An aggressive interaction rapidly increases brain androgens in a male songbird during the non-breeding season.

Aggression is a crucial behavior that impacts access to limited resources in different environmental contexts. Androgens synthesized by the gonads promote aggression during the breeding season. However, aggression can be expressed during the non-breeding season, despite low androgen synthesis by the gonads. The brain can also synthesize steroids ('neurosteroids'), including androgens, which might promote aggression during the non-breeding season. Male song sparrows, Melospiza melodia, are territorial year-round and allow the study of seasonal changes in the steroid modulation of aggression. Here, we quantified steroids following a simulated territorial intrusion (STI) for 10 min in wild adult male song sparrows during the breeding and non-breeding seasons. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined 11 steroids: pregnenolone, progesterone, corticosterone, dehydroepiandrosterone, androstenedione, testosterone, 5α-dihydrotestosterone, 17ß-estradiol, 17α-estradiol, estriol, and estrone. Steroids were measured in blood and 10 microdissected brain regions that regulate social behavior. In both seasons, STI increased corticosterone in the blood and brain. In the breeding season, STI had no rapid effects on androgens or estrogens. Intriguingly, in the non-breeding season, STI increased testosterone and androstenedione in several behaviorally-relevant regions, but not in the blood, where androgens remained non-detectable. Also in the non-breeding season, STI increased progesterone in the blood and specific brain regions. Overall, rapid socially modulated changes in brain steroid levels are more prominent during the non-breeding season. Brain steroid levels vary with season and social context in a region-specific manner and suggest a role for neuroandrogens in aggression during the non-breeding season.Significance Statement Steroids are important modulators of the brain and behavior. Steroids are secreted by glands into the bloodstream to exert their actions on target tissues. In addition, the brain itself can locally produce steroids ('neurosteroids'). We examined circulating and neural steroids across seasons and in response to an aggressive interaction in wild male song sparrows. Local androgen levels in the brain rapidly increase in response to an aggressive interaction in the non-breeding season, but not in the breeding season, indicating that neurosteroid production is dependent on the season and social context.

Effect of a Territorial Challenge on the Steroid Profile of a Juvenile Songbird.

Aggression is a social behavior that is critical for survival and reproduction. In adults, circulating gonadal hormones, such as androgens, act on neural circuits to modulate aggressive interactions, especially in reproductive contexts. In many species, individuals also demonstrate aggression before reaching gonadal maturation. Adult male song sparrows, Melospiza melodia, breed seasonally but maintain territories year-round. Juvenile (hatch-year) males aggressively compete for territory ownership during their first winter when circulating testosterone is low. Here, we characterized the relationship between the steroid milieu and aggressive behavior in free-living juvenile male song sparrows in winter. We investigated the effect of a 10 min simulated territorial intrusion (STI) on behavior and steroid levels in blood, 10 microdissected brain regions, and four peripheral tissues (liver, pectoral muscle, adrenal glands, and testes). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we quantified 12 steroids: pregnenolone, progesterone, corticosterone, 11-dehydrocorticosterone, dehydroepiandrosterone, androstenedione, testosterone, 5α-dihydrotestosterone, 17ß-estradiol, 17α-estradiol, estrone, and estriol. We found that juvenile males are robustly aggressive, like adult males. An STI increases progesterone and corticosterone levels in blood and brain and increases 11-dehydrocorticosterone levels in blood only. Pregnenolone, androgens, and estrogens are generally non-detectable and are not affected by an STI. In peripheral tissues, steroid concentrations are very high in the adrenals. These data suggest that adrenal steroids, such as progesterone and corticosterone, might promote juvenile aggression and that juvenile and adult songbirds might rely on distinct neuroendocrine mechanisms to support similar aggressive behaviors.

Brain and circulating steroids in an electric fish: Relevance for non-breeding aggression.

