Pubertal development of estradiol-induced hypothalamic progesterone synthesis.

In females, a hallmark of puberty is the luteinizing hormone (LH) surge that triggers ovulation. Puberty initiates estrogen positive feedback onto hypothalamic circuits, which underlie the stimulation of gonadotropin releasing hormone (GnRH) neurons. In reproductively mature female rodents, both estradiol (E2) and progesterone (P4) signaling are necessary to stimulate the surge release of GnRH and LH. Estradiol membrane-initiated signaling facilitates progesterone (neuroP) synthesis in hypothalamic astrocytes, which act on E2-induced progesterone receptors (PGR) to stimulate kisspeptin release, thereby activating GnRH release. How the brain changes during puberty to allow estrogen positive feedback remains unknown. In the current study, we hypothesized that a critical step in estrogen positive feedback was the ability for estradiol-induced neuroP synthesis. To test this idea, hypothalamic neuroP levels were measured in groups of prepubertal, pubertal and young adult female Long Evans rats. Steroids were measured with liquid chromatography tandem mass spectrometry (LC-MS/MS). Hypothalamic neuroP increases from pre-puberty to young adulthood in both gonad-intact females and ovariectomized rats treated with E2. The pubertal development of hypothalamic E2-facilitated progesterone synthesis appears to be one of the neural switches facilitating reproductive maturation.

Rapid Effects of an Aggressive Interaction on Dehydroepiandrosterone, Testosterone and Oestradiol Levels in the Male Song Sparrow Brain: a Seasonal Comparison.

Across vertebrates, aggression is robustly expressed during the breeding season when circulating testosterone is elevated, and testosterone activates aggression either directly or after aromatisation into 17ß-oestradiol (E2 ) in the brain. In some species, such as the song sparrow, aggressive behaviour is also expressed at high levels during the nonbreeding season, when circulating testosterone is non-detectable. At this time, the androgen precursor dehydroepiandrosterone (DHEA) is metabolised within the brain into testosterone and/or E2 to promote aggression. In the present study, we used captive male song sparrows to test the hypothesis that an acute agonistic interaction during the nonbreeding season, but not during the breeding season, would alter steroid levels in the brain. Nonbreeding and breeding subjects were exposed to either a laboratory simulated territorial intrusion (L-STI) or an empty cage for only 5 min. Immediately afterwards, the brain was rapidly collected and flash frozen. The Palkovits punch technique was used to microdissect specific brain regions implicated in aggressive behaviour. Solid phase extraction followed by radioimmunoassay was used to quantify DHEA, testosterone and E2 in punches. Overall, levels of DHEA, testosterone and E2 were higher in brain tissue than in plasma. Local testosterone and E2 levels in the preoptic area, anterior hypothalamus and nucleus taeniae of the amygdala were significantly higher in the breeding season than the nonbreeding season and were not affected by the L-STI. Unexpectedly, subjects that were dominant in the L-STI had lower levels of DHEA in the anterior hypothalamus and medial striatum in both seasons and lower levels of DHEA in the nucleus taeniae of the amygdala in the breeding season only. Taken together, these data suggest that local levels of DHEA in the brain are very rapidly modulated by social interactions in a context and region-specific pattern.

Neuronal Gonadotrophin-Releasing Hormone (GnRH) and Astrocytic Gonadotrophin Inhibitory Hormone (GnIH) Immunoreactivity in the Adult Rat Hippocampus.

