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Rapid social regulation of 3β-HSD activity in the songbird brain Pradhan, Devaleena S. 2008-08-25

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  i RAPID SOCIAL REGULATION OF 3?-HSD ACTIVITY IN THE SONGBIRD BRAIN  by  DEVALEENA S. PRADHAN BSc., University of British Columbia, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF    MASTER OF SCIENCE    in    The Faculty of Graduate Studies    (Zoology)     THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2008    ? Devaleena S. Pradhan 2008         ii ABSTRACT Rapid increases in plasma androgens are generally associated with short-term aggressive challenges in many breeding vertebrates.  However, some animals such as song sparrows (Melospiza melodia) are aggressive year-round, even during the non-breeding season, when gonads are regressed and systemic testosterone (T) levels are non-detectable.  In contrast, levels of the prohormone dehydroepiandrosterone (DHEA) are elevated year-round in the plasma and brain.  The local conversion of brain DHEA to potent androgens may be critical in regulating non-breeding aggression.  3?-hydroxysteroid dehydrogenase/?4-?5isomerase (3?-HSD) catalyzes DHEA conversion to androstenedione (AE) and the cofactor NAD+ assists in this transformation.  In this thesis, I asked whether brain 3?-HSD activity is regulated by social encounters in seasonally breeding male songbirds.  In Experiment 1, I looked at the long-term seasonal regulation of brain 3?-HSD activity.  3?-HSD activity was highest in the non-breeding season compared to the breeding season and molt.  In Experiment 2, I hypothesized that brain 3?-HSD activity is rapidly regulated by short-term social encounters during the non-breeding season.  A 30 min social challenge increased aggressive behavior.  Without exogenous NAD+, there was ~355% increase in 3?-HSD activity in the caudal telencephalon and ~615% increase in the medial central telencephalon compared to controls (p<0.05).  With exogenous NAD+, there was no effect of social challenge on 3?-HSD activity.  These data suggest that endogenous cofactors play a critical role in the neuroendocrine response to social challenges.  The increase in brain DHEA conversion to AE during social challenges may be a mechanism to rapidly increase local androgens in the non-breeding season, when there are many costs of systemic T.       iii TABLE OF CONTENTS  ABSTRACT........................................................................................................................ ii  TABLE OF CONTENTS.................................................................................................. iii  LIST OF TABLES .............................................................................................................iv  LIST OF FIGURES ............................................................................................................v  ACKNOWLEDGEMENTS ...............................................................................................vi  CO-AUTHORSHIP STATEMENT..................................................................................vii  REGULATION OF STEROIDOGENIC ENZYMES.......................................................1 Introduction.......................................................................................................................1 Long-term Regulation of Steroidogenic Enzymes in the Brain...........................................4 Short-term Regulation of Steroidogenic Enzymes in the Brain...........................................8 Objectives .......................................................................................................................11 References.......................................................................................................................14  SEASONAL AND SOCIAL REGULATION OF 3?-HSD ACTIVITY IN THE SONGBIRD BRAIN..........................................................................................................19 Introduction.....................................................................................................................19 Methods ..........................................................................................................................21 Results ............................................................................................................................27 Discussion.......................................................................................................................30 References.......................................................................................................................44  CONCLUSIONS AND GENERAL DISCUSSION..........................................................48 References.......................................................................................................................50           iv LIST OF TABLES  Table 2.1   Seasonal changes in plasma steroids in free-living adult male song sparrows.....................................................................................................................................36  Table 2.2   Effect of simulated territorial intrusions on brain 3?-HSD activity in free-living non-breeding song sparrows...........................................................................37  Table 2.3   Effect of simulated territorial intrusions on [3H]DHEA conversion to [3H]AE and [3H]AE metabolites in the brain in the absence of exogenously added NAD+..38                                         v LIST OF FIGURES  Figure 1.1 Simplified diagram of sex steroid synthesis....................................................12  Figure 1.2   Bidirectional relationship between physiology and behavior. .....................13  Figure 2.1  Timecourse of 3?-HSD activity with [3H]DHEA as a substrate in non-breeding adult male song sparrow brain..................................................................39  Figure 2.2   Representative HPLC chromatograph illustrating the peak and retention times of [3H]AE..........................................................................................................40  Figure 2.3   Seasonal profiles of baseline 3?-HSD activity in wild male song sparrows. 41  Figure 2.4   Effect of simulated territorial intrusions on brain 3?-HSD activity in free-living non-breeding song sparrows. ..........................................................................42  Figure 2.5   Hypothetical levels of systemic and local androgens in seasonally breeding male songbirds...........................................................................................................43                  vi ACKNOWLEDGEMENTS I offer my gratitude to many people who have supported me throughout this thesis.  First of all, I would like to thank my primary supervisor, Kiran Soma, who has guided me throughout the past three years.  He has always encouraged me to ask questions, express my ideas logically, and be persistent about my goals.  He has taught me all the techniques in the lab, and has always been available to discuss ideas.   I am grateful to my mentors during my undergraduate years, Wayne Goodey, Agnes Lacombe, and the late Jamie Smith, for introducing me to the areas of animal behavior and physiology.   I thank my co-supervisor, Jeff Richards, and the rest of my committee members, Trish Schulte and Liisa Galea, for stimulating questions and insightful comments.   I am deeply indebted to Amy Newman, who recruited me to work long hours in the field, for her invaluable friendship, motivating me when I doubted myself, and cheering for me all along.   A special thank you to Kim Schmidt for being a wonderful colleague and friend.   I am also grateful to Loretta Lau for helping me with assays and for her positive attitude.   Thank you to the rest of the past and current members of the Soma Lab: Thierry Charlier, Amit Shah, Eunice Chin, Roveena Sequeira, and Kelvin Po for interesting discussions and making the lab a wonderful place to work in.   Many thanks to Jennifer Barker and Jonathan Epp for their support and making the office so lively.   I also thank my dear friends, Sharon Shun, Kayla King, Neera Sharma, Nicole Hofs, and my cousin, Shamik Ghosh for always being there.   Finally I would like to thank my family, especially my parents, Ashok and Susmita Pradhan for their love and care, for always believing in me, and for teaching me to never give up.   vii CO-AUTHORSHIP STATEMENT  I, D.S. Pradhan, conducted a major portion of the data analyses for Experiment 1.  For Experiment 2, I assisted with the field work, and conducted all the laboratory work and data analyses myself.  The following co-authors are listed on the manuscript: Amy E. M. Newman, Douglas W. Wacker, B.A. Schlinger, J.C. Wingfield, and Kiran K. Soma.  Experiment 1 was designed and conducted primarily by K. K. Soma, with the assistance of D.W. Wacker, and under the guidance of B.A. Schlinger and J.C. Wingfield.  For Experiment 2, A.E.M. Newman primarily conducted the field work by collecting behavioral data and performing all the dissections.           1 REGULATION OF STEROIDOGENIC ENZYMES Introduction It is well established that a change in the physical or social component of an animal?s environment can influence its endocrine state to regulate behavior.  Steroids regulate behavior in both the long-term and short-term via genomic and non-genomic mechanisms, respectively (1-3).  In a dynamic environment, a stimulus may vary over time, and steroids are modulated to accommodate such changes.  Changes in steroid levels are the result of changes in steroid synthesis and metabolism (4).  Changes in steroidogenic enzymes may be crucial rate-limiting steps that govern behavioral transitions in changing environments (5, 6).    Enzymes involved in the biosynthesis of active steroid hormones are found in several organs such as the gonads, adrenal glands, liver, heart, placenta, and brain (7, 8).  Substantial advances have been made in understanding the expression of genes coding for steroidogenic enzymes, the structure of these enzymes, and the reactions catalyzed by them (8) (see Fig. 1.1 for steroid synthesis pathway).  