Steroids play a crucial role in modulating brain and behavior. While traditionally it is thought that the brain is a target of sex steroids produced in endocrine glands (e.g. gonads), the brain itself produces steroids, known as neurosteroids. Neurosteroids can be produced in regions involved in the regulation of social behaviors and may act locally to regulate social behaviors, such as reproduction and aggression. Our model species, the weakly electric fish Gymnotus omarorum, displays non-breeding aggression in both sexes. This is a valuable natural behavior to understand neuroendocrine mechanisms that differ from those underlying breeding aggression. In the non-breeding season, circulating sex steroid levels are low, which facilitates the study of neurosteroids. Here, for the first time in a teleost fish, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify a panel of 8 steroids in both plasma and brain to characterize steroid profiles in wild non-breeding adult males and females. We show that: 1) systemic steroid levels in the non-breeding season are similar in both sexes, although only males have detectable circulating 11-ketotestosterone, 2) brain steroid levels are sexually dimorphic, as females display higher levels of androstenedione, testosterone and estrone, and only males had detectable 11-ketotestosterone, 3) systemic androgens such as androstenedione and testosterone in the non-breeding season are potential precursors for neuroestrogen synthesis, and 4) estrogens, which play a key role in non-breeding aggression, are detectable in the brain (but not the plasma) in both sexes. These data are consistent with previous studies of G. omarorum that show non-breeding aggression is dependent on estrogen signaling, as has also been shown in bird and mammal models. Overall, our results provide a foundation for understanding the role of neurosteroids, the interplay between central and peripheral steroids and potential sex differences in the regulation of social behaviors.

Ultrasensitive Quantification of Multiple Estrogens in Songbird Blood and Microdissected Brain by LC-MS/MS.

Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows, Melospiza melodia, 17ß-estradiol (17ß-E2) promotes aggressive behavior, including during the nonbreeding season when circulating steroid levels are low. Estrogens are challenging to measure because they are present at very low levels, and current techniques often lack the sensitivity required. Furthermore, current methods often focus on 17ß-E2 and disregard other estrogens. Here, we developed and validated a method to measure four estrogens [estrone (E1), 17ß-E2, 17α-estradiol (17α-E2), estriol (E3)] simultaneously in microdissected songbird brain, with high specificity, sensitivity, accuracy, and precision. We used liquid chromatography tandem mass spectrometry (LC-MS/MS), and to improve sensitivity, we derivatized estrogens using 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). The straightforward protocol improved sensitivity by 10-fold for some analytes. There is substantial regional variation in neuroestrogen levels in brain areas that regulate social behavior in male song sparrows. For example, the auditory area NCM, which has high aromatase levels, has the highest E1 and 17ß-E2 levels. In contrast, estrogen levels in blood are very low. Estrogen levels in both brain and circulation are lower in the nonbreeding season than in the breeding season. This technique will be useful for estrogen measurement in songbirds and potentially other animal models.

Neuroendocrine Mechanisms Underlying Non-breeding Aggression: Common Strategies Between Birds and Fish.

Aggression is an adaptive behavior that plays an important role in gaining access to limited resources. Aggression may occur uncoupled from reproduction, thus offering a valuable context to further understand its neural and hormonal regulation. This review focuses on the contributions from song sparrows (Melospiza melodia) and the weakly electric banded knifefish (Gymnotus omarorum). Together, these models offer clues about the underlying mechanisms of non-breeding aggression, especially the potential roles of neuropeptide Y (NPY) and brain-derived estrogens. The orexigenic NPY is well-conserved between birds and teleost fish, increases in response to low food intake, and influences sex steroid synthesis. In non-breeding M. melodia, NPY increases in the social behavior network, and NPY-Y1 receptor expression is upregulated in response to a territorial challenge. In G. omarorum, NPY is upregulated in the preoptic area of dominant, but not subordinate, individuals. We hypothesize that NPY may signal a seasonal decrease in food availability and promote non-breeding aggression. In both animal models, non-breeding aggression is estrogen-dependent but gonad-independent. In non-breeding M. melodia, neurosteroid synthesis rapidly increases in response to a territorial challenge. In G. omarorum, brain aromatase is upregulated in dominant but not subordinate fish. In both species, the dramatic decrease in food availability in the non-breeding season may promote non-breeding aggression, via changes in NPY and/or neurosteroid signaling.

Profiling of systemic and brain steroids in male songbirds: Seasonal changes in neurosteroids.

Steroids are secreted by the gonads and adrenal glands into the blood to modulate neurophysiology and behaviour. In addition, the brain can metabolise circulating steroids and synthesise steroids de novo. Songbirds show high levels of neurosteroid synthesis. In the present study, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the measurement of 10 steroids in whole blood, plasma and microdissected brain tissue (1-2 mg) of song sparrows. Our assay is highly accurate, precise, specific and sensitive. Moreover, the liquid-liquid extraction is fast, simple and effective. We quantified steroids in the blood and brain of wild male song sparrows in both breeding and non-breeding seasons. As expected, systemic androgen levels were higher in the breeding season than in the non-breeding season. Brain androgens were detectable only in the breeding season; androstenedione and 5α-dihydrotestosterone levels were up to 20-fold higher in specific brain regions than in blood. Oestrogens were not detectable in blood in both seasons. Oestrone and 17ß-oestradiol were detectable in brain in the breeding season only (up to 1.4 ng g-1 combined). Progesterone levels in several regions were higher in the non-breeding season than the breeding season, despite the lack of seasonal changes in systemic progesterone. Corticosterone levels in the blood were higher in the breeding season than in the non-breeding season but showed few seasonal differences in the brain. In general, the steroid levels presented here are lower than those in previous reports using immunoassays, because of the higher specificity of mass spectrometry. We conclude that (i) brain steroid levels can differ greatly from circulating steroid levels and (ii) brain steroid levels show region-specific seasonal patterns that are not a simple reflection of circulating steroid levels. This approach using ultrasensitive LC-MS/MS is broadly applicable to other species and allows steroid profiling in microdissected brain regions.

