INTRODUCTION

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GLUCOCORTICOID SECRETION is often considered to be an obligatory response to noxious stimuli. Several psychological factors, such as the degree of control over the noxious stimulus (see Ref. 17 for review), can alter glucocorticoid secretion, but in general, stimuli applied in similar psychological contexts within the same species produce similar magnitudes of glucocorticoid secretion. Exogenous corticosteroids, when given in concentrations mimicking stress-induced increases, can blunt endogenous secretion [presumably through negative feedback (9, 10)], but stress-induced corticosteroid release is resistant to feedback effects. This is thought to occur through facilitation of the corticosteroid response so that release continues despite what would normally be a robust feedback signal (10, 13). Circadian timing can often affect stimulus-evoked corticosterone release (9, 15), but with the exceptions of pregnancy (31), early development (28), changes in social rank (27), and some pathological changes during aging (27), the magnitude of corticosteroid release to identical stimuli has proven remarkably consistent throughout an animal's life.

Recently, however, several bird species have been shown to seasonally modulate corticosterone secretion [the most important glucocorticoid in birds (14)] in response to an identical stressor (1, 2, 26, 34, 35, 37-40). In Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), for instance, peak corticosterone levels in response to the stressors of capture and restraint are lower during winter (26) and during molt when the birds are replacing all their feathers (1) than during the breeding season. Thus, in contrast to mammalian systems studied to date, these birds apparently regulate their corticosterone response to stress depending on the environmental and/or physiological context of the stimulus.

All species that have been demonstrated to seasonally modulate corticosterone release breed in either the desert (40) or the Arctic (38). Both habitats are typified by a short optimal breeding season and harsh weather conditions that likely place large demands on an animal's ability to respond to stressful stimuli. One species known to modulate corticosterone release is the arctic-breeding Lapland longspur (2, 39). Both this study and earlier studies focused on free-living Lapland longspurs, but we also tested whether captive birds seasonally modulate corticosterone release as well. Specifically, we tested whether corticosterone levels differ in free-living birds during breeding and while undergoing a prebasic molt, and in captive birds in the spring and while undergoing a prenuptial molt (Fig. 1). In Lapland longspurs, the prebasic molt (during which all feathers are replaced) is not equivalent to the prenuptial molt [during which only the plumage of the head and neck are replaced (8)].

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Yearly life history sequence of the Lapland longspur (approximate timing taken from personal observations and Ref. 8).

It is unclear what physiological changes in the hypothalamic-pituitary-adrenal (HPA) axis allow modulation of corticosterone release. HPA axis architecture is similar in birds and mammals, with pituitary ACTH controlling adrenal corticosterone release (20, 21). In addition, corticotropin-releasing factor (CRF), arginine vasotocin (AVT), and mesotocin (MT) are present in the median eminence of the hypothalamus (3, 20, 21) and are known to control ACTH secretion (6), presumably via the hypothalamic-pituitary portal blood. AVT and MT appear to play similar roles in ACTH secretion in birds as arginine vasopressin or oxytocin [the 2 other major putative ACTH-releasing factors (32)] do in mammals. Altering the pituitary's sensitivity to these releasing factors could facilitate the seasonal changes in corticosterone release by decreasing ACTH release. Alternatively or in conjunction, seasonal changes in the adrenals' sensitivity to ACTH could modulate corticosterone secretion. Data are presented here from experiments designed to test these two possibilities in free-living Lapland longspurs by injecting ACTH, CRF, AVT, or MT intravenously and assessing maximal corticosterone output.

