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Alterations in hypothalamic–pituitary–adrenal function associated with captivity in Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii)
Comparative Biochemistry and Physiology Part B 122 (1999) 13 – 20
Alterations in hypothalamic–pituitary–adrenal function associated with captivity in Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii ) L. Michael Romero *, John C. Wingfield Department of Zoology, Box 351800 Uni6ersity of Washington, Seattle, WA 98195, USA Received 14 January 1998; received in revised form 22 June 1998; accepted 31 August 1998
Abstract Gambel’s white-crowned sparrows were captured and brought into captivity in order to study seasonal changes in the function of the hypothalamic–pituitary–adrenal (HPA) axis in captive birds. 30 min of restraint elicited a rise in corticosterone titers that varied depending upon the season and physiological state of the birds. Restraint elevated corticosterone titers significantly more during the fall (within 2 weeks of capture from the wild) than during either the winter or during a prealternate or prebasic molt. We also examined what changes in the HPA axis could account for altered corticosterone levels. Exogenous ACTH significantly elevated corticosterone levels beyond the response to restraint during the fall, indicating a dramatic enhancement of the adrenal’s ability to secrete corticosterone. Exogenous ACTH was ineffective at other times, suggesting that the adrenal’s ability to release corticosterone often limits circulating levels. We further inferred the pituitary’s ACTH secretory ability by injecting exogenous corticotrophin-releasing factor, arginine vasotocin, and mesotocin and measuring corticosterone release. Pituitaries failed to respond to any exogenous releasing factor during the fall, suggesting that the pituitary may be the site in the HPA axis regulating corticosterone release at this time. When compared to wild-caught birds, these results suggest that captivity alters both adrenal and pituitary function during restraint in white-crowned sparrows, and that this change depends upon the season and/or physiological state of the animal. Captivity thus appears to have a profound affect on the function of the HPA axis, and these results reiterate the caution that must be used to extrapolate laboratory data to field conditions. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Birds; Corticosterone; Stress; Seasonal changes; Corticotrophin-releasing factor; Vasotocin; ACTH; Mesotocin; Hypothalamic–pituitary–adrenal axis
1. Introduction Recent work indicates that free-living Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii ) can seasonally alter their corticosterone responses to the stress of capture and handling [2,18 – 21]. Both unstressed (baseline) and stress-induced glucocorticoid titers are typically higher during the breeding season * Corresponding author. Present address: Department of Biology, Tufts University, Medford, MA 02155, USA; tel.: +1 617 6273378; fax: +1 617 6273805; e-mail: [email protected]
than at other times during the year. These previous studies, however, utilized free-ranging birds, which restricts the scope of potential studies. On the other hand, there are many potentially deleterious effects associated with captivity (e.g. the chronic stress of captivity itself). An earlier study, for example, demonstrated elevated baseline corticosterone levels in captive compared to free-ranging white-crowned sparrows [15]. In this study we tested whether stress-induced glucocorticoid levels are also affected by captivity, and whether the annual cycle in the glucocorticoid response to stress can be duplicated in captive birds.
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We explored various mechanisms that could account for alterations in adrenocortical responses associated with captivity. Corticosterone release primarily results from a cascade of signals collectively termed the hypothalamic–pituitary– adrenal (HPA) axis. Corticotrophin-releasing factor (CRF), arginine vasotocin (AVT), and mesotocin (MT) are secreted by the hypothalamus and delivered to the pituitary by the hypothalamic–pituitary portal blood [4,16,17]. These three ACTH releasing factors are known to regulate pituitary ACTH secretion [6,8]. ACTH in turn controls adrenal corticosterone release [16,17]. We determined the site in the HPA axis controlling changes in corticosterone release by injecting exogenous releasing factors. Insensitivity to an exogenous ACTH signal would indicate that seasonal changes in the adrenals’ sensitivity to ACTH could modulate corticosterone secretion. Alternatively or in conjunction, altering the pituitary’s sensitivity to CRF, AVT, and/or MT could facilitate changes in corticosterone release by decreasing ACTH release. This report tests these two possibilities.
2. Materials and methods
2.1. Birds Wild white-crowned sparrows were caught September 19–26, 1994 and September 14 – 22, 1995 near Sunnyside, WA (46.1°N, 119.5°W). Approximately 250 birds were trapped throughout the day using Japanese mist nets or seed-baited Potter traps and transported to the University of Washington. All birds were housed in 3.7 ×2.5×2.1 m outdoor aviaries (approximately 20 birds per aviary) under natural conditions except for one group of 24 birds. This group was placed in cages in photoperiod-controlled rooms on January 22, 1996 and shifted to long days (20L) until they completed a prebasic molt (April 22, 1996). All animals were fed a mixture of Mazuri Small Bird Maintenance Diet and Silversong Wild Bird Mix available ad libitum. Experiments were conducted in accord with NIH guidelines for the care and use of experimental animals and approved by the University of Washington Institutional Animal Care and Use Committee.
2.2. Preliminary Studies Differences in the corticosterone response to restraint between captive males and females were tested by subjecting four individuals of each sex to a 30 min restraint period (a period sufficient to induce maximal corticosterone secretion [12]). Individuals were sexed by laparotomy (birds were anesthetized with metofane, a small incision made in the flank, and the gonads identified visually). Initial blood samples were removed within 3
min of entering the aviary. Since corticosterone levels generally do not start to rise until 3 min after stress initiation [21], all samples taken within 3 min were considered to reflect baseline levels and were grouped together for statistical purposes. Sequential blood samples were then removed at 5, 10, and 30 min after entering the aviary. Birds were placed in opaque cloth bags between bleeds, and the entire sequence represents the restraint period. Each bleed consisted of approximately 60 ml of blood removed by a puncture of the alar vein in the wing. Blood was collected in heparinized microhematocrit capillary tubes (Fisher) and cotton stanched the blood flow. In order to determine an appropriate dose of ACTH to stimulate corticosterone release, 31 pugetensis whitecrowned sparrows (Zonotrichia leucophrys pugetensis, a different race of white-crowned sparrows) were injected with different doses of porcine ACTH (Sigma) dissolved in lactated Ringer’s solution (Baxter). Birds were removed from the aviary and injected with Ringer’s as a control or 10, 20, 100, or 250 IU kg − 1 body weight ACTH. All injections were in 10 ml. Birds were then placed in opaque cloth bags for 30 min, after which a blood sample was removed. We used the pugitensis race for determining optimal ACTH dose since birds of the Gambel’s race were unavailable at the beginning of the study.
