Mu Opioid Receptors and Neuronal Circuits of Addiction: Genetic Approaches in Mice

UNIVERSITÉ DE STRASBOURG

École Doctorale des Sciences de la Vie et de la Santé

IGBMC - CNRS UMR 7104 - Inserm U 964

THÈSE présentée par : Pauline CHARBOGNE

soutenue le : 9 juillet 2015

pour obtenir le grade de : Docteur de l’université de Strasbourg en Aspects Moléculaires et Cellulaires de la Biologie Mention Neurosciences

Mu opioid receptors and neuronal circuits of addiction: genetic approaches in mice

THÈSE dirigée par : Mme KIEFFER Brigitte L. Professeure, Université de Strasbourg/McGill University

RAPPORTEURS : M. BELIN David Maître de conférences, University of Cambridge Mme LE MOINE Catherine Directrice de recherche, Université de Bordeaux M. ZWILLER Jean Directeur de recherche, Université de Strasbourg

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S%]]CVIVJ :`7 V6]V`1IVJ s I8 M: V`1:Cs :JR MV .QRs 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 II8 RVs%C s 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 III8 D1sH%ss1QJ :JR ]V`s]VH 10Vs 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

P:` III7 T:`$V $C% :I: V`$1H `Q`VG`:1J I% `VHV] Q`s 1J :R%C I1HV

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8 T.V I% Q]1Q1R `VHV] Q` 1J $C% :I: V`$1H JV%`QJs 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 C:MKII $VJV 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 8 A1I Q` .V s %R77 :`$V $C% :I: V`$1H `Q`VG`:1J I% `VHV] Q`s 1J :R%C I1HV 888888888888888888888888888888888888888888888888888

II8 M: V`1:Cs :JR IV .QRs 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 III8 RVs%C s 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

8 IJ 010Q C`V :H 101 7 ]: V`J Q` .V :"#$$ RC`VER T IQ%sV C1JV 88888888888888888888888888888888888888888888888888888888888888888888888888 8( QJR1 1QJ:C(@JQH@Q% (Q`( .V(I%(`VHV] Q`(1J( :"#$$ RI% IQ%sV C1JV 88888888888888888888888888888888888888888888888888888888888888888

I8 D1sH%ss1QJ 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

P:` I7 C`V: V : C`V IQ%sV C1JV Q :`$V .V V6 VJRVR :I7$R:C:

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GENERAL DISCSSION 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

GVJV`:C :1I Q` .V .Vs1s 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 PV`s]VH 10V P:` s I II 8888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888 AJ1I:C IQRVCs7 `VCV0:JHV `Q` .%I:J `VsV:`H. 88888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

BIBLIOGRAPH 888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888888

RÉSMÉ EN FRANÇAIS 999999999999999999999999999999999999999999999999998

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GENERAL INTRODCTION

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Neuropharmacology 76 (2014) 204 e217

Contents lists available at ScienceDirect

Neuropharmacology

journal homepage: www.elsevier.com/locate/neuropharm

Invited review 15 years of genetic approaches in vivo for addiction research: Opioid receptor and peptide gene knockout in mouse models of drug abuse

Pauline Charbogne a, b, c, d, Brigitte L. Kieffer a, b, c, d, *, Katia Befort a, b, c, d a IGBMC Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS UMR 7104 e Inserm U964, Illkirch F-67404, France b CNRS, UMR7104, Illkirch F-67404, France c UdS Université de Strasbourg, CNRS UMR 7104 e Inserm U964, Illkirch F-67404, France d Inserm U964, Illkirch F-67404, France article info abstract

Article history: The endogenous opioid system is expressed throughout the brain reinforcement circuitry, and plays a Received 29 March 2013 major role in reward processing, mood control and the development of addiction. This neuromodulator Received in revised form system is composed of three receptors, mu, delta and kappa, interacting with a family of opioid peptides 19 August 2013 derived from POMC ( b-endorphin), preproenkephalin (pEnk) and preprodynorphin (pDyn) precursors. Accepted 23 August 2013 Knockout mice targeting each gene of the opioid system have been created almost two decades ago. Extending classical pharmacology, these mutant mice represent unique tools to tease apart the speci ﬁc Keywords: role of each opioid receptor and peptide in vivo , and a powerful approach to understand how the opioid Opioid receptors Opioid peptides system modulates behavioral effects of drugs of abuse. The present review summarizes these studies, Knockout mice with a focus on major drugs of abuse including morphine/heroin, cannabinoids, psychostimulants, Drugs of abuse nicotine or alcohol. Genetic data, altogether, set the mu receptor as the primary target for morphine and Addiction heroin. In addition, this receptor is essential to mediate rewarding properties of non-opioid drugs of Reward abuse, with a demonstrated implication of b-endorphin for cocaine and nicotine. Delta receptor activity reduces levels of anxiety and depressive-like behaviors, and facilitates morphine-context association. pEnk is involved in these processes and delta/pEnk signaling likely regulates alcohol intake. The kappa receptor mainly interacts with pDyn peptides to limit drug reward, and mediate dysphoric effects of cannabinoids and nicotine. Kappa/dynorphin activity also increases sensitivity to cocaine reward under stressful conditions. The opioid system remains a prime candidate to develop successful therapies in addicted individuals, and understanding opioid-mediated processes at systems level, through emerging genetic and imaging technologies, represents the next challenging goal and a promising avenue in addiction research. This article is part of a Special Issue entitled ‘NIDA 40th Anniversary Issue ’. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Snyder, 1973; Simon et al., 1973; Terenius, 1973 ). Met- and Leu-enkephalins were characterized in 1975, and altogether three families Opiates, including morphine, are potent analgesic compounds of endogenous opioid peptides precursors (pre-proenkephalin and represent major therapeutic drugs to treat severe pain. In pEnk, pre-prodynorphin pDyn and proopiomelanocortin POMC) addition, opiates induce strong euphoria and repeated exposure were identi ﬁed in the late 70 ’s ( Goldstein et al., 1979; Guillemin often leads to dependence and eventually opioid addiction. Mile- et al., 1976; Hughes et al., 1975; Li and Chung, 1976 ). Genes encod- stones in discoveries of the opioid system are shown in Fig. 1 . ing opioid peptide precursors were isolated in the early 80 ’s (pEnk Morphine, the most active component of opium, was isolated in (Comb et al., 1982; Gubler et al., 1982; Noda et al., 1982 ); pDyn 1805 by Serturner. Opioid receptors were described in 1973, based (Kakidani et al., 1982 ); POMC ( Nakanishi et al., 1979 )). The ﬁrst on opioid binding sites referred as mu, delta and kappa ( Pert and opioid receptor gene, encoding delta receptors, isolated by expression cloning in 1992 ( Evans et al., 1992; Kieffer et al., 1992 ), and the two other receptor genes were cloned by homology ( Mestek et al., * Corresponding author. Institut de Génétique et de Biologie Moléculaire et Cel- 1995; Simonin et al., 1994, 1995 ). Opioid receptors belong to the lulaire, Department of Neurobiology and Genetics, 1 Rue Laurent Fries, 67404 Ill- kirch, France. Tel.: þ33 (0) 3 88 65 56 93; fax: þ33 (0) 3 88 65 56 04. superfamily of G-protein coupled receptors ( Kieffer, 1995; Trigo E-mail address: [email protected] (B.L. Kieffer). et al., 2010 ), with coupling to Gi/Go proteins ( Law et al., 2000 ),

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Fig. 1. Milestone discoveries in opioid research . Opium is extracted from poppy seeds ( Papaver somniferum ) and consumed for several thousand years to relieve pain and produce euphoria. Morphine, the most active alkaloid extracted from opium, was the first opioid to be isolated (1805). Opiates act on the nervous system, where they speci fically activate receptors (1973), which are normally stimulated by a family of endogenous neurotransmitters, b-endorphin, enkephalins and dynorphins (1975). Several opioid receptors subtypes were further described based on receptor pharmacology (1976). Gene cloning occurred in early 80 ’s for peptide precursors (1979) and early 90 ’s for opioid receptors (1992). Opioid receptors genes ( Oprm1, Oprd1 and Oprk encoding mu-, delta- and kappa-opioid receptor; pomc , pEnk and pDyn encoding peptide precursors) were targeted in mice by homologous recombination, and mice lacking the mu receptor and enkephalins were available first (1996). Recently, re finement of in vivo targeted mutagenesis techniques led to the first conditional knockout mouse for the opioid system, with a delta receptor deletion restricted to primary afferent nociceptive neurons (2011). The 3D crystal structure of all three receptors was elucidated very recently (2012). OR: opioid receptor, KO: knockout mouse, cKO: conditional knockout mouse. Detailed references are in the text. and their structure was solved at high-resolution by X-ray crystal- opioid system, developed by others and us, have been extensively lography ( Granier et al., 2012; Manglik et al., 2012; Wu et al., 2012 ). studied and broadly shared within our research community. In this The opioid system is broadly expressed in the nervous system, review, we have gathered data from these KO mice that have particularly within the neurocircuitry of addiction ( Koob and accumulated in the past fifteen years (for previous reviews see Volkow, 2010 ). Both peptides and receptors are present in areas Contet et al., 2004; Kieffer and Gaveriaux-Ruff, 2002 ), and enabled associated with reward, motivation, learning and stress ( Le Merrer identi fication or clari fication of the speci fic role of each component et al., 2009; Mansour et al., 1995 ), and therefore play a key role in of the opioid system in drug reward and addiction. Note that the many aspects of addictive behaviors (see Lutz and Kieffer, 2013 ). opioid system plays a central role in pain processing, but this All the known drugs of abuse activate reinforcing brain cir- particular aspect will not be reviewed here (see recent reviews in cuitries ( Koob and Volkow, 2010 ). These drugs, however, recruit Bodnar, 2012; Gaveriaux-Ruff and Kieffer, 2011; Woolf, 2011 ). distinct molecular targets in the brain and show notable differences We will first summarize behavioral responses of null mutant in their pharmacological actions, which has led researchers and mice to opiates, then overview reports investigating the effects of physicians to classify them into distinct groups. Opiates, acting other drugs of abuse, including cannabinoids, psychostimulants directly at opioid receptors, produce sedative effects in addition to (cocaine, MDMA, amphetamine), nicotine and alcohol in these euphoria, and are therefore known as narcotics. In contrast, psy- mice, and finally conclude on the respective roles of opioid peptides chostimulants that include cocaine, amphetamine and metham- and receptors, and perspectives of opioid research in the area of phetamine, provide immediate euphoria with a feeling of drug abuse. Whereas data from receptor KO mice have unambig- intellectual and physical power, and indifference to pain and fa- uously clari fied receptor roles in vivo , data from peptide KO mice tigue, mainly via direct stimulation of dopaminergic transmission. are by essence more complex (low receptor selectivity) and the Nicotine, a major component of tobacco, is also considered a mild latter mutants still deserve further investigations. stimulant and a-nicotinic receptors constitute their molecular target. Relaxing and euphoric sensations searched by marijuana 2. Behavioral measures in the mouse users arise from the stimulation of CB1 receptors by cannabinoids, including the most active component delta9-tetrahydrocannabinol At present, behavioral paradigms to model distinct aspects of (THC). Finally, the most widely abused licit drug is alcohol, target- addiction (for a review see Everitt et al., 2008; Koob et al., 2009 ) in ing several receptors and ion channels in the brain and repre- rodents remain limited, particularly for mice (see Box ). Several senting a major health problem ( Hyman, 2008 ). It is now well well-described behavioral models in rats have nevertheless been established that the endogenous opioid system plays an important successfully adapted to mice, and largely applied to mutant ani- role in acute and chronic effects of all these drugs. The exact nature mals. Among these, voluntary/operant testing (two-bottle choice, of opioid receptor or peptide involved has been clari fied over the TBC and self-administration, SA) addresses some aspects of binge years, largely owing to genetic approaches, and this large set of data intoxication and/or excessive consumption, and conditioned place is overviewed here. preference (CPP) examines drug reward. Withdrawal and the Drug abuse is a major threat to public health ( Compton et al., negative effect of drug abstinence can be revealed by conditioned 2007; Gustavsson et al., 2011 ). For 40 years, NIDA has supported place aversion (CPA) and drug-induced physical withdrawal, and extensive research towards understanding molecular bases of drug preoccupation/anticipation can be tested by drug-, cue- or stress- abuse ( Everitt et al., 2008; Nestler, 2005; Pierce and Wolf, 2013 ), induced reinstatement of CPP. Finally locomotor activation by and developing innovative strategies for treatment ( Heilig et al., drugs of abuse, and sensitization to this effect upon repeated 2011; Kalivas and Volkow, 2011; Koob et al., 2009; Pierce et al., treatment, are also typical responses studied in rodents although 2012; Volkow and Skolnick, 2012 ). We are extremely grateful no human correlate exists for this behavior. Data from all these tests to NIDA for long-standing support to our efforts in developing ge- are summarized in Tables 1 e6, and main findings are summarized netic mouse models for opioid research. Knockout (KO) mice for the below.

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Box (Matthes et al., 1996; Nguyen et al., 2012a, 2012b; Sora et al., 2001 ) Behavioral measures in the mouse. and heroin ( Contarino et al., 2002 ) CPPs were abolished in mu KO mice at all the tested doses. Intravenous as well as intra-VTA in- fusions of the drug observed in wild type animals were also abol- Behavioral responses examined in mutant mice ( Tables 1 ished in mutants ( Sora et al., 2001; David et al., 2008 ). In another e6) are briefly explained below. study, mu KO mice self-administered morphine at levels lower than Conditioned place preference (CPP) or aversion (CPA) : control mice self-administering saline, perhaps unmasking a pavlovian conditioning based on capacity of the animal to kappa/dynorphin-mediated aversive state in these mutants ( Becker associate the drug effect with the context. If the drug has et al., 2000 ). Locomotor responses to morphine ( Tian et al., 1997; rewarding effects, mice explore the drug-paired compart- Sora et al., 2001; Chefer et al., 2003; Yoo et al., 2003, 2006; ment more than the vehicle-paired compartment, and thus Becker et al., 2000 ) and heroin administration ( Contarino et al., show a conditioned place preference (CPP). If the drug is aversive mice avoid the drug-paired box (Conditioned place 2002 ) were eliminated in mu KO animals (see Table 6 ). Together aversion or CPA). Reinstatement can be measured after a all the data demonstrate that mu receptors indeed represent the CPP paradigm: drug priming or stress can reinstate prefer- primary in vivo molecular target for both most clinically useful ence for the initially drug-paired box after extinction. This (morphine) and most largely abused (heroin) opiates. test models drug-seeking behavior ( Tzschentke, 2007 ). The role of delta receptor in reward is debated. Delta KO mice Self-administration (SA) : operant paradigms model several developed a place preference when morphine was paired with the elements of human drug consumption, and are therefore initially non-preferred compartment, but failed to do so when largely used in rodents. Drug SA in mice (except oral SA), paired to the preferred side of the apparatus ( Chefer and however, is technically difficult, and studies remain scarce. Shippenberg, 2009 ). The authors interpreted this result as a ceil- In drug SA models, the animal works to obtain the drug and ing effect in the biased CPP protocol that was used more than a learns an action/outcome association. Various aspects are decrease of rewarding properties of morphine. In another study, investigated: acquisition (under fixed ratio schedule); using unbiased CPP, delta KO animals did not develop place pref- motivation (under progressive ratio schedule and determi- erence to morphine ( Le Merrer et al., 2011 ). In the same study, nation of a breaking point, corresponding to the highest mutant mice showed impaired place conditioning to lithium, an response possible for a single delivery); extinction (response rate after end of drug-delivery); reinstatement (as aversive stimulus, and showed normal motivation to obtain for CPP). In addition to rewarding effects of the drug, this morphine in a SA paradigm ( Le Merrer et al., 2011 ). Together with a model enables investigation of motivational aspects of drug previous study showing intact intra-VTA SA in delta KO mice ( David intake ( Sanchis-Segura and Spanagel, 2006 ). et al., 2008 ), the data concur to indicate that morphine reward and motivation to obtain the drug are intact in these animals, however Two-bottle choice : In this test, mostly used for measuring alcohol consumption, the animal has access to a water- drug-context association is impaired. A subsequent study showed containing bottle and an alcohol-containing bottle. This that internal or external non-spatial cues (circadian, drug, auditory) access is either continuous (24 h/day) or intermittent (few predicting drug or food reward restored morphine CPP in delta KO hours a day or few days a week). The latter closely mimics mice, suggesting that only contextual learning is impaired in these binge drinking and can be used as a model of relapse by mice ( Le Merrer et al., 2012 ). Considering locomotor effects, the including phases of deprivation ( Crabbe et al., 2011 ). stimulant effect of acute morphine was unchanged in delta KO mice Locomotor effects and sensitization : Many drugs of (Chefer et al., 2003 ). However, sensitization or tolerance to this ef- abuse increase locomotor activity after acute treatment. fect, observed upon distinct regimen of chronic morphine admin- Repeated administration of the drug, classically increases istration, was enhanced and reduced respectively ( Chefer and this locomotor response, a phenomenon referred to as Shippenberg, 2009 ), indicating a role for delta receptors in these sensitization that may reflect the transition from voluntary adaptive responses to chronic morphine. Otherwise, physical intake to compulsive use ( Robinson and Berridge, 2008; dependence was unchanged in delta KO mice ( Nitsche et al., 2002 ). Vanderschuren and Pierce, 2010 ), or vulnerability to drug In conclusion, the delta receptor does not directly mediate addiction or drug-induced psychosis in humans ( Loweth morphine reward and likely facilitates contextual learning. Also, as and Vezina, 2011 ). many other systems, this receptor contributes to chronic morphine- Withdrawal : Chronic drug administration produces physical induced neuroplasticity. Mechanisms underlying a potential cross dependence, which is revealed after cessation of drug talk between delta receptor activity and mu opioid receptor exposure. Spontaneous withdrawal is difficult to detect and signaling in vivo remain unclear (see Pradhan et al., 2011; Stockton quantify in animals, therefore physical withdrawal is typi- and Devi, 2012 ). cally precipitated by treatment with an antagonist, followed by scoring of withdrawal signs. The latter vary with the drug b-endorphin KO animals compared with wild-type controls (ptosis, teeth chattering, tremor, paw tremor, wet-dog spent equal ( Niikura et al., 2008 ) or more ( Skoubis et al., 2005 ) time shakes, sniffing, jumping, diarrhea) and a global score is in the drug-paired compartment, depending on the dose and calculated to measure a general dependence index paradigm used. No modi fication of morphine CPP could be detected (Maldonado et al., 1996 ). in proenkephalin (pEnk) KO mice ( Skoubis et al., 2005 ), and physical dependence was either decreased ( Shoblock and Maidment, 2007 ) or enhanced in these mice ( Nitsche et al., 2002 ). These results suggest paradoxical negative modulatory roles for the two 3. Opioid system and opiate drugs endogenous peptides in morphine reward ( bend) and withdrawal (pEnk), or that compensatory mechanisms have developed in Morphine reward and withdrawal data are shown for the six KO knockout animals. lines in Table 1 . Locomotor effects of morphine are presented in Morphine CPP was unchanged in mice lacking the kappa opioid Table 6 together with stimulant effects of other drugs of abuse. receptor ( Simonin et al.,1998 ), as well as dynorphin ( Mizoguchi et al., Genetic studies have de finitely established that the mu opioid re- 2010; Zimmer et al.,2001 ). Prodynorphin KO mice showed unchanged ceptor is required for therapeutic effects as well as unwanted ef- (Mizoguchi et al., 2010; Zimmer et al., 2001 ) or increased hyper- fects of morphine (see Contet et al., 2004 ). Hence, morphine locomotor activity upon morphine administration ( Mizoguchi et al.,

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Table 1 Behavioral effects of morphine and heroin in opioid receptor and peptide knockout mice.

