Indiana University-Purdue University Indianapolis (IUPUI) / Some individuals with alcohol use disorder (AUD) continue to drink because they have developed a habit in which they are not considering the consequences of their actions. Habitual actions persist despite changes in reward and are often studied using devaluation procedures. Stress hormones, such as corticotropin releasing factor (CRF), have been linked to AUD when examining binge-like drinking and withdrawal in rodents. Stress has been examined in the switch from goal-directed to habitual behavior, and CRF has often mimicked the effects of stress exposure. This study looked at the possible direct effects of CRF on habit expression in rats using an operant paradigm. Finding possible novel mechanisms of habit could create an avenue for future novel treatment options. Female and male Long Evans rats were trained on a variable interval schedule using sucrose as a reward. Rats then underwent devaluation procedures including both sensory-specific satiety and conditioned taste aversion (CTA) to test for habitual behaviors. Prior to an extinction session post-CTA, animals were treated with either 20 mg/kg R121919, a CRF1 receptor antagonist, or vehicle. A second extinction session was conducted where animals received the alternative treatment. Lever presses were recorded as a measure of goal-directed or habitual behavior. Sensory-specific satiety devaluation tests revealed that animals were not sensitive to devaluation. This was further supported by both post-CTA extinction sessions. R121919 had no effect on lever pressing in either devalued or valued groups. Further research is needed to explore how a CRF receptor antagonist may affect habit formation or the transition from goal-directed to habit behaviors. Future studies should also examine any possible interaction effects CRF may have with alcohol or stress on habitual behaviors.

Graduation date: 1996

Aktivierte G-Protein-gekoppelte Rezeptoren (GPCR) werden schnell desensitisiert, internalisiert und anschließend entweder lysosomal degradiert oder zur Plasmamembran (PM) recycelt. Zur Resensitisierung der Zellen tragen neben recycelten auch neusynthetisierte Rezeptoren bei. Die Überlagerung beider Prozesse erschwert die Untersuchung des Rezeptorrecyclings. In dieser Arbeit sollte mit Hilfe des photokonvertierbaren Fluoreszenzproteins Kaede eine Technik entwickelt werden, mit der es möglich ist Recycling- von Neusyntheseprozessen zu trennen und das Recycling von GPCR mikroskopisch in Echtzeit zu beobachten. Als Modellproteine wurden der Vasopressin-1a-Rezeptor V1aR (recycelnder Rezeptor), der Vasopressin-2-Rezeptor V2R (degradierter Rezeptor) und der Corticotropin-Releasing Factor-Rezeptor Typ 1 (CRF1R) verwendet, wobei bei Letzterem untersucht werden sollte, ob er nach Stimulation zur PM zurücktransportiert wird. Da Kaede als fluoreszierendes Protein mit den GPCR fusioniert wird, wurde zunächst überprüft, ob es die Eigenschaften der Rezeptoren verändert und generell für Transportstudien geeignet ist. Eventuell könnte die bereits publizierte Tetramerisierung von Kaede seine Anwendung verhindern oder erschweren. Mittels Fluoreszenz-Korrelationsspektroskopie konnte gezeigt werden, dass Kaede nicht tetramerisiert, wenn es an ein Membranprotein fusioniert ist. Außerdem konnte in in vitro- und Zellkulturexperimenten belegt werden, dass die native und die photokonvertierte Form von Kaede gleichermaßen stabil sind. Darüber hinaus zeigten Kaede-fusionierte GPCR sowohl in Kolokalisationsstudien als auch in Agonistbindungs- und Rezeptoraktivierungsexperimenten die gleichen Eigenschaften wie CFP- bzw. die unfusionierte Rezeptoren. Lediglich die Expression der Kaede-fusionierten Rezeptoren war geringer. Parallel wurde anhand der bereits publizierten Kaede-Struktur