2.3. Stress protocol All birds were subjected to the identical stressor of restraint at four different times: during the fall after a 2-week ‘acclimation’ period to captivity (October 10– 19, 1994); during winter (December 18, 1994–January 13, 1995); during the early spring while birds were undergoing a prealternate molt (March 20–22, 1995); and during a prebasic molt after birds had been photoshifted (March 22–April 18, 1996). Experiments were initiated when photoperiod rooms (during prebasic molt) or aviaries (all other birds) were entered and birds quickly captured. Initial blood samples were taken from as many birds as possible within 3 min to measure baseline corticosterone levels (two–four birds). A maximum of ten birds were captured from each aviary and no bird was sampled more than once during each season. Immediately following initial blood sampling all birds were injected intrajugularly with various releasing hormones dissolved in lactated Ringer’s solution. Injection of Ringer’s served as a control and remaining birds received either 100 IU kg − 1 porcine ACTH, 3 mg kg − 1 ovine CRF (Sigma), 3 mg kg − 1 AVT (Bachem), 3 mg kg − 1 MT (Bachem), or 3 mg kg − 1 each of CRF and AVT. All injections were in 10 ml. The ACTH dose was determined from the preliminary study. Doses of CRF, AVT, and MT were determined by attempting to mimic
L.M. Romero, J.C. Wingfield / Comparati6e Biochemistry and Physiology, Part B 122 (1999) 13–20
the highest effective in vitro doses from two studies: Castro et al. [8] demonstration that CRF, AVT, and MT induce ACTH release from the isolated duck pituitary; and Carsia et al. [6] demonstration that CRF stimulates ACTH release from the isolated chicken pituitary. An equivalent in vivo dose of approximately 3 mg kg − 1 results from assuming a total blood volume of 15% body weight for each bird (unpublished observations). These doses were effective in elevating corticosterone levels in free-ranging white-crowned sparrows during the breeding season [19], but may not have maximally stimulated release in this study. Following injections, birds were placed in opaque cloth bags for a 30 min restraint period (post handling) and subsequently bled. Birds were then weighed and scored for fat. Fat scores consisted of an average of furcular and abdominal fat measured on a semi-quantitative scale of 0–5 with 5 being the fattest [20].
2.4. Sample processing and assays After collection of blood, microhematocrit tubes were sealed on one end with clay and centrifuged at approximately 400× g for 5 min. Plasma was then removed and frozen. Corticosterone levels were assayed by radioimmunoassay after extraction in dichloromethane (for details of the radioimmunoassay see [22]). Interassay and intraassay coefficients of variation were 15 and 8%, respectively (determined using plasma pools).
2.5. Statistical analysis Sex differences in the stress response were tested using a repeated measures ANOVA. Significant increases in corticosterone levels with different ACTH doses were determined by ANOVA followed by Fisher’s Protected Least Squares Difference (PLSD) posthoc tests. Baseline and 30 min samples (the stress response) within each season were tested using Mann– Whitney U. Seasonal differences in both baseline and 30 min samples, and comparisons between exogenous hormone injections at each season, were made using ANOVA followed by Fisher’s PLSD posthoc tests.
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Fig. 1. Serial changes in corticosterone levels in response to removal from the aviary and restraint in male and female captive Gambel’s white-crowned sparrows. All initial samples were taken within 3 min and are grouped together. Points represent the mean 9 S.E.M. for n =4 for each sex at each sampling time. Corticosterone levels increased significantly over time (p B 0.0001, F= 18.75, df =3), but there was no difference between the sexes (p = 0.72, F= 0.14, df = 1).
As little as 20 IU kg − 1 of exogenous ACTH significantly elevated stress-induced corticosterone levels (Fig. 2). The effect seemed to be saturated, however, by 100 IU kg − 1. Although this dose was determined in the pugitensis race, data on wild-caught Gambel’s suggest that 100 IU kg − 1 is a potent dose in this race as well [19]. This dose was consequently chosen for the remainder of the study. Baseline corticosterone levels did not vary (p=0.81, F= 0.33) throughout the study (Fig. 3). Corticosterone levels also increased significantly after 30 min of restraint in all captive birds (fall: pB 0.0001, Z=4.27; winter: pB 0.0005, Z= 3.58; prealternate molt: pB 0.0005, Z= 3.49; prebasic molt: pB 0.0001, Z=4.059).
3. Results Captive white-crowned sparrows respond to a 30 min restraint period with an increase in corticosterone (Fig. 1). Although the sample sizes were low, since there were no significant differences in the response between males and females (nor is there a difference in wild-caught sparrows [18]), sex was ignored for the rest of this study.
Fig. 2. Dose response for exogenous ACTH on corticosterone release in pugetensis white-crowned sparrows. Each bar represents the mean 9S.E.M. for the sample sizes indicated. * p B0.05 (F =2.72, df= 4) compared to ringers-injected controls.