Gene KO Drug of abuse Behavioral test Drug of abuse dose, route Genotype effect Ref

mu Morphine CPP 3 mg/kg, s.c. Abolished Matthes et al., 1996 CPP 10 mg/kg, s.c. Abolished Sora et al., 2001 CPP 10 mg/kg, s.c. Abolished Nguyen et al., 2012a CPP 10 mg/kg, s.c. Abolished Nguyen et al., 2012b þ challenge on d14 5 mg/kg, s.c. Abolished SA 2 or 4 mg/0.2 mL, i.c.v. FR1 Lower than Becker et al., 2000 saline groups SA 0.1 or 0.3 mg/kg/injection, i.v. FR4 Abolished Sora et al., 2001 VTA SA 50 or 100 ng/infusion Abolished David et al., 2008 Withdrawal 20 e100 mg/kg, i.p. (2x/d, 5d) Abolished Matthes et al., 1996 Heroin CPP 1 mg/kg, i.p. Abolished Contarino et al., 2002 delta Morphine CPP preferred side 10 mg/kg, s.c. Abolished Chefer and Shippenberg, 2009 CPP non-preferred side 10 mg/kg, s.c. Unchanged CPP drug free state 5 mg/kg, s.c. Abolished Le Merrer et al., 2011 CPP under morphine 5 mg/kg, s.c. Unchanged CPP without cue 10 mg/kg, s.c. Abolished Le Merrer et al., 2012 CPP with cue 5, 10 and 20 mg/kg, s.c. Unchanged/restored SA 0.25 or 0.5 mg/kg/infusion, i.v. FR1 Unchanged Le Merrer et al., 2011 0.25 mg/kg/infusion, i.v. PR Unchanged 0.5 mg/kg/infusion, i.v. PR Increased VTA SA 50 ng/infusion Unchanged David et al., 2008 Withdrawal 75 mg, pellet (3d) Unchanged Nitsche et al., 2002 kappa Morphine CPP 1 mg/kg, s.c. Unchanged Simonin et al., 1998 Withdrawal 20 e100 mg/kg, i.p. (2x/d, 6d) Abolished Simonin et al., 1998 bend Morphine CPP 10 mg/kg, s.c. Increased Skoubis et al., 2005 CPP 5 mg/kg, s.c. Unchanged Niikura et al., 2008 pEnk Morphine CPP 10 mg/kg, s.c. Unchanged Skoubis et al., 2005 Withdrawal 75 mg, pellet (3d) Increased Nitsche et al., 2002 Withdrawal jumping 20 mg/kg, s.c. (1 injection) Decreased Shoblock and Maidment, 2007 100 mg/kg, s.c. (2d) Abolished pDyn Morphine CPP 5 mg/kg, s.c. Unchanged Zimmer et al., 2001 CPP 3.5 mg/kg, s.c. Unchanged Mizoguchi et al., 2010 Withdrawal 20 e100 mg/kg, i.p. (2x/d, 5d) Unchanged Zimmer et al., 2001

2010 ), suggesting that dynorphin opposes mu receptor signaling for in agreement with pharmacological studies suggesting protective role the control of locomotor effects. Several signs of naloxone-induced of kappa receptor blockade in morphine dependence ( Wee and Koob, withdrawal were decreased in morphine-dependent kappa KO mice 2010 ). Involvement of this anti-reward system ( Koob and Le Moal, (Simonin et al., 1998 ), an effect that could not be observed in pDyn 2008 ) is overall better detected in knockout mice under conditions mutants ( Zimmer et al., 2001 ). A tonic role for the kappa/dynorphin of stress ( Bruchas et al., 2010 ) and in response to non-opioid drugs of system is therefore detected in dependent animals, at receptor level, abuse (see below).

Table 2 Behavioral effects of cannabinoid in opioid receptor and peptide knockout mice.

Gene KO Drug of abuse Behavioral test Drug of abuse dose, route Genotype effect Ref

mu THC CPP 1 mg/kg, i.p. Abolished Ghozland et al., 2002 CPA 5 mg/kg, i.p. Decreased Ghozland et al., 2002 Withdrawal 10 mg/kg, s.c. (5d) Unchanged Lichtman et al., 2001 30 or 100 mg/kg, s.c. (5d) Decreased Withdrawal 20 mg/kg, i.p. (2x/d, 6d) Unchanged Ghozland et al., 2002 delta THC CPP 1 mg/kg, i.p. Unchanged Ghozland et al., 2002 CPA 5 mg/kg, i.p. Unchanged Ghozland et al., 2002 Withdrawal 20 mg/kg, i.p. (2x/d, 6d) Unchanged Ghozland et al., 2002 mu delta THC CPP 1 mg/kg, i.p. Decreased Castane et al., 2003 Withdrawal 20 mg/kg, i.p. (2x/d, 6d) Decreased Castane et al., 2003 kappa THC CPP 1 mg/kg, i.p. Unchanged Ghozland et al., 2002 CPP without priming 1 mg/kg, i.p. Present, absent in WT CPA 5 mg/kg, i.p. Abolished Ghozland et al., 2002 Withdrawal 20 mg/kg, i.p. (2x/d, 6d) Unchanged Ghozland et al., 2002 pEnk THC Withdrawal 20 mg/kg, i.p. (2x/d, 6d) Decreased Valverde et al., 2000 pDyn THC CPA 5 mg/kg, i.p. Abolished Zimmer et al., 2001 Withdrawal 20 mg/kg, i.p. (2x/d, 6d) Decreased (trend) Zimmer et al., 2001 WIN SA 6.25 mg/kg/infusion, i.v. FR1 Increased Mendizabal et al., 2006 12.5 mg/kg/infusion, i.v. FR1 Abolished

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Table 3 Behavioral effects of psychostimulant in opioid receptor and peptide knockout mice.

Gene KO Drug of abuse Behavioral test Drug of abuse dose, route Genotype effect Ref

mu Cocaine CPP 5 e10 mg/kg i.p. Rightward shift Becker et al., 2002 CPP 10 mg/kg, i.p. Unchanged Contarino et al., 2002 CPP 5 mg/kg, s.c. Unchanged Hall et al., 2004 10 mg/kg, s.c. Decreased CPP 30 mg/kg, i.p. Unchanged Nguyen et al., 2012a SA 0.4, 0.8 or 1.6 mg/inf, i.v. FR1 Decreased Mathon et al., 2005 MDMA CPP 10 mg/kg, i.p. Unchanged Robledo et al., 2004 Amphetamine CPP 1 mg/kg, i.p. Unchanged Marquez et al., 2007 kappa Cocaine CPP Æ forced swim stress 15 mg/kg, s.c. Unchanged/no effect of stress McLaughlin et al., 2006a CPP 15 mg/kg, s.c. Unchanged Redila and Chavkin, 2008 Stress-induced reinstatement Abolished Cocaine prime test 15 mg/kg, s.c. Unchanged bend Cocaine CPP 30 e60 mg/kg i.p. Rightward shift Marquez et al., 2008 CPP 30 mg/kg, i.p. Abolished Nguyen et al., 2012a pDyn Cocaine CPP Æ forced swim stress 15 mg/kg, s.c. Unchanged/no effect of stress McLaughlin et al., 2003 CPP þ social defeat stress 15 mg/kg, s.c. Decreased McLaughlin et al., 2006b CPP 15 mg/kg, s.c. Unchanged Redila and Chavkin, 2008 Stress-induced reinstatement Abolished Cocaine prime test 15 mg/kg, s.c. Decreased

4. Opioid system and cannabinoids typically observed at a high dose of THC in wild-type mice, was abolished in both pDyn ( Zimmer et al., 2001 ) and kappa KO mice Both pharmacological studies and genetic approaches provide (Ghozland et al., 2002 ). The latter observations indicate that the considerable evidence suggesting that cannabinoid and opioid kappa/dynorphin system mediates aversive effects of THC, another systems interact bi-directionally to regulate both neurochemical facet of cannabinoid effects. This was further supported by facili- effects of drug and behavioral responses ( Trigo et al., 2010; Vigano tated self-administration of WIN, a cannabinoid agonist, in pDyn et al., 2005 ). Although mechanisms underlying functional in- KO mice ( Mendizabal et al., 2006 ). It has long been established that teractions remain unclear, receptors from the two systems show mu and kappa receptors oppositely regulate hedonic homeostasis overlapping distribution in various brain structures, and potential (Spanagel et al., 1992 ) and it is therefore possible that the same heterodimer formation between CB1 and mu opioid receptors has opposing activities of the two opioid receptors mediate the well- been suggested from in vitro studies ( Maldonado et al., 2011; known dual euphoric/aversive effects of cannabinoids. Notably, the Solinas et al., 2008 ). Data summarizing cannabinoid effects in KO delta receptor does not seem involved in all these THC effects, at mice for the opioid system are shown in Table 2 . least from knockout mice analysis ( Ghozland et al., 2002 ). THC-induced CPP was unchanged in delta or kappa KO mice THC withdrawal upon chronic THC treatment was reduced in (Ghozland et al., 2002 ), but was abolished in mu KO mutants pEnk KO mice ( Valverde et al., 2000 ) and double mu edelta KO mice (Ghozland et al., 2002 ) and the double mu edelta KO line ( Castane (Castane et al., 2003 ). Reduced THC withdrawal was also detected et al., 2003 ), suggesting that mu receptors mediate rewarding in mu KO animals, at high doses of THC ( Lichtman et al., 2001 ). properties of THC. Interestingly conditioned place aversion (CPA), Single mutants for pDyn ( Zimmer et al., 2001 ), mu, delta or kappa

Table 4 Behavioral effects of nicotine in opioid receptor and peptide knockout mice.

Gene KO Behavioral test Drug of abuse dose, route Genotype effect Ref

mu CPP 0.5 or 0.7 mg/kg, s.c. Abolished Berrendero et al., 2002 CPP 1 mg/kg, i.p. Abolished Walters et al., 2005 2 mg/kg, i.p. Unchanged Withdrawal 10 mg/kg/d, minipump (6d) Decreased Berrendero et al., 2002 delta CPP 0.17 mg/kg, s.c. Abolished Berrendero et al., 2012 SA 15 mg/kg/infusion 10d, i.v. FR1 Unchanged Berrendero et al., 2012 30 mg/kg/infusion 10d, i.v. FR1 Decreased 30 mg/kg/infusion, i.v. PR Decreased Withdrawal 8.77 mg/kg/d, minipump (6d) Unchanged Berrendero et al., 2012 bend CPP 0.5 mg/kg, s.c. Abolished Trigo et al., 2009 Withdrawal 10 mg/kg/d, minipump (6d) Unchanged Trigo et al., 2009 pEnk CPP 0.5 mg/kg, s.c. Abolished Berrendero et al., 2005 Withdrawal 25 mg/kg/d, minipump (6d) Decreased Berrendero et al., 2005 pDyn CPP 0.5 mg/kg, s.c. Unchanged Galeote et al., 2009 SA 5.2 e85.5 mg/kg/infusion, i.v. FR1 Leftward shift Galeote et al., 2009 5.2, 10.6, 21.3 or 85.5 mg/kg/infusion, i.v. PR Unchanged 42.7 mg/kg/infusion, i.v. PR Decreased Withdrawal 25 mg/kg/d, minipump (6d) Unchanged Galeote et al., 2009

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Table 5 Alcohol behavioral effects in opioid receptor and peptide knockout mice.

Gene KO Behavioral test Drug of abuse dose, route Genotype effect Ref

mu TBC limited access 10% Unchanged van Rijn and Whistler, 2009 TBC 10% Decreased Becker et al., 2002 TBC 2 e32% Decreased (female) Hall et al., 2001 TBC 10% Decreased Roberts et al., 2000 Oral SA 5 e10% Abolished Oral SA following TBC Abolished CPP 2 or 4 g/kg Unchanged Becker et al., 2002 CPP 2 g/kg Abolished (female) Hall et al., 2001 Withdrawal liquid diet 0.8 e5% Earlier signs Ghozland et al., 2005 delta TBC limited access 10% Increased van Rijn and Whistler, 2009; van Rijn et al., 2010 Oral SA 5 e10% Increased Roberts et al., 2001 TBC following SA 10% Increased kappa TBC limited access 10% Decreased van Rijn and Whistler, 2009 TBC 3 e12% Decreased Kovacs et al., 2005 bend TBC 7% Increased Grisel et al., 1999 TBC þ/À mild foot shock 8% Decreased/no effect of stress Racz et al., 2008 SA 75 mg/kg; 2h session/9d; i.v. FR3 Acquisition in KO but not WT Grahame et al., 1998 Oral SA 3 e6% Unchanged Hayward et al., 2004 Withdrawal forced drinking 16% Unchanged Racz et al., 2008 pEnk TBC 2 e10% Unchanged Koenig and Olive, 2002 TBC 8% Unchanged Racz et al., 2008 TBC þ foot shock Decreased (male) Oral SA 3 e6% Unchanged Hayward et al., 2004 CPP 2 g/kg, i.p. Unchanged Koenig and Olive, 2002 Withdrawal forced drinking 16% Unchanged Racz et al., 2008 pDyn TBC 2 e8% Increased Femenia and Manzanares, 2012 TBC 3 e12% Decreased (female) Blednov et al., 2006 TBC 8% Increased Racz et al., 2012 TBC þ foot shock Prolonged/WT TBC 4 e10% Unchanged Sperling et al., 2010 CPP 2 g/kg, i.p. Increased Femenia and Manzanares, 2012 CPP drug free state 2 g/kg, i.p. Unchanged (female) Nguyen et al., 2012c CPP priming 2 g/kg, i.p. challenge 1 g/kg Increased (female) CPP 2 g/kg, i.p. Unchanged Blednov et al., 2006 Conditioned taste aversion 2.5 g/kg, i.p. Unchanged Withdrawal 4 g/kg, p.o. Increased Femenia and Manzanares, 2012 4 g/kg, i.p. Unchanged Blednov et al., 2006

Data are shown for each knockout (gene KO) mouse line. Behavioral tests are detailed in the Box . Unchanged: no genotype effect; increased: KO shows higher response compared to wild-type (WT); decreased: KO shows lower response compared to WT; abolished: no response in KO; CPP: Conditioned Place Preference; d: day; SA: self- administration; TBC: two-bottle choice; FR: ﬁxed ratio. receptors ( Ghozland et al., 2002 ) otherwise showed normal THC Preference for the drug-paired compartment was maintained in withdrawal. The data together suggest that an endogenous enke- both animal models ( McLaughlin et al., 2006a, 2003; Redila and phalinergic tone, acting jointly at mu and delta receptors, con- Chavkin, 2008 ). In presence of stress, cocaine CPP is typically tributes to the development of physical dependence to THC. increased in wild type mice but remained unchanged in kappa and pDyn KO mice (forced-swim stress in McLaughlin et al. (2006a) ; 5. Opioid system and psychostimulants McLaughlin et al. (2003) ; social defeat stress in McLaughlin et al. (2006b) , indicating that the kappa/dynorphin system contributes Multiple studies have pointed out a role for opioid receptors and to the stress-mediated response. Within this line, stress-induced their endogenous ligands in psychostimulant e particularly reinstatement of extinguished cocaine CPP was decreased in cocaine-addiction (for a recent review, see Yoo et al., 2012 , and pDyn KO, although this was not observed in kappa KO mice ( Redila Table 3 ). Cocaine self-administration was dose-dependently and Chavkin, 2008 ). reduced in mu KO mice ( Mathon et al., 2005 ), and cocaine CPP Another well-known effect of psychostimulants is drug-induced was maintained ( Contarino et al., 2002; Hall et al., 2004; Nguyen hyperlocomotion ( Table 6 ). In some reports, the locomotor et al., 2012a ) or decreased ( Hall et al., 2004 ) depending on dose response to cocaine was reduced in mu KO mice ( Chefer et al., and experimental conditions (number of pairings, number and 2004; Yoo et al., 2006, 2003 ) as well as in bend KO mice duration of conditioning sessions). These data indicate that mu (Marquez et al., 2008 ), while in many other mu KO studies, this receptors mediate, at least in part, cocaine reward. A rightward shift cocaine effect was unchanged ( Becker et al., 2002; Chefer et al., of the CPP dose eresponse curve was observed in both mu ( Becker 2004; Contarino et al., 2002; Hall et al., 2004; Lesscher et al., et al., 2002 ) and b-endorphin ( Marquez et al., 2007 ) KO mice, 2005 ). Furthermore, sensitization to locomotor effects of cocaine suggesting decreased cocaine sensitivity in the two lines and a was reduced ( Yoo et al., 2006, 2003 ), maintained ( Lesscher et al., possible implication of mu/ bend signaling in cocaine reinforce- 2005 ), or enhanced ( Hummel et al., 2004 ), depending on the ment. Place preference studies were also conducted in mu KO for mouse genetic background ( Hummel et al., 2004 ) and the pattern amphetamine ( Marquez et al., 2007 ) and MDMA ( Robledo et al., of drug exposure (administration regimen and timing of injections) 2004 ) but no phenotype could be detected. (Allouche et al., 2013; Puig et al., 2012 ). In mu KO mice also, The rewarding properties of cocaine were examined using CPP methamphetamine-induced locomotion, was decreased at one in mice lacking either kappa receptors or preprodynorphin. dose, maintained in lower and higher doses, and no behavioral

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Table 6 Drugs of abuse locomotor effects in opioid receptor and peptide knockout mice.

Gene KO Drug of abuse Locomotor stimulation Drug of abuse dose, Genotype effect Ref route

mu Morphine Locomotion 2.3 mg/kg, i.p. Abolished Tian et al., 1997 Locomotion 5 or 10 mg/kg, s.c. Decreased/saline Becker et al., 2000 Locomotion 10 mg/kg, s.c. Abolished Sora et al., 2001 Locomotion 10 or 20 mg/kg, s.c. Abolished Chefer et al., 2003 Locomotor sensitization (6d inj) 10 mg/kg, s.c. d1 Abolished Yoo et al., 2003, 2006 locomotion þ challenge on day 12 10 mg/kg, s.c. d12 Abolished Heroin Locomotion 3 mg/kg, i.p. Abolished Contarino et al., 2002 THC Locomotor tolerance (2x/d, 5d) 20 mg/kg, i.p. Unchanged Ghozland et al., 2002 Cocaine Locomotion 20 or 40 mg/kg, i.p. Unchanged Becker et al., 2002 Locomotion 30 mg/kg, i.p. Unchanged Contarino et al., 2002 Locomotion 15 mg/kg, i.p. Abolished Yoo et al., 2003, 2006 Locomotion 10 mg/kg, i.p. Unchanged Chefer et al., 2004 20 mg/kg, i.p. Decreased Locomotion 20 mg/kg, s.c. Unchanged Hall et al., 2004 Locomotion 3, 10, 20, or Unchanged Lesscher et al., 2005 30 mg/kg i.p. Locomotor sensitization (6d inj) 15 mg/kg, i.p. Yoo et al., 2003, 2006 þ challenge on day 12 15 mg/kg, i.p. Decreased Locomotor sensitization (10d inj) 15 mg/kg, i.p. Hummel et al., 2004 þ challenge on day 17 15 mg/kg, i.p. Decreased in 129S6xC57BL/6J Increased in C57BL/6J Locomotor sensitization (5d inj) 20 mg/kg, s.c. Unchanged Hall et al., 2004 Locomotor sensitization (11d inj) 20 mg/kg, i.p. Lesscher et al., 2005 þ challenge on day 14 10 mg/kg, i.p. Unchanged Methamphetamine Locomotion 1.25 mg/kg, i.p. Unchanged Shen et al., 2010 2.5 mg/kg, i.p. Decreased 10 mg/kg, i.p. Unchanged Locomotor sensitization (7d inj) 0.62 mg/kg, i.p. Abolished Nicotine Locomotion 0.7, 1 or 3 mg/kg, s.c. Unchanged Berrendero et al., 2002 Locomotor sensitization (2x/d, 7d) 0.05 mg/kg, s.c. d1 No effect Yoo et al., 2004 (WT and KO) 0.05 mg/kg, s.c. d7 Abolished þ challenge on day 11 0.05 mg/kg, s.c. Abolished Locomotor sensitization (2x/d, 7d) 0.05 mg/kg, s.c. d1 No effect Yoo et al., 2005 (WT and KO) 0.05 mg/kg, s.c. d7 Abolished Alcohol Locomotion 0.75, 1.25 or 1.75 g/kg, i.p. Abolished Ghozland et al., 2005 Locomotion 0.5 or 1.2 g/kg, i.p. Decreased (trend) Hall et al., 2001 delta Morphine Locomotion 10 or 20 mg/kg, s.c. Unchanged Chefer et al., 2003 Locomotor sensitization (5d inj) 20 mg/kg, s.c. Unchanged, faster Chefer and Shippenberg, 2009 Challenge on day þ7 5 mg/kg, s.c. Increased Challenge on day þ33 5 mg/kg, s.c. Unchanged Locomotor tolerance (3d) 25 mg pellet, s.c. Decreased THC Locomotor tolerance (2x/d, 5d) 20 mg/kg, i.p. Unchanged Ghozland et al., 2002 Cocaine Locomotion 10 mg/kg, i.p. Increased Chefer et al., 2004 20 mg/kg, i.p. Unchanged Nicotine Locomotion 0.35, 1.05 or Unchanged Berrendero et al., 2012 2.10 mg/kg, s.c. mu delta THC Locomotion 20 mg/kg, i.p. Unchanged Castane et al., 2003 Cocaine Locomotion 5 or 15 mg/kg, i.p. Unchanged Chefer et al., 2005 10 mg/kg, i.p. Increased Locomotor sensitization (5d inj) 15 mg/kg, i.p. d1 Increased locomotion þ challenge on day 8 15 mg/kg, i.p. d8 Abolished kappa THC Locomotor tolerance (2x/d, 5d) 20 mg/kg, i.p. Decreased Ghozland et al., 2002 Cocaine Locomotion 5 or 15 mg/kg, i.p. Unchanged Chefer et al., 2005 10 mg/kg, i.p. Increased Locomotor sensitization (5d inj) 15 mg/kg, i.p. d1 Increased locomotion þ challenge on day 8 15 mg/kg, i.p. d8 Abolished bend Cocaine Locomotion 15, 30, or 60 mg/kg, i.p. Decreased Marquez et al., 2008 Nicotine Locomotion (horizontal) 1 or 3 mg/kg, s.c. Unchanged Trigo et al., 2009 Locomotion (vertical) 1 mg/kg, s.c. Increased 3 mg/kg, s.c. Unchanged Alcohol Locomotor sensitization (12d inj) 2 g/kg, i.p. Sharpe and Low, 2009 þ challenge on day 13 or 14 1.2 g/kg, i.p. Unchanged pEnk THC Locomotion 20 mg/kg, i.p. Unchanged Valverde et al., 2000 Nicotine Locomotion 1, 3 or 6 mg/kg, s.c. Unchanged Berrendero et al., 2005

