Substantial evidence suggests that serotonin (5-HT) activation within the brain modulates anxiety-like behavior. The bed nucleus of the stria terminalis (BNST) has been argued to mediate anxiety-like behavioral responding, and the activation of 5-HT systems may modulate anxiety-like behavior via the release of 5-HT within the BNST. Prior studies have suggested that the 5-HT1, 7 agonist 5-carboxyamidotrytamine (5-CT) is anxiolytic, which is consistent with a reduction in BNST activity via the activation of postsynaptic 5- HT1A receptors. However the anxiolytic effects of 5-CT could also have been mediated by 5-HT7 receptor activation. Hence, to isolate the effects of 5-HT1A on anxiety-like behavior, we infused the 5-HT1A antagonist WAY-100635 (0, 0.04, 0.4, and 4.0 μg/μl in saline vehicle) into the BNST of rats immediately before social interaction or acoustic startle testing. For social interaction testing pairs of rats were administered two 5-sec 1- mA footshocks immediately after infusion, removed from the chamber and measured for social interaction in a separate testing apparatus. For acoustic startle testing, rats were placed in boxes and measured for the percentage increase in test (post-infusion) startle from baseline (pre-infusion) startle. Anxiety levels were operationalized as the amount of social interaction per line cross and the percentage increase in startle following drug infusion. WAY-100635 dose dependently decreased social interaction, indicative of an anxiogenic effect. Interestingly, 0.4μg/μl of WAY-100635 decreased startle, indicative of an anxiolytic effect. These data suggest that activation of the 5-HT systems modulates anxiety-like behavior by altering activity within the BNST.

Females are disproportionately affected by stress- related mood disorders. Child abuse is the single greatest environmental risk factor for mood disorders. An animal model of child abuse, juvenile social subjugation (JSS), was used to determine whether males and females differentially process stress, specifically in the bed nucleus of the stria terminalis (BST). Rats (n=36) were randomly assigned to one of three conditions: JSS, Benign Control (BC) or Handled Control (HC). Following this procedure, brains were processed for Fos, which indicates neural activity. It was hypothesized that the JSS condition would evoke more neural activation than other conditions and would do so more in females. Across both sexes, we hypothesized there would be significantly more activation in the posterior BST than in the anterior BST. Based on earlier research, we hypothesized there would be and a sex difference in total neuron number, favoring males, in the posterior BST.

Females are disproportionately affected by stress- related mood disorders. Child abuse is the single greatest environmental risk factor for mood disorders. An animal model of child abuse, juvenile social subjugation (JSS), was used to determine whether males and females differentially process stress, specifically in the bed nucleus of the stria terminalis (BST). Rats (n=36) were randomly assigned to one of three conditions: JSS, Benign Control (BC) or Handled Control (HC). Following this procedure, brains were processed for Fos, which indicates neural activity. It was hypothesized that the JSS condition would evoke more neural activation than other conditions and would do so more in females. Across both sexes, we hypothesized there would be significantly more activation in the posterior BST than in the anterior BST. Based on earlier research, we hypothesized there would be and a sex difference in total neuron number, favoring males, in the posterior BST.