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There also appeared to be a seasonal difference in corticosterone release in response to exogenous ACTH coupled with restraint (Fig. 5). Elevated corticosterone titers as a result of exogenous ACTH nearly reached significance during the fall (Fig. 5(A)), but failed to further elevate corticosterone when compared to the Ringer’s-injected controls at any other time (Fig. 5(B)– (D)). In contrast, there was no significant response to any of the ACTH-releasing factors during any season (Fig. 5), although the ability to interpret responses during winter (Fig. 3(B)), prealternate molt (Fig. 3(C)), and prebasic molt (Fig. 5(D)) are limited because of the lack of a response to exogenous ACTH (see Section 4). Fig. 3. Seasonal baseline and 30 min post-restraint corticosterone levels in captive Gambel’s white-crowned sparrows. 30 min samples are from Ringer’s injected controls. Each bar represents the mean 9S.E.M. for the sample sizes indicated for each group. * p B 0.005 (F=9.54, df =3) compared to 30 min samples in other seasons.
Restraint resulted, however, in significantly higher corticosterone levels during the fall than at other times (pB 0.005, F = 9.54). This seasonal effect was mirrored in both fat reserves (Fig. 4(A)) and weight (Fig. 4(B)), with birds fattest and heaviest in the fall. Birds undergoing a prebasic molt showed a further reduction in fat stores, but no parallel decrease in weight or stimulated corticosterone release.
Fig. 4. Seasonal changes in fat score (A) and weight (B) in captive Gambel’s white-crowned sparrows. For fat score (5 is the fattest), ** pB 0.0001 compared to all other seasons, and * pB 0.005 compared to Prealternate Molt (F= 49.34, df= 3). For weight, * p B 0.005 compared to all other seasons (F = 11.39, df = 3).
4. Discussion Wild white-crowned sparrows continue to mount a corticosterone response to restraint while in captivity (Figs. 1 and 3). The magnitude of this stress response is consistent with previous studies on free-ranging members of this species [2,18,19,21]. In captivity, however, stress-induced corticosterone titers never match the 80– 100 ng ml − 1 levels reported during the breeding season in free-ranging animals [2,18,19,21], but instead correspond to levels reported at other times of the year (such as winter and during migration). This may not be surprising since these birds never bred while in captivity. A comparison of the laboratory and field data (Fig. 6) suggests that environmental cues may trigger increases and decreases of adrenocortical responses to stress in free-living birds. Studies have indicated that stress-induced corticosterone levels are dramatically lower during molt as compared to the breeding season in free-ranging whitecrowned sparrows [2,20] and several other species (unpublished data, [3]). We thus anticipated that maximal corticosterone levels would be lower during molt in captivity as well. This did not occur, since titers during molt were not significantly reduced compared to birds during the winter (Fig. 3). We chose to examine both a prebasic and a prealternate molt since they differ in both the timing and degree of molt [11]. The prebasic molt in this species takes place in the late summer and consists of replacing all body feathers, whereas the prealternate molt takes place in the spring prior to the breeding season and consists of a partial replacing of the feathers of the head and body. These two different kinds of molt, however, were not correlated with a decrease in stress-induced corticosterone titers. Furthermore, values from this study were nearly twice the levels reported in free-ranging molting birds [3]. Consequently, captivity appears to counteract molt-induced changes in corticosterone regulation. Baseline titers, however, were elevated by captivity. As part of another study, many free-ranging white-
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Fig. 5. Corticosterone responses to exogenous releasing factors in the fall (A), winter (B), and during a prealternate molt (C) and a prebasic molt (D). Each bar represents the mean 9 S.E.M. for the sample sizes indicated for each group. N.D., No Data. * p =0.08 compared to Ringers-injected controls.
crowned sparrows were bled within 3 min of capture before being brought into the lab for this study (i.e. in the fall; [19]). Mean baseline titers in the earlier study were 3.78 90.44 ng ml − 1 (n = 32), which increased in this study to 8.1090.75 (p B 0.0001, unpaired t-test, t = 5.48). Both these studies sampled from the same group of birds and only differed with the approximately 2 weeks of captivity. This increase is also consistent with another study showing that 35 days of captivity was insufficient to ‘acclimate’ baseline corticosterone levels in captive white-crowned sparrows to titers obtained in free-living animals [15]. Captivity thus appears to have a significant effect on baseline corticosterone levels (Fig. 6). We refer to baseline rather than basal
levels in this report because corticosterone concentrations may vary according to social status, etc. The approximate 2-fold increase in baseline corticosterone levels between free-ranging and captive birds was mirrored in an approximately 2-fold increase in stress-induced corticosterone titers as well [19]. Corticosterone titers after 30 min of restraint were 21.519 2.89 (n = 11) in free-ranging birds [19] compared to 40.989 4.13 in this study after 2 weeks of captivity (pB 0.001, unpaired t-test, t= 3.92). Even though baseline titers remain elevated in captivity, stress-induced corticosterone levels appear to ‘acclimate’ to captivity. After 2–3 months in captivity (during winter), corticosterone titers after 30 min of restraint had returned to
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Fig. 5. (Continued)
levels seen in free-ranging birds, and remained at these levels during both molts. As mentioned earlier, however, the huge spring increase in stress-induced corticosterone levels present in free-ranging birds is not seen in captivity (Fig. 6). The increase in stress-induced corticosterone levels during the fall corresponded to a dramatic increase in weight and fat stores (Fig. 4). Birds were generally lean when captured in the field [18] and they fed on the seed provided ad libitum in the aviaries. Some birds even became too fat to comfortably fly. Much of the initial over-feeding may be related to ad libitum food provided during a time of migratory fattening [13,14]. Alternatively, laboratory studies indicate that elevated
corticosterone titers can stimulate foraging behavior [1]. Although elevated corticosterone levels may account for the fattening and weight gain, it is not clear from this study whether elevated corticosterone stimulated foraging, or elevated fat stores altered adrenocortical functioning. Corticosterone levels during winter and both molts appear limited by the adrenal’s capacity to secrete corticosterone. At none of these times did the adrenals respond to an additional exogenous ACTH signal (Fig. 5(B)–(D)). This suggests that restraint maximally stimulates adrenal output, and that the adrenals are incapable of elevating corticosterone levels to those attained during the fall. The important regulation site of the
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Fig. 6. Comparison of baseline and stress-induced corticosterone levels in free-ranging and captive white-crowned sparrows. All dates are approximate and captive values are placed to coincide with the equivalent stage in free-ranging birds. Values in December are repeated at the end of the graph to portray the full yearly cycle. Note that the captive values in October are from the same free-ranging birds sampled 2 weeks earlier in September, and that captive ‘breeding’ values were from birds not actually breeding but sampled when they normally would be. All values from captive birds are from this study except for those sampled during the time when they would normally be breeding (unpublished data). All values from free-ranging birds are from reference [18] except during molt [2].
