Amy E. M. Newman

Aggressive interactions rapidly increase androgen synthesis in the brain during the non-breeding season.

In male song sparrows (Melospiza melodia), territorial challenges during the breeding season can rapidly increase circulating levels of testosterone (T). During the non-breeding season, male song sparrows are highly aggressive, but the gonads are regressed and plasma T levels are non-detectable and unaffected by territorial challenges. The pro-hormone dehydroepiandrosterone (DHEA) is elevated in song sparrow plasma and brain during the non-breeding season and may be locally converted to sex steroids in the brain to regulate aggression. The enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD) converts DHEA to androstenedione (AE) using the cofactor NAD(+), and this is a critical rate-limiting step. We predicted that brain 3beta-HSD activity varies seasonally and is rapidly modulated by aggressive challenges. In the first study, brain 3beta-HSD activity was highest in the non-breeding season in specific regions. In the second study, a simulated territorial challenge rapidly increased aggressive behavior in non-breeding song sparrows. Brain 3beta-HSD activity, when measured without exogenous NAD(+), increased by approximately 250 to 500% in telencephalic regions of challenged subjects. When brain 3beta-HSD activity was measured with exogenous NAD(+), these effects of territorial challenges were not observed. These data suggest that territorial challenges rapidly increase endogenous NAD(+) levels or increase 3beta-HSD activity specifically within a NAD-rich subcellular compartment. Together, these two studies suggest a shift from systemic to local sex steroid signaling in the non-breeding season. Local steroid signaling produces high spatial and temporal specificity of steroid signals and avoids the costs of high systemic T levels during the non-breeding season.

Dehydroepiandrosterone and Corticosterone Are Regulated by Season and Acute Stress in a Wild Songbird: Jugular Versus Brachial Plasma

Stress has well-known effects on adrenal glucocorticoid secretion, and chronic elevation of glucocorticoids can have detrimental effects on the brain. Dehydroepiandrosterone (DHEA), an androgen precursor synthesized in the adrenal glands or the brain itself, has anti-glucocorticoid properties, but little is known about the role of DHEA in the stress response, particularly in the brain. Here, we measured the effects of acute restraint on circulating corticosterone (CORT) and DHEA levels in wild song sparrows. Blood was collected from either the brachial or jugular vein. In songbirds, jugular plasma is enriched with neurally synthesized steroids, and therefore, jugular plasma is an indirect index of the neural steroidal milieu. Subjects were sampled during four times of year: breeding, molt, early nonbreeding, and mid-nonbreeding. Baseline CORT and DHEA levels showed similar seasonal changes; both steroids were elevated during the breeding season. Baseline CORT and DHEA levels were similar in jugular and brachial plasma. Acute stress had robust effects on CORT and DHEA that were season specific and vein specific. For CORT, during the molt, stress increased jugular CORT more than brachial CORT. For DHEA, during the breeding season, stress decreased jugular DHEA but not brachial DHEA. During the molt, stress increased jugular DHEA but not brachial DHEA. Acute stress did not affect brachial DHEA. These data suggest that acute stress specifically affects the balance between DHEA synthesis and metabolism in the brain. Furthermore, these results suggest that CORT and DHEA are locally synthesized in the brain during molt, when systemic levels of CORT and DHEA are low.

Stress, song and survival in sparrows

The stress response—increases in circulating glucocorticoids following a stressor—is typically considered adaptive, but few studies address the fitness consequences of individual variation in stress response. Generally, due to negative consequences of prolonged elevation of glucocorticoids, animals should have a transient stress response just sufficient to cope with the stressor. In rodents, stress responsiveness is affected by early developmental experience, and hyper-responsiveness to stress is linked to morbidity and mortality. We assessed individual variation in stress responses in free-living song sparrows, Melospiza melodia, in relation to fitness-related measures including song and overwinter survival. Birds with greater increases in corticosterone 30 min following restraint stress were less likely to return to breed the following year. Stress responsiveness was also correlated with song complexity: males with fewer syllables in their song repertoires had greater stress reactivity. Our findings support the hypothesis that developmental stressors both impair song development and affect the adult stress response. Thus, individual variation in the stress response may relate to variation in fitness.

