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This thesis is comprised of three parts. In the first part, we investigated zyklophin, a novel selective short-acting kappa opioid receptor (KOPR) antagonist, and its effects on scratching behaviors in Swiss-Webster mice. We investigated whether zyklophin was able to induce scratching in a dose-dependent fashion, and whether this scratching behavior could be blocked by pretreatment with nor-binaltorphimine (norBNI). We also used KOPR -/- mice to further clarify the role of the KOPR in this behavior. In the second part, we examined the role of the divergent amino acid at position 6.58 in the mu opioid receptor (MOPR) and the KOPR on the binding of beta-funaltrexamine ß-FNA). ß-FNA is an irreversible antagonist at the MOPR and a reversible agonist at the KOPR. Utilizing the recently published crystal structures of the MOPR and KOPR, we collaborated with Dr. Lei Shi, who employed molecular modeling to choose a residue in transmembrane helix 6 (TM6) to mutate at the same position in MOPR and KOPR. We then characterized the mutants by performing [3H]diprenorphine binding, competition binding by unlabeled β-FNA, irreversible ß-FNA binding and [35S]GTPγS binding. In the third part, we investigated the concept of functional selectivity, or ligand bias, at the KOPR. We studied 23 different KOPR agonists in vitro using [35S]GTPγS binding as a measure of G protein activation and the on-cell Western (OCW) as a measure of ß-arrestin-mediated receptor internalization at the human KOPR (hKOPR), and from the results, chose 13 ligands to study at the mouse KOPR (mKOPR). We then selected biased ligands from the in vitro mKOPR results and studied their effects on scratching behavior, inhibition of pain behaviors and dysphoria as measured by the conditioned place aversion (CPA) test. We predicted that the G biased ligand would produce analgesia and anti-scratching effects at lower doses than would produce aversion in the CPA test, since analgesia has been shown to be G protein mediated and CPA has been shown to be arrestin mediated. Our first set of studies revealed that zyklophin (0.1, 0.3 and 1 mg/kg, s.c., behind the neck), induced vigorous scratching in a dose-dependent manner. 0.3 mg/kg zyklophin induced 150 scratches over a 30 minute period. The scratching was not blocked by pretreatment with 20 mg/kg norBNI (i.p.) 18-20 hours before injection of 0.3 mg/kg zyklophin s.c. in the nape of the neck. The scratching also persisted in KOPR -/- mice, in which the absence of the KOPR was confirmed by [3H]U69,593 binding (2 nM). In our second set of studies, we mutated the lysine at position 303 in the MOPR to glutamic acid (K303E), and the glutamic acid at the equivalent position in the KOPR to lysine (E297K). We transfected these mutant receptors into mouse neuroblastoma (N2A) cells. We found that the mutations had no effect on [3H]diprenorphine binding affinity or competition binding with [3H]diprenorphine and β-FNA indicating a functional intact opioid receptor. The mutations also did not affect [35S]GTPγS binding EC50 or Emax values. The mutation K303E in the MOPR reduced irreversible binding by 2/3 compared to the wildtype MOPR. Finally, we found that there were several ligands that displayed bias at the hKOPR and the mKOPR. At the hKOPR, using dynorphin A as the reference ligand to calculate bias, ICI-199441 was the only G biased ligand, while enadoline, nalbuphine, pentazocine, salvinorin A, tifluadom and butorphanol were arrestin-biased. At the mKOPR, only salvinorin B methoxymethyl ether (MOM-SalB) was G-biased, and salvinorin B ethoxymethyl ether (EOM-SalB), ICI-199441, U50,488H, nalfurafine and 12-epi-salvinorin A (12epiSalA) were ß-arrestin-biased. Enadoline and salvinorin A were slightly arrestin biased with respect to dynorphin A. From the in vitro data at the mKOPR, we selected MOM-SalB as our G biased ligand, U50,488H as our arrestin biased ligand and additionally chose to investigate nalfurafine due to its use in clinical studies. We hypothesized that U50,488H and nalfurafine would produce aversion at lower doses than analgesia or anti-pruritic effects. We found that nalfurafine was the only ligand studied to have a separation between doses that produced analgesia and anti-scratching effects, with A50 values of 5.8 and 8 μg/kg, respectively, and only produced significant dysphoria at a dose of 20 μg/kg. U50,488H and MOM-SalB produced dysphoria at all doses tested (0.25-10 mg/kg and 0.01-0.3 mg/kg, respectively). U50,488H produced a dose-dependent analgesia and anti-scratching with A50 values of 0.58 mg/kg and 2.07 mg/kg, respectively. MOM-SalB was more potent than U50,488H in producing dose-dependent analgesia and anti-scratching, with A50 values of 0.017 mg/kg and 0.070 mg/kg, respectively. Therefore, we concluded that the in vitro bias is not able to accurately predict in vivo behaviors, and nalfurafine is the first selective full agonist at the KOPR to show ligand bias in vivo. / Pharmacology