Gonadotrophin-releasing hormone (GnRH) and gonadotrophin inhibitory hormone (GnIH) are neuropeptides secreted by the hypothalamus that regulate reproduction. GnRH receptors are not only present in the anterior pituitary, but also are abundantly expressed in the hippocampus of rats, suggesting that GnRH regulates hippocampal function. GnIH inhibits pituitary gonadotrophin secretion and is also expressed in the hippocampus of a songbird; its role outside of the reproductive axis is not well established. In the present study, we employed immunohistochemistry to examine three forms of GnRH [mammalian GnRH-I (mGnRH-I), chicken GnRH-II (cGnRH-II) and lamprey GnRH-III (lGnRH-III)] and GnIH in the adult rat hippocampus. No mGnRH-I and cGnRH-II+ cell bodies were present in the hippocampus. Sparse mGnRH-I and cGnRH-II+ fibres were present within the CA1 and CA3 fields of the hippocampus, along the hippocampal fissure, and within the hilus of the dentate gyrus. No lGnRH-III was present in the rodent hippocampus. GnIH-immunoreactivity was present in the hippocampus in cell bodies that resembled astrocytes. Males had more GnIH+ cells in the hilus of the dentate gyrus than females. To confirm the GnIH+ cell body phenotype, we performed double-label immunofluorescence against GnIH, glial fibrillary acidic protein (GFAP) and NeuN. Immunofluorescence revealed that all GnIH+ cell bodies in the hippocampus also contained GFAP, a marker of astrocytes. Taken together, these data suggest that GnRH does not reach GnRH receptors in the rat hippocampus primarily via synaptic release. By contrast, GnIH might be synthesised locally in the rat hippocampus by astrocytes. These data shed light on the sites of action and possible functions of GnRH and GnIH outside of the hypothalamic-pituitary-gonadal axis.

Effects of nutritional stress during different developmental periods on song and the hypothalamic-pituitary-adrenal axis in zebra finches.

In songbirds, developmental stress affects song learning and production. Altered hypothalamic-pituitary-adrenal (HPA) axis function resulting in elevated corticosterone (CORT) may contribute to this effect. We examined whether developmental conditions affected the association between adult song and HPA axis function, and whether nutritional stress before and after nutritional independence has distinct effects on song learning and/or vocal performance. Zebra finches (Taeniopygia guttata) were raised in consistently high (HH) or low (LL) food conditions until post-hatch day (PHD) 62, or were switched from high to low conditions (HL) or vice versa (LH) at PHD 34. Song was recorded in adulthood. We assessed the response of CORT to handling during development and to dexamethasone (DEX) and adrenocorticotropic hormone (ACTH) challenges during adulthood. Song learning and vocal performance were not affected by nutritional stress at either developmental stage. Nutritional stress elevated baseline CORT during development. Nutritional stress also increased rate of CORT secretion in birds that experienced stress only in the juvenile phase (HL group). Birds in the LL group had lower CORT levels after injection of ACTH compared to the other groups, however there was no effect of nutritional stress on the response to DEX. Thus, our findings indicate that developmental stress can affect HPA function without concurrently affecting song.

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.

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.

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.

Testosterone and aggression: Berthold, birds and beyond.

Berthold's classic study of domesticated roosters in 1849 demonstrated that testicular secretions are necessary for the normal expression of aggressive behaviour. Although this conclusion is undoubtedly correct, field studies of wild songbirds have yielded important modifications and limitations of Berthold's original hypothesis. For example, studies of the North American song sparrow (Melospiza melodia) during the breeding season reveal that not only does testosterone increase aggression, but aggressive interactions also increase plasma testosterone levels. Furthermore, in winter, nonbreeding song sparrows have low plasma testosterone levels but are very aggressive, and castration of nonbreeding song sparrows does not decrease aggression. Interestingly, an aromatase inhibitor (fadrozole) does decrease male aggression in the nonbreeding season, and the effects of fadrozole can be rescued with oestradiol. In winter, dehydroepiandrosterone (DHEA) from the periphery can be metabolised within the brain to supply oestradiol to specific neural circuits. Additionally, oestradiol might be synthesised de novo from cholesterol entirely within the brain. These mechanisms may have evolved to avoid the 'costs' of circulating testosterone in the nonbreeding season. Recent studies in tropical birds, hamsters, and humans suggest that these neuroendocrine mechanisms are important for the control of aggression in many vertebrate species.

