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Understanding the factors that limit and regulate wildlife populations requires insight into demographic and environmental processes acting throughout the annual cycle. Here, we combine multi-year tracking data of individual birds with a 26-year demographic study of a migratory songbird to evaluate the relative effects of density and weather at the breeding and wintering grounds on population growth rate. Our results reveal clear support for opposing forces of winter temperature and breeding density driving population dynamics. Above-average temperatures at the wintering grounds lead to higher population growth, primarily through their strong positive effects on survival. However, population growth is regulated over the long term by strong negative effects of breeding density on both fecundity and adult male survival. Such knowledge of how year-round factors influence population growth, and the demographic mechanisms through which they act, will vastly improve our ability to predict species responses to environmental change and develop effective conservation strategies for migratory animals.

Patterns of connectivity between breeding and wintering grounds can have important implications for individual fitness and population dynamics. Using light-level geolocators and stable hydrogen isotopes (δ(2)H) in feathers, we evaluated differential migration of Savannah sparrows (Passerculus sandwichensis) breeding on Kent Island in the Bay of Fundy, New Brunswick, Canada in relation to sex, age, and body size. Based on geolocators recovered from 38 individuals between 2012 and 2014, the winter distribution was centered in North Carolina (median latitude 34°, range 26°-41°), with males overwintering, on average, approximately 275 km further north than females. Based on analyses of tail feather samples collected from 106 individuals from the same population between 2008 and 2012, males and adults had more negative δ(2)H values than females and juveniles, respectively, providing additional evidence that males wintered north of females and that adults wintered north of juveniles. Winter latitude and δ(2)H values within each sex were not found to be related to body size. From geolocator data, males returned to the breeding grounds, on average, 14 days earlier than females. For males, there was some evidence that arrival date on the breeding grounds was negatively correlated with winter latitude and that individuals which arrived earlier tended to breed earlier. Thus, benefits for males of early arrival on the breeding grounds may have contributed to their wintering farther north than females. Social dominance may also have contributed to age and sex differences in winter latitude, whereby dominant males and adults forced subordinate females and juveniles further south.

Bacterial diversity within animals is emerging as an essential component of health, but it is unknown how stress may influence the microbiome. We quantify a proximate link between the oral microbiome and hypothalamic-pituitary-adrenal (HPA) axis activity using faecal glucocorticoid metabolites (FGM) in wild red squirrels (Tamiasciurus hudsonicus). Not only was bacterial diversity lower at higher levels of FGM, but also between capture periods a change in bacterial relative abundance was related to an increase in FGM. These linkages between the HPA axis and microbiome communities represent a powerful capacity for stress to have multi-dimensional effects on health., (© 2016 The Author(s).)

Conspecific density is widely recognized as an important ecological factor across the animal kingdom; however, the physiological impacts are less thoroughly described. In fact, population density is rarely mentioned as a factor in physiological studies on captive animals and, when it is infrequently addressed, the animals used are reared and housed at densities far above those in nature, making the translation of results from the laboratory to natural systems difficult. We survey the literature to highlight this important ecophysiological gap and bring attention to the possibility that conspecific density prior to experimentation may be a critical factor influencing results. Across three taxa: mammals, birds, and fish, we present evidence from ecology that density influences glucocorticoid levels, immune function, and body condition with the intention of stimulating discussion and increasing consideration of population density in physiology studies. We conclude with several directives to improve the applicability of insights gained in the laboratory to organisms in the natural environment., (Copyright © 2015 the American Physiological Society.)

Adult neuroplasticity is strongly influenced by steroids. In particular, corticosterone (CORT) and dehydroepiandrosterone (DHEA) can have opposing effects, where CORT reduces while DHEA increases neurogenesis and neuron recruitment. It has been previously shown that in adult male song sparrows, DHEA treatment increases neuron recruitment throughout the telencephalon, including the lateral ventricular zone, while the effect of CORT treatment is restricted to HVC, one of the song control regions. These data suggest that the two steroids may differentially affect proliferation, migration, differentiation, and/or survival of new neurons. To determine if CORT or DHEA alters the migration and differentiation of young neurons, we examined an endogenous marker of migrating immature neurons, doublecortin (DCX), in HVC and hippocampus of adult male song sparrows that were treated with CORT and/or DHEA for 28 days. In HVC, DHEA increased the number of DCX-labeled round cells, while CORT had no main effect on the number of DCX-labeled cells. Furthermore, DHEA increased the area covered by DCX immunoreactivity in HVC, regardless of CORT treatment. In the hippocampus, neither DHEA nor CORT affected DCX immunoreactivity. These results suggest that DHEA enhances migration and differentiation of young neurons into HVC while CORT does not affect the process, whether in the presence of DHEA or not., (Copyright © 2013 Wiley Periodicals, Inc., a Wiley company.)