Immune-brain-endocrine communication influences behaviour and contributes to the development of the central nervous system (CNS) in a sexually dimorphic manner. The bed nucleus of the stria terminalis (BST) is a highly sexually dimorphic brain region; in most mammalian species the male BST is larger than the female BST. Previously, our lab has shown that male and female mice lacking T cells due to knock out of the beta (b) and delta (d) chains of the T cell receptor (TCRb-/-d-/-) have reduced anxiety-like behaviour. This was shown with increased time spent in the open arms of the elevated plus maze by TCRb-/-d-/- mice compared to wild type (WT) mice of both sexes. T cell deficient mice also show differences in brain volume compared to WT, including a lack of sexual dimorphism in volume of the BST. The present study explored the impact of T cell deficiency on immune and endocrine factors implicated in sex differences of the CNS. The first analysis was of serum Anti-Müllerian hormone (AMH). AMH is a key determinant of the male phenotype during fetal development. It has also been shown by others to contribute to sexual dimorphic development of the BST. Our postnatal analysis of serum AMH using ELISA demonstrated an age and genotype effect, where a peak in serum AMH levels in WT mice of both sexes was absent in both male and female TCRb-/-d-/- mice at postnatal day (P) 7. These results suggest that T cells have an impact on the endocrine system in early life but the process does not appear to be sexually dimorphic. The present study also explored the impact of TCR knockout on microglia, the resident immune cells of the brain. Other have shown microglia contribute to sexual dimorphic brain development. This contribution occurs through interaction with endocrine factors, making them a key player in the immune-brain-endocrine crosstalk. Using immunohistochemistry and the microglial marker, anti-Iba1, microglia were examined in adult and P7 WT and TCRb-/-d-/ mice. To quantify microglia, soma were traced using AxioVision microscope software, and microglia cell number, perimeter, radius, feret ratio, and area in dorsal and ventral BST were assessed. Our results show sex differences in microglia number in dorsal BST in adult WT mice, where female WT mice had a lower number of microglia compared to WT males, however this difference was absent in TCRb-/-d-/- adult mice. There were no effects on microglia number in the ventral BST and morphology analysis did not reveal any effects in the dorsal or ventral BST. Furthermore, the difference in microglia number was absent in all groups of P7 mice and analysis of soma morphology did not reveal any significant effects. This study explored the impact of TCR knockout on the BST by exploring the immune and endocrine factors shown to contribute to its sexual dimorphic development. The results suggest a non-dimorphic impact on the endocrine system in the postnatal period and a dimorphic impact on microglia that is age and region-specific. The findings reveal a complex network emphasizing the importance of a systems-wide approach to the study of sex differences in the CNS. / Thesis / Master of Science (MSc)

The tendency of users to relapse severely hinders adequate treatment of addiction. Physical and psychological stressors often contribute to difficulties in maintaining behavior change, and may play a significant role in relapse. We have previously shown that the activation of pituitary adenylate cyclase activating peptide (PACAP) systems in the bed nucleus of the stria terminalis (BNST) mediates many consequences of chronic stressor exposure. Hence, chronic stress substantially increased BNST PACAP levels, intra-BNST PACAP infusions produced the behavioral and endocrine consequences of stressor exposure, and BNST PACAP antagonism blocked many of the consequences of chronic stress. In the present set of studies, we investigated the role of BNST PACAP in stress-induced reinstatement of cocaine seeking. Rats self-administered cocaine (3mg/ml; 0.5mg/ig/infusion, i.v.) for 1hr daily over 10 days, which was followed by extinction training in which lever pressing no longer resulted in cocaine delivery. In the first experiment we showed that intra-BNST PACAP infusion (1 μg; 0.5 μl per side) reinstated previously extinguished cocaine seeking behavior. In the second experiment intra-BNST infusions of the PAC1/VPAC2 antagonist, PACAP 6-38 (1 μg; 0.5 μl per side) blocked stress-induced reinstatement. Hence, stressor exposure (5 sec 2mA footshock) caused significant reinstatement of cocaine seeking behavior, which was blocked by intra-BNST PACAP6-38 infusion. Overall, these data suggest that BNST PACAP systems mediate stress-induced reinstatement to drug seeking. Understanding the neuropharmacology of BNST PACAP in stress-induced reinstatement and the role of PACAP systems may lead to viable targets for relapse prevention.

