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The profound programming effects of early life stress (ELS) on brain and behavior are thought to be primarily mediated by adrenal glucocorticoids (GCs). However, in mice, stressors are often administered between postnatal days 2 and 12 (PND2–12), during the stress hyporesponsive period (SHRP), when adrenal GC production is greatly reduced at baseline and in response to stressors. During the SHRP, specific brain regions produce GCs at baseline, but it is unknown if brain GC production increases in response to stressors. We treated mice at PND1 (pre-SHRP), PND5 (SHRP), PND9 (SHRP), and PND13 (post-SHRP) with an acute stressor (isoflurane anesthesia), vehicle control (oxygen), or neither (baseline). We measured a panel of progesterone and six GCs in the blood, hippocampus, cerebral cortex, and hypothalamus via liquid chromatography tandem mass spectrometry. At PND1, baseline corticosterone levels were high and did not increase in response to stress. At PND5, baseline corticosterone levels were very low, increases in brain corticosterone levels were greater than the increase in blood corticosterone levels, and stress had region-specific effects. At PND9, baseline corticosterone levels were low and increased similarly and moderately in response to stress. At PND13, blood corticosterone levels were higher than those at PND9, and corticosterone levels were higher in blood than in brain regions. These data illustrate the rapid and profound changes in stress physiology during neonatal development and suggest that neurosteroid production is a possible mechanism by which ELS has enduring effects on brain and behavior.

Hospitalized preterm infants experience painful medical procedures. Oral sucrose is the nonpharmacological standard of care for minor procedural pain relief. Infants are treated with numerous doses of sucrose, raising concerns about potential long-term effects. The objective of this study was to determine the long-term effects of neonatal oral sucrose treatment on growth and liver metabolism in a mouse model. Neonatal female and male mice were randomly assigned to one of two oral treatments ( n = 7–10 mice/group/sex): sterile water or sucrose. Pups were treated 10 times/day for the first 6 days of life with 0.2 mg/g body wt of respective treatments (24% solution; 1–4 μL/dose) to mimic what is given to preterm infants. Mice were weaned at age 3 wk onto a control diet and fed until age 16 wk. Sucrose-treated female and male mice gained less weight during the treatment period and were smaller at weaning than water-treated mice ( P ≤ 0.05); no effect of sucrose treatment on body weight was observed at adulthood. However, adult sucrose-treated female mice had smaller tibias and lower serum insulin-like growth factor-1 than adult water-treated female mice ( P ≤ 0.05); these effects were not observed in males. Lower liver S-adenosylmethionine, phosphocholine, and glycerophosphocholine were observed in adult sucrose-treated compared with water-treated female and male mice ( P ≤ 0.05). Sucrose-treated female, but not male, mice had lower liver free choline and higher liver betaine compared with water-treated female mice ( P < 0.01). Our findings suggest that repeated neonatal sucrose treatment has long-term sex-specific effects on growth and liver methionine and choline metabolism.

Glucocorticoids (GCs) are secreted by the adrenal glands and locally produced by lymphoid organs. Adrenal GC secretion at baseline and in response to stressors is greatly reduced during the stress hyporesponsive period (SHRP) in neonatal mice (postnatal day (PND) 2–12). It is unknown whether lymphoid GC production increases in response to stressors during the SHRP. Here, we administered an acute stressor (isoflurane anesthesia) to mice before, during, and after the SHRP and measured systemic and local GCs via mass spectrometry. We administered isoflurane, vehicle control (oxygen), or neither (baseline) at PND 1, 5, 9, or 13 and measured progesterone and six GCs in blood, bone marrow, thymus, and spleen. At PND1, blood and lymphoid GC levels were high and did not respond to stress. At PND5, blood GC levels were very low and increased slightly after stress, while lymphoid GC levels were also low but increased greatly after stress. At PND9, blood and lymphoid GC levels were similar at baseline and increased similarly after stress. At PND13, blood GC levels were higher than lymphoid GC levels at baseline, and blood GC levels showed a greater response to stress. These data demonstrate the remarkable plasticity of GC physiology during the postnatal period, show that local steroid levels do not reflect systemic steroid levels, provide insight into the SHRP, and identify a potential mechanism by which early-life stressors can alter immunity in adulthood.

Corticosterone is produced by the adrenal glands and also produced locally by other organs, such as the brain. Local levels of corticosterone in specific brain regions during development are not known. Here, we microdissected brain tissue and developed a novel liquid chromatography tandem mass spectrometry method (LC-MS/MS) to measure a panel of seven steroids (including 11-deoxycorticosterone (DOC), corticosterone, and 11-dehydrocorticosterone (DHC) in the blood, hippocampus (HPC), cerebral cortex (CC), and hypothalamus (HYP) of mice at postnatal day (PND) 5, 21, and 90. In a second cohort of mice, we measured the expression of three genes that code for steroidogenic enzymes that regulate corticosterone levels (Cyp11b1, Hsd11b1, and Hsd11b2) in the HPC, CC, and HYP. There were region-specific patterns of steroid levels across development, including higher corticosterone levels in the HPC and HYP than in the blood at PND5. In contrast, corticosterone levels were higher in the blood than in all brain regions at PND21 and PND90. Brain corticosterone levels were not positively correlated with blood corticosterone levels, and correlations across brain regions increased with age. Local corticosterone levels were best predicted by local DOC levels at PND5, but by local DHC levels at PND21 and PND90. Transcripts for the three enzymes were detectable in all samples (with highest expression of Hsd11b1) and showed region-specific changes with age. These data demonstrate that individual brain regions fine-tune local levels of corticosterone during early development and that coupling of glucocorticoid levels across regions increases with age.