	MATERIALS AND METHODS

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Subjects and field sites. Wild Lapland longspurs were caught on their breeding grounds on the Arctic tundra near Barrow, AK (71.1° N, 156.4° W) and near the Toolik Lake Research Station, located ~250 km south of Prudhoe Bay, AK (68° N, 149° W). Breeding longspurs were caught throughout the breeding season at both Barrow (from 12 May to 21 June 1994 and 1 June to 21 June 1995) and at Toolik Lake (from 19 May to 1 June 1995). Molting birds were caught at Barrow (from 6 to 14 August 1994 and from 30 July to 4 August 1995). Birds were trapped using Japanese mist nets, nest traps, or Potter traps baited with seeds. All experiments were initiated within 3 min of capture so that results are indicative of a free-living state. This avoided complications arising from acclimation to captive conditions. Previous studies of these two populations demonstrated no differences in the stress response between the sexes (2, 39), so sex was ignored in this study.

Stress protocol. Birds were captured and subjected to the stressors of capture and restraint. The stress paradigm was identical during both seasons. Within 3 min of capture (or 3 min of entering the aviary for the captive birds), ~60 µl of blood were removed by a puncture of the alar vein in the wing and prepared for baseline corticosterone measurements. Because corticosterone levels generally do not start to rise until 3 min after stress initiation (38), all samples taken within 3 min were considered to reflect baseline levels and were grouped together for statistical purposes. Blood was collected in heparinized microhematocrit capillary tubes (Fisher Scientific, Fair Lawn, NJ), and cotton stanched the blood flow.

Sample processing and assays. Capillary tubes containing blood were sealed on one end with clay and stored on ice. Samples were centrifuged for 6 min at ~400 g within 12 h, and plasma was removed and stored at 20°C and transported to the University of Washington for analysis. Corticosterone was extracted from plasma with dichloromethane and assayed by radioimmunoassay with an antibody from Endocrine Sciences (for details of the radioimmunoassay, see Ref. 40). Interassay and intra-assay variations were 15 and 8%, respectively. Corticosterone binding protein (CBP) capacity and affinity were determined by generating a steroid-free plasma and measuring radiolabeled corticosterone binding as described previously (36). Briefly, after creating a Scatchard plot using the bound/unbound ratio and correcting for nonspecific binding, we fitted a line to the curve (by least squares) and used the reciprocal of the slope to determine the dissociation constant (in nmol/l) and the intersection with the abscissa to determine capacity (in nmol/l). To collect sufficient plasma for CBP analysis, plasma samples from several animals were combined.

Statistical analysis. Differences between injection paradigms, seasonal baseline and maximal corticosterone levels, seasonal changes in weight and fat (for free-living birds), and CBP levels were compared by ANOVA followed by Fisher's protected least-significant difference (PLSD) post hoc tests. Seasonal changes in weight and fat for captives were assessed using paired t-tests. Comparisons between study sites of baseline and stress-induced corticosterone levels were made using a two-way ANOVA followed by Fisher's PLSD. Diel differences (differences associated with time of capture) in baseline corticosterone were compared using a second-order polynomial regression, and baseline corticosterone levels as a function of capture date were compared using simple regression. Spearman rank correlations were used to assess any relationships between corticosterone levels and fat and weight.

	RESULTS

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Several possible confounding factors associated with capturing wild birds, such as time and date of capture, proved not to be problematic and were ignored. Birds were captured throughout most of the 24 h of daylight (from 0400 to 2400) yet showed no diel differences (differences associated with time of capture) in baseline corticosterone levels during either season (r² = 0.022, P = 0.27 and r² = 0.104, P = 0.09 for breeding and molt, respectively). Neither did corticosterone levels vary over the course of a single season (r² = 0.004, P = 0.63 and r² = 0.015, P = 0.39 for breeding and molt, respectively).

Samples during the breeding season were collected from two different sites (Toolik Lake and Barrow). There was no significant difference in baseline corticosterone values between the two sites [7.60 ± 0.75 ng/ml at Barrow (n = 53) compared with 9.20 ± 0.45 ng/ml at Toolik Lake (n = 64); P = 0.060, F = 3.60]. Surprisingly, there was a large difference in weights [28.47 ± 0.27 g at Barrow (n = 58) compared with 26.86 ± 0.27 g at Toolik Lake (n = 75); P < 0.0001, F = 16.90] that was opposite a change in fat scores (1.18 ± 0.11 at Barrow compared with 1.52 ± 0.10 at Toolik Lake; P < 0.05, F = 5.07). However, there were no correlations between individual fat contents and either baseline or 30-min corticosterone levels at either site (Z < 0.8 and P > 0.16 for all tests).