versucht, die Tetramerisierung des Proteins durch den Austausch interagierender Aminosäuren zu unterbinden. Die eingeführten Mutationen bewirkten aber eine Fehlfaltung des Proteins und damit den Verlust der Fluoreszenz. Da zuvor gezeigt werden konnte, dass Kaede-fusionierte Membranproteine nicht tetramerisieren und nicht die Eigenschaften der fusionierten Proteine verändern, war monomerisiertes Kaede zur Untersuchung des Rezeptorrecyclings nicht notwendig. Im zweiten Teil der Arbeit wurde mit Hilfe von Kaede-Fusionsproteinen und mikroskopischer Testsysteme das noch unbekannte Recyclingverhalten des CRF1R untersucht. Hierfür wurden die Kaede-fusionierten Rezeptoren in eukaryotischen Zellen exprimiert und mit Agonisten internalisiert. Die internalisierten Rezeptoren wurden in Endosomen selektiv mit UV-Strahlung photokonvertiert. Anschließend wurde der Transport der photokonvertierten Form verfolgt. Sowohl beim CRF1R als auch beim V1aR wurden Signale in der PM detektiert, beim V2R hingegen nicht. Dies zeigt, dass es sich beim CRF1R um einen recycelnden Rezeptor handelt. Die als Kontrolle eingesetzten Rezeptoren verhielten sich in diesem Experiment wie erwartet: Der V1aR wurde zur PM zurücktransportiert, der V2R nicht. Diese Ergebnisse konnten mit Hilfe biochemischer und durchflusscytometrischer Experimente bestätigt werden. Die Internalisierung des CRF1R verläuft Clathrin-vermittelt in Anwesenheit von β-Arrestin. Je nach Stabilität der β Arrestin-Interaktion unterscheidet man zwei Klassen von Rezeptoren: Klasse A-Rezeptoren interagieren transient mit β Arrestin und können recyceln. Im Gegensatz dazu gehen Klasse B-Rezeptoren eine stabile Interaktion mit β Arrestin ein und werden nach Internalisierung degradiert. In mikroskopischen Untersuchungen konnte für die aktivierten CRF1R und V1aR eine Rekrutierung von β Arrestin zur PM und eine transiente Interaktion mit β Arrestin gezeigt werden (Klasse A-Rezeptoren). Für den V2R wurde dagegen eine stabile Interaktion mit β Arrestin beobachtet (Klasse B-Rezeptor). Diese Daten stützen die Ergebnisse des Kaede-basierten Recyclingversuchs und zeigen, dass der CRF1R ein recycelnder Rezeptor ist. Ferner wurde untersucht, ob der CRF1R zu den schnell oder langsam recycelnden Rezeptoren zählt. Schnell recycelnde Rezeptoren werden direkt aus frühen Endosomen, langsam recycelnde hingegen über das Trans-Golgi-Netzwerk (TGN) bzw. über Recycling-Endosomen zur PM transportiert. Als Marker für das TGN oder die Recycling-Endosomen wurde Rab11 verwendet. In Kolokalisationsstudien konnte gezeigt werden, dass der CRF1R den langsam recycelnden Rezeptoren zugeordnet werden kann. Zusammenfassend konnte in dieser Arbeit belegt werden, dass Kaede als Fusionspartner für Membranproteine genutzt werden kann um deren Transport in Echtzeit zu studieren. Damit wurde erstmals eine mikroskopische Methode etabliert, die es erlaubt recycelnde von neusynthetisierten Rezeptoren zu unterscheiden. Mit Hilfe dieser Methode war es möglich zu zeigen, dass der CRF1R ein recycelnder Rezeptor ist. / Upon ligand binding and receptor activation, G protein-coupled receptors (GPCR) are rapidly desensitized, internalized and subsequently degraded in lysosomes or recycled back to the plasma membrane. Resensitization of the cell is enabled by both recycling receptors and newly synthesized receptors. The overlap of recycling and synthesis processes largely complicates the study of GPCR recycling mechanisms. One aim of this thesis was to develop a new microscopic technique for real-time visualization of GPCR recycling using the photoconvertible Kaede protein allowing to differentiate newly synthesized from recycling receptors. As model proteins, the V1aR (recycling receptor), the V2R (degraded receptor) and the CRF1R were used. In the case of the CRF1R, it was unknown whether