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Table 6 (continued )

Gene KO Drug of abuse Locomotor stimulation Drug of abuse dose, Genotype effect Ref route

pDyn Morphine Locomotion 5 mg/kg, s.c. Unchanged Zimmer et al., 2001 Locomotion 4.2 mg/kg, s.c. Increased Mizoguchi et al., 2010 5 mg/kg, s.c. Unchanged THC Locomotion 20 mg/kg, i.p. Unchanged Zimmer et al., 2001 Cocaine Locomotion 10 or 15 mg/kg, i.p. Decreased Chefer and Shippenberg, 2006 Locomotor sensitization (14d inj) 15 mg/kg, i.p. d1 Unchanged Bailey et al., 2007 15 mg/kg, i.p. d3, 7 and 14 Increased Nicotine Locomotion 1, 3 or 6 mg/kg, s.c. Unchanged Galeote et al., 2009 Alcohol Locomotion 2 g/kg, i.p. Unchanged Nguyen et al., 2012c

Data are shown for each knockout (gene KO) mouse line. Measures of locomotor stimulation and sensitization are detailed in the Box . Unchanged: no genotype effect; increased: KO shows higher response compared to wild-type (WT); decreased: KO shows lower response compared to WT; abolished: no response in KO; d: day; inj: injection. sensitization was found ( Shen et al., 2010 ), therefore altogether, receptor also contributes to nicotine-induced locomotor sensitiza- evidence exists that mu receptor activity contributes to locomotor tion ( Yoo et al., 2005, 2004 ), see Table 6 . effects of cocaine, and the adaptive response to repeated exposure to the drug. 7. Opioid system and alcohol Cocaine-induced locomotion was also investigated in delta KO mice, showing an increased response to cocaine in these mutant Alcohol produces euphoria, among many other effects, and acts animals ( Chefer et al., 2004 ). Locomotion stimulation upon cocaine on several molecular targets in the brain. A recent analysis of 37 KO administration was maintained or increased ( Chefer et al., 2005 ) in mouse lines has provided evidence that alcohol consumption is kappa KO animals depending on the dose, and maintained ( Bailey controlled by multiple physiological systems ( Blednov et al., 2012 ). et al., 2007 ) or decreased ( Chefer and Shippenberg, 2006 ) in Among these, endogenous opioids represent an important neuro- pDyn KO mice, indicating contrasting effects of the kappa/dynor- biological component of alcohol intake and dependence phin system in this response. Similarly, locomotor sensitization was (Gianoulakis, 2009; Koob et al., 2003 ). Extensive research has abolished in kappa KO mice ( Chefer et al., 2005 ), and increased in implicated endogenous opioid peptide release in alcohol consump- pDyn KO animals ( Bailey et al., 2007 ), suggesting a dissociation of tion, and naltrexone, a general opioid antagonist, showed some ef- kappa receptors and dynorphins in the locomotor stimulant effect ficacy in the treatment of alcoholism ( Koob et al., 2009 ). Knockout of cocaine. mice have provided key insights into opioid mechanisms underlying alcohol-related behaviors (see Table 5 ). Mice lacking mu opioid re- 6. Opioid system and nicotine ceptors did not self-administer alcohol under several conditions, including oral self-administration and the two-bottle choice, and did Among psychostimulants, nicotine is the primary component not display conditioned place preference to alcohol ( Becker et al., of tobacco that maintains smoking habits. The drug acts as a 2002; Hall et al., 2001; Roberts et al., 2000 ), demonstrating that nicotinic acetylcholine receptor agonist to produce relaxation mu receptors are essential to consumption and motivation for and enhanced cognitive performance, and is strongly addictive. alcohol. mu receptor also plays a role in alcohol withdrawal as the Pharmacological and genetic studies have provided evidence for absence of mu receptor accelerated the progression of physical signs a critical role for the opioid system in nicotine addiction (for of withdrawal ( Ghozland et al., 2005 ). Finally, no locomotor stimu- recent reviews, see Berrendero et al., 2010; Drews and Zimmer, lation was observed following alcohol administration in mu KO mice 2010; Hadjiconstantinou and Neff, 2011; Tuesta et al., 2011 ), and (Ghozland et al., 2005 and Table 6 ), and altogether data show a knockout studies addressing nicotine reward and withdrawal are prominent role of mu receptors in many aspects of alcohol effects. summarized in Table 4 . Opposing mu receptor mutants, delta KO mice showed Rewarding properties of nicotine were altered in pEnk, bend, mu increased alcohol consumption in TBC ( Roberts et al., 2001; van Rijn and delta KO mice, as shown by decreased nicotine CPP in these et al., 2010; van Rijn and Whistler, 2009 ) and oral SA combined with mutant mice ( Berrendero et al., 2002, 2005, 2012; Trigo et al., 2009; TBC ( Roberts et al., 2001 ) paradigms and their innate anxiety Walters et al., 2005 ). In agreement, enhanced extracellular dopa- returned to wild-type levels after alcohol SA ( Roberts et al., 2001 ). mine induced by nicotine in the nucleus accumbens was attenuated Given the important role of delta in reducing emotional responses in mice lacking pEnk ( Berrendero et al., 2005 ) and delta receptors (Filliol et al., 2000 ), increased alcohol intake in these mutants may (Berrendero et al., 2012 ). Also, the acquisition of nicotine SA was re flect a self-medication approach to alleviate high levels of anxiety decreased in delta KO mice ( Berrendero et al., 2012 ), further sub- (for a recent review, see Chu Sin Chung and Kieffer, 2013 ). Inter- stantiating the notion that delta/pEnk receptor signaling contrib- estingly, pEnk KO animals showed intact rewarding effect of alcohol utes to reinforcing properties of nicotine. In contrast, self- and a normal pattern of alcohol consumption ( Koenig and Olive, administration of a low nicotine dose was increased in pDyn KO 2002 ), however alcohol drinking was modi fied in pEnk KO under mice ( Galeote et al., 2009 ) suggesting that, as for THC, dynorphin stressful conditions. The latter observation supports a role for delta/ may contribute to aversive effects of nicotine. It would be inter- pEnk signaling in regulating emotional responses that may impact esting to pursue similar experiments in kappa KO mice to con firm on alcohol consumption. b-endorphin may also be involved since this hypothesis. alcohol intake was reduced ( Racz et al., 2008 ), unchanged mu/pEnk signaling seems involved in nicotine dependence. (Hayward et al., 2004 ) or increased ( Grahame et al., 1998; Grisel Withdrawal signs of chronically nicotine-treated pEnk ( Berrendero et al., 1999 ) in bend KO mice. et al., 2005 ) and mu KO ( Berrendero et al., 2002 ) mice were Paradoxically, mice lacking the kappa receptor showed reduced attenuated, while no difference with wild-type controls was preference and alcohol consumption in TBC paradigms ( Kovacs et al., observed for pDyn ( Galeote et al., 2009 ), delta ( Berrendero et al., 2005; van Rijn and Whistler, 2009 ), which contrast with increased 2012 ) and bend ( Trigo et al., 2009 ) KO mice. Finally, the mu reinforcing effects of other drugs of abuse in these mice. Using similar

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TBC testing, pDyn KO mice showed increased voluntary consumption rewarding effects of non-opioid drugs of abuse, with a demon- (Femenia and Manzanares, 2012; Racz et al., 2012 ) suggesting that strated implication of bend for cocaine and alcohol, whereas the kappa receptor and dynorphins regulate alcohol intake via nicotine or cannabinoid reward has been little explored so far for distinct mechanisms. Alcohol CPP was unchanged ( Blednov et al., the two peptides. 2006; Nguyen et al., 2012c; Sperling et al., 2010 ) or increased (Femenia and Manzanares, 2012 ) in mice lacking pDyn. The latter 8.2. Role of kappa signaling in drug aversion observation is in agreement with the TBC data and the reported aversive-like activity of dynorphin peptides. pDyn KO mice otherwise The important role of kappa/dynorphin in dysphoric effects of showed normal increase in stress-induced alcohol preference ( Racz drugs of abuse has been reviewed recently ( Shippenberg et al., et al., 2012; Sperling et al., 2010 ), but developed stronger with- 2007; Wee and Koob, 2010 ). The set of data summarized here drawal signs after chronic alcohol ( Femenia and Manzanares, 2012 ). supports the notion that kappa receptors mainly interact with As mu receptors therefore, pDyn in ﬂuences several aspects of re- pDyn-derived peptides to limit drug reward and mediate dysphoric sponses to alcohol, and future studies will examine whether kappa/ aspects for some drugs (cannabinoids, nicotine). Moreover, and pDyn signaling indeed operates in alcohol abuse. only under stressful conditions, kappa/dynorphin activity increases sensitivity to cocaine reward. The kappa/dynorphin partnership 8. Discussion and concluding remarks regulating alcohol intake, however, requires further studies.

Knockout studies have highlighted very distinct roles for each 8.3. Role of delta signaling in drug reward component of the opioid system in drug reward and dependence: the mu receptor is a convergent molecular target mediating Data indicate that delta receptor activity reduces levels of anx- rewarding properties of all drugs of abuse, the kappa receptor op- iety and depressive-like behaviors, and that enkephalin is involved poses mu receptor signaling in the control of hedonic homeostasis, in this process ( Chu Sin Chung and Kieffer, 2013; Lutz and Kieffer, and also mediates aversive effects of cannabinoids and nicotine, 2012; Pradhan et al., 2011 ), and it is likely that delta/pEnk and the delta receptor most likely modulates drug consumption signaling also regulates alcohol intake through similar mechanisms. indirectly, by improving emotional states or facilitating drug- context association (see Lutz and Kieffer, 2012, 2013 ). Confronting 8.4. Clinical perspectives data from receptor KO and peptide KO mice is a dif ficult task, since ideally behavioral responses of the six knockout lines should be Many pharmacotherapies to treat addiction have been developed examined in parallel, using the same experimental setting. This was in the past decades, but have often shown modest ef ficacy or acted performed with the three receptor lines for some responses, but on sub-populations of patients ( Potenza et al., 2011; Volkow and was never achieved for the six lines together. Also studies from Skolnick, 2012 ). Clinical studies also showed reduced relapse rate constitutive gene deletions have sometimes yielded results which in patients receiving behavioral therapy (alcohol), and in general are discordant with behavioral pharmacology, often attributed to individual differences, including genetic vulnerability, need be compensatory mechanisms that may develop in genetically modi- considered ( Heilig et al., 2011 ). The question of whether novel opioid fied animals ( Kieffer and Gaveriaux-Ruff, 2002; Portugal and Gould, compounds could lead to more ef ficient treatments is under intense 2008 ). Altogether however, data analysis across the literature al- investigations. Naltrexone, a general opioid antagonist, was the lows identi fication of potential endogenous receptor/peptide sys- first opioid medication with FDA approval to reduce the level or tems operating in drug reinforcement processes, and reveals frequency of drug intake ( Pettinati and Rabinowitz, 2006 ). Metha- differing mechanisms across the distinct classes of drugs of abuse done treatment, targeting mu receptors, was a pioneering substitu- (Figs. 2 and 3). tion approach to treat heroin addiction, and a recent report describing eight compounds effective in the treatment of alcohol 8.1. Role of mu signaling in drug reward (acamprosate, naltrexone), opioid (buprenorphine, methadone, naloxone) and nicotine (nicotine, varenicline, bupropion) addiction, mu receptor is essential for rewarding effects of opiates as well shows that mu receptors remain a prime target in most successful as non-opiate drugs (cannabinoids, psychostimulants and alcohol). treatments for addiction ( Pierce et al., 2012 ). Delta agonists may be Both pEnk and bend ( Roth-Deri et al., 2008 ) are involved in ef ficient to limit disruption of emotional responses in addicted

Fig. 2. Involvement of opioid receptors in drug reward . The scheme summarizes data from receptor KO mice and highlights the role of each receptor in drug reward. The mu opioid receptor mediates rewarding properties of both opioid and non-opioid drugs of abuse. With the exception of nicotine, the delta receptor does not seem involved in drug reward. The kappa receptor mediates dysphoric effects of THC and favors cocaine reward after stress (red lines). The role of delta and kappa receptor in alcohol intake is under investigation (see text). Circles indicate euphoria (red/orange), no effect (white) or dysphoria (blue); n.d: not determined in receptor KO mice.

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Fig. 3. Distinct roles of opioid receptors and peptides in addiction-related effects of drugs of abuse . The upper left scheme summarizes known roles of opioid receptors in brain functions related to hedonic homeostasis and mood (from Lutz and Kieffer, 2012 ). In the ﬁve other panels, we propose mechanisms implicating opioid receptors and/or peptides in addiction liability of each class of drugs of abuse, as inferred from both receptor and peptide knockout mouse data reviewed here. “Reward ” and “drug-context association ” refer to CPP data, “aversive effects ” to CPA data, “motivation for the drug ” to SA experiments, and “dependence ” to scores of physical withdrawal under antagonist treatment. Data from locomotor studies are not included (see summary in Table 6 ). Opiates: peptide KO mice show paradoxical ( b-end/reward, pEnk/withdrawal) or no (pDyn/withdrawal) phenotype. THC: b-end KO mice not tested; cocaine: pEnk KO mice not tested; nicotine: b-end KO mice tested for reward but not withdrawal; alcohol: b-end KO mice show contrasting phenotypes and pEnk show a phenotype under stress. Altogether, data from peptide KO mice, combined with those from receptor KO mice, concur to substantiate involvement of a kappa/dynorphin system in dysphoric states associated to drugs of abuse, although this may not apply to alcohol. Data also suggest a role for mu/ b-end signaling in cocaine and nicotine reward, and implication of delta/pEnk signaling to regulate alcohol intake. individuals ( Lutz and Kieffer, 2012 ). Delta drugs have been developed research has de ﬁnitely established that kappa receptor activity plays to treat chronic pain and depression, and are currently being tested a role in addiction-related behaviors, with a prodepressant-like in the clinic, but their use in indications related to drug abuse has not activity (see review Lutz and Kieffer, 2013 ). Kappa antagonists are been considered, as yet ( Gaveriaux-Ruff and Kieffer, 2011 ). Preclinical therefore promising candidates for pharmacotherapies in stress- and

214 P. Charbogne et al. / Neuropharmacology 76 (2014) 204 e217 addiction-related disorders, and may attenuate compulsive drug neuronal populations in live animals ( Fowler and Kenny, 2012 ). For intake ( Wee and Koob, 2010 ) or speci fic symptoms of depressive example, light-mediated phasic activation of dopaminergic neu- disorders, depending on the administration time point ( Knoll and rons in the VTA produced a place preference in a CPP paradigm ( Tsai Carlezon, 2010 ). Finally, considering the growing evidence of co- et al., 2009 ) and the speci fic light-activation of cholinergic neurons morbidity between addiction and depression, possible improvement from nucleus accumbens reduced cocaine reward ( Witten et al., of addiction therapies may arise from the combination of substitu- 2010 ). The speci fic manipulation of mu, delta or kappa receptor tion treatments (mu agonists such as methadone, or partial agonists expressing neurons will be of great interest towards understanding such as buprenorphine) with kappa antagonists or delta agonists, for neuronal connectivity and plasticity while addiction develops. treating patients with comorbid conditions ( Lutz and Kieffer, 2013 ). Within this line, non-invasive neuroimaging and functional con- Further development of delta and kappa opioid drugs will join nectivity techniques, now developed in small rodents, offer the growing body of studies addressing other targets, such as promises in translational medicine ( Dalley et al., 2009; Jasinska gamma-aminobutyric acid receptors and voltage-gated ion chan- et al., 2013 ), and neuroimaging of opioid receptor and peptide ge- nels. These drugs will likely complete other non-pharmacological netic mutants may provide invaluable information towards un- therapies, including transcranial magnetic stimulation or behav- derstanding the human disease. ioral, cognitive therapies and group therapies considered very effective in long-term treatments ( Addolorato et al., 2012; Volkow 9.3. New animal models and Skolnick, 2012 ). Behavioral testing in mice is limited, however new models have 9. Future directions e addressing the neural circuit by genetic been developed to better characterize several stages of the addic- approaches tion cycle, or protracted abstinence and relapse (for example: Goeldner et al., 2011 ; for reviews see O’Brien and Gardner, 2005; 9.1. Conditional knockout Spanagel, 2003 ) Animal research is expanding in this direction for brain disorders in general ( Ahmed, 2010; Berton et al., 2012; Nestler Conventional knockout approaches have proved valuable to and Hyman, 2010 ). Also, automated multidimensional systems now tease apart respective contributions of opioid receptor and peptides enable recording behavior of mice living in social groups to char- in several aspects of drug abuse. Further important developments acterize novelty-seeking trait, anxiety, impulsivity, compulsivity in addiction research involve investigation of molecular mecha- and motivation, and such systems can be successfully applied to nisms operating at the level of neuronal circuits underlying the study behavioral adaptations to drugs of abuse ( Radwanska and distinct aspects of addiction ( Koob and Volkow, 2010 ). Therefore, Kaczmarek, 2011 ). Also, drosophila or zebra fish are model organ- genetic approaches targeted at speci fic brain sites or neuronal isms that allow rapid genetic screens and are being developed in populations are required ( Fowler and Kenny, 2012; Gaveriaux-Ruff the context of drug abuse ( Kaun et al., 2012; Klee et al., 2012; and Kieffer, 2007; Heldt and Ressler, 2009 ), among which condi- Stewart et al., 2011 ). tional gene knockout using the Cre/loxP system has received great Ultimately, the combination of emerging technologies at mo- attention ( Nagy, 2000 ). In the addiction field, several studies using lecular, circuit and behavioral levels holds enormous potential to this technology have provided invaluable insights into circuit discover novel mechanisms operating at integrated level. The mechanisms of drug reward. Site-speci fic deletion of a4-containing opioid system remains a prime candidate to develop successful nAChR ( McGranahan et al., 2011 ) as well as NMDA receptor NR1 therapies in addicted individuals, and understanding opioid- subunit ( Wang et al., 2010 ) has revealed involvement of NMDA mediated processes at systems levels represents a challenging receptors expressed in dopaminergic neurons in nicotine reward. goal in addiction research. Mice lacking CREB speci fically in the cerebral cortex were tested for cocaine self-administration and showed a role for CREB in medi- Acknowledgments ating cocaine reinforcement in this brain structure ( McPherson et al., 2010 ). A comprehensive analysis of behavioral and auto- We would like to thank Sandra Bour for her help with figure nomic effects of THC in several conditional lines has revealed preparation and Dominique Massotte for critical reading of the implication of the CB1 receptor expressed at the level of forebrain manuscript. This work was supported by CNRS, INSERM, and Uni- glutamatergic neurons (CB1CamKIIa-Cre mice), cortical gluta- versité de Strasbourg. We also thank the Mouse Clinical Institute matergic neurons (CB1NEX-Cre mice) and dopaminergic neurons (ICS, Illkirch, France), the European Union (Grant No. GENADDICT/ (CB1Drd1a-Cre mice), but not GABAergic neurons (CB1Dlx5/6-Cre FP6 005166), and the National Institutes of Health (National Insti- mice) ( Monory et al., 2007 ). Also a conditional knockout tute of Drug Addiction, grant #05010 and National Institute on approach using Pet1-Cre mice, targeting the transcription factor Alcohol Abuse and Alcoholism, grant #16658) for financial support. Lmx1b in developing serotonergic neurons of the hindbrain, showed that central serotonergic neurons modulate supraspinal References pain but are not involved in morphine reward ( Zhao et al., 2007 ). 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ABSTRACT