Steroid-converting enzymes are often studied at a highly reductionist level, focusing on parameters such as substrate affinity and specificity, maximum velocity, optimal temperature and pH, cofactor requirements, subcellular localization, and regulation by end products (9, 10).  This information can, in turn, allow integrative studies of how enzyme activities change as a function of age, endocrine condition, seasonal cues, stress, and social interactions (11, 12).         2 Gonadal Androgens and Behavior  Testosterone (T) is secreted by the testis, and, to a lesser extent, ovaries in many vertebrates and is important for the development of secondary sexual characteristics and activation of behavior.  In male Japanese quail (Coturnix japonica), castration completely eliminates copulatory behaviors, which can be restored by T treatment (13, 14).  Moreover, systemic T levels in male quail rapidly increase when presented with a receptive female or threatened by a conspecific male intruder (15, 16).  This increase in systemic T is mediated by the hypothalamic-pituitary-gonadal (HPG) axis (17).  Hypothalamic gonadotropin-releasing hormone stimulates the anterior pituitary to secrete luteinizing hormone (LH), which stimulates the gonads to secrete T.  In the Leydig cells of the testes, steroidogenic acute regulatory (StAR) protein transports cholesterol to the inner mitochondrial membrane to initite steroid synthesis (18).  However, the time frame in which T-synthesizing enzymes in the gonads are upregulated in the gonads remains unclear.    The traditional model of T action on behavior is that T secreted by the gonads is transported via the blood to distant target organs such as the brain, where T is metabolized to other steroids, such as estradiol (E2) to regulate behavior.  The aromatization model was formulated with two sets of discoveries.  First, E2 treatment can restore copulatory behavior in castrated quail (19).  Second, aromatase inhibitors block the behavioral effects of T (20).  Specifically, aromatase in the anterior hypothalamic-preoptic area (POA) is critical for reproductive behaviors (21).   Additionally, T also exerts its effects via conversion to other biologically active steroids.  For example, 5?-reduction of T leads to the formation of 5?-dihydrotestosterone (5?-DHT or androstane-17?-ol-3-one) and 5?-androstane-3?,17?-diol (5?-3?-diol) (22, 23).  In contrast, the 5?-reduced metabolites of T are considered biologically inactive.  In long-term castrated doves (Streptopelia risoria) there is a negative correlation between 5?-  3 reductase activity and aggression.  Administering E2 in castrated doves can restore aggressive behavior (24).  The reduction of T by 5?-reductase is considered an ?inactivation shunt? (25).  The balance between the concentrations of active and inactive metabolites of T regulates behavior (21).     In summary, conversion of gonadal T to E2 by brain aromatase is important for the regulation of behavior.  However, gonadal T secreted into the systemic circulation can reach many target organs.  In the periphery, T has other functions, such as spermatogenesis and development of accessory reproductive organs and secondary sexual characteristics, which may not be necessary (e.g., outside the breeding season).  Thus systemic increases in T are potentially costly (26, 27).  Neural Androgens and Behavior Local production of androgens within target tissues may be an effective mechanism to reduce the costs of systemic T (26, 27).  Androgens can be locally synthesized in the brain in at least two ways.  First, the circulating prohormone, dehydroepiandrosterone (DHEA), can be converted within the brain to the active and aromatizable androgen, androstenedione (AE).  Second, androgens can be synthesized de novo from cholesterol in the brain (Fig. 1.1).  All of the sex steroidogenic enzymes and StAR are expressed and are active in the brain of vertebrates, including birds (7, 28).  In song sparrows (Melospiza melodia), conversion of DHEA to active sex steroids within the brain may regulate non-breeding aggression (27, 29).  It is not clear whether the effects of DHEA on behavior are attributable to peripheral (e.g. adrenal) or neural sources of DHEA.  To understand how neural androgens regulate behavior, it is important to study the factors regulating steroidogenic enzymes.     4 Regulation of Steroidogenic Enzymes in the Brain Many studies have documented effects of steroids on behavior.  However, note that the reverse is also true: an organism?s behavior can also influence its steroid levels, via changes in the activities of steroidogenic enzymes (17).  For example, the degree of receptiveness of a female to a male dove?s displays can rapidly stimulate her reproductive endocrine system, which has been termed behavioral ?self-feedback? (30).   Additionally, in seasonal breeders, the reproductive system of the female is also influenced by environmental cues, such as slow changes in photoperiod.  Thus a combination of short-term and long-term regulation of steroidogenic enzymes is vital for the necessary feedback to affect behavior (Fig. 1.2).  For the remainder of this review, I will focus on aspects of steroidogenic enzyme regulation. Long-term Regulation of Steroidogenic Enzymes in the Brain Enzymes can be regulated in the long-term by a combination of factors of the external environment and endogenous rhythms.  Predictable changes in the environment, such as onset of seasons, which last for months, can stimulate transcription and translation of genes for steroidogenic enzymes (17).  This may be coupled with an upregulation in synthesis of other machinery required for steroid action, such as steroid receptors.  Steroid hormones released as a product of enzyme activity are secreted into the systemic circulation by endocrine glands.  These steroids travel to distant target cells and bind to intracellular receptors to affect gene transcription, generating a feedback system.  Genes continue to modulate steroidogenic enzyme expression over the course of predictable environmental changes.  This genomic regulation of enzyme expression and activity, which may take hours or days to occur, is termed ?long-term?, and prepares the organism to cope with predictable changes in the future.   5 Long-term effects of physical environment and life history stages on enzymes  Predictable seasonal changes in the physical environment, such as food availability, day length, temperature, and predator density have profound influences on the behavior and hormonal mileu of animals (31, 32).  Many vertebrates go through marked morphological and physiological cycles, including growth of muscle tissue, body fat, and gonads.  Seasonal growth of neural song control nuclei in songbirds is also regulated by changes in hormone levels across seasons (33).  Field studies allow us to study the effects of several integrated parameters, while laboratory studies can enable us to isolate these factors.   Field studies allow the study of seasonal changes in physiology and behavior in a natural environment.  For example, in the temperate zone, song sparrows go through seasonal changes in plasma T levels.  Plasma T peaks during the breeding season, and remains basal during the molt and non-breeding seasons (34).  Despite having low plasma T, song sparrows aggressively defend their territories during the non-breeding season.  Along with seasonal changes in plasma T are seasonal changes in the activities of steroid-converting enzymes in the brain.  Interestingly, brain aromatase activity shows region- and season-specific changes in these birds (35).  In male European starlings (Sturnus vulgaris), four T metabolizing enzymes (aromatase, 17?-hydroxysteroid dehydrogenase, 5?-reductase and 5?-reductase) also show seasonal changes in activity in the diencephalon and telencephalon (36).   Temporal patterns of enzymes also vary as a function of life history stage and mating system during the breeding cycle, but few studies have looked at this phenomenon.  In an Arctic breeding bird, the Lapland longspur (Calcarius lapponicus), activities of T metabolizing enzymes in the brain change within the short breeding season, which was correlated with changes in behavioral priorities (37).  In this species, the peak in systemic T level is very brief, about 1-4 days during the territorial display phase and is low even during the time that males mate-guard females.  Socially monogamous males who provide parental   6 care have different T levels during the breeding season compared to polygamous males who do not show parental care (38).  We can hypothesize that activity of brain steroidogenic enzymes in these animals also show specific patterns.   Animals respond to changes in photoperiod to adapt to seasons.  Day length and temperature can be easily adjusted in the laboratory to mimic photoperiod cues and study effects on enzyme activity and behavior.  For example, photo-inhibition (change from 16 hour long days to 12 hour days) affects aromatase activity, but not 5?-reductase or 17?-HSD in specific brain regions in Siberian hamsters (39).  Japanese quails exposed to long days, but not short days display sexual behavior (40).  In long days, 5?- and 5?-reductase activity are also affected in the pituitary (40).  In another experiment, in quail, exposure to long days increased brain aromatase activity, but not brain 5?- or 5?-reductase, in the mitochondrial and microsomal fractions (41).  Long-term effects of social environment on steroidogenic enzymes Presence of a reproductively mature female can affect hormone levels and behavior of males.  Plasma DHT levels increased in male mallards exposed to females over 10 days, compared to isolated males (42).   Repeated social stimulation over the course of days has been correlated with plasma hormone levels in other bird species (42-44).  Few studies have looked at the long-term effects of social stimulation on activities of T metabolizing enzymes in the brain.  In one study, male Japanese quails were exposed to a different social situation each day: a male was presented with a sexually receptive female for 5 min, or a stuffed female (for 1 min), or two males were allowed to interact aggressively for 1 min (15).  