this receptor recycles to the plasma membrane following agonist-promoted internalization. The study of the CRF1R recycling behaviour was another objective of this work. As the Kaede protein is fused C-terminally to the GPCRs, an influence on the pharmacological and trafficking properties of the receptors must be excluded. The previously published tetramerization of Kaede, for example, might hinder or even prevent its usability. To assess for the applicability of Kaede, fluorescence correlation spectroscopy experiments were performed and it was demonstrated that Kaede fused to membrane proteins cannot form tetramers in contrast to the soluble form. In vitro studies and experiments in cell culture revealed that both the native and the photoconverted Kaede are equally stable. Moreover Kaede-fused GPCR displayed the same pharmacological and trafficking properties as the untagged or CFP-tagged receptors. Only the expression levels of the Kaede fusion proteins were reduced, yet this did not affect the microscopic experiments. In parallel to these experiments, the interacting amino acids of the tetrameric Kaede were substituted according to the previously published crystal structure of the protein. Unfortunately, these mutations induced protein misfolding thereby causing loss of fluorescence functions. However, since it could be shown that membrane protein-fused Kaede cannot tetramerize, the monomerized Kaede was no more essential for the microscopic study of receptor recycling. In the second part of this work, Kaede-fusions were used to study the recycling behaviour of the CRF1R and the V1aR and V2R control proteins by the novel real-time recycling assay at the laser scanning microscope. To this end, HEK 293 cells expressing the Kaede-fused receptors were treated with agonist to induce receptor internalization. Internalized receptors were selectively photoconverted in endosomes using UV-irradiation and the subcellular fate of the new fluorescence signals was studied. In the case of the CRF1R, signals of the photoconverted receptors could be detected in the plasma membrane indicating that the CRF1R belongs to the family of recycling receptors. The control receptors showed the expected results: The V1aR recycled back to the plasma membrane whereas the V2R did not. These results were confirmed with biochemical and flow cytometry measurements. The CRF1R internalizes in a clathrin-dependent way via the adaptor protein AP2, dynamin and β arrestin. Depending on the stability of the resulting receptor-β-arrestin-complex, two classes of receptors can be differentiated. Class A receptors are recycling receptors undergoing a more transient β-arrestin interaction. In contrast, class B receptors stably interact with β-arrestin and are degraded after internalization. In the case of the CRF1R and V1aR, microscopic analyzes demonstrated that β arrestin transiently interacts with the stimulated CRF1R and V1aR indicating again that these receptors are recycling GPCRs (class A receptors). The V2R, in contrast, revealed a stable interaction (class B receptor). Moreover, it was studied whether the CRF1R recycles rapidly or more slowly to the plasma membrane. Rapidly recycling receptors are recruited out of early endosomes whereas slowly recycling receptors pass the trans-golgi-network or recycling endosomes before reaching the cell surface. Rab11 colocalization studies demonstrated that the CRF1R belongs to the family of slowly recycling receptors. In conclusion, a novel microscopic technique was established allowing to study GPCR recycling in real-time and to differentiate recycling and synthesis processes. Moreover, it was shown that the CRF1R belongs to the family of recycling receptors. The Kaede technique seems to be very well suited to study membrane protein trafficking in general.

