The measurement of stress hormone levels in wild free-living animals is becoming an increasingly effective method for examining proximate mechanisms of animal behavior and the physiological impacts of human activities on wildlife. In these studies I measured plasma levels of the stress hormone corticosterone in the endangered red-cockaded woodpecker (Picoides borealis) to determine their role in the reproductive behavior of individuals in this species, and whether they are affected by human disturbance. In chapter one, I provide an introduction to the vertebrate stress response and I describe the natural history of the red-cockaded woodpecker. In chapter two, I compare corticosterone and reproductive hormone levels between breeding males and helper males to examine hormonal mechanisms of reproductive suppression in helper males. No hormonal differences existed between breeding and helper males. However, baseline corticosterone levels were lower in all males living in groups with two or more helper males, suggesting that male helpers reduce the workload of all other group members. In chapter three, I compare corticosterone levels between birds living in clusters subject to two different training restriction regimes on a military installation. Males living in clusters without training restrictions had lower baseline corticosterone than those living in clusters with training restrictions, suggesting that males habituate to chronic disturbance by downregulating baseline corticosterone levels. / Master of Science

Graduation date: 2006 / Most vertebrates exhibit seasonality in many life history traits. Such seasonal rhythms are temporally organized via the transduction of environmental cues (e.g., photoperiod, temperature) into appropriate endocrine signals. However, among ectothermic vertebrates that undergo continuous winter dormancy, temperature is the only environmental cue available for synchronizing seasonal rhythms. Most intriguing is that in species where reproduction occurs immediately following spring emergence, the associated changes in neurophysiology and behavior that accompany reproduction likely occur during winter dormancy. The purpose of this dissertation research was to explore the mechanisms by which temperature cues affect the chronobiology and seasonal reproduction of red-sided garter snakes (Thamnophis sirtalis parietalis). Because of their roles in circadian organization and energy balance, melatonin and corticosterone are likely hormonal components of these time-keeping systems. I first characterized the interactions between melatonin and corticosterone to better understand the hormonal mechanisms facilitating temperature-induced reproduction. Melatonin and corticosterone additively inhibit reproductive behavior during the spring mating season. Experimental manipulations with a serotonin receptor antagonist suggest the mechanism underlying these effects involves a serotonin-regulated system. Although melatonin does not influence corticosterone responses to capture stress, capture stress significantly increases melatonin concentrations. To investigate the functional significance of these interactions in regulating temperature-induced reproduction, I measured body temperatures of snakes as well as circadian melatonin and corticosterone cycles during winter dormancy and spring emergence using a combination of field and laboratory experiments. Surprisingly, an increase in body temperature is not necessary for emergence from winter dormancy. Rather, critically low temperatures may serve as a zeitgeber entraining an endogenous circannual cycle that regulates emergence. Decreased environmental temperatures, in the absence of changing photoperiod cues, modulate circadian melatonin and corticosterone rhythms during hibernation. Such temperature-induced changes in hormone rhythms may facilitate seasonal reproductive behavior following spring emergence. Furthermore, a phase-shift in corticosterone rhythms during the mating season may regulate the seasonal transition between reproductive and non-reproductive states in red-sided garter snakes. Such studies investigating the environmental and hormonal mechanisms underlying time-keeping systems may provide valuable insight into the potential impact of environmental perturbations (e.g., climate change) on seasonal rhythms in physiology and behavior.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: This study: 1. Describes the localization of CYP11B1 in the Cape baboon adrenal gland using Western blot analysis. CYP11B1 was localized to the adrenal cortex and medulla. 2. Describes the catalytic activity of CYP11B1 towards 11-deoxycorticosterone and corticosterone in adrenal cortical- and medullary tissue homogenates. Aldosterone formation in the adrenal medulla was identified using an atmospheric pressure chemical ionization-mass spectrometry method, which was developed in our department. 3. Compares the catalytic activity of three recombinant Cape baboon CYP11B1 cDNAs, expressed in COS-1 cells, towards 11-deoxycorticosterone and 11-deoxycortisol. 4. Describes the determination of the Michaelis-Menten constants and maximum reaction rates of 11-deoxycorticosterone and 11-deoxycortisol utilization by two functional recombinant Cape baboon CYP11B1 cDNAs, respectively. 11-Deoxycorticosterone metabolites were quantified using an enzyme immunoassay kit. 11-Deoxycortisol metabolites were quantified using a liquid chromatography-mass spectrometry method, which was developed in our department. 5. Describes the homology modeling of two isoforms of Cape baboon CYP11B1 using CYP102 and CYP2C5 as structural templates. The influence of three amino acid residue substitutions, located in the predicted D-E helix, on the catalytic activity of the two CYP11B1 isoforms was examined. / AFRIKAANSE OPSOMMING: Hierdie studie: 1. Beskryf die lokalisering van CYP11B1 in die bynier van die Kaapse bobbejaan deur gebruik te maak van die Western kladtegniek. CYP11B1 is gelokaliseer tot die adrenale korteks en medulla. 