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Glucocorticoids (CORT) are well-known as important regulators of behaviour and cognition at basal levels and under stress. However, the precise mechanisms governing CORT action and functional outcomes of this action in the brain remain unclear, particularly in model systems other than rodents. In the present study, we investigated the dynamics of CORT regulation in the zebra finch, an important model system for vocal learning, neuroplasticity and cognition. We tested the hypothesis that CORT is locally regulated in the zebra finch brain by quantifying regional and stress-related variation in total CORT across brain regions. In addition, we used an ex vivo slice culture system to test whether CORT regulates target gene expression uniquely in discrete regions of the brain. We documented a robust increase in brain CORT across regions after 30 minutes of restraint stress but, interestingly, baseline and stress-induced CORT levels varied between regions. In addition, CORT treatment of brain slice cultures differentially affected expression of three CORT target genes: it up-regulated expression of FKBP5 in most regions and SGK1 in the hypothalamus only, whereas GILZ was unaffected by CORT treatment across all brain regions investigated. The specific mechanisms producing regional variation in CORT and CORT-dependent downstream gene expression remain unknown, although these data provide additional support for the hypothesis that the songbird brain employs regulatory mechanisms that result in precise control over the influence of CORT on glucocorticoid-sensitive neural circuits., (© 2020 British Society for Neuroendocrinology.)

Glucocorticoid (GC) levels vary with environmental conditions, but the functional interpretation of GC variation remains contentious. A primary function is thought to be metabolic, mobilizing body reserves to match energetic demands. This view is supported by temperature-dependent GC levels, although reports of this effect show unexplained heterogeneity. We hypothesized that the temperature effect on GC concentrations will depend on food availability through its effect on the energy spent to gather the food needed for thermoregulation. We tested this hypothesis in zebra finches living in outdoor aviaries with manipulated foraging conditions (i.e. easy versus hard), by relating within-individual differences in baseline GCs between consecutive years to differences in ambient temperature. In agreement with our hypothesis, we found the GC-temperature association to be significantly steeper in the hard foraging environment. This supports the metabolic explanation of GC variation, underlining the importance of accounting for variation in energy expenditure when interpreting GC variation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Glucocorticoid hormones (GCs) are often assumed to be indicators of stress. At the same time, one of their fundamental roles is to facilitate metabolic processes to accommodate changes in energetic demands. Although the metabolic function of GCs is thought to be ubiquitous across vertebrates, we are not aware of experiments which tested this directly, i.e. in which metabolic rate was manipulated and measured together with GCs. We therefore tested for a relationship between plasma corticosterone (CORT; ln transformed) and metabolic rate (MR; measured using indirect calorimetry) in a between- and within-individual design in captive zebra finches ( Taeniopygia guttata ) of both sexes. In each individual, CORT and MR were measured at two different temperature levels: 'warm' (22°C) and 'cold' (12°C). CORT and MR were both increased in colder compared with warmer conditions within individuals, but also across individuals. At the between-individual level, we found a positive relationship between CORT and MR, with an accelerating slope towards higher MR and CORT values. In contrast, the within-individual changes in CORT and MR in response to colder conditions were linearly correlated between individuals. The CORT-MR relationship did not differ between the sexes. Our results illustrate the importance of including variation at different levels to better understand physiological modulation. Furthermore, our findings support the interpretation of CORT variation as an indicator of metabolic needs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Early life stress has enduring effects on behavior and physiology. However, the effects on hormones and stress physiology remain poorly understood. In the present study, parents of zebra finches of both sexes were exposed to an increased foraging paradigm from 3 to 33days post hatching. Plasma and brains were collected from chicks at 3 developmental time points: post hatching days 25, 60 and adulthood. Plasma was assayed for testosterone (T), estradiol (E2), and corticosterone (CORT). The paraventricular nucleus of the hypothalamus was assessed for corticotrophin releasing factor (CRH) and glucocorticoid receptor (GR) expression. As expected, body mass was lower in nutritionally stressed animals compared to controls at multiple ages. Nutritionally stressed animals overall had higher levels of CORT than did control and this was particularly apparent in females at post hatching day 25. Nutritionally stressed animals also had a higher number of cells expressing CRH and GR in the paraventricular nucleus of the hypothalamus than did controls. There was an interaction, such that both measures were higher in control animals at PHD 25, but higher in NS animals by adulthood. Females, regardless of treatment, had higher circulating CORT and a higher number of cells expressing CRH than did males. Nutritionally stressed animals also had higher levels of T than did control animals, and this difference was greatest for males at post hatching day 60. There were no effects of nutritional stress on E2. These findings suggest that nutritional stress during development has long-lasting effects on testosterone and stress physiology., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The adrenal hormone corticosterone (CORT) acts on brain to mediate physiology and behavior. In songbirds, behavioral effects of CORT vary across species, environmental conditions, and life history stage, with several mechanisms proposed to account for these divergent results. Although blood CORT levels are well characterized, few studies measure CORT within the brain itself. Here we used in vivo microdialysis to measure CORT in two regions of the zebra finch brain, the hippocampus (HP) and caudal nidopallium (cNp). Our results show that we can successfully measure physiological levels of CORT in brain within 15- to 30-minute intervals of dialysate collection. Moreover, we found that levels in the cNp were generally lower than levels in the HP. Surprisingly, whereas plasma CORT levels increased in response to a standard stressor, no stress-induced surge was detected in the HP or cNp. In addition, although a diel CORT rhythm was observed in plasma, the rhythm in brain was attenuated and only observed when levels were integrated over a 4-hour time period. Regional differences in brain CORT levels were reflected in local mRNA expression levels of the CORT-inactivating enzyme 11β-hydroxysteroid dehydrogenase type 2 with levels elevated in the cNp relative to the HP. Region-specific CORT metabolism may therefore play a role in buffering the brain from CORT fluctuations.