Consumption of sucrose (table sugar) is high in much of the world. The effects of maternal sucrose intake on the placenta and fetal brain remain unknown. In rats, maternal consumption of sucrose at a human-relevant level alters the mother's physiology and steroids, as well as the adult offspring's brain and behavior. Effects in mothers are impaired glucose tolerance, increased liver lipids, increased adipose inflammation, and decreased corticosterone levels in the blood but not in the brain. In contrast, in adult female offspring, maternal sucrose intake increases corticosterone levels in the blood and the brain. In adult male offspring, maternal sucrose intake increases preference for high-sucrose and high-fat diets as well as motivation for sugar rewards in a progressive ratio task. In this study, we investigate the underlying mechanisms of the observed behavioral and endocrine effects in the adult offspring. We examine anti-inflammatory steroids, steroidogenic enzymes and cytokines in the placenta, amniotic fluid, fetal blood and brain. In our model, we feed rat dams either a high-sucrose diet (26% of kCal) or an isocaloric, matched, control diet (1% sucrose) 10 weeks prior to and during gestation. At embryonic day 19 (E19), we collected maternal serum, placenta, amniotic fluid, fetal blood, and fetal brain. We used Palkovits punch to microdissect the placenta and fetal brain. Next, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS), which is highly sensitive and specific, to measure multiple steroids (e. g. corticosterone, estrone, allopregnanolone). We examined multiple regions of the fetal brain (e. g. prefrontal cortex, nucleus accumbens, hypothalamus, hippocampus). We will also examine neuronal proliferation, microglia and tyrosine hydroxylase immunoreactivity in the fetal brain. Maternal high-sucrose diet increased 11-dehydrocorticosterone (inactive metabolite of corticosterone) and aldosterone in maternal serum and amniotic fluid. Testosterone and androstenedione were significantly higher in the amniotic fluid of male fetuses than female fetuses. Placental steroidogenic enzymes 3β-HSD and CYP19 were not affected by maternal diet or fetal sex; however, 3β-HSD activity was higher in the decidua than in the fetal part of the placenta. Steroid and cytokine data from the placenta, fetal blood and fetal brain are currently being analyzed. Ongoing analyses examine how a maternal high-sucrose diet affects brain development possibly by increasing inflammation. Presentation: No date and time listed

Traumatic brain injury (TBI) is a serious health concern and a leading cause of death. Emerging evidence strongly suggests that steroid hormones (estrogens, androgens, and progesterone) modulate TBI outcomes by regulating inflammation, oxidative stress, free radical production, and extracellular calcium levels. Despite this growing body of evidence on steroid-mediated neuroprotection, very little is known about the local synthesis of these steroids following injury. Here, we examine the effect of TBI on local neurosteroid levels around the site of injury and in plasma in adult male and female zebra finches. Using ultrasensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined estrogens, androgens, and progesterone in the entopallium and plasma of injured and uninjured animals. Three days after injury, elevated levels of 17β-estradiol (E

Behavioural flexibility is essential to adapt to a changing environment and depends on the medial prefrontal cortex (mPFC). Testosterone administration decreases behavioural flexibility. It is well known that testosterone is produced in the gonads, but testosterone is also produced in the brain, including the mPFC and other nodes of the mesocorticolimbic system. It is unclear how testosterone produced in the brain versus the gonads influences behavioural flexibility. Here, in adult male rats, we assessed the effects of the androgen synthesis inhibitor abiraterone acetate (ABI) and long-term gonadectomy (GDX) on behavioural flexibility in two paradigms. In Experiment 1, ABI but not GDX reduced the number of errors to criterion and perseverative errors in a strategy set-shifting task. In Experiment 2, with a separate cohort of rats, ABI but not GDX reduced perseverative errors in a reversal learning task. In Experiment 1, we also examined tyrosine hydroxylase immunoreactivity (TH-ir), and ABI but not GDX increased TH-ir in the mPFC. Our findings suggest that neurally-produced androgens modulate behavioural flexibility via modification of dopamine signalling in the mesocorticolimbic system. These results indicate that neurosteroids regulate executive functions and that ABI treatment for prostate cancer might affect cognition.

Neuroestrogens are synthesized within the brain and regulate social behavior, learning and memory, and cognition. In song sparrows,Melospiza melodia, 17β-estradiol (17β-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 17β-E2and disregard other estrogens. Here, we developed and validated a method to measure four estrogens [estrone (E1), 17β-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 E1and 17β-E2levels. 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.

