicotine, and can also be adapted to assess cue-induced relapse.

1.8.b Nicotine-conditioned Place Preference

An initially neutral environment can acquire incentive motivational properties by Pavlovian association with a reinforcing stimulus, modifying the subject's attributed value of the environment. Nicotine is sufficient in evoking this association, as assessed by conditioned place preference (CPP). The procedure allows us to measure the reinforcing value of a particular dose of nicotine, represented by the time spent in a nicotineassociated environment over a saline-associated one.

Procedure

The apparatus used for CPP is a box with two distinct chambers (floor texture, colour and/or shape) separated by movable partitions (figure 1.8.b). To test for potential predisposing bias, the animal is allowed to freely roam the two chambers (typically proceeding a 5min habituation in a closed central chamber), and the time spent in both chambers recorded (usually 15min). The mice are then separated into experimental (nicotine) and control (saline) groups. In the next session, mice receive an injection of nicotine or saline and are placed into a designated compartment (15-20mins). There are typically two sessions per day, with the saline group receiving saline in both sessions, and the nicotine group receiving nicotine in one session and saline in the other. Nicotine and saline compartment pairing is consistent for each mouse. After 2-3 days of conditioning the mice are tested, injection-free, in the apparatus. The time spent freely roaming between compartments is measured. The amount of time spent in the nicotinepaired environment compared to saline, or compared to pre-conditioning, is an indication of the positive or negative reinforcing properties of the particular dose used, in a particular set of subjects (e.g. WT or KO). Generally, a dose of 0.5mg/kg nicotine administered subcutaneously is sufficient to elicit CPP in the C57Bl/6J mouse, as repeatedly demonstrated by the laboratory of Dr. Damaj [START_REF] Jackson | Role of alpha5 nicotinic acetylcholine receptors in pharmacological and behavioral effects of nicotine in mice[END_REF][START_REF] Jackson | The Role of a6-Containing Nicotinic Acetylcholine Receptors in Nicotine Reward and Withdrawal[END_REF][START_REF] Jackson | Nicotine Reward and Affective Nicotine Withdrawal Signs Are Attenuated in Calcium/Calmodulin-Dependent Protein Kinase IV Knockout Mice[END_REF]Jackson et al. 2013;[START_REF] Sanjakdar | Differential Roles of α6β2* and α4β2* Neuronal Nicotinic Receptors in Nicotine-and Cocaine-Conditioned Reward in Mice[END_REF][START_REF] Kota | Nicotine Dependence and Reward Differ between Adolescent and Adult Male Mice[END_REF]Walters et al. 2006) and others [START_REF] Tapper | Nicotine Activation of alpha4* Receptors: Sufficient for Reward , Tolerance , and Sensitization[END_REF]). An example of this dose's lack of conditioning properties exists (Frahm et al. 2011). Genetic and pharmacological manipulation of nAChRs has highlighted the importance of nAChRs in nicotine-conditioned place preference, which is reviewed in section 1.6.

a b c

Modelling nicotine addiction processes CPP is therefore a useful experimental test, and provides an empirical measure of drug reinforcement. The paradigm is also used to explore associations of an environment with aversive stimuli (named conditioned place aversion, CPA). This can be assessed using, for example lithium chloride to induce a malaise [START_REF] Darcq | RSK2 signaling in brain habenula contributes to place aversion learning[END_REF]), but has also been widely used for the study of the environment aversion conditioned by nicotine withdrawal [START_REF] Jackson | The role of various nicotinic receptor subunits and factors influencing nicotine conditioned place aversion[END_REF][START_REF] Jackson | Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs[END_REF][START_REF] Jackson | The Role of a6-Containing Nicotinic Acetylcholine Receptors in Nicotine Reward and Withdrawal[END_REF]Jackson et al. 2013). The reinforcing properties of nicotine and its ability to induce positive contextual associations, as well as the negative contextual associations of nicotine withdrawal are both important factors in maintaining smoking behaviours (Dani et al. 2012).

1.8.c Two-bottle choice

The two-bottle choice method allows us to measure long term, voluntary nicotine consumption in rodents. Two measures can be extracted from this test; 1) preference for nicotine solution over vehicle solution, and 2) quantity of nicotine consumed.

Since self-administration is via an oral route, there is no need for catheter implants, which therefore removes the limitations imposed by surgery and long-term implantation.

Importantly, consumption of nicotine is voluntary as in human smokers. A caveat with this technique is that oral consumption does not evoke the same absorption dynamics as intravenous delivery. Bucal absorption of nicotine much depends on the pH of the nicotine-containing substance. At an acidic pH (as is the case in cigarettes) nicotine is minimally absorbed in the mouth, whereas when buffered to a basic pH (for example in chewing tobacco), nicotine is absorbed to the bloodstream supplying the mouth [START_REF] Benowitz | Nicotine Chemistry, Metabolism, Kinetics and Biomarkers[END_REF]. Unfortunately mention of pH is neglected in several recent nicotine two-bottle choice paradigms (Frahm et al. 2011;[START_REF] Levin | Nicotinic α7 or β2-Containing Receptor Knockout: Effects on Radial-Arm Maze Learning and Long-Term Nicotine Consumption in Mice[END_REF]Lee et al. 2015). In this two-bottle choice test, the solution stays in the mouth only momentarily before being swallowed and entering first-pass metabolism. The amount of nicotine that reaches the brain is therefore reduced, with an onset of approximately 30mins, which does not model the sub-minute nicotine absorption of smoked cigarettes [START_REF] Collins | Oral Nicotine Self-Administration in Rodents[END_REF].

Mice

Modelling nicotine addiction processes

As mentioned above, the two-bottle choice test is not a model of human-like nicotine consumption. It is, however, an appropriate paradigm for the measure of the appetitive nature of nicotine. It is also a useful tool for the investigation of the genetic factors on nicotine consumption, for example mutations in the β4 nAChR subunit (Slimak et al. 2014). Preference for nicotine-containing or control solutions allows us to study not only the positively reinforcing aspects of nicotine, but also its aversive effects (Frahm et al. 2011;Slimak et al. 2014).

1.8.d Commentary: The mouse as an appropriate model of addiction

Behavioural assessment of nicotine addiction processes was initially developed for the rat [START_REF] Parker | Rats are the smart choice: Rationale for a renewed focus on rats in behavioral genetics[END_REF]. A shift in preference towards the mouse was observed in the 1990's owing to the ability to manipulate its genome and generate knockout lines [START_REF] Parker | Rats are the smart choice: Rationale for a renewed focus on rats in behavioral genetics[END_REF]). This has allowed a genotype-to-phenotype research strategy in order to elucidate the function of a gene that result from its targeted mutation. A knockout line exists for all nAChR subunits, therefore rendering these mice readily available tools for the investigation of nAChR-mediated behavioural processes. We can model various processes that lead to nicotine dependence, or that are symptomatic of dependence. To deem a mouse "addicted", however, is difficult.

The acute neurological outcomes are well modelled in the mouse, including the necessary basis of addiction: nicotine reward and reinforcement. However, elucidating the neural mechanisms underlying the smoker's transition from infrequent tobacco use to habitual consumption and dependence is challenging in the mouse. A comparison of 10-day or 41-day nicotine i.v. self-administration in the rat shows that initial voluntary consumption is goal-directed, and that habit-formation ensues with extended training [START_REF] Clemens | Behavioral and neural substrates of habit formation in rats intravenously self-administering nicotine[END_REF]. A second important trait of human smoking behaviours is the escalation in consumption of nicotine. Only in the rat has this been demonstrated by chronic, intermittent nicotine exposure over several weeks [START_REF] Gilpin | Nicotine vapor inhalation escalates nicotine selfadministration[END_REF][START_REF] Cohen | Robust Escalation of Nicotine Intake with Extended Access to Nicotine Self-Administration and Intermittent Periods of Abstinence[END_REF]. Furthermore, extended access (21-hour) i.v. selfadministration sessions conducted over several weeks is sufficient to allow precipitated nicotine withdrawal [START_REF] Cohen | Robust Escalation of Nicotine Intake with Extended Access to Nicotine Self-Administration and Intermittent Periods of Abstinence[END_REF][START_REF] O'dell | Extended access to nicotine self-administration leads to dependence: Circadian measures, withdrawal measures, and extinction behavior in rats[END_REF]). This experimental procedure allowed a correlation between total nicotine intake and number of withdrawal signs to be established [START_REF] O'dell | Extended access to nicotine self-administration leads to dependence: Circadian measures, withdrawal measures, and extinction behavior in rats[END_REF]). The mouse presents a major obstacle for such protracted, extended access paradigms, due to the inability to maintain catheter patency for an extended period of time, and the inability to place the catheter in the second jugular vein when the first is blocked (as can be conducted in the rat).

Nicotine withdrawal in the mouse instead must follow chronic exposure via a sub cutaneous osmotic mini-pump over the course of several weeks.

Despite the aforementioned differences, the laboratory mouse remains a useful tool for the assessment of various nicotine addiction phenotypes. Substantial information can be extracted in mouse-adapted protocols, including measures of nicotine reinforcement (CPP and self-administration), motivation (breaking point for self-administration), and even relapse (cue-induced nicotine-seeking reinstatement). Teamed with its genetic malleability, the mouse boasts a significant advantage for the study of the cellular mechanisms underlying nicotine addiction traits.

Viral vectors as a research tool

The creation of KO mice has propelled research advances in the nicotinic receptor field.

It has allowed us to investigate the behavioural and physiological contribution of individual nAChR subunits that otherwise cannot be targeted by pharmacological molecules, such as the ⍺5 nAChR subunit. However, these gross KO models do not allow us to stratify the role of nAChR subtypes in a cerebro-regional dependent manner, which would normally be resolved by local infusions of pharmacological agents. To meet this demand, scientists have explored gene transfer as a means to selectively express a transgene (wild-type or mutant) into a tissue of interest onto a WT or KO background.

This technique has been heavily exploited by the laboratory of Dr Uwe Maskos, allowing us to compare the behavioural and physiological contribution of certain nAChR subunits in different brain structures, and most notably has revealed the action of nAChRs in the ventral tegmental area as being necessary in nicotine-related behaviours (Tolu et al. 2013;Morel et al. 2014;Orejarena et al. 2012;Pons et al. 2008;Exley et al. 2011;Maskos et al. 2005).

The method of gene transfer that shall be discussed is that of viral vectors, notably lentiviral vectors and adeno-associated viral vectors. These viruses have been exploited

for clinical gene therapy and basic research. They serve as biological mediators of transgene integration into living cells. By genetically modifying the genomes, scientists have been able to hinder replication and pathogenicity, yet maintain the desirable characteristics of these viruses: stable, long term expression of a transgene, and infection of post-mitotic cells. Viral-based methods of gene introduction are therefore a versatile tool for the creation of tailor-made in vivo models. We also have the means to further increase the resolution of our investigations, by targeting transgene expression to specific neuronal subpopulations within a brain region, for example dopaminergic neurons.

Adapted lentiviruses and adeno-associated viruses have both been exploited throughout this thesis. Their genomes, life cycle, and research applications shall be discussed, with particular focus on the lentivirus, which was used for expression of β4 nAChR subunits in vivo.

1.9.a The HIV-1 lentivirus and its genome

The lentivirus is a genus subclassification of the Retroviridae family, with a genetic vector of approximately 9kb. Retroviridae are Baltimore class VI viruses, characteristically presenting their genetic material as a single strand of RNA (ssRNA), alongside proteinic machinery required for reverse transcription of RNA into DNA. It is this retro, i.e. backwards, series of molecular events prior to protein synthesis that distinguishes this class of virus from DNA-containing viruses (e.g. class I). Further discriminating it, the lentivirus integrates its reverse-transcribed genetic material into the genome of the host cell for production of viral proteins, as opposed to remaining a separate entity in the nuclear compartment of the cell [START_REF] Trono | Lentiviral Vectors[END_REF].

The components and structure of the lentivirus are illustrated in figure 1.9.a.i. The viral genome is sufficient in encoding these components, and generates them with the help of cellular substrates. The viral genome is illustrated in figure 1.9.a.i, and its function is reviewed in table 1.9.a.ii.

RRE

Targeted by Rev for translocation to cytoplasm [START_REF] Malim | The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA[END_REF] polyA Signals polyadenylation for mRNA maturation [START_REF] Naldini | In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[END_REF] Ψ packaging and dimerisation signal [START_REF] Clever | Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation[END_REF] Table 1.9.a.ii. Overview of HIV-1 genome compartments and their function.

1.9.b Life cycle of HIV-1

The viral particle binds to a cell, exposing fusogenic proteins. The virus' glycoprotein envelope fuses with the cell's membrane, eventually forming a pore for the insertion of viral material, including its ssRNA-containing capsid. Once in the cell's cytoplasm, the capsid uncoats to expose its genetic material. In doing so, protein reordering occurs and the reverse transcription complex is formed. This complex has reverse transcriptase and integrase activity for the synthesis of viral cDNA and its integration into the host cell DNA, respectively. Unlike retroviruses such as onco-retroviruses, HIV-1 does not rely on host cell nuclear envelope breakdown for synthesis of viral cDNA, a process that occurs during mitosis. HIV-1 ssRNA can therefore enter the nucleus of non-dividing cells for synthesis of cDNA, and integration into the host cell genome, independent of cell cycle stage [START_REF] Trono | Lentiviral Vectors[END_REF].

Integration of viral cDNA into the host cell genome can occur using a cooperation of viral and host cell processes [START_REF] Trono | Lentiviral Vectors[END_REF]. Cellular transcription machinery is recruited It is also important to note that extensive genetic modifications have been made for the production of lentiviruses. An inspired strategy of segregating viral genes onto different vectors, allows production of viral components necessary for a progenitor virus, encapsidating the generated transgene-encoding ssRNA (Naldini, Blomer, Gage, et al. 1996;[START_REF] Naldini | In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[END_REF]. The ssRNA also encodes the proteins necessary for integration and expression of the transgene, but is not equipped with the genes for the generation of virus packaging components. The vectors used for lentivirus production are described below:

• The packaging vector contains the genes GAG, POL, REV and TAT, essential for the production of structural proteins and enzymes that are packaged into virions. The packaging vector lacks cis-acting elements as well as the packaging signal, Ψ.

Furthermore, the LTRs have been removed, impeding integration and replication of these genes. Instead, a cytomegalovirus (CMV) promoter is used to initiate transcription.

• The transfer vector contains a promoter and transgene(s) of interest, LTRs for integration of cDNA, RRE and central PPT. However, it lacks lacks GAG, POL, ENV, REV and TAT, preventing viral replication. New generation lentivirus constructs contain a woodchuck hepatitis B post-transcriptional regulatory element (WPRE) sequence to increase RNA stability and transgene expression. The U3 region of the 3'

LTR is deleted to impair LTR's promoter activity after reverse transcription, and preventing potential biological disarray in the presence of the transgene promoter. This is typical of a generation of self-inactivating, SIN, viruses.

Continued…

• The envelope vector segregates ENV onto a third vector, further improving the safety of lentivirus production (Naldini, Blomer, Gage, et al. 1996;[START_REF] Naldini | In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[END_REF]. ENV produces capsid glycoproteins, which will define the virus' infection selectivity. We use that of the vesicular stomatitis virus (VSV). VSV-G binds to phospholipids of cell membranes, allowing unbiased cell-type infection. The VSV-G capsid elicits infection of a cell via endocytosis, and not membrane fusion, as is the case with wild-type HIV-1 capsid. Alternatively, if specific infection of nerve terminals is desired, one can instead use a rabies-derived glycoprotein (CVS). Both of these glycoprotein coats have the advantage of stabilising the virus, even during the ultracentrifugation step of virus production.

All three vectors are transfected into HEK-293T cells in vitro to produce lentiviruses containing the transfer vector in ssRNA form (figure 1.9.c.i). A titre of viral particles is measured, and virus stored at -80°C in PBS pending use. The advantages and disadvantages of using lentiviruses for gene transfer are summarised below:

Advantages Disadvantages

Applicable to dividing and non-dividing cells

Integration allows for stable transgene expression

Integration into cell genome may cause deleterious mutation

Large cloning capacity

Broad spectrum of targetable cell types Immunogenic, limiting the amount of viral particles that can be injected in a tissue Table 1.9.c.ii. Advantages and disadvantages of lentivirus-based gene transfer.

1.9.d The adeno-associated virus as a research tool

In response to some of the drawbacks that are encountered with a lentivirus-based method of gene delivery, the use of other viruses has been explored such as the adenoassociated virus (AAV). The non-immunogenic nature of the virus is an acknowledged advantage in gene therapy. This lack of pathogenicity in fact hampered basic research into this virus, and delayed its consideration as a tool for gene therapy (Daya and Berns). The genome and life cycle of the wild-type AAV shall be briefly discussed, as well as its function as a gene transfer tool for in vivo studies.

The AAV contains a single stranded DNA genome of 4.

1.9.e The AAV lifecycle

The AAV attaches to, and infects a cell via membrane-associated heparin sulfate proteoglycan [START_REF] Summerford | Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions[END_REF]. The AAV enters the cell by endocytosis, and then escapes the endocytic vesicle to translocate into the nucleus where it uncoats to reveal its ssDNA genome. The subsequently formed dsDNA can integrate into the host cell genome and the virus enters a latent stage, with little replication. Helper viruses such as adenovirus [START_REF] Atchison | Adenovirus-associated defective virus particles[END_REF] and herpes simplex virus [START_REF] Buller | Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication[END_REF]) allow the AAV to leave the latent stage, and enter the lytic stage. The integrated provirus is excised from the host cell's DNA by a Rep-mediated mechanism, allowing more efficient gene transcription, genome replication, and production of virions to ensue. Exit from the cell is also dependent on helper virus-mediated cell lysis, highlighting the low potency of the AAV as an independent entity.

As with lentiviruses, AAVs can infect mature, non-dividing cells, including neurons. The AAV has therefore been heavily exploited in neuroscience research. The modifications to the genome for use as a gene transfer too shall now be discussed.

1.9.f AAV modifications for gene transfer

As with the lentivirus, the AAV genetic sequences have been segregated onto several vectors for safe, efficient synthesis of viral particles in vitro, with packaging of the replication-deficient recombinant AAV (rAAV) construct. The rAAV contains the viral ITRs, promoter of choice and gene of interest (figure 1.9.f). Cap and Rep are therefore delivered in trans on an AAV-RC vector, so are not present in the rAAV nor in the final AAV particles. Due to these modifications the rAAV genome cannot integrate into the host cell's genome. This is advantageous in that there is no risk of insertional mutagenesis, which may be the case with the lentivirus-based method. A third plasmid, helper vector, can be employed (although helper-free kits exist, Agilent Technologies), containing adenoviral genes (e.g. E1A, E1B, E2A and E4) that facilitate AAV-2

propagation [START_REF] Samulski | Adenovirus E1B 55-Mr polypeptide facilitates timely cytoplasmic accumulation of adeno-associated virus mRNAs[END_REF]. The three vectors are co-transfected into a mammalian cell line in vitro, in a similar manner to that described for lentivirus production (figure 1.9.f). One study documents the use of an AAV for research into the role of neuronal nAChRs, using the vector to express shRNA to knockdown β4 expression [START_REF] Mineur | Nicotine decreases food intake through activation of POMC neurons[END_REF].

Lentiviruses continue to curry favour in this domain, which may be due to the facility of lentivirus production over AAV production. An additional obstacle to the use of AAVs is the limitation in cloning capacity (4.4kbp) compared to that of the lentivirus (9kbp). This is a severe drawback when a co-marker is required for visualisation of virus infection (remember that no specific antibodies exist for in situ nAChR subunit labelling; [START_REF] Moser | Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures[END_REF]). With a promoter typically achieving several hundreds of base pairs (phosphoglycerate kinase promotor equaling 310 base pairs), plus 660bp of an antibiotic resistance gene, plus nAChR subunit lengths achieving approximately 1500 base pairs (β4 cDNA equaling 1488 base pairs), we have little room for manoeuvre to incorporate a fluorophore, such as the 714 bp green fluorescent protein sequence, plus an internal ribosome entry sequence (IRES) to enhance GFP's expression, standing at 590 base pairs. This demonstrated that 1) the lentivirus successfully infects cells and 2) the PGK promoter is suitable for expression and accumulation of the transgene. Verification of β4* nAChR expression in these cells by autoradiography was attempted, but was unsuccessful (data not shown).

Results

Lentivirus validation and stereotaxy procedures

The ensuing in vivo validation confirmed the successful expression of both the GFP and β4 subunit transgenes.

2.1.b Validation of the lentivirus in vivo

In order to determine whether the lentivirus delivery of the construct is functional in the mouse brain, in vivo stereotaxic injections of the ventral tegmental area were conducted.

This brain region was chosen for the ease of targeting and for comparison to other lentiviruses produced in the laboratory that successfully infect the VTA (Pons et al. 2008;Maskos et al. 2005;Orejarena et al. 2012;[START_REF] Reperant | Effect of the alpha4beta2* nicotinic acetylcholine receptor partial agonist varenicline on dopamine release in beta2 knock-out mice with selective re-expression of the beta2 subunit in the ventral tegmental area[END_REF]. The method of delivery (metal needle versus glass pipette, angle of injection), volume and concentration of lentivirus was verified for brain regions of interest (see section 2.1.c).

Immunofluorescene analysis allows us to assess the zone of GFP expression in the targeted brain region, a measure of lentivirus infection. It also allows us to determine whether the lentivirus, or the concentration used, is toxic as seen by the presence of tissue damage. This was not the case for the PGK-β4 lentivirus, deemed by the absence of autofluorescent cellular debris and the absence of holes at the site of infection. Figure The same production of lentivirus was used throughout this thesis, thanks to its high starting concentration (740ng P24/µl).

Validation of viral-mediated β4 expression in situ was conducted by autoradiographic assessment.

Mice were injected with the PGK-β4 lentivirus in the brain region of interest using a stereotaxic procedure (see Articles One and Two). After four weeks, the animals were sacrificed in a drug-free manner (by carbon dioxide asphyxiation or by cervical There is an unfortunate lack of specific antibodies against the various nAChR subtypes for in situ labelling. We overcome this by radio-labelling the nAChRs, which can be detected on Kodak Biomax MR films. The energy from gamma radiation, and even light, evokes a chemical reaction in the film allowing the visualisation of the source of radioactive emissions. By labelling the nAChR subtypes with an I 125 -coupled compound, we can therefore reveal the location of the nAChR subtypes in a tissue of interest.

There is no β4*-specific radio-labelled compound currently on the market. Instead, I 125epibatidine was used to label all heteromeric nAChRs (β2* and β4* nAChRs), and the molecule displaced from β2* nAChR, leaving I 125 -epibatidine on putatively β4* nAChRs.

This can be conducted using cytisine (Frahm et al. 2011), or the more β2-specific compound 5-iodo-A85380 (articles 1 and 2).

WT mice show high 5-iodo-A85380-resistant I 125 -epibatidine binding in the MHb and IPN, which is reduced in the β4 KO mouse and restored by lentivirus-delivery of β4 cDNA (figure 2.1.b.ii). The lentivirus is also capable of expressing β4 nAChR subunits (and β4* nAChRs) in the VTA. Epibatidine binds to the interface between ⍺ and β subunits (Hansen et al. 2005), therefore suggesting that virally-introduced β4 is able to assemble with endogenous ⍺(3)

subunits. This technique shows that viral β4 protein is accumulated in the targeted brain region after four weeks. Furthermore, in corroboration with immunohistofluorescence analysys, the volumes used during stereotaxic injections of the desired brain region are appropriate for specific targeting of these regions. However, VTA-targeted β4 nAChR subunit re-expression results in significant 5-iodo-A85380-resistant I 125 -epibatidine binding in the periphery of the IPN. The implications of diffusion of the PGK-β4 lentivirus from the VTA are considered in Article Two.

2.1.c Stereotaxically targeting the MHb

Intra-MHb injections had not been previously attempted in our team, whose experience lies notably in intra-VTA lentivirus injections.

Several modifications were made for intra-MHb stereotaxic targeting, following much trial and error. Vertical descent of a 35-gauge needle at ±0.3mm from the skull's midline was unsuccessful for multiple reasons. The cranial midline suture is variable among mice, even within an inbred strain such as the C57Bl/6J, and the margin of error created by this variability greatly diminished the success in reproducibly targeting the MHb, whose diameter is 0.5mm. Secondly, vertical descent of the needle proved to push against the epithelium between the habenula and dorsal hippocampus, impeding penetration by this blunt needle tip. Penetration of the MHb was further restricted by the large size of the needle (0.2mm). In order to overcome these issues, I decided to adapt the procedures used by Frahm and Fowler (Frahm et al. 2011;Fowler et al. 2011).

The principal adaptations to the system were to i) inject at an angle along the coronal plane, and ii) use pulled glass pipettes instead of a metal needle (figure 2.1.c.i). Injecting with a ±20° angle allowed me to use skull coordinates distal to the midline (±1.25mm instead of 0.3mm). Secondly, using a pulled glass pipette with a tip diameter of 35µm facilitated the penetration of the MHb, and minimised damage to the tissue that was otherwise caused by a 200µm metal needle

In order to improve the success of injecting both MHb, two injection pipettes were used.

This improved the success rate since both pipette tips were calibrated at precisely the same point on the bregma of the mouse's skull (figure 2.1.c.i). This is a useful strategy, since it guarantees that the injections on the left and right hand side will be at 2.5mm at the skull surface, with injection sites consistently separated by 0.8mm. The distance between the two MHb is stable between individuals.

Below is an example of a test injection of the MHb using methylene blue (figure 2.1.c.i).

The stereotaxic coordinates used were: anterior/posterior -1.80mm, medial ±1.25mm and dorsal/ventral -2.40mm from the bregma surface. The pressure applied for the injection was greater than that for lentivirus injections. The resultant pressure-induced damage to the tissue allows visualisation of the injection point. The mouse did not wake from this procedure, and was sacrificed for perfusion and analysis of the brain immediately after the injection.

Introductory comments

Recent human genetic studies have repeatedly shown that polymorphisms in the gene locus encoding ⍺5, ⍺3 and β4 nAChR subunits alter nicotine dependence risk (see section 1.7). This proposes the nAChRs containing ⍺5, ⍺3 and β4 subunits as potential therapeutic targets for an ever-necessary successful nicotine cessation aid.