HPA axis during these times thus appears to be the adrenals. (Interpreting the inability of ACTH-releasing factors to elevate corticosterone during winter and both molts is difficult. CRF, AVT, and MT generally stimulate corticosterone release through ACTH. Since exogenous ACTH fails to elevate corticosterone, elevating endogenous ACTH with exogenous CRF, AVT, or MT should also be ineffective at elevating corticosterone. Results of CRF, AVT, and MT injections during these seasons are included only to indicate that none of these releasing factors acts directly on the adrenals to alter normal corticosterone release.) On the other hand, elevated corticosterone levels during the fall appear to be partly a result of enhanced adrenal capacity to secrete corticosterone. Not only are fall corticosterone levels elevated, but the adrenals appear to respond further to an exogenous ACTH signal (Fig. 5(A)). This suggests that the adrenals of fall birds could respond further during stress if they received a higher endogenous ACTH signal. The fact that they don’t receive a higher ACTH signal seems a result of the pituitary placing a cap on ACTH secretion since none of the ACTH-releasing factors significantly elevated corticosterone (which presumably would have been elevated if they had stimulated further endogenous ACTH release). Corticosterone levels during the fall
(soon after being brought into captivity) thus appear to be primarily controlled by an enhanced capacity of the adrenals to secrete corticosterone, but the maximal levels achieved appear to be limited by the amount of ACTH released by the pituitary. It is possible that ACTH is not the only factor regulating corticosterone secretion. Prolactin and the thyroid hormones can alter adrenal responsiveness [5], although in birds prolactin appears to primarily alter corticosterone negative feedback [7], and to our knowledge seasonal effects of thyroid hormones on adrenal responsiveness have not been studied in passerines. The splanchnic nerve can also modulate glucocorticoid secretion [10], but it may be primarily involved in basal rather than stress-induced glucocorticoid release [9]. Pending further evidence, it thus appears that ACTH is the most important determinant of corticosterone levels. The enhanced corticosterone response to exogenous ACTH during the fall, it should be remembered, was in addition to stress-induced release. Restraint activated the HPA axis in every animal in this study (Figs. 1 and 3). We assume, however, that physiological constraints, rather than changes in the perceived stressfullness of the stimuli, underlie the seasonal modulation of corticosterone release (see also refs. [23–25]). CRF and
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AVT released from the hypothalamus control ACTH secretion, which will in turn control corticosterone secretion, in every animal in this study, but the site of the HPA axis that limits maximal corticosterone levels appears to change during captivity. Captivity thus appears to have a profound affect on the function of the HPA axis, and these results reiterate the caution that must be used to extrapolate laboratory data to field conditions.
Acknowledgements We thank Tom Hahn, Kathleen O’Reilly, Kiran Soma, and Anthony Tramontin for technical help. This work was supported by NIH 1RO1NS30240-01 and NSF OPP-9300771 to JCW and NSF IBN-9612534 to LMR.
References [1] Astheimer LB, Buttemer WA, Wingfield JC. Interactions of corticosterone with feeding, activity and metabolism in passerine birds. Ornis Scan 1992;23:355–65. [2] Astheimer LB, Buttemer WA, Wingield JC. Gender and seasonal differences in the adrenocortical response to ACTH challenge in an Arctic passerine, Zonotrichia leucophrys gambelii. Gen Comp Endocrinol 1994;94:33–43. [3] Astheimer LB, Buttemer WA, Wingfield JC. Seasonal and acute changes in adrenocortical responsiveness in an arctic-breeding bird. Horm Behav 1995;29:442–57. [4] Ball GF, Faris PL, Wingfield JC. Immunohistochemical localization of corticotropin-releasing factor in selected brain areas of the European starling (Sturnus 6ulgaris) and the song sparrow (Melospiza melodia). Cell Tissue Res 1989;257:155–61. [5] Carsia RV. Hormonal control of avian adrenocortical function: cellular and molecular aspects. Prog Comp Endocrinol, 1990:439 – 444. [6] Carsia RV, Weber H, Perez FMJ. Corticotropin-releasing factor stimulates the release of adrenocorticotropin from domestic fowl pituitary cells. Endocrinology 1986;118:143–8. [7] Carsia RV, Scanes CG, Malamed S. Polyhormonal regulation of avian and mammalian corticosteroidogenesis in vitro. Comp Biochem Physiol 1987;88A:131–40. [8] Castro MG, Estivariz FE, Iturriza FC. The regulation of the corticomelanotropic cell activity in Aves—II. effect of various peptides on the release of ACTH from dispersed, perfused duck pituitary cells. Comp Biochem Physiol 1986;83A:71–5.