Social isolation is an established risk factor for mental illness and impaired immune function. Evidence suggests that neuroinflammatory processes contribute to mental illness, possibly via cytokine-induced modulation of neural activity. We examined the effects of lipopolysaccharide (LPS) administration and social home cage environment on cognitive performance in the 5-Choice Serial Reaction Time Task (5CSRTT), and their effects on corticosterone and cytokines in serum and brain tissue. Male Long-Evans rats were reared in pairs or in isolation before training on the 5CSRTT. The effects of saline and LPS (150 µg/kg i.p.) administration on sickness behaviour and task performance were then assessed. LPS-induced sickness behaviour was augmented in socially-isolated rats, translating to increased omissions and slower response times in the 5CSRTT. Both social isolation and LPS administration reduced impulsive responding, while discriminative accuracy remained unaffected. With the exception of reduced impulsivity in isolated rats, these effects were not observed following a second administration of LPS, revealing behavioural tolerance to repeated LPS injections. In a separate cohort of animals, social isolation potentiated the ability of LPS to increase serum corticosterone and IL-6, which corresponded to increased IL-6 in the orbitofrontal and medial prefrontal cortices and the nucleus accumbens. Basal IL-4 levels in the nucleus accumbens were reduced in socially-isolated rats. These findings are consistent with the adaptive response of reduced motivational drive following immune challenge, and identify social isolation as an exacerbating factor. Enhanced IL-6 signalling may play a role in mediating the potentiated behavioural response to LPS administration in isolated animals.

Maternal diets can have dramatic effects on the physiology, metabolism, and behaviour of offspring that persist into adulthood. However, the effects of maternal sucrose consumption on offspring remain unclear. Here, female rats were fed either a sucrose diet with a human-relevant level of sucrose (25% of kcal) or a macronutrient-matched, isocaloric control diet before, during, and after pregnancy. After weaning, all offspring were fed a standard low-sucrose rodent chow. We measured indicators of metabolism (weight, adipose, glucose tolerance, and liver lipids) during development and adulthood (16–24 weeks). We also measured food preference and motivation for sugar rewards in adulthood. Finally, in brain regions regulating these behaviours, we measured steroids and transcripts for steroidogenic enzymes, steroid receptors, and dopamine receptors. In male offspring, maternal sucrose intake decreased body mass and visceral adipose tissue, increased preference for high-sucrose and high-fat diets, increased motivation for sugar rewards, and decreased mRNA levels of Cyp17a1 (an androgenic enzyme) in the nucleus accumbens. In female offspring, maternal sucrose intake increased basal corticosterone levels. These data demonstrate the enduring, diverse, and sex-specific effects of maternal sucrose consumption on offspring phenotype.

Androgens regulate behavioural flexibility, which is essential to adapt to a changing environment and depends on the medial prefrontal cortex (mPFC). Testosterone (T) administration decreases behavioural flexibility. It is well known that T is produced in the gonads, but T is also produced in the mesocorticolimbic system, which modulates behavioural flexibility. It is unclear how T produced in the brain versus the gonads influences behavioural flexibility. Here, we assess the effects of the androgen synthesis inhibitor abiraterone acetate (ABI) and long-term gonadectomy (GDX) on behavioural flexibility in two paradigms. In Experiment 1, ABI independent of GDX reduced the number of trials to criterion and perseverative errors in a strategy set-shifting task. Similarly, in Experiment 2, ABI but not GDX reduced perseverative errors in a reversal learning task. In subjects from Experiment 1, we also examined tyrosine hydroxylase immunoreactivity (TH-ir), and ABI but not GDX increased TH-ir in the mPFC. Our findings suggest that neurally-produced androgens modulate behavioural flexibility via modification of dopamine signalling in the mesocorticolimbic system. These results suggest novel roles for neurosteroids and possible side effects of ABI treatment for prostate cancer.

Despite the vast amount of metabolic information that can be captured in untargeted metabolomics, many biological applications are looking for a biology-driven metabolomics platform that targets a set of metabolites that are relevant to the given biological question. Steroids are a class of important molecules that play critical roles in many physiological systems and diseases. Besides known steroids, there are a large number of unknown steroids that have not been reported in the literature. The ability to rapidly detect and quantify both known and unknown steroid molecules in a biological sample can greatly accelerate a broad range of steroid-focused life science research. This work describes the development and application of SteroidXtract, a convolutional neural network (CNN)-based bioinformatics tool that can recognize steroid molecules in mass spectrometry (MS)-based untargeted metabolomics using their unique tandem MS (MS2) spectral patterns. SteroidXtract was trained using a comprehensive set of standard MS2 spectra from MassBank of North America (MoNA) and an in-house steroid library. Data augmentation strategies, including intensity thresholding and Gaussian noise addition, were created and applied to minimize data overfitting caused by the limited number of standard steroid MS2 spectra. The CNN model embedded in SteroidXtract was further compared with random forest and XGBoost using nested cross-validations to demonstrate its performance. Finally, SteroidXtract was applied in several metabolomics studies to demonstrate its sensitivity, specificity, and robustness. Compared to conventional statistics-driven metabolomics data interpretation, our work offers a novel automated biology-driven approach to interpreting untargeted metabolomics data, prioritizing biologically important molecules with high throughput and sensitivity.

