Electronic supplementary material
Methods
Study subjects
Birds were caught at bird feeders using mist nets and Potter traps. During breeding and
late breeding fledglings were determined using plumage features [1] and excluded from
sampling. All molting birds were molting primary feathers (P3-P9). Breeding stages were
confirmed by inspecting cloacal protuberances and beak color; gonads were also weighed during
sacrifice.
Tissue preparation and processing (All receptor techniques are described in greater detail in
Lattin et al. [2])
To reduce endogenous CORT that would otherwise interfere with receptor binding
assays, house sparrows were brought into the lab and immediately injected intramuscularly with
mitotane (ortho, para-DDD; 180 mg/kg body weight; Sigma Aldrich, St. Louis, MO) [3] ~36 and
~24 h prior to sacrifice. To measure the success of this treatment, immediately before sacrifice
birds were restrained in cloth bags for 30 min and ~30 ul of blood was taken. We determined
CORT concentrations in each sample using radioimmunoassay (RIA) following the methods of
Wingfield et al. [4]. Briefly, blood samples were stored on ice until centrifugation; plasma was
removed and stored at -20°C. Samples were assayed in duplicate using antibody B3-163
(Esoterix, Calabasas Hills, CA), with assay values corrected for individual recoveries following
extraction. In all cases, mitotane reduced stress-induced CORT; mean CORT was 1.7 ± 3.5
ng/ml, compared to normal values of ~20-30 ng/ml [5].
House sparrows were anesthetized with intramuscular injections of ketamine (~80 mg/kg
body weight; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (~20 mg/kg body weight;
Akorn, Inc., Decatur, IL), at doses shown to be appropriate for this species [6]. Birds were then
transcardially perfused with ice-cold heparinized saline, and belly skin (the ventral patch of skin
beginning halfway down the pectoralis muscle and ending above the cloaca) and bib skin (the
dorsal patch of skin beginning under the beak and ending halfway down the pectoralis muscle)
were removed, plucked of feathers and flash-frozen on dry ice. At each of the five different life
history stages, birds were sacrificed during the same time frame; that is, 10:00 - 16:00 EST.
Tissues were always taken in the same order, and the time to take all tissues recorded for each
bird (mean time = 13.5 ± 1.3 min). Tissues were stored at -80 C for up to 16 months until assay.
Radioligand binding assays
We used homogenization techniques, tissue to buffer ratios, incubation times and
temperatures previously optimized for house sparrow skin [2]. Binding in individual samples
was standardized per mg protein using Bradford assays. Each sample was run in triplicate, and
for each tissue, all individuals were run in the same assay to eliminate inter-assay variation.
Briefly, skin samples were homogenized in ice-cold buffer and spun in an ultracentrifuge to
separate soluble proteins (including MR and GR) from nuclear, mitochondrial and microsomal
proteins. Cytosol was incubated with 10 nM [3H]CORT (PerkinElmer, Waltham, MA) and
either 1) buffer, to measure total binding; 2) 1 μM unlabeled CORT (Sigma Aldrich, St. Louis,
MO), to measure non-specific binding; 3) 1 μM RU486 (mifepristone; Tocris Bioscience,
Minneapolis, MN), which only binds GR. After subtracting nonspecific binding, MR binding
can be calculated directly from test tubes containing RU486; GR binding can be calculated by
subtracting MR binding from total binding. Based on affinity estimates derived from previous
equilibrium saturation analyses [2], mass action predicts that 10 nM [3H]CORT should occupy
>95% of MR and ~63% of GR. Incubations were terminated by rapid filtration, and filter paper
was mixed with Ultima Gold scintillation fluid (PerkinElmer, Waltham, MA) and run on a
scintillation counter (TriCarb 1600, PerkinElmer/Packard, Waltham, MA).
Supplemental Fig 1. Photos of male house sparrows caught on the same day (10 Feb 2011) and
in the same place (Somerville, MA, USA), showing the natural variation in bib area that can
occur within one time of year. The male on the left has a relatively large bib compared to the
mean bib area for late winter sparrows; the male on the right, a relatively small bib.
1.
Lowther P.E., Cink C.L. 2006 House Sparrow (Passer domesticus). In The Birds of North
America Online (ed. Poole A.). Ithaca, Cornell Lab of Ornithology.
2.
Lattin C.R., Waldron-Francis K., Richardson J.W., de Bruijn R., Bauer C.M., Breuner
C.W., Romero L.M. 2012 Pharmacological characterization of intracellular glucocorticoid
receptors in nine tissues from house sparrow (Passer domesticus). General and Comparative
Endocrinology 179, 214-220.
3.
Breuner C.W., Orchinik M. 2001 Seasonal regulation of membrane and intracellular
corticosteroid receptors in the house sparrow brain. J Neuroendocrinol 13(5), 412-420.
4.
Wingfield J.C., Vleck C.M., Moore M.C. 1992 Seasonal changes of the adrenocortical
response to stress in birds of the Sonoran Desert. Journal of Experimental Zoology 264(4), 419428.
5.
Romero L.M., Cyr N.E., Romero R.C. 2006 Corticosterone responses change seasonally
in free-living house sparrows (Passer domesticus). General and Comparative Endocrinology
149(1), 58-65.
6.
Muresan C., Czirjak G.A., Pap P.L., Köbölkuti L.B. 2008 Ketamine and xylazine
anaesthesia in the House Sparrow. Veterinary Medicine 65(2), 193-195.