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INTRODCTION

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METHODS AND MATERIALS

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C:GQ`: Q`7 HQJR1 1QJs 11 . .V VI]V`: %`V I:1J :1JVR : ^ AC :JR .%I1R1 7 : ^Q8 M1HV 1V`V Vs VR R%`1J$ .V `1`s .Q%`s Q` .V R:`@ ].:sV Q` : `V0V`sVR C1$. LR:`@ H7HCV ^C1$. s Q`` : 8 . :JR QJ : 8 ._8 FQ` V6]V`1IVJ s Q` Q]V`:J HQJR1 1QJ1J$ I:1J :1JVR G7 H.QHQC: V5 I1HV 1V`V `QQRRRV]`10VR ^ Q Q` .V 1J1 1:C 1V1$. _ :JR 1: V` 1:s :0:1C:GCV :R C1G1 %I 8 AJ1I:C ]`QHVR%`Vs 1V`V HQJR%H VR 1J s `1H :HHQ`R:JHV 11 . .V $%1RVC1JVs Q` .V E%`Q]V:J CQII%J1 1Vs D1`VH 10V L LEEC `V$%C: 1J$ :J1I:C `VsV:`H. :JR 1V`V :]]`Q0VR G7 .V CQH:C V .1H:C HQII1 VV ^CEEARPRBB5 B:`HVCQJ:5 S]:1J_8

T`V: IVJ s T.V I% :$QJ1s s IQ`].1JV ^.7R`QH.CQ`1RV5 F`:JHQ]1:5 CV]1: S:JQ`15 F`:JHV_ :JR .V`Q1J ^R1:HV 7CIQ`].1JV .7R`QH.CQ`1RV5 @1JRC7 ]`Q01RVR G7 F`:JHQ]1:5 CV]1: S:JQ`15 F`:JHV_5 .V Q]1Q1R :J :$QJ1s J:CQ6QJV ^.7R`QH.CQ`1RV5 S1$I:RACR`1H.5 S LQ%1s5 SA_5 :JR .V H: VH.QC:I1JVR`VCV:s1J$ IQCVH%CV :I].V :I1JV ^DR:I].V :I1JV .VI1s%C`: V5 AR 5 S1$I:RACR`1H.5 S LQ%1s5 SA_ 1V`V %sVR 1J .V ]`VsVJ s %R78 FQ` :J:C$Vs1:5 ].7s1H:C RV]VJRVJHV5 CQHQIQ 1QJ5 H: :CV]s75 :JR HRFQs 1II%JQ`V:H 101 7 Vs s5 HQI]Q%JRs 1V`V 1J `:]V`1 QJV:CC7 ^18]8_ 1J=VH VR8 N:CQ6QJV 1J 11 .R`:1:C ]`QHVR%`V 1:s 1J=VH VR s%GH% :JVQ%sC7 ^s8H8_8 FQ` HQJR1 1QJVR ]C:HV ]`V`V`VJHV ^CPP_ V6]V`1IVJ s5 IQ`].1JV :JR .V`Q1J 1V`V :RI1J1s `: VR s8H8 IJ .V .V`Q1J sVC`R:RI1J1s `: 1QJ ]`QHVR%`V5 .V sQC% 1QJ 1:s RVC10V`VR G7 :J 1J `:0VJQ%s H: .V V` ^1808_8 ACC .V ].:`I:HQCQ$1H:C s%Gs :JHVs 1V`V R1ssQC0VR 1J N:CC 8 Q :JR :RI1J1s V`VR 1J : ILL@$ 0QC%IV8

GVJQ 7]1J$RPCR PCR :J:C7s1s QJ $VJQI1H DNA 1V`V ]V``QÌVR 1J Q`RV` Q $VJQ 7]V .V I1HV `Q` ]`VsVJHV Q` _ C`V `VHQIG1J:sV5 _ CQ6P s1 Vs :JR _ V6H1s1QJ Q` O]Ì 8 PCR 1:s :H.1V0VR QJ DNA ``QI IQ%sV R1$Vs VR R1$1 s:I]CV ^N:CC 8M6 T`1sRHCC IM ]H 8 6 EDTA IM6 SDS 8Q6 ]`Q V1J:sV K ^S1$I:_ I$LIL6 Q0V`J1$. : C_8 T.V C`V PCR `V:H 1QJ 1:s ]V``QÌVR G7 :RR1J$ 8 jL C7s: V Q 8 jL `V:H 1QJ I16 ^ PCR

G%``V` ^S1$I:_6 M$CC ^S1$I:_ 8 IM6 RNTPs 8 IM ^T.V`IQ SH1VJ 1`1H_6 TAQ DNA ]QC7IV`:sV 8 ^S1$I:_6 `Q`1:`R C`V ]`1IV` ^ ; RGAT CGC TGC CAG GAT ATA CGR ; _5 `V0V`sV C`V ]`1IV` ^ ; RCAT CGC CAT CTT CCA GCA GR ; _5 `Q`1:`R I7Qs1J $VJV ]`1IV` ^ ; RTTA CGT CCA TCG TGG ACA GCR ; _5 `V0V`sV I7Qs1J $VJV ]`1IV` ^ ; RTGG GCT GGG TGT TAG CCT TAR ; _ 8 jM_8 PCR `V:H 1QJ 1:s ]V``Q`IVR 11 . VI]V`: %`V H7HC1J$ ]:`:IV V`s HQJs1s 1J$ Q` 1J1 1:C RVJ: %`: 1QJ : C `Q` I1J `QCCQ1VR G7 H7HCVs Q` RVJ: %`: 1QJ : C `Q` I1J5 :JJV:C1J$ : C `Q` I1J5 V6 VJs1QJ : C `Q` I1J5 :JR : `1J:C 1JH%G: 1QJ : C `Q` I1J8

T.V CQ6P s1 Vs PCR `V:H 1QJ 1:s ]V``Q`IVR G7 :RR1J$ 8 jL C7s: V Q 8 jL `V:H 1QJ I16 ^

PCR G%``V` GQT:_ ^P`QIV$:_6 M$CC ^S1$I:_ IM6 RNTPs 8 IM ^T.VÌQ SH1VJ 1`1H_6 TAQ DNA ]QC7IV`:sV 8 ^P`QIV$:_6 `Q`1:`R I% `CQ6VR $VJV ]`1IV` ^ ; RGTT ACT GGA GAA TCC AGG CCA AGCR ; _5 `V0V`sV I% `CQ6VR $VJV ]`1IV` ^ ; RTGC TAG AAC CTG CGG AGC CAC AR ; _ jM_8 PCR `V:H 1QJ 1:s ]V``QÌVR 11 . VI]V`: %`V H7HC1J$ ]:`:IV V`s HQJs1s 1J$ Q` 1J1 1:C RVJ: %`: 1QJ : C `Q` I1J `QCCQ1VR G7 H7HCVs Q` RVJ: %`: 1QJ : C `Q` I1J5 :JJV:C1J$ : C `Q` I1J5 V6 VJs1QJ : C `Q` I1J5 :JR : `1J:C 1JH%G: 1QJ : C `Q` I1J8 T.V I% V6H1s1QJ PCR `V:H 1QJ 1:s ]V``QÌVR G7 :RR1J$ 8 jL C7s: V Q 8 jL `V:H 1QJ I16 ^

PCR G%``V` ^S1$I:_6 M$CC ^S1$I:_ 8 IM6 RNTPs 8 IM ^T.V`IQ SH1VJ 1`1H_6 TAQ DNA ]QC7IV`:sV 8 ^S1$I:_6 `Q`1:`R V6H1s1QJ ]`1IV` ^ ; RACC AGT ACA TGG ACT GGA TGT GCCR ; _5 `V0V`sV V6H1s1QJ ]`1IV` ^ ; RGAG ACA AGG CTC TGA GGA TAG TAA CR ; _5 `Q`1:`R I7Qs1J $VJV ]`1IV` ^ ; RTTA CGT CCA TCG TGG ACA GCR ; _5 `V0V`sV I7Qs1J $VJV ]`1IV` ^ ; RTGG GCT GGG TGT TAG CCT TAR ; _ 8 jM_8 PCR `V:H 1QJ 1:s ]V``Q`IVR 11 . VI]V`: %`V H7HC1J$ ]:`:IV V`s HQJs1s 1J$ Q` 1J1 1:C RVJ: %`: 1QJ : C `Q` I1J `QCCQ1VR G7 H7HCVs Q` RVJ: %`: 1QJ : C `Q` sVH5 :JJV:C1J$ : C `Q` sVH5 V6 VJs1QJ : C `Q` sVH5 :JR : `1J:C 1JH%G: 1QJ : C `Q` I1J8

T1ss%V HQCCVH 1QJ `Q` IRNA :J:C7s1s M1HV 1V`V s:H`1`1HVR G7 HV`01H:C R1sCQH: 1QJ8 B`:1Js 1V`V V6 `:H VR5 `1JsVR 1J HQCR PBS ^].Qs].: VRG%``V`VR s:C1JV sQC% 1QJ5 S1$I:_ :JR RII .1H@ sC1HVs 1V`V H% 11 . : s :1JCVss s VVC HQ`QJ:C G`:1J I: `16 H.1CCVR QJ 1HV ^H:`0:`R :]]:`: %s5 HQCC1s QJ5 MA5 SA_8 D1``V`VJ G`:1J `V$1QJs 1V`V HQCCVH VR ``QI Q I1HV ]V` $VJQ 7]V :JR `V: IVJ :HHQ`R1J$ Q .V s V`VQ :61H : C:s Q` IQ%sV G`:1J ^P:61JQs :JR F`:J@C1J5 _8 T.V H:%R: V ]% :IVJ ^CP%_ 1:s G1C: V`:CC7 ]%JH.VR %s1J$ : RII R1:IV V` 1ss%V HQ`V`6 NAH5 TA5 :I7$R:C:5 C: V`:C .7]Q .:C:I%s ^LH_ 1V`V G1C: V`:CC7 ]%JH.VR 11 . : 8RII 1ss%V HQ`V`6 ]`V``QJ :C HQ` V6 ^PCF_ :JR ]V`1:H_%VR%H:C $`V7 ^PAG_ 1V`V HVJ `:CC7 ]%JH.VR %s1J$ : RII R1:IV V` 1ss%V HQ`V`6 .:GVJ%C: ^HG_ :JR RQ`s:C `:].V J%HCV%s ^DRN_ 1V`V HVJ `:CC7 ]%JH.VR %s1J$ : 8RII R1:IV V` 1ss%V HQ`V`6 :JR .V .1]]QH:I]%s :JR s]1J:C HQ`R 1V`V R1ssVH VR8 S:I]CVs 1V`V 1IIVR1: VC7 ``Q

Q%:J 1 : 10V `V:CR 1IV PCR S:I]CVs 1V`V ]`QHVssVR Q V6 `:H Q :C RNA5 %s1J$ TRI$1:%$IV:%%`VR$11 .$ NDR N:JQD`Q] ^T.V`IQ F1s.V` SH1VJ 1`1H5 1CI1J$ QJ5 SA_ s]VH `Q].Q QIV V`8 RV0V`sV

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3 _ `Q` `1J:JH1:C s%]]Q` 8 This study was funded by the Intramural Programs of National Institute on

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GVJQ 7]V W D:7 F^ 5 _ Y 8 J8s8 F^5_ Y 8 J8s8

HQCV W D:7 F^ 5 _ Y 8 P \ 8 F^5_ Y 8 J8s8

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PART II

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ABSTRACT

M% Q]1Q1R `VHV] Q` @JQH@Q% I1HV Î% KO_ .:0V GVVJ s.Q1J Q `VH:]1 %C: V : `%CC s]VH `%I Q` :% 1s 1HR C1@V GV.:01Q`s5 .Q1V0V` JV%`:C H1`H%1 IVH.:J1sIs %JRV`C71J$ .1s ].VJQ 7]V .:0V JQ GVVJ V6]CQ`VR8 TQ 1RVJ 1`7 I% Q]1Q1R `VHV] Q`s `Vs]QJs1GCV `Q` .V :% 1s 1HRC1@V s7JR`QIV Q` Q :C I% KO I1HV5 1V :`$V VR .V O]Ì $VJV 1J GABAV`$1H `Q`VG`:1J JV%`QJs8 T.V HQJR1 1QJ:C DC6 L RC`V O]Ì `CL`C ^DC6R I%_ I1HV s.Q1VR s `QJ$C7 `VR%HVR `VHV] Q` V6]`Vss1QJ I:1JC7 1J s `1: %I :JR :I7$R:C:5 1J0QC0VR 1J sQH1:C `V1:`R :JR :J61V 78 V .VJ V6:I1JVR sQH1:C s@1CCs :JR :J61V 7RC1@V GV.:01Q`s5 `V]`VsVJ 1J$ I:1J HQ`V :JR sVHQJR:`7 s7I] QIs Q` :% 1sI s]VH `%I R1sQ`RV`s ÂSDs_8 As 1J Q%` ]`V01Q%s `V]Q` 5 sQH1:C 1J V`:H 1QJs 1V`V 1I]:1`VR 1J I% KO I1HV5 G% .V`V 1:s JQ RV`1H1 1J DC6RI% :J1I:Cs8 MQ`VQ0V`5 Q :C KO I1HV s.Q1VR 1JH`V:sVR CV0VCs Q` :J61V 7 1J GQ . I:`GCV G%`71J$ :JR JQ0VC 7Rs%]]`VssVR `VVR1J$ Vs s5 :s s.Q1J ]`V01Q%sC75 .Q1V0V` .1s RV`1H1 1:s :GsVJ 1J HQJR1 1QJ:C DC6RI% I1HV8 IJ :RR1 1QJ5 .V`V 1:s JQ RV VH :GCV ].VJQ 7]V 1J DC6RI% I1HV5 1.V .V` DC6RI% I1HV :JR HQJ `QCs 1V`V `:1sVR sV]:`: VC7 Q` Q$V .V`8 IJ HQJHC%s1QJ5 .V $VJV 1H RVCV 1QJ Q` I% Q]1Q1R `VHV] Q`s V6]`VssVR 1J GABAV`$1H `Q`VG`:1J 1s JQ s%``1H1VJ Q 1JR%HV :J :% 1s 1HRC1@V s7JR`QIV 1J I1HV8

KV71Q`Rs7 HQJR1 1QJ:C $VJV @JQH@Q% 5 I% Q]1Q1R `VHV] Q`5 GABAV`$1H `Q`VG`:1J JV%`QJs5 :% 1sI s]VH `%I R1sQ`RV`5 sQH1:C 1J V`:H 1QJ5 :J61V 7RC1@V GV.:01Q`8

INTRODCTION

T.V :% 1sI s]VH `%I R1sQ`RV` ^ASD_ 1s : JV%`QRV0VCQ]IVJ :C R1sQ`RV` H.:`:H V`1

1J GABAV`$1H JV%`QJs Q` .V `Q`VG`:1J 1JHC%R1J$ s `1: %I :JR :I7$R:C: ^C.:`GQ$JV V :C85 1J ]`V]:`: 1QJ5 P:` I_ :`V 1I]C1H: VR 1J sQIV Q` .V ASDRC1@V ].VJQ 7]Vs QGsV`0VR 1J .V I% KO I1HV8

MATERIALS AND METHODS

AJ1I:Cs E6]V`1IVJ s 1V`V ]V``QÌVR QJ I:CV :JR `VI:CV I1HV :$VR R 1VV@s : .V GV$1JJ1J$ Q` .V s %R78 DC6 L RC`V C1JV 1:s s%HHVss`%CC7 %sVR 1J ]`V01Q%s s %R1Vs Q HQJR1 1QJ:CC7 1J0:C1R: V H:JJ:G1JQ1R CB `VHV] Q`s ^MQJQ`7 V :C85 _ :JR RVC : Q]1Q1R `VHV] Q`s ^C.% S1J C.%J$ V :C85 _8 T.V I% `CQ6VR IQ%sV C1JV ^ O]Ì `CL`C _ .:s GVVJ ]`V01Q%sC7 RVsH`1GVR G7 Q%` $`Q%] ^V1GVC V :C85 _8 B`1V`C75 V6QJs :JR Q` .V I% `VHV] Q` $VJV O]Ì :`V `C:J@VR G7 CQ6P s1 Vs8 O]Ì `CL`C I1HV s.Q1 1J :H I% `VHV] Q` V6]`Vss1QJ ^V1GVC V :C85 _8 TQ $VJV`: V : HQJR1 1QJ:C @JQH@Q% `Q` O]Ì 1J GABAV`$1H `Q`VG`:1J JV%`QJs5 .V DC6 L RC`VRO]Ì RLR IQ%sV C1JV 1:s H`V: VR : .V ICSRIGBMC ÎJs 1 % CC1J1_%V RV C: SQ%`1s R IJs 1 % RV GXJX 1_%V V RV B1QCQ$1V MQCXH%C:1`V V CVCC%C:1`V5 ICC@1`H.5 F`:JHV_ G7 G`VVR1J$ .V DC6 L RC`V I1HV ^QG :1JVR ``QI BV: L% < C:GQ`: Q`75 IJs 1 % V Q` P.7s1QCQ$1H:C C.VI1s `75 JQ.:JJVs G% VJGV`$ J10V`s1 75 GVÌ:J7_ 11 . I% `CQ6VR I1HV8 RVs%C 1J$ C`V ]Qs1 10V ^C`VÛ_5 DC6 L RC`VRO]Ì RLR _ :J1I:Cs :`V H:CCVR DC6RI% :JR C`V JV$: 10V ^C`V^R_5 O]Ì `CL`C _ :`V CQJ `QC8 TQ Vs 1.V .V` CQJ `QC C1 VÌ: Vs .:0V :J 1J`C%VJHV QJ .V RV0VCQ]IVJ Q` ]Q VJ 1:C :% 1s 1HRC1@V ].VJQ 7]V 1J DC6RI% :J1I:Cs5 1V sV]:`: VR $VJQ 7]Vs GV`Q`V ]Qs J: :C R:7 8 NV1 GQ`J I1HV 1V`V $VJQ 7]VR _%1H@C7 :` V` G1` . :JR :ss1$JVR Q : sV]:`: VR ^QJC7 QJV $VJQ 7]V :IQJ$ ]%]s_ Q` I16VR ^.:C` CQJ `QCs :JR .:C` DC6R I%_ $`Q%]8 TQ :0Q1R s `Vss G1:s 1J sV]:`: VR $VJQ 7]Vs $`Q%]s5 1V :CsQ V6H.:J$VR .V I16VR ]%]s H:$Vs8 As : HQJ `QC V6]V`1IVJ s ^BVH@V` _5 1V :CsQ Vs VR I% Q]1Q1R `VHV] Q` @JQH@Q% ^KO_ :JR .V1` HQJ `QC ^J:IVR 11CR 7]V5 T_5 ]`V01Q%sC7 RVsH`1GVR 1J ^M: .Vs V :C85 _8 T.V C: V` I% :J s .:0V : R1``V`VJ $VJV 1H G:H@$`Q%JR ^ Q C BLL JR Q S0P:s_ HQI]:`VR Q DC6RI% :JR CQJ `QCs ^ Q C BLL JR Q S0P:s_8 ACC V6]V`1IVJ s 1V`V H:``1VR Q% :HHQ`R1J$ Q .V `VHQIIVJR: 1QJs Q` .V E%`Q]V:J CQII%J1 1Vs CQ%JH1C D1`VH 10V Q` SV] VIGV` 5 ^R1`VH 10V L LE_8 T.V s %R7 ]`Q QHQCs 1V`V :]]`Q0VR G7 .V CQH:C G1QV .1Hs HQII1 VV ^5QI1 X#R;8 .1_%V#]Q%`#C;:6]X`1IVJ : 1QJ#J" 1 % # CC1J1_%V RV C: SQ%`1s R IJs 1 % RV GXJX 1_%V V RV B1QCQ$1V MQCXH%C:1`V V CVCC%C:1`V5 ICC@1`H.5 F`:JHV_8 E6]V`1IVJ s 1V`V ]V``QÌVR QJ I:CV :JR `VI:CV I1HV LR1VV@ QCR : .V GV$1JJ1J$ Q` .V s %R75 .:G1 %: VR Q .V V6]V`1IVJ :C VJ01`QJIVJ :JR .:JRCVR `Q` R:7s GV`Q`V GV.:01Q`:C Vs 1J$8 E6]V`1IVJ :C `QQI C1$. 1:s sV : C%6 `Q` :CC .V Vs s ^V6HV] 1QJs :`V 1JR1H: VR 1J .V GV.:01Q`:C