It has been well established that the stress-related neurochemical systems corticotropin-releasing factor (CRF), noradrenaline (NA), and dopamine (DA) mediate stress-induced reinstatement of drug seeking. The three series of experiments presented in this dissertation constitute a further exploration of the role these neurochemical circuits play in reinstatement by providing the first direct exploration of whether central CRF and catecholamine (NA and DA) systems interact to influence reinstatement of cocaine seeking. The primary objective of the first series of experiments was to determine whether NA and CRF systems interact to mediate reinstatement of cocaine seeking and, if so, to determine the direction of this interaction. Results showed that central administration of NA induced reinstatement and up-regulated the expression of c-fos mRNA, a marker of neuronal activation, in brain regions involved in footshock-induced reinstatement. Pretreatment with a CRF antagonist blocked NA-induced reinstatement. In contrast, pretreatment with the α2-adrenoceptor agonist, clonidine, failed to block CRF-induced reinstatement. Taken together, these findings suggest a functional interaction between NA and CRF systems in mediating stress-induced reinstatement of cocaine seeking, whereby activation of CRF receptors occurs subsequent to, and downstream of, the sites of action of NA. A second series of experiments examined the role of D1- and D2-like receptors in CRF-induced reinstatement. Pretreatment with the D1- or D2-like receptor antagonists, SCH23390 and raclopride, respectively, dose-dependently blocked CRF-induced reinstatement of cocaine seeking. Taken together with previous findings, these results suggest that CRF-induced reinstatement of cocaine seeking likely involves DAergic signaling via D1- and D2-like receptors, subsequent to activation of CRF receptors. The final series of experiments investigated the neuropharmacology of yohimbine-induced reinstatement, focusing on the roles of α2-adrenoceptors, D1- and D2-like receptors. These experiments were prompted by an unexpected finding in the first series of experiments, in which a CRF antagonist failed to interfere in yohimbine-induced reinstatement of cocaine seeking. Results showed that pretreatment with the α2-adrenoceptor agonist, clonidine, or raclopride, prior to tests for yohimbine-induced reinstatement failed to influence responding. In contrast, pretreatment with SCH23390 blocked yohimbine-induced reinstatement. Taken together, these findings suggest that yohimbine may act through system(s) other than NA to have its effects.

It has been well established that the stress-related neurochemical systems corticotropin-releasing factor (CRF), noradrenaline (NA), and dopamine (DA) mediate stress-induced reinstatement of drug seeking. The three series of experiments presented in this dissertation constitute a further exploration of the role these neurochemical circuits play in reinstatement by providing the first direct exploration of whether central CRF and catecholamine (NA and DA) systems interact to influence reinstatement of cocaine seeking. The primary objective of the first series of experiments was to determine whether NA and CRF systems interact to mediate reinstatement of cocaine seeking and, if so, to determine the direction of this interaction. Results showed that central administration of NA induced reinstatement and up-regulated the expression of c-fos mRNA, a marker of neuronal activation, in brain regions involved in footshock-induced reinstatement. Pretreatment with a CRF antagonist blocked NA-induced reinstatement. In contrast, pretreatment with the α2-adrenoceptor agonist, clonidine, failed to block CRF-induced reinstatement. Taken together, these findings suggest a functional interaction between NA and CRF systems in mediating stress-induced reinstatement of cocaine seeking, whereby activation of CRF receptors occurs subsequent to, and downstream of, the sites of action of NA. A second series of experiments examined the role of D1- and D2-like receptors in CRF-induced reinstatement. Pretreatment with the D1- or D2-like receptor antagonists, SCH23390 and raclopride, respectively, dose-dependently blocked CRF-induced reinstatement of cocaine seeking. Taken together with previous findings, these results suggest that CRF-induced reinstatement of cocaine seeking likely involves DAergic signaling via D1- and D2-like receptors, subsequent to activation of CRF receptors. The final series of experiments investigated the neuropharmacology of yohimbine-induced reinstatement, focusing on the roles of α2-adrenoceptors, D1- and D2-like receptors. These experiments were prompted by an unexpected finding in the first series of experiments, in which a CRF antagonist failed to interfere in yohimbine-induced reinstatement of cocaine seeking. Results showed that pretreatment with the α2-adrenoceptor agonist, clonidine, or raclopride, prior to tests for yohimbine-induced reinstatement failed to influence responding. In contrast, pretreatment with SCH23390 blocked yohimbine-induced reinstatement. Taken together, these findings suggest that yohimbine may act through system(s) other than NA to have its effects.