2. Beskryf die metabolisme van 11-deoksikortikosteroon en kortikosteroon in adrenale korteks- and medulla weefsel preparate, onderskeidelik. Die produksie van aldosteroon in die medulla is geïdentifiseer deur gebruik te maak van ‘n atmosferiese druk chemiese ionisasie-massa spektrometrie metode wat in ons departement ontwikkel is. 3. Vergelyk die katalitiese aktiwiteit van drie rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11- deoksikortisol metabolisme. 4. Beskryf die bepaling van die Michaelis-Menten konstantes en maksimum snelhede van twee funksionele rekombinante Kaapse bobbejaan CYP11B1 cDNAs, getransfekteer in COS-1 selle, ten opsigte van 11-deoksikortikosteroon en 11-deoksikortisol metabolisme. 11-Deoksikortikosteroon metaboliete is gekwantifiseer deur gebruik te maak van ‘n ensiem immunotoets. 11-Deoksikortisol metaboliete is gekwantifiseer deur middel van ‘n vloeistofchromatografie-massaspektrometrie metode, ontwikkel in ons departement. 5. Beskryf die modelering van drie-dimensionele strukture van twee funksionele Kaapse bobbejaan CYP11B1 isoensieme deur CYP102 en CYP2C5 as template te gebruik. Die effek van drie aminosuurresiduveranderinge in die voorspelde D-E heliks op die katalitiese aktiwiteit van die twee CYP11B1 isoforme is bepaal.

Throughout North America, many species of aerial insectivorous birds – birds that capture flying insects – have exhibited sharp declines, with tree swallows (Tachycineta bicolor) experiencing a 2.8% annual decline in Canada since 1989. The timing of these declines coincides with major changes in agriculture, perhaps signaling a potential causal link. Declines may be influenced indirectly by agriculturally-driven reductions in the abundance of the swallow’s main diet of aerial insects through increased agrochemical use, wetland drainage and cropping intensity. My objectives were to determine how aerial insect abundance and biomass vary across an agricultural landscape in the Canadian Prairies, and determine how breeding tree swallows respond in terms of (i) foraging behaviours and return rates, (ii) body condition, and (iii) selected physiological responses (i.e. oxidative stress and feather corticosterone). Artificial nest-boxes were erected at each of 5 sites with varying levels of agricultural intensification in south-central Saskatchewan, 2012-2014. Insect abundance and biomass were monitored daily using passive aerial samplers at each of the sites throughout the breeding season. Tree swallows adults (n = 596) and nestlings (n = 1107) from each nest were captured, banded, measured and blood and feather sampled to determine condition and physiological status. In 2014, radio frequency identification (RFID) technology was employed at a subset of nest-boxes to determine adult foraging rates and durations. Despite strong differences in land use characteristics among sites, insect abundance and biomass varied by year but were similar between heavily cropped and grassland references sites. Regardless, birds responded to temporal changes in prey abundance and differences were found in measurements of foraging behavior and physiological measures of oxidative stress. Adult foraging rate (number of nest visits/hour) was primarily influenced by wind and nestling age, but was also positively related to daily insect biomass. Foraging intervals (i.e., length of time spent on each trip) differed between agricultural intensities; birds on agricultural sites spent more time away from the nest box, presumably foraging, suggesting reduced nest attentiveness. Return rates were primarily predicted by body condition and brood size from the previous breeding season, but were also higher on grassland sites, suggesting that agricultural sites may be less favorable. Older adults (based on banding records) and females displayed a higher body condition than did younger individuals and males, respectively. Measures of physiology (i.e. total antioxidant counts and oxidative damage) in both adult and nestling swallows were primarily driven by temporal changes in insect biomass and abundance. Similarly, adult body mass and physiology were related to local insect availability, but not land use (agriculture/grassland). Measures of nestling feather corticosterone were unrelated to any of the explanatory variables. These results provide valuable information pertaining to components of individual quality, foraging behaviour, and return status in response to prey availability under differing agricultural land use regimes, which may help to inform conservation strategies for a variety of insectivorous bird species of conservation concern.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressant effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the Glucocorticoid Receptor (GR), a cytosolic receptor that translocates to the nuclei upon ligand binding and alters gene transcription. Among a long list of genes activated by GCs is the Glucocorticoid Induced Leucine Zipper (GILZ). Although GC induced GILZ expression has been well established in lymphocytes, little is known whether cardiomyocytes respond to GCs by inducing GILZ. Unlike lymphocytes, in which GCs induce apoptosis and GILZ mediates GC induced apoptosis, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression pattern in cardiomyocytes in vivo and in vitro. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs and point to H9C2 cell line as a valid model for studying the biological function of GILZ in cardiomyocytes. I have also determined GILZ functions as GC induced cytoprotective protein against the known cardiac toxicant Doxorubicin. Finally I have determined GILZ stabilizes Bcl-xL pro-survival protein, providing a possible mechanism of cytoprotection in cardiomyocytes.