In fluctuating environments, mothers may enhance the fitness of their offspring by adjusting offspring phenotypes to match the environment they will experience at independence. In free-ranging red squirrels, natural selection on offspring postnatal growth rates varies according to population density, with selection favoring faster-growing offspring under high-density conditions. We show that exposing mothers to high-density cues, accomplished via playbacks of territorial vocalizations, led to increased offspring growth rates in the absence of additional food resources. Experimental elevation of actual and perceived density induced higher maternal glucocorticoid levels, and females with naturally or experimentally increased glucocorticoids produced offspring that grew faster than controls. Therefore, social cues reflecting population density were sufficient to elicit increased offspring growth through an adaptive hormone-mediated maternal effect.

The effects of chronic stressors on glucocorticoid levels are well described in laboratory rodents, but far less is known about the effects of chronic stressors on wild animals or on dehydroepiandrosterone (DHEA) levels. DHEA can be produced by the adrenal cortex and has prominent antiglucocorticoid properties. Here, we examined wild songbirds to elucidate the relationship between chronic predator pressure and plasma DHEA and corticosterone levels. We measured circulating steroid levels at baseline and after acute restraint in the breeding and nonbreeding seasons. During the breeding season, males in low predator pressure (LPP) environments had higher baseline DHEA levels than males in high predator pressure (HPP) environments. Also, acute restraint decreased DHEA levels in LPP males only but increased corticosterone levels in HPP and LPP males similarly. During the nonbreeding season, DHEA and corticosterone levels were lower than during the breeding season, and acute restraint decreased DHEA levels in both HPP and LPP males. Unlike males, breeding females showed no effect of predator pressure on baseline DHEA or corticosterone levels. These data suggest that naturalistic chronic and acute stressors affect circulating DHEA and corticosterone levels in wild animals and highlight the importance of using multiple endpoints when studying the physiological effects of chronic stress.

Stress-induced increases in glucocorticoids (GCs) modulate behavior and are key in directing energy reserves. The capture-restraint protocol was developed to experimentally stimulate and quantify the magnitude of the acute stress response by comparing baseline GC levels with those collected after restraining a subject for a period of time, typically 30 min. This protocol has been used extensively in the field and lab, yet few studies have investigated whether it parallels hypothalamic-pituitary-adrenal (HPA) activation under natural acute stressors. We examined the hypothesis that acute stress from the capture-restraint protocol accurately mimics the adrenocortical response induced by a natural acute stressor. Using wild-caught rock pigeons Columba livia in a repeated-measures design, we compared plasma corticosterone (CORT) concentrations at baseline, after exposure to acute capture-restraint (30 min in a cloth bag), after tethering in a harness (30 min), and after a real but nonlethal attack by a predatory raptor. As found in previous studies, the capture-restraint treatment significantly increased CORT levels of pigeons compared with baseline. However, we also found that when pigeons were exposed to an attack by a raptor, their CORT levels were more than twice as high compared with the capture-restraint treatment. Our results provide evidence that an authentic acute stressor can activate the HPA axis to a greater extent than the capture-restraint protocol and also suggest that real predation attempts can have a significant effect on acute stress levels of wild birds.

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., (© 2012 The Authors. Journal of Animal Ecology © 2012 British Ecological Society.)

The endocannabinoid (eCB) system is an important regulator of the stress response and mediates several stress-related behaviors, including anxiety. Despite anatomical evidence that eCBs interact with the principle stress peptide, corticotropin-releasing factor (CRF), few data exist that address functional interactions between these systems. Accordingly, we examined the effects of the CB1 receptor antagonist, AM251, on behavioral anxiety induced by (1) exogenous CRF, and (2) withdrawal from chronic cocaine exposure (mediated by CRF). After behavioral testing, we collected blood and assessed plasma corticosterone levels. In Experiment 1, male Long-Evans rats were pretreated with AM251 (0, 10, 100, or 200 μg, i.c.v.), followed by CRF (0 or 0.5 μg, i.c.v.), before testing for anxiety-like behavior in the elevated plus maze (EPM). In Experiment 2, rats were exposed to cocaine (20 mg/kg, i.p.) or saline for 14 consecutive days. Forty-eight hours following cocaine exposure, rats were pretreated with AM251 (0, 10, or 100 μg, i.c.v.) and tested in the EPM. AM251 produced an anxiogenic response at the highest dose, but reversed the behavioral anxiety induced by CRF and withdrawal from chronic cocaine in a dose-dependent manner. AM251 also increased plasma corticosterone levels, but did so irrespective of CRF treatment or cocaine preexposure. Our findings suggest that the anxiogenic effects of CRF and cocaine withdrawal are mediated, at least in part, by CB1 receptor transmission, and provide evidence in support of eCB-CRF interactions that are independent of the hypothalamic-pituitary-adrenal axis., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

