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Thesis

Reference

Disinhibition of ventral tegmental area dopamine neurons projecting to the nucleus accumbens drives heroin reinforcement

CORRE, Julie

Abstract

L'addiction est une neuropathologie chronique et généralement caractérisée par 1) un comportement compulsif de recherche et de prise de la substance, 2) la perte de contrôle sur la quantité consommée et 3) l'usage de la substance en dépit des conséquences négatives.

Aux États-Unis les abus et détournements des opioïdes sur prescription ont mené à une véritable épidémie d'overdoses et l'addiction aux opioïdes est aujourd'hui l'un des plus gros défis liés aux substances d'abus. L'hypothèse dopaminergique, formulée dans les années 1980, stipule que le point commun des substances addictives est leur faculté d'augmenter les niveaux méso-limbiques de dopamine, résultant en un renforcement du comportement d'auto-administration, amenant potentiellement à l'addiction. Un nombre considérable de données expérimentales obtenues avec les psychostimulants supportent cette hypothèse qui a cependant été questionnée pour une autre catégorie de drogues d'abus, les opioïdes. Pour l'héroïne en particulier, l'implication de la dopamine dans ces propriétés renforçantes demeure controversée, et [...]

CORRE, Julie. Disinhibition of ventral tegmental area dopamine neurons projecting to the nucleus accumbens drives heroin reinforcement. Thèse de doctorat : Univ. Genève et Lausanne, 2018, no. Neur. 230

DOI : 10.13097/archive-ouverte/unige:106828 URN : urn:nbn:ch:unige-1068282

Available at:

http://archive-ouverte.unige.ch/unige:106828

Disclaimer: layout of this document may differ from the published version.

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DOCTORAT EN NEUROSCIENCES des Universités de Genève

et de Lausanne

UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Professeur Christian Lüscher, directeur de thèse

TITRE DE LA THÈSE

DISINHIBITION OF VENTRAL TEGMENTAL AREA DOPAMINE NEURONS PROJECTING TO THE NUCLEUS ACCUMBENS DRIVES HEROIN REINFORCEMENT

THÈSE Présentée à la Faculté des sciences

de l’Université de Genève pour obtenir le grade de Docteure en Neurosciences

par

Julie CORRE

de Brest (France) Thèse N° 230

Genève

Editeur ou imprimeur : Université de Genève 2018

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ACKNOWLEDGMENTS

I would like to express my deepest gratitude to all the persons who contributed to these almost four years of PhD studies and have made this Swiss journey (mostly) fantastic.

First of all I would like to thank my thesis director, Prof. Christian Lüscher who gave me the

opportunity to work in his lab. Thanks for the patience and the support when needed, I will leave the CMU another person for sure.

I also would like to thank my thesis committee, Prof. Anthony Holtmaat, Prof. Alan Carleton, Prof.

Ivan Rodriguez and Prof. Hans Zeilhofer for the advices on my work and putting time and effort to discuss it.

From the bottom of my heart I thank all my coworkers, from my lab and the neighboring ones, for their endless support, encouragements but mostly all the drinks they offered me. These moments with you have been incredible. Special thanks to Dr. Vincent Pascoli (a.k.a Jean-Pierre) my unofficial supervisor, I will miss not going with you at La cantine for some choucroute. Other special thanks to Agnès Hiver and Catherine Pham, for making things work and always listening to my stories. Also huge thanks to Petit Clément (de Dieu!), Sarah (I still will be your coach and physio!), Niniels (for introducing me to weird experimental music), Yves (the SSN 2016 was fabulous!), Ruud (my second non-official supervisor), Michaël, Meghan, Ridouane, Fabrice, and all the people I am, or have been, around in the Lüscher Lab, you guys are wonderful persons!

Very special thanks to the coffee machine, my loyal companion during the long nights in the lab.

I also would like to thanks all the non-Lüscher and non-CMU people, for the apéros, the laughs, the shared concerns, the crazy nights, the trainings and your loving kindness. Julie , Sopickle (Wubba Lubba Dub Dub au Connemara!), Toto (attaque talon and PTL 2019!), Grand Clément, Béa, Marco, Greta, Lucie, JP, Claudia, Karin, Tom, Rodriguo, Thomas, Julien, and all the other ones I am

(temporarily) forgetting. You make me grow as a person and you stand me and my voice, which are not easy tasks.

Thanks to my long-time friends, you have my infinite love, you saw me evolved and I hope I am making you proud. Marie-Martine-Morue ma sœur de cœur, Georges mon roux de secours, Loulou, Steph, Mouche, Aurélie, Céline, Katie, Braulio, Imran. Distance does not matter.

I need to thank my family, Nicolas and Boris my big brothers, my father, but especially my mother and my little brother Vincent for their unconditional love, their support and the person I am today.

Finally thanks to Antoine, for your incommensurable love, for being the amazing person you are.

I dedicate this thesis to my brother Boris, I would have loved you being there to see me becoming the second Dr. Corre of the family.

Thanks to you all.

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RÉSUMÉ

L’addiction est une neuropathologie chronique et généralement caractérisée par 1) un

comportement compulsif de recherche et de prise de la substance, 2) la perte de contrôle sur la quantité consommée et 3) l’usage de la substance en dépit des conséquences négatives. Aux États- Unis les abus et détournements des opioïdes sur prescription ont mené à une véritable épidémie d’overdoses et l’addiction aux opioïdes est aujourd’hui l’un des plus gros défis liés aux substances d’abus. L’hypothèse dopaminergique, formulée dans les années 1980, stipule que le point commun des substances addictives est leur faculté d’augmenter les niveaux méso-limbiques de dopamine, résultant en un renforcement du comportement d’auto-administration, amenant potentiellement à l’addiction. Un nombre considérable de données expérimentales obtenues avec les psychostimulants supportent cette hypothèse qui a cependant été questionnée pour une autre catégorie de drogues d’abus, les opioïdes. Pour l’héroïne en particulier, l’implication de la dopamine dans ces propriétés renforçantes demeure controversée, et notamment dans la phase initiale d’exposition à l’héroïne, c’est à dire avant qu’une dépendance s’établisse. Dans cette étude nous confirmons les propriétés renforçantes de l’héroïne à l’aide d’un modèle murin d’auto-administration. Nous avons ensuite contrôlé quels étaient les effets initiaux de l’héroïne sur la souris en utilisant des indicateurs calciques génétiquement encodés, un capteur fluorescent à la dopamine et le gène d’activation immédiate cFos. Nous avons cartographié le circuit impliqué dans ces effets initiaux en nous servant de traceurs neuronaux rétrogrades et nous avons observé une activation sélective des neurones dopaminergiques de la partie ventro-latérale de l’aire tegmentale ventrale (VTA), une population de neurones projetant sur le noyau accumbens. Nous avons ensuite tiré profit d’outils opto et

chémogénétiques, exprimés dans les neurones dopaminergiques ou GABAergiques de la VTA afin d’inhiber ou d’occlure l’activité neuronale dans le système méso-limbique. Ces expériences établissent un lien de causalité et démontrent de façon non équivoque le caractère nécessaire et suffisant d’une désinhibition des neurones dopaminergiques de la VTA dans la nature renforçante des opioïdes. Nos données confirment un mécanisme de désinhibition où l’héroïne cible les neurones GABAergiques de la VTA résultant en une augmentation de l’activité des neurones dopaminergiques.