We investigated licking and grooming (LG) levels received by each pup from their dams and the locomotor activity, anxiety-like behaviors, and stress reactivity in adult female offspring. We also investigated glucocorticoid receptor (GR) gene expression and its DNA methylation status in the hippocampus, comparing pups between and with-in litters. Rats that receive more LG than their siblings showed less anxiety-like behaviors and increased locomotor activity, regardless of their litter type. Higher licked pups also showed increased expression of the GR gene. Gene expression levels of the GR 17 splice variant were not significantly different as a function of dam LG or LG received, whereas DNA methylation levels at two CpG sites within GR17 promoter were significantly higher in high LG pups than low LG pups. Our results indicate that naturally occurring intra- and inter-litter differences in maternal LG have a lasting effect on the phenotypic outcomes of adult female offspring.

We investigated licking and grooming (LG) levels received by each pup from their dams and the locomotor activity, anxiety-like behaviors, and stress reactivity in adult female offspring. We also investigated glucocorticoid receptor (GR) gene expression and its DNA methylation status in the hippocampus, comparing pups between and with-in litters. Rats that receive more LG than their siblings showed less anxiety-like behaviors and increased locomotor activity, regardless of their litter type. Higher licked pups also showed increased expression of the GR gene. Gene expression levels of the GR 17 splice variant were not significantly different as a function of dam LG or LG received, whereas DNA methylation levels at two CpG sites within GR17 promoter were significantly higher in high LG pups than low LG pups. Our results indicate that naturally occurring intra- and inter-litter differences in maternal LG have a lasting effect on the phenotypic outcomes of adult female offspring.

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.

Estudos recentes associaram fortemente a fisiopatologia da Depressão ao estresse crônico e suas conseqüências, desde o comportamento alterado até a disrupção do eixo HPA e mudanças na expressão gênica. A depressão é uma doença que afeta milhões de pessoas ao redor do mundo, causando um grande impacto na qualidade de vida dos pacientes. Evidências sugerem o papel contributivo do stress de início da vida (ELS) para Depressão Maior (MD). O paradigma do estresse crônico variável é amplamente utilizado, mas seus impactos não foram estudados no início da vida. Portanto, para entender melhor esta condição, os filhotes de ratos Wistar machos (P1-P21) foram expostos ao paradigma Multimodal ELS. Foram avaliados os níveis plasmáticos de corticosterona (cort) e os órgãos relacionados ao eixo HPA. Adicionalmente, estes foram avaliados durante a idade adulta no teste de consumo de sacarose (TCS), no teste de nado forçado (TNF) e no teste da caixa claro-escuro (TCE). Os resultados indicam que os filhotes não se habituaram ao ELS multimodal. Em P21, o peso das glândulas adrenais dos animais ELS é significantivamente maior, e o timo e o peso corporal diminuíram, quando comparados com o grupo controle. O timo também se mantém significativamente reduzido quando comparado ao grupo controle em P90. Além disso, os ratos adultos submetidos ao protocolo ELS apresentaram menor ingestão de sacarose e maior latência para o compartimento claro na TCE, quando comparados ao grupo controle. Um outro modelo animal que apresenta estresse intermitente e tem sido amplamente utilizado é o modelo da restrição do material de ninho. Neste modelo a redução do material do ninho promove uma disrupção no cuidado materno, e gera alterações no desenvolvimento encefálico e comportamental da prole. O ELS leva a um surgimento precoce da inibição do medo contextual e à maturação acelerada do hipocampo. Nós postulamos que a corticosterona desempenha um papel funcional na regulação do tempo de maturação das regiões subjacentes à aprendizagem e expressão de ameaças, incluindo o hipocampo e a amígdala. Nossa hipótese é que alterações nos níveis plasmáticos de corticosterona podem afetar o tempo de processos de maturação cerebral e comportamental. Portanto nosso objetivo foi avaliar como um bloqueador da síntese de corticosterona altera o comportamento de medo condicionado e expressão de BDNF em animais controle ou submetidos ao modelo ELS de redução de material do ninho. Tanto nos grupos controle como ELS utilizamos animais Naïve, ou que receberam Veículo ou Metirapona (MET; 50mg/kg) em P12. Em P18, P21 ou P28 grupos independentes de camundongos foram expostos