Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

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.

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is an integral component of the mouse accessory olfactory system, and plays a key role in pheromonal processing. In males, this region is not only larger and contains more neurons than in females, but the cells are also larger. The present study examined the relationship between preference for opposite-sex odour and regional volume, cell number and cell size within the BNSTp of both male and female mice. No correlations were found between olfactory preferences and any of the three morphological BNSTp parameters. However, the olfactory preference task results were not congruent with previous research. Therefore, it remains inconclusive whether relationships exist between olfactory preference behaviour and morphology of the BNSTp.

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.

The principal nucleus of the bed nucleus of the stria terminalis (BNSTp) is an integral component of the mouse accessory olfactory system, and plays a key role in pheromonal processing. In males, this region is not only larger and contains more neurons than in females, but the cells are also larger. The present study examined the relationship between preference for opposite-sex odour and regional volume, cell number and cell size within the BNSTp of both male and female mice. No correlations were found between olfactory preferences and any of the three morphological BNSTp parameters. However, the olfactory preference task results were not congruent with previous research. Therefore, it remains inconclusive whether relationships exist between olfactory preference behaviour and morphology of the BNSTp.

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)

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-12-20T16:28:52Z No. of bitstreams: 2 Dissertação - Juliana Macêdo Silvestre - 2015.pdf: 815128 bytes, checksum: 3bea2ad6d4ab6f0e783b901523c0d838 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-12-26T14:12:10Z (GMT) No. of bitstreams: 2 Dissertação - Juliana Macêdo Silvestre - 2015.pdf: 815128 bytes, checksum: 3bea2ad6d4ab6f0e783b901523c0d838 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-12-26T14:12:10Z (GMT). No. of bitstreams: 2 Dissertação - Juliana Macêdo Silvestre - 2015.pdf: 815128 bytes, checksum: 3bea2ad6d4ab6f0e783b901523c0d838 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-05-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Cerrado is a biome that occupies about 25% of Brazilian territory. The seasonality, hot-humid and cold-dry periods, is one of the most striking features of the Cerrado. Reduced rainfall reduces the availability of water in the surface layers of the soil and increases the evaporative demand of air leading to the formation of litter cooperating in cycling soil nutrients. Another important factor in the Cerrado is the occurrence of fires that influence the dispersion, bloom, sprouting and diversity of certain species of plants. The relationship between seasonality, low fertility of soils and naturally occurring fires has shaped vegetative and reproductive strategies of plants from the Cerrado biome. Ichthyothere terminalis (Spreng.) Blake Asteraceae species occurring in the Cerrado, accumulates large amounts of fructans in the underground organs as reserve carbohydrates. Fructans are fructose polymers which are synthesized by the enzymes 1-SST (sucrose: sucrose fructosyltransferase) and FFT (fructan: fructan fructosyltransferase) and depolymerized by 1-FEH (exohidrolase fructan) and invertase. This study investigated the effect of freezing in the production of the enzymes 1-SST, 1-FEH and Invertase in I. terminalis. The partial characterization of these enzymes was performed. The samples were frozen immediately after collection (EB2) or after 6 hours of transport to the laboratory (EB1). The highest amount of protein was found in EB1 (97.81 μg.mL-1) as compared to EB2 (11,90 μg.mL-1). The results showed that the extraction method was efficient, since there was a significant reduction in reducing sugars and the activity of enzymes 1-SST, 1-FEH and Invertase were detected, mainly in the EB1. Enzymes 1-SST and Invertase showed a pH optimum between pH 6 and 7, while the 1-FEH enzyme had a greater spectrum ranging from pH 5 to 7. The three enzymes had an optimum temperature in the range 40 to 50 (°C). This work represents an important step towards understanding the role of these enzymes in the fructan metabolism in I. terminalis. / O Cerrado é um bioma que ocupa cerca de 25% do território brasileiro. A sazonalidade climática, períodos quente-úmido e frio-seco, é uma das características mais marcantes do Cerrado. A redução pluviométrica diminui a disponibilidade de água nas camadas superficiais do solo e eleva a demanda evaporativa do ar levando a formação de serrapilheira que coopera na ciclagem de nutrientes do solo. Outro fator importante no Cerrado é a ocorrência de queimadas que influenciam na dispersão, floração, rebrota e diversidade de certas espécies de plantas. A relação entre sazonalidade climática, baixa fertilidade dos solos e ocorrência natural de queimadas tem moldado estratégias vegetativas e reprodutivas das plantas do bioma Cerrado. Ichthyothere terminalis(Spreng.) Blake, espécie da família Asteraceae ocorrente no Cerrado, acumula grandes quantidades de frutanos em seus órgãos subterrâneos como carboidratos de reserva. Os frutanos são polímeros de frutose sintetizados pelas enzimas 1-SST (sacarose: sacarose frutosiltransferase) e FFT (frutano:frutano frutosiltransferase) e despolimerizados pela 1-FEH (frutano exohidrolase) e invertase. O presente trabalho objetivou estudar o efeito do tempo de congelamento na produção das enzimas 1-SST, 1-FEH e Invertase em I. terminalis. Além disso, a caracterização parcial destas enzimas foi realizada. As amostras coletadas foram congeladas imediatamente após a coleta (EB2) ou após 6 horas de transporte até o laboratório (EB1). A análise da quantidade de proteína extraída mostrou que EB1 apresentou maior quantidade de proteínas (97,81 μg.mL-1) quando comparada com EB2 (11,90 μg.mL-1). O método de extração foi eficiente, pois houve uma redução significativa em açúcares redutores e a atividade das enzimas 1-SST, 1-FEH e Invertase foram detectadas, principalmente no EB1. As enzimas 1-SST e Invertase apresentaram um pH ótimo entre pH 6 e 7, ao passo que a enzima 1-FEH teve um espectro maior variando de pH 5 a 7. As três enzimas apresentaram uma temperatura ótima na faixa de 40 a 50 (°C). Este trabalho representa um passo importante para o entendimento do papel destas enzimas no metabolismo de frutanos em I. terminalis.

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.