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[9] Charlton BG. Noradrenergic innervation to the adrenal cortex is responsible for control of basal glucocorticoid secretion: a model. Med Hypotheses 1995;44:214 – 6. [10] Edwards AV, Jones CT. Autonomic control of adrenal function. J Anat 1993;183:291 – 307. [11] Gill FB. Ornithology. 2nd ed. New York: W.H. Freeman, 1995. [12] Keller-Wood ME, Shinsako J, Dallman MF. Integral as well as proportional adrenal responses to ACTH. Am J Physiol 1983;245:R53 – 9. [13] King JR. On the regulation of vernal premigratory fattening in the white-crowned sparrow. Physiol Zool 1961;34:145 –57. [14] King JR, Farner DS. Premigratory changes in body weight and fat in wild and captive male white-crowned sparrow. Condor 1959;61:315 – 24. [15] Marra PP, Lampe KT, Tedford BL. Plasma corticosterone levels in two species of Zonotrichia sparrows under captive and free-living conditions. Wilson Bull 1995;107:296 – 305. [16] Mikami S. Immunocytochemistry of the avian hypothalamus and adenohypophysis. Int Rev Cytol 1986;103:189 – 248. [17] Mikami S, Yamada S. Immunohistochemistry of the hypothalamic neuropeptides and anterior pituitary cells in the Japanese quail. J Exp Zool 1984;232:405 – 17. [18] Romero LM, Ramenofsky M, Wingfield JC. Season and migration alters the corticosterone response to capture and handling in an arctic migrant, the white-crowned sparrow (Zonotrichia leucophrys gambelii ). Comp Biochem Physiol 1997;116C:171–7. [19] Romero LM, Wingfield JC. Seasonal changes in adrenal sensitivity alter corticosterone levels in Gambel’s white-crowned sparrows (Zonotrichia leucophyrys gambelii ). Comp Biochem Physiol 1998;119C:31 – 6. [20] Wingfield JC, Farner DS. The annual cycle in plasma irLH and steroid hormones in feral populations of the white-crowned sparrow, Zonotrichia leucophrys gambelii. Biol Reprod 1978;19:1046 – 56. [21] Wingfield JC, Smith JP, Farner DS. Endocrine responses of white-crowned sparrows to environmental stress. Condor 1982;84:399 – 409. [22] Wingfield JC, Vleck CM, Moore MC. Seasonal changes of the adrenocortical response to stress in birds of the Sonoran Desert. J Exp Zool 1992;264:419 – 28. [23] Wingfield JC, DeViche P, Sharbaugh S, Astheimer LB, Holberton R, Suydam R, Hunt K. Seasonal changes of the adrenocortical responses to stress in redpolls, Acanthis flammea, in Alaska. J Exp Zool 1994;270:372 – 80. [24] Wingfield JC, Suydam R, Hunt K. The adrenocortical responses to stress in snow buntings (Plectrophenax ni6alis) and Lapland longspurs (Calcarius lapponicus) at Barrow, Alaska. Comp Biochem Physiol 1994;108B:299– 306. [25] Wingfield JC, O’Reilly KM, Astheimer LB. Modulation of the adrenocortical responses to acute stress in Arctic birds: a possible ecological basis. Am Zool 1995;35:285 – 94.
Alterations in hypothalamic–pituitary–adrenal function associated with captivity in Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii ) L. Michael Romero *, John C. Wingfield Department of Zoology, Box 351800 Uni6ersity of Washington, Seattle, WA 98195, USA Received 14 January 1998; received in revised form 22 June 1998; accepted 31 August 1998
Abstract Gambel’s white-crowned sparrows were captured and brought into captivity in order to study seasonal changes in the function of the hypothalamic–pituitary–adrenal (HPA) axis in captive birds. 30 min of restraint elicited a rise in corticosterone titers that varied depending upon the season and physiological state of the birds. Restraint elevated corticosterone titers significantly more during the fall (within 2 weeks of capture from the wild) than during either the winter or during a prealternate or prebasic molt. We also examined what changes in the HPA axis could account for altered corticosterone levels. Exogenous ACTH significantly elevated corticosterone levels beyond the response to restraint during the fall, indicating a dramatic enhancement of the adrenal’s ability to secrete corticosterone. Exogenous ACTH was ineffective at other times, suggesting that the adrenal’s ability to release corticosterone often limits circulating levels. We further inferred the pituitary’s ACTH secretory ability by injecting exogenous corticotrophin-releasing factor, arginine vasotocin, and mesotocin and measuring corticosterone release. Pituitaries failed to respond to any exogenous releasing factor during the fall, suggesting that the pituitary may be the site in the HPA axis regulating corticosterone release at this time. When compared to wild-caught birds, these results suggest that captivity alters both adrenal and pituitary function during restraint in white-crowned sparrows, and that this change depends upon the season and/or physiological state of the animal. Captivity thus appears to have a profound affect on the function of the HPA axis, and these results reiterate the caution that must be used to extrapolate laboratory data to field conditions. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Birds; Corticosterone; Stress; Seasonal changes; Corticotrophin-releasing factor; Vasotocin; ACTH; Mesotocin; Hypothalamic–pituitary–adrenal axis
1. Introduction Recent work indicates that free-living Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii ) can seasonally alter their corticosterone responses to the stress of capture and handling [2,18 – 21]. Both unstressed (baseline) and stress-induced glucocorticoid titers are typically higher during the breeding season * Corresponding author. Present address: Department of Biology, Tufts University, Medford, MA 02155, USA; tel.: +1 617 6273378; fax: +1 617 6273805; e-mail: [email protected]
than at other times during the year. These previous studies, however, utilized free-ranging birds, which restricts the scope of potential studies. On the other hand, there are many potentially deleterious effects associated with captivity (e.g. the chronic stress of captivity itself). An earlier study, for example, demonstrated elevated baseline corticosterone levels in captive compared to free-ranging white-crowned sparrows [15]. In this study we tested whether stress-induced glucocorticoid levels are also affected by captivity, and whether the annual cycle in the glucocorticoid response to stress can be duplicated in captive birds.