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 17β-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.

Sucrose consumption is associated with type 2 diabetes, cardiovascular disease, and cognitive deficits. Sucrose intake during pregnancy might have particularly prominent effects on metabolic, endocrine, and neural physiology. It remains unclear how consumption of sucrose affects parous females, especially in brain circuits that mediate food consumption and reward processing. Here, we examine whether a human-relevant level of sucrose before, during, and after pregnancy (17–18 weeks total) influences metabolic and neuroendocrine physiology in female rats. Females were fed either a control diet or a macronutrient-matched, isocaloric sucrose diet (25% of kcal from sucrose). Metabolically, sucrose impairs glucose tolerance, increases liver lipids, and increases a marker of adipose inflammation, but has no effect on body weight or overall visceral adiposity. Sucrose also decreases corticosterone levels in serum but not in the brain. Sucrose increases progesterone levels in serum and in the brain and increases the brain:serum ratio of progesterone in the mesocorticolimbic system and hypothalamus. These data suggest a dysregulation of systemic and local steroid signalling. Moreover, sucrose decreases tyrosine hydroxylase (TH), a catecholamine-synthetic enzyme, in the medial prefrontal cortex. Finally, sucrose consumption alters the expression pattern of FOSB, a marker of phasic dopamine signalling, in the nucleus accumbens. Overall, chronic consumption of sucrose at a human-relevant level alters metabolism, steroid levels, and brain dopamine signalling in a female rat model.

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.

Steroid fatty acyl esters (FAEs) are a class of steroid conjugates that are abundant in circulation, have long half-lives, and are stored in lipid-rich tissues. Steroid-FAEs are present in many species, but their functions are poorly understood. They can be metabolized to active, unconjugated steroids and therefore may act as a reservoir of steroids. Dehydroepiandrosterone (DHEA) is an androgen precursor that can be conjugated to various fatty acids. DHEA also modulates aggression in several species, including songbirds, rodents and humans. Recent studies suggest that DHEA-FAEs might be present in songbird blood and/or brain, in part, to regulate aggression. Here, we (1) investigated the abundance of multiple fatty acids in songbird blood and (2) developed an indirect method to measure DHEA-FAEs in songbird blood and brain. First, preliminary work demonstrated high circulating levels of total (esterified and non-esterified) fatty acids, especially oleic, linoleic, and palmitic acids. These data, in conjunction with previous research, suggest that these fatty acids might be conjugated to steroids, including DHEA. Second, we successfully developed a saponification technique to indirectly measure DHEA-FAEs. Saponification cleaves the bond between the steroid molecule and the fatty acid, and we then measure the unconjugated steroid. DHEA-FAEs were incubated in 0.5M potassium hydroxide in ethanol for 30 min at room temperature, and steroids were subsequently extracted twice with dichloromethane. Unconjugated DHEA was quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS), the gold standard in steroid measurement. DHEA recovery was 88% using reference standards in neat solution. We validated this method with song sparrow plasma and chicken serum and obtained recoveries of 94-105% with intra-assay variation of 2.6%. Future research will directly measure specific DHEA-FAEs (e.g. DHEA-oleate) in blood and brain using LC-MS/MS. This research will elucidate the possible roles of steroid-FAEs in brain function and the regulation of steroid-dependent behavior. This work may also clarify the identities, levels and functions of steroid-FAEs in other species, including rodent models and humans. These data have implications for basic and clinical neuroendocrinology, offering insights into a possible storage system for steroids that may influence social behaviour.

Rheumatoid arthritis (RA) is a chronic inflammatory condition with variable clinical presentation and disease progression. Importantly, animal models of RA are widely used to examine disease pathophysiology/treatments. Here, we exploited known vendor colony-based differences in endocrine/immune responses to gain insight into inflammatory modulators in arthritis, utilizing the adjuvant-induced arthritis (AA) model. Our previous study found that Sprague-Dawley (SD) rats from Harlan develop more severe AA, have lower corticosteroid binding globulin, and have different patterns of cytokine activation in the hind paw, compared to SD rats from Charles River. Here, we extend these findings, demonstrating that Harlan rats show reduced hypothalamic cytokine responses to AA, compared to Charles River rats, and identify colony-based differences in cytokine profiles in hippocampus and spleen. To go beyond individual measures, probing for networks of variables underlying differential responses, we combined datasets from this and the previous study and performed constrained principal component analysis (CPCA). CPCA revealed that with AA, Charles River rats show activation of chemokine and central cytokine networks, whereas Harlan rats activate peripheral immune/hypothalamic-pituitary-adrenal networks. These data suggest differential underlying disease mechanism(s), highlighting the power of evaluating multiple disease biomarkers, with potential implications for understanding differential disease profiles in individuals with RA.