In many species, including Syrian hamsters, male reproductive behavior depends on the perception of odor cues from conspecifics in the environment. Volatile odor cues are processed primarily by the main olfactory system, whereas non-volatile cues are processed primarily by the accessory olfactory system. Together, these two chemosensory systems mediate appetitive reproductive behaviors, such as attraction to female odors, and consummatory reproductive behaviors, such as copulation, in male Syrian hamsters. Main and accessory olfactory information are first integrated in the medial amygdala (MA), a limbic nucleus that is critical for the expression of reproductive behaviors. MA is densely interconnected with other ventral forebrain nuclei that receive chemosensory information and are sensitive to steroid hormones. Specifically, several lines of evidence suggest that MA may generate behavioral responses to socio-sexual odors via functional connections with the posterior bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA). It is unknown, however, how these three nuclei act as functional circuit to adaptively regulate appetitive and consummatory reproductive behaviors. Therefore, the overarching goal of this dissertation was to determine how BNST and MPOA function, both uniquely and as a circuit with MA, to generate attraction to female odors and copulatory behaviors in male Syrian hamsters. We found that BNST is required for attraction to female odors, but not for copulation, in sexually-naïve males. In contrast, MPOA is required for both attraction to female odors and for copulation in sexually-naïve males. Surprisingly, prior sexual experience mitigated the requirement of BNST and MPOA for these behaviors. Next, we found that MA preferentially transmits female odor information to BNST and to MPOA, whereas BNST relays female and male odor information equivalently to MPOA. Finally, we found that the functional connections between MA and BNST are required for attraction to female odors but not for copulation, whereas the functional connections between MA and MPOA are required for copulation but not for attraction to female odors. Ultimately, these data may uncover a fundamental mechanism by which this ventral forebrain circuit regulates appetitive and consummatory reproductive behaviors across many species and modalities.

Social conflict is a part of everyday life, and it can be a potent stressor for both humans and other animals. In the laboratory, when two Syrian hamsters (Mesocricetus auratus) compete for territory, a dominance hierarchy is quickly formed. Becoming subordinate is a significant stressor resulting in increased release of adrenocorticotropic hormone, β-endorphin, and cortisol. Defeated hamsters will also subsequently fail to display territorial aggression in future social encounters and will instead display increased submissive behavior, even in the presence of a smaller, non-aggressive intruder. This change in behavior is consistent and long-lasting and has been termed conditioned defeat (CD). Corticotropin releasing factor (CRF) is an important neuropeptide in the control of the hypothalamo-pituitary-adrenal (HPA) axis response to stress. It is also involved in a number of behaviors such as anxiety, stress responding, food intake, learning, and memory. The widespread distribution of CRF, CRF-like peptides, and CRF receptors, particularly in brain sites related to anxiety, fear, and stress responses, suggests a role for CRF and CRF-like peptides in modulating emotional responses other than via HPA axis activity. It has also been shown that CRF may have a role in the acquisition and expression of CD. Non-specific and CRF type 2-specific CRF antagonists reduce the acquisition and expression of CD in male hamsters while injection of a CRF type 1-specific antagonist does not. Therefore, the goal of this dissertation was to investigate the role of CRF type 1 and 2 receptors in CD in hamsters and to identify neuroanatomical locations where CRF may be acting. It was found that non-specific or CRF type 1 receptor specific agonists enhance the expression, but not acquisition, of CD. Further, these agonists appear to enhance aggressive behavior in animals that were not previously defeated, suggesting a modulatory role for CRF type 1 receptors in agonistic behavior that depends on an animal’s previous social experience. Further, localization of CRF receptors was determined in hamster brain in sites thought important for CD and agonistic behavior, but changes in receptor binding following defeat were not observed. Implications of these results and future directions are discussed.

In Syrian hamsters, social behavior is mediated exclusively by chemosensory cues and circulating gonadal steroid hormones. Where these two signals are processed in the brain is unknown, but the posterior bed nucleus of the stria terminalis (pBNST) has been suggested as a candidate site. Therefore, we tested male hamsters’ preference for opposite-sex odors following excitotoxic lesions of the pBNST. Lesions of the pBNST (pBNST-X) eliminated male hamsters’ preference for opposite-sex odors. Furthermore, pBNST-X males spent significantly less time investigating female odors than clean odors and significantly less time investigating female odors than control males did. Lesions of the pBNST did not change male hamsters’ investigation of male odors. The deficits observed in pBNST-X males were not due to a failure to discriminate between odors, as pBNST-X males were able to distinguish between odors. Together, these data suggest the pBNST is critical for opposite-sex odor preference in male hamsters.