Ce scénario est applicable pour un stage très précoce d’exposition à l’héroïne et non pas uniquement quand la consommation est devenue chronique et les souris dépendantes.

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ABSTRACT

Addiction is a chronic brain disease generally characterized by 1) compulsion to seek and take the drug 2) loss of control over the quantity and 3) use of the substance despite negative consequences.

In the United States abuses and misuses of prescribed opioids have led to an epidemic of drug overdose and opiates addiction is one of the biggest drug problem today. The dopamine hypothesis, proposed in the 1980s, states that the defining commonality of addictive drugs is an increase of mesolimbic dopamine levels, which reinforces self-administration eventually leading to addiction.

Much experimental evidence obtained with psychostimulants supports this hypothesis, but it has been challenged for opioids. For heroin in particular, the involvement of dopamine in the reinforcing properties remains controversial, especially for the initial phase of heroin exposure before

dependence is established. Here we confirm the reinforcing properties of heroin with a self- administration (SA) model in mice. We then monitor the initial effects of heroin in mice with

genetically encoded calcium indicators, a fluorescent DA sensor and the immediate early gene cFOS.

We map the circuit involved in these initial effects using retrograde tracers and we observe the selective activation of DA neurons in the ventrolateral part of the ventral tegmental area (VTA), a population projecting to the nucleus accumbens (NAc). We then use chemogenetic and optogenetic effectors expressed in VTA DA or GABA neurons to mutually inhibit and occlude neural activity in the mesolimbic system. These experiments establish causality and unambiguously demonstrate necessity and sufficiency of VTA DA neuron disinhibition for the opioid reinforcement. The data confirm a disinhibitory mechanism whereby heroin targets GABA neurons that then leads to an increase of DA neurons activity. This scenario applies from the very early stage of drug exposure and not only when opioid intake has become chronic and the mice are dependent.

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TABLE OF CONTENTS

TABLE OF CONTENTS ... 5

LIST OF ABBREVIATIONS ... 8

LIST OF FIGURES ... 11

INTRODUCTION ... 13

1. A brief history of opioids ... 13

1.1 Early history of opium use and abuse ... 13

1.2 From opium poppy to heroin, the era of synthetic opioids ... 15

1.3 The discovery of opioid receptors and opioid peptides, an endogenous machinery to process opioids ... 18

1.3.1 The three families of opioid peptides ... 19

1.3.2 The three families of opioids receptors ... 20

1.4 From miraculous medicine to drug of abuse, the downfall of heroin ... 21

1.4.1 Heroin as a miraculous medicine ... 21

1.4.2 Abuse of heroin, the heroin act ... 21

1.4.3 The “opiophobia” time, America declares war on opioids ... 22

1.5 The opioid crisis in the US, an enduring issue that still has no solution ... 23

1.5.1 Opioids as the main treatment for pain... 23

1.5.2 Dramatic increase of abuse and misuse of prescribed opioids ... 24

1.6 Toward a better use of opioids ... 25

2. The mesolimbic dopamine (DA) system ... 27

2.1 Overview of the mesolimbic DA system ... 27

2.2 Neuroanatomy of the mesolimbic DA system ... 28

2.2.1 The Nucleus Accumbens (NAc) ... 28

2.2.2 The Ventral Tegmental Area (VTA) ... 30

2.3 The functional role of the dopaminergic neurons of the VTA ... 32

2.4 The behavioral functions of the mesolimbic DA system ... 34

2.5 Interaction between mu opioid receptors (MORs) and dopaminergic transmission in the mesolimbic DA system ... 35

3. Animal behavioral models in addiction research ... 38

3.1 Modeling the binge/intoxication stage of addiction ... 38

3.1.1 Conditioned Place Preference (CPP) ... 38

3.1.2 Self-administration (SA) ... 40

3.2 Modeling relapse, animal models of the withdrawal/negative stage of the addiction cycle 41 3.3 Modeling relapse, animal models of the preoccupation/anticipation stage of addiction 42 3.3.1 Cue-induced reinstatement ... 42

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3.4 Summary of animal models of addiction ... 43

4. Main theories of addiction ... 43

4.1 Overview of the current theories of addiction ... 43

4.1.1 Aberrant learning theories of addiction ... 43

4.1.2 Frontostriatal-dysfunction (or impulsivity) theory of addiction ... 44

4.1.3 The opponent-process theory of addiction ... 44

4.1.4 Incentive-sensitization theory of addiction ... 45

4.1.5 Psychomotor-stimulant theory of addiction ... 45

4.2 Focus on the dopamine hypothesis of addiction ... 45

5. Opioid addiction and the mesolimbic DA system... 47

5.1 Effects of acute opioids on NAc neurons ... 48

5.2 Effects of chronic opioids on NAc neurons ... 49

5.3 Effects of acute opioids on VTA neurons ... 51

5.4 Effects of chronic opioids on VTA neurons ... 52

6. The dopamine hypothesis of opioid reward, challenged ... 53

6.1 The deprived/non-deprived hypothesis of opioid reinforcement ... 55

7. Aims of the project ... 58

Project: “Disinhibition of ventral tegmental area dopamine neurons projecting to the nucleus accumbens drives heroin reinforcement.” ... 60

1. Abstract ... 60

2. Introduction ... 60

3. Materials and Methods ... 61

3.1 Animals ... 61

3.2 Stereotaxic injections and optic fiber cannulation ... 62

3.3 Implantation of jugular vein catheter ... 62

3.4 Self-administration apparatus ... 63

3.5 Drug self-administration acquisition ... 63

3.6 Test of cue-associated drug seeking ... 64

3.7 Fiber photometry ... 64

3.7.1 Setup ... 64

3.7.2 VTA Calcium activity ... 64

3.7.3 Striatal dopamine dynamics ... 64

3.7.4 Fiber Photometry analysis ... 65

3.8 Immunostaining and cell counting ... 65

3.9 DA neuron self-stimulation/inhibition acquisition ... 66

3.10 Effect of heroin on locomotor activity ... 67

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3.11 Effect of chemogenetic inhibition of VTA DA neurons on self-administration behavior ... 67

4. Results ... 67

4.1 Behavioral model of heroin self-administration ... 67

4.2 Heroin increases NA DA levels and VTA DA activity in drug-naïve animals ... 69

4.3 Lateroventral VTA DA neurons projecting to the NAc drive heroin-induced increased level in DA 72 4.4 Chemogenetic inhibition of VTA DA neurons decreases heroin self-administration ... 76

4.5 Heroin occludes optogenetic self-stimulation of VTA DA neurons ... 78

4.6 Heroin occludes reinforcing effects of inhibition of VTA GABA neurons ... 80

5. Discussion ... 82

5.1 Function of subpopulation located in the lateroventral VTA ... 82

5.2 Role of GABA neurons in the VTA ... 84

5.3 Critique of the TPP model... 85

5.4 Critique of pharmacological challenges ... 87

5.5 Critique of genetic challenges ... 88

5.6 Implications for opioid addiction and its treatment ... 88

6. General conclusions ... 89

7. References ... 92

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LIST OF ABBREVIATIONS

AC = Adenylate cyclase

AMPA = α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid AP = Antero-Posterior