As such, we aimed to elucidate the contribution of β4-containing (β4*) nAChRs to nicotine's appetitive properties in an intravenous nicotine self-administration test. This was conducted in wild-type (WT) and β4 knockout (KO) mice, revealing that β4* nAChRs do indeed contribute to the positive reinforcing properties of nicotine that lead to its repeated consumption (7.5-60µg/kg/infusion). This is an appropriate tool for replying to Objective 1.

We demonstrate that nicotine-elicited responses of VTA DAergic neurons, and nicotine- sends neuronal efferents to co-release glutamate and acetylcholine onto GABAergic and serotonergic neurons of the IPN [START_REF] Ren | Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes[END_REF][START_REF] Groenewegen | Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat[END_REF]. IPN neurons project to the raphe nucleus and dorsal tegmentum (Hsu et al, 2013), which provide neurological input to the ventral tegmental area (VTA) (Geisler and Zahm, 2005). Thus far, the MHb and IPN have been linked to anxiety, impulsivity, nicotine withdrawal, and primary reinforcement (Hsu et al, 2014;Kobayashi et al;Zhao-Shea et al).

The role of β4* nAChRs has been investigated in certain nicotine-mediated behaviors, including nicotine-elicited seizures (Salas et al., 2004), aversion (Frahm et al, 2011;Slimak et al), anxiety (Salas et al, 2003), and withdrawal (Salas et al, 2004;Zhao-Shea et al, 2013), supporting the involvement of this subunit in regulating the aversive properties of nicotine (Fowler and Kenny, 2013). However, recent findings provide evidence for their role in the habenula-interpeduncular pathway in nicotine reinforcement. Notably, pharmacological inhibition of β4* nAChRs reduces nicotine-conditioned place preference and nicotine self-administration (Glick et al, 2011;Jackson et al).

Developing on this body of work, we aimed to elucidate the role of β4* nAChRs in the MHb and IPN in a segregated manner. To do this, we used a combination of transgenic mouse lines, lentivirus technology, and assessment of nicotine-mediated behavioral and physiological outcomes. This allowed us to establish the role of β4* nAChRs in the IPN independently of the β4-rich MHb.

MATERIALS AND METHODS

Animals

Experiments were conducted in male C57Bl/6J mice (wild-type, WT), and constitutive knockout (KO) mice lacking β4 nAChR subunits (Xu et al, 1999) was dissolved in physiological saline (0.9%), and the pH adjusted to 7.4 with sodium hydroxide.

Nicotine doses, reported as free base concentrations, were either contingently administered by intravenous route in the self-administration test and electrophysiological experiments (0-90 µg/kg/infusion) or by subcutaneous injection in the microdialysis experiments (0, 0.17, 0.24, 0.33 mg/kg).

Food and nicotine self-administration experiments

The self-administration experiments were conducted in mouse operant chambers (Model ENV-307A-CT; Med Associates Inc., Georgia, VT, USA) equipped with two holes, one randomly selected as the active hole and the other as the inactive hole. A cue light located above the active hole (intensity of 20 mA) was paired contingently with the delivery of the reinforcer (see supplementary material). β4KO and WT controls were first trained to respond for standard food pellets (Testdiet, Richmond, IN, USA) on a fixed ratio (FR) 1 schedule of reinforcement until stable responding was obtained and then on a FR3 schedule. An intra-jugular catheter was then implanted under anesthesia. After recovery from surgery, mice were re-trained on food responding and then trained to self-administer nicotine at the dose of 30 µg/kg/infusion during 6 days on a FR3 schedule of reinforcement. Subsequently, in β4KO and WT controls, a progressive ratio paradigm was performed in order to test the motivation of the animals to work for nicotine at this dose. Following stable responding, all mice underwent dose-response experiments performed according to a within-subjects design, including saline and nicotine at the doses of 7.5, 15, 30 and 60 µg/kg/infusion. Doses of nicotine were presented in ascending order, and saline was given last. All doses were available during 3 consecutive days.

Viral expression vectors

The lentivirus vector is based on that previously described (Maskos et al), obtaining a bicistronic cassette that concomitantly expresses mouse β4 and GFP under a phosphoglycerate kinase promoter (PGK-β4). Control lentiviral vectors induced GFP expression only (PGK-GFP).

Adeno-associated virus 2/1 serotype (pAAV.hSynap.Flex.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40, Vector Core, Pennsylvania) was used for histological analysis. The AAV construct contains a synapsin promotor upstream of a GCaMP5 sequence flanked by loxP and loxP2272 sites, allowing expression of the fluorophore GCaMP5 in crerecombinase expressing cells only.

Stereotaxic delivery of lentiviral vector

Stereotaxic delivery of viruses was performed as previously (Tolu et al, 2013) with the following modifications. The MHb was targeted at -1.70 and -1.90mm posterior to bregma, ±1.25 mm lateral to the midline and -2.40 mm ventral to bregma skull surface. Injections were conducted using pulled glass pipettes at an angle of ±20°. Two 0.5 µl volumes of lentivirus were delivered bilaterally at 0.2 µl/min (PGK-β4 300ng/µl, PGK-GFP 75 ng/µl p24 protein). The IPN was targeted at -3.60 mm posterior to bregma, -1.60 mm lateral to the midline and -4. We verified that slices were intact and that the autoradiographic puncta corresponded to the targeted brain region.

Immunofluorescence

Mice were deeply anaesthetized with ketamine:xylazine solution (i.p.) and perfused intracardially with PBS and 4% PFA. Coronal brain slices of 60 µm thickness were obtained using a vibratome (Leica Microsystems, Wetzlar, Germany) and incubated in 10% normal horse serum (NHS) and 0.2% triton-X in PBS for 4 h. Antibody incubations were conducted in 2% NHS and 0.04% triton-X solution. Primary antibodies used: rabbit anti-GFP (Life Technology, OR USA), sheep antityrosine hydroxylase (Millipore, CA USA), goat anti-choline acetyl Transferase (Chemicon, CA USA), goat anti-somatostatin (Santa Cruz Biotechnology, CA), 1:500 dilution, incubation overnight at 4°C. Sections were rinsed and incubated in the antibody solution along with secondary antibodies for 4 h at room temperature. Images were taken using a Zeiss fluorescent microscope. Colocalization was quantified using Image J software.

Microdialysis experiments

β4KO and WT mice were anesthetized with a ketamine:xylazine (5:1; 0.10 ml/10 g, i.p.) solution and placed in a stereotaxic apparatus. Unilateral microdialysis probes (CMA7: 1 mm, CMA Microdialysis, Stockholm, Sweden) were directly implanted vertically in the nucleus accumbens (NAC, Anterior +1.5; a ±0.8 lateral; ventral -4.8 mm from bregma and skull surface according to the coordinates of Paxinos and Franklin (Paxinos and Franklin), and then fixed to the skull with dental cement. Two days after surgery, animals were habituated to the microdialysis environment overnight. The following morning, probes were perfused with a ringer solution (NaCl: 148 mM, KCl: 2.7 mM, CaCl2:1.2 mM and MgCl2: 0.8 mM, pH 6.0) at a constant rate of 1

1 Cnd MgC MgC. Two days after surgery, animals were habituated to the microdialysis environment overnight. The following morning, probes were perfused with a ringer solution (NaCl: 148 mM, KCl: 2.7 mM, CaCl2:1.2 d by a nicotine administration (0.17, 0.24, 0.33 mg/kg, s.c.). Four samples were collected after saline and 8 samples after nicotine administration (see supplementary material).

Electrophysiology experiments

Mice were anaesthetized with chloral hydrate (8%), 400mg/kg via intra peritoneal route, and positioned in a stereotaxic frame with a catheter inserted into the mouse's saphenous vein for intravenous administration of drugs. Body temperature was maintained at 37°C. Glass recording electrodes (tip diameter of 1-2mm and impedance of 4-8 MΩ) were placed in the VTA: -2.90 to -3.9mm relative to bregma, ±0.24 to ±0.96mm mediolateral with respect to the midline, and -3.5 to -4.5mm from the cortical surface according to brain atlas coordinates (Paxinos et al).

Extracellular identification of dopamine (DA) neurons was based on their location as well as on the set of unique electrophysiological properties that characterize these cells in vivo [START_REF] Ungless | Are you or aren't you? Challenges associated with physiologically identifying dopamine neurons[END_REF]. After a baseline recording of 10-20 min, 10 ml saline (0.9% NaCl) was injected into the saphenous vein, and after 5-10min, 10-20µl of nicotine solution was administered in an identical manner. DA cell firing was analyzed with respect to the average firing rate and the percentage of spikes within a burst as previously described (Morel et al, 2014). To quantify nicotine effects, we determined the maximum of fluctuation on a 3-min period before and after injection.

Statistical Analysis 143

The self-administration data were analyzed using two-way repeated measures ANOVA (Statistical Package for the Social Sciences, SPSS and GraphPad Prism 6) with dose as the intra-subject factor for the different genotypes followed by estimation of parameters post-hoc test (Tukey's multiple comparison). The within-subjects dose-response data was analyzed by twoway repeated measures ANOVA with hole and dose as intra-subject factors followed by the estimation of parameters post-hoc test (Šídák multiple comparison). Statistical analysis of nicotine consumption was performed using one-way ANOVA with Tukey's post-hoc test.

Analysis of autoradiographic data was performed using unpaired t-test. The Wilcoxon paired test was used to analyze DA cell's activation from baseline from electrophysiological recordings.

Statistical significance was set at p<0.05.

144

RESULTS

Food self-administration in β4KO and WT mice

β4KO and WT mice were first trained to respond for food reward (Fig. 1A). For the amount of pellets earned on FR1, a significant effect of training day was revealed (F(7,805)=128.054, p<0.001), but no significant effects of genotype or interaction between factors were observed.

For number of pellets earned on FR3, a significant effect of day (F(5,575)=8.858, p<0.001), and genotype (F(1,115)=4.136, p<0.05) was revealed. Post-hoc analysis revealed that KO mice earned less pellets than WT mice on days 1, 5 and 6 (p<0.05). Following surgery, mice were retrained on responding for food on FR3 during 3 days. A significant effect of day was observed (F (2,198)=10.229, p<0.001), but no significant effects of genotype or interaction between factors were revealed. Active and inactive responding for food pellets in both genotypes is shown in supplementary figure 1A.

Nicotine self-administration acquisition

Acquisition of nicotine intravenous self-administration (IVSA) commenced with the dose of 30 µg/kg/infusion on a FR3 schedule of reinforcement (criteria are described in the supplementary materials). 77.27% of WT mice (n=22) acquired this behavior at the end of 10 days of training, while only 13.33 % (n=15) of β4KO mice acquired nicotine self-administration (Fig. 1B). For active and inactive nose-poke activity to self-administer this dose of nicotine, significant differences were observed between β4KO and WT mice (genotype effect, F(1,35)=8.498, p<0.0). A significant effect of day (F(9,315)=2.233, p<0.05), hole (F(1,35)=88.764, p<0.001) and significant interaction between hole x genotype (F(1,35)=9.105, p<0.01) and day x hole interaction (F(9,315)=2.369, p<0.05) were observed, but no significant day x genotype nor day x hole x genotype interactions were revealed. Post-hoc analysis showed that WT mice responded more at the active nose-hole than KO mice on days 4, 5, 7 8 9 and 10 (p<0.05). Furthermore, a significant decrease in the area under the curve (AUC) for β4KO mice was revealed (F(1,36)=8.770, p<0.01) compared to WT mice (Supp. Fig. 1B).

Progressive Ratio Paradigm and IVSA Dose-response curve

Following acquisition, mice were trained on a progressive schedule of reinforcement in order to evaluate their motivation to work for the drug (Fig. 1C). A significant decrease in breaking point was revealed for β4KO mice (F( The acute administration of 0.17 mg/kg nicotine did not significantly modify DA extracellular levels at any time point in either group (Fig. 2B). 0.24 mg/kg nicotine significantly increased DAmine outflow in the NAC with respect to baseline in KO, but not in WT mice (Fig. 2C). A significant main effect of time (F(8,64)=5.596, p<0.001), and an interaction between factors was observed (F(8,64)=2.459, p<0.05). Post-hoc analysis showed significant differences between genotypes 15, 75 and 90 min after nicotine administration (p<0.05). In contrast, at 0.33 mg/kg nicotine, DA outflow increased in WT but not in KO mice (Fig. 2D). A significant main effect of time (F(8,88)=15.887, p<0.001), and an interaction between factors was observed (F(8,88)=3.007, p<0.01). Post-hoc analysis comparing pretreatment at each time point showed significant differences between genotypes 15 min after nicotine administration only (p<0.05).

VTA DAergic neurons of β4KO mice demonstrate an increased sensitivity to nicotine

To address the role of the β4* nAChRs in VTA DA cells' response to nicotine, we performed in vivo electrophysiological recordings in WT and β4KO mice. No differences were observed in the spontaneous activity of DA cells in WT and β4KO mice (Fig. 3A,3B). In both groups, a systemic injection of nicotine strongly increased DA cells' firing and bursting activity (Fig. 3C, Supp. Fig. 2A). 15 µg/kg nicotine was required to increase DA cell activity in WT mice (p<0.05), yet 5 µg/kg was sufficient in increasing the change in frequency from baseline (p<0.01) and the proportion of spikes within bursts (p<0.05) amongst β4KO mice (figure 3C, Supp. Fig. 2A).

Re-expression of β4* nAChRs in the MHb partially rescues nicotine IVSA

In situ autoradiography revealed the MHb and IPN as major hosts of β4* nAChR expression in WT mice, and that 5-I-A85380-resistant I 125 -epibatidine binding was reduced in β4KO mice (Fig. 4C). Residual binding was observed in the MHb of nicotine-exposed KO-β4 MHb mice (11.7%, ±3.6% SEM). PGK-β4 lentivirus injections resulted in restoration of radioligand-binding sites, demonstrating that exogenous β4 subunits are able to assemble with endogenous nAChR subunit partners to form the obligatory inter-subunit epibatidine binding site (Fig. 4A, 4C;Hansen et al). GFP fluorescence demonstrated that the majority of the MHb was infected and that these neurons project to the the IPN (Fig. 4B and Supp. Fig. 2). Viral GFP in the terminals colocalizes with the cholinergic marker anti-choline acetyl transferase (ChAT), and extends to the ChATnegative lateral IPN. Virally induced expression of MHb β4 also rescues β4* nAChR expression in the terminals supplying the IPN (Supp. Fig. 2B).

Autoradiographic analysis of KO-β4 MHb brains revealed the heterogenous nature of β4 reexpression amongst individuals. The levels of MHb β4* nAChRs expression were therefore systematically verified. A minimum of 38% 5-I-A85380-resistant I 125 -epibatidine binding was revealed to be necessary for nicotine IVSA (see supplementary results). The nicotine IVSA responses amongst those above the threshold, and the WT and KO controls are described below.

WT mice were injected with control lentivirus (WT-GFP MHb ) and β4KOs injected with control (KO-GFP MHb ) or PGK-β4 lentivirus (KO-β4 MHb ) in the MHb and nicotine IVSA assessed (Fig. 5A, Supp. Fig. 3). All groups demonstrated specific nose-poking at the active hole (Supp. Fig. 4, Supp. Table 1). Assessment of the number of infusions earned by WT-GFP MHb , KO-GFP MHb and KO-β4 MHb mice during nicotine IVSA revealed a significant group (F(2, 21)=6.06, p<0.01) and dose effect (F(4, 84)=27.25, p<0.0001), but no effect at the interaction (Fig. 3A). Nicotine consumed between groups was similar at the doses of 7.5 and 60.0 µg/kg/infusion, whereas a difference between WT-GFP MHb and KO-GFP MHb is observed at 15.0 (F(2,20)=6.211, p<0.01) and 30.0 µg/kg/infusion (F(2,20)=4.719, p<0.05, Supp. Fig. 5). KO-β4 MHb mice consumed an intermediate amount of nicotine at all nicotine doses, with no statistical difference to WT-GFP MHb or KO-GFP MHb .

No significant differences between genotypes were observed in operant behavior to obtain food pellets during re-training following catheter surgery (mean number of pellets obtained in 1 h by WT,n=9,68.81 ±6.89;n=15,49.13 ±6.42; KO-β4 MHb , n=7; 50.0 ±6.14; one-way ANOVA n.s.).

observed at the dose of 60.0 µg/kg/infusion or saline. This was reflected in the resultant nicotine consumed, however KO-β4 IPN mice consumed similar quantities to WT-GFP IPN and KO-GFP IPN groups at 30 µg/kg/infusion nicotine (Supp. Fig. 6). No significant differences between genotypes were observed in operant behavior to obtain food pellets during re-training following catheter surgery (mean number of pellets obtained in 1 h by WT,n=6,36.94 firing frequency in these KO-β4 IPN mice (p<0.05, Fig. 5E).

DISCUSSION

Several recent human genetic studies have highlighted the polymorphic nature of the CHRNA5-CHRNA3-CHRNB4 gene cluster, and its contribution to smoking behaviors and dependence risk. Notably, variants in CHRNB4's coding region reduce the risk for nicotine dependence [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF], and variants in CHRNB4's regulatory domain decrease the age of onset for tobacco consumption (Schlaepfer et al, 2008). The present study aimed to elucidate the role of β4* nAChRs in nicotine reinforcement in mice, and the underpinning physiological correlates.

For this purpose, we first evaluated operant nicotine IVSA behavior in WT and β4KO mice. This experimental procedure has been shown to be a reliable method to model the addictive potential of several drugs of abuse, including nicotine (Orejarena et al, 2012;[START_REF] Sanchis-Segura | Behavioural assessment of drug reinforcement and addictive features in rodents: an overview[END_REF]. This paradigm incorporates various aspects of human smoking behaviors such as the volitional aspects, rapid nicotine delivery via systemic route, and chronic exposure to the drug.

β4KO mice show a deficit in nicotine self-administration at all doses tested (7.5, 15, 30 and 60 µg/kg/infusion) as compared to WT controls, as well as a decrease in motivation to selfadminister nicotine. This is not due to cognitive incapacity in the task since WT and β4KO mice alike self-administer food successfully during operant training. Potential underlying neural mechanisms were explored in order to elucidate the physiological correlates resolving the nicotine self-administration deficit. The mesolimbic DA system is an established substrate for the rewarding properties of nicotine (Di [START_REF] Chiara | Dopamine and drug addiction: the nucleus accumbens shell connection[END_REF], therefore we investigated whether the β4KO's behavioral deficit was reflected in the mesolimbic DA response to nicotine in vivo with neural networks intact, including the β4* nAChR-loaded habenulo-interpeduncular pathway.

Microdialysis experiments in the NAC revealed higher basal extracellular levels of DA in β4KO versus WT mice. A reinforcing dose of nicotine (0.33 mg/kg) was effective at eliciting an increase in NAC DA outflow in WT controls, but not in β4KO mice (Walters et al, 2006). These results are coherent with previous data showing that conditioned place preference induced by a rewarding dose of nicotine is blunted by pharmacological blockade of β4* nAChRs (Jackson et al, 2013), known to depend upon intact mesolimbic responses (Fields et al, 2007). Unexpectedly, a subeffective dose of nicotine amongst WT mice (0.24 mg/kg) induced an increase in DA extracellular levels in the NAC of β4KO mice, indicating a leftward shift in sensitivity to nicotineinduced DA activation in these animals. Basal responding was unaltered in in vivo electrophysiological assessment of VTA DAergic neurons of β4KO mice, however these neurons demonstrated an increase in sensitivity to acute doses of nicotine. Conversely, over-expression of the CHRNA5-CHRNA3-CHRNB4 cluster in transgenic mice increases nicotine IVSA acquisition, and reduces VTA DAergic activation by nicotine (Gallego et al, 2012). This altered sensitivity to nicotine amongst β4KO mice may be facilitating the downward shift of the doseresponse curve in the nicotine IVSA paradigm, although it remains to be determined whether these responses represent those occurring during nicotine IVSA. This idea is in agreement with a previous study showing that enhancing DA activity in the NAC core increased satiety-like responses to intravenously self-administered cocaine [START_REF] Suto | Satiating effects of cocaine are controlled by dopamine actions in the nucleus accumbens core[END_REF]. All together, these data indicate that β4* nAChRs are crucial for sustaining correct VTA and NAC responses, and for regulating the reinforcing properties of nicotine.

The habenulo-interpeduncular pathway is the principal host to β4* nAChR expression.

Conditional expression of a fluorophore in β4-expressing IPN neurons allowed us to visualize β4 subunit expression without the masking effects of β4 from MHb terminals. This strategy located β4 expression to the dorsal portion of the IPN, with colocalization to somatostatin-positive interneurons. We next pursued a stratification of β4* nAChR's region-specific role in our investigations. Lentivirus-mediated gene delivery to the MHb restored β4 nAChR subunit expression on a β4KO background, forming putative ⍺3β4* nAChRs in MHb soma and terminals innervating the IPN. MHb β4* nAChRs are required for nicotine-evoked excitatory responses (Dao et al, 2014;Görlich et al, 2013;Hsu et al, 2013), and β4* nAChRs on MHb terminals are required for nicotine-evoked acetylcholine release (Beiranvand et al, 2014;Grady et al, 2009). The present study builds upon our knowledge of the β4* nAChRs and the habenulointerpeduncular pathway, previously attributed to modulating nicotine aversion and nicotine withdrawal (Frahm et al, 2011;Salas et al, 2007). Here we provide evidence for a critical role of these receptors in the habenulo-interpeduncular pathway in modulating nicotine reinforcement and meso-accumbal DA responses to nicotine. Re-expressing β4* nAChRs in the MHb would restore responses to nicotine and allow activation of non-β4* nAChRs in the IPN by MHb

SUPPLEMENTARY MATERIALS SUPPLEMENTARY METHODS

IVSA

During food training, β4KO and WT controls were food deprived to 85-90% of their ad-libitum weight. Subsequently, free-feeding mice were implanted with indwelling intravenous silastic catheters in the right jugular vein under anaesthesia (ketamine/xylazine 5:1; 0.10 ml/10 g, i.p.), as previously reported [START_REF] Soria | Lack of CB1 cannabinoid receptor impairs cocaine self-administration[END_REF]. After recovery from surgery, mice were allowed to respond for food pellets again during three consecutive days in order to guarantee no residual effects of the surgery on learning processes. The next day, mice were trained to self-administer nicotine at the dose of 30 µg/kg/infusion. Nicotine was infused via a syringe that was mounted Nose poking on the active hole resulted in a nicotine infusion (23.5 µl over 2 sec), while nose poking on the inactive hole had no consequences. Each nicotine infusion was followed by a 10sec time-out period, as previously reported (Martin- [START_REF] Martin-Garcia | New operant model of nicotine-seeking behaviour in mice[END_REF]. Stable acquisition of nicotine IVSA behavior was achieved when mice showed, during three consecutive sessions, less than 20% deviation from the mean of the total number of reinforces (80% of stability), at least 65% responding on the active hole, and a minimum of five reinforcers per session. The intravenous catheter patency was maintained with a daily flush of heparin solution (30 UI/ml), and the patency was evaluated at the end of the experiment by infusing 0.1 ml of thiopental (5 mg/ml) through the catheter. If prominent signs of anaesthesia were not apparent within 3 s of the infusion, the mouse was removed from the experiment.

administration. We therefore subdivided the KO-β4 MHb animals into two groups depending on the extent of β4* nAChRs re-expression. Mice expressing greater than 38% of WT MHb β4* nAChRs levels at least unilaterally were classed as high expressers (n=7, 58.6% ±8.4% SEM, figure 5B); mice expressing greater than KO-GFP MHb but less than 38% of WT MHb levels were classed as low expressers (n=8, 26.0% ±3.6% SEM, Supp. Fig. 4B); mice with KO-like 5-I-A85380-resistant I 125 -epibatidine binding were removed from the data.

The low expressers, which express statistically greater levels of β4* nAChRs than KO-GFP MHb (t-test, p<0.001, Supp. Fig. 4B), demonstrated a statistically KO-GFP MHb -like behavior in the nicotine IVSA test. 2-way repeated measures ANOVA revealed a dose (p<0.0001, F(4, 88) = 25.97), group (p<0.001, F(2,22) = 12.32) and interaction (p<0.01, F(8,88) = 3.49) effect between WT-GFP MHb , KO-GFP MHb and KO-β4 MHb low expressers. Tukey's post-hoc analysis revealed a significant reduction of nicotine infusions at all doses tested amongst the KO-GFP MHb and KO-β4 MHb low expressers compared to WT-GFP MHb (Supp. Fig. 4A). There was no statistically significant difference in nicotine infusions earned between KO-GFP MHb and KO-β4 MHb low expressers at any dose, nor between all groups during saline self-administration.

aversive dose of nicotine will reinforce navigation towards the neutral arm in order to avoid self-administration (<5/10 infusions/session).

In response to Objective 1, we submitted WT, β4 KO and β4 over-expressing (TABAC) mice to the intra-VTA self-administration test. We show aberrant inhibitory motivation amongst β4 KO and TABAC mice. WT mice avoided intra-VTA self-administration of 600 and 1200ng/50nl infusions of nicotine, however these doses were shown to be positively reinforcing in β4 KO mice who continued to self-administer these otherwise aversive concentrations of nicotine. TABAC mice showed amplified aversive responses to these doses of nicotine as observed by a marked avoidance of nicotine self-administration, and longer latencies during the task.

INTRODUCTION

Tobacco use represents a serious social and public health problem. As a result of its noxious health effects, tobacco use is the greatest cause of preventable morbidity and mortality worlwide (World Health Organization, http://www.who.int/tobacco/statistics/tobacco_atlas/en/). Faced with the absence of satisfactory smoking cessation medication, identification of the neurobiological mechanisms involved in this disorder, far from being resolved, is of increasing importance. Nicotine, the main psychoactive compound responsible for tobacco addiction, interacts with the nicotinic acetylcholine receptors (nAChRs) in the central nervous system, composed of eight α (α2-α7 and α9-α10) and three β (β2-β4) subunits.