O sistema endocanabinoide é composto por ligantes endógenos, enzimas responsáveis pela síntese e degradação desses ligantes, além de receptores específicos. As duas principais moléculas endógenas, anandamida (AEA) e 2- araquidonilglicerol (2-AG), após sintetizadas, são difundidos para a fenda sináptica e agem retrogradamente em receptores canabinóides do tipo 1 e/ou 2 (CB1 e CB2, respectivamente). A ação da AEA termina após processo de internalização seguido por hidrólise através da enzima FAAH (fatty acid amid hydrolase; amidohidrolase de acidos graxos), presente no neurônio pós-sináptico. O 2-AG, por sua vez, é degradado pela MAGL (monoacilglicerol lipase), localizada pré-sinapticamente. O receptor CB1 modula negativamente a liberação de diversos neurotransmissores no sistema nervoso central. A participação dos endocanabinoides é amplamente descrita em diversas estruturas cerebrais envolvidas na expressão de respostas relacionadas aos comportamentos de ansiedade e medo, tendo seus efeitos mediados principalmente por CB1 e CB2. Entretanto, o papel dos endocanabinoides em algumas estruturas ainda não está completamente elucidado. Dentre essas, destaca-se o Núcleo Leito da Estria Terminal (NLET), uma estrutura límbica com importante papel na integração de informações associadas com controle autonômico, endócrino e comportamental durante situações aversivas. Há poucas evidências da presença e do envolvimento do sistema endocanabinoide no NLET sobre a modulação de respostas de ansiedade. Desta forma, o objetivo do presente estudo foi avaliar o envolvimento do sistema endocanabinoide presente no NLET na modulação de respostas aversivas inata e aprendida. Para isso ratos Wistar (240g - 270g) foram implantados com cânulas guia bilateralmente no NLET para administração de drogas. Foram utilizados: veículo (DMSO 10%), antagonista de receptores CB1, o AM251 nas doses de 0,1 a 0,3 nmol/100nL e um inibidor da enzima FAAH, URB597 nas doses de 0,01 e 0,1 nmol/100nL. Cinco dias após a cirurgia estereotáxica, os animais foram submetidos ao teste de resposta inata do Labirinto em cruz elevado por 5 minutos. Avaliou-se o número de entradas e o tempo gasto nos braços abertos e fechados. Dois a três dias após, os animais foram submetidos ao protocolo do medo condicionado ao contexto, realizado em 3 dias consecutivos. No dia do teste, respostas comportamentais (tempo de congelamento) e autonômicas (Pressão arterial média; Frequência cardíaca, e Temperatura cutânea da cauda) foram continuamente avaliadas durante 10 min. Nossos resultados demonstraram que o bloqueio ou ativação de receptores CB1 no NLET não alterou as respostas de medo inato. Entretanto, no medo aprendido, o antagonismo de receptores CB1 no NLET aumentou o componente comportamental e pressórico. Ainda, a inibição da FAAH no NLET, via CB1, diminuiu o componente comportamental e pressórico da resposta emocional condicionada. Desta maneira, podemos sugerir que o sistema endocanabinoide presente no NLET modula respostas de medo aprendido, sem intervir nas respostas de medo inato. / The cannabinoid system is composed by two main endogenous ligands, enzymes responsible for synthesis and degradation of these ligands, and specific receptors. The two main endogenous molecules, anandamide (AEA) and 2-arachidonoylglycerol (2- AG), after synthesized post-synaptically, are released into the synaptic cleft, where they activate cannabinoid receptors type 1 and 2 (CB1 and CB2, respectively). AEA action terminates after internalization followed by enzymatic hydrolysis via FAAH (fatty acid amid hydrolase) located in the postsynaptic neuron. Meanwhile, 2-AG is degraded presynaptically by MAGL (monoacylglycerol lipase). CB1 receptor negatively modulates the release of several neurotransmitters in central nervous system. The endocannabinoid system is widely present in several brain structures involved on fear expression and anxiety-related responses, mostly mediated via CB1 and CB2 