Sex diversity in the 21st century: Concepts, frameworks, and approaches for the future of neuroendocrinology.

Sex is ubiquitous and variable throughout the animal kingdom. Historically, scientists have used reductionist methodologies that rely on a priori sex categorizations, in which two discrete sexes are inextricably linked with gamete type. However, this binarized operationalization does not adequately reflect the diversity of sex observed in nature. This is due, in part, to the fact that sex exists across many levels of biological analysis, including genetic, molecular, cellular, morphological, behavioral, and population levels. Furthermore, the biological mechanisms governing sex are embedded in complex networks that dynamically interact with other systems. To produce the most accurate and scientifically rigorous work examining sex in neuroendocrinology and to capture the full range of sex variability and diversity present in animal systems, we must critically assess the frameworks, experimental designs, and analytical methods used in our research. In this perspective piece, we first propose a new conceptual framework to guide the integrative study of sex. Then, we provide practical guidance on research approaches for studying sex-associated variables, including factors to consider in study design, selection of model organisms, experimental methodologies, and statistical analyses. We invite fellow scientists to conscientiously apply these modernized approaches to advance our biological understanding of sex and to encourage academically and socially responsible outcomes of our work. By expanding our conceptual frameworks and methodological approaches to the study of sex, we will gain insight into the unique ways that sex exists across levels of biological organization to produce the vast array of variability and diversity observed in nature.

Sex differences in aggressive intensities and brain steroids during status resolution in a sex changing fish, Lythrypnus dalli.

For vertebrates living in social hierarchies, the neuroendocrine system regulates temporal aspects of aggressive interactions during status establishment. In teleost fishes, the sex steroids 17ß-estradiol (E2) and 11-ketotestosterone (KT), and the glucocorticoid, cortisol (CORT) are associated with aggression in distinct phases of their life history. Bluebanded gobies, Lythrypnus dalli, exhibit bidirectional sexual plasticity by responding to changes in their social structure by escalating aggression associated with neural changes that precede gonadal reorganization to the opposite sex. Here, we used a novel experimental design to investigate systemic (waterborne) and neural steroids associated with the earliest behavioral changes associated with feminization and masculinization during protandrous and protogynous sex change respectively. In stable social groups of wild-caught L. dalli comprising of one male and two females, we disrupted hierarchy by adding or removing a male, providing a social context for intrasexual aggression. Within only 30 min, males exhibited high rates of physical aggression inside the nest to maintain their territory, while females exhibited high rates of chases outside the nest to reestablish social status. During this period of instability, while waterborne steroids were not affected, brain E2 was higher in all fish and CORT was lower in male brains. Brain KT was higher in males who emerged as dominant compared to dominant females. Overall, a combination of differences in brain E2, CORT, and KT were important in the regulation of hierarchy re-establishment and maintenance. Rapid responses during conspecific aggressive encounters are likely mediated by neural steroid synthesis that precede changes in systemic steroids.

Leveraging individual power to improve racial equity in academia.

Academia in the United States continues to grapple with its longstanding history of racial discrimination and its active perpetuation of racial disparities. To this end, universities and academic societies must grow in ways that reduce racial minoritization and foster racial equity. What are the effective and long-lasting approaches we as academics should prioritize to promote racial equity in our academic communities? To address this, the authors held a diversity, equity, and inclusion (DEI) panel during the Society for Behavioral Neuroendocrinology 2022 annual meeting, and in the following commentary synthesize the panelists' recommendations for fostering racial equity in the US academic community.

Uncovering the seasonal brain: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a biochemical approach for studying seasonal social behaviors.

