Alcohol Use Disorders (AUDs) have devastating economic, mortality, and public health implications on society. Repeated cycles of alcohol intoxication and abstinence are known to induce neuroplastic alterations in specific brain regions, alterations which in turn trigger and sustain excessive alcohol drinking. The Bed Nucleus of the Stria Terminalis (BNST) has been proposed as a critical brain site for neuroadaptations induced by chronic alcohol. The Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP) system highly expressed in the BNST, has been proposed to be a master regulator of the stress response. These experiments aimed to investigate the role of the PACAP system of the BNST in alcohol drinking. Using a two-bottle choice chronic intermittent ethanol paradigm, we demonstrated that excessive intermittent alcohol consumption causes a marked increase in PACAP immunoreactivity in the BNST of mice. In addition, we observed a significant higher PACAP expression in the BNST of female, compared to male mice. These data lay the foundation for more extensive studies which may lead to the identification of a neuropeptide system with a critical role in heavy alcohol drinking. A deeper understanding of the specific neuroadaptations produced by chronic alcohol will be essential for the discovery of novel therapeutic agents to alleviate alcoholism. / 2019-07-11T00:00:00Z

Alcohol use disorder (AUD) costs the U.S. billions of dollars each year and is a leading cause of preventable death. AUD leads to many health complications, and those who suffer from AUD will often have stress and anxiety disorder comorbidities. To better understand this connection between AUD and stress and anxiety disorders, restraint stress (RS) and chronic-intermittent ethanol exposure (CIE) procedures were used on rats to analyze neuroinflammation via ELISA in the infralimbic and prelimbic cortices, and the bed nucleus of the stria terminalis (BNST). Levels of proinflammatory cytokines, TNF-α and IL-1β, were found to be elevated across different tests in both males and females. This study builds upon previous work in the BNST and offers new information for future studies of stress and alcohol in the region.

Thesis advisor: Alexa H. Veenema / The poorly understood, but robust sex differences in prevalence, symptom severity, and treatment responses of many psychiatric disorders characterized by social dysfunction signifies the importance of understanding the neurobiological mechanisms underlying sex differences in the regulation of social behaviors. One potential system involved is the oxytocin (OT) system. OT is an evolutionarily conserved neuropeptide that has been implicated in the regulation of a variety of social behaviors in rodents and humans. This thesis aims to clarify the role of OT in sex-specific regulation of social behavior and brain function in rats. Study 1 characterized sex differences in the OT system in the brain, and found that males show higher OT receptor (OTR) binding densities in several forebrain regions compared to females. Studies 2 and 3 then determined the relevance of these sex differences in OTR binding densities for the sex-specific regulation of social behavior using pharmacological manipulations of the OTR and in vivo measurement of OT release. Study 2 focused on the function of the OT system in the posterior bed nucleus of the stria terminalis (BNSTp), because this region showed the largest sex difference in OTR binding density, and is part of the core social behavior network. Results show that endogenous OT in the BNSTp is important for social recognition in both sexes, but that exogenous OT facilitated social recognition in males only. Furthermore, social recognition in males, but not in females, was associated with higher endogenous OT release in BNSTp. This study is the first to provide a link between sex differences in OTR binding density and OT release with sex-specific regulation of social recognition by OT. Study 3 focused on amygdala subregions because these regions were found to show sex-specific correlations of OTR binding density with social interest. Results show that the OT system modulates social interest in the central amygdala (CeA), but not the medial amygdala, in sex-specific ways, with activation of the OTR in the CeA facilitating social interest in males, but not in females. These results provide evidence that the CeA is a brain region involved in the sex-specific processing of social stimuli by the OT system. Finally, Study 4 examined whether sex differences in OTR binding densities in forebrain regions lead to sex-specific brain activation in response to OT. Functional magnetic resonance imaging was used to examine blood oxygen level-dependent (BOLD) activation in awake male and female rats following central or peripheral administration of OT. Central OT administration induced sex differences in BOLD activation in numerous brain regions (including several regions with denser OTR binding in males), in which males showed predominantly higher activation compared to females. Peripheral OT administration also induced sex differences in BOLD activation, but in fewer brain regions and in different brain regions compared to central OT, indicating that the pattern and the magnitude of sex differences in neural activation induced by OT strongly depend on the route of administration. Together, outcomes of this thesis provide novel insight into the sexual dimorphic structure and function of the OT system in rats, and highlights the fact that research seeking a full understanding of the role of the OT system in behavioral and brain responses is incomplete without the inclusion of both sexes. These results may be informative given the increasing popularity of the use of OT as a potential therapeutic agent in the treatment of social dysfunction in sex-biased psychiatric disorders. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Psychology.