Sexual behavior and frequency of crows were recorded, T levels in plasma were measured before and after social stimulation, and brain and cloacal tissue were collected at the end of the experiment.  The first group of males was stimulated with each social   7 condition once before being sacrificed after 6 days.  The second group was stimulated with each social condition twice after being sacrificed after 9 days.  There was a strong positive correlation between social behavior and active products of in vitro metabolism of T in the anterior hypothalamus, and negatively correlation between behavior and 5?-reduced (inactive) metabolites (15).   Exposure to social interaction via stimulation of visual or tactile stimuli with females can also affect brain aromatase.  Hutchison and Steimer performed a study (45), were they divided sexually mature male doves into 3 groups.  In Group A, visually isolated males could hear, but not see other doves; in Group B, males were paired with a sexually receptive female; and Group C consisted of males who could see and hear Group B pairs.  In Experiment 1, animals were socially stimulated for 2 and in Experiment 2 animals were socially stimulated for 4 days.  Male doves given an opportunity to interact with a female had higher aromatase activity in the POA compared to isolated males or males who were only stimulated visually by a female.  Interestingly, the increase in aromatase activity was specific to brain regions implicated in reproductive behavior.  Moreover, aromatase activity increased irrespective of the duration of social stimulation.  Other T metabolizing enzymes were not affected by sexual interaction (45).   Long-term effects of steroids on steroidogenic enzymes  Several studies have investigated the regulation of brain steroroidogenic enzymes by endocrine factors such as steroids.  Studies involving treatment of gonadectomized animals with steroids will provide insight into the function of neurosteroids.   For example, in female rats, neurally produced progesterone has been implicated in follicular maturation.  In the female rat, estradiol benzoate increases brain 3?-HSD mRNA expression 24 and 44 hr after treatment and 3?-HSD activity after 44 hours (46).   Note that 3?-HSD converts   8 pregnenolone to progesterone (Fig. 1.1).   In another study, in vivo treatment of castrated zebra finches with T for 10 days increases aromatase activity (47) and mRNA in the brain (48).  In an in vitro study of zebra finch brain cultures, long term (72 hour) E2 treatment decreased aromatase activity and expression, but had no effect on 5?- and 5?-reductase (49).  Short-term Regulation of Steroidogenic Enzymes in the Brain Steroidogenic enzymes can be regulated in the short-term by sudden, unpredictable changes in the environment, which may require an organism to rapidly modify its behavior.  Rapid changes in steroidogenic enzyme activity lead to rapid changes in steroid synthesis, resulting in behavioral change.  Short-term Changes in enzyme activity are likely to involve non-genomic mechanisms since the behavioral switch occurs very quickly, within 15 to 30 min      (50).   However, some experiments have looked at shorter times, but there are constraints of the ability to reliably measure steroidogenic enzyme activity in a very short time-scale.  Short-term effects of steroids on steroidogenic enzymes  The regulation of steroid biosynthesis in the gonads and adrenals has been studied in great detail (4, 8).  The discovery that steroid biosynthesis can be rapidly regulated in the brain, however, is fairly recent.  Neurally produced steroids could limit their own synthesis via local negative feedback loops (10) and this might be important for creating short steroid pulses at particular synapses.  In zebra finch brain, within only 10 min, E2 inhibits 3?-HSD activity in a dose-dependent manner.  Moreover, there is a sex difference in this E2 effect, with a greater effect in females than in males (51).  Similar to studies on subcellular localization of brain aromatase (41), we are currently investigating the subcellular localization of 3?-HSD in the zebra finch brain (L. Lau, D. Pradhan, and K. Soma   9 unpublished).  Treatment of specific subcellular compartments with E2 will help elucidate the regulation of brain 3?-HSD.   Short-term effects of stress on steroidogenic enzymes Predators can be a short-term stressor for animals in their natural environment.  Stress activates the hypothalamic-pituitary-adrenal axis (HPA), stimulating the adrenals to release glucocorticoids.  DHEA, released by the adrenal glands, is also regulated by stress in humans and song sparrows (52, 53).  Since DHEA has anti-glucocorticoid effects in the nervous system (54), neural metabolism of DHEA active sex steroids may be beneficial (55).  Interestingly, brain 3?-HSD activity decreases with 10 min restraint stress in zebra finches (56).  In contrast, in quail, brain aromatase activity increases with 15 min restraint (57).  The functional significance of these neurochemical changes in different species remains unclear.  Current studies are examining how brain 3?-HSD and aromatase are affected by 30 min restraint stress across different seasons (D. Pradhan, A. Newman, L. Lau, and K. Soma).  This is will be an excellent system to study both long-term and short-term effects of stress in wild animals.  Thus, scientists working with animals on behavior-endocrine relationships must pay attention to rapid effects of handling stress on steroidogenic enzymes.  Short-term effects of social interactions on enzymes In male-male social encounters, there have been reports of rapid increases in circulating androgens (presumably from the gonads) in many vertebrate species such as songbirds (58, 59), fish (60, 61), amphibians (62) and mammals (63).  The mechanisms by which gonadal T synthesis is rapidly upregulated are not clear, but it could involve StAR or phosphorylation of P450c17 (64, 65).   10 There have been few studies documenting the rapid effects of social interaction on brain steroidogenic enzyme activity.  In one study, intact male Japanese quail were either handled (control), or allowed to see a female or allowed to physically interact with a female for 1, 5, or 15 min.  In the POA, aromatase activity significantly decreased at 5 min after interaction, but not at 1 or 15 min.  Moreover, dopaminergic activity decreased after 1 min and reached baseline levels after 5 min (66).  This suggests that the rapid change in dopamine precedes changes in aromatase.  Moreover, there is evidence that dopamine regulates aromatase activity in the quail brain (67). In another study, in sex changing fish, there was no significant change in brain or gonadal aromatase activity of females within 10, 20, or 30 min after male removal, even though aggressive behavior rapidly increased after male removal (M. Grober, submitted).  However, there was a significant decrease in brain aromatase one day after male removal (68).  These results indicate that the time-scale for rapid changes depend upon the type of stimulus and the particular behavior being measured.  Mechanisms for short-term regulation of brain steroidogenic enzymes Over the past decade, considerable progress has been made in understanding the mechanisms underlying the rapid regulation of brain aromatase.  The aromatase protein has multiple phosphorylation sites, and there is evidence that Ca2+ dependent phosphorylation can rapidly downregulate aromatase activity in brain homogenates (69).  Catecholamines such as dopamine are rapidly released and can also rapidly affect aromatase activity (70).  These mechanisms could be applied to understand the rapid regulation of other steroidogenic enzymes.  For example, similar to aromatase, preliminary studies show that 3?-HSD protein consists of multiple phosphorylation sites (T. Charlier and K. Soma, unpublished).  In the brain, 3?-HSD protein is necessary to synthesize active sex steroids from DHEA.  In   11 summary, sudden changes in the environment could cause rapid release of neurotransmitters (e.g., dopamine, GABA) in the brain, which could act via kinases to increase Ca2+ mediated phosphorylation of steroidoidogenic enzymes.  Increased activity of these enzymes would change levels of steroids to influence behavior.  Objectives The goal of this thesis is to examine the potential for social regulation of brain DHEA metabolism through changes in 3?-HSD activity during the non-breeding season in wild male song sparrows.  Through preliminary studies (Experiment 1), we found that 3?-HSD activity is modulated by seasonally.  Based on these results, I performed Experiment 2, where I investigated whether a short-term, 30 min social challenge can alter 3?-HSD activity.  Objectives of Experiment 2 are to determine: 1.  Regional differences in brain 3?-HSD activity during the non-breeding season. 2.  Effects of short-term social challenges on brain 3?-HSD activity. Findings from this study can serve as a framework for future experiments to look at the behavior-endocrine dynamics over the course of an aggressive challenge.  It can also be applied to better understand the neuroendocrine control of behavior in species that undergo seasonal changes in gonadal hormones, or have low levels of circulating hormones throughout the year, such as tropical species (71).         12 Figure 1.1 Simplified diagram of sex steroid synthesis.                            Steroids: PREG, pregnenolone; PROG, progesterone; AE, androstenedione.  Enzymes: CYP11A1, cytochrome P450 side chain cleavage; CYP17, cytochrome P450 17?-hydroxylase/C17,20 lyase, 3?-HSD, 3?-hydroxysteroid dehydrogenase.  The enzyme 3?-HSD metabolizes DHEA into AE.  AE can then be converted to other steroids: 5?-A, 5?-androstanedione; 5?-A, 5?-androstanedione; E1, estrone; E2, estradiol; T, testosterone, DHT, dihydrotestosterone.                           13 Figure 1.2   Bidirectional relationship between physiology and behavior.                     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Baillien M, Cornil CA, Ball GF & Balthazart J (2004) Sexual performance and stress rapidly alter the preoptic aromatase activity in quail. Soc Neurosci Abstr  58. Harding CF & Follett BK (1979) Hormone changes triggered by aggression in a natural population of blackbirds. Science 203, 918-920.   