INTRODUCTION: For many years, stress and anxiety disorders have taken a heavy toll on the American population. Affecting approximately 40 million individuals over the age of 18, the discovery of treatment options is very important. Ever since the 1950s, a wide variety of compounds have been discovered and proven to have antagonistic properties for such disorders. For the last three decades, however, researchers have focused on a specific peptide that was discovered in 1981 by Dr. Wylie Vale and his colleagues at the Salk Institute in San Diego, California, corticotropin releasing factor (CRF). CRF is a 41 amino acid peptide that has been shown to play a very important role in an organism's endocrine response to stress through the activation of the hypothalamic–pituitary–adrenal (HPA) axis. Ever since its discovery, the identification and characterization of the CRF receptors and family members have allowed for the development of novel peptide and non–peptide antagonists. Unfortunately, these compounds have been unsuccessful in the progression to later stage clinical trials that could lead to promising therapeutics. There are two receptor subtypes for this family of peptides known as CRFR1 and CRFR2. While there have been many compounds identified that can block CRFR1, currently, there are no known selective non–peptide antagonists for the CRFR2 subtype. As the two receptor subtypes share 70% sequence identity, close observation of the functional properties of antagonist ligands for CRFR1 may lead to the development of such ligands for CRFR2. METHODS: In our current study, we focused on two residues in transmembrane domains (TMD) 3 (His199) and 5 (Met276) of CRFR1 that have proven to be important for the function of the highly selective small molecule antagonist antalarmin. In order to further prove the importance of these sites, we have mutated the two corresponding amino acids in CRFR2β to those of CRFR1: V215H in TMD 3 and V292M in TMD 5. In addition, we mutated a third amino acid residue, M293I, in order to avoid the positioning of two adjacent methionine amino acids. With this mutant construct, CRE–luciferase and cyclic AMP radioimmunoassay methodologies were used to observe the function of antalarmin on CRFR1, the mutant and wild type CRFR2β. The accumulation of cAMP was measured intracellularly following stimulation by the CRF receptor peptide agonists sauvagine, isolated from frog, and urocortin 1, isolated from rat. RESULTS: For the initial CRE–luciferase functional assay, we used the CRF receptor agonist sauvagine on our mutant CRFR2β to indirectly measure the accumulation of intracellular cAMP through the enzyme luciferase. In the presence or absence of the antagonist antalarmin, there were no significant changes on the function of the mutant CRFR2β. On the other hand, when directly measuring the accumulation of intracellular cAMP via radioimmunoassay, antalarmin successfully showed a functional inhibitory effect on the mutant CRFR2β receptor. As expected, Ucn1 stimulation of CRFR1 in the presence of antalarmin indicated a decrease in the EC50 for the peptide agonist, and thus an inhibitory effect by antalarmin. Compared to CRFR1, we observed a similar effect for Ucn1 stimulation of the mutant CRFR2β receptor in the presence of antalarmin. While the presence or absence of antalarmin did not have a significant inhibitory effect on the wild type CRFR2β, it can be concluded that the mutant CRFR2β receptor possessed similar properties to the CRFR1 receptor with respect to antalarmin antagonist activity. CONCLUSION: In our study, we were able to further support the importance of the two amino acid residues in TMD 3 and 5 of CRFR1 for the function of small molecule antagonists. In addition, we were able to show that antalarmin, a small molecule antagonist known to be highly selective for CRFR1, can have a functional inhibitory effect on the mutant CRFR2β. The progressive study of these discrete differences between the two CRF receptor subtypes may enable the discovery of novel selective non–peptide CRFR2β receptor antagonists.

Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI) and the active ingredient in Prozac®, is found in the environment and disrupts feeding and metabolism in exposed fish. The objective of this research was to investigate the mechanisms involved in the feeding and metabolism disruption in the model goldfish (Carassius auratus). Two short-term waterborne fluoxetine exposures (7- and 14-days) were performed using two environmentally relevant doses of fluoxetine (0.5 and 1 μg/L) and metabolic effects at the level of the brain, liver, serum and bile in goldfish were investigated. Abundances of mRNA transcripts coding for six feeding neuropeptides were examined to determine which may be involved in the initial neural changes associated with decreased appetite in goldfish. The 7-day fluoxetine exposure at 1 μg/L caused corticotropin-releasing factor (CRF) mRNA levels to increase by 2-fold in female hypothalamus and telencephalon, indicating that CRF may be one of the first of the feeding neuropeptides to be altered. Six hepatic miRNAs were also evaluated in the goldfish liver that were previously associated with fluoxetine exposure in zebrafish (Danio rerio). Following the 7-day exposure at 1 μg/L, miR-22b, miR-140, miR-210, miR-301a and miR-457b levels increased in the female goldfish liver by 4-6 fold. The 14-day fluoxetine exposure at 1 μg/L caused 2-fold increases in miR-210, miR-301a, miR-457b and let-7d in male goldfish liver. These miRNAs were associated with the down-regulation of anabolic metabolic pathways in zebrafish, indicating a conservation of miRNA and fluoxetine effect between fish species. Serum and bile metabolite profiles of fluoxetine exposed goldfish were evaluated using ultra performance liquid chromatography coupled to quadrupole time of flight mass spectrometry. Following the 14-day exposure at 1 μg/L, the bile metabolite profiles of male goldfish were significantly different from controls as detected by cluster analysis and fluoxetine was tentatively identified in the serum. No other discriminant metabolites were identified as of yet. The data presented suggest that fluoxetine causes metabolic disruption in goldfish at multiple organ levels. Because of the widespread detection of fluoxetine and other emerging SSRIs in the aquatic environment, future research is required to firmly establish this pharmaceutical class as a metabolic and endocrine disrupting chemical.