During the nonbreeding season, when gonadal androgen synthesis is basal, recent evidence suggests that neurosteroids regulate the aggression of male song sparrows. In particular, dehydroepiandrosterone (DHEA) is rapidly converted in the brain to androgens in response to aggressive interactions. In other species, aggressive encounters increase systemic glucocorticoid levels. However, the relationship between aggression and local steroid levels is not well understood. Here, during the breeding and nonbreeding seasons, we tested the effects of a simulated territorial intrusion (STI) on DHEA and corticosterone levels in the brachial and jugular plasma. Jugular plasma is enriched with neurosteroids and provides an indirect index of brain steroid levels. Further, during the nonbreeding season, we directly measured steroid levels in the brain and peripheral tissues. Both breeding and nonbreeding males displayed robust aggressive responses to STI. During the breeding season, STI increased brachial and jugular corticosterone levels and jugular DHEA levels. During the nonbreeding season, STI did not affect plasma corticosterone levels, but increased jugular DHEA levels. During the nonbreeding season, STI did not affect brain levels of corticosterone or DHEA. However, STI did increase corticosterone and DHEA concentrations in the liver and corticosterone concentrations in the pectoral muscle. These data suggest that 1) aggressive social interactions affect neurosteroid levels in both seasons and 2) local steroid synthesis in peripheral tissues may mobilize energy reserves to fuel aggression in the nonbreeding season. Local steroid synthesis in brain, liver or muscle may serve to avoid the costs of systemic increases in corticosterone and testosterone., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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 (E(2)) 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 E(2). 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. E(2) concentrations were much higher in the brain than the plasma. STI did not affect circulating levels of E(2) but rapidly reduced E(2) 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 E(2) concentrations in the brain, nor were aromatase activity or brain E(2) correlated with aggressive behaviour or plasma hormone levels. This is one of the first studies to measure E(2) 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 E(2) concentrations in specific brain regions., (© 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd.)

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.

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.

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

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.

The gonadal hormone testosterone (T) regulates aggression across a wide range of vertebrate species. Recent evidence suggests that the adrenal prohormone dehydroepiandrosterone (DHEA) may also play an important role in regulating aggression. DHEA can be converted into active sex steroids, such as T and estradiol (E(2)), within the brain. Previous studies show that circulating DHEA levels display diurnal rhythms and that melatonin increases adrenal DHEA secretion in vitro. Here we examined serum DHEA and T levels in long-day housed Siberian hamsters (Phodopus sungorus), a nocturnal species in which melatonin treatment increases aggression. In Experiment 1, serum DHEA and T levels were measured in adult male hamsters during the day (1200 h, noon) and night (2400 h, midnight). In Experiment 2, aggression was elicited using 5-min resident-intruder trials during the day (1800 h) and night (2000 h) (lights-off at 2000 h). Serum DHEA and T levels were measured 24 h before and immediately after aggressive encounters. In Experiment 1, there was no significant difference in serum DHEA or T levels between noon and midnight, although DHEA levels showed a trend to be lower at midnight. In Experiment 2, territorial aggression was greater during the night than the day. Moreover, at night, aggressive interactions rapidly decreased serum DHEA levels but increased serum T levels. In contrast, aggressive interactions during the day did not affect serum DHEA or T levels. These data suggest that nocturnal aggressive encounters rapidly increase conversion of DHEA to T and that melatonin may play a permissive role in this process.

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 10x lower in brain than in jugular plasma. In contrast, DHEA levels were up to 5x 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.

In 1849, Berthold demonstrated that testicular secretions are necessary for aggressive behavior in roosters. Since then, research on the neuroendocrinology of aggression has been dominated by the paradigm that the brain receives gonadal hormones, primarily testosterone, which modulate relevant neural circuits. While this paradigm has been extremely useful, recent studies reveal important alternatives. For example, most vertebrate species are seasonal breeders, and many species show aggression outside of the breeding season, when gonads are regressed and circulating testosterone levels are typically low. Studies in birds and mammals suggest that an adrenal androgen precursor-dehydroepiandrosterone (DHEA)-may be important for the expression of aggression when gonadal testosterone synthesis is low. Circulating DHEA can be metabolized into active sex steroids within the brain. Another possibility is that the brain can autonomously synthesize sex steroids de novo from cholesterol, thereby uncoupling brain steroid levels from circulating steroid levels. These alternative neuroendocrine mechanisms to provide sex steroids to specific neural circuits may have evolved to avoid the "costs" of high circulating testosterone during particular seasons. Physiological indicators of season (e.g., melatonin) may allow animals to switch from one neuroendocrine mechanism to another across the year. Such mechanisms may be important for the control of aggression in many vertebrate species, including humans.