A fundamental question in the neurobiological study of drug addiction concerns the mechanisms mediating the motivational effects of chronic drug withdrawal. According to one theory, drugs of abuse activate opposing motivational processes after both acute and chronic drug use. The negative experience of withdrawal is the opponent process of chronic drug use that drives relapse to drug-seeking and -taking, making the identification of the neurobiological substrates mediating withdrawal an issue of central importance in addiction research. In this thesis, I identify the involvement of the neurotransmitters dopamine (DA) and corticotropin-releasing factor (CRF) in the opponent motivational a- and b-processes occurring after acute and chronic nicotine administration. I report that acute nicotine stimulates an initial aversive a-process followed by a rewarding opponent b-process, and chronic nicotine stimulates a rewarding a-process followed by an aversive opponent b-process (withdrawal). These responses can be modeled using a place conditioning paradigm. I demonstrate that the acute nicotine a-process is mediated by phasic dopaminergic activity and the DA receptor subtype-1 (D1R) but not by tonic dopaminergic activity and the DA receptor subtype-2 (D2R) or CRF activity, and the opponent b-process is neither DA- nor CRF-mediated. I also demonstrate that the chronic nicotine a-process is DA- but not CRF-mediated, and that withdrawal from chronic nicotine (the b-process) decreases tonic but not phasic DA activity in the ventral tegmental area (VTA), an effect that is D2R- but not D1R-mediated. I show that a specific pattern of signaling at D1Rs and D2Rs mediates the motivational responses to acute nicotine and chronic nicotine withdrawal, respectively, by demonstrating that both increasing or decreasing signaling at these receptors prevents the expression of the conditioned motivational response. Furthermore, I report that the induction of nicotine dependence increases CRF mRNA in VTA DA neurons, and that blocking either the upregulation of CRF mRNA or the activation of VTA CRF receptors prevents the anxiogenic and aversive motivational responses to withdrawal from chronic nicotine. The results described in this thesis provide novel evidence of a VTA DA/CRF system, and demonstrate that both CRF and a specific pattern of tonic DA activity in the VTA are necessary for the aversive motivational experience of nicotine withdrawal.

A fundamental question in the neurobiological study of drug addiction concerns the mechanisms mediating the motivational effects of chronic drug withdrawal. According to one theory, drugs of abuse activate opposing motivational processes after both acute and chronic drug use. The negative experience of withdrawal is the opponent process of chronic drug use that drives relapse to drug-seeking and -taking, making the identification of the neurobiological substrates mediating withdrawal an issue of central importance in addiction research. In this thesis, I identify the involvement of the neurotransmitters dopamine (DA) and corticotropin-releasing factor (CRF) in the opponent motivational a- and b-processes occurring after acute and chronic nicotine administration. I report that acute nicotine stimulates an initial aversive a-process followed by a rewarding opponent b-process, and chronic nicotine stimulates a rewarding a-process followed by an aversive opponent b-process (withdrawal). These responses can be modeled using a place conditioning paradigm. I demonstrate that the acute nicotine a-process is mediated by phasic dopaminergic activity and the DA receptor subtype-1 (D1R) but not by tonic dopaminergic activity and the DA receptor subtype-2 (D2R) or CRF activity, and the opponent b-process is neither DA- nor CRF-mediated. I also demonstrate that the chronic nicotine a-process is DA- but not CRF-mediated, and that withdrawal from chronic nicotine (the b-process) decreases tonic but not phasic DA activity in the ventral tegmental area (VTA), an effect that is D2R- but not D1R-mediated. I show that a specific pattern of signaling at D1Rs and D2Rs mediates the motivational responses to acute nicotine and chronic nicotine withdrawal, respectively, by demonstrating that both increasing or decreasing signaling at these receptors prevents the expression of the conditioned motivational response. Furthermore, I report that the induction of nicotine dependence increases CRF mRNA in VTA DA neurons, and that blocking either the upregulation of CRF mRNA or the activation of VTA CRF receptors prevents the anxiogenic and aversive motivational responses to withdrawal from chronic nicotine. The results described in this thesis provide novel evidence of a VTA DA/CRF system, and demonstrate that both CRF and a specific pattern of tonic DA activity in the VTA are necessary for the aversive motivational experience of nicotine withdrawal.