Alcohol is one of the most common drugs of choice among adolescents. Normally, the method of consumption is drinking large quantities of alcohol in short periods of time, otherwise known as “binge drinking.” Corticotropin releasing factor (CRF) stress peptide producing cells in central nucleus of the amygdala (CeA) has been implicated in behavioral responses to stress and addiction. The goals of this thesis were to determine the effects of voluntary binge drinking in adolescence and vapor-induced alcohol dependence in adulthood on CRF cells in the CeA. These studies were done using an operant model of voluntary binge drinking in rodents in which adolescent animals are allowed to orally self-administer sweetened alcohol intermittently (or sweetened water for controls) during early adolescence. The current findings demonstrate that binge drinking during adolescence decreases the number of CRF-ir cells in the CeA. This decrease in cell number is long-term, lasting well into adulthood and dependence does not exacerbate this effect. A second goal was to determine whether certain behaviors could be used as a predictive measure for adolescent binge drinking. The current findings indicated that frequency of self-grooming, can be used as a predictive measure for adolescent binge drinking. Specifically, increased frequency of self-grooming predicts lower alcohol self administration during adolescence.

La prise illicite de drogues opiacées est un problème majeur de santé publique dans le monde. L’apparition du syndrome de sevrage aux opiacés (SAO) suite à l’arrêt de prise d’opiacés est considérée comme élément clef dans la vulnérabilité associée à la rechute de prise d’opiacés. En effet le syndrome de SAO est caractérisé par des altérations comportementales et neurobiologiques en réponse au stress qui sont déterminantes dans le phénomène de dépendance aux opiacés. Le système corticotropin-releasing factor (CRF) est un coordinateur central des circuits de réponse au stress par l’intermédiaire de ses deux récepteurs le: CRF1 et CRF2. L’objectif de cette thèse est de déterminer le rôle du récepteur CRF2 dans l’apparition des états affectifs négatifs et des désordres motivationnels impliqués dans la rechute de la consommation de drogues opioïdes lors du SAO.Nous avons démontré par une série d’expériences conduite chez des souris invalidées au récepteur CRF2 (CRF2-/-), que la délétion génétique du récepteur CRF2-/- éliminait les états dysphoriques ainsi que les altérations moléculaires induites par le SAO sans détériorer les réponses neuroendocriniennes qui sont primordiales lors des adaptations aux stress associées au sevrage. De surcroît, nous avons trouvé que les souris CRF2-/- entrainées dans une procédure de tâche opérante dirigée vers l’obtention d’une nourriture palatable, montraient une diminution des troubles motivationnels induits par le SAO. Plusieurs rapports montrent que chez l’Homme, les évènements stressants apparaissant lors d’une période de sevrage provoquent une rechute de la consommation d’alcool ou de drogues. Nous avons développé un modèle murin qui montrait un rétablissement de recherche de nourriture palatable suite à une procédure de stress appliquée pendant une période de SAO. Par ailleurs, nous avons observé un dimorphisme sexuel du rôle du récepteur CRF2 dans le rétablissement de recherche de nourriture palatable, suite au stress, longtemps après un SAO.Les résultats de ce travail de thèse nous permettent de mettre en avant le récepteur CRF2 comme possible cible thérapeutique dans le traitement de la dépendance aux opiacés. / Opiate illicit use represents one of the most severe sanitary problems throughout the world. Among humans, the emergence of the opiate withdrawal (OW) syndrome after cessation of opiate intake is considered as one of the key motivational elements that lead to the vulnerability to opiates relapse. Therefore, the OW is characterized by a various alterations of the behavioral and neurobiological homeostasis responses to stress which are determinants in opiate dependence. The Corticotropin-releasing factor (CRF) system is the major coordinator