The abuse of and addiction to drugs of abuse, such as tobacco, alcohol, opioids, and illicit drugs, are growing global problems that affect the welfare of individuals and societies worldwide. The National Institute of Drug Abuse estimates the annual cost of substance abuse to be over $740 billion in costs related to drug intoxication, withdrawal and relapse. A primary challenge in the treatment of substance abuse is the tendency of users to relapse following acute or extended periods of abstinence; on average over 60% of substance abusers will return to drug use within a year of receiving treatment, many relapsing following stressful life events. Central to the successful treatment of drug addiction is understanding the cellular mechanisms by which relapse episodes occur. Current data suggest that the activation of pituitary adenylate cyclase activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) is an important event underlying stress-induced reinstatement to drug-seeking in a rodent model of stress-induced relapse. In conjunction with immunohistochemistry and pharmacological treatments, we used this behavioral model of stress-induced relapse to evaluate PACAP and PACAP type-1 receptor (PAC1-R) signaling in stress-induced reinstatement to cocaine seeking. Activation of the PAC1 receptor appears to be critical to stress-induced reinstatement, as the selective PAC1-R agonist, maxadilan, produced reinstatement behaviors in the absence of stress. Moreover, BNST pretreatment with either mitogen activated protein kinase-ERK (MEK) or endocytosis inhibitors to block extracellular signal-related kinase (ERK) signaling attenuated stress-induced reinstatement. Furthermore, BNST phosphorylated ERK (pERK) expression, mediated by PAC1-R activation, is substantially potentiated in cocaine-experienced animals after stressor exposure, in a manner that is dependent on endosomal signaling and MEK activity. These data suggest that the activation of a PAC1 signaling cascade is a key event underlying stress-induced reinstatement. Furthermore, this data may suggest a permanent change in the BNST PACAP system (sensitization) following cocaine exposure.

Chronic or repeated exposure to stressful stimuli can result in several maladaptive consequences, including increased anxiety-like behaviors and altered peptide expression in brain structures involved in emotion. Among these structures, the bed nucleus of the stria terminalis (BNST) has been implicated in emotional behaviors as well as regulation of hypothalamic-pituitary-adrenal (HPA) axis activity. In rodents, chronic variate stress (CVS) has been shown to increase BNST pituitary adenylate cyclase activating polypeptide (PACAP) and its cognate PAC1 receptor transcript, and BNST PACAP signaling may mediate the maladaptive changes associated with chronic stress. In order to determine whether chronic stress would potentiate the behavioral and/or endocrine response to subthreshold BNST PACAP infusion, rats were exposed to a 7 day CVS paradigm previously shown to upregulate BNST PAC1 receptor transcripts; control rats were not stressed. Twenty-four hours following the last stressor, stressed and control rats were bilaterally infused into the BNST with 0.5 µg PACAP. Startle response to intra-BNST PACAP infusion was assessed post-infusion in Experiment 1. In Experiments 2 and 3, blood was sampled via a tail nick 30 min following PACAP infusion to assess the corticosterone response to PACAP following CVS. We found an increase in startle amplitude and an increase in plasma corticosterone levels 30 minutes following BNST PACAP infusion only in rats that had been previously exposed to CVS. These results were likely mediated via PAC1 receptors, as equimolar infusion of the VPAC1/2 receptor ligand vasoactive intestinal polypeptide (VIP) had no effect on plasma corticosterone levels. These results suggest that repeated exposure to stressors sensitizes the neural circuits underlying the behavioral and endocrine responses to BNST PACAP infusion and BNST PACAP/PAC1 receptor signaling likely plays a critical role in mediating stress responses.