IV .QR sVH 1QJ_8 ACC GV.:01Q`:C Vs 1J$ 1:s ]V``Q`IVR 11 . .V QGsV`0V` GC1JR Q .V $VJQ 7]V8 ACC :J1I:Cs 1V`V .Q%sVR 1J : `QQI I:1J :1JVR : ^ C :JR ^ Q .%I1R1 75 11 . : . C1$. RR:`@ H7HCV8 FQQR :JR 1: V` 1V`V :0:1C:GCV :R C1G1 %I 8

GVJQ 7]1J$RPCR PCR :J:C7s1s QJ $VJQI1H DNA 1V`V ]V``QÌVR 1J Q`RV` Q $VJQ 7]V .V I1HV `Q` ]`VsVJHV Q` _ C`V `VHQIG1J:sV5 _ CQ6P s1 Vs :JR _ V6H1s1QJ Q` O]Ì 8 PCR 1:s H:``1VR Q% QJ DNA DNA QG :1JVR ``QI .V HQCCVH VR IQ%sV R1$1 s R1$Vs VR 11 . P`Q V1J:sV K ^N:CC 8M6 T`1sRHCC IM ]H 8 6 EDTA IM6 SDS 8Q6 ]`Q V1J:sV K ^S1$I:_ I$LIL_ Q0V`J1$. : C8 T.V C`V PCR `V:H 1QJ 1:s ]V``QÌVR G7 :RR1J$ 8 jL C7s: V Q 8 jL `V:H 1QJ I16 ^ PCR

PCR G%``V` ^S1$I:_6 M$CC ^S1$I:_ 8 IM6 RNTPs 8 IM ^T.V`IQ SH1VJ 1`1H_6 TAQ DNA ]QC7IV`:sV 8 ^S1$I:_6 `Q`1:`R V6H1s1QJ ]`1IV` ^ ; RACC AGT ACA TGG ACT GGA TGT GCCR; _5 `V0V`sV V6H1s1QJ ]`1IV` ^ ; RGAG ACA AGG CTC TGA GGA TAG TAA CR ; _5 `Q`1:`R I7Qs1J $VJV ]`1IV` ^ ; RTTA CGT CCA TCG TGG ACA GCR ; _5 `V0V`sV I7Qs1J $VJV ]`1IV` ^ ; RTGG GCT GGG TGT TAG CCT TAR ; _ 8 jM_8 PCR `V:H 1QJ 1:s ]V``Q`IVR 11 . VI]V`: %`V H7HC1J$ ]:`:IV V`s HQJs1s 1J$ Q` 1J1 1:C RVJ: %`: 1QJ : C `Q` I1J `QCCQ1VR G7 H7HCVs Q` RVJ: %`: 1QJ : C `Q` sVH5 :JJV:C1J$ : C `Q` sVH5 V6 VJs1QJ : C `Q` sVH5 :JR : `1J:C 1JH%G: 1QJ : C `Q` I1J8

BV.:01Q`:C :ss:7s SQH1:C 1J V`:H 1QJ8 SQH1:C GV.:01Q` 1:s ]V``Q`IVR 1J V_%:C s_%:`V :`VJ:s ^ 6 HI_ sV]:`: VR G7 HIR.1$. Q]:_%V $`V7 PCV61$C:s 1:CCs Q0V` : 1.1 V PCV61$C:s ]C: `Q`I ^1V1 PQ1J 5 L7QJ5 F`:JHV_8 M1HV %sVR `Q` sQH1:C 1J V`:H 1QJ ^ >1J V`:H 1J$, I1HV?_, 1V`V RR1VV@RQCR $VJRV`RI: H.VR $`Q%]VRR.Q%sVR 11CRR 7]V I1HV5 sQH1:CC7 J:10V :JR %J`:I1C1:` Q .V V6]V`1IVJ :C :J1I:Cs8 OJ R:7 5 :CC .V :J1I:Cs 1V`V .:G1 %: VR Q .V :`VJ: R%`1J$ : RI1J sVss1QJ8 OJ R:7 5 GQ . 1J V`:H 1J$ :JR V6]V`1IVJ :C I1HV :`V ]C:HVR 1J Q .V Q]VJ `1CVR `Q` I1J :JR J%IGV` Q` JQsV :JR ]:1 HQJ :H s ^H`:1C1J$ Q0V`5 IQ%J 1J$5 s V]]1J$ QJ5 ]%s.1J$_5 $`QQI1J$ ^Q0V`:CC Q` ]`VH1sVC7 :` V` sQH1:C V0VJ _ :JR `QCCQ11J$5 :s 1VCC :s Q :C 1IV s]VJ 1J HCQsV HQJ :H ^JQsV :JR ]:1 HQJ :H s_5 1V`V sHQ`VR QJ 01RVQ `VHQ`R1J$s8 M:`GCV G%`71J$ Vs 8 M1HV 1V`V ]C:HVR QJ : HCV:` .QIV H:$V `1CCVR Q` RHI RVV] ``Vs. s:1R%s 5 HQJ :1J1J$ I:`GCVs :JR HQ0V`VR 11 . : `1C V`1J$ C1R `Q` I1J8 L1$. 1J VJs1 7 1:s sV : C%68 T.V J%IGV`,Q`,I:`GCV5,G%`1VR,^` Q_ 1J s:1R%s 1:s sHQ`VR8 NQ0VC 7Rs%]]`VssVR `VVR1J$8 M1HV 1V`V `1`s `QQRRRV]`10VR . :JR 1sQC: VR 1J : JV1 .QIV H:$V I1J ]`1Q` Vs 1J$8 L1$. 1J VJs1 7 1J .V V6]V`1IVJ :C `QQI 1:s sV : C%68 T.`VV H.V1 1V`V ]C:HVR 1J .V HVJ V` Q` : 1.1 V s_%:`VR 1ss%V CV` 1J .V,I1RRCV,Q`, .V,:`VJ:,^5VV,>5QH1:C,1J V`:H 1QJ?_5, HQ0V`VR Q` HI Q` ``Vs. s:1R%s 8 T.V :J1I:Cs 1V`V ]C:HVR 1J .V Q]VJ `1VCR :JR .V C: VJH7 Q `VVR 1s IV:s%`VR5 11 . : H% RQ`` 1IV Q` I1J8 T.V IQ%sV 1:s `:Js`V``VR G:H@ Q .V VI] 7 .QIV H:$V 1IIVR1: VC7 :` V` `V:H.1J$ :JR V: 1J$ .V `QQR ]VCCV 8 T.V IQ%sV 1:s :CCQ1VR Q V: R%`1J$ I1J 1J .1s HQJR1 1QJ :JR `QQR HQJs%I] 1QJ 1:s 1V1$.VR8 T.V 1IV C1JV 1s `V]`VsVJ VR 1J F1$%`V 8

S : 1s 1H:C :J:C7s1s ACC s : 1s 1H:C Vs s 1V`V ]V``Q`IVR %s1J$ P`1sI ^G`:].P:R SQ` 1:`V_8 T.V V``VH Q` $VJQ 7]V 1:s :J:C7sVR G7 QJVR1:7 ANOA8 S1$J1`1H:J $VJQ 7]V V``VH 1:s `QCCQ1VR G7 I%C 1]CV HQI]:`1sQJs Vs 8 .VJ QJC7 1Q $VJQ 7]Vs 1V`V HQI]:`VR5 1V %sVR 1QR :1CVR R Vs s8 V %sVR ;`%GG5;, V5 Q RV VH :JR V6HC%RV Q% C1V`s8

RESLTS

M% Q]1Q1R `VHV] Q` RVCV 1QJ 1J `Q`VG`:1J GABAV`$1H JV%`QJs 1s JQ s%``1H1VJ Q 1I]:1` sQH1:C 1J V`:H 1QJ F1`s 5 1V Vs VR 1.V .V` I% Q]1Q1R `VHV] Q`s 1J .V `Q`VG`:1J GABAV`$1H JV%`QJs HQJ `1G% V Q sQH1:C 1J V`:H 1QJs8 TQ RQ sQ5 1V V6:I1JVR sQH1:C :G1C1 1Vs Q` T :JR Q :C KO 1J .V sQH1:C 1J V`:H 1QJ Vs ^F1$%`V A _8 S %RVJ R Vs s `V0V:CVR .: HQJs 1 % 10V KO I1HV `Q` .V I% `VHV] Q` s.Q1 s1$J1`1H:J C7

CVss JQsV HQJ :H s HQI]:`VR Q T ` ^_ Y8 5 ]Y8 a :s 1V `Q%JR ]`V01Q%sC7 1J BVH@V` V :C85 8

V :CsQ `Q%JR : VJRVJH7 `Q` s.Q` V` 1IV 1J HCQsV HQJ :H ` ^_ Y8 5 ]Y8a :JR `Q` .1$.V` $`QQI1J$

V0VJ s ` ^_ Y8 5 ]Y8a8 T.VJ5 1V V6:I1JVR sQH1:C :G1C1 1Vs Q` HQJR1 1QJ:C DC6RI% I1HV5 :JR `Q%JR JQ R1``V`VJHVs GV 1VVJ HQJ `QC :JR DC6R I%,I1HV,^JQ ,5.Q1J_8,>;?I: Q,5 %R1V5,5%$$V5 , .: ,51GC1J$5,H:J, 1J`C%VJHV .V IQ%sV ].VJQ 7]V ^]V`sQJ:C HQII%J1H: 1QJ_5 1J ]:` 1H%C:` R1``V`VJHVs 1J sQH1:C 1J V`:H 1QJs GV 1VVJ I% :J :JR HQJ `QCs I:7 GV `VR%HVR 1.VJ s1GC1J$s ``QI .V 1Q $VJQ 7]Vs RV0VCQ] 11 .1J : I16VR $`Q%]5 :s 1s .V H:sV 1J Q%` G`VVR1J$ sH.VIV ^sVV IV .QRs_8 V .V`V`Q`V $VJQ 7]VR ]%]s : P :JR `VRH`V: VR s1GC1J$ $`Q%]s ``QI .V s:IV $VJQ 7]V8 V .VJ V6:I1JVR :R%C IQ%sV GV.:01Q`s 11 . s1GC1J$s .:`GQ%`1J$ V1 .V` Q .V s:IV $VJQ 7]V ^CQJ `QC sV]:`: VR :JR DC6R I% sV]:`: VR $`Q%]s_ Q` I16VR QR Q $VJQ 7]Vs ^CQJ `QC I16VR :JR DC6RI% I16VR $`Q%]s_8 OJVR1:7 ANOA R1R JQ s.Q1 :J7 R1``V`VJHVs 1J sQH1:C GV.:01Q` GV 1VVJ $`Q%]s5 JV1 .V` `Q` .V J%IGV` Q` JQsV

HQJ :H s ` F^5 _ Y8 5 ]Y8 a5 J%IGV` Q` $`QQI1J$ V0VJ s `F ^5 _ Y8 5 ]Y8 a JQ` .V Q :C 1IV s]VJ

1J HCQsV HQJ :H `F ^5 _ Y8 5 ]Y8 a ^ F1$%`V B _8 SVCVH 10V RVCV 1QJ Q` .V I% `VHV] Q` 1J `Q`VG`:1J GABAV`$1H JV%`QJs5 .V`V`Q`V5 RQVs JQ :C V` sQH1:C 1J V`:H 1QJs5 :JR .1s GV.:01Q` 1s JQ IQR1`1VR V0VJ 1.VJ .V HQJR1 1QJ:C @JQH@Q% :`V sV]:`: VR ``QI CQJ `QC s1GC1J$s8

M% Q]1Q1R `VHV] Q` RVCV 1QJ 1J `Q`VG`:1J GABAV`$1H JV%`QJs 1s JQ s%``1H1VJ Q 1I]:1` :J61V 7RC1@V GV.:01Q`s T.VJ5 1V s %R1VR 1.V .V` .V I% Q]1Q1R `VHV] Q` 1J `Q`VG`:1J GABAV`$1H JV%`QJs 1s 1J0QC0VR 1J .V :J61V 7RC1@V GV.:01Q` .: 1V ]`V01Q%sC7 QGsV`0VR 1J Q :C I% KO I1HV8 S]VH1`1H:CC75 1V V6:I1JVR :J61V 7RC1@V GV.:01Q` %s1J$ .V I:`GCV G%`71J$5 : RV`VJs10V :J61V 7 Vs 5 :JR JQ0VC 7 s%]]`VssVR `VVR1J$ ^NSF_ Vs s5 : HQJ`C1H Vs ^ F1$%`V A _8 IJ .V I:`GCV G%`71J$ V6]V`1IVJ 5 .V J%IGV` Q` I:`GCVs G%`1VR 1J : RI1J sVss1QJ 1:s IV:s%`VR8 F1`s 5 1V QGsV`0VR .V :J61V 7 C1@V GV.:01Q` 1J T :JR Q :C KO :J1I:Cs8 As 1V `Q%JR ]`V01Q%sC7 1J BVH@V` V :C85 5 s %RVJ R Vs s.Q1VR : s : 1s 1H:CC7 .1$.V`

J%IGV` Q` I:`GCVs G%`1VR 1J I% `VHV] Q` KO I1HV HQI]:`VR Q T ` ^_ Y85 ]Y8 a ^ F1$%`V B _8 IJ

.V JQ0VC 7Rs%]]`VssVR `VVR1J$ ]:`:R1$I5 R Vs `V0V:CVR : CQJ$V` C: VJH7 Q `VVR 1J KO HQI]:`VR Q

T :J1I:Cs ` ^_ Y 8 5 ]\8a8 V .VJ HQI]:`VR CQJ `QC :JR DC6RI% I1HV5 I16VR :JR sV]:`: VR

^F1$%`V C _8 IJ .V I:`GCV G%`71J$ Vs 5 QJVR1:7 ANOA `V0V:CVR JQ $VJQ 7]V V``VH `F ^5 _ Y8 5

]Y8a8 IJ .V NSF Vs 5 QJVR1:7 ANOA s.Q1VR JQ $VJQ 7]V V``VH `F ^5 _ Y85 ]Y8a8 M% `VHV] Q` @JQH@Q% 1J `Q`VG`:1J GABAV`$1H JV%`QJs5 .V`V`Q`V5 RQVs JQ sVVI Q HQJ `1G% V Q :J61V 7R`VC: VR GV.:01Q`s8

DISCSSION

AC Q$V .V`5 Q%` R: : s.Q1 .: $VJV 1H RVCV 1QJ Q` .V I% Q]1Q1R `VHV] Q` 1J `Q`VG`:1J GABAV`$1H JV%`QJs RQVs JQ ]`QR%HV :J7 RV VH :GCV sQH1:C Q` :J61V 7 RV`1H1 5 1.1H. :`V Q .V`11sV QGsV`0VR %]QJ HQI]CV V $VJV KO ^BVH@V` V :C85 6 ORR1 V :C85 _8 CQJsV_%VJ C75 .1s ]:` 1H%C:` I% Q]1Q1R `VHV] Q` s%G]Q]%C: 1QJ RQVs JQ sVVI Q HQJ `1G% V Q .V RV0VCQ]IVJ Q` ASDRC1@V s7I] QIs8 DC6RI% I1HV s.Q1 GC%J VR I% Q]1Q1R `VHV] Q` V6]`Vss1QJ .`Q%$.Q% .V s `1: %I ^H:%R: V ]% :IVJ :JR J%HCV%s :HH%IGVJs_5 :s 1VCC :s `VR%HVR `VHV] Q` J%IGV` : .V CV0VC Q` .V :I7$R:C:8 T.QsV `V$1QJs :`V :CC 1J0QC0VR 1J sQH1:C GV.:01Q`s ^C.V0:CC1V` V :C85 _8 SQH1:C ]C:7 GV.:01Q` 1J `: s5 1.1H. 1s .1$.C7 `V1:`R1J$ ^T`V<<: V :C85 :_5 1JR%HVs V6]`Vss1QJ Q` .V I:`@V` Q` HVCC%C:` :H 101 7 HR FQs 1:s 1JH`V:sVR 1J .V ]`V``QJ :C HQ` V65 RQ`s:C :JR 0VJ `:C s `1: %I5 C: V`:C :I7$R:C:5 sQIV .:C:I1H J%HCV15 RQ`s:C `:].V :JR ]VJR%JH%CQ]QJ 1JV V$IVJ :C J%HCV%s ^0:J KV`@.Q` V :C85 _8 F%` .V`5 .V`V 1s V01RVJHV `Q` : `QCV Q` I% Q]1Q1R `VHV] Q`s 1J sQH1:C `V1:`R : .V CV0VC Q` 0VJ `:C s `1: %I8 SQH1:C ]C:7 1J :RQCVsHVJ `: s 1s 1JH`V:sVR G7 1J `:RNAH 1J`%s1QJ Q` IQ`].1JV :JR I% `VHV] Q` :$QJ1s `DRAC: 5NR MVP.V 5GC7 RQCaVJHV].:C1J ^DAMGO_5 :JR RVH`V:sVR G7 1J `:RNAH 1J`%s1QJ Q` I% `VHV] Q` :J :$QJ1s

C7sRT7`RDRT`]RA`$RT.`RPVJRT.`RNH ^CTAP_ ^T`V<<: V :C85 G_8 MQ`VQ0V`5 CTAP 1J`%s1QJ 1J .V NAH ]`V0VJ s .V RV0VCQ]IVJ Q` sQH1:CR]C:7 HQJR1 1QJVR ]C:HV ]`V`V`VJHV ^T`V<<: V :C85 G_8 IJ ]`:1`1V 0QCVs5 IQR%C: 1QJ Q` .V I% `VHV] Q` G7 :J :$QJ1s s 1J R1``V`VJ s%G`V$1QJs Q` .V s `1: %I s%$$Vs s R1s 1JH `QCVs Q` .V RQ`s:C s `1: %I5 NAH HQ`V :JR s.VCC 1J ]:` JV` ]`V`V`VJHV5 ]:1` GQJR `Q`I: 1QJ :JR I: 1J$ ^RVsVJRV< V :C85 _ :JR :H 10: 1QJ Q` I% `VHV] Q`s 1J .V RQ`s:C s `1: %I :]]V:`VR : @V7 VCVIVJ Q` :R%C sQH1:C : :H.IVJ 1J ]`:1`1V 0QCVs ^B%`@V V :C85 _8 T.V C:H@ Q` HQJsV_%VJHVs Q` I% `VHV] Q` $VJV KO 1J .V NAH5 .V`V`Q`V5 1:s s%`]`1s1J$8 T.1s 1s %JC1@VC7 R%V Q 1J:]]`Q]`1: V GV.:01Q`:C Vs 1J$ HQJR1 1QJs Q` sVJs1 101 75 s1JHV .V sQH1:C RV`1H1 ].VJQ 7]V 1:s 1VCC RV VH VR 1J Q :C KO I1HV %JRV` .V s:IV V6]V`1IVJ :C HQJR1 1QJs8 R: .V`5 `VI:1J1J$ I% `VHV] Q` ]Q]%C: 1QJs V6]`VssVR : Q .V`