The zebra finch is a common model organism in neuroscience, endocrinology, and ethology. Zebra finches are generally considered opportunistic breeders, but the extent of their opportunism depends on the predictability of their habitat. This plasticity in the timing of breeding raises the question of how domestication, a process that increases environmental predictability, has affected their reproductive physiology. Here, we compared circulating steroid levels in various "strains" of zebra finches. In Study 1, using radioimmunoassay, we examined circulating testosterone levels in several strains of zebra finches (males and females). Subjects were wild or captive (Captive Wild-Caught, Wild-Derived, or Domesticated). In Study 2, using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined circulating sex steroid profiles in wild and domesticated zebra finches (males and females). In Study 1, circulating testosterone levels in males differed across strains. In Study 2, six steroids were detectable in plasma from wild zebra finches (pregnenolone, progesterone, dehydroepiandrosterone (DHEA), testosterone, androsterone, and 5α-dihydrotestosterone (5α-DHT)). Only pregnenolone and progesterone levels changed across reproductive states in wild finches. Compared to wild zebra finches, domesticated zebra finches had elevated levels of circulating pregnenolone, progesterone, DHEA, testosterone, androstenedione, and androsterone. These data suggest that domestication has profoundly altered the endocrinology of this common model organism. These results have implications for interpreting studies of domesticated zebra finches, as well as studies of other domesticated species.

Research on neural androgen receptors (ARs) has traditionally focused on brain regions that regulate reproductive and aggressive behaviors, such as the hypothalamus and amygdala. Although many cells in the prefrontal cortex (PFC) also express ARs, the number of ARs per cell appears to be much lower, and thus, AR immunostaining is often hard to detect and quantify in the PFC. Here, we demonstrate that biotin tyramide signal amplification (TSA) dramatically increases AR immunoreactivity in the rat brain, including critical regions of the PFC such as the medial PFC (mPFC) and orbitofrontal cortex (OFC). We show that TSA is useful for AR detection with both chromogenic and immunofluorescent immunohistochemistry. Double-labeling studies reveal that AR+ cells in the PFC and hippocampus are NeuN+ but not GFAP+ and thus primarily neuronal. Finally, in gonadally intact rats, more AR+ cells are present in the mPFC and OFC of males than of females. Future studies can use TSA to further examine AR immunoreactivity across ages, sexes, strains, and different procedures (e.g., fixation methods). In light of emerging evidence for the androgen regulation of executive function and working memory, these results may help understand the distribution and roles of ARs in the PFC.

As males age, systemic testosterone (T) levels decline. T regulates executive function, a collection of cognitive processes that are mediated by the mesocorticolimbic system. Here, we examined young adult (5 months) and aged (22 months) male Fischer 344 × Brown Norway rats, and measured systemic T levels in serum and local T levels in microdissected nodes of the mesocorticolimbic system (ventral tegmental area (VTA), nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and orbitofrontal cortex (OFC)). We also measured androgen receptor (AR) immunoreactivity (-ir) in the mesocorticolimbic system. As expected, systemic T levels decreased with age. Local T levels in mesocorticolimbic regions – except the VTA – also decreased with age. Mesocorticolimbic T levels were higher than serum T levels at both ages. AR-ir was present in the VTA, NAc, mPFC, and OFC and decreased with age in the mPFC. Taken together with previous results, the data suggest that changes in androgen signaling may contribute to changes in executive function during aging.

Premature infants ( Disclosure C.Y. Ramirez Contreras: None. E. Mussai: None. A.M. Wiedeman: None. A. Mehran: None. M. Salehzadeh: None. N. Boonpattrawong: None. M. Ranger: None. K.K. Soma: None. L. Holsti: Other Relationship; Self; Provincial Health Services Association. A.M. Devlin: None. Funding British Columbia Children's Hospital Research Institute

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.

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.

Glucocorticoids (GCs) are steroids produced by the adrenal glands and also by lymphoid organs such as bone marrow, thymus, and spleen (Taves et al., 2015). GCs are critical regulators of immune system development. During early development (postnatal day (PND) 2 to 12), mice show decreased adrenal GC secretion at baseline and in response to stressors, which is termed the stress hyporesponsive period (SHRP) (D’Amato et al., 1992). Lymphoid organs locally produce GCs, particularly during the SHRP, suggesting that these organs might increase local GC production in response to stress. Here, using PND1, PND5, PND9, and PND13 mice, we administered 5% isoflurane (an anesthetic) in 21% oxygen as an acute stressor, 21% oxygen as a vehicle control, or neither (baseline). For both isoflurane and oxygen treated groups, pups were removed from their home cage and placed in an induction chamber with a heating pad and nesting material from their home cage. Animals were given either given isoflurane for 3min followed by 27min oxygen or 30min continuous oxygen and rapidly euthanized. Baseline animals were given neither isoflurane nor oxygen and were rapidly euthanized in less than 3min. We then measured a panel of 7 steroids, including corticosterone, in the blood and lymphoid organs using liquid chromatography tandem mass spectrometry (LC-MS/MS). Note that PND1 is pre-SHRP, PND5 and PND9 are within the SHRP, and PND13 is post-SHRP. At PND1, corticosterone levels were generally high in both blood and lymphoid organs and did not differ with treatment. At PND5, corticosterone levels were generally very low, but increased with stress in a tissue-dependent fashion, showing the greatest increase in bone marrow and the least increase in blood. At PND9, baseline corticosterone levels were very low, but increased in both blood and lymphoid organs in response to a stressor. At PND13, corticosterone levels were higher in blood than in lymphoid organs for all treatments and increased with stress. Taken together, these novel data indicate that during the SHRP, an acute stressor produces little to no increase in corticosterone levels in blood but produces a 100-fold increase in bone marrow and a 20-fold increase in thymus and spleen. These data support the exciting possibility that mouse lymphoid organs can locally produce corticosterone, even when the adrenal glands produce little corticosterone. More generally, it is important to recognize that local steroid levels often do not match systemic steroid levels and that local steroid synthesis is particularly important in certain developmental and physiological contexts.