BLA = Basolateral Amygdala

BNST = Bed Nucleus of the Stria Terminalis BSA = Bovine Serum Albumin

Ca2+=Calcium

cAMP = Cyclic adenosine 3’ 5’–monophosphate ChR2 = Channelrhododopsin-2

CNO = Clozapine-N-Oxide CNS = Central Nervous System CPA = Conditioned Place Aversion CPP = Conditioned Place Preference CTB = Cholera Toxin Subunit B D1R = Dopamine Receptor 1 D2R = Dopamine Receptor 2 DA = Dopamine

DOR = δ Opioid Receptor

DREADDs = Designer Receptors Exclusively Activated by Designer Drugs DRN = Dorsal Raphe Nucleus

DV = Dorso-Ventral FR = Fixed Ratio

GABA = γ-aminobutyric acid

GIRKs = G protein-coupled inwardly rectifying K+ channels GluR1 = Glutamate receptor 1

GP = Globus Pallidus

GPCR = G-Protein Coupled Receptor

IC50 = half maximal inhibitory concentration i.p. = Intraperitoneal injection

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i.v. = Intravenous injection K+ = Potassium

KOR = κ Opioid Receptor

LDT = Lateral Dorsal Tegmentum LDTg = Laterodorsal Tegmental Nucleus LTD = Long Term Depression

LTP = Long Term Potentiation LP = Lever Presse

LS = Laser Stimulation ML = Medio-Lateral MOR = μ Opioid Receptor mPFC = medial Prefrontal Cortex MSN = Medium-sized Spiny Neuron NAc = Nucleus Accumbens

NERT = Norepinephrin Transporter NMDA = N-methyl-D-aspartate OFC = OrbitoFrontal Cortex OD = Overdose

PAG = Periaqueductal Gray PBS = Phosphate Buffered Saline PF = Prefrontal Cortex

PPTg = Pedunculopontine Tegmental Nucleus RMTg = Rostro Medial Tegmetum

ROI = Region of Interest SA = Self Administration SERT = Serotonin Transporter SI = Self-Inhibition

SN = Substancia Nigra

SNc = Substancia Nigra pers compacta STN = Subthalamic Nucleus

TH = Tyroxine Hydroxylase

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vHippo = ventral Hippocampus VP = Ventral Pallidum

VStr = Ventral Striatum VTA = Ventral Tegmental Area WT = Wild-Type

6-OH-DA = 6-hydroxydopamine

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LIST OF FIGURES

Figure 1. A brief history of opiates, from the Sumerians 3400 B.C. to now Figure 2. Morphine, chemical structure

Figure 3. Heroin, chemical structure

Figure 4. Bayer heroin bottle (left) and advertisement (right)

Figure 5. Distribution of opioid peptides and their receptors in the brain Figure 6. Characteristics of the three families of opioid receptors.

Figure 7. Retail sales of opioid medications (grams of medication) from 1997 to 2007 Figure 8. Deaths from unintentional drug overdoses in the U.S. between 1997 and 2007 Figure 9. Major connections of the mesolimbic DA system in the brain

Figure 10. Distribution of DA neuron cell groups in the adult rodent brain Figure 11. Confirmed inputs to and outputs from VTA neurons

Figure 12. VTA DA neurons code for reward prediction error

Figure 13. Interaction between MORs and mesolimbic dopamine system at the NAc level Figure 14. Interaction between MORs and mesolimbic dopamine system at the VTA level Figure 15. Schematic of an operant box for self-administration session, acquisition phase Figure 16. Schematic of an operant box for cue-induced reinstatement session

Table 1. The mechanistic classification of addictive drugs Figure 17. Effects of acute opioids on NAc neurons Figure 18. Effects of chronic opioids on NAc neurons Figure 19. Effects of acute opioid on VTA neurons Figure 20. Effects of chronic opioid on VTA neurons Figure 21. The nondeprived/deprived hypothesis

Figure 22. The deprived/non-deprived hypothesis: an integrated model of opioid reinforcement in the VTA

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Project: Disinhibition of ventral tegmental area dopamine neurons projecting to the nucleus accumbens drives heroin reinforcement.

Figure 1. Behavioral model of heroin self-administration Figure 2. Heroin increase VTA DA activity in drug-naive animals

Figure 3. Latero-ventral VTA DA neurons projecting to the NAc drive heroin-induced increased level in DA.

Figure 4. Chemogenetic inhibition of VTA DA neurons decreases heroin self-administration Figure 5. Heroin occludes optogenetic self-stimulation of VTA DA neurons

Figure 6. Heroin occludes reinforcing effects of inhibition of VTA GABA neurons

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INTRODUCTION

1. A brief history of opioids

Figure 1. A brief history of opiates, from the Sumerians 3400 B.C. to now. From the earliest civilizations to the present day, opioid use has always been about finding a balance between their strong analgesic properties and their euphoric effects, which can lead to misuse and abuse.

1.1 Early history of opium use and abuse

“Presently she cast a drug into the wine of which they drank to lull all pain and anger and bring forgetfulness of every sorrow.”

The Odyssey, Homer (Ninth century B.C)

Opioid misuse and addiction is a highly topical issue of major importance today, but unfortunately it is not a novel one. Although it is difficult to know exactly when the opium poppy began to be

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cultivated and used, there is evidence that its use dates back to the Antiquity 1. Despite the sometimes very metaphorical language of ancient writings that makes them quite difficult to interpret, a clear picture of opium use emerges from them. Indeed, some divinities of the ancient Greeks are portrayed crowned with poppies or holding them in their hands. Interestingly, the divinities associated with opium are Hypnos (the god of sleep), Nyx (the goddess of night), and Thanatos (the god of death), showing the already ambivalent perception of opium as a powerful drug, as able to heal as to kill 2.

The Sumerians (inhabitants of today’s Iraq) were cultivating poppies and extracting opium from the seeds as early as 3000 B.C. At that time, opium was called “gil”, meaning “joy”, and the poppy was called the “Hul Gil”, meaning the “plant of joy”.

Opium was used for spiritual and religious purposes and was thus used only by priests during ceremonies. However, it was not long before opium use shifted toward more medicinal purposes, given its analgesic and sedative properties. The Sumerians passed along this culture of medicinal opium use to the Babylonians and later to the Egyptians.

An example of the use of opium as a medicine can be found in The Ebers Papyrus (ca. 1550 B.C.), in which a recipe made from poppy seeds and excretion from flies is described as a “remedy to prevent excessive crying of children”. By 1300 B.C., the Egyptians are cultivating their own poppy fields and trading opium all along the Mediterranean.

Eight centuries later, Hippocrates, “the father of medicine”, records the magical attributes of opium and establishes its place as a strong legitimate medicine.

But these early days of opium use also came with problems, and even then, some “physicians” were concerned about this substance. Depending on how the mixtures were made and administered, the potency and the rate of absorption could drastically change, making these remedies dangerous or even fatal. Despite these concerns, opium spread to China and India (c. 8 A.D.).

During the Holy Inquisition (c. 1300 A.D.) in Europe, opium disappeared from the records for almost two centuries, possibly because at the time, everything from the East was considered evil.

The situation in the Orient was totally different, where the opium trade led to widespread addiction.

Already in the 16th century, manuscripts describe drug abuse and tolerance in many countries, including Turkey, Egypt, China (but also Germany and England). Opium addiction developed especially quickly in China where it was mostly smoked or orally ingested and was still prepared directly from the poppy plant. In 1799, facing an opium crisis, the Chinese Emperor Kia King prohibited opium totally, making any opium trade or growing illegal 3.