Many animals show pronounced changes in physiology and behavior across the annual cycle, and these adaptations enable individuals to prioritize investing in the neuroendocrine mechanisms underlying reproduction and/or survival based on the time of year. While prior research has offered valuable insight into how seasonal variation in neuroendocrine processes regulates social behavior, the majority of these studies have investigated how a single hormone influences a single behavioral phenotype. Given that hormones are synthesized and metabolized via complex biochemical pathways and often act in concert to control social behavior, these approaches provide a limited view of how hormones regulate seasonal changes in behavior. In this review, we discuss how seasonal influences on hormones, the brain, and social behavior can be studied using liquid chromatography-tandem mass spectrometry (LC-MS/MS), an analytical chemistry technique that enables researchers to simultaneously quantify the concentrations of multiple hormones and the activities of their synthetic enzymes. First, we examine studies that have investigated seasonal plasticity in brain-behavior interactions, specifically by focusing on how two groups of hormones, sex steroids and nonapeptides, regulate sexual and aggressive behavior. Then, we explain the operations of LC-MS/MS, highlight studies that have used LC-MS/MS to study the neuroendocrine mechanisms underlying social behavior, both within and outside of a seasonal context, and discuss potential applications for LC-MS/MS in the field of behavioral neuroendocrinology. We propose that this cutting-edge technology will provide a more comprehensive understanding of how the multitude of hormones that comprise complex neuroendocrine networks affect seasonal variation in the brain and behavior.

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.

Preparing to migrate: expression of androgen signaling molecules and insulin-like growth factor-1 in skeletal muscles of Gambel's white-crowned sparrows.

Migratory birds, including Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii), exhibit profound modifications of skeletal muscles prior to migration, notably hypertrophy of the pectoralis muscle required for powered flight. Muscle growth may be influenced by anabolic effects of androgens; however, prior to spring departure, circulating androgens are low in sparrows. A seasonal increase in local androgen signaling may occur within muscle to promote remodeling. We measured morphological parameters, plasma and tissue levels of testosterone, as well as mRNA expression levels of androgen receptor, 5α-reductase (converts testosterone to 5α-dihydrotestosterone), and the androgen-dependent myotrophic factor insulin-like growth factor-1. We studied the pectoralis muscle as well as the gastrocnemius (leg) muscle of male sparrows across three stages on the wintering grounds: winter (February), pre-nuptial molt (March), and pre-departure (April). Testosterone levels were low, but detectable, in plasma and muscles at all three stages. Androgen receptor mRNA and 5α-reductase Type 1 mRNA increased at pre-departure, but did so in both muscles. Notably, mRNA levels of insulin-like growth factor-1, an androgen-dependent gene critical for muscle remodeling, increased at pre-departure in the pectoralis but decreased in the gastrocnemius. Taken together, these data suggest a site-specific molecular basis for muscle remodeling that may serve to enable long-distance flight.

11ß-HSD Types 1 and 2 in the Songbird Brain.

Glucocorticoid (GC) hormones act on the brain to regulate diverse functions, from behavior and homeostasis to the activity of the hypothalamic-pituitary-adrenal axis. Local regeneration and metabolism of GCs can occur in target tissues through the actions of the 11ß-hydroxysteroid dehydrogenases [11 beta-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and 11 beta-hydroxysteroid dehydrogenase type 2 (11ß-HSD2), respectively] to regulate access to GC receptors. Songbirds have become especially important model organisms for studies of stress hormone action; however, there has been little focus on neural GC metabolism. Therefore, we tested the hypothesis that 11ß-HSD1 and 11ß-HSD2 are expressed in GC-sensitive regions of the songbird brain. Localization of 11ß-HSD expression in these regions could provide precise temporal and spatial control over GC actions. We quantified GC sensitivity in zebra finch (Taeniopygia guttata) brain by measuring glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) expression across six regions, followed by quantification of 11ß-HSD1 and 11ß-HSD2 expression. We detected GR, MR, and 11ß-HSD2 mRNA expression throughout the adult brain. Whereas 11ß-HSD1 expression was undetectable in the adult brain, we detected low levels of expression in the brain of developing finches. Across several adult brain regions, expression of 11ß-HSD2 covaried with GR and MR, with the exception of the cerebellum and hippocampus. It is possible that receptors in these latter two regions require direct access to systemic GC levels. Overall, these results suggest that 11ß-HSD2 expression protects the adult songbird brain by rapid metabolism of GCs in a context and region-specific manner.