18 59. Wingfield JC (1985) Short-term changes in plasma levels of hormones during establishment and defense of a breeding territory in male song sparrows, Melospiza melodia. Horm Behav 19, 174-187. 60. Oliveira RF, Hirschenhauser K, Carneiro LA & Canario AV (2002) Social modulation of androgen levels in male teleost fish. Comp Biochem Physiol B Biochem Mol Biol 132, 203-215. 61. Remage-Healey L & Bass AH (2005) Rapid elevations in both steroid hormones and vocal signaling during playback challenge: A field experiment in gulf toadfish. Horm Behav 47, 297-305. 62. Greenberg N & Crews D (1990) Endocrine and behavioral responses to aggression and social dominance in the green anole lizard, Anolis carolinensis. Gen Comp Endocrinol 77, 246-255. 63. Cavigelli SA & Pereira ME (2000) Mating season aggression and fecal testosterone levels in male ring-tailed lemurs (Lemur catta). Horm Behav 37, 246-255. 64. Miller WL (2005) Minireview: Regulation of steroidogenesis by electron transfer. Endocrinology 146, 2544-2550. 65. Tee MK, Dong Q & Miller WL (2008) Pathways leading to phosphorylation of p450c17 and to the posttranslational regulation of androgen biosynthesis. Endocrinology 149, 2667-2677. 66. Cornil CA, et al (2005) Rapid decreases in preoptic aromatase activity and brain monoamine concentrations after engaging in male sexual behavior. Endocrinology 146, 3809-3820. 67. Balthazart J, Baillien M & Ball GF (2002) Interactions between aromatase (estrogen synthase) and dopamine in the control of male sexual behavior in quail. 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Gen Comp Endocrinol 117, 20-33.      19 SEASONAL AND SOCIAL REGULATION OF 3?-HSD ACTIVITY IN THE SONGBIRD BRAIN1 Introduction Pioneering studies investigating inter-male aggression in song sparrows, Melospiza melodia, have provided support for the ?challenge hypothesis?, which predicts an increase in plasma testosterone (T) levels during periods of social instability (1).  The challenge hypothesis applies to many vertebrates, including humans (2).  In song sparrows, plasma T level is high during the breeding season, compared to other times of the year, and transiently increases further during social challenges (1).  However, the link between aggression and androgens is less clear during the non-breeding season.  Song sparrows and several other species maintain high levels of territorial aggression outside the breeding season despite low levels of circulating T (3-7).  One hypothesis is that local androgens within the brain regulate non-breeding aggression (8).  This may be a mechanism of local, rather than systemic, steroid signaling, in order to circumvent the ?costs? of high systemic T during the non-breeding season (8-10). Song sparrows are an excellent species to investigate the mechanisms that regulate aggressive behavior during the non-breeding season.  They maintain year-round territories in the Pacific Northwest and respond similarly vigorously to simulated territorial intrusions (STI) during the breeding and non-breeding seasons (4).  Intriguingly, during the non-breeding season, their gonads are completely regressed, they have low systemic T levels, and castration does not affect aggression (4).  Importantly, aggressive challenges do not increase systemic androgens in the molt and non-breeding season.                                                   1 A version of this chapter will be submitted for publication.  Pradhan, D.S., A.E.M. Newman, D.W. Wacker, B.A. Schlinger, J.C. Wingfield, K.K. Soma, 2008.  Seasonal and social regulation of 3?-HSD activity in the songbird brain.    20 Systemic levels of the prohormone dehydroepiandrosterone (DHEA) are high in non-breeding song sparrows (11).  DHEA implants during the non-breeding season increase territorial singing and the size of a song control nucleus (HVC) by almost 50% (12).  DHEA has no known intracellular receptor (13, 14) and is considered an inactive neurosteroid that must be converted to active sex steroids such as T and estradiol (E2) (15).  Reducing estrogen synthesis in wintering song sparrows significantly reduces aggressive behavior and HVC size (16).  These data suggest that DHEA is metabolized to active sex steroids such as E2 locally in the brain to regulate non-breeding aggression. Steroidogenic enzymes in the brain may be a link between the environment and behavior.  The songbird brain expresses all the enzymes necessary for the downstream metabolism of DHEA to active sex steroids (17, 18).  3?-hydroxysteroid dehydrogenase/?4-?5 isomerase (3?-HSD) catalyzes the conversion of DHEA to androstenedione (AE), an active and aromatizable androgen (19), and uses NAD+ as a cofactor.  Thus conversion of brain DHEA to AE via brain 3?-HSD may be critical for regulating territorial behavior during the non-breeding season.   In Experiment 1, we studied the long-term seasonal regulation of brain 3?-HSD activity by measuring the conversion of DHEA to AE during the breeding, molt, and non-breeding seasons.  In Experiment 2, we examined the short-term social regulation of brain 3?-HSD activity by social encounters during the non-breeding season.         21 Methods Experiment 1: Seasonal regulation of brain 3?-HSD activity Field Protocol. Free-living wild male song sparrows (n=18 total) were captured near Seattle, WA, with 3.58 ? 0.41 min conspecific song playback during the breeding (May 4-5), molt (Aug 30-31), and non-breeding (November 14-16) seasons of 2002.  Experimental protocols complied with University of Washington Animal Care Committee.  Within 3.08 ? 0.37 min of capture, the subject was rapidly sacrificed, and trunk blood was collected.  While in the field, blood was kept on wet ice, and the brain was quickly dissected according to (20) and stored on dry ice.  Upon returning to the laboratory, blood was centrifuged and plasma samples were stored at -20?C, and brain tissues were stored at -80?C until assayed.  Hormone assays.  Briefly, DHEA and T were extracted from plasma using dichloromethane and separated using diatomaceous earth/glycol columns and measured via radioimmunoassay as described in (11).  All the samples were run in a single assay, and the intra-assay variability was 3.25% for DHEA and 7.1% for T.  The antibodies used were: DHEA (Endocrine Sciences, Calabasas, CA D7-421) and T (Wien Laboratories, Saccasunna, NJ, T3003).  3?-HSD activity assay with exogenous NAD+.   We measured the in vitro conversion of [3H]DHEA to [3H]AE and [3H]5?-androstanedione (5?-A).  The assay procedure was followed from Soma et al (21), with few modifications.  First, a timecourse was performed to determine a suitable incubation time.  We used whole brain homogenates pooled from 2-3 animals and compared 3?-HSD activity in the breeding and non-breeding seasons.  Incubation times ranged from 60 to 300 min.  For samples from both seasons, AE was the major product formed, and AE formation increased linearly from 120 to 240 min (data not   22 presented) and we chose 180 min as the appropriate incubation duration.  Next, we measured 3?-HSD activity in different brain regions and processed a maximum of three regions at a time.  Each region was homogenized in 200 ?L sucrose phosphate (SPO4) buffer, and 180 ?L was incubated at 41?C with 200 nM [1,2,6,7-3H]DHEA (specific activity = 74 Ci/mmol), 1.5 mg AE cold-trap, 20 ?L NAD+ were added.  Tritiated AE was recrystalized to constant specific activity, which confirmed identity of the metabolites.  Trilostane, a 3?-HSD inhibitor, decreased [3H]AE formation by 98.5%, while fadrozole, an aromatase inhibitor, specifically and completely abolished [3H]estrone (E1) + [3H]E2 formation.  Experiment 2: Social regulation of brain 3?-HSD activity Field Protocol.  Free-living song sparrows (n=18 total) were studied during the non-breeding season, November 23 ? December 8, 2005 near Vancouver, BC (49? 12?N, 123? 01?W).  Experimental protocols complied with University of British Columbia Animal Care and the Canadian Council for Animal Care permits.  Individual territories of resident song sparrows were mapped at least 2 days prior to the study.  Prior to each trial, a mist net was installed, furled and camouflaged near the ground.  The resident was then exposed to a simulated territorial intrusion (STI) for 30 min.  The STI consisted of a live caged conspecific decoy and conspecific song playback placed in the approximate center of the territory.  Control animals were exposed to similar conditions; however, the cage was empty and no playback was used.  Territorial singing and defense displays made by the resident bird were recorded for 30 min.  Immediately after, the mist net was unfurled, and the resident was captured using conspecific playback.  STI stimulated animals were captured within 1.64 ? 0.53 min and controls were captured within 1.36 ? 0.6 min (t=0.332, p=0.744).  Blood samples were then taken from the brachial and jugular veins   23 (results to be presented separately).  Control subjects were sacrificed 4.19 ? 0.24 min after capture and STI subjects were sacrificed 4.13 ? 0.27 min after capture (t=0.184, p=0.857).   In the field, the brain was rapidly dissected into left and right regions and immediately frozen on dry ice.  All the dissection procedures were same as (20).  The following tissues were collected 1) rostral, medial central, lateral central, caudal, dorsal (contains HVC), and ventromedial telencephalon (contains taenial amygdala). 2) caudomedial neostriatum (NCM) (\ song perception); 3) hippocampus (learning and memory); 4) rostral and caudal diencephalon (motivated behaviors); 5) cerebellum.  Cerebellum and hippocampus served as control regions.  Tissue was stored at -80?C until assayed.  Experiment 2A: 3?-HSD activity assay with exogenous NAD+.  We determined 3?-HSD activity by measuring the in vitro conversion of [3H]DHEA to [3H]AE, and added NAD+ exogenously as a cofactor.  This facilitated the production and accumulation of [3H]AE.  In these experiments, we were also able to measure low quantities of [3H]5?-A.  All the samples were processed as in (21, 22).  Briefly, first, using pooled samples, we performed a time course where we compared 3?-HSD activity in brain homogenates and supernatants (homogenates centrifuged at 1000 x g at 4?C for 30 min) of non-breeding animals, using incubation times from 10 to 180 min (data not shown).  