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In 1527, Paracelsus, a Swiss astrologer, alchemist, and physician reintroduced opium in the Occident via an opium tincture he created called Laudanum (from the Latin verb laudare, to praise). His mixture contained opium, spices, and amber, among other substances and was used as a pain-killer.

Paracelsus’ Laudanum remained rarely used, but in 1680 the English apothecary Thomas Sydenham created his own version of Laudanum that he called Sydenham’s Laudanum and sold it as a remedy for numerous diseases. Today, Laudanum can still be purchased with a prescription in the U.S.A. and in the U.K. 4,5!

1.2 From opium poppy to heroin, the era of synthetic opioids

Figure 2. Morphine chemical structure. Morphine is an opiate alkaloid extracted from the plant Papaver somniferum. Morphine is the principal active compound in opium. Morphine acts as a full opioid agonist. Morphine acts on the entire central nervous system but also on smooth muscles.

In 1806, Friedrich Sertürner, a German pharmacist, isolated the active component in opium by dissolving it in acid. He named it morphine after Morpheus, the Greek god of dreams 1. In the 1850s, morphine began to be used as an anesthetic for minor surgeries and chronic pain. French

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physiologist and pioneer in anesthesia research Claude Bernard was the first to investigate the effects of morphine on animals. In 1874, he discovered that morphine potentiated the effects of chloroform and thus when the two were used in combination, allowed for reduced inhalation of chloroform, which had been responsible for numerous deaths 6.

Morphine was also used as a cure for opium addiction. Although the addictive properties of opium had been well known for some time, the same properties of morphine were not yet known. Opium addiction was becoming a public health issue, and in 1821 Thomas de Quincy published his

autobiographical testimony of opium addiction “Confessions of an English Opium-eater”. By 1830, the problem was such that approximately one ton of opium was being imported from Turkey and India by the UK to satisfy the demand for both medicinal and recreational use 1!

It quickly became obvious that morphine was as addictive as opium, leading to attempts to synthetize an opioid that would still have analgesic effects but would not lead to addiction.

Figure 3. Heroin chemical structure. Diacetylmorphine hydrochloride (heroin) is the hydrochloride salt of a diacetyl derivative of morphine. It is more lipid soluble than morphine and quicker to enter the brain. Heroin is metabolized into 6-monoacetylmorphine and morphine. Morphine is the active compound responsible for its effects on the central nervous system and on its peripheral targets.

In 1874, Charles Romley Alder Wright, an English chemist and physicist working at the St. Mary’s Hospital Medical School in London, synthetized heroin from morphine by isolating the acetylated

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derivatives of morphine. He called the compound that would later be known as heroin (or diacetylmorphine) "Tetra acetyl morphine". At first, diacetylmorphine did not interest most researchers or pharmaceutical companies, and twenty years passed before it gained recognition.

Starting in the 1890s, several investigators began to look more closely at the effects of

diacetylmorphine, and interest in it as a medication started to spread. Positive reports from these investigators about diacetylmorphine resulted the German company “Bayer” producing the drug on a commercial scale in 1898 7. They named their product “heroin”, probably after the German words

“heroisch”, which means “large, powerful, or extreme”. The word “heroin” is now used as a common name for the diacetylmorphine molecule.

Figure 4. Bayer heroin bottle (left) and advertisement (right). In the early 1900s, heroin was sold as a remedy for morphine addiction, cough, and other respiratory diseases.

Interestingly, heroin was not commercialized as an analgesic, but rather as a cough-suppressant. At that time, tuberculosis was prevalent, so there was a large demand for effective treatments. Heroin was welcomed as a wonder drug and received great enthusiasm from the medical community.

Heroin was also used to treat morphine and codeine addiction. It was presented as a safer, non- addictive, and more potent opioid than morphine and codeine. The 20th century saw the discovery of several opioids, many still in use today. In 1939, meperidine was synthetized, followed in 1946 by the discovery of methadone, a μ opioid receptor (MOR) agonist with similar properties to morphine.

However, methadone has a slower onset than morphine and its effects last longer. Additionally, methadone is orally active, dramatically reducing risk of diseases linked to i.v. injections. For these

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reasons, methadone has been used as a substitution treatment for heroin addicts for years, and patients taking methadone can lead a quite normal life 8,9.

1.3 The discovery of opioid receptors and opioid peptides, an endogenous machinery to process opioids

Why are opioids so potent and what is the molecular basis underlying their strong effects? In part, the answer lies in the existence of an endogenous opioid system consists of three families of peptides and three families of receptors. The endogenous opioid peptides and their receptors have a widespread, but selective, distribution throughout the nervous system. The endogenous opioid system is involved in the regulation and modulation of many important functions, such as analgesia, reward-related mechanisms, and stress response 10.

In order to identify effective treatments for addiction and to develop effective, non-addictive pain medications, precise dissection and characterization of this system is crucial.

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1.3.1 The three families of opioid peptides

Figure 5. Distribution of opioid peptides and their receptors in the brain, from Bennaroch (2012).

Distribution of the endogenous peptides (left), β-endorphin, enkephalins, and dynorphin, and their 3 receptors (right) μ, δ, and κ, in the central nervous system (CNS). The larger dots represent more expression of the peptides or the receptors. Neurons expressing β-endorphin are mainly found in the arcuate nucleus of the hypothalamus and the nucleus tractus solitaries. Enkephalin- and dynorphin- expressing neurons are primarily located in the striatum in both D1 and D2 medium spiny neurons (MSN). Opioid receptors are widely expressed throughout the brain. Notice the strong expression of MORs in areas known to be involved in reward such as the medial prefrontal cortex (mPFC), the nucleus accumbens (NAc), and the ventral tegmental area (VTA).

In 1975, Kosterlitz and Waterfield were working on the guinea pig ileum and noticed that brain extracts inhibited the release of acetylcholine 11. When they applied naloxone, an opioid receptor antagonist, they could reverse the effects of these brain extracts. In the same year, the components responsible for these effects on acetylcholine were identified as two pentapeptides, Met- and Leu-

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enkephalin 12. Further investigation made clear that the sequence of Met-enkephalin was also present in another molecule which had already long since been identified, β-endorphin 13,14. These two families, the enkephalins and endorphins, were found to share a high affinity for brain opioid receptors. In 1981, the third family of opioid peptides was identified and called dynorphins 15–17. These endogenous opioids come from large proteins called precursors that undergo proteolytic cleavage. Proopiomelanocortin is the precursor to β-endorphin, preproenkephalin is the precursor to both Met and Leu-enkephlin, and preprodynorphin is the precursor to dynorphin. As represented in figure 5, the different types of endogenous opioids do not necessarily share expression patterns in the CNS 10.

1.3.2 The three families of opioids receptors

Figure 6. Characteristics of the three families of opioid receptors. Three families of opioid receptors have been identified and characterized. Opioid receptors are differentially expressed throughout the brain, and their respective activations result in a wide range of behavioral effects.

After the discovery of the peptides, researches focused on identifying which receptors they were binding to. Pharmacological studies allowed for the identification of three families of receptors.

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Mu receptors (MOR) are activated by β-endorphins, δ receptors (DOR) are activated by both enkephalins and β-endorphins, and κ receptors (KOR) are activated by dynorphins. As shown in figure 5, the three families of receptors are strongly expressed, but not similarly distributed, in the CNS 10.