Reprint of "Concepts derived from the Challenge Hypothesis".

The Challenge Hypothesis was developed to explain why and how regulatory mechanisms underlying patterns of testosterone secretion vary so much across species and populations as well as among and within individuals. The hypothesis has been tested many times over the past 30years in all vertebrate groups as well as some invertebrates. Some experimental tests supported the hypothesis but many did not. However, the emerging concepts and methods extend and widen the Challenge Hypothesis to potentially all endocrine systems, and not only control of secretion, but also transport mechanisms and how target cells are able to adjust their responsiveness to circulating levels of hormones independently of other tissues. The latter concept may be particularly important in explaining how tissues respond differently to the same hormone concentration. Responsiveness of the hypothalamo-pituitary-gonad (HPG) axis to environmental and social cues regulating reproductive functions may all be driven by gonadotropin-releasing hormone (GnRH) or gonadotropin-inhibiting hormone (GnIH), but the question remains as to how different contexts and social interactions result in stimulation of GnRH or GnIH release. These concepts, although suspected for many decades, continue to be explored as integral components of environmental endocrinology and underlie fundamental mechanisms by which animals, including ourselves, cope with a changing environment. Emerging mass spectrometry techniques will have a tremendous impact enabling measurement of multiple steroids in specific brain regions. Such data will provide greater spatial resolution for studying how social challenges impact multiple steroids within the brain. Potentially the Challenge Hypothesis will continue to stimulate new ways to explore hormone-behavior interactions and generate future hypotheses.

Phenotypic flexibility of glucocorticoid signaling in skeletal muscles of a songbird preparing to migrate.

Glucocorticoids are commonly associated with responses to stress, but other important functions include homeostatic regulation, energy metabolism and tissue remodeling. At low circulating levels, glucocorticoids bind to high-affinity mineralocorticoid receptors (MR) to activate tissue repair and homeostasis (anabolic pathways), whereas at elevated levels, glucocorticoids bind to glucocorticoid receptors (GR) to activate catabolic pathways. Long distance migrations, such as those performed by Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), require modification of anatomy, physiology and behavior. Plasma corticosterone (CORT) increases in association with impending departure and flight and may promote muscle-specific anabolic states. To test this idea, we explored glucocorticoid signaling in the pectoralis (flight) and gastrocnemius (leg) muscles of male sparrows on the wintering grounds at three stages leading up to spring departure: winter (February), pre-nuptial molt (March), and pre-departure (April). CORT was detected in plasma and in both muscles, but measures of CORT signaling differed across muscles and stages. Expression of 11ß-hydroxysteroid dehydrogenase (11ß-HSD) Type 2 (inactivates CORT) increased in the pectoralis at pre-departure, whereas 11ß-HSD Type 1 (regenerates CORT) did not change. Neither of the two 11ß-HSD isoforms was detectable in the gastrocnemius. Expression of MR, but not GR, was elevated in the pectoralis at pre-departure, while only GR expression was elevated at pre-nuptial molt in gastrocnemius. These data suggest that anabolic functions predominate in the pectoralis only while catabolic activity is undetected in either muscle at pre-departure.

Concepts derived from the Challenge Hypothesis.

The Challenge Hypothesis was developed to explain why and how regulatory mechanisms underlying patterns of testosterone secretion vary so much across species and populations as well as among and within individuals. The hypothesis has been tested many times over the past 30years in all vertebrate groups as well as some invertebrates. Some experimental tests supported the hypothesis but many did not. However, the emerging concepts and methods extend and widen the Challenge Hypothesis to potentially all endocrine systems, and not only control of secretion, but also transport mechanisms and how target cells are able to adjust their responsiveness to circulating levels of hormones independently of other tissues. The latter concept may be particularly important in explaining how tissues respond differently to the same hormone concentration. Responsiveness of the hypothalamo-pituitary-gonad (HPG) axis to environmental and social cues regulating reproductive functions may all be driven by gonadotropin-releasing hormone (GnRH) or gonadotropin-inhibiting hormone (GnIH), but the question remains as to how different contexts and social interactions result in stimulation of GnRH or GnIH release. These concepts, although suspected for many decades, continue to be explored as integral components of environmental endocrinology and underlie fundamental mechanisms by which animals, including ourselves, cope with a changing environment. Emerging mass spectrometry techniques will have a tremendous impact enabling measurement of multiple steroids in specific brain regions. Such data will provide greater spatial resolution for studying how social challenges impact multiple steroids within the brain. Potentially the Challenge Hypothesis will continue to stimulate new ways to explore hormone-behavior interactions and generate future hypotheses.