G`:1J s1 Vs5 1JHC%R1J$ HQ` V6 1.V`V `VHV] Q` V6]`Vss1QJ 1s :CIQs 1J :H Q` I1RL.1JRG`:1J s `%H %`Vs5 I:7 GV s%``1H1VJ Q ]`QHVss `V1:`R1J$ s 1I%C1 :JR I:1J :1J JQÌ:C CV0VCs Q` sQH1:C GV.:01Q`s8 AC V`J: 10VC75 I% Q]1Q1R `VHV] Q`s V6]`VssVR 1J JQJRGABAV`$1H JV%`QJs 1J .V `Q`VG`:1J I:7 HQJ `1G% V Q sQH1:C `V1:`R5 : .7]Q .Vs1s .: 1Q%CR RVsV`0V `%` .V` 1J0Vs 1$: 1QJ8 AJ61V 7RC1@V GV.:01Q` IV:s%`VR 1J .V I:`GCV G%`71J$ Vs 1s .1$.V` 1J Q :C I% KO I1HV .:J T :J1I:Cs5 :s `V]Q` VR 1J Q%` ]`V01Q%s `V]Q` ^BVH@V` V :C85 _5 G% :J61V 7 CV0VCs 1J DC6RI% :J1I:Cs 1V`V s1I1C:` Q .V1` HQJ `QC C1 VÌ: Vs8 M:`GCV G%`71J$ 1s % 1C1;?I: Q,]`Q]Q5VR, .: ,51GC1J$5,H Q%CR 1J`C%VJHV,:,IQ%5V,].VJQ 7]V,^>;?I: Q5,]V`5QJ:C,HQII%J1H: 1QJ_5,G:5VR,QJ, .V,QG5V`0: 1QJ, .: ,?F>, ]: 1VJ s VJ.:JHV .V1` HQII%J1H: 1QJ s@1CCs G7 .V`:]7 1J0QC01J$ sV0V`:C `QÌs Q` sQH1:C V6]Qs%`V ^V1 C:%` V :C85 _8 IJ Q%` s %R75 s1GC1J$ V``VH s QJ sQH1:C :JR :J61V 7R`VC: VR GV.:01Q`s HQ%CR JQ GV RV VH VR8

TQ HQJHC%RV5 Q%` R: : s%$$Vs .: I% Q]1Q1R `VHV] Q`s 1J GABAV`$1H JV%`QJs Q` .V `Q`VG`:1J RQ JQ HQJ `QC ASDRC1@V GV.:01Q`s8 F%` .V` s %R1Vs 11CC GV JVHVss:`7 Q RV V`I1JV 1.1H. I% Q]1Q1R `VHV] Q` ]Q]%C: 1QJs :`V 1I]Q` :J 1J ASDRC1@V GV.:01Q`s8

REFERENCES

As:J5 E85 S V1J@V5 M85 LVsH.5 K8P85 8 SV`Q QJV`$1H 1JJV`0: 1QJ Q` .V :I7$R:C:7 T:`$V s5 `VHV] Q`s5 :JR 1I]C1H: 1QJs `Q` s `Vss :JR :J61V 78 H1s QH.VI8 CVCC B1QC8 5 T 8 RQ178 Ls R R R

B`1:JR5 L8 :85 H1C:`1Q5 M85 DQ15 H8C85 B`QR@1J5 E8S85 BCVJR75 J8 :85 BV` QJ5 O85 8 MQ%sV MQRVC Q` OPRM ^A G_ PQC7IQ`].1sI IJH`V:sVs SQH1:G1C1 7 :JR DQI1J:JHV :JR CQJ`V`s RVs1C1VJHV Q SQH1:C DV`V: 8 J8 NV%`QsH18 5 T 8 RQ178 LJNEROSCI8 R8

B`1R$Vs5 R8S85 G`1II5 C8T85 8 RV0V`s:C Q` IQ`].1JV R1s`%] 1QJ Q` I: V`J:C GV.:01Q` G7 HQJH%``VJ `V: IVJ 11 . .V Q]1: V :J :$QJ1s J:CQ6QJV 5 T 8

B%`@V 5 J8P85 S]1V$VC5 L8L85 IJQ%V5 K85 M%`].75 A885 Q%J$5 L8J85 8 AH 10: 1QJ Q` -RO]1Q1R RVHV] Q`s 1J .V DQ`s:C S `1: %I 1s NVHVss:`7 `Q` AR%C SQH1:C A :H.IVJ 1J MQJQ$:IQ%s P`:1`1V QCVs8 NV%`Q]s7H.Q].:`I:HQCQ$7 5 T8 RQ178 LJ]]88

CVCCQ 5 G85 C.V`%G1J15 E85 8 GABAV`$1H s1$J:C1J$ :s .V`:]V% 1H :`$V `Q` :% 1sI s]VH `%I R1sQ`RV`s8 F`QJ 8 PVR1: `8 5 8 RQ178 L`]VR88

C.V0:CC1V`5 C85 KQ.Cs5 G85 T`Q1:J15 85 B`QR@1J5 E8S85 SH.%C <5 R8T85 8 T.V sQH1:C IQ 10: 1QJ .VQ`7 Q` :% 1sI8 T`VJRs CQ$J8 SH18 5 T 8 RQ178 L=8 1Hs888

I1J_%V5,I85,KQJR1@15,F85,BRR15,>85,>1,IV` Q5,M8;85,M:`1JVCC15,F85,N`Q1515,:5,MQCV55,:5,>;?I: Q5,C8O85,8, MQRVC1J$ sQH1:CC7 :J.VRQJ1H s7JR`QIVs7 $VJV 1H :JR ].:`I:HQCQ$1H:C I:J1]%C: 1QJ Q` Q]1Q1R JV%`Q `:JsI1ss1QJ 1J I1HV8 T`:JsC8 Ps7H.1: `7 5 V 8 RQ178 L ]88

Hs%5 D8T85 S:J`Q`R5 B8J85 MV7V`s5 K8K85 LQ0V5 T8M85 H:

-RQ]1Q1R s7s VI Q sQH1:C `V=VH 1QJ :JR :HHV] :JHV8 MQC8 Ps7H.1: `7 5 T 8 RQ178 LI]88

KQI: s%5 H85 O.:`:5 A85 S:s:@15 K85 AGV5 H85 H: Q`15 H85 H:CC5 F8S85 .C5 G8R85 SQ`:5 I85 8 DVH`V:sVR `Vs]QJsV Q sQH1:C RV`V: s `Vss 1J -RQ]1Q1RR`VHV] Q` @JQH@Q% I1HV8 P.:`I:HQC8 B1QH.VI8 BV.:08 5 T 8 RQ178 L=8]GG88 8

M1`:JR:RP:10:5 C8M85 R1GV1`QRB:`GQs:5 E8R85 C:J V`:s5 N8S85 FVC1H1Q5 L8F85 8 A `QCV `Q` .V ]V`1:_%VR%H :C $`V7 1J Q]1Q1RV`$1H 1J.1G1 1QJ Q` I: V`J:C GV.:01Q%`8 E%`8 J8 NV%`QsH18 5 T 8 RQ178 L=8 R 88 86

MQCV55,?85,A1V``V`5,S8T85,>; AI: Q5 F8R85 8 DV`1H1 1J : :H.IVJ GV.:01Q` 1J I1HV C:H@1J$ .V I%RQ]1Q1R `VHV] Q` $VJV8 SH1VJHV 5 T 8 RQ178 LsH1VJHV8

BRR15, >85, I`%51Q5, 8V85, >;?I: Q5, C8O85, K1V `Q]:QCQ5, F85 8 MQJQ$VJ1H IQ%sV IQRVCs Q` sQH1:C R7s`%JH 1QJ7 II]C1H: 1QJs `Q` :% 1sI8 BV.:08 B`:1J RVs8 5 T 8 RQ178 L=8GG`888

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A B C

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C D

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PART III

T:`$V $C% :I: V`$1H `Q`VG`:1J I% `VHV] Q`s 1J :R%C I1HV

I8 IJ `QR%H 1QJ

8 T.V I% Q]1Q1R `VHV] Q` 1J $C% :I: V`$1H JV%`QJs

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8 C:MKII $VJV

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A B

F1$%`V 8 II:$Vs Q` C:MKII RGFP :R%C IQ%sV G`:1J8 ^ A_ S:$1 :C 01V18 CQJ`QH:C 1I:$V IQJ :$V s.Q1s GFP 1J : -I s:$1 :C sVH 1QJ ^C: V`:C8II_8 ^ B_ CQ`QJ:C 01V18 II:$V Q` GFP 1J : jI HQ`QJ:C sVH 1QJ ^B`V$I: R 8 II_8 SH:CV G:`s Y -I8 AR:] VR ``QI :J$ V :C85 8

AGG`V01: 1QJs7 J5 `:H1:C J%HCV%s `QQ 6 N5 `:H1:C J%HCV%s6 AHG5 :HH%IGVJs J%HCV%s6 AHQ5 :J V`1Q` HQII1ss%`V6 AM5 :I7$R:C:` J%HCV%s5 IVR1:C6 AL5 :I7$R:C:` J%HCV%s5 C: V`:C6 AJ$5 :J$%C:` .:C:I1H J%HCV%s6 AON5 :J V`1Q` QC`:H Q`7 J%HCV%s6 A`HD5 :`H%: V .7]Q .:C:I1H J%HCV%s5 RQ`s:C ]:` 6 A`HL5 :`H%: V .7]Q .:C:I1H J%HCV%s5 C: V`:C ]:` 6 A%5 :%R1 Q`7 HQ` V66 CA5 `1VCR CA6 CA5 `1VCR CA6 CG5 HV`VGVCC%I6 CEAI5 HVJ `:C :I7$R:C:` J%HCV%s5 C: V`:C6 CL5 HVJ `:C C: V`:C J%HCV%s Q` .V .:C:I%s6 CM5 HVJ `:C IVR1:C J%HCV%s Q` .V .:C:I%s6 CP%5 H:%RQ]% :IVJ6 DG5 RVJ : V $7`%s6 DM5 RQ`s:C IVR1:C J%HCV%s Q` .V .7]Q .:C:I%s6 EH 5 VH Q`.1J:C HQ` V66 F`5 `:sH1H%C%s `V `Q`CV6%s6 FÀ5 ``QJ :C :ssQH1: 1QJ HQ` V66 GV 5 $VC: 1JQ%s C:7V` Q` .V H:%R:C s]1J:C `1$VI1J:C J%HCV%s6 $`5 $`:J%CV C:7V` Q` HV`VGVCC%I6 G`DG5 $`:J%C:` C:7V` Q` RVJ : V $7`%s6 HPF5 .1]]QH:I]:C `QÌ: 1QJ6 H5 .7]Q .:C:I%s6 IC5 1J`V`1Q` HQCC1H%CC%s6 IMD5 1J`V` IVR1QRQ`s:C J%HCV%s Q` .V .:C:I%s6 LDDM5 C: V`QRQ`s:C .:C:I1H J%HCV%s5 RQ`sQIVR1:C ]:` 6 LDL5 C: V`QRQ`s:C .:C:I1H J%HCV%s5 0VJ `QC: V`:C ]:` 6LPMR5 C: V`:C ]Qs V`1Q` .:C:I1H J%HCV%s5 IVR1Q`Qs `:C ]:` 6 LH5 C: V`:C .7]Q .:C:I%s :`V:6 LIQC5 s `: %I C:H%JQs%IIQCVH%C:`6 L` 5 C: V`:C `V 1H%C:` J%HCV%s6 LSO5 C: V`:C s%]V`1Q` QC10V6 MB5 I1RG`:1J6 MO5 IQ Q` HQ` V66 MDC5 IVR1QRQ`s:C J%HCV%s Q` .V .:C:I%s5HVJ `:C ]:` 6 MRD5 IVR%CC:`7 `V 1H%C:` J%HCV%s5 RQ`s:C ]:` 6 MDL5 IVR1QRQ`s:C J%HCV%s Q` .V .:C:I%s5 RQ`s:C ]:` 6 MDM5 IVR1QRQ`s:C J%HCV%s Q` .V .:C:I%s5 IVR1:C ]:` 6 ME5 IVR1:J VI1JVJHV6 II 5 I:II1CQ .:CI1H `:H 6 MQ 5 IQ Q` `1$VI1J:C J%HCV%s6 MOB5 I:1J QC`:H Q`7 G%CG6 MQDG5 RVJ : V $7`%s5 IQCVH%C:` C:7V`6 MRN5 I1RG`:1J `V 1H%C:` J%HCV%s6 M5 MVR%CC:6 MV5 IVR1:C 0Vs 1G%C:` J%HCV%s6 O] 5 Q] 1H `:H 6 O`5 s `: %I Q`1VJs6 P5 ]QJs6 P:5 ]:`:0VJ `1H%C:` .7]Q .:C:I1H J%HCV%s6 PARN5 ]:`01 HVCC%C:` `V 1H%C:` J%HCV%s6 PAG5 ]V`1:_%VR%H :C $`:76 PC5 ]:`:HVJ `:CJ%HCV%s6 P1`5 ]1`1`QÌ HQ` V66 PH5 ]%`@1J=V HVCC C:7V` Q` HV`VGVCC%I6 PLCQ5 ]Qs V`QC: V`:C HQ` 1H:C :I7$R:CQ1R J%HCV%s6 PQ5 ]Qs V`1Q` HQI]CV6 Q` .V .:C:I%s6 PQDG5 ]QC7IQ`]. C:7V` Q` .V RVJ : V $7`%s6 PR.5 ]V`1`.1J:C HQ` V66 R:R5 s `: %I `:R1: %I6 RV5 J%HCV%s Q` `V%J1QJs6 RPO5 `Qs :C ]V`1QC10:`7 `V$1QJ6 RSG5 `V `Qs]CVJ1:C $`:J%C:` HQ` V66 RT5 `V 1H%C:` J%HCV%s Q` .V .:C:I%s6 S5 ]`1I:`7 sQI: QsVJsQ`7 HQ` V66 SC5 s%]V`1Q` HQCC1H%C%s6 SC%5 s `: %I C%H1R%I6 SNR5 s%Gs :J 1: J1$`:5 `V 1H%C:` ]:` Q` :I7$R:CQ1R :`V:6 S] 5 s]1J:C `1$VI1J:C J%HCV%s6 SPC5 s]1J:C J%HCV%s Q` .V `1$VI1J:C5 H:%R:C ]:` 6 S `5 s `1: %I VÌ1J:Cs6 S%G5 s%G]:`:`:sH1H%C:` J%HCV%s6 TH5 .:C:I%s6 T%5 QC`:H Q`7 %GV`HCV6 IS5 01s%:C HQ` V66 L5 0VJ `QC: V`:C J%HCV%s Q` .:C:I%s6 O5 0VJ `:C Q`G1 :C HQ` V66 PM5 0VJ `:C ]Qs V`Q IVR1:C J%HCV%s Q` .:C:I%s6 PL5 0VJ `:C ]Qs V`QC: V`:C J%HCV%s Q` .:C:I%s6 I5 1.1 VI: V`8 ]7`:I1R:C JV%`QJs :JR GABA Q GV HQJ :1JVR QJC7 1J JQJR]7`:I1R:C HVCCs Q` .V G:sQC: V`:C :I7$R:C: ^MHDQJ:CR V :C85 _8 IJ .V `Q`VG`:1J5 C:MKII 1s `Vs `1H VR Q V6H1 : Q`7 $C% :I: V`$1H JV%`QJs :JR :GsVJ ``QI GABARHQJ :1J1J$ JV%`QJs ^BVJsQJ V :C85 6 JQJVs V :C85 _8 SQIV s %R1Vs .:0V RVIQJs `: VR V6HV] 1QJs Q` JQJR HQJ0VJ 1QJ:C,R15 `1G% 1QJ,Q`,I:MA]],1J,1.1H.,I:MA]],15,JQ ,`V5 `1H VR, Q V6H1 : Q`7 HVCCs 1J :CC .V G`:1J `V$1QJs8 IJRVVR5 1J .V IQ%sV QC `:H Q`7, G%CG5, I:IA]], 1II%JQ`V:H 101 7 1:s ]Qs1 10V 1J .V GABAV`$1H $`:J%CV HVCCs5 :JR 1:s ]Qs1 10V 1J $C% :I: V`$1H JV%`QJs 1J .V ]1`1`QÌ HQ` V6 ^Q% V :C85 _8 RVHVJ C75 : C:MKII FRET sVJsQ` 1:s RV0VCQ]VR5 ]VÌ1 1J$ IQ`V ]`VH15V, CQH:C1<: 1QJ, Q`, .V, ]`Q V1J;5, :H 101 7, ^S.1G: : V :C85 _ 8, N.V, $VJV`: 1QJ, Q`, :, I:MA]] RGFP IQ%sV C1JV .:s GVVJ 0V`7 %sV`%C Q s %R7 .V `V$1QJ:C :JR HVCC%C:` R1s `1G% 1QJ Q` .V @1J:sV ^:J$ V :C85 _ ^ F1$%`V _8 NQ Q0V`C:] GV 1VVJ GFP :JR GABA 1II%JQ`V:H 101 7 1:s `Q%JR 1J .V JVQHQ` V65 .:C:I%s5 CA :JR Q` .V .1]]QH:I]%s5 ]1`1`QÌ HQ` V65 H:%R: V ]% :IVJ :JR .7]Q .:C:I%s5 G% : 0V`7 .1$. Q0V`C:] 1:s QGsV`0VR 1J .V $`:J%CV HVCCs Q` .V QC`:H Q`7 G%CG5 :s ]`V01Q%sC7 RVIQJs `: VR ^Q% V :C85 _8 T.%s5 .V %J1_%V R1s `1G% 1QJ Q` I:MA]] Vs :GC1s.Vs .1s $VJV :s :J V6HVCCVJ QQC Q s %R7 $C% :I: V`$1H HVCC s]VH1`1H ]Q]%C: 1QJs8

8 A1I Q` .V s %R77 :`$V $C% :I: V`$1H `Q`VG`:1J I% `VHV] Q`s 1J :R%C I1HV

TQ s %R7 .V `QCV Q` I% Q]1Q1R `VHV] Q`s 1J $C% :I: V JV%`QJ:C ]Q]%C: 1QJs5 1V RV0VCQ]VR : JV1 IQ%sV C1JV 1J 1.1H. I`V,`VHQIG1J:5V,15,V6]`V55VR,%JRV`, .V,HQJ `QC,Q`, .V,I:MA]],$VJV,]`QIQ V`, .: :`$V s $C% :I: V`$1H `Q`VG`:1J JV%`QJs8 T.1s C`V 1s `%sVR 11 . ER T ]`Q V1J :JR ]V`I1 s V6]`Vss1QJ Q` .V VJ<7IV QJC7 :` V` :IQ61`VJ `V: IVJ 8 TQ V0:C%: V .1s JV1 IQCVH%C:` QQC5 I H.:`:H V`1