receptors. Nevertheless, the role of the endocannabinoids system role in specific brain structures is not yet completely elucidated. One structure in particular, is the Bed Nucleus of Stria Terminales (BNST), which is a limbic structure responsible for integration of autonomic, neuroendocrine and behavioral information during aversive situations. There is little evidence about the presence and involvement of endocannabinoid system in the BNST on anxiety responses modulation. Therefore, the aim of the present study was to evaluate the role of the endocannabinoid system in the BNST on the modulation of innate and learned aversive responses. Male Wistar rats (240 - 270g) were submitted to stereotaxic surgery for bilateral guide cannula implantation directed to the BNST, for drug administration. Animals received local injections of vehicle, AM251 (CB1-antagonist; 0,1 - 0,3 nmol/100nL), URB597 (an inhibitor of FAAH; 0,01 - 0,1 nmol/100nL). Five days after the stereotaxic surgery, animals were submitted to the innate response test, the elevated plus maze, for 5 minutes. The percentage of entries and time spent in open and the number of enclosed arms entries were analyzed. After two to three days, animals were submitted to the contextual fear conditioning protocol, performed in three consecutive days. On test day, behavioral (freezing) and autonomic responses (mean arterial pressure, heart rate and tail cutaneous temperature) were recorded for 10 min. Our data suggest that after CB1 receptors blocking or activation in the BNST do not promote changes in innate fear responses. However, during fear learning, CB1 receptor antagonism in the BNST increased freezing behavior and mean arterial pressure. In addition, FAAH inhibition in the BNST, via CB1, reduced freezing behavior and mean arterial pressure in the emotional conditioned response. These results suggest that endogenous cannabinoid system in the BNST can modulate defensive responses in fear learning, but not innate fear responses.

O sistema endocanabinoide é composto por ligantes endógenos, enzimas responsáveis pela síntese e degradação desses ligantes, além de receptores específicos. As duas principais moléculas endógenas, anandamida (AEA) e 2- araquidonilglicerol (2-AG), após sintetizadas, são difundidos para a fenda sináptica e agem retrogradamente em receptores canabinóides do tipo 1 e/ou 2 (CB1 e CB2, respectivamente). A ação da AEA termina após processo de internalização seguido por hidrólise através da enzima FAAH (fatty acid amid hydrolase; amidohidrolase de acidos graxos), presente no neurônio pós-sináptico. O 2-AG, por sua vez, é degradado pela MAGL (monoacilglicerol lipase), localizada pré-sinapticamente. O receptor CB1 modula negativamente a liberação de diversos neurotransmissores no sistema nervoso central. A participação dos endocanabinoides é amplamente descrita em diversas estruturas cerebrais envolvidas na expressão de respostas relacionadas aos comportamentos de ansiedade e medo, tendo seus efeitos mediados principalmente por CB1 e CB2. Entretanto, o papel dos endocanabinoides em algumas estruturas ainda não está completamente elucidado. Dentre essas, destaca-se o Núcleo Leito da Estria Terminal (NLET), uma estrutura límbica com importante papel na integração de informações associadas com controle autonômico, endócrino e comportamental durante situações aversivas. Há poucas evidências da presença e do envolvimento do sistema endocanabinoide no NLET sobre a modulação de respostas de ansiedade. Desta forma, o objetivo do presente estudo foi avaliar o envolvimento do sistema endocanabinoide presente no NLET na modulação de respostas aversivas inata e aprendida. Para isso ratos Wistar (240g - 270g) foram implantados com cânulas guia bilateralmente no NLET para administração de drogas. Foram utilizados: veículo (DMSO 10%), antagonista de receptores CB1, o AM251 nas doses de 0,1 a 0,3 nmol/100nL e um inibidor da enzima FAAH, URB597 nas doses de 0,01 e 0,1 nmol/100nL. Cinco