a uma única sessão de condicionamento do medo, seguido 24 h depois por um único teste de contexto. A análise do comportamento de congelamento no teste de contexto revelou que o tratamento com MET bloqueou a aceleração na inibição do medo contextual em camundongos fêmeas submetidos ao ELS. Curiosamente, em filhotes machos controles tratados com MET, houve um atraso na curva de desenvolvimento de medo contextual. Observamos que houve um correlato do efeito do tratamento com Metirapona na expressão do BDNF em regiões límbicas (hipocampo ventral e amígdala basolateral). Com base nos presentes resultados, a CORT provavelmente desempenha um papel importante no momento do desenvolvimento típico e das mudanças associadas ao ELS no comportamento e na maturação do cérebro. A epilepsia é uma condição neurológica crônica caracterizada pela predisposição persistente a gerar crises epilépticas, e pelas consequências neurobiológicas, cognitivas, psicológicas e sociais desta condição. A depressão é uma comorbidade psiquiátrica muito comum em pacientes com epilepsia. Cada vez mais dados sugerem que a epilepsia, depressão e outros possíveis distúrbios psiquiátricos como a ansiedade compartilham mecanismos patogênicos. Nesse sentido, foram encontradas anormalidades na linhagem WAR (Wistar Audiogenic Rat) que a tornam um modelo interessante para o estudo do estresse, a Epilepsia e as comorbidades neuropsiquiátricas envolvidas. Com base nisso, o presente estudo teve comoxiv Godoy, L.D. objetivo avaliar na linhagem WAR: 1. Cuidados maternos na linhagem WAR nas condições controle e sob estresse 2. Comportamentos de tipo depressivo basal e após o kindling audiogênico (crises convulsivas crônicas- KAu). Não houve diferença no tempo em postura de amamentação ativa, licking/grooming ou tempo da mãe no ninho, e no número de ataques e comportamentos agressivos. Observamos um aumento na latência de ratas WAR para recuperar os filhotes após separação materna, e enquanto 100% das ratas Wistar agruparam toda a ninhada, nas fêmeas WAR foi observado apenas 40%. 2. Para avaliar os comportamentos de tipo depressivo, os ratos foram submetidos a 20 estímulos acústicos duas vezes ao dia (KAu) (Wistar-KAu, WAR-KAu), enquanto os respectivos grupos controle permaneceram sem estímulo (Wistar, WAR). Posteriormente, os grupos foram submetidos ao TCS e TNF Não houve diferença entre o grupo WAR e Wistar no TCS , porém o grupo WAR-KAu grupo apresentou um aumento significativo em relação aos grupos controles. No TNF ambos os grupos WAR e WAR-KAu apresentaram redução significativa de escalada na sessão teste quando comparados ao Wistar. Tomados em conjunto, esses achados indicam que o ELS pode gerar suceptibilidade às comorbidades psiquiátricas associadas ao estresse, e que os modelos experimentais em questão permitem investigar como os efeitos dos glicocorticoies se relacionam com o neurodesenvolvimento, especificamente na maturação do comportamento de medo e de estruturas límbicas. Além disso, dados preliminares indicam que, embora a linhagem WAR não apresente diferenças comportamentais maternas em condições basais, pode apresentar alterações sob o efeito de eventos estressantes. Também, com base nos achados, a susceptibilidade de crises convulsivas pode estar relacionada às alterações nas estratégias comportamentais em situações estressantes na vida adulta, que também pode constituir vulnerabilidade às comorbidades psiquiátricas. / Recent studies have strongly associated the pathophysiology of depression with chronic stress and its consequences, from altered behavior to HPA axis disruption and changes in gene expression. Depression is a disease that affects millions of people around the world, causing a major impact on the quality of life of patients. Evidence suggests the contributory role of early life stress (ELS) for Major Depression (MD). The variable chronic stress paradigm is widely used, but its impacts were not studied early in life. Therefore, to better understand this condition, male Wistar rat pups (P1-P21) were exposed to the Multimodal ELS paradigm. Plasma levels of corticosterone (CORT) and organs related to the HPA axis were evaluated. Additionally, these were evaluated during adulthood in the sucrose consumption test (SCT), the forced swimming test (FST) and the light-dark box test (LDT). The results indicate that pups did not habituate to multimodal ELS. In P21, the weight of the adrenal glands of the ELS animals is significantly greater, and the thymus and body weight decreased, when compared with the control group. The thymus also remains significantly reduced when compared to the control group in P90. In addition, adult rats submitted