Neurosteroids and female reproduction: estrogen increases 3beta-HSD mRNA and activity in rat hypothalamus.

A central event in mammalian reproduction is the LH surge that induces ovulation and corpus luteum formation. Typically, the LH surge is initiated in ovariectomized rats by sequential treatment with estrogen and progesterone (PROG). The traditional explanation for this paradigm is that estrogen induces PROG receptors (PR) that are activated by exogenous PROG. Recent evidence suggests that whereas exogenous estrogen is necessary, exogenous PROG is not. In ovariectomized-adrenalectomized rats, estrogen treatment increases hypothalamic PROG levels before an LH surge. This estrogen-induced LH surge was blocked by an inhibitor of 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD), the proximal enzyme for PROG synthesis. These data indicate that estrogen induces de novo synthesis of PROG from cholesterol in the hypothalamus, which initiates the LH surge. The mechanism(s) by which estrogen up-regulates neuro-PROG is unknown. We investigated whether estrogen increases 1) mRNA levels for several proteins involved in PROG synthesis and/or 2) activity of 3beta-HSD in the hypothalamus. In ovariectomized-adrenalectomized rats, estrogen treatment increased 3beta-HSD mRNA in the hypothalamus, as measured by relative quantitative RT-PCR. The mRNAs for other proteins involved in steroid synthesis (sterol carrier protein 2, steroidogenic acute regulatory protein, and P450 side chain cleavage) were detectable in hypothalamus but not affected by estrogen. In a biochemical assay, estrogen treatment also increased 3beta-HSD activity. These data support the hypothesis that PROG is a neurosteroid, produced locally in the hypothalamus from cholesterol, which functions in the estrogen positive-feedback mechanism driving the LH surge.

Avoiding the 'costs' of testosterone: ecological bases of hormone-behavior interactions.

A combination of laboratory and field investigations of birds has shown that expression of behavior such as territorial aggression can occur throughout the year in many species and in different life history stages. Although it is well known that testosterone regulates territorial aggression in males during the breeding season, the correlation of plasma testosterone and aggression appears to be limited to periods of social instability when a male is challenged for his territory by another male, or when mate-guarding a sexually receptive female. How essentially identical aggression is modulated in non-breeding life history stages is not fully resolved, but despite low circulating levels of testosterone outside the breeding season, expression of territorial aggression does appear to be dependent upon aromatization of testosterone and an estrogen receptor-mediated mechanism. There is accumulating evidence that prolonged high levels of circulating testosterone may incur costs that may potentially reduce lifetime fitness. These include interference with paternal care, exposure to predators, increased risk of injury, loss of fat stores and possibly impaired immune system function and oncogenic effects. We propose six hypotheses to explain how these costs of high testosterone levels in blood may be avoided. These hypotheses are testable and may reveal many mechanisms resulting from selection to avoid the costs of testosterone. It should also be noted that the hypotheses are applicable to vertebrates in general, and may also be relevant for other hormones that have a highly specialized suite of actions in one life history stage (such as breeding), but also have a limited action in other life history stages when the full spectrum of effects would be inappropriate.

Dehydroepiandrosterone in songbird plasma: seasonal regulation and relationship to territorial aggression.

Many male animals are territorial in the breeding season, when plasma testosterone (T) levels are high, and nonterritorial in the nonbreeding season, when plasma T levels are basal. In contrast to this common pattern, male song sparrows (Melospiza melodia morphna) are territorial year-round, except briefly during molt. Song sparrows are highly aggressive in the nonbreeding season (autumn and winter), even though plasma T, 5 alpha-dihydrotestosterone, androstenedione (AE), and 17beta-estradiol levels are undetectable (

Hippocampal volume does not change seasonally in a non food-storing songbird.