dias após a cirurgia estereotáxica, os animais foram submetidos ao teste de resposta inata do Labirinto em cruz elevado por 5 minutos. Avaliou-se o número de entradas e o tempo gasto nos braços abertos e fechados. Dois a três dias após, os animais foram submetidos ao protocolo do medo condicionado ao contexto, realizado em 3 dias consecutivos. No dia do teste, respostas comportamentais (tempo de congelamento) e autonômicas (Pressão arterial média; Frequência cardíaca, e Temperatura cutânea da cauda) foram continuamente avaliadas durante 10 min. Nossos resultados demonstraram que o bloqueio ou ativação de receptores CB1 no NLET não alterou as respostas de medo inato. Entretanto, no medo aprendido, o antagonismo de receptores CB1 no NLET aumentou o componente comportamental e pressórico. Ainda, a inibição da FAAH no NLET, via CB1, diminuiu o componente comportamental e pressórico da resposta emocional condicionada. Desta maneira, podemos sugerir que o sistema endocanabinoide presente no NLET modula respostas de medo aprendido, sem intervir nas respostas de medo inato. / The cannabinoid system is composed by two main endogenous ligands, enzymes responsible for synthesis and degradation of these ligands, and specific receptors. The two main endogenous molecules, anandamide (AEA) and 2-arachidonoylglycerol (2- AG), after synthesized post-synaptically, are released into the synaptic cleft, where they activate cannabinoid receptors type 1 and 2 (CB1 and CB2, respectively). AEA action terminates after internalization followed by enzymatic hydrolysis via FAAH (fatty acid amid hydrolase) located in the postsynaptic neuron. Meanwhile, 2-AG is degraded presynaptically by MAGL (monoacylglycerol lipase). CB1 receptor negatively modulates the release of several neurotransmitters in central nervous system. The endocannabinoid system is widely present in several brain structures involved on fear expression and anxiety-related responses, mostly mediated via CB1 and CB2 receptors. Nevertheless, the role of the endocannabinoids system role in specific brain structures is not yet completely elucidated. One structure in particular, is the Bed Nucleus of Stria Terminales (BNST), which is a limbic structure responsible for integration of autonomic, neuroendocrine and behavioral information during aversive situations. There is little evidence about the presence and involvement of endocannabinoid system in the BNST on anxiety responses modulation. Therefore, the aim of the present study was to evaluate the role of the endocannabinoid system in the BNST on the modulation of innate and learned aversive responses. Male Wistar rats (240 - 270g) were submitted to stereotaxic surgery for bilateral guide cannula implantation directed to the BNST, for drug administration. Animals received local injections of vehicle, AM251 (CB1-antagonist; 0,1 - 0,3 nmol/100nL), URB597 (an inhibitor of FAAH; 0,01 - 0,1 nmol/100nL). Five days after the stereotaxic surgery, animals were submitted to the innate response test, the elevated plus maze, for 5 minutes. The percentage of entries and time spent in open and the number of enclosed arms entries were analyzed. After two to three days, animals were submitted to the contextual fear conditioning protocol, performed in three consecutive days. On test day, behavioral (freezing) and autonomic responses (mean arterial pressure, heart rate and tail cutaneous temperature) were recorded for 10 min. Our data suggest that after CB1 receptors blocking or activation in the BNST do not promote changes in innate fear responses. However, during fear learning, CB1 receptor antagonism in the BNST increased freezing behavior and mean arterial pressure. In addition, FAAH inhibition in the BNST, via CB1, reduced freezing behavior and mean arterial pressure in the emotional conditioned response. These results suggest that endogenous cannabinoid system in the BNST can modulate defensive responses in fear learning, but not innate fear responses.