The endogenous opioid system is a crucial actor in drug addiction. It is a key component of the brain reward system, consisting of an interconnected neural network that mediates the

rewarding effects of drugs of abuse and of natural rewards like food and sex (Merrer et al. 2009).

1.4 From miraculous medicine to drug of abuse, the downfall of heroin 1.4.1 Heroin as a miraculous medicine

The history of heroin is quite unique. It went from being seen as a miracle drug, extoled and revered by the medical community, to being seen as a very dangerous substance only used by social outcasts.

To understand how that happened, let’s go back a bit in time. We are at the beginning of the 1900s, and heroin is incredibly popular. Hailed and praised as a wonder drug, heroin is massively prescribed.

It acquired such a positive image in the medical community that in the U.S, the philanthropic Saint James Society is shipping heroin samples through the mail to morphine addicts who are trying to give it up 19! Several studies, mostly on respiratory diseases (Manges, Wood, Jacobi and many others) 20, will confirm the benefits of heroin as a cough-suppressant, especially compared to morphine. Even at the turn of the century, there is almost no recognition of any danger in using heroin, despite the worries of some researchers and physicians who were concerned about this massive use and the potential deleterious effects of heroin 19. It took a lot of time before there was general, widespread agreement that heroin was a dangerous and highly addictive substance. Furthermore, there was no other medication that could effectively replace heroin for some medical indications, which meant that extensive heroin use persisted, despite the harm that could be done.

1.4.2 Abuse of heroin, the heroin act

Before long, the enthusiasm generated by heroin began to fade. As early as 1902, Jarrige 21 criticized the use of heroin to treat “morphinism”, worrying that the “morphinist” would instead become the

“heroinist”, trading one addiction for another. He also reported that withdrawal from heroin was even more painful than from morphine. He concluded that rather than preventing the use of

narcotics, heroin treatment was responsible for an increase in the numbers of people suffering from

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addiction. Several physicians echoed these sentiments, reporting cases of “heroinism”, painful withdrawal, and poisoning. In 1909, heroin addiction was rising to alarming rates in the U.S. The government decided to intervene and passed the Opium Exclusion Act, forbidding importation of opium for the purpose of smoking. This is considered the “first shot” in the American war on opioids.

In 1913, the number of heroin addicts was skyrocketing, and as it became obvious that a total ban was imminent, Bayer decided to stop producing heroin. On December 17th 1914, the U.S.A went a step further and passed the Harrison Narcotics Act, aimed at restraining drug use (cocaine and heroin). Under this law, physicians, pharmacists, or any other person who prescribes narcotics now has to register and pay a tax. More than just a way to control the distribution of narcotics, the Harrison Act was the first legislative attempt to prohibit drugs of abuse 19. But even as heroin was being regulated, the interest in opioids remained. In 1916, the same year that State Public Hospitals in the US stopped dispensing heroin at their ’relief stations’, a new type of opioid was synthesized.

Freund and Speyer 22 from the University in Frankfurt in Germany synthetized oxycodone, hoping it would be characterized by strong analgesic properties but without the addictive effects of its cousins, heroin and morphine.

1.4.3 The “opiophobia” time, America declares war on opioids

Opioid addiction (and drug addiction in general) is an international problem, but the way it has been handled and considered differ considerably depending on the country. In the U.S., it seems that heroin addiction has been a major issue, eclipsing other countries’ problems with addiction. Therefore, the U.S will be the focus of this section.

1924 represents a big turn in the way U.S.A. sees opioids. The Heroin Act made the production, importation, and possession of heroin completely illegal. Even the medicinal use of heroin was banned.

But the prohibition of heroin did not solve the problem of heroin addiction, and heroin addicts were forced to buy it illegally on the streets. Prohibition opened the door for the emergence of a black market for heroin which would continue to grow during the next decades. Demand for heroin was met by smugglers from Southeast Asia's Golden Triangle (Laos, Thailand and Burma) but also from the French Connection, made up of Corsican gangsters with ties to the mafia who refinied raw opium from Turkey in Marseille laboratories. During the Second World War, the illegal opium trade routes were shut down, curbing the opioid addiction epidemic. However, this was only temporary, and as soon as the war ended, smugglers were back in business, and there was a rebound explosion of heroin addicts.

This effect was then amplified by the Vietnam War. Indeed, American involvement in Vietnam can be

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blamed for a massive increase in the quantity of illegally imported heroin into the U.S. Fifty years after the Heroin Act was signed, the government finally began to seriously consider real efforts to control the drug addiction problem. In 1970, the Controlled Substances Act passed and began to define schedules to prescribed narcotics. This scheduling was based on several criteria including, the medicinal value of the substance, its danger, and its potential to be abused. There were five different schedules, and number one included the most dangerous drugs, which are no longer allowed to be prescribed. On July 1st 1973, President Nixon created the DEA (Drug Enforcement Administration) in order to concentrate all the forces combatting drug trafficking into one agency and to send a strong message that America had declared war on opioids. In a speech, he said, “America has the largest number of heroin addicts of any nation in the world. Heroin addiction is the most difficult to control and the most socially destructive form of addiction in America today.” The 1980s would be the culmination of the “opiophobia” period in the U.S. It becomes such a national cause that President Reagan calls on the population to join him in a crusade of zero tolerance for drugs. Paradoxically, at the same time, some researchers were investigating the use of narcotics/opioids to treat chronic pain, which ultimately resulted in massive numbers of prescriptions for opioids in America. In 2017, the crusade is still not over, yet the situation has gotten so out of control that the Americans refer to it as the “opioid crisis”.

1.5 The opioid crisis in the US, an enduring issue that still has no solution

Opioid addiction is strongly associated in people’s minds with illegally obtained drugs and life as an outcast, but actually over the past two decades, most of the deaths caused by opioids (principally motor vehicles crashes and suicides by overdoses) are due to prescribed narcotic analgesics. A culture among physicians in the U.S. of prescribing opioids for any indication of pain led to a dramatic increase in the number of prescriptions. Increased availability allowed for increased misuse of these drugs, leading to the current opioid crisis 23,24.

1.5.1 Opioids as the main treatment for pain

From the 1920s to the late 1990s, opioids did not have a good reputation and were used with extreme caution. They were principally used to treat chronic cancer pain or to relieve dying patients.

In the late 1990s, a change in use can be observed. Laws regulating opioid prescriptions for use as general pain killers were loosened, resulting in an escalated use of therapeutic opioids, especially

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oxycodone. Different factors can explain these changes in the use of opioids and what looks like a step backwards. Indeed, despite the fact that the long-term utility of opioids in the treatment of pain has been questioned countless times prescriptions for opioids are still on the rise. In part, the

softening of regulations on opioid prescriptions is due to new pain-management guidelines set by the Joint Commission on the Accreditation of Health Care Organizations (JCAHO) in 2000 and the

increasing demand for better pain treatment. In addition, strong pharmaceutical lobbies promoting the use of opioids laid the groundwork for the “opioid come back”. Opioids became the “standard treatment” for pain, and the increase in opioid prescriptions went hand in hand with a dramatic increase in deaths due to abuse or misuse of the prescribed opioids 23–25.