to the ELS protocol presented lower intake of sucrose and greater latency to light compartment in the LDT when compared to the control group. Another animal model that presents intermittent stress and has been widely used is the constraint model of nest material. In this model, the reduction of nest material promotes disruption in maternal care and causes changes in the encephalic and behavioral development of offspring. ELS leads to an early onset of contextual fear inhibition and accelerated maturation of the hippocampus. We postulate that CORT plays a functional role in regulating the maturation time of the regions underlying the learning and expression of threats, including the hippocampus and amygdala. Our hypothesis is that changes in plasma CORT levels may affect the timed brain and behavioral maturation processes. Therefore, our objective was to evaluate how a CORT synthesis blocker (Metyrapone; MET) alters conditioned fear behavior and BDNF expression in animals reared under control conditions or submitted to the ELS model of limited nesting bed. Both in control and ELS groups included Naïve animals or animals that received vehicle or MET; 50 mg/kg in P12. In P18, P21 or P28 independent groups of mice were exposed to a single session of fear conditioning, followed 24 hours later by a single context test. Analysis of the freezing behavior in the context test revealed that MET treatment blocked the acceleration of contextual fear suppression at P22 induced by ELS, with no difference between the P19 or P29 groups. This effect was observed mainly in female mice. Interestingly, in control male pups treated with MET, there was an altered contextual fear developmental curve. MET showed significant reduction in freezing in PND22, increase in freezing in PND29 followed by delayed suppression in P39. We observed that MET behavioural data were further supported by BDNF expression in limbic regions (ventral hippocampal and basolateral amygdala). Based on the current results, CORT probably plays an important role at the time of typical development and changes associated with ELS in the behavior and maturation of the brain. Epilepsy is a chronic neurological condition characterized by a persistent predisposition to epileptic seizures, and by neurobiological, cognitive, psychological and social consequences. Depression is a very common psychiatric comorbidity in patients with epilepsy. Increasingly data suggest that epilepsy, depression and other possible psychiatric disorders such as anxiety share pathogenic mechanisms. In this sense, abnormalities in the WAR strain (Wistar Audiogenic Rat) have been found that make it an interesting model for the study of stress, Epilepsy and neuropsychiatric comorbidities involved. Based on this, the present study aimed to evaluate in the WAR strain: 1. Maternal care under control and under stress conditions 2. Basal depressive type behaviors and after audiogenic kindling (chronic seizures-KAu).xvi Godoy, L.D. There was no difference for active breastfeeding posture, licking/grooming or time of mother in the nest and in the number of attacks and aggressive behaviors in maternal aggression test. WAR rats showed a higher latency to recover pup in pup retrieval test, whereas 100% of the Wistar rats grouped the entire litter, and in WAR it was only 40%. 2. In order to evaluate the depressive type behaviors, the rats were submitted to 20 acoustic stimuli twice a day (AuK) (Wistar-AuK, WAR-AuK), while the respective control groups remained without stimulus (Wistar, WAR). Subsequently, the groups were submitted to sucrose consumption test (SCT) and forced swim test (FST). There was no difference between the WAR and Wistar groups in the SCT, however the WAR-KAu group presented an increase in sucrose consumption in relation to the control groups 3 or 13 dasy after the AuK. In FST, both WAR and WARKAu groups showed a significant decrease in the test session when compared to Wistar. Taken together, these findings indicate that ELS may induce susceptibility to psychiatric comorbidities associated with stress, and that the current experimental models allow investigating how the effects of glucocorticoids are related to neurodevelopment, specifically in the maturation of fear behavior and limbic structures. In addition, preliminary data indicate that, although the WAR strain does not show maternal behavioral differences at baseline, it may present changes under stressful events. Also, based on the findings, the susceptibility of seizures may be related to changes in behavioral strategies in stressful situations in adult life, which may also constitute vulnerability to psychiatric comorbidities.