Activity in supernatants was 6x the homogenates and was non-linear by 10 min.  Hence we chose supernatants for the subsequent assays and examined earlier time-points in the next assay.  We measured 3?-HSD activity from 2.5 to 60 min; [3H]AE increased linearly from 2.5 to 15 min (Fig. 2.1A) and hence we chose 5 min as the appropriate duration for reactions with NAD+.   24 Brain tissue from either the right or left side (randomized) of each region was assayed for 3?-HSD activity. Brain tissue was homogenized on ice, (2 second bursts/pulses, 3 to 6 times) in 300 ?L ice-cold sugar-phosphate buffer, and centrifuged at 1000 g at 4?C for 30 min.  180 ?L of the supernatant was incubated with 200 nM [1,2,6,7-3H]DHEA (specific activity 74.5 Ci/mmol) and 20 ?L NAD+ (final concentration, 1 mM) at 41?C, with shaking (speed 90 RPM).  After 5 min, the reaction was terminated by snap-freezing in methanol/dry ice.  Samples were then wrapped in parafilm and stored at -20?C.  To determine procedural losses, tubes containing a known amount of repurified  [1,2,6,7-3H]AE were processed in parallel.  Control tubes contained everything but tissue.  Steroids were extracted by adding 3 mL diethyl ether to each sample and each sample was vortexed for 30 sec and centrifuged at 2100 RPM, at 4?C for 5 min.  After snap-freezing in methanol:dry ice bath, the supernatant was decanted into a separate tube.  This procedure was repeated two times, after which the samples were dried down under nitrogen. The dried residues obtained after ether extraction were resuspended in dichloromethane:methanol (1:1), and radioinert DHEA, AE, and 5?-A were added as markers.  Samples were spotted onto silica gel plates, and run in chloroform:ethyl acetate (4:1) for 18 min (2 times).  Steroids were visualized under UV light after spraying with primulin.  The appropriate bands were scraped from the plates, tritiated steroids were eluted from the silica with 900 ?L methanol:water (8:1), and 200 ?L aliquots were counted in a scintillation counter (BeckmanCoulter LS 6500).  The dpm were corrected for background values and recovery (average = 80%), and all data are presented as fmole per mg protein per min.  Lastly, to confirm the identity of products formed, the eluates from TLC were injected through HPLC and the retention time for [3H]AE was noted (Fig. 2.2).    25 Experiment 2B: 3?-HSD activity assay without exogenous NAD+.  In some telencephalic regions, we did not add exogenous NAD+.  In these preparations, the forward reaction of [3H]DHEA conversion to [3H]AE by 3?-HSD was driven by endogenous NAD+.  This assay allowed us to measure the production of [3H]AE, and also allowed the conversion of the formed [3H]AE to [3H]5?-A, [3H]5?-A, [3H]E1, and [3H]E2.  Thus we were also able to assess activities of 5?-reductase, 5?-reductase, and aromatase.  In a timecourse, [3H]AE increased linearly from 45 to 120 min of incubation and we chose 90 min as the appropriate duration (Fig. 2.1B).   Tissues from the rostral and caudal telencephalon were homogenized in 300 ?L buffer, and medial central telencephalon was homogenized in 500 ?L buffer and centrifuged.  The medial central telencephalon was split between the two assays, with and without NAD+.  200 ?L of the supernatant from each region was then incubated with 200 nM [3H]DHEA for 90 min with shaking, after which the reaction was terminated.   Following the extraction using diethyl ether (2 times), androgens and estrogens were separated first using phenolic partitioning as described in (23).  One microliter of CCl4 and 1 ?L NaOH (1N) were added to the dried extracts.  Samples were then centrifuged at 2100 RPM, 4?C for 5 min, with no brake.  Following this, 700 ?L of the estrogen containing NaOH top layer was collected and transferred to a new tube.  An equal volume of CCl4 was added to the new tube, and centrifuged as above, before 400 ?L of NaOH layer was collected and transferred to a new tube.  An equal volume of dH2O and 2 mL ethyl acetate was added to the NaOH.  Samples were vortexed for 20 sec and the NaOH was transferred to a new tube with a Pasteur pipette.  This procedure was repeated twice more to ensure the complete transfer of estrogens from NaOH to ethyl acetate.  The ethyl acetate tubes corresponding to the sample sample were combined and dried down.  One milliliter dH2O was added to the   26 tubes containing the remaining androgens and centrifuged as above.  One and a half milliliter of the CCl4 containing the androgen layer was extracted.  All of these procedures were conducted at 4?C and the samples were then dried down with nitrogen at 40?C.  Both estrogens and androgens were then resuspended in dichloromethane:methanol (1:1) and radioinert steroids of interest.  Resulting samples were spotted onto TLC plates for separation.  Estrogens were run twice for 23 min in 3:1 ether:hexane tanks, and plates were then kept under iodine fumes for visualizing [3H]E1 and [3H]E2, which were then scraped off the plates.  Androgens were run twice for 18 min in 4:1 chroloroform:ethyl acetate tanks.  [3H]AE was the major androgen metabolite, [3H]5?-A was the minor metabolite, and [3H]5?-A was non-detectable.  Tritiated silica was then resuspended with 800 ?L methanol and 100 ?L dH2O, vortexed for 30 sec and centrifuged at 2800 RPM, 4?C for 10 min.  Four hundred microlitres of the supernatant was counted in 5 mL scintillation fluid for 2 min per sample.  Recoveries for estrogens were 14 % for estrogens and 85 % for androgens.  Statistics.  All data are expressed as mean ? SEM. Data were transformed where appropriate, and analyzed using SPSS 11.0 or Prism 4.0 for Mac OS X.  For Experiment 1, we first performed the one-way analysis of variance (ANOVA) to compare seasonal changes in plasma hormones and body condition, followed by Fisher?s protected least significant difference (LSD) test as a post hoc.  For each brain region, differences among the three seasons were analyzed using one-way ANOVA.  When the ANOVA was significant, we performed a Fisher?s LSD test to determine differences between the seasons.  For Experiment 2, to investigate the differences in 3?-HSD activity between control and STI stimulated samples, each brain region was analyzed using separate t tests.  For samples without exogenously added NAD+, we performed correlation analysis between aggressive behavior   27 and 3?-HSD activity.  Errors for percentage increase in 3?-HSD activity were calculated according to (24).  Values of p < 0.05 (two-tailed) were considered significant.  Results Experiment 1: Seasonal regulation of brain 3?-HSD activity In this experiment, we investigated the seasonal regulation of brain 3?-HSD activity in a seasonally breeding songbird.  Male song sparrows were captured during the breeding, molt, and non-breeding seasons (n=18 total).  First, we examined the plasma levels of DHEA and T in each season (Table 2.1).  Overall, there were significant effects of season on plasma DHEA and T (DHEA: F2, 18=3.205, p=0.048; T: F2, 18=24.063, p<0.0001).  Plasma DHEA levels were highest in the breeding season, reduced during molt, and increased again during the non-breeding season.  Plasma T levels were highest in the breeding season and basal during the molt and non-breeding season.  Testis volume and the androgen-dependent cloacal protuberance were regressed outside of the breeding season (Table 2.1).  Next, we measured 3?-HSD activity in brains collected in different seasons by quantifying the conversion of [3H]DHEA to [3H]AE and [3H]5?-A in brain homogenates (Fig. 2.3).  3?-HSD activity changed seasonally in the central medial telencephalon (F2, 14=6.098, p=0.012), being lowest in the breeding season compared to molt (Fisher LSD, p=0.008) and non-breeding season (p=0.012).  3?-HSD activity also significantly differed among the three seasons in the caudal telencephalon (F2, 15=3.841 p=0.045), and was higher in the non-breeding season compared to molt (Fisher LSD, p=0.033) and breeding season (Fisher LSD, p=0.027).   Seasonal changes in 3?-HSD activity were also observed in the ventromedial telencephalon (F2, 15=3.857, p=0.045) and caudal diencephalon (F2, 15= 4.266, p=0.034), and was highest in the non-breeding season for both (p<0.05).  In the rostral   28 diencephalon, no significant change in 3?-HSD activity was observed (F2, 14=1.037, p=0.380).  Taken together, these data suggest that brain 3?-HSD activity is preferentially upregulated during the non-breeding season.    Experiment 2: Social Regulation of brain 3?-HSD activity The results from Experiment 1 raised the hypothesis that high DHEA metabolism during the non-breeding season may be a mechanism to locally elevate androgen levels in the brain to mediate aggression.  We further examined this hypothesis by testing the prediction that aggressive interactions would increase 3?-HSD activity.  During the non-breeding season of 2005, we exposed free-living male song sparrows to a simulated territorial intrusion (STI) for 30 min.  Stimulated animals responded strongly, by singing, wing-waving, and flying close to the decoy (25).  The response latency of STI stimulated birds was 1.23 ? 0.47 min and the song latency was 2.78 ? 2.37 min, indicating the rapid effect of STI on territorial behavior.  Experiment 2A: 3?-HSD activity assay with exogenous NAD+ Similar to Pradhan et al (22), we measured 3?-HSD activity in supernatants prepared from brain homogenates, because 3?-HSD activity in supernatants was 6x higher than in homogenates (see methods).  In Experiment 2A, we added 1 mM NAD+ exogenously, which maximized [3H]AE formation and inhibited [3H]AE conversion by aromatase (21).  Under these in vitro assay conditions, there was no significant effect of STI on 3?-HSD activity in any brain region, except for the cerebellum, in which STI decreased 3?-HSD activity (Table 2.2).  Note that 3?-HSD activity is ~8x higher in the rostral diencephalon than caudal diencephalon.  Similar to previous studies, 5?-A was a minor product formed (21, 22).  To assess 5?-reductase activity, we expressed [3H]5?-A as a percentage of total 3?-HSD   29 metabolites ([3H]AE + [3H]5?-A) (Table 2.1).  STI significantly increased 5?