0305-0491/99/$ - see front matter © 1999 Elsevier Science Inc. All rights reserved. PII S0305-0491(98)10161-X
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We explored various mechanisms that could account for alterations in adrenocortical responses associated with captivity. Corticosterone release primarily results from a cascade of signals collectively termed the hypothalamic–pituitary– adrenal (HPA) axis. Corticotrophin-releasing factor (CRF), arginine vasotocin (AVT), and mesotocin (MT) are secreted by the hypothalamus and delivered to the pituitary by the hypothalamic–pituitary portal blood [4,16,17]. These three ACTH releasing factors are known to regulate pituitary ACTH secretion [6,8]. ACTH in turn controls adrenal corticosterone release [16,17]. We determined the site in the HPA axis controlling changes in corticosterone release by injecting exogenous releasing factors. Insensitivity to an exogenous ACTH signal would indicate that seasonal changes in the adrenals’ sensitivity to ACTH could modulate corticosterone secretion. Alternatively or in conjunction, altering the pituitary’s sensitivity to CRF, AVT, and/or MT could facilitate changes in corticosterone release by decreasing ACTH release. This report tests these two possibilities.
2. Materials and methods
2.1. Birds Wild white-crowned sparrows were caught September 19–26, 1994 and September 14 – 22, 1995 near Sunnyside, WA (46.1°N, 119.5°W). Approximately 250 birds were trapped throughout the day using Japanese mist nets or seed-baited Potter traps and transported to the University of Washington. All birds were housed in 3.7 ×2.5×2.1 m outdoor aviaries (approximately 20 birds per aviary) under natural conditions except for one group of 24 birds. This group was placed in cages in photoperiod-controlled rooms on January 22, 1996 and shifted to long days (20L) until they completed a prebasic molt (April 22, 1996). All animals were fed a mixture of Mazuri Small Bird Maintenance Diet and Silversong Wild Bird Mix available ad libitum. Experiments were conducted in accord with NIH guidelines for the care and use of experimental animals and approved by the University of Washington Institutional Animal Care and Use Committee.
2.2. Preliminary Studies Differences in the corticosterone response to restraint between captive males and females were tested by subjecting four individuals of each sex to a 30 min restraint period (a period sufficient to induce maximal corticosterone secretion [12]). Individuals were sexed by laparotomy (birds were anesthetized with metofane, a small incision made in the flank, and the gonads identified visually). Initial blood samples were removed within 3
min of entering the aviary. Since corticosterone levels generally do not start to rise until 3 min after stress initiation [21], all samples taken within 3 min were considered to reflect baseline levels and were grouped together for statistical purposes. Sequential blood samples were then removed at 5, 10, and 30 min after entering the aviary. Birds were placed in opaque cloth bags between bleeds, and the entire sequence represents the restraint period. Each bleed consisted of approximately 60 ml of blood removed by a puncture of the alar vein in the wing. Blood was collected in heparinized microhematocrit capillary tubes (Fisher) and cotton stanched the blood flow. In order to determine an appropriate dose of ACTH to stimulate corticosterone release, 31 pugetensis whitecrowned sparrows (Zonotrichia leucophrys pugetensis, a different race of white-crowned sparrows) were injected with different doses of porcine ACTH (Sigma) dissolved in lactated Ringer’s solution (Baxter). Birds were removed from the aviary and injected with Ringer’s as a control or 10, 20, 100, or 250 IU kg − 1 body weight ACTH. All injections were in 10 ml. Birds were then placed in opaque cloth bags for 30 min, after which a blood sample was removed. We used the pugitensis race for determining optimal ACTH dose since birds of the Gambel’s race were unavailable at the beginning of the study.
2.3. Stress protocol All birds were subjected to the identical stressor of restraint at four different times: during the fall after a 2-week ‘acclimation’ period to captivity (October 10– 19, 1994); during winter (December 18, 1994–January 13, 1995); during the early spring while birds were undergoing a prealternate molt (March 20–22, 1995); and during a prebasic molt after birds had been photoshifted (March 22–April 18, 1996). Experiments were initiated when photoperiod rooms (during prebasic molt) or aviaries (all other birds) were entered and birds quickly captured. Initial blood samples were taken from as many birds as possible within 3 min to measure baseline corticosterone levels (two–four birds). A maximum of ten birds were captured from each aviary and no bird was sampled more than once during each season. Immediately following initial blood sampling all birds were injected intrajugularly with various releasing hormones dissolved in lactated Ringer’s solution. Injection of Ringer’s served as a control and remaining birds received either 100 IU kg − 1 porcine ACTH, 3 mg kg − 1 ovine CRF (Sigma), 3 mg kg − 1 AVT (Bachem), 3 mg kg − 1 MT (Bachem), or 3 mg kg − 1 each of CRF and AVT. All injections were in 10 ml. The ACTH dose was determined from the preliminary study. Doses of CRF, AVT, and MT were determined by attempting to mimic
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the highest effective in vitro doses from two studies: Castro et al. [8] demonstration that CRF, AVT, and MT induce ACTH release from the isolated duck pituitary; and Carsia et al. [6] demonstration that CRF stimulates ACTH release from the isolated chicken pituitary. An equivalent in vivo dose of approximately 3 mg kg − 1 results from assuming a total blood volume of 15% body weight for each bird (unpublished observations). These doses were effective in elevating corticosterone levels in free-ranging white-crowned sparrows during the breeding season [19], but may not have maximally stimulated release in this study. Following injections, birds were placed in opaque cloth bags for a 30 min restraint period (post handling) and subsequently bled. Birds were then weighed and scored for fat. Fat scores consisted of an average of furcular and abdominal fat measured on a semi-quantitative scale of 0–5 with 5 being the fattest [20].
2.4. Sample processing and assays After collection of blood, microhematocrit tubes were sealed on one end with clay and centrifuged at approximately 400× g for 5 min. Plasma was then removed and frozen. Corticosterone levels were assayed by radioimmunoassay after extraction in dichloromethane (for details of the radioimmunoassay see [22]). Interassay and intraassay coefficients of variation were 15 and 8%, respectively (determined using plasma pools).
2.5. Statistical analysis Sex differences in the stress response were tested using a repeated measures ANOVA. Significant increases in corticosterone levels with different ACTH doses were determined by ANOVA followed by Fisher’s Protected Least Squares Difference (PLSD) posthoc tests. Baseline and 30 min samples (the stress response) within each season were tested using Mann– Whitney U. Seasonal differences in both baseline and 30 min samples, and comparisons between exogenous hormone injections at each season, were made using ANOVA followed by Fisher’s PLSD posthoc tests.