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 11β-hydroxysteroid dehydrogenase (11β-HSD) Type 2 (inactivates CORT) increased in the pectoralis at pre-departure, whereas 11β-HSD Type 1 (regenerates CORT) did not change. Neither of the two 11β-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.

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.

Consumption of sucrose (table sugar) is high in much of the world. The effects of a maternal diet high in sucrose on the placenta and fetal brain remain unknown. In rats, maternal consumption of sucrose at a human-relevant level has effects on the mother’s physiology and steroids, as well as long-lasting and sex-specific effects on the adult offspring’s brain and behavior. In the mothers, there are metabolic effects of sucrose intake, such as impaired glucose tolerance, increased liver lipids, and increased adipose inflammation. In rat dams, sucrose intake also decreases corticosterone levels in the blood but not in the brain. In the adult male offspring, preference for a high-sucrose diet and a high-fat diet increases due to maternal sucrose intake. In addition, maternal sucrose intake increases motivation for sugar rewards in a progressive ratio schedule of reinforcement in adult male offspring. In adult female offspring, corticosterone levels increase in the blood and brain as a result of maternal sucrose intake. In this study, we investigated the underlying mechanisms of the observed behavioral and endocrine effects in the adult offspring. Here, we examined cytokines and anti-inflammatory steroids in the placenta, amniotic fluid, and fetal blood and brain. In our model, we feed rat dams either a high-sucrose diet (26% of kCal) or an isocaloric, matched, control diet (1% sucrose) 10 weeks prior to and during gestation. At embryonic day 19 (E19), we collected maternal blood, placenta, amniotic fluid, fetal blood, and fetal brain. We use Palkovits punch to microdissect the placenta and fetal brain. Next, we use a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, which is highly precise and specific, to measure multiple steroids (e.g. corticosterone, progesterone, estradiol, allopregnanolone). The method is highly sensitive, and we can measure neurosteroids in multiple regions of the fetal brain (e.g. prefrontal cortex, nucleus accumbens, hypothalamus, hippocampus). Moreover, we will examine steroidogenic enzymes and cytokines in the fetal brain and placenta. Preliminary data show distinct steroid patterns in amniotic fluid and fetal blood, as well as in different parts of the placenta.

Estrogens are steroid hormones that affect many aspects of brain function, including cognition, social behavior, and neuroprotection. It is well-known that estrogens are synthesized in the ovaries. Estrogens are also synthesized in the brain, where aromatase is expressed in specific regions. Importantly, estrogens play crucial roles in the brain, even at extremely low levels. Current assays lack the necessary sensitivity and/or specificity to measure brain-synthesized estrogens. Furthermore, current methods focus on only 17β-estradiol and generally disregard other estrogens that are synthesized in the brain. Here, we developed a method to measure several estrogens simultaneously, with high sensitivity and specificity. To improve sensitivity, we derivatized estrogens with 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS). We used liquid chromatography tandem mass spectrometry (LC-MS/MS) to examine a panel of eight estrogens: 17β-estradiol, 17α-estradiol, estrone, estriol, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 4-methoxyestradiol. After derivatization, we have improved sensitivity 20-fold, detecting as little as 0.01 pg per sample, demonstrating that our method is extremely sensitive. For each analyte, we have identified a distinct retention time as well as 2 scheduled multiple reaction monitoring (sMRM) transitions that were used as quality control criteria for clear identification. Therefore, we are able to distinguish each estrogen (even stereoisomers) by the chromatographic separation and the sMRM, demonstrating that our method is highly specific. This method has been applied to microdissected brain samples. Initially, we used a songbird model because songbirds have high levels of aromatase and 17β-estradiol in specific brain regions. We were able to simultaneously quantify multiple estrogens in small amounts of brain sample (1-2 mg). We examined seasonal changes of estrogens in the brain and blood. Future work will apply this method to mouse, rat, and human samples and expand the panel of estrogens examined. Our ultra-sensitive assay is essential for small animal models, where estrogen measurement is extremely challenging because of the limited amount of brain tissue. This novel technique will also have wide-ranging applications for basic research and clinical testing, including estrogen measurement in humans with low estrogen levels, such as men, pre-pubertal children, and post-menopausal women.