These subunits co-assemble into pentameric, ligand-gated ion channels in various combinations [START_REF] Dani | Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system[END_REF][START_REF] Changeux | Nicotinic receptors, allosteric proteins and medicine[END_REF]2008). The use of genetically modified animals allowed for significant progress in our understanding of nAChR function, with particular focus on β2-nAChRs [START_REF] Maskos | Emerging concepts: novel integration of in vivo approaches to localize the function of nicotinic receptors[END_REF]Changeux, 2010;[START_REF] Stoker | Unraveling the neurobiology of nicotine dependence using genetically engineered mice[END_REF]. nAChRs present in the mesolimbic circuitry are thought to play a critical role in the initiation and maintenance of tobacco use (see [START_REF] Koob | Neurocircuitry of addiction[END_REF]Dani et al., 2011, for review). The role of the mesolimbic VTAdopaminergic neurons targeting the nucleus accumbens in the reinforcing properties of nicotine has been well established [START_REF] Chiara | Role of dopamine in the behavioural actions of nicotine related to addiction[END_REF]. Previous studies revealed a crucial role of two subunits partner of the high-affinity widely expressed receptor (α4β2containing receptors, α4β2* nAChRs) in mediating nicotine's reinforcing properties (Picciotto et al., 1998;[START_REF] Besson | Genetic dissociation of two behaviors associated with nicotine addiction: beta-2 containing nicotinic receptors are involved in nicotine reinforcement but not in withdrawal syndrome[END_REF], particularly at the level of the ventral tegmental area (VTA) (Maskos et al., 2005;Orejarena et al., 2012;Tolu et al., 2013), and a more ambiguous contribution of the α6 subunit, another β2 partner, in this structure (Pons et al., 2008;Exley et al., 2011).

Recent human genetic studies have revealed that human coding and non-coding polymorphisms for α3, α5 and β4 subunits (locus 15q25) are correlated with smoking and/or lung cancer [START_REF] Amos | Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1[END_REF][START_REF] Hung | A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25[END_REF][START_REF] Thorgeirsson | A variant associated with nicotine dependence, lung cancer and peripheral arterial disease[END_REF][START_REF] Chen | Nicotine dependence and comorbid psychiatric disorders: examination of specific genetic variants in the CHRNA5-A3-B4 nicotinic receptor genes[END_REF]. These receptors are expressed in discrete regions of the central nervous system. Beside the VTA (Klink et al., 2001;[START_REF] Azam | Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons[END_REF], the medial part of the habenula (MHb) and its main output the interpeduncular nucleus (IPN; habenulointerpeduncular pathway), are two of the few regions that co-expresses the α3, α5 and β4 subunits (Grady et al., 2009;Salas et al., 2009 ;Scholze et al., 2012;Hsu et al., 2013;Shih et al., 2014). The VTA appears as a heterogeneous brain structure that could mediate reward-but also aversive-related behaviors depending on distinct circuits and cell sub-populations (Piciotto and [START_REF] Picciotto | Neuronal systems underlying behaviors related to nicotine addiction: neural circuits and molecular genetics[END_REF]Lammel et al., 2012;Ilango et al., 2014;Lammel et al., 2014;Eddine et al., 2015). Whereas recent evidence has pointed to the lateral part of the habenula (LHb) as a brain region critically involved in the control of reward system activity [START_REF] Russo | The brain reward circuitry in mood disorders[END_REF][START_REF] Proulx | Reward processing by the lateral habenula in normal and depressive behaviors[END_REF]Antolin-Fontes et al., 2014;Velasquez et al., 2014;[START_REF] Quina | Efferent pathways of the mouse lateral habenula[END_REF], little research has focused on the role of MHb in drug reward (Kobayashi et al., 2013;[START_REF] Viswanath | The medial habenula: still neglected[END_REF]. The MHb may trigger either reward (Hsu et al., 2014) or aversive properties (Lee et al., 2015). The MHb/IPN-α5 and -β4 subunits are involved in nicotine withdrawal and anxiety-like behavior, as well as nicotine-induced hypolomotion and seizures (Salas et al., 2004;Salas et al., 2009;Baldwin et al., 2011;Velasquez et al., 2014). The few seminal findings concerning the VTA-and MHb-α5 subunits involvement in inhibition of aberrant nicotine consumption seems to be convergent (Fowler et al., 2011;Morel et al., 2014). However, genetic and pharmacological studies provide more controversial arguments to the role of β4 subunits in the MHb-IPN axis (Glick et al., 2011;Frahm et al., 2011;McCallum et al., 2012;Slimak et al., 2014). Involvement of the β4* nAChR in the habenulo-interpeduncular pathway is likely to be important, since this subunit participates in nicotine-induced MHb-IPN activity (Shih et al., 2014) and is the sole nAChR subtype allowing stimulation of acetylcholine release in the IPN (Grady et al., 2009).

Thus, we aim here to identify the brain region-specific involvement of the β4* nAChR in the regulation of nicotine reinforcing properties. We focused on subchronic effects of nicotine, considered as the initial step of the addiction process [START_REF] Wise | Dopamine, learning and motivation[END_REF].

Self-administration is a powerful behavioral paradigm to investigate these early steps toward nicotine addiction [START_REF] Corrigall | Nicotine self-administration in animals as a dependence model[END_REF][START_REF] Rose | Nicotine self-administration in animals and humans: similarities and differences[END_REF]. As described previously for nicotine (Maskos et al., 2005;David et al., 2006), inctra-cranial selfadministration (ICSA) experiments were conducted in a Y-maze using a choice-based, spatial discrimination task. Choice-based paradigms are one of the most relevant methods to assess reward effectiveness [START_REF] Mason | Preference paradigm: provides better selfstimulation reward discrimination than a rate-dependent paradigm[END_REF]. Discrimination tasks appear to constitute a well adapted paradigm for detecting reward-related deficits in mutant mice [START_REF] David | Brain regional Fos expression elicited by the activation of mu-but not delta-opioid receptors of the ventral tegmental area: evidence for an implication of the ventral thalamus in opiate reward[END_REF], particularly for assessing reward value in mice lacking or re-expressing nAChRs (Maskos et al., 2005;[START_REF] Besson | Genetic dissociation of two behaviors associated with nicotine addiction: beta-2 containing nicotinic receptors are involved in nicotine reinforcement but not in withdrawal syndrome[END_REF][START_REF] Maskos | Emerging concepts: novel integration of in vivo approaches to localize the function of nicotinic receptors[END_REF]Besson et al., 2011 ;Tolu et al., 2013). An advantageous feature of the ICSA test is to allow the concurrent assessment of aversive, or inhibitory motivational effects (Gavello-Baudy et al., 2008), with the role of the nAChRs in the aversive effect of nicotine being suspected. ICSA allows region-specific delivery of nicotine for targeting specifically brain structures such as the VTA [START_REF] Wise | Brain reward circuitry: insights from unsensed incentives[END_REF]David et al., 2006). Identifying, in vivo, the neural circuitry underlying the acquisition of nicotine self-administration would provide essential clues to our understanding of nicotine addiction, and its treatment. A major obstacle to elucidating the role of nAChR subtypes is the varied and widespread expression of nAChRs. Targeted lentiviral re-expression of nAChR subunits has proven to be an efficient tool for assessing specific structures and nAChR-dependent effects on nicotine-seeking behavior (Maskos et al., 2005;[START_REF] Maskos | Emerging concepts: novel integration of in vivo approaches to localize the function of nicotinic receptors[END_REF]Tolu et al., 2013;Fowler et al., 2011;Slimak et al., 2014). We thus investigated nicotine self-administration in transgenic mice that do not express (knockout, KO) or over-express (TABAC) the β4 subunit, and in KO mice in which this receptor was re-expressed into reward-relevant brain regions, namely the VTA, MHb and IPN.

MATERIALS AND METHODS

Animals and ethical statement

All surgical and experimental procedures were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).

We used β4* nAChR knockout mice (β4-/-mice), bacterial artificial chromosome (BAC) transgenic mice over-expressing the β4 subunit (TABAC) and their wild-type counterparts (WT, C57BL/6J strain). All were males and raised at Charles River, France.

Mice were housed with ad-libitum access to food and water in a temperature-controlled room (23ºC) with a light-dark cycle (12 h -12 h, light on at 8.00 a.m.) and sawdust bedding changed weekly. Following surgery for ICSA preparation, mice were housed individually. The animals were aged 3-4 months (27-30 g) at the beginning of the experiments.

Material and experimental protocol

Lentivirus expression vector

A lentivirus vector was generated based on that described by Maskos and colleagues (Maskos et al., 2005). Viral production is as described previously (Tolu et al., 2013). The construct contains β4 mouse cDNA and green fluorescent protein (GFP) genetic sequences, whose expression are driven by the phosphoglycerate kinase (PGK)

promoter (PGK-β4). The β4-containing lentiviral construct was obtained by subcloning strategies. The control lentivirus induces GFP expression only (PGK-GFP, Tolu et al., 2013; Fig. 1A).

Intracranial stereotaxic procedure for the lentiviral re-expression

Wild-type (WT) and β4 knockout (KO) mice were anesthetized by intraperitoneal ketamine and xylazine (5:1; 0.10 ml/10g) and placed with the skull exposed in a stereotaxic frame (Stoelting Co., Wood Dale, IL). The VTA was targeted as previously described (Tolu et al., 2013). The following coordinates were used for injections into the medial habenula (MHb): -1.70 and -1.90mm posterior to bregma, ± 1.25mm lateral to the midline (±20°), and -2.40mm ventral relative to bregma skull surface. Mineral oil-filled pulled glass pipettes were used to deliver two 0.5µl volumes of lentivirus bilaterally at 0.2µl/min (PGK-β4 300ng/µl, PGK-GFP 75ng/µl p24 protein). Coordinates for the IPN are as follows: -3.60mm posterior to bregma, -1.60mm lateral to the midline, and -4.50mm ventral relative to bregma skull surface, at an angle of -20°. A metal 35 gauge needle was used to deliver a single injection of 2µl lentivirus to the central region of the IPN (PGKβ4 75ng/µl, PGKGFP 25ng/µl p24 protein). Transgene expression is optimal after four weeks post injection.

Quantification of β4* nAChR expression

Mice were euthanised by a drug-free method. The brains were dissected and frozen to -80°C. 20µm coronal sections were obtained using a cryostat at -20°C and thaw mounted onto microscope slides (Menzel Glaser SuperFrost Plus). Radioligand binding was performed as previously (Frahm et al., 2011), with the use of the β2-specific 5-Iodo-A-85380 dihydrochloride (25nM, A85380, Tocris Bioscience, Bristol UK) as the cold ligand. Following the binding procedure, slides were exposed to a Biomax MR film (Kodak) for 16 hours. Developed films were scanned at 1600dpi for analysis using Fiji software. The inverse luminosity of the resultant autoradiographic puncta were measured, as were three background points per brain section.

Immunohistofluorescence procedure

Deeply anaesthetised mice (ketamine:xylazine solution i.p.) were intracardially perfused with PBS and 4% PFA. 60µm-thick coronal brain sections were obtained using a vibratome (Leica Microsystems, Wetzlar, Germany). Sections were incubated in 10% normal horse serum (NHS) plus 0.2% tritonX-100 in PBS for three hours. Proceeding incubations were conducted in 2% NHS and 0.04% tritonX-100. Primary antibodies used: rabbit anti-GFP (Life Technology, OR USA), sheep anti-tyrosine hydroxylase (Millipore, CA USA), sheep anti-tyrosing hydroxylase, 1:500 dilution. Corresponding apparatus, which provided, via a micro-vernier system, a precise and highly reproducible descent of the microsyringe piston. Each self-injection (50 nl) lasted 4 s. The movement of the animals in the Y-maze was detected by an optical system and the injector rotated to avoid tangling of the catheter and to maintain the mouse's locomotor liberty. The number triggered self-injections within the nicotine-reinforced arm per daily session (number of self-administrations) and the time between the opening of the starting box door and triggering of self-injections (self-injection latency) or choosing neutral arm (no self-injection latency) for each subject were recorded by a computer running a program specifically develop for the ICSA procedure (YAA; Imetronic, Pessac, France). The nicotine-associated arm is assigned randomly and alternatively to each animal and remains constant through the experiments.

Behavioral protocol

Before the beginning of the task, all animals were submitted to a habituation session during which they were connected to self-administration apparatus and placed in the maze for 10 min of free roaming (no injections). During a second habituation session, the mice were also allowed to carry out 10 trials as described above without any injection, to assure lack of spatial bias in the apparatus.

The first experiment was conducted at a well-established optimum dose for nicotine self-administration (100 ng/50 nl/ injection; David et al., 2006) during an acquisition phase lasting 7 days (1 session/day). The ICSA behavior in the Y-maze was also investigated in wild-type mice (WT) and β4-/-mice lacking the β4 subunit (referred to as KO). A group of WT mice received aCSF when self-injected. The results are summarized in Fig. 3A and3B.

Four experiments were also conducted using high doses of nicotine. The protocol consisted of two consecutive phases (Fig. 4567): 7 sessions with nicotine at 600 ng/50 nl/injection available, followed by 7 sessions with nicotine at 1200 ng/50 nl/injection in:

1) wild-type mice (WT), β4-/-mice lacking the β4 subunit (KO), and bacterial artificial chromosome (BAC) transgenic mice overexpressing the β4 subunit (TABAC mice);

2) control lentivirus vectorized WT and KO mice (referred to as WT-GFP VTA and KO-GFP VTA , respectively) and b4-expressing lentivirus vectorized mice (KO-β4 VTA ) at the level of the VTA;

3) control lentivirus vectorized WT and KO mice (WT-GFP MHb and KO-GFP MHb , respectively) and β4-expressing lentivirus vectorized mice (KO-β4 MHb ) at the level of the MHb;

4) control lentivirus vectorized WT and KO mice (WT-GFP IPN and KO-GFP IPN , respectively) and β4-expressing lentivirus vectorized mice (KO-β4 IPN ) at the level of the IPN.

An acquisition session comprised the following steps. To begin a trial, a mouse was placed in the start box and after 1 min the door to the stem and target arms were opened. In each group, half of animals were assigned to go to the right arm to trigger the injection of nicotine whereas the remainders were assigned to the left arm. Each daily session was composed of 10 trials separated by a 1-min intertrial interval. Therefore a maximum of 10 injections could be obtained by each subject per daily session (chance level: 50%). During the first four trials of the first session only, if an animal had chosen the neutral arm, it was immediately allowed to access to the arm enabling an injection of nicotine. From the fifth trial onward, the mouse was removed after 30 sec and replaced directly into the start box for the following trial.

RESULTS

Similar acquisition of nicotine ICSA in b4-/-and WT mice at an optimally rewarding dose

Figure 3A shows the number of nicotine self-administrations into the VTA in wildtype (WT, n=7) and β4-/-mice (KO, n=8) at the well-established, positively reinforcing dose for the WT animals (100 ng/50 nl; David et al., 2006) across seven days of acquisition in the Y-maze. We previously observed an absence of reinforced-arm discrimination performance in β2-/-mice [START_REF] Besson | Genetic dissociation of two behaviors associated with nicotine addiction: beta-2 containing nicotinic receptors are involved in nicotine reinforcement but not in withdrawal syndrome[END_REF]. In contrast, KO mice, as well as WT mice, exhibited significant preference for the nicotine-reinforced arm of the Y-maze. A global two-way ANOVA with one repeated factor (session) revealed no difference between WT and KO groups performances at this dose (genotype effect:

F(1,13)=0,67, p>0.05; session effect: F(6,78)=12, p<0.0001).

Dose-dependent limitation of nicotine ICSA in wild-type mice

Nicotine ICSA dose-response in wild-type mice

The dose-response curve (Fig. 3B) represents the difference in the number of nicotine self-injections in the last vs. the first session of nicotine-reinforced arm choices, according to nicotine doses self-injected by WT mice (100 n=7, 600 n=8 and 1200 ng/50 nl n=9). The same parameter in a group of aCSF-injected mice (n=9) is also reported in the graph (mean of number of self-administrations through the acquisition: 4,71 ± 0,01; differential of self-administrations: 0 ± 0,27).

Among all the doses tested, 600 ng/50 nl seems to be a threshold dose, as shown by the decreased differential of performances compared to that of the optimum known dose (100 ng/50 nl) (unpaired t test, p<0,01). The highest dose (1200 ng/50 nl) decreased the number of nicotine self-administration as shown by the lowest and negative differential performance (lower than that of the aCSF mice one, unpaired t test, p<0.05).

Elevated latencies and non-reinforced arm choice in wild-type mice

Pooled self-injection (nicotine-reinforced arm choices) and no self-injection (nonreinforced arm choices) latencies in WT (n=30) and KO (n=31) mice from the four studies conducted at 600 ng and 1200ng /50 nl are illustrated in figure 3C. All animals exhibited a decrease in mean latencies to complete a trial (nicotine-reinforced or not)

over the sessions at 600 ng/50 nl (ANOVA session effect: F(6,342)=43,28, p<0.001). In contrast, when the dose available was increased (1200 ng/50 nl), only WT mice showed increased latencies, as compared to KO mice. WT mice displayed significantly higher latencies when choosing the neutral arm than during the reinforced arm choice, suggesting a decreased motivation at the dose of 1200 ng/50 nl (ANOVA genotype effect: F(1,57)=9.26 p<0.001; session effect: F(6,342)=2.36 p<0.05; post hoc WT selfinjection vs. WT no self-injection, p<0.05; WT self-injection vs. KO self-injection, p<0.05;

WT self-injection vs. KO no self-injection, p<0.05; WT no self-injection vs. KO selfinjection, p<0.001; WT no self-injection vs. KO no self-injection, p<0.0001).

Impact of β4 levels on nicotine self-administration

We then investigated the acquisition of intra-VTA nicotine self-administration in WT (n=10), KO (n=9) and TABAC (n=7) mice, over fourteen sessions at two different doses (7 days at 600ng/50nl/injection followed by 7 days at 1200ng/50nl/injection). The number of self-administrations (Fig. 4A) and the latencies to self-injected nicotine (Fig.

4B

) or to choose the neutral arm (Fig. 4C) were analyzed.

Dose of 600 ng/50 nl. Both WT and KO mice exhibited a weak preference for the nicotine-reinforced arm of the Y-maze, which remained stable across the sessions with no significant difference between these two groups. In contrast, TABAC mice displayed a rapid decrease in the reinforced-arm choice, which remained below the chance level and significantly lower than in KO mice over all sessions except session 2. These observations are supported by a global two-way ANOVA with one repeated factor (session) (genotype effect: F(2,23)=3.75 p<0.05; session effect: F(6,138)=0.62 ns; genotype x session interaction: F(12,138)=0.60 ns; post hoc TABAC vs. KO mice: p<0.05). A global two-way ANOVA revealed that latencies to self-inject nicotine (session effect: F(6,138)=7.5 p<0.001) or to choose the non-reinforced arm (session effect: F(6,138)=4.67 p=0.0003) were decreased in the three groups of mice, but more so in KO mice.

Dose of 1200 ng/50 nl. Whereas nicotine self-administrations increased over sessions in β4 null mutants, WT mice displayed a decrease in their nicotine reinforcedarm choices, which remained closed or below to chance level. Over-expression of β4

(TABAC mice) led to a decrease in nicotine preference, which remained significantly below chance level for the duration of the experiment. These observations are supported by a global two-way ANOVA (genotype effect: F(2,23)=9.47 p=0.001; session effect: F(6,138)=1.41 ns; genotype x session interaction: F(12,138)=1.38 ns). Post hoc analysis revealed that KO mice preferred the nicotine-reinforced arm, while TABAC mice preferred the non-reinforced arm in comparison to WT mice (post hoc WT vs. KO, p<0.05; WT vs. TABAC, p<0.05; TABAC vs. KO mice: p=0.0002). Two-way ANOVA of mean latencies to self-inject nicotine (genotype effect: F(2,23)=5.10 p<0.05; session effect: F(6,138)=1.55 ns) revealed no evolution of the latencies at 1200 ng/50 nl over sessions. Post hoc analysis of the nicotine self-injected latencies revealed that TABAC mice were much slower than the KO mice (TABAC vs. KO: p<0.01). Interestingly, post hoc analysis revealed that not only TABAC mice, but also WT mice became slower than KO mice when completing a non-reinforced trial (WT vs. KO, p<0.05; TABAC vs. KO, p<0.01). Co-occurrence of a preference for the neutral arm and elevated latencies as displayed by TABAC mice is typically associated with aversive effects of drugs of abuse (Gavello-Baudy et al., 2008).

Lentivirus-mediated expression of b4 in the VTA increases nicotine ICSA

We next investigated the effect of re-expression of β4 nAChR subunits into the VTA on nicotine ICSA. Two groups of mice were injected with the control lentivirus (WT-GFP VTA , n=8 and KO-GFP VTA , n=8), whereas one group received the β4-expressing lentivirus (KO-β4 VTA , n=8) into the VTA.

Lentiviral re-expression of β4 in the VTA

Immediately after completion of the intra-VTA nicotine self-administration test, brains were used to determine VTA expression of lentivirus vector-introduced β4 using autoradiographic assessment of I 125 -epibatidine binding sites. Lentiviral transfer of β4 cDNA into the VTA of KO mice (n=7) successfully increased A85380-resistant I 125epibatidine binding sites in this brain region (Fig. 5A). Since β4* nAChRs are undetectable in the VTA of WT mice, a quantification of relative β4* nAChR restoration was not conducted. KO-β4 VTA mice, however, show intermediate β4* nAChR expression to that of WT and TABAC mice (Fig. 5A). Radioligand binding was also detected in the periphery of the neighboring IPN.

WT-GFP MHb vs. KO-b4 MHb high, p<0.05). Overall, these data demonstrated that the number of nicotine self-administrations is inversely correlated to the level of β4 reexpression within the MHb.

Lentiviral-mediated expression of b4 in the IPN decreases nicotine ICSA

Finally, we studied the effects of β4 re-expression within the IPN on nicotine ICSA.

Two groups of mice were injected with the control lentivirus (WT-GFP IPN , n=6 and KO-GFP IPN , n=6), and one group received the lentivirus for β4 re-expression (KO-β4 IPN , n=6), in the IPN.

Lentiviral re-expression of β4 in the IPN

Nine KO-β4 IPN mice completed the intra-VTA nicotine self-administration test, of which seven were assessed for putative β4* nAChR expression at the site of lentivirus delivery, the IPN. Quantification of A85380-resistant I125-epibatidine autoradiographic intensity shows that non-β2* heteromeric nAChR (arguably β4*) expression is rescued to 64.2% that of WT-GFP IPN controls (+-9.7% SEM, fig. 7A). Radio-ligand binding is almost abolished in the KO-GFP IPN controls (2.3% +-0.6% SEM).

Nicotine ICSA response

WT-GFP IPN and KO-GFP IPN exhibited similar performances to WT and KO mice presented above (non-vectorized, WT-GFP VTA and KO-GFP VTA , WT-GFP MHb , KO-GFP MHb ). WT-GFP IPN performances were closed to chance level. In contrast, as previously observed KO-GFP IPN showed a dose-dependent preference for the nicotinereinforced arm, particularly at the dose of 1200 ng/50 nl (Fig. 7B). Interestingly however, KO-β4 IPN animals demonstrated a significant decrease in the number of nicotine selfadministrations at 600 and 1200 ng/50 nl as compared to their GFP-infused controls. There was no significant difference in self-injection latency between the three groups of mice (Fig. 7C). The global two-way ANOVA revealed a session-dependent decreased of latencies at the dose of 600 ng/50 nl (Session effect: F(6,90)=6.89 p<0.001). At 1200 ng/50 nl, latencies to self-injected nicotine remained stable and short (ANOVA, Session effect: F(6,90)=0.36 ns). These results suggest that β4 re-expression within the IPN altered nicotine preference in a manner similar to the re-expression in the MHb.

DISCUSSION

Dose-dependent effects of β4 subunit deletion on nicotine reinforcement

We previously established the dose-effect curve for nicotine ICSA in WT mice.

Among the three doses tested (10, 100 and 200 ng/injection), the 100 ng (4mM) constitutes an optimal dose allowing the acquisition of steady intra-VTA nicotine selfadministration [START_REF] Besson | Genetic dissociation of two behaviors associated with nicotine addiction: beta-2 containing nicotinic receptors are involved in nicotine reinforcement but not in withdrawal syndrome[END_REF]David et al., 2006). At this dose, we have revealed the essential roles of different nAChR subunits in reinforcing properties of nicotine, such as the widely distributed and high-affinity β2* nAChRs [START_REF] Besson | Genetic dissociation of two behaviors associated with nicotine addiction: beta-2 containing nicotinic receptors are involved in nicotine reinforcement but not in withdrawal syndrome[END_REF]Maskos et al., 2005;Tolu et al., 2013), α4* nAChRs and, although less critically, α6* nAChRs (Exley et al., 2011). In contrast, we observed WT-like nicotine-seeking behavior (as measured by increased preference for the nicotine reinforced-arm in a Y-maze) in β4

null mutants (KO), demonstrating that this subunit is not necessary for nicotine ICSA at a highly appetitive dose of nicotine. Information about the β4 subunit's role in nicotine effects are still rare, yet pharmacological studies reported that α3β4* nAChRs antagonists potently blocked self-administration in rats [START_REF] Glick | 18-MC reduces methamphetamine and nicotine self-administration in rats[END_REF][START_REF] Toll | AT-1001: a high affinity and selective α3β4 nicotinic acetylcholine receptor antagonist blocks nicotine self-administration in rats[END_REF].

Here we show that behavioral consequences of the deletion of the β4 subunit gene are very distinct from a β2 KO, as previously evidenced in other nicotine addiction features such as withdrawal symptoms [START_REF] Besson | Genetic dissociation of two behaviors associated with nicotine addiction: beta-2 containing nicotinic receptors are involved in nicotine reinforcement but not in withdrawal syndrome[END_REF]Salas et al., 2009;Jackson et al., 2013). β4*-nAChRs are likely involved in an unknown mechanism which strictly regulates nicotine intake. Consistently, oral consumption of high doses of nicotine and free-choice drinking is drastically reduced in β4 over-expressing TABAC mice (Frahm et al., 2011). These data raises the question of whether the β4 subunit may be more specifically involved in the aversive effects of nicotine. In a second experiment, we analyzed nicotine ICSA using high doses of nicotine in WT, KO and TABAC mice that constitutively do not-or over-express the β4 subunit, respectively. WT mice exhibited a slight increase in the number of self-injections, as well as a decrease in latencies, suggesting a weakly positive motivational state. However, not only did WT animals fail to acquire a steady ICSA response at 600 ng (as compared to 100 ng), they also progressively diminished their nicotine preference below the chance level when the dose was increased. This behavior did not resemble the well-characterized profile observed in non-reinforced, aCSF control animals (alternation and rapidly increasing latency; David et al., 2006), but suggest instead the development of a titration behavior to avoid cumulative/aversive effects of nicotine. In sharp contrast, β4 KO displayed a strong selfadministration habit as measured by both a rapidly increasing choice of the nicotinereinforced arm, concomitant with a decrease in latency to trigger self-injection over time.