Seasonal differences in hippocampal morphology have been reported in food-storing birds. Non food-storing species have not been investigated however. It is therefore unclear whether seasonal changes in the hippocampus are specifically related to food-storing or reflect a more general seasonal mechanism that occurs in both food-storing and non food-storing birds alike. We determined the volumes of the hippocampal formation and remaining telencephalon in the non-storing male song sparrow (Melospiza melodies morphna) in two experiments comparing birds collected in the spring and fall of 1992-94 (Experiment 1) and 1997 (Experiment 2). Although pronounced seasonal changes in song control nuclei such as the HVC and RA were previously reported for the same brains used in Experiment 1, we found that hippocampal volume did not change with season in either Experiment 1 or 2 for these song sparrow brains. These results suggest that seasonal changes in the hippocampus do not occur in this non food-storing species and may be specific to food-storing birds.

Neurosteroids and brain sexual differentiation.

There is new evidence that the brain of developing songbirds can synthesize estradiol de novo. In males, this neurally derived estrogen might masculinize a connection within the neural song system. These results challenge traditional concepts about mechanisms of brain sexual differentiation and reveal a significant function for neurosteroids.

Acute and chronic effects of an aromatase inhibitor on territorial aggression in breeding and nonbreeding male song sparrows.

Many studies have demonstrated that male aggression is regulated by testosterone. The conversion of testosterone to estradiol by brain aromatase is also known to regulate male aggression in the breeding season. Male song sparrows (Melospiza melodia morphna) are territorial not only in the breeding season, but also in the nonbreeding season, when plasma testosterone and estradiol levels are basal. Castration has no effect on nonbreeding aggression. In contrast, chronic (10 day) aromatase inhibitor (fadrozole) treatment decreases nonbreeding aggression, indicating a role for estrogens. Here, we show that acute (1 day) fadrozole treatment decreases nonbreeding territoriality, suggesting relatively rapid estrogen effects. In spring, fadrozole decreases brain aromatase activity, but acute and chronic fadrozole treatments do not significantly decrease aggression, although trends for some behaviors approach significance. In gonadally intact birds, fadrozole may be less effective at reducing aggression in the spring. This might occur because fadrozole causes a large increase in plasma testosterone in intact breeding males. Alternatively, estradiol may be more important for territoriality in winter than spring. We hypothesize that sex steroids regulate male aggression in spring and winter, but the endocrine mechanisms vary seasonally.

Oestrogen regulates male aggression in the non-breeding season.

Extensive research has focused on territorial aggression during the breeding season and the roles of circulating testosterone (T) and its conversion to 17beta-oestradiol (E2) in the brain. However, many species also defend territories in the non-breeding season, when circulating T-levels are low. The endocrine control of non-breeding territoriality is poorly understood. The male song sparrow of Washington State is highly territorial year-round, but plasma T is basal in the non-breeding season (autumn and winter). Castration has no effect on aggression in autumn, suggesting that autumnal territoriality is independent of gonadal hormones. However, non-gonadal sex steroids may regulate winter territoriality (e.g. oestrogen synthesis by brain aromatase). In this field experiment, we treated wild non-breeding male song sparrows with a specific aromatase inhibitor (fadrozole, FAD) using micro-osmotic pumps. FAD greatly reduced several aggressive behaviours. The effects of FAD were reversed by E2 replacement. Treatment did not affect body condition or plasma corticosterone, suggesting that all subjects were healthy These data indicate that E2 regulates male aggression in the non-breeding season and challenge the common belief that aggression in the non-breeding season is independent of sex steroids. More generally, these results raise fundamental questions about how sexual and/or aggressive behaviours are maintained in a variety of model vertebrate species despite low circulating levels of sex steroids or despite castration. Such non-classical endocrine mechanisms may be common among vertebrates and play an important role in the regulation of behaviour.

Testosterone and year-round territorial aggression in a tropical bird.