3ß-HSD expression in the CNS of a manakin and finch.

The prohormone, dehydroepiandrosterone (DHEA) circulates in vertebrate blood with the potential for actions on target tissues including the central nervous system (CNS). Many actions of DHEA require its conversion into more active products, some of which are catalyzed by the enzyme 3ß-hydroxysteroid-dehydrogenase/isomerase (3ß-HSD). Studies of birds show both expression and activity of 3ß-HSD in brain and its importance in regulating social behavior. In oscine songbirds, 3ß-HSD is expressed at reasonably high levels in brain, possibly linked to their complex neural circuitry controlling song. Studies also indicate that circulating DHEA may serve as the substrate for neural 3ß-HSD to produce active steroids that activate behavior during non-breeding seasons. In the golden-collared manakin (Manacus vitellinus), a sub-oscine bird, low levels of courtship behavior are displayed by males when circulating testosterone levels are basal. Therefore, we asked whether DHEA circulates in blood of manakins and whether the brain expresses 3ß-HSD mRNA. Given that the spinal cord is a target of androgens and likely important in regulating acrobatic movements, we also examined expression of this enzyme in the manakin spinal cord. For comparison, we examined expression levels with those of an oscine songbird, the zebra finch (Taeniopygia guttata), a species in which brain, but not spinal cord, 3ß-HSD has been well studied. DHEA was detected in manakin blood at levels similar to that seen in other species. As described previously, 3ß-HSD was expressed in all zebra finch brain regions examined. By contrast, expression of 3ß-HSD was only detected in the manakin hypothalamus where levels were greater than zebra finches. In spinal cord, 3ß-HSD was detected in some but not all regions in both species. These data point to species differences and indicate that manakins have the substrate and neural machinery to convert circulating DHEA into potentially active androgens and/or estrogens.

Sex differences in neuromuscular androgen receptor expression and sociosexual behavior in a sex changing fish.

Androgen signaling, via receptor binding, is critical for regulating the physiological and morphological foundations of male-typical reproductive behavior in vertebrates. Muscles essential for male courtship behavior and copulation are highly sensitive to androgens. Differences in the distribution and density of the androgen receptor (AR) are important for maintaining dimorphic musculature and thus may provide for anatomical identification of sexually selected traits. In Lythrypnus dalli, a bi-directional hermaphroditic teleost fish, both sexes produce agonistic approach displays, but reproductive behavior is sexually dimorphic. The male-specific courtship behavior is characterized by rapid jerky movements (involving dorsal fin erection) towards a female or around their nest. Activation of the supracarinalis muscle is involved in dorsal fin contributions to both agonistic and sociosexual behavior in other fishes, suggesting that differences in goby sexual behavior may be reflected in sexual dimorphism in AR signaling in this muscle. We examined sex differences in the local distribution of AR in supracarinalis muscle and spinal cord. Our results demonstrate that males do express more AR in the supracarinalis muscle relative to females, but there was no sex difference in the number of spinal motoneurons expressing AR. Interestingly, AR expression in the supracarinalis muscle was also related to rates of sociosexual behavior in males, providing evidence that sexual selection may influence muscle androgenic sensitivity to enhance display vigor. Sex differences in the distribution and number of cells expressing AR in the supracarinalis muscle may underlie the expression of dimorphic behaviors in L. dalli.

Ancestral androgenic differentiation pathways are repurposed during the evolution of adult sexual plasticity.