TABAC mice exhibited an early and quick avoidance of the nicotine-associated arm over sessions and an increase in latency to respond, especially when the dose was increased (1200 ng / inj). These observations are clearly consistent with the decreased nicotine oral consumption at high doses previously reported in TABAC mice (Frahm et al., 2011), but locate this inhibition of consumption to the VTA. Conversely, KO mice exhibited a strong positive response to high doses of nicotine, whereas TABAC showed instead an aversion-like response at doses, which were rewarding in the null mutant strain. We thus demonstrated that the level of β4 subunit expression inversely correlates with nicotine intake. Overall, our data suggest that the β4 subunit play a regulatory role limiting nicotine intake below a normally aversive dose.

Region-dependent effects of β4 re-expression in nicotine reinforcement

To elucidate the respective roles of β4*-AChR populations located in the VTA, MHb and IPN in nicotine's effects, we used lentivirus-mediated re-expression of β4 subunits in KO mice. It is important to note that control lentiviral vector injections did not modify nicotine-induced responses of WT and KO animals, independently of brain structures targeted (VTA, MHb and IPN).

The negative correlation between the level of β4* nAChRs and nicotine intake observed in the present study, as well as the neuro-specific expression of this receptor (expressed in a high proportion of GABA neurons as compared to other nAChRs; [START_REF] Azam | Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons[END_REF], suggest a potential role of β4 in mediating aversive properties of nicotine in the VTA. However, our results show instead an increase in nicotine ICSA when expression of the β4 subunit was restored in the VTA of KO-β4 VTA mice, as compared to WT-GFP VTA and KO-GFP VTA controls. This observation strongly suggests that limiting/aversive effects of nicotine involve β4*-nAChR-rich brain structures other than the VTA itself. Conversely, a recent study revealed that deletion of the α5 subunit resulted in increased nicotine intake, a behavior restored by the selective re-expression of α5 subunit in VTA DA cells. Therefore, α5 subunit in the VTA may be involved in mediating aversive properties of nicotine (Morel et al., 2014). Importantly, our results may be related to VTA over-expression of β4 subunits, since β4* nAChRs expression is moderate or undetectable in the VTA of WT mice, as revealed by autoradiographic assessment (present work) and by previous studies [START_REF] Zoli | Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems[END_REF][START_REF] Gahring | Neuronal and astrocyte expression of nicotinic receptor subunit beta4 in the adult mouse brain[END_REF]Baddick and Marcks 2011).

We then evaluated the role of the MHb and IPN β4* nAChRs in the modulation of the nicotine-induced activation of the reward system. Re-expression of the β4 subunit into either the MHb (KO-β4 MHb mice) or IPN (KO-β4 IPN mice) shifted the behavioral responses toward a WT-like behavior. Therefore, restricted expression of β4 subunit at the level of either the MHb or the IPN appears sufficient to reinstate reward-inhibiting effects on nicotine self-administration. These results are in agreement with a recent study reporting a decrease in nicotine consumption in mice over-expressing β4 subunit at the MHb level, using a two-bottle drinking test (Slimak et al., 2014). Pharmacological experiments in rats using the β4 subunit antagonist 18-MC showed an opposite effect on nicotine self-administration whether it was injected into the MHb (decrease in selfadministration) or the IPN (increase in self-administration) (Glick et al., 2011).

Accordingly, this antagonist reduced nicotine-evoked increases in accumbal dopamine when it was delivered in the MHb of rats (McCallum et al., 2012). These effects are not necessarily conflicting with results of the present study, as they likely depend on the dose of nicotine used. Interestingly, the α5 subunit in the MHb modulates inhibition of high doses of nicotine consumption systemically, with α5 null mutant mice selfadministering significantly more nicotine than their WT counterparts, without affecting the consumption at low doses. Re-expression of the α5 in the MHb of α5 KO mice reduced aberrant consumption of high doses of nicotine (Fowler et al., 2011). These studies, as well as our results, argue in favor of an involvement of the MHb-IPN axis function in aversive outcome of nicotine through β4 and α5 subunits. Yet interesting divergences between α5-and β4-dependent responses to high doses of nicotine can be noted. Whereas the α5 subunit seems to be involved in aversion-related processes regardless of its location, namely the MHb (Fowler et al., 2011) or the VTA (Morel et al., 2014); the present work suggest that the role of β4*-nAChRs appears to be structuredependent: i.e. reward-related in the VTA vs aversion-related in MHb. In this context, it may be recalled that contrasting and inverted levels of expression of α5 (strong in VTA and moderate in MHb) and β4 subunit (low in VTA and dense in MHb) are observed in WT animals (Hsu et al., 2013).

Activation of the MHb-IPN pathway limits VTA-elicited nicotine reinforcement

Our findings demonstrate that β4 subunit deletion leads to abnormal level of nicotine self-administration, specifically when high doses are presented. The activation of β4-containing nAChRs located along the habenulo-interpeduncular axis (MHb-IPN),

but not the VTA, act to limit nicotine reinforcement. Consistently, activity of the MHb-IPN axis is increased when the β4 subunit is over-expressed (TABAC mice; Frahm et al., 2011), and decreased when the β4 is absent (β4-/-mice) (Dao et al., 2014).

The dopaminergic input from VTA to the MHb could be involved (Phillipson and Pycock, 1982). Nicotine action in the VTA may also modulate β4*nAChR-expressing neuron activity of the MHb and IPN via an intermediary structure such as the septum.

Indeed, VTA dopaminergic neurons massively project to the medial and lateral part of the septum (Assaf and Miller, 1977;Kalivas et al., 1985;[START_REF] Oades | Ventral tegmental (A10) system: neurobiology. 1. Anatomy and connectivity[END_REF]Khakpai et al., 2013). The septal nuclei in turn send cholinergic inputs to the β4* nAChR-rich MHb and IPN [START_REF] Conrad | Efferents from medial basal forebrain and hypothalamus in the rat. II. An autoradiographic study of the anterior hypothalamus[END_REF]Contestabile and Fonnum, 1983;Sperlagh et al., 1998). There is evidence for nicotine-induced modification of neuronal currents [START_REF] Kodirov | Modulation of HCN channels in lateral septum by nicotine[END_REF] and increase of the c-fos expression [START_REF] Shram | Acute nicotine enhances c-fos mRNA expression differentially in reward-related substrates of adolescent and adult rat brain[END_REF] in the lateral septum. Reward (Gavello-Baudy et al., 2008) and anxiogenic [START_REF] File | Neurobiological mechanisms by which nicotine mediates different types of anxiety[END_REF] effects have been reported after septal injection of GABA A receptor agonist and nicotine, respectively. The MHb projects almost exclusively to the IPN, via the prominent glutamatergic-cholinergic pathway fasciculus retroflexus (Herkenham et al., 1979;[START_REF] Kawaja | Topographical and ultrastructural investigation of the habenulo-interpeduncular pathway in the rat: a wheat germ agglutinin-horseradish peroxidase anterograde study[END_REF][START_REF] Ren | Habenula "cholinergic" neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes[END_REF]. In turn the IPN sends GABAergic (and serotonergic) projections to the dorsal raphe nucleus (DRN), laterodorsal tegmentum (LDTg), pedunculopontine tegmentum (PPTg,) and septum (see Klemm et al., 2004;Qin and Luo, 2009, for review). These in turn provide critical serotonergic, dopaminergic and glutamatergic input to the VTA (see Faure et al., 2014;Lammel et al., 2014, for review).

The DRN is capable of reinforcing behavior primarily via non-serotonergic neurons, for which the main projection target is the VTA [START_REF] Mcdevitt | Serotonergic versus nonserotonergic dorsal raphe projection neurons: differential participation in reward circuitry[END_REF]. Excitatory input from

LDTg and PPTg appears to be critical for converting tonic firing of VTA DA cells to a burst firing pattern, known to be involved in reinforcing learning supported by dopamine release in the nucleus accumbens [START_REF] Lodge | The laterodorsal tegmentum is essential for burst firing of ventral tegmental area dopamine neurons[END_REF]. Future research should be aimed at connecting these different anatomical elements to provide a comprehensive overview of the neurocircuitry involved in the intriguing effects reported in the present study.

Conclusion

Our present work highlights the critical role β4*-nAChRs in regulating the reinforcing properties of nicotine through the MHb-IPN pathway. We first demonstrated that disruption of β4*-nAChR signaling extends the range of nicotine doses that promote nicotine reinforcement. Second, the level of β4* nAChR expression inversely correlates with nicotine self-administration and modulates the rewarding/aversive proprieties of nicotine by respectively maintaining/abolishing the positive response to high doses.

Finally, the activation of β4* nAChRs in the MHb-IPN axis mediates an inhibitory motivational signal limiting nicotine intake. This identifies β4*-nAChRs as a key element controlling the balance between negative and positive motivational states. Therefore, genetic mutations or polymorphisms that would compromise the efficiency of β4*-nAChR function may be considered as key genetic factors contributing to an altered risk of nicotine addiction. Arrows indicate the location of the fasiculus retroflexus (f.r.) and the interpeduncular nucleus (IPN).

This model was used for illustrative purposes. The ChAT-cre line was eventually crossed with β4 KO mice to obtain β4-/-; Chat-Cre Tg/+ mice.

f.r. The present thesis has depended upon cold ligand (5-iodo-A85380) displacement of I 125 -epibatidine binding from β2-specific nAChRs sites in order to reveal putative β4* nAChRs in situ. To clarify the nature of the 5-iodo-A85380-resistant I 125 -epibatidine binding, a second cold ligand could be used to further displace I 125 -epibatidine binding.

Using a β4-specific ligand, ⍺-conotoxin AuIB, in addition to 5-iodo-A85380, it would be possible to determine whether the 5-iodo-A85380-resistant I 125 -epibatidine binding is due to a single, β4* nAChR population. With this aim, the following protocol was used:

Protocol:

The protocol described in Article One was employed, with some modifications. Briefly, 20µm thick coronal brain sections were obtained from freshly frozen brains. On microscope slides, the sections were incubated for 30mins in 50mM TRIS (pH7. Poor levels of cDNA in the sample will give a Ct value approaching 40 cycles, whereas high expression levels will provide a Ct value nearing 20. ⍺3, ⍺5 and β4 nAChR subunit expression is greatest in HEK cells transfected with ⍺3, ⍺5 and β4 plasmids, respectively. Expression is also high in the HEK sample transfected with all three plasmids. This validates the primers, as well as the vectors used in transfection.

Data were obtained from a single experiment. A repeat is necessary for statistical interpretation.

These preliminary data show that the PGK-β4 plasmid used for transcription is capable of producing β4 mRNA, as shown by an increase in 1/Ct value in PGK-β4-transfected HEK cells (figure 2.4.c). This is a complementary validation to the in vitro HEK cell infection with the PGK-β4 lentiviral vector, expressing GFP (section 2.1.a). q-PCR analysis of MHb WT and β4 KO samples suggests that β4 is expressed at greater levels in the WT than the KO. Observations of this graph also suggest that ⍺3 and ⍺5 levels do not differ with genotype. This is in contrast to the reduced ⍺3 and ⍺5 protein levels in the MHb of β4 KO mice (Scholze et al. 2012).

Several crucial caveats must be brought to attention in this study. Firstly, beta-actin expression levels are low in HEK cells (low 1/Ct value, figure 2.4.c, left) because the primers target murin beta-actin, and not the human cDNA sequence. Secondly, HEK cell data and MHb data are from a single experiment, and must be repeated for statistical interpretation. Thirdly, these preliminary data were obtained without conducting serial dilutions of samples, which is necessary for the generation of a standard curve, and the calculation of efficiency of the reaction.

2.4.d Attempted antibody detection of β4 nAChR subunit protein in the mouse brain

As mentioned previously, there exist no commercially available antibodies that can specifically label nAChR subtypes in situ [START_REF] Moser | Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures[END_REF]). However, we obtained an antibody from Dr. Petra Scholze that had been previously validated in immunoprecipitation studies as specifically binding to β4 nAChR subunits (clone S04, unpublished). This antibody was tested in an immunohistochemistry protocol on MHbcontaining sections, a region known to express high levels of β4.

Protocol

Brains from PFA-perfused mice are sliced at 50µm along the coronal plane. Sections are incubated in aldehyde neutralising solution (30mins), followed by two hours in a blocking/permeabilising solution (10% NHS + 0.2% tritonX-100 in PBS) after PBS rinses.

The sections are rinsed in PBS then allowed to incubate in antibody solution (2% NHS + 0.04% tritonx-100) + 8µg/ml rabbit anti-β4 overnight at 4°C. The sections are once again rinsed, incubated in antibody solution + alkaline phosphatase anti-rabbit (1/100, Vactor Labs) for two hours. Once rinsed in PBS, the Vector Labs alkaline phosphatase In the positive control (β4 over-expressing mouse) we see specific staining of the ventral two thirds of the MHb, coherent with β4's established localisation (Shih et al. 2014). The negative control (β4 KO) showed no immunostaining above background. The difference between anti-β4 immunostaining in the WT and β4 KO was less convincing (figure 2.4.d.ii).

β4 KO β4 over-expr. The benefit of having a specific anti-β4 antibody is to be able to validate lentivirusmediated β4 protein expression. However, as demonstrated by autoradiography (see Article One), β4 re-expression reaches levels intermediate to the β4 KO and β4 WT mice. Whilst the S04 rabbit anti-β4 antibody seems adapted to detecting differences in β4 expression between the β4 over-expressing mouse and the KO, it is inefficient for sub-WT detection of β4. Further use was therefore not pursued.

β4 KO β4 WT β4 over-expr.

2.4.e Conditional expression of β4: lentivirus

The PGK-β4 lentivirus used throughout this thesis is useful in targeting β4 nAChR subunit expression to specific brain regions. However, studies have shown that β4 exhibits an expression bias within neuronal subpopulations of the VTA (Klink et al. 2001) and the IPN (Article One). Using lentiviruses, we can target transgene expression to neuronal subpopulations including dopaminergic and GABA-ergic neurons, as demonstrated by our laboratory [START_REF] Tolu | A versatile system for the neuronal subtype specific expression of lentiviral vectors[END_REF]Tolu et al. 2013;Morel et al. 2014).

The strategy involves a cre-lox system, and is explained in the illustration below (image 2.4.e.i).

Image 2.4.e.i. Illustrative description of a cre-lox system for cell-type specific expression of β4. The construct contains many of the features of the PGK-β4 lentivirus mentioned previously. Differences include the promoter (platelet-derived growth factor beta, PDGF-β) and the proceeding loxP-mCherry-STOP-loxP sequence prior to β4-IRES-eGFP. The example illustrated above is the effect of lentivirus delivery to the VTA of a GAD-cre mouse. This mouse line expresses cre recombinase in GABAergic neurons. In the absence of cre recombinase, translation ceases at the stop-rich sequence downstream of mCherry, preventing β4 expression (red dopaminergic neuron). In the presence of cre recombinase the mCherry-stop sequence is excised and ligation occurs at the loxP sites allowing β4 and GFP expression (green GABA neuron). Some GFP may be expressed in cre-negative neurons due to the IRES sequence upstream of GFP, which stabilises ribosome entry to this locus.

The lentivirus genes (e.g. 5' and 3'LTR) are still present in this construct and the viral coat is also equivalent to that of the PGK-β4 lentivirus used throughout this thesis. The As explained in section 1.9, the space for insertion of numerous transgenes is very limiting. This therefore meant that the GFP sequence, which allows visualisation of cell infection in the lentivirus, could not be introduced into this AAVifloxβ4 construct.

However, unlike many other nAChRs, β4* nAChRs can be detected specifically in a radioligand binding protocol, therefore visualising the location of AAV vector-inserted β4 subunits.

The AAVifloxβ4 construct (figure 2.4.f.ii) was generated by subcloning strategies. NheI and AscI restriction enzyme sites were introduced to the 5' and 3' end of a β4 cDNA, respectively. NheI and AscI double digestion allowed insertion of the β4 fragment into an pAAV5-EF1α-DIO-ChETA-eYFP (AAVifloxYFP) plasmid, replacing the YFP sequence. The AAV particles were synthesised at the Laboratoire d'Amplification de Vecteurs -CHU Nantes and concentrated to 3.6x10 10 infectious particles/ml. 262

The validation of this AAVifloxβ4 vector in vivo is inconclusive. Autoradiographs appear to show a greater than KO level of 5-iodo-A85380-resistant I 125 -epibatidine binding in the targeted IPN, however this is statistically insignificant. A disadvantage with the described loxP strategy of inversion is that, in the continued presence of cre recombinase, the inversion of the loxP-flanked segment can be reversed repeatedly. This may underly the low levels of 5-iodo-A85380-resistant I 125 -epibatidine binding observed in AAVifloxβ4injected brains. Double loxP pair strategies have been developed to prevent second inversion, which is the case in Article One's AAV for fluorophore expression (pAAV.hSynap.Flex.GCaMP5G(GCaMP3-T302L.R303P.D380Y).WPRE.SV40, Vector Core, Pennsylvania).

This alternative method of cre-dependent β4 expression was pursued in the aim of inducing a greater level of β4 nAChR expression than the lentivirus PDGFfloxβ4. Whilst the in vivo validation of the two viral vectors remains preliminary, it appears that the AAVifloxβ4 vector is much less successful (see previous subchapter).

2.4.g Assessment of MHb-IPN symmetry

The habenulo-peduncular pathway is highly conserved amongst vertabrates. In zebrafish, however, the habenula is not delineated into lateral and medial subnuclei, but instead into dorsally and ventrally. The fish's left dorsal habenula (equivalent to left mammalian MHb), projects to the entirity of the IPN, whereas the right dorsal habenula innervates only the ventral IPN [START_REF] Kuan | Neuropilin asymmetry mediates a left-right difference in habenular connectivity[END_REF]). Furthermore, it appears that in most species' brains, including the human, the size of the two habenula nuclei are not equal [START_REF] Hikosaka | The habenula: from stress evasion to value-based decision-making[END_REF]. With this in mind, an investigation of the left-right projection (a)symmetry of the mouse MHb was initiated.

Two tracing methods were attempted. The first was to use DiI on dissected postfixed brains; the other was to conduct in vivo injections of lentiviruses expression of mCherry or GFP. The DiI method proved unsuccessful (data not shown). The lentivirus method of fluorophore expression, however, was a useful tracing method.

Neuronal tracing protocol

The left and right MHb of the anaesthetised mouse were stereotaxically targeted for delivery of a PGK-GFP or PGK-mCherry lentivirus vector for expression of GFP and mCherry, respectively. Coordinates are as described in section 2.1.c. Four weeks postinjection, the mice were deeply anaesthetised and intracardially perfused with PBS and 4% PFA. PFA post-fixed brains were sliced to 60µm for immunofluorescence analysis.

After a three-hour blocking and permeabilisation step (10% NHS, 0.2% tritonX-100), half of the sections were incubated in polyclonal rabbit anti-GFP (1/500) and the other half in rabbit anti-RFP (1/500) primary antibodies (in 2% NHS + 0.04% tritonX-100). A greater number of correctly injected mice is necessary for a statistical analysis of the relevance of the projection patterns.

2.4.h Neuronal subpopulations of the IPN

The IPN contains somatostatin-expressing GABAergic interneurons (Article One supplementary information, Zhao-Shea et al. 2013). It was postulated that other GABAergic interneurons may be present in the IPN circuit. There are a plethora of different subtypes of GABAergic interneurons in the brain that are distinguished by electrophysiological profile, morphology and biomarker expression [START_REF] Molgaard | Immunofluorescent visualization of mouse interneuron subtypes[END_REF]. Taking advantage of the biomarker features (as for the localisation of somatostatin-expressing interneurons), antibodies against parvalbumin were used to determine the presence, and localisation, of parvalbumin-expressing GABAergic interneurons (figure 2.4.h). In this analysis, the hippocampus was used as a positive control, since it is known to contain somatostatin-and parvalbumin-expressing neuronal populations [START_REF] Nusser | Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells[END_REF][START_REF] Taniguchi | Genetic dissection of GABAergic neural circuits in mouse neocortex[END_REF][START_REF] Molgaard | Immunofluorescent visualization of mouse interneuron subtypes[END_REF].

Protocol

Deeply anaesthetised mice were intracardially perfused with PBS and 4% PFA. Brains were dissected, post-fixed in 4% PFA (4°C) and a vibratome used to collect 50µm coronal sections. Sections were incubated in blocking and permeabilisation reagent (10% NHS + 0.2% tritonX-100), and then incubated in 1/500 goat anti-somatostatin (Santa Cruz) plus 1/2000 rabbit anti-parvalbumin (Calbiochem), in 2% NHS + 0.04% tritonW-100, over three nights at 4°C. After rinsing in PBS, sections were incubated in secondary antibody solution at RT, rinsed, and mounted onto microscope slides.

In the presented immunostaining analysis there appears to be aberrant colocalisation of the two antibodies in the hippocampus. Furthermore, dense parvalbumin staining has been previously reported in the dentate girus [START_REF] Cabezas | Molecular and functional characterization of GAD67-expressing, newborn granule cells in mouse dentate gyrus[END_REF], which is not observed in the single immunofluorescence analysis conducted here (figure 2.4.h). This suggests a lack of antibody (primary or secondary) specificity in this structure, which has been highlighted in a recent study regarding commercially available antibodies against interneurons [START_REF] Molgaard | Immunofluorescent visualization of mouse interneuron subtypes[END_REF]. A single experiment was conducted on one mouse for this study, therefore sub-optimal experimental conditions cannot be ruled out. Further testing under different conditions is warranted. It would also be judicious to test different secondary antibodies in case of cross-reactivity. IPN parvalbumin interneurons were previously described in the cat (de [START_REF] De Leon | Distribution of parvalbumin immunoreactivity in the cat brain stem[END_REF].

We therefore show for the first time that the mouse IPN contains parvalbumin interneurons, but this must be confirmed with a greater number of samples. The presence of multiple inhibitory interneuron subtypes adds to the complexity of the IPN circuit. At present, the somatostatin interneurons have been shown to express β4 nAChR subunits (Article One), postulated to inhibit glutamate release from MHb terminals (Zhao-Shea et al. 2013). It remains to be determined whether the parvalbuminexpressing interneurons express nAChRs and their contribution to IPN-regulated phenotypes, such as nicotine withdrawal.

2.4.i Histological analysis of cannula guide-implanted brains

The intra-VTA nicotine self-administration test (section 1.8.a.ii) relies on correct implantation of the cannula guide so that nicotine can reach the VTA. Besson and colleagues [START_REF] Besson | Alpha7-nicotinic receptors modulate nicotine-induced reinforcement and extracellular dopamine outflow in the mesolimbic system in mice[END_REF]) demonstrated that a cannula inserted to the neighbouring substantia nigra does not support nicotine self-administration at the dose of 35 ng/infusion. It is therefore important to systematically verify the correct implantation of the cannula guide in mice subjected to the self-administration test (Article Two), and eliminate subjects from the behavioural data with incorrect cannula guide implantation.

Discussion

Overview of the results

Tobacco is consumed by an estimated 1 billion people worldwide. With 6 million deaths per year as a result of its detrimental health effects, tobacco consumption is the primary cause of preventable morbidity and mortality (WHO 2013). Human genetic studies have highlighted the polymorphic nature of the CHRNA5-CHRNA3-CHRNB4 genomic cluster, coding for subunits α5, α3 and β4, and its implication in smoking behaviours (section 1.7). Furthermore, variants in the CHRNB4 regulatory domain reduce the age of tobacco initiation, whilst gain-of-function variants in CHRNB4 reduce the risk of nicotine dependence (Schlaepfer et al. 2008;Saccone et al. 2009;[START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF].

The contribution of β2* nAChRs in nicotine addiction processes dominates the literature, with little investigation into the role of β4* nAChRs. With such strong genetic evidence pointing to the β4 nAChR subunit as having an important function in nicotine dependence risk, the β4* subpopulation of nAChRs therefore revealed itself as meriting further investigation.

So far, the β4* nAChRs had been principally investigated in the context of nicotine withdrawal processes [START_REF] Salas | Decreased signs of nicotine withdrawal in mice null for the beta4 nicotinic acetylcholine receptor subunit[END_REF]Jackson et al. 2013) and nicotine aversion (Frahm et al. 2011;Slimak et al. 2014), with only minor representation in nicotine self-administration studies (Glick et al. 2011;McCallum et al. 2012). The present thesis aimed to determine whether β4* nAChRs indeed contribute to nicotine consumption. Assessment of i.v. nicotine self-administration (0-60µg/kg/infusion, Article One), and intra-VTA nicotine self-administration (100-1200 ng/infusion, Article Two) demonstrated that β4* nAChRs are necessary for nicotine's positive and negative reinforcing properties. In order to further define the contribution of these receptors, lentivirus technology was used to stratify the cerebro-regional role of β4* nAChRs in consumption of appetitive (Article One) and aversive (Article Two) doses of nicotine.