Submitted by Daniele Amaral (daniee_ni@hotmail.com) on 2016-09-13T18:42:57Z No. of bitstreams: 1 DissLAO.pdf: 6690751 bytes, checksum: ad771d53381565a9c810eb4f7a21c348 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-15T13:32:59Z (GMT) No. of bitstreams: 1 DissLAO.pdf: 6690751 bytes, checksum: ad771d53381565a9c810eb4f7a21c348 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-15T13:33:08Z (GMT) No. of bitstreams: 1 DissLAO.pdf: 6690751 bytes, checksum: ad771d53381565a9c810eb4f7a21c348 (MD5) / Made available in DSpace on 2016-09-15T13:33:28Z (GMT). No. of bitstreams: 1 DissLAO.pdf: 6690751 bytes, checksum: ad771d53381565a9c810eb4f7a21c348 (MD5) Previous issue date: 2015-04-10 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / The corticotropin-releasing factor (CRF) is involved in behavioral and physiological responses to emotional stress through its action in several limbic structures, including the bed nucleus of the stria terminalis (BNST). Nevertheless, the role of CRF1 and CRF2 receptors in the BNST in autonomic adjustments during aversive threat is unknown. Therefore, in the present study we investigated the involvement of CRF receptors within the BNST in autonomic responses evoked by the acute restraint stress in rats. For this, we evaluated the effects of bilateral treatment of the BNST with selective agonists and antagonists of either CRF1 or CRF2 receptors in the arterial pressure and heart rate increase and the decrease in tail skin temperature induced by restraint stress. Microinjection of the selective CRF1 receptor antagonist CP376395 into the BNST reduced the pressor and tachycardiac responses caused by restraint. Conversely, BNST treatment with the selective CRF1 receptor agonist CRF increased restraint-evoked arterial pressure and HR responses and reduced the fall in tail skin temperature response. All effects of CRF were inhibited by local BNST pretreatment with CP376395. The selective CRF2 receptor antagonist antisauvagine-30 reduced the arterial pressure increase and the fall in tail skin temperature. The selective CRF2 receptor agonist urocortin-3 increased restraint-evoked pressor and tachycardiac responses and reduced the drop in cutaneous temperature. All effects of urocortin-3 were abolished by local BNST pretreatment with antisauvagine-30. These findings indicate an involvement of both CRF1 and CRF2 receptors in the BNST in autonomic adjustments during emotional stress. / O fator liberador de corticotropina (CRF) está envolvido em respostas comportamentais e fisiológicas ao estresse emocional por meio de sua ação em várias estruturas límbicas, incluindo o núcleo leito da estria terminal (NLET). No entanto, o papel dos receptores CRF1 e CRF2 no NLET nas respostas autonômicas durante situações aversivas é desconhecido. Portanto, no presente estudo nós investigamos o envolvimento de receptores de CRF do NLET nas respostas autonômicas evocadas pelo estresse de restrição agudo em ratos. Para isso, foram avaliados os efeitos do tratamento bilateral do NLET com agonistas e antagonistas seletivos dos receptores CRF1 ou CRF2 nas respostas de aumento da pressão arterial e frequência cardíaca e de diminuição da temperatura cutânea da cauda induzidas pelo estresse por restrição agudo. Microinjeção do antagonista seletivo do receptor CRF1, CP376395, no NLET reduziu as respostas de aumento da pressão arterial e frequência cardíaca evocadas pelo estresse por restrição. Por outro lado, o tratamento do NLET com o agonista seletivo do receptor CRF1, CRF, causou um aumento das respostas pressora e taquicárdica e reduziu a resposta de queda de temperatura cutânea da cauda. Os efeitos do CRF foram inibidos pelo pré-tratamento do NLET com CP376395. O antagonista seletivo do receptor CRF2, antisauvagine-30, reduziu o aumento da pressão arterial e a queda da temperatura cutânea da cauda induzidas pelo estresse por restrição. O agonista seletivo do receptor CRF2, urocortina-3, potenciou as respostas pressora e taquicárdica e reduziu a queda na temperatura cutânea da cauda. Todos os efeitos da urocortina-3 foram abolidos pelo pré-tratamento local no NLET com antisauvagine-30. Esses resultados indicam um envolvimento de ambos os receptores CRF1 e CRF2 no NLET nos ajustes autonômicos durante o estresse emocional.