-reductase activity in the dorsal telencephalon (p=0.036) and significantly decreased 5?-reductase activity in the rostral diencephalon (p=0.022).     Experiment 2B: 3?-HSD activity assay without exogenous NAD+ Next, we focused on telencephalic regions and did not add NAD+ exogenously.  These assay conditions permitted us to measure the activities of other steroidogenic enzymes that convert AE, such as aromatase, 5?- and 5?-reductase (21).  In the central medial telencephalon, STI significantly increased 3?-HSD activity 615 ? 118 % (t15=2.998, p=0.009; Fig. 2.4).  Similarly, in the caudal telencephalon, STI significantly increased 3?-HSD activity 355 ? 18 % (t13=3.721, p=0.003; Fig. 2.2).  Note that both these regions have androgen and estrogen receptors (26) and also contain parts of the limbic system (central medial telencephalon contains the septum).  There are also neuronal tracks that connect to the diencephalon from these regions.  In contrast, there was no effect of STI on 3?-HSD activity in the rostral telencephalon, which has not been implicated in the control of aggression (control = 0.97 ? 0.72 fmole/mg protein/min; STI = 0.88 ? 0.34 fmole/mg protein/min; t16=0.119, p=0.907).  Further, 3?-HSD activity was positively correlated with the time the subject spent within 1 m of the decoy (central medial telencephalon: Pearson correlation, r=0.56, p=0.0204; caudal telencephalon, Pearson correlation, r=0.73, p= 0.0021).  More time spent in close proximity to the intruder reflects a more aggressive response (27).   Lastly, we expressed each downstream product of 3[H]DHEA as a percentage of total metabolites, to calculate the indices of 5?-reductase, 5?-reductase, and aromatase activities (21, 22).  Percentage of 3[H]AE was higher in STI stimulated subjects in the central medial   30 telencephalon (p=0.038) and the caudal telencephalon (p=0.043) (Table 2.3).  We expressed 3[H]E1 + 3[H]E2 as a percentage of total 3[H]DHEA products to evaluate aromatase activity.  Surprisingly, aromatase activity significantly decreased in the central medial telencephalon (p=0.018) and caudal telencephalon (p=0.008).  Similarly, we expressed 3[H]5?-A as a percentage of total 3[H]DHEA metabolites to evaluate 5?-reductase activity.  There was no effect of STI on 5?-reductase activity on any region and no 5?-A was produced in any region, indicating very low 5?-reductase activity.  Taken together, these results indicate that during the STI, there was a net increase in active androgens.  Discussion Taken together, the results from this study suggest a mechanism for regulation of aggression during the non-breeding in free-living song sparrows, when plasma T levels are low.  First, wintering song sparrows have higher systemic DHEA compared to T.  Second, elevated brain 3?-HSD activity during the non-breeding season suggests increased conversion of DHEA to active sex steroids.  Third, 3?-HSD activity is rapidly (within 30 min) regulated by social challenges during the non-breeding season, consistent with non-genomic control mechanisms (28), and this was specific to behaviorally relevant forebrain regions.  The conversion of DHEA to active androgens in local regions of the brain appears critical during the non-breeding season, indicating a shift from systemic to local signaling during the winter (8).  Lastly, we also show that caution should be exercised in interpreting data from studies in which saturating concentrations of cofactor are exogenously added to maximize product formation.      31 Seasonal regulation of brain androgen synthesis Compared to the breeding season and molt, 3?-HSD activity was highest during the non-breeding season in several behaviorally relevant brain regions (Fig 2.3).  3?-HSD activity is a critical first step in converting the prohormone DHEA to active androgens and estrogens (29).  In song sparrows, aromatase activity also changes seasonally (30).  A combination of elevated 3?-HSD and aromatase activity can increase E2 levels locally, since E2 regulates aggression during the non-breeding season (16).     Other hormones, such as the pineal hormone melatonin, that undergo seasonal changes may also affect non-breeding aggression.  For example, chronic melatonin treatment (mimicking short-days) increases aggression in Siberian hamsters (31).  Our preliminary evidence suggests that long-term (10 days) melatonin treatment to song sparrows increases 3?-HSD activity, perhaps via gene transcription (K. Soma, unpublished).  There are seasonal changes in steroidogenic enzyme activities in other species as well.  For example, in Lapland longspurs, brain aromatase activity changes across the breeding season (32).  Interestingly, these changes are most pronounced in the caudal telencephalon, suggesting that this region is particularly sensitive to seasonal and behavioral regulation (32) (see Fig. 2.4).  In male European starlings, four T converting enzymes (aromatase, 17?-hydroxysteroid dehydrogenase, 5?-reductase and 5?-reductase) also show seasonal changes in activity in the diencephalon and telencephalon (33).  Additionally, studies in goldfish have revealed that there is a dramatic increase in aromatase activity in the anterior hypothalamus/preoptic area during gonadal maturation and the spawning season (34).       32 Social regulation of brain androgen synthesis Rapid, non-genomic mechanisms of steroid action in the brain have been implicated in several recent studies.  Here, we stimulated wild songbirds to a STI for 30 min and measured 3?-HSD activity in brain regions that contain parts of the social behavior network and regulate motivated behaviors such as aggression and reproduction (35, 36).  In STI stimulated subjects, 3?-HSD activity rapidly increased ~615% in the medial central telencephalon and ~355% in the caudal telencephalon. These rapid changes in 3?-HSD activity are much greater than previously reported rapid changes in aromatase activity (37-39).  Rapid increases in aromatase activity lead to increases in E2; an increase in 3?-HSD activity early in the steroidogenic pathway may be a mechanism to rapidly and transiently increase E2 at important synapses (38), which would then regulate behavior.  Relevant to our present findings, studies have shown that E2 rapidly increases aggression in mice housed in short days, but not long days (40).   However, contrary to our expectation that aromatase activity will increase as a result of aggressive encounters, surprisingly, our results show that the opposite is true (Table 2.3).  The data indicate that 30 min of STI causes a decrease in estrogens and a build-up of AE, which may signal via androgen receptors to increase aggression.  One explanation is that, social interactions are dynamic, and so is the steroid environment within the brain as a result of steroidogenic enzyme activity.  Over the 30 min of stimulation, the intensity of aggressive behavior can vary over time, depending upon the perception of threat.  Once the intruder leaves the territory, and the threat subsides, it might be useful to rapidly terminate active steroids and upregulate inactivating enzymes.   Rapid release and re-uptake of steroids at specific synapses could be mechanisms to transiently increase local steroid levels (28).  This hypothesis could be tested by measuring 3?-HSD and aromatase activities in shorter time intervals, and also post-STI.  All these   33 studies could also be compared with parallel studies in the breeding season.  Previous studies have found that after the removal of the decoy, wintering song sparrows are less persistent during the post-STI period compared to the breeding season.  When treated with T, non-breeding song sparrows maintain aggression post-STI (4).  Thus, we predict that in the spring, post-STI aggressive behavior will be coupled with increased 3?-HSD activity, while in the winter, decreased aggressive behavior post-STI will be coupled with decreased 3?-HSD activity.  Possible mechanisms for short-term regulation of brain 3?-HSD There are several possible mechanisms by which 3?-HSD activity may be modulated rapidly.  In this study, we found that 3?-HSD was regulated only when no exogenous NAD+ was added.  This suggests that 3?-HSD may be regulated via bioavailability of endogenous NAD+, and this hypothesis must be tested in future studies.  In most in vitro enzyme activity assays, cofactors are exogenously added to maximize the particular reaction.  In vivo, cells maintain ~ 1 mM NAD+ concentration compared to ~1 nM steroid concentrations (41).  Cellular redox states are maintained by NAD+/NADH concentration gradients, which in turn determine the direction of the steroid conversion (41).  Thus cofactors may play a critical role in the rapid regulation of 3?-HSD activity.  However, regulation of intermediate steps of metabolism and regeneration of cofactors are poorly understood and in need of further investigation (42).    In this experiment, centrifugation of homogenized brain tissue at 1000 g generated supernatants enriched with mitochondria, synaptosomes, and microsomes (43).  Since mitochondria recycle NAD+, and contain NAD+, we may have measured 3?-HSD activity specifically in these organelles.  Previous studies have reported that 3?-HSD activity is low   34 or non-detectable in microsomal fractions without exogenous NAD+ (44, 45) (L. Lau, D. Pradhan, K. Soma, unpublished results).  Addition of exogenous NAD+ however, maximized mitochondrial and microsomal 3?-HSD activity, causing a ceiling effect, which may have diluted the effects of regulation of  mitochondrial 3?-HSD activity.  Since synaptosomes also contain high levels of aromatase (46, 47), this assay provides evidence for the potential for rapid regulation of androgen synthesis at a very local level.   3?-HSD may also be regulated by mechanisms similar to aromatase.  Like aromatase, 3?-HSD amino acid sequence contains several possible phosphorylation sites (T. Charlier and K. Soma, unpublished), and studies from quail indicate that rapid changes in aromatase occur via Ca2+ mediated phosphorylation by protein kinases (38).  In addition, aromatase can be rapidly (within 5 min) modulated by specific glutamate receptor agonists (kainate and AMPA) (48).  