Previous studies have highlighted the MHb, principal host for β4* nAChR expression, as modulating nicotine aversion (Frahm et al. 2011;Slimak et al. 2014). In corroboration with this, Article Two demonstrates that β4* nAChRs in the MHb is necessary for nicotine's aversive properties. This is also the case of the IPN, which we show to express β4 nAChR subunits. Further adding to our knowledge of the habenulo- from MHb terminals (Shih et al. 2014;Beiranvand et al. 2014;Grady et al. 2009;[START_REF] Girod | Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors[END_REF]. WT mice show self-administer nicotine. Nicotinic activation of the MHb and IPN is abrogated in the KO mouse (Dao et al. 2014;[START_REF] Mulle | Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system[END_REF], but nicotinic activation of non-β4* nAChRs may permit Glu release from MHb terminals [START_REF] Girod | Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors[END_REF]. KO mice do not self-administer nicotine. suggesting that the VTA innervates the MHb (Phillipson and Pycock 1982). Retrograde the rat [START_REF] Lanca | The pedunculopontine tegmental nucleus and the role of cholinergic neurons in nicotine self-administration in the rat: a correlative neuroanatomical and behavioral study[END_REF]. Furthermore, the positive reinforcing properties of intra-VTA 24nmol nicotine in the mouse are reversed by lesions of the PPTg, as assessed in a CPP paradigm [START_REF] Laviolette | Lesions of the Tegmental Pedunculopontine Nucleus Block the Rewarding Effects and Reveal the Aversive Effects of Nicotine in the Ventral Tegmental Area[END_REF]. The inversion of motivational valence, from positive to negative, by PPTg lesions in the mouse may also play a role in Article Two's observed aversion deficit in the β4 KO mouse. The impact of β4 genetic depletion on PPTg function warrants investigation. It is possible, by altered activity of the IPN in absence of β4* nAChRs, that baseline PPTg electrophysiology is altered. This may be relayed onto the VTA, with potential consequences on responses to high doses of nicotine. The current thesis presents two pieces of evidences that support, and conflict the hypothesis that the MHb and IPN indirectly modulate VTA electrophysiological tone. The first is that baseline electrophysiological activity of VTA dopaminergic neurons in the β4 KO mouse is not statistically different to that of WT (Article One). These recordings were conducted in vivo, with the entire brain network intact, therefore allowing us to observe the VTA's integration of input from upstream neurological systems. However, the fact that mice were anaesthetised during recordings may mask any baseline differences in VTA dopaminergic neuron firing patterns. This is highlighted in in vivo microdialysis recordings of WT and β4 KO awake mice (Article One). Using this technique, we observed baseline hyperdopaminergia in the nucleus accumbens. This may have a functional impact on the VTA's response to aversive doses of nicotine, but remains to be determined experimentally. A proposed indirect functional loop between the VTA, MHb and IPN is via the septal nuclei. The septal nuclei are subdivided into the posterior, medial and lateral nuclei, with further subdivisions delineated within each of these regions [START_REF] Swanson | The connections of the septal region in the rat[END_REF]. The septum exerts important functions in learning, memory, anxiety and fear (Khakpai et al. 2013). The septal nuclei (in particular the posterior septum) send massive cholinergic input to the β4* nAChR-rich MHb and IPN (Contestabile and Fonnum 1983). The dorsal, apical portion of the IPN (where β4* nAChR-expressing interneurons are located, Article One) relays neuronal communication back to the medial septum [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]. The septum is also involved in rewardrelated behaviours, as demonstrated by intra-septal self-administration of a GABA A receptor agonist (Gavello-Baudy et al. 2008). This self-administration is abolished by intra-VTA dopamine 1 receptor antagonism (Gavello-Baudy et al. 2008). Indeed, there is an intimate connection between the brain's reward network and the septal nuclei, with the VTA sending dopaminergic and non-dopaminergic projections to the lateral and medial septa [START_REF] Kalivas | A catecholaminergic projection from the ventral tegmental area to the diagonal band of Broca: modulation by neurotensin[END_REF]Assaf and Miller 1977). This VTAseptum communication is reciprocated (Geisler and Zahm 2005).

In the study of the role of nAChRs in the MHb and IPN, the septum becomes an attractive point of influence in hypotheses to be drawn. It may relay information from the VTA, via cholinergic transmission to the MHb and IPN. Furthermore, this relay from the VTA to the IPN can occur independently of the MHb. This connectivity may underlie the independent impact of MHb and IPN β4* nAChR on intra-VTA nicotine aversion, and is summarised in figure 3.2.iv. The proposed hypotheses are conjectural. It would be necessary to conduct further experiments in order to elucidate the neural trajectories responsible for VTA, MHb and perhaps not indicative of the responses invoked in smokers' brains, who undergo prolonged nicotine exposure, with a plasma half-life in humans of 2-3h [START_REF] Benowitz | Pharmacology of nicotine: addiction and therapeutics[END_REF].

In dependent smokers, drawing on a cigarette does not cause a spike in brain nicotine concentration as in the non-dependent smoker [START_REF] Rose | Kinetics of brain nicotine accumulation in dependent and nondependent smokers assessed with PET and cigarettes containing 11C-nicotine[END_REF], highlighting the need to consider smoker-relevant nicotine kinetics and their neurological effects.

The temporal stimulation kinetics has a profound impact on receptor state, expression and pharmacological response. As described in section 1.2.a, nAChR expression increases in the brain following prolonged nicotine exposure, and in vitro after several hours of nicotine exposure. However, I would like to introduce a second important concept in altered nAChR pharmacology that can occur during sub-chronic nicotine exposure, which is more pertinent to the self-administration paradigms conducted over 30min-1h sessions in Articles One and Two. The physiological event that shall be broached is that of receptor desensitisation.

Prolonged exposure (in the order of seconds to >1h) to acetylcholine or nicotine stabilises the desensitised conformation of nAChRs, reducing the probability to transition to the active state (see section 1.b). Desensitisation is a reversible process, with increased probability for recovery occurring in the absence of agonist. In the desensitised conformation, the ligand is bound but the channel does not conduct ions, resulting in a decreased response of events that depend on nAChR activation, for example striatal dopamine release and VTA GABA release [START_REF] Picciotto | It is not "either/or": activation and desensitization of nicotinic acetylcholine receptors both contribute to behaviors related to nicotine addiction and mood[END_REF].

nAChR desensitisation also has a functional impact on other systems, enhancing the effects of muscarinic acetylcholine receptors, as reviewed by Wang (Wang and Sun AChRs to be expressed on the cell surface. The fraction of AChRs remaining activatable after desensitization was assayed using 1 mM ACh". The grey region shows overlap between activating and desensitising conditions. The dotted curve denotes the response of smouldering activation (presence of both active and desensitised states within the receptor population). The dark grey portion of this illustrates the response that is relevant to smokers' brain nicotine concentrations according to Kuryatov [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]. However, 1.3µM nicotine has been observed in a heavy smoker, therefore a greater portion of this smouldering activity response curve shall be considered.

Nicotine concentrations are transient in the non-dependent smoker, whereas the dependent smoker shows no cigarette puff-elicited spikes in brain nicotine concentrations [START_REF] Rose | Kinetics of brain nicotine accumulation in dependent and nondependent smokers assessed with PET and cigarettes containing 11C-nicotine[END_REF]). In the non-dependent smoker, we may observe greater effects of nAChR activation, due to "washout" of nicotine between cigarettes and return to the activatable resting state. The dependent smoker, however, may be under the effects of smouldering activity of ⍺4β2⍺5 and ⍺3β2⍺5 nAChRs, and to a lesser shown to be similar in this study [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].

An additional consideration is the desensitisation dynamics of ⍺3β4 nAChRs in the absence of ⍺5 subunits. The ⍺3β4⍺5 nAChR desensitises faster than the ⍺3β4 receptor [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]. This underscores the heterogeneity of nAChR pharmacological profiles, and the importance of their investigation in vivo in specific brain regions. Notably, ⍺5 nAChR subunits are almost non-existent in the ⍺3β4rich MHb, whereas ⍺3β4⍺5 nAChRs are highly represented in the IPN (Scholze et al. 2012;Beiranvand et al. 2014). It is possible therefore that β4* nAChRs in the MHb desensitise more slowly than those in the IPN, and have a greater contribution to smoking-related behaviours.

3.3.b Physiological relevance of β4* nAChRs in the nicotine-consuming mouse

The question of nAChR desensitisation and activation is pertinent to Articles One and Two. The aforementioned study conducted by Kuryatov and colleagues, investigating the smouldering activation potential of ⍺5* nAChRs following 1.5h of nicotine preexposure, is relevant not only to the human smoker, but also, in part, to the mice submitted to nicotine i.v. self-administration (Article One). In the human cigarette smoker, nicotine reaches the brain in less than 20 seconds [START_REF] Benowitz | Pharmacology of nicotine: addiction and therapeutics[END_REF], and i.v.

nicotine delivery exactly mimics the smoker's time course of blood nicotine concentrations [START_REF] Matta | Guidelines on nicotine dose selection for in vivo research[END_REF]. In order to estimate the accumulation of nicotine in the brain, one must also consider the species difference in nicotine clearance. Nicotine's half-life in the human is in the order of two hours (cigarette delivery), whereas in the mouse it is at approximately 5 minutes (i.v. delivery). During the one hour sessions, WT mice self-administered approximately 7 (WT) to 15 (WT-GFP MHb and WT-GFP IPN ) infusions in a one hour session when 7.5-30.0µg/kg/inf. nicotine was available (Article One). In the latter case, it is conceivable that nicotine is consumed at a rate that stabilises brain nicotine levels, since the rate of i.v. nicotine bolus delivery approximates the nicotine half-life. The actual concentration of brain nicotine during self-administration was not assessed, precluding possible interpretation of the nAChR conformational state during nicotine self-administration. However, the nicotine levels achieved in the brain are sufficient in recruiting β4* nAChRs, as demonstrated by altered self-administration phenotypes in WT and β4 KO mice. The same self-administration paradigm has also been used to assess the role of β2* nAChRs, achieving comparable self-administration rates as the WT mice in this thesis (Orejarena et al. 2012). Again, with a phenotypic difference in self-administration amongst WT and β2 KO mice, one can deduce that the doses used in this paradigm are relevant to activation and/or desensitisation of neuronal nAChRs.

Article Two presents an intra-VTA self-administration test, conducted over ten-trial choice sessions, typically lasting 20 minutes. Nicotine is delivered immediately to the VTA upon arrival in the nicotine-paired arm of the apparatus, faster than the 10-20 seconds delivery in the smoker. The half-life of this 50nl nicotine bolus is not known, nor is its area of dissipation. We also have not determined whether nicotine is accumulated, or whether its concentration is transient during the experiment. The intra-VTA selfadministration test does not attempt to model the nicotine pharmacokinetics observed in the human. Instead, it serves to stratify the contribution of an individual brain region in nicotine reinforcement. However, a quantitative understanding of the conformational states of the nAChRs at play in this paradigm is crucial.

There is a conspicuous difference in intra-VTA self-administration of 600ng and 1200ng of nicotine in WT and β4 KO mice, yet these two concentrations (24mM and 48mM, respectively) are both above the saturating concentration of nAChR activation. The application of a 50nl nicotine dose is presumed to diffuse in the brain tissue, resulting in a concentration gradient around the site of injection as well as over time. It is possible that the non-salient effect of 600ng nicotine and the aversive effect of 1200ng of nicotine are due to the difference in concentration gradients across the VTA (and possibly the neighbouring IPN). Electrophysiological recordings of VTA responses in vitro at these concentrations are warranted.

In order to determine whether the effect on self-administration is due to activation or desensitisation of nAChRs, we could experimentally inhibit desensitisation. This is possible, in the case of ⍺7 nAChRs, by pre-incubation with a type II positive allosteric modulator (PAM), which delays desensitisation of the receptor. dFBr is a PAM of ⍺4β2 and ⍺2β2 nAChRs, proving to enhance responses to ACh and alter nAChR desensitisation [START_REF] Kim | Synthesis of desformylflustrabromine and its evaluation as an alpha4beta2 and alpha7 nACh receptor modulator[END_REF]). These may prove useful tools in dissecting the contribution of nAChR activation and desensitisation on nicotine reward.

withdrawal amongst β4 KOs previously published (Salas et al. 2007). However, β4 T91I and T375I are shown to increase nicotine-elicited current amplitude [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF], as does increased β4 expression (Frahm et al. 2011). This highlights the need to assess the role of β4 T91I and T375I variants in vivo, since the evidence from altered expression of WT β4 in the mouse is inappropriate in modelling the effect of these β4

variants.

The β4 nAChR subunit also has an implication in the 37%-43% of Europeans and those of Middle Eastern origin who carry the ⍺5 rs16969968 SNP (D398N), risk factor for tobacco dependence [START_REF] Bierut | Variants in nicotinic receptors and risk for nicotine dependence[END_REF]). An in vivo study in the mouse shows that overexpression of β4 nAChR subunits results in aversion to nicotine in a two-bottle choice test (Frahm et al. 2011). This effect is reduced by expression of mouse ⍺5 397N locally in the MHb (Frahm et al. 2011). Over-expression of β4 nAChR subunits increases nicotine current amplitude, and is placated by the introduction of ⍺5 397N (Frahm et al. 2011). It is possible that a constellation of variants in the CHRNA5-CHRNA3-CHRNB4

gene cluster is consequential on smoking behaviours and addiction outcomes.

This leads us to discuss whether β4* nAChRs are appropriate targets for smoking cessation therapies.

Are β4* nAChRs an appropriate therapeutic target for smoking cessation?

The present thesis has described the differential, and sometimes overlapping, roles of various nAChR subtypes in nicotine addiction process. This complexity is a barrier for the development of a panacean treatment for nicotine cessation. Instead, it may be possible to use the complexity that nature has afforded and strategically target different nAChR subtypes in a symptomatic approach, for example appeasing affective withdrawal symptoms by targeting β2* nAChRs (see section 1.6.a). Article One demonstrates that genetic depletion of β4 nAChR subunits results in reduced nicotine self-administration at smoking-relevant concentrations of nicotine. Are β4* nAChRs therefore worth pursuing clinically as smoking cessation aids? Some convincing, and some conflicting, pre-clinical evidence for this approach have been presented throughout this thesis, and shall now be discussed.

β4 KO mice show reduced nicotine self-administration (Article One) and reduced somatic signs of withdrawal [START_REF] Salas | Decreased signs of nicotine withdrawal in mice null for the beta4 nicotinic acetylcholine receptor subunit[END_REF]. However, intra-IPN infusion of a β4* nAChR antagonist precipitates somatic withdrawal symptoms in nicotine-exposed WT mice (Zhao-Shea et al. 2013). Further pre-clinical assessment would be required to find a dosing regimen that would reduce somatic withdrawal signs, and not increase them. A second consideration is the fact that smokers with a gain-offunction variant in the CHRNB4 show a reduced risk for nicotine dependence (section 1.7), therefore proposing β4* nAChR agonism as a potential approach for a nicotine cessation therapy.

As introduced in the preamble, two pharmacological agents are approved as aids for nicotine cessation: varenicline and bupropion. Varenicline was developed as an ⍺4β2 partial agonist for the treatment of tobacco addiction, whereas the antidepressant bupropion was found serendipitously to aid in tobacco cessation, and later identified as a nAChR antagonist. Two contributing factors to varenicline's and buprion's mild success in increasing sustained remission rates is the fact that they reduce cravings and relapse (see preamble). It is unknown whether β4* nAChRs contribute to these phenotypes. This could be investigated by assessment of β4* receptor agonism or antagonism in the rat during cue-induced reinstatement of nicotine seeking.

Preclinical tests using the naturally occurring alkaloid ibogaine and its synthetic congener 18-MC, with known ⍺3β4 nAChR antagonism, were shown to reduce selfadministration of drugs of abuse in the rodent, including nicotine [START_REF] Koenig | The anti-addiction drug ibogaine and the heart: a delicate relation[END_REF]. Preliminary clinical investigations also indicate that ibogaine has "anti-addictive" properties in the human, alleviating drug craving and impeding relapse [START_REF] Koenig | The anti-addiction drug ibogaine and the heart: a delicate relation[END_REF]. However, the severe side effects of ibogaine have precluded further clinical pursuit of this substance as a suitable nicotine cessation therapy. Notably, ibogaine can induce cardiac arrhythmia via an unknown action [START_REF] Koenig | The anti-addiction drug ibogaine and the heart: a delicate relation[END_REF]. A second reason for the lack of clinical pursuit of β4* nAChRs for nicotine cessation therapies may come from the fact that high levels of β4* nAChRs are expressed in the autonomic ganglia (Xu et al. 1999). There may therefore be a cost (unwanted autonomic side effects) versus benefit (promoting nicotine cessation) imbalance that may be too problematic for immediate clinical pursuit.

Future perspectives

The present thesis has investigated the role of β4* nAChRs in important nicotine addiction processes. However, some information is lacking, as highlighted during this discussion.

A significant question is raised in Article One: is the lack of nicotine i.v selfadministration in β4 KO mice due to reduced nicotine reinforcement, or due to increased aversion? The nature of the i.v. self-administration paradigm does not allow us to resolve this question, since ambivalence or aversion to nicotine are both observed as reduced self-administration. In order to investigate this, a nicotine-conditioned place preference test in WT and β4 KO mice was envisaged. Initial attempts in WT mice failed,

showing no preference for the nicotine-paired (0.5mg/kg s.c.) compartment. A second problem encountered was a statistical baseline preference for one of the compartments.

This technique must therefore be optimised before re-attempting the experiment.

In Article Two, we demonstrate that β4* nAChRs at extra-VTA sites are important in regulating the VTA-mediated nicotine aversion of high doses of nicotine. How β4* nAChRs in the habenulo-interpeduncular pathway, in the absence of nicotinic stimulation, perform this regulation is an open debate. In section 3.2, the septum was raised as a potential intermediary in this communication. Its role could be confirmed by lesion the septal nuclei or optogenetic silencing, and the effect on the avoidance of intra-VTA nicotine self-administration investigated. A second, fundamental, issue in this study concerns the pharmacological effect of such high doses of nicotine (24mM and 48mM) on the VTA. This could be resolved initially by in vitro assessment of such concentrations in order to quantitatively determine the nicotine-elicited changes in current. Another issue raised with these concentrations of nicotine is the effects of diffusion of the 50nl bolus across the VTA, and potentially into neighbouring brain

regions. An optogenetic strategy would be appropriate to assess this, allowing a specific activation or inhibition of neurons in the VTA or the IPN during an intra-VTA optogenetic self-stimulation task.

Articles One and Two exploit the PGK-β4 lentivirus for ubiquitous cell infection at the site of injection. In order to further stratify the cerebro-regional role of β4* nAChRs, one could re-express β4 nAChR subunits in a neuron subpopulation-specific manner, using the cre-dependent lentivirus described in section 2.4.e. Four Cre mouse lines were developed with this aim -β4 KO ChAT-Cre; β4 KO DAT-Cre; β4 KO GAD-Cre; β4 +/-β4-Cre -for cre-recombinase expression in cholinergic, dopaminergic, GABAergic and β4-expressing neurons, respectively. Unfortunately β4-Cre+/-mice on a β4 KO background were never obtained, perhaps due to high, lethal, stimulation of the cre's β4

promoter in the absence of β4 nAChR subunit expression. This would have been especially useful, in the absence of a β4 KO x somatostatin-Cre line, for targeting β4 nAChR subunit re-expression to endogenous sites in the IPN. The role of β4* nAChRs in VTA dopaminergic and GABAergic neurons in intra-VTA or i.v. nicotine self-

Concluding statement

With a limited expression profile in the central nervous system and a pharmacology that overlaps little with the nicotine concentrations found in the smoker, β4* nAChRs were This thesis lies within this genetics-driven approach. The body of work presented demonstrates that β4* nAChRs in the MHb and IPN modulate the positive and negative reinforcing properties of nicotine. This enlarges the scope for future research into this subtype of nAChR, whose implication in nicotine addiction processes was previously limited to nicotine withdrawal and nicotine withdrawal-conditioned aversion. I hope that this work will contribute to the further stratification of nicotine addiction aetiology, and have a therapeutic impact in the future.

1 .

 1 Introduction ................................................................................................................. 21 1.a Nicotine, acetylcholine, and the nicotinic acetylcholine receptor .......................... 21 1.b Structure and function of the nicotinic acetylcholine receptor: a cys-loop receptor .................................................................................................................................... 25 1.2. Chronic nicotine exposure .................................................................................... 35 1.2.a Chronic nicotine exposure: upregulation ........................................................ 35 1.2.b Chronic nicotine exposure: sensitisation and tolerance ................................. 38 1.3 Nicotinic acetylcholine receptor localisation in the rodent brain ............................ 44 1.4 Neural circuits of nicotine reward .......................................................................... 49 1.4.1 The ventral tegmental area ............................................................................. 50 1.4.2 The medial habenula ...................................................................................... 58 1.4.3 The interpeduncular nucleus .......................................................................... 65 1.5. Neural circuits of nicotine aversion and withdrawal ............................................. 70 1.5.1 The ventral tegmental area and aversion ....................................................... 70 1.5.2.a The medial habenula and aversion ............................................................. 72 1.5.2.b The medial habenula and withdrawal .......................................................... 72 1.5.3 The interpeduncular nucleus and withdrawal ................................................. 74 1.5.4 Commentary ................................................................................................... 75 1.6. Knock-out mice: tools for studying the role of nAChRs in vivo ............................. 76 1.6.a The β2 KO mouse .......................................................................................... 76 1.6.b The β3 KO mouse .......................................................................................... 77 1.6.c The β4 KO mouse ........................................................................................... 78 1.6.d The ⍺2 KO mouse .......................................................................................... 81 1.6.e The ⍺3 KO mouse .......................................................................................... 81 1.6.f The ⍺4 KO mouse ........................................................................................... 82 1.6.g The ⍺5 KO mouse .......................................................................................... 83 1.6.h The ⍺6 KO mouse .......................................................................................... 85 1.6.i The ⍺7 KO mouse ........................................................................................... 86 1.7. Human genetic studies of nicotinic acetylcholine receptor genes and smoking .. 89 1.8 Behavioural models of nicotine addiction in the mouse ........................................ 92 1.8.a.i Intra venous nicotine self-administration ...................................................... 92 1.8.a.ii Intra-cranial nicotine self-administration ...................................................... 96 1.8.b Nicotine-conditioned Place Preference .......................................................... 98 1.8.c Two-bottle choice .......................................................................................... 101 1.8.d Commentary: The mouse as an appropriate model of addiction .................. 103 1.9 Viral vectors as a research tool ........................................................................... 105 1.9.a The HIV-1 lentivirus and its genome ............................................................ 106Publications and communicationsArticle One: β4* nAChRs in the habenulo-peduncular pathway regulate intra venous nicotine self-administration, submission pending Lauriane Harrington, Andrea Harrera-Solis, Africa Flores, Carol Morel, Stefania Tolu, Philippe Faure, Rafael Maldonado, Uwe Maskos, Patricia Robledo Article Two: β4-containing nicotinic receptors in the habenulo-peduncular pathway mediate a stop signal on nicotine reinforcement, submission pending Marianna Husson, Lauriane Harrington, Inés Ibañez-Tallon, Uwe Maskos, Vincent David Mediterranean Neuroscience Society Conference, Sardinia Italy. June 2015. The role of nicotinic acetylcholine receptor subunits in nicotine addiction. Oral presentation. Departmental seminar, Pasteur Institute, April 2015. Work in Progress: β4 subunitcontaining nicotinic acetylcholine receptors in the medial habenula regulate nicotine reinforcement and aversion. Oral presentation. Annual Mini-symposium of the Graduate School of Systemic Neurosciences (GSN), Munich Germany. The role of the β4 nicotinic acetylcholine receptor subunit in nicotine addiction. Poster presentation. Federation of Neurosciences Societies (FENS) Conference 2014, Milan Italy. The role of the β4 nicotinic acetylcholine receptor subunit in nicotine addiction. Poster presentation. Doctoral school retreat, École Doctorale Cerveau Cognition Comportement, 2014. Roscoff, France. The role of the β4 nAChR subunit in nicotine addiction. Oral presentation. Paris School of Neuroscience, student seminar 2013. Paris, France. The role of the β4 nAChR subunit in nicotine addiction. Oral presentation. Students at Pasteur retreat, June 2013. Valjoly, France. The role of the β4 nAChR subunits in nicotine addiction: an in vivo model. Oral presentation. Paris School of Neuroscience Days 2011-2013, France. The role of the β4 nicotinic acetylcholine receptor subunit in nicotine addiction. Poster presentations Common abbreviations: α -alpha-type subunit of nAChR β -beta-type subunit of nAChR ACh -acetylcholine EC50 -half maximal effective concentration ECD -extracellular domain nAChR -nicotinic acetylcholine receptor x* nAChR -x subunit-containing nAChR. Other subunits may be present GABA -gamma-aminobutyric acid GWAS -genome-wide association study

Figure 1 .

 1 Figure 1. Obtained from the WHO tobacco report 2013 (WHO 2013), showing the countries around the world that have implemented strict legislation to obtain completely smoke-free public environments.

Figure 1 .

 1 Figure 1.a.i. Left. Structural formula of nicotine. Right. Structural formula of acetylcholine

Figure 1 .

 1 Figure 1.b.i. Diagrammatic representations of the stoichiometry and structure of nAChRs. From (Changeux 2010). A) View of the pentameric nAChR assembly in the plasma membrane. B) Illustration of a single nAChR subunit, showing its four transmembrane (M1-M4) domains. C) Top-down view of the homomeric (⍺7) and heteromeric (⍺4β2) nAChR and its potential acetylcholine binding sites. D) Model of the ⍺7 nAChR, with nicotine molecules in grey.

Figure 1 .

 1 Figure 1.b.ii. Time-course of elucidation of 3D structure of various ligand-gated ion channels. The species from which the structures are derived are noted in parentheses. GluCl -glutamate-gated chloride ion channel; AChBP -acetylcholine binding protein; ELIC -Erwinia ligand-gated ion channel; GLIC -Gloeobacter ligand-gated ion channel. Provided by Pierre-Jean Corringer.

Figure 1 .

 1 Figure 1.b.iii A structural model of the ACh binding site in nAChR. The loops A-G contributing to the binding pocket and transmission of binding to the rest of the receptor structure, are illustrated. Loops A-C are provided by the principal subunit, and loops D-G are provided by the complementary subunit. Figure provided by P-J Corringer (Corringer et al. 2012).

Figure 1 .

 1 Figure 1.b.iv. Illustrative summary of pLGIC allosteric states. Top-down view. The conformational states of the receptor are in equilibrium between resting (R), active (A), R-A intermediate (I) and desensitised (D) states. This equilibria are altered upon binding of an agonist (blue dot). Blue dot -agonist. Green circlessubunits. Grey dot -closed channel. Absence of central grey dot denotes open channel. Arrowspathways of transition in equilibrium. Thickness of line denotes directional preference in the presence of agonist (unbound in R and bound in I, A, D).