Measurement of 11-dehydrocorticosterone in mice, rats and songbirds: Effects of age, sex and stress.

Glucocorticoids (GCs) are secreted into the blood by the adrenal glands and are also locally-produced by organs such as the lymphoid organs (bone marrow, thymus, and spleen). Corticosterone is the primary circulating GC in many species, including mice, rats and birds. Within lymphoid organs, corticosterone can be locally produced from the inactive metabolite, 11-dehydrocorticosterone (DHC). However, very little is known about endogenous DHC levels, and no immunoassays are currently available to measure DHC. Here, we developed an easy-to-use and inexpensive immunoassay to measure DHC that is accurate, precise, sensitive, and specific. The DHC immunoassay was validated in multiple ways, including comparison with a mass spectrometry assay. After assay validations, we demonstrated the usefulness of this immunoassay by measuring DHC (and corticosterone) in mice, rats and song sparrows. Overall, corticosterone levels were higher than DHC levels across species. In Study 1, using mice, we measured steroids in whole blood and lymphoid organs at postnatal day (PND) 5, PND23, and PND90. Corticosterone and DHC showed distinct tissue-specific patterns across development. In Studies 2 and 3, we measured circulating corticosterone and DHC in adult rats and song sparrows, before and after restraint stress. In rats and song sparrows, restraint stress rapidly increased circulating levels of both steroids. This novel DHC immunoassay revealed major changes in DHC concentrations during development and in response to stress, which have important implications for understanding GC physiology, effects of stress on immune function, and regulation of local GC levels.

Rapid effects of 17ß-estradiol on aggressive behavior in songbirds: Environmental and genetic influences.

Contribution to Special Issue on Fast effects of steroids. 17ß-estradiol (E2) has numerous rapid effects on the brain and behavior. This review focuses on the rapid effects of E2 on aggression, an important social behavior, in songbirds. First, we highlight the contributions of studies on song sparrows, which reveal that seasonal changes in the environment profoundly influence the capacity of E2 to rapidly alter aggressive behavior. E2 administration to male song sparrows increases aggression within 20 min in the non-breeding season, but not in the breeding season. Furthermore, E2 rapidly modulates several phosphoproteins in the song sparrow brain. In particular, E2 rapidly affects pCREB in the medial preoptic nucleus, in the non-breeding season only. Second, we describe studies of the white-throated sparrow, which reveal how a genetic polymorphism may influence the rapid effects of E2 on aggression. In this species, a chromosomal rearrangement that includes ESR1, which encodes estrogen receptor α (ERα), affects ERα expression in the brain and the ability of E2 to rapidly promote aggression. Third, we summarize studies showing that aggressive interactions rapidly affect levels of E2 and other steroids, both in the blood and in specific brain regions, and the emerging potential for steroid profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS). Such studies of songbirds demonstrate the value of an ethologically informed approach, in order to reveal how steroids act rapidly on the brain to alter naturally-occurring behavior.

Testosterone and Corticosterone in the Mesocorticolimbic System of Male Rats: Effects of Gonadectomy and Caloric Restriction.

Steroid hormones can modulate motivated behaviors through the mesocorticolimbic system. Gonadectomy (GDX) is a common method to determine how steroids influence the mesocorticolimbic system, and caloric restriction (CR) is often used to invigorate motivated behaviors. A common assumption is that the effects of these manipulations on brain steroid levels reflects circulating steroid levels. We now know that the brain regulates local steroid levels in a region-specific manner; however, previous studies have low spatial resolution. Using ultrasensitive liquid chromatography tandem mass spectrometry, we examined steroids in microdissected regions of the mesocorticolimbic system (ventral tegmental area, nucleus accumbens, medial prefrontal cortex). We examined whether GDX or CR influences systemic and local steroids, particularly testosterone (T) and steroidogenic enzyme transcripts. Adult male rats underwent a GDX surgery and/or CR for either 2 or 6 weeks. Levels of T, the primary steroid of interest, were higher in all brain regions than in the blood, whereas corticosterone (CORT) was lower in the brain than in the blood. Importantly, GDX completely eliminated T in the blood and lowered T in the brain. Yet, T remained present in the brain, even 6 weeks after GDX. CR decreased both T and CORT in the blood and brain. Steroidogenic enzyme (Cyp17a1, 3ß-hydroxysteroid dehydrogenase, aromatase) transcripts and androgen receptor transcripts were expressed in the mesocorticolimbic system and differentially affected by GDX and CR. Together, these results suggest that T is synthesized within the mesocorticolimbic system. These results provide a foundation for future studies examining how neurosteroids influence behaviors mediated by the mesocorticolimbic system.