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.

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.

It is a common practice to extract steroids from plasma, serum, or tissue samples prior to steroid measurement by radioimmunoassay (RIA) or enzyme immunoassay (EIA). Steroid extraction is critical because it can remove substances that interfere with the RIA or EIA. Steroid extraction is commonly achieved using organic solvents, such as diethyl ether or dichloromethane. However, organic solvent extractions can suffer from low recovery, imprecise recovery, or incomplete removal of assay interference. Here, we describe validations of a simple protocol to extract steroids (e.g., dehydroepiandrosterone, corticosterone, and estradiol) from avian plasma, serum, and brain tissue using solid phase extraction (SPE) with commercially available C18 columns. We compare various methods for (1) eluting steroids from columns, (2) drying eluates, and (3) resuspending dried eluates prior to RIA. The SPE method yields high and consistent recoveries. The SPE method also effectively separates steroids from interfering substances, even when extracting steroids from lipid-rich plasma and brain tissue. These data indicate that SPE is superior to organic solvent extraction on several measures. SPE should be broadly useful for extracting steroids from plasma or tissue samples.

Following transfection of murine fibroblasts, the lymphotropic strain of minute virus of mice (MVMi) does not efficiently produce progeny single-strand DNA (ssDNA). However, changing a single nucleotide in the MVMi 3' splice site to that found in the fibrotropic strain MVMp enabled full DNA replication and production of ssDNA. This change enhanced excision of the large intron and the production of NS2, likely by improving interaction, in fibroblasts with the branch point-binding U2 snRNA. One function of NS2 involves interaction with the nuclear export protein Crm1. The defect in production of MVMi ssDNA in fibroblasts can also be overcome by introducing a mutation in MVMi NS2 that enhances its interaction with Crm1. Although MVMi contains a 3' splice site that performs poorly in fibroblasts, MVMi generated at least as much R2 and NS2 in murine lymphocytes as did MVMp in fibroblasts. Therefore, it appears that MVMp has acquired a mutation that improves the excision of the large intron, as it adapted to fibroblasts to accommodate the need for NS2 for replication in these cells, and that the ratio of NS1 to NS2 may play a larger role in the host range of MVM than previously appreciated.

Synaptic transmission is a critical mechanism for transferring information from the nervous system to the body. Environmental stress, such as extreme temperature, can disrupt synaptic transmission and result in death. Previous work on larval Drosophila has shown that prior heat-shock exposure protects synaptic transmission against failure during subsequent thermal stress. This induced thermoprotection has been ascribed to an up-regulation of the inducible heat-shock protein, Hsp70. However, the mechanisms mediating natural thermoprotection in the wild are unknown. We compared synaptic thermosensitivity between D. melanogaster and a desert species, D. arizonae. Synaptic thermosensitivity and the functional limits of the related locomotor behavior differed significantly between closely related, albeit ecologically distinct species. Locomotory behavior of wandering third instar D. arizonae larvae was less thermosensitive and the upper temperature limit of locomotory function exceeded that of D. melanogaster by 6 degrees C. Behavioral results corresponded with significantly lower synaptic thermosensitivity at the neuromuscular junction in D. arizonae. Prior heat-shock protected only D. melanogaster by increasing relative excitatory junctional potential (EJP) duration, the time required for EJP failure at 40 degrees C, and the incidence of EJP recovery following heat-induced failure. Hsp70 induction profiles following heat-shock demonstrate up-regulation of inducible Hsp70 in D. melanogaster but not in D. arizonae. However, expression of Hsp70 under control conditions is greater in D. arizonae. These results suggest that the mechanisms of natural thermoprotection involve an increase in baseline Hsp70 expression.

Ventilation is a crucial motor activity that provides organisms with an adequate circulation of respiratory gases. For animals that exist in harsh environments, an important goal is to protect ventilation under extreme conditions. Heat shock, anoxia, and cold shock are environmental stresses that have previously been shown to trigger protective responses. We used the locust to examine stress-induced thermotolerance by monitoring the ability of the central nervous system to generate ventilatory motor patterns during a subsequent heat exposure. Preparations from pre-stressed animals had an increased incidence of motor pattern recovery following heat-induced failure, however, prior stress did not alter the characteristics of the ventilatory motor pattern. During constant heat exposure at sub-lethal temperatures, we observed a protective effect of heat shock pre-treatment. Serotonin application had similar effects on motor patterns when compared to prior heat shock. These studies are consistent with previous studies that indicate prior exposure to extreme temperatures and hypoxia can protect neural operation against high temperature stress. They further suggest that the protective mechanism is a time-dependent process best revealed during prolonged exposure to extreme temperatures and is mediated by a neuromodulator such as serotonin.