1.5.2 Dramatic increase of abuse and misuse of prescribed opioids

Figure 7. Retail sales of opioid medications (grams of medication) from 1997 to 2007, from Manchikanti and al., (2012). Between 1997 and 2007, the sales of methadone, hydrocodone, and oxycodone dramatically increased by 1293%, 280%, and 866%, respectively. Overall, the sales of prescribed opioids have increased by 149%.

The numbers speak for themselves, and between 1999 and 2010 opioids sales quadrupled! Sales of hydrocodone skyrocketed with a 280% increase between 1997 and 2007, but the incontestable

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winner is oxycodone with an 866% increase in sales. Data on production and sales of opioids

demonstrate that there is enough to supply every adult American with 5 mg of hydrocodone every 6 hours for 45 days! Of course, this dramatic increase in opioid production, sales, and use comes with adverse consequences.

Figure 8. Deaths from unintentional drug overdoses in the U.S. between 1997 and 2007, from Manchikanti and al., (2012). Surprisingly, cocaine and heroin are not the main causes of deaths from overdose. Indeed, opioid analgesic abuse and misuse causes more deaths than heroin and cocaine combined.

On adverse consequence is the spike in deaths by overdose. In 2007, quite shockingly, there were more deaths by overdose due to prescribed opioids than due to heroin and cocaine combined! In addition to accidental ODs, the number of suicides by opioid overdose drastically increased.

Emergency room visits related to opioids are also following this trend and increasing dramatically.

Furthermore, prescription opioids are not only abused, but are also misused. Data collected from emergency rooms show that prescribed opioids are often taken in larger amounts than prescribed, or even taken solely for recreation. Abuse and misuse are at such critical levels that the illicit use of prescribed opioids is now more problematic than that of non-prescribed opioids. The drug dealers are not on the streets anymore, they’re in doctor’s offices with prescription pads. And the ones pounding the pavement are no longer the primary source of abused opioids.

1.6 Toward a better use of opioids

The opioid crisis, like every crisis, is of course multi-factorial. The lack of knowledge about the consequences of opioid abuse and misuse among patients, the aberrant patterns of prescription

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delivery by physicians, and the perceived safety of prescribed opiates due to misleading marketing are a few reasons that explain this escalation of the use, abuse, and misuse of opioids over the past two decades.

So how can we curb the opioid crisis which is currently a plague on American society? The Food and Drug Administration decided to tackle the problem by focusing on proper education and drug

monitoring programs at every step of the process, e.g. from the manufacturers, to the prescribers, to the consumers. Closing the heroin trade routes during WWII was responsible for a drastic drop in opioid addiction at the time. Today, rather than prohibiting opiates, which leads to the development of a black market and thus circulation of hazardous, very impure substances, the FDA aims to encourage manufacturers to improve their products by making the substances more difficult to abuse. They can do this by working on the physical and chemical barriers around the active component to avoid diversion of the product from an initially oral drug to an injectable drug, for example. Another way to avoid diversion, and thus abuse, is to develop the product as a prodrug, which will be processed and activated only when inside the gastrointestinal tract. The drug is thus unattractive to snoring or injection. Another possible solution is to add an opioid antagonist to the product to remove the feeling of euphoria. Finally, another axis of research to avoid diversion, abuse, and misuse is to work on the delivery system of the drug in order to make it less easy to transform, for example prescribing an implant rather than pills.

Beyond altering the drugs themselves, prescribers and patients need improved education about the dangers of opiates. Indeed, one of the biggest issues contributing to the opioid crisis is the number of inappropriate prescriptions given, mainly due to a lack of knowledge about opioid safety and pain treatment.

As we saw with this condensed time travel, opioid use has always gone hand in hand with misuse, abuse, and addiction. Nowadays, opium is not considered to be a magical substance, and opioids are not thought of as miraculous drugs, yet we still lack a lot of knowledge about the proper use of opioids. Indeed, the molecular and cellular bases of opioid addiction are still debated in the field. A better understanding of the mechanisms of action of opioids and of opioid-induced neural changes is crucial to developing safer medications and better treatments for addiction.

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2. The mesolimbic dopamine (DA) system 2.1 Overview of the mesolimbic DA system

Figure 9. Major connections of the mesolimbic DA system in the brain. On this schematic diagram of the brain are depicted the DA projections (red), the glutamatergic (excitatory) projections (blue), and the GABAergic projections (green). BLA= Basolateral Amygdala, D1 = D1 MSN, D2 = D2 MSN, LHb=

Lateral Habenula, LDT = Laterodorsal Tegmentum, mPFC = Medial Prefrontal Cortex, NAc = Nucleus Accumbens, vHippo = Ventral Hippocampus, VTA = Ventral Tegmental Area, VP = Ventral Pallidum, RMTg = Rostromedial Tegmentum (Modified with permission from Lüscher C: Emergence of circuit model for addiction. Ann Rev Neurosci 2016; 39:257.).

The mesolimbic DA pathway is generally referred to as the “reward system” in the brain. We will focus on its two main structures, the NAc and the VTA. Considering how important this system is for species survival, it is not surprising that it has been extensively conserved throughout evolution. In normal conditions, this system controls responses to natural rewards such as food, social

interactions, and sex. It is generally acknowledged that the hijacking of this system by drugs of abuse eventually leads to addiction 26–31.

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2.2 Neuroanatomy of the mesolimbic DA system 2.2.1 The Nucleus Accumbens (NAc)

Heimer and Morgenson first discovered the nucleus accumbens, which is adjacent to the septum and distinct from the more dorsal compartments of the striatum 32,33. The NAc is an area of the brain where many of the pathways involved in reward processing and goal-directed behaviors converge.

Therefore, the NAc is more than just a “reward center”; it would be more accurate to describe it as a

“hub” involved in the regulation of action selection, integrating signals from limbic and cortical regions. The NAc changes its pattern of activity to influence both appetitive and aversive behaviors and is thus a critical convergent node and interface between the limbic and motor systems. For this reason, Floresco elegantly termed the NAc a “servant to many masters” in his detailed review about this brain structure 29.

The NAc is a flat, rounded region in the basal forebrain, which combined with the olfactory tubercle, makes up the ventral striatum. The ventral striatum is one of the components of the basal ganglia, a group of subcortical nuclei that also includes the dorsal striatum (caudate nucleus and putamen), the globus pallidus (GP), the ventral pallidum (VP), the substantia nigra (SN), and the subthalamic nucleus (STN). During embryonic development, the prosencephalon is subdivided into the diencephalon and the telencephalon, and the rostral portions of the latter then become the NAc and the olfactory tubercle (Salgado and Kaplitt 2015; Das and Altman 1970; Marchand and Lajoie 1986).

As is seen in dorsal striatal tissue, the NAc is characterized by a “patchwork-like” patch-matrix organization that consists of a mosaic pattern of two distinct compartments, the patch and the matrix 37,38. Quite interestingly these two compartments are characterized by differences in MOR expression densities. The patches are MOR-rich, while the matrices are poor in MOR binding sites 39–

41.