Thus, the social environment could rapidly modulate endogenous neurotransmitters such as dopamine, GABA, and various neuropeptides, increasing Ca2+ mediated phosphorylation of 3?-HSD via kinases.   Conclusions Together, our findings suggest that 3?-HSD activity is high during the non-breeding season in male song sparrows, and is increased even further by short-term conspecific social challenges.  Local androgen levels in the brain may be under an opposite regulatory pattern compared to systemic androgen levels (Fig. 2.5).  We propose that the increased baseline activity of brain 3?-HSD might be a mechanism to prime the steroidogenic response, so that local levels of androgens can be rapidly increased during periods of social instability. Owing to the numerous ?costs? of high levels of systemic T (49), there is considerable evidence that alternate mechanisms are necessary to regulate aggressive behavior in several vertebrates.  Regulation may involve rapid physiological changes localized within the brain   35 itself, and include multiple molecular and biochemical machinery such as cofactors, receptors, substrates, and enzymes expression and activity.  Moreover, both long- and short-term endocrine control mechanisms are important, depending on behavioral ecology, life history strategies, and seasonal cues.  These factors would collectively ensure the necessary hormonal feedback for maintaining or reducing aggressive behavior locally, without involving peripheral androgen levels.                       36 Table 2.1   Seasonal changes in plasma steroids in free-living adult male song sparrows.          Breeding   Molt  Non-breeding  F   p Plasma T (ng/ml)  4.82 ? 1.64a  0.28 ? 0.06b  0.20 ? 0.03b  24.063  <0.0001 Plasma DHEA (ng/ml)  0.48 ? 0.06a  0.31 ? 0.02b  0.42 ? 0.04ab  3.205  0.048 Testis volume (mm3)  2913.31 ? 350.48a  73.28 ? 58.58b  6.91 ? 0.63c  65.403  <0.0001 Cloacal protuberance (mm)  7.48 ? 0.29a  5.07 ? 0.37b  3.66 ? 0.22c  40.643  <0.0001   Note: Within rows, values with different superscripts differ significantly from each other. n=6 per group. Testosterone (T), plasma dehydroepiandrosterone (DHEA), testes volume, and cloacal protuberance length.                                 37  Table 2.2   Effect of simulated territorial intrusions on brain 3?-HSD activity in free-living non-breeding song sparrows.       Total metabolites fmole AE + 5?-A / mg protein / min             Control                              STI                       p 5?-A / Total %      Control                     STI                  p Rostral telencephalon  156.66 ? 30.63 (9)  127.35 ? 18.52 (9)  0.235  1.06 ? 0.69   1.78 ? 1.38  0.649 Central medial telencephalon  107.61 ? 27.86 (9)  119.84 ? 23.52 (9)  0.757  2.53 ? 0.98   3.38 ? 1.58  0.656 Central lateral telencephalon  20.14 ? 2.48 (9)  31.49 ? 8.00 (9)  0.101  7.96 ? 4.18   6.29 ? 1.56  0.713 Caudal telencephalon  44.53 ? 7.02 (7)  65.66 ? 7.23 (7)  0.519  0.33 ? 0.31   1.30 ? 0.36  0.067 Dorsal telencephalon  19.71 ? 5.96 (9)  21.6 ? 5.43 (8)  0.232  20.48 ? 8.65  53.14 ? 11.49  0.036* Ventromedial Telencephalon  42.04 ? 11.55 (9)  37.37 ? 21.91 (9)  0.188  32.00 ? 11.30  10.27 ? 6.8  0.158 NCM  70.80 ? 11.35 (7)  60.03 ? 12.88 (9)  0.606  2.31 ? 2.00   5.19 ? 2.3  0.377 Hippocampus  22.58 ? 5.81 (8)  11.22 ? 4.94 (9)  0.082  54.18 ? 15.51  37.18 ? 12.25  0.398              Rostral diencephalon  1175.19 ? 537.20 (9)  600.75 ? 2226.25 (9)  0.339    1.30 ? 0.51  0.18 ? 0.07  0.022* Caudal diencephalon  126.52 ? 27.01 (8)  184.42 ? 75.57 (8)  0.483    3.34 ? 1.78  1.46 ? 0.82  0.351 Cerebellum  164.92 ? 45.42 (9)  70.65 ? 16.66 (9)  0.002**    0.67 ? 0.37  11.74 ? 11.04  0.331   Note: [3H]DHEA was added as a substrate and NAD+ was exogenously added as a cofactor. The sum of the metabolites is an index of 3?-HSD activity and 5?-A % is an index of 5?-reductase activity. Numbers in parenthesis denote sample sizes.  Values in boldface indicate a significant difference between groups.  *p<0.05, **p<0.01.                        38 Table 2.3   Effect of simulated territorial intrusions on [3H]DHEA conversion to [3H]AE and [3H]AE metabolites in the brain in the absence of exogenously added NAD+.                  Control                        STI                          p Rostral telencephalon            AE/Total (%)  16.6 ? 10.98  62.84 ? 15.88  0.077      5?-A/Total (%)  12.05 ? 10.93  6.45 ? 3.48  0.889      E1+E2/Total (%)  60.23 ? 15.93  30.71 ? 15.22  0.189 Central medial telencephalon            AE/Total (%)  48.59 ? 15.56  89.23 ? 8.55  0.038*      5?-A/Total (%)  14.16 ? 8.45  0.68 ? 0.68  0.108      E1+E2/Total (%)  37.24 ? 13.12  10.08 ? 8.62  0.018* Caudal telencephalon            AE/Total (%)  92.76 ? 1.45  96.11 ? 0.56  0.043*      5?-A/Total (%)  1.35 ? 0.76  2.32 ? 0.54  0.130      E1+E2/Total (%)  5.90 ? 1.82  1.37 ? 0.40  0.008**   Note: Formed [3H]AE was converted to 5?-A, E1 and E2.  No 5?-A was produced.   p values are from t tests. Boldface indidcates p ? 0.05.                        39 Figure 2.1  Timecourse of 3?-HSD activity with [3H]DHEA as a substrate in non-breeding adult male song sparrow brain. A.                    B.                        (A) Androgens measured with the addition of 1 mM NAD+, from 2.5 to 60 min, n = 3 replicates per timepoint. (B) Androgens measured without addition of NAD+, from 10 to 360 min. n = 2 replicates per timepoint.  (Inset) Estrogens measured without addition of NAD+.   40 Figure 2.2   Representative HPLC chromatograph illustrating the peak and retention times of [3H]AE.                           Non-breeding male song sparrow brain supernatants were incubated with 200 nM [3H]DHEA for 5 min.  Steroids were separated using TLC and dried eluates were resuspended with methanol and injected through HPLC.                   [3H]AE % Mobile Phase   41 Figure 2.3   Seasonal profiles of baseline 3?-HSD activity in wild male song sparrows.   0.000.050.100.150.200.250.300.350.400.450.500.55BreedingMoltNon-breedingCentral medialtelencephalon     Caudaltelencephalon Ventromedialtelencephalon     Rostraldiencephalon     Caudaldiencephalon*******     [3H]DHEA was converted to [3H]AE and [3H]5?-A.  3?-HSD activity was significantly higher in several regions (n=6). *p<0.05, **p<0.01.               42 Figure 2.4   Effect of simulated territorial intrusions on brain 3?-HSD activity in free-living non-breeding song sparrows.     01234ControlSTICentral medialtelencephalonCaudaltelencephalon***    [3H]DHEA was added as a substrate and no NAD+ was added exogenously.  3?-HSD activity rapidly increased in the central medial telencephalon (n=8 control, n=8 STI) and caudal telencephalon (n=7 control, n=9 STI). *p<0.05, ** p<0.01      43 Figure 2.5   Hypothetical levels of systemic and local androgens in seasonally breeding male songbirds.                 Patterns of systemic (solid line) and local (dashed line) levels of androgens in seasonally breeding male songbirds. The portion between levels A and B represents the approximate region for baseline androgen levels in the brain throughout the year.  Level A, the non-breeding baseline of systemic androgens; Level B, the breeding baseline of systemic androgens; Level C, transient increases in systemic androgens during the breeding season, and transient increases in local androgens during the non-breeding season, in response to social challenge.  The duration of increases can be highly variable, depending upon environmental and physiological factors (1).  Dotted lines for A, B, and C represent approximate, rather than absolute levels.       C B A Jan Feb  Mar Apr  May  Jun  Jul    Aug   Sep   Oct   Nov   Dec systemic androgens  local ens   44 References 1. Wingfield JC, Hegner RE, Dufty AM & Ball GF (1990) The challenge hypothesis - theoretical implications for patterns of testosterone secretion, mating systems, and breeding strategies. Am Nat 136, 829-846. 2. Archer J (2006) Testosterone and human aggression: An evaluation of the challenge hypothesis. Neurosci Biobehav Rev 30, 319-345. 3. Caldwell GS, Glickman SE & Smith ER (1984) Seasonal aggression independent of seasonal testosterone in wood rats. Proc Natl Acad Sci 81, 5255-5257. 4. Wingfield JC & Hahn TP (1994) Testosterone and territorial behavior in sedentary and migratory sparrows. Anim Behav 47, 77-89. 5. Hau M, Wikelski M, Soma KK & Wingfield JC (2000) Testosterone and year-round territorial aggression in a tropical bird. Gen Comp Endocrinol 117, 20-33. 6. Lynn SE & Wingfield JC (2008) Dissociation of testosterone and aggressive behavior during the breeding season in male chestnut-collared longspurs, calcarius ornatus. Gen Comp Endocrinol 156, 181-189. 7. Soma KK, Scotti MA, Newman AE, Charlier TD & Demas GE (2008) Novel mechanisms for neuroendocrine regulation of aggression. Front Neuroendocrinol in press 8. Schmidt KL, et al (2008) Neurosteroids, immunosteroids, and the balkanization of endocrinology. Gen Comp Endocrinol 157, 266-274. 9. Wingfield JC, Lynn S & Soma KK (2001) Avoiding the 'costs' of testosterone: Ecological bases of hormone-behavior interactions. Brain Behav Evol 57, 239-251. 10. Soma KK (2006) Testosterone and aggression: Berthold, birds and beyond. J Neuroendocrinol 18, 543-551. 11. Soma KK & Wingfield JC (2001) Dehydroepiandrosterone in songbird plasma: Seasonal regulation and relationship to territorial aggression. Gen Comp Endocrinol 123, 144-155. 12. Soma KK, Wissman AM, Brenowitz EA & Wingfield JC (2002) Dehydroepiandrosterone (DHEA) increases territorial song and the size of an associated brain region in a male songbird. Horm Behav 41, 203-212. 13. Mellon, S.H. and Griffin, L.D. (2002) Neurosteroids: Biochemistry and clinical significance. Trend Endocrinol and Metabol 13, 35-43. 14. Widstrom RL & Dillon JS (2004) Is there a receptor for dehydroepiandrosterone or dehydroepiandrosterone sulfate?. Semin Reprod Med 22, 289-298. 15. Soma KK, Sullivan K & Wingfield J (1999) Combined aromatase inhibitor and antiandrogen treatment decreases territorial aggression in a wild songbird during the nonbreeding season. Gen Comp Endocrinol 115, 442-453.   45 16. Soma KK, Tramontin AD & Wingfield JC (2000) Oestrogen regulates male aggression in the non-breeding season. Proc Biol Sci 267, 1089-1096. 17. Vanson A, Arnold AP & Schlinger BA (1996) 3?