The incidence of inflammatory bowel diseases (IBDs) has steadily increased in recent decades—a phenomenon that cannot be explained by genetic mutations alone. Other factors, including the composition of the intestinal microbiome, are potentially important contributors to the increased occurrence of this group of diseases. Previous reports have shown a correlation between early-life antibiotic (Abx) treatment and an increased incidence of IBD. In this report, we investigated the effects of early-life Abx treatments on the pathogenicity of CD4+ T cells using an experimental T cell transfer model of IBD. Our results show that CD4+ T cells isolated from adult mice that had been treated with Abx during gestation and in early life induced a faster onset of IBD in Rag1-deficient mice compared with CD4+ T cells of untreated mice. Ex vivo functional analyses of IBD-inducing CD4+ T cells did not show significant differences in their immunologic potential ex vivo, despite their in vivo phenotype. However, genome-wide gene-expression analysis revealed that these cells displayed dysregulated expression of genes associated with cell-cycle regulation, metabolism, and cellular stress. Analysis of Abx-treated CD4+ T cell donors showed systemically elevated levels of the stress hormone corticosterone throughout life compared with untreated donors. The cohousing of Abx-treated mice with untreated mice decreased serum corticosterone, and a consequent transfer of the cells from cohoused mice into Rag1-deficient mice restored the onset and severity of disease to that of untreated animals. Thus, our results suggest that early-life Abx treatment results in a stress response with high levels of corticosterone that influences CD4+ T cell function.

Glucocorticoids (GCs) are circulating adrenal steroid hormones that coordinate physiology, especially the counter-regulatory response to stressors. While systemic GCs are often considered immunosuppressive, GCs in the thymus play a critical role in antigen-specific immunity by ensuring the selection of competent T cells. Elevated thymus-specific GC levels are thought to occur by local synthesis, but the mechanism of such tissue-specific GC production remains unknown. Here, we found metyrapone-blockable GC production in neonatal and adult bone marrow, spleen, and thymus of C57BL/6 mice. This production was primarily via regeneration of adrenal metabolites, rather than de novo synthesis from cholesterol, as we found high levels of gene expression and activity of the GC-regenerating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), but not the GC-synthetic enzyme CYP11B1. Furthermore, incubation with physiological concentrations of GC metabolites (11-dehydrocorticosterone, prednisone) induced 11β-HSD1- and GC receptor-dependent apoptosis (caspase activation) in both T and B cells, showing the functional relevance of local GC regeneration in lymphocyte GC signaling. Local GC production in bone marrow and spleen raises the possibility that GCs play a key role in B cell selection similar to their role in T cell selection. Our results also indicate that local GC production may amplify changes in adrenal GC signaling, rather than buffering against such changes, in the immune system.

Aging is associated with changes in executive functioning and the mesocorticolimbic dopamine system. However, the effects of aging on different forms of behavioral flexibility are not fully characterized. In young (∼5 months) and aged (∼22 months) male Fischer 344 × brown Norway rats, we assessed spatial working memory and different forms of behavioral flexibility using operant tasks: strategy set-shifting (study 1) or probabilistic reversal learning (study 2). We also assessed dopaminergic markers using immunohistochemistry. Compared with young rats, aged rats displayed impairments in working memory. Aged rats also showed nonperseverative impairments in set-shifting, with a subset also showing impairments in initial discrimination learning. In probabilistic reversal learning, aged rats completed more reversals, driven by an increased sensitivity to recent reward and negative feedback. Tyrosine hydroxylase (TH) showed region-specific changes with aging and was correlated with several measures of behavioral flexibility. These data suggest that age-related changes prefrontal cortical function and dopamine synthesis contribute to changes in executive functioning during aging.

Contribution to Special Issue on Fast effects of steroids. 17β-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.

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.

Across invertebrates and vertebrates, steroids are potent signaling molecules that affect nearly every cell in the organism, including cells of the nervous system. Historically, researchers have focused on the genomic (or "nuclear-initiated") effects of steroids. However, all classes of steroids also have rapid non-genomic (or "membrane-initiated") effects, although there is far less basic knowledge of these non-genomic effects. In particular, steroids synthesized in the brain ("neurosteroids") have genomic and non-genomic effects on behavior. Here, we review evidence that estradiol has rapid effects on aggression, an important social behavior, and on intracellular signaling cascades in relevant regions of the brain. In particular, we focus on studies of song sparrows (Melospiza melodia) and Peromyscus mice, in which estradiol has rapid behavioral effects under short photoperiods only. Furthermore, in captive Peromyscus, estrogenic compounds (THF-diols) in corncob bedding profoundly alter the rapid effects of estradiol. Environmental factors in the laboratory, such as photoperiod, diet, and bedding, are critical variables to consider in experimental design. These studies are consistent with the hypothesis that locally-produced steroids are more likely than systemic steroids to act via non-genomic mechanisms. Furthermore, these studies illustrate the dynamic balance between genomic and non-genomic signaling for estradiol, which is likely to be relevant for other steroids, behaviors, and species.