The main characteristic of the NAc, which is unique and not found in the rest of the striatum, is the presence of a central core surrounded by a shell 39,42. The shell and core both share “typical” striatal characteristics- around 90% of neurons in both regions are GABA medium spiny neurons expressing either D1R, D2R, or both. The rest of the neurons are local interneurons, including cholinergic and parvalbumin cells 43. These two regions can be differentiated according to various histochemical, electrophysiological and cellular morphological criteria, but also by looking at their projection patterns and functions 44. Moreover, the distribution of neurotransmitters in these regions is also different. The shell is rich in serotonin and dopamine receptors, substance P, and calretinin, while the core contains more enkephalin and GABAA receptors 45–50. The immunohistochemical characteristics of the NAc shell have actually led it to be thought of as a transitional zone between the striatum and

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the extended amygdala 51. The NAc shell and core are thus clearly two distinct areas, and this distinction should be considered when studying the NAc.

The main feature of the NAc that justifies it being considered a hub in the basal ganglia network is that it receives so many inputs, both from diverse locations and from a variety of cell types.

Dopaminergic, excitatory (i.e. glutamatergic), and inhibitory (i.e. GABAergic) afferents converge in the NAc. The dopaminergic afferents come from the mesolimbic DA pathway (i.e. the VTA and the SNc) 33,52,53, and the glutamatergic inputs come from the PFC, the hippocampus, and the BLA 49,52,54–56. Finally, the VP, the VTA, and the SNc send GABAergic inputs to the NAc 57,58. Throughout the shell and core, there is a distinct topographical organization of these various inputs. This has led to the

hypothesis that the ventral striatum is a collection of “neuronal ensembles”, composed of distinct clusters of cells involved in different functions, depending on the inputs they receive.

MSNs of the NAc project to various areas of the mesencephalon, but they also project back to some nuclei of the basal ganglia. The major projection targets of the NAc are the VP, the SN, the VTA, the hypothalamus, and the brainstem 59–62. Similar to what is observed with the afferents to the NAc, a differential pattern of projections can be detected between the shell and the core and between the patch and the matrix.

The “take-home message” from all of these connectivity studies is that the NAc is a heterogeneous structure that acts as an interface between the limbic and motor systems. Although thinking about the NAc only in terms of reward is quite reductive, the VTA-NAc pathway is the best characterized reward circuit in the brain.

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2.2.2 The Ventral Tegmental Area (VTA)

Figure 10. Distribution of DA neuron cell groups in the adult rodent brain. In the early 1960s, Carlsson and colleagues 63–65 discovered that the midbrain dopaminergic neurons can be separated into three cell groups. The A8 cells in the retrorubral field, the A9 cells in the substantia nigra, and the A10 cells in the five subregions which compose the ventral tegmental area and related nuclei, which is the largest group of dopaminergic cells (Björklund and Dunnett 2007).

Although the VTA is mostly composed of dopamine neurons, it also contains other neuronal populations. Indeed, the dopamine neurons represent around 60-65% of the total neuronal population 67,68, while GABAergic neurons represent most of the remaining 30-35% 67–70.

Glutamatergic neurons make up only around 2% of VTA neurons 67, but there are also mixed neurons that are counted as dopaminergic neurons, which co-release DA and glutamate, DA and GABA, and even GABA and glutamate 71. Moreover, these three neuronal populations can also be further

subdivided. Among the VTA DA neurons, one subpopulation displays the classical electrophysiological properties associated with DA neurons (i.e. large hyperpolarization-activated cation current, or Ih,,

long action potential durations, and an inhibition by D2R agonists), while other DA neurons are heterogeneous in their pharmacological and electrophysiological properties 71–73. VTA GABA neurons also present some heterogeneity, while glutamatergic neurons seem to constitute a quite

homogeneous group 71.

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Figure 11. Confirmed inputs onto and outputs from VTA neurons. See text for legend. From 71

Considering the extremely diverse neuronal composition of the VTA, it took a considerable amount of work to map the structures that innervate the VTA and to identify its targets in a cell-specific manner. Margolis and Morales 71 summarize this map very nicely, as shown in figure 11. VTA dopamine neurons receive both glutamatergic and GABAergic inputs from both extrinsic brain regions and from local circuits. The excitatory inputs largely come from the mPFC, the PPTg, the LDTg, the LHb, the BNST, and the DRN. The inhibitory inputs largely come from the RMTg, the LHT, and the VP. The DRN and the PAG send both inhibitory and excitatory inputs to the VTA.

The GABAergic neurons also receive both glutamatergic and GABAergic inputs. The two major sources of excitatory inputs are the LHb and the mPFC, while the major source of inhibitory inputs is NAc, specifically the D1 MSNs of the NAc. The PAG, the BNST, and the DRN send both glutamatergic and GABAergic inputs to the VTA GABA neurons. Unlike dopaminergic neurons, the GABAergic neurons do not receive any local inputs.

GABAergic neurons of the VTA project to the cholinergic interneurons of the NAc and to the LHb, while glutamatergic neurons of the VTA project to the parvalbumin interneurons of the NAc and to

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the LHb. Finally, the dopamine neurons of the VTA project to the MSN of the NAc. To summarize, all types of VTA neurons project to its main target, the NAc.

2.3 The functional role of the dopaminergic neurons of the VTA

The pioneering work of Olds and Milner in the 1950s has been the basis of our modern

understanding of the brain reward system 74. They implanted rats with electrodes in several brain areas and then gave them the option to self-administer electrical currents to these various brain regions. They showed that the stimulation of specific regions elicited a persistent self-stimulation, often at the expense of others behaviors. The main region they identified was the septum. In the 1980s and 90s, studies 75–77 using lesions, receptor antagonists, and electrical self-stimulation established the mesolimbic DA system as a key component in reward processing and learning.

However, despite a general agreement about the involvement of VTA DA neurons in reward- processing, there were still controversies about the exact role of these neurons. Elements of an answer to this question came in 1997, when Schultz and colleagues 78 demonstrated that the dopamine signal was actually encoding the reward prediction error and was not a reward signal per se. In this now classic study, (fig.12) monkeys were rewarded with drops of fruit juice. When the reward was not predicted but was delivered, DA neurons were activated by the delivery of the reward. However, when the delivery of the reward was paired with a cue (conditioned stimulus), which predicted reward delivery, the activation of the DA neurons switched from the reward delivery to the cue onset. If the reward was expected, due to presentation of the conditioned stimulus, but not delivered, the activity of the DA neurons was depressed at the moment when the reward should have been delivered. This function of DA neurons has also been observed in both humans 79 and rodents 80,81.

The recent developments in mouse genetics, virus-mediated gene transfer technology, and the development of new tools such as optogenetics have allowed us to study the specific cell-types in reward-related behavior. The initial study, led by the Deisseroth group 82, used the light-activated cation channel, channelrhodopsin (ChR2) to show that stimulation of VTA DA neurons is sufficient to elicit intracranial self-stimulation in rats. This confirmed some of the pioneering work on DA neurons without having to rely on electrophysiological properties of the cells, which can lead to

mischaracterization of neurons.

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Figure 12. VTA DA neurons code for reward prediction error. Top, dopamine neurons are activated when an unpredicted appetitive reward is delivered. (Middle) After repeated pairings of

visual/auditory cues and reward delivery, dopamine neuron activation switches from the reward delivery to the cue onset. (Bottom) In trials where the reward is not delivered at the expected time after the cue, dopamine neurons decrease their activity below their basal firing rates at the time that the reward delivery should have occurred. CS = conditioned stimulus. R= reward. 78.

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2.4 The behavioral functions of the mesolimbic DA system

Regulation of midbrain DA neuron activity is able to induce a broad range of behavioral effects, from euphoria and approach in the case of increased activity, to dysphoria and aversion in the case of decreased activity.