-hydroxysteroid Dehydrogenase/isomerase and aromatase activity in primary cultures of developing zebra finch telencephalon: Dehydroepiandrosterone as substrate for synthesis of androstenedione and estrogens. Gen Comp Endocrinol 102, 342-350. 18. London SE, Monks DA, Wade J & Schlinger BA (2006) Widespread capacity for steroid synthesis in the avian brain and song system. Endocrinology 147, 5975-5987. 19. Schlinger BA, Pradhan DS & Soma KK (2008) 3?-HSD activates DHEA in the songbird brain. Neurochem Int 52, 611-620. 20. Soma KK, Bindra RK, Gee J, Wingfield JC & Schlinger BA (1999) Androgen-metabolizing enzymes show region-specific changes across the breeding season in the brain of a wild songbird. J Neurobiol 41, 176-188. 21. Soma KK, Alday NA, Hau M & Schlinger BA (2004) Dehydroepiandrosterone metabolism by 3?-hydroxysteroid dehydrogenase/?5-?4 isomerase in adult zebra finch brain: Sex difference and rapid effect of stress. Endocrinol 145, 1668-1677. 22. Pradhan DS, Yu Y & Soma KK (2008) Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain. J Neurochem 104, 244-253. 23. Tam H & Schlinger BA (2007) Activities of 3?-HSD and aromatase in slices of developing and adult zebra finch brain. Gen Comp Endocrinol 150, 26-33. 24. Skoog DA, Holler JF & Neiman TA (1998) Principles of Instrumental Analysis, (Harcourt Brace College Publishers, Philadelphia). 25. Wingfield JC & Soma KK (2002) Spring and autumn territoriality in song sparrows: Same behavior, different mechanisms?. Integr Comp Biol 42, 11-20. 26. Ball GF, et al (2004) Seasonal plasticity in the song control system: Multiple brain sites of steroid hormone action and the importance of variation in song behavior. Ann N Y Acad Sci 1016, 586-610. 27. Nice MM (1943) Studies in the life history of the song sparrow. II. the behavior of the song sparrow and other passerine birds.6, 1-328. 28. Cornil CA, Ball GF & Balthazart J (2006) Functional significance of the rapid regulation of brain estrogen action: Where do the estrogens come from?. Brain Res 1126, 2-26. 29. Schlinger BA, Pradhan DS & Soma KK (2008) 3?-HSD activates DHEA in the songbird brain. Neurochem Int 52, 611-620. 30. Soma KK, Schlinger BA, Wingfield JC & Saldanha CJ (2003) Brain aromatase, 5?-reductase, and 5?-reductase change seasonally in wild male song sparrows: Relationship to aggressive and sexual behavior. J Neurobiol 56, 209-221.   46 31. Demas GE, Polacek KM, Durazzo A & Jasnow AM (2004) Adrenal hormones mediate melatonin-induced increases in aggression in male Siberian hamsters (Phodopus sungorus). Horm Behav 46, 582-591. 32. Soma KK, Bindra RK, Gee J, Wingfield JC & Schlinger BA (1999) Androgen-metabolizing enzymes show region-specific changes across the breeding season in the brain of a wild songbird. J Neurobiol 41, 176-188. 33. Riters LV, et al (2001) Seasonal variation in androgen-metabolizing enzymes in the diencephalon and telencephalon of the male European starling (Sturnus vulgaris). J Neuroendocrinol 13, 985-997. 34. Pasmanik M & Callard GV (1988) Changes in brain aromatase and 5 alpha-reductase activities correlate significantly with seasonal reproductive cycles in goldfish (Carassius auratus). Endocrinology 122, 1349-1356. 35. Newman SW (1999) The medial extended amygdala in male reproductive behavior. A node in the mammalian social behavior network. Ann N Y Acad Sci 877, 242-257. 36. Goodson JL, Saldanha CJ, Hahn TP & Soma KK (2005) Recent advances in behavioral neuroendocrinology: Insights from studies on birds. Horm Behav 48, 461-473. 37. Black MP, Balthazart J, Baillien M & Grober MS (2005) Socially induced and rapid increases in aggression are inversely related to brain aromatase activity in a sex-changing fish, Lythrypnus dalli. Proc R Soc B 272, 2435-2440. 38. Balthazart J, et al (2006) Rapid changes in production and behavioral action of estrogens. Neuroscience 138, 783-791. 39. Taziaux M, Keller M, Bakker J & Balthazart J (2007) Sexual behavior activity tracks rapid changes in brain estrogen concentrations. J Neurosci 27, 6563-6572. 40. Trainor BC, Lin S, Finy MS, Rowland MR & Nelson RJ (2007) Photoperiod reverses the effects of estrogens on male aggression via genomic and nongenomic pathways. Proc Natl Acad Sci USA 104, 9840-9845. 41. Agarwal AK & Auchus RJ (2005) Minireview: Cellular redox state regulates hydroxysteroid dehydrogenase activity and intracellular hormone potency. Endocrinology 146, 2531-2538. 42. Agarwal AK & Auchus RJ (2005) Minireview: Cellular redox state regulates hydroxysteroid dehydrogenase activity and intracellular hormone potency. Endocrinology 146, 2531-2538. 43. Rohmann KN, Schlinger BA & Saldanha CJ (2007) Subcellular compartmentalization of aromatase is sexually dimorphic in the adult zebra finch brain. Dev Neurobiol 67, 1-9. 44. Chapman JC & Sauer LA (1979) Intracellular localization and properties of 3?-hydroxysteroid dehydrogenase/isomerase in the adrenal cortex. J Biol Chem 254, 6624-6630.   47 45. Cherradi N, Defaye G & Chambaz EM (1994) Characterization of the 3?-hydroxysteroid dehydrogenase activity associated with bovine adrenocortical mitochondria. Endocrinology 134, 1358-1364. 46. Peterson RS, Yarram L, Schlinger BA & Saldanha CJ (2005) Aromatase is pre-synaptic and sexually dimorphic in the adult zebra finch brain. Proc Biol Sci 272, 2089-2096. 47. Rohmann KN, Schlinger BA & Saldanha CJ (2007) Subcellular compartmentalization of aromatase is sexually dimorphic in the adult zebra finch brain. Dev Neurobiol 67, 1-9. 48. Balthazart J, Baillien M & Ball GF (2006) Rapid control of brain aromatase activity by glutamatergic inputs. Endocrinology 147, 359-366. 49. Wingfield JC, Lynn S & Soma KK (2001) Avoiding the 'costs' of testosterone: Ecological bases of hormone-behavior interactions. Brain Behav Evol 57, 239-251.                     48 CONCLUSIONS AND GENERAL DISCUSSION  The goal of this thesis was to study the potential regulation of steroidogenic enzymes in the brain using an integrative approach.  In the field, we manipulated the behavior of a songbird in its natural environment and collected tissue for neurochemistry analyses in the laboratory.  The data from Experiment 1 provide an example of long-term regulation of plasma DHEA and T levels, as well as steroidogenic enzymes in different regions of the brain as a function of season.  In Experiment 2, I studied animals from the non-breeding season only, to examine the short-term regulation of steroidogenic enzymes by male-male conspecific interactions.  Previous studies had ruled out the role of sex steroids in regulating non-breeding aggression, based on their low levels in systemic circulation (1).  By measuring activity of steroidogenic enzymes in the brain, I challenged the accepted notion.  These results suggest a seasonal shift in the physiological mechanisms regulating behavior, from being primarily systemic in the breeding season, to primarily local during the non-breeding season (2).    In summary, regulation of brain 3?-HSD activity by social encounters is rapid and region-specific.  As reported in previous studies, these data indicate that the endocrine environment in the brain is dynamic and probably involve multiple regulatory mechanisms (3).  The conclusions about the neuroendocrine regulation of non-breeding aggression in song sparrows are limited.  In vivo measurement of brain DHEA, T, and E2 levels in discrete regions will complement studies on steroidogenic capacity of those tissues.  We can predict that decrease in DHEA levels in STI stimulated birds will parallel with the present data suggesting increased metabolism, while the increase in levels of active androgens would parallel with their increased synthesis.   It would also be useful to measure the regulation of   49 brain steroidogenic enzymes and steroid levels by social encounters during the breeding season. In the present study, aromatase activity decreased in the telencephalon.  This could be a reflection of the dynamic steroid environment and measuring additional timepoints earlier than 30 min may help understand the timecourse.  Further, we decided to measure 3?-HSD activity by adding NAD+ exogenously in all the brain tissues a priori, based on previous studies (4, 5).  It would have been useful to divide the tissue appropriately to measure 3?-HSD activity in the absence of NAD+ in other brain regions in as well.  For example, the diencephalon is very important in the control of motivated behaviors and has been reported to have high aromatase activity.     Here, we performed gross dissections to study conversion of DHEA to AE via 3?-HSD in different brain regions.  However, measurement of enzyme activity in large chunks of tissue may not be reflective of the true steroidogenic capacity of the specific region, due to dilution by non-steroidogenic tissue.  Measuring 3?-HSD activity in minute amounts of brain tissue by using laser guided micro-dissection or Palkovits Punch technique will provide better spatial resolution.     Finally, ongoing studies must clarify the cellular mechanisms involved in the rapid regulation of 3?-HSD activity. These might involve compounds such as catecholamines, endozepines and neuropeptides, which are known to act as neuromodulators and neurotransmitters.  We could also perform long-term social stimulation studies by performing multiple simulated territorial intrusions over days.  These studies will be critical in understanding the regulation of DHEA metabolism in the adult brain.      50 References 1. Wingfield JC (1994) Control of territorial aggression in a changing environment. Psychoneuroendocrinol 19, 709-721. 2. Schmidt KL, et al (2008) Neurosteroids, immunosteroids, and the balkanization of endocrinology. Gen Comp Endocrinol 157, 266-274  3. Soma KK, Bindra RK, Gee J, Wingfield JC & Schlinger BA (1999) Androgen-metabolizing enzymes show region-specific changes across the breeding season in the brain of a wild songbird. J Neurobiol 41, 176-188. 4. Soma KK, Alday NA, Hau M & Schlinger BA (2004) Dehydroepiandrosterone metabolism by 3?-hydroxysteroid dehydrogenase/?5-?4 isomerase in adult zebra finch brain: Sex difference and rapid effect of stress. Endocrinol 145, 1668-1677. 5. Pradhan DS, Yu Y & Soma KK (2008) Rapid estrogen regulation of DHEA metabolism in the male and female songbird brain. J Neurochem 104, 244-253.   

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