Although early exposure to androgens is necessary to permanently organize male phenotype in many vertebrates, animals that exhibit adult sexual plasticity require mechanisms that prevent early fixation of genital morphology and allow for genital morphogenesis during adult transformation. In Lythrypnus dalli, a teleost fish that exhibits bi-directional sex change, adults display dimorphic genitalia morphology despite the absence of sex differences in the potent fish androgen 11-ketotestosterone. Based on conserved patterns of vertebrate development, two steroid-based mechanisms may regulate the early development and adult maintenance of dimorphic genitalia; local androgen receptor (AR) and steroidogenic enzyme expression. Consistent with the ancestral pattern of AR expression during the multipotential phase of differentiation, juvenile differentiation into either sex involved high mesenchymal AR expression. In adults, AR expression was high throughout the male genitalia, but low or absent in females. Consistent with the hypothesis that adult sexual plasticity repurposes pathways from primary differentiation, we show that adults with transitioning genitalia also exhibited higher AR expression relative to females. Local androgen biosynthesis may also participate in genitalia transformation, as transitioning adults had greater 11ß-HSD-like immunoreactivity in the epithelial layer of the dorsal lumen compared to both sexes. By administering an AR antagonist to adult males, we show AR is necessary to maintain male-typical morphology. In a species that is resistant to early sexual canalization, early androgenic differentiation mechanisms are consistent with other vertebrates and the tissue-specific regulation of AR expression appears to be repurposed in adulthood to allow for transitions between sexual phenotypes.

Region-specific rapid regulation of aromatase activity in zebra finch brain.

Recent studies demonstrate that rapid modulation of the estrogen synthetic enzyme aromatase, regulates hypothalamic (HYP) estrogen production, and subsequent neurophysiology and reproductive behavior. In songbirds, in addition to expression in the HYP, aromatase is expressed at high levels in several brain regions notably in the hippocampus (HP) and caudomedial nidopallium (NCM), where estrogens affect learning and memory and auditory processing, respectively. Previous studies, largely in quail HYP, show that aromatase activity is acutely down-regulated by Ca2+ -dependent phosphorylation. Here, using zebra finches (Taeniopygia guttata), we ask if similar mechanisms are at work in the songbird HYP and if there are sex as well as regional differences in aromatase modulation. Using in vitro assays to measure activity in homogenates or in partially purified supernatants containing microsomes and synaptosomes of the HP, HYP, and NCM, we examined effects of Ca2+ , Mg2+ , ATP, NADPH, and an inhibitor of kinase activity. We report a rapid down-regulation of aromatase activity in the presence of phosphorylating conditions across all three brain regions and both sexes. However, regional differences were seen in response to some phosphorylating factors, some of which were improved by partial purification of the homogenates. Furthermore, while low concentrations of ATP inhibited aromatase activity, unexpectedly, inhibition was no longer seen with high ATP concentrations. These results provide evidence for a regional and temporal specificity in the rapid modulation of aromatase activity that may bear on local neuroendocrine function. Aromatase activity in male and female zebra finch hippocampus, hypothalamus, and caudomedial nidopallium is rapidly regulated by Ca2+ -dependent phosphorylation. Low ATP and Mg2+ decrease activity, whereas nicotinamide adenine dinucleotide phosphate (NADPH), high ATP, and inhibition of protein kinase C increase activity. Evidence suggests this may occur at the synapse. These results provide a mechanism for rapid regulation of behavior via brain estrogen synthesis.

Agonistic reciprocity is associated with reduced male reproductive success within haremic social networks.

While individual variation in social behaviour is ubiquitous and causes social groups to differ in structure, how these structural differences affect fitness remains largely unknown. We used social network analysis of replicate bluebanded goby (Lythrypnus dalli) harems to identify the reproductive correlates of social network structure. In stable groups, we quantified agonistic behaviour, reproduction and steroid hormones, which can both affect and respond to social/reproductive cues. We identified distinct, optimal social structures associated with different reproductive measures. Male hatching success (HS) was negatively associated with agonistic reciprocity, a network structure that describes whether subordinates 'reciprocated' agonism received from dominants. Egg laying was associated with the individual network positions of the male and dominant female. Thus, males face a trade-off between promoting structures that facilitate egg laying versus HS. Whether this reproductive conflict is avoidable remains to be determined. We also identified different social and/or reproductive roles for 11-ketotestosterone, 17ß-oestradiol and cortisol, suggesting that specific neuroendocrine mechanisms may underlie connections between network structure and fitness. This is one of the first investigations of the reproductive and neuroendocrine correlates of social behaviour and network structure in replicate, naturalistic social groups and supports network structure as an important target for natural selection.