Rapid effects of estradiol on aggression depend on genotype in a species with an estrogen receptor polymorphism.

The white-throated sparrow (Zonotrichia albicollis) represents a powerful model in behavioral neuroendocrinology because it occurs in two plumage morphs that differ with respect to steroid-dependent social behaviors. Birds of the white-striped (WS) morph engage in more territorial aggression than do birds of the tan-striped (TS) morph, and the TS birds engage in more parenting behavior. This behavioral polymorphism is caused by a chromosomal inversion that has captured many genes, including estrogen receptor alpha (ERα). In this study, we tested the hypothesis that morph differences in aggression might be explained by differential sensitivity to estradiol (E2). We administered E2 non-invasively to non-breeding white-throated sparrows and quantified aggression toward a conspecific 10 min later. E2 administration rapidly increased aggression in WS birds but not TS birds, consistent with our hypothesis that differential sensitivity to E2 may at least partially explain morph differences in aggression. To query the site of E2 action in the brain, we administered E2 and quantified Egr-1 expression in brain regions in which expression of ERα is known to differ between the morphs. E2 treatment decreased Egr-1 immunoreactivity in nucleus taeniae of the amygdala, but this effect did not depend on morph. Overall, our results support a role for differential effects of E2 on aggression in the two morphs, but more research will be needed to determine the neuroanatomical site of action.

Building the case for a novel teleost model of non-breeding aggression and its neuroendocrine control.

In vertebrates, aggression has been traditionally associated with high levels of circulating androgens in breeding males. Nevertheless, the centrality of androgens as primary modulators of aggression is being reconsidered in at least in two particular cases: (1) territorial aggression outside the breeding season, and (2) aggression by females. We are developing the weakly electric fish, Gymnotus omarorum, as a novel, advantageous model system to address these two alternative forms of aggression. This species displays a short, escalated contest, after which a clear hierarchical status emerges. Subordination of individuals involves three sequential decisions: interruptions of their electric discharges, retreats, and chirps. These decisions are influenced by both size asymmetry between contenders and aggression levels of dominants. Both females and males are aggressive, and do not differ in fighting ability nor in the value placed on the resource. Aggression is completely independent of gonadal hormones: dominance status is unrelated to circulating androgen and estrogen levels, and gonadectomy in males does not affect aggression. Nevertheless, estrogenic pathways participate in the modulation of this non-breeding aggression. Our results parallel those put forth in other taxa, heightening the value of G. omarorum as a model to identify commonalities in neuroendrocrine strategies of vertebrate aggression control.

Extra-gonadal steroids modulate non-breeding territorial aggression in weakly electric fish.

The neuroendocrine control of intraspecific aggression is a matter of current debate. Although aggression in a reproductive context has been associated with high levels of circulating androgens in a broad range of species, it has also been shown to occur during the non-breeding season when gonads are regressed and plasma steroid hormone levels are low. In mammals and birds the aromatization of androgens into estrogens plays a key role in the regulation of aggression in both the breeding and non-breeding seasons. This is the first study in a teleost fish to explore the role of steroids in the modulation of non-breeding aggression. Gymnotus omarorum is a highly aggressive teleost fish that exhibits aggression all year-round. We analyzed male-male non-breeding agonistic behavior, compared circulating 11-Ketotestosterone (11-KT) levels between dominants and isolated males, assessed the regulatory role of aromatization of androgens into estrogens, and evaluated the gonads as a source of these sex steroids. We found that high levels of aggression occurred in the non-breeding season despite low plasma 11-KT levels, and that there was no difference in 11-KT levels between dominant and isolated males. We demonstrated that acute aromatase inhibition decreased aggression, distorted contest dynamics, and affected expected outcome. We also found that castrated individuals displayed aggressive behavior indistinguishable from non-castrated males. Our results show, for the first time in teleost fish, that territorial aggression of G. omarorum during the non-breeding season depends on a non-gonadal estrogenic pathway.