Testosterone (T) regulates avian behaviors such as song and aggression during the breeding season. However, the role of T in year-round territorial birds is still enigmatic, especially in tropical birds. Spotted antbirds (Hylophylax n. naevioides) defend territories in the Panamanian rainforest year-round but have low plasma T levels (0.1-0.2 ng/ml), except during brief periods of social challenges. We manipulated T action in captive male Spotted antbirds to test whether this hormone is involved in the regulation of song and aggression. T-implants increased plasma androgen levels (T and dihydrotestosterone) and enhanced song in nonbreeding males. During a staged male-male encounter, T-implanted males sang more and were more aggressive than controls. In a second experiment, we blocked the two known T actions: its binding to androgen receptors and its conversion into estradiol by the enzyme aromatase. For this, we administered the androgen receptor antagonist flutamide (Flut) in combination with the aromatase inhibitor 1-4-6 androstatrien-3, 17-dione (ATD) to birds in breeding condition. ATD + Flut treatment significantly elevated plasma levels of luteinizing hormone, presumably via the lack of T feedback from its receptors. ATD + Flut-treated birds gave fewer spontaneous songs than control-implanted males. During staged male-male encounters, ATD + Flut-treated males did not sing at all and showed reduced aggressive behavior. Our data indicate that T can regulate aggressive behavior in these tropical birds. Although plasma T levels can be low year-round, Spotted antbirds may use T either by secreting it briefly during social challenges, by having a high sensitivity to T action, or by enzymatically converting circulating T precursors directly at the site of action. We hypothesize that plasma T levels are kept low in these year-round territorial birds to avoid potentially detrimental effects of tonically elevated T. Future treatment of nonbreeding birds with ATD + Flut will reveal whether T is indeed involved year-round in regulating aggressive behavior.

Androgen-metabolizing enzymes show region-specific changes across the breeding season in the brain of a wild songbird.

The Lapland longspur (Calcarius lapponicus) is an arctic-breeding songbird that shows rapid behavioral changes during a short breeding season. Changes in plasma testosterone (T) in the spring are correlated with singing but not territorial aggression in males. Also, T treatment increases song but not aggression in this species. In contrast, in temperate-zone breeders, song and aggression are highly correlated, and both increase after T treatment. We asked whether regional or temporal differences in androgen-metabolizing enzymes in the longspur brain explain hormone-behavior patterns in this species. We measured the activities of aromatase, 5alpha-reductase and 5beta-reductase in free-living longspur males. Aromatase and 5alpha-reductase convert T into the active steroids 17beta-estradiol (E(2)) and 5alpha-dihydrotestosterone (5alpha-DHT), respectively. 5beta-Reductase deactivates T via conversion to 5beta-DHT, an inactive steroid. We examined seven brain regions at three stages in the breeding season. Overall, aromatase activity was high in the hypothalamus, hippocampus, and ventromedial telencephalon (containing nucleus taeniae, the avian homologue to the amygdala). 5beta-Reductase activity was high throughout the telencephalon. Activities of all three enzymes changed over time in a region-specific manner. In particular, aromatase activity in the rostral hypothalamus was decreased late in the breeding season, which may explain why T treatment at this time does not increase aggression. Changes in 5beta-reductase do not explain the effects of plasma T on aggressive behavior.

Combined aromatase inhibitor and antiandrogen treatment decreases territorial aggression in a wild songbird during the nonbreeding season.