Contextual modulation of social and endocrine correlates of fitness: insights from the life history of a sex changing fish.

Steroid hormones are critical regulators of reproductive life history, and the steroid sensitive traits (morphology, behavior, physiology) associated with particular life history stages can have substantial fitness consequences for an organism. Hormones, behavior and fitness are reciprocally associated and can be used in an integrative fashion to understand how the environment impacts organismal function. To address the fitness component, we highlight the importance of using reliable proxies of reproductive success when studying proximate regulation of reproductive phenotypes. To understand the mechanisms by which the endocrine system regulates phenotype, we discuss the use of particular endocrine proxies and the need for appropriate functional interpretation of each. Lastly, in any experimental paradigm, the responses of animals vary based on the subtle differences in environmental and social context and this must also be considered. We explore these different levels of analyses by focusing on the fascinating life history transitions exhibited by the bi-directionally hermaphroditic fish, Lythrypnus dalli. Sex changing fish are excellent models for providing a deeper understanding of the fitness consequences associated with behavioral and endocrine variation. We close by proposing that local regulation of steroids is one potential mechanism that allows for the expression of novel phenotypes that can be characteristic of specific life history stages. A comparative species approach will facilitate progress in understanding the diversity of mechanisms underlying the contextual regulation of phenotypes and their associated fitness correlates.

A mechanism for rapid neurosteroidal regulation of parenting behaviour.

While systemic steroid hormones are known to regulate reproductive behaviour, the actual mechanisms of steroidal regulation remain largely unknown. Steroidogenic enzyme activity can rapidly modulate social behaviour by influencing neurosteroid production. In fish, the enzyme 11ß-hydroxysteroid dehydrogenase (11ß-HSD) synthesizes 11-ketotestosterone (KT, a potent teleost androgen) and deactivates cortisol (the primary teleost glucocorticoid), and both of these steroid hormones can regulate behaviour. Here, we investigated the role of neurosteroidogenesis in regulating parenting in a haremic bidirectionally hermaphroditic fish, Lythrypnus dalli, where males provide all requisite parental care. Using an in vitro assay, we found that an 11ß-HSD inhibitor, carbenoxolone (CBX), reduced brain and testicular KT synthesis by 90% or more. We modulated neurosteroid levels in parenting males via intracerebroventricular injection of CBX. Within only 20 min, CBX transiently eliminated parenting behaviour, but not other social behaviour, suggesting an enzymatic mechanism for rapid neurosteroidal regulation of parenting. Consistent with our proposed mechanism, elevating KT levels rescued parenting when paired with CBX, while cortisol alone did not affect parenting. Females paired with the experimental males opportunistically consumed unattended eggs, which reduced male reproductive success by 15%, but some females also exhibited parenting behaviour and these females had elevated brain KT. Brain KT levels appear to regulate the expression of parenting behaviour as a result of changes in neural 11ß-HSD activity.

3ß-HSD in songbird brain: subcellular localization and rapid regulation by estradiol.