CQ6P CQ6P $VJV ]`QIQ V` $VJV STOP L:H C:MKII C`V ER T ROSA IQ%sV C:MKII C`VER T IQ%sV

C`V ^R_ T:IQ61`VJ :JR V.1HCV C`V ^U_ T:IQ61`VJ C`V ^U_ V.1HCV

C`V `VHQIG1J:sV V6]`Vss1QJ JQ C`V `VHQIG1J:sV V6]`Vss1QJ

L:H STOP L:H

RG:C RG:C

BC%V ]`VH1]1 : V NQ s :1J1J$

F1$%`V 8 C:MKII C`VER T ROSA IQ%sV C1JV Q s %R7 .V C`V `VHQIG1J:sV :H 101 78 V G`VR .V C`V `VHQIG1J:sV `V]Q` V` ROSA I1HV 11 . C:MKII C`VER T I1HV Q QG :1J C:MKII C`VER T ROSA IQ%sV C1JV8 C`V `VHQIG1J:sV ]Qs1 10V :J1I:Cs 1V`V `V: VR 11 . :IQ61`VJ ^ I$LR:75 R:7s_ Q 1JR%HV C`V `VHQIG1J:sV :H 10: 1QJ5 CV:R1J$ Q V6H1s1QJ Q` .V s Q] sV_%VJHV8 T.V L:H $VJV 11CC .VJ GV V6]`VssVR :JR 11CC HQRV `Q` .V R$:C:H Qs1R:sV VJ<7IV .: ]`QR%HVs : GC%V ]`VH1]1 : V 1J ]`VsVJHV Q` 1 s s%Gs `: V RG:C 1J C`VRV6]`Vss1J$ HVCCs8

II8 M: V`1:Cs :JR IV .QRs

AJ1I:Cs E6]V`1IVJ s 1V`V ]V``QÌVR QJ I:CV :JR `VI:CV I1HV :$VR 1VV@s : .V GV$1JJ1J$ Q` .V s %R78 I:MA]] RC`VER T C1JV 1:s s%HHVss`%CC7 %sVR 1J : `VHVJ s %R7 Q HQJR1 1QJ:CC7 1J0:C1R: V N:V $VJV ^Q$C V :C85 _8 TQ $VJV`: V : HQJR1 1QJ:C @JQH@Q% `Q` O]Ì 1J $C% :I: V`$1H `Q`VG`:1J JV%`QJs5 .V I:MA]] RC`VER T RO]Ì RLR IQ%sV C1JV 1:s H`V: VR : .V ICSRIGBMC ÎJs 1 % CC1J1_%V RV C: SQ%`1s R IJs 1 % RV GXJX 1_%V V RV B1QCQ$1V MQCXH%C:1`V V CVCC%C:1`V5 ICC@1`H.5 F`:JHV_ G7 G`VVR1J$ .V I:MA]] RC`VER T I1HV ÎCS_ 11 . I% `CQ6VR I1HV8 T.V I% `CQ6VR IQ%sV C1JV ^ O]Ì `CL`C_ .:s GVVJ ]`V01Q%sC7 RVsH`1GVR G7 Q%` $`Q%] ^V1GVC V :C85 _8 B`1V`C75 V6QJs :JR Q` .V I% `VHV] Q` $VJV O]Ì 1V`V `C:J@VR G7 CQ6P s1 Vs8 O]Ì `CL`C I1HV s.Q1 1J :H I% `VHV] Q` V6]`Vss1QJ ^V1GVC V :C85 _8 RVs%C 1J$ C`V ]Qs1 10V ^C`VÛ_5 I:MA]] RC`VER T RO]Ì RLR _ :J1I:Cs :`V H:CCVR I:MA]] RI% :JR C`V JV$: 10V ^C`V^R_5 O]Ì `CL`C _ :`V CQJ `QCs8 I:MA]] RI% :JR C1 VÌ: V HQJ `QCs .:0V .V s:IV $VJV 1H G:H@$`Q%JR ^ Q C BLL J R Q C BLL N R Q S0P:s_8 CMRC`VRO]Ì RLR ^CMRI%_ I1HV 1V`V %sVR :s Q :C @JQH@Q% `Q` .V I% `VHV] Q` $VJV5 G7 G`VVR1J$ O]Ì `CL`C I1HV 11 . CMRC`V I1HV5 V6]`Vss1J$ .V C`V `VHQIG1J:sV %JRV` .V HQJ `QC Q` .V H7 QIV$:CQ01`%s ^CM5 %G1_%1 Q%s_ ]`QIQ V` ^MV <$V` :JR C.:IGQJ5 _8 T.1s C1JV .:s : Q C BLL JR Q S0P:s G:H@$`Q%JR8 FQ` .V C`V `VHQIG1J:sV :H 101 7 `V]Q` V` C1JV5 1V H`V: VR .V I:MA]] RC`VER T RROSA IQ%sV C1JV8 V QG :1JVR .1s JV1 C1JV G7 H`Qss1J$ .V I:MA]] RC`VER T I1HV ÎCS_ 11 . .V C`V :H 101 7 `V]Q` V` `:Js$VJ1H C1JV ROSA ^SQ`1:JQ5 _8 ACC V6]V`1IVJ s 1V`V H:``1VR Q% :HHQ`R1J$ Q .V `VHQIIVJR: 1QJs Q` .V NIH G%1RV `Q` C:`V :JR sV Q` L:GQ`: Q`7 AJ1I:Cs8 T.V s %R7 ]`Q QHQC 1:s :]]`Q0VR G7 .V LQH:C B1QV .1Hs CQII1 VV ^S `:sGQ%`$5 F`:JHV_8 ACC :J1I:Cs 1V`V .Q%sVR 1J : `QQI I:1J :1JVR : ^ C :JR ^ Q .%I1R1 75 11 . : . C1$. RR:`@ H7HCV8 FQQR :JR 1: V` 1V`V :0:1C:GCV :R C1G1 %I 8

L:H s :1J1J$ T.V C`VRV6]`Vss1QJ ]: V`J 1J C:MKII RI% I1HV 1:s H.:`:H V`1

LV1H:_ Q QG :1J HQ`QJ:C RjI .1H@ sVH 1QJs8 T.V7 1V`V s%GsV_%VJ C7 HQCCVH VR QJ sC1RVs ^M1H`Q SC1RVs P`VHCV:JVR R N`:,?R.V510V5,F%`$1]: ._,:JR,@V] ,: R C %J 1C %sV8 A` V` RI1J 1:Ì %] : `QQI VI]V`: %`V5 sC1RVs 1V`V 1JH%G: VR 1J : `16: 10V sQC% 1QJ ^`QÌ:CRV.7RV Q6 $C% :`:CRV.7RV 8Q6 T1VVJ 8Q 1J PBS_ `Q` : I1J% V :JR .VJ 1:s.VR : `QQI VI]V`: %`V I1J 1J PBST ^ PBS6 T1VVJ 8Q_8 SC1RVs 1V`V 1JH%G: VR : C 1J .V R:`@ 1J : s :1J1J$ sQC% 1QJ ^]Q :ss1%I `V``1H7:J1RV IM6 ]Q :ss1%I .V6:H7:JQ`V``: V IM6 M$CC IM5 R G:C I$LIL Ê%`QIVRV6_ 1J PBS_ %J 1C : GC%V HQCQ` RV0VCQ]VR8 NV6 5 sC1RVs 1V`V s%HHVss10VC7 1:s.VR 1IVs ^ I1J6 ._ 1J PBST : `QQI VI]V`: %`V8 SC1RVs 1V`V .VJ 1IIV`sVR 1J RVI1JV`:C1

T`V: IVJ C`V `VHQIG1J:sV :H 101 7 1:s 1JR%HVR G7 : RR:7 `V: IVJ Q` jL :IQ61`VJ ^ I$LIL5 18]85 11HV R:1C7_8 T:IQ61`VJ ]Q1RV` 1:s R1ssQC0VR 1J QRV .:JQC HQJ :1J1J$ s%J`CQ1V` Q1C8 T.V V6:H s:IV sQC% 1QJ 11 .Q% :IQ61`VJ 1:s %sVR :s : HQJ `QC ^0V.1HCV `V: VR :J1I:Cs_8

GVJQ 7]1J$RPCR PCR :J:C7s1s QJ $VJQI1H DNA 1V`V ]V``QÌVR 1J Q`RV` Q $VJQ 7]V .V I1HV `Q` ]`VsVJHV Q` _ C`V `VHQIG1J:sV5 _ CQ6P s1 Vs :JR _ V6H1s1QJ Q` O]Ì 8 PCR 1:s H:``1VR Q% QJ DNA QG :1JVR ``QI .V HQCCVH VR IQ%sV R1$1 s R1$Vs VR 11 . P`Q V1J:sV K ^N:CC 8M6 T`1sRHCC IM ]H 8 6 EDTA IM6 SDS 8Q6 ]`Q V1J:sV K ^S1$I:_ I$LIL6_ Q0V`J1$. : C8 T.V C`V PCR `V:H 1QJ 1:s ]V``QÌVR G7 :RR1J$ 8 jL C7s: V Q 8 jL `V:H 1QJ I16 ^ PCR

PCR G%``V` GQT:_ ^P`QIV$:_6 M$CC ^S1$I:_ IM6 RNTPs 8 IM ^T.V`IQ SH1VJ 1`1H_6 TAQ DNA

]QC7IV`:sV 8 ^P`QIV$:_6 `Q`1:`R I% `CQ6VR $VJV ]`1IV` ^ ; RGTT ACT GGA GAA TCC AGG CCA AGCR ; _5 `V0V`sV I% `CQ6VR $VJV ]`1IV` ^ ; RTGC TAG AAC CTG CGG AGC CAC AR ; _ jM_8 PCR `V:H 1QJ 1:s ]V``Q`IVR 11 . VI]V`: %`V H7HC1J$ ]:`:IV V`s HQJs1s 1J$ Q` 1J1 1:C RVJ: %`: 1QJ : C `Q` I1J `QCCQ1VR G7 H7HCVs Q` RVJ: %`: 1QJ : C `Q` I1J5 :JJV:C1J$ : C `Q` I1J5 V6 VJs1QJ : C `Q` I1J5 :JR : `1J:C 1JH%G: 1QJ : C `Q` I1J8 T.V I% V6H1s1QJ PCR `V:H 1QJ 1:s ]V``Q`IVR G7 :RR1J$ 8 jL C7s: V Q 8 jL `V:H 1QJ I16 ^

T1ss%V HQCCVH 1QJ `Q` IRNA :J:C7s1s M1HV 1V`V s:H`1`1HVR G7 HV`01H:C R1sCQH: 1QJ8 B`:1Js 1V`V V6 `:H VR5 `1JsVR 1J HQCR PBS ^].Qs].: VRG%``V`VR s:C1JV sQC% 1QJ5 S1$I:_ :JR RII .1H@ sC1HVs 1V`V H% 11 . : s :1JCVss s VVC HQ`QJ:C G`:1J I: `16 H.1CCVR QJ 1HV ^H:`0:`R :]]:`: %s5 HQCC1s QJ5 MA5 SA_8 D1``V`VJ G`:1J `V$1QJs 1V`V HQCCVH VR ``QI Q I1HV ]V` $VJQ 7]V :JR `V: IVJ 5 :HHQ`R1J$ Q .V s V`VQ :61H : C:s Q` IQ%sV G`:1J ^P:61JQs :JR F`:J@C1J5 _8 T.V CP% ^H:%R: V ]% :IVJ_ 1:s G1C: V`:CC7 ]%JH.VR %s1J$ : RII R1:IV V` 1ss%V HQ`V`6 NAH ^J%HCV%s :HH%IGVJs_5 BNST ^GVR J%HCV%s Q` .V s `1: V`I1J:C1s_5 EC ^VJ Q`.1J:C HQ` V6_5 :I7$R:C:5 :JR LH ^C: V`:C .7]Q .:C:I%s_ 1V`V G1C: V`:CC7 ]%JH.VR 11 . : 8RII HQ`V`6 PCF ^]`V``QJ :C HQ` V6_5 C$ ^H1J$%C:` HQ` V6_5 MH ^0VJ `QIVR1:C .7]Q .:C:I1H J%HCV%s_5 .:C:I%s :JR PAG ^]V`1:H_%VR%H:C $`V7_ 1V`V HVJ `:CC7 ]%JH.VR %s1J$ : RII R1:IV V` 1ss%V HQ`V`6 MPO ^0VJ `QIVR1:C ]`VQ] 1H J%HCV%s_5 A`H ^:`H%: V .7]Q .:C:I1H J%HCV%s_5 HG ^.:GVJ%C:_5 IP ^1J V`]VR%JH%C:` J%HCV%s_ :JR DRN ^RQ`s:C `:].V J%HCV%s_ 1V`V HVJ `:CC7 ]%JH.VR %s1J$ : 8RII R1:IV V` 1ss%V HQ`V`6 :JR .V H] ^.1]]QH:I]%s_5 SC ^s]1J:C HQ`R_5 :1C :JR sI:CC 1J Vs 1JV 1V`V R1ssVH VR8 S:I]CVs 1V`V 1IIVR1: VC7 ``Q

Q%:J 1 : 10V `V:CR 1IV PCR S:I]CVs 1V`V ]`QHVssVR Q V6 `:H Q :C RNA5 %s1J$ TRI

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III%JQ.1s QH.VI1s `7 `Q` `sRC`VRVGFP I1HV M1HV 1V`V :JVs .V 1

A E 5 E 5

B NAHH :H BNST CVA CA

PP :H

:H P

:H :H P

P :H :H

F1$%`V 8 `s V6]`Vss1QJ ]: V`J 1J .V RV0VCQ]1J$ VIG`7Q :JR 1J .V IQ%sV G`:1J R%`1J$ ]Qs RJ: :C RV0VCQ]IVJ G7 1J s1 % .7G`1R1<: 1QJ 8 ^A_ NQ V6]`Vss1QJ Q` .V FS $VJV 1s RV VH :GCV R%`1J$ VIG`7QJ1H RV0VCQ]IVJ 5 :s s.Q1J 1J .V s :$Vs E8 :JR E 8 ^s:$1 :C sVH 1QJs_8 ^B_ `s V6]`Vss1QJ 1J .V IQ%sV G`:1J : R1``V`VJ ]Qs RJ: :C s :$Vs ^HQ`QJ:C sVH 1QJs_8 MQRV`: V CV0VCs Q` .V `s `:JsH`1] :`V RV VH VR 1J .V EA :JR .V CA s1JHV ]Qs RJ: :C R:7 8 T.V V6]`Vss1QJ Q` `s `%` .V` 1JH`V:sVs 11 . 1IV :JR 1s HQI]:`:GCV Q :R%C V6]`Vss1QJ : ]Qs RJ: :C R:7 8 :H5 :J V`1Q` HQII1ss%`V6 BNST5 GVR J%HCV%s Q` .V s `1: V`I1J:C1s6 CA5 `1VCR CA Q` .1]]QH:I]%s6 CVA5 HVJ `:C :I7$R:CQ1R J%HCV%s6 NAHH5 J%HCV%s :HH%IGVJs8

^].Qs].: V G%``V` 8 M6 T`1 QJ R 8 Q_5 sVH 1QJs 1V`V 1JH%G: VR `Q` .Q%`s : `QQI VI]V`: %`V 11 . .V sVHQJR:`7 :J 1GQR7 GARRACV6: ^MQCVH%C:` ]`QGVs_ R1C% VR : L 1J GCQH@1J$ sQC% 1QJ8 F1J:CC75 .V ``VV `CQ: 1J$ G`:1J sVH 1QJs 1V`V 1:s.VR 6 I1J% Vs 1J 1:s.1J$ sQC% 1QJ : `QQI VI]V`: %`V :JR IQ%J VR QJ S%]V`F`Qs sC1RVs 11 . MQ11QC ^C:CG1QH.VI_ :JR DAPI ^S1$I:RACR`1H._ ^7_8 B`:1J sVH 1QJs 1V`V QGsV`0VR %JRV` :J V]1`C%Q`VsHVJ I1H`QsHQ]V ^LV1H:_ :JR 1I:$Vs 1V`V `VHQ`RVR %s1J$ : CCD H:IV`: ^CQQCSNAP5 RQ]V` SH1VJ 1`1H_ :JR .V CQQCSNAP sQ` 1:`V8

III%JQ.1s QH.VI1s `7 `Q` s.FS RVGFPRC`VER T :JR s.FS RVGFPRTARC`VER T I1HV M1HV 1V`V :JVs .V 1

III8 RVs%C s

E:`C7 ]`QIQ V` :H 101 7 I:7 `1$$V` RV0VCQ]IVJ :C HQI]VJs: 1QJs Q` I:7 CV:R Q : CV .:C ].VJQ 7]V 1` .V :`$V $VJV 1s VssVJ 1:C `Q` RV0VCQ]IVJ 8 MQ`VQ0V`5 V:`C7 ]`QIQ V` :H 101 7 H:J CV:R Q 1J:RV_%: V :JRLQ` 11RVs]`V:R `VHQIG1J: 1QJ 1` .V ]: V`J Q` V6]`Vss1QJ 1s I:`@VRC7 R1``V`VJ GV 1VVJ VIG`7QJ1H :JR :R%C s :$Vs8 T.V`V`Q`V5 C: V ]`QIQ V`s :`V ]`V`V``VR Q s %R7 $VJV `%JH 1QJ 1J .V :R%C

ATG V6 8 @G ]`QIQ V` V6 VJRQ$VJQ%s 1J `QJ S ]QC7A

F1$%`V 8 SH.VI: 1H `V]`VsVJ : 1QJ Q` .V HQJs `%H %sVR Q $VJV`: V .V `:Js$VJ1H FSRC`VRVGFP IQ%sV C1JV A s.Q` `:Js$VJV HQJ :1J1J$ : HDNA VJHQR1J$ : `C%Q`VsHVJ C`VRVGFP `%s1QJ ]`Q V1J %JRV` .V HQJ `QC Q` 8 @G `s ]`QIQ V` 1:s HQJs `%H VR :JR `%` .V` I1H`Q1J=VH VR 1J Q `V` 1C1

F1$%`V 8 ^A_ CQI]:`1J$ V6]`Vss1QJ ]: V`Js Q` `s IRNA ^T I1HV_ 11 . .V C`VRVGFP `:Js$VJV ^s.FSR C`VRVGFP I1HV_ 8 T.V Q] ]:JVCs RV]1H G`:1J sVH 1QJs Q` T I1HV :` V` 1J s1 % .7G`1R1<: 1QJ %s1J$ : `s ]`QGV 1.V`V:s .V GQ QI ]:JVCs RV]1H 1I:$Vs Q` G`:1J sVH 1QJs Q` s.FSRC`VRVGFP I1HV :` V` 1II%JQ.1s QH.VI1s `7 %s1J$ :J :J 1RGFP :J 1GQR78 II:$Vs A Q D HQ``Vs]QJR Q R1``V`VJ HQI]QJVJ s Q` .V EA5 1I:$V E Q .V PN5 1I:$V F .V R :JR 1I:$V G Q .V CA `1VCR Q` .V .1]]QH:I]%s8 BLA5 G:sQC: V`:C :I7$R:CQ1R J%HCV%s5 :J V`1Q` ]:` 6 BST5 GVR J%HCV%s Q` .V s `1: V`I1J:C1s6 CA5 `1VCR CA Q` .1]]QH:I]%s6 CVA5 HVJ `:C :I7$R:CQ1R J%HCV%s6 IPAC5 1J V`s 1 1:C J%HCV%s Q` .V ]Qs V`1Q` C1IG Q` .V :J V`1Q` HQII1ss%`V6 NAHC5 J%HCV%s :HH%IGVJs HQ`V6 NAHS5 J%HCV%s :HH%IGVJs s.VCC6 PN5 ]:`:0VJ `1H%C:` .:C:I1H J%HCV%s6 R 5 `V 1H%C:` .:C:I1H J%HCV%s8