In free-living birds, there were no differences between breeding and molt in weight (Fig. 2A), but birds had smaller fat stores during molt (Fig. 2B). Surprisingly, captive birds increased their fat scores during a prenuptial molt (Fig. 2B) that corresponded to an increase in weight (Fig. 2A). The overall increase in fat stores in captive birds relative to free-living birds likely reflects easy access to food.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Seasonal weight (A) and fat stores (B) in free-living and captive Lapland longspurs. Each bar represents the mean ± SE for sample sizes indicated at bottom of bars for each group. * P < 0.0005 (t = 5.61) for weight and * P < 0.005 (t = 3.68) for fat in captives compared with spring and * P < 0.005 (t = 3.04) for free-living birds compared with breeding birds.

Response to stress. Lapland longspurs increased corticosterone in response to the stress of capture and restraint during both seasons (Fig. 3). The response to capture and restraint, however, was dramatically reduced during molt. Although CBP levels also fall during molt (Fig. 4), only maximal corticosterone titers during breeding ever reach concentrations that saturate the binding capacity of CBPs. The change in CBP levels does not appear to be accompanied by production of different proteins because the binding affinity remained constant during both seasons (K_d = 4.9 × 10⁷ M during breeding and K_d = 5.0 × 10⁷ M during molt; P = 0.93, F = 0.006).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Changes in corticosterone levels in response to capture and restraint during both breeding and molt in free-living Lapland longspurs. All baseline samples taken before 3 min are grouped together, and 30-min samples are from Ringer-injected controls. Points represent means ± SE for each group at each sampling time; n = 117 and 52 for baseline levels in breeding and molting birds, respectively (P < 0.0001, F = 49.28), and n = 23 and 8 for levels after 30 min in breeding and molting birds, respectively (P < 0.0001, F = 21.09).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Capacity of corticosterone binding proteins (CBP) during breeding and molt in free-living Lapland longspurs. To compare capacity with circulating levels, data from Fig. 2 have been converted to nmol/l. Each bar represents mean ± SE for each group for n = 7 (combination of 12 animals) and n = 3 (combination of 19 animals) for breeding and molting birds, respectively. Cort, corticosterone. * P < 0.025 (F = 7.67) compared with breeding CBP capacity.

Adrenal and pituitary responses. There was a seasonal difference in corticosterone release in response to exogenous ACTH coupled with capture and handling. The adrenals in breeding Lapland longspurs failed to further elevate corticosterone levels with two doses of exogenous ACTH compared with the Ringer-injected control (Fig. 5A). On the other hand, during molt, the lower dose of exogenous ACTH produced a fourfold elevation of corticosterone compared with Ringer-injected controls (Fig. 5B). Additionally, in no molting bird did ACTH-stimulated corticosterone reach Ringer-injected control levels during breeding.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Adrenal and pituitary sensitivities to their respective releasing factors during breeding (A) and molt (B) in free-living Lapland longspurs. Each bar represents mean ± SE of corticosterone titers at 30 min for sample sizes indicated for each group; sample sizes are at bottom of bars. Doses are per kg body wt. CRF, corticotropin-releasing factor; AVT, arginine vasotocin; MT, mesotocin. * P < 0.05 (F = 2.83) compared with Ringer injection.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Corticosterone response to restraint and blood sampling and adrenal sensitivity to 100 IU/kg body wt ACTH in captive Lapland longspurs. Each bar represents the mean ± SE for sample sizes indicated at bottom of bar for each group. * P < 0.0005 (F = 15.82 for spring and F = 16.51 for prenuptial molt) compared with Ringer injection in same season. There were no seasonal differences.