The enzyme 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3ß-HSD) catalyzes the conversion of dehydroepiandrosterone to androstenedione, thereby playing a key role in sex steroid synthesis. In peripheral tissues, 3ß-HSD is membrane-bound, is present in both mitochondria and microsomes, and is regulated differentially in these two subcellular compartments. In the brain, 3ß-HSD is present, but its subcellular compartmentalization is unknown. Here, in Study 1, we examined the subcellular localization of 3ß-HSD in the brain of a songbird, the zebra finch. In Study 2, in males and females, we determined whether 3ß-HSD activity in different subcellular compartments is rapidly regulated by in vitro treatment with estradiol (E(2) ), which has many rapid effects on the brain. Brain 3ß-HSD was enriched primarily in microsomes and secondarily in mitochondria and synaptosomes. In both males and females, E(2) treatment rapidly (within 5 min) inhibited 3ß-HSD activity in both mitochondria/synaptosomes and microsomes, with greater inhibition in microsomes. We also assessed the activity of 5ß-reductase, which acts on androstenedione. E(2) rapidly inhibited 5ß-reductase activity in microsomes only. This is the first study to examine the subcellular localization of 3ß-HSD in the brain, and the data demonstrate the importance of subcellular localization for the regulation of steroidogenic enzymes in the brain.

Aggressive interactions rapidly increase androgen synthesis in the brain during the non-breeding season.

In male song sparrows (Melospiza melodia), territorial challenges during the breeding season can rapidly increase circulating levels of testosterone (T). During the non-breeding season, male song sparrows are highly aggressive, but the gonads are regressed and plasma T levels are non-detectable and unaffected by territorial challenges. The pro-hormone dehydroepiandrosterone (DHEA) is elevated in song sparrow plasma and brain during the non-breeding season and may be locally converted to sex steroids in the brain to regulate aggression. The enzyme 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD) converts DHEA to androstenedione (AE) using the cofactor NAD(+), and this is a critical rate-limiting step. We predicted that brain 3beta-HSD activity varies seasonally and is rapidly modulated by aggressive challenges. In the first study, brain 3beta-HSD activity was highest in the non-breeding season in specific regions. In the second study, a simulated territorial challenge rapidly increased aggressive behavior in non-breeding song sparrows. Brain 3beta-HSD activity, when measured without exogenous NAD(+), increased by approximately 250 to 500% in telencephalic regions of challenged subjects. When brain 3beta-HSD activity was measured with exogenous NAD(+), these effects of territorial challenges were not observed. These data suggest that territorial challenges rapidly increase endogenous NAD(+) levels or increase 3beta-HSD activity specifically within a NAD-rich subcellular compartment. Together, these two studies suggest a shift from systemic to local sex steroid signaling in the non-breeding season. Local steroid signaling produces high spatial and temporal specificity of steroid signals and avoids the costs of high systemic T levels during the non-breeding season.

Neurosteroids, immunosteroids, and the Balkanization of endocrinology.

Traditionally, the production and regulation of steroid hormones has been viewed as a multi-organ process involving the hypothalamic-pituitary-gonadal (HPG) axis for sex steroids and the hypothalamic-pituitary-adrenal (HPA) axis for glucocorticoids. However, active steroids can also be synthesized locally in target tissues, either from circulating inactive precursors or de novo from cholesterol. Here, we review recent work demonstrating local steroid synthesis, with an emphasis on steroids synthesized in the brain (neurosteroids) and steroids synthesized in the immune system (immunosteroids). Furthermore, recent evidence suggests that other components of the HPG axis (luteinizing hormone and gonadotropin-releasing hormone) and HPA axis (adrenocorticotropic hormone and corticotropin-releasing hormone) are expressed locally in target tissues, potentially providing a mechanism for local regulation of neurosteroid and immunosteroid synthesis. The balance between systemic and local steroid signals depends critically on life history stage, species adaptations, and the costs of systemic signals. During particular life history stages, there can be a shift from systemic to local steroid signals. We propose that the shift to local synthesis and regulation of steroids within target tissues represents a "Balkanization" of the endocrine system, whereby individual tissues and organs may become capable of autonomously synthesizing and modulating local steroid signals, perhaps independently of the HPG and HPA axes.

Dehydroepiandrosterone and corticosterone are regulated by season and acute stress in a wild songbird: jugular versus brachial plasma.