Corticosterone and dehydroepiandrosterone have opposing effects on adult neuroplasticity in the avian song control system

Chronic elevations in glucocorticoids can decrease the production and survival of new cells in the adult brain. In rat hippocampus, supraphysiological doses of dehydroepiandrosterone (DHEA; a sex steroid precursor synthesized in the gonads, adrenals, and brain) have antiglucocorticoid properties. With male song sparrows (Melospiza melodia), we examined the effects of physiological doses of corticosterone, the primary circulating glucocorticoid in birds, and DHEA on adult neuroplasticity. We treated four groups of nonbreeding sparrows for 28 days with empty (control), corticosterone, DHEA, or corticosterone + DHEA implants. Subjects were injected with BrdU on days 3 and 4. In HVC, a critical song control nucleus, corticosterone and DHEA had independent, additive effects. Corticosterone decreased, whereas DHEA increased, HVC volume, NeuN+ cell number, and BrdU+ cell number. Coadministration of DHEA completely reversed the neurodegenerative effects of chronic corticosterone treatment. In an efferent target of HVC, the robust nucleus of the arcopallium (RA), DHEA increased RA volume, but this effect was blocked by coadministration of corticosterone. There were similar antagonistic interactions between corticosterone and DHEA on BrdU+ cell number in the hippocampus and ventricular zone. This is the first report on the effects of corticosterone treatment on the adult song control circuit, and HVC was the most corticosterone‐sensitive song nucleus examined. In HVC, DHEA is neuroprotective and counteracts several pronounced effects of corticosterone. Within brain regions that are particularly vulnerable to corticosterone, such as the songbird HVC and rat hippocampus, DHEA appears to be a potent native antiglucocorticoid. J. Comp. Neurol. 518:3662–3678, 2010.

Rapid Effects of Aggressive Interactions on Aromatase Activity and Oestradiol in Discrete Brain Regions of Wild Male White-Crowned Sparrows

Testosterone is critical for the activation of aggressive behaviours. In many vertebrate species, circulating testosterone levels rapidly increase after aggressive encounters during the early or mid‐breeding season. During the late breeding season, circulating testosterone concentrations did not change in wild male white‐crowned sparrows after an aggressive encounter and, in these animals, changes in local neural metabolism of testosterone might be more important than changes in systemic testosterone levels. Local neural aromatisation of testosterone into 17β‐oestradiol (E2) often mediates the actions of testosterone, and we hypothesised that, in the late breeding season, brain aromatase is rapidly modulated after aggressive interactions, leading to changes in local concentrations of E2. In the present study, wild male white‐crowned sparrows in the late breeding season were exposed to simulated territorial intrusion (STI) (song playback and live decoy) or control (CON) for 30 min. STI significantly increased aggressive behaviours. Using the Palkovits punch technique, 13 brain regions were collected. There was high aromatase activity in several nuclei, although enzymatic activity in the CON and STI groups did not differ in any region. E2 concentrations were much higher in the brain than the plasma. STI did not affect circulating levels of E2 but rapidly reduced E2 concentrations in the hippocampus, ventromedial nucleus of the hypothalamus and bed nucleus of the stria terminalis. Unexpectedly, there were no correlations between aromatase activity and E2 concentrations in the brain, nor were aromatase activity or brain E2 correlated with aggressive behaviour or plasma hormone levels. This is one of the first studies to measure E2 in microdissected brain regions, and the first study to do so in free‐ranging animals. These data demonstrate that social interactions have rapid effects on local E2 concentrations in specific brain regions.

Plasma DHEA levels in wild, territorial red squirrels : Seasonal variation and effect of ACTH

In many species, territorial behavior is limited to the breeding season and is tightly coupled to circulating gonadal steroid levels. In contrast, both male and female red squirrels (Tamiasciurus hudsonicus) are highly aggressive in both the breeding and non-breeding seasons in defense of food stores on their individual territories throughout the boreal and northern forests of North America. Dehydroepiandrosterone (DHEA), an androgen precursor, is secreted from the adrenal cortex in some mammals, and DHEA has been linked to aggression in non-breeding songbirds. Here, we examined plasma DHEA levels in a natural population of red squirrels in the Yukon, Canada. Plasma DHEA levels in both males and females reached high concentrations (up to 16.952 ng/ml in males and 14.602 ng/ml in females), markedly exceeding plasma DHEA concentrations in laboratory rats and mice and similar to plasma DHEA concentrations in some primates. Circulating DHEA levels showed both seasonal and yearly variation. Seasonal variation in male plasma DHEA levels was negatively correlated with testes mass. Yearly variation in male DHEA levels was positively correlated with population density. In both males and females, circulating DHEA rapidly increased after ACTH treatment, implying an adrenal origin. This is the first examination of plasma DHEA concentrations in a wild rodent and the first field experiment on the regulation of plasma DHEA in any wild mammal. These data lay the foundation for future studies on the role of DHEA in non-breeding territoriality in this species and other mammals.

Corticosterone and dehydroepiandrosterone in songbird plasma and brain: effects of season and acute stress.