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Fig. 1. Serial changes in corticosterone levels in response to removal from the aviary and restraint in male and female captive Gambel’s white-crowned sparrows. All initial samples were taken within 3 min and are grouped together. Points represent the mean 9 S.E.M. for n =4 for each sex at each sampling time. Corticosterone levels increased significantly over time (p B 0.0001, F= 18.75, df =3), but there was no difference between the sexes (p = 0.72, F= 0.14, df = 1).
As little as 20 IU kg − 1 of exogenous ACTH significantly elevated stress-induced corticosterone levels (Fig. 2). The effect seemed to be saturated, however, by 100 IU kg − 1. Although this dose was determined in the pugitensis race, data on wild-caught Gambel’s suggest that 100 IU kg − 1 is a potent dose in this race as well [19]. This dose was consequently chosen for the remainder of the study. Baseline corticosterone levels did not vary (p=0.81, F= 0.33) throughout the study (Fig. 3). Corticosterone levels also increased significantly after 30 min of restraint in all captive birds (fall: pB 0.0001, Z=4.27; winter: pB 0.0005, Z= 3.58; prealternate molt: pB 0.0005, Z= 3.49; prebasic molt: pB 0.0001, Z=4.059).
3. Results Captive white-crowned sparrows respond to a 30 min restraint period with an increase in corticosterone (Fig. 1). Although the sample sizes were low, since there were no significant differences in the response between males and females (nor is there a difference in wild-caught sparrows [18]), sex was ignored for the rest of this study.
Fig. 2. Dose response for exogenous ACTH on corticosterone release in pugetensis white-crowned sparrows. Each bar represents the mean 9S.E.M. for the sample sizes indicated. * p B0.05 (F =2.72, df= 4) compared to ringers-injected controls.
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There also appeared to be a seasonal difference in corticosterone release in response to exogenous ACTH coupled with restraint (Fig. 5). Elevated corticosterone titers as a result of exogenous ACTH nearly reached significance during the fall (Fig. 5(A)), but failed to further elevate corticosterone when compared to the Ringer’s-injected controls at any other time (Fig. 5(B)– (D)). In contrast, there was no significant response to any of the ACTH-releasing factors during any season (Fig. 5), although the ability to interpret responses during winter (Fig. 3(B)), prealternate molt (Fig. 3(C)), and prebasic molt (Fig. 5(D)) are limited because of the lack of a response to exogenous ACTH (see Section 4). Fig. 3. Seasonal baseline and 30 min post-restraint corticosterone levels in captive Gambel’s white-crowned sparrows. 30 min samples are from Ringer’s injected controls. Each bar represents the mean 9S.E.M. for the sample sizes indicated for each group. * p B 0.005 (F=9.54, df =3) compared to 30 min samples in other seasons.
Restraint resulted, however, in significantly higher corticosterone levels during the fall than at other times (pB 0.005, F = 9.54). This seasonal effect was mirrored in both fat reserves (Fig. 4(A)) and weight (Fig. 4(B)), with birds fattest and heaviest in the fall. Birds undergoing a prebasic molt showed a further reduction in fat stores, but no parallel decrease in weight or stimulated corticosterone release.
Fig. 4. Seasonal changes in fat score (A) and weight (B) in captive Gambel’s white-crowned sparrows. For fat score (5 is the fattest), ** pB 0.0001 compared to all other seasons, and * pB 0.005 compared to Prealternate Molt (F= 49.34, df= 3). For weight, * p B 0.005 compared to all other seasons (F = 11.39, df = 3).
4. Discussion Wild white-crowned sparrows continue to mount a corticosterone response to restraint while in captivity (Figs. 1 and 3). The magnitude of this stress response is consistent with previous studies on free-ranging members of this species [2,18,19,21]. In captivity, however, stress-induced corticosterone titers never match the 80– 100 ng ml − 1 levels reported during the breeding season in free-ranging animals [2,18,19,21], but instead correspond to levels reported at other times of the year (such as winter and during migration). This may not be surprising since these birds never bred while in captivity. A comparison of the laboratory and field data (Fig. 6) suggests that environmental cues may trigger increases and decreases of adrenocortical responses to stress in free-living birds. Studies have indicated that stress-induced corticosterone levels are dramatically lower during molt as compared to the breeding season in free-ranging whitecrowned sparrows [2,20] and several other species (unpublished data, [3]). We thus anticipated that maximal corticosterone levels would be lower during molt in captivity as well. This did not occur, since titers during molt were not significantly reduced compared to birds during the winter (Fig. 3). We chose to examine both a prebasic and a prealternate molt since they differ in both the timing and degree of molt [11]. The prebasic molt in this species takes place in the late summer and consists of replacing all body feathers, whereas the prealternate molt takes place in the spring prior to the breeding season and consists of a partial replacing of the feathers of the head and body. These two different kinds of molt, however, were not correlated with a decrease in stress-induced corticosterone titers. Furthermore, values from this study were nearly twice the levels reported in free-ranging molting birds [3]. Consequently, captivity appears to counteract molt-induced changes in corticosterone regulation. Baseline titers, however, were elevated by captivity. As part of another study, many free-ranging white-
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Fig. 5. Corticosterone responses to exogenous releasing factors in the fall (A), winter (B), and during a prealternate molt (C) and a prebasic molt (D). Each bar represents the mean 9 S.E.M. for the sample sizes indicated for each group. N.D., No Data. * p =0.08 compared to Ringers-injected controls.