L’addiction est un désordre psychobiologique caractérisé par des prises de drogue répétées, une incapacité à en contrôler la consommation et une tendance chronique à la rechute. Dans le cas des opiacés (morphine, héroïne), l’arrêt de la consommation de drogue induit un syndrome de sevrage qui peut être associé de manière forte et durable à l’environnement dans lequel il est vécu. Cette association est telle que même après une longue période d’abstinence, la simple réexposition à cet environnement peut faire émerger un état émotionnel aversif qui pourrait favoriser la rechute. Dans le cadre de la dépendance aux opiacés, il est de plus en plus clair que la réactivation des mémoires affectives associées au sevrage joue un rôle dans la motivation à rechercher de la drogue. Au plan neurobiologique, il a été montré au laboratoire que le processus de plasticité synaptique se met en place lors du conditionnement des stimuli conditionnés au sevrage des opiacés, au sein de structures limbiques impliquées à différents titres dans le processus d’apprentissage associatif. Il a été proposé que les effets de la réexposition aux stimuli conditionnés au sevrage soient dus à la réactivation spécifique de ces structures limbiques. Dans ces travaux, les stimuli environnementaux étaient associés à la fois à l’état aversif précoce du sevrage et à des symptômes somatiques, ce qui permet une première avancée dans la compréhension des processus cellulaires impliqués dans la formation et le rappel de la mémoire du sevrage. Cependant afin de mieux comprendre comment cette mémoire pourrait exercer un rôle dans la rechute, il est nécessaire d’analyser les substrats neuronaux mis en jeu de manière plus spécifique dans les effets conditionnés de la seule composante aversive précoce du sevrage. En effet, cette composante dite « motivationnelle » joue un rôle majeur chez l’individu dépendant dans le besoin de continuer à consommer la drogue, et potentiellement chez l’individu abstinent dans sa vulnérabilité à la rechute.L’objectif de mon travail de thèse a consisté à préciser les substrats neurobiologiques impliqués dans le sevrage des opiacés et dans le rappel des mémoires aversives associées notamment à la composante motivationnelle du sevrage.Dans un premier temps, nous avons développé une approche d’hybridation in situ fluorescente (catFISH) dont le principal avantage est de préciser la dynamique des activations neuronales induites par une stimulation. Nous avons validé l’utilisation du catFISH en caractérisant la dynamique d’activation neuronale dans le cortex préfrontal (CPF), le noyau accumbens (Nac), le noyau central (CeA) et basolatéral (BLA) de l’amygdale lors de la précipitation d’un syndrome de sevrage des opiacés. Nos résultats montrent que le catFISH permet de révéler des activations neuronales durables et que le CeA et le Nac présentent une dynamique d’activation différente en réponse à la précipitation du sevrage des opiacés.Dans une deuxième partie nous avons étudié les substrats neuronaux impliqués lorsque le rappel des mémoires du sevrage des opiacés exerce un effet sur un comportement opérant dirigé vers la nourriture, et ceci en fonction de l’intensité du sevrage. L’utilisation du catFISH nous a permis de différencier les activations neuronales induites par la réexposition au contexte du sevrage ou par la présentation du stimulus conditionné au sevrage. Nos résultats montrent que le CPF et le Nac shell sont impliqués dans le rappel des mémoires contextuelles du sevrage et que le CPF ainsi que le Nac core et le BLA sont activés par le rappel du stimulus conditionné au sevrage.Enfin, nous avons analysé dans un protocole d’aversion de place conditionnée les substrats neurobiologiques recrutés par le rappel des mémoires associées au syndrome de sevrage motivationnel des opiacés. Nos résultats indiquent que le Nac shell et le BLA sont les deux structures cérébrales les plus sensibles au rappel des mémoires du sevrage. L’ensemble de ce travail a permis de faire ressortir le rôle crucial du Nac shell et du BLA au sein du réseau de substrats neuronaux impliqués dans le traitement des mémoires émotionnelles aversives associées au sevrage des opiacés. Ces structures pourraient représenter les substrats communs au traitement des mémoires émotionnelles associées aux effets de drogues d’abus. L’ensemble de ces résultats devra être mis en perspective avec des travaux débutés lors de ma thèse en électrophysiologie in vivo sur animal se comportant. Ces travaux consisteront à étudier de façon longitudinale les dynamiques du réseau CPF/Nac/BLA lors de la formation et