Figure 1 . 3 .

 13 Figure 1.3. Overview of nAChR subtype expression in the rodent brain, illustrated in the sagittal plane. ⍺4β2* and ⍺7 nAChRs, show widespread brain localisation. Illustration as by Taly and colleagues (Taly et al. 2009).

Figure 1 .

 1 Figure 1.4.1.a.i Diagrammatic representation of the anatomical location of the meso-limbic and mesocortical neural networks. Saggital plane of the human brain. Arrows denote direction of direct neural communication between structures. VTA -ventral tegmental area; NuAcc -nucleus accumbens; PFCprefrontal cortex.

Figure 1 .

 1 Figure 1.4.1.a.ii From Faure et al. (2014). VTA dopamine neuron firing profiles as revealed by in vivo electrophysiological recordings in the anaesthetised mouse(Faure et al. 2014).

  cellular location, refer to figure1.4.1.b.

Figure 1 .

 1 Figure 1.4.1.b.. Diagrammatic representation of the nicotinic acetylcholine receptors modulating the ventral tegmental area and nucleus accumbens. Adapted from a review by J-P Changeux (Changeux 2010). VTA -ventral tegmental area; NuAcc -nucleus accumbens; LDTg -laterodorsal tegmentum; PPTg -pedunculopontine tegmentum; PFC -prefrontal cortex; Glu -glutamatergic neuron; GABA -GABAergic neuron; DA -dopaminergic neuron; ACh -cholinergic neuron. Location of nAChRs denote their function at pre-or post-synaptic sites.

Figure 1 .

 1 Figure 1.4.2.a. MHb afferents and efferents in the mouse brain. The MHb receives input from the septum, and some input from the VTA, locus coeruleus (LC), and dorsal raphe nucleus (DR). The MHb projects its neurons tot he IPN and the LHb. The thickness of arrows indicates the extent of projections between brain regions. The representations of the orange and purple arrows shall be described in figure 1.4.3.a. Adapted from a recent review (Antolin-Fontes et al. 2014).

Figure 1 .

 1 Figure 1.4.2.b describes the proportion of different nAChR types found in the rat

Figure 1 .

 1 Figure 1.4.2.b. Proportion of receptor composition of the rat habenula according to Scholze and colleagues(Scholze et al. 2012). This was determined by immunoprecipitation studies.

Figure 1 .

 1 Figure 1.4.3.a. Diagrammatic representation of the afferent and efferent network of the rodent IPN. Adapted from a recent review (Antolin-Fontes et al. 2014), and a continuation of the previous figure 1.4.2.a. The IPN's principal input is from the MHb (red) and posterior nuclei (purple); dorsal raphe nucleus (DR), median raphe nucleus (MnR), LDTg, dorsal tegmentum (DTg), locus coeruleus (LC). This direct communication is reciprocated (orange). The IPN also receives reciprocal innervation from the septum, periaqueductal gray (PAG) and hypothalamus (Hyp). HC -hippocampys; LV, 3V, 4V -ventricles; ECentorhinal cortex; NI -nucleus incertus. Thickness of arrows indicates the extent of projections between brain regions.

Figure 1 .

 1 Figure 1.6. Nicotinic acetylcholine receptor subunits are involved in a multitude of processes contributing to addiction processes, from reward and positive reinforcement that drive consumption and increase cue valence important in relapse, to aversion, and withdrawal symptoms that impede successful abstinence.

Figure 1 .

 1 Figure 1.8.a.i. An example apparatus for intra venous self-administration. The box contains two magazines that are randomly assigned as active (nose-poking here for drug delivery) or inactive (nosepoking here does not deliver drug). The activity of the mouse at these magazines is detected and drug delivered in an automatic fashion. A light cue above the nose hole illuminates upon infusion of the drug.

Figure 1 .

 1 Figure 1.8.a.ii. Intra-cranial drug self-administration. Left. Illustratative representation of the apparatus for intra-cranial drug self-administration, adapted from Tolu (Tolu et al. 2013). Mice begin the trial in the start arm, and are allowed to move to the left or right arm. One of which is paired with nicotine. Upon reaching the chosen arm, the mouse breaks the photo beam and nicotine is delivered (or not). The mouse is enclosed in the chosen arm for 25 seconds before being guided back to the start arm to begin a new trial.

Figure 1 .

 1 Figure 1.8.b. Illustrative representation of the CPP procedure, consisting of a pre-conditioning session, three days of conditioning sessions, and a final test session. a) The mouse (or rat) is injected with nicotine and enclosed in a designated compartment for 15 minutes. b) Several hours later the mouse is injected with saline and placed in the saline-paired compartment where it is also confined for 15 minutes. The two compartments are often distinguishable by shape (not shown) or floor texture. Control groups receive saline in both sessions (a and b). c) After typically three days of conditioning, the partitions of the apparatus are open and the animal is free to explore the space. The amount of time spent in each compartment is recorded. Baseline preferences are also assessed this way, prior to conditioning (preconditioning test).

  are housed individually for the duration of the session or length of entire experiment (up to several weeks). Two bottles are present in the cage and the rodent can choose to consume nicotine or water. Nicotine-laced water has a bitter taste, so to control for this one must either add a bitter element to the control solution, or sweeten the nicotine-containing and control solutions (typically to 2% saccharine). The volume consumed from each bottle is measured daily or weekly. Typical nicotine doses tested in this paradigm range from 1-40mg/l[START_REF] Collins | Oral Nicotine Self-Administration in Rodents[END_REF], with the mid-point evoking preference amongst mice (Vieyra-Reyes,[START_REF] Picciotto | It is not "either/or": activation and desensitization of nicotinic acetylcholine receptors both contribute to behaviors related to nicotine addiction and mood[END_REF] Frahm et al. 2011) and rats[START_REF] Halder | The multiple bottle effect is overridden in male and female rats by simultaneous presentation of two oral nicotine solutions[END_REF], although some strain differences have been observed[START_REF] Vieyra-Reyes | Voluntary oral nicotine intake in mice down-regulates GluR2 but does not modulate depression-like behaviors[END_REF][START_REF] Marks | Genetic Matters: Thirty years of progress using mouse models in nicotinic research[END_REF]).

Figure 1 .

 1 Figure 1.9.a.i. Illustrative representation of an HIV-1 lentivirus (top) and the HIV-1 genome (bottom) Top) ssRNA, along with enzymes protease, reverse transcriptase and integrase are contained in a nucleocapside. This is surrounded by matrix proteins, and in turn by a glycoprotein coat (capsid). Proteins encoded by the virsus' open reading frames gag, pol and env are noted. Bottom) Diagrammatic representation of viral ssRNA according to Trono (Trono et al. 2002). LTR -long terminal repeat; TAR -Tat activation region; PBS -primer binding site; SD -splice donor site; Ψ -packaging and dimerisation signal; GAG -open reading frame; PRO -open reading frame: POL -open reading frame; PPTpolypurine tracts; VIF -accessory gene; R -Vpr gene; TAT -Tat gene, transcription regulator; REV -Rev gene, posttranscription regulator; U -Vpu gene; ENV -open reading frame; RRE -Rev response element; NEF -Nef gene; polyA -polyadenylation signal.

  for transcription of viral genes, enhanced by viral Tat protein, which binds to cis-acting sequences in the long terminal repeat sequence (LTR). As with cellular mRNA, viral RNA can undergo alternative splicing for the eventual synthesis of viral proteins. This translation occurs in the cytoplasm and in the rough endoplasmic reticulum. Viral RNA destined for new viral genomes remains unspliced and is transported to the plasma membrane via the cytoplasm. In order to exit the nucleus, the RNA recruits viral Rev protein, powered by Ran-GTP hydrolysis, for transport to the cytoplasm[START_REF] Trono | Lentiviral Vectors[END_REF].Gag and Pol structural proteins anchor to the plasma membrane where they are joined by viral ssRNA, glycosylated proteins and accessory proteins before budding off the membrane to form progenitor viral particles. The budding process is concomitant with maturation of proteins present. Proteases are activated to cleave Gag and Gag-Pol precursor proteins to form mature matrix protein, capsid protein, nucleocapsid protein, p6 protein, reverse transcriptase and integrase.1.9.c Genetically modified HIV-1 as a research toolHIV-1 was genetically modified for research applications, with the principal aim of preventing pathogenic viral replication post initial infection. Early attempts to modify HIV-1 for use as a genetic tool produced viruses with infection characteristics similar to that of wild-type HIV-1, i.e. successful infection almost limited to immune cells. Today, thanks to subsequent genetic modifications, HIV-1 based lentiviruses can infect a wide variety of cell types and efficiently transduce cells in various dynamic or arrested cell cycle stages.

Figure 1 .

 1 Figure 1.9.c.i. Diagrammatic representation of lentivirus production. Three vectors are co-transfected into HEK-293T cells in vitro. The packaging vector provides the genetic code for the production of viral proteins, essential for packaging of the transfer vector (in ssRNA form) in to viral capsids (encoded by the envelope vector). Resultant virions are are purified and stored at -80°C.

  7 kilo base pairs (kbp), typified by 145-bp inverted terminal repeats (ITRs) flanking two open reading frames. The ITRs are important in DNA replication and transcription, AAV genome packaging, and containRep binding elements (RBEs) and a terminal resolution site (TRS). RBEs and TRS allow processing of dsDNA intermediates for transcription in the infected cell. dsDNA is also the basis for production of single-stranded DNA for packaging into new viruses. The ITRs are the origins of replication and serve to self-prime DNA synthesis in the absence of primase, by formation of hairpins[START_REF] Daya | Gene therapy using adeno-associated virus vectors[END_REF]. The viral regulatory proteins, Rep, positively and negatively modulate transcription[START_REF] Pereira | The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection[END_REF], and are required for genome replication as well as for packaging of ssDNA into virions. The cap open reading frame is a template for capsid structural proteins VP1-3[START_REF] Daya | Gene therapy using adeno-associated virus vectors[END_REF]. Figure1.9.d illustrates the AAV's genome.

Figure 1 .

 1 Figure 1.9.d. Illustration of the AAV single-stranded DNA genome. Inverse terminal repeat sequences (ITR, sense left, antisense right) flank rep and cap open reading frames, who themselves contain promoters (p5, p19, p40). A poly adenylation (polyA) signal lies downstream of the cap open reading frame.

Figure 1 .

 1 Figure1.9.f. A recombinant AAV (rAAV) vector contains viral ITRs plus an exogenous promoter, a gene of interest (GOI), and typially a polyadenylation signal (polyA). A WPRE sequence for increased stability of the mRNA product can be included in the vector. The AAV-RC vector contains AAV replication (rep) and capsid (cap) genes to provide the trans-acting factors for replication and packaging of the AAV in the cell line. The helper vector contains adenovirus genes (E2A and E4) to increase production yield.

2. 1 .

 1 a Validation of the PGK-β4 lentivirus in vitro A lentivirus construct was synthesised by restriction enzyme digestion and ligation, according to manufacturer's instructions (figure 2.1.a.i). β4 mouse cDNA was digested and ligated into a plasmid containing a kanamycin-resistant gene, an internal ribosome entry sequence (IRES) and an enhanced green fluorescent protein (eGFP) sequence.Subsequent BsrGI, XhoI and SalI partial digestions liberated a β4-IRES-eGFP fragment that was ligated into a lentivirus construct. The latter was an available α7-containing vector. The plasmid's α7-IRES-eGFP sequence was replaced by that of β4-IRES-eGFP.

Figure 2 .

 2 Figure 2.1.a.i Lentiviral vector, herein named PGK-β4. Sub-cloning by SalI/XhoI/BsrGI restriction enzyme digestion. ∆U3 -deletion in U3 of 3'LTR; LTR -long terminal repeat; PGK -phosphoglycerate kinase promotor; β4 -β4 WT mouse cDNA sequence; IRES -internal ribosome entry sequence; eGFPenhanced green fluorescent protein; WPRE -woodchuck hepatitis B virus posttranscriptional response element.

Figure 2 .

 2 Figure 2.1.a.ii. In vitro infection of a HEK cell line with the PGK-β4 lentivirus results in GFP expression.Two days was sufficient for GFP (green) to accumulate these cells (DAPI). Cells were grown on sterile glass cover slips and infected with 460ng lentivirus in the well of a 12-well culture plate. Two days later, the cells were rinsed (PBS) and momentarily fixed in 4% PFA. The cell-adhered glass slip was removed and mounted onto a microscope slide using ProlongGold mounting medium (with DAPI) and images acquired with a Zeiss fluorescent microscope.

  123

2. 1

 1 .b.i demonstrates the successful infection, and extent of diffusion of the lentivirus in the VTA, IPN and MHb.

Figure 2 .

 2 Figure 2.1.b.i. Successful PGK-β4 lentivirus infection in vivo. Immunofluorescence analysis of lentivirus GFP expression in coronal sections of the mouse brain. Left, The VTA was injected with 60ng/µl PGK-β4. 2µl/VTA bilaterally. Middle, The IPN was injected with a single injection of 2µl of PGK-β4 at 75ng/µl. Right, The MHb was injected with two 0.5µl volumes of 200ng/µl PGK-β4 per MHb. Green -anti-GFP, blue -DAPI, red -anti-ChAT.

  the brains dissected, and frozen to -80°C. The brain was sectioned (20µm coronal) and thaw-mounted onto microscope slides.

Figure 2 .

 2 Figure 2.1.b.ii. The PGK-β4 lentivirus vector restores β4* nAChR expression on a KO background. Autoradiography of WT and KO mice injected with control (GFP) and test (β4) virus in the MHb (left), IPN (middle), or VTA (right). Total I 125 -epibatidine autoradiography indicates localisation of heteromeric nAChRs. 5-iodo-A85380 (A85380) displacement of I 125 -epibatidine reveals putatively β4* nAChRs. Mice were euthanised for autoradiographic assessment several months post-injection.

Figure 2 .

 2 Figure 2.1.c.i. Stereotaxic strategy for lentivirus injections to the MHb. a. The cranial sutures of the mouse viewed from above (Rynkowski et al, 2008). B -bregma, L -lamda. Stereotaxic coordinates are described as distance along the x (medial-lateral), y (anterior-posterior) and z (dorsal-lateral) axes from bregma. The bregma-lamda suture is used as the midline. b. Image of MHb injected bilaterally with methylene blue using the adapted strategy for stereotaxic targeting of the MHb illustrated in c. Illustration of the injection strategy using pulled glass pipettes, injecting at an angle of 20°C. Only one pipette is illustrated here for aesthetic reasons. In reality both MHb are injected simultaneously. Injecting a small (0.5µl) volume of lentivirus per injection (two injections per MHb) results in local infection (see section 2.1.c).

Figure 2 .Figure 2 1 . 3 .

 2213 Figure 2.1.c.ii. An optimised stereotaxic delivery of lentivirus vector to the MHb allows longitudinal diffusion of the virus (green). Green -anti-GFP; Red -ant-NeuN.

  evoked increases in NuAcc DA release of β4 KO mice are impaired in vivo. This was revealed by in vivo electrophysiological recordings and in vivo microdialysis studies, which reply to Objective 2.Before embarking on Objective 4, we wished to confirm the site of β4* nAChR expression in the mouse brain. Consistent with previous literature, we demonstrate that β4* nAChRs are richly expressed in the MHb and IPN. Transgenic β4-GFP mice have been used to characterise β4-expressing neurons in the MHb(Shih et al. 2014), but had not been conducted in the IPN. Using a cre-lox system we were able to illustrate the location of β4-expressing IPN neurons without the masking effect of β4-rich MHb terminals. Revealing an important post-synaptic expression of the nAChR subunit, in response to Objective 3, we embarked upon the stratification of the brain region-specific contribution of β4* nAChRs in nicotine self-administration. Lentivirus-driven restoration of β4* nAChRs on a KO background revealed the MHb and IPN as integral to nicotine self-administration, both of which serving to at least partially rescue the deficit observed in KO mice. This is reflected as an appeasement of the VTA dopamine neuronINTRODUCTIONTobacco is consumed by an estimated 1 billion people worldwide. With 6 million deaths per year as a result of its detrimental health effects, tobacco consumption is the primary cause of preventable morbidity and mortality[START_REF] Who | WHO report on the global tobacco epidemic[END_REF]. Nicotine is the principal psychoactive compound in tobacco, and stimulates the neurological reward system to drive its repeated consumption. The maintenance of nicotine consumption impedes cessation, and current therapies display weak support for abstinence(Hartmann-Boyce et al, 2013;[START_REF] Messer | Smoking cessation rates in the United States: A comparison of young adult and older smokers[END_REF].It is therefore important to elucidate the neural mechanisms underlying nicotine addiction to aid in the development of an efficient smoking cessation therapy.Nicotine exerts its effects by binding to nicotinic acetylcholine receptors (nAChRs). These transmembrane receptors are composed of alpha (⍺1-10) or alpha plus beta (β2-4) subunits, forming a diverse variety of homo-and hetero-pentameric ligand-gated ion channels endogenously activated by acetylcholine. Human genetic studies have highlighted the polymorphic nature of the CHRNA5-CHRNA3-CHRNB4 genomic cluster, encoding subunits α5, α3 and β4, and its implication in smoking behaviors. Furthermore, variants in the β4 regulatory domain reduce the age of tobacco initiation, whilst gain of function variants in CHRNB4 reduce the risk of nicotine dependence[START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF] Saccone et al, 2009; Schlaepfer et al). Whilst cholinergic responses are unaffected in CHRNB4 coding variants, nicotine-elicited currents are increased(Haller et al), which is reflected in nicotine-related behaviors in mice(Slimak et al) and humans(Haller et al). These studies have shown that the β4-containing (β4*) subpopulation of nAChRs merits further investigation.The pharmacological properties of β4* nAChRs are distinct with respect to other nAChR subtypes, demonstrating lower sensitivity to nicotine compared to ⍺4β2* nAChRs, and slower desensitization in the case of ⍺3β4(Fenster et al). Unlike the promiscuously expressed β2* nAChRs, β4* nAChRs demonstrate a discreet expression profile in the rodent central nervous system. The medial habenula (MHb) and interpeduncular nucleus (IPN) are the major hosts to β4* nAChRs. The MHb receives input from the posterior septum(Yamaguchi et al, 2013) and

  hydrogen tartrate salt [(-)-1-methyl-2(3-pyridyl)pyrrolidine] (Sigma, Madrid, Spain)

  50 mm ventral relative to bregma skull surface, at an angle of -20°. A metal 35-gauge needle was used to deliver a single injection of 2 µl lentivirus to the central region of the IPN (PGK-β4 75ng/µl, PGK-GFP 25 ng/µl p24 protein), or 0.5-1.0 µl (10 11 GC) of AAV. Lentivirus-injected mice were submitted to subsequent experiments 4 weeks post-injection. AAV-injected mice were sacrificed 2-4 weeks post-injection for immunohistological analysis.Verification and quantification of β4* expression in the MHb and IPNIn situ autoradiography was conducted as previously(Frahm et al) with the use of 25 nM 5-Iodo-A-85380 dihydrochloride (5-I-A85380, Tocris Bioscience, Bristol UK) as the cold ligand for displacement of 220 pM I 125 -epibatidine (2200Ci/mmol specific activity, PerkinElmer, Inc., Waltham MA) from β2* nAChR sites. Once dry, slides were exposed to a Kodak MR film for 16 h, developed (Kodak X-OMAT 2000), and the film scanned at 1600 dpi. I 125 -epibatidine binding localizes heteromeric nAChRs, whereas 5-I-A85380-resistant I 125 -epibatidine binding localizes putatively β4* nAChRs. The 5-I-A85380-resistant I 125 -epibatidine images were used for quantification using Image J (1/luminosity minus mean background over 3 points). Slides were stained with 0.1% cresyl violet and progressively dehydrated in 50-100% EtOH and histoclear.

  Despite the re-expression of β4* nAChRs along the length of MHb neurons, there is only a partial rescue of the nicotine self-administration deficit. β4 itself is rate-limiting in this behavioral outcome, with KO-β4 MHb mice expressing less than 38% of WT-GFP MHb β4* receptor levels maintaining a KO-GFP MHb -like nicotine self-administration phenotype. It is interesting to consider this finding in the context of human genetic studies: a variant in CHRNB4's 3' untranslated region reduces the age of onset of habitual smoking(Schlaepfer et al) and alters gene expression(Gallego et al, 2013), a known limiting factor for ⍺3β4 receptor activity(Frahm et al, 2011). Re-expressing β4 on IPN neurons had a more potent effect on nicotine IVSA, with KO-β4 IPN mice showing a full rescue of self-administration at the doses of 7.5 and 15.0 µg/kg/infusion. Whilst the VTA DAergic neurons of KO mice showed an increased sensitivity to nicotine-elicited changes in firing pattern, a dose of 30 µg/kg was required in the KO-β4 IPN mouse to deviate the VTA DAergic neurons' firing frequency from baseline. This suggests that β4* nAChRs in the IPN regulate VTA DAergic responses to nicotine, and may be responsible for the observed restoration of nicotine IVSA.

Figure 1 .

 1 Figure 1. Operant responding for food pellets and intravenous nicotine in WT and β4KO mice.

Figure 2 .

 2 Figure 2. Basal and nicotine-stimulated extracellular DA levels in the NAC of β4KO and WT

Figure 3 .

 3 Figure 3. Nicotine-elicited VTA DAergic neuron responses in vivo. A) DAergic neurons'

Figure 4 .

 4 Figure 4. Endogenous and exogenous expression of β4 in the mouse brain. A) PGK-β4 lentiviral

Figure 5 .

 5 Figure 5. The effect of β4* nAChR re-expression in the MHb and IPN on nicotine IVSA and VTA

  on a microinfusion pump (PHM-100A; Med Associates) and connected via Tygon tubing (0.96 mm o.d., Portex Fine Bore Polythene Tubing, Portex Ltd, Kent, UK) to a single-channel liquid swivel (Instech Laboratories, Plymouth Meeting, PA, USA), and to the mouse intravenous catheter. At the beginning of each training session, mice received one free nicotine infusion.

  This self-administration test located inhibitory motivational properties of nicotine to the VTA. However, suspecting an interplay between different brain regions on the behavioural outcome, we explored the individual contribution of β4-expressing brain regions in this task by lentivirus-mediated re-expression of β4* nAChRs to the VTA, MHb and IPN. Lentivirus vector-mediated expression of β4 nAChR subunits in the VTA of KO mice, resulting in WT-TABAC intermediate levels of β4* nAChRs, perpetuated the KO phenotype, showing high levels of nicotine ICSA. This suggests that the increased intra-VTA self-administration aversion observed amongst TABAC mice is not due to nicotine acting at VTA β4* nAChRs. Re-expressing β4* nAChRs in the MHb or the IPN on a KO background restored WT-like avoidance of nicotine ICSA at high doses of nicotine. For the first time, we show that nAChRs at extra-VTA sites modulate the outcome of VTA-mediated nicotine self-administration, with the MHb-IPN axis serving as addictive properties of nicotine, which are highly relevant in defining pharmacological strategies for smoking cessation.

Figure 1 .

 1 FIGURES LEGENDS

Figure 2 .

 2 Figure 2. Representative photomicrograph of a thionine-stained frontal brain sections

Figure 3 .

 3 Figure 3. A: Acquisition of intra-VTA nicotine self-administration in wild-type (WT, n=7)

Figure 5 .

 5 Figure 5. A. Lentivirus re-expression of β4*-nAChRs in the VTA. Representative I 125staining in coronal sections of WT-GFP VTA , KO-GFP VTA , TABAC and KO-β4 VTA brains, at the level of the VTA. The left column shows total I 125 -epibatidine binding, illustrative of

Figure 6 .

 6 Figure 6. A. Lentivirus re-expression of β4*-nAChRs in the MHb. KO-β4 MHb mice were

Figure 7 .

 7 Figure 7. A. Lentivirus re-expression of β4*-nAChRs in the IPN. Left) I 125 -staining in coronal sections of WT-GFP IPN , KO-GFP IPN and KO-β4 IPN brains, at the level of the IPN.The left column shows total I 125 -epibatidine binding, illustrative of heteromeric nAChRs.

Figure 2 .

 2 Figure 2.4.a. Mapping of cholinergic neurons in the mouse brain. Top, confocal microscope mosaic of a ChAT-cre Tg/+; Tom +/-mouse. Fluorescence is Tdtomato, without immunoamplification. Sagittal section at approximately 0.3mm from the midline. Below, illustrative map of a mouse sagittal section that corresponds to the above image. The red box delineates the area that is shown in fluorescence above.

IPN 2 .

 2 4.b ⍺-conotoxin AuIB displacement of I 125 -epibatidine.

  3), or 50mM TRIS + 5-iodo-A85380 (25nM), or 50mM TRIS + 5-iodo-A85380 + ⍺-conotoxin AuIB (0.5µM). A one-hour incubation in 220pM I 125 -epibatidine (2200Ci/mmol specific activity) with the corresponding cold ligands ensued. Slides were rinsed in TRIS and water. Once dry, slides were exposed to MR films (Kodak) for 16h, the slide developed, and the resultant autoradiograph scanned at 800dpi for analysis (figure 2.4.b).

Figure 2 .

 2 Figure 2.4.b. Autoradiography of I 125 -epibatidine labelling of heteromeric nAChRs in situ. MHb-containing 20µm sections of a single WT mouse were submitted to three radioligand binding protocols. The first shows total I 125 -epibatidine labelling (left), illustrating the localisation of heteromeric nAChRs indiscriminately. The second condition (middle), shows 5-iodo-A85380-resistant I 125 -epibatidine labelling, indicative of non-β2* heteromeric nAChRs. The third condition (right), tests the effect of 5-iodo-A85380 and ⍺-conotoxin AuIB displacement of I 125 -epibatidine binding. The first two conditions, used throughout this thesis, show expected I 125 -epibatidine labelling in this portion of the mouse brain. The third condition, however, appears no different to that of the second.

Figure 2 .