Prolonged increases in plasma glucocorticoids can exacerbate neurodegeneration. In rats, these neurodegenerative effects can be reduced by dehydroepiandrosterone (DHEA), an androgen precursor with anti‐glucocorticoid actions. In song sparrows, season and acute restraint stress affect circulating levels of corticosterone and DHEA, and the effects of stress differ in plasma collected from the brachial and jugular veins. Jugular plasma is an indirect index of the neural steroidal milieu. Here, we directly measured corticosterone and DHEA in several brain regions and jugular plasma, and examined the effects of season and acute restraint stress (30 min) (n = 571 samples). Corticosterone levels were up to 10× lower in brain than in jugular plasma. In contrast, DHEA levels were up to 5× higher in brain than in jugular plasma and were highest in the hippocampus. Corticosterone and DHEA concentrations were strongly seasonally regulated in plasma but, surprisingly, not seasonally regulated in brain. Acute stress increased corticosterone levels in plasma and brain, except during the molt, when stress unexpectedly decreased corticosterone levels in the hippocampus. Acute stress increased DHEA levels in plasma during the molt but had no effects on DHEA levels in brain. This is the first study to measure (i) corticosterone or DHEA levels in the brain of adult songbirds and (ii) seasonal changes in corticosterone or DHEA levels in the brain of any species. These results highlight several critical differences between systemic and local steroid concentrations and the difficulty of using circulating steroid levels to infer local steroid levels within the brain.

Timing of breeding carries over to influence migratory departure in a songbird: an automated radiotracking study.

1. Determining how events interact across stages of the annual cycle is critical for understanding the factors that affect individual fitness. However, there is currently little information detailing how breeding events influence migratory behaviour. 2. Using an automated digital telemetry array and an isolated island-breeding population of Savannah sparrows Passerculus sandwichensis, we provide the first direct evidence that the timing of breeding events carries over to influence the timing of migration in a songbird and assess for the first time how weather conditions on the breeding grounds also affect departure dates. 3. Date of migratory departure between September and October was strongly influenced by date of breeding completion in adults and fledging date in juveniles from June to July. 4. With respect to weather, adults departed during the first half of high-pressure systems, while juveniles departed throughout the entirety of high-pressure systems (including rainy evenings on the western edge of systems). 5. By combining both ecological and weather data, we could explain almost all variation in departure date for adults (95%), but weather conditions were not a good predictor of departure date for juveniles. 6. Our results provide strong evidence that the timing of breeding events is an important driver of migration timing and that exact departure dates are fine-tuned according to local weather conditions in adults, but not in juveniles.

Multiple measures elucidate glucocorticoid responses to environmental variation in predation threat

Predator-induced changes in the glucocorticoid responses of prey have been proposed to mediate indirect predator effects on prey demography. Ambiguities exist, however, as to whether differences in predation threat in the environment at large affect the mean glucocorticoid response in wild birds and mammals, and whether this is likely to affect reproduction. Most studies to date that have examined glucocorticoid responses to environmental variation in predation threat have evaluated just one of the several potential measures of the glucocorticoid response, and this may be the source of many ambiguities. We evaluated multiple measures of the glucocorticoid response [plasma total CORTicosterone, corticosteroid binding globulin (CBG) and free CORT] in male and female song sparrows (Melospiza melodia) sampled at locations differing in predation threat in the environment at large, where we have previously reported reproductive differences suggestive of indirect predator effects. Total CORT varied markedly with predation threat in males but not females whereas the opposite was true for CBG, and both sexes demonstrated the same moderately significant free CORT response. Considering all three indices, a glucocorticoid response to environmental variation in predation threat was evident in both sexes, whereas there were ambiguities considering each index singly. We conclude that collecting multiple physiological measures and conducting multivariate analyses may provide a preferable means of assessing glucocorticoid responses to environmental variation in predation threat, and so help clarify whether such glucocorticoid changes affect reproduction in wild birds and mammals.

17β-Estradiol levels in male zebra finch brain: Combining Palkovits punch and an ultrasensitive radioimmunoassay

Local aromatization of testosterone into 17beta-estradiol (E(2)) is often required for the physiological and behavioral actions of testosterone. In most vertebrates, aromatase is expressed in a few discrete brain regions. While many studies have measured brain aromatase mRNA or activity, very few studies have measured brain E(2) levels, particularly in discrete brain regions, because of technical challenges. Here, we used the Palkovits punch technique to isolate 13 discrete brain nuclei from adult male zebra finches. Steroids were extracted via solid phase extraction. E(2) was then measured with an ultrasensitive, specific and precise radioimmunoassay. Our protocol leads to high recovery of E(2) (84%) and effectively removes interfering brain lipids. E(2) levels were high in aromatase-rich regions such as caudal medial nidopallium and hippocampus. E(2) levels were intermediate in the medial preoptic area, ventromedial nucleus of the hypothalamus, lateral and medial magnocellular nuclei of anterior nidopallium, nucleus taeniae of the amygdala, and Area X. E(2) levels were largely non-detectable in the cerebellum, HVC, lateral nidopallium and optic lobes. Importantly, E(2) levels were significantly lower in plasma than in the caudal medial nidopallium. This protocol allows one to measure E(2) in discrete brain regions and potentially relate local E(2) concentrations to aromatase activity and behavior.