Parasites can negatively affect the evolutionary fitness of their hosts by eliciting physiological stress responses. Parasite-induced stress can be monitored by measuring changes in the adrenal steroid hormone corticosterone. We examined the effect of an invasive parasite on the corticosterone concentrations of a common species of Darwin's finch, the medium ground finch (Geospiza fortis). Philornis downsi (Diptera: Muscidae) is a parasitic nest fly recently introduced to the Galapagos Islands, where it feeds on the blood of nestlings and breeding adult female finches. Previous work shows that P. downsi significantly reduces the reproductive success of several species of finches. We predicted that the effect of P. downsi on host reproductive success is mediated by stress responses in breeding female finches. High stress levels could reduce the ability of females to invest in offspring, thus decreasing their reproductive success. To test this hypothesis, we experimentally manipulated the abundance of P. downsi in nests, then measured baseline and acute stress-induced corticosterone levels, body condition, and hematocrit (red blood cell content). Acute stress-induced corticosterone levels increased over baseline levels, but this response did not differ significantly with parasite treatment. There was also no significant difference in the body condition or hematocrit of females from parasitized versus non-parasitized nests. Our results suggest that the lower reproductive success of females from parasitized nests is not mediated by a physiological stress response., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Assessment of costs accompanying activation of immune system and related neuroendocrine pathways is essential for understanding the selective forces operating on these systems. Here we attempted to detect such costs in terms of disruption to redox balance and interference between different immune system components in captive wild-caught greenfinches (Carduelis chloris). Study birds were subjected to an endotoxin-induced inflammatory challenge and temporary exposure to a psychological stressor (an image of a predator) in a 2*2 factorial experiment. Injection of bacterial endotoxin resulted in up-regulation of two markers of antioxidant protection - erythrocyte glutathione, and plasma oxygen radical absorbance (OXY). These findings suggest that inflammatory responses alter redox homeostasis. However, no effect on markers of oxidative damage to proteins or DNA in erythrocytes could be detected. We found no evidence that the endotoxin injection interfered with antibody production against Brucella abortus antigen or the intensity of chronic coccidiosis. The hypothesis of within-immune system trade-offs as a cost of immunity was thus not supported in our model system. We showed for the first time that administration of endotoxin can reduce the level of corticosterone deposited into feathers. This finding suggests a down-regulation of the corticosterone secretion cascade due to an endotoxin-induced immune response, a phenomenon that has not been reported previously. Exposure to the predator image did not affect any of the measured physiological parameters.