 2 Figure 2.4.c. Validation of nAChR subunit ⍺3, ⍺5 and β4 primers in transfected HEK cells (left), and preliminary estimate of nAChR subunit expression in the MHb of WT and β4 KO mice (right). Results are expressed as 1/Ct (cycle threshold). Gaps in the graph indicate undetectable levels of cDNA targeted.

  photomicrographs are depicted in figure2.4.d.i.

Figure 2 .

 2 Figure 2.4.d.i. Immunohistochemical analysis of β4 nAChR subunit expression is the β4 knockout (KO, left) and β4 over-expressing (over-expr., right) mice. Blue -hydrolysis products from alkaline phosphatase reaction coupled to the secondary antibody.

Figure 2 .

 2 Figure 2.4.d.i. Photomicrographs of MHb-containing sections of β4 KO (left), β4 WT (middle) and β4 overexpressing (right) mice. Primary antibodies used: 2µg/ml rabbit anti-β4 and 1/500 mouse anti-GFP. Bluehydrolysis products from alkaline phosphatase reaction demonstrative of anti-β4 staining. Brownperoxidase-catalyzed products demonstrative of anti-GFP staining. An anti-GFP reaction was conducted due to the fact that the β4 over-expressing mouse also expresses GFP under the ⍺3 promoter(Frahm et al. 2011).

Figure 2 .

 2 Figure 2.4.e.iii. PDGFfloxβ4 lentivirus infection of the VTA of a DAT-cre mouse, expressing cre recombinase in dopaminergic (blue, anti-tyrosine hydroxylase, TH) neurons. mCherry (red, anti-RFP) immunostaining indicates a large diffusion area of the lentivirus. Coherent with the model, mCherry labelling does not colocalise with dopaminergic neurons. Green fluorescence was deemed non-specific, as auto-fluorescence is often observed near the trace of the injection needle.

Figure 2 .

 2 Figure 2.4.e.iv. The PDGFfloxβ4 lentivirus vector increases putatively β4* nAChR expression in the IPN of KO mice, but not the VTA. Autoradiographic analysis of heteromeric nAChR expression (left column) and non-β2* nAChR expression (principally β4* nAChR, right column). The WT control shows strong 5-iodo-A85380 (A85380)-resistant I 125 -epibatidine labelling in the IPN, and no detectable levels in the VTA. 5iodo-A85380-resistant I 125 -epibatidine labelling is reduced in the KO control, and in the KO-DATcre+ mouse injected with the PDGFfloxβ4 lentivirus to the VTA. Less than WT-like levels of I 125 -epibatidine binding are restored in the IPN of the KO-GADcre+ mouse injected in this region with the PDGFfloxβ4 lentivirus. This I 125 -epibatidine binding assay was conducted four weeks post injection.

Figure 2 .

 2 Figure 2.4.e.v. The PDGFfloxβ4 lentivirus vector does not restore β4* nAChR expression in the MHb of β4 KO ChATcre +/-mice. Autoradiographic analysis of heteromeric nAChR expression (left) and non-β2* nAChR expression (principally β4* nAChR). The WT control (WT-GFP MHb ) shows strong 5-iodo-A85380 (A85380)-resistant I 125 -epibatidine labelling in the MHb. 5-iodo-A85380-resistant I 125 -epibatidine labelling is reduced in the KO control (KO-GFP MHb ). KO-like I 125 -epibatidine binding is observed in the β4KO ChATcre +/-mouse injected with the PDGFfloxβ4 lentivirus in the MHb. Autoradiographic assessment was conducted four weeks post injection.

Figure 2 .

 2 Figure 2.4.f.ii. AAV construct for cre-dependent β4 nAChR subunit expression. The AAVifloxβ4 construct was based on a commercially available pAAV5-EF1α-DIO-ChETA-eYFP virus (Marlin et al. 2015). The EF1⍺ promoter is appropriate for neuronal expression of transgenes, as validated in the CNS (Yizhar et al. 2011), the VTA (van Zessen et al. 2012) and the IPN (Zhao-Shea et al. 2013). ITR -inversed terminal repeats; high polyA -polyadenylation signal; β4 -mouse wildtype β4 cDNA sequence; AscI and NheIrestriction enzyme digestion sites used for subcloning. Arrow denotes 5'-3' directionality of the β4 sequence.

Figure 2 .

 2 Figure 2.4.f.iii. Preliminary autoradiographic assessment of AAVifloxβ4. Left) I 125 -staining in IPNcontaining coronal brain sections of IPN AAVifloxβ4-injected β4KO GAD-cre +/-mice. WT and KO controls are from a previous experiment, and are PGK-GFP IPN-injected. The left column shows total I 125epibatidine binding, illustrative of heteromeric nAChRs. The right column shows I 125 -epibatidine binding of adjacent brain sections after displacement from β2* nAChR sites by 5-iodo-A85380 (A85380, 25nM), revealing putative β4* nAChRs. Right) Quantification of 5-iodo-A85380-resistant I 125 -epibatidine autoradiography. β4 KO GAD-cre +/-mice n=3; KO controls n=2. Mean inverse luminosity of autoradiographic puncta are expressed relative to WT controls (n=2). Unpaired t-test. A caveat with the interpretation of the quantification is that controls were nicotine-exposed, whereas KO-GADcre+_AAVifloxβ4 mice were nicotine-naive. This may have an effect on nAChR expression and resultant I 125 -epibatidine binding.

Figure 2 .

 2 Figure 2.4.g. Assessment of topographical innervation of the IPN from the left (LHS) and right (RHS) MHb. Two lentiviral vectors were used to express GFP (green) in LHS MHb neurons, and mCherry (red) in RHS MHb neurons. Immuno-amplified flourescence (⍺-GFP, ⍺-RFP) and endogenous fluorescence (GFP, mCherry) were recorded in the MHb (a), and the IPN (b-d). Images b-d are from sequential 60µm brain sections arranged from most anterior (b) to most posterior (d). GFP-expressing and mCherry-expressing MHb terminals reach the IPN (b-d).

Figure 2 .

 2 Figure 2.4.h. Localisation of GABAergic interneurons. A-C. The hippocampus contains somatostatin (A) and parvalbumin (B) interneurons in CA3. Dense somatostatin (D) is observed in the dorsal point of the IPN (D) and diffuse parvalbumin staining in the dorsal half (E). G, H and I are magnifications of images D, E and F, respectively.

  interpeduncular pathway, we show for the first time that extra-VTA nAChRs, activated by endogenous signalling mechanisms, can alter the behavioural outcome of nicotine locally in the VTA. We also reveal a role of β4* receptors in the MHb and IPN in selfadministration of nicotine intravenously, a paradigm that models many aspects of human smoking behaviours (Article One). We propose that the β4* receptors in the MHb are necessary, but β4* nAChRs in the IPN are sufficient, in modulating the positively reinforcing properties of nicotine in this i.v. self-administration test. Figure 3.1 illustrates this hypothesis, and pieces together information from various ex vivo studies presented throughout this thesis (figure 3.1).

Figure 3 . 1 .

 31 Figure 3.1. Diagrammatic representation of β4* nAChR expression in WT mice, knockout (KO) mice, and KO mice re-expressing β4* receptors in the medial habenula (KO-β4 MHb ) or interpeduncular nucleus (KO-β4 IPN ), and their effects on nicotine intravenous self-administration (IVSA). In the WT mouse, nicotine activates the MHb and IPN soma, and stimulates glutamate (Glu) and acetylcholine (ACh) release

  Restoring β4* nAChR expression to MHb neurons may rescue nicotine-evoked increases in firing of action potentials and neurotransmitter release in the IPN. KO-β4 MHb mice demonstrate a partial rescue of nicotine IVSA. Restoring β4* nAChR expression to IPN neurons may rescue WT-like nicotinic stimulation of IPN neurons, sufficient to restore WT-like nicotine IVSA at 7.5 and 15.0µg/kg/infusion. mesolimbic system, with β4 KO mice showing increased sensitivity to nicotine-evoked responses at the VTA dopaminergic soma. β4 KO mice also demonstrate altered sensitivity to nicotine-elicited accumbal dopamine outflow, showing blunted responses to nicotine concentrations known to condition place preference. Once again, extra-VTA β4* nAChRs were shown to be sufficient in modulating VTA responses, with the increased sensitivity to nicotine placated when β4* nAChRs were restored in the IPN of KO mice.The present thesis not only adds to the literature concerning β4* nAChRs, but it also highlights the intimate link between the habenulo-interpeduncular pathway and the mesolimbic pathway. Many more experiments are required to elucidate how β4* nAChRs contribute to the communication between these neuronal pathways, which shall be addressed in the proceeding sections.

3. 2

 2 Anatomical connection between the habenulainterpeduncular pathway and the ventral tegmental areaOptogenetic manipulation of the VTA can produce aversion phenotypes, as previously discussed (section 1.5.1). Article Two, however, demonstrates that genetic manipulation of nAChRs at extra-VTA sites modulates nicotine aversion in an intra-VTA selfadministration task. This section reviews the afferent and efferent connections from the habenulo-interpeduncular system that may contribute to the aversive outcome of high doses of nicotine at the VTA. Three hypothetical mechanisms shall be described. 1) Nicotine-evoked changes in VTA neuronal firing directly alter MHb and IPN activity. 2) β4* nAChRs in the MHb and IPN modulate VTA tone and its response to nicotine (i.e. MHb and IPN β4 having an effect upstream of VTA). 3) Nicotine-evoked changes in VTA neuronal firing indirectly alters MHb and IPN activity thus eliciting nicotine aversion (i.e. MHb and IPN β4* nAChRs having an effect downstream of VTA). 1) Nicotine-evoked changes in VTA neuronal firing directly alters MHb and IPN activity It is possible that the nicotine's effects at the VTA are directly communicated to the MHb and IPN (figure 3.2.i). A single study has demonstrated that the MHb receives dopaminergic input that is removed by a 6-hydroxydopamine lesion of the VTA,

  Re-expressing β4 nAChR subunits in the MHb (KO-β4 MHb ) or IPN (KO-β4 IPN ) of KO mice rescues the nicotine aversion deficit in β4 KO controls. MHb electrophysiological function and neurotransmission is reduced in the absence of β4 (see section 1.4.2.b), which may be restored in the KO-β4 MHb mouse, allowing activation of the IPN via glutamate receptors and non-β4* nAChRs. KO-β4 IPN mice also show restored intra-VTA nicotine aversion despite the loss of MHb function in the absence of β4. We must therefore consider another cholinergic input to the IPN that would activate its β4* nAChRs.This leads me to believe that the MHb and IPN modulate intra-VTA nicotine aversion via converging systems, with the MHb modulating nicotine aversion via input to the IPN (its almost sole direct target), and the IPN of KO-β4 IPN mice receiving cholinergic input from an alternative cholinergic source, for example the cholinergic LDTg or the septum.Common to these proposed pathways in the KO-β4 MHb and KO-β4 IPN models is modulation of the IPN with potential consequences on the IPN's efferent targets, which may in turn modulate the aversive outcome of intra-VTA nicotine administration. 3) Nicotine-evoked changes in VTA neuronal firing indirectly alters MHb and IPN activity The outcome of intra-VTA nicotine stimulation is a reflection of the state of nAChR expression in the MHb and IPN, with β4 nAChR deletion invoking aberrant nicotine selfadministration at normally aversive concentrations. It is possible, instead of the MHb and IPN predetermining the behavioural outcome of VTA nicotinic stimulation, that the MHb and IPN integrate nicotinic information from the VTA. This places the MHb and IPN downstream of the VTA, which, as discussed in hypothesis one, would necessarily occur via an indirect neuronal connection (figure 3.2.iii).

Figure 3 . 2 .

 32 Figure 3.2.iii. Illustrative description of the hypothesis 3. The MHb (red) and IPN (purple) integrate information from nicotinic stimulation of the VTA (grey) via intermediate brain regions (yellow). MHbmedial habenula; IPN -interpeduncular nucleus; VTA -ventral tegmental area.

Figure 3 . 2 .

 32 Figure 3.2.iv. Overview of the septal, habenulo-interpeduncular, and VTA anatomical

Figure 3 .

 3 Figure 3.3.a. Adapted from (Kuryatov, Berrettini, and Lindstrom 2011). Activation and desensitisation responses to nicotine in different ⍺5* nAChRs of transfected HEK cells. "Desensitization was measured after 1.5 h… allowing enough time for equilibrium desensitization but not enough time for up-regulated

  figure3.3.a). The maximum responses between ⍺3β4⍺5 and ⍺4β2⍺5 nAChRs were

  disfavoured in basic research to the advantage of β2* nAChR investigations. However, recent human genetic studies have robustly highlighted the polymorphic nature of the CHRNA5-CHRNA3-CHRNB4 cluster and its contribution to risk and onset of nicotine dependence. For the first time in the nicotine research domain, human genetic data is moulding the direction of basic research, and placing ⍺5, ⍺3 and β4 nAChR subunits into the limelight.

  . The pore is water-filled in the open conformation. Monovalent cations permeate all ⍺/β nAChR conformations, and demonstrate varying degrees of Ca 2+

  function envisaged by the authors, does not represent the physiological state. Later studies will hopefully resolve whether this tight, hydrophobic junction occurs in the uncleaved receptor and the type of mechanical signalling required for it to relax with

	channel opening.
	It is also important to consider the conformational changes that occur during receptor
	activation and inactivation. Monod, Wyman, and Changeux (MWC) proposed a model of

allosteric regulation based on ligand-induced changes in equilibrium constants to favour one particular conformation (e.g. active, open channel) over another (e.g. inactive, closed channel; Changeux 2013). Proposing that an oligomeric protein is in equilibrium between active and inactive states, the MWC model suggests that nAChRs may spontaneously activate even in the absence of an agonist, and that agonist binding serves to increase the probability of channel opening by thermodynamically stabilising the active state. This idea has been validated by single channel recordings of muscle nAChRs

[START_REF] Purohit | Unliganded gating of acetylcholine receptor channels[END_REF]

.

Developing on the generic MWC model, it is now known that the nAChR, and other pLGIC, enter into a third, desensitisation state that precedes the receptor's return from an active to inactive conformation. An additional intermediate state between inactive and active has also been demonstrated. This is summarised in figure

1.b.iv.

β4KO mice demonstrate basal NAC hyperdopaminergia, and deregulated nicotine- induced increase in NAC DA release

	1,36)=6.856, p<0.05). Mice were then tested on a within-subject
	design dose-response curve for nicotine reinforcement. Significant effects of dose
	(F(4,72)=16.683, p<0.001), genotype (F(1,18)=74.533, p<0.01), and interaction were observed
	(F(4,72)=4.443, p<0.01). Post-hoc comparisons revealed that β4KO mice self-administered less
	nicotine than WT mice at all the doses tested except saline (7.5, 30.0 and 60.0 µg/kg/infusion,
	Basal extracellular levels of DA in the NAC were statistically higher in β4KO (3.46 ±0.41 pg/15
	µl) than WT controls (2.30 ±0.23 pg/15 µl; genotype: F(1,24)=6.392, p<0.05; Fig. 2A). A
	challenge injection of saline did not change the levels of DA with respect to baseline in either
	group (WT = 2.37 ±0.26 pg/15 µl, KO = 3.61 ±0.47 pg/15 µl).

p<0.01; 15 µg/kg/infusion, p<0.05; Fig.

1D

).

  nAChR expression to the IPN attenuates the VTA's increased sensitivity to nicotine observed in KO mice, as observed in electrophysiological recordings of VTA DAergic neurons of KO-β4 IPN mice. A dose of 30 µg/kg nicotine was required to increase VTA DA cells'

	±3.50; KO-GFP IPN ,
	n=9, 31.26 ±2.29; KO-β4 IPN , n=9, 30.70 ±4.04, one-way ANOVA n.s.).
	Restoring β4*

  Their performances were not different from WT-GFP IPN mice (ANOVA at 600 ng/50 nl: Post hoc analysis revealed that at the dose of 1200 ng/50 nl, KO-GFP IPN mice self-injected more nicotine than the WT-GFP IPN and KO-β4 IPN mice (KO-GFP IPN vs WT-GFP IPN : p<0.01; KO-GFP IPN vs. KO-β4 IPN : p<0.01).

	Genotype effect: F(2,15)=3.56 p=0.05; Session effect: F(6,90)=1.70 ns; Genotype x
	Session interaction: F(12,90)=0.60 ns; ANOVA at 1200 ng/50 nl: Genotype effect:
	F(2,15)=7.45 p<0,01; Session effect: F(6,90)=1.12 ns; Genotype x Session interaction:
	F(12,90)=0.87 ns).

  p<0.05, **p<0.01, ***p<0.001 (vs WT); # p<0.05, ## p<0.01 (vs KO). Two-way ANOVA followed by post hoc Newman-Keuls test; significant global analysis p<0.05. H:

	habituation session.
	high, n=6; KO-β4 MHb low, n=6) over 7 training sessions (10 daily trials) at 600 ng nicotine
	as salt per self-administration followed by 7 training sessions at 1200ng nicotine as salt
	per self-administration. Mean number (± SEM.) of self-administrations. ANOVA at 600
	ng (session effect, p<0,01); at 1200 ng (genotype effect, p<0,01; post hoc: WT-GFP MHb
	vs. KO-GFP MHb , p<0,01; WT-GFP MHb vs. KO-b4 MHb low, p<0,01; KO-GFP MHb vs. KO-
	β4 MHb high, p<0,01; KO-β4 MHb high vs. KO-b4 MHb low, p<0.05). C: Mean (± SEM) latencies
	(in seconds) to trigger the injection (self-injection latency). ANOVA at 600 ng (session
	effect, p<0.0001); at 1200 ng (genotype effect, p<0.05; post hoc WT-GFP MHb vs. KO-
	GFP MHb , p<0,01; WT-GFP MHb vs. KO-b4 MHb high, p<0.05).

*

hyperactivity observed in KO mice. We therefore reveal the importance of a subset of nAChRs, previously attributed to nicotine aversion (Frahm et al. 2011; Slimak et al. 2014) and withdrawal (Salas, Pieri, and De Biasi 2004; Zhao-Shea et al. 2013), at sites outside of the ventral tegmental area as contributing to voluntary nicotine selfadministration. ACKNOWLEDGMENTS This work was supported by the Spanish Instituto de Salud Carlos III (RD06/001/001 and PI10/01708), FEDER funds, and Ministerio de Ciencia e Innovación (SAF2007-64062), the Catalan Government (SGR2009-00131), an FP7 ERANET program (NICO-GENE), the ICREA Foundation (ICREA Academia-2008), and a post-doctoral fellowship from CONACyT to AHS. Work in Paris was supported by the Institut Pasteur, Centre National de la Recherche Scientifique CNRS UMR 3571, the Agence Nationale pour la Recherche (ANR Neuroscience), and FP7 ERANET program (NICO-GENE), Grant agreement n009 BLANC 20092009BLANC 20 NeuroCypres" project), the École des Neurosciences de Paris (ENP), Fondation EDF, and the Fondation des Treilles. We would like to thank Martine Soudant, Stephanie Pons and Dulce Real for technical support and discussions, and Julien Dykmans for illustrations. a brake on consumption of high doses of nicotine. Whilst not a model of human smoking behaviours, this ICSA procedure and the lentivirus strategy used, in response to Objective 2, allows us to discriminate nicotinic and cholinergic modulation of nicotine self-administration.

Author contributions

• Harrington generated the mouse models by creating a novel lentivirus and developing stereotaxic targeting of the MHb and IPN. Harrington quantified β4* receptor expression and characterised β4-expressing neurons in the IPN. Harrington designed the research, wrote the article, and conducted the statistical analyses of the behavioural and nAChR expression data;

• Viñals, Herrera-Solís and Flores conducted intravenous nicotine self-administration;

• Morel and Tolu performed in vivo electrophysiology and its analysis;

• Maldonado, Faure and Maskos designed and supervised the research;

• Robledo performed in vivo microdialysis, intravenous nicotine self-administration, analysis of behavioural data, designed the research and wrote the article.

ABSTRACT

Nicotine exerts its psychopharmacological effects by activating the nicotinic acetylcholine receptor (nAChR), composed of alpha and/or beta subunits, giving rise to a diverse population of receptors with a distinct pharmacology. β4-containing (β4*) nAChRs are located almost exclusively in the habenulo-interpeduncular pathway. We examined the role of β4* nAChRs in the medial habenula (MHb) and the interpeduncular nucleus (IPN) in nicotine reinforcement using behavioral, electrophysiological and molecular techniques in transgenic mice. Nicotine intravenous self-administration (IVSA) was abrogated in constitutive β4 knockout (KO) mice at all doses tested (7.5, 15, 30 and 60 µg/kg/infusion) compared to wild-type (WT) mice. In vivo microdialysis showed that β4KO mice have higher extracellular dopamine (DA) levels in the nucleus accumbens, and exhibit a leftward shift in sensitivity to nicotine-induced DA outflow.

Furthermore, electrophysiological recordings in the ventral tegmental area (VTA) demonstrated that DA neurons of β4KO mice are more sensitive to nicotine. Viral-mediated rescue of β4* nAChRs in the MHb, including at MHb terminals in the IPN, partially restored the IVSA deficit of β4KO mice. Re-expression of β4* nAChRs in IPN neurons, however, fully restored nicotine IVSA, and attenuated the increased sensitivity of VTA DA neurons to nicotine. Considering established anatomical and functional links, these findings suggest that β4* nAChRs in the MHb are necessary, but that β4* nAChRs in the IPN are sufficient for maintaining nicotine IVSA. This work highlights the importance of the habenulo-interpeduncular pathway in positive nicotine reinforcement.

Re-expression of β4* nAChRs in the IPN rescues nicotine IVSA and VTA DAergic responses β4cre mice, expressing cre-recombinase under a β4 promoter, were injected with an AAV in the IPN for conditional expression of the fluorophore GCaMP5 (Fig. 4D). Immunohistofluorescence analyses revealed that these neurons colocalize with somatostatin immunoreactivity (95%, Supp. Fig. 8). β4cre neurons occupy the dorsal part of the anterior IPN at positions -3.3 to -3.5mm from bregma, and progressively invade the more ventral portion of the IPN towards -3.8mm from bregma (n=8).

The role of β4 on neurons of the IPN in nicotine IVSA was investigated. WT mice were injected in the IPN with a control lentivirus (WT-GFP IPN ) and KOs with control (KO-GFP IPN ) or PGK-β4 lentivirus (KO-β4 IPN ), and submitted to the nicotine IVSA test. GFP fluorescence and 5-I-A85380-resistant I 125 -epibatidine binding was detected in the IPN and the laterodorsal tegmentum (Fig. 4C, Supp. Fig. 9). Assessment of radioligand binding was conducted in all but two KO-β4 IPN brains. On-target β4* nAChR re-expression was observed in all brains assessed (KO-β4 IPN 63.7%, ±11.8%; KO-GFP IPN 1.3%, ±0.8%; Fig. 5D). All subjects were therefore included in the behavioral data. Discriminatory nose-poking at the active hole was observed amongst all groups (Supp. Table 2). Comparing the number of infusions earned shows an effect of group (F(2, 21)=10.15, p<0.001), dose (F(4, 84)=26.61, p<0.0001), matched subjects (F(21, 84)=9.76, p<0.0001), and interaction (F(8, 84)=5.39, p<0.0001, Fig. 5C). Post-hoc analysis reveals that KO-β4 IPN self-administer nicotine at WT-GFP IPN -like levels at the dose of 7.5 µg/kg/infusion and 15.0 µg/kg/infusion, where WT-GFP IPN is statistically different to KO-GFP IPN (p<0.0001, Fig. 5C). KO-β4 IPN nicotine IVSA at the dose of 30.0 µg/kg/infusion was statistically different to WT-GFP IPN (p<0.05) and KO-GFP IPN (p<0.05) who themselves show statistical divergence (p<0.0001, Fig. 5C). No statistical difference between groups was acetylcholine release and systemic nicotine. Restoring IPN β4* nAChR expression, whilst not rescuing MHb cholinergic input, would allow direct nicotinic activation of the structure (Supp. Fig. 12). It appears that β4* nAChR function in nicotine IVSA is more potent in the IPN than MHb, leading us to propose that these receptors in the MHb and IPN are necessary and sufficient, respectively, for nicotine IVSA. With a lack of direct innervation of the IPN to the VTA (Antolin-Fontes et al, 2014), systemic nicotine acting at IPN β4* nAChRs presumably engages an indirect pathway between the two structures. The relay may occur via the dorsal raphe nucleus, laterodorsal tegmentum and pedunculopontine tegmentum (Antolin-Fontes et al, 2014). This merits further investigation.

FUNDING AND DISCLOSURE

The authors declare no conflict of interest.

HPLC and probe detection

Dialysates (20 µl) were injected without any purification into an HPLC system that consisted of a pump linked to an automatic injector (Agilent 1100, Palo Alto, USA), a reverse-phase column (Zorbax SB C18, 5 mm, 150 x 4.6 mm, Agilent Technologies, Palo Alto, USA), and a coulometric detector (Coulochem II, ESA Inc., Chelmsford, USA) with a 5011A analytical cell. DA was quantified as previously described [START_REF] Robledo | The rewarding properties of MDMA are preserved in mice lacking mu-opioid receptors[END_REF]). Briefly, the first electrode was fixed at -100 mV and the second electrode at + 300 mV. The gain of the detector was set at 10 nA. The composition of the mobile phase was 50 mM NaH2PO4, 0.1 mM Na2EDTA, 0.65 mM octyl sodium sulfate and 15 % (vol/vol) methanol, pH 3.5. The flow rate was set at 1 µl/min and the sensitivity of the assay was 0.2 pg/20 µl. At the end of the experiments, mice were sacrificed and brains cut using a cryostat. Serial coronal sections (20 µm) were then processed with Cresyl Violet (Sigma-Aldrich, Madrid, Spain). Only those mice with correct probe placements were used in the study.