Tesis por compendio / La exposición crónica a sustancias de abuso lleva a cambios adaptativos en el cerebro que implican alteraciones en la expresión génica. Se ha propuesto que los factores de transcripción CREB y deltaFosB serían dianas moleculares para la regulación de la plasticidad, la cual lleva a la adicción.En este trabajo hemos estudiado los cambios en la activación de CREB, en PVN y NTS, y las quinasas que mediarían su activación durante la dependencia y síndrome de abstinencia a morfina, así como la respuesta del eje HHA durante dicho síndrome. También se investigó la posibilidad de que la activación de CREB y su coactivador transcripcional TORC1 dependan de la activación de receptores adrenérgicos. Además se evaluaron las posibles modificaciones en la expresión de FosB/deltaFosB en diferentes áreas cerebrales implicadas en la adicción, así como los cambios neuroendocrinos/neuroquímicos responsables de las alteraciones metabólicas observadas durante el tratamiento crónico con morfina. / Chronic exposure to opioids and other abused drugs results in adaptive changes in the brain involving alterations in gene expression. It is proposed that the transcription factors CREB and deltaFosB be molecular targets for the regulation of plasticity, which leads to addiction.In this work we studied changes in activation of the cAMP-response element binding protein (CREB) in PVN and NTS and the kinases that may mediate this activation during dependence and morphine withdrawal and the HPA axis response after naloxone-induced morphine withdrawal. We also investigated the possibility that the activation of CREB and the transcriptional coactivator of CREB, TORC1, arises from the activation of adrenergic receptors. We also evaluated the possible modifications in FosB/deltaFosB expression in several brain areas involved in addiction and neuroendocrine/neurochemical changes that are responsible for the metabolic alterations seen during chronic morphine treatment.

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Ciliary neurotrophic factor (CNTF) is produced by astrocytes which have been implicated in regulating stress responses. We found that CNTF in the medial amygdala (MeA) promotes despair or passive coping, i.e., immobility in an acute forced swim stress, in female mice, while having no effect in males. Neutralizing CNTF antibody injected into the MeA of wildtype females reduced activation of downstream STAT3 (Y705) 24 and 48 h later. In concert, the antibody reduced immobility in the swim test in females and only after MeA injection, but not when injected in the central or basolateral amygdala. Antibody injected into the male MeA did not affect immobility. These data reveal a unique role of CNTF in female MeA in promoting despair or passive coping behavior. Moreover, 4 weeks of chronic unpredictable stress (CUS) increased immobility in the swim test and reduced sucrose preference in wildtype CNTF+/+, but not CNTF-/- littermate, females. Following CUS, 10 min of restraint stress increased plasma corticosterone levels only in CNTF+/+ females. In males, the CUS effects were present in both genotypes. Further, CUS increased CNTF expression in the MeA of female, but not male, mice. CUS did not alter CNTF in the female hippocampus, hypothalamus and bed nucleus of stria terminalis. This suggests that MeA CNTF has a female-specific role in promoting CUS-induced despair or passive coping, behavioral anhedonia and neuroendocrine responses. Compared to CNTF+/+ mice, CNTF-/- mice did not show differences in CUS-induced anxiety-like behavior and sensorimotor gating function as measured by elevated T-Maze, open field and pre-pulse inhibition of the acoustic startle response. Together, this study reveals a novel CNTF-mediated female-specific mechanism in stress responses and points to opportunities for developing treatments for stress-related disorders in women.