	DISCUSSION

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Stress response. Free-living Lapland longspurs modulate corticosterone release seasonally in response to an identical stressor (Fig. 3), consistent with previous studies in Lapland longspurs (2, 39) and several other bird species (1, 26, 30, 37-40). It is not clear, however, which season represents the more usual levels because samples were not collected at other times during the year. This phenomenon, however, may disappear during captivity (Fig. 6).

Adrenal response. The damped corticosterone secretion during molt in free-living birds does not appear to be mediated solely at the adrenal level. All molting birds further increased corticosterone release after exogenous ACTH injection (Fig. 5B). This suggests that the adrenals could respond further during stress if they received more endogenous ACTH. Corticosterone synthesis by the adrenals is thus not the limiting step serving to reduce stress-induced corticosterone levels during molt. In contrast, the very-high-stress-induced corticosterone levels in breeding birds, coupled with the inability to respond to exogenous ACTH, suggest that adrenals in breeding birds are responding maximally to stress-induced ACTH release. This study does not address, however, whether other hormones, such as prolactin and the thyroid hormones, alter corticosterone synthesis (4).

Pituitary response. The HPA axes in birds and mammals are similar. The avian hypothalamus connects to the anterior pituitary via a portal blood system beginning in the median eminence (20). The avian median eminence is further divided into distinct anterior and posterior portions, with only anterior vessels projecting to the anterior pituitary (20, 21). Both CRF and AVT immunoreactive fibers terminate in the external zone of the anterior median eminence (3, 16, 20), consistent with a role in ACTH secretion. MT fibers are only found in the internal zone and may not release MT into the portal vasculature (21). Castro et al. (6), however, found that CRF, AVT, and MT can induce ACTH release from the isolated duck pituitary. Castro et al. (6) also report that AVT and MT are more potent than CRF at stimulating ACTH release in the duck. CRF is likely important, however, because CRF content decreases in the median eminence after unilateral adrenalectomy (20) and CRF stimulates ACTH release from the isolated chicken pituitary (5). Thus CRF, AVT, and perhaps MT, contribute to ACTH release in birds.

Captive responses. In contrast to free-living Lapland longspurs, corticosterone responses in captive birds do not appear to be seasonally modulated. There are at least four possible explanations for this result. First, captivity may prevent initiation of breeding, thereby preventing the expression of breeding corticosterone levels. In at least one other species (white-crowned sparrows), corticosterone levels during molt, rather than during breeding, are more representative of yearly levels (26). Second, providing shelter and food ad libitum may obviate any necessity for modulating corticosterone seasonally. Third, chronic stress can dramatically alter HPA axis function (27) and may be a serious problem in captive animals. Fourth, different control mechanisms may operate during prenuptial compared with prebasic molt. Regardless, the corticosterone response does not change during prenuptial molt in captive birds, and exogenous ACTH injections suggest that sites higher than the adrenal are controlling corticosterone levels.

Seasonal modulation. Taken together, results from injecting exogenous releasing factors suggest that mechanisms underlying seasonal modulation consist of both decreased adrenal capacity to secrete corticosterone (indicated by the reduced responsiveness to exogenous ACTH during molt vis-à-vis breeding) and decreased hypothalamic capacity to secrete CRF, AVT, and MT (indicated by the ability of exogenous ACTH-releasing factors to further elevate corticosterone). The purpose for modulating corticosterone release, however, remains controversial. Several species of vertebrates, including birds and amphibians (24), can modulate glucocorticoid release. All known instances appear related to the animal's physiological state, such as breeding or molt. Several bird species reduce corticosterone release during breeding, which is thought to facilitate parental behavior in the face of disruptive severe weather (39), but parental behavior does not occur during molt. Instead, corticosterone's ability to mobilize protein may dictate a decreased response at this time because protein mobilization could be detrimental during the energetically costly molt [daily energy costs for molt are 58% of the basal metabolic rate in white-crowned sparrows (22)].