Stress in the wild: chronic predator pressure and acute restraint affect plasma DHEA and corticosterone levels in a songbird.

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.

Antagonism of cannabinoid 1 receptors reverses the anxiety-like behavior induced by central injections of corticotropin-releasing factor and cocaine withdrawal.

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.

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 17ß-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.

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.

Analysis of steroids in songbird plasma and brain by coupling solid phase extraction to radioimmunoassay.

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.