The strongest evidence for the role of mesolimbic DA system in reward-related and motivated behaviors comes from studies on drugs of abuse, most often psychostimulant drugs like cocaine or amphetamine. Indeed, the administration (self-administration, systemic administration, or local, intracranial administration) of drugs of abuse dramatically increases extracellular DA concentrations in the striatum and especially in the NAc. This shared property of addictive drugs seems to be crucial for the rewarding/reinforcing effects of the drugs to occur. Self-administration of drugs can be modified by altering DA neurotransmission. High doses of DA receptor antagonists or 6-OH-DA lesions of the VTA terminals in the ventral striatum (VStr) diminish self-administration of drugs of abuse. More drastic reduction of DA activity will even induce aversion 83.

More recent studies using optogenetics and transgenic rodent lines unequivocally confirm the rewarding effects of VTA DA neuron activity. Stimulation of these neurons induces conditioned place- preference and self-stimulation behavior 84,85. As observed in the pharmacological studies,

optogenetic inhibition of the VTA DA neurons leads to anhedonia, aversion, and hypoactivity 86. Another study even went one step further and showed that stimulation of VTA DA neurons was sufficient to induce the behavioral and cellular characteristics of addiction 87.

These two opposite outcomes (reward and aversion) resulting from the regulation of DA

transmission can be explained by the nature of the main neuronal populations of the NAc. As already discussed, most of the striatal neurons (95% in rodents) are GABAergic medium-sized spiny neurons (MSNs), and they can be differentiated according to the dopamine receptors they express, specific expression of different neuropeptides, and projection targets 88. D1-MSNs express the D1 receptor, M4 cholinergic receptors, dynorphin, and substance P, whereas D2-MSNs the D2 receptor, A2a adenosine receptors, enkephalin, and neurotensin. In the dorsal striatum, these two cell types are very well segregated by projection target, but in the NAc, both D1- and D2-MSNs project to the VP, and only D1-MSN project to the SN and the VTA. Optogenetics studies have shown that these two cell types have opposing roles in learning. Stimulation of the D1-MSNs induces persistent

reinforcement, while stimulation of D2-MSNs causes transient punishment 89.

The NAc can therefore be thought of as an integrator of the DA signal received and the stimuli associated with that signal 90. Moreover, the NAc has been shown to be a convergent point for

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excitatory limbic and cortical afferents 91. The NAc then selects and integrates the information arriving from these afferents and sends a signal to the motor system in order to guide the appropriate behavior 90.

Because it is the relevant system for our work, we focus on the mesolimbic DA system. However, it is important to acknowledge that even though the mesolimbic DA system is a key player in motivation- and reward-related behavior, more and more studies are highlighting the importance of the

nigrostriatal DA system as well, i.e. DA neurons localized in the SNc and projecting to the caudate putamen 92,93.

2.5 Interaction between mu opioid receptors (MORs) and dopaminergic transmission in the mesolimbic DA system

In the first section, we described the endogenous opioid system and saw that opioid receptors are widely distributed throughout the CNS. The MORs especially are densely expressed in the NAc and the VTA 10. It has also been shown that the opioid system is involved in reward-related mechanisms and associated pathologies like drug addiction 94. Considering this information, the existence of a crosstalk between the endogenous opioid system and the mesolimbic dopamine system seems logical.

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Figure 13. Interaction between MORs and the mesolimbic dopamine system at the NAc level. In the NAc, MORs are localized both pre (on GABA and glutamatergic terminals)-and postsynaptically (on the GABAergic MSNs). MORs are G-protein coupled, and activate inhibitory G-proteins. Upon MOR activation, G protein α and βγ subunits interact with downstream effector systems to inhibit adenylyl cyclase and voltage-gated Ca2+ channels and stimulate G protein-activated inwardly rectifying K+

channels (GIRKs) and phospholipase Cβ. Adapted from 95.

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Figure 14. Interaction between MORs and the mesolimbic dopamine system at the VTA level. In the VTA, MORs are localized only pre-synapically on both glutamatergic and GABAergic terminals

synapsing on the DA neurons. Adapted from 95.

MORs are widely expressed throughout the brain and can be found basically everywhere. They can be localized both post-synaptically on dendrites and cell bodies and pre-synaptically on axons terminals 96. MORs are coupled to Gαi proteins, and their activation induces an intracellular cascade, leading to the inhibition of adenylyl cyclase (AC), voltage-gated Ca2+ channels, inwardly rectifying K+

channels (GIRKs), and phospholipase C. Because of their cellular localization and the effectors they switch on or inhibit, MOR activation regulates neuronal excitability, activation of downstream transduction pathways, or inhibition of neurotransmitter release.

In the NAc, MORs are coupled to inhibitory Gαi proteins and can be found both pre- and post-

synaptically, on both glutamatergic and GABAergic terminals, and on D1 MSNs. In the VTA, MORs are found on both glutamatergic and GABAergic terminals but are not observed post-synaptically on the VTA DA neurons 97–99.

MOR activity and DA neurotransmission are tightly linked. Indeed, it has been shown that rodents will self-administer opioids directly into both the NAc 100 and the VTA 101. Moreover, the NAc receives

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dopaminergic inputs from the VTA, which contains dopamine cell bodies. The rewarding effects of opioids are thought to occur via disinhibition of the VTA DA neurons. Indeed, opioids increase DA release in the NAc 102 by inhibiting VTA GABA neurons, reducing their inhibitory tone onto dopaminergic neurons 103. This is generally called the “disinhibition model”.

When opiates are present chronically and in large quantity, as in the chronic use typical of opioid addiction, drastic, long-term adaptations occur in both the NAc and the VTA. Adaptions will also occur if opioids are suddenly out of the system as in withdrawal.

3. Animal behavioral models in addiction research

Although animal behavioral models will never be able to fully recapitulate the human condition, especially for psychiatric disorders where several components, such as genetic, environment, social background, etc., are tangled. Nevertheless, they are key tools required to study, untangle, and understand core components of disorders, and it has been very successful with drug addiction.

Addiction is commonly defined by compulsive use of a substance, characterized by a loss of control over drug-taking despite negative consequences 104. Addiction can be seen as a cycle and broken down into several components that make up the different stages of the disease. Different behavioral models can then be used to study each of these different components. Animal models have been developed that have either face validity (i.e. appear mimic the human condition) or construct validity (i.e. have the same underlying etiology as the human condition)105 for the commonly defined three stages of addiction. For example, self-administration is used to study the initial stage of addiction, the binge and intoxication stage. Place conditioning, cue-induced seeking, and self-stimulation are models used to focus on the second stage of addiction, which is characterized by withdrawal and negative effects. Finally, the third stage is one of preoccupation and anticipation, where everything is done solely to get the drug regardless of any negative consequences. Animal models of resistance to punishment are used to study this phase of addiction 105,106. In an attempt to mimic the broad range of possible options that humans face, some models provide an alternative to drug intake, such as food or social interaction 107. We will briefly review the most commonly used rodent models in addiction research and detail the paradigms that are relevant for our project.

3.1 Modeling the binge/intoxication stage of addiction 3.1.1 Conditioned Place Preference (CPP)

CPP is probably the most used behavioral paradigm to investigate the motivational effects of drugs of abuse in animal research, even though this paradign is also used to study negative emotional states

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