le rappel des mémoires du sevrage et permettront de mieux définir les rôles spécifiques que jouent les substrats neurobiologiques que nous avons étudiés dans le traitement des mémoires du sevrage des opiacés. / Addiction is a psychobiological disorder that is characterized by repeated drug intakes, inability to control its consumption and a chronic tendency to relapse. Concerning opiate addiction (heroin, morphine), cessation of drug consumption induces a withdrawal syndrome, which can be strongly and persistently associated with the environment in which it is experimented. This association is so tight that a single re-exposure to this specific environment is enough to provoke a negative emotional state, which may promote drug relapse. In opiate dependence, it becomes clearer and clearer that reactivation of the affective memories associated with drug withdrawal play a major role in drug seeking. In terms of neurobiological processes, previous works conducted in the lab have shown that synaptic plasticity takes place during the conditioning of stimuli to opiate withdrawal, in limbic structures known to be involved in associative learning. It has been suggested that the consequences of the re-exposition to withdrawal conditioned stimuli are due to the reactivation of these specific limbic regions. In theses studies, environmental stimuli were both associated to the early aversive state of withdrawal and to somatic symptoms. This represents a first step in the understanding of the cellular processes involved in the formation and retrieval of withdrawal memories. However, in order to better understand how these memories could play a role in relapse, it is necessary to analyze the neuronal substrates involved in the conditioned effects of the sole early aversive motivational component of opiate withdrawal. Indeed, this motivational component is considered as exerting a strong influence on the maintaining of drug consumption, and eventually on the vulnerability to relapse in abstinent addicts. The aim of my work was to specify the neurobiological substrates involved in opiate withdrawal and in the retrieval of the aversive memories especially the memories associated with the motivational component of withdrawal. We first developed an in situ hybridization approach (catFISH) whose main advantage is to add a dynamical dimension to the neuronal activations induced by a stimulation. We validated the use of the catFISH method by studying the dynamics of neuronal activations in the prefrontal cortex (PFC), the nucleus accumbens (Nac), the central (CeA) and basolateral (BLA) nucleus of the amygdala as a consequence of the precipitation of opiate withdrawal. Our results show that catFISH allows determining persistent neuronal activations and that the CeA and the Nac have a different dynamics of activation in response to opiate withdrawal. In the second part, we studied the neuronal substrates involved when the retrieval of opiate withdrawal memories modifies an operant goal-directed behaviour, according to the withdrawal intensity. The use of catFISH allowed us to differentiate the neuronal activations induced by the re-exposition to the withdrawal context or to the conditioned stimuli. Our results show that the PFC and the Nac shell are involved in the retrieval of contextual memories of withdrawal and that PFC, Nac core and BLA are activated by the retrieval of more specific conditioned stimuli.Lastly, we analysed, using a conditioned place aversion protocol, the neuronal structures recruited by the retrieval of the memories associated with the motivational component of opiate withdrawal. Our results suggest that the Nac shell and the BLA are the brain structures that are the most sensible to the retrieval of the memories of opiate withdrawal.Overall, our work emphasized the crucial role played by the Nac shell and the BLA within a network of neuronal substrates involved in the processing of aversive emotional memories associated with opiate withdrawal. These structures could be considered as the common substrates to the processing of emotional memories associated with the effects of drugs of abuse. These results will be compared with an in vivo electrophysiology on behaving animals’ approach that we initiated during my PhD. This study will consist of detailing longitudinally the dynamics of the PFC/Nac/BLA network during the formation and the retrieval of the memories of opiate withdrawal. This study will also provide more details on the specific functions of the previously studied neuronal substrates in the processing of opiate withdrawal memories.