of stress-responsive circuitry. Through its two receptors CRF1 and CRF2, the CRF system has recently emerged as major contributor in the development of components of the OW syndrome. The aim of this thesis is to determine the role of CRF2 receptor in the negative affective states and motivational disorders implicated in opiate relapse during OW.Behavioral and biological experiments were conducted in CRF2 receptor-deficient mice (CRF2-/-). We reported that genetic deletion of the CRF2 receptor eliminates dysphoria and molecular alterations elicited by OW without impairing brain, neuroendocrine and autonomic stress-coping responses to withdrawal. Using behavioral approaches of operant responding to highly palatable food (HPF) we found that CRF2-/- reduces motivational disorders induced by intermittent morphine injections and withdrawal. Finally, we described a mouse model of stress-induced food reinstatement seeking behavior during prolonged OW. Furthermore, we reported a gender dimorphism in the role of the CRF2 receptor in the stress-induced reinstatement of HPF seeking behavior long-lasing after opiate treatment.These findings underscore the importance of CRF2 receptor as possible effective treatment of the critical problem of opiate dependence.

Les travaux de cette thèse visent à étudier le role du système corticotropin-releasing factor (CRF) dans les dysfonctions cognitives, les altérations du comportement social et la vulnérabilité au stress associées à l’addiction aux drogues. Les effets de la délétion génétique du récepteur CRF1 ou CRF2 sont examinés dans les tests de reconnaissance d’objet et de préférence sociale après une exposition chronique et pendant le sevrage à la cocaine. Le rôle du récepteur CRF2 dans la vulnérabilité au stress qui pourrait précipiter l’apparition de déficits cognitifs et sociaux pendant le sevrage prolongé à la cocaine est également étudié. / Stimulant-related disorders are characterized by emotional-like, cognitive and social dysfunction that may contribute to the maintenance of the disease. In addition, stimulant use and withdrawal may alter brain stress systems. The corticotropin-releasing factor (CRF) system is a major stress coordinator hypothesized to contribute to substance-related disorders. CRF signalling is mediated by two receptor types, named CRF1 and CRF2. The specific role of each of the CRF receptors in negative affective-like, cognitive and social dysfunction associated with stimulant administration and withdrawal remains largely unknown. The present study demonstrates that the CRF1 receptor-deficiency increases the anxiety-like behaviour induced by intermittent administration of escalating doses of cocaine (5-20 mg/kg, i.p.), as assessed by the elevated plus maze. In addition, the same cocaine regimen induces novel object recognition (NOR) and sociability deficits, which are unaffected by CRF2 receptor-deficiency. However, CRF2 receptor-deficiency effectively shortens the duration of the NOR and sociability deficit induced by cocaine withdrawal. Furthermore, following the apparent recovery of NOR and sociability performances during relative long-term (42 days) cocaine withdrawal, CRF2 receptor-deficiency eliminates the stress-induced re-emergence of NOR and sociability deficit. Stressed cocaine-withdrawn mice show a genotype-independent higher c-fos mRNA expression in the perirhinal cortex, a brain region mediating NOR performance, than stressed drug-naïve mice. However, neither genotype nor drug withdrawal affects the expression of tyrosine hydroxylase in the ventral tegmentale area and the locus coeruleus, CRF in the amygdala and the paraventricular nucleus of the hypothalamus and dynorphin in the nucleus accumbens shell. The latter results suggest that stress vulnerability during long-term cocaine withdrawal is not due to alterations in stress-coping mechanisms. The present study provides initial evidence of a critical role for the CRF system in cognitive and sociability deficits and vulnerability induced by stimulant administration and withdrawal, suggesting new therapeutic strategies for substance-related disorders.