G`:1J ^G:0X`1:%6RR%`` :JR K1V``V`5 _8 IJ Q`RV` Q RV VÌ1JV .V QJsV Q` .V FS ]`QIQ V`5 1V ]V``QÌVR 1J s1 % .7G`1R1<: 1QJ V6]V`1IVJ s QJ sVH 1QJs Q` 11CR 7]V IQ%sV 1ss%Vs ^VIG`7Qs Q` G`:1J_ HQCCVH VR R%`1J$ VIG`7QJ1H RV0VCQ]IVJ :JR : R1``V`VJ ]Qs RJ: :C s :$Vs ^ F1$%`V _8 NQ V6]`Vss1QJ Q` FS 1:s RV VH :GCV G7 1J s1 % .7G`1R1<: 1QJ : :J7 Q` .V VIG`7QJ1H RV0VCQ]IVJ :C s :$Vs Vs VR5 1JHC%R1J$ E8 5 E8 5 E 8 :JR E8 8 A ]Qs RJ: :C R:7 ^P_5 1V:@ Q IQRV`: V `s V6]`Vss1QJ 1s RV VH :GCV 1J .V NAH5 BNST5 CVA :JR CA8 LV0VCs Q` `s V6]`Vss1QJ QGsV`0VR : C: V` ]Qs RJ: :C s :$Vs ^P5 P :JR P_ 1V`V .1$.V`8 A .V P s :$V5 `s CV0VC Q` V6]`Vss1QJ 1s HQI]:`:GCV Q V6]`Vss1QJ QGsV`0VR 1J :R%C I1HV ^s :` 1J$ P _8 T.VsV `Vs%C s 1JR1H: V .: .V FS ]`QIQ V` :H 101 7 1s RV VH :GCV s :` 1J$ :`Q%JR G1` . :JR .: .1s V6]`Vss1QJ 1JH`V:sVs 11 . 1IV5 s :G1C1<1J$ : ]Qs RJ: :C R:7 8 T.VsV `Vs%C s HQJ`1ÌVR .QsV ``QI : s %R7 s.Q11J$ .: FS 1s QJC7 1V:@C7 V6]`VssVR 1J .V IQ%sV G`:1J : .V R:7 Q` G1` . :JR .: .1s V6]`Vss1QJ 1JH`V:sVs 11 . : ]V:@ : P ^K:1:JQ V :C85 _8 AC Q$V .V`5 .VsV R: : RVIQJs `: V .: .V FS ]`QIQ V` 1s : C: V ]`QIQ V` :JR 1s .V`V`Q`V : s%1 :GCV H:JR1R: V Q R`10V C`V V6]`Vss1QJ Q .V EA HVCCs 1.1CV :0Q1R1J$ RV0VCQ]IVJ :C HQI]VJs: 1QJs8 TQ ]`Q01RV : QQC :CCQ11J$ .V s %R7 Q` $VJV `%JH 1QJ s]VH1`1H:CC7 1J EA5 1V $VJV`: VR .V s.FSRC`VRVGFP `:Js$VJ1H IQ%sV IQRVC8 A s.Q` `:Js$VJV HQJ :1J1J$ : HDNA VJHQR1J$ : `C%Q`VsHVJ C`VRVGFP `%s1QJ ]`Q V1J ^C:CIVCs V :C85 _ %JRV` .V HQJ `QC Q` 8 @G FS ]`QIQ V` 1:s HQJs `%H VR ^ F1$%`V _8 T.1s HQJs `%H 1:s I1H`Q1J=VH VR 1J Q .V ]`QJ%HCV%s Q` `V` 1C1

P P B

NAH

F1$%`V 8 ^B_ P: V`J Q` C`VRVGFP V6]`Vss1QJ R%`1J$ .V ]Qs RJ: :C G`:1J RV0VCQ]IVJ 8 AJ 1RGFP 1II%JQ.1s QH.VI1s `7 `V0V:CVR .: C`VRVGFP ]`Q V1J 1s JQ 7V ]`VsVJ 1J `sRC`VREGFP I1HV : P s :$V5 G% 1s V6]`VssVR 1J JV%`QJs Q` .V NAH :JR CA : s :$V P 8 CA5 `1VCR CA Q` .1]]QH:I]%s6 NAH5 J%HCV%s :HH%IGVJs8

ATG V6 VJRQ$VJQ%s 1J `QJ V6 8 @G ]`QIQ V` S ]QC7A

F%s1QJ VGFPRC`VER T

F1$%`V 8 SH.VI: 1H `V]`VsVJ : 1QJ Q` .V HQJs `%H %sVR Q $VJV`: V .V `:Js$VJ1H s.FSRVGFPRC`VER T IQ%sV C1JV

F1$%`V 8 SH.VI: 1H `V]`VsVJ : 1QJ Q` .V HQJs `%H %sVR Q $VJV`: V .V `:Js$VJ1H s.FSR VGFPRTAR C`VER T IQ%sV C1JV

NV6 5 1V ]V``Q`IVR 1II%JQ.1s QH.VI1s `7 V6]V`1IVJ s : R1``V`VJ ]`V :JR ]Qs RJ: :C s :$Vs ^E8 5 P5 :JR P _ Q H.:`:H V`1

B`V$I: 8 II B`V$I: 8 II

B`V$I: R8 II B`V$I: R8 II B`V$I: R8 II

F1$%`V 8 C`VRVGFP V6]`Vss1QJ ]: V`J `V0V:CVR G7 :J 1RVGFP 1II%JQ.1s QH.VI1s `7 1J .V `:Js$VJ1H s.FS RVGFPRC`VER T IQ%sV C1JV ^HQ`QJ:C sVH 1QJs_8 :H:5 :J V`1Q` HQII1ss%`V5 :J V`1Q` ]:` 6 AHGCLS.5 :HH%IGVJs J%HCV%s5 HQ`VLs.VCC6 :H]5 :J V`1Q` HQII1ss%`V5 ]Qs V`1Q` ]:` 6 BLA5 G:sQC: V`:C :I7$R:CQ1R J%HCV%s5 :J V`1Q` ]:` 6 BSTLP5 GVR J%HCV%s Q` .V s `1: V`I1J:C1s5 C: V`:C R101s1QJ5 ]Qs V`1Q` ]:` 6 CALL5 `1VCR CALL Q` .1]]QH:I]%s6 CVL5 HVJ `:C :I7$R:CQ1R J%HCV%s5 C: V`:C R101s1QJ6 DG5 RVJ : V $7`%s6 1H5 1J V`J:C H:]s%CV6 LGP5 C: V`:C $CQG%s ]:CC1R%s6 R 5 `V 1H%C:` .:C:I1H J%HCV%s6 s 5 s `1: V`I1J:C1s6 PL5 0VJ `:C ]Qs V`QC: V`:C .:C:I1H J%HCV%s8

:JR C1JVs `Q` TA HQJs `%H 8 V H.:`:H V`1

I8 D1sH%ss1QJ :JR ]V`s]VH 10Vs

MV .QRs %s1J$ s1 VRs]VH1`1H C`VRIVR1: VR `VHQIG1J: 1QJ :`V 1J0:C%:GCV Q RVH1].V` $VJV `%JH 1QJ 1J :`$V VR HVCC 7]Vs5 `V$1QJs Q` H1`H%1 s8 MQ`VQ0V`5 .V %sV Q` ]`QIQ V`s .: R`10V $VJV @JQH@Q% C: V 1J .V RV0VCQ]IVJ ]VÌ1 s Q :0Q1R VIG`7QJ1H CV .:C1 7 Q` VJ :ssQH1: VR 11 . $CQG:C $VJV @JQH@Q% 8 FQ` sQIV ]`QIQ V`s5 `:Js1VJ V6]`Vss1QJ Q` .V `VHQIG1J:sV R%`1J$ $VÌC1JV Q` VIG`7QJ1H RV0VCQ]IVJ I:7 QHH%` ^1J@VCV` V :C85 _5 1J 1.1H. H:sV .V %sV Q` 1JR%H1GCV 0V`s1QJs Q` .V C`V `VHQIG1J:sV 1s IQs :]]`Q]`1: V8 A IQs 11RVC7 %sVR 1JR%H1GCV `QÌ Q` C`V `VHQIG1J:sV 1s C`VER T 5 1.V`V $VJV RVCV 1QJ QHH%`s QJC7 :` V` :IQ61`VJ `V: IVJ ^B`QH:`R V :C85 _5 :C .Q%$. :IQ61`VJ `V: IVJ 1s `:1`C7 1JV``1H1VJ Q 1JR%HV C`V :H 101 7 1J .V G`:1J R%V Q ]QQ` GCQQR G`:1J G:``1V` ]VJV `:JHV ^C:s:JQ0: V :C85 _8 T.V %sV Q` 1JR%H1GCV C`VER T 1s R1sH%ssVR 1J .V ]`V01Q%s H.:] V` ^P:` III_8 HV`V5 1V .:0V $VJV`: VR 1Q 01:GCV IQ%sV C1JVs 11 . C`VER T V6]`Vss1QJ IQs C7 `Vs `1H VR Q .V EA8 T.V JV6 s V] 11CC 1J0QC0V G`VVR1J$ Q` .V `:Js$VJ1H R`10V` C1JV 11 . I1HV .:`GQ%`1J$ `CQ6VR :CCVCVs `Q` .V $VJV Q` 1J V`Vs 8 B`VVR1J$ 1s %JRV`1:7 11 . I% Q]1Q1R `VHV] Q` `CQ6VR ^ O]Ì LLL_ :JR

B`V$I: 8 II B`V$I: 8 II

:H: BSTLP AHG :H]

B`V$I: R8 II B`V$I: R8 II B`V$I: R8 II

CA CVL PL CA R

BLA CA

F1$%`V 8 C`VRVGFP V6]`Vss1QJ ]: V`J `V0V:CVR G7 :J 1RVGFP 1II%JQ.1s QH.VI1s `7 1J .V `:Js$VJ1H s.FS RVGFPRTARC`VERT IQ%sV C1JV ^HQ`QJ:C sVH 1QJs_8 :H:5 :J V`1Q` HQII1ss%`V5 :J V`1Q` ]:` 6 AHG5 :HH%IGVJs J%HCV%s5 HQ`VLs.VCC6 :H]5 :J V`1Q` HQII1ss%`V5 ]Qs V`1Q` ]:` 6 BLA5 G:sQC: V`:C :I7$R:CQ1R J%HCV%s5 :J V`1Q` ]:` 6 BSTLP5 GVR J%HCV%s Q` .V s `1: V`I1J:C1s5 C: V`:C R101s1QJ5 ]Qs V`1Q` ]:` 6 CALL5 `1VCR CALL Q` .1]]QH:I]%s6 CVL5 HVJ `:C :I7$R:CQ1R J%HCV%s5 C: V`:C R101s1QJ6 R 5 `V 1H%C:` .:C:I1H J%HCV%s6 PL5 0VJ `:C ]Qs V`QC: V`:C .:C:I1H J%HCV%s8

GPR `CQ6VR ^ G]` LLL_ I1HV5 :s ]`V01Q%sC7 RQJV `Q` .V JQJR1JR%H1GCV FSRC`V C1JV8 T.V :J:C7s1s Q` :IQ61`VJR :JR 0V.1HCVR `V: VR :R%C I1HV 11CC RV V`I1JV 1.V .V` O]`I :JR G]` :`V V``1H1VJ C7 1J:H 10: VR s]VH1`1H:CC7 1J .V EA8 S.Q%CR .1s GV s%HHVss`%C5 .V 1Q C1JVs 1Q%CR `V]`VsVJ : .1$.C7 0:C%:GCV IQ%sV QQC Q :`$V :J7 $VJV Q` 1J V`Vs 1J .V EA5 1J ]:` 1H%C:` $VJVs ]Q VJ 1:CC7 1J0QC0VR 1J .V JV$: 10V :``VH 10V s : V HQJsV_%VJ Q G1J$V 1J Q61H: 1QJ 11 . R`%$s Q` :G%sV5 R`%$ H`:01J$ Q` RV]`Vss10V s : Vs .: H.:`:H V`1

GENERAL DISCSSION

GVJV`:C :1I Q` .V .Vs1s

T.V I% Q]1Q1R `VHV] Q` 1s Q` ]`1IV 1J V`Vs 1J JV%`QsH1VJHV :JR .V:C .H:`V8 T.V I% `VHV] Q` 1s .V ]`1I:`7 IQCVH%C:` :`$V `Q` IQ`].1JV 1J 010Q :JR IVR1: Vs 1 s I%C 1]CV V``VH s 1JHC%R1J$ :J:C$Vs1:5 QCV`:JHV5 RV]VJRVJHV5 `Vs]1`: Q`7 RV]`Vss1QJ5 HQJs 1]: 1QJ8 ACsQ5 1J ].7s1QCQ$75 .V I% `VHV] Q` IQR%C: Vs H:`R1Q0:sH%C:` :JR $:s `Q1J Vs 1J:C `%JH 1QJs5 JQH1HV] 1QJ5 CQHQIQ 1QJ :JR J: %`:C `V1:`Rs s%H. :s sQH1:C GV.:01Q`5 sV6%:C :H 101 7 :JR `QQR HQJs%I] 1QJ8 T.V %sV Q` Q :C @JQH@Q% IQ%sV IQRVC `Q` .V I% `VHV] Q`5 :s 1VCC :s CQH:C ].:`I:HQCQ$75 1:s : ]Q1V``%C ]`Q$`Vss 1J Q]1Q1R `VsV:`H.8 HQ1V0V`5 .V ]`VH1sV `QCV Q` I% `VHV] Q`s : .V CV0VC Q` JV%`:C ]: .1:7s `VI:1Js %J@JQ1J8 TQ .1s :1I5 1V :`$V VR sVCVH VR I% `VHV] Q` ]Q]%C: 1QJs %s1J$ C`VRCQ6P G:sVR @JQH@Q% VH.JQCQ$7 :JR V6:I1JVR IQCVH%C:`5 HVCC%C:` :JR GV.:01Q`:C `Vs]QJsVs Q` HQJR1 1QJ:C $VJV 1H I% :J I1HV8 IJ P:` I5 I 1J0Vs 1$: VR .V `QCV Q` I% Q]1Q1R `VHV] Q`s V6]`VssVR 1J GABAV`$1H `Q`VG`:1J JV%`QJs 1J Q]1Q1R V``VH s %s1J$ DC6RI% I1HV8 AJ: QI1H:C R1s `1G% 1QJ Q` .V O]`I IRNA 1J .1s I% :J s.Q1VR : @JQH@Q% 1J .V s `1: %I5 :I7$R:C: :JR .1]]QH:I]%s8 BV.:01Q`:C :ss:7s s.Q1VR JQ RV VH :GCV `QCV Q` .V :`$V VR I% `VHV] Q`s 1J :J:C$Vs1H V``VH s :JR ].7s1H:C RV]VJRVJHV 1JR%HVR G7 IQ`].1JV5 :JR `V1:`R1J$ ]`Q]V` 1Vs Q` Q]1: Vs 1V`V I:1J :1JVR8 IJ V`Vs 1J$C7 .Q1V0V`5 .V :J:C7s1s Q` :H 101 7 :JR Q]V`:J GV.:01Q`s `V0V:CVR .: GABAV`$1H `Q`VG`:1J I% `VHV] Q`s :`V VssVJ 1:C `Q` CQHQIQ Q` V``VH s Q` .V`Q1J :JR 1J.1G1 IQ 10: 1QJ Q $V .V`Q1J :JR H.QHQC: V8 IJ P:` II5 I V6:I1JVR .V `QCV Q` I% `VHV] Q`s 1J GABAV`$1H `Q`VG`:1J JV%`QJs 1J :% 1s 1HRC1@V GV.:01Q`s8 M% KO `VH:]1 %C: V :J :% 1s 1HRC1@V ].VJQ 7]V ^BVH@V` V :C85 _8 I V0:C%: VR HQ`V :JR sVHQJR:`7 s7I] QIs Q` :% 1sI s]VH `%I R1sQ`RV`8 NV1 .V` sQH1:C GV.:01Q` ^HQ`V s7I] QI_ JQ` CV0VCs Q` RV`VJs10V :JR HQJ`C1H :J61V 7 ^sVHQJR:`7 s7I] QIs_ 1V`V IQR1`1VR 1J .V HQJR1 1QJ:C I% :J s8 T.1s s %R7 .V`V`Q`V 1JR1H: Vs .: RVCV 1QJ Q` .V I% Q]1Q1R `VHV] Q` 1J `Q`VG`:1J GABAV`$1H JV%`QJs 1s JQ s%``1H1VJ Q ]`QR%HV sQH1:C :JR :J61V 7RC1@V GV.:01Q`s5 H.:`:H V`1

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Pauline CHARBOGNE Mu opioid receptors and neuronal circuits of addiction: genetic approaches in mice

Résumé

Le récepteur opioïde mu est responsable des propriétés analgésiques et addictives puissantes de la !"#$%&'(')(*'(+R$ "!&'/( 0%1(1!&( !*'(*R02)%!&((+R 2$'++'(*'1(2%"24%)1(&'4"!&045('1)( 0+(2!&&4(')(0( été peu étudié par des approches génétiques. Le récepteur mu est largement exprimé dans le système nerveux, essentiellement dans des neurones GABAergiques. Le premier objectif de mon projet a été dR%&02)%6'" le gène codant pour le récepteur mu dans les neurones GABAergiques du cerveau antérieur ')(*R en étudier les conséquences comportementales. Notre étude montre que ces récepteurs ne sont pas impl %74 1(*0&1(+R0&0+8 1%'(')(+0(* #'&*0&2'(#$91%74'((+0( !"#$%&'/( 0%1(74R%+1(1!&)('11'&)%'+1((+R'::')( $9#'"+!2! !)'4"(*'(+R$ "!&';(<'(#+41/(&!1(" 14+)0)1(%&*%74'&)(74'(2'1(" 2'#)'4"1(+% %)'&)(+0( !)%60)%!&(( 2!&1! '"( *'( +R$ "!&'( ')( *4( 2$!2!+0)/( " 6 +0&) un rôle entièrement nouveau pour cette population particulière de récepteurs (Manuscrit 1 : Mu opioid receptors in GABAergic forebrain neurons are necessary for heroin hyperlocomotion and reduce motivation for heroin and palatable food ). Aussi, cette population de récepteurs mu &R'1) pas responsable du syndrome autistique décrit chez les souris knockout totales (Manuscrit 2 : Mu opioid receptors in GABAergic forebrain neurons are not involved in autistic-like symptoms ). Enfin, nous avons développé un nouveau modèle transgénique visant +R%&02)%60)%!&(8 & )%74'(*4(" 2'#)'4"( 4(*0&1(+'1(&'4"!&'1(8+4)0 0)'"8%74'1/( 0%1(74%(&R0(#01(0=!4)%(( 4&(>&!2>!4)(2!&*%)%!&&'+(* )'2)0=+';(?!41(06!&1(0411%(%&%)% (+0(2" 0)%!&(*R4&'(+%8& '()"0&18 &%74'(@"'( #!4"(+R%&02)%60)%!&(*'(8 &'1(*R%&) ")(*0&1(+R0 98*0+'( )'&*4'/(74%(#'" '))"0(&!)0 '&)(*R )4*%'"(+'("+'( du récepteur mu dans ce microcircuit.

Mots-clés : récepteur opioïde mu, souris knockout conditionnelles, récompense, nociception, trouble autistique, amygdale étendue.

Résumé en anglais

Mu opioid receptors mediate the strong analgesic and addictive properties of morphine and heroin; however mu receptor function at circuit levels is not well understood and has been poorly studied by genetic approaches. These receptors are widely expressed throughout the nervous system, essentially in GABAergic neurons. The first aim of my project was to genetically inactivate the mu receptor gene in GABAergic forebrain neurons and study the behavioral consequences. Our study shows that these mu receptors are not implicated in morphine-induced analgesia and physical dependence, but are essential for locomotor effects of heroin. Moreover, our data show that these receptors inhibit motivation to consume heroin and chocolate, revealing an entirely new role for this particular population of mu receptors (Manuscript 1: Mu opioid receptors in GABAergic forebrain neurons are necessary for heroin hyperlocomotion and reduce motivation for heroin and palatable food ). Also, mu receptors expressed in forebrain GABAergic neurons are not responsible for the autistic syndrome described in total mu receptor knockout mice (Manuscript 2: Mu opioid receptors in GABAergic forebrain neurons are not involved in autistic-like symptoms ). Finally, we developed a new transgenic model targeting the mu receptor gene in glutamatergic neurons, but receptor deletion was not detectable in conditional mice. We also initiated the creation of a transgenic Cre driver line to knockout genes of interest in the extended amygdala, and this tool will enable us to study mu receptor function within this microcircuit.

Key words: mu opioid receptor, conditional knockout mice, reward, nociception, autism spectrum disorder, extended amygdala.