Stress has well-known effects on adrenal glucocorticoid secretion, and chronic elevation of glucocorticoids can have detrimental effects on the brain. Dehydroepiandrosterone (DHEA), an androgen precursor synthesized in the adrenal glands or the brain itself, has anti-glucocorticoid properties, but little is known about the role of DHEA in the stress response, particularly in the brain. Here, we measured the effects of acute restraint on circulating corticosterone (CORT) and DHEA levels in wild song sparrows. Blood was collected from either the brachial or jugular vein. In songbirds, jugular plasma is enriched with neurally synthesized steroids, and therefore, jugular plasma is an indirect index of the neural steroidal milieu. Subjects were sampled during four times of year: breeding, molt, early nonbreeding, and mid-nonbreeding. Baseline CORT and DHEA levels showed similar seasonal changes; both steroids were elevated during the breeding season. Baseline CORT and DHEA levels were similar in jugular and brachial plasma. Acute stress had robust effects on CORT and DHEA that were season specific and vein specific. For CORT, during the molt, stress increased jugular CORT more than brachial CORT. For DHEA, during the breeding season, stress decreased jugular DHEA but not brachial DHEA. During the molt, stress increased jugular DHEA but not brachial DHEA. Acute stress did not affect brachial DHEA. These data suggest that acute stress specifically affects the balance between DHEA synthesis and metabolism in the brain. Furthermore, these results suggest that CORT and DHEA are locally synthesized in the brain during molt, when systemic levels of CORT and DHEA are low.

Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain.

In the songbird brain, dehydroepiandrosterone (DHEA) is metabolized to the active and aromatizable androgen androstenedione (AE) by 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Thus, brain 3beta-HSD plays a key role in regulating the steroidal milieu of the nervous system. Previous studies have shown that stress rapidly regulates brain 3beta-HSD activity in a sex-specific manner. To elucidate endocrine regulation of brain 3beta-HSD, we asked whether 17beta-estradiol (E(2)) regulates DHEA metabolism in adult zebra finch (Taeniopygia guttata) and whether there are sex-specific effects. Brain tissue was homogenized and centrifuged to obtain supernatant lacking whole cells and cell nuclei. Supernatant was incubated with [(3)H]DHEA and radioinert E(2)in vitro. Within only 10 min, E(2) significantly reduced 3beta-HSD activity in both male and female brain. Interestingly, the rapid effects of E(2) were more pronounced in females than males. These are the first data to show a rapid effect of estrogens on the songbird brain and suggest that rapid estrogen effects differ between male and female brains.

3beta-HSD activates DHEA in the songbird brain.

Dehydroepiandrosterone (DHEA) is an abundant circulating prohormone in humans, with a variety of reported actions on central and peripheral tissues. Despite its abundance, the functions of DHEA are relatively unknown because common animal models (laboratory rats and mice) have very low DHEA levels in the blood. Over the past decade, we have obtained considerable evidence from avian studies demonstrating that (1) DHEA is an important circulating prohormone in songbirds and (2) the enzyme 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD), responsible for converting DHEA into a more active androgen, is expressed at high levels in the songbird brain. Here, we first review biochemical and molecular studies demonstrating the widespread activity and expression of 3beta-HSD in the adult and developing songbird brain. Studies examining neural 3beta-HSD activity show effects of sex, stress, and season that are region-specific. Second, we review studies showing seasonal and stress-related changes in circulating DHEA in captive and wild songbird species. Third, we describe evidence that DHEA treatment can stimulate song behavior and the growth of neural circuits controlling song behavior. Importantly, brain 3beta-HSD and aromatase can work in concert to locally metabolize DHEA into active androgens and estrogens, which are critical for controlling behavior and robust adult neuroplasticity in songbirds. DHEA is likely secreted by the avian gonads and/or adrenals, as is the case in humans, but DHEA may also be synthesized de novo in the songbird brain from cholesterol or other precursors. Irrespective of its source, DHEA seems to be an important prohormone in songbirds, and 3beta-HSD is a key enzyme in the songbird brain.