Glucocorticoids (GCs) are produced in the adrenal glands and also in extra-adrenal sites, including immune organs and brain. Here, we examined regulation of systemic GC levels in plasma and local GC levels in immune organs and brain during development. We conducted two studies and examined a total of 462 samples from 70 subjects. In study 1, we determined corticosterone and cortisol levels in the plasma, immune organs, and brain of wild European starlings on posthatch day 0 (P0) and P10 (at baseline and after 45 min of restraint). Baseline corticosterone and cortisol levels were low in the immune organs and brain at P0 and P10, providing little evidence for local GC synthesis in starlings. At P0, restraint had no significant effects on corticosterone or cortisol levels in the plasma or tissues; however, there was a trend for restraint to increase both corticosterone and cortisol in the immune organs. At P10, restraint increased corticosterone levels in the plasma and all tissues, but restraint increased cortisol levels in the plasma, thymus, and diencephalon only. In study 2, we directly compared GC levels in European starlings and zebra finches at P4. In zebra finches but not starlings, cortisol levels were higher in the immune organs than in plasma. This difference in immune GC levels might be due to evolutionary lineage, life history strategy, or experiential factors, such as parasite exposure. This is the first study to measure immune GC levels in wild animals and one of the first studies to measure local GC levels after restraint stress.

Post-natal stress has been shown to have important short and long term effects on many adult traits in birds. During stress, metabolic alterations often result in the mobilization of energy away from energy-sensitive functions such as growth, which could have significant implications for developing animals. However, little is known about the implications of stress hormones for energy consumption in growing individuals. We experimentally increased corticosterone (CORT) levels in nestling zebra finches via oral administration, between the ages of 7 and 18 days. The standard metabolic rate (SMR) of birds was measured twice overnight when birds were between 11-13 and 55-65 days of age. Developmental CORT administration significantly elevated overnight variability in SMR (sd) in nestling birds (during the treatment period), but not at 55-65 days (5-6 weeks after the treatment period). The effect on variability was seen more prominently in birds from larger brood sizes and in females. We found no effects of our treatments on mean SMR overnight. However, brood size and sex had interactive effects, with males from larger brood sizes having higher SMR at 55-65 days of age. These results suggest that stress hormones can have significant effects on energy metabolism and possibly nocturnal arousal and sleep fragmentation. However, there were no detectable long term effects of our treatments on SMR, suggesting that these effects are only short-lived, in order to maintain homeostasis in the short term.

Glucocorticoids (GCs) have profound effects on the immune and nervous systems during development. However, circulating GC levels are low neonatally and show little response to stressors. This paradox could be resolved if immune and neural tissues locally synthesize GCs. Here, we measured baseline corticosterone and cortisol levels in plasma, immune organs, and brain regions of developing zebra finches. Steroids were extracted using solid phase-extraction and quantified using specific immunoassays. As expected, corticosterone was the predominant GC in plasma and increased with age. In contrast, cortisol was the predominant GC in immune tissues (bursa of Fabricius, thymus, spleen) and decreased with age. Cortisol levels in immune tissues were higher than cortisol levels in plasma. In the brain, corticosterone and cortisol levels were similarly low, providing little evidence for local synthesis of GCs in the brain. This is the first study to measure 1) cortisol in the plasma of songbirds, 2) corticosterone or cortisol in the brain of songbirds, and 3) corticosterone or cortisol in the immune system of any species. Despite the prevailing dogma that corticosterone is the primary GC in birds, these results indicate that cortisol is the predominant GC in the immune system of developing zebra finches. These results raise the hypothesis that cortisol is synthesized de novo from cholesterol in the immune system as an "immunosteroid," analogous to neurosteroids synthesized in the brain. Local production of GCs in immune tissues may allow GCs to regulate lymphocyte selection while avoiding the costs of high systemic GCs during development.