SUPPLEMENTARY RESULTS

Nose-pokes responses amongst WT and β4 KO mice

For active and inactive nose-poke activity to obtain food reinforcement on a FR1, no significant differences were observed between β4 KO and WT mice (genotype effect F(1,115) = 2.033, NS). A significant effect of day ([F(7,805)=64.651, p < 0p<0.001), hole (F(1,115)=1281.479, p<0.001) and significant interaction between day x hole interaction (F(7,805)=183.849, p<0.001) were observed, indicating that both genotypes acquired this behavior in a similar manner (Figure 1). However, no significant day x genotype, hole x genotype nor day x hole x genotype interactions were revealed. For active and inactive nose-poke activity to obtain food reinforcement on a FR3, no significant differences were observed between β4 KO and WT mice (genotype effect F(1,115)=3.948). A significant effect of day (F(5,575)=9.546, p<0.001), hole (F(1,115)=2592.30, p<0.001) and significant interaction between hole x genotype (F(1,115)=4.299, p<0.05) and day x hole interaction (F(5,575)=7.943, p<0.001) were observed, but no significant day x genotype nor day x hole x genotype interactions were revealed.

Nose poke responses at the active and inactive hole amongst WT-GFP MHb , KO-GFP IPN , and KO-β4 MHb WT-GFP MHb KO-GFP MHb and KO-β4 MHb groups all demonstrated specific nose-poking at the active hole, as demonstrated by two-way repeated measures ANOVA (WT-GFP MHb F(1,8)=59.32, p<0.0001; KO-GFP MHb F(1,7)=34.09, p<0.001,;KO-β4 MHb F(1,6)=23.40, p<0.01).

Statistical analysis also revealed an effect of the dose on nose-poking (WT-GFP MHb F(4,32)=21.88, p<0.0001; KO-GFP MHb F(4,28)=6.94, p<0.001; KO-β4 MHb F(4,24)=6.14, p<0.01), and dose x hole interaction (WT-GFP MHb F(4,32)=16.46, p<0.0001; KO-GFP MHb F(4, 8)=6.06, p<0.01; KO-β4 MHb F(4,24)=6.40, p<0.01). Sidak's post-hoc analysis reveals statistically significant nose-poking at the active hole at all doses tested for WT-GFP MHb (all doses p<0.0001), KO-GFP MHb (7.5 p<0.0001; 15.0 p<0.01; 30.0 p<0.05; 60.0µg/kg/inf. p<0.01) and KO-β4 MHb (7.5 and 15.0 p<0.0001; 30.0 p<0.01; 60.0µg/kg/inf. p<0.05). There was no statistical difference between nose-poking at the active and inactive hole during saline self-administration.

Comparison between groups of nose-poking at the active hole revealed a statistically significant effect of group (F(2,21)=6.18, p<0.01,), dose (F(4,84)=27.89, p<0.0001), matched subjects (F(21,84)=4.54, p<0.0001) but not dose x group interaction (2-way repeated measures ANOVA). These data are also represented in tabular form (Supp. Table 1).

The effect of β4* nAChR re-expression in the MHb on nicotine IVSA

In a qualitative correlation assessment between levels of β4* re-expression and behavioral outcome, we observed a threshold effect of β4* nAChRs expression on nicotine self-

Article Two

Introductory comments

In this article we demonstrate the pivotal role of β4-containing (β4*) nAChRs in the modulation of nicotine consumption at high, aversive doses.

Using a choice-based intra-cranial self-administration (ICSA) paradigm, we are able to measure the appetitive and aversive properties of nicotine (section 1.8.a.ii). During tentrial sessions, mice are allowed to navigate towards an arm in a Y maze that is either paired to nicotine, resulting in nicotine delivery, or towards a neutral arm where no substance is delivered. An appetitive dose of nicotine will reinforce navigation towards the nicotine-paired arm for nicotine self-administration (>5/10 infusions/session). An β4-containing nicotinic receptors in the habenulo-interpeduncular pathway mediate a stop signal on nicotine reinforcement Marianne Husson 1,2* , Lauriane Harrington 3,4* , Inés Ibanez-Tallon 5, 6 , Uwe Maskos 3,4 § andVincent David 1,2 § 1 Université de Bordeaux, avenue des facultés, Talence, France Fax: 33(0)5 4000 8743 E-mail : vincent.david@u-bordeaux.fr Keywords: nicotine self-administration, lentiviral vector, β4 nicotinic acetylcholine receptor, ventral tegmental area, habenulo-interpeduncular pathway.

ABSTRACT

Tobacco consumption is the principal cause of preventable mortality and morbidity worldwide. Nicotine, the main psychoactive compound responsible for tobacco addiction, interacts with nicotinic acetylcholine receptors (nAChRs), widely expressed in the central nervous system. However, the relative contribution of these different nAChRs in the nicotine addiction development is not yet clear. While most of the studies focused on the involvement of the high-affinity α4β2-containing receptors in the nicotine's reinforcing effects, recent human genetic studies revealed that human polymorphism in the CHRNA5-CHRNA3-CHRNB4 gene cluster coding for α5-, α3-and β4-subunits are correlated with smoking and/or lung cancer. The investigation of the respective role of these poorly known subunits on nicotine addiction seems to be crucial and urgent. Here, we investigated the region-specific role of the β4 subunit in the reinforcing/aversive properties of nicotine in three β4 subunit-expressing brain structures linked to the reward system: the ventral tegmental area (VTA), the medial part of the habenula (MHb) and the interpeduncular nucleus (IPN). We first analyzed intra-VTA nicotine selfadministrations in mice that did not express (knock-out, KO) or over-express (TABAC) the β4 subunit. We show that the level of β4 subunit inversely correlates with nicotine consumption as seen in KO and TABAC mice. We then assess the effects of lentivirusmediated re-expressions of this subunit in these brain structures of KO mice on nicotine self-administration. Lentiviral re-expressions revealed a dual role of the β4 subunit: i.e. mediating reward within the VTA and inhibitory control in the MHb-IPN axis. The MHband IPN-β4 subunit can be considered as mediators for an inhibitory motivational signal that limits nicotine intake. These data provide new insights into the understanding of the AlexaFluor antibodies were used for localisation of targeted proteins. Images were obtained using a Zeiss fluorescent microscope (Fig. 1B).

Intracranial stereotaxic procedure for nicotine self-injections

The subjects were anaesthetized with a mixed xylazine / ketamine solution and lidocaine HCl was applied locally both before opening the scalp and trepanation. The animals were implanted unilaterally and in a counterbalanced left and right order. The tip of the guide-cannula (outer diameter 0.460 mm; inner diameter 0.255 mm) was positioned 1.5 mm above the VTA. The stereotaxic coordinates used were the following: VTA: 0.40 mm anterior to the interaural line; ± 0.30 mm lateral to the sagittal line and 3.30 mm vertically below the surface of the skull. The incisor bar was level with the interaural line. Mice were allowed to recover from surgery for at least one week.

Intracranial self-administration procedure

Apparatus and general procedure

The mouse model of intracranial self-administration (ICSA) has been previously described extensively (Maskos et al., 2005;David et al., 2006;Tolu et al., 2013). It is based on choice behavior in a Y-maze discrimination task between a nicotine-reinforced arm and a neutral arm. By interrupting the photocell beam in one of the two target arms, mice could trigger a self-injection of nicotine (100ng, 600 ng or 1200 ng nicotine tartrate in 50 nl of artificial cerebrospinal fluid (aCSF), resulting in a nicotine concentration of 4mM, 24mM or 48mM, respectively); the other arm being neutral (no injection).

Intracranial injections were carried out using an automated computer-controlled All groups were constituted of at least n=6 up to n=10 subjects per group. Only mice with self-injection cannula into the VTA (see Fig. 2 for histological control of stereotaxic implantation) and with appropriate b4 lentiviral re-expression levels were included in results and statistical analysis.

Statistical analyses

Statistical analysis of A8380-resistant I125-epibatidine autoradiography was conducted by unpaired t-test, or one-way ANOVA with Tukey's post hoc test, as appropriate. To analyze the acquisition of nicotine self-administration, a repeated measures ANOVA of the mean number of nicotine self-injections and mean self-injection (and no self-injection) latency were conducted with "genotype" as a between-subjects factor and "session" (or training day) as a within-subjects repeated measure, using StatView 5.0 (Abacus Concepts, Piscataway, NJ, USA). Significant effects were further analyzed using post hoc Newman-Keuls test. Student t tests were carried out to compare the differential of the number of self-administrations at 0 (ACSF), 100, 600 and 1200 ng/50 nl of nicotine. A significance level of p<0.05 was used for all statistical analyses.

Nicotine ICSA response

VTA GFP-vectorized WT and KO mice exhibited behavior profiles similar to the non-vectorized WT and KO mice. WT-GFP VTA and KO-GFP VTA displayed a weak preference for the nicotine-reinforced arm at the dose of 600 ng/50 nl (Fig. 5B). When nicotine dose was increase to 1200 ng/50 nl, WT-GFP VTA progressively switched from the nicotine-reinforced arm to the neutral arm, as shown by the steady decrease in selfadministrations below the chance level. In contrast, KO-GFP VTA mice increased their preference for the nicotine-associated arm. Interestingly, KO-β4 VTA exhibited a rapid and strong increase in the number of nicotine self-administrations starting at the dose of 600 ng/50 nl, which remain significantly higher than WT-GFP VTA and KO-GFP VTA mice over sessions using the dose of 1200 ng/50 nl. These observations are supported by a twoway ANOVA conducted for the 600 ng/50 nl dose (genotype effect: F(2,21)=3.79 p<0.05; session effect: F(6,126)=5.38 p<0.0001; genotype x session interaction: F(12,126)=1.65 ns; post hoc WT-GFP VTA vs. KO-β4 VTA , p<0.05), and for the 1200 ng/50 nl dose (genotype effect: F(2,21)=24,76, p<0.0001; session effect: F(6,126)=0,35, p>0.05; genotype x session interaction: F(12,126)=4,69, p<0.001; post hoc WT-GFP VTA vs. KO-GFP VTA , p<0,001; WT-GFP VTA vs. KO-β4 VTA , p<0.001; KO-GFP VTA vs. KO-β4 VTA , p<0.05).

Self-injection latency decreased similarly in the three groups of mice over sessions at 600 ng/50 nl (ANOVA, session effect: F(6,126)=12.79 p<0.001; fig. 5C). At 1200 ng/50 nl, this parameter remained stable (ANOVA, session effect: F(6,126)=1,52, p>0.05). However, self-injection latency increased in the WT-GFP VTA mice over sessions at this dose (post hoc WT-GFP VTA vs. KO-β4 VTA , p<0.05).

Combination of a strong preference for the nicotine-reinforced arm and short selfinjection latencies in KO-β4 VTA mice suggests that specific re-expression of the β4 subunit within the VTA maintains a high motivational value of these nicotine doses.

Lentivirus-mediated re-expression of b4 in the MHb decreases nicotine ICSA

We next investigated nicotine ICSA with high doses of nicotine when the expression of β4* nAChRs was rescued at the MHb. Two groups of mice were injected with the control lentivirus (WT-GFP MHb , n=6 and KO-GFP MHb , n=8), and one group received the β4-expressing lentivirus (KO-β4 MHb , n=12) into the MHb.

Lentiviral re-expression of β4 in the MHb

Stereotaxically targeting the MHb is technically challenging considering their small size and the fact that they flank the dorsal third ventricle. The levels of β4* nAChR reexpression were therefore systematically verified amongst mice that had completed the intra-VTA nicotine self-administration test. A85380-resistant I125-epibatidine binding demonstrates that the lentiviral vector successfully increases putative β4*-nAChR binding sites in the MHb (Fig. 6A). After quantification of radiographic intensity, we subcategorized mice as high expressers (autoradiographic intensity >30% of WT-GFP MHb ) and low expressers (autoradiographic intensity <30% of WT-GFP MHb ). The KO-β4 MHb high expressers demonstrated a mean MHb autoradiographic intensity of 53.6% that of WT-GFP MHb controls (+-6.5%SEM), which was statistically greater than the levels found in KO-GFP MHb mice (p<0.0001, Fig. 6A). KO-β4 MHb low expressers demonstrated 14.9% radiographic intensity compared to WT-GFP MHb (+-2.5%), that was not statistically different to KO-GFP MHb (15.0% +-6.5% SEM).

Nicotine ICSA response

At the dose of 600 and 1200 ng/50 nl, expression of GFP at the MHb of did not impact the number of nicotine self-administrations in either WT or KO mice, as compared with the non-vectorized WT and KO or the WT-GFP VTA and KO-GFP VTA mice.

When the dose was raised to 1200 ng/50 nl, performances of WT-GFP MHb mice decreased below the chance level, whereas KO-GFP MHb mice still displayed a strong preference for the nicotine-reinforced arm (Fig. 6B). Interestingly, nicotine ICSA response in KO-β4 MHb was found to depend strictly on the level of β4 re-expression.

Whereas low expressers (n=6) exhibited an ICSA profile similar to KO-GFP MHb animals, KO-β4 MHb high expressers (n=6) closely followed the WT-GFP MHb performance. These observations are supported by a two-way ANOVA conducted with the 600 ng/50 nl dose (genotype effect: F(2,22)=2.3 ns; Session effect: F(6,132)=3.62 p<0,01; Genotype x Session interaction: F(12,132)=1.09 ns). At the dose of 1200 ng/50 nl, the ANOVA yielded a Genotype effect: F(2,22)=6.60 p<0,01; Session effect: F(6,132)=1.55 ns; Genotype x Session interaction: F(12,132)=0.85 ns). Post hoc analysis demonstrated that at 1200 ng/50 nl, KO-β4 MHb high expressers self-injected as much nicotine as WT-GFP MHb mice, whereas KO-β4 MHb low expressers self-injected as much nicotine as KO-GFP MHb mice (WT-GFP MHb vs. KO-GFP MHb , p<0,01; WT-GFP MHb vs. KO-β4 MHb low, p<0,01; KO-GFP MHb vs. KO-β4 MHb high, p<0,01; KO-β4 MHb high vs. KO-β4 MHb low, p<0.05).

Self-injection latency decreased over sessions in all animals at the dose of 600 ng/50 nl (Session effect: F(6,132)=14.4 p<0.001; fig. 6C). At 1200 ng/50 nl, this parameter remained stable (ANOVA, Genotype effect: F(6,132)=3.56 p<0.05). However, latencies of the WT-GFP MHB mice increased significantly as compared to KO-GFP MHb mice (post hoc WT-GFP MHb vs. KO-GFP MHb p<0.01), and of the KO-β4 MHb high (post hoc

Preliminary data 2.4.a Mapping the cholinergic network in the central nervous system

Two transgenic mouse lines available in the team, when crossed together, allowed visualisation of cholinergic neurons (figure 2.4.a). The first line is a ChAT-cre line, expressing the cre recombinase enzyme under the promoter of choline acetyltransferase. The second line is a floxed Tdtomato line, with a CAG promoter upstream of a loxP-STOP-loxP site, followed by a tomato fluorophore sequence. In the presence of cre recombinase, the STOP sequence of the Tdtomato's transgene is excised and tomato fluorescence is expressed (see section 2.4.f for explantation of crelox system).

2.4.c Quantification of β4 mRNA expression: quantitative PCR

A real time, quantitative polymerase chain reaction (q-PCR) was conducted in vitro to 1) assess the expression capacity of the PGK-β4 lentivirus construct and 2) to validate the primers for analysis of ⍺3, ⍺5 and β4 mRNA expression in the MHb of the β4 KO mouse.

Human embryonic kidney (HEK) cells were transfected with the PGK-β4 construct, PGK-⍺5 construct (Morel et al. 2014), or a pCS2+ plasmid containing the ⍺3 mouse cDNA sequence. A fourth culture of HEK cells was transfected with all three plasmids, and a fifth culture of HEK cells was submitted to the same transfection procedure, without the addition of the aforementioned plasmids. Transfection was conducted using lipofectamine (Life Technologies) and solution plus reagent (Invitrogen). Cells were returned to their DMEM growth medium 12 hours after transfection. Cells were incubated in a 37°C chamber at 5% CO 2 .

Four days later, cells were liberated from their culture supports and centrifuged to remove the growth medium. RNA extraction proceeded. Cells were mechanically homogenised in Trizol reagent, centrifuged, and phenol chloroform added to the resultant supernatant. The solution was centrifuged to obtain an RNA-containing upper aqueous phase, to which isopropanol was added to remove residual phenol chloroform.

An additional ethanol-cleaning step proceeded. After centrifugation, ethanol was removed and the RNA pellet resuspended in DEPC-treated water. The MHb of seven WT mice and four β4 KO mice were dissected from fresh brains of mice euthanised using a drug-free method (cervical dislocation). The RNA was extracted using the Ambion mirVana RNA extraction kit (Life Technologies) according to manufacturer's instructions. The quality of RNA recuperated, and concentration, were assessed using a Bioanalyser.

RNA was reverse-transcribed (RT) using a Vilo reverse transcription kit, according to manufacturer's instructions (Life Technologies). SYBR Green q-PCR was conducted on the reverse transcription products, ensuring to use equivalent amounts of RT product per sample (estimated from quantity of cDNA in the RT reaction).

Primers were designed and pre-validated in silico. Primers flank cDNA exon boundaries in order to avoid amplification of contaminant genomic DNA. The sequences are as follows. ⍺3 for: CGAGTCCCTGCAGAGAAGAT, rev: CCAGGTCGATCTTTGCCTTG. ⍺5 for: CGTCTGGTTGAAGCAGGAA, rev: GAGAGTCTGAAGGAACACGTATAATTT. β4 for: CGCTACAACAACCTGATCCG, rev: CTCGCTTTGCAGGAATCCTC. Beta-actin was used as a control, with primers kindly provided by Baptiste Libé.

The results of the qPCR reaction are illustrated in figure 2.4.c. fundamental differences are as follows: 1) difference in promoter, 2) loxP-mCherry-STOP-loxP sequence between the promoter and the β4-IRES-eGFP sequence. The promoter used in this system is the platelet-derived growth factor β (PDGF-β) promoter. This has been validated as an appropriate promoter for expression of the viral mCherry and GFP four weeks post-injection in the mouse brain [START_REF] Tolu | A versatile system for the neuronal subtype specific expression of lentiviral vectors[END_REF]. The β4containing viral construct for cre-dependent expression of β4 was synthesised by various sub-cloning steps, as described below (figure 2.4.e.ii). The PDGF-β promoter is barely functional in HEK cells, which poses a barrier to in vitro validation of the lentivirus (data not shown) as classically conducted for PGK promotercontaining lentivirus. In vivo validation was therefore necessary.

The first step in in vivo validation was to verify the expression of mCherry and GFP in cre-expressing mouse lines. For the ease of validation, a mouse line expressing crerecombinase under the dopamine transporter (DAT) promoter (DAT-cre) was employed.

The dopaminergic nucleus, the VTA, of the DAT-cre mouse was stereotaxically targeted, and 2µl of lentivirus delivered bilaterally (50-100ng/µl). Four weeks post injection, the mouse was deeply anaesthetised and perfused intracardially with PBS and 4% PFA.

The brain was dissected and post-fixed in 4% PFA prior to sectioning using a vibratome.

Immunohistofluorescence analysis was conducted on the 60µm sections (figure 2.4.e.iii). This analysis was inconclusive, showing great variability amongst batches of lentivirus. Figure 2.4.e.iii shows no evidence of GFP and tyrosine hydroxylase (dopamine neuron marker) colocalisation, and even shows no cell-specific GFP expression (as determined by observation of morphology).

In vivo validation

The GABAergic IPN was deemed an appropriate brain region to validate the AAVifloxβ4 vector, with a readily available colony of β4 KO GAD-cre +/-mice. 1µl (undiluted, or 1/10 diluted) of the AAVifloxβ4 vector was stereotaxically injected into the IPN of the anaesthetised mouse. 6 weeks post-injection, the three injected mice were euthanised in a drug-free manner (cervical dislocation) and the fresh brains frozen to -80°C.

Radioligand binding was conducted on 20µm coronal brain sections as previously described (e.g. Article One). The 5-iodo-A85380-resistant I 125 -epibatidine autoradiography is low in the IPN of AAVifloxβ4-injected mice (11.8% +-4.7% SEM), and not statistically greater than KO controls (p>0.05, figure 2.4.f.iii).

Corresponding Alexafluor secondary antibodies were used. Out of four mice tested, one was successfully injected bilaterally with the two lentiviruses (figure 2.4.g).

Protocol

Injection sites were visualised by cresyl violet staining of coronal brain sections. 50-60µm sections of PFA-fixed brain or 20µm sections of frozen brain (cannula guide removed prior to dissection) were mounted onto Superfrost microscope slides. The slides were incubated in 0.1% cresyl violet for 3-5 minutes before progressive dehydration in 50-100% ethanol and finally in histoclear (approximately one minute).

VectaMount mounting medium was applied to the dried slides, and a glass coverslip applied. The trace of the cannula and guide is clearly visible as viewed under a light microscope (figure 2.4.i). tracing of the IPN revealed neuronal input from the caudal VTA, of neurons of equivalent morphology to anterior IPN cells [START_REF] Groenewegen | Cytoarchitecture, fiber connections, and some histochemical aspects of the interpeduncular nucleus in the rat[END_REF]. However, the functional implications of the VTA-MHb and VTA-IPN connections have not been explored. It is conceivable that neuronal projections from the VTA, whose activity may be altered by intra-VTA application of nicotine, may modulate the activity of the MHb and the IPN.

Article Two describes a functional impact of the presence, or absence, of β4* nAChRs in the MHb and IPN on intra-VTA nicotine self-administration, restoring aversion when β4* nAChRs are restored in these structures. With a lack of cholinergic neurons from the VTA, it is therefore not plausible that VTA neurotransmission could activate MHb and IPN nAChRs for modulation of the observed aversion phenotype. This leads to hypotheses two and three, based on indirect modulation between these structures. The IPN projects to nuclei known to modulate VTA activity. Namely, the IPN sends GABAergic and serotonergic projections to the dorsal raphe nucleus, laterodorsal tegmentum (LDTg) and pedunculopontine tegmentum (PPTg, see section 1.4.3.a).

These in turn provide critical input to the VTA (Faure et al. 2014;[START_REF] Lammel | Reward and aversion in a heterogeneous midbrain dopamine system[END_REF]. Intravenous nicotine self-administration is reduced by PPTg ablation in IPN functional connections via β4* nAChRs. Future perspectives shall be discussed in a proceeding section.

3.3.a Physiological relevance of β4* nAChRs in the smoker

The present thesis has reviewed the function of β4* nAChR subtypes in in vitro and in vivo rodent models. It is important now to discuss the relevance of these findings in the smoker.

Nicotine typically reaches concentrations of 50-200nM in the smoker's brain, however a recent study showed a concentration range of 6-215ng/ml in cerebrospinal fluid, which is the equivalent of up to 1.3µM nicotine [START_REF] Malkawi | Nicotine exposure can be detected in cerebrospinal fluid of active and passive smokers[END_REF]. Many of the studies cited in the present thesis are conducted using concentrations of nicotine that can be attained in the brains of heavy smokers. MHb neurons are activated by an acute application of 1µM nicotine (Dao et al. 2014), and also show increasing responses over the course of a tenminute application of 81nM (Görlich et al. 2013). β4-dependent nicotine-evoked noradrenaline release occurs at 1µM nicotine in mouse IPN synaptosomes, however ACh release requires 3µM nicotine (Beiranvand et al. 2014). The IPN is also activated by physiologically relevant levels of nicotine, with 0.5-1µM nicotine inducing fura2labelled calcium transients at pre-and post-synaptic sites [START_REF] Girod | Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors[END_REF].

Physiological recordings conducted under acute, millisecond nicotinic stimulation are 2005). This may have important repercussions in smokers, or those under acetylcholine esterase inhibitor treatment, whose nervous system have a prolonged exposure to nicotine or ACh.

The various conformational states of the nAChR exhibit different affinities for agonist.

Notably, the desensitised state has a greater apparent affinity for the agonist than the active state [START_REF] Wang | Desensitized nicotinic receptors in brain[END_REF]. As such, long exposures to low (non-activating) agonist concentrations may lead to the desensitisation of the receptor, and therefore can prevent subsequent activation by high concentrations of the agonist. The nicotine IC 50 values for this inhibitory desensitisation are typically 100-fold lower than the nicotine EC 50 values (Fenster et al. 1997), resulting in a narrow window of concentrations where prolonged exposure to the agonist leads to a significant "smouldering" current (figure 3.3.a). The concentrations of nicotine in the smokers' brains fall within this window, suggesting that there is equilibrium between active and desensitised states that transiently shifts with quantity of nicotine consumed, and delay between smoking.

In the context of human genetic studies

Articles One and Two show that β4 expression in the MHb is rate limiting for the outcome of positive and negative nicotine reinforcement, respectively. This is interesting to consider in the context of the rs1948 (C→T DNA) single nucleotide variant in β4's 3' UTR regulatory domain, which is associated with early initiation of alcohol and tobacco use (Schlaepfer et al. 2008). This sequence's modulatory role on gene expression was modelled in vitro using a dual-luciferase reporter assay, demonstrating that fragments of human CHRNB4 containing the rs1948 risk allele indeed significantly alter gene expression (Gallego et al. 2013). The expression-enhancing or -impairing properties of the single nucleotide variant were dependent on the size of the CHRNB4 3'UTR fragment that was used in the assay. An in vivo investigation of the variant's impact on β4 expression is merited in order to determine whether rs1948 reduces the age of tobacco initiation by increases or decreases in β4 expression. This would allow us to determine whether our observations in KO-β4 MHb mice, who show a KO-like phenotype when β4* nAChR re-expression is below approximately 40% that of WT MHb levels, are coherent with a subpopulation of smokers carrying the rs1948 allele.

A second point of consideration is the gain-of-function missense variants in β4 causing amino acid substitutions at positions 91 (T91I, rs12914008) and 375 (T375I rs61737499). Carriers of these mutations show a reduced risk of nicotine dependence, smoking fewer cigarettes per day than non-carriers, and reduced signs of withdrawal [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF]). One the one hand, this is consistent with the β4 KO mice who selfadminister less nicotine via an intravenous route (Article One), and the reduced nicotine administration may be investigated in the future. More than 40% β4* nAChR restoration is required in the MHb of β4 KO mice in order to observe WT-like nicotine selfadministration (Articles One and Two), and the PDGFfloxβ4 lentivirus does not appear to successfully restore β4* nAChR binding sites in the MHb of β4 KO ChAT-Cre mice (section 2.4.e). It is therefore unlikely that the PDGFfloxβ4 lentivirus will be used for targeting of MHb cholinergic neurons for in vivo studies.