crowned sparrows were bled within 3 min of capture before being brought into the lab for this study (i.e. in the fall; [19]). Mean baseline titers in the earlier study were 3.78 90.44 ng ml − 1 (n = 32), which increased in this study to 8.1090.75 (p B 0.0001, unpaired t-test, t = 5.48). Both these studies sampled from the same group of birds and only differed with the approximately 2 weeks of captivity. This increase is also consistent with another study showing that 35 days of captivity was insufficient to ‘acclimate’ baseline corticosterone levels in captive white-crowned sparrows to titers obtained in free-living animals [15]. Captivity thus appears to have a significant effect on baseline corticosterone levels (Fig. 6). We refer to baseline rather than basal
levels in this report because corticosterone concentrations may vary according to social status, etc. The approximate 2-fold increase in baseline corticosterone levels between free-ranging and captive birds was mirrored in an approximately 2-fold increase in stress-induced corticosterone titers as well [19]. Corticosterone titers after 30 min of restraint were 21.519 2.89 (n = 11) in free-ranging birds [19] compared to 40.989 4.13 in this study after 2 weeks of captivity (pB 0.001, unpaired t-test, t= 3.92). Even though baseline titers remain elevated in captivity, stress-induced corticosterone levels appear to ‘acclimate’ to captivity. After 2–3 months in captivity (during winter), corticosterone titers after 30 min of restraint had returned to
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Fig. 5. (Continued)
levels seen in free-ranging birds, and remained at these levels during both molts. As mentioned earlier, however, the huge spring increase in stress-induced corticosterone levels present in free-ranging birds is not seen in captivity (Fig. 6). The increase in stress-induced corticosterone levels during the fall corresponded to a dramatic increase in weight and fat stores (Fig. 4). Birds were generally lean when captured in the field [18] and they fed on the seed provided ad libitum in the aviaries. Some birds even became too fat to comfortably fly. Much of the initial over-feeding may be related to ad libitum food provided during a time of migratory fattening [13,14]. Alternatively, laboratory studies indicate that elevated
corticosterone titers can stimulate foraging behavior [1]. Although elevated corticosterone levels may account for the fattening and weight gain, it is not clear from this study whether elevated corticosterone stimulated foraging, or elevated fat stores altered adrenocortical functioning. Corticosterone levels during winter and both molts appear limited by the adrenal’s capacity to secrete corticosterone. At none of these times did the adrenals respond to an additional exogenous ACTH signal (Fig. 5(B)–(D)). This suggests that restraint maximally stimulates adrenal output, and that the adrenals are incapable of elevating corticosterone levels to those attained during the fall. The important regulation site of the
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Fig. 6. Comparison of baseline and stress-induced corticosterone levels in free-ranging and captive white-crowned sparrows. All dates are approximate and captive values are placed to coincide with the equivalent stage in free-ranging birds. Values in December are repeated at the end of the graph to portray the full yearly cycle. Note that the captive values in October are from the same free-ranging birds sampled 2 weeks earlier in September, and that captive ‘breeding’ values were from birds not actually breeding but sampled when they normally would be. All values from captive birds are from this study except for those sampled during the time when they would normally be breeding (unpublished data). All values from free-ranging birds are from reference [18] except during molt [2].
HPA axis during these times thus appears to be the adrenals. (Interpreting the inability of ACTH-releasing factors to elevate corticosterone during winter and both molts is difficult. CRF, AVT, and MT generally stimulate corticosterone release through ACTH. Since exogenous ACTH fails to elevate corticosterone, elevating endogenous ACTH with exogenous CRF, AVT, or MT should also be ineffective at elevating corticosterone. Results of CRF, AVT, and MT injections during these seasons are included only to indicate that none of these releasing factors acts directly on the adrenals to alter normal corticosterone release.) On the other hand, elevated corticosterone levels during the fall appear to be partly a result of enhanced adrenal capacity to secrete corticosterone. Not only are fall corticosterone levels elevated, but the adrenals appear to respond further to an exogenous ACTH signal (Fig. 5(A)). This suggests that the adrenals of fall birds could respond further during stress if they received a higher endogenous ACTH signal. The fact that they don’t receive a higher ACTH signal seems a result of the pituitary placing a cap on ACTH secretion since none of the ACTH-releasing factors significantly elevated corticosterone (which presumably would have been elevated if they had stimulated further endogenous ACTH release). Corticosterone levels during the fall
(soon after being brought into captivity) thus appear to be primarily controlled by an enhanced capacity of the adrenals to secrete corticosterone, but the maximal levels achieved appear to be limited by the amount of ACTH released by the pituitary. It is possible that ACTH is not the only factor regulating corticosterone secretion. Prolactin and the thyroid hormones can alter adrenal responsiveness [5], although in birds prolactin appears to primarily alter corticosterone negative feedback [7], and to our knowledge seasonal effects of thyroid hormones on adrenal responsiveness have not been studied in passerines. The splanchnic nerve can also modulate glucocorticoid secretion [10], but it may be primarily involved in basal rather than stress-induced glucocorticoid release [9]. Pending further evidence, it thus appears that ACTH is the most important determinant of corticosterone levels. The enhanced corticosterone response to exogenous ACTH during the fall, it should be remembered, was in addition to stress-induced release. Restraint activated the HPA axis in every animal in this study (Figs. 1 and 3). We assume, however, that physiological constraints, rather than changes in the perceived stressfullness of the stimuli, underlie the seasonal modulation of corticosterone release (see also refs. [23–25]). CRF and
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AVT released from the hypothalamus control ACTH secretion, which will in turn control corticosterone secretion, in every animal in this study, but the site of the HPA axis that limits maximal corticosterone levels appears to change during captivity. Captivity thus appears to have a profound affect on the function of the HPA axis, and these results reiterate the caution that must be used to extrapolate laboratory data to field conditions.
Acknowledgements We thank Tom Hahn, Kathleen O’Reilly, Kiran Soma, and Anthony Tramontin for technical help. This work was supported by NIH 1RO1NS30240-01 and NSF OPP-9300771 to JCW and NSF IBN-9612534 to LMR.
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