Male song sparrows (Melospiza melodia morphna) defend territories throughout the year in western Washington State. In the nonbreeding season (autumn and winter), aggression and song are robustly expressed but plasma testosterone (T) levels are basal. Also, castration does not decrease nonbreeding territoriality. In this field experiment, we asked whether nonbreeding aggression is independent of T. T can act via androgen receptors or T can be aromatized to 17beta-estradiol (E(2)) and act via estrogen receptors. We treated free-living nonbreeding birds with an aromatase inhibitor (ATD) and an androgen receptor antagonist (flutamide) in combination. We then challenged subjects with a live decoy and playback of tape-recorded songs. ATD+flutamide treatment decreased several aggressive behaviors. However, ATD+flutamide treatment did not affect body condition, suggesting that subjects were healthy and that foraging behavior was not reduced. As expected, ATD+flutamide treatment increased plasma T, probably by blocking negative feedback on luteinizing hormone. Surprisingly, ATD+flutamide treatment increased plasma E(2). Most other studies using aromatase inhibitors have not measured plasma E(2). However, it is possible that ATD+flutamide treatment decreased local E(2) levels in the brain but not in plasma. Finally, ATD+flutamide treatment increased plasma corticosterone, perhaps in response to increased plasma T or E(2). To our knowledge, these are the first data to suggest that nonbreeding territoriality is regulated by endogenous steroid hormones. Nongonadal production of sex steroids may support aggression in the nonbreeding season.

Seasonal changes in androgen receptor immunoreactivity in the song nucleus HVc of a wild bird.

In seasonally breeding songbirds, song behavior and neural morphology change seasonally. Song control nuclei are larger during the breeding season, as determined by multiple cytological labels. Seasonal changes in song nuclei are regulated by testosterone (T), and several song nuclei contain intracellular androgen receptors (AR). Changes in AR levels may interact with changes in plasma T levels to regulate song nuclei morphology. We measured seasonal changes in AR-immunoreactive cells in the telencephalic song nucleus HVc using the affinity-purified PG21 antibody to rat AR. We caught wild adult male Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) during spring breeding in Alaska and during autumn migration in Washington State. To enhance PG21 labeling, animals were treated with T for 90 minutes (as in Smith et al. [1996] J. Histochem. Cytochem. 44:1075-1080). AR+ cells were found in HVc and other song nuclei, hippocampus, nucleus taeniae (homologue to mammalian amygdala), and the hypothalamus. HVc volume was larger in spring (S) than autumn (A), in both the PG21- and Nissl-stained sections (S:A = 1.9 and 1.7, respectively). In spring, but not autumn, PG21 and Nissl measurements were slightly different (PG21:Nissl = 1.07), perhaps because PG21 labeled the most caudal extent of HVc more clearly. In HVc, AR+ cell density and number were greater in spring. The percentage of AR+ cells was also increased in spring. Qualitatively, the staining intensity of individual cells was higher in spring. In time course studies, the T injection enhanced PG21 staining within 15 minutes, suggesting that it increases labeling via AR translocation to and concentration in the cell nucleus.

Changes in pituitary and adrenal sensitivities allow the snow bunting (Plectrophenax nivalis), an Arctic-breeding song bird, to modulate corticosterone release seasonally.

Several free-living avian species have recently been shown to seasonally modulate corticosterone release in response to capture and restraint. We examined possible mechanisms underlying seasonal adrenocortical modulation in snow buntings (Plectrophenax nivalis), a species that breeds and molts (the energetically costly replacement of feathers) in the Alaskan Arctic Snow buntings dramatically reduced baseline and maximal corticosterone titers during molt compared to the breeding season. This effect is not explained by changes in either corticosterone binding protein capacity or the overall condition of the bird (assessed by weight and fat storage). Although the adrenal's capacity to secrete corticosterone is reduced during molt, adrenal insensitivity does not fully explain reduced maximal output since exogenous adrenocorticotropic-hormone enhanced corticosterone release during both seasons. In contrast, no exogenous adrenocorticotropic hormone releasing factor (corticotrophin-releasing factor, arginine vasotocin or mesotocin) enhanced corticosterone secretion during molt. This suggests that the pituitary's endogenous adrenocorticotropic secretion was maximal in response to capture and handling, making the pituitary an important site regulating corticosterone levels. Taken together, these results indicate that seasonal modulation of corticosterone release in this species is controlled at both the adrenal and pituitary glands.