17β-Estradiol ( E 2 ) acts in the brain via genomic and non-genomic mechanisms to influence physiology and behavior. There is seasonal plasticity in the mechanisms by which E 2 activates aggression, and non-genomic mechanisms appear to predominate during the non-breeding season. Male song sparrows ( Melospiza melodia ) display E 2 -dependent territorial aggression throughout the year. Field studies show that song sparrow aggression during a territorial intrusion is similar in the non-breeding and breeding seasons, but aggression after an intrusion ends differs seasonally. Non-breeding males stop behaving aggressively within minutes whereas breeding males remain aggressive for hours. We hypothesize that this seasonal plasticity in the persistence of aggression relates to seasonal plasticity in E 2 signaling. We used a non-invasive route of E 2 administration to compare the non-genomic (within 20 min) effects of E 2 on aggressive behavior in captive non-breeding and breeding season males. E 2 rapidly increased barrier contacts (attacks) during an intrusion by 173% in non-breeding season males only. Given that these effects were observed within 20 min of E 2 administration, they likely occurred via a non-genomic mechanism of action. The present data, taken together with past work, suggest that environmental cues associated with the non-breeding season influence the molecular mechanisms through which E 2 influences behavior. In song sparrows, transient expression of aggressive behavior during the non-breeding season is highly adaptive: it minimizes energy expenditure and maximizes the amount of time available for foraging. In all, these data suggest the intriguing possibility that aggression in the non-breeding season may be activated by a non-genomic E 2 mechanism due to the fitness benefits associated with rapid and transient expression of aggression.

The transition between the breeding and nonbreeding states is often marked by a shift in energy balance. Despite this well-known shift in energy balance, little work has explored seasonal differences in the orexigenic neuropeptides that regulate food intake in wild animals. Here we tested the hypothesis that free-living male song sparrows (Melospiza melodia) show seasonal changes in energetic state, circulating steroids, and both neuropeptide Y (NPY) and orexin (OX) immunoreactivity. Nonbreeding song sparrows had more fat and muscle, as well as a ketone and triglyceride profile suggesting a greater reliance on lipid reserves. Breeding birds had higher plasma androgens; however, nonbreeding birds did maintain androgen precursors in circulation. Nonbreeding birds had more NPY immunoreactivity, specifically in three brain regions: lateral septum, bed nucleus of the stria terminalis, and ventral tegmental area. Furthermore, nonbreeding birds had more OX immunoreactivity in multiple brain regions. Taken together, the data indicate that a natural shift in energy balance is associated with changes in NPY and OX in a region-specific manner.

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, 3β-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.

Glucocorticoids (GCs) are produced by the adrenal glands and circulate in the blood to coordinate organismal physiology. In addition, different tissues may independently regulate their local GC levels via local GC synthesis. Here, we find that in the mouse, endogenous GCs show tissue-specific developmental patterns, rather than mirroring GCs in the blood. Using solid-phase extraction, HPLC, and specific immunoassays, we quantified endogenous steroids and found that in tissues of female and male mice, (1) local GC levels can be much higher than systemic GC levels, (2) local GCs follow age-related patterns different from those of systemic GCs, and (3) local GCs have identities different from those of systemic GCs. For example, whereas corticosterone is the predominant circulating adrenal GC in mice, high concentrations of cortisol were measured in neonatal thymus, bone marrow, and heart. The presence of cortisol was confirmed with liquid chromatography-tandem mass spectrometry. In addition, gene expression of steroidogenic enzymes was detected across multiple tissues, consistent with local GC production. Our results demonstrate that local GCs can differ from GCs in circulating blood. This finding suggests that steroids are widely used as local (paracrine or autocrine) signals, in addition to their classic role as systemic (endocrine) signals. Local GC regulation may even be the norm, rather than the exception, especially during development.

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.

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.

Hypothalamic gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) are vital to reproduction in all vertebrates. These neuropeptides are also present outside of the hypothalamus, but the roles of extra-hypothalamic GnRH and GnIH remain enigmatic and widely underappreciated. We used immunohistochemistry and PCR to examine whether multiple forms of GnRH (chicken GnRH-I (GnRH1), chicken GnRH-II (GnRH2) and lamprey GnRH-III (GnRH4)) and GnIH are present in the hippocampus (Hp) of adult zebra finches (Taeniopygia guttata). Using immunohistochemistry, we provide evidence that GnRH1, GnRH2 and GnRH4 are present in hippocampal cell bodies and/or fibers and that GnIH is present in hippocampal fibers only. There are regional differences in hippocampal GnRH immunoreactivity, and these vary across the different forms of GnRH. There are also sex differences in hippocampal GnRH immunoreactivity, with generally more GnRH1 and GnRH2 in the female Hp. In addition, we used PCR to examine the presence of GnRH1 mRNA and GnIH mRNA in micropunches of Hp. PCR and subsequent product sequencing demonstrated the presence of GnRH1 mRNA and the absence of GnIH mRNA in the Hp, consistent with the pattern of immunohistochemical results. To our knowledge, this is the first study in any species to systematically examine multiple forms of GnRH in the Hp or to quantify sex or regional differences in hippocampal GnRH. Moreover, this is the first demonstration of GnIH in the avian Hp. These data shed light on an important issue: the sites of action and possible functions of GnRH and GnIH outside of the HPG axis.